Occurrence of Citrus leprosis virus in Llanos Orientales, Colombia.

In Colombia, citrus is cultivated in mostly small plantings that total 55,000 ha by approximately 25,000 farmers. Production includes 1,200 tons of fresh fruits and 60 tons of juice for domestic consumption, resulting in a net worth of US$650,000 per year. Most of the production comes from areas located between the Cordillera Occidental and Cordillera Central mountain ranges (departments of Antioquia, Caldas, Quindio, and Risaralda) near coffee plantations. The departments of Meta and Casanare, located at the east plains (Llanos Orientales), include a zone parallel (4 to 5°N, 72 to 74°W) to the east mountain range and generate approximately 10% of the total Colombian citrus production. Suspected citrus leprosis symptoms on leaves and fruits of sweet oranges (Citrus sinensis (L.) Osb.) were first observed by plant pathologists for CORPOICA (Colombian National Agricultural Research Organization) in citrus orchards in Casanare in 2003, and later in 2004, in Meta. To confirm the visual identification, leaves and fruits from Valencia sweet orange exhibiting typical lesions of leprosis were collected from several locations in the departments of Casanare (Yopal, Aguazul) and Meta (Guamal, Villavicencio, and Cumaral). Samples were fixed in cacodylate-buffered paraformaldehyde/glutaraldehyde solution and subsequently processed for examination in thin sections using electron microscopy. Samples were processed and examined at the Citrus Research and Educational Center (CREC) of the University of Florida, Lake Alfred, and the Agricultural College (ESALQ) of the Universidade de São Paulo at Piracicaba, SP, Brazil. Some leaf samples collected in Meta were also dried and used for detection of Citrus leprosis virus, cytoplasmic type (CiLV-C) by reverse transcription-polymerase chain reaction (RT-PCR) at the Centro APTA Citros Sylvio Moreira at Cordeirópolis (CAPTACSM). The RT-PCR was performed with primers that specifically amplify a fragment of the viral genome that codes for the putative cell-to-cell movement protein (1). Locations at CREC and ESALQ each observed, using electron microscopy, cell changes characteristic of CiLV-C that include short bacilliform particles in the endoplasmic reticulum and dense, vacuolated, and irregularly shaped viroplasm in the cytoplasm (2) in samples from Casanare and Meta. RT-PCR amplified cDNA fragments of the expected size for samples collected in Meta and one of the amplicons was sequenced (GenBank Accession No. DQ272491). The sequence obtained was found to have 98% nucleotide sequence identity to the Brazilian CiLV-C isolate (GenBank Accession No. AY289190.1). Mites collected from affected plants from the department of Meta were identified at ESALQ as Brevipalpus phoenicis (Geijskes), a known principal vector of CiLV-C (2). These several lines of evidence confirmed that the symptoms observed in sweet oranges at Meta and Casanare are due to the infection by CiLV-C. To our knowledge, this is the first report of this virus in Colombia. 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.

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.

Iridovirus in the root weevil Diaprepes abbreviatus.

Invertebrate iridescent virus 6 (IIV6) was evaluated for mode of transmission and ability to cause infection in the root weevil, Diaprepes abbreviatus (L.). This is the first evidence of IIV6 infection in D. abbreviatus, which caused both patent and sub-lethal covert infections in both larvae and adults. Adults and larvae were successfully infected with IIV6 by puncture, injection and per os. Transmission of IIV6 was demonstrated between infected and healthy individuals regardless of gender. Virus was detected in egg masses produced by virus-infected females suggesting IIV6 is transmitted transovarially. Virus particles were observed in the cytoplasm of weevil cells, and were shown to infect fat bodies, muscle, and nerve tissues, as visualized using transmission electron microscopy. Patent infections resulted in death of individuals within 3 to 4 days post infection. Individuals with covert infections tested positive for virus infection on day 7 by polymerase chain reaction analysis. Sequencing of PCR amplicons confirmed virus infection. Discovery of new pathogens against root weevils may provide new management tools for development of control strategies based on induced epizootics. This is the first report of a virus infecting D. abbreviatus.

Cucumber mosaic virus Diagnosed in Desert Rose in Florida.

Desert rose (Adenium obesum (Forssk) Roem. & Schult.) is a member of the family Apocynaceae and characterized by fleshy leaves and stems and colorful flowers. This popular, exotic ornamental, originally from southeastern Africa, is propagated vegetatively and is a perennial in warm climates. Virus-like foliar symptoms, including a mosaic with dark green islands surrounding the veins and chlorosis on the leaf margins, were observed on desert rose samples from two southwest Florida nurseries in November 2002. Cucumber mosaic virus (CMV) was identified in symptomatic plants by serological testing for the presence of CMV coat protein with a commercially available ImmunoStrip test (Agdia, Elkhart, IN). A third sample expressing similar symptoms was observed in southeastern Florida in February 2003. The presence of CMV was confirmed by serological detection with a commercially available double-antibody sandwich enzyme-linked immunosorbent assay (Agdia). An agent was mechanically transmitted from the third sample to Chenopodium quinoa, resulting in the formation of chlorotic local lesions. Examination of inoculated C. quinoa leaves by double-stranded (ds) RNA analysis and electron microscopy (leaf dips) revealed the presence of a typical cucumovirus dsRNA profile and spherical virions ~28 nm in diameter, respectively, providing additional confirmation of a CMV infection. A possible satellite RNA of ~350 nucleotides was also observed by dsRNA analysis. To our knowledge, this represents the first report of CMV infection of desert rose.

Discovering new insect viruses: whitefly iridovirus (Homoptera: Aleyrodidae: Bemisia tabaci).

Adult whiteflies, Bemisia tabaci (Gennadius), collected from the field were screened for viral pathogens using a cell line from the silverleaf whitefly, B. tabaci, B biotype (syn. B. argentifolii). Homogenates from the field-collected whiteflies were applied to cell cultures and checked for cytopathic effects (CPE). Cells were observed to develop cytoplasmic inclusions and to have a change in morphology. Cells displaying CPE were observed using a transmission electron microscope and found to be infected with a virus. The virus particles had an icosahedral shape and an approximate size of 120-130 nm. The virus was observed in defined areas of the cytoplasm adjacent to the cell nucleus. Analysis using polymerase chain reaction, Southern blot hybridization, and DNA sequencing confirmed that the virus discovered infecting the whitefly cell cultures was an iridovirus. Sequence analysis showed that the amplimer (893 bp) had a 95% homology to the invertebrate iridescent virus type 6 major capsid protein gene. Discovery of new viruses of whiteflies may provide renewed interest in using pathogens in the development of innovative management strategies. This is the first report of an iridescent virus isolated from whiteflies, B. tabaci, collected from the field.

Anatomical characteristics of roots of citrus rootstocks that vary in specific root length.

Among citrus rootstocks, higher specific root length (SRL, root length/d. wt) has been linked to several specific morphological and physiological traits, including smaller average root diameter, higher root hydraulic conductivity and higher rates of root proliferation. In this study, thickness of the outer tangential exodermal (hypodermal) wall and its suberin layer, number of passage cells, presence of epidermis, and stelar anatomy were examined and related to variation in root diameter of field roots of known maximum age. We also compared root morphology and anatomy of young roots in the field with those of potted rootstock seedlings in the glasshouse. Fibrous roots were measured separately from pioneer (framework) roots. Among the fibrous roots, only the first-order (root links having a root tip) and second-order (root links bearing first-order roots) laterals were examined. Among first-order field roots, larger root diameter was caused by larger rather than more numerous cells in the cortex. Root diameter of first-order roots was positively correlated with both number of passage cells in the exodermis and thickness of the secondary walls of the exodermis in both field and potted plants. Exodermal walls were about 80% thicker in field- than pot-grown roots. In the field, in more than 50% of the first-order roots examined less than 30% of the root surface was still covered by epidermis, with few differences among rootstocks. In contrast, in roots of 19-wk-old glasshouse plants generally 70-100% of the epidermis was still intact. There was no evidence of secondary xylem development in second-order fibrous roots in the field; in seedling, pot-grown rootsystems, 75-97% of second-order roots had formed secondary xylem despite their small diameter (<0.8 mm). It is argued that there can be suites of physiological, morphological and anatomical traits in roots that co-vary with specific root length. Investigations of how root morphology and anatomy are linked to root function, moreover, need to recognize trait variability and the potentially important differences between field- and pot- grown (seedling) roots.