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Subject: Agricultural Biotechnology × Author: Ahmed, Reshma × Start date: 2020 × Type: Thesis ×
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    ThesisItemOpen Access
    Characterization of the Sinapis alba-Alternaria brassicicola interaction and, identification of associated defense response genes
    (2021) Ahmed, Reshma; Bhorali, Priyadarshini
    Alternaria blight caused by Alternaria brassicicola is one of the most devastating and widespread fungal diseases of the oilseed mustards. It causes yield losses up to 47% and has been reported from various parts of the country including the state of Assam. The non-host Sinapis alba, wild relatives of Brassicaceae, has been reported to have resistance against Alternaria blight disease. In order to understand this non-host mechanism, isolation and identification of pathogen, morphopathological, screening for resistance both in vitro and in vivo, histopathological study by using Scanning Electron Microscopy (SEM) and global gene expression using next generation sequencing method(NGS), RNA-seq, has been done to develop resistance variety of rapeseed mustard against Alternaria blight. For this, infected leaves, siliques and stems of the Toria variety TS-38, showing the initial conspicuous characteristic symptoms of Alternaria blight, were collected for isolation of the pathogen. The infected plant parts were surface sterilized and inoculated in petriplates containing Potato Dextrose Agar (PDA) medium under optimal conditions for fungal growth and sporulation. The mycelial growth of the fungus was observed after 3days of inoculation and the hyphal growth covered the petriplates within 15days of inoculation. On the basis of the conidial morphology as observed through microscopic studies, the pathogen was identified as Alternaria brassicicola. Purification of the fungal pathogen was done by single spore isolation method. Further, molecular detection of the fungus was successfully carried out by amplification of the fungal genomic DNA using reported ITS primers (ITS1/ITS2 & ITS2/ITS4). Sequencing of the fungal ITS region also confirmed the pathogen at the genus level. Further confirmation upto species level has been done with A. brassicicola specific primers (ABS28). The primary screening test both in vitro detached leaf assay showed the development of infection by showing cholorotic region after 72hpi with no chlorosis in S. alba. The light microscopy study of the infected portion showed the appearance of increase number fungal filaments in B. rapa with a few filaments in S. alba. The trypan blue staining showed the increase level of necrosis in B. rapa in comparison with S. alba. The SEM analysis also showed a similar type of result with light microscopy with a less hyphal penetration with a few spores in comparison with B. rapa by the presence of mass of hyphal filament with increased number of spores. Further Pathogenecity test was performed in vivo that showed successful development of disease in B. rapa after 72hpi whereas the infection developed after 7dpi in S. alba. The disease progression study by morphological study showed a slow and restricted with little infection in S. alba after 10dpi whereas in B. rapa with severe infection with complete death of the inoculated leaves within that period. The disease scoring has been studied from 0hrs to 10 days. Leaf disease incidence percentage (LDI%) was calculated that analyzed the number of incidence of spot after infection. LDI% for B. rapa was found to be 82.50% while in S. alba it only showed 15% after 10dpi which is very less and showed its resistance against A. brassicicola. Global transcritome profile has been done at early infection period viz. 48 and 72 hours post inoculation to identify the genes responsible for its resistance by deferential expression study of genes in both the cultivars. A mapping percentage of 79.41% and 78.46% was found in S. alba after 48 and 72hpi and of 96.63% and 92.87% was found in B. rapa after 48 and 72hpi. DEG analysis showed a large number of genes were up-regulated at 48hpi than 72hpi. Validation of transcriptome data was performed by selecting 12 defense related genes that showed a high fold change in S. alba in comparison with B. rapa which showed a similar trend that confirms the validation of the targeted data set.