RESUMO Em 2016 em tomateiro tutorado para consumo in natura, foram observadas plantas com sintomas graves de necrose da medula e morte prematura da planta, em áreas de cultivo comercial nos estados do Paraná e Minas Gerais, Brazil, similares aos causados por Pseudomonas corrugata. Quatro cepas bacterianas foram isoladas a partir de amostras de tecido infectado e caracterizadas como Gram negativas, crescimento aeróbico e fluorescimento em meio de cultura King B. Testes LOPAT foram realizados e o isolado UFUH120 foi classificado no grupo Vb (+ + - + -) e isolados UFU H3, UFU H6 e UFU H21 no grupo Va (- + - + -). A patogenicidade dos isolados foi confirmada pela inoculação artificial em tomate Santa Cruz cv. Kada Gigante e os postulados de Koch realizados com sucesso. O sequenciamento da região do gene 16S rRNA dos isolados foi realizado e as sequências obtidas comparadas com as depositadas no GenBank, o isolado UFU H120 foi alinhado com Pseudomonas fluorescens (99.45% de similaridade) e os isolados UFU H3, UFU H6 e UFU H21 com Pseudomonas putida (similaridades de 99,2, 99,53 e 99,64%, respectivamente). Pseudomonas fluorescens e P. putida são mais conhecidas como bactérias saprofíticas presentes no solo, embora as duas espécies tenham sido relatadas na Itália, causando doença no tomateiro. No entanto, até o momento não há relatos que essas bactérias sejam fitopatogênicas no tomate ou em qualquer cultura no Brasil. Sendo este, por parte do conhecimento dos autores, o primeiro relato da ocorrência dessas espécies causando necrose da medula em lavouras de tomateiro no país.

ABSTRACT In 2016, several reports emerged of fresh-market staked tomato plants with severe symptoms of pith necrosis and premature death in commercial cultivation areas in the states of Paraná and Minas Gerais, Brazil, which are similar to those caused by Pseudomonas corrugata. Four bacterial strains were isolated from infected tissue samples and characterized as Gram-negative, aerobic, and fluorescent on King’s B Medium. LOPAT tests were performed and the isolate UFU H120 was classified into group Vb (+ + − + −) and the isolates UFU H3, UFU H6, and UFU H21 into group Va (− + − + −). The pathogenicity of the isolates was confirmed by artificial inoculation on tomato plants of Santa Cruz type, cv. Kada Gigante, and Koch’s postulates were accomplished successfully. The isolates sequences of their 16S rRNA gene region were compared with those deposited in GenBank, the isolate UFU H120 aligning with Pseudomonas fluorescens (99,45% similarity) and the isolates UFU H3, UFU H6, and UFU H21 with Pseudomonas putida (99.2, 99.53 and 99.64% similarity, respectively). Notably, P. fluorescens and P. putida are most known as saprophytic bacteria normally present in the soil, although both of these species have already been reported to infect tomato plants in Italy. However, so far, there have not been any reports of such bacteria being phytopathogenic on tomato or any crop in Brazil. Thus, to our knowledge, this is the first report of pith necrosis of such occurrence in Brazil.

Melon and watermelon bacterial fruit blotch, incited by Acidovorax citrulli, is limited to some areas in Brazil but causes important losses, mainly in melon-producing regions. Although genetic diversity has been observed among strains belonging to the species, they are considered a homogeneous group based on the fact that they show only slight physiological or nutritional differences. The objective of this study was to compare Brazilian strains from melon and watermelon by means of biochemical, pathogenicity, serological and molecular assays. Fifteen biochemical tests, cross inoculation between strains and hosts, ELISA and repetitive sequence analysis (rep-PCR) with the primers REP, ERIC and BOX were conducted. No differences were revealed by nutritional characterization or serology, but cross inoculation showed different pathogenicity groups, which could explain high aggressiveness of the bacteria to melon crops in some regions. Molecular analysis by BOX-PCR clustered strains according to their geographical origin, while ERIC- and REP-PCR, analyzed together, indicated genetic diversity, but without geographical or host origin relationships. One test that could be used to verify the pathogenicity of strains by inoculating detached leaf petioles, showing results in 36 h, is proposed here.

A combinação do uso do citômetro de fluxo (FCM) e de anticorpo policlonal (imuno-FCM) foi comparada à microscopia de imunofluorescência (IF) e ao plaqueamento em meio de cultura semi-seletivo, para a detecção de Xanthomonas axonopodis pv. phaseoli (Xap) em sementes de feijão. Concentrações de Xap variando de 10³ a 10(7) CFU/mL foram adicionados aos extratos de sementes. Um método de separação pelo citômetro de fluxo foi desenvolvido para a detecção de Xap em extratos de semente e posterior confirmação por PCR. Para avaliação da viabilidade das células foram usadas sondas fluorescentes, iodeto de propídio (PI)/carboxi diacetato de fluoresceína (cFDA) e PI/SYTO 9 e também, a combinação de imuno-FCM e PI. Em meio semi-seletivo e IF foram detectadas 10³-10(6) UFC/mL e no FCM 10(4)-10(6) UFC/mL, em extratos de sementes artificialmente infestados. Xap somente foi detectada em extratos de sementes por PCR, após o processo de separação pelo FCM. Foi possível pelo FCM a quantificação e identificação de células viáveis (verde fluorescente) e células mortas (vermelho fluorescente) de Xap, pelas sondas cFDA/SYTO 9 e PI, respectivamente. A combinação de immuno-FCM e PI poderá ser uma técnica promissora e segura para a detecção deste patógeno em sementes.

Flow cytometric analysis of immuno-stained cells (immuno-FCM) was compared to immunofluorescence microscopy (IF) and dilution plating on a semi-selective medium, for quantitative detection of Xanthomonas axonopodis pv. phaseoli (Xap) in bean seed extracts. Cell concentrations of Xap between 10³-10(7) CFU/mL were added to healthy bean seed extracts. A flow cytometry sorting procedure was developed to separate immuno-stained Xap cells from crude seed extracts and confirming by PCR. FCM was evaluated for direct viable counting (DVC) of Xap using combinations of propidium iodide (PI) and carboxy fluorescein diacetate (cFDA) or PI and SYTO 9 and also the combination of immuno-FCM and PI. Dilution plating and IF allowed detection of Xap in bean seed extracts in a range of 10³-10(6) CFU/mL and immuno-FCM from 10(4)-10(6) CFU/mL. Sorted cells could be detected in crude seed extracts by PCR without further extraction. FCM also allowed quantification of viable cells of Xap after DVC procedures; the red fluorescent dye propidium iodide was used to identify dead cells in combination with the green fluorescent dyes cFDA or SYTO 9, these identifying live cells. The combination of immuno-FCM and PI could be more promising and reliable to detect this pathogen in seeds.

Para a detecção de Xanthomonas axonopodis pv. phaseoli (Xap) em extratos de sementes inteiras e moídas de feijão, naturalmente contaminadas e sadias foram utilizados os meio de cultura semi-seletivos XCP1 e MT e o não seletivo 523 de Kado & Hesket. Os extratos de sementes foram também inoculados em folhas primárias de feijoeiro. O meio de cultura semi-seletivo XCP1 foi o mais eficiente na quantificação e detecção de Xap em extratos de sementes inteiras de feijão. Os extratos de sementes contaminadas, inoculados nas folhas primárias de feijoeiro produziram sintomas do crestamento bacteriano comum.

To detect Xanthomonas axonopodis pv. phaseoli (Xap) in whole and crushed bean seed extracts, naturally contaminated and healthy bean seeds were used in the semi-selective media XCP1 and MT and in the non-selective medium 523. Seed extracts were also inoculated in the first leaves of bean plants. The semi-selective medium XCP1 was more efficient in the quantification and detection of Xap in whole bean seed extracts. Naturally contaminated seed extracts, inoculated in the first leaves of bean plants, showed the symptoms of bacterial blight.