Novel tactics for controlling insect pests in perennial fruit and nut crops are needed because target pests often display decreased susceptibility to chemical controls due to overreliance on a handful of active ingredients and regulatory issues. As an alternative to chemical controls, entomopathogenic fungi could be utilized as biological control agents to manage insect pest populations. However, development of field ready products is hampered by a lack of basic knowledge. Development of field ready products requires collecting, screening, and characterizing a greater variety of potential entomopathogenic fungal species and strains. Creation of a standardized research framework to study entomopathogenic fungi will aid in identifying the potential mechanisms of biological control activity that fungi could possess, including antibiotic metabolite production; strains and species best suited to survive in different climates and agroecosystems; and optimized combinations of entomopathogenic fungi and novel formulations. This mini review therefore discusses strategies to collect and characterize new entomopathogenic strains, test different potential mechanisms of biocontrol activity, examine ability of different species and strains to tolerate different climates, and lastly how to utilize this information to develop strains into products for growers.

Entomopathogenic fungi are valuable sources of biological pesticides, with conidial yield and quality being pivotal factors determining their broad applications. AzaR, a fungus-specific zinc-cluster transcription factor, is known to regulate the biosynthesis of polyketone secondary metabolites in Aspergillus niger; however, its role in pathogenic fungi remains unclear. This study investigated the role of MaAzaR in the growth, development, and environmental tolerance of Metarhizium acridum. MaAzaR deletion slowed down conidial germination rate, caused reduction in conidial yield, lowered fungal tolerance to UV radiation, did not affect fungal heat-shock tolerance, and increased fungal sensitivity to the cell-wall-destructive agent calcofluor white. Furthermore, MaAzaR deletion transformed microcycle conidiation to normal conidiation on the microcycle conidiation medium. Transcription profile analysis demonstrated that MaAzaR could regulate transformation of the conidiation pattern by controlling the expression of genes related to cell division, mycelium growth and development, and cell wall integrity. Thus, this study identified a new gene related to fungal conidiation and environmental tolerance, enriching our understanding of the molecular mechanism of microcycle conidiation and providing theoretical support and genetic resources for the development of high-yielding strains.

This study aimed to isolate and characterize a native strain of Beauveria bassiana, coded as Bv065, showcasing its potential as a biological control agent targeting the palm weevil Dynamis borassi. Originating from a naturally infected D. borassi specimen collected in southwestern Colombia, the fungus underwent molecular identification and was identified as B. bassiana, exhibiting high sequence similarity with known reference strains. The physiological characterization revealed that Bv065 thrived within a temperature range of 25 to 30 °C and a pH range of 6 to 9. Moreover, the key carbon sources that allow optimal growth of the strain were identified through metabolic profiling, including sucrose, D-mannose, and γ-amino-butyric acid. These findings offer strategic insights for scalability and formulation methodologies. Additionally, enzymatic analyses unveiled robust protease activity within Bv065, crucial for catalysing insect cuticle degradation and facilitating host penetration, thus accentuating its entomopathogenic potential. Subsequent evaluations exposed Bv065\'s pathogenicity against D. borassi, causing significant mortality within nine days of exposure, albeit exhibiting limited effectiveness against Rhynchophorus palmarum. This study underscores the importance of understanding optimal growth conditions and metabolic preferences of B. bassiana strains for developing effective biopesticides. The findings suggest Bv065 as a promising candidate for integrated pest management strategies in neotropical regions, particularly for controlling palm weevil infestations in coconut and peach palm cultivation. Future research avenues include refining mass production methodologies, formulating novel delivery systems, and conducting comprehensive field efficacy trials to unlock the full potential of Bv065 in fostering sustainable pest management practices. Overall, this study contributes to the growing body of knowledge on entomopathogenic fungi and their pivotal role in biological control, offering nuanced perspectives on eco-friendly alternatives to conventional insecticidal interventions.

Greenhouse whitefly (Trialeurodes vaporariorum) is a major global pest, causing direct damage to plants and transmitting viral plant diseases. Management of T. vaporariorum is problematic because of widespread pesticide resistance, and many greenhouse growers rely on biological control agents to regulate T. vaporariorum populations. However, these are often slow and vary in efficacy, leading to subsequent application of chemical insecticides when pest populations exceed threshold levels. Combining chemical and biological pesticides has great potential but can result in different outcomes, from positive to negative interactions. In this study, we evaluated co-applications of the entomopathogenic fungi (EPF) Beauveria bassiana and Cordyceps farinosa and the chemical insecticide spiromesifen in laboratory bioassays. Complex interactions between the EPFs and insecticide were described using an ecotoxicological mixtures model, the MixTox analysis. Depending on the EPF and chemical concentrations applied, mixtures resulted in additivity, synergism, or antagonism in terms of total whitefly mortality. Combinations of B. bassiana and spiromesifen, compared to single treatments, increased the rate of kill by 5 days. Results indicate the potential for combined applications of EPF and spiromesifen as an effective integrated pest management strategy and demonstrate the applicability of the MixTox model to describe complex mixture interactions.

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.

(1) Background: The mycophagous mite, Tyrophagus putrescentiae, was found to feed on entomopathogenic fungi (EPF) in our previous experiments, which seriously impacted the culture and preservation of fungal strains. Therefore, it is necessary to investigate the biological characteristics of the occurrence and damage to EPF. (2) Methods: The mite\'s growth and development and feeding preference were surveyed by comparative culture and observation; also, optical and electronic microscopies were employed. (3) Results: T. putrescentiae could survive normally after being fed on seven EPF species, including Purpureocillium lilacinum, Marquandii marquandii, Cordyceps fumosorosea, Beauveria bassiana, Metarhizium flavoviride, Lecanicillium dimorphum, and Metacordyceps chlamydosporia. The first four fungi were the mite\'s favorites with their greater feeding amount and shorter developmental duration. Interestingly, the mite could also feed on Metarhizium anisopliae and Metarhizium robertsii, but this led to the mite\'s death. After feeding on M. anisopliae and M. robertsii, the mites began to die after 24 h, and the mortality rate reached 100% by 72 h. Observation under optical microscopy and scanning electron microscopy revealed that the conidia of M. anisopliae and M. robertsii adhered to the mite\'s surface, but there was no evidence of penetration or invasion. However, dissection observation indicated that the two Metarhizium species germinate and grow within the mite\'s digestive tract, which implies that Metarhizium generalists with broad-spectrum hosts and the production of destruxins have acaricidal activity toward the mycophagous mites.

Entomopathogenic fungi have been considered potential biological control agents against the fall armyworm Spodoptera frugiperda (J. E. Smith), the world\'s most important pest of maize. In this study, we evaluated the natural infection, molecular characteristics, and biological activity of Metarhizium rileyi (Farlow) isolated from S. frugiperda larvae of this insect, collected from maize crops in five Mexican locations. Natural infection ranged from 23% to 90% across all locations analyzed. Twenty-four isolates were evaluated on S. frugiperda second instars at a concentration of 1.0 × 108 conidia/mL, causing 70% to 98.7% mortality and 60.5% to 98.7% sporulation. Isolates T9-21, Z30-21, PP48-21, and L8-22 were selected to determine their phylogenetic relationships by β-tubulin gene analysis and to compare median lethal concentration (CL50), median lethal time (LT50), and larval survival. These isolates were grouped into three clades. The T9-21, PP48-21, and J10-22 isolates were closely related (clade A), but phylogenetically distant from Z30-21 (clade B) and L8-22 (clade C) isolates. These genetic differences were not always reflected in their pathogenicity characteristics since no differences were observed among the LC50 values. Furthermore, isolates T9-21, J10-22, and L8-22 were the fastest to kill S. frugiperda larvae, causing lower survival rates. We conclude that native M. rileyi isolates represent an important alternative for the biocontrol of S. frugiperda.

Thrips biocontrol research in greenhouse crops has focused primarily on western flower thrips (WFT; Frankliniella occidentalis). However, recent outbreaks of onion thrips (OT; Thrips tabaci) in Ontario, Canada, demonstrate that biocontrol-based IPM programs for WFT do not control OT sufficiently to prevent crop losses. A lack of comparative studies makes it difficult to determine which program components for WFT are failing for OT. We conducted several laboratory trials examining the extent to which commercial biocontrol products kill OT compared to WFT. These included phytoseiid mites (Amblyseius swirskii, Neoseiulus cucumeris, Amblydromalus limonicus, Iphiseius degenerans), a large generalist predator (Orius insidiosus), an entomopathogenic fungus (Beauveria bassiana strain GHA), and entomopathogenic nematodes (Steinernema feltiae, S. carpocapsae, Heterorhabditis bacteriophora). In no-choice trials, A. swirskii and O. insidiosus consumed more OT than WFT (first instars and adults, respectively). In choice trials, A. swirskii, N. cucumeris, and O. insidiosus consumed more OT than WFT. Steinernema feltiae caused higher mortality in OT than WFT. There was no difference in mortality between thrips species exposed to other biocontrol agents. This suggests available tools have the potential to manage OT as well as WFT. Possible explanations why this potential is not realized in commercial settings are explored.

Entomopathogenic fungi, often acknowledged primarily for their insecticidal properties, fulfill diverse roles within ecosystems. These roles encompass endophytism, antagonism against plant diseases, promotion of the growth of plants, and inhabitation of the rhizosphere, occurring both naturally and upon artificial inoculation, as substantiated by a growing body of contemporary research. Numerous studies have highlighted the beneficial aspects of endophytic colonization. This review aims to systematically organize information concerning the direct (nutrient acquisition and production of phytohormones) and indirect (resistance induction, antibiotic and secondary metabolite production, siderophore production, and mitigation of abiotic and biotic stresses) implications of endophytic colonization. Furthermore, a thorough discussion of these mechanisms is provided. Several challenges, including isolation complexities, classification of novel strains, and the impact of terrestrial location, vegetation type, and anthropogenic reluctance to use fungal entomopathogens, have been recognized as hurdles. However, recent advancements in biotechnology within microbial research hold promising solutions to many of these challenges. Ultimately, the current constraints delineate potential future avenues for leveraging endophytic fungal entomopathogens as dual microbial control agents.

(1) Background: In addition to the microclimate, host availability, and tick microbiota, soil environmental microorganisms can affect tick populations. This study aimed to (1) determine the presence and diversity of entomopathogenic fungi (EF) in forests, where ticks are abundant, and (2) estimate the effectiveness of the isolated EF strains against Ixodes ricinus. (2) Methods: EF were isolated using the trap insect method from soil collected from tick sites. A bioassay was used to estimate the effectiveness of EF against ticks. (3) Results: The presence of EF was found in all tested forest habitat types. A total of 53 strains belonging to the genera Metarhizium, Beauveria, and Isaria were isolated. All the six strains subjected to the bioassay showed potential efficacy against both adult and nymphal stages of I. ricinus; however, the strains differed in their effectiveness. The most effective isolate against I. ricinus was the soil environmental strain of Metarhizium anisopliae. (4) Conclusion: The study indicates that tick habitats can be the source of entomopathogenic fungi, which have a lethal effect on ticks, as demonstrated in preliminary laboratory tests with I. ricinus. However, for practical use, extensive field tests and further research on application methods and long-term effects are necessary to develop effective and sustainable tick management strategies.