Development of a Large-Scale Soil DNA Extraction Method for Molecular Quantification of Fusarium oxysporum f. sp. fragariae in Soil.

The most common soilborne diseases affecting the strawberry industry in California include Verticillium wilt due to Verticillium dahliae, charcoal root rot due to Macrophomina phaseolina, and Fusarium wilt due to Fusarium oxysporum f. sp. fragariae. Detection of these pathogens in soil is an important facet of disease management and fumigation recommendations. Whereas the soil populations of both M. phaseolina and V. dahliae can be readily quantified with quantitative PCR (qPCR) assays using DNA extractions with 500 mg of soil, the single-cell nature of the F. oxysporum chlamydospore does not provide enough pathogen DNA from 500-mg extractions to be reliably quantified. Here, we describe an improved DNA extraction protocol from 10 to 15 g of soil that allows for the quantification of F. oxysporum f. sp. fragariae populations below 10 CFU/g. The relationship between results from the TaqMan qPCR assay and pathogen population density in soil was determined by using this extraction method in pathogen-free soils artificially infested with a hygromycin-resistant strain of F. oxysporum f. sp. fragariae to facilitate accurate colony counts when plated on a selective medium. Although the protocol was developed for F. oxysporum f. sp. fragariae, it is applicable for detection and quantification of other soilborne pathogens.

Karyotype variation, spontaneous genome rearrangements affecting chemical insensitivity, and expression level polymorphisms in the plant pathogen Phytophthora infestans revealed using its first chromosome-scale assembly.

Natural isolates of the potato and tomato pathogen Phytophthora infestans exhibit substantial variation in virulence, chemical sensitivity, ploidy, and other traits. A chromosome-scale assembly was developed to expand genomic resources for this oomyceteous microbe, and used to explore the basis of variation. Using PacBio and Illumina data, a long-range linking library, and an optical map, an assembly was created and coalesced into 15 pseudochromosomes spanning 219 Mb using SNP-based genetic linkage data. De novo gene prediction combined with transcript evidence identified 19,981 protein-coding genes, plus about eight thousand tRNA genes. The chromosomes were comprised of a mosaic of gene-rich and gene-sparse regions plus very long centromeres. Genes exhibited a biased distribution across chromosomes, especially members of families encoding RXLR and CRN effectors which clustered on certain chromosomes. Strikingly, half of F1 progeny of diploid parents were polyploid or aneuploid. Substantial expression level polymorphisms between strains were identified, much of which could be attributed to differences in chromosome dosage, transposable element insertions, and adjacency to repetitive DNA. QTL analysis identified a locus on the right arm of chromosome 3 governing sensitivity to the crop protection chemical metalaxyl. Strains heterozygous for resistance often experienced megabase-sized deletions of that part of the chromosome when cultured on metalaxyl, increasing resistance due to loss of the sensitive allele. This study sheds light on diverse phenomena affecting variation in P. infestans and relatives, helps explain the prevalence of polyploidy in natural populations, and provides a new foundation for biologic and genetic investigations.

A Cas12a-based gene editing system for Phytophthora infestans reveals monoallelic expression of an elicitor.

Phytophthora infestans is a destructive pathogen of potato and a model for investigations of oomycete biology. The successful application of a CRISPR gene editing system to P. infestans is so far unreported. We discovered that it is difficult to express CRISPR/Cas9 but not a catalytically inactive form in transformants, suggesting that the active nuclease is toxic. We were able to achieve editing with CRISPR/Cas12a using vectors in which the nuclease and its guide RNA were expressed from a single transcript. Using the elicitor gene Inf1 as a target, we observed editing of one or both alleles in up to 13% of transformants. Editing was more efficient when guide RNA processing relied on the Cas12a direct repeat instead of ribozyme sequences. INF1 protein was not made when both alleles were edited in the same transformant, but surprisingly also when only one allele was altered. We discovered that the isolate used for editing, 1306, exhibited monoallelic expression of Inf1 due to insertion of a copia-like element in the promoter of one allele. The element exhibits features of active retrotransposons, including a target site duplication, long terminal repeats, and an intact polyprotein reading frame. Editing occurred more often on the transcribed allele, presumably due to differences in chromatin structure. The Cas12a system not only provides a tool for modifying genes in P. infestans, but also for other members of the genus by expanding the number of editable sites. Our work also highlights a natural mechanism that remodels oomycete genomes.

Metalaxyl Resistance in Phytophthora infestans: Assessing Role of RPA190 Gene and Diversity Within Clonal Lineages.

Prior work has shown that the inheritance of resistance to metalaxyl, an oomycete-specific fungicide, is complex and may involve multiple genes. Recent research indicated that a single nucleotide polymorphism (SNP) in the gene encoding RPA190, the largest subunit of RNA polymerase I, confers resistance to metalaxyl (or mefenoxam) in some isolates of the potato late blight pathogen Phytophthora infestans. Using both DNA sequencing and high resolution melt assays for distinguishing RPA190 alleles, we show here that the SNP is absent from certain resistant isolates of P. infestans from North America, Europe, and Mexico. The SNP is present in some members of the US-23 and US-24 clonal lineages, but these tend to be fairly sensitive to the fungicide based on artificial media and field test data. Diversity in the level of sensitivity, RPA190 genotype, and RPA190 copy number was observed in these lineages but were uncorrelated. Controlled laboratory crosses demonstrated that RPA190 did not cosegregate with metalaxyl resistance from a Mexican and British isolate. We conclude that while metalaxyl may be used to control many contemporary strains of P. infestans, an assay based on RPA190 will not be sufficient to diagnose the sensitivity levels of isolates.