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.

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.

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.

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.

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.

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.

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.

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.

Developing a list of invasive alien species likely to threaten biodiversity and ecosystems in the European Union.

The European Union (EU) has recently published its first list of invasive alien species (IAS) of EU concern to which current legislation must apply. The list comprises species known to pose great threats to biodiversity and needs to be maintained and updated. Horizon scanning is seen as critical to identify the most threatening potential IAS that do not yet occur in Europe to be subsequently risk assessed for future listing. Accordingly, we present a systematic consensus horizon scanning procedure to derive a ranked list of potential IAS likely to arrive, establish, spread and have an impact on biodiversity in the region over the next decade. The approach is unique in the continental scale examined, the breadth of taxonomic groups and environments considered, and the methods and data sources used. International experts were brought together to address five broad thematic groups of potential IAS. For each thematic group the experts first independently assembled lists of potential IAS not yet established in the EU but potentially threatening biodiversity if introduced. Experts were asked to score the species within their thematic group for their separate likelihoods of i) arrival, ii) establishment, iii) spread, and iv) magnitude of the potential negative impact on biodiversity within the EU. Experts then convened for a 2-day workshop applying consensus methods to compile a ranked list of potential IAS. From an initial working list of 329 species, a list of 66 species not yet established in the EU that were considered to be very high (8 species), high (40 species) or medium (18 species) risk species was derived. Here, we present these species highlighting the potential negative impacts and the most likely biogeographic regions to be affected by these potential IAS.

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.

Strong host specialization in fungus genus Strongwellsea (Entomophthorales).

The genus Strongwellsea (Entomophthorales) has a unique pathobiology. Infected adult dipteran hosts develop a large hole in their abdomens, through which conidia are actively discharged while the hosts are still alive. We analyzed the IST II region of Strongwellsea from 29 infected hosts representing 15 dipteran species from Anthomyiidae, Fanniidae, Muscidae, and Scathophagidae. Each genotype was found on only one host species or a few closely related host species. Strongwellsea genotypes infecting flies from Anthomyiidae represented a monophyletic lineage, including the species Strongwellsea castrans, while genotypes infecting Muscidae were very diverse and clustered at different places. All three host species from Fanniidae were infected with the same Strongwellsea genotype, namely the species Strongwellsea magna. It appears that members of the genus Strongwellsea are strongly adapted to their host species and have co-evolved.

Exploring virulence of new and less studied species of Metarhizium spp. from Brazil for two-spotted spider mite control.

The two-spotted spider mite Tetranychus urticae is an important pest of strawberry crops in Brazil and many other countries. Focus for biocontrol studies involving entomopathogenic fungi has been on three species from the genus Metarhizium: M. anisopliae sensu stricto (s.s.), M. brunneum and M. robertsii. Also, the species Beauveria bassiana has been studied for spider mite control and one isolate (ESALQPL63) is commercially available in Brazil. New and undescribed Metarhizium species have been found recently in Brazil and provide a pool of isolates with potential for biocontrol in Brazil and probably also elsewhere. The mortality of adult females of T. urticae when exposed to four new Brazilian species of Metarhizium was compared to the mortality when exposed to M. anisopliae s.s., M. brunneum, M. pingshaense, M. robertsii and Beauveria bassiana ESALQPL63. Fungal suspensions were sprayed onto mites at 107 conidia/mL with 0.05% Tween 80 in laboratory bio-assays. We measured total mortality and percentage sporulating cadavers 10 days after exposure and calculated median lethal time (LT50). The lowest LT50 (4.0 ± 0.17) was observed for mites treated with Metarhizium sp. Indet. 1 (ESALQ1638), which also performed well with respect to mortality after 10 days and capacity to sporulate from cadavers. Among the other little studied species tested, M. pingshaense (ESALQ3069 and ESALQ3222) and Metarhizium Indet. 2 (ESALQ1476) performed well and were comparable to B. bassiana (ESALQPL63). The new Metarhizium isolates and species thus showed potential for biological control.

Comparative transcriptomics reveal host-specific nucleotide variation in entomophthoralean fungi.

Obligate parasites are under strong selection to increase exploitation of their host to survive while evading detection by host immune defences. This has often led to elaborate pathogen adaptations and extreme host specificity. Specialization on one host, however, often incurs a trade-off influencing the capacity to infect alternate hosts. Here, we investigate host adaptation in two morphologically indistinguishable and closely related obligate specialist insect-pathogenic fungi from the phylum Entomophthoromycota, Entomophthora muscae sensu stricto and E. muscae sensu lato, pathogens of houseflies (Musca domestica) and cabbage flies (Delia radicum), respectively. We compared single nucleotide polymorphisms within and between these two E. muscae species using 12 RNA-seq transcriptomes from five biological samples. All five isolates contained intra-isolate polymorphisms that segregate in 50:50 ratios, indicative of genetic duplication events or functional diploidy. Comparative analysis of dN/dS ratios between the multinucleate E. muscae s.str. and E. muscae s.l. revealed molecular signatures of positive selection in transcripts related to utilization of host lipids and the potential secretion of toxins that interfere with the host immune response. Phylogenetic comparison with the nonobligate generalist insect-pathogenic fungus Conidiobolus coronatus revealed a gene-family expansion of trehalase enzymes in E. muscae. The main sugar in insect haemolymph is trehalose, and efficient sugar utilization was probably important for the evolutionary transition to obligate insect pathogenicity in E. muscae. These results support the hypothesis that genetically based host specialization in specialist pathogens evolves in response to the challenge of using resources and dealing with the immune system of different hosts.

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.

Life cycle assessment of edible insects for food protein: a review.

Compared to their vertebrate counterparts in traditional husbandry, insects are extremely efficient at converting organic matter into animal protein and dietary energy. For this reason, insects for food and feed show great potential as an environmentally friendly choice in future food systems. However, to obtain a true assessment of this, more information is needed about the production systems. Currently, only six studies applying the life cycle assessment (LCA) method to insect production systems have been published. The studies are heterogenous and thus difficult to compare. The aim of this paper was to establish a versatile reference framework that would allow for the selection of standardized settings for LCA applications in insect production systems, taking both the peculiarity of each system and the latest developments in food LCA into account. It is recommended that future LCAs of insect production systems take the following into account: (1) clear definition of the insect species and life stages included in the LCA, (2) use of at least two of the following types of functional units: nutritional, mass, or economic-based, (3) collection of empirical data in situ (e.g., on farms/production sites), (4) comparative analysis where production systems produce products that are realistic alternatives to the insect species under investigation, (5) inclusion of additional or previously unconsidered unit processes, such as processing and storage and waste management, and (6) use of a wide range of impact categories, especially climate change, resource consumption, nutrient enrichment potential, acidification potential, and impacts on land and water consumption in order to allow for comparison between studies.

Transcriptome of an entomophthoralean fungus (Pandora formicae) shows molecular machinery adjusted for successful host exploitation and transmission.

Pandora formicae is an obligate entomopathogenic fungus from the phylum Entomophthoromycota, known to infect only ants from the genus Formica. In the final stages of infection, the fungus induces the so-called summit disease syndrome, manipulating the host to climb up vegetation prior to death and fixing the dead cadaver to the surface, all to increase efficient spore dispersal. To investigate this fascinating pathogen-host interaction, we constructed interaction transcriptome libraries from two final infection stages from the material sampled in the field: (1) when the cadavers were fixed, but the fungus had not grown out through the cuticle and (2) when the fungus was growing out from host cadaver and producing spores. These phases mark the switch from within-host growth to reproduction on the host surface, after fungus outgrowth through host integument. In this first de novo transcriptome of an entomophthoralean fungus, we detected expression of many pathogenicity-related genes, including secreted hydrolytic enzymes and genes related to morphological reorganization and nutrition uptake. Differences in expression of genes in these two infection phases were compared and showed a switch in enzyme expression related to either cuticle breakdown or cell proliferation and cell wall remodeling, particularly in subtilisin-like serine protease and trypsin-like protease transcripts.

Root isolations of Metarhizium spp. from crops reflect diversity in the soil and indicate no plant specificity.

Metarhizium spp. have recently been shown to be associated with the roots of different plants. Here we evaluated which Metarhizium species were associated with roots of oat (Avena sativa), rye (Secale cereale) and cabbage (Brassica oleracea), common crop plants in Denmark. Thirty-six root samples from each of the three crops were collected within an area of approximately 3ha. The roots were rinsed with sterile water, homogenized and the homogenate plated onto selective media. A subset of 126 Metarhizium isolates were identified to species by sequencing of the 5' end of the gene translation elongation factor 1-alpha and characterized by simple sequence repeat (SSR) analysis of 14 different loci. Metarhizium brunneum was the most common species isolated from plant roots (84.1% of all isolates), while M. robertsii (11.1%) and M. majus (4.8%) comprised the remainder. The SSR analysis revealed that six multilocus genotypes (MLGs) were present among the M. brunneum and M. robertsii isolates, respectively. A single MLG of M. brunneum represented 66.7%, 79.1% and 79.2% of the total isolates obtained from oat, rye and cabbage, respectively. The isolation of Metarhizium spp. and their MLGs from roots revealed a comparable community composition as previously reported from the same agroecosystem when insect baiting of soil samples was used as isolating technique. No specific MLG association with a certain crop was found. This study highlights the diversity of Metarhizium spp. found in the rhizosphere of different crops within a single agroecosystem and suggests that plants either recruit fungal associates from the surrounding soil environment or even govern the composition of Metarhizium populations.