Entomopathogenic Fungus Metarhizium Brunneum Research Articles

The Identification of the Mitochondrial DNA Polymerase γ (Mip1) of the Entomopathogenic Fungus Metarhizium brunneum

Replication of the mitochondrial (mt) genome in filamentous fungi is under-studied, and knowledge is based mainly on data from yeasts and higher eukaryotes. In this study, the mitochondrial DNA polymerase γ (Mip1) of the entomopathogenic fungus Metarhizium brunneum is characterized and analyzed with disruption experiments and its in silico interactions with key proteins implicated in mt gene transcription, i.e., mt RNA polymerase Rpo41 and mt transcription factor Mtf1. Disruption of mip1 gene and its partial expression influences cell growth, morphology, germination and stress tolerance. A putative in silico model of Mip1-Rpo41-Mtf1, which is known to be needed for the initiation of replication, was proposed and helped to identify potential amino acid residues of Mip1 that interact with the Rpo41-Mtf1 complex. Moreover, the reduced expression of mip1 indicates that Mip1 is not required for efficient transcription but only for replication. Functional differences between the M. brunneum Mip1 and its counterparts from Saccharomyces cerevisiae and higher eukaryotes are discussed.

No interaction of fluctuating or constant temperature and virulence of entomopathogenic fungi in two noctuid species

AbstractThe potential interaction of fluctuating temperature and the virulence of entomopathogens has implications for biological control. The objective of this study was to investigate the effect of entomopathogenic fungi (Metarhizium brunneum, Beauveria bassiana) on noctuid pest caterpillars (Spodoptera littoralis, Heliothis virescens) under constant and fluctuating temperature regimes. The results revealed similar virulence of entomopathogenic fungi under fluctuating temperature (20–30 °C or 15–35 °C) compared with constant temperature (25 °C). Experiments with cotton leaves as food confirmed that S. littoralis was less susceptible to entomopathogenic fungi (M. brunneum) than H. virescens. Results of additional experiments with H. virescens larvae, B. bassiana, and artificial diet were comparable to experiments with M. brunneum and cotton leaves, despite that susceptibility to B. bassiana was three orders of magnitude lower than to M. brunneum. The fact that both fungus species showed reduced growth on medium under fluctuating temperatures when compared to constant temperatures did not translate to an interaction of temperature regime and the virulence against noctuid larvae. Our study implies that virulence studies with noctuid larvae under constant temperatures using plant material or artificial diet might be fair models also for environments with fluctuating temperatures.

Enhancing Soil Organic Matter Transformation through Sustainable Farming Practices: Evaluating Labile Soil Organic Matter Fraction Dynamics and Identifying Potential Early Indicators

The growing global population and increasing demand for agricultural products have exerted significant pressure on agricultural systems. As a result, soil organic matter depletion and degradation have become prevalent issues, including in regions such as South Bohemia, Czech Republic, where conventional farming practices are predominant. Soil organic matter (SOM) plays a critical role in soil health, crop productivity, and the sustainability of agricultural systems, with changes occurring in both the total and labile fractions of the organic matter pools. However, changes in the total soil organic matter carbon pool (TOC) resulting from agricultural practices occur gradually and may become evident only after several years, posing challenges for timely management adjustments. Therefore, the identification of early indicators of SOM dynamics is crucial for implementing prompt corrective actions. The aim of this study was to evaluate the effects of sustainable management practices, such as cultivated crops (Pisum sativum and Lupinus albus), selected entomopathogenic and myco parasitic fungi (MEHA) (Trichoderma virens and Metarhizium brunneum), and lactic acid bacteria (LAB) on the labile fraction of the SOM pool (CLSOM) and identify potential early indicators. Our findings demonstrated that the type of crop and applied microorganisms (treatments) significantly affected the CLSOM in peas, and the crop growth stages affected the TOC in both pea and lupin. Growth stages also showed an impact on the CLSOM in lupin. Moreover, in both crops, the change in CLSOM correlated with changes in the SOM oxidation rate constant (k), carbon lability index (LI), carbon management index (CMI), and carbon enrichment ratio (ER). Conversely, changes in the TOC did not exhibit significant correlations, except for LI and CMI, which showed a positive correlation with the TOC in peas (p < 0.05). Furthermore, the separate application of MEHA and LAB on seeds or leaves resulted in increased SOM carbon pools compared with the combined application. The application of these beneficial microorganisms in pea and lupin crops showed potential in maintaining or increasing CLSOM, which can be assessed early through indicators such as k, LI, CMI, and ER. Contributing to the development of sustainable soil management strategies, future research should further investigate different crops and microorganisms—and the mechanisms underlying their observed relationships—and explore additional early indicators to refine and optimize sustainable agricultural practices.

Behavioral response of three subterranean pests (Agriotes lineatus, Diabrotica virgifera virgifera, Phyllopertha horticola) to the fungal volatile organic compounds 1-octen-3-ol and 3-octanone

The volatile organic compounds (VOCs) 1-octen-3-ol and 3-octanone produced by the entomopathogenic fungus Metarhizium brunneum are known to have pesticidal properties at high doses against a range of invertebrate pests. Very little is known about their behavior-modifying (semiochemical) properties. This study focused on investigating the behavioral responses of three subterranean crop pests, wireworm (Agriotes lineatus), western corn rootworm (Diabrotica virgifera virgifera), and garden chafer (Phyllopertha horticola), to relatively low doses of 1-octen-3-ol and 3-octanone. The behavior of wireworms and corn rootworms were slightly influenced by the VOCs, yet not significantly. Western corn rootworms appeared to be slightly attracted by 100 µl and 200 µl 1-octen-3-ol and 100 µl dose of 3-octanone, respectively but slightly repelled by the higher dose of 3-octanone. Wireworms appeared to be slightly repelled by 1-octen-3-ol and high dose 3-octanone, but slightly attracted by the 100 µl dose of 3-octanone. The VOCs had no significant impact on garden chafer. In silico studies showed that corn rootworm odorant binding proteins (OBPs) had a strong binding affinity of 1-octen-3-ol and high dose 3-octanone, indicating that these VOCs can be detected and recognized by corn rootworm. OBPs are well conserved between species; thus, wireworm and garden chafer OBPs should also be able to bind with the VOCs. Further trials will be done to confirm that VOCs could be used as semiochemicals. Appropriate formulation of the VOCs should increase their efficacy and prevent rapid dissipation of the VOCs.

Effects of tomato inoculation with the entomopathogenic fungus Metarhizium brunneum on spider mite resistance and the rhizosphere microbial community.

Entomopathogenic fungi have been well exploited as biocontrol agents that can kill insects through direct contact. However, recent research has shown that they can also play an important role as plant endophytes, stimulating plant growth, and indirectly suppressing pest populations. In this study, we examined the indirect, plant-mediated, effects of a strain of entomopathogenic fungus, Metarhizium brunneum on plant growth and population growth of two-spotted spider mites (Tetranychus urticae) in tomato, using different inoculation methods (seed treatment, soil drenching and a combination of both). Furthermore, we investigated changes in tomato leaf metabolites (sugars and phenolics), and rhizosphere microbial communities in response to M. brunneum inoculation and spider mite feeding. A significant reduction in spider mite population growth was observed in response to M. brunneum inoculation. The reduction was strongest when the inoculum was supplied both as seed treatment and soil drench. This combination treatment also yielded the highest shoot and root biomass in both spider mite-infested and non-infested plants, while spider mite infestation increased shoot but reduced root biomass. Fungal treatments did not consistently affect leaf chlorogenic acid and rutin concentrations, but M. brunneum inoculation via a combination of seed treatment and soil drenching reinforced chlorogenic acid (CGA) induction in response to spider mites and under these conditions the strongest spider mite resistance was observed. However, it is unclear whether the M. brunneum-induced increase in CGA contributed to the observed spider mite resistance, as no general association between CGA levels and spider mite resistance was observed. Spider mite infestation resulted in up to two-fold increase in leaf sucrose concentrations and a three to five-fold increase in glucose and fructose concentrations, but these concentrations were not affected by fungal inoculation. Metarhizium, especially when applied as soil drench, impacted the fungal community composition but not the bacterial community composition which was only affected by the presence of spider mites. Our results suggest that in addition to directly killing spider mites, M. brunneum can indirectly suppress spider mite populations on tomato, although the underlying mechanism has not yet been resolved, and can also affect the composition of the soil microbial community.

From lab to field: biological control of the Japanese beetle with entomopathogenic fungi.

The Japanese beetle, Popillia japonica, is an invasive scarab and listed as quarantine organism in many countries worldwide. Native to Japan, it has invaded North America, the Azores, and recently mainland Europe. Adults are gregarious and cause agricultural and horticultural losses by feeding on leaves, fruits, and flowers of a wide range of crops and ornamental plants. Larvae feed belowground and damage grassland. To date, no efficient and environmentally friendly control measure is available. Larval populations of other scarab species such as Phyllopertha horticola and Melolontha melolontha are controlled by applying spores of the entomopathogenic fungi Metarhizium brunneum and Beauveria brongniartii to larval habitats. Here, we tested this control strategy against Japanese beetle larvae in grasslands, as well as spore spray applications against adults in crops. Using both, large-scale field experiments and inoculation experiments in the laboratory, we assess the efficacy of registered fungal strains against Japanese beetle larvae and adults. Metarhizium brunneum BIPESCO 5 established and persisted in the soil of larval habitats and on the leaves of adult's host plants after application. However, neither larval nor adult population sizes were reduced at the study sites. Laboratory experiments showed that larvae are not susceptible to M. brunneum ART 212, M. brunneum BIPESCO 5, and B. brongniartii BIPESCO 2. In contrast, adults were highly susceptible to all three strains. When blastospores were directly injected into the hemolymph, both adults and larvae showed elevated mortality rates, which suggests that the cuticle plays an important role in determining the difference in susceptibility of the two life stages. In conclusion, we do not see potential in adapting the state-of-the-art control strategy against native scarabs to Japanese beetle larvae. However, adults are susceptible to the tested entomopathogenic fungi in laboratory settings and BIPESCO 5 conidiospores survived for more than three weeks in the field despite UV-radiation and elevated temperatures. Hence, control of adults using fungi of the genera Beauveria or Metarhizium is more promising than larval control. Further research on efficient application methods and more virulent and locally adapted fungal strains will help to increase efficacy of fungal treatments for the control of P. japonica.

When Competitors Join Forces: Consortia of Entomopathogenic Microorganisms Increase Killing Speed and Mortality in Leaf- and Root-Feeding Insect Hosts.

Combining different biocontrol agents (BCA) is an approach to increase efficacy and reliability of biological control. If several BCA are applied together, they have to be compatible and ideally work together. We studied the interaction of a previously selected BCA consortium of entomopathogenic pseudomonads (Pseudomonas chlororaphis), nematodes (Steinernema feltiae associated with Xenorhabdus bovienii), and fungi (Metarhizium brunneum). We monitored the infection course in a leaf- (Pieris brassicae) anda root-feeding (Diabrotica balteata) pest insect after simultaneous application of the three BCA as well as their interactions inside the larvae in a laboratory setting. The triple combination caused the highest mortality and increased killing speed compared to single applications against both pests. Improved efficacy against P. brassicae was mainly caused by the pseudomonad-nematode combination, whereas the nematode-fungus combination accelerated killing of D. balteata. Co-monitoring of the three BCA and the nematode-associated Xenorhabdus symbionts revealed that the four organisms are able to co-infect the same larva. However, with advancing decay of the cadaver there is increasing competition and cadaver colonization is clearly dominated by the pseudomonads, which are known for their high competitivity in the plant rhizosphere. Altogether, the combination of the three BCA increased killing efficacy against a Coleopteran and a Lepidopteran pest which indicates that this consortium could be applied successfully against a variety of insect pests.

A novel polymycovirus infecting the entomopathogenic fungus Metarhizium brunneum.

In this study, at least four distinct double-stranded RNA viruses, including two partitiviruses, one bipartite virus, and a novel polymycovirus, were detected in the entomopathogenic fungus Metarhizium brunneum strain RCEF0736. We describe the characterization of one of these viruses, a novel polymycovirus, which we have named "Metarhizium brunneum polymycovirus 1" (MbPmV1). The genome of MbPmV1 has four dsRNA segments, ranging from 1153 to 2421 bp in length. dsRNA1, 2, and 3 of MbPmV1 each contain a single large open reading frame (ORF), while dsRNA4 has two ORFs. BLASTp analysis indicated that dsRNA1, 2, 3, and 4 of MbPmV1 have a high degree of similarity to the putative RdRp (59.45%), serine protease (44.22%), putative methyltransferase (48.76%), and proline-alanine-serine-rich protein (PASrp) (52.80%), respectively, of Phaeoacremonium minimum tetramycovirus 1 (PmTmV1). MbPmV1 was grouped in a cluster with members of the genus Polymycovirus and was most closely related to PmTmV1 in the phylogenetic tree. Thus, we propose that MbPmV1 represents a novel species of the genus Polymycovirus, family Polymycoviridae. To our knowledge, this is the first report of a polymycovirus in a member of the genus Metarhizium.

Molecular characterization of a novel double-stranded RNA virus infecting the entomopathogenic fungus Metarhizium brunneum.

There are four dsRNAs segments present in the entomopathogenic fungus Metarhizium brunneum strain RCEF0766. The genomic segments dsRNA1 and dsRNA3 are of a novel virus, "Metarhizium brunneum bipartite mycovirus 1" (MbBV1), while dsRNA2 and dsRNA4 are the components of the Metarhizium brunneum partitivirus 2 (MbPV2), a member in genus Gammapartitivirus of the family Partitiviridae based on molecular analysis and RT-PCR. This suggests that the strain RCEF0766 was co-infected by two different mycoviruses. The complete genome sequence of MbBV1 was elucidated by high-throughput sequencing and RLM-RACE. MbBV1 consists of two dsRNAs (1987 and 1642bp) encode open-reading frames (ORFs). The ORF1 in dsRNA 1 encode is a putative RNA-dependent RNA polymerase (RdRp) with the molecular weight of 68.08kDa, while ORF2 in dsRNA 2 encodes a hypothetical protein with the molecular weight of 33.07kDa. The deduced proteins of ORF1 and ORF2 have the highest identity to those of Erysiphe necator-associated bipartite virus 1 (76.88% and 65.30%). Based on the amino acid sequence of RdRp, MbBV1 is phylogenetically clustered together with the unassigned mycoviruses and represents a distinct lineage. Our study proposes that MbBV1 is a novel mycovirus with bisegmented dsRNA genomes and should be considered a new member of the unassigned group.

Stress-Mediated Responses of Aedes aegypti (Diptera: Culicidae) Larvae When Exposed to Metarhizium brunneum (Hypocreales: Clavicipitaceae) and Toxorhynchites brevipalpis (Diptera: Culicidae)

Aedes aegypti mosquitoes are capable of vectoring a wide range of diseases including dengue, yellow fever, and Zika viruses, with approximately half of the worlds’ population at risk from such diseases. Development of combined predator–parasite treatments for the control of larvae consistently demonstrates increased efficacy over single-agent treatments, however, the mechanism behind the interaction remains unknown. Treatments using the natural predator Toxorhynchites brevipalpis and the entomopathogenic fungus Metarhizium brunneum were applied in the laboratory against Ae. aegypti larvae as both individual and combined treatments to determine the levels of interaction between control strategies. Parallel experiments involved the removal of larvae from test arenas at set intervals during the course of the trial to record whole body caspase and phenoloxidase activities. This was measured via luminometric assay to measure larval stress factors underlying the interactions. Combined Metarhizium and Toxorhynchites treatments were seen to drastically reduce lethal times as compared to individual treatments. This was accompanied by increased phenoloxidase and caspase activities in combination treatments after 18 h (p < 0.001). The sharp increases in caspase and phenoloxidase activities suggest that combined treatments act to increase stress factor responses in the larvae that result in rapid mortality above that of either control agent individually. This work concludes that the underlying mechanism for increased lethality in combined parasite–predator treatments may be related to additive stress factors induced within the target host larvae.

Efficacy of Metarhizium brunneum and Beauveria bassiana isolates against the European tent caterpillar, Malacosoma neustria Linnaeus, 1758 (Lepidoptera: Lasiocampidae)

BackgroundThe European tent caterpillar Malacosoma neustria Linnaeus, 1758 (Lepidoptera: Lasiocampidae), a worldwide pest, feeds on a wide variety of woody and shrub-like plants in its larval stage and causes extensive economic losses. In the fight against this species, environmentally friendly biological control methods should be preferred instead of chemical control. The aim of this study was to evaluate the efficacy of entomopathogenic fungi (EPF) Metarhizium brunneum (ORP-13) and Beauveria bassiana (GOPT-301-2) isolates against the fourth instar larvae of M. neustria under laboratory conditions.ResultsM. neustria eggs were collected from the Kızılırmak Delta of Samsun Province, Turkey, and the fourth instar larvae were used in the experiment. Larvae in the control group were fed on sterilized leaves of Eleagnus rhamnoides. Both fungal isolates were applied onto the larvae at 2 ml for each concentration (1 × 106, 1 × 107, and 1 × 108 conidia ml−1). Ten larvae were placed in each group, and sterilized E. rhamnoides leaves were offered to the larvae. The study was carried out in 9 replicates for each group, and the larvae were observed for 14 days. As a result of the study, it was found that the survival rates of the larvae decreased as concentration increased. It was determined that both isolates caused 100% mortality at 1 × 108 conidia ml−1 concentration. The lowest LC50 value was found in larvae exposed to the ORP-13 isolate.ConclusionIt has been suggested that M. brunneum and B. bassiana isolates were virulent for M. neustria larvae and can be used for biological control of this species.

Fumigation of three major soil pests (Agriotes lineatus, Diabrotica virgifera virgifera, Phyllopertha horticola) with 3-octanone and 1-octen-3-ol enantiomers

ABSTRACT New pest management solutions are needed to control soil invertebrates (insects, nematodes, mollusks) in order to implement the goals of the European Green Deal. Natural volatile organic compounds (VOCs) such as 1-octen-3-ol and 3-octanone, emitted by the entomopathogenic fungus Metarhizium brunneum could be part of the solution. Three major crop pests, Agriotes lineatus (wireworm), Diabrotica virgifera virgifera (corn rootworm) and Phyllopertha horticola (garden chafer), were susceptible to fumigation with 1-octen-3-ol and 3-octanone. The toxicity of the VOCs was tested in soil-free arenas and in soils which differed in moisture content and porosity. The mortality rates were dependent on the interaction between the VOC treatment, dose and pest species. The insects differed in their sensitivity to these VOCs. A dose of 1.25 µl of 1-octen-3-ol applied in a closed glass tube was sufficient to kill D. v. virgifera and P. horticola in soil trials whereas 5 µl was needed to kill A. lineatus. The highest dose (20 µl) was highly toxic to all insects. Soil moisture content slightly influenced mortality rates whereas porosity had no obvious impact. The mode of action of the VOCs is unknown but the compounds are likely to cause tissue damage and loss of body fluids. This may explain the shrivelled appearance of corn rootworm and garden chafer and melanisation in wireworm. Both 1-octen-3-ol and 3-octanone show promise as biofumigants.

Antimicrobial Volatiles of the Insect Pathogen Metarhizium brunneum.

Fungal volatile organic compounds (VOCs) represent promising candidates for biopesticide fumigants to control crop pests and pathogens. Herein, VOCs produced using three strains of the entomopathogenic fungus Metarhizium brunneum were identified via GC-MS and screened for antimicrobial activity. The VOC profiles varied with fungal strain, development state (mycelium, spores) and culture conditions. Selected VOCs were screened against a range of rhizosphere and non-rhizosphere microbes, including three Gram-negative bacteria (Escherichia coli, Pantoea agglomerans, Pseudomonas aeruginosa), five Gram-positive bacteria (Micrococcus luteus, Staphylococcus aureus, Bacillus subtilis, B. megaterium, B. thuringiensis), two yeasts (Candida albicans, Candida glabrata) and three plant pathogenic fungi (Pythium ultimum, Botrytis cinerea, Fusarium graminearum). Microbes differed in their sensitivity to the test compounds, with 1-octen-3-ol and isovaleric acid showing broad-spectrum antimicrobial activity. Yeasts and bacteria were inhibited by the same VOCs. Cryo-SEM showed that both yeasts and bacteria underwent some form of “autolysis”, where all components of the cell, including the cell wall, disintegrated with little evidence of their presence in the clear, inhibition zone. The oomycete (P. ultimum) and ascomycete fungi (F. graminearum, B. cinerea) were sensitive to a wider range of VOCs than the bacteria, suggesting that eukaryotic microbes are the main competitors to M. brunneum in the rhizosphere. The ability to alter the VOC profile in response to nutritional cues may assist M. brunneum to survive among the roots of a wide range of plant species. Our VOC studies provided new insights as to how M. brunneum may protect plants from pathogenic microbes and correspondingly promote healthy growth.

A fungus infected environment does not alter the behaviour of foraging ants

Eusocial insects are exposed to a wide range of pathogens while foraging outside their nest. We know that opportunistic scavenging ants are able to assess the sanitary state of food and to discriminate a prey which died from infection by the entomopathogenic fungus Metarhizium brunneum. Here, we investigate whether a contamination of the environment can also influence the behaviour of foragers, both at the individual and collective level. In a Y-maze, Myrmica rubra ants had the choice to forage on two prey patches, one of which containing sporulating items. Unexpectedly, the nearby presence of sporulating bodies did not deter foragers nor prevent them from retrieving palatable prey. Ant colonies exploited both prey patches equally, without further mortality resulting from foraging on the contaminated area. Thus, a contamination of the environment did not prompt an active avoidance by foragers of which the activity depended primarily on the food characteristics. Generalist entomopathogenic fungi such as M. brunneum in the area around the nest appear more to be of a nuisance to ant foragers than a major selective force driving them to adopt avoidance strategies. We discuss the cost–benefit balance derived from the fine-tuning of strategies of pathogen avoidance in ants.

Preventive field application of Metarhizium brunneum in cover crops for wireworm control

Wireworms, the larvae of click beetles (Coleoptera: Elateridae), can cause substantial losses in marketable yield of potatoes, yet control options are limited. The entomopathogenic fungus Metarhizium brunneum (Ascomycota; Hypocreales) isolate ART2825 is highly virulent against two of the most detrimental wireworm species, Agriotes obscurus L. and A. lineatus L., but field application of this isolate during potato cultivation has never succeeded. In this study, we integrated the fungus into the agricultural crop rotation prior to potato cultivation, with the aim of better adapting the application strategy to the fungus’ ecological and environmental requirements. Application preceded sowing of cover crops in late summer. We hypothesized that higher temperatures and undisturbed development for several months would support the establishment of the entomopathogen and enhance biocontrol efficiency in the following season. In two subsequent seasons, we quantified (1) fungal establishment in the soil, (2) efficiency of treated soils against wireworms in vitro, and (3) levels of wireworm damage in field potatoes. Spore concentration was enhanced in treated plots and we recovered the released Metarhizium isolate from all mycosed, field-collected wireworms. Treated soils increased wireworm mortality in the laboratory, but a statistically significant reduction of potato damage was only achieved in two out of ten field trials. The application strategy shows potential for fungal enhancement and opens new avenues for biological wireworm control.

Susceptibility of Agriotes spp. larvae (Coleoptera: Elateridae) to stress-and-kill strategies using spinosad and the entomopathogenic fungus Metarhizium brunneum

Susceptibility of Agriotes spp. larvae (Coleoptera: Elateridae) to stress-and-kill strategies using spinosad and the entomopathogenic fungus Metarhizium brunneum

RNAi-mediated suppression of insect metalloprotease inhibitor (IMPI) enhances Galleria mellonella susceptibility to fungal infection

The co-evolutionary arms race between disease-causing agents and their insect victims is ancient and complex – leading to the development of specialised attack and defence strategies. Among such strategies is the capacity of fungal and oomycete pathogens to deploy degradative enzymes, notably proteases, to facilitate infection directly across the integument. To counter these proteases, insects such as the greater wax moth Galleria mellonella release metalloprotease inhibitors and other immune factors to thwart the invading fungus. To date, molecular-based confirmation of insect metalloprotease inhibitor's incontrovertible role in antifungal defence has been lacking. We targeted the IMPI gene for suppression using RNAi and exposed those insects to the entomopathogenic fungus Metarhizium brunneum ARSEF4556. Levels of IMPI were reduced significantly in the integument (10-fold) and fat body (5-fold) of RNAi-treated insects when compared to control larvae, and displayed a significantly higher mortality rate. We also surveyed candidate immune/detoxification gene expression levels (e.g., DOPA decarboxylase, galiomycin) in three tissues (integument, midgut, fat body) in order to gauge any potential non-target effects of RNAi. The loss of IMPI via RNAi compromises antifungal defences and leaves G. mellonella vulnerable to infection.

A novel gammapartitivirus from the entomopathogenic fungus Metarhizium brunneum.

Here, we report a novel partitivirus infecting Metarhizium brunneum, which was designated "Metarhizium brunneum partitivirus 2" (MbPV2). The complete genome of MbPV2 consists of two segments, dsRNA1 and dsRNA2, with each dsRNA possessing a single open reading frame (ORF). dsRNA1 (1,775 bp) encodes a conserved RNA-dependent RNA polymerase (RdRp) with the highest sequence similarity to Plasmopara viticola associated partitivirus 1 (PvAPV1), while dsRNA2 (1,568 bp) encodes a coat protein (CP) with the highest sequence similarity to Colletotrichum partitivirus 1 (CtParV1). Phylogenetic analysis based on RdRp sequences showed that MbPV2 is a new member of the genus Gammapartitivirus, family Partitiviridae. This is the first report of a gammapartitivirus that infects the entomopathogenic fungus Metarhizium brunneum.

Volatile organic compounds of Metarhizium brunneum influence the efficacy of entomopathogenic nematodes in insect control

The entomopathogenic fungus (EPF) Metarhizium brunneum occupies the same ecological niche as entomopathogenic nematodes (EPN), with both competing for insects as a food source in the rhizosphere. Interactions between these biocontrol agents can be antagonistic or synergistic. To better understand these interactions, this study focussed on investigating the effect of M. brunneum volatile organic compounds (VOCs), 1-octen-3-ol and 3-octanone, on EPN survival and behaviour. These VOCs proved to be highly toxic to the infective juveniles (IJs) of the EPN Steinernema carpocapsae, Steinernema feltiae and Heterorhabditis bacteriophora with mortality being dose dependent. Chemotaxis studies of H. bacteriophora IJs in Pluronic F127 gel revealed significant preference for the VOCs compared with controls for all tested concentrations. The VOCs also impacted on the test insects in a dose-dependent manner with 3-octanone being more toxic to Galleria mellonella, Cydia splendana and Curculio elephas larvae than 1-octen-3-ol. Mortality of C. splendana and G. mellonella larvae was significantly higher when exposed to relatively high doses (>25%) of 3-octanone. Lower doses of 3-octanone and 1-octen-3-ol immobilised test insects, which recovered after exposure to fresh air for 2 hrs. In depth studies on H. bacteriophora showed that exposure of IJs to > 10% concentration of 3-octanone or 1-octen-3-ol negatively affected infectivity whereas exposure to lower doses (0.1%, 0.01%) had no effect. The VOCs affected IJs, reducing penetration efficacy and the number of generations inside G. mellonella but they failed to inhibit the bacterial symbiont, Photorhabdus kayaii. The ecological significance of VOCs and how they could influence EPF-EPN insect interactions is discussed.

Development of a fluid-bed coating process for soil-granule-based formulations of Metarhizium brunneum, Cordyceps fumosorosea or Beauveria bassiana.

Granule-based products of solid state fermented micro-organisms are available for biocontrol. Because liquid fermentation has several advantages, we investigated fluid-bed coating with liquid fermented biomass. Biomass containing mycelium or mycelium and submerged spores of the entomopathogenic fungi Metarhizium brunneum, Cordyceps fumosorosea and Beauveria bassiana were produced in liquid culture, separated and different biomass concentrations were adjusted. Based on the examined thermo-tolerance, we defined fluid-bed coating adjustments and investigated granule colonization and sporulation on granules. Granule colonization depended on the biomass concentration and strain. For C. fumosorosea and B. bassiana, concentrations of 0·003%dry weight resulted in nearly 100% granule colonization, for M. brunneum with concentrations of 0·7%dry weight in only 50%. The conidiation on granules in sterile soil was highly influenced by the moisture content. Because the granule colonization of M. brunneum was unsatisfactory, we pre-coated nutrients followed by coating with biomass, submerged spores or conidia. Malt extract had a positive effect on the granule colonization for biomass and submerged spores. Furthermore, aerial conidia can also be coated. Fluid-bed coating of fungal biomass is suitable for the development of granules. With this technology, cost-efficient biocontrol products can be developed.