Development of species-specific PCR based detection assay for Ceratocystis fimbriata, mango wilt pathogen

Autor: Nidhi Kumari, P. K. Shukla, Anju Bajpai, Haripal Singh, Tahseen Fatima
Rok vydání: 2021
Předmět:
Zdroj: Indian Phytopathology. 75:215-220
ISSN: 2248-9800
0367-973X
DOI: 10.1007/s42360-021-00438-9
Popis: A PCR-based detection method was developed for Ceratocystis fimbriata detection, the main causal agent of mango wilt in India. Two primer pairs (one forward and two reverse primers) targeting the ITS region of the fungal ribosomal DNA were developed which proved to be highly specific and sensitive to C. fimbriata. The primer pairs produced the amplification product of 473 bp and ~ 350 bp, respectively while no amplification was observed in the case of C. paradoxa, Berkeleyomyces basicola (member of Ceratocystidaceae family), and other common soil-inhabiting fungi viz., Fusarium oxysporum and Trichoderma harzianum as well as other mango pathogens viz., Colletotrichum gloeosporioides, Diaporthe phyllanthicola, and Botryosphaeria dothidea. The primers were able to detect the pathogen at DNA concentration of 0.1 pg/25 μl both directly from pure fungal culture as well as rhizospheric soil. The protocol developed was further validated in the field by detecting C. fimbriata from the rhizospheric soil of partially and completely wilted mango trees. The absence of amplification in the rhizospheric soil samples of healthy mango trees, as well as other horticultural and vegetable crops, ruled out the possibility of false positives. Though, we failed to detect the pathogen from trunk wood samples of wilted mango trees despite the isolation of decent quality as well as quantity of DNA possibly due to the presence of many PCR inhibitors in wood samples. The method developed in the present study can be applied in the field to detect the pathogen from the soil which is a major infection pathway and thus can aid in the better management of this disease.
Databáze: OpenAIRE
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