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Serological detection of Didymella lycopersici (Kleb.) Kimani, Esther Wairimu 1990

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SEROLOGICAL DETECTION OF DIDYMELLA LYCOPERSICI (Kleb.) By ESTHER WAIRIMU KIMANI B. S c . , University of Nairobi, 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Plant Science) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1990 © E s t h e r Wairimu Kimani, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely 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 or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date "3*->ws. DE-6 (2/88) 11 ABSTRACT P o l y c l o n a l a n t i s e r a produced aga ins t spo res , s o l u b l e p r o t e i n and the whole mycelium f r a c t i o n s of D idymel la 1vcope rs i c i reac ted w i th the homologous and he tero logous an t i gens . The most s e n s i t i v e ant iserum was tha t r a i s e d aga ins t the whole mycel ium, the so l ub l e p r o t e i n and the spore , i n dec reas ing order of s e n s i t i v i t y . Using the ant iserum r a i s e d aga ins t the whole mycelium i t was p o s s i b l e to de tec t D. 1 v c o p e r s i c i on d iseased p l an t s and i n f e s t e d seeds . Cross r e a c t i v i t y was observed between the a n t i s e r a produced to D. 1 v c o p e r s i c i and D. app lana ta . D. bryoniae and other tomato fungal pathogens i n c l u d i n g Fusar ium spp. and B. c i n e r e a . ELISA was most s e n s i t i v e and r e l i a b l e compared to double immunodi f fus ion, and l a t e x t e s t s . No r e a c t i o n s were ob ta ined us ing the l a t e x a g g l u t i n a t i o n procedure and no ant iserum de tec ted spores in double d i f f u s i o n t e s t s . P r o t e i n p r o f i l e s on SDS-PAGE revea led tha t the t o t a l number o f p r o t e i n bands decreased w i th inc reased age of c u l t u r e s of D. 1 v c o p e r s i c i incubated in l i q u i d media. Western b l o t s probed w i th the ant iserum r a i s e d aga ins t the whole mycelium showed tha t p r o t e i n bands from e x t r a c t s o f both D. 1 v c o p e r s i c i and D. app lanata were a n t i g e n i c . i i i Table of Contents Page Ti t l e Page i Abstract i i Table of Contents. i i i Lis t of Tables v i i List of Figures v i i i Acknowledgement x i i i Dedication xiv 1. Introduction and Literature Review 1 Fungal Sporulation 2 Fungal Serology 3 ELISA 3 Immunodiffusion 4 Tests on Seeds 6 Objectives 8 2. Materials and Methods 9 2.1. Cultures, Media and Incubation Conditions 9 2.1.1. The influence of Culture Medium on Sporulation of D. 1 vcopersici 9 2.1.2. The Influence of Light on Sporulation of D. 1 vcopersici 10 Effect of Light Intensity 10 iv E f f e c t o f Time of Exposure at Optimum L igh t I n t e n s i t i e s 10 E f f e c t o f L igh t Q u a l i t y 11 2 . 1 . 3 . Growth of D. 1 v c o p e r s i c i i n L i q u i d Media 12 2 . 2 . Total Protein Profi les of D. 1 vcopersici — 14 2 . 3 . Serological Studies 15 Ant iserum Produc t ion Aga ins t D. 1 v c o p e r s i c i 15 Ant iserum P u r i f i c a t i o n 16 Western B l o t t i n g o f P ro te i ns Ex t rac ted from C u l t u r e s o f D. 1 v c o p e r s i c i 16 S e n s i t i v i t y Tests on A n t i s e r a Produced Aga ins t D. 1 v c o p e r s i c i 17 Enzyme-Linked Immunosorbent Assay (ELISA) 17 A n t i s e r a T i t r a t i o n s 18 Tes ts on S e n s i t i v i t y of A n t i s e r a Aga ins t Homologous and Heterologous Ant igens 18 Immunodiffusion Tes ts 18 S p e c i f i c i t y Tests 19 E f f e c t o f Adding Sap on S e n s i t i v i t y o f A n t i s e r a 21 P r e p a r a t i o n and Tes t i ng of Ant igens from Diseased P l a n t s . 21 Tes ts on In fes ted Seeds 22 E f f e c t o f Ant igen E x t r a c t i o n Method 22 Latex A g g l u t i n a t i o n Test 22 V 3 . Results 3.1. Culturing of D. 1 vcopersici 24 3 . 1 . 1 . In f luence of Cu l tu re Medium on S p o r u l a t i o n 24 3 . 1 . 2 . In f l uence o f l i g h t on s p o r u l a t i o n 24 In f luence of L igh t I n t e n s i t y on S p o r u l a t i o n 24 In f luence of t ime o f exposure to l i g h t on s p o r u l a t i o n o f D. 1 v c o p e r s i c i 28 In f l uence o f l i g h t q u a l i t y on s p o r u l a t i o n o f D. 1 v c o p e r s i c i 28 3 . 1 . 3 . C u l t u r a l Cond i t i ons f o r D. 1 v c o p e r s i c i i n L i q u i d Media 32 3 . 2 . Protein Profi les Obtained from Extracts of D. 1vcopersici 38 3 . 3 . Serological Studies. Western B l o t t i n g 43 Enzyme-Linked Immunosorbent Assay (ELISA) 43 A n t i s e r a T i t r a t i o n s 43 T e s t i n g f o r homologous and hetero logous an t igens 47 Immunodif fusion Tests 51 S p e c i f i c i t y Tests 51 E f f e c t o f Adding P lan t Sap 55 Symptoms and Tests on Diseased P l a n t s 55 Tes ts on In fes ted Seeds 60 Tes ts on the E f f e c t o f the Method of An t i gen E x t r a c t i o n 62 Latex A g g l u t i n a t i o n Test 62 v i D i s c u s s i o n 67 L i t e r a t u r e C i t e d 75 Appendix 79 v i i List of Tables. Table 1. A l i s t o f t reatment combinat ions used in the n u t r i e n t supplement experiment 13 Table 2. Sources o f fungi used in s p e c i f i c i t y , t e s t s o f ant iserum aga ins t D. 1 v c o p e r s i c i 20 Tab le 3 . Resu l t s o f the experiment on i n f l u e n c e of l i g h t i n t e n s i t y on s p o r u l a t i o n o f D. 1 v c o p e r s i c i 26 Tab le 4 . R e s u l t s o f the experiment on e f f e c t o f t ime o f exposure to optimum l i g h t i n t e n s i t y dur ing a 4 week pe r i od on s p o r u l a t i o n o f D. 1 v c o p e r s i c i 29 Table 5. R e s u l t s o f the experiment on e f f e c t o f growth i n the dark p r i o r to exposure to l i g h t on s p o r u l a t i o n o f D. 1 v c o p e r s i c i 30 Tab le 6. R e s u l t s o f the experiment on e f f e c t o f l i g h t q u a l i t y on s p o r u l a t i o n o f D. 1 v c o p e r s i c i 33 Tab le 7. Mean m y c e l i a l f r esh weight o f D. 1 v c o p e r s i c i grown i n d i f f e r e n t l i q u i d media at 20 + 2 C 34 vi i i List of Figures. F igu re 1. Formation of p y c n i d i a by D. 1 v c o p e r s i c i on ly around i n d i v i d u a l f l a k e s of oat f l a k e s medium 25 F igu re 2. Mature p y c n i d i a o f D. 1 v c o p e r s i c i c u l t u r e p l a t e s w i th and wi thout spores ooz ing out 27 F igu re 3 . Growth o f D. 1 v c o p e r s i c i incubated under f a r r e d , and whi te l i g h t 31 F igu re 4. I n t e r a c t i o n between Fe and the method o f i ncuba t i on on the growth of D. 1 v c o p e r s i c i i n l i q u i d media 35 F igu re 5. I n t e r a c t i o n between yeast and the method o f i n c u b a t i o n on the growth of D. 1 v c o p e r s i c i 36 F igu re 6. T h r e e - f a c t o r i n t e r a c t i o n among Fe , Zn and method o f i ncuba t i on on the growth of D. 1 v c o p e r s i c i 37 F i gu re 7. Sodium dodecy lsu lpha te po lyac ry lamide gel e lec t rophore togram of e x t r a c t s from c u l t u r e s o f D. 1 v c o p e r s i c i grown on d i f f e r e n t media 39 ix F igu re 8. Sodium dodecy lsu lpha te po lyacry lamide gel e lec t rophoretogram of s o l u b l e p r o t e i n e x t r a c t e d from c u l t u r e s o f D. 1vcope rs i c i grown f o r d i f f e r e n t l eng ths o f t ime and s ta ined w i th e i t h e r Coomassie Blue or s i l v e r s t a i n s 40 F igu re 9 . Sodium dodecy lsu lpha te po lyacry lamide gel e lec t rophore togram of s o l u b l e p r o t e i n and whole mycelium f r a c t i o n s ex t rac ted from c u l t u r e s o f D. 1 v c o p e r s i c i grown f o r d i f f e r e n t lengths o f t ime 41 F igu re 10. Sodium dodecy lsu lpha te po lyacry lamide gel e lec t rophore togram of s o l u b l e p r o t e i n and whole mycelium f r a c t i o n s ex t rac ted from c u l t u r e s o f D. 1 v c o p e r s i c i 42 F igu re 11. Western b l o t o f an SDS-PAGE gel o f p r o t e i n e x t r a c t s from 1 week o l d c u l t u r e s o f D. 1 v c o p e r s i c i and D. app lanata 44 F igu re 12. T i t r a t i o n by ELISA, o f a n t i s e r a r a i s e d aga ins t the spo res , s o l u b l e p r o t e i n and whole mycelium o f D. 1 v c o p e r s i c i 46 F igu re 13. De tec t i on o f D. 1 v c o p e r s i c i spores by an i n d i r e c t ELISA, us ing a n t i s e r a r a i s e d aga ins t s o l u b l e p r o t e i n , whole mycelium and spores o f D. 1 v c o p e r s i c i 48 X F igu re 14. De tec t i on of D. 1 v c o p e r s i c i s o l u b l e p r o t e i n s by an i n d i r e c t ELISA, us ing a n t i s e r a r a i s e d aga ins t s o l u b l e p r o t e i n , whole mycelium and spores of D. 1 v c o p e r s i c i 49 F igu re 15. De tec t i on o f whole mycelium of D. 1vcope rs i c i by an i n d i r e c t ELISA us ing a n t i s e r a r a i s e d aga ins t spo res , s o l u b l e p r o t e i n and whole mycelium of D. 1vcope rs i c i 50 F igu re 16. Double immunodif fusion a n a l y s i s o f spo res , s o l u b l e p r o t e i n and whole mycelium of D. 1vcope rs i c i aga ins t a n t i s e r a r a i s e d aga ins t spo res , s o l u b l e p r o t e i n and whole mycelium of D. 1 v c o p e r s i c i 52 F igu re 17. De tec t i on o f whole myce l i a l f r a c t i o n s of o ther fung i by an i n d i r e c t ELISA, us ing ant iserum r a i s e d aga ins t the whole mycelium of D. 1 v c o p e r s i c i 53 F igu re 18. Double immunodif fusion of a n t i s e r a r a i s e d aga ins t spo res , s o l u b l e p r o t e i n and whole mycelium of D. 1vcope rs i c i aga ins t o ther fungi 54 F igu re 19. The e f f e c t o f adding p lan t sap on the d e t e c t i o n o f D. 1 v c o p e r s i c i by an i n d i r e c t ELISA 56 x i F igu re 20. Crown l e s i o n and root format ion by tomato p l a n t s i n f e c t e d wi th D. 1 v c o p e r s i c i 57 F igu re 21 . Tomato p lan t i n f e c t e d wi th D. 1 v c o p e r s i c i showing severe w i l t i n g and death 58 F igu re 22. De tec t i on o f D. 1vcope rs i c i from e x t r a c t s o f d i seased crowns, by an i n d i r e c t ELISA 59 F igu re 23. De tec t i on o f D. 1 v c o p e r s i c i by an i n d i r e c t ELISA on seeds a r t i f i c i a l l y i n f e s t e d w i th spores and m y c e l i a l suspensions 61 F igu re 24. Double immunodif fusion a n a l y s i s o f a n t i s e r a r a i s e d aga ins t D. 1 v c o p e r s i c i aga ins t e x t r a c t s from seeds a r t i f i c i a l l y i n f e s t e d w i th mycelium 63 F igu re 25. The e f f e c t o f the method o f ant igen p repa ra t i on on d e t e c t i o n o f D. 1 v c o p e r s i c i by i n d i r e c t ELISA, us ing a n t i s e r a r a i s e d aga ins t spo res , s o l u b l e p r o t e i n and whole mycelium 64 x i i ACKNOWLEDGEMENTS I wish to acknowledge the members o f my supe rv i so ry committee f o r t h e i r adv ice and c r i t i c i s m . My deepest g r a t i t u d e goes to my s u p e r v i s o r Dr . R . J . Copeman f o r h i s s u p e r v i s i o n , encouragement and h e l p , throughout t h i s s tudy . H is sense o f humour was apprec ia ted dur ing tough moments. I am indebted to the Kenyan government f o r g ran t i ng me a study leave and the Canadian I n t e r n a t i o n a l Development Agency f o r p rov id i ng the funds . My s i n c e r e thanks go to Leroy f o r t e c h n i c a l a d v i c e , Meiq in and Morven f o r t h e i r f r i e n d s h i p and h e l p . Las t but not l e a s t , my thank fu lness to God f o r H is l o v i n g c a r e . • • • XI I I DEDICATION Th is t h e s i s i s ded ica ted to my pa ren ts , Wanjiku and K iman i , and a l l my f a m i l y f o r t h e i r pa t ience tha t knows no measure, du r ing the t ime tha t I was away from home to do t h i s work. 1 INTRODUCTION AND LITERATURE REVIEW The D idymel la stem ro t o f tomatoes in g lasshouses and i n the f i e l d , has long been f a m i l i a r to growers in Europe and in New Zealand (Mar t inson and Hogenboom 1968; Channon 1972; Cheah and Soteros 1983; Watterson 1986; H o l l i d a y and Pun i tha l ingam 1970). The d i sease was r e l a t i v e l y unknown in North Amer i ca , u n t i l 1983 when an outbreak r e s u l t i n g i n economic y i e l d l o s s was repor ted on the lower F raser V a l l e y , i n B r i t i s h Columbia . The d i s e a s e occurs s p o r a d i c a l l y , caus ing economic l o s s e s , and both seedborne (Hickman 1946; P h i l l i p s 1956 (a ) ; Derbysh i re 1961) and s o i l b o r n e (Hickman 1946; P h i l l i p s 1956 (b)) i n f e c t i o n s have been r e p o r t e d . Symptoms o f D idyme l la stem ro t on tomatoes have been f u l l y desc r i bed by P h i l l i p s (1956) and these i nc l ude dark brown l e s i o n s at the base of the stem, which e v e n t u a l l y g i r d l e the p l a n t . The epidermis and the co r t ex become s o f t and r o t t e n . The v a s c u l a r t i s s u e becomes brown and t h i s browning may extend above and below the l e s i o n . New roots form above the l e s i o n , he l p i ng the p l an t to s u r v i v e f o r seve ra l weeks before i t w i l t s and d i e s . Les ions occur main ly on the stem, but they may sometimes a l so be found on leaves and f r u i t s . Symptoms caused by Didymel la 1vcope rs i c i K l e b . on i n f e c t e d tomato p l a n t s , can be confused w i th those caused by B o t r v t i s c i n e r e a ( F l e t c h e r 1973; Watterson 1986), un less the c h a r a c t e r i s t i c b lack p y c n i d i a o f D. 1 v c o p e r s i c i are present on the l e s i o n s . Th is f r u s t r a t e s any e f f o r t s to e r a d i c a t e the d i s e a s e and the need f o r f a s t and r e l i a b l e d e t e c t i o n methods f o r t h i s pathogen, a r i s e s . 2 Fungal sporulation Fungal s p o r u l a t i o n i s important to p lan t p a t h o l o g i s t s and medical myco log i s t s s i nce spores are the major v e h i c l e f o r d i s s e m i n a t i o n o f d i s e a s e s . Our l a c k o f b a s i c understanding of the process i s d i f f i c u l t to e x p l a i n (Dalbergh and Van Et ten 1982). With the d i v e r s i t y o f f u n g i , i t has been d i f f i c u l t to g e n e r a l i s e about any aspect o f t h e i r morphology, phys io logy or b i o c h e m i s t r y . C h a r a c t e r i s t i c s desc r ibed f o r one fungal spore do not always app ly to o ther spo res , hence t h e i r use in taxonomy (Dalbergh and Van Et ten 1982). The i n f l u e n c e o f l i g h t on s p o r u l a t i o n has been s tud ied f o r a number o f f u n g i . In A s p e r g i l l u s n idu lans the a c q u i s i t i o n o f s p o r u l a t i o n competence has been found to be i n t e r n a l l y c o n t r o l l e d and i s not a f f e c t e d by c u l t u r e medium. C o l o n i e s must be a c e r t a i n age before they can s p o r u l a t e (Dalbergh and Van Et ten 1982). Rao and Singhal (1978) s tud ied the i n f l u e n c e o f l i g h t on s p o r u l a t i o n o f B o t r v o d i p l o d i a theobromae P a t , . They demonstrated tha t a minimum exposure t ime was s u f f i c i e n t to induce s p o r u l a t i o n , p rov ided tha t the c u l t u r e s were i n the ' r e c e p t i v e s tage ' o f t h e i r development. The on l y repor ted work on the growth c o n d i t i o n s o f D. 1 v c o p e r s i c i was c a r r i e d out by Maider and Burge (1979). I n v e s t i g a t i n g the i n f l u e n c e o f media and l i g h t on s p o r u l a t i o n o f D. 1 v c o p e r s i c i . they found tha t spores were on ly present on Czapek-Dox agar , Coon's agar and Brown's agar , o f the media t e s t e d . The s i z e o f p y c n i d i a was found to i nc rease w i th i nc reased i l l u m i n a t i o n . They a l s o found tha t when c u l t u r e s were incubated i n the dark , the re was improved growth ra te by as much as 19.5 % on some media. 3 Fungal Serology Fungi are a heterogenous group of organisms w i th g rea t comp lex i t y and v a r i a b i l i t y (Longbottom and Austwick 1986) and t h i s c rea tes problems f o r the p r e p a r a t i o n and s t a n d a r d i s a t i o n o f c e l l u l a r or e x t r a c e l l u l a r me tabo l i t es the e s s e n t i a l t o o l s f o r a n a l y s i s of the immunological responses . Fur thermore, most s e r o l o g i c a l t e s t s r e l y on the s o l u b l e e x t r a c t e d ma te r i a l o f c e l l u l a r or e x t r a c e l l u l a r metabo l i c o r i g i n and one o f the b igges t problems i s the r e p r o d u c i b i l i t y o f such e x t r a c t s . Even w i th a l l c o n d i t i o n s o f growth, h a r v e s t i n g and p repa ra t i on being kept cons tan t , v a r i a t i o n s occur from batch to b a t c h , which are p o s s i b l y more q u a n t i t a t i v e than q u a l i t a t i v e (Longbottom and Aus tw ick 1986). However, us ing d i f f e r e n t fungal an t i gens , a n t i b o d i e s have been s u c c e s s f u l l y r a i s e d and used in d e t e c t i o n o f va r ious fung i us ing d i f f e r e n t s e r o l o g i c a l t echn iques . Due to t h e i r s e n s i t i v i t y , s e r o l o g i c a l techn iques have found wide use in the diagnoses of d i seases caused by mic roorgan isms. None o f the s e r o l o g i c a l techniques i s f u l l y s u f f i c i e n t by i t s e l f and i t i s always advantageous to use severa l methods, tha t compliment each o t h e r . Enzyme-Linked Immunosorbent Assay (ELISA) By f a r the most w ide ly used technique i s ELISA. I t i s s i m p l e , s e n s i t i v e and can e a s i l y be automated making i t very use fu l f o r r o u t i n e d i a g n o s i s o f p l an t d i s e a s e s . A remarkable c h a r a c t e r i s t i c o f ELISA i s the a b i l i t y to de tec t p l a n t pathogens occu r r i ng in much lower concen t ra t i ons than i s p o s s i b l e by c l a s s i c a l immunoprec ip i ta t ion methods. A second c h a r a c t e r i s t i c 4 i s the a b i l i t y to de tec t ant igens of very d i f f e r e n t s i z e and morphology and the p o t e n t i a l f o r q u a n t i f y i n g the r e s u l t s ob ta ined ( C l a r k 1981). However, most o f the work on the sero logy of fungi us ing ELISA, has been c a r r i e d out on c l a s s i f i c a t i o n and spec ies d i f f e r e n t i a t i o n r a t h e r than on d iagnoses . D i a g n o s t i c k i t s are however a v a i l a b l e f o r P^thium and Phytophthora spec ies (H .S . P e p i n , personal communicat ion). In ELISA t e s t s , the ant iserum q u a l i t y i s the most important v a r i a b l e and best r e s u l t s are ob ta ined us ing high t i t r e a n t i s e r a (C la r k 1981). An ant iserum r a i s e d aga ins t the so l ub l e p r o t e i n e x t r a c t was used to de tec t hyphae o f V. d a h l i a e K l e b . i n co t ton root t i s s u e w i th a mod i f i ed i n d i r e c t ELISA (Ger ik et a l . 1987). Immunodif fusion The double gel d i f f u s i o n , developed by Ouchter lony (1968), i s based on the p r i n c i p l e t ha t both reac tan ts (ant igen and ant ibody) move through an i n e r t media and form a p r e c i p i t i n l i n e . The p r e c i p i t i n l i n e i s the s t a b l e an t i gen -an t i body complex formed at the zone of equ iva lence (Cawley et a l . 1978). The t e s t depends on the s p e c i f i c p r e c i p i t a t i o n o f the complexes at the proper r a t i o s and on the s o l u b i l i t y o f the r e a c t a n t s . The s e n s i t i v i t y of the t e s t depends on the c o n c e n t r a t i o n , t h i c kness and v i s c o s i t y o f the gel employed (W i l l i ams and Chase 1971). However, a p r e r e q u i s i t e f o r e v a l u a t i o n o f the t e s t i s tha t the reac tan ts be at an opt imal r a t i o , and sometimes an imbalance i s revea led as a d u p l i c a t i o n or m u l t i p l i c a t i o n o f p r e c i p i t i n l i n e s . Hence in m u l t i p l e p r e c i p i t i n l i n e s , i t i s d e s i r a b l e to use seve ra l d i l u t i o n s o f r e a c t a n t s to e s t a b l i s h the i n d i v i d u a l i t y o f each an t i gen -an t i body system 5 present (Axe lsen 1983). I t should a l so be noted tha t when the an t igen i s too concen t r a ted , minute amounts o f ant ibody are not d e t e c t e d . Sometimes r e f i l l i n g the w e l l s to prov ide a cont inuous d i f f u s i o n o f e i t h e r r e a c t a n t , permi ts d e t e c t i o n o f low c o n c e n t r a t i o n s , though t h i s procedure can produce u n d e s i r a b l e a r t i f a c t s . Dense broad l i n e s may obscure weaker l i n e s and two or more l i n e s can p o s s i b l y migrate at the same ra te and f a i l to r e s o l v e , thereby p r o v i d i n g i n c o r r e c t i n t e r p r e t a t i o n of r e s u l t s (W i l l i ams and Chase 1971). Us ing double d i f f u s i o n , an ant iserum produced to an e x t r a c e l l u l a r p r o t e i n - 1 i p o p o l y s a c c h a r i d e (PLP) ant igen de tec ted the an t igen in homogenates o f t u b e r s , stems and leaves of potato p lan t s i n f e c t e d w i th V e r t i c i l ! i u m  d a h l i a e but not i n e x t r a c t s o f hea l thy potato t i s s u e (Nachmias et al_. 1982). The PLP was prepared from c u l t u r e f i l t r a t e s of 21 d a y - o l d c u l t u r e s o f V. dahl i a e . The ant iserum to PLP was found to be more r e l i a b l e than tha t prepared aga ins t m y c e l i a l an t i gens . Ant iserum r a i s e d aga ins t mycelium was found to c ross reac t w i th o ther V e r t i c i l l i u m . Fusarium and C o l l e t o t r i c h u m spec ies (Nachmias et a l . 1982). Cross r e a c t i v i t y i s not uncommon in fungal s e r o l o g y . A n t i b o d i e s r a i s e d aga ins t r ibosomes of Fusarium moni l i fo rme were found to reac t s t r o n g l y w i th the homologous ant igens and weakly w i th corn r ibosomes in immunodi f fus ion t e s t s (Marsha l l and Pa t r i dge 1981). F i t z e l l e t a l - (1980) observed c ross r e a c t i v i t y i n the ant iserum r a i s e d aga ins t whole c e l l s o f V e r t i c i l l i u m  d a h l i a e w i th o ther V e r t i c i l l i u m spec ies us ing immunodi f fus ion t e s t s . They were on ly ab le to d i s t i n g u i s h V. d a h l i a e and V. a lbo-a t rum us ing c ross -abso rbed conjugate i n immunofluorescence t e s t s . A n t i s e r a r a i s e d aga ins t the s o l u b l e and the c e l l wa l l f r a c t i o n s reac ted e q u a l l y we l l w i th c o n i d i a , c e l l w a l l s and hyphal t i p s . 6 Tests on Seeds Most seed t e s t i n g l a b o r a t o r i e s do not r o u t i n e l y t e s t seeds f o r fungal d i s e a s e s . One reason may be tha t procedures f o r enumerat ing fungal pathogens on seeds are l a b o u r - i n t e n s i v e and t ime consuming. In the most w ide l y used p rocedure , the agar p l a t e b ioassay , seeds are incubated f o r 7-10 days on agar p l a t e s a f t e r which the fungi are d i s t i n g u i s h e d from one another on the bas i s o f morpho log ica l t r a i t s (Gleason et a l . 1987). Seed c e r t i f i c a t i o n s tandards f o r seedborne pathogens are l a c k i n g because the data about the r o l e o f seedborne inoculum on the d i sease development and subsequent y i e l d l o s s are o f ten l a c k i n g . In most cases the seedborne nature o f pathogens i s known, but the t r a n s m i s s i o n ra te and spread i n the f i e l d i s not known. Y e t , the l e v e l o f seed i n f e c t i o n governs the t r ansm iss ion of p l an t pathogens through seeds (Agarwal and S i n c l a i r 1987). The methods of t e s t i n g seeds , e s p e c i a l l y f o r c e r t i f i c a t i o n purposes, should be r e l i a b l e , f a s t and r e p r o d u c i b l e . The I n t e r n a t i o n a l Seed Tes t i ng A s s o c i a t i o n (ISTA) i s c u r r e n t l y work ing on deve lop ing and s t a n d a r d i s i n g s imple methods f o r seed hea l t h t e s t i n g , f o r i n t e r n a t i o n a l a p p l i c a t i o n (Agarwal and S i n c l a i r 1987). ELISA has been used to de tec t seedborne fung i i n t a l l fescue (Johnson et a l . 1982) and soybean (Shelby 1985; Gleason et al_. 1987) seeds . In soybean, an ant iserum r a i s e d aga ins t l y o p h y l i s e d mycelium o f Phomopsis l o n g i c o l l a . was used to de tec t i n f e c t i o n s on symptomless i n f e c t e d seeds us ing an i n d i r e c t ELISA and a mod i f i ed immunoblot assay . E x t r a c t s o f seeds w i thout the seed coa ts were found to produce a n o n s p e c i f i c background r e a c t i o n tha t obscured the s p e c i f i c r e a c t i o n . S ince ELISA does not d i s c r i m i n a t e between l i v e and 7 dead fungus, the seed immunoblot assay was found to be a b e t t e r i n d i c a t o r o f pathogen a c t i v i t y on seeds a f t e r p l a n t i n g (Gleason et a l - 1987). However, the assay does not account f o r r e s t i n g spores or s c l e r o t i a t ha t may not germinate w i t h i n the assay p e r i o d . Such propagules may be missed as c o n t r i b u t o r s o f i n f e c t i o n s in the f i e l d un less o ther t e s t s l i k e ELISA are used to de tec t them on seeds. Reports on whether the Didymel la stem ro t fungus can be c a r r i e d on seeds have been c o n f l i c t i n g (Derbysh i re 1961). However i t i s c u r r e n t l y he ld tha t seeds may be p o s s i b l e sources of i n f e c t i o n ( P h i l l i p s 1956 ( a ) ; F l e t c h e r 1973; Watterson 1986; Agarwal and S i n c l a i r 1987). P h i l l i p s (1956 ( a ) ) , repor ted tha t a l though few seeds may be i n f e c t e d , these may not g i ve r i s e to d i seased s e e d l i n g s but may cause secondary spread l a t e r i n the season. He a l s o concluded tha t the fungus i s c a r r i e d on the seed but the percentage of i n f e s t e d seeds i s smal l i n most c a s e s . Derbysh i re (1961) , worked w i th seeds from i n f e c t e d f r u i t and succeeded in ob ta in i ng the d i sease i n the greenhouse. She however d i d not determine the l e v e l s of contaminat ion o f the seeds used. Probab ly because of the sporad ic occurrence and l i m i t e d geograph ica l d i s t r i b u t i o n , r e l a t i v e l y l i t t l e in fo rmat ion on the b i o l ogy of D. 1 v c o p e r s i c i has been documented. The d i sease may be c a r r i e d on the seed and t h i s can r e s u l t i n spread of the d i sease to areas tha t are p r e s e n t l y f r e e of the d i s e a s e . The symptoms caused on i n f e c t e d p lan t s may be confused w i th those caused by o ther fungi and there are no known r e s i s t a n t tomato v a r i e t i e s . In view of t h i s i n f o r m a t i o n , the present study was undertaken to eva lua te the use o f se ro logy as a d i a g n o s t i c too l f o r D. 1 v c o p e r s i c i . 8 The objectives of the study were: 1. To study the p r o t e i n p r o f i l e s o f c u l t u r e e x t r a c t s o f D. 1 v c o p e r s i c i grown f o r d i f f e r e n t leng ths o f t ime and in d i f f e r e n t media. 2. To produce an ant iserum aga ins t the spo res , s o l u b l e p r o t e i n and the whole mycelium f r a c t i o n o f D. 1 v c o p e r s i c i . 3. To eva lua te the s e n s i t i v i t y o f the a n t i s e r a r e l a t i v e to homologous and hetero logous a n t i g e n s , d i seased p l a n t s and i n f e s t e d seeds. 4. To eva lua te the s p e c i f i c i t y o f the a n t i s e r a r e l a t i v e to o ther tomato fungal pathogens. 9 MATERIALS AND METHODS 1. Cultures, Media and Incubation Conditions The c u l t u r e o f D idvmel la 1 v c o p e r s i c i K l e b . used i n t h i s study was ob ta ined as No. 24638 from the American Type C u l t u r e C o l l e c t i o n , Mary land , USA and was mainta ined on Czapek-Dox agar ( D i f c o ) . 1.1. The influence of Culture Medium on Sporulation of !L lycopersici C u l t u r e s o f D. l y c o p e r s i c i were grown on each o f th ree agar media: Coon's m o d i f i e d , oat f l a k e s , or Czapek-Dox medium. Coon's mod i f i ed medium con ta ined 7.0 g ma l tose , 2.0 g KNO. 2.7 g KH PO , 1.2 g MgSO and 20.0 g agar 3 2 4 4 i n 1 1 o f water . The medium was autoc laved f o r 20 min at 110.9 kPa (15 p s i ) (Kn ight 1960). Oat f l a k e s medium was prepared as f o l l o w s . Oat f l a k e s (15-20) were p laced in P e t r i d i shes and a drop of water added before a u t o c l a v i n g f o r 15 min at 110.9 kPa (15 p s i ) . Water agar was prepared s e p a r a t e l y by d i s s o l v i n g 15 g o f agar i n 1 1 water . A f t e r a u t o c l a v i n g , the agar when coo led to 10 C, was poured i n t o the p l a t e s c o n t a i n i n g the f l a k e s and a l lowed to cool ( R . J . Bandoni , personal communicat ion) . Czapek-Dox medium con ta ined 2.0 g NaNO,, 1.0 g KH P O . 0.5 g MgSO , . 7 H O , 0 .5 g KC1, 0.01 3 2 4 4 2 g FeS0 4 . 7H 2 0 , 15.0 g Sucrose and 15.0 g agar i n 1 1 o f water (Tu i t e 1969). Four p l a t e s o f each medium, seeded w i th two 3 mm d i s k s cut from a 10-day-o ld co lony o f D. 1 v c o p e r i c i , were p laced 30 cm from the l i g h t source and 10 examined weekly f o r p y c n i d i a fo rmat ion . L igh t was supp l i ed by coo l whi te f l u o r e s c e n t tubes ( P h i l l i p s 20 W). The experiment was c a r r i e d out t w i c e . 1.2. The Influence of Light on Sporulation of D. lycopersici Effect of Light Intensity C u l t u r e s o f D. l y c o p e r s i c i were grown on Czapek-Dox agar as desc r i bed above. The c u l t u r e p l a t e s were incubated f o r 3 weeks in a growth chamber at l i g h t i n t e n s i t i e s as f o l l o w s ; 28; 39; 43; 44; 47; 81 ; 84; 105; u E / m 2 / s . The l i g h t was s u p p l i e d by two cool whi te f l u o r e s c e n t tubes ( P h i l l i p s 20 W) and the p l a t e s were p laced 10 cm below the l i g h t sou rce . The l i g h t i n t e n s i t y was measured at d i f f e r e n t po in t s a long the leng th o f the f l u o r e s c e n t tubes us ing a photometer (L i Cor , Inc . Model L1-185B) . Temperature i n the growth chamber was main ta ined at 20 +2 C. There were two c u l t u r e p l a t e s f o r each l i g h t i n t e n s i t y . The experiment was repeated th ree t imes . At the end of the exper iment spore concen t ra t i ons were determined by suspending spores in 1 ml s t e r i l e water and count ing w i th a haemocytometer at the app rop r i a t e d i l u t i o n s . Effect of Time of Exposure at Optimum Light Intensities The l i g h t i n t e n s i t i e s tha t gave the h ighes t spore count from the above exper iment were used to i n v e s t i g a t e the e f f e c t o f exposure t ime on s p o r u l a t i o n . D u p l i c a t e c u l t u r e p l a t e s (on Czapek-Dox medium) f o r each set were incubated as f o l l o w s : one set was exposed to l i g h t f o r 1 week and then 11 p laced in the dark f o r 3 weeks; another set was exposed f o r 2 weeks and then p laced i n the dark f o r 2 weeks; another set o f p l a t e s was grown i n the dark f o r 2 weeks and then exposed to l i g h t f o r 2 weeks and another se t o f p l a t e s was grown in the dark f o r 3 weeks before exposing f o r 1 week. Two more se ts o f c u l t u r e p l a t e s were e i t h e r exposed to l i g h t or grown i n the dark f o r 4 weeks con t i nuous l y a f t e r which the spore count was determined as above. The experiment was c a r r i e d out three t imes . To e s t a b l i s h the c r i t i c a l per iod of exposure t ime , one se t o f p l a t e s was grown in the dark f o r one week and then exposed to l i g h t f o r 0 . 5 , 1, 2 , 3 , 4, 5, 6, 7, 8 , 9 or 10 days then incubated in darkness . Another se t o f p l a t e s was grown in the l i g h t f o r 0 . 5 , 1, 2 , 3 , 4 , 5, 6 , 7, 8 , 9 and 10 days and then put i n da rkness . A l l p l a t e s were examined a f t e r 4 weeks from the date o f seed ing . The experiment was c a r r i e d out t w i c e . Effect of Light Quality Three c u l t u r e p l a t e s each were incubated f o r 7 days under r e d , f a r red and wh i te l i g h t sources and then p laced in darkness f o r 3 weeks. The spores were counted , as above, at the end of the exper iment . The red l i g h t was ob ta ined by f i l t e r i n g l i g h t from f i v e c o o l - w h i t e f l u o r e s c e n t tubes (40 W, General E l e c t r i c , F 40 CW) through a 3 mm t h i c k red f i l t e r (Rohm and Hass P l e x i g l a s s No. 2423) . Far red l i g h t was obta ined by f i l t e r i n g l i g h t from e i g h t incandescent bulbs (100 W, Westinghouse) through s i n g l e l a y e r s o f red P l e x i g l a s and Rosculux No. 95 medium b lue-green p l a s t i c f i l t e r s (Nolan 1989). The exper iment was c a r r i e d out t w i c e . 12 1 .3. Growth of D. lycopersici in Liquid Media M y c e l i a l d i s c s (10) cut from 10-day-o ld c u l t u r e s o f D. l y c o p e r s i c i were used to i n o c u l a t e 250 ml Erlenmeyer f l a s k s each c o n t a i n i n g 50 ml s t e r i l e c u l t u r e b r o t h . The media t es ted inc luded Czapek-Dox and Coon 's b r o t h , both o f which d i f f e r e d from t h e i r r e s p e c t i v e agar media in t ha t the bro th con ta ined no agar . Each medium was supplemented w i th m ine ra l s t ha t comprised the microelement s o l u t i o n o f L i l l y and Ba rne t t , 1951 (Tu i t e 1969) . For each medium, c u l t u r e s were incubated w i th or wi thout yeas t e x t r a c t o r supplemented w i th t r a c e e lements . To each f l a s k e i t h e r 0.2 mg ZnS0 4 -7H 2 0, 0.1 mg MnSO . 7 H 0 , 0.2 mg [ FeNH, (SO,) . 12H 0 1 per l i t r e o f water ( f i n a l 4 2 4 4 2 2 c o n c e n t r a t i o n ) or a mix ture o f the th ree minera l sources was added (Czapek-Dox medium conta ined Fe i n the b a s i c r e c i p e ) . Con t ro l f l a s k s con ta ined the b a s i c medium wi th no supplements. Treatment combinat ions are l i s t e d on Table 1. A l l c u l t u r e s were incubated as e i t h e r s t i l l o r shake c u l t u r e s and main ta ined at 20 ±2 C. At the end o f the experiment the f l a s k contents were c o l l e c t e d on a Whatman No. 1. f i l t e r paper on a Buchner funnel under vacuum. A f t e r the l i q u i d media was d ra ined o f f , the f r e s h weights were t a k e n . The experiment was a f a c t o r i a l des igned in a s p l i t p l o t w i th t h ree r e p l i c a t i o n s over t ime. Sub p l o t s were the method o f i ncuba t i on w i th t reatments arranged in a comple te ly randomized d e s i g n . An a n a l y s i s o f v a r i a n c e (ANOVA) was c a r r i e d out on the y i e l d s . The 26 degrees o f freedom were p a r t i t i o n e d i n t o main e f f e c t s and i n t e r a c t i o n s . Tes ts f o r p a r a l l e l i s m were c a r r i e d out on the i n t e r a c t i o n s us ing the F - t e s t . 13 Table 1. The t reatment combinat ions used to determine the e f f e c t o f supplements on the growth of D. l y c o p e r s i c i i n l i q u i d c u l t u r e media Medium Treatment 1. Coon's Cont ro l 2 . Coon 's Z inc (Zn) 3 . Coon's Manganese (Mn) 4 . Coon 's I ron (Fe) 5 . Coon's Zn + Mn 6. Coon 's Zn + Fe 7. Coon's Mn + Fe 8. Coon 's Zn + Mn + Fe 9. Coon's Yeast E x t r a c t 10. Czapek-Dox Cont ro l 11. Czapek-Dox Zn 12. Czapek-Dox Mn 13. Czapek-Dox Zn + Mn 14. Czapek-Dox Yeast E x t r a c t 14 2. Total Protein Profiles of D. lycopersici C u l t u r e s o f D. l y c o p e r s i c i were grown in l i q u i d shake c u l t u r e s f o r 1, 2, 3 , and 4 weeks in both Coon's and Czapek-Dox b r o t h . Each f l a s k c o n t a i n i n g 50 ml o f c u l t u r e medium, was i nocu la ted w i th 3 X 10 7 spores and main ta ined at 22 + 2 C. At the end of each incuba t ion per iod the conten ts o f the f l a s k s were c o l l e c t e d on a Whatman No. 1. f i l t e r paper on a Buchner funnel under vacuum and washed w i th phosphate buf fered s a l i n e (PBS) . PBS con ta ined 8.0 g N a C l , 0.2 g KH PO, 2.9 g N a H P 0 . 1 2 H 0 and 0.2 g KC1 i n 1 1 o f wa te r . The 3 2 4 3 2 4 2 3 samples o f harvested myce l i a l mats were ex t rac ted by g r i n d i n g w i th a mortar and p e s t l e i n a minimum amount o f PBS. A p r o t i n i n (Sigma) was used as a pro tease i n h i b i t o r (10 5 un i t s i n 50 ml PBS) . Each sample was d i v i d e d i n t o two, the f i r s t subsamples were kept and des ignated as whole mycel ium f r a c t i o n s , the second subsamples were c e n t r i f u g e d at 30000 g f o r 30 min , the supernatant r e t a i n e d as the so l ub l e f r a c t i o n and the p e l l e t d i s c a r d e d . S o l u b l e f r a c t i o n samples were concent ra ted by d i a l y s i n g aga ins t 10 % po l ye thy lene g l y c o l (PEG MW 8000) at 4 C to a concen t ra t i on o f 2.0 mg/ml. P r o t e i n concen t ra t i on was determined by the Coomassie Blue B ind ing method us ing reagent (a) (Scopes 1982). P ro te i n p r o f i l e s were determined by the d i s c o n t i n u o u s sodium dodecyl su lphate po lyacry lamide gel e l e c t r o p h o r e s i s (SDS-PAGE) system of Laemmli (1970). The Bio-Rad M i n i - P r o t e i n II v e r t i c a l ge l u n i t was used in accordance wi th the opera t ing i n s t r u c t i o n s . The g e l s employed were each 0.75 mm t h i c k and cons i s t ed of a s t a c k i n g and a r e s o l v i n g gel o f 4 % and 12 % acry lamide r e s p e c t i v e l y . The B io-Rad low mo lecu la r weight p r e - s t a i n e d SDS-PAGE standards used ranged from 17000 to 130000 da . 15 Gels were run at a constant 100 V and s ta ined w i th e i t h e r Coomassie Blue or s i l v e r s t a i n . The Coomassie Blue s t a i n i n g procedure (Bio-Rad L a b o r a t o r i e s ) was c a r r i e d out by submerging the g e l s i n a s o l u t i o n o f 0.1 % Coomassie B r i l l i a n t Blue R-250 in f i x a t i v e (10 % a c e t i c ac i d and 40 % methanol) on a shaker f o r 0 .5 h. Th i s was fo l l owed by d e s t a i n i n g wi th a s o l u t i o n o f 10 % methanol and 7.5 % a c e t i c a c i d in d i s t i l l e d water . The s i l v e r s t a i n procedure o f Wray et a l . (1981) was employed and a step by step procedure i s o u t l i n e d i n Appendix 1. 3. Serological Studies Antiserum Production Against D. lycopersici P o l y c l o n a l an t i bod ies were produced aga ins t spo res , the s o l u b l e p r o t e i n f r a c t i o n and the whole myce l i a l f r a c t i o n o f D. l y c o p e r s i c i . S o l u b l e and whole mycelium ant igens were prepared in the same manner as the f r a c t i o n s f o r SDS-PAGE wh i l e the spore ant igens were prepared by suspending spores from c u l t u r e p l a t e s in d i s t i l l e d s t e r i l e water . Ant igen c o n c e n t r a t i o n s used to immunize r a b b i t s were 10 7 s p o r e s / m l , 2.0 mg/ml o f the s o l u b l e f r a c t i o n and A ^ = 2.0 f o r the whole mycelium f r a c t i o n . P r o t e i n c o n c e n t r a t i o n o f the 280 nm s o l u b l e f r a c t i o n was determined by the Coomassie Blue B ind ing techn ique us ing Reagent (a) (Scopes 1982). One ml o f each ant igen was e m u l s i f i e d w i th 1 ml F reund 's incomplete adjuvant (Bac to ) . Three, 10- to 12-week-o ld White New Zealand r a b b i t s were i n j e c t e d i n t r amuscu la r l y w i th 1 ml o f each ant igen p r e p a r a t i o n on days 1, 10, 20, 31 , 56, 90 and 111. The r a b b i t s were b led on days 30 , 67, and 89 dur ing which 3 ml o f blood was c o l l e c t e d from the 16 ears f o r t i t r a t i o n purposes. The f i n a l b leed ing was 2 weeks a f t e r the f i n a l boos ter i n j e c t i o n . The blood was p laced at 35 C f o r 1 h and then at 4 C ove rn igh t to encourage c l o t t i n g . The serum was separated from the c l o t by low speed c e n t r i f u g a t i o n (10000 g) and s to red in a l i q u o t s at -18 C. Antiserum Purif icat ion One ml of ant iserum was added to 9 ml sa tu ra ted (NH) SO, s o l u t i o n and 4 2 4 l e f t to s tand f o r 1 h at room temperature. The s o l u t i o n was c e n t r i f u g e d f o r 5 min at 5000 X g , the supernatant d i sca rded and the p r e c i p i t a t e d i s s o l v e d in 2 ml h a l f s t reng th PBS. Th is was d i a l y s e d th ree t imes (1 h each t ime) aga ins t h a l f s t reng th PBS before pass ing through a DE-22 c e l l u l o s e (Whatman) p r e - e q u i l i b r a t e d column. The e f f l u e n t was monitored at 280 nm and the f i r s t peak c o l l e c t e d and s to red at -18 C in s i l i c o n i s e d tubes (Van Regenmortel 1982). Western Blots of Proteins Extracted from Cultures of D. lycopersici The s o l u b l e and the whole mycelium f r a c t i o n s were e l ec t ropho resed on an SDS-PAGE system as above and the ge l s were used f o r western b l o t s . T rans fe r o f p r o t e i n s from the ge l s onto n i t r o c e l l u l o s e membrane was c a r r i e d out us ing the B io -Rad mini T r a n s - B l o t e l e c t r o p h o r e t i c t r a n s f e r c e l l and accord ing to the manu fac tu re r ' s i n s t r u c t i o n s . The t r a n s f e r bu f f e r con ta ined 25 mM T r i s , and 192 mM g l y c i n e pH 8 . 3 . B l o t t i n g was c a r r i e d out at 30 V, 40 mA f o r 1 h. P r o t e i n was de tec ted by a mod i f ied immunoblot assay procedure o f Ward and De Boer (1989). A f t e r t r a n s f e r , the b l o t was b locked by submerging in 2 % 17 b l o t t o (100 g Carna t ion Ins tant M i l k Powder/1 o f water) i n PBS f o r 1 h at room temperature wi th a g i t a t i o n . The b l o t was then incubated in p u r i f i e d s p e c i f i c ant iserum (3 mg/ml) d i l u t e d to 1:400 in PBS p lus 0 .5 % b l o t t o (PBS-BL) f o r 2 h at room temperature, w i th a g i t a t i o n . A f t e r wash ing, the b l o t was incubated f o r 1 h in g o a t - a n t i - r a b b i t phosphatase con jugate (Sigma) d i l u t e d 1:1000 in PBS-BL, washed and incubated in s u b s t r a t e . A s tep by step procedure i s o u t l i n e d in Appendix 2. Sensi t ivi ty Tests on Antisera Produced Against D. lycopersici The f o l l o w i n g s e r o l o g i c a l techniques were employed to eva lua te the s e n s i t i v i t y o f D. 1 v c o p e r s i c i a n t i s e r a produced, to homologous and he tero logous an t i gens , d iseased p lan ts and i n f e s t e d seeds. Enzyme-Linked Immunosorbent Assay (ELISA) The mod i f i ed i n d i r e c t ELISA was used to t i t r e the a n t i s e r a and to de tec t the an t igen i n subsequent t e s t s i n t h i s s tudy . Sample an t igens (100 u l ) were p laced in each we l l o f f l a t -bo t tomed m i c r o t i t r e p l a t e s (L inb ro ) and incubated at 37 C f o r 1 h. The p l a t e s were then washed i n tap water f o r 15 s and tapped d r y . Wel ls were b locked by p l a c i n g 200 ul o f PBS p lus 2% b l o t t o . A f t e r i ncuba t i on f o r 1 h at 37 C, contents dumped and p l a t e s tapped d r y . S p e c i f i c a n t i s e r a were d i l u t e d in 0.5 % b l o t t o p lus PBS/Tween (0 .5 ml Tween 20 i n 1 1 PBS) and 100 ul p laced in each w e l l . A f t e r i ncuba t i on f o r 1 h f o l l owed by washing, 100 ul o f g o a t - a n t i - r a b b i t phosphatase conjugate d i l u t e d 1:1000 in PBS/Tween/Blo t to was added to each we l l and incubated f o r 1 h. 18 P l a t e s were then washed and the subs t ra te added. The s u b s t r a t e c o n s i s t e d of 24 mg p -n i t ropheny l d isodium phosphate (Sigma) d i s s o l v e d in 40 ml subs t ra te b u f f e r (97 ml d ie thanolamine in 1 1 d i s t i l l e d water pH 9 . 8 ) . The p l a t e s were read at 405 nm on a T i t e r t e k Mu l t i s kan photometer 30 min and 1 h a f t e r the a d d i t i o n o f the s u b s t r a t e . Antisera Titrations and Heterologous Tests The t i t r e o f each ant iserum was determined by ELISA. Two- fo ld d i l u t i o n s o f each ant iserum were prepared, s t a r t i n g from 1:250 to 1:64000. Non-immunised serum d i l u t e d at 1:500 was used as a c o n t r o l . For both the whole mycelium and the s o l u b l e f r a c t i o n ant igens t w o - f o l d d i l u t i o n s s t a r t i n g from 1:10 to 1:1280 were prepared. The und i l u ted an t igen had an o p t i c a l d e n s i t y read ing o f 2.0 at 280 nm. Two- fo ld d i l u t i o n s s t a r t i n g from 4 X 10 7 to 5.0 X 10 5 spores/ml were prepared f o r the spore a n t i g e n s . An t igens and a n t i s e r a used in hetero logous t e s t s were the same as those used i n t i t r a t i o n s and at the same c o n c e n t r a t i o n s . Spores , s o l u b l e p r o t e i n and whole mycelium were each tes ted aga ins t the th ree a n t i s e r a . A s e r i e s o f d i l u t i o n s f o r both the ant igens and the a n t i s e r a were prepared as used i n t i t r a t i o n s . Immunodiffusion Tests The Ouchter lony double d i f f u s i o n technique (W i l l i ams and Wi lson 1984) was c a r r i e d out i n p l a s t i c P e t r i d i shes w i th 1.5% agar . The we l l pa t t e rn c o n s i s t e d o f a c e n t r a l we l l surrounded by s i x p e r i p h e r a l w e l l s . The ant igens 19 were e x t r a c t e d in the same way as f o r e l e c t r o p h o r e s i s o u t l i n e d above. Spores , s o l u b l e p r o t e i n and whole mycelium were t es ted aga ins t the th ree a n t i s e r a and the pre-immunised s e r a . I n fes ted seeds were prepared by d ipp ing seeds in m y c e l i a l o r spore (1 X 10 7 spo res /m l ) suspens ions . M y c e l i a l suspensions were prepared by homogenising a 10-day o l d c u l t u r e p l a te in 40 ml s t e r i l e d i s t i l l e d water us ing a mortar and p e s t l e . D i f f e r e n t l e v e l s o f seed i n f e s t a t i o n were prepared by adding 10, 100, 200 and 500 i n f e s t e d seeds to un i n f es ted seeds to make up a sample s i z e o f 1000 seeds. Each seed l o t was homogenised i n 10 ml PBS w i t h a mortar and p e s t l e . Und i l u t ed an t igen t e s t samples (10 u l ) were app l i ed i n each o f the p e r i p h e r a l w e l l s . Und i lu ted a n t i s e r a were app l i ed to the c e n t r a l w e l l s . In a l l cases the p u r i f i e d a n t i s e r a were used. S p e c i f i c i t y Tests To t e s t the s p e c i f i c i t y o f the a n t i s e r a produced, seve ra l common tomato fungal pathogens and two o ther spec ies o f D idymel la (Table 2) were t e s t e d us ing the ELISA and Ouchter lony double d i f f u s i o n t e s t s as o u t l i n e d above. Each o f these fung i was grown in l i q u i d c u l t u r e s and an t igens were e x t r a c t e d i n the same way as p r e v i o u s l y desc r ibed f o r D. l y c o p e r s i c i . From fungal homogenates A „ o n = 2 . 0 , d i l u t i o n s o f 1:10, 1:100, 1:1000 and 1:10000 were 280 mn prepared and t es ted aga ins t a 1:1000 d i l u t i o n o f the whole mycelium ant iserum us ing ELISA. For double d i f f u s i o n t e s t s und i l u t ed samples o f the an t igens were t e s t e d aga ins t each of the und i l u ted a n t i s e r a . Samples o f 10 u l were a p p l i e d to each w e l l . 20 Table 2 . Sources of fungi used in s p e c i f i c i t y t e s t s o f a n t i s e r a f o r D. 1 v c o p e r s i c i Pathogen Host Source D. l y c o p e r s i c i D. app lanata D. bryon iae  B o t r v t i s c i n e r e a  Fusarium oxvsporum f . s p . r a d i c i s - l v c o p e r s i c i (FORL) F. oxvsporum f . s p . l y c o p e r s i c i (FOL) Tomato ATCC 24638 Raspberry H.S. P e p i n , Ag . Canada, Vancouver B .C . Cucumber E.W. Kimani Tomato E.W. Kimani Tomato A. B u o n a s s i s s i , BCMAF, C l o v e r d a l e , B .C . Tomato R . J . Copeman, P lan t Sc i Dept. UBC 21 Effect of Adding Sap on Sensitivity of Antisera A m y c e l i a l homogenate (A „„„ = 2.0) was d i l u t e d 1:2 w i th e i t h e r PBS or 280 nm p lan t sap . E x t r a c t s from crown s e c t i o n s con ta i n i ng l e s i o n s on d i seased p l a n t s and hea l thy p lan t sap (see below) were t es ted on the same m i c r o t i t r e p l a t e . A s e r i e s o f t e n - f o l d d i l u t i o n s o f each ant igen s t a r t i n g from 1:10 to 1:100000, i n PBS, was prepared and tes ted aga ins t a 1:1000 d i l u t i o n o f the whole mycelium an t i se rum. Con t ro l s c o n s i s t e d o f w e l l s c o n t a i n i n g an t igens w i th no ant iserum added and w e l l s con ta in i ng ant iserum but no a n t i g e n . Preparation and Testing of Antigens from Diseased Plants Ant igens from d iseased p lan t s were prepared i n the f o l l o w i n g manner. Tomato seeds ( v a r i e t y Dombito) were grown i n pots i n the greenhouse. Four weeks a f t e r sowing the seed l i ngs were i nocu la ted w i th spore (1 X 10 7 spores /m l ) or m y c e l i a l suspensions o f D. l y c o p e r s i c i . M y c e l i a l suspens ions were prepared by homogenising a 10-day o l d c u l t u r e p l a t e i n 40 ml s t e r i l e d i s t i l l e d water us ing a mortar and p e s t l e . Each p lan t was i n o c u l a t e d w i th 1 ml o f e i t h e r suspension watered around the base of the stem. The p l a n t s were r e - i n o c u l a t e d tw ice at weekly i n t e r v a l s . S e c t i o n s 5 cm long were cut from the crowns w i th v i s i b l e l e s i o n s . One gram o f each f r e s h sample was homogenised in 0 .5 ml PBS us ing a mortar and p e s t l e , and the p lan t deb r i s removed by c e n t r i f u g a t i o n (10000 g ) . A s e r i e s o f two - f o l d d i l u t i o n s were prepared from crown an t igens s t a r t i n g from u n d i l u t e d to 1:32 and tes ted aga ins t two - f o l d d i l u t i o n s o f ant iserum 22 from 1:500 to 1:128000. Heal thy p lan ts were homogenised in the same manner and used as c o n t r o l s . Tests on Infested Seeds Seeds at d i f f e r e n t l e v e l s o f i n f e s t a t i o n were e x t r a c t e d as above and t e s t e d w i th the th ree a n t i s e r a at t h e i r r e s p e c t i v e optimum c o n c e n t r a t i o n s (1:500 f o r spore ant iserum and 1:1000 f o r the two o ther a n t i s e r a ) . Effect of Antigen Extraction Method One batch o f m y c e l i a l c u l t u r e s was harvested and ground i n PBS i n a mortar and p e s t l e . Another batch o f c u l t u r e s were harves ted i n the same way and ground i n l i q u i d n i t rogen to a f i n e powder to which PBS was added. A s e r i e s o f t e n - f o l d d i l u t i o n s o f each e x t r a c t was prepared in PBS and t es ted aga ins t the th ree a n t i s e r a at d i l u t i o n s o f 1:1000 f o r both whole mycelium and s o l u b l e p r o t e i n a n t i s e r a and 1:500 f o r spore an t i se rum. Latex Agglutination Test The l a t e x a g g l u t i n a t i o n procedure o f Khan and S l a c k (1978) was employed i n t h i s s tudy . A s e r i e s o f t e n - f o l d d i l u t i o n s o f p u r i f i e d a n t i s e r a were prepared s t a r t i n g from 1:10 to 1:512000 and used to s e n s i t i z e l a t e x beads ( p o l y s t y r e n e , 0 .8 urn diam. Sigma). Ant igens were prepared i n the same way as those used i n ELISA t e s t s . Two- fo ld d i l u t i o n s from 1:10 to 1:5120 were prepared f o r both the s o l u b l e and whole mycelium f r a c t i o n s . Spores were 23 d i l u t e d from 3 X 10 7 to 10 3 spo res / m l . Using 100-lambda m i c r o p i p e t t e (Clay-Adams, 1 mm X 127 mm), 10 ul o f s e n s i t i z e d l a t e x f o l l owed by 20 ul o f an t igen was drawn by c a p i l l a r y a c t i o n . The p i p e t t e s were r o t a t ed at 7 rpm, f o r the conten ts to mix, and examined every 15 min. 24 RESULTS 1. Culturing of D. lycopersici 1.1. Influence of Culture Medium on Sporulation C u l t u r e s grown on a l l media in t h i s experiment took a long t ime to produce spores and f o r t h i s reason the spore counts were not de termined. P y c n i d i a were observed a f t e r 6 weeks on both Coon's and Czapek-Dox media and 5 weeks on oat f l a k e s medium. The oat f l a k e s medium induced p y c n i d i a fo rmat ion 1 week e a r l i e r than the o ther media. The p y c n i d i a were on ly l i m i t e d to the areas around the f l a k e s ( F i g . 1) and would t h e r e f o r e r e q u i r e a maximum number o f f l a k e s in order to ob ta in a maximum number o f spores from one p l a t e . P y c n i d i a format ion was uni form on the o ther p l a t e s (not shown). 1.2. Influence of l ight on sporulation The Influence of Light Intensity on Sporulation A r e d u c t i o n in both the number o f spores and the s i z e o f p y c n i d i a was observed w i th h igh and very low l i g h t i n t e n s i t i e s (Table 3 ) . Ex tens i ve condensat ion observed on the c u l t u r e p l a t e s d i d not seem to a f f e c t growth as would have been expec ted . No c o r r e l a t i o n between condensat ion and l i g h t i n t e n s i t y was observed s i nce l e s s condensat ion was observed at both medium and h igh l i g h t i n t e n s i t i e s . P y c n i d i a w i th ooz ing spores were observed at low l i g h t i n t e n s i t i e s but not at high l i g h t i n t e n s i t i e s ( F i g . 2 ) . Desp i te the 25 Figure 1. Formation of pycnidia by Didymella lycopersici only around individual flakes of oat flakes medium. 26 Table 3 . In f luence o f l i g h t i n t e n s i t y on s p o r u l a t i o n o f D. 1 v c o p e r s i c i S t a t i o n L i gh t I n t e n s i t y (uE/mVs) Avg. Spores per ml (X 10 7 ) Avg. P y c n i d i a leng th Shape (u) Notes A 28 1.3 126 S p h e r i c a l Aggregated p y c n i d i a No condensat ion Oozing on p l a t e edges B 39 4.6 156 S p h e r i c a l No condensat ion Ex tens i ve ooz ing C 43 6.1 198 S p h e r i c a l No condensat ion Ex tens i ve ooz ing 0 47 5.2 192 Elongate Ex tens i ve condensat ion Few ooz ing p y c n i d i a E 84 1.9 192 Elongate L i t t l e condensat ion No ooz ing pycn id i F 105 1.3 120 Elongate L im i ted condensat ion L im i ted ooz ing 27 Figure 2. Mature pycnidia of D. lycopersici culture plates with ( A ) and without ( B ) spores oozing out. Plates were incubated at different light intensities at 20 * 2 C, 10 cm from the light source. 28 l a c k o f v i s i b l e p y c n i d i a w i th ooz ing spores , high spore counts were recorded at a l i g h t i n t e n s i t y o f 47 uE/m 2 /s (Table 3 ) . Th is suggests tha t spores may have been washed o f f i n the ex tens ive condensa t ion , remain ing suspended in the wate r . No attempts were made to break up the p y c n i d i a to r e l e a s e spores in a l l c u l t u r e p l a t e s . Influence of time of exposure to l ight on sporulation of D. lycopersici C u l t u r e s tha t were incubated i n cont inuous darkness d i d not produce any spores (Table 4) but there was l i t t l e improvement i n s p o r u l a t i o n w i th i nc reased exposure t ime . However, when c u l t u r e s were incubated in the dark f o r 1 week p r i o r to exposure to l i g h t , a 12 h l i g h t pe r i od was s u f f i c i e n t to induce s p o r u l a t i o n (Table 5 ) . Cu l t u res incubated d i r e c t l y to the l i g h t d i d not produce spores u n t i l a f t e r s i x days. A few p y c n i d i a were formed around the cen t re o f the p l a t e s a f t e r 4 days but these were devo id o f spo res . I t appeared tha t c u l t u r e s of D. l y c o p e r s i c i r e q u i r e a c e r t a i n ma tu r i t y pe r iod before they can respond to pho to induc t i on . The number o f p y c n i d i a on each p l a t e and the spore counts were not determined. Influence of l ight quality on sporulation of D. lycopersici Under the f a r red l i g h t , myce l i a l growth was g r e a t l y i n h i b i t e d ( F i g . 3 A) and consequent ly no spores were produced. The red l i g h t d i d not i n h i b i t growth but f a i l e d to induce spore format ion ( F i g . 3 B ) . Under the whi te 29 Table 4. E f f e c t o f t ime of exposure to optimum l i g h t i n t e n s i t y du r ing a 4 week pe r iod on s p o r u l a t i o n o f D. l y c o p e r s i c i Exposure Time No. o f Spores (X 10 6 spores /m l ) 2 wk l i g h t / 2 wk dark 2 wk da rk /2 wk l i g h t 1 wk l i g h t / 3 wk dark 3 wk dark /1 wk l i g h t 4 wk dark 4 wk 1 igh t 2.6 5.0 1.3 2.9 0 30 30 Table 5 . E f f e c t o f 7 days growth in the dark p r i o r to exposure to l i g h t on s p o r u l a t i o n o f D. l y c o p e r s i c i Exposure Time P y c n i d i a Present (+) or Absent (-) i n (Days) D i r e c t Exposure Dark P re i ncuba t i on 0 .5 - + 1 - + 2 - + 3 - + 4 + + 5 - + 6 + + 7 + + 8 + + 9 + + 10 + + 31 Figure 3. Growth of Didymella lycopersici incubated under far red light (A) - sparse growth; red light (B); white light (C); for 7 days prior to incubation in the dark for 3 weeks. 32 l i g h t the re was ex tens i ve myce l i a l growth and numerous p y c n i d i a bear ing pycn id i ospo res ( F i g . 3 C ) . The l i g h t i n t e n s i t y under the f a r red and the red i l l u m i n a t i o n was qu i t e low when compared w i th the i n t e n s i t y under wh i te l i g h t (Table 6 ) . 1.3. Cultural Conditions for D. lycopersici in Liquid Media An a n a l y s i s o f va r iance (ANOVA) (Appendix 3) was c a r r i e d out on y i e l d s (Table 7) and the 26 degrees of freedom f o r the t reatments were p a r t i t i o n e d i n t o main e f f e c t s and i n t e r a c t i o n s . The a d d i t i o n o f yeas t e x t r a c t to the b a s i c media inc reased the y i e l d of D. l y c o p e r s i c i s i g n i f i c a n t l y as d i d the a d d i t i o n o f z i n c . Among the two - fac to r i n t e r a c t i o n s on ly two were s i g n i f i c a n t at P=0.05 conf idence l i m i t s , ( i n t e s t s f o r p a r a l l e l i s m ) ; the Fe X i ncuba t i on method ( F i g . 4) and the Z inc X incuba t ion method ( F i g . 5) i n t e r a c t i o n s . In the absence of Fe, shake c u l t u r e s y i e l d e d more than the s t i l l c u l t u r e s . A d d i t i o n o f Fe reduced the y i e l d o f a l l c u l t u r e s but more so f o r those incubated as shake c u l t u r e s . The inc reased y i e l d o f s t i l l c u l t u r e s compared to shake c u l t u r e s in the presence of Z inc was s i g n i f i c a n t l y d i f f e r e n t . The t h r e e - f a c t o r i n t e r a c t i o n i n v o l v i n g Fe, Zn and i ncuba t i on method f a c t o r s was s i g n i f i c a n t at P=0.05 conf idence l i m i t s ( F i g . 6 ) . From t h i s i n t e r a c t i o n , the Zn X incuba t ion method i n t e r a c t i o n was d i f f e r e n t i n the presence or absence of Fe. In the absence of Fe, the shake c u l t u r e s i n the absence o f Zn y i e l d e d more than the s t i l l c u l t u r e s wh i l e i n the presence of Zn the reve rse was t r u e . However in the presence of Fe the d i f f e r e n c e s in y i e l d between shake and s t i l l c u l t u r e s in absence of Zn were reduced compared to the same d i f f e r e n c e s in absence of Fe. 33 Table 6. E f f e c t o f l i g h t q u a l i t y on s p o r u l a t i o n o f D . l v c o p e r s i c i L igh t Observa t ions Spectrum I n t e n s i t y (uE/m 2 /s ) Far red l i g h t 1.8 no m y c e l i a l growth Red l i g h t 6.8 ex tens i ve m y c e l i a l growth no spores White l i g h t 47.0 ex tens i ve m y c e l i a l growth 5.09 X 10 7 spores/ml 34 Table 7. Mean m y c e l i a l f r esh weight o f D. l y c o p e r s i c i grown in d i f f e r e n t l i q u i d media at 20 + 2 C Medium Supplement Mean* m y c e l i a l f r e s h weight (g) S t i l l Shake Coon 's None 0.7 1.5 Coon's Mn 0.6 1.9 Coon 's Fe 2.1 1.7 Coon's Zn 5.0 2.2 Coon's Zn + Mn 4.6 3.0 Coon's Mn + Fe 0.6 1.7 Coon 's Zn + Fe 3.4 2.6 Coon 's Zn + Mn + Fe 3.2 3.3 Coon 's Yeast 4 .6 4 .4 Czapek None 0.7 2.3 Czapek Mn 1.0 2.3 Czapek Zn 3.3 4 .4 Czapek Zn + Mn 4.2 5.8 Czapek Yeast 5.3 4 .8 * = mean of 3 r e p l i c a t e s 35 Figure 4. Interaction between Fe (0.2 mg/l) and the method of incubation on the growth of Didymella lycopersici in liquid media. 36 Figure 5. Interaction between Zinc and method of incubation on the growth of Didymella lycopersici in liquid media. 37 Figure 6. Three-factor interaction among Fe ( 0.2 mg/l ), Zinc ( 0.2 mg/i ) and method of incubation on the growth of Didymella lycopersici in liquid media. ( A) without Fe ; (B) with Fe. 38 2. Protein Profi les Obtained from Extracts of D. lycopersici The p r o t e i n pa t te rns obta ined from c u l t u r e s grown in the two d i f f e r e n t media (Coon's and Czapek-Dox) were not d i f f e r e n t ( F i g . 7 ) . A l l p r o t e i n bands were o f low mo lecu la r weight ranging from 17000 to 130000 da w i th most o f them w i t h i n the range of 17000 to 50000 da . The s i l v e r s t a i n i n i n g techn ique d i d not seem to be more s e n s i t i v e than the Coomassie B r i l l i a n t Blue ( F i g . 8 ) . Apparent d i f f e r e n c e s in the F igures 8 A and B r e f l e c t the d i f f i c u l t y i n t a k i n g p i c t u r e s o f the Coomass ie-s ta ined g e l . When the s o l u b l e f r a c t i o n s of c u l t u r e s grown f o r 1, 2 , 3 and 4 weeks were compared ( F i g . 9 A ) , a decrease in the number of s t a i ned p r o t e i n bands was observed as c u l t u r e age i nc reased . Cu l t u res grown f o r 1 and 2 weeks produced the h ighes t number of p r o t e i n bands. These bands s ta i ned more i n t e n s e l y than those from 3 and 4 week-o ld c u l t u r e s . The bands from 4 week-o ld c u l t u r e s were p a r t i c u l a r l y f a i n t and attempts to i n t e n s i f y them by c o n c e n t r a t i n g the samples were not s u c c e s s f u l . A s i m i l a r t rend was observed f o r the whole mycel ium f r a c t i o n , ( F i g . 9 B) but more p r o t e i n bands were observed in the whole mycelium than in the so l ub l e f r a c t i o n . The e x t r a bands in the whole mycel ium f r a c t i o n s were in the high molecu lar weight r e g i o n . When p r o t e i n bands from the whole mycelium f r a c t i o n and the s o l u b l e f r a c t i o n were compared s i d e by s i d e ( F i g . 10) , the whole mycelium f r a c t i o n con ta ined more bands of the range 39000 to 50000 da . Th is i s c o n s i s t e n t w i th the e x p e c t a t i o n tha t the h igher mo lecu la r weight po lypep t ides would p r e c i p i t a t e du r ing c e n t r i f u g a t i o n and the re fo re be absent from the s o l u b l e f r a c t i o n . However, the s o l u b l e f r a c t i o n conta ined some c h a r a c t e r i s t i c high mo lecu la r weight 39 Figure 7. Sodium dodecylsulphate polyacrylamide gel electrophoretogram of extracts from cultures of Didymella lycopersici grown on different media. Lane 1 Bio-Rad pre-stained SDS-PAGE low molecular weight standards. Lane 2 Soluble protein from cultures grown for 1 week in Coon's medium. Lane 3 Soluble protein from cultures grown for 1 week in Czapek-Dox medium. Lane 4 Soluble protein from cultures grown for 2 weeks in Coon's medium. Lane 5 Soluble protein from cultures grown for 2 weeks in Czapek-Dox medium. 40 1 2 3 4 75000 50000 39000 27000 17 000 Figure 8. Sodium dodecylsulphate polyacrylamide gel electrophoretogram of whole mycelium extracted from cultures of Didymella lycopersici grown for different lengths of time in Czapek-Dox liquid media and stained with (A) Coomassie Blue and (B) Silver stain. Lane 1 Bio-Rad SDS-PAGE low molecular weight pre-stained standards. Lane 2 Cultures grown for 1 week. Lane 3 Cultures grown for 2 weeks. Lane 4 Cultures grown for 3 weeks. Lane 5 Cultures grown for 4 weeks. 41 A 1 2 3 4 5 1 2 3 4 75000 50000 39000 27000 17000 Figure 9. Sodium dodecylsulphate polyacrylamide gel electrophoretograms of soluble protein ( A ) and whole mycelium ( B ) extracted from cultures of Didymella lycopersici grown for different lengths of time in Czapek-Dox liquid medium. Lane 1 Bio-Rad SDS-PAGE weight pre-stained low molecular standards Lane 2 Cultures grown for 1 week. Lane 3 Cultures grown for 2 weeks. Lane 4 Cultures grown for 3 weeks. Lane 5 Cultures grown for 4 weeks. 42 Figure 10. Sodium dodecylsulphate polyacrylamide gel electrophoretogram of soluble protein and whole mycelium extracts from cultures of Didymella lycopersici grown over different lengths of time in Czapek-Dox liquid medium. Lane 1 Bio-Rad low molecular weight pre-stained SDS-PAGE standards. Lane 2 Soluble protein from cultures grown for 1 week. Lane 3 Whole mycelium from cultures grown for 1 week. Lane 4 Soluble protein from cultures grown for 2 weeks. Lane 5 Whole mycelium from cultures grown for 2 weeks. Lane 6 Soluble protein from cultures grown for 3 weeks. Lane 7 Whole mycelium from cultures grown for 3 weeks. 43 bands in the range of 130000 da tha t were absent i n the whole mycelium f r a c t i o n . Th is suggests that c e n t r i f u g a t i o n had an e f f e c t on the s i z e o f p o l y p e p t i d e s , probably r e s u l t i n g in l i n kages or aggregat ion at low temperature (4 C) to form high molecu lar weight p o l y p e p t i d e s . 3 . Serological Studies Western Blotting A l l p r o t e i n bands from e x t r a c t s o f D. l y c o p e r s i c i were a n t i g e n i c i n western b l o t s ( F i g . 11) . Th is was not unexpected because the ant iserum used con ta ined a n t i b o d i e s which are produced aga ins t seve ra l e p i t o p e s . D. 1 v c o p e r s i c i and D. applanata share some p r o t e i n bands o f s i m i l a r mo lecu la r weight i n the low molecu la r weight reg ion as revea led by SDS-PAGE g e l s . The p r o t e i n bands from D. applanata were a l so a n t i g e n i c , and s t a i n e d e q u a l l y we l l when the b l o t s were probed w i th the ant iserum r a i s e d aga ins t the whole mycel ium e x t r a c t o f D. l y c o p e r s i c i . D. l y c o p e r s i c i and D. app lana ta have i d e n t i c a l morphologies and based on the present s tudy , appear to be q u i t e c l o s e l y r e l a t e d , at l e a s t s e r o l o g i c a l l y . Enzyme-Linked Immunosorbent Assay (ELISA) Antisera Titrations The spore ant iserum d i l u t e d 1:32000 gave a v i s i b l e r e a c t i o n (A 4 Q 5= 0.23) w h i l e f u r t h e r d i l u t i o n s gave a read ing s i m i l a r to the background va lues ( F i g . 12 A ) . The t i t r e o f the spore ant iserum was t h e r e f o r e e s t a b l i s h e d as 44 Figure 11. Western blot of an S D S - P A G E gel of protein ex t rac ts from 1 week old cul tures of Didymella lycopersici and D. applanata, probed wi th polyc lonal ant iserum ra ised against the whole mycel ia l f ract ion of D. lycopersici. Lane 1. Whole mycel ia l f ract ion of D. applanata Lane 2. Whole mycel ia l f ract ion of D. lycopersici. Lane 3 . So lub le f ract ion of D. lycopersici. Lane 4. B i o - R a d S D S - P A G E low molecular weight p re - s ta i ned s tandards. 4 5 Figure 12. Titration by ELISA, of dilution series of antisera raised against the spores (A), soluble protein (B) and whole mycelium (C) fractions of Didymella lycopersici. For both the soluble protein and the whole mycelium, the antigen concentrations were prepared from a standardised preparation of A 2 8 0 -2.0. 46 1 250 - 1 - 1 500 1 1000 -e- 1 2000 -X- 1 4000 -0- 1 8000 1 16000 1 32000 SPORE CONC. ( X 10 Spores / ml ) 625 1:100 ANTIGEN 1:1000 1:10000 CONCENTRATION 1:10000 - I - 1 500 -*- 1 1000 - B - 1 2000 -X- 1 4000 -e- 1 8000 - A - 1 16000 - s - 1 32000 1000 1:2000 1:4000 1:8000 1:16000 1:32000 1:64000 1:12800 ANTIGEN CONCENTRATION 47 1:32000. The lowest v i s i b l e reading f o r the s o l u b l e p r o t e i n ant iserum (A 4 Q 5= 0.27) corresponded to an ant iserum d i l u t i o n o f 1:16000 ( F i g . 12 B ) . Th is an t iserum t i t r e was the re fo re e s t a b l i s h e d as 1:16000. The whole mycelium ant iserum t i t r e was e s t a b l i s h e d as 1:256000 s i n c e the lowest read ing (A 4 Q 5= 0.33 corresponded to an ant iserum d i l u t i o n o f 1:256000 ( F i g . 12 C ) . Testing for homologous and heterologous antigens When the d e t e c t i o n o f spores by the th ree a n t i s e r a was e v a l u a t e d , the spore ant iserum was l e a s t s e n s i t i v e o f the three a n t i s e r a ( F i g . 13) , i n d e t e c t i n g spores at the optimum a n t i s e r a d i l u t i o n s determined from t i t r a t i o n s . The s o l u b l e f r a c t i o n ant iserum had in te rmed ia te s e n s i t i v i t y to the spo res . The whole mycelium ant iserum was most s e n s i t i v e i n d e t e c t i n g spores and showed no reduc t i on in s e n s i t i v i t y w i th i nc reased ant iserum d i l u t i o n . Th i s would suggest tha t there was an ant igen excess f o r t h i s an t iserum even though the ant igen d i l u t i o n used here was optimum f o r the spore an t i se rum. S i m i l a r r e s u l t s were obta ined when the s o l u b l e f r a c t i o n was t e s t e d aga ins t the th ree a n t i s e r a ( F i g . 14) . The spore ant iserum was l e a s t s e n s i t i v e wh i l e the whole mycelium ant iserum remained most s e n s i t i v e . There was a l s o an i n d i c a t i o n o f an t igen excess r e l a t i v e to the whole mycelium an t i se rum. The spore ant iserum was l e a s t s e n s i t i v e to the whole mycelium f r a c t i o n ( F i g . 15) compared to read ings obta ined w i th the o ther a n t i s e r a . The s o l u b l e f r a c t i o n ant iserum had a good s e n s i t i v i t y to the whole mycelium 48 1.5 -1 -0.5 -0 CONTROL 1:500 1:1000 1:2000 1:4000 1:8000 ANTISERUM CONCENTRATION Figure 13. Detection of D. lycopersici spores ( 1 X 10 7 /ml ) by an indirect ELISA, using antisera raised against spores: &3 ; soluble protein: H ; whole mycelium: EE! ; of D. lycopersici. CONTROL 1:500 1:1000 1:2000 1:4000 1:8000 ANTISERUM CONCENTRATION Figure 14. Detection of soluble protein extracted from Didymella lycopersici by an indirect ELISA using antisera raised against spores: s?g ; soluble protein: w ; whole mycelium: rr~3; of Didymella lycopersici. An antigen dilution of 1:100 was prepared from a standardised extract of A 2 8 0 "2 and used for the tests. 50 2 1.5 r 1 -0.5 CONTROL 1:500 1:1000 1:2000 1:4000 ANTISERUM CONCENTRATION 1:8000 Figure 15. Detection of whole mycelium of Didymella lycopersici by an indirect ELISA, using antisera raised against spores: ; soluble protein: W ; whole mycelium: LZ3 ; of D. lycopersici. A dilution of 1:1000 of whole mycelium was prepared from a standard A 2 8 0 -2.0 preparation and used for the tests. 51 f r a c t i o n . The whole mycelium ant iserum showed c o n s i s t e n t l y b e t t e r s e n s i t i v i t y at a l l a n t i s e r a d i l u t i o n s . Immunodiffusion Tests P r e c i p i t i n l i n e s o f i d e n t i t y were observed f o r both the whole mycelium and the s o l u b l e p r o t e i n when tes ted aga ins t t h e i r r e s p e c t i v e a n t i s e r a ( F i g . 16) . Th i s r e s u l t showed that the s o l u b l e p r o t e i n and the whole mycel ium shared common a n t i g e n i c de terminants . There were no l i n e s formed between the spores and any of the a n t i s e r a , or between any of the an t igens and the pre- immunised s e r a . L ikewise there were no l i n e s formed between the spore ant iserum and e i t h e r the so l ub l e p r o t e i n or the whole mycelium f r a c t i o n s . Specif ici ty Tests When m y c e l i a l p repara t ions o f o ther fungi were s i m i l a r l y prepared and t e s t e d us ing ELISA, c ross r e a c t i o n s were observed at an an t igen d i l u t i o n o f 1:10 ( F i g . 17) us ing the whole mycelium an t i se rum. However at lower d i l u t i o n s , on ly the Didymel la spp. were de tec tab le wh i l e the Fusarium spp. and B o t r v t i s spp. were v i r t u a l l y unde tec tab le . At very h igh d i l u t i o n s (1 :10000) , D. l y c o p e r s i c i remained de tec tab le (A 4 Q 5= 0.84) over the o ther D idvme l l a spec ies (A 4 Q 5= 0.24 and 0 . 3 4 ) . L ines o f i d e n t i t y ( F i g . 18) i n immunnodi f fus ion t e s t s demonstrated tha t D. l y c o p e r s i c i and D. app lanata c o n t a i n common a n t i g e n i c de te rminants . None of the o ther fung i reac ted w i th any o f the a n t i s e r a i n double d i f f u s i o n t e s t s . 52 Figure 16. Double diffusion patterns depicting the reaction of spores (A) ; soluble protein (B); whole mycelium (C); of D. lycopersici, with the antisera raised against spores (1); soluble protein (2) and whole mycelium (3). Wells 4, 5 and 6 contain pre-immune sera from rabbits immunised with spores, soluble protein and whole mycelium of D. lycopersici, respectively. Immunodiffusion was carried out in 1.5 % agar for 3 days at room temperature. 53 2.5 Figure 17. Detection of whole mycelial fractions of other fungi by an indirect ELISA using an antiserum raised against the whole mycelium of Didymella lycopersici (1:1000). All mycelia were prepared in the same way as for D. lycopersici and dilutions prepared from an initial A 2 8 0 «2.0. HI D. lycopersici; D. applanata; { j D. bryoniae; H Botrytis spp.; Fusarium oxysporum f. sp. radicis~lycopersici; WL9 F. oxysporum f. sp. lycopersici. Figure 18. Double diffusion patterns depicting the reactions of antisera raised against spores ( A ), soluble protein ( B ) and whole mycelium ( C ) of D. lycopersici, against B. cinerea ( 1 ), D. lycopersici ( 2 ), D. applanata ( 3 ), D. bryoniae ( 4 ), FORL ( 5 ) and FOL ( 6 ) whole mycelia of 10- day old cultures. Immunodiffusion was carried out in 1.5 % agar for 3 days at room temperature. 55 Effect of Adding Plant Sap P lan t sap proved to have the e f f e c t of reduc ing the s e n s i t i v i t y o f the ant iserum ( F i g . 19) . S e n s i t i v i t y to the ant igens from d i seased p l a n t s was much lower than to the myce l i a l suspensions ( F i g . 19) d i l u t e d i n e i t h e r p lan t sap or b u f f e r . For a l l the ant igens t e s t e d , there was a reduced s e n s i t i v i t y o f the ant iserum wi th inc reased ant igen d i l u t i o n s . With t h i s ant iserum d i l u t i o n (1 :1000) , there was s a t i s f a c t o r y d e t e c t i o n o f a l l the an t igens t e s t e d even at the very high d i l u t i o n s (1:10000) o f the a n t i g e n s . Symptoms and Tests on Diseased Plants The f i r s t symptoms of stem ro t were observed 3 weeks a f t e r i n o c u l a t i o n . C h a r a c t e r i s t i c dark brown sunken l e s i o n s were present at the base o f the stem ( F i g : 20 ) : the l e s i o n s g i r d l e d the stem as the d i sease p rog ressed , caus ing death ( F i g . 21) o f the p l a n t s . A d v e n t i t i o u s roo ts ( F i g s . 20 and 21) formed above the l e s i o n s and extended up the stem as the d i s e a s e p rog ressed . E x t r a c t s from the l e s i o n e d crowns were t es ted aga ins t the th ree a n t i s e r a us ing ELISA, w i th p o s i t i v e r e s u l t s ( F i g . 22) . However, the read ings obta ined w i th the spore ant iserum were very low. The ant iserum tha t proved to be most s e n s i t i v e and consequent ly use fu l i n d e t e c t i n g an t igens from d i s e a s e d p lan t s was the whole mycelium ant iserum. Al though there were i n c o n s i s t e n s i e s w i th d u p l i c a t e d r e s u l t s , there was a tendency towards inc reased s e n s i t i v i t y w i th i nc reased d i l u t i o n s o f the ant igens f o r both the s o l u b l e f r a c t i o n and the whole mycelium a n t i s e r a ( F i g . 22 ) . When tes ted w i th the s o l u b l e p r o t e i n an t i se rum, absorbance read ings f o r ant igen d i l u t i o n s o f 1:16 and 1:32 were 56 2.5 Figure 19. The effect of adding plant sap on detection of D. lycopersici by an indirect ELISA using an antiserum raised against the whole mycelium, 1:1000. Purified mycelium; Mycelium + sap; rj diseased tissue; ^ healthy plant sap. Figure 20. Crown lesion and adventitious root formation by tomato plants infected with D. lycopersici. 58 59 0.5 -0.4 -Healthy Undiluted 1 : 2 1 : 4 1 : 8 1 : 16 1 : 32 ANTIGEN CONCENTRATION Figure 22. Detection of D. lycopersici from extracts of of diseased crowns, by an indirect ELISA using antisera raised against spores: 1:500; soluble protein: H ,1:1000 ; whole mycelium: , 1:1000 ; antisera. 60 h igher than f o r lower ant igen d i l u t i o n s ( r e s u l t s not shown). Tes ts w i th the whole mycelium ant iserum showed h igher read ings f o r an t igen d i l u t i o n s o f 1:8 than w i th the lower d i l u t i o n s i n c l u d i n g the u n d i l u t e d . When the e x t r a c t s from d i seased crowns were t es ted in immunodif fusion t e s t s , no p r e c i p i t i n l i n e s were formed. Tests on Infested Seeds Seeds i n f e s t e d wi th spores of D. l y c o p e r s i c i were poo r l y de tec ted by ELISA r e g a r d l e s s o f the ant iserum used ( F i g . 23 A ) . Weakly p o s i t i v e r e a c t i o n s (A„ B„ =0.89, 0.36 and 0.25 f o r spore , s o l u b l e p r o t e i n and whole mycel ium a n t i s e r a , r e s p e c t i v e l y ) were obta ined w i th seed l o t s i n which 50 % of the seeds had been a r t i f i c i a l l y i n f e s t e d w i th 10 7 spores /ml o f D. l y c o p e r s i c i . The r e s u l t s were u n r e l i a b l e even at the h igh read ings obta ined w i th the spore ant iserum s ince the same ant iserum had proved to be much l e s s s e n s i t i v e i n e a r l i e r t e s t s . The f a i l u r e to de tec t an t igens from these seeds may have been as a r e s u l t of poor adhesion of the spores on the su r f ace of seeds i n a d d i t i o n to the d i l u t i o n e f f e c t o f the volume o f seed ma te r i a l as compared to the few spores tha t may have been c a r r i e d on the seed s u r f a c e . De tec t i on o f D. l y c o p e r s i c i on seeds i n f e s t e d w i th mycel ium was b e t t e r than on s p o r e - i n f e s t e d seeds ( F i g . 23 B ) . The spore ant iserum was not s e n s i t i v e to these an t i gens . Both the s o l u b l e p r o t e i n and the whole mycelium a n t i s e r a showed s a t i s f a c t o r y de tec t i on f o r seed l o t s w i th 20 % and 50 % i n f e s t a t i o n l e v e l s . In immunodif fusion t e s t s , samples from i n f e s t e d seeds f a i l e d to produce p r e c i p i t i n l i n e s except the seed l o t c o n t a i n i n g 50 % m y c e l i a l - i n f e s t e d seeds. A very weak p r e c i p i t i n l i n e was formed when t h i s 61 HEALTHY 1 % 10 % 20 % 50 % ANTIGEN CONCENTRATION (% INFESTED SEEDS) HEALTHY 1 % 10 % 20 % 50 % ANTIGEN CONCENTRATION (% INFESTED SEEDS) Figure 23. Detection of D. lycopersici by an indirect ELISA on seeds artificially infested with spores ( A ) and mycelia ( B ). Concentrations of antisera were; spore antiserum: %M , 1:500; soluble protein antiserum: H i , 1:1000; whole mycelium antiserum: E l , 1:1000. 62 seed l o t was t e s t e d aga ins t the whole mycelium ant iserum ( F i g . 24 ) . The p r e c i p i t i n l i n e f a i l e d to show up when the g e l s were photographed. Tests on the Effect of the Method of Antigen Extraction When samples ex t rac ted e i t h e r w i th or w i thout l i q u i d n i t r o g e n were tes ted w i th the th ree a n t i s e r a , inc reased s e n s i t i v i t y was observed i n a l l cases f o r the samples ground i n l i q u i d n i t r o g e n . The spore ant iserum showed marked improvement i n s e n s i t i v i t y ( F i g . 25 A) at both h igh and low an t igen c o n c e n t r a t i o n s . The o ther a n t i s e r a showed some improvements at h igh c o n c e n t r a t i o n s of ant igens but there was s u b s t a n t i a l improvement i n s e n s i t i v i t y at low ant igen concen t ra t i ons ( F i g s . 25 B and C ) . The improvement may be a t t r i b u t e d to the un i f o rm i t y o f an t igen p a r t i c l e s and consequent ly un i form b ind ing of ant igens onto the m i c r o t i t r e p l a t e s . Th is suggested tha t the poor s e n s i t i v i t y o f the spore ant iserum to d i seased p l a n t s , i n f e s t e d seeds and even the whole mycelium p r e p a r a t i o n , was due to the poor b ind ing of ant igens onto m i c r o t i t r e p l a t e s . In t ha t c a s e , the a n t i s e r a w i th the h igher t i t r e ( so l ub le p r o t e i n and whole mycelium a n t i s e r a ) would r e a c t w i th the few ant igen p a r t i c l e s bound onto p l a t e s , g i v i n g a m p l i f i e d r e s u l t s compared to the ant iserum wi th a low t i t r e . Latex Agglutination Test When seve ra l d i l u t i o n s o f both the ant igens and the s e n s i t i s e d l a t e x p a r t i c l e s were t e s t e d , no p o s i t i v e r e a c t i o n s were observed . C l e a r i n g was observed on a l l m i c r o c a p i l l a r y tubes , i n c l u d i n g the c o n t r o l s , when the tubes Figure 24. Double diffusion patterns depicting the reaction of antisera raised against spores (A), soluble protein (B), and whole mycelium (C) of D. lycopersici, with extracts from healthy ( 1 ); 1 % ( 2 ); 10 % ( 3 ); 20 % ( 4 ); 50 % ( 5 ); mycelial-infested tomato seeds. Immunodiffusion was carried out in 1.5 % agar for 3 days at room temperature. 64 Figure 25. The effect of the method of antigen preparation on detection by an indirect ELISA using the antiserum raised against the spores ( A ) , 1:500 ; soluble protein ( B ), 1:1000; and whole mycelia ( C ), 1:1000 of D. lycopersici. The antigens were prepared by grinding in a mortar and pestle, with - * - ; or without liquid nitrogen. 65 UNDILUTED 1:10 1:100 1:1000 1:10000 1:10000 ANTIGEN CONCENTRATION ANTIGEN CONCENTRATION 66 were l e f t on the sp inn ing wheel ove rn igh t . D i f f e r e n t s i z e s o f m i c r o c a p i l l a r y tubes repea ted l y gave the same negat ive r e s u l t s . 67 4. D i s c u s s i o n I t must be noted tha t experiments c a r r i e d out on c u l t u r a l c o n d i t i o n s of D. l y c o p e r s i c i were done because such in fo rmat ion i s l a c k i n g in the l i t e r a t u r e and ye t was necessary f o r the subsequent exper iments (an t igen e x t r a c t i o n and c u l t u r e propagat ion) i n the s tudy. Much of the work here i s t h e r e f o r e p r e l i m i n a r y and even where i t would have been app rop r i a t e to c a r r y the exper iments f u r t h e r , t h i s was not done. S p o r u l a t i o n I t i s commonly accepted by fungal p h y s i o l o g i s t s tha t c o n d i t i o n s favour ing r a p i d m y c e l i a l growth hamper s p o r u l a t i o n and tha t spore fo rmat ion occurs when the growth ra te i s reduced. Thus, environmental c o n d i t i o n s such as l i g h t and n u t r i e n t type and composi t ion i n f l uence fungal s p o r u l a t i o n (Dalberg and Van Et ten 1982). For D. l y c o p e r s i c i . l i g h t appeared to be more important than n u t r i e n t compos i t i on . L igh t i n t e n s i t y had an i n f l u e n c e on s p o r u l a t i o n (Table 3 ) . By reduc ing the d i s t ance from the l i g h t source from 30 cm to 10 cm, p y c n i d i a fo rmat ion occured a f t e r 10 days i ns tead of f ou r weeks. D i f f e r e n t media d i d not make any d i f f e r e n c e in the t im ing of p y c n i d i a p r o d u c t i o n . A f t e r t e s t i n g severa l media, Maider and Burge (1979) were on ly ab le to ob ta in spores from c u l t u r e s grown on Brown's agar , Coon's agar and Czapek-Dox agar . In t h e i r experiment the p l a t e s were p laced 60 cm from the l i g h t source but the l i g h t i n t e n s i t y was not repo r ted . When the same c o n d i t i o n s were employed in t h i s s tudy , us ing Oat f l a k e s , Coon's and Czapek-Dox media no p y c n i d i a were observed a f t e r 3 weeks as repor ted by Maider and Burge. P y c n i d i a were on ly 68 observed a f t e r 5 weeks and t h i s was too long f o r the nature o f subsequent exper iments in the present s tudy . For many f u n g i , l i g h t of d i f f e r e n t wavelengths i s r equ i r ed to t r i g g e r s p o r u l a t i o n (Dahlberg and Van Et ten 1982). Phvcomvces b lakes leeanus sporangiophore format ion i s a f f ec ted by blue l i g h t (Dahlberg and Van Et ten 1982). At tempts to determine the s p e c i f i c wavelength tha t t r i g g e r s s p o r u l a t i o n i n D. l y c o p e r s i c i were i n c o n c l u s i v e . C u l t u r e p l a t e s incubated under the f a r red l i g h t f a i l e d to grow or grew poor l y ( F i g . 3 ) . The c o n s t r u c t i o n o f the l i g h t boxes permi t ted a b u i l d up o f heat i n s i d e the boxes e s p e c i a l l y i n the f a r red l i g h t box. Thus, temperature may have been r e s p o n s i b l e f o r the growth i n h i b i t i o n o f D. 1 v c o p e r s i c i c o n s i d e r i n g tha t i t ' s optimum temperature requirements l i e between 19 and 20 C ( H o l l i d a y and Pun i tha l ingam 1970). I t would the re fo re be i napp rop r i a t e to a s s o c i a t e the growth i n h i b i t i o n w i th the f a r red l i g h t source w i thout f u r t h e r exper iments . Because i t was not p o s s i b l e to prov ide equal l i g h t i n t e n s i t y o f each t ype , the f a i l u r e to ob ta in spores under the red l i g h t may be a t t r i b u t e d to the low l i g h t i n t e n s i t y as observed in the l i g h t i n t e n s i t y exper iment . The dependence on l i g h t f o r spore product ion in c e r t a i n fung i has f r e q u e n t l y been demonstrated. Rao and S inghal (1978) demonstrated tha t a minimum o f l i g h t exposure t ime was s u f f i c i e n t to induce s p o r u l a t i o n in B o t r v o d i p l o d i a theobromae P a t . , prov ided tha t the c u l t u r e s were i n the ' r e c e p t i v e s t a g e ' o f t h e i r development. In A s p e r g i l l u s n i d u l a n s . the a c q u i s i t i o n o f s p o r u l a t i o n competence i s an i n t e r n a l phenomenon and i s not a f f e c t e d by medium. Co lon ies o f t h i s organism must be at l e a s t 20 h o l d be fore they are competent to s p o r u l a t e . L ikewise Tr ichoderma v i r i d e r e q u i r e s 16 h to become competent to respond to pho to induc t ion (Dahlberg and 69 Van E t ten 1982). Th is competence phenomenon may we l l be t rue o f D. 1 v c o p e r s i c i . Maider and Burge (1979) repor ted an i nc rease i n spore p roduc t ion w i th inc reased l i g h t exposure t ime. The longes t exposure t ime in t h i s case was 2 weeks. There are two p o s s i b i l i t i e s ; e i t h e r the c u l t u r e s responded to the long exposure t ime (2 wk), or c u l t u r e s had grown to the r e c e p t i v e stage dur ing the exposure p e r i o d . I f the former was t r u e , then i t would be expected tha t dark grown c u l t u r e s would not form p y c n i d i a un less exposed to l i g h t f o r what would be the c r i t i c a l exposure t ime . They repor ted tha t growth ra te improved f o r c u l t u r e s grown in the dark but the e f f e c t o f t h i s improved growth on s p o r u l a t i o n when c u l t u r e s were exposed to l i g h t was not r e p o r t e d . In the present s tudy , c u l t u r e s grown in the dark p r i o r to be ing exposed to l i g h t produced p y c n i d i a sooner than c u l t u r e s exposed d i r e c t l y to l i g h t soon a f t e r seeding (Table 5 ) . C u l t u r e s exposed d i r e c t l y to l i g h t f o r upto 4 days d i d not form p y c n i d i a in the dark , w h i l e c u l t u r e s p re incuba ted in the dark f o r 1 week formed p y c n i d i a in the da rk , a f t e r being exposed f o r 12 h (Table 5 ) . Except the c u l t u r e s exposed f o r 9 and 10 days , none of the o ther c u l t u r e s had formed p y c n i d i a by the t ime they were t r a n s f e r e d to the dark . Th is i n d i c a t e s tha t there was a ' c a r r y over e f f e c t ' , i n t ha t the l i g h t exposure e f f e c t was r e s p o n s i b l e f o r p y c n i d i a p roduc t i on in the da rk . Th i s i s the f i r s t pub l i shed repor t o f attempts to grow D. 1 v c o p e r s i c i i n l i q u i d media. Whi le c u l t u r e s grew we l l i n agar media, the re was minimal growth when the fungus was t r a n s f e r r e d to the l i q u i d form o f the same media, d e r i v e d by ommision o f agar . I t became necessary to determine what f a c t o r s were necessary f o r such growth. The a d d i t i o n o f z i n c to the b a s i c c u l t u r e l i q u i d media (Table 7) proved to be as good as the a d d i t i o n o f yeas t e x t r a c t 70 f o r the growth of D. l y c o p e r s i c i . Because a chem ica l l y de f i ned medium was r e q u i r e d , c u l t u r e s in subsequent experiments were grown i n Czapek-Dox or Coon 's l i q u i d media supplemented wi th z i n c . Yeast e x t r a c t was not cons idered to be a s u i t a b l e media supplement because of the p o s s i b i l i t y o f i t being immunogenic (Treuhaf t et a l . 1981). Spores were used to s t a r t c u l t u r e s to be used f o r an t igen e x t r a c t i o n and f o r e l e c t r o p h o r e s i s purposes in o rder to avo id the agar media ca r ryove r tha t would be exper ienced when us ing m y c e l i a l d i s c s . When spores were used to s t a r t c u l t u r e s , the c u l t u r e s were grown on a shaker , t h i s having the advantage that ae ra t i on was e q u a l l y a v a i l a b l e i n the c u l t u r e s . Fur thermore, shake c u l t u r e s have proved more r e p r o d u c i b l e in t h e i r a n t i g e n i c con ten t , f o r some fungi ( A j e l l o et a l . 1959). P r o t e i n P r o f i l e s E l e c t r o p h r e t i c a n a l y s i s o f fungal e x t r a c t s o f D. l y c o p e r s i c i conf i rmed tha t p r o t e i n content changed w i th the age of c u l t u r e s . Th i s had i m p l i c a t i o n s f o r both ant iserum product ion and pathogen d e t e c t i o n . A n t i s e r a r a i s e d aga ins t young c u l t u r e s may be i n s e n s i t i v e to o l d e r myce l i a from d i seased p l a n t s and e s p e c i a l l y f o r monoclonal an t i bod ies or p o l y c l o n a l a n t i b o d i e s r a i s e d aga ins t p r o t e i n bands e lu ted from e l e c t r o p h o r e t i c g e l s . Dur ing long growth pe r i ods c e l l u l a r a u t o l y s i s almost c e r t a i n l y o c c u r s , and i n o ther systems t h i s has prov ided the most h i g h l y a n t i g e n i c c u l t u r e f i l t r a t e e x t r a c t s (Longbottom and Austwick 1986). For c u l t u r e s grown beyond the optimum growth p e r i o d , i t would be d e s i r a b l e to use c u l t u r e f i l t r a t e s as an t igens f o r an t i serum p r o d u c t i o n , r a the r than the mycelium e x t r a c t s . Us ing c u l t u r e f i l t r a t e s as a n t i g e n s , Dewey et a l . (1984) were ab le to produce spec ies -71 s p e c i f i c a n t i s e r a to Phaeolus s c h w e i n i t z i i ( F r . ) P a t . The i n f l u e n c e of c u l t u r e age on a n t i g e n i c i t y has been s tud ied in Mvcropolvspora f a e n i . an act inomycete (Treuhaf t et a l . 1981). In t h e i r s tudy , as the c u l t u r e s aged, appearance of p r o t e o l y t i c a c t i v i t y in c u l t u r e f i l t r a t e s c o i n c i d e d w i th a decrease in p r e c i p i t a t i n g ant igen con ten t . Th is was a s s o c i a t e d w i th a decrease i n t o t a l p r o t e i n and numbers o f p r o t e i n bands de tec ted on po l yac ry lam ide gel e l e c t r o p h o r e s i s . Se ro logy A n t i s e r a r a i s e d to the spores , so l ub l e p r o t e i n and whole mycel ium f r a c t i o n s of D. l y c o p e r s i c i reac ted w i th the homologous and he tero logous an t igens i n ELISA t e s t s . The whole mycelium ant iserum was most s e n s i t i v e in both homologous and hetero logous r e a c t i o n s . Th is ant iserum de tec ted the spo res , s o l u b l e p r o t e i n and the whole mycelium in p u r i f i e d p r e p a r a t i o n s , as we l l as myce l i a from i n f e s t e d seeds and d iseased p l a n t s . Th i s f i n d i n g was h e l p f u l because i t i s much e a s i e r to prepare ant igens (whole myce l ium) , f o r use in immunisat ion schedules and secondly the ant iserum cou ld de tec t o ther forms o f the fungus. S ince d iseased p lan t s are bound to c o n t a i n spores and/or mycelium tha t would be r espons ib l e f o r secondary spread i n the f i e l d , an ant iserum tha t can de tec t any of these propagules would be u s e f u l . The double d i f f u s i o n technique was l e s s s e n s i t i v e compared to ELISA, and requ i red very h igh concen t ra t i ons of both the ant igens and the a n t i s e r a . The t e s t a l s o had the d isadvantage in tha t the leng th o f t ime taken f o r p r e c i p i t i n bands to form was 2-3 days . V i s i b l e bands formed a f t e r 24 h but were not i n tense enough e s p e c i a l l y f o r photography. I t was however use fu l i n 72 i l l u s t r a t i n g the s e r o l o g i c a l re la tedness between the s o l u b l e p r o t e i n and the whole mycel ium. Using double d i f f u s i o n , F i t z e l l e t a l . (1980) found tha t the ant iserum r a i s e d aga ins t the c e l l wa l l was more s p e c i f i c than tha t r a i s e d aga ins t the s o l u b l e and whole c e l l f r a c t i o n s . They repor ted tha t V. d a h l i a e cou ld not be d i s t i n g u i s h e d from V. t r i c o r p u s and V. a lbo-a t rum i n f l u o r e s c e n t ant ibody t e s t s us ing a n t i s e r a to the so l ub l e and the c e l l wa l l f r a c t i o n s , a d i s t i n c t i o n tha t e a r l i e r workers repor ted making i n double d i f f u s i o n t e s t s w i th a n t i s e r a r a i s e d aga ins t c o n i d i a . From the present s tudy , a n t i s e r a produced to d i f f e r e n t a n t i g e n i c f r a c t i o n s e x h i b i t e d d i f f e r i n g degrees o f s e n s i t i v i t i e s us ing ELISA and double d i f f u s i o n . The spore ant iserum reac ted i n ELISA but not i n double d i f f u s i o n . I t would t h e r e f o r e appear t ha t s p e c i f i c i t y tha t i s a t t r i b u t e d to d i f f e r e n t ant igens may not be s p e c i f i c i t y but r a t h e r va ry i ng degrees of s e n s i t i v i t y of d i f f e r e n t techn iques and a n t i s e r a to d i f f e r e n t an t i gens . I t should a l s o be noted tha t the s e n s i t i v i t y o f the double d i f f u s i o n t e s t depends on the opt imal c o n c e n t r a t i o n s o f the r e a c t a n t s as we l l as t h e i r s o l u b i l i t y (Wi l l i ams and Chase 1971). Fur thermore, the d i f f i c u l t i e s in s t anda rd i z i ng fungal an t igens have been expressed (Longbottom and Austwick 1986). The l a t e x a g g l u t i n a t i o n t e s t was employed in t h i s study w i thou t success . Latex beads (Sigma) i n d i v i d u a l l y s e n s i t i z e d w i th the th ree d i f f e r e n t a n t i s e r a f a i l e d to de tec t even t h e i r homologous an t i gens . S e r i e s o f d i l u t i o n s o f both the an t igens and s e n s i t i z e d l a t e x beads were repea ted ly prepared and t e s t e d . The on l y exp lana t i on may be that the p u r i f i e d a n t i s e r a used were not s u i t a b l e s i n c e not a l l IgG p repara t ions obta ined by ammonium su lpha te p r e c i p i t a t i o n are e q u a l l y e f f e c t i v e f o r s e n s i t i z i n g l a t e x beads (Van Regenmortel 1982). In the l a t e x t e s t , e i t h e r ant igen or ant ibody can be adsorbed onto l a t e x 73 p a r t i c l e s . The use of ant igens to s e n s i t i z e l a t e x beads would be i n a p p r o p r i a t e f o r purposes of rou t i ne d i a g n o s i s , and f o r t h i s reason t h i s a l t e r n a t i v e was not eva lua ted . The a n t i s e r a were not s p e c i f i c to D. l y c o p e r s i c i but reac ted e q u a l l y we l l w i th D. app lanata and to a l e s s e r extent w i th D. b ryon iae in ELISA t e s t s . At h igh concen t ra t i ons Fusarium oxvsporum l y c o p e r s i c i . F. oxvsporum  r a d i c i s - l y c o p e r s i c i and B o t r y t i s spp. were d e t e c t e d . I t would however be u n l i k e l y t ha t these pathogens would occur i n mixed i n f e c t i o n s at such high c o n c e n t r a t i o n s . Cross r e a c t i v i t y o f a n t i s e r a w i th hetero logous spec ies i s not uncommon. A ldwe l l et al_. (1983) repor ted c ross r e a c t i v i t y i n a n t i s e r a r a i s e d to VAM fungi but tha t t h i s was reduced by us ing the s o l u b l e f r a c t i o n o f hyphal i s o l a t e s as the ta rge t an t i gen . Banowetz et a l . (1984) repor ted the same phenomenon in two spec ies o f wheat bunt fungi us ing monoclonal and p o l y c l o n a l a n t i b o d i e s . Hearn and MacKenzie (1980) repor ted c r o s s r e a c t i v i t y among fungal genera us ing po l yc l ona l a n t i s e r a . F i t z e l l e t a l . (1980) exper ienced c ross r e a c t i v i t y among four spec ies o f V e r t i c i l l i u m . Ge r i k et a l . (1987) observed c ross r e a c t i v i t y between a n t i s e r a aga ins t V. d a h l i a e w i th F. oxvsporum in western b l o t s . Much of the c ross r e a c t i v i t y observed between fung i i s based on the a n t i g e n i c s i m i l a r i t y among the major p o l y s a c c h a r i d e an t igens tha t are w ide ly d i s t r i b u t e d among fungi (Longbottom 1986). However success has been repor ted on the use of a n t i s e r a f o r the d i f f e r e n t i a t i o n o f s e l e c t e d races and forms of Fusarium oxvsporum (Buxton et a l . 1961). De tec t i on o f e i t h e r the spores or the mycelium of D. l y c o p e r s i c i from i n f e s t e d seeds was not very s a t i s f a c t o r y . The d e t e c t i o n was on ly p o s i t i v e f o r myce l i um- in fes ted seeds and at high l e v e l s o f i n f e s t a t i o n (20 % and 50 %). Such high l e v e l s o f i n f e s t a t i o n s would be de tec ted i n the f i e l d we l l 74 before h a r v e s t i n g . Negat ive r e s u l t s f o r the s p o r e - i n f e s t e d seeds suggested poor adhesion of spore on the sur face of seeds, probably because o f the smal l s i z e o f spores (12-15 x 5 u) compared to the m y c e l i a l f ragments . Us ing ELISA i t was p o s s i b l e to de tec t ant igens from d iseased p l an t s at q u i t e low c o n c e n t r a t i o n s ( F i g . 19) . In t h i s study p lan t sap d i d reduce the s e n s i t i v i t y o f the a n t i s e r a . The same phenomenon would be a t t r i b u t e d to the i n s e n s i t i v i t y o f the l e s s s e n s i t i v e a n t i s e r a to de tec t an t igens from i n f e s t e d seeds . Working w i th v i r u s e s , C l a r k and Adams (1977) found tha t sap c o n s t i t u e n t s o f herbaceous hosts had l i t t l e apparent e f f e c t on the ELISA t e s t . Whi le the ant iserum r a i s e d aga ins t the whole mycelium e x t r a c t o f D. 1 v c o p e r s i c i had a good s e n s i t i v i t y to spores , s o l u b l e p r o t e i n e x t r a c t and the homologous an t i gens , the ant iserum was not s p e c i f i c to D. 1 v c o p e r s i c i . Th i s put a l i m i t a t i o n to the use fu lness of the ant iserum f o r d i a g n o s t i c purposes e s p e c i a l l y i n s i t u a t i o n s where mixed i n f e c t i o n s occu r . Cross r e a c t i v i t y i s widespread in fungi and t h i s has slowed the development o f se ro logy in the f i e l d o f mycology. The use of monoclonal a n t i b o d i e s (MCA) has helped to e l i m i n a t e many of the problems assoc i a t ed w i th p o l y c l o n a l a n t i s e r a , i n c l u d i n g the v a r i a b i l i t y in serum q u a l i t y and l a c k o f s p e c i f i c i t y (Halk and De Boer 1985). MCAs may be the answer to a n t i s e r a s p e c i f i c i t y i n the d iagnoses of D. l y c o p e r s i c i desp i t e the f a c t tha t they have been produced to on ly a few fungal p lan t pathogens and used wi th l i m i t e d success (Halk and De Boer 1985). 75 L i t e r a t u r e c i t e d 1. Agarwa l , V . K . and S i n c l a i r , J . B . 1987. P r i n c i p l e s o f Seed Pa tho logy . V o l . I & I I . CRC P r e s s . Boca Raton, F l o r i d a . 2. A j e l l o , L . , W a l l s , K. Moore, J . C . and Fa lcone , R. 1959. Rapid p roduc t ion o f complement f i x a t i o n ant igens f o r systemic mycot ic d i s e a s e s . 1. C o c c i d i o c i n : i n f l u e n c e of media and mechanical a g i t a t i o n on i t s development. J . B a c t e r i o l . 77:753-755. 3 . A l d w e l l , F . E . B . , H a l l , I .R. and Smi th , J . M . B . 1983. Enzyme-Linked Immunosorbent Assay ( ELISA ) to i d e n t i f y endomycorrh iza l f u n g i . S o i l B i o l . Biochem. 15:377-378. 4 . A x e l s e n , N.H. 1983. Handbook of Immunoprec ip i t a t i on - i n - ge l Techn iques. B l ackwe l l S c i e n t i f i c . 383 pp. 5. Banowetz, G . M . , T r i o n e , E . J . and K r y g i e r , B .B . 1984. Immunological comparisons of t e l i o s p o r e s of two wheat bunt f u n g i , T i l l e t i a s p e c i e s , us ing monoclonal an t i bod ies and a n t i s e r a . Myco log ia 76 :51 -62 . 6. Buxton, E.W., C u l b r e t h , W. and E s p o s i t o , R.G. 1961. S e r o l o g i c a l sepa ra t i on o f forms and p h y s i o l o g i c a l races o f pathogenic Fusar ium  oxysporum. Phytopathology 51:575. (Abs t r . ) 7. Cawley, L . P . , M inard , B. and Penn, G.M. 1978. E l e c t r o p h o r e s i s & Immunochemical React ions in G e l s . Techniques and I n t e r p r e t a t i o n s . Second Ed. American S o c i e t y o f C l i n i c a l P a t h o l o g i s t s , Ch icago . 93 pp. 8 . Channon, A . G . 1972. Cont ro l of tomato stem ro t (D idymel la l y c o p e r s i c i K l e b . ) w i th Benomyl. Hor t . Res. 12:89-96. 9 . Cheah, L . H . and So te ros , J . J . 1983. Cont ro l o f D idyme l la stem canker on g lasshouse tomatoes. P roc . 36th N.Z. Weed and Pest Con t ro l Conf . 125-127. 10. C l a r k , M.F. 1981. Immunosorbent assays in p lan t pa tho logy . Ann. Rev. P h y t o p a t h o l . 19:83-106. 11. C l a r k , M.F. and Adams, A . N . 1977. C h a r a c t e r i s t i c s o f the m i c r o p l a t e method of Enzyme-Linked Immunosorbent Assay f o r the d e t e c t i o n o f p lan t v i r u s e s . J . gen. V i r o l . 34:475-453. 12. Da lbe rg , K.R. and Van E t t e n , J . L . 1982. Phys io logy and b iochemis t r y o f fungal s p o r u l a t i o n . Ann. Rev. Phy topa tho l . 20 :281-301. 13. D e r b y s h i r e , D.M. 1961. A study of seed-borne i n f e c t i o n o f tomato by D idymel la l y c o p e r s i c i K l e b . P roc . In t . Seed T e s t . A s s . 26 :61 -67 . 14. Dewey, F . M . , B a r r e t , D .K . , Vose, I.R. and Lamb, C . J . 1984. Immunofluorescence microscopy f o r the d e t e c t i o n and i d e n t i f i c a t i o n o f propagules o f Phaeolus s c h w e i n i t z i i i n i n f e c t e d s o i l . Phytopathology 74:291-296. 76 15. F i t z e l l , R. , Fahy, P .C . and Evans, G. 1980. S e r o l o g i c a l s t u d i e s on some A u s t r a l i a n i s o l a t e s of V e r t i c i l l i u m spp. Aus t . J . B i o l . S c i . 33 :115-124. 16. F l e t c h e r , J . T . 1973. D i seases . Pages 196-208 In : The U.K. Tomato Manual . Ed . Kingham, H.G. Grower Books, London. 196-208 pp. 17. G e r i k , J . S . , Lommel, S .A . and Huisman, O.C. 1987. A s p e c i f i c s t a i n i n g procedure f o r V e r t i c i l l i u m d a h l i a e in co t ton root t i s s u e . Phytopathology 77 :261-265. 18. G leason , M . L . , G h a b r i a l , S .A . and F e r r i s s , R .S . 1987. S e r o l o g i c a l d e t e c t i o n o f Phomopsis l o n q i c o l l a i n soybean seeds . Phytopathology 77:371-375. 19. Ha lk , E . L . and De Boer , S . H . 1985. Monoclonal a n t i b o d i e s i n p l a n t - d i s e a s e r e s e a r c h . Ann. Rev. Phy topa tho l . 23 :321-350. 20. Hearn, V .M. and MacKenzie, D.W.R. 1980. The p repa ra t i on and p a r t i a l p u r i f i c a t i o n o f f r a c t i o n s from m y c e l i a l fungi w i th a n t i g e n i c a c t i v i t y . M o l . Immunol. 17:1097-1103. 21 . Hickman, C . J . 1946. I n f e c t i o n of outdoor tomato crops by D idyme l la  l y c o p e r s i c i . J . Pomol. Hor t . S c i . 22 :69-75 . 22. H o l l i d a y , P. and Pun i tha l ingam, E. 1970. C . M . I . D e s c r i p t i o n o f pathogenic fung i and b a c t e r i a . No. 272. The Eas te rn Press L t d . , London. 23. Johnson, M . C . , P i r o n e , T . P . , S i e g e l , M.R. and Varney, D.R. 1982. De tec t i on o f Ep ich loe t yph ina in t a l l fescue by means o f enzyme- l inked immunosorbent assay . Phytopathology 72:647-650. 24. Khan, A . M . and S l a c k , S . A . 1978. S tud ies on the s e n s i t i v i t y o f a l a t e x t e s t f o r the s e r o l o g i c a l d e t e c t i o n o f potato v i r u s S and pota to v i r u s X in W i s c o n s i n . Amer. Potato J . 55:627-637. 25 . K n i g h t , D .E . 1960. S tud ies on D idymel la l y c o p e r s i c i K l e b . , the causa l organism of stem ro t d i sease of tomatoes. T rans . B r i t , myco l . Soc . 43 :519-522. 26. Laemmli , U.K. 1970. Cleavage of s t r u c t u r a l p r o t e i n s du r i ng assembly o f the head o f T4. Nature 227:680-685. 27. Longbottom, J . L . 1986. A p p l i c a t i o n s o f immunological methods i n mycology. 121 pp In : Handbook of Exper imental Immunology. V o l . IV. Eds. Wei r , D .M. , Harzenberg, L . A . , B l a c k w e l l , C. and Herzenberg, Leonore A. B l a c k w e l l S c i e n t i f i c . Ox fo rd . 28. Longbottom, J . L . and Aus tw ick , P . K . C . 1986. Fungal a n t i g e n s . 7 pp In : Handbook o f Exper imental Immunology. V o l . 1 . Eds. We i r , D .M . , Harzenberg, L . A . , B l a c k w e l l , C. and Herzenberg, Leonore A. B l ackwe l l S c i e n t i f i c . Ox fo rd . 77 29. Ma ide r , G.H. and Burge, M.N. 1979. In f luence of l i g h t and medium on pycnid ium and pycn id iospore product ion in D idymel la l y c o p e r s i c i . T rans . B r . myco l . Soc. 72:504-506. 30 . M a r s h a l l , M.R. and P a t r i d g e , J . E . 1981. Immunochemical i d e n t i f i c a t i o n o f Fusarium mon i l i fo rme ribosomes from d iseased corn ( Zea mays L.) s t a l k t i s s u e . P h y s i o l . P lan t P a t h o l . 19:277-288 3 1 . M a r t i n s o n , V . A . and Hogenboom, N.G. 1968. Screen ing young tomato s e e d l i n g s f o r r e s i s t a n c e to D idymel la f o o t - and s temro t . Euphy t i ca 17:173-182. 32 . Nachmias, A . , Buchner, V. and K r i k u n , J . 1982. D i f f e r e n t i a l d i a g n o s i s o f V e r t i c i l 1ium d a h l i a e in potato w i th a n t i s e r a to p a r t i a l l y p u r i f i e d pathogen-produced e x t r a c e l l u l a r an t i gens . Potato Res. 25 :321-328. 33. No lan , D.G. 1989. Seed germinat ion c h a r a c t e r i s t i c s o f Centaurea d i f f u s a and C. maculosa. M.Sc. T h e s i s . U n i v e r s i t y o f B r i t i s h Co lumbia . 207pp. 34. Ouch te r l ony , 0 . 1968. Handbook of Immunodiffusion and Immunoelect rophores is . Ann Arbor Sc ience Inc . Ann Arbor 215 pp. 35 . P h i l l i p s , D.H. 1956 a . Tomato seed t r ansm iss ion of D idyme l l a l y c o p e r s i c i K l e b . T rans . B r i t , myco l . Soc. 39:319-329. 36. P h i l l i p s , D.H. 1956 b. S o i l - b o r n e i n f e c t i o n o f tomatoes by D idyme l la  l y c o p e r s i c i K l e b . T rans . B r i t , myco l . Soc. 39 :330-340. 37. Rao, P .V . and S i n g h a l , G .S . 1978. C h a r a c t e r i s a t i o n o f l i g h t dependent, synchronous p y c n i d i a l p roduct ion in B o t r v o d i p l o d i a theobromae. T rans . B r . myco l . Soc. 70:121-129. 38 . Scopes, R. 1982. P r o t e i n P u r i f i c a t i o n . P r i n c i p l e s and P r a c t i c e . S p r i n g e r - V e r l a g , New York. 329 pp. 39 . She lby , R. 1985. Use of enzyme- l inked immunosorbent assay ( ELISA ) as an a i d in d e t e c t i o n o f Diapor the phaseolorum v a r . c a u l i v o r a . P r o c . Sou th . Soybean D i s . Workers 12:70. 40. T reuha f t , M.W., Rober ts , R . C . , Hackbar th , C. and Marx, J . J . 1981. C h a r a c t e r i s a t i o n o f s y n t h e t i c medium ant igens of Mvcropo lvspora faen i and Thermoactinomvces cand idus . J . A l l e r g y c l i n . Immunol. 67 :375-387. 41 . T u i t e , J . 1969. P lan t Pa tho log i ca l Methods. Fungi and B a c t e r i a . Burgess Pub. M i n n e a p o l i s . 239 pp. 42 . Van Regenmorte l , M.H.V. 1982. Sero logy and Immunochemistry o f P lan t V i r u s e s . Academic P r e s s . New York London. 267 pp. 43 . Ward, L . J . and De Boer, S . H . 1989. C h a r a c t e r i s a t i o n o f a monoclonal ant ibody aga ins t a c t i v e pec ta te l yase from E rw in i a c a r o t o v o r a . Can. J . M i c r o b i o l . 35 :651-655. 78 44. Watterson, J . C . 1986. Diseases. Pages 443-484 In: The Tomato Crop: a sc ien t i f i c basis for improvement. Eds. J . G . Atherton and J . Rudich. Chapman and Hal l , London. New York. 661 pp. 45. Williams, B.L. and Wilson, K. 1984. A Biologist 's Guide to Principles and Techniques of Practical Biochemistry. Second Ed. Edward Arnold, London. 318 pp. 46. Williams, C A . and Chase, M.W. 1971. Methods in Immunology and Immunochemistry. Vol.I I I . Academic Press, New York and London. 515 pp. 47. Wray, W., Boulikas, T . , Wray, V.P. and Hancock, R. 1981. Si lver staining of proteins in polyacrylamide gels. Anal. Biochem. 118:197-203. 79 o Appendix 1. A step by step procedure of the s i l v e r s t a i n i n g techn ique of Wray et a l . (1981) as used in t h i s study to s t a i n SDS-PAGE g e l s o f p r o t e i n ex t rac ted from c u l t u r e s o f D. l y c o p e r s i c i . 1. Soak the gel f o r 45 min in a s o l u t i o n o f 0.1 ml formaldehyde (38 %), 50 ml methanol and 50 ml d i s t i l l e d de ion i sed (dd) wa te r . 2 . Wash the gel w i th dd water f o r 20 min. 3 . Make up the s t a i n s o l u t i o n - MUST BE USED WITHIN 5 MIN. a . 0.4 g s i l v e r n i t r a t e in 2 ml dd water . b. 2 ml (0.2 M) NaOH, 0.450 ml 12 M (NH) OH. With cons tan t s t i r r i n g add (a) dropwise to (b) and make up to 50 ml w i th dd wate r . 4 . S t a i n the gel f o r 15 min w i th constant a g i t a t i o n . 5. Wash w i th dd water f o r 5 min. 6 . Make up the deve loper - USE WITHIN 15 MIN. 0.625 ml 1 % c i t r i c a c i d , 0.063 ml 38 % formaldehyde. Make up to 125 ml w i th dd water . 7. Soak the gel i n the deve loper . 8 . Wash i n a s o l u t i o n o f 50 % methanol and 10 % HoAC. 80 Appendix 2. A mod i f ied step by step procedure of the immune assay used to probe p ro te i ns o f D. l y c o p e r s i c i t r a n s f e r r e d to n i t r o c e l l u l o s e paper (Ward and De Boer 1989). 1. B lock the b l o t by f l o a t i n g on 2 % b l o t t o i n PBS f o r 5 min and submerging i t f o r 1 h at 37 C w i th a g i t a t i o n . 2. Incubate the b l o t in pr imary ant ibody d i l u t e d i n PBS p lus 0 .5 % b l o t t o (PBS-BL) f o r 2-4 h at 37 C w i th a g i t a t i o n . 3 . Wash the b l o t 3-5 t imes wi th a g i t a t i o n at room temperature f o r 15 min i n PBS-BL. 4 . Incubate the b l o t i n secondary ant ibody/enzyme con jugate d i l u t e d in PBS-BL f o r 1 h. 5. Wash b l o t i n PBS-BL as above and then once i n 50 mM T r i s - H C l , pH 8 . 0 . 6 . Submerge b l o t i n subs t ra te [ 3.3 mg Naphthol As-MX phosphate (S igma) , 50 mg Fast Red TR s a l t (Sigma), 10 ml 50 mM T r i s - H C l pH 8 . 0 ] . Incubate w i th a g i t a t i o n in the dark f o r 5-35 min . 7. Reac t i ve ant igens are v i s i b l e as red bands on b l o t s tha t correspond to an t igen bands on the o r i g i n a l SDS-PAGE g e l . 81 A p p e n d i x 3 . A n a l y s i s o f v a r i a n c e (ANOVA) c a r r i e d o u t o n t h e y i e l d o f D i d y m e l l a l y c o p e r s i c i c u l t u r e s grown on two m e d i a s u p p l e m e n t e d w i t h d i f f e r e n t m i n e r a l e l e m e n t s and y e a s t e x t r a c t S o u r c e d f SS MS F (% o f T o t a l T o t a l 83 2 6 1 . 8 0 1 0 0 . 0 0 B l o c k 2 1 1 . 6 8 5 . 8 4 1 . 3 9 4 . 4 6 S h a k e / S t i l l 1 0 . 9 1 0 . 9 1 0 . 2 2 0 . 3 5 E r r o r (a) 2 8 . 4 0 4 . 2 0 4 . 8 4 3 . 2 1 T r e a t m e n t 13 1 6 1 . 1 8 Y e a s t / None 1 4 7 . 4 6 4 7 . 4 6 5 4 . 6 7 1 8 . 1 3 * * C I / C2 no Y e a s t 1 0 . 8 6 0 . 8 6 1 . 0 0 0 . 3 3 F e / N o n e 1 0 . 7 3 0 . 7 3 0 . 8 4 0 . 2 8 C 1 / C 2 w i t h Y e a s t 1 5 . 3 8 5 . 3 8 6 . 2 0 2 . 06 * . Mn 1 0 . 8 1 0 . 8 1 0 . 9 3 0 . 3 1 Z n 1 9 5 . 5 0 9 5 . 5 0 1 1 0 . 0 0 3 6 . 4 8 * * Mn X Z n 1 2 . 1 4 2 . 1 4 2 . 4 6 0 . 8 2 F e / N o n e X Mn 1 0 . 0 0 0 . 0 0 0 . 00 0 . 00 F e / N o n e X Z n 1 0 . 3 7 0 . 3 7 0 . 4 3 0 .14 F e / N o n e X Mn X Z n 1 0 . 9 5 0 . 9 5 1 . 1 0 0 . 3 6 C 1 / C 2 s Y X Mn 1 2 . 5 2 2 . 5 2 2 . 9 0 0 . 9 6 C 1 / C 2 sY X Z n 1 4 . 4 7 4 . 4 7 5 . 1 5 1 . 7 1 * . C 1 / C 2 sY X Mn X Z n 1 0 . 0 0 0 . 00 0 . 0 0 0 . 00 S X T r e a t m e n t 13 3 4 . 4 9 S X Y e a s t / None 1 0 . 8 5 0 . 8 5 0 . 9 7 0 . 3 2 S X C I / C2 1 0 . 1 1 0 . 1 1 0 . 1 3 0. 04 S X F e / N o n e 1 6 . 7 2 6 . 7 2 7 . 7 4 2 . 5 7 * * S X C 1 / C 2 1 5 . 9 7 5 . 9 7 6 . 8 8 2 .28 * . S X Mn 1 2 . 4 3 2 . 4 3 2 . 8 0 0 . 9 3 S X Z n 1 8 . 2 0 8 . 2 0 9 . 4 5 3 .13 * * S X Mn X Z n 1 0 . 1 7 0 . 1 7 0 . 1 9 0. 06 S X F e / N o n e X Mn 1 0 . 0 9 0 . 0 9 0 . 1 0 0 . 0 3 • • S X F e / N o n e X Z n 1 8 . 4 5 8 . 4 5 9 . 7 4 3 . 2 3 * * S X F e / N o n e X Mn 1 0 . 0 5 0 . 0 5 0 . 0 6 0 . 0 2 S X C 1 / C 2 X Mn 1 0 . 8 2 0 . 8 2 0 . 9 4 0 . 3 1 S X C 1 / C 2 X Z n 1 0 . 2 2 0 . 2 2 0 . 2 5 0 . 08 S X C 1 / C 2 X Mn X 1 0 . 4 1 0 . 4 1 0 . 4 7 0 . 1 6 E r r o r (b) 52 4 5 . 1 4 0 . 8 7 1 7 . 2 4 * — * * * S= I n c u b a t i o n m e t h o d * = P = 0 . 0 5 C I = C o o n s medium * * = P = 0 . 0 1 C2 = C z a p e k - D o x medium 

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