Development of a Multiplex PCR Method to Detect Fungal Pathogens for Quarantine on Exported Cacti

Cho, Hyun Ji; Hong, Seong Won; Kim, Hyun-Ju; Kwak, Youn‐Sig

doi:10.5423/ppj.nt.09.2015.0184

Cited by 24 publications

(9 citation statements)

References 12 publications

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“…Multiplex PCR for pathogen diagnosis permits simultaneous detection of several plant pathogens in a single reaction mixture and facilitates cost‐effective diagnosis (Hu et al ; Cho et al ). Multiplex PCR protocols have been developed to detect two to several Phytophthora spp.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2019

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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.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2019

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“…The samples were centrifuged at 13,000 rpm at 4°C for 5 minutes. Supernatant was removed by washing with 500 μl of 70% ethanol ( Cho et al, 2016 ). Ethanol was subsequently removed and pellets dried at room temperature for 30 minutes.…”

Section: Methodsmentioning

confidence: 99%

Epidemiology and Control of Strawberry Bacterial Angular Leaf Spot Disease Caused by Xanthomonas fragariae

Kim

Gang

Jeon

et al. 2016

The Plant Pathology Journal

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Strawberry bacterial angular leaf spot (ALS) disease, caused by Xanthomonas fragariae has become increasingly problematic in the strawberry agro-industry. ALS causes small angular water-soaked lesions to develop on the abaxial leaf surface. Studies reported optimum temperature conditions for X. fragariae are 20°C and the pathogen suffers mortality above 32°C. However, at the nursery stage, disease symptoms have been observed under high temperature conditions. In the present study, results showed X. fragariae transmission was via infected maternal plants, precipitation, and sprinkler irrigation systems. Systemic infections were detected using X. fragariae specific primers 245A/B and 295A/B, where 300-bp and 615-bp were respectively amplified. During the nursery stage (from May to August), the pathogen was PCR detected only in maternal plants, but not in soil or irrigation water through the nursery stage. During the cultivation period, from September to March, the pathogen was detected in maternal plants, progeny, and soil, but not in water. Additionally, un-infected plants, when planted with infected plants were positive for X. fragariae via PCR at the late cultivation stage. Chemical control for X. fragariae with oxolinic acid showed 87% control effects against the disease during the nursery period, in contrast to validamycin-A, which exhibited increased efficacy against the disease during the cultivation stage (control effect 95%). To our knowledge, this is the first epidemiological study of X. fragariae in Korean strawberry fields.

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“…This problem has been resolved using multiplex PCR assays. It was noted that the diagnosing tool was sufficient to detect and identify these quarantine fungal pathogens in grafted cacti ( Cho et al., 2016 ). Though multiplex PCR assays are quick and reliable in nature, the assays are potentially expensive and resource-intensive, and decreased sensitivity associated with the multiplex methods ( Pallás et al., 2018 ).…”

Section: Molecular Tools For Detection Of Fungimentioning

confidence: 99%

Recent Advances in Molecular Diagnostics of Fungal Plant Pathogens: A Mini Review

Hariharan¹,

Prasannath²

2021

Front. Cell. Infect. Microbiol.

118

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Phytopathogenic fungal species can cause enormous losses in quantity and quality of crop yields and this is a major economic issue in the global agricultural sector. Precise and rapid detection and identification of plant infecting fungi are essential to facilitate effective management of disease. DNA-based methods have become popular methods for accurate plant disease diagnostics. Recent developments in standard and variant polymerase chain reaction (PCR) assays including nested, multiplex, quantitative, bio and magnetic-capture hybridization PCR techniques, post and isothermal amplification methods, DNA and RNA based probe development, and next-generation sequencing provide novel tools in molecular diagnostics in fungal detection and differentiation fields. These molecular based detection techniques are effective in detecting symptomatic and asymptomatic diseases of both culturable and unculturable fungal pathogens in sole and co-infections. Even though the molecular diagnostic approaches have expanded substantially in the recent past, there is a long way to go in the development and application of molecular diagnostics in plant diseases. Molecular techniques used in plant disease diagnostics need to be more reliable, faster, and easier than conventional methods. Now the challenges are with scientists to develop practical techniques to be used for molecular diagnostics of plant diseases. Recent advancement in the improvement and application of molecular methods for diagnosing the widespread and emerging plant pathogenic fungi are discussed in this review.

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Development of a Multiplex PCR Method to Detect Fungal Pathogens for Quarantine on Exported Cacti

Cited by 24 publications

References 12 publications

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

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

Epidemiology and Control of Strawberry Bacterial Angular Leaf Spot Disease Caused by Xanthomonas fragariae

Recent Advances in Molecular Diagnostics of Fungal Plant Pathogens: A Mini Review

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