SCAR marker forPhytophthora nicotianaeand a multiplex PCR assay for simultaneous detection ofP. nicotianaeandCandidatusLiberibacter asiaticus in citrus

Abstract

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 (CaLas) in citrus roots using the developed SCAR marker and a previously published 16SrDNA‐based CaLas‐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 µl−1 of mycelial DNA. Furthermore, the specific SCAR primers coupled with a previously reported CaLas‐specific primer set were used effectively in developing a multiplex PCR assay to detect P. nicotianae and CaLas 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 CaLas 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 CaLas. The present technique developed would also be useful in monitoring disease epidemiology and phytosanitary surveillance.

Infection and distribution of Candidatus Liberibacter asiaticus in citrus plants and psyllid vectors at the cellular level

“…Recent studies on CLas have primarily focused on the identification of pathogens, improvement of the detection system and technique sensitivity, analysis of omics and preparation of antibodies (Ding et al ., 2015 , 2016 , 2017 ; Zheng et al ., 2016 ; Kruse et al ., 2017 ; Selvaraj et al ., 2018 ; Das et al ., 2019 ; Li et al ., 2021 ). The mechanisms underlying the interactions among CLas, citrus and D. citri have also gradually been revealed (Mann et al ., 2012 , 2018 ; Ammar et al ., 2016 ; Ghanim et al ., 2016 ; Clark et al ., 2018 ; Wu et al ., 2018 ).…”

Phylogenetic Analysis and Development of Molecular Tool for Detection of Diaporthe citri Causing Melanose Disease of Citrus

“…SCAR-marker was developed to detect Pseudofabraes citricarpa, a fungus causing target spot on Satsuma mandarin (Citrus unshiu) and kumquat (Fortunella margarita) in China [58]. Similarly, SACR-marker derived from random amplified polymorphic DNA (RAPD) was used to simultaneously detect Phytophthora nicotianae and Candidatus Liberibacter asiaticus, the causal agents of citrus roots rot and greening [59]. Pereira, et al [60] developed a multiplex real-time PCR assay to detect Colletotrichum abscissum and Colletotrichum gloeosporioides, the causal agents of citrus post-bloom fruit drop.…”

Rapid and Efficient Molecular Detection of Phytophthora nicotianae Based on RPA-CRISPR/Cas12a

“…Currently, traditional morphological identification methods are time-consuming, have low sensitivity, require highly skilled technicians, and often produce inaccurate results [11]. Several molecular detection techniques are available for the identification of P. nicotianae, including duplex PCR [12], multiplex PCR [13,14], and real-time PCR [15,16]. However, these techniques have limitations such as a long reaction time and the need for specialized equipment, which restrict their use in field disease diagnosis [17].…”