OBSERVATIONS ON THE POPULATION BIOLOGY OF THE GRAPE POWDERY MILDEW FUNGUS UNCINULA NECATOR

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1 Journal of Plant Pathology (2003), 85 (2), Edizioni ETS Pisa, OBSERVATIONS ON THE POPULATION BIOLOGY OF THE GRAPE POWDERY MILDEW FUNGUS UNCINULA NECATOR M. Miazzi, H. Hajjeh and F. Faretra Dipartimento di Protezione delle Piante e Microbiologia applicata, Università degli Studi di Bari, Via Amendola 165/A, Bari, Italy SUMMARY Random Amplified Polymorphic DNA (RAPD) analysis and mating type distribution were used to investigate the population biology of the phytopathogenic fungus Uncinula necator (Schw.) Burr., the causal agent of powdery mildew of grapevine. It is known that the fungus has two overwintering strategies, mycelium and conidia in dormant buds or cleistothecia, but their relative importance in disease epidemiology is still undetermined. Recently, the existence has been hypothesized of two genetically separated biotypes in U. necator that would be related with its overwintering modes: a biotype overwintering as conidia and mycelium in buds would infect shoots and leaves early in the season; the other biotype would overwinter as cleistothecia and infect bunches. RAPD analysis was carried out on 374 isolates of U. necator collected in Southern Italy. Statistical analysis of variation clustered the isolates into two major groups according to the time in the season when they were sampled in vineyards, confirming the existence of two different biotypes into the fungal species. Similar proportions of the two mating types were found in the pathogen s populations, as well as into each biotype, even in single vineyards. Therefore, the two mating types of the fungus are not separated either in space or time. Sexual crosses between isolates belonging to the two biotypes were fertile and yielded viable ascospore progeny. These results suggest that meiotic recombination can be an important source of genetic variation in U. necator and cleistothecia can play an important role in its overwintering. Key words: Uncinula necator, grapevine, RAPD analysis, sexual behaviour, genetic variability, epidemiology. INTRODUCTION Powdery mildew, caused by Uncinula necator (Schw.) Burr. [teleomorph of Oidium tuckeri Berk.; recently the Corresponding author: F. Faretra Fax: faretra@agr.uniba.it fungus was renamed Erysiphe necator Schw. and placed into the section Uncinula of the genus) (Braun and Takamatsu, 2000)], is a worldwide economically important fungal disease of the grapevine. It costs millions dollars annually to vine growers, due to crops losses and an intensive usage of fungicides for its control. Only few genetic studies are available in U. necator due to its obligate biotrophism. The fungus cannot be grown on artificial media and observations in the field can be carried out only in a limited period of the year. Field- or greenhouse-grown vines and detached leaves have been used to study different aspects of the disease (Doster and Schnathorst, 1985; Chellemi and Marois, 1991). For molecular investigations, however, these methods are subject to the possible interference of other epiphytic micro-organisms. Although more laborious, the usage of dual culture for growing U. necator colonies on in vitro-grown grape material under aseptic conditions is a more reliable technique (Klempka et al., 1994; Evans et al., 1996; Miazzi et al., 1997). U. necator has been reported to overwinter as mycelium or conidia in dormant buds (Sall and Wrysinsky, 1982; Pearson and Gärtel, 1985; Gemmrich and Seidel, 1996) and/or as cleistothecia on infected tissues, on the bark of vines or in the soil (Diehl and Heintz, 1987; Pearson and Gadoury, 1987; Gadoury and Pearson, 1988, 1991; Viccinelli et al., 1996; Cortesi et al., 1997). However, the role of the sexual process in the pathogen s life cycle and epidemiology of powdery mildew, and its importance in the generation of new recombinant genotypes has been neglected for a long time. In Europe, cleistothecia were not observed for 40 years after the introduction of the pathogen from America, so that its overwintering was believed to be sustained exclusively by dormant mycelium and conidia. The sexual system of the fungus is a bipolar heterothallism coded by the major gene MAT1 with its two alleles (likely idiomorphs, based on what known on other Ascomycetes) MAT1-1 and MAT1-2 (Gadoury and Pearson, 1991; Miazzi et al., 1997, 2002). The initial introduction of only one mating type was probably the reason of the delay of cleistothecia discovery in Europe (Couderc, 1893; Yarwood, 1957) and, more recently, in Peru (Dongo and Aréstegui, 1973) and Australia (Wicks et al., 1985). Genetic markers are crucial for studying the genetic

2 124 Population biology of Uncinula necator Journal of Plant Pathology (2003), 85 (2), structure of pathogen populations and the epidemiology of the incited disease; molecular markers can be particularly useful for biotrophic fungi, such as U. necator (Milgroom, 1997). Information on population genetics and epidemiology can result in deeper knowledge of population biology that could be essential for developing more effective and environmental-friendly disease control strategies (Milgroom, 2001). Random Amplified Polymorphic DNA (RAPD) analysis (Williams et al., 1990), a fingerprinting technique based on Polymerase Chain Reaction (PCR), has been successfully applied to study variation in many fungal pathogens, including U. necator (Délye et al., 1995, 1997, 1998; Evans et al., 1996; Stummer et al., 1999, 2000). Délye et al. (1997, 1998), through RAPD analysis, distinguished two biotypes in European powdery mildew populations having different overwintering modes. One biotype was constituted by isolates overwintering as conidia or mycelium in buds and causing the typical symptoms on young shoots known as flag shoots, early in the season. The other biotype would overwinter as cleistothecia and be responsible of late infections mostly on bunches. The two biotypes were thought to be genetically separated because in sexual crosses they did not yield fertile cleistothecia and viable ascospores (Délye et al., 1997, 1998). The aim of the present work was to investigate the population biology of U. necator in some vineyards of Southern Italy, using mating type and RAPD markers. Special attention was paid to the pathogen s sexual behaviour and the occurrence of distinguishable biotypes in its natural populations. MATERIALS AND METHODS U. necator isolates. Thirty-one vineyards were selected as representatives of the most important grape-growing areas in six regions of Southern Italy (Abruzzo, Basilicata, Calabria, Molise, Puglia and Sicilia). Samples of naturally infected bunches, leaves and canes were collected between April and September from 1998 to Isolates from typical flag shoots were sampled immediately after their appearance (April-May). An extensive sampling (140 isolates) was carried out in an experimental vineyard made up by randomly repeated plots of eight cultivars, recording the exact position of each sample. Plantlets of the very susceptible table grape cv. Baresana were grown and dual cultures of U. necator on grape leaves established as described by Miazzi et al. (1997). Briefly, young expanded leaves were collected from in vitro-grown grapevine shoots and placed singly on a 2-mm thick layer of HB medium (Hoos and Blaich, 1988), with half strength of salts, in 55-mm-diam Petri dishes, taking care that their stalks were immersed into the medium. Leaves were seeded with small amounts of conidia collected from naturally infected samples with single eyelashes glued to glass pipettes. Dishes were sealed with Parafilm and kept at 21±1 C with a 16 h photoperiod from a combination of 3 Osram L36W lamps and 3 Sylvania Grolux F36W lamps. After one week, single conidia were collected and transferred onto new leaves to obtain pure cultures. Isolates were maintained by transferring conidia from single chains onto new leaves at 4-5 week intervals (Fig. 1). Fig. 1. A dual culture of Uncinula necator on a leaf from in vitro-grown grape plantlets. Mating type. The strains UN52 (MAT1-1) and UN68 (MAT1-2) were used as references to assess the mating type of newly collected isolates (Miazzi et al., 1997). Each isolate was paired with both reference strains on single leaves that were kept under above described conditions. Cleistothecia appearance was checked at oneweek interval for 6-7 weeks with the aid of a stereomicroscope. Mature cleistothecia were collected with a needle, squashed in a drop of a vital stain (1 µl of a stock solution containing 2 mg ml -1 fluorescein diacetate in acetone diluted in 1 ml of water) on a glass slide (Widholm, 1972) and observed with the aid of an epifluorescence microscope (Photomicroscope III, Zeiss, equipped with a HBO 100 W/2 mercury lamp; excitation filter BP436/8, beam-splitter FT460, barrier filter LP470) to assess ascospore viability. DNA extraction and RAPD amplifications. Mycelium and conidia (approximately 4 mg fresh weight) were collected from 2-3-week-old colonies by scraping the surface of infected grape leaves with a disposable lancet. DNA was extracted by using InstaGene Matrix (Bio-Rad Laboratories, Hercules, CA, USA), following the manufacturer s instructions. DNA solutions were kept at -20 C until usage. Samples of DNA from two strains of U. necator, kindly supplied by Prof. Corio- Costet (National Institute of Agricultural Research, Bordeaux, France), were used as representatives of the two

3 Journal of Plant Pathology (2003), 85 (2), Miazzi et al. 125 Table 1. Origin of Uncinula necator isolates. Vineyard No. Location Cultivar No. of isolates Isolate code Sampling time Source 1 Valenzano (Bari) Several (a) 140 V-isolates July 1998 Bunches 2 Locorotondo (Bari) Several (b) 3 304,305,309 August 1999 Bunches 3 Sannicandro (Bari) Regina bianca 3 290,293,294 July 1999 Bunches 4 Brindisi (Brindisi) Negroamaro May 1999 Flag shoots 5 Cellino (Brindisi) Malvasia nera 3 47,48,49 June 1999 Flag shoots 6 Cellino (Brindisi) Negroamaro ,57 June 1999 Flag shoots 7 Cellino (Brindisi) Negroamaro June 1999 Flag shoots 8 Oria (Brindisi) Negroamaro 28 UO1-UO28 May-June 2000 Flag shoots 9 Trinitapoli (Foggia) Michele Palieri May 1999 Flag shoots 10 Cannole (Lecce) Negroamaro 2 314,315 September 1999 Bunches 11 Galatone (Lecce) Malvasia nera ,39-44,46 May 1999 Flag shoots 12 Ginosa (Taranto) Italia July 1999 Leaves 13 Vinosa (Taranto) Trebbiano July 1999 Bunches 14 Ginosa (Taranto) Italia 56 G1-G55, GG5 July 1999 Bunches 15 Massafra (Taranto) Victoria 3 251,256,260 July 1999 Bunches 16 Castellana (Pescara) Michele Palieri , July 1999 Bunches 17 Collalto (Pescara) Montepulciano , July 1999 Bunches 18 S. Martino (Termoli) Montepulciano July 1999 Bunches 19 S. Martino (Termoli) Montepulciano July 1999 Bunches 20 S. Martino (Termoli) Montepulciano 3 239,245,246 July 1999 Bunches 21 Nova Siri (Matera) Pinot bianco ,110, June 1999 Leaves 22 Cirò marina (Cosenza) Gaglioppo 5 76,80,82,85,86 June 1999 Flag shoots 23 Cirò marina (Cosenza) Gaglioppo June 1999 Flag shoots 24 Cirò marina (Cosenza) Gaglioppo June 1999 Leaves 25 Cirò marina (Cosenza) Gaglioppo June 1999 Leaves 26 Cirò marina (Cosenza) Gaglioppo 4 126,129,130,132 June 1999 Leaves 27 Cirò marina (Cosenza) Gaglioppo June 1999 Leaves 28 Chiaramonte (Ragusa) Italia 2 150,155 July 1999 Leaves 29 Cannizzaro (Ragusa) Italia July 1999 Bunches 30 Chiaramonte (Ragusa) Italia July 1999 Bunches 31 Primosole (Catania) Italia 2 159,160 July 1999 Bunches (a) Alphonse Lavallèe, Baresana, Italia, Negroamaro, Regina bianca, Sangiovese, Trebbiano, Uva di Troia. (b) Malvasia nera, Montonico nero. biotypes: F1 belonged to the flag shoot biotype and F3 to the ascospore biotype (Délye et al., 1998). RAPD amplifications were carried out in 25 µl containing 10 mm Tris-HCl, ph 9.0; 50 mm KCl; 0,1% Triton X-100; 2 mm MgCl 2 ; 75 µm each of datp, dctp, dgtp and dttp (Promega, Madison, WI, USA); 0,5 µm primer; 100 ng of target DNA and 2 U of Taq DNA Polymerase (Promega, Madison, WI, USA). Reactions were carried out in a thermal cycler (Geneamp PCR System 9700; Perkin Elmer, Norwalk, USA) programmed as follows: 4 min at 95 C (initial denaturation); 40 cycles of 30 s at 94 C, 30 s at 35 C, 30 s at 72 C; and a conclusive extension phase of 7 min at 72 C. Sixty-three 10-mer primers (Kit A, B, D; Operon Technologies, Alameda, CA, USA) were used for DNA amplification from a first set of 28 isolates. Twenty-three primers revealing higher polymorphism were used to amplify DNA extracted from the remaining isolates. Two independent amplifications were done for each isolate and primer combination. Aliquots (10 µl) of reaction mixtures were loaded on 1,4% Amplisize agarose gel (Bio-Rad Laboratories, Hereules, CA, USA) and run in 0,5xTBE buffer (45 mm Tris-borate, 1 mm Na-EDTA; ph 8) at 110 V for 110 min (Sub-Cell TM, Bio-Rad Laboratories, Hercules, CA, USA). A 100-bp DNA Ladder (New England Bio- Labs, Beverly, USA), giving 12 bands between 100 and 1,500 bps, was used as a standard for molecular sizes. Gels were stained with 1 µg ml -1 ethidium bromide for 40 min, destained in water for 10 min, and gel images were digitalised with a Gel Doc 1000 System (Bio-Rad Laboratories, Hercules, CA, USA).

4 126 Population biology of Uncinula necator Journal of Plant Pathology (2003), 85 (2), Data analysis. Manipulation of gel images and elaboration of data were carried out using the software package Diversity Database (Ver. 2.1 for Windows; Bio- Rad Laboratories, Hercules, CA, USA). The banding patterns of each isolate was scored for the presence or absence of each marker. To provide a quantitative measure of relatedness among isolates, genetic similarity between all pairs of isolates was estimated according to the formula 2bij/(bij + bi + bj), where bij is the number of amplicons shared by two isolates i and j, and bi and bj are the number of unshared bands (Dice, 1945; Nei and Li, 1979). Similarity matrices were then used to construct a phylogenetic tree according to the Unweighted Pair Group Method using Arithmetic Averages (UPGA- MA). Data were also submitted to Principal Components Analysis. RESULTS AND DISCUSSION A collection of 374 isolates of U. necator was established from samples of naturally infected grape tissues collected in 31 vineyards located in 6 regions of South Italy (Abruzzo, Basilicata, Calabria, Molise, Puglia and Sicilia). All the isolates of U. necator were singly mated with two reference strains of known mating type, UN52 (MAT1-1) and UN68 (MAT1-2). The number of cleistothecia in fertile crosses was broadly variable depending on isolates and crosses; some crosses yielded numerous cleistothecia, scattered all over the fungal colonies, while others yielded only few cleistothecia. All isolates proved fertile with only one of the reference strains. No isolates were able to differentiate cleistothecia in crosses with both the reference strains, or when they were grown alone on grapevine leaves. These results corroborate previous findings on the sexual behaviour and mating system of U. necator that can be ascribed to a bipolar heterothallism under the control of a single major gene, MAT1, with its two alleles MAT1-1 and MAT1-2 (Gadoury and Pearson, 1991; Miazzi et al., 1997, 2002). The two mating types, MAT1-1 and MAT1-2, were present in similar proportions in all pathogen populations, although the former prevailed slightly over the latter: 199 isolates were MAT1-1 and 175 were MAT1-2 (Table 2). The two mating types were present in similar proportions even in single vineyards (Table 2). Numerous isolates (140) were sampled in an experimental vineyard made up by vines of 8 cultivars. Mating types were evenly present on all the cultivars and showed an aggregated distribution in the vineyard (Fig. 2). In preliminary RAPD experiments carried out with 28 fungal isolates, 23 out of 63 tested primers generated from 1 to 10 amplicons that separated in agarose gel into discrete bands corresponding to molecular sizes ranging from 200 to 1,300 bps (Fig. 3). RAPD profiles Table 2. Distribution of mating types in Uncinula necator isolates of different origin. Vineyard No. No. of tested isolates No. of isolates MAT Total MAT1-2 Fig. 2. Distribution of mating types, MAT1-1 (dark grey) and MAT1-2 (light grey), in the population of Uncinula necator in an experimental vineyard made up by 8 grape cultivars. Each cell represents a vine: I = Italia ; S = Sangiovese ; L = Alphonse Lavallèe ; R = Regina ; N = Negroamaro ; U = Uva di Troia ; B = Baresana ; T = Trebbiano.

5 Journal of Plant Pathology (2003), 85 (2), Miazzi et al. 127 were unchanged even when PCR reaction was carried out on DNA extracted from the same isolates one year later, i.e. about after 12 asexual generations by colony transfer. Contamination by grapevine DNA was never detected in RAPD amplifications (data not shown). Fig. 3. Examples of banding patterns obtained using two single 10-mer primers (OPD5, left; OPD8, right) to amplify DNA from in vitro-grown isolates of Uncinula necator. Arrows indicate markers discriminating isolates belonging to the two hypothesized biotypes of the pathogen. The 23 selected primers were used with the whole collection of 374 isolates of U. necator. These yielded 137 amplicons, 55 of which were polymorphic. Polymorphic markers were used to establish genetic relatedness among fungal isolates. RAPD analysis showed a limited genetic variation in U. necator, the mean value of genetic similarity in the whole population being The UPGMA cluster analysis discriminated two major groups of isolates (Fig. 4). Group I included all the 53 isolates collected from flag shoots and 30 isolates sampled from leaves or bunches early in the season, from May to the beginning of June; intra-group mean genetic similarity was Group II included the remaining 291 isolates collected later in the season; intragroup mean genetic similarity was Inter-group genetic similarity was 0.82, only slightly lower than that of the whole population. Usually, into each of the two major groups, isolates sampled in a same vineyard clustered together (Fig. 4). Two French reference strains of the biotype flag shoot and ascospore (Délye et al., 1997) clustered in Group I and II, respectively. The existence of the two distinguishable groups of isolates was also confirmed by Principal Components Analysis (Fig. 5). A F Fig. 4. Phylogenetic tree derived by UPGMA analysis of RAPD data: Group I: isolates derived from flag shoots (A indicates isolates from flag shoots from a single vineyard). Group II: isolates derived from leaves and bunches after may; letters B to F indicate sub-groups of the Group II, formed in the experimental vineyard N. 1. Letter G indicates isolates from the vineyard N. 14. Fig. 5. Principal component analysis of variation in U. necator isolates on the ground of RAPD markers. Notice the discrimination of isolates from flag shoots (F) from those collected later in the season and likely deriving from ascospores (A).

6 128 Population biology of Uncinula necator Journal of Plant Pathology (2003), 85 (2), These results corroborate previous findings on the existence of two distinct biotypes in U. necator (Délye et al., 1997, 1998). Isolates belonging to Group I were found only until June. This suggests that such variant of the pathogen, which is prevalent early in the season, tends to disappear, showing a very low, if any, frequency into the fungal population later in the season. To explain the phenomenon, Délye et al. (1997) hypothesized that the active stage of the flag shoot biotype is limited to spring, when it infects primarily shoots and leaves; then it would survive in latent form into infected buds until the next spring, when new infections occur. Mating types were similarly distributed into the two major groups of isolates: 37 and 46 isolates were, respectively, MAT1-1 and MAT1-2 in Group I ( flag shoot biotype); 162 and 129 isolates were, respectively, MAT1-1 and MAT1-2 in Group II ( ascospore biotype). Délye et al. (1998) crossed isolates belonging to the two different biotypes and observed differentiation of cleistothecia but not of viable ascospores; hence, they concluded that the two biotypes of the fungus are genetically isolated since gene recombination allowed by the sexual process is prevented. On the contrary, in our study, crosses between isolates of opposite mating types recognized as belonging to the flag shoot and the ascospore biotype were fertile, forming normal cleistothecia and ascospores within 7 weeks. Ascospores proved normally viable when stained with the vital stain fluorescein diacetate. Cortesi et al. (personal communication) obtained similar results in crosses among U. necator isolates from Tuscany. Therefore, genetic separation of the two biotypes is not due to sexual sterility, and the sexual process can occur in nature. Likely, the different ecological niches they occupy result in a temporal separation preventing mating. The flag shoot biotype, active only early in the season, surviving in infected buds and in a quiescent state in late summer, might be impaired in recombining with the ascospore biotype that is active later in the season when the sexual process occurs and cleistothecia are differentiated in vineyards. Further observations on this aspect are in progress in the hope that more environment- and consumer-friendly crop protection strategies can be designed with a deeper knowledge on U. necator population biology. For instance, spraying early in the season could be useless if the flag shoot biotype is not really responsible of infection of bunches. Biotype-specific markers have been used for developing SCAR (Sequence Characterised Amplified Regions) primers for epidemiological studies (Délye et al., 1997; Hajjeh et al., unpublished results). These should allow following the evolution of the composition of U. necator populations during the grapevine-growing season and to understand the roles of the two biotypes in the pathogen s life cycle and in the epidemiology of grape powdery mildew. The combination of mating types and RAPD markers proved effective in the study of the genetic structure of the pathogen s population present in an experimental vineyard with 8 cultivars. In this experimental vineyard, RAPD analysis revealed the existence of 5 groups of isolates (B-F) characterised by intra-group genetic similarity higher than 0.94; they showed a clear aggregation in the vineyard, resembling that of mating types (data not shown). Mating types were distributed in the 5 groups as follows: 6 and 23; 23 and 1; 12 and 16; 10 and 19; 24 and 6; respectively for MAT1-1 and MAT1-2. Both types of markers showed that similar isolates showed an aggregated distribution in the vineyard, independently from cultivars. This result supports the hypothesis formulated by Bulit and Lafon (1978) and Willoquet (1994), according to which, whatever the prevalent overwintering mode, the spreading of U. necator clones in vineyards would be rather limited and at short distance, in contrast with the nature of its winddispersed conidia. The dissemination of the fungus over long distances would be more likely due to man s activities (Délye et al., 1997). The similar distribution of mating types observed in the pathogen s populations in Southern Italy indicates that there are no obstacles, neither geographical nor temporal, that prevent matching of sexually compatible individuals. Hence, the occurrence of the sexual process, leading to differentiation of cleistothecia and ascospores, depends exclusively on the occurrence of permissive environmental conditions, and may play an important role in the population biology of U. necator, acting as a source of release of new recombinant genotypes and increasing the inoculum responsible of primary infections in the next season. ACKNOWLEDGEMENTS Research granted by the Minister of Agricultural and Forestry Politics, project Genetic variability of fungal pathogens and selection for resistance of grapevine to biotic stress (Coordinated Research Assisted selection for resistance to biotic stress and qualitative improvement of fruit trees, National Programme on Plant Biotechnology), by the University of Bari, project Epidemiology and genetics of phytopathogenic microorganisms, and by CEGBA (Centro di Eccellenza in Genomica Comparata in Campo Biomedico e Agrario), Research Line n. 7. We thank Prof. M.F. 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