In vitro efficacy of Metarhizium anisopliae sensu lato against unfed Amblyomma parvum (Acari: Ixodidae).

Amblyomma parvum Aragão (Acari: Ixodidae) is a tick species found with wide distribution in the Neotropical region. Even though it is a wildlife-related tick, it is also a frequent parasite of domestic animals, is aggressive to human beings and may harbor pathogenic microorganisms. Therefore, it is a target species for control on domestic animals, particularly those at the rural-wildlife interface. Herein, the efficacy of two isolates (E9 and IBCB 425) of an entomopathogenic fungus, Metarhizium anisopliae sensu lato, already evaluated for ticks that parasitize domestic animals, was tested against unfed A. parvum adults. Both isolates displayed high acaricidal efficacy after immersion in fungal conidial suspensions for 5 min. Isolate E9 killed all ticks by the 7th day post-treatment, and isolate IBCB 425 did so by the 11th day. Tick mortality of 80 and 90% was achieved as early as the 3rd and 4th days, respectively, with both treatments. Thus, if a commercial M. anisopliae s.l. acaricide against domestic animal ticks is developed, it would also be effective against A. parvum.

Compatibility of Metarhizium anisopliae with liposoluble photoprotectants and protective effect evaluation against solar radiation / Compatibilidade de Metarhizium anisopliae com fotoprotetores lipossolúveis e avaliação do efeito protetor contra a radiação solar

The aim of this study was to analyze the compatibility of the Metarhizium anisopliae JAB 68 isolate with liposoluble photoprotectants and the photoprotection conferred to its conidia. The fungus was cultivated in a medium with the photoprotectants Neo Heliopan® BB, Neo Heliopan® E 1000, Eusolex® 6007 and Neo Heliopan® AV in various concentrations. Vegetative growth, sporulation and germination were assessed to determine compatibility. Then, conidia were suspended in oil containing the compatible photoprotectants, in their respective concentrations, and exposed to 0-7 hours of light from a solar simulator operating at 1,000 W m-2. Neo Heliopan BB® and Neo Heliopan® E 1000 were compatible with M. anisopliae at 2% and 1%, respectively. Eusolex® 6007 and Neo Heliopan® AV were compatible up to 8% a 7%, respectively. Conidia exposed to radiation from the solar simulator, with photoprotectant-free oil as its carrier, presented germination of 78.12% after five hours. When adding any photoprotectant to the conidia oily suspension, the germination percentage was always higher than that obtained in the control. Neo Heliopan® BB at 1 and 2% and Neo Heliopan® AV at 0.5% increased the germination. After 7 hours of exposure the germination percentages were 93.77, 94.55 and 98.82%, respectively. Eusolex® 6007 was not as efficient as previous products, but in the same exposure period was able to protect the conidia (88.60% germination) when used at a 1% concentration. Adding photoprotectants in oily formulations of M. anisopliae protects against the harmful effects of UV radiation contributing to the fungus efficiency in the field.

O objetivo desse trabalho foi analisar a compatibilidade do isolado JAB 68 de Metarhizium anisopliae com fotoprotetores lipossolúveis e a fotoproteção conferida aos seus conídios. O fungo foi cultivado em meio contendo os fotoprotetores Neo Heliopan® BB, Neo Heliopan® E 1000, Eusolex® 6007, Neo Heliopan® AV em várias concentrações. O crescimento vegetativo, a esporulação e a germinação foram avaliados para determinar a compatibilidade. Conídios foram suspensos em óleo contendo os fotoprotetores compatíveis, em suas respectivas concentrações, e submetidos por 0 a 7 horas à luz de um simulador solar na potência de 1.000W m-2. Neo Heliopan BB® e Neo Heliopan® E 1000 foram compatíveis com M. anisopliae à 2% e 1%, respectivamente. Eusolex® 6007 e Neo Heliopan® AV foram compatíveis até 8% e 7%, respectivamente. Conídios de M. anisopliae expostos à radiação do simulador solar, tendo óleo isento de fotoprotetor como veículo, apresentaram germinação de 78,12% após cinco horas. Adicionando-se qualquer fotoprotetor à suspensão oleosa de conídios a porcentagem de germinação foi sempre maior que a obtida no controle. Neo Heliopan® BB a 1 e 2% e Neo Heliopan® AV a 0,5% incrementaram a germinação. Após 7 horas de exposição as porcentagens de germinação foram de 93,77, 94,55 e 98,82%, respectivamente. Eusolex® 6007 não foi tão eficiente quanto os produtos anteriores, mas no mesmo período de exposição foi capaz de proteger os conídios (88,60% de germinação) quando usado na concentração de 1%. A adição de fotoprotetores em formulações oleosas de M. anisopliae confere proteção aos raios UV, contribuindo com a eficiência do fungo no campo.

Compatibilidade de isolados de Beauveria bassiana a inseticidas, herbicidas e maturadores em condições de laboratório / Compatibility of Beauveria bassiana in the laboratory for insecticides, herbicides and ripeners

Foram avaliadas as diferenças de toxicidade de inseticidas, herbicidas e maturadores para os isolados AM 09, JAB 07, IBCB 07 e JAB 46 de Beauveria bassiana, por intermédio de experimentos realizados em meio de cultura. Os isolados foram inoculados em meio de batata, dextrose e ágar (BDA) contendo os agroquímicos. Foram avaliados a germinação, o crescimento e a esporulação e, com base nesses parâmetros, fez-se a classificação toxicológica dos agroquímicos para cada isolado. O inseticida Actara 250 WG(r) foi considerado compatível com AM 09, moderadamente tóxico para JAB 07 e tóxico para IBCB 07 e JAB 46. O Regente WG 800(r) foi considerado compatível com todos os isolados, já o Temik 150(r) se mostrou tóxico. Quanto aos herbicidas, o produto Glifosato Nortox(r) foi considerado compatível com AM 09, moderadamente tóxico para IBCB 07 e JAB 46 e tóxico para JAB 07. Plateau(r) foi classificado como moderadamente tóxico para AM 09 e JAB 07 e tóxico para IBCB 07 e JAB 46. Contain(r), DMA(r), Karmex(r), Sencor 480(r) e Velpar-K(r) foram classificados como tóxicos para todos os isolados. Tratando-se dos maturadores, tanto Curavial K(r) quanto Moddus(r) se mostraram tóxicos para todos os isolados. Os resultados revelaram que há variação entre os isolados de B. bassiana quanto à toxicidade dos agroquímicos. AM 09 foi o mais tolerante à ação tóxica dos produtos. A germinação sofreu menor efeito do que o crescimento e a esporulação, e JAB 07 foi o isolado menos afetado pelos agroquímicos com relação a esse parâmetro.(AU)

The differences in the toxicity of pesticides, herbicides and ripeners for isolates AM 09, JAB 07, IBCB 07 e JAB 46 of Beauveria bassiana were evaluated through experiments conducted in culture medium. The isolates were cultured on potato dextrose agar (PDA) containing agrochemicals. Germination, growth and sporulation were evaluated and, based on these parameters, the toxicological classification of pesticides for each isolate was made. The insecticide Actara(r) was considered compatible with AM 09, moderately toxic for JAB 07 and toxic for IBCB 07 and JAB 46. Regente WG 800(r) was considered compatible with all isolates, while Temik 150(r) showed to be toxic. In relation to the herbicides, the product Glifosato Nortox(r) was considered compatible with AM 09, moderately toxic for IBCB 07 and JAB 46 and toxic for JAB 07. Plateau(r) was classified as moderately toxic for AM 09 and JAB 07, and toxic for IBCB 07 and JAB 46. Contain(r), DMA(r), Karmex(r), Sencor 480(r) e Velpar-K(r) were classified as toxic for all isolates. In the case of ripeners, both Curavial K(r) and Moddus(r) showed to be toxic for all the isolates. The results revealed that there is variation between isolates of B. bassiana regarding the toxicity profile of agrochemicals. AM 09 was the most tolerant to the toxic action of the products. Germination suffered minor effect than growth and sporulation, and JAB 07 was the isolate less affected by agrochemicals with regard to this parameter.(AU)

Sensibilidade de isolados de fungos entomopatogênicos às radiações solar, ultravioleta e à temperatura / Sensibility of isolates of entomopathogenic fungi to solar radiation, ultraviolet rays and temperature

Este trabalho teve por objetivo avaliar a sensibilidade de isolados dos fungos Metarhizium anisopliae (Metsch.) Sorok. e Beauveria bassiana (Bals). Vuill. ao efeito das radiações solar e ultravioleta e da temperatura. Conídios dos isolados foram expostos, por vários períodos, aos raios de um simulador solar em diversas irradiâncias e a uma lâmpada de raios ultravioleta germicida. Os conídios do isolado de M. anisopliae foram também expostos às temperaturas de 19,5; 24,2 e 31,0ºC, e os do isolado de B. bassiana a 19,4; 20,8 e 28,3ºC, e 18,7; 23,8 e 30,9ºC. Avaliou-se a germinação de conídios pelo teste de viabilidade. Os isolados dos fungos se mostraram bastantes sensíveis aos raios do simulador solar e aos raios ultravioleta. A germinação de ambos sofreu significativa redução a partir de 30 minutos de exposição à radiação do simulador solar. O efeito mais severo foi evidenciado pelo isolado de B. bassiana, com grande redução da germinação dos conídios em todas as irradiâncias testadas. A sensibilidade à radiação ultravioleta também foi grande, pois ocorreu acentuada redução da germinação dos conídios do isolado de M. anisopliae (38,2%) e de B. bassiana (65%) já aos 30 segundos de exposição. A temperatura afetou a viabilidade de ambos os fungos. Temperaturas entre 23,8 e 31ºC favoreceram a germinação dos conídios, enquanto temperaturas próximas de 20ºC dificultaram a germinação.(AU)

This study aimed to access the sensibility of isolates of the fungus Metarhizium anisopliae (Metsch.) Sorok. and Beauveria bassiana (Bals.) Vuill. to the effect of solar and ultraviolet radiation and temperature. Conidia were exposed for various periods to the rays from a solar simulator at various irradiances, and to light germicidal ultraviolet rays. Conidia of the isolate of M. anisopliae were also exposed to temperatures of 19.5, 24.2 and 31.0ºC and the isolate of B. bassiana to 19.4, 20.8 and 28.3ºC, and also to 18.7, 23.8 and 30.9ºC. The germination of conidia was evaluated by the viability test. The fungal isolates showed to be very sensitive to the solar simulator and ultraviolet rays. Germination of both was significantly decreased starting from 30 minutes of exposure to the rays of the solar simulator. The most severe effect was evidenced by the isolate of B. bassiana with great reduction in conidia germination in all the tested irradiances. Sensitivity to ultraviolet radiation was also great, showing a marked reduction in the germination of M. anisopliae (38.2%) and B. bassiana (65%) conidia after 30 seconds of exposure. The temperature affected the viability of both fungi. Temperatures ranging of 23.8 to 31ºC favor the germination of conidia while temperatures around 20ºC constrained germination.(AU)

Effect of Metarhizium anisopliae fungus on off-host Rhipicephalus (Boophilus) microplus from tick-infested pasture under cattle grazing in Brazil.

This work aimed to assess the effect of the fungus Metarhizium anisopliae on off-host Rhipicephalus (Boophilus) microplus from tick-infested Brachiaria decumbens pasture undergoing cattle grazing. For this purpose a naturally tick-infested Brachiaria decumbens pasture of 60 m × 100 m with twelve grazing Holstein Friesian-Nelore (Bos indicus) cross breed bovines was sprayed 12 times, 21 days apart with an aqueous conidial suspension of the E9 isolate of M. anisopliae fungus. Control pasture was treated with conidial suspension vehicle only. Efficacy of treatment was evaluated by tick larvae counts on the pasture and that of engorged female ticks on grazing cattle. Fungus persistence on grass stems as well as soil pasture was assessed after each spray. Efficacy of fungus against ticks was also measured by an in vitro immersion test. Whereas in vitro test showed a clear pathogenic effect of the fungus on ticks, no pathogenic effect over R. (B.) microplus ticks was detected in the field trial by spraying pasture with fungal suspension. Fungus from the suspension could be recovered from both the grass stem as well as the soil of sprayed pasture, even though the numbers obtained varied distinctly and could only be recovered shortly after spray. Pasture environments with exposure to sun and rain, seem to be very detrimental to the fungus, thus suspensions which provide fungus with protection or more resistant isolates to these should be looked for. In order to achieve high fungal efficacy against tick under field conditions, accurate laboratory assays, optimization of application strategy and knowledge of interactions between fungal strains and environmental factors are warranted.

Reaction of Bipolaris euphorbiae to environmental factors and its tolerance to solar and ultraviolet radiation.

The determination of the ideal cultivation conditions and the knowledge of solar radiation tolerance are important aspects that must be investigated for the use of Bipolaris euphorbiae Muchovej and Carvalho as a biocontrol agent. The present study was done to assess the growth rate, sporulation and viability of the fungus cultivated under different conditions of the initial pH value of the growth medium, temperature and photoperiod. The tolerance of this fungus' conidia to light from a source simulating solar and ultraviolet radiation was also assessed. B. euphorbiae was affected by different pH values of the growth medium being 6.0 the adequate pH for fungus cultivation. Incubation of the fungus under temperatures of 22, 25 and 28°C enhanced mycelium growth, while sporulation and viability of conidia were better at 22 and 16°C. Different regimens of exposure to light of fungal cultures only affect sporulation. Conidia exposed to solar and ultraviolet radiation for 8 h and 90 min, respectively, presented viabilities higher than 92%, indicating high tolerance levels to radiation exposure.

Entomopathogenic fungal activity against pupae and adult Haematobia irritans (Diptera: Muscidae).

The horn fly Haematobia irritans is one of the most important ectoparasites associated with grazing bovines. This study investigated the pathogenic activity of Metarhizium anisopliae (E9, IBCB425 and IBCB159), Beauveria bassiana (JAB06, JAB07 and AM09), Isaria fumosorosea (=Paecilomyces fumosoroseus) (IBCB133 and CB75) and Isaria farinosa (=Paecilomyces farinosus) (CG189 and CG195) fungi isolates on pupae and adult H. irritans. Groups of 20 pupae and 30 adult flies were respectively bathed and sprayed with fungal isolate suspensions containing 10(6), 10(7) and 10(8) conidia ml(-1) in bioassays conducted in laboratories. In both assays the adult flies were fed bovine blood for 15 days, and death rates were assessed daily. The E9 and IBCB425 M. anisopliae isolates caused pupae death at concentrations of 10(7) and 10(8) conidia ml(-1), and the JAB07 and AM09 B. bassiana isolates caused higher pupae mortality at a concentration of 10(8) conidia ml(-1). I. farinosa isolates were the most effective considering pupae mortality, with the CG195 inducing more deaths (56.6%) in the 10(8) conidia ml(-1) concentration suspension. Adult flies were more susceptible to the fungi's pathogenic action, since the E9 isolate of M. anisopliae and all of the B. bassiana induced death in 100% of the flies at the 10(8) conidia ml(-1) concentration suspension. The I. fumosorosea and I. farinosa isolates, on the other hand, were less effective in controlling adult flies. In both stages, but mostly the adult phase, pathogenicity was great at higher conidial concentrations.

Efficiency of entomopathogenic fungi in the control of eggs and larvae of the horn fly Haematobia irritans (Diptera: Muscidae).

The present study assessed the pathogenic effect of isolates E9, IBCB425 and IBCB159 of the Metarhizium anisopliae fungus, JAB06, JAB07 and AM09 of Beauveria bassiana, IBCB133 and CB75 of Isaria fumosorosea (=Paecilomyces fumosoroseus) and CG189 and CG195 of Isaria farinosa (=Paecilomyces farinosus) against eggs and larvae of the horn fly Haematobia irritans. Eggs were inoculated with suspensions containing 10(6), 10(7) and 10(8)conidiaml(-1) of the fungal isolates and observed after 48h to determine viability. In the larvae study, eggs were allowed to hatch into fresh bovine feces that had been treated with 10(8), 10(7) or 10(6)conidiamgfeces(-1). In both studies, 5 days after initial procedures, all formed pupae were transferred to an incubator at 27+/-0.5 degrees C until the emergence of the adult flies was complete. The M. anisopliae isolates did not cause the death of H. irritans eggs, but they did promote the death of larvae that hatched from treated eggs, and therefore increased the total mortality. Isolate E9 promoted 100% mortality of treated larvae at a concentration of 10(8)conidiaml(-1). For the B. bassiana isolates, no activity was observed against insect eggs or larvae. Both I. fumosorosea isolates promoted significant mortality (p<0.05) of eggs at every concentration of conidia. Isolate CG195 of I. farinosa increased the mortality of larvae and pupae that hatched from treated eggs and promoted significant total mortality (p<0.05) of the insect at every concentration of conidia.

Susceptibility of adult and larval stages of the horn fly, Haematobia irritans, to the entomopathogenic fungus Metarhizium anisopliae under field conditions.

The efficacy of M. anisopliae strain E9 as a biological insecticide for the adult and larval stages of H. irritans was assessed under field conditions. To assess larvicidal activity, nine heifers were randomly divided into three groups, which were maintained separated from each other. The first group ingested fungal spores encapsulated in alginate pellets. The second group ingested in natura spores that were grown on sterilized rice. In both groups, each animal received three meals a day, with each meal containing 2 x 10(10)conidia. The third group received no treatment and was used as a control. Fecal samples from manure and whole dung pats were collected from each of the three separate pastures on the day that the animals were allocated and on days 1, 3, 6, 9 and 12 afterwards. The fecal samples were tested for the presence of fungal colony forming units (CFU), and the emergence of horn flies was observed in the dung pats. Significantly less (P<0.01) adult horn flies were found in dung pats of the group treated with encapsulated fungi (11.7) than in those from the heifers treated with conidia in natura (27.9) or from the control group (29.5). The fecal samples of the treated animals presented significantly higher numbers of M. anisopliae CFUs then those from the untreated controls. We found that on day 9 fecal samples from animals given microencapsulated conidia had significantly higher CFUs than those from animals treated with conidia in natura. To assess adulticide activity, four heifers were sprayed with a suspension of 3 x 10(10)conidial(-1) of M. anisopliae, and four control animals were sprayed with the same solution without conidial content. Four sprayings were done at five-day intervals, and all animals were photographed daily to observe the quantity of flies present. After the second spraying, we observed an average of 22.9 flies per animal; untreated heifers had an average of 43 flies per animal; thus, the treatment significantly (P<0.05) decreases fly infestation. The results obtained from both tests show that M. anisopliae strain E9 has a pathogenic effect on H. irritans larvae in bovine manure when administered orally and on adult fly infestations when applied as a spray on the hosts.

Influence of culture media in viability test of conidia of entomopathogenic fungi

O presente trabalho objetivou investigar se meios de cultura utilizados em teste de viabilidade afetam a germinação de conídios de cinco isolados de Lecanicillium lecanii, cinco de Beauveria bassiana e quatro de Paecilomyces fumosoroseus. Os testes foram realizados em lâminas de microscopia contendo um dos seguintes meios de cultura: Agar-água (AA), Meio Mínimo (MM), Batata, dextrose e ágar (BDA), Batata, dextrose, ágar e 1% de extrato de levedura (BDAL), Sabouraud, dextrose, ágar e extrato de levedura (SDAL) e Meio Completo (MC). Delimitaram-se três áreas por lâmina e em cada uma aplicou-se 0,05mL de uma suspensão com concentração de 5,5 x 105 conídios ml-1. Para cada isolado foi realizado um bioensaio, com seis tratamentos e cinco repetições. Avaliou-se a germinação 15 horas após incubação, a 26±0,5ºC. Os meios de cultura influíram na capacidade de germinação das três espécies estudadas, ocorrendo variações inter e intraespecíficas. Verificou-se que os meios Completo e BDA proporcionaram as maiores porcentagens de germinação dos isolados de L. lecanii, sendo que e as menores foram obtidas nos meios SDAL e AA. Os meios ricos em nutrientes (BDA, BDAL e Completo) favoreceram a germinação dos isolados de B. bassiana, o que não ocorreu com os meios pobres (AA e MM). Nos meios Completo e BDA foram obtidas as maiores porcentagens de germinação dos isolados de P. fumosoroseus. As menores percentagens, por sua vez, foram obtidas no meio SDAL. Entretanto, alguns isolados apresentaram alta germinação em meios pobres em nutrientes (AA e MM).