Quantification of Live ‘Candidatus Liberibacter asiaticus’ Populations Using Real-Time PCR and Propidium Monoazide

Abstract

Huanglongbing (HLB) is a devastating citrus disease. It is associated with a phloem-restricted bacterium, ‘Candidatus Liberibacter asiaticus’, and primarily transmitted by Asian citrus psyllid in Florida. Because Liberibacter cannot be cultured, early diagnosis of HLB relies on DNA-based polymerase chain reaction (PCR), including real-time quantitative (q)PCR. Although estimating genomes from live bacteria (GLB) is critical for HLB research, PCR does not distinguish between live and dead cells and, thus, does not estimate GLB in hosts. Propidium monoazide (PMA), a novel DNA-binding dye, has been successfully used on many bacterial pathogens to effectively remove DNA from dead cells but there is no report of its use on uncultured bacteria. In this study, PMA-qPCR protocols were first optimized to work with plant and psyllid samples, respectively. Both TissueLyser treatment and plant tissue were demonstrated to have an insignificant impact on the GLB detected by PMA-qPCR. Finally, a standard curve for GLB determination was successfully established between PMA-qPCR results and microscopic counts and then applied in two studies with different greenhouse plant samples. This rapid qPCR method provides a more accurate way to determine GLB in HLB hosts which, in turn, should benefit disease epidemiology studies and serve as a crucial component in HLB management.

Live Population Dynamics of ‘Candidatus Liberibacter asiaticus’, the Bacterial Agent Associated with Citrus Huanglongbing, in Citrus and Non-Citrus Hosts

“…In order to monitor the dynamic change of the live 'Ca. L. asiaticus' population inside host plants, some of the successfully inoculated plants were tested monthly by PMA-qPCR (14). These plants included three Valencia sweet orange (C. sinensis Valencia) and three S. buxifolia plants inoculated by either grafting or psyllid.…”

“…The monitoring lasted 20 months, from August 2010 to April 2012. PMA pretreatrnent on plant samples was conducted following the protocol published by Hu et al (14). Briefly, about 120 mg of finely chopped midrib or petiole tissue was pulverized with the TissueLyser II (Qiagen) with liquid nitrogen to make a homogenized fissue pool, and two portions of the 50-mg fissue powder from this pool were weighed out and received differential treatments.…”

“…Current HLB diagnosis and research mainly uses DNA-based methods such as real-time quantitative PCR (qPCR). qPCR alone cannot differentiate between live and dead cells (14). The bacterial population detected by qPCR is the total genome present (TGP) in the sample; that is, the sum of live bacteria, dead bacteria (with integrity-compromised cell structure), and naked DNA.…”

“…The bacterial population detected by qPCR is the total genome present (TGP) in the sample; that is, the sum of live bacteria, dead bacteria (with integrity-compromised cell structure), and naked DNA. The detection of tbe TGP may fulfill diagnostic puipose but the information obtained from TGP is limited (14), especially if the genomes of live bacteria (GLB) is the focus. There are several studies on bacterial populations in HLB-affected citrus hosts.…”

“…L. asiaticus' population in citrus hosts was reported to be from September to December (15), whereas Wang et al (34) reported that September was one of tbe months with lowest bacterial population. With a novel cell membrane-impermeant DNA-binding dye, propidium monoazide (PMA), the DNA from dead bacteria can be removed in a pretreatment before the total DNA extraction, which leaves only the DNA from live bacteria for detection in tbe subsequent qPCR (14,24). PMA-qPCR has been successfully applied for many bacterial pathogens to detect the live bacterial populations (18,21,25,35) but there are no systemic applications, except for the initial published method with the HLB-associated bacteria (14).…”