A gene regulating ergot alkaloid biosynthesis in Metarhizium brunneum.

Ergot alkaloid synthesis (eas) gene clusters found in several fungi encode biosynthesis of agriculturally and pharmaceutically important ergot alkaloids. Although the biosynthetic genes of the ergot alkaloid pathway have been well characterized, regulation of those genes is unknown. We characterized a gene with sequence similarity to a putative transcription factor and that was found adjacent to the eas cluster of Metarhizium brunneum, a plant symbiont and insect pathogen. Function of the novel gene, easR, was explored by CRISPR-Cas9-derived gene knockouts. To maximize potential for ergot alkaloid accumulation, strains of M. brunneum were injected into larvae of the insect Galleria mellonella. Larvae infected with the wild type contained abundant ergot alkaloids, but those infected with easR knockouts lacked detectable ergot alkaloids. The easR knockout strains had significantly reduced or no detectable mRNA from eas cluster genes in RNAseq and qualitative RT-PCR analyses, whereas the wild-type strain contained abundant mRNA from all eas genes. These data demonstrate that the product of easR is required for ergot alkaloid accumulation and provide evidence that it has a role in the expression of ergot alkaloid biosynthesis genes. Larvae infected with an easR knockout survived significantly longer than those infected with the wild type (P < 0.0001), indicating a role for EasR, and indirectly confirming a role for ergot alkaloids, in the virulence of M. brunneum to insects. Homologs of easR were found associated with eas clusters of at least 15 other ergot alkaloid-producing fungi, indicating that EasR homologs may contribute to regulation of ergot alkaloid synthesis in additional fungi. IMPORTANCE: Ergot alkaloids produced by several species of fungi are important as contaminants of food and feed in agriculture and also as the foundation of numerous pharmaceuticals prescribed for dementia, migraines, hyperprolactinemia, and several other disorders. Information on control of the ergot alkaloid pathway may contribute to strategies to limit their production in agricultural settings or increase their yield for pharmaceutical production. Our results demonstrate that a previously uncharacterized gene clustered with the ergot alkaloid synthesis genes is required for the sufficient transcription of the ergot alkaloid biosynthesis genes. This observation suggests the gene encodes a factor regulating transcription of those biosynthetic genes.

In search of lost ergots: phylogenetic re-evaluation of Claviceps species in Japan and their biogeographic patterns revealed.

Claviceps (Clavicipitaceae, Hypocreales) was erected in 1853, although ergotism had been well-known for a much longer time. By 2000, about 70 taxa had been described in Claviceps, of which eight species and six varieties were based on Japanese type or authentic specimens. Most of these Japanese Claviceps taxa are based on lost specimens or have invalid names, which means many species practically exist only in the scientific literature. The ambiguous identities of these species have hindered taxonomic resolution of the genus Claviceps. Consequently, we sought and collected more than 300 fresh specimens in search of the lost Japanese ergots. Multilocus phylogenetic analyses based on DNA sequences from LSU, TEF-1α, TUB2, Mcm7, and RPB2 revealed the phylogenetic relationships between the Japanese specimens and known Claviceps spp., as well as the presence of biogeographic patterns. Based on the phylogenetic analysis, host range and morphology, we re-evaluated Japanese Claviceps and recognised at least 21 species in Japan. Here we characterised 14 previously described taxa and designated neo-, lecto- and epi-types for C. bothriochloae, C. imperatae, C. litoralis, C. microspora, C. panicoidearum and C. yanagawaensis. Two varieties were elevated to species rank with designated neotypes, i.e. C. agropyri and C. kawatanii. Six new species, C. miscanthicola, C. oplismeni, C. palustris, C. phragmitis, C. sasae and C. tandae were proposed and described. Taxonomic novelties: New species: Claviceps miscanthicola E. Tanaka, Claviceps oplismeni E. Tanaka, Claviceps palustris E. Tanaka, Claviceps phragmitis E. Tanaka, Claviceps sasae E. Tanaka, Claviceps tandae E. Tanaka; New status and combination: Claviceps agropyri (Tanda) E. Tanaka, Claviceps kawatanii (Tanda) E. Tanaka; Typifications (basionyms): Lecto- and epitypification: Claviceps yanagawaensis Togashi; Neotypifications: Claviceps purpurea var. agropyri Tanda, Claviceps bothriochloae Tanda & Y. Muray, Claviceps imperatae Tanda & Kawat., Claviceps microspora var. kawatanii Tanda, Claviceps litoralis Kawat., Claviceps microspora Tanda, Claviceps panicoidearum Tanda & Y. Harada; Resurrection: Claviceps queenslandica Langdon. Citation: Tanaka E, Tanada K, Hosoe T, Shrestha B, Kolarík M, Liu M (2023). In search of lost ergots: phylogenetic re-evaluation of Claviceps species in Japan and their biogeographic patterns revealed. Studies in Mycology 106: 1-39. doi: 10.3114/sim.2022.106.01.

Revisiting Metarhizium and the description of new species from Thailand.

Over the last two decades the molecular phylogeny and classification of Metarhizium has been widely studied. Despite these efforts to understand this enigmatic genus, the basal lineages in Metarhizium are still poorly resolved. In this study, a phylogenetic framework is reconstructed for the Clavicipitaceae focusing on Metarhizium through increased taxon-sampling using five genomic loci (SSU, LSU, tef, rpb1, rpb2) and the barcode marker ITS rDNA. Multi-gene phylogenetic analyses and morphological characterisation of green-spored entomopathogenic Metarhizium isolates from Thailand and soil isolates of M. carneum and M. marquandii reveal their ecological, genetic and species diversity. Nineteen new species are recognised in the Metarhizium clade with narrow host ranges: two new species are found in the M. anisopliae complex - M. clavatum on Coleoptera larvae and M. sulphureum on Lepidoptera larvae; four new species are found in the M. flavoviride complex - M. biotecense and M. fusoideum on brown plant hoppers (Hemiptera), M. culicidarum on mosquitoes, M. nornnoi on Lepidoptera larvae; three new species M. megapomponiae, M. cicadae, M. niveum occur on cicadas; five new species M. candelabrum, M. cercopidarum, M. ellipsoideum, M. huainamdangense M. ovoidosporum occur on planthoppers, leafhoppers and froghoppers (Hemiptera); one new species M. eburneum on Lepidoptera pupae; and four new species M. phuwiangense, M. purpureum, M. purpureonigrum, M. flavum on Coleoptera . Of these 19 new species, seven produce a sexual morph (M. clavatum, M. eburneum, M. flavum, M. phuwiangense, M. purpureonigrum, M. purpureum, and M. sulphureum) and asexual morphs are found in the remaining new species and also in M. sulphureum, M. purpureonigrum and M. purpureum. Metarhizium blattodeae, M. koreanum and M. viridulum are new records for Thailand. An alternative neotype for Metarhizium anisopliae is proposed based on multi-gene and 5'tef analyses showing that CBS 130.71 from Ukraine is more suitable, being from a much closer geographical location to Metchnikoff's Metarhizium anisopliae. This isolate is distinct from the neotype of Metarhizium anisopliae var. anisopliae proposed by M. Tulloch from Ethiopia (ARSEF 7487). Six new genera are established for monophyletic clades subtending the core Metarhizium clade, including Keithomyces, Marquandomyces, Papiliomyces, Purpureomyces, Sungia, and Yosiokobayasia. Metarhizium carneum, M. aciculare, and M. neogunnii are combined in Keithomyces and one new combination for M. marquandii in Marquandomyces is proposed. Purpureomyces is introduced for species producing purple stromata including a new combination for M. khaoyaiense and two new species P. maesotensis and P. pyriformis. Papiliomyces contains two new combinations for M. liangshanense and Metacordyceps shibinensis. The genus Sungia is proposed for the Korean species M. yongmunense on Lepidoptera pupa and Yosiokobayasia for the Japanese species M. kusanagiense also on Lepidoptera pupa. A synoptic and dichotomous key to the accepted taxa is provided together with tables listing distinguishing morphological characters between species, host preferences, and geography.

Fungal pathogens occurring on Orthopterida in Thailand.

Two new fungal genera and six species occurring on insects in the orders Orthoptera and Phasmatodea (superorder Orthopterida) were discovered that are distributed across three families in the Hypocreales. Sixty-seven sequences generated in this study were used in a multi-locus phylogenetic study comprising SSU, LSU, TEF, RPB1 and RPB2 together with the nuclear intergenic region (IGR). These new taxa are introduced as Metarhizium gryllidicola, M. phasmatodeae, Neotorrubiella chinghridicola, Ophiocordyceps kobayasii, O. krachonicola and Petchia siamensis. Petchia siamensis shows resemblance to Cordyceps mantidicola by infecting egg cases (ootheca) of praying mantis (Mantidae) and having obovoid perithecial heads but differs in the size of its perithecia and ascospore shape. Two new species in the Metarhizium cluster belonging to the M. anisopliae complex are described that differ from known species with respect to phialide size, conidia and host. Neotorrubiella chinghridicola resembles Torrubiella in the absence of a stipe and can be distinguished by the production of whole ascospores, which are not commonly found in Torrubiella (except in Torrubiella hemipterigena, which produces multiseptate, whole ascospores). Ophiocordyceps krachonicola is pathogenic to mole crickets and shows resemblance to O. nigrella, O. ravenelii and O. barnesii in having darkly pigmented stromata. Ophiocordyceps kobayasii occurs on small crickets, and is the phylogenetic sister species of taxa in the 'sphecocephala' clade.

Chemical study of the strain Cordyceps spp. from cell fusion between Cordyceps militaris and Cordyceps cicadae.

Chemical investigation on the cultures of the fungus Cordyceps spp., a strain from cell fusion between Cordyceps militaris and Cordyceps cicadae, resulted in the isolation of 13 compounds including 2 new ones named 2-(5-(3-oxobutyl) furan-2-yl) acetic acid (1) and cordycepone (2). Their structures were elucidated from the analysis of 1D/2D NMR and CD data. Among them, compounds 1, 7-9, at a concentration of 50 µg/ml, showed weak inhibitory activity against AChE. Moreover, compounds 6, 9, and 11 showed moderate inhibitory activity against the nematode Panagrellus redivivus with mortality ratio of 79.0, 71.7, and 72.3% at 2.5 mg/ml, respectively.

Evolutionary history of ergot with a new infrageneric classification (Hypocreales: Clavicipitaceae: Claviceps).

The ergot, genus Claviceps, comprises approximately 60 species of specialised ovarial grass parasites famous for the production of food toxins and pharmaceutics. Although the ergot has been known for centuries, its evolution have not been resolved yet. Our approach combining multilocus phylogeny, molecular dating and the study of ecological, morphological and metabolic features shows that Claviceps originated in South America in the Palaeocene on a common ancestor of BEP (subfamilies Bambusoideae, Ehrhartoideae, Pooideae) and PACMAD (subfamilies Panicoideae, Aristidoideae, Chloridoideae, Micrairoideae, Arundinoideae, Danthonioideae) grasses. Four clades described here as sections diverged during the Paleocene and Eocene. Since Claviceps are parasitic fungi with a close relationship with their host plants, their evolution is influenced by interactions with the new hosts, either by the spread to a new continent or the radiation of the host plants. Three of the sections possess very narrow host ranges and biogeographical distributions and have relatively low toxicity. On the contrary, the section Claviceps, comprising the rye ergot, C. purpurea, is unique in all aspects. Fungi in this section of North American origin have spread all over the world and infect grasses in all subfamilies as well as sedges, and it is the only section synthesising toxic ergopeptines and secalonic acids. The evolutionary success of the Claviceps section members can be explained by high toxin presence, serving as feeding deterrents and playing a role in their protective mutualism with host plants. Closely related taxa Neoclaviceps monostipa and Cepsiclava phalaridis were combined into the genus Aciculosporium.

Metarhizium alvesii sp. nov.: A new member of the Metarhizium anisopliae species complex.

A strain within the Metarhizium anisopliae species complex was isolated in 2009 from a soil sample in a banana plantation in the municipality of Quixeré, Northeastern region of Brazil. Previous studies showed that this insect-pathogenic strain does not fit with any current taxon within the M. anisopliae species complex, as determined by both genomic and by mass spectrometric analyses. In the present study, CG1123 (=ARSEF 13308) is shown to be morphologically indistinguishable from most species in this cosmopolitan species complex, whereas multilocus phylogeny confirmed its uniqueness and supports its recognition as a new species, Metarhizium alvesii, in honor of Sérgio Batista Alves, one of the founders of insect pathology in Brazil.

Characterization of Metarhizium viride Mycosis in Veiled Chameleons (Chamaeleo calyptratus), Panther Chameleons (Furcifer pardalis), and Inland Bearded Dragons (Pogona vitticeps).

Metarhizium viride has been associated with fatal systemic mycoses in chameleons, but subsequent data on mycoses caused by this fungus in reptiles are lacking. The aim of this investigation was therefore to obtain information on the presence of M. viride in reptiles kept as pets in captivity and its association with clinical signs and pathological findings as well as improvement of diagnostic procedures. Beside 18S ribosomal DNA (rDNA) (small subunit [SSU]) and internal transcribed spacer region 1 (ITS-1), a fragment of the large subunit (LSU) of 28S rDNA, including domain 1 (D1) and D2, was sequenced for the identification of the fungus and phylogenetic analysis. Cultural isolation and histopathological examinations as well as the pattern of antifungal drug resistance, determined by using agar diffusion testing, were additionally used for comparison of the isolates. In total, 20 isolates from eight inland bearded dragons (Pogona vitticeps), six veiled chameleons (Chamaeleo calyptratus), and six panther chameleons (Furcifer pardalis) were examined. Most of the lizards suffered from fungal glossitis, stomatitis, and pharyngitis or died due to visceral mycosis. Treatment with different antifungal drugs according to resistance patterns in all three different lizard species was unsuccessful. Sequence analysis resulted in four different genotypes of M. viride based on differences in the LSU fragment, whereas the SSU and ITS-1 were identical in all isolates. Sequence analysis of the SSU fragment revealed the first presentation of a valid large fragment of the SSU of M. viride According to statistical analysis, genotypes did not correlate with differences in pathogenicity, antifungal susceptibility, or species specificity.

Horizontal acquisition of toxic alkaloid synthesis in a clade of plant associated fungi.

Clavicipitaceae is a fungal group that comprises species that closely interact with plants as pathogens, parasites or symbionts. A key factor in these interactions is the ability of these fungi to synthesize toxic alkaloid compounds that contribute to the protection of the plant host against herbivores. Some of these compounds such as ergot alkaloids are toxic to humans and have caused important epidemics throughout history. The gene clusters encoding the proteins responsible for the synthesis of ergot alkaloids and lolines in Clavicipitaceae have been elucidated. Notably, homologs to these gene clusters can be found in distantly related species such as Aspergillus fumigatus and Penicillium expansum, which diverged from Clavicipitaceae more than 400 million years ago. We here use a phylogenetic approach to analyze the evolution of these gene clusters. We found that the gene clusters conferring the ability to synthesize ergot alkaloids and loline emerged first in Eurotiomycetes and were then likely transferred horizontally to Clavicipitaceae. Horizontal gene transfer is known to play a role in shaping the distribution of secondary metabolism clusters across distantly related fungal species. We propose that HGT events have played an important role in the capability of Clavicipitaceae to produce two key secondary metabolites that have enhanced the ability of these species to protect their plant hosts, therefore favoring their interactions.

Unveiling chemical defense in the rice stalk stink bug against the entomopathogenic fungus Metarhizium anisopliae.

Eggs, nymphs (1st-5th instar) and adults of Tibraca limbativentris were challenged by conidial suspensions of its major fungal pathogen Metarhizium anisopliae in order to assess their susceptibility. The role of chemical defensive compounds from exocrine secretions produced by both nymphs and adults were examined for their participation on M. anisopliae infection. Although insect susceptibility to M. anisopliae followed a dose-dependent manner, adults followed by older nymphs displayed the highest resistance. Eggs were highly susceptible showing >96% fungal infection. Crude extracts isolated from metathoracic scent gland and dorsal abdominal glands of adults and nymphs, respectively, showed fungistatic effects by impairing spore germination, vegetative growth and sporulation. Gas chromatography-mass spectrometry analysis of these extracts revealed that the major components were short-chain hydrocarbons (C10-13) and unsaturated aldehydes. In vitro tests with the corresponding synthetic standards indicated compounds with greater antifungal activity including (E)-2-hexenal, (E)-2-octenal, and (E)-2-decenal, with the latter being the most deleterious to fungal fitness. We demonstrated that differential susceptibility of the rice stalk stink bug to M. anisopliae infection is age-specific and partly mediated by fungistatic properties of aldehydes, which are produced by scent glands of both nymphs and adults.

Two distinct Epichloë species symbiotic with Achnatherum inebrians, drunken horse grass.

Achnatherum inebrians, colloquially known as drunken horse grass, is associated with livestock toxicity in northern China. Epichloë gansuensis (Eg) was described from endophyte isolates from A. inebrians in Sunan County, Gansu Province, whereas a morphologically distinct variety, E. gansuensis var. inebrians (Ei), was described based on two isolates from A. inebrians seeds collected in Urumqi County, Xinjiang Province. Genome sequencing and alkaloid analyses also distinguish these taxa; the Ei isolates produce neurotropic lysergic acid amides (ergot alkaloids), and an Eg isolate produces paxilline (an indole-diterpene alkaloid). To better elucidate the taxonomic diversity of Epichloë spp. symbiotic with A. inebrians, we surveyed eight populations in Xinjiang, Gansu and Inner Mongolia provinces of China and analyzed their genotypes by multiplex PCR for alkaloid biosynthesis genes and mating-type genes. Genotypes consistent with Ei were present in all eight populations, of which they dominated seven. The Ei isolates were all mating type A and tested positive for the ergot alkaloid gene, dmaW. In contrast Eg isolates were all mating type B and had the indole-diterpene gene, idtG. The genome was sequenced from an Ei isolate from seeds collected in Xiahe County, Gansu, and compared to that of the varietal ex type isolate from Urumqi. Alkaloid genes and four different housekeeping genes were nearly identical between the two sequenced Ei isolates and were distinct from a sequenced Eg isolate. Phylogenetic analysis placed Ei, Eg and Epichloë sibirica into respective subclades of a clade that emanated from the base of the Epichloë phylogeny. Given its chemotypic, genotypic, morphological and phylogenetic distinctiveness, its widespread occurrence in rangelands of northern China, and its importance in livestock toxicity, we propose raising Ei to species rank as Epichloë inebrians.

Phylogenetic and chemotypic diversity of Periglandula species in eight new morning glory hosts (Convolvulaceae).

Periglandula ipomoeae and P. turbinae (Ascomycota, Clavicipitaceae) are recently described fungi that form symbiotic associations with the morning glories (Convolvulaceae) Ipomoea asarifolia and Turbina corymbosa, respectively. These Periglandula species are vertically transmitted and produce bioactive ergot alkaloids in seeds of infected plants and ephemeral mycelia on the adaxial surface of young leaves. Whether other morning glories that contain ergot alkaloids also are infected by Periglandula fungi is a central question. Here we report on a survey of eight species of Convolvulaceae (Argyreia nervosa, I. amnicola, I. argillicola, I. gracilis, I. hildebrandtii, I. leptophylla, I. muelleri, I. pes-caprae) for ergot alkaloids in seeds and associated clavicipitaceous fungi potentially responsible for their production. All host species contained ergot alkaloids in four distinct chemotypes with concentrations of 15.8-3223.0 µg/g. Each chemotype was a combination of four or five ergot alkaloids out of seven alkaloids detected across all hosts. In addition, each host species exhibited characteristic epiphytic mycelia on adaxial surfaces of young leaves with considerable interspecific differences in mycelial density. We sequenced three loci from fungi infecting each host: the nuclear rDNA internal transcribed spacer region (ITS), introns of the translation factor 1-α gene (tefA) and the dimethylallyl-tryptophan synthase gene (dmaW), which codes for the enzyme that catalyzes the first step in ergot alkaloid biosynthesis. Phylogenetic analyses confirmed that these fungi are in the family Clavicipitaceae and form a monophyletic group with the two described Periglandula species. This study is the first to report Periglandula spp. from Asian, Australian, African and North American species of Convolvulaceae, including host species with a shrub growth form and host species occurring outside of the tropics. This study demonstrates that ergot alkaloids in morning glories always co-occur with Periglandula spp. and that closely related Periglandula spp. produce alkaloid chemotypes more similar than more distantly related species.

Fertilization of Epichloe typhina stromata by mycophagous slugs.

Epichloë typhina, a fungal endophyte of cool season grasses, is heterothallic and an obligate out-crosser. In areas of endemism, its spermatia are moved between stromata of the two opposite mating types through egg-laying activities of Botanophila flies. In western Oregon, where the fungus was inadvertently introduced into seed-production fields of Dactylis glomerata (= orchardgrass, cocksfoot), flies do not appear to be the sole vectors for E. typhina fertilization. Here we examined the role of the common agricultural slug pest Deroceras reticulatum and mycophagous slug species Prophysaon andersoni and Arion subfuscus in E. typhina spermatia transfer. Frass from P. andersoni, A. subfuscus and D. reticulatum fed stromata of one mating type was transferred to stromata of the opposite mating type, resulting in 100%, 93% and 25% stromata fertilization respectively. An experiment designed to mimic field conditions examined stromata fertilization on E. typhina-infected plants of opposite mating type in the presence of slugs. Treatments with P. andersoni and D. reticulatum had greater stromata fertilization compared to the no-slug control, but the slug treatments were not different. This appears to be the first report of mollusks vectoring viable spermatia leading to the cross fertilization of stromata of different mating types.

MALDI-TOF mass spectrometry applied to identifying species of insect-pathogenic fungi from the Metarhizium anisopliae complex.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a powerful tool for taxonomic resolution of microorganisms. In this proof-of-concept study, we assessed the effectiveness of this technique to track the current gene sequence-based phylogenetic classification of species in the Metarhizium anisopliae complex. Initially the phylogenetic analysis of 5' strains by sequencing of the 59' end of the TEF-1α gene region revealed seven species within M. anisopliae sensu lato and two varieties outside this complex. Because initial studies on MS profiles from different cell types showed that mycelial fragments or conidia produced on nutrient-poor medium may yield too much background noise, all subsequent spectrometric analyses were performed with acidhydrolyzed conidia from 10-12 d old PDA cultures. The initial MALDI-TOF reference library included protein spectral profiles from nine taxonomically distinct, molecularly identified isolates sharing high genetic homology with the ex-type or ex-epitype isolates of these taxa in Metarhizium. A second reference library added one isolate each for M. anisopliae sensu stricto and M. robertsii. The second, larger reference library (including 11 taxa) allowed nearly perfect MALDI-TOF matching of DNA-based species identification for the 40 remaining isolates molecularly recognized as M. anisopliae sensu stricto (n = 19), M. robertsii (n = 6), M. majus (n = 3), M. lepidiotae (n = 1), M. acridum (n = 3), M. flavoviride var. pemphigi (n = 1), plus seven unidentified strains (six of them phylogenetically close to M. anisopliae sensu stricto and one outside the Metarhizium pingshaense-anisopliae-robertsii-brunneum clade). Due to the increasing frequency of phylogenetically (genomically) based taxonomic revisions of fungi, this approach is especially useful for culture collections, because once the protein profiles of Metarhizium isolates are obtained taxonomic updating of MALDI-TOF library data is easily accomplished by comparing stored profiles with those of newly proposed taxa.

Five undescribed cyclopeptides from Cordyceps militaris.

Ustiloxins I-M (1-5), five undescribed cyclopeptides bearing a 15-membered macrocyclic skeleton, were isolated from Cordyceps militaris. The structures of 1 and 5 were identified by spectroscopic and crystallographic methods, whereas the structures of 2-4 were assigned by spectroscopic and computational approaches. Biological evaluation of all the compounds toward human triple-negative breast cancer cells revealed that compounds 4 and 5 are toxic with IC50 values of 64.29 µM and 28.89 µM, respectively.

Water Stress and Black Cutworm Feeding Modulate Plant Response in Maize Colonized by Metarhizium robertsii.

Plants face many environmental challenges and have evolved different strategies to defend against stress. One strategy is the establishment of mutualistic associations with endophytic microorganisms which contribute to plant defense and promote plant growth. The fungal entomopathogen Metarhizium robertsii is also an endophyte that can provide plant-protective and growth-promoting benefits to the host plant. We conducted a greenhouse experiment in which we imposed stress from deficit and excess soil moisture and feeding by larval black cutworm (BCW), Agrotis ipsilon, to maize plants that were either inoculated or not inoculated with M. robertsii (Mr). We evaluated plant growth and defense indicators to determine the effects of the interaction between Mr, maize, BCW feeding, and water stress. There was a significant effect of water treatment, but no effect of Mr treatment, on plant chlorophyl, height, and dry biomass. There was no effect of water or Mr treatment on damage caused by BCW feeding. There was a significant effect of water treatment, but not Mr treatment, on the expression of bx7 and rip2 genes and on foliar content of abscisic acid (ABA), 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), and gibberellin 19 (GA19), whereas GA53 was modulated by Mr treatment. Foliar content of GA19 and cis-Zeatin (cZ) was modulated by BCW feeding. In a redundancy analysis, plant phenology, plant nutrient content, and foliar DIMBOA and ABA content were most closely associated with water treatments. This study contributes toward understanding the sophisticated stress response signaling and endophytic mutualisms in crops.

New insights on the effectiveness of Metarhizium anisopliae formulation and application against Aedes aegypti eggs.

Increasing needs for innovative control tools against the dengue vector Aedes aegypti have prompted investigations into the development of specific mycoinsecticides. The entomopathogenic fungus Metarhizium anisopliae attacks both larval and adult stages, but its ovicidal activity against A. aegypti is still little explored. This study reports important findings about the effectiveness of conidia formulated in water and oil-in-water emulsions and of direct and indirect application techniques against A. aegypti eggs. The ovicidal activity of M. anisopliae increased with higher conidial concentrations regardless of the application technique, and larvae elimination concentrations were lowest with oil-in-water-formulated conidia (LEC50 ≤ 4·8 × 10(3) conidia cm(-2) and LEC90 ≤ 1·9 × 10(5) conidia cm(-2), respectively). Conidia eventually stimulated larval eclosion. Consequently, the indirect application of oil-based fungal formulations onto substrates where oviposition will later occur appears to be a more efficient means to infect those eggs than the direct fungal application to previously deposited eggs.

The Epichloe endophytes associated with the woodland grass Hordelymus europaeus including four new taxa.

Epichloë endophytes (Clavicipitaceae, Ascomycota), including asexual forms placed in Neotyphodium, are common in cool-season grasses. Here we characterize the endophytes of the European woodland grass Hordelymus europaeus based on growth characteristics, morphology of conidiophores and conidia and phylogenetic relationships. Of the six different taxa found on H. europaeus, four are new, for which we propose the species names E. hordelymi, E. disjuncta, E. danica and subspecies E. sylvatica subsp. pollinensis. The other two are assigned to previously described E. bromicola and E. sylvatica. E. hordelymi, E. disjuncta and E. danica are asexual interspecific hybrids, while the other taxa are haploid. Only E. sylvatica subsp. pollinensis was found to reproduce sexually on H. europaeus. The high diversity of endophytes may be explained by repeated host jumps to H. europaeus with and without subsequent interspecific hybridizations.

Morphological and phylogenetic characterisation of two new soil-borne fungal taxa belonging to Clavicipitaceae (Hypocreales, Ascomycota).

The fungal taxa belonging to the Clavicipitaceae (Hypocreales, Ascomycota) are widely distributed and include diverse saprophytic, symbiotic and pathogenic species that are associated with soils, insects, plants, fungi and invertebrates. In this study, we identified two new fungal taxa belonging to the family Clavicipitaceae that were isolated from soils collected in China. Morphological characterisation and phylogenetic analyses showed that the two species belong to Pochonia (Pochoniasinensissp. nov.) and a new genus for which we propose Paraneoaraneomycesgen. nov. in Clavicipitaceae.

The complete mitogenome of the entomopathogenic fungus Metarhizium pinghaense 15R.

In this study, the complete mitogenome of the entomopathogenic fungus Metarhizium pinghaense 15 R, which is highly virulent to aphids and was isolated from Korean soil, was assembled and annotated for three ATP synthase subunits (atp6, atp8, and atp9), three cytochrome oxidase subunits (cox1, cox2, and cox3), apocytochrome b (cob), seven subunits of NADH dehydrogenase (nad1, nad2, nad3, nad4, nad4L, nad5, and nad6), two ribosomal RNAs (rnl and rns), and 19 tRNA genes. Five genes were carrying a total of eight introns, and they may encode ribosomal protein S3, LAGLIDADG and GIY-YIG endonucleases. Phylogenetic analysis based on the mitochondrial nucleotide sequence confirmed that the M. pinghaense 15 R is a member of the Clavicipitaceae, and is closely related to the species M. anisopliae, M. robertsii, and M. brunneum. The mtDNA base sequence of the M. pinghaense 15 R strain reported in this study is thought to be useful for biological resource genetic data.