Perfil genético e fenotípico de linhagens Xanthomonas citri isoladas de pomares com diferentes incidências de cancro cítrico
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Data
2022-01-19
Autores
Alleoni, Natália
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Universidade Estadual Paulista (Unesp)
Resumo
O Brasil é o maior produtor de laranjas doce do mundo, e a citricultura constitui uma das atividades agrícolas mais importantes para nosso país. A citricultura é ameaçada pelo cancro cítrico, uma doença infecciosa e sem cura, que acomete todas as variedades de citros de importância comercial. O agente etiológico desta doença é a bactéria Gram-negativa Xanthomonas citri subsp. citri (X. citri), que se encontra presente e em caráter endêmico no estado de São Paulo, o maior produtor de laranjas doce do mundo. Este estado foi declarado área de mitigação de risco (SMR), com o uso de sprays regulares de formulações a base de cobre. Tais medidas, embora eficazes, são paliativas, pois a resistência ao cobre já foi documentada em X. citri, além do fato de ser este um metal bio-cumulativo e tóxico. Assim o objetivo foi verificar variações fenotípicas e/ou genotípicas em isolados de X. citri de áreas diferentes. Foram obtidos 390 isolados de Xanthomonas citri subsp. citri e foram testadas 89 para susceptibilidade a cobre. Os isolados foram classificados em: 79 como sensíveis (CuS), 8 tolerantes (CuT) e 1 como mais tolerantes (CuT+); não foram obtidos isolados resistentes ao cobre. Com exceção dos controles, nenhuma das linhagens testadas apresentaram amplificação para os genes copLAB e copABCD, indicando ausência dos plasmídeos de resistência. Ainda, as linhagens foram testadas quanto a sua susceptibilidade aos antibióticos comerciais: ácido nalidíxico, canamicina, rifampicina e cloranfenicol. Estas não apresentaram crescimento nas concentrações de canamicina e rifampicina capazes de inibir a linhagem CuS X. citri 306 - usada como controle neste teste - porém, mostraram-se resistentes a clorafenicol crescendo em todas as concentrações testadas. O ácido nalidíxico foi capaz de inibir o crescimento de todas as linhagens, com execeção a CuT+ CAJ 45 que apresentou crescimento na concentração mais baixa (7,5 μg/mL). Das linhagens testadas, apenas 3 (CAJ 28, CAJ 73 e AM 52) apresentaram alterações na motilidade, crescendo similarmente ao controle CuS X. citri 306, já os demais isolados apresentaram crescimento semelhante aos controles CuR, indicando que uma maior tolerância não altera a motilidade. No teste de biofilme apenas o isolado CuT+ CAJ 45 apresentou uma redução significativa na formação de biofilme, contrariando o esperado que uma linhagem mais tolerante produz mais biofilme.
Brazil is the largest sweet orange producer in the world, and citriculture represents an important economic activity in the country. The sector is threatened by citrus canker, an infectious and incurable disease that affects all commercially important citrus varieties. The causing agent of this disease is the Gram-negative bacteria Xanthomonas citri subsp. citri (X. citri), which is present and endemic in the state of São Paulo. São Paulo was declared an area of risk mitigation (SMR), with the use of regular sprays of copper formulations. Such measures, although effective, seem palliative, as copper resistance has already been documented in X. citri, in addition to the fact that this is a bio-accumulative and toxic metal. Thus, the objective was to verify phenotypic and/or genotypic variations in X. citri isolates from different areas. 390 isolates of Xanthomonas citri subsp. citri were obtained and 89 were tested for their copper susceptibility. The isolates were then classified accordingly, as: 79 as sensitive (CuS), 8 tolerant (CuT) and 1 as more tolerant (CuT+), no isolates resistant to copper were obtained. With the exception of the positive controls, none of the isolates tested amplified bands for the genes copLAB and copABCD, remarking the absence of resistance plasmids. Furthermore, the isolates were tested for their susceptibility to commercial antibiotics: nalidixic acid, kanamycin, rifampicin and chloramphenicol. The isolates did not grow under concentrations of kanamycin and rifampicin capable of inhibiting the CuS X. citri 306 strain - used as a control in this test - however, they were resistant to chloramphenicol, growing on all tested concentrations. Nalidixic acid was able to inhibit the growth of all isolates, with the exception of CuT+ CAJ 45, which showed growth at the lowest concentration (7.5 μg/mL). Of the strains tested, only 3 (CAJ 28, CAJ 73 and AM 52) showed changes in motility, growing similarly to the CuS X. citri 306 control, while the other isolates showed similar growth to the CuR controls, indicating that a greater tolerance does not change motility. In the biofilm test, the isolate CuT+ CAJ 45 showed a significant reduction in biofilm formation, contrary to the expected that a more tolerant strain produces more biofilm.
Brazil is the largest sweet orange producer in the world, and citriculture represents an important economic activity in the country. The sector is threatened by citrus canker, an infectious and incurable disease that affects all commercially important citrus varieties. The causing agent of this disease is the Gram-negative bacteria Xanthomonas citri subsp. citri (X. citri), which is present and endemic in the state of São Paulo. São Paulo was declared an area of risk mitigation (SMR), with the use of regular sprays of copper formulations. Such measures, although effective, seem palliative, as copper resistance has already been documented in X. citri, in addition to the fact that this is a bio-accumulative and toxic metal. Thus, the objective was to verify phenotypic and/or genotypic variations in X. citri isolates from different areas. 390 isolates of Xanthomonas citri subsp. citri were obtained and 89 were tested for their copper susceptibility. The isolates were then classified accordingly, as: 79 as sensitive (CuS), 8 tolerant (CuT) and 1 as more tolerant (CuT+), no isolates resistant to copper were obtained. With the exception of the positive controls, none of the isolates tested amplified bands for the genes copLAB and copABCD, remarking the absence of resistance plasmids. Furthermore, the isolates were tested for their susceptibility to commercial antibiotics: nalidixic acid, kanamycin, rifampicin and chloramphenicol. The isolates did not grow under concentrations of kanamycin and rifampicin capable of inhibiting the CuS X. citri 306 strain - used as a control in this test - however, they were resistant to chloramphenicol, growing on all tested concentrations. Nalidixic acid was able to inhibit the growth of all isolates, with the exception of CuT+ CAJ 45, which showed growth at the lowest concentration (7.5 μg/mL). Of the strains tested, only 3 (CAJ 28, CAJ 73 and AM 52) showed changes in motility, growing similarly to the CuS X. citri 306 control, while the other isolates showed similar growth to the CuR controls, indicating that a greater tolerance does not change motility. In the biofilm test, the isolate CuT+ CAJ 45 showed a significant reduction in biofilm formation, contrary to the expected that a more tolerant strain produces more biofilm.
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Microorganismos fitopatogênicos, Biologia molecular, Cobre, Phytopathogenic microorganisms, Molecular biology, Copper