Population genomics of an outbreak of the potato late blight pathogen, Phytophthora infestans, reveals both clonality and high genotypic diversity.

An outbreak of the potato late blight pathogen Phytophthora infestans in Denmark was characterized in order to resolve the population structure and determine to what extent sexual reproduction was occurring. A standard set of microsatellite simple sequence repeats (SSRs) and single nucleotide polymorphism (SNP) markers generated using restriction site-associated DNA sequencing (RAD-seq) were employed in parallel. A total of 83 individuals, isolated from seven different potato fields in 2014, were analysed together with five Danish whole-genome sequenced isolates, as well as two Mexican individuals used as an outgroup. From a filtered dataset of 55 288 SNPs, population genomics analyses revealed no sign of recombination, implying clonality. In spite of this, multilocus genotypes were unique to individual potato fields, with little evidence of gene flow between fields. Ploidy analysis performed on the SNPs dataset indicated that the majority of isolates were diploid. These contradictory results with clonality and high genotypic diversity may suggest that rare sexual events likely still contribute to the population. Comparison of the results generated by SSRs vs SNPs data indicated that large marker sets, generated by RAD-seq, may be advised going forward, as it provides a higher level of genetic discrimination than SSRs.

In Planta Quantification of Plasmodiophora brassicae Using Signature Fatty Acids and Real-Time PCR.

Until now, molecular and biochemical methods have only been used to show whether or not Plasmodiophora brassicae is present in plant or soil samples but not to what extent. Here, in planta quantification of P. brassicae by whole-cell fatty acid (WCFA) measurements and real-time polymerase chain reaction (PCR) was evaluated. Arachidonic acid (ARA, 20:4) was the most abundant fatty acid in resting spores and was only found in infected roots, which indicates a potential of ARA as a biomarker for P. brassicae. A real-time PCR assay was developed using primers designed from the internal transcribed spacer region of the ribosomal DNA. Using these primers, it was possible to detect P. brassicae in infected roots 10 days after germination of plants sown in infested soil. A bioassay showed that the amounts of ARA found by WCFA analysis and the DNA found by real-time PCR in infected plants were well correlated. These measurements also correlated with the soil spore content and the assessed disease incidence and disease severity scores. Therefore, we conclude that WCFA analysis and real-time PCR are good tools for P. brassicae quantification that can be applied to basic studies of the pathogen and in resistance screens.

Interactions between indigenous arbuscular mycorrhizal fungi and Aphanomyces euteiches in field-grown pea.

This is the first reported study of the interactions between indigenous arbuscular mycorrhizal fungi (AMF) and Aphanomyces euteiches in pea under field conditions. A. euteiches was applied to the soil by adding oospores produced in vitro. Attempts were made to create a non-mycorrhizal control by incorporating carbendazim (Derosal Fl) in the topsoil before sowing. However, all carbendazim-treated plants showed approximately 20% root colonisation with AMF. Pea plants not treated with carbendazim showed a wide variation in AMF colonisation of 35-70% at the full flowering stage. In these control plots, root length infected with oospores of A. euteiches and colonisation by AMF were negatively correlated. Application of carbendazim increased the percent root length infected with oospores by 50-70%, depending on inoculum density of A. euteiches. Despite the lower levels of AMF colonisation in these treated plots, a negative correlation with oospore-containing root length was still observed. No correlation was found between AMF colonisation and disease severity, disease incidence or pathogen enzymatic activity (glucose-6-phosphate dehydrogenase). Thus, AMF do not seem to influence the vegetative stage of pathogen development during which cortical root rotting takes place, but rather the reproductive stage when oospores are produced. The results of this study underline the importance of field experiments for validating the significance of mycorrhizal fungi for plant health.

Bacterial populations associated with mycelium of the arbuscular mycorrhizal fungus Glomus intraradices.

Abstract The influences of the arbuscular mycorrhizal fungus Glomus intraradices on the culturable aerobic-heterotrophic bacterial communities in the rhizosphere and hyphosphere of cucumber plants (Cucumis satvius) were investigated. Mycorrhizal and non-mycorrhizal plants were grown in compartmentalised growth units, each with a root compartment and two lateral root-free compartments. Samples representing rhizosphere, root-free soil, root-free sand and washed sand extract were collected 52 days after sowing from treatments both with and without mycorrhiza. No significant difference in total bacterial number was observed between the mycorrhizal and non-mycorrhizal treatment. Fourteen hundred bacterial colonies were isolated and identified by fatty acid methyl ester analysis using the Sherlock system (MIDI Inc.), 87 species within 48 genera were identified with a similarity index >0.30. Pseudomonas, Arthrobacter and Burkholderia were the genera most frequently encountered. Large differences in bacterial community structure were observed between rhizosphere soil, root-free soil/sand and washed sand extract, whereas major differences between mycorrhizal and non-mycorrhizal treatments were observed for a few bacterial species only. Isolates identified as Paenibacillus spp. were more frequently found in the mycorrhizal treatment and especially in the washed sand extract with mycelium of G. intraradices, indicating that bacteria within this genus may live in close association with mycelium of these fungi.

Population structure and pathogenicity of members of the Fusarium oxysporum complex isolated from soil and root necrosis of pea (Pisum sativum L.).

Forty-nine strains of the Fusarium oxysporum complex were isolated from five different sample locations within two neighboring pea fields. Of these, 39 strains were isolated from soil and 10 from pea plants showing symptoms of root rot. Twenty-eight of the isolates were tested for pathogenicity towards pea. Based on percentage discoloration of the roots and stem base, the isolates were divided into three groups: seven strains were pathogenic, 14 strains were weakly pathogenic, and seven strains were non-pathogenic towards pea. To assess the genetic relatedness of all 49 strains, gene genealogies were constructed from aligned DNA sequences from part of the translation elongation factor, nitrate reductase, beta tubulin, and mitochondrial small subunit rDNA. Maximum parsimony analysis of the combined data set yielded a single most-parsimonious tree containing three strongly supported clades which may represent cryptic species. No correlation was observed between the multigene phylogeny and pathogenicity toward pea, strain geographic origin and substrate (soil or plant) from which the strains were isolated. Strains that were non-pathogenic, weakly pathogenic or pathogenic sometimes shared the same multilocus genotype. These results suggest that strains pathogenic and putatively non-pathogenic to pea are very closely related genetically.

Interactions between pea root-inhabiting fungi examined using signature fatty acids.

• Interactions are investigated between the arbuscular mycorrhizal fungus, Glomus mosseae, and the root pathogen, Aphanomyces euteiches, on pea (Pisum sativum) roots, as arbuscular mycorrhiza are known to suppress a broad range of root pathogens. • Phospholipid (PLFA) and neutral lipid (NLFA) fatty acids were used as indicators of biomass and energy reserves, respectively, of A. euteiches and G. mosseae in inoculated roots of pot-grown pea seedlings. • Symbiosis between pea and G. mosseae had no effect on the severity of disease caused by A. euteiches, which decreased pea shoot and root dry weight. However, the presence of G. mosseae in pea roots reduced both biomass and energy reserves of A. euteiches, indicated by a reduction in PLFA 14 : 0 and both NLFAs 14 : 0 and 14 : 1ω9. Similarly, a reduction in PLFA and NLFA 16 : 1ω5 indicated reduced biomass and energy reserves of G. mosseae in A. euteiches-infected roots. • Signature fatty acids can be used to quantify biomass and energy reserves of G. mosseae and A. euteiches simultaneously, in pea root; this appears to be a promising method for studying interactions between arbuscular mycorrhizal fungi and root pathogens in planta.