Establishment of the entomopathogenic fungus Beauveria bassiana as an endophyte in Capsicum annuum and its effects on the aphid pest Myzus persicae (Homoptera: Aphididae) / Establecimiento del hongo entomopatógeno Beauveria bassiana como endófito en Capsicum annuum y sus efectos sobre la plaga del áfido Myzus persicae (Homoptera: Aphididae)

Introduction: Entomopathogenic fungi have been used as biological control agents of insect pests and have demonstrated a promising ability to colonize as endophytes a wide range of plant species. Objective: The aims of this study were to evaluate, in pepper plants, the most effective Beauveria bassiana inoculation techniques. In order to evaluate the presence of natural endophytes and its interaction with B. bassiana. Also to assess the effect of B. bassiana on the aphid Myzus persicae pepper leaf feeding preference and growth. Methods: Three inoculation techniques were evaluated: leaf spraying, seed immersion and root dipping. The pepper plants were inoculated with a conidia suspension adjusted to 1×108 conidia/ml. The presence of B. bassiana in the seedlings was evaluated at 7, 15 and 28 days after inoculation. Results: We found significant statistical differences between inoculation techniques. Foliar spraying showed the highest B. bassiana colonizationfrequency. The natural endophytes presented higher presence in the treatments in which B. bassiana had a low presence. This might indicate the occurrence of antifungal activity that interferes with the establishment of the entomopathogen in pepper tissues. We did not find statistical differences between inoculated and control pepper leaves with regard to the mortality and reproduction of the aphid pest M. persicae. However, the inoculation with B. bassiana showed a tendency to increase the mortality of adults and to decrease the offspring (nymphs). We did not find significant differences in the feeding preference between inoculated and control leaves, although there was a higher number of individuals on control leaves compared to those inoculated. Conclusions: The strain B. bassiana LPSC 1067 was established effectively as an endophyte in the pepper tissues. The most effective inoculation technique was leaf spraying. We did not find differences of statistical significance with regard to the mortality and reproduction of the aphid pest M. persicae. However, the inoculation with B. bassiana showed a tendency to increase the mortality of adults and to decrease the offspring (nymphs). Future studies evaluating the indirect effects of endophytic entomopathogens on both plants and herbivorous insects are needed to further explore their potential use in the management of insect pests.

Introducción: Los hongos entomopatógenos se han utilizado como agentes de control biológico de plagas de insectos y han demostrado una capacidad prometedora para colonizar una amplia gama de especies de plantas como endófitos. Objetivo: Los objetivos de este estudio fueron: evaluar las técnicas de inoculación más efectivas para B. bassiana en plantas de pimiento; evaluar la presencia de endófitos naturales y su interacción con B. bassiana y evaluar el efecto de B. bassiana sobre la preferencia de alimentación de la hoja de pimiento y el crecimiento de la plaga de áfidos M. persicae. Métodos: Se evaluaron tres técnicas de inoculación: aspersión foliar, inmersión de semillas e inmersión de raíces. Las plantas de pimiento se inocularon con una suspensión de conidias ajustada a 1x108 conidias/ml. La presencia de B. bassiana en las plántulas se evaluó a los 7, 15 y 28 días después de la inoculación. Resultados: Se encontraron diferencias significativas entre las técnicas de inoculación. La aspersión foliar mostró la frecuencia más alta. Los endófitos naturales presentaron frecuencias más altas en los tratamientos en los que B. bassiana tuvo una frecuencia baja, esto podría indicar la presencia de actividad antifúngica que interfiere con el establecimiento del entomopatógeno en los tejidos de pimiento. No se encontraron diferencias significativas en la mortalidad, reproducción y preferencia alimentaria de M. persicae. Conclusiones: Se demostró que la cepa B. bassiana LPSC 1067 se estableció efectivamente como endófito en los tejidos de pimiento. La técnica de inoculación más efectiva fue la aspersión foliar. No se encontraron diferencias significativas con respecto a la mortalidad, reproducción y preferencia alimentaria de M. persicae. Sin embargo, la inoculación con B. bassiana mostró una tendencia a aumentar la mortalidad de los adultos y a disminuir la descendencia (ninfas). Se necesitan estudios futuros que evalúen los efectos indirectos de los entomopatógenos endofíticos tanto en plantas como en insectos herbívoros para explorar más a fondo su posible uso en el manejo de insectos plaga.

Effect of surfactants and temperature on germination and vegetative growth of Beauveria bassiana

Three non-ionic surfactants: Tween20, Tween80 and Breakthru® were screened for their effects on spore germination and mycelial growth rates and for their influence on three isolates of Beauveria bassiana spore germination at various temperatures. Tween20 and Tween80 were compatible with all the B. bassiana isolates in the germination studies, but inhibited germination at higher surfactant concentrations, irrespective of the conidial concentrations. Breakthru® had an inhibitory effect on germination even at the lowest concentration of 0.1% on all the B. bassiana isolates. The effects of the surfactants on spore germination did not correspond with their effects on colony growth. Conidial viability within the same formulation declined significantly with increases in temperature, irrespective of the surfactant. The optimal temperature for conidial germination of B. bassiana isolates was approximately 25 °C with an upper limit at 30 °C. Isolate 7320 was identified as the least affected by the different surfactants. This isolate was able to germinate rapidly in a broad temperature range of 25–30 °C after 24 h, this characteristic being an essential factor in controlling house fly populations in poultry houses.

Lethal effects of a Mexican Beauveria bassiana (Balsamo) strain against Meccus pallidipennis (Stal)

The entomopathogenic fungus Beauveria bassiana (Balsamo 1835) Vuillemin is an effective alternative control agent against some agricultural pests and biological vectors of important diseases such as Chagas disease. In this work we studied an isolate of Beauveria bassiana from of the town of San Antonio Rayón, Puebla, Mexico and its entomopathogenic effects on Meccus pallidipennis (Stal 1872). Phylogenetic analysis using molecular comparison of the ITS and EF1α genes, showed that the resulting cladogram places the BUAP 04 strain with a relationship closer to the AFAO 9-6 strain, within the diversity of the B. bassiana sensu lato group. Although there was the possibility that BUAP 04 strain was a direct descendant of strains used in campaigns of biologic control, molecular study allowed us to recognize that it was a different fungus due to numerous inserts. A strain isolated from a T. dimiata was evaluated for pathogenicity against another triatoma (Meccus pallidipennis) species obtaining an LC50 of 4.16 x 10(6) spores/mL, confirming that the BUAP 04 strain is virulent for M. pallidipennis and could be a good prospect for formulations to control M. pallidipennis.

Estudio histopatológico y ultraestructural de la infección de Beauveria bassiana (Ascomycota) sobre Rhodnius prolixus (Triatominae) / Histopathological and ultraestructural study of Beauveria bassiana (Ascomycota) infection on Rhodnius prolixus (Triatominae)

Se realizó un estudio para indagar aspectos histopatológicos y ultraestructurales de la interacción entre un aislamiento nativo (LF14) del hongo entomopatógeno Beauveria bassiana (Ascomycota, Hypocreales) y ninfas de quinto estadio y adultos de Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae). En los primeros estadios de la infección se observaron las conidias adheridas al exoesqueleto y en el momento de su penetración del tubo germinal. Después de la penetración vía tegumento y traqueolas del hemocele, las hifas invadieron virtualmente todos los tejidos y órganos, con el subsiguiente fallecimiento del insecto. En el último estadio del proceso infeccioso, se detectó la infección masiva del hifomiceto penetrando todo el cuerpo de adentro hacia aberturas del tegumento, para finalmente esporular, observándose numerosos conidios desprendidos de los conidióforos. La Microscopía Electrónica de Transmisión (MET) reveló una invasión masiva de hifas y micelios fúngicos en todo el hemocele, con hemocitos fagocitando, encapsulando y melanizando al hongo. Durante la penetración tisular y tegumentaria de las hifas, se observan áreas electrotransparentes que sugieren actividad enzimática.

A histopathological and ultrastructural study was used to describe the mode of interaction between a native isolate (LF14) of entomopathogenic fungus Beauveria bassiana (Ascomycota, Hypocreales) and nymphs/adults of Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae). Conidia germinate on the surface of triatomine bugs and immediately begin penetration of the cuticle and spiracles by germinal tube. Once invade the haemocele, the fungus multiplies extensively whole insect tissues and organs, leading to triatomine death. When insect cadaver is in mummification stage, outgrowths of fungal hyphae occur first and most extensively in the intersegmental regions of triatomines, emerging at the surface, where they initiate sporulation process. Electron Transmission Microscopy (ETM) studies revealed abundant growth of fungal hypahe and micelar into haemocele, with haemocytic infiltration during fagocitation, encapsulation and melanization process of fungal structures. Areas of low electron-density were frequently observed, suggesting the secretion of enzymes by hyphae to weaken and penetrating cuticle and tissues.

Produção semissólida de Metarhizium anisopliae e Beauveria bassiana em diferentes substratos e efeito da radiação ultravioleta e da temperatura sobre propágulos desses entomopatógenos / Semi-solid production of Metarhizium anisopliae (Metsch) Sorok and Beauveria bassiana (Bals) Vuill on different media and the effects of ultraviolet radiation and temperature on the infective propagules of these fungi

O trabalho teve os seguintes objetivos: 1) avaliar diferentes substratos para a produção semissólida dos fungos Metarhizium anisopliae e Beauveria bassiana; 2) verificar a tolerância dos conídios produzidos ao efeito da radiação ultravioleta e da temperatura; e 3) avaliar a patogenicidade dos conídios a Diatraea saccharalis. Para ambos os fungos, foram utilizadas 6 repetições para cada tratamento: amido de milho, arroz integral, arroz parboilizado tipo1, arroz tipo 1 e 2, aveia em flocos, canjiquinha, farelo de trigo, farinhas de mandioca crua, de milho amarela e de trigo especial, fubá, milho em grãos, polvilho azedo, soja em grãos, trigo moído e turfa. Os conídios foram quantificados em câmara de Neubauer e a determinação da viabilidade foi realizada através da observação em microscópio, dos conídios germinados e não germinados, após espalhamento da suspensão fúngica em placas de Petri contendo BDA. No ensaio com radiação os fungos foram expostos à radiação por 25 e 50 segundos e foi considerado um tratamento sem exposição; para temperatura os fungos foram expostos a 20, 25, 30 e 35o C. Utilizando a torre de Potter, 2 mL de cada suspensão fúngica dos tratamentos foram pulverizados em lagartas de D. saccharalis. As maiores concentrações de conídios de M. anisopliae e B. bassiana foram encontradas nos tratamentos com arroz parboilizado tipo 1, arroz tipo 1 e 2, farinha de milho amarela, fubá e trigo moído e a viabilidade dos conídios produzidos superou os 94%. Quanto maior o tempo de exposição à radiação menor foi o número de conídios viáveis; também ocorreu perda significativa da viabilidade dos conídios quando expostos à temperatura de 35o C. Os fungos foram patogênicos para D. saccharalis.

The present study was aimed to evaluate different (semi-solid) media for the production of Metarhizium anisopliae and Beauveria bassiana propagules, and to evaluate the tolerance of these propagules to ultraviolet radiation and temperature. The experiments were performed at the Biological Control Laboratory of the Instituto Biológico at Campinas, São Paulo, Brazil. For both fungi, 6 repetitions were performed for each of the 17 treatments: corn starch, full rice, parboiled rice, type-1 rice, type-2 rice, oat flakes, canjiquinha [grits], wheat flour, raw cassava flour, yellow corn flour, special wheat flour, corn flour, corn in grains, cassava starch, soy in grains, crushed wheat, and turf. The viability analysis was done in plastic plates containing BDA. For the bioassays involving exposure to ultraviolet light and temperature, BDA was also used for viability analysis, and each treatment was exposed to the UV radiation for 0, 25 and 50 seconds, the temperature exposure being at 20, 25, 30 and 35º C. Using a Potter tower, 2 mL of fungus suspension from each treatment was inoculated to the Diatraea saccharalis caterpillars. Regarding the sporulation, the largest concentrations of M. anisopliae and B. bassiana were found for the treatments with parboiled rice, type-1 rice, type-2 rice, yellow corn flour, corn flour and crushed wheat. The viability of all treatments was superior to 94.00%. Also, the longer the duration of the exposition to the UV, the 2Universidade Estadual Paulista, Faculdade de Ciências Agronômicas, Botucatu, SP, Brasil.

Cuticle-degrading proteases produced by the entomopathogenic fungus Beauveria bassiana in the presence ofcoffee berry borer cuticle / Proteases degradadoras de cutícula produzidas pelo fungo entomopatogênico Beauveria bassiana em presença de cutícula de broca do café

A Brazilian isolate of Beauveria bassiana (CG425) that shows high virulence against the coffee berry borer (CBB) was examined for the production of subtilisin-like (Pr1) and trypsin-like (Pr2) cuticle-degrading proteases. Fungal growth was either in nitrate-medium or in CBB cuticle-containing medium under both buffered and unbuffered conditions. In unbuffered medium supplemented with cuticle, the pH of cultures dropped and Pr1 and Pr2 activities were detected in high amounts only at a pH of 5.5 or higher. In buffered cultures, Pr1 and Pr2 activities were higher in medium supplemented with cuticle compared to activities with nitrate-medium. The Pr1 and Pr2 activities detected were mostly in the culture supernatant. These data suggest that Pr1 and Pr2 proteases produced by strain CG425 are induced by components of CBB cuticle, and that the culture pH influences the expression of these proteases, indicating the occurrence of an efficient mechanism of protein secretion in this fungus. The results obtained in this study extend the knowledge about protease production in B. bassiana CG425, opening new avenues for studying the role of secreted proteases in virulence against the coffee berry borer during the infection process.

O isolado brasileiro de Beauveria bassiana (CG425) que apresenta alta virulência contra a broca do café (CBB) foi analisado quanto à produção de proteases degradadoras de cutícula, tipo-subtilisina (Pr1) e tipo-tripsina (Pr2). O crescimento fúngico foi realizado em meio contendo nitrato e em meio contendo cutícula da broca em condições de pH tamponado e não tamponado. Em meio não tamponado, suplementado com cutícula, o pH da cultura caiu e as atividades de Pr1 e Pr2 foram detectadas somente em valores de pH igual ou superior a 5,5. Em culturas tamponadas, as atividades Pr1 e Pr2 foram superiores em meio suplementado com cutícula, comparativamente as atividades em meio contendo nitrato. As atividades Pr1 e Pr2 ocorreram predominantemente no sobrenadante de cultivo. Os dados obtidos sugerem que Pr1 e Pr2 produzidas pelo isolado CG425 são induzidas por componentes da cutícula da broca do café (CBB), e que o pH da cultura influencia a expressão destas proteases, indicando a ocorrência de um mecanismo eficiente de secreção por este fungo. Os resultados obtidos neste estudo aumentam o conhecimento a respeito da produção de proteases por B. bassiana CG425, abrindo novos caminhos para o estudo do papel de proteases na virulência contra a broca do café durante o processo de infecção.

Desenvolvimento dos fungos Metarhizium anisopliae (Metschnikoff, 1879) Sorokin, 1883 E Beauveria bassiana (Balsamo) Vuillemin, 1912 sobre Ctenocephalides felis felis (Bouché, 1835) / Development of the fungi Metarhizium anisopliae (Metschnikoff, 1879) Sorokin, 1883 and Beauveria bassiana (Balsamo) Vuillemin, 1912 on the Ctenophephalides felis felis (Bouché, 1835)

A pulga Ctenocephalides felis felis é um parasita causador dermatites alérgicas e também pode transmitir diversos agentes etiológicos aos animais domésticos e aos homens. O objetivo deste trabalho foi verificar o desenvolvimento do fungo sobre a cutícula da pulga, através da microscopia eletrônica de varredura. Os isolados fúngicos testados foram o Metarhizium anisopliae 959 e Beauveria bassiana 986, ambos na concentração 10(8) conídios/ml. Após a exposição dos isolados fúngicos no período de duas, 15, 26 e 96 horas , o material foi processado para a microscopia eletrônica de varredura. Com a obtenção das micrografias, pode-se observar que com 2 horas após exposição aos fungos, os conídios estavam aderidos por toda a cutícula, situando-se preferencialmente nas membranas intersegmentais do abdome. Com 15 horas observou-se a formação do tubo de germinação e a cabeça do apressório e após 26 horas foi possível observar as ramificações e o engrossamento das hifas sobre a cutícula das pulgas. Os resultados indicam que os fungos testados foram capazes de se desenvolver sobre a cutícula de C. f. felis.

The flea Ctenocephalides felis felis is a parasite that causes allergic dermatitis and also may transmit etiologic agents to domestic animals and humans. This study investigated by scanning electron microscopy the development of entomopathogenic fungi on flea cuticle. Fleas were exposed to conidia (10(8) ml-1) of Metarhizium anisopliae (isolate 959) or Beauveria bassiana (isolate 986). Following standard protocols for electron microscopy, the specimens were prepared 2, 15, 26 and 96 h after infection. The micrography revealed that 2 h after fungus exposure, conidia attachments encompassed the entire flea cuticle, especially on abdominal intersegmental membranes. The emergence of germ tubes and appressoria formation occurred at 15 h, thickening and branching of hyphae on the flea cuticle was noted at 26 h. Therefore, both of these fungal isolates were able to develop on cuticular surfaces of C. f. felis.