Emulsifiable oil-formulated Beauveria bassiana competes with imidacloprid for seasonal control of cereal aphids in Zhejiang, China.

BACKGROUND: Alternatives to neonicotinoids against cereal aphids are needed to mitigate aphid resistance and non-target effects. The emulsifiable oil formulations of two Beauveria bassiana strains, namely Bb registered as a mycoinsecticide and TBb overexpressing an endogenous virulence factor, were tested for seasonal control of cereal aphids at the elongating (April 7) to milk ripening (May 12) stages of winter wheat crop in Yuhang, Zhejiang. Each of three field trials consisted of blank control and the treatments (three randomized 100-m2 plots per capita) of each fungal strain sprayed biweekly at rates of 1.0 × 1013 and 1.5 × 1013 conidia ha-1 and 10% imidacloprid WP sprayed biweekly at a label rate. RESULTS: Tiller infestation percentage and aphid density in the 5-week field trials after the first spray were reduced to 18.7-22.4% and 9.1-12.4 aphids per tiller in the fungal treatments, and 12.8-25.3% and 2.8-20.9 aphids per tiller in the chemical treatment, contrasting with 49.2-60.3% and 37.1-108.5 aphids per tiller in the control. Percent control efficacies (±SD) computed with weekly aphid densities over the period averaged 84.0 ± 1.6 and 85.3 ± 1.8 versus 78.0 ± 4.0 and 79.9 ± 3.2 in the high-rate versus low-rate treatments of Bb and TBb, respectively, and 84.5 ± 7.8 in the chemical treatment. Imidacloprid showed faster kill action but more variable efficacy than the fungal treatments throughout the trials. CONCLUSION: Either Bb or TBb formulation competes with imidacloprid in reducing percent infestation and aphid density. The overall efficacy was significantly higher in the treatments of TBb than of Bb. © 2024 Society of Chemical Industry.

Imperfect match between radiation exposure times required for conidial viability loss and infective capacity reduction attenuate UV-B impact on Beauveria bassiana.

BACKGROUND: UV-B radiation represents a significant challenge for the widespread use of entomopathogenic fungi in pest management. This study focused on research of the asynchronous response between virulence and conidial viability against Ceratitis capitata adults using specific statistical models. Moreover, it was also investigated whether the observed differences in susceptibility to UV-B radiation in in vitro assays among three selected isolates of Beauveria bassiana were reflected in the above-mentioned asynchrony. RESULTS: While the irradiation of the three isolates of B. bassiana was associated with a significant loss of conidial viability, their virulence was not significantly affected compared to nonirradiated treatments when exposed to 1200 mW m-2 for 6 h before or after the inoculation of C. capitata. In fact, the irradiation time needed to reduce the mortality to 50% compared to the controls was 34.69 h for EABb 10/225-Fil, 16.36 h for EABb 09/20-Fil, and 24.59 h for EABb 09/28-Fil. Meanwhile, the irradiation time necessary to reduce conidial viability to 50% was 9.89 h for EABb 10/225-Fil, 8.74 h for EABb 09/20-Fil, and 4.71 h for EABb 09/28-Fil. CONCLUSION: These results highlight the importance of modeling the response of entomopathogenic fungi virulence and conidial susceptibility when exposed to UV-B radiation for the selection of environmentally competent isolates, regardless of the results obtained in previous in vitro assays on conidial germination. This strategic approach is critical in overcoming the challenges posed by UV-B radiation and holds the key to realizing the full potential of entomopathogenic fungi in pest management. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Impact of endophytic colonization by entomopathogenic fungi on the behavior and life history of the tobacco peach aphid Myzus persicae var. nicotianae.

Entomopathogenic fungi can adopt an endophytic lifestyle and provide protection against insect herbivores and plant pathogens. So far, most studies have focused on Beauveria bassiana to increase plant resistance against abiotic and biotic stresses, while only little is known for other entomopathogenic fungi. In this study, we investigated whether root inoculation of sweet pepper (Capsicum annuum L.) by the entomopathogenic fungi Akanthomyces muscarius ARSEF 5128 and B. bassiana ARSEF 3097 can improve resistance against the tobacco peach aphid Myzus persicae var. nicotianae. First, dual-choice experiments were performed to test the hypothesis that the fungi deter aphids via modifying plant volatile profiles. Next, we tested the hypothesis that endophytic colonization negatively affects aphid life history traits, such as fecundity, development and mortality rate. Aphids were significantly attracted to the odor of plants inoculated with A. muscarius over non-inoculated plants. Plants inoculated with A. muscarius emitted significantly higher amounts of ß-pinene than non-inoculated plants, and significantly higher amounts of indole than B. bassiana-inoculated and non-inoculated plants. Inoculation with the fungal strains also caused significantly higher emission of terpinolene. Further, both aphid longevity and fecundity were significantly reduced by 18% and 10%, respectively, when feeding on plants inoculated with A. muscarius, although intrinsic rate of population increase did not differ between inoculated and non-inoculated plants. Sweet pepper plants inoculated with B. bassiana ARSEF 3097 did not elicit a significant behavioral response nor affected the investigated life history traits. We conclude that endophytic colonization by entomopathogenic fungi has the potential to alter olfactory behavior and performance of M. persicae var. nicotianae, but effects are small and depend on the fungal strain used.

Entomopathogenic fungi and Schinus molle essential oil: The combination of two eco-friendly agents against Aedes aegypti larvae.

Aedes aegypti transmits arbovirus, which is a public health concern. Certain filamentous fungi have the potential to control the disease. Here, the effects of Metarhizium anisopliae s.l. CG 153, Beauveria bassiana s.l. CG 206 and Schinus molle L. were investigated against Aedes aegypti larvae. In addition, the effect of essential oil on fungal development was analyzed. Fungal germination was assessed after combination with essential oil at 0.0025 %, 0.0075 %, 0.005 %, or 0.01 %; all of the oil concentrations affected germination except 0.0025 % (v/v). Larvae were exposed to 0.0025 %, 0.0075 %, 0.005 %, or 0.01 % of the essential oil or Tween 80 at 0.01 %; however, only the essential oil at 0.0025 % achieved similar results as the control. Larvae were exposed to fungi at 107 conidia mL-1 alone or in combination with the essential oil at 0.0025 %. Regardless of the combination, M. anisopliae reduced the median survival time of mosquitoes more than B. bassiana. The cumulative survival of mosquitoes exposed to M. anisopliae alone or in combination with essential oil was 7.5 % and 2 %, respectively, and for B. bassiana, it was 75 % and 71 %, respectively. M. anisopliae + essential oil had a synergistic effect against larvae, whereas B. bassiana + essential oil was antagonistic. Scanning and transmission electron microscopy, and histopathology confirmed that the interaction of M. anisopliae was through the gut and hemocoel. In contrast, the mosquito's gut was the main route for invasion by B. bassiana. Results from gas chromatography studies demonstrated sabinene and bicyclogermacrene as the main compounds of S. molle, and the in-silico investigation found evidence that both compounds affect a wide range of biological activity. For the first time, we demonstrated the potential of S. molle and its interaction with both fungal strains against A. aegypti larvae. Moreover, for the first time, we reported that S. molle might be responsible for significant changes in larval physiology. This study provides new insights into host-pathogen interplay and contributes to a better understanding of pathogenesis in mosquitoes, which have significant consequences for biological control strategies.

Fungal consortium of two Beauveria bassiana strains increases their virulence, growth, and resistance to stress: A metabolomic approach.

The use of two or more microorganisms in a microbial consortium has been increasingly applied in the biological control of diseases and pests. Beauveria bassiana is one of the most widely studied fungal species in biological control, yet little is known about its role in fungal consortiums. In a previous study, our group found that a consortium formed by two strains of B. bassiana had significantly greater biocontrol potential against the polyphagous caterpillars Duponchelia fovealis (Lepidoptera: Crambidae) than either strain on its own. In this study, we use GC-MS and LC-MS/MS to evaluate and discuss the metabolomics of the consortium. A total of 21 consortium biomarkers were identified, corresponding to 14 detected by LC-MS/MS and seven by GC-MS. Antioxidant and anti-inflammatory mechanisms are the main properties of the metabolites produced by the consortium. These metabolites can depress the insect's immune system, increasing its vulnerability and, hence, the fungal virulence of the consortium. In light of these results, we propose an action model of insect mortality due to the metabolites secreted by the consortium. The model includes the inhibition of defense mechanisms such as pro-inflammatory interleukin secretion, cell migration, cell aggregation, Dif, Dorsal and Relish gene transcription, and JAK/STAT and JNK signaling pathways. It also promotes the cleaning of oxidative molecules, like ROS, NOS, and H2O2, and the induction of virulence factors.

Optional strategies for low-risk and non-risk applications of fungal pesticides to avoid solar ultraviolet damage.

BACKGROUND: Solar ultraviolet (UV) irradiation is harmful to formulated conidia as active ingredients of fungal pesticides and hence restrains their field application in sunny days of summer, a season requiring frequent pest controls. This conflict makes it necessary to explore optimal strategies for the application of fungal pesticides to suppress pest populations but avoid solar UV damage during summer. RESULTS: The conidia of Beauveria bassiana, a wide-spectrum fungal pesticide, were tolerable to UVB (major solar UV wavelengths) damage of ≤0.5 J cm-2 . The damage of this upper limit caused a loss of conidial viability and infectivity if not photoreactivated by light exposure after irradiation. Intriguingly, the light exposure resulted in a high photoreactivation rate of UVB-inactivated conidia and an insignificant or marginal difference in insecticidal activity between normal conidia and those photoreactivated. Modeling analysis of solar UVB intensity recorded hourly over the daylight of five sunny summer days from 5:00 am to 7:00 pm at 30° 17'57'' N and 120°5'7'' E revealed a variation of daily accumulated UVB dose from 2.07 to 2.78 J cm-2 , which was far beyond the upper limit. A more tolerable dose of ~0.2 J cm-2 appeared between 3:00 pm and 5:00 pm, and no harmful dose accumulated between 5:00 pm and 7:00 pm. CONCLUSION: Fungal UVB tolerance, fungal photoreactivation capability and the daily accumulation pattern of solar UV irradiation are based to propose an optional strategy for low-risk or non-risk application of fungal pesticides after 3:00 or 5:00 pm during summer. © 2022 Society of Chemical Industry.

Synergistic Effect of Beauveria bassiana and Trichoderma asperellum to Induce Maize (Zea mays L.) Defense against the Asian Corn Borer, Ostrinia furnacalis (Lepidoptera, Crambidae) and Larval Immune Response.

Ostrinia furnacalis, is the major pest of maize causing significant yield losses. So far, many approaches have been used to increase the virulence of entomopathogenic fungal isolates. The current study is an attempt to estimate synergistic effect of Beauveria bassiana and Trichoderma asperellum in order to explore larval immune response through RNA sequencing and differentially expression analysis. In vivo synergism was examined in seven proportions (B. bassiana: T. asperellum = 1:1, 1:2, 1:3, 1:4, 4:1, 3:1, 2:1) and in the in vitro case, two inoculation methods were applied: seed coating and soil drenching. Results revealed significant decrease in plant damage and high larval mortality in fungal treatments. Fungal isolates mediated the plant defense by increasing proline, superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO) and protease activities. Seed coating method was proved to be the most effective in case of maize endophytic colonization. In total, 59 immune-related differentially expressed genes DEGs were identified including, cytochrome P450, heat shock protein, ABC transporter, cadherin, peptidoglycan recognition protein (PGRP), cuticlular protein, etc. Further, transcriptomic response was confirmed by qRT-PCR. Our results concluded that, coculture of B. bassiana and T. asperellum has the synergistic potential to suppress the immune response of O. furnacalis and can be used as sustainable approach to induce plant resistance through activation of defense-related enzymes.

Colonization of Beauveria bassiana 08F04 in root-zone soil and its biocontrol of cereal cyst nematode (Heterodera filipjevi).

Cereal cyst nematodes cause serious yield losses of wheat in Hunaghuai winter wheat growing region in China. Beauveria bassiana 08F04 isolated from the surface of cysts is a promising biological control agent for cereal cyst nematodes. As the colonization capacity is a crucial criteria to assess biocontrol effectiveness for a microbial agent candidate, we aimed to label B. bassiana 08F04 for efficient monitoring of colonization in the soil. The binary pCAM-gfp plasmid containing sgfp and hph was integrated into B. bassiana 08F04 using the Agrobacterium tumefaciens-mediated transformation. The transformation caused a significant change in mycelial and conidial yields, and in extracellular chitinase activity in some transformants. The cultural filtrates of some transformants also decreased acetylcholinesterase activity and the survival of Heterodera filipjevi second-stage juveniles relative to the wild-type strain. One transformant (G10) had a growth rate and biocontrol efficacy similar to the wild-type strain, so it was used for a pilot study of B. bassiana colonization conducted over 13 weeks. Real-time PCR results and CFU counts revealed that the population of G10 increased quickly over the first 3 weeks, then decreased slowly over the following 4 weeks before stabilizing. In addition, the application of wild-type B. bassiana 08F04 and transformant G10 significantly reduced the number of H. filipjevi females in roots by 64.4% and 60.2%, respectively. The results of this study have practical applications for ecological, biological and functional studies of B. bassiana 08F04 and for bionematicide registration.

The Effects of Endophytic Beauveria bassiana Inoculation on Infestation Level of Planococcus ficus, Growth and Volatile Constituents of Potted Greenhouse Grapevine (Vitis vinifera L.).

Endophytic entomopathogenic fungi are being explored for the management of phytophagous insect pests. The effects of Beauveria bassiana (Hypocreales) inoculation of grape plants on the infestation level of P. ficus, tissue nutrient contents, and growth and volatile constituents of potted grape plants were assessed. Grapevine plants were individually inoculated with a suspension of 1 × 108 conidia mL-1 of B. bassiana by drenching before experimentally infesting each of them with thirty adult females of P. ficus. At four weeks post-treatment, the fungus was re-isolated from leaves of 50% of the fungus-exposed plants. However, no significant difference (p > 0.05) was observed in all the plant growth parameters measured in the fungus-treated and control plants. Plant tissue analysis revealed markedly higher contents of calcium (Ca) and magnesium (Mg) in the leaf tissue of plants exposed to the B. bassiana relative to the control. Gas chromatography mass spectrometry (GC-MS) analyses showed that a significantly (X² = 5.1; p < 0.02) higher number of known anti-insect volatile compounds (nine) were present among fungus treated plants compared to the control plants (five). Naphthalene, which is toxic to insects and humans, was detected only in the volatiles of the fungus-exposed plants. B. bassiana did not have any significant effect on total polyphenol, alkaloid, and flavonoids. Overall, treatment with fungus did not inhibit the infestation by P. ficus. In conclusion, these findings shed light on some of the mechanisms involved in endophytic fungus-plant-insect interactions.

Persistence of Metarhizium (Hypocreales: Clavicipitaceae) and Beauveria bassiana (Hypocreales: Clavicipitaceae) in Tobacco Soils and Potential as Biocontrol Agents of Spodoptera litura (Lepidoptera: Noctuidae).

Entomopathogenic fungi (EPF), such as Metarhizium spp. and Beauveria bassiana, are widely used in the biocontrol of many species of insect pests. Tobacco is an economically important crop in Guangdong Province of China, but insect pests, such as Spodoptera litura Fabricius, are a major threat to production. Here, we tested the persistence of five Metarhizium species and B. bassiana in glasshouse pot and field experiments and assessed their long-term efficacy against S. litura. We found that the colony forming units of these EPF decreased by c. 93% by 180 d in the pot soils declines tended to be exponential. In contrast, declines of c. 99% in field soils were more gradual (linear), occurring throughout the 360 d experiment. Metarhizium anisopliae Ma09 had the longest estimated half-life of 41 d, while that of B. bassiana was the shortest (9 d). Fungal density in the upper soil layer (0-5 cm) decreased rapidly and was undetectable after 150 d, whereas density was consistently greatest in the mid-layer (10-15 cm). At 180 d after inoculation, strain Ma09 elicited highest rates of mortality in S. litura. We conclude that soils in Guangdong Province are all suitable for the use of Metarhizium as a biocontrol agent, where M. anisopliae Ma09 offers greatest residual activity.

Efectividad de Beauveria bassiana (Baubassil®) sobre la garrapata común del ganado bovino Rhipicephalus microplus en el Departamento de la Guajira, Colombia / Effectiveness of Beauveria bassiana (Baubassil®) on the common cattle tick Rhipicephalus microplus in the Department of Guajira, Colombia

Se evaluó la efectividad de Baubassil® (Beauveria bassiana) sobre la garrapata Rhipicephalus (Boophilus) microplus. En la fase in vitro, se determinaron la temperatura óptima de germinación del hongo mediante microcultivos a 25, 30 y 35° C, y la mortalidad de las garrapatas. En campo, en un diseño aleatorio, se utilizaron 30 vacas cebuinas; 15 vacas se trataron con Baubassil® una vez por semana con 1 x 10(6) conidias/ml y un grupo control se dejó sin tratamiento. Se observó que a temperaturas de 25, 30 y 35 °C, la germinación del entomopató-geno fue del 97,5, el 94,7 y el 6,3%, respectivamente. En los animales tratados con Baubassil® se obtuvo una efectividad corregida del 96,8%. En el Caribe Seco, Baubassil® constituiría una alternativa de control del ectoparásito, pues no existen registros anteriores en La Guajira del control sostenible de esta garrapata.

The effectiveness of Baubassil® (Beauveria bassiana) on the tick Rhipicephalus (Boophilus) microplus was evaluated. In the in vitro phase, the optimal germination temperature of the fungus was determined by microcultures at 25, 30 and 35 °C and the mortality of ticks. In the field, in a randomized design, 30 cebuine cows; 15 cows were treated with Baubassil® once a week at a 1 x 10(6) conidia/ml, and a control group was left untreated. It was observed that at temperatures of 25, 30 and 35 °C, germination of the entomopathogen was 97.5, 94.7 and 6.3%, respectively. In animals treated with Baubassil® obtained a corrected effectiveness of 96.8% on ticks in the fifth week of application. The use of Baubassil® would be a valid alternative for controlling the cattle tick under the agroecological conditions of the Dry Caribbean.

The Beauveria bassiana Gas3 ß-Glucanosyltransferase Contributes to Fungal Adaptation to Extreme Alkaline Conditions.

Fungal ß-1,3-glucanosyltransferases are cell wall-remodeling enzymes implicated in stress response, cell wall integrity, and virulence, with most fungal genomes containing multiple members. The insect-pathogenic fungus Beauveria bassiana displays robust growth over a wide pH range (pH 4 to 10). A random insertion mutant library screening for increased sensitivity to alkaline (pH 10) growth conditions resulted in the identification and mapping of a mutant to a ß-1,3-glucanosyltransferase gene (Bbgas3). Bbgas3 expression was pH dependent and regulated by the PacC transcription factor, which activates genes in response to neutral/alkaline growth conditions. Targeted gene knockout of Bbgas3 resulted in reduced growth under alkaline conditions, with only minor effects of increased sensitivity to cell wall stress (Congo red and calcofluor white) and no significant effects on fungal sensitivity to oxidative or osmotic stress. The cell walls of ΔBbgas3 aerial conidia were thinner than those of the wild-type and complemented strains in response to alkaline conditions, and ß-1,3-glucan antibody and lectin staining revealed alterations in cell surface carbohydrate epitopes. The ΔBbgas3 mutant displayed alterations in cell wall chitin and carbohydrate content in response to alkaline pH. Insect bioassays revealed impaired virulence for the ΔBbgas3 mutant depending upon the pH of the media on which the conidia were grown and harvested. Unexpectedly, a decreased median lethal time to kill (LT50, i.e., increased virulence) was seen for the mutant using intrahemocoel injection assays using conidia grown at acidic pH (5.6). These data show that BbGas3 acts as a pH-responsive cell wall-remodeling enzyme involved in resistance to extreme pH (>9).IMPORTANCE Little is known about adaptations required for growth at high (>9) pH. Here, we show that a specific fungal membrane-remodeling ß-1,3-glucanosyltransferase gene (Bbgas3) regulated by the pH-responsive PacC transcription factor forms a critical aspect of the ability of the insect-pathogenic fungus Beauveria bassiana to grow at extreme pH. The loss of Bbgas3 resulted in a unique decreased ability to grow at high pH, with little to no effects seen with respect to other stress conditions, i.e., cell wall integrity and osmotic and oxidative stress. However, pH-dependent alternations in cell wall properties and virulence were noted for the ΔBbgas3 mutant. These data provide a mechanistic insight into the importance of the specific cell wall structure required to stabilize the cell at high pH and link it to the PacC/Pal/Rim pH-sensing and regulatory system.

Entomopathogenic fungal infection leads to temporospatial modulation of the mosquito immune system.

Alternative methods of mosquito control are needed to tackle the rising burden of mosquito-borne diseases while minimizing the use of synthetic insecticides, which are threatened by the rapid increase in insecticide resistance in mosquito populations. Fungal biopesticides show great promise as potential alternatives because of their ecofriendly nature and ability to infect mosquitoes on contact. Here we describe the temporospatial interactions between the mosquito Aedes aegypti and several entomopathogenic fungi. Fungal infection assays followed by the molecular assessment of infection-responsive genes revealed an intricate interaction between the mosquito immune system and entomopathogenic fungi. We observed contrasting tissue and time-specific differences in the activation of immune signaling pathways and antimicrobial peptide expression. In addition, these antifungal responses appear to vary according to the fungal entomopathogen used in the infection. Enzyme activity-based assays coupled with gene expression analysis of prophenoloxidase genes revealed a reduction in phenoloxidase (PO) activity in mosquitoes infected with the most virulent fungal strains at 3 and 6d post-fungal infection. Moreover, fungal infection led to an increase in midgut microbiota that appear to be attributed in part to reduced midgut reactive oxygen species (ROS) activity. This indicates that the fungal infection has far reaching effects on other microbes naturally associated with mosquitoes. This study also revealed that despite fungal recognition and immune elicitation by the mosquito, it is unable to successfully eliminate the entomopathogenic fungal infection. Our study provides new insights into this intricate multipartite interaction and contributes to a better understanding of mosquito antifungal immunity.

Synergistic Combinations of a Pyrethroid Insecticide and an Emulsifiable Oil Formulation of Beauveria bassiana to Overcome Insecticide Resistance in Listronotus maculicollis (Coleoptera: Curculionidae).

The annual bluegrass weevil, Listronotus maculicollis (Kirby), is a major pest of golf course turf in eastern North America and has become particularly problematic owing to widespread development of insecticide resistance. As an alternative option to manage resistant adult L. maculicollis, we explored combinations of the pyrethroid insecticide bifenthrin with an emulsifiable oil formulation of the entomopathogenic fungus Beauveria bassiana strain GHA (Bb ES). Combinations synergistically enhanced mortality in both insecticide-susceptible and insecticide-resistant L. maculicollis adults in the laboratory when bifenthrin was used at LC50s for each population. To determine the component behind the synergism, technical spores of B. bassiana GHA and the emulsifiable oil carrier in the fungal formulation were tested separately or in combination with bifenthrin. In both separate and combined applications, the emulsifiable oil carrier was responsible for high mortality within 3 d after treatment and interacted synergistically with bifenthrin, whereas fungus-induced mortality started later. Strong synergism was also observed in three field experiments with a relatively resistant L. maculicollis population. Combinations of Bb ES and bifenthrin hold promise as an effective L. maculicollis management tool, particularly of pyrethroid-resistant populations.

Activation of Aedes aegypti prophenoloxidase-3 and its role in the immune response against entomopathogenic fungi.

Serine protease cascade-mediated melanization is an important innate immune response in insects and crustaceans, which involves the proteolytic activation of prophenoloxidase (PPO). In this study, we investigated the role of Aedes aegypti PPO3 in antifungal immune defence. We expressed and purified recombinant PPO3 (rPPO3) in Escherichia coli and demonstrated that rPPO3 was activated by ethanol and, to a lesser extent, by cetylpyridinium chloride. In the presence of Cu2+ , rPPO3 exhibited enzyme activity. Immunoblot results revealed that the rPPO3 was cleaved by the haemolymph from immune-challenged mosquitoes or purified Ostrinia furnacalis serine protease 105 in vitro. The cleaved rPPO3 converted dopamine to toxic intermediates that killed fungal conidia of Beauveria bassiana in vitro. In mosquitoes challenged with Be. bassiana, cleavage of rPPO3 produced a 50 kDa phenoloxidase (PO) fragment. Further analysis revealed that the survival rate of mosquitoes with fungal infection increased significantly following injection of rPPO3 into the haemocoel. Taken together, our results suggest that proteolytic cleavage of the mosquito PPO3 plays an important role in the antifungal immune response. This has led to a better understanding of the mechanism of PPO activation in the mosquito and the role of melanization in the antifungal immune response.

Suppression of a methionine synthase by calmodulin under environmental stress in the entomopathogenic fungus Beauveria bassiana.

Methionine synthase (MetE, EC 2.1.1.14) catalyses the final step in the methionine biosynthetic pathway. Methionine biosynthesis plays a major role in protein biogenesis and is the source of S-adenosyl methionine (SAM), the universal donor of methyl groups. In this study, we demonstrated that BbMetE acts as a typical MetE enzyme in the entomopathogenic fungus Beauveria bassiana. In addition, we found that BbMetE binds to calmodulin (CaM) in vitro and in vivo. The functional role of CaM binding to BbMetE was to negatively regulate BbMetE activity in B. bassiana. Our proton-nuclear magnetic resonance data revealed that CaM inhibitor W-7 increases methionine content in B. bassiana, suggesting that CaM negatively regulates the BbMetE activity. Environmental stress stimuli such as salt, H2 O2 and heat suppressed BbMetE activity in B. bassiana. W-7 reversed this effect, suggesting that the inhibitory mechanism is mediated through stimulation of CaM activity. Therefore, this work suggests that BbMetE plays an important role in methionine biosynthesis, which is mediated by environmental stress stimuli via the CaM signalling pathway.

IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF TWO SERINE PROTEASES AND THEIR POTENTIAL INVOLVEMENT IN PROPHENOLOXIDASE ACTIVATION IN Plutella xylostella.

The proteolytic activation of prophenoloxidase (proPO) is a humoral defense mechanism in insects and crustaceans. Phenoloxidase (PO) is produced as an inactive precursor namely, proPO and is activated via specific proteolytic cleavage by proPO-activating proteinase. The current research reports two novel serine proteinase genes (PxSP1-768 bp and PxSP2-816 bp) from Plutella xylostella, encoding 255 and 271 amino acid residues, respectively. Tissue distribution analyses by semiquantitative reverse transcription-PCR (RT-PCR) revealed the resultant genes to be primarily expressed in the hemocytes, while quantitative-RT-PCR (qRT-PCR) assay showed that transcription level of PxSP1 and PxSP2 increased significantly after injection of the fungal pathogen Beauveria bassiana. Purified recombinant fusion proteins of PxSP2 and PxSP1 were injected to New Zealand white rabbits and polyclonal antibodies were generated with the titers of 1:12,800. After silencing the expression of PxSP2 by RNAi, the PO activity decreased significantly. The results show that PxSP2 is involved in prophenoloxidase activation in P. xylostella.

Expression profiles of astakine-like transcripts in the tarnished plant bug, Lygus lineolaris, exposed to fungal spores of Beauveria bassiana.

Astakines are hematopoietic cytokines originally isolated from crustaceans. We identified three astakine-like transcripts in the tarnished plant bug (Lygus lineolaris), LlAst-1, LlAst-2 and LlAst-3, containing prokineticin domains. Quantitative real-time PCR showed variation in expression patterns of astakines in different tissues and between sexes. Relative expression levels of LlAst-1 were highest in the fat bodies of females, while LlAst-2 expression was highest in the fat bodies of both males and females. LlAst-3 expression was higher in male legs compared with the female legs, but lower in all other tissues. Infection with the entomopathogenic fungus Beauveria bassiana slightly elevated LlAst-1 expression 48 h after infection in both males and females. In contrast, the expression levels of LlAst-2 and LlAst-3 were not significantly changed in males and females. Compared with 12:00 h, LlAst-1 level was higher in both sexes at 18:00 h and 00:00 h (midnight). By 6:00 h, the LlAst-1 level in females was significantly reduced while that in males remained high. LlAst-2 and -3 had highest relative expression levels in females at midnight but were significantly lower than in males at midnight and in both sexes at 18:00 h and 6:00 h. This is the first report of expression of astakine-like cytokines from insects.

Ostrinia furnacalis serpin-3 regulates melanization cascade by inhibiting a prophenoloxidase-activating protease.

Serine protease cascade-mediated prophenolxidase activation is a prominent innate immune response in insect defense against the invading pathogens. Serpins regulate this reaction to avoid excessive activation. However, the function of serpins in most insect species, especially in some non-model agriculture insect pests, is largely unknown. We here cloned a full-length cDNA for a serpin, named as serpin-3, from Asian corn borer, Ostrinia furnacalis (Guenée). The open reading frame of serpin-3 encodes 462-amino acid residue protein with a 19-residue signal peptide. It contains a reactive center loop strikingly similar to the proteolytic activation site in prophenoloxidase. Sequence comparison indicates that O. furnacalis serpin-3 is an apparent ortholog of Manduca sexta serpin-3, a defined negative regulator of melanization reaction. Serpin-3 mRNA and protein levels significantly increase after a bacterial or fungal injection. Recombinant serpin-3 efficiently blocks prophenoloxidase activation in larval plasma in a concentration-dependent manner. It forms SDS-stable complexes with serine protease 13 (SP13), and prevents SP13 from cleaving prophenoloxidase. Injection of recombinant serpin-3 into larvae results in decreased fungi-induced melanin synthesis and reduced the expression of attacin, cecropin, gloverin, and peptidoglycan recognition protein-1 genes in the fat body. Altogether, serpin-3 plays important roles in the regulation of prophenoloxidase activation and antimicrobial peptide production in O. furnacalis larvae.

Subcellular localization of six thioredoxins and their antioxidant activity and contributions to biological control potential in Beauveria bassiana.

Thioredoxins (Trx) can detoxify sulfide or act as electron donors in the reduction of disulfide and dithiol to protect yeast cells from ROS damage but remain poorly explored in filamentous fungi. Here we show more Trx homologs in Beauveria bassiana than in many other fungi and examine their functions. This filamentous entomopathogen has six Trx homologs, including four (Txr1-4) evidently localized in cytoplasm, one (Trx5) in nuclear membrane and another (Trx6) in mitochondria. Deletion of each trx had no effect on radial growth on rich or minimal medium but resulted in remarkable transcriptional up-regulation of other partners for compensation. Compared with wild-type, only Δtrx2 was significantly more sensitive to menadione whereas none of six Δtrx mutants was responsive to other oxidants including H2O2. Intriguingly, Δtrx2 showed uniquely a significant increase in total Trx activity in normal cultures but a remarkable decrease in total SOD activity in the cultures grown normally or co-cultivated with menadione. The ratio of reduced/oxidized glutathione accumulated in hyphal cells stressed with menadione decreased to only 0.4 in Δtrx2 from ∼1.0 observed in wild-type and other mutants. The six Δtrx mutants displayed one or more phenotypic changes associated with the fungal biocontrol potential, including conidiation, and germination, thermotolerance, UV-B resistance and virulence of their conidia. All the changes were restored by trx complementation. Taken together, the greater Trx diversity evolutionarily gained by B. bassiana could help it to maintain cellular redox homeostasis and infect insect hosts in diverse habitats.