Efficacy of Purpureocillium lilacinum AUMC 10149 as biocontrol agent against root-knot nematode Meloidogyne incognita infecting tomato plant / Eficácia de Purpureocillium lilacinum AUMC 10149 como agente de biocontrole contra o nematoide Meloidogyne incógnita que infecta plantas de tomate

Abstract Root-knot nematode Meloidogyne incognita is among the biotic factors which has greatly affected both the yield and the quality of the tomato crop. The egg parasitic nematode, Purpureocillium lilacinum (Pl) is considered as one of the most promising agents in controlling and overcoming this plant pathogen. The nematicidal effect of the native isolate Pl AUMC 10149 on second stage juvenile's survival and egg hatching of M. incognita at different times of exposure was tested in vitro. The obtained data showed that Pl gave a maximum percentage of J2 mortality (97.6%) and egg hatching inhibition (79.8%) after 72 hours of exposure. The potentiality of Pl as well as Bio-Nematon to control M. incognita infecting tomato was conducted using different times of application in vivo. Nine treatments with five replicates were used for such bioagents compared with the nematicide Oxamyl. Each seedling was inoculated with 1000 J2s of nematode/pot and 10 mL of Pl (1x1010 CFU/mL) or Bio-Nematon spore suspension (1x108 CFU/mL) 10mL/pot. The results indicated that the most effective treatments in reducing nematode population, number of galls and egg masses of M. incognita in plant roots was performed with treatment by Pl pre-planting and post-infection with Pl (Rf 1.9) giving a significant enhancement in plant length (64.9%), fresh weight (72.52%) and shoot dry weight (163.41%) without negatively impacting environment. Therefore, the present study confirmed that using P. lilacinum AUMC 10149 can be used as a practical supplement to environmentally friendly disease management of root-knot nematodes in Egypt.

Resumo O nematoide-das-galhas Meloidogyne incognita está entre os fatores bióticos que afetaram enormemente a produção e a qualidade da cultura do tomate. O nematoide parasita de ovos, Purpureocillium lilacinum (Pl), é considerado um dos mais promissores agentes no controle e superação desse fitopatógeno. O efeito nematicida do isolado nativo Pl AUMC 10149 na sobrevivência de juvenis de segundo estágio e na eclosão dos ovos de M. incognita em diferentes momentos de exposição foi testado in vitro. Os dados obtidos mostraram que o Pl deu um percentual máximo de mortalidade de J2 (97.6%) e inibição da eclosão dos ovos (79.8%) após 72 horas de exposição. A potencialidade de Pl e de Bio-Nematon para controlar M. incognita infectando tomate foi conduzida em diferentes tempos de aplicação in vivo. Nove tratamentos com cinco repetições foram usados ​​para tais bioagentes em comparação com o nematicida Oxamyl. Cada muda foi inoculada com 1.000 J2s de nematoide / vaso e 10 mL de Pl (1×1010 CFU/mL). Ou suspensão de esporos Bio-Nematon (1×108 CFU/mL) 10mL/pot. Os resultados indicaram que os tratamentos mais eficazes na redução da população de nematoides, número de galhas e desovas de M. incognita nas raízes das plantas foram realizados com Pl pré-plantio e pós-infecção com Pl (Rf 1.9), dando um aumento significativo no comprimento da planta (64.9%), massa fresca (72.52%) e massa seca da parte aérea (163.41%) sem impactar negativamente o meio ambiente. Portanto, o presente estudo confirmou que o uso de P. lilacinum AUMC 10149 pode ser usado como um suplemento prático para o manejo ecologicamente correto de nematoides-das-galhas no Egito.

Purpureocillium lilacinum and Trichoderma virens for biological control of trichostrongylid parasites of sheep: an in vitro evaluation / Purpureocillium lilacinum e Trichoderma virens no controle biológico de tricostrongilídeos de ovinos: uma avaliação in vitro

Abstract The study evaluated the ovicidal activity of enzymatic extracts of Purpureocillium lilacinum and Trichoderma virens against trichostrongylid eggs from sheep. Filtered extract (FE) and macerated crude extract (MCE) were prepared from fungal cultures in minimal broth. In the experiment, 100 trichostrongylid eggs, obtained from the feces of naturally infected sheep, were exposed to fungal extracts for 24 and 48 hours/25°C. In the control group, eggs were incubated in minimal broth. The number of L1 larvae was ascertained. Each treatment consisted of four repetitions and the experiment was repeated five times. It was observed that the effect of FE and MCE of P. lilacinum and T. virens on egg hatchability differed from that of the control group. MCE of T. virens and P. lilacinum showed higher ovicidal activity than FE over both periods and at 48 hours of exposure, respectively. From the percentage reductions in hatchability of the eggs, MCE was shown to be superior to FE for both fungi. This study demonstrated the ovicidal potential of these fungi against trichostrongylid eggs. However, further studies are needed in order to identify the molecules responsible for the ovicidal effects, and to evaluate the behavior of fungal extracts in biotic and abiotic interactions.

Resumo O estudo avaliou a atividade ovicida de extratos enzimáticos de Purpureocillium lilacinum e Trichoderma virens sobre ovos de tricostrongilídeos de ovinos. Extrato filtrado (EF) e extrato macerado bruto (EMB) foram preparados a partir de culturas fúngicas em caldo mínimo. No ensaio experimental, 100 ovos de tricostrongilídeos, obtidos de fezes de ovinos naturalmente infectados, foram expostos durante 24 e 48 horas/25ºC aos extratos dos fungos. No grupo controle, os ovos foram incubados em caldo mínimo. O número de larvas L1 foi determinado. Cada tratamento consistiu em quatro repetições e o experimento foi repetido cinco vezes. Observou-se que o efeito ovicida do EF e EMB de P. lilacinum e T. virens diferiu do grupo controle. O EMB de T. virens e P. lilacinum evidenciou atividade ovicida superior ao EF em ambos os períodos avaliados e em 48 horas de exposição, respectivamente. O percentual de redução de eclodibilidade evidenciou que o EMB foi superior ao EF em ambos os fungos. Este estudo demonstra o potencial ovicida desses fungos sobre ovos de tricostrongilídeos. No entanto, estudos adicionais são necessários para identificar as moléculas responsáveis pelo efeito ovicida, bem como avaliar o comportamento dos extratos fúngicos em interações bióticas e abióticas.

Exposure of Toxocara canis eggs to Purpureocillium lilacinum as a biocontrol strategy: an experimental model evaluation / Exposição de ovos de Toxocara canis a Purpureocillium lilacinum como estratégia de biocontrole: uma avaliação em modelo experimental

Abstract Purpureocillium lilacinum is a nematophagous fungus used in biological control against some parasites, including Toxocara canis. This study researched the infectivity of embryonated T. canis eggs after exposure to the fungus P. lilacinum. T. canis eggs were exposed to P. lilacinum for 15 or 30 days and subsequently administered to Swiss mice (n=20). Control group consisted of mice who received T. canis embryonated eggs without fungal exposure. Forty-eight hours after infection, heart, lung, and liver from animals of each group were collected to assess larval recovery. The organs of mice that received embryonated eggs exposed to the fungus showed a lower average larval recovery (P<0.05) suggesting that exposure of T. canis eggs to P. lilacinum was able to reduce experimental infection. Under the evaluated conditions, the interaction time between the fungus and the parasite eggs was not a significant factor in larvae recovery. P. lilacinum may be considered a promising T. canis biological control agent. However, further studies are needed to determine a protocol for the use of this fungus as a biological control agent.

Resumo Purpureocillium lilacinum é um fungo nematófago com potencial para uso no controle biológico de parasitos, incluindo Toxocara canis. Este estudo pesquisou a infectividade de ovos de T. canis embrionados após exposição ao fungo P. lilacinum . Ovos de T. canis foram expostos ao fungo por 15 ou 30 dias e subsequentemente administrados a camundongos Swiss (n=20). O grupo controle consistiu de camundongos que receberam ovos embrionados do parasita sem exposição ao fungo. Quarenta e oito horas após a infecção, coração, pulmão e fígado dos camundongos foram coletados para avaliar a recuperação larval. Os órgãos dos animais que receberam ovos embrionados expostos ao fungo apresentaram menor média de recuperação larval (P<0,05) do que os infectados com ovos sem exposição ao fungo, sugerindo que a exposição dos ovos de T. canis a P. lilacinum foi capaz de reduzir a infecção experimental. Nas condições avaliadas, o tempo de interação entre o fungo e os ovos do parasito não foi um fator significativo na recuperação das larvas. P. lilacinum pode ser considerado um promissor agente de controle biológico de T. canis, no entanto, mais estudos são necessários para avaliar o emprego deste fungo como um agente de controle biológico.

Brote de fungemias por Sarocladium kiliense en ocho hospitales públicos por contaminación intrínseca de ondansetrón intravenoso / Outbreak of fungemias by Sarocladium kiliense in eight public hospitals per intrinsic contamination of ondansetron intravenous

Resumen Introducción: Sarocladium kiliense es un hongo saprófito que puede generar infecciones oportunistas asociadas a procedimientos invasores. Se informa un brote multicéntrico nosocomial de fungemias de fuente común por este agente. Luego del reporte de cinco casos en pacientes en tres hospitales al Programa de Control de Infecciones del Ministerio de Salud de Chile en julio de 2013, se estudiaron a nivel nacional todos los pacientes con hemocultivo positivo para este agente. Se trató de cuadros clínicos leves a moderados, sin muertes atribuibles. El estudio identificó 65 casos en 8 hospitales, en su mayoría pacientes pediátricos en quimioterapia. Estudios iniciales de 94 muestras de cuatro fármacos y dispositivos usados en todos los casos resultaron negativas hasta que, en un segundo análisis de lotes seleccionados por criterios epidemiológicos y su matriz farmacéutica, se identificó la contaminación intrínseca de ampollas de ondansetrón de un productor específico, que se usó en todos los casos. Se realizó un retiro nacional de las ampollas de los tres lotes contaminados del fármaco, después de lo cual se contuvo el brote. La vigilancia de infecciones en los hospitales y el programa nacional coordinado con los laboratorios de microbiología fueron claves para identificar un brote multicéntrico de fuente común por contaminación de un fármaco por un hongo inusual.

Sarocladium kiliense is a saprophyte fungus that can cause opportunistic infections associated to invasive procedures. We report a multi-hospital nosocomial outbreak of fungemias due to this agent. Patients with positive blood culture to this agent were studied after six bloodstream infections identified in three Chilean hospitals in July 2013 were reported to Ministry of Health National Infection and Prevention Control Program. In general, there were mild clinical manifestations, without deaths attributable to the infection. Epidemiological and micro-biological study identified 65 cases in 8 hospitals, mostly pediatric patients in chemotherapy. Initial studies of 94 different drugs and medical devices had negative results, until a second analysis of specific blisters and their pharmaceutical matrix selected by epidemiological criteria identified an intrinsic contamination of ondansetron blisters from a specific producer used in all the patients. A recall of contaminated ondansetron blisters was performed in all the country, after which the outbreak was contained. Surveillance and response of local and national infection prevention and control programs and laboratory support were key to control of a national multi-hospital common source outbreak due to contamination of a drug by an unusual fungus.

Statistical tools application on dextranase production from Pochonia chlamydosporia (VC4) and its application on dextran removal from sugarcane juice

ABSTRACT The aim of this study was to optimize the dextranase production by fungus Pochonia chlamydosporia (VC4) and evaluate its activity in dextran reduction in sugarcane juice. The effects, over the P. chlamydosporia dextranase production, of different components from the culture medium were analyzed by Plackett-Burman design and central composite design. The response surface was utilized to determine the levels that, among the variables that influence dextranase production, provide higher production of these enzymes. The enzymatic effect on the removal of dextran present in sugarcane juice was also evaluated. It was observed that only NaNO3 and pH showed significant effect (p<0.05) over dextranase production and was determined that the levels which provided higher enzyme production were, respectively, 5 g/L and 5.5. The dextranases produced by fungus P. chlamydosporia reduced by 75% the dextran content of the sugarcane juice once treated for 12 hours, when compared to the control treatment.

Producción de unidades infectivas de Isaria fumosorosea (Hypocreales: Cordycipitaceae) a partir de aislados nativos del noreste de México mediante 3 estrategias de propagación / Production of infectious units of Isaria fumosorosea ( Hypocreales: Cordycipitaceae) from different indigenous isolates of northeastern Mexico using 3 propagation strategies

El objetivo del presente estudio fue evaluar la producción de blastosporas y conidios de diferentes aislados nativos de México del hongo entomopatógeno Isaria fumosorosea y de una cepa de colección mediante diferentes técnicas de propagación. En la producción de blastosporas se utilizaron 2 medios de cultivo líquidos (sumergidos), uno a base de casaminoácidos y el otro a base de peptona de colágeno como fuentes de nitrógeno, con glucosa como fuente de carbono en ambos. Para la producción de conidios, los hongos se cultivaron en agar papa dextrosa, a partir de esos cultivos se prepararon suspensiones de 1 x 10(6) conidios/ml para inocular matraces con caldo dextrosa Sabouraud, para iniciar así la fase líquida del cultivo bifásico, denominado también precultivo. Posteriormente con el precultivo y las suspensiones de conidios se inocularon bolsas con granos de arroz, que se incubaron durante 14 días para el cultivo bifásico y para la fermentación sólida, respectivamente. El aislado HIB-23 fue el que logró la más elevada concentración de blastosporas obtenida en el cultivo sumergido: 4,90 x 10(8) blastosporas/ml en el medio casaminoácidos; y en el medio con peptona de colágeno se obtuvieron 2,15 x 10(8) blastosporas/ml. La máxima producción de conidios en fermentación sólida la logró la cepa Pfr-612 (1,58 x 10(9) conidios/g), mientras que la máxima en cultivo bifásico correspondió al aislado HIB-30 (9,00 x 10(6) conidios/g). La fermentación sólida resultó ser el método más efectivo, con un promedio de 1,09 x 10(9) conidios/g, mientras que el cultivo bifásico fue el menos efectivo, con un promedio de 2,76 x 10(6) conidios/g. Para la producción de blastosporas en los medios sumergidos no se obtuvo diferencia significativa alguna.

The aim of this study was to evaluate the production of blastospores and conidia of different native isolates and a strain of Isaria fumosorosea using different propagation techniques. Two liquid culture media of casamino acids and peptone as nitrogen sources and glucose as carbon source for both media cultures were respectively used in the production of blastospores, while for the production of conidia, the fungi were grown in potato dextrose agar; from these cultures, solutions of conidia to a concentration of 1 x 10(6) per milliliter were prepared to inoculate flasks with Sabouraud dextrose broth for the liquid phase of the biphasic culture, also known as preculture. Subsequently, rice grain bags were inoculated with the preculture and the conidia solutions, which were incubated for 14 days for solid fermentation and biphasic culture, respectively. The HIB-23 isolate recorded a concentration of 4.90 x 10(8) blastospores/ml in the casamino acid medium, while a concentration of 2.15 x 10(8) blastospores/ml was obtained in the peptone collagen medium. For the Pfr-612 strain, the conidia production in solid-state fermentation was 1.58 x 10(9) conidia/g, and for HIB-30 in the biphasic culture of 9.00 x 10(6) conidia/g. Solid-state fermentation proved to be the most effective method with an average of 1.09 x 10(9) conidia/g, whereas the biphasic culture was the least effective method with 2.76 x 10(6) conidia/g; no significant difference was reported for the submerged production media.

Endophytic fungus Purpureocillium sp. A5 protect mangrove plant Kandelia candel under copper stress

Abstract Mangrove is an important ecosystem in the world. Mangrove ecosystems have a large capacity in retaining heavy metals, and now they are usually considered as sinks for heavy metals. However, the mechanism of why the soil of mangrove ecosystems can retain heavy metal is not certain. In this research, endophytic fungus Purpureocillium sp. A5 was isolated and identified from the roots of Kandelia candel. When this fungus was added, it protected the growth of K. candel under Cu stress. This can be illustrated by analyzing chlorophyll A and B, RWC and WSD to leaves of K. candel. Purpureocillium sp. A5 reduces uptake of Cu in K. candel and changes the pH characterization of soil. Furthermore, A5 increase the concentration of Cu complexes in soil, and it enhanced the concentration of carbonate-bound Cu, Mn-Fe complexes Cu and organic-bound Cu in soil. Nevertheless, a significant reduction of the Cu ion was noted among A5-treated plants. This study is significant and illustrates a promising potential use for environmental remediation of endophytes, and also may partially explain the large capacity of mangrove ecosystems in retaining heavy metals.

Contribution to the knowledge of pathogenic fungi of spiders in Argentina: Southernmost record in the world / Aporte al conocimiento de los hongos patógenos de arañas en Argentina: El registro más austral del mundo

The aim of this study was to identify entomopathogenic fungi infecting spiders (Araneae) in a protected area of Buenos Aires province, Argentina. The Araneae species identified was Stenoterommata platensis. The pathogens identified were Lecanicillium aphanocladii Zare & W. Gams, Purpureocillium lilacinum (Thom) Luangsa-ard, Houbraken, Hywel Jones & Samson and Ophiocordyceps caloceroides (Berk & M.A. Curtis). This study constitutes the southernmost records in the world and contributes to expanding the knowledge of the biodiversity of pathogenic fungi of spiders in Argentina.

El objetivo de este estudio fue identificar hongos entomopatógenos de arañas en un área protegida de la provincia de Buenos Aires, Argentina. La especie de araña identificada fue Stenoterommata platensis. Los patógenos identificados fueron Lecanicillium aphanocladii Zare y W. Gams, Purpureocillium lilacinum (Thom) Luangsa-ard, Houbraken, Hywel Jones y Samson y Ophiocordyceps caloceroides (Berk y M.A. Curtis). Este estudio constituye el registro más austral del mundo y contribuye a ampliar el conocimiento de la biodiversidad de hongos patógenos de arañas en Argentina.

Predatory activity of Butlerius nematodes and nematophagous fungi against Haemonchus contortus infective larvae / Atividade predatória de nematóides Butlerius e fungos nematófagos contra larvas infectantes de Haemonchus contortus

Abstract The purpose of this study was to evaluate the predatory activity of the nematode Butlerius spp. and fungal isolates of Duddingtonia flagrans, Clonostachys rosea, Arthrobotrys musiformis and Trichoderma esau against H. contortus infective larvae (L3) in grass pots. Forty-eight plastic gardening pots containing 140 g of sterile soil were used. Panicum spp. grass seeds (200 mg) were sown into each pot and individually watered with 10 mL of tap water. Twelve days after seeding, the pots were randomly divided into 6 groups (n=8). Two thousand H. contortus infective larvae (L3) were added to each group. Additionally, the following treatments were established: Group 1 – 2000 Butlerius spp. larvae; group 2 – A. musiformis (1x107 conidia); group 3 – T. esau (1x107 conidia); group 4 – C. rosea (1x107 conidia), group 5 – D. flagrans (1x107conidia) and Group 6 – no biological controller (control group). The larval population of H. contortus exposed to Butlerius spp. was reduced by 61.9%. Population reductions of 90.4, 66.7, 61.9 and 85.7% were recorded in the pots containing A. musiformis, T. esau, C. rosea and D. flagrans, respectively. The results of this study indicate that the predatory nematode Butlerius spp. and the assessed fungi display an important predatory activity can be considered suitable potential biological control agents.

Resumo O objetivo deste estudo foi avaliar a atividade predatória do nematoide Butlerius spp. e isolados fúngicos de Duddingtonia flagrans, Clonostachys rosea, Arthrobotrys musiformis e Trichoderma esau contra larvas infectantes (L3) de Haemonchus contortus em vasos plantados com Panicum spp. Foram utilizados quarenta e oito potes plásticos de jardinagem contendo 140 g de solo estéril, 200 mg de sementes de Panicum spp.. Cultivar colonião, foi semeado em cada vaso e, diariamente, molhados com 10 mL de água da torneira. Doze dias após, os vasos foram divididos em 6 grupos (n = 8), e duas mil L3 de H. contortus foram adicionadas a cada vaso. Foram estabelecidos os seguintes tratamentos: Grupo 1 - 2.000 larvas de Butlerius spp.; Grupo 2 - A. musiformis (1x107 conídios); grupo 3 - T. esau (1x107 conídios); grupo 4 - C. rosea (1x107 conídios); grupo 5 - D. flagrans (1x107conidia); e Grupo 6 – somente L3 de H. contortus que serviu como controle negativo. A população de L3 de H. contortus expostas a Butlerius spp. foi reduzida em 61,9%. Redução populacional de 90,4, 66,7, 61,9 e 85,7% foram observadas nos vasos contendo A. musiformis, T. esau, C. rosea e D. flagrans, respectivamente. Os resultados deste estudo indicaram que o nematoide Butlerius spp. e os fungos avaliados exibiram importante atividade predatória e podem ser considerados como agentes de controle biológico.

Classification and infection mechanism of entomopathogenic fungi / Classificacão e mecanismo de infecção dos fungos entomopatogênicos

Entomopathogenic fungi are important biological control agents throughout the world, have been the subject of intensive research for more than 100 years, and can occur at epizootic or enzootic levels in their host populations. Their mode of action against insects involves attaching a spore to the insect cuticle, followed by germination, penetration of the cuticle, and dissemination inside the insect. Strains of entomopathogenic fungi are concentrated in the following orders: Hypocreales (various genera), Onygenales (Ascosphaera genus), Entomophthorales, and Neozygitales (Entomophthoromycota).(AU)

Os fungos entomopatogênicos são importantes agentes de controle biológico em todo o mundo e têm sido objeto de intensa pesquisa por mais de 100 anos, infectando artrópodes na natureza e podendo ocorrer em níveis enzoóticos ou epizoóticos em suas populações de hospedeiros. O seu mecanismo de infecção envolve a fixação do esporo à cutícula do inseto, seguido da germinação, penetração da cutícula e disseminação interna no inseto. As linhagens dos fungos entomopatogênicos estão concentradas nas ordens: Hypocreales (vários gêneros), Onygenales (gênero Ascosphaera), Entomophthorales e Neozygitales (Entomophthoromycota).(AU)

Molecular diagnosis and intraspecific genetic variability of root pathogens of arid legumes in Western Rajasthan, India / Diagnóstico molecular y la variabilidad genética intraespecífica de patógenos de raíces en leguminosas resistentes a sequías del oeste de Rajasthan, India

Abstract:The productivity of arid legumes, such as Clusterbean (Cyamopsis tetragonoloba), Cowpea (Vigna unguiculata), Moth bean (Vigna aconitifolia) and Horse gram (Macrotyloma uniflorum), may remain stagnant over decades because of their high susceptibility to root diseases. Besides, there is a limitation on the information about molecular diagnosis and intraspecific genetic variability of root pathogens in arid legumes. To contribute in this field, we assessed a total of 52 isolates from 88 root samples that were found infected with fungal pathogens in Jodhpur, Jaipur and Bikaner Districts of Rajasthan. Diseased roots samples were analyzed following standard microbiological methods for fungus extraction and purification, and for genetic studies. Irrespective of the geographical location from where the diseased samples were collected, all pathogen isolates were clustered in RAPD dendrograms as per their respective genera. Phylogram, based on multiple sequence alignment, revealed that different genera (i.e. Fusarium, Neocosmospora and Syncephalastrum), separated from each other, and species within the same genera, clustered together with their reference sequences with apreciable bootstrap values. Out of 20 representative isolates representing each cluster and all outgroups sequenced, eight were molecularly identified as Neocosmospora vasinfecta, five as Fusarium solani, two as Neocosmospora striata, two as Fusarium acutatum, one as Syncephalastrum monosporum, one as Fusarium oxysporum and one as Fusarium species. The root pathogens of the arid legumes were found neither restricted to a geographical location nor were host specific in nature. Fusarium solani wilt in cowpea and seedling rot in moth bean, F. oxysporum wilt in moth bean, F. acutatum damping off in cowpea and Clusterbean, Fusarium sp. seedling rot in Clusterbean, Neocosmospora striata root rot in cowpea and wilt in Clusterbean and Syncephalastrum monosporum root rot in Clusterbean were molecularly identified as new fungal records as pathogens causing root diseases in arid legumes. Rev. Biol. Trop. 64 (4): 1505-1518. Epub 2016 December 01.

Resumen:La producción de leguminosas resistentes a sequías como Cyamopsis tetragonoloba, Vigna unguiculata, Vigna aconitifolia y Macrotyloma uniflorum, puede permanecer inactiva durante décadas debido a su alta susceptibilidad a enfermedades en las raíces. Además, hay información limitada relacionada con el diagnóstico molecular y la variabilidad genética intraespecífica de patógenos de raíces en estas leguminosas resistentes a sequías. Para contribuir en esta área, evaluamos un total de 52 extractos de 88 raíces infectadas con patógenos fúngicos en los distritos de Jodhpur, Jaipur y Bikaner de Rajastán. Las muestras de raíces infectadas se analizaron siguiendo los métodos estándar de microbiología para extracción y purificación de hongos y para estudios genéticos. Independientemente del sitio donde se recolectaron las muestras contaminadas, todos los extractos patógenicos se agruparon en dendrogramas RAPD en cada uno de sus respectivos géneros. El filograma, basado en alineamiento de secuencias múltiples reveló que distintos géneros (Fusarium, Neocosmospora y Syncephalastrum) separados entre ellos y especies del mismo género se agrupan con sus secuencias de referencia con valores de bootstrap significativos. De cada 20 extractos representantes de cada agrupamiento y todos los grupos externos secuenciados, ocho fueron identificados molecularmente como Neocosmospora vasinfecta, dos como Fusarium acutatum, una como Syncephalastrum monosporum, una como Fusarium oxysporum y una como Fusarium. Los patógenos de estas leguminosas resistentes a sequías no están restringidos por la localidad ni por un hospedero específico. Fusarium solani que marchita el frijol de vaca y pudre la semilla de Vigna aconitifolia, F. oxysporum que marchita a Vigna aconitifolia, F. acutatum que marchita a Vigna unguiculata y Cyamopsis tetragonoloba, Fusarium sp. que pudre la semilla de Cyamopsis tetragonoloba, Neocosmospora striata que pudre la raíz de Vigna unguiculata y marchita a Cyamopsis tetragonoloba y, Syncephalastrum monosporum que pudre la raíz en Cyamopsis tetragonoloba, fueron identificados molecularmente como nuevos registros de patógenos fúngicos que causan daños en las raíces de leguminosas resistentes a sequías.

Microbial control of the invasive spiraling whitefly on cassava with entomopathogenic fungi

Abstract The entomopathogenic fungi Beauveria bassiana, Metarhizium anisopliae, Lecanicillium lecanii and Isaria fumosorosea were tested for their efficacy in managing the exotic spiraling whitefly Aleurodicus dispersus (Hemiptera, Aleyrodidae) on cassava (Manihot esculenta) during 2 seasons (2011-2012 and 2012-2013). The fungi I. fumosorosea and L. lecanii exhibited promising levels of control (> 70% mortality of the A. dispersus population). The percent mortality increased over time in both seasons. Application of I. fumosorosea was highly pathogenic to A. dispersus in both seasons compared to the other entomopathogenic fungi. Analysis of the percent mortality in both seasons revealed differences in efficacy between 3 and 15 days after treatment. The season also influenced the effects of the fungi on the A. dispersus population. Thus, entomopathogenic fungi have the potential to manage A. dispersus infestation of cassava.

Compatibilidad in vitro de un bioplaguicida a base de Lecanicillium lecanii (Hypocreales: Clavicipitaceae) con agroquímicos empleados en los cultivos de algodón y berenjena / In vitro compatibility of the biopesticide based on Lecanicillium lecanii (Hypocreales: Clavicipitaceae) with agrochemicals used in cotton and eggplant crops

El aislamiento colombiano de Lecanicillium lecanii Vl026 formulado como un granulado dispersable WG, ha demostrado una alta eficiencia para el control de Bemisia tabaci en los cultivos de algodón y berenjena. Teniendo en cuenta el potencial de este bioinsumo, el objetivo del siguiente fue determinar la compatibilidad in vitro del bioplaguicida a base de L. lecanii con agroquímicos (insecticidas y fungicidas) que se utilizan con mayor frecuencia en dichos cultivos. La compatibilidad in vitro se estableció determinando el porcentaje de germinación (%) y las Unidades Formadoras de Colonia (UFC) en presencia de los plaguicidas y estimando la concentración inhibitoria 10 (CI10). Cada plaguicida se evaluó a la dosis recomendada, a la mitad y a un cuarto de ésta. Los fungicidas químicos (Benomil®, Vitavax®, Ridomil® y Manzate®) no fueron compatibles con L. lecanii, ya que inhibieron la germinación y las Unidades Formadoras de Colonia del hongo en las tres dosis evaluadas. En cuanto a los insecticidas químicos, el producto Confidor® no inhibió la viabilidad en comparación con el tratamiento control, considerándose compatible con el bioplaguicida. Cuando se evaluó la dosis completa de los demás insecticidas (Oportune®, Actara®, Match® y Malathion®) se obtuvieron germinaciones inferiores al 80%, por lo que dichos productos se clasificaron como no compatibles con el bioplaguicida a base de L. lecanii. El único agroquímico que fue compatible en condiciones in vitro con L. lecanii fue el producto Confidor®. Sin embargo, se recomienda evaluar el efecto in vivo de los productos químicos habitualmente utilizados por los agricultores sobre L. lecanii, con el propósito de desarrollar y establecer estrategias de manejo integrado de la mosca blanca Bemisia tabaci.

A colombian isolate of Lecanicillium lecanii formulated as dispersible granules WG, has shown high efficiency to control Bemisia tabaci in cotton and eggplant crops. Considering that, the objetive of this work was to determine the in vitro compatibility of biopesticide based on L. lecanii with agrochemicals (insecticides and fungicides) that are most frequently used in tobacco and eggplant crops. In vitro compatibility of L. lecanii with agrochemicals was determinated by germination (%) and Colony Forming Units (CFU) in the presence of pesticides and also estimating the inhibitory concentration 10 (IC10). Each agrochemical was evaluated at the recommended dose, a half and a quarter of it. For the three doses tested (Benomyl®, Vitavax®, Ridomil® and Manzate®) were not compatible with L. lecanii, because these inhibited germination and Colony Forming Units of the fungus. Confidor® did not inhibit viability compared to control treatment, and it was considered compatible with the biopesticide. When the recommended dose (Oportune®, Actara®, Match® and Malathion®)was used, the germination of the L. lecanii was lower than 80%, then these products were classified as non-compatible with the biopesticide based on L. lecanii. The only agrochemical that was compatible in vitro with L. lecanii was Confidor®. However, is necesary to evaluate the in vivo effect of agrochemicals commonly used by farmers on L. lecanii, in order to develop and establish integrated management strategies for the control of the whitefly Bemisia tabaci.

Controle de Meloidogyne javanica com Pochonia chlamydosporia e esterco bovino / Control of Meloidogyne javanica with Pochonia chlamydosporia and cow manure

A aplicação conjunta de agentes de controle biológico e matéria orgânica pode potencializar o controle do nematoide das galhas em hortaliças. Desta forma, o objetivo deste estudo foi avaliar o efeito da aplicação de Pochonia chlamydosporia (Hypocreales: Clavicipitaceae) (PC) e esterco bovino na redução populacional de Meloidogyne javanica (Tylenchida: Heteroderidae) em tomateiro. Os tratamentos foram constituídos da incorporação ao solo de 20g de canjiquinha colonizada pelo fungo (PC), 20g de canjiquinha não-colonizada (CNC), 70g de esterco bovino + 20g de canjiquinha colonizada (EBPC) e apenas 70g de esterco bovino (EB). Ao solo de cada vaso foi adicionado o respectivo tratamento e 5.000 ovos de M. javanica, seguido pelo transplantio das mudas. A incorporação ao solo de EBPC, seguido de EB, aumentou a biomassa da parte aérea e das raízes de tomateiros. A maior redução no número de galhas e de ovos de M. javanica foi observada após a aplicação de EB, seguido por EBPC. Não houve diferença significativa na aplicação apenas do fungo em relação à testemunha considerando a avaliação do número de galhas e de ovos.

Concomitant application of biological control agents and organic amendment can enhance the control of the root-knot nematode on vegetables. Thus, the objective of this study was to evaluate the effect of the application of Pochonia chlamydosporia (Hypocreales: Clavicipitaceae) (PC) and cow manure on the reduction of the Meloidogyne javanica (Tylenchida: Heteroderidae) on tomato. The treatments were: 20 g of milled corn colonized by the fungus (PC), 20 g of non-colonized milled corn (NCMC), 70g of cow manure + 20g of PC-colonized milled corn (CMPC) and 70g of cow manure alone (CM). Into the soil of each pot was added the respective treatment and 5,000 eggs of M. incognita and M. javanica, separately, followed by the transplanting of the seedlings. Soil amendment with CMPC, followed by CM, increased the tomato aboveground and root system biomass. Highest reduction on the number of galls and eggs was observed after soil amendment with CM, followed by CMPC. No significant reduction on the number of galls and eggs was observed by the application of PC.

Predatory activity of chlamydospores of the fungus Pochonia chlamydosporia on Toxocara canis eggs under laboratory conditions / Atividade predatória de clamidósporos do fungo Pochonia chlamydosporia sobre ovos de Toxocara canis em condições laboratoriais

The objective of this study was to use chlamydospores of the fungus Pochonia chlamydosporia (isolates VC1 and VC4) against Toxocara canis eggs in a 15-day in vitro assay. One thousand T. canis eggs were placed in Petri dishes containing 2% water agar medium with different concentrations of chlamydospores (1,000, 10,000 or 100,000) of each fungal isolate of P. chlamydosporia (treated groups) and 1,000 eggs in Petri dishes without fungus (control group). Egg counts were performed to determine the ovicidal activity, which was classified as three effect levels: type 1, type 2 and type 3. Significant differences (P < 0.01) in egg destruction were found in comparison with the control group. The highest percentage of egg destruction was found in plates containing 100,000 chlamydospores (68.5% for VC1 and 70.5% for VC4). Chlamydospores of P. chlamydosporia were effective in destroying T. canis eggs and may contribute in the future towards combating the eggs of this parasite.

O objetivo do trabalho foi utilizar clamidósporos do fungo Pochonia chlamydosporia (isolados VC1 e VC4) na destruição de ovos de Toxocara canis, num ensaio in vitro, realizado no intervalo de 15 dias. Em cada placa de Petri com ágar-água 2% foram vertidos 1.000 ovos de T. canis em 1.000, 10.000 ou 100.000 clamidósporos de cada isolado do fungo (grupos tratados). Foram realizadas as contagens para verificar a atividade ovicida, classificada em três níveis de efeito: tipo 1, tipo 2 e tipo 3. Os resultados demonstraram que houve diferença significativa (P < 0,01) na destruição dos ovos em relação aos ovos observados nas placas do grupo controle. O maior percentual de ovos destruídos foi observado nas placas contendo 100.000 clamidósporos (68,5% para VC1 e 70,5% para VC4). Clamidósporos do fungo P. chlamydosporia foram efetivos na destruição dos ovos de T. canis podendo contribuir no futuro para o combate aos ovos deste parasito.

Pathogenic and enzyme activities of the entomopathogenic fungus Tolypocladium cylindrosporum (Ascomycota: Hypocreales) from Tierra del Fuego, Argentina

Tolypocladium cylindrosporum is an entomopathogenic fungi that has been studied as a biological control agent against insects of several orders. The fungus has been isolated from the soil as well as from insects of the orders Coleoptera, Lepidoptera, Diptera and Hymenoptera. In this study, we analyzed the ability of a strain of T. cylindrosporum, isolated from soil samples taken in Tierra del Fuego, Argentina, to produce hydrolytic enzymes, and to study the relationship of those activities to the fungus pathogenicity against pest aphids. We have made the traditional and molecular characterization of this strain of T. cylindrosporum. The expression of hydrolase activity in the fungal strain was estimated at three incubation temperatures (4ºC, 12ºC and 24ºC), on different agar media supplemented with the following specific substrates: chitin azure, Tween ® 20, casein, and urea for chitinase, lipase, protease, and urease activity, respectively. The hydrolytic-enzyme activity was estimated qualitatively according to the presence of a halo of clarification through hydrolase action, besides was expressed semi-quantitatively as the ratio between the hydrolytic-halo and colony diameters. The pathogenicity of the fungus was tested on adults of the aphid Rhopalosiphum padi at three temperatures of incubation (4ºC, 12ºC and 24ºC). The suspension was adjusted to a concentration of 1x10 7 conidia/ml. In pathogenicity assays at seven days post-inoculation, the fungus caused the mortality of adults of Ropalosiphum padi at different temperatures also showed a broad ability to grow on several agar-culture media, supplemented with different carbon sources at the three incubation temperatures tested. Although, the growth was greater with higher incubation temperatures (with maximum levels at 24°C), the fungus reached similar colony diameters after 15 days of incubation on the medium supplemented with Tween® 20 at the lower two incubation temperatures of 4°C or 12°C. In accordance with the results on colony diameters, the fungus revealed an ability to degrade casein, chitin derivatives, Tween® 20, and urea as evidenced by the appearance of a halo around the fungal colony. Because of its origin and temperature tolerance, this Argentine strain has great potential for use as a biocontrol agent for insect pest control in cold and temperate environments. Rev. Biol. Trop. 60 (2): 833-841. Epub 2012 June 01.

El hongo entomopatógeno Tolypocladium cylindrosporum ha sido estudiado como un agente de control biológico contra insectos de varios órdenes. Esta especie fue aislada del suelo, así como de insectos de los órdenes Coleoptera, Lepidoptera, Diptera e Hymenoptera. En el presente trabajo hemos analizado la capacidad de una cepa de T. cylindrosporum (LPSC Nº1065) aislada del suelo en Tierra del Fuego, Argentina, para producir enzimas hidrolíticas y determinar la relación de esta actividad con la patogenicidad del hongo para combatir la plaga de los áfidos en diferentes temperaturas (4º, 12º y 24ºC). En los ensayos de patogenicidad, siete días posteriores a la inoculación, se registró mortalidad en los adultos del áfido Ropalosiphum padi a diferentes temperaturas y también se demostró una amplia capacidad de crecer en varios medios de cultivos complementados con diferentes fuentes de carbono bajo las tres temperaturas de incubación ensayadas. Debido a su origen y a la tolerancia que tiene a bajas temperaturas esta cepa, presenta un gran potencial para su uso como agente de control biológico para las plagas de insectos de ambientes fríos y templados.

Ação ovicida do extrato bruto enzimático do fungo Pochonia chlamydosporia sobre ovos de Ancylostoma sp / Ovicidal action of a crude enzymatic extract of fungus Pochonia chlamydosporia against Ancylostoma sp eggs

INTRODUÇÃO: Ancylostoma sp é um geo-helminto potencialmente zoonótico. MÉTODOS: O objetivo deste trabalho foi avaliar in vitro a ação do extrato bruto enzimático de Pochonia chlamydosporia (VC4) sobre ovos de Ancylostoma sp, em meio ágar-água 2 por cento e em cultura de fezes. RESULTADOS: Observou-se um percentual de redução na eclosão dos ovos de Ancylostoma sp, de 76,8 por cento na placas de Petri do grupo tratado em relação ao grupo controle. CONCLUSÕES: O extrato bruto enzimático de Pochonia chlamydosporia foi eficiente na redução da eclosão dos ovos de Ancylostoma sp, podendo ser utilizado como controlador biológico desse nematoide.

INTRODUCTION: Ancylostoma sp is a potentially zoonotic geohelminth. METHODS: This study aimed to evaluate in vitro the action of crude enzyme extract of Pochonia chlamydosporia (VC4) on eggs of Ancylostoma sp in 2 percent water-agar and in fecal cultures. RESULTS: The percentage reduction in Ancylostoma sp egg eclosion was 76.8 percent in Petri dishes of the treated group compared to the control group. CONCLUSIONS: The crude enzyme extract of Pochonia chlamydosporia was effective at reducing Ancylostoma sp egg eclosion and can be used as biological control of this nematode.

Aucula magnifica (Schaus, 1904) (Lepidoptera: Noctuidae: Agaristinae): morphology of egg and last instar larvae / Aucula magnifica (Schaus, 1904) (Lepidoptera: Noctuidae: Agaristinae): morfologia do ovo e da larva de último ínstar

Aiming to contribute to knowledge of the immature instars of Neotropical Lepidoptera, this study details the morphology of the egg and last instar larvae of Aucula magnifica (Schaus, 1904) (Lepidoptera: Noctuidae: Agaristinae), emphasising the structures of the corium and the chaetotaxy. There is also a report of the occurrence of entomopathogenic action of Nomuraea rileyi (Farlow) Samson fungi on the larva.

Este estudo objetiva contribuir para o conhecimento dos estágios imaturos dos lepidópteros neotropicais. Nele é feito o detalhamento da morfologia das fases de ovo e de larva de Aucula magnifica (Schaus, 1904) (Lepidoptera: Noctuidae: Agaristinae), dando ênfase ao estudo das estruturas do córion e da quetotaxia da larva de último ínstar. Além disso é relatada a ocorrência da ação entomopatogênica do fungo Nomuraea rileyi (Farlow) Samson sobre suas larvas.

Impact of moisture on survival of Aedes aegypti eggs and ovicidal activity of Metarhizium anisopliae under laboratory conditions

The effect of relative humidity (43 percent, 75 percent, 86 percent and > 98 percent) on Aedes aegypti eggs treated with Metarhizium anisopliae or water only was tested for up to a six months exposure at 25ºC. Survival of larvae inside eggs was clearly affected by the lowest humidity (43 percent) tested, and eclosion diminished at all humidities after increasing periods of exposure. M. anisopliae showed to have a strong ovicidal activity only at humidity close to saturation. No difference of activity was found between conidia and hyphal bodies tested. This fungus affected larvae inside eggs and has potential as a control agent of this important vector in breeding sites with high moisture.