Juliana Freitas-Astúa

Taxonomy of the order Mononegavirales: update 2016

In 2016, the order Mononegavirales was emended through the addition of two new families (Mymonaviridae and Sunviridae), the elevation of the paramyxoviral subfamily Pneumovirinae to family status (Pneumoviridae), the addition of five free-floating genera (Anphevirus, Arlivirus, Chengtivirus, Crustavirus, and Wastrivirus), and several other changes at the genus and species levels. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Expression Profiling of Virulence and Pathogenicity Genes of Xanthomonas axonopodis pv. citri

DNA macroarrays of 279 genes of Xanthomonas axonopodis pv. citri potentially associated with pathogenicity and virulence were used to compare the transcriptional alterations of this bacterium in response to two synthetic media. Data analysis indicated that 31 genes were up-regulated by synthetic medium XVM2, while only 7 genes were repressed. The results suggest that XVM2 could be used as an in vitro system to identify candidate genes involved in pathogenesis of X. axonopodis pv. citri.

Development of a Molecular Tool for the Diagnosis of Leprosis, a Major Threat to Citrus Production in the Americas

Citrus leprosis virus (CiLV), a tentative member of the Rhabdoviridae family, affects citrus trees in Brazil, where it is transmitted by mites Brevipalpus spp. It also occurs in other South American countries and was recently identified in Central America. This northbound spread of CiLV is being considered a serious threat to the citrus industry of the United States. However, despite its importance, difficulties related to the biology of CiLV have hindered much of the progress regarding its accurate detection, leaving both the analyses of symptoms and electron microscopy as the only tools available. An attempt to overcome this problem was made by constructing a cDNA library from double-stranded RNA extracted from leprosis lesions of infected Citrus sinensis (sweet orange) leaves. After cloning and sequencing, specific primers were designed to amplify putative CiLV genome regions with similarity to genes encoding the movement protein and replicase of other plant viruses. RNA from infected citrus plants corresponding to different varieties and locations were amplified by reverse transcription-polymerase chain reaction (RT-PCR) using the two pairs of primers. Amplified products were purified, cloned in pGEM-T, and sequenced. The sequences confirmed the genomic regions previously associated with CiLV. The results demonstrate that RT-PCR was specific, accurate, rapid, and reliable for the detection of CiLV.

Candidatus Liberibacter americanus induces significant reprogramming of the transcriptome of the susceptible citrus genotype

BackgroundCitrus huanglongbing (HLB) disease is caused by endogenous, phloem-restricted, Gram negative, uncultured bacteria named Candidatus Liberibacter africanus (CaLaf), Ca. L. asiaticus (CaLas), and Ca. L. americanus (CaLam), depending on the continent where the bacteria were first detected. The Asian citrus psyllid vector, Diaphorina citri, transmits CaLas and CaLam and both Liberibacter species are present in Brazil. Several studies of the transcriptional response of citrus plants manifesting HLB symptoms have been reported, but only for CaLas infection. This study evaluated the transcriptional reprogramming of a susceptible genotype of sweet orange challenged with CaLam, using a customized 385K microarray containing approximately 32,000 unigene transcripts. We analyzed global changes in gene expression of CaLam-infected leaves of sweet orange during the symptomatic stage of infection and compared the results with previously published microarray studies that used CaLas-infected plants. Twenty candidate genes were selected to validate the expression profiles in symptomatic and asymptomatic PCR-positive leaves infected with CaLas or CaLam.ResultsThe microarray analysis identified 633 differentially expressed genes during the symptomatic stage of CaLam infection. Among them, 418 (66%) were upregulated and 215 (34%) were down regulated. Five hundred and fourteen genes (81%) were orthologs of genes from Arabidopsis thaliana. Gene set enrichment analysis (GSEA) revealed that several of the transcripts encoded transporters associated with the endomembrane system, especially zinc transport. Among the most biologically relevant gene transcripts in GSEA were those related to signaling, metabolism and/or stimulus to hormones, genes responding to stress and pathogenesis, biosynthesis of secondary metabolites, oxidative stress and transcription factors belonging to different families. Real time PCR of 20 candidate genes validated the expression pattern of some genes in symptomatic and asymptomatic leaves infected with CaLam or CaLas.ConclusionsMany gene transcripts and biological processes are significantly altered upon CaLam infection. Some of them had been identified in response to CaLas infection, while others had not been previously reported. These data will be useful for selecting target genes for genetic engineering to control HLB.

Dichorhavirus: a proposed new genus for Brevipalpus mite-transmitted, nuclear, bacilliform, bipartite, negative-strand RNA plant viruses

Abstract Orchid fleck virus (OFV) is an unassigned negative-sense, single-stranded (−)ssRNA plant virus that was previously suggested to be included in the family Rhabdoviridae, order Mononegavirales. Although OFV shares some biological characteristics, including nuclear cytopathological effects, gene order, and sequence similarities, with nucleorhabdoviruses, its taxonomic status is unclear because unlike all mononegaviruses, OFV has a segmented genome and its particles are not enveloped. This article analyses the available biological, physico-chemical, and nucleotide sequence evidence that seems to indicate that OFV and several other Brevipalpus mite-transmitted short bacilliform (−)ssRNA viruses are likely related and may be classified taxonomically in novel species in a new free-floating genus Dichorhavirus.

An annotated list of ornamentals naturally found infected by Brevipalpus mite-transmitted viruses

The first cases of ornamental plants found infected by Brevipalpus transmitted viruses (BTV) were described in the 1990s from the region of Piracicaba, State of Sao Paulo, Brazil; subsequent cases were from other regions in the country and other American countries. Currently, 37 ornamental plant species (for the sake of simplicity, orchids being considered as a single species), belonging to 18 families of dicotyledons, have been reported hosting BTV. Because of the non systemic type of infection of these viruses, the localized diseases they cause are unimportant usually, but they have the potential to cause economic losses if severe outbreaks of Brevipalpus mite populations occur. Some ornamentals may serve as reservoirs to BTV known to cause serious damage to food crops as Citrus leprosis virus- cytoplasmic type (CiLV-C), passion fruit green spot virus (PFGSV) and Coffee ringspot virus (CoRSV).

The citrus leprosis pathosystem

Citrus leprosis is considered the main viral disease for the Brazilian citrus production, particularly for the State of Sao Paulo, due to the high costs spent for the chemical control of its vector, the tenuipalpid mite Brevipalpus phoenicis. In addition, its global importance has significantly increased in the last years, with the dissemination of the virus to new countries in South and Central America. In Brazil, despite its economical importance and occurrence for more than seven decades, the most significant advances towards understanding the pathosystem interactions have been obtained only in the last ten years. This review focuses on various aspects of the disease, beginning with a historical view, its main characteristics, alternatives for its control, its increasing economical importance in Brazil and abroad, and the new data on the search for understanding the interactions amongst the mite vector, the virus, and the plant host.

Evaluation of the effects of Candidatus Liberibacter asiaticus on inoculated citrus plants using laser-induced breakdown spectroscopy (LIBS) and chemometrics tools

This study investigated the organic and inorganic constituents of healthy leaves and Candidatus Liberibacter asiaticus (CLas)-inoculated leaves of citrus plants. The bacteria CLas are one of the causal agents of citrus greening (or Huanglongbing) and its effect on citrus leaves was investigated using laser-induced breakdown spectroscopy (LIBS) combined with chemometrics. The information obtained from the LIBS spectra profiles with chemometrics analysis was promising for the construction of predictive models to identify healthy and infected plants. The major, macro- and microconstituents were relevant for differentiation of the sample conditions. The models were then applied to different inoculation times (from 1 to 8 months). The models were effective in the classification of 82-97% of the diseased samples with a 95% significance level. The novelty of this method was in the fingerprinting of healthy and diseased plants based on their organic and inorganic contents.

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. Pera 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.

In situ observation of the Cardinium symbionts of Brevipalpus (Acari: Tenuipalpidae) by electron microscopy

Brevipalpus (Acari: Tenuipalpidae) mites are important pests on a variety of host plant species. The mites damage their hosts directly by feeding and some species also serve as vectors of plant viruses. Among more than 200 described Brevipalpus species, three are recognized as vectors of plant viruses: B. phoenicis, B. californicus and B. obovatus. These species occur worldwide in subtropical and tropical regions. Brevipalpus mites reproduce mostly by thelytokous parthenogenesis and this condition was attributed to a bacterial endosymbiont, recently characterized as a member of the genus Cardinium. The same symbiont infects many other arthropods and is capable of manipulating their host reproduction in various ways. Generally the presence of Cardinium is determined by molecular, PCR based, techniques. In the current work we present visual evidence for the presence of these bacteria by transmission electron microscopy as a complement of previous detection by PCR. Cardinium is easily identified by the presence of a unique array of microtubule-like structures (ML) in the cell. Symbionts have been observed in several organs and eggs from different populations of all three Brevipalpus species known as vector of plant viruses. Cardinium cells were always immersed directly within the cytoplasm of infected cells. Bacteria were observed in all females of all instars, but were absent from all males examined. Females from some Brevipalpus populations were observed to be uninfected by Cardinium. This observation confirmed previous PCR-based results that these populations were aposymbiotic. The observed distribution of the bacteria suggests that these bacteria could have other functions in the mite biology beside feminization.