Search

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. [ABSTRACT FROM AUTHOR]

Trap crops and entomopathogenic fungi can provide partial solutions for integrated pest management, by attracting and killing insect pests, respectively, but both solutions have some limitations restricting their practical field applications. Both solutions have been tested against a major soil-borne pest of brassicaceous vegetables, the cabbage root fly Delia radicum. Chinese cabbage is very attractive to this pest, but it is also a high-quality host plant for developing larvae of D. radicum, which limits the application as a trap crop in the field. The entomopathogenic fungus Metarhizium brunneum can infect D. radicum larvae in the soil, but M. brunneum has not proved to be sufficiently effective in reducing damages caused by cabbage root fly. In the present work, we evaluated whether the entomopathogenic fungus M. brunneum can be used to regulate D. radicum populations together with trap crops by inoculating Chinese cabbage and broccoli plants at sowing with M. brunneum colonized rice grains before transplantation of small plants to field soil. The evaluation was performed under natural fly infestation. In both plant treatments, D. radicum infestations were high with no or only moderate effect of the fungus inoculation on the number of larvae and pupae recorded, despite evidence of successful fungal infections. On broccoli plants, our results clearly demonstrated that the M. brunneum application was inefficient in reducing number of D. radicum stages in the soil and resulting plant mortality. However, in the trap crop, Chinese cabbage, M. brunneum inoculation reduced the number of D. radicum imagos emerging from the plants by 36%. Hence, the strategy is likely to have effects on the next D. radicum generation. This result is the first to indicate complementarity between the 'attract' and 'kill' strategies to control pest development inside a favorable trap crop and prevent future pest population outbreaks. Also, from both plant inoculation treatments, some emerging D. radicum imagos developed M. brunneum infection, which may assist the transmission of the entomopathogenic fungus among adult populations. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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]

The application of fungal entomopathogens, such as Metarhizium brunneum, has the potential to continue to impact targeted host populations through secondary cycling. In the present study, we examined the horizontal transmission of M. brunneum conidia from conspecific mycosed cadavers or soil on which the cadavers sporulated to Agriotes obscurus beetles. Close contact between the susceptible hosts and sporulating cadavers or contaminated soil was required for increased mortality of hosts and subsequent production of mycosed cadavers. [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

Abstract Polyvinyl alcohol (PVA) is a biodegradable, water-soluble polymer with excellent film forming properties, commonly studied or used as tablet coating, food packaging or controlled release fertilizers. Attract-and-kill (AK) beads are sustainable, microbial alternatives to synthetic soil insecticides, whose onset of lethal effect largely depend on how fast the encapsulated entomopathogenic fungus forms virulent conidia. Therefore, the objective of this study was to develop a water-soluble coating accelerating the kill effect of AK beads by immediately releasing virulent Metarhizium brunneum CB15-III blastospores. We assessed three PVA types (PVA 4-88, 8-88, 10-98) which differed in their degree of hydrolysis or molecular weight for their ability to release viable blastospores from thin films after drying at 60–40 °C, and examined how polyethylene glycol and soy-lecithin impact the blastospore survival. Finally, we evaluated the effectiveness of coated AK beads in a bioassay against Tenebrio molitor larvae. The blastospore release rate quadrupled within the first 5 min with decreasing molecular weight and degree of hydrolysis, with PVA 4-88 releasing 79 ± 19% blastospores. Polyethylene glycol and soy-lecithin significantly increased the blastospore survival to 18–28% for all three PVA types. Coated beads exhibited a uniform, 22.4 ± 7.3 µm thin coating layer, with embedded blastospores, as confirmed by scanning electron microscopy. The blastospore coating increased the mortality rate of T. molitor larvae over uncoated AK beads, decreasing the median lethal time from 10 to 6 days. Consequently, the blastospore coating accelerated the kill effect of regular AK beads. These findings pave the way to enhanced pest control efficacy from coated systems such as beads or seeds. Graphical Abstract

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.

(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]

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.

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]

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. [ABSTRACT FROM AUTHOR]

Polyvinyl alcohol (PVA) is a biodegradable, water-soluble polymer with excellent film forming properties, commonly studied or used as tablet coating, food packaging or controlled release fertilizers. Attract-and-kill (AK) beads are sustainable, microbial alternatives to synthetic soil insecticides, whose onset of lethal effect largely depend on how fast the encapsulated entomopathogenic fungus forms virulent conidia. Therefore, the objective of this study was to develop a water-soluble coating accelerating the kill effect of AK beads by immediately releasing virulent Metarhizium brunneum CB15-III blastospores. We assessed three PVA types (PVA 4-88, 8-88, 10-98) which differed in their degree of hydrolysis or molecular weight for their ability to release viable blastospores from thin films after drying at 60–40 °C, and examined how polyethylene glycol and soy-lecithin impact the blastospore survival. Finally, we evaluated the effectiveness of coated AK beads in a bioassay against Tenebrio molitor larvae. The blastospore release rate quadrupled within the first 5 min with decreasing molecular weight and degree of hydrolysis, with PVA 4-88 releasing 79 ± 19% blastospores. Polyethylene glycol and soy-lecithin significantly increased the blastospore survival to 18–28% for all three PVA types. Coated beads exhibited a uniform, 22.4 ± 7.3 µm thin coating layer, with embedded blastospores, as confirmed by scanning electron microscopy. The blastospore coating increased the mortality rate of T. molitor larvae over uncoated AK beads, decreasing the median lethal time from 10 to 6 days. Consequently, the blastospore coating accelerated the kill effect of regular AK beads. These findings pave the way to enhanced pest control efficacy from coated systems such as beads or seeds. Key points: PVA enables immobilization of virulent blastospores on alginate beads. Blastospore coating accelerates kill effect of attract-and-kill beads. PEG4000 and soy lecithin improve blastospore survival in PVA films. [ABSTRACT FROM AUTHOR]

Abstract Background The pine processionary moth Thaumetopoea wilkinsoni Tams, 1926 (Lepidoptera: Notodontidae), causes severe skin reactions to animals and humans. The insect also destroys pine ecosystems by feeding on pine leaves at its larval stage. Instead of chemical control, eco-friendly biological control methods should be preferred to combat this species. Results The purpose of this study was to evaluate the efficacy of Beauveria bassiana isolate (GOPT-331) and Metarhizium brunneum isolates (ORP-13 and ORP-18) against the second and fourth larval instars of T. wilkinsoni under laboratory conditions. T. wilkinsoni eggs were collected from pine trees at Ondokuz Mayıs University in Samsun, Turkey, in 2021, and the second and fourth larval instars were used for the experiment. Two milliliters of a spray of the three fungal isolates was applied to the larvae for each concentration (1 × 105–1 × 108 conidia ml−1). The mortality rates of GOPT-331, ORP-13, and ORP-18 were changed between 91.1 and 100% for the second-instar larvae and between 86.6 and 97.7% for the fourth-instar larvae at 1 × 108 conidia ml−1. The ORP-13 isolate showed the lowest LC50 values. Conclusion In conclusion, it is suggested that all the three isolates were virulent to T. wilkinsoni and can be used for biological control of this species. The promising results from the study were obtained from trials conducted under controlled laboratory conditions, and it will be critical to explore the potential of these promising entomopathogenic fungi in field conditions.

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]

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

Background: The pine processionary moth Thaumetopoea wilkinsoni Tams, 1926 (Lepidoptera: Notodontidae), causes severe skin reactions to animals and humans. The insect also destroys pine ecosystems by feeding on pine leaves at its larval stage. Instead of chemical control, eco-friendly biological control methods should be preferred to combat this species. Results: The purpose of this study was to evaluate the efficacy of Beauveria bassiana isolate (GOPT-331) and Metarhizium brunneum isolates (ORP-13 and ORP-18) against the second and fourth larval instars of T. wilkinsoni under laboratory conditions. T. wilkinsoni eggs were collected from pine trees at Ondokuz Mayıs University in Samsun, Turkey, in 2021, and the second and fourth larval instars were used for the experiment. Two milliliters of a spray of the three fungal isolates was applied to the larvae for each concentration (1 × 105–1 × 108 conidia ml−1). The mortality rates of GOPT-331, ORP-13, and ORP-18 were changed between 91.1 and 100% for the second-instar larvae and between 86.6 and 97.7% for the fourth-instar larvae at 1 × 108 conidia ml−1. The ORP-13 isolate showed the lowest LC50 values. Conclusion: In conclusion, it is suggested that all the three isolates were virulent to T. wilkinsoni and can be used for biological control of this species. The promising results from the study were obtained from trials conducted under controlled laboratory conditions, and it will be critical to explore the potential of these promising entomopathogenic fungi in field conditions. [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.

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]

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. [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]

Abstract Background Pathogenicity of the entomopathogenic fungi (EPF), isolated from soil samples collected from Ordu Province, Turkey, was evaluated on the second-instar larvae of the cotton leaf worm Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) under laboratory conditions. Results Firstly, single-concentration response tests were conducted in order to determine the efficacy of the 64 isolates on S. littoralis larvae at the concentration of 1 × 108 conidia/ml. The five isolates displaying the highest mortality rates in single-concentration response tests, ORU-50, ORM-40, ORP-13, ORP-27 and ORM-48 (which included Beauveria bassiana, Metarhizium brunneum and Clonostachys rogersoniana), were subjected to concentration–response tests at the concentrations of 1 × 105–1 × 109 conidia/ml. The lowest LC50 and LC90 values were recorded at ORP-27 with 1.68 × 107 and 4.60 × 108 conidia/ml, respectively, followed by ORP-13 and ORM-40. Conclusions Accordingly, it was found that M. brunneum isolates were more effective than B. bassiana and C. rogersoniana against S. littoralis larvae. ORP-27, ORP-13 and ORM-40 of M. brunneum isolates can be a potential biological control agent used against S. littoralis larvae.

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.

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. [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]

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

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.

Malacosoma neustria is a pest for many plant species. Instead of chemical control, eco-friendly biological control methods should be used to combat this species. The purpose of this study was to evaluate the efficacy of isolates of two entomopathogenic fungi, Metarhizium brunneum (ORP-18) and Beauveria bassiana (GOPT-331), used as both oral and spray application on the 4th instar larvae of M. neustria under laboratory conditions. In addition, it was determined how the infection affected the amounts of malondialdehyde, NADPH oxidase and glutathione of the larvae. Both fungal isolates were placed in separate containers at 2 ml for each concentration (1x105, 1x106, 1x107 and 1x108 conidia/ml) in the oral group and contaminated all over the Elaeagnus rhamnoides leaves and then the contaminated leaves were offered to ten larvae in each container. For the spray group, 2 ml of spray was applied to every ten larvae placed in the containers for each concentration. As a result, it was found that both isolates killed the larvae. The ORP-18 isolate caused higher mortality rates than the GOPT-331 isolate. The spray application was more lethal than the oral application. With increasing conidial concentrations of both isolates, the amounts of malondialdehyde and NADPH oxidases increased, while the glutathione amounts decreased, which indicated the adverse effects of reactive oxygen species (ROS) on larval survival. On the basis of the results, it is recommended to use the ORP-18 isolate for the control of larvae of M. neustria. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [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]

Background: Pathogenicity of the entomopathogenic fungi (EPF), isolated from soil samples collected from Ordu Province, Turkey, was evaluated on the second-instar larvae of the cotton leaf worm Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) under laboratory conditions. Results: Firstly, single-concentration response tests were conducted in order to determine the efficacy of the 64 isolates on S. littoralis larvae at the concentration of 1 × 108 conidia/ml. The five isolates displaying the highest mortality rates in single-concentration response tests, ORU-50, ORM-40, ORP-13, ORP-27 and ORM-48 (which included Beauveria bassiana, Metarhizium brunneum and Clonostachys rogersoniana), were subjected to concentration–response tests at the concentrations of 1 × 105–1 × 109 conidia/ml. The lowest LC50 and LC90 values were recorded at ORP-27 with 1.68 × 107 and 4.60 × 108 conidia/ml, respectively, followed by ORP-13 and ORM-40. Conclusions: Accordingly, it was found that M. brunneum isolates were more effective than B. bassiana and C. rogersoniana against S. littoralis larvae. ORP-27, ORP-13 and ORM-40 of M. brunneum isolates can be a potential biological control agent used against S. littoralis larvae. [ABSTRACT FROM AUTHOR]

There is a critical need to develop effective, high-quality, fungal-based biopesticides for use against ticks. Dermacentor albipictus, the winter tick, is a one-host species that can cause mortality in large ungulates, particularly moose (Alces alces). The larval stage of this species aggregates on the ground in a prolonged, quiescent state during the summer and then quests for hosts in autumn. This behaviour offers an opportunity to treat a vulnerable life stage with entomopathogenic fungi in an environment where both are acclimated. We produced granular formulations of Metarhizium brunneum (Petch) and Metarhizium anisopliae (Metschn.) Sorokin, and tested their efficacy against winter tick larvae under semi-natural conditions within the laboratory. When granules were applied at rates of 5.0 × 105 and 1.5 × 107 conidia/cm2, mortality ranged from 53–91% and 58–98% after nine weeks, respectively. Overall, the granular formulations had a similar ability to cause mortality to winter tick larvae regardless of application rate. Fungal granules persisted for nine weeks post application at concentrations of 9 × 104–6 × 105 CFU/g. These results demonstrate the potential of granular formulations of Metarhizium to persist and kill a one-host tick whose larvae have a prolonged off-host stage. [ABSTRACT FROM AUTHOR]

Aim of study: Entomopathogenic fungi (EPF) are biocontrol agents, plant growth promoters, and increase tolerance to biotic-abiotic stresses. In this study we investigated the factors associated to the application method, which are crucial for the interaction between the fungus and the host plant at initial crop growth stages. Area of study: The study was performed in Cordoba (Spain) Material and methods: Three experiments were performed to investigate: (i) the effect of different concentrations of the surfactant Tween® 80 (0, 0.5, 1, 5, and 10%) on wheat seed coating with conidia of Metarhizium brunneum and seed and conidia viability; (ii) the performance of wheat seedlings at first growth stages after their inoculation with Beauveria bassiana or M. brunneum via seed coating or soil drenching; and (iii) the role of soil sterilization and seed disinfection on leaf concentration of chlorophyll (SPAD) and B. bassiana or M. brunneum colonization. Main results: Tween® 80 concentration linearly improved seed coating (up to 127%) without altering wheat seeds and fungal conidia germination. Seedling length of inoculated plants was significantly increased with B. bassiana and M. brunneum (67% and 46%, respectively) via seed coating. Seed disinfection was key to achieve an enhancement in wheat SPAD (10-18%) with B. bassiana or M. brunneum concerning Control, that combined with sterilization of soil showed the highest endophyte colonization rates (up to 83.3% with both fungi) Research highlights: The surfactant concentration, application method, seed disinfection, and soil sterilization are key parameters to improve the potential benefits on the EPF-plant relationship.

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.

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.

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.

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. [ABSTRACT FROM AUTHOR]

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. [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]

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.

The use of entomopathogenic fungi for pest control is gaining increasing attention. These fungi act as contact biological insecticides but also via endophytic colonization of targeted crops. In addition, the joint use of entomopathogenic fungi and natural enemies hold potential in many pest control programs. Here, we evaluated in vitro and in planta multitrophic interactions among the endophytic fungus Metarhizium brunneum colonizing melon (Cucumis melo) plant, the parasitoid Hyposoter didymator and the pest Spodoptera littoralis. In all experiments, total mortality of S. littoralis larvae was significantly affected by the presence of the parasitoid; the treatments including the parasitoid achieved the highest mortality rates both in vitro and in planta. Simultaneous exposure to the fungus and the parasitoid did not significantly increase the total mortality of S. littoralis larvae than the parasitoid alone. The time between exposure to fungus and parasitoid attack did not affect S. littoralis mortality, nor parasitism (parasitoid) or infection rates (fungus). However, the parasitoid showed a significant preference for larvae fed on control plants (24.4% parasitism) compared with larvae fed on fungus-colonized plants (4.4%). A histological study of S. littoralis larvae simultaneously attacked by H. didymator and M. brunneum showed, for the first time, the coexistence of both agents within the same host; parasitoid larvae developed inside the host despite fungal colonization. This provides key information about intra-host interactions between two important biological control agents when used together for S. littoralis control. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]