Antagonistic activity of bacteria from wild honey against Colletotrichum musae, and testing of wild honey as biopesticide spray to control banana anthracnose

Anthracnose is a foliar and fruit disease caused by Colletotrichum spp. affecting a wide range of crops. Infection occurs early followed by quiescence in fruits, such as in banana, where chemical-based pesticides are used as a dependable fungal control for many years. There is an increasing need for a safe control and as implicated in the Organic Agriculture Act of 2010 (RA 10068) in the Philippines. This scenario drove the use of alternative pest control such as the use of biologicals and natural products. In this study, seven bacteria were isolated from wild honey, produced by Apis mellifera, wherein four (BC2, BC3, BC6 and BC7) were found to be an effective antagonist against Colletotrichum musae in in vitro conditions. These bacteria were identified to belong to the genus Lactobacillus spp. (BC2, BC3, BC7) and Bacillus spp. (BC6) based on sugar utilization tests, morphological and cultural growth in PDPA. For the in vivo test, different dilutions of wild honey were used and it was found out that lower concentrations were effective as biopesticide spray to prevent anthracnose infection. Lastly, we report herewith the first isolation of bacteria with biological control potential from wild honey, and to apply the raw or natural product as biopesticide in postharvest fruits.

Cgl-SLT2 is required for appressorium formation, sporulation and pathogenicity in Colletotrichum gloeosporioide

The mitogen-activated protein (MAP) kinase pathways has been implicated in the pathogenicity of various pathogenic fungi and plays important roles in regulating pathogenicity-related morphogenesis. This work describes the isolation and characterization of MAP kinase gene, Cgl-SLT2, from Colletotrichum gloeosporioides. A DNA sequence, including 1,633 bp of Cgl-SLT2 open-reading frame and its promoter and terminator regions, was isolated via DNA walking and cloned. To analyze gene function, a gene disruption cassette containing hygromycin-resistant gene was constructed, and Cgl-SLT2 was inactivated via gene deletion. Analysis on Cgl-slt2 mutant revealed a defect in vegetative growth and sporulation as compared to the wild-type strain. When grown under nutrient-limiting conditions, hyperbranched hyphal morphology was observed in the mutant. Conidia induction for germination on rubber wax-coated hard surfaces revealed no differences in the percentage of conidial germination between the wild-type and Cgl-slt2 mutant. However, the percentage of appressorium formation in the mutant was greatly reduced. Bipolar germination in the mutant was higher than in the wild-type at 8-h post-induction. A pathogenicity assay revealed that the mutant was unable to infect either wounded or unwounded mangoes. These results suggest that the Cgl-SLT2 MAP kinase is required for C. gloeosporioides conidiation, polarized growth, appressorium formation and pathogenicity.

Atividade antifúngica de extratos de Momordica charantia L. sobre Colletotrichum musae / Antifungal activity of Momordica charantia L. extracts against Colletotrichum musae

Os objetivos do presente trabalho foram avaliar os efeitos de extratos de Momordica charantia sobre o crescimento micelial e a germinação de conídios de Colletotrichum musae, e a eficiência destes extratos no controle da antracnose, causada por C. musae, em bananas. Extratos aquoso e hidroetanólico, obtidos de folhas e ramos, na concentração de 50 por cento em relação ao volume adicionado, em meio sólido, proporcionaram 71 e 65 por cento de inibição do crescimento micelial, respectivamente, enquanto que em meio líquido, a inibição do crescimento micelial foi de 86 e 81 por cento, respectivamente. Somente o extrato aquoso e o tiofanato metílico, nas concentrações de 50 por cento e 1000 µg mL-1 respectivamente, proporcionaram 100 por cento de inibição da germinação de esporos de C. musae. Os extratos metanólico e aquoso inibiram em 80 e 70 por cento, respectivamente, o desenvolvimento das lesões em bananas, quando aplicados até dois dias antes da inoculação do fungo. Estes resultados foram semelhantes ao tratamento com tiofanato metílico, que inibiu 80 por cento do desenvolvimento das lesões. Confirma-se a presença de substância antifúngica nos extratos de M. charantia e outros estudos devem ser realizados para viabilizar seu uso no controle da antracnose da banana.

The aims of the present work were to evaluate the effects of Momordica charantia extracts on mycelial growth and conidial germination of Colletotrichum musae, as well as the efficiency of these extracts in controlling anthracnose caused by C. musae in bananas. Water and hydroethanol extracts were obtained from leaves and branches at 50 percent concentration relative to the added volume. In solid medium, extracts led to 71 and 65 percent mycelial growth inhibition, respectively, whereas in liquid medium the mycelial growth was inhibited at 86 and 81 percent, respectively. Only water extract and thiophanate-methyl, at 50 percent and 1000 µg mL-1, respectively, resulted in 100 percent inhibition of C. musae spore germination. Methanol and water extracts inhibited by 80 and 70 percent, respectively, the development of lesions in bananas when applied until two days before fungal inoculation. These results were similar to those of the treatment with thiophanate-methyl, which inhibited 80 percent development of lesions. The presence of antifungal substance was confirmed in M. charantia extracts. Future studies must be performed to make its use viable for the control of anthracnose in bananas.

Ensayos de inoculación in vitro de Colletotrichum gloeosporioides aislado de hojas asintomáticas de Citrus limon / In vitro inoculation assays of Colletotrichum gloeosporioidesisolated from symptomless leaves of Citrus limon

Los hongos endofíticos pueden colonizar la parte interna de los tejidos sin causar daños aparentes al hospedador. Colletotrichum gloeosporioides, agente causal de la antracnosis, Es la especie más frecuentemente aislada de plantas sanas de Citrus limon en Tucumán. El objetivo de este trabajo fue estudiar, mediante técnicas de inoculación in vitro, la capacidad de estos aislamientos de producir infecciones sintomáticas o asintomáticas en plantines de Citrange ‘Troyer’ (Poncirus trifoliata x Citrus sinensis) y C. Limon. Se determinó el poder germinativo de conidios de loa aislamientos Nº328 y 797 obtenidos de hojas asintomáticas, y el patogénico Nº. N1. Platines en tubos de ensayo y macetas fueron inoculados con una suspensión de 105 conidios/ml y mantenidos en cámara de crecimiento a 25-28ºC. Los conidios de todas las cepas alcanzaron el máximo de germinación (100 por ciento) entre 6 y 7 horas de la siembra. Los aislamientos Nº 328 y 797 indujeron síntomas de antracnosis (clorosis de las hojas, necrosis del tallo y hojas, y defoliación) únicamente en los platines en tubos de ensayo en macetas no desarrollaron la enfermedad. El aislamiento patogénico produjo síntomas de enfermedad en los platines en tubos y macetas. La frecuencia de infección asintomática de los platines en tubo de ensayo fue del 12,5 por ciento para las cepas Nº 328 y 797. Los síntomas de antracnosis producidos por estas 2 cepas en platines en tubos y no en macetas, indicarían que la falta de preservación de condiciones naturales, favorables al hongo, no resultan las apropiadas para determinar el tipo de simbiosis que ocurre en la naturaleza. Consideramos que la utilización de mutantes endofíticas y la realización de los ensayos en plantas de limón a campo, permitirían estudiar mejor el comportamiento de estos hongos en los tejidos de la planta.