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Y. Ben-Yephet (1977) Phytoparasitica 5(2): 10%113.
SPORULATION OF V E N T U R I A
P I R I N A ON G R O W T H M E D I A By
Y. BEN-YEPHET* A synthetic medium suitable for abundant sporulation of Venturia pirina, was developed. It contained trace elements, macroelements, vitamins, fructose, and ammonium sulfate; the pH was adjusted to 6 and the incubation temperature was 20~ Sporulation of the various isolates was different: a strain that originated from natural bulk spores was 3.5 to 11 times lower than that of single spore isolates grown under the same conditions. KEY WORDS: Carbon sources; nitrogen sources. INTRODUCTION Pear scab, caused by Venturia pirina Aderh,, is prevalent in the pear-growing regions of Israel. Of five physiological races so far identified (2), only race 2 is of e c o n o m i c i m p o r t a n c e because it infects the main cultivars - S p a d o n a and Gentile. A b u n d a n t sporulation of the fungus o c c u r r e d on pear fruits or leaves, whereas sporulation of V. pirina on the culture m e d i u m p o t a t o - d e x t r o s e agar ( P D A ) (3.9%) or 1% P D A (2) was much less. T h e purpose of this study was to determine the conditions and m e d i u m composition suitable for sporulation of Venturia pirina. MATERIALS
Fungus Venturia pirina (race 2), f r o m several pear orchards, was originally provided by E. Shabi, T h e Volcani Center, Div. of Plant Pathology. Single spore isolates of the fungus were separated by microscopic observation and designated as 0, 1, 2, 4, 6, 7, and 8. Isolate 0 was used for the experiments on sporulation of the fungus in this work; the other isolates were used to c o m p a r e sporulation on the various media.
Publication of the Agricultural Research Organization. 1977 series, no. 234-E. Received May 19, 1977; received in final form Sept. 15, 1977. *Div. of Plant Pathology, ARO, The Volcani Center, Bet Dagan.
Phytoparasitica 5:2, 1977
Media Mineral salts (macroelements, trace elements) and vitamins according to Vogel (3) were used as a basis for developing the medium for the sporulation of V. pirina. Fructose, glucose, sorbose and mannose were tested as carbon sources. Ammonium sulfate, ammonium nitrate and sodium nitrate were tested as nitrogen sources. Mineral salts and a carbon source were individually sterilized to prevent carmelization of sugars. Sugars were added to the medium after sterilization. Vitamins were sterilized with filtration and added before pouring the medium into petri dishes. Different degrees of pH in the medium were achieved by titration with KOH or HCI solutions following sterilization of the medium. Potato-dextrose agar, 3.9% or 1% (2), and V-8 (1) were also evaluated for sporulation of V. pirina.
Inoculum preparation Venturia pirina was grown on slants following modification of a minimal medium (3) that contained vitamins, and fructose instead of glucose. After 10-12 days of incubation at 20~ a thin layer of agar with fungus hyphae was taken and introduced into a tissue homogenizer containing 2-3 ml sterile distilled water. Then, 0.1 ml of the homogenate (spores and hyphal fragments) was spread on 10 ml of the test medium in each petri dish and incubated at the indicated temperature. Ten days after incubation, the mycelial layer in each petri dish was divided into eight parts with a spatula and transferred into a 100 ml Erlenmeyer flask containing 10 ml distilled water with 0.2 ml Tween-20 per liter. The Erlenmeyer flasks were then thoroughly shaken by hand for 30 seconds and the spore suspensions were transferred into another set of flasks. Another 10 ml of distilled water was added to each one of the first set of Erlenmeyer flasks containing the fungi, shaken for 10 seconds, and the spore suspensions were again poured into the second set of flasks. Ninety-five percent of the spores which developed in the medium were transferred into 20 ml of distilled water in this manner. Spore density was then determined with a haemacytometer. RESULTS
Effect of medium composition, incubation temperature and pH on sporulation of Venturia pirina As carbon sources for the growth and sporulation of the fungus, fructose, glucose, sorbose and mannose were evaluated, using 5 g each per liter of medium. Sporulation on a medium with fructose or mannose as carbon sources was significantly higher than that with glucose or sorbose. Maximal sporulation occurred at 20~ though growth of hyphae at 15~ was more abundant than at 20~ Increasing the temperature of incubation to 25~ resulted in the suppression of both hyphal growth and spore production. Maximal sporulation was achieved on a medium with pH 5. An increase in the pH of the medium corresponded to a decrease in hyphal growth and spore production (Table 1).
TABLE 1 E F F E C T O F C A R B O N S O U R C E S , I N C U B A T I O N T E M P E R A T U R E S A N D pH OF T H E M E D I U M O N S P O R U L A T I O N OF V E N T U R I A P I R I N A (Each figure represents the m e a n of three experiments, each consisting of four replicates. Mineral salts and vitamins according to Vogel (3) were used and p H of the m e d i u m was 5.8.)
Carbon source ~
Spores per petri dish
Incubation temperature b
(xlO0 None Fructose Glucose Mannose Sorbose
Spores per petri dish
1.6 174 59 142 12
Spores per petri dish
15 20 25 28
59 179 10 c
5.0 5.5 6.0 6.5 7.0 8.0
430 346 160 12 -r _d
qncubation temperature was 20~ "Fructose was used as the carbon source. ~Hyphal growth was very poor. aHyphal growth could not be detected.
The effect of trace elements, vitamins and citrate on sporulation of the fungus was tested. The results (Table 2) showed the following. On a medium without citrate, maximal sporulation was attained at pH 6.0. An increase of medium pH to 7 corresponded to a significant decrease in hyphal growth and sporulation of the fungus. On the other hand, a decrease of medium pH to 4.5 caused an increase in hyphal growth but a decrease in TABLE 2 EFFECT
O F S U P P L E M E N T S T O T H E M E D I U M ON SPORULATION OF VENTURIA PIRINA (Each figure represents the m e a n of two experiments, each consisting of four replicates. Two h a e m a c y t o m e t e r s were counted in each replicate.)
4.5 4.8 5.0 5,5 6.0 7.0
Number of spores per petri dish (xllY) Complete m e d i u m
401 455 530 441 351
343 441 697 62
"T=trace elements, C=citrate, V=vitamins.
Phytoparasitica 5:2, 1977
sporulation of about 30%. When citrate was added to the medium, maximal sporulation occurred at pH 5. Sporulation in a medium without citrate at pH 6.0 was about 30% higher than that in a medium with citrate at pH 5.0. Trace elements, when present in the medium, were found to increase sporulation by about 60%. Vitamins had no effect on sporulation of isolate 0 of V. pirina, but they improved sporulation in other isolates of the fungus. Fructose, ammonium nitrate, ammonium sulfate and sodium nitrate were tested at various concentrations; the pH of the medium was always adjusted to 6.0. Maximal sporulation of the fungus occurred within the range of 5-20 g of fructose per liter, and 2 g of ammonium sulfate per liter as a nitrogen source. Based on these results, a medium for sporulation is suggested hereunder, and will be designated as fructose medium: 10 g fructose, 2 g (NH4)2SO 4, 5 g KHzPO 4, 0.2 gMgSO4"7H20 , 0.l g CaC12, trace elements and vitamins, according to Vogel (3). The pH of the medium was adjusted to 6.0 before sterilization.
Comparison of sporulation between Venturia pirina isolates in different media A comparison of sporulation between (a) isolates originating from single spores, (b) a mixture of the same isolates, and (c) an isolate that
TABLE 3 OF SPORULATION BETWEEN VENTURIA P I R I N A I S O L A T E S IN V A R I O U S M E D I A
(Each figure represents the mean of three replicates, in each of which three haemacytometers were counted.)
'Figures represent results of sporulation on ordinary PDA or poor PDA. A=Modification of minimal medium. B= Fructose medium.
Phytoparasitica 5:2, 1977
originated from a natural bulk of spores collected from ten pear orchards, was made. The isolates were seeded on the following media: fructose (pH 6.0), modification of minimal (pH 5) (3), potato dextrose agar (pH 5.5) (2), and V-8 (pH 6) (1). In order to compare the sporulation ability of each isolate in different media, the same number of hyphal fragments and spores (about 2.8x105 per petri dish) from each isolate or mixture was used. After 10 days of incubation at 20~ spores were counted. The results (Table 3) showed the following. The abilities of single spore isolates to sporulate differ from each other. Sporulation of the isolate that originated from natural bulk spores was 3-11 times lower than that of single spore isolates. Sporulation of the isolate mixture (with isolates 1 and 2, or without them) was also lower than the theoretical sporulation mean. Sporutation of all isolates which originated from single spores was higher on fructose medium and 2.5-4 times that on PDA or poor PDA (2), and 0.35-0.9 times that on modified minimal medium. Sporulation on V-8 (1) was very poor in all isolates and adjustment of the medium pH to 5 or 7 did not improve sporulation. DISCUSSION In this study it has been shown that sporulation of V. pirina strains originating from single spores is very sensitive to different conditions, such as incubation temperature and pH of the medium. By increasing the incubation temperature from 20~ to 25~ a sharp decrease in sporulation of the fungus occurred. The same effect was achieved when the pH was increased from 6 to 7 in the fructose medium. As a carbon source, the ketone forms of sugar (fructose or mannose) were more suitable for sporulation than the aldehyde forms (glucose or sorbose), and ammonium sulfate more suitable than ammonium nitrate or sodium nitrate. When sporulation of an isolate originating from natural bulk spores or from mixtures of isolates (0, 1, 2, 4, 6, 7 and 8) was tested, it was found to be low and almost equal in all the media. A possible explanation for this would be that antagonism between the isolates caused a decrease in fungus sporulation. REFERENCES 1.
Miller, P.M. (1955) V-8 juice agar as a general purpose medium for fungi and bacteria. Phytopathology 45: 461-462.
Shabi, E., Rotem, J. and Loebenstein, G. (1973) Physiological races of Venturia pirina on pear. Phytopathology 63: 41-43.
Vogel, H.J. (1956) A convenient growth medium for Neurospora (Medium N). Microbiol. Genet. Bull. t3: 42-43.