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UBC Theses and Dissertations

Ultrastructure of Achlya bisexualis Coker and Couch Ricker, Nancy Anne 1971

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THE ULTRASTRUCTURE OF Achlya bisexualis COKER AND COUCH by Nancy Anne R i c k e r B .A. , U n i v e r s i t y o f C a l i f o r n i a S an t a B a r b a r a , 1963 M . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTORATE OF PHILOSOPHY i n t h e Depar tment o f Bo tany We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA J u l y , 1971 In presenting th i s thes i s in pa r t i a l f u l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f r ee l y ava i l ab le for reference and study. I fu r ther agree tha permission for extensive copying of th i s thes is for scho lar ly purposes may be granted by the Head of my'Department or by his representat ives. It is understood that copying or pub l i ca t ion of th i s thes is fo r f i nanc ia l gain shal l not be allowed without my wr i t ten permission. Department of "tBoTfl/VV  The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada Date A o u y VSH For my husband and my p a r e n t s , a l l o f whom have made t h i s s t u d y p o s s i b l e . i A b s t r a c t The u l t r a s t r u c t u r e o f t h e v e g e t a t i v e hyphae and o f a s e x u a l and s e x u a l r e p r o d u c t i v e s t a g e s o f Achlya bisexualis C oke r and Couch i s s t u d i e d and i s compared w i t h t h a t o f o t h e r Oomycetes . A l s o c o n s i d e r e d a r e p o s s i b l e s t r u c t u r a l mechanisms of c e l l u l a r g r o w t h , s e p t a t i o n , z o o s p o r o g e n e s i s and s e x u a l r e p r o d u c t i o n . The v e g e t a t i v e hyphae a r e c h a r a c t e r i z e d by an a p i c a l g r ow ing z o n e , a s u b - a p i c a l m i t o c h o n d r i a l zone and a d i s t a l m u l t i - o r g a n e l l e z o n e . The a p i c a l g r o w i n g zone c o n t a i n s numerous v e s i c l e s , u s u a l l y t o t h e e x c l u s i o n of o t h e r o r g a n e l l e s and i n c l u s i o n s . The s u b - a p i c a l zone c o n s i s t s of morpho-l o g i c a l l y s i m i l a r v e s i c l e s , bu t m i t o c h o n d r i a and m i c r o b o d i e s a r e abundant i n i t s c e n t r a l p r o t o p l a s m . The d i s t a l m u l t i - o r g a n e l 1 e zone i s c h a r a c t e r i z e d by a v a r i e t y of o r g a n e l l e s and i n c l u s i o n s : d i c t y o s o m e s , E .R. , m i t o c h o n d r i a , r i b o s o m e s , m i c r o b o d i e s , m u l t i - v e s i c u l a r b o d i e s , l i p i d d r o p l e t s , n u c l e i and c e n t r i o l e s . The d i c t y o s o m e s o c c u r i n a s s o c i a t i o n w i t h E.R. and/o r n u c l e i ; c e n t r i o l e s appea r nea r m o d i f i e d r e g i o n s of n u c l e a r e n v e l o p e s ; r i b o s omes a r e most abundant i n p r o t o p l a s m n e a r e s t a c t i v e l y g r o w i n g r e g i o n s ; and l i p i d d r o p l e t s a c c u m u l a t e i n o l d e r a r e a s . L iposomes a l s o become e v i d e n t as hyphal d i f f e r e n t i a t i o n o c c u r s , and v a c u o l a t i o n of t h e p r o t o p l a s m i s common. The w a l l s o f t he v e g e t a t i v e hyphae a r e composed of a two phase sy s tem i n w h i c h f i b r i l s a r e embedded i n an amorphous m a t r i x . They a l s o a r e o f t e n c h a r a c t e r i z e d by p o c k e t s o f e l e c t r o n dense g r a n u l a r and v e s i c u l a r m a t e r i a l w h i c h f o rm d u r i n g a p p o s i t i o n d e p o s i t i o n of w a l l c o n s t i t u e n t s . The gemmae po s se s s t h e same v a r i e t y of o r g a n e l l e s and i n c l u s i o n s as o b s e r v e d i n v e g e t a t i v e hyphae. However, t h e i r p r o t o p l a s m i s more dense and zones of m o r p h o l o g i c a l o r g a n i z a t i o n a r e no t a p p a r e n t . The w a l l s a r e i i e x t r e m e l y t h i c k . The z o o s p o r a n g i a appea r as t e r m i n a l r e g i o n s o f v e g e t a t i v e hyphae w h i c h have become d e l i m i t e d f r om s u b t e n d i n g p r o t o p l a s m by s e p t a . They each u s u a l l y d e v e l o p a s i n g l e a p i c a l p a p i l l a t h r o u g h w h i c h p r i m a r y z o o -s p o r e i n i t i a l s e s c a p e . The i n i t i a l s a r e fo rmed by t h e c l e a v a g e o f t h e p r o -t o p l a s m - a p r o c e s s e f f e c t e d by numerous s t r u c t u r a l c hange s . The re i s t h e deve l opmen t o f p a r a s t r a s o m e s ; n u c l e i become p y r i f o r m and e q u i d i s t a n t l y spaced f r o m each o t h e r and f rom t h e s p o r a n g i a ! w a l l ; p a i r s o f c e n t r i o l e s d i f f e r e n t i a t e i n t o b a s a l b o d i e s ; b a s a l b o d i e s g i v e r i s e t o s h o r t axonemes w h i c h e x t e n d i n t o e x p a n d i n g axonemal v e s i c l e s ; a n d , o t h e r o r g a n e l l e s and i n c l u s i o n s become o r i e n t e d abou t t h e n u c l e i - t h e c l e a v a g e v e s i c l e s p a r t i -c u l a r l y becoming a l i g n e d and f u s i n g i n p l a n e s abou t each o f t h e s e a g g r e -ga ted masses o f p r o t o p l a s m to f o rm z o o s p o r e i n i t i a l s . E n c y s t e d p r i m a r y z o o -s po re s d i f f e r e n t i a t e f rom t h e i n i t i a l s and c h a r a c t e r i s t i c S a p r o l e g n i a l e s b i f l a g e l l a t e s e conda r y z o o s p o r e s d e v e l o p f r o m t h e p r i m a r y z o o s p o r e p r o t o -p l a s t s . The f l a g e l l a t e a p p a r a t u s i n each z o o s p o r e i s c o n n e c t e d w i t h t h e o u t e r membrane o f t h e n u c l e a r e n v e l o p e and i t c o n s i s t s o f an axoneme, b a s a l body and r o o t l e t s . The r o o t l e t s a r e t h o u g h t t o be f o r m e d , d i r e c t l y o r i n d i r e c t l y , by p a r a s t r a s o m e s . The gametang ia a r e m o r p h o l o g i c a l l y d i s t i n c t f r o m each o t h e r . A n t h e r i -d i a a r e t h i n - w a l l e d and po s se s s a s i m i l a r p a t t e r n o f s t r u c t u r a l o r g a n i z a t i o n t o t h a t o f v e g e t a t i v e hyphae. T h e i r m i t o c h o n d r i a m o s t l y appea r as r o d s w i t h t e r m i n a l o r s u b - t e r m i n a l i n v a g i n a t i o n s ; l i p o s o m e s a r e se ldom p r e s e n t i n t h e i r p r o t o p l a s m . The o o g o n i a a r e g l o b o s e c e l l s and t h e i r p r o t o p l a s m c o n s i s t s p r e d o m i n a n t l y o f l i p o s o m e s and l i p i d d r o p l e t s . The n u c l e i i n bo th a n t h e r i d i a and o o g o n i a d i v i d e m e i o t i c a l l y . D u r i n g s e x u a l r e p r o d u c t i o n , a n t h e r i d i a encompass o o g o n i a and bo th i i i c e l l t y p e s r e s p e c t i v e l y a r e d e l i m i t e d f r om hypha l p r o t o p l a s m by s e p t a . F e r t i l i z a t i o n t u b e s , f o rmed by a n t h e r i d i a , p e n e t r a t e t h e w a l l s o f o o g o n i a and e f f e c t f e r t i l i z a t i o n i n p r e - c l e a v e d and t h i n - w a l l e d o o s p h e r e s . The f e r t i l i z e d oo sphe re s d e v e l o p t h i c k w a l l s and d i f f e r e n t i a t e i n t o o o s p o r e s . The p r o t o p l a s m o f t h e o o s p o r e s i s dense and i n each c e l l t h e r e i s a s i n g l e l a r g e n u c l e u s . E l o n g a t i o n and/o r e x p a n s i o n o f v e g e t a t i v e hyphae and o f a s e x u a l and s e x u a l r e p r o d u c t i v e s t r u c t u r e i s t h o u g h t t o o c c u r by t h e f u -s i o n o f d i c t y o s o m e - d e r i v e d s e c r e t o r y v e s i c l e s w i t h t h e p lasma membrane -a mechans/i)m p r o v i d i n g f o r bo th an i n c r e a s e i n s u r f a c e a r e a o f t h e p lasma membrane and f o r t h e d e p o s i t i o n o f w a l l c o n s t i t u e n t s and o t h e r m o l e c u l e s o u t w a r d l y . S e p t a t i o n and a p p o s i t i o n a l w a l l f o r m a t i o n s i m i l a r l y o c c u r e x c e p t t h a t p o c k e t s o f e l e c t r o n dense g r a n u l a r and v e s i c u l a r m a t e r i a l o f t e n a r e encased i n d e p o s i t e d w a l l m a t e r i a l . i v TABLE OF CONTENTS INTRODUCTION - - - - - — — — - — .— . 1 MATERIALS AND METHODS O rgan i sm — - - . — _ - - - - - - . - - - _ — — _ _ • _ _ _ — . — _ _ . ______ . 7 L i g h t M i c r o s c o p y T e c h n i q u e s — _ - _ _ — _ — 7 U l t r a - t h i n S e c t i o n T e c h n i q u e s - E l e c t r o n M i c r o s c o p y - - - 8 . F r e e z e - E t c h i n g T e c h n i q u e s •- ____. 8 S h a d o w - C a s t i n g T e c h n i q u e s ___________ . .__.______. 9 RESULTS . V e g e t a t i v e Hyphae — _ _ _ _ _ _ _ — _ _ _ _ _ _ _ _ _ _ _ _ _ _ .-..10 A p i c a l G row ing Zone ________ • IQ S u b - a p i c a l M i t o c h o n d r i a l Zone — — - _ _ - l l D i ' s t a T M u l t i - O r g a n e l l e Z o n e — — - - - - — — — : 13 Hypha l M a t u r a t i o n and D i f f e r e n t i a t i o n — . — . - - ' 19 Gemmae — — — 21 Z o o s p o r a n g i a L i g h t M i c r o s c o p y O b s e r v a t i o n s 26 The F i n e S t r u c t u r e o f Z o o s p o r a n g i a l Deve lopment — . • 28 E n c y s t e d P r i m a r y Z o o s p o r e s — ' • - — _ . - - - — " - - , 35-F T a g e l l a t e d S e c o n d a r y Z o o s p o r e s — — _ - . — 4 0 E n c y s t e d S e c o n d a r y Z o o s p o r e s . — - - - — - - - - - — _ _ • — _ • . 42 • Germ Tube F o r m a t i o n — — _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ — _ _ _ _ — 45 A n t h e r i d i a ! and O o g o n i a l Deve lopment . L i g h t M i c r o s c o p y O b s e r v a t i o n s ' — - - - — 45 V F i n e S t r u c t u r e o f A n t h e r i d i a l Hyphae — — - — : — 46 F i n e S t r u c t u r e o f Opgon ia — - — - - - — . ' - , . . v v . - . - , . . . - 52 Oosphere F i n e S t r u c t u r e - - — - - _ - - _ , — _ — _ — _ — , — 57 Oospore s — ; — _ _ — — _ — _ — — _ „ _ _ _ _ ,__ 58 DISCUSSION F i n e S t r u c t u r e o f Aohlya b-teexualis C e l l W a l l s — - — - - - - — — — — — - - - - - — - - — — — - . — ,-. 60 P l a sma Membrane - — — - 63 Lomasomes and LomasomeT l i ke C o n f i g u r a t i o n s — - . _ - — — — 6 5 ' P e l l i c l e - — - — _ - 67 M i t o c h o n d r i a — — - — — 69 L iposomes — - . — — -. 71 P a r a s t r a s o m e s — . — - - _ 73 D i c t y o s o m e s — — , — . - . — — - — . 74 M i c r o b o d i e s — ________ 75 M i c r o t u b u l e P a c k e t s and M i c r o t u b l e V e s i c l e s •- 78 N u c l e u s - - - - - — — — — — — — — — — - — 79 C e n t r i o l e s —~------—--r .-- 83 F l a g e l l a ______ _ _________ 8 5 C e l l u l a r Growth and A p p o s i t i o n a l Wa l l F o r m a t i o n 88 Gi S e p t a l S t r u c t u r e and Deve l opmen t — ' • ..91; Z o o s p o r o g e n e s i s S e p t a t i o n - - - - - - - — — ______ P a p i l l a F o r m a t i o n — - — — . . — • 94 B a s a l Body and F l a g e l l a Deve l opment — • 95 P r o t o p l a s m i c C l e a v a g e — , — 95 v i S e x u a l R e p r o d u c t i o n A n t h e r i d i a ! B r a n c h I n i t i a t i o n s - T - - ^ 96 O o g o n i a ! I n i t i a l Deve l opmen t - - - - - - - - - - - - - - - - - - - - - - - - - - - - — 9 8 ' • A n t h e r i d i a i n C o n t a c t w i t h Oogon ia - - ' — • • — 99 S e p t a t i o n .———————— — - - . — - — - - - - — - — 9 9 F e r t i l i z a t i o n Tube F o r m a t i o n and P e n e t r a t i o n — — 100 Oogon i a ! C l e a v a g e - - - - - - - T - - — — 1 0 0 M e i o s i s _._=— : _ _ l o i ; CONCLUSION — r — — — ! 0 4 BIBLIOGRAPHY ... 1 0 6 APPENDIX — — — — — — — - . — T - 1 1 6 v-i-A L IST OF DIAGRAMS D iag ram I D iag ram I I D iag ram I I I D iag ram IV D iag ram V D iag ram VI ; C y t o p l a s m i c Reg i on s o f t h e V e g e t a t i v e Hyphae Mo rpho logy o f an A c t i v e l y G row ing Hypha l T i p C e n t r i o l e S t r u c t u r e Ba s a l Body Mo rpho l o g y Mo rpho l ogy o f t h e F l a g e l l a t e d Se conda r y Zoo spo re M i t o c h o n d r i a l Mo rpho l o g y v i i LIST OF TABLES TABLE I M a t e r i a l s f o r L i g h t and E l e c t r o n Microscopy Pro-cedures •• — - — 116 TABLE II F i x a t i o n , Dehydration and Embedding Procedures f o r E l e c t r o n Microscopy ;_________________ u g TABLE III T h i n - S e c t i o n , L i g h t Microscopy Procedures —-- 121 TABLE IV Hyphal Growth. Rates of -Aohlya b i s e x u a l i s 122 TABLE V Wall Morphology — _ — — — — _ 124 TABLE VI Lomasome Morphology and D i s t r i b u t i o n —---- 132 TABLE VII Mitochondrial Morphology and D i s t r i b u t i o n — — 138 TABLE VIII Microbody-Morphology and D i s t r i b u t i o n 149 TABLE IX Dictyosome Morphology and D i s t r i b u t i o n --— 153 TABLE .X Microtubule Packet Morphology and D i s t r i b u t i o n 159 TABLE0(1 Parastrasome Morphology and D i s t r i b u t i o n 164 TABLE-XII Liposome Morphology and D i s t r i b u t i o n — 167 TABLE .XIIL L i p i d D r o plet Morphology and D i s t r i b u t i o n 175 TABLE .Xiy C e n t r i o l e Morphology and D i s t r i b u t i o n — — 181 TABLE ;XV F l a g e l l a r Morphology and D i s t r i b u t i o n — 186 TABLE .XVI Nuclear Morphology and D i s t r i b u t i o n ------------------ 194 TABLE XVII Fine S t r u c t u r a l Changes Observed During Sexual Reproduction —•--— •— 97 v i i i P l a t e One P l a t e Two P l a t e T h r e e P l a t e Four P l a t e F i v e P l a t e S i x P l a t e Seven P l a t e E i g h t P l a t e N i n e P l a t e Ten P l a t e E l e v e n P l a t e Twe l ve P l a t e T h i r t e e n P l a t e F o u r t e e n P l a t e F i f t e e n P l a t e S i x t e e n P l a t e S e v e n t e e n P l a t e E i g h t e e n L I ST OF PLATES F i g u r e s 1-2 F i g u r e s 3-8 F i g u r e s 9-13 F i g u r e s 14 -19 F i g u r e s 20-24 F i g u r e s 25 -29 F i g u r e 30 F i g u r e s 31 -33 F i g u r e 34 F i g u r e s 35 -40 F i g u r e 41 F i g u r e 42 F i g u r e s 43 -44 F i g u r e 45 F i g u r e s 46-47 F i g u r e s 48 -50 F i g u r e s 51-53 F i g u r e s 54-57 C u l t u r e s o f Achlya bisexua-lis G e r m i n a t i o n o f e n c y s t e d s e -c o n d a r y z o o s p o r e s V e g e t a t i v e Hyphae (LM) V e g e t a t i v e Hyphae (LM) V e g e t a t i v e Hyphae (LM) V e g e t a t i v e Hyphae (LM) A p i c a l g r o w i n g zone o f v e -g e t a t i v e hypha A p i c a l g r ow ing zone o f v e -g e t a t i v e hypha S u b - a p i c a l m i t o c h o n d r i a l zone o f v e g e t a t i v e hypha S t r u c t u r e s common to s u b - a p i c a l m i t o c h o n d r i a l zone o f v e g e -t a t i v e hypha D i s t a l m u l t i - o r g a n e l l e zone o f v e g e t a t i v e hypha V a c u o l a r deve l opment i n d i s -t a l m u l t i - o r g a n e l l e zone o f v e g e t a t i v e hypha V a c u o l a t i o n i n d i s t a l m u l t i -o r g a n e l l e zone V a c u o l a t i o n i n d i s t a l m u l t i -o r g a n e l l e zone O ld v a c u o l a t e d r e g i o n o f v e -g e t a t i v e hyphae Hyphal w a l l mo rpho logy Hyphal w a l l mo rpho l ogy M i t o c h o n d r i a i n v e g e t a t i v e hyphae i x P l a t e N i n e t e e n P l a t e Twenty P l a t e Twenty -one P l a t e Twenty - two P l a t e T w e n t y - t h r e e P l a t e T w e n t y - f o u r P l a t e T w e n t y - f i v e P l a t e T w e n t y - s i x P l a t e Twen t y - s e ven P l a t e T w e n t y - e i g h t P l a t e T w e n t y - n i n e P l a t e T h i r t y P l a t e T h i r t y - o n e P l a t e T h i r t y - t w o P l a t e T h i r t y - t h r e e P l a t e T h i r t y - f o u r P l a t e T h i r t y - f i v e P l a t e T h i r t y - s i x P l a t e T h i r t y - s e v e n F i g u r e s 58-62 F i g u r e s 63-64 F i g u r e s 65-68 F i g u r e s 69-72 F i g u r e s 73 -76 F i g u r e s 77-83 F i g u r e 84 F i g u r e s 85 -86 F i g u r e 87 F i g u r e s 88 -89 F i g u r e s 90-93 F i g u r e s 94-97 F i g u r e 98 F i g u r e 99 F i g u r e s 100-101 F i g u r e s 102-103 F i g u r e s 104-109 F i g u r e s 110-112 F i g u r e s 113-114 D i c t yo somes i n t h e d i s t a l m u l t i - o r g a n e l l e zone D i c t y o s omes i n o l d e r and/o r d i f f e r e n t i a t i n g v e g e t a t i v e hyphae D i c t y o s omes i n d i f f e r e n t i a t i n g v e g e t a t i v e hyphae L iposome morpho logy and d e -ve l opmen t L iposome and m u l t i - v e s i c u l a r b o d i e s i n hyphal p r o t o p l a s m M i c r o b o d i e s i n d i f f e r e n t i a -t i n g v e g e t a t i v e hyphae N u c l e u s i n hypha l p r o t o p l a s m N u c l e i i n d i s t a l m u l t i - o r g a n -e l l e zone M i t o t i c a l l y d i v i d i n g n u c l e u s i n hypha l p r o t o p l a s m N u c l e i i n o l d hypha l p r o t o -p la sm N u c l e a r s u b - s t r u c t u r e and c e n -t r i o l e s i n v e g e t a t i v e hyphae Gemmae (LM) Gemma i n l o n g i t u d i n a l s e c t i o n Gemma i n l o n g i t u d i n a l s e c t i o n Gemma apex and w a l l s t r u c t u r e S ep t a d e l i m i t i n g gemmae M i c r o b o d i e s and m i t o c h o n d r i a i n gemma p r o t o p l a s m D i c t yo somes i n gemma,p ro to -p la sm D i c t yo somes i n gemma p r o t o -p lasm P l a t e T h i r t y - e i g h t P l a t e T h i r t y - n i n e P l a t e F o r t y P l a t e F o r t y - o n e P l a t e F o r t y - t w o P l a t e F o r t y - t h r e e P l a t e F o r t y - f o u r P l a t e F o r t y - f i v e P l a t e F o r t y - s i x P l a t e F o r t y - s e v e n P l a t e F o r t y - e i g h t P l a t e F o r t y - n i n e P l a t e F i f t y P l a t e F i f t y - o n e P l a t e F i f t y - t w o P l a t e F i f t y - t h r e e P l a t e F i f t y - f o u r x F i g u r e s 115-116 F i g u r e 117 F i g u r e s 118 -119 F i g u r e s 120-123 F i g u r e s 124-132 F i g u r e s 133 -139 F i g u r e s 140-141 F i g u r e . 142 F i g u r e s 143-144 F i g u r e s 145-147 F i g u r e s 148-150 F i g u r e s 151-154 F i g u r e s 155-158 F i g u r e s 159-163 F i g u r e s 164 -169 F i g u r e s 170-174 F i g u r e s 175-177 V a c u o l a r m e t a b o l i t e s and l i p o -somes i n gemma p r o t o p l a s m N u c l e u s i n gemma N u c l e u s and n u c l e a r s u b - s t r u c -t u r e i n gemma p r o t o p l a s m C e n t r i o l e s i n gemmae Z o o s p o r a n g i a l d e v e l o p m e n t (LM) Z o o s p o r a n g i a and e n c y s t e d p r i -mary z o o s p o r e s (LM) L o n g i t u d i n a l s e c t i o n o f u n -c i eaved ' z o o s p o r a n g i a l p r o t o p l a s m nea r t i p L o n g i t u d i n a l . s e c t i o n o f z o o -spo rang i um p r i o r t o p r o t o -p l a s m i c c l e a v a g e C r o s s - s e c t i o n o f z oo spo r ang i um p r i o r t o p r o t o p l a s m i c c l e a -v a g e ; septum d e l i m i t i n g z oo spo r ang i um f r o m hypha; E a r l y s t a g e s o f p r o t o p l a s m i c c l e a v a g e i n z o o s p o r a n g i a . . F i n a l , c l e a v a g e s t a g e i n z o o s p o r -a n g i a l - d e v e l o p m e n t . - p r i m a r y z o o s p o r e i n i t i a l f o r m a t i o n P a p i l l a de ve l opmen t i n z o o s p o r -a n g i a C l e a v a g e p l a n e s i n z o o s p o r e d i f f e r e n t i a t i o n M i t o c h o n d r i a and m i c r o t u b u l e p a c k e t s i n z o o s p o r a n g i a l p r o -t o p l a s m P a r a s t r a s o m e s , l i p o s o m e s and m i c r o b o d i e s i n z o o s p o r a n g i a l p r o t o p l a s m D i c t yo somes i n z o o s p o r a n g i a l p r o t o p l a s m N u c l e u s . a n d n u c l e a r s u b - s t r u c -t u r e i n z o o s p o r a n g i a l p r o t o -p l a sm x i P l a t e F i f t y - f i v e P l a t e F i f t y - s i x P l a t e F i f t y - s e v e n P l a t e F i f t y - e i g h t -P l a t e F i f t y - n i n e P l a t e S i x t y P l a t e S i x t y - o n e P l a t e S i x t y - t w o P l a t e S i x t y - t h r e e . P l a t e S i x t y - f o u r P l a t e S i x t y - f i v e P l a t e S i x t y - s i x P l a t e S i x t y - s e v e n P l a t e S i x t y - e i g h t P l a t e S i x t y - n i n e P l a t e S e v e n t y F i g u r e s , 178-183 F i g u r e s 184-189 F i g u r e 190 F i g u r e s 191-192 F i g u r e s 193-196 F i g u r e s 197-199 F i g u r e s 200-202 F i g u r e s 203-205 F i g u r e s 206-211 F i g u r e s 212-214 F i g u r e 215 F i g u r e s 216-218 F i g u r e s 219 -220 F i g u r e s 221-228 F i g u r e s 229-232 F i g u r e s 233-235 B a s a l body mo rpho l ogy i n z o o -s p o r a n g i a l p r o t o p l a s m Ba s a l body m o r p h o l o g y : i n z o o -s p o r a n g i a l p r o t o p l a s m E n c y s t e d p r i m a r y z o o s p o r e E n c y s t e d p r i m a r y z o o s p o r e s M u l t i v e s i c u l a r b o d i e s , l i p o -somes, and w a l l s o f p r i m a r y z o o s p o r e s M i t o c h o n d r i a and o t h e r o r g a n -e l l e s i n p r i m a r y z o o s p o r e s V a c u o l e s i n p r i m a r y z o o s p o r e s M i c r o t u b u l e p a c k e t s and v e s i -c l e s i n p r i m a r y z o o s p o r e s L i po i - da l s t r u c t u r e s i n p r i m a r y z o o s p o r e p r o t o p l a s m and t h e i r deve l opmen t D i c t y o s o m e s i n p r i m a r y z o o -s p o r e s D i c t y p s o m e - n u c l e u s a s s o c i a t i o n i n . p r i m a r y z o o s p o r e N u c l e u s and n u c l e a r s u b - s t r u c -t u r e i n p r i m a r y z o o s p o r e N u c l e o l i i n p r i m a r y z o o s p o r e , n u c l e i S e c t i o n s o f p r i m a r y z o o s p o r e b a s a l b o d i e s B a s a l b o d i e s and a s s o c i a t e d s t r u c t u r e s i n e n c y s t e d p r i -mary z o o s p o r e s B a s a l b o d i e s , . r o o t l e t s and n u c l e i - . t h e i r i n t e r - a s s o -c i a t i o n i n p r i m a r y z o o s p o r e s X I 1 P l a t e S e v e n t y - o n e P l a t e S e v e n t y - t w o P l a t e S e v e n t y - t h r e e PI a t e S e v e n t y - f o u r P l a t e S e v e n t y - f i v e P l a t e S e v e n t y - s i x P l a t e S e v e n t y - s e v e n P l a t e . S e v e n t y - e i g h t P l a t e S e v e n t y - n i n e P l a t e E i g h t y P l a t e E i g h t y - o n e P l a t e E i g h t y - t w o P l a t e E i g h t y - t h r e e P l a t e E i g h t y - f q u r P l a t e E i g h t y - f i v e P l a t e E i g h t y - s i x P l a t e E i g h t y - s e v e n F i g u r e s 236-238 F i g u r e s 239-243 F i g u r e s 244-247 F i g u r e s 248 -250 F i g u r e s 251-255 F i g u r e s 256-264 F i g u r e s 265-266 F i g u r e s 267-271 F i g u r e s 272-275 . F i g u r e s 276 -280 F i g u r e s 281-285 F i g u r e s 286-290 F i g u r e s 291-296 F i g u r e s 297-301 F i g u r e s 302-303 F i g u r e s 304 -306 F i g u r e s 307-311 P a r a s t r a s o m e - b a s a l body r e l a -t i o n s h i p i n p r i m a r y z o o s p o r e s P a r a s t r a s o m e morpho l ogy and deve l opment i n p r i m a r y z o o -s p o r e s . P a r a s t r a s o m e s o f p r i m a r y z o o -s p o r e s • E n c y s t e d p r i m a r y z o o s p o r e s p r i o r t o g e r m i n a t i o n D i c t y o s o m e s o f ' e n c y s t e d , p r i -m a r y - z o o s p o r e s p r i o r t o g e r m i -n a t i o n and o f f l a g e l l a t e d s e -c o n d a r y z o o s p o r e s G e r m i n a t i o n o f e n c y s t e d p r i -mary z o o s p o r e s and morpho logy o f f l a g e l l a t e d s e c o n d a r y z o o -s p o r e s (LM) S e conda r y z o o s p o r e s P e l l i c l e o f s e c o n d a r y z o o s p o r e s F l a g e l l a r morpho logy o f s e c o n -d a r y z o o s p o r e s Seconda r y z o o s p o r e f r l a g e l l a Ency s tmen t o f s e c o n d a r y z o o -s p o r e s G e r m i n a t i o n o f e n c y s t e d s e c o n -d a r y z o o s p o r e s Germ t u b e morpho logy A n t h e r i d i a and o o g o n i a (LM) L o n g i t u d i n a l s e c t i o n o f young a n t h e r i d i a l hypha a t g r ow ing t i p A n t h e r i d i a p r i o r t o o o g o n i a l c o n t a c t M i t o c h o n d r i a i n a n t h e r i d i a l hyphae x i i i P l a t e E i g h t y - e i g h t P l a t e E i g h t y - n i n e P l a t e N i n e t y P l a t e N i n e t y - o n e P l a t e N i n e t y - t v i o P l a t e N i n e t y - t h r e e P l a t e N i n e t y - f o u r P l a t e N i n e t y - f i v e P l a t e N i n e t y - s i x P l a t e N i n e t y - s e v e n P l a t e N i n e t y - e i g h t P l a t e N i n e t y - . n i n e P l a t e One hundred P l a t e One hundred one P l a t e One hundred two P l a t e One hundred t h r e e P l a t e One hundred f o u r P l a t e One hundred f i v e F i g u r e s 312-314 F i g u r e s 315-318 F i g u r e s 319 -320 F i g u r e s 321 -322 F i g u r e s 323 -325 F i g u r e s 326-328 F i g u r e s 329-331 F i g u r e s 332 -335 F i g u r e s 336 -340 F i g u r e s 341-344 F i g u r e 345 F i g u r e s 346-347 F i g u r e s 348-351 F i g u r e s 352.-353 F i g u r e s 354-356 F i g u r e s 357 -359 F i g u r e s 360-364 F i g u r e s 365 -370 N u c l e i o f a n t h e r i d i a ; , m o r - • p h o l o g y o f a n t h e r i d i a i n c o n t a c t w i t h oogonium S e p t a l f o r m a t i o n i n a n t h e r i -d i a l hyphae S e p t a i n a n t h e r i d i a l hyphae S e p t a i n a n t h e r i d i a l hyphae M o r p h o l o g y . o f young oogonium Oogonium p r i o r t o a n t h e r i d i a l c o n t a c t D i c t y o s omes and o t h e r o r g a n -e l l e s c h a r a c t e r i s t i c o f o o g o n i a L iposome deve l opmen t i n o o g o n -i a l p r o t o p l a s m S e p t a d e l i m i t i n g o o g o n i a f r o m v e g e t a t i v e h yphae ; m i t o -c h o n d r i a and m i c r o b o d i e s o f o o g o n i a ! p r o t o p l a s m A n t h e r i d i a l - o o g o n i a l c o n t a c t A n t h e r i d i a l - o o g o n i a l c o n t a c t A n t h e r i d i a l - o o g o n i a l c o n t a c t A n t h e r i d i a l - o o g o n i a l c o n t a c t M a t u r a t i o n o f o o g o n i a l p r o t o -p lasm I n t e r p h a s e n u c l e i i n o o g o n i a M e i o t i c n u c l e i i n a n t h e r i d i a l and o o g o n i a l p r o t o p l a s m S y n a p t i n e m a l complex f o r m a t i o n C e n t r i o l a r a s s o c i a t i o n s w i t h i n t e r p h a s e and m e i o t i c n u -c l e i i n a n t h e r i d i a l and o o -g o n i a l p r o t o p l a s m ; m e i o t i c t e l o p h a s e x i v P l a t e One Hundred s i x P l a t e One Hundred s even P l a t e One hundred e i g h t P l a t e One Hundred n i n e P l a t e One hundred t e n P l a t e One hundred e l e v e n P l a t e One.Hundred t w e l y e P l a t e One hundred t h i r t e e n F i g u r e s 371-374 F i g u r e s 375 -376 F i g u r e s 377-378 F i g u r e s 379 -382 F i g u r e s 383 -386 F i g u r e s . 3 8 7 - 3 9 0 F i g u r e s 391 -393 F i g u r e s 394-396 N u c l e i d u r i n g anaphase N u c l e i d u r i n g t e l o p h a s e II o f m e i o s i s P e n e t r a t i o n o f o o g o n i a by f e r t i l i z a t i o n t u b e s A n t h e r i d i a l f e r t i l i z a t i o n t u b e p e n e t r a t i o n i n t o o o -g o n i a Oospheres and t h e i r n u c l e i L ipo somes and v e s i c l e s common to o o s p h e r e s Oospores i n oogonium L i p o s o m e , n u c l e u s and w a l l mo rpho logy o f oo spores . XV ACKNOWLEDGEMENTS The a u t h o r g r e a t l y a p p r e c i a t e s and w i s h e s t o t hank t h e f o l l o w i n g p e r s o n s f o r t h e i r a s s i s t a n c e and a d v i c e d u r i n g t h e p r e p a r a t i o n o f t h i s t h e s i s . D r . T. B i s a l p u t r a , A s s o c i a t e P r o f e s s o r and s u p e r v i s o r f o r t h i s s t u d y . H i s p a t i e n c e , a d v i c e , and p r o v i s i o n o f m a t e r i a l and equ ipment d u r i n g t h e p a s t s e v e r a l y e a r s i s s i n c e r e l y a p p r e c i a t e d . D r . R. B a n d o n i ; D r . D. F r a n c i s ; D r . G. Hughes; D r . C. P e r s o n ; D r . G. Rouse ; D r . R. S c a g e l , Depar tment Head ; , D r . G. H. N. Towe r s . A l l have o f f e r e d a d v i c e and u s e f u l c r i t i c i s m as members o f t h e a u t h o r ' s s u p e r v i s i n g c o m m i t t e e . M r s . Va l P r e u t e r and M r s . J a n W e l t z . T h e i r t i m e and p a t i e n c e i n t h e t y p i n g o f t h i s m a n u s c r i p t i s a p p r e c i a t e d . My p a r e n t s and many f r i e n d s . They have g i v e n mora l s u p p o r t and have h e l p e d i n t h e moun t i n g o f t h e m i c r o g r a p h s and i n p r o o f - r e a d i n g o f t h i s p a p e r . . Mr . K a r l R i c k e r . H i s encou ragemen t , a s s i s t a n c e and p a t i e n c e d u r i n g t h e p a s t s e v e r a l y e a r s i s i n v a l u a b l e . S p e c i a l c o n s i d e r a t i o n i s awarded t o h im . The r e s e a r c h p o r t i o n o f t h i s s t u d y was s u p p o r t e d l a r g e l y by t h e N a t i o n a l R e s e a r c h C o u n c i l o f Canada and t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a ; o t h e r e x p e n d i t u r e s i n c u r r e d d u r i n g t h i s p a s t y e a r were p a i d by Mr. K a r l R i c k e r . INTRODUCTION The m o r p h o g e n e t i c p r o c e s s e s o f hypha l g r o w t h , s e p t a l d e v e l o p m e n t , and a s e x u a l and s e x u a l r e p r o d u c t i o n i n f u n g i a l l a r e dynamic s y s tems w h i c h a r e r e f l e c t e d i n t h e o r g a n i s m s ' c y t o l o g y . However , t h e r e a r e few f u n g i i n w h i c h most o f t h e s e p r o c e s s e s have been s t u d i e d by e l e c t r o n m i c r o s c o p y , e s p e c i a l l y i n t h e Oomycetes where hormones have been found t o c o o r d i n a t e s e v e r a l s t a g e s o f hypha l d i f f e r e n t i a t i o n and deve l opmen t and where doub t s t i l l e x i s t s as t o w h e t h e r t h e i r l i f e - c y c l e i s p r e d o m i n a n t l y h a p l o i d o r d i p l o i d > -G r o v e , . B r a c k e r and M o r r e / ( 1 9 6 7 , 1970) and Grove and B r a c k e r (1970) have o b s e r v e d t h a t bo th g r o w i n g and n o n - g r o w i n g r e g i o n s o f Oomycete h yphae , s p e c i f i c a l l y t h o s e o f Pythivm ultimum Trow and Pythivm aphanidermatum (Edson ) F i t z p a t r i c k , a r e s i m i l a r s t r u c t u r a l l y t o hyphae o f many f u n g i . The hyphae a r e c h a r a c t e r i z e d by an a p i c a l zone c o n s i s t i n g a l m o s t e x c l u s i v e l y o f v e s i c l e s , a s u b - a p i c a l n o n - v a c u o l a t e and m u l t i - o r g a n e l l e z o n e , and a d i s t a l zone o f v a c u o l a t i o n . . G r o v e , B r a c k e r and M o r r e ' ( l 9 7 0 ) h y p o t h e s i z e t h a t t he mechanism o f hypha l g r owth i n v o l v e s a f u n c t i o n a l l y c o n t i n u o u s , e n -domembrane s y s tem i n w h i c h s e c r e t o r y v e s i c l e s c o n t a i n i n g w a l l c o n s t i t u e n t s a r e f o r m e d . The v e s i c l e s t h e n m i g r a t e t o t h e hypha l t i p s where t h e y f u s e w i t h t h e p la sma membrane t o d e p o s i t t h e i r c o n t e n t s t owa rd t h e w a l l . D i f f e r e n t i a t i o n o f hyphae i n t o d i r e c t and i n d i r e c t g e r m i n a t i n g z o o s p o r a n g i a a l s o has been s t u d i e d by e l e c t r o n m i c r o s c o p y i n s e v e r a l Oomy-c e t e s . Hohl and Hamamoto (1967) n o t e t h a t i n Phytophthora parasitica D a s t . t h e i r j t i c l e l . ; b g c d m e ^ p y f f f b V i r t ' i a^Ki . o r i e n t e d a t p o i n t s e q u i d i s t a n t bo th f r o m each o t h e r and f r o m t h e s p o r a n g i a ! w a l l . They a l s o r e p o r t t h a t b a s a l b o d i e s , w h i c h l a t e r e l o n g a t e t o f o rm f l a g e l l a r axonemes, r a p i d l y d i f f e r e n -t i a t e f r o m c e n t r i o l e s a t t h e na r r ow p o l e o f t h e n u c l e i a b o u t w h i c h o t h e r - 2 -o r g a n e l l e s and i n c l u s i o n s become o r i e n t e d . C l e a v a g e v e s i c l e s , however , become a l i g n e d i n p l a n e s e q u i d i s t a n t between a d j a c e n t n u c l e i and t h e s p o r -a n g i a ! w a l l , and t h e y g r a d u a l l y f u s e t o g e t h e r t o c l e a v e t h e p r o t o p l a s m i n t o numerous u n i n u c l e a t e naked c e l l s . S i m i l a r mechanisms o f z o o s p o r e f o r m a t i o n have been d e s c r i b e d f o r Phytophthora oapsioi L e o n i a n ( W i l l i a m s and W e b s t e r , 1 9 7 0 ) , Phytophthora infestans (Mon t . ) de Ba ry ( E i s n e r , H o r t o n a n d ' B o w e n , . 1967 ' ) , Phytophthora erythroseptioa P e t h y b . (Chapman and V u j i c i c , 1965) and Saprolegnia ferax ( G r u i t h u i s e n ) T h u r e t (Gay and Greenwood, 1 9 6 6 ) . Chapman and V u j i c i c (1965) i n d i c a t e t h a t i n i n d i r e c t l y g e r m i n a t i n g z o o s p o r a n g i a t he W a l l a l s o becomes t r i p l e - l a y e r e d , and t h e y s u g g e s t t h a t t h e c e n t r a l v e s i -c u l a r l a y e r p o s s i b l y f u n c t i o n s i n t h e r e l e a s e o f z o o s p o r e s . Hemmes and Hohl (1969) have o b s e r v e d p o c k e t s o f s i m i l a r m a t e r i a l i n w a l l s o f d i r e c t l y g e r m i n a t i n g z o o s p o r a n g i a ; however , t h e y i n t e r p r e t t h e v e s i c u l a r e l e m e n t s t o be e x c e s s membrane m a t e r i a l w h i c h has been e n t r a p p e d w i t h i n w a l l c o n -s t i t u e n t s d e p o s i t e d d u r i n g a p p o s i t i o n a l g r o w t h . O the r i n v e s t i g a t o r s (Hohl and Hamamoto, 1967 ; W i l l i a m s and W e b s t e r , 1970) r e p o r t . tha t t h e w a l l s o f z o o s p o r a n g i a a r e fo rmed o f o n l y two l a y e r s - an o u t e r l a y e r w h i c h i s c o n t i n u o u s a b o u t t h e a p i c a l p a p i l l a e and an i n n e r l a y e r w h i c h t a p e r s o f f nea r t h e p a p i l l a e . These a u t h o r s a l s o n o t e t h a t t h e p a p i l l a e a r e f i l l e d w i t h f i b r i l l a r m a t e r i a l w h i c h i s d e p o s i t e d . u p o n t h e r e l e a s e o f t h e z o o -s p o r e s . The z o o s p o r e s examined by e l e c t r o n m i c r o s c o p y a r e a l l b i f l a g e l l a t e a n d , as i s c h a r a c t e r i s t i c o f Oomycete s , t h e f l a g e l l a a r e o f two t y p e s -t i n s e l and w h i p l a s h . D e s j a r d i n s , Zentmyer and Reyno l d s ( 1 9 6 9 ) , H e a t h , Greenwood and G r i f f i t h s ( 1 9 7 0 ) , Man ton , C l a r k e and Greenwood (1951 , 1 9 5 2 ) , R e i c h l e ( 1 9 6 9 a ) , and V u j i c i c , Chapman and Co l houn (1965) have shown t h a t t h e t i n s e l f l a g e l l u m , o r a n t e r i o r l y d i r e c t e d f l a g e l l u m i n t h e s e c o n d a r y - 3 -s t a g e z o o s p o r e , p o s s e s s e s numerous l a t e r a l h a i r s w i t h a b r u p t l y t a p e r e d end s . R e i c h l e (1969a) a l s o n o t e s t h a t t h e l a t e r a l h a i r s po s se s s a c e n t r a l c a n a l w h i c h e a s i l y i s v i s i b l e i n t h i n s e c t i o n s . H e a t h , Greenwood and G r i f f i t h s (1970) s u g g e s t t h a t t h e h a i r s a r e m i c r o t u b u l e s w h i c h a r e fo rmed w i t h i n t h e e n d o p l a s m i c r e t i c u l u m and a r e r e l e a s e d d u r i n g f l a g e l l a r g e n e s i s , p o s s i b l y i n a manner s i m i l a r t o t h a t o b s e r v e d i n . Pythium middletonii by B r a c k e r , He i n t z . . and Grove ( 1 9 7 0 ) . The w h i p l a s h f l a g e l l u m , o r p o s t e r i o r l y - d i r e c t e d f l a g e l l u m i n t he s e c o n d a r y s t a g e z o o s p o r e s , a l s o has been r e p o r t e d t o have numerous f i n e l a t e r a l h a i r s w h i c h a r e s h o r t e r , s m a l l e r i n d i a m e t e r , and no t t a p e r e d as a r e t h o s e on t h e t i n s e l - t y p e f l a g e l l u m ( D e s j a r d i n s , Z e n t -myer and R e y n o l d s , 1 9 6 9 ; Man ton , C l a r k e and Greenwood, 1 9 5 1 ; R e i c h l e , 1969a ; V u j i c i c , Chapman and C o l h o u n , 1 9 6 5 ) . The i n t e r n a l s t r u c t u r e o f bo th t y p e s o f f l a g e l l a i s s i m i l a r t o o t h e r e u c a r y o t i c f l a g e l l a ( B e r l i n and Bowen, 1964 ; B r a c k e r , 1967 ; Hohl and Hamamoto, 1967 ; Ho, Z a c h a r i a h . and H i c k -man, 1 968 ; K o l e , 1965 ; R e i c h l e , 1 969a ; W i l l i a m s and W e b s t e r , 1 9 7 0 ) . Ho, Z a c h a r i a h and Hickman (1968) and R e i c h l e (1969a) n o t e t h a t t h e f l a g e l l a c l i e a d j a c e n t t o t h e beaked -end o f t h e n u c l e i and i n c l o s e a s s o c i a t i o n w i t h a l a r g e v a c u o l e . The v a c u o l e m i g h t be c o n t r a c t i l e and c o n t a i n m a t e r i a l t r a n s f e r r e d f r o m nea rby d i c t y o s o m e s e c r e t o r y v e s i c l e s ( B r a c k e r , H e i n t z and G r o v e , 1 9 7 0 ) . Most o t h e r z o o s p o r e c o n s t i t u e n t s have been f ound t o r e -semble t h e i r r e s p e c t i v e fo rms i n t h e z o o s p o r a n g i a l p r o t o p l a s m e x c e p t fp>":-t h e f l a t v e s i c l e s w h i c h ; ' R e i c h l e (1969a) o b s e r v e d l i e p a r a l l e l w i t h t h e c e l l s ' p l a sma membrane. The e n c y s t m e n t o f t h e z o o s p o r e s , as o b s e r v e d by e l e c t r o n m i c r o s c o p y , o c c u r s e i t h e r upon t h e r e t r a c t i o n p f t h e f l a g e l l a r axonemes. ( R e i c h l e , 1969a) o r upon t h e c o i l i n g up and s h e d d i n g o f t h e axonemes w i t h i n t h e i r s h e a t h s (Ho, Z a c h a r i a h and H i c k m a n , 1 9 6 7 ) . L i g h t m i c r o s c o p y s t u d i e s a l s o i n d i c a t e - 4 -t h a t a w a l l i s d e p o s i t e d abou t t he c e l l s ( B r a c k e r , 1967; Ho and H i c k m a n , 1 9 6 7 ) ; howeve r , t h i s does no t appea r t o have been no ted i n any f i n e s t r u c -t u r a l s t u d i e s t o d a t e . S e xua l r e p r o d u c t i o n i n t h e Oomycetes has been s t u d i e d e x t e n s i v e l y by l i g h t m i c r o s c o p y and i t has been shown t h a t f e r t i l i z a t i o n i s e f f e c t e d by t h e p e n e t r a t i o n o f o o s p h e r e s w i t h i n o o g o n i a by f e r t i l i z a t i o n t ube s f o r m -ed by a n t h e r i d i a ( B r y a n t and Howard, 1969 ; C o k e r and C o u c h , 1927 ; R a p e r , 1960; Sansome, 1 9 6 1 , 1963 , 1965 ; S t e v e n s , 1899 , 1 9 0 1 ; T row, 1 895 , 1899 , 1904; Z i e g l e r , 1953),. S e v e r a l i n v e s t i g a t o r s , i n d i c a t e t h a t m e i o s i s o c c u r s s ub sequen t t o f e r t i l i z a t i o n and i s z y g o t i c ( S t e v e n s , 1 9 0 1 ; S c h r a d e r , 1901 - s ee B r y a n t and Howard, 1969 ; Z i e g l e r , 1 9 5 3 ) ; however , o t h e r s t u -d i e s r e p o r t t h a t m e i o s i s o c c u r s d u r i n g g ametogene s i s ( B r y a n t and Howard, 1969; F l a n a g a n , 1970; Sansome, 1961 , 1963 , 1965 ; S t e v e n s , 1899 ; T r o w , . 18 9 5 , 18 9 9 , , 1 9 0 4 ) . Raper ( 1 939a , 1939b, 1942a , 1942b, 1 950a , 1950b, 1960) and o t h e r s (Rape r and Haagen S m i t , 1 942 ; B a r k s d a l e , 1963) a l s o have demon-s t r a t e d t h a t s e x u a l . r e p r o d u c t i o n i s a f f e c t e d by hormones , as r e p r o d u c e d i n t he d i a g r a m below.- z \ - 5 -HORMONE PRODUCED BY: ACTION A, A 2 ? T h a l l u s I n i t i a t i o n of a n t h e r i d i a l f i l a m e n t s on t h a l l u s A 1, A 3 c? T h a l l u s A''" augments the a c t i o n of A and A ; A^ tends to suppress t h i s a c t i o n B cf T h a l l u s ( a f t e r s t i m u l a t i o n by A's) Development of oogonial i n i t i a l s on t h a l l u s C Oogonial i n i t i a l s Regulates d i r e c t i o n a l growth of a n t h e r i d i a l f i l a m e n t s ; d e l i m i t a t i o n of a n t h e r i d i a a f t e r contact D A n t h e r i d i a D e l i m i t a t i o n of oogonia and d i f f e r e n t i a t i o n of oospheres From An E v o l u t i o n a r y Survey of the Plant Kingdom by Scagel, R., R. Bandoni, G. Rouse, W. S c h o f i e l d , J . S t e i n and T. Ta y l o r The re a r e , howeve r , few s t u d i e s d e s c r i b i n g t h e f i n e s t r u c t u r e o f s e xua l r e p r o d u c t i o n . Ma r chan t (1968) has shown i n P. ultimum.that t h e a n t h e r i d i a l hyphae become c o n s t r i c t e d s l i g h t l y a t t he p o i n t o f p e n e t r a t i o n t h r o u g h t h e o o g o n i a l w a l l , and he s u g g e s t s t h a t a n t h e r i d i a l p e n e t r a t i o n i s e f f e c t e d by t h e p h y s i c a l and c h e m i c a l a c t i v i t i e s o f t h e hyphae t h e m -s e l v e s , p o s s i b l y by some f u n c t i o n o f t h e o o g o n i a l p r o t o p l a s m . The o o g o n i a , he n o t e s , p o s s e s s n u c l e i , numerous smal1 v a c u o l e s and membrane-bounded g l o b u l e s . S i m i l a r p r o t o p l a s m i c c o n s t i t u e n t s and a l a r g e c e n t r a l v a c u o l a r f r a c t i o n , t h e o o l y m p h , have been o b s e r v e d i n o o g o n i a o f Soprolegnia ter-restris Cookson (Moore and Howard, 1 9 6 8 ) . Howard and Moore ( s ee A l d r i c h and M ims, 1970) no te t h a t some o f t h e n u c l e i a l s o po s se s s s i n g l e a x i a l e l e m e n t s t y p i c a l o f u n p a i r e d m e i o t i c chromosomes. The o o s p o r e s o f bo th P. ultimum and S. terrestris have been d e s -c r i b e d as h a v i n g t h i c k w a l l s w h i c h a r e i r r e g u l a r i n p r o f i l e , and a p r o t o -p lasm w h i c h i s composed p r e d o m i n a n t l y o f s t o r a g e o r g a n e l l e s ( M a r c h a n t , ' - 6 -1968; Moore and Howard, 1 9 6 8 ) . In P. ultimum.Marchant (1968) n o t e s t h a t t h e i r n u c l e i a r e l o c a t e d c e n t r a l l y w i t h i n t he c e l l s ; howeve r , i n s. terres tris Moore ,and Howard (1968) i n d i c a t e t h a t t h e n u c l e i o c c u r i n t h e oop lenum a s u b - c e n t r i c s h e l l o f p e r i p h e r a l o o s p o r e p r o t o p l a s m . In t h i s s t u d y t h e u l t r a s t r u c t u r e o f t h e m a j o r d e v e l o p m e n t a l s t a g e s i n t he l i f e - c y c l e o f Aohlya bisexualis Goker and Couch , a d i o e c i o u s Oomy-c e t e w h i c h can be grown e a s i l y under l a b o r a t o r y c o n d i t i o n s , i s d e s c r i b e d f o r t h e f i r s t t i m e . I t i s compared w i t h t h a t o f o t h e r Oomycetes i n w h i c h f i n e s t r u c t u r a l i n f o r m a t i o n i s f r a g m e n t a r y . . A l s o c o n s i d e r e d a r e the. p o s -s i b l e mechanisms o f hypha l g r o w t h , septum d e v e l o p m e n t , z o o s p o r o g e n e s i s , as w e l l as t h o s e w h i c h f u n c t i o n d u r i n g s e x u a l r e p r o d u c t i o n where a n t h e r i d i a l and o o g o n i a l de ve l opmen t i s c o n t r o l l e d by hormones. - 7 -MATERIALS AND METHODS The o r g a n i s m used i n t h i s s t u d y was Aohlya bisexualis Coker and Couch , a d i o e c i o u s P h y c o m y c e t e , o b t a i n e d f r o m C e n t r a a l b u r e a u v o o r S c h i m m e l -c u l t u r e s , B a a r n , N e t h e r ! a n d . The ma le (CBS - 102 .62 ) and f e m a l e (CBS -101.62 ) s t r a i n s were m a i n t a i n e d s e p a r a t e l y on h e a t - s t e r i l i z e d sesame seeds i n a p p r o x i m a t e l y 50 ml s t e r i l e , c a r b o n - f i l t e r e d , d i s t i l l e d w a t e r i n 10 cm Kimax p e t r i - d i s h e s a t room t e m p e r a t u r e ( ^ 2 2 - 2 3 0 C ) ( F i g . 1 ) . S e x u a l r e p r o d u c t i o n o c c u r r e d w i t h i n t h r e e t o f o u r days a f t e r p l a c i n g v e g e t a t i v e hyphae f r o m ma le and f e m a l e c u l t u r e s i n t o t h e same p e t r i - d i s h c o n t a i n i n g t h e g r owth medium m e n t i o n e d above ( F i g . 2 ) . L i g h t M i c r o s c o p y T e c h n i q u e s L i v i n g and p r e s e r v e d m a t e r i a l was examined and pho tog r aphed w i t h a Z e i s s P h o t o m i c r o s c o p e u s i n g b r i g h t - f i e l d , p h a s e - c o n t r a s t , N o m a r k s i , and u l t r a v i o l e t ( e x c i t e r f i l t e r UG 5/3 w i t h a t r a n s m i s s i o n r ange f r o m 220 -420 my) i l l u m i n a t i o n . The l i v i n g m a t e r i a l was mounted i n d i s t i l l e d w a t e r on g l a s s s l i d e s w i t h c o v e r s l i p s or. on d e p r e s s i o n s l i d e s w i t h c o v e r s l i p s . The m a t e r i a l w h i c h had been f i x e d f o r one hour a t room t e m p e r a t u r e ( /~ 22 - 23°C ) i n 2 . 0 - 2 . 5 % g l u t a r a l d e h y d e b u f f e r e d w i t h 0.1 M sod ium c a c o d y l a t e (pH 7.2) was mounted i n b u f f e r . M a t e r i a l w h i c h had been f i x e d and embedded i n M a r a -g l a s f o r e l e c t r o n m i c r o s c o p y was s e c t i o n e d a t 0.5-1 .0 u t h i c k n e s s e s w i t h g l a s s k n i v e s on a P o r t e r Blum MT-1 m i c r o t o m e f o r l i g h t m i c r o s c o p y ( f o r d e t a i l e d p r o c e d u r e , see A p p e n d i x , T a b l e I I ) . The s e c t i o n s were t r a n s f e r r e d t o g l a s s s l i d e s , d r i e d o v e r an a l c o h o l l a m p , and s t a i n e d w i t h F lemming t r i p l e t s t a i n (Conn et a £ . , . 1 9 6 0 ) o r 0 . 1% t o l u i d i n e b l u e w i t h bo r ax (Conn et al. t 1960) ( f o r d e t a i l e d p r o c e d u r e , s ee A p p e n d i x , T a b l e I ) . - 8 -N i l e B l u e and Sudan IV were used as c y t o c h e m i c a l t e s t s f o r l i p i d l o c a l i z a t i o n i n 1 i v i n g m a t e r i a l ( f o r d e t a i l e d p r o c e d u r e s , see A p p e n d i x , T a b l e I ) . U l t r a - t h i n S e c t i o n T e c h n i q u e s - E l e c t r o n M i c r o s c o p y B o t h v e g e t a t i v e and s e x u a l r e p r o d u c t i v e m a t e r i a l w a t e f i x e d f o r one hour a t room t e m p e r a t u r e ( ~ 2 2 - 2 3 ° C ) i n a 1:1 m i x t u r e o f 2% o s m i c a c i d and 5% g l u t a r a l d e h y d e b u f f e r e d w i t h 0.1 M sod ium c a c o d y l a t e a t pH 7 . 2 . Then t h e m a t e r i a l was .washed t h o r o u g h l y w i t h t h e sod ium c a c o d y l a t e b u f f e r . P r i m a r y , s e c o n d a r y and g e r m i n a t i n g s p o r e s were c o n c e n t r a t e d by c e n t r i f u g a -t i o n a f t e r each o f t he above s o l u t i o n c h a n g e s , and a f t e r t h e f i n a l wash i n b u f f e r , t h e y were embedded i n one d r op o f 4% a g a r . The d r op was s u b d i v i d e d i n t o a p p r o x i m a t e l y 1 mm c u b e s . A l l t h e m a t e r i a l was d e h y d r a t e d i n a g r aded s e r i e s o f e t h a n o l and p r o p y l e n e o x i d e s o l u t i o n s and embedded i n M a r a g l a s ( B i s a l p u t r a and W e i e r , 1 9 6 3 ; f o r d e t a i l e d p r o c e d u r e on f i x a t i o n , d e h y -d r a t i o n and embedd ing , see A p p e n d i x , T a b l e s . I and I I I ) . P o l y m e r i z a t i o n o f t h e M a r a g l a s was a c h i e v e d a f t e r 18 -24 hour s a t 65°C i n a vacuum o v e n . The m a t e r i a l was s e l e c t e d and t r immed f o r s e c t i o n i n g b y - e x a m i n i n g t h e b l o c k s w i t h a N i k o n d i s s e c t i n g m i c r o s c o p e a t X60 m a g n i f i c a t i o n . U l t r a - t h i n s e c -t i o n s were o b t a i n e d u s i n g a P o r t e r - B l u m MT-1 m i c r o t o m e and a duPont d iamond k n i f e . The s e c t i o n s were mounted on f o r m v a r - c o a t e d c oppe r g r i d s and p o s t -s t a i n e d 4 0 - 6 0 . m i n u t e s . i n u r a n y l a c e t a t e ( s a t u r a t e d s o l u t i o n i n 70% m e t h a n o l ) f o l l o w e d by 6-7 m i n u t e s i n l e a d c i t r a t e ( R e y n o l d s , 1 9 6 3 ) . A l l m a t e r i a l was examined w i t h a Z e i s s EM 9-A e l e c t r o n m i c r o s c o p e . F r e e z e - E t c h i n g T e c h n i q u e s Sma l l amounts o f t h e o r g a n i s m i n d i s t i l l e d w a t e r were p l a c e d i n g o l d r immed d i s c s . E x c e s s w a t e r a round t h e spec imens was drawn o f f w i t h - 9 -f i l t e r p a p e r . P h y s i c a l s t a b i l i z a t i o n and e n z y m a t i c i n a c t i v a t i o n were i n -s u r e d by q u i c k l y immer s i n g t h e d i s c s w i t h m a t e r i a l i n t o l i q u i d F reon 22 ( - 1 5 0 ° C ; Moor and M u h l e t h a l e r , .1963). A f t e r 5-8 s e c o n d s , t h e d i s c s were t r a n s f e r r e d t o l i q u i d n i t r o g e n ( - 190°C ) and t h e n t o t he spec imen t a b l e o f a B a l z e r s F r e e z e - E t c h u n i t (BA 360M) w h i c h was . m a i n t a i n e d e i t h e r a t -100°C o r -11.0e-C. The m a t e r i a l was s e c t i o n e d , s u b l i m a t e d f o r o n e . m i n u t e a t -100°C o r t h r e e m i n u t e s a t - 1 1 0 ° C , and c o a t e d w i t h a p l a t i n u m and c a r b o n f i l m . The s pec imen s t h e n were thawed and t h e r e p l i c a s f l o a t e d on to t h e s u r f a c e o f d i s t i l l e d w a t e r . The r e p l i c a s were t r a n s f e r r e d , u s i n g p l a t i n u m w i r e l o o p s , t o 70% s u l p h u r i c a c i d (1-1 1/2 h o u r s ) , d i s t i l l e d w a t e r (10 m i n u t e s ) , sod ium h y p o c h l o r i t e (1 h o u r ) , and d i s t i l l e d w a t e r ( s e v e r a l changes a t 10 -15 m i n u t e s per c h a n g e ) . The r e p l i c a s were mounted on f o r m v a r - c o a t e d c o p p e r . g r i d s and examined w i t h a Z e i s s EM 9-A e l e c t r o n m i c r o s c o p e . S h a d o w - c a s t i n g T e c h n i q u e s Drops o f d i s t i l l e d w a t e r c o n t a i n i n g f l a g e l l a t e d s e c o n d a r y z o o s p o r e s were p l a c e d on f o r m v a r - c o a t e d c o p p e r g r i d s . The w a t e r was drawn o f f w i t h f i l t e r paper and t h e g r i d s w i t h t h e z o o s p o r e s were p r e p a r e d f o r e l e c t r o n m i c r o s c o p y by u n i d i r e c t i o n a l s h a d o w - c a s t i n g . P a l l a d i u m - g o l d ( 80 - 20 ) was e v a p o r a t e d on to t h e spec imens a t . a p p r o x i m a t e l y a 3 0 ° a n g l e . The s p e c -imens were examined w i t h an AEI 801 e l e c t r o n m i c r o s c o p e . - . 1 0 -RESULTS VEGETATIVE HYPHAE V e g e t a t i v e hyphae o f b o t h t h e ma le and f e m a l e s t r a i n s o f Achlya bisexualis Coke r and Couch d e v e l o p r a p i d l y by a p i c a l e l o n g a t i o n , e x p a n s i o n and b r a n c h i n g o f germ t u b e s t h a t a r e u s u a l l y f o rmed by t h e g e r m i n a t i o n o f e n c y s t e d s e c o n d a r y z o o s p o r e s ( F i g s . 3 - 8 ) . They grow r a p i d l y f o r s e v e r a l days u n t i l e n v i r o n m e n t a l c o n d i t i o n s f a v o u r t h e i r d i f f e r e n t i a t i o n i n t o a s e x u a l and/o r s e x u a l r e p r o d u c t i v e s t r u c t u r e s ( f o r hypha l g r owth r a t e s , see A p p e n d i x , T a b l e I V ) . The v e g e t a t i v e hyphae measure 4 5 - 120 y and 30 -70 y i n d i a m e t e r i n t h e ma le and f e m a l e s t r a i n s , r e s p e c t i v e l y . M o r p h o l o g i c a l l y , t h e hyphae o f b o t h . s t r a i n s a r e s i m i l a r . T h e i r . c y t o p l a s m i s d i f f e r e n t i a t e d , i n te rms o f t h e r e l a t i v e abundance and d i s -t r i b u t i o n o f o r g a n e l l e s and i n c l u s i o n s , i n t o an a p i c a l g r o w i n g zone ( A ) , a s u b a p i c a l m i t o c h o n d r i a l zone ( S A ) , and a d i s t a l m u l t i - o r g a n e l l e zone (D) w h i c h becomes p r o g r e s s i v e l y more v a c u o l a t e d i n o l d e r hypha l r e g i o n s ( D i a -gram I; F i g s . 9 - 1 9 ) . A p i c a l G row ing Zone The a p i c a l g r o w i n g zone o f t h e v e g e t a t i v e hyphae , as o b s e r v e d by p h a s e - c o n t r a s t and Nomarsk.i m i c r o s c o p y , a p p e a r s a t a 5-20 y den se c y t o p l a s -m ic cap o f r a p i d l y mov ing g r a n u l e s ( F i g s . 9 - 1 9 ) . Sometimes a r e f r a c t i l e o r dense a r e a a t t h e t i p a l s o i s d i s c e r n i b l e ( F i g s . 1 4 - 1 5 ) ; however , a " S p i t z e n k o r p e r " , as f i r s t d e s c r i b e d by B r u n s w i c k ( 1924 ; see B u r n e t t , 1 9 6 8 ) , i s n o t v i s i b l e . The f i n e s t r u c t u r e o f t h e a p i c a l g r o w i n g zone i s s i m i l a r t o t h a t o b -s e r v e d i n c e r t a i n o t h e r f u n g i ( B r e n n e r and C a r r o l l , 1968 ; G i r b a r d t , 1969 ; G r o v e , B r a c k e r and M o r r e ' 1 967 , 1970; M c C l u r e , Pa r k and R o b i n s o n , 1 9 6 8 ) . - l i -l t i s c h a r a c t e r i z e d by a t h i n - w a l l e d c y t o p l a s m w h i c h u s u a l l y c o n s i s t s o f o v o i d t o i r r e g u l a r l y - s h a p e d v e s i c l e s (D iag rams I and I I ; F i g s . 3 0 - 3 3 ) . O the r o r g a n e l l e s and i n c l u s i o n s se ldom a r e o b s e r v e d i n t h e a p i c a l c y t o -p l a s m . The v e s i c l e s c o r r e s p o n d t o t h e r a p i d l y mov ing g r a n u l e s v i s i b l e by l i g h t m i c r o s c o p y and a r e 30 -300 my i n d i a m e t e r . They c o n t a i n f i b r i l l a r and amorphous m a t e r i a l o f v a r y i n g e l e c t r o n d e n s i t y and each i s bound by a s i n g l e membrane, 70-80A t h i c k ( F i g s . 3 1 - 3 3 ) . F u s i o n o f t h e v e s i c l e s w i t h each o t h e r and w i t h t h e p lasma membrane i s common, as i n d i c a t e d by i t s c r e n u l a t e a p p e a r a n c e ( F i g s . 3 1 - 3 3 ) . The p lasma membrane a l s o i s 70-80A t h i c k .and f r e q u e n t l y e l a b o r a t e d t o f o rm lomasomes ( F i g s . 3 0 - 3 1 ) . The lomasomes appea r as a g g r e g a t e s o f i r r e g u l a r l y - s h a p e d v e s i c l e s l o c a t e d between t h e p lasma membrane a n d t h e h y -pha l w a l l , and t h e y s t r u c t u r a l l y r e s e m b l e t h o s e i n Syncephalastrum s p . ( F i g . 6 , Moore and M c A l e a r , 1 9 6 1 ) , . Phytophthora infestans ( E h r l i c h a'nd E h r l i c h , 1 9 6 8 ) , and Pythium ultimum ( F i g . 2 2 , M a r c h a n t , P e a t and B a n b u r y , 1 9 6 7 ) . . The w a l l a b o u t t h e a p i c a l g r o w i n g zone a p p e a r s a s an i r r e g u l a r l a y e r o f e l e c t r o n t r a n s p a r e n t , amorphous m a t e r i a l , 30 -70 my i n t h i c k n e s s ( F i g s . 3 0 - 3 1 ) . S u b - A p i c a l M i t o c h o n d r i a l Zone, L i g h t m i c r o s c o p i c . s t u d i e s r e v e a l t h a t a p p r o x i m a t e l y 5-20 y f r om t h e hypha l t i p s , t h e c y t o p l a s m c o n t a i n s numerous m i t o c h o n d r i a and g r a n u l e s ( F i g s . 9 - 1 9 ) . The m i t o c h o n d r i a a r e d i s t r i b u t e d t h r o u g h o u t t h e c e n t r a l p a r t o f t h e h yphae , whereas t h e g r a n u l e s u s u a l l y a r e l o c a t e d i n t h e p e r i p h e r a l r e g i o n ( F i g s . 1 0 - 1 1 ) . Sometimes o v o i d t o s p i n d l e - s h a p e d n u c l e i a r e v i s i b l e i n t he s u b - a p i c a l p r o t o p l a s m , bu t t h e y u s u a l l y a r e no t a p p a r e n t u n t i l 2 0 - 80 CYTOPLASMIC REGIONS of the VEGETATIVE HYPHAE of ACHLYA BISEXUALIS Sub—Apical /Mitochondrial Zone Distal Multi —Organelle Zone S j DIAGRAM X MORPHOLOGY of an ACTIVELY GROWING HYPHAL TIP of ACHLYA BISEXUALIS Apical Growing Zone Sub-Apical Mitochondrial Zone Distal Multi-Organelle Zone DIAGRAM H - 12 -y be low t h e hypha l t i p s ( F i g s . 1 0 - 1 8 ) . The f i n e s t r u c t u r e o f t h e s u b - a p i c a l zone c o r r e s p o n d s t o t h a t o b -s e r v e d by l i g h t m i c r o s c o p y . V e s i c l e s and r o d - s h a p e d m i t o c h o n d r i a a r e p r e -d o m i n a n t , but r i b o s o m e s , e n d o p l a s m i c r e t i c u l u m ( E . R . ) and d i c t y o s o m e - 1 i k e c i s t e r n a e a l s o a r e obse rved , i n t h e p r o t o p l a s m (D iag rams I and I I ; F i g . 3 4 ) . Most v e s i c l e s a r e c o n c e n t r a t e d i n t h e p e r i p h e r a l p r o t o p l a s m and a r e m o r p h o l o g i c a l l y t h e same as t h o s e i n t h e a p i c a l g r ow ing zone ( F i g s . 3 4 - 3 6 ) . However , t h e r e l a t i v e p r o p o r t i o n o f t h e s m a l l e r v e s i c l e s t o t h e l a r g e ones i s g r e a t e r t h a n o b s e r v e d p r e v i o u s l y . A l s o , t h e s m a l l e r v e s i c l e s f r e q u e n t l y a r e c l u s t e r e d about one o r two d i s c o n t i n u o u s c i s t e r n a e w h i c h r e s e m b l e t h e " r u d i m e n t a r y d i c t y o s o m e s " o b s e r v e d i n hypha l t i p s o f o t h e r f u n g i . ( G i r b a r d t , 1969 ; G r o v e , B r a c k e r and M o r r e ' 1970 ; F i g s . 3 7 - 3 8 ) . The c i s t e r n a e o f t h e " r u d i m e n t a r y d i c t y o s o m e - 1 i k e b o d i e s " a r e bound by a s i n g l e membrane w h i c h a p p e a r s t o be abou t 70-80A t h i c k . M o r p h o l o g i c a l l y d i s t i n c t d i c t y o -somes a r e no t e v i d e n t i n t h e p r o t o p l a s m . O t h e r v e s i c l e s w h i c h r e s e m b l e m i c r o b o d i e s a n d . o f t e n o c c u r i n c l o s e a s s o c i a t i o n w i t h v e s i c u l a r ER a r e s c a t t e r e d t h r o u g h o u t t h e c e n t r a l hypha l p r o t o p l a s m ( F i g . 3 4 ) . They a r e o y o i d t o s l i g h t l y i r r e g u l a r i n p r o f i l e and 150 -400 my i n d i a m e t e r ; o c c a s i o n a l l y r e n i f o r m - s h a p e d ones a r e o b s e r -v a b l e ( F i g . 3 4 ) . The v e s i c l e s c o n t a i n e l e c t r o n - d e n s e g r a n u l a r m a t e r i a l a n d , • s o m e t i m e s , c o n c e n t r i c l a m e l l a r b a n d s o r t u b u l e s ( F i g s . 3 4 , 3 9 - 4 0 ) . A s i n g l e membrane, 60-7.0A t h i c k , encompasses t h e e l e c t r o n - d e n s e m a t e r i a l . The m i t o c h o n d r i a a r e m o s t l y r o d - s h a p e d and a r e a l i g n e d w i t h t h e i r l o n g axes p a r a l l e l t o t h a t o f t h e hyphae ( F i g . 3 4 ) . They measure 0 . 3 - 0 . 6 y i n d i a m e t e r and a p p r o x i m a t e l y 1 . 5 - 3 . 5 y i n l e n g t h . The o u t e r and i n n e r membranes o f t h e m i t o c h o n d r i a a r e each abou t 60A t h i c k , and t h e a r e a b e -tween t he two membranes . i s 5 0 - 7 0 A . i n t h i c k n e s s . The i n n e r membrane i n -v a g i n a t e s t o f o r m numerous t u b u l a r c r i s t a e , each 50 -60 my i n d i a m e t e r ( F i g s . 3 4 , 5 5 ) . The c r i s t a e e x t e n d toward t h e c e n t r a l a x i s o f . e a c h m i t o -c h o n d r i o n and t h e y se l dom o v e r l a p , one a n o t h e r . W i t h i n t h e c r i s t a e a r e e l e c t r o n - d e n s e f i b r i l s , 80 -100A i n d i a m e t e r , and e l e c t r o n - l i g h t amorphous m a t e r i a l ( F i g s . 3 4 , 5 5 ) . The m a t r i x o f a m i t o c h o n d r i o n i s g r a n u l a r and much d e n s e r t h a n e i t h e r t h e s u r r o u n d i n g c y t o p l a s m o r t h e m a t e r i a l i n t h e c r i s t a e . E n d o p l a s m i c r e t i c u l u m ( E . R . ) i s p r e s e n t i n t h e s u b - a p i c a l c y t o p l a s m , bu t i t i s n o t abundant o r e x t e n s i v e l y d e v e l o p e d ( F i g . 3 4 ) . I t appea r s f.as f l a t t e n e d s a c s o r v e s i c l e s d e l i m i t e d by a membrane, 50-60A t h i c k , R i b o -somes, 120-140A i n d i a m e t e r , u s u a l l y a r e ' • v i s i b l e - o n i t s s u r f a c e . The p la sma membrane appea r s c r e n u l a t e and f r e q u e n t l y i s e l a b o r a t e d t o f o rm lomasomes ( F i g s . 3 4 - 3 6 ) . The w a l l i s 60 -100 my i n t h i c k n e s s and a l s o i s s i m i l a r t o t h a t d e s c r i b e d p r e v i o u s l y . D i s t a l M u l t i - O r g a n e l l e Zone The d i s t a l m u l t i - o r g a n e l l e z o n e , as o b s e r v e d by l i g h t m i c r o s c o p y , i s d i s t i n g u i s h a b l e 20-80/-y f r om t h e hypha l t i p s ( F i g s . 9 - 1 8 ) . I t c o n t a i n s v e s i c l e s , r o d - s h a p e d m i t o c h o n d r i a , E .R. , n u c l e i , 1 i p i d d r o p l e t s and v a c -u o l e s . The v a c u o l e s u s u a l l y . a r e s m a l 1 , o r l a c k i n g , i n t h e p r o t o p l a s m imme-d i a t e l y b e h i n d t h e s u b - a p i c a l z o n e ; howeve r , as t h e d i s t a n c e f r o m t h e hyphal , t i p s i n c r e a s e s , v a c u o l a t i o n i n c r e a s e s u n t i l most o f t h e p r o t o p l a s m c o n s i s t s o f v a c u o l e s ( F i g s . 9 - 1 6 ) . The re a l s o i s an i n c r e a s e i n t h e number o f l i p i d d r o p l e t s i n t h e p r o t o p l a s m as v a c u o l a t i o n o c c u r s . . T h i s i s p a r -t i c u l a r l y e v i d e n t i n hyphae s t a i n e d w i t h ; Sudan IV o r M i l e B l u e where t h e l i p i d d r o p l e t s a ppea r o r a n g e - r e d o r f l u o r e s c e g o l d e n - y e l l o w under u l t r a -v i o l e t l i g h t , r e s p e c t i v e l y ( F i g s . 2 5 - 2 9 ) . The m i t o c h o n d r i a and n u c l e i c o n s e q u e n t l y appea r as t h e p r e d o m i n a n t o r g a n e l l e s i n t h e p r o t o p l a s m imme-- 14 -d i a t e l y b a s i p e t a l t o t he s u b - a p i c a l z o n e . The m i t o c h o n d r i a a r e t h e same as t h o s e d e s c r i b e d p r e v i o u s l y e x c e p t t h a t t h e y u s u a l l y a r e l o n g e r - mea-s u r i n g 6-7 p i n l e n g t h ( F i g s . 2 0 - 2 4 ) . Ovo i d to s p i n d l e - s h a p e d n u c l e i a r e o r i e n t e d p a r a l l e l t o t h e l o n g a x i s o f t h e hyphae , as a r e t h e m i t o c h o n d r i a ( F i g s . 2 0 - 2 4 ) . They a r e 1 . 0 -1 .4 y i n w i d t h and 5 . 0 - 6 . 0 y i n l e n g t h . Op-t i c a l l y dense b o d i e s a r e d i s c e r n i b l e abou t t h e p e r i p h e r y o f t h e n u c l e i ( F i g s , 2 3 - 2 4 ) . The E.R. appea r s as l o n g f i l a m e n t s t h r o u g h o u t t h e p r o t o -p lasm,, and i t a l s o i s . o r i e n t e d w i t h t h e l o n g a x i s o f t h e hyphae ( F i g s . 2 3 2 2 4 ) . The f i n e s t r u c t u r e o f t h e d i s t a l . m u l t i - o r g a n e l l e zone i s s i m i l a r t o t h a t o b s e r v e d by l i g h t m i c r o s c o p y a n d . i s shown i n D iag ram I. I t i s c h a r a c t e r i z e d by n u c l e i , c e n t r i o l e s , m o r p h o l o g i c a l l y d i s t i n c t d i c t y o s o m e s , l i p i d d r o p l e t s , m u l t i - v e s i c u l a r b o d i e s and v a c u o l e s ( F i g . 4 1 ) . I t a l s o c o n s i s t s o f m i t o c h o n d r i a , v e s i c l e s , E . R . a n d r i bo somes ( F i g . 4 1 ) . A n d , as o b s e r v e d by J ' i g h t m i c r o s c o p y , the, m i t o c h o n d r i a , n u c l e i and v e s i c l e s a r e t h e most p r edom inan t o r g a n e l l e s i n t h e p r o t o p l a s m n e a r e s t t h e a p i c a l g r ow-i n g z o n e , whereas l i p i d d r o p l e t s and v a c u o l e s a r e most abundant i n t h e d i s t a l h ypha l r e g i o n . The w a l l s u r r o u n d i n g t h e p r o t o p l a s m o f t h e d i s t a l m u l t i - o r g a n e l l e zone v a r i e s f r o m 60-100 my i n t h i c k n e s s nea r t h e a p i c a l g r o w i n g zone t o 1 3 0 -150 my i n t h i c k n e s s i n t h e o l d e r , h i g h l y v a c u o l a t e d a r e a s ( F i g s . 4 8 , 5 1 ) . I t i s r e l a t i v e l y e l e c t r o n - l i g h t e x c e p t f o r i t s o u t e r s u r f a c e w h i c h i s d e n - , s e r and s l i g h t l y f r a y e d i n appea r ance ( F i g s . 4 8 , 5 1 - 5 3 ) . As i n o t h e r Oomy-c e t e s ( A r o n s o n , . i 9 6 5 ; Cooper and A r o n s o n , 1967 ; G r o v e , B r a c k e r and M o r r e ' 1 970 ; Manocha and C o l v i n , 1968 ; P a r k e r , P r e s t o n and Fogg , 1963) t h e w a l l i s composed o f an amorphous m a t r i x i n w h i c h f i b r i l s a r e embedded ( J i g s . - 15 -4 8 - 5 1 ) . The f i b r i l s a r e 40-70A i n d i a m e t e r . In t h i n s e c t i o n s , f i b r i l l a r -l i k e e l e m e n t s a l s o a r e d i s t i n g u i s h a b l e and a p p e a r as two p a r a l l e l o r c u r v e d l i n e s o f u n d e t e r m i n e d l e n g t h ( F i g s . 48 , . .51 ) . In a l m o s t c o m p l e t e l y v a c u o -l a t e d hypha l r e g i o n s ; a r e a s o f t h e w a l l a r e l a y e r e d and p o c k e t s o f e l e c t r o n -dense v e s i c u l a r a n d . m e m b r a n e - l i k e m a t e r i a l o c c u r among t h e l a y e r s ( F i g s . 5 2 - 5 3 ) . The p o c k e t s o f m a t e r i a l a p p e a r t o be fo rmed by t h e e n t r a p m e n t o f v e s i c l e s i n w a l l m a t e r i a l by l o m a s o m e - 1 i k e . c o n f i g u r a t i o n s o f t h e p lasma membrane ( F i g . 3 6 ) . The v e s i c l e s , w h i c h c h a r a c t e r i z e t h e p e r i p h e r a l p r o t o p l a s m o f t h e s u b - a p i c a l z o n e , a l s o a r e common t h r o u g h o u t t h e d i s t a l m u l t i - o r g a n e l l e z o n e . They a r e most abundant i n t h e p r o t o p l a s m n e a r e s t t h e s u b - a p i c a l zone a n d , as s u g g e s t e d by o t h e r i n v e s t i g a t o r s . ( G i r b a r d t , 1969 ; G r o v e , B r a c k e r and M o r r e ' 1 9 7 0 ) , t h e y appea r t o o r i g i n a t e f r om d i c t y o s o m e s ( F i g s . 5 8 - 6 2 ) . The d i c t y o s o m e s o c c u r i n c l o s e a s s o c i a t i o n w i t h E.R. and n u c l e i ( F i g s . 5 8 - 6 8 , 8 8 - 8 9 ) . They each c o n s i s t o f a s t a c k o f 2-5 c i s t e r n a e and v e s i c l e s ( F i g s . 5 8 - 68 ) . : . The c i s t e r n a e appea r as m e m b r a n e - l i m i t e d d i s c s w i t h t u b u l a r p e r i p h e r i e s . They a r e 0 . 4 - 0 . 8 y i n d i a m e t e r and t h e t h i c k n e s s o f t h e i r l i m i t i n g membranes and lumens a r e abou t 60-70A and 80?-120A, r e s -p e c t i v e l y . In t h e a r e a between t he c i s t e r n a e and t h e E.R. and/o r n u c l e a r e n v e l o p e s a r e v e s i c l e s m e a s u r i n g 300-400A i n d i a m e t e r ( F i g s . 5 8 - 6 8 , 8 8 - 8 9 ) . They a p p e a r t o o r i g i n a t e as b l e b s f r om t h e c l o s e l y a s s o c i a t e d E.R. and n u -c l e a r membranes ( F i g s . 5 8 - 6 8 , 8 8 - 8 9 ) . They t hen appea r t o f u s e and fo rm t h e p r o x i m a l o r f o r m i n g f a c e c i s t e r n a e o f d i c t y o s o m e s , as d e s c r i b e d by M o l l e n h a u e r and Mo| r re / ( 1966 ) . V e s i c l e s , w h i c h m o r p h o l o g i c a l l y a r e s i m i l a r t o t h o s e i n t h e p e r i p h e r a l c y t o p l a s m , a l s o appea r t o be fo rmed by t h e d i s -c o n t i n u i t y o f c i s t e r n a e on t h e o p p o s i t e o r d i s t a l f a c e o f d i c t y o s o m e s ( F i g s . 5 8 - 6 2 ) . - 16 -The E.R. c o n s i s t s o f membrane-bound c i s t e r n a e w h i c h a r e a p p r o x i -m a t e l y 250-600A t h i c k ( F i g s . 5 8 , 6 1 ) . T h e i r 1 i m i t i n g membrane i s 50-60A t h i c k and t h e i n t r a - c i s t e r n a l lumens a r e 100-450A i n t h i c k n e s s . R i bo somes , 140-150A i n d i a m e t e r , a r e o b s e r v e d on t h e o u t e r s u r f a c e o f t h e membranes e x c e p t where t h e E.R. i s i n c l o s e a s s o c i a t i o n w i t h m i c r o b o d i e s o r t h e f o r m i n g f a c e c i s t e r n a e o f d i c t y o s o m e s . The E.R. t h e n i s r i b o s o m e - f r e e and o f t e n ^ d i l a t e d ( F i g s . 6 3 - 6 5 , 7 7 - 7 9 ) . The smooth s u r f a c e o f j u x t a - d i c t y p -somal E.R. may d e v e l o p b l e b s w h i c h become p i n c h e d o f f t o f o r m v e s i c l e s ( F i g s . 6 3 - 6 5 ) . E.R. i s n o t e x t e n s i v e l y d e v e l o p e d i n t h e v e r y young hypha l p r o -t o p l a s m , b u t , a s t h e hyphae become o l d e r and b e g i n t o d i f f e r e n t i a t e , t h e E.R. becomes, more d e v e l o p e d ( F i g s . 6 3 - 6 5 ) . M i t o c h o n d r i a , w h i c h s t r u c t u r a l l y a r e t h e same as t h o s e i n t h e s u b -a p i c a l z o n e , a r e e x t r e m e l y abundant i n t h e young hypha l p r o t o p l a s m o f t h e d i s t a l m u l t i - o r g a n e l 1 e zone ( F i g s . 4 0 - 4 1 ) . They a r e d i s t r i b u t e d r andomly t h r o u g h o u t t h e p ro top la sm^ a n d , as o b s e r v e d by l i g h t m i c r o s c o p y , t h e y a r e o r i e n t e d p a r a l l e l t o t h e l o n g a x i s o f t h e hyphae ( F i g s . 4 0 - 4 1 ) . As v a c u o -l a t i o n o c c u r s , t h e m i t o c h o n d r i a become d i s t r i b u t e d i n t h e p e r i p h e r a l h y -pha l p r o t o p l a s m ( F i g s . 43-4.5); t h e y i n c r e a s e i n l e n g t h , and t h e i r m a t r i x becomes l e s s den se ( F i g s . 5 5 - 5 6 ) . In t h e o l d and a l m o s t c o m p l e t e l y v a c u o -l a t e d p r o t o p l a s m , t h e m i t o c h o n d r i a o f t e n appea r t o have f r a g m e n t e d as t h e y se ldom a r e l o n g e r t h a n 3-4 y. A l s o , a r e a s v o i d o f g r a n u l e s a r e common i n t h e i r m a t r i x ( F i g s . 5 1 , 5 7 ) . V e s i c l e s c o n t a i n i n g e l e c t r o n - d e n s e g r a n u l a r m a t e r i a l and r e s e m b l -i n g m i c r o b o d i e s i n s i z e and g e n e r a l mo rpho l o g y a r e s c a t t e r e d t h r o u g h o u t t h e hypha l p r o t o p l a s m ( F i g s . 4 0 - 4 1 ) . , They a r e s p h e r i c a l t o o v o i d i n p r o -f i l e and 200 -450 m ^ i n d i a m e t e r ( F i g s . 4 0 - 4 1 , 7 7 - 8 0 ) . They a r e l i m i t e d by a membrane w h i c h i s o f a p p r o x i m a t e l y t h e same t h i c k n e s s as t h a t o f t h e - 17 -p la sma membrane. The m a t r i x o f the m i c r o b o d y - 1 i k e v e s i c l e s c o n s i s t s m o s t -l y o f e l e c t r o n - d e n s e g r a n u l e s , b u t c o n c e n t r i c m e m b r a n e - l i k e f r a g m e n t s and t u b u l e s a l s o a r e v i s i b l e i n some o f t h e v e s i c l e s ( F i g s . 7 8 , 8 0 - 8 1 , 5 9 ) . The t u b u l e s a r e 150-200A i n d i a m e t e r . The m i c r o b o d y - 1 i k e v e s i c l e s se ldom a p -pea r t o be i n c l o s e a s s o c i a t i o n w i t h t h e E.R. nea r t h e a p i c a l g r o w i n g zone ( F i g . 4 1 ) , but i n o l d e r and/o r d i f f e r e n t i a t i n g h yphae , t h e y o c c u r a d j a c e n t t o n o n - r i b o s o m a l a r e a s o f t h e E.R. ( F i g s . 7 7 - 8 0 ) . The m u l t i - v e s i c u l a r b o d i e s a r e o v o i d t o i r r e g u l a r l y - s h a p e d v e s i c l e s i n t he p r o t o p l a s m ( F i g s . 7 5 - 7 6 ) . They a r e 300 -600 my i n d i a m e t e r , a t t h e i r w i d e s t p o i n t , and a r e bound by o n e - o r two membranes - each membrane mea -s u r i n g 70-80A i n t h i c k n e s s . S m a l l e r v e s i c l e s , 40 -120 my i n d i a m e t e r , a r e p r e s e n t . w i t h i n [ t h e m u l t i - v e s i c u l a r b o d i e s ( F i g s . 7 5 - 7 6 ) . T h e y a r e s p h e r i -c a l j , o v o i d , o r i r r e g u l a r i n p r o f i l e and u s u a l l y c o n t a i n e l e c t r o n - d e n s e g r a n -u l a r m a t e r i a l . Numerous s m a l l v a c u o l e s , a r e d i s c e r n i b l e i n young hypha l p r o t o p l a s m , bu t t h e y r a p i d l y expand and c o a l e s c e t o f o rm l a r g e r v a c u o l e s w h i c h become o r i e n t e d a l o n g t h e c e n t r a l a x i s p f t h e hyphae ( F i g s . 4 3 - 4 7 ) . The v a c u o l e s v a r y i n s i z e and s h a p e , b u t u s u a l l y t h e y a r e e l o n g a t e and i r r e g u l a r i n p r o f i l e . A . s i n g l e membrane, t h e t o n o p l a s t , encompa s se s s t he v a c u o l a r s a p . The l i p i d d r o p l e t s a r e s p h e r i c a l t o o v o i d - s h a p e d g l o b u l e s wh i ch v a r y f r o m 0 .4 -1 .2 . y i n d i a m e t e r ( F i g s . 4 1 - 4 7 ) . As d e s c r i b e d p r e v i o u s l y , t h e y b e c o m e . i n c r e a s i n g l y abundant as t h e d i s t a n c e f r om t h e a p i c a l g r o w i n g zone i n c r e a s e s . They a r e n o t membrane-bound and t h e y appea r t o be h i g h l y s a t u r a t e d compounds because o f t h e i r l ow e l e c t r o n - d e n s i t y w i t h a f i x a t i v e c o n t a i n i n g osmium t e t r o x i d e . R i bo somes , 140-150A i n d i a m e t e r , a r e abundant t h r o u g h o u t t h e d i s -t a l m u l t i - o r g a n e l l e zone e x c e p t i n o ld, - , h i g h l y v a c u o l a t e d p r o t o p l a s m ( F i g s . - 18 -4 1 - 4 7 ) . They u s u a l l y o c c u r o n . t h e s u r f a c e o f E.R. e x c e p t where t h e E;R. i s i n c l o s e a s s o c i a t i o n w i t h d i c t y o s o m e s and m i c r o b o d y - 1 i k e v e s i c l e s ( F i g s . 58 , 61 , 6 3 - 6 7 , 7 7 - 7 9 ) . Ovo id t o s p i n d l e - s h a p e d n u c l e i , 3 . 0^5 ;0 y i n l e n g t h and 1 .2 -1 .3 y i n w i d t h , a r e d i s p e r s e d t h r o u g h o u t t h e p e r i p h e r a l p r o t o p l a s m - t h e i r l o n g a x i s b e i n g o r i e n t e d p a r a l l e l t o t h a t o f t h e hyphae t F i g s . 4 1 , 8 4 - 8 9 ) . They a r e encompassed by a n u c l e a r e n v e l o p e w h i c h c o n s i s t s o f an o u t e r and an i n n e r membrane. The membranes each a r e 60-70A t h i c k e x c e p t i n t h e a r e a a d -j a c e n t t o t h e c e n t r i o l e s where t h e i n n e r membrane becomes 100-120A t h i c k -( F i g s . 9 2 - 9 3 ) . The i n t e r v e n i n g s pace between t he membranes i s 120-170A t h i c k . A n n u l i , 400-600A i n d i a m e t e r , o c c u r t h r o u g h o u t t h e n u c l e a r e n v e -l o p e e x c e p t i n t h e r e g i o n a d j a c e n t t o t h e c e n t r i o l e s ( F i g s . 8 4 - 9 0 , 9 2 - 9 3 ) . They a r e f o rmed o f an u n d e t e r m i n e d number o f s u b - u n i t s a b o u t a dense c o r e ( F i g . 9 0 ) . The n u c l e o l u s i s e c c e n t r i c a l l y l o c a t e d w i t h i n each n u c l e u s and i s a p p r o x i m a t e l y 0 . 7 - 1 . 0 y i n d i a m e t e r ( F i g s . 8 4 , 9 1 ) . I t c o n s i s t s o f a w e l l - d e f i n e d pars .fibrosa and pars granulosa ( J i g . 9 1 ) . The papp-fibrosa, i s composed m o s t l y o f f i n e f i l a m e n t s , 40-50A i n d i a m e t e r ; how-e v e r , .a few g r a n u l e s w h i c h a r e s i i g h t l y 1 a r g e r i n d i a m e t e r a r e v i s i b l e among t h e dense f i b r i l s . The pars- granulosa c o n s i s t s l a r g e l y o f g r a n u l e s , 100-140A i n d i a m e t e r and i t i s v i s i b l e a b o u t t h e pars fibrosa ( F i b s . 8 5 , 9 1 ) . Le s s dense r e g i o n s , somet imes r e f e r r e d t o as n u c l e o l a r v a c u o l e s (Brown and B e r t k e , 1969) o r t h e pars-amorpha (Bus.ch and Smetana,,. 1970) a l s o a r e common i n t h e n u c l e o l i ( F i g . 9 1 ) . A n d , as d e t e r m i n e d by s e r i a l s e c t i o n s , t h e y a r e c o n t i n u o u s w i t h t h e s u r r o u n d i n g n u c l e o p l a s m . Some n u c l e i a l s o appea r t o be i n a s t a t e o f d i v i s i o n ( F i g s . 8 7 , 9 2 - 9 3 ) . S p i n d l e f i b r i l s r a d i a t e f r o m e c c e n t r i c p o l a r r e g i o n s o f t h e t h i c k -ened i n n e r n u c l e a r membrane a d j a c e n t t o c e n t r i o l e s . . They a r e each 1 8 0 -- 19 -200A i n d i a m e t e r and each c o n s i s t s o f a dense r i m and a l e s s den se c o r e . Some o f t h e s p i n d l e f i b r i l s e x t e n d f r om p o l e t o p o l e w h i l e o t h e r s appea r t o t e r m i n a t e nea r e l e c t r o n - d e n s e c h r o m a t i n r e g i o n s , as o b s e r v e d i n o t h e r f u n g i (Hea th and Greenwood, ,1968; I c h i d a and F u l l e r , 1 968 ) . O c c a s i o n a l l y m e t a - . phase o r e a r l y a n a p h a s e - 1 i k e c o n f i g u r a t i o n s a r e a p p a r e n t ( F i g . 8 7 ) . The c e n t r i o l e s o c c u r i n p a i r s and 1 i e a p p r o x i m a t e l y 400 -500 f r o m the m o d i f i e d a r e a o f t h e n u c l e a r e n v e l o p e ( F i g s . 9 2 - 9 3 ) . They a r e c o m p a r a b l e , bo th i n s t r u c t u r e and d i m e n s i o n , t o c e n t r i o l e s i i i most o t h e r Phycomycetes ( B e r l i n and Bowen, ,1964 ; F u l l e r , .1966; F u l l e r and R e i c h l e , 1965 ; I c h i d a and F u l l e r , 1968 ; Heath and Greenwood, 1 9 6 8 ) . They a r e each 160-165:.,mu i n l e n g t h and d i a m e t e r , and t h e y c o n s i s t o f n i n e t r i p l e t s i n c y l i n d r i c a l a r r angement ( F i g . 9 2 ) . The n i n e t r i p l e t s a r e c o n n e c t e d t o a c e n t r a l t u b u l e by r a d i a l f i l a m e n t s , t h u s f o r m i n g a c a r t w h e e l . c o n f i g u r a -t i o n ( F i g s . 9 2 a - b ) . C e n t r i o l a r s a t e l l i t e s o r i n t e r c o n n e c t i o n s w i t h t h e n u c l e a r e n v e l o p e a r e no t d i s c e r n i b l e . The d e t a i l s a n d - d i m e n s i o n s o f c e n -t r i o l e s t r u c t u r e a r e shown i n D iag ram I I I . Hypha l M a t u r a t i o n and D i f f e r e n t i a t i o n A f t e r s e v e r a l d a y s * t he g r owth r a t e o f many hyphae d e c r e a s e s and z o n a t i o n o f t h e p r o t o p l a s m i n t h e hypha l t i p s becomes l e s s a p p a r e n t - o f t e n o n l y t h e a p i c a l g r o w i n g zone and t h e d i s t a l m u l t i - o r g a n e l l e zone b e i n g d i s t i n g u i s h a b l e ( F i g . 1 9 ) . An i n c r e a s e i n t h e o p t i c a l d e n s i t y o f t h e p r o t o -p l a sm o c c u r s and numerous p a r t i c l e s , r e c o g n i z a b l e as l i p o s o m e s by e l e c t r o n m i c r o s c o p y , become d i s c e r n i b l e t h r o u g h o u t t h e hyphae ( F i g . 1 9 ) . A l s o , as r e s o l v e d by e l e c t r o n m i c r o s c o p y , .the E:R. becomes more e x t e n s i v e l y d e v e l -oped and l e s s dense v e s i c u l a r r e g i o n s appea r w i t h i n m i c r o b o d i e s ( F i g s . 4 2 -4 5 ) . O the r o r g a n e l l e s and i n c l u s i o n s appea r s t r u c t u r a l l y t h e same as t h e i r - 20 -r e s p e c t i v e f o rms i n t h e younge r hypha l p r o t o p l a s m . The l i p o s o m e s a r e o v o i d t o s l i g h t l y i r r e g u l a r membrane-bound b o d i e s i n t h e p r o t o p l a s m ( F i g s . 4 2 - 4 5 , 6 9 - 7 3 ) . They a r e 0.3*1 .0 p I n - d i a m e t e r , and i n i t i a l l y c o n t a i n a homogeneous m i x t u r e o f amorphous and f i n e g r a n u l a r m a t e r i a l ( F i g s . 6 7 - 6 9 ) . The amorphous m a t e r i a l i s e l e c t r o n l i g h t , whereas t h e g r a n u l e s a r e a l m o s t as den se as t h e hypha l g round c y t o p l a s m . Osmio -p h i l i c g l o b u l e s r a p i d l y appea r w i t h i n t h e l i p o s o m e s a n d , e x c e p t i n e x t r e m e -l y o l d hypha l p r o t o p l a s m ( F i g . 7 3 ) , l a y e r i n g o f t h e e l e c t r o n - 1 i g h t m a t e r i a l a bou t t h e g l o b u l e s o c c u r s ( F i g s . 6 9 - 7 2 ) . The l a y e r s o f e l e c t r o n - l i g h t m a t -e r i a l a r e each a b o u t 90-100A t h i c k and a r e s e p a r a t e d f r o m each o t h e r by e l e c t r o n - d e n s e band s , 50-11 OA t h i c k . L a y e r i n g c o n t i n u e s u n t i l , t h e l i p o V somes a r e f i l l e d a l m o s t c o m p l e t e l y by . e l e c t r o n ^ i g h t and d a r k bands a b o u t t h e o s m i o p h i l i c g l o b u l e s ( F i g . 7 2 ) . The membrane a b o u t t h e l i p o s o m e s i s a p p r o x i m a t e l y 50-60A t h i c k , i . e . , 1 t h e same as t h a t o f E.R. The E.R. i s s t r u c t u r a l l y s i m i l a r t o t h a t o b s e r v e d i n y ounge r hyphal. p r o t o p l a s m ; however , i t becomes more e x t e n s i v e l y d e v e l o p e d as t h e hyphae d i f f e r e n t i a t e i n t o a s e x u a l and s e x u a l r e p r o d u c t i v e s t r u c t u r e s ( F i g s . 4 2 , 6 4 ) . D i l a t i o n o f t h e E.R. somet imes i s o b s e r v e d a n d , as d e s c r i b e d i n o o g o n i a l d e v e l o p m e n t , l i p o s o m e s a p p e a r t o d i f f e r e n t i a t e f r o m t h i s d i l a t e d E.R. ( F i g . 6 7 ) . The m i c r o b o d i e s u s u a l l y e x h i b i t t h e same s t r u c t u r a l f e a t u r e s a s t h o s e d e s c r i b e d p r e v i o u s l y ( F i g s . 7 8 - 8 0 ) . However , t h e y a l s o may become c h a r a c t e r i z e d by l e s s dense v e s i c u l a t e a r e a s w i t h i n t h e i r d e n s e r m a t r i x ( F i g s . 7 7 , 8 1 - 8 3 ) . I n d i c a t i o n s o f c o n s t r i c t i o n o r budd ing o f m i c r o b o d i e s a r e e v i d e n t , as d u m b - b e l 1 - 1 i k e fo rms o c c u r o c c a s i o n a l l y i n t h e p r o t o p l a s m ( F i g s . 7 7 , 8 2 ) . - 21 -GEMMAE In b o t h t h e ma le and f e m a l e s t r a i n s , t e r m i n a l and i n t e r c a l a r y gemmae a r e o b s e r v e d f o u r t o s i x days a f t e r i n i t i a t i o n o f hypha l deve l opmen t ( F i g s . t 9 4 - 9 7 ) . T h e y u s u a l l y o c c u r s i n g l y on t h e hyphae ( J i g s . 9 6 - 9 7 ) , bu t o c c a -s i o n a l l y c a t e n u l a t e fo rms d e v e l o p ( F i g s . 9 4 - 9 5 ) . Most.gemmae a r e f i l i f o r m o r d o l i o f o r m i n p r o f i l e and a r e d e l i m i t e d f r om t h e v e g e t a t i v e hyphae by c o n c a v e - s h a p e d s e p t a ( F i g s . 9 4 - 9 6 ) . T h e i r d i a m e t e r i s s l i g h t l y g r e a t e r t h a n t h a t o f t h e hyphae and t h e i r l e n g t h i s v a r i a b l e , r a n g i n g f r o m one hundred t o s e v e r a l hundred m i c r o n s . S p h e r i c a l o r g l o b o s e - s h a p e d gemmae a l s o a r e fo rmed i n t h e f e m a l e s t r a i n ( F i g . 97B. T h e i r d i a m e t e r i s a l m o s t t w i c e t h a t o f t h e hyphae. The gemmae o f bo th t h e ma le and f e m a l e s t r a i n s a r e m o r p h o l o g i c a l l y s i m i l a r ( F i g s . 98.-99). T h e i r p r o t o p l a s t s a r e bound by a w a l l fo rmed b a s i -c a l l y o f two l a y e r s - an o u t e r l a y e r w h i c h , i s a c o n t i n u a t i o n o f t h e o u t e r hypha l w a l l , and an i n n e r l a y e r w h i c h encompasses t h e gemmae p r o t o p l a s m ( F i g s . 1 0 0 - 1 0 3 ) . The o u t e r l a y e r c o m p r i s e s a p p r o x i m a t e l y o n e - f o u r t h o f t h e t o t a l w a l l t h i c k n e s s and c o n s i s t s o f amorphous and f i b r i l l a r m a t e r i a l ( F i g s . 1 0 0 - 1 0 1 ) . I t s s u r f a c e u s u a l l y a p p e a r s a s a l o o s e r e t i c u l a t i o n o f e l e c t r o n - d e n s e f i b r i l s w h i c h s l o u g h o f f and e f f e c t i v e l y remove b a c t e r i a and/o r o t h e r p a r t i c l e s o r m i c r o - o r g a n i s m s w h i c h have adhe red t o i t ( F i g s . 9 8 , 1 0 0 ) . The i n n e r l a y e r o f t h e w a l l i s s t r u c t u r a l l y s i m i l a r t o , and o f t e n d i f f i c u l t t o d i s t i n g u i s h f r o m , t h e o u t e r l a y e r ( F i g s . 98 , 1 0 0 - 1 0 3 ) . I t a l s o i s composed o f a m o r p h o u s . m a t e r i a l and f i b r i l s , bu t f i n e s t r i a t i o n s , i n d i c a t i v e o f l a y e r i n g , u s u a l l y a r e c h a r a c t e r i s t i c o f t h i s i n n e r w a l l m a t -e r i a l ( F i g s . 1 0 1 - 1 0 3 ) . P o c k e t s o f e l e c t r o n - d e n s e g r a n u l a r and v e s i c u l a r m a t e r i a l a r e d i s t i n g u i s h a b l e between t h e l a y e r s o f t h e w a l l o r w i t h i n t h e - 22 -i n n e r w a l l l a y e r i n some hyphae ( F i g . 1 0 0 ) . The t h i c k n e s s o f t h e gemmae w a l l s i s v a r i a b l e ( F i g s . 98 , 1 0 0 - 1 0 3 ) ; however , t h e o l d e r and/o r l a r g e r gemmae u s u a l l y p o s s e s s t h i c k e r w a l l s t h a n do t h e y o u n g e r and/o r s m a l l e r gemmae. The w a l l s measure 500-950 my i n t h i c k n e s s . C o m p l e t e s e p t a s e p a r a t e gemmae f rom t h e r e s t o f t h e hypha l p r o t o -p la sm and f r om each o t h e r ( F i g s . 1 0 2 - 1 0 3 ) . The s e p t a e a r e c o n c a v e i n p r o -f i l e and a r e s t r u c t u r a l l y s i m i l a r t o t h o s e w h i c h d e l i m i t z o o s p o r a n g i a , a n t h e r i d i a and o o g o n i a f r om h y p h a l p r o t o p l a s m . They a r e f o rmed o f two w a l l l a y e r s - one b e i n g t h e i n n e r l a y e r o f t h e gemma w a l l and t h e o t h e r b e i n g c o n t i n u o u s . w i t h t h e i n n e r l a y e r o f t h e hypha l w a l l ( F i g s . 102-.103). The l a t t e r l a y e r i s t h i n n e r t h a n t h e f o r m e r o n e , but i t i s m o r p h o l o g i c a l l y s i m i l a r ( F i g . 1 0 3 ) . E l e c t r o n dense m a t e r i a l and v e s i c u l a r e l e m e n t s a r e ' p r e s e n t between t h e two s e p t a l w a l l l a y e r s ( F i g s . 1 0 2 * 1 0 3 ) ; t h e y a r e s i m i l a r t o t h o s e o b s e r v e d i n t h e i n n e r w a l l l a y e r i n s p o r a n g i a o f Ph.yto-ph.-r. thova erytkroseptioa (Chapman and V u j i c i c , 1965) and t o t h e v e s i c u l a r e l e -ments i n t h e w a l l s o f P. parasitioa (Hemmes and H o h l , 1 9 6 9 ) . O c c a s i o n a l p r o l i f e r a t i o n s and e l a b o r a t i o n s o f t h i s m a t e r i a l and o f t h e w a l l l a y e r s o c c u r and i r r e g u l a r i t y i n t h e s e p t a l p r o f i l e r e s u l t s ( F i g . 1 0 2 ) . I f s e -p a r a t i o n o f t h e gemmae f r om t h e hyphae o r f r o m each o t h e r o c c u r s , t h e f r a c -t u r e zone a p p e a r s t o be i n t h i s v e s i c u l a r e l e m e n t a r e a between t h e two w a l l l a y e r s . . The p la sma membrane i s s i m i l a r t o t h a t d e s c r i b e d f o r t h e v e g e t a t i v e hyphae , e x c e p t t h a t i t se ldom becomes e l a b o r a t e d t o f o rm lomasomes ( F i g s . 9 8 , 1 0 1 ) . O n l y i r r e g u l a r l y - s h a p e d , v e s i c l e s appea r t o be c l o s e l y a s s o c i a t -ed w i t h , o r somet imes f u s e d w i t h , t h e membrane ( F i g s . 98 , 1 0 0 - 1 0 1 ) . The v e s i c l e s , w h i c h measure a p p r o x i m a t e l y 120-200 my i n d i a m e t e r , c o n t a i n m a t -- 23 -e r i a l o f a l m o s t t h e same e l e c t r o n d e n s i t y as t h a t o f t h e w a l l . And , u n -l i k e t h e c o n d i t i o n o b s e r v e d . i n v e g e t a t i v e hyphae , t h e r e i s n o t t h e accumu-l a t i o n o f t h e s e . v e s i c l e s t o t h e e x c l u s i o n o f o t h e r o r g a n e l l e s i n t h e a p i c a l p r o t o p l a s m ( F i g s . 9 8 , 1 0 0 ) . U s u a l l y t h e r e i s np c o n c e n t r a t i o n o f v e s i c l e s a t t h e a p i c e s , as compared w i t h t h e p r o x i m a l p e r i p h e r i e s ( F i g . 1 0 0 ) . V e s i - . c l e s a r e s c a t t e r e d t h r o u g h o u t t h e p r o t p l a s m . W i t h i n t h e p r o t op l a sm.numerou s o t h e r o r g a n e l l e s and i n c l u s i o n s a r e r andomly d i s t r i b u t e d a b o u t a l a r g e c e n t r a l v a c u o l a r s y s t em w h i c h i s a com-p o s i t e o f s e v e r a l s m a l l e r v a c u o l e s ( f i g - 9 9 ) - The v a c u o l a r s y s t e m , o r v a c u o l e s , ; o f t e n a t t a i n s a d i a m e t e r o f 6-25 u o r l a r g e r . I t s l e n g t h i s v a r i a b l e , d e p e n d i n g on t h e gemmae. The t o n o p l a s t , w h i c h f r e q u e n t l y a p p e a r s r u p t u r e d , i s 90-100A i n t h i c k n e s s - t h e same as i n t h e v e g e t a t i v e hyphae ( F i g s . 99 , 1 1 8 ) . The v a c u o l a r sap. a l s o i s s i m i l a r t o t h a t o b s e r v e d i n t h e v e g e t a t i v e hyphae ; however , s p h e r i c l e s o f a g g r e g a t e d m e t a b o l i t e s a r e d i s c e r n i b l e i n some v a c u o l e s ( F i g s . 99 , 1 1 5 ) . The s p h e r i c l e s a r e 1 . 0 - 3 . 0 ji i n d i a m e t e r and a r e l e s s e l e c t r o n - d e n s e t h a n t h e s u r r o u n d i n g v a c u o l a r s a p . S m a l l e r v a c u o l e s a l s o a r e common t h r o u g h o u t t h e p r o t o p l a s m ( F i g s . 9 8 - 9 9 ) . M i t o c h o n d r i a a r e abundant i n t h e p r o t o p l a s m o f t h e gemmae ( F i g s . 9 8 - 9 9 ) . They a r e r andom l y o r i e n t e d , bu t t h e y o f t e n a r e i n c l o s e a s s o c i a -t i o n o r i n c o n t a c t w i t h l i p o s o m e s ( F i g s . 9 8 - 9 9 , 1 0 8 - 1 0 9 ) . The shapes and s i z e s . o f t h e m i t o c h o n d r i a a r e v a r i a b l e . O v o i d , r o d , d u m b - b e l l and i r r e -g u l a r f o rms a l l a r e common, and t h e y r ange f r o m 0 . 6 - 2 . 4 y i n l e n g t h t o 0 . 3 - 0 . 6 u ; i n w i d t h ( F i g s . 9 8 - 9 9 , 1 0 8 - 1 0 9 ) . The g r e a t v a r i a t i o n i n l e n g t h and t he c o n s t r i c t e d a p p e a r a n c e o f some fo rms i n d i c a t e t h a t many m i t o c h o n -d r i a p r o b a b l y have r e p l i c a t e d r e c e n t l y , w h i l e o t h e r s a r e i n t h e p r o c e s s o f d o i n g s o . S t r u c t u r a l l y , t h e m i t o c h o n d r i a a r e s i m i l a r t o t h o s e i n t h e v e g e t a t i v e hyphae e x c e p t t h a t t he c r i s t a e a r e l o n g e r and u s u a l l y o v e r - . , l a p each o t h e r ( F i g s . 9 8 - 9 9 , 1 0 8 - 1 0 9 ) . - 2 4 -G r a n u l a r E.R. i s p r e s e n t i n t h e p r o t o p l a s m , b u t i t i s n o t abundant no r i s i t as ea sy t o d i s c e r n a s . i n o t h e r d e v e l o p m e n t a l s t a g e s ( F i g s . 110 , 1 14 , ,117). I t o f t e n i s a s s o c i a t e d w i t h t h e f o r m i n g f a c e o f d i c t y o s o m e s and w i t h t h e o u t e r membrane o f n u c l e a r e n v e l o p e s ( F i g s . 110 , 1 14 , 1 1 8 ) , The E.R. a s s o c i a t e d w i t h t h e f o r m i n g f a c e c i s t e r n a e o f d i c t y o s o m e s . u s u a l l y e x -h i b i t s s l i g h t h y p e r t r o p h y and l a c k s r i b o s o m e s on i t s j u x t a - d i c t y o s o m a l s u r f a c e ( F i g s . 110 , 1 1 4 ) . I n d i c a t i o n s o f b l e b b i n g on i t s n o n - r i b o s o m a l s u r f a c e a l s o a r e e v i d e n t ( F i g . 1 1 0 ) . The g r a n u l a r E.R. w h i c h a p p e a r s in.-* dependen t o f o t h e r o r g a n e l l e s and i n c l u s i o n s i s no t as e x t e n s i v e l y d e v e l o p -ed as i n t h e v e g e t a t i v e hyphae ( F i g . 1 1 7 ) . M i c r o b o d i e s ( F i g s . 98 , 1 0 4 - 1 0 7 ) , l i p o s o m e s ( J i g s . 9 8 - 9 9 , 1 1 6 ) , l i p i d d r o p l e t s ( F i g s . 98 -99) ; and r i b o s omes ( F i g s . 98 -99 ) a r e a l l common i n t h e p r o t o p l a s m o f gemmae. They a l l a r e s i m i l a r i n a p p e a r a n c e t o t h e i r r e s p e c t i v e f o rms i n t he v e g e t a t i v e hyphae ; however , a f ew d i f f e r e n c e s a r e d e t e c t a b l e . On l y h i g h l y d i f f e r e n t i a t e d , l i p o s o m e s a r e p r e s e n t i n f u l l y -fo rmed gemmae ( F i g s . 9 8 - 9 9 , 1 1 6 ) . Younge r , l e s s d e v e l o p e d f o rms a r e no t d i s t i n g u i s h a b l e . They a r e o f t e n p a r t i a l l y s u r r o u n d e d by o r i n c l o s e c o n -t a c t w i t h m i t o c h o n d r i a ( F i g s . 1 0 8 - 1 0 9 ) . T h e i r membranes c o m e . i n t o d i r e c t c o n t a c t w i t h , b u t do no t f u s e w i t h , t h e o u t e r m i t o c h o n d r i a l membranes. L i p i d d r o p l e t s a r e more abundant i n gemmae t h a n i n t h e v e g e t a t i v e hyphae . A l s o , t h e y a r e i r r e g u l a r i n p r o f i l e , m e a s u r i n g 0 . 7 - 1 . 2 u a t t h e i r g r e a t e s t p o i n t ( F i g s . 9 8 - 9 9 ) . D i c t y o s o m e s . a r e o b s e r v e d i n t h e same p o s i t i o n a l a s s o c i a t i o n w i t h E.R. and t h e o u t e r membrane o f n u c l e a r e n v e l o p e s as t h e y a r e i n t h e hyphae ( F i g s . 1 1 0 - 1 1 4 ) . A l s o , t h e y e x h i b i t t h e same b a s i c s t r u c t u r a l o r g a n i z a t i o n as t h e y do i n t h e o t h e r d e v e l o p m e n t a l s t a g e s ( J i g s . 110-11.4). However , t h e c i s t e r n a e o f t e n appea r t o be d i s c o n t i n u o u s and numerous v e s i c l e s , 6 0 0 -- 25 -750A i n d i a m e t e r , a r e v i s i b l e between t h e f o r m i n g f a c e c i s t e r n a e and t h e n u c l e a r e n v e l o p e o r E.R. ( F i g s . 1 1 0 - 1 1 2 ) . The v e s i c l e s w h i c h a r e p i n c h e d o f f f r o m t h e c i s t e r n a l p e r i p h e r i e s a t t h e d i s t a l f a c e a r e s i m i l a r t o t h e v e s i c l e s s c a t t e r e d t h r o u g h o u t t h e p r o t o p l a s m ( F i g s . 9 8 - 1 0 1 ) . N u c l e i o f v a r i o u s c o n f i g u r a t i o n s ' a r e d i s t r i b u t e d r andom l y t h r o u g h -o u t t h e c y t o p l a s m ( F i g . 9 8 ) . T h e i r m o r p h o l o g i c a l c h a r a c t e r i s t i c s a r e s i m i -l a r t o t h o s e no ted p r e v i o u s l y f o r n u c l e i i n , t h e v e g e t a t i v e hyphae; however , s e v e r a l d i f f e r e n c e s a r e d i s c e r n i b l e . The n u c l e i u s u a l l y a r e s h o r t e r t h a n t h o s e o f t h e hyphae - t h e l a r g e s t ones m e a s u r i n g 4 . 0 y i n l e n g t h ( F i g s . 1 1 7 - 1 1 9 ) . The e n v e l o p e membranes a r e more c r e n u l a t e i n p r o f i l e ( F i g s . 1 1 7 - 1 1 9 ) . The n u c l e o l i o f t e n a r e l a r g e r , a t t a i n i n g d i a m e t e r s a s g r e a t a s 1.4 And t h e y a r e composed o f a w e l l d i f f e r e n t i a t e d pars fibrosa--' and pars granulosa, t h e l a t t e r o f w h i c h f o rms a d i f f u s e r e t i c u l a t i o n abou t t he par J fibrosa . ( F i g s . 117 , 1 1 9 ) . The pars ahromasqma^ so i s , d i s c e r -n i b l e i n a few n u c l e i ( F i g . 1 1 9 ) . J u x t a - n u c l e a r , p a i r e d c e n t r i o l e s a r e p r e s e n t i n t h e gemmae o f A. bisexualis ( F i g s . 1 2 0 - 1 2 3 ) . They a r e a l i g n e d e n d - t o - e n d and l i e p a r a l l e l t o a m o d i f i e d a r e a o f t h e n u c l e a r e n v e l o p e , ( F i g s . 1 2 0 - 1 2 3 ) . A l s o , t h e y o c c u r a d j a c e n t t o o r i n c l o s e p r o x i m i t y t o t h e n u c l e o l u s o f each n u c l e u s . ( F i g . 1 2 1 ) . The c e n t r i o l e s s t r u c t u r a l l y r e s e m b l e t h o s e i n t h e v e g e t a t i v e hyphae and t h o s e o b s e r v e d i n p u b l i s h e d m i c o r g r a p h s o f Aohlya s p . (Brown and B e r t k e , 1969 , pp. 3 9 5 ) . However , two p a i r s o f c e n t r i o l e s u s u a l l y a r e a s s o c i a t e d w i t h each n u c l e u s and each p a i r may be i n t e r c o n n e c t e d by one o f t he p e r i p h e r a l t r i p l e t t u b u l e s ( F i g . 1 2 0 ) . A l s o , t h e c e n t r a l r e g i o n i n each c e n t r i o l e a p p e a r s a s a d o u b l e t ( F i g . 1 2 3 ) . - 26 -ZOOSPORANGIA L i g h t M i c r o s c o p y O b s e r v a t i o n s o f Z o o s p o r a n g i a l Deve lopment I n d i c a t i o n s o f z o o s p o r a n g i a l d e v e l o p m e n t a r e o b s e r v e d two t o t h r e e day s s ub sequen t t o hypha l d e v e l o p m e n t . A d e c r e a s e i n t h e hypha l g r owth r a t e o c c u r s and t h e r e i s an i n c r e a s e i n t h e o p t i c a l d e n s i t y o f t h e p r o t o -p l a s m . The p r o t o p l a s m i n t h e hypha l t i p s becomes d e l i m i t e d f r om t h e r e s t o f t he p r o t o p l a s m by t h e deve l opmen t o f a s ep tum, w h i c h a p p e a r s c o n c a v e i n p r o f i l e , and t e r m i n a l z o o s p o r a n g i a a r e fo rmed ( F i g - 1 2 5 ) . The z o o s p o r a n -g i a a r e l o n g and c y l i n d r i c a l - t h e i r d i a m e t e r s b e i n g s l i g h t l y g r e a t e r t h a n t h o s e o f s u b t e n d i n g hyphae ( F i g s . 1 2 5 - 1 2 9 ) . M o r p h o l o g i c a l l y , t h e z o o -s p o r a n g i a a r e s i m i l a r , t o t h e " n o r m a l " t y p e d e s c r i b e d by Ro t her t (.1892), They a r e c h a r a c t e r i z e d by t h i c k p a r i e t a l p r o t o p l a s m a b o u t a c e n t r a l v a c -u o l e ( F i g s . 1 2 5 - 1 2 7 , 1 2 9 - 1 3 0 ) . O c c a s i o n a l l y , o n l y a t h i n l a y e r o f p r o t o -plasm, i s p r e s e n t a b o u t t h e v a c u o l e ( F i g . 1 2 8 ) . The m a t u r a t i o n o f t h e z o o s p o r a n g i a i s s i m i l a r i n bo th t h e m a l e and f e m a l e s t r a i n s o f Aehlya bisexualis. I t o c c u r s by t h e p r o g r e s s i v e c l e a v a g e o f t h e p r o t o p l a s m , as d e s c r i b e d by o t h e r i n v e s t i g a t o r s ( R o t h e r t , 1892 ; S c h w a r z e , 1 9 2 2 ) . I n i t i a l l y t h e r e i s t h e a p p e a r a n c e o f u n d u l a t i o n s , o r f u r r o w s , a t t h e i n t e r f a c e o f t he p r o t o p l a s m and t h e c e n t r a l v a c u o l e ( F i g . 1 2 6 ) . The f u r r o w s d e v e l o p i n t o d i s t i n c t c l e f t s . t h a t e x t e n d toward t h e p e r i p h e r y o f t h e z o o s p o r a n g i a ( F i g . 1 2 7 ) . A f t e r 10 -15 m i n u t e s , com-p l e t e p r o t o p l a s m i c c l e a v a g e o c c u r s and numerous z o o s p o r e i n i t i a l s a r e d i s -c e r n i b l e ( F i g . 1 2 8 ) . , The z o o s p o r e i n i t i a l s each appea r s p h e r i c a l t o p o l y -g o n a l , a s seen i n s u r f a c e v i e w ( F i g . 1 2 8 ) . A l s o , t h e r e i s t h e deve l opmen t o f an a p i c a l o r , i n f r e q u e n t l y , a l a t e r a l p a p i l l a on each z oo spo r ang i um ( F i g s . 1 2 6 - 1 3 0 , 1 33 , 1 3 5 ) . R a r e l y , two p a p i l l a e a r e fo rmed on a s i n g l e z o o s p o r a n g i u m ( F i g . 1 3 4 ) . P r o t o p l a s m i s d i s c e r n i b l e i n t h e p a p i l l a e . Soon a f t e r t h e d e v e l o p m e n t o f z o o s p o r e i n i t i a l s , t h e z o o s p o r a n g i a l p r o t o -p l a sm r a p i d l y becomes homogeneous i n a p p e a r a n c e a g a i n ( F i g . 1 2 9 ) . S i m u l -t a n e o u s l y , a s l i g h t r e d u c t i o n i n t h e l e n g t h and d i a m e t e r o f t h e z o o s p o r a n -g i a o c c u r s and t h e s e p t a become l e s s c o n c a v e i n p r o f i l e . O the r i n v e s t i g a -t o r s (Gay and Greenwood, 1966; R o t h e r t , 1892 ; S c h w a r z e , 1922) have r e -p o r t e d s i m i l a r o b s e r v a t i o n s f o r c l o s e l y r e l a t e d f u n g i . C o n t r a c t i o n and r e - a p p e a r a n c e o f d i s t i n c t z o o s p o r e i n i t i a l s o c c u r ( F i g . 1 3 0 ) , t h e a p i c a l d i s c h a r g e p a p i l l a e r u p t u r e , and p r i m a r y z o o s p o r e s e s c ape f rom each z o o -spo rang i um ( F i g . 1 3 1 ) . The p r i m a r y z o o s p o r e s somet imes e x h i b i t s l i g h t amoebo id movement, b u t t h e y u s u a l l y e n c y s t i m m e d i a t e l y and r e m a i n nea r t h e r u p t u r e d p a p i l l a e u n t i l t h e y d e v e l o p i n t o f l a g e l l a t e d s e c o n d a r y z o o s p o r e s ( F i g s . 1 32 , 1 3 6 - 1 3 7 ) . I n d i c a t i o n s o f f l a g e l l a t i o n i n t he p r i m a r y z o o s p o r e i n i t i a l s a r e n o t o b s e r v e d d u r i n g t h e i r d e v e l o p m e n t . - 28 -The F i n e S t r u c t u r e o f Z o o s p o r a n g i a l Deve lopment The f i n e s t r u c t u r e o f z o o s p o r a n g i a l d e v e l o p m e n t i s s i m i l a r t o t h a t d e s c r i b e d f o r o t h e r c l o s e l y r e l a t e d Phycomycetes ( B e r l i n and Bowen, 1964 ; Chapman and V u j i c i ' c , 1965 ; Gay and Greenwood, .1966; Hemmes and H o h l , 1969; Hohl and Hamamoto, 1967; W i l l i a m s and W e b s t e r , 1 970 ) . Each young z oo spo r ang i um i s bound by a w a l l , 120 -200 my t h i c k . The w a l l c o n s i s t s o f an ,oute r and an i n n e r l a y e r , b o t h o f w h i c h a r e r e l a t i v e l y e l e c t r o n - l i g h t and f i n e l y s t r i a t e d ( F i g s . 140 , 144 , 1 5 7 ) . The o u t e r l a y e r appea r s t o be a c o n t i n u a t i o n o f t h e o u t e r hypha l w a l l ; t h e . i nne r l a y e r encompasses t h e z o o s p o r a n g i a l p r o t o p l a s m . P o c k e t s o f e l e c t r o n - d e n s e g r a n u -l a r and v e s i c u l a r m a t e r i a l somet imes a r e v i s i b l e between t h e two l a y e r s ( F i g . 1 4 3 ) . As z o o s p o r a n g i a l d i f f e r e n t i a t i o n p roceeds , , the t h i c k n e s s o f t h e w a l l i n c r e a s e s t o a p p r o x i m a t e l y 250 -300 my e x c e p t i n t h e a p i c a l r e g i o n where a n i p p l e - s h a p e d p a p i l l a d e v e l o p s ( F i g s . 1 5 1 - 1 5 4 ) . The p a p i l l a i s fo rmed e a r l y i n z o o s p o r a n g i a l d i f f e r e n t i a t i o n , and i t s d e v e l o p m e n t a p p e a r s t o be c o r r e l a t e d w i t h t h e p r e s e n c e o f v e s i c l e s i n t h e a p i c a l p r o t o p l a s m ( F i g s . 1 5 1 - 1 5 2 ) . The v e s i c l e s r e s e m b l e t h o s e i n t h e a p i c a l g r o w i n g zone o f t h e v e g e t a t i v e hyphae . They a r e p r e s e n t i n abundance p r i o r t o p a p i l l a d e v e l o p m e n t , b u t few o f t h e s e v e s i c l e s a r e v i s i b l e i n t h e p r o t o p l a s m o f t h e r e l a t i v e l y w e l l - d e v e l o p e d p a p i l l a ( F i g s . 1 5 3 - 1 5 4 ) . The w a l l o f t h e p a p i l l a i s 50 -100 my t h i c k . The septum d e l i m i t i n g t h e z o o s p o r a n g i a l p r o t o p l a s m f r o m t h e hypha l p r o t o p l a s m i s t h e same as t h a t d e s c r i b e d f o r gemmae ( F i g . 1 4 4 ) . On l y t h e t h i c k n e s s o f t h e s e p t a l w a l l l a y e r s d i f f e r s - b o t h l a y e r s u s u a l l y a r e o f t h e same o r s i m i l a r t h i c k n e s s . The p r o t o p l a s m o f t h e young z o o s p o r a n g i a i s e x t r e m e l y den se ( F i g s . - 29 -140-1.44). I t c o n s i s t s o f numerous r andomly s c a t t e r e d c l e a v a g e v e s i c l e s , m i t o c h o n d r i a , l i p o s o m e s and r i b o s o m e s . M i c r o b o d i e s , d i c t y o s o m e s , ^ e n d o p l a s -m i c r e t i c u l u m ( E . R . ) and d e v e l o p i n g p a r a s t r a s o m e s a r e p r e s e n t . C e n t r i o l e s o c c u r i n c l o s e a s s o c i a t i o n w i t h n u c l e i ; , t h e n u c l e i r a p i d l y become o r i e n t e d e q u i d i s t a n t t o each o t h e r . A l a r g e c e n t r a l v a c u o l a r s y s t e m , as w e l l as s m a l l e r p e r i p h e r a l v a c u o l e s , a r e a l s o p r e s e n t i n t h e p r o t o p l a s m . The c l e a v a g e v e s i c l e s a r e s p h e r i c a l t o i r r e g u l a r l y - s h a p e d membrane-bound b o d i e s ( F i g s . 1 4 1 - 1 5 0 ) . They a r e 100 -250 my a t t h e i r g r e a t e s t expanse and c o n t a i n amorphous to, g r a n u l a r m a t e r i a l . As z o o s p o r a n g i a l d e v e l o p m e n t o c c u r s , t h e s e v e s i c l e s become a l i g n e d i n p l a n e s w h i c h a r e e q u i d i s t a n t b e -tween a d j a c e n t n u c l e i ( F i g s . 1 4 5 - 1 4 7 ) . C o n c u r r e n t l y , m i t o c h o n d r i a become . o r i e n t e d abou t each o f t h e n u c l e i , 1 i p i d d r o p l e t s s u r r o u n d t h e m i t o c h o n d r i a , l i p o s o m e s e n c i r c l e t h e l i p i d d r o p l e t s , and d e v e l o p i n g p a r a s t r a s o m e s become a s s o c i a t e d w i t h p l a n e s o f c l e a v a g e v e s i c l e s and w i t h c e n t r i o l e s w h i c h a l -r e a d y have d i f f e r e n t i a t e d i n t o b a s a l b o d i e s ( F i g s . 1 4 8 - 1 5 0 ; 1 5 5 - 1 5 8 ) . The c l e a v a g e v e s i c l e s t hen f u s e w i t h each o t h e r and w i t h t h e v a c u o l e s a n d , as a r e s u l t , t h e p r o t o p l a s m c l e a v e s i n t o n u m e r o u s , u n i - n u c l e a t e p r i m a r y z o o -s p o r e i n i t i a l s ( F i g s . 1 4 5 - 1 5 0 ) . However , as no ted by Hohl and Hamamoto (1967) t h e p r o c e s s o f v e s i c u l a r f u s i o n does no t appea r t o p r o c e e d p e r f e c t -l y . F ragments o f c y t o p l a s m o f t e n become i s o l a t e d o r t r a p p e d between t h e z o o s p o r e i n i t i a l s and/o r t h e i n i t i a l s and t h e z o o s p o r a n g i a l w a l l ( F i g s . 1 4 8 - 1 5 0 ) , The m i t o c h o n d r i a a r e s i m i l a r t o t h o s e d e s c r i b e d f o r t h e v e g e t a t i v e hyphae , but s h o r t e r , m e a s u r i n g 0 . 5 - 0 , 7 v i n d i a m e t e r and 0 . 8 - 3 . 0 y i n l e n g t h , and more v a r i a b l e i n p r o f i l e - s p h e r i c a l , o v o i d and c u p - s h a p e d fo rms b e i n g o b s e r v e d i n some z o o s p o r a n g i a ( F i g . 1 4 3 ) . As no ted b e f o r e , d u r i n g z o o s p o r a n g i a l d i f f e r e n t i a t i o n , t h e m i t o c h o n d r i a become o r i e n t e d a b o u t each o f t h e n u c l e i , t h e i r , d e n s i t y a p p e a r s t o i n c r e a s e , and t h e i r - 30 -c r i s t a e e l o n g a t e and become f l e x u o u s i n p r o f i l e ( F i g s . 1 4 5 - 1 5 0 ) . In t h e f e m a l e s t r a i n , one c r i s t a i n each m i t o c h o n d r i o n a l s o becomes d i l a t e d ( F i g s . ' 1 5 9 - 1 6 0 ) . The c r i s t a e c o n t a i n e l e c t r o n - l i g h t amorphous m a t e r i a l and e l e c t r o n - d e n s e f i b r i l s - t h e l a t t e r u s u a l l y e x t e n d i n g t h e l e n g t h o f each c r i s t a ( F i g s . 1 5 9 - 1 6 2 ) . The l i p o s o m e s appea r as s p h e r i c a l t o i r r e g u l a r l y - s h a p e d . o s m i o p h i l i c b o d i e s ; i n t h e p r o t o p l a s m ( F i g s . 1 6 7 - 1 6 8 ) . They measure 0 . 4 - 1 . 2 v i n d i a -me te r and a r e encompassed by a 60-70A t h i c k membrane. One, and somet imes two o r t h r e e , g l o b u l e s a r e s u r r o u n d e d by numerous a l t e r n a t i n g l a y e r s o f e l e c t r o n - l i g h t and e l e c t r o n - d e n s e amorphous - m a t e r i a l , 90-100A and 40 -60A t h i c k , r e s p e c t i v e l y . G r a n u l e s , 110-200A i n d i a m e t e r , a l s o a r e p r e s e n t i n some o f t h e l i p o s o m e s ( F i g ; , 1 6 7 ) . As no ted p r e v i o u s l y , t h e l i p o s o m e s a t l a t e r s t a g e s o f z o o s p o r a n g i a l d i f f e r e n t i a t i o n become o r i e n t e d abou t t h e l i p i d d r o p l e t s and m i t o c h o n d r i a w h i c h have c l u s t e r e d a b o u t each o f t h e n u c l e i i n t u r n ( F i g s . 1 4 5 - 1 5 0 ) . The m i c r o b o d i e s , 1 i p i d - d r o p l e t s .and r i b o s o m e s a l l r e s e m b l e t h e i r r e s p e c t i v e fo rms as d e s c r i b e d f o r t h e v e g e t a t i v e hyphae ( F i g s . 1 4 0 - 1 5 8 ) . However j t h e l i p i d d r o p l e t s and r i b o s omes a r e r e l a t i v e l y more abundant t h a n was o b s e r v e d p r e v i o u s l y and o r i e n t a t i o n o f t h e l i p i d d r o p l e t s a b o u t t he m i t o c h o n d r i a becomes e v i d e n t d u r i n g p r o t o p l a s m i c c l e a v a g e ( F i g s . 1 45 -1 5 0 ) . The d i c t y o s o m e s , a l s o a r e s i m i l a r t o t h o s e d e s c r i b e d f o r t h e v e g e t a -t i v e hyphae ( F i g s . 1 4 1 , 1 7 0 - 1 7 4 ) . They, each c o n s i s t o f a s t a c k o f 4 - 6 c i s -t e r n a e , t h e l a t t e r o f t e n b e i n g d i s c o n t i n u o u s i n t h e u n c l e a v e d p r o t o p l a s m bu t becoming h i g h l y d e v e l o p e d as c l e a v a g e o c c u r s ( F i g s . 1 7 0 - 1 7 4 ) . They e x h i b i t p o l a r i t y and appea r i n t e r - a s s o c i a t e d w i t h E.R. o r n u c l e i and s e c -r e t o r y v e s i c l e s , as d e f i n e d by G r o v e , B r a c k e r and M o r r e / ( 1 9 6 8 , 1 9 7 0 ) . In . - 31 -young z o o s p o r a n g i a , t h e p r o x i m a l d i c t y o s o m e c i s t e r n a e u s u a l l y a r e a s s o -c i a t e d w i t h E.R. , bu t as i n d i v i d u a l p r i m a r y z o o s p o r e i n i t i a l s become d e -l i m i t e d , t h e d i c t y o s o m e s become c l u s t e r e d a b o u t t h e b a s a l body r e g i o n o f each o f t h e n u c l e i ( F i g s . 1 75 - 176 , 1 7 8 ) . The s e c r e t o r y , v e s i c l e s a s s o c i a t e d w i t h t h e d i s t a l d i c t y o s o m e c i s t e r n a e r e s e m b l e b o t h t h e c l e a v a g e v e s i c l e s and t h e v e s i c l e s f u s i n g t o f o r m t h e axonemal s h e a t h s ( F i g s . 1 4 1 , .170).. They a r e s p h e r i c a l t o i r r e g u l a r i n p r o f i l e , 100-300 my i n d i a m e t e r , and t h e y c o n t a i n e l e c t r o n - l i g h t amorphous m a t e r i a l . They a r e e x t r e m e l y abundant i n e a r l y z o o s p o r a n g i a l d e v e l o p m e n t , bu t as c l e a v a g e o c c u r s t h e v e s i c l e s b e -come l e s s d i s t i n c t ( F i g s . 1 7 0 - 1 7 4 ) . The d e v e l o p i n g p a r a s t r a s o m e s , a r e p a r t i c u l a r l y c h a r a c t e r i s t i c o f d i f f e r e n t i a t i n g z o o s p o r a n g i a l p r o t o p l a s m . They appea r i n hypha l p r o t o p l a s m j u s t p r i o r t o t h e d e l i m i n a t i o n o f z o o s p o r a n g i a by s e p t a , and t h e y become i n c r e a s i n g l y abundant as p r o t o p l a s m i c c l e a v a g e o c c u r s . - o f t e n c o n c e n t r a t i n g i n t h e p r o t o p l a s m nea r b a s a l b o d i e s and p l a n e s o f c l e a v a g e v e s i c l e s (.Figs. 1 5 5 - 1 5 8 ) . The p a r a s t r a s o m e s a r e s p h e r i c a l t o o v o i d i n p r o f i l e and 1 7 0 -350 m y i n d i a m e t e r . Each c o n s i s t s o f a c o r e o f e l e c t r o n - l i g h t amorphous m a t e r i a l r immed by a l a y e r o f den se f i b r o u s t o t u b u l a r m a t e r i a l , and f r e -q u e n t l y some o f t h e t u b u l a r o r . f i b r o u s e l e m e n t s e x t e n d r andomly i n t o t h e c o r e . ( F i g s . 1 6 4 - 1 6 6 ) . The membrane a b o u t t h e p a r a s t r a s o m e s i s 65-75A t h i c k . As c l e a v a g e o c c u r s , p a c k e t s o f m i c r o t u b u l e s r e s e m b l i n g t h o s e o b -s e r v e d i n s p o r a n g i a o f Pythium middletonii ( B r a c k e r , H e i n t z and G r o v e , 1970) and Phytophthora capsiai ( W i l l i a m s and W e b s t e r , 1970) a l s o become e v i d e n t i n t h e p r o t o p l a s m ( F i g s . 1 6 1 - 1 6 3 ) . The p a c k e t s appea r as l o n g r o d - s h a p e d s t r u c t u r e s , 100 -130 nry i n d i a m e t e r and up t o 1.7 y i n l e n g t h . They u s u a l l y l i e a d j a c e n t t o m i t o c h o n d r i a and t h e y c o n t a i n numberous t u -- . 3 2 -bules. The tubules are each 85-100A in diameter and are characterized by an electron-l ight core and an electron-dense periphery. They are orient-ed parallel to one another and they appear to extend the length pf the packets. Sometimes tubules also are discernible in spherical to ovoid-shaped vesicles; however, as observed in the encysted primary zoospores (Figs. 203-205), the 1imi ting membrane of the vesicles is thicker (about 80-90A thick) than i t is for the rod-shaped packets of rubules (60-70A thick) . Also,, i t appears as a single osmiophilic layer of material about the packets of tubules; whereas, the vesicular l imit ing membrane is t r ipar t i te in prof i le . The nuclei in zoosporangia i n i t i a l l y are similar to those described for the vegetative hyphae; however, they rapidly become oriented within the protoplasm so that adjacent nuclei are aligned equidistant to each other (Fig. 143). They also become pyriform with their narrower, or anter-i o r , pole being oriented toward the zoosporangial wall and the nucleoli becoming localized in the larger posterior region (Figs. 145-150). The nucleoli are 1-1.4 u in diameter and they consist of a well -differentiated pars'fibrosa- peripheral ized by pars granulosa (Figs. 175, 177). Less dense areas, the pars- amovphd, also are distinguishable in many of the nucleoli (Fig. 177). The surrounding nucleoplasm is homogeneously granular to f i b r i l l a r in appearance except near the. anterior pole where osmiophilic part ic les, 200-300A in diameter, are clustered (Fig. 176). The particles closely resemble those shown in published micrographs of P. parasitica nu-c le i (Hohl and Hamamoto, 1967). The nuclear envelope also is similar in size and general morphology to those previously described (Figs. 175-176)}.. however, the thickened juxta-centriolar region of the envelopes always appears at the anterior end of the nuclei (Figs. 176, 185-186). - 33 -Several investigators (Berlin and Bowen, 1964; Gay and Greenwood, 1966; Renaud and Swift, 1964; Williams and Webster, 1970) have noted that in Phycomycetes the transformation of centrioles into f lagel lar basal bodies occurs early in the development of either gametes or zoospores. Similar results are obtained for A. bisexualis. Pairs of centrioles loca-ted adjacent to the anteriorly-thickened region of nuclear envelopes rap-idly develop into basal bodies prior to protoplasmic cleavage in the zoo-sporangia (Figs. 178-189). The basal bodies are oriented at an angle of about 120-135° to.each other and they each are approximately 160-165 mu in diameter and 650-680 my in length. Each one consists of nine c y l i n d r i -cally-arranged and equally-spaced tubules - the tubules appearing as either doublets or t r ip lets (see Diagram IV - Basal Body Morphology; Figs. 184-1:89). The doublets pass through the basal plate at the distal end of each, basal body, whereas the t r ip lets are located at the proximal end. The t r ip lets are connected by fine filaments to a central tubule, 200-220A in diameter and 160-165 my in length. The central tubule does not extend the entire length of the basal bodies; instead, i t is v is ib le only in the area nearest the nucleus.. The distal , or terminal, plates of the basal bodies are characterized by several osmiophilic bands that pass through the central axis of the structure (Fig. 182). The bands appear to be several hundred angstroms in length. Microtubules also arise from the periphery.of the basal bodies and radiate into the surrounding protoplasm -usually remaining in close proximity to the nuclei with which the basal bodies are associated (Figs. 178-179). However, some microtubules ap-pear as . highly-structured units which inter-connect the basal bodies of each pair (Fig. 179). The number and exact arrangement of these units is BASAL BODY MORPHOLOGY Measurements -Peripheral Doublet -Tubular Disc Area - S u b -f ibr i l A -Sub - f ib r i l B -Terminal Plate -Sub- f ibr i l C -Peripheral Triplet •Central Tubule •Radial Link •-Centriolar Region -Rootlet DIAGRAM E2 - 34 -d i f f i c u l t to determine from the sections obtained in this study. The axonemal sheaths which are formed in association with the d i s -tal pole of the'basal bodies are similar to those described by Williams and Webster (1970) (Figs. 179-189). They are formed during early cleavage of the protoplasm and they become connected with the terminal plate of the basal bodies by an electron-dense, band (Figs. 181-183). The doublets on the sheath side of the terminal plate then elongate'to form short f l a -gellar shafts and the sheaths coalesce with the plasma membrane (.Figs. 179-183). - 35 -ENCYSTED PRIMARY ZOOSPORES Clusters of primary zoospores usually are v is ib le near the rup-tured papillae where spore protoplasts encyst after their release from zoosporangia (Figs. 132, 136-137). The encysted zoospores are spherical, 7.0-11.0 y in diameter, and reniform or irregular in prof i le . As observed by l ight microscopy, they are uninucleate, often vacuolated, and dense granules are common within the protoplasm. The fine structure of the encysted primary zoospores is similar to that of the zoospore i n i t i a l s (Figs. 190-192). However, as implied by the term "encysted", a wall surrounds each of the protoplasts. Tt is 40-55 my in thickness and is composed of.a single layer of electron-1ight amor-phous and f i b r i l l a r material (Figs. 195-196). The protoplasm.contains: num-erous mitochondria, liposomes, microtubule packets and vesicles, small parastrasomes, and microbodies - a l l of which are scattered randomly within the cel ls (Figs. 190-192). Lipid droplets, multi -vesicular bodies, endo-plasmic reticulum (E.R.) and l ipoidal rosettes also are present in the pro-toplasm (Figs. 190-192). Dictyosomes occur in close association with E.R. and/or the basal body region of pyriform nuclei (Figs. 214-215). The basal bodies l i e at the narrower pole of the nuclei and are attended by one, or sometimes, two large parastrasomes (Figs. 236-238). And, as ob-served by l ight microscopy, the encysted primary zoospores may possess vacuoles (Figs. 192, 200-201). The mitochondria are spherical, ovoid or rod- l ike in profi le (Figs. 190-192, 197-198). They measure 0.4-0.7 u in diameter and 0.7-2.0 y in length. Their cristae appear attentuated and, as noted previously, osmio-phi l ic f i b r i l s are present within the electron-l ight intracr istal lumen (Figs. 197-198). - 36 -The liposomes appear as .membrane-!imited vesicles, 0.3-0.9 y in diameter (Figs. 196-197, 199). They are spherical to irregular in shape and they contain alternating layers of electron-l ight and dense material in association with osmiophilic globules. However, as noted by Williams and Webster (1970) in their study on zoospore formation in Phyiophthora capsici, electron transparent, regions develop within the liposomes, par-t icu lar ly within the osmiophilic globules (Figs. 196-197, 199). The microtubule packets and microtubule vesicles are similar to those observed in the zoospore i n i t i a l s (Figs. 197, 203-205). However, they us-ually are more common within the primary zoospore protoplasts, with 4-7 packets lying in close association with one another. The packets and ves i -cles of microtubules both appear randomly distributed within the c e l l s , but they seldom .1ie adjacent to mitochondria as observed previously. Also, 80-100 hexagonally-arranged tubules with center-to-center spacing of 250-300A are evident within the thinner membrane-bound packets and there is a decrease in diameter of the terminal 0.2-0.3 y of some tubules, as noted in Diotyuohus sterile Coker and Saprolegnia ferax spores (Heath, Greenwood, and Gr i f f i ths , 1970).. The tapered end of the tubules is 75-85A in thick-ness. Ribosomes.often may be distinguishable on the surface of the packets. The multi-vesicular bodies, 1ipid droplets and microbodies a l l re -semble their respective forms in primary zoospore i n i t i a l s except that they are not as numerous as they were in the protoplasm previously (Figs. 190-194) except prior to germination. Then, the 1 ipid droplets and microbodies become extremely common in the ce l ls (Figs. 248-249). The parastrasomes in primary zoospores are extremely variable, both in size and morphology. They may appear as smaller bodies randomly scat-tered throughout the protoplasm or they may be present as large ovoid to - 37 -spherical structures which always are associated with the basal bodies (Figs. 239-247). The smaller spherically-shaped parastrasomes measure 250-500 mp in diameter. They are membrane-bound and each .contains a core of less dense amorphous material rimmed by a 25-35 my thick layer of osmio-phi l ic material (Figs. 239-240). Tubules, 140-160A in diameter, traverse randomly from the osmiophilic rim into the core. As the parastrasomes i n -crease in s ize , the tubules become aligned parallel to one another and f re -quently appear to become inter-connected with one another by osmiophilic filaments (Figs. 240-241). Amorphous regions also develop within the cores. Eventually one or two large parastrasomes (0.6-0.7 u in diameter and 1.0-1.2 y in length) form within each primary zoospore (.Figs. 236-238, 243-247). They l i e adjacent to the basal bodies, and rootlets part ia l ly encompass their l imit ing membrane (Figs. 236-238, 243). The cores of the large parastra-somes are mostly electron-dense except near one end where tubules traverse less dense material. Tubules also are discernible sometimes within the denser matrix. The rims of the parastrasomes remain unchanged. Irregularly-shaped vacuoles, possibly formed by the di lat ion of E.R. also are present in some spores (Figs. 200-202). And, alternating layers of E.R. and thick-membraned cisternae and/or vesicles often are associated wi.th the vacuoles. The vacuoles are irregular in profi le and are bounded by a 60-70A thick tonoplast membrane. The vacuolar sap is electron-l ight and similar to that in vacuoles of the vegetative hyphae. The E.R., which appears to form the vacuoles and which alternates with layers of cisternae and/or vesicles, .also, is similar to the E.R. observed in the vegetative hyphae (Figs. 201-202). The vesicles and the thick-membraned cisternae -the latter being formed by the fusion of the vesicles - are limited by a 90-100A thick membrane and they contain electron-l ight amorphous material. - 38 -The Tipoidal rosettes are characteristic particularly of primary zoospores as they have not,been observed in any other developmental stage in the l i fe - cyc le of A. bisexualis* They f i r s t appear as single non-mem-brane encompassed bars of electron-l ight material, often in association with vesicles containing material of a similar density (Fig. 206). The bars are usually convex or concave-shaped and their ends are tapered (Figs. 206-207). They then become aggregated about l i p i d droplets and eventually 1.0-1.5 P wide rosettes are formed (Figs. 207-211). Usually one "or two rosettes are observed in any one cel l and they do not appear to be associated with any given region or structure in the c e l l . The dictyosomes are morphologically the same as those in the primary zoospore i n i t i a l s (Figs. 212-215). Each consists of a stack of 3-5 c i s t e r - , nae; each cisterna consists of a central disc and peripheral anastomosing tubules (Fig. 213); blebs from the outer nuclear membrane or from the. E.R. form the proximal cisternae (Figs. 214-215), and secretory.vesicles are f o r m -ed by the distal cisternae (Figs. 212, 214). The secretory vesicles are similar to vesicles near the plasma membrane and near developing l ipoidal bodies (Figs. 195-196, 206-209). They also resemble those which fuse to form the cisternae that alternate with layers of E.R. near vacuoles or near the cell wall (Figs. 199-202). Immediately prior to germination of the en-cysted primary zoospores, numerous secretory vesicles are produced by the distal cisternae (Figs. 248-254). These vesicles contain mostly globules of electron-l ight amorphous material, but dense granules and microtubules are v is ib le within a few of their l imit ing membranes. The vesicles then aggregate near the distal pole of basal bodies (Figs. 226-228, 272)* or there is the fusion of many of the vesicles to form large irregularly-shaped vacuoles in the protoplasm (Figs. 248-254). - 39 -The pyriform nuclei too are similar to those previously described except that clusters of osmiophilic granules are not discernible within their beaked end (Figs. 215-220). Instead, a striated osmiophilic sheath is associated eccentrically about their narrower end and i t is attached to the outer nuclear membrane by 35-45A thick osmiophilic filaments (Figs. 229-232). Electron-dense f i b r i l s also inter-connect the sheath with the basal bodies via the groupsof osmiophilic f i b r i l s and rootlets (Figs. 231-232, 234-235). The basal bodies, which morphologically correspond to those in the primary zoospore i n i t i a l s , l i e at an angle of 120-135° to each other at the narrow end of the pyriform nuclei (Figs. 129-132, 22T-235; see Diagram IV - Basal Body Morphology). They are inter-connected by groups of os-miophilic f i b r i l s (Figs. 231-232, 233, 235). The osmiophilic f i b r i l s are attached speci f ical ly to 190-200A thick tubule which radiates from sub-f i b r i l C of each peripheral t r ip le t (Figs. 233, 235). The f i b r i l s also are attached directly to the sheath about the nucleus and to the rootlets of the basal bodies (Figs. 233-235, 238). The rootlets each consist of a single plane of approximately 7-9 microtubules (Figs. 230, 235-238) which extend into the protoplasm near a closely associated parastrasome (Figs. 236-238). Immediately prior to germination, there is a rapid elon-gation of the rootlets and often they appear 3-5 p in length (Fig. 273). - 40 -FLAGELLATED SECONDARY ZOOSPORES The differentiation of secondary zoospores occurs as primary zoo-spore protoplasts emerge from their spore cases (Figs. 256-261). Upon emergence, the protoplasts are spherical ; however, they rapidly become amoeboid and differentiate into biflagellated cel ls (Figs. 262-264). The biflagellated secondary zoospores, as observed by l ight micro-scopy, are similar to zoospores of Phytophthora megasperma var. sojae (Ho, Zachariah and Hickman, 1967; Ho, Hickman and Telford, 1968). They are ovoid with one bluntly-pointed end (Figs. 262-264). One side of the zoospores- also may appear flattened (Fig. 264). The f lagel la of the zoo-spores arise near the mid-point of a groove that runs longitudinally along each c e l l . One flagellum is directed anteriorly, whereas the other f l a -gellum is oriented posteriorly.(Figs. 262-264). The anterior flagellum is s l ight ly shortly than the posterior one. The secondary spores usually are motile for 10-30 minutes; they then become sluggish and encyst. The encysted secondary zoospores almost immediately form germ tubes that elongate into vegetative hyphae (Figs. 3-8) The fine structure of the flagellated protoplasts is similar to that described for Phytophthora zoospores (Desjardins, Zentmyer.and Rey-nolds, 1969; Ho, Zachariah and Hickman, 1968; Reichle, 1969a, 1969b; see Diagram.V - Morphology of the Flagellated Secondary Zoospore; Figs. 265-280). The secondary zoospores are encompassed by an 80A thick plasma membrane which immediately is underlaid by large globose to flattened ves i -cles (Figs. 265-269). The vesicles occur throughout the peripheral proto-MORPHOLOGY of the FLAGELLATED SECONDARY ZOOSPORE DIAGRAM 2 - 4 1 -plasmexcept at the mid-point of the longitudinal groove where the pyr i -form nucleus, basal bodies and vacuolar region is located (Figs. 265-266; 274-275). They often appear to fuse with the plasma membrane and empty their electron-1ight amorphous and denser f i b r i l l a r contents to the outside of the cel l (Figs. 270-271). The l imit ing membrane of the ves i -cles is of the same thickness as that of the plasma membrane. Mitochondria, l i p i d droplets, liposomes, microtubule packets and microbodies are scattered throughout the ribosome-dehse protoplasm of the zoospores (Fig. 265). Morphologically they are similar to those in the encysted primary zoospores. Dictyosomes,also are conspicuous in the zoo-spore protoplasm, and they usually are oriented about the narrow end of the pyriform nucleus (Fig. 255). A large irregular-shaped vacuole l ies adjacent to the dictyosomes in each zoospore {Fig. 266). The pyriform nucleus appears to undergo l i t t l e morphological change during the morphogenesis of the secondary zoospores (Fig. 265). It is located centrally within each cel l with i t s narrow end oriented towards the mid-point of the groove. The basal bodies of the f lagel la l i e adja-cent to and are directly associated with the pointed end of the nucleus (refer to Figs. 233-235). The basal bodies of the f lagel la l i e at an angle of 120-135° to each other, as described previously for those in the primary zoospores (Figs. 274-275). Each is formed of nine doublet-tr iplet f i b r i l s . The t r ip le t f i b r i l s characterize the proximal end of the basal bodies; the doublets, which are formed by the extension of two of,the three sub- f ibr i l s of the t r i p l e t s , pass through the terminal plate at the proximal end of the basal bodies. Their terminal plate is connected by osmiophilic bands to the plasma membrane. Rootlets and inter-connecting f i b r i l s also are associated with the proximal end of the basal bodies. The rootlets pass - 42 -from the basal bodies into the protoplasm, near several large parastrasomes (Figs. 274-275). They then become aligned near the plasma membrane along the longitudinal axis of the c e l l ; however, in the sections examined, the rootlets do not appear to run to the ends of the c e l l , as reported by Reichel (1969a) in P. -parasitica zoospores. Other microtubules, each 200-230A in diameter, radiate from a common point near the proximal end.of the basal bodies toward the nucleus (Fig. 275). The axonemes of the f lagel la consist of nine peripheral doublets and two single central tubules (Figs. 274-275). The peripheral doublets appear as extensions of the basal body doublets, but the two central tu -bules stop short of the terminal plate. The axonemes also are bound by an 80A thick membrane. The anterior axoneme sheath is covered with num-erous tubules, as characterize anterior f lagel la of other Oomycetes (Des-jardins, Zentmyer and Reynolds, 1969; Heath, Greenwood and Gr i f f i ths , 1970; Manton, Clarke and Greenwood, 1951, 1952; Reichle, 1969a. The tubules are 2.0-3.0. p long and each is 240-280A in diameter, except for the terminal 0.3-0.6 p which is 130-140A in diameter (Figs. 278-280). The posterior whip-lash f lagella;" also are covered with hairs; however, these hairs are finer and are usually shed i f l i v ing zoospores are a i r -dried directly onto a grid instead of f i r s t being fixed (Figs. 276-277). The f iner hairs are about 120-150A in diameter and 300-450A in length. The hairs, or tubules, do not occur at the tapered ends of either the whip-lash or tinsel f lage l la . Encysted Secondary Zoospores The encysted secondary zoospores frequently are observed in the same sections,as the flagellated protoplasts; however, they can be d i s -tinguished easily fromfthe latter cel ls by their wall. (Figs. 281-282, 285), The wall is 25r40 mp thick and is morphologically similar to the - 43 -wall of primary zoospores. Remnants of the f lagel lar axonemes and axonemal beads also are v is ib le near the wall of the encysted protoplasts (Figs. 283-284). The remnants may appear only as naked axonemes or f l a -gellar sheaths, but they usually consist of membrane-encompassed axonemes in which one or more f i b r i l s , often the central f i b r i l s , are lacking (Fig. 284). The axonemal beads consist of several axoneme sections within a common membrane, (Fig. 283). The axonemal beads are similar to those des-cribed for P. megasperma:var. sojae zoospore f lagel la (Ho, Zachariah and Hickman, 1967). The protoplasm of the encysted secondary zoospores also is easy to distinguish from that of the flagellated ce l ls (Figs. 281-282). It contains large vacuoles, numerous rod-shaped mitochondria, microbodies in association with E.R., and microtubule,packets. The microtubule packets often occur near dictyosomes, and the dictyosomes l i e adjacent to E.R. and/or nuclei . The nuclei are irregular to spinale-shaped and there are usually several nuclei in each protoplast. Centrioles are oriented paral-le l to the thickened and-siightly depressed region of each nuclear enve-lope. Liposomes and parastrasomes are not present in the encysted secon-dary zoospores. The fine structure of the fore-mentioned organelles and inclusions, excepting the microtubule packets, is similar to that described for their respective forms in young vegetative hyphae. The microtubule packets resemble those observed ,in encysted primary zoospores and flagellated se-condary zoospores; ; however, they usually occur near dictyosomes, as -previously .noted. Germ Tube Formation One or more germ tubes usually develop from each protoplast soon - 44 -after the encystment of secondary zoospores (Figs. 3-8). The germ tubes grow by apical elongation and expansion to form vegetative hyphae. And, as revealed by electron microscopy, the fine structure of the germ tubes and their development is similar to that of the actively growing hyphae (Figs. 286-296). Numerous vesicles, 100-200 my in diameter, .aggregate, near the plasma membrane of encysted secondary zoospores (Fig. 286). They are1 spherical to irregular in prof i le , and they each are 1imited by a 75-80A thick membrane. The matrix of the vesicles consists mostly of electron-l ight amorphous material, but membrane-like fragments also are v is ib le within the vesicles. The plasma membrane adjacent to the vesicles is crenulate. As germ tube formation occurs, the vesicles are maintained at the growing t ip (Figs. 287-292).. They usually occur to the exclusion of other organelles and inclusions and theyoften appear to fuse with each other or with the plasma membrane. The wall at the.t ip of the germ tubes is extremely th in , seldom exceeding 10-50 my in thickness (Figs. 287, 291-292). The other organelles and inclusions appear in the germ tubes immediately behind the apical vesicular zone (Figs. 287-290, 293-294). However, mitochondria, ribosomes and vacuoles are the most predomi-nant structures. - 45 -ANTHERIDIAL AND OOGONIAL..DEVELOPMENT Light Microscopy Observations The development of antheridia and oogonia and the process of sex-ual reproduction is as described by Raper (I960). Soon after vegetative hyphae of the male and female strains have been placed together in the same petri d ish, there is respective differentiation of antheridia and oogonia. The antheridia appear f i r s t as highly branched knob-Tike hyphae 20-80 u in diameter(Fig. 297). Fine granules are common in their growing tips and the rest of the antheridial protoplasm is characterized by vacuo-les , mitochondria and nuclei . Lipid droplets also are discernible in the protoplasm after antheridia have been stained with Sudan IV or Nile blue. The antheridia then induce oogonial d i f ferent iat ion, as demonstra-ted by Raper (1939a, 1939b, 1942a, 1942b, 1950a, 1950b) and Barksdale (1963). The oogonia usually develop as short lateral branches which are oriented at right angles to the female hyphae and whose tips become spher-ical to globose in shape (Fig. 298). The oogonia are 40-180 y in diameter and their protoplasm is extremely dense so that few organelles and/or i n -clusions are distinguishable. During oogonial development, the antheridia grow toward and gra-dually envelope the young c e l l s . Frequently it;appears that the entire surface of an oogonium is surrounded by several antheridia (Figs. 299-300). Delimitation of antheridial and oogonial protoplasm by septation then occurs and antheridia fe r t i l i za t ion tubes begin to penetrate the oogonial walls (Figs. 299-300). The septa delimiting both the antheridial and oogonial protoplasm appear to be similar to those delimiting asexual reproductive structures - 46 -from hyphal protoplasm as seen by l ight and electron microscopy (Figs. 299-300, 319-322, 336-337). In longitudinal section, they appear to consist of three layers - one layer is continuous about the sex cel l pro-toplasm, another layer l i es adjacent to the hyphal protoplasm and is con-tinuous.with the hyphal wal l , and a third layer appears as independent intervening material (Figs. 299-300, 337). The protoplasm of the delimited antheridia and oogonia., as seen in section, however, d i f fers . In oogonia, there is a large central vacuole surrounded by numerous spherical to ovoid-shaped inclusions (Figs. 299-300). Most of the inclusions are about 1.0 y in diameter and are s l ight -ly .denser than is the vacuolar sap. However, a few randomly-scattered inclusions are smaller and stain dark blue-violet or purple with t o l u i -dine blue with borax or Flemming's t r ip le s ta in , respectively. Their walls appear relat ively smooth except where antheridial ce l ls penetrate them. The antheridial hyphae also are vacuolated and their surrounding proto-plasm is particulate in appearance (Figs. 299-300). However, dark-staining bodies seldom are v is ib le within their c e l l s . As antheridial fe r t i l i za t ion tubes penetrate the oogonia, the oo-gonial protoplasm becomes cleaved and usually 5-15 oospheres are formed. The oospheres are each 12-40 y in diameter and, as noted for other Oomy-cetes (Bryant and Howard, 1969; Sansome, 1961 , 1963), their protoplasm is extremely dense. The fe r t i l i za t ion tubes then penetrate the oospheres, and thick-walled oospores develop as a result of fe r t i l i za t ion (Fig. 301). The antheridia then appear highly vacuolated or empty. Fine Structure of Antheridial Hyphae Although the fine structure of antheridia is d is t inct ive , i t basi -ca l ly is similar.to that described for young, vegetative hyphae. The ac t i ve - 1 - 47 -ly growing tips are thin-walled and their protoplasm is characterized by numerous ovoid to s l ight ly irregularly-shaped vesicles which correspond to the fine granules observed by 1ight-microscopy (Fig. 302). Ribosomes sometimes are v is ib le in the protoplasm, but other organelles and inclusions, seldom occur in the actively growing t ips . The vesicles are mostly 130-300 my in diameter, but a few vesicles are smaller, measuring 30-60 my in diameter (Fig. 302). They are similar to those in the apical growing zone of the vegetative hyphae, but their" matrix is more electron-dense and granular. Less dense amorphous material usually is not v is ib le in the vesicles unti l the antheridia have come into contact with oogonia (Figs. 377-378). Fusion of the vesicles with each other and with the plasma membrane, both prior to oogonial contact and during oogonial penetration, is apparent (Figs. 302, 377-378). The plasma membrane and the wall about the growing tips also re-semble that of the vegetative hyphae. The antheridial wall, i s , however, . s l ight ly more electron-dense .(Fig. 302), and i t becomes almost ind is t in -guishable from the oogonial wall during.penetration (Figs. 377-378). Few 1omasome-like configurations are v is ib le about the growing apices. The sub-apical protoplasm is characterized by numerous organelles and inclusions - vesicles morphologically corresponding to those in the vegetative apical zone, mitochondria, l i p i d droplets, multi-vesicular bodies, dictyosomes, vacuoles and nuclei being the most conspicuous,struc-tures (Figs. 303-306). Ribosomes, endoplasmic reticulum (E.R.), micro-bodies, centrioles and liposomes also are present. The vesicles are scattered throughout the protoplasm of the young antheridia (Figs. 303-306). The larger vesicles usually are commonly located near the plasma membrane and the smaller ones nearer the distal dictyosome cisternae from which they appear to be derived (Figs. 304-306). - 48 -As the antheridia encompass oogonia, the vesicles become concentrated near the cel ls ' , surface in the v ic in i ty of contact with the oogonial walls (Figs. 341-345). They then appear to fuse with each other and with the plasma membrane, releasing their contents toward, the appressing sur-faces (Figs. 342-345, 378). Vesicles also become concentrated at distal , regions in the antheridia whereby their fusion results in, the formation of septa (Figs. 315-322). Ultrastructurally the septa are similar to those described pre-viously (Figs. 319-322). They consist of two wall layers of similar thick- -ness.andan intervening space which is formed by the entrapment, compaction and condensation of vesicles and possibly other cytoplasmic material during septal development (Figs. 315-322). The walls about the sub-apical antheridial protoplasm are the same as the hyphal walls except they seldom are more than 40-90. m.p thick (Figs. 303-306, 319-322). They usually appear to be composed of a single layer of amorphous to, f ib r i l la r imater ia l except near septa where two layers may be distinguishable. Pockets of electron-dense, granular and vesicular mat-ter also are v is ib le in the walls, particularly in areas where antheridia have come into contact with oogonia (Figs. 314, 345). The mitochondria exhibit a complex of forms which are dist inct ive and serve as avwaluable indicator of antheridial protoplasm (Figs. 303-311). The simplest forms are ovoid to rod-shaped mitochondria, but the predominant type is rod-shaped with a terminal or sub-terminal invagina-tion (Fig. B, Diagram VI - Mitochondrial Morphology). The invaginations are dist inct ,if the mitochondria are sectioned in plane c - c' or as seen in face view (Fi,g. B, Diagram VI; Figs. 305, 307, 309). However, i f mitochondria have been sectioned through planes a - a' or b - b' (Fig. MITOCHONDRIAL MORPHOLOGY DIAGRAM 3ZI - 49 -B, Diagram VI), they appear as vesicles (Figs. 303-305, 309). If sections do not pass through invaginated regions, the mitochondria then appear similar to the simplest forms, being spherical, ovoid or rod- l ike in pro-f i l e as determined by the section plane (Figs. 303-306). The invaginations are formed by the progressive and concurrent infolding of the outer and inner membranes unti l they are in close proximity to the opposing membranes. Consequently, cristae seldom are formed in the regions of invaginations.. The cytoplasmic matrix within-the invaginations usually is granular and less electron-dense than is the rest of the cytoplasm; however, i t also may contain ribosomes, vesicles, osmiophilic bodies and relat ively un-differentiated ,1 iposomes (Figs. 303, 305, 307, 309). Cup-shaped and dumbbell-shaped mitochondria are present in antheri-d ia , but they are indistinguishable from other forms unless serial sec-tions are obtained (Figs. 303, 305, 307, 311). The cup-shaped mitochon-dria appear U-like i f observed in median-longitudinal section (Figure'A, face view or plane c - c ' , Diagram VI; Figs. 307, 311); donut-like con-figurations are common i f they are seen in cross or oblique section (Fig. A, planes a - a 1 and b - b' , Diagram VI; Figs. .303, 305). The dumbbell-shaped mitochondria are recognizable only i f sectioned median longitudi-nally (Fig. C, plane a - a 1 or as shown in face view, Diagram VI; Figs. 314,. 344). In any other sectional plane, they would appear spherical, ovoid, or rod-shaped (Fig. C, planes b - b1 and c - c ' , Diagram VI; Fig. 308). The mitochondria are 0.3-0.9 p in diameter and 0.8-2.5 p in length - the cup-l ike forms usually having the greatest diameter, the dumbbell and rod-shaped mitochondria being the longest. Their structure is the same as .that described for mitochondria in younger vegetative hyphae (Figs. - 50 -302-311). Their cristae are elongated, rarely extending completely across. a mitochondrion, and their matrix is dense. Occasionally the mitochondria are encompassed by one or more membranes and appear to undergo progressive degradation to form multi -vesicular bodies, as described by Brown and Bertke (1969) (Fig. 308). The multi-vesicular bodies, dictyosomes, l i p i d droplets,, micro-bodies, and ribosomes a l l are similar to. their respective forms in the vegetative hyphae (Figs. 302-305, 312-314). . However, E.R. may be either vesiculate or cisternal (Figs. 303-306, 312-314). The vesiculate form usually is present in the protoplasm immediately behind the apical growing zone, whereas the cisternal E.R. often is oriented about the vacuoles and is the form with which microbodies and dictyosomes generally are associated. Ribosomes are present on both types of E.R. The vacuoles resemble those in germ tubes and in young vegetative hyphae. They are relat ively small in the. newly formed sub-apical protoplasm, but they rapidly enlarge and become aligned centrally within the more distal regions of-the hyphae (Figs. 304-306). They sometimes, contain osmiophilic globules\ -similar to those in liposomes (Fig. 302). After the penetration of oogonia, antheridial hyphae become highly vacuolated and l i t t l e protoplasm is l e f t in .their cel ls (Figs. 379-380).. Liposomes are scattered throughout the sub-apical protoplasm, but they are not common or highly-differentiated structures as in other de-velopmental stages of Aohlya (Figs. 302, 312-313, 345). Their matrix con-sists of an osmiophilic globule and less dense granular and amorphous material. There rarely is layering of material about the globule. The nuclei in young antheridia usually are ovoid to spindle-shaped, and they are structurally similar to those in vegetative hyphae (Figs. 312-313). They measure 1.5-2.3 u in diameter and 3.0-4.7 ji in - 51 -length. However, as the antheridia encompass and penetrate oogonia, many nuclei appear to divide meiotically (Figs. 358-365). Meiotic division is intra-nuclear, as is mitosis, and the nucleoli remain as dist inct en-t i t i e s during the various divisional stages; however, they often are smaller (0.6-0.8 y in diameter) and consist only of pars f i b r o s a and pars amorpha - the pars granulosa seldom being distinguishable (Figs. 361-365). In prophase I, the nuclei become irregular in profi le and single axial elements, similar to those-in other nuclei (Franchi and Mandl, 1963; Lu, 1967) become v is ib le within their nucleoplasm (Figs. 358-360). The single axial elements often are associated with the inner nuclear envelope membrane and they each are about 400-500A thick. Their exact dimensions are d i f f i c u l t to determine, however, as.many chromatin f i b r i l s extend as lateral projections, from their central axis. The axial elements then be-gin to pair and there is condensation of electron-dense chromatin-1 ike material about them (Figs. 361-365). Complete pairing of the elements to form synaptinemal complexes is not shown, but synaptinemal complexes were seen -in some of the nuclei during the early stages of this study., The process from metaphase I to telophase II of meiosis appears to occur rapidly as only a few anaphase I and II and telophase I and II figures have been observed in the antheridia (Figs. 365-366, 369-376). During anaphase I and II , intra-nuclear spindles are present and homolo-gous chromosomes or chromatids move to opposite poles of respective^nuclei (Figs. 365-366, 371-374). The spindles usually are eccentric and consist of 200A diameter tubules which radiate from thickened polar regions of the inner envelope membrane adjacent to centrioles (Figs. 365-368, 37.1-374). The tubules extend from pole to pole or. they terminate on chromosomes or - 52 -chromatids, depending on the stage of d iv is ion. The chromatin bodies are similar to those in other fungi, appearing as relat ively dense granu-lar to f i b r i l l a r structures in the nucleoplasm. In telophase I and II , the nuclei usually appear elongated or dumbbell-shaped and chromatin material is clumped near their poles (Figs. 375-376). Individual chromosomes seldom are v is ib le during telophae. The clumped chromatin material usually is associated with extremely short spindle tubules which are in contact with the thickened region of the inner envelope membrane. Spindle tubules also extend from pole to pole (Figs. 375-376). At the end of .meiosis, the nuclei formed are relat ively smaller than the ones observed prior to the division process (Figs. 369-370, 378), They seldom are larger than 1.4-2.0 y in diameter and 2-0-3.2 y in length. Bryant and Howard (1969) also have shown that the nuclei formed by meio-sis contain about one half the amount of DNA of the parent nuclei ; how-ever, they appear structurally similar to the nuclei observed in the ve-getative hyphae. The meiotic nuclei then migrate to the growing tips of the antheridia which are penetrating oogonia (Fig. 378). The centrioles are the same as those, described previously (Figs. 365-368). They occur in pairs, often with the members of each pair lying end-to-end. Also, they usually l i e in well-defined pockets of the nu-clear envelope (Figs. 365-366). Sometimes flaps of E.R. cover the pockets (Fig. 366). Fine Structure of Oogonia Oogonia usually are dist inct ive when observed by l ight microscopy, . but their f ine structure closely resembles that described for other develop-. - 53 -mental stages in the l i fe - cyc le of Achlya. The young oogonia are similar to differentiating vegetative hyphae. They are comparatively thin-walled and their protoplasm consists of numerous.vesicl es, mitochondria, d i f f e r -entiating liposomes, endoplasmic reticulum (E.R.) , dictyosomes and nuclei (Figs. 323-325). Lipid droplets, vacuoles, microbodies, ribosomes and centrioles also are present (Figs. 323-325). Older oogonia are characterized by similar protoplasmic const i -tuents; however, there is greater differentiation and development of cer-tain of the organelles and inclusions (Figs. 326-329, 341-353). There also is an increase in wall thickness about the oogonia, and septa, structurally the same as those described, for gemmae, are formed subsequent to antheri-dial contact (Figs. 336-337). Eventually protoplasmic cleavage occurs and thin-walled oospheres are formed within mature oogonia (Figs. 382-383). The vesicles in the oogonia are similar to those in the apical growing zone of vegetative hyphae and in germ tubes (Figs. 323, 325-331). They are ovoid to irregular in profi le and 30-300 mjj in diameter. The smaller vesicles mostly occur in close association with the dictyosomes, from which they appear to be,.formed (Figs. 323, 325, 329-331). The larger forms usually are conspicuous near the .plasma membrane, especially adja-cent to areas of antheridial contact (Figs. 341-347). The matrix of the vesicles is composed:'• mostly of electron-l ight amorphous to granular material which appears to be deposited toward the wall upon the fusion of the vesicles with the plasma membrane (Figs. 327-330). Sometimes smaller vesicles or membranes also are present in the matrix and are deposited toward the wall (Figs. 346-347). Continued deposition of material appears to account for the increase in wall thickness during the maturation of the oogonia (Figs. 323, 326, 329, 341-351). - 54 -The walls are 60-80 my thick about young oogonial protoplasm, but they increase to 1.5-2.0 u in thickness about mature oogonia (Figs. 323, ' 326, 329, 341-351, 377-378). Structurally they are the same as described previously except that pockets of electron-dense granular and vesicular material rarely are present in their walls. The mitochondria are similar to those of the vegetative hyphae. They are ovoid to rod-shaped'and are 0.3-0.5 y in diameter and 0.7-4.0 y in length (Figs. 323-329, 338). The longer forms usually are characteris-t i c of the ce l ls (Figs. 323-325); the shorter rod and ovoid-shaped mito-chondria are more common in s l ight ly older and in mature oogonia where they become distributed randomly about a large central vacuolar system (Figs. 326, 348-351). The matrix of the mitochondria consists mostly of dense granular material and the cristae contain electron-l ight amoprhous mater-ia l except for several osmiophilic f i b r i l s which usually extend along their central axes (Fig. 338). The vacuolar complex develops soon after oogonial formation (Figs. 323-326). Small vacuoles are scattered throughout differentiating oogon-ial protoplasm, but they rapidly enlarge and often coalesce to form a large central vacuolar complex within the globose or spherical c e l l s . The small vacuoles are irregular in profi le and appear to be formed from undifferen-tiated liposomes or thin-membraned cisternae (Figs. 323-325). They con-tain homogeneously granular sap which usually is less dense than the sur-rounding protoplasm (Figs. 323-325). The large central vacuole, or vacuo-lar complex, also containsfine granular sap, or oolymph as termed by Moore and Howard (1968) (Figs. 326, 348-351). However, osmiophilic glo-bules, similar to those in differentiating liposomes, are present in the vacuoles (Fig. 326). The tonoplast is about 90A thick. - 55 -The 1iposomes are extremely abundant in oogonial protoplasm and they appear to be formed possibly .from dilated endoplasmic reticulum or from secretory vesicles of the dictyosomes with which dilated endoplasmic r e t i -culum often is associated (Figs. 323^326, 332-333). However, the latter process does not seem probable as' the 1imiting membrane of the liposomes is usually less thick than is the l imit ing membrane of the secretory ves i - . c les. The liposomes -are formed mostly in young differentiating oogonia and they appear as irregularly-shaped vesicles, .250-400 mp at their great-est point (Figs. 323-325). Their matrix consists of electron-dense granules and electron-1ight amoprhous material. The liposomes then enlarge and as-sume an ovoid shape (Figs. 326-329)_. They are 0.4-1.2 p in diameter and 0.8-1,5 y in length. Concurrently, one or more osmiophilic globules/devel-op within their matrix and much of the electron-l ight amorphous material becomes concentrated about their periphery (Figs. 329, 334). It is at this time that the oogonia become encompassed by antheridia and that septa are formed to delimit oogonial protoplasm from the vegetative hyphae (Figs. 336, 341-347). Continued liposome development results in layering of the electron-1 ight amorphous material about the globules unti l the vesicles are similar to those described for gemmae, encysted primary zoospores • and flagellated secondary zoospores (Figs. 335, 348-353). Upon liposome maturation, antheridial penetration begins to occur and oogonial nuclei divide meioti.cally (Figs. 348-351 ). There also i s .a great increase in the 1ipid droplets and 1iposomes.become relegated to small areas amongthe l ip idbod ies (Figs. 348-353). The 1ipid droplets are similar to those in the vegetative hyphae. The microbodies and dictyosomes also resemble their respective forms as described for vegetative hyphae., and they usually appear in close - 56 -association with smooth surfaces of ribosome-coated endoplasmic ret icu -lum (Figs. 323-325, 329-331, 339-340). The endoplasmic reticulum is abun-dant in oogonia, particularlyr-Muring liposome formation whe^e i t often is di lated. The nuclei in oogonia are scattered throughout the protoplasm and there may be several to ten nuclei v is ib le in any one section (Figs. 326-328, 348-351). In young differentiating oogonia, they usually are elon-gated or spindle-shaped and their envelope membranes are crenulate (Figs. 323-324); however, the nuclei often become ovoid to lobed upon the en-compassing of the oogonia by antheridia and their membranes also become smoother in prof i le (Figs. 326-329, 355-357). They appear to undergo l i t t l e change morphologically unti l the beginning of antheridial penetration when the oogonial protoplasm is extremely dense and composed of masses of l i p i d . droplets and highly-structured liposomes; the nuclei then divide meioti-cal ly (Figs. 348-351). Before meiosis, the nuclei vary from 1.5-2.3 y. in diameter to 3.0-4.7 y in length. They each possess a nucleolus, about, 1.0 y i n diameter, and their nucleoplasm is,uniformly granular to f i b r i l -lar in appearance (Figs. 355-356). The nucleolus l i es adjacent to the modified centriolar region of the nuclear envelope and i t consists of a well-defined pars f i b r o s a and pars granulosa, as described for nucleoli in the vegetative hyphae (Figs. 356). During meiosis, the nuclei undergo a series of changes which closely resemble those noted for meiotically dividing antheridial nuclei (Figs. 348-351, 357-364).. However, the second phase of meiotic division in oogonial nuclei has not been observed, and the author suspects that i t must occur immediately prior to or during the cleavage of the protoplasm in oosphere formation. The cleavage process has not been observed in A. b i s e x u a l i s . - 57 -The centr ioles, both structurally and posit ional ly , are the same as those in the antheridia (Figs. 324, 327-328, 354-356). Oosphere Fine Structure Oospheres are relat ively thin-walled spherical protoplasts within oogonia (Fig? 382). They are 12-40 y in diameter and their protoplasm consists predominantly of l i p i d droplets and'liposomes; ' however, vesicles, mitochondria, dictyosomes and other organelles common tp the oogonia prior, to cleavage also are present in areas among the l i p i d droplets and l i p o -somes (Figs. 383-384, 387-390). Each oosphere possesses a single nucleus, but vacuoles seldom.are distinguishable in i t s protoplasm. The wall about each oosphere is about.30-150 my thick and i t is similar to that about encysted secondary zoospores (Figs. 383-384). How-ever, the wall often is coated with a loosely-bound network of more electron-dense granular to f i b r i l l a r material (Fig. 383). The l i p i d droplets d i f fer l i t t l e from those in the oogonia. They usually are spherical to ovoid-shaped and 0.7-1.7 y in diameter; however, they often become isodiametric in profi le when they are appressed against each other((Fig. 383). Also, the l i p i d droplets sometimes.coalesce and form massive irregularly-shaped bodies within the protoplasm, but this condition usually occurs in only abortive-appearing oospheres where the cel l i t s e l f is irregular in shape (Fig. 384). The liposomes usually are larger, 1.0-1.2 y at their widest point, and more irregular in profi le than those in other developmental stages in the l i f e - cyc le of'A. b i s e x u a l i s (Fig. 389). Also, each is characterized by several osmiophilic globules about which the electron-Tight amorphous material is layered.. The l imit ing membrane, where i t is distinguishable, is of a similar thickness to the membrane about dictyosome secretory ves i -- 58 -cles - the latter appearing as probable precursors of the liposomes (Fig. 385). The secretory vesicles are mostly spherical, 100-200 my in diameter, and they each contain a single osmiophilic body surrounded by less dense amorphous material (Figs. 387-388). Mitochondira, endoplasmic reticulum and dictyosomes a l l appear to resemble their respective forms in older oogonial protoplasm; however, microbodies rarely are v is ib le in the ce l ls and nuclei are d i f f i c u l t to distinguish. The nuclei appear to be located accentrically within the oospheres and they usually are irregular in prof i le , varying from 1.4-2.0 y in diameter to 2.0-3.2 y in length (Figs. 385-386). Their nucleo-plasm consists mostly of granular material, but denser areas, suggestive of chromatin material, also are dispersed throughout the nuclei (Figs. 385-386). Nucleoli have not been observed in any of the sections, but complete serial sections through the nuclei have not been obtained by the author and i t is s t i l l possible that they are present.,;' Centrioles occur in close association with the nuclei , and they appear to be structurally similar to those adjacent to oogonial nuclei (Fig. 390). Oospores The oospores also are 12-40 .y in diameter and are formed upon fer -t i l i z a t i o n of oospheres, effected by the penetration of antheridia through oogonia and oosphere walls respectively (Figs. 378-381, 390). They be-come extremely thick-walled and,their protoplasm is dense (Figs. 390-392). Structural ly, they resemble oospores formed by other Oomycetes (Marchant, 1968; Moore and Howard, 1968). Their wall is several layers thick and s i ight ly undulate in prof i le . It usually consists of 0.2-0.6 y wide, electron-l ight inner layer separated - 59 -from a thinner electron-l ight outer layer by dense granular material (Figs. 390-393). The outer layer appears to correspond to the old oosphere wall except that pockets of dense granular and vesicular material often are v is ib le in the outer layer (Figs. 390, 392). Their protoplasm consists mostly of 1ipid .droplets and liposomes. The l i p i d droplets are extremely large, measuring 1.3-3.5 y in diameter; the liposomes are similar to those in the oospheres (Figs. 390-391, 393-394). Mitochondria and nuclei also are present in the c e l l s , but other organelles are indistinguishable because of the density of the protoplasm (Figs. 390-392,, 393-395). The mitochondria are ovoid to rod-shaped and 0.2-0.4 y in diameter and 0.3-1.0 y in length (as determined from electron micrographs). Structural ly, they appear to resemble those described pre-viously. The nuclei are ovoid to irregular in prof i le , and-they appear to be much larger than those in the oospheres (Figs. 395). They are approxi-mately 1.0-1.4 y in diameter and 3.0-4.5 JJ in length. Their nucleoplasm consists of two phases: a peripheral phase which is mostly granular in appearance and a central phase which is composed of osmiophilic f i b r i l s within less dense granular to amorphous material (Fig. 391). The f i b r i l s are about 100-300A thick. - 60 -DISCUSSION FINE STRUCTURE OF Achlya bisexualis Cell Walls The results of this study indicate that ce l l walls of Aehyla bisexualis structurally are, similar to cel l walls of other Oomycetes (see Appendix, Table V - Wall Morphology and Composition) and of most other fungi (see Aronson* 1965). , They consist of a two phase system in which- f i b r i l s are embedded within an amorphous matrix (Fig. 50). The f i b r i l s in Oomycete cel l walls are thought to be formed of ce l lu lose - l ike polymers, while the. amorphous matrix is formed of other polysaccharides (particularly glucans which mask the f i b r i l s ) , proteins and inorganic constituents (Aronson, 1965; Aronson, Cooper and Ful ler , 1967; Bartnicki-Garcia, 1966; Cooper and Aronson, 1967; Frey, 1950; Manocha and Colvin, 1968; . Parker, Preston and Fogg, 1963). The structural organization of the walls of vegetative hyphae and of asexual and sexual reproductive structure which have directly differentiated from the hyphae of A. bisexualis is s imilar - to that described for the walls in respective developmental stages of other Oomycetes (see Appendix, Table V - Wall Morphology and Composition). The walls about actively growing regions of the vegetative hyphae (Figs. 30-31, 34), germ tubes (Figs. 286-296), discharge papillae of zoosporangia (Figs. 151, 154), and antheridial and f e r t i l i z a t i o n tubes (Figs. 303, 377-378) appear as single thin layers of material, but in more distal and older regions of these respective cel ls and of gemmae and oogonia, the walls are usually thickerand often appear to be formed of at . least two layers of material, particularly in areas of septati.on (Figs. 102-103, 144, 319-322, 336). Thus, the pattern - 61 -of structural organization of these cel l walls is compatible with evidence that Oomycete hyphal walls are formed of at. least two differently-oriented layers of f i b r i l s except within young apical regions where only one layer of randomly-oriented f i b r i l s is continuous (Manocha and Colyin, 1968). It also indicates that the outer layer of wall constituents is formed apically during hyphal growth while the. inner layer(s) is (are) formed later by appositional deposition of wall material, as suggested by other investigators (see Aronson, 1965;. Hawker, 1965; Manocha and Colvin, 1968). The pockets of electron dense granular and vesicular material which are often present in the walls of almost a l l developmental stages of A. bisexualis (Figs. 52-53, 100, 143, 377) are similar to the vesicular aggregates, observed in zoosporangial walls of Phytophthora (Chapman and Vujici.Cj 1965; Hemmes and Hohl, 1968; Hohl and Hamamoto, 1967; Vuj ic ic , Chapman and Col noun, 1965). They also are morphologically the same as those in the septa of A. bisexualis (Figs. 102-103, 144,,315-322, 336) and in the • basal plugs seen in Phytophthora zoosporangia (Chapman and Vuj ic ic , 1965; Hemmes and Hohl, 1969; Hohl and Hamamoto, 1968; Williams and Webster, 1970). The results of this study, however, do not indicate thatthe pockets of- : electron dense granular and vesicular material in the walls possess enzyme act iv i ty which would result in papil lar degradation during the zoospore discharge process as postulated by Chapman and Vujicic (1965). If such enzyme, act iv i ty were characteristic of these pockets, the following morphological features would not be expected: 1. The consistent absence of vesicular aggregates in zoosporangial discharge papillae walls of A, bisexualis (Figs. 151-154). And, 2. The consistent presence of vesicular aggregates in walls of other developmental stages of A. bisexualis (Figs. 52-53, 100, 143, 377). This does not negate the idea, however, that other types of enzymes (e.g. - -- 62 -wal1 polymer-coupling enzymes) may be present in the vesicular pockets i f they are formed by the "entrapment" mechanism as is discussed later (see Septal Structure and Development, Cellular Growth and Appositional Wall Formation). The th in , single-layered walls of the encysted zoospores, as seen in this study* are similar to those.of the hyphae (Figs. 190-192, 286-290). It would thus appear that they also are composed of a two phase system of f i b r i l s embedded within an amorphous matrix. This is further supported by the fact that no fine structural difference is distinguishable in the region of differentiation between the walls of germinating encysted zoospores and developing germ tubes. However, to the author's knowledge, the basic structure of encysted zoospore walls is yet unknown. • The presence of a dist inct oosphere wall (Fig. 383), which structurally is similar to the walls of encysted, zoospores of A. bisexualis, is observed. This is in contrast to observations by others (Moore and Howard, 1969; Flanagan, 1970) where the presence of a wall about oosphere protoplasm in other members of the Saprolegniaceae is questioned. This difference in the apparent development and/or thickness of the oosphere wall of these different species of the Saprolegniaceae might ref lect inherent differences within the organisms themselves or in conditions under which the organisms were growing at the time of oosphere development. The.oospore wall of A. bisexualis, as indicated in this study± is multi-layered. (Figs. 391-394). This is in accord with evidence of other investigators (see Dick, 1969) who indicate that the saprolegnian oospore wall is-formed of an epispore (or outer) layer and of several inner (or endospore) layers. The epispore layer, "as suggested by DeBary (1887 - see Dick, 1969) and as supported by observations in this study, is synonymous with the original oosphere wall formed prior to the fe r t i l i za t ion process. Corres-- 63 -pondingly, the endospore layers represent those layers which have been deposited subsequent to f e r t i l i z a t i o n . The mechanism by which the wal;l layers are formed has not been observed in this study; however, the presence of pockets of dense granular and vesicular material in the oospore walls (Figs. 393) and the basic structural s imilar i ty of the wall layers in the oospore to that in walls of the other developmental stages of A.bisexualis-suggest that oospore walls are formed by the same type of vesicle-plasma membrane fusion mechanism by which the other cel lu lar walls are formed and as is discussed later (see Cellular Growth and Appositional Wall Formation). This type of mechanism for oospore wall formation is compatible with obser-vations which reveal that dictyosomes are present in oospore protoplasm. Plasma Membrane The plasma membrane is a tenuous structure which regulates molecu-lar and ionic exchange between the cel l and i ts surrounding environment. In almost a l l stages of development of A. bisexualis and of most other fungi (see Hawker, 1965), the membrane is adpressed to the cel l wall except in areas where there are lomasomes, or lomasome-1ike configurations, and where in older cel ls the protoplasts have pulled away from the wal l . It then appears irregularly invaginated in prof i le . In the flagellated zoospores where there are no encompassing wal ls , the plasma membrane usually is smooth in profi le and i t may occur in association with large flattened saccules or vesicles to form a p e l l i c l e . The plasma membrane in ce l ls of A.bisexualis and in cel ls of most other organisms (Brown and Bertke, 1969; DuPraw, 1968) represents a "unit membrane" as defined by Robertson (1959). It is 70-85 A thick and appears as two opaque lines separated from each other by a region of low electron density (Figs. 51, 269). The opaque lines were thought to each correspond to a molecular layer of protein, while the central region represents a - 64 -bimolecular layer of phospholipids. However, recent studies (see.DuPraw, 1968) indicate that carbohydrates and possibly other types of molecules may also be associated with the plasma membrane and that i t is not the simple protein-1ipid-protein sandwich as or iginal ly proposed by.Davson and Daniell i (1935 - see DuPraw, 1968). Growth and development of the plasma membrane in cel ls of A. bisexualis .and in cel ls of other Oomycetes (Grove and Bracker, i 9 7 0 ; Grove, Bracker and Mo.r.re, 1970; Hemmes and Hohl, 1968; Hohl and Hamamoto, 1967; Williams and Webster, 1970) consistently appear to occur by the fusion of vesicle membranes with each other and/or with pre-existing plasma membrane* This is particularly evident during zoosporogenesis where cleavage vesicles become aligned in planes equidistant about the nuclei and the membranes of the vesicles coalesce with each other to form the plasma membranes of the zoospores (Figs. 145-150; Hohl and Hamamoto, 1967; Williams and Webster, 1970). It is also apparent in young vegetative hyphal tips (Figs. 30-31; Grove and Bracker, 1970; Grove, Bracker and Morre, 1970; Hemmes and Hohl, 1968), in ends of germ tubes (Figs. 287, 291), in antheridial apices (Figs. 303, 378), in enlarging oogonial cel ls (Figs. 323, 326), in developing zoosporangial discharge papillae (Figs. 151-152) and in areas of septation (Figs. 315-316). In a l l of these growing or differentiating regions of c e l l s , vesicles.are present in the protoplasm adjacent to the plasma membrane which in i t s e l f is crenulate in p ro f i le , suggestive of previous vesicular fusions with i t . Evidence which further supports the concept that growth and development of the plasma membrane is effected by vesicular membrane incorporation is that both membranes are of the same morphological type: possessing the same overall thickness, stain intensity and substructural - 65 -patterns of stain deposition (Grove, Bracker and Morre, 1968). Also, Girbardt (1969) has shown.that the reverse process of vesicle formation from the plasma membrane is not probable. Lomasomes and Lomasome-1ike Configurations The lomasome-1ike configurations in cel ls of A. bisexualis structurally resemble those f i r s t reported by Girbardt (1.958, 1961) and as named by Moons and McAlear (1961). They are also similar to those lomasomes which have been described for many of the other Oomycetes (see Appendix, Table VI - Lomasome Morphology and Distribution). They appear as groups of vesicles or membranes within a wal1-1 ike matrix between the plasma membrane and the wall and they usually occur in areas of protoplasmic vesiculation - near actively elongating hyphal and antheridial t i p s , in fe r t i l i za t ion tubes and in oogonia in association with appositional deposition of wall constituents. Both of these factors ( i . e . - their morphology and distribution) thus suggest that many of the lomasome-1ike configurations in cel ls of A:• -bisexualis probably develop during cel l wall formation when several to many vesicles, or vesicle fragments, become delimited from the protoplasm by the fusion of more centrally-located vesicles with the plasma'membrane. This type of mechanism is compatible with the presence of the vesicular components and wa l l - l i ke matrix as observed in most of the configurations in this study. There is no morphological evidence to indicate that the lomasome-1ike configurations in A. bisexualis are formed, by any other mechanisms as have been suggested in other studies: (1) intrusion and subsequent evagination of the plasma membrane (Robards and Kidwai, 1969), (2) incorporation of multi-vesicular bodies between the plasma membrane and cel l wall (Marchant, Peat and - 66 -Banbury, 1967), or (3) elaboration of the plasma membrane (Peyton and Bowen, 1963). It is tempting to further hypothesize that the larger lomasome-like configurations, as seen in figure 36, eventually become compacted and condensed to form pockets.of electron dense granular and vesicular material as are distinguishable in older areas of hyphal walls of many of the asexual and sexual reproductive cel ls (Figs. 52-53, 100, 314). Alternatively, the positional relationship of the lomasome-like configurations with "growing" regions of the cel ls of A. bisexualis could indicate that the configurations function in wall formation as has been suggested for lomasomes in other fungi (Wilsenach and Kessel, 1965) and for other plant cel ls (Barton, 1965; Crawley, 1965; Hendy, 1966). However, the involvement or function of the configurations in such a process is doubtful for two basic reasons. 1. Lomasome-like configurations are not a consistent feature in areas of wall formation. They seldom are discernable in germ tubes, and they have not been observed in enlarging zoosporangial discharge papillae or in gemmae where wall formation is conspicuous. And, 2. Wall formation consistently appears to occur by the.deposition of wal1,constituents from dictyosome-derived vesicles and their fusion with-the plasma membrane. It seems unlikely that in some cells of -A. bisexualis two. mechanisms of wall formation exist while in others there is only a single functional mechanism. There is also l i t t l e or no evidence to indicate that the lomasome-like configurations, as observed in this study, (1) serve to increase the surface area of the plasma membrane as proposed by Girbardt (1961), (2) induce alterations in host cel l walls during infection as suggested by - 67 -Ehr l ich, Schafer and Ehrlich (1968), or (3) function in membrane elaboration during nuclear division as noted by Robb (personal communication). Similar ly , . they do not appear to act directly in cel lu lar absorption or secretion as communicated by other investigators (respectively Peyton and Bowen, 1963; Moore and McAlear, 1961). It would appear more l ike ly that i f most of the lomasome-1ike configurations in A. bisexualis represent aggregates of vesicles which have been delimited by "mistake" during, c e l l . wall formation, then they eventually might be converted into wal 1 material. However, until further studies are carried out might their fate or function be known.• . Pe l l i c le In this study and in one other (Reichle, 1969a), f lagellated zoospores of Oomycetes are described as being naked cel ls in which their plasma membrane is immediately-underlaid by large flattened saccules or vesicles. It is suggested that the association of the plasma membrane and saccules in the flagellated zoospores of.these-Oomycetes act as a "pe l l ic le" , ' s imilarly to those which have been observed in some protozoans (Pi te lka, 1965; V iv ier , Devauchelle, Petitprez, Porchet-Hennere, Prensier, Schrevel and Vinckier, .1970). They thus may serve to protect the underlying cortical protoplasm of the zoospores from physical and/or chemical insult by functioning as an insulating layer and by governing the amplitude of interaction between the cel l and i t s surrounding environment. Also, the "pe l l i c le " of the zoospores may function as a skeletal layer, providing form and some support to the cel l in much the same way as form is conveyed to many modern day vinyl and plastic objects by the inf lat ion of certain patterned chambers with ai r or with other materials. This latter property of the pel l ic les then might explain the appearance of the - 68. -longitudinally-oriented groove in each zoospore where there are no large saccules or vesicles in association with the plasma membrane. If the cel ls are surrounded by a type of skeleton except along part of one side, the plasma membrane in this latter area might be subjectto certain mechanical stress which would result in a trough or groove being formed - the actual length, shape and arrangement of the groove being governed by the arrangement and physical condition of the saccules themselves. Changes in the saccules then could induce changes in the shapes of the c e l l s . This l ine of reasoning is in accord with observations indicating that (1) the physical state of saccules is altered within the flagellated protoplasts, and (2) f lagellated zoospores change in shape during their l i fe -span. In zoospores of A. bisexualis and of P. parasitica (Reichle, 1969a), the large flattened saccules or vesicles often appear to coalesce with the plasma membrane and to deposit material outwardly. The vesicles, however, do npt appear to remain fused with the plasma membrane as there is no accompanying overall increase in the size of the zoospores during their period of existence. Thus,, it seems probable that their membranes re-coalesce and that material is again accumulated in the saccules for re-deposition as indicated by the saccules different thicknesses. The shapes of the zoospores vary from ovoid, being bluntly pointed at one or both ends, to ovoid with one side' flattenedj to, reniform (Crump and Branton, 1966;. Ho and Hickman, 1967; Ho, Hickman and Tel ford, 1968; Sparrow, 1960). The development of the pe l l i c le in zoospores of A. bisexualis occurs prior to the escape of f lagellated secondary protoplasts from encysted protoplasts from: encysted primary zoospores (Figs. 249-250). However, the mechanism by which the. saccules develop is yet uncertain. The migration, alignment and fusion of dictyosome-1ike secretory vesicles - 69 -adjacent to the plasma membrane in encysted protoplasts (Fig. 199) suggest that the flattened saccules or vesicles develop in this manner. Evidence in accord with this concept is that the plasma membrane and dictyosome-1ike secretory vesicles are of the same thickness and of the same type (Grove, Bracker and Morre, 1968). Alternatively, the deposition of degraded 1iposome-1ike elements" from the saccules in secondary zoospores (Figs. 270-271 ), indicate that.the saccules may be formed by the fusion and flattening of liposomes (Figs. 249-250). However, because the membranes of the liposomes and of the saccules are different in thickness and in stain intensity, the latter mechanism seems doubtful. Mitochondria The mitochondria of A.:bisexualis, as observed in this study, are morphologically similar to those of other Oomycetes (see Appendix, Table XVII - Mitochondrial Morphology and Distribution). They appear as double membrane-limited organelles with tubular cristae (Figs. 54-57, 159, 197, 307-311). The distribution of the mitochondria in A. bisexualis corresponds to that observed in most other Oomycetes (see Appendix, Table VII - Mitochondrial Morphology and Distribution). The mitochondria are usually most abundant in area of growth, this being especially evident in hyphal tips (Figs. 30-31; Grove and Bracker, 1970; Grove, Bracker and Morre, 1970; Hemmes and Hohl, 1969; see Cellular Growth and Appositional Wall Formation). Also, to fac i l i ta te cleavage, zoospores in zoosporangia, the mitochondria become oriented about the nuclei (Figs. 147, 143; see Appendix, Table VII -Mitochondrial Morphology and Distribution). The external form of the mitochondria in A. bisexualis may be - 70 -globose, ovoid, rod-shaped, rod-shaped with or without terminal, or sub-terminal invaginations, cup-shaped and dumb-bell shaped. This morphology indicates the metabolic state of the adjacent protoplasmic mileau. Those forms which exhibit l i t t l e external morphological elaboration may be characteristic of more high energy compounds^  The forms possessing greater external morphological elaboration indicate a higher rate of cytoplasmic-mitochondrial exchange and less production of high energy compounds. Thus, the surface (which is in contact with the cytoplasm) to volume ratio of the external membrane suggests something of the metabolic state of the c e l l , as has been suggested by Bagshaw, Brown and Yeoman (1969). However, the external morphology ( i . e . - surface to volume ratio) may not reflect degree of metabolic act iv i ty or cytoplasmic interdependence in dumb-bell-shaped forms. The dumb-bell-like forms, as occasionally observed in cel ls of A. bisexualis (Fig. 314) and as have been observed in other Oomycete protoplasm (Reichle, 1969a, 1969b) may represent binary f iss ion of the. mitochondria, as thought to occur in other cel lu lar systems (see DePraw, 1968; Brown and Bertke, 1969; Armentrout, Smith and Wilson, 1968);' The internal morphology as personified by density, relative length of cristae and di lat ion of a central major cr ista are equally valid c r i te r ia for extent of mitochondria act iv i ty and mitochondria interaction. The matrix of mitochondria is extremely dense in young or actively growing regions of the cel ls (Figs. 34, 41, 303). The cristae become proportionally longer during zoosporogenesis (Figs. 159-160, 163; Williams and Webster, 1970; Ehrlich and Ehrl ich, 1966). Dilation of the central cristae occurs during active respiration (Lehninger, 1970) and may or may not be related to the observed di lat ion occurring during zoosporogenesis and secondary zoospore stages in the female strain of A. bisexualis (Figs. 159-160) or in the - 71 -asexual spores of P. megasperma var. sojae as observed by Ho, Zachariah and Hickman (1968). This di lat ion which is different from that observed by Lehninger (1970) - only one atypical central cr ista is dilated (Figs. 159-160) - may be indicative of storage. The morphology of the mitochondria of Oomycetes is probably indicative of the functional relation between cytoplasm and these organelles. Thus, the table (see Appendix, Table VII - Mitochondrial Morphology and Distribution) which gives the morphological relation of the mitochondria in Oomycetes l ike ly expresses the functional relationship of these mitochondria in differentiating developmental stages of each organism. Liposomes These bodies, although they possibly contain l i t t l e l i p i d , are morphologically the same as those inclusions referred to as liposomes in other ultrastructural studies (see Williams and Webster, 1970). . Thus, this study for.convenience perpetuates the same terminology. The liposomes in-A. bisexualis,- as shown in this study, are morphologically similar to those observed in other Oomycetes (see Appendix, Table XII - Liposome Morphology and Distribution). They are a single membrane-limited organelles possessing an. osmiophilic globule surrounded by alternating layers of electron dense and l ight material (Figs. 73-76, 98, 194, 352). Thus, they also resemble vesicles which contain osmiophilic globules in other fungi (Fuller, 1966; Reichle and Ful ler , 1967). The results of this study indicate a probabi1ity of protein within the organelles, possibly in the.absence of l i p i d , in contrast with other reports which indicate a quantity of l i p i d (Williams and Webster, 1970; Moore and Howard, 1968; Zalokar; 1965). Liposomes (described as membrane-bound food storage bodies by Marchant, 1968) occur in oogonia and seldom in antheridia - 72 - . (Fig. 345;- Marchant, 1968; Moore and Howard, 1968). Thus, the bodies stained in the oogonia and absent in the. antheridia are l ike ly liposomes (Figs. 299, 300). This staining evidence suggests the presence of protein. The organelles reacted positively for nucleoproteins with Flemming t r ip le stain (Fig. 300) and toluidine blue with borax (Fig. 299) thus indicating that they are formed of proteins. The liposomes did not appear to fluoresce under u l t ra -v io let l ight after treatment with Nile blue. This suggests that , their major constituent is not l i p i d . Liposomes, then, may contain protein, but they do not appear to contain l i p i d . Liposomes in A. bisexualis probably are .derived from dilated endoplasmic reticulum as explained in the section on sexual reproduction. They appear to be formed during nutrient excess, as this formation occurs simultaneously with l i p i d droplet formation (Figs. 42, .323). Their ultimate fate, which bespeaks their- function, differs in different tempera! and structural stages of A. bisexualis and, thus, i t is d i f f i c u l t to.determine whether-they are primarily waste storage and/or nutrient storage organelles. In several developmental stages (e.g. -pr imary and secondary zoospores), the liposomes are degraded, .thus, possibly being ut i l i zed (Figs. 196, 265). In the flagellated zoospores, electron microscopy studies intimate that liposome degradation in i t iates in the protoplasm and these degrading organelles then fuse with the flattened vesicles or saccules of the p e l l i c l e . The degraded products are subsequently released, from the cel l (Figs. 270-271). In a few stages, particularly the oogonia, the liposomes appear to fuse with or form vacuoles (Fig. 326) and the vacuoles retain their contents (Fig. 326). Therefore, in f lagel late zoospores, the liposomes may function as waste storage organelles, but in oogonia they appear to serve as a nutrient or structural storage organelle as indicated in other studies (Moore and Howard 1968; King, Colhoun and Butler, 1968; Marchant, 1968). '.. - 73 -Parastrasomes The results of this study indicate that parastrasomes function as a source for microtubule constituents in f lagel lar rootlet formation in A. bisexualis. A number of observations support this conclusion: 1. Rootlets always develop adjacent to large parastrasomes (Figs. 273, 275), . 2. Microtubules or f i b r i l s in the parastrasomes are of the same diameter as those-in the rootlets (Figs. 245-247, 275), 3. The one or two extremely .large parastrasomes which develop within zoospores are always in close association with the basal bodies of the f lagel la (Figs. 233-238), 4. The parastrasome l imit ing membrane frequently appears altered or traversed by microtubule - l ike elements adjacent to differentiating rootlets (Figs. 243, 247),. 5. The one or two large parastrasomes usually are s l ight ly smaller after rootlet formation has ceased (Figs. 273, 275); and 6. The composition of the parastrasomes appears to be largely proteinaceous, as indicated in enzymatic degradation studies presently being conducted by this author. There also is other evidence which supports this hypothesis. In Phytophthora (see Appendix, Table XI - - Parastrasome Morphology and Composition), the parastrasomes observed in the sporangia have the same morphology as those observed in the encysted primary zoospores of A. bisexualis. Thus, the stages of development leading to f lagel lat ion in both of these species have similar type parastrasomes. Further, the morphology of parastrasomes in sporangia of A. bisexualis has no observed, by this author, correlate as - 74 -indicated by published micrographs in Phytophthora (see Williams and Webster, 1970). This is suggestive of two things: f i r s t , parastrasomes are in some way connected with- f lagel lar apparatus formation; and, second, the parastrasomes seen in A. b i s e x u a l i s sporangia are less highly differentiated forms of those seen in sporangia of P. oapsioi (see Williams and Webster, 1970). Upon further investigation, other functions of the parastrasomes may be uncovered. However, unti l this time, this author postulates that parastrasomes function as a source for microtubule constituents in f lagel lar rootlet formation in A., b i s e x u a l i s . . Dictyosomes; The dictyosomes in A. b i s e x u a l i s morphologically correspond to those observed in other Oomycetes (see Appendix, Table IX - Dictyosome Morphology and Distribution). They also resemble those in other plant cel ls (see Mollenhauer and Morref 1966). They each consist of a stack of flattened cisternae and vesicles, and they each occur in close proximity to ribosome-free areas of endoplasmic reticulum (Figs. 63-67, 110) or of the nuclear envelope (Figs. 214-215). The face.of the cisternae which occur in association with and appear to be formed by blebs or vesicles from the endoplasmic reticulum.(Figs. 67, 110) or outer nuclear membrane (Figs. 214-215) usually is referred to as the proximal, or forming, face cisternae. Those cisternae most distant from the endoplasmic reticulum or nuclear envelope are termed the d i s t a l , or maturing, face cisternae (Figs. 63-67, 214). Vesicles often appear to.be formed from the latter cisternae and represent the secretory vesicles of the dictyosomes (Figs. 63-67, 110, 214). The relationship of endoplasmic reticulum, or nuclear envelope, with - 7 5 -vesicles, with dictyosome cisternae, with secretory vesicles suggests both a structural and. function transport.mechanism in the c e l l s . ' In Oomycetes, such an endomembrane transport system is i l lucidated by Grove, Bracker and Worre' 0968) and Bracker, Heintz and Grove (1970). In' A. bisexualis, as well as most other Oomycetes, (see Appendix., Table IX - Dic.tyosome.Morphology and Distr ibution), a similar morphological and.functional relationship is apparent; however, t ransit ions' in membrane thicknesses: (probably a reflection of post-staining procedure \:as 'previously indicated by Grove, Bracker and Morre'', 1968) and in contents are not so evident' (Figs. 63-67, 110, 214). The function of the dictyosomes in .4. bisexualis, as indicated in . this study.and in mostother Oomycetes (see Appendix, Table IX - Dictyosome Morphology and Composition):, appears to be extremely variable. The dictyosomes may function as a.transport.mechanism for (1) wall polymers and polymer coupling enzymes during growth (see Cellular Growth and Appositional Wall Formation)., (2) exoenzymes ( i . e . - wall degradative enzymes - see Sexual Reproduction), (3) hormones (see Sexual Reproduction), (4) axonemal sheath processes for tinsel-type, flagellum (see Microtubule Packet and Microtubule Vesicles), and (5) possibly 1iposome material (see Sexual Reproduction). Concurrently, i t also often'serves as a source of membranes which usually .appear.to fuse with, and. increase the surface area of, the plasma membrane as discussed previously (see Plasma Membrane). Dictyosomes, in A. bisexualis and in other Oomycetes (see Appendix, Table IX - Dictyosome Morphology and Distribution) thus seem to have potentially a dual role in morphogenesis - one involving mainly their contents, the other the i r membranes. Microbodies The single membrane-1imited inclusions occurring in close association • - 76 -with ribosome-free surfaces of endoplasmic reticulum as observed in most developmental forms of A. bisexualis in this- study.arei similar to microbodies observed in other Oomycetes (see Appendix, Table VIII -Microbody Morphology and Distribution) and in cel ls of other eucaryotic organisms (see Frederick et a l . , 1968; Hruban and Rechcigl, 1969; Mollenhauer, Morre'and Kelley, 1966). Also, these inclusions do not fluoresce with Nile blue (as is evident in the sub-apical zone of vegetative hyphae where they are known to be extremely abundant as seen by electron microscopy) (Fig. 28-29) and have the appropriate size (see Hruban and Rechcigl, 1969) for microbodies. It seems most l ike ly then that these inclusions are microbodies (as defined by Mollenhauer, Morre'and Kelley, 1966). The morphology of these microbodies is as follows: They have an extremely dense matrix which is often characterized by fine tubules or dense concentric lamellar bands (Figs. 79-80i 83, 121 \ as observed in microbodies of other Oomycetes (see Appendix, Table VIII - Microbody Morphology and Distr ibution). They are, as previously mentioned, limited by a single membrane (Figs. 77-78). And, the microbodies occur in extremely close association with ribosome-free areas of endoplasmic reticulum (Fig. 79); however, in the sub-apical and in young areas of the distal multi-organelle zones, this association with the endoplasmic reticulum is not as evident as in more distal hyphal regions or in other developmental stages of the organism (Figs. 38-40). The microbodies usually are common throughout'the protoplasm of almost a l l developmental stages of 4. bisexualis (Figs. 34, 41-42; 104^1-07; see Hruban and Rechcigl, 1969; Frederick et a l . , 1968; Mollenhauer, Morre'and Kelley, 1966), but they are particularly evident in growing regions, being most conspicuous.in the sub-apical mitochondrial zone of vegetative hyphae - 77 -(Fig. 34). In these regions, there is usually a concurrent absence of l i p i d droplets and a relative excess of mitochondria. This suggests that microbodies function in l i p i d metabolism. Other studies CBreidenbach et a l . , 1968 - see Hruban and Rechcigl, 1969) also indicate this to be the case. Microbodies also thought to function in photorespiration CKisaki and Tolbert, 1968 - see Hruban and Rechcigl, 1969), but probably not in this organism as A. bisexualis is not a photosynthesizer. Other functions of the microbodies may; exist,' but to the author's knowledge, they do not appear evident.in this study. - 78 -Microtubule Packets and Microtubule Vesicles The packets and vesicles of microtubules in zoosporangia and zoospores of .A. bisexualis correspond to those observed in other Oomycetes (see Appendix, Table X - Microtubule Packet Morphology and Distribution) and in other organisms which have cel ls with tinsel-type f lagel la (Bisalputra, personal communication; Bouch, 1969; Ful ler , 1966; Gibbs, 1962; Heath, Greenwood and Gr i f f i ths , 1970; Koch and Schnepf, 1967; Leadbeater, 1969; Leedale,' Leadbeater and Massalski, 1970). The morphological association of these-structures suggests a functional relationship as indicated in several studies (Heath, Greenwood and Gr i f f i ths , 1970; Bouch, 1969; Leedale, Leadbeater and Massalski, 1970) and as i l lucidated by Bracker, Heintz and Grove (1970). Their relationship ( i .e . - packets (endoplasmic ret icu lum)— dictyosomes > vesicles > axonemal membrances and the processes of the tinsel type f lagel la) is supported in this study by the following evidence. 1. Packet membranes are of approximately the same thickness as that of the endoplasmic reticulum (Figs. 203-205). 2. Ribosomes are often v is ib le on the outer surface of the packet membranes, thus indicating that the packets are morphologically comparable to granular endoplasmic reticulum (Fig. 205). 3. Microtubules in the packets are of a similar morphology and length to those of the axonemal processes (Figs. 162-163, 205). 4. Vesicles of microtubules, the latter of which are of the same diameter as those in the packets and in independent - 79 -vesicles, are sometimes v is ib le in continuum with distal dictyosome cisternae (Fig. 251). And, 5. The membrane thicknesses of the vesicles are the same as that of the axonemal sheath (Figs. 203, 251). This study contradicts the proposed relationship in observing a discrepancy in microtubule diameter versus axonemal process diameter (Figs. 205, 276-280). This discrepancy may ar ise, however, as a result of air-drying the l iv ing zoospores in preparation for shadowing. The association of the packets with mitochondria in the zoospore stage in A. bisexualis and in other Oomycetes (Bracker, Heintz and Grove, 1970; Williams and Webster, 1970) suggests that microtubule formation . within the packets is an energy requiring process. Nucleus The fine structureoof the nucleus in A. bisexualis in most developmental stages of the l i fe - cyc le reveals the nucleus to be similar to that of other Oomycetes (see Appendix, Table X>VI - Nucl ear .Morphology and Distribution). It has the same shape and basic morphology with regard to nuclear envelope, nucleolus and chromatin material. It is in most respects a typical fungal nucleus and a typical eucaryotic nucleus. The nucleus exhibits an external morphology and a plast ic nature similar to that of.other Oomycetes" (see Appendix, Table XVI - Nuclear Morphology and Distr ibution). It appears ovoid to spindle-shaped i n . vegetative hyphae (Figs. 41, 84), ovoid to irregular to comma-shaped in gemmae (Figs. 117-118), pyriform in zoosporangia and zoospores (Fig. 175), and ovoid to irregular in profi le in most.other developmental stages (Figs. 312-313, 354-356). The nuclei are bounded by a typical double - 80 - - . membrane which is sometimes"eontinuous with the endoplasmic reticulum (Figs. 118, .217). It is now generally accepted on the basis of membrane thickness and other evidence (DuPraw, 1968) that the nuclear envelope is specialized or.modified endoplasmic reticulum. There i s no reason to believe that this is not true for A. bisexualis. The nuclear envelope displays typical nuclear pores, which in some organisms have been shown to occupy up to ten percent of the surface area of the nuclear envelope (see DuPraw, 1968). The annuli (thought to regulate nuclear-cytoplasmic exchange) observed in this study are typical ly composed of seven to eight sub-units (Fig. 218) and exhibit a.complex morphology as frequently seen in other organisms. The nuclear envelope is thickened on the inner surface in areas adjacent to the centrioles (Figs. 121, 123) and in the zoospore stages is connected to a sheath which is continuous with f lagel la (Figs. 234^235). Both of these "features appear to be typical of the Oomycetes (see Appendix, Table -XVI - Nuclear Morphology and Distribution). The fine structure of the nucleolus in A. bisexualis. is similar to that observed in other organisms (see Hay, 1968; Busch and Smetana, 1970). The central region of the nucleolus is represented mostlyby -pars fibrosa, a meshworkof f i b r i l s of approximately 40-50A (Figs.91, 119, 219). It is interspersed with groups of electron opaque granules, the pars granulosa, of a diameter of 100-140A (Figs. 91, 119, 219). The granules often may appear inter-connected by fine filaments (Figs. 91, 119, 219) and are usually located in most preparations near the periphery.of the nucleolus, although there is no clear dist inction between, the pars fibrosa and the more particulate periphery. It is suggested (Bernhard, 1959 - see DuPraw, 1968; Bernhard and Granboulan, ,1968 - see Busch and Smetaria, 1970) that the granules in the nucleolus ( i . e . - mostly the .pars granulosa), are ribonucleoprotein: C.RNP), similar to those in cytoplasmic ribosomes.. The filaments on which the nucleolar granules seem to reside are thought to be continous with intranucleplar and perinucleolar chromatin f i b r i l s , ,the pars, chromosoma, and with i l l -def ined filaments of the pars fibrosa (Hay, 1968; Hay and Revel, ,1963; Narayan et al_., 1966 - see Busch and Smetana, 1970). The pars fibrosa, or meshwork of f i b r i l s usually within the central region of the nucleolus, i s thought to also be formed of RNP f i b r i l s which l i e within a proteinaceous matrix (Bernhard and Granboulan, 1968 - see Busch and Smetana, 1970; Stevens, 1964). The pars ohromosoma3 which is f inely / f ib r i l l a r as seen in thin-section,, corresponds chromatin, or DNA, constituents (Busch and Smetana., .1970; Hay 1966). As noted by numerous.investigators (see Hay, 1968) and as observed in this study (Fig. 119), i t often is d i f f i c u l t to distinguish the chromatin f i b r i l s from other nucleoplasmic. material. The pars amorpha, as f i r s t described in l ight microscopic studies by Estable and Sotelo (1951 - see Hay, 1968) represents the pars fibrosa as observed in this study. However, i tsalso is used, to distinguish electron l ight areas within nucleol i , as seen by electron microscopy (Busch and Smetana., 1970). In nucleoli of A. bisexualis, the pars amorpha appears to correspond to nuclepplasmic constituents ( i . e . - proteins) in which chromatin material is probably suspended (Fig? 220). It can be seen that the nucleolus in nuclei of A. bisexualis. appears as a morphologically typical nucleolus characteristic of other eucaryotic organisms. The chromatin is also typicaT'in that i t is not easily distinguishable from nucleoplasm in interphase nuclei (.Figs. 84, 219). The chromatin only - 82 - . becomes dist inct during mitotic or meiotic d iv is ion , where i t then appears as more dense granules and f i b r i l s in the nucleoplasm (Figs. 87, .357-376). It is clear from ultrastructural evidence that the A. bisexualis nucleus during most stages of development is a typical Oomycete nucleus and may be taken as. a representative eucaryotic nucleus. The division of the nuclei (both mitotic and meiotic) in A. bisexualis is intranuclear and usually eccentric (Figs. 87, 357-376). It thus closely resembles mitotic division in Saproleghia ferax as described by Heath and Greenwood (1968). However, the appearance of single axial elements and the in i t ia t ion of synaptinemal. complex formation are dist inct ive of only the meiotically dividing nuclei and are discussed later (see Meiosis). - 83 -Centrioles The centrioles in cel ls of A. bisexualis are structurally similar to those observed in other Oomycetes (see Appendix, Table XIV - Centriole Morphology and Distribution) and in most other eucaryotic organisms (see Brown and Bertke, 1969; DuPraw, 1968; Bracker, 1967). They usually occur in pairs and each member consists of nine peripheral f i b r i l s in cyl indrical and cartwheel-1 ike arrangement about a central spoke or "tubular- l ike" element as seen in cross-section (Figs. 92-93, 120-123, 365-370). The peripheral f i b r i l s each are formed of three sub- f ibr i l s (Figs. 92, 122). As characteristic of A. bisexualis and of other Oomycetes (see Appendix, Table XIV - Centriole Morphology and Distribution);, the dimensions of the centrioles are generally less than those of centriolesin most other eucaryotic c e l l s . Similar ly , they also are usually oriented end-to-end, not at 90-130° angles to each other as are characteristic of centrioles in most other systems (see Brown and Bertke, 1969; DuPraw, 1968). The mechanism of centriolar repl icat ion, as indicated in this study, clearly does, not resemble the pro-centriolar mechanism characteristic of numerous other organisms (see Dirksen and Crocker, 1965). No pro-centriolar mass or pro-centriolar body, as described in the Oomycete, P.- parasitica (Hohl and Hamamoto, 1968), has been observed in any cel ls of- A. bisexualis. Instead, what is seen is compatible with evidence that centrioles contain DNA and are capable of se l f - repl icat ion (see DePraw, 1968). The inter-connections which sometimes are distinguishable between peripheral f i b r i l s of adjacent centrioles and the association of such a pair with another l ike pair suggests that centriolar replication occurs by the elongation and subsequent binary f iss ion of pre-existing centrioles - 84 -(Figs. 120-123). This has been indicated or implied in studies on other-Oomycetes (see Appendix, Table XIV - Centriole Morphology and Distribution). There.is no morphological evidence in this study.to support any other form of centriolar replication as indicated for other organisms (see Brown and Bertke, 1969; DuPraw, 1968). The functions of the centrioles in A. bisexualis appear to be' basically twofold: F i r s t l y , in the zoosporangial and zoospore stages of . development, the centrioles give r ise to the basal bodies of the f lagel lar apparatus (Figs. 226-228; see Flagellar Development, Zoosporogenesis). A similar situation in fungi was f i r s t observed in the fungus Allomyces avbusoula (Renaud and Swift, 1964) and subsequently in many other f lagellated fungi (see Appendix, Table XV - Flagellar Morphology and Distr ibution; see Bracker, 1967; Kole, 1965). Secondly, the centrioles appear to regulate orientation and.possibly act iv i ty of the spindle apparatus during mitotic and meiotic nuclear division (Figs. 87, 365-367, 371-374). Such regulation by centrioles has been noted in numerous reports on other organisms (see Brown and Bertke, 1969; DuPraw, 1968). However, in fungi where centrioles are present usually no direct connection between the centrioles and the spindle f i b r i l s has been observed (Heath and Greenwood, 1968). In A., bisexualis, thin filaments sometimes appear to traverse the nuclear envelope between the peripheral t r ip lets of the centriole and the terminus of the spindle f i b r i l s on the thickened inner nuclear membrane (Fig. 366). - 85 -Flagellar Apparatus The f lagel la of secondary zoospore of A. .bisexualis morphologically are similar to those f lagel la of other motile cel ls of Oomycetes (see Appendix, Table XV - - F l a g e l l a r Morphology and Distribution) and of f lagellated or c i l ia ted protoplasts of most other eucaryotic organisms (see Fawcett, 1961; Kole, 1965). They each consist of an axoneme, basal body and rootlets (Figs. 224-275). The axoneme is enclosed within a membrane (the axonemal sheath) which is continuous with the plasma membrane of the cel l (Figs. 274). It is formed of an axis of eleven f i b r i l s arranged in a "9 + 2" pattern: two single central microtubules of c ircular cross-section are surrounded by and are inter-associated with a cylinder of nine equidistantly-spaced double microtubules, each with a f igure-of-eight cross-section. The basal body, from which the axoneme differentiates, s imilarly consists of nine microtubules in cyl indric arrangement (Figs. 221-225). However, each of the microtubules appears as a t r ip le t except near the distal ly - located terminal, or basal, plate. The microtubules then appear double and continuous with those of the axoneme (Figs. 221-222, 274-275). The most proximal region of the basal body is comparable with that of the centr iole, from which the basal body develops (Figs. 226-227). The rootlets of the f lagel la similarly are associated with each basal body and are formed of microtubules (Figs. 274-275; see Appendix, Table XV - - F l a g e l l a r Morphology and Distribution). In A. bisexualis zoospores, as observed in this study, the rootlets morphologically resemble those in zoospores of P. parasitica as described by Reichle (1969a) and as observed in other Oomycetes (see Appendix, Table XV - - F l a g e l l a r Morphology and Distribution). Only the microtubules which arise at right angles from - 86 -the long rootlet , their " l ^ " and "R3" as seen in figure 15, are not evident (Figs. 273-275). However, that these osmiophilic f i b r i l s are in direct continuity, via a microtubule, to sub - f ib r i l C of each of nine peripheral f i b r i l s of the basal body (Figs. 233, 235) i s , to the author's knowledge, shown for the f i r s t time. Simi lar ly , that these same f i b r i l s are directly inter-connected with, the nucleus by a sheath is shown in this study (Figs. 234-235, 238). This inter-association of the basal bodies and the nucleus in each zoospore suggests that the nucleus may serve as a control mechanism and/or co-ordinator of f lagel lar act iv i ty . S imi lar ly , i t seems reasonable to assume that i t , as do the root lets , functions as an anchor and/or possible stabi l i zer for the f lagel lar apparatus. The results of this study also are compatible with ultrastructural evidence (Desjardins, Zentmyer and Reynolds, 1969; Mantqn, Clarke and Greenwood, 1961; Reichle, 1969a) showing that Oomycete zoospores possess two types of f lagellar whip-lash and t inse l . The tinsel-type flagellum is characterized by numerous lateral processes with tapered ends, referred to as hairs or mastigonemes, which extend from the axonemal sheath, except at the terminal region where the axonemal axis is tapered (Figs. 280; Desjardins, Zentmyer and Reynolds, 1969; Manton, Clarke and Greenwood, 1961; Reichle, 1969a). Also, as noted by Reichle (1969a) and as observed in this study (Figs. 278-280), each of the processes appear hollow. This latter feature, thus, is in accord with the hypothesis that the processes are microtubules as presented by Heath, Greenwood and Gr i f f i ths (1970) and others (Bouch, 1969; Leedale, Leadbeater and Massalski, 1970) and which has been discussed previously (see Microtubule Packets and Microtubule Vesicles). The whip-lash type flagellum similar ly possesses processes along their lateral axis except at their,ends where they are absent (Figs. 276-277; Desjardins, - 87 -Zentmyer and Reynolds, 1969; Manton, Clarke and Greenwood, 1961; Reichle, 1969a); however, the processes are shorter, thinner and not tapered as those of the tinsel-type flagellum. Also, as revealed by this study, these processes of the tinsel flagellum are more easily disrupted from the surface of the axonemal sheath, especially upon air -dry treatment of the zoospores (Figs. 276-277). This difference in sensit iv i ty to air-drying suggests that either the processes are coupled with, or attached to , the axonemal sheath by a different mechanism or that other chemical and/or physical differences exist between the two types of processes of the . f lagel la . The results of this study also are in accord with, on the basis of f lagel lar morphology, the concept that uniflagellate fungi (e.g. - - - the chytridiomycetetous fo.rms) are derived from, or related to, the bif lagellated forms (see Olson and Ful ler , .1968). No vestigial centrioles or basal bodies have been observed in association with either of the f lagel lar basal bodies. - 88 -Cellular Growth and Appositional Wall Deposition Grove, Bracker and Morre (1970) recently proposed that hyphal t ip growth in the Oomycete, Pythiym ultimum, occurs by the fusion of dictyosome-derived vesicles with the plasma membrane - - a mechanism which is particularly attractive because i t provides for both an increase in the surface area of the plasma membrane and a deposition of constituents to the expanding surface. The results of this study support their hypothesis and suggest that this -type of mechanism is not only functional in hyphal t ip growth in A. bisexualis (Figs. 30-33, .58-64), but that i t is also operational in germ tube elongation (Figs.,286-296), zoosporangial discharge papil la formation (Figs. 303, 377-378) antheridial and fe r t i l i za t ion tube development (Figs. 303, 377-378), and oogonial expansion (Figs. 323, 325-330). In a l l of these preceding areas where cel l elongation and/or expansion is known to occur, the protoplasm contains numerous vesicles which are morphologically similar to those which are in direct continuity with, distal dictyosome cisternae in each of the ce l l s . The vesicles l i e in close association with the plasma membrane which is usually crenulate and sometimes characterized by one to several dist inct vesicle fusion prof i les. The walls adjacent.to these areas of vesiculation also are relat ively irregular in prof i le . Thus, i t seems reasonable to assume, in l ight of evidence presented by Grove, Bracker and Morre (1970) in support of their hypothesis, that cel lu lar growth in A. bisexualis occurs when dictyosome-derived vesicles migrate to the plasma membrane and fuse with i t . The pattern of vesicle distribution in growth of the hyphal t i p , as seen in figure 30 corresponds to.that described for other Oomycetes by Grove and Bracker (1970). S imi lar ly , as postulated by the previous - 89 -. investigators, this pattern may explain the lack of a "Spitzenkorper" in hyphae examined by l ight microscopy (Figs. 9-19). The pattern of mitochondrial and microbody distr ibut ion, as shown in figures 34 and 41 and as observed in young vegetative hyphae in other studies (Brenner and Carro l l , 1968; Grove, Bracker and Morre, 1970; McClure, Park and Robinson, 1968), indicates that cel l growth is a high energy requiring process as has been demonstrated in numerous other systems (see Brown and Bertke, 1969; DuPraw, 1968; DeRobertis, Nowinski and Saez, 1970). Because the microbodies (which are thought, to be active in l i p i d metabolism - - Breidenbach et eQ.., 1968 - see Hruban and Rechcigl, 1969) are most abundant in the sub-apical and young distal multi-organelle zones (Figs. 34, 41) and a conspicuous lack of l i p i d droplets exists, one might postulate that l i p i d metabolism is occurring. This hypothesis concurs with the high energy required as indicated by the mitochondrial distribution Simi lar ly , high act iv i ty in other metabolic processes (e.g. - protein synthesis) is evident in the sub-apical and younger areas of the distal multi-organelle zone as is indicated by organelle distribution (e.g. -ribosomes) both in this study (Figs. 34, 41) and in others ( Brenner and Carro l l , 1968; Girbardt, 1969; Grove, Bracker and Morre, 1967, 1970; McClure, Park and Robinson, 1968; Rosen et a l . , 1964; Thornton, 1968; Zalokar, 1959). Appositional wall deposition appears to occur by the same mechanism as noted for cel lu lar growth (Figs. 48, 98, 100-101, 326-330). However, as suggested by Hemmes and Hohl (1968), the pockets of electron dense granular and vesicular material which are seen in the walls may occur by the entrapment mechanism as are discussed later in this paper (see Septal Structure and Development). - 90 -If cellular.growth and appositional wall formation in A. bisexualis and in other fungi does occur by the mechanism as proposed by Grove, Bracker and Morre (1970), the contents of the vesicles logical ly must contain structural wall polymers and polymer-coupling enzymes. This is supported by the results of Bartnicki-Garcia and Lippman (1969) who report the apical deposition of the structural wall polymer, N-acteyl-d-(GL-H ) glucosamine, in rapidly growing-Mucor rouxii germ tubes. They also note that the posit ion, smallness and steepness of the ..active regions of hyphal wall synthesis suggest a minute sub-cellular organelle, less than 1.0 u in diameter, to be responsible for;apical growth. Similar ly , I.B. Heath (1969 - see Grove and Bracker* 1970) f inds, using siIver hexamine cytochemical determinations, that both the vesicles and the walls in Pythium aphanidermatum hyphae give a similar positive reaction for polysaccharides. It -correspondingly seems reasonable to assume that enzymes which would allow for plast ic izat ion of the wall about the growing areas must be present in the vesicles, as previously indicated by Grove, Bracker and Morre, 1970. Girbardt (1969) suggests that exoenzymes probably are released by the vesicles. This lat ter property then would explain the ab i l i t y of the antheridial hyphae to penetrate the extremely thick wall of oogonia and the thinner wall of oosphere during sexual reproduction. Also, hormones, possible as those detected by Raper. (1939a, 1939b, 1940a, 1940b, 1950a, 1950b, 1960) and Barksdale (1963) might be released from some of the secretory vesicles upon their fusion with the plasma membrane. - 91 -Septal Structure and Development The septa of Aohlya bisexualis are similar to the "basal plugs" which function as partitions between zoosporangia and respective vegetative hyphae as have been described for other Oomycetes (Chapman and Vuj ic ic , 1965; Hemmes and Hohl, 1969; Hohl and Hamamoto, 1968; Williams and Webster, 1970). They each consist of two layers of wall material, each of which is continuous with the inner lateral hyphal or reproductive eel 1 wall and is separated from the other by a region of electron dense granular and vesicular material. Thus, the septa of A. bisexualis and of other Oomycetes are structurally d is -t inct from those characteristic of other fungi as described by Moore (1965). The development of this type of septum, as indicated by observations in this study (Figs. 102-103, 144, 315-322, 336), occurs by a mechanism simi-Jar to that suggested by Hemmes and Hohl (1969): 1. Dictyosome-derived secretory vesicles concentrate in the hyphal protoplasm, particularly about a common band of plasma membrane. 2. The vesicles fuse with the plasma membrane and concurrent de-position of vesicular material and centripetal growth of the plasma membrane occurs. During this process vesicles and protoplasmic ground material are also, separated from the pro-toplasm by the enclosure of.these structures between developing septa and preformed plasma membrand. And, 3. Compaction and condensation of the vesicular aggregates within the deposited, material occurs, and cel l division is completed upon the fusion of the plasma membrane across the center of the hypha. In consideration of the f i r s t process of-septal development, the presence of stacks of cisternae in association with ribosome-free regions of endoplas-mic reticulum and in association with the outer envelope membrane of nuclei - . 9 2 -in A. bisexualis and in most other Oomycetes (see Appendix, Table IX — Dictyosome Morphology and Distribution) is in accord with the presence of dictyosomes in the protoplasm. Similar ly , that the vesicles adjacent to the plasma membrane originate from dictyosomes is compatible with evidence that (1) the membranes of the vesicles and of mature dictyosome cisternae are of the same-type .(Grove, Bracker and Morre, 1968), (2) the contents of the vesicles both near the plasma membrane and adjacent to or in continuity with distal dictyosome cisternae are similar (Figs. 304-305), and (3) l ike migrational patterns have been suggested for dictyosome-derived vesicles in other studies on Oomycetes (Grove and Bracker, 1970; Grove, Bracker and Morre, 1970; Hemmes and Hohl, 1969; Hohl and Hamamoto, 1968; Manocha and Colvin, 1968). The second process in septation is supported by the fact that a number of morphological features appear-concurrently. The plasma membrane is crenulate and there are occasionally dist inct vesicle-plasma membrane fusion profiles (Figs. 315-316). - The membranes of the dictyosome secretory vesicles and of the plasma membrane have been shown to be of the same type (Grove, Bracker and Morre, 1968). And, plasma membrane constrictions characteristic of incomplete septal growth are evident (Figs. 315-316). Further, the entrapment of cytoplasmic constituents into developing septa is compatible with evidence given by Bouch and Galston (1967). The latter investigators note that large pockets of -flattened sacs, similar to the pockets, of electron dense granular and vesicular material in the septa of A. bisexualis, frequently become incorporated in walls of pea stem internode cel ls which are not actively elongating after treatment with an auxin analog which inhibits ce l l growth without inhibiting wall synthesis. This entrapment process thus appears to provide a mechanism negating membrane surplus in accordance with the interpr.eation of Hemmes and Hohl (1969). - 93 -The increase in electron density and the concurrent' reduction in size of the entrapped granular and vesicular material (Figs. 317-322) lends evidence to the third postulated process in septal development. Simultaneous with the completion of cel l d iv is ion, the entrapment phenomenon ceases, probably because of a reduction in the number of vesicles in adjacent protoplasm as is indicated in this study (Figs. 102-103, 144,,319-322, 336). - 94 - . Zoosporogenesis TLoosporogenesis in A. bisexualis, as revealed in this study, is a dynamic process which is characterized by numerous fine structural changes, many of which are manifest in mechanisms of-zoosporangial delimitation from hyphal protoplasm (e.g. - septation), discharge papil la formation, basal body and f lagel lar development and protoplasmic cleavage. ; Septation The delimitation of zoosporangia from hyphal protoplasm appears to occur rapidly, as seldom are intermediate stages of septation observed. The mechanism of septal development and the structure of the septa of A. bisexualis is similar to that described by Hemmes and Hohl (1969) for basal plugs in zoosporangia of P. -parasitica and has been discussed previously (see Septal Structure and Development). The change which occurs in septal profi le during zoosporogenesis (Figs. 128-131, 138-139) appears to correspond with the completion of zoospore cleavage, as noted for other members of the Saprolegniales (Gay and Greenwood, 1966; Hartog, 1888; Rothert, 1892; Schwarz, 1922). Rothert (1892) and Hartog.(1888) suggest that this change is associated with a rapid loss in turgor pressure during zoosporogenesis which Gay and Greenwood (1966) attribute to leakage of vacuolar sap. Papil la Formation The mechanism of papilla development, as indicated in this study, is the dynamic process of vesicle fusion with the plasma membrane with concurrent deposition of wall constituents, as previously described (Figs. 151-152; see Cellular Growth and Appositional Wall Formation). The fine structure of the mature zoosporangial discharge papil la in A. bisexualis (a representative of the Saprolegniaceae), ..as shown for the f i r s t time, differs from that observed in other Oomycete papillae which - 95 -have been described to date (Chapman and Vuj ic ic , 1965; Hemmes and Hohl, 1969; Hohl and Hamamoto, 1968; Williams and Webster, 1970; Vuj ic ic , Chapman and Colhoun, 1965). These differences are: f i r s t l y , protoplasm and cleaved primary zoospore i n i t i a l s occur within the zoospore discharge papillae of A. bisexualis next to their wal l ; secondly, the fibrous plug, as observed in zoosporangial discharge papillae of Phytophthora is absent. (Figs. 154). Thus, the fine structure of the papillae of A. bisexualis is d ist inct from those of others yet described and the development of the papillae resembles that which characterizes other ."growing" or "forming" systems. Basal Body and Flagellar Development. Basal body and f lagel lar development during zoosporogenesis in A. bisexualis closely res.embles that previously described for other Oomycetes (Berlin and Bowen, 1965;. Eisner, Horton and Bowen, 1967; Hemmes and Hohl, 1969; Williams and Webster, 1970) and for most other bif lagellated stages of fungi (see Bracker, 1967; Kole, 1965; Hawker, 1965) - - this formation being through the differentiation of centrioles (Figs. 178-189). Protoplasmic Cleavage Protoplasmic cleavage during zoosporogenesis is fundamentally the same as previously reported in other Oomycetes (Chapman and Vuj ic ic , 1965; Hemms and Hohl, 1969; Williams and Webster, 1970). However, the work of Gay and Greenwood (1966) shows the formation of the zoospores to be by centr i -fugal cleavage producing a structly peripheral layer of c e l l s . Cleavage of zoospores in A. bisexualis occurs uniformly, thereby producing a greater depth of cel ls in the sporangium. This difference in the cleavage patterns is pro-bably relative to the ratio of protoplasmic to vacuolar volume. - 96 -Sexual Reproduction Raper (1960) has observed by the use of .light microscopy that sexual reproduction in A. bisexualis is a dynamic system that is effected by f ive major processes: (1) Ini t iat ion of antheridial branches; (2) Development of oogonial i n i t i a l s on female thal lus; (3) Growth of antheridial filaments to oogonial i n i t i a l s and delimitation of these .reproductive : structures by septa; (4)_ Cleavage of oogonial protoplasm to form oospheres; and (5) Formation of fe r t i l i za t ion tubes by antheridia and subsequent penetration by the tubes of oogonia and oosphere walls respectively, eventually effecting transfer of male nuclei to. oospheres. . The results, of this study are in accord with the observations of Raper (1960) except that f ine structural observations reveal that f e r t i l i z a t i o n tube penetration.is in i t iated prior to cleavage of the oogonial protoplasm (Figs. 349, 351, 377-378). They also indicate, except for process (4) which was, not observed, that each of these major processes is usually accompanied by numerous f ine structural changes. One of these .changes indicates that meiosis is gametic. That i s , meiosis is observed to occur, prior to , or during, f e r t i l i z a t i o n tube penetration into oogonia. The correlation of the ultrastructural changes observed in this study with the processes as outlined by. Raper (1960) is shown in the following table (see Table XVII - - Fine Structural Changes Observed During Sexual Reproduction). As this is the f i r s t time that the fine structure of many of,these processes in sexual reproduction have; to the author's knowledge, been observed in A. .bisexualis or in any Oomycete, many of the changes are i11ucidated as follows. Antheridial Branch Ini t iat ion The change in electron density which occurs simultaneously in i FINE -STRUCTURAL CHANGES Sexual Reproductive Processes Antheridial Branch In i t iat ion Oogonial In i t ia l Development Antheridial Contact with Oogonia TABLE XVII OBSERVED DURING SEXUAL REPRODUCTION Ultrastructural Changes Marked increase in electron density of .ves ic les - in apical growing zone (Fig. .303).-Increase in electron density of .distal dictyosome cisternae (Figs. 304-305). Development of terminal and sub-terminal invaginations in rod-shaped mitochondria and concurrent appearance of cup-shaped forms. (Figs. 302, 304-307, 309-311). Consistent,and uniform vesicle region appears in peripheral protoplasm (Figs. 326-329). Dilation of endoplasmic reticulum (Figs. 323-324). Rapid liposome development (Figs. 323-324). Rapid l ip id droplet accumulation (Figs. 323-329). Aggregation of dictyosome-derived vesicles at points of contact with concurrent decrease in the electron density of,the vesicles in antheridial protoplasm (Figs. .342-345). Increased l ip id droplet formation in oogonial protoplasm (Figs. 341-345, 348-353). Increased liposome differentiation in oogonial protoplasm (Figs.,343-345). Appearance of meiotic division figures in the nuclei (Figs. .357-364, 371-376). TABLE XVII (CONTINUED) Sexual Reproductive Processes U l t r a s t r u c t u r a l Changes S e p t a t i o n Accumulation and apparent f u s i o n of dictyosome-d e r i v e d v e s i c l e s w i t h band of plasma membrane ( F i g s . 315-316, see S e p t a l S t r u c t u r e and Develop-ment ). F e r t i l i z a t i o n ' T u b e Formation A g g r e g a t i o n of v e s i c l e s i n d e v e l o p i n g f e r t i l i -and P e n e t r a t i o n z a t i o n tube a p i c e s ( F i g s . 377-378). Apparent d e g r a d a t i o n of o o g o n i a l w a l l at p o i n t of a n t h e r i d i a l c o n t a c t ( F i g s . 377-378). . C o n s t r i c t i o n of f e r t i l i z a t i o n tube at p o i n t of p e n e t r a t i o n ( F i g s . 379-380). - 98 -both the vesicles and the dictyosomes, from which the, vesicles are .probably derived, indicates a change in content. It may be that this change is mediated by hormonal changes as shown by Raper (1939a, 1939b, 1942a, 1942b, 1950a, 1950b, 1960), Raper and Haagen Smit (1942), and Barksdale (1963). It is also possible that the vesicles and dictyosomes.are agents of hormonal secretion. Other fine structural changes (e.g. - terminal and sub-terminal invagination of rod-shaped mitochondria and the appearance of cup-shaped mitochondria) .may also be hormone dependent or a r e s u l t of metabolic control. As the antheridial branches are young and vigorous, these changes (e.g. - - mitochondrial invagination) are not l i ke l y a result of starvation as suggested by Palade (1959). Oogonial In i t ia l Development The appearance of .the vesicular region in the peripheral protoplasm is indicative of the vesicle-plasma membrane fusion mechanism which results in both over-al l growth of the oogonia and an increase in their wall thickness as discussed previously (see Cellular Growth and Appositional Wall Formation). The concurrent d i lat ion of the endoplasmic reticulum and increase in both size and number of liposomes in the oogonial i n i t i a l s suggest that the liposomes may be derived from the dilated endoplasmic reticulum. This . is further supported by the s imi lar i ty in membrane thickness and of the similar appearance in their contents (Figs. 323-325, 332). However, i t is possible -that the liposomes are derived from vesicles which originate from dictyosomes in close association with the dilated endoplasmic reticulum (Figs. 323, 333), but membrane thicknesses do not support this latter concept. - 99 -The rapid increase in both the number of liposomes and l i p i d droplets in oogonial protoplasm (Figs. 341-353), as also observed in other developmental stages of A. bisexualis (Figs. 98,- 140-141), suggests that there is an excess of nutrients in the protoplasm at the time of oogonial i n i t i a l development. The l i p i d droplets might thus serve as a local store of energy and a source of short carbon chains which could be used in the synthesis of l ipid-containing structural components (e.g. - - membrane) or in the elaboration of specific secretory products as suggested by Fawcett (1966). The liposomes may function in a similar way; however, as described previously (see Liposomes), they probably function as a source of protein i n the eel 1. Antheridia in Contact with Oogonia The concurrent aggregation of vesicles at the points of contact between the antheridia and oogonia, and the simultaneous decrease in electron density in the vesicles of antheridial protoplasm imply an influence which may be hormonal and/or enzymatic-The continued thickening of the oogonial wall suggests that walls become a protective barrier for the developing .oospheres and oospores, as indicated by other investigators (see Dick, 1969). Other morphological changes, which are particularly dist inct ive of only antheridia and oogonia subsequent to contact, are observed in the nuclei. These changes are indicative of gametic meiosis and are discussed later (see Meiosis). Septation As observed by Raper (1960), septation f i r s t occurs in antheridia, subsequently in oogonia. The structure of the septa and the mechanism by which the septa develop, delimiting gametangial protoplasm from subtending hyphal protoplasm, has been discussed previously (see Septal Structure and Development). - 100 -Fert i l i zat ion Tube Formation and Penetration Fert i l i zat ion tube formation and penetration is probably mediated by antheridial vesicular ac t i v i t y . The vesicle-plasma membrane fusion mechanism, as previously discussed (see Cellular Growth and Appositional Wall Formation) may be responsible for f e r t i l i z a t i o n tube formation. Dissolution of the oogonial wall (and 1ikely oosphere wall) is probably effected by an enzyme(s) released upon vesicular fusion with the plasma membrane. The fact that oogonial wall dissolution occurs only on the antheridial side at the point of contact (Figs. 377-378) suggests the action of an antheridial enzyme. In a similar system of apical growth, Girbardt (1969) suggests'.'that other exoenzymes are probably mediated vesicularly'i Also, Hawker and Gooday (1969) observed that an enzyme or enzymes are responsible for gametangial wall dissolution. Thus, i t seems l ike ly that the apical fusion vesicles in the fe r t i l i za t ion tubes are responsible for both tube formation and tube penetration. Oogonial Cleavage Oogonial cleavage has not been observed by electron microscopy in this study; however, i t seems l ike ly that i t is effected by basical ly the same mechanism as is functional in cleavage of. zoosporangial protoplasm. This is, supported by the fact that the organelles are basically the same in both systems. - 101 -MEIOSIS The results of this study concur with most recent evidence (Barksdale, 1966, 1968; Bryant and Howard, 1969; Flanagan,.1970; Moore:and Howard - see Aldrich and 'Minis', 1970; .Sansome, 1961 , 1963, 1965; Sansome and Harris, 1962) indicating that meiosis in Saprolegnian.fungi is. gametic. In antheridia and/or oogonia, prior to f e r t i l i z a t i o n , the following features dist inct ive of meiosis are indicated: (1) Two successive nuclear divisions with,a corresponding reduction in^nucTear size (Figs. 375-376, 378). (2) Single axial elements in association with the inner envelope membrane of prophase I nuclei (Figs. 357-359). And, (3) Pairing of single-axial elements dist inctive of i n i t i a l stages of synaptinemal complex formation" (Figs. 360-361 , 364). . . . . . . In evidence of the f i r s t feature of meiosis in A. bisexualis, the following morphological features have been observed: (1) Nuclei in antheridia and oogonia prior to contact are generally larger (see Appendix, Table XVI -Nuclear Morphology and Distribution) than those observed in association iwth fe r t i l i za t ion tubes or in oospheres formed by cleavage of the oogonial protoplasm (Figs. 312-313, 354-356, 378, 385-386). . (2) Nuclei in various stages of division are extremely common in the antheridial and oogonial protoplasm subsequent to contact and prior to fe r i l i za t ion (Figs. 357-376). (3) Frequently two nuclei which are in the same stage of division are closely associated with each other (Figs. 375-376). - 102 -(4) It appears that there is an overall increase in number of nuclei in antheridia which is more than double. The cumulative effect .of these observations is suggestive of two successive nuclear divisions with a corresponding reduction in nuclear s i ze , indicative of meiosis. This assumption is in accord with evidence showing a reduction in DNA content from 4C to 1C in nuclei of respective developmental stages in the Oomycete, Sapvolegnia terrestris (Bryant and Howard, 1969). Also, i t is compatible with the observations of Sansome (1961, 1963, 1965) and Sansome and Harris (1962) for other Oomycetes. The single axial elements, as observed in only the oogonial and antheridial nuclei of A. bisexualis, morphologically correspond to those observed in meiotically dividing nuclei of other organisms (Lu, 1967; Moses, 1968). They each appear as relat ively straight filaments which usually are in association with the inner nuclear membrane (Figs. 358-359). . Also, microf ibr i ls appear to extend perpendicularly from the axis of the filaments (Figs. 358-359). The combination of these characteristics consistently is.associated with only meiotically dividing nuclei (Moses, 1968). Lu (1967) and Moses (1968) both equate the single axial elements to chromosome axes which then pair to form synaptinemal complexes in only meiotic nuclei . Indications of this pairing have been observed in this study (Fig. 364), as well as partially-formed synaptinemal complexes (undocumented by micrographs). Thus, these features ( i . e . - s ing le axial elements and indications of pairing of these elements to form synaptinemal complexes) further indicate that meiosis is gametic. A further feature supporting this conclusion (e.g.. - meiosis is - 103 -gametic) is a difference between the nuclei observed in the oogonia and antheridia (i.e. meiotic) and those other developmental, stages (mitotic). , The centrioles in association with the oogonial and antheridial nuclear envelopes during division'are frequently sheathed by E.R. (Fig.. 365). This may be an indicator of meiosis in this .organism. A lack of nucleolar protein synthesis has been noted to occur during nuclear division (see Busch and Smetana, 1970). This study observes an apparent disappearance of the pars granulosa during meiosis (Figs. 360-362). It may tentatively be concluded then that the pars granulosa is in some way responsible for nucleolar protein synthesis. Thus, the morphological differences of the nucleolus appear to ref lect the functional state. - 104 -CONCLUSION The ;.fine structure of the developmental stages of A. bisexualis is that of most Oomycetes. Cellular growth and apposition wall formation occurs by the vesicle-plasma membrane fusion mechanism as hypothesized by Grove, Bracker and Morre 0970). " Septation is mediated by the vesicle-plasma membrane fusion mechanism, as functions for cel lu lar growth and apposition wall formation. Zoosporogenesis in.A. bisexualis is similar to that previously described for other Oomycetes; Zoosporangia are delimited from subtending vegetative hyphal protoplasm septation; papil la development occurs; basal body and f lagel la are formed; and, cleavage of protoplasm occurs. Parastrasomes are single membrane-limited inclusions of complex morphology in zoosporangia and zoospores which probably serve as a source of microtubule constituents in rootlet formation. Sexual reproduction is effected by the same basic processes as defined by Raper (1960): in i t ia t ion of anthenidial branches; development of oogonial i n i t i a l s on female thai!us; growth of antheridial filaments to oogonial i n i t i a l s and delimitation of these reproductive structures by septa; cleavage of oogonial protoplasm to form oosphere; and, formation of fe r t i l i za t ion tubes of oogonia and oosphere wal ls , respectively, eventually effecting transfer of male - 105 -nuclei to oospheres. 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Zeigler, A.W. 1953. Meiosis in the Saprolegniaceae. Am. J. Botany 40: 60-66. • APPENDIX - 116 -TABLE I MATERIALS FOR LIGHT AND ELECTRON MICROSCOPY PROCEDURES Section A: Sodium Cacodylate Buffer (Sodium Dimethyl Arsenate) 2.14 g sodium cacodylate 100 ml d i s t i l l e d water Adjust pH to 7.2 with hydrochloric acid Section B: Glutaraldehyde and Osmium Tetroxide Fixative Solution A: 1 cc 25% glutaraldehyde (stock .solution) 5 cc sodium cacodylate buffer (pH 7.2) Solution B: 2% osmium tetroxide Mix solution A in equal parts with solution B. Allow to stand 15-20 minutes unti l mixture turns purple. Section C: Maraglas (Bisalputra and Weier, 1963) 18.3 g maraglas 7.2 g cardolite .8 g DMP Mix wel l . This makes approximately. 25 ml. Section D: . Uranyl Acetate Saturated.'solution in 70% methanol. Section E. Lead Citrate (Reynolds, 1963) 1.33 g lead nitrate 1.76 g sodium citrate 30 ml d i s t i l l e d water 1. Place above in .50 ml, volumetric f lask. 2. Shake vigorously for one minute. 3. Allow suspension to stand with intermittent shaking for 30 minutes to insure complete conversion to lead c i t ra te . ' -4. Add 8 ml IN sodium hydroxide which should be as car-bonate free as possible. 5. Dilute suspension to 50 ml with d i s t i l l e d water. 6. Mix by inversion. -Note: Solution is ready for use. The pH.should be approximately 12 and the solution should be clear. - 117 -Section F: Formvar .3 g formvar 100 ml ethylene dichloride • Shake to dissolve and allow to stand for 30 minutes be-fore use. Section G: Nile Blue (Jensen, .1962) - modified Nile Blue (1%): 0.5 g Nile Blue (cc) 50 ml d i s t i l l e d water Section H: 1 3. 4, Stain l iv ing material in 1% Nile Blue for 10 minutes (cc) at 37°C. Differentiate in 1% acetic acid at 37°C for 30 seconds. Washiin d i s t i l l e d water. Mount in d i s t i l l e d water on glass s l ide . Results: Lipids fluoresce golden yellow under u l t ra -violet i l lumination. Fats, o i l s , and waxes (neutral l ip ids) stain red as.observed under br ight - f ie ld i l lumina-t ion; free fatty.acids and phospholipids (acidic l ip ids) stain blue. However, the blue dye binds at. other sites in the cyto-plasm as i t also is a basic dye. Sudan IV (Jensen, 1962) Saturate 70% ethyl alcohol with Sudan IV. F i l t e r . 1. Place l iv ing material in 50% ethyl alcohol for 2-3 minutes. 2. Stain material for 20 minutes in saturated Sudan IV solution. 3. Differentiate in 50% ethyl alcohol for 1 minute. 4. Mount in d i s t i l l e d water on glass s l ide . Results: Fats, o i l s , and waxes stain red; fatty acids are unstained. Section I:. Flemming Triple Stain (Conn et al. , 1960) Safranin (1%): 0.5 g safranin 0 (cc) 50 ml d i s t i l l e d water Crystal Violet (1%): 1 gm crystal v iolet (cc) 20 ml 95% ethanol 80 ml d i s t i l l e d water - 118 -Dissolve the crystal violet in the ethanol. Add the d i s t i l l e d water and mix. Orange G (.25%): 0.13 g orange G (cc) 50 ml clove o i l 1. Stain preserved material 40 minutes in 1% aqueous safranin 0 (cc). 2. Rinse in d i s t i l l e d water. 3. Stain material 20 minutes in 1% aqueous crystal v iolet (cc). 4. Rinse in d i s t i l l e d water. 5. Dehydrate in graded series of ethanol. 6. Differentiate about 15 minutes in 0.25% orange G (cc) in clove o i l . Results: Chromosomes - red; Nucleoli - red; Metabolic Chromatin- deep purple; Cytoplasm - orange. Section J : Toluidine Blue with Borax (Conn e t al. , 1960) 0.1 g toluidine blue 0 (cc) • 0. 1 gm borax 100 ml d i s t i l l e d water 1. Stain preserved material 1-2 minutes in 0.1% t o l u i -dine blue 0 (cc) with borax. 2. Rinse with d is t i l l ed , water. Results: Certain acidic carbohydrates - metachromatic pink to red or v io le t ; . Nuclei and cytoplasm -orthochromatic blue. V - 119 -TABLE II FIXATION, DEHYDRATION AND EMBEDDING PROCEDURES FOR ELECTRON MICROSCOPY Part A: Vegetative Hyphae, Anteridia, Oogonia and Gemmae Step 1. Fix material one hour in glutaraldehyde and osmium tetroxide f ixat ive . (See Appendix, Table I, Section B.) Step 2. Wash material 3-4 times, 10 minutes per wash, in 0.1M sodium cacodylate buffer at pH 7.2. Step 3. Dehydrate: 20. minutes in 30% ethanol 20 minutes in 50% ethanol 20 minutes in 70% ethanol 20 minutes in 90% ethanol 20 minutes in 100% ethanol (2-3 changes) 20 minutes in 1:1 mixture of 100% ethanol and.propylene oxide Step 4. Add 3-4 drops of propylene oxide every 10-15 minutes unti l propylene oxide concentration is 90-95% and the 100% ethanol concentration is 5-10%. Step 5. Change material to 100% propylene oxide for 10-15 minutes (2-3 changes). Step 6. Add 4-5 drops of maraglas every 10-15 minutes unti l maraglas concentration is 90-95% and propylene oxide concentration i s 5-10%. Step 7. Transfer material into 100% maraglas for 5-10 minutes (2-3 changes). Step 8. Embed.material in 100% maraglas. Polymerize mara-glas block or capsule at 65°C in vacuum oven for 18-24. hours. Part B. Primary, Secondary and. Germinating Spores Step 1. Concentrate spores by centrifugation. Step 2. Pour off the growth medium (d is t i l led water). Step 3. Add f ixat ive and allow to stand for one hour. Step 4. Reconcentrate spores by centrifugation, discard f ixat ive . - 120 -Step 5 . , Wash material 3-4 times with buffer, each time re-concentrating spores by centrifugation and d i s -carding buffer. Step 6. Add 1-2 drops of 4% water agar to keep spores con-centrated in a pel let . Step 7. Cut agar pellet into approximately 1 mm cubes. Step 8. Continue dehydration and embedding procedures as described in Part A, Steps 3-8. - 121 -TABLE III THIN-SECTION, LIGHT MICROSCOPY PROCEDURES Step 1: Section Maraglas embedded material in 0.5-1.0 y thickness using glass knives and a Porter Blum MT-1 microtome. Step 2.:'. With a platinum wire loop, pick up sections .from the boat of the glass knife and place sectionsson glass s l ide . Step 3: Allow slide to a i r dry. Then, gently heat sl ide over a l -cohol lamp to increase the adhesion of the sections to the s l ide . Step 4: Allow slide to cool and place 1-2 drops of ethylene dich-loride on sections for one minute. Step 5: Rinse slide well with d i s t i l l e d water. Step 6: Rinse slide with 50% acetone. Step 7: Reheat sl ide over alcohol lamp unti l dry. Step 8: Stain sections with desired stain or stains as directed in Table I, Sections G and H. - 122 -TABLE IV HYPHAL GROWTH RATES OF ACHLYA BISEXUALIS Male Strain (CBS - 102.62) Culture Diameter of Colony in mm 24.hrs. 48 hrs. 72 hrs. 96hrs . 1 7 17 26 34 2 6 17 26 35 3 7 16 27 35 4 7 17 26 34 5 7 17 26 38 6 5 14 24 35 7 6 16 27 36 8 5 15 25 35 9 7 17 27 38 10 6 . 16 27 37 Average growth rate 8.9 mm/24 ,hrs.. .37 mm/hr.~ Female Strain (CBS - 101.62) Culture Diameter of Colony in mm 24 hrs. 48 hrs. 72 hrs. 96 hrs. 1 4' 12 20 28 2 4' 12 23 30 3 6 13 22 29 4 5 12 23 29 5 5 15 23 30 6 6 15 22 '• 27 7 5 15 22 27 8 5 12 22 27 9 5 12 23 28 10 4 12 23 26 Average growth rate 7.1 mm/24 hrs. .29 mm/hr. Note: Conditions under which organism was growing are described in Materials and Methods. - 123 -LEGEND FOR TABLES V - XVI •= absent = as determined from published micrographs of' other investigators. NP = not published; however, cited by other inves-tigators or based on personal communication. NS = not distinguishable in published micrographs and not described by respective author or authors. TABLE V WALL MORPHOLOGY AND COMPOSITION Cell Type Organism Thickness Morphological Description Chemical Composition Method Reference Vegetative Hyphae Aahlya sp. Aahlya bisexualis ApJianomyces euteiohes Peronosvora manshuriaa Phytophthora , oinnamoni Apical growing zone - 30-70 Sub-apical zone - 60-100 my Distal mu l t i -organelle zone - 60-200 my 40-250 my* 0.15-0.4 Appears, as i rregular layer of e lectron- l ight amorphous material in younger hyphae and as a regularly uniform layer in older hyphal regions; two layers distinguishable near septa; composed of an amor-phous matrix in which f i b r i l s are inter-twined; pockets of vesicles and dense granular matter sometimes v i s ib le with -in wal 1. Electron-transparent zone that was generally amorphous - with evidence of laminar structure in older hyphal areas. Thick wall which exhibits f ine ly granular dense layer (20-40 my thick) on i t s external surface. Cellulose Cellulose Cellulose I X-ray IKI-H 2S0 4 Thin-section electron microscopy (glutar-aldehyde-osmium tetroxide f ixat ion) Freeze-etch Thin-section electron microscopy (glutar-aldehyde f i xat ion with osmium tetroxide post-f ixation Thin section-electron microscopy (osmium tetroxide f i x a t i o n ; KMn0» f i xa t ion ; gluteraldehyde f i x -ation with osmium tetroxide pos t - f i x -ation) , Chemical analysis - of isolated walls Frey (1950 -see Aronson, 1965) Cooper and Aronson, 1967 Ricker (this report) Shat la, Yang and M i t c h e l l , 1966 Peyton and Bowen, 1963 Bar tn ick i -Garcia, 1966 TABLE V continued Cell Type Organism Thickness WALL MORPHOLOGY AND COMPOSITION Chemical Morphological Description Composition Method Reference Vegetative Phytophthora Hyphae infestans continued Phytophthora parasitica 90-100 niji* 100-200 m»* Pythium debaryanum 80-200 rati 0 .1 -0 .3 Consists of single amorphic zone. Consists of single amorphic zone. Represented by narrow electron transparent zone Composed of amorphous matrix (mostly glucans) in which cel lulose micor f ib r i l s em-bedded Consists of two phases - (1) M i c r o f i b r i l l a r phase of which there are two layers -an outer layer of randomly oriented f i b r i l s and an inner layer of microf ibr i l s or ient -ed paral le l to hyphal ax is ; (2) Amorphous phase which has branching system of pores. . Layers of dif ferent electron density apparent in thin sections of iso lated, sonic-treated, f u l l y extracted wal ls . Cellulose I M i c r o f i b r i l l a r cel lulose and other polysac-charides, pro-tein and inor-ganic material Presence of ce l lu -lose not con-firmed. Thin-section electron microscopy (KMnO. f i xa t ion ; osmium tetroxide f ixat ion with KMnO. post-f ixat ion) Thin-section electron microscopy (KMnO. f i x a t i o n ; osmium tetroxide f ixat ion with KMnO. post-f ixat ion) Chemical analysis of isolated wal ls . Thin-section electron microscopy (Luft 's permanganate f ixation) IKI-H„S0 4 test ; X- • ray d i f f rac t ion ; Electron microscopy transmission) on chemically isolated walls X-ray d i f f r a c t i o n ; thin-section e lec -tron microscopy (KMnO. f i xa t ion ; glutaraldehyde1 f ixat ion with -KMnO, post - f i xa -tion) Ehrl ich and Ehr l i ch , 1965, 1966 Eisner, Horton and Bowen, 1967 Ehrl ich and Ehr l i ch , 1966 Bar tn ick i -Garcia, 1966 Hawker and Abbott, 1963 Cooper and Aronson, 1967 Manocha and Colv in , 1968 Cell Type Vegetative Hyphae continued Organism Thickness Pythium ultimum 30-200 my Saprolegnia ferax . 50 my Different ia - Achlya bisexualis 100-200 my ting Vege-tative Hyphae Aphanomyaes 40-250 my .euteiahes Gemmae Aahlya bisexualis 500-950 my (thinner near apex) TABLE V continued WALL MORPHOLOGY AND COMPOSITION Morphological Description Chemical Composition Method Reference Comprised of two-phase system in which network of f i b r i l s is . embedded in amorphous matrix; irregular, or lumpy prof i le at hyphal apex and along l a t -eral walls where vesicles in close association Homogeneous layer Usually appears as a re la -t ive ly uni form layer of amor-phous material in which f i -b r i l l a r - l i k e elements v i -s i b l e ; two layers d i s -cerned near septa. Electron-transparent zone that was generally amorphous with evidence of laminar structure in older hyphal areas. Formed basically of two layers - an outer layer which is continuation of outer hyphal wal l , . an inner layer which encompasses protoplasm; Outer layer comprises one-fourth total wall thickness; s t r iat ion v is ib le in inner ; layer; appears to be com-posed of amorphous and f i b r i l -lar material. Thin-section electron microscopy (glutar-aldehyde f i xa t ion ; gluteraldehyde-acro-le in f i xa t ion ; os-mium tetroxide f i x -at ion ; KMn0d f i x a -tion) Thin-section electron microscopy (osmium tetroxide f ixation) Thin-section electron microscopy (glutar-aldehyde-osmium tetroxide f ixation) McConnel1, Grove and Bracker, 1968 Grove, Bracker and Morre, 1970 Gay and Greenwood, 1966 Ricker (this report) Thin-section electron Shatla, Yang microscopy (glutar- • and Mitchell aldehyde f ixat ion with 1966 osmium tetroxide.post-f ixat ion) Thin-section electron microscopy (glutsr-aldehyde-osmium tetroxide f ixation) Ricker (this report) TABLE V c o n t i n u e d WALL MORPHOLOGY AND COMPOSITION C e l l Type O r g a n i s m T h i c k n e s s M o r p h o l o g i c a l D e s c r i p t i o n C h e m i c a l C o m p o s i t i o n . Method R e f e r e n c e S p o r a n g i a Achlya bisexualis Aphanomyces euteiches Phytophthora capsiai Phytophthora erythroseptica 2 5 0 - 3 0 0 mp e x -c e p t a b o u t a p i c a l p a p i l -l a where i t i s 5 0 - 1 0 0 my 4 0 - 2 5 0 mp NS a p i c a l p a p i l l a - 4 . 5 . x 10 p NS O u t e r 1 a y e r -2 0 0 - 3 0 0 mp e x c e p t i n o l d s p o r a n g i a , t h e n 1 5 0 - 2 0 0 . m t h i c k . I n n e r l a y e r v a r i a -b l e i n t h i c k -n e s s . Formed b a s i c a l l y o f two l a y e r s - an o u t e r l a y e r w h i c h i s c o n t i n u a t i o n o f o u t e r hypha l w a l l , an i n n e r l a y e r w h i c h encompasses p r o t o p l a s m . B o t h l a y e r s a r e f i n e l y s t r i a t e . P o c k e t s o f e l e c t r o n dense g r a n u l a r and v e s i c u l a r m a t -t e r sometimes v i s i b l e b e -tween t h e two l a y e r s . E l e c t r o n - t r a n s p a r e n t zone t h a t was g e n e r a l l y amorphous - w i t h e v i d e n c e of. l a m i n a r s t r u c t u r e i n o l d e r hypha l a r e a s . Composed o f two l a y e r s . - o u t e r l a y e r s u r r o u n d s p a p i l l a ; i n n e r ' l a y e r nar rows and d i s -a p p e a r s near c r o w n ; p a p i l l a a p p e a r as f i b r o u s t h i r d l a y e r . C o n s i s t s o f o u t e r homogenous e l e c t r o n - t r a n s p a r e n t l a y e r and h e t e r o g e n e o u s v e s i c u l a r , l a y e r , p r o m i n e n t i n a p i c a l r e g i o n . I n n e r homogeneous l a y e r formed i n i n d i r e c t l y g e r m i n a t i n g s p o r a n g i a . C o n s i s t s o f o u t e r homogeneous l a y e r and i n n e r v e s i c u l a r l a y e r e x c e p t i n o l d s p o r a n g i a w h i c h c a n n o t g e r m i n a t e i n d i r e c t l y -t h e n no i n n e r v e s i c u l a r l a y e r p r e s e n t . An i n n e r homogeneous l a y e r formed i n i n d i r e c t l y g e r m i n a t i n g s p o r a n g i a . . T h i n - s e c t i o n e l e c t r o n m i c r o s c o p y ( g l u t e r -a l d e h y d e - o s m i u m t e t r o x i d e f i x a t i o n ) R i c k e r ( t h i s r e p o r t ) . T h i n - s e c t i o n e l e c t r o n m i c r o s c o p y ( g l u t a r -a l d e h y d e f i x a t i o n w i t h osmium t e t r o x i d e p o s t - f i x a t i o n ) . S h a t l a , Yang and M i t c h e l l , 1965 T h i n - s e c t i o n e l e c t r o n . W i l l i a m s and m i c r o s c o p y (KMnO, f i x a t i o n ; g l u t a r a l -dehyde f i x a t i o n w i t h osmium t e t r o x i d e p o s t -f i x a t i o n ) T h i n - s e c t i o n e l e c t r o n m i c r o s c o p y T h i n - s e c t i o n e l e c t r o n m i c r o s c o p y (osmium t e t r o x i d e f i x a t i o n ; KMnO^ f i x a t i o n ) W e b s t e r , 1970 V u j i c i c , Chapman and C a l h o u n , 1965 Chapman and V u j i c i c , 1965 TABLE V continued Cell Type Organism Thickness WALL MORPHOLOGY AND COMPOSITION Morphological Description Chemical Composition Sporangia • continued Phytophthora parasitica Saprolegnia ferax Outer layer 0.2 y Inner layer 1.5 p Outer layer 0.2 y Inner layer 1.5 y 30-45 mp* Consists of two layers - outer layer appears finely granular-, and continuous over t ip of sporangium; inner layer of homogeneous matrix with faint longitudinal st r iat ion - ex-tends to apical plug where i t tapers off to form neck of plug. Plug composed of f i -brous material running appro-ximately at right angles to sporangial axis. Consists of two layers as des-cribed by Hohl and Hamamoto (1967). A new inner layer de-posited on existing wall which extends within germ tube; vesicular aggregates encased within new wall layer. NS Asexual Spores Achlya bisexualis 40-55 mp Appears as homogeneous layer of electron- l ight amorphous material about each primary zoospore protoplast. F la -gellated secondary zoospores lack wal1. Method Reference Thin-section electron, microscopy (osmium tetroxide f i x a t i o n ; KMnO. f i x a t i o n ; g lu -taraldehyde f ixat ion with osmium tetro-xide or KMnO, post-f i xa t ion . Hohl and Hamamoto, 1967 Thin-section electron microscopy (glutar- . aldehyde f ixat ion with osmium tetroxide or KMnO* post - f i xa -tion) Hemmes and Hohl , 1969 Thin-section electron-microscopy (KMnO» f i xa t ion ; g lu tara l -dehyde f ixat ion with osmium tetroxide post-f ixat ion) Thin-section electron microscopy (gluter-aldehyde-osmium tetroxide f ixat ion) Gay and Greenwood, 1966 Ricker (this report) ro co. Cell Type .Organism Thickness TABLE V continued WALL MORPHOLOGY AND COMPOSITION Morphological Description Chemical Composition. Method. Reference Antheridia Achlya bisexualis Pythium ultimum. Oogonia Achlya bisexualis 40-90 mp thick , except in forms which penetrate oogonia during f e r t i l i z a t i o n - then 15-30 my thick NS Usually appears as single layer of f inely striated material except about growing t ip where i t is thin and irregular in pro-f i l e ; two layers are d i s -tinguishable near septa; pockets of dense granular a and vesicular material present'in wal ls , pa r t i -cularly in region in con-tact with oogonia. Thin wal1 Oogonia- 60-80 mp thick in early stage of . d i f ferent ia -t ion , increa-sing to 1.5-2.0 p in thick-ness at time of oosphere for -mation. Oospheres -130-180 mp thick Oogonial wall formed of two f inely striated layers; outer layer a continuation of outer hyphal wall layer, inner layer surrounds proto-plasm; in young oogonia wall layers usually d i f f i c u l t to distinguish from one another except near septa; outer wall layer increases in elec-tron density as oogonial de-velopment occurs and i s . easily recognizable in oogonia at time of f e r t i l i z a t i o n . Dense granular and f i b r i l l a r material appears to slough from i t s surface. Thin-section electron microscopy (g lutar - . aldehyde-osmium tetroxide f ixation) Thin-section electron microscopy (KMnO, f i x a t i o n ; g lu tara l -dehyde f ixat ion with osmium tetroxide post-f i x a t i o n ; acrolein f ixat ion with osmium tetroxide post - f i xa -tion) Thin-section electron microscopy (glutflr-aldehyde-osmium tetroxide f ixation) Ricker (this report) Marchant, 1968 Ricker (this report) Cell Type. . Organism. Thickness Oogonia Achlya bisexualis continued continued Pythium ultimum • NS Oospore Achlya bisexualis 0.7-1.1 p thick TABLE V continued WALL MORPHOLOGY AND COMPOSITION Chemical , Morphological Description Composition Method Reference Oosphere wall formed of a single layer of electron-l ight amorphous material: dense f i b r i l l a r and granular network often d i s t i n -guishable on outer surface of wal1 materia]. Relatively thin about young unfert i l ized protoplasm; becomes thicker after fe r -t i l i z a t i o n . Loose and poorly-organized plug of material forms septum separating oogonial proto-plasm from hyphal proto-plasm. Wall s l ight ly undulate in prof i le and formed of 3-4 layers; innermost layer is thickest and is f inely s t r ia te ; dense, f inely granular layer is next to innermost layer; outer layer.or layers is (are) same as oosphere wa l l ; pockets of electron-dense vesicular and gra-nular matter often present between the outer layers Thin-section electron microscopy (KMnO, f i xa t ion ; g lu tara l -dehyde f ixat ion with osmium tetroxide post - f i xat ion ; acrolein, f ixat ion with osmium tetroxide post-f ixation) Thin-section electron microscopy (glutar-aldehyde-osmium tetroxide f ixat ion) Marchant, 1968 ' Ricker (this report) co o Cell Type Organism Thickness TABLE V continued WALL MORPHOLOGY AND COMPOSITION Morphological Description Chemical .Composition,. Method Reference Oospore continued Pythium ultimum NS Thickened wall with i r r e -gular outer surface. Saprolegnia terrestris NS Wrinkled wall becomes smoother. Thin-section electron Marchant, microscopy (KMnO- 1968 f i xa t ion ; g lu tara l -dehyde f ixat ion with osmium tetroxide post-f i x a t i o n ; acrolein f ixat ion with osmium tetroxide pos t - f i xa -tion) Thin-section electron Moore and microscopy (KMnO, Howard, f ixat ion) 1968 TABLE VI LOMASOME MORPHOLOGY AND DISTRIBUTION Cell Type .Organism Si ze Shape Distribution. Morphological Description. .Reference Vegetative Aahlya bisexualis variable Hyphae Albugo aandida NS Aphanomyoes euteiohes Peronospora manshuriaa NS NS irregularly-shaped pockets in plasma membrane irregularly-shaped pockets in the plasma membrane irregularly-shaped pockets in plasma membrane hemispherical-shaped* pockets in the plas-ma membrane - some-times irregular in prof i le common in apical growing, zone, sub-apical .mito-chondrial zone, and younger d is ta l mu l t i -organelle zone. . seldom observed in older vacuolated area. random NS random Aggregates of i r r e -gularly-shaped ve-s ic les located in . pockets between the plasma membrane and hyphal wa l l . Glutaraldehyde-osmium tetroxide f i xa t ion . a system o f unit-mem-brane tubules and ves ic les , apparently elaborations of the plasma membrane. KMnO. f i x a t i o n ; glu-taraldehyde f ixat ion with osmium tetroxide post - f ixat ion. Lomasomes - as des-cribed by Moore and McAlear (1961). GIutara1dehyde-fix-ation with osmium tetroxide post - f ixat ion. Ricker (this report) Berl in and . Bowen, 1964 Shatla, Yang Mi tche l l , 1966 A system of unit mem-brane tubules and vesicles which appear to be elaborations of the plasma membrane. Osmium tetroxide f i x a -t ion ; KMnO. f i xa t ion ; glutaraldehyde f i x a -tion with osmium tetroxide post - f i xa -t ion . Peyton and Bowen, 1963 . TABLE VI continued LOMASOME MORPHOLOGY AND DISTRIBUTION Cell Type Organism Size Shape Distr ibut ion. Vegetative Phytophthora some mas-. irregularly-shaped* Hyphae infestans sive pockets in plasma-continued membrane • frequent Phytophthora some mas- irregularly-shaped* parasitica sive pockets in plasma' membfane frequent NS irregular-shaped pockets of ves i -cles and tubules NS Pythium debar-yanum NS irregularly-shaped* pockets in plasma' membrane in young vegetative NS NS NS / .Morphological Description Reference Invaginations of the . plasma .membrane KMnO. f i x a t i o n ; os-jnium tetroxide f i x a -. tion with KMnO» post - f ixat ion. Lomasomes. ,KMnO^ f i x a t i o n ; os . mium tetroxide f i x a -tion with KMnO. post - f ixat ion. Composed of tubular and yesicular e le -ments which are less orderly in appear-ance in pockets of the plasmalemma. Glutaraldehyde f i x a -tion with osmium tetroxide post-f ixat ion . Ehrl ich and Ehrl i c h , 1965,1966 Ehrlich and Ehr l ich , . 1966 Hemmes and Hohl, 1969 Lomasomes; contain amorphous matter and are s imilar to those shown by Moore and McAlear (1961). Luft 's permanganate f i xa t ion . Hawker and Abbott, 1963 Network of f ine tu - Hendy, bules; bounded on 1966 inner side by mem-brane continuous with plasma membrane and on outer side by c e l l wal l . Glutaraldehyde-fixation with osmium tetroxide post - f ixat ion. Cell Type Organism Size TABLE VI continued LOMASOME MORPHOLOGY AND DISTRIBUTION Shape Distr ibut ion. Vegetative Hyphae . continued Pythium debar-yanum con11 NS irregular-shaped* pockets in plasma membrane NS-Pythium ultimum irregular irregular regularly produced in abundance both at t ip and in older hypha; regions NS irregularly-shaped in hyphal t ips pockets in plasma membrane Di f ferent ia - Ashlya bisexualis variable irregularly-shaped occasionally observed ting Vege- pockets in plasma along length of tative membrane hyphae Hyphae .Morphological Description Reference Groups of tubules or Manocha and . vesicles between Colyin, the plasma membrane 1968 and wal1; may be produced in cyto-plasm, then become enclosed in enve-lope and migrate to surface of eel 1. KMnO, f i xa t ion ; glu-taraldehyde f ixat ion with KMnO. post - f i xa -t ion . Plasmalemma elabora- Marchant, ations; tubular ap- Peat and pearance; granular 1967 electron-opaque contents. Acrolein f ixat ion with osmium tetroxide post - f ixat ion. Lomasome-like conf i - Grove, gurations; not a Bracker and consistent feature . Morre', 1970 of hyphal t ips . Aggregate of i r r e - Ricker (this gularly-shaped ves i - report) cles located in pock-ets between the plasma membrane and hyphal wa l l . \ Glutaraldehyde-osmium' tetroxide f i xa t ion . TABLE VI continued LOMASOME MORPHOLOGY AND DISTRIBUTION • Morphological Cell Type ...Organism . Size Shape Distribution . . Description . . . . Reference Differen- . • Aplianomyaes . NS ' : irregularly-shaped* FTS \ : ~ ~ '. ' Lomasomes - as des- Shatla,. t ia t ing euteiahes pockets in plasma cribed by Woore Yang and Vegeta- membrane and McAlear (1961) M i t c h e l l , t ive Glutaraldehyde f i x a - 1966 Hyphae • tion with osmium cont tetroxide post-f i xa t ion . Gemmae . Aahlya bisexualis - - - - Ricker (this report) Sporangia Aahlya bisexualis - - - ' - Ricker (this report) Aphanomyoes NS irregularly-shaped* NS Lomasomes - as des- Shatla, euteiahes pockets in plasma cribed by Moore ' Yang and membrane and McAlear (1961) M i t c h e l l , Glutaraldehyde f i x a - • 1966 tion with osmium tetroxide post-f i xa t ion . Phytophthora NS irregularly-shaped* Type 1 - at base of ' Two types of loma- Hemmes and parasitica pockets in plasma apical plug where germ somes - (1) aggre Hohl, membrane tube w i l l penetrate gates of small ves- . 1969 Type 2 - in sporangia teles or tubules of uniform diam. (approx. 300A) in pocket of plasmalemma; (2) tubular and ves i -cular elements which are larger and less orderly in appear-ance, in pockets of the plasmalemma.-Glutaraldehyde f i x a -tion with osmium tetroxide post-f i xa t ion . Cell Type Organism Sporangia continued Phytophthora parasitica Size NS Asexual Spores Achlya bisexualis variable Antheridia Achlya bisexualis variable Pythium ultimum NS TABLE VI continued LOMASOME MORPHOLOGY AND DISTRIBUTION Shape . Distr ibution irregularly-shaped*. . NS pockets in plasma" membrane Morphological Description Reference. Numerous lomasomes . Hohl and Osmium tetroxide Hamamoto, f i x a t i o n ; , KMnO. _ 1967 f i xa t ion ; glutar-aldehyde f ixat ion with osmium tetro-xide post - f i xat ion ; glutaraldehyde f i x a -tion with KMnO, post-f i xa t ion . irregularly-shaped pockets in the plas-ma membrane random Present in only ency-sted primary spores; are aggregates of vesicles in irregu-larly-shaped, pockets between the plasma mem-brane and spore wa l l . Glutaraldehyde-osmium tetroxide f i xa t ion . Ricker (this report) irregularly-shaped pockets in plasma membrane few associated with grow-ing t i p ; sometimes in antheridial area which has penetrated oogonium Aggregates of vesicles in irregularly-shaped pockets between the plasma membrane and wal l . Glutaraldehyde-osmium tetroxide f ixat ion Ricker (this report) co cn irregularly-shaped at tips which have Elaborations of the Marchant, pockets of tubular- penetrated oogonia plasmalemma 1968 . l ike elements KMnO* f i x a t i o n ; g lu -taraldehyde f ixat ion with osmium tetroxide post - f i xat ion ; acro-le in f ixat ion with osmium tetroxide post - f i xat ion . TABLE y i continued LOMASOME MORPHOLOGY AND DISTRIBUTION Cell Type Organism Size Shape Distr ibution Morphological Description Reference Oogonia Aahlya bisexualis seldom greater irregularly-shaped than 0.3 JJ pockets in plasma membrane random in young, pre-cleayed oogonia Aggregates of sev-eral yesicles in pockets between the . plasma membrane and wa l l . GIutaraldehyde-osmi um tetroxide f i xa t ion . Ricker (this report) Oospores Aahlya bisexualis Not observed in sections examined. Ricker (this report) TABLE y i l Cell Type MITOCHONDRIAL .MORPHOLOGY AND DISTRIBUTION Organism; Size Shape. . Distr ibution .'. Morphological .Description '. . . . Reference Vegetative '. Hyphae Achlya bisexualis 0.3-0.6 ja in diameter 1.5-6.0 ji . in length rod-shaped lacking in apical grow-ing, zone; scattered throughout central hyphal protoplasm in sub-apical zone; be-come concentrated in peripheral hyphal pro-toplasm in distal multi -organelle zone as vacuolation occurs) Rod-shaped mitochon- Ricker (this dria with tubular report)' . . c r i s tae ; matrix dense except in older . hyphal regions where less dense areas are common in mitochon-d r i a ; cr istae seldom overlap each other and f i b r i l s usually v i s ib le in their lumens. Mitochondria often ap-pear fragmented in older vacuolated hyphal proto-plasm. Glutaraldehyde-osmium tetroxide f i xa t ion . Aphanomyces 1 euteiches NP rod to .probably cup-shaped* Phytophthora infestans NP NP mostly in the peripher-al protoplasm* MP Mitochondria bound by a double membrane; the inner one of which was invaginated to form markedly swol-len c r is tae . Mito-chondria with tubular cr istae which gener-al ly do not appear to overlap; cr istae abundant. Glutaraldehyde f ixat ion with osmium tetroxide f i c a t i o n . Mitochondria typical in structure and have tubular c r i s -tae. Also present in the hyaloplasm of the haustoria. KMnO. f i x a t i o n ; os-mium tetroxide f i x a -tion and KMnO^  post-f i xa t ion . • Shatla, Yang and Mi tche l l , 1966 Ehrlich and Ehr l ich , 1965 / Cell Type Organism. Size.. TABLE Y U - continued MITOCHONDRIAL MORPHOLOGY AND DISTRIBUTION Shape . . . . Distr ibution. , Morphological Description Reference yegetatiye Hyphae continued Phytophthora parasitica NP elongate located mainly at peri*-phery of the protoplasm Pythium debar-yanum NP oyoid with a regular out-l ine mostly in the peripheral protoplasm* Pythium ultimum 0.5-0.7 y in* diameter ex-cept in older zone of. vacuo-la t ion ; up to 5 y in length ovoid to rod-shaped* usually lacking in apical zone; abundant in sub-apical zone, present in zone of vacuolation Numerous raitochon- Hemmes and dria behind ves i - Hohl, 1959 • cular zone at hyphal t i p ; their ' ' .' long axis oriented paral le l to the axis of the hypha. Glutaraldehyde f i x a -tion and KMnO, post- • f i xa t ion . Mitochondria are sur- Hawker and rounded by a mem- Abbott, brane appearing as 1963 two dark layers en-closing a wider 1ight one. The inner dark layer projects into the inter ior to form more or less tubular, irregularly-shaped cr is tae . Outer mem-brane may appear con-tinuous with ER and apparently gives r ise to small vesic les , matrix resembles en-doplasm. Luft 's permanganate f i xa t ion . Mostly rod-shaped mi to- Grove, Brack-^ chondria with tubular ' er and Morre, c r i s tae ; cr istae 1970 abundant; matrix dense except in mito-chondria in older zone of vacuolation. Glutaraldehyde f i x a -t i o n ; glutsraldehyde-acrolein f i x a t i o n ; osmium tetroxide f i x a -t i o n ; or KMnO^  f i x a -t ion . co Cell Type Organism. Size TABLE VII - continued MITOCHONDRIAL MORPHOLOGY AND DISTRIBUTION Shape . . . Distribution Morphological .Description Reference Vegetative Hyphae continued Pythium ultimum continued 0.5 -0 .6 ji in 'diameter* length un-determinable ,NP jnostly in the peripheral cytoplasm* Mitochondria with tubular cristae*. Acrolein f i xa t ion . Marchant, Peat and Banbury, 1967 Saprolegnia ferax 0.5 y. in d i a -meter; up to . 10 JJ in length cyl indr ical aligned with the axis of the hyphae Mic rov iH i of mito-chondria extend to the centres of the p ro f i l es . Osmium tetroxide f i x a -t ion ; KMnO. f i x a t i o n ; glutaraldehyde f i x a -tion and osmium tetroxide post - f i xa -t ion . Gay and Green-wood, 1966 NP NP NP Mitochondria with tu-* bular cr istae and dense matrix. Glutaraldehyde f i x a -tion with osmium tetroxide post-f i xa t ion . Heath and Greenwood, 1968 4*. O Saprolegnia NP spherical terrestris Oifferen- Achlya bisexualis 0 .3 -0 .6 y. in rod-shaped t ia t ing diameter Vegetative 3 .5 -6 .0 M Hyphae in length . NP in peripheral hyphal protoplasm Mitochondria with bleb-1 ike cr is tae. KMnO^  f i xat ion . Moore and Howard, 1968 Rod-shaped mitochondria Ricker (this with tubular c r i s -tae; matrix dense; cr istae sometimes overlap each other; f i b r i l s usually v i s -ib le in the cr istae. GIuteraldehyde-osmi um tetroxide f i xat ion . report) l TABLE VII - continued Cell Type -. .Organism. Size MITOCHONDRIAL MORPHOLOGY AND DISTRIBUTION . . Shape . Distribution Morphological Description Reference. Differen- Aphanomyces t ia t ing euteiches Vegetative Hyphae continued • Gemmae Achlya bisexualis NP 0.3-0.6 JJ in diameter;. 0.6-2.4 j i in length rod to probably ' cup-shaped* NP ovoid, rod, dumb-bell and i r r e -gular forms random; sometimes in close association with l i p o -somes Mitochondria bound by a double .mem-brane; the inner one of which was invagi-nated to form marked-ly swollen c r i s tae . Glutaraldehyde f i x a -tion with osmium tetroxide post - f ixat ion Mitochondria with tubu-lar cristae which of-ten overlap each other; f i b r i l s d i s -. cernable in c r i s tae ; constricted forms indi cative o f - rep l i cat ion . Glutaraldehyde-osmium tetroxide f i xa t ion . Shatla, Yang and M i t c h e l l , 1966 : .. Ricker (this report) Sporangia Achlya bisexualis 0.3-0.4 v in ovoid to rod-shaped; random prior to cleavage; Mitochondria with long Ricker (this' diameter; occasionally cup- oriented about nuclei tubular cristae which report) 0.8-3.0 ji in l ike forms observed during cleavage and prior overlap each other; length to zoospore release; in in female strain one peripheral protoplasm.of c r is ta often becomes f lagel lated secondary aligned along central zoospores axis of each mitochon-dr ion; i t is dialated and contains electron-l ight amorphous mater-ia l ; f i b r i l s v i s ib le in almost al1 the c r i s tae ; matrix dense. Glutaraldehyde-osmium tetroxide f i xa t ion . Cell Type Organism Size Sporangia Aphanomyces Np continued euteiches TABLE y i l - continued MITOCHONDRIAL MORPHOLOGY AND DISTRIBUTION Shape Distribution rod-shaped* NP Morphological Description Reference Mitochondria bound Shatla, Yang by a double mem- and M i t c h e l l , brane; the inner one . 1966 of which was invagi -nated to form marked-ly swollen cr is tae. GlutBraldehyde f ixat ion with osmium tetroxide post - f i xat ion . Phytophthora capsici Phytophthora eryihroseptica 0.25-0.3 n in* rod-shaped diamter; up to 1.3 ji in length uniformly distributed in cytoplasm of non-cleav-ing sporangia; accumu-late around nuclei, during cleavage 0.2-0.3 ji in* diameter; 0.3-1.3 ji in length various shapes scattered throughout* cytoplasm; abundant Mitochondria have an enclosing membrane, with the inner layer forming tubular c r i s -tae; cr istae stubby in non-cleaving spor-angia but become atten-uated as cleavage prog-resses; cr istae long and flexuous by time zoospores formed. KMnO, f ixat ion or g lu -taraldehyde f ixat ion with osmium tetroxide post - f ixat ion. The inner of the two 1imiting mitochon-dr ia l membranes i n -folded to form c r i s -tae which are short and stumpy in young • non-germinating spor-angia and elongate in young sporangia s t i -mulated to germinate d i rec t l y . Osmium tetroxide f i x a - . t ion or KMnO^  f i xa t ion . Williams and Webster, . 1970 Chapman and Vu j i c i c , 1965 / Cel l Type Organism Size TABLE VU - continued MITOCHONDRIAL MORPHOLOGY AND DISTRIBUTION Morphological Shape Distr ibution Description Reference Phytophthora erythroseptioa continued Phytophthora infestans Phythphthora parasitica NP 0.5-1.4 a* NP spherical to el-on-gate 0.4-0.6 u in* diameter; • up to 2.0 p in length mostly rod-shaped* NP mostly in electron-dense cort ical sleeve of hyaloplasm uniformly distributed* throughout cytoplasm prior to cleavage; be-come distributed about nuclei during cleavage Indirect germination . of sporangia results in the marked elonga-tion of mitochondrial cr is tae. y u j i c i c , • Chapman and Colhoun. 1965 Mitochondrial boundary Ehrlich and consists of an inner Ehr l ich , ..'" and outer electron- 1966 dense membrane sepa-rated by a narrow per i -mi tochondrial space; the outer membrane . sometimes not complete; cr istae are short bul -bous tubules which elon-gate somewhat as hyphae age; some cristae may form loops; some mito-chondria with rings. KMnO. f ixat ion or os-mium tetroxide f i xa t ion . Mitochondria with tu - Hohl and bular cr is tae. Hamamoto,. Osmium tetroxide f i x a - 1967 ' t i o n , KMnO. f ixat ion or glutaraldehyde.fixa-tion with osmium te t -roxide or KMnO. post-f i xa t ion . co Cell Type Organism Size TABLE VII - continued MITOCHONDRIAL MORPHOLOGY AND DISTRIBUTION Shape . . . D i s t r i b u t i o n . . . Morphological Description Reference Sporangia continued Pytophthora .parasitica continued Saprolegnia ferax 0.5-1.4 J J* in diameter spherical to.elon-gate jnostly in electrons-dense cort ical sleeve of hyaloplasm 0.6-0.8 p in* diameter; up to 1.6 p in length ovoid to rod-shaped* frequently accumulate at cytoplasmic periphery mostly 1.0-1.5 p ovoid to cyl indr ical in length; s e l -dom greater than 2.0 p in length mostly in cytoplasm* near nuclei . Mitochondrial boun- . Ehrlich and dary consists of Ehr l ich , an inner and outer 1966 electron-dense mem-brane separated by a narrow perimitochon-d r ia l space; the out-er membrane sometimes not complete; cr istae are short bulbous tubules which elongate somewhat as hyphae age; some cristae may form loops;some mitochondria with rings. KMnO, f ixat ion or os-mium tetroxide f i xa t ion . Outer membrane of the Hemmes and mitochondria often Hohl, touch plasmalemma but 1969 never fuse with i t . They may have con- . tained looped tubules. Glutaraldehyde f ixat ion with osmium tetroxide post - f ixat ion or KMnO^ , post - f ixat ion. M i c r o v i l l i of the Gay and mitochondria set Greenwood, close together and 1966 are rather straight and uniform in length; often do not extend to center of the mito-chondria. Some round-ing of mitochondria may occur. KMnO. f i x a t i o n , osmium tetroxide f ixat ion or glutaraldehyde f ixat ion with osmium tetroxide post - f ixat ion. / Cell Type Organism, Size TABLE VII - continued MITOCHONDRIAL .MORPHOLOGY AND DISTRIBUTION Shape Distr ibut ion. .. Morphological Description Reference" Asexual Spores Aahlya bisexualis Phytophthora capsiai. Phytophthora megasperma var. sojae 0.4-0;7j i in . 'diameter; 0.7-2.0 ji length 0.3-0.5 y* in diameter; up to 1.3 y in length 0.3-0.5 u in* diameter; up to 1.2 p in 1ength spherical , oyoid t o c y l i n d r i c random rod-shaped around nuclei spherical to oblong or i r -regular in shape abundant in peripheral* cytoplasm Mitochondria with long tubular c r i s -tae which overlap each other; one c r i s t a e , which is dialated and contains electron- l ight amor-. phous mater ial , may be aligned along cen-tral axis of some mito-chondria, of female s t ra in ; f i b r i l s are v i s ib le in almost a l l c r i s tae ; matrix dense. Glutaraldehyde-osmium tetroxide f i xa t ion . Mitochondria having an enclosing mem-brane with the inner layer forming tubu-lar c r i s tae ; c r i s -tae longand flexuous. KMnO. f ixat ion or glu-taraldehyde f ixat ion with osmium tetroxide post - f ixat ion. Mitochondria are bound-ed by a double mem-brane and contain many narrow, elongate c r i s -tae lying paral le l to one another. \ Mitochondria may ap-* pear to each possess • one swollen or enlar-ged cr is ta as obser-ved in Figure 18. Glutaraldehyde f ixat ion with osmium tetroxide post - f ixat ion. Ricker (this report) Williams and Webster, 1970 Ho, Zachariah and Hickman, 1968 Cell Type Organism Size TABLE VII - continued MITOCHONDRIAL MORPHOLOGY AND DISTRIBUTION Shape Distribution Morphological Descri ption Reference Asexual Spores Continued Phytophthora parasitica Phytophthora parasitica • var. nico-tiana Saprolegnia ferax 0.6-0.8 p in* diameter; up to 1.6 p. in length ovoid to rod-shaped* frequently accumulate at cytoplasmic periphery 0.25-0.3 p in* diameter; 0,9-1.0 p in length usually 1.0-1. p. in length; seldom more than 2.0 p in length round, oblong or dumb-bell shaped ovoid to cy l indr i -cal mostly l i e close to the periphery of the cel l mostly in cytoplasm near* nuclei Mitochondria occa- Hemmes and sionally contain Hohl, 1969 small inclusions which are aggregates 1 of granular material which stain dark with lead c i t r a t e ; may be .. glycogen. Glutaraldehyde f ixat ion with, osmium tetroxide or KMnO^  post - f ixat ion. Mitochondria with tu - Reichle, bular c r i s tae ; c r i s - 1969a, tae elongate and tend 1969b to overlap one another. Glutaraldehyde f ixat ion with osmium tetroxide post - f ixat ion. M ic rov i l l i of mi to- Gay and chondria set close Greenwood, together and are r a - 1966 ther straight and uni -form in length; often do not extend to cen-ter of the organelle. KMn0» f i xa t ion ; os-. mium tetroxide f ixat ion or glutaraldehyde f i x a -tion with osmium tetro -xide post - f ixat ion. / TABLE VII - continued Cell Type Organism Size MITOCHONDRIAL MORPHOLOGY AND DISTRIBUTION Shape Distribution Morphological Description Reference Antheridia. Aahlya bisexualis Pythium ultimum Oogonia Saprolegnia terrestris Achlya bisexualis 0.3-0.9 u in diameter; 0.8-2.5 » in length 0.3-0.6 v in* diameter; up • to 1.2 JJ in length NP 0.3-0.5 y in diameter; 0.7-4.0 ji length ovoid; rod; modified rod- l ike forms with terminal or sub-ter-minal invaginations; cup-shaped; dumb-bell-shaped' ovoid to rod-shaped* spherical ovoid to rod-shaped random or in peripheral hyphal protoplasm i f vacuolation great mostly in peripheral* . cytoplasm; oriented with their long axis par-a l l e l to that of the hyphae NP ranaom Mitochondria with long tubular c r i s -tae which often over-lap each other; sub-terminal or terminal invaginations common in some mitochondria; matrix dense. GIutaraldehyde-osmi um tetroxide f i xat ion . Numerous mitochondria (with tubular c r i s -tae). KMnO. f i x a t i o n ; g lu -taraldehyde f ixat ion with osmium tetroxide post - f i xat ion ; acro-le in f ixat ion with osmium tetroxide post-f i xa t ion . Mitochondria with b leb - l ike cr istae. KMnO^  f i xa t ion . Rod-shaped mitochon-dria with tubular cr istae which often overlap each other; f i b r i l s present in c r i s tae ; matrix dense. v Glutaraldehyde-bsmium tetroxide f i xat ion . Ricker (this report) Marchant, 1968. . Moore and Howard,. 1968 Ricker (this report) Cell Type Organism Size TABLE VII - continued MITOCHONDRIAL MORPHOLOGY AND DISTRIBUTION Shape Distribution Morphological Description Reference Oogonia Pythium ultimum 0.5-0.7 y in* diameter spherical uniformly distributed* throughout cytoplasm prior to cleavage Oospores Saprolegnia NP terresiris Achlya bisexualis 0.2-0.4 y in diameter; 0.3-1.0 y 'in length globose or cup- NP l ike Pythium ultimum NP ovoid to rod- l ike NP random relegated to small areas between the storage organelles Non-iden-t i f i e d ce l ls • Saprolegnia terresiris Achlya sp. NP NS globose or cup-l ike in prof i le rod-shaped NP NS Mitochondria contain large number of tubul i . KMnO^  f i x a t i o n ; glu-taraldehyde f ixat ion with osmium tetroxide post - f i xat ion ; acro-le in f ixat ion with osmium tetroxide post - f i xat ion . Mitochondria with b leb - l ike cr is tae. KMnO^  f i xa t ion . Mitochondria with tubular c r i s tae . GIutsraldehyde-os-mi um tetroxide f i x a -t ion . NP KMnO, f i xa t ion ; g lu - . taraldehyde f ixat ion with osmium tetroxide post - f i xat ion ; acro-le in f ixat ion with osmium tetroxide post - f ixat ion. Mitochondria with bleb-1ike cr istae. KMnO^  f ixat ion. , Rod-shaped mitochon-dr ia with tubular c r i s tae ; matrix dense. Fixation not given. . Marchant, 1968 Moore and Howard, 1968 Ricker (this report) Marchant, 1968 ^ Moore and Howard, 1968 Turner (see Figure 19-2, Brown and Bertke, 1969) / TABLE VIII Ce l l Type Organism Size MICROBODY MORPHOLOGY AND DISTRIBUTION Shape : D i s t r i b u t i o n . Vegetative Hyphae Aohlya bisexualis .150-450 JUJJ in diameter s p h e r i c a l , oyoid occas ional l y r e n i -form, to s l i g h t l y i r regu la r in mostly centra l hyphal protoplasm' in s u b - a p i -cal zone; random and usual ly c l o s e l y asso -c ia ted with E.R. i n d i s t a l m u l t i - o r g a n e l l e zone Pythium ultimum 0.2-0.3 J J * in ovoid to spherical usual ly in assoc ia t ion diameter' with r ibosome-free sur -faces of E.R. / Morphological Descr ip t ion Reference-Membrane-bounded Ricker ( th is yes i c les c o n t a i n - report ) ' , ing e lect ron-dense granular m a t e r i a l , sometimes tubules and/or concent r ic lamel lar bands; l ess dense v e s i c l e s a lso v i s i b l e in some; in d i s t a l m u l t i - o r g a n e l l e zone they appear c l o s e -l y associated with non-ribosomal areas of granular E.R. Gl u - twal dehyde-osmi um tet rox ide f i x a t i o n . Membrane-bound with ves i c les whose con-tents may be amor-phous and homogen-eous, or they may contain c r y s t a l s , a marginal p l a t e , or small tubules . Mem-brane of microbody is not as th ick as plasma membrane. Glutaraldehyde f i x a -t i o n ; g l u t a r a l d e -hyde -acro le in f i x a -t i o n ; osmium tet rox ide f i x a t i o n ; or KMnOy, f i x a t i o n . Grove, Bracker and Morre, 1970 (Bracker , 1969 -personal communica-t ion) Cell Type Organism Size.. . TABLE VIII continued MICROBODY MORPHOLOGY AND DISTRIBUTION Shape Distribution Differen-t ia t ing Vegeta-t ive Hyphae : Achlya bisexualis _ 150-450 .my in. diameter spherical , ovoid to' dumb-bell shaped random Gemmae Achlya bisexualis 150-450 my spherical to in diameter ovoid random Sporangia Achyla bisexualis 150-450 my spherical to random in diameter ovoid Morphological Description . Reference Membrane-bounded ves i - Ricker (this-cles containing e lec - report) tron-dense granular material and some-times tubules; 1 ess dense vesicles also present in some micro- . bodies; usually occur in close association with non-ribosomal areas of granular E.R. Glutaraldehyde-osmium tetroxide f i xa t ion . Membrane-bounded ves i - Ricker (this cles containing elec- report) ' tron-dense granular material and sometimes tubules; less dense vesicles also present in some microbodies; usually occur in close association with non-ribosomal areas of granular E.R. Glutaraldehyde-osmium tetroxide f i xa t ion . Membrane-bounded ves i - Ricker (this cles containing elec- report) tron-dense granular mater ial , sometimes tubules and/or con-centric lamellar bands; usually occur in close association with non-ribosomal areas of granular E.R. Glutaraldehyde-osmium tetroxide f i xa t ion . TABLE.VIII - continued MICROBODY MORPHOLOGY AND DISTRIBUTION Cell Type... Organism .. Size Shape . .Distribution •Phytophthora 0.5 ji in spherical to NP parasitica diameter e l l ipsoidal Asexual Achlya bisexualis 150-450 mp spherical , ovoid random Spores in diameter and sometimes reniform Phytophthora megasperma var. sojae 320-450 my* in diameter spherical peripheral region of cytoplasm Morphological Description Reference Microbodies sur- Hemmes and rounded by a.single Hohl, 1969 unit membrane and -have homogeneously granular contents; some contain small sets of concentric lamellar material . Glutaraldehyde f i x a -tion with osmium tetroxide or KMn0« post - f i xat ion . Membrane-bounded ves i - Ricker (this cles containing elec- report) tron-dense granular mater ia l , sometimes tubules and/or con-centric lamellar bands; usually occur in closs asso-c iat ion with non-ribosomal areas of granular E.R. Scarce in encysted primary zoospores; numer-ous in f lagel lated secondary zoospores. Gluteraldehyde-os-mium tetroxide f i xa t ion . Membrane-bound bodies Ho, Zachariah containing f ine ly and Hickman, granular, evenly- 1968 dispersed, contents; occasionally contain one or a few bars of extremely dense material . Glutaraldehyde-fixation with osmium tetroxide post - f i xat ion . TABLE VIII - continued MICROBODY MORPHOLOGY AND DISTRIBUTION Cell Type Organism Size Shape Distr ibution Antheridia Aahlya bisexualis 150-450 my spherical to oyoid random in diameter Oogonia Aahlya bisexualis 150-450 my spherical to ovoid random in diameter Oospores Aahlya bisexualis Not observed in sections examined / Morphological Description Reference Membrane-bounded vesi' cles containing eleo tron-dense granular mater ia l , sometimes tubules and/or con-centric lamellar bands; usually occur in close asso-c iat ion with non-r i bosomal areas of granular E.R. Glutaraldehyde-osmium tetroxide f i xa t ion . Membrane-bounded vesi' cles containing eleo tron-dense granular mater ial , sometimes tubules and/or con-centric lamellar bands; usually occur in close asso-c iat ion with' non-ribosomal areas of granular E.R. Glutaraldehyde-osmium tetroxide f i xa t ion . Ricker (.this report) Ricker (this report) Ricker (this report) Cel1 Type Organism Size TABLE LX DICTYOSOME MORPHOLOGY AND DISTRIBUTION Distribution Morphological Description Reference Vegetative Hyphae [Achlya bisexualis 1- 2 cisternae/dic- adjacent to ER and/ tyosome in sub^ or nuclei apical mitochondrial zone. 2- 5 cisternae/dic- -tyosome in distal multi-organelle zone. Dictyosomes consist of stack of cisternae and ves ic les , s imilar to those described by Grove, Bracker and Morre', 1970; d iscont in -uous cisternae in sub-apical zone. Glutaraldehyde-osmium tetroxide f i xa t ion . Ricker (this-report) Aphanomyces euteiches Phytophtlwra infestans 3-4 cisternae/* dictyosome NS near ER and nuclei* perinuclear Typical dictyosome or Golgi body as described by Moore and McAlear, 1953. Glutaraldehyde f ixat ion with osmium tetroxide post - f ixat ion. Golgi dictyosomes. Fixation not given. Shatla, Yang and Mitchell 1966 Ehrlich and Ehr l ich , 1965 Phytophthora parasitica Plasmoa'iophora • brassicae Pythium debary-anum 4-5 cisternae/* dictyosome 4-5.cisternae/* dictyosome 3-4 cisternae/* dictyosome 4-5 cisternae/* dictyosome • 4 cisternae/* dictyosome near ER near ER frequently near nucl ei near E.R and nuclei* near ER and nuclei* Golgi dictyosome. KMnO^  f i x a t i o n ; osmium tetroxide f ixat ion witn KMnO^  post - f ixat ion. Golgi dictyosomes. KMnO. f i x a t i o n ; osmium tetroxide f ixat ion witn KMnO^  post - f ixat ion. Dictyosomes of the Golgi apparatus. Glutaraldehyde f ixat ion with osmium tetroxide post - f ixat ion. Golgi bodies or dictyosomes. Luft 's permanganate f i xa t ion . Golgi bodies with numerous vesic les . KMn04 f i xa t ion . Ehrlich and Ehr l ich , 1966 Ehrlich and Ehr l ich , . 1966 Williams and Yukawa, 1967 Hawker and Abbott, 1963 Manocha and Colvin, 1963 Co TABLE LX continued DICTYOSOME MORPHOLOGY' AND DISTRIBUTION Cell Type Organism Size Distribution. .Morphological Description Reference Vegetative Hyphae continued Pythium ultimum' 4-6 cisternae/* dictyosome 4-7 cisternae/ dictyosome 2-7 cisternae/ dictyosome bapro legm-a fevax NS 3-4 cisternae/ dictyosome usually associated with, nuclear enve^ lope and/or ER adjacent to ER or nuclear enve> lope adjacent to ER or nuclear enve-* lope NS adjacent nucleus Dictyosomes exhibit structural po lar i ty ; their cisternae immediately adjacent to nuclear envelope or ER are discontinuous; cisternae in median position continuous and tend to be compressed; cisternae at d istal pole discontinuous but more swollen , than those at proximal pole. Osmium tetroxide f i x a t i o n ; glutaraldehyde f ixat ion with osmium tetroxide post - f i xa -t ion ; KMnO^  f i xa t ion . Dictyosomes exhibit t ransit ion in membrane morphology across stacks of cisternae (.from ER - l i k e at one pole to plasma membrane-like at opposite pole); possible sites of mem-brane inter-conversion. Glutaraldehyde f ixat ion with osmium tetroxide • post - f i xat ion . s Dictyosomes in apical region have few d i s -continuous cisternae loosely-organized into a stack; dictyosomes in sub-apical zone exhibit structure as described above; secretory vesicles migrate to and fuse with plasma membrane. Glutaraldehyde f i x a t i o n ; gluteraldehyde-acrolein f i xa t ion ; osmium tetroxide f i xa t ion ; or KMnO^  f i xa t ion . Golgi areas; many smaller vesicles and tubular elements with characterist ic association. Osmium tetroxide f i x a t i o n ; KMnO. f i xa t ion ; gluteraldehyde. f ixat ion and osmium \ tetroxide post - f i xat ion . NS Gluteraldehyde f ixat ion with osmium tetroxide post - f i xat ion . Groye, Morre and Bracker, 1967 Grove, Bracker and Morre', 1968 Grove, Bracker and Morre", 1970 4*. Gay and Greenwood, 1966 Heath and Greenwood, 1968 Cell Type Organism Size Vegetative . Hyphae continued Saprolegnia• terrestris often massive Gemmae Aohlya bisexualis 3-6 cisternae/ dictyosome Sporangia. Aohlya bisexualis. 4-6 cisternae/ dictyosome Aphanomyces 3-4 cisternae/* euteiches dictyosome Phytophthora 3-5 cisternae/* capsici dictyosome TABLE IX - continued DICTYOSOME MORPHOLOGY AND DISTRIBUTION Distribution Morphological Description Reference frequently continuous Golgi dictyosome is highly fenestrated; with, outer membrane often massive, of the nuclear envelope KMnO^  fixation. adjacent to ER and/ or nuclei adjacent to ER and/ or nuclei near ER or nuclei often present near nuclei where they 1ie adjacent to centrioles and kinetosomes Dictyosomes similar to those in vegetative hyphae, but cisternae often appear discon-tinuous; numerous vesicles visible about forming face cisternae. Glutaraldehyde-osmium tetroxide fixation. Dictyosome cisternae often appear discon-tinuous and numerous secretory vesicles associated with distal face cisternae prior to protoplasmic cleavage; cisternae be-come highly developed and stacks of c is - . ternae cluster about anterior pole of nuclei as zoosporangial differentiation occurs. Glutaraldehyde-osmium tetroxide fixation. Dictyosome or Golgi apparatus as described by Moore and McAlear, 1963. Glutaraldehyde fixation with osmium tetroxide post-fixation. Dictyosomes of Golgi apparatus present in all stages of zoosporangial devel-opment; especially well-developed next to nuclei participating in flagellum genesis; numerous small vesicles appear to be released from the dicty-osomes. i . KMnO, fixation; glutaraldehyde fixation with osmium tetroxide post-fixation. " Moore and Howard, 1968 Ricker (this report) Ricker (this", report). Shatla, Yang and Mitchel 1, 1966 . Williams and Webster, 1970 cn TABLE IX - continued DICTYOSOME MORPHOLOGY'.AND DISTRIBUTION ^ell Type Organism Size. Distr ibut ion. Morphological Description Reference Sporangia continued Phytophthora erythroseptica Phytophthora parasitica 5-6 cisternae/* dictyosome NS 4-7 cisternae/* dictyosome 3-6 cisternae/* dictyosome Pythium middle-tor.ii 5-6 cisternae/* dictyosome usually in close proximity to nuclei . often in proximity to nuclei adjacent to ER often in perinu-clear posit ion; one end often associated with ER continuous with ER Golgl dictyosomes took characterist ic form of stack of close-packed flattened cisternae associated with smaller v e s i -c les ; closely resemble Golgi dictyosomes. in higher plants and other fungi . Osmium tetroxide f i x a t i o n ; Golgi dictyosomes. Osmium tetroxide f i xa t ion . KMnO^  f i xa t ion . Dictyosomes appear to pinch off vesicles in large numbers; vesicles become c lea -vage vesicles. Osmium tetroxide f i xa t ion ; KMn04 f i x a t i o n ; glutaraldehyde f ixat ion with osmium tetro -xide or KMnO^  post - f ixat ion. Dictyosomes exhibit polar ity s imilar to that described by Bracker (1967); secre-tory vesicles f i l l e d with substance of mottled texture; similar vesicles ac-cumulate at periphery of sporangium and at t ip of growing hyphae. Glutaraldehyde f ixat ion with osmium tetroxide or KMnO,, post - f i xat ion . Clusters of dictyosomes produce copious quantities of vesicles and cisternae that coalesce to fora the cleavage apparatus; . 100A diameter tubules observed within cisternae as well as ER; vesicles con-taining tubules liberated from distal pole during cleavage. Direct membrane continuity between ER at proximal pole and 1st cisternae as well as in ter - \. connections between 1st and 2nd c i s t e r -nae. Membranes of d ista l cisternae re -semble plasma membrane; membrane of proximal cisternae resemble ER. Glutaraldehyde or glutaraldehyde-formal-dehyde f ixat ion with osmium tetroxide post - f i xat ion . Chapman and V u j i c i c , 1965 • Vujicic', Chap-man and Col noun, 1965 Hohl and Hamamoto, 1967 Hemmes and Hohl, 1969 Bracker, Heiritz and Grove, 1970 C e l l Type • Organism • ••. S i z e Sporangia • ,': Saprolegnia 4 - 5 c is ternae/* continued ferax dictyosome Asexual : . . Aohlya.bisexualis 4 -6 c is ternae/ .Spores . . . . dictyosome Phytophthora . NS . eryt'ttroseptica Phytophthora 3-6 c is ternae/ megasperma dictyosome var . sojae Phytophthora 3 -5 c is ternae/* parasitica dictyosome Saprolegnia 4 -5 c is ternae/* ferax dictyosome' A n t h e r i d i a Aohlya bisexualis 2-4 c i s te rnae/ dictyosome TASLE U - continued DICTYOSOME MORPHOLOGY' AND DISTRIBUTION D i s t r i b u t i o n '. Morphological D e s c r i p t i o n Reference near ER and/or* n u c l e i usua l l y c lus te red near an te r io r pole of nucleus, usual -l y adjacent to nucleus but may be associated with ER of ten in proximity to nucle i near nuclei in area* of basal bodies adjacent to ER and/ or nucle i in area of basal bodies near ER and/or* nuc le i near ER and/or nucle i Golgi bodies with, p a r a l l e l c i s t e r n a e ex -panded at the margins, sometimes with. . attached or assoc iated v e s i c l e s . KMnOy, f i x a t i o n ; g lutaraldehyde f i x a t i o n w i th osmium t e t r o x i d e p o s t - f i x a t i o n . Dictyosomes appear h i g h l y - d i f f e r e n t i a t e d ; numerous v e s i c l e s assoc iated with d i s t a l c i s t e r n a e , e s p e c i a l l y i n f l a g e l l a t e d secondary zoospores. GlutBraldehyde-osmium t e t r o x i d e f i x a t i o n . Golgi dictyosome-. Osmium t e t r o x i d e f i x a t i o n . NS Glutaraldehyde f i x a t i o n with osmium t e t r o x i d e p o s t - f i x a t i o n . Golgi complex. Glutaraldehyde f i x a t i o n wi th osmium t e t r o x i d e p o s t - f i x a t i o n ; KMnO^ f i x a t i o n . Golgi bodies w i th p a r a l l e l c i s t e r n a e ex -panded at the margins, sometimes with a t -tached or assoc iated v e s i c l e s . KMnO, f i x a t i o n ; g lutara ldehyde f i x a t i o n witn osmium t e t r o x i d e p o s t - f i x a t i o n . Dictyosomes morpho log ica l l y s i m i l a r to those in vegetat i ve hyphae; not abundant i n ' , protoplasm. Glutaraldehyde-osmium t e t r o x i d e f i x a t i o n . Gay and Greenwood, 1966 R icker ( t h i s repor t ) V u j i c i c , Chap-man and C c l -houn, 1965 Ho, Zachariah and Hickman, 1968 R e i c h l e , 1 969a Gay and Greenwood, 1966 R icker ( t h i s repor t ) cn TABLE LX r. continued DICTYOSOME MORPHOLOGY AND DISTRIBUTION Cell Type Organism Size. Distribution. Morphological Description Reference Antheridia continued Pythium ultimum Saprolegnia terresiris often massive Oogonia Aahlya bisexualis 2-5 cisternae/ dictyosome Pythium ultimum frequently continu-ous with outer membrane of nuclear .envelope adjacent to ER and/or nuclei No dictyosomes found; only one structure resembling dictyosomes observed and is probably a chance configuration of endo-plasmic reticulum. KMriOy, f i xa t ion ; glutaraldehyde f ixat ion witn osmium tetroxide post - f i xat ion ; acrolein f ixat ion with osmium tetroxide post - f ixat ion. Golgi dictyosome is highly fenestrated KMnO^  f i xa t ion . Dictyosomes abundant, especially in young oogonial protoplasm; morphologically simi-lar to those in vegetative hyphae; numer-ous secretory vesicles associated with distal cisternae. Glutaraldehyde-osmium tetroxide f i xa t ion . No dictyosomes found; only one structure resembling dictyosomes observed and is probably a chance configuration of endo-plasmic reticulum. KMnO. f i xa t ion ; glutaraldehyde f ixat ion with osmium tetroxide post - f i xat ion ; acrolein f ixat ion with osmium tetroxide post - f ixat ion. Marchant, 1968 Moore and Howard, 1968 Ricker (this report) Marchant, 1958 CO Oospores Saprolegnia NS terresiris Aahlya bisexualis near nuclei Typical Golgi dictyosomes, less than ly in diameter. KMnO^  f i xa t ion . Dictyosomes not discernible in densely- 1 packed protoplasm; probably present. Glutaraldehyde-osmium tetroxide f i xa t ion . Moore and howard, 1968 Ricker (this report) Unidentified . Aahlya sp. Cells 3-4 cisternae/* dictyosome near ER* NS Fixation not given. Turner uee Figure 19-2, Brown and Bertke. 1969) Cell lype Organism Size TABLE X MICROTU.BULAR PACKET MORPHOLOGY AND DISTRIBUTION Shape Distribution Vegetative Hyphae Differen-t iat ing Vegetative Hyphae Gemmae Aahlya bisexualis Aahlya'bisexualis Achlya bisexualis Sporangia Aahlya bisexualis vesicles 100-300 my in diameter; packets 100-300.mp in diameter; up to 1.7 p in length spherical to ovoid-shaped vesicles; rod- l ike packets often occur in close association, with mitochondria Phytophthora 80 mp wide rectangular, oval , in close association aapsiai 2-3 p long or irregular in ' with mitochondria; cross-section; ER always in asso-long (see size) ciation / Morphological Description Reference Ricker (this report) Ricker (this report) Ricker (this report) Membrane-bound units Ricker (this of tubules; mem- report) braise of microtu-bule vesicles 80-90 A thick; mem- ••' brane of microtu-bule packets th in -ner, 50-60 A . Tu-bules each about 85-100 A thick; each with electron-dense periphery and electron-1ight core. Glutaraldehyde-os-mium tetroxide f i x a -t ion . Microtubular packets Williams which are surrounded and Webster, by a membrane. , 1970 Appear during late cleavage stages. Individual microtu-bules are 90A in diameter and have an electron transparent core with an osmio-ph i l i c r ind . Glutaraldehyde f ixat ion and.osmium tetroxide KMnO- f ixat ion destroys the microtubules. TABLE .X - continued, MICROTUBULAR PACKET MORPHOLOGY AND DISTRIBUTION Cel l Type Organism. Size. Shape Distribution Morphological Description ' Reference Sporangia continued Pythium middle-tonii Asexual Spores Aahlua bisexualis 50-75 my in diameter; at least 1.0. y long ER cisternae are* cyl indrical to rec-tangular; vesicles spherical to ovoid vesicles 150- spherical to ovoid-500 mp in d i a - shaped vesic les; meter; packets rod- l ike packets 100-300 my in diameterj up to 1.7 p in. length ER cisternae with tubu-les in close association with mitochondria prior . to cleavage; become associated with proximal dictyosome cisternae during cleavage random, but become close-ly associated with dicty -osomes upon encystment of secondary zoospores ER cisternae contain cluster or fasc ic le of narrow tubules (approx. 100A in -diameter).; cisternae continuous with pro-ximal dictyosome c i s -ternae after sporan-gia induced to form zoospores. Tubules observed in vesicles and cisternal f rag -ments that, are l i b - , erated from d is ta l distyosome pole during protoplas-mic cleavage. Glutaraldehyde f i x a -tion with osmium tetroxide post - f i xa -t ion ; . 1:1 formalde-hyde: glutaralde-hyde f ixat ion with osmium tetroxide post - f ixat ion. Membrane-bound units of tubules; membrane cf microtubule ves i -cles 80-90A th ick ; membrane of micro-tubule packets th in - , ner, 50-60A. >. Tu-. bules each about 85-100A thick, each with electron-dense per i -phery and electron-l ight core; diameter of packet tubules de-crease near terminal (cont'd) Bracker, Heintz and Grove, 1970 o Ricker (this report) TABLE X - continued MICROTUBULAR PACKET MORPHOLOGY.AND DISTRIBUTION Ce i l Type . Organism. S ize Shape D i s t r i b u t i o n . Morphological ' . 'Description Reference Asexual Spores continued Aahlya bisexualis continued Diatyuahus s t e r i l e 0 . 2 - 0 . 6 y wide; not greater than 1.6 y, i n length u s u a l l y rectangular but may be s p h e r i -cal to i r r e g u l a r of ten assoc iated w i th nuclear envelope 0 . 2 - 0 . 3 y , then . measure'75-85A t h i c k . Osmio-p h i l i c i n t e r - c o n -n e c t i o n , . o r l i n k s , v i s i b l e among some . tubu les . Glutaraldehyde-osmium t e t r o x i d e f i x a t i o n . Membrane-bounded ves i c l e , f requent l y studded wi th r i b o -somes and may be con-t inuous, wi th nuclear envelope or f l a t t e n e d c i s t e r n a e of RER; conta in groups of t u - . bu les . Tubules 1 3 . 5 -18.1 my i n d iameter ; c e n t e r - t o - c e n t e r spa -c ing of 25 my; may show hexagonal p a c k i n g ; tubules of a group f requent l y terminate at same l e v e l at both ends, each tubule tapered f o r .3 y in l e n g t h ; l i e a n t i - p a r -a l l e l . Tubules r e -semble and probably form f 1 imrner h a i r s . KMnO. f i x a t i o n ; . o s -mium t e t r o x i d e f i x a -t i o n ; g lutera ldehyde f i x a t i o n wi th osmium t e t r o x i d e p o s t - f i x a -t i o n . Heath , . . Greenwood and G r i f f i t h s , 1970 . / Cell Type... .'. Organism. Size • ^TABLE X - continued MICROTUBULAR PACKET MORPHOLOGY AND DISTRIBUTION Shape Distr ibution Morphological .Description . Reference Asexual Spores continued • Phytophthora,-, . parasitica var.. nicotiana. Rhizidomyces apophysatus Saprolegnia . ferax 0.2-0.4 p* wide, 0.5-1.4 p long rectangular interspersed 0.3-1.0 p wide* rectangular c a . 1.3 ji long posterior end of c e l l * 0.2-0.6 p wide not greater . than 1.6 p in length usually rectangular often associated with nuclear envelope Membrane-bound yes i -cle containing fine tubules. Glutaraldehyde f i x a -tion with osmium tetroxide post-f i xa t ion . . Membrane-bound or-ganelle f i l l e d with.tubules 16-18 mp in diameter. Osmium tetroxide f i xa t ion . Membrane-bound ves i -c l e , frequently studded with r ibo-. somes and may be con-tinuous with nuclear envelope or flattened cisternae of RER; contain groups of tu -bules. Tubules about 12.0-21.0 mp . in diameter (ca. 7.0-10.0 mp in diameter as deter-mined by micro-densitometer); may show hexagonal pack-ing; frequently ter -minate at same level at both ends and each tubule tapered'; l i e a n t i - p a r a l l e l . Tu-bules resemble and probably form flimmer hairs. KMn0« f i x a t i o n ; osmium tetroxide f i x a t i o n ; glutaraldehyde f i x a -tion with osmium tetroxide post - f i xa -t ion . Reichle, . 1969a Fu l le r , 1966 Heath, Greenwood and G r i f f i t h s , 1970 TABLE X - continued MICROTUBULAR PACKET MORPHOLOGY' AND DISTRIBUTION Morphological . Cell .Type. . . . . Organism Size .' . . Shape Distribution . Description Reference NP NP . Np Similar to those pb- Crump (see . . served in Shizidio- . p. 78 myoes apophysatus. Fu l le r , Observed in encysted 1966) primary zoospores. Fixation not indicated. Antheridia Aohlya bisexualis - . - . . - - . ' Ricker (this report) Oogonia Aohlya bisexualis - - . - - Ricker (this report) Oospores Aohlya bisexualis - - - - Ricker (this report) i co Asexual „ , • Spores - ' ' Saprolegma continued , . •a / TABLE XI PARASTRASOME MORPHOLOGY AND DISTRIBUTION Cell Type Organism Size Shape Distr ibution Vegetative Aahlya bisexualis - -Hyphae Dif ferent ia- Aahlya bisexualis - -ting Vege-tative Hyphae Phytophthora - - -. aapsiai Gemmae Aahlya bisexualis - - -Sporangia Aahlya bisexualis 170-350 my spherical to random in precleavage in diameter ovoid protoplasm near basal . 1 bodies and cleavage vesicles during and after protoplasmic cleavage Phytophthora aapsiai 0.25 x 0.36 y ovoid usually associated with vacuoles, lying between liposomes and vacuoles Morphological Description Reference Ricker (this report) - Ricker (this report) Williams and . Webster, 1970 Ricker (this report) Membrane-bounded ves i - Ricker ( th is , cles containing report) . core of electron-1 ight material sur-rounded by a 1ayer of more dense mat-er ial ; fibrous e le -ments v i s ib le throughout electron-l ight zone. Glutaraldehyde-os-mium tetroxide f i xat ion . Ovoid inclusions enclosed by a mem-brane which has occasional gaps; dark l ine trans-verses one side of inclusions and may have several chevrons running diagonally from i t ; appear in preclea-vage stage. Glutaraldehyde f i x a -tion with osmium tetroxide post-f i xat ion . Williams, and Webster, 1970 . TABLE XI - continued . PARASTRASOME MORPHOLOGY AND DISTRIBUTION Cell Type. Organism Size Shape Distr ibution Asexual Aahlya bisexualis smaller forms spherical to oyoid random distr ibut ion of Spores 250-500 m^  smaller forms; larger in diameter bodies always found in larger forms close association with near basal basal bodies bodies . 0.6-0.7 x 1.0-1.2 v Phytophthora 0.25 x 0.36 p ovoid usually associated with aapsiai vacuoles, lying between liposomes and vacuoles Phytophthora parasitica var. niaotiana 0.3-0.4 a* spherical to i r re -* gular peripheral region of the cytoplasm Morphological Description Reference Membrane-bounded Ricker (this vesicles containing report) . core of electron-l ight material .'sur-rounded by a.layer •: of more dense mater-ia l - dense bands develop within the electron- l ight cores; one. or two vesicles near basal bodies en-large and a re lat ive ly dense matrix in which f i b r i l s are discer-nable develope within them; f lage l la r root-lets par t ia l l y encom-. pass larger vesicles. Glutaraldehyde-osmium tetroxide f i xa t ion . Ovoid inclusions enclosed by a mem-brane which has occasional gaps; dark l ine trans-verses one side of inclusions and may have several chev-rons running diagon-a l l y from i t . Glutaraldehyde f i x a -tion with osmium tetroxide post - f i xa -t ion . Williams and Webster, 1970 Vesicles with bar- Reich!e, shaped structures 1969a,1969b and membranes within them. Glutaraldehyde f i x a -tion with osmium tetroxide post-f i xa t ion . Cell Type . Organism Size TABLE XI - continued PARASTRASOME MORPHOLOGY AND DISTRIBUTION Shape •. Distr ibution Morphological Description Reference Antheridia Aohlya bisexualis Oogonia Oospores Aohlya bisexualis Achlya bisexualis Ricker (this report) Ricker (this report) Ricker (this '. report) av TABLE XII LIPOSOME MORPHOLOGY AND DISTRIBUTION Cell Type Organism Size Shape.. . . . D i s t r i b u t i o n Vegetative Aohlya bisexualis - -Hyphae Aphanomyces 0.7-1.1 y* ovoid to i r re -* random* euteiches gular . Phytophthora NS NS NS parasitica Dif ferent ia - . Achlya bisexualis 0.4-1.1 y • ovoid to i r r e - random ting Vegata- gular t ive Hyphaa Morphological Description . Reference Ricker (this report) Crystals with a speci - Shatla, Yang f i c laminar structure, and Mi tche l l , Glutaraldehyde f ixat ion 1966 with osmium tetroxide post - f i xat ion . Vesicles with a cry- Reichle, s t a l l i n e content. 1969a Glutaraldehyde f i x a -tion with osmium tetroxide post-f i xa t ion . Liposomes i n i t i a l l y . Ricker (this appear as membrane- report) bound bodies contain-ing homogeneous mix-ture of electron -l ight amorphous mat-er ia l and electron-dark granules. Osmiophilic globules develop and layering of the amorphous mat-er ia l about the os-miophil ic globules occurs. Electron-l ight bands 90-100 A th ick; darker bands 50-1-1 OA thick. Glutaraldehyde-osmium tetroxide f i xa t ion . Cell Type Organism Size TABLE Xll - continued LIPOSOME MORPHOLOGY' AND DISTRIBUTION Shape Distribution Morphological Description Reference Aphanomyces euteiahes 0.7-1.1 JJ ovoid to i r re -* gular random* Gemmae Aahlya bisexualis 0.5-1.2 y i rregular random; however, some in close association with mitochondria . Sporangia Aahlya bisexualis 0.4-1.2 y irregular random in uncleaved proto-plasm; oriented among the cleavage vesicles and mitochondria during and after protoplasmic cleavage Crystals with a speci f ic laminar structure. In hyphae di f feren-t ia t ing into zoo- . sporangia. Glutaraldehyde f i x a -tion with osmium tetroxide post-f i xa t ion . Wei 1-differentiated, 1iposomes contain-ing osmiophilic glo-bules surrounded by layers of electron-1ight amorphous material . Electron-l ight bands 90-100 A th ick; darker bands 50-110 A thick. Glutaraldehyde-os-mium tetroxide f i x a -t ion . Wei 1-di f ferent i ated liposomes containing osmiophilic globules surrounded by layers of e lectron- l ight amor phous material . Elec-t ron- ! ight bands 90-100 A thick; darker bands 40-50 A'.thick. Glutaraldehyde-os-mium tetroxide f i xa t ion . Shatla, Yang . and Mi tche l l , 1966 Ricker (this report) Ricker (this report) TABLE .XII - continued LIPOSOME MORPHOLOGY AMD DISTRIBUTION Cell Type ..Organism . . . . . S i z e . . . Shape.. . .Distr ibution Sporangia Aphanomyces 1.0-2.Op* ovoid to irregu-* • random* • continued euteiahes lar Phytophthora, ca. 0.5 p. spherical ' random* aapsiai Phytophthora ' erthroseptioa 0.3-0.8 p irregular random* .Morphological Description. . Reference Crystals with a speci - Shatla, Yang f i c laminar struc- and Mi tche l l , ' ture. 1965 . Glutaraldehyde f i x a -tion with osmium , tetroxide post - f i xa -t ion . Vacuoles are uniform- Williams and ly osmiophilic in Webster, precleavage stage, 1970 but become less opa-que at the margins during cleavage un-t i l s t r iat ions dev-elop and encompass a l l or most of the 1 iposomes. Electron-transparent regions develop and encompassing mem-brane may disappear as zoospore cleavage occurs. Glutaraldehyde f i x a -tion ana osmium tetroxide post-f ixat ion or KMnO, f i xa t ion . Lipid inclusions . Chapman which possess a fine and Vu j i c i c , lamellar structure 1965 with a periodicity of 60-70A. Characteristic of young and old non-germinating sporan-g ia . • Osmium tetroxide f i x a -tion or KMnO^  f i x a -t i o n . . Cell Type . Organism. Size TABLE Xll - continued LIPOSOME MORPHOLOGY AND DISTRIBUTION Shape Distr ibut ion. . . Sporangia , continued Phytophthora-.: infestans NS NS NS Phytophthora parasitica 0.7-1.4 p* spherical , ovoid, to s l ight ly irregular random* Pythium ultimum* . 0.3-0.7 p spherical to oyoid random Saprolegnia ferax 0.4-C.9 p* spherical random* Asexual Achlya bisexualis 0.3-0.9 P . spherical to i r r e - random Spores • " gular Morphological Description. Reference yacuoles with mye-l i n - ! ike 1ipoidal inclusions Fixation not given. Vesicles which con-tain f i b r i l l a r mat-e r i a l . Glutaraldehyde f i x a -. t ion and osmium tetroxide post-f ixat ion . . NP Eisner, Horton and , Bowen, 1967 Hohl and Hamamoto, 1967 Grove, Bracker and Morre^ 1970 Vesicles contain dense bodies which may. have an outer zone of f ine concentric l ines with a period of 9.5 nip. Glutaraldehyde f ixat ion and osmium tetroxide post - f i xat ion ; KMnO '^ f i x a t i o n ; or osmium' tetroxide f i xat ion . Sometimes a clear center develops after KMnO. f i xa t ion . Wei 1-differentiated liposomes containing osmiophilic globules surrounded by layers of e lectron- l ight arnor phous material . Elec-t ron- l ight bands 90" 100A thick; darker Gay and Greenwood, 1966 Ricker (this report) cont'd TABLE _XII - continued LIPOSOME MORPHOLOGY AND DISTRIBUTION Cell Type • Organism Size Shape Distr ibution Asexual • Achlya bisexualis Spores continued continued Blasiocladiella emersonii 0.3-0.7 p spherical to* s l ight ly irregular often near end of st r iated-root let channels or in peri phery of ce l l Monoblephaj'ella ca. 1.1 ji sp. spherical to* s l ight ly irregular anterior end of ce l l Phytophthora 0.4-0.9 ja* spherical NS .capsici Phytophthora megasperma var. sojae 0.5-1.3 p* ovoid to i r r e - random* .Morphological Description. Reference bands 50-11 OA thick. Degradation of 1ipo-somes evident, es-pecial ly in f l a g -ellated secondary zoospores. GIutaraldehyde-osmium tetroxide f i xa t ion . Vesicles are enclosed by unit membrane and contain electron-opaque material . KMnO^  f i xa t ion . Vesicles with osmio-* ph i l i c inclusion. Glutaraldehyde f i x a -tion and osmium tetroxide post - f i xa -t ion . Reichle and-Fu l le r , 1957 Ful ler. and Reichle, 1968 Vacuoles with osmio-ph i l i c globules which are encompassed by s t r ia t ions . Glutaraldehyde f i x a -tion with osmium tetroxide post - f i xa -tion or KMnO^  f i x a -t ion . Williams and Webster, 1970 Vesicles contain crys- Ho, Zachariah, t a l l i n e material which and Hickman, is either in the. form 1968 of an irregular net-work or an uniform dense mass. Glutaraldehyde f i x a -tion and osmium tetroxide post - f i xa -t ion . TABLE XII - continued •LIPOSOME MORPHOLOGY" AND DISTRIBUTION. Cell Type '. Organism. . . . . Size Shape D is t r ibut ion . . . . . . ^Spores •'Phytophthora - " 0.6-1.4 ; ; * spherical , ovoid random* :ontinued S p o r e s parasitica " to s l ight l y -irregular Phytophthora .0.5-1.3 spherical to s l ight -* random* parasitica ly irregular var. nicotiana Shizidiomyaes. 0.5-1.3 J J irregular near periphery of •apophysaius* ce l l Saprolegnia ferax 0.4-0.9 y* spherical random* Antheridia Achlya' bisexualis 0.4-0.8 n spherical , ovoid to irregular random in mitochon-dr ia l invagination Morphological Description. . Reference Yesicles with f i b r i l - Hohl and '. lar material . Hamamoto, Glutaraldehyde f i x a - 1967 tion with osmium tetroxide post - f i xa -tion or KMnO, post-f i xa t ion ; KPInO. f i x a -tion ; or osmiQm tetroxide f i xat ion . Vesicles with a crys-t a l ! ine content. Glutaraldehyde f i x a -tion and osmium tetroxide post - f i xa -t ion. Reichle, 1969a 1969b . , Vacuole. Fu l le r , 1966 Osmium tetroxide f i x a -t ion . Vesicles contain e lect - Gay and tron-dense bodies Greenwood, which may have an 1966 outer zone of f ine concentric l ines with a period of 9.5 m.y. Glutaraldehyde f i x a -tion and osmium tet -roxide post - f ixat ion. Sometimes a clear center develops after KMnO^  fixation.' . Liposomes seldom pre- Ricker (this sent; i f present report) liposomes appear as membrane-bound bodies containing osmiophilic globules in a homo-geneous matrix of cont'd Cell Type Organism Size TABLE XII - continued LIPOSOME MORPHOLOGY AND DISTRIBUTION Shape Distribution Antheridia . continued. Aahlya bisexualis continued Pythium ultimum* Oogonia Achlya bisexualis 0.2-1.2 p ovoid to irregu- random 1 ar 'Pythium ultimum ca 0.2-0.6 y* spherical to* random* ovoid Morphological Description Reference electron- l ight amor-phous material and electron-dense granular material . Glutaraldehyde-osmium tetroxide f i xa t ion . Marchant, 1968 Liposome i n i t i a l l y Ricker (th appear as di lated report) . ER containing homo-geneous mixture of e lectron- l ight amor-phous material and electron-dense gra-nules; d i f ferent iate into membrane-bound bodies in which os-miophilic globules develop; layering of e lectron- l ight amor-phous material about globules occurs and w e l l -defined 1iposomes develop. GI utaraldehyde-osmium tetroxide f i xa t ion . Membrane-bounded or- Marchant, ganelles containing 1968 storage material . KMnO, f i xa t ion ; g lu -taraldehyde f ixat ion . with osmium tetroxide post - f ixat ion or acrolein f i xat ion with osmium tetro -xide post - f ixat ion. . • TABLE XII - continued LIPOSOME MORPHOLOGY AND DISTRIBUTION - C e l l Type Organism. . . .Size. Shape . . . Distr ibution Oospores . Achlya bisexualis 0.3-1.2.• _p . spherical to i r r e - random yu 1 ar Pythium ultimum' ca. 0.2-0.6 31* spherical to cyoid* random* Unidentified Achlya sp. * NS irregular* NS Cells Morphological Description . Reference Wei 1-differentiated forms common in protoplasm; often contain three to f i ve osmiophiiic globules/1 iposome; Glutaraldehyde-os-mium tetroxide f i x a -t ion . Ricker (this report) Generally a continuous system of membrane-bounded storage or -ganel 1 es.. 'KMn0A f i x a t i o n ; g lu -taraldehyde f ixat ion with osmium tetroxide post - f i xat ion ; or acrolein f ixat ion • with osmium tetro-xide post - f i xat ion . NS Marchant, i 968 Turner (see figure 19-2 Brown and Bertke, 1969 . —i Cel 1 Type •.'Organism i i z e . • TABLE m i LIPID DROPLET MORPHOLOGY AND DISTRIBUTION Shape. .Distr ibution. Morphological Description Reference Vegetative Hyphae - . Achlya bisexualis 0.4-1.2 y spherical to ovoid usually lacking in api^ cal protoplasm - random throughout distal m u l t i -organelle zone Lipid droplets non-membrane bound; pro-bably re lat ive ly sat -urated compounds; become more abundant as distance from grow-ing t ip increases. GlutBraldehyde-osmium tetroxide f i xa t ion . Ricker -(this report) Phytophthora infestans NP NP in hyaloplasm NP Ehrlich and Ehr l ich , 1965 1966 Phytophthora varasitica .Pythium 0.3-0.6 y* 0.3-0.7 y* spherical to i r re -* random* gular spherical* random* Lipid bodies abundant in older hyphae. KMnO^  f i xa t ion . NP Acrolein f i xa t ion . Ehrlich and Ehr l ich , 1966 Marchant, Peat and Banbury, 1967. 0.6-1.6 y* Saprolegnia ferax Up tO 1 .0 y spherical to ovoid in non-vacuolated sub-in sub-apical zone. apical zone-irregular in "mature" random within vacuolated hyphal region zone spherical NS Lipid bodies. Glutaraldehyde f i x a -t ion ; glutaraldehyde-acrolein f i xa t ion ; osmium tetroxide f i xa t ion ; or KMnO, f i xa t ion . Droplets of cytoplas-mic l i p i d . Uniform-ly pale except, for a denser out l ine; Osmium tetroxide-f i xa t ion . Grove, Bracker and Morre' . 1970-Gay and Greer wood, 1966 TABLE XIII - continued LIPID DROPLET MORPHOLOGY AND DISTRIBUTION Cell Type Organism Size Shape Distribution Morphological Description Reference Differentiating Aahlya bisexualis 0.5-1.7 y Vegetative , Hyphae Gemmae Sporangta Aahlya bisexualis 0.7-1 .2 y Achlua bisexualis 0.5-1.2 Aphanoinydss* euteiahes 1.5-2.7 y spherical tp ovoid irregular spherical to ovoid spherical random random random in uncleaved protoplasm; scattered in peripheral protoplasm during and after c l e a -vage - l i e between mitochondria and c l e a -vage plane periphery of developing spore Lipid droplets; non-membrane bound; probably re lat i ve ly saturated compounds; become increasingly abundant with age. Glutaraldehyde-osmium tetroxide f i x a t i o n . Lipid droplets non-membrane bound; pro-bably re lat ive ly saturated compounds; abundant. Glutaraldehyde-osmium tetroxide f i x a t i o n . . Lipid droplets; non-membrane-bound; pro-bably re lat ive ly saturated compounds; abundant. Glutaraldehyde-osmium tetroxide f i x a t i o n . NP Ricker (this." • report) Ricker (this report) Ricker (this report) Shatla, Yang and Mitchel l . 1966 phytophthora erythroseptica 0.3-0.8 y irregular NS Small electron-trans-parent bodies of i r -regular shape with an intensely electron-opaque periphery. Mere numerous in older sporangia. Osmium tetroxide f i x a -t ion or KMnO^  f i xa t ion . Chapman and Vu j i c i c , 1965 . TABLE XIII - continued . LIPID DROPLET MORPHOLOGY AND DISTRIBUTION Cell Type Organism Size Shape Distribution Morphological Description Reference Sporangia Phytopht'fiora parasitica 0.7-1.3 u* Pythium ultimum 0.8-1.2 y spherical to* ovoid spherical to irregular in peripheral cytoplasm* of most forming spores . NS Saprolegnia ferax up to 1.0 y spherical MP Asexual Spores Aohlya bisexualis 0.4-0.8 y in spherical encysted random Lipid inclusions. . Hohl and Glutaraldehyde f i x a - Hamamoto, t ion with osmium 1967 tetroxide post - f i xa -t ion . Lipid body. Grove, Bracker Glutaraldehyde f i x a - and Morre, t ion ; gluteraldehyde- 1970 acrolein f i xa t ion ; osmium tetroxide f i x a -t ion ; or KMnO^  f i xa t ion . Droplets of cytoplasmic Gay and Green-1 i p i d ; uniformly pale except for a dense outl ine. Osmium tetroxide f i x a -t ion . Lipid droplets; noh- •  membrane bound; pro-bably re lat ive ly saturated compounds; not abundant in en-cysted primary zoo-spores unti l shortly before f lagel lated secondary zoospore released; abundant in f lagel lated secon-dary .zoospores.\ Glutaral dehyde-osmium tetroxide f i xa t ion . wood, .1960 Ricker (this report) TABLE .XI11 - continued LIPID DROPLET MORPHOLOGY AND DISTRIBUTION Cell Type Organism Size Shape Distribution Asexual Spores . continued .. Phytophthora • parasitica var. niootiana spherical to oval clustered Plasmodiophova 0.1-0.8 p brassioae spherical NP Antheridia Aahlya bisexualis 0.6-1.3 p spherical to ovoid random. Pythium ultimum 0.2-0.6 y* spherical* random* Morphological Description Reference Lipid droplets. Do not have 1 imi t -ing .'membranes. Gluteraldehyde f i x a -tion with osmium tetroxide post - f i xa -t ion . Lipid droplets. Abun-dant. Gluteraldehyde f i x a -tion with osmium tetroxide post - f i xa -t ion . Lipid droplets;, non-membrane bound; pro - , bably re la t i ve ly saturated compounds; common but not abun-dant. GIuteraldehyde-osmium tetroxide f i xa t ion . Reichle, 196Sa, , 1959b Williams and Yukawa, 1967 Ricker (this report) Lipid droplets. Glutaraidehyde-f i xa -tion with, osmium tetroxide post-f i x a t i o n ; acro-le in f ixat ion with osmium tetroxide post - f i xat ion , i 'larchant, 1963 TABLE 7,111 - continued LIPID DROPLET MORPHOLOGY AND DISTRIBUTION Cell Type /.Organism Size Shape ."".".".".".".'"Distribution Oogonia .. Aohlya bisexualis 0.5-1,2 _p in spherical to.ovoid.. random young oogonia . . . 0.7-1.7 p'in . oospheres Pythium ultimum 0.4-1.0 p* spherical* random but generally Saprolegnia NP NP terrestris Oospores Aohlya bisexualis 0.1-3.5 p spherical to highly irregular in ooplenium random Morphological Description. Reference Lipid drop!ets; non- . membrane-bound; probably re lat ive ly saturated compounds; bbecome increasingly abundant in older oogonia. Glutaraldehyde-os- . mium tetroxide f i xa t ion . Bicker (this report) . Lipid droplets Abundant. KMn04 f ixat ion or glutaraldehyde f ixat ion with os-mium tetroxide post - f i cat ion ; acrolein f ixat ion with osmium tetro -xide post - f ixat ion. Marchant, 1953 Membrane-bound stor -age material which probably is 1 ip id . Not preserved with KMn0„. Moore and Howard, 1958 Lipid droplets often coalesce to form large i r regular ly -shaped globules; non-membrane-bound; pro-bably re lat ive ly saturated compounds; extremely abundant. Glutaraldehyde-osmium tetroxide f i xa t ion . Ricker (this report) TABLE llll - continued .• . LIPID DROPLET MORPHOLOGY AND DISTRIBUTION Cell Type . Organism. Size Shape Distr ibution .Oospores .•• Pythium ultimum 0.4r0.7 -u* spherical* about periphery continued _ oospore Plasmodia Plasmodiophora 0.1-0.8 JJ brassiaa spherical random .Morphological Description . Reference Lipid droplets. Marchant, Abundant'. 1968 Glutaraldehyde f i x a -tion with osmium tetroxide post - f i xa -tion or acrolein f ixat ion with os-mium tetroxide - - -post - f ixat ion. Lipid droplets. . Williams and Abundant. Yukawa, Glu taral dehyde-f i xa - 1967 tion with osmium tetroxide post-f i xa t ion . co o TABLE CENTRIOLE MORPHOLOGY AND DISTRIBUTION Cell Type Organism Size Shape Distr ibution Vegetative . Aohlya bisexualis ca. 165 mp cyl indrical one pair of centrioles Hyphae • long,., adjacent to modified 165 mp in area of nuclear enve-diam. lopes Albugo Candida ca. 200 mp cyl indrical perinuclear in length, 200 mp in . diam. Pythium ultimum ca. 230 mp cyl indrical perinuclear in diam.* length -.NS / Morphological Description Reference Each centr iole con - ' Ricker (this • s ists of nine p e r i - report) pheral t r i p l e t tubules oriented about a single , •' I central tubule in a cartwheel pattern; radial filaments in ter - ..; connect central tubule to peripheral t r i p l e t s ; inter-connections be-tween centrioles and i n -tra-nuclear spindle f i b r i l s are indiscer -nable. Glutaraldehyde-osmiun; tetroxide f i xat ion . Consist of nine t r i p -lets (three appres-sed tubules) connect-ed to a central tu -bule by radial e le -ments in a cartwheel arrangement; paired centrioles arranged end-to-end with l i t t l e or no angle between them. Glutaraldehyde f i x a -tion with osmium tetroxide post - f ixa -t ion . Berl in and Bowen, 1964 Composed of nine even- Grove - see ly spaced groups of Bracker, t r i p l e t microtubules. 1967 Glutaraldehyde f ixat ion Grove, Bracker with osmium tetroxide and Morref post f i xa t ion . 1970 Cell Type Organism Size TABLE XIV - continued CENTRIOLE MORPHOLOGY AND DISTRIBUTION ShaDe Distribution Morphol ogical Description Reference Vegetative. Hyphae continued Differentia-ting Vege-.tative Hyphae Saprolegnia . ferax Achlya bisexualis Gemmae Achlya bisexualis Sporangia Achy la bisexualis Albugo Candida ca 200 mp in length, 200 mp in diam. ca. 165 mp ' long, 165 nvp in diam. cylindrical cylindrical ca. 165 mp long, 165 mp in diam. cylindrical adjacent to nucleus at pole of intra-nuclear spindle. one pair of centrioles l ie adjacent to modified area of nuclear enve-lope two pairs of centrioles 1ie adjacent to modi -fied area of nuclear envelope (see TABLE Xy - Flagellar Morphology and Distribution) (see TABLE XV - Flagellar Morphology and Distribution) As described by Ber-l in and Bowen (1964) Glutaraldehyde f ixa- . tion with osmium tetroxide post-fixa-tion. Each centriole consists of nine peripheral triplet tubules orient-ed about a single cen-tral tubule in a cart-wheel pattern; radial filaments inter-connect central tubule to peri-pheral tr iplets. Glutaraldehyde-osmium tetroxide fixation. Each centriole consists of nine.peripheral triplet tubules orient-ed about two central tubules in a cartwheel pattern; radial f i l a -ments interconnect central tubules to peripheral triplets. Glutaraldehyde-osmium tetroxide fixation. Heath and Greenwood, ' 1968 Ricker (this report) Ricker (this report) co ro / Cell Type Organism Size TABLE JCIV - continued CENTRIOLE MORPHOLOGY AND DISTRIBUTION Shaoe Distr ibution Morphological Description Reference Sporangia continued Phytophthora aapsioi 0.25-0.34 p in diameter, length - NS cyl indr ical paired centrioles l i e next to narrow poles of . nuclei Phytophthora parasitica 0.5 p in length 0.2 n in diam. tube-1ike pairs near nuclei Saprolegnia ferax ca. 200 my in diameter cyl indrical one pair of centrioles in close proximity to each nucleus Display typical nine-f i b r i l arrangement with, a t r i f i b r i l i a r structure; rapidly elongate to form kinetosomes ( i . e . , f l age l la r basal bodies - see TABLE XV -F lagel lar Morphology, and Distr ibut ion) . KMnO^  f i x a t i o n ; g lu -taraldehyde f ixat ion with osmium tetroxide post - f i xat ion . Tube-like structures with one f l a t end and the other round-ed; have an electron-dense wall with fine longitudinal str ip ing and a less-dense homogeneous content -represent procen-t r io les - lack nine peri pher'al ly-arranged f i b r i l s . Osmium.tetroxide f i x a -t i o n ; KKnO^ f i x a t i o n ; glutaraideiiyde f i x a -tion with osmium te t -roxide or KMnO^  post-f i xa t ion . \ Composed of nine per i -pheral f i b r i l s ; cen-t r io les at r ight angles. Osmium tetroxide f i x a -t i o n ; KMn0« f i xa t ion ; glutaraldehyde f i x a -tion and osmium te t -roxide post - f ixat ion. Williams and Webster, 1970 Hohl and Hamamoto, 1957 Gay and Greenwood, 1966 Cell Type Organism Size TABLE XIV - continued CENTRIOLE MORPHOLOGY AND DISTRIBUTION Shape Distr ibution Morphological Description Reference' Asexual Spores Aahlya bisexualis (see TABLE XV - Flagellar Morphology and Distr ibution) (see TABLE XV - Flagellar Morphology and Distribution) Antheridia Phytophthora aapsiai Phytophthora . megasperma var. sojae Phytophthora • parasitica Phytophthora parasitica var. niootiana Achlya bisexualis Pythium ultimum (see TABLE XV - Flagellar Morphology and Distribution) (see TABLE XV - Flagellar Morphology and Distr ibution) (see TABLE XV - Flagellar Morphology and Distribution) ca. 165 mp cyl indrical long, 165 mp in diam. one or two centr iolar pairs, usually l i e in depression of modified area of nuclear enve-lopes 180-190 mp in diam.* length - NS NS adjacent to a depression in nuclear envelope Each centriole con-s is ts of nine pe r i -pheral t r i p l e t tubu-les oriented about a single central tubule in a cartwheel pat-tern; radial f i l a -ments interconnect central tubule to peripheral t r i p l e t s ; intra-nuclear spindle f i b r i l s penetrate nuclear envelope and attach to centr iolar s a t e l l i t e s . GIutsraldehyde-osmi um tetroxide f i xa t ion . NS ' . KMn04 f i x a t i o n ; g lu -taraldehyde f ixat ion with osmium tetroxide post - f i xat ion ; acro-le in f ixat ion with osmium tetroxide post-f i xa t ion . Ricker (this report) co -F=> Marchant, 1958 . Call Type Organism Size TABLE .XIV - continued CENTRIOLE MORPHOLOGY AND DISTRIBUTION Shape Distribution Oogonic Aohlya bisexualis ca. 165 mp long, 165 .mp in diam. cyl indrical one or two centr iolar pairs l i e adjacent to modified area of nuclear envelopes Oospores • Aohlya bisexualis NS NS NS Unidentified Aohlya sp. NS cyl indr ical l ies adjacent to modi-Cells : f ied nuclear envelope Morphological Description Reference Each centriole con-s ists of nine pe r i -pheral t r i p l e t tubu-les oriented about a single central tubule, in a cartwheel pattern; radial f i l a -. ments interconnect central tubule to peripheral t r i p l e t s ; intra-nuclear spin-dle, f i b r i l s penetrate nuclear envelope and attach to centr iolar s a t e l l i t e s . Gl utaraldehyde-osmium tetroxide f i xa t ion . NS Consists of two parts interrupted at the' center; shows nine t r i p l e fibers and cartwheel center; nuclear envelope adjacent to centriole is modified inside and outside. Fixation not given. Ricker (this report) . Ricker (this report) Turner -see Figure 19.2 . Brown and Bertke, 1969. •• •• TABLE .Xy :Cell Type Organism Size FLAGELLAR MORPHOLOGY AND DISTRIBUTION Shape . Distribution Morphological Description Reference Vegetative Hyphae Differentiating Vegetative . Hyphae Gemmae Sporangia See Table XIV - Centriole Morphology and Distribution See Table XIV See Table XIV Aahlya bisexualis -Cent r io le Morphology and Distribution - Centriole Morphology and Distribution basal body cyl indr ical 160-165 my • in diameter; 650-680 my in length at- anterior pole of pyriform-shaped nuclei - near zoosporangial wall in peripherally-oriented nuclei Formed of nine car t -wheel and c y l i n d r i -cally-arranged, equal-ly-spaced tubules -tubules appearing as doublets or t r i p l e t s . Doublets pass through basal plate at d i s -tal end of basal bod-ies ; t r ip le ts char-acterize proximal end. Microtubules radiate from basal bodies into surround-ing protoplasm -usually remaining in close proximity to closely-associated nucleus. Groups of microtubules also inter-connect basal bodies of each pair ; axonemal sheath be-comes associated with distal ends of the basal bodies. Glutaraldehyde-osmium tetroxide f i xa t ion . Ricker (this report) Cell Type Organism Size TABLE Xy - continued FLAGELLAR MORPHOLOGY AND DISTRIBUTION Shape Distr ibution Morphological Description Reference Sporangia . continued Albugo Candida basal body 160-165 mu in diameter; 550-680 mp in length cyl indrical on sides of nuclei near-est sporangia! wall Olpidium . brassicae NS cyl indr ical between perinuclear membrane system and nucleus Phytophthora capsioi kinetosome -0.25-0.34 u in diameter; 0.65 v in length cyl indrical adjacent to narrow pole of nuclei Consists of nine doub- . Berl in and • lets around two cen- Bowen,. t ra l tubules; one 1964 flage!1 um develops from each of two basal bodies ; basal plate separates flagellum ' •• • from basal body; spur f ibers and rhizo-plasts associated with basal body. • • Glutaraldehyde f i x a -tion with osmium tetroxide post-f i xa t ion . One of two centrioles Temmink becomes a kinetosome and Camp-and elongates; pushes b e l l , 1969 ectbplast into c lea - , . •  vage vacuole; axone-mal sheath formed. Glutaraldehyde f i x a -t ion with osmium tetroxide post - f i xa -t i o n ; Karnovsky's f ixat ion with osmium tetroxide post-f i x a t i o n ; KMnO^  f i xa t ion . Centrioles with nine- Williams f i b r i l arrangement . and Web-with t r i f i b r i l l a r s ter , 197C structure elongated to form kinetosomes; la t te r extends through cytoplasm toward re-ceptive axonemal ves i -c l e s ; axonemes form in ves ic les , possess cont'd 0 0 Cell Type Organism Size TABLE XV - continued . FLAGELLAR MORPHOLOGY AND DISTRIBUTION Shape Distr ibution Morphological Description Reference Sporangia continued Phytophthora aapsiai continued Phytophthora parasitica basal bodies 0.2 y in diameter; 0.7 y in length see above tube-l ike see above Asexual Spores Aahlya bisexualis basal bodies 160-165 my ir, diameter; 650-680 my in length cyl indrical in close association with pointed end of nucl ei see above at anterior pole of pyriform-shaped nuc le i ; in secondary zoospore two f lage l la are insert -ed in groove along side of ce l l - t insel f l a -. gellum directed anter-i o r l y , whip-lash f l a -gellum directed pos-te r io r l y typical 9 + 2 morphology and ter -minal plate; nucleus . connected to kinetid base via microtubules; f l age l la develop early in zoosporangial d i f -ferent iat ion. KMnO, f i x a t i o n ; gluter- . aldehyde f ixat ion with osmium tetroxide post-f i x a t i o n . NS Osmium tetroxide f i x a -t i o n ; KMnO^  f ixat ion with osmium tetroxide or KMnO^  post-f ixation FIagella form but de- . generate. Glutaraldehyde f i x a -tion with osmium tetroxide or KMnO^  post - f i xat ion . Flagella formed of basal body, axoneme, and root lets ; basal body and rootlets pre-sent in primary zoo-spore, axoneme formed upon di f ferent iat ion of secondary zoospore. . Basal bodies morpho-log ica l l y same as those in zoosporangial pro-toplasm. Axoneme formed by elongation of doublet tubules; . membrane about axo-neme formed from Hohl and Ham-amoto, 1967 Hemmes and Hohl ,1959 Ricker (this report) cont'd TABLE XV - continued FLAGELLAR MORPHOLOGY AND DISTRIBUTION Cell.Type Organism Size Shape . . .Distr ibut ion Asexual ••' ... • Achlya bisexualis Spores continued .. . • • •. continued. Olvidium brassi- axonemal f i - cy l indr ical* NS . a a e b r i l s 160-170 . mp in d i a -meter i Morphological Description Reference axonemal vesicles . Rootlets formed of groups of microtu-bules which are at - . tached to basal body and pass into proto-plasm near parastra- ' somes - p a r t i a l l y surrounding the 1 at -ter structure. Whip-lash flagellum with tapered distal region; f ine hairs 300-450A long, and 120-150A in diameter,. often d i s -cernable on surface of axoneme. Tinsel flagellum has hairs which are 240-250A' in diameter for approx. 3/4 their length and 120-130A in diameter the terminal 1/4 length; total length of mastigonems 2-3 p. Glutaraldehyde-osmium tetroxide f i xa t ion ; shadow-casting. 9 + 2 axonemal f i b - Temmink r i l s ; retraction and Campb occurs; f i b r i l s not 1969 surrounded by membrane in cytoplasm and gra-dually become d i s t o r t - , ed; axonemal sheath pulled in and breaks to form coiled mem-branes. Glutaraldehyde f ixat ion with osmium tetroxide post - f ixat ion (Sabatini et al.); Karnovsky's Cell Type Organism Size TABLE Xy - continued FLAGELLAR MORPHOLOGY- AND DISTRIBUTION Shape . .Distr ibution Morphological Description' Reference Asexual Spores continued Phytophthora erytiirosepiioa Phytophthora megasperma var. sojae NS kinetosome -* 190-200 my in diameter; ca. 600 my in length axoneme -(excluding sheath) 190-200 my in diameter as above cyl indr ic to tubular • NS cyl indr ic to tubular two f lage l la inserted in groove running along longitudinal axis as aoove as above Thicker part of f l a -gellum bears f ine hairs less than 60A in diameter; knob-l i k e structures - con-s i s t of coiled-up flagellum axis within sheath, present on ends of f lage l la in aged zoospores. Shadowed. Axoneme made up of peripheral r ing of nine double f i b r i l s and two central f i b -r i l s ; surrounded by sheath which is con-tinuous with unit membrane; mastigonemes present on one f l a g e l -lum; central f i b r i l s stop at basal plate; peripheral f i b r i l s continue as kineto-some where they ap-pear as inwardly i n -clined t r i p l e t s . Glutaraldehyde f ixat ion with osmium tetroxide post - f i xat ion . Beads present on f l a -gella prior to zoo-spore encystment; axoneme coiled with f inely f i b r i l l a r matrix of sheath; axoneme d i s -plays 9 + 2 arrangement GlutHraldehyde f ixat ion with osmium tetroxide post - f ixat ion. Vu j i c i c , • Chapman and Colhouris 1965 . Ho, Zachar-iah and Hickman, • 1968 Ho, Zachar-iah and Hickman, 1967 Cell Type Organism. Size TABLE .Xy continued FLAGELLAR MORPHOLOGY AND DISTRIBUTION Shape Distr ibution . Morphological Descript ion. . Reference Asexual Spores . Continued Phytophthora : palmivora ca. 200 m y*. in diameter cy l indr ic to tu-bular NS Phytophthora . parasitica var. nicotiana ca. 200 my in diameter cyl indr ic to tu-bular two f lage l la inserted in rai sed area of a de-pression running in longitudinal direction on side of spore, in close association with nucleus Desjardins, Zentmyer, and Reynolds, 1969 Whiplash flagellum bears f ine la te ra l . hairs 500 my long and 15 my wide; lateral hairs on f u l l length of t insel f l a -gellum 2 y long -measure 30 my for 3/4 of i t s length and 13 my in apical 1/4 of i t s length. Osmium tetroxide f i x a -t ion ; gluteraldehyde f ixat ion - unidirec-tional and rotary sha-dowcasting techniques at angles of 3:1 and 4 :1 . Kinetosomes l i e 90° to Reichle, each other; formed 1969a of nine sets of doublet-t r i p l e t f ibers ; doub-lets pass through ter -minal plate into f l a -gellum proper; has one . short and one long root let ; long-rootlet formed of eight micro-' tubules, transverses 1/2 of zoospore from which microtubules arise at 90° angle in r ib . - l ike fashion; other microtubules originate near proximal end of kinetosome and radiate out into cytoplasm. Anterior of flagellum has mastigonemes which are of uniform th ick -ness (173-190A in d i a -cont'd lABLE _XV - continued FLAGELLAR MORPHOLOGY AND DISTRIBUTION Cell Type Organism . •. .. Size. Shape Distr ibution Asexual • Spores continued Phytophthora parasitioa v a r . . n i c o t i a n a • continued see above see above see above Saprolegnia ferax ca. 250 mp in diameter long, cylinder or tube front and 'hind f l a gel la meter) for 2/3 their length, the terminal third thinner (20-56A in diameter) and con-s ists of two substrands twisted around one another. Posterior flagellum is whiplash; has minute hairs 230-300 my long and 30A thick. Glutaraldehyde f ixat ion with osmium tetroxide post - f i xat ion ; nega-tive staining. Retracted f lage l la r Reichle, axonemes in freshly 1969b ; encysted spores. Glutaraldehyde-osmium tetroxide f i xa t ion . "Flimmer hai rs" , 2-3 y long, arranged in two rows of front f l a -gel Turn; sheath about flagel 1 um - with 11 f i b r i 1 s , the two cen-tral f i b r i l s being more undulate and easi ly broken; hind flagellum devoid of "Flimmer" and is covered by sheath. Short hairs 0.'5 y long sometimes v i s ib le on sheath. Osmium tetroxide f i x a -tion and staining. Manton, Clarke and Greenwood, 1951 , 1952 Cell Type Organism Size.. TABLE X)l - continued FLAGELLAR MORPHOLOGY AND DISTRIBUTION .Shape . . . . . . Distr ibut ion. . Morphological .Description . . . .Reference Antheridia Oogonia Oospores See Table XIV - Centriole Morphology and Distr ibution See Table XIV - Centriole Morphology and Distribution see Table XIV - Centriole Morphology and Distribution CO 1 \ TABLE .XVI Cell Type Organism Size NUCLEAR MORPHOLOGY AND DISTRIBUTION Shape • Distr ibution Morphological Description Reference Vegetative Aahlya bisexualis .1.2-1.3 JJ Hyphae in diameter; 3.0-5.0 p in length ovoid to spindle-shaped Aphanomyces euteiahes ca. 4.2 v in* diameter irregular Phytophthora infestans ca. 1.2 p in* diameter spherical to* irregular usually absent in apical crowing zone ana sub-apical mitochondrial -zone; in peripheral protoplasm of d ista l multi-organelle zone throughout mycelium; usually along the hyphae; sometimes in clusters of two to several in central core of hyalo-plasm; absent in haustoria Nuclei bounded by nuclear envelope which becomes struc-tura l ly modified in area of centr iolar association - is thicker and appears to form s l ight depres-sion in p r o f i l e ; spin-dle f i b r i l s sometimes radiate from the thickened area of the envelope. Nucleolus consists of wel1-de-fined pars fibrosa and pars granulosa; pars chromosoma dist inguish-able in a few nucle i . Glutaraldehyde-osmium tetroxide f i xa t ion . Ricker (this report). Numerous nuc le i ; their envelope interrupted by nuclear pores; nucleolus appeared as electron-dense area in nucleus. Glutaraldehyde f i x a -tion and osmium tetroxide post - f i xa -t ion . Bound by c lassical unit membrane in ter -rupted by pores'. KMnO. f i xa t ion ; os-mium tetroxide f i x a -tion with KMnO. post-f i xa t ion . Shatla, Yang and Mi tche l l , 1966 Ehrlich and Ehr l ich , 1965, 1966 / Cell Type Organism Size TABLE iyi - continued . NUCLEAR MORPHOLOGY AND DISTRIBUTION Shape Distr ibution Vegetative Hyphae continued Phytophthora parasitica 1.6 JJ in* diameter spherical to* irregular in hyphae but absent in haustoria Pythium debary-anum irregular irregular NS Pythium ultimum ca. 6.2 J J * spherical , ovoid* absent from apical zone to irregular in and in anterior 15-20 JJ prof i le of sub-apical, zone; distributed throughout rest of hyphae Morphological Description Reference. Surrounded by t y p i - Ehrl ich and cal nuclear enve- Ehr l ich , lope consisting of 1966 two membranes separ-ated from one another by perinuclear space; pores present in en-velope. KMnO^  f i xa t ion ; osmium tetroxide f ixat ion with KMnO^  post-f i xa t ion . Nuclear membrane of Hawker and three layers - an Abbott, inner l ight layer and 1963 two enclosing thin dark layers; numer-ous pores in membrane and occasionally gaps; may be connected with ER. KMnO^ , f i xa t ion . Nuclear envelope struc-tura l ly inter -asso-ciated with ER, d ic ty -osomes and associated vesicles. Osmium tetroxide f i x a -t i o n ; KMnO« f i x a t i o n ; glutSraldenyde arid acrolein f ixat ion with osmium tetroxide post-f i xa t ion ; glutaralde-hyde f ixat ion with os-mium tetroxide post-f i xa t ion . Grove, Bracker, and Morre, 1970 Cell . Type .Organism Size TABLE XVI - continued NUCLEAR MORPHOLOGY' AND DISTRIBUTION Shape Distribution Morphological Description Reference Vegetative Hyphae continued Saprolegnia ferax ca. 2.7 .„* irregular* present throughout ap i -cal regions of hyphae NS Osmium t ion . :etroxide f i x a -Gay and Greenwood, 1968 . NS Gemmae Saprolegnia terresiris Aohlya bisexualis NS 4.0-4.5 a in length; 1.2-1.5 u in diameter bporangia Achlya bisexualis ca. 1.5-2.0 i, in diameter; ca. 4.0-4.5 .n in length irregular to dumb-* NS bell-shaped globose various config-urations -shaped, ovoid, irregular i n i t i a l l y ovoid to spindle-shaped, but rapidly become pyriform not part icular ly associated with ER random random in young zoo-sporangia, but rapidly become oriented equi-distant to each other -anterior pole is aligned toward zoo-sporangial wall Somatic nuclei in state of d i v i s ion ; intranuclear spindle of about 40 micro-tubular f i b r i l s ; convergent to poles close to inner mem-brane, with atten-dant centr io les . Gluteraldehyde f i x a -tion with osmium tet -roxide post - f i xat ion . NS Morphologically s imi -lar to those in ve- . getative hyphae ex-cept that envelope membranes are more . crenulate in prof i le and nucleoli are larger. Glutaraldehyde-osmium tetroxide f i xa t ion . Morphologically s imi -lar to those in vege-tative hyphae; how-ever, when pyriform-shaped the nucleolus becomes oriented in posterior pole of nuc le i ; modified thickened envelope Heath and. Greenwood, 1968 Moore and Howard, 1968 Ricker (this report) . ID CTl Ricker (this report) cont'd TABLE XVI - continued NUCLEAR MORPHOLOGY AND DISTRIBUTION Cell Type . Organism. Size Shape Distribution .Morphological Description Reference Sporangia continued Aohlya bisexualis continued Aphanomyoes euteiches NS irregular NS Phytophthora NS capsici obovate-pyri-form (nuclei adjacent to f lagel la) narrow pole toward peri -phery of sporangium at anterior pele of nuc le i ; cluster of osmiophilic granules also present in nu-cleoplasm at anter-ior pole. Glutaraldehyde-osmium tetroxide f i xa t ion . Numerous nucle i ; their envelope interrupted by nuclear pores; nucleolus appeared as electron-dense area in nucleus. Glutaraldehyde f i x a -tion with osmium tetroxide post - f i xa -t ion . No dividing nuclei observed in sporan-gia mature enough to develop zoospores; surrounded by t r i p l e -layered membrane con-taining numerous pores; membrane sub-units about 50A in diameter; nucleo-plasm compact and grainy - is composed of numerous tubules and f ib res . Definite chromosome structure not seen. KMnO, f i xa t ion ; g lu -taraldehyde f ixat ion with osmium tetroxide post - f i xat ion . Shatla, Yang and Mi tche l l , 1966 Williams and Webster, 1970 AO TABLE XVI - continued NUCLEAR MORPHOLOGY AND DISTRIBUTION Cell Type Organism Size Shape Distr ibution Sporangia Phytophthora 1.5-3.0 JJ continued . . erythi-oseptioa in diameter roughly e l l i p s o i - near periphery of* dal with minor sporangium i r regular i t ies in out! ine Morphological Description Reference Possess a homogen- Chapman and eous f inely-granular Vu j ic ic , appearance; nuclear 1965 envelope comprises double membrane t r a -versed by numerous pores; continuity between nuclear mem-brane and membranous organelles in c y t o - . plasm. Osmium tetroxide f i x a t i o n ; KMnQ. f i xa t ion . TABLE XVI - continued NUCLEAR .MORPHOLOGY AND DISTRIBUTION Ceil Type Organism Size Shape Distr ibution Morphological Description Reference Sporangia continued. Phytophthora parasitica Saprolegnia . ferax Asexual Spores Achlya bisexualis ca. 2.3-3.0 . •> in diameter; ca. 3 .2 -4 .0 in length 2.0-3.5 * ca. 1.5-2.0 in diameter; ca. 4 .0 -4 .5 in length Phytophthora erytiirosepiica NS spheroid in f u l l y -expanded resting sporangium; pear~ shaped during zoospore formation pyriform to irregu-* lar . pyriform become equidistant from each other in lobes of peripheral protoplasm . central to sub-central in encysted primary zoospore; anterior pole in association with basal bodies secon-dary zoospore oriented towards lateral groove NS Nucleus becomes pear-shaped and surround-ed by layer of c l e a -vage yes ic les ; basal bodies l i e at pointed end of nuclei . Osmium tetroxide f i x a -t i o n ; KMnO, f i x a t i o n ; glutaraldehyde f i x a -t ion with KMnO,, or osmium tetroxide post - f i xat ion . Nuclei with porous double membrane; nucleoli and chroma-t in material resemble in general those of interphase nucle i . Osmium tetroxide f i x a -t i o n ; KMnO. f i x a t i o n ; glutaraldehyde f i x a -tion with osmium tet -roxide post - f ixat ion. Nuclei s imilar to those in primary zoospore i n i t i a l s ; nucleolus in poster-ior region; anterior pole par t ia l l y bound-ed by f lage l la r sheath - cluster of osmiophilic granules not present in nucleo-plasm of anterior pol e. Glutaraldehyde-osmium tetroxide f i xa t ion . Golgi dictyosomes of -ten found in proximi-ty, to nucl e i . Fixation not given. Hon! ana Hamamoto, 1967 '. Gay and Greenwood, 1966 Ricker (this report) .Vuj ic ic , Chap-man and Col -houn, 1965 Cel 1 Type .Organ is::; . . . . . . Size. Shape TABLE .XVI - continued NUCLEAR MORPHOLOGY' AND DISTRIBUTION Distr ibut ion . . . . . . . 'Morphological .Description Reference Asexual Spores continued Phytophthora : megasperma . var. sojae 3.0-3.6 u* pear-shaped narrow end oriented towards groove region Phytophthora parasitica 3.2-3.5 u * pear-shaped narrow end oriented towards groove region but in center of spores •Antheridia Achlya sp. NS NS Achlya ambi-sexualis NS NS MS Large nucleus with, conspicuous nucleo-lus ; bounded by double nucl ear mem-brane; concentric rings of rough ER around nucleus. Glutaraldehyde f i x a -tion with osmium tetroxide pos t - f i xa -t ion . Nuclei with pointed t ip with which f l a -gel lar kinetosomes are associated. Glutaraldehyde f ixat ion with osmium tetroxide f i xa t ion ; KMnO« f i x a -t ion . Nuclei divide by meiosis; metaphase plates observed; se-cond div is ion not ob-served . Light microscopy -alcohol: acetic acid (3:1) f ixat ion Even's modification of Carnoy's f i x a -t ion . Nuclei divide by -meiosis during gam-etogensis; stages . associated with f i r s t nuclear d i v i -sion d i f fe r from those in second d i v i s i o n ; number of chromosomes Ho, Zachariah. and Hickman, 1968 . ..• -Reichle, 1969a, 1969b Sansome, 1965 ho o o Barksdale, 1968 (see Bryant .and Howard, 1959) cont'd Cell Type Organism TABLE XVI - continued NUCLEAR MORPHOLOGY AND DISTRIBUTION Size.. . Shape Distr ibut ion. Morphological Description Reference Antheridia continued Aahlya . 'arribi-sexualis continued Aahlya bisexualis Aahlya oolorata 1.5-2.3 y in diameter; 3 .0 -4.7 y in length prior to and during prophase I of meiosis; 1.4-2.0 y in diameter; 2.0-3.2 y in length after meiosis spindle-shaped to irregular dumb-bell shaped during telophase of nuclear d i v i -sion in peripheral proto-plasm except in actively growing t ips or during f e r t i l i z a t i o n when nuclei are near t ip NS. NS NS at poles at end of. each div is ion is three. Fixation not given. Nuclei bounded by nu- Ricker ( th is ; Clear envelope which report) becomes structural ly modified in area of centr iolar associa-t ion , then becomes thicker and s l ight l y depressed in p r o f i l e ; spindle f i b r i l s r a -diate from thickened area of envelope during nuclear d iv is ion . . Prophase I, early ana-phase, telophase stages of meiosis observed -acentric to centric intra-nuclear spindle of microtubules vi% si bl e; nucleolus persistent; numerous nucl ei prior to f e r -t i l i z a t i o n but many degenerate after fe r -t i l i z a t i o n . Glutaraldehyde-osmium tetroxide f i xa t ion . Nuclei divide by meio- Sansome, s i s ; metaphase plates 1955 observed; second d i v i -sion not observed. Light microscopy - a l -cohol :acetic acid (3:1.) f i x a t i o n , Evan's modi-f i cat ion of Carnoy's f i xa t ion . . . . TABLE XVI - continued NUCLEAR MORPHOLOGY AND DISTRIBUTION Cell Type Organism Size' Shape Distr ibution ..Antheridia . Phytophthora NS NS NS continued', ' caotorum Phytophthora NS NS NS erythroseptioa PyiTiium debar-yanum NS NS near growing t ip* Morphological Description Reference Numerous nuc le i ; i n - Sansome, crease in size and 1965 enter prophase simul-taneously , followed by metaphase in which chromosomes most d i s - : -t i n c t , then anaphase; •. second div is ion f o l -lows; one nucleus passes into oogonium, others degenerate; num-ber of bivalents approximately nine. Light microscopy -alcohcl :acet ic acid (3:1) f i x a t i o n ; Evan's modification of Carnoy's f i xa t ion . Observations in accor-dance with those of P. oaatorum; approx-imately nine bivalents Light microscopy - alec hoi :acetic acid (3:1) f i x a t i o n ; Evan's modification of Car-noy's f i xa t ion . One or sometimes two Sansome, nuclei/antheridium; 1951 haploid number of chromosomes approx. twenty; meiotic d i v i -s ions, metaphase f i -gures observed. Light microscopy - aceto-orcein squash prepara-t ion . Sansome,-ho 1965 g Cel1 Type Organism Size TABLE xn - continued NUCLEAR MORPHOLOGY AND DISTRIBUTION Shape Distr ibution Antheridia continued Pythium debar-yanum continued NS NS NS Pythium ultimum ca. 1.0 y. in* diameter; 2-2.3 p in length ovo\a* NS Saprolegnia terrestris NS globose NS ca. 2.5 y in diameter in early prophase; 0.7-1.0 y in diameter after double d i v i -sion spherical , ovoid* to s l ight ly irregular random* / Morphological Description Reference Association of four . Sansome, chromosomes in d i v i - 1963 ding nuc le i , ind ica -t ive of meiotic d i v i -s ion; nuclei undergo two divisions - resul t -ing nuclei appro*, half the size of vegetative nuc le i ; metaphase figures observed. Light microscopy - aceto-orcein squash prepara-t ions; alcohol :acetic acid (3:1) f i xa t ion ; Evan's modification of Carnoy's f i xa t ion . Several nuclei v i s i -ble in single section. KMnO* f i x a t i o n ; g lu -taraldehyde f ixat ion with osmium.tetroxide post - f i xat ion ; acro-le in f ixat ion with osmium tetroxide post - f ixat ion. Marchant, 1968 Nuclei not part icu-la r l y associated with ER. KMnO^  f i xa t ion . Microspectrophotometer analysis reveals pre-div is ional nuclei as 4C; subsequent re -duction div is ion pro-duces 1C nuclei . Light microscopy -ethyl alcohol ; acetic acid (3:1) f i xa t ion ; stained with Sch i f f ' s base.. Moore and Howard, 1963 Bryant and Howard, 1959 Cel1 Type Organism Size TABLE XVI - continued NUCLEAR MORPHOLOGY AND DISTRIBUTION Distribution Shape Oogonia Achlya sp. NS NS NS Achlya bisexualis 1.5-2.3 JJ in. diameter; ; 3.0-4.7 u in length prior to and during prophase I of meiosis, 1.4-2.0 p i n d ia -meter, 2.0-3.2 y i n length after meiosis, as seen in oospheres spindle-shaped, ovoid to irregular; random prior to vacua!a-t ion ; in peripheral . protoplasm after vacuolation Morphological Description . Reference Nuclei divide by meiosis; meta-phase plates observed; second div is ion not observed. Light microscopy •-alcohol:acetic acid (3:1) f i x a t i o n ; Evan's modification of Carnoy's f i xa t ion . Nuclei bounded by nu-clear envelope which becomes modified in area of centr iolar association, then becomes thicker and s l ight l y depressed in p r o f i l e ; spindle f i -b r i l s radiate from thickened area of envelope during nu-clear d iv is ion . Interphase and pro-phase stages of meio-sis observed - acen-t r i c to centric i n t r a -nuclear spindle of microtubules v i s i b l e ; chromosomes v i s i b l e , nucleolus persistent; numerous nuclei appear to degenerate during oosphere formation. Gluteraldehyde-osmium tetroxide f i xa t ion . Sansome, 1965 Ricker (this report) o TABLE XVI - continued NUCLEAR MORPHOLOGY AND DISTRIBUTION .Cell Type Organism Size Shape . Distr ibution .Oogonia , Achlya colorata NS NS NS ; continued •' Phytophthora NS NS NS . cactorum -Phytophthora NS NS NS erythroseptica Morphological Description Reference Nuclei divide by meio- Sansome, s i s ; metaphase plates 1965 observed; second div is ion not observed. Light microscopy -alcohol :acetic acid (3:1) f i x a t i o n ; Evan's modification of Carnoy's f i x a t i o n . Numerous nucle i ; i n - Sansome, crEi.se in size and 1955 enter prophase sim-ultaneously, followed by metaphase in which chromosomes most d i s -t i n c t , then anaphase; second div is ion f o l -lows; number of b i -valents approximately nine. Light microscopy -alcohol :acetic acid (3:1) f i x a t i o n ; Evan's modification of Car-noy's f i xa t ion . Observations in accor- Sansome, dance with those of 1965 P.cactorum; approxi-mately nine bivalents. Light microscopy -alcohol :acetic acid (3:1) f i x a t i o n ; Evan's modification of Carnoy's f i x a t i o n . Cel T Type Organism TABLE x n - continued NUCLEAR MORPHOLOGY AND DISTRIBUTION Size Shape- Distr ibution Morphological Description . Reference Oogonia .' continued' Pythium -• debaryanum Pythium ultimum Saprolegnia terresiris NS NS. NS nuclei at end of 1st d i v i -sion ca. same size as somatic nuclei ; nuclei at end of 2nd division appre-ciably smaller. NS NS ca. 2 u in* diameter spherical to* si ightly irregular in peripheral region of* oogonium 2-3 • JJ in d i a -meter lobate in ooplasm, the peri -pheral fraction Two-six nuclei under- Sansome, . going division/oo- 1961 gonium; haploid num-ber of chromosomes is about 20;. meio-t i c d iv is ions ; meta-phase figures observed. Light microscopy -Aceto-orcein squash preparations. Young oogonia with Sansome, more nuclei than 1961 oogonia with d i v i d -ing nucle i ; nucleus increases in size prior to d i v i s ion ; prophase, metaphase - with chromosome number = 18, ana-phase stages ob-. served. Several nuclei v i s i - Marchant, ble in each section. 1968 KMnO^  f i xa t ion ; g lu -taraldehyde f ixat ion with osmium tetroxide f i x a t i o n ; acrolein f ixat ion with osmium tetroxide f i xa t ion . Nuclei surrounded by Moore and several layers of Howard, ER elements. - 1968 KMnO^  f i xa t ion . I\3 O cn TABLE XYI - continued NUCLEAR MORPHOLOGY AND DISTRIBUTION Morphological . • Cell Type' Organism Size ' Shape Distr ibution • Description Reference Oogonia. continued Saprolegnia terrestris continued ca. 2.5 y in diameter in earlv prophase; 0.7-1.0 y in diameter after double d i v i -sion spherical , ovoid* to si ightly irregular NS Microspectrophoto- " Bryant and metric analysis re - Howard, veals pre-divisional 1969 nuclei as 4C; subse-. quent reduction d i v i -sion produces i t nu-c l e i . . • Light microscopy - ethyl alcohol:acetic acid (3:1) f i x a t i o n ; s ta i n - . ed with Schi f f 's . base. ovoid to i rregu- sub-centric Nuclei bounded by nu- Ricker (this Tar clear envelopes; groups report) of microtubules in cross-section) some-times v i s ib le in nu-cleoplasm; f i b r i l l a r elements in central region of nucleus, granular material . near periphery of nucleus. Glutaraldehyde-osmium tetroxide f i xa t ion . Aohlya colorata NS spherical to ovoid eccentric Pairing and fusion of Ziegler , (as diagrammed) sexual nuclei in 1953 young oospores; hap-lo id number of chro-mosomes = 3; d ip lo id number - 5; meiosis seems to occur during germination. Light microscopy - chro-mic acid-formalin f i x a t i o n ; gram stained. Oospores Aohlya bisexualis 1.0-1.4 y in diameter; 3.0-4.5 v in length TABLE XVI - continued NUCLEAR MORPHOLOGY AMD DISTRIBUTION Cell Type Organism Size . Shape Distr ibut ion. . Oospores' Aahlya- NS • spherical to ovoid centric to subcentric continued. • ' msgaspevma (as diagrammed) Achlya reaurva NS spherical , ovoid, eccentric tear-shaped and irregular (as diagrammed) Morphological Description Reference Pairing and fusion Ziegler , of sexual nuclei in 1953. young oospores; l ep -. totene stage nucleus with d is t inc t nucleo-lus and ten leptotene threads; nucleolus fades in. pachytene; haploid number of chromosomes = 5 ; diploid number = 10. Light microscopy -chromic acid-forma-tion f i x a t i o n ; gram stained. Nucleus with d i s t i n c t , Z iegler , round, deeply-stain- 1953 ing nucleolus surround-ed by clear nucleo-plasm; few threads radiate from nucleo-lus to nuclear enve-lope; pairing and fusion of sexual nu-cl ei in young oospore; nuclei divide by meio-sis upon germination. Light microscopy -chromic-acid formalin f i x a t i o n ; gram stained. TABLE .XVI - continued NUCLEAR MORPHOLOGY AND DISTRIBUTION Cell Type Organism Size Shape Distribution Oospores• . continued Isoaahlya intermedia NS spherical to ovoid (as diagrammed) sub-centric Morphological Description Nuclei s imilar to others reported in this paper; haploid number of chromo-somes = 6. . Light microscopy-chromic acid-forma-l i n f i x a t i o n ; gram stained. Reference Ziegl er , 1953 Pythium ultimum 3-12 u in diameter NS centrally-placed Saprolegnia NS l i t o r a l i s spherical to ovoid centric to sub-centric* Nucleus occupies 1arge proportion of volume of oospore. KMnO» f i x a t i o n ; g lu -teraldehyde f ixat ion with osmium tetroxide post - f i xat ion ; acro-le in f ixat ion with osmium tetroxide post - f ixat ion Light microscopy -Feulgen stained. Nuclear pairing and fusion occurs; hap-lo id number of chro-mosomes = 7; pachy-tene stage observed. Light microscopy -chromic acid-forma-1 in f i x a t i o n ; gram stained. Marchant, 1968 ro o Ziegler , 1953 Saprolegnia terrestris 2-3 in lobate sub-centric diameter Nuclei surrounded by Moore and several layers - of Howard, ER elements. 1968. KMnO^  f i xa t ion . TABLE 1^1 -. continued NUCLEAR MORPHOLOGY AND JJISTRIBUTI Cell Type Organism Size Shape Distr ibution Oospores Thrausiotheoa NS continued primoachlya spherical to oyoid (as diagrammed) sub-centric / Morphological Description Reference Nuclear fusion occurs, Ziegler , d iploid nucleus form- 1953 ed; only pachytene stage of nuclear d i -v ision observed - 5 chromosomes present; mitosis also observ-ed upon germination. Light microscopy -chromic acid-formalin f i x a t i o n ; gram s ta i n -ed. PLATE ONE Vegetative hyphae of the male ( left) and female (right) strains of Aahlya bisexualis growing on ster i l i zed sesame seeds in d i s t i l l e d water. Three days growth. X 0.7 Hyphae of the male and female strains of Achyla bi-sexualis which are producing sexual reproductive structures after growing on ster i l i zed sesame seeds, in d i s t i l l e d water for three days. X 0.7 PLATE TWO Encysted secondary zoospore prior to germination. Nomarski microscopy. X 760 Encysted secondary zoospore germinating by a single germ tube. Nomarski microscopy. X 700 Encysted secondary zoospores germinating by two (figures 5,6,7) or three (figure 8) germ tubes. Note the development of branches on several of the germ tubes. Nomarski microscopy. Figures>5 and 6 - X 650 Figures 7 and 8 - X 700 PLATE THREE Figure 9 A vegetative hypha of Aohlya bisexualis (female strain) . A = apical growing zone; SA = sub-apical mitochondrial zone; D = distal mult i -organelle zone. Phase-contrast microscopy. X 240 Figure 10-13 A series of micrographs taken at 30-second inter-vals to show apical growth of the hypha as ob-served at greater magnification. Note the change in shape of the t ip as growth occurs. A = apical growing zone; SA = sub-apical mito4 , chondrial zone; D = distal multi-organelle zone. Phase-contrast microscopy. X 750 PLATE FOUR Figures 14-16 A series of micrographs taken at 30-second inter-vals showing apical growth in a vegetative hypha of Aohlya bisexualis (male strain) . Note the change in shape of the hyphal t i p . A = apical growing zone; SA = sub-apical mito-chondrial zone; D = distal multi-organelle zone. Phase-contrast microscopy. X 700 Figures 17-18 Apices of actively growing hyphae of A. bisexualis as observed at greater magnifications. The apical growing zone (A) appears as.;a conical cap of fine granules about the sub-apical mitochondrial zone (SA). Rod-like mitochondria are v is ib le in the sub-apical zone, and other organelles and inc lu -sions become discernable in the protoplasm of the distal multi-organelle zone (D). Phase-contrast microscopy. Figure 17 .- X 1,6.00 Figure 18 - X 825 Figure 19 The t ip of a -differentiating vegetative hypha. The apical growing zone (A) is distinguishable, but the sub-apical zone is not distinguishable from, the distal multi-organelle zone (D). Numerous particles with dense centers, probably corresponding to l ipo -somes as observed by electron microscopy, are ev i -dent throughout the protoplasm. Phase-contrast microscopy. X 550 PLATE FIVE Figures 20-22 Vegetative hyphae of Aohlya bisexualis. Ovoid to spindle-shaped nuclei and most rod- l ike mitochon-dria are aligned with the long axis of the hyphae. The end-to-end association of several of the nuclei is suggestive of recent nuclear d iv is ion. Phase-contrast microscopy. Figure 20 - X 880 Figure 21 - X 880 Figure 22 - X 1,4.00 Figures 23-24 V.egetatiye hyphae of Achlya bisexualis. Opti-cal ly dense spots (arrows; are v is ib le about the nuclei and f i b r i l s , which may be endoplasmic reticulum, are present in the protoplasm. Phase-contrast microscopy. Figure 23 - X 3,400 Figure 24 - 1 3,500 PLATE SIX A vegetative hypha of Aohlya bisexualis which has been stained with Sudan IV to determine the presence and distribution of l i p i d s . The l ip ids appear as spherical, red-orange bodies (visible as highly refractive bodies in this micrograph) throughout the protoplasm. They usually are lacking in the apical (A) and sub-apical (SA) zones, but they become increasingly abundant throughout the d is -tal multi-organelle zone (D). Note the increase in the number of l i p i d bodies as the distance from the hyphal t ip increases. Phase-contrast microscopy. X 360 An increased magnification of the hyphal t ip stain-ed with Sudan IV. Note that almost no l i p i d bodies are discernable in the f i r s t few hundred microns of the hyphal protoplasm, Phase-contrast microscopy. X 810 An increase magnification of an older region of a hypha stained with Sudan IV. The l i p i d bodies are extremely abundant throughout the protoplasm. Phase-contrast microscopy. X 1,125 Hyphal tips which have been stained with Nile Blue to detect the presence of l i p i d s . Lipid bodies are not detectable in the hyphal tips (top of micrographs), but as the distance from the tips increases, l i p i d bodies become v is ib le and f luor -esce as golden-yellow ;spheres (visible as l ight bodies against the dark background). Ultra-v iolet microscopy - exciter f i l t e r UG 5/3 with a transmission range from 220 - 420 my. X 450 PLATE SEVEN Apical growing zone of an actively growing hypha of Achlya bisexualis. Numerous vesicles (Ve) occur to the exclusion of other organelles in the protoplasm. The plasma membrane (PM) is crenulate to irregular in profi le and frequently forms lomasome-like (Lo) configurations. The hyphal wall (W) appears as a single layer of electron l ight material. Glutaraldehyde-osmium tetroxide f ixat ion. X 25,000 PLATE EIGHT Figure 31 An enlargement of the t ip of the apical growing zone showing the crenulate prof i le of the plasma membrane (PM) and the irregularity in wall thickness. Lomasomes (Lo) also are common about the hyphal t i p . Glutaraldehyde-osmium tetroxide f ixat ion. X 40,625 Figures 32-33 Enlargements of areas within the apical growing zone showing the fusion of vesicles with each other (arrows). Glutaraldehyde-osmium tetroxide f ixat ion. Figure 32 - X 34,500 Figure 33 - X 37,000 PLATE NINE The sub-apical mitochondrial zone of an actively growing- hypha of Aahlya bisexualis. Rod-shaped mitochondria (M), vesicles containing electron-dense material (Ve-2), and vesicles (Ve-1) which are the same as those in the apical growing zone appear as the predominant organelles and/or i n -clusions in this zone. 'Gluteraldehyde-osmium tetroxide f ixat ion. X 18,000 PLATE TEN Figure 35 The wall about the sub-apical protoplasm appears as a homogeneous layer of electron l ight material which is deposited by vesicles in the protoplasm. The wall also is less irregular in prof i le than i t is about the apical growing zone, except in areas ad-jacent to lomasomes. The plasma membrane usually is irregular in profi le - suggestive of vesicu-lar fusion. Glutaraldehyde-osmium tetroxide f ixat ion. X 33,000 Figure 36 The lomasome-1ike configuration of the plasma mem-brane is common to the sub-apical mitochondrial zone. Numerous vesicles, which morphologically are the same as those in the apical growing zone, occur in close association with and appear to become incor-porated into the lomasome. The wall is irregular in prof i le and is not uniformly dense adjacent to the lomasome. ; Glutaraldehyde-osmium tetroxide f ixat ion. X 34,000 Figure 37 A cluster of small vesicles common in the peripher-al hyphal protoplasm of the sub-apical zone. Glutaraldehyde-osmium tetroxide f ixat ion. X 36,000 Figure 38 A "rudimentary dictyosome-1ike body", characteris-t i c of the sub-apical mitochondrial protoplasm. One or two discontinuous cisternae are surrounded by smaller-sized vesicles as are observed in the apical growing zone. Glutaraldehyde-osmium tetroxide f ixat ion. X 33,500 Figures 39-40 Microbody-1ike vesicles common in the central hy-phal protoplasm of the sub-apical zone. Glutaraldehyde-osmium tetroxide 'f ixation. Figure 39 Figure 40 - X 35,450 - X 37,500 PLATE ELEVEN The distal multi-organelle zone of a vegetative hypha of Aahlya bisexualis as viewed near the growing t i p . Rod-shaped mitochondria (M) and spindle-shaped nuclei (N) are oriented parallel to the long axis of the hypha. Vesicles (Ve) are concentrated about the dictyosomes (D) which are closely associated with nuclei . The vesicles also are common in the protoplasm near the plasma membrane (PM) and they re-semble those in the apical growing zone. Micro-body-like vesicles(Mi), l i p i d droplets (L), multi-vesicular bodies (MVB) and ribosomes are y is ib le in the protoplasm. Glutsraldehyde-osmium tetroxide f ixat ion. X 29,200 PLATE TWELVE Protoplasm of the distal multi-organelle zone which is characterized by an increase in the number of 1ipid droplets, a more extensive development of ER, and vacuolation. Lipo-somes (Li) begin to appear - indicating hyphal maturation and dif ferent iat ion. (ER = endoplasmic reticulum; L = l i p i d droplets; Li - liposome;. M = mitochon-drion; MVB = multivesicular body; Va = vacuole). Glutaraldehyde-osmium tetroxide f ixat ion. X 27,200 PLATE THIRTEEN Vacuolation of the protoplasm in the distal mult i -organelle zone as is characteristic of both young and differentiating vegetative hyphae. ER appears to become dialated and aligned along the central axis of the hypha to form a complex of vacuoles. Fusion of many vacuoles probably occurs, as i n -dicated^by the close association and i n t e r - d i g i -tation of vacuolar membranes. Glutaraldehyde-osmium tetroxide f ixat ion. X 17,325 An enlargement of the above figure showing the inter -digi tat ion of vacuolar membranes (arrows). Dialated ER-like elements also are discernable in the protoplasm. Glutaraldehyde-osmium tetroxide f ixat ion. X 26 ,375 PLATE FOURTEEN Figure 45 Vacuolation of protoplasm in the distal mult i -organelle zone as is characteristic of both young and differentiating vegetative hyphae. Vacuolation occurs along the central axis of the hypha and is a complex of many smaller vacuoles. Glutaraldehyde-osmium tetroxide f ixat ion. X 11,100 PLATE FIFTEEN Figures 46-47 The distal multi-organelle zone in o ld , highly vacuolated protoplasm. Striations are v is ib le in the thickened wall (W), the plasma membrane (PM) is smooth in p rof i le , vesicles are scarce or absent in the protoplasm, few ribosomes are present, and the ER appears as small fragments throughout the protoplasm. The vacuole appears as a large, single central vacuole instead of a complex of smaller vacuoles as observed in the younger protoplasm of the distal mult i -organelle zone. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 46 - X TB.750 Figure 47 - X 52,000 PLATE SIXTEEN Figure 48 The hyphal wall about the distal multi-organelle zone as viewed by thin-section electron micro-scopy. F ibr i l s (arrows) appear to be inter -twined within an electron l ight amorphous ma-t r i x . Fraying of the outer wall surface also is evident. Vesicles containing wal1-1 ike mat-er ial l i e adjacent to the plasma membrane (PM). Glutaraldehyde-osmium tetroxide f ixat ion. X 58,250 Figures 49-50 F ib r i l s and pores (arrows) are evident in the wall as viewed in freeze-etched preparation. The f i b r i l s appear to be randomly oriented and are inter-twined within amorphous-like material. Figure 49 -Figure 50 -X 51,525 X 38,350 PLATE SEVENTEEN Figure 51 The wall surrounding o ld , highly vacuolated hyphal protoplasm. It appears f inely str iate - indica-tive of being layered - and fraying of i ts sur-face is evident. The plasma membrane is smooth in profi le and few vesicles are v is ib le near the membrane. Glutaraldehyde-osmium tetroxide f ixat ion. X 57,350 Figures 52-53 Areas of the wall are layered and pockets of elec-tron dense granular and yesicular material are v is ib le among the layers. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 52 - X 24,050 Figure 53 - X 39,830 PLATE EIGHTEEN Figures 54-57 Rod-shaped mitochondria characteristic of the vege-tative hyphae. They appear extremely dense in the sub-apical zone (figure 54), but they become less dense as the distance from the hyphal tips i n -creases (figures 55-57). In o ld , highly vacuo-lated protoplasm, electron l i gh t , granular free areas are common in the mitochondria (figure 57). The mitochondria in the d i s t a l , multi-organelle zone (figure 55) and of differentiating hyphae (figure 56) are the same. F ibr i l s (arrows) ex-tend along the central axis of the cristae ( f i -gures 54-57). Glutsraldehyde-osmium tetroxide f ixat ion. Figure 54 - X 33,600 Figure 55 - X 42,150 Figure 56 - X 25,100 Figure 57 - X 25,200 PLATE NINETEEN Figures 58-62 Dictyosomes characteristic of the d i s t a l , mult i -organelle zone in young hyphal protoplasm. They occur in close association with ER and nuclei (N), and they each are composed of a stack of 2-3 cisternae. The cisternae exhibit progres-sive dialation from the proximal face (G-p) to the distal face (C-d). Secretory vesicles (Ve), which, morphologically are similar to those in the apical growing zone and sub-apical mito-chondrial zone, are v is ib le about the distal dictyosome cisternae. They vary in size -usually the smaller ones being closer to the cisternae. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 58 - X 39,630 Figure 59 - X 43,200 Figure 60 - X 51 ,400 Figure 61 - X 40,000 Figure 62 - X 39,430 PLATE TWENTY Figures 63-64 Dictyosomes characteristic of the distal mult i -organelle zone in older and/or differentiating hyphal protoplasm. The dictyosomes occur in close association with the smooth face of d ia -lated ER. Vesicles (Ve-f) appear to bleb (arrows) from the ER and then fuse together to form the proximal dictyosome cisternae (C-p). Larger vesicles (Ve-m) containing electron-l ight amor-phous material occur in close assiation with the distal face cisternae (C-d). Glutaraldehyde-osmium tetroxide f ixat ion. Figure 63 - X 49,920 Figure 64 - X 34,375 PLATE TWENTY-ONE Figures 65-68 Dictyosomes (D) characteristic of differentiating hyphal protoplasm. They occur in close associa-tion with the smooth face of dialated ER and nuclei (N). Structurally they are the same as those in figures 64 and 65. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 65 - X 44,520 Figure 66 - X 39,000 Figure 67 - X 37,680 Figure 68 - X 46,100 PLATE TWENTY-TWO Figures 69-72 Liposome morphology and development in maturing and/or differentiating vegetative hyphae. Lipo-somes f i r s t appear as membrane-bound bodies con-taining a relatively homogeneous mixture of electron-l ight amorphous material and electron-dark granules (figure 69 r» upper le f t bodies; figure 70 - upper central body). Osmiophilic globules develop within the liposomes (figure 69 - upper central bodies) and layering of the electron-l ight amorphous material about the osmiophilic globules occurs (figures 69-72). Layering continues unti l the liposomes are f i l l e d almost completely (figure 72). The electron-l ight bands are approximately 80-100 A thick. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 69 - X 38,900 Figure 70 - X 61,000 Figure 71 - X 38,890 Figure 72 - X 50,150 PLATE TWENTY-THREE Figure 73 A liposome in older hyphal protoplasm. Its encompassing membrane appears fragmented, and layering of the electron-l ight amorphous mat-erial about the osmiophilic globule is barely v i s ib le . Glutaraldehyde-osmium tetroxide f ixat ion. X 37,600 Figure 74 An unidentified membrane-bound body in o ld , highly vacuolated hyphal protoplasm. Membrane-l ike fragments and electron-dense granules are v is ib le within the body. GlutBraldehyde-osmium tetroxide f ixat ion. X 40,710 Figures 75-76 Multi -vesicular bodies in young and differentiating hyphal protoplasm. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 75 - X 42,500 Figure 76 - X 46,800 PLATE TWENTY-FOUR Figures 77-80 Microbodies in differentiating hyphal proto-plasm. They occur adjacent to ribosome-free areas of granular ER. Tubules (arrows) and less dense vesicles (Fig. 78) are v is ib le in some microbodies. Constriction or budding of a microbody is shown in Figure 78. Gluteraldehyde-osmium tetroxide f ixat ion. Figure 77 - X 37,140 Figure 78. - X 38,930 Figure 79 - X 36,925 Figure 80 - X 40,560 Figures 81-83 Microbodies possessing less dense vesicles and/ or tubules [arrows) in their granular matrix. Glutsraldehyde-osmium tetroxide f ixat ion. Figure 81 - X 46,740 Figure 82 - X 39,110 Figure 83 - X 50,575 PLATE TWENTY-FIVE A spindle-shaped nucleus - characteristic of both young and differentiating hyphal proto- , plasm. It is encompassed by a crenulate nuclear envelope (NE) in which annuli (A) are v i s ib le . A single nucleolus (Nu) is located acentrically within ther;nucleus. (A = annul us; NE = nuclear envelope; Nu -nucleolus).. Glutaraldehyde-osmium tetroxide f ixat ion. X 45,000 PLATE TWENTY-SIX Figures 85-86 Spindle-shaped nuclei in the distal mult i -organelle zone of young hyphal protoplasm. ER is closely associated with the nuclei , and annuli (A) are v is ib le in the nuclear envelope (NE). (A = annuli ; ER = endoplasmic reticulum; N = nucleus; NE = nuclear envelope) Glutaraldehyde-osmium tetroxide f ixat ion. Figure 85 Figure 86 - X 40,000 - X 40,000 PLATE TWENTY-SEVEN Figure 87 Longitudinal to oblique section through a meta-phase to early anaphase nucleus in vegetative hypha. Spindle f i b r i l s (SF) radiate from thick-ened inner envelope membrane (NE) adjacent to polar centrioles (Ce). Several dense granular regions also are suggestive of chromatin material (Ch). (Ce = centr iole; Ch =.chromatin material; NE = nuclear envelope; SF = spindle f i b r i l ) . Glutaraldehyde-osmium tetroxide f ixat ion. X 95,300 PLATE TWENTY-EIGHT Figures 88-89 Nuclei (N) in old vacuolated hyphal protoplasm. They appear less dense than the nuclei in younger . hyphal protoplasm, but they structurally are similar . ER and dictyosomes (D) are closely asso-ciated with the nuclei , and blebs (arrows) from the nuclear envelope (NE) appear to give r ise to the vesicles (Ve) which fuse together to form the proximal dictyosome cisternae (C-p) (Fig. 89). Glutaraldehyde-osmium tetroxide f ixat ion. Figure 88 -Figure 89 -X 39,300 X 57,770 PLATE TWENTY-NINE Figure 90 Annuli, as seen in surface view. They appear to be formed of sub-units (arrows) about a central dense core. Glutaraldehyde-osmium tetroxide f ixat ion. X 44,540 Figure 91 The nucleolus of a nucleus in young hyphal proto-plasm. It is composed of the pars fibrosa (PF), the pars granulosa (PG) and the pars chromosoma (PC). Less dense areas, sometimes referred to as the pars amorpha (PA), also are v is ib le within the nucleolus. Glutaraldehyde-osmium tetroxide f ixat ion. X 68,580 Figures 92a-92b Cross-sectional view of centrioles in young hyphal protoplasm. The centrioles each consist of nine t r ip lets in cyl indrical arrangement about a single central tubule. ,and radial filaments connect the central tubule to the nine peripheral t r i p le t s , spindle f i b r i l s (SF) radiate from the thickened inner membrane of the nuclear envelope, but direct connections from the spindle f i b r i l s to the cen-tr io les are not discernible. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 92a - X 60,150 Figure 92b - X 51,430 Figure 93 A longitudinal section through a pair of centrioles. The centrioles (Ce) are aligned end-to-end and l i e parallel to the modified area of the nuclear enve-lope (NE). Spindle f i b r i l s (SF) radiate from the thickened inner nuclear membrane. Glutaraldehyde-osmium eteroxide f ixat ion. X 45,500 PLATE THIRTY A chain of intercalary, flask-shaped gemmae. They appear as optical ly dense cel ls which possess one or more large vacuoles (less dense areas in each c e l l ) . A concave-shaped septum separates the chain of gemmae from the vege-tative hypha (arrow). Living material. Br ight - f ie ld microscopy. X 240 A chain of catenulate-shaped gemmae. Live material. Br ight - f ie ld microscopy. X 240 A single, terminal gemmae which is delimited from the vegetative hypha by a concave-shaped septum. Living material. Br ight - f ie ld microscopy. X 400 A terminal, globose gemma as formed by the female st ra in . Living material. Br ight - f ie ld microscopy. X 250 PLATE THIRTY-ONE A longitudinal section through the t ip of a gemma. A thick wall (W) encompasses the gemma's dense protoplasm. Its outer sur-face appears to be sloughing off (arrow). Th protoplasm contains numerous vesicles (Ve), mitochondria (M), irregularly-shaped l i p i d droplets (L), liposomes (Li) and small vacuoles (Va). A nucleus (N) and dictyosome (D) are also v is ib le in the protoplasm. Glutaraldehyde-osmium tetroxide f ixat ion. X 9,800 PLATE THIRTY-TWO A longitudinal section through the vacuolar region of a gemma. Several smaller vacuoles appear to be closely associated with each other to form a large, central vacuolar sys-tem. Spheric!es of aggregated metabolites (Sp) are present in the vacuoles. Numerous l i p i d droplets (L), mitochondria (M), and liposomes (Li) are present in the protoplasm about the vacuolar system (Va). Glutaraldehyde-osmium tetroxide f ixat ion. X 9,150 PLATE THIRTY-THREE Micrograph of the t ip of a gemma. Irregularly shaped vesicles are common in the protoplasm near the t i p , but they do not occur to the exclusion of other organelles or inclusions The wall consists of two layers of amorphous and f i b r i l l a r (arrows) material. The outer layer (OL) appears s l ight ly more dense than the inner layer (IL), and sloughing of f i -b r i l l a r - ! ike material is evident on i ts outer surface. A pocket of electron-dense vesicu-lar and granular material is v is ib le near or between the outer and inner wall layers. Glutaraldehyde-osmium tetroxide f ixat ion. X 20,000 The gemma wal l . The outer wall layer (OL) appears globular and irregular in prof i le . The inner layer (IL) comprises almost three-fourths the total thickness of the wal l . Fine striations are indicative of layering of material in the inner wal l . F ibr i l s (arrows) are evident in both the inner and the outer layers. Glutaraldehyde-osmium tetroxide f ixat ion. X .39,600 PLATE THIRTY-FOUR A septum delimiting the protoplast of a gemma from that of the hypha. It is concave, but i r regular, in profi le and is formed of two wall layers which are separated from each other by electron-dense vesicular and granular material Note that the gemma's inner wall layer (IL) is much thicker than either i ts outer wall layer (OL) or the inner wall layer of the hyphae. GlutBraldehyde-osmium tetroxide f ixat ion. X 5,990 An enlargement of a part of a septum. S t r ia -t ions, indicative of layering of material, are evident in the thick inner wall layer (IL). Also note that the outer wall layer (OL) is a continuation of the outer hyphal wall layer. Glutsraldehyde-osmium tetroxide f ixat ion. X 24,000 103 PLATE THIRTY-FIVE Figures 104-107 Microbodies in gemmae protoplasm. Spherical and ovoid forms are common, but indications of tubular forms are v is ib le (Fig. 106). They are characterized by an electron-dense granular matrix in which tubules (arrows) often are ob-served. Less dense vesicles also are present in some microbodies (Fig. 104). The microbodies occur in close association with non-ribosomal areas of granular ER; however, this association with the ER is not shown in these sections. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 104 - X 53,150 Figure 105 - X 43,140 Figure 106 - X 50,700 Figure 107 - X 42,165 Figures 108-109 Mitochondria in gemma protoplasm. They occur in close association with liposomes, but fusion of liposome encompassing membranes and outer .mitochondrial membranes is not evident. The. mitochondrial cristae often appear to overlap each ^other. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 108 - X 47,500 Figure 109 - X 39,350 PLATE THIRTY-SIX Figures 110-112 Dictyosomes in gemma protoplasm. They possess the same basic structural organization as do dictyosomes. in the vegetative hyphae and other developmental stages. However, their cisternae often appear discontinuous (Fig. 110) and numer-ous vesicles (Ve-f) are associated with the pro-ximal dictyosome face (C-p). The vesicles are relat ively uniform in diameter, and they appear to originate from blebs off dialated ER (Fig. 110). The ER is inter-associated with the outer membrane of the nuclear envelope (NE). Larger vesicles (Ve-m) also are v is ib le about the periphery of the cisternae and near distal dictyosome c i s -ternae (C-d). Glutaraldehyde-osmium tetroxide f ixat ion. Figure 110 - X 63,300 Figure 111 - X 21,000 Figure 112 - X 40,655 PLATE THIRTY-SEVEN Figures 113-114 Dictyosomes in gemma protoplasm. The dictyo-somes are closely associated with the outer nuclear membrane (Fig. 113) and with dialated ER which structurally is inter-associated with the nuclear envelope (Fig. 114). (ER = endoplasmic reticulum; C-d= distal dictyosome cisternae; C-p= proximal dictyo-some cisternae; N = nucleus; NE = nuclear envelope; —£ = bleb). Glutaraldehyde-osmium tetroxide f ixat ion. *-Figure 113 - X 58,460 Figure 114 - X 47,300 PLATE THIRTY-EIGHT Figure 115 Sphericle of aggregated metabolites (Sp) in the vacuolar sap of gemma protoplasm. The tonoplast seldom appears intact - only frag-ments of the membrane are v i s ib le . Glutaraldehyde-osmium tetroxide f ixat ion. X 28,000 Figure 116 Liposomes in gemma protoplasm. They each are f i l l e d almost completely with layers of electron l ight amorphous material about an osmiophilic globule. Glutaraldehyde-osmium tetroxide f ixat ion. X 50,410 PLATE THIRTY-NINE Figure 117 Nucleus in gemma protoplasm. The nucleolus is associated closely with the nuclear envelope (arrows) and consists of a well defined pars fibrosa (PF) and pars granulosa (PG). Chroma-t i n - l i k e filaments (Ch) are closely bound with the pars granulosa. The nuclear envelope (NE) is crenulate in profi le and annuli (A) are pre-sent throughout i ts structure. Glutsraldehyde-osmium tetroxide f ixat ion. X 54,975 PLATE FORTY Figure 118 Ovoid-shaped nucleus in gemma protoplasm. Struc-tural ly i t is similar to the nucleus in Figure 117; however, ER forms almost a continuous sheet about the nucleus. (ER = endoplasmic reticulum; NE = nuclear envelope; Nu = nucleolus; T = tonoplast; Va = vacuolar sap). Glutaraldehyde-osmium tetroxide f ixat ion. X 31,300 Figure 119 Nucleolus in the nucleus of gemma protoplasm. It is closely associated with the nuclear enve-lope (arrow) and consists of the pars fibrosa (PF) and the pars granulosa (PG). Chromatin-l ike filaments (Ch) appear as less dense regions among the pars granulosa. Glutaraldehyde-osmium tetroxide f ixat ion. X 48,000 PLATE FORTY-ONE Figures 120-122 Two pairs of centrioles aligned end-to-end and adjacent to the structurally modified area of nuclei. In longitudinal section, they exhibit similar characteristics to those in hyphal protoplasm except that each pair sometimes ap-pears to be inter-connected by one of the per i -pheral t r ip le t tubules (arrow - Fig. 120). Glutaraldehyde-osmium tetroxide f ixat ion. Figure 120 - X 55,750 'Figure 121 - X 49,640 Figure 122 - X 54,250 Figure 123 The cross-section of a centriole in gemma proto-plasm showing nine peripheral t r ip lets in their characteristic cartwheel arrangement. Two cen-tral tubules (doublet) appear inter-connected to each of the nine peripheral t r ip lets by rad-ial filaments. Glutaraldehyde-osmium tetroxide f ixat ion. X 50,500 PLATE FORTY-TWO Young cylindric-shaped zoosporangium. Its dense protoplasm is delimited from the hyphal proto-plasm by a concave-shaped septum. Br ight - f ie ld microscopy. X 170 Zoosporangia in which cleavage of the protoplasm is occurring. They resemble the "normal" type des cribed by Rothert (.1892) - possessing thick • parietal protoplasm about a central vacuole. Br ight - f ie ld microscopy. Figure 126 - X 125 Figure 127 - X 200 Zoosporangium in which cleavage of the protoplasm into numerous primary zoospores has occurred. Only a thin layer of protoplasm is v is ib le about the large central vacuole. Br ight - f ie ld microscopy. X 220 Zoosporangium in which individual zoospore i n i t i a l have become indiscernible (Fig. 129), but contrac-tion and reformation of the i n i t i a l s then has re-occurred (Fig. 130). Br ight - f ie ld microscopy. X 255 The release of numerous primary zoospores through the ruptured apical papil la of the zoosporangium. The zoospores encyst immediately outside the papi l la . Br ight - f ie ld microscopy. Figure 131 - X 255 Figure 132 - X 300 PLATE FORTY-THREE Figure 133 Single apical papil la of differentiating zoo-sporangium. Br ight - f ie ld microscopy. X - 700 Figure 134 Differentiating zoosporangium with two apical papil lae. Phase-contrast microscopy. X - 355 Figure 135 Zoosporangium with single lateral papi l la, Br ight - f ie ld microscopy. X -340 Figures 136-137 Encysted primary zoospores about ruptured papi l -lae of zoosporangia. Phase-contrast microscopy. Figure 136 - X450 Figure 137 - X400 Figure 138 Concave-shaped septum separating differentiating zoosporangial protoplasm from hyphal- protoplasm. Br ight - f ie ld microscopy. X - 350 Figure 139 Convex-shaped septum separating hyphal protoplasm from empty zoosporangium. Phase-contrast microscopy. X - 475 PLATE FORTY-FOUR Longitudinal section through t ip of zoosporan-gium prior to protoplasmic cleavage. Numerous mitochondria (M), liposomes (L i ) , and l i p i d droplets (L) are distributed randomly through-out the thin-walled (W) zoosporangium. Vesicles (Ve) are common in the apical proto-plasm. Gluteraldehyde-osmium tetroxide f ixat ion. X 6,825 An enlargement of the uncleaved zoosporangial protoplasm. Numerous vesicles (Ve) are closely associated with dictyosomes (D). Parastrasomes (arrows) are scattered throughout the proto-plasm; liposomes (L i ) , mitochondria (M) and' l i p id droplets (L) are abundant. Gluteraldehyde-osmium tetroxide f ixat ion. X 13,280 PLATE FORTY-FIVE Longitudinal section through young, uncleaved zoosporangium. Vacuoles (Va) are oriented most! along the central axis of the zoosporangium, and cleavage vesicles (Ve), mitochondria (M), l i p i d droplets (L), and liposomes (Li) are scattered throughout the protoplasm. Nuclei (N) also are v i s ib le . Glutaraldehyde-osmium tetroxide f i xat ion . . X 5,720 PLATE FORTY-SIX Cross-section of zoosporangium prior to proto-plasmic cleavage. Nuclei (N) appear equidis-tant from each other; a large central vacuo-lar complex (Va), as well as smaller peripher-ally-oriented vacuoles (Va) are v i s ib le ; nu-merous' cleavage vesicles (Ve-C), mitochondria (M), liposomes (L i ) , and l i p i d droplets (L) are scattered throughout the protoplasm. A large pocket of electron-dense granules and vesicles appears within the wall (W). Glutaraldehyde-osmium tetroxide f ixat ion. X - 4,130 Septum separating uncleaved zoosporangial pro-toplasm from more highly vacuolated protoplasm. It is formed of two wall layers separated from each other by electron dense granular and vexi -cular material. Glutaraldehyde-osmium tetroxide f ixat ion. X 6,850 PLATE FORTY-SEVEN Figures 145-146 Longitudinal sections through a differentiating zoosporangium in which protoplasmic cleavage is v i s ib le . Numerous cleavage vesicles (Ve) are aligned in planes equidistant between ad-jacent nuclei (N) and adjacent to the plasma membrane. Mitochondria (M), l i p i d droplets (L), and liposomes (Li) also are oriented about each nucleus in the protoplasm. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 145 - X 5,600 Figure, 146 -*X 5,950 Figure 147 An enlargement showing cleavage of a single zoospore i n i t i a l . Dictyosomes (D) occur in close association with the nucleus (N); mitochondria CM) surround the nucleus; l i p i d droplets (L) and liposomes (Li) are scattered about the mitochondria. Planes of cleavage vesicles border the liposomes and l i p i d drop-le ts . Parastrasomes (arrows) and micro-bodies (Mi) also are v is ib le in the proto-plasm. Glutaraldehyde-osmium tetroxide f ixat ion. X 9,250 PLATE FORTY-EIGHT Figure 148 Cross-section through zoosporangium in which there is complete delimitation of primary zoospore i n i t i a l s . Arrows depict cleavage planes. Gluteraldehyde-osmium tetroxide f ixat ion. X 4,920 Figures 149-150 Individual uninucleate primary zoospore i n i -t i a l s . G.lutBraldehyde-osmium tetroxide f ixat ion. Fig. 149 - X 7,630 Fig. 150 - X 8,080 PLATE FORTY-NINE Tangential section through apex of zoosporan-gium prior to papil la development. Vesicles, structurally similar to those in the apical growing zone of vegetative hyphae, are common in the protoplasm adjacent to the plasma mem-brane. Glutaraldehyde-osmium tetroxide f ixat ion. X 17,280 Cross-section through developing papil la of young zoosporangium. Vesicles are common throughout the protoplasm. GlutaraTdehyde-osmium tetroxide f ixat ion. X 9,150 Sl ightly tangential section through a re la -t ively well-developed papi l la . Mostly cleavage vesicles are present in the proto-plasm. Glute-raldehyde-osmium tetroxide f ixat ion. X 9,305 Longitudinal section through a well-formed api cal papi l la . The wall is extremely thin near the apex and only cleavage vesicles are v is ib l in the protoplasm. Glutaraldehyde-osmium tetroxide f ixat ion. X 13,000 PLATE FIFTY Figures 155-156 Zoosporangial protoplasm in which alignment and fusion of cleayage vesicles are v i s ib le . Larger vacuoles, formed by the coalescence of vesicles, also appears flattened along the same planes as the cleavage vesicles are aligned. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 155 - X 17,500 Figure 156 - X 18,750 Figures 157-158 Zoosporangial protoplasm upon completion of cleayage - the plasma membrane of the primary zoospore i n i t i a l s being formed by the fusion of the cleavage vesicle membranes. Para-strasomes (arrows) occur in close association with the cleavage planes. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 157 Figure 158 - X 19,300 - X. 15,000 PLATE FIFTY-ONE Figures 159-160 Mitochondria in zoosporangia of female strain during protoplasmic cleavage. They each pos-sess a single cr ista which is dilated and contains electron-l ight amorphous material. Their other cristae are elongated and simi-lar to those in the male strain (see Figure 163). Dense f i b r i l s also are v is ib le in many of the cristae. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 159 - X 48,000 Figure 160 - X 55,750 Figures 161-163 Microtubule packets in zoosporangia during protoplasmic cleavage. They often appear in close association with mitochondria (Figures 162-163) and the individual microtubules (arrows) in each packet are straight to undu-late in prof i le . Glutsraldehyde-osmium tetroxide f ixat ion. Figure 161 - X 63,325 Figure 162 - X 36,000 Figure 1 6 3-1 47,380 PLATE FIFTY-TWO Figures 164-166 Developing parastrasomes in zoosporangial proto-plasm. A core of electron l ight amorphous mat-erial rimmed by a layer of dense fibrous to tu -bular material is v is ib le in the parastrasomes. Some tubules or fibrous elements also appear to extend into the amorphous core material. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 164 - X 39,800 Figure 165 - X 65,800 Figure 166 - X 65,800 Figures 167-168 Liposomes common to zoosporangial protoplasm. They contain osmiophilic globules and layers of electron l ight amorphous material. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 167 - X 54,285 Figure 168 - X 37,700 Figure 169 Microbodies characteristic of zoosporangial protoplasm. Glutaraldehyde-osmium tetroxide f ixat ion. X 44,000 PLATE FIFTY-THREE Figure 170 Dictyosomes in young precleaved zoosporangial protoplasm. The cisternae appear discontinuous and numerous vesicle containing amorphous mat-erial are v is ib le about the distal dictyosome face. Glutaraldehyde-osmium tetroxide f ixat ion. X 50,890 Figure 171 Dictyosome in zoosporangium during early proto-plasmic cleavage. The cisternae are mostly con-tinuous and fewer vesicles are present about the distal face. Glutaraldehyde-osmium tetroxide f ixat ion. X 33,780 Figures 172-174 Dictyosomes in late and post-cleavage stages of zoosporangia. They occur in close association with the nuclei and their cisternae are highly-developed. Few vesicles are v is ib le about their distal face. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 172 - X 42,150 Figure 173 - X 34,000 Figure 174 - X 34,000 PLATE FIFTY-FOUR Tangential section of a nucleus in zoosporangial protoplasm. A highly-differentiated nucleolus (Nu) is v is ib le in the nucleus (N), and dicty -osomes (D) occur in close association with the nuclear envelope (NE). Glutaraldehyde-osmium tetroxide f ixat ion. X 35,200 Anterior pole of a nucleus in zoosporangial protoplasm. A cluster of osmiophilic granules (G) are v is ib le at the apex of the nucleus, near the area of basal body association. Dictyo-somes (D) are"visible about the nuclear enve-lope (NE). Glutaraldehyde-osmium tetroxide f ixat ion. X 36,000 Nucleolus in zoosporangial nucleus. The pars fibrosa (PF) is perpheralized by the pars granu-losa (PG), and pars amorpha (PA) is common throughout the nucleolus. Glutaraldehyde-osmium tetroxide f ixat ion. X 25,750 PLATE FIFTY-FIVE Figures 178-^ 179 Basal body region in zoosporangial protoplasm. Microtubules (arrows) radiate from the basal bodies (BB) and become aligned about the an-terior pole of the closely associated nucleus (N). Glutaraldehyde-osmium tetroxide f ixat ion. Figure 178 - X 35,290 ' Figure 179 - X 33,000 Figures 180-181 Longitudinal sections of basal bodies in d i f -ferentiating primary zoospore i n i t i a l s . Devel-oping axonemal vesicles ( A Ye) occur in close association with the distal pole of the basal bodies. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 180 - X 43,590 Figure 181 - X 43,000 Figures 182-183 Longitudinal to, tangential sections of basal bodies. Osmiophilic bands (arrows) appear to inter-connect the terminal plate of the basal bodies with the membrane of the axonemal ves i -cles (A Ve). In Figure 182, the axonemal ves i -cular membrane is continuous with the plasma membrane (PM). Glutaraldehyde-osmium tetroxide f ixat ion. Figure 182 - X 46,140 Figure 183 - X 50,425 PLATE FIFTY-SIX Figure 184 Cross-section of basal body at distal pole near the terminal plate. The nine peripheral f i b r i l s appear as doublets. The axonemal sheath is v is ib le about the periphery of the basal body. Glutaraldehyde-osmium tetroxide f ixat ion. X 35,415 Figures 185-187 Cross-sections of basal bodies in area immediate-ly proximal to the terminal plate. Nine per i -pheral t r ip lets appear in cyl indrical cartwheel arrangement and sub- f ibr i l A of each t r ip le t is oriented toward the central axis of the basal bodies. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 185 - X 50,300 Figure 186 - X 49,200 Figure 187 - X 59,700 Figures 188-189 Cross-sections through the proximal poles of basal bodies. Nine peripheral t r ip lets appear in cy-l indr ica l arrangement about a single central tubule. Groups of microtubules also are v is ib le near the margins of the basal bodies (Figure 189). . Glutaraldehyde-osmium tetroxide f ixat ion. Figure 188 - X 47,500 Figure 189 - X 47,500 PLATE FIFTY-SEVEN An encysted primary zoospore. (Li = liposome; Lo - lomasome; M = mito-chondrion; Mt-Ve - microtubule packet; N = nucleus; Nu = nucleolus; P = para-strasome; PM = plasma membrane; RER = granular endoplasmic reticulum; Ve = vesic le; W = wall)" Glutaraldehyde-osmium tetroxide f ixat ion. X 22,500 PLATE FIFTY- EIGHT Figures 191-192 Encysted primary zoospores. (C = cisternae; Li - liposome; M = mito-chondrion; Mt-Ve = microtubule packet; N = nucleus; Nu = nucleolus; P = para-strasome; PM = plasma membrane; Va = vacuole; W = wal1). Glutaraldehyde-osmium tetroxide f ixat ion. Figure 191 - X 18,000 Figure 192 = X 16,800 PLATE FIFTY-NINE Figures 193-194 Mult i -ves icular - l ike vesicles in the protoplasm of encysted primary zoospores. GIuteraldehyde-osmium tetroxide f ixat ion. Figure 193 - X 30,570 Figure 194 - X 80,000 Figures 195-196 Cross-sections of the wall about encysted p r i -mary zoospores. The wall appears as a single layer about the protoplasm. It consists of amorphous material and fine f i b r i l s (arrows). Similar material is v is ib le in vesicles (Ve). which appear to originate from dictyosomes (D) (Figure 195). Liposomes (Li.) containing re la -t ively few layers of alternating electron-l ight and osmiophilic material also are present in the protoplasm. Glutaraldehyde-osmium. tetroxide f ixat ion. Fibure 195 - X 42,380 Figure 196 - X 43,700 PLATE SIXTY Protoplasm of encysted primary zoospore con-taining mitochondria (M), microtubules packets (Ve-Mt), liposomes (L i ) , and adjacent layers of ER and coalescing vesicles (Ve). Osmio-phi l ic f i b r i l s are v is ib le in the mitochon-drial cr istae. GluttJraldehyde-osmium tetroxide f ixat ion. X 29,850 Mitochondrion in encysted primary zoospore protoplasm.- Less dense areas, indicative of DNA regions, are v is ib le in the matrix-;and osmiophilic f i b r i l s are present in the c r i s -tae. Glutaraldehyde-osmium tetroxide f ixat ion. X 54,940 Alternating layers of ER and coalescing ves i -cles near wall of an encysted primary zoo-spore. Irregularly-shaped liposomes also are v is ib le in the protoplasm. Glutaraldehyde-osmium tetroxide f ixat ion. ' X 46,440 PLATE SIXTY-ONE Figures 200-202 Vacuoles in encysted primary zoospores. The vacuoles (Va) are irregular in profi le and often appear to be formed by the di lat ion of endoplasmic reticulum (ER-Di). Alternating layers of vesicles (Ve), cisternae (C), and other endoplasmic reticulum (ER) usually oc-cur in close association with the vacuoles. Glutaraldehyde-osmium tetroxide f ixat ion. Figure Figure Figure 200 -201 -202 -X 29,140 X 34,390 X 40,390 PLATE SIXTY-TWO Microtubule packets and microtubule vesicles in encysted primary zoospores. The packets appear blunt-ended and parallel oriented microtubules extend the length of the packets. Osmiophilic filaments (arrows) also appear to inter-connect some of the microtubules. The thicker membraned-bound vesicles contain numerous convoluted microtubules. (Li= liposome; M = mitochondrion; P-mt = microtubule packet; Ve-mt - microtubule vesicle) . Gluturaldehyde-osmium tetroxide f ixat ion. X 39,100 Cross-section of microtubule packet.- The microtubules are hexagonally arranged within the packets and filaments (arrows) appear to inter-connect some of the microtubules. Glutaraldehyde-osmium tetroxide f ixat ion. X 51,000 Longitudinal to oblique sections of microtu-bule packets. The microtubules appear to be s l ight ly tapered near the blunt-end of the packets and osmiophilic filaments (arrows) inter-connect the microtubules. Ribosome (circles) sometimes are v is ib le on the sur-face of the packets. Glutaraldehyde-osmium tetroxide f ixat ion. X 57,000 PLATE SIXTY-THREE Figures 206-209 Differentiating l ipoidal bodies in encysted primary zoospore protoplasm. Vesicles appear in close association with the bodies. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 206 - X 31 ,100 Figure 207 - X 38,000 Figure 208 - X 43,700 Figure 209 - X 47,500 Figures 210-211 Highly-developed l ipoidal bodies in encysted primary zoospore protoplasm. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 210 - X 52,250 Figure 211 - X 54,760 PLATE SIXTY-FOUR Dictyosomes, as viewed in cross-section, in encysted primary zoospores. Smaller ves i -cles (Ve-f) are v is ib le about the proximal cisternae (C-p) and larger vesicles (Ve-m) containing amorphous material are common about the distal cisternae (C-d). Glutaraldehyde-osmium tetroxide f ixat ion. X 42,250 Dictyosomes, as seen in face-view, in encysted primary zoospores. The cisternae each con-s is t of a dense central disc from which anas-tomosing i tubules radiate. Glutaraldehyde-osmium tetroxide f ixat ion. X 57,000 Blebs (arrows) on the outer nuclear membrane appear to give r ise to the vesicles (Ve-f) associated with the proximal dictyosome c i s -ternae. Glutaraldehyde-osmium tetroxide f ixat ion. X 54,150 PLATE SIXTY-FIVE Figure 215 Tangential section through an encysted primary zoospore nucleus (N). Vesicles (Ve-f) which are closely associated with the forming face cisternae of dictyosomes (D) appear to arise from the nuclear envelope (NE). The sheath (Sh) interconnecting the nuclear envelope to the basal body of the flagellum also is v i s ib le . Glutaraldehyde-osmium tetroxide f ixat ion. X 68,900 PLATE SIXTY-SIX The nucleus of an encysted primary zoospore. The nucleolus (Nu) appears at the posterior end of the nucleus; whereas, dictyosomes (D) occur inclose association with the an-terior pole of the nucleus. Pores (P) 7or annuli , are discernable throughout the nuclear envelope (NE). Glutaraldehyde-osmium tetroxide f ixat ion. X 35,680 Enlargement of the nuclear envelope. ER (arrows) appear continuous with the outer membrane of the nuclear envelope (NE), and pores (P) also are v is ib le in the envelope Glutaraldehyde-osmium tetroxide f ixat ion. X 57,000 Surface-view of annuli in the nuclear enve-lope. Dense granules are v is ib le in the center of the pores and sub-units (arrows) appear to form the annuli. Glutaraldehyde-osmium tetroxide f ixat ion. X 88,670 PLATE SIXTY-SEVEN Figures 219-220 Nucleoli in encysted primary zoospore nuclei . The pars fibrosa (PF) forms the major con-stituent of each nucleolus and i t is sur-rounded by the pars granulosa (PG). Less dense areas, sometimes referred to as the pars amorpha (PA), are common in many of the nucleol i . The nucleoli occur in close, association with the;nuclear envelope (NE). Glutaraldehyde-osmium tetroxide f ixat ion. Figure 219 - X 67,500 Figure 220 - X 70,480 PLATE SIXTY-EIGHT Cross-section of a basal body immediately below the terminal plate. The nine peripheral f i -br i ls appear as doublets and an osmiophilic disc is v is ib le in the central region of.the basal body. Glutaraldehyde-osmium tetroxide f ixat ion. X 48,850 Cross-section of a basal body approximately 300-400A below the terminal plate. The nine peripheral f i b r i l s s t i l l appear as doublets, an osmiophilic disc or tubule is present in the central region, and spur fibers (arrows) are arranged in a cartwheel pattern about the periphery of the basal body. Glutaraldehyde-osmium tetroxide f ixat ion. X 57,000 Cross-section of a basal body in region where peripheral f i b r i l s become t r ip lets (arrow). A central disc or tubule is lacking. Glutaraldehyde-osmium tetroxide f ixat ion. X 64,500 Cross-section of a basal body in area of root-let attachment. Several of the nine peripheral t r ip lets are attached to a common rootlet. Glutaraldehyde-osmium tetroxide f ixat ion. X 66,500 An oblique section of a basal body in the cen-t r io la r region. The peripheral t r ip lets are equally spaced about a single central tubule and are closely associated with a common root-l e t . Glutaraldehyde-osmium tetroxide f ixat ion. X 71,250 PLATE SIXTY-EIGHT (cont'd) Figures 226-228 Longitudinal sections through a basal body. A tubule-l ike disc (D) is v is ib le in central region of the terminal plate (TP); spur fibers (S) radiate from the peripheral f i b r i l s near the terminal plate; rootlets (R) are attached near the distal centriolar region of the basal body. Parastrasomes (P) ap-pear in close association with the rootlet region of the basal body, and vesicles (Ve-Ax), which probably would fuse and form an axone-mal sheath, are v is ib le near the distal pole of the basal body. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 226 - X 51 ,540 Figure 227 - X 57,140 Figure 228 - X 60,000 PLATE SIXTY-NINE Figures 229, Basal body region in encysted primary zoo-231-232 spores. Two basal bodies (BB) l i e at an angle of 120-135° to each other and are connected proximally with one another by a group of microtubules (arrows). The basal bodies also are associated with the anterior pole of nuclei by a sheath (Sh) which l i es adja-cent to the other nuclear membrane. Cross-striations appear to connect the sheath with the nuclear envelope. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 229 - X 33,500 Figure?231 - X 56,700 Figure 232 - X 48,200 Figure 230 Surface view of the sheath which part ia l ly sur-rounds the anterior end of nuclei . Parallel striations are v is ib le in the sheath (Sh). A basal body (BB) and rootlet (R) also are v is ib le in the section. The rootlet appears to be formed of at least 7 microtubules (arrows). Glutaraldehyde-osmium tetroxide f ixat ion. A..54,000 PLATE SEVENTY Figures 233-235 Sections of encysted primary spores showing the inter-association of basal bodies, root-l e t s , and nuclear envelope. F i b r i l s , or tubules, appear to connect each of the nine peripheral t r ip lets to closely associated rootlets (Figures 233 and 235). One of the rootlets also appears to be directly con-nected with a sheath which part ia l ly enve-lopes the anterior pole of each nucleus (Figures 234-235). (N = nucleus; Sh = nuclear sheath; P = parastrasome; R = .rootlet). Glutaraldehyde-osmium tetroxide f ixat ion. Figure 233 Figure 234 Figure 235 - X 60,400 - X 73,625 - X 67,870 PLATE SEVENTY-ONE Figures 236-238 The association of parastrasomes and rootlets of basal bodies in encysted primary zoospores. Groups of microtubules radiate in a single plane from the basal bodies (Figure 238) into the surrounding protoplasm - part ia l ly encompassing the closely-associated para-strasomes. Rootlets also form a lateral sheet or cap about the anterior pole of the nuclei . Glutaraldehyde-osmium tetroxide f ixat ion. Figure 236 - X 34,730 Figure 237 - X 35,000 Figure 238 - X 51 ,600 PLATE SEVENTY-TWO Figure 239 Parastrasomes prior to their association with basal bodies. Numerous randomly-oriented tubular elements in the electron-l ight core are rimmed by a layer of osmiophilic mater-i a l . Glutaraldehyde-osmium tetroxide f ixat ion. X 54,000 Figure 240 Parastrasomes exhibiting greater morphologi-cal organization. Groups of tubular elements are aligned parallel to each other and there is.the development of an accentric amor-phous area in each parastrasome. Glutaraldehyde-osmium tetroxide f ixat ion. X 54,625 Figures 241-242 The enlargement and continued differentiation of parastrasomes. They appear ovoid in pro-f i l e , containing tubular elements near their broad pole and amorphous to granular mat-erial in the rest of their core. A dense rim of material is v is ib le about the periphery of the parastrasomes. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 241 - X 52,780 Figure 242 - X 49,400 Figure 243 A highly-differentiated parastrasome near the basal body region of an encysted primary zoospore. Rootlets (arrows) of the basal body barely are discernable near the small pole of the parastrasome. Glut-era!dehyde-osmium tetroxide f ixat ion. X 46,440 PLATE SEVENTY-THREE Figures 244-247 Highly-differentiated parastrasomes in en-cysted primary zoospores. Smaller para-strasomes often appear near the larger forms. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 244 - X 54,000 Figure 245 - X 59,600 Figure 246 - X 71 ,250 Figure 247 - X 67,000 PLATE SEVENTY-FOUR Figures 248-250 Encysted primary zoospores prior to germina-t ion. An increase in vesicles (Ve), vacuoles (Va), and 1ipid droplets (L) is common. (BB = basal body; D = dictyosome; L = 1ipid droplet; Li - liposome; M = mito-chondrion; N = nucleus; Nu - nucleolus; P =' parastrasome; P-mt - microtubule packet; Va = vacuole; Ve = vesic le; Ve-mt = microtubule vesic le; W = wall . ) Glutaraldehyde-osmium tetroxide f ixat ion. Figure 248 Figure 249 Figure 250 - X 15,750 - X 8,050 - X 7,500 PLATE SEVENTY-FIVE Figures 251-254 Dictyosomes in encysted primary zoospores immediately,prior to germination and in f l a -gellated secondary zoospores. Numerous secretory vesicles containing electron-l ight amorphous material and a few tubular elements (arrows) are formed by the distal cisternae. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 251 - X 41,170 Figure 252 - X 43,170 Figure 253 - X 41,170 Figure 254 - X 34,460 Figure 255 Dictyosome in an encysted secondary zoospore. Large secretory vesicles no longer are common about the distal cisternae. GIutaraldehyde-osmium tetroxide f ixat ion. X 40,560 PLATE SEVENTY-SIX Figures 256-261 Germination of encysted primary zoospore. The protoplast of f lagellated secondary zoospore emerges through pore in wal l . Phase-contrast microscopy. Figures 256-261 - X 700 Figures 262-264 Flagellated secondary zoospores. Phase-contrast microscopy. Figures 262 - 264 - X 725 o 256 257 258 P 259 260 261 o 262 2 63 2S4 PLATE SEVENTY-SEVEN Cross-section of flagellated secondary zoospores. (L = l i p i d droplet; Li = liposome; M = mito-chondrion; Mi = microbody; N = nucleus; Nu = nucleolus; PM = plasma membrane; S = saccule.) Glutaraldehyde-osmium tetroxide f ixat ion. X 9,270 Vacuolar region in flagellated secondary zoo-spores. Large vesicles,or saccules, are not present near the vacuole. (D = dictyosome area; PM = plasma membrane; Va = vacuole.) ? Glutaraldehyde-osmium tetroxide f ixat ion. X 38,200 PLATE SEVENTY-EIGHT Figures 267-269 Pe l l i c le of f lagellated secondary zoospores. It consists of flattened to dialated saccules, or vesic le, which immediately underlie the plasma membrane. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 267 - X 17,340 Figure 268 - X 34,660 Figure 269 - X 50,000 Figures 270-271 The release of microtubule-1ike fragments from flagellated secondary zoospores. Vesi-cles containing the fragments appear to have fused with the plasma membrane. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 270 Figure 271 - X 70,000 - X 71,500 PLATE SEVENTY-NINE Figure 272 Longitudinal section of a basal body in ger-minating primary zoospore. A developing set of rootlets is v is ib le at the proximal pole of the basal body, and numerous axonemal vesicles are common about the distal pole of the basal body. Glutaraldehyde-osmium tetroxide f ixat ion. X 37,200 Figure 273 Rootlets in germinating primary zoospore. They appear as groups of microtubules ( R - l , R-2) which originate near the proximal pole of the basal bodies. One group of microtu-bules (R-l) passes near the parastrasome. Glutaraldehyde-osmium tetroxide f ixat ion. X 14,000 Figures 274-275 Longitudinal sections of a whip-lash flagellum. ". - •; Osmiophilic bands (arrows) connect the termi-nal plate of the basal body region with the plasma or axonemal membrane. Rootlets (R) radiate from the proximal region of the basal bodies and numerous individual microtubules (Mt) also radiate into the protoplasm. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 274 - X 30,400 Figure 275 - X 35,450 PLATE EIGHTY Figures 276-277 Whip-lash f lagel la of secondary zoospores. Their terminal axis appear tapered - mea-suring about one-third to one-fourth the diameter of the rest of the axis. Palladium-gold shadowed. Figure. 276 - X 35,000 Figure 277 - X 23,000 Figures 278-279 Enlargements of mastigonemes of tinsel f lage l la . A s l ight ly dense core or canal (arrows) barely is v is ib le in each of the mastigonemes. Palladium-gold shadowed. Figure 278 - X 49,500 Figure 279 - X 36,000 Figure 280 Tinsel flagellum of secondary zoospore. Its end is tapered and lacks mastigonemes. The thick part of the flagellum is characterized by mastigonemes, and arrows indicate fine ha i r - l ike extensions on the ends of the mastigonemes. Palladium-gold shadowed.. X 28,800 PLATE EIGHTY-ONE Figures 281-282 Encysted secondary zoospores. (D = dictyosome; L = l i p id droplet; M = . mitochondrion; Va = vacuole; Ve-mt = microtubule packet; W = wall . ) Glutaraldehyde-osmium tetroxide f ixat ion. Figure 281 - X 15,000 Figure 282 - X 11,560 Figure 283 Section of discarded f lagel lar bead. The axonemal sheath (AxS) surrounds several sec-tions of the 9+2 f i b r i l l a r association. Glutaraldehyde-osmium tetroxide f ixat ion. X 28,700 Figure 284 Remnants of a discarded flagellum. Central f i b r i l s are lacking in part of the flagellum and fragmentation (arrows) of the axonemal sheath is apparent. Glutaraldehyde-osmium tetroxide f ixat ion. X 29,460 Figure 285 An encysted secondary zoospore prior to germ tube formation. Several muclei are v is ib le in the protoplasm. (D = dictyosome; L = l i p i d droplet; M = mitochondrion; Mi = microbody; N = nucleus; Va = vacuole; Ve-mt - microtubule packet; W = wal1.) Gluteraldehyde-osmium tetroxide f ixat ion. X 9,520 PLATE EIGHTY-TWO Figure 286 Germ tube formation region of encysted secon-dary zoospore. Vesicles become concentrated in protoplasm where germ tube wi l l be formed. Glutaraldehyde-osmium tetroxide f ixat ion. X 26,000 Figure 287 Longitudinal section of apical growing zone of germ tube. Numerous vesicles are present in t ip of thin-walled protoplasm. Glutaraldehyde-osmium tetroxide f ixat ion. X 25,900 Figures 288-290 Longitudinal sections of germ tubes formed by encysted secondary zoospores. Vesicles (Ve) are common in t ip of germ tubes and mito-chondria (M) and.small vacuoles (Va) are conspicuous throughout rest of protoplasm. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 288 Figure 289 Figure 290 - X 10,130 - X 3,4.00 - X 11,435 PLATE EIGHTY-THREE Figures 291-296 Selected serial sections through germ tube formed by encysted secondary zoospore. Vesicles are common in most of the apical protoplasm (Figures 290-292), but mitochon-dr ia , vacuoles and other organelles and inclusions become apparent as the distance from the t ip of germ tube increases (Figures 292-295). Glutaraldehyde-osmium tetroxide f ixat ion. Figure: 291 Figure 292 Figure 293 Figure 294 Figure 295 Figure 296 - X 37,440 - X 37,440 - X 31 ,780 - X 30,400 - X 27,730 - X 24,000 PLATE EIGHTY-FOUR Figure 297 Young antheridial hypha prior to oogonial con-tact. A septum has not yet been formed de-l imit ing antheridial protoplasm from the ve-getative hypha. Phase-contrast microscopy. X 840 Figure~298 A developing oogonium. A septum has not yet been formed to delimit the oogonium from the vegetative hypha. Phase-contrast microscopy. X 800 Figures 299-300 Longitudinal to s l ight ly oblique sections of oogonia encompassed by antjieridia. The oo-gonia are larger than the antheridia and a regular to irregularly-shaped septum de-l imits the oogonial protoplasm from the hyphal protoplasm. Note that numerous dark blue granules, probably liposomes, are d is -tinguishable in the oogonial protoplasm after Toluidine blue with borax staining (Figure 298). The dark-blue staining gra-nules are lacking in the antheridia. L i t t l e or no difference in the staining properties of the granules was observed after sections were treated with Flemming's t r ip le stain. Br ight - f ie ld microscopy. Figure 299 - X 1400 Figure 300 - X 1200 Figure 301 Oospores in a fe r t i l i zed oogonium. Phase-contrast microscopy. X 840 PLATE EIGHTY-FIVE Figure 302 Sl ightly oblique section of young antheridium. Numerous vesicles (Ve) containing osmiophilic gra-nules are scattered throughout the protoplasm and many mitochondria (M) are characterized by termi-nal invaginations. Multivesicular bodies (MVB) and l i p id droplets (L) also are conspicuous in the antheridium. Glutaraldehyde-osmium tetroxide f ixat ion. X 20,400 Figure 303 Longitudinal section of the growing t ip of a young antheridium. Numerous vesicles containing osmio-phi l ic granules are concentrated in the apical protoplasm near the plasma membrane which is crenulate to irregular in prof i le . The wall about the growing t ip also is very irregular in prof i le . Glutaraldehyde-osmium tetroxide f ixat ion. X 32, 140 PLATE EIGHTY-SIX Figure 304 Longitudinal section of a young antheridium. Numerous vacuoles (VaJ) are oriented along the central axis of the hypha to form a large central vacuolar complex and other organelles and inclusions are scattered throughout the peripheral protoplasm. (D = dictyosome; M = mitochondrion; Va = vacuole; Ve = osmiophilic vesicle; W = wall) Glutaraldehyde-osmium tetroxide f ixat ion. X 20,5.70 Figures 305-306 Cross-section of young antheridia. Vesicular invaginations are conspicuous in many of the mitochondria (M) and vesicles (Ve) containing osmiophilic granules appear most abundant in the peripheral protoplasm. CD = dictyosome; L - l i p i d droplet; M = mitochondrion; Va = vacuole; Ve = vesicle containing osmiophilic granules; W = wall) Glutaraldehyde-osmium tetroxide f ixat ion. Figure 305 - X 10,500 Figure 306 - X 7,275 PLATE EIGHTY-SEVEN Figures 307-311 Mitochondria, in antheridial protoplasm. Vesicular invaginations containing osmiophilic granules, liposomes, or small vesicular particles are pre-sent at the ends of several of the mitochondria (Figs. 307, 309 and 310, respectively). Other mitochondria appear highly constricted (Figs. 307, 310) or are encompassed by multi -vesicular bodies (Fig. 308). Glutaraldehyde-osmium tetroxide f ixat ion. Figure 307 - X 27,740 Figure 308 - X 36,000 Figure 309 - X 39,000 Figure 310 - X 43,700 Figure 311 - X 26,500 PLATE EIGHTY-EIGHT Figures 312-313 Nuclei in young antheridial protoplasm. Note that only a few poorly-developed liposomes (Li) are v is ib le in the hyphae and that sev-eral mitochondria (M) possess terminal ves i -cular invaginations. (ER = endoplasmic reticulum; L = l i p i d drop-l e t ; Li = liposome; Lo = lomasome; M = mitochondrion; MVB = multi-vesicular body; N = nucleus; Va = vacuole; W = wall) Glutaraldehyde-osmium tetroxide f ixat ion. Figure 312 - X 12,000 Figure 313 - X 12,510 figure 314 Antheridial hyphae in contact with the oogo-nial c e l l . Mitochondria and granular ER are abundant in the protoplasm and pockets of dense granules and vesicular elements are v is ib le in the antheridial walls. (Ce = centriole; ER = endoplasmic ret icu -lum; M = mitochondrion; Mi = microbody; N = nucleus;. Va = vacuole; W-a = antheridial wal l ; W-o = oogonial wal l ) . Glutaraldehyde-osmium tetroxide f ixat ion. X 16,665 PLATE EIGHTY-NINE Figures 315-316 Septal formation in antheridial hyphae. Vesi-cles containing dense granular material aggre-gate and fuse together near the hyphal wal l . GlutBraldehyde-osmium tetroxide f ixat ion. Figure 315 - X 37,180 Figure 316 - X 37,180 Figure 317 Pockets of s l ight ly condensed osmiophilic granular and vesicular material appear to form several small septa within antheridial protoplasm. Glutaraldehyde-osmium tetroxide f ixat ion. X 17,365 Figure 318 An oblique section of a developing septum in an antheridial hypha. Greater condensation of the dense granular and vesicular material is evident. Glutaraldehyde-osmium tetroxide f ixat ion. X 16,255 PLATE NINETY Figures 319-320 Septa in antheridial hyphae. Variable amounts of dense granular and vesicular material appear condensed between two wall layers. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 319 - X 16,600 Figure 320 - X 24,000 PLATE NINETY-ONE Figures 321-322 Septa in antheridial hyphae. Variable amounts of dense vesicular material is condensed within layers of wall material in each septum. The septa appear re la -t ively uniform in profi le (Figure 321) or extremely irregular (Figure 322). Glutaraldehyde-osmium tetroxide f ixat ion. Figure 321 - X 14,280 Figure 322 - X 22,200 PLATE NINETY-TWO Section of a young oogonium. Rod-shaped mito-chondria (M), liposomes (Li) in various stages of development, dilated granular ER, l i p i d droplets (L), and a nucleus (N) are conspi-cuous within the thin-walled protoplasm. Glutaraldehyde-osmium tetroxide f ixat ion. X 20,400 Sl ightly oblique section of a nucleus in young oogonial protoplasm. The nuclear envelope is crenulate in profi le except adjacent to the centriole where i t is de-pressed. Microbodies (Mi), dilated gra-nular ER, and dictyosomes (D) also are v is ib le in the surrounding protoplasm. Glutaraldehyde-osmium tetroxide f ixat ion. X 20,400 Dictyosome in young oogonial protoplasm. It occurs in close association with the nucleus (N) and numerous irregularly-shaped vesicles are v is ib le about the cisternae. Vesicles also appear near the plasma mem-brane and fusion of the vesicles with the membrane is evident (arrows). Glutaraldehyde-osmium tetroxide f ixat ion. X 23,300 PLATE NINETY-THREE Figure 326 Cross-section of a young oogonial protplast. A thin peripheral protoplasm consisting of numerous 1ipid droplets (L), liposomes (L i ) , mitochondria (M), several nuclei (N), and vesicles (Ve) containing wa l l - l i ke material surrounds a large central vacuole (Va). A wall (W) with a rough surface encompasses the oogonium. Glutaraldehyde-osmium tetroxide f ixat ion. X 4,420 Figures 327-328 Enlargements of the oogonial protoplmas. Juxta-nuclear centrioles (Ce), dictyosomes (D), and microbodies (Mi) also are evident in the proto-plasm. The vesicles (Ve) containing wa l l - l i ke material mostly occur near the plasma membrane, and several vesicles appear to have fused with the plasma membrane (arrows). Glutaraldehyde-osmium tetroxide f ixat ion. Figure 327 - X 6,600 Figure 328 - X 11,250 PLATE NINETY-FOUR Figure 329 Cross-section of a maturing oogonium. Numerous vesicles containing wall-1 ike material are concentrated near the plasma membrane (PM) and appear to fuse with i t (arrows). The vesicles appear to arise from secretory vesicles formed from nearby dictyosomes (D). Liposomes (L i ) , l i p id droplets (L), and mitochondria also are conspicuous in the protoplasm. Glutaraldehyde-osmium tetroxide f ixat ion. X 15,600 Figures 330-331 Dictyosomes in oogonial protoplasm. Note the s imilar i ty in the secretory vesicles and those vesicles adjacent to the plasma mem-brane. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 330 - X 26,400 Figure 331 - X 33,000 PLATE NINETY-FIVE Figures 332-333 Liposome development. Dialated ER (Figures 332-333) and/or secretory vesicles formed by dictyosomes (Figure 332) appear to d i f f e r -entiate into liposomes. (D = dictyosome; ER = dialated endoplasmic reticulum; Ve = secretory vesicle) Glutaraldehyde-osmium tetroxide f ixat ion. Figure 332 - X 38,275 Figure 333 - X 45,000 Figures 334-335 Differentiating liposomes in oogonial proto-plasm. Figure 334 - X 34,200 Figure 335 .- X 38,000 PLATE NINETY-SIX Figure 336 A section showing a septum which delimits. oogonial protoplasm from hyphal protoplasm. The septum consists of an irregular layer of osmiophilic granules and vesicles com-pressed between two electron-l ight layers of wall material. One wall layer is consid-erably thicker than the other and i t also is continuous about the oogonial protoplasm. The other layer l ies adjacent to the hyphal protoplasmic region. Glutaraldehyde-osmium tetroxide f i xa t ion . ' X 24,000 Figure 337 Section of an oogonial septum. Both optical ly l ight layers of the septum are distinguishable, especially the layer.adjacent to the oogonial protoplasm. Also note that dark-blue staining bodies are v is ib le in oogonial and hyphal protoplasm, but are lacking in antheridial cel ls (Toluidine blue with borax stained). Br ight - f ie ld microscopy. X 2,800 Figure 338 Mitochondria in young oogonial protoplasm. Gluteraldehyde-osmium tetroxide f ixat ion. X 36,000 Figures 3.39-340 Microbodies common to young oogonial proto-plasm. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 339 - X 54,460 Figure 340 - X 39,500 PLATE NINETY-SEVEN Figure 341 Section showing i n i t i a l contact of oogonium by antheridial hyphae. Vesicles (arrows) in both cel l types are distributed randomly within the peripheral protoplasm. Also note that liposomes are abundant within the oogo-nial cel l but are lacking in the antheridia. Glutaraldehyde-osmium tetroxide f ixat ion. X 4,210 Figures 342-344 Sections showing later stages of antheridial and oogonial contact. Vesicles (Ve) are concentrated near the plasma membrane in both cel l types at the regions of contact. Few vesicles are present in other areas of the protoplasm. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 342 Figure 343 Figure 344 - X 7,435 - X 12,300 - X 14,775 PLATE NINETY-EIGHT Section showing the encompassing of an oogonium by antheridial hyphae. Vesicles (Ve) are concentrated near the plasma membrane in both cel l types at the regions of contact. Also, the antheridial cel ls are characterized by mitochondria with terminal vesicles or i n -vaginations and the oogonium contains num-erous liposomes (Li) and l i p i d droplets^(L). (L = l i p id droplet; Li - liposome; M = mitochondrion; N = nucleus; Va = vacuole; Ve = vesicle) Glutaraldehyde-osmiun tetroxide f ixat ion. X 7,040 PLATE NINETY-NINE Figures 346-347 Antheridial and oogonial contact. Vesicles in both cel l types become concentrated near the contact surfaces, and fusion (arrows) of several vesicles to the plasma membrane is apparent. The walls of the antheridia and oogonium appear to "cement" together. (Ce = centriole; L = l i p id droplet; Li = liposome; M = mithchondrion; MVB = multi -vesicular body; N = nucleus; Nu = nucleolus; Ve = vesic le; W-a _=• antheridial wal l ; W-o = oogonial wall) Glutaraldehyde-osmium tetroxide f ixat ion. Figure 346 - X 16,665 Figure 347 - X 18,750 PLATE ONE HUNDRED Figures 348-351 Antheridial and oogonial contact in Aahlya bisexualis. Antheridial hyphae almost com-pletely surround an oogonium. Concurrently, nuclei (N) in both cel l types appear in various stages of meiosis. (L = l i p i d droplet; Li = liposome; M = mitochondrion; N = nucleus; Va = vacuole; W-a = antheridial wa l l ; W=o = oogonium wal 1) Glutaraldehyde-osmium tetroxide f ixat ion. Figure 348 - X 3,790 Figure 349 - X 3,790 Figure 350 - X 3,790 Figure 351 - X 3,790 PLATE ONE HUNDRED AND ONE Figures 352-353 Maturing oogonial protoplasm. An extremely thick wall (W) formed of two major layers, surrounds the dense protoplasm. Lipid droplets (L) and liposomes (Li) are extremely abundant and several nuclei (N) appear randomly scattered within the c e l l . Other organelles and inclusions are more easily recog-nizable at greater magnifications. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 352 - X 4,080 Figure 353 - X 4,080 PLATE ONE HUNDRED AND TWO Figures 354-356 Nuclei in the interphase state in oogonial protoplasm.. They are irregular in profi le and they each possess a single nucleolus which l ies adjacent to the centriolar re-gion of the nuclear envelope. (Ce = centriole; NE = nuclear envelope; Nu = nucleolus) Glutaraldehyde-osmium tetroxide f ixat ion. Figure 354 - X 23,370 Figure 355 - X 33,400 Figure 356 - X 45,700 PLATE ONE HUNDRED AND THREE Figures 357-359 Antheridial and oogonial nuclei in prophase I of meiosis. Single axial elements, which often are associated (arrows) with the nu-clear envelope, are v is ib le within the nucleoplasm. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 357 - X 33,775 Figure 358 - X 20,530 Figure 359 - X 33,500 PLATE ONE HUNDRED AND FOUR Figures 360-364 Synaptinemal complex formation in antheridial and oogonial nuclei. Chromatin material is closely associated with the nuclear envelope and spindle fibers (arrows) become conspicuous within the nucleoplasm. The spindle fibers radiate from the thickened inner-membrane of the envelope. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 360 - X 32,170 Figure 361 - X 32,800 Figure 362 - X 31 ,385 Figure 363 - X 38,000 Figure 364 - X 21,000 PLATE ONE HUNDRED AND FIVE Figures 365-366 Centrioles in association with nuclei in anaphase-of meiosis. The centrioles l i e in a depression of the nuclear envelope and spindle f i b r i l s ra -diate to chromosomes from the thickened inner en-velope membrane adjacent to the centrioles. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 365 - X 36,000 Figure 366 - X 36,500 Figure 367 Centrioles in association with meiotic telophase nucleus. Spindle f i b r i l s appear short and are associated with mass of chromatin-1ike material. Individual chromosomes are not distinguishabe. Glutaraldehyd,e-osmium tetroxide f ixat ion. X 42,000 Figure 368 Cross-section of centriole showing the 9 peripheral t r ip lets in cartwheel arrangement about central "tubule". . Glutaraldehyde-osmium tetroxide f ixat ion. X 47,000 Figures 369-370 Nuclei at end of telophase I of meiosis. Spindle f i b r i l s are barely discernible in area adjacent to centrioles. Nucleoli present in both newly-formed nuclei. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 367 - X 20,100 Figure 368 - X 29,750 PLATE ONE HUNDRED AND SIX Figures 371-374 Serial sections of meiotic anaphase. Chromosomes are beginning to separate and move to opposite intranuclear poles. Juxta-nuclear centrioles l i e adjacent to the poles. Spindle - f ibr i ls extend from pole-to-pole or appear attached to one of the chromosomes. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 371. - X 22,220 Figure 372 - X 20,400 Figure 373 - X 20,400 Figure 374 - X 25,500 Figures 373(a)- Increased magnifications of Figures 374 and 375, 374(a) respectively. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 373(a) - X 38,700 Figure 374(a) - X 48,750 PLATE ONE HUNDRED AND SEVEN Figures 375-376 Nuclei during telophase II of meiosis in antheridial protoplasm. Kinesis of the two daughter nuclei (N-1 and N-2) is v i s ib le . (Ce = centr iole; N-1 and N-2 = nuclei ; Nu = nucleolus; SF = spindle f i b r i l ) . Glutaraldehyde-osmium tetroxide f ixat ion. Figure 375 - X 25,200 Figure 376 - X 24,065 PLATE ONE HUNDRED AND EIGHT Figures 377-378 Penetration of oogonia by antheridial f e r t i l i z a -tion tubes. Numerous vesicles are v is ib le at the point of penetration and the wall of the antheridium become almost undistinguishable from the oogonial wal l . (L = l i p i d droplet; M = mitochondrion; N = nucleus; Ve - vesicles; W-a = antheridial wal l ; W-o = oogonial wal l ) . Glutaraldehyde-osmium tetroxide f ixat ion. Figure 377 - X 29,000 Figure 378 - X 15,000 PLATE ONE HUNDRED AND NINE Figures 379-380 Penetration of oogonia by antheridial f e r t i l i z a -tion tubes. Constriction of the thin-walled antheridial hyphae at the point of penetration is apparent. Glutaraldehyde-osmium tetroxide f ixat ion. Figure 379 - X 11 ,425 Figure 380 - X 18,800 Figure 381 Fert i l i zat ion tube in contact with an oospore. Glutaraldehyde-osmium tetroxide f ixat ion. X 13,710 Figure 382 Old fe r t i l i za t ion tube. Glutaraldehyde-osmium tetroxide f ixat ion, X 13,200 PLATE ONE HUNDRED AND TEN Figure 383 Oosphere within an oogonium (L = l i p i d droplet; M = mitochondrion; N = nucleus; W = wal l ) . Glutaraldehyde-osmium tetroxide f ixat ion. X 23,300 Figure 384 Oosphere within an oogonium. Many l i p i d drop-lets in the oopshere protoplasm have coalesced. Old fe r t i l i za t ion tubes are v is ib le about the oosphere. (A = antheridial hyphae; L = l i p i d droplet; W = oosphere wal1). Glutaraldehyde-osmium tetroxide f ixat ion. X 8,500 Figures 385-386 Oosphere nuclei . Dense chromatin-1ike material (arrows) is v is ib le within the nuclei . Glutaraldehyde-osmium tetroxide f ixat ion. Figure 384 Figure 385 - X 38,250 - X 64,000 PLATE ONE HUNDRED AMD ELEVEN Figures 387-388 Dictyosome regions in oosphere protoplasm. Numerous vesicles containing osmiophilic cores (arrows) are v is ib le near the dictyosomes. (D = dictyosome; Li = liposome; M = mito-chondrion; N = nucleus). Glutaraldehyde-osmium tetroxide f ixat ion. Figure 386 - X 42,160 Figure 387 - X 36,800 Figure 389 Liposomes in oosphere protoplasm. They frequently contain several osmiophilic bodies which are bounded by alternating layers of electron-l ight and electron-dense material. Glutaraldehyde-osmium tetroxide f ixat ion. X 41,560 Figure 390 Centrioles in oosphere protoplasm. (Ce = centr iole; M = mitochondrion; N = nucleus). Glutaraldehyde-osmium tetroxide f i xat ion . X 46,800 PLATE ONE HUNDRED AND TWELVE Oospores within an old oogonium which is encom-passed by empty antheridial fe r t i l i za t ion tube. (AH = antheridial hyphae; L - l i p i d droplet; Li = liposome; N = nucleus; OW = oospore wal l ; PP = penetration pore; W-o = oogonial wal l ) . Glutaraldehyde-osmium tetroxide f ixat ion. X 5,230 Section of an oospore with a fusion nucleus (N-), Lipid droplets (L) and liposomes (Li) are conspicuous in the protoplasm. Glutaraldehyde-osmium tetroxide f ixat ion. X 8,400 Section showing the walls of an antheridium, the oogonium and the oospore. Four layers are distinguishable in the oospore wal l . (W-a = antheridium wal l ; W-o = oogonium wal l ; 1 , 2, 3 , 4 = oospore wall layers). Glutaraldehyde-osmium tetroxide f ixat ion. X 13,680 PLATE ONE HUNDRED AND THIRTEEN Enlargement of oospore wall and surrounding proto-plasm. The wall consists of three layers, the inner layer is thickest and appears electron- l ight. It is separated from the outer layer by a thin dense middle layer which is irregular in prof i le . Lipid droplets (L) and liposomes (Li) are v is ib le in the protoplasm. Glutaraldehyde-osmium tetroxide f ixat ion. X 24,360 Liposome bounded by l i p i d droplets in oospore proto-plasm. (L = l i p i d droplet; Li = liposome). Glutaraldehyde-osmium tetroxide f ixat ion. X 35,930 A possible fusion nucleus in oospore protoplasm. F i b r i l - l i k e elements are v is ib le within i ts nucleoplasm, and l i p id droplets and liposomes surround the nucleus. (L = l i p i d droplet; Li = liposome; N = nucleus). Glutaraldehyde-osmium tetroxide f ixat ion. X 21,300 

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