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. 2016 Feb;32(1):53-7.
doi: 10.5423/PPJ.NT.09.2015.0184. Epub 2016 Feb 1.

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

Hyun Ji Cho  1 , Seong Won Hong  2 , Hyun-Ju Kim  3 , Youn-Sig Kwak  2
Affiliations

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

Hyun Ji Cho et al. Plant Pathol J. 2016 Feb.

Abstract

Major diseases in grafted cacti have been reported and Fusarium oxysporum, Bipolaris cactivora, Phytophthora spp. and Collectotrichum spp. are known as causal pathogens. These pathogens can lead to plant death after infection. Therefore, some European countries have quarantined imported cacti that are infected with specific fungal pathogens. Consequently, we developed PCR detection methods to identify four quarantined fungal pathogens and reduce export rejection rates of Korean grafted cacti. The pathogen specific primer sets F.oF-F.oR, B.CF-B.CR, P.nF-P.nR, and P.cF-P.CR were tested for F. oxysporum, B. cactivora, P. nicotinae, and P. cactorum, respectively. The F.oF-F.oR primer set was designed from the Fusarium ITS region; the B.CF-B.CR and P.nF-P.nR primers respectively from Bipolaris and Phytophthora ITS1; and the P.cF-P.CR primer set from the Ypt1protein gene region. The quarantine fungal pathogen primer pairs were amplified to the specific number of base pairs in each of the following fungal pathogens: 210-bp (F. oxysporum), 510-bp (B. cactivora), 313-bp (P. nicotinae), and 447-bp (P. cactorum). The detection limit for the mono- and multiplex PCR primer sets was 0.1 ng of template DNA under in vitro conditions. Therefore, each primer set successfully diagnosed contamination of quarantine pathogens in export grafted cacti. Consequently, our methodology is a viable tool to screen contamination of the fungal pathogen in exported grafted cacti.

Keywords: PCR detection; grafted cactus; quarantine pathogen.

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Figures

Fig. 1
Fig. 1
Sensitivity testing of different genomic DNA quantities (100 ng, 50 ng, 25 ng, 10 ng, 5 ng, 2.5 ng, 1 ng, 0.1 ng, 0.01 ng/ul). DNA was amplified with specific primers and amplicons detected different DNA quantities. Fungal pathogen sensitivity level is identified in agarose gels. (A) F. oxysporum sensitivity level 0.01 ng. (B) B. cactivora sensitivity level 0.01 ng. (C) P. nicotianae sensitivity level 0.1 ng. (D) P. cactorum sensitivity level 1 ng.
Fig. 2
Fig. 2
Multiplex PCR, four sets of primers were combined in a single tube to identify the four fungal pathogens (100 ng, 50 ng, 25 ng, 10 ng, 5 ng, 2.5 ng, 1 ng, 0.1 ng, 0.01 ng/ul). (A) P. nicotianae sensitivity 0.1 ng DNA. (B) P. cactorum 1 ng DNA. (C) B. cactivora 0.01 ng DNA. (D) F. oxysporum 0.01 ng DNA.
Fig. 3
Fig. 3
Multiplex PCR in planta conditions. After cutting inoculated cactus (109, 108, 107, 106, 105, 104, 103, 102, 101 spore/ml), genomic DNA was extracted for testing multiplex PCR. Nine cacti were inoculated with spore stocks (109, 108, 107, 106, 105, 104, 103, 102, 101 spore/ml) and plant genomic DNA was extracted. (A) F. oxysporum, (B) B. cactivora, (C) P. nicotianae, (D) P. cactorum.
Fig. 4
Fig. 4
Multiplex PCR in spore-stock conditions. After inoculating spore stocks on cactus (109, 108, 107, 106, 105, 104, 103, 102, 101 spore/ml), total genomic DNA was extracted using cotton swab to test multiplex PCR. Nine cacti were inoculated with spore stocks (109, 108, 107, 106, 105, 104, 103, 102, 101 spore/ml) and plant genomic DNA was extracted. (A) F. oxysporum multiplex PCR showed spore stock ranged from 104 to 109. (B) Cacti inoculated with B. cactivora showed 104 to 109. (C) P. nicotianae multiplex PCR sensitivity is 103 to 109 (D) P. cactorum sensitivity is ranged from 104 to 109 spore stock detected by multiplex PCR in cacti.
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