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Entomopathogenic fungi can achieve important innovative outcomes for integrated mosquito control especially of Aedes aegypti, the key vector of arboviruses to humans in the tropics and subtropics. This study sought to design and to develop a simple dissemination device to attract and to infect gravid A. aegypti adults with a granular formulation of the ascomycete Metarhizium humberi IP 46, and to validate this device in the laboratory as well as in semi-field and field conditions. Hydrogel (polyacrylamide potassium polyacrylate) was confirmed to be a suitable substitute for water used in the device that attracted gravid females under field conditions. Females laid eggs on black polyethylene terephthalate carpet fixed in the device that also proved to be a suitable substrate for a granular formulation of fungal microsclerotia and/or conidia. The plastic device (29.5 cm high) was divided into a lower closed compartment with a water reservoir and an upper, laterally open but covered compartment with continuously hydrated gel and the fungal formulation attached to the carpet. The uppermost compartment permitted free circulation of mosquito adults. The device attracted both male and female A. aegypti. The fungal formulations of IP 46 propagules tested in the device were effective against adults in laboratory, semi-field, and field settings. Findings in the laboratory, semi-field, and especially in field conditions strengthen the value and utility of this innovative device for focal applications of a mycoinsecticide against this important mosquito vector. Key points • Low-cost and simple disseminating device for focal control of Aedes aegypti. • Granulized Metarhizium humberi IP 46 and hydrogel yield extended control. • Findings in field tests strengthen benefit of the device for focal application.

The impact of ambient relative humidity (RH) on conidial production of Metarhizium humberi IP 46 microsclerotia (MS) formulated in pellets or granules was investigated, and a promising granular formulation was tested against Aedes aegypti adults to confirm its efficacy. Microcrystalline cellulose (MC) and diatomaceous earth (DE) or a combination of vermiculite (VE), DE and silicon dioxide (SD) were tested as carriers in granular formulations containing MS. A range of 93-96.5% RH was critical for fungal development, and at least 96.5-98.5% RH was required for high conidial production on pellets or granules. Conidial production was clearly higher on pellets and granules prepared with VE than MC as the main carrier. VE granules containing MS were highly active against A. aegypti adults. Most mosquitoes were killed within 6 days after treatment regardless of the exposure time of adults to the formulation (1 min-24 h) or ambient humidity (75 or >98%). Production of conidia on dead adults varied between 7.3 × 106 and 2.2 × 107 conidia/individual, when exposed to MS granules for 12 h and 1 min, respectively. Granular formulations containing VE as the main carrier and MS as the active ingredient of M. humberi have strong potential for use against A. aegypti. KEY POINTS: • High conidial production on granular microsclerotial formulations at >96.5% RH • Vermiculite is more appropriate as a carrier than microcrystalline cellulose • Granules with IP 46 microsclerotia are highly active against Aedes aegypti adults.

The susceptibility of 1st to 4th instars of Bemisia tabaci (Gennadius, 1989) (Hemiptera: Aleyrodidae) to three isolates of Cordyceps sp. (Hypocreales: Cordycipitaceae) was evaluated in screenhouse experiments under variable temperatures and moisture conditions. No differences in susceptibility to the Cordyceps sp. isolates were observed among 1st, 2nd, and 3rd instar nymphs with respect to median lethal time (LT50) values. Confirmed mortalities ranged from 63.7 to 87.8% when the isolates were tested at 5 × 107 conidia mL−1. The 4th instar was the least susceptible to the fungal isolates (≤ 36.6% mortality). However, 60.0 to 99.5% of the adults that emerged from 4th instar nymphs previously treated with the fungus succumbed to the infection. Temperature was more detrimental to Cordyceps sp. virulence towards B. tabaci nymphs than relative humidity (RH). At similar RH, median LT50 for 1st instar (9.4 days) was higher than for 3rd instar (5.3 days) when the fungus was tested at 5 × 107 conidia mL−1; minimal temperatures of ≥ 12.6°C compared to ≥ 17.0°C were registered for experiments with 1st and 3rd instars, respectively. However, temperatures ≥ 35°C for 4 to 6 h daily did not affect the efficacy of the fungus against nymphs. Cordyceps sp. showed high virulence to all life stages of B. tabaci at relatively low RH, and an ability to grow extensively over the leaf surface and to produce high amounts of conidia on infected hosts. These attributes certainly boost its potential as an important pest control component of B. tabaci biotype B, especially for management of populations resistant to synthetic insecticides.

Entomophthoralean fungi with pathogenic abilities to infect social insects are rare. Here, we describe a fungus isolated from leafcutter ants. Morphologically, the fungus has spherical primary conidia and two types of microconidia: one with the same shape as the primary conidia and another with an elliptical to half-moon shape. The fungus also produces villose conidia known previously only from Conidiobolus coronatus. A multilocus phylogenetic analysis was performed with nuc rDNA sequences from three regions (28S, 18S, and internal transcribed spacer [ITS]). Our isolates are distinguished as a new species, described here as Conidiobolus lunulus, and is more closely related to C. brefeldianus than to C. coronatus, despite the greater morphological resemblance to the latter. Morphological differences, unique phylogenetic placement, and isolation from an altogether new host support this finding. This is the first record of an entomophthoralean species isolated from leafcutter ants.

Two new species from the genus Strongwellsea (Entomophthorales: Entomophthoraceae) that infect adult flies from the genus Helina (Muscidae) are described: Strongwellsea selandia Eilenberg & Humber infecting adult Helina evecta (Harris), and Strongwellsea gefion Eilenberg & Humber infecting adult Helina reversio (Harris). The descriptions are based on pathobiological, phenotypical and genotypical characters. The new species differ from other described members from the genus Strongwellsea by a) pathobiology as revealed by natural host species, b) morphology of primary conidia, c) color of resting spores, and d) genotypical clustering based on analysis of ITS2. The two new species have only been documented from North Zealand, Denmark.

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.

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 × 10

This article provides an outline of the classification of the kingdom Fungi (including fossil fungi. i.e. dispersed spores, mycelia, sporophores, mycorrhizas). We treat 19 phyla of fungi. These are Aphelidiomycota, Ascomycota, Basidiobolomycota, Basidiomycota, Blastocladiomycota, Calcarisporiellomycota, Caulochytriomycota, Chytridiomycota, Entomophthoromycota, Entorrhizomycota, Glomeromycota, Kickxellomycota, Monoblepharomycota, Mortierellomycota, Mucoromycota, Neocallimastigomycota, Olpidiomycota, Rozellomycota and Zoopagomycota. The placement of all fungal genera is provided at the class-, order- and family-level. The described number of species per genus is also given. Notes are provided of taxa for which recent changes or disagreements have been presented. Fungus-like taxa that were traditionally treated as fungi are also incorporated in this outline (i.e. Eumycetozoa, Dictyosteliomycetes, Ceratiomyxomycetes and Myxomycetes). Four new taxa are introduced: Amblyosporida ord. nov. Neopereziida ord. nov. and Ovavesiculida ord. nov. in Rozellomycota, and Protosporangiaceae fam. nov. in Dictyosteliomycetes. Two different classifications (in outline section and in discussion) are provided for Glomeromycota and Leotiomycetes based on recent studies. The phylogenetic reconstruction of a four-gene dataset (18S and 28S rRNA, RPB1, RPB2) of 433 taxa is presented, including all currently described orders of fungi., Nalin N. Wijayawardene thanks Mushroom Research Foundation and National Science Foundation of China (No. NSFC 31950410558) for financially supporting this project. Kevin D. Hyde acknowledges the Foreign Experts Bureau of Yunnan Province, Foreign Talents Program (2018; grant no. YNZ2018002), Thailand Research grants entitled Biodiversity, phylogeny and role of fungal endophytes on above parts of Rhizophora apiculata and Nypa fruticans (grant no: RSA5980068), the future of specialist fungi in a changing climate: baseline data for generalist and specialist fungi associated with ants, Rhododendron species and Dracaena species (grant no: DBG6080013), Impact of climate change on fungal diversity and biogeography in the Greater Mekong Subregion (grant no: RDG6130001). H.T. Lumbsch thanks support by the Grainger Bioinformatics Center. E. Malosso is grateful to CAPES for financial support (grant no. 88881.062172/2014-01). B.T. Goto, G.A. Silva and K. Jobim, L.C. Maia acknowledges CNPq (Brazilian Scientific Council, grants no. 465.420/2014-1, 307.129/2015-2 and 408011/2016-5) and CAPES for support. The study was partially supported by the National Science Centre, Poland, under Grants No. 2015/17/D/NZ8/00778 and 2017/25/B/NZ8/00473 to Julia Pawłowska. The research of Martin Kukwa received support from the National Science Centre (NCN) in Poland (project no 2015/17/B/NZ8/02441). Alan J.L. Phillips acknowledges the support from UID/MULTI/04046/2019 Research Unit grant from FCT, Portugal to BioISI. H. Zhang is financially supported by the National Natural Science Foundation of China (Project ID: NSF 31500017). S. Boonmee would like to thank the Thailand Research Fund (Project No. TRG6180001). Dong-Qin Dai and Li-Zhou Tang would like to thank the National Natural Science Foundation of China (No. NSFC 31760013, NSFC 31260087, NSFC 31460561), the Scientific Research Foundation of Yunnan Provincial Department of Education (2017ZZX186) and the Thousand Talents Plan, Youth Project of Yunnan Provinces for support. R. Phookamsak, M. Doilom, D. N. Wanasinghe, S.C. Karunarathna and J.C. Xu express sincere appreciations to Key Research Program of Frontier Sciences of the Chinese Academy of Sciences (grant no. QYZDY-SSW-SMC014) for research financial support. R. Phookamsak thanks the Yunnan Provincial Department of Human Resources and Social Security (grant no. Y836181261), Chiang Mai University and National Science Foundation of China (NSFC) project code 31850410489 for research financial support. S.C. Kaunarathna thanks CAS President’s International Fellowship Initiative (PIFI) for funding his postdoctoral research (No. 2018PC0006) and the National Science Foundation of China (NSFC) for funding this work under the project code 31851110759. S. Tibpromma would like to thank the International Postdoctoral Exchange Fellowship Program (number Y9180822S1), CAS President’s International Fellowship Initiative (PIFI) (number 2020PC0009), China Postdoctoral Science Foundation and the Yunnan Human Resources, and Social Security Department Foundation for funding her postdoctoral research. Yuri S. Tokarev, Elena S. Nassonova and Irma V. Issi are indebtful to Yuliya Y. Sokolova (Institute of Cytology RAS, St. Petersbug, Russia) and Anastasia V. Simakova (Tomsk State University, Tomsk, Russia) for kind permission of reproduction of electron microscopy images of Metchnikovella incurvata and Crepidulospora beklemishevi, respectively. Yuri S. Tokarev and Irma V. Issi thank Russian Foundation of Basic Research, grant number 17-04-00871 (taxonomy of Rozellomycota). Elena S. Nassonova thank Russian Foundation of Basic Research, grant number 18-04-01359 (early evolution of Microsporidia, phylogeny of Metchnikovellida). Adam Flakus and Pamela Rodriguez-Flakus are greatly indebted to all staff of the Herbario Nacional de Bolivia, Instituto de Ecología, Universidad Mayor de San Andrés, La Paz and the SERNAP (http://sernap.gob.bo), for their generous cooperation providing permits, assistance and facilities support for scientific studies. The research of AF and PRF were financially supported by the National Science Centre (NCN) in Poland (DEC-2013/11/D/NZ8/03274). Adam Flakus and Pamela Rodriguez-Flakus received additional support under statutory funds from the W. Szafer Institute of Botany, Polish Academy of Sciences, Krakow, Poland. The authors would like to thank Yunnan Innovation Platform for Development and Utilization of Symbiotic Fungi Resources for finance support. Li-Fang Zhang would like to thank grant-in-aid from Science and Technology Department of Yunnan Province (2018FD080) for finance support. Chun-Ying Deng thanks the Biodiversity Survey and Assessment Project of the Ministry of Ecology and Environment, China (2019HJ2096001006). Yingqian Kang would like to thank Guizhou Scientific Plan Project [(2019) 2873]; Excellent Youth Talent Training Project of Guizhou Province [(2017) 5639]; Guiyang Science and Technology Project [(2017) No. 5-19]; Talent Base Project of Guizhou Province, China [FCJD2018-22]; Research Fund of Education Bureau of Guizhou Province, China [(2018) 481]. D. N. Wanasinghe would like to thank the CAS President’s International Fellowship Initiative (PIFI) for funding his postdoctoral research (number 2019PC0008), the National Science Foundation of China and the Chinese Academy of Sciences for financial support under the following grants: 41761144055, 41771063 and Y4ZK111B01. Yuri K. Novozhilov and Oleg N. Shchepin acknowledge support from the Russian Foundation of Basic Research, project 18-04-01232 А. Ivana Kušan, Neven Matočec, Armin Mešić and Zdenko Tkalčec are grateful to Croatian Science Foundation for their financial support under the project grant HRZZ-IP-2018-01-1736 (ForFungiDNA). K. Tanaka would like to thank the Japan Society for the Promotion of Science (JSPS 19K06802)

We studied phylogenetic and taxonomic features of isolates identified as Lagenidium giganteum recovered from six different species of mosquito larvae. The isolates grew vigorously at 25 C, moderately at 30 C, and not at all at 37 C and developed submerged, white colonies with few short, hyaline, aerial hyphae. Cultures displayed phenotypic plasticity, with broad, hyaline hyphae strongly constricted at septa that developed oval, spherical, or amorphous segments. These developed into sporangia producing one or two exit tubes, from which evanescent gelatinous vesicles containing zoospores developed. Three isolates developed oogonia consistent with features previously described for L. giganteum. Phylogenetic analysis of nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS = ITS) and cytochrome c oxidase subunit II (COXII) sequences of L. giganteum consistently grouped into eight clusters. Four of the investigated isolates grouped with sequences of an unnamed Lagenidium species infecting nematodes. Based on phenotypic and phylogenetic data, we describe the latter isolates as L. juracyae, sp. nov. In addition, we also investigated a species of Paralagenidium from a dog with lagenidiosis and describe it as new, Paralagenidium ajellopsis, sp. nov.

Granular microsclerotial formulations of entomopathogenic fungi deserve attention because of their post-application, in situ production of new conidia that enhance and prolong mycoinsecticidal efficacy against a target pest insect. Because high ambient moisture is a crucial condition to induce fungal development and conidiogenesis on granules, we tested the impacts of the additions of three humectants-glycerin, propylene glycol, and polyethylene glycol 400-on water absorption by pellets incorporating microsclerotia of Metarhizium humberi IP 46 with microcrystalline cellulose or vermiculite carriers, and on the production of infective conidia of IP 46 microsclerotia in ambient humidities suboptimal for routine conidiogenesis. Glycerin facilitated greater and faster absorption of water than the other humectants. Microcrystalline cellulose absorbed low quantities of water without any added humectant whereas vermiculite did not. IP 46 did not grow or sporulate on pellets prepared with or without glycerin at 86% relative humidity (RH) or on control pellets without glycerin at 91% RH; conidial production on pellets prepared with vermiculite or microcrystalline cellulose and 10% glycerin reached 1.1 × 105 conidia/mg and 1 × 105 conidia/mg, respectively, after 20 days of exposure at 91% RH. Hence, these results strongly support glycerin as a suitable humectant for granular microsclerotial formulations of this fungus.

The entomopathogenic fungus Metarhizium humberi affects Aedes aegypti adults, larvae and eggs, but its ovicidal activity is not yet well documented. Conidia of this fungus adhered to the chorion, initiated germination within 12 h, and germinating conidia were detected for up to 10 d after contact with the egg. Germ tubes either penetrated the chorion directly or formed appressoria at the end of a short hypha (5 μm) or, subsequently, on longer, branched hyphae. Thin layers of what was most probably a fungal mucilaginous excretion were detected on the chorion adjacent to germ tubes, appressoria and hyphae. After 5 d eggs frequently appeared shriveled with ruptures in the chorion, and with the interior filled with hyphae that eventually produced mycelium and new conidia on the egg surfaces. Findings demonstrated that this fungus can infect A. aegypti eggs and subsequently recycle on their surface by producing large numbers of new conidia that should be infective for further generations of eggs, larvae and adults.

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.

Two new species from the genus Strongwellsea (Entomophthorales: Entomophthoraceae) are described: Strongwellsea tigrinae from adult Coenosia tigrina (Diptera: Muscidae) and Strongwellsea acerosa from adult Coenosia testacea. The descriptions are based on pathobiological, phenotypical and genotypical characters. Further, the circumscription of the genus Strongwellsea is emended. Our findings suggest that Strongwellsea harbors a high number of species, of which now only five have been described.

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.

The aquatic fungal genus Culicinomyces attacks dipteran larvae but little is known about its efficacy against Aedes aegypti. Here we report on the activity of both described species–Culicinomyces clavisporus and Culicinomyces bisporalis–on larvae, eggs and adults, and on trans-stadial transmission. Ten C. clavisporus isolates (ARSEF 372, 582, 644, 706, 964, 1260, 2471, 2478, 2479 and 2480) and C. bisporalis ARSEF 1948 were screened against larvae of this important vector of viral diseases. ARSEF 644, 964 and 2479 had the lowest LC50 (≤3.6 × 105 conidia/ml) after a 3-day exposure and shortest LT50 (≤1.3 days) at 106 conidia/ml against larvae; none of these isolates affected either eggs or adults treated topically with conidia. However, adults fed on a conidial (106 conidia/ml) suspension in 10% sucrose died (≤26.6 ± 3.3% mortality, 5 days after feeding) but no fungal development was detected on dead adults. No pupae or adults obtained following treatment of fourth instar larvae with 105 or 106 conidia/ml showed any indication of fungal presence. C. clavisporus–especially ARSEF 644, 964 and 2479–is the first choice for control of A. aegypti and has high potential in control strategies targeting aquatic larvae.

Compared to the higher fungi (Dikarya), taxonomic and evolutionary studies on the basal clades of fungi are fewer in number. Thus, the generic boundaries and higher ranks in the basal clades of fungi are poorly known. Recent DNA based taxonomic studies have provided reliable and accurate information. It is therefore necessary to compile all available information since basal clades genera lack updated checklists or outlines. Recently, Tedersoo et al. (MycoKeys 13:1–20, 2016) accepted Aphelidiomycota and Rozellomycota in Fungal clade. Thus, we regard both these phyla as members in Kingdom Fungi. We accept 16 phyla in basal clades viz. Aphelidiomycota, Basidiobolomycota, Blastocladiomycota, Calcarisporiellomycota, Caulochytriomycota, Chytridiomycota, Entomophthoromycota, Glomeromycota, Kickxellomycota, Monoblepharomycota, Mortierellomycota, Mucoromycota, Neocallimastigomycota, Olpidiomycota, Rozellomycota and Zoopagomycota. Thus, 611 genera in 153 families, 43 orders and 18 classes are provided with details of classification, synonyms, life modes, distribution, recent literature and genomic data. Moreover, Catenariaceae Couch is proposed to be conserved, Cladochytriales Mozl.-Standr. is emended and the family Nephridiophagaceae is introduced.

A new species from the genus Strongwellsea (Entomophthorales: Entomophthoraceae) is described: Strongwellsea crypta Eilenberg & Humber from adult Botanophila fugax (Meigen) (Diptera: Anthomyiidae). The description is based on pathobiological, phenotypical and genotypical characters. The abdominal holes in infected hosts develop rapidly and become strikingly large and edgy, almost rhomboid in shape. The new species S. crypta differs from S. castrans, the only described species infecting flies from Anthomyiidae, by: (a) naturally infecting another host species, (b) by having significantly longer primary conidia, and (c) by genotypical clustering separately from that species when sequencing ITS2.

Mitochondrial DNA is generally regarded to evolve faster than nuclear DNA in animals, whereas if this is also true in fungi remains unclear. Herein, we annotate the first complete mitochondrial genome (mitogenome) of the cyclosporin-producing fungus Tolypocladium inflatum and report the genome-wide sequence variations among five isolates originating from distantly separated localities. We found that T. inflatum has among the most compact of fungal mitogenomes; its 25 kb DNA molecule encodes all standard fungal mitochondrial genes and harbors only one intron. Transcriptional analyses validated the expression of most conserved genes. We found several uncommon repetitive elements and evidence of gene transfer from the mitochondrion to the nucleus. Phylogenetic analyses confirmed the placement of T. inflatum in the fungal order Hypocreales although there was uncertainty on its family-level affiliation. Comparative genomic analyses among the five isolates identified an overall lower level of intraspecific variation in mitogenomes than in nuclear genomes; however, both the nuclear and mitochondrial genomes revealed similar isolate relationships, not correlating with geographic sources of these isolates. Our study shed new insights into the evolution of the medicinally important ascomycete T. inflatum.

Culicinomyces spp. (Hypocreales: Cordycipitaceae) are facultative fungal pathogens affecting the larval stages from a range of mosquito species and are especially notable in their ability to infect hosts through the digestive tract after conidial ingestion. While Culicinomyces spp. were studied mainly in the 1980s, little is yet known about inter- and intraspecific variability of the in vitro development of these fungi at different temperatures, and nothing is known about the impact of serial host-passage on the development or virulence against Aedes aegypti larvae. The development of ten isolates of C. clavisporus (ARSEF 372, 582, 644, 706, 964, 1260, 2471, 2478, 2479 and 2480) and one of C. bisporalis (ARSEF 1948) was assessed on solid SDAY/4 and liquid SDY/4 at 15, 20, 25, 30 and 35 °C. Based on the results of these assays, three isolates were selected (ARSEF 644, 964 and 2479) for three serial host-passage/reisolation cycles, and comparison of the reisolates with the original stock isolates for their virulence, vegetative growth and conidiogenesis. The highest germination rates (≥95%) after 48 h incubation were obtained at 25 and 20 °C, and the lowest germination (≤12%) at 35 °C after the same time. The optimal temperature for radial growth was 25 °C (≥11.8 mm), followed by 20 °C for all isolates. ARSEF 706, 582 and 372 showed the greatest vegetative growth (≥20 mm). In general, there was little radial growth of colonies at 30 °C (≤2.5 mm), and none at 35 °C. Isolates, especially ARSEF 964, 2479, and 644, generally produced the highest numbers of conidia at 25 °C (≥1.42 × 105 conidia/plate) after 15 days. After two host-passages, conidiogenesis increased significantly on SDAY/4 for ARSEF 2479 but not for ARSEF 644 or 964. All larvae exposed to these three isolates of C. clavisporus died within 7 days regardless of the concentration or host-passage; C. bisporalis was not tested in these experiments. The virulence of ARSEF 964 increased at lower concentrations (106–3 × 105 conidia/ml) after the first host-passage.

The cypress aphid (Cinara cupressi) is listed among the hundred most important invasive pests in the world. In Chile, it was first detected in 2003 and currently is present throughout the country. In the course of a survey of their natural enemies in Chile, three strains of entomopathogenic fungi were isolated. The isolates were identified and tested against the aphid in laboratory experiments. Two further entomopathogenic fungi (ARSEF 5126 and 5128), formulated in the mycoinsecticides Vertalec® and Mycotal®, were used as reference strains. The three Chilean isolates were identified genomically as Lecanicillium attenuatum and were pathogenic to third-instar nymphs. The isolate ARSEF 13279 yielded the lowest overall lethal concentration (LC50), 3 × 105 conidia ml−1 at four days post-inoculation, and the shortest lethal time (LT50), 3.7 days after inoculation with 106 conidia ml−1. The results indicate that the isolates have considerable potential as microbial control agents of the invasive cypress aphid.

In several surveys in the tropical forests in Thailand, specimens that looked morphologically similar to Metarhizium martiale and Cordyceps variegata, as well as other Metarhizium species were collected and cultured in vitro. A combined phylogeny of several genes including the small (18S) and large (28S) subunits of the ribosomal DNA, elongation factor 1-α (TEF), RNA polymerase II subunits 1 and 2 (RPB1, RPB2) genes has shown these to be new taxa in the Clavicipitaceae. Nigelia is described as a new genus closely related to Metarhizium, to the scale insect pathogens Aschersonia (Hypocrella), Samuelsia and Moelleriella, and to plant pathogens in Claviceps and Balansia, and other relatives. Nigelia comprises M. martiale and a new species Nigelia aurantiaca, which has been found infecting lepidopteran larvae and which produces pseudoimmersed, obliquely arranged, obpyriform perithecia with curved or bent ostioles and with whole (non-separating) cylindric ascospores. Metarhizium chaiyaphumense, M. kalasinense, M. prachinense, M. samlanense, and M. takense are described as new species of Metarhizium. Metarhizium martiale is transferred to Nigelia, and Paecilomyces reniformis is transferred to Metarhizium.

We studied phylogenetic and taxonomic features of isolates identified as

Future progress in research with entomopathogenic fungi depends on a number of diverse considerations that help to stabilize the state of knowledge while supporting research about the documentation of the biodiversity and systematics of these fungi as well as those studies about their actions as pathogens of major and minor pests, and even as biological curiosities rather than as serious agents for use in biological control. This review considers (1) the role of service culture collections in culturing, preserving, and providing essential germplasm resources of these fungi for any and all research purposes; (2) whether there is too much stability in the current spectrum of entomopathogenic fungi actually being used in a practical sense and of possible alternative strategies to exploit more fungal entomopathogens; and (3) the diverse and far-reaching impacts of new nomenclatural rules that are constricting the pool of names applicable to entomopathogenic fungi while also stripping away their underlying taxonomic concepts that have long guided our interpretation and understanding of these fungi at a time when so many more taxa are being recognized. Some urgent problems underlying the shift from traditional to genomically based taxonomic approaches and about issues about the rapidly growing mass of genomic data are also discussed.

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 Aruana, Goias 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 24 h of exposure to the pathogen, and increased to 100% as quickly as 3 days after inoculation (with the highest conidial concentration tested, 8.3 × 104 conidia/cm2). Repeated attempts to obtain genomic sequence data failed despite confirmations that the DNA extraction methods were themselves successful.

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.

The genus Neozygites has been known in Brazil until now only on mites, and this is its first report on aphids in Brazil. Tree-dwelling aphids (Cinara sp.) on a cypress tree were regularly monitored for entomopathogenic fungi in the city of Terezópolis de Goiás in Central Brazil between July 2014 and April 2015. During the survey, mycosed aphids were found attached to twigs. The fungus was identified morphologically as Neozygites osornensis. No in vitro cultures were established from infected aphids. The finding reported here suggests that special attention should be paid in the future for the possible occurrences of Neozygites species wherever cinaran aphids occur around the globe.

Two types of secondary conidia and their formation are described from six species of Strongwellsea infecting hosts from Anthomyiidae, Muscidae and Fanniidae. We used a simple device allowing secondary conidia to be produced under very moist or comparatively dry conditions. Ellipsoid type secondary conidia, which are formed under very moist conditions, have never been reported before from the genus Strongwellsea, and they are unique for Entomophthorales; these are broadly ellipsoidal with a clearly pointed basal papilla and are actively discharged. Subglobose type secondary conidia are, for the first time, described from several species in the genus Strongwellsea; they are subglobose to almost bell-shaped with a flattened papilla and are actively discharged. Subglobose type secondary conidia are formed under more dry conditions. A general pattern of the formation of secondary conidia in Strongwellsea and the ecological roles of primary conidia and of the two types of secondary conidia are discussed.

Entomopathogenic fungi routinely kill their hosts before releasing infectious spores, but select species keep insects alive while sporulating, which enhances dispersal. Transcriptomics and metabolomics studies of entomopathogens with post-mortem dissemination from their parasitized hosts have unraveled infection processes and host responses, yet mechanisms underlying active spore transmission by Entomophthoralean fungi in living insects remain elusive. Here we report the discovery, through metabolomics, of the plant-associated amphetamine, cathinone, in four Massospora cicadina-infected periodical cicada populations, and the mushroom-associated tryptamine, psilocybin, in annual cicadas infected with Massospora platypediae or Massospora levispora, which appear to represent a single fungal species. The absence of some fungal enzymes necessary for cathinone and psilocybin biosynthesis along with the inability to detect intermediate metabolites or gene orthologs are consistent with possibly novel biosynthesis pathways in Massospora. The neurogenic activities of these compounds suggest the extended phenotype of Massospora that modifies cicada behavior to maximize dissemination is chemically-induced.

AIMS To survey and to characterize entomopathogenic fungi as natural enemies of mosquitoes in Central Brazil. METHODS AND RESULTS Tolypocladium cylindrosporum (Hypocreales, Ophiocordycipitaceae) was isolated for the first time in South America by using Aedes aegypti (Diptera, Culicidae) as sentinel larvae in a stagnant mosquito breeding site in a secondary tropical forest. Two isolates were morphologically and molecularly identified, and their activity tested against A. aegypti eggs, larvae and adults. CONCLUSIONS Findings emphasize the importance of T. cylindrosporum as a natural fungal antagonist of mosquitoes. SIGNIFICANCE AND IMPACT OF THE STUDY Both isolates affected larvae and adults but were less effective against eggs; both have potential for development as a mycoinsecticide especially against larvae of A. aegypti the main vector of dengue, chikungunya, Zika and urban yellow fever.

The air-breathing snail Biomphalaria glabrata proliferates in stagnant freshwater, and nothing is known about the survival of eggs in intermittently (rather than perpetually) wet habitats. In the present study their egg masses matured, and juveniles subsequently eclosed and were mobile in a stable water film of transitory habitats simulated by two different simple test devices described here. The viability of eggs maintained in an unstable film however, was diminished. The maturation of egg masses in a water film or in water was significantly prevented by the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae. The efficiency depended on the fungal propagule and test environment. Hyphal bodies were more effective against egg masses than conidia. This appears to be a first report of activity of either entomopathogen against a mollusc. Both devices offer accurate and reproducible conditions to test both biological questions and the effects of substances or pathogens against B. glabrata egg masses in water films.

Beside the taxonomic features displayed by Crypticola clavulifera in culture and unpublished data on its phylogeny, little is known about the main phylogenetic features of this unusual mosquito pathogen. We have PCR amplified the 18S SSU rDNA, ITS, and the partial coding regions of COXII and COXI to study the phylogenetic features of this pathogen within the tree of life. Our phylogenetic data showed C. clavulifera clustered among homologous DNA sequences of the Oomycota class Saprolegniomycetes. Our study support previous taxonomic and unpublished molecular analysis, placing this unusual mosquito larvae pathogen as part of the earliest diverging cluster within the Saprolegniomycetes.

A project from 2013 to 2017 sought to discover pathogenic fungi and oomycetes from dipteran species that are vectors of major diseases of humans and animals in central Brazil and to begin evaluating the potential of these pathogens as potential biological control agents concentrated on mosquito larvae. Some collecting sites proved to be especially productive for pathogens of naturally occurring mosquito species and for placements of healthy sentinel larvae of Aedes aegypti in various sorts of containers in a gallery forest in the Santa Branca Ecoturismo Private Reserve of Natural Patrimony (RPPN) near Terezópolis de Goiás (GO). Collections during May-April of 2016 and February 2017 yielded a few dead mosquito larvae of an undetermined Onirion sp. (Culicidae: Sabethini) whose hemocoels contained many ovoid, thick-walled, yellow-golden to golden-brown, ovoid thick-walled resistant sporangia, 38.3 ± 4 × 22.8 ± 2.3 µm, decorated by numerous, closely and randomly spaced punctations of variable size and shape. These were the first indisputable collections from Brazil of any Coelomomyces species. Comparisons of the morphology of these sporangia with those of other species of Coelomomyces, confirmed that this Brazilian fungus represented a new species that is described here as Coelomomyces santabrancae. Fil: Rueda Páramo, Manuel Enrique. Universidade Federal de Goiás; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina Fil: Montalva, C.. Universidade Federal de Goiás; Brasil. Universidad Austral de Chile; Chile Fil: Arruda, W.. Universidade Federal de Goias. Instituto de Ciencias Biologicas; Brasil Fil: Fernandes, Éverton. Universidade Federal de Goiás; Brasil Fil: Luz, Wolf Christian. Universidade Federal de Goiás; Brasil Fil: Humber, Richard A.. United States Department of Agriculture. Agricultural Research Service; Argentina

A strain within the Metarhizium anisopliae species complex was isolated in 2009 from a soil sample in a banana plantation in the municipality of Quixere, 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 Sergio Batista Alves, one of the founders of insect pathology in Brazil.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a powerful tool for taxonomic resolution of microorganisms. In this proof-of-concept study, we assessed the effectiveness of this technique to track the current gene sequence-based phylogenetic classification of species in the Metarhizium anisopliae complex. Initially the phylogenetic analysis of 5' strains by sequencing of the 59' end of the TEF-1α gene region revealed seven species within M. anisopliae sensu lato and two varieties outside this complex. Because initial studies on MS profiles from different cell types showed that mycelial fragments or conidia produced on nutrient-poor medium may yield too much background noise, all subsequent spectrometric analyses were performed with acidhydrolyzed conidia from 10-12 d old PDA cultures. The initial MALDI-TOF reference library included protein spectral profiles from nine taxonomically distinct, molecularly identified isolates sharing high genetic homology with the ex-type or ex-epitype isolates of these taxa in Metarhizium. A second reference library added one isolate each for M. anisopliae sensu stricto and M. robertsii. The second, larger reference library (including 11 taxa) allowed nearly perfect MALDI-TOF matching of DNA-based species identification for the 40 remaining isolates molecularly recognized as M. anisopliae sensu stricto (n = 19), M. robertsii (n = 6), M. majus (n = 3), M. lepidiotae (n = 1), M. acridum (n = 3), M. flavoviride var. pemphigi (n = 1), plus seven unidentified strains (six of them phylogenetically close to M. anisopliae sensu stricto and one outside the Metarhizium pingshaense-anisopliae-robertsii-brunneum clade). Due to the increasing frequency of phylogenetically (genomically) based taxonomic revisions of fungi, this approach is especially useful for culture collections, because once the protein profiles of Metarhizium isolates are obtained taxonomic updating of MALDI-TOF library data is easily accomplished by comparing stored profiles with those of newly proposed taxa.

Fil: Manfrino, Romina Guadalupe. Instituto Nacional de Tecnologia Agropecuaria. Centro Regional Santa Fe. Estacion Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico La Plata. Centro de Estudios Parasitologicos y de Vectores (i); Argentina

The elongation factor 1-alpha (EF1-α) and the internal transcribed spacer (ITS) regions ITS1 and ITS2 (ITS1-5.8S-ITS2) sequences were used to characterize and to identify Isaria isolates from Argentina, Mexico, and Brazil, as well as to study the phylogenetic relationships among these isolates and other related fungi from the order Hypocreales. The molecular characterization, which was performed by PCR-RFLP of EF1-α and ITS1-5.8-ITS2 genes, was useful for resolving representative isolates of Isaria fumosorosea, Isaria farinosa, and Isaria tenuipes and to confirm the taxonomic identity of fungi from Argentina, Mexico, and Brazil. The phylogenetic analyses showed three clades corresponding to three families of Hypocreales. The genus Isaria was confirmed as polyphyletic and in family Cordycipitaceae, Isaria species were related to anamorphic species of Beauveria, Lecanicillium, and Simplicillium and to teleomorphic Cordyceps and Torrubiella. Therefore, EF1-α and ITS1-5.8S-ITS2 genes were found to be powerful tools for improving the characterization, identification, and phylogenetic relationship of the Isaria species and other entomopathogenic fungi.

Entomophthoromycota is one of six major phylogenetic lineages among the former phylum Zygomycota. These early terrestrial fungi share evolutionarily ancestral characters such as coenocytic mycelium and gametangiogamy as a sexual process resulting in zygospore formation. Previous molecular studies have shown the monophyly of Entomophthoromycota, thus justifying raising the taxonomic status of these fungi to a phylum. Multi-gene phylogenies have identified five major lineages of Entomophthoromycota. In this review we provide a detailed discussion about the biology and taxonomy of these lineages: I) Basidiobolus (Basidiobolomycetes: Basidiobolaceae; primarily saprobic); II) Conidiobolus (Entomophthoromycetes, Ancylistaceae; several clades of saprobes and invertebrate pathogens), as well as three rapidly evolving entomopathogenic lineages in the family Entomophthoraceae centering around; III) Batkoa; IV) Entomophthora and allied genera; and V) the subfamily Erynioideae which includes Zoophthora and allied genera. Molecular phylogenic analysis has recently determined the relationships of several taxa that were previously unresolved based on morphology alone: Eryniopsis, Macrobiotophthora, Massospora, Strongwellsea and two as yet undescribed genera of Basidiobolaceae.

Summary In Chile, the invasive and noxious pest Vespula vulgaris (L.) (Hymenoptera: Vespidae) was first reported in 2011 in the Araucania region and is currently distributed between Araucania and Magallanes regions. In Mar 2015 (autumn), during an ongoing monitoring of funnel traps by the Servicio Agricola y Ganadero, a fungus-infected individual was collected. The fungus was identified morphologically as a species of Hirsutella (Hypocreales: Ophiocordycipitaceae). This is the first report of any Hirsutella species on V. vulgaris in Chile. No in vitro cultures were successfully established from the infected insect.