Rice blast disease is caused by the filamentous fungus Magnaporthe oryzae and is the most destructive disease of cultivated rice. It was the first plant pathogenic fungus to have its genome sequence published which opened up the opportunities to discern the principal genetic components that confer pathogenicity on the fungus. The availability of the genome sequence has also presented fresh challenges in terms of converting sequence data into meaningful biological information. Functional genomics studies involve the generation of genome-wide mutant collections and comprehensive screens with potential to identify novel pathogenicity determinants. In this study I utilized Agrobacterium tumefaciens mediated random insertional mutagenesis to study the infection mechanism of M. oryzae. A collection 10,200 M. oryzae T-DNA insertion mutants were generated as part of this study and pathogenicity was assayed by high-throughput disease screening. From the primary qualitative screening I obtained 200 mutants that were reduced or lacking in pathogenicity. Quantitative re-screening allowed selection of 71 T-DNA mutants, including 9 non-pathogenic and 63 reduced virulence mutants exhibiting at least a 50% reduction in disease symptoms. Finally, we selected 8 non-pathogenic mutants for detailed phenotypic and gene functional analysis. A novel approach was used to retrieve T-DNA tagged genes from mutants of interest. Next generation DNA sequencing (NGS) was used to retrieve T-DNA flanking sequences in a high-throughput manner. The efficiency of NGS to facilitate the high-throughput large scale insertional mutagenesis was therefore demonstrated. Out of 8 selected mutants, I identified three novel genes that putatively encode a transcription factor, a PH domain containing signalling protein and a MAP kinase. I also provided evidence that, MGG_05343 is a functional C6 zinc finger transcription factor involved in conidiogenesis. The PH domain containing protein MGG_12956 is involved in vegetative growth, condiogenesis and virulence. The novel kinase MGG_15325 is a S. cerevisiae IME2 homolog that belongs to the Ime2 class of non-classical MAP kinase subfamily. Intriguingly, M. oryzae IME2 seems to have an essential role in growth in planta because the mutant was able to penetrate and colonize plant tissue but failed to cause necrotic rice blast lesions. Identification of these novel genes will allow us greater insight into the processes required for condiogenesis, vegetative and invasive growth and a more integrated understanding of the post-penetration phases of plant tissue colonization. Interestingly, I identified two mutants tagged with T-DNA insertion in the autophagy genes ATG2 and ATG3, reaffirming the importance of infection-associated autophagy in plant infection by M. oryzae and we characterized the ATG3 gene. In addition, I generated a resource of 63 unidentified T-DNA mutants which can potentially lead to identification of more novel determinants of pathogenicity in rice blast disease.

Statistical analyses were used to develop predictive models of rice blast and to relate the favorability of environment to disease incidence and severity on different rice cultivars at five sites in Asia. The WINDOW PANE program was used to search for weather factors highly correlated with blast. Stepwise and r-square linear regression procedures were then applied to generate the predictive models at each site. Models developed at Icheon, South Korea included relative humidity and rainfall factors as the most important predictors of disease. Temperature, rainfall, wind speed, and relative humidity factors were components of models at Cavinti and the IRRI blast nursery in the Philippines. Rainfall, temperature, and solar radiation factors were important at Gunung Medan and Sitiung, Indonesia. Model validation was done to verify accuracy of models for predictions. Model predictions were also used to determine the effects on blast of sowing time, nitrogen amount, and increase in temperature. Limitations of the models are discussed. Path coefficient analysis was used to identify direct and indirect influences exerted by weather factors on blast. The largest direct influence on disease was exerted by humidity factors at Icheon; temperature, rainfall, and wind speed factors at Cavinti; temperature and humidity factors at IRRI; rainfall factors at Gunung Medan; and temperature factors at Sitiung. Although path coefficient values (Py) were estimated from the decomposition of correlation coefficients, factors that had a high correlation with disease parameters did not always give high Py. Multivariate analysis was used to determine the effects of sowing times on proneness of tropical rice to blast. Cluster analysis of 24 hypothetical sowing months at Cavinti, the IRRI blast nursery, and Sitiung sites revealed three blast proneness groups. Principal component analysis showed that IR50 cultivar would be susceptible at Cavinti at any time of the year. Sowing C22 cultivar at Cavinti in Group I and III months would make it prone to panicle and leaf blast, respectively. At the IRRI blast nursery, leaf and panicle infections on IR50 would be probable only in Group I and II months. This trend was also observed for C22 at Sitiung, although some months in Group III at this site had moderate to high degree of proneness to leaf blast. / Graduation date: 1994

Thesis (Ph. D.)--University of Wisconsin--Madison, 1984. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographies.

Master of Science / Department of Plant Pathology / Erick D. DeWolf / Wheat blast, caused by Magnaporthe oryzae pathotype triticum, has emerged as a serious problem for wheat production in South America and recently emerged as a threat to wheat production in Bangladesh. To prepare for the possible introduction of wheat blast in to the United States, it would be helpful to identify areas of the country most at risk for blast epidemics. Because wheat blast occurs primarily in tropical and subtropical regions of the world, cold winter temperatures may restrict the establishment of the blast pathogen in the United States. Therefore, the first objective of this research was to quantify the freeze-thaw tolerance of the wheat blast pathogen in naturally infected wheat rachises from Bolivia and to measure the viability of the conidia after exposure to various treatments. The results indicate that exposing the fungus in moist residue to multiple freeze-thaw cycles is more damaging than exposing the fungus in moist residue to longer, single freezes. When in dry residue, the fungus was not harmed by the freeze-thaw cycles. Freezing and thawing of the wheat blast fungus in moist residue significantly affected its ability to produce viable conidia. The second objective of this research was to identify environmental conditions that could be conducive for wheat blast epidemics by examining historical epidemics of rice blast, caused by Magnaporthe oryzae pathotype oryza. The dataset used in this analysis consisted of 60 site-years of historical observations of rice blast levels from Arkansas, Louisiana, and Texas. These observations were coupled with monthly and weekly summaries of hourly weather variables based on temperature, relative humidity, precipitation, and regional moisture indices. Classification trees and logistic regression were used to identify variables associated with rice blast epidemics. The results indicate that rice blast epidemics are favored by cooler April temperatures and higher levels of precipitation in June. Preliminary models for rice blast based on these variables were able to correctly classify epidemic years with >75% accuracy. In the future, the results of this project will be used as part of a risk assessment for a wheat blast introduction and establishment in the United States.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 91-101).

No description available.

No description available.

Rice blast disease is caused by the ascomycete fungus Magnaporthe oryzae and is of economic importance worldwide, due to its wide geographical distribution and the severe yield losses it causes on cultivated rice. Understanding the population structure of M. oryzae is key to sustainable management of blast disease. In this study, a total of 290 M. oryzae isolates were collected from rice growing regions in Kenya including Central Kenya (Mwea irrigation scheme), Western Kenya (Ahero and Maugo irrigation schemes in Ahero and Homa-Bay respectively) and Coastal Kenya (Kwale). Initially, I undertook genotyping of a subset of Kenyan isolates by DNA sequence analysis of the internal transcribed spacer regions (ITS 1 and ITS 2) of the rRNA-encoding gene unit and by DNA fingerprinting using the Pot2 repetitive DNA element. Phylogenetic analyses based on ITS sequences clustered together isolates from Western and Coastal Kenya which were distinct from Central Kenya isolates. Cluster analysis based on 80% DNA fingerprint similarity, identified five clonal lineages designated KL1, KL2, KL3, KL4 and KL5 with most isolates belonging to lineages KL2, KL3, KL4. The clustering of isolates was region specific with Western and Coastal isolates closely related to each other and distinct from Central Kenya isolates. Distribution of mating type gene loci (MAT1.1 and MAT1.2) was determined using mating type gene specific primers. My results indicate that MAT1.1 is the predominant mating type and is distributed in all the rice growing regions of Kenya. MAT1.2 isolates were identified only in Coastal Kenya. I further undertook high throughput next-generation DNA sequencing of the genomes of 27 M. oryzae isolates from sub-Saharan Africa (SSA), including Kenya, Uganda, Tanzania, Benin, Togo, Nigeria and Burkina Faso and compared them to other sequenced strains from China, India, USA, Philippines, Thailand, Korea, Japan, France and French Guiana. Single nucleotide polymorphisms (SNPs) indicated that majority of East African isolates of M. oryzae clustered separately from West African isolates. African isolates clustered with isolates from India and China, indicating that rice blast in SSA may have originated from Asia. Pathotype analysis of Kenyan isolates was undertaken using a set of monogenic differential rice varieties, collectively harbouring 24 disease resistance genes. Rice blast resistance gene Pi-z5 conferred resistance to all the isolates tested. Other resistance genes that conferred resistance to majority of isolates tested include Pi-9, Pi-12(t), Pi-ta, Pi-ta2 and Pi-z. These resistance genes are suitable candidates for introgressing into commercially grown varieties in Kenya in combinations. I also investigated the population of M. oryzae isolates to identify cognate avirulence gene loci, including novel genes not yet reported. Finally, I evaluated rice varieties grown in Kenya for resistance to indigenous rice blast isolates under laboratory conditions. Rice variety Basmati 370 was susceptible to rice blast with varieties IR2793-80-1, BW 196, NERICA 1, NERICA 4, NERICA 10, and NERICA 11 showing some disease resistance. Varieties ITA 310 and Duorado Precoce were moderately tolerant to rice blast. This information is being used to develop a durable blast resistance strategy in sub-Saharan Africa.

Doctor of Philosophy / Department of Plant Pathology / Barbara S. Valent / The hemibiotrophic rice blast fungus Magnaporthe oryzae undergoes complex morphological development throughout its infection cycle. From 8-20 hours after a fungal spore lands on a leaf surface, the fungus differentiates a complex appressorium that punctures the host cuticle. By ~24 hours post inoculation (hpi), the fungus grows inside an epidermal cell as a primary hypha, and by 36 hpi the fungus has differentiated specialized biotrophic invasive hyphae (IH) that are filling the first-invaded cell and moving into neighbor cells. Throughout its life cycle, IH invade living rice cells although invaded cells appear dead when the fungus moves into the next cell. Biotrophic invasion must be mediated by fungal effectors, proteins that pathogens secrete inside live host cells to control them. However, little is known about blast effectors, and the low fungal biomass in early infection stages complicates identification of effector genes, as well as identification of rice genes controlled by effectors. The characterized AVR-Pita effector gene is specifically expressed in planta, but it was not clear how its gene expression pattern changed in different infection stages. We found that AVR-Pita is first expressed around the time of penetration. AVR-Pita is highly expressed in IH developing in asymptomatic tissue from 36 hpi to as late as 7 days post inoculation when lesions are maturing. Using inoculated rice sheaths, we successfully enriched for infected tissue RNA that contained ~20% IH RNA at 36 hpi. We compared IH gene expression to expression in mycelium from pure culture using a whole-genome M. oryzae oligoarray, and we compared infected rice gene expression to expression in mock-inoculated tissue using a rice oligoarray. Rice genes that were induced >50-fold during infection were enriched for genes involved in transferring information from sensors to cellular responses. Fungal genes that were induced >50-fold in IH included known effectors and many IH-specific genes encoding hypothetical secreted proteins that are candidate effectors. Gene knock-out analyses of three putative effector genes failed to show major effects on pathogenicity. Details of the blast interaction transcriptome will provide insights on the mechanisms of biotrophic plant disease.

Appressorium-mediated plant infection is a common strategy used by many plant pathogenic fungi. Understanding the underlying genetic network that controls cellular differentiation of appressorium is therefore pivotal to design durable resistance strategies for these devastating pathogens. This thesis describes four published studies, which investigate the role of septin GTPases in infection and the role of secretion during plant tissue invasion by the rice blast pathogen Magnaporthe oryzae. Appressorium development involves a series of morphogenetic changes that are tightly regulated by cell cycle checkpoints. Entry into mitosis allows differentiation of an appressorium, while penetration peg emergence appears to require progression through subsequent cell cycle checkpoints and cytokinesis. The studies presented here show that symmetry-breaking events that occur during appressorium differentiation are mediated by scaffold proteins, named septins. Septin GTPases recruit actomyosin ring components during septation and define the site of cytokinesis. They also recruit a toroidal cortical F-actin network to the appressorium pore that provides cortical rigidity to facilitate plant infection. Septins act as diffusion barriers for proteins that mediate membrane curvature necessary for penetration peg formation. Repolarization of the F-actin cytoskeleton at the appressorium pore is essential for plant penetration and is controlled by cell polarity regulators, such as Cdc42 and Chm1. Septin-mediated plant infection is regulated by NADPH oxidase (Nox) dependent generation of reactive oxygen species (ROS). The Nox2/NoxR complex is essential for septin organization at the appressorium pore. Septins are therefore key determinants of appressorium repolarization. I also report an investigation of fungal secretory processes during tissue invasion and present evidence that distinct pathways are involved in effector secretion by Magnaporthe oryzae. A BrefeldinA-sensitive pathway is necessary for secretion of apoplastic effectors, such as Bas4 and Slp1, while a BrefeldinA-insensitive pathway is necessary for secretion of effectors destined for delivery to rice cells.