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In Scandinavia, the bird cherry-oat aphid Rhopalosiphum padi overwinter as eggs on the bird cherry tree Prunus padus. Branches of P. padus were collected at the late February / early March from 17 locations in Norway over a three-year period. We found 3599 overwintering aphid eggs, 59.5% of which were dead. Further, a total of 879 overwintering fungus-killed cadavers were observed. These cadavers were found close to bud axils, where overwintering eggs were also usually attached. Cadavers were infected with either Zoophthora cf. aphidis or Entomophthora planchoniana. All the fungal-killed cadavers were filled with overwintering structures of Z. cf. aphidis (as resting spores) or E. planchoniana (as modified hyphal bodies). We found a significant negative correlation between eggs and cadavers per branch. However, both numbers of eggs and cadavers varied greatly between years and among tree locations. This is the first report of E. planchoniana overwintering in R. padi cadavers as modified hyphal bodies. We discuss whether P. padus may act as an inoculum reservoir for fungi infecting aphids in cereals in spring., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Pandora neoaphidis and Entomophthora planchoniana are widespread and important specialist fungal pathogens of aphids in cereals (Sitobion avenae and Rhopalosiphum padi). The two aphid species share these pathogens and we compare factors influencing susceptibility and resistance. Among factors that may influence susceptibility and resistance are aphid behavior, conspecific versus heterospecific host, aphid morph, life cycle, and presence of protective endosymbionts. It seems that the conspecific host is more susceptible (less resistant) than the heterospecific host, and alates are more susceptible than apterae. We conceptualize the findings in a diagram showing possible transmission in field situations and we pinpoint where there are knowledge gaps., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Entomophthoralean fungi are insect pathogenic fungi and are characterized by their active discharge of infective conidia that infect insects. Our aim was to study the effects of temperature on the discharge and to characterize the variation in the associated temporal pattern of a newly discovered Pandora species with focus on peak location and shape of the discharge. Mycelia were incubated at various temperatures in darkness, and conidial discharge was measured over time. We used a novel modification of a statistical model (pavpop), that simultaneously estimates phase and amplitude effects, into a setting of generalized linear models. This model is used to test hypotheses of peak location and discharge of conidia. The statistical analysis showed that high temperature leads to an early and fast decreasing peak, whereas there were no significant differences in total number of discharged conidia. Using the proposed model we also quantified the biological variation in the timing of the peak location at a fixed temperature., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Molecular methods were used to determine the generic placement of two species of Entomophthorales known only from resting spores. Historically, these species would belong in the form-genus Tarichium, but this classification provides no information about phylogenetic relationships. Using DNA from resting spores, Zoophthora independentia, infecting Tipula (Lunatipula) submaculata in New York State, is now described as a new species and Tarichium porteri, described in 1942, which infects Tipula (Triplicitipula) colei in Tennessee, is transferred to the genus Zoophthora. We have shown that use of molecular methods can assist with determination of the phylogenetic relations of specimens within the form-genus Tarichium for an already described species and a new species for which only resting spores are available., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The selection of suitable reference genes is crucial for accurate quantification of gene expression and can add to our understanding of host-pathogen interactions. To identify suitable reference genes in Pandora neoaphidis, an obligate aphid pathogenic fungus, the expression of three traditional candidate genes including 18S rRNA(18S), 28S rRNA(28S) and elongation factor 1 alpha-like protein (EF1), were measured by quantitative polymerase chain reaction at different developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae), and under different nutritional conditions. We calculated the expression stability of candidate reference genes using four algorithms including geNorm, NormFinder, BestKeeper and Delta Ct. The analysis results revealed that the comprehensive ranking of candidate reference genes from the most stable to the least stable was 18S (1.189), 28S (1.414) and EF1 (3). The 18S was, therefore, the most suitable reference gene for real-time RT-PCR analysis of gene expression under all conditions. These results will support further studies on gene expression in P. neoaphidis., (Copyright © 2015 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

The natural occurrence of entomophthoralean fungi pathogenic towards aphids on cereal and potato crops was investigated in the years 2009, 2010, and 2011. Infected aphids were sampled in three bioclimatic zones in Tunisia (Beja, Cap bon, and Kairouan) and fungal species were determined based on morphological characters such as shape, size, and number of nuclei in the primary conidia. Polymerase Chain Reaction (PCR) on the internal transcribed spacer 1 region (ITS1) was used to verify morphological determination. Both methods gave consistent results and we documented for the first time the natural occurrence of two fungal species from the order Entomophthorales (phylum Entomophthoromycota), Pandora neoaphidis and Entomophthora planchoniana. Both fungi were recorded on the aphid species Sitobion avenae and Myzus persicae on barley ears and potato leaves, respectively. Moreover, natural mixed infections by both species (P. neoaphidis and E. planchoniana) were documented on the target aphids. This investigation provides basic information of entomopathogenic fungi infecting economically important aphids in Tunisia.

The entomopathogenic fungus Pandora neoaphidis is a recognized pathogen of aphids, causes natural epizootics in aphid populations, and interacts and competes with aphid predators and parasitoids. Survival of entomophthoralean fungi in periods of unsuitable weather conditions or lack of appropriate host insects is accomplished mainly by thick-walled resting spores (zygospores or azygospores). However, resting spores are not known for some entomophthoralean species such as P. neoaphidis. Several hypotheses of P. neoaphidis winter survival can be found in the literature but so far these hypotheses do not include the presence of resting spores. Resting spores were found in an aphid population where P. neoaphidis was the only entomophthoralean fungus observed during surveys conducted in organic horticultural crops in greenhouses and open fields in Buenos Aires province, Argentina. This study sought to use molecular methods to confirm that these resting spores were, in fact, those of P. neoaphidis while further documenting and characterizing these resting spores that were produced in vivo in aphid hosts. The double-walled resting spores were characterized using light and transmission electron microscopy. The Argentinean resting spores clustered together with P. neoaphidis isolates with bootstrap values above 98 % in the small subunit ribosomal RNA (SSU rRNA) sequence analysis and with bootstrap values above 99 % the Internal Transcribed Spacer (ITS) II region sequence analysis. This study is the first gene-based confirmation from either infected hosts or cultures that P. neoaphidis is able to produce resting spores., (Copyright © 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Three DNA regions (ITS 1, LSU rRNA and GPD) of isolates from the insect-pathogenic fungus genus Entomophthora originating from different fly ( Diptera) and aphid ( Hemiptera) host taxa were sequenced. The results documented a large genetic diversity among the fly-pathogenic Entomophthora and only minor differences among aphid-pathogenic Entomophthora. The evolutionary time of divergence of the fly and the aphid host taxa included cannot account for this difference. The host-driven divergence of Entomophthora, therefore, has been much greater in flies than in aphids. Host-range differences or a recent host shift to aphid are possible explanations. [ABSTRACT FROM AUTHOR]

Abstract: Isolates of the entomopathogenic fungus Entomophthora muscae were obtained from houseflies sampled at five stables on Zealand during an epidemic in fall 2005. DNA fingerprints were generated from single conidia from 40 E. muscae isolates using the PCR-based method of Inter-Simple Sequence Repeats (ISSR). This resulted in fingerprint patterns consisting of about 50 fragments, of which, 14 were polymorphic. From the polymorphic loci we estimated the reproductive mode, genetic differentiation and gene diversity of E. muscae populations using the statistics of index of association, Weir & Cockerhams theta and Nei''s analysis of gene diversity in subdivided populations. Our results revealed no significant differences in allele frequencies among the five populations. The index of association test rejected the null hypothesis of random mating, but the test for paired locus compatibility showed weak or no linkage between loci indicating recombination or homoplasy in the dataset. From this study we cannot exclude the possibility that the population genetic structure underlying E. muscae epidemics could be panmictic consisting of several lineages with a high level of reciprocal migration. [Copyright &y& Elsevier]

Abstract: Entomopthora muscae sensu lato is a complex of morphologically similar fungal species pathogenic to evolutionarily advanced flies (Cyclorrhapha). To reach an operational species definition and recognition of species within this complex, the values of host range, morphological and genetic characteristics are reconsidered. Within the E. muscae species complex morphological and nuclear characteristics of the primary conidia are taxonomically important. In this study we compared the dimensions and nuclear numbers of the primary conidia of isolates from their original (natural) hosts and after being transferred to alternative hosts (cross-transmission) in order to check the stability of these characteristics. The conidial characteristics change substantially when produced in alternative host species, but their overall range in variability still fit within the traditional morphological species circumscriptions. The phylogenetic analyses of the ITS II and LSU rRNA gene sequences, revealed three distinct lineages within the complex: E. schizophorae, E. muscae and E. syrphi. Within each of these lineages sequence divergence was seen between isolates originating from different host species. Our studies on the physiological host range showed that several isolates were able to infect alternative dipteran species. Musca domestica was a particularly good receptor. The ecological host range of any individual isolate seems, however, to be limited to one host species evidenced by the occurrence of distinct genotypes within each natural infected host species shown by RAPD. The high host specificity of these fungi emphasizes the importance of identifying the host taxon at species level in the recognition of Entomophthora species. We recommend that morphological characteristics of fungal structures and host taxon, together with molecular data, serve as criteria for species determination in future studies on members of the E. muscae complex. [Copyright &y& Elsevier]