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Petersenia pollagaster (Oomycetes) : an invasive pathogen of Chondrus crispus (Rhodophyceae) Molina, Francis I. 1986

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PETERSENIA POLLAGASTER (OOMYCETES): AN INVASIVE PATHOGEN OF CHONDRUS CRISPUS (FHODOPHYCEAE) by FRANCIS I . MOLINA M . S c , U n i v e r s i t y of the P h i l i p p i n e s , 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPBY i n THE FACULTY OF GRADUATE STUDIES (Department of Botany) We accept t h i s f h - e s i s as confor m i n q t o the r/equired s t a n d a r d . THE UNIVERSITY OF BRITISH COLUMBIA 30 June 1986 °Francis I . M o l i n a , 1986 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. F r a n c i s I . M o l i n a Department of B o t a n y  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date 23 J une 1986 DE-6 (3/81) - i i -ABSTRACT Cytochemistry, v i t a l s t a i n i n g , l i g h t - and t r a n s m i s s i o n e l e c t r o n microscopy were employed to study the h o s t - p a r a s i t e r e l a t i o n s h i p between a pathogenic oomycete and a m a r i c u l t u r e d gametophytic s t r a i n of Chondrns c r i s p u s Stackhouse ( I r i s h moss). I n o c u l a t i o n of healthy a l g a l t h a l l i with i n f e c t i v e f u n g a l zoospores was performed at f i v e temperatures and the highest i n f e c t i o n r a t e was obtained at 20C. The c y c l e c o n s i s t i n g of i n f e c t i o n of healthy t h a l l i , e n d o b i c t i c development, and r e l e a s e of zoospores i s completed i n 48-72 h at t h i s temperature. The p a r a s i t e i n i t i a l l y invades c o r t i c a l c e l l s of the host and spreads to the s u b - c o r t i c a l and medullary t i s s u e s . I t i s e n d o b i o t i c , simple, h o l c c a r p i c , and produces heterokont zoospores which are r e l e a s e d v i a a s i n g l e e x i t tube. Cytochemical s t a i n i n g procedures employing C a l c o f l u o r White, Congo Red, and Zinc C h l o r i o d i d e r e v e a l e d the presence of c e l l u l o s e i n s p o r a n g i a l w a l l s . The pathogen i s a s s i g n e d to P e t e r s e n i a p o l l a g a s t e r (Petersen) Sparrow. Four fluorochroraes were i n i t i a l l y screened f o r f l u o r e s c e n c e d i f f e r e n t i a t i o n between l i v i n g and dead host c e l l s . The most c o n s i s t e n t r e s u l t s were obtained with 4',6-diamidino-2-phenylindole (DAPI). Incubation of newly i n o c u l a t e d host t i s s u e i n t h i s fluorochrome r e s u l t e d i n the f l u o r e s c e n c e of host c e l l s around i n f e c t i o n s i t e s , showing - i i i e x t e n s i v e h o s t t i s s u e damage. U l t r a s t r u c t u r a l s t u d i e s r e v e a l e d t h a t z o o s p o r e s r e t r a c t t h e i r f l a g e l l a d u r i n g e n c y s t m e n t cn t h e h o s t a l g a . They s o o n become i n v e s t e d w i t h a t h i n w a l l and a f u z z y c e l l c o a t w h i c h may have a r o l e i n h o s t r e c o g n i t i o n and a t t a c h m e n t . The d e v e l o p m e n t i s m a i n l y i n t r a c e l l u l a r . The p a r a s i t e i n i t i a l l y d e v e l o p s w i t h i n t h e h o s t as a p r o t o p l a s t . I t s w a l l - l e s s v e g e t a t i v e t h a l l u s p r o l i f e r a t e s v i a a s y m p l a s t i c r o u t e and c a u s e s t h e f u s i o n o f h o s t c e l l s t h r o u g h t h e r e m o v a l o f p i t p l u g s . Damage t o h o s t t i s s u e i s m i n i m a l d u r i n g t h e e a r l y s t a g e s o f d e v e l o p m e n t and s u g g e s t s t h e p a r a s i t e ' s need f o r a l i v i n g h o s t . M o b i l i z a t i o n o f f l o r i d e a n s t a r c h g r a i n s i n t h e h o s t c y t o p l a s m i s i n d i c a t e d by t h e i r a p p a r e n t d i s s o l u t i o n . D eath o f h o s t c e l l s , c h a r a c t e r i z e d by d i s i n t e g r a t i o n o f t h e plasmalerama, a p p e a r s t o t r i g g e r z c o s p o r c g e n e s i s . C l e a v a g e o f th e s p o r o g e n i c c y t o p l a s m i s a c c o m p l i s h e d by t h e f u s i o n o f G o l g i - d e r i v e d c i s t e r n a e and s m a l l v a c u o l e s . F o r m a t i o n o f z o o s p o r e s i s c o m p l e t e d w i t h i n t h e s p o r a n g i u m . The d e a t h o f h o s t c e l l s a t i n f e c t i o n s i t e s and t h e demise o f some z o o s p o r e s i n s i d e t h e a l g a l t h a l l u s i m p l i e s a h y p e r s e n s i t i v e h o s t r e s p o n s e . - i v -TABLE OF CONTENTS Fage ABSTRACT i i TABLE OF CONTENTS . i v L I S T OF TABLES v i i LIS T OF FIGURES v i i i ACKNOWLEDGEMENTS x i i INTRODUCTION A. F u n g i as s y m b i o n t s o f a l g a e 1 B. S t a g e s i n t h e d e v e l o p m e n t o f p a r a s i t i c Oomycetes .. 2 1. B o s t r e c o g n i t i o n and s e t t l e m e n t o f z o o s p o r e s .. 3 2. C y s t g e r m i n a t i o n 6 3. Z o o s p o r o g e n e s i s 7 C. Host r e s p o n s e s t o i n f e c t i o n 12 D. Changes i n the h o s t - p a r a s i t e i n t e r f a c e 16 E. P r e v i o u s s t u d i e s on d i s e a s e s o f c u l t i v a t e d seaweeds 17 F. B a c k g r o u n d o f t h e p r o b l e m 1. C h o ndrus c r i s o u s S t a c k h o u s e 19 2. F u n g a l i n f e c t i o n s o f £ . c r i s p u s 20 3. P e t e r s e n i a p o l l a o a s t e r ( P e t e r s e n ) Sparrow 21 G. O b j e c t i v e s o f t h e p r e s e n t s t u d y 22 MATERIALS AND METHODS A. C o l l e c t i o n of m a t e r i a l s 24 B. L a b o r a t o r y c u l t u r e s o f t h e h o s t 24 C. C r o s s - i n f e c t i o n e x p e r i m e n t s 25 - v -page D. C u l t u r i n g of the p a r a s i t e 26 E. L i g h t microscopy 27 F. E l e c t r o n microscopy 31 RESULTS AND OBSERVATIONS A. P.. p o l l a g a s t e r : zoospores and c e l l w a l l c y t o c h e m i s t r y 32 B. c u l t u r i n g of p.. p o l l a g a s t e r 33 C. O b s e r v a t i o n s on pathogenesis 33 D. U n i n f e c t e d £ . c r i s p u s 1. L i g h t microscopy . 35 2. U l t r a s t r u c t u r e 36 E . Development of j». p o l l a g a s t e r 1. L i g h t microscopy 38 2. U l t r a s t r u c t u r e a. P o s t m o t i l e spores and c y s t s 41 b. V e g e t a t i v e phase 44 c. Sporangium for m a t i o n and zoosporogenesis 46 F. E f f e c t s of i n f e c t i o n on host t i s s u e 1. F l u o r e s c e n t v i t a l s t a i n i n g 50 2. S w e l l i n g of p e n e t r a t e d host c e l l s : morphometric a n a l y s i s 52 3. Changes i n host ul t r a s t r ucture 53 DISCUSSION A. Taxonomic a f f i n i t y of the pathogen 55 B. C u l t u r i n g of the pathogen 57 - vi -page C. Observations on pathogenesis and factors that favor the spread and development of the parasite .. 58 D. Uninfected £. crispus: l ight and electron microscopy 60 E. Development of £. pollaaaster 1. Postmotile stage, encystment, and vegetative phase 62 2. Zoosporogenesis 69 F. Effects of infection on the host 1. Fluorescence induced by DAPI and swelling of host ce l l s 74 2. Changes in host ultrastructure and extent of host response 76 GENERAL DISCUSSION AND SUMMARY 82 TABLES 86 KEY TO FIGURES 89 FIGURES 91 LITERATURE CITED 129 APPENDICES 147 L I S T OF TABLES TABLE PAGE I B o s t r a n g e and d i s t r i b u t i o n o f P.. p o l l a g a s t e r 86 and £ . lQbata I I S t a i n i n g t e c h n i q u e s u s e d f o r g e n e r a l 87 h i s t o l o g i c a l and c y t o c h e m i c a l s t u d i e s I I I t - t e s t c o m p a r i s o n o f c r o s s - s e c t i o n a l a r e a s o f u n i n f e c t e d and i n f e c t e d s u b c o r t i c a l c e l l s 88 - v i i i -LIST OF FIGURES FIGURE page 1 L o c a t i o n of Marine C o l l o i d s ' c u l t u r e f a c i l i t i e s 92 2 & 3 zoospores of P.. p o l l a g a s t e r 94 4 C a l c o f l u o r White s t a i n i n g of s p o r a n g i a l w a l l 94 5 F l u o r e s c e n c e of s p o r a n g i a l w a l l with Congo red 94 6 Sporangia of P_. p o l l a g a s t e r p e n e t r a t i n g host c u t i c l e , C a l c o f l u o r White s t a i n i n g 94 7 Zinc C h l o r i o d i d e r e a c t i o n of fu n g a l w a l l 94 8 I n f e c t e d £ . c r i s p u s ; e a r l y stage 96 9 Late stage of the d i s e a s e 96 10 H e a v i l y i n f e c t e d host t h a l l u s 96 11 U n i n f e c t e d host 96 12 S c a t t e r diagram of number of l e s i o n s at f i v e 98 temperatures showing the best f i t curve 13 U n i n f e c t e d £ . c r i s p u s : c r o s s - s e c t i o n showing 100 c u t i c l e and three t i s s u e types 14 U n i n f e c t e d h o s t : m e d u l l a r y c e l l s 100 15 Fine s t r u c t u r e of u n i n f e c t e d host c u t i c l e 100 16 C o r t i c a l c e l l of u n i n f e c t e d host 100 17&18 S u b c o r t i c a l c e l l s of £ . c r i s p u s 102 19 S t a r c h g r a i n s i n the cytoplasm of a c o r t i c a l c e l l 102 20 Me d u l l a r y c e l l of u n i n f e c t e d host 102 21 P i t c o n n e c t i o n beween s u b c o r t i c a l c e l l s 102 22 I n f e c t e d c o r t i c a l c e l l , l i g h t microscopy 104 i x -FIGURE PAGE 23 Wall l e s s t h a l l u s of £. p o l l a g a s t e r showing 104 s y m p l a s t i c development 24&25 Sporangia i n s u b c o r t i c a l and med u l l a r y t i s s u e s 104 of the host 26&28 M u l t i n u c l e a t e sporangium of £ . p o l l a g a s t e r 106 27 I n t r a c e l l u l a r sporangium of the p a r a s i t e 106 29 E x i t tube of sporangium forming i n a 106 pre-forroed channel 30 Emptied sporangium showing a f u l l y - f o r m e d 106 zoospore 31 Released sporangia of £. p o l l a g a s t e r 106 32&34 U l t r a s t r u c t u r e of p o s t - m o t i l e spore 108 35 R e t r a c t e d axoneroe, l o n g i t u d i n a l s e c t i o n 108 36 Basal bodies r e s u l t i n g from the breakdown 108 of the axoneme 37 Cyst of the p a r a s i t e on host c u t i c l e 110 38 Petersenia-Chondrus i n t e r f a c e : c y s t stage 110 39 P a r t l y d i s s o c i a t e d c y s t showing d i s s o l u t i o n 110 of host c u t i c l e 40 I n v a g i n a t i o n of host membrane by the 110 p a r a s i t e p r o t o p l a s t 41 Petersenia-Chondrus i n t e r f a c e : v e g e t a t i v e stage 110 42 I n t r a c e l l u l a r p r o t o p l a s t of R. p o l l a g a s t e r 112 43 B i n u c l e a t e p r o t o p l a s t with prominent 112 n u c l e o l i and p e r i p h e r a l m itochondria - x -FIGURE PAGE 44 Formation of a narrow c y t o p l a s m i c p r o c e s s p r i o r 112 to i n v a s i o n of adjacent host c e l l s 45 P r o t o p l a s t G o l g i i n zone of e x c l u s i o n 112 46 M i g r a t i o n of mito c h o n d r i a d u r i n g s y m p l a s t i c 112 spread of the p a r a s i t e 47 I n t r a c e l l u l a r sporangium i n n e c r o t i c host 112 c e l l 48 N e c r o t i c host c e l l s a d jacent to sporangium 114 49&50 E a r l y s p o r a n g i a l stages of P.. p o l l a g a s t e r 114 51 Sporangium with c e n t r a l v acuole 114 52 M o b i l i z a t i o n of dense bodies 114 53&54 Nuclear e n v e l o p e - t r a n s i t i o n v e s i c l e s - G o l g i 116 r e l a t i o n s h i p 55 M i c r o b o d y - l i k e s t r u c t u r e s 116 56 C o n c e n t r i c dense membranes i n 116 s p o r a n g i a l cytoplasm 57 A s s o c i a t i o n of p a i r e d b a s a l bodies with 116 the nucleus 58 I n t r a c e l l u l a r sporangium with v a r i o u s 118 types of i n c l u s i o n s 59 Fragmentation of the t h a l l u s 118 60&61 Cleavage by G o l g i - d e r i v e d c i s t e r n a e 118 62 F u s i o n of vacuoles with cleavage c i s t e r n a e 120 63 A s s o c i a t i o n of nucleus with m i t o c h o n d r i a , 120 G o l g i , and b a s a l bodies - xi -FIGURE PAGE 64 Mobilization of crenate vesicle inclusions 120 65 Synthesis of raastigonemes in dilated ER 120 cisternae 66&67 Exerted f lage l la between zoospore i n i t i a l s 122 68 Post-cleavage sporangial cytoplasm 122 69 Flagella associated with pyriform nucleus 122 70 Post-cleavage sporangial cytoplasm 124 71&72 Moribund zoospores in sporangium 124 73 Fluorochroming with DAPI: infected specimen 124 74 Fluorochroming with DAPI: formaldehyde- 124 treated control 75 Dilatation of a proximal interband region 126 in host cut ic le 76 Directly penetrated host c e l l : loss of 126 wall f i b r i l l a r structure 77 Chloroplasts and mitochondria of infected 126 host c e l l 78 Symplastic movement of parasite protoplast 126 79 Pit plug between infected and adjacent 126 host ce l l 80 Formation of compound host ce l l as a 126 result of symplastic development 81 Changes in PetersenAa-ChondKUS inter fac ia l 128 zone - x i i -ACKNOWLEDGEMENTS The author g r a t e f u l l y acknowledges the generous support and guidance p r o v i d e d by Dr. G i l b e r t C. Hughes, h i s re s e a r c h s u p e r v i s o r . T h i s study was funded by c o n t r a c t 080-198/0-6316 from the N a t i o n a l Research C o u n c i l (G. C. Hughes, i n v e s t i g a t o r ) . A d d i t i o n a l funds came from the N a t u r a l Sciences and E n g i n e e r i n g Research C o u n c i l (Grant A-2561) and Sigma X i , the S c i e n t i f i c Research S o c i e t y . Dr. T. B i s a l p u t r a ' s l a b o r a t o r y f a c i l i t i e s and i n s i g h t s i n the i n t e r p r e t a t i o n of micrographs have been e s s e n t i a l to t h i s i n v e s t i g a t i o n . The author a l s o wishes t o thank Mr. A. Burns f o r h i s a s s i s t a n c e i n some b a s i c a spects of e l e c t r o n microscopy and Dr. J . S. C r a i g i e f o r h i s help i n o b t a i n i n g specimens. T h i s study was undertaken while the author was on study leave from h i s post at the Marine Sciences I n s t i t u t e , U n i v e r s i t y of the P h i l i p p i n e s . - 1 -INTRODUCTION A. Fungi as symbionts of algae Fungal symbionts of algae ( a l g i c o l o u s species) i n c l u d e members of the Chytridiomycetes, Bypochytridiomycetes, Oomycetes, Ascomycotina, and Fungi I m p e r f e c t i (Andrews 1976, Kohlmeyer and Kohlroeyer 1979, Goff and Glasgow 1980). The occurrence of z o o s p o r i c f u n g i on or i n marine algae was known as e a r l y as 1905 through the i n v e s t i g a t i o n s of Petersen i n Denmark. Subsequent s t u d i e s , c o n c e n t r a t i n g l a r g e l y on the taxonomy of the species i n v o l v e d , have been reviewed by Meyers (1957), Wilson (1960), Kohlmeyer (1974), Kohlmeyer and Kohlmeyer (1979) and Jones (1976) . Johnson and Sparrow (1961) d e s c r i b e d the occurrence, l i f e h i s t o r i e s , and d i s t r i b u t i o n of the lower groups i n a d d i t i o n to t h e i r taxonomy. More r e c e n t l y , K a r l i n g (1981) p u b l i s h e d a t r e a t i s e on the simple, h o l o c a r p i c , b i f l a g e l l a t e Phycomycetes that i n c l u d e d t h e i r taxonomy, morphology, and host ranges. Despite the abundance of l i t e r a t u r e on the occurrence of fungi on algae, l i t t l e i s known of t h e i r exact n u t r i t i o n a l r e l a t i o n s h i p s and the evidence for t h e i r p a r a s i t i c e x i s t e n c e has been l a r g e l y presumptive (Andrews 1976, 1979). In the absence of s t u d i e s under c o n t r o l l e d l a b o r a t o r y c o n d i t i o n s , i t i s even more d i f f i c u l t to a s c e r t a i n whether these species are n e c r o t r o p h i c ( i . e . , those that contact t h e i r hosts, excrete t o x i c substances which k i l l the host c e l l s , and u t i l i z e the . n u t r i e n t s released) or b i o t r o p h i c , - 2 -b e i n g a b l e t o o b t a i n n u t r i e n t s from t h e i r h o s t s w i t h l i t t l e or no apparent harm ( B a r n e t t and B i n d e r 1 9 7 3 ) . In r e c e n t y e a r s , w i t h the development of advanced m a r i c u l t u r e t e c h n i q u e s and w i t h the growing commercial i m p o r t a n c e of seaweeds, the need has become apparent f o r r e s e a r c h on a l l a s p e c t s of h o s t - p a r a s i t e i n t e r a c t i o n s t h a t may p r o v i d e the b a s i s f o r a r a t i o n a l approach t o d i s e a s e c o n t r o l . Such s t u d i e s might i n c l u d e l i f e h i s t o r y , p a t h o g e n e s i s , e p i d e m i o l o g y , f u n g a l growth w i t h i n the h o s t , and the e x t e n t of host response t o the i n v a s i o n (Andrews 1979, Kohlmeyer and Kohlmeyer 1979) . B. Stages i n t h e development of p a r a s i t i c Oomycetes H e l d (1973) d i v i d e d the i n t e r a c t i o n s of h o l o c a r p i c , m o n o c e n t r i c , e n d o p a r a s i t i c , z o o s p o r i c f u n g i w i t h t h e i r h o s t s i n t o t h r e e major s t a g e s : 1) host r e c o g n i t i o n by means of z o o s p o r e a t t r a c t i o n or t h e i r c o n t a c t w i t h the host s u r f a c e , l e a d i n g t o t h e i r a t t a c h m e n t , e n cystment, and subsequent g e r m i n a t i o n , 2) p e n e t r a t i o n of the host c y t o p l a s m , and 3) i n t r a c e l l u l a r development and m a t u r a t i o n of the p a r a s i t e . He s t r e s s e d an i m p o r t a n t d i s t i n c t i o n between the s e " o l p i d i o i d " f u n g i and most t e r r e s t r i a l p l a n t pathogens: the former d e v e l o p c o m p l e t e l y i n s i d e the host c y t o p l a s m whereas the l a t t e r u s u a l l y form h a u s t o r i a . Complete development, and hence s p r e a d of the p a r a s i t e , i s dependent on the s u c c e s s of a l l the d e v e l o p m e n t a l s t a g e s . - 3 -In the f o l l o w i n g review of l i t e r a t u r e , the trends and developments i n the study of z o o s p o r i c f u n g i are c o n s i d e r e d . A background of the f u n g a l d i s e a s e problem which developed i n the c u l t u r e f a c i l i t i e s of Acadian S e a p l a n t s , L t d . ( f o r m e r l y Marine C o l l o i d s , Ltd.) i n Nova S c o t i a i s a l s o g i v e n . References to s p e c i e s that are found on v a r i o u s other s u b s t r a t a are made whenever a p p r o p r i a t e . 1. Host r e c o g n i t i o n and s e t t l e m e n t of zoospores The l i f e c y c l e s of e n d o p a r a s i t i c , z o o s p o r i c f u n g i t y p i c a l l y s t a r t with the s e t t l e m e n t of zoospores on the host (Held 1973). As i n other c e l l - c e l l i n t e r a c t i o n s , s u c c e s s f u l r e c o g n i t i o n and l o c a t i o n of the host may depend on chemotaxis and/or the presence of c e l l s u r f a c e r e c e p t o r s (Keen 1982). V a r i o u s types of c e l l - s u r f a c e sugars have been i d e n t i f i e d i n zoospores and c y s t s i n both t e r r e s t r i a l and a q u a t i c pathosystems. B i n d i n g of Concanavalin A (Con A), a l e c t i n s p e c i f i c f o r alpha-d-glucosyl/alpha-d-mannosyl r e s i d u e s , has been r e p o r t e d f o r zoospores of Phytophthora palmivora ( B u t l . ) B u t l . (Sing and B a r t n i c k i - G a r c i a 1975) and B l a s t o c l a d i e l l a  e m e r s o n i i Cantino et Hyatt (Jen and Haug 1979). B a c i c et a l . (1985) examined the nature of the sugars on the s u r f a c e of Phytophthora cinnamomi Rands zoospores and c y s t s using v a r i o u s i o d i n a t e d l e c t i n s . Zoospores bound Con A, but d i d not bind any of the other l e c t i n s t e s t e d . S i m i l a r r e s u l t s were obtained by Hardham (1985) u s i n g f l u o r e s c e n t - l a b e l l e d l e c t i n s . C y s t s , on - 4 -the other hand, bound both Con A and soybean a g g l u t i n i n (SBA), a l e c t i n t h a t binds to a l p h a - d - g a l a c t o s y l and n - a c e t y l - d - g a l a c t o s a m i n o s y l r e s i d u e s . He concluded t h a t changes i n a c c e s s i b l e s u g a r - c o n t a i n i n g molecules at the c e l l s u r f a c e accompany the t r a n s i t i o n from a p r o t o p l a s t (zoospore) to a w a l l - e n c a p s u l a t e d p r o t o p l a s t ( c y s t ) . P o s i t i v e chemotaxis of £ . palmivora zoospores has been demonstrated i n v i t r o by Cameron and C a r l i l e (1978, 1985). Although these s t u d i e s do not c o n c l u s i v e l y show the involvement of chemical a t t r a c t i o n and c e l l s u r f a c e r e c e p t o r s i n host r e c o g n i t i o n , they o f f e r a p o s s i b l e mechanism f o r host l o c a t i o n . More d i r e c t c y t o l o g i c a l evidence f o r the r o l e of c e l l s u r f a c e r e c e p t o r s such as g l y c o p r o t e i n s comes from the s t u d i e s of Sing (1974) who found t h a t the adhesive m a t e r i a l which glues the £. palmivora zoospore to a s o l i d substratum i s r i c h i n mannose, g l u c o s e , and g a l a c t o s e . He t h e o r i z e d t h a t the contents of p e r i p h e r a l v e s i c l e s may have the dual r o l e of m i c r o f i b r i l c r o s s - l i n k i n g d u r i n g c y s t w a l l s y n t h e s i s and b i n d i n g of the c e l l t o an e x t e r n a l s u r f a c e . The p o s s i b l e involvement of these p e r i p h e r a l f i b r o u s v e s i c l e s i n c y s t w a l l s y n t h e s i s and adhesion i s suggested by the f i n d i n g s of Bland and Amerson (1973) f o r Laoenidium c a l l i n e c t e s Couch. They observed that the f i b r o u s v e s i c l e s fused with the plasmalemma, d e l i v e r i n g t h e i r c o n t e n t s . Overton et a l . (1983) o b t a i n e d s i m i l a r r e s u l t s i n H a l i p h t h o r o s  m i l f o r d e n s i s V i s h n i a c . L i k e w i s e , secondary c y s t s of Lagenisma  c o s c i n o d i s c i Drebes become a t t a c h e d to the f r u s t u l e of C o s c i n o d i s c u s g r a n i i Gough by means of a s e c r e t e d , amorphous - 5 -substance (Schnepf et a l . 1978b). Encystrnent, a process by which the unwalled f l a g e l l a t e zoospore i s transformed i n t o a round w a l l e d c y s t , occurs e i t h e r through shedding or withdrawal of the f l a g e l l a i n t o the zoospore body (Koch 1968) . Although f l a g e l l a r detachment i s r a r e i n u n i f l a g e l l a t e zoospores i t has n e v e r t h e l e s s been observed (Koch 1968). More commonly, however, f l a g e l l a are r e t r a c t e d by one of four methods d e s c r i b e d by Koch (1968): l a s h - a r o u n d , body-twist, s t r a i g h t - i n , and v e s i c u l a r r e t r a c t i o n . F l a g e l l a r r e t r a c t i o n a l s o occurs i n b i f l a g e l l a t e s p e c i e s . In Phytophthora i n f e s t a n s (Mont) de Bary, r e s o r p t i o n of f l a g e l l a o c curs at higher temperatures b e f o r e sporangia germinate d i r e c t l y ( E i s n e r et a l . 1970). The newly r e l e a s e d zoospores of i . c o s c i n o d i s c i r e t r a c t t h e i r f l a g e l l a and undergo two d i f f e r e n t c y s t stages before i n f e c t i n g c e l l s of C o s c i n o d i s c u s sp. (Schnepf et a l . 1978b). Primary c y s t s are i n v e s t e d with a two-layered w a l l whereas the i n f e c t i v e secondary c y s t s have t h i c k e r , f i n e - f i b r i l l a r w a l l s of low e l e c t r o n d e n s i t y . F l a g e l l a r r e t r a c t i o n and rounding up of the spore i s accompanied by a c e n t r a l p o s i t i o n i n g of most o r g a n e l l e s (Schnepf et a l . 1978b). Thus, both mechanisms of f l a g e l l a r l o s s d u r i n g encystrnent, shedding and r e s o r p t i o n , have been observed i n u n i f l a g e l l a t e and b i f l a g e l l a t e s p e c i e s . B a r t n i c k i - G a r c i a and Hemmes (1974) concluded that both b i f l a g e l l a t e and u n i f l a g e l l a t e zoospores - 6 -"have a preformed complement of encystment enzymes, they s y n t h e s i z e a w a l l d_£ novo from i n t e r n a l r e s e r v e s , and they become adhesive p r i o r to or d u r i n g encystment." Although f l a g e l l a r r e s o r p t i o n may be a means of i n c r e a s i n g spore l o n g e v i t y as suggested by E i s n e r et a l . (1970), i t may a l s o be a mechanism f o r r e c y c l i n g t u b u l i n . I t i s not immediately apparent why zoospores of some s p e c i e s or even some zoospores i n the same p o p u l a t i o n (e.g. £. p a l m i v C E a [=£. p a r a s i t i c a ] ) shed t h e i r f l a g e l l a ( R e i c h l e 1969) . 2. Cyst g e r m i n a t i o n The second major developmental stage, c y s t g e r m i n a t i o n , i s commonly e f f e c t e d by the f o r m a t i o n of a l a r g e e x p u l s i o n v a c u o l e i n the d i s t a l r e g i o n of the c y s t . Expansion of t h i s v a c u o l e serves as a mechanism to i n t r o d u c e the p a r a s i t e p r o t o p l a s t i n t o the host through a p e r f o r a t i o n on the host c e l l w a l l (Held 1973). E c t r o g e l l a p e r f o r a n s Petersen uses such a mode of i n g r e s s i n t o c e l l s of Licmophora h y a l i n a Agardh (Kumar 1980a). P e n e t r a t i o n may occur by enzymatic d e g r a d a t i o n of host c e l l w a l l c o n s t i t u e n t s or by mechanical means. During e n t r y of Olpidium b r a s s i c a e (Wor.) Dang. (Temmink and Campbell 1969) i n t o cabbage root c e l l s , the l a y e r s of the host w a l l show no s i g n of d i s r u p t i o n . T h i s l e d Bracker and L i t t l e f i e l d (1973) to suggest that enzymatic d i s s o l u t i o n i s i n v o l v e d i n host p e n e t r a t i o n . A s i m i l a r i n f e c t i o n mechanism was r e p o r t e d i n L . c o s c i n o d i s c i by Schnepf et a l . (1978b). Working with m y c o p a r a s i t e s , Hoch and F u l l e r (1977) d e s c r i b e d the c o n t a c t i n t e r f a c e of Pythium acanthicum Drechs. and Phycomyces blgkgglgegnus B u r g e f f . Host p e n e t r a t i o n by p. acanthicum appears to i n v o l v e enzymatic d i g e s t i o n i n a d d i t i o n to mechanical p r e s s u r e , as suggested by the occurrence of i r r e g u l a r , e l e c t r o n - t r a n s p a r e n t areas i n the host w a l l where the p a r a s i t e had made cont a c t at p o t e n t i a l p e n e t r a t i o n s i t e s . I t i s p o s s i b l e that the l y t i c enzymes are d e l i v e r e d to the p a r a s i t e c e l l s u r f a c e by G o l g i - d e r i v e d v e s i c l e s . F o l l o w i n g s u c c e s s f u l p e n e t r a t i o n of the h o s t , h o l o c a r p i c f u n g a l s p e c i e s t y p i c a l l y develop as a w a l l - l e s s v e g e t a t i v e t h a l l u s (Kumar 1978, 1980b,c, Schnepf et a l . 1978a,b, Pueschel and van der Meer 1985). O b s e r v a t i o n s of the h o s t - p a r a s i t e i n t e r f a c e of Laaenisma gogcinodiscj, and Cggcinodiscu.s sp. r e v e a l e d t h a t the i n t e g r i t y of the host and the p a r a s i t e plasmalemma i s r e t a i n e d and t h a t e x o c y t o s i s of l y t i c enzymes and e n d o c y t o s i s of d i g e s t e d m a t e r i a l are not i n v o l v e d (Schnepf et a l . 1978a,b). T h i s l e d the authors to conclude that " i n t e r a c t i o n s between the fungus and the a l g a take p l a c e on the m o l e c u l a r l e v e l o n l y " (Schnepf et a l . 1978b). 3 . Zoosporogenesis The i n t r a c e l l u l a r development of z o o s p o r i c f u n g i - 8 -culminates i n w a l l formation and cleavage of the protoplasm i n t o zoospores. Olson et a l . (1981) reviewed the known p a t t e r n s of spo r o g e n e s i s i n z o o s p o r i c f u n g i and d e s c r i b e d two model systems based on B l a s t o c l a d i e l l a and A c h l y a . In S a p r o l e g n i a f a c e n t r a l vacuole expands g r a d u a l l y between the u n i n u c l e a t e masses of protoplasm thereby d e l i m i t i n g zoospore i n i t i a l s . The t o n o p l a s t e v e n t u a l l y fuses with the plasmalemma (Gay and Greenwood 1966, Gay et a l . 1971). The i n c r e a s e i n s i z e and osmotic p o t e n t i a l of the c e n t r a l v a c u o l e was a t t r i b u t e d by these authors to the "dense b o d i e s " i n the s p o r a n g i a l cytoplasm. The second type of c y t o p l a s m i c cleavage d e s c r i b e d by Olson and co-workers (1981) i s through the f u s i o n of v e s i c l e s . T h i s mode of cleavage has been r e p o r t e d i n oomycetous s p e c i e s such as Phytophthora palmivora (Bohl and Hamamoto 1967) , P_. j n f g s t a n s ( E i s n e r et a l . 1970), bagenid ium c a l l i n g c t e s (Bland and Amerson 1973, G o t e l l i 1974b), Pythiuro p r o l i f e r u m de Bary (Lunney and Bland 1976), S a p r o l e a n i a ferax. Coker et Couch ( B a r t n i c k i - G a r c i a and Hemmes 1974) and Achlya b i sexua l i s (Bicker 1971). Cleavage through the f u s i o n of v e s i c l e s has a l s o been r e p o r t e d i n c h y t r i d i o m y c e t o u s s p e c i e s such as Harpochytrium sp. (T r a v l a n d and Whis l e r 1971) and Allomyces  c a t e n o i d e s Sparrow (Olson et a l . 1981). The cleavage v e s i c l e s are b e l i e v e d to a r i s e from the v e s i c u l a t i o n of G o l g i c i s t e r n a e (Hohl and Bamamoto 1967, E i s n e r et a l . 1970). In t h e i r study of zoosporogenesis i n L. c a l l i n e c t e s Couch, Bland and Amerson - 9 -(1973) found that the sporogenic cytoplasm i s d i s c h a r g e d from the hyphal p o r t i o n of the sporangium i n t o an e x t e r n a l v e s i c l e i n d i s c r e t e c y t o p l a s m i c u n i t s connected i n sequence by c y t o p l a s m i c t h r e a d s . Cleavage of the sporogenic cytoplasm then proceeds through the f u s i o n of s m a l l cleavage v a c u o l e s (Bland and Amerson 1973). In comparing zoosporogenesis i n S a p r o l e g n i a sp. and i n Allomyces sp., Olson et a l . (1981) suggested that d e s p i t e the taxonomic d i f f e r e n c e between these organisms, the dense bodies found i n the cytoplasm may have the same f u n c t i o n : the g e n e r a t i o n of cleavage v e s i c l e s . Bowever, i t i s u n l i k e l y t h a t the electron-opaque i n c l u s i o n s i n s i d e the v e s i c l e s c o n t r i b u t e d i r e c t l y to membrane growth as they had suggested. I t may be th a t t h e i r c o n t r i b u t i o n to d_£ novo membrane s y n t h e s i s i s an i n d i r e c t one, i n v o l v i n g t h e i r f u s i o n with v a c u o l e s t h a t have a lysosomal f u n c t i o n , thus d e l i v e r i n g t h e i r c o n t e n t s . Subsequent breakdown of the i n c l u s i o n would r e l e a s e monomeric s u b u n i t s which can d i f f u s e i n t o the cytoplasm and be u t i l i z e d at the l e v e l of the ER. A s i m i l a r r o l e has been a s c r i b e d to the e l e c t r o n - d e n s e bodies which fuse with the c e n t r a l v acuole i n SaprolegnAa sp. (Heath 1976). Although two b a s i c zoosporogenesis p a t t e r n s have been d e s c r i b e d by Olson and co-workers (1981), i t appears that v a r i a n t s of these p a t t e r n s e x i s t and that the sporogenic protoplasm may be c l e a v e d by a l t e r n a t i v e mechanisms. H e i n t z (1971) r e p o r t e d t h a t both c y t o p l a s m i c v e s i c l e s and c i s t e r n a e produced by the dictyosome c o a l e s c e and fuse with the - 10 -plasmalemma d u r i n g zoosporogenesis i n Pythium n)idd3 .9toni A Sparrow. Coalescence of cleavage v e s i c l e s i s not i n v o l v e d i n primary spore f o r m a t i o n i n Aphanomyces e u t e i c h e s Drechs.; the e x i s t i n g plasmalemma and t o n o p l a s t are u t i l i z e d (Hoch and M i t c h e l l 1972) . L i k e w i s e , cleavage seems to proceed through i n f o l d i n g s of the plasmalemma i n B las tu l A d i u m poedophthorum Perez (Manier 1976). That no new membrane i s s y n t h e s i z e d d u r i n g the process (Hoch and M i t c h e l l 1972) i s d i f f i c u l t to e s t a b l i s h i n the absence of morphometric d a t a . I t a l s o c o n t r a d i c t s the c u r r e n t concept of membrane flow and c y c l i n g which i n v o l v e s the turnover of membrane components. A unique mode of cleavage which i s q u i t e d i s t i n c t from those d e s c r i b e d by Olson and co-workers (1981) has been observed i n Lagenisma c o s c i n o d i s c i . i n t h i s organism, the p r o t o p l a s t d i v i d e s by means of G o l g i - d e r i v e d c i s t e r n a e which fuse with each other and the plasmalemma (Schnepf et a l . 1978c) . S e p a r a t i o n v e s i c l e s then form between zoospore i n i t i a l s but e v e n t u a l l y d i s i n t e g r a t e . Cleavage i n another l a g e n i d i o i d s p e c i e s , Petersen Aa palPiarjagf a l s o proceeds through f u s i o n of c i s t e r n a e (Pueschel and van der Meer 1985). The mode and t i m i n g of f l a g e l l a r axoneme f o r m a t i o n v a r i e s i n d i f f e r e n t s p e c i e s . F l a g e l l a r development i s c l o s e l y c o o r d i n a t e d with p r o t o p l a s m i c cleavage i n many Oomycetes ( e . g . Phytophthora: Hohl and Hamamoto 1967, W i l l i a m s and Webster 1970 , L . c a l l i n e c t e s ; Bland and Amerson 1973, Pythj,u.m: - 11 -Lunney and Bland 1976, £. palmariaet Pueschel and van der Meer 1985). In P. p a l m a r l a e . zoosporogenesis i s c h a r a c t e r i z e d by the simultaneous f o r m a t i o n of mastigonemes i n d i l a t e d endoplasmic r e t i c u l u m (EE) p r o f i l e s and f u s i o n of cleavage c i s t e r n a e (Pueschel and van der Meer 1985). M a t u r a t i o n of mastigonemes i s thought to occur i n p e r i p h e r a l g o l g i c i s t e r n a e (Bouck 1969, Schnepf et a l . 1978c). Mastigoneme-containing EB c i s t e r n a e are t y p i c a l l y a s s o c i a t e d with m i t o c h o n d r i a i n oomycetous taxa such as Pythium p r o l i f e r u m (Lunney and Bland 1976) and Laaenidium ca 3,1 Anect<35 (Bland and Amerson 1973, G o t e l l i 1974). filastulAdAuro sp. (Manier 1976) and LagenAsma c o s c i n o d i s c i i (Schnepf and Deichgraber 1978c) appear to be e x c e p t i o n s i n that the development of axonemes i s delayed u n t i l completion of c l e a v a g e . Furthermore, the e l o n g a t i o n of b a s a l bodies i n t o f l a g e l l a without the p a r t i c i p a t i o n of a f l a g e l l a r v e s i c l e appears to be unique to kagenisma c o s c i n o d j s c i A . F o l l o w i n g complete cleavage of the protoplasm, the zoospores are r e l e a s e d from the sporangium. T h i s p r o c e s s i s accomplished i n one of s e v e r a l ways i n the Oomycetes. In S a p r o l e g n i a spp. an a p i c a l p a p i l l u m develops as a l o c a l i z e d e x t e n s i o n of the sporangium w a l l and then i s broken down e n z y m a t i c a l l y (Heath et a l . 1970). In Phytophthora s p e c i e s (Hemmes and Hohl 1969, W i l l i a m s and Webster 1970), an a p i c a l plug i s formed and i s subsequently pushed out. - 12 -C. Host responses to i n f e c t i o n In compatible h o s t - p a r a s i t e i n t e r a c t i o n s , the i n f e c t i o n p r o c e s s i s i n v a r i a b l y accompanied by profound p h y s i o l o g i c a l and c y t o l o g i c a l changes i n the h o s t . These changes may be l o c a l i z e d or s y s t e m i c . A p p o s i t i o n s t h a t i n c r e a s e the host w a l l mass through d e p o s i t i o n of m a t e r i a l on i t s inner s u r f a c e may be formed around the areas of s u c c e s s f u l p e n e t r a t i o n or may prevent p e n e t r a t i o n a l t o g e t h e r (Bracker and L i t t l e f i e l d 1973). In some s p e c i e s , they appear to be necessary fo r s u c c e s s f u l p e n e t r a t i o n . U l t r a s t r u c t u r a l examination of Allomyces a r b u s c u l a B u t l e r , a c h y t r i d i n f e c t e d with R o z e l l a a l l o m y c i s F o r r e s t , r e v e a l e d t h a t e n d o b i o t i c hyphae always t r a v e r s e d a p a p i l l a ( = a n i p p l e - l i k e a p p o s i t i o n ) whereas such a p p o s i t i o n s were absent from s i t e s where nonpenetrated c y s t s had a t t a c h e d (Held 1972). In a study of the h o s t - p a r a s i t e i n t e r f a c e between Spathulospora sp. and B a l l i a sp., Walker et a l . (1979) observed the f o r m a t i o n of a p p o s i t i o n a l m a t e r i a l i n the inner r e g i o n of the host c e l l w a l l d u r i n g the e a r l y stages of the p a r a s i t e ' s development. The w a l l a p p o s i t i o n s were d e p o s i t e d beneath the p o i n t of a p p r e s s o r i a l attachment and assumed the form of a p a p i l l a d u r i n g p e n e t r a t i o n ; they were c o l l a r - l i k e where hyphae emerge. The nature of t h i s h o s t - p a r a s i t e i n t e r f a c e i s r e m i n i s c e n t of t e r r e s t r i a l pathosystems (Bracker and L i t t l e f i e l d 1973) except that the hyphae continue to develop w i t h i n the host and e v e n t u a l l y emerge from the c e l l to form the e x t e r n a l t h a l l u s (Walker et a l . 1979). Formation of - 13 -a p p o s i t i o n s has a l s o been r e p o r t e d i n Aphelidium s p e c i e s (Schnepf 1972). and Olpidium b r a s s i c a e (Temmink and Campbell 1969). In t h e i r study of raycoparasitic i n t e r a c t i o n s , Hoch and F u l l e r (1977) concluded that "a p a p i l l a ( = c a l l o s i t y ) w i l l l i k e l y be formed i n n e c r o t r o p h i c r e l a t i o n s h i p s as w e l l as i n some b i o t r o p h i c r e l a t i o n s h i p s . " They c o n s i d e r e d p a p i l l a f o r m a t i o n as a g e n e r a l response of the host c e l l to wounding or i r r i t a t i o n r a t h e r than a mechanism to r e s i s t i n f e c t i o n . S e v e r a l other d i s e a s e symptoms have been d e s c r i b e d i n marine a l g a e . C e l l s of Licmophora h y a l i n a which are i n f e c t e d with E c t r o g e l l a peyforans are h y p e r t r o p h i e d and c h l o r o t i c and have a g r a n u l a r and dark cytoplasm i n l a t e r stages (Petersen 1905, Sparrow 1934, 1936, 1960, Aleem 1950a,b 1953). Hypertrophy and h y p e r p l a s i a are l i k e w i s e induced by EugychasruidiHrt) tmnefacjgps (Magnus) Sparrow i n the a p i c a l or nodal c e l l s of Ceramium sp. (Sparrow 1936, Dixon 1960). Eurychasma d i c k s o n i i (Wright) Magnus was r e p o r t e d to cause severe damage to fronds of G i f f o r d i a g r a n u l o s a (Sm.) Bamel f=Ectocarpus g r a n u l o s u s ! i n England (Aleem 1950c) and S t r i a r i a  a t t e n u a t a G r e v i l l e i n Sweden (Aleem 1953) , with symptoms ranging from d i s t o r t i o n and d i s c o l o r a t i o n to e x t e n s i v e hypertrophy (Wright 1869, P a t t r a y 1885, Sparrow 1934,1960). I n f e c t i o n s may be g e n e r a l i z e d or l o c a l i z e d . Thus, almost a l l c e l l s of Ectocarpus s i l i c u l o s u s (Dillwyn) Lyngbye are i n f e c t e d with Q l p i d i o p s i s andyeej (Lagerheim) K a r l i n g (Sparrow 1936). T h a l l i of Ectocarpus sp. which are i n f e c t e d with A n i s o l p i d i u m - 14 -e c t o c a r p i i K a r l i n g show s i m i l a r symptoms: both v e g e t a t i v e c e l l s and p l u r i l o c u l a r sporangia are invaded ( K a r l i n g 1943, Johnson 1957, Johnson and Sparrow 1961). In c o n t r a s t , i n f e c t i o n s of S e i r o s p o r a sp. a s s o c i a t e d with the l a g e n i d i o i d fungus P e t e r s e n i a l o b a t a (Petersen) Sparrow are c o n f i n e d to host sporangia (Feldmann and Feldmann, 1940). Thompson (1981) s t u d i e d the anatomy and h i s t o c h e m i s t r y of Fucus d i s t i c h u s L. i n f e c t e d with Pythium species and found that n e c r o t i c l e s i o n s develop i n the most recent dichotomy. Fungal hyphae are c o n f i n e d to the c o r t i c a l and medullary regions and appear to penetrate host c e l l w a l ls through enzymatic d i s s o l u t i o n of the a l g i n i c a c i d and c e l l u l o s i c components. The fungus p a r a s i t i z e s s e v e r a l host c e l l s by d i s s o l v i n g " p i t connections" (Thompson 1981). Symptoms a s s o c i a t e d with i n f e c t i o n s of marine algae by s p e c i e s of Ascoraycotina and Fungi I m p e r f e c t i have been d e s c r i b e d and reviewed by Andrews (1976) , Kohlmeyer (1974) , Kohlmeyer and Kohlmeyer (1979) , and Goff and Glasgow (1980) . S e v e r a l species cause d i s c o l o r a t i o n i n red algae - for example, Lul w o r t h i a kniepii Kohlmeyer, Didymella g l o j o p e l t j j d i s , (Miyabe et Tokida) Kohlmeyer et Kohlmeyer, and Chadefaudia marina G. Feldmann (Miyabe and Tokida 1948, Bauch 1936, Kohlmeyer 1974). The most common fungus on brown algae i s probably Phycomelaina  l a m i n a r i a e (Postrup) Kohlmeyer which forms black patches of stromata on the s t i p e s of Laminaria species or "black dots" disease (Sutherland 1915; Kohlmeyer 1968). The ascomycete - 15 -p r o l i f e r a t e s i n t r a c e l l u l a r l y w i t h i n the host cortex and e v e n t u a l l y causes d i s o r g a n i z a t i o n and death of l a r g e areas (Kohlmeyer 1968). Schatz (1980) noted n e c r o s i s i n Laminaria  s a c c h a r i n a (L.) Lamour i n f e c t e d with t h i s fungus. G a l l formation has been observed i n s p e c i e s of the phaeophycean genera C y s t o s e i r a . flalidrys, Sarga.ssu.rn,> and CystQPhQKa i n f e c t e d with the ascomycetes H a l o g u i a n a r d i a and Massarina (Kohlmeyer and Kohlmeyer 1979). The s i g n i f i c a n c e and r o l e of host c e l l death, which has been s t u d i e d mainly i n t e r r e s t r i a l systems, v a r i e s depending on the p a r t i c u l a r fungus-plant i n t e r a c t i o n (Ingram 1982). Stakman (1915) f i r s t used the term " h y p e r s e n s i t i v e response" f o r the r a p i d death of c e l l s around p e n e t r a t i o n s i t e s i n v a r i e t i e s of wheat and other c e r e a l s r e s i s t a n t to P u c c i n i a graminis Pers. He d e s c r i b e d the sequence of events during the h y p e r s e n s i t i v e response to be: i n v a s i o n by the fungus, death of p l a n t c e l l s , f o l l o w e d by death of the fungus. A c l a s s i c example i s the r e s i s t a n c e of c e r t a i n cowpea v a r i e t i e s to the rust fungus TJromyces p h a s e d i var. vianae (Rab.) Wint. that i s a s s o c i a t e d with the i n d u c t i o n of c e l l death i n the mesophyll f o l l o w i n g formation of rudimentary h a u s t o r i a (Heath 1979). Probably the only known occurrence of a h y p e r s e n s i t i v e response i n marine algae i s i n £. d i s t i c h u s i n f e c t e d with Pythium s p e c i e s . Thompson (1981) found that c e l l s i n advance of the Pythium hyphae aut o l y z e d and that p h e n o l i c compounds accumulated i n the host matrix. He suggested that accumulation of these - 16 -substances t r i g g e r s the formation of an a b s c i s s i o n zone by which the i n f e c t e d p o r t i o n s are dropped out of the a l g a l t h a l l u s . Although the same response could not be d u p l i c a t e d under l a b o r a t o r y c o n d i t i o n s , t h i s i s probably the only report of a h y p e r s e n s i t i v e response i n a^marine a l g a . D. Changes i n the h o s t - p a r a s i t e i n t e r f a c e The h o s t - p a r a s i t e i n t e r f a c e has s p a t i a l and temporal c h a r a c t e r i s t i c s . D i f f e r e n t c e l l u l a r components may be i n v o l v e d i n the e p i b i o t i c and i n the e n d o b i o t i c stages of a p a r a s i t e ' s development. Bracker and L i t t l e f i e l d (1973) reviewed the d i f f e r e n t host-symbiont i n t e r f a c e types that have been observed and how they change i n space and time as development proceeds. T h e i r review concentrated on i n t r a c e l l u l a r r e l a t i o n s h i p s and emphasized the importance of examining the i n t e r f a c e components for a more complete understanding of how hosts and symbionts i n t e r a c t . In summary, the i n t e r a c t i o n s of e n d o b i o t i c , z o o s p o r i c , a l g i c o i o u s f u n g i with t h e i r hosts can be d i v i d e d i n t o three major stages: 1) host r e c o g n i t i o n and adhesion, 2) p e n e t r a t i o n of the host cytoplasm, and 3) i n t r a c e l l u l a r development and maturation (Held 1973) . Host r e c o g n i t i o n may i n v o l v e chemotaxis and/or c e l l s u r f a c e r e c e p t o r s . There are two b a s i c modes of encystment: f l a g e l l a r shedding and r e t r a c t i o n . Both have been observed i n chytridiomycetous and oomycetous s p e c i e s . - 17 P e n e t r a t i o n , a process whereby the p a r a s i t e i s i n j e c t e d i n t o the host cytoplasm, i s accomplished by mechanical pressure or enzymatic d i g e s t i o n of host c e l l components. At t h i s stage, the host may already show one or s e v e r a l disease symptoms which can be recognized on the gross morphological and m i c r o s c o p i c l e v e l s . H o l o c a r p i c s p e c i e s t y p i c a l l y have a w a l l - l e s s v e g e t a t i v e t h a l l u s which e v e n t u a l l y becomes converted i n t o a sporangium. Zoosporogenesis commonly proceeds through the expansion of a c e n t r a l vacuole or the coalescence of v e s i c l e s which serves to d i v i d e the protoplasm i n t o u n i n u c l e a t e zoospore i n i t i a l s . In two l a g e n i d i o i d s p e c i e s , L . c o s c i n o d i s c i (Schnepf et a l . 1978c) and P.. palmariae (Pueschel and van der Meer 1985), i t occurs through the f u s i o n of G o l g i - d e r i v e d c i s t e r n a e . Formation of f l a g e l l a i s c l o s e l y a s s o c i a t e d with cleavage, although i t i s delayed u n t i l completion of cleavage i n B l a s t u U d i U P l sp. (Manier 1976) and Laaenisma c o s c i n o d i s c i i (Schnepf et a l . 1978c). Zoospores are then r e l e a s e d for the next round of i n f e c t i o n . The development of a p a r a s i t i c fungus i s commonly accompanied by changes i n the nature of the h o s t - p a r a s i t e i n t e r f a c e . E. Previous s t u d i e s on d i s e a s e s of c u l t i v a t e d seaweeds Probably the only r e p o r t s of fungal i n f e c t i o n s of commercially grown species reaching epidemic p r o p o r t i o n s are i n m a r i c u l t u r e d Porphyra spp. A s e r i o u s fungal disease c a l l e d "Akagusare" has been reported i n Japan by A r a s a k i and - 18 -co-workers (Arasaki 1947, 1962, A r a s a k i et a l . 1968). The disease was a t t r i b u t e d to Pythium porphyrae Takahashi, I c h i n o t a n i et S a s a k i . In a l a t e r i n v e s t i g a t i o n , F u j i t a (1978) obtained z o o s p o r u l a t i o n of t h i s fungus by al l o w i n g i t s oospores 1 to germinate i n v i t r o . He confirmed the presumed p a t h o g e n i c i t y of the zoospores by i n o c u l a t i n g healthy fronds which l a t e r showed c h a r a c t e r i s t i c red r o t symptoms. Two other d e s t r u c t i v e pathogens were reported i n c u l t u r e d Porphyra by M i g i t a (1969, 1973): an e n d o b i o t i c , h o l o c a r p i c p a r a s i t e which he assigned to the genus O l p i d i o p s i s and a ChyUjcUuffl s p e c i e s which causes l e a c h i n g of pigments of the *Conchocelis' stage. For obvious economic reasons, the Japanese group have concentrated on f i n d i n g c o n t r o l measures that might prevent f u r t h e r spread and development of the p a r a s i t e , i n c l u d i n g exposure to the atmosphere for as long as three days (Goff and Glasgow 1980) . L i m i t e d i n f o r m a t i o n i s p r e s e n t l y a v a i l a b l e on the u l t r a s t r u c t u r e of e n d o b i o t i c development i n zo o s p o r i c a l g i c o i o u s f u n g i . Kazama and F u l l e r (1970) s t u d i e d the i n f e c t i o n of Porphyra p e r f o r a t a J . Ag. by Pythium marinum Sparrow. They found that the fungus penetrates c e l l s of the host as a t h i n - w a l l e d hypha without forming h a u s t o r i a . Only those c e l l s that are d i r e c t l y penetrated showed d i s r u p t i o n of or g a n e l l e s and d i s s o l u t i o n of s t a r c h g r a i n s . In P e t e r s e n i a  palmariae van der Meer et Pueschel, the t h a l l u s develops i n t r a c e l l u l a r ^ and causes f u s i o n of host c e l l s (Pueschel and van der Meer 1985). The t h a l l u s resembles that of Laoenisma - 19 -CQScinc-disgi (Schnepf et a l . 1978a,b) and E c t E o g g l l a p e r f o r a n s (Kumar 1978, 1980a,b) i n being w a l l - l e s s . Single-membraned i n t r a c e l l u l a r Plasmodia have a l s o been d e s c r i b e d f o r the c h y t r i d i o m y c e t e s R o z e l l a a l l o m y c i s (Held 1972) and CTpidium b r a s s i c a e (Temmink and Campbell 1969). An understanding of the b i o l o g y of p a r a s i t i c a l g i c o l o u s f u n g i , e s p e c i a l l y those which p a r a s i t i z e e c o n o m i c a l l y important s p e c i e s , and the f a c t o r s that i n f l u e n c e t h e i r development w i l l c l e a r l y f a c i l i t a t e the f o r m u l a t i o n of a p p r o p r i a t e c o n t r o l measures. F. Background of the problem 1. Chondrus c r i s p u s Stackhouse C. c r i s p u s i s a g i g a r t i n a l e a n a l g a which has an i somorphic, d i p l o b i o n t i c l i f e h i s t o r y ( T a y l o r and Chen 1973). I t i s one of the e c o n o m i c a l l y important seaweeds on both s i d e s of the North A t l a n t i c and i s commonly known as I r i s h moss. The i n d u s t r y a s s o c i a t e d with t h i s seaweed o r i g i n a t e d i n I r e l a n d and was i n t r o d u c e d i n t o America i n 1835 (Chapman and Chapman 1980). Q. c r i s p u s i s extremely v a r i a b l e i n morphology and a l a r g e number of forms have been d e s c r i b e d (Turner 1802, Lyngbye 1819, Posenvinge 1931, Thomas 1938). I t i s a major source of the a l g a l h y d r o c o l l o i d carrageenan, a term a p p l i e d to a group of g a l a c t a n p o l y s a c c h a r i d e s . Carrageenans are e x t r a c t e d from red seaweeds and have an e s t e r s u l p h a t e content of at l e a s t 18%. They are a l t e r n a t e l y alpha-1,3; beta-1,4 g l y c o s i d i c a l l y l i n k e d 20 -(Moirano 1977). Carrageenans are g e n e r a l l y used i n the d a i r y , c o n f e c t i o n e r y and phar m a c e u t i c a l i n d u s t r i e s because of t h e i r p h y s i c a l f u n c t i o n s i n g e l a t i o n , v i s c o u s b e h a v i o r , and s t a b i l i z a t i o n of emulsions (Chapman and Chapman 1980). D i f f e r e n t types of carrageenans are r e c o g n i z e d , based on t h e i r p h y s i c o - c h e m i c a l p r o p e r t i e s , e.g. KC1 p r e c i p i t a b i l i t y , IP s p e c t r a , and g e l s t r e n g t h s (Mackie and Pres t o n 1974) . The p r i n c i p a l Chondrus-producing r e g i o n s are I r e l a n d , France, Canada, and the United S t a t e s . U n t i l 1971 Canadian p r o d u c t i o n formed 80-85% of the world's t o t a l h a r v e s t . Since then t h i s v a l u e has dropped to 30-35%, having been superseded by p r o d u c t i o n from the P h i l i p p i n e s (W. Yaphe, p e r s o n a l communication). P r o d u c t i o n i n Nova S c o t i a comes mainly from 700 h a r v e s t e r s who crop 65 d i s t i n c t beds ( P r i n g l e 1979) as w e l l as the m a r i c u l t u r e f a c i l i t i e s of Marine C o l l o i d s , Inc. (now Acadian S e a p l a n t s , L t d . ) . 2. Fungal i n f e c t i o n s of C. c r i s p u s Fungal i n f e c t i o n s i n n a t u r a l p o p u l a t i o n s of £. c r i s p u s were f i r s t noted by Postrup i n 1889 who d e s c r i b e d the pyrenomycetous fungus as Leptpsphaef ia marina E l l i s et E v e r h a r t . Wilson and Knoyle (1961) suggested that i t s c o r r e c t name i s Didymosphaeria danica a f t e r examining m a t e r i a l from England, S c o t l a n d , Wales, and the U.S.A. No dis e a s e outbreak has h e r e t o f o r e been r e p o r t e d i n m a r i c u l t u r e d Chondrus spp. - 21 -An e p i p h y t o t i c , presumably of f u n g a l o r i g i n , was r e p o r t e d i n the C. c r i s p u s c u l t u r e f a c i l i t i e s of Marine C o l l o i d s , L t d . i n lower East Pubnico, Nova S c o t i a i n the autumn of 1980. The i n f e c t e d a l g a l s t r a i n was a high k-carrageenan-producing male gametophyte (Guiry 1981) which i s propagated v e g e t a t i v e l y through c u t t i n g s and i s d e s i g n a t e d as "T4 n. Disease outbreaks have o c c u r r e d r e g u l a r l y d u r i n g the autumn season from 1981 through to 1984. The i n f e c t i o n i s c h a r a c t e r i z e d by n e c r o t i c l e s i o n s on the t i p s of the a l g a l t h a l l u s . P r e l i m i n a r y examination of i n f e c t e d t h a l l i r e v e a l e d that the fungus a s s o c i a t e d with the d i s e a s e was simple, h o l o c a r p i c , e n d o b i o t i c , and b i f l a g e l l a t e and might be a s s i g n e d to P e t e r s e n i a  p o l l a g a s t e r (Petersen) Sparrow (G. C. Hughes, u n p u b l . ) . 3 . Pe te rsen ia p o l l a g a s t e r (Petersen) Sparrow £. p o l l a g a s t e r i s one of seven s p e c i e s i n the genus P e t e r s e n i a which i n c l u d e s t h r e e a l g i c o l o u s s p e c i e s . I t was f i r s t d e s c r i b e d as P l e o t r a c h e l u s on fronds of Ceramium rubrum (Huds.) C. Agardh by Petersen i n 1905 from s e v e r a l Danish l o c a l i t i e s . Subsequently, the fungus has been r e p o r t e d only t w i c e : Sparrow r e - c o l l e c t e d the s p e c i e s i n Denmark i n 1934 on the same red a l g a l host and t r a n s f e r r e d i t to J E . p o l l a g a s t e r based on the b i f l a g e l l a t e nature of the zoospores; Johnson and Howard (1968) found i t on £. rubrum from I c e l a n d . I t i s d e l i n e a t e d from the c l o s e l y r e l a t e d s p e c i e s £. l o b a t a (Petersen) Sparrow mainly on the b a s i s of t h a l l u s l o b u l a t i o n - 22 -and has not been r e p o r t e d from North America p r i o r to i t s appearance i n the Chondrus c u l t u r e s i n Nova S c o t i a . The host range and d i s t r i b u t i o n of £. p o l l a g a s t e r and £. l o b a t a are given i n Table I I . Very l i t t l e i s known of\ the b i o l o g y , l i f e h i s t o r y , or ontogeny of £. p o l l a g a s t e r and even l e s s i s known of i t s c y t o l o g i c a l e f f e c t s on the host algae ( e i t h e r Ceramium or Chondrus). Since p r e v i o u s r e p o r t s of the fungus were based on l i m i t e d q u a n t i t i e s of i n f e c t e d a l g a l t h a l l i , no attempts have been made p r e v i o u s l y to e s t a b l i s h i t s presumed p a t h o g e n i c i t y . G. O b j e c t i v e s of the presen t study The o b j e c t i v e s of t h i s t h e s i s a r e : 1. To confirm the i n i t i a l taxonomic d i a g n o s i s of the ChondKUS c r i s p u s pathogen as be l o n g i n g to P e t e r s e n i a p o l l a g a s t e r . 2. To i n v e s t i g a t e p a t h o g e n i c i t y and some of the f a c t o r s t h a t favor the spread of the p a r a s i t e under l a b o r a t o r y c o n d i t i o n s , 3. To d e s c r i b e seme of the developmental stages of P.. p o l l a g a s t e r using l i g h t and e l e c t r o n microscopy, and - 23 -4. To examine the h o s t - p a r a s i t e i n t e r f a c e and determine the extent of host response to the i n v a s i o n using v a r i o u s c y t o l o g i c a l t e c h n i q u e s . - 24 -MATERIALS AND METHODS A. C o l l e c t i o n of M a t e r i a l s D i seased t h a l l i of Chondrus c r i s p u s ' T 4 * were c o l l e c t e d from the m a r i c u l t u r e f a c i l i t i e s of Acadian S e a p l a n t s , L t d . l o c a t e d at Pubnico Harbour, Nova S c o t i a ( L a t . 4 3 ^ 4 2 f N , Long. 65°48'W, see F i g . 1) i n the f a l l of 1981 to 1 9 8 4 . U n i n f e c t e d fronds were l i k e w i s e c o l l e c t e d from a nearby f a c i l i t y for comparative purposes. Specimens were p l a c e d i n p l a s t i c bags, l o o s e l y packed i n i c e , and shipped to Vancouver, B r i t i s h Columbia by a i r . They were promptly t r a n s f e r r e d to a sea water medium upon r e c e i p t and sub-samples were processed f o r l i g h t and e l e c t r o n microscopy. B. L a b o r a t o r y c u l t u r e s of the Host Upon a r r i v a l i n Vancouver, a l g a l c u l t u r e s were maintained at 15C i n an a r t i f i c i a l sea water medium supplemented with n i t r a t e , phosphate, and o r g a n i c m i c r o n u t r i e n t s as d e s c r i b e d by Chen and T a y l o r (1978) . The c u l t u r e medium had a s a l i n i t y of 30%o and i t s composition i s given i n Appendix A. Approximately f i f t y grams (wet weight) of the specimen were weighed using a M e t t l e r t o p - l o a d i n g balance and p l a c e d i n a r e c t a n g u l a r g l a s s j a r h o l d i n g 4 . 0 l i t e r s of sea water. The i r r a d i a n c e l e v e l was kept at 38 .0juE cm -2 s e c ~ l f o r 8 h and a l t e r n a t e d with a dark - 25 -p e r i o d of 16 h. C u l t u r e s were a e r a t e d and the medium was changed once every week. C. C r o s s - i n f e c t i o n Experiments In order to have a continuous supply of i n f e c t e d m a t e r i a l , experiments were conducted to determine i f i n f e c t i o n c o u l d be induced i n the l a b o r a t o r y . Some of the f a c t o r s t h at f a v o r the spread and development of the p a r a s i t e were a l s o i n v e s t i g a t e d . I n f e c t i o n of h e a l t h y t h a l l i was accomplished using one of two methods: 1 ) , u n i n f e c t e d t i p s measuring 1.0-2.0 cm were e x c i s e d and seeded i n t o g l a s s d i s h e s (75x100 mm) with d i s e a s e d fronds i n 100 ml of s t e r i l e c u l t u r e medium, or 2 ) , 1.0 ml of z o o s p o r e - c o n t a i n i n g medium (approximately 5 zoospores/250X microscope f i e l d ) was i n t r o d u c e d i n t o c u l t u r e s of u n i n f e c t e d t h a l l i i n 100 ml of sea water medium. Although c u l t u r e s were not a x e n i c , care was taken to minimize contamination by f i r s t washing both the u n i n f e c t e d t i p s which were used to "seed" d i s e a s e d specimens and the i n f e c t e d fronds with a s t r o n g stream of s t e r i l e sea water i n a laminar flow hood. C u l t u r e s were incubated i n a " P e r c i v a l " c o n t r o l l e d environment chamber under the same ph o t o p e r i o d used f o r l a b o r a t o r y c u l t u r e s of the h o s t . C r o s s - i n f e c t i o n experiments were performed at 5, 10, 15, 20, and 25C using the second method d e s c r i b e d above to determine the e f f e c t s of temperature on the spread and development of the p a r a s i t e . A l l other c u l t u r e parameters - 26 -( i . e . composition of the medium, s a l i n i t y , and i r r a d i a n c e ) were i d e n t i c a l . The optimum temperature was determined by counting the number of l e s i o n s i n t e r c e p t e d by a randomly p l a c e d 2.0-mm eyepiece micrometer l i n e under a m a g n i f i c a t i o n of 25X a f t e r i n c u b a t i o n f o r 3 days using e i g h t measurements from d i f f e r e n t a l g a l t i p s . I n o c u l a t e d t i p s which d i d not show l e s i o n s at the end of four days were reseeded and r e t u r n e d to 20C to determine i f propagules remained v i a b l e . Gross m o r p h o l o g i c a l changes i n i n o c u l a t e d t h a l l i were monitored every 6-12 h using a Wild stereo-zoom microscope. P i c t u r e s were taken with a Wild MPS 11 camera f i t t e d with a MPS 15 Semiphotomat. For s t a t i s t i c a l a n a l y s i s , homogeneity of v a r i a n c e s was f i r s t t e s t e d on an Apple l i e computer using a program w r i t t e n f o r t h i s purpose by P. E. de Wreede. A s c a t t e r diagram of the i n d i v i d u a l measurements was then prepared and the best f i t t i n g curve c o n s t r u c t e d u s i n g the POLYFIT program (Spain 1982). Relevant s t a t i s t i c a l parameters, i n c l u d i n g the c o e f f i c i e n t of d e t e r m i n a t i o n ( P2) and F v a l u e , were computed. D. C u l t u r i n g of the P a r a s i t e Attempts were made to c u l t u r e the p a r a s i t e using three b a s i c i s o l a t i o n t e c h n i q u e s : 1) d i r e c t p l a t i n g , where i n f e c t e d t i p s were e x c i s e d , washed three times with 15-25 ml volumes of s t e r i l e sea water, and p l a t e d on v a r i o u s c u l t u r e media i n c l u d i n g those d e s c r i b e d by V i s h n i a c (1956), F u l l e r et a l . - 27 -(1964), and G o t e l l i (1974); 2) membrane f i l t r a t i o n : washed, i n f e c t e d t i s s u e was homogenized i n a s t e r i l e Waring seroi-microblender, washed over a membrane f i l t e r , and p l a t e d on a s o l i d medium; 3) p l a t i n g on s o f t agar as d e s c r i b e d by Whiffen (1941). Attempts were a l s o made to grow the fungus on h e a t - k i l l e d u n i n t e c t e d t h a l l i . E. L i g h t Microscopy Zoospores of P e t e r s e n i a p o l l a g a s t e r were photographed with a L e i t z Ortholux microscope equipped with phase c o n t r a s t o p t i c s and a Nikon M i c r o f l e x AFM using I l f o r d HP5 f i l m r a t e d at 800 ASA. For b r i g h t f i e l d microscopy, specimens were f i x e d i n a mixture of 5% g l u t a r a l d e h y d e and 4% formaldehyde (Karnovsky 1965) i n 0.2M phosphate or sodium c a c o d y l a t e b u f f e r at pH 7.4 fo r 3 h at room temperature s i n c e t h i s method y i e l d e d the best r e s u l t f o r e l e c t r o n microscopy. F i x a t i o n was f o l l o w e d by th r e e r i n s e s i n b u f f e r and specimens were dehydrated i n a graded methanol s e r i e s i n increments of 10%. A f t e r two changes with 100% methanol, the s o l u t i o n was r e p l a c e d with c a t a l y z e d JB-4 s o l u t i o n A (a water s o l u b l e r e s i n , P o l y s c i e n c e s , I n c . ) . I n f i l t r a t i o n was c a r r i e d out on a TAAB 2 rpm r o t a t o r at room temperature f o r 24 h a f t e r which the c a t a l y z e d s o l u t i o n A was decanted and r e p l a c e d with f r e s h s o l u t i o n . Specimens were l e f t i n t h i s s o l u t i o n f o r another 24 h f o r complete i n f i l t r a t i o n . They were then p l a c e d i n p l a s t i c molds and embedded i n a mixture of 1 pa r t JB-4 s o l u t i o n B to 24 p a r t s f r e s h l y c a t a l y z e d - 28 -s o l u t i o n A. Blocks were allowed to polymerize o v e r n i g h t at room temperature. Semi-thin s e c t i o n s (1 .0-1.5_um) were cut with g l a s s knives on a S o r v a l l JB-4 microtome, f l o a t e d on d i s t i l l e d water, and c o l l e c t e d on g l a s s s l i d e s . They were then d r i e d onto the s l i d e s on a hot p l a t e to e f f e c t attachment. M e t h a c r y l a t e s e c t i o n s were s t a i n e d with 1% T o l u i d i n e Blue 0 i n 1% aqueous sodium borate (pH 4.4) f o r r o u t i n e l i g h t m icroscopy. For the d e t e c t i o n of c e l l u l o s e , they were t r e a t e d with the f o l l o w i n g s t a i n s : 1) aqueous C a l c o f l u o r White ST (4, 4*-bis [ 4 - a n i l i n o - 6 - b i s (2-hydroxyethyl) a m i n o - s - t r i a z i n - 2 -x y l a m i n o ] - 2 , 2 ' - s t i l b e n e d i s u l f o n i c a c i d ) , 2) Congo Fed, and 3) Zinc C h l o r i o d i d e . Some s e c t i o n s were a l s o s t a i n e d with A c r i d i n e Orange f o r the v i s u a l i z a t i o n of n u c l e a r f l u o r e s c e n c e . For C a l c o f l u o r White s t a i n i n g , specimens were t r e a t e d f o r 1 min with a 0.1% aqueous s o l u t i o n of t h i s f l u o r e s c e n t b r i g h t e n e r , r i n s e d with d i s t i l l e d water, and viewed with a L e i t z D i a l u x 20EB e p i f l u o r e s c e n c e microscope equipped with a f i l t e r block g i v i n g an e x c i t a t i o n range of 340 to 380 nm and t r a n s m i t t i n g at over 430 nm. Other s e c t i o n s were s t a i n e d with a 0.1% s o l u t i o n of Congo Red i n 50% ethanol f o l l o w e d by b r i e f d i f f e r e n t i a t i o n i n 0.2% e t h a n o l i c potassium hydroxide. They were then examined us i n g a f l u o r e s c e n c e e x c i t a t i o n range of 530-560 nm with the f i l t e r b l o ck e m i t t i n g at 580 nm and above. As a f i n a l h i s t o c h e m i c a l t e s t f o r c e l l u l o s e , s e c t i o n s were s t a i n e d f o r 3 min with Zinc C h l o r i o d i d e f o l l o w i n g the procedure given by Stevens (1974) . The c o l o r r e a c t i o n s of these s t a i n i n g - 29 -techniques are given i n Table I I . S l i d e s of JB4-embedded specimens which were s t a i n e d f o r b r i g h t f i e l d microscopy were made permanent by p l a c i n g a drop of Permount over the s e c t i o n s and a p p l y i n g a cover s l i p . For f l u o r e s c e n c e s t u d i e s , Harleco n o n - f l u o r e s c e n t mounting medium was s u b s t i t u t e d f o r the Permount. The pathogen was i d e n t i f i e d to the genus l e v e l using keys p r o v i d e d by Sparrow (1960, 1973a,b). K a r l i n g ' s (1981) t r e a t i s e on h o l o c a r p i c b i f l a g e l l a t e s and o r i g i n a l s p e c i e s d e s c r i p t i o n s were l i k e w i s e c o n s u l t e d . Measurements of host and p a r a s i t e c e l l s were made using a L e i t z - W e t z l a r 12.5x micrometer eyepiece under two o b j e c t i v e m a g n i f i c a t i o n s : 40x f o r fu n g a l sporangia and medullary c e l l s of the alga and lOOx f o r the w a l l - l e s s stages of the p a r a s i t e and the outer t i s s u e l a y e r s of the hos t . Means and standard d e v i a t i o n s were computed f o r 50 separate measurements. Morphometric s t u d i e s were a l s o undertaken to determine i f s w e l l i n g of host c e l l s i s a s s o c i a t e d with the i n f e c t i o n . O u t l i n e s of 80 i n f e c t e d s u b c o r t i c a l c e l l s l o c a t e d 25 to 35 micrometers from the c u t i c l e ( f i f t h to s i x t h c e l l l a y e r ) were drawn on white paper with the a i d of a L e i t z - W e t z l a r 60° t r a c i n g a i d at lOOOx m a g n i f i c a t i o n . These images were i n turn t r a c e d on a Hipad d i g i t i z e r l i n k e d to an IBM Personal Computer (PC). T o t a l areas, means, and standard d e v i a t i o n s were computed using the "Trace Area" and s t a t i s t i c s f u n c t i o n s of the Hipad Drawing and Measurement Program (Houston Instruments, - 30 -I n c . ) . Areas of s u b c o r t i c a l c e l l s from u n i n f e c t e d c o n t r o l s were a l s o determined f o r comparison. Mean areas of h e a l t h y and i n f e c t e d c e l l s were compared using the Student's t - t e s t with the l e v e l of s i g n i f i c a n c e at 0.05. To determine i f host c e l l death i s a s s o c i a t e d with e a r l y stages of the i n f e c t i o n , three s t a i n s were i n i t i a l l y screened f o r f l u o r e s c e n c e d i f f e r e n t i a t i o n between l i v i n g and dead c e l l s : A c r i d i n e Orange, F l u o r e s c e i n D i a c e t a t e , and 4',6 Diamidino-2-p h e n y l i n d o l e (DAPI). S i n c e DAPI gave the most c o n s i s t e n t s t a i n i n g r e s u l t s , i t was employed i n the r e s t of the f l u o r e s c e n t v i t a l s t a i n i n g experiments. I n o c u l a t e d t i p s were incubated o v e r n i g h t i n t h i s fluorochrome d i s s o l v e d i n the c u l t u r e medium to g i v e a f i n a l c o n c e n t r a t i o n of l.OLjg m l " 1 . Freehand s e c t i o n s were made the f o l l o w i n g day, r i n s e d 3 times f o r 15 min to remove excess DAPI, and mounted i n f i l t e r e d sea water. A second s e t c o n s i s t i n g of u n i n f e c t e d specimens was f i x e d with 5% formaldehyde i n phosphate b u f f e r at pH 7.4, washed 4 times f o r 15 min with b u f f e r , and incubated i n the same fluorochrome o v e r n i g h t to serve as c o n t r o l s . F i n a l l y , a t h i r d set of h e a l t h y , l i v i n g Chondrus t h a l l i was l e f t incubated i n DAPI to determine i f c e l l s w i l l i n c o r p o r a t e the fluorochrome without p r e v i o u s f i x a t i o n . Specimens were viewed with a L e i t z D i a l u x 20EB e p i f l u o r e s c e n c e microscope using the same f i l t e r block as that used f o r c a l c o f l u o r white f l u o r o c h r o m i n g . - 31 -G. E l e c t r o n microscopy D i f f e r e n t specimen p r e p a r a t i o n r o u t i n e s i n v o l v i n g v a r i o u s permutations of f i x a t i o n time and temperature, f i x a t i v e types and c o n c e n t r a t i o n s , v e h i c l e s , embedding media, and i n f i l t r a t i o n times were t e s t e d for e l e c t r o n microscopy. The procedure f i n a l l y adapted i n v o l v e s s e q u e n t i a l f i x a t i o n with a mixture of 5% g l u t a r a l d e h y d e and 4% formaldehyde (Karnovsky 1965) i n 0.2M phosphate or sodium c a c o d y l a t e b u f f e r and osmium t e t r o x i d e . A r t i f i c i a l l y i n f e c t e d specimens were e x c i s e d i n a drop of the aldehyde mixture a f t e r 6, 12, 24, 48, and 72 h from the time of i n o c u l a t i o n . They were then t r a n s f e r r e d to a v i a l c o n t a i n i n g the same f i x a t i v e and allowed to f i x f o r 3 h at room temperature. A f t e r four r i n s e s i n b u f f e r , they were p o s t - f i x e d i n 1% osmium t e t r o x i d e for 48 h at room temperature ( p r e v i o u s l y determined to be the optimum p o s t - f i x a t i o n t i m e ) . Fixed t i s s u e s were washed i n b u f f e r four times, taken to 100% propylene oxide from a graded methanol s e r i e s i n 14 steps, and i n f i l t r a t e d with Epon or Polybed 812 for 8 days. The composition of the epoxy r e s i n employed f o r e l e c t r o n microscopy i s given i n Appendix B. Blocks were polymerized at 60C overnight and s i l v e r s e c t i o n s were cut with a Dupont diamond k n i f e on a R i c h e r t OMU2 ultramicrotome. They were then c o l l e c t e d on formvar-coated g r i d s , s t a i n e d f o r 15-30 min i n s a t u r a t e d raethanolic uranyl a cetate and 6-10 min i n Sato's (1967) l e a d c i t r a t e and viewed with a Z e i s s EM 9A e l e c t r o n microscope. - 32 -RESULTS AND OBSERVATIONS A. P e t e r s e n i a p o l l a g a s t e r : zoospores and c e l l w a l l cytochemistry The zoospores of P.. p o l l a g a s t e r are l a t e r a l l y b i f l a g e l l a t e . They are broadly reniform to elongate and measure 3.0 to 3.5 pin wide by 4.0 to 4.5 jura long ( F i g s . 2 and 3 ) . Two heterokont f l a g e l l a a r i s e from a groove that runs along the length of the spore. Prolonged i l l u m i n a t i o n of the planont caused the f l a g e l l a to be resorbed. Treatment of methacrylate s e c t i o n s with C a l c o f l u o r White ( F i g . 4) and Congo Red ( F i g . 5) r e s u l t e d i n the f l u o r e s c e n c e of fungal walls . Examination of unstained c o n t r o l s r e v e a l e d only a f a i n t a u t o f l u o r e s c e n c e of fungal w a l l s and host t i s s u e s which could e a s i l y be d i s t i n g u i s h e d from fluorochrome-induced f l u o r e s c e n c e . S i m i l a r l y s t a i n e d freehand s e c t i o n s of u n f i x e d specimens showed the same f l u o r e s c e n c e c h a r a c t e r i s t i c s of fungal w a l l s ( F i g . 6) . Zinc C h l o r i o d i d e , a commonly used s t a i n f o r oomycete w a l l s , l i k e w i s e gave a p o s i t i v e s t a i n i n g r e a c t i o n ( F i g . 7 ) . The combination of these s t a i n i n g r e a c t i o n s i s i n d i c a t i v e of the presence of c e l l u l o s e . - 33 -B . c u i t u r i n g of Pe tersen ia p o l l a g a s t e r Attempts to grow the pathogen i n v a r i o u s c u l t u r e media for m u l a t e d by V i s h n i a c (1956), F u l l e r et a l . (1964), and G o t e l l i (1974) y i e l d e d n e g a t i v e r e s u l t s . L i k e w i s e , h e a t - k i l l e d u n i n f e c t e d Chondrus t h a l l i were not c o l o n i z e d by the fungus. C. O b s e r v a t i o n s on pathogenesis The p a r a s i t e invades the growing p o i n t s of the a l g a l host which are on the most d i s t a l b i f u r c a t i o n s . I n f e c t i o n i s i n d i c a t e d by the f o r m a t i o n o f n e c r o t i c l e s i o n s a f t e r i n c u b a t i o n f o r 48 h at 20C. F i g s . 8 and 9 shew e a r l y and l a t e stages of the i n f e c t i o n . Small l e s i o n s measuring 0.15 mm i n diameter developed i n the t i p s w i t h i n 48 h of i n o c u l a t i o n and i n c r e a s e d to 0.4 mm at the end of 72 h. In h e a v i l y i n f e c t e d t h a l l i ( F i g . 10), there can be hundreds of s m a l l l e s i o n s per cro2. F i g . 11 shows an u n i n f e c t e d specimen which i s i n c l u d e d f o r comparison. I n o c u l a t i o n of p r e v i o u s l y u n i n f e c t e d t h a l l i at the f i v e temperatures t e s t e d showed t h a t temperature extremes are u n f a v o r a b l e to pathogenesis under the l a b o r a t o r y c o n d i t i o n s used. I n f e c t i o n d i d not occur w i t h i n 72 h at 5 and 10C as shown by the absence of l e s i o n s and c o u l d not be d e t e c t e d even a f t e r 7 days. Absence of i n f e c t i o n was confirmed f u r t h e r by the l a c k of p a r a s i t e c e l l s i n freehand s e c t i o n s of t h a l l i t h a t - 34 -were i n o c u l a t e d at these temperatures. However, zoospores remained v i a b l e at IOC and c o u l d be induced to i n f e c t host t h a l l i by t r a n s f e r r i n g c u l t u r e s to the p e r m i s s i v e temperature of 20C. F i g u r e 12 g i v e s a s c a t t e r diagram of the number of l e s i o n s formed under the f i v e temperatures t e s t e d . The best f i t t i n g curve was o b t a i n e d with the p o l y n o m i a l equation given i n the e x p l a n a t i o n to the f i g u r e . The curve g i v e s a c o e f f i c i e n t of d e t e r m i n a t i o n of 0.943 and a r e g r e s s i o n c o e f f i c i e n t of 0.971. A t e s t f o r the s i g n i f i c a n c e of the r e g r e s s i o n c o e f f i c i e n t y i e l d e d F [ i , 3 0 ] = 586.28 which g r e a t l y exceeds the c r i t i c a l l i m i t of 4.17 at the 0.05 p r o b a b i l i t y l e v e l , showing t h a t a s i g n i f i c a n t r e g r e s s i o n e x i s t s . . The optimum temperature range f o r the spread and development of £. p o l l a g a s t e r under the l a b o r a t o r y c o n d i t i o n s used was 15 to 20C. At these temperatures the c y c l e c o n s i s t i n g of i n f e c t i o n of h e a l t h y t h a l l i , e n d o b i o t i c development, and emergence of zoospores was completed i n 48 to 72 h. Although d e t a i l s of zoospore r e l e a s e c o u l d not be observed owing to the massive nature of the host, completion of the p a r a s i t e ' s l i f e c y c l e i s i n d i c a t e d by the appearance of protuberances i n the n e c r o t i c l e s i o n s a f t e r 48 h which c o i n c i d e s with the f o r m a t i o n of e x i t tubes. At the end of 72 h a pore i s formed at the center of each l e s i o n ( F i g . 8) i n d i c a t i n g t h a t most of the sporangia have a l r e a d y d i s c h a r g e d t h e i r zoospores. - 35 -D . U n i n f e c t e d Chondrus c r i s p u s 1. L i g h t microscopy A c r o s s - s e c t i o n through the most d i s t a l b i f u r c a t i o n of £. c r i s p u s shows an e x t e r n a l c u t i c l e which i s approximately 1.0 pro t h i c k . Below t h i s c u t i c l e the t h a l l u s i s o r g a n i z e d i n t o t hree r e g i o n s : the c o r t e x , the subcortex, and the medulla ( F i g . 13). The c o r t i c a l c e l l s which are mostly found i n the f i r s t 25 pra immediately below the c u t i c l e are round to b r o a d l y elongate and measure 3.5 to 5.0 pm by 3.5 to 7.0 pm (mean: 3.9 + 0.9 by 6.3 + 0.6 pm). T h e i r o r g a n e l l e s are d i f f i c u l t to d i s t i n g u i s h as they are very c l o s e l y packed i n s i d e each i n d i v i d u a l c e l l . There i s very l i t t l e i n t e r c e l l u l a r m a t r i x . The c o r t i c a l c e l l s comprise the f i r s t 4 to 5 c e l l l a y e r s beneath the c u t i c l e . The subcortex c o n s i s t s of c e l l l a y e r s found 25 to 70 pra from the c u t i c l e ( F i g . 13). S u b c o r t i c a l c e l l s (=outer m e d u l l a r y c e l l s , C o t t i e r 1971) are more elongate and l a r g e r than the outer c o r t i c a l c e l l s . They measure 6.5 to 9.5 jum wide by 7.0 to 11.5 jum long (mean: 7.9 + 1.2 by 9.0 + 1.4 jum) and have abundant i n t e r c e l l u l a r m a t r i x . C e l l s i n the subcortex are more l o o s e l y packed than those i n the subcortex and have lobed c h l o r o p l a s t s as w e l l as many p i t c o n n e c t i o n s . With T o l u i d i n e Blue 0 s t a i n i n g the area of most i n t e n s e r e d d i s h metachromasia occurs i n areas immediately o u t s i d e the c e l l w a l l s ( F i g . 13). - 36 -The h i g h l y e l ongate medullary c e l l s ( F i g . 14) comprise t i s s u e l a y e r s l o c a t e d 70 to 150 urn from the c o r t e x and are the l a r g e s t of the t h r e e c e l l t y p e s . They e x h i b i t a much l e s s dense i n t e r n a l packing of o r g a n e l l e s than the c o r t i c a l and s u b c o r t i c a l c e l l s . They are h i g h l y v a r i a b l e i n s i z e and measure 12.0 to 22.5 jam wide by 16.0 to 48.0 jam long (mean: 18.8 +3.4 jum by 28.8 + 6.3 jam). T h e i r cytoplasm i s l o c a t e d at the p e r i p h e r y of the c e l l . As i n s u b c o r t i c a l c e l l s , areas of most i n t e n s e T o l u i d i n e Blue O metachromasia are found immediately o u t s i d e the c e l l w a l l s . 2. D l t r a s t r u c t u r e O l t r a s t r u c t u r a l s t u d i e s of Chondrus c r i s p u s have been done by C o t t i e r (1971) and Gordon and McCandless (1973) who ob t a i n e d r e s u l t s s i m i l a r to those found i n the present i n v e s t i g a t i o n . A t r a n s v e r s e s e c t i o n through the most d i s t a l b i f u r c a t i o n of the a l g a l t h a l l u s r e v e a l s t h a t the c u t i c l e which ranges from 1.0 to 1.25 ;um t h i c k i s made up of 7 to 12 e l e c t r o n - d e n s e bands separated by e l e c t r o n - t r a n s p a r e n t r e g i o n s ( F i g . 1 5 ) . The electron-opaque bands are approximately 420 nm t h i c k and are more c l o s e l y appressed i n the proximal l a y e r s . The i n t e r p o s i n g e l e c t r o n - t r a n s p a r e n t l a y e r s c o n t a i n g r a n u l a r m a t e r i a l which resembles t h a t found d i r e c t l y beneath the c u t i c l e . - 37 -C e l l s of the co r t e x ( F i g . 16) are c l o s e l y packed and c o n t a i n very l i t t l e i n t e r c e l l u l a r m a t r i x . T h e i r immediate w a l l s c o n t a i n m i c r o f i b r i l s t h a t are o r i e n t e d p a r a l l e l to the c e l l s u r f a c e and e n c i r c l e the protoplasm. A l l the c o r t i c a l c e l l s examined had only one nucleus and a s i n g l e c h l o r o p l a s t p r o f i l e . There i s a r a t h e r t i g h t packing of c e l l u l a r o r g a n e l l e s , and m i t o c h o n d r i a are f r e q u e n t l y found adjacent to the n u c l e u s . The cytoplasm c o n t a i n s ribosomes a s s o c i a t e d with endoplasmic r e t i c u l u m . S u b - c o r t i c a l c e l l s ( F i g s . 17 & 18) are l a r g e r and are l e s s c l o s e l y packed than those i n the c o r t e x . They are separated from each other by l a r g e i n t e r c e l l u l a r spaces which exceed s e v e r a l times the diameter of the c e l l s . As i n c o r t i c a l c e l l s , a f i b r i l l a r w a l l surrounds the protoplasm and i t s f i b e r s are o r i e n t e d p a r a l l e l t o the c e l l s u r f a c e . The c h l o r o p l a s t i s h i g h l y d i v i d e d and has c h a r a c t e r i s t i c a l l y unstacked t h y l a k o i d s , with the outermost l a m e l l a e n c i r c l i n g the inner ones. M i t o c h o n d r i a are o f t e n found between the p e r i p h e r a l lobes of the c h l o r o p l a s t which surround the n u c l e u s . Fibosomes and el e c t r o n - d e n s e membranous whorls which might be i n v o l v e d i n v a c u o l e f o r m a t i o n are d i s p e r s e d i n the c y t o s o l ( F i g . 1 8 ) . S t a r c h g r a i n s , when pr e s e n t , are s u b f u s i f o r m i n shape and are d e p o s i t e d i n the cytoplasm, o u t s i d e the c h l o r o p l a s t ( F i g . 1 9 ) . C e l l s of the medulla ( F i g . 20) are elongate and are 2 to 3 times as long as wide. As i n c e l l s of the c o r t e x and the - 38 -subcortex, they are surrounded by a m i c r o f i b r i l l a r w a l l . Much of the c e l l ' s volume i s occupied by a l a r g e c e n t r a l v acuole surrounded by the p e r i p h e r a l protoplasm. The c h l o r o p l a s t i s a l s o h i g h l y d i s s e c t e d and i t s lobes are l o c a t e d towards the p e r i p h e r y of the c e l l . I t s t h y l a k o i d s are c h a r a c t e r i s t i c a l l y unstacked. There can be one to s e v e r a l n u c l e i i n each me d u l l a r y c e l l . P i t c o n n e c t i o n s are o f t e n found between c e l l s of the a l g a l t h a l l u s and are most abundant i n s u b c o r t i c a l and medullary t i s s u e . They are dumbbell-shaped and are surrounded by a membrane ( F i g . 21). An e l e c t r o n - d e n s e m a t e r i a l f i l l s t h e i r i n t e r i o r . E . Development of Pe tersen ia p o l l a g a s t e r 1. L i g h t microscopy Although the very e a r l y stages of p e n e t r a t i o n were not observed, i t i s apparent that the p a r a s i t e p r o t o p l a s t breaches the host c u t i c l e and develops i n i t i a l l y as a w a l l - l e s s c e l l which s t a i n s i n t e n s e l y with T o l u i d i n e Blue 0 ( F i g s . 22 & 23). The l a c k of C a l c o f l u o r White s t a i n i n g and Congo Red r e a c t i v i t y c o n f i r m s the absence of a w a l l d u r i n g the e a r l y ( i . e . , f i r s t 24 h) stages of e n d o b i o t i c development. Being c l o s e s t to the s u r f a c e , the c o r t i c a l c e l l s of £. c r i s p u s are the f i r s t to be - 39 -p e n e t r a t e d by the p a r a s i t e ( F i g . 22). The v e g e t a t i v e t h a l l u s c o n t i n u e s to p r o l i f e r a t e w i t h i n host c e l l s . I t i n f e c t s other c e l l s i n the u n d e r l y i n g s u b c o r t i c a l and m edullary t i s s u e s v i a a s y m p l a s t i c route ( F i g . 22) thereby promoting f u s i o n of host c e l l s . Each nucleus has a prominent n u c l e o l u s d u r i n g the w a l l - l e s s stage ( F i g . 23). Most of the i n f e c t e d host c e l l s observed were i n the s u b c o r t i c a l and medullary r e g i o n s . There i s no m o r p h o l o g i c a l l y r e c o g n i z a b l e host defense mechanism or r e a c t i o n to the i n f e c t i o n i n the e a r l y stages of development of the p a r a s i t e . Comparison of i n f e c t e d specimens ( F i g s . 22 and 23 ) with u n i n f e c t e d c o n t r o l s ( F i g . 13) shows tha t the g e n e r a l host t i s s u e o r g a n i z a t i o n i s m a i n t a i n e d d u r i n g • the e a r l y stages of fungal development. Formation of a l i g h t l y s t a i n i n g w a l l by the p a r a s i t e probably marks the onset of zoosporogenesis and commonly occurs i n the subcortex and medulla ( F i g s . 24 and 25). The w a l l e d t h a l l u s of p. p o l l a g a s t e r e x h i b i t s v a r y i n g degrees of l o b u l a t i o n and spans s e v e r a l host c e l l l a y e r s ( F i g s . 24,25,26). Sporangia measure 17.0 to 44.0 pm wide by 40.0 to 78.5 pm long (mean: 30.0 + 12.7 Jim by 51.3 + 12.3 pm) and may show g r e a t l y i n f l a t e d l o b u l a t i o n s ( F i g . 26) or may only be s l i g h t l y lobed ( = o l p i d i o i d , K a r l i n g 1981). The presence of more than 100 n u c l e i i n a s i n g l e s e c t i o n of a sporangium ( F i g s . 24,26,28) i n d i c a t e s the p o t e n t i a l f o r p r o d u c t i o n of a l a r g e number of p a r a s i t e zoospores. A s e c t i o n made f u r t h e r i n t o the host - 40 -t h a l l u s shows that the sporangium i s c o n t a i n e d w i t h i n the invaded host c e l l s and i s t h e r e f o r e i n t r a c e l l u l a r ( F i g . 27). L a t e r i n i t s development the sporangium produces an e x i t tube which i s 20.0 to 40.0 pm long ( F i g s . 28 and 30). That the sporangium produces only one e x i t tube was determined through p a r t i a l t h r e e - d i m e n s i o n a l r e c o n s t r u c t i o n of s e r i a l s e c t i o n s and by viewing whole, i n t a c t sporangia i n fluorochromed freehand s e c t i o n s with the e p i f l u o r e s c e n c e microscope. The e x i t tube e v e n t u a l l y p e n e t r a t e s the host c u t i c l e and produces a sho r t e x t e r n a l beak ( F i g . 6 ) . At t h i s stage, damage to host t i s s u e i s more apparent as c e l l s i n the immediate v i c i n i t y of the sporangium have c o l l a p s e d ( F i g s . 28 & 30). The host t i s s u e appears to be pushed upward d u r i n g e x i t tube f o r m a t i o n and e l o n g a t i o n as suggested by the presence of a l o c a l i z e d protuberance on the host s u r f a c e ( F i g s . 26,28,30). The e x i t tube appears to pass through a channel t h a t c o u l d have been formed d u r i n g e a r l y p e n e t r a t i o n ( F i g . 29). Cleavage of the s p o r a n g i a l protoplasm and f o r m a t i o n of zoospores are presumably completed i n the sporangium as r e v e a l e d by s e c t i o n s through p a r t i a l l y r e l e a s e d sporangia which show c e l l s t h a t are presumably f u l l y formed zoospores ( F i g s . 30 & 31). F i g . 30 shows c a sporangium that has r e l e a s e d most of i t s contents except f o r a s i n g l e remaining zoospore that was " f r o z e n " by the f i x a t i o n p r o c e s s . Most of the zoosporangia have a l r e a d y d i s c h a r g e d at the end of 72 h ( F i g . 31). - 41 -2. P e t e r s e n i a p o l l a g a s t e r ! U l t r a s t r u c t u r e of developmental stages a. P o s t m o t i l e spores and c y s t s R e s u l t s of u l t r a s t r u c t u r a l s t u d i e s g e n e r a l l y c o n f i r m and supplement those o b t a i n e d from l i g h t microscopy. The e a r l i e s t stage i n the development of £ . p o l l a g a s t e r observed was that of the p o s t m o t i l e spore on the host c u t i c l e ( F i g . 32). The a f l a g e l l a t e c e l l i s br o a d l y elongate and i s of the same s i z e and shape as the planonts d e s c r i b e d i n S e c t i o n 1. That the f l a g e l l a have been resorbed can be deduced by the occurrence i n the cytoplasm of p r o f i l e s showing the 9+2 arrangement of axonemal m i c r o t u b u l e s ( F i g . 32.,insert).. ' A very t h i n w a l l seems to be a l r e a d y present immediately o u t s i d e the plasma membrane and s m a l l v e s i c l e s measuring 85-180 nm are found i n the cytoplasm. S e v e r a l vacuoles c o n t a i n i n g f i b r o u s m a t e r i a l occur p e r i p h e r a l l y i n the c e l l . Some of them show c o n c e n t r i c c o n v o l u t e d membranes ( F i g s . 33 & 34). Ribosomes are d i s p e r s e d i n the v a c u o l a t e cytoplasm. A s i n g l e nucleus measuring 1.2 pm i n i t s narrowest diameter i s prese n t near the c e n t e r . Adjacent to t h i s nucleus t h e r e i s a l a r g e l i p i d g l o b u l e a s s o c i a t e d with s e v e r a l m i t o c h o n d r i a having t u b u l a r c r i s t a e . Another c l a s s of c y t o p l a s m i c i n c l u s i o n i s r e c o g n i z a b l e i n the p o s t m o t i l e stage: an e l e c t r o n - d e n s e , membrane-bound i n c l u s i o n ( h e r e a f t e r r e f e r r e d to as c r e n a t e v e s i c l e i n c l u s i o n ) measuring 130 nm i n diameter. The e l e c t r o n - d e n s e m a t e r i a l i s probably u n s a t u r a t e d l i p i d and - 42 -has an e v i d e n t s u b - s t r u c t u r e ( F i g s . 32,33,34). The u t i l i z a t i o n of t h i s i n c l u s i o n i s i n d i c a t e d by the f u s i o n of crenate v e s i c l e s with the vacuoles and the subsequent l o c a l i z a t i o n of t h e i r contents i n the v a c u o l a r lumen. F i g . 35 shows another p o s t m o t i l e spore with a r e t r a c t e d axoneme i n l o n g i t u d i n a l s e c t i o n . As i n F i g . 32, axonemal m i c r o t u b u l e s comprising the c e n t r a l s i n g l e t and the p e r i p h e r a l doublets are e v i d e n t . The axoneme soon breaks down to the l e v e l of the t e r m i n a l p l a t e of the b a s a l body a f t e r the f l a g e l l a have been withdrawn ( F i g . 36) . Encystrnent of the p o s t m o t i l e spore i s accompanied by rounding up of the c e l l and s y n t h e s i s of a t h i n , e l e c t r o n - t r a n s p a r e n t , amorphous w a l l , averaging 170 nm i n t h i c k n e s s ( F i g . 37). A f l u f f y coat which resembles the f i b r o u s c o ntents of the s m a l l p e r i p h e r a l vacuoles observed i n the p o s t m o t i l e spore i s present on the c e l l s u r f a c e , e x t e r n a l to the w a l l . D i s r u p t i o n of the c u t i c l e ' s f i n e s t r u c t u r e i s not e v i d e n t d u r i n g e a r l y encystrnent. The h o s t - p a r a s i t e i n t e r f a c e at t h i s stage c o n s i s t s of the c l o s e l y appressed c y s t w a l l and the outermost l a y e r of the host c u t i c l e s eparated only by what appears to be a t h i n l a y e r of the f l u f f y coat ( F i g . 38). The c y s t w a l l and the outermost l a y e r of the host c u t i c l e are p a r a l l e l . A p p a r e n t l y , c y s t s are a b l e to e f f e c t a l o c a l i z e d d i s s o l u t i o n of the outermost l a y e r of the host c u t i c l e . F i g u r e 39 shows a cyst t h at might have been p a r t l y d i s s o c i a t e d from the host c u t i c l e d u r i n g specimen p r e p a r a t i o n . The outermost - 43 -l a y e r s of the c u t i c l e adjacent to the c y s t e x h i b i t a s t r u c t u r a l change: from a more or l e s s even, e l e c t r o n - d e n s e l a y e r ( F i g . 15) to an i r r e g u l a r network of l o o s e l y arranged f i b e r s . Cysts e x h i b i t the same complement of o r g a n e l l e s and i n c l u s i o n s as the p o s t m o t i l e spore: a s i n g l e n u c l e u s , a l i p i d g l o b u l e a s s o c i a t e d with m i t o c h o n d r i a having t u b u l a r c r i s t a e , s e v e r a l vacuoles some of which c o n t a i n c o n c e n t r i c c o n v o l u t e d membranes, and ribosomes d i s p e r s e d i n the c y t o s o l ( F i g . 37) . The c r e n a t e v e s i c l e i n c l u s i o n s p r e v i o u s l y observed i n p o s t m o t i l e spores e x h i b i t a v a r i e t y of forms ( F i g . 3 7 ). Some of them appear to fuse with a va c u o l e ( F i g s . 32 and 38) so t h a t the i n c l u s i o n becomes l o c a l i z e d i n the v a c u o l a r sap ( F i g . 35). In a l l s e c t i o n s examined, the l i p i d g l o b u l e was always l e s s than 0.5 jjm i n diameter, about h a l f the s i z e of that present i n the p o s t m o t i l e spor e. - 44 -b. V e g e t a t i v e phase Although the very e a r l y stages of host p e n e t r a t i o n were not observed, the fungus a p p a r e n t l y breaches the c u t i c l e and invades host c e l l s as a w a l l - l e s s p r o t o p l a s t whose g e n e r a l e l e c t r o n d e n s i t y d i f f e r s markedly from the c y s t protoplasm ( F i g s . 40 to 46). The d i f f e r e n t i a l e l e c t r o n d e n s i t y between p a r a s i t e and host c e l l s f a c i l i t a t e s d i s t i n c t i o n between the two c e l l t y p e s . C o r t i c a l c e l l s are the f i r s t to be invaded. The p a r a s i t e becomes c l o s e l y a s s o c i a t e d with the host c e l l cytoplasm and i n v a g i n a t e s a host membrane. Thus, i t becomes surrounded by two c l o s e l y appressed membranes: i t s own plasmalemma and another of host o r i g i n ( F i g . 40). Measurements of the s u r f a c e and the i n v a g i n a t e d host membranes r e v e a l t h a t o they have a common t h i c k n e s s of approximately 95A. The c l o s e p r o x i m i t y of host and p a r a s i t e membranes was e x h i b i t e d by a l l stages observed p r i o r to sporangium f o r m a t i o n . The two membranes are s e p a r a t e d only by an e l e c t r o n - t r a n s p a r e n t r e g i o n which i s approximately 180 nm t h i c k ( F i g . 41). Granular m a t e r i a l i s o f t e n p r e s e n t i n t h i s r e g i o n between the two p r o t o p l a s t s . The f u n g a l p r o t o p l a s t e x h i b i t s v a r i o u s degrees of l o b u l a t i o n ranging from elongate and branching ( F i g . 42) to h i g h l y lobed ( F i g . 43). E a r l y i n i t s e n d o b i o t i c development the t h a l l u s i s u n i n u c l e a t e and has w e l l - d e v e l o p e d rough endoplasmic r e t i c u l u m . Each nucleus measures from 3.5 to 4.0 - 45 -pm i n diameter and has a very prominent n u c l e o l u s ( F i g s . 43 & 44). G o l g i bodies are found i n a zone of e x c l u s i o n adjacent to the n u c l e a r envelope ( F i g . 45). Convoluted membranes are present i n some of the v a c u o l e s . S e v e r a l m i t o c h o n d r i a with t u b u l a r c r i s t a e are found at the p e r i p h e r y of the c e l l , underneath the plasmalemma ( F i g s . 43 & 44). They have i r r e g u l a r o u t l i n e s and vary i n shape and s i z e depending on the plane of s e c t i o n i n g , the l a r g e s t ones measuring 0.9 jum i n l e n g t h . At some p o i n t s on the p r o t o p l a s t , the p a r a s i t e appears to endocytose p o r t i o n s of host cytoplasm as i n d i c a t e d by the presence i n the fu n g a l plasmalemma of i n p o c k e t i n g s c o n t a i n i n g host c e l l m a t e r i a l ( F i g . 41). Small v a c u o l e s are present i n the f u n g a l cytoplasm. L a t e r i n i t s development, the p a r a s i t e spreads to adjacent host c e l l s v i a a s y m p l a s t i c r o u t e . P e n e t r a t i o n of n e i g h b o r i n g host c e l l s w i t h i n the same l a y e r as w e l l as i n u n d e r l y i n g t i s s u e i s preceded by the fo r m a t i o n of narrow c y t o p l a s m i c processes by the p a r a s i t e ( F i g . 44). M i t o c h o n d r i a then migrate and aggregate i n s i d e these p r o c e s s e s ( F i g . 46). - 46 -c. Sporangium formation and zoosporogenesis The s h i f t from a v e g e t a t i v e to a r e p r o d u c t i v e phase i n the p a r a s i t e i s a s s o c i a t e d with host c e l l death. The fungal t h a l l u s remains l o c a l i z e d i n a n e c r o t i c host c e l l ( F i g . 47). Examination of c e l l s a d jacent to f u n g a l s p o r a n g i a showed that they had c o l l a p s e d and had a c q u i r e d a very dense cytoplasm ( F i g . 4 8 ). T h e i r i n t e r n a l membranous o r g a n i z a t i o n i s d i s r u p t e d and the i n d i v i d u a l o r g a n e l l e s become d i f f i c u l t to d i s t i n g u i s h . The f i r s t stage i n sporangium f o r m a t i o n i s the s y n t h e s i s of a w a l l by the naked p r o t o p l a s t . The e a r l i e s t s p o r a n g i a l stage observed had a t h i n , amorphous w a l l which measured approximately 0.2 u^m ( F i g . 49). Examination of the w a l l ' s s t r u c t u r e under higher m a g n i f i c a t i o n r e v e a l s t h a t i t i s a c t u a l l y composed of two amorphous l a y e r s : a moderately e l e c t r o n - d e n s e outer l a y e r which measures approximately 40 nm t h i c k and a wider e l e c t r o n - t r a n s p a r e n t inner l a y e r which i s 3 to 4 tiroes the t h i c k n e s s of the outer l a y e r ( F i g . 64). Remnants of the host c e l l are p r e s e n t immediately o u t s i d e the w a l l . The f u n g a l cytoplasm has numerous s m a l l vacuoles a v e r a g i n g 0.8 pm i n diameter f i l l e d with f i n e g r a n u l a r m a t e r i a l . Some of these v a c u o l e s c o n t a i n c o n c e n t r i c c o n v o l u t e d membranes; o t h e r s have an e l e c t r o n - d e n s e , l u n a t e i n c l u s i o n ( h e r e a f t e r r e f e r r e d to as dense body) c l o s e l y appressed to one s i d e of the t o n o p l a s t ( F i g . 49). The morphology of the i n c l u s i o n s appears to change as development of the p a r a s i t e - 47 -proceeds. S p h e r i c a l n u c l e i measuring approximately 1.5 pm are di s p e r s e d i n the cytoplasm and have n u c l e o l i which are much smaller than those found i n the w a l l - l e s s stage (0.4 pm= compared to 3.0 jjm) . Eibosomes a s s o c i a t e d with endoplasmic r e t i c u l u m are a l s o present and are e s p e c i a l l y abundant at the per i p h e r y of the nuclear envelope ( F i g . 50) . Mitochondria with t u b u l a r c r i s t a e and measuring 0.4 x 0.9 pm i n l o n g i t u d i n a l s e c t i o n are a l s o d i s p e r s e d i n the cytoplasm. They are v a r i a b l e i n s i z e and shape and do not show any s p e c i a l a s s o c i a t i o n with the n u c l e i at t h i s stage. C i s t e r n a e of rough endoplasmic r e t i c u l u m e n c i r c l e the nucleus ( F i g . 50) . Later i n s p o r a n g i a l development a c e n t r a l vacuole i s formed ( F i g . 51). Membrane components are of t e n present i n s i d e the vacuole. Although s a t i s f a c t o r y p r e s e r v a t i o n of the tono p l a s t was d i f f i c u l t to o b t a i n , the images obtained suggest the f u s i o n of the dense bodies with the va c u o l a r membrane ( F i g . 52). Thus, t h e i r electron-dense contents e v e n t u a l l y become l o c a l i z e d i n the vacuolar sap. Two other types of cy t o p l a s m i c i n c l u s i o n s develop: 1) an e l e c t r o n dense, membrane bound i n c l u s i o n which resembles the crenate v e s i c l e i n c l u s i o n s observed i n p o s t m o t i l e spores and c y s t s and 2) a l a r g e r , moderately e l e c t r o n dense i n c l u s i o n (to be r e f e r r e d to as l i p i d g l o b u l e ) which ranges from 0.3 to 0.7 pm i n diameter ( F i g . 54). Intense s y n t h e t i c a c t i v i t y i s i n d i c a t e d by the abundance of rough endoplasmic r e t i c u l u m . G o l g i p r o f i l e s are commonly found adjacent to the n u c l e i ( F i g . 53). The involvement of the - 48 -nuclear envelope i n the s y n t h e s i s of p r o t e i n s i s suggested by the presence of t r a n s i t i o n v e s i c l e s between i t and the c i s (=forming) face of the G o l g i ( F i g . 53). Mitochondria which were p r e v i o u s l y d i s p e r s e d i n the cytoplasm a s s o c i a t e with, and become c l o s e l y appressed to the n u c l e a r envelope ( F i g . 54). Single-membrane bounded i n c l u s i o n s (to be c a l l e d m i c r o b o d y - l i k e s t r u c t u r e s ) are often found adjacent to a mitochondrion or a l i p i d body and a s s o c i a t e d with endoplasmic r e t i c u l u m ( F i g . 55). They measure approximately 0.18 pm i n diameter and have granular c o n t e n t s . Numerous dense bodies are a l s o p r e s e n t . A greater p r o p o r t i o n of the pre-cleavage sporangium becomes occupied by other p r o t o p l a s m i c components as the vacuole decreases i n s i z e ( F i g . 52). C o n c e n t r i c convoluted membranes a l s o become l o c a l i z e d i n the v a c u o l e s . L a t e r , a p a i r of b a s a l bodies measuring 0.15 x 0.6 pxa and o r i e n t e d at r i g h t angles to each other appears adjacent to the nuclear envelope ( F i g . 56 & 57). Each b a s a l body has a prominent t e r m i n a l p l a t e . S e q u e n t i a l s e c t i o n i n g showed that some of the n u c l e i are already p y r i f o r m at t h i s stage. That the sporangium i s i n t r a c e l l u l a r i s shown by s e c t i o n s showing p i t connections between the compound c e l l i t occupies and adjacent host c e l l s ( F i g . 58). O c c a s i o n a l l y , extremely c o n s t r i c t e d p o r t i o n s of the sporangium are present, g i v i n g the impression that a small part of the t h a l l u s i s s e p a r a t i n g from the main sporangium. F i g . 59 shows a c o n d i t i o n where the s e p a r a t i n g p i e c e and the parent - 49 -sporangium are connected only by a very narrow segment of cytoplasm that i s l e s s than 0.3 um and i n d i c a t e s fragmentation of the t h a l l u s . During cleavage, c i s t e r n a e p r o l i f e r a t e between u n i n u c l e a t e blocks of cytoplasm ( F i g s . 60 and 61). These cleavage c i s t e r n a e are probably d e r i v e d from the t r a n s (=maturing) face of the G o l g i as i n d i c a t e d by the p r o x i m i t y of the two membrane systems and the absence of ribosomes from t h e i r s u r f a c e . Measurements of membrane t h i c k n e s s showed that they are both approximately 90 nm. Small vacuoles a l s o appear to be i n v o l v e d i n cleavage as shown by t h e i r f u s i o n with the agranular c i s t e r n a e ( F i g . 62). They become f l a t t e n e d as development proceeds ( F i g . 63). The electron-dense i n c l u s i o n s i n s i d e the crenate v e s i c l e s e x h i b i t v a r i o u s forms of i n t e r n a l s u b s t r u c t u r e ranging from s t r i a t e d to having a l e s s electron-dense outer r i n g . They are apparently m o b i l i z e d during cleavage as shown by t h e i r f u s i o n with vacuoles ( F i g . 64) . Mastigonemes measuring 10 nm i n diameter are s y n t h e s i z e d i n d i l a t e d c i s t e r n a e of rough endoplasmic r e t i c u l u m ( F i g . 65). Ribosomes are present on the cytoplasmic s i d e of these c i s t e r n a e except where the membrane i s very c l o s e l y appressed to a mitochondrion. Formation of f l a g e l l a i s brought about by the d i s t a l e l o n g a t i o n of the basal b o d i e s . The e l o n g a t i n g axonemes are surrounded by a double membrane ( F i g s . 66 & 67). - 50 -Presumably, the inner membrane forms the sheath of the f l a g e l l a r s h a f t and the outer one fuses with the plasma membrane. F l a g e l l a become exerted between the u n i n u c l e a t e zoospore i n i t i a l s (Fig.66 & 67). As a r e s u l t of cleavage, the s p o r a n g i a l protoplasm becomes d i v i d e d i n t o c l o s e l y appressed u n i n u c l e a t e zoospore i n i t i a l s ( F i g . 68). Or g a n e l l e s which were p r e v i o u s l y observed i n the po s t m o t i l e spore are now prese n t : a group of small vacuoles, a nucleus, s e v e r a l mitochondria with t u b u l a r c r i s t a e , a l i p i d g l o b u l e c l o s e l y appressed to the mitochondria, crenate v e s i c l e i n c l u s i o n s , rough endoplasmic r e t i c u l u m , and b a s a l bodies ( F i g s . 69 & 70). C o n c e n t r i c dense membranes ( F i g s . 69 & 70) become a prominent f e a t u r e of the post-cleavage sporangium. G o l g i bodies were not observed at t h i s stage. Although s e c t i o n s showing f u l l y formed zoospores were not obtained, i t i s c l e a r that zoosporogenesis i s completed i n s i d e the sporangium. Examination of moribund zoospores i n sporangia which f a i l e d to escape showed that the planonts are indeed a l r e a d y f u l l y formed ( F i g s . 71 and 72). The u l t r a s t r u c t u r e of the remaining o r g a n e l l e s i n moribund c e l l s c l o s e l y resembles that of zoospore i n i t i a l s . The beaked nucleus and mitochondria with t u b u l a r c r i s t a e are e s p e c i a l l y prominent. F. E f f e c t s of i n f e c t i o n on host t i s s u e 1. F l u o r e s c e n t v i t a l s t a i n i n g - 51 -Of the three fluorochromes screened for f l u o r e s c e n c e d i f f e r e n t i a t i o n between l i v e and dead c e l l s , DAPI gave the most r e l i a b l e r e s u l t . The o v e r l a p i n the emission wavelength of A c r i d i n e Orange (AO) i n presumably n e c r o t i c c e l l s and c h l o r o p l a s t a u t o f l u o r e s c e n c e precluded accurate d e t e c t i o n of the expected a c r i d i n e orange-induced c y t o p l a s m i c reddening i n host c e l l s . The other fluorochrome, F l u o r e s c e i n D i a c e t a t e (FDA), could not be detected i n l i v i n g , u n i n f e c t e d t i s s u e and was t h e r e f o r e of no value i n d i s t i n g u i s h i n g between l i v e and dead host c e l l s . I n o culated host t h a l l i , incubated overnight i n 1.0 jjg m l ~ l DAPI, showed severe damage to the c o r t i c a l and s u b c o r t i c a l t i s s u e s as shown by the f l u o r e s c e n c e of c e l l s i n areas surrounding p o i n t s of i n f e c t i o n ( F i g . 73). Focussing on d i f f e r e n t planes w i t h i n the freehand s e c t i o n r e v e a l e d f l u o r e s c e n c e of s e v e r a l host c e l l l a y e r s . These regions presumably g i v e r i s e to the n e c r o t i c l e s i o n s . T h i s f l u o r e s c e n c e of host c e l l s i n the areas of i n f e c t i o n was s i m i l a r , to that e x h i b i t e d by f o r m a l d e h y d e - k i l l e d u n i n f e c t e d c o n t r o l s incubated i n the same fluorochrome ( F i g . 74). In c o n t r a s t , l i v e healthy t h a l l i t r e a t e d with DAPI f a i l e d to show f l u o r e s c e n c e , i n d i c a t i n g that v i t a l c e l l s do not take up the s t a i n . Thus, i n f e c t i o n of host t i s s u e by the p a r a s i t e and f i x a t i o n with formaldehyde appear to induce a s i m i l a r a l t e r a t i o n : the p e r m e a b i l i t y of c e l l s to DAPI. Host c e l l death apparently occurs before completion of zoosporogenesis. A - 52 -protuberance on the host s u r f a c e i s absent, i n d i c a t i n g t h a t the e x i t tube has not y e t been formed. By comparing the area of host t i s s u e damage as r e v e a l e d by f l u o r e s c e n t v i t a l s t a i n i n g ( F i g . 73) with the extent of i n f e c t i o n i n m e t h a c r y l a t e s e c t i o n s ( F i g . 28), i t can be deduced that c e l l death i s e x t e n s i v e . Even those c e l l s which are not d i r e c t l y p e n e t r a t e d by the p a r a s i t e become n e c r o t i c . 2. S w e l l i n g of p e n e t r a t e d host c e l l s : morphometric a n a l y s i s Comparison of s e m i - t h i n s e c t i o n s of h e a l t h y and d i s e a s e d t h a l l i shows a g e n e r a l i n c r e a s e i n the s i z e of d i r e c t l y p e n e t r a t e d host c e l l s . T able I I I summarizes the r e s u l t s of morphometric a n a l y s i s performed with the a i d of the Hipad Drawing and Measurement Program. The area of s u b c o r t i c a l c e l l s i n u n i n f e c t e d c o n t r o l s range from 71.1 to 126.9 jum2 (mean: 94.5 + 14.5 jjm2) . i n c o n t r a s t , s u b c o r t i c a l c e l l s which had been p e n e t r a t e d by the p a r a s i t e were 42% l a r g e r ; they measured 95.3 to 167.2 LMI2 (mean: 135.0 + 16.3 riiri2) . A p p l i c a t i o n of the Student's t - t e s t to the mean areas y i e l d e d a value of 16.61 which exceeds the c r i t i c a l l i m i t of 1.98 a s s o c i a t e d with the 0.05 p r o b a b i l i t y l e v e l with 120 degrees of freedom. The high t v a l u e o b t a i n e d i n d i c a t e s t h a t there i s a s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e between the mean c r o s s - s e c t i o n a l areas of i n f e c t e d and u n i n f e c t e d s u b c o r t i c a l c e l l s . - 53 -3. Changes i n host u l t r a s t r u c t u r e The roost notable change that occurs i n the host upon encystment of the p a r a s i t e i s the l o c a l i z e d d i l a t a t i o n of one of the proximal interband regions of the c u t i c l e . Beneath the po i n t where the spore has s e t t l e d and encysted on the host s u r f a c e , the two proximal electron-dense l a y e r s of the c u t i c l e separate, forming a gap ( F i g . 75). T h i s l o c a l i z e d d i l a t a t i o n , apparently caused by s e c r e t i o n and d e p o s i t i o n of a f i n e g ranular m a t e r i a l , was not observed i n u n i n f e c t e d c o n t r o l s ( F i g . 16). The u l t r a s t r u c t u r e of host c e l l s e x h i b i t a few a l t e r a t i o n s during the e a r l y stages of the p a r a s i t e ' s e n d o b i o t i c development. Walls of d i r e c t l y penetrated host c e l l s appear to los e t h e i r f i b r i l l a r s t r u c t u r e ( F i g . 76), making i t d i f f i c u l t to a s c e r t a i n the outer l i m i t s of the c e l l . D i s s o l u t i o n of s t a r c h granules i s a l s o a s s o c i a t e d with the i n f e c t i o n . Whereas they are subfusiforra and uniformly e l e c t r o n - t r a n s p a r e n t i n u n i n f e c t e d host c e l l s that have them ( F i g . 19), s t a r c h g r a i n s i n invaded Chondrus c e l l s are i r r e g u l a r l y shaped and show electron-dense cores ( F i g s . 42 , 44, &78) . Organelles such as mitochondria and c h l o r o p l a s t s ( F i g s . 40, 41, & 77) do not show r e c o g n i z a b l e changes r e l a t i v e to u n i n f e c t e d c o n t r o l s ( F i g s . 17 to 19). Although some c h l o r o p l a s t s show sinuous and i r r e g u l a r l y spaced t h y l a k o i d s ( F i g . 43), t h i s e f f e c t does not appear to be g e n e r a l i z e d . Except for the d i s s o l u t i o n of t h e i r - 54 -s t a r c h g r a i n s , adjacent host c e l l s which are not penetrated do not e x h i b i t any d i s r u p t i o n i n f i n e s t r u c t u r e during the e a r l y stages of i n f e c t i o n . During the i n t r a c e l l u l a r p r o l i f e r a t i o n of the p a r a s i t e , p i t connections between i n f e c t e d c e l l s and those which are soon to be penetrated appear to be d i s s o l v e d . Such d i s s o l u t i o n i s suggested by the disappearance of the electron-dense m a t e r i a l i n s i d e the p i t connections. In i t s p l a c e , a l o o s e l y packed f i n e f i b r i l l a r m a t e r i a l remains i n the p i t connection's i n t e r i o r ( F i g . 80). Symplastic l a t e r a l and v e r t i c a l spread of the v e g e t a t i v e t h a l l u s then r e s u l t s i n the i n v a s i o n of c e l l s i n the three t i s s u e l a y e r s , l e a d i n g to t h e i r f u s i o n and the formation of a compound host c e l l ( F i g . 79). Examination of c e l l s adjacent to fungal sporangia showed that they have c o l l a p s e d and a c q u i r e d a very dense cytoplasm ( F i g . 48). T h e i r i n t e r n a l membranous o r g a n i z a t i o n i s d i s r u p t e d and the i n d i v i d u a l o r g a n e l l e s become unrecognizable. - 55 -DISCUSSION A. Taxonomic a f f i n i t y of the pathogen The nature of c e l l w a l l p o l y s a c c h a r i d e s i s an important c r i t e r i o n i n fungal c l a s s i f i c a t i o n (Aronson 1967, Garraway and Evans 1984). The p a r a s i t e ' s s p o r a n g i a l w a l l s s t a i n e d p o s i t i v e l y with C a l c o f l u o r White, Congo Fed, and Zinc C h l o r i o d i d e . The combination of these r e a c t i o n s i n d i c a t e the presence of c e l l u l o s e . The f l u o r e s c e n t b r i g h t e n e r C a l c o f l u o r White i s known to have an a f f i n i t y for b e t a - l i n k e d glucans (Maeda and I s h i d a 1967, Berth and Schnepf 1980, B e s l o p - H a r r i s o n and B e s l o p - H a r r i s o n 1980, Fulcher and Wong 1982) and has been used to l a b e l c e l l u l o s e (Berth and Schnepf 1980, C o l v i n and Witter 1983) . Since Oomycete c e l l w a l l s are composed of a mixture of c e l l u l o s e and glucans with mixed beta l i n k a g e s (Aronson et a l . 1967, B a r t n i c k i - G a r c i a 1968, Zevenhuisen and B a r t n i c k i - G a r c i a 1969), the p o s s i b i l i t y that the f l u o r e s c e n c e obtained with C a l c o f l u o r White may be due to glucans other than c e l l u l o s e cannot be excluded. However, two other cytochemical s t a i n i n g procedures for c e l l u l o s e i n v o l v i n g Congo Red and Zinc C h l o r i o d i d e a l s o gave p o s i t i v e s t a i n i n g r e a c t i o n s and s t r o n g l y suggest the presence of t h i s polymer. These r e s u l t s combined with the b i f l a g e l l a t e nature of the zoospores c l e a r l y e s t a b l i s h the oomycetous nature of the pathogen. - 56 -Two types of planonts are found i n the Oomycetes (Sparrow 1960): primary zoospores which are ovoid to p y r i f o r m and are b i f l a g e l l a t e with p o s t e r i o r l y d i r e c t e d but a n t e r i o r l y attached f l a g e l l a , and secondary zoospores which are reniform to elongate and are l a t e r a l l y b i f l a g e l l a t e . Only heterokont zoospores of the secondary type were observed i n the case of the Chondrus c r i s p u s pathogen. The monomorphic c h a r a c t e r i s t i c of the zoospore combined with the h o l o c a r p i c and e n d o b i o t i c nature of i t s t h a l l u s warrant i t s assignment to the order L a g e n i d i a l e s . Since only a s i n g l e e x i t tube i s produced and the sporogenic cytoplasm i s not emptied i n t o a v e s i c l e p r i o r to zoospore maturation, the organism can be assigned to P e t e r s e n i a  p o l l a g a s t e r (Petersen) Sparrow. Measurements of sporangia and zoospore type, s i z e , and f l a g e l l a t i o n a l l agree with the o r i g i n a l d e s c r i p t i o n given by Sparrow (1934). The genus P e t e r s e n i a was o r i g i n a l l y p l a c e d by Sparrow (1934) i n the O l p i d i o p s i d a c e a e . On the other hand, K a r l i n g (1981) t r e a t e d the genus i n the S i r o l p i d i a c e a e based on the s i m i l a r i t y of the t h a l l u s of P e t e r s e n i a spp. with SiEQlpidjum spp. and the fragmentation of the t h a l l u s . D l t r a s t r u c t u r a l evidence obtained from the present study shows that the t h a l l u s may indeed fragment and d i s a r t i c u l a t e i n t o smaller t h a l l i supports K a r l i n g ' s view. Johnson (1977) l i k e w i s e observed that the t h a l l u s of another P e t e r s e n i a s p e c i e s , £. i r r e g u l a r e . may separate i n t o s e v e r a l segments as i n other s i r o l p i d i a c e o u s genera. - 57 -P r i o r to i t s appearance i n the Chondrus c u l t u r e s i n Nova S c o t i a , P . p o l l a g a s t e r had been reported only from Ceramium  rubrum (Sparrow 1934, Johnson and Howard 1968, as P l e o t r a c h e l u s by Petersen 1905). Likewise, the c l o s e l y r e l a t e d s p e c i e s p . l o b a t a which may be c o n s p e c i f i c with p . p o l l a g a s t e r (Aleem 1953, A. A. Aleem, pe r s . comm.) has been observed only on a l g a l hosts i n the Ceramiaceae and Ehodomelaceae. The present i n v e s t i g a t i o n t h e r e f o r e c o n s t i t u t e s the f i r s t d e t a i l e d r e p o r t on a s e r i o u s f u n g a l e p i p h y t o t i c i n m a r i c u l t u r e d £. c r i s p u s . I t a l s o extends the known host range for the genus P e t e r s e n i a . Except for the study of Pueschel and van der Meer (1985) on p . palmariae. very l i t t l e i n f o r m a t i o n i s p r e s e n t l y a v a i l a b l e on the u l t r a s t r u c t u r e of P e t e r s e n i a s p e c i e s . Since p r e v i o u s r e p o r t s of p . p o l l a g a s t e r (Sparrow 1934, Johnson and Howard 1968) d i d not deal with i t s u l t r a s t r u c t u r e , a d e t a i l e d comparison of the Chondrus pathogen with the Ceramium symbionts i s not p o s s i b l e . B . C u l t u r i n g of the Pathogen P e t e r s e n i a p o l l a g a s t e r could not be grown away from the host with the v a r i o u s c u l t u r e media and i s o l a t i o n techniques employed i n the present i n v e s t i g a t i o n . Likewise, h e a t - k i l l e d u n i n f e c t e d t h a l l i were not c o l o n i z e d by the fungus, i n d i c a t i n g that the pathogen may have a s t r i c t requirement for a l i v i n g h o s t. 58 -C. O b s e r v a t i o n s on pathogenesis and f a c t o r s t h a t f a v o r the spread and development of the p a r a s i t e B esides m o i s t u r e , temperature i s probably the most important p h y s i c a l f a c t o r i n f l u e n c i n g the spread and development of z o o s p o r i c p l a n t pathogens (Dunniway 1983, T eakle 1983) . R e s u l t s of the r e g r e s s i o n a n a l y s i s p r o v i d e s e m p i r i c a l evidence i n support of t h i s g e n e r a l i z a t i o n . The l a r g e r e g r e s s i o n c o e f f i c i e n t (0.971) i n d i c a t e s a s t r o n g a s s o c i a t i o n between temperature and number of l e s i o n s ( i n f e c t i o n r a t e ) . T h i s o b s e r v a t i o n c o r r e l a t e s w e l l with the e x c l u s i v e occurrence of the e p i p h y t o t i c i n the l a t e summer to autumn p e r i o d when water temperature may exceed 15C (J.S. C r a i g i e , p e r s o n a l communication). I n f e c t i o n r a t e f e l l s h a r p l y to l e s s than h a l f the number of l e s i o n s per sampling l i n e at the s u p r a o p t i m a l temperature of 25C. Thus, under the growth c o n d i t i o n s used i n the p r e s e n t study, s u c c e s s f u l i n f e c t i o n of £ . c r i s p u s by £,. p o l l a g a s t e r occurs only w i t h i n a narrow temperature range. However, f u n g a l propagules a p p a r e n t l y remained v i a b l e even a f t e r s e v e r a l days at 10C as they c o u l d be induced to i n f e c t the host when c u l t u r e s were t r a n s f e r r e d to a p e r m i s s i v e temperature. These o b s e r v a t i o n s may be e x p l a i n e d by the e f f e c t of temperature on zoospore behavior r e p o r t e d i n other z o o s p o r i c s p e c i e s . Fry and Campbell (1966) found t h a t the cabbage root p a r a s i t e , Olpidium bjcassicae m u l t i p l i e d b e t t e r from 10 to 16C than at 22 or 27C. Zoospore m o t i l i t y was r e t a i n e d f o r a much longer p e r i o d at temperatures below 20C than above i t (Teakle - 59 -1962, 1 9 8 3 ) . In Phytophthora s p e c i e s , i n c r e a s i n g temperatures r e s u l t i n decreased m o t i l i t y of zoospores ( E i b e i r o 1978) . Maintenance of m o t i l i t y has an obvious advantage to the pathogen which must s u c c e s s f u l l y l o c a t e and i n f e c t a s u i t a b l e host i n order to complete i t s development (Held 1973) . That zoospores d i d not s u r v i v e the low temperature extreme of 5C can be deduced from the absence of l e s i o n s on the host even a f t e r i n o c u l a t e d c u l t u r e s were t r a n s f e r r e d to 20C. T h i s o b s e r v a t i o n r a i s e s the q u e s t i o n of how P.. p o l l a g a s t e r o v e r w i n t e r s . Water temperatures drop below 5C i n the commercial c u l t i v a t i o n tanks d u r i n g the w i n t e r . I t i s p o s s i b l e t h a t the p a r a s i t e forms t e m p e r a t u r e - r e s i s t a n t r e s t i n g s p o r e s , although t h i s has yet to be demonstrated. Sparrow (1934) observed s e v e r a l t h i c k - w a l l e d , s p h e r i c a l bodies which he thought might be r e s t i n g spores of t h i s s p e c i e s . B e s t i n g spores have l i k e w i s e been r e p o r t e d i n other P e t e r s e n i a s p e c i e s i n c l u d i n g £. i r r e g u l a r e (Constantineanu) Sparrow ( 1 9 3 4 ) , £ . u t r i c u l o b a M i l l e r ( 1 9 6 2 ) , £ . c a t e n o p h l y c t i d i s Sundaram ( 1 9 6 8 ) , and £. p a n i c i o l a Thirumalachar and Lacy ( 1 9 5 1 ) . I t i s a l s o p o s s i b l e t h a t £ . p o l l a g a s t e r can i n f e c t a l t e r n a t e hosts where i t may form r e s i s t a n t spores d u r i n g the c o l d e r months. Ceramium rubrum which i s the host i n areas where the p a r a s i t e was o r i g i n a l l y r e p o r t e d from (as P l e o t r a c h e l u s by Petersen i n 1905; Sparrow 1934) i s known to occur i n the v i c i n i t y of Acadian S e a p l a n t s ' c u l t u r e f a c i l i t i e s i n Nova S c o t i a . - 60 -Although frequency of i n f e c t i o n was not q u a n t i f i e d under c o n d i t i o n s of vigorous a e r a t i o n and very low crowding d e n s i t i e s , i t would seem that these f a c t o r s may have p o t e n t i a l 1 f o r disease c o n t r o l . Very few l e s i o n s were formed on the host under these growth c o n d i t i o n s . These i n h i b i t o r y parameters could be combined with sodium dodecyl s u l f a t e treatment which has been used to sharply reduce i n f e c t i o n r a t e under m a r i c u l t u r e c o n d i t i o n s ( C r a i g i e 1984). D. U n i n f e c t e d £. c r i s p u s : l i g h t and e l e c t r o n microscopy Results of l i g h t and e l e c t r o n microscopy of u n i n f e c t e d £. c r i s p u s g e n e r a l l y agree with those p r e v i o u s l y d e s c r i b e d by C o t t i e r (1971) and Gordon and McCandless (1973) except that measurements obtained i n the present study were g e n e r a l l y l a r g e r . I t i s d i f f i c u l t to make d i r e c t comparisons s i n c e these authors d i d not s p e c i f y t h e i r sample s i z e . However, the v a r i a t i o n from p r e v i o u s l y reported values may be because of the d i f f e r e n c e s i n the s t r a i n s examined. With T o l u i d i n e Blue 0 s t a i n i n g the most intense metachromatic s t a i n i n g occurs immediately o u t s i d e the w a l l surrounding s u b c o r t i c a l and medullary c e l l s . T h i s s t a i n i n g r e a c t i o n i s thought to be caused mainly by the presence of s u l f a t e d p o l y s a c c h a r i d e s (McCully 1966, McCully et a l . 1978). Using f l u o r e s c e n t a n t i b o d i e s prepared against s p e c i f i c carrageenans, Gordon and McCandless (1973) obtained s p e c i f i c b i n d i n g of the a n t i - k - 61 -carrageenan antibody to the areas corresponding to these metachromatic bands. The f i n e s t r u c t u r e of £. c r i s p u s resembles that of other g i g a r t i n a l e a n a l gae. Walls of c e l l s i n the c o r t i c a l , s u b c o r t i c a l , and medullary t i s s u e s have a l a y e r e d f i b r i l l a r appearance t y p i c a l of the Fhodophyceae (Duckett and Peel 1977) . The f i b r i l s are b e l i e v e d to be a p o l y s a c c h a r i d e framework embedded i n an amorphous matrix (Gordon and McCandless 1973) . As i n other red algae (Sheath 1977, Duckett and Peel 1977) , c h l o r o p l a s t s have s i n g l e - t h y l a k o i d l a m e l l a e with at l e a s t one e n c i r c l i n g t h y l a k o i d . The storage p o l y s a c c h a r i d e , f l o r i d e a n s t a r c h , i s de p o s i t e d i n the cytoplasm rather than w i t h i n the p l a s t i d . P i t connections occur between adjacent c e l l s and c o n s i s t of lens-shaped plugs which f i t ' i n t o the w a l l a p e r t u r e . As i n other g i g a r t i n a l e a n algae, the p i t plug l a c k s the cap found i n many s p e c i e s (Pueschel and Cole 1982). Plasmalemmavilli are f i n g e r - l i k e e v a g i n a t i o n s of the plasma membrane that may extend i n t o the immdediate w a l l . They have been p r e v i o u s l y r e p o r t e d i n £. c r i s p u s ( C o t t i e r 1971, Gordon and McCandless 1973) and i n other red algae, e.g. Nemalion sp. (Duckett and Peel 1978). They were a l s o observed i n the present i n v e s t i g a t i o n , although they were much s h o r t e r than those observed by Gordon and McCandless (1973) . The nature and r o l e of these s t r u c t u r e s are u n c e r t a i n . Pueschel (1980b) obtained evidence that at l e a s t i n Palmaria palmata - 62 -they may be a r t i f a c t u a l because they were absent i n w e l l - f i x e d specimens. E. Development of Petersenia p o l l a g a s t e r 1. P o s t m o t i l e stage, encystrnent, and v e g e t a t i v e phase In the Mastigoraycotina, encystrnent of zoospores i s accompanied by c e l l u l a r changes i n c l u d i n g l o s s of m o t i l i t y , settlement, disappearance of f l a g e l l a , and rounding o f f of the c e l l (Waterhouse 1962, Hickman 1970, Held 1973, B a r t n i c k i - G a r c i a and Heromes 1974). In P e t e r s e n i a p o l l a g a s t e r f l a g e l l a r l o s s i s accomplished by r e t r a c t i o n of the axoneme. Such f l a g e l l a r r e s o r p t i o n has been observed i n s e v e r a l oomycetous s p e c i e s (Phytophthora i n f e s t a n s : E i s n e r et a l . 1970, Phytophthora palmivoga: R e i c h l e 1969, fl. m i l f o r d e n s i s : Overton et a l . 1983; P e t e r s e n i a palmariae; Pueschel and van der Meer 1985) although i n t h i s group f l a g e l l a r l o s s i s q u i t e v a r i a b l e . In Aphanomyces euteiches (Hoch and M i t c h e l l 1972) and Phytophthora cinnamomi (Hardham 1985) the f l a g e l l a are detached i n s t e a d of being resorbed. Both mechanisms of f l a g e l l a r disappearance have been observed i n the same p o p u l a t i o n of Phytophthora palmivora ( P e i c h l e 1969). R e t r a c t i o n , detachment, and d i s i n t e g r a t i o n have been proposed to account for f l a g e l l a r l o s s i n the Oomycetes (Kole 1965, Colhoun 1966). F l a g e l l a r - 63 -r e s o r p t i o n has a l s o been observed i n Olpidium b r a s s i c a e (Temmink and Campbell 1969) and appears to be the norm i n u n i f l a g e l l a t e s (Bracker 1967, Koch 1968, Beld 1973). Sing and B a r t n i c k i - G a r c i a (1975) concluded that encystrnent i s f u n c t i o n a l l y s i m i l a r i n b i f l a g e l l a t e and u n i f l a g e l l a t e s p e c i e s . R e t r a c t i o n of f l a g e l l a a l s o occurs i n P e t e r s e n i a EalmaJLiaie, a s p e c i e s r e l a t e d to Petersenia p o l l a g a s t e r . In the l a t t e r , zoospores have been observed to encyst even before they e x i t from the sporangium (Pueschel and van der Meer 1985). F l a g e l l a r r e s o r p t i o n i n p. p o l l a g a s t e r and other z o o s p o r i c s p e c i e s may be a means of r e c y c l i n g m i crotubule and a s s o c i a t e d p r o t e i n s . S t i l l u n c e r t a i n i s the mode of f l a g e l l a r d e g r a d a t i o n . I t i s remarkable that b a s a l bodies of both f l a g e l l a p e r s i s t and remain a s s o c i a t e d with the nucleus even a f t e r degradation of the axonemes. I t may be that these b a s a l bodies can f u n c t i o n as c e n t r i o l e s as has been suggested f o r the r e l a t e d l a g e n i d i o i d s p e c i e s , fl. m i l f o r d e n s i s (Overton et a l . 1983). Except f o r the absence of dense bodies and a d i s t i n c t G o l g i , the complement of i n c l u s i o n s and o r g a n e l l e s i n the p o s t - m o t i l e spore of p. p o l l a g a s t e r i s very s i m i l a r to the s p o r a n g i a l cytoplasm. Prominent f e a t u r e s i n c l u d e the nucleus, which has a much smaller n u c l e o l u s than the v e g e t a t i v e phase, and a s i n g l e l i p i d g l o b u l e that i s very c l o s e l y appressed to at l e a s t one mitochondrion. Presumably, the l i p i d body i s a - 64 -reserve product u t i l i z e d i n zoospore m o t i l i t y and encystment. The adhesiveness of zoospores only becomes evident during e a r l y encystment and i s of short d u r a t i o n ( B a r t n i c k i - G a r c i a and Hemmes 1974, R i b e i r o 1978). The p o s t m o t i l e spore of P.. p o l l a g a s t e r apparently a c q u i r e s adhesiveness during i t s settlement on the c u t i c l e of £. c r i s p u s . Although the h o s t - p a r a s i t e i n t e r f a c e at t h i s stage does not c l e a r l y show a cementing s t r u c t u r e such as the electron-dense m a t e r i a l which glues Phytophthora palmivora zoospores to a s o l i d substratum (Sing 1974, Sing and B a r t n i c k i - G a r c i a 1975), the attachment of cy s t s must be c o n s i d e r a b l y f i r m as i t withstands the d i f f e r e n t p r e p a r a t i v e manipulations f o r e l e c t r o n microscopy. I t i s easy to account f o r spore adhesiveness i n s p e c i e s which s e c r e t e a w e l l - d e f i n e d adhesive m a t e r i a l (£. palmivora: B a r t n i c k i - G a r c i a and Hemmes 1974, Sing 1974, Sing and B a r t n i c k i - G a r c i a 1975; Laoenisma c o s c i n o d i s c i : Schnepf et a l . 1978c). B a r t n i c k i - G a r c i a and Hemmes (1974) found that i n P.. palmivora the cementing substance was r i c h i n mannose, glucose, and g a l a c t o s e . The fuzzy coat of £. p o l l a g a s t e r c y s t s may serve a s i m i l a r f u n c t i o n . Reports on the presence of a fuzzy or mucilaginous c e l l coat on c y s t s of z o o s p o r i c f u n g i are r a r e . Heath (1970) observed patches of amorphous m a t e r i a l on the su r f a c e s of secondary c y s t s of £. f erax. Likewise, Hegnauer and Hohl (1973) noted a f l u f f y coat of v a r i a b l e t h i c k n e s s on cyst w a l l s - 65 -of £. palmivora. That t h i s s u r f a c e coat i s e a s i l y removed by the specimen p r e p a r a t i o n steps for e l e c t r o n microscopy (Hegnauer and Bohl 1973) may e x p l a i n why i t has not been observed more of t e n i n z o o s p o r i c f u n g i . In £. p o l l a g a s t e r the contents of the small p e r i p h e r a l vacuoles i n the p o s t m o t i l e spore c l o s e l y resembles the s t r u c t u r e of the fuzzy coat on the cyst w a l l . Fusion of some of these vacuoles with the cyst plasmalemma suggests that the f i b r o u s contents are d e l i v e r e d to the c e l l s u r f a c e v i a an e x o c y t o t i c p r o c e s s . A s i m i l a r mechanism has been proposed for S a p r o l e g n i a £e_r_a_x. by Heath (1970). Overton et a l . (1983) noted that i n fl_. m i l f o r d e n s i s l a r g e f i b r o u s v e s i c l e s may o c c a s i o n a l l y fuse with the cyst plasma membrane and extrude t h e i r contents during cyst w a l l formation. S i m i l a r o b s e r v a t i o n s were p r e v i o u s l y reported i n another l a g e n i d i o i d s p e c i e s , fragenidigm c a l l i n e c t e s by Bland and Amerson (1973) and i n Phytophthora cinnamomi by Zentmyer (1980). The chemical composition of the cyst w a l l and the mechanism of host r e c o g n i t i o n remains unknown. I t may be that s p e c i f i c host s u r f a c e r e c e p t o r s are recognized by the p a r a s i t e zoospore as suggested by Held (1973) . The v e g e t a t i v e development of £. p o l l a g a s t e r e x h i b i t s a remarkable resemblance to other h o l o c a r p i c Oomycetes which have an i n t r a c e l l u l a r , e n d o b i o t i c stage ( E c t r o g e l l a perfor/ans: Kumar 1980a,b; Lagenisma c o s c i n o d i s c i ; Schnepf et a l . a , b ; Petersgnia palmariae: Pueschel and van der Meer 1985). L i k e P e t e r s e n i a  u t r i c u l o b a ( M i l l e r 1962), £. i r r e c u l a r e (Milanez and Do V a l - 66 -1969), and P.. palmariae (Pueschel and van der Meer 1985), i t s e a r l y developmental stage i s w a l l - l e s s . The p a r a s i t e t h a l l u s becomes c l o s e l y a s s o c i a t e d with the host c e l l by i n v a g i n a t i n g the host plasmalemma. A s i m i l a r c o n d i t i o n has been observed i n Aphelidium sp. (Schnepf et a l . 1971, Schnepf 1972) and Lagenisma c o s c i n o d i s c i (Schnepf et a l . 1978b) where the host plasmalemma completely surrounds the e n d o b i o t i c p r o t o p l a s t . I n v a g i n a t i o n of the host plasma membrane has a l s o been noted i n the c h y t r i d Olpidium b r a s s i c a e (Temmink and Campbell 1969) and the plasmodiophoromycete PlasmodiOPhora b r a s s i c a e (Williams and McNabola 1970). However, u n l i k e Aphelidium sp. and £. b r a s s i c a e f compound plasmalemmata are not formed i n the case of the P e t e r s e n i a p o l l a g a s t e r i n f e c t i o n . Father, there i s a prolonged a s s o c i a t i o n between the host and p a r a s i t e plasma membranes. In t h i s r e s p e c t , P.. p o l l a g a s t e r resembles the p r o t o p l a s t of L> c o s c i n o d i s c i which i n f e c t s c e l l s of Coscinodiscus sp. (schnepf et a i . 1978b), and E c t r o g e l l a p e r f o r a n s f a p a r a s i t e of the marine diatom, Licmophora h y a l i n a (Kumar 1980b). However, i n £. perforans the u n i n u c l e a t e fungal t h a l l u s becomes surrounded by four to s i x electron-dense membranes of which the innermost i s the fungal plasmalemma and the outermost i s the in v a g i n a t e d host plasma membrane (Kumar 1980b). As suggested by Schnepf et a l . (1971), having the host plasmalemma around i t may enable the p a r a s i t e to a l t e r the p e r m a b i l i t y of the host and determine the flow of n u t r i e n t s . In n u c l e i of the v e g e t a t i v e phase of £. p o l l a g a s t e r . much of the nucleoplasm i s occupied by a l a r g e n u c l e o l u s . I t s s i z e and - 67 -the p r o l i f e r a t i o n of G o l g i around the nucleus i n d i c a t e intense p r o t e i n s y n t h e t i c a c t i v i t y which must accompany the growth and s y m p l a s t i c spread of the p a r a s i t e t h a l l u s . Studies of h o s t - p a r a s i t e i n t e r a c t i o n s have h e r e t o f o r e d e a l t mainly with t e r r e s t r i a l systems and the r e l a t i o n s h i p of the fungal haustorium with the host c e l l (Bracker and L i t t l e f i e l d 1973, A i s t 1976). The h o s t - p a r a s i t e i n t e r f a c e of P . p o l l a g a s t e r and £. c r i s p u s d i f f e r s markedly from that of h a u s t o r i a l p a r a s i t e s i n the absence of h a u s t o r i a and p a p i l l a e i n the host. Instead of forming h a u s t o r i a for a b s o r p t i o n of n u t r i e n t s , the naked p a r a s i t e t h a l l u s becomes h i g h l y lobed and d i s s e c t e d . T h i s e f f e c t i v e l y i n c r e a s e s the c e l l s u rface area and probably enhances n u t r i e n t procurement. The c l o s e a p p o s i t i o n of host and p a r a s i t e membranes may a l s o have a f u n c t i o n a l s i g n i f i c a n c e : i t may enable the p a r a s i t e to a l t e r the host p e r m e a b i l i t y to i t s advantage (Schnepf et a l . 1971, Kumar 1980b) . The s y m p l a s t i c spread of p. p o l l a g a s t e r i s reminiscent of P . palmariae which b r i n g s about c e l l f u s i o n i n Palmaria m o l l i s through the d i s s o l u t i o n of p i t plugs (Pueschel and van der Meer 1985). However, p e n e t r a t i o n of adjacent c e l l s by these two s p e c i e s may d i f f e r c o n s i d e r a b l y i n that the two-layered plug cap present i n Palmaria but not i n Chondrus i s an extra b a r r i e r that has to be d i g e s t e d . Although s p e c i f i c h ydrolases have not been demonstrated e i t h e r i n p. p o l l a g a s t e r or p. pa,lma.ria,g, the - 68 -d i s r u p t i o n and l o s s of e l e c t r o n d e n s i t y of the p i t plugs' contents would seem to i n d i c a t e that d i s s o l u t i o n was indeed t a k i n g p l a c e . I t may be that h y d r o l y t i c enzymes d i r e c t e d against compounds making up the plug are exocytosed by the p a r a s i t e p r o t o p l a s t s during i n v a s i o n of adjacent host c e l l s . Proteases are most l i k e l y i n v o l v e d s i n c e rhodophycean p i t plugs are l a r g e l y proteinaceous (Pueschel 1980a). A s i m i l a r s y m p l a s t i c spread and development was observed i n Pythium i n f e c t i o n s of Fucus d i s t i c h u s (Thompson 1981) where the fungus p a r a s i t i z e s s e v e r a l neighboring host c e l l s " v i a d i g e s t i o n of the p i t connection" (Thompson 1981), although plasmodesmata may be the more l i k e l y avenue for such an i n t e r n a l p r o l i f e r a t i o n . The m i g r a t i o n and aggregation of mitochondria i n t o c y toplasmic extensions has not been reported i n the Oomycetes and seems to be unique to £ . p o l l a g a s t e r . Such l o c a l i z e d c l u s t e r i n g of mitochondria has been observed i n the developing germ tube t i p of Neurospora c r a s s a Shear et Dodge by T u r i a n and G e i s s l e r (1984). They p o s t u l a t e d that mitochondria are i n v o l v e d i n the d i r e c t i o n a l d e l i v e r y of protons i n t o the germ Tube which a c t s as an a c i d i c sink a t t r a c t i n g a p i c a l v e s i c l e s . The aggregation of mitochondria of £. p o l l a g a s t e r p r i o r to i n v a s i o n of adjacent host c e l l s may serve a s i m i l a r f u n c t i o n . - 69 -2. Zoosporogenesis Host c e l l death appears to t r i g g e r sporangium formation i n P e t e r s e n i a p o l l a g a s t e r . P o s s i b l y , the demise of the a l g a l c e l l s prevents f u r t h e r t r a n s f e r of n u t r i e n t s from the host to the p a r a s i t e . S i m i l a r o b s e r v a t i o n s have been made i n both u n i c e l l u l a r (Coscjnodiscus sp.: Schnepf et a l . 1978a,c; Licmophora sp.: Kumar 1980) and m u l t i c e l l u l a r (Palmaria m o l l i s : Pueschel and van der Meer 1985) h o s t s . In P.. p o l l a g a s t e r . the amorphous s p o r a n g i a l w a l l i s continuous and completely surrounds the cytoplasm. In c o n t r a s t , the developing f i b r i l l a r w a l l of the r e l a t e d s p e c i e s , £. palmariae (Pueschel and van der Meer 1985) i s penetrated by membranous t u b u l e s , suggesting that i n t h i s s p e c i e s the sporangium may not be i s o l a t e d from the host c e l l u n t i l l a t e r i n i t s development. During e a r l y sporangium formation, a c e n t r a l vacuole i s formed and i t decreases i n s i z e as development proceeds. T h i s change i n the c e n t r a l vacuole i s d i s t i n c t from those Oomycetes which undergo zoosporogenesis using the expanding c e n t r a l vacuole p a t t e r n (e.g. S a p r o l e g n i a : Gay and Greenwood 1966, Gay et a l . 1971). Formation of a l a r g e c e n t r a l vacuole p r i o r to the cleavage phase has a l s o been reported i n L. c o s c i n o d i s c i (Schnepf et a l . 1978c). The presence of membrane p r o f i l e s and v a r i o u s types of i n c l u s i o n s i n the c e n t r a l vacuole of £. p o l l a g a s t e r suggests that i t may have a lysosomal f u n c t i o n . Presumably, breakdown of macromolecules i n s i d e the vacuole - 70 -re l e a s e s b u i l d i n g - b l o c k subunits which are then u t i l i z e d f o r s y n t h e t i c processes i n the cytoplasm. The v a r i o u s e l e c t r o n -dense i n c l u s i o n s may have an important r o l e i n sporogenesis; some are apparently m o b i l i z e d during s p o r a n g i a l development while others become packaged with the zoospores. Kumar (1980b) has suggested that the accumulation of l i p i d i n the sporangium as the host c e l l d i e s represents an e n e r g y - s t o r i n g a c t i v i t y which precedes zoosporogenesis. The s t o r e d . l i p i d might serve as a source of s t r u c t u r a l components or energy. The dense bodies may be u t i l i z e d during zoosporogenesis and for zoospore m o t i l i t y . T h i s can be deduced from the presence of these i n c l u s i o n s i n the cytoplasm and i n the c e n t r a l vacuole of the pre-cleavage sporangium and t h e i r absence i n p o s t - m o t i l e spores and c y s t s . The p r o l i f e r a t i o n of mi c r o b o d y - l i k e s t r u c t u r e s during zoosporogenesis a l s o suggests the p o s s i b l e u t i l i z a t i o n of l i p i d s . M o b i l i z a t i o n of electron-dense products during zoosporogenesis has a l s o been suggested f o r Phytophthora c a p s i c i by W i l l i a m s and Webster (1970) and for Sap r o l e o n i a by Gay et a l . (1971) . D e l i m i t a t i o n of zoospore i n i t i a l s i n many Oomycetes i s acccoroplished by the coalescence of v e s i c l e s that are "presumably d e r i v e d from the G o l g i (£. palmivora: Hohl and Hamamoto 1967, £. i n f e s t a n s ; E i s n e r et a l . 1970 , Lagenidiuin: Bland and Amerson 1973, G o t e l l i 1974, Pythium p r o l i f e r u m : Lunney and Bland 1976, Achlya b i s e x u a l i s ; P i c k e r 1971, Saprol e g n i a f e r a x : B a r t n i c k i - G a r c i a and Bemmes 1974). Fusion - 71 -of v e s i c l e s during zoosporogenesis has a l s o been reported i n some chytridiomycetous s p e c i e s (Travland and Whisler 1971, Olson et a l . 1981). A l t e r n a t i v e l y , cleavage of the sporogenic cytoplasm may be accompished by a c e n t r a l vacuole which expands g r a d u a l l y between the u n i n u c l e a t e blocks of protoplasm and e v e n t u a l l y fuses with the plasma membrane (e.g. S a p r o l e a n i a : Gay and Greenwood 1966, Gay et a l . 1971; Aphanomyces e u t e i c h e s : Boch and M i t c h e l l 1972) . Zoosporogenesis i n P e t e r s e n i a  p o l l a g a s t e r d i f f e r s markedly from most other Oomycetes i n that the protoplasm i s cleaved i n t o zoospore i n i t i a l s by the f u s i o n of G o l g i - d e r i v e d c i s t e r n a e and small v a c u o l e s . The p a r t i c i p a t i o n of G o l g i c i s t e r n a e i n cleavage has been observed i n two other s p e c i e s i n the L a g e n i d i a l e s which have been examined on the u l t r a s t r u c t u r a l l e v e l : L. c o s c i n o d i s c i (Schnepf et a l . 1978c) and P.. palmar iae (Pueschel and van der Meer 1985). The taxonomic and p h y l o g e n e t i c u t i l i t y of t h i s c h a r a c t e r must await f u r t h e r examination and comparison of other l a g e n i d i o i d Oomycetes. F l a g e l l a r development in £. p o l l a g a s t e r d i f f e r s from other Oomycetes i n the timing of axoneme formation. Whereas i n many spe c i e s (e.g. of Phytophthora: Bohl and Bamamoto 1967, W i l l i a m s and Webster 1970, Laoenidium c a l l i n e c t e s : Bland and Amerson 1973, Pythium sp.: Lunney and Bland 1976) the development of f l a g e l l a i s c l o s e l y c o o r d i n a t e d with p r o t o p l a s m i c cleavage, i n P. p o l l a g a s t e r f u s i o n of cleavage c i s t e r n a e occurs even before the d i s t a l extension of basal bodies i n t o axonemes. The - 72 -formation of cleavage membranes before axonemal extension has a l s o been reported i n B l a s t u l i d i u r o poedophthorum (Manier 1976) and L . c o s c i n o d i s c i (Schnepf et a l . 1978c). In c o n t r a s t , the f u s i o n of cleavage c i s t e r n a e , s y n t h e s i s of roastigonemes, and formation of axonemes occur almost simultaneously i n £. palmariae (Pueschel and van der Meer 1985). In both £. p o l l a g a s t e r and £. palmariae. mastigonemes are s y n t h e s i z e d i n d i l a t e d RER c i s t e r n a e which are c l o s e l y appressed to a mitochondrion. The c l o s e a s s o c i a t i o n of mitochondria with mastigoneme-containing ER c i s t e r n a e has a l s o been observed i n other Oomycetes (Laoenidium: Bland and Amerson 1973, G o t e l l i 1974; Pythium p r o l i f e r u m : Lunney and Bland 1976). The mitochondria i n £. p o l l a g a s t e r . as i n most Oomycetes (Bracker 1967) , c o n t a i n t u b u l a r c r i s t a e throughout the l i f e c y c l e . That f l a g e l l a are formed by the d i s t a l e x tension of b a s a l body microtubules i n t o an axonemal v e s i c l e i n £. p o l l a g a s t e r could be deduced by the presence of double membranes around the axonemes. F l a g e l l a r formation i n v o l v i n g axonemal v e s i c l e s has a l s o been reported i n other oomycetous s p e c i e s such as Achlya  b i s e x u a l i s (Ricker 1971) and Aphanomycgg eute iches (Hoch and M i t c h e l l 1972) and i n the c h y t r i d Allomyces catenoides (Olson et a l . 1981). In c o n t r a s t , b a s a l bodies elongate i n t o f l a g e l l a without the p a r t i c i p a t i o n of a f l a g e l l a r v e s i c l e i n 1. c o s c i n o d i s c i (Schnepf et a l . 1978c). - 73 -During l a t e zoosporogenesis, when the f l a g e l l a are already f u l l y formed, the n u c l e i of p . p o l l a g a s t e r assume a p y r i f o r m shape. T h i s change i n nuclear morphology appears to be widespread i n the b i f l a g e l l a t e f u n g i and has been reported i n widely d i s p a r a t e taxa (e.g. Achlya b i s e x u a l i s ; Ricker 1971, H a l i p h t h o r o s m i l f o r d e n s i s ; Overton et a l . 1983). As a r e s u l t of zoosporogenesis, the zoospores must come to c o n t a i n a l l the requirements for s u r v i v a l . Just e x a c t l y how each planont i s assured a f u l l complement of o r g a n e l l e s i s s t i l l u n c l e a r . In S a p r o l e a n i a (Heath and Greenwood 1971) i t has been p o s t u l a t e d that cytoplasmic microtubules which are formed around the nucleus of each spore i n i t i a l may guide the movement of cleavage v e s i c l e s through r e l a t i v e l y "weaker" areas of cytoplasm. The mechanism for the d e l i m i t a t i o n of zoospore i n i t i a l s i n p . p o l l a g a s t e r i s more d i f f i c u l t to e x p l a i n owing to the apparent absence of microtubule a r r a y s around the n u c l e i . I t may be that these microtubules were not preserved by the f i x a t i o n method employed i n the present i n v e s t i g a t i o n . Such m i c r o t u b u l e s , i f present, could c o n c e i v a b l y d e l i m i t c y t o p l a s m i c domains where cleavage c i s t e r n a e cannot t r a v e r s e and hence may e s t a b l i s h the boundaries of zoospore i n i t i a l s . Although s a t i s f a c t o r y p r e s e r v a t i o n of r e l e a s e d p . p o l l a g a s t e r zoospores was not obtained, s e v e r a l c o n c l u s i o n s can be made regarding t h e i r nature. Some of the s e c t i o n e d sporangia showed moribund zoospores, i n d i c a t i n g that they are - 74 -f u l l y formed i n the sporangium. The s i m i l a r i t y of t h e i r s t r u c t u r e with that of the sporogenic cytoplasm and the dimensions of the o r g a n e l l e s s t r o n g l y suggest that only one fungal s p e c i e s was i n v o l v e d and e l i m i n a t e s the p o s s i b i l i t y that the f l a g e l l a t e c e l l s observed were j u s t contaminants. E s p e c i a l l y prominent were the crenate v e s i c l e i n c l u s i o n s which were present i n both the sporogenic cytoplasm and the zoospores. F. E f f e c t s of i n f e c t i o n on the host 1. Fluorescence induced by DAPI and s w e l l i n g of host c e l l s DAPI proved to be the most u s e f u l of a l l the t e s t e d dyes for demonstrating the e f f e c t s of fungal i n f e c t i o n on the host a l g a . R e s u l t s of f l u o r e s c e n c e v i t a l s t a i n i n g with t h i s fluorochrome show that host c e l l death i s a s s o c i a t e d with the i n f e c t i o n . S i m i l a r r e s u l t s have been obtained i n animal systems by van der Linden and Deelder (1984) who employed the dye as a probe fo r i n v i t r o death of schistosomula. DAPI was a l s o found to enter r a p i d l y i n t o dead lymphocytes and bind with DNA (Tanke et a l . 1982). The longer time r e q u i r e d f o r t h i s fluorochrome to penetrate dead or moribund c e l l s of £. c r i s p u s may be caused by the presence of c e l l w a l l s and abundant i n t e r c e l l u l a r matrix which i t must i n i t i a l l y t r a v e r s e . - 75 -V i t a l s t a i n i n g techniques such as the one used i n the present i n v e s t i g a t i o n are based on membrane p e r m e a b i l i t y changes (Bowen et a l . 1985). The entry of DAPI i n t o dead c e l l s i s s a i d to be dependent on damage of the plasmalemma (Tanke et a l . 1982). However, the q u e s t i o n a r i s e s whether a l t e r e d host c e l l p e r m e a b i l i t y r e s u l t s from the i n f e c t i o n i t s e l f or the fluorochrome treatment. The absence of f l u o r e s c e n c e i n u n i n f e c t e d c o n t r o l s which were l i k e w i s e incubated i n DAPI e s s e n t i a l l y e l i m i n a t e s the second p o s s i b i l i t y . The c o n c e n t r a t i o n of DAPI used i n the present study (1.0 pg/ml-l) has a l s o been shown to have no t o x i c e f f e c t e i t h e r on i n d i v i d u a l lymphocytes (Tanke et a l . 1982) or on schistosomula (van der Linden and Deelder 1984) even a f t e r 48 h i n c u b a t i o n . T h i s fluorochroming technique should t h e r e f o r e be a p p l i c a b l e i n the study of other h o s t - p a r a s i t e i n t e r a c t i o n s , e s p e c i a l l y for the demonstration of c e l l death i n the host. Morphometric a n a l y s i s of c r o s s - s e c t i o n a l areas of u n i n f e c t e d and di s e a s e d host c e l l s r evealed a general s w e l l i n g of s u b c o r t i c a l c e l l s e a r l y i n the i n f e c t i o n p r o c e s s . D i r e c t l y penetrated c e l l s were s i g n i f i c a n t l y l a r g e r than the c o n t r o l s by 42% (p < 0.05). This s w e l l i n g of host c e l l s may i n d i c a t e accommodation of the growing p a r a s i t e p r o t o p l a s t by the penetrated c e l l s . I t could a l s o be an i n i t i a l stage i n induced c e l l death or n e c r o s i s (Bowen 1985). - 76 -2. Changes i n host u l t r a s t r u c t u r e and extent of host response The f i r s t r e c o g n i z a b l e change i n host u l t r a s t r u c t u r e occurs during the encystment of zoospores on the a l g a l t h a l l u s . A l o c a l i z e d d i l a t a t i o n i s formed i n one of the proximal interband regions beneath the p o i n t of encystment. T h i s d i l a t a t i o n may be caused by the d e p o s i t i o n of a granular electron-dense m a t e r i a l by the u n d e r l y i n g host c e l l s v i a e x o c y t o s i s . That s e c r e t o r y products may become l o c a l i z e d i n the l i g h t bands of the c u t i c l e a f t e r they are produced by c o r t i c a l c e l l s of £. c r i s p u s i s suggested by the s t u d i e s of Gordon and McCandless (1973) . They observed that the e l e c t r o n - d e n s e , granular m a t e r i a l i n the interband regions c l o s e l y resembled the contents of the s e c r e t o r y v e s i c l e s found i n c o r t i c a l c e l l s . The l o c a l i z e d d i l a t a t i o n i n the host c u t i c l e may be analogous to c e l l w a l l a p p o s i t i o n s which are f r e q u e n t l y formed i n p l a n t c e l l s during i n f e c t i o n by f u n g i , b a c t e r i a , and v i r u s e s (Akai et a l . 1971, Goodman et a l . 1977, Kimmins 1977, Sequeira et a l . 1977) as w e l l as i n m y c o p a r a s i t i c r e l a t i o n s h i p s (Hoch and F u l l e r 1977). However, the term "wall a p p o s i t i o n " (Bracker and L i t t l e f i e l d 1967) i s not a p p l i c a b l e i n the case of the c u t i c u l a r d i l a t a t i o n i n £. c r i s p u s s i n c e i t dees not represent an a d d i t i o n of m a t e r i a l to the inner s u r f a c e of the c e l l w a l l s . Whatever the nature of the m a t e r i a l found i n the l o c a l i z e d - 77 -d i l a t a t i o n , i t may p o t e n t i a l l y prevent p e n e t r a t i o n and may be part of a defense mechanism. A s i m i l a r f u n c t i o n i s g e n e r a l l y a s c r i b e d to v a r i o u s c e l l w all m o d i f i c a t i o n s (Misaghi 1 9 8 2 ) . Whereas p a r a s i t i c Oomycetes of the order Peronosporales t y p i c a l l y invade host c e l l s by means of h a u s t o r i a that remain surrounded by the c e l l w a l l of the p a r a s i t e (Kajiwara 1973, Shimony and F r i e n d 1975), the i n t r a c e l l u l a r t h a l l u s of £. p o l l a g a s t e r p r o l i f e r a t e s as a w a l l - l e s s p r o t o p l a s t . Other than the i n v a g i n a t e d plasmalemma, d i r e c t l y penetrated host c e l l s e x h i b i t few u l t r a s t r u c t u r a l changes. The l o s s i n f i b r i l l a r s t r u c t u r e of t h e i r w a l l s may be caused by the s e c r e t i o n of w a l l - l o o s e n i n g enzymes by the p a r a s i t e which would permit not only i t s own expansion but a l s o that of the penetrated host c e l l . The d i s s o l u t i o n of f l o r i d e a n s t a r c h granules i n i n f e c t e d £. c r i s p u s i s reminiscent of Pythium marinum i n f e c t i o n s of Porphyra p e r f o r a t a (Kazama and F u l l e r 1 9 7 0 ) . In t h i s a l g a , the d i s s o l u t i o n of s t a r c h g r a i n s leaves a r e t i c u l a t e remnant at s i t e s which they formerly occupied. Kazama and F u l l e r (1970) suggested that the p a r a s i t e p a r t i c i p a t e s i n the d i s s o l u t i o n of s t a r c h g r a i n s through the pr o d u c t i o n of e x t r a c e l l u l a r h y d r o l y t i c enzymes. However, i n the case of the Chondrus pathogen, the pr o d u c t i o n of exoenzymes and t h e i r subsequent h y d r o l y s i s of the f l o r i d e a n s t a r c h reserves i s q u e s t i o n a b l e s i n c e i n t h i s pathosystem, the i n v a g i n a t e d host membrane may - 78 -prevent d i r e c t access of fungal exoenzymes to the s u b s t r a t e . I t may be that d i s s o l u t i o n of s t a r c h granules i n i n f e c t e d c e l l s of £. c r i s p u s represents the m o b i l i z a t i o n of endogenous r e s e r v e s . The p r o t o p l a s t of p . p o l l a g a s t e r invades adjacent c e l l s v i a a s y m p l a s t i c route. Although cytochemical t e s t s were not performed to e s t a b l i s h the presence of h y d r o l a s e s , d i s s o l u t i o n of p i t plugs r e s u l t i n g i n the formation of a c o n f l u e n t host c e l l i s a l i k e l y mechanism that allows the spread and f u r t h e r development of the p a r a s i t e . The spread of the pathogen resembles that of p . palmariae, which i n i t i a l l y i n f e c t s c o r t i c a l c e l l s of p . moll i s and develops i n t r a c e l l u l a r l y as a naked p r o t o p l a s t (Pueschel and van der Meer 1985). A s i m i l a r route of development has been d e s c r i b e d i n p . undulatum i n f e c t i o n s of £. d i s t i c h u s by Thompson (1981). However, h i s micrographs i n d i c a t e that the filamentous hyphae may i n i t i a l l y be paramural, a s i t u a t i o n that has not been observed i n P e t e r s e n * ? s p e c i e s . In the Petersenia-Chondrus pathosystem, there i s a r e l a t i v e l y prolonged p e r i o d when d i s r u p t i o n of host c e l l o r g a n e l l e s i s minimal. In c o n t r a s t , the d e s t r u c t i o n of o r g a n e l l e s i n Porphyra p e r f o r a t a during i t s e a r l y p e n e t r a t i o n by the hypha of Pythium marinum i s almost immediate (Kazama and F u l l e r 1970). S i m i l a r l y , the cytoplasm of Fucus d i s t i c h u s c e l l s soon c o l l a p s e s a f t e r they are penetrated by hyphae of - 79 -P v t h i u m undulatum (Thompson 1981). D i r e c t l y p e n e t r a t e d c e l l s of Chondrus c r i s p u s a l s o do not appear to r e a c t t o the i n f e c t i o n by p r o d u c i n g a sheath or w a l l m a t e r i a l around the fungus as i s f r e q u e n t l y the case w i t h h a u s t o r i a l p a r a s i t e s ( B r a c k e r 1967). Encasement of the h a u s t o r i u m i n an amorphous, e l e c t r o n - o p a q u e sheath i s thought t o be r e s p o n s i b l e f o r a b a l a n c e d , a l b e i t temporary, h o s t - p a r a s i t e r e l a t i o n s h i p (Bracker 1967) . Rapid d e s t r u c t i o n of i n f e c t e d c e l l s i n Porphyra  p e r f o r a t a has been a t t r i b u t e d t o the i n a b i l i t y of the h o s t - p a r a s i t e complex t o form a s h e a t h i n g membrane (Kazama and F u l l e r 1 9 7 0). However, my r e s u l t s and those o b t a i n e d by Kumar (1980a) f o r the E c t r o o e l l a p e r f o r a n s - L i c m o p h o r a hya l i na system argue a g a i n s t the need f o r such a sheath i n b a l a n c e d p a r a s i t i s m . M a l l a p p o s i t i o n s and sheaths are a l s o absent i n i n f e c t i o n s caused by O l p i d i u m b r a s s i c a e (Temmink and Campbell 1969) and Plasmodiophora b r a s s i c a e ( W i l l i a m s and Yukawa 1967) and can be i n t e r p r e t e d t o mean t h a t the host i s c o m p l e t e l y s u s c e p t i b l e (Kumar 1980a). M i n i m a l i n i t i a l d i s r u p t i o n of host c e l l s might a l s o i n d i c a t e t h a t £. p o l l a g a s t e r and o t h e r s p e c i e s not c a u s i n g severe damage t o the host d u r i n g the i n i t i a l s t a g e s of i n f e c t i o n are o b l i g a t e l y p a r a s i t i c and r e q u i r e a l i v i n g , u n d i s t u r b e d host (Held 1973, Kumar 1980a). What i n i t i a l l y appears t o be b a l a n c e d p a r a s i t i s m of Chondrus c r i s p u s by £. p o l l a g a s t e r i s e v i d e n t l y s h o r t - l i v e d as host c e l l s d i e a p p r o x i m a t e l y 24 h from the time of i n o c u l a t i o n . At t h i s s tage the i n v a g i n a t e d host plasmalemma s u r r o u n d i n g the - 80 -v e g e t a t i v e p a r a s i t e t h a l l u s d i s i n t e g r a t e s and i n d i v i d u a l host c e l l o r g a n e l l e s become i n d i s t i n g u i s h a b l e . S i m i l a r o b s e r v a t i o n s have been made i n E c t r o g e l l a p erforans i n f e c t i o n s of Licroophora  h y a l i n a by Kumar (1980a). She found that the seemingly d e l i c a t e balance maintained between the host and p a r a s i t e ended with the breakdown of the host plasma membrane and i r r e v e r s i b l e changes l e a d i n g to c e l l death. Likewise, Schnepf et a l . (1978c) observed that p r i o r to death of the i n f e c t e d c e l l s of C o s c i n o d i s c u s sp., the host plasmalemma becomes d i f f i c u l t to preserve by f i x a t i o n which might w e l l i n d i c a t e i t s breakdown. H o s t - p a r a s i t e r e l a t i o n s h i p s i n v o l v i n g P e t e r s e n i a s p e c i e s d i f f e r s i g n i f i c a n t l y from i n t e r a c t i o n s of h o l o c a r p i c oomycetes with u n i c e l l u l a r hosts (Lagenisma c o s c i n o d i s c i ; Schnepf et a l . 1978a,b,c ; E c t r o a e l l a p e r f o r a n s ; Kumar 1980a,b,c). Although not d i r e c t l y penetrated, adjacent c e l l s i n m u l t i c e l l u l a r hosts may p a r t i c i p a t e i n the i n t e r a c t i o n by a l t e r i n g the p h y s i o l o g y of the i n f e c t e d c e l l s and/or the p a r a s i t e (Mansfield 1985). In P e t e r s e n i a i n f e c t i o n s of £. c r i s p u s f u n i n f e c t e d host c e l l s around the s i t e s of i n f e c t i o n d i e . On the u l t r a s t r u c t u r a l l e v e l , death of these host c e l l s i s i n d i c a t e d by the d i s r u p t i o n of t h e i r i n t e r n a l membranous o r g a n i z a t i o n such that i n d i v i d u a l o r g a n e l l e s become unrecognizable. Further evidence f o r t h e i r demise comes from p o s i t i v e fluorochroming with DAPI. The involvement of neighboring host c e l l s i n some t e r r e s t r i a l h o s t - p a r a s i t e i n t e r a c t i o n s i s t y p i f i e d by the - 81 -s o - c a l l e d h y p e r s e n s i t i v e response, c h a r a c t e r i z e d by the r a p i d death of c e l l s around p e n e t r a t i o n s i t e s . Stakman (1915) considered the major events of h y p e r s e n s i t i v i t y to be: p e n e t r a t i o n by the fungus, death of p l a n t c e l l s , and eventual death of the fungus. Although widespread i n t e r r e s t r i a l p l a n t systems, the h y p e r s e n s i t i v e response has been documented i n f r e q u e n t l y i n a q u a t i c systems. The occurrence of a h y p e r s e n s i t i v e response has been reported only once i n marine macroalgae: Thompson (1981) observed that £. d i s t i c h u s c e l l s i n advance of the invading hypha of Pythium sp. aut o l y z e d and that an a b s c i s s i o n zone i s formed around the p o i n t s of i n f e c t i o n . Although the h o s t - p a r a s i t e r e l a t i o n s h i p d e a l t with here i s mainly compatible, the demise of £. c r i s p u s c e l l s around those which are d i r e c t l y penetrated by the £. p o l l a g a s t e r t h a l l u s can be i n t e r p r e t e d as a form of h y p e r s e n s i t i v i t y . - 82 -GENERAL DISCUSSION AND SUMMARY The present i n v e s t i g a t i o n d e t a i l s the only known occurrence of P e t e r s e n i a p o l l a g a s t e r on a g i g a r t i n a l e a n alga and extends the host range f o r the genus P e t e r s e n i a . Laboratory o b s e r v a t i o n s of pathogenesis and f a c t o r s that enhance i t show why the disease i s so d e s t r u c t i v e to c u l t i v a t e d Chondrus c r i s p u s . The fungus invades the meristematic apices of the host t h a l l u s thereby p r e v e n t i n g i t s growth. R e l a t i v e l y warm temperatures (15-20C) favor the spread and development of the pathogen; t h i s c o r r e l a t e s w e l l with outbreaks of the disease i n the warm summer months. Studies of h c s t - p a r a s i t e i n t e r a c t i o n s must consider the changes i n the host-pathogen i n t e r f a c i a l zone (=interface) as development of the p a r a s i t e proceeds. Bracker and L i t t l e f i e l d (1973) c o n s i d e r e d the h o s t - p a r a s i t e i n t e r f a c i a l zone as a dynamic, three-dimensional region which c o n s i s t s not only of s t r u c t u r e s that are i n d i r e c t c o n t a c t , but a l s o those which are s p e c i f i c a l l y i n v o l v e d i n the i n t e r a c t i o n . These i n c l u d e a l l substances between the plasmalemma of the i n t e r a c t i n g p r o t o p l a s t s (Bracker and L i t t l e f i e l d 1973) . In the Petersenia-Chondrus pathosystem, the p r o g r e s s i o n of developmental stages i n the p a r a s i t e i s accompanied by changes i n the nature of the h o s t - p a r a s i t e i n t e r f a c i a l zone. These changes are diagrammed i n F i g . 81 using a m o d i f i c a t i o n of the - 83 -scheme o r i g i n a l l y developed by Bracker and L i t t l e f i e l d (1973) . The i n i t i a l s i t e of h o s t - p a r a s i t e i n t e r a c t i o n i s commonly at or on the host s u r f a c e (Dickinson and Lucas 1982). The f i r s t i n t e r f a c i a l zone i n v o l v i n g £. p o l l a g a s t e r and £. c r i s p u s observed c o n s i s t e d of the newly s e t t l e d , p o s t m o t i l e spore on the c u t i c l e of the host a l g a . That the zoospore i s the i n f e c t i v e stage i s proven by experiments where i n o c u l a t i o n with zoospore suspensions produced disease symptoms i n the host. Evidence from e l e c t r o n microscopy r e v e a l that l i k e most z o o s p o r i c p l a n t pathogens (Held 1973), planonts of P.. p o l l a g a s t e r encyst on the host s u r f a c e p r i o r to i n f e c t i o n . Encystrnent i s accompanied by c e l l u l a r changes i n c l u d i n g rounding up of the c e l l and l o s s of zoospore m o t i l i t y by f l a g e l l a r r e t r a c t i o n . Apparently, there i s a very b r i e f p e r i o d when the p o s t - m o t i l e spore has not yet s y n t h e s i z e d a w a l l and there i s no i n t e r v e n i n g matrix between i t and the host. T h i s i s a t r a n s i t o r y i n t e r f a c e as a c y s t w a l l i s soon formed ( F i g . 81, IT1). A t h i n f l u f f y c e l l coat i s a l s o s y n t h e s i z e d and becomes i n t e r p o s e d between the p a r a s i t e ' s w a l l and the host c u t i c l e (IT2). L a t e r , there i s a d i l a t a t i o n of a proximal interband r e g i o n of the host c u t i c l e which may be due to the s e c r e t i o n and d e p o s i t i o n of an amorphous, e l e c t r o n - t r a n s p a r e n t m a t e r i a l ( I T 3 ) . T h i s s t r u c t u r e may be analogous to w a l l a p p o s i t i o n s commonly observed i n i n f e c t i o n s caused by h a u s t o r i a l p a r a s i t e s (Bracker 1967, Bracker and L i t t l e f i e l d 1973, Hoch and F u l l e r 1977). - 84 -The i n i t i a l e n d o b i o t i c stage of P.. p o l l a g a s t e r i s a p r o t o p l a s t . During p e n e t r a t i o n of the host c e l l , the w a l l l e s s p a r a s i t e i n v a g i n a t e s the host plasmalemma and p r o l i f e r a t e s v i a a s y m p l a s t i c route. As i n most o b l i g a t e l y p a r a s i t i c f u n g a l s p e c i e s , d i s r u p t i o n of host c e l l s i s minimal during e a r l y development (Beld 1973, Kumar 1980a) i n d i c a t i n g that the p a r a s i t e might have a s t r i c t requirement for a l i v i n g host. Further support f o r t h i s c o n c l u s i o n comes from i t s i n a b i l i t y to grow e i t h e r on a r t i f i c i a l media commonly used f o r the i s o l a t i o n of Phycomycetes or h e a t - k i l l e d host t h a l l i . The Chondrus pathogen induces the f u s i o n of host c e l l s through the removal of p i t p l u g s . The h o s t - p a r a s i t e i n t e r f a c e at t h i s stage i n c l u d e s the two c l o s e l y appressed plasmalemmata of of the w a l l - l e s s endosymbiont and the host separated by a very narrow matrix (IT4). D i s s o l u t i o n of f l o r i d e a n s t a r c h g r a i n s i s a s s o c i a t e d with the i n f e c t i o n and may i n d i c a t e p a r a s i t e - i n d u c e d m o b i l i z a t i o n of reserve m a t e r i a l . F l u o r e s c e n t v i t a l s t a i n i n g and u l t r a s t r u c t u r a l s t u d i e s r e v e a l e d that host c e l l death, c h a r a c t e r i z e d by d i s r u p t i o n of f i n e s t r u c t u r e and l e a k i n e s s of the plasmalemma, occurs during the l a t e r stages of i n f e c t i o n . T h i s i n d i c a t e s the p a r t i c i p a t i o n of adjacent host c e l l s i n the h o s t - p a r a s i t e i n t e r a c t i o n . Death of host c e l l s i n i n f e c t i o n s i t e s and the presence of moribund zoospores and c e l l u l a r d e b r i s i n some sporangia s t r o n g l y suggest h y p e r s e n s i t i v i t y . - 85 -As host c e l l s d i e and zoosporogenesis commences, the i n v a g i n a t e d host membrane d i s i n t e g r a t e s and a w a l l i s l a i d down between the p a r a s i t e and the remaining n e c r o t i c host cytoplasm. The s p o r a n g i a l w a l l of the p a r a s i t e then comes i n con t a c t with the dead host c e l l cytoplasm (IT5) . Upon completion of \ zoosporogenesis the planonts are r e l e a s e d f o r another round of i n f e c t i o n . The mechanism of host l o c a t i o n and r e c o g n i t i o n i n the Petersenia-Chondrus pathosystem remains to be e l u c i d a t e d . I t may be that chemotaxis and c e l l s u r f a c e r e c e p t o r s are i n v o l v e d , as i n other c e l l - c e l l i n t e r a c t i o n s (Keen 1982). C e l l s u r f a c e sugars which might be prese n t i n zoospores and c y s t s of P.. p o l l a g a s t e r are l i k e l y i n v o l v e d i n the i n t e r a c t i o n . I t would be i n t e r e s t i n g to e s t a b l i s h the presence of c e l l s u r f a c e r e c e p t o r s i n t h i s s p e c i e s with the use of l e c t i n l a b e l i n g . T h i s technique has been s u c c e s s f u l l y used i n the study of other z o o s p o r i c p l a n t pathogens (Jen and Haug 1979, B a c i c et a l . 1985, Hardham 1985). Such s t u d i e s may p r o v i d e c l u e s on the mechanism of host r e c o g n i t i o n . O l i g o s a c c h a r i n s , c e l l w a l l fragments t h a t serve as r e g u l a t o r y molecules, may w e l l t r i g g e r the e v e n t u a l death of host c e l l s at i n f e c t i o n s s i t e s (Albersheim and D a r v i l l 1985). Cytochemical and b i o c h e m i c a l s t u d i e s need to be made and c o r r e l a t e d with m o r p h o l o g i c a l o b s e r v a t i o n s f o r a complete understanding of t h i s h o s t - p a r a s i t e r e l a t i o n s h i p . - 86 -TABLE I : Bost range and d i s t r i b u t i o n of P e t e r s e n i a p o l l a g a s t e r and £. l f i & A l A Bost F a a i l y Country/Region Reference £. p o l l a g a s t e r Ceraaium rubrum Ceramiaceae Denmark Petersen (1905) Sparrow (1934) Johnson and Howard (1968) £. lo b a t a Antithamnion (?) C a l l i t h a m n i o n h o o h e i i £. roeeum C a l l i t h a m n i o n sp. Ceramium sp. £. g e d i c e l l a t u a £. c o r t i c a t u l u m Spermotfiamnion t u r n e r i S. repens var. t u r n e r ! 5. reaena S e l r o s B o r a I n t e r r u s t a fi. a p i c u l a t a £. g r i f f i t h e i a n a Ceramiaceae Denmark Denmark U. S. A. Mediter-ranean Sweden Ice l a n d Sweden M Denmark France Mediter-ranean France Petersen (as P l e o t r a c h e l u s . 1905) Petersen (1905) Sparrow (1936) Aleem (1950c) Aleem (1950b) Aleem (1953) Johnson and Howard (1968) Aleem (1953) Aleem (1953) Petersen (as P l e o t r a c h e l u s . 1905) Sparrow (1934) Feldmann (1954) Feldmann and Feldmann (1940) Feldmann and Feldmann (1940) (Feldmann 1954) Berposiphonia t e n e l l a P o l y s i p h o n i a u r c e o l a t a Bhodomelaceae Mediter-ranean " Sweden Aleem (1950c) Aleem (1953) - 37 -TABLE I I : S t a i n i n g techniques used for general h i s t o l o g i c a l and cytochemical studies Staining Method Compounds/ Components I d e n t i f i e d Color Reaction Reference(s) Acridine orange Sulfated poly-saccnarldes; n u c l e i c acids Fluorescence under UV i l l u m i n a t i o n Cooke 1982 Calcofluor white -1,3 and -1,4 glucans Bluish-white fluorescence Haeda and Ishida 1967, Haigler 1980, Beslcpp-Barrison and Beslcpp-Barrison 1980 , Berth and Schnepf 1980 Congo red -1,3 glucans Bright red fluorescence Wood 1980 Toluidine blue 0 (PH 4.4) Sulfated palysaccharides Carboxylated polysaccnandes Nuclei and cytoplasm Pink Red to blue Blue McCully 1970, ClarK 1981 Zinc c h l o r i o d i d e C e l l u l o s e Bluish-purple Stevens 1974 - 88 -TABLE I I I : Comparison of c r o s s - s e c t i o n a l areas of u n i n f e c t e d and i n f e c t e d c o r t i c a l c e l l s using the t - t e s t U n i n f e c t e d (u) I n f e c t e d ( i ) 94.5 + 14.5 pm2 135.0 + 16.3 pm SS 16609.75 20989.51 210.25 265 .69 n 80 80 S2p = 237.97 S2 u - x , = 2.439 df = 158 t = 16.61 > 1.98 (p = 0.05) (Reject H Q) - 89 -ABBREVIATIONS USED IN FIGURES axoneme b a s a l body c o r t e x c e l l coat c h l o r o p l a s t c i s t e r n a c u t i c l e c e n t r a l v acuole c r e n a t e v e s i c l e i n c l u s i o n dense membrane dense body endoplasmic r e t i c u l u m f l a g e l l u m f l o r i d e a n s t a r c h f i b r o u s v a c u o l e G o l g i (body) host i n t e r c e l l u l a r m atrix l i p i d body medulla mitochondrion roast igoneme m i c r o b o d y - l i k e s t r u c t u r e - 90 -N - nucleus ne - n u c l e a r envelope Nu - n u c l e o l u s P - p a r a s i t e pm - plasma membrane py - p y r i f o r m nucleus r - ribosome rer - rough endoplasmic r e t i c u l u m S - sporangium SC - Subcortex tp - t e r m i n a l p l a t e tv - t r a n s i t i o n v e s i c l e V - vacuole W - w a l l - 91 -F i g . 1. L o c a t i o n of Acadian S e a p l a n t s ' c u l t u r e f a c i l i t i e s i n Pubnico Harbor, Nova S c o t i a ( c i r c l e d a r e a ) . ( M o d i f i e d from Chapman and Chapman 1980). - 92 -- 93 -F i g s . 2 & 3. L a t e r a l l y b i f l a g e l l a t e zoospores of P e t e r s e n i a  p o l l a g a s t e r . Arrowhead p o i n t s to the second f l a g e l l u r n behind the spore body. (phase c o n t r a s t o p t i c s ) . x2,000 . F i g s . 4 - 6 . Cytochemical procedures fo r the d e t e c t i o n of c e l l u l o s e i n s p o r a n g i a l w a l l s with the use of f l uorochromes. F i g . 4. F l u o r e s c e n c e of s p o r a n g i a l w a l l s a f t e r treatment with C a l c o f l u o r White. x2,000. F i g . 5. F l u o r e s c e n c e of s p o r a n g i a l w a l l s : Congo Fed s t a i n i n g . x 900. F i g . 6. Freehand s e c t i o n t r e a t e d with C a l c o f l u o r White to show p e n e t r a t i o n of host c u t i c l e by e x i t tubes of sporangia (arrows). x 500. F i g . 7. P o s i t i v e s t a i n i n g r e a c t i o n of s p o r a n g i a l w a l l to Zinc C h l o r i o d i d e . x 800. - 95 -F i g s . 8 & 9. Stages i n the development of the f u n g a l d i s e a s e i n Chondrus c r i s p u s . N e c r o t i c l e s i o n s are l i m i t e d to the two most d i s t a l b i f u r c a t i o n s of the t h a l l u s . F i g . 8. E a r l y stage: 48 h a f t e r i n o c u l a t i o n with i n f e c -t i v e zoospores. Small l e s i o n s develop on the growing t i p s of the t h a l l u s . x 15. F i g . 9. Late stage (72 h a f t e r i n o c u l a t i o n ) . Arrows p o i n t to pores at the center of the l e s i o n s formed by e x i t tubes d u r i n g zoospore r e l e a s e . x 15. F i g . 10. H e a v i l y i n f e c t e d t h a l l u s . The number of l e s i o n s exceed 100 cm~2. x 12. F i g . 11. U n i n f e c t e d c o n t r o l . x 12. - 9 6 -- 97 -Regression curve f o r the number of l e s i o n s formed at the f i v e temperatures t e s t e d using 8 r e p l i c a t e s . V a r i a n c e s are homogeneous from 15 to 25 C; the best p r e d i c t i v e a b i l i t y can be expected at t h i s i n t e r v a l . Curve f i t was obtained with the polynomial e q u a t i o n : Y = AO + Al (X) + A2(X2) + A3(X3) + A4 (X4) where AO = 18.317 Al = -6.858 A2 = 0.801 A3 = -0.035 A4 = 5.144 The c o e f f i c i e n t of d e t e r m i n a t i o n , R2 i s 0.943. The r e g r e s s i o n c o e f f i c i e n t , r i s 0.971. F [ l , 3 1 ] = 586.28, p < ..05. - 99 -F i g s . 13-16. U n i n f e c t e d C. c r i s p u s . F i g . 13. C r o s s - s e c t i o n through the roost d i s t a l b i f u r c a t i o n showing c u t i c l e , c o r t e x , subcortex, and medulla. TBO s t a i n i n g . x 700. F i g . 14. M e d u l l a r y c e l l s . Note the p e r i h e r a l cytoplasm. The r e g i o n s of most i n t e n s e roetachromasia occur o u t s i d e the immediate w a l l s . x 1,500. F i g . 15. Fine s t r u c t u r e of u n i n f e c t e d host c u t i c l e showing el e c t r o n - d e n s e bands with i n t e r p o s i n g e l e c t r o n -t r a n s p a r e n t r e g i o n s . xl5.000. F i g . 16. C o r t i c a l c e l l . Most of the cytoplasm i s occupied by the nucleus and a s i n g l e c h l o r o p l a s t p r o f i l e . The c e l l w a l l c o n s i s t s of m i c r o f i b r i l s t h a t are o r i e n t e d p a r a l l e l to the c e l l s u r f a c e . x21,000. -too-- 101 -F i g s . 17-21. U l t r a s t r u c t u r e of u n i n f e c t e d £. c r i s p u s . F i g s . 17&18. S u b c o r t i c a l c e l l s showing lobed c h o r o p l a s t s with unstacked t h y l a k o i d s and f i b r i l l a r w a l l . Dense membranes are commonly found i n the cytoplasm. F i g . 17. x 14,000. F i g . 18. x 14,000 F i g . 19. S t a r c h g r a i n s and dense membranes i n cytoplasm of a c o r t i c a l c e l l . x 38,000. F i g . 20. M e d u l l a r y c e l l showing l a r g e c e n t r a l v acuole and p e r i p h e r a l cytoplasm. x 2,800. F i g . 21. P i t co n n e c t i o n between medullary c e l l s . The plug i s surrounded by a s i n g l e membrane and i t s ends c o n t a i n an e l e c t r o n - d e n s e m a t e r i a l . x 32,000. - 103 -V e g e t a t i v e t h a l l u s of P.. p o l l a g a s t e r i n c e l l s of £ . c r i s p u s , 12 h a f t e r i n o c u l a t i o n . The p a r a s i t e c e l l l a c k s a w a l l at t h i s s t a g e . TBO s t a i n i n g . Spread of the p a r a s i t e p r o t o p l a s t from a c o r t i c a l c e l l t o u n d e r l y i n g t i s s u e l a y e r s . x 3,500. Development v i a a s y m p l a s t i c r o u t e . Note the l a r g e n u c l e o l u s i n each nucleus (arrowheads). x 3,500. M u l t i n u c l e a t e sporangia formed 48 h a f t e r i n o c u l a t i o n showing d i f f e r e n t degrees of l o b u l a t i o n . E n d o b i o t i c development takes p l a c e mainly i n the s u b c o r t i c a l and med u l l a r y t i s s u e . TBO s t s i n i n g . x 2,000. x 1,500. - 105 -F i g . 26. Deeply lobed sporangium of £. p o l l a g a s t e r forming an e x i t tube (arrowhead), 48 h a f t e r i n o c u l a t i o n . The presence of numerous n u c l e i i n d i c a t e s the p o t e n t i a l f o r producing a vast number of i n f e c t i v e zoospores. A c r i d i n e Orange s t a i n i n g . x 1 ,000 . F i g s . 27-31. P a r a s i t e s p o r a n g i a . TBO s t a i n i n g . F i g . 27. S e c t i o n made f u r t h e r i n t o the sporangium 24 h from the time of i n o c u l a t i o n showing t h a t the t h a l l u s i s s t i l l i n t r a c e l l u l a r . x 3,000. F i g . 28. M u l t i n u c l e a t e sporangium with a s i n g l e e x i t tube (arrowhead). The d i s r u p t i o n of host t i s s u e o r g a n i z a t i o n i s now more evident (48 h a f t e r i n o c u l a t i o n ) . x 1,000. F i g . 29. E x i t tube of sporangium p a s s i n g through a pre-formed channel which was probably formed e a r l i e r i n the i n f e c t i o n p r o c e s s . x 3,000. F i g . 30. Discharged sporangium at the end of 72 h showing a s i n g l e remaining zoospore (arrowhead), x 1,500. F i g . 31. Sporangia at the end of 72 h showing a few moribund zoospores. x 3,000. - 107 -F i g s . 32-36. P o s t m o t i l e spores 6 h from time of i n o c u l a t i o n . F i g s . 32-34. Fine s t r u c t u r e of post m o t i l e spores showing l a r g e l i p i d g l o b u l e and p e r i p h e r a l f i b r o u s v a c u o l e s . Arrowheads p o i n t to axonemal p r o f i l e s . F i g . 32. Post m o t i l e spore with c r o s s s e c t i o n s of the r e t r a c t e d axoneme. x 28,000. I n s e r t : higher m a g n i f i c a t i o n showing the 9+2 arrangement of m i c r o t u b u l e s and a c r e n a t e v e s i c l e i n c l u s i o n . x 56,000. F i g . 33. C e l l coat on the s u r f a c e of post m o t i l e spore. Note s i m i l a r i t y to contents of f i b r o u s v a c u o l e s . x 27,000. F i g . 34. C o n c e n t r i c . c o n v o l u t e d membranes and c v i ' s i n post m o t i l e spore. Crenate v e s i c l e s appear to fuse with the f i b r o u s vacuoles and thus may c o n t r i b u t e to t h e i r c o n t e n t s . x 32,000. F i g . 35. R e t r a c t e d axoneme i n l o n g i t u d i n a l s e c t i o n . M i c r o t u b u l e s comprising the c e n t r a l s i n g l e t s and p e r i p h e r a l doublets are e a s i l y r e c o g n i z a b l e . x 34,000. F i g . 36. Basal bodies remaining a f t e r the axonemes have broken down to the l e v e l of the t e r m i n a l p l a t e . x 50,000. - 109 -F i g s . 37-39. Cysts of P_. p o l l a g a s t e r on the host c u t i c l e (observed approximately 6 h from time of i n o c u l a t i o n ) . F i g . 37. F l u f f y c e l l coat e x t e r n a l to the c y s t w a l l (arrows). There i s no a l t e r a t i o n i n the f i n e of the host c u t i c l e at t h i s s t a g e . x 21,000. F i g . 38. Cyst - host c u t i c l e i n t e r f a c e c o n s i s t i n g of the c l o s e l y appressed c y s t w a l l and the outermost l a y e r of the host c u t i c l e , s eparated by a t h i n l a y e r of the c e l l c o a t . x 38,000. F i g . 39. P a r t l y d i s s o c i a t e d c y s t showing d i s s o l u t i o n of the outermost c u t i c l e l a y e r (arrowhead). x 20 ,000 .. F i g . 40&41. P a r a s i t e p r o t o p l a s t - host i n t e r f a c e , 12 h from time of i n o c u l a t i o n . Note the f i n e l y g r a n u l a r m a t e r i a l i n narrow matrix between the two c l o s e l y appressed plasmalemmata. F i g . 40. Part of p a r a s i t e p r o t o p l a s t surrounded by an i n v a g i n a t e d host membrane, p o s s i b l y the host plasmalemma. The two membranes are separated by a narrow matrix showing the d e p o s i t i o n of f i n e g r a n u l a r m a t e r i a l . x 38,000. F i g . 41. P r o t o p l a s t - host i n t e r f a c e showing i n p o c k e t i n g s of the f u n g a l plasmalemma and numerous ribosomes. The i n f o l d i n g s of the plasmalemma may i n d i c a t e e n d o c y t o s i s . x 37,000. - I l l -F i g s . 42-45. V e g e t a t i v e f u n g a l t h a l l u s i n host c e l l s at the end of 12 h. F i g . 42. Elongate p r o t o p l a s t with numerous m i t o c h o n d r i a . The f l o r i d e a n s t a r c h granules have a c q u i r e d a dense core i n d i c a t i n g d i s s o l u t i o n . x 8,000. F i g . 43. B i n u c l e a t e f u n g a l p r o t o p l a s t with w e l l - d e v e l o p e d endoplasmic r e t i c u l u m , p e r i p h e r a l m i t o c h o n d r i a , and prominent n u c l e o l i . The development of the EF, G o l g i , and the l a r g e n u c l e o l u s i n d i c a t e i n t e n s e p r o t e i n s y n t h e t i c a c t i v i t y . x 9,000. F i g . 44. Formation of a narrow c y t o p l a s m i c p r o c e s s by fun-g a l p r o t o p l a s t p r i o r to i n v a s i o n of adjacent host c e l l . x. 3,000. F i g . 45. G o l g i i n zone of e x c l u s i o n around the p r o t o p l a s t n u c l e u s . T h e i r p r o x i m i t y to the n u c l e a r envelope i n d i c a t e s t h e i r p o s s i b l e involvement i n the flow of products from the NE to the plasma membrane. x 24,000. F i g . 46. Aggregation and m i g r a t i o n of m i t o c h o n d r i a d u r i n g p e n e t r a t i o n of n e i g h b o r i n g host c e l l s . x 9,000. F i g . 47. Sporangium i n a n e c r o t i c host c e l l . L i p i d g l o b u l e s have accumulated i n the f u n g a l cytoplasm, a p p a r e n t l y i n p r e p a r a t i o n f o r c l e a v a g e . x 9,000. - 1 1 2 -- 113 -P i g . 48. Host c e l l s adjacent to a sporangium. Note the c o l l a p s e d n e c r o t i c cytoplasm and the d i s r u p t i o n of the g e n e r a l c e l l u l a r o r g a n i z a t i o n . x 3,000. F i g s . 49&50. E a r l y stages i n sporangium fo r m a t i o n (observed approximately 24 h from time of i n o c u l a t i o n ) . N u c l e i and m i t o c h o n d r i a are d i s p e r s e d i n the cytoplasm and show no s p e c i a l a s s o c i a t i o n . Remnants of host c e l l s are v i s i b l e around the s p o r a n g i a . F i g . 49. x 9,000. F i g . 50. x 6,000. F i g s . 51&52. Formation of c e n t r a l v acuole, a p p a r e n t l y t a k i n g p l a c e a f t e r 24 h from i n o c u l a t i o n . F i g . 51. Dense membranes i n c e n t r a l vacuole of sporangium. x 5,000. F i g . 52. Apparent f u s i o n of dense bodies with c e n t r a l v a c u o l e . At t h i s s tage, a l a r g e r p r o p o r t i o n of the s p o r o g e n i c mass becomes occupied by p r o t o p l a s m i c components. x 28,000. - 115 -F i g s . 53-57. Stages i n the development of P.. p o l l a g a s t e r : a p p a r e n t l y formed a f t e r 24 h from time of i n o c u l a t i o n . F i g s . 53&54. Nuclear envelope - t r a n s i t i o n v e s i c l e - G o l g i a s s o c i a t i o n , i n d i c a t i n g the involvement of the G o l g i i n i n t r a c e l l u l a r flow of p r o d u c t s . M i c r o b o d y - l i k e s t r u c t u r e s and mi t o c h o n d r i a with t u b u l a r c r i s t a e t y p i c a l of the Oomycetes are a l s o p r e s e n t . F i g . 53. x 34,000. F i g . 54. x 10,000. F i g . 55. M i c r o b o d y - l i k e s t r u c t u r e s adjacent to mitochon-d r i a , x 29 ,000 ., F i g s . 56&57 P a i r e d b a s a l b o d i e s , o r i e n t e d at 90° to each o t h e r , become a s s o c i a t e d with a beaked n u c l e u s . S e q u e n t i a l s e c t i o n i n g showed t h a t some of the n u c l e i were^ a l r e a d y p y r i f o r m at t h i s s t a g e . C o n c e n t r i c dense membranes are a l s o p r e s e n t . F i g . 56. x 14,000. F i g . 57. x 43,000. - 117 -F i g s . 58-61. Developmental stages a p p a r e n t l y formed a f t e r 24 F i g . 58. Completely i n t r a c e l l u l a r sporangium showing the c l o s e a s s o c i a t i o n between mito c h o n d r i a and n u c l e i and v a r i o u s types of e l e c t r o n - d e n s e i n c l u s i o n s . A p i t c o n n e c t i o n i s present (arrowhead), x 6,000. F i g . 59. Fragmentation of a sporangium i n t o s m a l l e r t h a l l i . x 6,000. F i g s . 60&61. P r o l i f e r a t i o n of G o l g i - d e r i v e d c i s t e r n a e around zoospore i n i t i a l s d u r i n g cleavage of the spo r o g e n i c cytoplasm. F i g . 60. x 24,000. F i g . 61 . x 25 ,000. - 119 -F i g s . 62-65. Developmental stages observed a f t e r 24 h from time of i n o c u l a t i o n . F i g . 62. F u s i o n of s m a l l vacuoles with G c l g i - d e r i v e d c i s t e r n a e d u r i n g c l e a v a g e . Note m i c r o b o d y - l i k e s t r u c t u r e s and c v i ' s . x 31,000. F i g . 63. U n i n u c l e a t e block of cytoplasm showing the a s s o c i a t i o n of n u c l e u s , m i c t o c h o n d r i a , G o l g i , and b a s a l b o d i e s . A s l i g h t l y d i l a t e d cleavage c i s t e r n a i s a l s o p r e s e n t . x 33,000. F i g . 64. F u s i o n of c r e n a t e v e s i c l e s with s m a l l v a c u o l e s , s u g g e s t i n g t h e i r u t i l i z a t i o n d u r i n g s p o r o g e n e s i s . The s p o r a n g i a l w a l l i s now t h i c k e r and appears to c o n s i s t of two l a y e r s . x 38,000. F i g . 65. S y n t h e s i s of mastigonemes i n d i l a t e d EF c i s t e r n a e . Eibosomes are present on the c y t o p l a s m i c s i d e of the mastigoneme-bearing c i s t e r n a e except where the l a t t e r are c l o s e l y appressed to a m i t o c h o n d r i o n . x 35,000. - 121 -L a t e z o o s p o r o g e n e s i s ; s t a g e s o b s e r v e d 48 h from time of i n o c u l a t i o n . E x e r t e d f l a g e l l a between zoospore i n i t i a l s . At t h i s s t a g e , most of the n u c l e i a r e a l r e a d y p y r i f o r m . Note d o u b l e membrane around axonemes. x 15 ,000. x 12,000. P o s t - c l e a v a g e s p o r a n g i a showing i t s p a r t i t i o n i n t o u n i n u c l e a t e b l o c k s o f c y t o p l a s m . Zoospore i n i t i a l s a r e v e r y c l o s e l y a p p r e s s e d i n F i g . 68. x 11,000. x 15,000. - 123 -F i g . 70. P o s t - c l e a v a g e s p o r a n g i a l cytoplasm (approximately 48 h a f t e r i n o c u l a t i o n ) . A p a i r of b a s a l b o d i e s , i n d i f f e r e n t planes of s e c t i o n i n g , i s a s s o c i a t e d i s a s s o c i a t e d with each p y r i f o r m n u c l e u s . x 8,000. F i g s . 71&72. Moribund zoospores i n sporangia at the end of 72 h. F i g . 71 . x 2,000. F i g . 72. x 12,000. F i g . 73. F l u o r e s c e n c e of i n f e c t i o n s i t e a f t e r i n c u b a t i o n i n DAPI, i n d i c a t i n g plasmalemmal damage. x 300. F i g . 74. F l u o r e s c e n c e of f o r m a l d e h y d e - t r e a t e d c o n t r o l s . x 400 . - 125 -F i g . 75. D i l a t a t i o n of a proximal i n t e r b a n d r e g i o n of the host c u t i c l e at the s i t e of zoospore encystment (approximately 6h from time of i n o c u l a t i o n ) . Note f i n e g r a n u l a r d e p o s i t . The d i l a t a t i o n might be analogous to w a l l a p p o s i t i o n s i n t e r r e s t r i a l sys-tems. I t s t r a n s i t o r y nature i s suggested by i t s disappearance i n l a t e r stages of the p a r a s i t e ' s development. x 15,000. F i g s . 76-80. Changes o c c u r r i n g i n the host a f t e r 12 h from the time of i n o c u l a t i o n . F i g . 76. Decrease i n g e n e r a l e l e c t r o n d e n s i t y and f i b r i l l a r s t r u c t u r e of d i r e c t l y p e n e t r a t e d host c e l l wall.. x 3,000. F i g . 77. C h l o r o p l a s t s and m i t o c h o n d r i a of p e n e t r a t e d host c e l l , showing l i t t l e change r e l a t i v e to u n i n f e c -ted c o n t r o l s ( F i g s . 16-19) . F i g . 78. S y m p l a s t i c development of p a r a s i t e p r o t o p l a s t . Note the d i s s o l u t i o n of s t a r c h g r a i n s i n the adjacent host c e l l . x 3,000. F i g . 79. Compound host c e l l r e s u l t i n g from the s y m p l a s t i c development of the p a r a s i t e . x 2,000. F i g . 80. Apparent d i s s o l u t i o n of a p i t c o n n e c t i o n between d i r e c t l y p e n e t r a t e d and a d j a c e n t host c e l l . The bounding plasmalemma has d i s i n t e g r a t e d and the e l e c t r o n - d e n s e m a t e r i a l f i l l i n g the i n t e r i o r has been reduced to a loose f i b r i l l a r network. x 24,000. - 127 -Changes i n the Petersenia-Chondrus i n t e r f a c i a l zone (based on a m o d i f i c a t i o n of Bracker and L i t t l e f i e l d ' s [3973] scheme). Key to symbols: P a r a s i t e cytoplasm Host cytoplasm Host c u t i c l e P a r a s i t e c e l l w a l l ^ Host c e l l w a l l P a r a s i t e plasma membrane Host plasma membrane I n t e r - or e x t r a c e l l u l a r matrix -d e r i v e d from p a r a s i t e or host or both Inter-band s e c r e t o r y product Moribund or dead host protoplasm Aqueous environment - 128 -IT 1 P H Live, walled post motile spore on host c u t i c l e ; host e x t r a c e l l u l a r matrix and c e l l wall interposed between host protoplasm and the parasite. IT 2 P H E x t r a c e l l u l a r material (adhesive f l u f f y coat) present between the parasite (cyst) wall and host c u t i c l e ; other components as in IT 1. IT 3 P H Granular substance (presumably a host secretory product) deposited i n a proximal interband region of the host c u t i c l e ; other components as i n IT2. 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Biochem. 8: 1496-1502 . - 147 -APPENDIX A: C u l t u r e medium f o r c u l t i v a t i o n of £. c r i s p u s 'Instant Ocean" 36 g/1000 ml d e i o n i z e d water N H 4 N O 3 0.1 mmol N/1 (NH 4) 2BP04 0.1 mmol P / l Organic roicronutrients: 25 mg thiamine HCl, 5 mg 1.0 ml n i c o t i n i c a c i d , 5 mg Ca-pantothenate, 50 jjg b i o t i n , 100 ug f o l i c a c i d , 250 ug thymine and 50 ijg cobalamine i n 100 ml d i s t i l l e d water pH ( a d j u s t e d with 0.IN'HCl) 8.0 - 148 -APPENDIX B: Epoxy r e s i n used f o r e l e c t r o n m i c r o s c o p y PolyBed 812 25.8 g ( P o l y s c i e n c e s , Inc.) Dodecenyl s u c c i n i c a n h y d r i d e 12.9 g (D D S A) Nadic methyl a n h y d r i d e 13.2 g 2 , 4 , 6 - T r i ( d i m e t h y l a m i n o - 0.7 ml m e t h y l ) - p h e n o l 

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