B. M. Bashyal

Mapping quantitative trait loci responsible for resistance to Bakanae disease in rice.

BackgroundBakanae or foot rot disease caused by Fusarium fujikuroi [teleomorph: Gibberella fujikuroi (Sawada) Ito] is emerging as a serious disease in rice. The disease causes both quantitative and qualitative losses to the grains under the field conditions. Breeding for resistance to Bakanae disease is a promising strategy to manage this emerging disease. In this study, we used a population of 168xa0F14 recombinant inbred lines (RILs) derived from two indica rice parents Pusa 1342, a highly resistant variety and Pusa Basmati 1121, a highly susceptible variety to map quantitative trait loci (QTLs) governing resistance against Bakanae disease.ResultsThe disease reaction of 168xa0F14 RILs were measured on the seedlings inoculated using Fusarium fujikuroi culture using high-throughput screening protocol under glasshouse conditions. Utilizing inclusive composite interval mapping, three QTLs governing resistance to Bakanae were identified, namely qBK1.1, qBK1.2 and qBK1.3 which accounted 4.76, 24.74 and 6.49xa0% of phenotypic variation, respectively. The major effect QTL designated qBK1.2 was mapped in 0.26xa0Mb region between RM5336 and RM10153. A total of 55 annotated genes were identified within the identified QTL region qBK1.2.ConclusionsThe novel QTLs identified in this study are useful resource for efficiently breeding rice cultivars resistantxa0to Bakanae disease. This is the first report on identification of QTLs governing resistance against Bakanae in rice using inclusive composite interval mapping strategy in a RIL population.

Occurrence, identification and pathogenicity of Fusarium species associated with bakanae disease of basmati rice in India

The incidence of bakanae disease of rice was studied in the basmati growing states of Punjab, Haryana, Uttar Pradesh and Uttarakhand in India. Maximum disease incidence (20xa0%) was observed from the Karnal district of Haryana and Bulandshar, Ghaziabad and Mathura districts of Uttar Pradesh in rice variety Pusa Basmati 1121 and Pusa Basmati 1509. Disease was also observed in other popular basmati rice varieties of the country including Pusa Basmati 6, Pusa 2511, CSR 30, Pakistani Basmati and Sarbati with the incidence of 0.5xa0% to 10xa0%. One hundred and twenty-six isolates of Fusarium spp. were isolated from symptomatic plants and were characterized morphologically and 42 isolates were characterized at the molecular level. Out of 42 isolates characterized based on translation elongation factor 1 α (TEF- 1α), 41 were identified as Fusarium fujikuroi. Virulence analysis performed on rice variety Pusa Basmati 1509 categorized the isolates as moderately virulent (37.3xa0%), virulent (34.1xa0%) and highly virulent (28.6xa0%). Results indicated that F. fujikuroi is predominant in symptomatic diseased plants and a management programme should be developed by considering the prevalence and virulence of the pathogen in the area of intended cultivation.

Pathogenicity, Ecology and Genetic Diversity of the Fusarium spp. Associated with an Emerging Bakanae Disease of Rice ( Oryza sativa L.) in India

Bakanae disease is one of the emerging diseases of rice (Oryza sativa L.). Gibberella fujikuroi species complex were detected in popularly grown rice varieties of India with infection percentage ranging from 1 to 24 %. Pathogenicity test of Fusarium spp. was performed in susceptible rice variety Pusa 1121, which showed reduced seed germination and possessed varying ability to cause symptoms. On the basis of internal transcribed spacer (ITS) and Translation Elongation Factor (TEF), three Fusarium spp., viz. F. verticillioides, F. fujikuroi and F. proliferatum were found associated with bakanae disease of rice in India. Maximum numbers of slender and chlorotic leaves were produced by F. fujikuroi (90 %), whereas crown rot and stem rot was produced by F. verticillioides (50 %). F. proliferatum produced both elongation and rotting symptoms. Information on the bakanae disease, its distribution, characterization and identification in India could be helpful for the development of management strategies.

Development of high throughput screening protocol and identification of novel sources of resistance against bakanae disease in rice (Oryza sativa L.)

Bakanae or foot rot disease caused by Fusarium fujikuroi (teleomorph: Gibberella fujikuroi, Sawada, Wollenweber) is emerging as a serious disease of rice. A simple, reliable and high-throughput method for screening the disease would enable rapid screening of germplasm aimed at identifying resistance sources, mapping QTLs/genes and developing resistant rice cultivars. In the present study, a highthroughput, reliable bioassay to screen rice germplasm for resistance to bakanae disease was developed and compared with the conventional screening technique. This technique involves soaking of rice seeds in fungal spore suspension (1.0x106 spores ml–1) for 24 hours at room temperature. Seedling growth at 30°/25° (±3)°C day/night temperature and 60/80(±10)% day/night relative humidity in glasshouse gave the best results. The new protocol described here produces consistent and reproducible bakanae disease symptoms and enables screening of hundreds of rice germplasm within 15 days without any loss of precision in screening of rice genotypes against bakanae disease. The resistant and susceptible genotypes can be used for developing mapping population and identification of QTLs/genes conferring resistance to bakanae disease.

SINGLE AND COMBINED EFFECTS OF THREE FUSARIUM SPECIES ASSOCIATED WITH RICE SEEDS ON THE SEVERITY OF BAKANAE DISEASE OF RICE

Fusarium spp. associated with susceptible rice variety Pusa Basmati 1121 were isolated. Based on translation elongation factor 1α (TEF-1α) Fusarium spp. were identified as F. fujikuroi, F. proliferatum, F. verticillioides (F. moniliforme), F. sacchari, F. pseudonygamai, F. thapsinum and F. andiyazi. Pathogenicity test conducted on susceptible variety Pusa Basmati 1121 confirmed that only F. fujikuroi, F. proliferatum and F. verticillioides produced bakanae disease as elongation, rotting or both types of symptoms. Gibberellic acid was exclusively extracted from the isolates of F. fujikuroi and it ranged from 450 μg/g to 4361 μg/g of mycelium. Gibberellic acid content was positively correlated (r=0.79) with elongation symptom of the disease. Fusaric acid was present in all the species of Fusarium, ranging from 60 μg/g to 2920 μg/g of mycelium. However, significant correlation between fusaric acid content and disease severity could not be established. Co-inoculations of pathogens showed significant reduction in percent seed germination and increased disease severity compared to pathogen inoculated individually. Therefore, present investigation will be helpful to understand the effect of different Fusarium spp. in bakanae disease etiology.

Screening and identification of new sources of resistance to sheath blight in wild rice accessions

A total of 218 wild rice accessions including O. rufipogon and O. nivara were evaluated under artificial inoculation for resistance to sheath blight disease for two years under field conditions. The germplasm identified as resistant to sheath blight were further evaluated through artificial inoculation for an additional year under glass house conditions. Based on three years of testing, two Oryza rufipogon accessions, namely, IC336719 and IC336721 were identified as resistant to sheath blight disease. In vitro characterization of resistant genotypes revealed that the size of sheath blight lesion formation was small along with comparatively lesser number of infection cushions and penetration pegs as compared to the susceptible checks. The O. rufipogon accessions identified in the present study are very valuable genetic resource, which can be utilized in the development of introgression lines and mapping QTL(s) governing resistance to sheath blight of rice.

Whole Genome Sequencing of Fusarium fujikuroi Provides Insight into the Role of Secretory Proteins and Cell Wall Degrading Enzymes in Causing Bakanae Disease of Rice

Fusarium fujikuroi causing bakanae disease has emerged as one of the major pathogen of rice across the world. The study aims to comparative genomic analysis of Fusarium fujikuroi isolates and identification of the secretary proteins of the fungus involved in rice pathogenesis. In the present study, F. fujikuroi isolate “F250” was sequenced with an assembly size of 42.47 Mb providing coverage of 96.89% on reference IMI58289 genome. A total of 13,603 protein-coding genes were predicted from genome assembly. The average gene density in the F. fujikuroi genome was 315.10 genes per Mb with an average gene length of 1.67 kb. Additionally, 134,374 single nucleotide polymorphisms (SNPs) are identified against IMI58289 isolate, with an average SNP density of 3.11 per kb of genome. Repetitive elements represent approximately 270,550 bp, which is 0.63% of the total genome. In total, 3,109 simple sequence repeats (SSRs), including 302 compound SSRs are identified in the 8,656 scaffolds. Comparative analysis of the isolates of F. fujikuroi revealed that they shared a total of 12,240 common clusters with F250 showing higher similarity with IMI58289. A total of 1,194 secretory proteins were identified in its genome among which there were 356 genes encoding carbohydrate active enzymes (CAZymes) capable for degradation of complex polysaccharides. Out of them glycoside hydrolase (GH) families were most prevalent (41%) followed by carbohydrate esterase (CE). Out of them CE8 (4 genes), PL1 (10 genes), PL3 (5 genes), and GH28 (8 genes) were prominent plant cell wall degrading enzymes families in F250 secretome. Besides this, 585 genes essential for the pathogen–host interactions were also identified. Selected genes were validated through quantitative real-time PCR analyses in resistant and susceptible genotypes of rice at different days of inoculation. The data offers a better understanding of F. fujikuroi genome and will help us enhance our knowledge on Fusarium fujikuroi–rice interactions.

Emergence of Aggressive Population in the Bipolaris sorokiniana of Barley (Hordeum vulgare L.) Through Anastomosis

Bipolaris sorokiniana is a hemibiotrophic pathogenic fungus responsible for spot blotch of wheat and barley. Evolution process of most virulent population of B. sorokiniana of barley was studied through colony morphology, melanin content, spore production, pathogenicity, nuclear condition and anastomosis. Bridge (H) type and hyphal tip anastomosis were observed between black and white isolates. Maximum number of nuclei (4.14xa0nucleixa0cell−1) were present in mixed isolates followed by white isolate (2.43xa0nucleixa0cell−1) and black isolate (1.14xa0nucleixa0cell−1). Results indicated that aggressive population in B. sorokiniana was evolved by the variations in the nucleus due to anastomosis.

First report of Sclerotinia rot of pigeonpea caused by Sclerotinia sclerotiorum (Lib.) de Bary in India

Abstract Sclerotinia stem rot caused by Sclerotinia sclerotiorum (Lib.) de Bary was observed for the first time on pigeonpea in Pusa, Bihar (India) during the years 2012–2014. Typical symptoms of the disease included blighting of twigs, stem lesions, and development of cottony white mycelium and numerous large sclerotia on diseased plant parts. The fungus was identified on the basis of cultural and morphological characteristics, and its pathogenicity was also established. The internal transcribed spacer (ITS) region of pathogen was amplified with primers ITS1 and ITS4, which revealed 100% query coverage along with 99% identity with S. sclerotiorum recovered from different hosts. The highest disease incidence was recorded on cultivar ‘ICPL151’, whereas cultivars ‘MAL13’ and ‘Kudrat’ were observed to have significantly less disease under natural conditions.

Isolation and Identification of Defense Responsive Genes in Wheat During Incompatible Interaction with Bipolaris sorokiniana (Cochliobolus sativus) using SSH Technique

During the initial phase of host pathogen interaction, induction of defense genes is very critical for the incompatible interactions. In order to isolate the defense responsive genes during interaction of wheat with spot blotch fungus, Bipolaris sorokiniana, suppressive subtraction hybridization (SSH) technique was used. Four hundred and eighty clones were obtained from cDNA subtraction library and out of these 36% (172) were of high quality which were sequence analysed using n BLAST from NCBI database. Forty nine differentially expressed clones were obtained which were classified into seven classes based on their putative functions. Twelve clones were classified as plant defense responsive genes based on their putative functions observed in NCBI database. Clone SSH_Wh 135 matched with putative puroindoline b proteins from Triticum aestivum which are encoded by puroindoline gene (pin B gene). This gene contains tryptophan rich hydrophobic domain, which is responsible for grain hardness and also has antimicrobial properties having lipid binding sites.