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Morphogenesis of the wheat stem rust uredospore Wisdom, Carolyn Jean 1978

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MORPHOGENESIS OF THE WHEAT STEM RUST UREDOSPORE by CAROLYN JEAN WISDOM Honours B . S c , Brock U n i v e r s i t y , S t . Ca tha r i ne s , 1971 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF T H F THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE . in THE FACULTY OF GRADUATE STUDIES (Department of P lant Science) We accept t h i s t he s i s as conforming to the requ i red standard THE UNIVERSITY OF BRITISH COLUMBIA October 1977 Carolyn Jean Wisdom, 1977 In presenting t h i s thesis in p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make it f r e e l y available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for f i n a n c i a l gain shall not be allowed without my written permission. n . f PLANT SCIENCE Department of The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date A P R I L 2 8> 1 9 7 8 ABSTRACT The u r e d i a l stage of P u c c i n i a graminis Pers. f. sp. t r i t i c i E r i k s s . and Henn. is o b l i g a t e l y p a r a s i t i c on wheat in nature. The t r a n s i t i o n from the dormant uredospore to the v e g e t a t i v e s t a t e takes place on the plant by the sequential development of a germ tube and a s e r i e s of s p e c i a l i z e d i n f e c t i o n s t r u c t u r e s (appressoriurn, peg, v e s i c l e , and i n f e c t i o n hypha). Conditions ifavouring uredospore germination are d i f f e r e n t from those promoting d i f f e r e n t i a t i o n . I n f e c t i o n s t r u c t u r e s w i l l only develop in response to a d e l i c a t e l y timed sequence of s p e c i f i c environmental s t i m u l i . Thus, the p r e c i s e timing of c e l l u l a r events o c c u r r i n g during d i f f e r e n t i a t i o n could be c r u c i a l f o r rust s u r v i v a l . The timing of nuclear development, DNA, RNA, and p r o t e i n synthesis was i n v e s t i g a t e d c y t o l o g i c a 1 l y in the d i f f e r e n t i a t i n g uredospore apart from the host p l a n t . For the in v i t r o production of i n f e c t i o n s t r u c t u r e s , a l i q u i d medium was developed, c a l l e d MPG, which consisted of a Ca-K-PO^ b u f f e r , D-glucose, and 'Evans' peptone. There was s i g n i f i c a n t l y greater i n f e c t i o n s t r u c t u r e formation on t h i s medium than on the Ca-K-PO^ b u f f e r alone. Nuclear behaviour in d i f f e r e n t i a t e d s p o r e l i n g s was found to d i f f e r from that in s p o r e l i n g s which were germinated without forming i n f e c t i o n s t r u c t u r e s . The young germ tube was d i k a r y o t i c . Nuclei of germinating spores remained r e l a t i v e l y unchanged during development. D i v i s i o n s r a r e l y occurred, and when they did bn some older germ tubes, four was the maximum nuclear number. Septation soon followed to r e s t o r e the b i n u c l e a t e c o n d i t i o n . During d i f f e r e n t i a t i o n , on the other hand, the nuclear number increased due to d i v i s i o n s both in the appressori.um and i i n t t h e w . e s i c l e . The mature i i i appressorium normally had k n u c l e i , the mature v e s i c l e 7 or 8. This number was subsequently reduced in the i n f e c t i o n hypha to 1 or 2. In a d d i t i o n , two types of nuclear formations were r e g u l a r l y seen in the v e s i c l e ; b i l a t e r a l nuclear clumps and s p e c i f i c migration patterns. Results of experiments using metabolic i n h i b i t o r s i n d i c a t e d that the sy n t h e t i c requirements f o r morphogenesis of the germ tube and the i n f e c t i o n s t r u c t u r e s were a l s o d i f f e r e n t . The germ tube d i d not appear to require e i t h e r DNA, RNA, or p r o t e i n s y n t h e s i s , whereas the i n f e c t i o n s t r u c t u r e s required a l l three. RNA s y n t h e s i s , e s s e n t i a l f o r the appressorium, was found to occur during the f i r s t 2 hours of germination even before the heat stimulus to induce i n f e c t i o n s t r u c t u r e s was a p p l i e d . The r o l e of i n f e c t i o n s t r u c t u r e s i s s t i l l poorly understood. Therefore, st u d i e s were undertaken to determine the e f f e c t of d i f f e r e n t i a t i o n both on i n f e c t i o n of the host and on continued growth of the rust in axenic c u l t u r e . Spores that were heat-shocked to induce d i f f e r e n t i a t i o n gave a markedly higher i n f e c t i o n count when placed on exposed host mesophyll than those which were only pre-germinated without heat shock. This suggested that the i n f e c t i o n s t r u c t u r e s might be e s s e n t i a l f o r plant i n f e c t i o n , not merely f o r stomatal p e n e t r a t i o n . Attempts were made to produce v e g e t a t i v e colonies from s i n g l e 2 uredospores. P h y s i c a l l y separate, thinly-seeded spores ( l to 10 spores/mm ) f a i l e d to i n i t i a t e c o l o n i e s on a defined AXENIC medium which normally supported growth i f thickly-seeded (1000 to 2000 spores/mm ). When t h i n l y -seeded spores were f i r s t germinated or d i f f e r e n t i a t e d iin MPG medium for periods of from 2 hours to k days, then t r a n s f e r r e d to the AXENIC medium, i V co lon ie s were induced, each a r i s i n g from a s i n g l e uredospore. Colonies which had o r i g i n a t e d from d i f f e r e n t i a t e d spo re l i ng s sus ta ined growth f o r a longer per iod than those from germinated ones, suggest ing that i n f e c t i o n s t r u c tu re s are important f o r vege ta t i ve growth. When the above two-stage medium was used w i th s i n g l e uredospores, each in a separate w e l l of a p l a s t i c micnotest p l a t e , no vege ta t i ve growth occur red . Germ tubes were sho r te r and d i f f e r e n t i a t i o n r a r e l y occurred in the i s o l a t e d s i n g l e spore c ond i t i o n as compared to p h y s i c a l l y separate spores in a common medium. Th i s d i f f e r e n c e was independant of the volume of medium per spore. Attempts to use media, cond i t i oned f o r va ry ing time i n t e r v a l s w i th large numbers of d i f f e r e n t i a t i n g spores, as a s t a r t i n g medium f o r s i n g l e spores , proved un succe s s fu l . Attempts to i s o l a t e the d i f f e r e n t i a t i o n s t i m u l a t o r to apply i t to s i n g l e spores a l s o f a i l e d , although a germinat ion i n h i b i t o r and s t i m u l a t o r were de tec ted . Resu l t s of f i n a l experiments suggested that the use of g las s v i a l s as conta iner s f o r s i n g l e spores might y i e l d more promis ing r e s u l t s . V TABLE OF CONTENTS PAGE ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES i x ACKNOWLEDGEMENTS x i i INTRODUCTION 1 Main o b j e c t i v e s 5 LITERATURE REVIEW 6 F a c t o r s a f f e c t i n g g e r m i n a t i o n and d i f f e r e n t i a t i o n 6 C y t o l o g y o f e a r l y development 16 M a c r o m o l e c u l a r s y n t h e s i s d u r i n g morphogenesis... 18 D i f f e r e n t i a t i o n and f u r t h e r growth 22 MATERIALS AND METHODS.. 25 P r o d u c t i o n o f u r e d o s p o r e s on p l a n t 25 G e r m i n a t i o n and d i f f e r e n t i a t i o n i n v i t rorMMPG<med i..um — . 29 A x e n i c c u l t u r e , 34 D i s t i 1 1 at i on o f crude g e r m i n a t i o n i n h i b i t o r 40 N u c l e a r s t a i n i ng: 'Feubgen 43 M e t a b o l i c i n h i b i t o r s 44 D i f f e r e n t i a t i o n and ho s t i n f e c t i o n .... 45 M i c r o s c o p y .. .. 49 RESULTS , 50 A s u i t a b l e d i f f e r e n t i a t i o n medium: rMBG 50 The t i m i n g o f u r e d o s p o r e morphogenesis <i)n MPG mediurn. ............... 56 N u c l e a r s t a i n i n g . . . . 61 N u c l e a r development d u r i n g d i f f e r e n t i a t i o n . . 63 T i m i n g o f e s s e n t i a l DNA, RNA, and p r o t e i n synthes.i s . . . . : 92 E f f e c t o f d i f f e r e n t i a t i o n on h o s t i n f e c t i o n 109 E f f e c t o f d i f f e r e n t i a t i o n on a x e n i c c u l t u r e growth 120 I s o l a t e d s i n g l e s p o r e s v s . s p o r e s i n common medium 131 E f f e c t o f ' c o n d i t i o n e d ' media on s i n g l e s p o r e development 135 v i E f f e c t of ' d i f f e r e n t i a t i o n s t i m u l a t o r ' on s i n g l e spore development 135 E f f e c t of spore concent ra t i on and spore number on d i f f e r e n t i a t i o n 141 DISCUSSION 145 Factors a f f e c t i n g morphogenesis 145 Cy t o l o g i c events dur ing morphogenesis 147 Role of d i f f e r e n t i a t i o n in host i n f e c t i o n 158 D i f f e r e n t i a t i o n and axen ic growth :SS hngdessporecdev.etopment... . 160 Summary 164 CONCLUSIONS 165 LITERATURE CITED 167 v i i LIST OF TABLES TABLE PAGE B-1 Reports of s e l f - i n h i b i t i o n and s e 1 f - s t i m u l a t ion of rust uredospores 10 C-1 B5 medium f o r exc i sed wheat leaves 28 C-2 Substrates te s ted dur ing d i f f e r e n t i a t i o n t r i a l s 31 C-3 MPG :~d i f,f e:rent,i:at:i,onmmed;ium 33 C-4 AXENIC c u l t u r e medium.. 36 C-5 Feulgen reagents . . 42 D-1 E f f e c t s of Ca-K-P0^ bu f f e r and W i l l i a m ' s n u t r i e n t medium on d i f f erent i at i on . . . .54 D-2 E f f e c t s of v a r i a t i o n s of Ca-K-PO^ bu f f e r and W i l l i a m ' s medium on d i f f e r e n t i a t i o n 54 G-l Development of i n f e c t i o n s t r u c t u r e s a f t e r i n h i b i t o r t reatment s , expressed as a % of t o t a l spores 96 G-2 Development a f t e r i n h i b i t o r treatments,%%• of tota1 spores expressed as a % of c o n t r o l . . 96 G-3 Development of i n f e c t i o n s t r u c t u r e s a f t e r i n h i b i t o r t reatments , expressed as a% of the preceding s t r u c t u r e . . . . . . 98 G-4 Development a f t e r i n h i b i t o r treatments ,?.%oof;. preceding s t r u c t u r e expressed as a % of c on t r o l 98 H-1 I n f e c t i on of wheat leaves by pre-developed s p o r e l i n g s . . . . . . . . 117 1-1 Growth of t h i n l y - seeded spo re l i ng s in axenic c u l t u r e . . 123 J-1 Development of i s o l a t ed_ s i n g l e - s po r e s versus p h y s i c a l l y separate spores in a common -mediurn. 134 J-2 Development of i s o l a t e d s i n g l e spores versus p h y s i c a l l y separate spores in a common medium w i th the same volume of medium per spore..... 134 ,K-1 E f f e c t o f ' c o n d i t i o n e d ' MPG medium on germinat ion and d i f f e r e n t i a t i o n of s t e r i l e i s o l a t e d s i n g l e spores 137 v i i i TABLE . PAGE K-2 E f f e c t of ' c o n d i t i o n e d ' g las s d i s t i l l ed.. H^ O on germinat ion of non-s ter i le i s o l a t e d s i n g l e spores 137 L-1 E f f e c t of germinat ion i n h i b i t o r - d i s t i 1 l a t e on germinat ion and d i f f e r e n t i a t i o n of i s o l a t e d s i n g l e spores in g lass d i s t i l i e d .H.O. . 1 40 L-2 E f f e c t of germinat ion i n h i b i t o r d i s t i l l a t e on' germ tube length of i s o l a t e d s i n g l e spores in g l a s s d i s t i l l e d h^O 140 L-3 E f f e c t of germinat ion i n h i b i t o r d i s t i l l a t e on germinat ion and d i f f e r e n t i a t i o n of i s o l a t e d s i n g l e spores in.MPG... 140 M-1 E f f e c t of spore number and spore concent ra t i on on germinat ion and d i f f e r e n t i a t i o n of uredospores in g las s v i a l s 144 ix LIST OF FIGURES FIGURE PAGE A-1 Uredospore penet ra t i on of wheat l ea f 3 B-1 S t r u c t u r a l formula of germinat ion s e l f - i n h i b i t o r of P_. graminis t r i t i c i (methyl c i s - f e r u l a t e ) 13 B-2 S t r u c t u r a l formula of germinat ion s e l f - i n h I b i t o r of Uromyces phaseol i (methyl c i s~3 , 4 - d i methoxyc i nnamate) 13 C-1 P reparat ion of a s ep t i c wheat l ea f f o r i n f e c t i o n exper iment s . . . 4 8 D-1 Uredospore morphogenesis on Ca-K-PO^ bu f f e r 52 D-2 Uredospore d i f f e r e n t i a t i o n on MPG medium 52 E-1 Temporal sequence of i n f e c t i o n s t r u c t u r e format ion on MPG to medium 58 "E-i 4 E-15 Uredospore i n f e c t i o n s t r u c t u r e s a f t e r 20 hours 60 F-1 Two nuc l e i in germ tube a f t e r 2 hours of development 65 F-2 Two nuc l e i enclosed by a septum in germ tube at 16 hours 65 F-3 Nuclear d i v i s i o n in germ tube a f t e r 12 hours of development... 67 F - 4 Completion of nuc lear d i v i s i o n in germ t u b e - p r i o r to s e p t a t i o n 67 F-5 Two nuc l e i in germ tube a f t e r format ion of appressorium 70 F-6 Four nuc l e i in germ tube a f t e r format ion of appressorium 70 F-7 Two nuc l e i in app re s s o r i a l i n i t i a l a f t e r 3 1/2 hours of development 72 F-8 Two nuc l e i in appressorium a f t e r 4 hours of development 72 F-9 Appressorium w i th 3 nuc l e i 72 F-10 Appressorium w i th one of i t s 3 nuc l e i in the process of di v i s ion 72 F-11 Appressorium at 5 1/2 hours w i th 4 n u c l e i arranged in p a i r s . . . 72 X 71 GURE PAGE F-12 Nuclei which have migrated i n t o v e s i c l e i n i t i a l at 6 hours.. 72 F-13 Nuclei in appressorium and v e s i c l e at 10 hours ~]k F-14 Nuclear d i v i s i o n in v e s i c l e ~/k F-15 Enlargement of v e s i c l e nucleus showing m i t o t i c prophase lh F-16 Nuclear d i v i s i o n in v e s i c l e at 12 hours showing p a r a l l e l p a i r i n g arrangement 77 F-17 Nuclear d i v i s i o n in v e s i c l e at 12 hours showing bead-like p a i r i n g and r i n g s t r u c t u r e s 77 F-18 Enlargement of nuclear p a i r i n g arrangement in v e s i c l e 77 F-19 Enlargement of bead-like nuclear s t r u c t u r e and nuclear ri n g s t r u c t u r e in v e s i c l e 77 F-20 Enlargement of v e s i c l e nucleus showing m i t o t i c anaphase 77 F-21 Nuclei showing paired arrangement of chromosomes migrating i n t o v e s i c l e 79 F-22 Enlargement of paired arrangement of chromosomes in v e s i c l e . 79 F-23 V e s i c l e w i t h 5 n u c l e i at 7 1/2 hours 81 F-24 V e s i c l e with 6 n u c l e i at 11 hours 81 F-25 V e s i c l e with 7 n u c l e i 81 F-26 V e s i c l e with 8 n u c l e i at 10 hours 81 F-27 Nuclear 'pools' in the v e s i c l e arranged as b i l a t e r a l clumps. 83 F-28 B i l a t e r a l nuclear clumps in v e s i c l e at 10 hours with k n u c l e k p e r clump 83 F-29 B i l a t e r a l nuclear clumps in v e s i c l e , each with a d i f f e r e n t number of n u c l e i 83 F-30 B i l a t e r a l nuclear clumps in v e s i c l e a f t e r 12 hours of development 83 F-31 Stages in the development of nuclear migration patterns as to n u c l e i move from the v e s i c l e i n t o the developing i n f e c t i o n F-36 hypha 86 x i FIGURE PAGE F-36A I n fec t i on hypha con ta in i ng 8 nuc l e i at 14 hours 89 F-37 Developing i n f e c t i o n hypha at 14 hours con ta in ing a s i n g l e lead ing nucleus fo l l owed by others 86 F-38 Two nuc l e i in develop ing i n f e c t i o n hypha at 14 hours 86 F-39 A s i n g l e nucleus in i n f e c t i o n hypha a f t e r 16 hours 91 F-40 A s i n g l e nucleus in i n f e c t i o n hypha a f t e r 20 hours 91 F-41 Coa lesc ing nuc l e i atcti.poof, i n f e c t i o n , hypha-a f ter 20 hours of development 91 F-42 A s i n g l e nucleus at t i p of i n f e c t i o n hypha a f t e r 20 hours of development 91 :G — 1 E f f e c t of i n h i b i t o r s of RNA synthes i s on uredospore morphogenesis 101 G-2 E f f e c t of puromycin on uredospore morphogenesis 103 G - 3 E f f e c t of 5 - f 1uo rou rac i 1 on uredospore morphogenesis 106 G-4 E f f e c t of cyc lohex imide on uredospore morphogenesis 108 H-1 Two areas of wheat l e a f used f o r i n f e c t i o n counts 112 H-2 I n f ec t i on of wheat leaves by pre-deve1 oped spo re l i ng s 115 H~7 Rust i/co;l ony. sporu 1 at-:i ng o n l i y i i n i a r e a of • i .ntact-epidermi s and oh wheat l e a f 119 He 8 1-1 Growth of t h i n l y - s eeded spo re l i ng s in axenic c u l t u r e a f t e r 10 days . . 126 1-2 Growth of t h i n l y - seeded spo re l i ng s in axenic c u l t u r e a f t e r 2 months 128 1-3 Colony i n i t i a t i o n by s i n g l e d i f f e r e n t i a t e d uredospore and a f t e r 10 days 130 I-4 N-1 Summary of the t iming of e s s e n t i a l DNA, RNA, and p r o t e i n synthes i s dur ing uredospore morphogenesis 153 x i i ACKNOWLEDGEMENTS I would l i k e to g ive s p e c i a l thanks to Dr. Michael Shaw fo r h i s cont inu ing support and encouragement dur ing the course of the present s tudy. . He always had an open mind, but never an empty one. It was a p r i v i l e g e to be h i s s tudent. I would a l s o l i k e to thank Dr. V.C. Runeckles and Dr. R.J . Copeman fo r t h e i r s i n ce re i n t e r e s t and adv i ce . My app rec i a t i on goes to Dr. G.W. Eaton f o r help w i th the s t a t i s t i c a l analyses and to Dr. C.A. Hornby f o r h e l p f u l d i s cu s s i on on nuc lear d i v i s i o n . I a l s o wish to thank the other members of Dr. Shaw's research group f o r t h e i r mot i va t ing d i s cu s s i on s and the f o l l o w i n g people who were always ready to help when needed; Mr. I D e r i c s , Mr. A. Herath, Mr. L. Scrubb, and Mr. A Hoda. Th i s study was supported by grants from the Nat i onal .'Research. Counci 1 to Dr. Michael Shaw. 1 INTRODUCTI ON Rust fungi are o b l i g a t e p lant p a r a s i t e s , producing d iseases on many economica l l y important crops. One such d i sease is the b lack stem rust of wheat caused by Pucc i n i a graminis Pers . f . sp. t r i t i c i E r i k k s . and Henn. The l i f e c y c l e of t h i s fungus inc ludes f i v e spore forms and two hos t s , but the repeat ing u r e d i a l stage is by f a r the most d e s t r u c t i v e . The u r e d i a l stage begins when a mature uredospore lands on a s u s cep t i b l e wheat p l a n t . Leaf penet ra t i on and the i n i t i a t i o n of vege ta t i ve growth occurs in two main s tages ; germinat ion and d i f f e r e n t i a t i o n s . This is shown s chemat i ca l l y in F igure A - l . Germination invo lves the hydrat ion of the spore and the outgrowth and e longa t i on of the germ tube along the l ea f s u r f ace . When the guard c e l l s of a stomatal opening are reached, tube e longa t i on ceases and d i f f e r e n t i a t i o n beg ins. This invo lves the sequent ia l formation of i n f e c t i o n s t r u c t u r e s which con s i s t of the f o l l o w i n g ; the appressor ium, the i n f e c t i o n peg, the substomatal v e s i c l e , and the i n f e c t i o n hypha. The appressorium i s formed as a s w e l l i n g d i r e c t l y above the stoma. A small p r o j e c t i o n , the i n f e c t i o n peg, then a r i s e s from the appressorium and penetrates between the guard c e l l s reaching the substomatal c a v i t y where the v e s i c l e i s produced. Subsequently, andirhfieetion hypha p r o j e c t s from the v e s i c l e . Th is i s normal ly cons idered to be the end of the penet ra t i on phase. The i n f e c t i o n s t r u c t u r e s formed dur ing t h i s phase are the main sub ject of the present t h e s i s . The next phase i s i n f e c t i o n . During t h i s t ime, h o s t - p a r a s i t e r e l a t i o n s are e s t ab l i s hed by the formation of a haustorium w i t h i n a host mesophyll c e l l . As a p r e r e q u i s i t e to t h i s , the i n f e c t i o n hypha cuts o f f a h a u s t o r i a l 2 F igure A-1 Diagrammatic rep re senta t i on of uredospore penet ra t i on of wheat l e a f . Whi le penet ra t ing the l e a f , spore undergoes morphogenesis. Ea r l y stages dur ing morphogenesis are germinat ion and d i f f e r e n t i a t i o n . The l a t t e r inc ludes format ion of the f o l l o w i n g s t r u c t u r e s ; appressorium (3), i n f e c t i o n peg (4), substomatal v e s i c l e (5), and i n f e c t i o n hypha (6). Approx. magn i f i c a t i on X 800. U r e d o s p o r e P e n e t r a t i o n o f W h e a t L e a f D e v e l o p m e n t a l S e q u e n c e 1. U r e d o s p o r e 2. G e r m t u b e 3. A p p r e s s o r i u m 4. I n f e c t i o n p e g 5. S u b s t o m a t a l v e s i c l e 6. I n f e c t i o n h y p h a 7. H a u s t o r i a l m o t h e r 8. H a u s t o r i u m c e l l 4 mother c e l l which d i s s o l ve s away both i t s own and the host c e l l w a l l . This a l lows the mother c e l l t o invag inate the host p r o t o p l a s t . The fungus cont inues to spread throughout the l ea f by means of i n t e r c e l l u l a r hyphae and hau s to r i a u n t i l the s p o r u l a t i o n phase. At the end of t h i s t ime, a new crop of uredospores is produced. The whole c y c l e takes approx imately nine days. Germination and d i f f e r e n t i a t i o n are two of the e a r l i e s t morphogenetic events. They occur w i t h i n the f i r s t twenty- four hours of development, when the rust fungus i s attempting to gain ent ry i n t o the host. This i s the most c r i t i c a l time f o r fungus s u r v i v a l , s i n ce r e l a t i o n s between the host and p a r a s i t e are not yet f i r m l y e s t a b l i s h e d . The re fo re , t h i s would be the idea l time f o r a p p l i c a t i o n of c on t r o l measures. But f i r s t , a d e t a i l e d understanding of the fungus at t h i s stage is necessary. There are a few important areas which need i n v e s t i g a t i o n . F i r s t l y , f a c t o r s a f f e c t i n g uredospore morphogenesis are not complete ly understood. I n i t i a l d i f f e r e n t i a t i o n apart from the hos t , as we l l as f u r t h e r growth in axen ic c u l t u r e , are o f ten d i f f i c u l t to ach ieve , and when ob ta i ned , may not be r ep roduc i b l e . In a d d i t i o n , colony growth does not occur from s i n g l e uredospores. This makes genet i c s tud ie s almost imposs ib le wi thout the host. Secondly, c y t o l o g i c event s , p a r t i c u l a r l y the t iming of uredospore gene a c t i on such as nuc lear development and the synthes i s of e s s e n t i a l macromolecules, are poor ly understood as they r e l a t e to the d i f f e r e n t i a t i o n process. Ye t , fungus s u r v i v a l may depend on the p rec i s e t im ing of these events . 5 F i n a l l y , the s i g n i f i c a n c e of i n f e c t i o n structures is s t i l l in question. They appear, at least, to play a role in host penetration. However, they may also be necessary for continued fungal growth. Attempts were made during the present study to investigate these problems. The main objectives were: 1) to determine the factors a f f e c t i n g d i f f e r e n t i a t i o n so the inf e c t i o n structures could be consistently produced apart from the host 2) to study the timing of the c y t o l o g i c a l processes occurring during d i f f e r e n t i a t i o n , s p e c i f i c a l l y nuclear development and the synthesis of DNA, RNA, and protein 3) to determine the role of inf e c t i o n structures for survival of the fungus both on the host and in axenic culture k) to culture the uredial stage of the fungus from single uredospores 6 LITERATURE REVIEW FACTORS AFFECTING GERMINATION AND DIFFERENTIATION: Uredospore penet ra t i on behaviour i s c o n t r o l l e d by a complex i n t e r a c t i o n between the hos t , the pathogen, and the environment (Brown and Shipton 1964). Gene ra l l y , c ond i t i on s favour ing germinat ion on the host are d i f f e r e n t from those promoting d i f f e r e n t i a t i o n . For Pucc i n i a gramin i s t r i t i c i , optimum germinat ion takes p lace in the dark (or l e s s than 300 f t - c l i g h t ) w i th temperatures of 60-75°F and high r e l a t i v e humid i ty . D i f f e r e n t i a t i o n , on the other hand, p a r t i c u l a r l y the v e s i c l e s tage, requ i re s at l ea s t 500-1500 f t - c , increased temperatures of 85°F, and d r i e r atmospheric cond i t i on s (Sharp e _ a _ . 1958, Rowell e_t_ al_. 1958). Despite the o b l i g a t e nature o f rust p a r a s i t i s m , germinat ion can r e a d i l y take p lace on water apart from the host. Un l i ke the c o n i d i a of many saprophyt ic f u n g i , the uredospore i s ab le to germinate by u t i l i z i n g stored re se rve s , p r i m a r i l y l i p i d s in the form of f a t t y ac ids such as c i s -9 ,10 -epoxyoctadecanoic a c i d , but a l s o sugar a l c oho l s such as mannitol (Shu et a 1. 1954, Daly et_ a_l_. 1967) • D i f f e r e n t i a t i o n in the absence of the host does not occur so r e a d i l y . Hurd-Karrer and Rodenhiser (1947) were the f i r s t to report the product ion of i n f e c t i o n s t r u c t u r e s on a r t i f i c i a l media. D i f f e r e n t i a t i o n of va r i ou s ru s t s pec i e s , i n c l ud i n g Pucc i n i a graminis t r i t i c i , took p lace at room temperature on an H20 agar base con ta i n i ng mineral s a l t s and D-glucose. S t ruc tu re s were not produced on the H^ O agar a lone. Even on the n u t r i e n t agar, t h e i r frequency of formation was extremely low: only one in 1000 spores d i f f e r e n t i a t e d . 7 It is now known that i n f e c t i o n s t r u c t u r e s w i l l on ly develop in response to a d e l i c a t e l y timed sequence of environmental s t i m u l i , s p e c i f i c f o r each rust spec ie s . Substrate sur face p r o p e r t i e s , temperature and l i g h t c o n d i t i o n s , s e l f - i n h i b i t o r s and s t i m u l a t o r s , gaseous atmospheric components, and n u t r i e n t s have a l l been imp l i ca ted as t r i g g e r s f o r d i f f e r e n t i a t i o n . The e f f e c t of membrane sur faces on uredospore morphogenesis was f i r s t s tud ied by D ick inson (1949). I n fec t i on s t r u c t u r e s , c h a r a c t e r i s t i c of the var ious rus t s employed, were formed on membranes made from mixtures of c e l l w a l l s , p a r a f f i n wax, and c o l l o d i o n , when these membranes were f l o a t e d on water. The presence or absence of i n f e c t i o n s t r u c tu re s could be c o n t r o l l e d by the congeal ing po int of the waxes used in making the membranes: f o r £_. t r i t ic i na , s t r u c t u r e s were not produced on waxes w i th a low congeal ing po int (36°C) , but were formed on those w i th a higher congea l ing po int of 52°C. D i c k i n son , t h e r e f o r e , concluded that d i f f e r e n t i a t i o n was a th igmotrop ic response to the s u r f ace . Pavgi and Dickson (1961) obta ined s i m i l a r r e s u l t s w i th P_. sorgh i . V e s i c l e formation was g reate r on ce l lophane membranes over water agar than on water agar a lone. Wynn (1976), l i k e w i s e , a t t r i b u t e d d i f f e r e n t i a t i o n to a contact s t imulus r e a c t i o n . He e l im ina ted the p o s s i b i l i t y of chemical f a c t o r s , such as sur face waxes, by us ing p l a s t i c to make copies of the lea f c u t i c l e . I n fec t i on s t r u c t u r e s developed in response to the p ro t rud ing l i p s of the p l a s t i c copies of guard c e l l s . The f o l l o w i n g rus t s have been found to be most responsive to membrane sur face p r o p e r t i e s ; Uromyces phaseol i , P_. hel i an th i , and F\ an t i r r h i n i . In c o n t r a s t , P_. grami n i s t r i t ic i , a lthough a f f e c t e d a l i t t l e by host c u t i c l e , is not respons ive to a r t i f i c i a l membranes at a l l (Maheshwari 1966). 8 The morphogenesis of _. grami n i s t r i t i c i i s c o n t r o l l e d in v i t r o , as on the host , by v a r i a t i o n s in l i g h t or temperature. The f i r s t report that a s i g n i f i c a n t p ropor t i on of uredospores from t h i s spec ies could be induced to d i f f e r e n t i a t e wi thout the host came from Emge (1958). Spores were placed on subs t ra tes of acid-washed f i 1 t e r paper, ce l lophane d i a l y s i s membrane, or m i l l i p o r e f i l t e r , each o v e r l a i d on 1 % water agar, or on 1 % water agar a lone. Optimum i n f e c t i o n s t r u c t u r e formation occurred when spores, which had been germinated fo r 2 hours in the, dark at 70-75°F, were exposed to 6 hours of s un l i g h t at 2000-5000 f t - c and temperatures of 85°F. Heat and l i g h t together gave best r e s u l t s , although sepa ra te l y they d id induce some d i f f e r e n t i a t i o n . It was subsequently found that up to 95 percent of uredospores would form i n f e c t i o n s t r u c t u r e s a f t e r heat shock a lone (Maheshwari e t a l . 1967a). Spores were f l o a t e d in the dark on a Ca-K-P0^ bu f f e r f o r 2 hours at 20°C, shocked fo r 1 1/2 hours at 30°C, then returned to 20°C fo r 12-16 hours. The du ra t i on of the heat shock per iod could not be a l t e r e d ; sho r te r times y i e l d e d a t y p i c a l s t r u c t u r e s , whereas w i th longer pe r i od s , the spo re l i ng s d i e d . Uredospore morphogenesis i s a l s o known to be c o n t r o l l e d by the s i z e of the spore popu l a t i on . Th is phenomenon i s thought to be due to the i n f l uence of s e l f - i n h i b i t o r s and s t i m u l a t o r s . These compounds are re leased from the spores themselves and are a c t i v e at extremely low concent ra t i ons (hormone l e v e l s ) . There appear to be at l ea s t seven d i f f e r e n t popu l a t i on -dependent e f f e c t s on uredospores (Table B-1). A l l are e i t h e r known or suspected of being due to growth r e gu l a t i n g compounds. Reports inc lude the i n h i b i t i o n and s t i m u l a t i o n of both germinat ion and d i f f e r e n t i a t i o n . Only the germinat ion i n h i b i t o r has been i d e n t i f i e d to date . For P. gramin?s 9 Table B-1 Reports of s e 1 f - i n h i b i t i o n and se1f-st imu1 a t i on of rust u redospores. Reports of Inhibition or Stimulation of Rust Uredospores T A B L E B-l CATEGORY EFFECT ON DEVELOPMENT APPROX. SPORE CONC. WHEN EFFECT SHOWN COMPOUND OR HOW ISOLATED REPORTED BY Germi nat ion 1nhi bi tor -inhibits % of spores germinating -% of germinating spores decreases with increasing spore concentration 1 mg spores/ml 3 x 103 to 3 x 105 spores/ml -crude inhibitor-float spores on H20 -has been identified as cis-methyl ferulate for P. graminis t r i t i c i -has been identified as cis-methyl S.'i-dimethoxycinnamate for Uromyces phaseoli P. helianthi-\ P. antirrhini f_. sorghi J Allen 1955 P. graminis t r i t i c i Macko et a l . 1971 Macko et a l . 1970 Macko et a l . 1972 Germination Stimulator -overcomes action of germination . inhibitor in large population of spores -increases % germination 1 mg spores/ml -disti l lation of crude germination inhibitor at 100 C: f i rst 10-20 % of dist i l late Allen 1957 P. graminis t r i t i c i Germ tube 1nh ib i tor -inhibits length of germ tubes -germ tubes shorter when germination % not inhibited ? -disti l lation of crude germination Inhibitor as above -not isolated Allen 1957 P. graminis t r i t i c i Yarwood 1956 Uromyces phaseoli Germ tube St imulator -stimulates length of germ tubes -length of germ tubes increases with increasing spore concentration -germ tubes longer when germination % inhibited 50-400 spores/drop 25 or more spores/mm3 -not isolated -not isolated Ezekiel 1930 P. graminis t r i t i c i Yarwood 1956 Uromyces phaseoli Differentiation 1 nh i bi tor -inhibits % of spores differentiating -% of differentiating spores decreases with increasing spore concentration (specifically affects appressorial stage) 2-5 mg spores/ml -not isolated Dunkle and Allen 1971 P. graminis t r i t i c i Differentiation St imulator -stimulates % of spores differentiating ? -disti l lation of crude germination inhibitor as above Allen 1957 P. graminis t r i t i c i Infection hypha 1nhibi tor -inhibits length of Infection hypha more than 2.5 mg spores/ml -not isolated Dunkle and Allen 1971 P. graminis t r i t i c i 11 t r i t i c i , i t i s methyl c i s - f e r u l a t e (Macko e_t aj_. 1 971 , A l l e n 1 9 7 2 ) . The s t r u c t u r a l formula i s shown in F igure B -1 . It ac t s on the spore "during the imbibat ion of water and complete ly prevents germination.; y e t , i t has no e f f e c t on d i f f e r e n t i a t i o n . The crude i n h i b i t o r can be obta ined s imply by f l o a t i n g l a rge popu la t ions of spores on a small volume of water ( A l l e n 1955, Forsyth 1 955 ) . If the crude i n h i b i t o r i s then steam d i s t i l l e d , the r e s u l t i n g v o l a t i l e f r a c t i o n s w i l l not i n h i b i t ge rminat ion , but have a new e f f e c t . They we l l s t imu l a t e germinat ion by overcoming the o r i g i n a l i n h i b i t o r a c t i v i t y in a large popu la t ion of spores. These same f r a c t i o n s w i l l a l s o induce the format ion of i n f e c t i o n s t r u c t u r e s and i n h i b i t the e longat ion of germ tubes (A l l en 1957, French et_a_L 1 9 5 7 ) . It i s not known how many compounds are i n vo l ved . The germinat ion i n h i b i t o r f o r ru s t s such as Uromyces phaseol i (Macko et_ aj_. 1 9 7 0 ) , IP. hel i a n th i , P_. an t i r r h i n i , and f_. sorghi (Macko et_ al_. 1972) has a l s o been i d e n t i f i e d . It i s the c i s form of 3 ,k-dimethoxycinnamate ( F i g . B - 2 ) , a compound c l o s e l y r e l a t e d to methyl f e r u l a t e . Thus, r u s t s can be d i v i ded i n to two major groups depending on t h e i r d i f f e r e n t i a t i o n behaviour. The f i r s t group inc ludes P_. graminis t r i t i c i and a s soc ia ted rus t s which are respons ive to heat and l i g h t , not membranes, and which have methyl f e r u l a t e as t h e i r germinat ion i n h i b i t o r . The second inc ludes Uromyces phaseol i and others (mentioned above) which d i f f e r e n t i a t e in response to the contact s t i m u l i of membranes. In t h i s group, germinat ion i s i n h i b i t e d by 3,4-dimethoxycinnamate. In a d d i t i o n to the germinat ion i n h i b i t o r and i t s d e r i v a t i v e s which s t imu l a te d i f f e r e n t i a t i o n , there are repor t s of u n i d e n t i f i e d substances 12 F i g u r e B-1 S t r u c t u r a l f o r m u l a o f methyl, c i s - f e r u l a t e . T h i s compound i s the g e r m i n a t i o n s e 1 f - i n h i b i t o r o f P. g r a m i n i s t r i t i c i . F i g u r e B-2 S t r u c t u r a l f o r m u l a o f methyl c i s - 3 , h-dimethoxycinnamate. T h i s compound i i s tfhejgeinmiiinati.on^seff-tifhhiliibi.torv.of J , <.<. U romyces p h a s e o l i , P_. h e l i a n t h i , P_. ant i r r'h i n ? > and P_. sorghli,. 13 Germination Self-Inhibitors F ig . B-l Methyl cis-4-hydroxy-3 methoxycinnamate (Methyl c i s - f e ru l a te ) C H 3 O C H 3 O F ig . B-2 Methyl cis-3,4-dimethoxycinnamate 14 which i n h i b i t d i f f e r e n t i a t i o n in P_. grami ni s t r i t i c i . Dunkle and A l l e n (1971) found that i f the germinat ion medium was replaced before a p p l i c a t i o n o f the heat s t i m u l u s , the percentage o f spores forming i n f e c t i o n s t r u c t u r e s markedly inc reased, but changing the medium a f t e r heat shock had l i t t l e e f f e c t . Th is i nd i ca ted to them that there was an i n h i b i t o r in the germinat ion medium which was s p e c i f i c a l l y d i r e c t e d toward the app re s s o r i a l stage. Atmospheric c o n s t i t u e n t s , other than the v o l a t i l e s t imu lan t s j u s t de s c r i bed , are a l s o known to i n f l uence d i f f e r e n t i a t i o n . For example, 5 % CC>2 (normal a i r has approx imately 0.033 % CO^) has been reported to induce the format ion o f appres so r i a when the spores of P_. s t r i i formi s are f l o a t e d on de ion i zed water at 5°C (Macko and Fuchs 1970). Uredospore germinat ion does not requ i re the presence of exogenous n u t r i e n t s because the spore i s ab le to u t i l i z e s to red reserves . On the other hand, a lthough i t i s not known whether n u t r i e n t s are mandatory f o r d i f f e r e n t i a t i o n , they do have a s i g n i f i c a n t e f f e c t . Hurd-Karrer and Rodenhiser (1947) s ta ted that i n f e c t i o n s t r u c t u r e s d id not develop on water agar, but d id on a n u t r i e n t agar. Sharpband Smithi-(1952),', working w i th JP. co ronata , s i m i l a r l y found l i t t l e or no v e s i c l e product ion on water agar w i t h z i n c , but up to 40 % v e s i c l e s on a subs t ra te of 3 % g e l a t i n p lus z i n c . Couey and Smith (1961) determined that ca t i on s in the g e l a t i n in f luenced the a c t i o n of z i n c on d i f f e r e n t i a t i o n . Z inc would not induce v e s i c l e format ion on de ion i zed g e l a t i n , but d id on whole g e l a t i n or when e i t h e r Ca o r Mg was added. In t h e i r attempts to c u l t u r e P. graminis t r i t i c i , Fuchs and Gaertner (1958) d id not ob ta in v e s i c l e formation on g e l a t i n w i t h or wi thout the a d d i t i o n o f z i n c , a lthough 10 percent o f the spores d i d form v e s i c l e s 15 on a s i l i c a gel medium. V e s i c l e format ion was f u r t h e r enhanced by the a d d i t i o n of c y s t e i n to t h i s medium in the presence of Fe. F i n a l l y , Na i to e_ aJL (1964), us ing P_. co ronata , found that a n u t r i e n t agar medium, c on t a i n i n g peptone and sucrose as w e l l as other mineral s a l t s , gave good i n f e c t i o n s t r u c t u r e fo rmat ion . These i n ve s t i g a t o r s concluded that i t was the peptone and sucrose components that were r e spon s i b l e . I n te rac t i on between var ious e x t e r n a l l y app l i ed s t i m u l i may a l s o a l t e r d i f f e r e n t i a t i o n behaviour. Maheshwari (1966) found that phy s i ca l and chemical s t i m u l i had a s y n e r g i s t i c e f f e c t . L i t t l e or no d i f f e r e n t i a t i o n o f Uromyces phaseo l i occurred on c o l l o d i o n membranes unless hydrocarbon or e t h e r - s o l u b l e con s t i t uen t s of c u t i c l e wax or p a r a f f i n o i l were i ncorpora ted. For an A u s t r a l i a n form of P_. graminis t r i t i c i , a n u t r i e n t s t imulus has been found to be more e f f e c t i v e in combinat ion w i t h a heat shock (Wi11iams 1971). In t h i s case, i n f e c t i o n s t r u c t u r e s were r a r e l y formed on water agar. A p p l i c a t i o n of a heat shock d i d not s i g n i f i c a n t l y increase t h e i r frequency. However, on a n u t r i e n t agar c on t a i n i n g peptone, g lucose , and minera l s a l t s , a p p l i c a t i o n of a heat s t imulus r e su l t ed in a d i f f e r e n t i a t i o n increase from one s i x t h in the unshocked c o n d i t i o n to over three quar ter s in the shocked. Exogenously app l i ed s t i m u l i are not the on ly f a c t o r s a f f e c t i n g morphogenesis. The spore i t s e l f determines i t s own development. D i f f e r e n t races of F\ graminis t r i t i c i vary in t h e i r c apac i t y to d i f f e r e n t i a t e . For example, the A u s t r a l i a n races 126-6,7 and 2 1 - 1 , 2 , 3 , 7 both d i f f e r e n t i a t e more r e a d i l y than race 111 -E -2 . Spores c o l l e c t e d on d i f f e r e n t days a f t e r the i n i t i a t i o n o f s p o r u l a t i o n a l s o vary in p o t e n t i a l to form i n f e c t i o n s t r u c t u r e s . D i f f e r e n t i a t i o n i s h ighest i n spores c o l l e c t e d on the second 16 day a f t e r the opening of the uredosorus, then dec l i n e s p r og re s s i v e l y as the uredosorus ages (Wi l l iams 1971). It is not known whether these d i f f e r e n c e s in morphogenetic p o t e n t i a l are l i nked w i th s e l f - i n h i b i t o r s or st imu la to r s . The repor t s descr ibed here reveal that germinat ion and d i f f e r e n t i a t i o n can be t r i g ge red by a v a r i e t y of s t i m u l i : those a f f e c t i n g germinat ion are d i f f e r e n t and o f ten complete ly oppos i te to those i n f l u e n c i n g d i f f e r e n t i a t i o Morphogenesis i s a l s o cond i t i oned by u n i d e n t i f i e d f a c t o r s in the spore i t s e l f . Un f o r t una te l y , even though a number of s tud ie s have been c a r r i e d ou t , e a r l y development, p a r t i c u l a r l y d i f f e r e n t i a t i o n , i s o f ten not c on s i s t en t or rep roduc ib le apart from the host p lant (Weise and Daly 1967). CYTOLOGY OF EARLY DEVELOPMENT: Key c y t o l o g i c events o c cu r r i n g dur ing morphogenesis, such as nuc lear development, are l i t t l e understood. The u r e d i a l stage i s cons idered to be the ' i m p e r f e c t ' or non-sexual stage o f the rust l i f e c y c l e s i nce karyogamy and meios i s are not known to take p lace at t h i s t ime. Nuclear d i v i s i o n s do occu r , but the exact nature of the d i v i s i o n process has not been determined, Sporadic re ferences in the l i t e r a t u r e i n d i c a t e that there i s an increase in nuc lear number dur ing the d i f f e r e n t i a t i o n of var ious r u s t s , but report s vary as to the p rec i s e f i g u r e and the d e t a i l s of d i v i s i o n . The f i r s t mention of these s po re l i n g n u c l e i was by Sapp in-Trouf fy (1896) f o r P_. g r am in i s , even before i n f e c t i o n s t r u c t u r e s were named as such. As uredospores were germinat ing on water , two nuc l e i were seen to pass 17 from the spore to the germ tube. Then in the "premiere v e s i c u l e " (appressor ium), the two nuc l e i d i v i ded to y i e l d f ou r . Evans (1907) in h i s c l a s s i c study of the d i f f e r e n t i a t i o n of var ious rust spec ies on t h e i r hos t s , s ta ted that the substomatal v e s i c l e of P_. glumarum was "crammed w i th n u c l e i " . A l l e n (1923) noted in her s tud i e s on host i n f e c t i o n , that the appressorium o f P_. graminis t r i t i c i had k n u c l e i , whereas the young i n f e c t i o n hypha had two. For P_. t r i t i c i n a , the v e s i c l e sometimes had 4, sometimes 8, and the i n f e c t i o n hypha had 6 n u c l e i ( A l l en 1926). When i n f e c t i o n s t r u c t u r e s of f_. t r i t i c i n a were d i f f e r e n t i a t e d on a r t i f i c i a l membranes, D ick inson (1949) reported that two or three nuc lear d i v i s i o n s took p lace in the appressorium and subsequently e i t h e r 4 or 8 nuc l e i migrated in to the v e s i c l e . In v e s i c l e s developed on waxes o f high congeal ing p o i n t s , up to 20 n u c l e i were counted. For P_. grami ni s, the maximum nuc lear number that was seen was fou r . Maheshwari et a l . (1967b) were the f i r s t and on ly ones to study the e n t i r e sequence of nuc lear development dur ing d i f f e r e n t i a t i o n apart from the host. Using bean r u s t , they found that the appressor ium normal ly had 8 n u c l e i , wh i l e up to 16 nuc le i were observed in the i n f e c t i o n hypha. There have been even fewer s tud ie s of nuc lear form. S a v i l l e (1939), as part of a d e t a i l e d study of the complete l i f e c y c l e of numerous rust s pec i e s , desc r ibed the nuc l e i of uredospores germinat ing on t h i n agar f i l m s . Un fo r t una te l y , i n f e c t i o n s t r u c t u r e s were not mentioned. Nuc le i were desc r ibed as e i t h e r "expanded" or "unexpanded". The mature uredospore had an "expanded" type of nucleus in which a l l the chromatin lay in an outer sphere, the " e c to sphe re " . Upon ge rminat i on , the nuc l e i reverted to the "unexpanded" s t a t e , that i s , they decreased in s i z e and l o s t t h e i r 18 "endospheres " . These two nuc lear forms were a l s o r e f e r r ed to by C r a i g i e (1959) when he germinated spores of P_. hel i an th i . Nuc le i were "expanded", then changed to arrowhead and "unexpanded" forms. Th i s l a t t e r form was i n t e rp re ted as being r e g u l a r l y a s soc i a ted w i t h nuc lear d i v i s i o n . MACROMOLECULAR SYNTHESIS DURING MORPHOGENESIS: The i n i t i a t i o n of development from the dormant s t a t e i s accompanied by increased s y n t h e t i c a c t i v i t y of the c e l l , p a r t i c u l a r l y of macromolecules such as DNA, RNA, and p r o t e i n . In teres t has r e cen t l y increased in these a c t i v i t i e s as they r e l a t e to the c o n t r o l o f development in the germinat ing and d i f f e r e n t i a t i n g uredospore. Uredospores possess a l l the necessary requirements f o r germinat ion apart from the host. Dormant bean rust spores conta in RNA wi th the p rope r t i e s of messenger RNA. This RNA, w i th a sedimentat ion value between 4-19S-j s t imu la te s amino ac id i n co rpo ra t i on in a c e l l - f r e e system from E_. cojl_i_ depleted of messenger (Ramakr i shnan and Stap les 1970). Funct iona l polyr ibosomes and a c t i v a t i n g enzymes are a l s o present in the r e s t i n g spore (Stapl es et a 1. 1968). As expected, germinat ing uredospores can synthes i ze , p r o t e i n , but they do so a t low l e v e l s compared w i t h sap rophy t i c fungi (S tap les e t a_l_. 1962, S tap les 1968). The i n co rpo ra t i on of l 2 t C - l e u c i n e occurs at a l i n e a r r a t e ; degradat ion ra tes are s i m i l a r . In a d d i t i o n , there i s no increase in t o t a l p r o t e i n n i t r ogen content dur ing ge rminat ion , whereas f o r saprophytes, such as A. n i g e r , increases are l o g a r i t h m i c . F i n a l l y , exogenous amino ac id s do not s t imu l a te p r o t e i n synthes i s as they do f o r saprophyt i c f u n g i . These are a l l i n d i c a t i o n s t h a t , dur ing ge rminat ion , 19 p ro te i n is only synthes i zed by turnover of e x i s t i n g amino ac ids such that there i s no ' ne t i n c r e a s e ' in s yn the s i s . In the bean rust uredospore, the p ro te i n s yn the s i z i n g machinery breaks down a f t e r approximately 4-6 hours as determined by the i nco rpo ra t i ng a c t i v i t y of ribosomes ex t r ac ted from spores a f t e r var ious stages of germinat ion (Staples 1968) - This d e c l i n e in a c t i v i t y is thought to be c o n t r o l l e d by e i t h e r the na tu ra l messenger RNA or a f a c t o r a s soc ia ted w i th the ribosome such as an enzyme. C e l l - f r e e p ro te i n synthes i s (measured by using amino ac id i n co rpo ra t i on by microsomes from germinat ing spores) dec l i n ed from 2 hours when na tu ra l messenger was used, but d i d not change when i n co rpo ra t i on of pheny la lan ine was measured us ing a s y n t h e t i c messenger (poly U). This suggests a d e c l i n e in natu ra l m-RNA. As j u s t s t a t e d , microsome i n co rpo ra t i ng a c t i v i t y remained f a i r l y constant dur ing e i gh t hours of germinat ion when poly U was used. However, i f microsomes were f i r s t washed w i th sod i urn "deoxy.chb l a t e - (D0G):i andcthe resu.lfi.ng ribosomes supplemented w i th s o l ub l e t rans fe ra se s from dormant spores, pheny la lan ine i n co rpo ra t i on dec l i ned l i n e a r l y dur ing the e i gh t hour germinat ion per iod (Yaniv and Staples 1969) - This po in t s to a f a c t o r , present in germinat ing spores, but not in dormant ones, which i f removed, causes ribosome a c t i v i t y to dec l i n e . R i bonuc le i c ac id is a l s o synthes i zed dur ing ge rmina t i on , but at a constant rate as determined by u r i d i n e l a b e l l i n g . Most i s in the form of r-RNA and t-RNA. It i s thought that RNA, l i k e p r o t e i n , is synthes i zed under ' t u r n o v e r ' c o n d i t i o n s , s i n ce ribosomes do not accumulate dur ing germinat ion (Staples et a 1. 1971). 20 The p a t t e r n s o f RNA s y n t h e s i s i n g e r m i n a t i n g and d i f f e r e n t i a t i n g s p o r e s a r e d i f f e r e n t . Even though the dormant s p o r e has t e m p l a t e a c t i v i t y (mentioned a b o v e ) , some new t e m p l a t e RNA i s s y n t h e s i z e d d u r i n g g e r m i n a t i o n . However, when the a c t i v i t y . o f b u l k RNA, e x t r a c t e d from s p o r e s a f t e r v a r i o u s s t a g e s of development, i s t e s t e d u s i n g a c e l l - f r e e system from E. c o 1 i d e p l e t e d o f messenger, t e m p l a t e a c t i v i t y d e c l i n e s a f t e r 4 hours i n g e r m i n a t i n g s p o r e s , but i n c r e a s e s between 4 and 6 hours i n s p o r e s induced t o form a p p r e s s o r i a ( S t a p l e s ejt_ a_l_. 1971. Ramakrishnan and S t a p l e s 1970). P u 1 s e - 1 a b e 1 1 i n g e x p e r i m e n t s w i t h g e r m i n a t i n g and d i f f e r e n t i a t i n g s p o r e s a l s o show t h a t t h e r e i s i n c r e a s e d s y n t h e s i s o f t e m p l a t e RNA from 4-5 hours when a p p r e s s o r i a l f o r m a t i o n o c c u r s . In a d d i t i o n , the i n c o r p o r a t i o n o f H - u r i d i n e i n t o RNA o f d i f f e r e n t i a t i n g s p o r e s i s 3 times t h a t o f g e r m i n a t i n g ones. T h i s RNA i s thought t o be a messenger type s i n c e i t i s l o c a t e d i n the 4-19S r e g i o n o f s u c r o s e d e n s i t y g r a d i e n t s . S e c o n d l y , when RNase i s added t o t h e r a d i o a c t i v e RNA, the polysomes a r e c o n v e r t e d t o monosomes and a t the same tim e they l o s e r a d i o a c t i v i t y . However, r a d i o a c t i v i t y i s not r e c o v e r e d i n t h e monosome f r a c t i o n . T h e r e f o r e , i t i s thought t o be i n t h e m-RNA (Ramakrishnan and S t a p l e s 1970b). The c o l l o d i o n membrane s u r f a c e ^ o n - w h i c h bean r u s t u r e d o s p o r e s a r e g e r m i n a t e d and d i f f e r e n t i a t e d has now been shown t o have an e f f e c t at the moleeuilac." l e v e l . U r e d o s p o r e s g e r m i n a t i n g on membranes i n c o r p o r a t e d t h r e e 14 t i m e s more C-pheny1 a 1 any 1 t-RNA than s p o r e s g e r m i n a t i n g on w a t e r , i f n a t u r a l messenger was used. D i f f e r e n t i a t e d s p o r e s had f u r t h e r i n c r e a s e s i n a c t i v i t y . However, ribosomes were e q u a l l y a c t i v e r e g a r d l e s s of the mode of g e r m i n a t i o n when a s y n t h e t i c messenger was used. I n d i c a t i o n s were t h a t the membrane was r e s p o n s i b l e f o r i n c r e a s i n g the number o f ribosomes a t t a c h e d t o 21 the na tu ra l m-RNA. This would r e s u l t in increased i n i t i a t i o n of p r o t e i n chains (Yaniv and Stap les 1974). With regard to the wheat rust fungus, membranes are not requi red f o r d i f f e r e n t i a t i o n , and in agreement w i th t h i s , i t waslfound that membranes were not needed to increase ribosomal a c t i v i t y (Staples et_ a]_. 1972) . Studies on DNA synthes i s have only r e cen t l y begun. Never the le s s , i t has a l ready been shown w i th Uromyces p h a s e o l i , using the i nco rpo ra t i on of 3 H-adenosii>ne i n t o DNA f r a c t i o n e d on CsCl gna'diiients, t t h a t t ' t h e t t ype r o f DNA synthes i s is i n f luenced by whether the spores are germinat ing or 1 : " d i f f e r e n t i a t i n g (Staples 1974). During ge rminat i on , r a d i o a c t i v i t y bands at a dens i t y c h a r a c t e r i s t i c of m i tochondr i a l DNA. If i n f e c t i o n s t r u c t u r e s are not induced, m i tochondr i a l DNA synthes i s cont inues . There is l i t t l e or no nuc lear DNA s yn the s i s . However, i f i n f e c t i o n s t r u c t u r e s are induced, the synthes i s of nuc lear DNA begins when the appres so r i a s t a r t to form, so that both types of syntheses take p lace concomi tant l y . Then, dur ing the per iod of v e s i c l e f o rmat i on , m i tochondr i a l s ynthes i s s tops , leav ing on ly the nuc lear component (Staples 1974). The report s descr ibed here show that uredospores s yn thes i ze DNA, RNA, and p r o t e i n dur ing morphogenesis and that these a c t i v i t i e s occur dur ing s p e c i f i c developmental s tages. In a d d i t i o n , the patterns of syntheses d i f f e r in germinat ing and d i f f e r e n t i a t i n g s p o r e l i n g s . However, the t iming of syntheses, e s s e n t i a l f o r the var ious developmental s tages , as opposed to ac tua l s y n t h e s i s , i s poor ly understood. As w e l l , most of the work to date has been done w i th Uromyces phaseo l i (bean r u s t ) . L i t t l e is known concerning the wheat rust organism. 22 DIFFERENTIATION AND FURTHER GROWTH: What is the role of infection structures for further growth, both on the host plant and apart from the host in axenic culture? Are infection structures always produced during host penetration? Are they necessary for fungal growth and infection of the host plant? These are questions which have not been f u l l y answered to date. The fact that infection structures are seen on the host during penetration of the epidermis, does not necessarily show that they are essential for survival beyond the penetration phase during the infection period. Two attempts have been made to study this issue usingetherhost plant. Dickinson (19^9) placed membranes, containing spores of P_. t r i t i c i n a which had either germinated or di f f e r e n t i a t e d , face down on the exposed mesophyll of wheat leaves from which the epidermis had been removed. Haustoria were produced in host mesophyll c e l l s from only those spores which had formed vesicles. There was no sign of mesophyll penetration from spores of the other group. This suggested that vesicles were required for infection. Wfie dormant to (ungermi nated) spores were used and were placed at the junction of epidermis and mesophyll, when only part of the host epidermis was removed, infection structures and haustoria were produced, but only on the epidermis side. On the exposed mesophyll side, vesicles were not formed and penetration of mesophyll was not observed. Experiments of Charkravarti (1966) d i r e c t l y contradicted this last point. Spores of P_. graminis t r i t i c i , placed on exposed mesophyll, formed appressoria and did infect the host as evidenced by the production of haustoria and 23 s p o r u l a t i o n a f t e r 8 days. The r o l e of morphogenesis in determin ing the growth of the rus t fungus in axenic c u l t u r e without the host a l s o remains a ques t i on . The major components of uredospores; t r e h a l o s e , sugar a l c o h o l s , amino, f a t t y , and organ ic a c i d s , malonic a c i d , and the intermediates of the TCA c y c l e , are a l l s i m i l a r to those of saprophyt i c fungi (Shaw 1963). Indeed, the wheat rust fungus can now be cu l t u r ed a x e n i c a l l y on a r t i f i c i a l media (Wi l l iams et_ aj_. 1966). In f a c t , P_. gramin i s t r i t i c i has r e cen t l y been grown on ' c h e m i c a l l y d e f i n e d ' subs t ra tes (Foudin and Wynn 1972, Bose and Shaw 197^a). However, the complete u r e d i a l s tage, i n c l ud i ng s p o r u l a t i o n , i s r a r e l y ob ta ined , p a r t i c u l a r l y in the case of the North American races. The f a c t o r s c o n t r o l l i n g the t r a n s i t i o n from the spo re l i n g to the axen ic colony are not understood. Growth cannot always be ob ta i ned , even f o r the A u s t r a l i a n races , and i f growth does occur , i t is o f ten e r r a t i c f o r reasons which cannot be i d e n t i f i e d (Kuhl et a 1. 1971)• A l l repor t s of the axen ic c u l t u r e of P_. gramin i s t r i t i c i agree on one major p o i n t , that the success of colony i n i t i a t i o n is r e l a ted to the dens i t y of the spores used f o r seed ing. Cu l tu re of s i n g l e spores has not been achieved to date. The higher the inoculum d e n s i t y , the greater the chance of colony i n i t i a t i o n (Wi l l iams et a 1. 1966, Kuhl et a 1. 1971)• In a d d i t i o n , races of P_. graminis t r i t i c i d i f f e r in the minimum dens i t y of spores they requ i re to produce v i s i b l e c o l o n i e s . Ha r t l ey and Wi l l i ams (1971a) found that some A u s t r a l i a n races such as 126-ANZ-6,7 needed 2 approx imately 200 spores/cm , whereas <for others such as 111, amounts as 5 2 high as 10 spores/cm were necessary. The reason f o r such large numbers i s 24 not known. However, the p o s s i b i l i t y e x i s t s that e i t h e r phy s i ca l or chemical contact between spo re l i ng s is e s s e n t i a l f o r f u r t h e r development. For example, Bose and Shaw (1971) found t h a t , f o r P_. graminis t r i t i c i , anastomosis of germ tubes was a p r e r e q u i s i t e f o r colony f o rmat i on . Kuhl et  a 1 . (1971) reported that colony i n i t i a t i o n of th in1y-seeded spores , i nocu la ted on one s i de of an agar b l o ck , increased two to t h r e e - f o l d i f t h i c k spore seedings were app l i ed to the oppos i te agar f a ce . There have a l s o been repor t s of c e r t a i n races s t i m u l a t i n g the growth of others when inocu la ted in mixed c u l t u r e s (Hart ley and W i l l i ams 1971b). It has not been e s t ab l i s hed how, or i f , d i f f e r e n t i a t e d spores i n f l uence axeni.c growth. Few report s even i n d i c a t e whether i n f e c t i o n s t r u c tu re s are produced in the c u l t u r e medium. SSomeaaUthors i j idd i i catetthat i lifif ect-i)on s t r u c t u r e s have been r o u t i n e l y ppdduced as part of colony i n i t i a t i o n (Har t ley and W i l l i ams 1971a), others that they have not (Bose and Shaw 1974a 1974a), and others that co l on i e s were produced sometimes from -u n d i f f e r e n t i a t e d and sometimes from d i f f e r e n t i a t e d spores (Wi l l iams and Ha r t l e y 1971). W i l l i ams (1971) has concluded that d i f f e r e n t i a t i o n is necessary to ob ta in d i k a r y o t i c mycelium and that co l on i e s e s t ab l i s hed from germ tubes are monokaryot ic. However, the nuc lear number in these s tud ie s appears to be q u i t e v a r i a b l e . Further s tud ie s are necessary. 25 MATERIALS AND METHODS UREDOSPORE PRODUCTION ON HOST: Uredospores used f o r the present study were of race 15B-2 Pucc i n i a  graminis t r i t i c i E r i k s s . and Henn. and were obtained from the USDA Co-operat i ve Rust Laboratory , U n i v e r s i t y of Minnesota. Spores were r o u t i n e l y propagated on wheat seed l ings (T r i t i cum aestivum L. va r . L i t t l e C l ub ) . 1) NON-STERILE UREDOSPORES: P lant s were grown in a P e r c i v a l growth chamber w i t h a 16 hour photoper iod of 900 f t - c and temperatures of 25°C l i g h t , 18°C dark. Leaves were inocu la ted by su r face contact e i gh t days a f t e r the p lants were sown. A mixture of t a l c and spores was used in a r a t i o of 3:1, made i n to a paste by the a d d i t i o n of tap water. To f o s t e r high humid i ty , p lant pots were set on t rays con ta in i ng 2 cm water , covered w i th p l a s t i c , and incubated at 18°C f o r 30 hours. The p l a s t i c was then removed and the p lant s were returned to t h e i r normal photoper iod. Spores were harvested beginning on the 8th to the 12th day a f t e r i n o c u l a t i o n by l i g h t l y tappingt'theldieav.esooverr.a-jpiece, of weighing paper so that only mature spores would f a l l . Harvested spores were subsequently s tored at k°C f o r no longer than 12 hours p r i o r to exper imental use. Germination was u s u a l l y 90-100 percent . 26 2) ASEPTIC UREDOSPORES: The technique f o r r a i s i n g s t e r i l e spores was the same as that f o r n o n - s t e r i l e spores up u n t i l the f l e c k i n g stage (6 days a f t e r i n o c u l a t i o n ) . At t h i s t ime , the leaves were exc i sed and su r face s t e r i l i z e d in 20 % Chemtech bleach w i t h 5.25 % a v a i l a b l e c h l o r i n e , f o r 6 minutes, fo l l owed by washing in 3 changes of s t e r i l e d i s t i l l e d r^O. The a d d i t i o n of a few drops of Tween 80 per l i t r e of b leach helped to wet the e n t i r e leaf s u r f ace . A f t e r being d r i ed w i th a s t e r i l e paper t owe l , each leaf was p laced v e r t i c a l l y i n t o a t e s t tube con ta i n i n g B5 medium (Gamborg et a 1. 1968) w i th 1 % agar (Table C - l ) . The cut end was in ser ted 5 mm below the medium su r f ace . Tubes were incubated at 18°C under continuous f l u o r e s cen t l i g h t of 400 f t - c u n t i l abundant uredospore product ion was ev ident (8 to 10 days ) . Spores were c o l l e c t e d by tapping the leaf w i th s t e r i l e forceps over a s t e r i l e p e t r i e d i s h . Storage was at 4°C, as f o r n o n - s t e r i l e spores. A s e p t i c uredospores d id not have as great a c apac i t y f o r germinat ion as n o n - s t e r i l e ones. Germination va r i ed from 55 to 90 percent . UREDOSPORE GROWTH IN VITRO: 1) GERMINATION AND DIFFERENTIATION: I n i t i a l d i f f e r e n t i a t i o n t r i a l s were done us ing the method of Maheshwari et a 1. (1967a). Spores were d i spersed on 2 ml 0.01 M Ca-K-P0^ b u f f e r , pH 7-0, in the cent re w e l l of a Conway d i f f u s i o n c e l l . In the outer w e l l , k ml 10 M nonyl a l coho l were p l aced . Dishes were covered and 27 Table C-1 B~5 medium, .composition and p repa ra t i on . This medium is used to support s t e r i l e wheat leaves as they produce a new crop of a s e p t i c uredospores. 5 M e d i u m 28 (Gamborg et a 1. 1968) Stock s o l u t i o n - 1 0 X normal concen t ra t i on T A B L E C - l INGREDIENT AMOUNT/LITRE DISTILLED H 0 N a h y C y h ^ O 1.5 g KNO 3 25 g ( N H 4 ) 2 S 0 4 1.34 g MgS0 i t.H 20 25 g C a C l 2 . 2 H 2 0 1.5 g Sequestrene 330.Fe 0.28 g N i c o t i n i c Ac i d 0.01 g Thiamine-HCL 0.1 g Pyr idox ine-HCL 0.01 g m-1nos i t o l 1 9 MnSO^.h^O 0.1 g H 3 B0 3 0.03 g ZnS0 2 >.7H 20 0.02 g Na 2Mo0^.2H 20 2.5 mg CuSO^ 0.25 mg CoCl 2.6H 20 0.25 mg Kl 7-5 mg Sucrose 200 g 2,4-D 0.02 g -pH of s tock s o l u t i o n ad ju s ted to 5.5 p r i o r to au toc l a v i n g -when ready to use, B d i l u t e d 10 X, 1 % agar added, au toc laved aga in 29 set in darkness under the f o l l o w i n g temperature regime; 20°C f o r the f i r s t 2 hours, 30°C f o r the next 1 1/2 hours, and 20°C f o r the remaining 18 hours. Although germinat ion on t h i s medium was good, d i f f e r e n t i a t i o n was poor, ranging from 0-6 % of the germinated spores. S ince t h i s was not cons idered adequate, an a l t e r n a t i v e d i f f e r e n t i a t i o n medium was developed. Var ious subs t ra tes were tes ted (Table C-2). The pH of a l l media was adjusted p r i o r to au toc l a v i ng at 15 ps i f o r 15 minutes. A l l t e s t s were c a r r i e d out as above us ing Conway d i f f u s i o n c e l l s . Only the sub s t ra te in the cen t re we l l was changed. MPG MEDIUM: The se l ec ted med i urn, rea 1 dtedMMPG , was a l i q u i d , pH 6.8, c o n s i s t i n g of a Ca-K-P0^ bu f f e r w i th the a d d i t i o n of ' Evans i peptone and D-glucose (Table C-3) • D i f f e r e n t i a t i o n on t h i s medium was good: approx imate ly 20 to 70 % of the germinated spores formed i n f e c t i o n s t r u c t u r e s , depending upon the spore l o t used. To min imize v a r i a t i o n , a spore l o t was never changed w i t h i n an exper iment, that is on ly one l o t was used. PROCEDURE: Uredospores were rout i ne 1 yggermi.nated3 and' 'd i f f e r e n t i ated accord ing to the f o l l o w i n g procedure. Fresh uredospores were d i spersed in 1 ml double g las s d i s t i l l e d h^O in a po ly s ty rene weighing boat, k\ mm square. A uniform su r face f i l m of spores was e s s e n t i a l and. was produced w i th a rap id back and f o r t h motion of a p lat inum i no cu l a t i n g loop. A f t e r 1-2 minutes, a l oop fu l of spores was t r an s fe red from the boat to 1.5 ml MPG medium in the inner w e l l of a Pyrex Conway d i f f u s i o n c e l l and was v i g o r ou s l y mixed w i th the medium. The loop had a diameter of 1 cm. The 30 Table C-2 Media tes ted dur ing d i f f e r e n t i a t i o n t r i a l s , composit ion and p r epa r a t i on . Two bas ic media were used: Ca-K-P0, bu f f e r and W i l l i a m ' s n u t r i e n t medium. Ingredients were e i t h e r added to or subt racted from each of the ba s i c media. M e d i a f o r D i f f e r e n t i a t i o n T r i a l s TABLE C -2 MEDIUM DERIVED FROM L"^> Ca-K-P0^. buffer Q.0J..M, pH 7.0 . (Matteshwari. J967a) Will iams nutr ient medium pH 6.4 (Williams 1971) NAME OF HEDIUH _ ^ Buffer Buffer •HUagar Buffer Buffer +KCL MPG Wi11iams Wi11iams -KUagar Wi 11 iams -Peptone -G1 ucose W i l l i ams -G1ucose W i l l i ams -MgSO^ Wi 11iams -FeSO^ INGREDIENT jg/1 g lass +NaN03 1 d i s t . H 0 0.025 + + + + + K.HP0,, 1 .Iks + + + + + + + + + + 0.449 + + + + KCL 0.25 + + + + + + + Peptone (Evans 5.0 + + + + + + D-glucose 30.0 + . + + + + NaN03 3.0 + + + + + + + HgS0 4.7H 20 0.25 + + + + F e S O ^ H ^ 0.005 + + + + N a 3 C 6 H 5 0 7 . 2H 2 0 1.5 + + + + + + Agar (Bacto) 0.1 + + pH adjusted to 6.8 wi th: Phosph A c i d -KOH Phosph A c i d -KOH HNO -NaOH KCL-KOH Phosph HCL-ftcid- M nu KOH HCL-NaOH HCL-NaOH HCL-NaOH HCL-NaOH HCL-NaOH 32 Table C-3 MPG, the d i f f e r e n t i a t i o n med i urn,, compos i t i on and p repa ra t i on . Nonyl a l coho l was a l s o used to ensure good ge rmina t i on , but was not necessary w i th f r e sh spores. 33 T A B L E C - 3 D i f f e r e n t i a t i o n M e d i u m INNER WELL OF CONWAY CELL OUTER WELL OF CONWAY CELL MPG medium -4 1.5 x 10 M Nonyl a l coho l ( V o l a t i l e germinat ion s t imu lan t ) C a ( H 2 P 0 Z ( ) 2 . H 2 0 0.025 g STOCK SOLUTION: K HPO. 1.145 g 0.0247 ml of 3.5 M nonyl a l coho l was added to 1 l i t r e g la s s d i s t . KH 2 P0 i f 0.449 g H 20 us ing a m i c r o l i t r e s y r i n ge , c apac i t y 0.05 ml . Peptone 5 g mixture was son icated using an D-g1ucose 30 g u l t r a s o n i c a t o r f o r 15 minutes or volume made to u n t i l a l c oho l d rop l e t s d i spe r sed . 1000 ml g la s s d i s t . H 20 Stock s o l u t i o n d i l u t e d 4 X f o r use The Ca(H 2 P0^) 2 H 20 was mixed in D i s t . H 2 0 w i t h KH P0. . The K^PO^ was added unt i1 pH 6.8 was reached. Peptone and g lucose were added. M i x tu re was s t i r r e d w h i l e heat ing u n t i l peptone d i s s o l v e d . pH was read and ad jus ted as necessary w i t h phosphor ic a c i d o r KOH. Media was poured i n t o tubes and au toc l a ved . A s l i g h t a l t e r a t i o n in c o l ou r was noted a f t e r a u t o c l a v i n g and was normal. 34 f i n a l concent ra t i on of spores va r i ed w i th the type of exper iment, but was 2 approximately 50-100 spores/mm (20,000-40,000 spores/ml). Three ml 1.5 x 10 ^ M nonyl a l coho l (see Table 0 3 f o r p reparat ion ) were then t r a n s f e r r e d to the outer chamber of the d i f f u s i o n c e l l . The e n t i r e procedure was c a r r i e d out w i t h the dishes on i c e . The Conway ves se l s were sealed by app ly ing s i l i c o n stopcock grease (Dow Corning) to the rim and press ing the f l a t top i n t o the grease. They were then covered w i t h aluminum f o i l to exclude l i g h t . For ge rminat i on , the Conway dishes were incubated at 18°C f o r 17-18 hours without i nterntiptii>on . Tilioi liiridueedd i f feueiiit i at<iion , they were incubated at 18°C f o r 2 hours, fo l l owed by a heat shock at 30°C f o r 1 1/2 hours, then back to 18°C f o r 14-15 hours. 2) AXENIC CULTURE: Two bas i c media were used f o r axenic c u l t u r e t r i a l s ; MPG n u t r i e n t medium (Table C~3) and a chemica l l y def ined l i q u i d sub s t ra te (Bose and Shaw 1974b) r e f e r r e d to here as AXENIC medium (Table C-4). For most exper iments, a two - s tage medium was used. Spores were i n i t i a l l y placed i n to the s t a r t i n g medium, e i t h e r MPG or AXENIC. Some treatments were heat shocked. A f t e r a s u i t a b l e i n t e r v a l (2 hours to 4 days ) , the medium was rep laced. This f i n a l medium was e i t h e r the same as the s t a r t i n g medium or the a l t e r n a t i v e s ub s t r a t e . At p e r i o d i c i n t e r v a l s of 3 - 4 weeks, the f i n a l medium was replaced w i th f re sh s ub s t r a te . Ca-K-PO^ b u f f e r , d i s t i l l e d H^O, and tap water were a l s o used as t e s t media where s p e c i f i e d . A sep t i c uredospores were used f o r a l l axenic c u l t u r e exper iments. 35 Table C-4 AXENIC c u l t u r e medium, composition and preparation. 36 TABLE C-4 AXENIC CULTURE MEDIUM AXENIC MEDIUM M I CRONUTRIENTS Medium B + (Bos Glutamine, C y s t e i n e , Na e and Shaw 1974b) c i t r a t e (Turel 1969) INGREDIENT AMOUNT/LITRE h^O INGREDIENT AMOUNT/200 ml D-glucose 30 g Seques trene (13 %) 10 g KH 2P0 A 1 9 MnSO^.h^O 0.447 g KCL 0.5 g Kl 0.01 g Na c i t r a t e 1.5 g Ni C l 2 . 6 H 2 0 0.018 g NaN03 2 g CoCl 2.6H 20 0.018 g Mg S O ^ ^ O 0.5 g 0 0.042 g FeS0 i f.7H 20 0.01 g ZnS0 r7H 20 0.0623 g L-g]utami ne (42 mM) 6.136 g CuSO^.H 0 0.015 g L-cyste i ne (3 mM) 0.363 g - BeSO^ 0.02 g C a ( N 0 3 ) 2 2 g H 3 P 0 4 0.01 g mi c r o n u t r i e n t s 0.8 ml H 2S0^ (cone. ). 0.2 ml Ingredients were d i s s o l v e d without heat. Ca(N0,) 5 was added l a s t to avoid p r e c i p i t a t i o n and the pH then adjusted t o 5.5 w i t h NaOH. Medium was f i l t e r e d though a Nalgene m i l l i p o r e f i l t e r , pore s i z e 0.2 u (Sybron Corp. Rochester USA) Mi c r o n u t r i e n t s do not d i s s o l v e completely i n t h i s amount of HO Therefore, shake mixture vigour-ously before use. 37 S t e r i l e techniques were observed^and work.was c a r r i e d out in a laminar a i r f low cab i ne t . Two methods of i n o c u l a t i o n were used. The f i r s t method was employed f o r a l l 'en masse 1 exper iments. 'En masse' r e fe r s to the f a c t that spores were grown w i th no phy s i ca l b a r r i e r s p laced between them. In these t r i a l s , the inoculum dens i t y v a r i ed 2 from 1 to 10 spores/mm (300 to 3000 spores/ml) depending upon the exper i ment. Spores were f i r s t d i spersed as a t h i n f i l m on the su r face of h ml medium in a small p l a s t i c p e t r i d i s h (60mm x 15mm, 5cm d iameter ) . This was used as the inoculum source f o r a l l treatments w i th the same s t a r t i n g medium. One l oop fu l of spores was then t r a n s f e r r e d to 2.5 ml s t a r t i n g medium in a treatment p e t r i d i s h ( p l a s t i c , 35mm x 10mm, 3•7cm d iameter ) . O c c a s i o n a l l y , 50 ml erlenmeyer f l a s k s (9ml medium) and Pyrex Conway d i f f u s i o n c e l l s (2ml medium) were used. Uredospores were v i go rou s l y mixed w i th the medium and any p la te s showing clumps were d i s ca rded . The second i n o c u l a t i o n technique was employed f o r a l l ' s i n g l e spore ' exper iments. They were so named because uredospores were inocu la ted s i n g l y , and unless o therwi se s t a t e d , each spore was grown in i t s own compartment i s o l a t e d from the o the r s . Spores were tapped i n to an empty p l a s t i c p e t r i d i s h and shaken l a t e r a l l y to produce a sparse f i l m on the d i sh bottom. Excess spores were t ipped out . Each spore^wassihddivi'dua.li 1 yytransferreddtootheemediurn us ing a s i n g l e s t e r i l e eyebrow h a i r j o i n ed to a wooden a p p l i c a t o r . The spore adhered we l l to the ha i r and was re leased immediately upon contact w i th the 38 medium. Un fo r tuna te l y , uredospores could not be removed again from the medium us ing t h i s method. The e n t i r e t r a n s f e r operat ion was c a r r i e d out under a Cenco d i s e c t i n g microscope which had been sur face s t e r i l i z e d . Although t ed i ou s , t h i s i n o c u l a t i o n method was super io r in that spore numbers cou ld be s t r i c t l y c o n t r o l l e d . P r e l im i na r y s i n g l e spore t r i a l s were done by spo t t i ng d rop le t s of medium onto the bottom of a p l a s t i c p e t r i d i s h and subsequently i n o cu l a t i n g one spore per d r o p l e t . However, the convex meniscus permitted the spore to f a l l from the top middle of the d r o p l e t , down to the bottom edge, making v iewing d i f f i c u l t . P l a s t i c m i c ro te s t p l a te s ( # 3034 Falcon P l a s t i c s , Oxnard, Ca)^were found to be an acceptab le a l t e r n a t i v e and were r o u t i n e l y used f o r s i n g l e spore exper iments. A p l a t e had 60 w e l l s , each con ta in i ng 10 u l medium. A s i n g l e spore was placed i n to each w e l l . Viewing was ea s i e r s i n ce the spore f r e q u e n t l y remained in the cent re of the concave meniscus. On occa s i on , g la s s v i a l s (10mm diameter x 12mm high) were a l s o used. Each was f i l l e d w i th 0.08 ml medium and was capped w i th aluminum f o i l . Media f o r a l l axen ic experiments were kept at 4°C u n t i l i n o c u l a t i o n was complete. A f t e r i n o c u l a t i o n , a l l d ishes and t e s t ve s se l s were sea led w i th p a r a f i l m and were incubated as descr ibed f o r germinat ion or d i f f e r e n t i a t i o n . Dishes were kept from 2 days to 3 months depending on the treatment and were viewed at i n t e r v a l s of from 1 to 7 days . ; 39 3) CRITERIA FOR ASSESSMENT OF IN VITRO DEVELOPMENT: Three developmental stages were assessed; ge rm ina t i on , d i f f e r e n t i a t i o n , and myce l i a l growth. For the f i r s t two s tages , the f o l l o w i n g c r i t e r i a were used. A spore was cons idered to be germinated i f the length of the germ tube was equal t o , or g rea te r than, i t s own spore d iameter. A l l others were counted as not germinated. For d i f f e r e n t i a t i o n , the complete development of the appressor ium, peg, and v e s i c l e was r equ i r ed , although the i n f e c t i o n hypha was normal ly not cons idered necessary. Component i n f e c t i o n s t r u c t u r e s ( app re s so r i a , pegs, v e s i c l e s , hyphae) were i n d i v i d u a l l y assessed in many exper iments. A l l s po re l i ng s which had been inocu lated, ' ennmasse ' i n to a common medium were counted as f o l l o w s . For each r e p l i c a t e v e s s e l , 3 separate areas were m i c r o s c o p i c a l l y viewed us ing a l i ned ocu l a r i n se r t which de l i nea ted an area 310 u square. The number of t o t a l spores , germinated spores, and d i f f e r e n t i a t e d spores were counted w i t h i n each square. For ' s i n g l e spore ' exper iments, a l l w e l l s were assessed i n d i v i d u a l l y and counts recorded. D i f f e r e n t i a t i o n was normal ly c a l c u l a t e d as a percentage of the t o t a l spores, but was based occasiona11y on germinated spores. ! M y c e l i a l growth, the t h i r d s tage, r e f e r r ed to development beyond germinat ion and d i f f e r e n t i a t i o n or to any hypha1 growth v i s i b l e to the naked eye. Th i s was d i f f i c u l t to measure a ccu ra te l y in terms of spore numbers because of v a r i a t i o n s in inoculum dens i t y w i th 'en masse' seedings: consequent ly , no attempt was made to weigh the mycelium. Never the le s s , w i t h i n each exper iment, presence or absence of growth was determined and treatments were visuadi ly compared. 40 DISTILLATION OF QRUDE GERMINATION INHIBITOR: Attempts were made to i s o l a t e the d i f f e r e n t i a t i o n s t i m u l a t o r w i th the aim of improving d i f f e r e n t i a t i o n percentages of s i n g l e spores. The crude germi nat ion i nh i b i t o r was f i r s t obta i ned as s t a r t i ng materia1 us i ng the method of A l l e n (1957)• Twenty mg of 2 week o ld uredospores were placed i n to 7.0 ml double g l a s s - d i s t i 1 led H^O in a round bottom f ]a sk (50ml c a p a c i t y ) . The mixture was v i g o rou s l y shaken, then incubated at 18°C in the dark f o r 5 hours. A f t e r a 1 ml a l i q u o t of crude i n h i b i t o r was removed, the re s t was d i s t i 1 led and c o l l e c t e d in 1 ml f r a c t i o n s in an ice bath. A l l f r a c t i o n s were placed in pyrex tubes t i g h t l y sealed w i th ground g la s s s topper s , l u b r i c a t e d w i th s i l i c o n grease, and s tored at 4°C f o r 24 hours p r i o r to use. MORPHOLOGICAL AND CYT0L0GICAL. STUDIES: 1) STAINING: Sta ins were employed f o r both morphological and nuc lear development s t u d i e s . To f o l l o w morphological development, phloxine-KOH proved most e f f e c t i v e . Development was fo l l owed in the c u l t u r e ves se l i t s e 1 f , usua11y a Conway d i f f u s i o n c e l l or p e t r i d i s h . A f t e r the medium was removed, 5 to 10 drops of 5 % KOH were added, fo l lowed by 1 to 3 drops of 0.5 % ph lox i ne . A l l s t r u c t u r e s were s ta ined a b r i gh t red by t h i s method. Nuclear development of d i f f e r e n t i a t i n g uredospores was fo l lowed by s t a i n i n g w i th a mod i f ied Feulgen technique (Tirjilifr G^S')™ 'Theufcechn i que -41 Table C-5 Reagents used in the Feulgen s t a i n i n g technique. FEULGEN TECHNIQUE T A B L E C-5 P R E P A R A T I O N OF S L I D E S P R E - F I X A T I V E F I X A T I V E S C H I F F ' S R E A G E N T 1) S l i d e s c l e a n e d i n c h r o m a t e c l e a n i n g s o l u t i o n - d i s s o l v e 1 0 0 g p o t a s s i u m b i c h r o m a t e i n 8 5 0 m l H 2 0 - s l o w l y a d d 1 0 0 m l c o n c e n t r a t e d ^ S O ^ wh i 1 e s t i r r i n g - s o a k s l i d e s o v e r n i g h t i n s o l u t i o n - w a s h s l i d e s 3 h o u r s i n r u n n i n g t a p H^O - r i n s e i n d i s t . h^O 2 ) S l i d e s d i p p e d i n g e l a t i n c o a t i n g - d i s s o l v e 2 . 5 g g e l a t i n i n 5 0 0 ml w a r m d i s t . H 2 0 - a d d 0 . 2 5 g c h r o m i u m p o t a s s i u m s u l p h a t e - m a y b e n e c e s s a r y t o f i l t e r t o r e m o v e b u b b l e s - a d d 8 g p - d i c h l o r o b e n z e n e c r y s t a l s t o 2 0 0 ml d i s t . H 2 0 - h e a t and- s t i r - p o u r i n t o w a r m b o t t l e w h e n m o s t o f c r y s t a l s h a v e . d i s s a p e a r e d - s h a k e we 11 - k e e p a t 6 0 ° C f o r a f e w h o u r s t o o v e r n i g h t - f i 1 t e r w h i l e w a r m - l e t c o o l s l o w l y u n t i l r o o m t e m p e r a t u r e i s r e a c h e d - u s e i m m e d i a t e l y -3 p a r t s a b s o l u t e e t h a n o 1 -1 p a r t g l a c i a l a c e t i c a c i d - 2 p a r t s c h l o r o f o r m - b o i 1 2 0 0 m l d i s t . H20 - c o o l t o 7 5 ° C - a d d 3 g b a s i c f u c h s i n (BDH) - g r i n d t h i s f i r s t i n m o r t a r a n d • p e s t l e - s t i r u n t i1 d i s s o l v e d - c o o l t o 25°C - d i s s o l v e 6 g p o t a s s i u m m e t a b i s u l p h i t e i n 60 m l N HCL - a d d t h i s t o f u c h s i n s o l u t i o n - s h a k e - s t a n d 3 h o u r s t o o v e r n i g h t - d o n o t m a k e f l a s k a i r t i g h t - a d d 2 g a c t i v a t e d c h a r c o a l - s h a k e w e l l (10-20 m i n u t e s ) - l e t i t s t a n d f o r 30 m i n u t e s t h e n f i 1 t e r - s t o r e a t VC i n t i g h t l y s t o p p e r e d b o t t l e 43 was developed a f t e r t r i a l s us ing the standard Feulgen s t a i n (Maheshwari et  a 1 . 1967b) as we l l as a number of other nuc lear dyes such as methyl g reen -py ron in , methyl green a lone, haematoxy1 in, and a c e t o - o r c e i n , a l l y i e l d e d u n s a t i s f a c t o r y r e s u l t s . To improve the Feulgen procedure, experiments were run to t e s t ways of prepar ing S c h i f f ' s reagent, f i x a t i v e s , times of ac id h y d r o l y s i s , s t a i n i n t e n s i f i e r s , and c oun te r s t a i n s . The f o l l o w i n g Feulgen procedure was found to g i ve super io r r e s u l t s and was r o u t i n e l y used f o r nuc lear s t a i n i n g . Germ tubes and i n f e c t i o n s t r u c t u r e s , a f t e r var ious stages of growth were p r e f i x ed in saturated aqueous p-d ich lorobenzene (Meyer 1949) f o r 4 hours. P r e f i x a t i o n was done at 18°C in the c u l t u r e vesse l i t s e l f . T i s sue was then r insed b r i e f l y in d i s t i l l e d H 2 0, t r a n s f e r r e d to g e l a t i n coated s l i d e s (Jensen 19&2 p. 199) using a large p lat inum loop, and d r i ed on a s l i d e warmer set at 38°C. S l i d e s were subsequent-<ly placed in Carnoy ' s f i x a t i v e (Maheshwari 1966) f o r 10-12 hours and were q u i c k l y rehydrated in a descending a l coho l s e r i e s at room temperature. T i s sue was hydro lyzed w i th e i t h e r 5 N HCL f o r 20 minutes at room temperature of 1 N HCL f o r 5_6" minutes at 60°C. M a t e r i a l was r insed in co ld H2O and s ta ined w i th S c h i f f ' s reagent (modif ied from Gurr 1962: Table C-5) f o r 2 hours in the dark, f o l l owed by 6 changes' of H^O. At t h i s s tage, the t i s s u e was r o u t i n e l y mounted in g l y c e r i n e and H2O, mixed in a 1:1 r a t i o , O c c a s i o n a l l y , t i s s u e was f i r s t placed in 1 % t o l u i d i n e b1ue f o r 30 minutes, fo l l owed by 6 changes of H^ O and was subsequently mounted. Nuc le i were s ta ined from a pale pink to magenta w i t h Feulgen alone and magenta to deep blue w i th the Feu 1gen- to lu id ine blue method. Table C-5 shows the procedure 44 for preparation of various reagents used in the staining procedure; prefixative, fixative, Schiff's reagent, and gelatin coating for the slides. 2) METABOLIC INHIBITORS: The timings of DNA, RNA, and protein syntheses were studied with the aid of metabolic inhibitors known to specifically inhibit one or more of the above syntheses. The following inhibitors were used; 5-fluorouracil 100 ug/ml, actinomycin-D 5 ug/ml, a-amanitin 40 ug/ml, puromycin 100 ug/ml, and cycloheximide 10 ug/ml. All were obtained from Sigma corporation except for a-amanitin which was purchased from Calbiochem Inc, San Diego, Ca. MPG medium was added to the inner wells of Conway diffusion c e l l s , either with or without inhibitor. Spores were inoculated 'en masse' in amounts of approximately 50 spores/mm (20,000 spores/ml). Each inhibitor in MPG medium, was either added to, or removed from the Conway cells after various stages of uredospore growth. The time schedule is outlined on page 93 of the result section. To avoid disturbance of the sporelings, the medium was removed with a 1 ml syringe. After each medium change, sporelings were washed with several changes of fresh medium. Al l treatments were transferred to 4°C upon completion of the experiment (after 18 to 20 hours) and then stained with ph1oxine-K0H. All inhibitor experiments were repeated on three separate occasions except for those with a-amanitin and cycloheximide which were only run twice. Each experiment had from two to five replicate dishes. Three 45 sample count ings were taken from each r e p l i c a t e . Spore counts of each component s t r u c t u r e of the d i f f e r e n t i a t i o n stage were made as descr ibed e a r l i e r . A synthes i s was cons idered e s s e n t i a l i f morphologic development was prevented by the presence of the corresponding i n h i b i t o r . Data were analyzed by ' A na l y s i s of V a r i a n c e ' . Treatments were compared to the c o n t r o l f o r each morphologic category using Dunnett ' s Test (Steel and T o r r i e 1960 p. 111) to determine s i g n i f i c a n t d i f f e r e n c e s at the 0.05 and 0.01 l e ve l s of conf idence. DIFFERENTIATION AND HOST INFECTION: Experiments were c a r r i e d out to t e s t the a b i l i t y of spo re l i ng s to i n f e c t the host p lant a f t e r 30 hours of e i t h e r germinat ion or d i f f e r e n t i a t i o n . The exper imental plan i s shown on page 110-of the Re su l t s . For some t reatments , a po r t i on of l ea f epidermis was removed p r i o r to s p o r e l i n g i n o c u l a t i o n . I n i t i a l t e s t s were c a r r i e d out w i t h n o n - s t e r i l e spo re l i ng s and p l a n t s . However, these were u n s a t i s f a c t o r y due to , contaminat ion of the d i f f e r e n t i a t i o n medium and lea f nec ros i s at s i t e s of epidermis removal. Tibeeefjoke , i s . t e r i! l:essipor;es aan'd pp Lants Awere used f o r . a l l f u r t h e r exper iments. P l an t s were prepared as f o l l o w s . Wheat seeds (var . L i t t l e Club) were su r face s t e r i 1 i z e d in 20 % Chemtech bleach f o r 10 minutes fo l lowed by 3 changes of s t e r i l e H^O. Each seed was placed at one end of a deep square p l a s t i c p e t r i d i s h con ta in i ng 2 % agar in tap water. P l a te s were poured so that there was an a d d i t i o n a l b u i l d up of agar at one end f o r the seed. They 46 were then placed under a 16 hour photoperiod at an angle which encouraged lea f growth along the agar su r f ace . A f t e r 9 days, p lant s were prepared f o r i n o c u l a t i o n . An area of epidermis 20mm x 2mm was removed from the centre abax i a l su r face of some leaves. To accomplish t h i s , a sur face wound was made at the d i s t a l end of the lea f and the epidermis peeled back using f i n e f o r cep s . Each leaf was anchored h o r i z o n t a l l y aga inst the agar w i th a g las s t r i a n g l e so that the abax i a l lea f su r face was face up. The s ides of each t r i a n g l e enclosed the l ea f area from which the epidermis had been removed (see F i g . C-1). The t r i a n g l e s were made from 6 mm diameter g lass rods and had e q u i l a t e r a l s ides approximately 4 mm in length. Spore l ings were grown in MPG medium p r i o r to being inocu lated onto the p l a n t . Three d ishes were run f o r each t reatment, approximately 3200 spores per d i s h . Spore l ings were then washed in s t e r i l e g la s s d i s t i l l e d H^ O and inocu la ted onto the 20mmxx22mm area of l ea f su r face s i t u a t e d w i t h i n the t r i a n g l e . Approximately 400 spores were placed on each l e a f . Undeveloped spores were a l s o used and were placed i n to MPG medium fo r 2 minutes p r i o r to washing in the H^O. P l a te s were incubated h o r i z o n t a l l y under a 16 hour photoperiod f o r 12 days and the r e s u l t i n g number of i n f e c t i o n s i t e s counted. The experiment was performed twice on separate occas ions . Twenty- f ive separate leaves, each in i t s own d i s h , were inocu lated f o r each treatment g i v i n g a t o t a l of 200 d i shes per experiment. hi Figure C-1 Preparation of a s e p t i c wheat leaf f o r i n f e c t i o n ;xp• tv „ r experiments. Glass t r i a n g l e holds l e a f in place against 2 % FLO agar. 48 49 MICROSCOPY: Morpho log ica l observat ions of s po re l i n g and axenic growth were made w i th a Wi ld inver ted microscope. This microscope made d i r e c t observat ion w i t h i n the unopened c u l t u r e ves se l p o s s i b l e at much higher magn i f i c a t i on s that those of any d i s e c t i n g scope. A Bentax camera was attached f o r photographic purposes. For s i n g l e spore exper iments, a su r face s t e r i l i z e d Cenco # 60918-2 d i s e c t i n g microscope was r o u t i n e l y used. Nuclear s tud ie s were c a r r i e d out w i th a Car l Ze iss photomicroscope II w i th neo f l ua r o b j e c t i v e lenses . P i c t u r e s werettakenvowit-htthebbuilt in automatic 35mm camera. Kodak co l ou r photomicrography f i l m # 2483 or Kodachrome co lour s l i d e f i l m was used f o r co lour s l i d e s . Colour p r i n t s were made from s l i d e s by e i t h e r f i r s t making an in te rnegat I ve or re-photographing p r i n t s made d i r e c t l y from s l i d e s . Some p r i n t s were made us ing Kodakcolour II negat ive f i l m . For b lack and whi te photography, Kodak panatomic-X or Kodak high con t ra s t copy f i l m was used. Negatives were developed w i th e i t h e r •. microdol -X or D-19- Kodak ectamat ic A p r i n t i n g paper was used f o r b lack and wh i te p r i n t s . 50 RESULTS UREDOSPORE DIFFERENTIATION IN VITRO: 1) A SUITABLE DIFFERENTIATION MEDIUM: P re l im ina r y t r i a l s to produce i n f e c t i o n s t r u c tu re s on 0.01 M Ca-K-PO^ b u f f e r sub s t ra te (Maheshwari et_ aj_. 1967a; Table C-2) were u n s a t i s f a c t o r y . Attempts were, t h e r e f o r e , made to f i n d a more s u i t a b l e d i f f e r e n t i a t i o n medium. The Ca-K-PO^ bu f f e r had the f o l l o w i n g drawbacks. F i r s t l y , d i f f e r e n t i a t i o n percentages were too low, approx imately 5 %> Secondly, spores tended to aggregate in clumps and produced long, t w i s t e d , matted germ tubes. This made v iewing and count ing of i n d i v i d u a l spo re l i ng s extremely d i f f i c u l t ( F i g . D-1). Maheshwari ' s Ca-K-PO^ b u f f e r and a n u t r i e n t medium (Wi l l iams 1971) were were both tes ted in l i q u i d form and w i th the a d d i t i o n of 1 % agar, a t o t a l of 3 subs t ra tes in a d d i t i o n to the l i q u i d b u f f e r . A l l three subst rates showed marked improvement over the l i q u i d bu f f e r in that d i f f e r e n t i a t i o n percentages were h igher and spore aggregat ion was e l im ina ted (Table D-1). However, i t was f e l t that a l i q u i d medium such as Wil Harm's would be p re fe r red to the other two s o l i d agar media. Un fo r t una te l y , i n f e c t i o n s t r u c t u r e format ion on W i l l i a m ' s l i q u i d was a t y p i c a l . Germ tubes were e x ce s s i v e l y short and i n f e c t i o n s t r u c t u r e s were o f ten formed without a tube at a l l . O c c a s i o n a l l y , v e s i c l e s were present without any p r e r e q u i s i t e pegs or app re s so r i a : in these case, v e s i c l e s appeared to a r i s e d i r e c t l y from the spores themselves. 51 F igure D-1 F igure D-2 Uredospore morphogenesis in 0.01 M Ca-K-P0. l i q u i d b u f f e r . Spores were heat shocked. Note long matted germ tubes. There are no i n f e c t i o n s t r u c t u r e s . X 511. Uredospore morphogenesis in MPG l i q u i d medium. Spores were heat shocked. Note abundant i n f e c t i o n s t r u c t u r e fo rmat ion . X 467. 53 Table D-1 E f f e c t s of 0.01 M Ca-K-PO^ bu f f e r and Wi1 l i am 1 s ; n u t r i e n t medium on diiif fe rent-rat ion t : oMed i a were te s ted both w i th and wi thout the add i t i o n of 1 % agar. Table D-2 E f f e c t s of component v a r i a t i o n s of 0.01 M Ca-K-P0, bu f fe r and W i l l i a m ' s n u t r i e n t medium on d i f f e r e n t i a t i o n . Ingredients were added to or removed from the two bas i c . med i a. 54 EFFECTS OF MEDIA ON DIFFERENTIATION TABLE D-1 S U B S T R A T E 0 . 0 1 M C a - K - P O ^ b u f f e r pH 6 . 8 N u t r i e n t m e d i u m ( W i l l i a m ' s ) S O L I D OR L I Q U I D l i q u i d + 1 % a g a r l i q u i d + 1 % a g a r % D I F F E R E N T I A T I O N S P O R E A G G R E G A T I O N C O N D I T I O N OF GERM T U B E S 6 1 2 9 % y e s n o l o n g m a t t e d g e r m t u b e s g e r m t u b e s s h o r t e r t h a n o n l i q u i d b u f f e r 21 % 2 5 % n o n o s h o r t g e r m g e r m t u b e s t u b e s , o c c a - l o n g e r t h a n o n s i c n a l l y n o n e l i q u i d n u t r i e n t med i um -TABLE D-2 BASE SUBSTRATE Maheshwari's Ca-K -PO^ buffer pH 6.8 Wi l l i am's Nutrient medium pH 6.8 Nutrie nts added to base buffer Nutrients subtracted from base medium NUTR1ENT COMPOSITION Buffer G.-i i y +Peptone +•3!ucose +NaN03 1 J +KCL +1% agar Complete med i um -Peptone -Glucose -Glucose - M g S O ^ % DIFFERENTIATION SPORE AGGREGATION TYPICAL INFECTION STRUCTURES LENGTH OF GERM TU6ES 2 yes yes long 5 3 no yes 6 yes yes long 8 yes yes long 19 no yes med 22 no no short 1 yes yes med 7 no no med 9 no no short 12 no not recorded short — - — ~ — — — — — — — ^ — 55 Effects of further component variations in the buffer and nutrient medium are shown in Table D-2. The highest d i f f e r e n t i a t i o n percentages were recorded for the Ca-K-PO^ buffer with the addition of peptone and D-glucose (MPG medium). On this substrate, germ tubes were of medium length, in f e c t i o n structures developed normally, and infec t i o n hypha were longer than those on any other substrate (Fig. D-2). In addition, the peptone prevented spore aggregation so that sporelings were evenly d i s t r i b u t e d over the medium. They could be indiiividuaTlyuseen and counted (Fig. D-2). Further attempts were made to improve the MPG medium. Mineral s a l t s of William's medium were added to MPG with the exception of K^HPO^ and KCL. As a r e s u l t , the test medium was iden t i c a l to William's except for the buffer component which was that of MPG. D i f f e r e n t i a t i o n was not improved, but was comparable to that in William's medium. The addition of mineral components i n d i v i d u a l l y to MPG had the following e f f e c t s : NaNO^ or MgSO^ resulted in shorter i n f e c t i o n hyphae, NaCitrate resulted in less germination, shorter germ tubes, and atypical d i f f e r e n t i a t i o n s i m i l a r to that on William's medium. With FeSO^, d i f f e r e n t i a t i o n was s i m i l a r to, but s l i g h t l y higher than on MPG alone. These tests suggested that improvements to MPG might be possible. NaCitrate in concentrations less than that used in William's medium (l/10th concentration) might result in s l i g h t l y shorter germ tubes and increased d i f f e r e n t i a t i o n percentages without the disadvantages of the atypical form. Further tests with FeSO^ might also prove b e n e f i c i a l . However, these p o s s i b i l i t i e s were not f u l l y tested. 56 2) THE TIMING OF UREDOSPORE MORPHOGENESIS: The sequential timing of i n fec t ion s t ructure formation was determined as i t occurred on MPG medium (Figs. E-1 to E-15) • This was done to e s tab l i sh a reference guide fo r f u r t h e r , more d e t a i l e d , staging experiments. Spores were f i r s t placed at 18°C. A f te r 2 hours, germination was e s s e n t i a l l y complete (Figs. E-2 and E-3), although tube elongation did continue a f te r this time. A d i f f e r e n t i a t i o n stimulus was then given in the form of a heat shock at 30°C for 1 1/2 hours, fol lowed by a return to 18°C. Some appressoria began to form during the heat shock i t s e l f (F ig . E-h) , but the majority did not develop u n t i l from 30 minutes to 1 hour a f te r the end of th i s temperature treatment (at 3 1/2 to h 1/2 hours a f te r the s ta r t of development). Figure E-5 shows a typ ica l appressorium a f te r h hours of development. The majority of appressoria had formed in fec t ion pegs by 5 1/2 hours, although pegs were regu lar ly seen e a r l i e r than t h i s , at 5 hours (F ig . E-6). Figure E-7 shows the beginning of the next stage, the v e s i c l e i n i t i a l , which developed around 6 hours a f t e r the s t a r t . However, the development of the v e s i c l e (Figs. E-8 to E-10) was not usua l ly complete u n t i l 8 hours (F ig . E — 11). At approximately 10 hours, the in fect ion. hypha began to form as a protrus ion from one end of the ve s i c l e and i t continued to elongate 0(F<jgs . EET J!3aaridEET<H) uunt i 1 .t-ermi nat ion of the study. Figure E-15 shows a complete set of in fec t ion s tructures with the in fec t ion hypha a f te r 20 hours of development. Attempts to speed up or improve the d i f f e r e n t i a t i o n process by a l t e r i n g the t iming, durat ion , or number of heat shock s t imul i were unsuccess fu l . If the heat shock was not given a f te r 2 hours for the 57 F i g u r e E-1 Temporal sequence o f i n f e c t i o n s t r u c t u r e f o r m a t i o n i n MPG t o E-14 medium, from 1 1/2 hours t o 13 hours o f development. S t a i n e d w i t h ph1oxine-KOH. X 370. F i g u r e E-1 t o E-3 Germ tube F i g u r e E-4 F i g u r e E-5 F-iigure E-6 F i g u r e E~7 t o E-10 A p p r e s s o r i a l i n i t i a l M a ture a p p r e s s o r i u m I n f e c t i o n peg Development o f v e s i c l e F i g u r e E-11 Mature v e s i c l e and E-12 F i g u r e E-13 Development of i n f e c t i o n hypha and E-14 59 F i g u r e E-15 Uredospore w i t h complete s e t o f i n f e c t i o n s t r u c t u r e s a f t e r 20 hours o f development i n MPG medium. Note long i n f e c t i o n hypha. S t a i n e d w i t h phloxine-KOH. X 1280. 2 0 H o u r s 61 necessary dura t ion of 1 1/2 hours, the d i f f e r e n t i a t i o n was e i t h e r extremely poor, or d i d not occur at a l l . 3) CYTOLOGY OF THE DIFFERENTIATING SPORES: C y t o l o g i c a l s tud ie s were conducted t o f i n d out more about the t iming of c r u c i a l c e l l u l a r events accompanying the d i f f e r e n t i a t i o n process. S p e c i f i c a l l y chosen was the express ion of uredospore gene a c t i o n in terms of both nuc lear development and the synthes i s of e s s e n t i a l macromolecu 1es. NUCLEAR STAINING':' A technique was developed using the DNA s p e c i f i c Feulgen r eac t i on as a nuc lear s t a i n , but var ious other nuc lear dyes were a l s o t r i e d . Methyl g reen^pyron in, methyl green a lone, haematoxy1in , and aceto-orce in,aa11 y i e l d e d u n s a t i s f a c t o r y r e s u l t s . I n i t i a l s t a i n i n g t r i a l s w i th Feulgen (Maheshwari 1967b) , :1 ikewi se , f a i l e d to demonstrate the presence of nuc l e i in rust s p o r e l i n g s , yet wheat t i s s u e s ta ined in p a r a l l e l had prominent n u c l e i . Numerous t e s t s were run which r e su l t ed in a s e r i e s of m o d i f i c a t i o n s to the o r i g i n a l procedure. The f i n a l technique (page 43 of M a t e r i a l s and Methods, a l s o Table C-5) c o n s i s t e n t l y produced spo re l i n g nuc l e i w i th e i t h e r a p ink , magenta, or deep b lue co lou r and l e f t l i t t l e or no s t a i n in other c e l l u l a r a reas . Although there were many c o n t r i b u t i n g f a c t o r s , the f o l l o w i n g three changes were the ones d i r e c t l y re spons ib le f o r the success of the method: 1) S c h i f f ' s reagent (Gurr 1962) was modi f ied by t r i p l i n g the q u a n t i t i e s of bas i c f u c h s i n , potassium metab i su1ph i te , and N HCL. This r e s u l t e d in a s i g n i f i c a n t l y b r i g h t e r nuc lear s t a i n . Even doubl ing of the ing red ient s 62 improved results 2) The hydrolysis time was reduced from 12 minutes at 60 C with N HCL to 6 minutes. Times of less than 5 - 6 minutes resulted in paler n u c l e i . Times greater than this gave a s l i g h t l y darker s t a i n , but the number of v i s i b l e nuclei s t e a d i l y decreased u n t i l , at 12 minutes, nuclei were seldom seen 3) P.ref jxat.ion in p-dichlorbbenzene also increased the intensity of the nuclear s t a i n . The use of 1 % t o l u i d i n e blue as a counters tain further improved nuclear contrast, although i t was not a necessary step. Different developmental stages varied in t h e i r response to this treatment. T o r example, the counterstain was not e f f e c t i v e for the early stages prior to 6 hours. Other t h i a z i n dyes such as methylene blue and azur A had an e f f e c t simi 1ar to t o l u i d i n e blue except that the hyphae appeared grey in colour and had a more granular appearance. Thiiss .was. less desi rable because i t detracted from the nuclear stain i t s e l f . The intensity of nuclear st a i n was also affected by the brand of basic fuchsin used. BDH was superior in terms of staining power over the other brands. Na t i ona 1 An i 1 i ne , Fisher, and ESBE swet5eea 1 1 t r i e d and are l i s t e d in decreasing order of effectiveness. However, i t is possible that these differences were due to d i f f e r e n t dye lots and not the brand of s ta i n. Certain treatments, normally standard as part of the Feulgen technique, were eliminated because they were not e f f e c t i v e . D i f f e r e n t i a t i o n of the nuclear s t a i n with 2 % NaBisu1phite was one of these. The use of i n t e n s i f i e r s such as prppionocarmine with either k % f e r r i c ammonium sulphate or 0.01 %.FeCl^-was also discontinued. They did not . in t e n s i f y the 63 nuc lear s t a i n : i n s t ead , the hyphae were s ta ined n o n - s e l e c t i v e l y . In a d d i t i o n , counte r s ta in s such as orange G and l i g h t green were not worthwhi le . L i ght green. impaired nuc1 ear v i s i b i l i t y , wh i l e orange G, which o f fe red some c o n t r a s t , faded w i t h i n 2 days a f t e r use. NUCLEAR DEVELOPMENT: For s tud ie s of nuc lear development, uredospores were p.laced i n t o MPG medium. Uredospore nuc l e i were then s ta ined a f t e r var ious developmental stages from the beginning of germinat ion to the end of i n f e c t i o n hypha fo rmat ion . The dormant rust uredospore was b i n u c l e a t e . During d i f f e r e n t i a t i o n . , the number of nuc l e i was found to increase d r a m a t i c a l l y due to d i v i s i o n s both in the appressorium and in the v e s i c l e . Th is number was subsequently reduced in the i n f e c t i o n hypha. Spore l ings which were germinated,. but were not heat shocked to induce format ion of i n f e c t i o n s t ructures, , remained b i nuc l ea te except in ra re cases when a s i n g l e nuc lear d i v i s i o n took p l a ce . Two hours a f t e r the s t a r t of ge rm ina t i on , the two nuc l e i had migrated i n t o the germ tube ( F i g . F-1). These young germ tube nuc l e i were un i fo rmly s p h e r i c a l or s l i g h t l y arrowhead in shape. They were c l o s e l y a s s o c i a t ed , one behind the o t he r , approx imately 1.2 u~apa rtearidwwer_e, removed _ at l ea s t 30 u from the hyphal t i p . Most u n d i f f e r e n t i a t e d spo re l i ng s remained b i n u c l e a t e throughout the f o l l o w i n g per iod from 2 t o 20 hours ( F i g . F-2) . However, in some rare cases, a s i n g l e nuc lear d i v i s i o n d id occur in germ tubes over 10 hours of age (F ig s . F-3 and F-4). In d i f f e r e n t i a t i n g s p o r e l i n g s , the nuc1ei norma 11y remained in the germ tube u n t i l 3 1/2 hours when the bulbous appressorium i n i t i a l had been 64 F igure F-1 Two nuc l e i in young germ tube a f t e r 2 hours of development in MPG. Sta ined w i th feu l gen . X 2200. F igure F-2 Two nuc l e i enclosed by a septum in o l de r germ tube a f t e r 16 hours of development in MPG. Sta ined w i th f eu l gen . X 2,f80. 65 66 F igure F-3 Germ tube nuc le i undergoing d i v i s i o n a f t e r 12 hours of development. Sta ined w i th feu l gen . X 3810. F igure F-4 Germ tube nuc l e i at the end of the d i v i s i o n process p r i o r to septum fo rmat ion . Germ tubes are 14 hours Sta ined w i th f e u l g e n - t o l u i d i n e b lue . X 1650. j u s t o l d . 67 4 68 formed ( F i g . F-5) . They then t r a v e l l e d to the appressor ium. On rare occas ions , the nuc l e i d i v i ded prematurely in the germ tube before the appressorium had been reached ( F i g . F-6). But u s u a l l y , by k hours , the 2 nuc l e i had reached the appressorium wi thout a d i v i s i o n and became o r i en ted s ide by s ide (F i gs . FF:?7aand F-8) . Nuclear d i v i s i o n began soon a f t e r , at about k 1/2 hours. The nuc l e i d id not appear to d i v i d e synchronously and appres so r ia w i th 3 nuc l e i were r e g u l a r l y seen ( F i g . F-9). F i gu re F-10 shows an appressorium w i t h one of i t s three nuc l e i undergoing d i v i s i on. Mature app re s so r i a each had k n u c l e i arranged in pa i r s ( F i g . F — 1 1) . This pa i red arrangement was maintained dur ing nuc lear mig ra t ion i n t o the v e s i c l e . At approximately 6 hours, one p a i r moved in to the v e s i c l e i n i t i a l w h i l e the other p a i r remained in the appressorium ( F i g . F-12). The remaining pa i r a l s o migrated, but- the time f o r t h i s v a r i ed anywhere from 7 to 11 hours. In F igure F-13» a nuc lear p a i r and the surrounding cytoplasm are s t i l l v i s i b l e in the appressorium a f t e r 10 hours, whereas in F igure F-23, the appressorium is empty and 5 n u c l e i a re a l ready in the v e s i c l e a f t e r only 7 1/2 hours. It i s po s s i b l e that some nuc lear pa i r s d id not migrate i n t o the v e s i c l e at a l l , but t h i s was not ab s o l u t e l y determined. Feulgen p o s i t i v e areas , located e i t h e r in the appressorium or in the germ tube region adjacent to the appressorium ,were seen in a few l a t e stage v e s i c l e p reparat ions ( F i g . F-37)• Once a nuc lear p a i r reached the v e s i c l e , d i v i s i o n began ( F i g . F-14). The t iming of t h i s process was qu i t e v a r i a b l e and i t occurred from 7 to 13 hours a f t e r the s t a r t of development. During d i v i s i o n , nuc lear f i g u r e s 69 F i g u r e F-5 Two n u c l e i i n germ tu b e . A p p r e s s o r i u m i s a l r e a d y formed. S p o r e l i n g i s k hours o l d . S t a i n e d w i t h f e u l g e n . X 2300. F i g u r e F-6 Four n u c l e i i n germ tube. A p p r e s s o r i u m i s a l r e a d y formed. N u c l e i have d i v i d e d p r e m a t u r e l y , p r i o r t o r e a c h i n g t h e a p p r e s s o r i u m . S t a i n e d w i t h f e u l g e n - t o l u i d i n e b l u e . X 2510. 7 0 71 Figure F-7 Two n u c l e i in a p p r e s s o r i a l i n i t i a l a f t e r 3 1/2 hours of development. Stained with feulgen. X 3480. Figure F-8 Two n u c l e i in appressorium a f t e r Stained with feulgen. X 3440. 4 hours of development. Figure F-9 Appressoriurn w i t h 3 n u c l e i . I n f e c t i o n form. Stained w i t h feulgen. X 3110. peg i s beginning to Figure F - 1 0 Appressorium with 3 process of d i v i s i o n X 3790. n u c l e i . One of the n u c l e i i s in the Stained w i t h feu 1gen-toluidine blue. Figure F-11 Appressorium with 4 n u c l e i in a paired arrangement. V e s i c l e i n i t i a l i s forming. Sporeling is 5 1/2 hours Stained with feu 1gen-toluidine blue. X 3650. o l d , Figure F-12 Two n u c l e i in appressorium and 2 n u c l e i in v e s i c l e i n i t i a l a f t e r 6 hours of development. Nuclei have migrated i n t o the v e s i c l e in p a i r s . Stained.with feu 1gen-toluidine blue. X 3420. 73 Figure F-13 Nuclei in v e s i c l e . A nuclear p a i r a l s o s t i l l in . appressorium a f t e r 10 hours of development. Stained with f e u l g e n - t o l u i d i n e blue. X 2310. Figure F-1A Nuclear d i v i s i o n in v e s i c l e . Nuclei are a l s o in appressorium. Sporeling is 12 hours o l d . Stained with feulgen. X 231.0. Figure F-15 M i t o t i c prophase of v e s i c l e nucleus. Enlargement of nucleus in F i g . F-13 - Stained with f e u l g e n - t o l u i d i n e blue. X 14„80O. 75 c h a r a c t e r i s t i c of m i t o s i s could n o t be fo l lowed w i th c e r t a i n t y , a lthough stages s i m i l a r to prophase, metaphase, and anaphase were seen (F i g s . F-15» F-18, and F - 2 0 r e s p e c t i v e l y ) . E l i p t i c a l l y - s h a p e d chromosome s t r u c t u r e s were a l s o apparent ( F i g . F-19). In a d d i t i o n , p a r a l l e l p a i r i n g arrangements s i m i l a r to m e i o t i c d i p l o t ene (F igs . F F T jli 6 F F T<11 8 , F -21 , and F-22) and bead7 1 l u k e s s t riuctu i=essstiirh i har t tocch rJomomer.esoof 1 ilsepbot-ene [l(F j 'gs . : F -1 7- and F-19) were v i s i b l e in the v e s i c l e . L i ke that in the appressor ium, d i v i s i o n in the v e s i c l e was not synchronous and numbers of 5, 6, and 7 nuc l e i were r e g u l a r l y seen (F i g s . F-23, F-24, and F-25 r e s p e c t i v e l y ) . The mature v e s i c l e normal ly had 8 nuc l e i ( F i g . F-26) , never more than t h i s , however, 7 nuc l e i were not uncommon. In some cases, nuc l e i appeared to lose t h e i r i n d i v i d u a l i t y in whatt 1 ookedd 1 ikeeaa 1 argeschhomosomeeppoil)! ( F i g s : F-27 and . F-31). Two phenomena, in a d d i t i o n to nuc lear d i v i s i o n , were Regu la r l y seen in the v e s i c l e ; f i r s t l y , the format ion of b i l a t e r a l nuc lear clumps, and second ly , the format ion of nuc lear m ig ra t i on pa t t e rn s . Both were s p e c i f i c nuc lear o r i e n t a t i o n s r e l a t i v e to the v e s i c l e s t r u c t u r e i t s e l f , as opposed to the open arrangement of nuc l e i a l s o seen. The clumps were v i s i b l e as two areas of nuc lear aggregat ion, symmet ica l ly located on e i t h e r s i de of the i n f e c t i o n peg, in the middle of the v e s i c l e ( F i g s . F F T 2 8 to F-30). The number of nuc l e i present in each clump, when d i s c e r n a b l e , was always e i t h e r h or l e s s . In a d d i t i o n , the chromatin of each group always appeared to be matched in c o n d i t i o n or stage to that of i t s s i s t e r clump. These clumps were u s u a l l y found at approx imately 13 hours of development when d i v i s i o n in the v e s i c l e was Nuclear d i v i s i o n in v e s i c l e at 12 hours. Note p a r a l l e l p a i r i n g arrangement of chromosomes in centre of v e s i c l e . Nuclei are of various s i z e s and shapes. Stained with Feulgen. X 2500. Nuclear d i v i s i o n in v e s i c l e . Note p a r a l l e l p a i r i n g arrangement in centre of v e s i c l e . Stained w i t h feulgen. X 2390. P a r a l l e l p a i r i n g arrangement in v e s i c l e s i m i l a r to meiotic d i p l o t e n e (lower r i g h t ) . Note a l s o nuclear arrangement s i m i l a r to metaphase ( f a r l e f t ) . Enlargement of F i g . F-16 Stained with feulgen. X 7250. P a r a l l e l arrangement of bead-like chromosomes in v e s i c l e s i m i l a r to chromomeres of mei o t i c leptotene ( r i g h t ) . Note e11iptica11y-shaped incomplete r i n g s t r u c t u r e at bottom l e f t . Enlargement of F i g . F — 17- Stained with feulgen. X 7350. Nuclear arrangement in v e s i c l e s i m i l a r to m i t o t i c anaphase Enlargement of F i g . F-16. Stained with feulgen. X 8450. 78 Figure F-21 Nuclei migrating i n t o v e s i c l e . S i n g l e leading nucleus in i n f e c t i o n hypha followed by 2 n u c l e i w i t h chromosomes in a p a r a l l e l paired arrangement. Nuclei appear j o i n e d by f e u l g e n - p o s i t i v e thread. Stained with feulgen. X 2310. Figure F-22 P a r a l l e l paired chromosome-1ike s t r u c t u r e s in v e s i c l e . Enlargement of F i g . F-21. Stained with feulgen. X 8270. 79 80 Figure F-23 Five to 6 n u c l e i in v e s i c l e . Sporeling is 7 1/2 hours o l d , t o l u i d i n e blue. X 17lt0. Appressorium i s empty, Stained with feulgen-Figure F-2k Six n u c l e i in v e s i c l e a f t e r 11 hours of development, Stained w i t h f e u l g e n - t o l u i d i n e blue. X 2270. Figure F-25 Seven n u c l e i in v e s i c l e a f t e r 11 hours of development, Note nuclear clump (far l e f t ) with 3 n u c l e i . It i s composed of one leading nucleus flanked by two others as i t moves in t o the i n f e c t i o n hypha. Stained with f e u l g e n - t o l u i d i n e blue. X 2670. Figure F-26 Eight n u c l e i in v e s i c l e a f t e r 10 hours of development, Stained with f e u l g e n - t o l u i d i n e blue. X 2656. Nuclei in v e s i c l e a f t e r 9 hours of development. The nuc l e i have l o s t t h e i r i n d i v i d u a l i t y and appear in the form of 'chromosome pools'. Note a l s o the b i l a t e r a l arrangement of the n u c l e i . Stained with feulgen. X 2330. Eight n u c l e i in v e s i c l e at 10 hours. Nuclei are arranged in b i l a t e r a l clumps symmetrically located on e i t h e r side of the i n f e c t i o n peg in the centre of the v e s i c l e . Each clump contains 4 n u c l e i . Sta i ned'.w.i th' feu 1 gen-tol u i dii ne blue. X 3370. B i l a t e r a l nuclear clumps in v e s i c l e at 11 hours. One clump has 4 n u c l e i , the other 3. Stained with feulgen. X 3490. Nuclei in v e s i c l e a f t e r 12 hours of development. Nuclei are in'the form of-b i 1 a't'era 1 clumps.- rThe nuclear . number cannotgbe d i s t i ngu i shed Sta i ned^wi t h ~ f eud.gen-to.l u i d i ne blue.3 X 3380. 83 8k complete (F i g s . F-28 and F-29)• However, nuc l e i in other d i v i s i o n a l stages were a l s o seen in clumps ( F i g s . F-27 and F-30). These aggregat ions were present in s l i g h t l y e a r l i e r p reparat ions than those w i th mig ra t ion pa t t e r n s , p r i o r t o any i n d i c a t i o n of the po l a r outgrowth of the i n f e c t i o n hypha. The i n f e c t i o n hypha of P_. grami n i s t r i t i c i race 15 -B, u n l i k e that of some other r u s t s , only grows from one end of the v e s i c l e , J_e_. the v e s i c l e has p o l a r i t y . In t h i s s tudy, the o r i e n t a t i o n of nuc lear m ig ra t ion patterns was r e l a t ed to the p o l a r i t y of the v e s i c l e . Once growth of the i n f e c t i o n hypha was i n i t i a t e d , the f i r s t nuc lear m ig ra t ion pa t te rn was ev i den t . The nuc l e i formed the mig ra t ion pat te rn as they l i ned up at r i gh t angles to t h i s new outgrowth (F ig s . F-31 and F~3k). The shape of the pat te rn changed as the nuc l e i moved toward the p o t e n t i a l hypha. The nuc l e i formed an a rc w i th i t s convex s ide f a c i ng the v e s i c l e ' s hyphal end (F i g s . F-32 and F-35)-As the nuc l e i began moving i n t o the developing i n f e c t i o n hypha, the arc became c r e s cen t , then arrowhead in shape (F i g s . F-33 and F-36). The i n i t i a t i o n of the nuc lear m ig ra t i on p a t t e r n , l i k e c lumping, d id not appear to be d i r e c t l y l i nked to the t iming of d i v i s i o n s i nce m ig ra t i on toward the hypha occurred dur ing var ious stages of the d i v i s i o n a l process (F ig s . F-32 and F-35). However, d i v i s i o n in most cases was complete by the time the rni'.grat ing nucleiinmovedddown • thee i n fec t iohnhyphay <.sometI me between 13 and 18 hou r s . As the nuc l e i moved along the i n f e c t i o n hyph'a, the shape of the mig ra t i on a rc changed to that of a horseshoe w i t h long t r a i l i n g s ides •(F.Tg.) - F-33) v 1 - <Sometimes , ton lyTt lhe i t-ra'Pl I'ng nuc l e i were seen. They were w e l l s t a i ned i n i t i a l l y , but f u r t h e r along in the o l de r hyphae, they became weakly s t a i n i n g bodies (Figs.FF53 '9 and E>r40.) and;:pften disappeared Stages in the development of nuclear migration patterns as n u c l e i move from the v e s i c l e i n t o the developing i n f e c t i o n hypha. Nuclear 'pool' in v e s i c l e . I n d i v i d u a l n u c l e i cannot be d i s t i n g u i s h e d . Nuclear 'pool' i s in the form of a crescent-shaped migration arc as i t moves i n t o the developing i n f e c t i o n hypha. Stained w i t h feulgen. X 1260. Nuclear migration pattern in v e s i c l e at 13 hours. I n d i v i d u a l n u c l e i cannot be d i s t i n g u i s h e d . Note, there are at l e a s t two groups of n u c l e i ; a leading group in the i n f e c t i o n hypha i n t i a l ( f a r l e f t ) , and a nuclear arc s t i l l in v e s i c l e . Stained with f e u l g e n - t o l u i d i n e blue. X 1260. Nuclear migration p a t t e r n . M i g r a t i o n arc has changed to an arrowhead form. Stained with f e u l g e n - t o l u i d i n e blue. X 12-80. F i r s t sign of a nuclear migration pattern in v e s i c l e . Three n u c l e i have l i n e d up at r i g h t angles to the p o t e n t i a l i n f e c t i o n hypha. Stained with feulgen. X 1550. Nuclear migration pattern in v e s i c l e at 13 hours. Note two groups of n u c l e i ; a leading group of 3 moving i n t o the i n f e c t i o n , hypha i n t i a l and a group of 4 n u c l e i in the form of an arc s t i l l in the v e s i c l e . The f i r s t nuclear group of 3 has a s i n g l e leading nucleus flanked by two others. Stained w i t h f e u l g e n - t o l u i d i n e blue. X 1290. Eight n u c l e i have moved i n t o the developing i n f e c t i o n hypha at 14 hours. Note the s i n g l e leading nucleus. Stained with feulgen. X 1390. Nuclear migration in the i n f e c t i o n hypha at 14 hours. Note s i n g l e i n t e n s l e y f e u l g e n - p o s i t i v e nucleus followed by an undetermined number of others. Stained with f e u l g e n - t o l u i d i n e blue. X 1730. Two small n u c l e i entering the developing i n f e c t i o n hypha at 14 hours. Other n u c l e i appear to have d i s s o l v e d . In the o r i g i n a l p r e p a r a t i o n , a f a i n t l y s t ained blue area could be seen adjacent to the 2 dark n u c l e i . Stained with f e u l g e n - t o l u i d i n e blue. X 14+00. 86 31 t 34 32 35 0 , 33 ^ ^ ^ ^ ^ * *f- V •" *•*/*• ' *' ^  36 87 a l t o g e t h e r ( F i g . F-38). They were normally preceded by a s i n g l e i n t e n s e l y f e u l g e n - p o s i t i v e nucleus ( F i g s . F-37 and F-40), and o c c a s i o n a l l y two (F i g . F-38). This leading nucleus was extremely small and s p h e r i c a l l y compact when compared with a l l the other nuclear stages of d i f f e r e n t i a t i o n . Thus, the horseshoe pattern described, appeared to be i n i t i a l l y a combination of at l e a s t three n u c l e i ; a s i n g l e small leading nucleus flanked by two or more elongated ones. This formation could be d i s t i n g u i s h e d as such during many migration stages from 11 to 18 hours. Figure F-34 shows 3 n u c l e i j u s t before t h e i r movement i n t o the hypha: Figure F-37 i s a l a t e r stage. The leading nucleus continued to move to the extreme t i p of the growing hypha, u n t i l i t was approximately 5 u from the end ( F i g . F-h2). Young i n f e c t i o n hypha n u c l e i were, f o r the most p a r t , e i t h e r poorly s t a i n e d , or not stained at a l l , w i t h the exception of the s i n g l e leading granule. This made nuclear counts during migration d i f f i c u l t . Sometimes, however, a l l the n u c l e i were c l e a r l y v i s i b l e ( F i g s . F-36 and F - 36A ). Thus, i t appears that on c e r t a i n occasions, at l e a s t , the f u l l complement of 7 or 8 n u c l e i do enter the i n f e c t i o n hypha. In most instances though, the number seen to reach the beginning of the i n f e c t i o n hypha was less than t h i s ( F i g . F - 3 8 ) . What happens to the rest of the n u c l e i i s not c e r t a i n , since mature i n f e c t i o n hyphae only have e i t h e r one or two nu c l e i at t h e i r t i p ( F i g . F-42). It is p o s s i b l e that they d i s s o l v e as appears to be the case in Figure F-38 or coalesce as in Figure F-41. Most, but not a l l , v e s i c l e s and i n f e c t i o n hyphae examined showed the nuclear migration patterns j u s t described. In some, the nuc l e i appeared to be moving toward and i n t o the i n f e c t i o n hypha in a r e l a t i v e l y loose f a s h i o n . 88 F igure F-36A Developing i n f e c t i o n hypha at 14 hours w i th 8 n u c l e i . Note s i n g l e leading nuc leus . X 4450. 90 F igure F-39 F igure F-40 S i ng le f e u l g e n - p o s i t i v e nucleus in i n f e c t i o n hypha a f t e r 16 hours of development. Sta ined w i th f eu l gen . X 2750. S i ng le i n ten se l y s ta ined nucleus in i n f e c t i o n hypha a f t e r 20 hours of development. Th is nucleus is fo l lowed by p o s i t i v e l y s ta ined t r a i l i n g threads . Sta ined w i th f e u l g e n - t o l u i d i n e b lue . X 2770. F igure F-41 Nuc le i at t i p of i n f e c t i o n hypha a f t e r 20 hours. Nuc le i appear to have coa le sced . Sta ined w i th f e u l g e n - t o l u i d i n e b lue . X 2760. F igure F-42 S i ng l e feu 1 gen-pos i t i v e nucleus at tjip of i n f e c t i o n hypha a f t e r 20 hours. Nucleus i s fo l l owed by t r a i l i n g threads of feu 1gen-pos i t i ve m a t e r i a l . Stained w i th f eu l gen . x 4460. 91 42 92 In o t h e r s , d i s o r i e n t e d horseshoe patterns were seen. It is not known whether these patterns represent d i f f e r e n t nuclear stages, or r e f l e c t a l t e r a t i o n s in timing of those already described. Nuclei in u n d i f f e r e n t i a t e d germ tubes, in c o n t r a s t to those which were induced to produce i n f e c t i o n s t r u c t u r e s , f a i l e d to e x h i b i t any of the changes described above. The two n u c l e i were not seen to change t h e i r s i z e and shape appreciably during the p e r i o d , 0 to 20 hours, s t u d i e d . This behaviour was very d i f f e r e n t from that in the d i f f e r e n t i a t i n g s p o r e l i n g where many nuclear shapes and s i z e s were v i s i b l e . Nuclear d i v i s i o n in u n d i f f e r e n t i a t e d germ tubes r a r e l y occurred, wheireasninninfeetiiion s t r u c t u r e s , i t was a regular phenomenon. In a d d i t i o n , large numbers of n u c l e i , common in v e s i c l e s , were not seen in germ tubes. Nuclei only d i v i d e d once, y i e l d i n g a t o t a l of 4 n u c l e i . Septa were l a i d down soon a f t e r d i v i s i o n . Figure F-2> shows a germ tube a f t e r 16 hours. The 2 n u c l e i are enclosed in a septate c e l l . These c e l l s in no way resembled the unique s t r u c t u r e s formed during d i f f e r e n t i a t i o n . TIMING OF ESSENTIAL DNA, RNA, AND PROTEIN SYNTHESES: The timing of DNA, RNA, and p r o t e i n syntheses was i n v e s t i g a t e d with the use of metabolic i n h i b i t o r s . I n h i b i t o r s known to block one or more of the above synthesesv.wereeeacheaddedtt 'O (orrRemoved*f &orh) tthecd i ff:erent;iat:i;ng ' in v i t r o uredospore according to a s p e c i f i e d time schedu l e j t f c b idetermine stages at wh;i.ch-essentia 1 syntheses might take place. A p a r t i c u l a r synthesis was considered e s s e n t i a l i f normal morphologic development f a i l e d to occur in the presence of the i n h i b i t o r . Ea.-i • r n ! ! i 5 1 »• p. 'ese. i d c -93 Each i n h i b i t o r was present dur ing the f o l l o w i n g time i n t e r v a l s . 18°C 30°C 18°C TIME 0 2 3-5 18 INTERVAL Hours, r.-. .-.-Hours-.-;-. -. .- rHours . .- •...-. i Hours TREATMENT Contro l 1 2 3 k — 5 6 INHIBITOR PRESENCE Development of morpholog ica l s t r u c t u r e s was recorded us ing the f o l l o w i n g c a t e g o r i e s ; germ tube, appressor ium, i n f e c t i o n peg, v e s i c l e , and i n f e c t i o n hypha. For each spore in the sample, i n d i v i d u a l s t r u c tu re s were counted as e i t h e r developed or not developed. Treatments were - • compared to the con t r o l f o r each morphologic category by Ana l y s i s of Var iance (see page kS of M a t e r i a l s and Methods). The q u a l i t a t i v e e f f e c t s of the i n h i b i t o r s on d i f f e r e n t i a t i o n were a l s o noted fo r each d i s h . The mean development percentages f o r each morphologic s t r u c t u r e f o r the combined experiments of each i n h i b i t o r are shown in Tables G-1 to Q-k. The data are presented in two major ways. F i r s t l y , s t r u c tu re s are expressed as a percentage of the t o t a l spore number f o r each t reatment, 3k as in Tables G-1 and G-2. Secondly, s i nce i n f e c t i o n s t r u c t u r e s develop in sequence, each is dependent upon the immediately preceding s t r u c t u r e f o r i t s fo rmat ion . There fo re , the data are a l s o expressed as a percentage of the preceding s t r u c t u r e , as in Tables G~3 and G-4. Treatment values are a l s o l i s t e d as percentages of the c o n t r o l values (Tables G-2 and G -4 ) . A l l the i n h i b i t o r s had some e f f e c t on the process of development. Th is e f f e c t v a r i ed w i t h the i n h i b i t o r , the time of i n h i b i t o r a p p l i c a t i o n , and the p a r t i c u l a r morphogenetic s t r u c t u r e being cons idered. Act inomycin-D and a-amanit in are both known i n h i b i t o r s of RNA s yn the s i s . In t h i s s tudy, r e s u l t s using each of these i n h i b i t o r s were s i m i l a r . Ne i ther actimomycin-D nor a-amanit in prevented normal ge rminat i on , but both stopped d i f f e r e n t i a t i o n (treatment 1: Tables G-1 to G -4 ) . When e i t h e r i n h i b i t o r was only present f o r the f i r s t two hours of development (treatment 2 : Tables G-1 to G - 4 ) , app re s s o r i a l format ion was markedly a f f e c t e d . Not only was the number ofeappressdrnladg beat 1 y reduced by comparison w i th the c o n t r o l , approximately 20 % (Table G -2 ) , but those that were seen, were merely smal l te rmina l s w e l l i n g s , not t rue app re s so r i a . Some small t rue appres so r i a began to form when a.ctinomycin was e l im i na ted fo r the f i r s t two hours, but was present dur ing the heat shock (treatment 3 T a b l e s G-1 to G -4 ) . However , some i n h i b i t o r e f f e c t was s t i l l ev ident in t h i s treatment (tbea.tment 3 ) - In the case of ac t i nomyc in , the app re s s o r i a l number was about one t h i r d of the c on t r o l (Table G -2 ) , and in a -amanit in p repa ra t i on s , t rue appre s so r i a were not present at a l l , a lthough some te rmina l swe l l i n g s were v i s i b l e . I n h i b i t o r e f f e c t was v i r t u a l l y e l imiinated : |i,f t^he w i nh ib i tg j jdwas .wi thejd unt i 1 ^ .af;£§r the-heat-shock process 95 T a b l e G-1 Development o f i n d i v i d u a l components o f i n f e c t i o n s t r u c t u r e s a f t e r v a r i o u s i n h i b i t o r t r e a t m e n t s . Development i s e x p r e s s e d as a mean % of the t o t a l number of s p o r e s (,c,>e. .v ( d e v e l o p e d o r undeveloped) counted p e r t r e a t m e n t . T a b l e G-2 Development f o r each t r e a t m e n t i n T a b l e G-1 e x p r e s s e d as a p e r c e n t a g e o f the c o n t r o l t r e a t m e n t . H4it Shock Development as a mean 9b of total spores 18 C Hours 0- 2 -18 T A B L E G-1 1 N H I B I T O R Actinomycin-D 5 ugAnl GERM APPRE PEG VESIC HYPHA a-Amanitin 40 ug/ml GERM APPRE PEG VESIC HYPHA Puromycin 100 ug/ml GERM APPRE . PEG' VESIC HYPHA' 5-Fluorouracil 100 ug/rnl GERM APPRE PEG VESIC HYPHA Cycloheximide 10 u y n l | GERM APPRE PEG VESIC HYPHA CONTROL 96.5 46,4 42.7 "33:2 -29.4 95.6 9 J y y H 97.9 8.8 2J) CL4 0.2 96.3 15L7 y y y 9518 34J 29JL }6J 5 J 95.0 y y y y 96.3 21.0 6.7 1.0 0.0 88.5 62.5 60.6 56.J 43.3 93.1 IgJJ 2A gj) 0J) 95.2 14J 4.8 0J) OJ) 87.5 U U O J U 91.0 3 8 J UA OJ: O J 95.2 10J 4.8 O j OJ) 92.2 8.7 3.9 0.0 0.0 :98."4 :42."1 37.1 28.4 15.9 97.6 52.5 19.1 OJ] 0^0 97.3 49.9 44.1 37.4 26.3 97.7 35.5 31.1 24.9 18.7 98.4 41.8 33.4 gj) 0_J. 96.1 53.6 50.7 HJ) 2jL_7 97.8 42.1 30.8 0.0 0.0 96.2 39.0 37.8 32.9 24.6 98.0 55.9 41.9 |4Jj jLJ. 97.9 43.1 36.0 22^ 5 16.4 99.7 43.5 36.3 22Jj 12.2 99.1 44.8 40.9 2JL| LJ. 98.1 50.7 40.3 J6j2 .7.4 97.6 47.9 39.2 16.8 2.1 99.5 50.1 47.9 35.4 24.7 43.4 0J) £ J OJ) 0.0 63.8 O J O j y 95.8 &^2_ 3.6 0__0 CU) 96.7 39.1 12J O J 0J_' 65.6 3 J O J O J OJ) 96.9 2.5 0.0 0.0 0.0 Development mean % expressed as a % of control GERM APPRE PEG VESIC HYPHA 99.1 2Q.J O J O J O J 101.5 IJLP u y y 99.8 33J3 16Ji 2J OJ 99.2 74Jj 6_8Jl 5JJJ 18.4 98.4 31.5 0.0 OJ) JJJ 99.8 45J3 35.7 ?•_? 0 0 GERM APPRE PEG VESIC HYPHA 105.3 17J2 4 J O J O J 107.6 23J y OJ O J 98.9 9Jt IJ) O J O J 102.9 M J . 2JL4 O J OJ . 107.7 16J5 7Jj O J O J 104.3 32^ 9 6 J OJ) 0.0 GERM APPRE PEG VESIC HYPHA 99.1 124.7 51.4 OJ) 0J_ 98.9 118.5 119.0 431.9 T65J 99.3 84.3 84.0 87.8 117.7 100.0 '.' 99.3 . 90.2 : 0.0 0.0 97.6 127.2 1JL7 165.1 180.3 99.4 100.0 83.0 OJ) GERM APPRE PEG VESIC HYPHA 101.9 143.3 110.8 43J 2J> 101.8 110.4 95.3 6JJf 66.6 103.6 111.5 96.2 U J 4?J 103.0 114.8 108.3 67_J| 2 § J 102.0 130.0 106.7 4_U 30J. 101.5 122.8 103.8 §OJ £ J TABLE G"2 GERM APPRE PEG VESIC HYFKA 43.6 0.0 O J O J 0 J _ 64.1 5.4 U C J OJ 96.2 y y 7 j y y 97.2 77.9 2|J_ 0_J. QJj 65.9 H y y y 97.4 5.0 0.0 0.0 0.0 TREATMENTS SIGNIFICANTLY DIFFERENT FROM CONTROL AT - .05 LEVEL OF SIG. ~ -01 LEVEL OF SIG. = <-0 cn 97 Table G-3 Development of i n d i v i d u a l components of i n f e c t i o n s t r u c t u r e s a f t e r various i n h i b i t o r treatments. Each s t r u c t u r e w i l l only form i f the preceding s t r u c t u r e has f i r s t developed. Therefore, development i s expressed as a mean % of the number of s t r u c t u r e s ..immed i a t e l y preceding the one being assessed. Table G-A Development f o r each treatment in Table G~3 expressed as a percentage of the c o n t r o l treatment. HEAT SHOCK 18 C / ^ O ^ I S C Hours 0 2 3.5 18 Development as a mean Sb of preceding structure I N H I B; | T O R T A B L E G-3 Actinomycin-D 5 ug^nl GERM APPRE PEG VESIC HYPHA a-Amanitin 40 ugAnl GERM APPRE PEG VESIC HYPHA Puromycin 100 ug*n| GERH APPRE PEG VESIC HYPHA 5-Fluorouracil ' 100 ug/M GERM APPRE PEG VESIC HYPHA Cycloheximide 10 ug^nl GERM APPRE PEG VESIC HYPHA 96.5 48.5 93.7 75.3 8--.6 95.6 10.3 3.3 33.3 00.0 97.9 9.0 26.3 17.7 33.3 96.3 j6.ii 45.7 30.2 8.3 95.8 36.4 83.6 53.9 39.9 95-0 5.8 00.0 00.0 00.0 96.3 22.3 37.6 32.5 00.0 88.5 70.7 96.3 93.7 76.3 93.3 33.6 27.3 00.0 00.0 95.2 35.2 33.3 00.0 00.0 87.5 6.5 39.9 00.0 00.0 93.0 42.0 3 2.3 0 0.0 00.0 95.2 33.0 45.5 00.0 00.0 92.2 9.4 44.4 00.0 00.0 98.4 42.3 88.7 73.3 67.3 97.6 53-8 43.6 00.0 00.0 97.3 53.5 88.7 83.5 73.3 97.7 36.2 86.7 78.9 80.3 98.4 42.6 79.8 00.0 00.0 96.3 55.8 94.5 85.5 73.3 97.8 42.9 74.8 00.0 00.0 96.2 40.9 97.3 86.7 70.3 98.0 57.0 75.8 33.3 2.5 97.9 43.9 83.3 59.1 65.2 99.7 43.6 82.7 60.7 52.1 99.1 45.2 90.3 49.9 32.5 98.I 51.8 80.9 37.6 43.5 97.6 49.3 80.4 37.1 8.2 99.5 50.3 95.9 72.1 72.8 43.4 00.0 00.0 00.0 00.0 63.8 4.0 1 2.5 00.0 00.0 95.8 8.5 26.9 00.0 00.0 96.7 40.7 29.2 00.0 00.0 65.6 5.5 6.2 OO.O 00.0 96.9 2.6 00.0 00.0 00.0 Development mean % (above) expressed as a % of control GERM APPRE PEG VESIC HYPHA 99.1 20.5 2.01 110.6 00 .0 101.5 17-3 24.4 23.5 42.6 99.8 35.1 45.0 12.9 25.9 99.2 73-3 89.1 73.4 28.1 98.4 11.6 00.0 00.0 00.0 99.8 46.3 3 6 . 7 13-8 00.0 GERM APPRE PEG VESIC HYPHA 105.3 36.4 28.3 00^0 00.0 107.6 21.5 34.3 00.0 00.0 98.9 9.2 20.5 00.0 00.0 102.9 59.4 33.3 00.0 00.0 307.7 35.6 46.9 00.0 00.0 104.3 13.4 45.8 00.0 00.0 GERM APPRE PEG VESIC HYPHA 99.1 325.9 46.6 00.0 00.0 98.9 323.7 302.8 317.2 333.0 99-3 88.9 96.8 109.3 326.3 300.0 99.0 93.3 00.0 00.0 97.6 132.4 107.5 318.3 109.5 99.4 100.6 83.0 00.0 00.0 CERM APPRE PEG VESIC HYPHA 103.9 342.7 78.1 34 .6 4.0 301.8 310.7 85.3 67.6 92.8 303.6 138.4 86.5 63.2 77-7 303.O 318.3 : 92.5 58.7 50.5 302.0 333.6 82.2 42.6 66.8 101.5 126.2 83.1 ^ . g 13.5 T A B L E G-4 GERH APPRE PEG VESIC HYPHA 43.6 00.0 00.0 00.0 00.0 64.3 35.4 25.9 00.0 00.0 96.2 27-5 53.3 00.0 00.0 97.2 78.9 30.6 00.0 00.0 65.9 37.1 32.8 00.0 00.0 97.4 9.4 00.0 00.0 00.0 OO 99 (treatment 4: Tables G-1 to G-h). In t h i s case, t rue appres so r i a were abundant in the presence of both i n h i b i t o r s . V e s i c l e s were abundant, as w e l l , in the presence of ac t inomyc in . A l l of these r e s u l t s , us ing i n h i b i t o r s of RNA s yn the s i s , are summarized in F igure G-1. They i n d i c a t e that RNA synthes i s does not appear to be e s s e n t i a l f o r ge rminat i on , but i s necessary f o r d i f f e r e n t i a t i o n . Formation of the appressorium requi res RNA synthes i s p r i m a r i l y dur ing the i n i t i a l 2 hour germinat ion pe r i od . V e s i c l e s and i n f e c t i o n hyphae a l s o appear to requ i re RNA s yn the s i s ; the v e s i c l e requ i res i t sometime a f t e r the s t a r t of heat shock, and the i n f e c t i o n hypha a f t e r the end of the shock process. One of the two i n h i b i t o r s of p r o t e i n synthes i s used in t h i s study was puromycin. Uredospores germinated normal ly and r e a d i l y formed appres so r i a in the presence of puromycin. Complete development of normal i n f e c t i o n s t r u c t u r e s took p lace (treatments 2, 3, and 5 "• Tables G-1 to G-4) except when puromycin was added a f t e r the heat shock (treatment h) , thereby prevent ing the v e s i c l e stage. Resu l t s w i th puromycin are summarized in F igure G-2. These r e s u l t s i n d i c a t e that p r o t e i n synthes i s may not be requ i red f o r germinat ion or app re s so r i a l f o rmat ion , but is necessary sometime a f t e r heat shock f o r the v e s i c l e s tage. 5-F1uorouraci1 (5 -FU) is a s p e c i f i c i n h i b i t o r of DNA s yn the s i s . When present throughout development (treatment 1: Tables G-1 to G-4), 5-FU d id not markedly a f f e c t development u n t i l the l a t e v e s i c l e and i n f e c t i o n hypha s tages. V e s i c l e s were sma l le r than normal, and i n f e c t i o n hyphae, in the ma jo r i t y of treatment 1 exper iments, were not seen at a l l . I den t i c a l 100 F i g u r e G-1 E f f e c t o f i n h i b i t o r s o f RNA s y n t h e s i s o n r u s t s p o r e l i n g m o r p h o g e n e s i s . I n h i b i t o r s a r e A c t i n o m y c i n - D a n d a - A m a n i t i n . I n h i b i t o r p r e s e n c e i s i n d i c a t e d b y t h e s o l i d b l a c k b a r . N o t e t h a t d i f f e r e n t i a t i o n i s p r e v e n t e d i f i n h i b i t o r s a r e p r e s e n t f o r o n l y t h e f i r s t t w o h o u r s o f d e v e l o p m e n t . 102 F igure G-2 E f f e c t of a p r o t e i n synthes i s i n h i b i t o r on rust s po re l i n g morphogenesis. I n h i b i t o r is Puromycin. Presence of puromycin is i nd i ca ted by the s o l i d b lack bar. Note that v e s i c l e development is i n h i b i t e d when puromycin is present a f t e r the heat shock process. 104 r e s u l t s were obta ined when the i n h i b i t o r was present con t i nuou s l y , du r i n g , as w e l l as a f t e r heat shock (treatment 6: Tables G-1 to G-4) . For a l l other t reatments , t y p i c a l v e s i c l e s and i n f e c t i o n hyphae were present (see summary F i g . G-3). Thus, the i n f e c t i o n hypha appears to be the only s t r u c t u r e t o t a l l y r equ i r i n g DNA s yn the s i s , and t h i s s ynthes i s takes p lace sometime du r i ng , as w e l l as a f t e r the heat shock process. There was a l s o some i n d i c a t i o n that v e s i c l e complet ion requ i res DNA synthes i s dur ing the same time frame (treatment 6: Tables G 12 and G-4) . Cyc lohex imide, the l a s t i n h i b i t o r used in the present s t u d i e s , prevents the synthes i s of DNA, RNA, and p r o t e i n . Cycloheximide was the only i n h i b i t o r which had a notable e f f e c t on ge rminat i on , even when present throughout the f u l l development per iod (treatment 1: Tables G-2 and G-4). It d id not complete ly prevent i t , but d id reduce the germinat ion rate by more than one h a l f . Germ tubes were cons ide rab ly sho r te r and s t r a i g h t e r than normal. In a d d i t i o n to t h i s , the i n h i b i t o r had a profound e f f e c t on the d i f f e r e n t i a t i o n process. V e s i c l e s and i n f e c t i o n hyphae were not seen in any of the treatments (treatments 1 to 6: Tab l e s . G-1 _to<.G-4) . Development of app re s so r i a was prevented p r i m a r i l y when cyc lohex imide was present dur ing the i n i t i a l 2 hours before the heat shock st imulus (treatment 2: Tables G-1 to G-4). This i nd i ca te s f u r t h e r that events c r u c i a l to app re s s o r i a l format ion do occur dur ing the germinat ion pe r i od . Resu l t s summarizing the e f f e c t of cyc lohex imide are shown in F igure G-4. 105 F igure G-3 E f f e c t of an i n h i b i t o r of DNA synthes i s on uredospore morphogenesis. I n h i b i t o r i s 5 - F1uo rou rac i 1 . I n h i b i t o r presence is i nd i ca ted by the s o l i d b lack bar. Note that development of i n f e c t i o n hypha is i n h i b i t e d only i f 5"FU is present both dur ing and a f t e r heat shock. o 107 F igure Q-k E f f e c t of an i n h i b i t o r of DNA, RNA, and p r o t e i n s yn thes i s on uredospore morphogenesis. I n h i b i t o r i s cyc lohex imide. I n h i b i t o r presence i s i nd i ca ted by the s o l i d b lack bar. Note that d i f f e r e n t i a t i o n is prevented i f cyc lohex imide is present f o r the f i r s t two hours of development a lone. 109 THE EFFECT OF DIFFERENTIATION ON HOST INFECTION: There i s s t i l l a q u e s t i o n c o n c e r n i n g the b a s i c f u n c t i o n o f i n f e c t i o n s t r u c t u r e s . However, i t i s thought t h a t they a r e i n some way n e c e s s a r y f o r i n f e c t i o n o f the h o s t p l a n t . T h i s i d e a came from an e x p e r i m e n t done by D i c k i n s o n (1949), the o n l y one of i t s k i n d t o d a t e . He g e r m i n a t e d and d i f f e r e n t i a t e d u r e d o s p o r e s o f P_. t r i t i c i na on w a x - c o l l o d i o n membranes, then p l a c e d the membranes o f each t r e a t m e n t s e p a r a t e l y on wheat l e a v e s from w h i c h the e p i d e r m i s had been removed. H i s c r i t e r i o n f o r i n f e c t i o n was the f o r m a t i o n of h a u s t o r i a i n the h o s t m e s o p h y l l c e l l s . H a u s t o r i a were o n l y d e t e c t e d i n the c a s e of the d i f f e r e n t i a t e d s p o r e l i n g s . A s i m i l a r e x p e r i m e n t was done f o r the p r e s e n t s t u d y . However, a more s u i t a b l e index o f u r e d o s p o r e i n f e c t i o n was used, J_e. t h e number of p u s t u l e s ( o r v e g e t a t i v e c o l o n i e s ) produced d u r i n g s p o r u l a t i o n . In a d d i t i o n , t h e p o s s i b l e i n t e r f e r i n g e f f e c t s o f membranes were a v o i d e d . F u r t h e r m o r e , treatmentsrwereerun-'toocheekkwhether: spp red ings , . a;l ready, g e r m i n a t e d and d i f f e r e n t i a t e d , c o u l d p e n e t r a t e t h r o u g h the l e a f e p i d e r m i s . S t e r i l e u r e d o s p o r e s were e i t h e r g e r m i n a t e d o r d i f f e r e n t i a t e d by heat shock i n MPG medium. At the end^of 30 h o u r s , sample c o u n t s were made t o d e t e r m i n e the p e r c e n t a g e g e r m i n a t i o n and d i f f e r e n t i a t i o n i n both c a s e s . These d e v e l o p e d s p o r e l i n g s were then p l a c e d on s t e r i l e wheat l e a v e s . The e p i d e r m i s had been removed from some l e a v e s : o t h e r s were s t i l l i n t a c t . Mature 1undeve1 oped s p o r e s were a l s o used as c o n t r o l t r e a t m e n t s . 110 A summary of the e x p e r i m e n t a l p l a n i s as f o l l o w s . TREATMENT 1 (Cont r o l ) 2 ( C o n t r o l ) 3 4 5 6 9 10 CONDITION OF UREDOSPORES undeveloped undeve1 oped d i f f e r e n t i a t e d g e r m i n a t e d o n l y d i f f e r e n t i a t e d g e r m i n a t e d o n l y no s p o r e s no s p o r e s CONDITION OF LEAVES n o r m a l - i n t a c t e p i d e r m i s removed e p i d e r m i s removed e p i d e r m i s removed normal - i n t a c t norma 1 - i n t a c t normal - i n t a c t e p i d e r m i s removed I n f e c t i o n c o u n t s were made on two d i f f e r e n t a r e a s o f each l e a f ; t h e 2cm x 2mm c e n t r e a r e a l a c k i n g an e p i d e r m i s w h i c h was o r i g i n a l l y i n o c u l a t e d , as w e l l as the i n t a c t l e a f r e g i o n s u r r o u n d i n g t h i s . T h i s second a r e a was w i t h i n t h e 2cm l e n g t h , but extended p a s t the exposed m e s o p h y l l l a t e r a l l y t o t he l e a f edge ( F i g . H-1). Counts were t a k e n here because o f the p o s s i b i l i t y o f i n f e c t i o n t h r o u g h the r e m a i n i n g i n t a c t e p i d e r m i s . O r i g i n a l l y , i t had been i n t e n d e d t o use t h e number of u r e d o s p o r e p u s t u l e s as an i n d i c a t i o n o f i n f e c t i o n . However, s p o r u l a t i n g p u s t u l e s were r a r e l y seen on t h o s e t r e a t m e n t s fnorm whhch t h e e e p i d e r m i s " h a d been removed. Those p u s t u l e s t h a t were seen w e r e - e i t h e r r o n f o r ^ e x t r e m e l y c l o s e t o , the ar e a s 111 F igure H-1 Areas of wheat leaf used f o r i n f e c t i o n counts. Two l ea f areas were assessed; the 2 mm x 2 cm centre area of exposed mesophyll (1) and the„area of i n t a c t epidermis surrounding t h i s (2). leaf e x p o s e d mesophyl l intact ep idermi s 113 of i n t a c t ep idermi s . On the areas of exposed mesophyll f o r these t reatments , f l u f f y vege ta t i ve myce l i a l c o l on i e s were present i n s tead . F igures H - 7 and H - 8 show t h i s e f f e c t of epidermis on s p o r u l a t i o n . Each f i g u r e shows a s i n g l e i n fec ted area b i sec ted by the edge of the ep idermi s . On the epidermal s i d e , the fungus i s in the form of a s po ru l a t i n g pu s tu l e : on the exposed mesophyll s i d e , the rust is growing as a vege ta t i ve co lony. In a d d i t i o n to the lack of s po r u l a t i n g pustu les on exposed mesophy l l , high humidity i n s i de the p e t r i p l a t e environment f o s te red the growth of vege ta t i ve hyphae from the l ea f over any area where the epidermis was broken or l a c k i n g , even from a l ready s po ru l a t i n g areas. As a r e s u l t , the number of t o t a l growths, not j u s t pu s tu l e s , was used as an i n d i c a t o r of i n f e c t i o n . Growths inc luded s po r u l a t i n g pu s tu l e s , pustu les overgrown w i th hyphae, and vege ta t i ve c o l o n i e s . Resu l t s of the var ious treatments are shown in F igure H-2 (treatments 1 to 6) and in Table H-1. Twenty - f i ve 1eaves.had.been . inoeu1ated f o r each treatment. Mature undeveloped uredospores, when placed on leaves w i th the epidermis removed (treatment 2: F i g . H-2), producedHsppru;!at i ng pustu les on the i n t a c t epidermis of a l l 25 leaves and co lon ie s on the exposed mesophyll of 19 leaves (treatment 2: Table-,H-1). However, these leaves were so overun w i th colony growth that i t was imposs ib le to t e l l whether spores had i n i t i a t e d the co l on ie s from the area of exposed mesophyll or from the area w i th the i n t a c t ep idermi s . Spore l ings which had developed in MPG p r i o r to being placed on the exposed mesophyll a l s o produced i n fec t i ons.. (treatments-3 and k: F i g . H-2). Those which 'were heat shocked to induce the format ion of i n f e c t i o n 114 F i g u r e H-2 I n f e c t i o n o f wheat l e a v e s by p r e - d e v e l o p e d s p o r e l i n g s . The 6 t r e a t m e n t s a r e as f o l l o w s : mature undeve1 o p e d . s p o r e s - i n t a c t e p i d e r m i s (1) mature undeveloped s p o r e s - e p i d e r m i s removed (2) s p o r e s d e v e l o p e d on MPG medium and heat shocked t o induce d i f f e r e n t i a t i o n - e p i d e r m i s (3) removed s p o r e s g e r m i n a t e d on MPG medium w i t h o u t /.\ heat s h o c k - i n t a c t e p i d e r m i s s p o r e s d e v e l o p e d ori^MPG-med i urruand- heat shocked t o induce d i f f e r e n t i a t i o n - i n t a c t (5) ep i de rmi s s p o r e s g e r m i n a t e d on MPG medium w i t h o u t <v\ heat s h o c k - i n t a c t e p i d e r m i s Approx. m a g n i f i c a t i o n X 4. Note m y c e l i a l growth on a r e a s o f exposed m e s o p h y l l ( t r e a t m e n t s 2 and 3) 116 Table H-1 I n fec t i on of leaves by pre-developed s p o r e l i n g s . Two areas of i n f e c t i o n were assessed. The f i r s t was the area of exposed mesophyll w i t h i n an area 2 mm x 2 cm in the cent re of the l e a f , c a l l e d 'WITHIN AREA'. The second was the area of i n t a c t epidermis surrounding t h i s , c a l l e d 'OUTSIDE AREA'. Growth occu r r i ng at the j u n c t i o n of these two areas i s r e f e r r ed to 'ON EDGE'. Growths ' ' o n edge ' -were a 1 so inc luded in the,. 'w i t h i n ' c a t e go r y . ' c u t e - r ' • -. -Infection of Leaves by Germinated and Differentiated Sporelings TABLE H-l TREATMENT NO. OF L E A V E S I N F E C T E D w i t h i n a r e a NO. OF L E A V E S I N F E C T E D o u t s i d e a r e a TOTAL NO. OF GROWTHS MEAN COLONY S I Z E w i t h i n a r e a o u t s i d e a r e a o n j u n c t i o n ( a l s o c o u n t e d w i t h i n a r e a ) mm. 1 UNDEVELOPED SPORE 1NTACT LEA F 23 25 145 216 N/A 1.22 2 UNDEVELOPED SPORE RBMOVED E P I D E R M I S 19 25 46 152 27 1.80 3 D I F F E R E N T I A T E D SPORE REMOVED E P I D E R M I S 13 5 23 7 3 2.65 4 GERMINATED SPORE REMOVED E P I D E R M I S 1 3 1 3 1 1.41 C D I F F E R E N T I A T E D SPORE 0 INTACT L E A F 5 4 6 5 N/A 1.82 6 GERMINATED SPORE INTACT L E A F 3 5 5 6 N/A 1.41 MEAN % DEVELOPMENT I N SOURCE CULTURE D I S H E S -HEAT SHOCKED SPORES Z^GERMI NAT 10N= 73.2 % E X P R E S S E D A S A % OF TOTAL SPORES * D I F F E R E N T I AT 10N= 17.1 -UNSHOCKED SPORESZSGERM I NATI 0N= 85.4 % Y D I F F E R E N T I A T ION= 1 % 118 F i g u r e H-7 I n f e c t i o n o f wheat l e a f . A s t r i p o f m e s o p h y l l i s exposed i n c e n t r e o f l e a f . E p i d e r m i s i s i n t a c t on a r e a s u r r o u n d i n g t h i s . The j u n c t i o n o f t h e s e two a r e a s runs t h r o u g h a r u s t c o l o n y . On the exposed m e s o p h y l l s i d e , the c o l o n y i s gro w i n g v e g e t a t i v e l y : on the e p i d e r m a l s i d e , t he c o l o n y i s s p o r u l a t i n g ( f a r r i g h t ) . Approx. m a g n i f i c a t i o n X 6. F i g u r e H-8 I n f e c t i o n of wheat l e a f as above. Colony i s s p o r u l a t i n g on a r e a of i n t a c t e p i d e r m i s . Note myce 1 ia.l growth on ar e a o f exposed„mesophy11. Approx. m a g n i f i c a t i o n X 23. 120 s t r u c t u r e s gave a markedly h igher i n f e c t i o n count than those spo re l i ng s which were not shocked. The shocked spo re l i ng s (treatment 3' Table H-1) i n f e c ted 13 leaves w i t h i n the area of exposed mesophyl1, y i e l d i n g a t o t a l of 23 growths; whereas, f o r the unshocked spo re l i ng s (treatment h: Table H-1), only one growth was counted on a s i n g l e i n fec ted l e a f . This one growth was on the edge of the i n t a c t ep idermi s . When developed spo re l i ng s were placed on i n t a c t leaves (treatments 5 and 6: F i g . H-2) , they a l s o produced pu s tu le s . Th is ra i se s the p o s s i b i l i t y that t h i s one i n f e c t i o n f o r treatment k cou ld have occurred through the epidermis i t s e l f . D i f f e r e n t i a t i o n percentages f o r heat shocked spores , developed in MPG medium, were extremely low: only 17 % of the t o t a l spores d i f f e r e n t i a t e d (Table H-1). S ince 27 % of the spores d id not germinate, t h i s meant that 56 % of the heat shocked spores had germinated without producing i n f e c t i o n s t r u c t u r e s . Because of t h i s , there was no way of knowing whether the s p o r e l i n g s , i n f e c t i n g the exposed mesophyll f o r treatment 3, were germinated or d i f f e r e n t i a t e d ones. Spore l ings in the unshocked treatments a l s o developed i n f e c t i o n s t r u c t u r e s in a few cases (1 % of the t o t a l spores d i f f e r e n t i a t e d ) ; As a r e s u l t , the s i n g l e i n f e c t i o n on the exposed mesophy l l , f o r the unshocked treatment k, may have been caused by d i f f e r e n t i a t e d spores. EFFECT OF DIFFERENTIATION ON AXENIC CULTURE GROWTH: Experiments were a l s o done to determine the r o l e of i n f e c t i o n s t r u c tu re s f o r the growth of the fungus in axenic c u l t u r e . Although P_. graminis t r i t i c i has been cu l t u r ed a x e n i c a l l y , extremely large amounts 121 of inoculum are requ i red f o r i n i t i a t i o n and estab l i shment of co l on i e s (Kuhl et a l . 1971, Bushnel l and Stewart 1971 , Bose and Shaw 1974a), ranging from 2 500-2000 spores/mm . S ince la rge numbers of s t e r i l e spores are d i f f i c u l t to o b t a i n , t h i s method of rust c u l t u r e i s i n e f f i c i e n t . The re fo re , f o r present-;:experhments f cat-tempts r were made ~totr'educe Kthe ; number.' of spores used as s t a r t i n g inoculum. The o b j e c t i v e was to produce co l on ie s from s i n g l e uredospores. There are two ser ious f laws in any axenic experiment performed to date w i th rust organisms. The f i r s t is the i n a b i l i t y to know p r e c i s e l y how many rust propagules i n i t i a t e d a s i n g l e co lony , the second i s i n s u f f i c i e n t knowledge concerning the genet i c o r i g i n s of the co lony. If co l on ie s could be i n i t i a t e d from s i n g l e spores, these problems would be so l ved . Experiments were designed to grow the rust under axenic c o n d i t i o n s , up to and beyond the i n f e c t i o n s t r u c t u r e stage. For these p re l im ina r y t r i a l s , extremely t h i n seedings of uredospores were used, only 1 to 10 2 spores per mm . Clumping was avoided and the spores were, f o r the most p a r t , p h y s i c a l l y separate from one another. Two media were used, medium 1 the MPG d i f f e r e n t i a t i o n s ub s t r a t e , and medium 2 an-iAXENIC c u l t u r e medium (Bose and Shaw 1974b| see Table C-4). Spores were i n i t i a 11y p1aced in e i t h e r medium 1 or 2. Some were g iven a heat shock t o induce _ d i f f e r e n t i a t i o n . Then the subs t ra tes were a l l changed at 30 hours, e i t h e r to the same medium in which the spores were s t a r t e d , or to the a l t e r n a t i v e s ub s t r a te . The exper imental plan is summarized in Table 1-1. Three separate experiments were done, each w i th s u c ce s s i v e l y fewer spores. The two a d d i t i o n a l experiments were s i m i l a r to the f i r s t except 122 Table 1 - 1 Growth of th in l y - seeded spo re l i ng s in axen ic c u l t u r e . E f f e c t of d i f f e r e n t i a t i o n and a ' two - s tage ' medium. * The two-stage medium re fe r s to two subs t ra tes used in succe s s i on ; MPG medium fo l lowed by AXENIC medium. 123 GROWTH IN AXENIC CULTURE-THIN SEEDING R a c e 1 5 - B P u c c i n i a g r a m i n i s t r i t i c i ( 1 - 1 0 u r e d o s p o r e s / m m ^ ) MEDIUM " MPG: D i f f e r e n t i a t i o n m e d i u m pH 6.7 ( p e p t o n e , g l u c o s e , i n o r g a n i c s a l t s ) MEDIUM 2 A X E N I C : C h e m i c a l l y d e f i n e d a x e n i c c u l t u r e m e d i u m pH 5 . 5 ( B o s e a n d Shaw 197A ) TABLE l-l MEDIUM SUMMARY A X E N I C GROWTH NO. D I SHES WITH HEALTHY GROWTH -2-3 MO. TREATMENT I N I T I A L T I M E 0 h o u r s F I N A L T IME 30 h o u r s HEAT SHOCK 3 WEEKS 2 MONTHS EXP 1 EXP 2 EXP 3 A 1 MPG 2 A X E N I C + + + 3 It 6 B - + - 0 0 6 C 2 A X E N I C 2 A X E N I C + - - 0 0 0 D - • - - 0 0 0 E 1 MPG 1 MPG + - - 0 0 0 124 that in the second exper iment, the medium was changed a f t e r 2 hours and 4 days instead of 30 hours. A f t e r d ishes w i th contaminated media were d i scarded and random s e l e c t i o n was made to ensure equal sample s i z e s , 6 r e p l i c a t e d ishes were kept f o r each treatment per experiment. D i f f e r e n t i a t i o n did not occur in the AXENIC medium even though a heat shock was app l i ed (treatment C: F i g . 1-1). This lack of d i f f e r e n t i a t i o n was not due to d i f f e r e n c e s in pH between the AX ENT C and MPG media. I n fec t i on s t r u c t u r e s were c o n s i s t e n t l y produced in MPG media of both pH 5-75 and 6.75, and repeatedly f a i l e d to form in AXENIC media of pH 5.75 and 6.75. Even though heat shock d id not induce d i f f e r e n t i a t i o n in the AXENIC medium, i t d id have an e f f e c t on growth morphology. Th is was not e a s i l y seen under the microscope, but was apparent w i th the naked eye at about 10 days. There was some growth in a l l treatments up to 10 days ( F i g . 1-1). At t h i s t ime, growth in most treatments ceased, wh i l e in o t he r s , co l on ie s began to form. These t h i n l y seeded, p h y s i c a l l y separate uredospores did not i n i t i a t e vege ta t i ve co l on ie s on e i t h e r medium 1 MPG (treatment E: F i g . 1-1 and I-2) or medium 2 AXENIC (treatments C and D: F i g . 1-1 and I-2) which normal ly supports growth at h igher spore concen t r a t i on s . But when the MPG medium was changed a f t e r 2 hours, 30 hours, or 4 days, to the AXENIC medium, f u r t h e r growth d id occur (treatments A and B: F i g . 1-1 and 1-2). On t h i s two-stage medium, co l on i e s were i n i t i a t e d from small groups as w e l l as i n d i v i d u a l spo re l i ng s a f t e r 10 to 20 days, depending upon the o r i g i n a l spore c oncen t r a t i on . F igures I-3 and 1-4 show these s i n g l e spo re l i ng s each i n i t i a t i n g a co lony . 125 F igure 1-1 Growth of th in l y - seeded spo re l i ng s in axenic c u l t u r e . Spore l ings are 10 days o l d . Treatments are as f o l l o w s : MPG medium changed to AXENIC medium /.•> at 30 hours: w i th heat shock MPG medium changed to AXENIC medium at 30 hours: no heat shock AXENIC medium: w i th heat shock (C) AXENIC.medium: no heatbshock (D) MPG medium: w i t h heat shock (E) M a g n i f i c a t i o n X 87. 127 F igure I-2 Growth of t h i n l y - seeded spo re l i ng s in axenic c u l t u r e . Spore l ings are 2 months o l d . Co lon ies from treatment A are the only ones s t i l l a l i v e . The treatments are as fol1ows: MPG medium changed to AXENIC medium /.\ at 30 hours: w i th heat shock MPG medium changed to AXENIC medium /„•> at 30 hours: no heat shock AXENIC medium: w i th heat shock (C) AXENIC medium: no heat shock (D) MPG medium: w i th heat shock (E) Approx. magn i f i c a t i on X 0.98. 129 F igure I-3 S i ng le d i f f e r e n t i a t e d s po re l i n g i n i t i a t i n g a colony a f t e r and \-h 10 days growth. Spore l ings were grown in a medium common to other spores , but were p h y s i c a l l y separate from them. Treatment A was used: i n i t i a l MPG medium was changed to AXENIC a f t e r 30 hours.-" Spores-were-heat--.shocked. I X -450 . \ 130 131 Colony growth was followed m i c r o s c o p i c a l l y f o r several months. A f t e r one month, col o n i e s on the two-stage medium were s t i l l growing whether or not a heat shock had been given. Colonies from the d i f f e r e n t i a t e d s p o r e l i n g s , however, were smaller and more d i s t i n c t than those from the germinated ones, which tended to be la r g e r and to spread out over the di s h l i k e a mat. A f t e r 2 to 3 months, c o l o n i e s formed from the germinated spores had died (treatment B: F i g . 1-2), w h i l e those formed from i n f e c t i o n s t r u c t u r e s were s t i l l growing in a healthy manner (treatment A: F i g . 1-2). This was the case f o r the f i r s t two experiments. There was, n e v e r t h e l e s s , some v a r i a b i l i t y in these r e s u l t s s i n c e , in the t h i r d experiment, c o l o n i e s from both the germinated and d i f f e r e n t i a t e d s p o r e l i n g s were s t i l l a l i v e a f t e r 3 months. Whether t h i s behaviour was due to the lower inoculum concentrations of the t h i r d experiment is not known. Results of a l l treatments are summarized in Table 1-1. Thus, there were two main r e s u l t s . F i r s t l y , i n d i v i d u a l s p o r e l i n g s produced c o l o n i e s on AXENIC medium i f they were f i r s t placed in MPG medium fo r periods as b r i e f as the f i r s t 2 hours of development. Secondly, there were i n d i c a t i o n s that the development of i n f e c t i o n s t r u c t u r e s was necessary to s u s t a i n growth at low spore c o n c e n t r a t i o n s . ISOLATED SINGLE SPORES VS. SPORES IN COMMON MEDIUM: Subsequently, attempts were made to develop axenic c o l o n i e s from i s o l a t e d s i n g l e uredospores, each placed in a separate w e l l of a micrsotest p l a t e f i l l e d w ith media according to the same plan as described in Table 1-1. Germination and d i f f e r e n t i a t i o n percentages were extremely low, and no f u r t h e r growth or branching took p1 ace,aaftertthermediacchange, in any of 132 the t reatments . Consequently, f u r t h e r experiments were c a r r i e d out to compare germinat ion and d i f f e r e n t i a t i o n behaviour in the i s o l a t e d s i n g l e spore c ond i t i o n (each spore in a separate w e l l ) versus that of t h i n l y seeded, p h y s i c a l l y separate spores in a common medium. Spores in the common medium were inocu la ted in two ways. In the f i r s t exper iment, these s pones swerve. th'rniliyyseededdusingnann i ihoouilat i:rig j 1 oop,., I the .second exper iment, spore numbers were s t r i c t l y c o n t r o l l e d . Each uredospore was i n d i v i d u a l l y p l a ced , w i thout m ix ing , i n to a p e t r i d i sh con ta in i ng the same volume of medium per spore as in the i s o l a t e d s i n g l e spore c ond i t i o n (each spore in a separate;:we.l 1) . Resu l t s of the two experiments are shown in Tables J-1 and J - 2 . The data were analyzed using a Binomial Ana l y s i s of Var iance and the Chi Square Test of Independence f o r unequal sample s i z e s . There was no d i f f e r e n c e at the 0.05 l e ve l of s i g n i f i c a n c e between germinat ion percentages of spores in the common medium and those of i s o l a t e d s i n g l e spores. In both cases, germinat ion percentages were h ighest in the MPG medium and lowest in tap water. However, in a l l t reatments , germ tubes were -cons ide rab l y sho r te r in the singde spore c o n d i t i o n . Th is d i f f e r e n c e in germ tube length was most no t i c eab l e in d i s t i l l e d H^ O and b u f f e r , and l ea s t obvious in MPG medium. D i f f e r e n t i a t i o n percentages were low in a l l cases. Never the le s s , there was c o n s i s t e n t l y g rea te r v e s i c l e format ion By those spores in the common med i um,' both ",i n* the shocked and unshocked t reatments , than in the i s o l a t e d s ing.le-spore c o n d i t i o n . I n fec t i on hyphae were not even seen in the s ingde spore p l a t e s . Nonyl a l c o h o l , a germinat ion stimu1 ant , twas a l s o t e s t e d . It had no e f f e c t on germinat ion f requency, germ tube l eng th , or d i f f e r e n t i a t i o n of 133 Table J-1 Germination and d i f f e r e n t i a t i o n of i s o l a t e d s i n g l e spores versus p h y s i c a l l y separate spores in a common medium. Var ious subs t ra tes were used. Note that d i f f e r e n t i a t i o n was reduced in the i s o l a t e d s i n g l e spore c o n d i t i o n . Table J-2 Germination and d i f f e r e n t i a t i o n of i s o l a t e d s i n g l e spores versus p h y s i c a l l y separate spores in a common medium. Subst rate was MPG. The same volume of medium per spore was used f o r both the i s o l a t e d spores and those in a common medium. Note that d i f f e r e n t i a t i o n was reduced in the i s o l a t e d s i n g l e spore c o n d i t i o n . Isolated Single Spores VS. Spores in Common Medium TABLE J- l % of total spores EXPERIMENT 1 GERMINATION DIFFERENTIATION MEDIUM TREATMENT HEAT SHOCK COMMON MEDIUM SINGLE SPORES COMMON MEDIUM SINGLE SPORES NO. MPG 1 + 9 7 8 9 12 7 2 - 9 2 8 7 0 0 AXENIC 3 + 70 63 0 7 4 - 82 6 5 0 0 Ca-K-P0«4 5 + 6 8 59 0 0 BUFFER 6 - 73 64 0 0 TAP WATER 7 + 0 0 0 0 8 - 0 4 0 0 GLASS DIST. 9 + 4 7 36 0 0 H 20 6 6 10 35 0 0 TABLE J-2 EXPERIMENT 2 % of total spores TREATMENT TREATMENT NO. HEAT SHOCK GERMINATION DIFFERENTIATION 100 33 Petrie 1 + 100 23 Dish 98 16 60 spores 97 8 98 2 (in common 2 94 2 med1 urn) 98 4 98 3 98 2 MIcrotest 3 + 96 0 Plate 99 0 98 0 60 spores 100 0 (1 per 4 97 0 well) 97 0 95 0 INITIAL MEDIUM-MPG-CHANGED TO AXENIC AFTER 3 6 HOURS 135 single spores in MPG medium. EFFECT OF CONDITIONED MEDIUM ON SINGLE SPORE DEVELOPMENT: Experiments were carried out to test the p o s s i b i l i t y that a growth stimulator, normally e f f e c t i v e at higher spore concentrations, but not at lower ones, might be connected with poor development in the isolated single spore condition. MPG medium was conditioned by using i t to d i f f e r e n t i a t e quantities of uredospores (100-2000sppoes/mmm)";1 ennmassei: Aliquots of this medium were then removed af t e r various time intervals and used as the s t a r t i n g medium for the development of isolated single spores. The single spores were also given a heat shock to induce d i f f e r e n t i a t i o n . Results are presented in Table K-1. Data were analyzed by the Chi Square Test of Independence. Treatments were not d i f f e r e n t at the 0.05 level of s i g n i f i c a n c e . Neither germination nor d i f f e r e n t i a t i o n was improved in conditioned MPG medium. Because of the p o s s i b i l i t y that the MPG medium might be masking the e f f e c t s of germination, or other growth stimulators, the e f f e c t of conditioned glass d i s t i l l e d H^ O on germination was tested. Conditioned water also had no e f f e c t on germination at the 0.05 level of s i g n i f i c a n c e (Table K-2). EFFECT OF 'DIFFERENTIATION STIMULATOR' ON SINGLE SPORE GROWTH: An attempt was made to i s o l a t e the d i f f e r e n t i a t i o n stimulator with the aim of us i ng M 11->to improve i nfect ion, st ructure. format i on o f - i s o l a t e d single spores. Crude germination i nh.lbii tor .was obtainedufrom uredospores as.described e a r l i e r (page kO of Materials and Methods). Six 1 ml fractions were co l l e c t e d a f t e r steam d i s t i l l a t i o n of the ,crude l i q u i d . Fractions 5 and 6 136 Table K-1 E f f e c t of ' c o n d i t i o n e d ' MPG medium on germinat ion and d i f f e r e n t i a t i o n of s t e r i l e i s o l a t e d s i n g l e spores. ' C o n d i t i o n e d ' medium had no e f f e c t on morphogenesis at the 0.05 l e ve l of s i g n i f i c a n c e . Table K-2 E f f e c t of ' c o n d i t i o n e d ' g las s d i s t i l l e d hLO on germinat ion of n o n - s t e r i l e i s o l a t e d s i n g l e spores. ' C ond i t i o ned ' H^ O had no e f f e c t on morphogenesis at the 0.05 l e ve l of s i g n i f i c a n c e . EFFECT OF 'CONDITIONED 1 MPG MEDIUM ON GERMINATION AND DIFFERENTIATION OF STERILE ISOLATED SINGLE SPORES TABLE K-1 EFFECT OF 'CONDITIONED' GLASS DISTILLED HO ON GERMINATION OF NON-STERILE ISOLATED SINGLE SPORES TABLE K-2 138 were combined to g i ve a new f r a c t i o n 5. Of the r e s u l t i n g f i v e tubes , numbers 1, 4, and 5 had a strong f i s h y odour. There was no odour in tubes 2 and 3 or in the crude i n h i b i t o r . Each of the f i v e f r a c t i o n s was tes ted fo r i t s e f f e c t on morphogenesis of i s o l a t e d s i n g l e spores , both in g la s s d i s t i l l e d H^ O and in MPG medium. A l l treatments were heat shocked to induce the format ion of i n f e c t i o n s t r u c t u r e s . P r i o r to t e s t i n g , f r a c t i o n s were d i l u t e d 6 times w i th the appropr i a te s ub s t r a te . Crude i n h i b i t o r was cen t r i f uged at 7000 rpm f o r 20 minutes to r i d i t of spores and d i l u t e d 1:2 w i t h g las s d i s t i l l e d H 2 0. The e f f e c t of these f r a c t i o n s on spore morphogenesis in H^ O is shown in Table L-1. None of the o r i g i n a l uredospores; ,used to obta in the crude i n h i b i t o r , germinated during t h e i r incubat ion per iod in the presence of the crude l i q u i d . However, when t h i s crude i n h i b i t o r was tested f o r i t s e f f e c t on i s o l a t e d s i n g l e spore ge rminat i on , i t was comparable to the c o n t r o l : the crude l i q u i d had no i n h i b i t o r y p r o p e r t i e s . On the other Hand1, the f i r s t 17-20 % of the d i s t i l l a t e gave complete i n h i b i t i o n of s i n g l e spore germinat ion in H2O ( F rac t i on 1: Table L-1). Th is i n h i b i t i o n was e f f e c t i v e at e a r l y germinat ion stages s i n ce none of the spores had any i n i t i a l germ tube outgrowths whatsoever. In con t ra s t to t h i s , the l a s t 45-50 % of the d i s t i l l a t e gave marked s t i m u l a t i o n of germinat ion (F rac t i ons IV and V: Table L-1) . Th is s t imulus appeared not .on ly to increase the number of spores germinat ing i n i t i a l l y , but a l s o to be p a r t i c u l a r l y e f f e c t i v e at l a t e r stages of germinat ion i n vo l v i n g tube e l onga t i on . Germ tubes were s i g n i f i c a n t l y longer in these treatments (F rac t ions IV and V: Table L-2). Those f r a c t i o n s of the d i s t i l l a t e which had e i t h e r an i n h i b i t o r y or s t imu l a t o r y e f f e c t , were a l s o the same ones which had a 139 Table L-1 E f f e c t of crude germinat ion i n h i b i t o r d i s t i l l a t e on germinat ion and d i f f e r e n t i a t i o n of i s o l a t e d s i n g l e spores in g las s d i s t i l l e d H O . F r a c t i o n I i n h i b i t e d germinat ion . F r a c t i on s IV and V s t imu la ted germinat ion . Table L-2 E f f e c t of crude germinat ion i n h i b i t o r d i s t i l l a t e on germ tube length of i s o l a t e d s i n g l e spores in g las s d i s t i l l e d H o0. F r ac t i on s IV and V s t imu la ted germ tube e l onga t i on . Table L-3 E f f e c t of crude germinat ion i n h i b i t o r d i s t i l l a t e on germinat ion and d i f f e r e n t i a t i o n of i s o l a t e d s i n g l e spores in MPG medium. No e f f e c t was observed in MPG medium. EFFECT OF CRUDE INHIBITOR DISTILLATE ON GERMINATION AND DIFFERENTIATION 140 OF SINGLE SPORES IN GLASS DISTILLED H 20 % of tota1 spores FRACTION GERMINATION DIFFERENTIATION Control H 20 45 0 Crude Inhibitor 6 2 0 Fraction l-fishy 0 0 Fraction II 53 0 Fraction 111 38 0 Fraction IV-fishy 90 0 Fraction V-fishy 89 0 TABLE L-l EFFECT OF CRUDE INHIBITOR DISTILLATE ON GERM TUBE LENGTH OF SINGLE SPORES IN GLASS DISTILLED H20 % of total spores FRACTION Considered not qerm. Considered qerm. WITH NO GERM TUBE WITH TUBE < SPORE D1AM. WITH TUBE =. SPORE DIAM. WITH TUBE> SPORE DIAM. Control H 20 20 35 2 7 18 Crude Inhibitor 6 32 38 25 Fraction l-fishy 100 0 0 0 Fraction II 13 35 18 35 Fraction 111 34 28 34 k Fraction IV-fishy 5 5 20 70 Fraction V-fishy 8 3 26 63 EFFECT OF CRUDE INHIBITOR DISTILLATE ON GERMINATION AND DIFFERENTIATION OF SINGLE SPORES IN MPG MEDIUM % of total spores FRACTION GERMINATION DIFFERENTIATION Control H 20 77 0 Fraction l-fishy 77 0 Fraction II 85 0 Fraction 111 6 3 0 Fraction IV-fishy 77 0 Fraction V-fishy 6 9 0 TABLE L-3 141 f i s h y odour. The odour seemed to be the same despi t e the d i f f e r e n c e s in e f f e c t between the two parts of the d i s t i l l a t e . None of the f r a c t i o n s which a f f e c t e d germination of s i n g l e spores when tested in water, had any e f f e c t when tested in the presence of MPG medium (Table L~3). This i n d i c a t e s that the MPG medium tends to overcome the e f f e c t s of germination i n h i b i t o r s or s t imul ators^.on uredospore morphogenesis. The spores had been tre a t e d with both the appropriate heat shock, as we l l as the i n h i b i t o r d i s t i l l a t e , yet none of the spores d i f f e r e n t i a t e d . These r e s u l t s i n d i c a t e that the d i f f e r e n t i a t i o n s t i m u l a t o r was not i s o l a t e d , o r , i f present, was not detected. However, a germination i n h i b i t o r (tube outgrowth), and a germination s t i m u l a t o r (tube outgrowth and elongation) were detected during t h i s study. EFFECT OF SPORE CONCENTRATION AND SPORE NUMBER ON DIFFERENTIATION: An attempt was made to c l a r i f y the basic e f f e c t s of both spore concentration and spore number on d i f f e r e n t i a t i o n . Three experiments were designed. These experiments were intended to d i s t i n g u i s h between the e f f e c t s of spore numbers and the e f f e c t s of spore numbers per unit volume of medium (spore c o n c e n t r a t i o n ) . The f i r s t experiment was planned to t e s t the e f f e c t of both spore numbers and spore concentration on percentage d i f f e r e n t i a t i o n , the second to t e s t the e f f e c t of spore numbers o n l y , and the t h i r d , the e f f e c t of concentration only. Plans of the experiments are as f o l l o w s . In the f i r s t experiment, in c r e a s i n g numbers of spores are placed i n t o a constant volume of medium. In the second, in c r e a s i n g numbers of spores 142 are placed into increasing volumes of media such that the spore concentration remains the same, but the spore numbers increase. In the th i r d experiment, constant'spore-numbers are placed into increasing volumes of media such that the spore concentration v a r i e s , while the spore number remains the same in a l l preparations. Unfortunately, only the f i r s t experiment was carried out. Results of the f i r s t experiment are shown in Table M-1. Each of the spores had been i n d i v i d u a l l y placed into the appropriate container. Small s t e r i l e glass v i a l s (0.08 ml medium) were used here, instead of the p l a s t i c microtest plates. D i f f e r e n t i a t i o n was unusually high for isolated single spores. There was no s i g n i f i c a n t difference in germination or d i f f e r e n t i a t i o n with simultaneously increasing spore numbers and spore concentrations. One explanation for this could be the use of glass containers. Because of these f i n d i n g s , i t may be possible to culture rust from single sporeshif gJiass containers are used. Another explanation could be the increased volume of medium per spore over that used in the p l a s t i c microtest plates. However, further experiments are needed. 143 Table M-1 Effect of spore number and spore concentration on germination and d i f f e r e n t i a t i o n of uredospores in glass v i a l s . i 144 EFFECT OF SPORE NUMBER AND SPORE CONCENTRATION ON GERMINATION AND DIFFERENTIATION OF UREDOSPORES IN GLASS VIALS TABLE M-1 TREATMENT NO. NO. DISHES NO. SPORES PER DISH GERMINATION % DIFFERENTIATION % o f t o t a l spores % o f germ s p o r e s 1 40 1 88 31 36 2 20 2 83 17 21 3 10 k 71 21 29 4 5 8 93 13 14 5 3 16 85 12 9 6 1 32 87 13 15 7 1 64 79 14 18 8 1 128 80 20 25 9 1 256 69 12 17 EACH SPORE INDIVIDUALLY PLACED INTO DISH CONTAINING 0.08 ml MPG 145 DISCUSSION R e s u l t s o f the p r e s e n t s t u d y s u p p o r t the v i e w t h a t morphogenesis o f the wheat stem r u s t u r e d o s p o r e i s due t o a p r e c i s e l y timed sequence o f even t s o c c u r r i n g w i t h i n t h e d e v e l o p i n g s p o r e l i n g . C o r r e c t t i m i n g and c o - o r d i n a t i o n o f th e s e e v e n t s i s e s s e n t i a l i f normal development i s t o t a k e ^ p l a c e . T h i s development f o l l o w s two d i s t i n c t s t a g e s : the f i r s t l e a d s t o the p r o d u c t i o n o f the germ tube and the second leads t o t h e f o r m a t i o n o f the i n f e c t i o n hypha. Even though t h e s e two r e s u l t i n g s t r u c t u r e s a r e morpholgg i.ca 1 1 y s i mi .l.ar, t hey -are phys i o l o g i c a 1 1 y d i f f e r e n t . They a r e d i f f e r e n t i n terms o f f a c t o r s a f f e c t i n g t h e i r development, c y t o l o g i c e v e n t s o c c u r r i n g d u r i n g t h e i r f o r m a t i o n , and the r o l e t h a t each p l a y s f o r f u r t h e r growth and s u r v i v a l o f the fu n g u s . FACTORS AFFECTING MORPHOGENESIS: F a c t o r s a f f e c t i n g the g e r m i n a t i o n s t a g e o f P_. g r a m i n i s t r i t i c i a r e d i f f e r e n t and o f t e n c o n t r a d i c t o r y t o those r e q u i r e d f o r d i f f e r e n t i a t i o n . Thus, i n agreement w i t h Maheshwari et_ aj_. (1967a), low t e m p e r a t u r e s f a v o u r e d g e r m i n a t i o n , whereas .fo'igher'" t e m p e r a t u r e s ' i nt the form o f .a p r e c i s e l y timed heat s h o c k , promoted the f o r m a t i o n o f i n f e c t i o n s t r u c t u r e s . However, c o n t r a r y t o t h e i r r e p o r t s t h a t 95 % d i f f e r e n t i a t i o n o c c u r r e d on a Ca-K-PO^ b u f f e r , 7 % was the h i g h e s t count r e c o r d e d i n many t r i a l s d u r i n g the p r e s e n t s t u d y , even though g e r m i n a t i o n was ove r 90 % on t h i s b u f f e r . The rac e used by Maheshwari e t a 1. (1967a) was not r e p o r t e d , but Dunkle e t a 1. (1969), u s i n g the same t e c h n i q u e , r e p o r t e d 80-90 % d i f f e r e n t i a t i o n w i t h 1 4 6 r a c e 5 6 . The r a c e used f o r the p r e s e n t s t u d y was 15B-2. I t i s a l r e a d y known t h a t r a c e s v a r y i n t h e i r c a p a c i t y t o d i f f e r e n t i a t e on a g i v e n s u b s t r a t e (page 15 o f L i t e r a t u r e R e v i e w ) . The d i s c r e p a n c y i n t h e p r e s e n t o b s e r v a t i o n s l e a d s one t o s p e c u l a t e t h a t r a c e s may a l s o d i f f e r as t o the type o f s t i m u l u s r e q u i r e d t o a c h i e v e d i f f e r e n t i a t i o n . T h i s w a r r a n t s f u r t h e r i n v e s t i g a t i o n . W i t h 15B-2, i n f e c t i o n s t r u c t u r e s were formed o n l y when n u t r i e n t s were added t o the medium ( T a b l e s D-1 and D-2). S p o r e l i n g s were p a r t i c u l a r l y r e s p o n s i v e t o the a d d i t i o n o f 0 . 5 % peptone and 3 % g l u c o s e (MPG medium, F i g . D-2). In a d d i t i o n t o the n u t r i e n t s t i m u l u s , peptone may have a l s o s a t i s f i e d t he need f o r a s u r f a c t a n t s i n c e i t pr e v e n t e d s p o r e l i n g a g g r e g a t i o n . D i f f e r e n t i a t i o n was a l s o i n c r e a s e d by the pr e s e n c e o f a g a r , p o s s i b l y due t o i t s n u t r i e n t o r s u r f a c e p r o p e r t i e s . On MPG medium, g e r m i n a t i o n o f 100 % was not uncommon, however o n l y about 70 % o f the s p o r e s formed i n f e c t i o n s t r u c t u r e s even under o p t i m a l c o n d i t i o n s . The amount o f d i f f e r e n t i a t i o n a c h i e v e d was q u i t e v a r i a b l e , e s p e c i a l l y between e x p e r i m e n t s . S f a n d a r d u c o n d i t i o n s were m a i n t a i n e d f o r t h e ' p r o d u c t ] o n o f sp o r e s on t h e ^ p l a n t ; t h e age o f the. s p o r e s c o l l e c t e d , and the d i f f e r e n t i a t i o n p r o c e d u r e . Y e t , f a c t o r s a f f e c t i n g t h i s v a r i a t i o n c o u l d not be i d e n t i f i e d . E i t h e r t he i n t e n s i t y o f the s t i m u l u s was not equal i n the m i c r o e n v i r o n m e n t f o r a l l s p o r e l i n g s , o r s p o r e l i n g s v a r i e d i n t h e i r m o r p h o g e n e t i c p o t e n t i a l as suggested by W i l l i a m s (1971). I f the l a t t e r i s t r u e , i t means t h a t the s t r e n g t h o f the s t i m u l u s r e q u i r e d t o e l i c i t t he d i f f e r e n t i a t i o n r e sponse may v a r y w i t h t h e p a r t i c u l a r s p o r e l i n g i n q u e s t i o n . In a d d i t i o n , i t i s thought t h a t a l l s p o r e l i n g s have t h e c a p a c i t y t o form i n f e c t i o n s t r u c t u r e s . T h i s may not be t r u e . 147 Factors a f f e c t i n g germinat ion and d i f f e r e n t i a t i o n of s i n g l e spores are d i scussed on pages 161 to 163 of the D i s cu s s i on . CYTOLOGIC EVENTS DURING MORPHOGENESIS: The c y t o l o g i c events tak ing p lace dur ing d i f f e r e n t i a t i o n were more complex and v a r i ed than those occu r r i ng dur ing germinat ion . Nuclear d i v i s i o n took p lace in both germinat ing and d i f f e r e n t i a t i n g spores. In d i f f e r e n t i a t i n g s p o r e l i n g s , d i v i s i o n was a regu lar event and i t s stages fo l lowed a timed sequence. It was i n i t i a t e d in the appressorium dur ing e a r l y morphogenesis at about 3~5 hours ( F i g s . F-9 to F-11) , and in t i m i n g , was c l o s e l y a s soc ia ted w i th the development of i n f e c t i o n s t r u c t u r e s . In c o n t r a s t , d i v i s i o n dur ing germinat ion was rare and sporad ic and was only observed in germ tubes over 10 hours of age (F i g s . F-3 and F-4). V a r i a t i o n s in nuc lear s i z e and nuc lear boundaries were a l s o much more common dur ing d i f f e r e n t i a t i o n than germinat ion . There was only one nuc lear d i v i s i o n in the germ tube, wh i l e there were two in the i n f e c t i o n s t r u c t u r e s dur ing the same time frame; the f i r s t in the appressorium ( F i g . F-11), and the second in the v e s i c l e ( F i g . F-26). Ye t , the f i n a l nuc lear number was o f ten less in the i n f e c t i o n hypha (one nuc leus , F i g . F-40) than in the germ tube (two n u c l e i , F i g . F-2) . The mechanics of the d i v i s i o n process a l s o appeared to d i f f e r between germinat ing and d i f f e r e n t i a t i n g s p o r e l i n g s . Un f o r t una te l y , the type of d i v i s i o n , whether m i t o s i s or me i o s i s , could not be determined w i t h c e r t a i n t y . Chromosomes were seen, but being s m a l l , were d i f f i c u l t to r e so l ve . Never the le s s , the f o l l o w i n g phenomena were i d e n t i f i e d as being 148 c h a r a c t e r i s t i c o f n u c l e a r b e h a v i o u r i n d i f f e r e n t i a t i n g s p o r e l i n g s and were not o b s e r v e d d u r i n g g e r m i n a t i o n . 1) P a r a l l e l p a i r i n g o f chromosome-1ike s t r u c t u r e s : P a r t s o f whole n u c l e i were o c c a s i o n a l l y seen as two p a r a l l e l s t r a n d s ( F i g . F-22) . In some c a s e s , i t l o o k e d as though e n t i r e chromosomes were j o i n e d end t o end. McG i nn i s i (1953) r e p o r t e d a s i m i l a r phenomenon i n t h e g e r m i n a t i n g P_. g ram i n i s b a s i d i o s p o r e . In a d d i t i o n , r i n g s t r u c t u r e s and s p i r a l arrangements s i m i l a r t o t h o s e d e s c r i b e d by M c G i n n i s (1956) were seen (F i gs .rF-*19'=and F-18 r e s p e c t i v e l y ) . R i n g s t r u c t u r e s have a l s o been r e p o r t e d f o r o t h e r f u n g i such as Pen i c i 1 1 i um sp. (Robinow 1965) as w e l l as h i g h e r p l a n t s ( D u b i n i n and Nemtseva 1969). 2) Seeming breakdown of n u c l e a r i d e n t i t y and e s t a b l i s h m e n t o f chromosome-1ike p o o l s : T h i s p r o c e s s was e v i d e n t i n the v e s i c l e s ( F i g . F-27). Large a r e a s of feu1 g e n - p o s i t i v e m a t e r i a l c o u l d be s e e n , but i n d i v i d u a l n u c l e i c o u l d n o t . 3) S p e c i f i c n u c l e a r o r i e n t a t i o n s : These were s p a t i a l o r i e n t a t i o n s r e l a t i v e t o the v e s i c l e and p o t e n t i a l i n f e c t i o n hypha. Some were seen as a g g r e g a t i o n s (Figs.'F - 2 7 t o F-30) : o t h e r s were seen as m i g r a t i o n p a t t e r n s ( F i g s . F-31 t o F-36) , t h e l a t t e r d u r i n g the e x p r e s s i o n o f p o l a r i t y . 4) R e d u c t i o n i n n u c l e a r number i n the i n f e c t i o n hypha: The mature i n f e c t i o n hypha o n l y had e i t h e r one o r two n u c l e i (Figs..F - ;40 and F-42) . What happened t o the o t h e r 5 t o 7 n u c l e i ? From o b s e r v a t i o n s made h e r e , i t i s l i k e l y t h a t e i t h e r some, o r a l l , d i s s o l v e d ( F i g . -F r 3 8) i n a manner s i m i l a r t o the p r o c e s s o f megasporogenesis im f l o w e r i n g p l a n t s , o r t h a t f u s i o n took p l a c e ( F i g . F-41), o r both o f t h e s e a l t e r n a t i v e s . R e s u l t s i n d i c a t e d U 9 t h a t from the i n i t i a l 7 or 8 n u c l e i i n t h e v e s i c l e , u s u a l l y o n l y 3 o r k t r a v e l l e d down the i n f e c t i o n hypha, one l e a d i n g compact n u c l e u s f l a n k e d by two o t h e r s t r a i l i n g b e h i n d i t . The q u e s t i o n a r i s e s as t o what advantage i s g a i n e d by n u c l e a r d i v i s i o n i n t h e v e s i c l e and subsequent r e d u c t i o n i n t h e hypha. M e i o s i s and r e d u c t i o n d i v i s i o n t a k e s p l a c e i n t h e t e l i o s p o r e ( S a p p i n - T r o u f f y 1896). Subsequent d i k a r y o t i z a t i o n o c c u r s w i t h n u c l e i o f d i f f e r e n t m ating t y p e s i n the aecium on the a l t e r n a t e h o s t ( C r a i g i e 1927, 1959). C o n s e q u e n t l y , the r u s t u r e d o s p o r e i s n o r m a l l y c o n s i d e r e d t o be a h a p l o i d h e t e r o k a r y o t i c d i k a r y o n ( f o r t e r m i n o l o g y see J i n k s and Simchen 1966). However, M c G i n n i s (1953, 1954, and 1956) proposed t h a t the genus P u c c i n i a was p o l y p l o i d . He s u g g e s t e d t h a t t h e b a s i c chromosome number was 3 and t h a t b a s i d i o s p q r e n u c l e i were a u t o o r a l i o t e t r a p l o i d . T h i s c o m p l i c a t e s the i s s u e somewhat and means t h a t a p r o c e s s s i m i l a r t o meihos i s cannot be r u l e d out i n t h e d i f f e r e n t i a t i n g r u s t u r e d o s p o r e . In f a c t ' ; beadr 1 i ke s t r u c t u r e s s i m i l a r t o th e chromomeres o f the m e i o t i c l e p t o t e n e ( F i g . F-19) and p a i r i n g arrangements such as thos e o f d i p l o t e n e ( F i g . F-18 and F-22) were v i s i b l e d u r i n g d i f f e r e n t i a t i o n . N e v e r t h e l e s s , the e n t i r e m a t t e r d e s e r v e s more d e t a i l e d s t u d y s i n c e t h e r e i s not enough i n f o r m a t i o n f o r d e f i n i t e c o n c l u s i ons. S e v e r a l q u e s t i o n s remain unanswered. 1) Is the d i v i s i o n m i t o s i s o r m e i o s i s , o r v a r i a t i o n s o f e i t h e r ? Many f u n g i do not have a m i t o t i c p a t t e r n t h a t can be f i t t e d i n t o a c l a s s i c c a t e g o r y ( B u r n e t t 1968). 2) Of what s i g n i f i c a n c e a r e the n u c l e a r a g g r e g a t i o n s ? A r e t h e y m i l i e u x 150 f o r p r o c e s s e s such as g e n e t i c r e c o m b i n a t i o n ? Do a l l the n u c l e i o f a s i n g l e b i l a t e r a l clump b e l o n g t o the progeny of a s i n g l e m ating t y p e ? Do the m i g r a t i o n p a t t e r n s have d i v i s i o n a l s i g n i f i c a n c e o r a r e they s t r i c t l y f o r the c o n t r o l of m i g r a t i o n ? 3) What i s the p l o i d y of the i n f e c t i o n hypha n u c l e u s ? I f n u c l e a r f u s i o n s o c c u r , how many n u c l e i a r e i n v o l v e d ? I f n u c l e i d i s s o l v e , what d e t e r m i n e s w h i c h ones w i l l do so? D e s p i t e t h e s e unanswered q u e s t i o n s , a d e f i n i t e p a t t e r n of n u c l e a r development e x i s t s i n the d i f f e r e n t i a t i n g u r e d o s p o r e . R e s u l t s of t h e s e n u c l e a r development s t u d i e s a r e s u p p o r t e d by the p r e s e n t e x p e r i m e n t s w i t h m e t a b o l i c i n h i b i t o r s . S i n c e n u c l e a r d i v i s i o n s t a k e p l a c e i n the a p p r e s s o r i u m and i n the v e s i c l e , one would e x p e c t DNA s y n t h e s i s i m m e d i a t e l y p r i o r t o the d i v i s i o n p r o c e s s . Data from the s t u d i e s w i t h 5 - f 1 u o r o u r a c i 1 s u g g e s t - t h a t e s s e n t i a l DNA-synthesl's does'" t a k e p l a c e b o t h - d u r i n g and a f t e r h e a t ^ s h o c k l t > " J . « 5 - F l u o r o u r a c i 1 (5 _FU) , an a nalogue of u r a c i l , i s a s p e c i f i c i n h i b i t o r of DNA s y n t h e s i s . It i s m e t a b o l i z e d i n v i v o t o 5~f1uorodeoxy UMP w h i c h i s a s p e c i f i c i n h i b i t o r of t h y m i d y l a t e s y n t h e t a s e , the enzyme which c a t a l y z e s the i n t e r c o n v e r s i o n o f p y r i m i d i n e n u c l e o t i d e s i e . the c o n v e r s i o n o f deoxy UMP t o deoxy TMP. Enzyme i n h i b i t i o n r e s u l t s i n a s h o r t a g e of thymine n u c l e o t i d e s and i m p a i r s DNA s y n t h e s i s (Roy-Burman 1970 p. 49 -52). I t has been s u g g e s t e d t h a t 5~FU a l s o i n h i b i t s RNA s y n t h e s i s . However, a l t h o u g h the analogue can be i n c o r p o r a t e d i n t o RNA i n the p l a c e of u r a c i l , t h e s e 5 -FU c o n t a i n i n g RNAs a r e known t o behave i n normal f a s h i o n (Gale et_ aj_. 1972 p. 181) . 151 The t i m i n g of e s s e n t i a l s y n t h e s e s d u r i n g morphogenesis as d e t e r m i n e d by a l l the i n h i b i t o r s , i n c l u d i n g 5 _FU , i s summarized i n F i g . N - 1 . DNA s y n t h e s i s :is shown by the t r i a n g l e i n g r e e n . The base of each t r i a n g l e i n d i c a t e s the t i m e t h a t the i n h i b i t o r was e f f e c t i v e i n p r e v e n t i n g l a t e r development; i n o t h e r words, the time d u r i n g w h i c h e s s e n t i a l s y n t h e s i s took p l a c e . The top of each t r i a n g l e shows the s p e c i f i c s t r u c t u r e a f f e c t e d by the i n h i b i t o r . In the c a s e of 5 _FU, t h i s s t r u c t u r e was the i n f e c t i o n hypha. The r e s u l t s o f the s t u d i e s w i t h 5 -FU r e v e a l e d t h a t e s s e n t i a l DNA s y n t h e s i s d i d not o c c u r w i t h i n the f i r s t two hours and was not n e c e s s a r y f o r g e r m i n a t i o n . There was, l i k e w i s e , no e v i d e n c e of n u c l e a r d i v i s i o n w i t h i n the f i r s t two hours of development. DNA s y n t h e s i s d i d o c c u r a f t e r t h i s , b o th d u r i n g and f o l l o w i n g the heat shock. In agreement w i t h t h i s , n u c l e a r d i v i s i o n began i n t h e l a t e a p p r e s s o r i a l s t a g e j u s t a f t e r the end of the shock p r o c e s s . DNA s y n t h e s i s was found t o be e s s e n t i a l f o r i n f e c t i o n hypha f o r m a t i o n . T h i s leads one t o s p e c u l a t e t h a t n u c l e a r d i v i s i o n might a l s o be e s s e n t i a l . 5 -FU d i d not c o m p l e t e l y p r e v e n t the. development of s t r u c t u r e s o t h e r than the i n f e c t i o n hypha. The i n f e c t i o n hypha may be t h e s t r u c t u r e i m p o r t a n t f o r r u s t s u r v i v a l s i n c e 5-FU has a l s o been shown t o be i n h i b i t o r y i n low c o n c e n t r a t i o n s t o r u s t development on bean l e a v e s ( H e i t e f u s s 1966). The e f f e c t s of 5"FU on i n v i t r o d i f f e r e n t i a t i o n , as r e p o r t e d here f o r r ace 1 5 B - 2 , were d i f f e r e n t from those r e p o r t e d f o r r a c e 56 i n a s i m i l a r s t u d y by Dunkle et_ a_l_. (1969). D u r i n g the p r e s e n t i n v e s t i g a t i o n , 5 _FU had no r e a l e f f e c t on the.developmeht of: s t r u c t u r e s - o t h e r than t h e -Figure N-1 The timing of essential DNA, RNA, and protein synthesis during uredospore morphogenesis. The base of each t r i a n g l e indicates the time during which essential synthesis takes place. The top of each t r i a n g l e points to the s p e c i f i c structure affected by the lack of synthesis. HEAT SHOCK i — 1 C T C O E S S E N T I A L S Y N T H E S E S D U R I N G G E R M I N A T I O N A N D D I F F E R E N T I A T I O N 154 i n f e c t i o n hypha. I n fec t i on hypha development was prevented only i f 5 _FU was present cont inuous ly du r i n g , as we l l as a f t e r , heat shock. In the experiments of Dunkle et a 1., however, i n f e c t i o n s t r u c t u r e development ( i n c l ud i n g the appressorium) was t o t a l l y prevented i f 5 -FU was present , e i t h e r f o r the i n i t i a l 2 hours before heat shock,oordduf imgtthe heat shock i t s e l f . I n fec t i on s t r u c t u r e s developed only i f the i n h i b i t o r was w i t h e l d u n t i l a f t e r thetheat shock. The i n t e r p r e t a t i o n of Dunkle et a l . was a l s o d i f f e r e n t because they assumed that 5 -FU was i n h i b i t i n g RNA, not DNA, s yn the s i s . In support of t h i s was the f a c t that u r a c i l (200 ug/ml) overcame the i n h i b i t i o n of d i f f e r e n t i a t i o n by 5 - FU. However, d e t a i l s of the u r a c i l a p p l i c a t i o n were not s p e c i f i e d . For the present s tudy, u r a c i l was not t e s t e d . Resu l t s of other i n h i b i t o r s used dur ing the present study f u r t h e r comp 1 etedptheepattern ' -of cthehsynthet i c requirements dur ing d i f f e r e n t i a t i o n . The t imings of o t he r ee s s en t i a l syntheses, such as RNA, were d i f f e r e n t from those f o r DNA s yn the s i s . In a d d i t i o n , the s t r u c t u r e s r e q u i r i n g such syntheses were d i f f e r e n t . RNA synthes i s was found to be e s s e n t i a l f o r a l l stages of d i f f e r e n t i a t i o n , but not f o r germinat ion ( F i g . N-1. orange t r iang l e s ) . Act inomycin-D and a-amanit in are both known i n h i b i t o r s of RNA syntheses. Actinomycin-D binds to the deoxyguanosine res idues of DNA in the narrow groove of the B h e l i x . Due to i t s s t e r i c e f f e c t on the DNA, RNA polymerase is blocked and chain e l onga t i on ceases (Goldberg et a l . 1962, Goldberg and Reich 1964, Sobe l l et_ al_. 1971). a - Aman i t i n , l i k e w i s e , i n h i b i t s chain e l o n g a t i o n , but d i f f e r s in that i t i n h i b i t s RNA polymerase 1 5 5 I I or B d i r e c t l y , the polymerase responsible f o r the synthesis of 1DNA-1i ke 1 RNA of eukaryotic c e l l s (Horgen and G r i f f i n 1971, Kedinger et_ a]_. 1971). It binds to and forms a s t a b l e complex with the polymerase i t s e l f (Meihlac e_t_ a_l_. and F a u l s t i c h et_ a_l_. 1970). During the present study, i t was discovered that i f e i t h e r actinomycin or a-amanitin were present before heat shock, appiressoria 1 development and thus the re s t of d i f f e r e n t i a t i o n was prevented, even though germination was unaffected. This i s noteworthy because the heat shock stimulus to t r i g g e r apppessorial formation does not occur u n t i l a f t e r t h i s 2 hour germination period. In a d d i t i o n , the appressorium i t s e l f does not form u n t i l between 3 and k hours. This means that events c r u c i a l f o r d i f f e r e n t i a t i o n take plaee during germination before the a p p l i c a t i o n of any known stimulus to induce i n f e c t i o n s t r u c t u r e formation. It would be h e l p f u l i f the exact time during which these events take place could be pinpointed. Thus, one might look to the germination stage f o r ways of improving d i f f e r e n t i a t i o n in v i t ro. It i s p o s s i b l e that the a p p l i c a t i o n or removal of s p e c i f i c s t i m u l i during the f i r s t two hours of germination could increase d i f f e r e n t i a t i o n , even though the e f f e c t s of these treatments may not be v i s i b l e u n t i l a l a t e r time. The above r e s u l t s with actinomycin again disagree-with those of the study done by Dunkle et a 1 . (1969). They found that there was no e f f e c t on d i f f e r e n t i a t i o n i f actinomycin was present before the .heat shock. However, they used race 56, whereas 15B-2 was used here. This suggests the need f o r study of d i f f e r e n t races. One could speculate that v a r i a t i o n s e x i s t in the timing of e s s e n t i a l syntheses between d i f f e r e n t races. This 156 may have something t o do w i t h t h e i r d i f f e r e n c e s i n p o t e n t i a l i n f e c t i v i t y . In a c c o r d a n c e w i t h i n v i t ro i n h i b i t i o n by a c t i n o m y c i n r e p o r t e d h e r e , t h i s a n t i b i o t i c a l s o i n h i b i t s the development o f P_. g r a m i n i s t r i t i c i on detacheddwheat l e a v e s , but o n l y i f a p p l i e d e a r l i e r than 48 hours a f t e r i n o c u l a t i o n ( H e i t e f u s s 1970). C o n c e n t r a t i o n s w h i c h i n h i b i t t h i s development on the ho s t a r e 0 .5 - 2 ppm. Y e t , g e r m i n a t i o n i s not a f f e c t e d by c o n c e n t r a t i o n s o f 40 ppm. In a d d i t i o n , a c t i n o m y c i n p r e v e n t s 14 3 i n c o r p o r a t i o n o f C - o r o t i c a c i d and H - u r i d i n e i n t o both t he ho s t and p a r a s i t e ( H e i t e f u s s 1970). There have a l s o been r e p o r t s o f s i m i l a r i n h i b i t i o n on the ho s t f o r both bean ( H e i t e f u s s 1966) and f l a x r u s t (Shaw 1967). D i f f e r e n c e s were a l s o found between germ tubes and i n f e c t i o n s t r u c t u r e s i n t h e i r response t o puromycin. Puromycin i n h i b i t s p r o t e i n s y n t h e s i s . I t a c t s on both p r o k a r y o t i c and e u k a r y o t i c l a r g e r i b o s o m a l s u b u n i t s (50 o r 60S ) . T h i s a n t i b i o t i c i s a s t r u c t u r a l a n alogue o f am i n o a c y l a d e n o s i n e , t he 3 ' end of am i n o a c y l t-RNA. It r e a d i l y b i n d s t o the am i n o a c y l s i t e (A s i t e ) o f t he l a r g e r i b o s o m a l s u b u n i t and forms a complex w i t h the e l o n g a t i n g p e p t i d e c h a i n . The p e p t i d y 1 - p u r o m y c i n complex soon de t a c h e s from the ribosome t h e r e b y i n t e r r u p t i n g c h a i n e l o n g a t i o n (Gale e t a l . 1972 p. 283 and 322). G e r m i n a t i o n was not a f f e c t e d by the pr e s e n c e o f p u r o m y c i n , whereas the v e s i c l e s t a g e was. R e s u l t s s u g g e s t e d t h a t , u n l i k e RNA s y n t h e s i s , p r o t e i n s y n t h e s i s was not r e q u i r e d u n t i l a f t e r the heat shock ( F i g . N - 1 , b l a c k t r i a n g l e ) . T h i s was i n complete agreement w i t h the r e s u l t s o f Dunkle et a 1. (1969). An a d d i t i o n a l e f f e c t was noted when puromycin was p r e s e n t b e f o r e heat shock o r u n t c l t h e end'ioff.thecshock per>i:bdpsrInstead o f p r e v e n t i n g 157 development, there was 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 increase in percentage d i f f e r e n t i a t i o n over the c o n t r o l (Table G-2). The presence of puromycin might serve to check unecessary p r o t e i n synthesis such t h a t , when the block i s removed at a time when synthesis i s re q u i r e d , there i s greater synchrony e i t h e r w i t h i n or between s p o r e l i n g s . Staples et a l . (1361, 1962) found that puromycin (80 ppm) induced accumulation of r a d i o a c t i v e amino acids when the inco r p o r a t i o n of e i t h e r r a d i o a c t i v e sodiumsacetate or leucine w a s t e s t e d . Therefore, i t may be that these amino acids are more r e a d i l y a v a i l a b l e f o r immediate use when the block to synthesis i s removed. The most potent i n h i b i t o r used during the present study was cycloheximide. It i n h i b i t s the synthesis of DNA, RNA, and p r o t e i n . It acts only on the p r o t e i n s y n t h e s i z i n g machinery of eu k a r y o t i c c e l l s , and has no e f f e c t on p r o k a r y o t i c c e l l s or mitochondrial p r o t e i n s y n t h e s i s . Cycl ohexi mi de |imhf biits phote bnaehai.nrel ongatiionsby 1 p revent i ngP the"movement of ribosomes along the m-RNA. Part of t h i s e f f e c t i s due to i t s i n h i b i t i o n of t r a n s f e r f a c t o r II which catal y z e s the h y d r o l y s i s of GTP, a step necessary f o r the t r a n s l o c a t i o n of the p e p t i d y l t-RNA from the A s i t e to the R s i t e in the 80S ribosomal complex (Gale et_ aj_. 1972 p. 257). Cycloheximide a l s o has an immediate e f f e c t on DNA sy n t h e s i s . In a d d i t i o n , i t i s thought tbaprevent ribosomal RNA synthesis since cycloheximide i n h i b i t s RNA polymerase I in molds and rat l i v e r (Timberlake et a 1. 1972) Cycloheximide t o t a l l y abolished any sign of d i f f e r e n t i a t i o n , but did not completely prevent germination. D i f f erent i at i.on was prevented when cycloheximide was present during the i n i t i a l 2 hours before the heat- shock sti m u l u s . This strengthens the idea that events c r u c i a l f o r d i f f e r e n t i a t i o n 158 do occur dur ing the e a r l y germinat ion pe r i od . In summary, these r e s u l t s i n d i c a t e that the synthes i s of e s s e n t i a l macromolecu 1es fo l l ows a s p e c i f i c p a t t e r n . The t iming of t h i s e s s e n t i a l s yn thes i s v a r i e s w i th the type of macromolecule? If the t iming of the synthes i s is i n t e r f e r e d w i t h , normal development does not resume. In a d d i t i o n , the s y n t h e t i c requirements of the germ tube and i n f e c t i o n hypha appear to be very d i f f e r e n t . The germ tube does not requ i re e i t h e r DNA, RNA, or p r o t e i n s y n t h e s i s , whereas the i n f e c t i o n hypha requ i res a l l th ree. However, germinat ion and d i f f e r e n t i a t i o n cannot be separated in terms of development, s i nce i t appears that the synthes i s of RNA e s s e n t i a l f o r d i f f e r e n t i a t i o n commences dur ing germinat ion even before the heat shock s t imulus i s a p p l i e d . This i s fo l lowed by DNA and then p r o t e i n s yn the s i s . D e f i n i t e conc lus ions regarding each stage using i n h i b i t o r s alone should be avo ided. The problem of e s s e n t i a l synthes i s versus ac tua l s ynthes i s remains l a r g e l y unreso lved. A complete time study of the k i n e t i c s , using l a b e l l e d nuc leos ides and amino a c i d s , w i th and wi thout added i n h i b i t o r s , is needed f o r c l a r i f i c a t i o n . THE ROLE OF DIFFERENTIATION IN INFECTION: The f a c t that i n h i b i t o r s of DNA and RNA synthes i s i n h i b i t rust development on the host , as we l l as d i f f e r e n t i a t i o n in v i t ro , is compatible w i th present r e s u l t s concerning the r o l e of i n f e c t i o n s t r u c tu re s f o r f u r t h e r growth and host i n f e c t i o n . The r e s u l t s presented in t h i s t he s i s suggest that d i f f e r e n t i a t i o n may be a process requ i red not j u s t f o r 159 penet ra t i on through the ep ide rm i s , but f o r i n f e c t i o n of the host p l a n t . Only heat shocked spo re l i ng s i n fec ted exposed host mesophyll (treatment 3: F i g . H-2) ; unshocked spo re l i ng s d id not (treatment k: F i g . H-2) . S ince heat shocked spo re l i ng s had a higher d i f f e r e n t i a t i o n percentage than unshocked ones, i t is po s s i b l e that only the d i f f e r e n t i a t e d spo re l i ng s were capable of i n f e c t i n g the exposed mesophy l l . However, the proof is not conc l u s i v e . Never the le s s , these r e s u l t s are in agreement w i th those of D ick inson (19^9). Mature undeveloped spores ( v i ab l e but ungerminated) were a l s o found to i n f e c t exposed mesophyll (treatment 2: F i g . H-2). One cannot r u l e out the p o s s i b i l i t y of some penet ra t i on through the epidermis of areas surrounding the exposed mesophy l l . However, in view of the large amount of growth obta ined on the mesophyll i t s e l f , t h i s appears u n l i k e l y . This i n t e r p r e t a t i o n is in agreement w i th the work done by Chakravar t i (1966) who, Hi (likewise-, 3obta;hnedn i n f e c t i o n iwiiith mature^dormant, spores 1 of P j_gj;am j n i s t r i t i c i when the epidermis was removed. This suggests that the epidermis i s not necessary f o r host i n f e c t i o n . These r e s u l t s are u n l i k e those of D ick inson (19^9) and Ros se t t i and Morel (1958) who f a i l e d to obta in i n f e c t i o n under s i m i l a r c i rcumstances . When dormant spores were placed on the j u n c t i o n of exposed mesophyll and i n t a c t ep ide rm i s , s p o r u l a t i o n on ly occurred in those areas w i th an i n t a c t epidermis (F i gs . !-<HT7::aridHH-8) . Th i s Vindicates that the epidermis plays an e s s e n t i a l r o l e in the s p o r u l a t i o n process. Whether s p o r u l a t i o n i s due to some chemical p r ope r t i e s of the ep ide rmi s , or whether i t would occur in response to a r t i f i c i a l membranes i s not known. These r e s u l t s 160 s u g g e s t t h e p o s s i b i l i t y o f c o n t r o l l i n g w h e a t r u s t s p o r u l a t i o n i n a x e n i c c u l t u r e b y t h e u s e o f s u c h m e m b r a n e s , e i t h e r r e a l o r a r t i f i c i a l . I t w a s a l s o f o u n d t h a t s p o r e s a l r e a d y g e r m i n a t e d o r d i f f e r e n t i a t e d f o r p e r i o d s o f 30 h o u r s , c o u l d i n f e c t p l a n t s w i t h a n i n t a c t e p i d e r m i s ( t r e a t m e n t s 5 a n d 6: F i g . H -2 ) . U n f o r t u n a t e l y , 100 % o f t h e s p o r e s h a d n o t g e r m i n a t e d w h e n t h e s p o r e l i n g s w e r e p l a c e d o n t h e e p i d e r m i s . T h e r e f o r e , t h e r e a r e t w o p o s s i b i l i t i e s . F i r s t l y , t h o s e s p o r e s t h a t h a d r e m a i n e d u n g e r m i n a t e d i n t h e o r i g i n a l c u l t u r e m e d i u m may h a v e g e r m i n a t e d w h e n p l a c e d o n t h e h o s t e p i d e r m i s . T h i s i s l i k e l y , s i n c e i n f e c t i o n t h r o u g h i n t a c t e p i d e r m i s b y t h e d e v e l o p e d ( g e r m i n a t e d o r d i f f e r e n t i a t e d ) s p o r e l i n g p r e p a r a t i o n s w a s m u c h l e s s t h a n i n f e c t i o n b y m a t u r e d o r m a n t s p o r e s . T h i s w o u l d b e e x p e c t e d i f o n l y a s m a l l n u m b e r o f o r i g i n a l l y u n g e r m i n a t e d s p o r e s w e r e t h e o n e s r e s p o n s i b l e f o r t h e i n f e c t i o n . T h e s e c o n d p o s s i b i l i t y i s t h a t g e r m i n a t e d o r d i f f e r e n t i a t e d s p o r e s , a f t e r a l a g p e r i o d i n v i t r o , p e n e t r a t e d t h r o u g h t h e e p i d e r m i s . T h i s i s l e s s l i k e l y b e c a u s e t h e t o t a l n u m b e r o f i n f e c t i o n s i t e s w a s m u c h l o w e r h e r e t h a n t h e n u m b e r f o r d e v e l o p e d s p o r e l i n g s i n f e c t i n g w i t h o u t t h e e p i d e r m i s ; ( t r e a t m e n t 3'- T a b l e H -1 ) . D I F F E R E N T I A T I O N AND A X E N I C GROWTH: S I N G L E S P O R E D E V E L O P M E N T A t t e m p t s t o p r o d u c e u r e d o s p o r e s c o l o n i e s f r o m i s o l a t e d s i n g l e u r e d o s p o r e s w e r e u n s u c c e s s f u l . H o w e v e r , p h y s i c a l l y s e p a r a t e s i n g l e s p o r e s p r o d u c e d a x e n i c c o l o n i e s w h e n p l a c e d i n a common m e d i u m ( F i g s . 1-2, l _ 3 , . a n d 1-4). T h i s g r o w t h , a t m u c h l o w e r s p o r e c o n c e n t r a t i o n s t h a n n o r m a l l y r e q u i r e d , w a s a c c o m p l i s h e d b y t h e u s e o f a t w o - s t a g e m e d i u m , MPG ( T a b l e C-3) f o l l o w e d b y A X E N I C m e d i u m ( T a b l e C -4 ) . T h e MPG m e d i u m h a d a n e f f e c t on 161 l a t e r growth i f i t was present f o r per iods as b r i e f as the f i r s t two hours of development. Other observat ions (pages Sh, 99, and 104 of Resu l t s ) i n d i c a t e that e s s e n t i a l synthes i s a l s o occurs dur ing t h i s same time pe r i od . These f a c t s suggest t h a t , although germinat ion w i l l r e a d i l y take p lace dur ing the f i r s t two hours on water a l one , n u t r i t i o n a l events at t h i s e a r l y stage may be of key importance f o r f u tu re rust growth and s u r v i v a l . The two-stage medium supported the growth of both germinated and d i f f e r e n t i a t e d s p o r e l i n g s . However, c o l on i e s from d i f f e r e n t i a t e d treatments surv ived f o r a longer per iod than those from germinated ones, although there was some v a r i a t i o n in these experiments (Table 1-1). Bas ic d i f f e r e n c e s in germinat ion and d i f f e r e n t i a t i o n behaviour were a l s o noted in the case of i s o l a t e d s i n g l e spores when compared w i th p h y s i c a l l y separate spores in a common con ta i ne r . I n fec t i on s t r u c t u r e s produced in the i s o l a t e d s i n g l e spore c ond i t i o n were reduced in number over those in a common medium, whereas germ tubes were not , i e . germinat ion percentages were the same in both cases (Tables J-1 and J-2) . Th is was thought to be due to the (lack of a d i f f e r e n t i a t i o n s t i m u l a t o r . In support of t h i s was the f a c t that i n d i v i d u a l s i n g l e spores would form co lon ie s on the two-stage medium, only i f they were in the same medium in a common c o n a t i n e r , and not i f i s o l a t e d in m ic ro te s t w e l l s . It seemed u n l i k e l y that an i n h i b i t o r was invo lved because the concent ra t ions ( lO^ul medium/spore) at which the spores were developing was much lower than that normal ly required f o r i n h i b i t o r e f f e c t of e i t h e r the germinat ion or d i f f e r e n t i a t i o n type. However, the ' c o n d i t i o n e d ' media f a i l e d to s t imu l a te s i n g l e spore development (Tables K-1 and K-2) . Kuhl et_ al_. (1971) a l s o had d i f f i c u l t y in demonstrat ing a growth s t imu lan t in l i q u i d s o l u t i o n s , yet they d id so 162 w i th agar. The reason f o r t h i s i s unknown at present . The r e s u l t s presented in t h i s t he s i s do not support , but do not e l i m i n a t e the p o s s i b i l i t y that the f unc t i on of a growth s t i m u l a t o r i s impaired in the i s o l a t e d s i n g l e spore c o n d i t i o n . Fur ther work is needed. Media, ' c o n d i t i o n e d ' by spore masses ,ccourl dube app l i ed to s i n g l e spores at e xa c t l y the same stage of development. The use of nurse c u l t u r e s i s another promis ing a l t e r n a t i v e . A l l e n ' s procedure (1957) was fo l lowed when attempting to i s o l a t e the d i f f e r e n t i a t i o n s t i m u l a t o r f o r i t s a p p l i c a t i o n to s i n g l e spores. However, u n l i k e A l l e n , i s o l a t e d s i n g l e spores were used instead^of spores 'en masse' to detect the presence of growth r e gu l a t o r s . Resu l t s of the present study (Tables L-1, L-2, and L-3) d i f f e r from A l l e n ' s in more than one re spec t : 1) No d i f f e r e n t i a t i o n s t i m u l a t o r was detected as a r e s u l t of d i s t i11 at i on 2) Crude germinat ion i n h i b i t o r d id not i n h i b i t the germinat ion of i s o l a t e d s i n g l e spores 3) Potent germinat ion i n h i b i t o r was recovered from the f i r s t 17 _20 % of the d i s t i 11 ate 4) Germination stimu 1 ator:(s) was recovered from the l a s t 45 - 50 % of the d i s t i l l a t e . The a p p l i c a t i o n of t h i s s t imu lant r e su l t ed in an increase in both germinat ion percentage and tube e l onga t i o n . These d i f f e r e n c e s could be due to the use of i s o l a t e d s i n g l e spores instead of spore masses as b i o d e t e c t o r s . The concent ra t ions used by A l l e n to detect the d i f f e r e n t i a t i o n s t i m u l a t o r were not c l e a r l y def ined (1957, 1955). Another reason f o r the d i f f e r e n c e s could be because of race. A l l e n (1957) and French et a l . (1957) both used race 56 to i s o l a t e the s t i m u l a t o r . 163 Why was a d i f f e r e n t i a t i o n s t i m u l a t o r not detected? F i r s t l y , the proper concent ra t ions of the d i s t i l l a t e might not have been used to g i ve the appropr i a te e f f e c t . If e i t h e r of the two growth r egu l a t i n g compounds that were detected (germinat ion i n h i b i t o r and s t imu l a t o r ) had been f u r t h e r d i l u t e d , they might have s t imu la ted d i f f e r e n t i a t i o n . It has been suggested that the s t i m u l a t o r induces d i f f e r e n t i a t i o n on ly in a narrow range of concent rat ions (Macko and Stap les 1973). Secondly, the d i f f e r e n t i a t i o n s t i m u l a t o r may have been present , but was masked in e f f e c t by other growth regu l a t i ng compounds. Further d i s t i l l a t i o n and separat ion may have been necessary. T h i r d l y , there may not have been a d i f f e r e n t i a t i o n s t i m u l a t o r component inxthe spores. When g la s s conta iner s were used to d i f f e r e n t i a t e i s o l a t e d s i n g l e spores , d i f f e r e n t i a t i o n percentages were h igher than usual (Table M-1). Glass has not been known to improve d i f f e r e n t i a t i o n , but the a d d i t i o n of g las s beads to germinat ion media has been reported to r e s u l t in improved germinat ion percentages (A l l en 1955). Glass apparent ly adsorbs the germinat ion i n h i b i t o r . It is not c l e a r why in t h i s case the use of g las s conta iner s proved more e f f e c t i v e than p l a s t i c , s ince a s i n g l e spore in 0.01 ml medium (the amount used in each p l a s t i c m i c ro te s t w e l l ) i s much below the concent ra t i on cons idered necessary f o r the germinat ion or d i f f e r e n t i a t i o n i n h i b i t o r to be e f f e c t i v e . Never the le s s , these r e s u l t s could be exp la ined i f a d i f f e r e n t i a t i o n s e 1 f - i n h i b i t o r was i n vo l ved . In a d d i t i o n , the r a t i o of the volume of medium/spore in the g lass conta ine r s was 8 times that used in the p l a s t i c w e l l s . This could^have-reduced an i n h i b i t o r ' s e f f e c t i v e n e s s through d i l u t i o n . If i n h i b i t o r presence had been the reason that s i n g l e spores d id not d i f f e r e n t i a t e in p l a s t i c m ic ro te s t w e l l s , instead 164 of the lack of a d i f f e r e n t i a t i o n s t i m u l a t o r , i t would e xp l a i n why ' c o n d i t i o n e d ' media f a i l e d to s t imu l a t e d i f f e r e n t i a t i o n in the p l a s t i c m i c ro te s t p l a t e s . The present r e s u l t s us ing g lass conta iner s should be repeated. SUMMARY: There has been a tendency f o r researchers to over look the importance of the d i f f e r e n t i a t i o n process as i t r e l a t e s to rust development and to cons ider that the i n f e c t i o n hypha is a mere extens ion of the germ tube (D i ck in son , 1955, was an e x cep t i on ) . This view i s changing. Resu l t s presented here show that these two s t r u c tu re s are p h y s i o l o g i c a l l y d i f f e r e n t and that the format ion of the i n f e c t i o n hypha is important f o r rust growth. Never the le s s , the germ tube a l s o plays a major r o l e in the development process . Morphogenesis occurs as a p rec i s e sequence of events . Cor rect t'imi.ngeof these e a r l y events is e s s e n t i a l . Events tak ing p lace dur ing the f i r s t two hours of germinat ion may be the most c r i t i c a l f o r rust s u r v i v a l . 165 CONCLUSIONS 1) I n f e c t i o n s t r u c t u r e s of P_. graminis t r i t i c i , race 15B-2, w i l l form in abundance in y i t r o on a Ca-K-PO, l i q u i d b u f f e r with the a d d i t i o n of 'Evans' peptone and D-glucose (MPG medium). 2) Morphogenesis of P_. graminis t r i t i c i occurs as a p r e c i s e l y timed s e r i e s of events. If these events are i n t e r r u p t e d , normal development' wi11 not occur. 3) Germ tubes and i n f e c t i o n hyphae, although morphologically s i m i l a r , are p h y s i o l o g i c a l l y d i f f e r e n t s t r u c t u r e s . k) Nuclear d i v i s i o n in the d i f f e r e n t i a t i n g spore follows a timed se r i e s , of events r e l a t e d to the morpholgical development of i n d i v i d u a l i n f e c t i o n s t r u c t u r e s . There are two nuclear d i v i s i o n s , one in the appressorium and one in the v e s i c l e , y i e l d i n g , a t o t a l of 8 n u c l e i . 5) The number of nu c l e i are reduced from 8 in the v e s i c l e to 1 or 2 in the i n f e c t i o n hypha. V e s i c l e n u c l e i e i t h e r d i s s o l v e or coalesce w i t h one another. 6) Nuclei form s p e c i f i c o r i e n t a t i o n patterns during development in the d i f f e r e n t i a t i n g spore. V- T : - . ~ 7) DNA, RNA, and p r o t e i n synthesis are a l l e s s e n t i a l f o r d i f f e r e n t i a t i o n . Germination does not require any of"these.syntheses . 8) The synthesis of macromolecu 1es e s s e n t i a l f o r various morphological s t r u c t u r e s f o l l o w s a p r e c i s e l y timed sequence. E s s e n t i a l RNA synthesis occurs throughout the development p e r i o d , but is p a r t i c u l a r l y important f o r l a t e r d i f f e r e n t i a t i o n as i t occurs during the f i r s t 2 hours of germination.' E s s e n t i a l DNA synthesis begins during heat shock (from 2 to 3 1/2 hours) and i s a l s o apparent a f t e r the heat shock process. P r o t e i n synthesis is not required u n t i l a f t e r the shock period. 9) I n f e c t i o n s t r u c t u r e s may be necessary, not only f o r p e n e t r a t i o n , but a l s o f o r the i n f e c t i o n process. Sporelings which are heat shocked to induce the formation of i n f e c t i o n s t r u c t u r e s subsequently infect, exposed host mesophyll, whereas unshocked sp o r e l i n g s do not. 166 10) The presence of s p e c i f i c n u t r i e n t s dur ing the f i r s t few hours of - s p o r e l i n g development is of key importance f o r format ion of co l on ie s in axenic c u l t u r e . Th in l y - seeded , s i n g l e uredospores wi11 not i n i t i a t e vege ta t i ve co l on ie s on a chemica l l y def ined AXENIC medium unless spores are. f i r s t placed i n t o MPG medium f o r per iods of 2 hours to 4 days (two-stage medium). 11) I n fec t i on s t r u c t u r e s may be important f o r susta ined axenic growth at low spore concen t r a t i on s . Axenic co l on i e s o r i g i n a t i n g f r o m . t h i n l y -seeded d i f f e r e n t i a t e d spo re l i ng s s u r v i ve f o r a longer time than those from th i n l y - seeded germinated spores. 12) S i ng le spores , each i s o l a t e d in i t s own w e l l of a p l a s t i c m ic ro te s t p l a t e , do not i n i t i a t e axen ic co l on ie s on the two-stage medium. 13) The percentage of i n f e c t i o n s t r u c t u r e s and germ tube length are both reduced in the i s o l a t ed s i n g l e spore c o n d i t i o n «(pl as (feme- w e l l s ) as opposed .tocsponesmciiriiamcommon medium. 14) A germinat ion i n h i b i t o r and a germinat ion s t i m u l a t o r can be i s o l a t e d from spores of P_. graminis t r i t-i c i race 15B-2, by steam d i s t i l l a t i o n of the crude spore leachate. 167 LITERATURE CITED A l l e n , Paul J.. 1972. S p e c i f i c i t y of the cis-lsomers of Inhibitors of Uredospore Germination in the Rust Fungi. Proc. Nat. Acad. 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