Quadruplex and q-PCR based diagnostic assay to delineate the major quarantine and other seed-borne fungal pathogens of soybean

• Published in: World journal of microbiology & biotechnology Vol. 39; no. 9; p. 233
• Main Authors: Tripathi, Aradhika, Akhtar, Jameel, Kalaiponmani, K., Dubey, Sunil C., Chalam, Vasimalla Celia
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01-09-2023; Springer Nature B.V
• Subjects: Actin; Airborne microorganisms; Applied Microbiology; Biochemistry; Biomarkers; Biomedical and Life Sciences; Biotechnology; Blight; COX2 protein; Diagnostic systems; Downy mildew; Dry rot; Environmental Engineering/Biotechnology; Fungi; Germplasm; Glycine max - microbiology; Histones; International trade; Life Sciences; Microbiology; Pathogens; Plant Diseases - microbiology; Polymerase chain reaction; Polymerase Chain Reaction - methods; Quarantine; Root rot; Seeds; Seeds - microbiology; Soybeans; Stem blight; India; Fungal pathogen; Quadruplex PCR; Quarantine; qPCR Soybean; Diagnostics; Detection
• ISSN: 0959-3993; 1573-0972
• DOI: 10.1007/s11274-023-03683-4

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.