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, 33 (3), 307-317

Development of Multiplex PCR for Simultaneous Detection of Citrus Viruses and the Incidence of Citrus Viral Diseases in Late-Maturity Citrus Trees in Jeju Island

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Development of Multiplex PCR for Simultaneous Detection of Citrus Viruses and the Incidence of Citrus Viral Diseases in Late-Maturity Citrus Trees in Jeju Island

Jae Wook Hyun et al. Plant Pathol J.

Abstract

Satsuma dwarf virus (SDV) or Citrus mosaic sadwavirus (CiMV) were not consistently detected in RT-PCR assay with the primer sets based on gene of Japan isolates. SDV and CiMV isolates were distinctively divided into two groups based on phylogenetic analysis of PP2 gene cloned from 22 Korean isolates, and the Korean CiMV and SDV isolates shared 95.5-96.2% and 97.1-97.7% sequence identity with Japanese isolate, respectively. We developed PP2-1 primer set based on the PP2 gene sequence of Korean isolates to simultaneously and effectively detect SDV and CiMV. And CTLV-2013 and CTV-po primer sets were newly designed for detection of Citrus tatter leaf virus (CTLV) and Citrus tristeza virus (CTV), respectively. Using these primer sets, a new multiplex PCR assay was developed as a means to simultaneously detect 4 citrus viruses, CTV, CTLV, SDV, and CiMV. The degree of detection by the multiplex PCR were consistent with those of uniplex RT-PCR for detection of each of the viruses. Therefore, the new multiplex PCR provides an efficient method for detecting 4 citrus viruses, which will help diagnose many citrus plants at the same time. We verified that 35.2% and 72.1% of 775 trees in 155 orchards were infected with SDV or CiMV (SDV/CiMV) and CTV by the multiplex-PCR assay, respectively, and CTLV was not detected in any of the trees tested.

Keywords: PCR; citrus virus; detection; multiplex.

Figures

Fig. 1
Fig. 1
Typical symptoms of citrus viral infection. (A) Citrus tristeza virus (CTV) on yuju (Citrus junos). (B) Citrus tatter leaf virus (CTLV) on early satsuma mandarin (Citrus unshiu ‘Miyagawa Wase’). (C) Satsuma dwarf virus (SDV) on ‘Setoka’ ([Citrus unshiu × C. sinensis] × C. reticulate). (D) Citrus mosaic sadwavirus (CiMV) on very early satsuma mandarin (Citrus unshiu ‘Miyamoto Wase’).
Fig. 2
Fig. 2
Primers used in multiplex PCR to detect Satsuma dwarf virus (SDV) and Citrus mosaic sadwavirus (CiMV), derived from the PP2 gene.
Fig. 3
Fig. 3
Phylogenetic tree resulting from maximum parsimony analysis of sequences of the PP2 gene. Bootstrap values are based on 1,000 replicates.
Fig. 4
Fig. 4
Simultaneous detection of citrus viruses using multiplex PCR. Lane M: 100-bp DNA ladder; lanes 1–7: virus isolate. SDV, Satsuma dwarf virus; CiMV, Citrus mosaic sadwavirus; CTLV, Citrus tatter leaf virus; CTV, Citrus tristeza virus.
Fig. 5
Fig. 5
Comparison of detection sensitivity between multiplex PCR and uniplex PCR for each of Citrus mosaic sadwavirus (CiMV), Citrus tristeza virus (CTV), and Citrus tatter leaf virus (CTLV). (A) Multiplex PCR. (B–D) Uniplex PCR for each of CiMV, CTLV, and CTV, respectively. Lane M: 100-bp DNA ladder; lanes 1–5: 10, 50, 100, 500, and 1,000 times dilution of cDNA from CSM-1 isolate.
Fig. 6
Fig. 6
Detection of 4 citrus viruses from ‘Kanpei’ citrus trees in orchards by multiplex PCR assay. Lane M: 100-bp DNA ladder; lane P: positive control; lanes 1–15: isolates from ‘Kanpai’ citrus trees.

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