Metschnikowia cf. typographi and other pathogens from the bark beetle Ips sexdentatus - Prevalence, histological and ultrastructural evidence, and molecular characterization.

Ips sexdentatus (six-spined engraver beetle) from Austria and Poland were dissected and examined for the presence of pathogens. Specimens collected in Austria were found to contain the ascomycetous fungus Metschnikowia cf. typographi. Infection rates ranged from 3.6% to 26.8% at different collection sites. M. cf. typographi infected midguts were investigated by histological, ultrastructural and molecular techniques. Extraordinary ultrastructural details are shown, such as ascospores with bilateral flattened flanks resembling alar rims at both sides of their attenuating tube-like ends. These have not yet been described in other yeast species. Molecular investigations showed a close phylogenetic relationship to the fungi Metschnikowia agaves and Candida wancherniae. Presence of the entomopathogenic fungus Beauveria bassiana found in Austria was confirmed both morphologically and molecularly. The eugregarine Gregarina typographi was diagnosed most frequently. Infection rates of all I. sexdentatus specimens ranged from 21.4% to 71.9% in Austria and 54.1% to 68.8% in Poland. Other entomopathogenic protists, bacteria, or viruses were not detected.

Diversity within the entomopathogenic fungal species Metarhizium flavoviride associated with agricultural crops in Denmark.

BACKGROUND: Knowledge of the natural occurrence and community structure of entomopathogenic fungi is important to understand their ecological role. Species of the genus Metarhizium are widespread in soils and have recently been reported to associate with plant roots, but the species M. flavoviride has so far received little attention and intra-specific diversity among isolate collections has never been assessed. In the present study M. flavoviride was found to be abundant among Metarhizium spp. isolates obtained from roots and root-associated soil of winter wheat, winter oilseed rape and neighboring uncultivated pastures at three geographically separated locations in Denmark. The objective was therefore to evaluate molecular diversity and resolve the potential population structure of M. flavoviride. RESULTS: Of the 132 Metarhizium isolates obtained, morphological data and DNA sequencing revealed that 118 belonged to M. flavoviride, 13 to M. brunneum and one to M. majus. Further characterization of intraspecific variability within M. flavoviride was done by using amplified fragment length polymorphisms (AFLP) to evaluate diversity and potential crop and/or locality associations. A high level of diversity among the M. flavoviride isolates was observed, indicating that the isolates were not of the same clonal origin, and that certain haplotypes were shared with M. flavoviride isolates from other countries. However, no population structure in the form of significant haplotype groupings or habitat associations could be determined among the 118 analyzed M. flavoviride isolates. CONCLUSIONS: This study represents the first in-depth analysis of the molecular diversity within a large isolate collection of the species M. flavoviride. The AFLP analysis confirmed a high level of intra-specific diversity within the species and lack of apparent association patterns with crop or location indicates limited ecological specialization. The relatively infrequent isolation of M. flavoviride directly from crop roots suggests low dependence of root association for the species.

Root isolations of Metarhizium spp. from crops reflect diversity in the soil and indicate no plant specificity.

Metarhizium spp. have recently been shown to be associated with the roots of different plants. Here we evaluated which Metarhizium species were associated with roots of oat (Avena sativa), rye (Secale cereale) and cabbage (Brassica oleracea), common crop plants in Denmark. Thirty-six root samples from each of the three crops were collected within an area of approximately 3ha. The roots were rinsed with sterile water, homogenized and the homogenate plated onto selective media. A subset of 126 Metarhizium isolates were identified to species by sequencing of the 5' end of the gene translation elongation factor 1-alpha and characterized by simple sequence repeat (SSR) analysis of 14 different loci. Metarhizium brunneum was the most common species isolated from plant roots (84.1% of all isolates), while M. robertsii (11.1%) and M. majus (4.8%) comprised the remainder. The SSR analysis revealed that six multilocus genotypes (MLGs) were present among the M. brunneum and M. robertsii isolates, respectively. A single MLG of M. brunneum represented 66.7%, 79.1% and 79.2% of the total isolates obtained from oat, rye and cabbage, respectively. The isolation of Metarhizium spp. and their MLGs from roots revealed a comparable community composition as previously reported from the same agroecosystem when insect baiting of soil samples was used as isolating technique. No specific MLG association with a certain crop was found. This study highlights the diversity of Metarhizium spp. found in the rhizosphere of different crops within a single agroecosystem and suggests that plants either recruit fungal associates from the surrounding soil environment or even govern the composition of Metarhizium populations.

Molecular diversity of the entomopathogenic fungal Metarhizium community within an agroecosystem.

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