The Detection and Surveillance of Asian Citrus Psyllid (Diaphorina citri)—Associated Viruses in Florida Citrus Groves

Abstract

The plant pathogenic bacterium Candidatus Liberibacter asiaticus (CLas), the causal agent of the citrus disease Huanglongbing (HLB), and its insect vector, the Asian citrus psyllid (ACP; Diaphorina citri), have been devastating the Florida citrus industry. To restore the competitive production presence of Florida in the worldwide citrus market, effective and sustainable control of HLB and the ACP needs to be identified. As alternatives for resistance-inducing insecticides, viruses are currently being considered for biological control of the ACP. To identify possible biological control candidates, we conducted one of the most comprehensive surveys of natural ACP populations in major citrus production regions spanning 21 counties in Florida. By optimizing PCRs and RT-PCRs, we were able to successfully detect and monitor the prevalence of five previously identified ACPassociated RNA and DNA viruses throughout Florida citrus groves, which include: Diaphorina citri-associated C virus (DcACV), Diaphorina citri flavi-like virus (DcFLV), Diaphorina citri densovirus (DcDNV), Diaphorina citri reovirus (DcRV), and Diaphorina citri picorna-like virus (DcPLV). Adult and nymph ACP populations from 21 of Florida's major citrus-producing counties were collected each month during approximately 18 consecutive months. RNA extracts used for these viral screens were also regionally combined and subjected to High Throughput Sequencing (HTS) to reveal a more comprehensive picture of known and unknown viruses in Florida ACP populations. We discovered that DcACV was the most prevalent ACP-associated virus throughout nymph and adult ACP populations in Florida, detected in more than 60% of all samples tested, followed by DcPLV and DcFLV. HTS allowed us to identify a novel ACP-associated reolike virus and a picorna-like virus. The putative reo-like virus, tentatively named Diaphorina citri cimodo-like virus, was later surveyed and detected back in seasonal adult and nymph ACP samples collected in Florida during this study. HTS generated data also revealed that the most abundant virus in Florida ACP populations was Citrus tristeza virus (CTV), which is not an ACP-associated virus, suggesting persistent presence of CTV infection in citrus throughout Florida groves. Collectively, information obtained from our study may be able

Partial Genome Sequence of a Novel Reo-Like Virus Detected in Asian Citrus Psyllid (Diaphorina citri) Populations from Florida Citrus Groves

“…Insect-specific viruses (ISVs) can offer potential biological alternatives as lethal and efficient viral vectors toward their respective invertebrate hosts ( 3 ). To identify ISVs of Florida ACP populations, high-throughput sequencing (HTS) was conducted on ACPs collected from Florida citrus groves ( 4 ). Subsequent bioinformatic analyses revealed the presence of sequences belonging to a novel reo-like virus with consistent closest protein similarity to the Cimodo virus (CMDV), a reovirus isolated from African mosquitoes ( 5 ).…”

“…HTS preparation for identification of this novel reo-like virus was previously described by Britt et al ( 4 ). Briefly, total RNAs were extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions, from a pool of adult and nymph ACPs ( n = ∼30) that had been collected from Florida citrus groves in 2020.…”

Crowdsourced Identification of Potential Target Genes for CTV Induced Gene Silencing for Controlling the Citrus Greening Vector Diaphorina citri

“…Although many dsRNA molecules caused mortalities at a high dose, we selected only dsRNA molecules that exhibited dose response RNA interference. Because CTV is the most abundant virus in Florida citrus and is found where D. citri populations affect citrus (Britt et al, 2020) we engineered these molecules into attenuated CTV and expressed them in citrus leaves to protect against D. citri. We also report that targeting ETC membrane proteins by dsRNA in artificial diet is not as effective as targeting the soluble ETC subunit Rieske.…”

Preliminary Report on the Acquisition, Persistence, and Potential Transmission of Citrus tristeza virus by Diaphorina citri

“…Recently, a study revealed that CTV was the most abundant virus in Florida D. citri populations by high-throughput sequencing [58]. The results in this study indicate a high prevalence (40% to 100%) of CTV among D. citri collected from orchards.…”