Cucurbit Genetics Cooperative Report 23:30-31 (article 9) 2000
Variability Among Israeli Isolates of Sphaerotheca fuliginea: Virulence Races, DNA Polymorphism, and Fatty Acid Profiles
N. Katzir1, R. Cohen1, R. Greenberg1, S. Shraiber1, G. Tzuri1, I.S. Ben-Zeev2 and O. Yarden3
Department of Vegetable Crops, Agricultural Research Organization, Newe Ya'ar Research Center, P.O. Box 1021, Ramat Yishay, 30095;2 Plant Protection and Inspection Services, Ministry of Agriculture and Rural Development, P.O. Box 78, Bet Dagan 50250, Israel;3 Department of Plant Pathology and Microbiology, Faculty of Agricultural. Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100. Israel
Introduction. Powdery mildew is a limiting factor for the production of cucurbits worldwide (Sitterly, 1978). In Israel, Sphaerotheca fuliginea is the causal agent of the disease, whereas Erysiphe cichoracearum, though it occurs on cichory, has yet to be found in cucurbits (Cohen and Eyal, 1995). Different races of S. fuliginea have been identified on the basis of differential host specificity (Thomas, 1978, Bardin et al., Pitrat et al. 1998). However, S. fuliginea race determination can be difficult, as plant response to inoculation may differ for a number of reasons including environmental conditions, genetic variability within a fungal population and shifts in pathogen populations.
The application of molecular markers has been demonstrated as a powerful tool for the study of populations of pathogenic fungi. Therefore, the aim of our study was to assess three techniques, RAPD, ISSR and FA analyses, as additional tools for the identification of powdery mildew races.
Materials and Methods. Five melon cultivars were used to identify S. fuliginea races based on their response to inoculation (Cohen et al., 1996). Sphaerotheca fuliginea isolates were collected from various cucurbit species during different seasons and at selected locations in Israel.
To obtain sufficient quantities (30-50 mg) of conidia for DNA or fatty acid analyses, cucumber plants at the age of 2-3 true leaves were inoculated with the different isolates (prepared from cultures originating from single spores). Two weeks postinoculation, conidia were harvested by washing infected leaves with sterile water. The conidial suspensions were filtered through a glass fiber filter (GFA, Whatman) over a Buchner funnel. After filtration, conidia were vacuum dried, collected to 1.5 ml microfuge tubes and maintained at -80˚C until analyzed.
Fungal DNA was isolated as described by Danin-Poleg et al. (1998). This protocol was found to be efficient for DNA extraction from the 30-50 mg of spores collected from each isolate. RAPD analysis was performed according to Williams et al. (1990) and ISSR analysis was performed according to Danin-Poleg et al. (1998).
Profiles of fatty acids: conidia (30-60 mg) from each isolate were processed as descriobed by Ben-Ze'ev et al., (1997) for fatty acid extraction. fatty acid profiles were identified by gas chromatography.
Results. RAPD and ISSR analyses were applied to detect polymorphism among 26 isolates of S. fuliginea from Israel. Only 2% of the 440 RAPD primers that were tested on isolates of S. fuliginea yielded reproducible polymorphic patterns. The nine primers that detected polymorphism were UBC primers: 807, 834, 840, 841, 861, 873. Cluster analysis of RAPD and ISSRT products did not result in grouping related to biological races.
The fatty acid composition of the 25 S. fuliginea isolates was analyzed and profiles were obtained using the method and library 'Fungi' (MIDI, 1992; 1993). They clustered in two groups of 12 and 14 isolates, with the larger cluster consisting of 2 subgroups. The 2 subgroups linked at <9 Euclidean distance units (EDU) while the 2 groups linked at ~27 EDU. Such distances would indicate two subspecific entities within one of two congeneric species (Greenberg, 1997). More detailed information will be published elsewhere.
Discussion. Traditionally, races of S. fuliginea were identified on the basis of differential host specificity. Three races of S. fuliginea have been identified in the US (Thomas, 1978) and recently six races have been described in France (Pitrat et al., 1998). This implies that the number of possible or pathotypes is larger than could be identified by differential plants. Additional approaches for race identification and for assessment of genetic variability have therefore been tested.
In general, using RAPD and ISSR analyses, a low level of polymorphism was detected among isolates of S. fuliginea that were collected from a narrow geographic range, in which sexual mating occurs rarely, if at all. ISSR was slightly more efficient than RAPD in detecting polymorphism among the isolates (10% versus 2% of the primers detected polymorphism). However, the RAPD and ISSR profiles were not useful in distinguishing among S. fuliginea races, confirming the previous observations by Bardin et al. (1997) who employed RAPD and RFLP analyses. Fatty acid profiles obtained for the same isolates were found to be more promising for the distinction among races. The fatty acid profiles as belonging to two races definitely belonged to two different FA subgroups. Further studies are required to confirm this observation.
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