Multiplex detection of candidatus liberibacter asiaticus and Spiroplasma citri by qPCR and droplet digital PCR

Authors: MAHESHWARI, Y - Foreign Agricultural Service (FAS, USDA); SEVARAJ, V - Foreign Agricultural Service (FAS, USDA); GODFREY, K - University Of California, Davis; HAJERI, S - Central California Tristeza Eradication Agency; Yokomi, Raymond - Ray

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/3/2021
Publication Date: 3/17/2021
Citation: Maheshwari, Y., Sevaraj, V., Godfrey, K., Hajeri, S., Yokomi, R.K. 2021. Multiplex detection of “Candidatus Liberibacter asiaticus” and Spiroplasma citri by qPCR and droplet digital PCR. PLoS ONE. 16(3):e0242392. https://doi.org/10.1371/journal.pone.0242392.
DOI: https://doi.org/10.1371/journal.pone.0242392

Interpretive Summary: Huanglongbing (HLB) and citrus stubborn disease (CSD) are caused by phloem-limited bacteria transmitted by psyllids and leafhoppers, respectively. HLB is a devastating citrus disease and is becoming established in residential properties in citrus in southern California. HLB-infected trees are eradicated to reduce threat of spread by psyllids to commercial citrus plantings. CSD is endemic in commercial citrus orchards and has symptoms that can be confused with HLB. To facilitate eradicative efforts for HLB, a multiplex real-time PCR (qPCR) assay and a duplex droplet digital PCR (ddPCR) assay were developed for simultaneous detection of HLB- and CSD-affected citrus trees. Both assays used conserved multicopy genes for pathogen detection to maximize sensitivity. The multiplex qPCR included an internal control for citrus DNA and was as sensitive as singleplex tests. The ddPCR was more sensitive than qPCR for detection of HLB and had higher resilience to PCR inhibitors and yielded highly reproducible results. The qPCR and ddPCR assays developed have the benefit of testing both pathogens simultaneously to reduce testing costs without sacrificing sensitivity and can augment the regulatory-approved protocol for HLB detection in California.

Technical Abstract: “Candidatus Liberibacter asiaticus” (CLas) and Spiroplasma citri are phloem-limited bacteria that infect citrus and are transmitted by insect vectors. In California, S. citri causes citrus stubborn disease (CSD) and is vectored by the beet leafhopper. CLas is associated with the devastating citrus disease known as 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 confused with HLB. Consequently, a multiplex qPCR and duplex droplet digital PCR (ddPCR) were developed for simultaneous detection of CLas and S. citri. The multiplex qPCR assay was aimed to detect multicopy genes of CLas - RNR (5 copies), 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 had greater resilience to inhibitors and yielded highly reproducible results. The multiplex qPCR assay has the benefit of testing both pathogens at reduced costs and can serve to augment official regulatory protocols 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.