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Axenic culture of wheat stem rust fungus Bose, A. (Amitava) 1970

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AXENIC CULTURE OF WHEAT STEM RUST FUNGUS by AMITAVA BOSE B . S c . ( A g r . ) , B h a g a l p u r U n i v e r s i t y , 1962 M . S c . ( A g r . ) , B h a g a l p u r U n i v e r s i t y , 1965 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e Department o f P l a n t S c i e n c e We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1970 In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree l y ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th i s thes is for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It i s understood that copying or pub l i ca t ion of th i s thes is fo r f inanc ia l gain sha l l not be allowed without my writ ten permission. Department of The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada I ABSTRACT The development of our knowledge of the metabolism, nutrition., and physiology of the rust fungi has been res-t r i c t e d because of our i n a b i l i t y to grow these fungi i n axenic culture. Most of the physiological and biochemical Investigations on rust fungi have been l i m i t e d to studies of the germination and development of germ tubes. I t i s not known, whether f a i l u r e of the rusts to grow axenically i s associated with a metabolic block i n essential biochemical pathways. The u r e d i a l stage of an Australian i s o l a t e (ANZ 126-6,7) of wheat stem rust was grown on a r e l a t i v e l y simple medium. Successful i n v i t r o growth was obtained on a medium containing Czapek's minerals, glucose and an organic nitrogen source v i z . yeast extract, peptone or BSA. Vegetative growth producing a 'discrete colony appeared to be dependent upon high density seeding, when conventional dry seeding techniques were followed. When a spore suspen-sion i n g e l a t i n was used f o r inoculation consistent, reproducible vegetative growth at low density seeding was obtained. Water spore suspensions f a i l e d to support any growth on the same ser i e s of media, under i d e n t i c a l conditions. Sodium c i t r a t e , host extract, and host protein f r a c t i o n s i n general i n h i b i t e d growth whereas pectin increased the l a g phase. i i Spore-like bodies were obtained inside the colony, which were devoid of any pigmentation. It i s suggested that the combination of physical and chemical factors surrounding the germinating spore play an important role in the in vitro development of rust fungi. Supervisor i i i TABLE OF CONTENTS Pa fie ABSTRACT i TABLE OF CONTENTS i i i LIST OF TABLES iv LIST OF FIGURES v INTRODUCTION 1 LITERATURE REVIEW 3 Section I. Dual membered culture systems. 4 Section I I . Growth of mycelium directly from spores. 11 MATERIALS AND METHODS 15 A. Preparation of inoculum 15 B. Aseptic leaf culture to obtain un-contaminated uredospores 16 C. Cultural studies 22 D. Inoculation 27 E. Incubation 29 F. Disposal of materials 30 G. Microtechnique 30 EXPERIMENTAL RESULTS 34 Section I. Effect of various organic compounds on in vitro development of rust using dry seeding techniques. 34 A. Effect of yeast extract 34 B. Effect of Ewan's peptone 38 C. Effect of unfractionated host extract 40 D. Effect of wheat leaf protein fraction 40 E. Effect of bovine serum albumin 41 F. Effect of sterols 43 Section II. Effect of spore suspension techniques on growth and development of rust. 45 DISCUSSION 56 SUMMARY 63 REFERENCES 65 i v LIST OF TABLES Table Page I E f f e c t of spore suspension inoculation technique on the development of rust on media containing various organic s a l t s . 46 V LIST OF FIGURES Figure Page 1 Schematic representation of the system used i n c u l t i v a t i n g rust fungi. 21 2 Flow sheet f o r wheat leaf f r a c t i o n s . 28 '3 Germination of uredospores on Czapek's medium containing yeast extract. 35 4 Germ tube showing a t y p i c a l swollen t i p on Czapek's medium containing yeast extract. 36 5 Germ tube showing s p i r a l growth on Czapek's medium containing yeast extract. 36 6 Rust mycelium formed on Czapek's minerals and yeast extract medium. 37 7 • A hypha from the colony i n F i g . 6 showing septation. 37 8 Profuse mycelial growth on Czapek's medium containing Evan's peptone. 39 9 Cross section of the stroma produced i n basic medium containing BSA from dry seeding. 42 10 A hypha showing two nu c l e i at centre f i e l d . 42 11 Globular bodies on a e r i a l hyphae i n the basic medium containing ^ - s i t o s t e r o l . 44 12 Typical clumping of spores on the medium sur-face when water suspensions of uredospores were used to inoculate p e t r i dishes. 47 13 Uniform d i s t r i b u t i o n of uredospores on the medium surface when ge l a t i n suspensions of uredospores were used to inoculate p e t r i dishes. 47 14 Growth of mycelium on basic medium con-t a i n i n g yeast extract and BSA. 48 15 P e t r i dish showing two colonies developed on basic medium containing BSA where spores were clumped together from water suspension i n -oculation. 48* 16 E f f e c t of adding pectin to the basic medium containing yeast extract and BSA. 50 Colony development at 26* days a f t e r the basic medium plus BSA was inoculated with a g e l a t i n spore suspension. Series of i r r e g u l a r bodies around the gel matrix on basic medium containing BSA. Cross section of the stroma developed on the basic medium containing BSA. Uredospore-like bodies formed near the stroma i n the basic medium containing BSA. Ae r i a l hyphae from a colony showing d i -karyotic hypha, stained with iron haematoxylin. A highly vacuolated terminal hypha show-ing a single nucleus i n the terminal c e l l . v i i ACKNOWLEDGMENTS I take this opportunity to express my deep sense of gratitude to Dr. Michael Shaw for suggesting the problem and for guidance throughout the course of the research and in the preparation of the manuscript. Thanks are also extended to Dr, M.D. Coffey for active cooperation in the later part of the investigation and to Gayle Thompson for typing the manuscript. I am grateful to Dr, B. van der Kamp for permitting use of the laminar airflow. I thank Dr. C.O. Person and Mr. B. Sivak for help with the photomicrography and many other people who have enriched my work. This research was supported by a grant to Dr. M. Shaw from the National Research Council of Canada. 1 INTRODUCTION The technique of axenic culture - i . e . the growth of a single species i n the absence of l i v i n g organisms or l i v i n g c e l l s of any other species (Scott and Maclean 1 9 6 9 ) - o f f e r s a number of pot e n t i a l advantages f o r the study of obligate fungal parasites l i k e the rust fungi. Many papers have been published on the comparative metabolism of rust infected and uninfected t i s s u e s . These include studies on r e s p i r a t i o n , photosynthesis, tran s l o c a t i o n , nitrogen metabolism and protein synthesis, hormone le v e l s and u l t r a s t r u c t u r a l changes. Many of these studies have to a large extent f a i l e d to indicate the r e l a t i v e contributions of host and fungus to pos t - i n f e c t i o n a l changes. Much information has also been accumulated about the factors governing the germination of rust spores and the conditions under which i n f e c t i o n w i l l occur. P r a c t i c a l l y nothing i s known about the s p e c i f i c metabolites which these parasites obtain from t h e i r hosts. Mycelium produced i n axenic culture may be useful for studying the phys i o l o g i c a l and biochemical relationships of host and parasite. The technique may provide information re-garding the series of evolutionary changes among the primitive r u s t - l i k e basidiomycetes, from saprophytism through various stages of f a c u l t a t i v e parasitism to s t r i c t obligate parasitism, provided one assumes that such mutations have occurred and been conserved. Evidence concerning the metabolic pathways i n the obligate plant parasites may indicate the basis of physiologic s p e c i a l i z a t i o n and host resistance. On the 2 practical side the technique may provide a suitable screening method for the development of systemic fungicides for rust control. The d i f f i c u l t i e s encountered in establishing axenic cultures of obligatory parasites pose intriguing problems. The solutions of these problems have been sought using two approaches: l ) growth of the parasite on tissue cultures of the host and 2 ) mycelial growth directly from spores. It i s believed that when a dual-membered tissue culture i s established, vigorous growth may permit expression of any mutation in the pathogen which allows i t to grow independently of i t s host. Another view i s that after the obligate parasites have been "trained" in dual-membered cultures they may develop adaptive enzymes which may permit them to grow axenically. Recent studies on in vitro growth of Puccinia graminis  t r i t i c i (Eriks. and Henn.), Australian race ANZ-126-6,7 by Williams et a l . (1966) and i t s subsequent partial confirma-tion by Bushnell (1968) indicated the f e a s i b i l i t y of establish-ing the rust fungi in axenic culture. The specific objectives of the present study were to develop a suitable technique for growing substantial amounts of mycelium in axenic culture with Various organic substrates and to confirm the findings of Williams et a l . (1966). To this end the experimental work was concerned with the effect of various organic substrates and the method of inoculating the medium with uredospores on the growth and development of the fungus. 3 LITERATURE REVIEW A number of techniques has been employed in attempts to grow rusts in axenic culture. Before describing these, i t w i l l be helpful to the reader to consider the terminology em-ployed in the literature in this f i e l d . An organism i s considered to be an obligate parasite when i t can grow only by securing i t s food through continued association with another l i v i n g organism (Brown 1936, Butler and Jones 1919, DeBary and Gurnsey 1887, Gaumann and Brierly 1950). In other words, obligate parasites are those organisms which cannot be cultured routinely on non-living substrates. It has been recognized since the time of DeBary and Gurnsey (1887) that the vegetative stages of such pathogens do not continue independent growth apart from the cells of their re-spective hosts. Dougherty (1950) has defined axenic culture as the growth of one organism free of a l l others, the term 'axenic culture* as employed by Yarwood (1956) refers to the culture of a rust isolated from i t s host and i t s growth in the mycelial condition on a synthetic medium in the complete absence of a l l other organisms. A tissue culture i s defined as a mass of c e l l s , derived from any part of a plant, growing aseptically in vitro under conditions where subsequent growth is relatively undifferen-tiated. This i s synonymous with White's concept of callus culture (White 1942). 4 In the earliest attempts to culture rusts, various media were enriched with host extracts and with supplementary growth factors in attempts to determine the precise factors responsi-ble for the obligate parasitism of the Uredinales. These ex-periments indicated that the usual methods employed for the isolation and culture of facultative parasites were inadequate for the growth of these obligates. The literature on the subject may be divided into two sections. The f i r s t section i s devoted to the tissue culture approach ending in successful axenic culture. The second sec-tion deals with the axenic cultures obtained directly from fungal spores. Section I. Dual-Membered Culture Systems Morel (1948) f i r s t reported successful establishment of downy mildew of grape in callus cultures and later described intensive investigations resulting in the cultivation of some other obligate parasites on tissue cultures. Hotson and Cutter (1951) grew Gymnosporangium iuniperi-virginianae-infected cedar galls in tissue cultures After several months, they observed a fungus growing on the agar substrate away from the host tissue. When this fungus was transferred to a standard medium (PDA), i t continued to grow as a reddish mass. Inoculation of the alternate host (Crataegus  coccinea) with this saprophytic mycelium was successful. Cutter (1959) further reported that 3 additional strains of G. .1 uniperi-virginianae had been isolated in axenic culture by 5 the same technique and were maintained by repeated subcul-ture for 6 months without loss of their a b i l i t y to reinfect the alternate hosts. Altogether, seven strains were isolated from tissue culture grown in axenic culture. A l l were able to survive continuous transfer on various solid media. After axenic culture, a l l of these strains proved able to reinfect their alternate rosaceous hosts in tissue culture and under f i e l d conditions. Four of them, which presumably were dikary-otic , were also capable of infecting Juniperus. Cutter found that one strain was atypical in attacking both Crataegus and Pyrus and in showing the characteristics of G, globossum when parasitizing Crataegus. Plausible explanations for this were (i) axenic culture had modified the genotype and i t s host spe c i f i c i t y had been broadened; ( i i ) the strain represented a mixed culture of G. globossum and G. juniperi-virginianae. Since the emergence of mycelium was extremely rare, Hotson'and Cutter suggested that the in vitro growth arose by mutation from the parasitic mycelium. Another possibility was that only an occasional rust c e l l was capable of independent existence. Due to the exhaustiveness of the experiment, i t was possible to screen out or select potential saprobic clones from the entire culture population. Perhaps a more logical explanation i s that, after a period of culture, the callus and fungus caused chemical changes in the medium that allow the growth of the fungus into the medium. Unfortunately, Turel and Ledingham (1957) have not been able to establish G, .juniperi-virginianae on a r t i f i c i a l media, to confirm this 6 work. Maheshwari, Hildebrandt and Allen (1967) were able to culture red cedar galls induced by cedar apple rust. Although callus formation occurred readily, rust mycelium did not i n -vade the newly formed tissues. Cutter (I960) further reported successful callus cul-tures from corms of Arisaema trlphyllum which were systemically infected with Uromyces a r i - t r i p h y l l i for several years. Aecial stages were produced on the callus tissues. Altogether five strains of the rust were isolated and grown in axenic culture. These isolated strains were maintained in solid media and a l l were capable of reinfecting Arisaema in tissue culture and under f i e l d conditions gave rise to sori with viable spores. These results are similar to those obtained in his previous work. Very few clones, which survived apart from the host, were selected out from a large population. Heim and Gries (1953) obtained some growth of Erysiphe  cichoracearum on sunflower tumor tissue, but did not carry the cultures through a series of transfers. Successful infection was achieved by inoculating spores from two-membered cultures on seedlings. Establishment of the mildew occurred only on tumor tissue and not on callus tissue, but only two out of about one hundred t r i a l s were successful. The success re-ported was with tumor tissue which was growing rapidly, in agreement with evidence that active growth of the host favors these parasites. 7 Turel and Ledingham (1957) were able to produce dense, f e l t - l i k e growth of mycelium of Melampsora l i n i by surface-sterilizing the rust-infected cotyledons of flax on a modified Knop's medium containing fresh, ripe, coconut milk, sucrose and Difco Bacto agar. The rust hyphae did not show any tendency to grow axenically and developed only where the leaf was not in contact with the nutrient medium. Upon senescence of the leaf tissue, the growth of the mycelium stopped. Nozzolillo and Craigie (I960) obtained growth of Puccinia  helianthi on tissue cuiktures of i t s host. When rust-infected cotyledons, hypocotyls and stem sections of sunflower seedlings were cultured on nutrient agar media, profuse callus growth was obtained from hypocotyl and stem sections. Infections arising from basidiospores produced an abundant tuft-like growth of haploid surface mycelium. Growth of surface mycelium was almost entirely lacking from infections arising from uredo-spores. Copious aeciospore production occurred in some cul-tures where two basidiospore infections of different mating types coalesced. Later, such compound infections produced uredospores and teliospores. Tufts of mycelium developed on the new callus growth of some cultures. However, the fungal mycelium never showed any tendency to invade the medium apart from i t s host. Maheshwari, Hildebrandt and Allen (1967) were able to obtain callus formation of rust-infected sunflower cotyledons. The rust mycelium from the explant, however, never grew into the proliferating callus tissue. 8 Milholland ( 1 9 6 2 ) was able to grow L i t t l e Club wheat callus tissue in 4 days. The medium used was basal potato dextrose agar ( P D A ) substituted with napthalene acetic acid ( N A A ) , coconut milk, calcium pantothenate, and 2,4-D, Sur-face ste r i l i z e d wheat seeds were germinated in PDA. After 43 hr scutellar node tissue was excised, inoculated in the substituted medium mentioned above and incubated under #50 f t -c light at two different temperatures (25C and 20C). In both cases 10-12 mm of callus was produced. L i t t l e Club wheat leaves inoculated with rust race 11 were used to infect the callus. At the flecking stage, 1 cm leaf bits were surface ster i l i z e d and the lower epidermis removed. The infected meso-phyll was placed in direct contact with 2 week old callus. On removal of the leaf a small mass of aerial hyphae was observed at the point of inoculation. Histological studies on the i n -fected callus showed that large haustoria and runner hyphae had penetrated the callus up to 4 cells deep. Further evidence i s lacking to assess the advantage of this type of system. Nakamura (1965) was able to infect healthy turnip root callus tissue with Peronospora parasitica. He raised uncon-taminated spores (conidia) by infecting slices of healthy root kept in a moist chamber. Surface contaminants from these roots were removed by using a 1% solution of sodium hypochlorite. After removing injured surfaces, sound remaining parts were reincubated i n moist petri dishes. Uncontaminated spores produced after 24 hrs were used in the form of a suspension 9 to inoculate turnip root callus tissue grown in an agar medium containing various inorganics, glucose, thiamine and NAA. Conidiophores bearing conidia were found on the callus tissue after 10 days. These conidia were able to infect healthy callus. None of the hyphae in his tissue cultures were able to invade the medium to give rise to axenic culture of the fungus. Maheshwari, Hildebrandt and Allen (1967) were able to induce profuse aerial mycelium of Puccinia antirrhini in i n -fected snap dragon leaf tissue cultured on a solid medium. Aerial mycelium was found to develop as white tufts, 1-3 mm long. These authors were unsuccessful in obtaining axenic cultures by transferring the mycelium to nutrient medium. Attempts to infect host tissue were also unsuccessful. In-oculation with uredospores of snap dragon tissue cultures covered by host leaf epidermis did not produce any infection. Experiments to infect callus tissue by placing i t in contact with uredospores germinated on collodion membranes also f a i l e d . The authors proved that the cause of failure of uredospores to infect callus tissues of snap dragon was the presence of an extracellular, heat-stable, dialyzable, non-ionic, ether-and ethanol-insoluble inhibitor. This could be leached out in agar or in water. Koenigs (1968) reported that Western white pine stem sections infected with Cronartlum ribicola can be grown in malt extract medium under 200-250 ft-c fluorescent illumination 10 at 20C. C_. ribi c o l a colonized 90% of the calluses sectioned. The fungus appeared to be alive in calluses up to 6 1/2 months after inoculation. Subcultures of infected and healthy callus remained alive for 3 months but did not pro-l i f e r a t e significantly in tissue culture. He held that auto-claving the malt extract medium apparently did not affect the maintenance of subcultures, but the lack of continuous callus formation i s a response previously reported with this medium when i t i s autoclaved (Loewenberg and Skoog 1952). Lack of vigorous calluslng by a l l subcultures suggested that growth regulators might have some important role. Ingram (1969) reported the growth of Plasmodiophora  brassicae in the explants of young tumor tissues of Brassica  rapa t B. napus and B. oleracea capitata. Infected tissue gave rapid callus growth in a medium containing 2,4-D and coconut milk. A l l stages of the parasitic l i f e cycle were detected in active callus. He could maintain the organism for 1 year, by transferring to new medium every # weeks. Infected tissue was found to develop callus tissue in media deficient in growth factors in contrast to uninfected ones which declined or died. His work was substantiated by Williams et_ a l . (1969), working in Wisconsin. Tewari and Arya (1969) were able to grow Sclerospora  graminlcola (Sacc.) & Chroet., the causal organism of downy mildew of pearl millet. The medium used was White's (1942) basal mineral salts plus glucose, casein hydrolyzate (Oxoid), vitamins and kinetin. Infected tissue gave proliferation of the pathogen to produce fru i t i n g bodies, which in turn infected 11 healthy c a l l u s . The fungus grew out i n the medium beyond the c a l l u s and survived two successive transfers i n the same medium. Sporangia and other abnormal f r u i t i n g bodies were formed. The sudden emergence of saprophytic growth of Sclerospora gramini-cola from infected c a l l u s t i s s u e on a r t i f i c i a l media i s pro-bably due to mutation or to changes i n the medium that allowed the fungus to grow out into i t . This work bears some resem-blance to the work of Cutter. Section I I . Growth of Mycelium D i r e c t l y from Spores Ray (1901) reported the axenic culture of rose rust on a g e l a t i n medium containing plant extract. He stated that te l i o s p o r e s of the rust were produced within the g e l a t i n medium. Such cultures of the p a r a s i t i c fungi, he said were l e s s v i r u l e n t than cultures on the hosts. This report was never substantiated and i s d i f f i c u l t to evaluate i n the l i g h t of present knowledge. GretschushniKoff (1936) reported the i n v i t r o growth of four species of Puccinia which were maintained f o r twelve days on a culture medium containing substances which adsorbed ammonia and urea produced by the growing mycelium. These com-pounds had been found to i n h i b i t development of rust spores, and s u s c e p t i b i l i t y was considered to depend upon t h e i r con-tinuous removal by the host plant. Gretschushnikoff"s almost casual claims have also never been ("confirmed. 12 McKay (1939) attempted to grow Peronospora destructor by adding onion extract to glucose potato agar medium. Old oospores were used to inoculate s l i d e s coated with media or water and incubation was done at 15-20C i n moist chambers. He reported more growth i n medium than i n water. Hyphae 1.5 -5"mm i n length were found within 24 hr and from 4 - 10;5 mm i n 48 hr. Maximum growth was reported to be 1.5 cm i n 6 days. Branching of the hyphae occurred but there was no i n d i c a t i o n of chlamydospore formation. Only a very small percentage of the inoculations grew and t h i s type of approach has not led to successful growth of mildew organisms i n axenic culture. This work i s d i f f i c u l t to evaluate as there i s no mention of contamination problems and the oospores were not surface-s t e r i l i z e d before use. Recently Williams et a l . (1966) reported the growth and sporulation i n v i t r o of Puccinia graminis t r i t i c i (race ANZ 126-6,7) on a culture medium containing Czapek fs minerals, sucrose and 0.1$ Difco yeast extract at pH 6.4. Uncontaminated uredo-spores f o r seeding the agar medium were raised by the method of aseptic leaf culture. More abundant vegetative growth was obtained by modifying the nutrient medium with yeast extract which was passed through Zeocarb 225 (ammonium form) and Dowex-1 (formate form) ion exchange re s i n s , the effluent being added to Czapek's medium. Pathogenicity and sporulation of the saprophytic c u l -ture of P. graminis t r i t i c i were reported i n 1967 by the same 13 group. Reinfection by uredospores formed i n a r t i f i c i a l c u l -ture was obtained only when spores were placed on exposed mesophyll of L i t t l e Club wheat. The application of uredo-spores to i n t a c t leaves under the usual conditions of inocula-t i o n resulted i n no v i s i b l e symptoms. Very few appressoria were formed over stomata and there was no further development. They concluded that the saprophytic rust had a low potential f o r independent growth during the i n i t i a l stages of i n f e c t i o n . Williams* group believes that the c u l t u r i n g problem i s essen-t i a l l y n u t r i t i o n a l . Colonies obtained by the group were ex-tremely small and mycelium never spread i n the medium from the o r i g i n a l inoculum. This i s probably due to the peculiar growth nature of t h i s p a r t i c u l a r rust fungus i n the a r t i f i c i a l medium. Bushnell (1968) was able to confirm Williams* observa-tions using a s i m i l a r system. He f a i l e d to get good growth by incorporating yeast extract, but when Evan's peptone and glu-cose were used i n the place of yeast extract and sucrose i n Czapek's medium he obtained small axenic cultures of P. graminis. Sporulation was both inconsistent and of a variable form. The presence of abnormal bodies resembling aeciospores and various intermediates of aeciospores and uredospores was observed. In both the Australian and the American work, mycelial growth was somewhat lim i t e d and did not spread very f a r from the o r i g i n a l seeding. Growth appeared to be slow and always occurred i n close proximity to the inoculated zone. Later 14 mycelium produced from seeded uredospores formed a stroma-l i k e structure as a res u l t of submerged growth i n agar. How-ever, the system used f o r axenic culture seems to be a great advancement on the ti s s u e culture systems described above and may lead to the culture of other fungal obligate para-s i t e s . In f a c t , Turel (1969) has been successful i n growing f l a x rust, Melampsora l i n i (Pers.) Lev, Race No. 3 using the same direct-seeding system mentioned above but on a d i f f e r e n t medium. She used a modified Knop's medium plus chelated iron and 0.1% Difco yeast extract. Growth of the mycelium was better when sucrose was used instead of glucose and rvjHPO^  was added to the medium. Only a small percentage of the colonies grew but sporulation occurred along with the forma-t i o n of abnormal structures i n the culture. An increased concentration of yeast extract and a reduction of the sugar concentration supported more sporulation. Turel r e a l i z e d the eff e c t of environmental factors i n determining the successful establishment of growth i n that a high temperature during seeding of the medium with uredospores reduced the percentage of growing colonies. The reports summarized above leave no doubt that the c u l t i v a t i o n of obligate parasites i n media containing inorganic s a l t s and an organic nitrogen source i s now f e a s i b l e . The answers to -questions concerning the nature, precise n u t r i t i o n a l requirement, e.g. mineral balance, and the method of maintaining saprophytic cultures of the rust fungi await further meticulous work. 15 MATERIALS AND METHODS A. Preparation of Inoculum Uredospores of stem rust , Puccinla graminis (Pers.) f . sp. t r i t l c i ( E r i k s s . & E. Henn.) race ANZ-126-6,7 were obtained from Dr. P.G. Williams, Department of Biochemistry, University of Sydney, A u s t r a l i a . The material obtained was a very small amount of le a f t i s s u e having a few dry pustules. V i a b i l i t y of the spores was found to be very low. L i t t l e Club wheat, Triticum aestivum s. sp. compacturn (host) Mackay was used throughout the experiments and gave the susceptible type IV reaction to the above mentioned Australian race of rus t . Wheat seeds (without any f u n g i c i d a l treatment) were sown i n a mixture of steam-sterilized loam s o i l , peat moss and sand (4:1:1) i n 8 inch pots or 16 x 8 x 4 inch wooden f l a t s . The plants were grown i n controlled environment cabinets at a l i g h t i n t e n s i t y of 500 f t - c on the plant surface f o r 16 hr and at 25 + IC followed by an 8 hr dark period at 19 ± IC. When the seedlings were about a week old, the f i r s t leaves were inocu-la t e d with a spore suspension i n d i s t i l l e d water. The plants were thoroughly sprayed with d i s t i l l e d water before and a f t e r a p p l i c a t i o n of the spore suspension to the leaves by hand. Inoculated plants were completely enclosed within a p l a s t i c bag to provide high humidity and incubated at 19C fo r 20-24 hr i n the dark. The bags were then removed and plants kept i n the 16 hr l i g h t regime (25 ± IC l i g h t and 19 + IC dark). Owing to the low v i a b i l i t y of the imported spore material, 16 only a few f l e c k s appeared a f t e r 10 days to give r i s e to uredopustules. However, successive inoculations from fresh uredospores raised i n t h i s fashion gave a progressively l a r g e r number of pustules per l e a f . After inoculation, i t . was found that fresh v i a b l e uredospores produced flecks a f t e r 7 days. Spores were collected a f t e r another week and stored at 5C. The v i a b i l i t y of these spores was tested from time to time and f r e s h l y produced spores were stored i n place of old ones. Spore v i a b i l i t y dropped 10% i n a month. B. Aseptic Leaf Culture to Obtain Uncontaminated Uredospores 1. Surface s t e r i l i z a t i o n Wheat leaves inoculated i n the manner described e a r l i e r showed d i s t i n c t whitish f l e c k s with a rudimentary pustule i n the centre under the epidermis a f t e r 6 to 7 days. These leaves were harvested and cut into 5 mm pieces and surface s t e r i l i z e d i n one of the following ways: ( i ) Mercuric chloride solution (0.001$ solution) - the le a f pieces were washed for 1 min i n t h i s solution and then washed three times i n s t e r i l e d i s t i l l e d water. ( i i ) Freshly prepared calcium hypochlorite solution (1% solu-tion) - l e a f pieces were rinsed i n f r e s h l y prepared solution ( f i l t e r e d before use) for about 5 min followed by three changes i n s t e r i l e d i s t i l l e d water (Bushnell 1968). ( i i i ) Commercial "chlorox* or *Javex ? bleach (5$ available chlorine) - 10% bleach was used to rinse the leaf pieces i n 17 two successive changes (5 min each). A surfactant, 0.001$ Tween 80 (Polyoxyethylene (20) Sorbitan mono-oleate), was added to the bleach before surface s t e r i l i z a t i o n . These leaves were washed i n three changes of s t e r i l e d i s t i l l e d water (Williams et a l . 1966). Bleach was found to be more sui t a b l e and convenient, because mercuric chloride and calcium hypochlorite produced necrotic zones around the pustules a f t e r incubation on the tissue culture medium. Bleach was there-fore used i n a l l subsequent work. 2, Size of ti s s u e i n r e l a t i o n to uredos'pore production Leaf pieces, 5 mm i n length, produced small pustules, sometimes causing a clumping of the spores i n a pustule so that they did not separate r e a d i l y . This occurred consistently and may be due to the germination of some uredospores i n s i t u (Bushnell 1968). Less b a c t e r i a l contamination was observed when small l e a f pieces were used and the tissue remained green up to 10 days a f t e r excision. When complete leaves bearing f l e c k s were placed on t i s s u e culture medium larger pustules with dry uredospores were obtained. Unfortunately, b a c t e r i a l contamination was greater and the system was therefore hard to handle. When complete leaves were placed i n the culture bottles with one end dipped into the medium, dry uncontaminated uredo-spores xirere produced but manipulation of these spores was found to be d i f f i c u l t i n the absence of a suitable inoculating hood. 18 The method f i n a l l y adopted was to use l e a f pieces about 3 cm long with one end inserted into the medium. Bac-t e r i a l contamination was minimal and uredosori were of reasonable s i z e and produced dry uredospores. The successful manipulation of these l e a f cultures was only possible a f t e r the laminar ai r f l o w bench became av a i l a b l e . This equipment provides an aseptic environment. 3. Tissue culture media Two tissue culture media were prepared according to the formulae given below: ( i ) Medium I - Modified Knop's suggested by Turel (1969) Ca(N0 3) 2 . 4H 20 KN03 MgS0 4 . 7H 20 KH PO, 2 4 NH4N03 Adenine sulphate Cysteine HC1 Thiamine Indole acetic acid Sucrose Coconut milk Berthelot solution Agar 1 . 5 0 g 0 . 2 5 0 g 0.250 g 0 . 2 5 0 g 0 . 0 2 0 g 0 . 0 1 0 g 0 . 0 1 0 g 0 . 0 0 1 g 0 . 1 g 4 0 g 1 0 0 ml (see below) 1 0 drops 1 5 g made up to 1 l i t e r with d i s t i l l e d water 19 Berthelot solution f o r trace elements (Gautheret 1942). H 20 200 ml F e 2 ( S 0 4 ) 3 10 g MnSO^ . H 20 0.448 g Kl 0.01 g M i C l 2 . 6H 20 0.018 g CaCl 2 . 6H 20 0.018 g Extraction of Coconut Milk The coconut milk used i n medium I was obtained from r i p e coconuts purchased from the l o c a l market. This was f i l t e r e d , heated up to 80C f o r 40 min i n a water bath, cooled to room temperature, and f i l t e r e d again. I f t u r b i d i t y persisted i n the preparation a f t e r cooling overnight i n the r e f r i g e r a t o r (at 5C), another f i l t r a t i o n was done to obtain a clear l i q u i d devoid of a l l p a r t i c l e s . This preparation was stored at -15C f o r routine use. ( i i ) Medium II - White's tissu e culture medium (White 1942). Salts Mg/Liter MgS03 360.0 Ca(N0 3) 2 200.0 KNO^ 80.0 KC1 65.0 NaH 2P0 4.H 20 16.5 F e 2 ( S 0 4 ) 3 2.5 MnSO^ 4.5 ZnSO^ 1.5 H 3B0 3 1.5 20 Medium II (Continued) Salts Mg/Liter KI 0.75 Sucrose 20,000.0 Glycine 3-0 Nicotinic Acid 0.5 Pyridoxine 0.1 Thiamine 0.1 Solidified with 1.5% agar (Difco Bacto). Both media were autoclaved for 15 min at 15 psi. In both the f i n a l pH was 5.2r-5.4 after autoclaving. 4. Incubation of aseptic leaf culture Inoculation of the media with surface-sterilized leaf-pieces was performed under aseptic conditions either inside a hood or in a horizontal laminar airflow cabinet. Plastic petri dishes about 9 cm in diam containing 15 ml of media were used for the purpose. Petri dishes were loosely covered and arranged on wooden trays. Each tray, size 20 x 15 x 5 inches, having 1/2 inch anhydrous CaSO^ (Drierite) at the bottom was com-pletely enclosed by a piece of polyethylene sheet to ensure dryness. Enclosed trays of petri dishes were incubated at 17C under white fluorescent light (350-400 f t - c ) . The system used for aseptic leaf culture and subsequent axenic culture i s rep-resented schematically in Figure 1, 21 Figure 1. Schematic representation of the system used i n c u l t u r i n g rust fungi. A. ^ e a t seedling with rust i n f e c t i o n at the f l e c k i n g stage. B. Aseptic l e a f culture - Surface s t e r i l i z e d l e a f pieces (3 cm long) with rudimentary uredia placed on tissue culture medium i n p e t r i dishes. P e t r i dishes were enclosed i n t r a y containing d r i e r i t e for incubation under l i g h t . C. Uncontaminated uredospores produced from uredia were suspended i n 15% gelatin to make inoculum. D. Inoculation of p e t r i dishes by uredospore suspension using a Pasteur pipette. E. Sealed p e t r i dishes wrapped i n aluminum f o i l for incubation i n dark. F. Rust colonies developed on s o l i d media a f t e r one month. 22 c . C u l t u r a l Studies The basic culture medium used i n d i f f e r e n t experiments was prepared according to a standard formula given below. P r i o r to the addition of agar and s t e r i l i z a t i o n the pH of the medium was adjusted to 6.4 using a Beckman glass electrode pH meter (Bushnell 1968). Glucose was used to replace sucrose i n Czapek»s medium as suggested by Williams (see Bushnell 1968). The composition of the medium was 'as follows: Glucose 30 g NaN03 2.0 g KH 2P0 4 1.0 g MgS0 4.7H 20 0.5 g KCl 0.5 g FeS0 4.7H 20 0.01 g D i s t i l l e d water 1000 ml Stock solutions (Czapek Dox) were prepared as follows: Solution A NaN03 200 g / l Solution B KCl 50 g / l MgS04.7H20 5 0 g / l Solution C KH 2P0 4 100 g / l Solution D FeS0 4.7H 20 1.0 g / l The medium was prepared by mixing the following: Glucose 30.0 g Solution A 10 ml Solution B 10 ml Solution C 10 ml Solution D 10 ml D i s t i l l e d water 1 l i t e r The constituents were dissolved i n glass d i s t i l l e d , deionized water and 1.5$ Difco Bacto agar was added f o r s o l i d i f i c a t i o n . The medium was then placed i n a b o i l i n g water bath to dissolve the agar. 23 Flasks of solid medium were sterilized at 121C and 15 psi for 20 min. After s t e r i l i z a t i o n , the medium was cooled to 45C and poured into either glass or disposable plas-tic, s t e r i l e petri dishes (internal diam 9 cm approximately). Medium prepared from stock solution was inoculated with spore suspensions in the large scale experiments. 1. Addition of yeast extract, peptone, sodium citrate and pectin Difco yeast extract 0.1$ w/v or 0.1$ w/v Evan's peptone (Evan's Medical Ltd., Speke, Liverpool, England) was always added to the medium before f i n a l adjustment of the pH. Sodium citrate 0.2$ w/v and pectin 0.2$ w/v were added before s t e r i l i z a t i o n . 2. Addition of bovine serum albumin Bovine serum albumin fraction V from various commercial sources viz. Nutritional Biochemicals, Commonwealth Serum Lab-oratory and Armour Chemical Company was defatted before use (Chen 1966). A weighed sample was dissolved in about 10 times (w/v) i t s amount of d i s t i l l e d water by s t i r r i n g with a magnetic s t i r r e r at room temperature, and mixed with l/2 i t s weight of solid Darco Charcoal. The mixture was stirred for 30 min i n an ice bucket after adjusting the pH to 3.0 with IN HC1. The charcoal was removed by centrifugation for 1 hr at 13,000 rpm (20,000 g). The supernatant (defatted BSA) was decanted and 24 i t s pH readjusted to pH 7.0 with IN NaOH. A pressure m i l l i -pore f i l t e r s i z e 0.22 u. was used to s t e r i l i z e the solution as an ordinary m i l l i p o r e f i l t r a t i o n denatured the protein. P r e s t e r i l i z e d defatted BSA was added a s e p t i c a l l y into the molten medium at exactly 45C to avoid any p r e c i p i t a t i o n , and to give a f i n a l BSA concentration of 1% w/v. The BSA (Cal-biochem., Ohio) was added to basic medium both with or without yeast extract. Twenty plates were poured f o r each batch of medium, A single zone was seeded on each p e t r i dish and the p e t r i dishes were examined a f t e r 4 weeks incubation at 170 i n darkness. Defatted BSA (Fraction V) from three d i f f e r e n t commer-c i a l sources was added separately to the basal medium of Czapek*s minerals and peptone. The eff e c t of yeast extract, pectin and sodium c i t r a t e when added separately or i n combina-t i o n to the basic medium plus defatted BSA (Armour), was examined i n the following media:-1. Czapek's minerals with peptone and BSA (Armour) and yeast extract (Difco). 2. Czapek's minerals with peptone and BSA (Armour) and 0.2% sodium c i t r a t e ( N u t r i t i o n a l Biochemicals). 3. Czapek's minerals with peptone and BSA (Armour) and 0.2% pectin ( N u t r i t i o n a l Biochemicals). 25 4. Czapek's minerals with peptone and BSA (Armour) and 0.1$ yeast extract, and 0.2$ sodium citrate and 0.2$ pectin. 5. Czapek's minerals with peptone and BSA (Armour) and yeast extract and 0.2$ sodium citrate. 6. Czapek's minerals with peptone and BSA (Armour) and 0.2$ sodium citrate and 0.2$ pectin. 7. Czapek's minerals with peptone and BSA (Armour) and yeast extract and 0.2$ pectin. Eighteen petri dishes from each treatment were seeded with a gelatin spore suspension and two plates with water sus-pension. Each droplet of spore suspension spread to form a circular zone measuring about 1 cm in diam by .orle,.:houroafter inoculation. The spore density was estimated under the micro-scope and ranged from 20-40 spores per mm . A sample batch containing one petri dish from each treatment was examined after 6 days and the remainder was kept undisturbed for exam-ination after incubation for 28 and 50 days. 3. Addition of sterols to the medium Five sterols v iz. ergosterol, stigmasterol, / ? - s i t o -sterol , cholesterol and sterol acetate (Nutritional Biochemi-cals) were separately added to the medium. Twenty mg of each sterol were dissolved in 1000 ml of diethyl ether and 2 ml of this solution (0.04 mg of sterol) were dispensed on the surface of 25 ml of Bushnell's medium (Bushnell 1968). The ether was allowed to evaporate overnight. A very thin deposition of 26 s t e r o l was thus obtained on the surface of the medium (Hendrix 1964). In order to provide a s t r i c t comparison f i v e control p e t r i dishes were s i m i l a r l y treated with ether. For each s t e r o l , f i v e p e t r i dishes were inoculated i n each of two aon.es and incubated undisturbed for 4 weeks. 4. Addition of host extract Leaves and stems from 1 week old wheat plants were cut i n t o small pieces and 250 g quantities were boiled i n 300 ml of d i s t i l l e d water f o r 30 min and f i l t e r e d . The-extract so obtained was made up to 1000 ml by adding the basic medium (Czapek's minerals, glucose and peptone). Agar (1.5$) was added f o r s o l i d i f i c a t i o n and the medium was autoclaved at-121C for 20 min. F i f t y p e t r i dishes were prepared, inoculated and incubated at 17C and examined a f t e r 4 weeks. 5. Addition of wheat l e a f f r a c t i o n s A l l operations were carried out at 0-4C. One week old plants were cut into pieces and homogenized with t r i s - s u c c i n a t e buffer (.05M, pH 7.0). The homogenate was f i l t e r e d through cheese clo t h to remove the plant debris. The f i l t r a t e thus-obtained was centrifuged at 13,000, rpm (20,000 g) f o r 2 hr. The p e l l e t (A) was extracted with buffer (.05M t r i s , pH 7.0) overnight and the soluble f r a c t i o n was used. An aliquot of the supernatant l i q u i d (B) was taken and protein was p r e c i p i -tated by slowly adding s o l i d (NH^gSO^ to a f i n a l concentra-t i o n of 40$. The mixture was allowed to stand f o r 30 min and then centrifuged at 13,000 rpm f o r 30 rain. The p e l l e t obtained 27 (C) was saved f o r further treatment. The supernatant l i q u i d was brought to 60$ (NH^^SO^ as above and was then c e n t r i -fuged f o r 30 min at 13,000 rpm (20,000 g). The p e l l e t (D) so obtained was saved along with supernatant (E); Fractions C, D, and E were dialyzed overnight against 4 l i t e r s of 0.00LM t r i s buffer at pH 7.0, with one change of buffer (see F i g . 2). Equal amounts (dry weight) of a l l the f r a c t i o n s were used i n the experiment. Various fractions were added to the basic medium (Czapek's minerals and peptone) at the rate of 1 mg/ml either before or a f t e r autoclaving. In each series replicated p e t r i dishes were used f o r each l e a f f r a c t i o n and p e t r i dishes containing basic medium only were used as controls. Treated and control p e t r i dishes were inoculated and incubated using normal procedures. D. Inoculation 1. Dry seeding - Uncontaminated dry uredospores produced as previously described were c a r e f u l l y taken onto a flattened i n o c u l a t i n g needle, which had been f l a m e - s t e r i l i z e d and cooled i n the medium to be inoculated. Care was taken not to touch the host tiss u e which i n v a r i a b l y contains bacteria. Two or four zones were seeded by gently swabbing the medium with the needle. 28 Figure 2. Flow sheet f o r wheat l e a f f r a c t i o n s . Wheat le a f with Tris-succinate homogenate buffer ( 0 . 0 5 M , pH 7 . 0 ) Cheese clo t h f i l t r a t i o n F i l t r a t e Centrifuge P e l l e t (A) (Extracted with buffer 0 . 0 5 M , pH 7 . 0 ) ( T r i s -succinate) overnight. S o l . Fraction used). 0-P e l l e t (C)< Supernatant (B) i b ammonium sulphate centrifuge Supernatant 40-P e l l e t (D) ammonium sulphate centrifuge Supernatant (E) Pe l l e t s C, D, and E were dialyzed overnight against 4 l i t e r s 0.05M, pH 7.0 (tris-succinate) buffer (changed once) before use. Fractionation was done at 0-4C. 29 2. Spore suspension inoculation - Either d i s t i l l e d water or 15% (w/v) g e l a t i n (Baker & Adamson) solution i n d i s t i l l e d water was s t e r i l i z e d i n the autoclave and cooled i n a r e f r i g -erator f o r making spore suspensions. Uredospores were c o l -l e c t e d by means of a flattened needle from 6-7 well developed pustules without touching the host tissue and placed i n a 10 ml conical f l a s k . A small quantity of water or g e l a t i n was added and the spore suspension prepared by s t i r r i n g with a cotton swab ( F i g . I c ) . Cotton-plugged, p r e - s t e r i l i z e d and cooled Pasteur pipettes were used to inoculate about 10 zones per p e t r i dish with approximately 0.5 ml of spore suspension. The inoculated zones were allowed to dry by leaving the covered, unsealed p e t r i dishes undisturbed for 1 hr i n the laminar a i r flow bench. E. Incubation P e t r i dishes were incubated i n a moist atmosphere (Desiccator with water i n the bottom section) and wrapped with aluminum f o i l at 17 + 0.1C. Contamination was reduced by sea l i n g the edge of each p e t r i dish with masking tape and wrapping the dishes i n batches of s i x i n aluminum f o i l f o r incubation i n the dark at 17 ± 0.1C ( F i g . I e ) . The p e t r i dishes were kept undisturbed as f a r as practicable f o r about 2 weeks. 30 F. Disposal of Materials Used plant materials, instruments and petri dishes were autoclaved at 15 psi for 20 min before disposal to pre-vent any possibility of the spread of the Australian race of rust in North America. G. Microtechnique 1. Fixation Pieces of the colonies were cut out of the agar and fixed in formalin-acetic acid-alcohol (50$ ethyl alcohol -90 ml, glacial acetic acid - 5 ml, commercial formalin - 5. ml), or in Carnoy's fixative (absolute alcohol - 3 :, glacial acetic acid - 1 : chloroform - 2 ) . Fixation times were varied between 24 and 36 hr in Carnoy's fixative at room temperature. 2. Embedding, sectioning and mounting After fixation, the tissue was thoroughly washed with running tap water. Fluffy mycelium was stained without em-bedding and sectioning. The stroma tissue was dehydrated in an alcohol series as follows: 20 min each of 10$, 30$ and 50$ ethanol. It was cleared and in f i l t r a t e d for 2 hr in each of five concentrations of the tertiary butyl alcohol series (50$, 70$, 85$, 95$ and 100$) prepared according to Jensen (1962). The stroma tissue was then transferred to 100$ tertiary butyl alcohol and l e f t for 4 hr before paraffin i n f i l t r a t i o n and embedding. 31 The t i s s u e was placed on the surface of a small v i a l of warm s o l i d i f i e d p a r a f f i n (melting range 5 4 - 5 6 C ) i n a mini-mal volume of the normal butanol-paraffin o i l mixture. The p a r a f f i n was allowed to melt i n the v i a l on the top shelf of an oven set at 5 6 + 2 C , As the p a r a f f i n melted, the tissue sank to the bottom of the v i a l a f t e r 5 hr. The p a r a f f i n was changed three times during 2 4 hr. The tissue was embedded i n p a r a f f i n i n p l a s t i c p e t r i dishes to f a c i l i t a t e hardening at 5C. A rotary microtome was used to cut sections 1,0 to 15 u. thick i n either the v e r t i c a l or horizontal plane of the t i s s u e . Sections were affixed to microscope s l i d e s using standard methods. 3. Staining ( i ) Sections selected f o r stroma staining were hydrated i n the following se r i e s ; 1 0 minin xylene, 5 min xylene-absolute ethanol (1:1); 5 min i n each of two changes of absolute ethan-o l and one change i n 9 5 $ ethanol, and 5 min i n each of 80$, 60$, 5 0 $ and 3 0 $ ethanol, followed by two rinses i n d i s t i l l e d water. Slides were stained i n a solution of 0 . 5 $ aqueous c r y s t a l v i o l e t (Jensen 1 9 6 2 ) . After dehydration i n an as-cending alcohol series, the stained sections were mounted i n permount or i n immersion o i l for microscopic observation. 32 ( i i ) For hyphal s t a i n i n g the s l i d e s were precoated i n ge l a t i n adhesive (Jensen 1962) and dried. F l u f f y mycelium fi x e d i n the Carnoy's f i x a t i v e was teased out c a r e f u l l y on a drop of water to separate the hyphae. The s l i d e s were allowed to dry at room temperature, leaving hyphae f i r m l y a f f i x e d to the glass. The s l i d e s were then placed i n d i s t i l l e d water at 60C f o r 5 min, hydrolyzed 10-12 min i n IN hydrochloric acid at 60C, rinsed three times i n d i s t i l l e d water and stained i n Feulgen reagent, prepared according to Swift (1955). The s l i d e s were then treated f o r 20 min with two changes of a c i d i f i e d potassium metabisulfite (Swift 1955). After a b r i e f water r i n s e , the s l i d e s were dehydrated to xylene. The material was f i n a l l y mounted i n immersion o i l . The following technique proved to be more sui t a b l e . The f l u f f y mycelium was teased out on a gel a t i n coated cover s l i p and stained as follows (Henderson and Lu 1968). Haema-t o x y l i n (2$ w/v) i n 50$ propionic acid was mixed with 0.5$ (w/v) i r o n alum ( f e r r i c ammonium sulfate) solution i n 50$ pro-pionic acid (1:1). The mixture was allowed to age f o r 24 hr at room temperature. After the hydrolysis of the material on a cover s l i p i n IN HC1 at 60G f o r 8 min, a drop of st a i n was applied to i t . The covers l i p was then inverted on a clean s l i d e and allowed to s i t f o r 5 min. The material was squashed c a r e f u l l y without breaking the coverslip before examination under microscope. 33 4 . Photomicrography Photomicrographs were taken with a Carl-Zeiss photo-microscope, using either a bright f i e l d or a phase contrast o i l immersion lens and built in a 3 5 mm camera. Improved resolution was obtained by using Ilford Pan-X black and white film rather than Polaroid 4 x 5 black and white, type 5 5 P/N. 34 E X P E R I M E N T A L R E S U L T S S E C T I O N 1. E f f e c t o f v a r i o u s o r g a n i c c o m p o u n d o n i n v i t r o d e v e l o p m e n t o f r u s t u s i n g d r y s e e d i n g t e c h n i q u e s . T h i s s e c t i o n d e s c r i b e s t h e e f f e c t o f a d d i n g v a r i o u s o r g a n i c c o m p o u n d s t o t h e b a s i c m e d i u m o n i n v i t r o d e v e l o p m e n t o f t h e r u s t f u n g u s . B a s i c m e d i u m c o n t a i n i n g C z a p e k ' s m i n e r a l s , g l u c o s e a n d p e p t o n e a s a n o r g a n i c n i t r o g e n s o u r c e w a s c h o s e n a f t e r p r e l i m i n a r y e x p e r i m e n t a t i o n . U n l e s s o t h e r w i s e s t a t e d , t h e d r y s e e d i n g t e c h n i q u e w a s u s e d f o r i n o c u l a t i o n o f t h e p e t r i d i s h e s i n t h i s s e r i e s o f e x p e r i m e n t s . E x p e r i m e n t A . E f f e c t o f y e a s t e x t r a c t Y e a s t e x t r a c t (0.1$ D i f c o ) w a s a d d e d t o t h e m e d i u m c o n -t a i n i n g C z a p e k ' s m i n e r a l s a n d g l u c o s e . F o r t y p e t r i d i s h e s w e r e i n o c u l a t e d w i t h d r y , u n c o n t a m i n a t e d u r e d o s p o r e s a t e a c h o f t w o s i t e s . Two p e t r i d i s h e s w e r e e x a m i n e d m i c r o s c o p i c a l l y a f t e r 2 d a y s o f i n c u b a t i o n . G e r m i n a t i o n w a s a b o u t 90$ a n d t h e g r o w t h o f t h e g e r m t u b e s a p p e a r e d t o b e n o r m a l ( F i g . 3). G e r m t u b e t i p s w e r e f r e q u e n t l y s w o l l e n ( F i g . 4). A f e w g e r m t u b e s s h o w e d s p i r a l g r o w t h ( F i g . 5). E x a m i n a t i o n o f t h e r e m a i n i n g p e t r i d i s h e s a f t e r 4 w e e k s s h o w e d t h a t 20$ o f t h e p e t r i d i s h e s w e r e c o n t a m i n a t e d w i t h b a c t e r i a . W h e r e t h e r e w a s n o c o n t a m i n a t i o n , t i n y , f l u f f y w h i t e c o l o n i e s d e v e l o p e d i n t h e s e e d e d z o n e s ( F i g . 6). T h e s e c o n s i s t e d o f d e n s e c l u s t e r s o f b r a n c h e d a e r i a l h y p h a e . When a s i n g l e h y p h a w a s e x a m i n e d m i c r o s c o p i c a l l y , i t w a s f o u n d t o b e s e p t a t e ( F i g . 7). On f u r t h e r i n c u b a t i o n t h e s e 35 Figure 3. Germination of uredospores on Czapek's medium containing yeast extract. Polaroid photograph after 2 days incubation (X 520). Figure 4. Germ tube showing a t y p i c a l swollen t i p on Czapek*s medium containing yeast extract. Photographed a f t e r 2 days of incubation (X 520). Figure 5. Germ tube showing s p i r a l growth on Czapek fs medium containing yeast extract. Photo-graphed a f t e r 2 days of incubation (X 480). Figure 6. Rust mycelium formed on Czapek's minerals and yeast extract medium. Photographed after 4 weeks of incubation (X 7 5 ) . Figure 7 . A hypha from the colony in Fig. 6 showing septation. Polaroid photograph (X I 6 5 6 ) . 38 colonies showed a tendency to collapse and never resumed growth. These observations p a r t l y confirm those of Williams et a l . ( 1 9 6 6 ) . Experiment B. E f f e c t of Evan Ts peptone Evan's peptone (0.1$ was added to the medium instead of yeast extract. After incubation f o r 4 weeks at 17C i n dark-ness, mycelial colonies (2 mm^ ) were found i n every inoculated zone. The a e r i a l mycelium was white, f l u f f y , and velvety at the surface (Fig. 8) but colonies were yellowish brown on the underside where they were i n contact with the substrate. In a few instances, undifferentiated germ tubes were found adjacent to mycelial colonies. Only a few germ tubes had swollen t i p s or showed s p i r a l growth. These never branched and showed no ten-dency to develop further. Colonies i n v a r i a b l y formed i n those places where the spore density was very high (spores i n contact with each other). After an additional two weeks, there was no further growth of a e r i a l hyphae and no increase i n diameter of the colonies. However, a leathery stroma of compact, intermeshed mycelium developed just below the surface of the medium i n the seeded zones. These stromata were i n i t i a l l y a l i g h t yellow i n color but turned dark brown by 8 weeks a f t e r seeding. Cross-sections of the stroma showed compact central pigmented, septate mycelium bordered by hyphae, which bore some resemblance to sporophores. C r i t i c a l examination did not reveal the forma-3 9 Figure 6*. Profuse mycelial growth on Czapek's medium containing Evan's peptone. Photographed after 4 weeks incubation (X 5 8 ) . 40 t i o n of spores at the t i p s of these hyphae. They did how-ever bear smooth-walled globose bodies which were much smaller than uredospores. Experiment C. E f f e c t of unfractionated host extract Microscopic examination of p e t r i dishes containing unfractionated host extract showed that about 90% of the uredospores developed normal germ tubes. However, growth was i n h i b i t e d at t h i s point and no a e r i a l hyphae were formed. This r e s u l t suggests that a substance or substances i n the leaf ex-t r a c t i n h i b i t e d growth a f t e r germination had occurred. Experiment D. E f f e c t of wheat le a f protein fra c t i o n s After incubation f o r 4 weeks at 17C i n darkness, the control p e t r i dishes containing only the basic medium developed f l u f f y white colonies. B a c t e r i a l contamination developed on those p e t r i dishes containing l e a f f r a c t i o n s which had not been autoclaved. No contamination developed on those con-t a i n i n g leaf f r a c t i o n s which had been autoclaved with the medium. Although uredospore germination (about 90%) occurred normally, no colonies developed i n the presence of any l e a f f r a c t i o n . Seven weeks a f t e r the o r i g i n a l seeding, the f l u f f y colonies on. basic medium alone shoxved evident collapse and contained degenerating hyphae. Examination of the p e t r i dishes containing basic medium plus tiss u e fractions showed that the germ tubes never developed into vegetative hyphae. These re s u l t s show c l e a r l y that the l e a f protein f r a c t i o n s 41 i n h i b i t e d rust development a f t e r germination had occurred. Experiment E. Ef f e c t of bovine serum albumin. After 4 weeks incubation, compact, c i r c u l a r colonies with entire margins were observed on a l l p e t r i dishes, both i n the presence and absence of yeast extract. In the pre-sence of yeast extract the colonies were l i g h t yellow at the centre and white at the periphery. The amount of a e r i a l growth was judged from v i s u a l observation to be greater than i n the treatments used i n the experiments described e a r l i e r . How-ever, there were no apparent differences i n the amount of growth i n the presence or absence of yeast extract. When another observation was made 40 days a f t e r the o r i g i n a l seed-in g , a leathery stroma was found to have developed. At t h i s time the a e r i a l mycelium showed a tendency to collapse. Stroma sections ( F i g . 9) did not reveal any sporulation but did show the presence of small globular bodies i n the colony. Staining with the Feulgen reagent showed that the septate my-celium was dikaryotic and highly vacuolated ( F i g . 10). How-ever, the r e s u l t s obtained with the Feulgen reagent were not considered to be p a r t i c u l a r l y s a t i s f a c t o r y because Feulgen p o s i t i v e p a r t i c l e s appeared to be generally d i s t r i b u t e d throughout the cytoplasm. 42 Figure 9. Cross section of the stroma produced i n basic medium containing PSA from dry seeding (X 150). Figure 10. A hypha stained with Feulgen reagent showing two nuclei at centre f i e l d . Note additional Feulgen p o s i t i v e s t a i n i n g p a r t i c l e s within the hypha. Phase contrast (X 1294). 43 Experiment F. Effect of sterols A l l petri dishes were examined after 4 weeks. Ergo-sterol and sterol acetate did not inhibit germination but prevented further development and the formation of hyphae. The aseptate germ tube found in the presence of these sub-stances frequently showed spiral growth with swollen ti p s . Normal colonies developed on the control petri dishes and in the presence of stigmasterol, -sitosterol and cholesterol. Globular bodies were formed on the marginal hyphae in the pre-sence of /Q -sitosterol. These bodies were smooth-walled, unpigmented and about 10 p. in diameter i.e. about half the size of normal uredospores (Fig. 11). By 6 weeks after seed-ing colonies on the control petri dishes developed a stroma. It was noted in this experiment that colonies developed from high density spore clumps. Normally, colonies did not develop from uredospores in areas of low density, unless they were close to a colony developing from a high density clump. After 6 weeks, about 30$ of the sterol-containing petri dishes were contaminated with bacteria. Rust colonies on un-contaminated petri dishes containing stigmasterol, -sito-sterol or cholesterol showed degeneration of the hyphae with the development of a small stroma but no spore formation. Thus the addition of the sterols used did not promote sporula-tion. Figure 11. Globular bodies on a e r i a l hyphae i n the basic medium containing z? - s i t o s t e r o l (x 2 6 0 ) . r 45 SECTION 2. E f f e c t of spore suspension techniques on growth and development of r u s t . This section describes the effect of yeast extract, pectin and sodium c i t r a t e on i n v i t r o growth of rust when added separately or i n combination to the basic medium plus defatted BSA. The p e t r i dishes were inoculated with spore suspensions, rather than by dry seeding. A l l the re s u l t s are summarized i n Table I. After 6 days, the germination of uredospores i n both water and g e l a t i n suspension was above 95$. Spore d i s t r i -bution was found to be d i f f e r e n t i n the two suspensions. Gelatin suspensions gave a more uniform spore d i s t r i b u t i o n than did water, i n which there was greater clumping of spores as shown i n Figures 12 and 13. The f i r s t observation, a f t e r 2& days, revealed that water inoculation supported no growth i n any treatment (Table I, F i g . 14b). Only a few colonies appeared i n those places where spores were clumped together ( F i g . 15). With g e l a t i n , however, luxurious growth as shown i n Figures 14a and 16 was obtained. Growth varied s l i g h t l y according to the treatments ( i e . media employed). The best growth was supported by basic medium plus BSA ( i e . treatments i , 2, & 3 i n Table I) and by basic medium plus BSA and yeast extract ( i e . treatment 4). Figure 17 i l l u s t r a t e s t y p i c a l luxuriant colonies which developed on these media. There were no differences i n growth (as judged by v i s u a l estimation) with the d i f f e r e n t samples of BSA ( i e . between treatments 1, 2 & 3). Addition of sodium c i t r a t e Table I, Effec t of spore suspension inoculation technique on the development of rust on media containing various organic s a l t s . Treat-ments Organic Compounds Yeast Sodium Pectin Extract C i t r a t e Gelatin spore suspension Growth a f t e r Growth a f t e r 28 days 5 0 days Dis- Undis- Dis- Undis-turbed turbed turbed turbed Water spore sus-pension Growth a f t e r 28 days 1 Basic medium + BSA (General Biochemicals) 2 Basic medium + BSA (Armour Pharmaceutical) 3 Basic medium + BSA (Commonwealth Serum Lab) 4 Basic medium + BSA(Armour) 5 Basic medium + BSA(Armour) 6 Basic medium + BSA(Armour) 7 Basic medium + BSA(Armour) 8 Basic medium + BSA(Armour) 9 Basic medium + BSA(Armour) 1 0 Basic medium + BSA(Armour) + + + + + + + + + + Organic compounds present - Organic compounds absent ++ F l u f f y mycelium growth present — F l u f f y mycelium growth absent +++ Stromata present. * Disturbed - means examined for germination at 6 days, ++ ++ ++ +++ +++ +++ ++ ++ ++ -p-47 Figure 12. Typical clumping of spores on the medium surface when water suspensions of uredo-spores were used to inoculate petri dishes (X 124). Figure 13. Uniform distribution of uredospores on the medium surface when gelatin suspensions of uredospores were used to inoculate petri dishes (X 124). WM Figure 14. Growth of mycelium on basic medium con-taining yeast extract and BSA. (a) Gelatin spore suspension inoculation, (b) T , Tater spore suspension inoculation. Figure 15. Petri dish showing two colonies developed on basic medium containing BSA where spores were clumped together from water suspension inoculation. 49 and pectin either alone (Treatments 5 & 6) or in combination with yeast extract (Treatments 7 & 10) caused inhibition of growth. Inhibition by sodium citrate and pectin was found to be stronger in the presence of yeast extract (Treatments 8, 9, and 10). Samples of disturbed petri dishes did not show the development of mycelium in any treatments (Fig. 16). When the petri dishes were examined after 50 days, marked changes in the character of the colonies was noted. On media containing BSA and BSA plus yeast extract (Treatments 1, 2, 3, and 4) the f l u f f y aerial mycelium had collapsed completely and a dark brown stroma had developed under the seeded circular zone. A peculiar feature noted was the diffusion of a brown pigment (alcohol soluble) into the agar around the colonies (Fig. 14a). This phenomenon was more evident in the media with yeast ex-tract (Treatment 4). Another interesting feature was that, wherever pectin was present, there developed a luxurious f l u f f y aerial mycelium which was white or light yellowish in color (Treatments 6, 7, 9, and 10) (Fig. 16b). Growth was never resumed when sodium citrate was used alone in the medium (Treat-ment 5). Samples from petri dishes which were examined in order to record germination (6 days after seeding) showed an inter-esting feature when the second observation was made after 50 days. There was no additional growth in media containing pectin (Treatments 6, 7, 8, 9, and 10), but where BSA alone (Treatments 1, 2, and 3) and BSA with yeast extract (Treatment 4) were used there was a complete recovery of growth. Colonies on these media attained f u l l size and produced a f l u f f y aerial mycelium (Fig. 16c). 50 Figure 16. Effec t of adding pectin to the basic medium containing yeast extract and BSA. Photo-graphed a f t e r 50 days. a,b - pectin added c ,d - pectin omitted a, c - disturbed a f t e r 6 days b. d - undisturbed during incubation. Figure 17. Colony development, 28 days a f t e r the basic medium plus BSA was inoculated with a gela-t i n spore suspension. 51 Microscopic observations were made 60 days afte r i n -cubation. At the periphery of the colonies the mycelium showed l i t t l e tendency to invade the medium. Rather, there were formations of small i r r e g u l a r l y shaped bodies arranged i n a chain. These bodies were hyaline, lacked verrucose walls, and ranged i n size from 5-10 u. i n diameter ( F i g . 18). Stroma sections were made and examined f o r possible spore formation. With the exception of some i r r e g u l a r bodies no f r u i t i n g struc-tures were found ( F i g . 19). When stroma pieces were teased and examined c a r e f u l l y , a group of pyriform, spore-like bodies were observed ( F i g . 20). These were very infrequent i n occur-rence. Such bodies were more or less hyaline i n color, devoid of any pigmentation, and lacked the d i s t i n c t sporophore of a t p y i c a l uredospore, produced i n vivo. However, some of them were s i m i l a r i n s i z e to uredospores and ranged from 26-36 p, i n length and 18-26 u. i n width. Iron haematoxylin preparations showed that the mycelium was septate with d i s t i n c t septal pores, and dikaryotic with large prominent nuclei (Figures 21 & 22), 52 Figure 18. Series of i r r e g u l a r bodies around the gel matrix on basic medium containing BSA (X 96). 53 Figure 19. Cross section of the stroma developed on the basic medium containing BSA. Inoculated with a g e l a t i n spore suspension (X 134). Figure 20. Uredospore-like bodies formed near the stroma in the basic medium containing BSA (X 480). 55 Figure 21. Aerial hyphae from a colony showing d i -karyotic hypha, stained with iron naema-toxylin. Phase contrast (X 1462). Figure 22. A highly vacuolated terminal hypha showing a single nucleus in the terminal c e l l (X 1450). 56 DISCUSSION The uredial stage of an Australian isolate (ANZ 1 2 6 - 6 , 7 ) of wheat stem rust has been cultured on a medium containing Czapek*s minerals, glucose, and an organic nitrogen source. This confirms the findings of Williams et a l . ( 1 9 6 6 ) . A new finding, possibly one of great significance, i s the effect of gelatin in promoting the growth and development of the cultures of this isolate. Difco yeast extract (unfractionated) added to the basic medium promoted vegetative growth under conditions of high density seeding (Experiment A). It was emphasized by Williams* group that Czapek*s medium, although i t contains sodium nitrate as a source of nitrogen, never supported any growth in the ab-sence of yeast extract. In the present study, growth appeared to be vigorous and uniform when yeast extract was replaced by Evan*s peptone (Experiment B). This corroborates the finding of Bushnell ( 1 9 6 8 ) , who reported that yeast extract failed to support any growth of the same rust isolate in the United States. It i s possible that amino acids produced from peptone by auto-claving were utilized by the fungus. The particular peptone used i s described as a proteolytic digest of muscle and contains free amino acids, together with small and large peptides. It has, however, been suggested by Bushnell (personal communica-tion) that peptides are not required for the growth of certain North American rust races. He has found that, with certain 57 races of P. graminis t casamino acids promote growth in vitro i n the absence of peptone. There i s a number of other factors such as mineral balance, the physical properties of the medium and the method of inoculation which may affect the growth in vitro. In high density populations of heterotrophic organisms, variation in response to growth conditions i s a common phenomenon. Turel has recently (1969) reported the axenic culture of Melampsora l i n i (Flax rust) race No. 3. She found that several dozen spores were necessary for the establishment of a colony, though stimulation of growth was not achieved by further increasing the density of the inoculum. There i s no evidence to date of establishment of rust cultures as a result of single hyphal tip transfer or of the establishment of single spore cul-tures. This raises the question whether the amount of endo-genous growth substances present in the inoculum plays an im-portant role in the i n i t i a l development of mycelium. These substances have been shown important in various animal somatic c e l l s developing in tissue culture, and in several slow grow-ing Basidiomycetes and Phycomycetes. Thus, for example, a high density of zoospores of Phytophthora infestans i s re-quired to produce a colony even in the most favourable medium (Clarke 1966). Evidence of self-stimulation in germinating uredospores was reported by Ezekiel (1930), who found that anastomosis of adjacent sporelings resulted in longer germ tubes. Bushnell (1968) refers to a similar phenomenon, 53 resulting from the fusion of hyphae originating from di f f e r -ent uredospores, as the formation of 'growth centres* (Fig. B, Bushnell 1968). The effect of anastomosis on vegetative growth needs further cytochemical investigation. The addition of host extract (Experiment C) and various protein fractions (Experiment D) from susceptible host tissue allowed normal germination, but inhibited subsequent growth. The toxicity of juice from susceptible tissue to parasites has been reported by several earlier-workers (Anderson 1934, Johnstone 1931). Host extracts were found to be highly toxic to Pseudoperonospora humuli (Newton and Yarwood 1930). Yarwood (1956) has suggested that improved extraction procedures may however yield interesting results. In the current experiments germination frequently produced spiral germ tubes or germ tubes with swollen ti p s . Vegetative growth probably originates from germ tubes having a long polar growth without the formation of infection structures. This point needs further investigation. Williams et a l . (1966) reported the formation of infection structures in vitro with a very low and variable frequency. They did not distinguish whether the hyphal growth for the de-velopment of colonies arose from infection structures or un-differentiated germ tubes. The addition of BSA apparently increased the colony size, as found by Williams and Bushnell (personal communication). This occurred both in the presence and absence of yeast extract (Experiment E) but the stroma did not produce spores. There 59 are now several investigations which show clearly that serum protein can provide specific substrates or cofactors in limited quantities for the growth of isolated plant and animal c e l l s (Harris 1964). It has been established that sterols induce sporulation i n certain fungi ( E l l i o t t , Hendrix, Knights & Parker 1964, Haskins, Tulloch & Micetich 1964, Heridrix 1965, Leal, Friend & Holliday 1964). Addition of sterols to the basic medium (Experiment E) indicated that ^ - s i t o s t e r o l can induce the formation of globular bodies at hyphal tip s , but not the pro-duction of uredospores. It i s probable that physical factors, e.g. light, may have some effect on sporulation in the presence of sterol, but this was not investigated in the present study. The results in Section II show that the best vegetative growth of the rust fungus was obtained when a 15$ gelatin sus-pension of uredospores was used to inoculate a series of media, the basic constituents of which were 3$ glucose, Czapek's minerals, peptone, and 1$ bovine serum albumin. Inoculation of the same media with a water suspension of uredospores at the same density (20-40 uredospores per mm^ ) did not result in good vegetative growth, although nearly 100$ of the uredospores germinated. The role of gelatin could be either physical or chemical or a combination of both. It i s possible that essen-t i a l metabolites or stimulators produced by the sporeling are retained and utiliz e d by the fungus when gelatin suspensions 60 are employed but l o s t to the medium when water suspensions are used. Perhaps ge l a t i n i n some way protects the c e l l mem-branes of the germ tubes. This i s , of course, pure specula- . t i o n . Addition of sodium c i t r a t e at the concentration used (see Table 1) was found to be i n h i b i t o r y . Pectin retarded development of the mycelium. Moreover, the presence of pec-t i n completely i n h i b i t e d the growth i n disturbed plates. This indicates the existence of a c r i t i c a l phase i n growth when the fungus i s very s e n s i t i v e to physical factors l i k e l i g h t and temperature. Growth c h a r a c t e r i s t i c s were found to be s l i g h t l y d i f f e r e n t i n colonies produced by dry seeding and those pro-duced from g e l a t i n spore suspensions. A e r i a l mycelium developed on the g e l a t i n crust with the formation of a stroma, but very few fungal hyphae invaded the medium outside the gel matrix. Chains of i r r e g u l a r bodies formed outside the matrix i n con-ta c t with the medium. These phenomena could be attributed to the formation of abnormal hyphae. Globular bodies s i m i l a r to uredospores formed i n very limited numbers. These apparently d i f f e r somewhat from those observed by the Australian workers. Bushnell (1968) reported the formation of variable spore forms by the same race of rust i n axenic culture. The d i f f u s i o n of brown pigment was not recorded by either the Australian workers or by Bushnell. However, there are reports of oxidase l i b e r a t i o n into the medium by a number of Basidiomycetes. Further analysis of the medium should help i n the i d e n t i f i c a t i o n of the pigment. 61 The overall growth and development of rust in vitro was found to be very slow by comparison with growth in vivo as described by Allen (1923). A plausible explanation for this may be in the special physico-chemical properties of the intercellular environment' as well as the supply of labile nutrients by the congenial host. It i s believed by many workers that the failure of net protein and nucleic acid synthesis in germinating uredospores i s probably at the root of the failure of the rust fungi to grow in axenic culture. Such deficiencies in biosynthetic systems could arise from a number of biochemical or fine structural lesions (Brian 1967). Shaw (1967) has speculated that i f the nucleolus i s absent from mature uredospores or cannot be reformed after nuclear division during germination, then subsequent proliferation and develop-ment of the fungus may depend on stimulation from the host directed at synthesis of ribosomes and protein i n the fungus. From the overall developmental pattern of the rust colony in axenic culture, i t can be concluded that a net synthesis of DNA occurs during the development of hyphae, triggered by manipulations effective in inducing their development. The net synthesis of nucleic acids could result i n i t i a l l y from the pool of metabolites present in the uredospore inoculum when adequate physico-chemical conditions (i.e. composition of medium, presence of surface active agents, micro-environment) for mycelial development are provided. This i s in contrast with conclusions of Staples et a l . (1962), that germination of 62 urediospores i s not accompanied by net nucleic acid syn-thesis. Their conclusion, however, was based on germination under different conditions where subsequent growth and nuclear divisions were restricted. Once a colony has been established stroma pieces can proliferate in the absence of the original spores after transfer to favourable medium containing tissue protein. The most plausible interpretation of growth stimulation by tissue proteins (peptone or BSA) would be either the inhibition of, or the compensation for, metabolite loss from rust hyphae in v i t r o , although the exact mechanism remains to be determined. 63 SUMMARY 1. Solid media containing Czapek's minerals, 3$ glucose, and 0.1$ Difco yeast extract supported the vegetative growth of the uredial stage of Puccinia graminis t r i t i c i race ANZ 126-6,7. 2. Mycelial growth was improved when yeast extract was replaced by Evan's peptone. 3. Addition of host extract to the basic medium did not support any vegetative growth. Various protein fractions from host tissue were found to be Inhibitory. 4. Addition of BSA to the basic medium gave a larger colony in the presence or absence of yeast extract. 5. Addition of sterols (stigmasterol, ^ - s i t o s t e r o l , and cholesterol) failed to stimulate sporulation, but did support vegetative growth. Ergosterol and sterol acetate were found to be inhibitory. 6. A suspension of uredospores in gelatin solution (15$ w/v) when seeded on a range of media containing Czapek's minerals, glucose, peptone, BSA and other organics gave consistent luxurious vegetative growth at low density 2 seedings (20-40 spores per mm ). A water suspension of uredospores under identical conditions failed to support any growth on the same series of media. 64 Among various organic chemicals tested, sodium c i t r a t e was found to be i n h i b i t o r y and pectin increased the i n i t i a l l a g phase f o r the development of the colony. 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