Diversity of fungi associated with macroalgae from an estuarine environment and description of Cladosporium rubrum sp. nov. and Hypoxylon aveirense sp. nov.

Fungal communities associated with macroalgae remain largely unexplored. To characterize algicolous fungal communities using culture dependent methods, macroalgae were collected from different sampling sites in the Ria de Aveiro estuary, Portugal. From a collection of 486 isolates that were obtained, 213 representative isolates were selected through microsatellite-primed PCR (MSP-PCR) fingerprinting analysis. The collection yielded 33 different genera, which were identified using the ITS region of the rDNA. The results revealed that the most abundant taxa in all collections were Acremonium-like species: Alternaria, Cladosporium, Leptobacillium and Penicillium. The fungal community composition varied with macroalgae species. Through multilocus phylogenetic analyses based on ITS, tub2, tef1-α and actA sequences, in addition to detailed morphological data, we propose Cladosporium rubrum sp. nov. (type strain=CMG 28=MUM 19.39) and Hypoxylon aveirense sp. nov. (type strain=CMG 29=MUM 19.40) as novel species.

Molecular phylogeny, identification and pathogenicity of Rhizopus oryzae associated with root rot of mulberry in India.

AIMS: Root rot caused by a group of fungi is a serious disease in mulberry. This study aims to identify and characterize Rhizopus oryzae and other fungal species associated with root rot of mulberry in India. METHODS AND RESULTS: Rotted root samples were collected from the mulberry gardens from four states of Southern India. The majority of the isolates identified were R. oryzae, and others were saprophytic fungi, less abundant to occasional. Two methods of inoculations were tested to confirm the pathogenicity of the selected isolates and R. oryzae was found to be pathogenic on susceptible mulberry genotypes RC2 and SRDC-1. Multi gene phylogenetic analyses using the internal transcribed spacer region (ITS), actin (ACT) and translation elongation factor 1-α (TEF), identified the isolates as R. oryzae. Additionally, Ovatospora brasiliensis, Amesia nigricolor, Gongronella butleri, Myrmecridium schulzeri, Scedosporium boydii, Graphium euwallacea, Clonostachys rosea andTalaromyces spp. were also identified. CONCLUSION: This study revealed the existence of eleven species of fungi including the first report of R. oryzae and the occurrence of weak pathogens or saprophytes that are associated with the root rot of mulberry in India. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of R. oryzae causing Rhizopus rot of mulberry in India. Moreover, the occurrence of saprophytes associated with root rot of mulberry was identified. Further studies should focus more on the ability of these species to generate secondary metabolites and extracellular lytic enzymes as they are beneficial for the management of root rot disease.

Purpureocillium roseum sp. nov. A new ocular pathogen for humans and mice resistant to antifungals.

BACKGROUND: Infectious keratitis is the main cause of preventable blindness worldwide, with about 1.5-2.0 million new cases occurring per year. This inflammatory response may be due to infections caused by bacteria, fungi, viruses or parasites. Fungal keratitis is a poorly studied health problem. OBJECTIVES: This study aimed to identify a new fungal species by molecular methods and to explore the possible efficacy of the three most common antifungals used in human keratitis in Mexico by performing in vitro analysis. The capacity of this pathogen to cause corneal infection in a murine model was also evaluated. METHODS: The fungal strain was isolated from a patient with a corneal ulcer. To identify the fungus, taxonomic and phylogenetic analyses (nrDNA ITS and LSU data set) were performed. An antifungal susceptibility assay for amphotericin B, itraconazole and voriconazole was carried out. The fungal isolate was used to develop a keratitis model in BALB/c mice; entire eyes and ocular tissues were preserved and processed for histopathologic examination. RESULTS AND CONCLUSION: This fungal genus has hitherto not been reported with human keratitis in Mexico. We described a new species Purpurecillium roseum isolated from corneal infection. P roseum showed resistance to amphotericin B and itraconazole and was sensitive to voriconazole. In vivo study demonstrated that P roseum had capacity to developed corneal infection and to penetrate deeper corneal tissue. The global change in fungal infections has emphasised the need to develop better diagnostic mycology laboratories and to recognise the group of potential fungal pathogens.

Discovery of two hypocrealean fungi infecting spotted lanternflies, Lycorma delicatula: Metarhizium pemphigi and a novel species, Ophiocordyceps delicatula.

In the eastern United States, populations of the invasive spotted lanternfly, Lycorma delicatula, can be infected by native fungal entomopathogens, including Batkoa major and Beauveria bassiana. In some areas of southeastern Pennsylvania, localized population collapses have been observed in L. delicatula populations to be caused by these pathogens. Two additional fungal pathogens were discovered infecting L. delicatula at low levels, and these were identified as Metarhizium pemphigi and Ophiocordyceps delicatula, a new species that has not been previously described. Therefore, four species of native entomopathogenic fungi have now been documented infecting this abundant, invasive planthopper that is spreading in the United States.

Morphological and molecular identification of four isolates of the entomopathogenic fungal genus Akanthomyces and their effects against Bemisia tabaci on cucumber.

The cotton whitefly, Bemisia tabaci Gen. (Hem., Aleyrodidae), is a key pest of many vegetables. Entomopathogenic fungi are promising microbial control agents against B. tabaci, but limited information is available concerning indigenous Iranian isolates. In this study, three isolates of Akanthomyces lecanii (PAL6, PAL7, and PAL8) and one isolate of A. muscarius (AGM5) were obtained from citrus hemipteran pests, Pulvinaria aurantii Cock. and Aphis gossypii Glover, in Mazandaran province, northern Iran. The isolates were then morphologically and molecularly identified. The efficacies of five different agar media for vegetative growth and conidiation of each isolate were determined. Potato dextrose agar was the medium on which the fungal mycelia developed at a relatively high rate. However, the highest rate of conidiation was found on Sabouraud dextrose agar. To determine the effects of the isolates on B. tabaci, a dose-response bioassay was carried out to estimate lethal concentration (LC50) and lethal time (LT50) values of each fungal isolate to second instar nymphs. The mean LC50 values of A. lecanii isolates ranged from 4.22 × 106 to 7.35 × 1013 conidia ml-1 at 5 to 7 days after the treatment. For A. muscarius, the values varied from 9.2 × 104 to 8.7 × 1010 conidia ml-1 at 5 to 7 days after the treatment. The lowest and the highest mean LC50 values were observed for A. mucarius (AGM5) and A. lecanii (isolate PAL6), respectively. The mean LT50 values of A. lecanii and A. muscarius isolates were 7.1-9.0 and 4.9-7.2 days, respectively. The LT50 values of A. muscarius were significantly lower than the other isolates. Overall, all isolates, especially A. muscarius (AGM5), exhibited appropriate potential as a biological control agent against B. tabaci.

Massive lateral transfer of genes encoding plant cell wall-degrading enzymes to the mycoparasitic fungus Trichoderma from its plant-associated hosts.

Unlike most other fungi, molds of the genus Trichoderma (Hypocreales, Ascomycota) are aggressive parasites of other fungi and efficient decomposers of plant biomass. Although nutritional shifts are common among hypocrealean fungi, there are no examples of such broad substrate versatility as that observed in Trichoderma. A phylogenomic analysis of 23 hypocrealean fungi (including nine Trichoderma spp. and the related Escovopsis weberi) revealed that the genus Trichoderma has evolved from an ancestor with limited cellulolytic capability that fed on either fungi or arthropods. The evolutionary analysis of Trichoderma genes encoding plant cell wall-degrading carbohydrate-active enzymes and auxiliary proteins (pcwdCAZome, 122 gene families) based on a gene tree / species tree reconciliation demonstrated that the formation of the genus was accompanied by an unprecedented extent of lateral gene transfer (LGT). Nearly one-half of the genes in Trichoderma pcwdCAZome (41%) were obtained via LGT from plant-associated filamentous fungi belonging to different classes of Ascomycota, while no LGT was observed from other potential donors. In addition to the ability to feed on unrelated fungi (such as Basidiomycota), we also showed that Trichoderma is capable of endoparasitism on a broad range of Ascomycota, including extant LGT donors. This phenomenon was not observed in E. weberi and rarely in other mycoparasitic hypocrealean fungi. Thus, our study suggests that LGT is linked to the ability of Trichoderma to parasitize taxonomically related fungi (up to adelphoparasitism in strict sense). This may have allowed primarily mycotrophic Trichoderma fungi to evolve into decomposers of plant biomass.

Comparative Analysis of Mitochondrial Genome Features among Four Clonostachys Species and Insight into Their Systematic Positions in the Order Hypocreales.

The mycoparasite fungi of Clonostachys have contributed to the biological control of plant fungal disease and nematodes. The Clonostachys fungi strains were isolated from Ophiocordyceps highlandensis, Ophiocordycepsnigrolla and soil, which identified as Clonostachyscompactiuscula, Clonostachysrogersoniana, Clonostachyssolani and Clonostachys sp. To explore the evolutionary relationship between the mentioned species, the mitochondrial genomes of four Clonostachys species were sequenced and assembled. The four mitogenomes consisted of complete circular DNA molecules, with the total sizes ranging from 27,410 bp to 42,075 bp. The GC contents, GC skews and AT skews of the mitogenomes varied considerably. Mitogenomic synteny analysis indicated that these mitogenomes underwent gene rearrangements. Among the 15 protein-coding genes within the mitogenomes, the nad4L gene exhibited the least genetic distance, demonstrating a high degree of conservation. The selection pressure analysis of these 15 PCGs were all below 1, indicating that PCGs were subject to purifying selection. Based on protein-coding gene calculation of the significantly supported topologies, the four Clonostachys species were divided into a group in the phylogenetic tree. The results supplemented the database of mitogenomes in Hypocreales order, which might be a useful research tool to conduct a phylogenetic analysis of Clonostachys. Additionally, the suitable molecular marker was significant to study phylogenetic relationships in the Bionectriaceae family.

Genomic and transcriptomic survey of an endophytic fungus Calcarisporium arbuscula NRRL 3705 and potential overview of its secondary metabolites.

BACKGROUND: Secondary metabolites as natural products from endophytic fungi are important sources of pharmaceuticals. However, there is currently little understanding of endophytic fungi at the omics levels about their potential in secondary metabolites. Calcarisporium arbuscula, an endophytic fungus from the fruit bodies of Russulaceae, produces a variety of secondary metabolites with anti-cancer, anti-nematode and antibiotic activities. A comprehensive survey of the genome and transcriptome of this endophytic fungus will help to understand its capacity to biosynthesize secondary metabolites and will lay the foundation for the development of this precious resource. RESULTS: In this study, we reported the high-quality genome sequence of C. arbuscula NRRL 3705 based on Single Molecule Real-Time sequencing technology. The genome of this fungus is over 45 Mb in size, larger than other typical filamentous fungi, and comprises 10,001 predicted genes, encoding at least 762 secretory-proteins, 386 carbohydrate-active enzymes and 177 P450 enzymes. 398 virulence factors and 228 genes related to pathogen-host interactions were also predicted in this fungus. Moreover, 65 secondary metabolite biosynthetic gene clusters were revealed, including the gene cluster for the mycotoxin aurovertins. In addition, several gene clusters were predicted to produce mycotoxins, including aflatoxin, alternariol, destruxin, citrinin and isoflavipucine. Notably, two independent gene clusters were shown that are potentially involved in the biosynthesis of alternariol. Furthermore, RNA-Seq assays showed that only expression of the aurovertin gene cluster is much stronger than expression of the housekeeping genes under laboratory conditions, consistent with the observation that aurovertins are the predominant metabolites. Gene expression of the remaining 64 gene clusters for compound backbone biosynthesis was all lower than expression of the housekeeping genes, which partially explained poor production of other secondary metabolites in this fungus. CONCLUSIONS: Our omics data, along with bioinformatics analysis, indicated that C. arbuscula NRRL 3705 contains a large number of biosynthetic gene clusters and has a huge potential to produce a profound number of secondary metabolites. This work also provides the basis for development of endophytic fungi as a new resource of natural products with promising biological activities.

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.

Clonostachys viticola sp. nov., a novel species isolated from Vitis vinifera.

A collection of fungal isolates obtained from crop plants, specifically grapevine and blueberry, in Peru were characterised through morphological and DNA sequence analyses of the nuclear ribosomal internal transcribed spacer (ITS), beta-tubulin (tub2) and translation elongation factor 1-alpha (tef-1α) regions. Isolates produced monomorphic and dimorphic conidiophores typical of members of the genus Clonostachys. Single- and multi-locus gene phylogenies confirmed the isolates as representing members of the genus Clonostachys, more closely related to species in the subgenus Bionectria. In phylogenetic analyses the isolates grouped in two separate clades, one corresponding to the species Clonostachys pseudochroleuca and the other one distinct from all known species of the genus Clonostachys. These isolates are recognized as representing a novel species species for which the name Clonostachys viticola is proposed.

Entomopathogenic effect of Trichothecium roseum (Pers.) Link (Hypocreales: Ascomycota) against Pauropsylla buxtoni (Psylloidea: Hemiptera) infesting Ficus carica leaves and its potential use as biocontrol agent of the insect.

AIMS: To isolate and characterize a native strain of Trichothecium roseum infecting the immatures of Pauropsylla buxtoni on fig leaves, to study the morphological features of the isolated strain, then to test the entomopathogenic effect of the isolated strain against the immatures of P. buxtoni on fig leaves. METHODS AND RESULTS: The isolated strain of T. roseum produced pink mycelial growth on culture medium with septate mycelium and conidiophores. It also produced two-celled conidia with elliptical to pyriform shape born at the tip of conidiophores. Molecular characterization of the isolated strain confirmed the identity of the strain as T. roseum. In bioassays, application of conidial suspension of the isolated strain against the 4th instar of P. buxtoni immatures infesting fig leaves showed an obvious entomopathogenic effect of the applied fungus strain against the targeted insect. This effect was exhibited by the death of treated P. buxtoni immatures with the fungus. The dead insects were characterized by the presence of pinkish mycelial growth on the outer surface which is characteristic to the fungus, in addition to the positive isolation of the fungus from internal tissues of treated insects after a proper external disinfection. Moreover, significant differences (at P < 0·018) were obtained between the means of mortality % of P. buxtoni immatures treated with different concentrations of conidial suspension of the fungus. CONCLUSIONS: The overall results confirm the entomopathogenic effect of T. roseum against P. buxtoni immatures infesting fig leaves. Significant mortalities of P. buxtoni immatures were obtained when the different concentrations of the fungus conidial suspension were bio-assessed against the insect. SIGNIFICANCE AND IMPACT OF THE STUDY: The tested strain of T. roseum can be applied as biocontrol agent of P. buxtoni on fig leaves within an integrated control programme to reduce the impact of pest on fig trees.

Sarocladium and Acremonium infections: New faces of an old opportunistic fungus.

The genera Acremonium and Sarocladium comprise a high diversity of morphologically and genetically related fungi generally found in the environment, although a few species, mainly Sarocladium kiliense and Acremonium egyptiacum, can also be involved in many human infections. Clinical management of opportunistic infections caused by these fungi is very complex, since their correct identification is unreliable, and they generally show poor antifungal response. More than 300 clinical cases involving a broad range of Acremonium/Sarocladium infections have so far been published, and with this review we aim to compile and provide a detailed overview of the current knowledge on Acremonium/Sarocladium human infections in terms of presentation, diagnosis, treatments and prognoses. We also aim to summarise and discuss the data currently available on their antifungal susceptibility, emphasising the promising results obtained with voriconazole as well as their impact in terms of animal infections.

Occurrence and Diversity of Black-Foot Disease Fungi in Symptomless Grapevine Nursery Stock in Spain.

In this study, 3,426 grafted grapevines ready to be planted from 15 grapevine nursery fields in Northern Spain were inspected from 2016 to 2018 for black-foot causing pathogens. In all, 1,427 isolates of black-foot pathogens were collected from the asymptomatic inner tissues of surface sterilized secondary roots and characterized based on morphological features and DNA sequence data of the nuclear ribosomal DNA-internal transcribed spacer region, histone H3, translation elongation factor 1-alpha and ß-tubulin genes. Eleven species belonging to the genera Dactylonectria, Ilyonectria, Neonectria, and Thelonectria were identified, including Dactylonectria alcacerensis, D. macrodidyma, D. novozelandica, D. pauciseptata, D. torresensis, Ilyonectria liriodendri, I. pseudodestructans, I. robusta, Neonectria quercicola, Neonectria sp. 1, and Thelonectria olida. In addition, two species are newly described, namely D. riojana and I. vivaria. Twenty-four isolates representing 13 black-foot species were inoculated onto grapevine seedlings cultivar 'Tempranillo'. The pathogenicity tests detected diversity in virulence among fungal species and between isolates within each species. The most virulent species was D. novozelandica isolate BV-0760, followed by D. alcacerensis isolate BV-1240 and I. vivaria sp. nov. isolate BV-2305. This study improves our knowledge on the etiology and virulence of black-foot disease pathogens, and opens up new perspectives in the study of the endophytic phase of these pathogens in grapevines.

Genome Sequence Resource for Ilyonectria mors-panacis, Causing Rusty Root Rot of Panax notoginseng.

Ilyonectria mors-panacis is the cause of a serious disease hampering the production of Panax notoginseng, an important Chinese medicinal herb, widely used for its anti-inflammatory, antifatigue, hepato-protective, and coronary heart disease prevention effects. Here, we report the first Illumina-Pacbio hybrid sequenced draft genome assembly of I. mors-panacis strain G3B and its annotation. The availability of this genome sequence not only represents an important tool toward understanding the genetics behind the infection mechanism of I. mors-panacis strain G3B but also will help illuminate the complexities of the taxonomy of this species.

The complete mitochondrial genome of the Chan-hua fungus Isaria cicadae: a tale of intron evolution in Cordycipitaceae.

Isaria cicadae is an entomogenous fungus of great medicinal value. Its nuclear genome has been reported, while its mitogenome remains unknown. Herein, we first described its mitogenome and then inferred intron evolution from both intraspecific and interspecific perspectives. The fungus represented the largest mitogenome (56.6 kb in strain CCAD02) known in Cordycipitaceae due to the presence of 25 introns interrupting nine genes. Comparison of three I. cicadae strains revealed intron presence/absence dynamics at six intron loci plus a few indels and single nucleotide polymorphisms. Phylogenetic analyses confirmed the placement of I. cicadae in Cordycipitaceae. Comparison of 10 Cordycipitaceae species revealed a high degree of synteny and conserved genetic content. They, however, varied in intron numbers (1-25 per species) with overall 34 intron loci identified, which resulted in more than twofold variations in mitogenome sizes (24.5-56.6 kb). An rnl intron encoding ribosomal protein S3 was present in all species, suggesting its early invasion in Cordycipitaceae, while further divergence occurred for this intron. The other introns identified in this study were present in some, but not all of the species and have undergone multiple gains and losses in Cordycipitaceae. This study greatly enhanced our understanding of intron evolution in Cordycipitaceae.

Memnoniella sinensis sp. nov., a new species from China and a key to species of the genus.

During a survey of fungal diversity in a deserted rocky area in Yunnan, PR China, a new species, Memnoniella sinensis, was identified. This new species is characterized by having phialidic conidiogenous cells with conspicuous collarettes, and aseptate, verrucose, ellipsoidal to sometimes ovoid, olivaceous brown to dark brown conidia. Morphologically, M. sinensis is similar to M. dichroa, but can be easily distinguished due to its hyaline conidiophores and smaller conidia. Phylogenetic analysis based on DNA sequences at five loci showed that our strain grouped together with M. dichroa and M. oenanthes. Here, the new species is described and illustrated, and a key to the species of the genus Memnoniella is provided.

Genome sequence of Isaria javanica and comparative genome analysis insights into family S53 peptidase evolution in fungal entomopathogens.

The fungus Isaria javanica is an important entomopathogen that parasitizes various insects and is effective for pest control. In this study, we sequenced and assembled the genomes (IJ1G and IJ2G) of two I. javanica strains isolated from different insects. The genomes were approximately 35 Mb in size with 11,441 and 11,143 protein-coding genes, respectively. Using a phylogenomic approach, we evaluated genome evolution across five entomopathogenic fungi in Cordycipitaceae. By comparative genome analysis, it was found that family S53 serine peptidases were expanded in Cordycipitaceae entomopathogens, particularly in I. javanica. Gene duplication events were identified based on phylogenetic relationships inferred from 82 S53 peptidases within six entomopathogenic fungal genomes. Moreover, we found that carbohydrate-active enzymes and proteinases were the largest secretory protein groups encoded in the I. javanica genome, especially chitinases (GH18), serine and aspartic peptidases (S53, S08, S10, A01). Pathogenesis-related genes and genes for bacterial-like toxins and secondary metabolites were also identified. By comparative transcriptome analysis, differentially expressed genes in response to insect nutrients (in vitro) were identified. Moreover, most S53 peptidases were detected to be significantly upregulated during the initial fungal infection process in insects (in vivo) by RT-qPCR. Our results provide new clues about understanding evolution of pathogenic proteases and may suggest that abundant S53 peptidases in the I. javanica genome may contribute to its effective parasitism on various insects.

Escovopsis kreiselii specialization to its native hosts in the fungiculture of the lower attine ant Mycetophylax morschi.

Parasite-host associations are widespread in nature and the fungus-growing ants are considered model organisms to study such interactions. These insects cultivate basidiomycetous fungi for food, which are threatened by mycotrophic fungi in the genus Escovopsis. Although recently described from colonies of the lower attine ant Mycetophylax morschi, the biology and pathogenicity of Escovopsis kreiselii are unknown. Herein, we evaluated the interaction of E. kreiselii with fungi cultivated by M. morschi (native hosts) and with a fungus cultivated by another attine ant species (non-native host). In addition, we examined the physical interactions between hypha of E. kreiselii and hypha from its native hosts using scanning electron microscopy. Escovopsis kreiselii inhibited the growth of fungal cultivars by 24% or more (with exception of one isolate), when compared to the fungal cultivars growing alone. Escovopsis kreiselii is attracted towards its native hosts through chemotaxis and inhibition occurs when there is physical contact with the hyphae of the fungal cultivar. As reported for Escovopsis parasites associated with leafcutter ants (higher attines), E. kreiselii growth increased in the presence of its native hosts, even before contact between both fungi occurred. In interactions with the fungal cultivar that is not naturally infected by E. kreiselii (non-native host), it caused inhibition but not at the same magnitude as in native hosts. Multiple lines of evidence suggest that E. kreiselii is an antagonist of the fungus cultivated by M. morschi and can chemically recognize such fungus.

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.

Comparative Genomic Analysis of Drechmeria coniospora Reveals Core and Specific Genetic Requirements for Fungal Endoparasitism of Nematodes.

Drechmeria coniospora is an obligate fungal pathogen that infects nematodes via the adhesion of specialized spores to the host cuticle. D. coniospora is frequently found associated with Caenorhabditis elegans in environmental samples. It is used in the study of the nematode's response to fungal infection. Full understanding of this bi-partite interaction requires knowledge of the pathogen's genome, analysis of its gene expression program and a capacity for genetic engineering. The acquisition of all three is reported here. A phylogenetic analysis placed D. coniospora close to the truffle parasite Tolypocladium ophioglossoides, and Hirsutella minnesotensis, another nematophagous fungus. Ascomycete nematopathogenicity is polyphyletic; D. coniospora represents a branch that has not been molecularly characterized. A detailed in silico functional analysis, comparing D. coniospora to 11 fungal species, revealed genes and gene families potentially involved in virulence and showed it to be a highly specialized pathogen. A targeted comparison with nematophagous fungi highlighted D. coniospora-specific genes and a core set of genes associated with nematode parasitism. A comparative gene expression analysis of samples from fungal spores and mycelia, and infected C. elegans, gave a molecular view of the different stages of the D. coniospora lifecycle. Transformation of D. coniospora allowed targeted gene knock-out and the production of fungus that expresses fluorescent reporter genes. It also permitted the initial characterisation of a potential fungal counter-defensive strategy, involving interference with a host antimicrobial mechanism. This high-quality annotated genome for D. coniospora gives insights into the evolution and virulence of nematode-destroying fungi. Coupled with genetic transformation, it opens the way for molecular dissection of D. coniospora physiology, and will allow both sides of the interaction between D. coniospora and C. elegans, as well as the evolutionary arms race that exists between pathogen and host, to be studied.