Susceptibility of All Nymphal Stages of Bemisia tabaci Biotype B (Hemiptera: Aleyrodidae) to Three Brazilian Isolates of Cordyceps sp. (Hypocreales: Cordycipitaceae) in a Screenhouse Under Variable Temperature and Moisture Conditions.

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.

Conidiobolus lunulus, a new entomophthoralean species isolated from leafcutter ants.

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.

Characterization of Tolypocladium cylindrosporum (Hypocreales, Ophiocordycipitaceae) isolates from Brazil and their efficacy against Aedes aegypti (Diptera, Culicidae).

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.

First report of Coelomomyces santabrancae sp. nov. (Blastocladiomycetes: Blastocladiales) infecting mosquito larvae (Diptera: Culicidae) in central Brazil.

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±2.3µm, 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.

Potential of Entomopathogenic Fungi as Biological Control Agents of Diamondback Moth (Lepidoptera: Plutellidae) and Compatibility With Chemical Insecticides.

The objectives were to evaluate the efficiency of entomopathogenic fungi against Plutella xylostella (L.) and the compatibility of the most virulent isolates with some of the insecticides registered for use on cabbage crops. Pathogenicity tests used isolates of Beauveria bassiana, Metarhizium rileyi, Isaria fumosorosea, Isaria sinclairii, and Lecanicillium muscarium standardized at a concentration of 10(7) conidia/ml. Cabbage leaf discs were immersed in these suspensions, and after evaporation of the excess water, were placed 10 second-instar larvae of P. xylostella, totaling 10 leaf discs per treatment. Mortality was assessed 7 d after treatment, and the isolates that caused mortality>80% were used to estimate LC50 and LT50. The compatibilities of the most virulent isolates and the insecticides were tested from the mixture of these into the culture medium, and after solidifying, the medium was inoculated with an aliquot of the isolated suspension. The following parameters were evaluated: growth of the colony, number and viability of conidia after 7 d. The isolated IBCB01, IBCB18, IBCB66, and IBCB87 of B. bassiana, LCMAP101 of M. rileyi, and ARSEF7973 of I. sinclairii caused mortality between 80 and 100%, with LC50 and LT50 between 2.504 to 6.775×10(4) conidia/ml and 52.22 to 112.13 h, respectively. The active ingredients thiamethoxam and azadirachtin were compatible with the entomopathogenic fungi. The results suggest that the use of these isolates is an important alternative in the pesticidal management of P. xylostella, with the possible exception of the associated use of chemical controls using the active ingredients thiamethoxam or azadirachtin.

Neozygites osornensis (Neozygitales: Neozygitaceae) Infecting Cinara sp. (Hemiptera: Aphididae) in Brazil.

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.

Phylogenetic lineages in Entomophthoromycota.

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.

First and southernmost records of Hirsutella (Ascomycota: Hypocreales) and Pandora (Zygomycota: Entomophthorales) species infecting Dermaptera and Psocodea.

We present the first and southernmost records of the fungi Hirsutella strigosa Petch, H. citriformis Speare (Ascomycota: Hypocreales), and Pandora nouryi (Remaudière & Hennebert) Humber (Zygomycota: Entomophthorales) infecting Doru lineare (Eschscholtz) (Dermaptera: Forficulidae), Ectopsocus californicus (Banks) (Psocodea: Ectopsocidae), and Heterocaecilius sp. (Psocodea: Pseudocaeciliidae), respectively. This is the first report of naturally occurring pathogenic fungi infecting Psocoptera, and it is also the first report of P. nouryi from any host outside of the Aphididae. The three fungal species were morphologically described from their host insects and from microscopic preparations. Attempts to obtain pure fungal isolates were unsuccessful but slides and photographs of these fungi were preserved and deposited in mycological collections as herbarium material.

First record of Clonostachys rosea (Ascomycota: Hypocreales) as an entomopathogenic fungus of Oncometopia tucumana and Sonesimia grossa (Hemiptera: Cicadellidae) in Argentina.

Clonostachys rosea (Link: Fries) Schroers, Samuels, Seifert, and Gams (Ascomycota: Hypocreales) has been reported as a mycoparasite of fungi and nematodes and as saprobe in soils, but this fungus has not been reported previously to be entomopathogenic. Many species of cicadellid leafhoppers cause economic damage to crops as vectors of plant pathogens. In the present work, we report the first record of C. rosea as an entomopathogenic fungus of two leafhoppers pest, Oncometopia tucumana and Sonesimia grossa (Hemiptera: Cicadellidae), in Argentina and evaluate the pathogenicity of C. rosea against them.

Description and analysis of two internet-based databases of insect pathogens: EDWIP and VIDIL.

In 1996, two searchable databases covering insect pathogens were posted on the World Wide Web: the Ecological Database of the World's Insect Pathogens (EDWIP) and the Viral Diseases of Insects in the Literature database (VIDIL). In this paper, we describe the format and contents of EDWIP and VIDIL on the World Wide Web. EDWIP contains over 9,400 pathogen-host association records, 677 negative test result or "no association" records, 4,454 host species, 2,285 pathogen species records, and 2,057 bibliographical references. Species of Coleoptera and Lepidoptera are the best represented groups in EDWIP. Lepidopteran species account for the most associations of any host order in EDWIP, over 2,500, or 27%. Of the pathogen groups, Protozoa (including microsporidia) accounted for nearly 66% of the pathogen species records and over 40% of the association records in EDWIP. Fungi account for only 18% of the pathogen species, but nearly 33% of the association records. Habitats dominated by human activities (e.g., crop, stored product, and human dwelling) account for most of the host habitats recorded in EDWIP. The United States and Japan are the most common locations and the Nearctic and Palearctic are the most common biogeographic regions reported in EDWIP. There are 4,801 annotated bibliographic records in VIDIL.

Polyketide synthase genes in insect- and nematode-associated fungi.

Production of polyketides is accomplished through complex enzymes known as polyketide synthases (PKS); these enzymes have highly conserved domains that might be useful in screens for PKSs in diverse groups of organisms. A degenerate PCR-based approach was used to amplify PKS fragments of the ketosynthase domain from genomic DNA of a group of insect- and nematode-associated fungi. Of 157 isolates (representing 73 genera and 144 species) screened, 92 isolates generated PCR products of predicted size (approximately 300 bp). The ability to detect PKS domains was a function of the number of different primer pairs employed in the screen. Cloning and sequencing revealed that 66 isolates had at least one unique PKS sequence; ten members of this set contained multiple PKS fragments, for a total of 76 unique PKS fragments. Since PKS genes appear to be widespread among fungi, a PCR-based screening system appears to be an efficient, directed means to identify organisms having the potential to produce polyketides.

Entomopathogenic fungi in flies associated with pastured cattle in Denmark.

Cattle flies, including Musca autumnalis, Haematobia irritans, and Hydrotaea irritans, are pests of pastured cattle. A 2-year study of the natural occurrence of entomopathogenic fungi in adult cattle flies and other flies associated with pastures showed that the four species included in the Entomophthora muscae species complex (E. muscae sensu lato) caused high infection levels in several species of flies. However, only a few specimens of cattle flies were infected by E. muscae sensu stricto despite the fact that cattle flies were observed to perch on spear thistles, which acted as transmission site for all four Entomophthora species. Transmission experiments with E. muscae s.l. supported the field data. Of the two species considered host specific, E. syrphi caused substantial infection in a muscid, and E. scatophagae likewise could be transmitted to a muscid. This emphasizes the need for a revision of the two species. Low prevalences were recorded of another entomophthoralean, Furia americana, and of the hyphomycetes Beauveria bassiana and Verticillium lecanii.

Preliminary survey for entomopathogenic fungi associated with Ixodes scapularis (Acari: Ixodidae) in southern New York and New England, USA.

Free-living larval, nymphal, and adult Ixodes scapularis Say were collected from scattered locales in southern New England and New York to determine infection rates with entomopathogenic fungi. Infection rates of larvae, nymphs, males, and females were 0% (571), 0% (272), 0% (57), and 4.3% (47), respectively. Two entomopathogenic fungi were isolated from field-collected I. scapularis females from Fire Island, NY. Isolates were identified as Verticillium lecanii (Zimmermann) Viegas and Verticillium sp. (a member of the Verticillium lecanii species complex).

Entomopathogenic potential of Verticillium and Acremonium species (Deuteromycotina: Hyphomycetes).

Hyphomycetes with conidia formed in slimy heads from hyaline mycelium were isolated from a range of insect hosts. Isolation on artificial medium and microscopic examination revealed that these fungi in many cases were not Verticillium lecanii despite a superficial resemblance to this common entomopathogen. The fungi were identified as Verticillium fusisporum, Verticillium psalliotae, Verticillium lamellicola, and species of Acremonium. Isolates of these fungi were bioassayed against the sweet-potato whitefly (Bemisia tabaci) and against the housefly (Musca domestica) to examine their entomopathogenicity. A test was also conducted with a coleopteran (lesser mealworm, Alphitobius diaperinus) to further evaluate the host range for some of the fungi. V. lamellicola was not pathogenic to the two species of insects treated, while varying levels of pathogenicity were shown for the other species. In general, V. lecanii was the most pathogenic species. Immature whiteflies appeared to be more susceptible to fungal infection than adult houseflies, and the host range for several of the fungi also included lesser mealworm.

Mycosis of bandedwinged whitefly (Trialeurodes abutilonea) (Homoptera: aleyrodidae) caused by Orthomyces aleyrodis gen. & sp. nov. (Entomophthorales: Entomophthoraceae).

A new genus and species of fungus, Orthomyces aleyrodis Steinkraus, Humber & Oliver gen. & sp. nov. (Entomophthorales: Entomophthoraceae) is described. In 1994 and 1995, this fungus caused epizootics in high populations of bandedwinged whitefly (Trialeurodes abutilonea) on cotton and cocklebur in Alabama. In 1996, T. abutilonea populations were lower and no infected specimens were found. This is the first report of an entomophthoralean infecting an aleyrodid in the New World and the first report of entomophthoralean epizootics in whiteflies.

RAPD-PCR for identification of Zoophthora radicans isolates in biological control of the potato leafhopper.

Biological control studies require the ability to distinguish released pathogens from locally occurring isolates of the same species. We have developed a technique that differentiates genotypes using random amplified polymorphic DNA (RAPD) for the apomictic species Zoophthora radicans (Zygomycota: Entomophthorales), a pathogen of the potato leafhopper, Empoasca fabae (Homoptera: Cicadellidae). RAPD analysis was performed on Z. radicans isolates released in test plots in 1990 and 1991 for leafhopper control; isolates later recovered from the same plots and diverse other isolates were included in the analysis. RAPD fragment profiles of five recovered isolates proved very similar to those of the released isolates and different from all other isolates tested; they are probable descendants of the released isolates. One of the recovered isolates had RAPD profiles similar to isolates derived from aphids and probably represents a population endemic at the release site. In addition to verifying the successful establishment of our experimental releases, RAPD analysis revealed clear relationships among isolates derived from the same host taxon. We propose that this simple and relatively inexpensive method will be valuable in determining the establishment and spread of organisms released in biological control studies.

Allozyme and restriction fragment length polymorphism analyses confirm Entomophaga maimaiga responsible for 1989 epizootics in North American gypsy moth populations.

In 1989, populations of North American gypsy moth, Lymantria dispar, in seven contiguous northeastern states were severely reduced by a fungal pathogen. Based on morphology, development, and pathology, this organism appeared to be Entomophaga maimaiga. We have now used allozyme and restriction fragment length polymorphism analyses to confirm this identification. Previously, this mycopathogen had been reported only from gypsy moth populations in Japan. During 1989, E. maimaiga occurred only in areas that had been initially defoliated by gypsy moth >10 years ago. E. maimaiga caused 60-88% mortality in late instar larvae on research sites in central Massachusetts.

Crypticola clavulifera gen. et sp. nov. and Lagenidium giganteum: oomycetes pathogenic for dipterans infesting leaf axils in an Australian rain forest.

The isolation of two entomopathogenic fungi from Forcipomyia marksae larvae collected in leaf axils of Colocasia macrorrhiza in northeastern Queensland rain forests is reported. An oomycete, in which the zoospores complete their development wholly within the sporangium and are discharged through a short papillar extension, is described as a new genus and species, Crypticola clavulifera. A strain of the well-known oomycete, Lagenidium giganteum, was isolated from F. marksae--the first record of this fungus infecting a member of the Ceratopogonidae. The pathogenesis and zoosporogenesis of the two fungi in mosquito and ceratopogonid larvae are described.

Equine zygomycosis caused by Conidiobolus lamprauges.

A 15-year-old Arabian mare from southern Louisiana with a 2-month history of periodic epistaxis and severe weight loss had a large, fibrosing, granulomatous mass containing numerous nodules ("kunkers") projecting dorsally into the nasopharynx, and was euthanized at the owner's request. In addition to these kunkers, the mass contained a single trematode tentatively identified as Fasciola hepatica. Several kunkers were removed, washed thoroughly in sterile water and embedded in nutrient agars; the fungus that grew out of them was identified as Conidiobolus lamprauges Drechsler (Entomophthorales: Ancylistaceae). This is the first report of C. lamprauges from any vertebrate mycosis, and only the third Conidiobolus species reported from vertebrates. Unlike many other entomophthoraleans, the fungus isolated from this mycosis grew well at 37 degrees C. The possible means by which Conidiobolus species may infect vertebrates is discussed. These mycoses probably result most often from chronic exposure during sleep to conidia discharged from fungal growth on decaying plant material in the bedding.