Prospecção de bactérias no controle biológico de nematoide-das-galhas (Meloidogyne incognita) e promoção de crescimento vegetal
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Data
2022-02-24
Autores
Amorim, Deucleiton Jardim
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Universidade Estadual Paulista (Unesp)
Resumo
Os nematoides-das-galhas, gênero Meloidogyne, causam prejuízos à agricultura, com perdas estimadas em mais de 100 bilhões de dólares em todo mundo. O manejo desses nematoides é um complicador para muitas culturas agrícolas. Assim, ao longo do tempo, muitos mecanismos foram adotados, a fim de minimizar os danos desses fitopatógenos, entre eles o controle biológico, além de outros. O objetivo deste trabalho foi selecionar bactérias, para possível controle biológico de Meloidogyne incognita e promoção de crescimento vegetal. Plantas de tomateiro Santa Cruz ‘Kada Gigante’ foram inoculadas com esse nematoide a partir de uma população pura. Após 60 dias, raízes dessas plantas foram processadas para obtenção de juvenis de segundo estádio (J2s). Cento e um isolados bacterianos foram inoculados em meio NB (Nutrient Broth), incubados a 180 rpm por 72 h, até chegarem, aproximadamente, 108 unidades formadoras de colônias (UFC). Os testes in vitro foram realizados em três etapas, a primeira para seleção de isolados com mortalidade igual ou a 50%. Na segunda etapa foi utilizado nove isolados com de 10% de concentração da suspensão bacteriana, com avaliações às 24, 48 e 72 h, após a inoculação), inoculando 100 J2 mL-1. Cinco isolados foram selecionados para uma terceira etapa, com sete concentrações (0; 2; 4; 7; 10; 15; 20%), adicionando-as em tubos de ensaio e depois, uma suspensão de 500 J2 mL-1, após homogeneização, foram inoculados em placas de Petri de 6 mm de diâmetro sobre 12 g de areia sílica esterilizada, com avaliação realizada às 92 h, após a inoculação. Todas as triagens seguiram um delineamento experimental inteiramente casualizado, com quatro repetições em duplicata, tendo como testemunhas, água destilada e meio NB. As etapas in vivo foram divididas em duas, a primeira para avaliação do controle do nematoide, após 60 dias da inoculação de 1.000 J2 mL-1. Os tratamentos foram compostos de 5 e 15% da concentração da suspensão bacteriana dos seguintes isolados: 195b, MA07, 287a, 321b e 311a, tendo como testemunha água e meio NB. O segundo experimento foi composto pelos mesmos tratamentos, nas mesmas concentrações, para avaliação de promoção de crescimento vegetal, sem nematoides. As avaliações ocorreram após 60 dias da inoculação, sendo as variáveis: nematoides por grama de raiz, altura da parte aérea, massa fresca e massa seca da parte aérea, crescimento, massa fresca e massa seca da raiz, aferidas. Os experimentos foram repetidos duas vezes, com quatro repetições cada tratamento. O “screening” de toda a diversidade de bactérias finalizou com 14% do total com mortalidade igual ou a 50% (20 isolados). Na segunda etapa cinco isolados foram eficientes, em uma concentração de 10% da suspensão bacteriana. Esses resultaram com mortalidade acima de 80%, já na terceira etapa, os cincos isolados nas diferentes concentrações atingiram mortalidade próxima de 100%. O isolado 287a foi o mais promissor no experimento de controle do nematoide, este demostrou ser um agente bacteriano potencial, para o controle de juvenis, tendo uma baixa concentração de ovos + J2, quando comparado com as testemunhas não tratadas com bactérias. Os isolados testados promoveram crescimento vegetal, pois as plantas inoculadas com bactérias cresceram mais que àquelas não tratadas. Esses resultados são promissores, pois indicam que foi possível selecionar isolados potenciais para o controle biológico de Meloidogyne incognita sob triagens in vitro e in vivo. Outros estudos em condições de campo necessitam ser realizados.
Root-knot nematodes, Meloidogyne, cause damage to agriculture, with losses estimated at more than 100 billion dollars worldwide. The management of this nematode is a complicating factor for many agricultural crops. Thus, over time, many mechanisms were adopted in order to minimize the damage of these phytopathogens, including biological control, among others. The objective of this research is to select bacteria for possible biological control of Meloidogyne incognita and plant growth-promoting. Tomato plants Santa Cruz 'Kada Gigante' were inoculated with this nematode from a pure population and after 60 days, roots of these plants were processed to obtain second-stage juveniles (J2s). Hundred of one bacterial isolates were inoculated in NB medium (Nutrient Broth), incubated at 180 rpm for 72 h, with approximately 108 colony-forming units (CFU). In vitro tests were performed in three stages, the first for selecting isolates with mortality equal to or 50%. In the second stage, nine isolates with a 10% concentration of bacterial suspension were used, with evaluations at 24, 48, and 72 after inoculation), inoculating 100 J2 mL-1. Five isolates were selected for a third stage, with seven concentrations (0; 2; 4; 7; 10; 15; 20%, evaluated at 92 h after inoculation), inoculating 500 J2 mL-1 in Petri dishes 6 mm in diameter on 12 g of sterile silica sand. All “screenings” followed a completely randomized experimental design, with four replicates in duplicate, with distilled water and NB medium as controls. The in vivo steps were divided into two, the first for evaluating the control of the nematode, 60 days after inoculation of 1,000 J2 mL-1. The treatments were composed of 5 and 15% of the bacterial suspension concentration of the following isolates: 195b, MA07, 287a, 321b, and 311a, with water and NB medium as a control. The second experiment consisted of the same treatments, at the same concentrations, to evaluate plant growth promotion without nematodes. The evaluations took place 60 days after inoculation, and the variables were: nematodes per gram of root, the height of shoot, fresh mass and dry mass of shoot, growth, fresh mass and dry mass of the root, measured. The experiments were repeated twice, with four repetitions for each treatment. The “screening” of all bacterial diversity ended with 14% of the total with mortality equal to or 50% (20 isolates). In the second step, five isolates were efficient, at a concentration of 10% of the bacterial suspension. These resulted in mortality above 80%, in the third stage, the five isolates at different concentrations reached mortality close to 100%. Isolate 287a was the most promising in the nematode control experiment, it proved to be a potential bacterial agent for the control of juveniles, having a low concentration of eggs + J2 when compared to controls not treated with bacteria. The tested isolates promoted plant growth, as plants inoculated with bacteria grew more than untreated controls. These results are promising, as they indicate that it was possible to select potential isolates for the biological control of Meloidogyne incognita under in vitro and in vivo “screenings”. Biochemical tests, metabolite stability, molecular identification, and experiments under field conditions with other agricultural crops need to be carried out.
Root-knot nematodes, Meloidogyne, cause damage to agriculture, with losses estimated at more than 100 billion dollars worldwide. The management of this nematode is a complicating factor for many agricultural crops. Thus, over time, many mechanisms were adopted in order to minimize the damage of these phytopathogens, including biological control, among others. The objective of this research is to select bacteria for possible biological control of Meloidogyne incognita and plant growth-promoting. Tomato plants Santa Cruz 'Kada Gigante' were inoculated with this nematode from a pure population and after 60 days, roots of these plants were processed to obtain second-stage juveniles (J2s). Hundred of one bacterial isolates were inoculated in NB medium (Nutrient Broth), incubated at 180 rpm for 72 h, with approximately 108 colony-forming units (CFU). In vitro tests were performed in three stages, the first for selecting isolates with mortality equal to or 50%. In the second stage, nine isolates with a 10% concentration of bacterial suspension were used, with evaluations at 24, 48, and 72 after inoculation), inoculating 100 J2 mL-1. Five isolates were selected for a third stage, with seven concentrations (0; 2; 4; 7; 10; 15; 20%, evaluated at 92 h after inoculation), inoculating 500 J2 mL-1 in Petri dishes 6 mm in diameter on 12 g of sterile silica sand. All “screenings” followed a completely randomized experimental design, with four replicates in duplicate, with distilled water and NB medium as controls. The in vivo steps were divided into two, the first for evaluating the control of the nematode, 60 days after inoculation of 1,000 J2 mL-1. The treatments were composed of 5 and 15% of the bacterial suspension concentration of the following isolates: 195b, MA07, 287a, 321b, and 311a, with water and NB medium as a control. The second experiment consisted of the same treatments, at the same concentrations, to evaluate plant growth promotion without nematodes. The evaluations took place 60 days after inoculation, and the variables were: nematodes per gram of root, the height of shoot, fresh mass and dry mass of shoot, growth, fresh mass and dry mass of the root, measured. The experiments were repeated twice, with four repetitions for each treatment. The “screening” of all bacterial diversity ended with 14% of the total with mortality equal to or 50% (20 isolates). In the second step, five isolates were efficient, at a concentration of 10% of the bacterial suspension. These resulted in mortality above 80%, in the third stage, the five isolates at different concentrations reached mortality close to 100%. Isolate 287a was the most promising in the nematode control experiment, it proved to be a potential bacterial agent for the control of juveniles, having a low concentration of eggs + J2 when compared to controls not treated with bacteria. The tested isolates promoted plant growth, as plants inoculated with bacteria grew more than untreated controls. These results are promising, as they indicate that it was possible to select potential isolates for the biological control of Meloidogyne incognita under in vitro and in vivo “screenings”. Biochemical tests, metabolite stability, molecular identification, and experiments under field conditions with other agricultural crops need to be carried out.
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Palavras-chave
Bacillus, Controle biológico, Meloidogyne, Pseudomonas, Biological control