Researching a local weapons: A potential native fungal biocontrol in Mexico

Researching a local weapons: A potential native fungal biocontrol in Mexico | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Researching a local weapons: A potential native fungal biocontrol in Mexico Humberto Javier López-Macías, Omar Fabián Hernández-Zepeda, Luis Enrique Flores-Pantoja, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6873622/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Entomopathogenic fungi´s (EPFs), represents a successful alternative against pests in various agroecosystems worldwide. However, Mexican agrifood production faces serious challenges due to the incidence of insect pests that have reduced crop yields, leading to the indiscriminate use of organosynthetic pesticides. Therefore, it is necessary to study the potential of native EPFs isolates as an alternative for Integrated Pest Management (IPM). The objective of this study was to sample agricultural soils in Venustiano Carranza and Pajacuarán, Michoacán, Mexico, to isolate EPFs with biocontrol potential and to characterize them pathogenically and at the molecular level. Results Morphological and molecular analyses indicate the genus of the isolate corresponds to Beauveria spp; Metarhizium sp. and Aspergillus spp. Interestingly in the phylogenic analysis, we observed a great genetic distance between Aspergillus isolates. This is consistent with the theory that this genus shows genetic robustness. Conclusions These results indicate the presence of native EPFs in the study areas, which provides a promising basis for the development of new biopesticides, offering a sustainable alternative for agricultural production. Aspergillus spp. Beauveria bassiana Biological control Integrated Pest Management Metarhizium anisopliae Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background About one million insect species are known in nature and of this extensive variety, only one percent are known to be pests in agricultural production (Hussain et al. 2022 ); however, this small percentage causes economic losses of about 10–60% in key food and feed crops such as rice ( Oryza sativa L., 1753), maize ( Zea mays L., 1753), soybean ( Glycine max Merr., 1917) and wheat ( Triticum aestivum L., 1753) (Mateos-Fernández et al. 2022 ). In addition, global warming has been predicted to increase arthropod pest populations and subsequently lead to resistance and further declines in global agronomic production yields, which will hamper current Integrated Pest Management (IPM) efforts, which will evidently impact on the skyrocketing application of organosynthetic pesticides at a rate of more than two million tons of active ingredient per year (Syafrudin et al. 2021 ). For these reasons, it is urgent to generate and adopt other strategies that contribute to the healthy and sustainable production of agricultural crops. Thus, an alternative with high potential is entomopathogenic fungi (EPF), which are cosmopolitan natural enemies of arthropod pests and are highly effective in favoring their decline in agroecosystems with a more ecological approach that includes the self-spreading technique (Gutiérrez-Cárdenas et al. 2019 ; Sharma and Sharma 2021 ). Thus, Beauveria bassiana (Balsamo-Crivelli) Vuillemin 1912, Hirsutella thompsonii FE Fisher 1950, Metarhizium anisopliae Metschnikoff 1879, and M . rileyi (Farl.) Kepler, S.A. Rehner & Humber 2015, are some of the most commonly used species in crop protection programs. All of the above has led to a high level of attention in the use of these agents for the generation of new bioinsecticides due to their high virulence, broad host spectrum and ease of isolation and multiplication. It has been documented that B . bassiana is able to infect more than 700 phytophagous species (Ranesi et al. 2024 ), while M . anisopliae can cause infection to more than 200 species, respectively (Bidochka et al. 2001 ). In fact, these EPFs were recorded into ENTOMOFUN 1.0 have already these data contains 1,791 records. Surprisingly, Aspergillus records were observed, showing the importance to incorporate a fungal that present changes in their ecological role (Gebremariam et al. 2021 ). However, one of the main challenges to overcome in global agroecosystems is that of the different EPF-based bioinsecticides available on the market, it has not been possible to adapt them to the different local agroecological conditions where the target pests are found, which has been the cause of failures in biological control by EPFs, and in the particular case of Mexico, there are several pest species under active surveillance such as the avocado twig borer Copturus aguacatae Kissinger, 1957 (Coleoptera: Curculionidae), blind hen Phyllophaga spp. (Coleoptera: Scarabaeidae), fall armyworm Spodoptera frugiperda (Smith & Abbot, 1797) (Lepidoptera: Noctuidae), corn earworm Helicoverpa armigera (Hübner, 1808) (Lepidoptera: Noctuidae) and red palm weevil Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera: Curculionidae), which require strong and sustainable strategies. Fortunately, this challenge could be overcome through the isolation and identification of native isolates obtained from local sampling for the management of these pests. In this regard, native isolates of EPFs are reported to be more adaptable to the agroecosystems under study, which presumably will have better effects on mortality of target pests on regional pest management (Gebremariam et al. 2021 ). In general, in several studies the most commonly used method for obtaining native isolates of EPFs is based on the sentinel larva technique using Galleria mellonella (Linnaeus, 1758) (Lepidoptera: Pyralidae) as bait (Mantzoukas et al. 2020 ) and once these isolates are obtained, it is necessary to characterize them pathogenically, taxonomically and molecularly as indispensable requirements to distinguish those isolates with the greatest potential to perform IPM with a higher degree of success. Several studies have used the internal transcribed spacer region ITS1-5.8S-ITS4 of ribosomal DNA (DNA-ITS) in molecular identification due to its high accuracy in differentiating isolates of the genus Beauveria spp. and Metarhizium sp. (Serna-Domínguez et al. 2018 ). In short, we perform a phylogenetic analysis for knows the genetic distances between the isolated fungus (Gebremariam et al. 2021 ). Nevertheless, the identification of the isolated fungus revealed three principal genus: Metarhizium sp., Beauveria spp., and Aspergillus spp., these genus were previously reported as an important fungus with invaluable “weapon” for biocontrol uses (Ayaz et al. 2023 ). In the specific case of Aspergillus fungus, recently reported by Chen et al. ( 2024 ) as a possible changes in ecological habits, pass to a phytopathogen and opportunistic habits to capable of infected insects, these news lights provide a direction for future investigations that permit deepened in the genomic contemplations. For these reasons, the aim of the present study was to carry out sampling in agricultural regions belonging of the municipalities of Pajacuarán and Venustiano Carranza, Michoacán, Mexico, to isolate and characterize entomopathogenic fungi at taxonomic and molecular level, as well as to evaluate their pathogenicity on G . mellonella larvae. Materials and methods Biological material Insect rearing and bioassays were carried out in the laboratories of Genómica Alimentaria at Universidad de La Ciénega del Estado de Michoacán de Ocampo (UCEMICH, Mexico). Experimental conditions were controlled at 25 ± 2 °C, with a relative humidity (RH) of 65 ± 10 % and a photoperiod of 16:8 h (light: dark), using a (RTOP Seris ® ) RTOP-260D camera (China). Establishment and rearing of Galleria mellonella Rearing of G. mellonella was obtained commercially from (PetMmal ® ) (State of Mexico, Mexico). Larvae were placed in transparent plastic jars with perforated lids to allow gas exchange (3.8 L) (Uline ® , Mexico) where they were fed ad libitum with the diet described by (Realpe et al. 2007) until the pupal stage. Subsequently, groups of 30 pupae were placed in the plastic containers described above to allow mating and egg laying. Soil sampling and isolation of entomopathogenic fungi Five soil samples were taken from maize, oats ( Avena sativa L., 1753) and alfalfa ( Medicago sativa L., 1753) crops in different fields in the Ciénega region of the state of Michoacán, recording the geographical coordinates and altitude with data provided by a GPS (Garmin ® ), eTrex 10, (Schaffhausen, Switzerland) ( Table 3 ). Samples were excavated to a depth of 10-15 cm and transported in (Ziploc ® ) bags to the Biological Control Laboratory where sentinel larval methodology was followed using five L 5 larvae (< 24 h from moult) of G . mellonella placed in 100 g of soil per sample (Hajek and Eilenberg 2018). The carcasses found were placed in humidity chambers (100 %) based on Petri dishes with distilled water to determine that the cause of mortality was due to EPFs (Gutiérrez-Cárdenas et al. 2019). From larvae infected by mycosis, a simple of the mycelium adhered to the cadaver was collected and initially inoculated in Bengal Rose Agar (RBA), (HIMEDIA ® , USA) medium for inhibit contaminants and facilitated the selective isolation of specific fungi. The cultures were then transferred to Petri dishes containing Potato Dextrose Agar (MCD LAB ® , Mexico) supplement with Yeast Extract one 1 % (PDA + YE) to promote optimal fungal growth and obtain pure cultures of EPFs. Pathogenic characterization of entomopathogenic fungal isolates Newly moulted larvae to L 5 of G . mellonella were inoculated with a concentration of 1 × 10 8 conidia/mL of the EPFs isolates based on the methodology described by (Grewal et al. 1994) which consists of using sterile 100 × 15 mm plastic Petri dishes (Velaquin ® , Mexico) where they were inoculated through spraying by applying six shots with the aid of a manual sprayer (Uline ® , Mexico) emulating the application of chemical control. At the bottom of the Petri dishes, a sterile filter paper disc (Whatman ® , China) was placed with 1 mL of sterile distilled water to maintain the necessary humidity conditions. Subsequently, the larvae were incubated at the conditions described above over the course of five days. During this process, mortality was assessed daily and the corpses obtained were placed in humidity chambers consisting of 150 × 20 mm Petri dishes (Pyrex ® , Germany) with sterile filter paper and moistened with sterilized water to favor fungal sporulation. Four replicates were prepared for each fungal isolate and each replicate contained 10 host larvae (N = 40 larvae per evaluated fungal). A negative control without fungus was added with 10 larvae per replicate (for a total of four replicates, respectively). Morphological identification For morphological characterization, microcultures were performed with blocks of PDA culture medium, which were inoculated with conidia and mycelial fragments collected from pure cultures of each isolated fungus. Subsequently, they were incubated at a temperature of 27-28 °C for 5-7 days. Once the fungus presented reproductive structures, their morphological characteristics were examined by preparing slides with cotton blue lactophenol (10:1). The samples were analyzed under an Axio Scope optical microscope A1 microscope (Carl Zeiss ® , Germany) to study their morphological characteristics, focusing on asexual reproductive structures (conidia and conidiophores) for taxonomic identification according to (Humber 2012). Images were captured with 40 X and 100 X objectives using an AxioCam ICc1 camera (Carl Zeiss ® Microscopy GmbH) and (AxionVision ® ) 4.9.1 software. For morphometric analysis, 30 conidia and 30 conidiophores were measured considering length and width. For data analysis, average values including maximum, minimum, mean and standard deviation were considered using (Microsoft Excel ® ) 2010 software. Molecular characterization. Biological samples and DNA extraction Cultures of the six EPFs strains were performed in (MCD LAB ® ), Mexico nutrient broth (8 g/L) to which 1 % yeast extract was added and incubated at the temperatures described above for 4-7 days. To perform molecular identification of candidate fungi, genomic DNA was first extracted using the DELLAPORTA method (Dellaporta et al. 1983), designed as an extraction method for EPF DNA with some modifications: A tissue sample of 100 μg was obtained, transferred to a tube (Eppendorf ® ) and 500 μL of β-mercaptoethanol (≥ 99.0 % purity, Aldrich ® , Germany) was added, and then vortexed for 5 min (Genie2 ® Scientific Industries, New York). Subsequently, 500 μL of SDS was added to the same tube and vortexed for another 5 min. The tube was incubated at 65 °C in a heating rack (Heidolph ® , Germany) for 20 min. 150 μL of 20 % potassium acetate was added and vortexed for 1 min and incubated at 4 °C for 10 min. Subsequently, it centrifuged at 4 °C for 10 min at 12,000 rpm. 500 μL of the middle part of the supernatant was removed, trying not to take the upper and lower part, and transferred to a new tube, which was centrifuged again at 4 °C for 10 minutes at 12,000 rpm. The supernatant was decanted and only the DNA pellet was left inside the tube. 500 μL of absolute ethanol was added and mixed by immersion and centrifuged again at the previously described conditions. The supernatant was decanted and 500 μL of molecular biology grade isopropanol (≥ 99.0 % purity, USP; Sigma-Aldrich ® , St. Louis, MO) was added. It was mixed by immersion and centrifuged under the same conditions as described above. The supernatant was decanted, and the pellet was resuspended in 100 μL of ultrapure water, molecular biology grade (Invitrogen ® , Grand Island, NY). Finally, the quality and quantity of DNA was checked using a spectrophotometer (NanoDrop Np80, Implen ® , Germany). To verify the DNA integrity, 1.1 % agarose gel electrophoresis was performed 1.1. g of agarose Invitrogen ® , Grand Island, NY) was dissolved in 100 mL of TAE 1X buffer Tris-Acetate-EDTA (TAE). The solution was heated until completely dissolved and 5 μL of ethidium bromide (0.5 μg/mL) (Sigma-Aldrich ® ) was added as a fluorescent dye for the DNA visualization under UV light. 10 μL of each DNA sample was loaded together with 5 μL of TAE loading buffer. A molecular weight marker (DNA-Ladder 1 kb plus, Invitrogen ® ) was used as a reference. Electrophoresis was carried out a 95 V, 400 mA for 35 minutes. The gel was visualized on a photo-documenter (Bio-Rad model Gel Doc ® -XR+). Amplification of ITS regions for molecular identification of fungi Target sequences were amplified by polymerase chain reaction (PCR). Universal primers were used for the amplification of (ITS1: 22.04 nmol, 154_1660837468, IDL7366, TCCGTAGGTGAACCTGCGG) and (ITS4, in concentration of 24.21 nmol, 154_1660837468, IDL7367, CCTCCGCTTATTGATAGC). These oligonucleotides were selected for their ability to amplify intergenic regions transcribed from various eukaryotic organisms. The design of the ITS1 and ITS4 primers is based on the conserved sequences acquired by T4 (OLIGO ® ), respectively with an amplification product of 556 bp (Toju et al. 2012). For to perform PCR we followed a manufacture instruction of (Thermo Scientific ® ) with some modifications: To make a 50 µL PCR mix, 25 µL of PCR Master mix (Thermo Scientific ® ), 5 µL Forward, 5 µL of Reverse, 5 µL of DNA Template, 10 µL of ultrapure water, molecular biology grade, were used. For isolates belonging to the genera Metarhizium sp., while for the fungus genus Beauveria spp. the following concentrations were used: 5 µL of PCR Master mix, 2 µL Forward, 2 µL of Reverse, 1 µL of DNA Template, and 5 µL of ultrapure water, molecular biology grade. An aliquot of DNA was taken and placed next to each pair of oligonucleotides and DNA polymerase (Invitrogen ® , Carlsban, CA, USA). PCR was performed using the following program: an initial denaturation cycle at 95 °C for 2 min, followed by 30 cycles of 95 °C for 1 min, alignment at 60 °C for 1 min, extension at 72 °C for 1 min and a final extension at 72 °C for 5 min. For each gene, the optimal hybridization temperature and number of hybridization cycles were determined to obtain results in the linear range of amplification. A (Bio-Rad ® ) Thermo Cycler C1000 thermal cycler designed for high precision PCR reactions was used. This device has interchangeable thermal blocks that allow working with different formats, such as 96- and 384-well plates or individual tubes. PCR products were separated by electrophoresis on 1.1 % agarose gels (Bio-Rad ® Laboratories Inc., CA, USA). The intensity levels of the bands in the electrophoresis gel were determined with (IMAGE LAB ® ) software (http://www.bio-rad.com/en-tu/product/image-lab-software http://www.bio-rad.com/en-ru/product/image-lab-software). To purify the PCR product, SpinBind filter units were used, five volumes of SpinBind solution were added to each PCR reaction (500 µL for a 100 µL reaction) and mixed by pipetting. If oil was present, the top layer of oil was avoided by transferring the mixture to a SpinBind filtration unit. The samples were centrifuged for 10-30 seconds at 10,000 × g and the flow-through was discarded. After reassembling the filter unit, 300 µl of SpinClean buffer was added and centrifuged again for 30 seconds. The flow through was discarded and a final 60 second centrifugation completely removed the residual liquid. The filter was transferred to a clean tube and 50 µL of sterile water was applied to the membrane, after centrifugation for 60 seconds at 10,000 × g, the filter was discarded, and the purified DNA was collected and stored at -20 °C. Sequencing and phylogram analysis Once the amplification was verified by 1.1 % agarose gel, the amplicons were sent to the Genomic Services Laboratory (LABSERGEN ® ) belonging to the National Laboratory of Genomics for Biodiversity (LANGEBIO, Mexico, CINVESTAV-Irapuato; https://portal.cinvestav.mx/uga-langebio/investigacion/servicios/laboratorio-de-servicios-gen243micos-1) where they were sequenced by the Sanger Dideoxynucleotides method in ABI 3,730-xl DNA Analyzer (Applied Biosystems ® ; www.appliedbiosystems.com). After obtaining the sequences of the EPFs amplifications, they were identified by BLASTN (GenBank, EMBL, DDBJ)- Envir. (http://unite.ut.ee/). All sequences were compared with other sequences from GenBank using BLAST and the best match was recorded and selected, with a maximum identity value (E-value). A sequence from B . bassiana ; Metarhizium spp. and Aspergillus spp. were used as a reference group. The sequences obtained were analyzed, eliminating the low-quality regions in the forward and reverse sequences. Contig assembly was performed using BioEdit software (version 7.0.52). The assembled contigs were subject to a BLAST search against the NCBI database to corroborate their identity and determine the degree of similarity to other previously reported sequences, selecting those with at least 98 % identity. These amplicon´s sequences were compared with five similar sequences of each, selected from public databases, specifically from NCBI, and represented by their GenBank identifiers (ID). The complete DNA sequences of six isolates were edited with Bioedit version 7.0.5.2. and subsequently deposited in the GenBank database, with accession numbers AMP-M2-R2-A; PQ835711, AMP-M2-R1-A; PQ835761, AMP-M7-R2-B; PQ835714, AMP-M2-R2-C; PQ835756, AMP-M7-R1-A; PQ835716, and AMP-M2-R2-B; PQ835762. For phylogenetic analysis of the EPF sequences, the program (MEGA 11 ® ) (Tamura et al. 2021) was used. Consensus trees were constructed with the Consensus program of the (PHYLIP ® ) package version 3.6 using the Neighbor-joining 1000 bootstrap method. Phylogenetic inferences were performed and displayed using MEGA 11. Statistical analysis Mortality data were analyzed by one-way analysis of variance (ANOVA). Prior to analysis, assumptions of homoscedasticity and normal distribution of the data were checked, and mean separations were estimated using a Tukey test ( P ≤ 0.05). To estimate the effects on survival of G . mellonella to EPFs isolates, mortality data were subjected to Kaplan-Meier analysis and Median Time to Death (MTD) values were calculated. All analyses were performed using SPSS for Windows version 24.0 (IBM ® , Inc., Armonk, NY). Results Pathogenic characterization of the native EPF isolates In the present study, out of six isolated EPF strains, two strains belonging to the genus Beauveria were found, one to the genus Metarhizium , and finally, three strains of the genus Aspergillus were found and the exposure of G . mellonella L 5 larvae (< 24 h from moult) to these native EPFs isolates showed significant differences between isolates (F 6,14 = 6.94; P < 0.014) and the percentages of cumulative mortality at the fourth day of evaluation showed the following mortality percentages: AMP-M2-R1-A (100 ± 0.0%), AMP-M2-R2-C (93.33 ± 5.77%), AMP-M2-R2-A (86.67 ± 23.09%), AMP-M7-R2-B (73.33 ± 30.55%), AMP-M7-R1-A (56.67 ± 32.14%) and AMP-M2-R2-B (53.33 ± 23.09%), all higher than those recorded in the negative control whose mortality was 6.67 ± 5.77%, respectively (Fig. 1 ). The obtained MTD values revealed significant differences in the mortality rate of the treatments (χ 2 = 22.672, df = 5, P < 0.001) (Table 1 ). The survival curve (Fig. 2 ) shows how the mortality of the isolates was shown with respect to time. It is worth mentioning that only Metarhizium sp. strain AMP-M2-R1-A causes 50% mortality in larvae within the first 3 days, indicating its rapid and highly virulent effect, which is in agreement with the ANOVA performed. In contrast, the other strains started to show larval mortality close to 50% after 3 days after treatment. The highest mortality rate in all isolates was recorded between 48 and 96 hours. Morphological identification The morphology of the Aspergillus spp. strains was characterized by colonies of velvety texture, with a light-yellow color in the center, followed by a yellowish green color. The mycelium was characterized as filamentous with septate hyphae, conidiophores of variable size, cone-shaped vesicle and smooth wall. Its phialides are uniseridate (single-ended), conidia in a chain, squinulate, globose to rounded, green in color. The dimensions of conidiophores and conidia of the obtained isolates are for AMP-M2-R2-C 8.95 × 3.60 µm and conidia 4.12 × 3.89 µm (Fig. 3 a, b), for AMP-M7-R2-B they were 8.49 × 3.54 µm and 4.54 × 4.23 µm (Fig. 3 c, d), and for AMP-M2-R2-B, 7.93 × 4.07 µm. 4.38 × 3.94 µm (Fig. 3 e, f). Metarhizium sp. (AMP-M2-R1-A), presented white colonies in its early stages of growth until reaching a mature stage of development, a dark olive green color, a powdery texture due to the production of conidia, these presented a cylindrical or ellipsoidal shape measuring 6.70 x 2.59 µm on average (Fig. 4 a). Beauveria spp. strains, presented a cottony white coloration, turning to cream with a slightly yellowish tone, with a cottony texture and on the reverse side of the colony it presented a cream color, light yellow, and an abundant and spongy aerial mycelium that at the end became powdery. The edges of the colonies were regular with slightly lobed parts. The smooth, globose-shaped conidia of AMP-M7-R1-A were 2.45 × 1.97 (Fig. 4 b) and AMP-M2-R2-A measured 2.33 × 1.97 µm (Fig. 4 c) (Table 2 ). Molecular identification and phylogenetic analysis For the PCR amplifications of the universal primers ITS-1 and ITS-4, before this we obtained amplicons from six fungal isolates (Table 3 ) (Fig. 5 ). The concentration of each amplicon was adequate for Sanger-Deoxy-sequencing. The Blast annotation revealed high identity with a principal EPF fungus ( B. bassiana ; Metarhizium sp.) isolated from soils. However, the phylogram analysis reveals three putative Aspergillus spp. fungus (Fig. 6 ). The Aspergillus isolates also agree with the morphology reported for these fungi (see Fig. 3 morphological analysis). In addition, in the phylogram we observe a diversity and different genetic distances between Aspergillus spp. genus. The mayor genetic distances were observed between AMP-M2-R2-A ( Beauveria spp.) and AMP-M2-R2-B ( Aspergillus spp.) with 52% of genetic distances in comparison with AMP-M7-R1-A ( Beauveria spp.) and AMP-M2-R2-B ( Aspergillus spp.) in which the genetic distances are lower by up to 31%. For the Phylogram analysis, we perform a tree obtained by the Maximum Likelihood method from the contig ITS region of the six isolates belonging to the genera Metarhizium , Aspergillus and Beauveria shows well-defined clades (groups), grouping the sequenced fungi together with reference species. However, the sequence of Akanthomyces dipterigenus (Petch) Spatafora, Kepler, Zare & B. Shrestha, 2017 (EF6791177.1) was also used as an out-group sequence reference. Discussion In the IPM, it is essential to carry out continuous bioprospecting aimed at isolating and identifying biocontrol agents of agricultural pests through the strategy of soil sampling and subsequent baiting with sentinel insects, which can be G. mellonella or Tenebrio molitor Linnaeus, 1758 (Coleoptera: Tenebrionidae); in turn, this approach has shown different levels of success on pests of fundamental importance in various countries worldwide (Fofana et al. 2023 ). For driven this approach, six isolates of EPF belonging to the genera Aspergillus spp., Beauveria spp. and Metarhizium sp. were isolated and identified in the present study, respectively. Similar studies in Mexico reveal that EPF species present in local agroecosystems belong to B. bassiana (Hernández-Domínguez et al. 2024 )d anisopliae (Alcantara-Vargas et al. 2020 ), respectively. In this study, the visual characters of isolates belonging to Beauveria spp. were determined, who showed a characteristic creamy white color of colonies and on host corpses, while in isolates belonging to the genus Metarhizium sp. their particular olive green color was determined, as described in other studies (Villamizar et al. 2021 ; Bhadani et al. 2022 ). In our bioassays, all EPF isolates evaluated were found to be pathogenic on G . mellonella L 5 larvae; however, there were significant differences in larval mortality, with isolate AMP-M2-R1-A showing the highest virulence (100%) on the fourth day of evaluation with differences with respect to isolating AMP-M2-R2-B whose mortality was 53.33%, respectively. These results coincide with those reported by (Baki et al. 2021 ), who reported a mortality of larvae of the Colorado potato beetle Leptinotarsa decemlineata (Say, 1824) (Coleoptera: Chrysomelidae) of between 91.7 and 100% at nine days of evaluation. In this case, the asexual structures observed correspond to the species Aspergillus flavus Link, 1809. However, the diameter of the conidia is larger than expected for this species, which has been documented in other species of the genus Aspergillus , such as A . niger and A . versicolor , whose conidia usually have diameters ranging between 2 and 3.5 µm (dos Santos et al. 2020 ). Isolate AMP-M7-R2-B shows these similarities, despite belonging to the species A . flavus . This atypical morphology could lead to confusion with other fungal genera that show conidia of similar size and shape, such as Penicillium spp. and Paecilomyces spp. However, these genera can be ruled out due to the distinctive features of Aspergillus , such as the presence of a well-defined apical vesicle and the arrangement of the phialides, which can be uniseriate or biseriate. Authors such as (dos Santos et al. 2020 ), attribute these changes to genetic variations in isolates from regions with high drought and extreme climates where, in these environments, the rounded shape of the conidia could be related to greater efficiency in their aerial dispersal, which would allow them to move more effectively in hostile ecosystems with low humidity and high temperatures. On the other hand, in relation to Beauveria spp. and Metarhizium sp. the results obtained in this study are in agreement with those previously reported (Ayala-Zermeño et al. 2015 ). The molecular characterization of the phylogenetic tree of the species Beauveria spp., and Metarhizium sp., clearly shows the significant evolutionary relationship in comparison to the genus Aspergillus spp., which shows a distinctive clade, suggesting significant genetic differences in comparison with the genera Beauveria and Metarhizium , which suggest a greater evolutionary affinity between these due to their common adaptive characteristics in their interaction of mortality with their hosts. Currently, a wide variety of native strains of EPFs have been reported in Asia, Europe, North America, and Latin America (Sharma and Sharma 2021 ), and this highlights their potential for biocontrol applications. Specifically, fungi of the genus Aspergillus spp. have shown genomic plasticity. This genus has been investigated for its robust mutation rate, with an average of 5.1 × 10 − 11 mutations and GC to AT changes per 4000 mitoses per asexual cell cycle (Álvarez-Escribano et al. 2019 ). Ecologically, these fungi play diverse roles, including plant pathogens, facultative fungi, and, more recently, entomopathogens. These recent studies demonstrate that even fungi not initially recognized as EPFs can change roles to ensure their fitness, so these findings represented in this work are in agreement with what was recently reported by (Chen et al. 2024 ), where it was observed that strains of A. flavus considered an opportunistic organism, are capable of infecting insects of Diaphania indica (Saunders, 1851) (Lepidoptera: Crambidae) with a mortality rate of up to 70%, demonstrating the high adaptability of the fungus. The isolates were applied at concentrations ranging from 10 4 -10 7 conidia/mL to the ant Dolichoderus thoracicus (Smith, 1860) (Hymenoptera: Formicidae), achieving high mortality levels (100%) by the seventh day with the 10 7 conidia/mL concentration. Furthermore, other authors like Kianifard et al. ( 2023 ), demonstrated that Aspergillus oryzae (Ahlb.) Cohn, 1884 can be transmitted transstadially from larvae to adults of the mosquito Anopheles stephensi Liston, 1901 (Diptera: Culicidae). In this case, it inhibited the formation of oocysts of the parasite Plasmodium berghei (Haemosporida: Plasmodiidae) by secreting antiplasmodial effector peptides within the mosquito's midgut. This opens the possibility of using this fungus to inhibit the development of malaria parasites in A. stephensi . Conclusions This study provides evidence of native EPFs with high potential for use in biocontrol. Furthermore, to our knowledge, we report for the first time in Mexican soils of the state of Michoacán, native strains of Aspergillus spp. with entomopathogenic activity. These strains demonstrate the potential of EPFs for biocontrol use and application in the Ciénega de Chapala region of Michoacán. Abbreviations (EPFs) Entomopathogenic fungi´s (IPM) Integrated Pest Management (PCR) Polymerase Chain Reaction (PDA + YE) Potato Dextrose Agar + Yeast Extract (RBA) Bengal Rose Agar (UCEMICH) Universidad de La Ciénega del Estado de Michoacán de Ocampo Declarations Funding This research was funded by Institute of Science, Technology and Innovation of the Government of Michoacán, Mexico grant number PICIR22-030-C. Thanks are also due to the Secretariat for Science, Humanities, Technology and Innovation. Author Contribution Humberto Javier López-Macías: Data curation; Investigation; Writing—original draft. Omar Fabián Hernández-Zepeda: Formal analysis; Methodology; Validation; Writing—original draft; Writing—review & editing. Luis Enrique Flores-Pantoja: Writing—review & editing; Resources; Software. Ma. Guadalupe Sánchez-Saavedra: Writing—review & editing; Resources; Software. Angélica María Berlanga-Padilla: Investigation; Formal analysis; Methodology; Validation; Writing—review & editing. Oscar Giovanni Gutiérrez-Cárdenas: Formal analysis; Methodology; Software; Validation; Writing—original draft; Writing—review & editing. Isaac Zepeda-Jazo: Funding acquisition; Investigation; Project administration; Resources; Supervision. All authors reviewed the manuscript. Acknowledgement The authors thank Nikté Huerta-Návez, for technical support and Dr. Roberto Montesinos-Matías, for technical support and text revision. References Alcantara-Vargas E, Espitia-López J, Garza-López PM, Angel-Cuapio A (2020) Producción y calidad de conidios de cepas de entomopatógenos del género Metarhizium anisopliae , aislados en zonas agrícolas del estado de México. Rev Mex Biodiv doi:10.22201/ib.20078706e.2020.91.2912. 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Chen C, Borham A, Chen X, Ling H, SU H (2024) Aspergillus flavus as an entomopathogen infecting Diaphania indica and control efficacy across different developmental stages. 1-13. doi:10.21203/rs.3.rs-4296110/v1. Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: Version II. Plant Mol Biol Rep 1:19-21. doi:10.1007/BF02712670. dos Santos RA, Steenwyk JL, Rivero-Menendez O, Mead ME, Silva LP, Bastos RW, Alastruey-Izquierdo A, Goldman GH, Rokas A (2020) Genomic and phenotypic heterogeneity of clinical isolates of the human pathogens Aspergillus fumigatus , Aspergillus lentulus , and Aspergillus fumigatiaffinis . Front Genet 11(2020):1-17. doi:10.3389/fgene.2020.00459. Fofana F, Descombes C, Kouamé AP, Lefort F (2023) Isolation, identification and evaluation of the effects of native entomopathogenic fungi from Côte d’Ivoire on Galleria mellonella . Microorganisms doi:10.3390/microoorganisms11082104. Gebremariam A, Chekol Y, Assefa F (2021) Phenotypic, molecular, and virulence characterization of entomopathogenic fungi, Beauveria bassiana (Balsam) Vuillemin, and Metarhizium anisopliae (Metschn.) Sorokin from soil samples of Ethiopia for the development of mycoinsecticide. Heliyon doi:10.1016/j.heliyon.2021.e07091. Grewal PS, Lewis EE, Gaugler R, Campbell JF (1994) Host finding behaviour as a predictor of foraging strategy in entomopathogenic nematodes. Parasitology 108:207-215. doi:10.1017/S003118200006830X. Gutiérrez-Cárdenas OG, Cortez-Madrigal H, Malo EA, Gómez-Ruíz J, Nord R (2019) Physiological and pathogenical characterization of Beauveria bassiana and Metarhizium anisopliae isolates for management of adult Spodoptera frugiperda . Southwest Entomol 44:409-421. doi:10.3958/059.044.0206. Hajek AE, Eilenberg J (2018) Fungal pathogens of invertebrates. In: Hajek AE, Eilenberg J (eds) Natural enemies, an introduction to biological control, Cambridge University Press, UK. Hernández-Domínguez C, Zamora-Bernardino C, Vázquez-Cruz F, Reyes-López D, Domínguez-Perales LA, Enríquez-García F (2024) Mortalidad de Galleria mellonella L. por Beauveria bassiana (Bálsamo) Vuill (Ascomycota: Hypocreales). Rev Mex Cienc Agríc 15:1-11. doi:10.29312/remexca.v15i4.3146. Humber RA (2012) Preservation of entomopathogenic fungal cultures. In:Lacey LA (ed)Manual of techniques in insect pathology, Academic Press, Washington. Hussain D, Asrar M, Khalid B, Hafeez F, Saleem M, Akhter M, Ahmed M, Ali I, Hanif K (2022) Insect pests of economic importance attacking wheat crop ( Triticum aestivum L.) in Punjab, Pakistan. Intl J Trop Insect Sci 42:9-20. doi:10.1007/s42690-021-00574-9. Khalil AMA, Hashem AH (2018) Morphological changes of conidiogenesis in two Aspergillus species. J Pure Appl Microbiol 12:2041-2048. doi:10.22207/JPAM.12.4.40. Kianifard L, Rafiqi AM, Akcakir O, Aly AS, Billingsley PF, Uysal S (2023) A recombinant Aspergillus oryzae fungus transmitted from larvae to adults of Anopheles stephensi mosquitoes inhibits malaria parasite oocyst development. Sci Rep 13:1-15. doi:10.1038/s41598-023-38654-0. Mantzoukas S, Lagogiannis I, Ntoukas A, Eliopoulos PA, Kouretas D, Karpouzas DG, Poulas K (2020). Trapping entomopathogenic fungi from vine terroir soil samples with insect baits for controlling serious pests. Appl Sci doi:10.3390/app10103539. Mateos-Fernández R, Petek M, Gerasymenko I, Juteršek M, Baebler Š, Kallam K, Moreno-Giménez E, Gondolf J, Nordmann A, Gruden K, Orzaez D, Patron NJ (2022) Insect pest management in the age of synthetic biology. Plant Biotechnol J 20:25-36. doi:10.1111/pbi.13685. Ranesi M, Vitale S, Staropoli A, Di Lelio I, Izzo LG, De Luca MG, Becchimanzi A, Pennacchio F, Lorito M, Woo SL, Vinale F, Turrà D (2024) Field isolates of Beauveria bassiana exhibit biological heterogeneity in multitrophic interactions of agricultural importance. Microbiol Res doi:10.1016/j.micres.2024.127819. Realpe FJ, Bustillo AE, López JC (2007) Optimización de la cría de Galleria mellonella (L.) para la producción de nematodos entomopatógenos parásitos de la broca del café. Cenicafé 58:142-157. Serna-Domínguez MG, Andrade-Michel GY, Arredondo-Bernal HC, Gallou A (2018) Two efficient methods for isolation of high-quality genomic DNA from entomopathogenic fungi. J Microbiol Methods 148:55-63. doi:10.1016/j.mimet.2018.03.012. Sharma R, Sharma P (2021) Fungal entomopathogens: A systematic review. Egypt J Biol Pest Control 31:1-13. doi:10.1186/s41938-021-00404-7. Syafrudin M, Kristanti RA, Yuniarto A, Hadibarata T, Rhee J, Al-onazi WA, Algarni TS, Almarri AH, Al-Mohaimeed AM (2021) Pesticides in drinking water — A review. Int J Environ Res Public Health doi:10.3390/ijerph18020468. Talaei-Hassanloui R, Kharazi-Pakdel A, Goettel M, Mozaffari J (2006) Variation in virulence of Beauveria bassiana isolates and its relatedness to some morphological characteristics. Biocontrol Science and Technology 16:525-34. doi:10.1080/09583150500532758. Tamura K, Stecher G, Kumar S (2021) MEGA11: Molecular evolutionary genetics analysis version 11. Mol Biol Evol 38:3022-3027. doi:10.1093/molbev/msab120. Toju H, Tanabe AS, Yamamoto S, Sato H (2012) High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples. PLoS ONE doi:10.1371/journal.pone.0040863. Villamizar LF, Barrera G, Hurst M, Glare TR (2021) Characterization of a new strain of Metarhizium novozealandicum with potential to be developed as a biopesticide. Mycology 12:261-278. doi:10.1080/21501203.2021.1935359. Tables Table 1 Estimated MTD values in G. mellonella L 5 larvae (24 h from moult) inoculated by spraying with the entomopathogenic fungal isolates at a concentration of 1 × 10 8 conidia/mL (< 24 h from molt) at a concentration of 1 × 10 8 conidia/mL. Treatment MTD 1 (days) 95 % confidence limits AMP-M2-R1-A 2.50 a 2.25 2.74 AMP-M2-R2-C 3.26 a 2.83 3.69 AMP-M7-R2-B 3.33 b 2.52 4.14 AMP-M2-R2-A 3.76 c 3.24 4.28 AMP-M7-R1-A 4.30 d 3.42 5.17 AMP-M2-R2-B 4.46 d 3.58 5.35 1 MTD: Median time to death. Values followed by different letters indicate statistically significant differences ( P ≤ 0.05) in G. mellonella L 5 mortality. Table 2 Morphometric analysis of Aspergillus spp., Beauveria spp., and Metarhizium sp. based on structures obtained from PDA medium-based microcultures. Acronym Conidia (average) Phialide taken from the base (average) Length Width Length Width AMP-M2-R2-A 2.33 ± 0.23 1.97 ± 0.18 7.84 ± 2.87 1.05 ± 0.34 AMP-M2-R1-A 6.70 ± 0.40 2.59 ± 0.13 5.69 ± 0.70 2.23 ± 0.33 AMP-M7-R2-B 4.54 ± 0.48 4.23 ± 0.42 8.49 ± 1.12 3.54 ± 0.38 AMP-M2-R2-C 4.12 ± 0.29 3.89 ± 0.28 9.39 ± 1.64 3.18 ± 0.42 AMP-M7-R1-A 2.45 ± 0.22 1.97 ± 0.19 7.69 ± 2.86 0.99 ± 0.34 AMP-M2-R2-B 4.38 ± 0.60 3.94 ± 0.53 8.42 ± 1.05 3.73 ± 0.79 Comparative studies Length Width Length Width (Talaei-Hassanloui et al. 2006) 2.4 ± 0.33 5.8 ± 1.0 Nd 1 Nd 1 (Ayala-Zermeño et al. 2015) 4.52 ± 0.28 1.8 ± 0.19 6.22 ± 0.65 2.15 ± 0.26 (Khalil and Hashem 2018) 2.50 2.50 6.75 2.25 1 Nd: Not described. Table 3 Sampling areas of entomopathogenic fungi in the Ciénega de Michoacán, Mexico. Geographic origin, coordinates and altitude expressed in meters above sea level are indicated. Strain Blast annotation NCBI ID Geographical origin Coordinates (N, W) Altitude (masl) AMP-M2-R2-A Beauveria sp . PQ835711 Venustiano Carranza 20°06.82822" 102°30.370” 1528 AMP-M2-R1-A Metarhizium sp . PQ835761 Pajacuarán 20°07.043" 102°30.384" 1527 AMP-M7-R2-B Aspergillus sp . PQ835714 Pajacuarán 20°06.636 102°45.158" 1573 AMP-M2-R2-C Aspergillus sp . PQ835756 Venustiano Carranza 20°06.371" 102°45.193" 1526 AMP-M7-R1-A Beauveria sp. PQ835716 Pajacuarán 20°04.951 102°27.556" 1533 AMP-M2-R2-B Aspergillus sp. PQ835762 Pajacuarán 20°07.043" 102°30.384" 1527 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6873622","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":476781724,"identity":"8e8cb17f-c3a9-43ab-8108-b54e91ed197a","order_by":0,"name":"Humberto Javier López-Macías","email":"","orcid":"","institution":"Universidad de La Ciénega del Estado de Michoacán de Ocampo, C. 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P. 59103","correspondingAuthor":true,"prefix":"","firstName":"Isaac","middleName":"","lastName":"Zepeda-Jazo","suffix":""}],"badges":[],"createdAt":"2025-06-11 16:08:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6873622/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6873622/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85659825,"identity":"5a53bfdb-c4e4-4bdf-8185-f4a74598d73c","added_by":"auto","created_at":"2025-06-30 11:26:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":71889,"visible":true,"origin":"","legend":"\u003cp\u003eCumulative larval mortality of \u003cem\u003eG. mellonella\u003c/em\u003e L\u003csub\u003e5\u003c/sub\u003e larvae (\u0026lt; 24 h from moult) spray-inoculated with the entomopathogenic fungal isolates at a concentration of 1 × 10\u003csup\u003e8\u003c/sup\u003e conidia/mL. Data are the mean (± standard error) of four replicates/treatment (n = 10). One-way ANOVA. Different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e ≤ 0.05).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6873622/v1/277ff63f256267252c26b1b4.jpg"},{"id":85660235,"identity":"8f19a1e6-c1aa-41d2-9a68-c3d385432adf","added_by":"auto","created_at":"2025-06-30 11:34:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":49755,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival curve of L\u003csub\u003e5 \u003c/sub\u003elarvae (\u0026lt; 24 h from moult) of \u003cem\u003eG. mellonella\u003c/em\u003e with the entomopathogenic fungal isolates at a concentration of 1 × 10\u003csup\u003e8\u003c/sup\u003e conidia/mL.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6873622/v1/c69ed370ee00d2a665180472.jpg"},{"id":85659820,"identity":"1fd25ee8-7245-4c9c-84d0-75540e3d8f3b","added_by":"auto","created_at":"2025-06-30 11:26:43","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":74560,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival curve of L\u003csub\u003e5 \u003c/sub\u003elarvae (\u0026lt; 24 h from moult) of \u003cem\u003eG. mellonella\u003c/em\u003e with the entomopathogenic fungal isolates at a concentration of 1 × 10\u003csup\u003e8\u003c/sup\u003e conidia/mL.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6873622/v1/14f9f2a6a6d7b6a965c43edc.jpg"},{"id":85659827,"identity":"87fee4a0-2e1a-44d3-aa30-7a2a0f28db67","added_by":"auto","created_at":"2025-06-30 11:26:43","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":62354,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eAspergillus\u003c/em\u003e spp. with complete morphological structure and vesicle with uniseriate phialides with chains of conidia. (A) and (B), Strain AMP-M2-R2-C (C), (D); Strain AMP-M7-R2-B, (E), (F) strain AMP-M2-R2-B. Bars figures (A, C and E) = 10 μm and figures (B, D and F) = 5 μm.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6873622/v1/25cbfd33a2dbab44e81dd118.jpg"},{"id":85660237,"identity":"47682d05-4db2-4ce5-976b-bd1ec5c10737","added_by":"auto","created_at":"2025-06-30 11:34:43","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":31918,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology of conidia of the genus. (A) \u003cem\u003eMetarhizium\u003c/em\u003e sp. isolate AMP-M2-R1-A; (B) and (C) \u003cem\u003eBeauveria\u003c/em\u003e spp. isolates AMP-M7-R1-A and AMP-M2-R2-A, respectively. Bars figures (A, B and C) = 5 μm.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6873622/v1/90634d510d3566262ae6c2da.jpg"},{"id":85659824,"identity":"834a87f8-caba-4115-ae68-8ee83aaaf621","added_by":"auto","created_at":"2025-06-30 11:26:43","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":92962,"visible":true,"origin":"","legend":"\u003cp\u003eImage of agarose gel stained at 1.1 % with ethidium bromide under ultraviolet light. Each band represents a DNA amplicon of the DNA amplicon of the ITS 1 region, in the first line is the 1 kb (thousand base pairs) molecular marker.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6873622/v1/a9198018b3eedb8ea47ce962.jpg"},{"id":87734214,"identity":"8b3eb0da-45e1-4038-b8c5-534cef186a65","added_by":"auto","created_at":"2025-07-28 12:09:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1404206,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6873622/v1/33662dc3-2fb1-4213-b861-372d082d0162.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Researching a local weapons: A potential native fungal biocontrol in Mexico","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAbout one million insect species are known in nature and of this extensive variety, only one percent are known to be pests in agricultural production (Hussain et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e); however, this small percentage causes economic losses of about 10\u0026ndash;60% in key food and feed crops such as rice (\u003cem\u003eOryza sativa\u003c/em\u003e L., 1753), maize (\u003cem\u003eZea mays\u003c/em\u003e L., 1753), soybean (\u003cem\u003eGlycine max\u003c/em\u003e Merr., 1917) and wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L., 1753) (Mateos-Fern\u0026aacute;ndez et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In addition, global warming has been predicted to increase arthropod pest populations and subsequently lead to resistance and further declines in global agronomic production yields, which will hamper current Integrated Pest Management (IPM) efforts, which will evidently impact on the skyrocketing application of organosynthetic pesticides at a rate of more than two million tons of active ingredient per year (Syafrudin et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor these reasons, it is urgent to generate and adopt other strategies that contribute to the healthy and sustainable production of agricultural crops. Thus, an alternative with high potential is entomopathogenic fungi (EPF), which are cosmopolitan natural enemies of arthropod pests and are highly effective in favoring their decline in agroecosystems with a more ecological approach that includes the self-spreading technique (Guti\u0026eacute;rrez-C\u0026aacute;rdenas et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sharma and Sharma \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Thus, \u003cem\u003eBeauveria bassiana\u003c/em\u003e (Balsamo-Crivelli) Vuillemin 1912, \u003cem\u003eHirsutella thompsonii\u003c/em\u003e FE Fisher 1950, \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e Metschnikoff 1879, and \u003cem\u003eM\u003c/em\u003e. \u003cem\u003erileyi\u003c/em\u003e (Farl.) Kepler, S.A. Rehner \u0026amp; Humber 2015, are some of the most commonly used species in crop protection programs. All of the above has led to a high level of attention in the use of these agents for the generation of new bioinsecticides due to their high virulence, broad host spectrum and ease of isolation and multiplication. It has been documented that \u003cem\u003eB\u003c/em\u003e. \u003cem\u003ebassiana\u003c/em\u003e is able to infect more than 700 phytophagous species (Ranesi et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), while \u003cem\u003eM\u003c/em\u003e. \u003cem\u003eanisopliae\u003c/em\u003e can cause infection to more than 200 species, respectively (Bidochka et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn fact, these EPFs were recorded into ENTOMOFUN 1.0 have already these data contains 1,791 records. Surprisingly, \u003cem\u003eAspergillus\u003c/em\u003e records were observed, showing the importance to incorporate a fungal that present changes in their ecological role (Gebremariam et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, one of the main challenges to overcome in global agroecosystems is that of the different EPF-based bioinsecticides available on the market, it has not been possible to adapt them to the different local agroecological conditions where the target pests are found, which has been the cause of failures in biological control by EPFs, and in the particular case of Mexico, there are several pest species under active surveillance such as the avocado twig borer \u003cem\u003eCopturus aguacatae\u003c/em\u003e Kissinger, 1957 (Coleoptera: Curculionidae), blind hen \u003cem\u003ePhyllophaga\u003c/em\u003e spp. (Coleoptera: Scarabaeidae), fall armyworm \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e (Smith \u0026amp; Abbot, 1797) (Lepidoptera: Noctuidae), corn earworm \u003cem\u003eHelicoverpa armigera\u003c/em\u003e (H\u0026uuml;bner, 1808) (Lepidoptera: Noctuidae) and red palm weevil \u003cem\u003eRhynchophorus ferrugineus\u003c/em\u003e (Olivier, 1790) (Coleoptera: Curculionidae), which require strong and sustainable strategies.\u003c/p\u003e \u003cp\u003eFortunately, this challenge could be overcome through the isolation and identification of native isolates obtained from local sampling for the management of these pests. In this regard, native isolates of EPFs are reported to be more adaptable to the agroecosystems under study, which presumably will have better effects on mortality of target pests on regional pest management (Gebremariam et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In general, in several studies the most commonly used method for obtaining native isolates of EPFs is based on the sentinel larva technique using \u003cem\u003eGalleria mellonella\u003c/em\u003e (Linnaeus, 1758) (Lepidoptera: Pyralidae) as bait (Mantzoukas et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and once these isolates are obtained, it is necessary to characterize them pathogenically, taxonomically and molecularly as indispensable requirements to distinguish those isolates with the greatest potential to perform IPM with a higher degree of success. Several studies have used the internal transcribed spacer region ITS1-5.8S-ITS4 of ribosomal DNA (DNA-ITS) in molecular identification due to its high accuracy in differentiating isolates of the genus \u003cem\u003eBeauveria\u003c/em\u003e spp. and \u003cem\u003eMetarhizium\u003c/em\u003e sp. (Serna-Dom\u0026iacute;nguez et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In short, we perform a phylogenetic analysis for knows the genetic distances between the isolated fungus (Gebremariam et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Nevertheless, the identification of the isolated fungus revealed three principal genus: \u003cem\u003eMetarhizium\u003c/em\u003e sp., \u003cem\u003eBeauveria\u003c/em\u003e spp., and \u003cem\u003eAspergillus\u003c/em\u003e spp., these genus were previously reported as an important fungus with invaluable \u0026ldquo;weapon\u0026rdquo; for biocontrol uses (Ayaz et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In the specific case of \u003cem\u003eAspergillus\u003c/em\u003e fungus, recently reported by Chen et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) as a possible changes in ecological habits, pass to a phytopathogen and opportunistic habits to capable of infected insects, these news lights provide a direction for future investigations that permit deepened in the genomic contemplations. For these reasons, the aim of the present study was to carry out sampling in agricultural regions belonging of the municipalities of Pajacuar\u0026aacute;n and Venustiano Carranza, Michoac\u0026aacute;n, Mexico, to isolate and characterize entomopathogenic fungi at taxonomic and molecular level, as well as to evaluate their pathogenicity on \u003cem\u003eG\u003c/em\u003e. \u003cem\u003emellonella\u003c/em\u003e larvae.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eBiological material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInsect rearing and bioassays were carried out in the laboratories of Gen\u0026oacute;mica Alimentaria at Universidad de La Ci\u0026eacute;nega del Estado de Michoac\u0026aacute;n de Ocampo (UCEMICH, Mexico). Experimental conditions were controlled at 25 \u0026plusmn; 2 \u0026deg;C, with a relative humidity (RH) of 65 \u0026plusmn; 10 % and a photoperiod of 16:8 h (light: dark), using a (RTOP Seris\u003csup\u003e\u0026reg;\u003c/sup\u003e) RTOP-260D camera (China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstablishment and rearing of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGalleria mellonella\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRearing of \u003cem\u003eG. mellonella\u003c/em\u003e was obtained commercially from (PetMmal\u003csup\u003e\u0026reg;\u003c/sup\u003e) (State of Mexico, Mexico). Larvae were placed in transparent plastic jars with perforated lids to allow gas exchange (3.8 L) (Uline\u003csup\u003e\u0026reg;\u003c/sup\u003e, Mexico) where they were fed \u003cem\u003ead libitum\u003c/em\u003e with the diet described by (Realpe et al. 2007) until the pupal stage. Subsequently, groups of 30 pupae were placed in the plastic containers described above to allow mating and egg laying.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSoil sampling and isolation of entomopathogenic fungi\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFive soil samples were taken from maize, oats (\u003cem\u003eAvena sativa\u003c/em\u003e L., 1753) and alfalfa (\u003cem\u003eMedicago sativa\u003c/em\u003e L., 1753) crops in different fields in the Ci\u0026eacute;nega region of the state of Michoac\u0026aacute;n, recording the geographical coordinates and altitude with data provided by a GPS (Garmin\u003csup\u003e\u0026reg;\u003c/sup\u003e), eTrex 10, (Schaffhausen, Switzerland) (\u003cstrong\u003eTable 3\u003c/strong\u003e). Samples were excavated to a depth of 10-15 cm and transported in (Ziploc\u003csup\u003e\u0026reg;\u003c/sup\u003e) bags to the Biological Control Laboratory where sentinel larval methodology was followed using five L\u003csub\u003e5\u003c/sub\u003e larvae (\u0026lt; 24 h from moult) of \u003cem\u003eG\u003c/em\u003e. \u003cem\u003emellonella\u003c/em\u003e placed in 100 g of soil per sample (Hajek and Eilenberg 2018). The carcasses found were placed in humidity chambers (100 %) based on Petri dishes with distilled water to determine that the cause of mortality was due to EPFs (Guti\u0026eacute;rrez-C\u0026aacute;rdenas et al. 2019).\u003c/p\u003e\n\u003cp\u003eFrom larvae infected by mycosis, a simple of the mycelium adhered to the cadaver was collected and initially inoculated in Bengal Rose Agar (RBA), (HIMEDIA\u003csup\u003e\u0026reg;\u003c/sup\u003e, USA) medium for inhibit contaminants and facilitated the selective isolation of specific fungi. The cultures were then transferred to Petri dishes containing Potato Dextrose Agar (MCD LAB\u003csup\u003e\u0026reg;\u003c/sup\u003e, Mexico) supplement with Yeast Extract one 1 % (PDA + YE) to promote optimal fungal growth and obtain pure cultures of EPFs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePathogenic characterization of entomopathogenic fungal isolates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNewly moulted larvae to L\u003csub\u003e5\u003c/sub\u003e of \u003cem\u003eG\u003c/em\u003e. \u003cem\u003emellonella\u003c/em\u003e were inoculated with a concentration of 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e conidia/mL of the EPFs isolates based on the methodology described by (Grewal et al. 1994) which consists of using sterile 100 \u0026times; 15 mm plastic Petri dishes (Velaquin\u003csup\u003e\u0026reg;\u003c/sup\u003e, Mexico) where they were inoculated through spraying by applying six shots with the aid of a manual sprayer (Uline\u003csup\u003e\u0026reg;\u003c/sup\u003e, Mexico) emulating the application of chemical control. At the bottom of the Petri dishes, a sterile filter paper disc (Whatman\u003csup\u003e\u0026reg;\u003c/sup\u003e, China) was placed with 1 mL of sterile distilled water to maintain the necessary humidity conditions. Subsequently, the larvae were incubated at the conditions described above over the course of five days. During this process, mortality was assessed daily and the corpses obtained were placed in humidity chambers consisting of 150 \u0026times; 20 mm Petri dishes (Pyrex\u003csup\u003e\u0026reg;\u003c/sup\u003e, Germany) with sterile filter paper and moistened with sterilized water to favor fungal sporulation. Four replicates were prepared for each fungal isolate and each replicate contained 10 host larvae (N = 40 larvae per evaluated fungal). A negative control without fungus was added with 10 larvae per replicate (for a total of four replicates, respectively).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphological identification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor morphological characterization, microcultures were performed with blocks of PDA culture medium, which were inoculated with conidia and mycelial fragments collected from pure cultures of each isolated fungus. Subsequently, they were incubated at a temperature of 27-28 \u0026deg;C for 5-7 days. Once the fungus presented reproductive structures, their morphological characteristics were examined by preparing slides with cotton blue lactophenol (10:1). The samples were analyzed under an Axio Scope optical microscope A1 microscope (Carl Zeiss\u003csup\u003e\u0026reg;\u003c/sup\u003e, Germany) to study their morphological characteristics, focusing on asexual reproductive structures (conidia and conidiophores) for taxonomic identification according to (Humber 2012). Images were captured with 40 X and 100 X objectives using an AxioCam ICc1 camera (Carl Zeiss\u003csup\u003e\u0026reg;\u003c/sup\u003e Microscopy GmbH) and (AxionVision\u003csup\u003e\u0026reg;\u003c/sup\u003e) 4.9.1 software. For morphometric analysis, 30 conidia and 30 conidiophores were measured considering length and width. For data analysis, average values including maximum, minimum, mean and standard deviation were considered using (Microsoft Excel\u003csup\u003e\u0026reg;\u003c/sup\u003e) 2010 software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular characterization. Biological samples and DNA extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCultures of the six EPFs strains were performed in (MCD LAB\u003csup\u003e\u0026reg;\u003c/sup\u003e), Mexico nutrient broth (8 g/L) to which 1 % yeast extract was added and incubated at the temperatures described above for 4-7 days. To perform molecular identification of candidate fungi, genomic DNA was first extracted using the DELLAPORTA method (Dellaporta et al. 1983), designed as an extraction method for EPF DNA with some modifications:\u003c/p\u003e\n\u003cp\u003eA tissue sample of 100 \u0026mu;g was obtained, transferred to a tube (Eppendorf\u003csup\u003e\u0026reg;\u003c/sup\u003e) and 500 \u0026mu;L of \u0026beta;-mercaptoethanol (\u0026ge; 99.0 % purity, Aldrich\u003csup\u003e\u0026reg;\u003c/sup\u003e, Germany) was added, and then vortexed for 5 min (Genie2\u003csup\u003e\u0026reg;\u003c/sup\u003e Scientific Industries, New York). Subsequently, 500 \u0026mu;L of SDS was added to the same tube and vortexed for another 5 min. The tube was incubated at 65 \u0026deg;C in a heating rack (Heidolph\u003csup\u003e\u0026reg;\u003c/sup\u003e, Germany) for 20 min. 150 \u0026mu;L of 20 % potassium acetate was added and vortexed for 1 min and incubated at 4 \u0026deg;C for 10 min. Subsequently, it centrifuged at 4 \u0026deg;C for 10 min at 12,000 rpm. 500 \u0026mu;L of the middle part of the supernatant was removed, trying not to take the upper and lower part, and transferred to a new tube, which was centrifuged again at 4 \u0026deg;C for 10 minutes at 12,000 rpm. The supernatant was decanted and only the DNA pellet was left inside the tube. 500 \u0026mu;L of absolute ethanol was added and mixed by immersion and centrifuged again at the previously described conditions. The supernatant was decanted and 500 \u0026mu;L of molecular biology grade isopropanol (\u0026ge; 99.0 % purity, USP; Sigma-Aldrich\u003csup\u003e\u0026reg;\u003c/sup\u003e, St. Louis, MO) was added. It was mixed by immersion and centrifuged under the same conditions as described above. The supernatant was decanted, and the pellet was resuspended in 100 \u0026mu;L of ultrapure water, molecular biology grade (Invitrogen\u003csup\u003e\u0026reg;\u003c/sup\u003e, Grand Island, NY). Finally, the quality and quantity of DNA was checked using a spectrophotometer (NanoDrop Np80, Implen\u003csup\u003e\u0026reg;\u003c/sup\u003e, Germany). To verify the DNA integrity, 1.1 % agarose gel electrophoresis was performed 1.1. g of agarose Invitrogen\u003csup\u003e\u0026reg;\u003c/sup\u003e, Grand Island, NY) was dissolved in 100 mL of TAE 1X buffer Tris-Acetate-EDTA (TAE). The solution was heated until completely dissolved and 5 \u0026mu;L of ethidium bromide (0.5 \u0026mu;g/mL) (Sigma-Aldrich\u003csup\u003e\u0026reg;\u003c/sup\u003e) was added as a fluorescent dye for the DNA visualization under UV light. 10 \u0026mu;L of each DNA sample was loaded together with 5 \u0026mu;L of TAE loading buffer. A molecular weight marker (DNA-Ladder 1 kb plus, Invitrogen\u003csup\u003e\u0026reg;\u003c/sup\u003e) was used as a reference. Electrophoresis was carried out a 95 V, 400 mA for 35 minutes. The gel was visualized on a photo-documenter (Bio-Rad model Gel Doc\u003csup\u003e\u0026reg;\u003c/sup\u003e-XR+).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAmplification of ITS regions for molecular identification of fungi\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTarget sequences were amplified by polymerase chain reaction (PCR). Universal primers were used for the amplification of (ITS1: 22.04 nmol, 154_1660837468, IDL7366, TCCGTAGGTGAACCTGCGG) and (ITS4, in concentration of 24.21 nmol, 154_1660837468, IDL7367, CCTCCGCTTATTGATAGC). These oligonucleotides were selected for their ability to amplify intergenic regions transcribed from various eukaryotic organisms. The design of the ITS1 and ITS4 primers is based on the conserved sequences acquired by T4 (OLIGO\u003csup\u003e\u0026reg;\u003c/sup\u003e), respectively with an amplification product of 556 bp (Toju et al. 2012).\u003c/p\u003e\n\u003cp\u003eFor to perform PCR we followed a manufacture instruction of (Thermo Scientific\u003csup\u003e\u0026reg;\u003c/sup\u003e) with some modifications: To make a 50 \u0026micro;L PCR mix, 25 \u0026micro;L of PCR Master mix (Thermo Scientific\u003csup\u003e\u0026reg;\u003c/sup\u003e), 5 \u0026micro;L Forward, 5 \u0026micro;L of Reverse, 5 \u0026micro;L of DNA Template, 10 \u0026micro;L of ultrapure water, molecular biology grade, were used. For isolates belonging to the genera \u003cem\u003eMetarhizium\u003c/em\u003e sp., while for the fungus genus \u003cem\u003eBeauveria\u003c/em\u003e spp. the following concentrations were used: 5 \u0026micro;L of PCR Master mix, 2 \u0026micro;L Forward, 2 \u0026micro;L of Reverse, 1 \u0026micro;L of DNA Template, and 5 \u0026micro;L of ultrapure water, molecular biology grade.\u003c/p\u003e\n\u003cp\u003eAn aliquot of DNA was taken and placed next to each pair of oligonucleotides and DNA polymerase (Invitrogen\u003csup\u003e\u0026reg;\u003c/sup\u003e, Carlsban, CA, USA). PCR was performed using the following program: an initial denaturation cycle at 95 \u0026deg;C for 2 min, followed by 30 cycles of 95 \u0026deg;C for 1 min, alignment at 60 \u0026deg;C for 1 min, extension at 72 \u0026deg;C for 1 min and a final extension at 72 \u0026deg;C for 5 min. For each gene, the optimal hybridization temperature and number of hybridization cycles were determined to obtain results in the linear range of amplification. A (Bio-Rad\u003csup\u003e\u0026reg;\u003c/sup\u003e) Thermo Cycler C1000 thermal cycler designed for high precision PCR reactions was used. This device has interchangeable thermal blocks that allow working with different formats, such as 96- and 384-well plates or individual tubes.\u003c/p\u003e\n\u003cp\u003ePCR products were separated by electrophoresis on 1.1 % agarose gels (Bio-Rad\u003csup\u003e\u0026reg;\u003c/sup\u003e Laboratories Inc., CA, USA). The intensity levels of the bands in the electrophoresis gel were determined with (IMAGE LAB\u003csup\u003e\u0026reg;\u003c/sup\u003e) software (http://www.bio-rad.com/en-tu/product/image-lab-software http://www.bio-rad.com/en-ru/product/image-lab-software).\u003c/p\u003e\n\u003cp\u003eTo purify the PCR product, SpinBind filter units were used, five volumes of SpinBind solution were added to each PCR reaction (500 \u0026micro;L for a 100 \u0026micro;L reaction) and mixed by pipetting. If oil was present, the top layer of oil was avoided by transferring the mixture to a SpinBind filtration unit. The samples were centrifuged for 10-30 seconds at 10,000 \u0026times; g and the flow-through was discarded. After reassembling the filter unit, 300 \u0026micro;l of SpinClean buffer was added and centrifuged again for 30 seconds. The flow through was discarded and a final 60 second centrifugation completely removed the residual liquid. The filter was transferred to a clean tube and 50 \u0026micro;L of sterile water was applied to the membrane, after centrifugation for 60 seconds at 10,000 \u0026times; g, the filter was discarded, and the purified DNA was collected and stored at -20 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequencing and phylogram analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOnce the amplification was verified by 1.1 % agarose gel, the amplicons were sent to the Genomic Services Laboratory (LABSERGEN\u003csup\u003e\u0026reg;\u003c/sup\u003e) belonging to the National Laboratory of Genomics for Biodiversity (LANGEBIO, Mexico, CINVESTAV-Irapuato; https://portal.cinvestav.mx/uga-langebio/investigacion/servicios/laboratorio-de-servicios-gen243micos-1) where they were sequenced by the Sanger Dideoxynucleotides method in ABI 3,730-xl DNA Analyzer (Applied Biosystems\u003csup\u003e\u0026reg;\u003c/sup\u003e; www.appliedbiosystems.com). After obtaining the sequences of the EPFs amplifications, they were identified by BLASTN (GenBank, EMBL, DDBJ)- Envir. (http://unite.ut.ee/). All sequences were compared with other sequences from GenBank using BLAST and the best match was recorded and selected, with a maximum identity value (E-value). A sequence from \u003cem\u003eB\u003c/em\u003e. \u003cem\u003ebassiana\u003c/em\u003e; \u003cem\u003eMetarhizium\u003c/em\u003e spp. and \u003cem\u003eAspergillus\u003c/em\u003e spp. were used as a reference group. The sequences obtained were analyzed, eliminating the low-quality regions in the forward and reverse sequences. Contig assembly was performed using BioEdit software (version 7.0.52). The assembled contigs were subject to a BLAST search against the NCBI database to corroborate their identity and determine the degree of similarity to other previously reported sequences, selecting those with at least 98 % identity. These amplicon\u0026acute;s sequences were compared with five similar sequences of each, selected from public databases, specifically from NCBI, and represented by their GenBank identifiers (ID). The complete DNA sequences of six isolates were edited with Bioedit version 7.0.5.2. and subsequently deposited in the GenBank database, with accession numbers AMP-M2-R2-A; PQ835711, AMP-M2-R1-A; PQ835761, AMP-M7-R2-B; PQ835714, AMP-M2-R2-C; PQ835756, AMP-M7-R1-A; PQ835716, and AMP-M2-R2-B; PQ835762. For phylogenetic analysis of the EPF sequences, the program (MEGA 11\u003csup\u003e\u0026reg;\u003c/sup\u003e) (Tamura et al. 2021) was used. Consensus trees were constructed with the Consensus program of the (PHYLIP\u003csup\u003e\u0026reg;\u003c/sup\u003e) package version 3.6 using the Neighbor-joining 1000 bootstrap method. Phylogenetic inferences were performed and displayed using MEGA 11.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMortality data were analyzed by one-way analysis of variance (ANOVA). Prior to analysis, assumptions of homoscedasticity and normal distribution of the data were checked, and mean separations were estimated using a Tukey test (\u003cem\u003eP\u003c/em\u003e \u0026le; 0.05). To estimate the effects on survival of \u003cem\u003eG\u003c/em\u003e. \u003cem\u003emellonella\u003c/em\u003e to EPFs isolates, mortality data were subjected to Kaplan-Meier analysis and Median Time to Death (MTD) values were calculated. All analyses were performed using SPSS for Windows version 24.0 (IBM\u003csup\u003e\u0026reg;\u003c/sup\u003e, Inc., Armonk, NY).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003ePathogenic characterization of the native EPF isolates\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eIn the present study, out of six isolated EPF strains, two strains belonging to the genus \u003cem\u003eBeauveria\u003c/em\u003e were found, one to the genus \u003cem\u003eMetarhizium\u003c/em\u003e, and finally, three strains of the genus \u003cem\u003eAspergillus\u003c/em\u003e were found and the exposure of \u003cem\u003eG\u003c/em\u003e. \u003cem\u003emellonella\u003c/em\u003e L\u003csub\u003e5\u003c/sub\u003e larvae (\u0026lt;\u0026thinsp;24 h from moult) to these native EPFs isolates showed significant differences between isolates (F\u003csub\u003e6,14\u003c/sub\u003e = 6.94; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.014) and the percentages of cumulative mortality at the fourth day of evaluation showed the following mortality percentages: AMP-M2-R1-A (100 \u0026plusmn; 0.0%), AMP-M2-R2-C (93.33 \u0026plusmn; 5.77%), AMP-M2-R2-A (86.67 \u0026plusmn; 23.09%), AMP-M7-R2-B (73.33 \u0026plusmn; 30.55%), AMP-M7-R1-A (56.67 \u0026plusmn; 32.14%) and AMP-M2-R2-B (53.33 \u0026plusmn; 23.09%), all higher than those recorded in the negative control whose mortality was 6.67 \u0026plusmn; 5.77%, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe obtained MTD values revealed significant differences in the mortality rate of the treatments (\u0026chi;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;22.672, df\u0026thinsp;=\u0026thinsp;5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The survival curve (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) shows how the mortality of the isolates was shown with respect to time. It is worth mentioning that only \u003cem\u003eMetarhizium\u003c/em\u003e sp. strain AMP-M2-R1-A causes 50% mortality in larvae within the first 3 days, indicating its rapid and highly virulent effect, which is in agreement with the ANOVA performed. In contrast, the other strains started to show larval mortality close to 50% after 3 days after treatment. The highest mortality rate in all isolates was recorded between 48 and 96 hours.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eMorphological identification\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe morphology of the \u003cem\u003eAspergillus\u003c/em\u003e spp. strains was characterized by colonies of velvety texture, with a light-yellow color in the center, followed by a yellowish green color. The mycelium was characterized as filamentous with septate hyphae, conidiophores of variable size, cone-shaped vesicle and smooth wall. Its phialides are uniseridate (single-ended), conidia in a chain, squinulate, globose to rounded, green in color. The dimensions of conidiophores and conidia of the obtained isolates are for AMP-M2-R2-C 8.95 \u0026times; 3.60 \u0026micro;m and conidia 4.12 \u0026times; 3.89 \u0026micro;m (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, b), for AMP-M7-R2-B they were 8.49 \u0026times; 3.54 \u0026micro;m and 4.54 \u0026times; 4.23 \u0026micro;m (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec, d), and for AMP-M2-R2-B, 7.93 \u0026times; 4.07 \u0026micro;m. 4.38 \u0026times; 3.94 \u0026micro;m (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee, f).\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eMetarhizium\u003c/em\u003e sp. (AMP-M2-R1-A), presented white colonies in its early stages of growth until reaching a mature stage of development, a dark olive green color, a powdery texture due to the production of conidia, these presented a cylindrical or ellipsoidal shape measuring 6.70 x 2.59 \u0026micro;m on average (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eBeauveria\u003c/em\u003e spp. strains, presented a cottony white coloration, turning to cream with a slightly yellowish tone, with a cottony texture and on the reverse side of the colony it presented a cream color, light yellow, and an abundant and spongy aerial mycelium that at the end became powdery. The edges of the colonies were regular with slightly lobed parts. The smooth, globose-shaped conidia of AMP-M7-R1-A were 2.45 \u0026times; 1.97 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb) and AMP-M2-R2-A measured 2.33 \u0026times; 1.97 \u0026micro;m (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec) (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eMolecular identification and phylogenetic analysis\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eFor the PCR amplifications of the universal primers ITS-1 and ITS-4, before this we obtained amplicons from six fungal isolates (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). The concentration of each amplicon was adequate for Sanger-Deoxy-sequencing. The Blast annotation revealed high identity with a principal EPF fungus (\u003cem\u003eB. bassiana\u003c/em\u003e; \u003cem\u003eMetarhizium\u003c/em\u003e sp.) isolated from soils. However, the phylogram analysis reveals three putative \u003cem\u003eAspergillus\u003c/em\u003e spp. fungus (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe \u003cem\u003eAspergillus\u003c/em\u003e isolates also agree with the morphology reported for these fungi (see Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e morphological analysis). In addition, in the phylogram we observe a diversity and different genetic distances between \u003cem\u003eAspergillus\u003c/em\u003e spp. genus. The mayor genetic distances were observed between AMP-M2-R2-A (\u003cem\u003eBeauveria\u003c/em\u003e spp.) and AMP-M2-R2-B (\u003cem\u003eAspergillus\u003c/em\u003e spp.) with 52% of genetic distances in comparison with AMP-M7-R1-A (\u003cem\u003eBeauveria\u003c/em\u003e spp.) and AMP-M2-R2-B (\u003cem\u003eAspergillus\u003c/em\u003e spp.) in which the genetic distances are lower by up to 31%.\u003c/p\u003e\n \u003cp\u003eFor the Phylogram analysis, we perform a tree obtained by the Maximum Likelihood method from the contig ITS region of the six isolates belonging to the genera \u003cem\u003eMetarhizium\u003c/em\u003e, \u003cem\u003eAspergillus\u003c/em\u003e and \u003cem\u003eBeauveria\u003c/em\u003e shows well-defined clades (groups), grouping the sequenced fungi together with reference species. However, the sequence of \u003cem\u003eAkanthomyces dipterigenus\u003c/em\u003e (Petch) Spatafora, Kepler, Zare \u0026amp; B. Shrestha, 2017 (EF6791177.1) was also used as an out-group sequence reference.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the IPM, it is essential to carry out continuous bioprospecting aimed at isolating and identifying biocontrol agents of agricultural pests through the strategy of soil sampling and subsequent baiting with sentinel insects, which can be \u003cem\u003eG. mellonella\u003c/em\u003e or \u003cem\u003eTenebrio molitor\u003c/em\u003e Linnaeus, 1758 (Coleoptera: Tenebrionidae); in turn, this approach has shown different levels of success on pests of fundamental importance in various countries worldwide (Fofana et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For driven this approach, six isolates of EPF belonging to the genera \u003cem\u003eAspergillus\u003c/em\u003e spp., \u003cem\u003eBeauveria\u003c/em\u003e spp. and \u003cem\u003eMetarhizium\u003c/em\u003e sp. were isolated and identified in the present study, respectively. Similar studies in Mexico reveal that EPF species present in local agroecosystems belong to \u003cem\u003eB. bassiana\u003c/em\u003e (Hern\u0026aacute;ndez-Dom\u0026iacute;nguez et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)d \u003cem\u003eanisopliae\u003c/em\u003e (Alcantara-Vargas et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), respectively.\u003c/p\u003e \u003cp\u003eIn this study, the visual characters of isolates belonging to \u003cem\u003eBeauveria\u003c/em\u003e spp. were determined, who showed a characteristic creamy white color of colonies and on host corpses, while in isolates belonging to the genus \u003cem\u003eMetarhizium\u003c/em\u003e sp. their particular olive green color was determined, as described in other studies (Villamizar et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Bhadani et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In our bioassays, all EPF isolates evaluated were found to be pathogenic on \u003cem\u003eG\u003c/em\u003e. \u003cem\u003emellonella\u003c/em\u003e L\u003csub\u003e5\u003c/sub\u003e larvae; however, there were significant differences in larval mortality, with isolate AMP-M2-R1-A showing the highest virulence (100%) on the fourth day of evaluation with differences with respect to isolating AMP-M2-R2-B whose mortality was 53.33%, respectively. These results coincide with those reported by (Baki et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), who reported a mortality of larvae of the Colorado potato beetle \u003cem\u003eLeptinotarsa decemlineata\u003c/em\u003e (Say, 1824) (Coleoptera: Chrysomelidae) of between 91.7 and 100% at nine days of evaluation.\u003c/p\u003e \u003cp\u003eIn this case, the asexual structures observed correspond to the species \u003cem\u003eAspergillus flavus\u003c/em\u003e Link, 1809. However, the diameter of the conidia is larger than expected for this species, which has been documented in other species of the genus \u003cem\u003eAspergillus\u003c/em\u003e, such as \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eniger\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eversicolor\u003c/em\u003e, whose conidia usually have diameters ranging between 2 and 3.5 \u0026micro;m (dos Santos et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Isolate AMP-M7-R2-B shows these similarities, despite belonging to the species \u003cem\u003eA\u003c/em\u003e. \u003cem\u003eflavus\u003c/em\u003e. This atypical morphology could lead to confusion with other fungal genera that show conidia of similar size and shape, such as \u003cem\u003ePenicillium\u003c/em\u003e spp. and \u003cem\u003ePaecilomyces\u003c/em\u003e spp. However, these genera can be ruled out due to the distinctive features of \u003cem\u003eAspergillus\u003c/em\u003e, such as the presence of a well-defined apical vesicle and the arrangement of the phialides, which can be uniseriate or biseriate. Authors such as (dos Santos et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), attribute these changes to genetic variations in isolates from regions with high drought and extreme climates where, in these environments, the rounded shape of the conidia could be related to greater efficiency in their aerial dispersal, which would allow them to move more effectively in hostile ecosystems with low humidity and high temperatures. On the other hand, in relation to \u003cem\u003eBeauveria\u003c/em\u003e spp. and \u003cem\u003eMetarhizium\u003c/em\u003e sp. the results obtained in this study are in agreement with those previously reported (Ayala-Zerme\u0026ntilde;o et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe molecular characterization of the phylogenetic tree of the species \u003cem\u003eBeauveria\u003c/em\u003e spp., and \u003cem\u003eMetarhizium\u003c/em\u003e sp., clearly shows the significant evolutionary relationship in comparison to the genus \u003cem\u003eAspergillus\u003c/em\u003e spp., which shows a distinctive clade, suggesting significant genetic differences in comparison with the genera \u003cem\u003eBeauveria\u003c/em\u003e and \u003cem\u003eMetarhizium\u003c/em\u003e, which suggest a greater evolutionary affinity between these due to their common adaptive characteristics in their interaction of mortality with their hosts. Currently, a wide variety of native strains of EPFs have been reported in Asia, Europe, North America, and Latin America (Sharma and Sharma \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and this highlights their potential for biocontrol applications.\u003c/p\u003e \u003cp\u003eSpecifically, fungi of the genus \u003cem\u003eAspergillus\u003c/em\u003e spp. have shown genomic plasticity. This genus has been investigated for its robust mutation rate, with an average of 5.1 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;11\u003c/sup\u003e mutations and GC to AT changes per 4000 mitoses per asexual cell cycle (\u0026Aacute;lvarez-Escribano et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Ecologically, these fungi play diverse roles, including plant pathogens, facultative fungi, and, more recently, entomopathogens. These recent studies demonstrate that even fungi not initially recognized as EPFs can change roles to ensure their fitness, so these findings represented in this work are in agreement with what was recently reported by (Chen et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), where it was observed that strains of \u003cem\u003eA. flavus\u003c/em\u003e considered an opportunistic organism, are capable of infecting insects of \u003cem\u003eDiaphania indica\u003c/em\u003e (Saunders, 1851) (Lepidoptera: Crambidae) with a mortality rate of up to 70%, demonstrating the high adaptability of the fungus.\u003c/p\u003e \u003cp\u003eThe isolates were applied at concentrations ranging from 10\u003csup\u003e4\u003c/sup\u003e-10\u003csup\u003e7\u003c/sup\u003e conidia/mL to the ant \u003cem\u003eDolichoderus thoracicus\u003c/em\u003e (Smith, 1860) (Hymenoptera: Formicidae), achieving high mortality levels (100%) by the seventh day with the 10\u003csup\u003e7\u003c/sup\u003e conidia/mL concentration. Furthermore, other authors like Kianifard et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), demonstrated that \u003cem\u003eAspergillus oryzae\u003c/em\u003e (Ahlb.) Cohn, 1884 can be transmitted transstadially from larvae to adults of the mosquito \u003cem\u003eAnopheles stephensi\u003c/em\u003e Liston, 1901 (Diptera: Culicidae). In this case, it inhibited the formation of oocysts of the parasite \u003cem\u003ePlasmodium berghei\u003c/em\u003e (Haemosporida: Plasmodiidae) by secreting antiplasmodial effector peptides within the mosquito's midgut. This opens the possibility of using this fungus to inhibit the development of malaria parasites in \u003cem\u003eA. stephensi\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study provides evidence of native EPFs with high potential for use in biocontrol. Furthermore, to our knowledge, we report for the first time in Mexican soils of the state of Michoac\u0026aacute;n, native strains of \u003cem\u003eAspergillus\u003c/em\u003e spp. with entomopathogenic activity. These strains demonstrate the potential of EPFs for biocontrol use and application in the Ci\u0026eacute;nega de Chapala region of Michoac\u0026aacute;n.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e(EPFs) \u0026nbsp; Entomopathogenic fungi\u0026acute;s\u003c/p\u003e\n\u003cp\u003e(IPM) \u0026nbsp;\u0026nbsp;Integrated Pest Management\u003c/p\u003e\n\u003cp\u003e(PCR)\u0026nbsp; \u0026nbsp;Polymerase\u0026nbsp;Chain\u0026nbsp;Reaction\u003c/p\u003e\n\u003cp\u003e(PDA + YE) \u0026nbsp; Potato\u0026nbsp;Dextrose\u0026nbsp;Agar\u0026nbsp;+ Yeast\u0026nbsp;Extract\u003c/p\u003e\n\u003cp\u003e(RBA)\u0026nbsp; \u0026nbsp;Bengal Rose\u0026nbsp;Agar\u003c/p\u003e\n\u003cp\u003e(UCEMICH) \u0026nbsp; Universidad de La Ci\u0026eacute;nega del Estado de Michoac\u0026aacute;n de Ocampo\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by Institute of Science, Technology and Innovation of the Government of Michoac\u0026aacute;n, Mexico grant number PICIR22-030-C. Thanks are also due to the Secretariat for Science, Humanities, Technology and Innovation.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHumberto Javier L\u0026oacute;pez-Mac\u0026iacute;as: Data curation; Investigation; Writing\u0026mdash;original draft. Omar Fabi\u0026aacute;n Hern\u0026aacute;ndez-Zepeda: Formal analysis; Methodology; Validation; Writing\u0026mdash;original draft; Writing\u0026mdash;review \u0026amp; editing. Luis Enrique Flores-Pantoja: Writing\u0026mdash;review \u0026amp; editing; Resources; Software. Ma. Guadalupe S\u0026aacute;nchez-Saavedra: Writing\u0026mdash;review \u0026amp; editing; Resources; Software. Ang\u0026eacute;lica Mar\u0026iacute;a Berlanga-Padilla: Investigation; Formal analysis; Methodology; Validation; Writing\u0026mdash;review \u0026amp; editing. Oscar Giovanni Guti\u0026eacute;rrez-C\u0026aacute;rdenas: Formal analysis; Methodology; Software; Validation; Writing\u0026mdash;original draft; Writing\u0026mdash;review \u0026amp; editing. Isaac Zepeda-Jazo: Funding acquisition; Investigation; Project administration; Resources; Supervision. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank Nikt\u0026eacute; Huerta-N\u0026aacute;vez, for technical support and Dr. Roberto Montesinos-Mat\u0026iacute;as, for technical support and text revision.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlcantara-Vargas E, Espitia-L\u0026oacute;pez J, Garza-L\u0026oacute;pez PM, Angel-Cuapio A (2020) Producci\u0026oacute;n y calidad de conidios de cepas de entomopat\u0026oacute;genos del g\u0026eacute;nero \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e, aislados en zonas agr\u0026iacute;colas del estado de M\u0026eacute;xico. 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Biocontrol Sci Technol 25:1192-1207. doi:10.1080/09583157.2015.1051878.\u003c/li\u003e\n\u003cli\u003eAyaz M, Li CH, Ali Q, Zhao W, Chi YK, Shafiq M, Ali F, Yu XY, Yu Q, Zhao JT, Yu JW, Qi RD, Huang WK (2023) Bacterial and fungal biocontrol agents for plant disease protection: Journey from lab to field, current status, challenges, and global perspectives. Molecules doi:10.3390/molecules28186735.\u003c/li\u003e\n\u003cli\u003eBaki D, Tosun HS, Erler F (2021) Efficacy of indigenous isolates of \u003cem\u003eBeauveria bassiana\u003c/em\u003e (Balsamo) Vuillemin (Deuteromycota: Hyphomycetes) against the colorado potato beetle, \u003cem\u003eLeptinotarsa decemlineata\u003c/em\u003e (Say) (Coleoptera: Chrysomelidae). Egypt J Biol Pest Control doi:10.1186/s41938-021-00406-5.\u003c/li\u003e\n\u003cli\u003eBhadani RV, Gajera HP, Hirpara DG, Kachhadiya HJ (2022) Characterization and bio-efficacy of entomopathogenic \u003cem\u003eBeauveria\u003c/em\u003e associated with cuticle-degrading enzymes to restrain sucking pest \u003cem\u003eBemisia tabaci\u003c/em\u003e. Parasitol Res 121:2019-2031. doi:10.1007/s00436-022-07557-w.\u003c/li\u003e\n\u003cli\u003eBidochka MJ, Kamp AM, Lavender TM, Dekoning J, De Croos JA (2001) Habitat association in two genetic groups of the insect-pathogenic fungus \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e: Uncovering cryptic species? Appl Environ Microbiol 67:1335-1342. doi:10.1128/AEM.67.3.1335-1342.2001.\u003c/li\u003e\n\u003cli\u003eChen C, Borham A, Chen X, Ling H, SU H (2024) \u003cem\u003eAspergillus flavus\u003c/em\u003e as an entomopathogen infecting \u003cem\u003eDiaphania indica\u003c/em\u003e and control efficacy across different developmental stages. 1-13. doi:10.21203/rs.3.rs-4296110/v1.\u003c/li\u003e\n\u003cli\u003eDellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: Version II. Plant Mol Biol Rep 1:19-21. doi:10.1007/BF02712670.\u003c/li\u003e\n\u003cli\u003edos Santos RA, Steenwyk JL, Rivero-Menendez O, Mead ME, Silva LP, Bastos RW, Alastruey-Izquierdo A, Goldman GH, Rokas A (2020) Genomic and phenotypic heterogeneity of clinical isolates of the human pathogens \u003cem\u003eAspergillus fumigatus\u003c/em\u003e, \u003cem\u003eAspergillus lentulus\u003c/em\u003e, and \u003cem\u003eAspergillus fumigatiaffinis\u003c/em\u003e. Front Genet 11(2020):1-17. doi:10.3389/fgene.2020.00459.\u003c/li\u003e\n\u003cli\u003eFofana F, Descombes C, Kouam\u0026eacute; AP, Lefort F (2023) Isolation, identification and evaluation of the effects of native entomopathogenic fungi from C\u0026ocirc;te d\u0026rsquo;Ivoire on \u003cem\u003eGalleria mellonella\u003c/em\u003e. Microorganisms doi:10.3390/microoorganisms11082104.\u003c/li\u003e\n\u003cli\u003eGebremariam A, Chekol Y, Assefa F (2021) Phenotypic, molecular, and virulence characterization of entomopathogenic fungi, \u003cem\u003eBeauveria bassiana\u003c/em\u003e (Balsam) Vuillemin, and \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e (Metschn.) Sorokin from soil samples of Ethiopia for the development of mycoinsecticide. Heliyon doi:10.1016/j.heliyon.2021.e07091.\u003c/li\u003e\n\u003cli\u003eGrewal PS, Lewis EE, Gaugler R, Campbell JF (1994) Host finding behaviour as a predictor of foraging strategy in entomopathogenic nematodes. Parasitology 108:207-215. doi:10.1017/S003118200006830X.\u003c/li\u003e\n\u003cli\u003eGuti\u0026eacute;rrez-C\u0026aacute;rdenas OG, Cortez-Madrigal H, Malo EA, G\u0026oacute;mez-Ru\u0026iacute;z J, Nord R (2019) Physiological and pathogenical characterization of \u003cem\u003eBeauveria bassiana\u003c/em\u003e and \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e isolates for management of adult \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e. Southwest Entomol 44:409-421. doi:10.3958/059.044.0206.\u003c/li\u003e\n\u003cli\u003eHajek AE, Eilenberg J (2018) Fungal pathogens of invertebrates. In: Hajek AE, Eilenberg J (eds) Natural enemies, an introduction to biological control, Cambridge University Press, UK.\u003c/li\u003e\n\u003cli\u003eHern\u0026aacute;ndez-Dom\u0026iacute;nguez C, Zamora-Bernardino C, V\u0026aacute;zquez-Cruz F, Reyes-L\u0026oacute;pez D, Dom\u0026iacute;nguez-Perales LA, Enr\u0026iacute;quez-Garc\u0026iacute;a F (2024) Mortalidad de \u003cem\u003eGalleria mellonella\u003c/em\u003e L. por \u003cem\u003eBeauveria bassiana\u003c/em\u003e (B\u0026aacute;lsamo) Vuill (Ascomycota: Hypocreales). Rev Mex Cienc Agr\u0026iacute;c 15:1-11. doi:10.29312/remexca.v15i4.3146.\u003c/li\u003e\n\u003cli\u003eHumber RA (2012) Preservation of entomopathogenic fungal cultures. In:Lacey LA (ed)Manual of techniques in insect pathology, Academic Press, Washington.\u003c/li\u003e\n\u003cli\u003eHussain D, Asrar M, Khalid B, Hafeez F, Saleem M, Akhter M, Ahmed M, Ali I, Hanif K (2022) Insect pests of economic importance attacking wheat crop (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.) in Punjab, Pakistan. Intl J Trop Insect Sci 42:9-20. doi:10.1007/s42690-021-00574-9.\u003c/li\u003e\n\u003cli\u003eKhalil AMA, Hashem AH (2018) Morphological changes of conidiogenesis in two \u003cem\u003eAspergillus\u003c/em\u003e species. J Pure Appl Microbiol 12:2041-2048. doi:10.22207/JPAM.12.4.40.\u003c/li\u003e\n\u003cli\u003eKianifard L, Rafiqi AM, Akcakir O, Aly AS, Billingsley PF, Uysal S (2023) A recombinant \u003cem\u003eAspergillus oryzae\u003c/em\u003e fungus transmitted from larvae to adults of \u003cem\u003eAnopheles stephensi\u003c/em\u003e mosquitoes inhibits malaria parasite oocyst development. Sci Rep 13:1-15. doi:10.1038/s41598-023-38654-0.\u003c/li\u003e\n\u003cli\u003eMantzoukas S, Lagogiannis I, Ntoukas A, Eliopoulos PA, Kouretas D, Karpouzas DG, Poulas K (2020). Trapping entomopathogenic fungi from vine terroir soil samples with insect baits for controlling serious pests. Appl Sci doi:10.3390/app10103539.\u003c/li\u003e\n\u003cli\u003eMateos-Fern\u0026aacute;ndez R, Petek M, Gerasymenko I, Juter\u0026scaron;ek M, Baebler \u0026Scaron;, Kallam K, Moreno-Gim\u0026eacute;nez E, Gondolf J, Nordmann A, Gruden K, Orzaez D, Patron NJ (2022) Insect pest management in the age of synthetic biology. Plant Biotechnol J 20:25-36. doi:10.1111/pbi.13685.\u003c/li\u003e\n\u003cli\u003eRanesi M, Vitale S, Staropoli A, Di Lelio I, Izzo LG, De Luca MG, Becchimanzi A, Pennacchio F, Lorito M, Woo SL, Vinale F, Turr\u0026agrave; D (2024) Field isolates of \u003cem\u003eBeauveria bassiana\u003c/em\u003e exhibit biological heterogeneity in multitrophic interactions of agricultural importance. Microbiol Res doi:10.1016/j.micres.2024.127819.\u003c/li\u003e\n\u003cli\u003eRealpe FJ, Bustillo AE, L\u0026oacute;pez JC (2007) Optimizaci\u0026oacute;n de la cr\u0026iacute;a de \u003cem\u003eGalleria mellonella\u003c/em\u003e (L.) para la producci\u0026oacute;n de nematodos entomopat\u0026oacute;genos par\u0026aacute;sitos de la broca del caf\u0026eacute;. Cenicaf\u0026eacute; 58:142-157.\u003c/li\u003e\n\u003cli\u003eSerna-Dom\u0026iacute;nguez MG, Andrade-Michel GY, Arredondo-Bernal HC, Gallou A (2018) Two efficient methods for isolation of high-quality genomic DNA from entomopathogenic fungi. J Microbiol Methods 148:55-63. doi:10.1016/j.mimet.2018.03.012.\u003c/li\u003e\n\u003cli\u003eSharma R, Sharma P (2021) Fungal entomopathogens: A systematic review. Egypt J Biol Pest Control 31:1-13. doi:10.1186/s41938-021-00404-7.\u003c/li\u003e\n\u003cli\u003eSyafrudin M, Kristanti RA, Yuniarto A, Hadibarata T, Rhee J, Al-onazi WA, Algarni TS, Almarri AH, Al-Mohaimeed AM (2021) Pesticides in drinking water \u0026mdash; A review. Int J Environ Res Public Health doi:10.3390/ijerph18020468.\u003c/li\u003e\n\u003cli\u003eTalaei-Hassanloui R, Kharazi-Pakdel A, Goettel M, Mozaffari J (2006) Variation in virulence of \u003cem\u003eBeauveria bassiana\u003c/em\u003e isolates and its relatedness to some morphological characteristics. Biocontrol Science and Technology 16:525-34. doi:10.1080/09583150500532758.\u003c/li\u003e\n\u003cli\u003eTamura K, Stecher G, Kumar S (2021) MEGA11: Molecular evolutionary genetics analysis version 11. Mol Biol Evol 38:3022-3027. doi:10.1093/molbev/msab120.\u003c/li\u003e\n\u003cli\u003eToju H, Tanabe AS, Yamamoto S, Sato H (2012) High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples. PLoS ONE doi:10.1371/journal.pone.0040863.\u003c/li\u003e\n\u003cli\u003eVillamizar LF, Barrera G, Hurst M, Glare TR (2021) Characterization of a new strain of \u003cem\u003eMetarhizium novozealandicum\u003c/em\u003e with potential to be developed as a biopesticide. Mycology 12:261-278. doi:10.1080/21501203.2021.1935359.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Estimated MTD values in \u003cem\u003eG. mellonella\u003c/em\u003e L\u003csub\u003e5\u003c/sub\u003e larvae (24 h from moult) inoculated by spraying with the entomopathogenic fungal isolates at a concentration of 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e conidia/mL (\u0026lt; 24 h from molt) at a concentration of 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e conidia/mL.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"529\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMTD\u003csup\u003e1\u0026nbsp;\u003c/sup\u003e(days)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e95 % confidence limits\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eAMP-M2-R1-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e2.50 a\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e2.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e2.74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eAMP-M2-R2-C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e3.26 a\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e2.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e3.69\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eAMP-M7-R2-B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e3.33 b\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e2.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e4.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eAMP-M2-R2-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e3.76 c\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e3.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e4.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eAMP-M7-R1-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e4.30 d\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e3.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e5.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eAMP-M2-R2-B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e4.46 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e3.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e5.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eMTD: Median time to death. Values followed by different letters indicate statistically significant differences (\u003cem\u003eP\u003c/em\u003e \u0026le; 0.05) in \u003cem\u003eG. mellonella\u003c/em\u003e L\u003csub\u003e5\u003c/sub\u003e mortality.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Morphometric analysis of \u003cem\u003eAspergillus\u003c/em\u003e spp., \u003cem\u003eBeauveria\u003c/em\u003e spp., and\u0026nbsp;\u003cem\u003eMetarhizium\u003c/em\u003e sp. based on structures obtained from PDA medium-based microcultures.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"708\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 216px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAcronym\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConidia\u003c/strong\u003e \u003cstrong\u003e(average)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhialide\u003c/strong\u003e \u003cstrong\u003etaken\u003c/strong\u003e \u003cstrong\u003efrom\u003c/strong\u003e \u003cstrong\u003ethe\u003c/strong\u003e \u003cstrong\u003ebase\u003c/strong\u003e \u003cstrong\u003e(average)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLength\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWidth\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLength\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWidth\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 216px;\"\u003e\n \u003cp\u003eAMP-M2-R2-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e2.33\u0026nbsp;\u0026plusmn;\u0026nbsp;0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1.97 \u0026plusmn; 0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e7.84 \u0026plusmn; 2.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e1.05\u0026nbsp;\u0026plusmn;\u0026nbsp;0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 216px;\"\u003e\n \u003cp\u003eAMP-M2-R1-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e6.70\u0026nbsp;\u0026plusmn;\u0026nbsp;0.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e2.59 \u0026plusmn; 0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e5.69\u0026nbsp;\u0026plusmn;\u0026nbsp;0.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e2.23\u0026nbsp;\u0026plusmn;\u0026nbsp;0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 216px;\"\u003e\n \u003cp\u003eAMP-M7-R2-B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e4.54\u0026nbsp;\u0026plusmn;\u0026nbsp;0.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e4.23\u0026nbsp;\u0026plusmn;\u0026nbsp;0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e8.49\u0026nbsp;\u0026plusmn;\u0026nbsp;1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e3.54\u0026nbsp;\u0026plusmn;\u0026nbsp;0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 216px;\"\u003e\n \u003cp\u003eAMP-M2-R2-C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e4.12\u0026nbsp;\u0026plusmn;\u0026nbsp;0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e3.89\u0026nbsp;\u0026plusmn;\u0026nbsp;0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e9.39\u0026nbsp;\u0026plusmn;\u0026nbsp;1.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e3.18\u0026nbsp;\u0026plusmn;\u0026nbsp;0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 216px;\"\u003e\n \u003cp\u003eAMP-M7-R1-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e2.45\u0026nbsp;\u0026plusmn;\u0026nbsp;0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1.97\u0026nbsp;\u0026plusmn;\u0026nbsp;0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e7.69\u0026nbsp;\u0026plusmn;\u0026nbsp;2.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e0.99\u0026nbsp;\u0026plusmn;\u0026nbsp;0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 216px;\"\u003e\n \u003cp\u003eAMP-M2-R2-B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e4.38\u0026nbsp;\u0026plusmn;\u0026nbsp;0.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e3.94\u0026nbsp;\u0026plusmn;\u0026nbsp;0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e8.42\u0026nbsp;\u0026plusmn;\u0026nbsp;1.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e3.73\u0026nbsp;\u0026plusmn;\u0026nbsp;0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 216px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eComparative studies\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLength\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWidth\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLength\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWidth\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 216px;\"\u003e\n \u003cp\u003e(Talaei-Hassanloui et al. 2006)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e2.4\u0026nbsp;\u0026plusmn;\u0026nbsp;0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e5.8\u0026nbsp;\u0026plusmn;\u0026nbsp;1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eNd\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003eNd\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 216px;\"\u003e\n \u003cp\u003e(Ayala-Zerme\u0026ntilde;o et al. 2015)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e4.52\u0026nbsp;\u0026plusmn;\u0026nbsp;0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1.8\u0026nbsp;\u0026plusmn;\u0026nbsp;0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e6.22\u0026nbsp;\u0026plusmn;\u0026nbsp;0.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e2.15\u0026nbsp;\u0026plusmn;\u0026nbsp;0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 216px;\"\u003e\n \u003cp\u003e(Khalil and Hashem 2018)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e2.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e2.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e6.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e2.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eNd: Not described.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e Sampling areas of entomopathogenic fungi in the Ci\u0026eacute;nega de Michoac\u0026aacute;n, Mexico. Geographic origin, coordinates and altitude expressed in meters above sea level are indicated.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"828\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlast annotation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNCBI ID\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 175px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGeographical origin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 181px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCoordinates (N, W)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAltitude (masl)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003eAMP-M2-R2-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cem\u003eBeauveria\u0026nbsp;\u003c/em\u003esp\u003cem\u003e.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003ePQ835711\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 175px;\"\u003e\n \u003cp\u003eVenustiano Carranza\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 181px;\"\u003e\n \u003cp\u003e20\u0026deg;06.82822\u0026quot; 102\u0026deg;30.370\u0026rdquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1528\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003eAMP-M2-R1-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cem\u003eMetarhizium\u0026nbsp;\u003c/em\u003esp\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003ePQ835761\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 175px;\"\u003e\n \u003cp\u003ePajacuar\u0026aacute;n\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 181px;\"\u003e\n \u003cp\u003e20\u0026deg;07.043\u0026quot; 102\u0026deg;30.384\u0026quot;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1527\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003eAMP-M7-R2-B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cem\u003eAspergillus\u0026nbsp;\u003c/em\u003esp\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003ePQ835714\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 175px;\"\u003e\n \u003cp\u003ePajacuar\u0026aacute;n\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 181px;\"\u003e\n \u003cp\u003e20\u0026deg;06.636 102\u0026deg;45.158\u0026quot;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1573\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003eAMP-M2-R2-C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cem\u003eAspergillus\u0026nbsp;\u003c/em\u003esp\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003ePQ835756\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 175px;\"\u003e\n \u003cp\u003eVenustiano Carranza\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 181px;\"\u003e\n \u003cp\u003e20\u0026deg;06.371\u0026quot; 102\u0026deg;45.193\u0026quot;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1526\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003eAMP-M7-R1-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cem\u003eBeauveria\u0026nbsp;\u003c/em\u003esp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u0026nbsp;PQ835716\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 175px;\"\u003e\n \u003cp\u003ePajacuar\u0026aacute;n\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 181px;\"\u003e\n \u003cp\u003e20\u0026deg;04.951 102\u0026deg;27.556\u0026quot;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1533\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003eAMP-M2-R2-B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cem\u003eAspergillus\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u0026nbsp; PQ835762\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 175px;\"\u003e\n \u003cp\u003ePajacuar\u0026aacute;n\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 181px;\"\u003e\n \u003cp\u003e20\u0026deg;07.043\u0026quot; 102\u0026deg;30.384\u0026quot;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1527\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Aspergillus spp., Beauveria bassiana, Biological control, Integrated Pest Management, Metarhizium anisopliae","lastPublishedDoi":"10.21203/rs.3.rs-6873622/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6873622/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eEntomopathogenic fungi\u0026acute;s (EPFs), represents a successful alternative against pests in various agroecosystems worldwide. However, Mexican agrifood production faces serious challenges due to the incidence of insect pests that have reduced crop yields, leading to the indiscriminate use of organosynthetic pesticides. Therefore, it is necessary to study the potential of native EPFs isolates as an alternative for Integrated Pest Management (IPM). The objective of this study was to sample agricultural soils in Venustiano Carranza and Pajacuar\u0026aacute;n, Michoac\u0026aacute;n, Mexico, to isolate EPFs with biocontrol potential and to characterize them pathogenically and at the molecular level.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eMorphological and molecular analyses indicate the genus of the isolate corresponds to \u003cem\u003eBeauveria\u003c/em\u003e spp; \u003cem\u003eMetarhizium\u003c/em\u003e sp. and \u003cem\u003eAspergillus\u003c/em\u003e spp. Interestingly in the phylogenic analysis, we observed a great genetic distance between \u003cem\u003eAspergillus\u003c/em\u003e isolates. This is consistent with the theory that this genus shows genetic robustness.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese results indicate the presence of native EPFs in the study areas, which provides a promising basis for the development of new biopesticides, offering a sustainable alternative for agricultural production.\u003c/p\u003e","manuscriptTitle":"Researching a local weapons: A potential native fungal biocontrol in Mexico","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-30 11:26:38","doi":"10.21203/rs.3.rs-6873622/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a6f3db8b-a62a-49b0-8a68-ad823ccf629f","owner":[],"postedDate":"June 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-28T12:08:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-30 11:26:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6873622","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6873622","identity":"rs-6873622","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}