Genetic Structure of the Rice Blast Pathogen (Magnaporthe oryzae) over a Decade in North Central California Rice Fields.

Rice blast, caused by the ascomycete Magnaporthe oryzae, is one of the most destructive rice diseases worldwide. Even though the disease has been present in California since 1996, there is no data for the pathogen population biology in the state. Using amplified fragment length polymorphisms and mating-type markers, the M. oryzae population diversity was investigated using isolates collected when the disease was first established in California and isolates collected a decade later. While in the 1990 samples, a single multilocus genotype (MLG) was identified (MLG1), over a decade later, we found 14 additional MLGs in the 2000 isolates. Some of these MLGs were found to infect the only rice blast-resistant cultivar (M-208) available for commercial production in California. The same samples also had a significant decrease of MLG1. MLG1 was found infecting the resistant rice cultivar M-208 on one occasion whereas MLG7 was the most common genotype infecting the M-208. MLG7 was identified in the 2000 samples, and it was not present in the M. oryzae population a decade earlier. Our results demonstrate a significant increase in genotypic diversity over time with no evidence of sexual reproduction and suggest a recent introduction of new virulent race(s) of the pathogen. In addition, our data could provide information regarding the durability of the Pi-z resistance gene of the M-208. This information will be critical to plant breeders in developing strategies for deployment of other rice blast resistance genes/cultivars in the future

Phylogenetic placement of the ectomycorrhizal genus Cenococcum in Gloniaceae (Dothideomycetes)

Cenococcum is a genus of ectomycorrhizal Ascomycota that has a broad host range and geographic distribution. It is not known to produce either meiotic or mitotic spores and is known to exist only in the form of hyphae, sclerotia and host-colonized ectomycorrhizal root tips. Due to its lack of sexual and asexual spores and reproductive structures, it has proven difficult to incorporate into traditional classification within Ascomycota. Molecular phylogenetic studies of ribosomal RNA placed Cenococcum in Dothideomycetes, but the definitive identification of closely related taxa remained elusive. Here we report a phylogenetic analysis of five nuclear loci (SSU, LSU, TEF1, RPB1, RPB2) of Dothideomycetes that placed Cenococcum as a close relative of the genus Glonium of Gloniaceae (Pleosporomycetidae incertae sedis) with strong statistical support. Glonium is a genus of saprobic Dothideomycetes that produces darkly pigmented, carbonaceous, hysteriate apothecia and is not known to be biotrophic. Evolution of ectomycorhizae, Cenococcum and Dothideomycetes is discussed.Keywords: fungi, ectomycorrhizae, Cenococcum, phylogenetics, Dothideomycetes, Gloniaceae, systematic

Two distinct viral suppressors of RNA silencing encoded by citrus tatter leaf virus

Two proteins of the citrus tatter leaf virus (CTLV), a strain of the apple stem grooving virus (ASGV), capable of inducing citrus bud union disorders on commercially important trifoliate and citrange rootstocks, were identified as viral suppressors of RNA silencing (VSR). Both the coat protein (CP) and the movement protein (MP) suppressed RNA silencing in GFP-transgenic Nicotiana benthamiana 16c plants in agrobacterium-mediated co-infiltration assays; the MP acted as a local VSR, while the CP acted as a systemic VSR. When the potato virus X (PVX) infectious vector harbored either the CTLV CP or MP gene, viral infection and symptom development were promoted in N. benthamiana. Deletions of amino acids in the CP sequence or the MP sequence resulted in failure to promote PVX infections as well as suppression of silencing in agrobacterium-mediated co-infiltration assays. Mass spectrometry-based immunoprecipitation proteomics showed that neither the CTLV CP nor the MP interacts with cellular components directly involved in host antiviral RNA silencing pathways. RNA immunoprecipitation (RIP) and RNA-protein pull-down assays indicated that the CTLV MP interacts with double-stranded RNA (dsRNA) presumably through a protein complex or proteins containing RNA binding domains. It is possible that the MP prevents dsRNA cleavage through this mechanism, leading to suppression of host antiviral RNA silencing. These findings confirmed that CTLV uses VSRs as part of its overall strategy to overcome host antiviral defenses and are indicative of the ability of ASGV and CTLV to infect a wide range of hosts including different species of woody and herbaceous plants

First report of the ectomycorrhizal status of boletes on the Northern Yucatan Peninsula, Mexico determined using isotopic methods

Despite their prominent role for tree growth, few studies have examined the occurrence of ectomycorrhizal fungi in lowland, seasonally dry tropical forests (SDTF). Although fruiting bodies of boletes have been observed in a dry tropical forest on the Northern Yucatan Peninsula, Mexico, their occurrence is rare and their mycorrhizal status is uncertain. To determine the trophic status (mycorrhizal vs. saprotrophic) of these boletes, fruiting bodies were collected and isotopically compared to known saprotrophic fungi, foliage, and soil from the same site. Mean δ15N and δ13C values differed significantly between boletes and saprotrophic fungi, with boletes 8.0‰ enriched and 2.5‰ depleted in 15N and 13C, respectively relative to saprotrophic fungi. Foliage was depleted in 13C relative to both boletes and saprotrophic fungi. Foliar δ15N values, on the other hand, were similar to saprotrophic fungi, yet were considerably lower relative to bolete fruiting bodies. Results from this study provide the first isotopic evidence of ectomycorrhizal fungi in lowland SDTF and emphasize the need for further research to better understand the diversity and ecological importance of ectomycorrhizal fungi in these forested ecosystems

Microsatellite characterization and marker development for the fungus Penicillium digitatum, causal agent of green mold of citrus.

Penicillium digitatum is one of the most important postharvest pathogens of citrus on a global scale causing significant annual losses due to fruit rot. However, little is known about the diversity of P. digitatum populations. The genome of P. digitatum has been sequenced, providing an opportunity to determine the microsatellite distribution within P. digitatum to develop markers that could be valuable tools for studying the population biology of this pathogen. In the analyses, a total of 3,134 microsatellite loci were detected; 66.73%, 23.23%, 8.23%, 1.24%, 0.16%, and 0.77% were detected as mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats, respectively. As consistent with other ascomycete fungi, the genome size of P. digitatum does not seem to correlate with the density of microsatellite loci. However, significantly longer motifs of mono-, di-, and tetranucleotide repeats were identified in P. digitatum compared to 10 other published ascomycete species with repeats of over 800, 300, and 900 motifs found, respectively. One isolate from southern California and five additional isolates from other countries ("global isolates") were used to initially screen microsatellite markers developed in this study. Twelve additional isolates, referred to as the "local isolates," were also collected from citrus at the University of California Riverside agricultural experiment station and were subsequently used to screen the primers that sequenced well and were polymorphic based on the global isolates. Thirty-six primers were screened, and nine trinucleotide loci and one hexanucleotide locus were chosen as robust markers. These loci yielded two to seven alleles and will be useful to study population genetic structure of P. digitatum populations

Phylogenetic placement of the ectomycorrhizal genus Cenococcum in Gloniaceae (Dothideomycetes)

Cenococcum is a genus of ectomycorrhizal Ascomycota that has a broad host range and geographic distribution. It is not known to produce either meiotic or mitotic spores and is known to exist only in the form of hyphae, sclerotia and host-colonized ectomycorrhizal root tips. Due to its lack of sexual and asexual spores and reproductive structures, it has proven difficult to incorporate into traditional classification within Ascomycota. Molecular phylogenetic studies of ribosomal RNA placed Cenococcum in Dothideomycetes, but the definitive identification of closely related taxa remained elusive. Here we report a phylogenetic analysis of five nuclear loci (SSU, LSU, TEF1, RPB1, RPB2) of Dothideomycetes that placed Cenococcum as a close relative of the genus Glonium of Gloniaceae (Pleosporomycetidae incertae sedis) with strong statistical support. Glonium is a genus of saprobic Dothideomycetes that produces darkly pigmented, carbonaceous, hysteriate apothecia and is not known to be biotrophic. Evolution of ectomycorhizae, Cenococcum and Dothideomycetes is discussed. © 2012 by The Mycological Society of America

Isolation source matters: sclerotia and ectomycorrhizal roots provide different views of genetic diversity in <i>Cenococcum geophilum</i>

<p><i>Cenococcum geophilum</i> forms sclerotia and ectomycorrhizas with host plants in forest soils. We demonstrated the differences in genetic diversity of <i>C. geophilum</i> between cultured isolates from sclerotia and those from ectomycorrhizal roots in the same 73 soil samples based on glyceraldehyde-3-phosphate dehydrogenase (<i>GAPDH</i>) gene sequences and newly developed microsatellite markers. Based on <i>GAPDH</i> sequences, 759 cultured isolates (553 from sclerotia and 206 from ectomycorrhizas) were classified into 107 “genotypes” with sequence variation of up to 8.6%. The total number of <i>GAPDH</i> genotypes per soil sample ranged from 1 to 9, but genotypes that were shared between sclerotia and ectomycorrhizas were uncommon (0–3 per soil sample). More than 50% of <i>GAPDH</i> genotypes were unique to one source in most soil samples. Unique <i>GAPDH</i> genotypes were detected from either scleotia or ectomycorrhizal roots in most of the soil samples. Multilocus analysis using nine microsatellite markers provided additional resolution to differentiate fungal individuals and supported the results of <i>GAPDH</i> genotyping. The results indicated that sampling both sclerotia and ectomycorrhizal roots maximizes the detection of diversity at the soil core scale. On the other hand, when all isolates were viewed together, 82 <i>GAPDH</i> genotypes were unique to sclerotia whereas only 6 <i>GAPDH</i> genotypes were unique to ectomycorrhizas. Rarefaction analysis indicated that <i>GAPDH</i> genotypic diversity is significantly higher in sclerotia than ectomycorrhizal roots and the diversity within sclerotia is nearly the same as that of both sclerotia and ectomycorrhizas together. These findings suggest that sampling sclerotia alone is likely to detect the majority of <i>GAPDH</i> genotypes in <i>Cenococcum</i> at the regional scale. When deciding whether to sample sclerotia, ectomycorrhizas, or both types of tissues from <i>Cenococcum</i>, it is critical to consider the spatial scale and also the main questions and hypotheses of the study.</p