D. Gobbin

Identification of microsatellite markers for Plasmopara viticola and establishment of high throughput method for SSR analysis

The Oomycete Plasmopara viticola is the causal organism of downy mildew on grapevine (Vitis spp.). In order to set up the techniques for investigating downy mildew disease dynamics and genetic structure, co-dominant, neutral, highly reproducible and polymorphic microsatellite markers for P. viticola were developed. Five markers, two with a (TC)n repeat (loci BER and ISA), two with a (TC)n(AC)n repeat (loci CES and REX) and one with a (CT)n(CTAT)n repeat (locus GOB), were selected. Simple sequence repeat (SSR) markers revealed different degrees of polymorphism within 190 oil spots (disease symptoms) collected from an infected Italian vineyard. The most polymorphic SSR marker GOB showed 43 alleles (Neis expected gene diversity He = 0.89) while CES, ISA, BER and REX showed 14 (He = 0.71), 4 (He = 0.57), 3 (He = 0.24) and 1 allele (He = 0), respectively. A high throughput DNA extraction method, that allowed molecular analysis of this obligate pathogen directly in the host without any isolation procedure, was developed. The quality and quantity of oil spots did not influence the SSR analysis. Amplified SSR loci were separated by electrophoresis on a Beckman–Coulter 2000XL sequencer and automatically analysed. The objective of this study was to develop molecular biological tools and methods that allow high throughput analysis of the downy mildew populations.

Development of a High-Throughput Method for Quantification of Plasmopara viticola DNA in Grapevine Leaves by Means of Quantitative Real-Time Polymerase Chain Reaction.

ABSTRACT Plasmopara viticola is a strictly biotrophic oomycete that causes downy mildew, which is one of the most important grapevine diseases. Control of the disease is most often achieved by fungicide applications, which may have severe environmental consequences. Therefore, alternative control strategies based on biocontrol agents (BCAs) are currently in development. Thousands of potential BCAs have to be screened for their antagonist efficacy against Plasmopara viticola. Evaluation of their effect on the pathogen can be achieved by detecting the amount of P. viticola DNA in leaves treated with potential antagonists and infected with the pathogen. In this study, a rapid high-throughput method was developed for relative quantification of P. viticola DNA directly from Vitis vinifera leaves by means of multiplex real-time quantitative polymerase chain reaction (PCR) with TaqMan chemistry. This method allows simultaneous amplification, but independent detection, of pathogen and host DNA by using species-specific primers and TaqMan probes that are labeled with different fluorescent dyes. Including detection of V. vinifera DNA in the tests is fundamental because it provides an endogenous reference and allows normalization for variations caused by sample-to-sample differences in DNA extraction, PCR efficiencies, and pipetting volumes. The developed method allows highly sensitive and specific detection of P. viticola DNA (minimal detectable quantity of 0.1 pg). Moreover, high precision and reproducibility of TaqMan assays were observed over a linear range of four orders of magnitude, confirming the reliability of the developed PCR assay. Potential applications range from screening for BCA efficiency to evaluation of fungicide efficacy, or assessment of host resistance.

Development and test of 21 multiplex PCRs composed of SSRs spanning most of the apple genome

A series of 21 multiplex (MP) polymerase chain reactions containing simple sequence repeat (SSR) markers spanning most of the apple genome has been developed. Eighty-eight SSR markers, well distributed over all 17 linkage groups (LGs), have been selected. Eighty-four of them were included in 21 different MPs while four could not be included in any MPs. The 21 MPs were then used to genotype approximately 2,000 DNA samples from the European High-quality Disease-Resistant Apples for a Sustainable agriculture project. Two SSRs (CH01d03 and NZAL08) were discarded at an early stage as they did not produce stable amplifications in the MPs, while the scoring of the multilocus (ML) SSR Hi07d11 and CN44794 was too complex for large-scale genotyping. The testing of the remaining 80 SSRs over a large number of different genotypes allowed: (1) a better estimation of their level of polymorphism; as well as of (2) the size range of the alleles amplified; (3) the identification of additional unmapped loci of some ML SSRs; (4) the development of methods to assign alleles to the different loci of ML SSRs and (5) conditions at which an SSR previously described as ML would amplify alleles of a single locus to be determined. These data resulted in the selection of 75 SSRs out of the 80 that are well suited and recommended for large genotyping projects.

Population genetic structure of Plasmopara viticola after 125 years of colonization in European vineyards

SUMMARY To examine the within- and among-population genetic structure of Plamopara viticola oosporic populations in Europe, 8991 lesions from 32 vineyard plots were collected and analysed. Four multi-allelic microsatellite markers were used to genotype the pathogen. All populations had high levels of gene and genotypic diversity. Most populations were in Hardy-Weinberg equilibrium and thus randomly mating. Among P. viticola populations, significant low to moderate genetic differentiation was observed, even between geographically close populations. This genetic differentiation was also evident in the neighbour-joining phylogenetic genetic distance tree, showing clear substructure and distinguishing mainly five clusters based on geographical origin. Significant isolation by distance was found in central European P. viticola populations, suggesting a step-wise migration model. No significant isolation by distance was found within Greek populations, most probably owing to natural geographical barriers such as the sea and mountains, as well as the frequent population bottlenecks occurring in these populations, preventing natural migration among populations. The high variability of P. viticola provides explanation for its successful infestation of the heterogeneous European vineyards in the last 125 years after its introduction.

Microsatellite-based characterization of the Castanea sativa cultivar heritage of southern Switzerland.

Southern Switzerland has a long tradition of chestnut cultivation as a staple food. Local inhabitants constantly selected varieties according to the ripening period, the type of use, and the adaptability to the territory. As a result, the panorama of chestnut varieties is very complex, as reflected by more than 120 different variety names in an area of 26,000 ha. Since 1994, 47 varieties have been conserved in the chestnut germplasm of southern Switzerland (CSS), including Marroni, Euro-Japanese, and French varieties. A selection of 164 individuals from the CSS was analysed by 8 SSR markers (4 of which were developed in this study). Microsatellite analysis indicated that the CSS was accurately established, as 86% of the individuals grafted were correctly labeled. The identification of 98 genotypes, 10 clonal chestnut groups, 4 synonym groups, and 12 homonym groups reflected the complex ethnogeographical structure of the chestnut distribution. The 17 Marroni individuals considered clustered in 2 differentiated genetic groups instead of only 1 as expected. The fundamental problem of the frequent cases of homonymy and synonymy is discussed, as is the need for criteria for discriminating between polyclonal varieties and distinct homonymous varieties.

Selection for fungicide resistance throughout a growing season in populations of Plasmopara viticola

A method for evaluating the potential threat of selection for resistance to organically-based fungicides in populations of P. viticola is needed to screen a large panel of products alternative to copper in organic viticulture. Populations from an unexposed plot were compared throughout one season with a population sprayed with azoxystrobin (Quadris), reported as engendering selection pressure and resistance, and a population sprayed with an organically-based fungicide (Mycosan). The evolution of the three populations was followed with neutral specific SSR markers and with the specific marker for strobilurin resistance, as control of selection for resistant mutants. A reduction in genetic diversity of the P. viticola population was observed in the population sprayed with azoxystrobin, consistent with directional selection toward higher resistance, confirmed by an enhanced frequency of resistant mutants with respect to the unexposed population. In contrast, a higher diversity and a reduced frequency of resistant mutants were observed in the population sprayed with the organically-based fungicide. Assessing a reduction of genotypic diversity allows the detection of selection for resistance and constitutes a valid instrument for screening a large panel of products with non-specific, different and possibly indirect modes of action.

Microsatellite based population structure of Plasmopara viticola at single vine scale

The genetic structure of a Plasmopara viticola population was characterized on five single vines, one for each cultivar Regent, Merlot, Isabella, Müller-Thurgau and Solaris, using four neutral specific polymorphic microsatellite markers. Five-hundred and seventy samples were collected at four dates in the period between the 10th of July and the 23rd of August 2006. On average over all five cultivars, 67% of the genotypes present on the single selected vines derived from primary infections and caused 37% of the lesions genotyped. Fifty-three percent of these genotypes occurred only once on the vine throughout the survey period, while 14% were able to asexually reproduce on the selected single vine throughout the survey period, causing 23% of the lesions. Thirty-three percent of the genotypes on the single vine derived from other vines, 28% from vines of other cultivars in the other rows, and 5% from vines of the same cultivar in the same row. New primary infections appear all along the sampling dates. The overwhelmingly quantitative role of primary infections at vineyard scale was known, however here we observed the phenomenon also at the single vine scale and the reduced contribution of secondary lesions to the populations present on more resistant cultivars compared to the susceptible cultivars. As the sampling extended almost to defoliation, the results are judged to be representative of a typical P. viticola epidemic.

Contribution of molecular studies to botanical epidemiology and disease modelling: grapevine downy mildew as a case-study

After being accidentally introduced from the USA at the end of the 19th century, downy mildew caused by Plasmopara viticola (Berk. et Curt.) Berlese et De Toni became one of the most damaging diseases affecting Vitis vinifera in Europe. Downy mildew causes both direct and indirect losses and can lead to severe reduction of yield. Our understanding of the life cycle and epidemiology of P. viticola has been recently altered by molecular studies that revealed that the overwintering inoculum (i.e., the oospores) does more than initiate disease, as was previously thought. A mechanistic model was developed for predicting the entire chain of processes leading to primary infections, and this primary infection model was linked to other models of secondary infection cycles. The model for primary infections defines the length of the primary inoculum season and a seasonal oospore dose consisting of several cohorts of oospores that progressively mature. The model was evaluated by means of Bayesian analysis in both Italy and eastern Canada, and showed high sensitivity, specificity, and accuracy both for potted plants and vineyards. Fungicide applications are necessary to control downy mildew because preventive agronomic practices are not very effective, including host resistance. The use of warning systems based on weather-driven models leads to a reduction in the use and cost of chemicals and a reduction in their environmental impact.

Prevalence of type III secretion system in effective biocontrol pseudomonads

Functional type III secretion system (T3SS) genes are needed for effective biocontrol of Pythium damping-off of cucumber by Pseudomonas fluorescens KD, but whether biocontrol Pseudomonas strains with T3SS genes display overall a higher plant-protecting activity is unknown. The assessment of 198 biocontrol fluorescent pseudomonads originating from 60 soils worldwide indicated that 32% harbour the ATPase-encoding T3SS gene hrcN, which was most often found in tomato isolates. The hrcN(+) biocontrol strains (and especially those also producing 2,4-diacetylphloroglucinol and displaying 1-aminocyclopropane-1-carboxylate deaminase activity) displayed higher plant-protecting ability in comparison with hrcN(-) biocontrol strains, both in the Pythium/cucumber and Fusarium/cucumber pathosystems.

Genetic Diversity of Armillaria spp. Infecting Highbush Blueberry in Northern Italy (Trentino Region)

Armillaria spp. are the causal agents of root rots of several woody plants, including highbush blueberry. Since 2003, highbush blueberry plants infected by Armillaria spp. have been found in Valsugana Valley, Trentino region, northern Italy. Our aim was to identify the Armillaria spp. involved in these infections, as well as possible sources of inoculum in blueberry fields. Samples of Armillaria spp. were collected from diseased blueberry plants in 13 infected blueberry fields, from bark spread along the blueberry rows, from infected trees in the vicinity of the fields, and from four forest locations. The identification of Armillaria spp. was accomplished using a species-specific multiplex polymerase chain reaction method and by sequencing the rDNA at a specific locus. The differentiation between genotypes was performed by using simple-sequence repeat analysis. Armillaria mellea and A. gallica were the most frequently observed species infecting blueberry in the Valsugana Valley. Three to eight Armillaria genotypes were identified in each blueberry field. No individual genotypes were found in more than one blueberry field. Two-thirds of the genotypes found colonizing trees in the immediate vicinity of infected fields and two-thirds of the genotypes found colonizing the bark spread in blueberry rows were also isolated from blueberry plants in the field, indicating that bark used as mulch and infected trees surrounding the fields may be important sources of inoculum.