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.

Comparative proteomics of two citrus varieties in response to infection by the fungus Alternaria alternata.

Alternaria brown spot (ABS) is a disease caused by the necrotrophic fungus Alternaria alternata, which induces necrotic lesions on fruits and young leaves due to the production of the host-specific ACT toxin by the fungus. To better understand the citrus-A. alternata interaction and to identify putative resistance proteins, as well as the receptor of the ACT toxin, citrus plants susceptible ('Minneola' mandarin) and resistant ('Clemenules' tangor) to A. alternata, infected or not (control) with the pathogen were analyzed by proteomics. Protein changes were observed between citrus genotypes after infection, and 150 candidate proteins were obtained. A general scheme of the metabolic processes involved in susceptible and resistant citrus-A. alternata interactions was designed. Susceptible plants presented a high level of proteins involved in stress response at the final stages of the infection, whereas resistant plants presented high level of ROS proteins, metabolic proteins, and proteins involved in the immune system process. Proteins like ferredoxin and cyclophilin are specific to the susceptible variety and may be good candidates as fungal effector-interacting proteins. This is the first citrus-A. alternata proteomics analysis, which has allowed a better understanding of the molecular bases of the citrus response to ABS disease.

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.

Laser-induced Fluorescence Spectroscopy (LIFS) for Discrimination of Genetically Close Sweet Orange Accessions ( Citrus sinensis L. Osbeck).

Although there is substantial diversity among cultivated sweet oranges genotypes with respect to morphological, physiological, and agronomic traits, very little variation at DNA level has been observed. It is possible that this low DNA molecular variability is due to a narrow genetic basis commonly observed in this citrus group. The most different morphological characters observed were originated through mutations, which are maintained by vegetative propagation. Despite all molecular tools available for discrimination between these different accessions, in general, low polymorphism has been observed in all groups of sweet oranges and they may not be identified by molecular markers. In this context, this paper describes the results obtained by using laser-induced fluorescent spectroscopy (LIFS) as a tool to discriminate sweet orange accessions ( Citrus sinensis L. Osbeck) including common, low acidity, pigmented, and navel orange groups, with very little variation at DNA level. The findings showed that LIFS combined with statistical methods is capable to discriminate different accessions. The basic idea is that citrus leaves have multiple fluorophores and concentration depends on their genetics and metabolism. Thus, we consider that the optical properties of citrus leaves may be different, depending on variety. The results have shown that the developed method, for the best classification rate, reaches an average sensitivity and specificity of 95% and 97.5%, respectively. An interesting application of this study is the development of an economically viable tool for early identification in seedling certification, in citrus breeding programs, in cultivar protection, or in germplasm core collection.

Positive relationship between citrus leaf miner and alternaria brown spot / Correlação positiva entre lagarta-minadora-dos-citros e mancha marrom de alternária

Alternaria brown spot, or ABS (Alternaria alternata), is the most prevalent fungal disease of tangerines in the world. Field observations have revealed ABS lesions on leaves wounded by the citrus leaf miner (CLM). Thus, the objective of this study was to assess the relationship between CLM and ABS. Leaves from young Murcott tangor plants and detached leaves from 16 different varieties of tangerines with and without lesions caused by CLM were inoculated with A. alternata. The symptoms of the plants were subsequently quantified by counting the number of lesions, and the lesion area was estimated using a diagrammatic scale. The presence of CLM damage aggravates the severity of Alternaria alternata fungus infections in susceptible tangerine varieties.

A mancha marrom de altenária, ou MMA (Alternaria alternata), é a doença fúngica mais importante das tangerinas no mundo. Observações de campo revelaram a ocorrência de lesões de MMA em folhas feridas pela lagarta-minador-dos-citros (LMC). Assim, o objetivo deste estudo foi avaliar a relação entre a LMC e MMA. Folhas jovens de plantas de tangor Murcott e folhas destacadas de 16 diferentes variedades de tangerinas com e sem lesões, causadas por CLM, foram inoculadas com A. alternata. Os sintomas da MMA foram subsequentemente quantificados através da contagem do número de lesões, e a área da lesão foi estimada utilizando uma escala diagramática. A presença de danos da LMC agrava a severidade de Alternaria alternata nas variedades de tangerina suscetíveis.

Screening of genomic libraries.

Microsatellites, or simple sequence repeats (SSRs), have proven to be an important molecular marker in plant genetics and breeding research. The main strategies to obtain these markers can be through genomic DNA and from expressed sequence tags (ESTs) from mRNA/cDNA libraries. Genetic studies using microsatellite markers have increased rapidly because they can be highly polymorphic, codominant markers and they show heterozygous conserved sequences. Here, we describe a methodology to obtain microsatellite using the enrichment library of DNA genomic sequences. This method is highly efficient to development microsatellite markers especially in plants that do not have available ESTs or genome databases. This methodology has been used to enrich SSR marker libraries in Citrus spp., an important tool to genotype germplasm, to select zygotic hybrids, and to saturate genetic maps in breeding programs.

Caracterização molecular de uma progênie de tangerineira 'Clementina Fina' e 'Montenegrina' / Molecular characterization of a progeny between tangerines 'Clementina Fina' and 'Montenegrina'

Os citros apresentam uma taxonomia muito complexa, principalmente com relação ao número de espécies que constituem o gênero Citrus e os gêneros afins. Genótipos classificados como espécies podem ter sido originados por hibridação interespecífica e preservados por meio da embrionia nucelar. O presente trabalho teve como objetivo caracterizar uma população de tangerineiras híbridas oriundas do cruzamento das tangerineiras 'Clementina Fina' (Citrus clementina Hort. ex Tan.), genitor feminino, e 'Montenegrina' (Citrus deliciosa Ten.), genitor masculino, utilizando marcadores do tipo microssatélites (SSR). Com 12 pares de primers, foi possível diferenciar 93 acessos do estudo e agrupar a F1 em indivíduos mais próximos do genitor feminino e do genitor masculino. O PIC (Conteúdo de Informação de Polimorfismo) dos primers variou de 0,27 a 0,65. Toda a progênie do cruzamento entre 'Clementina Fina' e 'Montenegrina' analisada neste estudo é híbrida, e os SSRs foram eficientes para identificar híbridos com maior similaridade genética em relação aos genitores, mostrando a existência de variabilidade genética entre as plantas da população estudada.

Citrus have a very complex taxonomy, especially considering the number of species included in genus Citrus and related genera. What is classified as a species may have originated by interespecific hybridization and preserved through nucellar embryony. This research aimed to characterize a population of hybrid tangerines, originated from the cross of 'Clementina Fina' (Citrus clementina Hort. ex Tan.) as female and 'Montenegrina' (Citrus deliciosa Ten.) as male parents, using microsatellite molecular makers. With 12 pairs of primers it was possible to differentiate 93 of the studied accessions and to group F1 individuals nearer to the male or to the female parent. The primers PIC (Polymorphism Information Content) ranged from 0.27 to 0.65. All the analyzed progeny between 'Clementina Fina' and 'Montenegrina' is hybrid, where SSR were efficient in identifying hybrids more similar to the genitors, showing genetic variability among plants of the studied population.

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.

Development of genetic maps of the citrus varieties 'Murcott' tangor and 'Pera' sweet orange by using fluorescent AFLP markers.

The progeny of 87 BC(1) hybrids of 'Murcott' tangor and 'Pera' sweet orange, genotyped with fluorescent amplified fragment length polymorphism (fAFLP) markers, was used for the construction of genetic maps for both citrus varieties. Mapping strategies, considering the progeny as a result of backcrossing and cross-pollination, were exploited in Mapmaker 2.0 (LOD score >or= 3.0 and or= 3.0 and theta

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.

Frequency and distribution of microsatellites from ESTs of citrus

Nearly 65,000 citrus EST (Expressed Sequence Tags) have been investigated using the CitEST project database. Microsatellites were investigated in the unigene sequences from Citrus spp. and Poncirus trifoliata. From these sequences, approximately 35 percent of the non-redundant ESTs contained SSRs. The frequencies of different SSR motifs were similar between Citrus spp and trifoliate orange. In general, mononucleotide repeats appeared to be the most abundant SSRs in the CitEST database, but we also identify di-, tri-, tetra-, penta- and hexanucleotide repeats. The AG/CT and AAG/CTT were the most common dinucleotide and trinucleotide motifs, with frequencies of 54.4 percent and 25.2 percent, respectively. Primer sequences flanking SSR motifs were successfully designed and synthesized. After in silico polymorphism analysis, a subset of sixty-eight primers was validated in different Citrus spp. and Poncirus trifoliata. PCR-amplification revealed polymorphism in citrus with all tested primer pairs and showed the potential of these markers for linkage mapping. Our study showed that the CitEST database can be exploited for the development of SSR markers that can amplify Citrus spp. and related genus for comparative mapping and other genetic analyses.

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.

Diversidade genética de porta-enxertos cítricos baseada em marcadores moleculares RAPD / Genetic diversity of citrus rootstocks based on RAPD marker analysis

Este trabalho teve como objetivo caracterizar a diversidade genética, através do marcador molecular RAPD, dos porta-enxertos da Coleção de Citros da Estação Experimental Agronômica da Universidade Federal do Rio Grande do Sul (EEA/UFRGS) e acessos de porta-enxertos cítricos coletados em viveiristas da Região do Vale do Rio Caí do estado do Rio Grande do Sul. Para tanto, coletaram-se folhas de nove porta-enxertos cítricos da EEA/UFRGS e de dez acessos de trifoliata (Poncirus trifoliata) de viveiristas. Com o uso de nove seqüências inicializadoras, foi possível separar os porta-enxertos cítricos em dois grupos principais, um formado pelo limoeiro æCravoÆ e outro pelo trifoliata e seus híbridos, apresentando alta dissimilaridade genética entre os grupos. Marcadores moleculares RAPD foram eficientes para caracterizar variedades de porta-enxertos de citros e para separar o porta-enxerto P. trifoliata de seus híbridos podendo serem utilizados para caracterização de plantas matrizes, análise de variabilidade genética entre genitores em programas de melhoramento genético de porta-enxertos e para identificar a origem sexual ou nucelar de mudas de trifoliata em viveiros comerciais.