ABSTRACT: Brevipalpus-transmitted viruses (BTV) are a taxonomically diverse group of plant viruses which severely affect a number of major crops. Members of the group can be sub-classified into cytoplasmic (BTV-C) or nuclear type (BTV-N) according to the accumulation sites of virions in the infected plant cells. Both types of BTV produce only local infections near the point of inoculation by viruliferous mites. Features of BTV-plant interactions such as the failure of systemic spread in their natural hosts are poorly understood. In this study we evaluated Arabidopsis thaliana, a model plant commonly used for the study of plant-virus interactions, as an alternative host for BTV. Infection of Arabidopsis with the BTV-N Coffee ringspot virus and Clerodendrum chlorotic spot virus, and the BTV-C Solanum violaefolium ringspot virus, were mediated by viruliferous Brevipalpus mites collected in the wild. Upon infestation, local lesions appeared in 7 to 10 days on leaves of, at least, 80 % of the assayed plants. Presence of viral particles and characteristic cytopathic effects were detected by transmission electron microscopy (TEM) and the viral identities confirmed by specific reverse-transcriptase polymerase chain reaction (RT-PCR) and further amplicon sequencing. The high infection rate and reproducibility of symptoms of the three different viruses assayed validate A. thaliana as a feasible alternative experimental host for BTV.

O objetivo deste trabalho foi buscar novas fontes de resistência à leprose-dos-citros, no Banco Ativo de Germoplasma do Centro APTA Citros Sylvio Moreira, Instituto Agronômico, em Cordeirópolis, SP. Foram utilizadas plantas obtidas por sementes de 26 acessos, infectadas com o vírus da leprose-dos-citros (Citrus leprosis virus - CiLV), por meio do seu vetor Brevipalpus phoenicis. O aparecimento de lesões, a partir de 21 dias após a inoculação, foi observado em 11 dos genótipos testados (42,3%). Quinze espécies, entre elas Citrus pennivesiculata e C. celebica, comportaram-se como altamente resistentes, enquanto outras, como C. keraji, foram mais suscetíveis que o padrão C. sinensis. Os dados mostraram grande variação de respostas de Citrus spp. à leprose, com elevado número de espécies resisentes, que podem ser utilizadas como fonte de resistência à doença em programas de melhoramento.

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 Agronômico, in Cordeirópolis, 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.

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.

Os objetivos deste trabalho foram avaliar a freqüência de híbridos de cruzamento entre tangerina 'Cravo' (Citrus reticulata Blanco) e laranja 'Pêra' (Citrus sinensis (L.) Osbeck), o uso de marcadores morfológicos e moleculares (RAPD) na identificação precoce de plantas zigóticas, e a variabilidade dos híbridos. A porcentagem de híbridos foi maior na população germinada em placas de Petri (19,4%). Verificou-se que quanto maior a competição entre os "seedlings" por espaço e nutrientes, menor a freqüência de plantas híbridas. A identificação dos híbridos não foi possível apenas com o uso de marcadores morfológicos. A análise morfológica dos híbridos revelou elevada variabilidade.

The objectives of this work were to evaluate the hybrid frequency from the cross between tangerine 'Cravo' (Citrus reticulata Blanco) and sweet orange 'Pêra' (Citrus sinensis (L.) Osbeck), the use of morphological and RAPD markers for early identification of zygotic plants between parents with similar phenotype, and the morphological variability among the hybrids. Plants germinated on Petri dishes showed the higher hybrid percentage (19.4%). Hybrid plant frequency was inversely proportional to the competition level for space and nutrients among the hybrids. Accurate hybrid identification is not possible using morphological markers alone. The hybrids selected showed high morphological variability.

A análise do polimorfismo isoenzimático foi usada para determinar a variabilidade genética entre espécies e híbridos de Citrus spp. e um acesso da espécie Poncirus trifoliata (L.) Raf. Dez diferentes sistemas enzimáticos foram analisados, incluindo aspartato aminotransferase (AAT), fosfatase ácida (ACP), leucina aminopeptidase (LAP), 6-fosfogluconato desidrogenase (6-PGD), isocitrato desidrogenase (IDH), fosfoglucose isomerase (PGI), fosfoglucomutase (PGM), diaforase (DIA), shiquimato desidrogenase (SKD) e peroxidase (PRX). Um total de 20 locos e 48 alelos foram identificados. Os cultivares de laranja doce (C. sinensis (L.) Osbeck) apresentaram um grande número de locos heterozigotos, mas similares entre eles, com exceção dos cultivares Westin e Lima graúda. Os cultivares de mandarim (C. reticulata Blanco) apresentaram diferentes padrões entre eles, enquanto que Poncirus trifoliata (L.) Raf. apresentou elevada diferenciação em relação a todas as espécies de Citrus e híbridos. Fenótipos exclusivos foram observados em alguns sistemas enzimáticos, sendo encontrados padrões similares entre os híbridos interespecíficos e seus possíveis parentais.

Isoenzymatic polymorphism analysis was used to determine genetic variability among species and hybrids of Citrus spp. and one accession of Poncirus trifoliata (L.) Raf. Ten enzymatic systems aspartate aminotransferase (AAT), acid phosphatase (ACP), leucine aminopeptidase (LAP), 6-phosphogluconate dehydrogenase (6-PGD), isocitrate dehydrogenase (IDH), phosphoglucoisomerase (PGI), phosphoglucomutase (PGM), diaphorase (DIA), shikimate dehydrogenase (SKD) and peroxidase (PRX) were analyzed. Twenty loci and 48 alleles were identified. Sweet orange cultivars (C. sinensis (L). Osbeck) showed the highest polymorphism with the largest number of heterozygous loci, although the alleles of those loci were the same in all cultivars, with the exception of Westin and Lima graúda. Mandarins (C. reticulata Blanco) exhibited diverse patterns, whereas Poncirus trifoliata (L.) Raf. showed high variability with all Citrus species and hybrids. Exclusive phenotypes were observed in some enzymatic systems, and similar patterns were found among interspecific hybrids and their putative parents.