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Abstract Fall armyworm, Spodoptera frugiperda (FAW) is a cosmopolitan crop pest species that has recently become established in sub-Saharan Africa and Southeast Asia. Current FAW control is almost entirely dependent on synthetic pesticides. Biopesticides offer a more sustainable alternative but have limitations. For example, pyrethrum is an effective botanical insecticide with low mammalian toxicity but is highly UV labile, resulting in a rapid loss of efficacy in the field. Beauveria bassiana is an entomopathogenic fungus that is more persistent, but there is a time lag of several days before it causes insect mortality and leads to effective control. The combination of these biopesticides could mitigate their drawbacks for FAW control. Here we evaluated the efficacy of pyrethrum and B. bassiana as individual treatments and in combination against 3rd instar FAW. Four different combinations of these two biopesticides were tested, resulting in an antagonistic relationship at the lowest concentrations of B. bassiana and pyrethrum (1 × 104 conidia mL−1 with 25 ppm) and an additive effect for the other 3 combined treatments (1 × 104 conidia mL−1 with 100 ppm and 1 × 105 conidia mL−1 with 25 ppm and 100 ppm pyrethrum). Additionally, a delay in efficacy from B. bassiana was observed when combined with pyrethrum as well as a general inhibition of growth on agar plates. These results appear to show that this particular combination of biopesticides is not universally beneficial or detrimental to pest control strategies and is dependent on the doses of each biopesticide applied. However, the additive effect shown here at specific concentrations does indicate that combining biopesticides could help overcome the challenges of persistence seen in botanical pesticides and the slow establishment of EPF, with the potential to improve effectiveness of biopesticides for IPM.

Little is known concerning terpenoids produced by members of the fungal order Ophiostomales, with the member Harringtonia lauricola having the unique lifestyle of being a beetle symbiont but potentially devastating tree pathogen. Nine known terpenoids, including six labdane diterpenoids (1–6) and three hopane triterpenes (7–9), were isolated from H. lauricola ethyl acetate (EtOAc) extracts for the first time. All compounds were tested for various in vitro bioactivities. Six compounds, 2, 4, 5, 6, 7, and 9, are described functionally. Compounds 2, 4, 5, and 9 expressed potent antiproliferative activity against the MCF-7, HepG2 and A549 cancer cell lines, with half-maximal inhibitory concentrations (IC50s) ~12.54–26.06 μM. Antimicrobial activity bioassays revealed that compounds 4, 5, and 9 exhibited substantial effects against Gram-negative bacteria (Escherichia coli and Ralstonia solanacearum) with minimum inhibitory concentration (MIC) values between 3.13 and 12.50 μg/mL. Little activity was seen towards Gram-positive bacteria for any of the compounds, whereas compounds 2, 4, 7, and 9 expressed antifungal activities (Fusarium oxysporum) with MIC values ranging from 6.25 to 25.00 μg/mL. Compounds 4, 5, and 9 also displayed free radical scavenging abilities towards 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide (O2−), with IC50 values of compounds 2, 4, and 6 ~3.45–14.04 μg/mL and 22.87–53.31 μg/mL towards DPPH and O2−, respectively. These data provide an insight into the biopharmaceutical potential of terpenoids from this group of fungal insect symbionts and plant pathogens.

Abstract Aphids are major pests affecting cereals, vegetables, fruit, forestry and horticultural produce. A multimodal approach may be an effective route to controlling this prolific pest. We assessed the individual and combined effect of eight insecticides and the entomopathogenic fungi, Metarhizium anisopliae (Metschin.) against the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae), under laboratory conditions. Six of the insecticides tested were found to be highly compatible (flonicamid, imidacloprid, nitenpyram, dinotefuran, pyriproxyfen and spirotetramat), showing positive integration with the fungus and were selected for bioassays. The combination mixtures (1:1 ratio of M. anisopliae: insecticide) were significantly more toxic to A. gossypii than individual treatments. Maximum mortality (91.68%) of A. gossypii was recorded with combination of flonicamid and M. anisopliae (2.4 × 106 cfu/ml) 72 h after application. While minimum mortality (17.08%) was observed with the individual treatment of M. anisopliae (2.4 × 106 cfu/ml). The insecticides revealed toxicity consistent with their compatibility with M. anisopliae, ranking for efficacy exactly as they did for compatibility. In addition, the synergy factor (SF) and co-toxicity coefficient (CTC) values indicated synergistic interactions at different time intervals. The synergistic efficacy revealed the potential of fungus-insecticide integration against sucking insect pests.

C-type lectins (CTLs) are a superfamily of Ca2+-dependent carbohydrate-recognition proteins, and an important pattern recognition receptor (PRR) in insect innate immunity which can mediate humoral and cellular immunity in insects. In this study, we report a novel dual carbohydrate-recognition domain (CRD) CTL from Plutella xylostella which we designate PxIML. PxIML is a protein with a 969 bp open reading frame (ORF) encoding 322 amino acids, containing a signal peptide and a dual-CRD with EPN (Glu124-Pro125-Asn126) and QPD (Gln274-Pro275-Asp276) motifs. The expression of PxIML mRNA in the fat body was significantly higher than in hemocytes and midgut. The relative expression levels of PxIML in the whole insect and the fat body were significantly inhibited after infection with Bacillus thuringiensis 8010 (Bt8010) at 18 h, while they were significantly upregulated after infection with Serratia marcescens IAE6 or Pichia pastoris. The recombinant PxIML (rPxIML) protein could bind to the tested pathogen-associated molecular patterns (PAMPs), and the bacteria of Enterobacter sp. IAE5, S. marcescens IAE6, Staphylococcus aureus, Escherichia coli BL21, and Bt8010 in a Ca2+-dependent manner, however, it showed limited binding to the fungus, P. pastoris. The rPxIML exhibited strong activity in the presence of Ca2+ to agglutinate Bt8010, Enterobacter sp. IAE5 and S. aureus, but it only weakly agglutinated with E. coli BL21, and could not agglutinate with S. marcescens IAE6 or P. pastoris. Furthermore, the rPxIML could bind to hemocytes, promote the adsorption of hemocytes to beads, and enhance the phenoloxidase (PO) activity and melanization of P. xylostella. Our results suggest that PxIML plays an important role in pathogen recognition and in mediating subsequent humoral and cellular immunity of P. xylostella.

The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), is a pest of many important crops globally. Effective control is challenging, with the pest exhibiting resistance to different synthetic pesticides across various groups. However, the mechanisms employed by resistant insects for overexpression of relevant detoxification genes remain unclear. The activity of detoxification enzymes was investigated in this study. Additionally, using RNA interference (RNAi), a functional analysis was completed of two P450s genes in an indoxacarb resistant population of fall armyworms. Elevated resistance levels (resistance ratio = 31.37-fold) in indoxacarb-selected populations of FAW were observed after 14 generations. The qRT-PCR showed higher expression of two cytochrome P450 genes, CYP321A7 and CYP6AE43, in this selected population compared to the control population. RNAi was applied to knock down the P450 dsCYP321A7 and dsCYP6AE43 genes in the FAW larvae. Droplet feeding of the dsRNAs (CYP321A7 and CYP6AE43) via an artificial diet significantly increased mortality rates in the indoxacarb treated population. A shorter larval developmental time of FAW was detected in all dsRNAs-fed larvae. Correspondingly, larval mass was reduced by dsRNAs in indoxacarb resistant populations of fall armyworm. Larval feeding assays demonstrate that dsRNAs targeting, specifically of CYP321A7 and CYP6AE43 enzymes, could be a beneficial technique in the management of indoxacarb resistant populations. Further study on the potential use of dsRNA and its application should be conducted in efforts to counter the development of resistance in FAW against various insecticides in the field.

The insect pathogenic fungus, Ascosphaera apis, is the causative agent of honeybee chalk brood disease. Amylases are secreted by many plant pathogenic fungi to access host nutrients through the metabolism of starch, and the identification of new amylases can have important biotechnological applications. Production of amylase by A. apis in submerged culture was optimized using the response surface method (RSM). Media composition was modeled using Box–Behnken design (BBD) at three levels of three variables, and the model was experimentally validated to predict amylase activity (R2 = 0.9528). Amylase activity was highest (45.28 ± 1.16 U/mL, mean ± SE) in media composed of 46 g/L maltose and1.51 g/L CaCl2 at a pH of 6.6, where total activity was ~11-fold greater as compared to standard basal media. The enzyme was purified to homogeneity with a 2.5% yield and 14-fold purification. The purified enzyme had a molecular weight of 75 kDa and was thermostable and active in a broad pH range (> 80% activity at a pH range of 7–10), with optimal activity at 55 °C and pH = 7.5. Kinetic analyses revealed a Km of 6.22 mmol/L and a Vmax of 4.21 μmol/mL·min using soluble starch as the substrate. Activity was significantly stimulated by Fe2+ and completely inhibited by Cu2+, Mn2+, and Ba2+ (10 mM). Ethanol and chloroform (10% v/v) also caused significant levels of inhibition. The purified amylase essentially exhibited activity only on hydrolyzed soluble starch, producing mainly glucose and maltose, indicating that it is an endo-amylase (α-amylase). Amylase activity peaked at 99.38 U/mL fermented in a 3.7 L-bioreactor (2.15-fold greater than what was observed in flask cultures). These data provide a strategy for optimizing the production of enzymes from fungi and provide insight into the α-amylase of A. apis.