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

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

Adult Diaphorina citri Biocontrol Using Hirsutella citriformis Strains and Gum Formulations.

Hirsutella citriformis Speare is the only entomopathogenic fungus that has been applied to control the hemipteran Diaphorina citri Kuwayama. However, the use of available commercial products under field conditions is limited due to conidia's shelf life and short environmental persistence. We have previously reported the citrus psyllid D. citri adults' biocontrol potential using H. citriformis strains. The aim of the present study was to evaluate different formulations based on H. citriformis (OP-Hir-3, OP-Hir-10, and OP-Hir-12 strains) conidia and gums as additives to improve D. citri adults' biocontrol, under laboratory, greenhouse, and field conditions, using Hirsutella gums as conidia stabilizers to improve their viability under environmental drought conditions and as insecticide. Laboratory bioassay results showed that the highest (p < 0.05) D. citri mortality was achieved using FOP-Hir-10GH (63.5%), followed by the Hirsutella gum control (42.2%). Under greenhouse conditions, adults' mortality reached up to 84.6% with FOP-Hir-12 and 49.0% with Hirsutella gum. In addition, we applied H. citriformis formulations under field conditions in a commercial citrus grove located in Tecomán, Colima, México, at 21.5 °C and 73.3% relative humidity (RH) in March and 25.7 °C and 72.5% RH in October 2022 and observed 67.3% and 94.0% mortality of D. citri adults, respectively. Hirsutella gum alone showed significant insecticidal activity against D. citri adults. In conclusion, this study demonstrated that Hirsutella gum functioned as additive to H. citriformis conidia formulations, improving D. citri adults' mortality and showing potential for this pest biocontrol in citrus orchards.

In Planta Detection of Beauveria bassiana (Ascomycota: Hypocreales) Strains as Endophytes in Bean (Phaseolus vulgaris L.).

Beauveria bassiana (B. bassiana) is a significant entomopathogenic fungus (EPF) in agriculture as a sprayable biocontrol agent. It has the potential to be established as an endophyte (ENP) in various crops, resulting in beneficial effects for the host plants, including resistance to pest insects and increased growth and yield. However, it is not known whether a B. bassiana strain has such a favorable impact on the plant, since it is a common soil microorganism. Therefore, techniques that allow strain monitoring will be advantageous. To date, methods for detecting or monitoring a specific EPF strain after external application are scarce. In the present study, an in planta nested PCR technique was standardized to differentiate between three B. bassiana strains (GHA, PTG4, and BB37) established as endophytes in bean plants under laboratory conditions by detecting the insertion profile of four group I introns located in the 28S gene of B. bassiana ribosomal DNA. This technique recognized a distinct pattern of bands of different sizes for each strain, with a sensitivity of 1 pg per 10 ng of plant DNA. This molecular approach may be more effective monitoring B. bassiana strains after application to evaluate their significance on crops.

Increased Attraction and Stability of Beauveria bassiana-Formulated Microgranules for Aedes aegypti Biocontrol.

Aedes aegypti (Linn.) incidence has increased in recent years, causing human viral diseases such as dengue, which are often fatal. Beauveria bassiana (Bals., Vuillemin) efficacy for Ae. aegypti biological control has been evidenced but it relies on host susceptibility and strain virulence. We hypothesized that B. bassiana conidia microgranular formulations (MGF) with the additives acetone, lactic acid, and sugar increase Ae. aegypti adult exposure, thus improving their biocontrol effectiveness. Beauveria bassiana strain four (BBPTG4) conidia stability was assessed after 0 d, 5 d, and 30 d storage at 25 °C ± 2 °C with additives or in MGF after 91 d of storage at 25 °C ± 2 °C or 4 °C ± 1 °C, whereas mortality was evaluated after adult exposure to MGF + conidia, using home-made traps. Additives did not show toxicity to conidia. In addition, we observed that sugar in MGF increased Ae. aegypti adults' attraction and their viability resulted in a 3-fold reduction after 5 d and 1- to 4-fold decrease after 30 d of storage, and formulations were less attractive (p < 0.05). Conidia stability was higher on MGF regardless of the storage temperature, losing up to 2.5-fold viability after 91 d. In conclusion, BBPTG4 infected and killed Ae. aegypti, whereas MGF attracting adults resulted in 42.2% mortality, increasing fungus auto dissemination potential among infected surviving adults. It is necessary to further evaluate MGF against Ae. aegypti in the field.

In Vitro Antitumor Activity of Endophytic and Rhizosphere Gram-Positive Bacteria from Ibervillea sonorae (S. Watson) Greene against L5178Y-R Lymphoma Cells.

Plant-associated microorganisms represent a potential source of new antitumor compounds. The aim of the present study was to isolate endophytic and rhizosphere Gram-positive bacteria from Ibervillea sonorae and produce extracts with antitumor activity. Methanol and ethyl acetate extracts were obtained from 28 d bacterial fermentation, after which murine L5178Y-R lymphoma cells growth inhibition was evaluated at concentrations ranging from 15.62 µg/mL to 500 µg/mL by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide reduction colorimetric assay. IC50 and the selectivity index (SI) were calculated and compared with healthy control human peripheral blood mononuclear cells (PBMC). Identification of the isolated strains was performed using the 16S ribosomal gene and by MALDI-TOF MS mass spectrometry. The endophytic and rhizosphere bacterial extracts from strains ISE-B22, ISE-B26, ISE-B27, ISS-A01, ISS-A06, and ISS-A16 showed significant (p < 0.05) L5178Y-R cell growth inhibition, compared with an untreated control. The rhizosphere Micromonospora echinospora isolate ISS-A16 showed the highest (90.48%) percentage of lymphoma cells growth inhibition and SI (19.1) for PBMC, whereas the Bacillus subtilis ISE-B26 isolate caused significant (p < 0.01) growth inhibition (84.32%) and a SI of 5.2. Taken together, results of the present study evidenced antitumor effects by I. sonorae endophytic and rhizosphere bacteria culture extracts. Further research will involve the elucidation of the compounds that exert the antitumor activity and their evaluation in pre-clinical studies.

In Vitro Tumor Cell Growth Inhibition Induced by Lophocereus marginatus (DC.) S. Arias and Terrazas Endophytic Fungi Extracts.

Endophytic fungi have become potential sources of antitumor agents, particularly against antineoplastic-resistant cancer cells, with marginal or nil adverse effects for the oncological patient. Endophytic fungi were isolated from stems of the Lophocereus marginatus cactus, commonly found in Mexico. Methanol extracts were then obtained from fungus liquid cultures and their effects on tumor cell growth against murine lymphoma (L5178Y-R), human colorectal adenocarcinoma (HT-29), and human breast cancer (MCF-7) cells were evaluated at concentrations ranging from 31 µg/mL to 250 µg/mL via the colorimetric 3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide reduction assay, using monkey kidney epithelial (MA-104) and human peripheral mononuclear (PBMC) cells as controls. Furthermore, we obtained the IC50 and the selectivity index (SI) was calculated from the IC50 ratio of normal and tumor cells. In addition, molecular identification of fungi showing cytotoxic activity was determined, using internal transcribed spacer molecular markers. PME-H001, PME-H002, PME-H005, PME-H007, and PME-H008 filamentous fungus strain extracts showed significant (p < 0.05) tumor cell growth inhibition. In particular, they significantly (p < 0.05) inhibited L5178Y-R cell growth, whereas the least susceptible cell line was HT-29. The endophytic strain PME-H008 of Cladosporium sp. caused the highest growth inhibition percentage against L5178Y-R and HT-29 cells with 96.6% (p < 0.01) and 42.5% (p < 0.05) respectively, and the highest SIs against L5178Y-R cells with 2.4 and 2.9 for MA-104 and PBMCs, respectively, whereas the PME-H005 extract showed SIs of 2.77 and 1.5 against MCF-7 and L5178Y-R cells, respectively, as compared with PBMCs. In addition, the endophytic strain PME-H007 of Metarhizium anisopliae caused the highest percentage of growth inhibition (p < 0.01) against MCF-7 cells with 55.8% at 250 µg/mL. We demonstrated in vitro antitumor effects of L. marginatus endophytic fungi. Further research will involve the isolation and in vivo testing of bioactive compounds.

In vitro antitumor, pro-inflammatory, and pro-coagulant activities of Megalopyge opercularis J.E. Smith hemolymph and spine venom.

Contact with stinging spines venom from several Lepidoptera larvae may result in skin lesions. In Mexico, envenomation outbreaks caused by Megalopyge opercularis were reported between 2015 and 2016. The aim of this study was to identify the venomous caterpillars in Nuevo Leon, Mexico and evaluate several biological activities of their hemolymph (HEV) and spine setae (SSV) venoms. M. opercularis was identified by cytochrome oxidase subunit (COI) designed primers. HEV and SSV extracts cytotoxic activity was assessed on the L5178Y-R lymphoma cell line. For apoptotic cells number and apoptosis, cells were stained with acridine orange/ethidium bromide and validated by DNA fragmentation. Human peripheral blood mononuclear cells (hPBMC) cytokine response to the extracts was measured by the cytometric bead array assay. Extracts effect on pro-coagulation activity on human plasma was also evaluated. HEV and SSV extracts significantly inhibited (p < 0.01) up to 63% L5178Y-R tumor cell growth at 125-500 µg/mL, as compared with 43% of Vincristine. About 79% extracts-treated tumor cells death was caused by apoptosis. Extracts stimulated (p < 0.01) up to 60% proliferation of resident murine lymphocytes, upregulated IL-1ß, IL-6, IL-8, and TNF-α production by hPBMC, and showed potent pro-coagulant effects. The pharmacological relevance of these venoms is discussed.

Bioactive Products From Plant-Endophytic Gram-Positive Bacteria.

Endophytes constitute plant-colonizing microorganisms in a mutualistic symbiosis relationship. They are found in most ecosystems reducing plant crops' biotic and abiotic stressors by stimulating immune responses, excluding plant pathogens by niche competition, and participating in antioxidant activities and phenylpropanoid metabolism, whose activation produces plant defense, structural support, and survival molecules. In fact, metabolomic studies have demonstrated that endophyte genes associated to specific metabolites are involved in plant growth promotion (PGP) by stimulating plant hormones production such as auxins and gibberellins or as plant protective agents against microbial pathogens, cancer, and insect pests, but eco-friendly and eco-safe. A number of metabolites of Gram-positive endophytes isolated from agriculture, forest, mangrove, and medicinal plants, mainly related to the Firmicutes phyla, possess distinctive biocontrol and plant growth-promoting activities. In general, Actinobacteria and Bacillus endophytes produce aromatic compounds, lipopeptides, plant hormones, polysaccharides, and several enzymes linked to phenylpropanoid metabolism, thus representing high potential for PGP and crop management strategies. Furthermore, Actinobacteria have been shown to produce metabolites with antimicrobial and antitumor activities, useful in agriculture, medicine, and veterinary areas. The great endophytes diversity, their metabolites production, and their adaptation to stress conditions make them a suitable and unlimited source of novel metabolites, whose application could reduce agrochemicals usage in food and drugs production.

Antimicrobial and Anti-Biofilm Effect of an Electrolyzed Superoxidized Solution at Neutral-pH against Helicobacter pylori.

The presence of Helicobacter pylori in the oral cavity has been associated to the failure of antimicrobial therapy in patients with gastrointestinal infection and the development of oral diseases. However, it has been reported that the maintenance of good oral hygiene can improve the therapeutic success rates, where the use of mouthwashes with anti-Helicobacter activity would help to achieve it. The aim was to evaluate the antimicrobial activity of OxOral® mouthwash against H. pylori and its effect on biofilm formation. The minimum inhibitory concentration (MIC) of OxOral® (pH = 6.4-7.5, ORP = 650-900 mV) against H. pylori was calculated testing serial dilutions 0.117-15 ppm against 1 × 108 CFU/mL of H. pylori (ATCC® 700824™) by broth microdilution method using 96-well plates. The H. pylori biofilm formation was determined by the optical density measurement at 600 nm from coverslips stained with 0.1% crystal violet. The gene expression of ureA, luxS, flaA, omp18, and lpxD were analyzed by RT-qPCR. OxOral® cytotoxicity was evaluated in a human gingival fibroblast cell line by MTT assay. MIC was of 3.75 ppm, with 99.7 ± 7.7% bacterial growth inhibition. In the negative control, the biofilm formation was observed, whereas when bacteria were treated with OxOral® at 0.234, 0.469, and 0.938 ppm, an inhibition of 35.5 ± 0.9%, 89.1 ± 1.2%, and 99.9 ± 5.5% were obtained, respectively. The gene expression analysis showed that flaA, omp18, and lpxD genes were down-regulated with OxOral® compared with control (p < 0.05). Low cytotoxicity of 16.5 ± 7.6% was observed at the highest dose (15 ppm); no significant differences were observed from 15 to 0.469 ppm compared to the control of untreated cells (p > 0.05). Our results reveal an important anti-Helicobacter activity of OxOral® and open the possibility of its therapeutic use new studies, which would increase the success rate of conventional therapies against H. pylori.

Alterations of Immune Parameters on Trichoplusia ni (Lepidoptera: Noctuidae) Larvae Exposed to Extremely Low-Frequency Electromagnetic Fields.

Worldwide mobile telephone and microwave use have resulted in an increasing presence of extremely low-frequency electromagnetic field radiations (ELF-EMFs) in ecosystems. ELF-EMFs have been associated with altered physiological processes that can adversely affect exposed organisms. In this study, Trichoplusia ni Hübner larvae were exposed for 24, 48, or 72 h to ELF-EMFs (60 Hz and 2.0 mT) to assess effects on immune response parameters and fertility. Trichoplusia ni life cycle and fertility were not affected by 24-h exposure. However, the number of apoptotic-like cells and cellular immune response significantly increased (P < 0.01) after 72-h exposure (2- and 1.1-fold, respectively), whereas hemolymph total protein and hemocyte cells were reduced (P < 0.01; 16 and 50%, respectively) after 48-h exposure. Hemocyte cell type analysis resulted in significantly (P < 0.01) higher granulocytes number in the unexposed (2-fold increase) and oenocytoids in the 72-h-exposed larvae (28.6-fold increase). Quantitative retrotranscription (RT-qPCR) showed that after 72-h ELF-EMF exposure, the antimicrobial peptides cecropin, lysozyme, gallerimycin, and pgrp were downregulated by 24,866.0, 2.69-, 119.1-, and 1.45-fold, respectively, whereas attacin and defensin were upregulated by 1.59- and 1.85-fold, respectively. The effect of ELF-EMFs on the T. ni larvae immune response and their potential impact on its physiology and susceptibility to pathogens are discussed. This information may provide new insight of ELF-EMFs on other pest species, as well as for the preservation of ecologically important species.

Regulation by gut bacteria of immune response, Bacillus thuringiensis susceptibility and hemolin expression in Plodia interpunctella.

Plodia interpunctella (Hübner) is an important stored grain insect pest worldwide, and the first lepidopteran with reported resistance to Bacillus thuringiensis (Bt) toxins. Since gut bacteria may affect Bt insecticidal activity, we evaluated whether P. interpunctella lacking gut bacteria had differences in immune responses and susceptibility to the Bt formulation, Bactospeine. In order to clear gut bacteria, third instar larvae were reared on artificial diet containing antibiotics, or were obtained from sterilized eggs and reared under sterile conditions, and larvae were fed diets with or without Bt. Mortality was significantly lower (p<0.05) in bacteria-free larvae treated with Bt, compared with Bt-treated larvae with unaffected gut bacteria. The number of hemocytes was lower in control and Bt-treated larvae, but was significantly higher (p<0.001) in larvae treated with antibiotics and Bt, and larvae from presterilized eggs and reared on sterile diet had the highest number of hemocytes. Phenoloxidase activity was significantly lower (p<0.05) in Bt-treated larvae from presterilized eggs reared on antibiotics for 24h or in larvae reared on antibiotic-treated diets prior to Bt introduction compared with those fed control diet. Hemolin gene expression was reduced in larvae fed Bt diets compared with control and was not detected in larvae treated with antibiotics. Larvae from sterilized eggs and fed sterile diet never reached the pupal stage. Therefore, the loss of gut bacteria in P. interpunctella larvae affected the host immune response and expression of the hemolin gene, and significantly reduced susceptibility to Bt.