Community composition of the entomopathogenic fungal genus Metarhizium in soils of tropical and temperate conventional and organic strawberry fields.

Studies on community composition and population structure of entomopathogenic fungi are imperative to link ecosystem functions to conservation biological control. We studied the diversity and abundance of Metarhizium spp. from soil of conventionally and organically farmed strawberry crops and from the adjacent field margins in two different climatic zones: Brazil (tropical) and Denmark (temperate), using the same isolating methods. In Brazilian strawberry soil, Metarhizium robertsii (n = 129 isolates) was the most abundant species, followed by M. humberi (n = 16); M. anisopliae (n = 6); one new taxonomically unassigned lineage Metarhizium sp. indet. 5 (n = 4); M. pingshaense (n = 1) and M. brunneum (n = 1). In Denmark, species composition was very different, with M. brunneum (n = 33) being isolated most commonly, followed by M. flavoviride (n = 6) and M. pemphigi (n = 5), described for the first time in Denmark. In total, 17 haplotypes were determined based on MzFG543igs sequences, four representing Danish isolates and 13 representing Brazilian isolates. No overall difference between the two climatic regimes was detected regarding the abundance of Metarhizium spp. in the soil in strawberry fields and the field margins. However, we found a higher Shannon's diversity index in organically managed soils, confirming a more diverse Metarhizium community than in soils of conventionally managed agroecosystems in both countries. These findings contribute to the knowledge of the indigenous diversity of Metarhizium in agricultural field margins with the potential to contribute to pest regulation in strawberry cropping systems.

Potential for Use of Species in the Subfamily Erynioideae for Biological Control and Biotechnology.

The fungal order Entomophthorales in the Zoopagomycota includes many fungal pathogens of arthropods. This review explores six genera in the subfamily Erynioideae within the family Entomophthoraceae, namely, Erynia, Furia, Orthomyces, Pandora, Strongwellsea, and Zoophthora. This is the largest subfamily in the Entomophthorales, including 126 described species. The species diversity, global distribution, and host range of this subfamily are summarized. Relatively few taxa are geographically widespread, and few have broad host ranges, which contrasts with many species with single reports from one location and one host species. The insect orders infected by the greatest numbers of species are the Diptera and Hemiptera. Across the subfamily, relatively few species have been cultivated in vitro, and those that have require more specialized media than many other fungi. Given their potential to attack arthropods and their position in the fungal evolutionary tree, we discuss which species might be adopted for biological control purposes or biotechnological innovations. Current challenges in the implementation of these species in biotechnology include the limited ability or difficulty in culturing many in vitro, a correlated paucity of genomic resources, and considerations regarding the host ranges of different species.

Pandora cacopsyllae Eilenberg, Keller & Humber (Entomophthorales: Entomophthoraceae), a new species infecting pear psyllid Cacopsylla pyri L. (Hemiptera: Psyllidae).

The new species Pandora cacopsyllae Eilenberg, Keller & Humber (Entomophthorales) is described. The fungus was found on infected pear psyllids Cacopsylla pyri (Hemiptera: Psyllidae) in a pear orchard in Zealand, Denmark. Morphological structures (conidia, rhizoids, cystidia) were described on the designated type host C. pyri. In addition, conidia from an in vitro culture were described. Pandora cacopsyllae differs from other Pandora species by a) C. pyri is the natural host; b) conidia are different from other Pandora species infecting Psylloidea; c) ITS differs from other Pandora species infecting Hemiptera. The fungus has a high potential for future use in biological control of Cacopsylla pest species as well as other psyllids.

Comparative transcriptomics of growth metabolism and virulence reveal distinct morphogenic profiles of yeast-like cells and hyphae of the fungus Metarhizium rileyi.

Metarhizium rileyiis an entomopathogenic fungus with a narrow host range which distinguishes it from other Metarhiziumspecies with broad host ranges. This species is also unique because the initial yeast-like growth on solid media is only observed in liquid culture in other Metharizium species. A lack of knowledge about the metabolism and genetic signatures of M. rileyiduring this yeast-like phase on solid and in liquid media is a bottleneck for its large-scale production as a commercial biocontrol agent.In this study wefound that M. rileyiyeast-like cells produced on solid medium infected and killed the important insect pest Spodoptera frugiperda with comparable efficiency as yeast-like cells grown in liquid medium. Secondly, we used comparative transcriptomic analysis to investigate theactive genes and genomic signatures of the M. rileyi yeast-like morphotypes produced on solid and in liquid media. Yeast-like cells grown in liquid medium had upregulated genes relating specifically to signal transduction andparticular membrane transporters. Thirdly, we compared the transcriptomic profiles of yeast-like phases of M. rileyi with those of M. anisopliae. The yeast-like phase of M. rileyi grown on solid medium upregulated unique genes not found in otherMetarhiziumspecies including specific membrane proteins and several virulence factors. Orthologous genes associated with heat shock protein, iron permease, membrane proteins and key virulence traits (e.g. collagen-like protein Mcl1) were upregulated in both species. Comparative transcriptome analyses of gene expression showed more differences than similarities between M. anisopliae and M. rileyi yeast-like cells.

Strongwellsea selandia and Strongwellsea gefion (Entomophthorales: Entomophthoraceae), two new species infecting adult flies from genus Helina (Diptera: Muscidae).

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.

Growth and Reproductive Performance of Edible Grasshopper (Ruspolia differens) on Different Artificial Diets.

Ruspolia differens (Serville) (Orthoptera: Tettigoniidae), also known as the 'edible grasshopper', 'African edible bush-cricket', and 'nsenene', is regarded as one of the most promising edible insect species that can be used for food, particularly in Sub-Saharan Africa. However, there is insufficient information on suitable diets and their effects on survival, adult weight, fecundity, and developmental time of this species, which are preconditions for large-scale production. In this study, we experimentally evaluated the effects of 12 diets (wheat bran, rice seed head, finger millet seed head, soya bran, maize bran, fresh maize comb, millet flour, chicken feed egg booster, simsim cake, sorghum seed head, powdered groundnut, and germinated finger millet), that are known to be accepted by R. differens, on their growth and reproductive parameters. The survival rate, developmental time, and adult weight varied considerably on the various diets. The highest nymphal survival rates, shortest development times, and highest adult weights were recorded for both sexes when fed fresh maize comb and germinated finger millet diet. Lifetime fecundity of females fed on germinated finger millet also was, on average, more than twice higher compared to other diets. The present study demonstrated that relatively inexpensive and locally available germinated finger millet, fresh maize seed (at the silking stage on the comb), sorghum seedhead, and finger millet seedhead could be successfully used to rear and sustain populations of R. differens. Our findings contribute to the future design of an effective mass-rearing system for this economically important edible insect.

Encapsulation of the psyllid-pathogenic fungus Pandora sp. nov. inedit. and experimental infection of target insects.

BACKGROUND: Pandora sp. nov. inedit. (Entomophthorales: Entomophthoraceae) (ARSEF 13372) is a recently isolated entomophthoralean fungus with potential for psyllid pest control. This study aimed to develop a formulation based on biocompatible hydrogel beads in order to transfer the fungus into an easily applicable form and to test the effects on psyllids. RESULTS: After encapsulation in calcium alginate beads, Pandora sp. nov. grew from the beads and discharged conidia over 12 days under optimal humidity conditions at 18 °C. Conidial number was increased 2.95-fold by the addition of skimmed milk as nutritional formulation adjuvant to the beads. The virulence of the encapsulated fungus was assessed with the two target psyllid species; the summer apple psyllid, Cacopsylla picta and the pear psyllid, Cacopsylla pyri. Beads containing skimmed milk as nutritional adjuvant led to the highest mortalities (48.3% on C. picta and 75.0% on C. pyri). In a second bioassay, survival time of C. pyri exposed to beads containing different concentration (10%, 20% or 40%) of Pandora sp. nov. was tested. The survival time of C. pyri was significantly reduced when exposed to beads containing 10% or 20% Pandora sp. nov. The median survival time was reached after 5-6 days past inoculation and the cumulative mortality for C. pyri treated with Pandora sp. nov. beads showed up to 89% mortality. CONCLUSION: The promising results of this study will ease the way for large-scale field application of a novel Pandora species in biological psyllid pest control.

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.

Development of novel spray-dried and air-dried formulations of Metarhizium robertsii blastospores and their virulence against Dalbulus maidis.

The present research addressed spray-drying and air-drying techniques applied to Metarhizium robertsii blastospores to develop wettable powder (WP) formulations. We investigated the effect of co-formulants on blastospore viability during drying and assessed the wettability and stability of formulations in water. The effect of oxygen-moisture absorbers was studied on the shelf life of these formulations stored at 26 °C and 4 °C for up to 90 days. Additionally, we determined the virulence of the best spray-dried and air-dried formulations against the corn leafhopper Dalbulus maidis. While sucrose and skim milk played an essential role as osmoprotectants in preserving air-dried blastospores, maltodextrin, skim milk, and bentonite were crucial to attain high cell survival during spray drying. The lowest wettability time was achieved with spray-dried formulations containing less Ca-lignin, while charcoal powder amount was positively associated with formulation stability. The addition of oxygen-moisture absorbers inside sealed packages increased from threefold to fourfold the half-life times of air-dried and spray-dried formulations at both storage temperatures. However, the half-life times of all blastospore-based formulations were shorter than 3 months regardless of temperature and packaging system. Spray-dried and air-dried WP formulations were as virulent as fresh blastopores against D. maydis adults sprayed with 5 × 107 blastospores mL-1 that induced 87.8% and 70.6% mortality, respectively. These findings bring innovative advancement for M. robertsii blastospore formulation through spray-drying and underpin the importance of adding protective matrices coupled to oxygen-moisture absorbers to extend cell viability during either cold or non-refrigerated storage. KEY POINTS: • Cost-effective wettable powder formulations of M. robertsii blastospores were developed. • Bioefficacy of formulations against the corn leafhopper was comparable to fresh blastospores. • Cold storage and dual oxygen-moisture absorber are critical for extended shelf life.

Image analysis-based quantification of fungal sporulation by automatic conidia counting and gray value correlation.

The present work describes a new computer-assisted image analysis method for the rapid, simple, objective and reproducible quantification of actively discharged fungal spores which can serve as a manual for laboratories working in this context. The method can be used with conventional laboratory equipment by using bright field microscopes, standard scanners and the open-source software ImageJ. Compared to other conidia quantification methods by computer-assisted image analysis, the presented method bears a higher potential to be applied for large-scale sample quantities. The key to make quantification faster is the calculation of the linear relationship between the gray value and the automatically counted number of conidia that has only to be performed once in the beginning of analysis. Afterwards, the gray value is used as single parameter for quantification. The fast, easy and objective determination of sporulation capacity enables facilitated quality control of fungal formulations designed for biological pest control.•Rapid, simple, objective and reproducible quantification of fungal sporulation suitable for large-scale sample quantities.•Requires conventional laboratory equipment and open-source software without technical or computational expertise.•The number of automatically counted conidia can be correlated with the gray value and after initial calculation of a linear fit, the gray value can be applied as single quantification parameter.

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.

Why has the authorization of microbial biological control agents been slower in the EU than in comparable jurisdictions?

The aim of this study was to identify reasons why the authorization of microbial pest control agents is lengthier under regulatory frameworks of the European Union (EU) than in comparable jurisdictions. A main conclusion is that although the EU's regulatory processes have strong scientific foundations, the most appropriate scientific concepts, knowledge and expertise have not been applied in the safety assessment of microorganisms and biological control. Tradition and conceptual legacies from assessments of conventional chemical pesticides have likely contributed to this by steering the evaluations of microorganisms in less appropriate directions. According to our investigation, the current framework for microbial plant protection products complies poorly with the principles that legislation should have legal predictability, proportionality, and that it should be non-discriminative, for instance in comparison to corresponding regulations in comparable jurisdictions. We also found that existing possibilities to take non-safety and ethical considerations into account can probably be used more. To rationalize the EU's authorization of microbial control products, both the basic legislation and the evaluations of agents and products need stronger rooting in fundamental microbiological science. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

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.

Comparative RNAseq Analysis of the Insect-Pathogenic Fungus Metarhizium anisopliae Reveals Specific Transcriptome Signatures of Filamentous and Yeast-Like Development.

The fungus Metarhizium anisopliae is a facultative insect pathogen used as biological control agent of several agricultural pests worldwide. It is a dimorphic fungus that is able to display two growth morphologies, a filamentous phase with formation of hyphae and a yeast-like phase with formation of single-celled blastospores. Blastospores play an important role for M. anisopliae pathogenicity during disease development. They are formed solely in the hemolymph of infected insects as a fungal strategy to quickly multiply and colonize the insect's body. Here, we use comparative genome-wide transcriptome analyses to determine changes in gene expression between the filamentous and blastospore growth phases in vitro to characterize physiological changes and metabolic signatures associated with M. anisopliae dimorphism. Our results show a clear molecular distinction between the blastospore and mycelial phases. In total 6.4% (n = 696) out of 10,981 predicted genes in M. anisopliae were differentially expressed between the two phases with a fold-change > 4. The main physiological processes associated with up-regulated gene content in the single-celled yeast-like blastospores during liquid fermentation were oxidative stress, amino acid metabolism (catabolism and anabolism), respiration processes, transmembrane transport and production of secondary metabolites. In contrast, the up-regulated gene content in hyphae were associated with increased growth, metabolism and cell wall re-organization, which underlines the specific functions and altered growth morphology of M. anisopliae blastospores and hyphae, respectively. Our study revealed significant transcriptomic differences between the metabolism of blastospores and hyphae. These findings illustrate important aspects of fungal morphogenesis in M. anisopliae and highlight the main metabolic activities of each propagule under in vitro growth conditions.

Growth kinetic and nitrogen source optimization for liquid culture fermentation of Metarhizium robertsii blastospores and bioefficacy against the corn leafhopper Dalbulus maidis.

The cosmopolitan entomopathogenic and root endophytic fungus Metarhizium robertsii has a versatile lifestyle and during liquid fermentation undergoes a dimorphic transformation from hyphae to conidia or microsclerotia, or from hyphae to blastospores. In all cases, these processes are mediated by environmental and nutritional cues. Blastospores could be used in spray applications to control arthropod pests above ground and may serve as an attractive alternative to the traditional solid-grown aerial conidial spores of Metarhizium spp. found in commercial products. Nitrogen is a vital nutrient in cell metabolism and growth; however, it is the expensive component in liquid cultures of entomopathogenic fungi. Our goals in this study were to optimize nitrogen sources and titers for maximum production of M. robertsii blastospores cultured in shake flasks at highly aerated conditions and to further determine their virulence against the corn leafhopper Dalbulus maidis, an important vector of serious pathogens in maize crops worldwide. Our fermentation studies revealed that the low-cost corn steep liquor (CSL) was the most suitable nitrogen source to improve blastospore growth in M. robertsii. The growth kinetic assays determined the optimal titer of 80 g L-1 and a yield up to 4.7 × 108 cells mL-1 within 5 days of cultivation (3 days preculture and 2 days culture), at a total cost of US$0.30 L-1. Moreover, the blastospore growth kinetic was strongly dependent on glucose and nitrogen consumptions accompanied by a slight drop in the culture pH. Insect bioassays evidenced a high virulence of these blastospores, either as dried or fresh cells, to D. maidis adults fed on maize plants. Our findings provide insights into the nutritional requirements for optimal and cost-efficient production of M. robertsii blastospores and elucidate the potential of blastospores as an ecofriendly tool against the corn leafhopper.

Psychoactive plant- and mushroom-associated alkaloids from two behavior modifying cicada pathogens.

Entomopathogenic fungi routinely kill their hosts before releasing infectious spores, but a few species keep insects alive while sporulating, which enhances dispersal. Transcriptomics- and metabolomics-based studies of entomopathogens with post-mortem dissemination from their parasitized hosts have unraveled infection processes and host responses. However, the 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 likely 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.

Entomopathogenic fungal conidia marginally affect the behavior of the predators Orius majusculus (Hemiptera: Anthocoridae) and Phytoseiulus persimilis (Acari: Phytoseiidae) foraging for healthy Tetranychus urticae (Acari: Tetranychidae).

We determined how conidia of arthropod-pathogenic fungi on leaves affected the behavior of two predators-Orius majusculus (Hemiptera: Anthocoridae) and Phytoseiulus persimilis (Acari: Phytoseiidae)-when offered a choice between preying on two-spotted spider mites, Tetranychus urticae (Acari: Tetranychidae), in the presence or absence of infective conidia of Metarhizium brunneum (Ascomycota: Hypocreales) and Neozygites floridana (Entomophthoromycota: Neozygitaceae). The results indicate no significant relation between the presence of conidia and predator behavior. The only indication of interference is between the generalists O. majusculus and M. brunneum, with a trend towards more time spent feeding and more prey encounters turning into feeding events on leaf discs without conidia than on leaf discs with conidia. Our results show that the presence of fungal conidia does not alter the preying behavior of the predators, and using predators and fungi together is not limited by any interference between organisms in the short term.