Pathogen-microbiome interactions and the virulence of an entomopathogenic fungus.

Bacteria shape interactions between hosts and fungal pathogens. In some cases, bacteria associated with fungi are essential for pathogen virulence. In other systems, host-associated microbiomes confer resistance against fungal pathogens. We studied an aphid-specific entomopathogenic fungus called Pandora neoaphidis in the context of both host and pathogen microbiomes. Aphids host several species of heritable bacteria, some of which confer resistance against Pandora. We first found that spores that emerged from aphids that harbored protective bacteria were less virulent against subsequent hosts and did not grow on plate media. We then used 16S amplicon sequencing to study the bacterial microbiome of fungal mycelia and spores during plate culturing and host infection. We found that the bacterial community is remarkably stable in culture despite dramatic changes in pathogen virulence. Last, we used an experimentally transformed symbiont of aphids to show that Pandora can acquire host-associated bacteria during infection. Our results uncover new roles for bacteria in the dynamics of aphid-pathogen interactions and illustrate the importance of the broader microbiological context in studies of fungal pathogenesis. IMPORTANCE: Entomopathogenic fungi play important roles in the population dynamics of many insect species. Understanding the factors shaping entomopathogen virulence is critical for agricultural management and for the use of fungi in pest biocontrol. We show that heritable bacteria in aphids, which confer protection to their hosts against fungal entomopathogens, influence virulence against subsequent hosts. Aphids reproduce asexually and are typically surrounded by genetically identical offspring, and thus these effects likely shape the dynamics of fungal disease in aphid populations. Furthermore, fungal entomopathogens are known to rapidly lose virulence in lab culture, complicating their laboratory use. We show that this phenomenon is not driven by changes in the associated bacterial microbiome. These results contribute to our broader understanding of the aphid model system and shed light on the biology of the Entomophthorales-an important but understudied group of fungi.

Entomophthoralean and hypocrealean fungal pathogens of the sugarcane aphid, Melanaphis sacchari (Hemiptera: Aphididae), on sorghum in Georgia.

The sugarcane aphid, Melanaphis sacchari, is a widely distributed insect that attacks grasses in different genera including Miscanthus, Saccharum, and Sorghum. The invasive aphid superclone was first discovered in the U.S. attacking grain sorghum in Texas in 2013. Since then, it has been found in at least 25 states including Georgia. We conducted a survey of naturally occurring fungal pathogens of sugarcane aphids on five farms in Georgia, and identified a hypocrealean fungus, Akanthomyces dipterigenus, and two entomophthoralean fungi, Neoconidiobolus spp. From 2018 to 2020, fungal activity differed across farms but at one farm both major fungal species, A. dipterigenus and N. thromboides, were found each of the 3 years infecting sugarcane aphids, attacking adults, both alatae and apterae, and nymphs.

Histologic findings of Massospora cicadina infection in periodical cicadas (Magicicada septendecim).

Massospora cicadina, an obligate fungal pathogen in the subphylum Entomophthoromycotina (Zoopagomycota), infects periodical cicadas (Magicicada spp.) during their adult emergence and modifies their sexual behavior to maximize fungal spore dissemination. In this study, 7 periodical cicadas from the Brood X emergence in 2021 infected by M. cicadina were histologically examined. In 7 of 7 cicadas, fungal masses replaced the posterior portion of the abdominal cavity, effacing portions of the body wall, reproductive organs, alimentary tract, and fat bodies. No appreciable inflammation was noted at the intersections of the fungal masses and host tissues. Fungal organisms were present in multiple morphologies including protoplasts, hyphal bodies, conidiophores, and mature conidia. Conidia were clustered into eosinophilic membrane-bound packets. These findings help uncover the pathogenesis of M. cicadina by suggesting there is evasion of the host immune response and by providing a more in-depth description of its relationship with Magicicada septendecim than previously documented.

Strongwellsea crypta (Entomophthorales: Entomophthoraceae), a new species infecting Botanophila fugax (Diptera: Anthomyiidae).

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.

Pathogenicity against hemipteran vector insects of a novel insect pathogenic fungus from Entomophthorales (Pandora sp. nov. inedit.) with potential for biological control.

A new but still unpublished entomopathogenic fungus (ARSEF13372) in the genus Pandora (Entomophthorales: Entomophthoraceae) was originally isolated from Cacopsylla sp. (Hemiptera: Psyllidae). Several species of the genus Cacopsylla vector phloem-borne bacteria of the genus 'Candidatus Phytoplasma', which cause diseases in fruit crops such as apple proliferation, pear decline and European stone fruit yellows. To determine Pandora's host range and biocontrol potential we conducted laboratory infection bioassays; Hemipteran phloem-feeding insects were exposed to conidia actively discharged from in vitro produced mycelial mats of standardized area. We documented the pathogenicity of Pandora sp. nov. to species of the insect families Psyllidae and Triozidae, namely Cacopsyllapyri L., C.pyricola (Foerster), C.picta (Foerster, 1848), C.pruni (Scopoli), C.peregrina (Foerster), and Trioza apicalis Foerster. The occurrence of postmortem signs of infection on cadavers within 10 days post inoculation proved that Pandora sp. nov. was infective to the tested insect species under laboratory conditions and significantly reduced mean survival time for C.pyri (summer form and nymph), C.pyricola, C.picta, C.pruni, C.peregrina and T.apicalis. Assessing a potential interaction between phytoplasma, fungus and insect host revealed that phytoplasma infection ('Candidatus Phytoplasma mali') of the vector C.picta and/or its host plant apple Malus domestica Borkh. did not significantly impact the survival of C.picta after Pandora sp. nov. infection. The results from infection bioassays were discussed in relation to Pandora sp. nov. host range and its suitability as biocontrol agent in integrated pest management strategies of psyllid pests, including vector species, in orchards.

Stochasticity and Infectious Disease Dynamics: Density and Weather Effects on a Fungal Insect Pathogen.

In deterministic models of epidemics, there is a host abundance threshold above which the introduction of a few infected individuals leads to a severe epidemic. Studies of weather-driven animal pathogens often assume that abundance thresholds will be overwhelmed by weather-driven stochasticity, but tests of this assumption are lacking. We collected observational and experimental data for a fungal pathogen, Entomophaga maimaiga, that infects the gypsy moth, Lymantria dispar. We used an advanced statistical-computing algorithm to fit mechanistic models to our data, such that different models made different assumptions about the effects of host density and weather on E. maimaiga epizootics (epidemics in animals). We then used Akaike information criterion analysis to choose the best model. In the best model, epizootics are driven by a combination of weather and host density, and the model does an excellent job of explaining the data, whereas models that allow only for weather effects or only for density-dependent effects do a poor job of explaining the data. Density-dependent transmission in our best model produces a host density threshold, but this threshold is strongly blurred by the stochastic effects of weather. Our work shows that host-abundance thresholds may be important even if weather strongly affects transmission, suggesting that epidemiological models that allow for weather have an important role to play in understanding animal pathogens. The success of our model means that it could be useful for managing the gypsy moth, an important pest of hardwood forests in North America.

Secondary conidia types in the insect pathogenic fungal genus Strongwellsea (Entomophthoromycotina: Entomophthorales) infecting adult Diptera.

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.

Strongwellsea tigrinae and Strongwellsea acerosa (Entomophthorales: Entomophthoraceae), two new species infecting dipteran hosts from the genus Coenosia (Muscidae).

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.

Human Pathogenic Entomophthorales.

The pathogenic entomophthoralean fungi cause infection in insects and mammalian hosts. Basidiobolus and Conidiobolus species can be found in soil and insect, reptile, and amphibian droppings in tropical and subtropical areas. The life cycles of these fungi occur in these environments where infecting sticky conidia are developed. The infection is acquired by insect bite or contact with contaminated environments through open skin. Conidiobolus coronatus typically causes chronic rhinofacial disease in immunocompetent hosts, whereas some Conidiobolus species can be found in immunocompromised patients. Basidiobolus ranarum infection is restricted to subcutaneous tissues but may be involved in intestinal and disseminated infections. Its early diagnosis remains challenging due to clinical similarities to other intestinal diseases. Infected tissues characteristically display eosinophilic granulomas with the Splendore-Höeppli phenomenon. However, in immunocompromised patients, the above-mentioned inflammatory reaction is absent. Laboratory diagnosis includes wet mount, culture serological assays, and molecular methodologies. The management of entomophthoralean fungi relies on traditional antifungal therapies, such as potassium iodide (KI), amphotericin B, itraconazole, and ketoconazole, and surgery. These species are intrinsically resistant to some antifungals, prompting physicians to experiment with combinations of therapies. Research is needed to investigate the immunology of entomophthoralean fungi in infected hosts. The absence of an animal model and lack of funding severely limit research on these fungi.

Mulching with coffee husk and pulp in strawberry affects edaphic predatory mite and spider mite densities.

Mulching of soil beds of strawberry fields is usually done with polyethylene film in southern Minas Gerais state, Brazil. This material is relatively expensive and difficult to discard after use. In some countries, mulching is done with the use of organic material that could have an advantage over the use of plastic for its easier degradation after use, and for favoring edaphic beneficial organisms. Predatory mites (especially Gamasina, Mesostigmata) may be abundant in the soil and could conceivably move to the soil surface and onto the short-growing strawberry plants at night, helping in the control or pest arthropods. The two-spotted spider mite, Tetranychus urticae Koch, is considered an important strawberry pest in that region, where the fungus Neozygites floridana (Weiser and Muma) has been found to infect it. Different mulching types could affect the incidence of this pathogen. Dehydrated coffee husk and pulp (DCHP) is a byproduct readily available in southern Minas Gerais, where could be used as organic mulching in strawberry beds. The temporary contact of that material with the soil of a patch of natural vegetation could facilitate its colonization by edaphic predatory mites helpful in the control of strawberry pests. The objective of this work was to study the effect of mulching type on the population dynamics of the two-spotted spider mite, associate mites and N. floridana, in a greenhouse and in the field. The use of DCHP increased the number of edaphic Gamasina on strawberry plants-Proctolaelaps pygmaeus (Müller) (Melicharidae) and Blattisocius dentriticus (Berlese) (Blattisociidae) were observed on strawberry leaflets, mainly in nocturnal samplings, indicating their possible daily migration from soil to plants. Lower levels of two-spotted spider mite occurred on plants from pots or soil beds mulched with DCHP instead of polyethylene film, possibly because of the slightly higher levels of mites of the family Phytoseiidae and infection by N. floridana. Adding DCHP onto the floor of natural vegetation did not result in higher diversity or levels of gamasine mites on DCHP. Complementary studies should be conducted to find ways to increase diversity and density of those organisms in strawberry beds, in an attempt to improve biological control of strawberry pests. The decision to use DCHP for mulching should also take into account other factors such as strawberry yield, costs and efficiency of weed management, to be evaluated in subsequent studies.

Pandora formicae, a specialist ant pathogenic fungus: New insights into biology and taxonomy.

Among fungi from the order Entomophthorales (Entomophthoromycota), there are many specialized, obligatory insect-killing pathogens. Pandora formicae (Humber & Balazy) Humber is a rare example of an entomophthoralean fungus adapted to exclusively infect social insects: wood ants from the genus Formica. There is limited information available on P. formicae; many important aspects of this host-pathogen system remain hitherto unknown, and the taxonomical status of the fungus is unclear. Our study fills out some main gaps in the life history of P. formicae, such as seasonal prevalence and overwintering strategy. Field studies of infection prevalence show a disease peak in late summer and early autumn. Typical thick-walled entomophthoralean resting spores of P. formicae are documented and described for the first time. The proportion of cadavers with resting spores increased from late summer throughout autumn, suggesting that these spores are the main overwintering fungal structures. In addition, the phylogenetic status of Pandora formicae is outlined. Finally, we review the available taxonomical literature and conclude that the name P. formicae should be used rather than the name P. myrmecophaga for ant-infecting fungi displaying described morphological features.

Fatal diseases and parasitoids: from competition to facilitation in a shared host.

Diverse parasite taxa share hosts both at the population level and within individual hosts, and their interactions, ranging from competitive exclusion to facilitation, can drive community structure and dynamics. Emergent pathogens have the potential to greatly alter community interactions. We found that an emergent fungal entomopathogen dominated pre-existing lethal parasites in populations of the forest defoliating gypsy moth,Lymantria dispar The parasite community was composed of the fungus and four parasitoid species that only develop successfully after they kill the host, and a virus that produces viable propagules before the host has died. A low-density site was sampled over 17 years and compared with 66 sites across a range of host densities, including outbreaks. The emergent fungal pathogen and competing parasitoids rarely co-infected host individuals because each taxa must kill its host. The virus was not present at low host densities, but successfully co-infected with all other parasite species. In fact, there was facilitation between the virus and one parasitoid species hosting a polydnavirus. This newly formed parasite community, altered by an emergent pathogen, is shaped both by parasite response to host density and relative abilities of parasites to co-inhabit the same host individuals.

Interactions between natural enemies: Effect of a predatory mite on transmission of the fungus Neozygites floridana in two-spotted spider mite populations.

Introducing the predatory mite Phytoseiulus persimilis into two-spotted spider mite, Tetranychus urticae, populations significantly increased the proportion of T. urticae infected with the spider mite pathogen Neozygites floridana in one of two experiments. By the final sampling occasion, the number of T. urticae in the treatment with both the predator and the pathogen had declined to zero in both experiments, while in the fungus-only treatment T. urticae populations still persisted (20-40 T. urticae/subsample). Releasing P. persimilis into crops in which N. floridana is naturally present has the potential to improve spider mite control more than through predation alone.

Detection of presumptive mycoparasites associated with Entomophaga maimaiga resting spores in forest soils.

The fungal pathogen Entomophaga maimaiga can provide high levels of control of the gypsy moth, Lymantria dispar, an important forest defoliator. This fungus persists in the soil as resting spores and occurs naturally throughout many areas where gypsy moth is established. Studies on the spatial dynamics of gypsy moth population have shown high variability in infection levels, and one possible biological factor could be the variable persistence of E. maimaiga resting spores in the soil due to attacks by mycoparasites. We surveyed presumptive mycoparasites associated with parasitized E. maimaiga resting spores using baiting and molecular techniques and identified an ascomycete (Pochonia sp.) and oomycetes (Pythium spp.).

Two-spotted spider mite and its natural enemies on strawberry grown as protected and unprotected crops in Norway and Brazil.

Cultivation of strawberry in plastic tunnels has increased considerably in Norway and in southeastern Brazil, mainly in an attempt to protect the crop from unsuitable climatic factors and some diseases as well as to allow growers to expand the traditional production season. It has been hypothesized that cultivation under tunnels could increase the incidence of one of its major pests in many countries where strawberry is cultivated, including Norway and Brazil, the two spotted spider mite, Tetranychus urticae. The objective of this study was to evaluate the effect of the use of tunnels on the incidence of T. urticae and on its natural enemies on strawberry in two ecologically contrasting regions, Norway (temperate) and southeastern Brazil (subtropical). In both countries, peak densities of T. urticae in tunnels and in the open fields were lower than economic thresholds reported in the literature. Factors determining that systematically seem to be the prevailing relatively low temperature in Norway and high relative humidity in both countries. The levels of occurrence in Norway and Brazil in 2010 were so low that regardless of any potential effect of the use of tunnel, no major differences were observed between the two cropping systems in relation to T. urticae densities. In 2009 in Norway and in 2011 in Brazil, increase in T. urticae population seemed to have been restrained mainly by rainfall in the open field and by predatory mites in the tunnels. Phytoseiids were the most numerous predatory mite group of natural occurrence on strawberry, and the prevalence was higher in Brazil, where the most abundant species on strawberry leaves were Neoseiulus anonymus and Phytoseiulus macropilis. In Norway, the most abundant naturally occurring phytoseiids on strawberry leaves were Typhlodromus (Anthoseius) rhenanus and Typhlodromus (Typhlodromus) pyri. Predatory mites were very rare in the litter samples collected in Norway. Infection rate of the pest by the fungus Neozygites floridana (Neozygitaceae) was low. The results of this work suggest that in Norway the use of tunnels might not affect the population densities of T. urticae on strawberry in years of lower temperatures. When temperature is not a limiting factor for the development of T. urticae in that country (apparently always the case in southern Brazil), strawberry cultivation in the tunnels may allow T. urticae to reach higher population levels than in open fields (because of the provided protection from the direct impact of rainfall), but natural enemies may prevent higher levels from being reached.

ENTOMOPATHOGENIC FUNGUS ENTOMOPHAGA MAIMAIGA AND INTEGRATED PEST MANAGEMENT IN SERBIA.

During the latest outbreak of the gypsy moth in Serbia (2009-2014), some areas of Central Serbia were particularly endangered, and one of them was Krusevac region, where the forests give way to orchards in the pattern resembling the tiger's skin. Since the number of the laid egg masses in the autumn 2013 guaranteed the defoliation of both forest tree species and agricultural crops, and the presence of E. maimaigo, in Central Serbia had already been determined, at 30 selected plots the assisted spread of it was performed, through the introduction of the infectious inoculum in the beech and oak forests which border the orchards. Since there was dealt with the living organism--fungus, which is particularly susceptible to the weather conditions (temperature and air humidity, as well as the precipitation), and under the conditions of the global warming and great drought, the special recipe for the preparation of inoculum was made. In the following year the mass epizootic of the gypsy moth caterpillars, of the younger instars (L2 and L3), occurred, which implies that E. maimaiga caused the crash of the outbreak of this most harmful species of the defoliating insects of the forests and orchards.

Confirmation of Neozygites floridana azygospore formation in two-spotted spider mite (Tetranychus urticae) in strains from tropical and temperate regions.

Neozygites floridana is an obligate fungal pathogen of mites in the family Tetranychidae and is an important natural enemy of the two-spotted spider mite (Tetranychus urticae). Until now, information about the formation of azygospores remained to be fully confirmed. In this study, we document the formation of azygospores by a Brazilian N. floridana strain and the formation of azygospores and zygospores by a Norwegian N. floridana strain, both in the host T. urticae. Evidence of both zygosporogenesis and azygosporogenesis was also found in the same individual in the Norwegian stains. Further we report the presence of immature azygospores with 1-3 nuclei for the Norwegian strains, immature resting spores (probably azygospores) with 1-8 nuclei for the Brazilian strain, and mature resting spores with 2 nuclei for both the Norwegian and the Brazilian strains (azygo- or zygospores). Our observations suggest that the immature resting spore (prespore) of both strains begins in a multinucleate condition but that the nuclear number is reduced during maturation until mature resting spore is binucleate regardless of its origin as a zygospore or azygospore.

Signatures of transposon-mediated genome inflation, host specialization, and photoentrainment in Entomophthora muscae and allied entomophthoralean fungi.

Despite over a century of observations, the obligate insect parasites within the order Entomophthorales remain poorly characterized at the genetic level. In this manuscript, we present a genome for a laboratory-tractable Entomophthora muscae isolate that infects fruit flies. Our E. muscae assembly is 1.03 Gb, consists of 7810 contigs and contains 81.3% complete fungal BUSCOs. Using a comparative approach with recent datasets from entomophthoralean fungi, we show that giant genomes are the norm within Entomophthoraceae owing to extensive, but not recent, Ty3 retrotransposon activity. In addition, we find that E. muscae and its closest allies possess genes that are likely homologs to the blue-light sensor white-collar 1, a Neurospora crassa gene that has a well-established role in maintaining circadian rhythms. We uncover evidence that E. muscae diverged from other entomophthoralean fungi by expansion of existing families, rather than loss of particular domains, and possesses a potentially unique suite of secreted catabolic enzymes, consistent with E. muscae's species-specific, biotrophic lifestyle. Finally, we offer a head-to-head comparison of morphological and molecular data for species within the E. muscae species complex that support the need for taxonomic revision within this group. Altogether, we provide a genetic and molecular foundation that we hope will provide a platform for the continued study of the unique biology of entomophthoralean fungi.

Emergent fungal entomopathogen does not alter density dependence in a viral competitor.

Population cycles in forest Lepidoptera often result from recurring density-dependent epizootics of entomopathogens. While these systems are typically dominated by a single pathogen species, insects are often infected by multiple pathogens, yet little is known how pathogens interact to affect host dynamics. The apparent invasion of northeastern North America by the fungal entomopathogen Entomophaga maimaiga some time prior to 1989 provides a unique opportunity to evaluate such interactions. Prior to the arrival of E. maimaga, the oscillatory dynamics of host gypsy moth, Lymantria dispar, populations were apparently driven by epizootics of a nucleopolyhedrovirus. Subsequent to its emergence, E. maimaiga has caused extensive mortality in host populations, but little is known about how it has altered multigenerational dynamics of the gypsy moth and its virus. Here we compared demographic data collected in gypsy moth populations prior to vs. after E. maimaiga's invasion. We found that the recently invading fungal pathogen virtually always causes greater levels of mortality in hosts than does the virus, but fungal mortality is largely density independent. Moreover, the presence of the fungus has apparently not altered the gypsy moth-virus density-dependent interactions that were shown to drive periodic oscillations in hosts before the arrival of the fungus.

Chytrid mycoparasitism of entomophthoralean azygospores.

Mycoparasitism - when one fungus parasitizes another - has been reported to affect Beauveria bassiana and mycorrhizal fungi in the field. However, mycoparasitism of any fungi in the Order Entomophthorales has never been reported before now. The majority of entomophthoralean species persist as resting spores (either zygospores or azygospores) in the environment and dormant entomophthoralean resting spores (whether formed as zygospores or azygospores) are thought to be especially well adapted for survival over long periods due to their thick double walls. Entomophthoralean resting spores can accumulate in the soil as large reservoirs of inoculum which can facilitate the onset and development of epizootics. We report parasitism of azygospores of the gypsy moth pathogen Entomophaga maimaiga caged in soil from southern Ohio by the chytrid fungus Gaertneriomyces semiglobifer. G. semiglobifer had previously been isolated from soil samples from North America, Europe and Australia or horse manure from Virginia. After isolation and identification of G. semiglobifer, azygospores of E. maimaiga exposed to zoospores of G. semiglobifer exhibited high levels of mycoparasitism and G. semiglobifer was subsequently reisolated from mycoparasitized resting spores. We discuss the importance of this finding to the epizootiology of insect diseases caused by entomophthoralean fungi.