Natural infection of Chiromyzinae larvae (Diptera: Stratiomyidae) in southern Chile by Tolypocladium valdiviae sp. nov.

A new species from the fungal genus Tolypocladium (Hypocreales: Ophiocordycipitaceae) that infects Stratiomyidae larva from the genus Hylorops is described: Tolypocladium valdiviae Gallardo-Pillancari, Montalva & González. The description is based on both genomic data and morphological characteristics. The sexual stage of T. valdiviae presents fleshy and visible stromata; unlike Tolypocladium ophioglossoides, it is smaller and emerges directly from its host and resembles Tolypocladium longisegmentis and Tolypocladium capitatum, both of which are parasites of deer truffle fungi of the genus Elaphomyces (Ascomycota: Eurotiales). In the anamorphic state, T. valdiviae presents conidiogenous cells similar in shape and arrangement to those of Tolypocladium inflatum, however T. valdiviae produces larger conidiogenous cells and, occasionally, produces chlamydospores. Phylogenetic evidence suggested that T. valdiviae is in a clade close to T. longisegmentis, T. inflatum and T. ophioglossoides, species also recognized to be parasites of fungi of the genus Elaphomyces. The new species is known so far only from Valdivia, southern Chile.

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.

Activity Against Musca domestica of Hypocrealean Fungi Isolated from Culicids in Central Brazil and Formulated in Vermiculite.

Musca domestica L. is a cosmopolitan nuisance of high sanitary importance. Entomopathogenic fungi are innovative and attractive tools for integrated control of the housefly to overcome insufficient levels of control caused by increasing resistance of this pest against chemical insecticides. High virulence of a fungal strain is a prerequisite to develop a mycoinsecticide, and the present study investigated the potential of hypocrealean fungi from the genera Beauveria, Clonostachys, Cordyceps, Akanthomyces, Metarhizium, and Tolypocladium, isolated from mosquitoes in Central Brazil against M. domestica. The highest mortalities (larvae, pupae, and adults) were caused by Metarhizium humberi IP 478 (98%) and IP 421 (90%), Metarhizium anisopliae IP 432 (85%), Beauveria bassiana IP 433 (82%), and Tolypocladium cylindrosporum IP 425 (68%) after a 23-day exposure of initially pre-pupating third instar larvae to conidia mixed with vermiculite. Lethal concentrations to kill 90% of adults of IP 433 and IP 478 were 5 × 107 and 108 conidia g-1 substrate, respectively. Fifty percent of adults were killed within 4 to 5 days of exposure initially as pupae close to emergence to substrate treated with conidia of IP 478 or IP 433 at 1.1 × 108 conidia g-1, respectively. The other fungal strains tested were less virulent. The results demonstrate high potentials for conidial preparations in vermiculite of IP 433 and IP 478 as candidates for the biological control of both pre-pupating larvae, pupae, and emerging adults of houseflies.

Simple method to detect and to isolate entomopathogenic fungi (Hypocreales) from mosquito larvae.

Entomopathogenic fungi are important agents for mosquito vector control. We report on the utility of a simple method to detect fungi on living larvae of Aedes aegypti that had been exposed to a fungal entomopathogen. Four species of the hypocrealean genera Metarhizium, Beauveria, Tolypocladium and Culicinomyces, known for their larvicidal activity against mosquito species, were tested. Living larvae previously exposed to a suspension of different conidial concentrations were set directly into the surface water film on non-nutritive agar supplemented with chloramphenicol, thiabendazole and crystal violet and then incubated. Except for C. clavisporus ARSEF 964 (which developed and produced conidia mostly inside the cadaver rather than on its surface in the present study), this method favored external fungal development and conidiogenesis on larvae of different instars after death. The dead larva on the water agar represents the unique and specific source of nutrition for the fungus that killed it. The technique facilitates the detection and posterior isolation of entomopathogenic fungi, and offers a compact, convenient, and rapid means to survey larval mosquito populations for fungal pathogens at the field.

Microbial Control of Invasive Forest Pests with Entomopathogenic Fungi: A Review of the Current Situation.

The health of the forestlands of the world is impacted by a number of insect pests and some of them cause significant damage with serious economic and environmental implications. Whether it is damage of the North American cypress aphid in South America and Africa, or the destruction of maple trees in North America by the Asian long horned beetle, invasive forest pests are a major problem in many parts of the world. Several studies explored microbial control opportunities of invasive forest pests with entomopathogenic bacteria, fungi, and viruses, and some are successfully utilized as a part of integrated forest pest management programs around the world. This manuscript discusses some invasive pests and the status of their microbial control around the world with entomopathogenic fungi.

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.

Pythium insidiosum isolated from infected mosquito larvae in central Brazil.

Pythium insidiosum is a straminopilan pathogen causing life threatening infections in mammals inhabiting temperate, tropical and subtropical areas of the world. The concept that P. insidiosum could also infect mosquitoes was mentioned earlier by investigators conducting phylogenetic analysis on available P. insidiosum isolates deposited at different culture collections. However, an official report and details on its pathological features in mosquitoes are not available. We are reporting the isolation of P. insidiosum from infected mosquito larvae during a survey conducted in central Brazil. At least three oomycotan isolates were recovered during the survey. Due to their ability to infect mosquito larvae the isolates were deposited in the U.S. Department of Agriculture, Agricultural Research Service Collection of Entomopathogenic Fungi (ARSEF; Ithaca, New York) as putative Lagenidium species. The investigated isolates developed very well at 37 °C, produced typical Pythium-like vesicles containing numerous biflagellate zoospores, hydrolyzed sucrose, and their cultured extracted proteins were recognized in serological analysis by anti-P. insidiosum antibodies. Phylogenetic analyses using ITS and partial COXII DNA sequences identified the isolates as P. insidiosum within the American Cluster I. This is the first official report of P. insidiosum recovered from infected mosquito larvae, indicating that this mammalian pathogen, in addition to plants, it could also use mosquito larvae to complete its life cycle in nature.

Transcriptomic insight into pathogenicity-associated factors of Conidiobolus obscurus, an obligate aphid-pathogenic fungus belonging to Entomopthoromycota.

BACKGROUND: Conidiobolus obscurus is a widespread fungal entomopathogen with aphid biocontrol potential. This study focused on a de novo transcriptomic analysis of C. obscurus. RESULTS: A number of pathogenicity-associated factors were annotated for the first time from the assembled 17 231 fungal unigenes, including those encoding subtilisin-like proteolytic enzymes (Pr1s), trypsin-like proteases, metalloproteases, carboxypeptidases and endochitinases. Many of these genes were transcriptionally up-regulated by at least twofold in mycotized cadavers compared with the in vitro fungal cultures. The resultant transcriptomic database was validated by the transcript levels of three selected pathogenicity-related genes quantified from different in vivo and in vitro material in real-time quantitative polymerase chain reaction (PCR). The involvement of multiple Pr1 proteases in the first stage of fungal infection was also suggested. Interestingly, a unique cytolytic (Cyt)-like δ-endotoxin gene was highly expressed in both mycotized cadavers and fungal cultures, and was more or less distinct from its homologues in bacteria and other fungi. CONCLUSION: Our findings provide the first global insight into various pathogenicity-related genes in this obligate aphid pathogen and may help to develop novel biocontrol strategy against aphid pests. © 2018 Society of Chemical Industry.

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.

A natural fungal infection of a sylvatic cockroach with Metarhizium blattodeae sp. nov., a member of the M. flavoviride species complex.

A wild, forest-dwelling cockroach from the subfamily Ectobiidae (order Blattodea) in a nature reserve in Cavalcante, in the state of Goiás, Brazil, was found to be infected by a new, genetically distinct species in the Metarhizium flavoviride species complex that we describe here as Metarhizium blattodeae. The status of this fungus as a new species is supported by both multigenic sequence comparisons and protein profiles generated by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry. This is one of the first reports of a naturally occurring fungal pathogen affecting any sylvatic (forest-dwelling) cockroach from any part of the world. M. blattodeae caused up to 96 % mortality of Periplaneta americana nymphs (a serious peridomestic cockroach species) after 10 d.

Pandora bullata (Entomophthoromycota: Entomophthorales) affecting calliphorid flies in central Brazil.

Fungi are where one finds them, and if one seeks fungal pathogens affecting flies, then a garbage dump may be an ideal place to find both persistent, abundant fly populations and their fungal pathogens. An obvious fungal epizootic affecting the oriental latrine fly, Chrysomya megacephala (Diptera: Calliphoridae), was observed over several days in mid-February 2015 at the local garbage dump adjacent to the city of Cavalcante, northern Goiás. This site harbored large populations of both C. megacephala and a Musca sp. (Diptera: Muscidae) but only the population of oriental latrine fly was affected by any fungal pathogen and presented unusually dense populations of fresh cadavers. The fungus was identifiable as Pandora bullata (Entomophthorales: Entomophthoraceae) only after a very small number of characteristically decorated resting spores were found in these flies two months later; this represents the first Brazilian (and South American) record of this species. P. bullata is known previously from a small number of North American, European and Australian collections, all of which have included relatively abundant production of resting spores. We cannot dismiss the possibility that the extremely sparse formation of resting spores at this Brazilian site may be due to abiotic factors such as latitude (13°46'40.53″S), day length, ambient temperatures, or even the precipitation patterns in this mid-tropical montaine site. Epizootic events affecting calliphorids in Brazil strengthen the interest in entomophthoran pathogens for biological control of flies.

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.

Molecular differentiation of recently described Neozygites osornensis (Neozygitales: Neozygitaceae) from two morphologically similar species.

Neozygites osornensis is a recently described aphid pathogen characterized by morphological criteria and compared with two morphologically close taxa, Neozygites cinarae and Neozygites turbinata. Neozygites species are traditionally classified based on morphological characteristics and molecular data that would confirm these taxa and permit investigation of their phylogenetic relationships are scarce. In this study, we successfully evaluated a genetic variation within partial 18S rDNA sequences among the three Neozygites species and supported the recent erection of N. osornensis as a new species. A phylogenic analysis was also included to support our results.