Studies on the genetic diversity of primary and secondary infections of Plasmopara viticola using RAPD/PCR

E-Mail: Martina.Stark@lvwo.bwl.de
            Walter.Kast@lvwo.bwl.de

Summary
In 1998 and 1999 isolates from primary and secondary infections of Plasmopara viticola (downy mildew) were sampled from several grapevine varieties. Fungal isolates were taken from the same vineyard located in Württem-berg/Germany. Using the random amplified polymorphic DNA (RAPD) technique as an initial screening for genetic variation within P . viticola isolates, different molecular fingerprint patterns were obtained. Twenty-seven (1998) and 29 (1999) different genotypes out of 48 isolates of primary infections were found. In contrast, variability of secondary infections was lower about 9 in 1998 and 14 in 1999 different genotypes out of 48 isolates. The high genotype diversity in primary infections and the low level of genotype diversity in secondary infections strongly suggests environmentally directed selection.

Introduction
Plasmopara viticola propagates asexually in summer by forming sporangia and zoospores, while in autumn sexual propagation starts and diploid oospores are produced in host tissues (Emmet et al . 1992; Vercesci et al . 1999). Primary infection occurs when water-borne sporangia or zoospores, produced by germinating oospores, are splashed onto wet vine foliage. Then motile zoospores move to stomata and penetrate the stomatal aperture via a germ tube. After an incubation period sporangiophores and sporangia are produced and grow out through stomatal pores during warm humid nights. Secondary infection follows wind or water-splash dispersal of these sporangia (Espino and Nesbitt 1982). Dependent on weather several circles of secondary infections can take place during one season.

RAPD markers were used to obtain more information about the biology and epidemiology of P. viticola . DNA-markers based on PCR are a powerful tool to study the genetics of fungal plant pathogens (Edel 1998). Moreover, random amplified polymorphic DNA (RAPD) has been successfully used in various genetic studies (Annamalai 1999; Castagnone-Sereno et al. 1994; Delye et al. 1997; Welsh and McClelland 1990). RAPD is a type of genetic marker based on DNA-amplification with single or multiple arbitrary primers requiring no knowledge of the target DNA sequence. Thus, amplification products are produced that often show size polymorphism within species. The reproducibility within one laboratory is usually satisfactory (Tommerup et al. 1995). Furthermore, Délye et al. (1995) have demonstrated that even two different DNA-polymerases or three different thermocyclers show similar RAPD-profiles of Uncinula necator . Therefore, the RAPD/PCR technique seemed to be suitable for estimation of the genetic diversity of a P. viticola population on a small scale.

The aim of this study was to compare the genetic variability of isolates deriving from primary infections to that of isolates deriving from secondary infections.

Material and Methods
In June 1998 and 1999 48 isolates of P. viticola were sampled from primary infections found on common V. vinifera varieties (Müller-Thurgau, Traminer, Samtrot and Weißburgunder) located in Wildeck (Baden-Wuerttemberg, Germany). Temperature was recorded by an Adcon device positioned at a distance of about 200 m in a height of 2.5m. Using the addVANTAGE program (Adcon, Austria) to determine oospore germination and possible infection with P. viticola the exact time of primary infection was determined. To ensure that each primary infection corresponded to one infecting zoospore, only singular, circular oil spots were taken. Each vine showing a primary infection was marked in order to sample secondary infections at the same location. Finally, at the end of the vegetation period in September / October 48 isolates from secondary infections were sampled. Samples were taken from the same grapevines in June and October, respectively. To obtain sufficient fungal material for DNA extraction, leaves containing oil spots were incubated for 48 h at 20 °C and almost 100 % r.h. Thereby P. viticola formed enough sporangia at the leaf surface which were harvested for DNA extraction. If too little sporangia were formed, P. viticola was propagated on detached leaves of the susceptible cv . Trollinger until sufficient material for DNA extraction was available.

DNA extraction and amplification was performed as described by Seidel et al. (1998). Genomic DNA was extracted from 0.5 mg sporangia according to the method of Zhu et al. (1993) using benzyl chloride for chemical cell wall disintegration. PCR was performed using a ready-to-go RAPD analysis kit from Pharmacia (Uppsala, Sweden). 25 pmol of primer, 30 ng of template DNA and distilled water were added to give a final volume of 25 µl. Random primers were obtained from Pharmacia and Roth (Karlsruhe, Germany). After screening of 26 different decamer primers, the primers Pha6 [5' CCCGTCAGCA 3'] (Pharmacia) and Roth 170-06 [5' GGACTCCACG 3'] (Roth) were used subsequently because they revealed a high level of polymorphism between isolates of Plasmopara viticola . Thus clear and reproducible RAPD-patterns were obtained. Samples were placed in a thermocycler from Progene (Techne, Cambridge, United Kingdom) and subjected to the following cycle profile: 1 cycle at 95°C for 5 min followed by 45 cycles at 95 °C for 1 min, 36 °C for 1 min and 72 °C for 2 min. 5 µl of the reaction mixture was analysed by electrophoresis on 7,5 % PAA-TBE gels. Gels were stained with silver (Budowle and Allen 1990) and documented with ImageMaster VDS, Pharmacia. Data were used to construct phenograms, using a program based on UPGMA (ImageMaster 1D software, Pharmacia). Analysis was repeated at least twice from extraction to electrophoresis of amplification products.

Resultsand Discussion
In 1998 and 1999 a population of 48 isolates deriving from primary infections and 48 isolates from secondary infections were screened with RAPD/PCR using different primers for DNA-amplification. Samples from primary infections revealed a high genetic variability according to their different fingerprint patterns ( Fig. 1 ). In 1998 27 different isolates of primary infections were observed, while isolates deriving from secondary infections showed less genetic variability ( Fig. 2 ): Nine groups out of 48 isolates tested exhibited different fingerprint patterns. In 1999 similar results were obtained. The phenogram of primary infections revealed 29 distinct groups ( Fig. 3 ). In contrast, the phenogram of secondary infections revealed 14 distinct groups. Moreover, the genotype of three isolates (Wi 52, Wi 53 and Wi 54) is much more abundant.

Although the isolates had been sampled on a small scale, they exhibited different fingerprint patterns indicating a high genetic variability. Moreover, even on one leaf different oil spots were found that showed different fingerprint patterns (data not shown).

We cannot totally exclude that single oil spots correspond to infections of different zoopores. However, this does not seem to be of great importance, since the experiments were repeated in different years. Furthermore it was shown that different primers reveal similar results.

High genetic variability has also been described for other populations of sexually reproducing fungi. E. g., in Gremmeniella abietina a surprising amount of variation can be present even on one single host tree (Wang et al. 1997). However, Plasmopara halstedii , which causes downy mildew in sunflower, lacked genetic variability (Roeckel-Drevet et al. 1997). Sampling isolates of P. viticola from secondary infections in October 1998 and 1999 we frequently found a low level of genotype variability. In contrast, Kump et al. (1998) described a high genetic variability of secondary infections in P. viticola , which is explained by numerous new primary infections in later phases of the epidemic.

Comparing variability of secondary infections of P. viticola to that of primary infections, the genetic variability was strongly reduced. It seems that during several cycles of secondary infections selection of the fittest isolates can occur. Selection is generally recognised to be a strong force in shaping pathogen population structures (Huang et al . 1995). When the limited dispersal and highly asexual reproduction of a fungal population is coupled with localised adaptive differentiation, the potential for selection of isolates adapted to very specific environmental conditions can be very high. E. g. it was found (Ennos and McConnell 1995) that small scale environmental variation had a larger effect on the relative performance of Crumenulopsis sororia than differences in the environment over longer periods.

The new characteristics of these isolates would be diluted after sexual reproduction with other less fit genotypes. Then, during the next asexual reproductive phase, again selection of the fittest isolates might occur. They will eventually dominate within the population between circles of sexual reproduction. Therefore, the most successful asexual isolates are likely to be the most frequent parents of most of the next season’s isolates (Duncan et al. 1998).

The high genetic variability of primary infections may be due to genetic recombination during genesis of oospores. Furthermore oospores which are able to persist in the soil for at least 5 years (HILL, per. comm.) might skip one or several vegetation periods thus contributing to the high genetic variability in spring.

The high level of genotype diversity in primary infections and the low level of genotype diversity in secondary infections strongly suggests that environmentally directed selection takes place.

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Dieser Artikel ist auch inVitis 39 (4) S. 163-166 publiziert.