Multidimensional discriminant analysis of species, strains and culture age of closely related entomopathogenic fungi using reflectance spectroscopy.

The diversity of fungal strains is influenced by genetic and environmental factors, growth conditions and mycelium age, and the spectral features of fungal mycelia are associated with their biochemical, physiological, and structural traits. This study investigates whether intraspecific differences can be detected in two closely related entomopathogenic species, namely Cordyceps farinosa and Cordyceps fumosorosea, using ultraviolet A to shortwave infrared (UVA-SWIR) reflectance spectra. Phylogenetic analysis of all strains revealed a high degree of uniformity among the populations of both species. The characteristics resulting from variation in the species, as well as those resulting from the age of the cultures were determined. We cultured fungi on PDA medium and measured the reflectance of mycelia in the 350-2500 nm range after 10 and 17 days. We subjected the measurements to quadratic discriminant analysis (QDA) to identify the minimum number of bands containing meaningful information. We found that when the age of the fungal culture was known, species represented by a group of different strains could be distinguished with no more than 3-4 wavelengths, compared to 7-8 wavelengths when the age of the culture was unknown. At least 6-8 bands were required to distinguish cultures of a known species among different age groups. Distinguishing all strains within a species was more demanding: at least 10 bands were required for C. fumosorosea and 21 bands for C. farinosa. In conclusion, fungal differentiation using point reflectance spectroscopy gives reliable results when intraspecific and age variations are taken into account.

Effect of Selected Entomopathogenic Fungal Species on Embryonic Development of Ascaris suum (Nematoda).

The aim of the study was to evaluate the potential of using five selected species of entomopathogenic fungi (Beauveria bassiana, B. brongniartii, Conidiobolus coronatus, Isaria fumosorosea, and Metarhizium robertsii) in the bioregulation of the dispersive stages of the parasitic nematode-Ascaris suum. Experimental cultures of each of the selected entomopathogenic fungi, as well as a control culture without fungi, were incubated with A. suum eggs at 26 °C for 28 days. Development of the A. suum eggs was observed using a light microscope on the 7th, 14th, 21st, and 28th days of incubation. The API-ZYM® test was used to determine, semiquantitatively, the activity of 19 hydrolytic enzymes from the entomopathogenic fungi. The cytotoxicity of the fungi was determined using tetrazole salt MTT. It was found that none of the five tested strains of entomopathogenic fungi showed an ovicidal effect, and none of them colonized the A. suum egg shells. However, ovistatic activity was observed mainly until the 14th day of incubation by I. fumosorosea, M. robertsii, and B. bassiana. In the MTT test, M. robertsii showed moderate cytotoxicity, while the other species showed low cytotoxicity. Among the strains tested, I. fumosorosea showed the highest spectrum of hydrolase production (13 out of 19 enzymes gave a positive reaction from 3 to 5; 20-40 nM or more). The absence of morphological changes in the A. suum egg shells suggests that the antagonistic effect of the studied entomopathogenic fungi may be due to their cytotoxicity, associated with the production of secondary metabolites-toxins (M. robertsii) and enzymatic activity (I. fumosorosea).

The impact of Beauveria species bioinocula on the soil microbial community structure in organic strawberry plantations.

Introduction: The multifunctionality of microorganisms, including entomopathogenic fungi, represents a feature that could be exploited to support the development, marketing, and application of microbial-based products for plant protection. However, it is likely that this feature could affect the composition and dynamics of the resident soil microorganisms, possibly over a longer period. Therefore, the methodology utilized to evaluate such impact is critical for a reliable assessment. The present study was performed to evaluate the impact of strains of Beauveria brongniartii and Beauveria bassiana on soil bacterial and fungal communities using an approach based on the terminal restriction fragment polymorphism (T-RFLP) analysis. Materials and methods: Soil samples in the vicinity of the root system were collected during a 3-year period, before and after the bioinocula application, in two organic strawberry plantations. Specific primers were used for the amplification of the bacterial 16S rRNA gene and the fungal ITS region of the ribosome. Results and discussion: Data of the profile analysis from T-RFLP analysis were used to compare the operational taxonomic unit (OTU) occurrence and intensity in the inoculated soil with the uninoculated control. With regard to the impact on the bacterial community, both Beauveria species were not fully consistently affecting their composition across the seasons and fields tested. Nevertheless, some common patterns were pointed out in each field and, sometimes, also among them when considering the time elapsed from the bioinoculum application. The impact was even more inconsistent when analyzing the fungal community. It is thus concluded that the application of the bioinocula induced only a transient and limited effect on the soil microbial community, even though some changes in the structure dynamic and frequency of soil bacterial and fungal OTUs emerged.

Effects of the entomopathogenic fungi Metarhizium robertsii, Metarhizium flavoviride, and Isaria fumosorosea on the lipid composition of Galleria mellonella larvae.

Galleria mellonella is a pest of the honey bee (Apis mellifera L.) and causes significant losses to the beekeeping industry; therefore, experiments are needed to decode the effects of entomopathogenic fungi on insect physiology. The gas chromatography-mass spectrometry (GC-MS) method was successful for the determination of the organic compounds of Galleria mellonella larvae, noninfected and infected by three fungal species: M. robertsii, M. flavoviride, and I. fumosorosea. A total of 46 compounds were identified in G. mellonella, including fatty acids, other acids, fatty acid methyl esters, monoacylglycerols, amino acids, sterols, and several other organic compounds. The lipids of G. mellonella larvae after M. robertsii, M. flavoviride, and I. fumosorosea exposure contained 40, 35, and 33 organic compounds, respectively. The following organic compounds, present in the noninfected larvae, were absent from the infected larvae: fatty acids C22:0 and C24:0, glutaric acid, urocanic acid, hydroxycinnamic acid, dihydroxycinnamic acid, 10-oxodecanoic acid, glycine, aspartic acid, glutamic acid, lysine, tyrosine, tryptophan, 2-aminobutyric acid, and tyramine. These compounds can be used by fungi as an exogenous source of carbon. The following organic compounds, present in the infected larvae, were absent from the noninfected larvae: fatty acids C10:0, C11:0, C13:0, and C20:0, suberic acid, phenylacetic acid, fatty acid methyl ester (FAME) C16:0, FAME C18:2, FAME C18:1, glycerol 2-monopalmitate, norvaline, proline, sitosterol, and 2-dekanal. These compounds can be synthesized as an insect response to fungal infection.

Biocontrol of Melolontha spp. Grubs in Organic Strawberry Plantations by Entomopathogenic Fungi as Affected by Environmental and Metabolic Factors and the Interaction with Soil Microbial Biodiversity.

The efficacy of two strains of two Beauveria species (B. bassiana and B. brongniartii), individually or as co-inoculants, to control Melolontha sp. grubs was assessed in two organic strawberry plantations in relation to the environmental conditions, their abundance after soil inoculation, and their in vitro chitinolytic activity, thereby also verifying their impact on soil microbial communities. A reduction of the grubs' damage to strawberry plants was observed when compared to the untreated control in one plantation, irrespective of the strain used and whether they were applied as single or as co-inoculum. The metabolic pattern expressed by the two fungi in vitro was different: B. bassiana showed a higher metabolic versatility in the use of different carbon sources than B. brongniartii, whose profile was partly overlapped in the co-inoculum. Similar differences in the chitinolytic activity of each of the fungi and the co-inoculum were also pointed out. A higher abundance of B. bassiana in the soils receiving this species in comparison to those receiving B. brongniartii, together with its in vitro metabolic activity, could account for the observed diverse efficacy of pest damage control of the two species. However, environmental and climatic factors also affected the overall efficacy of the two bioinocula. According to the monitoring of the two species in soil, B. bassiana could be considered as a common native species in the studied locations in contrast to B. brongniartii, which seemed to be a non-endemic species. Nevertheless, the inoculation with both species or the co-inoculum did not consistently affect the soil microbial (fungi and bacteria) biodiversity, as expressed by the operational taxonomic unit (OTU) number and Shannon-Wiener diversity index based on terminal restriction fragment length polymorphism (TRFLP) data. A small transient increase of the share of the inoculated species to the total fungal community was noted by the analysis of genes copy numbers only for B. brongniartii at the end of the third growing season.

Abundance of Entomopathogenic Fungi in Leaf Litter and Soil Layers in Forested Habitats in Poland.

This study aims to determine the species composition and density of colony-forming units (CFU) of entomopathogenic fungi (EPF) in leaf litter at different depths of the top layer of forest soils depending on the type of forest (coniferous, deciduous and mixed forest), and the date of sampling (spring, autumn). In each type of forest, leaf litter and soil were collected using a soil stick from four depths of soil: 0-5, 5-10, 10-15 and 15-20 cm. Entomopathogenic fungi were isolated by a soil or litter dilution plating method on a selective medium. Four fungal genera were found: Beauveria spp., Cordyceps spp., Metarhizium spp., and Lecanicillium spp. The density of EPF was usually higher in leaf litter than in the layers of soil below, and the most frequently isolated species from both environments were Beauveria spp. among soil samples from all forest types; Beauveria spp. were most abundant in the top layer (0-5 cm), and their density of CFUs gradually decreased deeper into the soil profile.

Identification of Entomopathogenic Fungi as Naturally Occurring Enemies of the Invasive Oak Lace Bug, Corythucha arcuata (Say) (Hemiptera: Tingidae).

The oak lace bug (OLB), Corythucha arcuata (Hemiptera: Tingidae), was first identified as an invasive pest in Europe in northern Italy in 2000 and since then it has spread rapidly, attacking large forested areas in European countries. The OLB is a cell sap-sucking insect that is native to North America, with Quercus spp. as its main host. Its rapid expansion, successful establishment in invaded countries, and observations of more damage to hosts compared to native areas are most likely due to a lack of natural enemies, pathogens and competitors. In its native area, various natural enemies of OLBs have been identified; however, little is known about the occurrence and impact of OLB pathogens. None of the pathogenic fungi found on OLBs in natural conditions have been identified until now. In this study, we provide evidence of four entomopathogenic fungi that are naturally occurring on invasive OLBs found in infested pedunculate oak forests in eastern Croatia. On the basis of their morphology and multilocus molecular phylogeny, the fungi were identified as Beauveria pseudobassiana, Lecanicillium pissodis, Akanthomyces attenuatus and Samsoniella alboaurantium. The sequences generated for this study are available from GenBank under the accession numbers MT004817-MT004820, MT004833-MT004835, MT027501-MT27510, and MT001936-MT0011943. These pathogenic species could facilitate biological control strategies against OLBs.

Effects of the entomopathogenic fungus Metarhizium flavoviride on the fat body lipid composition of Zophobas morio larvae (Coleoptera: Tenebrionidae).

Insects employ different defense strategies against fungal infections and chemicals. We aimed to identify the lipid compositions of the fat body of Zophobas morio larvae before and after fungal infection with the entomopathogenic fungus Metarhizium flavoviride. We used gas chromatography-mass spectrometry to analyze lipid extracts of the fat body isolated of Z. morio 2, 5, and 7 days after fungal infection (treatment group) and compared it with the lipid extracts in a control group injected with physiological isotonic saline. In all the samples, fatty acids were the most abundant compound found in the fat body extracts, with hexadecanoic acid/C16:0 being the most abundant lipid. However, the types and concentrations of lipids changed after fungal infection, likely as an immune response. The most considerable changes occurred in the concentrations of long-chain fatty acids, i.e., hexadecanoic acid/C16:0, octadecenoic acid/C18:1, and octadecanoic acid/C18:0. Contents of methyl ester increased significantly after infection, but that of other esters, especially octanoic acid decyl ester/OADE, decreased on the 5th day after infection. To the best of our knowledge, this is the first detailed analysis of the changes in the lipid composition of the fat body of Z. morio larvae as a result of fungal infection. Our results suggest that entomopathogenic fungal infection affects the internal lipid composition of insects, potentially as a way of adjusting to such infection. These results can help understand infection processes and defense strategies of insects against fungal infection. Ultimately, they can contribute to the creation of more effective chemicals against pest insects.

Application of HS-SPME-GC-MS for the analysis of aldehydes produced by different insect species and their antifungal activity.

In this study, a procedure was developed to determine aldehydes using headspace solid-phase microextraction (HS-SPME) followed by gas chromatography (GC) coupled with mass spectrometry (MS). The aldehydes selected for research had previously been identified in various species of insects. Minimal inhibitory concentrations of the compounds against strains of entomopathogenic fungi were also determined. At the outset, the best SPME extraction conditions were chosen for the analysis to obtain good chromatographic separation. The analysis was carried out using a BZ-5 column and different SPME fibers were used to isolate the aldehydes. DVB/CAR/PDMS fiber appeared to be the most efficient coating for undertaking the measurements. The best parameters of separation by HS-SPME and analysis by GC-MS were selected. In addition, the aldehydes were tested for their potential antifungal activity. A procedure was developed to determine the aldehydes using HS-SPME-GC-MS. Heptanal, 2,4-nonadienal, 2-decenal and undecanal were the most effective antifungal compounds against entomopathogenic fungi.

Comparison of volatile compounds released by entomopathogenic fungi.

Entomopathogenic fungi are fungal species which are used as a potential source of biopesticides. These fungi produce secondary metabolites which in insects can cause disruption in the normal functioning of their bodies, disease or even death. In order to fully characterize the physiology of entomopathogenic fungi we should identify the volatile organic compounds which are involved in this process. Therefore, we conducted a qualitative and quantitative analysis of volatile compounds produced by entomopathogenic fungi. Seven different species of fungi were analyzed: Metarhizium anisopliae, Metarhizium flavoviride, Pandora sp., Isaria fumosorosea, Hirsutella danubiensis, Batkoa sp. and Beauveria bassiana. The analyses were performed using the HS-SPME/GCMS technique. In the analyzed fungi, 63 volatile compounds were identified and classified into the following groups: aldehydes, ketones, alcohols, esters, acids, terpenes and others. The results show that entomopathogenic fungi produce a wide profile of secondary metabolites. Principal Components Analysis was used to determine whether separate classes of fungi can be distinguished from one another based on their metabolite profiles.

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.

New opportunities for the integration of microorganisms into biological pest control systems in greenhouse crops.

Biological pest control with mass-produced arthropod natural enemies is well developed in greenhouse crops and has often resulted in the evolution of complex ecosystems with persistent populations of multiple arthropod natural enemy species. However, there are cases where arthropod natural enemies are either not effective enough, not available, or their use is rather costly. For these reasons, biological control based on microorganisms, also referred to as 'microbials', represents a complementary strategy for further development. Although commercially available microbials have been around for quite some time, research on and the applied use of combinations of arthropod natural enemies and microbials have remained relatively under explored. Here, we review current uses of entomopathogenic fungi, bacteria and viruses, and their possible direct and indirect effects on arthropod natural enemies in European greenhouses. We discuss how microbials might be combined with arthropod natural enemies in the light of new methodologies and technologies such as conservation biological control, greenhouse climate management, and formulation and delivery. Furthermore, we explore the possibilities of using other microorganisms for biological control, such as endophytes, and the need to understand the effect of insect-associated microorganisms, or symbionts, on the success of biological control. Finally, we suggest future research directions to optimize the combined use of microbials and arthropod natural enemies in greenhouse production.

Utilization of 4-n-nonylphenol by Metarhizium sp. isolates.

Nonylphenol (4-NP) is a xenobiotic classified as an endocrine disrupting compound with an ability to interfere with hormonal systems of numerous organisms including humans. It is widely distributed not only in aquatic but also in terrestrial systems. The aim of this study was to evaluate the ability of cosmopolitan fungus Metarhizium (commonly persistent in soil as a facultative insect pathogen, controlling populations of arthropods in natural environment) to degrade 4-n-nonylphenol. All isolates examined in this work were identified to a species rank based on five, independent genetic markers. Among eight Metarhizium strains; six of them have been identified as M. robertsii, and two others as M. brunneum and M. lepidiotae. All investigated Metarhzium isolates were found to eliminate 4-n-NP with significant efficiency (initial xenobiotic concentration 50 mg L(-1)). The degradation process was very effective and at 24h of incubation 50-90% of 4-n-NP was eliminated by certain strains, while extended incubation resulted in further utilization of this compound. At the end of the experiments 64-99% of 4-n-NP was removed from the culture medium. Additionally, in all tested cultures three major metabolites were detected: 4-hydroxybenzoic acid; 2-(4-hydroxyphenyl)acetic acid and 4-hydroxyphenylpentanoic acid. The obtained results indicate that Metarhizium sp. possesses an ability to degrade NP and can serve as a potential candidate for further biodegradation studies.

Diversity of acaropathogenic fungi in Poland and other European countries.

The occurrence, species diversity and some aspects of taxonomical affinity and host selectivity of acaropathogenic fungi associated with phytophagous, saprotrophic and predacious mites in Poland and other European countries were investigated on wild and cultivated plants, in insect feeding sites under the bark and in decayed wood. From among 33 species of fungi affecting mites only five species of Entomophthorales were separated and the most numerous were Neozygites floridana mostly on Tetranychus urticae, N. abacaridis on a few eriophyid species, and Conidiobolus coronatus attacking gamasid mites most frequently of the genus Dendrolaelaps. The most frequent mite pathogens occurring in mite communities on plants and in wood infested by insects were of the genus Hirsutella. Until now 13 of their form-species have been recognized in these habitats, but only H. kirchneri, H. necatrix and H. thompsonii (including its variety synnematosa) can be treated as exclusive oligophagous pathogens of phytophagous mites, though their potential host range seems to embrace only selected eriophyid or tarsonemid mites. Taxonomical differentiation of fungal strains was based on close morphological observations and molecular analysis of ITS region sequences. Two new species of acaropathogenic fungi were described in these studies. Hirsutella danubiensis sp. nov. was found in the tetranychid T. urticae, whereas H. vandergeesti sp. nov. affected phytoseiid mites of the genera Amblyseius, Neoseiulus, Seiulus and Typhlodromus, and the tarsonemid Tarsonemus lacustris.

Laboratory trials to infect insects and nematodes by some acaropathogenic Hirsutella strains (Mycota: Clavicipitaceous anamorphs).

Laboratory assays have been carried out to artificially infect insect larvae of the birch bark-beetle (Scolytus ratzeburgi Jans.-Coleoptera, Scolytidae) and codling moth Cydia pomonella L. -Lepidoptera, Tortricidae) as well as the potato cyst nematode-Globodera rostochiensis Wollenweber, sugar beet nematode-Heterodera schachtii Schmidt and root-knot nematode-Meloidogyne hapla Chif (Nematoda, Heteroderidae), by the phialoconidia of some fungal species of the genus Hirsutella. From among four species tested on insects only H. nodulosa Petch infected about 20% of S. ratzeburgi larvae, whereas H. kirchneri (Rostrup) Minter, Brady et Hall, H. minnesotensis Chen, Liu et Chen, and H. rostrata Balazy et Wisniewski did not affect insect larvae. Only single eggs of the root-knot nematode were infected by H. minnesotensis in the laboratory trials, whereas its larvae remained unaffected. No infection cases of the potato cyst nematode (G. rostochiensis) and sugar beet nematode eggs were obtained. Comparisons of DNA-ITS-region sequences of the investigated strains with GenBank data showed no differences between H. minnesotensis isolates from the nematodes Heterodera glycines Ichinohe and from tarsonemid mites (authors' isolate). A fragment of ITS 2 with the sequence characteristic only for H. minnesotensis was selected. Two cluster analyses indicated close similarity of this species to H. thompsonii as sister clades, but the latter appeared more heterogenous. Insect and mite pathogenic species H. nodulosa localizes close to specialized aphid pathogen H. aphidis, whereas the phytophagous mite pathogens H. kirchneri and H. gregis form a separate sister clade. Hirsutella rostrata does not show remarkable relations to the establishment of aforementioned groups. Interrelated considerations on the morphology, biology and DNA sequencing of investigated Hirsutella species state their identification more precisely and facilitate the establishment of systematic positions.