Citrus canker, caused by Xanthomonas axonopodis Starr and Garces pv. citri (Hasse) Vauterin et al., is one of the main problems affecting citrus production. In order to obtain resistance to phytopathogenic bacteria, insect genes, coding for antimicrobial proteins, have been used in plant genetic transformation. In this study, transgenic Citrus sinensis (L.) Osb. `Hamlin' plants expressing the antimicrobial insect-derived attacin A gene (attA) were obtained by Agrobacterium tumefaciens (Smith and Towns.) Conn-mediated transformation. Initially, the cDNA clone was used to construct a binary plasmid vector (pCattA 2300). The construction included the native signal peptide (SP) responsible for directing the insect protein to the extracellular space where bacteria is supposed to accumulate in vivo. In order to investigate the native SP effectiveness in a plant model system, onion (Allium cepa L.) epidermal cells were transformed, via biobalistics, using plasmids containing the attA gene with or without SP, fused with the green fluorescent protein gene (pattA 1303 and pSPattA 1303). Fluorescence accumulation surrounding the cells was observed only in tissues transformed with the plasmid containing the gene with SP, indicating the protein secretion to the apoplast. Citrus transformation was confirmed by PCR and Southern blot hybridization analysis in 12 regenerated plants. Transcription of attA gene was detected by Northern blot analysis in all transgenic plants. Eight selected transgenic lines were propagated and inoculated with a 106 cfu/mL suspension of the pathogen X. axonopodis pv. citri. Compared to control (non-transformed plant), seven transgenic lines showed a significant reduction in susceptibility to citrus canker. The results obtained here indicate the potential use of antibacterial proteins to protect citrus from bacterial diseases.

This work reports the evaluation of citrus canker resistance in transgenic lines of four sweet orange cultivars Hamlin, Natal, Pera and Valencia expressing the rice Xa21 R-gene. The Agrobacterium-mediated genetic transformation was performed and transgene integration was confirmed by Southern blot analyses. These analyses showed that the plants had undergone 1-3 independent transgene integration events. The genetic transformation efficiency was 18AE6% for Hamlin, 8AE3% for Natal, 3% for Pera and 11AE3% for Valencia sweet orange. Transgene transcription was confirmed by northern blot analyses in most transgenic lines analysed. Transgenic lines of each of the cultivars were selected and propagated for resistance evaluation to Xanthomonas axonopodis pv. citri. Transgenic lines with a higher resistance to citrus canker were identified for cvs Hamlin, Natal and Pera. The lowest diseased leaf area values were detected for transgenic lines of Natal and Pera, cultivars which are considered moderately resistant to citrus canker in the field, indicating an influence of cultivar genetic background on the effectiveness of the Xa21 gene in controlling the pathogen. Transgenic lines in which transgene transcription was not detected did not show any improvement in resistance to the pathogen. These results show the integration and expression of the rice Xa21 gene in citrus and the possibility of using R-genes in controlling diseases in a heterologous genetic background.

Genetic transformation with genes that code for antimicrobial peptides has been an important strategy used to control bacterial diseases in fruit crops, including apples, pears, and citrus. Asian citrus canker (ACC) caused by Xanthomonas citri subsp. citri Schaad et al. (Xcc) is a very destructive disease, which affects the citrus industry in most citrus-producing areas of the world. Here, we report the production of genetically transformed Natal, Pera, and Valencia sweet orange cultivars (Citrus sinensis L. Osbeck) with the insect-derived attacin A (attA) gene and the evaluation of the transgenic plants for resistance to Xcc. Agrobacterium tumefaciens Smith and Towns-mediated genetic transformation experiments involving these cultivars led to the regeneration of 23 different lines. Genetically transformed plants were identified by polymerase chain reaction, and transgene integration was confirmed by Southern blot analyses. Transcription of attA gene was detected by Northern blot analysis in all plants, except for one Natal sweet orange transformation event. Transgenic lines were multiplied by grafting onto Rangpur lime rootstock plants (Citrus limonia Osbeck) and sprayinoculated with an Xcc suspension (10 6 cfu mL −1 ). Experiments were repeated three times in a completely randomized design with seven to ten replicates. Disease severity was determined in all transgenic lines and in the control (nontransgenic) plants 30 days after inoculation. Four transgenic lines of Valencia sweet orange showed a significant reduction in disease severity caused by Xcc. These reductions ranged from 58.3% to 77.8%, corresponding to only 0.16-0.30% of leaf diseased area as opposed to 0.72% on control plants. One transgenic line of Natal sweet orange was significantly more resistant to Xcc, with a reduction of 45.2% comparing to the control plants, with only 0.14% of leaf diseased area. Genetically transformed Pera sweet orange plants expressing attA gene did not show a significant enhanced resistance to Xcc, probably due to its genetic background, which is naturally more resistant to this pathogen. The potential effect of attacin A antimicrobial peptide to control ACC may be related to the genetic background of each sweet orange cultivar regarding their natural resistance to the pathogen.

The use of organic matter that improves the physical, chemical and biological soil properties has been studied as an inducer of suppressiveness to soilborne plant pathogens. The objective of this work was to evaluate the effect of different sources and concentrations of organic matter on tomato bacterial wilt control. Two commercially available organic composts and freshly cut aerial parts of pigeon pea (Cajanus cajan) and crotalaria (Crotalaria juncea) were incorporated, in concentrations of 10, 20 and 30 % (v/v), into soil infested with Ralstonia solanacearum. The soil with the fresh organic matter of pigeon pea and crotalaria was incubated for 30 and 60 days before planting. Tomato seedlings of cv. Santa Clara were transplanted into polyethylene bags with 3 kg of the planting substrate (infested soil + organic matter). The wilting symptoms and percentage of flowering plants were evaluated for 45 days. All evaluated concentrations with incorporation and incubation for 30 days of aerial parts of pigeon pea and crotalaria controlled 100% tomato bacterial wilt. With 60 days of incubation, only the 10 % concentration of pigeon pea and crotalaria did not control the disease. These results suggest that soil incorporation of fresh aerial parts of pigeon pea and crotalaria is an effective method for bacterial wilt control.

RESUMOEste trabalho teve como objetivo a caracterização física e físico-química de frutos de tomateiro oriundos de plantas enxertadas em comparação com plantas pés-franco. A enxertia foi realizada utilizandose o método de fenda cheia tendo o híbrido Hawaii 7996 como porta-enxerto, resistente à murcha bacteriana, e as cultivares Santa Clara e Santa Cruz Kada e o híbrido Débora Plus como enxerto. O delineamento experimental foi em blocos ao acaso, com seis tratamentos (três enxertados e três pés-franco) e cinco repetições de campo. Foram avaliadas as seguintes características: massa média dos frutos, diâmetros transversal e longitudinal, sólidos solúveis totais (SST), pH, acidez total titulável (ATT) e a relação SST/ATT. A enxertia não alterou as características acima descritas para os frutos de tomate, quando comparados aos frutos oriundos de plantas pés-franco, com exceção do pH para as duas cultivares utilizadas. O híbrido 'Hawaii 7996' pode ser utilizado como porta-enxerto com as cultivares comerciais Santa Clara e Santa Cruz Kada e o híbrido Débora Plus, sem prejuízo para a qualidade do fruto do tomateiro. Palavras-chave:Lycopersicon esculentum, enxertia, Hawaii 7996, qualidade do fruto. ABSTRACT QUALITY CHARACTERISTICS OF TOMATO FRUITS FROM GRAFTED PLANTSThis research was conducted with the objective of characterizing the physical and physical-chemical characteristics of tomato fruits from grafted plants as compared to those from own rooted seedlings. Cultivars Santa Clara, Santa Cruz Kada, and the hybrid Débora Plus were cleft grafted on to hybrid Hawaii 7996, resistant to bacterial wilting, as the rootstock. The experimental design was the completely randomized blocks, with six treatments (three grafted and three ungrafted), with five replications in the field. It were evaluated fruit weight, length and width, total soluble solids (TSS), pH, titratable total acidity (TTA), and the ratio TSS/TTA. Grafting did not affect the above characteristics, except for pH of the two cultivars. Thus, the hybrid 'Hawaii 7996' can be used as commercial rootstock for cultivars 'Santa Clara'; 'Santa Cruz Kada' and the 'Débora Plus' hybrid, without affecting fruit quality.

Avaliação da resistência a Xylella fastidiosa Wells et al. e Xanthomonas axonopodis pv. citri Vauterin et al. em plantas transgênicas de Citrus sinensis L. Osbeck expressando os genes atacina A ou Xa21 Suane Coutinho Cardoso Tese apresentada para obtenção do título de Doutor em Agronomia. Área de concentração: Fitotecnia Piracicaba 2008 Suane Coutinho Cardoso Engenheiro Agrônomo Avaliação da resistência a Xylella fastidiosa Wells et al. e Xanthomonas axonopodis pv. citri Vauterin et al. em plantas transgênicas de Citrus sinensis L. Osbeck expressando os genes atacina A ou Xa21 Orientador: Prof. Dr. FRANCISCO DE ASSIS ALVES MOURÃO FILHO Tese apresentada para obtenção do título de Doutor em Agronomia. Área de concentração: Fitotecnia Piracicaba 2008 Dados Internacionais de Catalogação na Publicação (CIP) DIVISÃO DE BIBLIOTECA E DOCUMENTAÇÃO-ESALQ/USP Cardoso, Suane Coutinho Avaliação da resistência a Xylella fastidiosa Wells et al. e Xanthomonas axonopodis pv. citri Vauterin et al. em plantas transgênicas de Citrus sinensis L. Osbeck expressando os genes atacina A ou Xa21 / Suane Coutinho Cardoso.-Piracicaba, 2008. 99 p. : il.

A técnica de enxertia é utilizada em algumas hortaliças, principalmente em áreas infestadas, para atribuir resistência a patógenos do solo, minimizando as perdas de produção de cultivares suscetíveis. Este trabalho teve como objetivo avaliar o desenvolvimento e a produtividade de plantas de tomateiro enxertadas em híbrido, tido como resistente a Ralstonia solanacearum, em comparação a pés-francos. O experimento foi desenvolvido em viveiro (produção de mudas e enxertia) e em céu aberto, no município de Cruz das Almas, Bahia, no período de outubro de 2003 a fevereiro de 2004. Utilizou-se o método de enxertia de fenda cheia, com o híbrido 'Hawaii 7996' como porta-enxerto e as cultivares Santa Clara, Santa Cruz Kada e Débora Plus, suscetíveis a R. solanacearum, como enxerto. O delineamento experimental foi em blocos ao acaso, com seis tratamentos (3 enxertados e 3 pés-francos), 5 repetições e 6 plantas por parcela útil. Avaliaram-se os caracteres: diâmetro do caule, altura da planta e da primeira inflorescência, distância entre inflorescências e produtividade comercial de frutos. O desenvolvimento das plantas, a produção total e a massa média dos frutos foram semelhantes para os tratamentos enxertados em relação a seus respectivos pés-francos e não houve incompatibilidade entre porta-enxerto e enxerto. A cultivar Santa Cruz Kada foi mais sensível às condições agroecológicas do município de Cruz das Almas, refletindo negativamente na produção e no peso médio dos frutos. Observou-se na enxertia com o híbrido 'Hawaii 7996' potencial para viabilizar a produção de tomateiros comerciais suscetíveis à murcha bacteriana, em áreas infestadas com R. solanacearum.

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