Overexpression of the CYP51A1 Gene and Repeated Elements are Associated with Differential Sensitivity to DMI Fungicides in Venturia inaequalis.

The involvement of overexpression of the CYP51A1 gene in Venturia inaequalis was investigated for isolates exhibiting differential sensitivity to the triazole demethylation inhibitor (DMI) fungicides myclobutanil and difenoconazole. Relative expression (RE) of the CYP51A1 gene was significantly greater (P < 0.0001) for isolates with resistance to both fungicides (MRDR phenotype) or with resistance to difenoconazole only (MSDR phenotype) compared with isolates that were resistant only to myclobutanil (MRDS phenotype) or sensitive to both fungicides (MSDS phenotype). An average of 9- and 13-fold increases in CYP51A1 RE were observed in isolates resistant to difenoconazole compared with isolates with MRDS and MSDS phenotypes, respectively. Linear regression analysis between isolate relative growth on myclobutanil-amended medium and log10 RE revealed that little to no variability in sensitivity to myclobutanil could be explained by CYP51A1 overexpression (R(2) = 0.078). To investigate CYP51A1 upstream anomalies associated with CYP51A1 overexpression or resistance to difenoconazole, Illumina sequencing was conducted for three isolates with resistance to difenoconazole and one baseline isolate. A repeated element, "EL 3,1,2", with the properties of a transcriptional enhancer was identified two to four times upstream of CYP51A1 in difenoconazole-resistant isolates but was not found in isolates with the MRDS phenotype. These results suggest that different mechanisms may govern resistance to different DMI fungicides in the triazole group.

Genome sequencing and mapping reveal loss of heterozygosity as a mechanism for rapid adaptation in the vegetable pathogen Phytophthora capsici.

The oomycete vegetable pathogen Phytophthora capsici has shown remarkable adaptation to fungicides and new hosts. Like other members of this destructive genus, P. capsici has an explosive epidemiology, rapidly producing massive numbers of asexual spores on infected hosts. In addition, P. capsici can remain dormant for years as sexually recombined oospores, making it difficult to produce crops at infested sites, and allowing outcrossing populations to maintain significant genetic variation. Genome sequencing, development of a high-density genetic map, and integrative genomic or genetic characterization of P. capsici field isolates and intercross progeny revealed significant mitotic loss of heterozygosity (LOH) in diverse isolates. LOH was detected in clonally propagated field isolates and sexual progeny, cumulatively affecting >30% of the genome. LOH altered genotypes for more than 11,000 single-nucleotide variant sites and showed a strong association with changes in mating type and pathogenicity. Overall, it appears that LOH may provide a rapid mechanism for fixing alleles and may be an important component of adaptability for P. capsici.

Overexpression of ShCYP51B and ShatrD in Sclerotinia homoeocarpa isolates exhibiting practical field resistance to a demethylation inhibitor fungicide.

We investigated genetic factors that govern the reduced propiconazole sensitivity of Sclerotinia homoeocarpa field isolates collected during a 2-year field efficacy study on dollar spot disease of turf in five New England sites. These isolates displayed a >50-fold range of in vitro sensitivity to a sterol demethylation inhibitor (DMI) fungicide, propiconazole, making them ideal for investigations of genetic mechanisms of reduced DMI sensitivity. The CYP51 gene homolog in S. homoeocarpa (ShCYP51B), encoding the enzyme target of DMIs, is likely a minor genetic factor for reduced propiconazole sensitivity, since there were no differences in constitutive relative expression (RE) values and only 2-fold-higher induced RE values for insensitive than for sensitive isolate groups. Next, we mined RNA-Seq transcriptome data for additional genetic factors and found evidence for the overexpression of a homolog of Botrytis cinerea atrD (BcatrD), ShatrD, a known efflux transporter of DMI fungicides. The ShatrD gene showed much higher constitutive and induced RE values for insensitive isolates. Several polymorphisms were found upstream of ShatrD but were not definitively linked to overexpression. The screening of constitutive RE values of ShCYP51B and ShatrD in isolates from two golf courses that exhibited practical field resistance to propiconazole uncovered evidence for significant population-specific overexpression of both genes. However, linear regression demonstrated that the RE of ShatrD displays a more significant relationship with propiconazole sensitivity than that of ShCYP51B. In summary, our results suggest that efflux is a major determinant of the reduced DMI sensitivity of S. homoeocarpa genotypes in New England, which may have implications for the emergence of practical field resistance in this important turfgrass pathogen.

Genetic diversity of Phytophthora capsici isolates from pepper and pumpkin in Argentina.

Phytophthora capsici is a soilborne oomycete plant pathogen that limits pepper production worldwide. The population structure varies significantly depending on the location (e.g. Peru vs. USA) and little is known about the diversity of P. capsici in Argentina. Our objective was to assess the diversity of P. capsici in Argentina at key pepper production areas. Forty isolates were recovered 2006-2009 from pepper and one isolate from pumpkin at 11 locations. Isolates were assessed for mating type, mefenoxam sensitivity and multilocus single nucleotide polymorphism (SNP) genotype profiles. Ten isolates with identical SNP profiles also were genotyped with amplified fragment length polymorphism (AFLP) markers. All 41 isolates had the A1 mating type and were sensitive to mefenoxam. Genotypic analysis using eight polymorphic SNP markers indicated 87% of the isolates had the same multilocus genotype, which is fixed for heterozygosity at seven of the eight SNP sites. AFLP analyses confirmed these findings, and overall it appears that clonal reproduction drives the population structure of P. capsici in Argentina. The implications for breeding resistant peppers and overall disease management are discussed.

Co-occurrence and genotypic distribution of Phytophthora species recovered from watersheds and plant nurseries of eastern Tennessee.

In 2008 statewide surveys of symptomatic foliage of nursery plants from Tennessee resulted in isolation of 43 isolates of Phytophthora spp. This sample set includes four described species (P. citrophthora, P. citricola, P. nicotianae, P. syringae), and a provisional species of Phytophthora ('P. hydropathica'). At the same time a stream-baiting survey was initiated to recover Phytophthora from eight watersheds in eastern Tennessee, some of which are near plant nurseries. Baiting was accomplished by submerging healthy Rhododendron leaves approximately 1 wk and isolation onto selective media. Six baiting periods were completed, and in total 98 Phytophthora isolates and 45 isolates of Pythium spp. were recovered. Three described species (P. citrophthora, P. citricola and P. irrigata) and the provisional species 'P. hydropathica' were obtained as well as three undescribed Phytophthora taxa and Pythium litorale. Isolates from both surveys were identified to species with morphology and the internal transcribed spacer (ITS) sequence. Isolates from species co-occurring in streams and nurseries (P. citricola, P. citrophthora and 'P. hydropathica') were characterized further with amplified fragment length polymorphism (AFLP) analyses and mefenoxam tolerance assays. Isolates representing a putative clonal genotype of P. citricola were obtained from both environmental and nursery sample sets.

Loss of heterozygosity in Phytophthora capsici after N-ethyl-nitrosourea mutagenesis.

Loss of heterozygosity (LOH) occurs in a variety of diploid organisms after chemical mutagenesis and was observed in the vegetable pathogen Phytophthora capsici after N-ethyl-nitrosourea (ENU) mutagenesis at three loci during reverse genetic screening. Our objectives were to determine (i) the frequency of LOH among mutants, (ii) the directionality of the LOH events and (iii) the length of the genomic tracts exhibiting LOH. Of the 1152 ENU mutants screened, LOH was most frequent at locus 3 (99 ENU mutants), with locus 1 (10 ENU mutants) and locus 2 (9 ENU mutants) undergoing LOH at similar frequencies. LOH was bidirectional for all three loci, with locus 3 mutants biased toward one haplotype. Analysis of upstream and downstream heterozygosity indicates that the LOH events spanned up to at least 4.6 kb. The implications of mitotic recombination and LOH for reverse genetics and natural variation in Phytophthora are discussed.

A pleiotropic drug resistance transporter is involved in reduced sensitivity to multiple fungicide classes in Sclerotinia homoeocarpa (F.T. Bennett).

Dollar spot, caused by Sclerotinia homoeocarpa, is a prevalent turfgrass disease, and the fungus exhibits widespread fungicide resistance in North America. In a previous study, an ABC-G transporter, ShatrD, was associated with practical field resistance to demethylation inhibitor (DMI) fungicides. Mining of ABC-G transporters, also known as pleiotropic drug resistance (PDR) transporters, from RNA-Seq data gave an assortment of transcripts, several with high sequence similarity to functionally characterized transporters from Botrytis cinerea, and others with closest blastx hits from Aspergillus and Monilinia. In addition to ShatrD, another PDR transporter showed significant over-expression in replicated RNA-Seq data, and in a collection of field-resistant isolates, as measured by quantitative polymerase chain reaction. These isolates also showed reduced sensitivity to unrelated fungicide classes. Using a yeast complementation system, we sought to test the hypothesis that this PDR transporter effluxes DMI as well as chemically unrelated fungicides. The transporter (ShPDR1) was cloned into the Gal1 expression vector and transformed into a yeast PDR transporter deletion mutant, AD12345678. Complementation assays indicated that ShPDR1 complemented the mutant in the presence of propiconazole (DMI), iprodione (dicarboximide) and boscalid (SDHI, succinate dehydrogenase inhibitor). Our results indicate that the over-expression of ShPDR1 is correlated with practical field resistance to DMI fungicides and reduced sensitivity to dicarboximide and SDHI fungicides. These findings highlight the potential for the eventual development of a multidrug resistance phenotype in this pathogen. In addition, this study presents a pipeline for the discovery and validation of fungicide resistance genes using de novo next-generation sequencing and molecular biology techniques in an unsequenced plant pathogenic fungus.

The Top 10 oomycete pathogens in molecular plant pathology.

Oomycetes form a deep lineage of eukaryotic organisms that includes a large number of plant pathogens which threaten natural and managed ecosystems. We undertook a survey to query the community for their ranking of plant-pathogenic oomycete species based on scientific and economic importance. In total, we received 263 votes from 62 scientists in 15 countries for a total of 33 species. The Top 10 species and their ranking are: (1) Phytophthora infestans; (2, tied) Hyaloperonospora arabidopsidis; (2, tied) Phytophthora ramorum; (4) Phytophthora sojae; (5) Phytophthora capsici; (6) Plasmopara viticola; (7) Phytophthora cinnamomi; (8, tied) Phytophthora parasitica; (8, tied) Pythium ultimum; and (10) Albugo candida. This article provides an introduction to these 10 taxa and a snapshot of current research. We hope that the list will serve as a benchmark for future trends in oomycete research.

Evaluation of partial tef1, rpb2, and nLSU sequences for identification of isolates representing Armillaria calvescens and Armillaria gallica from northeastern North America.

Armillaria calvescens and Armillaria gallica are two of the most closely-related species of Armillaria in North America and have been difficult to distinguish from one another using morphological and molecular techniques. In an attempt to better distinguish these two species, partial sequences of the elongation factor-1 alpha (tef1), RNA polymerase II (rpb2), and nuclear large subunit (nLSU) genes were generated for 32 total isolates; 12 isolates each for A. calvescens and A. gallica, along with two isolates each of Armillaria gemina, Armillaria mellea, Armillaria sinapina, and Armillaria solidipes. Within the tef1 amplicon, five single nucleotide polymorphisms (SNPs) were present between A. calvescens and A. gallica. Phylogenetic reconstruction using the maximum likelihood (ML) and maximum parsimony (MP) methods showed that tef1 was the only gene capable of distinguishing A. calvescens from A. gallica, and additionally, all isolates representing the six northeastern North American species. Partial tef1 sequences grouped A. calvescens into a strongly-supported, monophyletic clade with bootstrap support (BS) values of 98/98% (ML/MP), while A. gallica was grouped into a monophyletic clade with lower BS support (76/59%). The results illustrate the utility of partial tef1 sequences for the identification of field isolates of Armillaria from northeastern North America.