Marinês Bastianel

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.

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% 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% and 25.2%, 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.

Transferability and Level of Heterozygosity of Microsatellite Markers in Citrus Species

Microsatellite markers are a powerful tool for genetic studies, including germplasm conservation, cultivar identification, and integration of linkage maps. Several works have shown that primer pairs designed for one species can be used in related species to facilitate wider application because it reduces the costs for primer development. The objective of this study was to evaluate the transferability of microsatellite primers which was previously developed from the genomic library of Pêra sweet orange (Citrus sinensis L. Osbeck) and to determine the level of heterozygosity between citrus accessions and related genera. Twenty-four microsatellite loci were evaluated on 12 genotypes of Citrus, Poncirus, and an intergeneric hybrid. All analyzed markers were transferable across all genotypes. Seventeen loci were polymorphic, and the number of alleles per loci ranged from one to six. The lowest level of heterozygosity was observed for Poncirus trifoliata (L.) Raf. cultivars while the highest level was for Swingle citrumelo. In general, microsatellite markers showed wide genetic variation and demonstrated that they can be useful in citrus breeding programs.

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.

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

The progeny of 87 BC1 hybrids of ‘Murcott’ tangor and ‘Pêra’ 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 ≥ 3.0 and 9 ≤ 0.40) and JoinMap 3.0 software (LOD score ≥ 3.0 and 9 ≤ 0.25), respectively. Genetic map distances (in cM) between the linked fAFLPs were estimated, in both packages, by the Kosambi’s function. Maps of both parents were constructed in Mapmaker with 121 of the 202 fAFLP markers showing 1:1 Mendelian segregation rates (’Murcott’ map: 65 fAFLPs, average distance between them 29.5 cM, divided into 9 linkage groups (LGs), total size 1651.47 cM; ’Pêra’ map: 55 fAFLPs, average distance between them 31.9 cM, divided into 5 LGs, total size 1596.2 cM). The second ’Murcott’ map, constructed through linkage analysis of 347 fAFLP markers with 3:1 or 1:1 segregation rates by using JoinMap, resulted in the linkage of 227 markers with an average distance of 4.25 cM among them, divided into 9 LGs of 845 cM. fAFLP loci showing distorted segregation and/or clustered were observed in different LGs of the maps generated by all the software. The use of the ’Murcott’ tangor and ’Pêra’ sweet orange genetic maps in research on identification of citrus QRLs (quantitative resistance loci) toXylella fastidiosa and QTLs (quantitative trait loci) related to the productivity and quality of the juice, respectively, is discussed.

Breeding, genetic and genomic of citrus for disease resistance

Embora a citricultura seja uma das mais importantes atividades economicas no Brasil, ela esta apoiada em um pequeno numero de variedades. Este fato tem contribuido para sua vulnerabilidade, especial no que ser refere a fatores fitossanitarios. A obtencao de um maior numero de variedades, seja para a industria seja para o mercado de fruta fresca, tem sido um dos principais objetivos dos programas de melhoramento. O melhoramento genetico de citros tem sido ampliado nas ultimas decadas em funcao da possibilidade de associacao de ferramentas da biotecnologia e metodos classicos de melhoramento. O uso de marcadores moleculares para a selecao precoce de plântulas zigoticas em cruzamentos controlados ampliou a possibilidade de selecao de maior numero de novas combinacoes e, em consequencia, o estabelecimento de grande numero de hibridos em experimentos de campo. Quanto mais rapido novas ferramentas sejam incorporadas ao programa, mais rapido e a possibilidade de obter novos genotipos que possam ser testados como variedades. Caracteristicas adequadas podem ser incorporadas ou mantidas, enquanto caracteristicas indesejaveis podem ser excluidas ou minimizadas nos novos genotipos. Copas e porta-enxertos nao podem ser considerados separadamente, e a compatibilidade de enxertia, qualidade de fruta e produtividade sao caracteristicas essenciais a serem avaliadas nos ultimos estagios do programa. Mapeamento de caracteristicas quantitativas (QTLs) tem propiciado os programas de melhoramento de varias especies lenhosas perenes, e em citros e possivel mapear varias caracteristicas com heranca quantitativa e qualitativa. A existencias de mapas de ligacao e QTLs, o desenvolvimento de bibliotecas de ESTs e BACs, assim como o sequenciamento completo do genoma de citros, ampliam a urgencia para exploracao de tais dados para ampliar os conhecimentos sobre genetica do grupo. O volume crescente de informacoes de genomas de varios outros organismos tem ampliado novas abordagens procurando a integracao entre melhoramento, genetica e genoma. Selecao assistida por genoma envolve mais que sequenciamento de genes ou genomas completos e tem se tornado uma importante ferramenta nos programas de melhoramento de especies perenes e anuais. Um grande volume de informacoes pode ser derivada da analise de genomas. O uso e beneficio de tais informacoes dependera da base genetica do programa de melhoramento.

Detection of Brevipalpus-transmitted viruses in their mite vectors by RT–PCR

The diagnosis of plant diseases caused by Brevipalpus-transmitted viruses (BrTVs) has been done through the analyses of symptoms, transmission electron microscopy, and RT–PCR of infected plant tissues. Here, we report the detection of Citrus leprosis virus C, Orchid fleck virus, Clerodendrum chlorotic spot virus and Solanum violaefolium ringspot virus in their viruliferous vectors Brevipalpus spp. using specific primer pairs for each of the viruses. The efficiency of virus transmission by Brevipalpus mites is low, so the detection of these pathogens in their vectors could constitute an important tool for studies involving virus-vector relationships, transmission, and monitoring the pathogen prior to the appearance of symptoms in the field.

Citrus leprosis virus C Naturally Infecting Commelina benghalensis, a Prevalent Monocot Weed of Citrus Orchards in Brazil

There are two bacilliform, rhabdo-like viruses that cause citrus leprosis: Citrus leprosis virus C (CiLV-C), which accumulates in the cytoplasm of infected cells, and Citrus leprosis virus nuclear type (CiLV-N), which accumulates in their nucleus. The first one, the prototype of the new genus Cilevirus, is prevalent and occurs in several countries of the American continent, from Argentina to Mexico (1). The second type, still a tentative member of the Rhabdoviridae family, is of rare occurrence, with a few reports in Brazil and one in Panama (1). Leprosis is particularly important to the Brazilian citrus industry because of the 60 to 80 million dollars spent yearly for the control of Brevipalpus phoenicis (Geijskes, 1939) (Acari: Tenuipalpidae), the vector of the virus (1). For decades, the disease was considered unique to citrus plants; however, greenhouse experiments conducted in the 1990s demonstrated the mechanical transmission of CiLV-C to noncitrus plants (1). Years later, researchers were able to transmit the virus to nonrutaceous hosts using viruliferous mites (1,4). Recently, León et al. (2) reported the occurrence of the first noncitrus plant naturally infected by CiLV-C, the rutaceous Swinglea glutinosa Blanco (Merr.). Tropical spiderworts (Commelina benghalensis L.; Commelinaceae) are monocot weeds commonly found in citrus orchards in Brazil. In a survey conducted in orchards with high incidences of leprosis in the municipalities of Borborema and São José do Rio Preto, State of Sao Paulo, Brazil, tropical spiderworts were found exhibiting necrotic spots with a yellow halo in green leaves and green spots with necrotic center in senescent leaves. Since these symptoms are similar to those caused by CiLV-C in citrus, symptomatic plants were collected and subjected to transmission electron microscopy analyses and reverse transcription-PCR using primers that specifically amplify a region within the putative movement protein gene of the virus (3). Bacilliform virus particles and typical inclusions were seen in the lesions. Bands of the expected 344 bp size were seen in agarose gels of symptomatic samples only. The analysis of the consensus sequence showed 100% identity with CiLV-C sequence available in the GenBank (Accession No. YP_654542.1). Experimental transmission of CiLV-C by B. phoenicis reproduced the lesions in inoculated tropical spiderwort. Also, the virus could be easily transmitted back from C. benghalensis to sweet orange plants. Our data show that this widespread weed is the first monocot as a natural host for CiLV-C. Since tropical spiderwort is a host for B. phoenicis and symptomatic plants were found in two municipalities 130 km apart from each other, it is possible that this weed may serve as reservoir for the virus and play a relevant role in the spread of the disease in the field, but this issue still needs to be addressed in further experiments. References: (1) M. A. Bastianel et al. Plant Dis. 94:284, 2010. (2) M. G. A. Leon et al. Plant Dis. 90:682, 2008. (3) E. C. Locali-Fabris et al. Plant Dis. 87:1317. (4) M. A. Nunes et al. Plant Dis. Online publication. doi:10.1094/PDIS-06-11-0538, 2011.

Resposta de diferentes genótipos de tangerinas e seus híbridos à inoculação in vitro e in vivo de Alternaria alternata

ABSTRACT - The confirmation of a new disease in the Brazilian tangerine orchards, the alternaria brown spot, has been concerning citrus growers because of the serious damages to commercial crops of susceptible varieties, such as Ponkan tangerine and Murcott tangor. In this research, in vitro and in vivo inoculations of Alternaria alternata fungus were held in 54 different varieties of the tangerine group selected from the Citrus Germoplasm Bank of the Citros Center APTA Sylvio Moreira, from the Instituto Agronomico, in Cordeiropolis, State of Sao Paulo, Brazil, aiming to find varieties with more resistance. First, a pathogenicity test with ten different A. alternata isolates was made to select the most aggressive one. After this, inoculations in detached leaves and seedlings were held and, approximately two (in vitro) and three (in vivo) days after, the number of lesions in each leaf was counted, and the disease severity estimated with diagrammatic scale help (in vivo). More than a half of varieties showed symptoms of the disease, however with different levels of susceptibility. Varieties like South African tangerine and Orlando tangelo were the most susceptible; on the other hand the satsumas and willow leaf group and some tangerines, showed resistance, indicating new options to the Brazilian citriculture.Index terms: brown spot, Citrus reticulata, varietal resistance.

Resposta diferencial de espécies e de híbridos de citros à leprose

The objective of this work was to prospect for new sources of resistance to leprosis in the Citrus Germplasm Bank of Centro APTA Citros Sylvio Moreira, Instituto Agronomico, in Cordeiropolis, SP, Brazil. Plants obtained through seeds from 26 citrus accessions were mite-infected with Citrus leprosis virus (CiLV). Typical lesions started to appear in 11 genotypes (42.3% of the tested accessions) 21 days after inoculation. Fifteen species, among them Citrus pennivesiculata and C. celebica, behaved as highly resistant, while other genotypes, as C. keraji, were more susceptible than the standard C. sinensis. The data showed a wide variation in the response of Citrus spp. to leprosis, with a large number of resistant species, wich can be used as source of resistance to this disease, in breeding programs.