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In 2005, symptoms of watermelon vine decline (WVD) were observed on a 200-acre watermelon farm in Santa Isabel, on the south central coast of Puerto Rico. WVD symptoms included leaf curling, mosaics, and internode necrosis. In early growth stages of WVD, reduced vigor and plant stunting occurred. At flowering, symptoms progressed to necrosis and wilting of vines. A 2006 to 2007 survey demonstrated that fungal pathogens were not associated with the presence of WVD symptoms (3,4). By 2006, other watermelon fields were also affected. Field trials in 2007 and 2008 with insect-proof cages and insecticides suggested a role of whiteflies (Bemisia tabaci) in the transmission of a virus (3,4). Here, we report that watermelon and pumpkin plants were successfully infected in Puerto Rico by mechanical inoculation and through B. tabaci transmission assays, similarly to transmissions previously conducted in Florida with Squash vein yellowing virus (SqVYV) (1). In addition, plants of Cucurbita moschata exhibited vein clearing symptoms typical of SqVYV after mechanical inoculation with extracts from watermelon plants with WVD symptoms. In 2011, eight watermelon samples from plants exhibiting WVD syndrome were collected in Guánica, Santa Isabel, Juana Díaz, and Mayagüez, and two Momordica charantia samples were collected from Mayagüez. RNA was extracted from all 10 original samples, as well as from plants that were used in mechanical and vector transmission assays, using RNeasy Plant Mini Kit (Qiagen, Valencia, California), and all samples were found positive for SqVYV by reverse transcription-PCR, using previously described primers and methods (1,2). In all cases, a single ~1-kb PCR fragment was revealed, and PCR fragments from four samples were selected for direct sequencing. All sequences showed high levels (>99%) of nucleotide identity with SqVYV sequences from Florida (JF897989, JF897985, and JF897984). Sequences of the SqVYV CP gene from Puerto Rico were deposited in GenBank under accession numbers KC713961 to KC713964. To our knowledge, this is the first report of SqVYV in Puerto Rico associated with WVD syndrome in cucurbits, and thus has implications for management of viral diseases of watermelon in the Caribbean. This is also the first detection of SqVYV outside of the continental United States in both watermelon and a wild species, M. charantia (bitter gourd). References: (1) S. Adkins et al. Phytopathology 97:145, 2007. (2) S. Adkins et al. Plant Dis. 92:1119, 2008. (3) C. Estévez de Jensen et al. Phytopathology 98:S52, 2008. (4) L. Polanco-Florián. El marchitamiento súbito de la sandía [Citrullus lanatus (Thumb.) Matsum & Nakai]. M.S. thesis, University of Puerto Rico, Mayagüez, PR, 2009.

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The red palm mite (Raoiella indica), an invasive pest of coconut, entered the Western hemisphere in 2004, then rapidly spread through the Caribbean and into Florida, USA. Developing effective sampling methods may aid in the timely detection of the pest in a new area. Studies were conducted to provide and compare intra tree spatial distribution of red palm mite populations on coconut in two different geographical areas, Trinidad and Puerto Rico, recently invaded by the mite. The middle stratum of a palm hosted significantly more mites than fronds from the upper or lower canopy and fronds from the lower stratum, on average, had significantly fewer mites than the two other strata. The mite populations did not vary within a frond. Mite densities on the top section of the pinna had significantly lower mite densities than the two other sections, which were not significantly different from each other. In order to improve future sampling plans for the red palm mite, the data was used to estimate the variance components associated with the various levels of the hierarchical sampling design. Additionally, presence-absence data were used to investigate the probability of no mites being present in a pinna section randomly chosen from a frond inhabited by mites at a certain density. Our results show that the most precise density estimate at the plantation level is to sample one pinna section per tree from as many trees as possible.

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Citrus leprosis virus (CiLV) constitutes one of the most important viruses in citrus in the areas where it occurs. Two morphological types of virus particles have been described from associated leprosis symptoms, nuclear (CiLV-N) and cytoplasmic (CiLV-C) (4). The CiLV-C is more common, representing more than 99% of samples collected from South and Central America (E. W. Kitajima and J. C. V. Rodrigues, unpublished). Both virus types are associated with the mite vector, Brevipalpus phoenicis (Geijskes) (Acari: Tenuipalpidae). So far, CiLV-C has only been naturally transmitted by these mites to citrus (3). Plants of Solanum violaefolium Schott (Solanaceae) and ornamental and sweet orange seedlings were infested with viruliferous adult female mites, colony no. 61 (GenBank Accession No. AY320027) that were previously maintained on citrus seedlings infected with CiLV-C according to Rodrigues et al. (3). Fifteen days after the mites were transferred, spotted yellowish symptoms were observed on leaves of plants of S. violaefolium and similar symptoms were observed after 25 days on citrus leaves. The symptomatic tissues were studied using transmission electron microscopy. Particles typical of CiLV-C were observed in samples from both plant species. To our knowledge, this is the first reported case of mites transmitting CiLV-C to a noncitrus host. Subsequent experiments showed that mites were able to transmit the virus between plants of S. violaefolium. Attempts to transmit the virus by mites from S. violaefolium to citrus were unsuccessful. The dsRNA viral electrophoresis profile showed differences between the two host plants. Reverse transcription-polymerase chain reaction (RT-PCR) and sequencing assays with primers designed to detect CiLV-C (2) amplified DNA fragments of the expected size and base composition. These data suggest the loss or alteration of some viral components from the Solanum sp. host that might be essential for the transmission or infection in citrus. Such a mechanism may explain why, despite the ever-increasing number of Brevipalpus-transmitted viruses in a large number of different host plant species (1), cross transmission is not common. This information shows one of the potential routes for CiLV to invade citrus orchards, and suggests one alternative host plant that allows rapid multiplication of the virus for characterization. References: (1) E. W. Kitajima et al. Exp. Appl. Acarol. 30:135, 2003. (2) E. C. Locali et al. Plant Dis. 87:1317, 2003. (3) J. C. V. Rodrigues et al. Proc. Int. Org. Citrus Virol. 174, 2000 (4) J. C. V. Rodrigues et al. Exp. Appl. Acarol. 30:161, 2003.

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The citrus crop is rapidly expanding in the Province of Santa Cruz de la Sierra, Bolívia. Citrus, mostly planted by small growers, currently comprises approximately 15,000 ha. Sweet oranges (Citrus sinensis) and mandarins (C. reticulate) are the main citrus-types grown primarily for internal consumption. Recently, there has been an increase in incidence of leprosis-like symptoms (round to elliptic lesions on the leaves, chlorotic to necrotic lesions in young twigs, and depressed small lesions on the fruits). These symptoms were associated with infestations by the tenuipalpid mite Brevipalpus sp. To verify if Citrus leprosis virus was the causal agent of the observed symptoms, leaf and fruit samples (mostly from Valencia sweet orange) were collected from commercial groves in El Torno, 32 km south of Santa Cruz, and Yapacani and Colónia San Juan, 130 km northwest of Santa Cruz. Small fragments of these samples were placed immediately in a mixture of glutaraldehyde and paraformaldehyde in cacodylate buffer and later processed with transmission electron microscopy at ESALQ, Piracicaba, SP, Brazil. Some of the leaf samples were dried at 35°C and used for reverse transcription-polymerase chain reaction (RT-PCR) with primers that specifically amplify portions of the genome of Citrus leprosis virus, cytoplasmic type (CiLV-C) (1) at Centro APTA Citros, Cordeirópolis, SP, Brazil. Brevipalpus sp. mites were also collected and kept in 90% ethanol for further identification at the University of Florida, Gainesville and ESALQ. In the samples from the three surveyed areas, transmission electron microscopy confirmed the presence of short bacilliform particles within endoplasmic reticulum cisternae and electron dense viroplasms in the cytoplasm, typical of infection by CiLV-C (2). CiLV-C specific primers amplified DNA fragments of expected sizes in RT-PCR from dried leaf samples that came from these three localities. Direct sequencing of at least three amplicons of each sample confirmed the identity of the virus. The consensus sequence of the putative movement protein gene in samples from Yapacani and Colónia San Juan (GenBank Accessions Nos. AY960216 and AY960215, respectively) were identical and exhibited 99% nucleotide and 98% amino acid homology with the Brazilian isolate sequence available at GenBank (Accession No. AY289190). The consensus sequence of the putative replicase gene found in the sample from El Torno (GenBank Accession No. AY960214) exhibited 96 and 93% nucleotide and amino acid homology, respectively with the Brazilian isolate (GenBank Accession No. AY289191). Sampled mites were identified as B. phoenicis (Geijskes), the known vector of CiLV-C (2). The symptomatology, particle morphology and cytopathology, detection by molecular methods and the association with infestation by B. phoenicis, together indicate that the foliar, stem, and fruit lesions in sweet orange observed in the Santa Cruz region were caused by CiLV-C. To our knowledge, this is the first report of this virus in Bolivia. References: (1) E. C. Locali et al. Plant Dis. 87:1317, 2003. (2) J. C. V. Rodrigues et al. Exp. Appl. Acarol. 30:161, 2003.

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Brevipalpus phoenicis (Geijskes) (Acari: Tenuipalpidae) is recognized as the vector of citrus leprosis virus that is a significant problem in several South American countries. Citrus leprosis has been reported from Florida in the past but no longer occurs on citrus in North America. The disease was recently reported in Central America, suggesting that B. phoenicis constitutes a potential threat to the citrus industries of North America and the Caribbean. Besides B. phoenicis, B. obovatus Donnadieu, and B. californicus (Banks) have been incriminated as vectors of citrus leprosis virus and each species has hundreds of host plants. In this study, Brevipalpus mite specimens were collected from different plants, especially citrus, in the States of Florida (USA) and São Paulo (Brazil), and reared on citrus fruit under standard laboratory conditions. Mites were taken from these colonies for DNA extraction and for morphological species identification. One hundred and two Random Amplified Polymorphic DNA (RAPD) markers were scored along with amplification and sequencing of a mitochondrial cytochrome oxidase subunit I gene fragment (374 bp). Variability among the colonies was detected with consistent congruence between both molecular data sets. The mites from the Florida and Brazilian colonies were morphologically identified as belonging to B. phoenicis, and comprise a monophyletic group. These colonies could be further diagnosed and subdivided geographically by mitochondrial DNA analysis.

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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.

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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.

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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.

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Coffee ringspot is characterized by conspicuous ringspot symptoms on leaves, berries, and less frequently on twigs. It is caused by coffee ringspot virus (CoRSV), a short, bacilliform virus (40 nm x 100-110 nm). The virus is not seed borne and is transmitted by Brevipalpus phoenicis (Geijskes). Transovarial transmission within the mite does not occur. CoRSV has been mechanically transmitted to Chenopodium amaranticolor Coste and Reynaud, C. quinoa Wildenow, Beta vulgaris L., and Alternanthera tenella Colla resulting in local lesions. Systemic infection within both C. amaranticolor and C. quinoa occurs. Virions are found in the nucleus or cytoplasm of infected cells, commonly associated with membranes. Occasionally, membrane bounded particles are found within the cisternae of the endoplasmic reticulum. A characteristic electron lucent, nuclear inclusion is commonly found in many infected cells. These cytopathic effects place CoRSV among the nuclear type of Brevipalpus-borne viruses. The disease has been reported in several Brazilian states (São Paulo, Paraná, Minas Gerais, and Federal District) and recently found in Costa Rica. A similar disease is known in the Philippines, but no information exists about its relationship to CoRSV. Coffee ringspot had no economical significance until recently when a large scale infection was reported in Minas Gerais that resulted in yield loss.

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Passion fruit green spot disease was first identified in 1997 after a severe outbreak at Vera Cruz County, state of São Paulo, Brazil. Mature yellow fruits of Passiflora edulis Simms f. flavicarpa Degener showed characteristic green spots, 2-5 mm in diameter and patches of green tissues were present on senescent leaves. The devastating effect to passion flower is caused by necrotic lesions that encircle the stems and kill the plant. In severe cases, entire orchards of a few hectares in size have been completely destroyed. The disease was always preceded by heavy infestations of Brevipalpus phoenicis (Geijskes) (Acari: Tenuipalpidae). Transmission electron microscopy of affected tissues (fruits, leaves, and stems) consistently revealed the presence of short, bacilliform particles (50-70 nm x 100-120 nm) in the cisternae of the endoplasmic reticulum, as well as the presence of a dense viroplasm in the cytoplasm. This cytopathic effect has been found in several other Brevipalpus-transmitted or associated viruses and is classified as a cytoplasmic type of disease. Experimental reproduction of the leaf and stem symptoms was achieved by transferring B. phoenicis collected from affected field passion flower plants onto healthy plants. The evidence supports a viral etiology for the disease and the agent was named passion fruit green spot virus. Its relationship with other B. phoenicis related viruses continues to be studied. The disease was also found in the Brazilian states of Bahia, Sergipe, Rondonia, Minas Gerais, Rio de Janeiro, and in the Federal District. Use of one or more of the following acaricides (hexythiazox, fenbutatin-oxide, propargite, quinomethionate, or dicofol) has significantly reduced the incidence of the disease.

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Coffee ringspot virus (CoRSV) (family Rhabdoviridae) is transmitted by Brevipalpus phoenicis (Geijskes) (Acari: Tenuipalpidae). Coffee ringspot disease was first reported in coffee plants from Brazil in 1939 (1). In August 2000, severe symptoms of concentric ringspots and "oak leaf" patterns on coffee leaves (Coffea arabica L. cv. Catuai) were observed during field inspections conducted in two areas of San Gabriel de Desamparados, Costa Rica. The disease caused premature fruit and leaf drop in the affected plants. Some areas within the ringspot lesions remained green on senescent leaves. Because CoRSV particles remain restricted to lesion areas (1), this virus has not been purified, and antiserum for virus detection is not available. Therefore, leaves with symptoms were collected and examined by transmission electron microscopy. In ultrathin sections of symptomatic leaves, arrays of rhabdovirus-like particles were associated with the nucleus as described for CoRSV (2). Healthy tissues did not contain similar arrays of bacilliform and bullet-shaped particles. Twenty mites collected from the infected plants at the same locations and time were slide-mounted and identified as B. phoenicis. High populations of this mite were also observed infesting plants of Cajanus cajan L. that were intercropped with coffee at the same location. Sweet orange trees growing in the same fields as shade for the coffee did not show symptoms of citrus leprosis, a disease caused by another Brevipalpus-transmitted virus that was recently reported in Panama (3). To our knowledge, this is the first report of a virus similar to CoRSV in Costa Rica. The spread of this virus, presumably CoRSV, could seriously affect the coffee industry throughout Central America by increasing production costs. It may be necessary to apply one or more foliar acaricides to effectively control the mite vector. References: (1) A. Bitancourt. O. Biol. 5:33, 1939. (2) C. M. Chagas et al. Phytopathol. Z. 102:100, 1981. (3) F. S. Dominguez et al. Plant Dis. 85:228, 2001.