Control of post-harvest anthracnose papaya with yeast killer

Este trabalho objetivou isolar leveduras capazes de produzir e excretar a toxina killer a partir de frutos tropicais, para atuarem no controle biolÃgico de fitopatogenos em pÃs-colheita. Inicialmente, foram isoladas 580 leveduras a partir de 87 amostras de frutos tropicais (mamÃo, caju, sapoti, murici, manga e acerola), dentre as quais 29 exibiram o fenÃtipo killer. Todas as cepas killer foram identificadas pelo sequenciamento da regiÃo D1/D2 do 28S rRNA, em que ficou demonstrada a presenÃa de Candida aaseri, Wickerhamomyces anomalus, Pichia kluyveri, Meyerozyma guilliermondii, Kodamaea ohmeri. Cinco leveduras foram capazes de inibir em 100% a germinaÃÃo de conÃdios em meio lÃquido e reduzir o crescimento micelial, em meio sÃlido de Colletotrichum gloeosporioides in vitro, com destaque para M. guilliermondii (cepa 443) que foi capaz de reduzir o crescimento micelial do fitopatogeno em 60% em meio sÃlido. As duas leveduras com melhores resultados in vitro foram testadas in vivo, contra C. gloeosporioides, agente da antracnose em pÃs-colheita de mamÃo e outros frutos tropicais, W. anomalus (cepa 422) e M. guilliermondii (cepa 443). TambÃm foi investigada a ocorrÃncia de micoparasitismo como mecanismo de aÃÃo do antagonista por meio de microscopia eletrÃnica de varredura â MEV e detecÃÃo das enzimas hidrolÃticas, quitinase e β-1-3 glucanase. Os resultados demonstraram que, quando aplicada simultaneamente ao fitopatÃgeno e incubadas em cÃmara Ãmida (95% U.R.) a 28 ÂC, as leveduras W. anomalus (cepa 422) e M. guilliermondii (cepa 443) foram capazes de reduzir a ocorrÃncia da doenÃa em 24,62%, 7,0% e 20,68%, respectivamente, atà 06 dias apÃs a inoculaÃÃo. Verificou-se que o tempo de inoculaÃÃo da levedura teve significativa influÃncia sobre sua aÃÃo antagonista; a aplicaÃÃo dos agentes com 24 ou 12 horas de antecedÃncia em relaÃÃo ao fitopatÃgeno resultou em reduÃÃo de 30% e 13,75% para W. anomalus (cepa 422), em 40% e 35% para W. anomalus (cepa 440) e em, 41,17 e 31,35% para M. guilliermondii (cepa 443) respectivamente. A ocorrÃncia de micoparasitismo foi confirmada atravÃs de eletromicrografias que evidenciaram a uniÃo das leveduras Ãs hifas do C. gloeosporioides, provocando, em alguns casos, perda de turgidez e atà ruptura dessas; tudo isso associado à produÃÃo de β 1-3-glucanase. As leveduras killer, W. anomalus (cepa 422) e M. guilliermondii (cepa 443) que apresentaram melhores resultados nos testes in vivo foram testadas associaÃÃo com cinco diferentes veÃculos de aplicaÃÃo na proteÃÃo em pÃs-colheita de mamÃo. ApÃs 90 dias de incubaÃÃo a 4 ÂC, essas leveduras mantiveram-se viÃveis em todos os veÃculos de aplicaÃÃo testados e durante todo o perÃodo de incubaÃÃo, frutos tratados com formulaÃÃes (leveduras + veÃculos de aplicaÃÃo) apresentaram incidÃncia de doenÃa, pelo menos 30% menores, quando comparadas aos frutos nÃo tratados. Para W. anomalus (cepa 422), o tratamento que utilizou amido (2%) reduziu em 48,3% a ocorrÃncia de doenÃas, jà para M. guilliermondii, (cepa 443), os tratamentos mais eficientes no controle da doenÃa foram os que utilizaram gelatina e cera lÃquida de carnaÃba (2%) como veÃculos de aplicaÃÃo, ambos foram capazes de reduzir em 50% a ocorrÃncia de doenÃas em pÃs-colheita de mamÃes. Eletromicrograficas revelaram que todos os veÃculos de aplicaÃÃo foram eficientes em permitir a fixaÃÃo das leveduras na superfÃcie do fruto. Leveduras killer podem atuar no biocontrole em pÃs-colheita de mamÃo e estes microrganismos atuam atravÃs de uma variedade de mecanismos de aÃÃo, o que potencializa seu efeito protetor e amplia sua eficiÃncia de aÃÃo.This study aimed to isolate yeast able to produce and excrete the toxin from killer tropical fruits, to act in the biological control of plant pathogens in postharvest. A total of 580 yeasts strains, isolated from Ceara State of Brasil, were evaluated for their ability to produce killer toxin. Of these strains, 29 tested positive for the killer phenotype and were further evaluated for their ability to control Colletotrichum gloeosporioides germination in vitro. All yeast strains that expressed the killer phenotype were characterized by sequencing the D1/D2 regions of the large subunit of the rRNA gene. Five yeast strains provided a significant reduction in mycelial growth and conidial germination of C. gloeosporioides in vitro, especially Meyerozyma guilliermondii, which was able to reduce the fungal mycelial growth on solid medium (PDA) by 60% and block 100% of conidia germination in liquid media (PDB). Filtering and autoclaving the liquid cultures had no effect on the growth of the pathogen. These results indicate the potential use of antagonist yeasts isolated from tropical fruits in the control of anthracnose caused by C. gloeosporioides in papaya. Further elucidation of main mechanisms involved on anthracnose control by these yeasts could be helpful for the development of biocontrol techniques related to the management of this disease in tropical fruits. The efficiency of two killer yeast strains, with better results in vitro were tested in vivo against C. gloeosporioides, Wickerhamomyces anomalus (strain 422) and Meyerozyma guilliermondii (strain 443), as biocontrol agents against C. gloeosporioides, a postharvest anthracnose agent of papaya and other tropical fruits, was assessed. These strains were previously selected through in vitro assays, but in the present study, their in vivo action was assessed. In addition, the influence of phytopathogen inoculation time on the fruit in combination with the use of the biocontrol agent was also assessed. Through the use of scanning electron microscopy (SEM), we assessed mycoparasitism as an antagonistic mechanism of action. In addition, two hydrolytic enzymes, chitinase and β-1, 3 glucanase, were assayed. Our results indicated that W. anomalus (strain 422) and M. guilliermondii (strain 443) reduced the disease occurrence by 24.62% and 20.68%, respectively, for up to 6 days after inoculation, when applied 3 hours before the phytopathogen and incubated in a wet chamber (95% relative humidity) at 28ÂC. The time of yeast inoculation had a significant effect on its antagonistic action. Application of the yeasts 12 or 24 hours before the phytopathogen inoculation resulted in 13.75% and 30% of disease reductions for W. anomalus (strain 422) and 31.35% and 41.17% reductions for M. guilliermondii (strain 443), respectively. Electron micrographs confirmed mycoparasitism by clearly indicating the interaction of the yeasts with C. gloeosporioides hyphae, causing, in some cases, a loss of turgor and yeast penetration of walls with marked concavity formation on hypha cell walls. The efficiency of two killer yeasts, Wickerhamomyces anomalus (strain 422) and Meyerozyma guilliermondii (strain 443), which showed better results in tests in vtro and in vivo, associated with five different application vehicles was assessed for the protection of papayas postharvest. In this study, after 90 days of incubation at 4ÂC, W. anomalus (strain 422) and M. guilliermondii (strain 443) were viable with all application vehicles tested. Fruits treated with different formulations (yeasts + application vehicles) had a decreased incidence of disease (by at least 30%) compared with untreated fruits. The treatment of W. anomalus (strain 422) + 2% starch lowered disease occurrence by 48.3%. The most efficient treatments using M. guilliermondii (strain 443) were those with 2% gelatin or 2% liquid carnauba wax, both of which reduced anthracnose by 50% in postharvest papayas. Electron micrographs of the surface tissues of the treated fruits showed that all application vehicles provided excellent adhesion of yeast to the surface. The formulations based on starch (2%), gelatin (2%) and carnauba wax (2%) were the most efficient at controlling fungal diseases in postharvest papaya