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[Display omitted] 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. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Strongwellsea from 15 dipteran hosts were strongly associated with host species. • Strongwellsea genotypes infecting seven species from Anthomyiidae formed a monophyletic lineage. • Strongwellsea genotypes infecting four species from Muscidae clustered at different places. • All infections in three Fannidae species were caused by the same genotype of Strongwellsea magna. • Based on host species, conidial morphology and phylogeny, the genus Strongwellsea contains several undescribed species. Abstract 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. [ABSTRACT FROM AUTHOR]

[Display omitted] • Two new species from the fungal genus Strongwellsea (Entomophthorales) are described. • Strongwellsea selandia infects adult Helina evecta (Diptera). • Strongwellsea gefion infects adult Helina reversio (Diptera). • The two new species differ from other described species from the genus. • Differences include morphology of spores, genetic identity, and host species. 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. [ABSTRACT FROM AUTHOR]

Strongwellsea crypta Eilenberg & Humber. [Display omitted] • Strongwellsea crypta is a new species infecting its dipteran host adult Botanophila fugax. • The description includes symptoms on the infected host, and morphology of spores. 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. [ABSTRACT FROM AUTHOR]

Abstract: It is increasingly recognized that the biodiversity in agroecosystems deliver significant ecosystem services to agricultural production such as biological control of pests. Entomopathogenic fungi, specifically the anamorphic taxa Beauveria bassiana and Metarhizium anisopliae, Hypocreales (Ascomycota), are among the natural enemies of pests in agroecosystems and the fungi are candidates for future conservation biological control in temperate regions. Conservation biological control is a biological control strategy in which farming practices and environmental manipulations are adopted to enhance the living conditions for specific natural enemies of pests. However, in order to manipulate the environment for the benefit of populations of the entomopathogens, knowledge of fundamental aspects of the ecology of the fungi considered is necessary. This knowledge is still scarce despite the large bulk of recent research into inoculation and inundation biological control with these fungi. Here, we review the current knowledge of the ecology of indigenous populations of B. bassiana and M. anisopliae in agroecosystems of temperate regions, primarily Europe and North America. We suggest anamorphic life cycles of B. bassiana and M. anisopliae in these regions based on the literature of their natural occurrence and distribution in agroecosystems, population dynamics, and interactions with other organisms, environmental factors, and agronomical practices. [Copyright &y& Elsevier]

[Display omitted] • Laboratory infection bioassay with conidia shower from Pandora sp. nov. mycelial mat. • The entomopathogen infects insect families Psyllidae and Triozidae. • Survival times of fruit crop pests are significantly reduced after inoculation. • No interaction between phytoplasma infection of vector, host plant and the fungus. • Pandora sp. nov. has high potential to become a biocontrol agent for vector 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 Cacopsylla pyri 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. [ABSTRACT FROM AUTHOR]

[Display omitted] • In Brazilian strawberry soil, M.robertsii was the most abundant species. • M. brunneum was the most abundant species in Denmark. • 17 Metarhizium haplotypes were determined. • We found a higher Shannon's diversity index in organically managed soils. 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. [ABSTRACT FROM AUTHOR]

• Two new species from the genus Strongwellsea are described from dipteran hosts. • Strongwellsea tigrinae was found on the host Coenosia tigrina (Muscidae) • Strongwellsea acerosa was found on the host Coenosia testaceae. • The circumscription of the genus Strongwellsea is emended. 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. [ABSTRACT FROM AUTHOR]

• We described two types of actively discharged secondary conidia in species from the insect pathogenic fungus genus Strongwellsea. • Ellipsoid type secondary conidia are broadly ellipsoidal with a clearly pointed basal papilla. • Subglobose type secondary conidia are subglobose to almost bell-shaped with a flattened papilla. 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 s econdary 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. [ABSTRACT FROM AUTHOR]

Among fungi from the order Entomophthorales (Entomophthoromycota), there are many specialized, obligatory insect-killing pathogens. Pandora formicae (Humber & Bałazy) 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. [ABSTRACT FROM AUTHOR]

Abstract: The below- and aboveground communities of fungal entomopathogens (Ascomycota: Hypocreales) were investigated in an experimental conventional and organic vegetable cropping system over two seasons in Denmark. The experimental design allowed for evaluating differences between farming practices in the occurrence of soil-borne fungal entomopathogens and as natural infections aboveground in arthropod hosts. Belowground, Metarhizium anisopliae dominated the agricultural field with Beauveria bassiana, Metarhizium flavoviride, and Isaria fumosorosea being present at lower frequencies. Abundances of M. anisopliae were not different between conventional and organic soils. Aboveground, B. bassiana was the most common fungal entomopathogen in arthropod host cadavers. Infections of M. flavoviride, Isaria farinosa, and Gibellula spp. were also recorded. Most mycosed cadavers were recovered in August and September from conventionally farmed plots. Cadaver sizes ranged from 1.9 to 23.6mm with means of 3.7–4.1mm. Among host functional groups, predators dominated (47–63%) over herbivores, parasitoids, and nectar-feeders. No occurrence of M. anisopliae was recorded among >200 mycosed cadavers aboveground. The results therefore suggest that M. anisopliae is not involved in direct below- and aboveground interactions in the investigated agroecosystem. Conservation biological control strategies targeting M. anisopliae should thus be solely for controlling soil-dwelling pests in temperate regions as this fungus is unlikely naturally to infect aboveground hosts in the agroecosytem. [Copyright &y& Elsevier]

Abstract: Recent studies have shown that the entomopathogenic fungus Beauveria bassiana occurs naturally on the phylloplanes of several plants, including nettles. Insects could, by their activity, be contributing to this inoculum by dispersing it from other sites. The potential of nettle aphids Microlophium carnosum and their predator Anthocoris nemorum to disperse conidia of B. bassiana from soil to nettles and from sporulating cadavers in the nettle canopy was investigated in laboratory experiments. In petri dish assays, aphids showed potential to distribute B. bassiana from soil to nettle leaves. Predators dispersed inoculum from both soil and cadavers to nettle leaves in petri dishes. In microcosms, aphids did not disperse B. bassiana from the soil or from cadavers confined in the canopy, but A. nemorum were able to transfer inoculum from soil into the nettle canopy and to distribute conidia from cryptic cadavers. In some instances, infections were initiated in aphids and predators as a consequence of dispersal. [Copyright &y& Elsevier]

A two-year field study using a blend of entomopathogens in conjunction with chemical insecticides was carried out to determine to which extent they affect western corn rootworm (WCR), Diabrotica virgifera virgifera , survival, maize root damages, and grain yield and to assess the potential for side-effects on natural arthropod enemies. The products tested were conducted on a maize crop and included maize seeds dressed with the neonicotinoid clothianidin (Poncho™), Belem™ (ai: cypermethrin), dianem™ (entomopathogenic nematode: Heterorhabditis bacteriophora ), and a granular formulation of the insect pathogenic fungus Metarhizium brunneum . Trials were conducted at four (2014) and two (2015) sites heavily infested with WCR in the Southeast of Styria, Austria. The lowest number of WCR adults were recorded in 2015 at sites where the nematode and fungal biocontrol agents were used with clothianidin dressed maize seeds. The treatments did not significantly influence plant lodging and grain yield compared with the untreated (negative) control. The WCR population density rose during the investigation period and the average number of beetles in all treatments and in both study years far exceeded the stipulated economic threshold value for continuous maize of around one beetle per plant. Non-target evaluation using pitfall traps did not show any significant impact of the treatments on the abundance or diversity of indigenous predatory beetles and spiders. The lack of damage suggests that the WCR threshold is considerably higher than the stipulated economic threshold under favourable growing conditions, but a systematic crop rotation is recommended as an integral part of the WCR management both as a prophylaxis and as a background for the best treatment. [ABSTRACT FROM AUTHOR]

• Yeast-like cells from solid and liquid media have similar virulence. • Yeast-like cells of Metarhizium rileyi express specific genes related to virulence. • Yeast-like cells grown on solid medium express unique genes. • Few up-regulated orthologous genes in common between M. rileyi and M. anisopliae yeast-like cells. • Many unknown protein-coding genes up-regulated in the yeast-like cells from solid medium. Metarhizium rileyi is an entomopathogenic fungus with a narrow host range which distinguishes it from other Metarhizium species 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. rileyi during 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 we found that M. rileyi yeast-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 the active 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 and particular 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 other Metarhizium species 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. [ABSTRACT FROM AUTHOR]

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 3 ha. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The entomopathogenic fungal Metarhizium anisopliae lineage harbors cryptic diversity and was recently split into several species. Metarhizium spp. are frequently isolated from soil environments, but the abundance and distribution of the separate species in local communities is still largely unknown. Entomopathogenic isolates of Metarhizium spp. were obtained from 32 bulked soil samples of a single agroecosystem in Denmark using Tenebrio molitor as bait insect. To assess the Metarhizium community in soil from the agricultural field and surrounding hedgerow, 123 isolates were identified by sequence analysis of 5′ end of elongation factor 1-α and their genotypic diversity characterized by multilocus simple sequence repeat (SSR) typing. Metarhizium brunneum was most frequent (78.8%) followed by M. robertsii (14.6%), while M. majus and M. flavoviride were infrequent (3.3% each) revealing co-occurrence of at least four Metarhizium species in the soil of the same agroecosystem. Based on SSR fragment length analysis five genotypes of M. brunneum and six genotypes of M. robertsii were identified among the isolates. A single genotype within M. brunneum predominated (72.3% of all genotypes) while the remaining genotypes of M. brunneum and M. robertsii were found at low frequencies throughout the investigated area indicating a diverse Metarhizium community . The results may indicate potentially favorable adaptations of the predominant M. brunneum genotype to the agricultural soil environment. [ABSTRACT FROM AUTHOR]

Abstract: The discovery of novel secretome proteins can add to our understanding of host–pathogen interactions. Here we report a rich diversity of secreted proteins from the interaction between grain aphids (host, insect order Hemiptera) and fungi of the order Entomophthorales (insect pathogens). The proteins were identified using a unique method unbiased by known sequences or functions to screen a cDNA library constructed directly from field-sampled material. We show for the first time that fungi from the genera Pandora and Entomophthora are armed with a battery of hydrolytic enzymes for penetrating the host cuticle. This enables both access to the hemolymph and exit for sporulation. Further, they secrete enzymes, most notably a number of lipases, for digestion of easily accessible high-energy compounds in the hemolymph. In contrast, we identified only few host genes potentially involved in the interaction, indicating that aphids respond only weakly to the pathogens. These results support recent findings that aphids have a reduced immune repertoire. [Copyright &y& Elsevier]

The survival of Pandora neoaphidis was studied for both discharged primary conidia and hyphal bodies inside aphid cadavers after storage on moist soil at different temperatures. The activity of the inoculum was quantified by the ability to produce replicate conidia as well as the ability to infect aphids. No effect of inoculum type was found. Conidia were produced after storage for at least 32 days at 20 °C, 64 days at 10 °C, and 96 days at 5 °C. Inoculum retained the ability to initiate infections in aphids after storage for at least 14 days at 20 °C, 32 days at 10 °C, and 64 days at 5 °C. Morphological studies of the inoculum suggest that P. neoaphidis may survive unfavorable conditions as thick-walled conidia also known as loricoconidia. Furthermore, P. neoaphidis and Conidiobolus obscurus were documented for the first time in field-collected soil in early spring by baiting the soil with aphids. We hypothesize that germination of overwintering inoculum is stimulated by host-induced factors since inoculum apparently responded to the presence of aphids. [Copyright &y& Elsevier]

Neozygites fresenii-infected Aphis gossypii cadavers, containing dormant hyphal bodies of N. fresenii, were stored in 4 ml glass vials at −14 °C in a standard consumer-type refrigerator/freezer for 1, 21, 30, 43, 51, and 68 months to determine the effect of storage on fungal survival. When the cadavers were removed from the freezer and placed in 25±1 °C, 100% relative humidity, and 12:12 (L:D) conditions, N. fresenii survival, as shown by fungal sporulation from the cadavers, was high at all storage periods. The average percentage of cadavers from which the fungus sporulated were 93, 47, 100, 100, 80, and 60% from 1, 21, 30, 43, 51, and 68 months storage periods, respectively. The number of primary conidia discharged from each sporulating cadaver was estimated using a scale of 1 (low, ca. 1000 primary conidia), 2 (medium, ca. 2000 primary conidia) and 3 (high, ca. 3000 primary conidia). The median scores for the number of primary conidia produced per sporulating cadaver were 3, 2, 3, 3, 2.5, and 1 for 1, 21, 30, 43, 51, and 68 months, respectively. Therefore, except for the longest storage period, most cadavers produced medium to high numbers of primary conidia. Mean germination of primary conidia produced from N. fresenii-infected-aphid cadavers from each time period varied significantly from 66.3 to 86.1% in the 21 and 43 months categories, respectively. Infectivity of capilliconidia, produced from frozen N. fresenii, to live healthy cotton aphids varied significantly from 16.7 to 68.7% from cadavers stored 68 months and 1 month, respectively. Overall N. fresenii survived well in dried frozen cotton aphid cadavers for up to 6 years with little reduction in sporulation, numbers of spores produced, germination of primary conidia, or infectivity. [Copyright &y& Elsevier]

Microbial control agents (MCAs) are promising for use in sustainable agriculture. Nevertheless, they face a major hurdle in the registration process of the European Union, one reason being unclear data requirements on metabolites. This review examines the EU regulatory perspective on metabolites of MCAs for plant protection and identifies some key issues and concerns. Current data requirements for secondary metabolites of micro-organisms are based on degradation products/metabolites of chemicals. We conclude that this has contributed strongly to the current confusion regarding how to best evaluate potential production of toxic substances by MCAs. We suggest that data requirements should be revised and/or need guidance fit for purpose, in order to give the EU-regulation for MCAs a stronger base in microbiological knowledge. We also suggest implementation of a basic hazard assessment. If this is passed without any concerns, the production of unknown, potentially toxic, substances does not need to be further investigated. [ABSTRACT FROM AUTHOR]