Survey of the citrus leprosis vector (Brevipalpus yothersi) and phytoseiids in spontaneous plants of an organic citrus orchard.

Citrus leprosis (CL) is one of the most important viral diseases in sweet orange orchards in Latin America. It is caused by members of at least five species of the so-called Brevipalpus-transmitted viruses (BTV), and the prevalent is Citrus leprosis virus C (CiLV-C). This virus has the broadest host range amongst all CL-associated viruses and is transmitted by Brevipalpus yothersi, a polyphagous mite that can colonize a large variety of host plants, including some spontaneous ground cover plants. But if, on one hand, spontaneous plants can host CL virus and vector, on the other hand, they can offer alternative food for predators, equally common in organic citrus orchards. Brevipalpus yothersi and predator mites were surveyed in 33 spontaneous plants of a Westin sweet orange orchard conducted under organic production system in Brazil, from June 2010 to April 2011. Predatory mites were identified as phytoseiids, and Iphiseiodes zuluagai was the prevalent species, representing 58% of all predators. Other phytoseiids were considered accidental species in the area. Ageratum conyzoides and Alternanthera tenella were the most represented plant host species to predators, comprising 28 and 10% of the total surveyed plants, respectively. Brevipalpus yothersi specimens were detected on various spontaneous species: A. conyzoides, A. tenella, Amaranthus deflexus, Bidens pilosa, Ipomoea quamoclit, I. cairica, Merremia cissoides, Solanum americanum, Panicum maximum, and, predominantly, Commelina benghalensis. The latter has been previously reported as host of CiLV-C as well and, therefore, it is recommended to eliminate this species from citrus orchards.

Rescue of a Cilevirus from infectious cDNA clones.

Reverse genetics systems represent an important tool for studying the molecular and functional processes of viral infection. Citrus leprosis virus C (CiLV-C) (genus Cilevirus, family Kitaviridae) is the main pathogen responsible for the citrus leprosis (CL) disease in Latin America, one of the most economically important diseases of the citrus industry. Molecular studies of this pathosystem are limited due to the lack of infectious clones. Here, we report the construction and validation of a CiLV-C infectious cDNA clone based on an agroinfection system. The two viral RNA segments (RNA1 and RNA2) were assembled into two binary vectors (pJL89 and pLXAS). Agroinfiltrated Nicotiana benthamiana plants showed a response similar to that observed in the natural infection process with the formation of localized lesions restricted to the inoculated leaves. The virus recovered from the plant tissue infected with the infectious clones can be mechanically transmitted between N. benthamiana plants. Detection of CiLV-C subgenomic RNAs (sgRNAs) from agroinfiltrated and mechanically inoculated leaves further confirmed the infectivity of the clones. Finally, partial particle-purification preparations or sections of CiLV-C-infected tissue followed by transmission electron microscopy (TEM) analysis showed the formation of CiLV-C virions rescued by the infectious clone. The CiLV-C reverse genetic system now provides a powerful molecular tool to unravel the peculiarities of the CL pathosystem.

Circulative Transmission of Cileviruses in Brevipalpus Mites May Involve the Paracellular Movement of Virions.

Plant viruses transmitted by mites of the genus Brevipalpus are members of the genera Cilevirus, family Kitaviridae, or Dichorhavirus, family Rhabdoviridae. They produce non-systemic infections that typically display necrotic and/or chlorotic lesions around the inoculation loci. The cilevirus citrus leprosis virus C (CiLV-C) causes citrus leprosis, rated as one of the most destructive diseases affecting this crop in the Americas. CiLV-C is vectored in a persistent manner by the flat mite Brevipalpus yothersi. Upon the ingestion of viral particles with the content of the infected plant cell, virions must pass through the midgut epithelium and the anterior podocephalic gland of the mites. Following the duct from this gland, virions reach the salivary canal before their inoculation into a new plant cell through the stylet canal. It is still unclear whether CiLV-C multiplies in mite cells and what mechanisms contribute to its movement through mite tissues. In this study, based on direct observation of histological sections from viruliferous mites using the transmission electron microscope, we posit the hypothesis of the paracellular movement of CiLV-C in mites which may involve the manipulation of septate junctions. We detail the presence of viral particles aligned in the intercellular spaces between cells and the gastrovascular system of Brevipalpus mites. Accordingly, we propose putative genes that could control either active or passive paracellular circulation of viral particles inside the mites.

Impact of diseases and pests on premature fruit drop in sweet orange orchards in São Paulo state citrus belt, Brazil.

BACKGROUND: Despite technical improvements in the citrus chain and leadership in orange production achieved in the past decades, premature fruit drop remains a major component of crop loss in São Paulo state citrus belt, the largest sweet orange production area in the world. The present study aimed to determine, during five consecutive seasons, the impact of the diseases and pests on premature fruit drop in the orange belt. RESULTS: Fruit drop due to the main diseases and pests averaged approximately 11.0%, which corresponded to approximately 63% of the annual fruit drop. The average fruit drop rate due to fruit borer and fruit flies combined was 4.0%, Huanglongbing (HLB) 3.3%, black spot 2.6%, leprosis 1.0% and citrus canker 0.3%. The average amount of fruit drop (million 40.8 kg boxes) and value of crop losses (million US$ dollars), in five seasons, were 12.7 and 66.2 for fruit borer/fruit flies, 11.0 and 57.9 for HLB, 8.1 and 42.2 for black spot, 3.1 and 15.6 for leprosis, and 0.9 and 4.9 for citrus canker, respectively. CONCLUSION: Fruit borer and fruit flies (combined), HLB, black spot, leprosis and citrus canker are, in this order, the main diseases and pests in the orange belt of São Paulo state. All of these causes significantly increased the overall fruit drop rate in the evaluated seasons. The results will contribute to the development of the Brazilian citrus industry, while showing to other citrus-growing regions the potential that diseases and pests have to jeopardize production. © 2022 Society of Chemical Industry.

Citrus Leprosis Virus C Encodes Three Proteins With Gene Silencing Suppression Activity.

Citrus leprosis virus C (CiLV-C) belongs to the genus Cilevirus, family Kitaviridae, and is considered the most devastating virus infecting citrus in Brazil, being the main viral pathogen responsible for citrus leprosis (CL), a severe disease that affects citrus orchards in Latin America. Here, proteins encoded by CiLV-C genomic RNA 1 and 2 were screened for potential RNA silencing suppressor (RSS) activity by five methods. Using the GFP-based reporter agroinfiltration assay, we have not found potential local suppressor activity for the five CiLV-C encoded proteins. However, when RSS activity was evaluated using the alfalfa mosaic virus (AMV) system, we found that the p29, p15, and p61 CiLV-C proteins triggered necrosis response and increased the AMV RNA 3 accumulation, suggesting a suppressive functionality. From the analysis of small interfering RNAs (siRNAs) accumulation, we observed that the ectopic expression of the p29, p15, and p61 reduced significantly the accumulation of GFP derived siRNAs. The use of the RSS defective turnip crinkle virus (TCV) system revealed that only the trans-expression of the p15 protein restored the cell-to-cell viral movement. Finally, the potato virus X (PVX) system revealed that the expression of p29, p15, and p61 increased the PVX RNA accumulation; in addition, the p29 and p15 enhanced the pathogenicity of PVX resulting in the death of tobacco plants. Furthermore, PVX-p61 infection resulted in a hypersensitive response (HR), suggesting that p61 could also activate a plant defense response mechanism. This is the first report describing the RSS activity for CiLV-C proteins and, moreover, for a member of the family Kitaviridae.

Pest categorisation of Citrus leprosis viruses.

The EFSA Panel on Plant Health performed a pest categorisation of the Citrus leprosis viruses for the EU territory and identified five distinct viruses, Citrus leprosis virus C (CiLV-C), Citrus leprosis virus C2 (CiLV-C2), Hibiscus green spot virus 2 (HGSV-2), the Citrus strain of Orchid fleck virus (OFV) and Citrus leprosis virus N sensu novo (CiLV-N) as causing this severe disease, most significantly in sweet orange and mandarin. These viruses have in common that they do not cause systemic infections in their hosts and that they all are transmitted by Brevipalpus spp. mites (likely but not confirmed for HGSV-2). Mites represent the most important means of virus spread, while plants for planting of Citrus are only considered of minor significance. These well characterised viruses occur in South and Central America. Leprosis is currently regulated in directive 2000/29 EC and, together with its associated viruses, has never been recorded in the EU. All five viruses have the potential to enter into, establish in and spread within the EU territory, with plants for planting of non-regulated hosts, fruits of Citrus and hitch-hiking of viruliferous mites identified as the most significant pathways. Given the severity of the leprosis disease, the introduction and spread of the various viruses would have negative consequences on the EU citrus industry, the magnitude of which is difficult to evaluate given the uncertainties affecting the Brevipalpus spp. vectors (identity, distribution, density, transmission specificity and efficiency). Overall, leprosis and its five associated viruses meet all the criteria evaluated by EFSA to qualify as Union quarantine pests, but do not fulfil those of being present in the EU or of plants for planting being the main spread mechanism to qualify as Union regulated non-quarantine pests. The main uncertainties affecting this categorisation concern the Brevipalpus spp. mite vectors.

Production of monoclonal antibodies for detection of Citrus leprosis virus C in enzyme-linked immuno-assays and immunocapture reverse transcription-polymerase chain reaction.

Citrus leprosis virus C (CiLV-C) causes damage in citrus production in the South and Central America. Since closely related types of citrus viruses have recently been described monoclonal antibodies (MAbs) are needed for accurate and sensitive diagnosis of CiLV-C. In this study, MAbs to the expressed coat protein of CiLV-C were produced for serological detection of CiLV-C in crude extracts of infected tissues in double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA), dot blot immunosorbent assays (DBIA) and immuonocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) procedures. Monoclonal antibodies were developed in mice to the purified expressed coat protein of CiLV-C. The published standard protocols of DAS-ELISA, DBIA and IC-RT-PCR were followed for the detection of coat protein p29 of CiLV-C in the crude extracts of CiLV-C infected tissues. Two monoclonal antibodies, designated G10 and C11, were identified from four potential candidates for the specific and sensitive detection of coat protein p29 of CiLV-C in the crude citrus extracts of CiLV-C infected tissues in DAS-ELISA, whereas G10 was also selected based on performance for use in the DBIA and IC-RT-PCR diagnostic assays. Sensitivity analysis comparing the three methods for detection of coat protein p29 of CiLV-C determined that IC-RT-PCR was more sensitive than DAS-ELISA and DBIA. The creation of MAbs to CiLV-C allows for the sensitive and accurate detection of the virus from CiLV-C infected citrus leaf tissues. Successful detection of the virus in three diagnostic assays formats provides flexibility to diagnosticians who can use either ELISA or DBIA for screening large numbers of samples, and IC-RT-PCR for rapid, sensitive confirmation testing.