Improved control of Trialeurodes vaporariorum using mixture combinations of entomopathogenic fungi and the chemical insecticide spiromesifen.

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.

Isolation and characterization of a native strain of the entomopathogenic fungus Beauveria bassiana for the control of the palm weevil Dynamis borassi (Coleoptera: Curculionidae) in the neotropics.

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.

Metabolomic profiling of virulent and non-virulent Beauveria bassiana strains: insights into the pathogenicity of Tetranychus truncatus.

In this study, the pathogenicity of local Beauveria bassiana strains was elucidated using molecular and metabolomics methodologies. Molecular verification of the B. bassiana-specific chitinase gene was achieved via phylogenetic analysis of the Bbchit1 region. Subsequent metabolomic analyses employing UPLC-Q-TOF-MS revealed a different number of non-volatile metabolite profiles among the six B. bassiana strains. Bb6 produced the most non-volatile compounds (17) out of a total of 18, followed by Bb15 (16) and Bb12 (15). Similarly, Bb5, Bb8, and Bb21, three non-virulent B. bassiana strains, produced 13, 14, and 14 metabolites, respectively. But unique secondary metabolites like bassianolide and beauvericin, pivotal for virulence and mite management, were exclusively found in the virulent strains (Bb6, Bb12, and Bb15) of B. bassiana. The distinctive non-volatile metabolomic profiles of these strains underscore their pathogenicity against Tetranychus truncatus, suggesting their promise in bio-control applications.

Laboratory Investigations on the Potential Efficacy of Biological Control Agents on Two Thrips Species, Onion Thrips (Thrips tabaci Lindeman) and Western Flower Thrips (Frankliniella occidentalis (Pergande)).

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.

Natural Prevalence, Molecular Characteristics, and Biological Activity of Metarhizium rileyi (Farlow) Isolated from Spodoptera frugiperda (J. E. Smith) Larvae in Mexico.

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 ß-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.

Mycophagous Mite, Tyrophagus putrescentiae, Prefers to Feed on Entomopathogenic Fungi, except Metarhizium Generalists.

(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.

The Identification of the Mitochondrial DNA Polymerase γ (Mip1) of the Entomopathogenic Fungus Metarhizium brunneum.

Replication of the mitochondrial (mt) genome in filamentous fungi is under-studied, and knowledge is based mainly on data from yeasts and higher eukaryotes. In this study, the mitochondrial DNA polymerase γ (Mip1) of the entomopathogenic fungus Metarhizium brunneum is characterized and analyzed with disruption experiments and its in silico interactions with key proteins implicated in mt gene transcription, i.e., mt RNA polymerase Rpo41 and mt transcription factor Mtf1. Disruption of mip1 gene and its partial expression influences cell growth, morphology, germination and stress tolerance. A putative in silico model of Mip1-Rpo41-Mtf1, which is known to be needed for the initiation of replication, was proposed and helped to identify potential amino acid residues of Mip1 that interact with the Rpo41-Mtf1 complex. Moreover, the reduced expression of mip1 indicates that Mip1 is not required for efficient transcription but only for replication. Functional differences between the M. brunneum Mip1 and its counterparts from Saccharomyces cerevisiae and higher eukaryotes are discussed.

Compatibility of the Entomopathogenic Fungus Metarhizium anisopliae (Ascomycota: Hypocreales) and the Predatory Coccinellid Menochilus sexmaculatus (Col.: Coccinellidae) for Controlling Aphis gossypii (Hem.: Aphididae).

Metarhizium anisopliae (Ascomycota: Hypocreales) is an entomopathogenic fungus considered a key factor in developing integrated management of several insect pests on a variety of crops. The predatory coccinellid, Menochilus sexmaculatus (Col.: Coccinellidae), is also an important natural enemy that must be conserved for effective aphid control. Laboratory studies were conducted under controlled conditions to investigate the interaction between M. anisopliae isolate IRN. 1 and the coccinellid predator M. sexmaculatus in combating Aphis gossypii Glover (Hem.: Aphididae). The combined application of M. sexmaculatus and M. anisopliae led to significant reduction in aphid populations. The foraging behavior of M. sexmaculatus notably facilitated the dispersion of M. anisopliae conidia to uninfected plants, resulting 54 ± 1.3% decrease in aphid density after 10 days. In both choice and non-choice experiments, female adult M. sexmaculatus to fungus-infected aphids was offered as prey and avoided as a food source during all starvation periods. However, live and dead non-fungus-infected aphids were fed upon. The result revealed the compatibility between M. sexmaculatus and M. anisopliae, which may provide a sustainable strategy for the effective management of A. gossypii in a cropping system.

Survival dynamics of stick insect and the impact of environmental factors on natural fungal infection during the rainy season.

Phasmatodea, commonly known as stick insects, are recognized as noteworthy pests globally, impacting agriculture and forest ecosystems. Among them, the outbreak of Ramulus mikado has emerged as a notable concern in East Asian forests. Recently, Metarhizium phasmatodeae has been identified as utilizing stick insects as hosts. We have observed evidence of this entomopathogenic fungus infecting stick insects. Given the increase in these occurrences during the rainy period, this study investigated the relationship between the survival of R. mikado and the M. phasmatodeae infection during the rainy seasons of 2022 and 2023. We collected stick insects in two representative forests of the Republic of Korea and examined insect survival, fungal infection, and various environmental factors. No infections were detected in specimens collected in June before the rainy season, but from July onwards, both the mortality of R. mikado and the fungal infection substantially increased. By the last sampling date of each year, 75% (2022), 71.4% (2023) of the specimens were infected, and over 90% of the total individuals succumbed as a result. Fungi isolated from deceased R. mikado were successfully identified as M. phasmatodeae using morphological and taxonomic approaches. Various statistical analyses, including principal component analysis and modeling, revealed a robust association between fungal infection and the survival of stick insects. The results highlight the correlation between mass deaths of stick insects and fungal infection, particularly during the summer rainy season. These findings offer valuable insights for forecasting R. mikado population in the upcoming year and developing effective pest control strategies.

Plant-Entomopathogenic Fungi Interaction: Recent Progress and Future Prospects on Endophytism-Mediated Growth Promotion and Biocontrol.

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.

Molecular taxonomic characterization and infra-specific diversity of entomopathogenic Beauveria bassiana fungi from Argentina.

It has been the aim of this study to molecular-taxonomically identify 15 Beauveria isolates collected from different geographical regions and insect hosts in Argentina and to investigate the levels of inter- and intra-specific diversity across this set of isolates. Based on phylogenetic analyses of EF1A-RPB1-RPB2 concatenated genes and BLOC markers, all Beauveria strains were identify as Beauveria bassiana. Within the B. bassiana clades of both phylogenies, isolates from Argentina were not clustered according to geographic origin or host. The 15 fungal isolates were further analyzed by PCR amplification of the intron insertion hot spot region of the nuclear 28S rRNA encoding sequence. By intron sequence and position, seven different group-I intron combinations termed variants A, B1, B2, C, D, E and F were found in the 15 isolates under study. Variants B1/B2 consisting of a single 28Si2 intron were found in ten isolates, whereas variant A occurred twice and variants C through F were unique across the set of isolates under study. The determination of the different introns and intron combinations in the 28S rRNA gene is a powerful tool for achieving infraspecific differentiation of B. bassiana isolates from Argentina.

Complex nitrogen sources from agro-industrial byproducts: impact on production, multi-stress tolerance, virulence, and quality of Beauveria bassiana blastospores.

We investigated the impact of various complex organic nitrogen sources on the submerged liquid fermentation of Beauveria bassiana, a versatile entomopathogenic fungus known for producing hydrophilic yeast-like single cells called blastospores. Specifically, we examined yeast extract, autolyzed yeast, inactive yeast, cottonseed flour, corn bran, and corn gluten meal as nitrogen compounds with different carbon-to-nitrogen (C:N) ratios. Our comprehensive analysis encompassed blastospore production, tolerance to abiotic stresses, shelf stability after drying, and virulence against mealworm larvae, crucial attributes for developing effective blastospore-based biopesticides. Notably, cottonseed flour emerged as the optimal nitrogen source, yielding up to 2.5 × 109 blastospores/mL within 3 days in a bioreactor. These blastospores exhibited the highest tolerance to heat stress and UV-B radiation exposure. The endogenous C:N ratio in blastospore composition was also impacted by nitrogen sources. Bioassays with mealworm larvae demonstrated that blastospores from cottonseed flour were the most virulent, achieving faster lethality (lower LT50) and requiring a lower inoculum (LC50). Importantly, blastospores produced with cottonseed flour displayed extended viability during storage, surpassing the retention of viability compared to those from autolyzed yeast over 180 days at 4°C. Despite differences in storage viability, both nitrogen sources conferred similar long-term blastospore bioactivity against mealworms. In summary, this research advances our understanding of the crucial impact of complex organic nitrogen selection on the phenotypic traits of blastospores in association with their intracellular C:N ratio, contributing to the production of ecologically fit, shelf-stable, and virulent propagules for effective pest biocontrol programs. IMPORTANCE: Biological control through entomopathogenic fungi provides essential ecological services in the integrated management of agricultural pests. In the context of submerged liquid fermentation, the nutritional composition significantly influences the ecological fitness, virulence and quality of these fungi. This study specifically explores the impact of various complex organic nitrogen sources derived from agro-industrial byproducts on the submerged liquid fermentation of Beauveria bassiana, a versatile entomopathogenic fungus known for producing hydrophilic yeast-like blastospores. Notably, manipulating the nitrogen source during submerged cultivation can influence the quality, fitness, and performance of blastospores. This research identifies cottonseed flour as the optimal low-cost nitrogen source, contributing to increased production yields, enhanced multi-stress tolerance, heightened virulence with extended shelf life and long-term bioactivity. These findings deepen our understanding of the critical role of nitrogen compound selection in liquid media formulation, facilitating the production of ecologically fit and virulent blastospores for more effective pest biocontrol programs.

Novel report on soil infection with Metarhizium rileyi against soil-dwelling life stages of insect pests.

Entomopathogenic fungi are the most effective control remedy against a wide range of medical and agricultural important pests. The present study aimed to isolate, identify, and assess the virulence of Metarhizium rileyi against Spodoptera litura and Spodoptera frugiperda pupae under soil conditions. The biotechnological methods were used to identify the isolate as M. rileyi. The fungal conidial pathogenicity (2.0 × 107, 2.0 × 108, 2.0 × 109, 2.0 × 1010, and 2.0 × 1011 conidia/mL-1) was tested against prepupae of S. litura and S. frugiperda at 3, 6, 9, and 12 days after treatments. Additionally, the artificial soil-conidial assay was performed on a nontarget species earthworm Eudrilus eugeniae, using M. rileyi conidia. The present results showed that the M. rileyi caused significant mortality rates in S. litura pupae (61-90%), and S. litura pupae were more susceptible than S. frugiperda pupae (46%-73%) at 12 day posttreatment. The LC50 and LC90 of M. rileyi against S. litura, were 3.4 × 1014-9.9 × 1017 conidia/mL-1 and 6.6 × 105-4.6 × 1014 conidia/mL-1 in S. frugiperda, respectively. The conidia of M. rileyi did not exhibit any sublethal effect on the adult stage of E. eugeniae, and Artemia salina following a 12-day treatment period. Moreover, in the histopathological evaluation no discernible harm was observed in the gut tissues of E. eugeniae, including the lumen and epithelial cells, as well as the muscles, setae, nucleus, mitochondria, and coelom. The present findings provide clear evidence that M. rileyi fungal conidia can be used as the foundation for the development of effective bio-insecticides to combat the pupae of S. litura and S. frugiperda agricultural pests.

Toxicity of Beauveria bassiana to Bactrocera dorsalis and effects on its natural predators.

Entomopathogenic fungi (EPF) are economical and environmentally friendly, forming an essential part of integrated pest management strategies. We screened six strains of Beauveria bassiana (B1-B6) (Hypocreales: Cordycipitaceae), of which B4 was the most virulent to Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). We further assessed the biological characteristics of strain B4 and the environmental factors influencing its ability to infect B. dorsalis. We also evaluated the effects of B4 on two of the natural predators of B. dorsalis. We found that strain B4 was the most virulent to 3rd instar larvae, pupae, and adult B. dorsalis, causing mortality rates of 52.67, 61.33, and 90.67%, respectively. B4 was not toxic to B. dorsalis eggs. The optimum B4 effects on B. dorsalis were achieved at a relative humidity of 91-100% and a temperature of 25°C. Among the six insecticides commonly used for B. dorsalis control, 1.8% abamectin emulsifiable concentrate had the strongest inhibitory effect on B4 strain germination. B4 spraying affected both natural enemies (Amblyseius cucumeris and Anastatus japonicus), reducing the number of A. cucumeris and killing A. japonicus adults. We found a valuable strain of EPF (B4) that is virulent against many life stages of B. dorsalis and has great potential for the biological control of B. dorsalis. We also provide an important theoretical and practical base for developing a potential fungicide to control B. dorsalis.

Entomopathogenic Fungi in Forest Habitats of Ixodes ricinus.

(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.

The composition and function of bacterial communities in Bombyx mori (Lepidoptera: Bombycidae) changed dramatically with infected fungi: A new potential to culture Cordyceps cicadae.

Cordyceps cicadae (Hypocreales: Cordycipitaceae) is a renowned entomopathogenic fungus used as herbal medicine in China. However, wild C. cicadae resources have been threatened by heavy harvesting. We hypothesised that Bombyx mori L. (Lepidoptera: Bombycidae) could be a new alternative to cultivate C. cicadae due to the low cost of rearing. Bacterial communities are crucial for the formation of Cordyceps and for promoting the production of metabolites. To better understand the bacterial community structure associated with Cordyceps, three Claviciptaceae fungi were used to explore the pathogenicity of the silkworms. Here, fifth-instar silkworms were infected with C. cicadae, Cordyceps cateniannulata (Hypocreales: Cordycipitaceae) and Beauveria bassiana (Hypocreales: Cordycipitaceae). Subsequently, we applied high-throughput sequencing to explore the composition of bacterial communities in silkworms. Our results showed that all three fungi were highly pathogenic to silkworms, which suggests that silkworms have the potential to cultivate Cordyceps. After fungal infection, the diversity of bacterial communities in silkworms decreased significantly, and the abundance of Staphylococcus increased in mummified larvae, which may play a role in the death process when the host suffers infection by entomopathogenic fungi. Furthermore, there were high similarities in the bacterial community composition and function in the C. cicadae and C. cateniannulata infected samples, and the phylogenetic analysis suggested that these similarities may be related to the fungal phylogenetic relationship. Our findings reveal that infection with different entomopathogenic fungi affects the composition and function of bacterial communities in silkworms and that the bacterial species associated with Cordyceps are primarily host dependent, while fungal infection affects bacterial abundance.

Effect of entomopathogenic fungi on behavior and physiology of Solenopsis invicta (Hymenoptera, Formicidae).

In an ant colony, a large number of nestmates with a similar gene pool coexist, making them more vulnerable to pathogenic attacks. These pathogens influence the behavior and physiology of the fire ant Solenopsis invicta Buren. Here, we evaluated the impact of entomopathogenic fungi (EPF) Metarhizium anisopliae on the behavior (locomotion and foraging) and physiology (biological molecules, anti-fungal activity, and survival) of S. invicta. Distance traveled and velocity significantly decreased, while turn angle and angular velocity significantly increased in ants exposed to a higher concentration of M. anisopliae compared to ants exposed to control after 36 h, which showed disturbed locomotion. Fungus infection significantly affected the foraging behavior of ants. Fungus-exposed ants spent significantly less time in the food zone (area with food) than in the inner zone (area without food). The activities of 4 enzymes, peroxidase, glutathione-S-transferase, hydrogen peroxide (H2O2), and carboxylesterase were significantly decreased. In contrast, catalase and anti-fungal activities were increased after fungal exposure compared to the control. The activity of acetylcholinesterase, which hydrolyses the important neurotransmitter acetylcholine, also decreased after fungal application compared to the control. Survival of ants was also significantly reduced after fungus infection compared to the control. Our findings help to understand the influence of M. anisopliae on the behavior and physiology of S. invicta, which will help in the management of S. invicta using the EPF M. anisopliae.

Development of SCAR Markers for Genetic Authentication of Metarhizium acridum.

In this study, molecular typing using Randomly Amplified Polymorphic DNA (RAPD-PCR) was conducted on 16 original isolates of Metarhizium acridum obtained from locusts (Schistocerca piceifrons ssp. piceifrons.) in Mexico (MX). The analysis included reference strains of the genus Metarhizium sourced from various geographical regions. The isolates were identified by phenotypic (macro and micromorphology) and genotypic methods (RAPD-PCR and Amplified Fragment Length Polymorphisms (AFLP), through a multidimensional analysis of principal coordinates (PCoA) and a minimum spanning network (MST). Subsequently, Sequences-Characterized Amplified Region (SCAR) markers were developed for the molecular detection of M. acridum, these markers were chosen from polymorphic patterns obtained with 14 primers via RAPD-PCR. Phenotypic and genotypic characterization identified the MX isolates as M. acridum. Of all the polymorphic patterns obtained, only OPA04 and OPA05 were chosen, which presented species-specific bands for M. acridum, and further utilized to create SCAR markers through cloning and sequencing of the specific bands. The specificity of these two markers was confirmed via Southern hybridization. The SCAR markers (Ma-160OPA-05 and Ma-151OPA-04) exhibit remarkable sensitivity, detecting down to less than 0.1 ng, as well as high specificity, as evidenced by their inability to cross-amplify or generate amplification with DNAs from other strains of Metarhizium (as Metarhizium anisopliae) or different genera of entomopathogenic fungi (Cordyceps fumosorosea and Akanthomyces lecanii). These SCAR markers yield readily detectable results, showcasing high reproducibility. They serve as a valuable tool, especially in field applications.

The Combination of Enzymes and Conidia of Entomopathogenic Fungi against Aphis gossypii Nymphs and Spodoptera frugiperda Larvae.

The slow action of fungi is one of the biggest challenges in using entomopathogenic fungi. A promising alternative to reduce the time of action is to combine conidia with extracellular enzymes. This study aimed to characterize the production of Pr1 subtilisin protease and lipases by Beauveria bassiana and Metarhizium anisopliae in different culture media and to evaluate the efficiency of the enzymatic treatment against Aphis gossypii and Spodoptera frugiperda. The isolates were cultivated in five different liquid cultures, and, after 7 days, the culture was filtered and centrifuged, and the activity of the Pr1 and lipases was measured. The fungi cultured in a Luria-Bertani broth medium had the highest activity of proteases and lipases. The mortality of A. gossypii nymphs treated with conidia 7 days after the treatment was 39% (JEF-410), 76.5% (JEF-492), 74.8% (ERL-836), and 70.9% (JEF-214). The B. bassiana JEF-410 supernatant combined with conidia increased the fungal virulence at day 5 and day 6 after treatment. When S. frugiperda larvae were treated with B. bassiana JEF-492 conidia combined with its supernatant, the time of infection was shorter compared to the larvae treated with conidia only. Once the supernatant was incubated at 37 °C, the relative activity decreased from 100% to 80% after 2 h and to 45% after 24 h. The results suggest that the supernatant of entomopathogenic fungi may be formulated and used as a biopesticide in an efficient strategy for the biological control of pests.

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

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