Kevin King

Evolutionary Relationships between Rhynchosporium lolii sp. nov. and Other Rhynchosporium Species on Grasses

The fungal genus Rhynchosporium (causative agent of leaf blotch) contains several host-specialised species, including R. commune (colonising barley and brome-grass), R. agropyri (couch-grass), R. secalis (rye and triticale) and the more distantly related R. orthosporum (cocksfoot). This study used molecular fingerprinting, multilocus DNA sequence data, conidial morphology, host range tests and scanning electron microscopy to investigate the relationship between Rhynchosporium species on ryegrasses, both economically important forage grasses and common wild grasses in many cereal growing areas, and other plant species. Two different types of Rhynchosporium were found on ryegrasses in the UK. Firstly, there were isolates of R. commune that were pathogenic to both barley and Italian ryegrass. Secondly, there were isolates of a new species, here named R. lolii, that were pathogenic only to ryegrass species. R. lolii was most closely related to R. orthosporum, but exhibited clear molecular, morphological and host range differences. The species was estimated to have diverged from R. orthosporum ca. 5735 years before the present. The colonisation strategy of all of the different Rhynchosporium species involved extensive hyphal growth in the sub-cuticular regions of the leaves. Finally, new species-specific PCR diagnostic tests were developed that could distinguish between these five closely related Rhynchosporium species.

Comparative genomics to explore phylogenetic relationship, cryptic sexual potential and host specificity of Rhynchosporium species on grasses

BackgroundThe Rhynchosporium species complex consists of hemibiotrophic fungal pathogens specialized to different sweet grass species including the cereal crops barley and rye. A sexual stage has not been described, but several lines of evidence suggest the occurrence of sexual reproduction. Therefore, a comparative genomics approach was carried out to disclose the evolutionary relationship of the species and to identify genes demonstrating the potential for a sexual cycle. Furthermore, due to the evolutionary very young age of the five species currently known, this genus appears to be well-suited to address the question at the molecular level of how pathogenic fungi adapt to their hosts.ResultsThe genomes of the different Rhynchosporium species were sequenced, assembled and annotated using ab initio gene predictors trained on several fungal genomes as well as on Rhynchosporium expressed sequence tags. Structures of the rDNA regions and genome-wide single nucleotide polymorphisms provided a hypothesis for intra-genus evolution. Homology screening detected core meiotic genes along with most genes crucial for sexual recombination in ascomycete fungi. In addition, a large number of cell wall-degrading enzymes that is characteristic for hemibiotrophic and necrotrophic fungi infecting monocotyledonous hosts were found. Furthermore, the Rhynchosporium genomes carry a repertoire of genes coding for polyketide synthases and non-ribosomal peptide synthetases. Several of these genes are missing from the genome of the closest sequenced relative, the poplar pathogen Marssonina brunnea, and are possibly involved in adaptation to the grass hosts. Most importantly, six species-specific genes coding for protein effectors were identified in R. commune. Their deletion yielded mutants that grew more vigorously in planta than the wild type.ConclusionBoth cryptic sexuality and secondary metabolites may have contributed to host adaptation. Most importantly, however, the growth-retarding activity of the species-specific effectors suggests that host adaptation of R. commune aims at extending the biotrophic stage at the expense of the necrotrophic stage of pathogenesis. Like other apoplastic fungi Rhynchosporium colonizes the intercellular matrix of host leaves relatively slowly without causing symptoms, reminiscent of the development of endophytic fungi. Rhynchosporium may therefore become an object for studying the mutualism-parasitism transition.

Novel Technologies for the detection of Fusarium head blight disease and airborne inoculum

Many pathogens are dispersed by airborne spores, which can vary in space and time. We can use air sampling integrated with suitable diagnostic methods to give a rapid warning of inoculum presence to improve the timing of control options, such as fungicides. Air sampling can also be used to monitor changes in genetic traits of pathogen populations such as the race structure or frequency of fungicide resistance. Although some image-analysis methods are possible to identify spores, in many cases, species-specific identification can only be achieved by DNA-based methods such as qPCR and LAMP and in some cases by antibody-based methods (lateral flow devices) and biomarker-based methods (‘electronic noses’ and electro-chemical biosensors). Many of these methods also offer the prospect of rapid on-site detection to direct disease control decisions. Thresholds of spore concentrations that correspond to a disease risk depend on the sampler (spore-trap) location (whether just above the crop canopy, on a UAV or drone, or on a tall building) and also need to be considered with weather-based infection models. Where disease control by spore detection is not possible, some diseases can be detected at early stages using optical sensing methods, especially chlorophyll fluorescence. In the case of Fusarium infections on wheat, it is possible to map locations of severe infections, using optical sensing methods, to segregate harvesting of severely affected areas of fields to avoid toxins entering the food chain. This is most useful where variable crop growth or microclimates within fields generate spatially variable infection, i.e. parts of fields that develop disease, while other areas have escaped infection and do not develop any disease.

Loop-mediated isothermal amplification (LAMP) assays for rapid detection of Pyrenopeziza brassicae (light leaf spot of brassicas)

Pyrenopeziza brassicae (anamorph Cylindrosporium concentricum) is an ascomycete fungus that causes light leaf spot (LLS) disease of brassicas. It has recently become the most important pathogen of winter oilseed rape (Brassica napus) crops in the UK. The pathogen is spread by both asexual splash-dispersed conidia and sexual wind-dispersed ascospores. Such inoculum can be detected with existing qualitative and quantitative PCR diagnostics, but these require time-consuming laboratory-based processing. This study describes two loop-mediated isothermal amplification (LAMP) assays, targeting ITS or beta-tubulin DNA sequences, for fast and specific detection of P. brassicae isolates from a broad geographical range (throughout Europe and Oceania) and multiple brassica host species (B. napus, B. oleracea and B. rapa). Neither assay detected closely related Oculimacula or Rhynchosporium isolates, nor other commonly occurring oilseed rape fungal pathogens. Both LAMP assays could consistently detect DNA amounts equivalent to 100 P. brassicae conidia per sample within 30 minutes, although the beta-tubulin assay was more rapid. Reproducible standard curves were obtained using a P. brassicae DNA dilution series (100 ng – 10 pg) that enabled quantitative estimation of amounts of pathogen DNA in environmental samples. In planta application of the faster beta-tubulin sequence-based LAMP assay to individual oilseed rape leaves collected from the field found no statistically significant difference in the amount of pathogen DNA present in visually asymptomatic and symptomatic (visible LLS) parts of those leaves. The P. brassicae LAMP assays described here could have multiple applications, including detection of asymptomatic host infection and automated real-time monitoring of pathogen inoculum. This article is protected by copyright. All rights reserved.