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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. [ABSTRACT FROM AUTHOR]

(1) Background: The entomopathogenic fungus Metarhizium anisopliae sensu lato forms a species complex, comprising a tight cluster made up of four species, namely M. anisopliae sensu stricto, M. pinghaense, M. robertsii and M. brunneum. Unambiguous species delineation within this "PARB clade" that enables both the taxonomic assignment of new isolates and the identification of potentially new species is highly solicited. (2) Methods: Species-discriminating primer pairs targeting the ribosomal intergenic spacer (rIGS) sequence were designed and a diagnostic PCR protocol established. A partial rIGS sequence, referred to as rIGS-ID800, was introduced as a molecular taxonomic marker for PARB species delineation. (3) Results: PARB species from a validation strain set not implied in primer design were clearly discriminated using the diagnostic PCR protocol developed. Using rIGS-ID800 as a single sequence taxonomic marker gave rise to a higher resolution and statistically better supported delineation of PARB clade species. (4) Conclusions: Reliable species discrimination within the Metarhizium PARB clade is possible through both sequencing-independent diagnostic PCR and sequencing-dependent single marker comparison, both based on the rIGS marker. [ABSTRACT FROM AUTHOR]

Metarhizium brunneum is a soil-borne fungal entomopathogen that can be associated with plant roots. Previous studies have demonstrated that root colonization by beneficial fungi can directly affect soil-borne pathogens through competition and antibiosis and can activate a systemic response in plants, resulting in a primed state for a faster and/or stronger response to stressors. However, the mechanisms by which Metarhizium inoculation ameliorates symptoms caused by plant pathogens are not well known. This study evaluated the ability of M. brunneum to protect oilseed rape (Brassica napus L.) plants against the soil-borne pathogen Verticillium longisporum and investigated whether the observed effects are a result of direct interaction and/or plant-mediated effects. In vitro and greenhouse experiments were conducted to measure fungal colonization of the rhizosphere and plant tissues, and targeted gene expression analysis was used to evaluate the plant response. The results show that M. brunneum delayed pathogen colonization of plant root tissues, resulting in decreased disease symptoms. Direct competition and antibiosis were found to be part of the mechanisms, as M. brunneum growth was stimulated by the pathogen and inhibited the in vitro growth of V. longisporum. Additionally, M. brunneum changed the plant response to the pathogen by locally activating key defense hormones in the salicylic acid (SA) and abscisic acid (ABA) pathways. Using a split-root setup, it was demonstrated that there is a plant-mediated effect, as improved plant growth and decreased disease symptoms were observed when M. brunneum was in the systemic compartment. Moreover, a stronger systemic induction of the gene PR1 suggested a priming effect, involving the SA pathway. Overall, this study sheds light on the mechanisms underlying the protective effects of M. brunneum against soil-borne pathogens in oilseed rape plants, highlighting the potential of this fungal entomopathogen as a biocontrol agent in sustainable agriculture. [ABSTRACT FROM AUTHOR]

Important for the infection of an insect with an entomopathogenic fungus and its use as a plant protection agent are its growth, conidiation, germination, and virulence, which all depend on the environmental temperature. We investigated not only the effect of environmental temperature but also that of production temperature of the fungus. For this purpose, Metarhizium brunneum JKI-BI-1450 was produced and incubated at different temperatures, and the factors mentioned as well as conidial size were determined. The temperature at which the fungus was produced affects its subsequent growth and conidiation on granule formulation, the speed of germination, and the conidial width, but not its final germination or virulence. The growth and conidiation was at its highest when the fungus was produced at 25 °C, whereas when the germination was faster, the warmer the fungus was produced. The incubation temperature optimum of JKI-BI-1450 in relation to growth, speed of germination, and survival time was 25–30 °C and for conidiation 20–25 °C. Conidial length decreased with increasing incubation temperature. Although the fungus could not be adapted to unfavorable conditions by the production temperature, it was found that the quality of a biological control agent based on entomopathogenic fungi can be positively influenced by its production temperature. [ABSTRACT FROM AUTHOR]

Wireworm, the larval stages of click beetles, are a serious pest of tubers, brassicas and other important commercial crops throughout the northern hemisphere. No effective control agent has been developed specifically for them, and many of the pesticides marketed as having secondary application against them have been withdrawn from EU and Asian markets. Metarhizium brunneum, an effective entomopathogenic fungus, and its derived volatile metabolites are known to be effective plant biostimulants and plant protectants, although field efficacy has yet to be validated. Field validation of a combined M. brunneum and derived VOC treatments was conducted in Wales, UK, to assess the effects of each as a wireworm control agent and biostimulant. Plots were treated with Tri-Soil (Trichoderma atroviridae), M. brunneum, 1-octen-3-ol or 3-octanone, or combinations thereof. Treatments were applied subsurface during potato seeding (n = 52), and potatoes were harvested at the end of the growing season. Each potato was weighed individually and scored for levels of wireworm damage. Applications of both the VOCs and the M. brunneum individually were found to significantly decrease wireworm burden (p < 0.001). Combinations of M. brunneum and 3-octanone were also found to significantly decrease wireworm damage (p < 0.001), while no effect on yield was reported, resulting in an increased saleable mass over controls (p < 0.001). Herein, we present a novel 'stimulate and deter' wireworm control strategy that can be used to significantly enhance saleable potato yields and control wireworm populations, even under high pest pressure densities. [ABSTRACT FROM AUTHOR]

Metarhizium species can be mutualistic symbionts of plants. They are able to colonize roots, promote plant growth and provide protection against pests. We previously found Metarhizium robertsii and M. brunneum associated with coffee roots in a diversified coffee system. Here, we investigated whether these fungi, when inoculated in coffee seedlings, can associate with roots, improve seedling growth and indirectly protect against the coffee leaf miner (CLM) Leucoptera coffeella (Lepidoptera: Lyonetiidae). We performed a greenhouse experiment with coffee seedlings using suspensions of each Metarhizium species applied as soil drenches to potted seedlings. We also challenged these plants with CLM infestation (two adult couples per plant). We recovered Metarhizium spp. from most of the seedling roots 43 days after fungal inoculation. Plants inoculated with M. robertsii showed a 30% leaf area increase compared to the control. Both isolates promoted protection against CLM in coffee seedlings, reducing the percentual of leaf area mined and prolonging CLM development time by two days versus controls. Besides this protection provided by Metarhizium, M. robertsii also improves seedling growth. Therefore, these Metarhizium species could be considered for the development of inoculants for coffee seedlings. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Metarhizium brunneum is a highly effective entomopathogenic fungus that also functions as a plant biostimulant. It can act as both an endophyte and rhizosphere colonizer; however, the mechanisms driving biostimulation are multifactorial. In this work, oilseed rape (Brassica napus) seeds were grown in composts treated with different concentrations of M. brunneum strains ARSEF 4556 or V275, or the M. brunneum-derived volatile organic compounds 1-octen-3-ol and 3-octanone. Biostimulation efficacy was found to be strongly dose dependent. Concentrations of 1 × 106 conidia g−1 compost were found to be most effective for the M. brunneum, whereas dosages of 1 µL 100 g−1 compost were found to be efficacious for the volatiles. These optimized doses were assessed individually and in combined formulations with a hydrogel against oilseed rape (Brassica napus), sitka spruce (Picea sitchensis), maize (Zea mays) and strawberry (Fragaria annanassa). Both volatile compounds were highly effective biostimulants and were found to increase in biostimulatory efficiency when combined with M. brunneum conidia. Hydrogels were not found to interact with the growth process and may offer avenues for novel formulation technologies. This study demonstrates that Metarhizium-derived volatile organic compounds are actively involved in plant growth promotion and have potential for use in novel formulations to increase the growth of a wide range of commercially relevant crops. [ABSTRACT FROM AUTHOR]

Entomopathogenic fungi infect insects via spores but also live inside plant tissues as endophytes. Frequently, colonization by entomopathogens provides plants with increased resistance against insects, but the mechanisms are little understood. This study investigated direct, local, and systemic root-mediated interactions between isolates of the fungus Metarhizium brunneum and larvae of the cabbage root fly (CRF) Delia radicum attacking Brassica napus plants. All fungal isolates infected CRF when conidia were present in the soil, leading to 43–93% mortality. Locally, root-associated M. brunneum isolates reduced herbivore damage by 10–20% and in three out of five isolates caused significant insect mortality due to plant-mediated and/or direct effects. A split-root experiment with isolate Gd12 also demonstrated systemic plant resistance with significantly reduced root collar damage by CRF. LC-MS analyses showed that fungal root colonization did not induce changes in phytohormones, while herbivory increased jasmonic acid (JA) and glucosinolate concentrations. Proteinase inhibitor gene expression was also increased. Fungal colonization, however, primed herbivore-induced JA and the expression of the JA-responsive plant defensin 1.2 (PDF1.2) gene. We conclude that root-associated M. brunneum benefits plant health through multiple mechanisms, such as the direct infection of insects, as well as the local and systemic priming of the JA pathway. [ABSTRACT FROM AUTHOR]

Recent European Union regulations aim at reducing the use of chemical pesticides in agriculture. In sunflower production in Serbia, the use of synthetic insecticides for soil and seed treatments has become a prevailing practice for wireworm (Coleoptera: Elateridae) control. However, a number of compounds efficiently used as seed treatments have been phased out. This work aimed at assessing the potential of an environmentally friendly "Attract and Kill" strategy (A&K) for controlling wireworms in sunflower in comparison to conventional insecticides. The experiments were carried out in 2018, 2019 and 2021 involving (a) soil treatments with ATTRACAP® and a Force 1.5 G; (b) seed treatments with Sonido, Force 20 CS, Lumiposa and Buteo Start 480 FS; (c) controls (i.e., untreated seeds). The efficacy of the treatments was assessed based on the plant density and emerging plant damage (%). A damage rating scale (levels 0–5) was created, aiming for a more reliable and concrete interpretation of the results. Data were processed using binomial and multinomial regressions, followed by modelling of the damage and calculating the odds of damage occurrence, depending on the applied insecticide. In all experimental years and at all localities, the Force 20 CS and/or Force 1.5 G treatments resulted in the highest plant density and the lowest percentage of plant damage. ATTRACAP® showed good effectiveness, since plant density and percentage of damaged plants were at the same level of significance as the commonly used conventional insecticides in sunflower production (Sonido for seed treatment and Force 1.5 G for soil treatment). Thus, the A&K strategy was efficient in controlling wireworms at conditions of low abundance, based on three-year experimental results. Although the damage rating scale enabled a clear differentiation of plant damage caused by wireworms responsible for reduced plant density, more reliable models were obtained by binomial regressions, classifying plants as damaged or undamaged. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Growing global population and environmental concerns necessitate the transition from chemical to eco-friendly pest management. Entomopathogenic fungi (EPF) are rising candidates for this task due to their ease of growing, broad host range and unique disease process, allowing EPF to infect hosts directly through its cuticle. However, EPF’s requirement for high humidity negates their integration into conventional agriculture. To mitigate this problem, we formulated Metarhizium brunneum conidia in an oil-in-water Pickering emulsion. Conidia in aqueous and emulsion formulations were sprayed on Ricinus communis leaves, and Spodoptera littoralis larvae were introduced under low or high humidity. The following were examined: conidial dispersion on leaf, larval mortality, conidial acquisition by larvae, effects on larval growth and feeding, and dynamic of disease progression. Emulsion was found to disperse conidia more efficiently and caused two-fold more adhesion of conidia to host cuticle. Mortality from conidia in emulsion was significantly higher than other treatments reaching 86.5% under high humidity. Emulsion was also found to significantly reduce larval growth and feeding, while conferring faster fungal growth in-host. Results suggest that a Pickering emulsion is able to improve physical interactions between the conidia and their surroundings, while weakening the host through a plethora of mechanisms, increasing the chance of an acute infection. [ABSTRACT FROM AUTHOR]

The generalist entomopathogenic fungus, Metarhizium brunneum, has proved to have great potential as a versatile biological pest control agent. The gall midge Aphidoletes aphidimyza is a specialist predator that occurs naturally in Europe and has been successfully used for aphid suppression. However, the interaction between these two biological control organisms and how it may affect the biological control of aphids awaits further investigation. As part of the EU-supported project INBIOSOIL, this study was conducted in greenhouse conditions to assess the possible effects of combining both biological control agents. In a randomized complete block design, sweet corn (Zea mays var. saccharata) plants were grown in large pots filled with natural soil or natural soil inoculated with M. brunneum. At the third leaf stage, before being individually caged, plants were infested with Rhopalosiphum padi and A. aphidimyza pupae were introduced in the soil. Aphidoletes aphidimyza midge emergence, number of living midges and number of aphids were recorded daily. The presence of conidia in the soil and on leaves was assessed during the experiment. At the conclusion of the experiment, the number of live aphids and their developmental stage, consumed aphids, and A. aphidimyza eggs was assessed under stereomicroscope. This study’s findings showed that the presence of M. brunneum did not affect A. aphidimyza midge emergence. However, longevity was significantly affected. As the study progressed, significantly fewer predatory midges were found in cages treated with M. brunneum compared to untreated cages. Furthermore, by the end of the study, the number of predatory midges found in the Metarhizium-treated cages was four times lower than in the untreated cages. Both daily and final count of aphids were significantly affected by treatment. Aphidoletes aphidimyza applied alone suppressed the aphid population more effectively than M. brunneum applied alone. Additionally, the aphid population was most suppressed when both agents were combined, though the suppression was less than additive. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (Φ PSII ) was lower in M. brunneum -inoculated than in control plants, but not in B. bassiana -inoculated plants. After herbivory, however, Φ PSII 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 (Φ NPQ ) decreased in the inoculated plants, resulting in an increased fraction of nonregulated energy loss (Φ NO ) in M. brunneum . This indicates an increased singlet oxygen ( 1 O 2 ) 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.

Ants are the hosts of many microorganisms, including pathogens that are incidentally brought inside the nest by foragers. This is particularly true for scavenging species, which collect hazardous food such as dead insects. Foragers limit sanitary risks by not retrieving highly infectious prey releasing entomopathogenic fungal spores. This study investigates whether similar prophylactic strategies are also developed for food associated with weak or delayed risks of fungal contamination. We compared, in Myrmica rubra ant colonies, the retrieval dynamics of dead flies that were (1) conidia-free, (2) covered with a low amount of Metarhizium brunneum entomopathogenic conidia or (3) recently fungus-killed but not yet sporulating. Foragers mostly avoided fungus-killed prey and delayed the retrieval of conidia-covered flies. A second sanitary filter occurred inside the nest through a careful inspection of the retrieved prey. Ultimately, ants mostly consumed conidia-free and conidia-covered flies, but they relocated and discarded all fungus-killed prey outside of the nest. Our study confirms that, as a host of generalist entomopathogenic fungi, Myrmica rubra ants have developed a prophylactic avoidance and a differential management of prey depending on their infectious potential. We discuss the functional value as well as the possible cues underlying pathogen avoidance and prey discrimination in ants. [ABSTRACT FROM AUTHOR]

Entomopathogenic fungi are sprayed commercially for aphid control in greenhouses. Recently, their ability to grow endophytically within plants was discovered, offering the opportunity for systemic biological control. Endophytic colonization of host plants could also influence life-table parameters and behavior of herbivores. We investigated lethal and pre-mortality effects of Beauveria bassiana and Metarhizium brunneum on Aphis gossypii; aphids either received inoculum while feeding on recently sprayed leaves (surface inoculum and endophytically-colonized) or while feeding on unsprayed but endophytically-colonized leaves. We used choice assays to identify any preferences for endophytically-colonized or control plants. Volatile emissions from endophytically-colonized plants and control plants were also compared. Aphid mortality rates ranged between 48.2 and 56.9% on sprayed leaves, and between 37.7 and 50.0 on endophytically-colonized leaves. There was a significant effect of endophytic colonization on the rate of nymph production, but this did not result in an overall increase in the aphid population. Endophytic colonization did not influence host-plant selection even though there were qualitative and quantitative differences in the blend of volatiles released by endophytically-colonized and control plants. Although endophytic colonization did not change herbivore behavior, plants still benefit via indirect defense, resistance to plant pathogens or abiotic stress tolerance. [ABSTRACT FROM AUTHOR]