WALTER K. KAST 1 , MARTINA STARK-URNAU 1 , MARTIN SEIDEL 2 and ARMIN R. GEMMRICH 2 1 Staatliche Lehr- und Versuchsanstalt für Wein- und Obstbau
Six isolates of Plasmopara viticola, the downy mildew fungus on vine were collected from six different locations of Southern Germany and Switzerland. The isolates were tested for their virulence pattern on leaf disks from either field-resistant hybrid varieties like Johanniter (FR 177-68) and Regent or the highly susceptible variety Trollinger. On the highly susceptible variety Trollinger and also on the resistant hybrid variety Johanniter only small, however significant, differences were found between all six isolates. On the highly resistant variety Regent almost no growth of the isolates We03, We06, We07, and We08 was observed. However, infection of Regent with isolate We10 resulted in an extremely high percentage of diseased leaf area (65%) comparable to that found on Trollinger (69%). Isolate We11 also showed a high disease incidence on Regent (42%), though its disease incidence on Trollinger was much higher (64%). Thus isolates We10 and We11 showed extraordinary fitness, though not being specifically adapted to one grapevine variety. We conclude that resistance of the hybrid variety Regent might be dependent on the biological fitness and the genotype of the P. viticola – isolate. Untersuchungen zur unterschiedlichen Virulenz von Plasmopara viticola –Isolaten auf resistenten Hybridreben Von sechs verschiedenen Standorten in Süddeutschland und der Schweiz wurden sechs Isolate des falschen Mehltaupilzes der Weinrebe gesammelt. Die Virulenz der Isolate wurde auf Blattscheiben der feldresistenten Sorten Johanniter (FR 177-68) und Regent oder der hoch anfälligen Rebsorte Trollinger getestet. Auf der hochanfälligen Sorte Trollinger und auf der resistenten Rebsorte Johanniter zeigten alle sechs Isolate nur geringfügige, wenn auch bedeutende Unterschiede. Auf der resistenten Sorte Regent wurde fast kein Wachstum der Isolate We03, We06, We07 und We08 beobachtet. Die Infektion von Regent mit dem Isolat We10 führte zu einem Befall von 65%, vergleichbar mit dem Befall auf Trollinger (69%). Auch das Isolat We11 befiel die Sorte Regent. Die Befallsrate war mit 42% allerdings niedriger als auf Trollinger (64%). Sowohl das Isolat We10 als auch das Isolat We11 zeigten eine außerordentliche Fitness, die nicht auf eine Rebsorte beschränkt war. Die Resistenz der Hybridsorte Regent scheint folglich von der biologischen Fitness des Genotyps der P. viticola-Isolate abhängig zu sein. Etude des différences de la virulence de Plasmopara viticola (isolats) sur des cépages hybrides résistants. Six isolats de Mildiou de la vigne ont été effectué dans six endroits dans le sud de l`Allemagne et en Suisse. La virulence des isolats a été sur les feuilles des cépages résistants Johanniter (FR 177-68) et Régent ainsi que sur le cépage hypersensible Trollinger. Les six isolats montrèrent peu de différences même s'ils sont néanmoins notables sur Trollinger et Johanniter. Il fut observé que les isolats We03, We06, We07 et We08 ne sont pratiquement pas développés sur le cépage résistant Régent. L’infection du cépage Régent avec l’isolat We10 a conduit à une contamination de 65%, comparable avec la contamination du Trollinger (69%). L’isolat We11 s’est également propagé sur le cépage Régent. Le taux de contamination était de 42%, inférieur à celui sur Trollinger (64%). Aussi bien l’isolat We10 que l’isolat We11 possèdent une condition biologique exceptionnelle indépendante du cépage. La résistance du cépage hybride Régent semble dépendant de la condition biologique ainsi que du génotype des isolats de P. viticola. The obligate biotrophic parasitic fungus Plasmopara viticola is the causal agent of the downy mildew disease in Vitis . Being an oomycete P. viticola occurs throughout important wine growing regions in temperate climates and causes considerable yield losses. Symptoms appear as yellowish, oily spots on the abaxial surface and the characteristic signs are white sporangiophores and sporangia that emerge through the stomata (Emmet et al. , 1992). Given the economic importance of downy mildew, grape breeders in Germany and European countries are breeding field-resistant interspecific hybrids, using a recurrent back cross system. Some of those vine varieties are, concerning phenotype and wine quality, not distinguishable from established European varieties. However, in some cases in Southern Germany and Switzerland those normally highly field-resistant vine varieties were seriously attacked by downy mildew during the last years. This could be either due to extreme weather conditions or to a decrease of resistance as a result of fungal adaptation to the genes of these hybrid varieties (Johal et al. 1995, Robinson, 1976; Vanderplank, 1968). As resistance against downy mildew is assumed to be inherited polygenetically (Demichaelis, 1994; Diehl 1988; Li, 1988), a sudden break down of the resistance by specific virulent isolates seems to be unlikely; but in the long run a shift seems not to be impossible. Little is known about the diversity of strains of P. viticola although different ecotypes may exist and strains of varying virulence to hybrid varieties Vitis spp. have been found (Kast, 1996A and 1996B). The selection of isolates of P. viticola was not at random as isolates were sampled on severely diseased resistant hybrid varieties. Thus the chance of finding aggressive isolates with highly specific genotypes was increased. The aim of our study was to test under controlled conditions whether resistance of hybrid varieties can be overcome by especially virulent and aggressive isolates of P. viticola . Materials and Methods Fungal isolates were obtained from three different grapevine cultivars and from six different locations (s. Tab. 1). Tests were performed on highly susceptible Trollinger and on the field-resistant varieties Johanniter and Regent. Table 1: Isolates of P. viticola collected from different grapevine cultivars
The isolates were propagated on detached leaves of their respective host plant i. e. of the grapevine varieties Trollinger, Johanniter and Regent at 20°C and a 16h photoperiod. Prior to inoculation the leaves were put in petri dishes their abaxial surface facing up. Then a solution of 50 000 sporangia/ml was applied by using an air brush sprayer. For tests only newly formed sporangia (maximum age nine days after inoculation) were used. Test procedure: For each of the four experiments fifteen leaf disks (30 mm diameter) per fungal isolate were cut with a cork borer from leaves of fifteen different greenhouse grown grapevine cuttings of each variety e.g. Trollinger, Regent, and Johanniter. Only fully expanded leaves were selected from the forth to the sixth node from the base, as younger leaves generally provide more reliable results than older ones (Stein et al. , 1985; Reuveni, 1998). Inoculum was produced by rinsing infected leaves with water and adjusting to 30 000 sporangia/ml by using a hematocytometer. The inoculum was sprayed onto leaf disks by using a fixed air brush sprayer that was spraying the inoculum on the disks while they were sliding past below the air brush sprayer on a conveyor belt with a defined speed. Thus, 0.007ml/cm 2 inoculum was applied. After inoculation leaf disks were placed, abaxial surface up, on water saturated filter paper sheets laid over styropor plates that were floating on water in plastic containers (15 cm x 95 cm). The plastic containers were closed and placed at 18°C and at a 16h photoperiod to promote infection and sporulation. Eight days after inoculation leaf disks were rated for % leaf area with sporangiophores and sporangia on a scale of 0 to 100. Results and Discussion On the highly susceptible variety Trollinger only small, however significant, differences were found between all six isolates tested. The highest disease incidence showed the isolate We10 (fig. 1). The lowest disease incidence was obtained by the isolate We03 (53%) although this isolate was originally derived from Trollinger. This might be due to its reduced biological fitness, as this isolate has been cultivated in the laboratory since 1995. On the resistant hybrid variety Johanniter only small, however significant, differences were found between all six isolates tested: Isolate We03 from Trollinger and isolate We06 from Regent showed a reduced disease incidence (20%), while isolates collected from Johanniter (We07 and We08) or Regent (We10 and We11) had a disease incidence of about 40-50%. This could be a result of an adaptation to Johanniter or a consequence of extraordinary fitness of the isolates tested. On the resistant variety Regent considerable differences between the six isolates were observed: Infection with isolate We10 collected from Regent resulted in an extremely high disease incidence (65%) comparable to that found on Trollinger (69%). Thus isolate We10 showed extraordinary fitness, though not being specifically adapted to one grapevine variety. Isolate We11 originally found on Regent, also showed a comparably high disease incidence on Regent (42%). However, infection of Regent with the other four isolates resulted in a low disease incidence, although isolate We06 had been originally sampled from Regent in Baden/Germany. This finding can be explained by the early occurring primary infection and the extremely wet weather in that region in 1999. Thus a not really aggressive isolate, like We06, could also be found on Regent. The leaf disks assay proved to be an appropriate method for resistance screening of P. viticola (Denzer et al. 1995, Stein et al. 1985). By using leaf disks, we were able to test a large population of isolates on three different grapevine-cultivars, making a total of 270 leaf disks per trial. Brown et al. (1999) showed for P. viticola that there is a good correlation between the leaf disk technique and field studies and that this technique is more practical than greenhouse methods or field studies. The selection of isolates of P. viticola was not at random, as isolates were sampled on severely diseased resistant hybrid varieties. Thus the chance of finding aggressive isolates with highly specific genotypes was increased. Our study shows that there are significant host-parasite-interactions between fungal isolates and grapevine varieties. Disease incidence on the resistant hybrid variety Regent proved to be very variable in dependency of the fungal isolate tested; i. e. isolate We10 showed an extraordinary high disease incidence, while the other isolates did not. Generally, isolates showed in the mean different percentages of disease incidence which correlates with results of Li et al. (1986). The extended use of resistant hybrid varieties might promote the episodic selection of a highly fitted isolatclone of P. viticola from an originally heterogeneous population (Brasier, 1995). The heterogenity of primary infections of P. viticola has already been demonstrated by use of RAPD/PCR methods (Seidel et al. 1998; Stark-Urnau et al. 2000). It seems unlikely that distinct virulent isolates will survive from one vegetation period to the next, as no asexual reproduction of P. viticola occurs during the winter (Emmet 1992; Vercesi et al. 1999). However, virulence genes persist in the population even when oospores are formed in autumn. A complete and permanent break down of resistance due to aggressive isolates within a short period of time seems improbable. But the frequency of the genotype We10 would have to be closely related to the frequency of the genotype "Regent" in the host population. Theoretical considerations point to the fact that in this case a threshold for the frequency of the "Regent" resistance genes could exist (Leonard and Czochar, 1980, Geiger et al. 1980). If this threshold is passed the number of virulent genotypes should continuously increase. Extremely aggressive isolates of P. viticola without specific adaptation could cause problems because resistance of hybrid varieties is in most cases not complete (Kortekamp et al. 1998). Furthermore, the occurrence of aggressive isolates, such as We10 and We11, indicates that the pathogen population in Europe might change with the extended use of resistant hybrid varieties. A preventive strategy should be developed, that reduces the danger of overcoming of resistance. By employing different resistance genes in the varieties, the frequency of each gene could be kept low (Robinson, 1976). Furthermore the selection of virulent isolates should be suppressed by taking care that in years of high disease pressure and favourable weather conditions disease incidence is kept low. Fig. 1: Disease incidence of six different isolates of P. viticola on the vine varieties Trollinger, Johanniter, and Regent. Each value represents the mean of 60 data out of four different experiments. Standard deviation of all means was 2,2. Acknowledgements : Monika Seidel is gratefully acknowledged for fruitful discussions and for technical assistance. This work was kindly supported by the Ministerium für Ländlichen Raum Stuttgart, project-No. 25 - 8261. 02 / 009 E. References Brasier, C. M. 1995. Episodic Selektion as a force in fungal microevolution with special reference to clonal speciation and hybrid introgression. Can. J. 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