Abstract
Citrus huanglongbing (HLB) disease associated with the ‘Candidatus Liberibacter asiaticus’ (CLas) bacterium has caused significant financial damage to many citrus industries. Large-scale pathogen surveys are routinely conducted in California to detect CLas early in the disease cycle by lab-based qPCR assays. We have developed an improved reference gene for the sensitive detection of CLas from plants in diagnostic duplex qPCR and analytical digital droplet PCR (ddPCR) assays. The mitochondrial cytochrome oxidase gene (COX), widely used as a reference, is not ideal because its high copy number can inhibit amplification of small quantities of target genes. In ddPCRs, oversaturation of droplets complicates data normalization and quantification. The variable copy numbers of COX gene in metabolically active young tissue, greenhouse plants, and citrus relatives suggest the need for a non-variable, nuclear, low copy, universal reference gene for analysis of HLB hosts. The single-copy nuclear gene, malate dehydrogenase (MDH), developed here as a reference gene, is amenable to data normalization, suitable for duplex qPCR and ddPCR assays. The sequence of MDH fragment selected is conserved in most HLB hosts in the taxonomic group Aurantioideae. This study emphasizes the need to develop standard guidelines for reference genes in DNA-based PCR assays.
Highlights
Citrus huanglongbing (HLB or citrus greening disease) is associated with an alphaproteobacterium, “Candidatus Liberibacter asiaticus” (CLas), and vectored by Diaphorina citri Kuwayama (Asian citrus psyllid; ACP) [1,2,3]
The goals of the current study were: (1) to identify an alternate reference gene that has a low copy number in citrus hosts; (2) to develop assays using a region of the reference gene highly conserved in members of subfamily Aurantioideae so that all known natural plant hosts of HLB can be evaluated in diagnostic assays; (3) comparative analysis of the newly developed reference gene with cytochrome oxidase gene (COX) gene in HLB diagnostic assays and; (4) to develop the reference gene suitable for data normalization in qPCR and digital droplet PCR (ddPCR) assays, while analyzing plant samples of Citrus and other closely related genera
A gene fragment that is conserved in many citrus accessions and related taxa may be suitable as a reference gene for duplex qPCR assays and normalizing data in digital PCRs
Summary
Citrus huanglongbing (HLB or citrus greening disease) is associated with an alphaproteobacterium, “Candidatus Liberibacter asiaticus” (CLas), and vectored by Diaphorina citri Kuwayama (Asian citrus psyllid; ACP) [1,2,3]. Intensive efforts have been in place to detect, eradicate, and contain the spread of HLB. HLB finds in CA are monitored by intensive follow-up surveys, aggressive vector control, eradicating infected trees, and other quarantine measures. While the disease has spread widely in certain locations, in other areas in CA, the HLB-positive trees and CLas-positive ACP were restricted to a very small number of plants and/or insects with no apparent secondary spread from the foci of infection. In CA, HLB surveys are conducted by testing ACP, since they indicate disease incidence early in an area [6]. Subsequent intensive testing of surrounding trees is conducted using mature leaves [7] and young flush where ACP feeds, acquires, oviposits, and transmits the HLB pathogen [8]. As a result of the long pathogen incubation period in citrus trees infected with CLas [9], identification of CLas-infected but asymptomatic trees with low levels of the HLB-associated pathogen is essential for the prevention of the further spread of the pathogen
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