Occurrence of entomopathogenic hypocrealean fungi in mosquitoes and their larval habitats in Central Brazil, and activity against Aedes aegypti.

Collecting entomopathogenic fungi associated with mosquitoes and studies on their activity against mosquito developmental stages will improve the understanding of their potential as agents to control important mosquito vectors. Twenty-one strains of entomopathogenic fungi affecting mosquitoes in Central Brazil were studied: 7 of Beauveria bassiana, 7 of Metarhizium humberi, 3 of M. anisopliae, 2 of Cordyceps sp. and one each of Akanthomyces saksenae and Simplicillium lamellicola. These fungi were isolated from field-collected mosquito adults (3 strains) or larvae (a single strain); the other 17 strains were isolated from laboratory-reared Aedes aegypti sentinel larvae set out in partially immersed cages placed in diverse small- to middle-sized aquatic mosquito habitats in or close to areas with secondary tropical forest. The frequent recovery of normally soil-borne Metarhizium spp. and B. bassiana from aquatic habitats is notable. Our laboratory findings indicated that M. anisopliae IP 429 and IP 438 and M. humberi IP 421 and IP 478 were highly active against immature stages and, together with M. anisopliae IP 432, also against adults. These strains appear to be the most promising candidates to develop effective control strategies targeting the different developmental stages of A. aegypti, the most important vector of viral diseases in humans in the tropics.

Clonostachys spp., natural mosquito antagonists, and their prospects for biological control of Aedes aegypti.

Aedes aegypti (Linnaeus, 1762) is an important vector of arboviruses in the tropics and subtropics. New control strategies based on natural enemies such as entomopathogenic fungi are of utmost importance, and the present study reports the first isolation of Clonostachys spp. (Hypocreales: Bionectriaceae) from mosquitoes and their activity against A. aegypti. Entomopathogenic fungi were surveyed in central Brazil using A. aegypti larvae as sentinels and, also, a CDC light trap. Clonostachys eriocamporesii R.H. Perera & K.D. Hyde, 2020 (IP 440) and Clonostachys byssicola Schroers, 2001 (IP 461) were identified by sequence analysis of the nuclear ribosomal internal transcribed spacer gene, and tested against eggs, larvae, and adults. Both strains were highly active against A. aegypti third instar larvae, with mortalities ≥ 80% at 107 conidia/mL after 5 days but distinctly less active against eggs and adults. This is the first report of both C. eriocamporesii and C. byssicola as naturally occurring pathogens affecting mosquitoes, and IP 440 appears to be a promising control agent against aquatic stages of A. aegypti.

Metarhizium humberi sp. nov. (Hypocreales: Clavicipitaceae), a new member of the PARB clade in the Metarhizium anisopliae complex from Latin America.

A new species, Metarhizium humberi, from the M. anisopliae complex and sister lineage of the M. anisopliae s.str. in the PARB clade, including M. pingshaense, M. anisopliae, M. robertsii and M. brunneum, is described based on phylogenetic analyses [translation elongation factor 1-alpha (5'TEF and 3'TEF), RNA polymerase II largest subunit (RPB1a), RNA polymerase II second largest subunit (RPB2a) and ß-tubulin (BTUB)]. Metarhizium humberi was first collected in 2001 in the Central Brazilian state of Goiás, later found to be a common fungus in soils in Brazil, and since then has also been isolated from coleopteran, hemipteran and lepidopteran insects in Brazil and Mexico. This new species, named in honor of Richard A. Humber, a well-known insect pathologist and taxonomist of entomopathogenic fungi, is characterized by a high insecticidal activity against different developmental stages of arthropod pests with importance in agriculture and vectors of diseases to human and animals.

Metarhizium alvesii sp. nov.: A new member of the Metarhizium anisopliae species complex.

A strain within the Metarhizium anisopliae species complex was isolated in 2009 from a soil sample in a banana plantation in the municipality of Quixeré, Northeastern region of Brazil. Previous studies showed that this insect-pathogenic strain does not fit with any current taxon within the M. anisopliae species complex, as determined by both genomic and by mass spectrometric analyses. In the present study, CG1123 (=ARSEF 13308) is shown to be morphologically indistinguishable from most species in this cosmopolitan species complex, whereas multilocus phylogeny confirmed its uniqueness and supports its recognition as a new species, Metarhizium alvesii, in honor of Sérgio Batista Alves, one of the founders of insect pathology in Brazil.

Conidiobolus macrosporus (Entomophthorales), a mosquito pathogen in Central Brazil.

A new fungal pathogen of Culicinae (Diptera: Culicidae) adults, Conidiobolus macrosporus (Entomophthorales: Ancylistaceae), was detected and isolated during a survey of mosquito pathogens close to the city of Aruanã, Goiás State, in December 2014. The morphological characteristics of C. macrosporus are presented, and reasons for some uncertainty about this identification are discussed. The pathogenicity and high virulence of this fungus for Aedes aegypti were confirmed in laboratory conditions. Mortality of adults exposed to conidia was observed within 24h of exposure to the pathogen, and increased to 100% as quickly as 3days after inoculation (with the highest conidial concentration tested, 8.3×10(4)conidia/cm(2)). Repeated attempts to obtain genomic sequence data failed despite confirmations that the DNA extraction methods were themselves successful.

First report of Leptolegnia chapmanii (Peronosporomycetes: Saprolegniales) affecting mosquitoes in central Brazil.

Numerous isolates of an oomycete 'fungus', Leptolegnia chapmanii, are reported from Brazil for the first time. This aquatic pathogen was baited with Aedes aegypti sentinel larvae from stagnant, temporary bodies of water in selected locations under secondary tropical forest in and near the central Brazilian city of Goiânia and from more distant sites in the western and northern regions of the state of Goiás. Isolates were identified based on their morphological and developmental characters, comparative sequence data for the ITS and TEF loci, as well as their rapid activity against A. aegypti larvae. Taxonomic issues affecting the application of the name L. chapmanii and its typification are rectified. This study contributes to a better understanding of the presence and distribution of this oomycete in Brazil, its sequence-based identification, and of its potential as a biological agent against mosquito vectors.

Occurrence of Metarhizium spp. in Central Brazilian soils.

The biodiversity of entomopathogenic fungi in tropical ecosystems is still little investigated, and the objective of this study was to isolate and identify fungi of the entomopathogenic genus Metarhizium (Hypocreales: Clavicipitaceae) present in undisturbed soils of the Central Brazilian Cerrado. A total of 107 Metarhizium isolates was obtained from soils collected from Cerrado sites in the state of Goiás; gene sequences from 63 of these were obtained and compared. Among these, one was confirmed to be M. anisopliae sensu stricto; 53 were very closely allied to M. anisopliae but require more extensive genetic characterization to determine if they might represent a new taxon in the M. anisopliae species complex. Eight of these Cerrado isolates were referable to M. robertsii, and the remaining isolate is the first South American (and Southern Hemisphere) collection of M. flavoviride var. pemphigi. These findings underline the need for better characterization of the diversity of these widely distributed fungi in Brazil.

Utility of six fungicides for selective isolation of Evlachovaea sp. and Tolypocladium cylindrosporum.

The effects of the fungicides dodine, benomyl, thiabendazole, mancozeb, cupric sulfate, and copper oxychloride were examined in vitro upon germination and further development of Evlachovaea sp. and Tolypocladium cylindrosporum. Fungicidal activity depended on concentrations and varied among products, fungi and the strains tested. Depending on the fungicidal concentration, germination of conidia was induced but germlings produced neither mycelium nor new conidia. There was a good recovery of both Evlachovaea sp. and T. cylindrosporum from previously sterilized soils with fungicide-supplemented medium. Fungi were resistant to copper oxychloride up to 30 g/l, and this fungicide was found to have no utility for a selective medium. Minimal fungicide concentrations for successful isolations were 1 mg/l for benomyl, 200 mg/l for cupric sulfate, 50 mg/l for dodine, 100 mg/l for mancozeb, and 4 mg/l for thiabendazole. Thiabendazole, which is easy to obtain and can be used in low quantities, showed the greatest utility for a selective medium with these entomopathogenic fungi.

Record of Evlachovaea sp. (Hyphomycetes) on Triatoma sordida in the state of Goiás, Brazil, and its activity against Triatoma infestans (Reduviidae, Triatominae).

A fungal isolate was detected on a dead Triatoma sordida (Stål) collected in a peridomestic area in central Brazil. The fungus belongs to Evlachovaea Borisov and Tarasov, a new genus that was recently described in Russia. The isolate seems to be a third species and the second new and undescribed species from Brazil. The fungus was shown to be active against Triatoma infestans (Klug) third-instar nymphs at a humidity close to saturation. However, activity was reduced at a lower humidity (75%). Values of LC50 varied between 1.1 x 10(5) and 1.5 x 10(4) conidia/cm2 treated surface, 15 and 20 d after fungal application and incubation at humidity close to saturation. This new fungus may have a potential for biological control of peridomestic Chagas' disease vectors during the rainy season.

Morphology and molecular taxonomy of Evlachovaea-like fungi, and the status of this unusual conidial genus.

The entomopathogenic anamorphic genus Evlachovaea was described to differ from other fungi in forming its conidia obliquely to the axis of the conidiogenous cell and with successive conidia having alternate orientations with a zipper- or chevron-like arrangement resulting in flat, ribbon-like chains. Morphological and molecular studies of six Evlachovaea-like isolates baited from Central Brazilian soils using Triatoma infestans (a vector of Chagas disease) and of other entomopathogens with Evlachovaea-like conidiogenesis led to a re-evaluation of the status of this little known fungal genus. The Brazilian isolates formed two distinct groups based on gene sequences for both the internal transcribed spacer (ITS) and translation elongation factor (EF-1α) genes, morphology, and growth patterns; both groups also differed from the type species, Evlachovaea kintrischica. More detailed studies of these fungi indicated that the alternatingly oblique orientations of forming conidia are neither a stable nor invariant character (even on single phialides). Furthermore, the molecular cladistic analysis unambiguously placed the Evlachovaea isolates firmly within the genus Isaria (Hypocreales: Cordycipitaceae). The ITS sequences of E. kintrischica were very similar or even identical to those of Isaria amoenerosea and Isaria cateniobliqua, thereby suggesting that E. kintrischica is a synonym of one of these species, and that the genus Evlachovaea must be treated as a later synonym of Isaria, which must now be recognized to include several highly divergent modes of conidiogenesis. These taxonomic findings are discussed in the context of dramatic changes recently imposed on the nomenclatural standards used to determine the correct names of all pleomorphic fungi.

Diatomaceous earth and oil enhance effectiveness of Metarhizium anisopliae against Triatoma infestans.

Entomopathogenic fungi, especially Metarhizium anisopliae, have potential for integrated control of peridomestic triatomine bugs. However, the high susceptibility of these vectors to fungal infection at elevated ambient humidities decreases in the comparatively dry conditions that often prevail in their microhabitats. A formulation adapted to this target pest that induces high and quick mortality can help to overcome these drawbacks. In the present study diatomaceous earth, which is used against pests of stored grains or as an additive to mycoinsecticides, delayed but did not reduce in vitro germination of M. anisopliae s.l. IP 46 conidia after >24h agitation without affecting viability, and did not hamper the survival of Triatoma infestans nymphs exposed to treated surfaces. The settling behavior of nymphs on a treated surface in choice tests depended on the concentration of diatomaceous earth and ambient light level. Conidia formulated with diatomaceous earth and a vegetable oil synergized the insecticidal effect of the fungus in nymphs, and quickly killed all treated insects, even at 75% relative humidity (LT(90) 8.3 days) where unformulated conidia caused only 25% mortality after a 25 days exposure. The improved performance of a combined oil and desiccant dust formulation of this Metarhizium isolate raises the likelihood for its successful mycoinsecticidal use for triatomine control and, apparently, against other domestic insect pests.

Activity of Metarhizium spp. and Isaria spp. from the Central Brazilian Cerrado against Triatoma infestans nymphs.

Entomopathogenic fungi attack Chagas disease vectors, and there is a need of effective isolates for development of biological control. Ten isolates of Metarhizium spp., four of Isaria cateniannulata and two of an Isaria sp., all isolated with Triatoma infestans as a bait insect from soils collected in Central Brazil, and tested under laboratory conditions were effective against third instar nymphs of T. infestans. This is the first report of pathogenicity of M. flavoviride var. pemphigi, M. robertsii and I. cateniannulata against T. infestans. M. robertsii IP 34 and I. cateniannulata IP 141 are among the most virulent fungi reported, and both have potential for integrated control of triatomine vectors.

Activity of some hypocrealean fungi collected in a Cerrado ecosystem against Rhodnius spp. (Hemiptera: Reduviidae) under laboratory conditions.

The hypocrealean invertebrate-pathogenic fungi Lecanicillium psalliotae, Paecilomyces lilacinus and Pochonia chlamydosporia isolated from soils in Central Brazil were tested against palm-dwelling Rhodnius neglectus, R. nasutus, R. prolixus, and R. robustus; all of these insects are vectors of Chagas disease. Particularly R. neglectus and R. nasutus, which are both important secondary intruders of human habitations were susceptible to all three of the fungi tested, and fungi sporulated successfully on the cadavers. This is the first report of activity of L. psalliotae, P. lilacinus and P. chlamydosporia against Rhodnius spp., and these fungi have interest for integrated control of triatomine vectors.

A new resting trap to sample fungus-infected mosquitoes, and the pathogenicity of Lecanicillium muscarium to culicid adults.

Some soil-dwelling entomopathogenic fungi that are widely used in pest control are also able to reduce the survival of adult mosquito vectors under laboratory conditions. However, there is still little information about the naturally occurring fungal pathogens affecting culicid mosquitoes. As such, we hypothesized that fungi that already kill mosquitoes in realistic domestic environments could be effective against these vectors in human habitations. A simple, inexpensive, handmade, cylindrical kiln-fired clay pot (30 cm height, 24 cm inner diameter, 0.8-1cm wall thickness) was modified into a trapping device for resting adult mosquitoes and to sample fungus-infected moribund and dead individuals. The entomopathogenic fungus Lecanicillium muscarium was isolated from a dead culicid mosquito collected with this trap in southeastern Tanzania. This isolate is the first L. muscarium reported to occur naturally on adult culicids in Tanzania and was found to be pathogenic also to adults of Aedes aegypti, Anopheles arabiensis and Culex quinquefasciatus under laboratory conditions. The trapping device confirmed its efficacy to sample mosquito-specific fungi in domestic locations and that the isolated fungus might have potential for mosquito control.

Activity of oil-formulated Beauveria bassiana against Triatoma sordida in peridomestic areas in Central Brazil.

Field tests were carried out during the rainy season of 2001/2002 in São Luís de Montes Belos, Goiás, Brazil, to evaluate the potential of the entomopathogenic fungus, Beauveria bassiana, against peridomestic Triatoma sordida. An oil-water formulation of the isolate CG 14 (Embrapa) was applied in triatomine infested hen houses of four farms at a final concentration of 10(6) conidia/cm2. Numbers of T. sordida decreased over the next 25 days, after application of the fungus, and B. bassiana developed on dead insects in one hen house. A high number of B. bassiana colonies was detected in substrates collected in treated hen houses 24 h after application of CG 14. In the following three months the presence of B. bassiana declined to values found before treatment.

Activity of some hypocrealean fungi collected in a Cerrado ecosystem against Rhodnius spp. (Hemiptera: Reduviidae) under laboratory conditions

The hypocrealean invertebrate-pathogenic fungi Lecanicillium psalliotae, Paecilomyces lilacinus and Pochonia chlamydosporia isolated from soils in Central Brazil were tested against palm-dwelling Rhodnius neglectus, R. nasutus, R. prolixus, and R. robustus; all of these insects are vectors of Chagas disease. Particularly R. neglectus and R. nasutus, which are both important secondary intruders of human habitations were susceptible to all three of the fungi tested, and fungi sporulated successfully on the cadavers. This is the first report of activity of L. psalliotae, P. lilacinus and P. chlamydosporia against Rhodnius spp., and these fungi have interest for integrated control of triatomine vectors

Detection of entomopathogenic fungi in peridomestic triatomine-infested areas in central Brazil and fungal activity against Triatoma infestans (Klug) (Hemiptera: Reduviidae) / Detecção de fungos entomopatogênicos em áreas peridomiciliares infestadas por triatomíneos no Brasil central e atividade dos fungos contra Triatoma infestans (Klug) (Hemiptera: Reduviidae)

From 148 substrate samples collected in peridomestic triatomine-infested habitats of 24 farms in Central Brazil, 31 isolates of Metarhizium anisopliae (Metsch.) Sorok. and 15 isolates of Beauveria bassiana (Bals.) Vuill. were obtained. Most of the isolates were found in substrates that consisted of soil mixed with animal feces or soil only in areas of trees where poultry roost and pig houses. Fungi were detected with an insect bait method using Triatoma infestans (Klug), and combined in vitro and in vivo techniques with modified Chase medium and T. infestans. All isolates were highly virulent to T. infestans third instar nymphs at a relative humidity > 98 percent and temperature of 25 ± 0.5°C. However, activity against nymphs was reduced at 75 percent relative humidity. Results underline the potential of B. bassiana and M. anisopliae as agents for biological control of vectors of Chagas disease. Both species occur naturally in peridomestic habitats of triatomines in Central Brazil and may contribute to control these vectors and to reduce the risk of reinfestation of houses after eliminating domestic vector species.

Foram detectados 31 isolados de Metarhizium anisopliae (Metsch.) Sorok. e 15 isolados de Beauveria bassiana (Bals.) Vuill. em 148 amostras de substratos coletadas em habitats peridomiciliares infestados com triatomíneos de 24 fazendas localizadas no Centro do Brasil. A maioria dos isolados foi encontrada em solos misturados com fezes de animais ou solos de áreas de poleiros e chiqueiros. Os fungos foram detectados com um método de captura utilizando Triatoma infestans (Klug) como isca, e um método combinado in vitro e in vivo, com meio Chase modificado e T. infestans. Os primeiros estudos sobre a atividade dos fungos indicaram que todos os isolados foram patogênicos para T. infestans quando testados em umidade relativa acima de 98 por cento e 25 ± 0,5°C. Porém, a atividade foi reduzida em umidade relativa de 75 por cento. Os resultados ressaltam o potencial de B. bassiana e M. anisopliae como agentes de controle para os vetores da doença de Chagas. As duas espécies ocorrem naturalmente em habitats peridomiciliares de triatomíneos no Centro do Brasil e podem contribuir para o controle desses vetores e reduzir o risco de reinfestação das casas após eliminação de espécies domiciliares.

Activity of oil-formulated Beauveria bassiana against Triatoma sordida in peridomestic areas in central Brazil

Field tests were carried out during the rainy season of 2001/2002 in São Luís de Montes Belos, Goiás, Brazil, to evaluate the potential of the entomopathogenic fungus, Beauveria bassiana, against peridomestic Triatoma sordida. An oil-water formulation of the isolate CG 14 (Embrapa) was applied in triatomine infested hen houses of four farms at a final concentration of 10(6) conidia/cm². Numbers of T. sordida decreased over the next 25 days, after application of the fungus, and B. bassiana developed on dead insects in one hen house. A high number of B. bassiana colonies was detected in substrates collected in treated hen houses 24 h after application of CG 14. In the following three months the presenceof B. bassiana declined to values found before treatment.