STUDIES ON UROMYCES FABAE (PERS.) DE BARY PRODUCED IN AXENIC CULTURE (UREDOSPORES GERMINABILITY, ENZYMATIC ACTIVITY AND PATHOGENICITY)

Germinability of axenic uredospores of U. fabae isolates A and B was depended upon compsition of growth media. Percentag of germination was higher in uredospores of younger than older cultures and i t was inversed proportionally with spore concentration. Germination was better in 2% than 4 or 6% of sucrose or glucose solutions in case of uredospores produced o n MS-7 medium provided with extracts of broad bean leaves, meanwhile, uredospor e f rmed on MS-7 medium alone germinated better in 4 and 6% glucose solutio n than the same concentrations of sucrose. Dikaryotic germ tubes, sprouted like ye ast mycelium, oidiospores likestructures, germinated oidiospores, and infection s tructures i. e. appressoria, infection pegs, and infection hyphae were frequentl y observed during germination of uredospores. Isolates A and B were quitely varied in their abil ities to hydrolyzing starch and liquefying gelatin as well as in activities of pectulolytic and cellulolytic enzymes secreted in liquid growth media. The highes t activities of pectulolytic and cellulolytic enzymes were obtained at pH values 6 a nd 6.6, respectively. The activity of constitutive and induced pectulolytic a nd cellulolytic enzymes were higher in isolate A than isolate B. However, the r atio between induced and constitutive pectulolytic and cellulolytic enzymes of isolate A and pectulolytic enzymes only in isolate B were narrower in filtrate s of 14 and 21 compared with filtrates of 7 days old cultures. The opposite tren d was noticed, however, in isolate B. Under the IN VITRO conditions, the axenic uredospores of Uromyces fabae (Pers.) de Bary isolates A and B were able to infec t both broad bean calli forming intercellular septate mycelium and oidiospo res like structures and detached broad bean leaves forming oidiospores like structur es in their epidermal tissues, however, under greenhouse conditions the typical ru st pustule were frequently observed on inoculated leaves. Pathogenicity of ia lates A and B in relation to uredospore germination and activities of pectulolyt ic and cellulolytic enzymes was discussed. The isolate A produced few but larger co ncentric rust pustules surrounded by large numbers of minute pustules and clear yellowish halo zone meanwhile isolate B produced large numbers of small er rust pustules without yellowish halo. INTRODUCTION Resting or germinating spores, usually uredospores, have provided the only material for metabolic studies on the free-liv ing fungus ( Allen, 1965, Shaw, 1964, Staples and Wynn 1965). As sporelings have failed to initiate saprop hytic growth, most of these studies have been restricted to investigating biochemical and physiological events associated with spore germinat io and germ tube elongation. Annals Of Agric. Sc., Moshtohor, Vol. 37 (3): 1677-1693 (1998) 1678 However, if failure of the rusts to grow axenically were associated with a metabolic block in an essential pathway, it was hoped that th ese studies would reveal it. Because the axenic culture of an obligate parasite i an interesting biological problem in itself and the present knowle dg of the nutrition, metabolism, and physiology of the rusts has been restricted by the materials available for study, culturing the rusts are considered more important. In this regard, much work has been performed on the host-parasite complex ( Allen 1954 & 1959; Daly 1967; Hare 1966; Heitefuss 1966; Millerd and Scott 1962; Shaw 1963 and Yarwood 1967) and significant changes have been observed in the metabolism of infected plants. However, it has not been possible to distin guish unequivocally between the relative contribution of host and pathogen to these changes. Some metabolic studies have been performed on mycelium isolated from infec ted host tissues. To obtain definitive data on the nutrition and metabolism of rusts it seems necessary to grow these organisms axenically. When we know how the pa rasite breathes, feeds, excretes and uses, within its host and at its host’ s expense, whatever biochemical processes link it to that host we may learn to cont rol i . An exchange of nutrients, toxins, or both between the rust and host may deter min whether the host is resistant or susceptible. Saprophytic cultures shou ld prove useful in investigating these interactions, and so aid our understanding of mechanisms determining host resistance or susceptibility. Uromyces fabae (Pers.) DeBary, the causal of broad bean rust disea se, is one of the obligate parasites. There were no availa ble literature about axenic culture of this pathogen. The present work was aimed to inv estigate uredospore germination, enzymes activities and pathogenicity o f the resultant axenic uredospores. MATERIAL & METHODS Factos affecting urediospores germinability: 1-Effect of concentration of urediospores: Uredospores of 14 days-old cultures of U. fabae isolates A and B grown on MS-7 medium alone or provided with broad bean ex tracts were used. Numbers of Uredospores /ml in each particular stock of ured iospores suspension ( extracted in 10 ml distilled water / plate) were counted. Sto ck spore suspensions were diluted to contain 80, 60, 40, and 20% of the counted uredi ospores. Few drops of each urediospore suspension from a particular dilution w ere placed on sterilized glass slides (12 slides fore each particular treatment), raised on U-shaped glass rods in Petri dishes lined with moistened filter papers the n incubated for 6, 12, 18, and 24 hours at 25 C and 100% R.H. Percentages of germinat ion of urediospores for each particular treatment were calculated. 2-Effect of growth medium and age of cultures: Germinability of urediospores of the two isolates o f U. fabae produced on 5, 10, 15 and 20 days old cultures grown on MS-7 me dium alone or supplemented with broad bean leaf extract was tested as above me ntion d. 3-Effect of concentrations of sucrose and glucose: Urediospores produced from 5 days old cultures of t he two isolates of U. fabae grown on MS-7 medium with or without broad bean lea f xtract were used. Percentage of uredospore germinabilities in 0, 2, 4 , and 6% sucrose or glucose solutions were determined as described above. Annals Of Agric. Sc., Moshtohor, Vol. 37 (3): 1677-1693 (1998) 1679 Starch hydrolysis and Gelatin liquefaction: 1-Hydrolysis of starch (Amylase test): Uromyces faba isolates A and B were grown on nutrient agar-mediu m containing 0.2% soluble starch; beaf extract, 0.3% ; peptone, 0.5%; agar, 1.5%; and distilled water, 1000 ml (Taha, 1964). The medium w as sterilized as usual. The plates were inoculated with equal discs (6 mm diam. ) of fungal growths taken from 7 days old cultures (grown on MS-7 medium) and incu bated for different intervals at 25 °C. The resultant fungal growths were flooded with l ugol's iodine. The presence of clear zone outside the area of the grow th, indicating the extent of starch hydrolysis was measured and recorded. 2-Gelatin liquefaction: ( Gelatinase test): Equal discs of U. fabae isolates A and B were transferred from 7 days old cultures (grown on MS-7 medium) to plates of nutrie nt agar containing 0.4% of gelatin. The growth was flooded with 8-10 ml of sol ution of 15 g Hg Cl2. This reagent forms a white opaque precipitate with nativ e gelatin but a liquefied is surrounded by a clear zone (Smith et al, 1952) whic h was measured and recorded as indicator the ability of these fungal isolates to l iquefy gelatin. Pectulolytic and cellulolytic enzymes activities: Isolates A and B of U. fabae were left to grow for different periods at 25 C on liquid MS-7 medium (20 ml per each 100 cc flask) contained sucrose, 1.0% of pure citrus pectin or carboxsy methyl cellulose (CM ) as carbon sources. Five replicates for each particular treatment were used. The resultant fungal growths after each period were filtered through 3 layers of cheesecloth. The supernatant filtrates were frozen. until used for determinatio n f pectulolytic (PG) and cellulolytic (Cx) enzymes activities by using the m ethod described by Alexander (1954) as follow: 1-Effect of pH value on pectulolytic and cellulolytic enzymes activities: In this study filtrates of 14 days-old liquid cultu res of isolates A and B grown on MS-7 medium containing 1.0% of pure citru s pectin or carbosy methyl cellulose (CMC) as carbon sources were used. Effect of different pH values i.e.4.0, 4.6, 5.2, 5.6, 6/2, 6.8, 7.4 and 8.0 on polygalactu ronase (PG) and cellulase (Cx) enzymes activities of both isolates was determined by following the change in viscosity of l.2% pure citrus pectin or CMC, respec tively. Acetate buffer solution (0.1 M) was used for the first four pH values, whil e phosphate buffer solution (0.1 M) was used for the last four values. The reduction in viscosity was determined in the reaction mixture after 4 hours at 30 ?C. Enzyme activities were expressed as percentage of reduction in viscosity of the reactio n mixture. 2-Effect of reaction time on activities of pectulolytic ancellulolytic enzymes: Uromyces fabae isolates A and B were grown at 25 C for 21 days on a liquid MS-7 medium containing 1.0% pure citrus pect in or carbosy methyl cellulose (CMC) as sole sources of carbon then their filtrate s were obtained. The obtained filtrates were used for determination of Pectulolyt ic (PG) and cellulolytic (Cx) enzymes activities expressed as percentage of reduc tion in viscosity of the reaction mixture as affected by time reaction (after 5, 10, 3 50, 80, 120, 180 and 240 minutes) by using the method recorded by Alexander (1954). The pH values 6.2 and 6.8 were used for determining PG and Cx enzymes activities, respectively. 3-Effect of cultural age and substrate reaction on PG and Cx enzymes activities: Annals Of Agric. Sc., Moshtohor, Vol. 37 (3): 1677-1693 (1998) 1680 U. fabae isolates A and B were growfor 7, 14 and 21 days at 22C on a liquid MS-7 medium containing sucrose, citrus pecti n and carboxy methyl cellulose (CMC) as sole sources of carbon. Fungal filtrates w re