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Impatiens downy mildew (IDM) disease is a primary constraint on the production of Impatiens walleriana , a popular and economically important floriculture plant. IDM is caused by the biotrophic. oomycete Plasmopara destructor that emerged as a pathogen of I. walleriana in the 2000s. To enable P. destructor detection and quantification, a hydrolysis-probe-based quantitative PCR diagnostic assay was developed based on unique orientation and order of the mitochondrial cytochrome c oxidase subunit1 ( cox 1) and ATP synthase subunit alpha ( atp 1) genes in the genus Plasmopara . Nucleotide sequences and analysis of the cox 1/ atp 1 region distinguished P. destructor and its sister-species P. obducens , consistent with prior phylogenetic analyses using cox 2 and rDNA markers. Specificity for P. destructor was incorporated into a hydrolysis probe targeting the cox 1 gene and flanking primers that amplified across the cox 1/ atp 1 intergenic region. The limit of detection was 0.5 fg/μl of P. destructor DNA (∼100 plasmid copies/μl), with amplification efficiency = 0.95. The assay was validated against a panel of target and nontarget oomycetes, which showed that the primers were specific for Plasmopara spp., while the probe was specific for P. destructor infecting both I. walleriana and I. balsamina . Testing of Impatiens tissue collected from 23 locations across 13 states indicated all samples with IDM symptoms tested positive for P. destructor . Asymptomatic plants from two locations also tested positive for P. destructor .

The obligatory biotrophic oomycetes Plasmopara viticola is the causal agent of downy mildew, a destructive disease of grapevine worldwide. So far, chemical fungicides are widely employed to limit this pathogen, but their adverse effects are stimulating the quest for environmentally friendly alternative approaches. Here, we report on the search for mycoviruses that might be later developed as biocontrol agents for this pathogen. Symptomatic leaves were collected from various regions in Spain and Italy and mycelia associated to leaf lesions was harvested. Total RNA extractions were depleted of rRNA and metatranscriptomes were generated using a high-throughput sequencing approach. The virome associated to leaf lesions was then characterized through a bioinformatic pipeline relying on blast searches against current viral databases. Here, we present an inventory of 283 new RNA viruses: 222 positive strand RNA viruses, 29 negative strand RNA viruses, 27 double-stranded RNA viruses and 5 ORFan virus RdRP segments, which could not be reliably assigned to any existing group in the Riboviria . In addition to ORFan viruses, we found other surprising new evolutionary trajectories in this wide inventory of viruses. The most represented viruses in our collection are those in phylum Lenarviricota , and, among them, a group of mycovirus segments distantly related to narnaviruses, but characterized by a polymerase palm domain lacking subdomain C, with the putative GDD catalytic triad. We also provided evidence of a strict association between two RNA segments that form a new mycovirus clade of positive strand RNA in the phylum Kitrinoviricota , order Martellivirales . In the phylum Negarnaviricota , we report for the first time in the order Mononegavirales a clade of viruses that is ambisense, a feature that so far was present only in the order Bunyavirales . Furthermore, in the same phylum we detected the widespread occurrence and abundant accumulation in our libraries of a distinct mycovirus clade distantly related to the Muvirales and Goujanvirales orders, which so far include only viruses infecting invertebrates. Possible new oomycetes-specific virus clades are also described in the phylum Duplornaviricota . These data greatly expand the evolutionary history of mycoviruses adding new layers of diversity to the realm Riboviria ., (© The Author(s) 2020. Published by Oxford University Press.)

Cyclophilins (CYPs), a highly-conserved family of proteins, belong to a subgroup of immunophilins. Ubiquitous in eukaryotes and prokaryotes, CYPs have peptidyl-prolyl cis-trans isomerase (PPIase) activity and have been implicated as virulence factors in plant pathogenesis by oomycetes. We identified 16 CYP orthogroups from 21 diverse oomycetes. Each species was found to encode 15 to 35 CYP genes. Three of these orthogroups contained proteins with signal peptides at the N-terminal end, suggesting a role in secretion. Multidomain analysis revealed five conserved motifs of the CYP domain of oomycetes shared with other eukaryotic PPIases. Expression analysis of CYP proteins in different asexual life stages of the hemibiotrophic Phytophthora infestans and the biotrophic Plasmopara halstedii demonstrated distinct expression profiles between life stages. In addition to providing detailed comparative information on the CYPs in multiple oomycetes, this study identified candidate CYP effectors that could be the foundation for future studies of virulence.