Aims: To characterize the geo-distinct isolates of Ustilaginoidea virens for morpho-molecular and mating-type locus diversity. Methods and Results: Sixty-one isolates of U. virens collected from Southern India exhibited significant diversity in mycelial width (3Á45-5Á50 µm), colony colour (yellow, pale yellow, and white), and growth pattern (thick leather mat, raised-fluffy, flat-fluffy, and raised). Field-borne chlamydospores of each isolate were significantly smaller in size (3Á34-5Á26 µm 2 ) compared to those formed on culture media (18Á6-100Á89 µm 2 ). The phylogenetic study based on internal transcribed sequences revealed two clusters; however, most isolates (n = 54) were grouped in cluster-I, indicating common ancestral origin. We also identified 42 haplotypes; among them, Hap_3 has the highest number of isolates (n = 19). Mating-type locus (MAT1) analysis revealed all sixty-one isolates as heterothallic, wherein 37 and 24 isolates belonging to MAT1-1-1 and MAT1-2-1 heterothallic mating types, respectively. The microsynteny analysis of MAT1 loci of one of the Indian strain (Uv-Gvt) along with Uv-8b (China) strain revealed synteny conservation at MAT1 locus, which is flanked by conserved genes SLA2 and a hypothetical protein in the upstream and APN2, COX12 and APC5 in the downstream of the locus. Conclusions: Morpho-molecular study revealed the significant diversity among geo-distinct isolates, and MAT1 loci analysis indicated the distribution of heterothallic mating types in south Indian paddy fields. And also, complete synteny conservation between Indian and Chinese strain was observed at the MAT1 locus. Significance and Impact of the Study: This is the first report describing the sexuality of Indian strains of the U. virens, which would help better understand the genetic diversity of the U. virens prevailing in Southern India and aid in developing resistant rice cultivars against this pathogen population.

Aims:To investigate the diversity of eco-distinct isolates of Magnaporthe oryzae for their morphological, virulence and molecular diversity and relative distribution of five Avr genes. Methods and Results:Fifty-two M. oryzae isolates were collected from different rice ecosystems of southern India. A majority of them (n = 28) formed a circular colony on culture media. Based on the disease reaction on susceptible cultivar (cv. HR-12), all 52 isolates were classified in to highly virulent (n = 28), moderately virulent (n = 11) and less-virulent (13) types. Among the 52 isolates, 38 were selected for deducing internal transcribed spacer (ITS) sequence diversity. For deducing phylogeny, another set of 36 isolates from other parts of the world was included, which yielded two distinct phylogenetic clusters. We identified eight haplotype groups and 91 variable sites within the ITS sequences, and haplotype-group-2 (Hap_2) was predominant (n = 24). The Tajima's and Fu's Fs neutrality tests exhibited many rare alleles. Furthermore, PCR analysis for detecting the presence of five Avr genes in the different M. oryzae isolates using Avr gene-specific primers in PCR revealed that Avr-Piz-t, Avr-Pik, Avr-Pia and Avr-Pita were present in 73.68%, 73.68%, 63.16% and 47.37% of the isolates studied, respectively; whereas, Avr-Pii was identified only in 13.16% of the isolates.Conclusions: Morpho-molecular and virulence studies revealed the significant diversity among eco-distinct isolates. PCR detection of Avr genes among the M. oryzae population revealed the presence of five Avr genes. Among them, Avr-Piz-t, Avr-Pik and Avr-Pia were more predominant. Significance and Impact of the Study:The study documented the morphological and genetic variability of eco-distinct M. oryzae isolates. This is the first study demonstrating the distribution of the Avr genes among the eco-distinct population of M. oryzae from southern India. The information generated will help plant breeders to select appropriate resistant gene/s combinations to develop blast disease-resistant rice cultivars. 7 MF716846.1 M. oryzae isolate AMA UOC-01 8 KT693184.1 M. oryzae strain UPM-PO Malaysia 9 KM249937.1 M. oryzae strain PO-FA01 10 MW042178.1 P. oryzae strain Pos-3 China 11 MW042177.1 P. oryzae strain Pos-2 12 MT588875.1 P. grisea isolate DW-2016-2 13 MW042176.1 P. oryzae strain Pos-1 14 MG642970.1 M. e grisea 15 MF946553.1 Pyricularia sp. strain F2 United Kingdom 16 MN203028.1 P. oryzae isolate T49 17 FN555121.1 M. oryzae 18 JX469384.1 M. oryzae culture-collection NFCCI:2605 India (NE) 19 MF678836.1 M. oryzae isolate Gudalur India (TN) 20 MF669475.1 M. oryzae isolate Nagaranai 21 MF668695.1 M. oryzae isolate Aduthurai 22 MF668692.1 M. oryzae isolate Maduranthakam 23 KM816799.1 M. oryzae strain w11-65 18S Korea 24 KM816801.1 M. oryzae strain w11-110 25 KM816797.1 M. oryzae strain w11-58 26 KM816796.1 M. oryzae strain w11-23 27 AB269937.1 M. grisea genes Japan 28 MT103091.1 P. oryzae isolate Mf3 Iran 29 MT103090.1 P. oryzae isolate Mb3 30 LC552959.1

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Single spore isolation from a diseased sample is an essential step in obtaining a pure culture of a fungal pathogen. Rice blast disease caused by Magnaporthe oryzae is an inferior saprophytic competitor, and therefore, many fast-growing fungal or bacterial contaminants are predominant during its isolation. For isolation of M. oryzae, several methods are being followed; however, they are complex and often lead to contamination. In the present study, we have standardized an efficient method for rapid isolation of M. oryzae from the blast disease infected rice-leaf using single spore isolation by spore-drop technique. Following the spore-drop technique, pure culture for an isolate of M. oryzae was obtained quickly with the least contamination (4%), whereas the conventional spore-dilution and leaf-press method recorded 26.12 and 45.50% contamination, respectively. The spore-drop approach has yielded the single spore isolates in the shortest time (10 days) and can be used for regular rice blast pathogen isolation. This method can also be used for other sporulating pathogens successfully.