Multiplex detection of "Candidatus Liberibacter asiaticus" and Spiroplasma citri by qPCR and droplet digital PCR

PLoS One. 2021 Mar 17;16(3):e0242392. doi: 10.1371/journal.pone.0242392. eCollection 2021.

Abstract

"Candidatus Liberibacter asiaticus" (CLas) and Spiroplasma citri are phloem-limited bacteria that infect citrus and are transmitted by insect vectors. S. citri causes citrus stubborn disease (CSD) and is vectored by the beet leafhopper in California. CLas is associated with the devastating citrus disease, Huanglongbing (HLB), and is vectored by the Asian citrus psyllid. CLas is a regulatory pathogen spreading in citrus on residential properties in southern California and is an imminent threat to spread to commercial citrus plantings. CSD is endemic in California and has symptoms in citrus that can be easily confused with HLB. Therefore, the objective of this study was to develop a multiplex qPCR and duplex droplet digital PCR (ddPCR) assay for simultaneous detection of CLas and S. citri to be used where both pathogens can co-exist. The multiplex qPCR assay was designed to detect multicopy genes of CLas-RNR (5 copies) and S. citri-SPV1 ORF1 (13 copies), respectively, and citrus cytochrome oxidase (COX) as internal positive control. Absolute quantitation of these pathogens was achieved by duplex ddPCR as a supplement for marginal qPCR results. Duplex ddPCR allowed higher sensitivity than qPCR for detection of CLas and S. citri. ddPCR showed higher tolerance to inhibitors and yielded highly reproducible results. The multiplex qPCR assay has the benefit of testing both pathogens at reduced cost and can serve to augment the official regulatory protocol for CLas detection in California. Moreover, the ddPCR provided unambiguous absolute detection of CLas and S. citri at very low concentrations without any standards for pathogen titer.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Citrus / microbiology
  • DNA, Bacterial / analysis*
  • DNA, Bacterial / metabolism
  • Liberibacter / genetics*
  • Liberibacter / isolation & purification
  • Multiplex Polymerase Chain Reaction / methods*
  • Plant Diseases / microbiology
  • Real-Time Polymerase Chain Reaction / methods*
  • Reproducibility of Results
  • Spiroplasma citri / genetics*
  • Spiroplasma citri / isolation & purification

Substances

  • DNA, Bacterial

Grants and funding

RY 5300-191 Citrus Research Board RY Yok-18 California citrus Nursery Board RY 2034-22000-013-10D United States Department of Agriculture, Agricultural Research Service, Base Project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.