Effect of CO2 Concentrations on Entomopathogen Fitness and Insect-Pathogen Interactions.

Numerous insect species and their associated microbial pathogens are exposed to elevated CO2 concentrations in both artificial and natural environments. However, the impacts of elevated CO2 on the fitness of these pathogens and the susceptibility of insects to pathogen infections are not well understood. The yellow mealworm, Tenebrio molitor, is commonly produced for food and feed purposes in mass-rearing systems, which increases risk of pathogen infections. Additionally, entomopathogens are used to control T. molitor, which is also a pest of stored grains. It is therefore important to understand how elevated CO2 may affect both the pathogen directly and impact on host-pathogen interactions. We demonstrate that elevated CO2 concentrations reduced the viability and persistence of the spores of the bacterial pathogen Bacillus thuringiensis. In contrast, conidia of the fungal pathogen Metarhizium brunneum germinated faster under elevated CO2. Pre-exposure of the two pathogens to elevated CO2 prior to host infection did not affect the survival probability of T. molitor larvae. However, larvae reared at elevated CO2 concentrations were less susceptible to both pathogens compared to larvae reared at ambient CO2 concentrations. Our findings indicate that whilst elevated CO2 concentrations may be beneficial in reducing host susceptibility in mass-rearing systems, they may potentially reduce the efficacy of the tested entomopathogens when used as biological control agents of T. molitor larvae. We conclude that CO2 concentrations should be carefully selected and monitored as an additional environmental factor in laboratory experiments investigating insect-pathogen interactions.

Entomopathogenic Fungus-Related Priming Defense Mechanisms in Cucurbits Impact Spodoptera littoralis (Boisduval) Fitness.

Entomopathogenic fungi (EPF) exhibit direct and indirect mechanisms to increase plant resistance against biotic and abiotic stresses. Plant responses to these stresses are interconnected by common regulators such as ethylene (ET), which is involved in both iron (Fe) deficiency and induced systemic resistance responses. In this work, the roots of cucurbit seedlings were primed with Metarhizium brunneum (EAMa 01/58-Su strain), and relative expression levels of 18 genes related to ethylene (ET), jasmonic acid (JA), and salicylic acid (SA) synthesis, as well as pathogen-related (PR) protein genes, were studied by reverse transcription-quantitative PCR (qRT-PCR). Effects of priming on Spodoptera littoralis were studied by feeding larvae for 15 days with primed and control plants. Genes showed upregulation in studied species; however, the highest relative expression was observed in roots and shoots of plants with Fe deficiency, demonstrating the complexity and the overlapping degree of the regulatory network. EIN2 and EIN3 should be highlighted; both are key genes of the ET transduction pathway that enhanced their expression levels up to eight and four times, respectively, in shoots of primed cucumber. Also, JA and SA synthesis and PR genes showed significant upregulation during the observation period (e.g., the JA gene LOX1 increased 506 times). Survival and fitness of S. littoralis were affected with significant effects on mortality of larvae fed on primed plants versus controls, length of the larval stage, pupal weight, and the percentage of abnormal pupae. These results highlight the role of the EAMa 01/58-Su strain in the induction of resistance, which could be translated into direct benefits for plant development. IMPORTANCE Entomopathogenic fungi are multipurpose microorganisms with direct and indirect effects on insect pests. Also, EPF provide multiple benefits to plants by solubilizing minerals and facilitating nutrient acquisition. A very interesting and novel effect of these fungi is the enhancement of plant defense systems by inducing systematic and acquired resistance. However, little is known about this function. This study sheds light on the molecular mechanisms involved in cucurbits plants' defense activation after being primed by the EPF M. brunneum. Furthermore, the subsequent effects on the fitness of the lepidopteran pest S. littoralis are shown. In this regard, a significant upregulation was recorded for the genes that regulate JA, SA, and ET pathways. This increased expression of defense genes caused lethal and sublethal effects on S. littoralis. This could be considered an added value for the implementation of EPF in integrated pest management programs.

Elucidating the Effect of Endophytic Entomopathogenic Fungi on Bread Wheat Growth through Signaling of Immune Response-Related Hormones.

Entomopathogenic fungi (EF) provide a potent biocontrol tool; also, their endophytic behavior has broadened their contribution to integrated pest management (IPM) and crop production. In this work, Beauveria bassiana and Metarhizium brunneum were applied to bread wheat (Triticum aestivum) seedlings to elucidate how fungal colonization influences plant growth and the relative expression of 24 genes involved in hormonal syntheses and plant immune mechanisms. A preliminary assay was used to determine the time needed for fungal colonization and assess its effect on wheat growth. Then, plant material collected at various times after inoculation (viz., 2, 8, 20, and 36 h and 9 and 15 days) was used to investigate gene expression by quantitative reverse transcription PCR (RT-qPCR). During the colonization time, B. bassiana and M. brunneum caused strong downregulation of most genes associated with plant immunity and the synthesis of hormones like auxin, cytokinin, and gibberellin. This effect was concomitant with a slowdown of endophytic-colonization-related plant growth until 19 days postinoculation (dpi). However, the wheat started to recover at 15 dpi, simultaneously with upregulation of auxin- and gibberellin-related genes. The results suggest that the EF trigger induced systemic resistance rather than acquired systemic resistance during early plant-microbe cross talk in wheat. Also, they confirm that the hormone and immune responses of wheat triggered by EF inoculation influenced plant growth, which can be useful with a view to optimizing management of these microorganisms for sustainable agriculture. IMPORTANCE Microbial control of insect and mite pests is a key tool to develop integrated pest management (IPM) and sustainable agriculture. Entomopathogenic fungi (EF) may have associations with the plants, playing additional ecological roles in the rhizosphere, in the phylloplane, and as plant endophytes. Beauveria bassiana 04/01TIP and Metarhizium brunneum 01/58Su are two strains that showed very good results either in pest control or plant growth promotion and would be good candidates to develop mycoinsecticides as an alternative to pesticides. However, deep knowledge about their interaction with the plant would let farmers optimize their use and understand the plant response, enhancing and promoting their broader contribution to IPM and crop production.

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 1642 bp) encode open-reading frames (ORFs). The ORF1 in dsRNA 1 encode is a putative RNA-dependent RNA polymerase (RdRp) with the molecular weight of 68.08 kDa, while ORF2 in dsRNA 2 encodes a hypothetical protein with the molecular weight of 33.07 kDa. 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.

Mortality of various-age larval winter ticks, Dermacentor albipictus, following surface contact with entomopathogenic fungi.

The larval stage of the one-host tick, Dermacentor albipictus Packard (Acari: Ixodidae), the winter tick, is spent aggregated on the ground in summer until they quest for hosts in the autumn. Metarhizium brunneum (Petch) and Beauveria bassiana (Balsamo-Crivelli) Vuillemin are promising fungal biocontrol agents of ticks. The success of tick biocontrol using entomopathogenic fungi relies on infective propagule contact with a susceptible host at the right time within their life cycle and stage. The susceptibility of various-age D. albipictus larvae to commercial isolates of M. brunneum strain F52 (Mb-F52) and B. bassiana strain GHA (Bb-GHA) was assessed over a three-week period after contact with surfaces treated with 1.3 × 106 conidia/cm2. Larvae of four ages were examined (a) upon eclosion from eggs, (b) 2-weeks, (c) 1.5-months and (d) 3-months-old. Mortality of larvae exposed to fungus-treated surfaces ranged from 4 to 51% for Bb-GHA and 64-100% for Mb-F52 after three weeks. Significantly greater mortality was observed when larvae were exposed to Mb-F52 than Bb-GHA. Larval susceptibility was significantly greater within 2 weeks of eclosion from eggs and after 3 months of age. These results demonstrate the varying susceptibility of larvae to mycoacaricides at different times within their non-parasitic larval life stage.

Evaluation of the entomopathogenic fungus Metarhizium brunneum and the predatory mite Stratiolaelaps scimitus against Rhizoglyphus robini under laboratory conditions.

Rhizoglyphus robini Claparède (Acari: Acaridae) is a pest of bulbs, corms and tubers of several economically important crops. The biological control of R. robini has yet to be fully explored as an alternative to chemical pesticides. Entomopathogenic fungi in the genera Metarhizium (Hypocreales: Clavicipitaceae) are used for the biological control of several agricultural pests. The soil-dwelling predatory mite, Stratiolaelaps scimitus (Womersley) (Acari: Acaridae) is also frequently used alone or in combination with other biological control agents. There are few reports on the use of M. brunneum or S. scimutus against R. robini. The objectives of this research were to investigate the in vitro effect of different predatory mite ratios of S. scimitus on R. robini mortality and the combined use of a M. brunneum-based granule with S. scimitus as potential strategies to manage this pest. Mortality of R. robini in Petri dishes containing predators was significantly higher than without predators. When soil-filled containers containing R. robini were treated with both M. brunneum granules and S. scimitus, the lower densities of the bulb mite were obtained with the highest ratio of predator/prey mites. The number of bulb mites in the containers treated with only M. brunneum was significantly lower than the untreated control. These results demonstrate the potential for releasing of S. scimitus alone and in combination with M. brunneum granules to manage R. robini.

Comparative Study of Larvicidal Activity of Spinel Co3O4 Nanorods and Entomopathogenic Metarhizium brunneum Conidia against Culex pipiens.

Herein, we report the synthesis of spinel cobalt oxide nanorods (Co3O4 NRs) by a modified co-precipitation approach and examine their larvicidal activity against Culex pipiens. The structure and morphology of the as-prepared Co3O4 NRs were emphasized using X-ray diffraction (XRD), Raman spectroscopy, energy dispersive X-ray spectroscopy (EDAX), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). It was found that Co3O4 nanostructures have a face-centered spinel cubic crystal structure with a mean crystallite size of 38 nm. These nanostructures have a rod like shape with a mean diameter of 30 nm and an average length of 60 nm. The TGA measurements revealed the high stability of the formed spinel cubic structure at 400 °C. The optical behavior indicates the direct transition of electrons through an optical band gap in the range of 2.92-3.08 eV. These unique chemical and physical properties of Co3O4 NRs enabled them to be employed as a strong agent for killing the C. pipiens. A comparison study was employed between the as-prepared Co3O4 and the entomopathogenic fungus Metarhizium brunneum as a control agent of C. pipiens larvae. The results revealed that the as-prepared nanorods have higher mortality against C. pipiens larvae compared with the well-known M. brunneum.

Entomopathogenic fungi tested in planta on pepper and in field on sorghum, to control commercially important species of aphids.

Given the aphids high reproductive capacity, assessing their biocontrol by using entomopathogenic fungi is crucial; to determine their potential, fungi were tested in planta and in field conditions. Significant decrease of Myzus persicae (Sulzer) population was observed in planta after applying Beauveria bassiana (strain 7R), Trichoderma gamsii (strain Z) or Metarhizium brunneum (strain Meta Br1) at 1 × 107 or 1 × 108 conidia/mL on pepper plants. Significant differences of aphids' populations were detected between fungus concentration and control (F = 68.743, df = 6.980, P < 0.001), where M. brunneum at 1 × 108 conidia/mL reduced aphids population close to zero. At 20 °C, dead aphids' mycosis by B. bassiana and T. gamsii was 78% and 84%; at 25 °C was 83% and 88%; and at 30 °C was 75% and 79%, respectively. In field conditions, Mexican PTG4 and commercial GHA B. bassiana strains were tested [(1 × 106 conidia/mL + corn starch) seed treatments] against the Melanaphis sacchari (Zehntner) aphid populations, on naturally infested sorghum plants. Results showed that plant germination and emergence were not affected, whereas yield (grams of sugar/plant) was significantly higher among treated compared with untreated plants. The aphid population decreased in plants from PTG4 treated seeds; indeed, this treatment had a significant positive effect on the flowering index, whereas the stem fresh weight and juice volume was significantly increased among plants from GHA treated seeds. Taken together, tested strains can be used as a tool to control aphids' population on several crops such as pepper and even increase the yield in sorghum.

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

AIM: 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. METHODS AND RESULTS: 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. CONCLUSIONS: Fluid-bed coating of fungal biomass is suitable for the development of granules. SIGNIFICANCE AND IMPACT OF THIS STUDY: With this technology, cost-efficient biocontrol products can be developed.

Induction and Priming of Plant Defense by Root-Associated Insect-Pathogenic Fungi.

Plants evolved in close contact with a myriad of microorganisms, some of which formed associations with their roots, benefitting from carbohydrates and other plant resources. In exchange, they evolved to influence important plant functions, e.g. defense against insect herbivores and other antagonists. Here, we test whether a fungus, Metarhizium brunneum, which is mostly known as an insect pathogen, can also associate with plant roots and contribute to above-ground plant defense. Cauliflower (Brassica oleracea var. botrytis) seeds were sown together with M. brunneum-inoculated rice grains, and the resulting plants subjected to leaf herbivory by the specialist Plutella xylostella. Activity of myrosinases, the enzymes activating glucosinolates, was measured before and after herbivory; larval consumption and plant weight at the end of experiments. Metarhizium brunneum clearly established in the plant roots, and after herbivory myrosinase activity was substantially higher in M. brunneum-treated plants than in controls; before herbivory, M. brunneum-treated and control plants did not differ. Leaf consumption was slightly lower in the M. brunneum-treated plants whereas total biomass and allocation to above- or below-ground parts was not affected by the Metarhizium treatment. Thus, M. brunneum associates with roots and primes the plant for a stronger or faster increase in myrosinase activity upon herbivory. Consistent with this, myrosinase function has been suggested to be rate-limiting for induction of the glucosinolate-myrosinase defense system. Our results show that M. brunneum, in addition to being an insect pathogen, can associate with plant roots and prime plant defense.

Isolate-Specific Effect of Entomopathogenic Endophytic Fungi on Population Growth of Two-Spotted Spider Mite (Tetranychus urticae Koch) and Levels of Steroidal Glycoalkaloids in Tomato.

Entomopathogenic fungi (EPF) can be experimentally established in several plant species as endophytes. Ecological effects of EPF inoculations on plant growth and plant-herbivore interactions have been demonstrated, potentially by altering plant physiological responses. However, the role of these responses in plant-fungus-herbivore tripartite interactions has not been well elucidated. Steroidal glycoalkaloids (SGAs) are plant specialized metabolites with bioactive properties against arthropod herbivores. Here, the effects of seed treatments by three EPF isolates, representing Beauveria bassiana, Metarhizium brunneum, and M. robertsii, on population growth of two-spotted spider mites (Tetranychus urticae Koch) were evaluated on tomato (Solanum lycopersicum). The levels of two SGAs, α-tomatine and dehydrotomatine, were determined in tomato leaves by LC-MS with and without T. urticae infestations after EPF inoculations. Interestingly, the population growth of T. urticae was significantly highest with M. brunneum and lowest with M. robertsii and B. bassiana at 15 days after infestation. Overall there was a significant negative correlation between SGAs content and the number of T. urticae. The levels of SGAs were significantly induced by T. urticae presence in all treatments, while only M. robertsii showed significantly higher levels of SGAs than M. brunneum and control in one of two experiments. Contrastingly, the effects on SGAs accumulation and population growth of T. urticae did not directly correlate with EPF endophytic colonization patterns of the inoculated plants. This study suggests a link between ecological effects and physiological responses mediated by EPF inoculations and T. urticae infestation with potential implications for plant protection.

Root-Associated Entomopathogenic Fungi Modulate Their Host Plant's Photosystem II Photochemistry and Response to Herbivorous Insects.

The escalating food demand and loss to herbivores has led to increasing interest in using resistance-inducing microbes for pest control. Here, we evaluated whether root-inoculation with fungi that are otherwise known as entomopathogens improves tomato (Solanum lycopersicum) leaflets' reaction to herbivory by Spodoptera exigua (beet armyworm) larvae using chlorophyll fluorescence imaging. Plants were inoculated with Metarhizium brunneum or Beauveria bassiana, and photosystem II reactions were evaluated before and after larval feeding. Before herbivory, the fraction of absorbed light energy used for photochemistry (ΦPSII) was lower in M. brunneum-inoculated than in control plants, but not in B. bassiana-inoculated plants. After herbivory, however, ΦPSII increased in the fungal-inoculated plants compared with that before herbivory, similar to the reaction of control plants. At the same time, the fraction of energy dissipated as heat (ΦNPQ) decreased in the inoculated plants, resulting in an increased fraction of nonregulated energy loss (ΦNO) in M. brunneum. This indicates an increased singlet oxygen (1O2) formation not detected in B. bassiana-inoculated plants, showing that the two entomopathogenic fungi differentially modulate the leaflets' response to herbivory. Overall, our results show that M. brunneum inoculation had a negative effect on the photosynthetic efficiency before herbivory, while B. bassiana inoculation had no significant effect. However, S. exigua leaf biting activated the same compensatory PSII response mechanism in tomato plants of both fungal-inoculated treatments as in control plants.

Formalin-casein enhances water absorbency of calcium alginate beads and activity of encapsulated Metarhizium brunneum and Saccharomyces cerevisiae.

The control of root-feeding wireworms has become more challenging as synthetic soil insecticides have been progressively phased out due to environmental risk concerns. Innovative microbial control alternatives such as the so-called attract-and-kill strategy depend on the rapid and successful development of dried encapsulated microorganisms, which is initiated by rehydration. Casein is a functional additive that is already used in food or pharmaceutical industry due to its water binding capacity. Cross-linked forms such as formalin-casein (FC), exhibit altered network structures. To determine whether FC influences the rehydration of alginate beads in order to increase the efficacy of an attract-and-kill formulation for wireworm pest control, we incorporated either casein or FC in different alginate/starch formulations. We investigated the porous properties of alginate/starch beads and subsequently evaluated the activities of the encapsulated entomopathogenic fungus Metarhizium brunneum and the CO2 producing yeast Saccharomyces cerevisiae. Adding caseins altered the porous structure of beads. FC decreased the bead density from (1.0197 ± 0.0008) g/mL to (1.0144 ± 0.0008) g/mL and the pore diameter by 31%. In contrast to casein, FC enhanced the water absorbency of alginate/starch beads by 40%. Furthermore, incorporating FC quadrupled the spore density on beads containing M. brunneum and S. cerevisiae, and simultaneous venting increased the spore density even by a factor of 18. Moreover, FC increased the total CO2 produced by M. brunneum and S. cerevisiae by 29%. Thus, our findings suggest that rehydration is enhanced by larger capillaries, resulting in an increased water absorption capacity. Our data further suggest that gas exchange is improved by FC. Therefore, our results indicate that FC enhances the fungal activity of both fungi M. brunneum and S. cerevisiae, presumably leading to an enhanced attract-and-kill efficacy for pest control.

Modification of reproductive schedule in response to pathogen exposure in a wild insect: Support for the terminal investment hypothesis.

Trade-offs in the time and energy allocated to different functions, such as reproductive activities, can be driven by alterations in condition which reduce resources, often in response to extrinsic factors such as pathogens or parasites. When individuals are challenged by a pathogen, they may either reduce reproduction as a cost of increasing defence mechanisms or, alternatively, modify reproductive activities so as to increase fecundity thereby minimizing the fitness costs of earlier death, a behaviour consistent with the terminal investment hypothesis (TIH). The TIH predicts that individuals with decreased likelihood of future reproduction will maximize current reproductive effort, which may include shifts in reproductive timing. We examined how wild, adult female click beetles (Agriotes obscurus) responded after exposure to the fungal pathogen Metarhizium brunneum. Field-collected beetles exposed to a high concentration of M. brunneum died earlier and in greater numbers than those exposed to a low concentration. Using a multivariate approach, we examined the impact of pathogen challenge on lifespan and a suite of reproductive traits. Stepdown regression analysis showed that only female lifespan differed among the fungal treatments. Fungal-induced reductions in lifespan drove changes in the reproductive schedule, characterized by a decrease in preoviposition period. Moving the start of egg laying forward allowed the females to offset the costs of a shortened lifespan. These changes suggest that there is a threshold for terminal investment, which is dependent on strength of the survival threat. From an applied perspective, our findings imply that exposing adult click beetles to M. brunneum to reduce their population density might not succeed and is an approach that needs further investigation.

Evaluation of spray applications of Metarhizium anisopliae, Metarhizium brunneum and Beauveria bassiana against larval winter ticks, Dermacentor albipictus.

Dermacentor albipictus (Acari: Ixodidae), the winter tick, is a one-host tick that parasitizes large ungulates. They can dramatically affect moose, Alces alces (Artiodactyla: Cervidae), causing significant physiological and metabolic stress and mortality among heavily parasitized individuals. Entomopathogenic fungi in the genera Metarhizium (Hypocreales: Clavicipitaceae) and Beauveria (Hypocreales: Cordycipitaceae) are promising tick biological control agents. We examined the pathogenicity of experimental and commercially formulated isolates of M. anisopliae, M. brunneum and B. bassiana sprayed at concentrations of 106, 107 and 108 conidia/mL against the larval stage of D. albipictus and assessed the efficacy of spraying the commercial product Met52®EC, containing M. brunneum, strain F52, under laboratory conditions. Results showed larval D. albipictus mortality was significantly higher and occurred earlier when treated with M. anisopliae and M. brunneum isolates compared to B. bassiana at 106, 107 and 108 conidia/mL. Mortality was observed as early as 3 days in the M. anisopliae and M. brunneum treatments and after 6 days in the B. bassiana treatments. After 21 days, larval mortality ranged from 74-99% when ticks were treated with M. anisopliae and M. brunneum isolates at 106, 107 and 108 and conidia/mL. In contrast, mortality of ticks treated with B. bassiana ranged from 30 to 64%. When larvae were treated with the commercial product Met52, mortality was ~ 45% after 3 days and ~ 96% after 9 days. These results demonstrate the effectiveness of M. anisopliae and M. brunneum against D. albipictus.

Asian longhorned beetle bioassays to evaluate formulation and dose-response effects of Metarhizium microsclerotia.

Asian longhorned beetles (ALB; Anoplophora glabripennis), are invasive wood borers susceptible to Metarhizium brunneum. This fungus can be prepared as dried microsclerotia which, after rehydration, produce infective conidia within weeks. Wood samples coated with formulated microsclerotia were attached to trees in the Ohio USA ALB-eradication zone and collected after 4-week periods. Adult ALB exposed to these samples had 100% mortality. In an experiment comparing formulations with or without humectant hydrogel, hydrogel did not significantly increase mortality of exposed ALB. In a dose-response experiment with 5 application rates, ALB survival decreased with increasing application rate and conidial density.

Seed inoculation with endophytic fungal entomopathogens promotes plant growth and reduces crown and root rot (CRR) caused by Fusarium culmorum in wheat.

MAIN CONCLUSION: Fungal entomopathogens, Beauveria bassiana (NATURALIS) and Metarhizium brunneum (BIPESCO5), can promote the growth of wheat following their endophytic establishment within plants through seed treatment. Similar to endophytic B. bassiana which has already been reported as a disease antagonist by several previous studies, the present study demonstrates that M. brunneum can suppress disease pathogens following plant colonization as well. An upsurge of research hints at the ability of entomopathogenic fungi, almost exclusively considered and used as insect pathogens, to endophytically colonize the internal tissues of a wide array of host plants and subsequently confer numerous benefits including enhancement of plant growth and suppression of disease pathogens. Such an ability has mainly been investigated for Beauveria bassiana. Fewer studies have demonstrated plant growth promotion by Metarhizium brunneum colonization, whereas no studies have reported on the potential of endophytic M. brunneum as a plant disease antagonist. The present study was, therefore, conducted to investigate whether seed treatment with B. bassiana (NATURALIS) and M. brunneum (BIPESCO5) could result in their endophytic establishment in wheat and promote plant growth. The study further examines the effect of the fungal strains as endophytes against Fusarium culmorum, one of the main causal agents of crown and root rot (CRR) in wheat. Both B. bassiana and M. brunneum were able to systemically colonize roots and shoots of wheat, and promote several plant growth parameters (shoot height, root length, and fresh root and shoot weights). Moreover, endophytic colonization of wheat with either fungal entomopathogen resulted in a significant reduction in disease incidence, development and severity. These results support the notion of the multiple ecological roles that could further be played by entomopathogenic fungi. Bearing such additional roles in mind while developing these fungi as microbial agents could improve the value of many commercially available mycoinsecticides.

Investigating the potential of an autodissemination system for managing populations of vine weevil, Otiorhynchus sulcatus (Coleoptera: Curculionidae) with entomopathogenic fungi.

Vine weevil, also known as black vine weevil, (Otiorhynchus sulcatus) is an economically important pest affecting soft fruit and nursery stock in temperate regions. We used laboratory and polytunnel experiments to investigate a novel control system based on autodissemination of spores of an entomopathogenic fungus to populations of adult vine weevils. The fungus was applied as a conidial powder, used on its own or formulated with talc, to a simple plastic refuge for vine weevils. The potential for adult weevils to disseminate the fungus was investigated first in polytunnel experiments using fluorescent powders applied to the refuge in lieu of fungal conidia. In this system, 88% of adult weevils came in contact with the powder within 48 h. When the powder was applied to five adult weevils that were then placed within a population of 35 potential recipients, it was transmitted on average to 75% of the recipient population within 7 days. Three isolates of entomopathogenic fungi (Beauveria bassiana isolate codes 433.99 and 1749.11 and Metarhizium brunneum isolate code 275.86), selected from a laboratory virulence screen. These three isolates were then investigated for efficacy when applied as conidial powders in artificial refuges placed among populations of adult weevils held in experimental boxes in the laboratory at 20 °C. Under this regime, the fungal isolates caused 70-90% mortality of adult weevils over 28 days. A final polytunnel experiment tested the efficacy of conidial powders of M. brunneum 275.86 placed in artificial refuges to increase vine weevil mortality. Overall weevil mortality was relatively low (26-41%) but was significantly higher in cages in which the conidial powders were placed in refuge traps than in cages with control traps. The lower weevil mortality recorded in the polytunnel experiment compared to the laboratory test was most likely a consequence of the greater amounts of inoculum required to kill adult weevils when conditions fluctuate between favourable and unfavourable temperatures e.g. below 15 °C. The potential of an autodissemination system for entomopathogenic fungi as a means of controlling vine weevil as part of an integrated pest management programme is discussed.

Age, pathogen exposure, but not maternal care shape offspring immunity in an insect with facultative family life.

BACKGROUND: To optimize their resistance against pathogen infection, individuals are expected to find the right balance between investing into the immune system and other life history traits. In vertebrates, several factors were shown to critically affect the direction of this balance, such as the developmental stage of an individual, its current risk of infection and/or its access to external help such as parental care. However, the independent and/or interactive effects of these factors on immunity remain poorly studied in insects. RESULTS: Here, we manipulated maternal presence and pathogen exposure in families of the European earwig Forficula auricularia to measure whether and how the survival rate and investment into two key immune parameters changed during offspring development. The pathogen was the entomopathogenic fungus Metarhizium brunneum and the immune parameters were hemocyte concentration and phenol/pro-phenoloxidase enzyme activity (total-PO). Our results surprisingly showed that maternal presence had no effect on offspring immunity, but reduced offspring survival. Pathogen exposure also lowered the survival of offspring during their early development. The concentration of hemocytes and the total-PO activity increased during development, to be eventually higher in adult females compared to adult males. Finally, pathogen exposure overall increased the concentration of hemocytes-but not the total-PO activity-in adults, while it had no effect on these measures in offspring. CONCLUSIONS: Our results show that, independent of their infection risk and developmental stage, maternal presence does not shape immune defense in young earwigs. This reveals that pathogen pressure is not a universal evolutionary driver of the emergence and maintenance of post-hatching maternal care in insects.

Effects of four commercial fungal formulations on mortality and sporulation in house flies (Musca domestica) and stable flies (Stomoxys calcitrans).

The house fly Musca domestica L. (Diptera: Muscidae) and stable fly Stomoxys calcitrans (L.) (Diptera: Muscidae) are major pests of livestock. Biological control is an important tool in an integrated control framework. Increased mortality in filth flies has been documented with entomopathogenic fungi, several strains of which are commercially available. Three strains of Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Hypocreales: Cordycipitaceae) and one strain of Metarhizium brunneum (Petch) (Hypocreales: Clavicipitaceae) were tested in commercial formulations for pathogenicity against house flies and stable flies. There was a significant increase in mortality of house flies with three of the formulations, BotaniGard® ES, Mycotrol® O, and Met52® EC, during days 4-9 in comparison with balEnce™ and the control. In stable flies, mortality rates were highest with Met52® EC, followed by Mycotrol® O, BotaniGard® ES and, finally, balEnce™. There was a significant fungal effect on sporulation in both house flies and stable flies. Product formulation, species differences and fungal strains may be responsible for some of the differences observed. Future testing in field situations is necessary. These commercial biopesticides may represent important tools in integrated fly management programmes.