Juvenile hormone antagonistic activity of secondary metabolites from Streptomyces lactacystinicus and their insecticidal activity against Plutella xylostella

Due to their specificity to target insects and low toxicity to non-target organisms, insect growth regulators (IGRs) have been promising alternatives to neurotoxic insecticides. Actinobacteria produce a wide range of secondary metabolites with insecticidal and insect growth regulatory activities. In this study, the culture media of 25 actinobacteria isolates showing high juvenile hormone antagonist (JHAN) activity were assessed for their insecticidal activity to identify novel IGR compounds toxic to Plutella xylostella. Among them, four isolates exhibited high insecticidal activity against 3rd instar larvae of P. xylostella. Two isolates of IMBL-1412 and IMBL-1823 showing relatively high insecticidal activities (greater than90% mortality) were identified as Streptomyces lactacystinicus based on colony color on various International Streptomyces Project (ISP) media and nucleotide sequences of the 16S rRNA gene. The ethyl acetate fractions of both isolates showed high JHAN and insecticidal activities against P. xylostella larvae at a concentration of 100 ppm when the culture media of these two isolates were extracted sequentially using hexane, ethyl acetate, and butyl alcohol. These results suggested that secondary metabolites of these actinobacterial isolates could be efficiently applied to develop novel IGR insecticides for the control of P. xylostella.     

Mosquito larvicidal activities of farnesol and farnesyl acetate via regulation of juvenile hormone receptor complex formation in Aedes mosquito

Due to their target specificity and low-toxicity, insect growth regulators (IGRs) are regarded as promising alternatives to chemical insecticides. In this study, farnesol and farnesyl acetate exhibited juvenile hormone (JH)-based IGR activities. While farnesyl acetate showed JH agonist (JHA) activity in concentration-dependent manner, farnesol was identified as JH antagonist (JHAN) by interfering pyriproxyfen-mediated binding JH receptor complex. Both compounds showed mosquito larvicidal activities and caused retardation of ovarian growth of female Aedes albopictus by modulating the formation of JH receptor complex, expression of JH-inducible genes, and thereby disrupting JH-based endocrine regulations. These results suggested that farnesol and farnesyl acetate could be applicable for investigating underlying mechanisms of JH-regulated insect physiologies as well as developing novel eco-friendly insecticides.     

Insect growth regulatory and larvicidal activity of chalcones against Aedes albopictus

Widespread use of chemical insecticides has resulted in development of insect resistance and natural products with biological activities could become an attractive alternative to control insect pests. In order to find more effective insecticides for controlling mosquito, various mosquitocidal compounds are studied. Recently, juvenile hormone antagonists (JHANs) have been found to be to safe and effective insecticides for control of mosquito. In order to identify novel insecticidal compounds with JHAN activity, several chalcones were surveyed on their JHAN activities and larvicidal activities against Aedes albopictus larvae. Among them, 2′‐hydroxychalcone and cardamonin showed high levels of JHAN and mosquito larvicidal activities. These results suggested that chalcones with JHAN activity could be useful for control of mosquito larvae.     

Mosquitocidal activity of penfluridol as juvenile hormone antagonist

Juvenile hormone antagonists (JHANs) are known to interfere with the formation of juvenile hormone (JH) receptor complex. JHANs might be effective for control of target pests in larval stages at which stages high level of endogenous JH titer is maintained. In order to identify novel insecticidal compounds, 2352 compounds were surveyed on their JHAN activities using the yeast-two hybrid system. Among 53 compounds with JHAN activities, penfluridol showed high level of insecticidal activity against larvae of Aedes albopictus. JHAN activity was increased in proportion to the concentration of penfluridol. Larvicidal activity of penfluridol was 1.3–2.0 folds higher than that of pyriproxyfen. These results suggested that penfluridol could be useful for control of mosquito larvae.     

Characteristics of insecticidal substances from the entomopathogenic fungus Metarhizium pinghaense 15R against cotton aphid in Korea

Aphids (Hemiptera: Aphididae) are agricultural pests for a wide range of economically important crops worldwide. Entomopathogenic fungi have been studied and developed as a biological agents to control hard to control insect pests like the aphid. In particular, secondary metabolites produced by entomopathogenic fungi are being studied as insecticidal substances. This study evaluated the insecticidal activity and characteristics of insecticidal substances in culture filtrate against aphids to confirm the insecticidal substances produced by Metarhizium pinghaense 15R. After treating cotton aphids with the culture filtrate, strong insecticidal activity (100 % insect mortality) was observed on the third day of treatment, and it was confirmed that insecticidal substances in the culture filtrate had high thermal stability. In addition, the physicochemical properties of insecticidal substances in the culture filtrate were evaluated, and it was determined that the insecticidal substances were hydrophobic, with small molecules of 3 kDa or less. Based on these results, it was suspected that the insecticidal substances in the culture filtrate were destruxins (DTXs), which are representative secondary metabolites of Metarhizium known to have insecticidal activity. Through HPLC (High Performance Liquid Chromatography) analysis, the presence of DTX A and B was found in all active samples, and it was confirmed that the insecticidal substances of M. pinghaense 15R were DTXs. This study provides primary data for developing biochemical crop protection agents using these insecticidal substances, and we suggest the potential of using insecticidal substances of entomopathogenic fungi as biochemical pesticides in Korea.     

Management of pine wilt disease vectoring Monochamus alternatus adults using spray and soil application of Metarhizium anisopliae JEF isolates

Chemical control is widely used to control the Japanese pine sawyer beetle, Monochamus alternatus, but strong chemical regulations require an environmentally sound management strategy. In this work, we investigated the use of entomopathogenic fungi and their application as a means of practical pest management. Thirty-two diverse species of fungal isolates were assayed against adult pine sawyer beetles using a contact method under laboratory conditions, and four isolates showed over 70% virulence consequently. These isolates, two each of Beauveria bassiana and Metarhizium anisopliae were sprayed on the adult beetles at 1 × 10⁷ conidia/ml in plastic containers, respectively. The M. anisopliae-treated adult beetles showed 67% mortality. M. anisopliae isolates JEF-197 and JEF-279 demonstrated dosage-dependent insecticidal activity. Following the laboratory experiments, semi-field trials were conducted in young pine trees under high (RH 94%) and low (RH 35%) humidity conditions. In the high humidity conditions, most of the adult beetles stayed on the top of the branches. When the two M. anisopliae isolates were sprayed on the beetles, they showed ca. 50–70% insecticidal activity 11 days after application. In contrast, in low humidity conditions, the adult beetles tried to move off the branches and onto the soil. When the beetles reached the JEF-197 and JEF-279-treated soil, we measured >90% insecticidal activity. This work suggests that M. anisopliae was the most virulent entomopathogenic fungus against adult Japanese pine sawyer beetles, and this forest insect could be ecologically controlled by the spray and soil application of the M. anisopliae isolates.     

Simple and rapid screening of entomopathogenic fungi having radical-scavenging and anticancer activities

Entomopathogenic fungal metabolites exhibit a wide variety of insecticidal, antimicrobial, anticancer, antioxidant, and antiviral activities, and they have been suggested as potential candidates for the development of new bioactive agents. The rapid and simple screening of entomopathogenic fungi with such bioactivities is important for investigating bioactive substances. The radical-scavenging and anticancer activities of 28 species and 20 genera of 342 isolates of entomopathogenic fungi were evaluated using only culture filtrates. As a result, 23 isolates of Metarhizium anisopliae var. anisopliae, 1 isolate of Pochonia sp., and 1 isolate of Aspergillus sp. showed high radical-scavenging activity. Additionally, anticancer activity in HCT116 human colon cancer cells was also confirmed for selected culture filtrates. This simple screening method of entomopathogenic fungi using culture filtrates may be useful for the rapid detection of related substances with pharmacological activity from a large number of fungal samples.     

Design,synthesis and insecticidal evaluation of aryloxy dihalopropene derivatives

Plutella xylostella (P. xylostella) is a highly migratory, cosmopolitan species and one of the most important pest of cruciferous crops worldwide. Pyridalyl as a novel class of insecticides has good efficacy against P. xylostella. On the basis of the commercial insecticide pyridalyl, a series of new aryloxy dihalopropene derivatives were designed and synthesized by using Intermediate Derivatization Methods. Their chemical structures were confirmed by ¹H NMR, high-resolution mass spectrum (HRMS), and single-crystal X-ray diffraction analysis. The insecticidal activities of the new compounds against P. xylostella were evaluated. The results of bioassays indicated that most of the compounds showed moderate to high activities at the tested concentration, especially compounds 10e and 10g displayed more than 75% insecticidal activity against P. xylostella at 6.25 mg/L, while pyridalyl showed 50% insecticidal activity at the same concentration. The field trials result of the insecticidal activities showed that compound 10e as a 10% emulsifiable concentrate (EC) was effective in the control of P. xylostella at 75–150 g a.i./ha, and the mortality of P. xylostella for treatment with compound 10e at 75 g a.i./ha was equivalent to pyridalyl at 105 g a.i./ha.     

Characterisation and expression of a novel holotype crystal protein gene, <Emphasis Type="Italic">cry56Aa1</Emphasis>, from <Emphasis Type="Italic">Bacillus</Emphasis> <Emphasis Type="Italic">thuringiensis</Emphasis> strain Ywc2-8

Bacillus thuringiensis isolate Ywc2-8, from soil in Sichuan Basin in western China, contains a spherical crystal harbouring two insecticidal crystal proteins with masses of 70 kDa and 130 kDa. A novel cry-type gene, encoding a 664 amino acid protein with 34% homology to cry29Aa1, was found and cloned from this strain. This gene belongs to a novel holotype cry and is designated as cry56Aa1. It was expressed in E. coli. Insecticidal activity assays showed that recombinant Cry56Aa1 was toxic to both Dipteran (Aedes aegypti) and Lepidopteran (Plutella xylostella and Helicoverpa armigera) pests. Cloning of this gene may help to overcome the increasing resistance of pests to currently used insecticides.     

Soil and entomopathogenic fungi with potential for biodegradation of insecticides: degradation of flubendiamide in vivo by fungi and in vitro by laccase

Purpose

Flubendiamide is a highly toxic and persistent insecticide that causes loss of insect muscle functions leading to paralysis and death. The objective was to screen for filamentous fungi in soils where insecticides had been applied, to isolate entomopathogenic fungi from insect larva (Anticarsia gemmatalis) that infest soybean crops, and to use these in biodegradation of insecticides.

Method

Filamentous fungi were isolated from soils, and growth inhibition was evaluated on solid medium containing commercial insecticides, Belt® (flubendiamide) and Actara® (thiamethoxam). A total of 133 fungi were isolated from soil and 80 entomopathogenic fungi from insect larva. Based on growth inhibition tests, ten soil fungi, 2 entomopathogenic fungi, and Botryosphaeria rhodina MAMB-05 (reference standard) were selected for growth on commercial insecticides in solid media. Fungi were grown in submerged fermentation on media containing commercial insecticides and assayed for laccase activity.

Result

Isolates JUSOLCL039 (soil), JUANT070 (insect), and MAMB-05 performed best, and were respectively inhibited by 48.41%, 75.97%, and 79.23% when cultivated on 35 g/L Actara®, and 0.0, 5.42%, and 43.39% on 39.04 g/L Belt®. JUSOLCL039 and JUANT070 were molecularly identified as Trichoderma koningiopsis and Neurospora sp., respectively. The three fungal isolates produced laccase constitutively, albeit at low activities. Fungal growth on pure flubendiamide and thiamethoxam resulted in only thiamethoxam inducing high laccase titers (10.16 U/mL) by JUANT070. Neurospora sp. and B. rhodina degraded flubendiamide by 27.4% and 9.5% in vivo, while a crude laccase from B. rhodina degraded flubendiamide by 20.2% in vitro.

Conclusion

This is the first report of fungi capable of degrading flubendiamide, which have applications in bioremediation.

     

Cloning and expression of novel lipase from entomopathogenic fungi,Beauveria bassiana

Lipase, which catalyzes the hydrolysis of triacylglycerol, is one of the enzymes of interest in the bio-industry. In this study, strains of entomopathogenic fungi were screened for their lipase activity and the characteristics of lipase isolated from Beauveria bassiana JEF-351 strain showing high lipase activity were investigated. Lipase gene of B. bassiana JEF-351 strain, BBL351, was introduced into the genome of Autographa californica nucleopolyhedrovirus (AcMNPV) and recombinant lipases were expressed as non-secreted and secreted protein, respectively. Enzyme activity of BBL351 was higher when the protein was expressed as secreted form, demonstrating that post-translational modification such as glycosylation is crucial for enzyme activity. In addition, the enzyme activity of BBL351 was higher than that of CML, which is an sn-1(3) regioselective lipase reported from Cordyceps militaris. These results suggested that the lipase derived from the B. bassiana JEF-351 strain could be useful as biocatalyst in the biotechnological applications.     

Colonization of Metarhizium anisopliae on the surface of pine tree logs: A promising biocontrol strategy for the Japanese pine sawyer,Monochamus alternatus

We investigated the colonization potential of five Metarhizium anisopliae isolates on pine tree surfaces under laboratory conditions, determined the influence of the pine bark extract on fungal growth and evaluated the insecticidal activity following colonization on the Japanese pine sawyer. Finally, the effect of colonization on adults pine sawyer was evaluated using the top three performing isolates (JEF-197, JEF-271 and JEF-279) under laboratory and field conditions. As a result, isolate JEF-197 showed the highest conidial production on the pine surfaces, and five isolates, including JEF-197, showed higher hyphal growth on autoclaved pine bark extract agar, compared to a water agar. Pine bark treated with the isolates showed 40–70 % mortality of adults pine sawyer. Under mimicked overwintering conditions, in the JEF-197 treatment group, 40 % of the inserted larvae became adults and all were dead after 59 d. In a field test, colonized isolate JEF-197 also showed 37 % insecticidal activity against emerged adults from the pine logs as overwintering sites. This work suggests that M. anisopliae isolate JEF-197 possibly colonized the pine surface and application of a conidial suspension on the pine logs as overwintering sites could be an effective strategy to control the pine sawyer.     

Correlation of fruit tree rhizosphere soils with entomopathogenic fungi

Entomopathogenic fungi are microorganisms that control the density of host insects in nature; they are being studied as environmentally friendly alternatives to chemical insecticides for controlling insect pests. The main habitat of entomopathogenic fungi is soil, and the correlation between the distribution of entomopathogenic fungi and the physicochemical characteristics of soils planted with different trees, including vine (outdoor, greenhouse, and greenhouse shine musket), apple, peach, and pear, were analyzed. The entomopathogenic fungi of the genera Beauveria, Metarhizium, and Purpureocillium investigated in this study were all found in soil samples from vine-greenhouse, apple, and peach trees. Purpureocillium and Beauveria abundances were positively correlated with soil properties; however, Metarhizium abundances were not correlated with soil properties. The Metarhizium isolates discovered in this study showed pathogenicity to cotton aphids (an agricultural pest) and can be employed as sources for biological studies in the future. This study provides data on the diversity and abundance of entomopathogenic fungi related with soil properties, as well as the molecular, biological, and insecticidal characteristics of Metarhizium isolates.     

Insecticidal and antifeeding activity of Perilla frutescens‐derived material against the diamondback moth,Plutella xylostella L

Insecticidal and antifeeding activities against Plutella xylostella were observed using whole‐plant‐derived Perilla frutescens material. The active ingredient in P. frutescens was identified by spectroscopic analysis as the sesquiterpenoid α‐farnesene, which showed insecticidal activity against third‐instar larva of P. xylostella in a leaf‐dipping bioassay based on 24‐h LD₅₀ values (LD₅₀ = 53.7 ppm). The feeding inhibition rate of α‐farnesene was 82.98% against P. xylostella at 10 ppm, and the antifeeding responses were determined using an oscilloscope to detect electrophysiological responses. The electrophysiological responses of the medial styloconic sensillum (MSS) were approximately 7‐fold more sensitive at 100 ppm than those of the lateral styloconic sensillum (LSS). These results suggest that the insecticidal and antifeeding effect of α‐farnesene, which is a P. frutescens‐derived material, can be used as a potential control agent for P. xylostella.     

Entomopathogenic fungi-mediated biological control of the red palm weevil Rhynchophorus ferrugineus

The red palm weevil (RPW), Rhynchophorus ferrugineus, is an important pest of palms, and difficult to control by conventional methods. Therefore, microbial control is an alternative strategy for controlling RPW. Herein, a total of 15 entomopathogenic fungi (EPFs) were subjected to primary pathogenicity screening against last stage of RPW larvae. The preliminary data showed that four Beauveria bassiana isolates (JEF-484, 158, 462 and 507) and one Isaria fumosorosea isolate (JEF-014) resulted in 100 % mortality within 5–10 days post inoculation (d.p.i.), respectively. According to the time required for RPW mortality, JEF-484, 158, 462 and 014 were further subjected to bioassays using 10⁷ conidia/ml suspensions by spraying method. Based on the results, JEF-484 showed the highest mortality and shortest LT₅₀ on the last stage of RPW larvae, followed by JEF-158. The two isolates also showed good conidial production and high thermal stability compared to the other isolates. Therefore, JEF-484 and JEF-158 were selected for bioassays against RPW egg and the last larval stage with different concentrations of 10⁵, 10⁶ and 10⁷ conidia/ml conidial suspensions by spraying method. For the bioassay at the egg stage, JEF-158 showed a significantly higher ovicidal effect than JEF-484. In the larval bioassay, both EPF isolates showed a dosage-dependent effect on the RPW larvae. JEF-484 caused higher mortality in RPW larvae than JEF-158. In summary, the combination of the 2 promising EPF isolates might provide an opportunity for the practical microbial control of RPW at different life stages in palm tree fields.     

Insecticide susceptibility of Culex pipiens pallens (L.) and Aedes albopictus (Skuke) to five commonly used pesticides in the Republic of Korea

Field collected populations of two mosquito species, Culex pipiens pallens and Aedes albopictus, were tested for susceptibility to five pyrethroid insecticides using a topical application bioassay. The laboratory strain of Cx. pipiens pallens (KS‐CP) demonstrated the most and the lowest susceptibility to deltamethrin and d‐cis/trans prallethrine with LD₅₀ values of 0.00022 μg/♀ and 0.00178 μg/♀, respectively, while Ae. albopictus (KS‐AL) showed the greatest and the lowest susceptibility to phthalethrin and deltamethrin with LD₅₀ values of 0.00015 μg/♀ and 0.00085 μg/♀, respectively. Cx. pipiens pallens (JF‐CP) collected at a nearby rice field in Jeonju showed the greatest susceptibility to deltamethrin (LD₅₀ value of 0.0473 μg/♀) and the resistance ratios (RRs) and compared to the laboratory strain were 215 fold greater. JF‐CP demonstrated the lowest susceptibility to d‐cis/trans phenothrin (0.7697 μg/♀) with RRs 711.0 fold greater than the KS‐CP. An Ae. albopictus (JF‐AL) collected at a nearby forest in Jeonju showed the highest susceptibility to deltamethrin (0.00203 μg/♀) with RRs 2.4 fold greater than the KS‐AL. JF‐AL demonstrated the lowest susceptibility to d‐cis/trans prallethrine (LD₅₀ value 0.00646 μg/♀) with RRs 9.4 fold greater than the KS‐AL. JF‐AL demonstrated the greatest resistance to phthalethrin with RRs 39.3 fold greater than the KS‐AL. Resistance ratios of Cx. pipiens pallens between field and laboratory strains ranged from 215.0 to 711.0 and Ae. albopictus ranged from 2.4 to 39.3. These results indicate that the choice of effective insecticides to selected species of mosquito populations will result in more effective control against field strains of mosquito vectors and decrease environmental pollution.     

Interactions of Bacillus thuringiensis bioinsecticides and the predatory stink bug Podisus nigrispinus to control Plutella xylostella

The diamondback moth (DBM), Plutella xylostella (L.), is a major pest of brassica crops worldwide. Control of this pest is difficult because it rapidly develops resistance to synthetic and biological insecticides and because of the effects of insecticides on its natural enemies. Podisus nigrispinus (Dallas) is a predator that feeds on its prey, as well as on the host plants of its prey, and is an important biological control agent of DBMs. The aim of this study was to determine the susceptibility of P. xylostella larvae to two bioinsecticides: the HD1 strain of Bacillus thuringiensis (B. thuringiensis var. kurstaki) and the commercial product Agree^® (B. thuringiensis var. aizawai CG 91). In addition, the impact of these bioinsecticides on the P. nigrispinus consumption of DBM larvae and phytophagy was evaluated. Both the HD1 strain and Agree^® caused 100% mortality in P. xylostella larvae. P. nigrispinus nymphs fed only with kale leaves (Brassica oleracea var. acephala) sprayed with water, the HD1 strain, or Agree^® did not complete their nymphal development. When prey was also available, P. nigiripinus fed on kale leaves to obtain water. Both nymphs and adults of P. nigrispinus consumed greater numbers of DBM larvae, and fed less on kale leaves, when sprayed with the HD1 strain or Agree^®. These results suggest a positive interaction of B. thuringiensis‐based products and the predator P. nigrispinus in the control of P. xylostella larvae.     

Exposure of helices α4 and α5 is required for insecticidal activity of Cry2Ab by promoting assembly of a prepore oligomeric structure

Cry2Ab, a pore‐forming toxin derived from Bacillus thuringiensis, is widely used as a bio‐insecticide to control lepidopteran pests around the world. A previous study revealed that proteolytic activation of Cry2Ab by Plutella xylostella midgut juice was essential for its insecticidal activity against P. xylostella, although the exact molecular mechanism remained unknown. Here, we demonstrated for the first time that proteolysis of Cry2Ab uncovered an active region (the helices α4 and α5 in Domain I), which was required for the mode of action of Cry2Ab. Either the masking or the removal of helices α4 and α5 mediated the pesticidal activity of Cry2Ab. The exposure of helices α4 and α5 did not facilitate the binding of Cry2Ab to P. xylostella midgut receptors but did induce Cry2Ab monomer to aggregate and assemble a 250‐kDa prepore oligomer. Site‐directed mutagenesis assay was performed to generate Cry2Ab mutants site directed on the helices α4 and α5, and bioassays suggested that some Cry2Ab variants that could not form oligomers had significantly lowered their toxicities against P. xylostella. Taken together, our data highlight the importance of helices α4 and α5 in the mode of action of Cry2Ab and could lead to more detailed studies on the insecticidal activity of Cry2Ab.     

Impacts of fungal entomopathogens on survival and immune responses of Aedes albopictus and Culex pipiens mosquitoes in the context of native Wolbachia infections

Microbial control of mosquitoes via the use of symbiotic or pathogenic microbes, such as Wolbachia and entomopathogenic fungi, are promising alternatives to synthetic insecticides to tackle the rapid increase in insecticide resistance and vector-borne disease outbreaks. This study evaluated the susceptibility and host responses of two important mosquito vectors, Ae. albopictus and Cx. pipiens, that naturally carry Wolbachia, to infections by entomopathogenic fungi. Our study indicated that while Wolbachia presence did not provide a protective advantage against entomopathogenic fungal infection, it nevertheless influenced the bacterial / fungal load and the expression of select anti-microbial effectors and phenoloxidase cascade genes in mosquitoes. Furthermore, although host responses from Ae. albopictus and Cx. pipiens were mostly similar, we observed contrasting phenotypes with regards to susceptibility and immune responses to fungal entomopathogenic infection in these two mosquitoes. This study provides new insights into the intricate multipartite interaction between the mosquito host, its native symbiont and pathogenic microbes that might be employed to control mosquito populations.