Citrus Bright Spot Virus: A New Dichorhavirus, Transmitted by Brevipalpus azores, Causing Citrus Leprosis Disease in Brazil.

Citrus leprosis (CL) is the main viral disease affecting the Brazilian citriculture. Sweet orange (Citrus sinensis L. Osbeck) trees affected by CL were identified in small orchards in Southern Brazil. Rod-like particles of 40 × 100 nm and electron lucent viroplasm were observed in the nucleus of infected cells in symptomatic tissues. RNA extracts from three plants, which proved negative by RT-PCR for known CL-causing viruses, were analyzed by high throughput sequencing and Sanger sequencing after RT-PCR. The genomes of bi-segmented ss(-)RNA viruses, with ORFs in a typical organization of members of the genus Dichorhavirus, were recovered. These genomes shared 98-99% nt sequence identity among them but <73% with those of known dichorhavirids, a value below the threshold for new species demarcation within that genus. Phylogenetically, the three haplotypes of the new virus called citrus bright spot virus (CiBSV) are clustered with citrus leprosis virus N, which is a dichorhavirus transmitted by Brevipalpus phoenicis sensu stricto. In CiBSV-infected citrus plants, B. papayensis and B. azores were found, but the virus could only be transmitted to Arabidopsis plants by B. azores. The study provides the first evidence of the role of B. azores as a viral vector and supports the assignment of CiBSV to the tentative new species Dichorhavirus australis.

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.

Citrus leprosis resistance within the citrus group.

Reported in Brazil since the 1930's, citrus leprosis, caused mainly by citrus leprosis virus C, has been a major concern for the national sweet orange production. In recent years, the disease has spread to several other countries and it is now considered a worldwide threat. The occurrence of the disease has been studied almost exclusively in sweet oranges because other citrus genotypes are of secondary relevance in Brazil and in some other American countries where it occurs. Here we report 12 resistant citrus genotypes among 160 accessions evaluated. After 90 days of the infestation with viruliferous mites, asymptomatic genotypes were observed in sour orange, lemon, grapefruit, mandarins, tangelo, and tangor groups. The results revealed promising genotypes resistant to the disease, which can be incorporated in citrus breeding programs aiming to obtain varietal resistance, and confirmed the susceptibility of many citrus genotypes to CiLV-C. This assay ratify the already reported uneven level of susceptibility within the citrus group.

Effects of radiation (Cobalt-60) on the elimination of Brevipalpus phoenicis (Acari: Tenuipalpidae) Cardinium endosymbiont.

Brevipalpus phoenicis (Geijskes) (Acari: Tenuipalpidae) is a polyphagous mite with worldwide distribution and it is also a vector of several plant viruses. In citrus, B. phoenicis transmits Citrus leprosis virus (CiLV), the causal agent of leprosis, a disease that costs millions of dollars per year for its prevention and control. Brevipalpus phoenicis mites reproduce through thelytokous parthenogenesis, producing haploid females. This characteristic is attributable to the presence of an endosymbiont bacterium of the genus Cardinium; however, very little is known about the biological and ecological implications of the presence of this endosymbiont in Brevipalpus mites. In order to investigate the role of Cardinium in the transmission of CiLV to citrus plants, our goal was to eliminate the bacterium from the mite. We assessed the effectiveness of different doses of radiation from a Cobalt-60 source to cure B. phoenicis populations from Cardinium sp. The efficiency of irradiation on the elimination of the endosymbiont was determined by counting the number of females and males obtained in the F(1) generation after irradiation and confirming the presence of the endosymbiont by PCR. Both radiation treatments influenced the oviposition period and the number of eggs laid by irradiated females. Also, irradiation eliminated the Cardinium endosymbiont and increased the number of males in progeny of the exposed populations. Although macroscopic morphological abnormalities were not observed among the treated mites, the mortality was higher compared to the non-irradiated control group.

Differentially expressed stress-related genes in the compatible citrus-Citrus leprosis virus interaction

Leprosis, caused by Citrus leprosis virus, cytoplasmic type (CiLV-C), is the main viral disease in the Brazilian citrus industry. This occurs because of the widespread source of inoculum and the year-round presence of the vector, the tenuipalpid mite Brevipalpus phoenicis, in citrus plants. In addition, while some Citrus species are resistant to CiLV-C, C. sinensis, the main cultivated species in the country, is extremely susceptible to the disease. The main objective of this work was to identify genes in C. sinensis cv. Pêra plants that were differentially expressed after the host was challenged with CiLV-C. In order to accomplish that, cDNA libraries were constructed from healthy and CiLV-inoculated sweet orange leaves. Two hundred and fifty-four genes were found to differ significantly in terms of expression, with 193 of them induced and 61 repressed after inoculation. Here we discuss the possible roles of a sub-set of these genes involved in metabolism, energy, signaling and cell rescue, defense and virulence, and indicate which kind of response may take place in the initial steps of the disease. Although the symptoms induced by CiLV-C in its compatible interaction with sweet orange resemble those of hypersensitive response (HR) in incompatible interactions, our data indicate that, apparently, the manifestation of leprosis symptoms should not be considered HR.

Inheritance and heritability of resistance to citrus leprosis.

ABSTRACT The genetic inheritance of resistance to leprosis, the most important viral disease of citrus in Brazil, was characterized through the phenotypic assessment of 143 hybrids resulting from crosses between tangor 'Murcott' (Citrus sinensis x C. reticulata) and sweet orange 'Pêra' (C. sinensis), considered to be resistant and susceptible to the disease, respectively. All plants were grafted onto Rangpur lime (C. limonia) and inoculated with Citrus leprosis virus, cytoplasmic type through the infestation with viruliferous mites, Brevipalpus phoenicis. The experiments were arranged in a completely randomized block design with 10 replicates. Incidence and severity of the disease in leaves and stems as well as plant growth parameters (plant height and stem diameter) were recorded for 3 years after the infestation with the viruliferous mites. The average values of all variables were analyzed using principal component analysis, discriminant factorial analysis, estimation of the clonal repeatability coefficients, and frequency of the distributions of the average values for each measured variable. The principal component analysis resulted in the identification of at least two groups with resistance and susceptibility to leprosis, respectively. About 99% of all hybrids were correctly classified according to the discriminant factorial analysis. The broad-sense heritability coefficients for characteristics associated with incidence and severity of leprosis ranged from 0.88 to 0.96. The data suggest that the inheritance of resistance to leprosis may be controlled by only a few genes.