Metarhizium Brunneum Research Articles

Isolation and identification of native Chilean entomopathogenic fungi and their potential for the control of Drosophila suzukii

Drosophila suzukii is an invasive pest of berries and other soft-skinned fruits, was first detected in Chile in 2017, and has since spread over 2,800 km from north to south. Sustainable control of the spotted-wing drosophila (SWD) is essential due the negative attitude of the consumers toward the excessive use of insecticides. During a survey in Chile for biological control agents, thirty-two isolates of entomopathogenic fungi (EPF) were isolated from mycotized insects and soil samples, identified through sequence analysis, and tested against D. suzukii adults under laboratory conditions. The EPF identified are Akanthomyces muscarius, Beauveria bassiana, Beauveria pseudobassiana, Clonostachys rosea, Metarhizium alvesii, Metarhizium brunneum and Metarhizium robertsii. Six isolates caused 100 % of mortality of D. suzukii adults within ten days after the initial exposition to conidia; these included four B. bassiana isolates (LSB 110, LSB 114, LSB 122 and LSB 125), one M. robertsii isolate (LSB 115) and one M. brunneum isolate (LSB 127). LSB 122 and LSB 125 induced the shortest lethal time (LT50 4 and 4.2 days, respectively), while Akanthomyces and Clonostachys caused ≤36 % cumulative mortality of the adults at 10 days of exposure. These results indicate that isolates of B. bassiana, M. robertsii and M. brunneum have significant potential as microbial control agents against D. suzukii adults. This study marks a critical step forward in identifying and validating native entomopathogenic fungi in Chile for sustainable pest management. Future work will focus on further testing these isolates under laboratory, semi-field and field conditions to optimize their application in real-world agricultural settings.

Single-cell polymer coating improves the desiccation tolerance of Metarhizium brunneum blastospores

Equipping cells with artificial shells or coats has been explored throughout the last decade, with goals such as immunomasking, in vivo tracing, and imparting tolerance to various biotic and abiotic stressors. One stressor, however, drying, has curiously been overlooked. In an industrial setting, the drying of cells is relevant when a satisfactory product shelf life must be achieved at a low cost. The drying of entomopathogenic organisms for biocontrol is a prime example of this. Here, the thin-walled blastospores of the entomopathogenic fungus Metarhizium brunneum are a great model organism for testing whether thin-cell surface polyelectrolyte layers may increase desiccation tolerance. In this study, we coated single M. brunneum blastospores with alternating layers of chitosan and alginate and assessed their effects on blastospore viability after drying. The desiccation tolerance improved with increasing layer numbers from 6.9% to a maximum of 27.5%. In addition, as the polymer chain length decreased, the desiccation tolerance further increased to 33.1%. Furthermore, we provided visual proof of the coating surrounding the blastospores via the use of fluorescent polymers and scanning electron microscopy. Finally, an investigation of differences in water absorption into coated and uncoated cells revealed that water absorbed faster into coated cells when alginate was on the surface of the structure but slower when the outermost layer was composed of chitosan. We conclude that, via polyelectrolyte multilayering on thin-walled blastospores, desiccation tolerance can be significantly increased, but a deeper understanding is necessary to extract the full potential from this technique.Graphical

Transcriptional reprogramming in the entomopathogenic fungus Metarhizium brunneum and its aphid host Myzus persicae during the switch between saprophytic and parasitic lifestyles

BackgroundThe fungus Metarhizium brunneum has evolved a remarkable ability to switch between different lifestyles. It develops as a saprophyte, an endophyte establishing mutualistic relationships with plants, or a parasite, enabling its use for the control of insect pests such as the aphid Myzus persicae. We tested our hypothesis that switches between lifestyles must be accompanied by fundamental transcriptional reprogramming, reflecting adaptations to different environmental settings.ResultsWe combined high throughput RNA sequencing of M. brunneum in vitro and at different stages of pathogenesis to validate the modulation of genes in the fungus and its host during the course of infection. In agreement with our hypothesis, we observed transcriptional reprogramming in M. brunneum following conidial attachment, germination on the cuticle, and early-stage growth within the host. This involved the upregulation of genes encoding degrading enzymes and gene clusters involved in synthesis of secondary metabolites that act as virulence factors. The transcriptional response of the aphid host included the upregulation of genes potentially involved in antifungal activity, but antifungal peptides were not induced. We also observed the induction of a host flightin gene, which may be involved in wing formation and flight muscle development.ConclusionsThe switch from saprophytic to parasitic development in M. brunneum is accompanied by fundamental transcriptional reprogramming during the course of the infection. The aphid host responds to fungal infection with its own transcriptional reprogramming, reflecting its inability to express antifungal peptides but featuring the induction of genes involved in winged morphs that may enable offspring to avoid the contaminated environment.

How to control nasty scarabs? Effectiveness of the generalist entomopathogenic fungus Metarhizium brunneum against garden chafer larvae

Several scarab beetles (Scarabaeidae) cause major damage in agriculture, horticulture, and forestry. Especially root feeding scarab larvae cause substantial economic losses on crops, forage plants and recreational areas. In Europe, the entomopathogenic fungi (EPF) Beauveria brongniartii and Metarhizium brunneum are applied against the most problematic, native scarabs, the common cockchafer (Melolontha melolontha) and the garden chafer (Phyllopertha horticola). While the control of cockchafer larvae with B. brongniartii is well-researched, conclusive results from field applications for the control of the garden chafer with M. brunneum remain elusive. We therefore assessed the performance of commercially available fungal strains of M. brunneum against garden chafer larvae in pot and large-scale field experiments.The application of M. brunneum significantly increased the abundance of fungal propagules in the soil by approximately a factor of ten, irrespective of high levels of naturally occurring Metarhizium spp. Furthermore, the applied strains infected and propagated on the larvae and the mortality of garden chafer larvae was slightly increased due to the fungal treatments. We found three other EPF species frequently infecting garden chafer larvae (mean infection rates: 13–25%), including B. brongniartii which is considered to be a specific pathogen of the cockchafer. Thus, the applied fungal strains were only part of a consortium of natural enemies which reduces garden chafer populations strongly as a whole. Hence, we suggest that the application of EPF may be advisable on areas with reduced natural enemies such as golf courses but is probably redundant on meadows harboring a diverse consortium of antagonists.

A gene regulating ergot alkaloid biosynthesis in Metarhizium brunneum.

Ergot alkaloids produced by several species of fungi are important as contaminants of food and feed in agriculture and also as the foundation of numerous pharmaceuticals prescribed for dementia, migraines, hyperprolactinemia, and several other disorders. Information on control of the ergot alkaloid pathway may contribute to strategies to limit their production in agricultural settings or increase their yield for pharmaceutical production. Our results demonstrate that a previously uncharacterized gene clustered with the ergot alkaloid synthesis genes is required for the sufficient transcription of the ergot alkaloid biosynthesis genes. This observation suggests the gene encodes a factor regulating transcription of those biosynthetic genes.

Metarhizium-Inoculated Coffee Seeds Promote Plant Growth and Biocontrol of Coffee Leaf Miner.

Metarhizium (Hypocreales: Clavicipitaceae) has a multifunctional life cycle, establishing as a plant endophyte and acting as entomopathogenic fungi. Metarhizium robertsii and Metarhizium brunneum can be associated with coffee plants and provide enhanced protection against a major pest of coffee, the coffee leaf miner (CLM) (Leucoptera coffeella). This association would be an easily deployable biological control option. Here we tested the potential of inoculating coffee seeds with M. robertsii and M. brunneum collected from the soil of coffee crops in the Cerrado (Brazil) for control of the CLM and the enhancement of plant growth with a commonly used fungicide. We conducted the experiment in a greenhouse and after the seedlings grew, we placed them in a cage with two couples of CLMs. We evaluated the CLM development time, reproduction, and plant growth traits. We observed a longer development time of CLMs when fed on plants inoculated with both isolates. In addition, the CLMs laid fewer eggs compared to those fed on plants without fungal inoculation. Plant growth was promoted when seeds were inoculated with fungi, and the fungicide did not affect any evaluated parameter. Coffee seed inoculation with M. robertsii and M. brunneum appears to provide protection against CLMs and promote growth improvement.

Comparative genomics of Metarhizium brunneum strains V275 and ARSEF 4556: unraveling intraspecies diversity.

Entomopathogenic fungi belonging to the Order Hypocreales are renowned for their ability to infect and kill insect hosts, while their endophytic mode of life and the beneficial rhizosphere effects on plant hosts have only been recently recognized. Understanding the molecular mechanisms underlying their different lifestyles could optimize their potential as both biocontrol and biofertilizer agents, as well as the wider appreciation of niche plasticity in fungal ecology. This study describes the comprehensive whole genome sequencing and analysis of one of the most effective entomopathogenic and endophytic EPF strains, Metarhizium brunneum V275 (commercially known as Lalguard Met52), achieved through Nanopore and Illumina reads. Comparative genomics for exploring intraspecies variability and analyses of key gene sets were conducted with a second effective EPF strain, M. brunneum ARSEF 4556. The search for strain- or species-specific genes was extended to M. brunneum strain ARSEF 3297 and other species of genus Metarhizium, to identify molecular mechanisms and putative key genome adaptations associated with mode of life differences. Genome size differed significantly, with M. brunneum V275 having the largest genome amongst M. brunneum strains sequenced to date. Genome analyses revealed an abundance of plant-degrading enzymes, plant colonization-associated genes, and intriguing intraspecies variations regarding their predicted secondary metabolic compounds and the number and localization of Transposable Elements. The potential significance of the differences found between closely related endophytic and entomopathogenic fungi, regarding plant growth-promoting and entomopathogenic abilities, are discussed, enhancing our understanding of their diverse functionalities and putative applications in agriculture and ecology.

Isolation and identification of entomopathogenic fungi strains for Colorado potato beetle (Leptinotarsa decemlineata) control.

The Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) is the most widespread insect pest that causes major economic losses, especially on potatoes. Due to heavy insecticide use, this species now resists most pesticides, posing a significant control challenge. Frequent pesticide application also harms non-target organisms, the environment, and human health. Hence, utilizing biocontrol agents like entomopathogenic fungi (EPF) offers a viable alternative for pest management. The aim of this study was to identify and characterize new EPF strains isolated from soil samples and evaluate their efficacy against adult L. decemlineata under laboratory conditions. Soil samples were collected in potato fields or uncultivated areas adjacent to the field in the Czech Republic and the EPF strains were isolated using a modified Tenebrio bait method. A total of 20 fungal strains were isolated and identified using morphological and molecular markers based on the 28S rRNA, ITS, and elongation factor 1-alpha gene sequences as Beauveria bassiana (Bals.-Criv.) Vuill., Beauveria brongniartii (Sacc.) Petch, and Cordyceps fumosorosea (Wize) Kepler, B. Shrestha & Spatafora (Hypocreales: Cordycipitaceae), Purpureocillium lilacinum (Thom.) Luangsa-ard, Houbraken, Hywel-Jones & Samson (Hypocreales: Ophiocordycipitaceae), Metarhizium brunneum (Petch), and Metarhizium robertsii Bisch., Rehner & Humber (Hypocreales: Clavicipitaceae). The bioassays revealed high variability among virulence of these strains against L. decemlineata with the shortest median time to death (LT50=5 days) in M. robertsii strain MAN3b. Results shown that some EPF strains, particularly those of genera Metarhizium, can be promising biocontrol agents against the Colorado potato beetle.

Unraveling the interaction of co-encapsulated Saccharomyces cerevisiae and Metarhizium brunneum in calcium alginate-based attract-and-kill beads.

Attract-and-kill (AK) beads are biological, microbial insecticides developed as an alternative to synthetic soil insecticides. For wireworm control, beads are based on calcium alginate/starch co-encapsulating the carbon dioxide (CO2) producing yeast Saccharomyces cerevisiae H205 as the attract component, and the entomopathogenic fungus Metarhizium brunneum CB15-III as the kill component. However, the physicochemical processes inside beads during co-cultivation are still unclear. Here we reveal for the first time the spatiotemporal conditions of oxygen and pH inside AK beads measured with microelectrodes and describe the impact of S. cerevisiae on CO2 and conidia formation. Measurements revealed a steep oxygen gradient already 2 days after co-encapsulation, with an internal hypoxic zone. Encapsulating either S. cerevisiae or M. brunneum already decreased the average pH from 5.5 to 4.7 and 4.6, respectively. However, on day 3, co-cultivation lead to temporal strong acidification of beads down to pH 3.6 which followed the maximum CO2 productivity and coincided with the maximum conidiation rate. Decreasing the yeast load decreased the total CO2 productivity to half, and the conidial production by 93%, while specific productivities normalized to 1% yeast load increased eight-fold and three-fold, respectively, with day 3 being an exception. Our findings indicate a general beneficial interaction between M. brunneum and S. cerevisiae, but also suggest competition for resources. These findings will contribute to develop innovative co-formulations with maximum efficiency to save application rates and costs. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

Thermal ecology shapes disease outcomes of entomopathogenic fungi infecting warm-adapted insects

The thermal environment is a critical determinant of outcomes in host-pathogen interactions, yet the complexities of this relationship remain underexplored in many ecological systems. We examined the Thermal Mismatch Hypothesis (TMH) by measuring phenotypic variation in individual thermal performance profiles using a model system of two species of entomopathogenic fungi (EPF) that differ in their ecological niche, Metarhizium brunneum and M. flavoviride, and a warm-adapted model host, the mealworm Tenebrio molitor. We conducted experiments across ecologically relevant temperatures to determine the thermal performance curves for growth and virulence, measured as % survival, identify critical thresholds for these measures, and elucidate interactive host-pathogen effects. Both EPF species and the host exhibited a shared growth optima at 28 °C, while the host’s growth response was moderated in sublethal pathogen infections that depended on fungus identity and temperature. However, variances in virulence patterns were different between pathogens. The fungus M. brunneum exhibited a broader optimal temperature range (23–28 °C) for virulence than M. flavoviride, which displayed a multiphasic virulence-temperature relationship with distinct peaks at 18 and 28 °C. Contrary to predictions of the TMH, both EPF displayed peak virulence at the host's optimal temperature (28 °C). The thermal profile for M. brunneum aligned more closely with that of T. molitor than that for M. flavoviride. Moreover, the individual thermal profile of M. flavoviride closely paralleled its virulence thermal profile, whereas the virulence thermal profile of M. brunneum did not track with its individual thermal performance. This suggests an indirect, midrange (23 °C) effect, where M. brunneum virulence exceeded growth. These findings suggest that the evolutionary histories and ecological adaptations of these EPF species have produced distinct thermal niches during the host interaction. This study contributes to our understanding of thermal ecology in host-pathogen interactions, underpinning the ecological and evolutionary factors that shape infection outcomes in entomopathogenic fungi. The study has ecological implications for insect population dynamics in the face of a changing climate, as well as practically for the use of these organisms in biological control.

Effects of an entomopathogenic fungus on the reproductive potential of Drosophila males.

While mortality is often the primary focus of pathogen virulence, non-lethal consequences, particularly for male reproductive fitness, are less understood; however, they are essential for understanding how sexual selection contributes to promoting resistance. We investigated how the fungal pathogen Metarhizium brunneum affects mating ability, fertility, and seminal fluid protein (SFP) expression of male Drosophila melanogaster paired with highly receptive virgin females in non-competitive settings. Depending on sex and dose, there was a 3-6-day incubation period after infection, followed by an abrupt onset of mortality. Meanwhile, the immune response was strongly induced already 38 h after infection and continued to increase as infection progressed. Latency to mate somewhat increased during the incubation period compared to sham-treated males, but even on Day 5 post infection >90% of infected males mated within 2 h. During the incubation period, M. brunneum infection reduced male reproductive potential (the number of offspring sired without mate limitation) by 11%, with no clear increase over time. Approaching the end of the incubation period, infected males had lower ability to convert number of mating opportunities into number of offspring. After repeated mating, infected males had lower SFP expression than sham controls, more so in males that mated with few mates 24 h earlier. Overall, despite strong activation of the immune response, males' mating ability and fertility remained surprisingly little affected by the fungal infection, even shortly before the onset of mortality. This suggests that the selection for resistance acts mainly through mortality, and the scope for fertility selection to enhance resistance in non-competing settings is rather limited.

Minor impact of probiotic bacteria and egg white on Tenebrio molitor growth, microbial composition, and pathogen infection.

The industrial rearing of the yellow mealworm (Tenebrio molitor) for feed and food purposes on agricultural by-products may expose larvae and adults to entomopathogens used as biocontrol agents in crop production. Bacterial spores/toxins or fungal conidia from species such as Bacillus thuringiensis or Metarhizium brunneum could affect the survival and growth of insects. Therefore, the aim of this study was to investigate the potential benefits of a wheat bran diet supplemented with probiotic bacteria and dried egg white on larval development and survival and its effects on the gut microbiome composition. Two probiotic bacterial species, Pediococcus pentosaceus KVL B19-01 and Lactiplantibacillus plantarum WJB, were added to wheat bran feed with and without dried egg white, as an additional protein source, directly from neonate larval hatching until reaching a body mass of 20 mg. Subsequently, larvae from the various diets were exposed for 72 h to B. thuringiensis, M. brunneum, or their combination. Larval survival and growth were recorded for 14 days, and the bacterial microbiota composition was analyzed using 16S rDNA sequencing prior to pathogen exposure and on days 3 and 11 after inoculation with the pathogens. The results showed increased survival for T. molitor larvae reared on feed supplemented with P. pentosaceus in the case of co-infection. Larval growth was also impacted in the co-infection treatment. No significant impact of egg white or of P. pentosaceus on larval growth was recorded, while the addition of Lb. plantarum resulted in a minor increase in individual mass gain compared with infected larvae without the latter probiotic. On day 14, B. thuringiensis was no longer detected and the overall bacterial community composition of the larvae was similar in all treatments. On the other hand, the relative operational taxonomic unit (OTU) abundance was dependent on day, diet, and probiotic. Interestingly, P. pentosaceus was present throughout the experiments, while Lb. plantarum was not found at a detectable level, although its transient presence slightly improved larval performance. Overall, this study confirms the potential benefits of some probiotics during the development of T. molitor while underlining the complexity of the relationship between the host and its microbiome.

Greenhouse melon crop protection and production through the compatible use of a parasitoid with endophytic entomopathogenic ascomycetes

This study delves into the compatible use of a parasitoid with multifunctional endophytic Entomopathogenic Ascomycetes (EA) in IPM under greenhouse conditions. The parasitoid Hyposoter didymator was evaluated against Spodoptera littoralis in a multitrophic system with melon plants that were endophytically colonized by one of three EA strains (Metarhizium brunneum [one] or Beauveria bassiana [two]). In the first scenario, plants were inoculated by three different methods, and after infestation with noctuid larvae, the parasitoid was released at a 1:20 ratio. Microbiological and molecular techniques allowed the identification of progressive colonization throughout the whole plant life cycle, and for B. bassiana, approximately 20% of seeds from new fruits were colonized. The parasitoid was shown to be compatible with all strains and application methods, with total mortality rates ranging from 11.1 to 77.8%. Significant lethal and sublethal effects, a decrease in pupal weight and mortality of pupae showing abnormalities and an extension of the immature developmental times were observed for different strain–application method combinations. Additionally, the fungal treatments improved crop growth, as revealed by the significant gains in plant weight. In a second scenario (by inoculating plants with the fungi only by leaf spraying), and after infestation with noctuid larvae, the parasitoid was released at a 1:10 ratio, which revealed the remote fungal effect from the inoculation point and confirmed the compatibility of the parasitoid-EA-based strategy. These findings underscore the compatible use of a parasitoid with endophytic EA for S. littoralis control that can additionally exploit their multifunctionality for sustainable crop production.

Fungal infection alters collective nutritional intake of ant colonies

In animals, parasitic infections impose significant fitness costs.1,2,3,4,5,6 Infected animals can alter their feeding behavior to resist infection,7,8,9,10,11,12 but parasites can manipulate animal foraging behavior to their own benefits.13,14,15,16 How nutrition influences host-parasite interactions is not well understood, as studies have mainly focused on the host and less on the parasite.9,12,17,18,19,20,21,22,23 We used the nutritional geometry framework24 to investigate the role of amino acids (AA) and carbohydrates (C) in a host-parasite system: the Argentine ant, Linepithema humile, and the entomopathogenic fungus, Metarhizium brunneum. First, using 18 diets varying in AA:C composition, we established that the fungus performed best on the high-amino-acid diet 1:4. Second, we found that the fungus reached this optimal diet when given various diet pairings, revealing its ability to cope with nutritional challenges. Third, we showed that the optimal fungal diet reduced the lifespan of healthy ants when compared with a high-carbohydrate diet but had no effect on infected ants. Fourth, we revealed that infected ant colonies, given a choice between the optimal fungal diet and a high-carbohydrate diet, chose the optimal fungal diet, whereas healthy colonies avoided it. Lastly, by disentangling fungal infection from host immune response, we demonstrated that infected ants foraged on the optimal fungal diet in response to immune activation and not as a result of parasite manipulation. Therefore, we revealed that infected ant colonies chose a diet that is costly for survival in the long term but beneficial in the short term-a form of collective self-medication.

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.

Trap crops enhance the control efficacy of Metarhizium brunneum against a soil-dwelling pest

Soil-dwelling insect pests may cause considerable damage to crops worldwide, and their belowground lifestyle makes them hard to control. Amongst the most promising control agents for subterranean pests are soilborne entomopathogenic fungi (EPF) such as Metarhizium brunneum. Albeit EPF can be highly pathogenic to their target pest species under laboratory conditions, their efficacy in the field is often limited due to adverse environmental conditions. Here, we test for the first time if the efficacy of EPF can be improved when they are augmented with trap crops. In a field experiment, the M. brunneum strain ART2825 was combined with a trap crops mixture of six plant species and evaluated for its control effect of wireworms (Coleoptera: Elateridae). When both were combined in the main crop, potato damage was lowered on average by 42.5% and wireworm abundance by 50.8%. Single application of trap crops or EPF lowered damage/pest abundance only by 29.9%/15.89% and 34.7%/4.77%, respectively. Importantly, the strength of the synergistic pest control effect between trap crops and EPF increased disproportionately with increasing wireworm abundance. However, DNA-based gut content analysis showed that wireworms’ feeding preferences were not shifting toward the trap crops. Our findings demonstrate that combining trap crops with EPF improves the efficacy of the latter and leads to a synergistic control effect which magnifies with increasing wireworm abundance. Hence, the synergistic effect of EPF and trap crops might be a promising control strategy for soil-dwelling pests in general and significantly improve our abilities to manage soil pests environmentally friendly.

Compatibility of soil application of Metarhizium brunneum and cover crops against Ceratitis capitata soil-dwelling stages

Fruit fly ground-dwelling stages (late third instar larvae, pupae, and teneral adults) are susceptible to predation from generalist ground-dwelling predators and to infection by entomopathogenic fungi (EPF). The effect of predators can be enhanced with cover crops and that of EPF by augmentative releases. However, whether these two biological control methods could be combined has not been studied under field conditions yet. Here, we studied in the field whether the enhanced activity of predators against the medfly, Ceratitis capitata, already observed in a Lolium arundinaceum ground cover could be impaired by a soil application of Metarhizium brunneum. Our results show that C. capitata adult emergence was reduced by this EPF for up to three months after fungal application, with the combination of the cover and M. brunneum being the most effective at reducing C. capitata emergence relative to bare soil (92.5% reduction). Although M. brunneum reduced the activity density of ground-dwelling predatory beetles up to 93 days after application, it showed no clear negative effects on earwigs, no effects on spiders, and a positive effect on ants up to 65 days after application. Therefore, the combined use of a ground cover of L. arundinaceum and M. brunneum against the soil-dwelling stages of C. capitata seems to work synergistically and appears as a strong and sustainable control tactic against the medfly and other fruit orchard pests.

Limited effects of plant-beneficial fungi on plant volatile composition and host-choice behavior of Nesidiocoris tenuis.

Biological control using plant-beneficial fungi has gained considerable interest as a sustainable method for pest management, by priming the plant for enhanced defense against pathogens and insect herbivores. However, despite promising outcomes, little is known about how different fungal strains mediate these beneficial effects. In this study, we evaluated whether inoculation of tomato seeds with the plant-beneficial fungi Beauveria bassiana ARSEF 3097, Metarhizium brunneum ARSEF 1095 and Trichoderma harzianum T22 affected the plant's volatile organic compound (VOC) profile and the host-choice behavior of Nesidiocoris tenuis, an emerging pest species in NW-European tomato cultivation, and the related zoophytophagous biocontrol agent Macrolophus pygmaeus. Results indicated that fungal inoculation did not significantly alter the VOC composition of tomato plants. However, in a two-choice cage assay where female insects were given the option to select between control plants and fungus-inoculated plants, N. tenuis preferred control plants over M. brunneum-inoculated plants. Nearly 72% of all N. tenuis individuals tested chose the control treatment. In all other combinations tested, no significant differences were found for none of the insects. We conclude that inoculation of tomato with plant-beneficial fungi had limited effects on plant volatile composition and host-choice behavior of insects. However, the observation that N. tenuis was deterred from the crop when inoculated with M. brunneum and attracted to non-inoculated plants may provide new opportunities for future biocontrol based on a push-pull strategy.

Efficacy of Entomopathogenic Fungi as Prevention against Early Life Stages of the Red Palm Weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae) in Laboratory and Greenhouse Trials.

The red palm weevil (RPW) Rhynchophorus ferrugineus is a highly destructive invasive pest for palms whose management is mainly by application of synthetic pesticides. As a key pest of date palm plantations, it is necessary to integrate environmentally safe measures for its management. Entomopathogenic fungi (EPF) have been primarily studied as a preventative control measure due to the horizontal transfer of conidia within the RPW population. We previously demonstrated the horizontal transmission of fungal conidia from an egg-laying surface to the female weevil and then to the eggs and larvae. Based on that strategy, this study aimed to evaluate the virulence of commercial EPF products and laboratory EPF preparations to RPW females and their progeny, and their ability to protect palms against infestation. As such, it serves as a screening platform for field experiments. Mortality rates of females and eggs depended on the applied treatment formulation and fungal strain. Velifer®, a Beauveria bassiana product, and Metarhizium brunneum (Mb7) resulted in 60-88% female mortality. Mb7-as a conidial suspension or powder-resulted in 18-21% egg-hatching rates, approximately 3 times less than in the non-treated control. Treating palms with Mb7 suspension or dry formulation significantly inhibits infestation signs and results in protection. These results lay the foundation for investigating the protective rate of EPF products against RPW in date plantations.