Knockdown of the glucosamine-6-phosphate N-acetyltransferase gene by RNA interference enhances the virulence of entomopathogenic fungi against rice leaffolder Cnaphalocrocis medinalis.

Insect cuticle acts as a first line of defense and a physical protective barrier against entomopathogens. Chitin biosynthesis pathway plays a crucial role in chitin formation in the cuticle of insects. Glucosamine-6-phosphate N-acetyltransferase (GNA) is a key enzyme in insect chitin biosynthesis that regulate the chitin formation. However, how GNA-mediated cuticle metabolism influences virulence of entomopathogenic fungi is still unknown. In this study, CmGNA gene was cloned and characterized from the rice leaffolder Cnaphalocrocis medinalis. The CmGNA contains an open read frame (ORF) 600 nucleotides, encoding 199 amino acids with an isoelectric point of 8.65 and a molecular weight of 22.30 kDa. The expression profile showed that CmGNA was highly expressed in 4th instar larvae and in the cuticle. Here, we also reported the impact of CmGNA gene and entomopathogenic fungi, Metarhizium anisopliae and Beauveria bassiana, on expression pattern of chitin biosynthesis genes, feeding behavior, survival rate and average body weight of infected larvae, phenotypic deformities, rate of pupation, and adult emergence. Our results showed that knockdown of CmGNA and application of M. anisopliae and B. bassiana three days after RNA interference (RNAi) significantly decreased the expression of CmGNA and other associated genes, reduced feeding efficiency and survival rate, and caused loss of average body weight, less rate of pupation and adult emergence of infected larvae. Knockdown of CmGNA gene also increased the lethality of larvae caused by M. anisopliae and B. bassiana and resulted in significantly phenotypic deformities of infected larvae. Our findings illustrated that RNAi-mediated CmGNA knockdown disturbed the chitin synthesis genes that led to enhancing the virulence of M. anisopliae and B. bassiana, which can provide us new insights to develop novel biocontrol strategies against C. medinalis.

Marine Natural Products: The Important Resource of Biological Insecticide.

Due to the unique environmental conditions and vast territory, marine habitat breeds more abundant biological resources than terrestrial environment. Massive marine biological species provide valuable resources for obtaining a large number of natural products with diverse structure and excellent activity. In recent years, new breakthroughs have been made in the application of marine natural products in drug development. In addition, the use of marine natural products to develop insecticides and other pesticide products has also been widely concerned. Targeting marine plants, animals, and microorganisms, we have collected information on marine natural products with insecticidal activity for nearly decade, including alkaloids, terpenes, flavonoids and phenols fatty acids, peptides, and proteins, et al. In addition, some active crude extracts are also included. This review describes the insecticidal activities of marine natural products and their broad applications for future research in agriculture and health.

Biochemical and toxicological investigation of karanjin, a bio-pesticide isolated from Pongamia seed oil.

Karanjin, a furanoflavonol from Pongamia pinnata L is used in agricultural practices for its pesticidal, insecticidal and acaricidal activities. It is commercially available as a bio-pesticide targeting a wide variety of pests. The present study was intended to evaluate the biochemical interactions of karanjin with bovine serum albumin (BSA) and study its toxicological effects on mammalian and bacterial cell lines. Karanjin bound to BSA at a single site with a dissociation constant of 19.7 µM. Evaluation of BSA-karanjin interactions at three different temperatures indicated the involvement of static mode of quenching. Binding experiments in the presence of warfarin and computational docking analysis indicated that karanjin bound closer to the warfarin binding site located in the Subdomain IIA of BSA. Using Förster resonance energy transfer analysis the distance between TRP 213 of BSA and karanjin was found to be 20 Å. Collective results from synchronous fluorescence spectra analysis, differential scanning calorimetry, and circular dichroism analysis indicated that binding of karanjin induced conformational changes in the secondary structure of BSA. Karanjin exhibited low toxicity against human cervical cancer cells and normal mouse fibroblast L929 cells and modestly inhibited the growth of B. subtilis and E. coli cells. The data presented in this study provides insights for understanding the binding interactions of karanjin with BSA and its possible toxicological effects on mammalian cell lines and bacteria.

A new biopesticide from a local Bacillus thuringiensis var. tenebrionis (Xd3) against alder leaf beetle (Coleoptera: Chrysomelidae).

Use of chemical pesticides in agriculture harms humans, non-target organisms and environments, and causes increase resistance against chemicals. In order to develop an effective bio-pesticide against coleopterans, particularly against Agelastica alni (Coleoptera: Chrysomelidae) which is one of the serious pests of alder leaf and hazelnut, we tested the insecticidal effect of 21 Bacillus isolates against the larvae and adults of the pest. Bacillus thuringiensis var. tenebrionis-Xd3 (Btt-Xd3) showed the highest insecticidal effect based on screening tests. For toxin protein production and high sporulation of Xd3, the most suitable medium, pH and temperature conditions were determined as nutrient broth medium enriched with salts, pH 7 and 30 °C, respectively. Sporulated Btt-Xd3 in nutrient broth medium enriched with salts transferred to fermentation medium containing soybean flour, glucose and salts. After fermentation, the mixture was dried in a spray dryer, and spore count of the powder product was determined as 1.6 × 1010 c.f.u. g-1. Moisture content, suspensibility and wettability of the formulation were determined as 8.3, 86% and 21 s, respectively. Lethal concentrations (LC50) of formulated Btt-Xd3 were determined as 0.15 × 105 c.f.u. ml-1 for larvae at laboratory conditions. LC50 values were also determined as 0.45 × 106 c.f.u. ml-1 at the field condition on larval stage. Our results showed that a new bio-pesticide developed from B. thuringiensis tenebrionis (Xd3) (Btt-Xd3) may be valuable as a biological control agent for coleopteran pests.

Matrine and oxymatrine in corroborant plant extracts and fertilizers: HPLC/MS-MS method development and single-laboratory validation.

A reversed phase high-performance liquid chromatographic method (HPLC/MS-MS) has been developed and validated for detection of alkaloids matrine and oxymatrine in fertilizer with labeled enhancer plant defense activities. The analytical method was validated statistically. The results show a strong matrix effect, requiring quantification by standard addition method. The regression lines showed r(2) > 0.994. Recoveries ranging from 97 to 104% were obtained for the fortification level of 0.01% wt wt(-1) and the relative standard deviations ranged from 3 to 4% (n = 10). The limits of detection were below 0.0001% wt wt(-1), while the limits of quantification did not exceed 0.0004% wt wt(-1). The method is currently applied in ICQRF Laboratory of Catania on fertilized and corroborant plant extract collected in the Italian market in the frame of MIPAAF institutional quality control activity, with the aim to dectect these unpermitted active substances.

Pesticide mediated silent neurotoxicity and its unmasking: An update on recent progress.

Being human's one of the most protected organs, brain is yet most vulnerable to xenobiotics exposure. Though pesticide-mediated neurotoxicity is well-explored, the fraternity of neurotoxicologists is less focused on the phenomenon of "silent" or "clinically undetectable" neurotoxicity. Silent neurotoxicity defines continual trivial changes in the nervous system that do not manifest any overt signs of toxicity unless unmasked by any natural or experimental event. Although this perception is not novel, insufficient experimental and epidemiological evidence makes it an outlier among toxicological research. A report in 2016 highlighted the need to investigate silent neurotoxicity and its potential challenges. The limited existing experimental data unveiled the unique responsiveness of neurons following silent neurotoxicity unmasking. Concerned studies have shown that low-dose developmental exposure to pesticides sensitizes the nigrostriatal dopaminergic system towards silent neurotoxicity, making it vulnerable to advanced cumulative neurotoxicity following pesticide challenges later in life. Therefore, conducting such studies may explain the precise etiology of pesticide-induced neurological disorders in humans. With no updates on this topic since 2016, this review is an attempt to acquaint the neurotoxicologist with silent neurotoxicity as a serious threat to human health, and proof-of-concept through a narrative using relevant published data so far with future perspectives.

Essential oils from Acacia nilotica (Fabales: Fabaceae) seeds: May have insecticidal effects?

In the present study Acacia nilotica seed derived essential oils were tested against Spodoptera litura, Tenebrio molitor, Oxycarenus hyalinipennis, and Aphis fabae, as well as their effects on non-target species Eudrilus eugeniae and Artemia salina at 24 h post treatment. The seed essential oil produced insecticidal activity against A. fabae (LC50 = 41.679, LC90 = 75.212 µl/mL), O. hyalinipennis (LC50 = 37.629, LC90 = 118.485 µl/mL), T. molitor (LC50 = 56.796, LC90 = 201.912 µl/mL), and S. litura (LC50 = 62.215, LC90 = 241.183 µl/mL). Essential oils do not cause a remarkable effect on E. eugeniae and A. salina cytotoxicity. The essential oils produced a lower effect on Artemia salina (LC50 = 384.382, LC90 = 1341.397 µl/mL) and no lethal effects were observed on E. eugeniae. The histopathological evaluation showed no sub-lethal effects of essential oils on earthworm gut tissues. GC-MS analysis results revealed that the major chemical constituent was hexadecane (19.560%) and heptacosane (17.214%) and FT-IR analysis revealed the presence of alkanes and alkyles, aromatics, and amides functional groups that may be involved in insecticidal activity. Overall, the results showed that the seed derived essential oil has excellent insecticidal action against major agricultural insect pests and may therefore offer an environmentally benign alternative to conventional insecticide.

Efficacy of Mentha aquatica L. Essential Oil (Linalool/Linalool Acetate Chemotype) against Insect Vectors and Agricultural Pests.

In recent years, agrochemical industries have been focused on the development of essential oil (EO)-based biopesticides, which can be considered valuable alternatives to traditional chemical products. The genus Mentha (Lamiaceae) comprises 30 species characterized by a wide range of biological activities, and some of their EOs showed good potential as pesticidal agents. In this regard, the aim of this study was to evaluate the insecticidal activity of the EO obtained from a rare linalool/linalool acetate chemotype of Mentha aquatica L. The EO was found to be highly effective against Culex quinquefasciatus (Say) 2nd instar larvae, Metopolophium dirhodum (Walker) adults, Spodoptera littoralis (Boisduval) 2nd instar larvae, and Tetranychus urticae (Koch) adults, showing lethal concentrations (LC50) or doses (LD50) of 31.5 ± 2.2 µL L-1, 4.9 ± 0.8 mL L-1, 18.5 ± 2.1 µg larvae-1, and 3.3 ± 0.5 mL L-1, respectively. On the contrary, Musca domestica L. adults and 3rd instar larvae of C. quinquefasciatus and S. littoralis were moderately affected by the treatment (LC50 or LD50: 71.4 ± 7.2 µg adult-1, 79.4 ± 5.2 µL L-1, 44.2 ± 5.8 µg larvae-1, respectively). The results obtained in this work demonstrated that various insects and pests could be differently sensible to the same EO and may lead to the exploitation of this plant or its major volatile compounds as novel ingredients of botanical insecticides and pesticides.

Efficacy of Helicoverpa Armigera Nucleopolyhedrovirus on Soybean for Control of Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) in Arkansas Agriculture.

Helicoverpa armigera nucleopolyhedrovirus (HearNPV) is a naturally occurring virus commercially produced for control of Heliothines, including Helicoverpa zea. One drawback with using this virus for control has been the slower time to mortality compared with synthetic insecticides. However, a new formulation (Heligen®) has anecdotally been thought to result in quicker mortality than previously observed. The objective of this study was to evaluate percent defoliation, the efficacy of HearNPV on mortality for each H. zea larval instar, and the potential for control of a second infestation. Fourteen days after the first infestation, all plants were re-infested with a second instar larva to simulate a second infestation. Helicoverpa armigera nucleopolyhedrovirus was effective at killing 1st-3rd instars, resulting in 99% mortality over 4-6 days. However, 4th and 5th instar mortality only reached 35%. Second infestation larvae died between 3.4 and 3.8 days, significantly faster than the 1st infestation of 2nd instars, which had a mean time to mortality of 4.9 days. An increase in mortality rate is probably due to increasing viral concentrations after viral replication within the first hosts. Final defoliation percentages were significantly smaller in the treated plants versus the untreated plants. Only 3rd and 4th instar larvae caused percent defoliation to exceed the current Arkansas action threshold of 40%. Helicoverpa armigera nucleopolyhedrovirus in the Heligen formulation can control 1st-3rd instars within 4-6 days, while keeping defoliation below the action threshold of 40%.

Screening of Tomato Seed Bacterial Endophytes for Antifungal Activity Reveals Lipopeptide Producing Bacillus siamensis Strain NKIT9 as a Potential Bio-Control Agent.

The current study investigates the diversity pattern and fungicidal potential of bacterial endophytes isolated from two different organic varieties of tomato plants (V1 and V2). A total of seventy-five bacterial isolates identified by 16S rRNA gene sequencing revealed a majority of genus as Bacillus and one Planococcus, which were grouped into eight different species. The Shannon diversity H' (1.56), Simpson's index of diversity (0.93), Magalef' index (2.23), Evenness (0.96), and Species richness (7) indicated the high endophytic bacterial diversity in the V1 variety of the tomato. Bacterial endophytes isolated from both of the varieties were screened for their antifungal activity against five economically critical fungal pathogens (viz., Botrytis cinerea, Rhizoctonia solani, Fusarium solani, Verticillium lateritium, and Alternaria solani) of tomato crop through dual culture assay. The data revealed B. siamensis strain NKIT9 as the most potent antagonist, significantly (p < 0.05) inhibiting the mycelial growth between 75 to 90% against selected fungal pathogens. High bioactivity of lipopeptide extract of strain NKIT9 was recorded against R. solani with minimum IC50 value of 230 µg/ml. The Ultra Performance Liquid Chromatography-High Definition Mass Spectrometry (UPLC-HDMS) analysis of this lipopeptide extract revealed the presence of Surfactin and Bacillomycin D. Furthermore, in-vitro results showed that the selected bacterial strain significantly minimized the disease incidence in damping-off assay which makes this strain a promising antifungal bio-control agent. Moreover, in the pot experiment the NKIT9 increased the fruit yield by 59.2% compared with the untreated R. solani infested control.

Bacillus thuringiensis Spores and Vegetative Bacteria: Infection Capacity and Role of the Virulence Regulon PlcR Following Intrahaemocoel Injection of Galleria mellonella.

Bacillus thuringiensis is an invertebrate pathogen that produces insecticidal crystal toxins acting on the intestinal barrier. In the Galleria mellonella larvae infection model, toxins from the PlcR virulence regulon contribute to pathogenicity by the oral route. While B. thuringiensis is principally an oral pathogen, bacteria may also reach the insect haemocoel following injury of the cuticle. Here, we address the question of spore virulence as compared to vegetative cells when the wild-type Bt407cry- strain and its isogenic ∆plcR mutant are inoculated directly into G. mellonella haemocoel. Mortality dose-response curves were constructed at 25 and 37 °C using spores or vegetative cell inocula, and the 50% lethal dose (LD50) in all infection conditions was determined after 48 h of infection. Our findings show that (i) the LD50 is lower for spores than for vegetative cells for both strains, while the temperature has no significant influence, and (ii) the ∆plcR mutant is four to six times less virulent than the wild-type strain in all infection conditions. Our results suggest that the environmental resistant spores are the most infecting form in haemocoel and that the PlcR virulence regulon plays an important role in toxicity when reaching the haemocoel from the cuticle and not only following ingestion.

Expressing MicroRNA Bantam Sponge Drastically Improves the Insecticidal Activity of Baculovirus via Increasing the Level of Ecdysteroid Hormone in Spodoptera exigua Larvae.

Bantam is a conserved miRNA highly expressed in insects. We previously showed that the antisense inhibitor (antagomiR) of bantam improved the infection by baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) in Spodoptera exigua and S. litura larvae. Here, we constructed a recombinant AcMNPV (vPH-banS) expressing bantam sponge, an mRNA containing eight antisense binding sites for bantam. Infection with wild type AcMNPV (WT) or the control recombinant virus vPH resulted in a significant increase of bantam level, whereas infection with vPH-banS led to an approximately 40% reduction of bantam in both Sf9 cells and S. exigua larvae. Although, comparable production of budded virus and polyhedra were detected in vPH-banS-, vPH-, and WT-infected Sf9 cells, vPH-banS showed remarkably increased insecticidal activity in S. exigua larvae. The 50% lethal concentration and the median lethal time of vPH-banS was only 1/40 and 1/2, respectively, of both vPH and WT. Further analysis showed that the level of molting hormone 20-hydroxyecdysone (20E) was significantly higher in larvae infected with vPH-banS than those infected with vPH or WT. This was confirmed by the result that the larvae treated with bantam inhibitor also had a markedly increased 20E level. Moreover, feeding larvae with 20E increased the virus-mediated mortality, whereas feeding with juvenile hormone partially reverted the high insecticidal effect of vPH-banS. Together, our results revealed that vPH-banS infection suppresses the level of bantam, and in turn elevates level of 20E in infected insects, resulting in increased susceptibility to baculovirus infection. Our study provided a novel approach to improve a baculovirus bio-insecticide by interfering with a key homeostasis-regulating miRNA of the host.

Two-steps gas double-dynamic solid-state fermentation enhances growth and spore production of Conithyrium minitans.

Gas double-dynamic solid-state fermentation (SSF) is a promising strategy with the potential in transforming open-pattern fermentation into closed-pattern fermentation. This paper investigated gas double-dynamic SSF performance in cultivating Coniothyrium minitans (C. minitans), as well as its effect on physiology of C. minitans. Results showed that gas double-dynamic increased biomass content by 48.6%. High temperature impeded pycnidia formation and increased glycine production. More pycnidia formed in solid matrix at 20 °C, which was responsible for higher conidia production (1.5 (±0.03) × 1010 spores/g dry mass), indicating decisive role of high temperature in pycnidia formation of C. minitans in solid-state fermentation. Higher glycine content may be the response of high temperature stress which has close relationship with pycnidia and conidia production. Based on the findings, a two-step strategy for gas double-dynamic SSF was proposed and an satisfactory conidia production was obtained while fermentation period shortened.

The effects of insecticides on butterflies - A review.

Pesticides, in particular insecticides, can be very beneficial but have also been found to have harmful side effects on non-target insects. Butterflies play an important role in ecosystems, are well monitored and are recognised as good indicators of environmental health. The amount of information already known about butterfly ecology and the increased availability of genomes make them a very valuable model for the study of non-target effects of pesticide usage. The effects of pesticides are not simply linear, but complex through their interactions with a large variety of biotic and abiotic factors. Furthermore, these effects manifest themselves at a variety of levels, from the molecular to metapopulation level. Research should therefore aim to dissect these complex effects at a number of levels, but as we discuss in this review, this is seldom if ever done in butterflies. We suggest that in order dissect the complex effects of pesticides on butterflies we need to integrate detailed molecular studies, including characterising sequence variability of relevant target genes, with more classical evolutionary ecology; from direct toxicity tests on individual larvae in the laboratory to field studies that consider the potentiation of pesticides by ecologically relevant environmental biotic and abiotic stressors. Such integration would better inform population-level responses across broad geographical scales and provide more in-depth information about the non-target impacts of pesticides.

Bio-controlling Effect of Leaf Extract of Tagetes patula L. (Marigold) on Growth Parameters and Diseases of Tomato.

BACKGROUND: The genus Tagetes (Asteraceae) is native to Americas but some of its members (in particular T. erecta and T. patula) commonly known as marigolds were naturalized in the old world (India, North Africa and Europe) as early as in 16th century. The flowers of French marigold (Tagetes patula L.) are widely used in folk medicine, in particular for treating inflammation-related disorders. MATERIALS AND METHODS: This study investigated the potential use of marigold (Tagetes patula L.) flower aqueous flower extract by spaying method on tomato plants on a weekly basis and the data of growth, yield and disease of tomato pants were observed from 10th day onwards under field condition. RESULTS: The marigold flower extract showed significant increase in shoot height, number of branches, number of leaves, number of buds, number of flowers and number of fruits of tomato plant, while significant reduction in various diseases of tomato plants over control at probability level ***p<0.001. The percentage of reduction of disease was calculated after the spray of marigold flower aqueous extract on plants. The marigold flower extract was found effectively in controlling canker (62.82%), early blight (61.53%), wilt (18.42%), fruit spot (27.41%), blossom end rot (50.43%) and sun scald (26.44%) in comparison to controls under field condition. CONCLUSION: The findings are in line with the bio-controlling properties of marigold preparations as bio-pesticide confirmed in growth and yield of tomato plants. Thus, marigold can contribute in reducing use of chemical pesticides and act as a good alternative to synthetic pesticides.

Piperolein B and piperchabamide D isolated from black pepper (Piper nigrum L.) as larvicidal compounds against the diamondback moth (Plutella xylostella).

BACKGROUND: There is growing demand for the development of alternative pest control agents that are effective as well as non-toxic to human health and the environment. Plant protection products derived from plant extracts are an eco-friendly alternative to synthetic pesticides. The aim of this study was to identify larvicidal compounds isolated from a natural source against Plutella xylostella L. In a larvicidal activity assay, several solvent fractions from the methanol extract of Piper nigrum L. fruit showed larvicidal effects against P. xylostella. RESULTS: Screening results indicated that chloroform extract was the most effective against P. xylostella larvae. Two compounds with insecticidal activity in the chloroform fraction were identified as piperolein B and piperchabamide D by spectroscopic analyses, including mass spectrometry and NMR, and by comparison to published data. At applications of 0.1 mg mL-1 concentration, piperolein B and piperchabamide D, respectively, induced 96.7 ± 5.8% and 79.2 ± 16.6% mortality rates of P. xylostella larvae 4 days post-application. CONCLUSION: Our results demonstrate that piperolein B and piperchabamide D isolated from P. nigrum are the major constituents of the extract demonstrating insecticidal properties for the control of P. xylostella larvae. These plant-derived compounds should become useful alternatives to synthetic chemicals after studying their insecticidal mechanisms. © 2017 Society of Chemical Industry.

Toxicity and Nonrepellency of Spinosad and Spinetoram on Formosan Subterranean Termites (Isoptera: Rhinotermitidae).

Spinosyn products, spinosad and spinetoram, are widely used to control various agricultural pests. Spinosad has been tested on Kalotermitidae and Termitidae but not on Rhinotermitidae, the most destructive of termite families. In this study, we tested the effect of spinosad and spinetoram on Coptotermes formosanus Shiraki. Both no-choice and choice tests were conducted using three concentrations, 1 ppm, 25 ppm, and 50 ppm, of the spinosyn products Entrust, Tracer, and Radiant on three substrates, sand, soil, and filter paper. In the no-choice test in sand, >85% mortality was observed at 25 and 50 ppm after 1 d of exposure followed by 100% mortality at 7 d. Similarly, after 7 d at 25 and 50 ppm in soil and filter paper, 100% mortality was observed, but compared to sand at 1 d, mortality was low. In the two-choice test, observations before the onset of termite mortality showed that none of the products or concentrations was repellent. Likewise, in the multiple-choice test, there was no repellency or preference of termites among 1 ppm, 25 ppm, 50 ppm, control, and release chamber at all three concentrations, and the tunnel area in the control and treated choices were not significantly different. These findings support the nonrepellent attribute of spinosyns on C. formosanus .

Comparative bio-efficacy of nuclear polyhedrosis virus (NPV) and Spinosad against American bollwormm, Helicoverpa armigera (Hubner)

ABSTRACT American bollworm (ABW), Helicoverpa armigera (Hubner), is considered as a major pest of cotton, Gossypium hirsutum, all over the globe. Due to its destructive feeding nature and continuous consumption of the same chemicals, it devolved resistant against many insecticides. Therefore, a combined application of bio- and synthetic-pesticide need to evaluate against this pest. The entomopathogenic viruses like nuclear polyhedrosis virus (NPV), a member of baculoviruses, can be the potential candidates for better control against ABW. The present study was conducted to assess the comparative efficacy of NPV and Spinosad 240SC (with the concentration of 250 mL · ha-1) against ABW in the controlled environment. The ABW was treated with different concentrations of NPV and Spinosad separately and in a combination of NPV with 0.1% Spinosad. The results revealed that highest concentrations showed highest mortality (95%) followed by 95%, 92%, 84%, 82% and 78% mortality at 1 × 109, 1 × 108, 1 × 107, 1 × 106 and 1 × 105 POBs, respectively. Spinosad when mixed in diet give 100% mortality at 0.8% followed by 50.87%, 42.10%, 29.82%, 26.31% and 22.80% mortality at 0.4%, 0.2%, 0.1%, 0.5% and 0.025% respectively. The results of this study revealed that microbial control of ABW through NPV is an effective tool. The repeated use of synthetic pesticides caused the resurgence of many insect pests, and this study results would provide useful insight to build a framework for future investigations for the management of many major insect pests.