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SCAR marker for Phytophthora nicotianae and a multiplex PCR assay for simultaneous detection of P. nicotianae and Candidatus Liberibacter asiaticus in citrus
Author(s) -
Das A.K.,
Nerkar S.,
Gawande N.,
Thakre N.,
Kumar A.
Publication year - 2019
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.14392
Subject(s) - phytophthora nicotianae , primer (cosmetics) , rapd , biology , amplicon , multiplex , phytophthora , microbiology and biotechnology , genetics , botany , polymerase chain reaction , gene , population , genetic diversity , chemistry , demography , organic chemistry , sociology
Aims This study aimed to develop a random amplified polymorphic DNA (RAPD)‐based sequence characterized amplified region (SCAR) marker for species‐specific detection of Phytophthora nicotianae , a global plant pathogen. Another objective was to develop a multiplex PCR assay for simultaneous detection of P. nicotianae and huanglongbing‐causing bacterium, Candidatus Liberibacter asiaticus ( Ca Las) in citrus roots using the developed SCAR marker and a previously published 16SrDNA‐based Ca Las‐specific primer set. Methods and Results The RAPD primer, OPA4, amplified a specific fragment of c. 400 bp only in P. nicotianae isolates. The fragment was eluted, purified, cloned and sequenced. One set of SCAR primers (SCAR4F/SCAR4R1), developed from the sequence information of the fragment, was found specific to P. nicotianae and produced an amplicon of 330 bp size, and was found non‐specific to the five Phytophthora species ( P. citrophthora , P. palmivora , P. lacustris , P. boehmeriae and P. insolita ) and five other pathogens ( Mycosphaerella citri , Alternaria alternata , Septobasidium pseudopedicillatum , Phytopythium vexans and Colletotrichum gloeosporioides ) isolated from the citrus agroecosystem. The sensitivity of the primer pair was 5 pg µ l −1 of mycelial DNA. Furthermore, the specific SCAR primers coupled with a previously reported Ca Las‐specific primer set were used effectively in developing a multiplex PCR assay to detect P. nicotianae and Ca Las simultaneously in root tissues of citrus plants. Conclusions A rapid method using a RAPD‐based SCAR marker for the detection of P. nicotianae was developed. Furthermore, a multiplex PCR assay was established for simultaneous detection of P. nicotianae and Ca Las in citrus roots. Significance and Impact of the Study A RAPD‐SCAR marker‐based detection system and the one‐step multiplex PCR method developed in this study can be applied to index citrus trees infected (individually or conjointly) with P. nicotianae and Ca Las. The present technique developed would also be useful in monitoring disease epidemiology and phytosanitary surveillance.