Determining the Population Structure and Avirulence Gene Repertoire of the Rice Blast Fungus Magnaporthe oryzae in Kenya by Comparative Genome Analysis

Abstract

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.