Assessment of the Virulence Spectrum and Its Association with Genetic Diversity in Magnaporthe oryzae Populations from Sub-Saharan Africa.

A collection of 122 isolates of Magnaporthe oryzae, from nine sub-Saharan African countries, was assessed for virulence diversity and genetic relatedness. The virulence spectrum was assessed by pathotype analysis with a panel of 43 rice genotypes consisting of differential lines carrying 24 blast resistance genes (R-genes), contemporary African rice cultivars, and susceptible checks. The virulence spectrum among isolates ranged from 5 to 80%. Five isolates were avirulent to the entire rice panel, while two isolates were virulent to ∼75% of the panel. Overall, cultivar 75-1-127, the Pi9 R-gene donor, was resistant to all isolates (100%), followed by four African rice cultivars (AR105, NERICA 15, 96%; NERICA 4, 91%; and F6-36, 90%). Genetic relatedness of isolates was assessed by single nucleotide polymorphisms derived from genotyping-by-sequencing and by vegetative compatibility tests. Phylogenetic analysis of SNPs of a subset of isolates (n = 78) revealed seven distinct clades that differed in virulence. Principal component analysis showed isolates from East Africa were genetically distinct from those from West Africa. Vegetative compatibility tests of a subset of isolates (n = 65) showed no common groups among countries. This study shows that blast disease could be controlled by pyramiding of Pi9 together with other promising R-genes into rice cultivars that are adapted to East and West African regions.

Infection and Colonization of Several Bermudagrasses by Ophiosphaerella korrae.

Bermudagrass (Cynodon spp.) is the most commonly used turfgrass in the southern United States where it is severely affected by spring dead spot (SDS) caused by Ophiosphaerella herpotricha, O. korrae, and O. narmari. In this study, infection of bermudagrass roots and stolons by O. korrae was characterized using a transformant that expressed the red fluorescent protein tdTomato. Roots of interspecific hybrid cultivars Midlawn and Tifway 419, C. transvaalensis accessions Uganda and 3200, and C. dactylon cultivar U3 were inoculated and observed from 2 to 14 days postinoculation (DPI) while stolons were observed from 2 to 22 DPI. For all five cultivars tested, a similar level of root colonization was observed; however, differences were observed in the rate of necrosis development. Necrosis of Tifway 419 and Midlawn tissues was evident at 2 DPI, in Uganda and 3200 at 8 DPI, and in U3 necrosis was often absent as late as 14 DPI. The fungus rapidly penetrated the root epidermis and colonized the cortex of all cultivars by 4 DPI. Colonization of stele tissues by O. korrae was rare in hybrid cultivars but common in C. transvaalensis and C. dactylon accessions. On intact stolons, the fungus did not penetrate the epidermis 22 DPI though epidermal necrosis was evident on the surface of only the hybrid bermudagrasses. Wounded stolons became necrotic in all cultivars. Infection and colonization of various bermudagrasses by O. korrae was found to be similar to that by O. herpotricha, suggesting that host genetic resistance may be used for effective management of SDS caused by both species.

Influence of Temperature and Time of Year on Colonization of Bermudagrass Roots by Ophiosphaerella herpotricha.

The influence of temperature on the infection of bermudagrass seedlings by Ophiosphaerella herpotricha and colonization of plants in the field was investigated. Bermudagrass seedlings (cv. Jackpot) inoculated with O. herpotricha exhibited dark lesions after 8 days. Root lesion length was greatest at 17°C and was similar for all temperatures examined below 21°C. Seedlings grown at 25 or 30°C had small lesions that remained similar in size when evaluated at 8 and 10 days post inoculation. Colonization of bermudagrass roots from field plots were examined in July, October, and November of 2003 and 2004. In 2003, no differences between sampling dates were observed for plants sampled from the edge of the spring patch in 5.4-cm increments to a total distance of 21.6 cm. In 2004, July and October samples were similar; however, an increase in root colonization was found between the October and November samplings. These studies suggest that infection and colonization of bermudagrass roots by O. herpotricha occurs over a wide range of cool soil temperatures, occurs in the spring, and can be variable in the autumn.

Molecular identification of the turf grass rapid blight pathogen.

Rapid blight is a newly described disease on turf grasses, primarily found on golf courses using suboptimal water for irrigation purposes. On the basis of shared morphological characteristics, it has been proposed that the rapid blight pathogen belongs to a genus of stramenopiles, Labyrinthula, which had been known to cause disease of marine plants only. We have collected 10 isolates from four species of turf grass in five states and sequenced portions of the SSU (18S) rDNA gene from each to provide a definitive taxonomic placement for rapid blight pathogens. We also included sequences from Labyrinthuloides yorkensis, Schizochytrium aggregatum, Aplanochytrium sp., Thraustochytrium striatum, Achlya bisexualis and several nonturf-grass isolates of Labyrinthula. We found that rapid blight isolates indeed are placed firmly within the genus Labyrinthula and that they lack detectable genetic diversity in the 18S rDNA region. We propose that the rapid blight pathogens share a recent common ancestor and might have originated from a single, infected population.

Characterization of the protein processing and secretion pathways in a comprehensive set of expressed sequence tags from Trichoderma reesei.

Trichoderma reesei is a filamentous fungus widely used as an efficient protein producer and known to secrete large quantities of biomass degrading enzymes. Much work has been done aimed at improving the secretion efficiency of this fungus. It is generally accepted that the major bottlenecks in secretion are protein folding and ornamentation steps in this pathway. In an attempt to identify genes involved in these steps, the 5' ends of 21888 cDNA clones were sequenced from which a unique set of over 5000 were also 3' sequenced. Using annotation tools Gene Ontology terms were assigned to 2732 of the sequences. Homologs to the majority of Aspergillus niger's Srg genes as well as a number of homologs to genes involved in protein folding and ornamentation pathways were identified.

Global gene expression during nitrogen starvation in the rice blast fungus, Magnaporthe grisea.

Efficient regulation of nitrogen metabolism likely plays a role in the ability of fungi to exploit ecological niches. To learn about regulation of nitrogen metabolism in the rice blast pathogen Magnaporthe grisea, we undertook a genome-wide analysis of gene expression under nitrogen-limiting conditions. Five hundred and twenty genes showed increased transcript levels at 12 and 48 h after shifting the fungus to media lacking nitrate as a nitrogen source. Thirty-nine of these genes have putative functions in amino acid metabolism and uptake, and include the global nitrogen regulator in M. grisea, NUT1. Evaluation of seven nitrogen starvation-induced genes revealed that all were expressed during rice infection. Targeted gene replacement on one such gene, the vacuolar serine protease, SPM1, resulted in decreased sporulation and appressorial development as well as a greatly attenuated ability to cause disease. Data are discussed in the context of nitrogen metabolism under starvation conditions, as well as conditions potentially encountered during invasive growth in planta.

The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea.

Magnaporthe grisea, the causal agent of rice blast disease, differentiates a specialized infection cell, an appressorium, that is required for infection of its host. Previously, cAMP was implicated in the endogenous signaling pathway leading to appressorium formation. To obtain direct evidence for the role of cAMP in appressorium formation, the gene encoding the catalytic subunit of the cAMP-dependent protein kinase (cpkA) was cloned, sequenced, and disrupted. Polymerase chain reaction primers designed after highly conserved regions in the same gene from other organisms were used to amplify genomic DNA fragments. The cloned amplification products were used to identify genomic clones. DNA blot analysis indicated that cpkA is present as a single copy in the genome. cpkA consists of 1894 bp, including three short introns sufficient to encode a protein of 539 amino acids with a predicted molecular mass of 60.7 kD. The deduced peptide shares > 45% identity with other catalytic subunits and contains all functional motifs and residues with the addition of a glutamine-rich region at the N terminus. Two transformants, L5 and T-182, in which cpkA had been replaced with a hygromycin resistance gene cassette, were unable to produce appressoria, could not be induced to form appressoria by cAMP, and were nonpathogenic on susceptible rice, even when leaves were abraded. These results were confirmed by analysis of 57 progeny from a cross between transformant L5 and the wild-type laboratory strain 70-6. Other aspects of growth and development, including vegetative growth as well as asexual and sexual competence, were unaffected when measured in vitro. These results provide direct evidence that the cAMP-dependent protein kinase is necessary for infection-related morphogenesis and pathogenesis in a phytopathogenic fungus.