Mitogenome evolution in Trichoderma afroharzianum strains: for a better understanding of distinguishing genus.

Trichoderma afroharzianum (Hypocreales) is known as an important mycoparasite and biocontrol fungus and feeds on fungal material by parasitizing other fungi. Recent studies indicate that this species is also an ear rot pathogen in Europe. Here, the complete mitochondrial genome of three T. afroharzianum strains was sequenced using next-generation sequencing and comparatively characterized by the reported Trichoderma mitogenomes. T. afroharzianum mitogenomes were varying between 29 511 bp and 29 517 bp in length, with an average A + T content of 72.32%. These mitogenomes contain 14 core protein coding genes (PCGs), 22 tRNAs, two rRNAs, one gene encoding the ribosomal protein S3, and three or four genes including conserved domains for the homing endonucleases (HEGs; GIY-YIG type and LAGLIDADG type). All PCGs are initiated by ATG codons, except for atp8, and all are terminated with TAA. A significant correlation was observed between nucleotide composition and codon preference. Four introns belonging to the group I intron class were predicted, accounting for about 14.54% of the size of the mitogenomes. Phylogenetic analyses confirmed the positions of T. afroharzianum strains within the genus of Trichoderma and supported a sister group relationship between T. afroharzianum and T. simmonsii. The recovered trees also supported the monophyly of all included families and of the genus of Acremonium. The characterization of mitochondrial genome of T. afroharzianum contributes to the understanding of phylogeny and evolution of Hypocreales.

Entomophthoralean and hypocrealean fungal pathogens of the sugarcane aphid, Melanaphis sacchari (Hemiptera: Aphididae), on sorghum in Georgia.

The sugarcane aphid, Melanaphis sacchari, is a widely distributed insect that attacks grasses in different genera including Miscanthus, Saccharum, and Sorghum. The invasive aphid superclone was first discovered in the U.S. attacking grain sorghum in Texas in 2013. Since then, it has been found in at least 25 states including Georgia. We conducted a survey of naturally occurring fungal pathogens of sugarcane aphids on five farms in Georgia, and identified a hypocrealean fungus, Akanthomyces dipterigenus, and two entomophthoralean fungi, Neoconidiobolus spp. From 2018 to 2020, fungal activity differed across farms but at one farm both major fungal species, A. dipterigenus and N. thromboides, were found each of the 3 years infecting sugarcane aphids, attacking adults, both alatae and apterae, and nymphs.

The endophytobiome of wild Rubiaceae as a source of antagonistic fungi against the American Leaf Spot of coffee (Mycena citricolor).

AIMS: The American leaf spot, caused by Mycena citricolor, is an important disease of coffee (Coffea arabica), mostly in Central America. Currently, there are limited pathogen control alternatives that are environment friendly and economically accessible. The use of fungi isolated from the plant endomycobiota in their native habitats is on the rise because studies show their great potential for biological control. To begin to generate a green alternative to control M. citricolor, the objectives of the present study were to (i) collect, identify, screen (in vitro and in planta), and select endophytic fungi from wild Rubiaceae collected in old-growth forests of Costa Rica; (ii) confirm endophytic colonization in coffee plantlets; (iii) evaluate the effects of the endophytes on plantlet development; and (iv) corroborate the antagonistic ability in planta. METHODS AND RESULTS: Through in vitro and in planta antagonism assays, we found that out of the selected isolates (i.e. Daldinia eschscholzii GU11N, Nectria pseudotrichia GUHN1, Purpureocillium aff. lilacinum CT24, Sarocladium aff. kiliense CT25, Trichoderma rifaii CT5, T. aff. crassum G1C, T. aff. atroviride G7T, T. aff. strigosellum GU12, and Xylaria multiplex GU14T), Trichoderma spp. produced the highest growth inhibition percentages in vitro. Trichoderma isolates CT5 and G1C were then tested in planta using Coffea arabica cv. caturra plantlets. Endophytic colonization was verified, followed by in planta growth promotion and antagonism assays. CONCLUSIONS: Results show that Trichoderma isolates CT5 and G1C have potential for plant growth promotion and antagonism against Mycena citricolor, reducing incidence and severity, and preventing plant mortality.

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.

Isolation and evaluation of South African isolates of Beauveria bassiana (Hypocreales: Cordycipitaceae) on Rhipicephalus microplus (Acari: Ixodidae).

Beauveria bassiana (Hypocreales: Cordycipitaceae) is an entomopathogenic fungus that has shown promising results as a biocontrol agent of ticks. Locally isolated B. bassiana are better acclimatised to the natural conditions of their geographical origin; therefore, they are essential in developing effective biocontrol agents for ticks. The current study aimed to isolate native strains of B. bassiana that are pathogenic to Rhipicephalus microplus ticks. The virulence of the isolates was tested against R. microplus larvae using a formulation containing 15% avocado oil, 0.05% adjuvant and 108 conidia mL-1. The two best strains were further evaluated for various biological parameters on adult engorged female ticks. Breakthru® or Ballista® (adjuvant) was mixed with the formulation to compare their effect on the isolates' virulence. In total 61 entomopathogenic fungi were isolated from the 360 greater wax moth larvae, Galleria mellonella (Lepidoptera: Pyralidae) used. The virulence test identified Bb-40 and Bb-41 to be the most virulent isolates against R. microplus larvae with mortalities of 91 and 93% and LT50 values of 5.8 and 6.2 days, respectively. Compared to the control, both strains significantly affected all the measured biological parameters. The type of adjuvant also considerably affected the susceptibility of ticks to the fungi. In conclusion, the two isolates combined with adjuvants can be used as a biocontrol agent to control R. microplus.

Masters of the manipulator: two new hypocrealean genera, Niveomyces (Cordycipitaceae) and Torrubiellomyces (Ophiocordycipitaceae), parasitic on the zombie ant fungus Ophiocordyceps camponoti-floridani.

During surveys in central Florida of the zombie-ant fungus Ophiocordyceps camponoti-floridani, which manipulates the behavior of the carpenter ant Camponotus floridanus, two distinct fungal morphotypes were discovered associated with and purportedly parasitic on O. camponoti-floridani. Based on a combination of unique morphology, ecology and phylogenetic placement, we discovered that these morphotypes comprise two novel lineages of fungi. Here, we propose two new genera, Niveomyces and Torrubiellomyces, each including a single species within the families Cordycipitaceae and Ophiocordycipitaceae, respectively. We generated de novo draft genomes for both new species and performed morphological and multi-loci phylogenetic analyses. The macromorphology and incidence of both new species, Niveomyces coronatus and Torrubiellomyces zombiae, suggest that these fungi are mycoparasites since their growth is observed exclusively on O. camponoti-floridani mycelium, stalks and ascomata, causing evident degradation of their fungal hosts. This work provides a starting point for more studies into fungal interactions between mycopathogens and entomopathogens, which have the potential to contribute towards efforts to battle the global rise of plant and animal mycoses. Citation: Araújo JPM, Lebert BM, Vermeulen S, et al. 2022. Masters of the manipulator: two new hypocrealean general, Niveomyces (Cordycipitaceae) and Torrubiellomyces (Ophiocordycipitaceae), parasitic on the zombie ant fungus Ophiocordyceps camponoti-floridani. Persoonia 49: 171-194. https://doi.org/10.3767/persoonia.2022.49.05.

Telomere length de novo assembly of all 7 chromosomes and mitogenome sequencing of the model entomopathogenic fungus, Metarhizium brunneum, by means of a novel assembly pipeline.

BACKGROUND: More accurate and complete reference genomes have improved understanding of gene function, biology, and evolutionary mechanisms. Hybrid genome assembly approaches leverage benefits of both long, relatively error-prone reads from third-generation sequencing technologies and short, accurate reads from second-generation sequencing technologies, to produce more accurate and contiguous de novo genome assemblies in comparison to using either technology independently. In this study, we present a novel hybrid assembly pipeline that allowed for both mitogenome de novo assembly and telomere length de novo assembly of all 7 chromosomes of the model entomopathogenic fungus, Metarhizium brunneum. RESULTS: The improved assembly allowed for better ab initio gene prediction and a more BUSCO complete proteome set has been generated in comparison to the eight current NCBI reference Metarhizium spp. genomes. Remarkably, we note that including the mitogenome in ab initio gene prediction training improved overall gene prediction. The assembly was further validated by comparing contig assembly agreement across various assemblers, assessing the assembly performance of each tool. Genomic synteny and orthologous protein clusters were compared between Metarhizium brunneum and three other Hypocreales species with complete genomes, identifying core proteins, and listing orthologous protein clusters shared uniquely between the two entomopathogenic fungal species, so as to further facilitate the understanding of molecular mechanisms underpinning fungal-insect pathogenesis. CONCLUSIONS: The novel assembly pipeline may be used for other haploid fungal species, facilitating the need to produce high-quality reference fungal genomes, leading to better understanding of fungal genomic evolution, chromosome structuring and gene regulation.

Comparative Mitogenomics of Fungal Species in Stachybotryaceae Provides Evolutionary Insights into Hypocreales.

Stachybotrys chartarum is one of the world's ten most feared fungi within the family Stachybotryaceae, although to date, not a single mitogenome has been documented for Stachybotryaceae. Herein, six mitogenomes of four different species in Stachybotryaceae are newly reported. The S. chartarum mitogenome was 30.7 kb in length and contained two introns (one each in rnl and cox1). A comparison of the mitogenomes of three different individuals of S. chartarum showed few nucleotide variations and conservation of gene content/order and intron insertion. A comparison of the mitogenomes of four different Stachybotryaceae species (Memnoniella echinata, Myrothecium inundatum, S. chartarum, and S. chlorohalonata), however, revealed variations in intron insertion, gene order/content, and nad2/nad3 joining pattern. Further investigations on all Hypocreales species with available mitogenomes showed greater variabilities in gene order (six patterns) and nad2/nad3 joining pattern (five patterns) although a dominant pattern always existed in each case. Ancestral state estimation showed that in each case the dominant pattern was always more ancestral than those rare patterns. Phylogenetic analyses based on mitochondrion-encoded genes supported the placement of Stachybotryaceae in Hypocreales. The crown age of Stachybotryaceae was estimated to be approximately the Early Cretaceous (141-142 Mya). This study greatly promotes our understanding of the evolution of fungal species in Hypocreales.

Genomic characterization of Parengyodontium americanum sp. nov.

Modern genome analysis and phylogenomic methods have increased the number of fungal species, as well as enhanced appreciation of the degree of diversity within the fungal kingdom. In this context, we describe a new Parengyodontium species, P. americanum, which is phylogenetically related to the opportunistic human fungal pathogen P. album. Five unusual fungal isolates were recovered from five unique and confirmed coccidioidomycosis patients, and these isolates were subsequently submitted to detailed molecular and morphological identification procedures to determine identity. Molecular and morphological diagnostic analyses showed that the isolates belong to the Cordycipitaceae. Subsequently, three representative genomes were sequenced and annotated, and a new species, P. americanum, was identified. Using various genomic analyses, gene family expansions related to novel compounds and potential for ability to grow in diverse habitats are predicted. A general description of the genomic composition of this newly described species and comparison of genome content with Beauveria bassiana, Isaria fumosorosea and Cordyceps militaris shows a shared core genome of 6371 genes, and 148 genes that appear to be specific for P. americanum. This work provides the framework for future investigations of this interesting fungal species.

A new zombie ant behavior unraveled: Aggregating on tree trunks.

Hosts can be manipulated by parasites to move to locations advantageous for onward transmission. To investigate the role of behavioral manipulation in creating transmission hotspots, we studied the distribution of zombie turtle ants in the Amazon rainforest. The turtle ant Cephalotes atratus nests and mostly forages in the canopy, but is found at the base of trees when infected with the zombie ant fungus Ophiocordyceps kniphofioides. We found 626 infected cadavers on 14.8% of 162 trees sampled. Cadavers were highly aggregated on the surface of the trees, explained by behavioral observations indicating infected ants as slightly attracted to zombie ant cadavers on a tree. From 1,726 h of camera footage, we recorded the removal of three zombie ant cadavers by live ants. The number of removals compared to the density of infected individuals indicates the base of a tree as an escape from the evolved ability of social insects to recognize and treat disease inside the nest, allowing the parasite to continuously remain in the environment.

Illuminating type collections of nectriaceous fungi in Saccardo's fungarium.

Specimens of Nectria spp. and Nectriella rufofusca were obtained from the fungarium of Pier Andrea Saccardo, and investigated via a morphological and molecular approach based on MiSeq technology. ITS1 and ITS2 sequences were successfully obtained from 24 specimens identified as 'Nectria' sensu Saccardo (including 20 types) and from the type specimen of Nectriella rufofusca. For Nectria ambigua, N. radians and N. tjibodensis only the ITS1 sequence was recovered. On the basis of morphological and molecular analyses new nomenclatural combinations for Nectria albofimbriata, N. ambigua, N. ambigua var. pallens, N. granuligera, N. peziza subsp. reyesiana, N. radians, N. squamuligera, N. tjibodensis and new synonymies for N. congesta, N. flageoletiana, N. phyllostachydis, N. sordescens and N. tjibodensis var. crebrior are proposed. Furthermore, the current classification is confirmed for Nectria coronata, N. cyanostoma, N. dolichospora, N. illudens, N. leucotricha, N. mantuana, N. raripila and Nectriella rufofusca. This is the first time that these more than 100-yr-old specimens are subjected to molecular analysis, thereby providing important new DNA sequence data authentic for these names.

Characterization of Tolypocladium cylindrosporum (Hypocreales, Ophiocordycipitaceae) isolates from Brazil and their efficacy against Aedes aegypti (Diptera, Culicidae).

AIMS: To survey and to characterize entomopathogenic fungi as natural enemies of mosquitoes in Central Brazil. METHODS AND RESULTS: Tolypocladium cylindrosporum (Hypocreales, Ophiocordycipitaceae) was isolated for the first time in South America by using Aedes aegypti (Diptera, Culicidae) as sentinel larvae in a stagnant mosquito breeding site in a secondary tropical forest. Two isolates were morphologically and molecularly identified, and their activity tested against A. aegypti eggs, larvae and adults. CONCLUSIONS: Findings emphasize the importance of T. cylindrosporum as a natural fungal antagonist of mosquitoes. SIGNIFICANCE AND IMPACT OF THE STUDY: Both isolates affected larvae and adults but were less effective against eggs; both have potential for development as a mycoinsecticide especially against larvae of A. aegypti the main vector of dengue, chikungunya, Zika and urban yellow fever.

Current status and perspectives of fungal entomopathogens used for microbial control of arthropod pests in Brazil.

Entomopathogenic fungi play a central role in Brazil's biopesticide market. Approximately 50% of registered microbial biopesticides comprise mycoinsecticides and/or mycoacaricides consisting of hypocrealean fungi, with most based on Metarhizium anisopliae sensu stricto (s. str.) and Beauveria bassiana s. str. These fungi are mainly used to control spittlebugs in sugarcane fields and whiteflies in row crops, respectively, with annual applications surpassing three million hectares. Research also emphasizes the potential of fungal entomopathogens to manage arthropod vectors of human diseases. Most registered fungal formulations comprise wettable powders or technical (non-formulated) products, with relatively few new developments in formulation technology. Despite the large area treated with mycoinsecticides (i.e., approx. 2 million ha of sugarcane treated with M. anisopliae and 1.5 million ha of soybean treated with B. bassiana), their market share remains small compared with the chemical insecticide market. Nevertheless, several major agricultural companies are investing in fungus-based products with the aim at achieving more sustainable IPM programs for major pests in both organic and conventional crops. Government and private research groups are pursuing innovative technologies for mass production, formulation, product stability and quality control, which will support cost-effective commercial mycoinsecticides. Here, we summarize the status of mycoinsecticides currently available in Brazil and discuss future prospects.

Occurrence and diversity of entomopathogenic fungi (Beauveria spp. and Metarhizium spp.) in Australian vineyard soils.

Entomopathogenic Ascomycetes: Hypocreales fungi occur worldwide in the soil; however, the abundance and distribution of these fungi in a vineyard environment is unknown. A survey of Australian vineyards was carried out in order to isolate and identify entomopathogenic fungi. A total of 240 soil samples were taken from eight vineyards in two states (New South Wales and Victoria). Insect baiting (using Tenebrio molitor) and soil dilution methods were used to isolate Beauveria spp. and Metarhizium spp. from all soil samples. Of the 240 soil samples, 60% contained either Beauveria spp. (26%) or Metarhizium spp. (33%). Species of Beauveria and Metarhizium were identified by sequencing the B locus nuclear intergenic region (Bloc) and elongation factor-1 alpha (EFT1) regions, respectively. Three Beauveria species (B. bassiana, B. australis and B. pseudobassiana) and six Metarhizium species (M. guizhouense, M. robertsii, M. brunneum, M. flavoviride var. pemphigi, M. pingshaense and M. majus) were identified. A new sister clade made up of six isolates was identified within B. australis. Two potentially new phylogenetic species (six isolates each) were found within the B. bassiana clade. This study revealed a diverse community of entomopathogenic fungi in sampled Australian vineyard soils.

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.

Escovopsioides as a fungal antagonist of the fungus cultivated by leafcutter ants.

BACKGROUND: Fungus gardens of fungus-growing (attine) ants harbor complex microbiomes in addition to the mutualistic fungus they cultivate for food. Fungi in the genus Escovopsioides were recently described as members of this microbiome but their role in the ant-fungus symbiosis is poorly known. In this study, we assessed the phylogenetic diversity of 21 Escovopsioides isolates obtained from fungus gardens of leafcutter ants (genera Atta and Acromyrmex) and non-leafcutter ants (genera Trachymyrmex and Apterostigma) sampled from several regions in Brazil. RESULTS: Regardless of the sample locality or ant genera, phylogenetic analysis showed low genetic diversity among the 20 Escovopsisoides isolates examined, which prompted the identification as Escovopsioides nivea (the only described species in the genus). In contrast, one Escovopsioides isolate obtained from a fungus garden of Apterostigma megacephala was considered a new phylogenetic species. Dual-culture plate assays showed that Escovopsioides isolates inhibited the mycelium growth of Leucoagaricus gongylophorus, the mutualistic fungus cultivated by somes species of leafcutter ants. In addition, Escovopsioides growth experiments in fungus gardens with and without ant workers showed this fungus is detrimental to the ant-fungus symbiosis. CONCLUSIONS: Here, we provide clues for the antagonism of Escovopsioides towards the mutualistic fungus of leafcutter ants.

Identification of cyclosporin C from Amphichorda felina using a Cryptococcus neoformans differential temperature sensitivity assay.

We used a temperature differential assay with the opportunistic fungal pathogen Cryptococcus neoformans as a simple screening platform to detect small molecules with antifungal activity in natural product extracts. By screening of a collection extracts from two different strains of the coprophilous fungus, Amphichorda felina, we detected strong, temperature-dependent antifungal activity using a two-plate agar zone of inhibition assay at 25 and 37 °C. Bioassay-guided fractionation of the crude extract followed by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) identified cyclosporin C (CsC) as the main component of the crude extract responsible for growth inhibition of C. neoformans at 37 °C. The presence of CsC was confirmed by comparison with a commercial standard. We sequenced the genome of A. felina to identify and annotate the CsC biosynthetic gene cluster. The only previously characterized gene cluster for the biosynthesis of similar compounds is that of the related immunosuppressant drug cyclosporine A (CsA). The CsA and CsC gene clusters share a high degree of synteny and sequence similarity. Amino acid changes in the adenylation domain of the CsC nonribosomal peptide synthase's sixth module may be responsible for the substitution of L-threonine compared to L-α-aminobutyric acid in the CsA peptide core. This screening strategy promises to yield additional antifungal natural products with a focused spectrum of antimicrobial activity.

Zombie-ant fungi across continents: 15 new species and new combinations within Ophiocordyceps. I. Myrmecophilous hirsutelloid species.

Ophiocordyceps species infecting ants - the so-called zombie-ant fungi - comprise one of the most intriguing and fascinating relationships between microbes and animals. They are widespread within tropical forests worldwide, with relatively few reports from temperate ecosystems. These pathogens possess the ability to manipulate host behaviour in order to increase their own fitness. Depending on the fungal species involved the infected ants are manipulated either to leave the nest to ascend understorey shrubs, to die biting onto vegetation, or descend from the canopy to die at the base of trees. Experimental evidence has demonstrated that the behavioural change aids spore dispersal and thus increases the chances of infection, because of the existing behavioural immunity expressed inside ant colonies that limits fungal development and transmission. Despite their undoubted importance for ecosystem functioning, these fungal pathogens are still poorly documented, especially regarding their diversity, ecology and evolutionary relationships. Here, we describe 15 new species of Ophiocordyceps with hirsutella-like asexual morphs that exclusively infect ants. These form a monophyletic group that we identified in this study as myrmecophilous hirsutelloid species. We also propose new combinations for species previously described as varieties and provide for the first time important morphological and ecological information. The species proposed herein were collected in Brazil, Colombia, USA, Australia and Japan. All species could readily be separated using classic taxonomic criteria, in particular ascospore and asexual morphology.

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.

Mitochondrial genome of the nematode endoparasitic fungus Hirsutella vermicola reveals a high level of synteny in the family Ophiocordycipitaceae.

Ophiocordycipitaceae is a diverse fungal family comprising multiple ecologically, economically, medicinally, and culturally important fungal species; however, only four species of the family have available mitochondrial genomes (mitogenomes). In this study, the complete mitogenome of the nematode endoparasitic fungus Hirsutella vermicola in Ophiocordycipitaceae was sequenced, and a comparative mitogenomic analysis of Ophiocordycipitaceae was performed. We found that the 53,793-bp circular mitogenome of H. vermicola, except for standard fungal mitochondrial genes, harbors seven introns acquired possibly through lateral transfer from other fungi and three free-standing open reading frames (ORFs) coding for hypothetical proteins. Phylogenetic analysis based on concatenated mitochondrial protein sequences confirmed its placement in Ophiocordycipitaceae. Comparison on five mitogenomes of Ophiocordycipitaceae revealed great variation on their sizes, from 35.2 kb in Tolypocladium ophioglossoides to 157.5 kb in Ophiocordyceps sinensis, mainly due to variable numbers of introns (from 7 to 54) as well as variable lengths of intergenic regions. The five mitogenomes, however, are highly syntenic to each other in terms of gene order, the presence of an intronic ORF encoding ribosomal protein S3 within rnl, and the nad2/nad3 joining pattern. Our study is the first report of the mitogenome of H. vermicola and has facilitated the understanding of mitogenome evolution of Ophiocordycipitaceae.