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Biology and genetics of the grape powdery mildew fungus, Erysiphe necator

 

Phylogeography of native and introduced populations

Based on historical records and the relatively high resistance of North American Vitis species to powdery mildew, E. necator has been thought to be native to eastern North America, and introduced into all other grape-growing regions of the world.  Studies on genetic variation in E. necator in Europe and Australia have shown the presence of only two distinct genetic groups or lineages, A and B. To our knowledge, no comparable studies have been done on the diversity of E. necator populations in North America.  Therefore, we collected approximately 100 E. necator isolates from North America, and obtained DNA from both lineages from Europe and Australia.  We obtained a total 1803 bp of sequence from three gene regions in more than 150 isolates and found 37 single-nucleotide polymorphisms (SNPs). 

Based on phylogeographic analyses we found the following:  First, populations in the eastern US are much more diverse than in Europe and Australia.  Second, we found only two haplotypes, which differ by one SNP, in California and Oregon, both in the group B lineage, which predominates in Europe and Australia.  Third, we found isolates in the group A lineage in the southeastern US; no variation was found among group A isolates from Europe and Australia.  From these results we conclude that populations in the eastern US are the most likely source for introduction to the west coast of the US, Europe and Australia.

Relevant recent publications:

--Brewer MT, Milgroom MG, 2010. Phylogeography and population structure of the grape powdery mildew fungus, Erysiphe necator, from diverse Vitis species. BMC Evolutionary Biology, 10: 268. [full text]

 

Variation in pathogenicity and aggressiveness

We studied variation in aggressiveness of E. necator, particularly in populations sampled from wild Vitis species in the eastern US.  To study aggressiveness, we inoculated 50 isolates from five Vitis species onto detached leaves of V. vinifera and the V. labrusca hybrid cv. ‘Niagara’ and determined lesion size and latent period.  We found significant variation among isolates for both lesion area and latent period.  Differences among isolates were larger on ‘Niagara’ than on V. vinifera, as reported previously. 

Muscadine grapes (V. rotundifolia) commonly grow wild and are cultivated in the southeastern US.  Some taxonomic schemes have considered them to be different enough from other Vitis species that they are sometimes placed in their own genus (Muscadinia).  We found evidence of host specialization where only isolates collected from V. rotundifolia could form sporulating colonies on V. rotundifolia.  Interestingly, however, isolates collected from V. rotundifolia only differed from a common haplotype found on other Vitis species in the southeastern US by one or two SNPs.  To continue studying host specialization among other Vitis species, we also tested for evidence of genetic structure with respect to host species of origin (see below). 

Relevant recent publications:

-- Frenkel O, Brewer MT, Milgroom MG, 2010. Variation in pathogenicity and aggressiveness of Erysiphe necator from different Vitis species and geographic origins in the eastern United States. Phythopathology 100: 1185-1193. [abstract][full text].

 

Transcriptome sequencing and marker development

In collaboration with Lance Cadle-Davidson, USDA-ARS, Grape Genetics Research Unit, Geneva, NY, we sequenced the transcriptome of E. necator. The aim of this project is to develop tools for expression profiling and to develop additional genetic markers. The first draft of these sequences became available in June 2009 (Click here for more information about transcriptome sequences).  Additional sequences will be added to this initial assembly in 2011.  The preliminary transcriptome sequence has already aided in identifying differential expression of genes associated with sporulation (originally identified by differential cDNA-AFLPs), and identifying homologs to genes associated with DMI resistance in other fungi (see below). 

The transcriptome sequence has been useful for developing microsatellite (SSR) and mating-type markers (see below). An ongoing project is to discover a large number of single-nucleotide polymorphisms (SNPs) for genetic studies.  Therefore, we have resequenced seven additional isolates of E. necator.  These sequences will be compared to the reference transcriptome to find SNPs.

Relevant recent publications:

-- Wakefield, L., Gadoury, D. M., Seem, R. C., Milgroom, M. G., Sun, Q. and Cadle-Davidson, L. 2011. Differential gene expression during conidiation in the grape powdery mildew fungus, Erysiphe necator. Phytopathology (in press). [abstract][full text]

 

Mating-type genes in E. necator and other powdery mildews:

We identified the mating-type gene MAT1-2-1 in E. necator by searching in the genome sequence of the cereal powdery mildew fungus, Blumeria graminis f. sp. hordei, for the conserved HMG domain. Based on finding a marker for mating type MAT1-2, we chose an isolate of the opposite mating type for transcriptome sequencing. We identified the genes MAT1-1-1 and MAT1-1-3 at the mating-type locus in the transcriptome sequence of E. necator. With sequences of genes from both mating-type idiomorphs we developed a multiplex PCR marker to determine mating type in E. necator. We also developed degenerate primers to isolate conserved regions of both mating-type genes in other powdery mildew fungi, including B. graminis f. sp. hordei, B. graminis f. sp. tritici, Microsphaera syringae and Podosphaera xanthii. These markers will facilitate studies of mating types in populations of powdery mildew fungi.

Relevant recent publications:

--Brewer, M. T., Cadle-Davidson, L., Cortesi, P., Spanu, P. D., Milgroom, M. G., 2011. Identification and structure of the mating-type locus and development of PCR-based markers for mating type in powdery mildew fungi. Fungal Genetics and Biology doi:10.1016/j.fgb.2011.04.004.

 

Population structure in the eastern US assessed by microsatellites:

We used transcriptome sequences of E. necator to develop microsatellite markers (EST-SSRs) to study its relatively unexplored population structure in its center of diversity in eastern North America. Screening the transcriptome sequences revealed 116 contigs with candidate microsatellites, from which we developed 11 polymorphic microsatellite markers from 31 markers tested. Using eight of these markers, we genotyped 129 isolates from different regions and hosts in the eastern USA and compared them to samples from southern France and Italy. Genetic diversity in the eastern USA is much greater than in Europe. Bayesian cluster analyses showed that 10 isolates from North America have high affinities with, but differ from, European group A; these are referred to as A-like isolates. No isolates with close affinity to European group B were found in the eastern USA. Bayesian analyses also detected genetic differentiation between isolates from V. rotundifolia and isolates from other Vitis hosts. Genetic differentiation detected between the northeastern and southeastern USA was mostly attributable to the A-like isolates in the southeast, which are significantly more aggressive than the other populations. This research demonstrates that transcriptome sequencing of fungal pathogens is useful for developing genetic markers in protein-coding regions and highlights the role of these markers in population biology studies of E. necator.

Relevant recent publications:

--Frenkel O, Portillo I, Brewer MT, Péros J-P, Cadle-Davidson L, Milgroom MG, 2011. Development of microsatellite markers from the transcriptome of Erysiphe necator for analyzing population structure in North America and Europe. Plant Pathology (accepted with revision).

 

Gene expression correlated to DMI resistance

We have used the transcriptome sequences of E. necator to find homologs of ATP-binding cassette (ABC) transporter genes because of their potential role in DMI resistance.  In an effort to determine which of these genes contributes to the DMI resistance phenotype in E. necator, we have designed PCR primers for quantitative RT-PCR for use on cDNA.  We will quantify the expression of ABC transporter genes and also C14-demethylase (CYP51) from isolates that span a spectrum of resistance phenotypes to DMI fungicides to test for correlations.  These studies are currently in progress.

Additional publications on grape powdery mildew

grape powdery mildew on leaves

Signs and symptoms of grape powdery mildew (Photo from P. Cortesi).


grapes with PM

Grape cluster with powdery mildew, caused by Erysiphe necator (Photo from P. Cortesi).