ABSTRACT The transmission of tobamovirus by tomato and pepper seeds is an important mean of virus introduction in crops. Therefore, detecting its presence in the seed becomes essential for the preventive control of virus diseases. In this study, a method was proposed for the detection of Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) in tomatoes (Solanum lycopersicum) and Pepper mild mottle virus (PMMoV) in pepper (Capsicum annum) seeds. Seed lots with different levels of incidence were analyzed by biological, serological, and molecular methods. Using DAS-ELISA technique, it was possible to detect TMV up to the limit rate of 1:170 (1 contaminated seed: total seeds) and the ToMV up to 1:200 in tomato seeds; PMMoV in pepper seeds was detected up to 1:140. The IC-RT-PCR detected the TMV and ToMV up to the limit of 1:400 and PMMoV up to the limit of 1:300. The assembled lots containing only 1 contaminated seed in 1000 (1:1000) were combined into 30 sub lots for DAS-ELISA analysis and 10 sub lots for IC-RT-PCR analysis. Both techniques, DAS-ELISA and IC-RT-PCR, were efficient to detect the three viruses in all analyzed samples, but the detection of tobamoviruses with RT-PCR and biological tests was not reliable. Based on the results of this study, in which a combination of seeds in sub lots was made to reduce the number of tests performed, it is possible to make significant savings in the cost of the diagnostic methods routinely conducted in official laboratories, with high efficiency and reliability.

O alelo Ry adg é muito utilizado pelos melhoristas, por conferir resistência extrema a todas as estirpes do PVY. Entretanto, a estirpe necrótica possui grande capacidade de recombinação, de modo que, novos variantes genéticos capazes de provocar perdas consideráveis na produtividade, vêm surgindo recentemente. Até o momento, nem todas as estirpes recombinantes de PVY necrótico foram testados quanto à sua reação com o gene Ry. Neste estudo, objetivou-se identificar clones de batata, portadores do alelo de resistência e avaliar a sua reação aos seguintes isolados recombinantes: NTN (PVY NTN), Wilga (PVY N-Wi) e "top crespo" (PVY E). Clones avançados de batata, do programa de melhoramento da Universidade Federal de Lavras, foram avaliados por meio de um marcador molecular específico para o alelo Ry adg. Os clones portadores do alelo de resistência foram enxertados em plantas de fumo infectadas com o PVY N e três isolados recombinantes. Os clones portadores do alelo de resistência não foram infectados com nenhum dos recombinantes e nem com o PVY N no teste de enxertia. Esses resultados confirmam que o alelo Ry adg confere resistência também aos variantes testados.

The Ry adg allele is widely used by breeders to confer extreme resistance to all strains of PVY. However, the necrotic strain has increased recombination resulting in recent considerable losses in productivity. Thus far, not all necrotic recombinant strains of PVY have been tested for their reaction to the Ry gene. The objective of this study was to identify potato clones carrying the resistant allele and to assess their reaction to the following recombinant strains: NTN (PVY NTN), Wilga (PVY N-Wi), and "curly top" (PVY E). Advanced clones from the potato breeding program at Universidade Federal de Lavras were evaluated through a specific molecular marker for the Ry adg allele. The clones carrying the resistance allele were grafted on tobacco plants infected with necrotic recombinant strains of PVY. The clones carrying the allele for resistance to PVY were not infected with any of the recombinants during the grafting test. These results confirm that resistance to necrotic recombinant strains has not yet been overcome and that the Ry adg allele also confers resistance to the three recombinant strains tested.

O crestamento bacteriano comum, causado por Xanthomonas axonopodis pv. phaseoli (Xap), é a principal bacteriose do feijoeiro no Brasil, sendo transmitida principalmente por sementes. O presente trabalho teve como objetivo aperfeiçoar uma técnica, por meio de diferentes métodos de preparação do extrato, para a detecção de Xap, bem como sua detecção simultânea com Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) nos extratos de sementes de feijão,via PCR. A partir de amostras de sementes de feijão inoculadas artificialmente com Xap e lotes comerciais, foram avaliados: extrato bruto obtido diretamente das sementes, extrato concentrado por filtração em membrana de 0,22µM de diâmetro, extrato concentrado por centrifugação e extrato plaqueado em meio semi-seletivo XCP1 com e sem antibióticos (BIO-PCR). Avaliou-se a presença simultânea de Xap e Cff em 10 lotes comerciais de sementes de feijão através da reação multiplex, utilizandos-se os primers X4c, X4e, CffFOR2 e CffREV4. A partir do extrato bruto, do extrato concentrado por centrifugação e por filtragem em membrana Millipore® não foi possível a detecção de Xap nas sementes de feijão artificialmente contaminadas nem nos 47 lotes comerciais de sementes/ grãos de feijão. A técnica de BIO-PCR permitiu a detecção de Xap a partir de extratos de sementes de feijão artificialmente contaminadas e em 18 dos 47 lotes comerciais. A técnica de detecção simultânea de Xap e Cff no mesmo gel é viável, por amplificar fragmentos de DNA típicos de cada fitobactéria. O uso do meio de cultura XCP1 sem adição de antibióticos permitiu detectar Xap com período de incubação menor em um dia em comparação à detecção utilizando-se o meio de cultura com antibióticos

Common bacterial blight, caused by Xanthomonas axonopodis pv. phaseoli (Xap), is the major bacteriosis affecting bean plants in Brazil and is mainly transmitted by seeds. This study aimed to improve a technique, using different methods of extract preparation, for the detection of Xap alone and simultaneously with Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) in bean seed extracts, via PCR. Samples of bean seeds artificially inoculated with Xap and commercial batches were used to evaluate: crude extract obtained directly from the seeds, extract concentrated by filtration in 0.22ìM-diameter membrane, extract concentrated by centrifugation and extract plated on semiselective XCP1 medium with and without antibiotics (BIO-PCR). The simultaneous presence of Xap and Cff in 10 commercial batches of bean seeds was assessed through multiplex reaction, using the primers X4c, X4e, and CffFOR2 CffREV4. By using crude extract, extract concentrated by centrifugation and by filtration in millipore® membrane, detection of Xap in bean seeds artificially contaminated or in 47 commercial batches of bean seeds/grains was not possible. BIO-PCR technique allowed the detection of Xap in extracts from artificially contaminated bean seeds and in 18 of the 47 commercials batches. The technique of simultaneous detection of Xap and Cff on the same gel is feasible, for amplifying DNA fragments typical of each phytobacterium. The use of XCP1 culture medium without adding antibiotics allowed the detection of Xap with shorter incubation period in one day, compared to detection using the culture medium with antibiotics.

Blister spot (Colletotrichum gloeosporioides) is now widespread in most coffee producing states of Brazil, becoming a limiting factor for production. The lack of data relating to the reproduction of typical symptoms (light green, oily patches) leaves a gap within the pathosystem, forcing the search for new methodologies for monitoring the disease. Monitoring of genetically modified organisms has proven to be an effective tool in understanding the host x pathogen interactions. Thus, the present study was carried out to evaluate the effectiveness of two systems of genetic transformation in obtaining mutants using the gfp reporter gene. Using the two transformation systems (PEG and electroporation) revealed the efficiency of both, confirmed by fluorescence microscopy and resistance to the antibiotic hygromycin-B, when incorporated into the culture medium. The fungus maintained its cultural and morphological characteristics when compared to wild strains. When inoculated on coffee seedlings, it was found that the pathogenicity of the processed isolates had not changed.

O objetivo deste trabalho foi avaliar a flutuação populacional do ácaro Brevipalpus phoenicis, vetor do Coffee ringspot virus (CoRSV) - agente causal da mancha-anular do cafeeiro -, bem como a sua distribuição em plantas de café e o efeito de acaricidas no seu controle em lavouras cafeeiras. O experimento foi realizado no Município de Coromandel, na região do Alto do Paranaíba, MG, durante dois anos. No segundo ano, avaliou-se o progresso da doença, e determinou-se a relação entre o crescimento populacional do ácaro e a incidência da doença. Observou-se que a mancha-anular e o ácaro ocorreram durante todo o ano, com maiores populações e incidência da doença nos meses com menores índices pluviais e temperaturas amenas. As análises de Pearson mostraram correlação negativa entre os parâmetros climáticos e a população do ácaro. Na análise da incidência da doença em diferentes épocas, verificou-se correlação positiva entre a primeira avaliação e as avaliações subsequentes. Quanto à distribuição espacial, foi observada a presença do ácaro em toda a planta; porém, as folhas são preferidas pelos adultos, enquanto os frutos são preferidos para oviposição. Os acaricidas são eficientes para manter a população do ácaro em baixos níveis, com consequente diminuição da incidência da doença.

The objective of this work was to evaluate the population fluctuation of the mite Brevipalpus phoenicis, vector of the Coffee ringspot virus (CoRSV) - the causal agent of the coffee ringspot disease -, as well as its distribution in coffee plants, and the effect of acaricides in its control in coffee crops. The experiment was carried out at the municipality of Coromandel, in the region of Alto do Paranaíba, MG, Brazil, during two years. In the second year, the progress of the disease was evaluated, and the relationship between the mite population and disease incidence was determined. Ringspot disease and mites were detected throughout the year, with larger populations and disease incidence in the months with lower rainfall and mild temperatures. Pearson analyses showed negative correlation between the climatic parameters and the mite population. In the analysis of the disease incidence in different periods, a positive correlation was found among the first and subsequent evaluations. Regarding spatial distribution, mite presence was observed in the whole plant; however, leaves are preferred by the adults, whereas the fruit are preferred for oviposition. The acaricides are effective in keeping the mite population at low levels, with consequent reduction of disease incidence.

Atualmente, existe a necessidade de se desenvolver métodos sensíveis, baratos, reproduzíveis e rápidos para a detecção de fitobactérias em sementes. O objetivo deste trabalho foi otimizar um método de obtenção das células bacterianas e a técnica de PCR para detecção de Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), em sementes de feijão. Foi utilizado o primer CffFOR2-REV4, desenhado a partir do fragmento amplificado via PCR baseado na seqüência repetitiva (Rep-PCR). Avaliaram-se também quatro métodos de preparação dos extratos de sementes de feijão para a obtenção células de Cff : 1) extrato bruto de sementes; 2 ) extrato concentra do por filtração em membra na milipore (0,22Mu de diâmetro) e ressuspensão em água; 3) extrato concentrado por centrifugação a 10 .00 0 xg por 15 minutos no volume de 20 ou de 80 mL e 4) Bio-PCR. Dentre esses métodos, tanto a Bio-PCR quanto a concentração do extrato por centrifugação, seja no volume de 20 ou de 80 mL, possibilitaram amplificar o segmento de DNA de 306 pb, característico de Cff. Essas duas técnicas, além de detectar a bactéria, apresentam alta sensibilidade, detectando até 1 semente inoculada artificialmente artificialmente com Cff em 999 sementes sadi as. Analisaram-se dezessete lotes comerciais de sementes de feijão pelo método de concentração de extrato por centrifugação, sendo qu e em doze detectou-se a bactéria Cff, observado pela presença de bandas com 306 pb. Portanto, foi possível otimizar um método de obtenção das células bacterianas e técnica de PCR para detecção de Cff em sementes de feijão, que seja sensível, reproduzível e de fácil execução, que poderá ser utilizado rotineiramente em laboratórios de análises de sementes.

Currently, there is a need to develop sensitive methods, inexpensive, reproducible and rapid detection of phytobacteria seeds. The objective of this study was to optimize a method for obtaining bacterial cells and the PCR technique to detect Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), in bean seeds using PCR. It was used one pairs of primers CffFOR2-REV4, designed from an PCR amplified fragment of the conserved repetitive sequence (Rep-PCR). The primer pair CffFOR2-REV4 was selected in this study, because demonstrated higher reproducibility and efficacy in the detection of Cff strains in bean seeds. Four methods were also evaluated in the seed extract preparation for PCR: 1) rough seed extract; 2) millipore membrane filter (0.22Mu diameter) concentrated seed extract and subsequently ressuspended in water; 3) centrifuge concentrated seed extract 10.000 xg for 15 minutes for 20 and 80 mL; 4) Bio-PCR. Among those methods, either Bio-PCR or centrifuge concentrated extract, in a total 20 or 80 mL suspension volume, produced a 306bp DNA fragment, diagnostic for Cff. Those two techniques detected the bacterium and presented high sensitivity, detecting up to 1 Cff physiological conditioning artificially contaminated seed in a total 999 healthy ones. A total of seventeen commercial bean seed lots were analyzed by the method of concentration of the extract by centrifugation, and twelve detected in the bacterium Cff, observed by the presence of bands with 306 bp. Therefore, it was possible to optimize a method of obtaining the bacterial cells and PCR for detection of Cff in bean seeds, that is sensitive, reproducible and easy to perform, which can be used routinely in laboratories for seed.

The aim was to assess heterosis in a set of 16 summer-squash hybrids, and evaluate the combining capacity of the respective parental lines, which differed as to the degree of parthenocarpy and resistance to PRSV-W (Papaya Ringspot Virus-Watermelon strain). The hybrids were obtained using a partial diallel cross design (4 x 4). The lines of parental group I were 1 = ABX-037G-77-03-05-01-01-bulk, 2 = ABX-037G-77-03-05-03-10-bulk, 3 = ABX-037G77-03-05-01-04-bulk and 4 = ABX-037G-77-03-05-05-01-bulk, and of group II, 1' = ABX-037G-77-03-05-04-08-bulk, 2' = ABX-037G-77-03-05-02-11-bulk, 3' = Clarice and 4' = Caserta. The 16 hybrids and eight parental lines were evaluated for PRSV-W resistance, parthenocarpic expression and yield in randomized complete-block designs, with three replications. Parthenocarpy and the resistance to PRSV-W were rated by means of a scale from 1 to 5, where 1 = non-parthenocarpic or high resistance to PRSV-W, and 5 = parthenocarpic or high susceptibility to PRSV-W. Both additive and non-additive gene effects were important in the expression of parthenocarpy and resistance to PRSV-W. Whereas estimates of heterosis in parthenocarpy usually tended towards a higher degree, resistance to PRSV-W was towards higher susceptibility. At least one F1 hybrid was identified with a satisfactory degree of parthenocarpy, resistance to PRSV-W and high fruit-yield.

Foram avaliadas 20 linhagens de melancia, provenientes do cruzamento da cultivar comercial suscetível Crimson Sweet e da introdução PI 595201 resistente ao Watermelon mosaic virus (WMV) e Papaya ringspot virus (PRSV-W). As linhagens, e os parentais foram inoculados com o WMV ou com o PRSV-W em casa-de-vegetação distintas. Aos 35 e 49 dias após a primeira inoculação (DAI), as plantas foram avaliadas por meio de uma escala de notas, em que 1 (ausência de sintomas) a 5 (intenso mosaico e deformações foliares). Pelos resultados infere-se que, aos 35 DAI, as linhagens 1, 2 e 20 apresentaram resistência tanto para o WMV como para o PRSV-W, com médias de 1,95, 1,80 e 2,25 para o WMV, e de 2,50, 2,30 e 2,50 para o PRSV-W, respectivamente. As linhagens 5, 7 e 13 foram resistentes somente ao WMV e as plantas das linhagens 3, 10 e 18 para o PRSV-W. A reação das linhagens permaneceu em geral pouco alterada aos 49 DAI. A existência de linhagens resistentes somente ao WMV e somente ao PRSV-W, ao lado de linhagens resistentes a ambos os vírus, é indicativo de que as resistências ao WMV e ao PRSV-W não são controladas pelos mesmos genes.

Twenty advanced watermelon breeding lines, derived from the cross between cv. Crimson Sweet (susceptible) and PI 595201 (resistant to WMV and PRSV-W), were screened for resistance to both potyviruses. The twenty lines, among with Crimson Sweet and PI 595201, were inoculated with either WMV or PRSV-W, in two different greenhouse trials. Plants were evaluated for symptoms 35 and 49 days after the first inoculation (DAI), using a scale from 1 (no symptoms) to 5 (severe mosaic and foliar distortion). Evaluations at 35 DAI indicated that lines 1, 2 and 20 had good levels of resistance to both WMV and PRSV-W, with ratings of 1,95, 1,80 and 2,25 for WMV, and of 2,50, 2,30 and 2,50 for PRSV-W, respectively. Lines 5, 7 and 13 were resistant to WMV only, whereas lines 3, 10 and 18 were resistant to PRSV-W only. The reaction of the lines 49 DAI remained essentially unchanged. The existence of lines with resistance to WMV only and to PRSV-W only, along with lines with resistance to both viruses, indicates that resistance to WMV and PRSV-W are under control of different genes.

A mancha anular do cafeeiro, causada pelo Coffee ringspot virus (CoRSV) que é transmitido pelo ácaro Brevipalpus phoenicis (Geijskes) (Acari: Tenuipalpidae), tem sido observada em altas incidências em várias regiões cafeeiras do Estado de Minas Gerais. O CoRSV causa manchas cloróticas arrendondadas ou irregulares nas folhas, caules e frutos. Foi feita uma avaliação do efeito da infecção de frutos do cafeeiro pelo CoRSV na qualidade da bebida por meio de teste bioquímico e de degustação, e também na eventual perda de peso nos grãos. Testes revelaram que grãos provenientes de frutos de café infectados pelo CoRSV apresentavam menor teor de açúcares redutores e maior condutividade elétrica. Houve também depreciação na qualidade de bebida gerada pelos frutos infectados por meio do teste de degustação (teste de xícara). O peso médio dos grãos provenientes de frutos manchados foi cerca de 5% menor do que dos grãos de frutos sem sintomas.

Coffee ringspot virus (CoRSV), transmitted by the tenuipalpid mite Brevipalpus phoenicis, has been found in high incidences in several regions of the state of Minas Gerais. It induces chlorotic spots on the leaves and fruits and may induce severe fall of the leaves with implication in the yield. An evaluation was made on the effects of CoRSV-infected coffee berries on the beverage quality as well as on the weight of the beans. Infected beans had less reducing sugars and presented an increase in the electrical conductivity. The quality of the beverage prepared from infected fruits was lower than that of healthy fruits. There was a reduction of about 5% in the weight of beans from infected fruits.

Este trabalho foi realizado com onze isolados do Potato Vírus X (PVX), sendo seis do Centro de Indexação de Vírus Minas Gerais (VIC, LAV, UF, NR, SJ, UDI), um de São Paulo (VEL), dois do Rio Grande do Sul (P21 e P22), um de Santa Catarina (CAN) e um do Distrito Federal (BR). Os isolados de PVX foram inoculados mecanicamente, em duas épocas diferentes (setembro de 2001 e julho de 2002), em hospedeiras diversas, para verificação da sua agressividade e sintomatologia induzida nos hospedeiros, com a finalidade de se fazer sua diferenciação. A diagnose foi feita por inspeção visual e DAS-ELISA. Foram feitas também tentativas de recuperação dos vírus em plantas com DAS-ELISA negativo, por inoculação nas plantas indicadoras Gomphrena globosa e Nicotiana tabacum 'TNN'. Os sintomas induzidos pelos isolados de PVX na maioria das plantas foram semelhantes, com exceção de Nicotiana glutinosa e Physalis floridana, que se revelaram ótimas diferenciadoras para os isolados VEL e P21, respectivamente. Provavelmente os 11 isolados de PVX, estudados neste trabalho, possuem a mesma origem que os isolados dos grupos 1 e 3.

This work was accomplished with eleven PVX isolates: six from national and imported seeds analised at the Virus Indexation Center of Minas Gerais - Brazil (VIC, LAV, UF, NR, SJ, UDI), one from São Paulo state (VEL), two from Rio Grande do Sul state (P21 and P22), one from Santa Catarina state (CAN) and one from the Federal District (BR). The PVX isolates were mechanically inoculated in several hosts plant, in two diferent seasons (September, 2001 and July, 2002), to verify the severity and host symptoms. The diagnosis was done by visual inspection and DAS-ELISA. Attempts to recover the PVX isolates from DAS-ELISA negative plants were also done by inoculation in the indicator plants Gomphrena globosa and Nicotina tabacum TNN. The symptoms shown by the majority of host plants, when inoculated with the eleven isolates, were similar, but Nicotina glutinosa and Physalis floridana showed distinct symptoms when inoculated with P21 and VEL. Probably, the eleven PVX isolates studied in this work have the same origin of the isolates from groups 1 and 3.