Lack of pathogenicity and toxicity of the mycoinsecticide Metarhizium anisopliae var. acridum following acute gastric exposure in mice.

Metarhizium anisopliae var. acridum, monospore culture EH-502/8 (CNRCB MaPL40), isolated in Mexico from Schistocerca piceifrons ssp. piceifrons (Orthoptera: Acrididae) was tested for acute oral intragastric pathogenicity and toxicity in CD-1 mice. Animals were inoculated with one dose (10(8) conidia/animal) of viable (72 mice), non-viable (24 mice) conidia and compared to 18 control mice. Clinical observations were done daily; mycological and histological tests were performed during necropsies after the inoculation. No mice showed clinical symptoms of illness or died during the study. The fungus was able to persist in some organs until day 3, but did not cause any damage to the host. The gross pathology observed was splenomegaly in mice inoculated with viable and non-viable conidia. Non-germinated conidia, observed in several organs, suggest hematogenous spread, but without any histopathological tissue reaction. Results support the non-pathogenic and non-toxic status of this fungal strain when administered in a single intragastric dose to mice.

Virulence testing and extracellular subtilisin-like (Pr1) and trypsin-like (Pr2) activity during propagule production of Paecilomyces fumosoroseus isolates from whiteflies (Homoptera: Aleyrodidae).

To properly characterize several isolates of Paecilomyces fumosoroseus, a fungal entomopathogen of whiteflies (Homoptera: Aleyrodidae) and other insect pests for biocontrol purposes, virulence towards Trialeurodes vaporariorum, and subtilisin-like (Pr1) and trypsin-like (Pr2) protease activity during propagule production were investigated in monospore cultures (MCs). The virulence of three MCs towards second instar whiteflies was measured and expressed as lethal median concentration (LC50). Number and widthlength ratio of propagules (blastospores, hyphal bodies, short hyphae, submerged conidia) and extracellular proteolytic activity was determined simultaneously in liquid medium. Total protease activity was assayed with azocasein, Pr1 and Pr2 activity was determined with the substrates N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-Benzoyl-Phe-Val-Arg-pnitroanilide, respectively. Natural variability in virulence, propagule production and cuticle-degrading proteases among isolates was observed. Bioassays showed a LC50 of 1.1 x 1,000, 2.5 x 10,000 and 7.6 x 10,000 conidia/ml for MCs EH-506/3, EH-503/3 and EH-520/3, respectively, EH-506/3 being the most virulent isolate. Isolate EH-503/3 produced the highest yield of propagules (7.7 x 10000000 propagules/ml), followed by EH-520/3 with 6.4 x 10000000 and EH-506/3 with 1.0 x 10000000 propagules/ml. Subtilisin-like (Pr1) and trypsin-like (Pr2) activity was present in the three MCs. Subtilisin-like (Pr1) activity was highest (745.7 UPr1/ml at 120 h) in the most virulent isolate, EH-506/3, pointing at Pr1 as a phenotypic marker of virulence for P. fumosoroseus. EH-506/3 appears to be a good candidate for whitefly biocontrol due to its high virulence, Pr1 concentration and rapid transition to blastospores in submerged liquid medium.

Virulence testing and extracellular subtilisin-like (Pr1) and trypsin-like (Pr2) activity during propagule production of Paecilomyces fumosoroseus isolates from whiteflies (Homoptera: Aleyrodidae)

Con el objetivo de caracterizar aislamientos para el control biológico demosquita blanca (Homoptera: Aleyrodidae), se estudiaron tres cultivosmonospóricos (CMs) de Paecilomyces fumosoroseus, hongo entomopatógenode la mosquita blanca (Homoptera: Aleyrodidae). Se determinó la virulencia enninfas de segundo estadio de la mosquita blanca expresada comoconcentración letal media (CL50). Se determinó la producción de propágulosfúngicos (blastosporas, cuerpos hifales, hifas cortas, conidios sumergidos)en medio líquido, se midió la relación de largo y ancho de los propágulos,y la actividad proteolítica (total; tipo subtilisina, Pr1 y tipo tripsina, Pr2)simultáneamente con la producción de propágulos. La proteasa total sedeterminó con azocaseina, y las actividades de Pr1 y Pr2 con N-Succinil-Ala-Ala-Pro-Phe-p-nitroanilida y N-Benzoil-Phe-Val-Arg-p-nitroanilida comosustratos específicos, respectivamente. Se observó variabilidad entre los tresCMs en cuanto a la virulencia, producción de propágulos y proteasas. Losbioensayos mostraron una CL50 de 1,1 x 103, 2,5 x 104 y 7,6 x 104 conidios/mlpara los CMs EH-506/3, EH-503/3 y EH-520/3, respectivamente.El CM donde se observó el mayor número de propágulos en las condicionesensayadas fue EH-503/3 (7,7 x 107), luego EH-520/3 con 6,4 x 107 y EH-506/3con 1,0 x 107 propágulos/ml. Las actividades enzimáticas de Pr1 y Pr2 sedemostraron en los tres CMs. La actividad tipo subtilisina (Pr1) fue mayor enel aislado más virulento (EH-506/3) con 745,7 UPr1/ml a las 120 h, y señala aPr1 como un marcador fenotípico de virulencia para P. fumosoroseus.EH-506/3 es un buen candidato para el control biológico de la mosquitablanca en México, por su alta virulencia, elevada concentración de Pr1 y surápida transición a blastosporas en el medio líquido ensayado(AU)

To properly characterize several isolates of Paecilomyces fumosoroseus,a fungal entomopathogen of whiteflies (Homoptera: Aleyrodidae) and otherinsect pests for biocontrol purposes, virulence towards Trialeurodesvaporariorum, and subtilisin-like (Pr1) and trypsin-like (Pr2) protease activityduring propagule production were investigated in monospore cultures (MCs).The virulence of three MCs towards second instar whiteflies was measuredand expressed as lethal median concentration (LC50). Number andwidth–length ratio of propagules (blastospores, hyphal bodies, short hyphae,submerged conidia) and extracellular proteolytic activity was determinedsimultaneously in liquid medium. Total protease activity was assayed withazocasein, Pr1 and Pr2 activity was determined with the substratesN-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-Benzoyl-Phe-Val-Arg-pnitroanilide,respectively. Natural variability in virulence, propagule productionand cuticle-degrading proteases among isolates was observed. Bioassaysshowed a LC50 of 1.1 x 103, 2.5 x 104 and 7.6 x 104 conidia/ml for MCsEH-506/3, EH-503/3 and EH-520/3, respectively, EH-506/3 being the mostvirulent isolate. Isolate EH-503/3 produced the highest yield of propagules(7.7 x 107 propagules/ml), followed by EH-520/3 with 6.4 x 107 and EH-506/3with 1.0 x 107 propagules/ml. Subtilisin-like (Pr1) and trypsin-like (Pr2) activitywas present in the three MCs. Subtilisin-like (Pr1) activity was highest(745.7 UPr1/ml at 120 h) in the most virulent isolate, EH-506/3, pointing at Pr1as a phenotypic marker of virulence for P. fumosoroseus. EH-506/3 appears tobe a good candidate for whitefly biocontrol due to its high virulence,Pr1 concentration and rapid transition to blastospores in submerged liquidmedium(AU)

Acute oral intragastric pathogenicity and toxicity in mice of Paecilomyces fumosoroseus isolated from whiteflies.

Paecilomyces fumosoroseus, monospore culture EH-506/3, isolated in Mexico from Bemisia tabaci whitefly was tested for acute oral intragastric pathogenicity and toxicity in CD-1 mice. Animals were inoculated by gavage with only one dose (10(8) conidia/animal) of viable (72 mice), heat-killed (24 mice) fungus and compared to 18 control mice. Clinical observations were done daily; mycological and histological tests were performed during necropsies at days 3, 10, 17, and 21 after the inoculation. No mice were clinically ill or died. At the end of the study, their mean weight corresponded to healthy adults. Positive fungal cultures of feces were obtained only 24 h after inoculation. Positive cultures were found in 15 out of 360 organs (liver, spleen, kidney, brain, lung) in 12 of 72 mice inoculated with viable conidia. Gross pathology exhibited splenomegaly and liver paleness in mice inoculated with viable and heat-killed fungus. Non-germinated conidia were observed in studied organs, without any pathological tissue reaction, suggesting no mycological or histopathological evidence of fungal multiplication. The fungus was able to persist, but did not cause permanent damage to the host. This study supports the non-pathogenic/toxic status of P. fumosoroseus EH-506/3 when administered intragastrically in mice.

Estudio preliminar sobre la obtención y conservación de propágulos de Verticillium lecanii (Zimmermann) Viègas / Preliminary study on yield and storage of fungal propagules from Verticillium lecanii (Zimmermann) Viègas

Verticillium lecanii se cultivó en un medio sólido con granos de arroz hidratados y en un medio líquido con extracto de levadura y glucosa para determinar el tipo de propágulos fúngicos obtenidos y observar la conservación de su viabilidad a 4ºC en agua destilada, cristales gel de sílice y vermiculita después de períodos de tres, nueve meses y tres años. Los propágulos observados, a los 8-10 días de incubación a 27-27ºC, fueron conidios en el medio de arroz (6.8 x 10 6/ml) y blastoporas (3.3 x 19 7/ml) en el medio líquido, La viabilidad de los propágulos se mantuvo en todos los soportes ensayados durante tres y nueve meses, los preservados en gel de sílice perdieron la viabilidad a los 3 años

Actividades enzimáticas en levaduras de Histoplasma capsulatum / Enzymatic activities from Histoplasma capsulatum yeasts

Se determinaron actividades enzimáticas en 5 cepas de Histoplasma capsulatum en fase levaduriforme (EH46, EH 50, EH51, EH52, EH53) por el micrométodo del APIZYM. De las 19 enzimas probadas se encontraron actividades de fosfatasa alcalina, esterasa lipasa C8, fosfatasa ácida y fosfoamidasa en todas las cepas estudiadas, mientras que la lipasa C14 y la leucina arilamidasa, sólo se determinaron en 4 cepas. Estas últimas enzimas no aparecieron en la cepa EH53 de alta virulencia

Panorama general de los hongos Entomophthorales en el control biologico de la mosca pinta en México / General panorama of Entomophthorales fungi for biological control of the spittlebug in México

Desde 1876 se han producido en México sucesivas infestaciones por una plaga de insectos dañinos conocida como "mosca pinta" o "salivazo de los pastos", Aeneolamia albofasciata y Prosapia simulants, los cuales además de desnutrir los pastizales, afectan a cultivos de productos básicos de la agricultura como son el maíz y el arroz y por lo tanto producen importantes pérdidas económicas en el país. Un medio eficaz que podría controlar la "mosca pinta", sería el uso de hongos entomopatógenos como control biológico, entre los cuales están aquéllos pertenecientes al Orden de los Entomophthorales. En América Latina se han estudiado poco estos microorganismos, sin embargo, en México se ha venido desarrollando un programa de investigación que tiene como objetivo estudiar aquellos Entomophthorales que podrían coadyuvar al control de esta plaga de insectos a través de programas de lucha biológica. Se han realizado trabajos sobre inocuidad en animales de experimentación, estudios fisiológicos para conocer las condiciones ideales que propicien el crecimiento y producción de biomasa a nivel de laboratorio, así como estudios de esporulación y germinación que permitan sentar las bases para la producción de un insecticida fúngico. Debido a su difícil taxonomía, actualmente se trabaja también en la identificación inmunológica de Entomophthorales. Este trabajo presenta un panorama general sobre la plaga de la mosca pinta en México y las diversas investigaciones realizadas en la UNAM en torno a los hongos que pudieron servir como insecticidas biológicas para este problema