Unravelling the involvement of cilevirus p32 protein in the viral transport.

Citrus leprosis (CL) is a severe disease that affects citrus orchards mainly in Latin America. It is caused by Brevipalpus-transmitted viruses from genera Cilevirus and Dichorhavirus. Currently, no reports have explored the movement machinery for the cilevirus. Here, we have performed a detailed functional study of the p32 movement protein (MP) of two cileviruses. Citrus leprosis-associated viruses are not able to move systemically in neither their natural nor experimental host plants. However, here we show that cilevirus MPs are able to allow the cell-to-cell and long-distance transport of movement-defective alfalfa mosaic virus (AMV). Several features related with the viral transport were explored, including: (i) the ability of cilevirus MPs to facilitate virus movement on a nucleocapsid assembly independent-manner; (ii) the generation of tubular structures from transient expression in protoplast; (iii) the capability of the N- and C- terminus of MP to interact with the cognate capsid protein (p29) and; (iv) the role of the C-terminus of p32 in the cell-to-cell and long-distance transport, tubule formation and the MP-plasmodesmata co-localization. The MP was able to direct the p29 to the plasmodesmata, whereby the C-terminus of MP is independently responsible to recruit the p29 to the cell periphery. Furthermore, we report that MP possess the capacity to enter the nucleolus and to bind to a major nucleolar protein, the fibrillarin. Based on our findings, we provide a model for the role of the p32 in the intra- and intercellular viral spread.

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.

Bioactivity of an oxymatrine-based commercial formulation against Brevipalpus yothersi Baker and its effects on predatory mites in citrus groves.

The acaricidal bioactivity of an oxymatrine-based commercial formulation against Brevipalpus yothersi Baker (Acari: Tenuipalpidae), a vector mite of the Citrus leprosis virus (CiLV), and its impact on predatory mites were assessed. For this purpose, laboratory and field assays using bioacaricide concentrations ranging from 0.5 to 2.0 mg L-1 of oxymatrine were performed during the years from 2015 to 2016. Laboratory results showed that the oxymatrine-based commercial formulation does not cause deleterious effects on B. yothersi eggs; however, it causes high larval mortality. For adult females, the bioacaricide caused high acute toxicity and residual effect for at least 5 days after application. In the field, the bioacaricide exhibited high acaricidal activity against B. yothersi, with efficacy levels similar to that of synthetic acaricide spirodiclofen (48 mg L-1) until 49 days after the application. The application of the bioacaricide did not negatively affect the population levels of phytoseiid predatory mites. Therefore, our results suggest that the oxymatrine-based commercial formulation is an important tool for management of the citrus leprosis mite in citrus groves.

Reference genes for gene expression studies by RT-qPCR in Brevipalpus yothersi (Acari: Tenuipalpidae), the mite vector of citrus leprosis virus.

Quantitative reverse transcription PCR (RT-qPCR) is a high-throughput method to analyze the transcriptional expression of genes. Currently, no reference genes have been described for evaluating gene expression in Brevipalpus yothersi, the false spider mite, a polyphagous that act as vector of the citrus leprosis virus C (CiLV-C), an important citrus disease. This study aimed to identify the most stable reference genes in B. yothersi. The RT-qPCR expression data for selected genes were evaluated from three conditions: different developmental stages, plant hosts and acquisition of CiLV-C. To analyze the stability of the candidate reference genes we used ΔCq method, GeNorm, NormFinder, BestKeeper and RefFinder. Ubiq and GAPDH are best suited for normalizing gene expression data in viruliferous and non-viruliferous mites. Ubiq, EF1α and GAPDH are the most stable for different developmental stages. RPL13 and RPL32 are the best reference genes for approaches to B. yothersi in different host plants. Considering all the experimental conditions, Ubiq, EF1α, and GAPDH were the most stable genes. Here we developed an accurate and comprehensive RT-qPCR strategy for use in B. yothersi gene expression analysis. These results will improve the understanding of the biology of the false spider mites and their role as virus vectors.

Dichorhaviruses in their Host Plants and Mite Vectors.

A group of related bacilliform, nuclear viruses with a bisegmented negative-sense RNA genome that are transmitted by Brevipalpus mites likely in a circulative-propagative manner were recently classified in the new genus Dichorhavirus, family Rhabdoviridae. These viruses cause localized lesions on leaves, stems, and fruits of economically significant horticultural and ornamental plant species. Among its members, orchid fleck virus, citrus leprosis virus N, and coffee ringspot virus are most prominent. This chapter summarizes the current knowledge about these viruses, available detection techniques, and their interactions with their plant hosts and mite vectors.

Space-time variability of citrus leprosis as strategic planning for crop management.

BACKGROUND: Citrus leprosis is the most important viral disease of citrus. Knowledge of its spatiotemporal structure is fundamental to a representative sampling plan focused on the disease control approach. Such a well-crafted sampling design helps to reduce pesticide use in agriculture to control pests and diseases. RESULTS: Despite the use of acaricides to control citrus leprosis vector (Brevipalpus spp.) populations, the disease has spread rapidly through experimental areas. Citrus leprosis has an aggregate spatial distribution, with high dependence among symptomatic plants. Temporal variation in disease incidence increased among symptomatic plants by 4% per month. CONCLUSIONS: Use of acaricides alone to control the vector of leprosis is insufficient to avoid its incidence in healthy plants. Preliminary investigation into the time and space variation in the incidence of the disease is fundamental to select a sampling plan and determine effective strategies for disease management. © 2018 Society of Chemical Industry.

Identification and Characterization of Citrus Chlorotic Spot Virus, a New Dichorhavirus Associated with Citrus Leprosis-Like Symptoms.

Local chlorotic spots resembling early lesions characteristic of citrus leprosis (CL) were observed in leaves of two sweet orange (Citrus sinensis L.) trees in Teresina, State of Piauí, Brazil, in early 2017. However, despite the similarities, these spots were generally larger than those of a typical CL and showed rare or no necrosis symptoms. In symptomatic tissues, transmission electron microscopy revealed the presence of viroplasms in the nuclei of the infected parenchymal cells and rod-shaped particles with an average size of approximately 40 × 100 nm, resembling those typically observed during infection by dichorhaviruses. A bipartite genome of the putative novel virus, tentatively named citrus chlorotic spot virus (CiCSV) (RNA1 = 6,518 nucleotides [nt] and RNA2 = 5,987 nt), revealed the highest nucleotide sequence identity values with the dichorhaviruses coffee ringspot virus strain Lavras (73.8%), citrus leprosis virus N strain Ibi1 (58.6%), and orchid fleck virus strain So (56.9%). In addition to citrus, CiCSV was also found in local chlorotic lesions on leaves of the ornamental plant beach hibiscus (Talipariti tiliaceum (L.) Fryxell). Morphological characterization of mites recovered from the infected plants revealed at least two different types of Brevipalpus. One of them corresponds to Brevipalpus yothersi. The other is slightly different from B. yothersi mites but comprises traits that possibly place it as another species. A mix of the two mite types collected on beach hibiscus successfully transmitted CiCSV to arabidopsis plants but additional work is required to verify whether both types of flat mite may act as viral vectors. The current study reveals a newly described dichorhavirus associated with a citrus disease in the northeastern region of Brazil.

Citrus leprosis virus N: A New Dichorhavirus Causing Citrus Leprosis Disease.

Citrus leprosis (CL) is a viral disease endemic to the Western Hemisphere that produces local necrotic and chlorotic lesions on leaves, branches, and fruit and causes serious yield reduction in citrus orchards. Samples of sweet orange (Citrus × sinensis) trees showing CL symptoms were collected during a survey in noncommercial citrus areas in the southeast region of Brazil in 2013 to 2016. Transmission electron microscopy analyses of foliar lesions confirmed the presence of rod-like viral particles commonly associated with CL in the nucleus and cytoplasm of infected cells. However, every attempt to identify these particles by reverse-transcription polymerase chain reaction tests failed, even though all described primers for the detection of known CL-causing cileviruses and dichorhaviruses were used. Next-generation sequencing of total RNA extracts from three symptomatic samples revealed the genome of distinct, although highly related (>92% nucleotide sequence identity), viruses whose genetic organization is similar to that of dichorhaviruses. The genome sequence of these viruses showed <62% nucleotide sequence identity with those of orchid fleck virus and coffee ringspot virus. Globally, the deduced amino acid sequences of the open reading frames they encode share 32.7 to 63.8% identity with the proteins of the dichorhavirids. Mites collected from both the naturally infected citrus trees and those used for the transmission of one of the characterized isolates to Arabidopsis plants were anatomically recognized as Brevipalpus phoenicis sensu stricto. Molecular and biological features indicate that the identified viruses belong to a new species of CL-associated dichorhavirus, which we propose to call Citrus leprosis N dichorhavirus. Our results, while emphasizing the increasing diversity of viruses causing CL disease, lead to a reevaluation of the nomenclature of those viruses assigned to the genus Dichorhavirus. In this regard, a comprehensive discussion is presented.

Phylogenetic and Molecular Variability Studies Reveal a New Genetic Clade of Citrus leprosis virus C.

Citrus leprosis virus C (CiLV-C) causes a severe disease affecting citrus orchards in the Western hemisphere. This study reveals the molecular variability of the virus by analyzing four genomic regions (p29, p15, MP and RNA2-intergenic region) distributed over its two RNAs. Nucleotide diversity (π) values were relatively low but statistically different over the analyzed genes and subpopulations, indicating their distinct evolutionary history. Values of πp29 and πMP were higher than those of πp15 and πRNA2-IR, whereas πMP was increased due to novel discovered isolates phylogenetically clustered in a divergent clade that we called SJP. Isolate BR_SP_SJP_01 RNA1 and RNA2 sequences, clade SJP, showed an identity of 85.6% and 88.4%, respectively, with those corresponding to CiLV-C, the type member of the genus Cilevirus, and its RNA2 5'-proximal region was revealed as a minor donor in a putative inter-clade recombination event. In addition to citrus, BR_SP_SJP_01 naturally infects the weed Commelina benghalensis and is efficiently transmitted by Brevipalpus yothersi mites. Our data demonstrated that negative selection was the major force operating in the evaluated viral coding regions and defined amino acids putatively relevant for the biological function of cilevirus proteins. This work provides molecular tools and sets up a framework for further epidemiological studies.

Diversity and Genetic Variation among Brevipalpus Populations from Brazil and Mexico.

Brevipalpus phoenicis s.l. is an economically important vector of the Citrus leprosis virus-C (CiLV-C), one of the most severe diseases attacking citrus orchards worldwide. Effective control strategies for this mite should be designed based on basic information including its population structure, and particularly the factors that influence its dynamics. We sampled sweet orange orchards extensively in eight locations in Brazil and 12 in Mexico. Population genetic structure and genetic variation between both countries, among locations and among sampling sites within locations were evaluated by analysing nucleotide sequence data from fragments of the mitochondrial cytochrome oxidase subunit I (COI). In both countries, B. yothersi was the most common species and was found in almost all locations. Individuals from B. papayensis were found in two locations in Brazil. Brevipalpus yothersi populations collected in Brazil were more genetically diverse (14 haplotypes) than Mexican populations (four haplotypes). Although geographical origin had a low but significant effect (ca. 25%) on the population structure, the greatest effect was from the within location comparison (37.02 %). Potential factors driving our results were discussed.

History and Diversity of Citrus leprosis virus Recorded in Herbarium Specimens.

Leprosis refers to two diseases of citrus that present similar necrotic local lesions, often surrounded by chlorotic haloes on citrus. Two distinct viruses are associated with this disease, one that produces particles primarily in the nucleus of infected plant cells (Citrus leprosis virus nuclear type [CiLV-N]; Dichorhavirus) and another type that produces particles in the cytoplasm of infected plant cells (Citrus leprosis virus cytoplasmic type [CiLV-C]; Cilevirus). Both forms are transmitted by Brevipalpid mites and have bipartite, single-stranded, RNA genomes. CiLV-C and CiLV-N are present in South and Central America and as far north as parts of Mexico. Although leprosis disease was originally described from Florida, it disappeared from there in the 1960s. The United States Department of Agriculture-Agricultural Research Service maintains preserved citrus specimens identified at inspection stations 50 or more years ago with symptoms of citrus leprosis. We isolated RNA from these samples and performed degradome sequencing. We obtained nearly full-length genome sequences of both a typical CiLV-C isolate intercepted from Argentina in 1967 and a distinct CiLV-N isolate obtained in Florida in 1948. The latter is a novel form of CiLV-N, not known to exist anywhere in the world today. We have also documented the previously unreported presence of CiLV-N in Mexico in the mid-20th century.

Role Bending: Complex Relationships Between Viruses, Hosts, and Vectors Related to Citrus Leprosis, an Emerging Disease.

Citrus leprosis complex is an emerging disease in the Americas, associated with two unrelated taxa of viruses distributed in South, Central, and North America. The cytoplasmic viruses are Citrus leprosis virus C (CiLV-C), Citrus leprosis virus C2 (CiLV-C2), and Hibiscus green spot virus 2, and the nuclear viruses are Citrus leprosis virus N (CiLV-N) and Citrus necrotic spot virus. These viruses cause local lesion infections in all known hosts, with no natural systemic host identified to date. All leprosis viruses were believed to be transmitted by one species of mite, Brevipalpus phoenicis. However, mites collected from CiLV-C and CiLV-N infected citrus groves in Mexico were identified as B. yothersi and B. californicus sensu lato, respectively, and only B. yothersi was detected from CiLV-C2 and CiLV-N mixed infections in the Orinoco regions of Colombia. Phylogenetic analysis of the helicase, RNA-dependent RNA polymerase 2 domains and p24 gene amino acid sequences of cytoplasmic leprosis viruses showed a close relationship with recently deposited mosquito-borne negevirus sequences. Here, we present evidence that both cytoplasmic and nuclear viruses seem to replicate in viruliferous Brevipalpus species. The possible replication in the mite vector and the close relationship with mosquito borne negeviruses are consistent with the concept that members of the genus Cilevirus and Higrevirus originated in mites and citrus may play the role of mite virus vector.

A novel virus of the genus Cilevirus causing symptoms similar to citrus leprosis.

Citrus leprosis in Colombia was previously shown to be caused by cytoplasmic Citrus leprosis virus (CiLV-C). In 2011, enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction (RT-PCR)-based diagnostic methods failed to identify CiLV-C from citrus samples with symptoms similar to citrus leprosis; however, virions similar to CiLV-C were observed in the cytoplasm of the symptomatic leaves by transmission electron microscopy. Furthermore, the causal organism was transmitted by the false spider mite, Brevipalpus phoenicis, to healthy citrus seedlings. A library of small RNAs was constructed from symptomatic leaves and used as the template for Illumina high-throughput parallel sequencing. The complete genome sequence and structure of a new bipartite RNA virus was determined. RNA1 (8,717 nucleotides [nt]) contained two open reading frames (ORFs). ORF1 encoded the replication module, consisting of five domains: namely, methyltransferase (MTR), cysteine protease-like, FtsJ-MTR, helicase (Hel), and RNA-dependent RNA polymerase (RdRp); whereas ORF2 encoded the putative coat protein. RNA2 (4,989 nt) contained five ORFs that encode the movement protein (MP) and four hypothetical proteins (p7, p15, p24, and p61). The structure of this virus genome resembled that of CiLV-C except that it contained a long 3' untranslated terminal region and an extra ORF (p7) in RNA2. Both the RNA1 and RNA2 of the new virus had only 58 and 50% nucleotide identities, respectively, with known CiLV-C sequences and, thus, it appears to be a novel virus infecting citrus. Phylogenetic analyses of the MTR, Hel, RdRp, and MP domains also indicated that the new virus was closely related to CiLV-C. We suggest that the virus be called Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) and it should be unambiguously classified as a definitive member of the genus Cilevirus. A pair of CiLV-C2 genome-specific RT-PCR primers was designed and validated to detect its presence in citrus leprosis samples collected from the Casanare and Meta states in Colombia.

Brevipalpus mites (Acari: Tenuipalpidae): vectors of invasive, non-systemic cytoplasmic and nuclear viruses in plants.

Multi-directional interactions occur among plant hosts, Brevipalpus mites and the plant viruses they transmit. Such interactions should be considered when evaluating the severity of a disease such as citrus leprosis. The current understanding of Brevipalpus-transmitted viruses relies on the capability of the vector to transmit the disease, the persistence of the virus in the host plant and the ability of the disease to spread. Previously, we discussed the Citrus leprosis virus (CiLV) and its importance and spread over the past decade into new areas of South and Central America, most recently into southern Mexico and Belize. Here, we address key questions to better understand the biology of the mite vector, fitness costs, and the peculiarities of Brevipalpus mite reproduction, virus survival, transmissibility and spread, and the expansion of the host plant range of Brevipalpus species vectoring the disease.

Detection of Brevipalpus-transmitted viruses in their mite vectors by RT-PCR.

The diagnosis of plant diseases caused by Brevipalpus-transmitted viruses (BrTVs) has been done through the analyses of symptoms, transmission electron microscopy, and RT-PCR of infected plant tissues. Here, we report the detection of Citrus leprosis virus C, Orchid fleck virus, Clerodendrum chlorotic spot virus and Solanum violaefolium ringspot virus in their viruliferous vectors Brevipalpus spp. using specific primer pairs for each of the viruses. The efficiency of virus transmission by Brevipalpus mites is low, so the detection of these pathogens in their vectors could constitute an important tool for studies involving virus-vector relationships, transmission, and monitoring the pathogen prior to the appearance of symptoms in the field.

Brevipalpus mites Donnadieu (Prostigmata: Tenuipalpidae) associated with ornamental plants in Distrito Federal, Brazil.

Brevipalpus mites colonize a great number of fruit and ornamental plants. Mite species belonging to this genus have been associated with many plant viruses. Citrus leprosis (CiLV) is the most economically important virus transmitted by B. phoenicis mites. It has recently been shown that ornamental plant species can be alternative hosts of this virus. The high volume of trade and frequent movement of live ornamental plants make them efficient pest disseminators. Because of this, it is desirable to expand knowledge of potential pests aiming to guide the adoption of quarantine measures. This work reports ornamental plant hosts of Brevipalpus mites in the Distrito Federal (DF), as well the occurrence of symptoms consistent with Brevipalpus-borne plant viruses in these same hosts. Between July and September of 2005, five surveys were carried out in 14 localities within DF. Leaves and branches of fifty-five ornamental plant species were sampled. The species Pithecellobium avaremotemo Mart. is for the first time reported as a host for B. phoenicis (Geijskes), B. californicus Banks and B. obovatus Donnadieu species. Additionally, seven new species are reported as hosts for Brevipalpus within South America. New hosts are also listed for individual mite species. Typical symptoms of Brevipalpus-borne viruses were observed in Ligustrum sinense Lour., Pelargonium hortorum L.H. Bailey, Hibiscus rosa-sinensis L. and orchids (Dendrobium and Oncidium). The results of this work emphasize the potential role of the ornamental plants as vehicles for dissemination of Brevipalpus mites.

Brevipalpus-transmitted plant virus and virus-like diseases: cytopathology and some recent cases.

An increasing number of diseases transmitted by Brevipalpus mite species (Acari: Tenuipalpidae) is being identified that affect economically important plants such as citrus, coffee, passion fruit, orchids, and several ornamentals. All of these diseases are characterized by localized lesions (chlorotic, green spots, or ringspots) on leaves, stems, and fruits. Virus or virus-like agents are considered to be the causal agents, possibly transmitted in a circulative-propagative manner by Brevipalpus mites. The virus or virus-like particles are short, rod-like, or bacilliform, that induce two characteristic types of cell alteration: (1) 'Nuclear type'--nuclei of parenchyma and epidermal cells in the lesions often contain a large electron lucent inclusion. Short, naked, rod-like (40-50 nm x 100-110 nm) particles may be seen in the viroplasm or nucleoplasm and in the cytoplasm. These particles are commonly arranged perpendicularly on the membranes of the nuclear envelope or endoplasmic reticulum (ER). In a very few instances, they were found to be membrane-bound, within the ER cavities. (2) 'Cytoplasmic type'--short bacilliform particles (60-70 nm x 110-120 nm) are present within the cisternae of the ER and often have electron dense viroplasm of varied shapes present in the cytoplasm. Bacilliform particles may be seen budding into the ER lumen near the viroplasm. These particles resemble those of members of the Rhabdoviridae, but are shorter. The only sequenced virus of this group, orchid fleck virus (OFV), has a negative sense (bipartite) type ssRNA genome, but its organization is similar to known rhabdoviruses, which are monopartite. Both types of cytopathological effects have been found associated with citrus leprosis. In orchids, OFV has a 'nuclear type' of cytopathology, but in some species the 'cytoplasmic type' has been found associated with ringspot symptoms. In Hibiscus and Clerodendron, green spot symptoms have been associated with the cytoplasmic type of cell alteration, while chlorotic spots, in the same species, are associated with the nuclear type. In a few cases, both types of cytopathological effects have been found in the same tissue and cell.

Citrus leprosis virus vectored by Brevipalpus phoenicis (Acari: Tenuipalpidae) on citrus in Brazil.

Citrus leprosis is caused by Citrus leprosis virus (CiLV) that is transmitted by mites in the genus Brevipalpus (Acari: Tenuipalpidae). This disease directly reduces production and the life span of the citrus plant. The main symptoms of the disease include lesions on fruits, leaves, and twigs or small branches, causing premature fruit drop, defoliation, and death of the twigs or branches leading to serious tree decline. Leprosis is a highly destructive disease of citrus, wherever it occurs. The Brazilian citrus industry spends over 100 million US dollars annually on acaricides to control the vector, Brevipalpus phoenicis (Geijskes). This review contains information about the history of the etiology of citrus leprosis, its geographical distribution, host range, the role of the mite vectors, viral morphology and relationships with the infected cell, and transmissibility of the virus by the mite. In addition, data on the mite-virus-plant relationship, disease damage, and strategies for controlling disease spread are presented.

Citrus leprosis and its status in Florida and Texas: past and present.

According to published reports from 1906 to 1968, leprosis nearly destroyed the Florida citrus industry prior to 1925. This was supported with photographs showing typical leprosis symptoms on citrus leaves, fruit, and twigs. Support for the past occurrence of citrus leprosis in Florida includes: (1) presence of twig lesions in affected orange blocks in addition to lesions on fruits and leaves and corresponding absence of similar lesions on grapefruit; (2) yield reduction and die-back on infected trees; and (3) spread of the disease between 1906 and 1925. Transmission electron microscopy (TEM) examination of tissue samples from leprosis-like injuries to orange and grapefruit leaves from Florida in 1997, and fruits from grapefruit and sweet orange varieties from Texas in 1999 and 2000 did not contain leprosis-like viral particles or viroplasm inclusions. In contrast, leprosis viroplasm inclusions were readily identified by TEM within green non-senescent tissues surrounding leprosis lesions in two of every three orange leaf samples and half of the fruit samples obtained from Piracicaba, Brazil. Symptoms of leprosis were not seen in any of the 24,555 orange trees examined across Florida during 2001 and 2002. The authors conclude that citrus leprosis no longer exists in Florida nor occurs in Texas citrus based on: (1) lack of leprosis symptoms on leaves, fruit, and twigs of sweet orange citrus varieties surveyed in Florida: (2) failure to find virus particles or viroplasm inclusion bodies in suspect samples from both Florida and Texas examined by TEM; (3) absence of documented reports by others on the presence of characteristic leprosis symptoms in Florida; (4) lack of its documented occurrence in dooryard trees or abandoned or minimal pesticide citrus orchard sites in Florida. In view of the serious threat to citrus in the U.S., every effort must be taken to quarantine the importation of both citrus and woody ornamental plants that serve as hosts for Brevipalpus phoenicis (Geijskes), B. californicus (Banks), and B. obovatus Donnadieu (Acari: Tenuipalpidae) from countries where citrus leprosis occurs.