Search

Soybean is one of the most important crops grown worldwide and accounting for significant global trade including transgenic soybean. The crop is attacked by several seed-borne fungal pathogens and some of them are of quarantine concern for India. Keeping in view of the risks associated with movement of soybean seeds, sensitive and reliable molecular diagnostics have been developed for precise and simultaneous detection of three pathogens of quarantine concern for India namely, Diaporthe phaseolorum (stem blight), D. longicolla (seed decay), Peronospora manshurica (downy mildew), along with Macrophomina phaseolina causing dry root rot. The targeted pathogens after isolation from imported transgenic and non-transgenic soybean seeds were identified. Quadruplex and qPCR assays were developed targeting the sequences of different genes such as Histone-3 for detection of D. longicolla and M. phaseolina. The markers DlHisF2&R2 and MpHisF1&R1 produced 265 and 309 bp amplicons for D. longicolla and M. phaseolina, respectively. Actin gene based marker DpActF1&R2 was developed for D. phaseolorum which provided 113 bp amplicon whereas, COX2 based marker PmCoxF2&R2 was developed for P. manshurica with amplified product of 152 bp. During qPCR analysis, these markers proved highly specific and sensitive for detection of these pathogens up to 0.1 pg of template DNA. Quadruplex PCR protocol was also developed by combining these specific markers which could distinguish all the targeted pathogens simultaneously in a single reaction. The developed diagnostic protocols are extremely valuable for quarantine clearance and to ensure the safe transboundary exchange and healthy conservation of germplasm in the National Genebank. [ABSTRACT FROM AUTHOR]

During seed health testing of 160 accessions of safflower, we observed some fungal growth on seed surface of four accessions i.e., IC337954, IC338197, EC182199 and EC181301 which were received from Hyderabad, Telangana, India for long‐term conservation in the National Genebank, ICAR‐National Bureau of Plant Genetic Resources, New Delhi, India. The associated fungus was morphologically identified as Diaporthe helianthi. Later on, the identity was confirmed as Diaporthe helianthi (Desm.) Grove by the sequencing of internal transcribed spacer (ITS) region (GenBank Id: KM979906.1). During seed health testing, seed infection of 30.0 to 70.0 per cent was recorded with no seed germination in infected seeds. Previous findings revealed that D. helianthi is host‐specific pathogen causing stem canker to sunflower only and the fungus is seed‐borne. As a destructive pathogen causing substantial yield losses, especially to sunflower, it may be a serious threat to safflower cultivation in the country and anywhere else. Therefore, the detection and identification of D. helianthi can be helpful in alarming the threat at an early stage thereby minimizing the probable damage to safflower cultivation in near future. [ABSTRACT FROM AUTHOR]

The DNA barcodes were developed from ITS region for the identification of fungal plant pathogens namely, Alternaria alternata and A. tenuissima both causing leaf spots, Ascochyta rabiei causing Ascochyta blight, Fusarium oxysporum f. sp. ciceris causing wilt, Macrophomina phaseolina causing dry root rot, Rhizoctonia solani causing web blight and wet root rot, Sclerotium (Athelia) rolfsii causing collar rot, Sclerotinia sclerotiorum causing stem rot and Cercospora canescens and Pseudocercospora cruenta both causing leaf spots in pulse crops. Barcode compliance for A. alternata (DBTPQ001-18), A. tenuissima (DBTPQ002-18), A. rabiei (DBTPQ003-18), F. oxysporum f. sp. ciceris (DBTPQ004-18), M. phaseolina (DBTPQ005-18), R. solani (DBTPQ006-18), S. rolfsii (DBTPQ007-18), S. sclerotiorum (DBTPQ008-18), C. canescens (DBTPQ009-18) and P. cruenta (DBTPQ029-20) have been generated based on the Barcode of Life Data System (BOLD) system. In addition to ITS, other genomic regions were also explored and on the basis of sequence variation they were ranked as TEF-α > SSU > LSU > β-tubulin. These genes could be considered for secondary barcode and phylogenetic relatedness. ITS-based markers for the detection of A. alternata (BAA2aF and BAA2aR) and R. solani (BRS17cF and BRS17cR) were developed which provided 400 bp and 220 bp amplicons, respectively. While, for F. oxysporum f. sp. ciceris, COX1-based marker (FOCox1F and FOCox3R) was developed which amplified 150 bp. The markers proved highly specific and sensitive with detection limit of 0.0001 ng of template DNA using qPCR and simultaneously detected these three pathogens. The DNA barcodes and diagnostics developed are suitable for quick and reliable detection of these pathogens during quarantine processing and field diagnostics. [ABSTRACT FROM AUTHOR]

Maize is a versatile multi-purpose crop used as a feed and food crop and is one of the most extensively grown and traded crops globally. The maize cultivation is greatly influenced by several seed-borne fungal pathogens including Bipolaris maydis and Stenocarpella maydis. Keeping the potential risks associated with movement of seeds, a conventional and qPCR based reliable and sensitive method for detection of B. maydis and S. maydis was developed. Four sets of specific primers derived from kilbournase gene were developed for detection and quantification of S. maydis, whereas, six sets of specific primers were developed from internal transcribed spacer (ITS) region for detection of B. maydis. The specificity and sensitivity of the primers developed for both the fungal pathogens were tested and the primers were found specific to the target pathogens and are able to detect up to 0.1 pg of genomic DNA. A duplex PCR assay for simultaneous detection of these fungal pathogens was successfully established using highly specific and sensitive primer sets, BmayITS1F&5R specific to B. maydis and DMKB4F&4R specific to S. maydis. The primers produced specific size of amplicons in duplex as well as individually for respective target pathogens. The developed protocol in the present study could be utilized effectively for detection and quantification of these pathogens to facilitate the quick quarantine processing, monitor seed health and seed certification thereby preventing infected maize from entering into food and feed chain and also to facilitate their long-term safe conservation. • Maize, a versatile multi-purpose crop, is going to be the most extensively grown and traded crop globally, whose cultivation is greatly influenced by Bipolaris maydis and Stenocarpella maydis. • Duplex PCR and qPCR were developed using species-specific primers for detection and quantification of S. maydis targeting kilbournase gene , whereas, internal transcribed spacer (ITS) region derived primers were developed for B. maydis. • The protocol could be utilized to facilitate the quick quarantine processing, monitor seed health, seed certification and also to facilitate long-term safe conservation of healthy seeds. [ABSTRACT FROM AUTHOR]