Insecticidal Assay Research Articles

Chemical Composition, Antibacterial and Insecticidal Activities of the Essential Oil from the Pistacia terebinthus L. Spp. Palaestina (Boiss.) (Anacardiaceae)

Essential oil (EO) was hydrodistillated using a Clevenger-type apparatus and chemically determined using gas chromatography (GC) and GC/mass spectrometry. The analysis of the EO from the leaves of the Pistacia terebinthus has indicated that α-pinene (19.97%), sabinene (15.43%), β-pinene (8.57%) and terpinen-4-ol (9.65%) are the major compounds of the oil in concern. In the antibacterial disc diffusion assay, EO revealed a broad spectrum activity on the growth of both gram-positive and gram-negative bacteria when compared with the standard antibiotics. Likewise, in the insecticidal assay, significant activity was observed on two important pest insects, Acanthoscelides obtectus and Tribolium castaneum. A 100% mortality was found at 160 μL/L air dose for T. castaneum at 48-h and A. obtectus at 72-h exposure periods, respectively. Practical Applications Various parts of the Pistacia terebinthus L. belonging to the Anacardiaceae family are edible and contribute significant benefits with invaluable potentialities to the Mediterranean life. Leaves of this tree are consumed as either fresh or cooked. The biological potential of the essential oil (EO) from the leaves of P. terebinthus L. grown in the Eastern Mediterranean region has not been assessed so far. The present assays indicated that the EO of P. terebinthus contains bioactive compounds against bacteria and pest insects suggesting its potential use in food processing and preservation.

English

The aim of the current work was to screen the crude methanolic extract (Cr. MeOH Ext.) and various fractions of Hedera nepalensis for antibacterial, antifungal, brine-shrimp lethality, phytotoxicity, heamagglutination, insecticidal and nitric oxide free radical scavenging assay. The results revealed that chloroform (CHCl3) fraction of the plant possess good antibacterial activity (60%) against Staphylococcus aureus while the rest of the fractions and Cr. MeOH Ext. showed moderate (>40%) and low antibacterial activity (<40%). The Cr. MeOH Ext., n-hexane and ethyl acetate (EtOAc) fractions of the plant showed good phytotoxicity at higher concentration (1000 µg/ml) while low phytotoxicity was observed at lower concentrations (100 and 10 µg/ml), against Lamina minor. The Cr. MeOH Ext., n-hexane and CHCl3 fractions of the plant showed good nitric oxide free radical scavenging activity at a concentration of 1.5 mg/ml. Moderate brine shrimp lethality was also observed for Cr. MeOH Ext. and CHCl3 fraction at a concentration of 1000 µg/ml, against Artemisia salina. The plant materials (Cr. MeOH Ext. and various fractions) exhibited no antifungal, insecticidal or heamagglutination activity.   Key words: Antibacterial, antifungal, brine-shrimp lethality, phytotoxicity, heamagglutination, insecticidal and nitric oxide free radical scavenging assay.

Transgenic Rice Expressing a Fusion Protein of Cry1Ab and Cry9Aa Confers Resistance to a Broad Spectrum of Lepidopteran Pests

ABSTRACTThe truncated active Bacillus thuringiensis endotoxin genes, cry1Ab and cry9Aa, were fused to create a fusion gene, cry1Ab–cry9Aa, encoding a protein of about 146 kDa with Cry1Ab at the N terminus and Cry9Aa at the C terminus. The fusion protein expressed in Escherichia coli is highly active against some major lepidopteran pests—oriental armyworm [Mythimna separata (Walker)], Asian corn borer [Ostrinia furnacalis (Guenée)], cotton bollworm [Helicoverpa armigera (Hübner)], and tobacco cutworm [Spodoptera litura (Fabricius)]. This fusion gene under the control of the corn (Zea mays L.) polyubiquitin‐1 promoter was transformed into rice (Oryza sativa L.) via Agribacterium‐mediated transformation. Western blot analysis suggested that the transgenic lines, C19‐2 and C19‐25, were two high expressers among the 35 transgenic lines obtained. Southern blot analysis revealed that the line C19‐2 contains a single copy of the T‐DNA, while the C19‐25 has three copies. Insecticidal activity assays demonstrated that most of the transgenic rice lines expressing this fusion protein are highly resistant to rice stem borer [Chilo suppressalis (Walker)] and rice leaf folder [Cnaphalocrocis medinalis (Guenée)]. This study suggested that this fusion gene could be useful for engineering transgenic crops for the control of lepidopteran pests.

Resistance monitoring of Heliothis virescens to pyramided cotton varieties with a hydrateable, artificial cotton leaf bioassay

Proof of concept was demonstrated for a practical, off-the-shelf bioassay to monitor for tobacco budworm resistance to pyramided Bt cotton using plant filtrates. The bioassay was based on a previously described feeding disruption test using hydrateable artificial diet containing a blue indicator dye, a diagnostic dose of insecticide and novel assay architecture. Using neonate larvae from a Bt-susceptible, laboratory reared tobacco budworm strain, a diagnostic dose for Bollgard II and WideStrike cotton was obtained that limited neonate blue fecal production to 0–2 pellets in 24 h (Bt-resistant larvae produced >2 fecal pellets). The bioassay was tested with three different field populations of tobacco budworm collected from tobacco in central North Carolina (USA) and shown to accurately diagnose susceptibility to Bt. The diagnostic doses were also successfully evaluated with two Bt-resistant, laboratory reared tobacco budworm strains. Shelf life studies showed the assay could be stored for at least 6 months at room temperature (longer storage times were not studied). The application of the bioassay as an easy to use monitoring tool is discussed.

Cloning and characterization of truncated cry1Ab gene from a new indigenous isolate of Bacillus thuringiensis

The insecticidal crystal protein(s) encoded by cry gene(s) of Bacillus thuringiensis (Bt) have been used for insect control both as biopesticides and in transgenic plants. A new 3'-truncated cry1Ab gene was cloned from an indigenous isolate of Bt, A19-31. Nucleotide sequencing and homology search revealed that the deduced amino acid sequence of Cry1Ab toxin of Bt strain A19-31 had a variation of two amino acid residues with the holotype sequence, Cry1Ab1. Expression of the 3'-truncated cry1Ab gene was studied in an acrystalliferous strain of Bt (4Q7). SDS-PAGE and immunostrip analysis of spore-crystal mixture revealed a low level expression of the 3'-truncated cry1Ab gene. Insecticidal activity assay showed that the recombinant 3'-truncated cry1Ab gene product was toxic to larvae of both Helicoverpa armigera and Spodoptera litura.

Characterisation and expression of a novel haplotype cry2A-type gene from Bacillus thuringiensis strain JF19-2

The Bacillus thuringiensis isolate JF19-2, a native organism found in soil samples from the Sichuan Basin in western China, contains a bipyramidal crystal harbouring one insecticidal crystal protein (about 70 kDa). Interestingly, a novel cry2A-type gene, encoding a 635 amino acid protein with 92% homology to cry2Ab1, was found and cloned from this strain. According to the nomenclature principles adopted for B. thuringiensis insecticidal crystal proteins, this gene represents a novel haplotype cry gene and was designated as cry2Ag1 by the Bacillus thuringiensis Pesticide Crystal Protein Nomenclature Committee. Cry2Ag1 was expressed in Escherichia coli BL21(DE3)pLysS cells, and the results of insecticidal activity assays showed that Cry2Ag1 was toxic to both Dipteran (Aedes aegypti) and Lepidopteran (Plutella xylostella and Helicoverpa armigera) pests. These results strongly suggest that the cloning of the cry2Ag1 gene is not only of interest to researchers studying insecticidal crystal genes, but may ultimately serve as a solution to the increasing resistance of pests to currently used insecticides.

Amplifikasi Gen CRY1 dan Analisis Genom Isolat Bacillus thuringiensis Lokal

A study on amplification of Cry1 gene and genome analysis of local isolate of Bacillus thuringiensis TU1 has been done. Amplification was done for cryI gene by PCR-technique. Genome analysis was performed using pulsed-field gel electrophoresis. Insecticidal specificity assay of the isolate to larvae was done in larvae of Heliothis armigera, Plutella xylostella, Aedes aegypti, and Culex sp. Isolate of commercial strain, B. thuringiensis var kurstaki strain HD-7, was used as control for amplification of cryI gene, genome analysis, and insecticidal specificity assay. The result showed that insecticidal specificity assay of the isolates of both TU1 and commercial have similar spectrum of toxicity to the larvae. Amplification of cry1 gene resulted to a fragment of approximately 550 bp in both TU1 and commercial. Genome profiles of TU1 and commercial strain were similar.

Characterisation and expression of a novel holotype crystal protein gene, cry56Aa1, from Bacillus thuringiensis 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.

Mixtures of Complete andpif1- andpif2-Deficient Genotypes Are Required for Increased Potency of an Insect Nucleopolyhedrovirus

The insecticidal potency of a nucleopolyhedrovirus population (SfNIC) that infects Spodoptera frugiperda (Lepidoptera) is greater than the potency of any of the component genotypes alone. Occlusion bodies (OBs) produced in mixed infections comprising the complete genotype and a deletion genotype are as pathogenic as the natural population of genotypes from the field. To test whether this increased potency was due to the deletion or to some other characteristic of the deletion variant genome, we used the SfNIC-B genome to construct a recombinant virus (SfNIC-B Delta 16K) with the same 16.4-kb deletion as that observed in SfNIC-C and another recombinant (SfNIC-B Delta pifs) with a deletion encompassing two adjacent genes (pif1 and pif2) that are essential for transmission per os. Mixtures comprising SfNIC-B and SfNIC-B Delta 16K in OB ratios that varied between 10:90 and 90:10 were injected into insects, and the progeny OBs were fed to larvae in an insecticidal potency assay. A densitometric analysis of PCR products indicated that SfNIC-B was generally more abundant than expected in mixtures based on the proportions of OBs used to produce the inocula. Mixtures derived from OB ratios of 10, 25, or 50% of SfNIC-B Delta 16K and the corresponding SfNIC-B proportions showed a significant increase in potency compared to SfNIC-B alone. The results of potency assays with mixtures comprising various proportions of SfNIC-B plus SfNIC-B Delta pifs were almost identical to the results observed with SfNIC-B Delta 16K, indicating that deletion of the pif gene region was responsible for the increased potency observed in mixtures of SfNIC-B and each deletion recombinant virus. Subsequently, mixtures produced from OB ratios involving 10 or 90% of SfNIC-B Delta 16K with the corresponding proportions of SfNIC-B were subjected to four rounds of per os transmission in larvae. The composition of each experimental mixture rapidly converged to a common equilibrium with a genotypic composition of approximately 85% SfNIC-B plus approximately 15% SfNIC-B Delta 16K. Nearly identical results were observed in peroral-passage experiments involving mixtures of SfNIC-B plus SfNIC-B Delta pifs. We conclude that (i) the deletion of the pif1 and pif2 region is necessary and sufficient to explain the increased potency observed in mixtures of complete and deletion genotypes and (ii) viral populations with decreased ratios of pif1- and pif2-deficient genotypes in the virus population increase the potency of genotypic mixtures and are likely to positively influence the transmission of this pathogen.

Solubilization, Activation, and Insecticidal Activity ofBacillus thuringiensisSerovar thompsoni HD542 Crystal Proteins

Cry15Aa protein, produced by Bacillus thuringiensis serovar thompsoni HD542 in a crystal together with a 40-kDa accompanying protein, is one of a small group of nontypical, less well-studied members of the Cry family of insecticidal proteins and may provide an alternative for the more commonly used Cry proteins in insect pest management. In this paper, we describe the characterization of the Cry15Aa and 40-kDa protein's biochemical and insecticidal properties and the mode of action. Both proteins were solubilized above pH 10 in vitro. Incubation of solubilized crystal proteins with trypsin or insect midgut extracts rapidly processed the 40-kDa protein to fragments too small to be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the Cry15 protein yielded a stable product of approximately 30 kDa. Protein N-terminal sequencing showed that Cry15 processing occurs exclusively at the C-terminal end. Cry15 protein showed in vitro hemolytic activity, which was greatly enhanced by preincubation with trypsin or insect gut extract. Larvae of the lepidopteran insects Manduca sexta, Cydia pomonella, and Pieris rapae were susceptible to crystals, and presolubilization of the crystals enhanced activity to P. rapae. Activity for all three species was enhanced by preincubation with trypsin. Larvae of Helicoverpa armigera and Spodoptera exigua were relatively insensitive to crystals, and activity against these insects was not enhanced by prior solubilization or trypsin treatment. The 40-kDa crystal protein showed no activity in the insects tested, nor did its addition or coexpression in Escherichia coli increase the activity of Cry15 in insecticidal and hemolytic assays.

Transgenic Bt plants decompose less in soil than non- Bt plants

Bt plants are plants that have been genetically modified to express the insecticidal proteins (e.g. Cry1Ab, Cry1Ac, Cry3A) from subspecies of the bacterium, Bacillus thuringiensis ( Bt), to kill lepidopteran pests that feed on corn, rice, tobacco, canola, and cotton and coleopteran pests that feed on potato. The biomass of these transgenic Bt plants ( Bt+) was decomposed less in soil than the biomass of their near-isogenic non- Bt plant counterparts ( Bt−). Soil was amended with 0.5, 1, or 2% (wt wt −1) ground, dried (50 °C) leaves or stems of Bt corn plants; with 0.5% (wt wt −1) ground, dried biomass of Bt rice, tobacco, canola, cotton, and potato plants; with biomass of the near-isogenic plants without the respective cry genes; or not amended. The gross metabolic activity of the soil was determined by CO 2 evolution. The amounts of C evolved as CO 2 were significantly lower from soil microcosms amended with biomass of Bt plants than of non- Bt plants. This difference occurred with stems and leaves from two hybrids of Bt corn, one of which had a higher C:N ratio than its near-isogenic non- Bt counterpart and the other which had essentially the same C:N ratio, even when glucose, nitrogen (NH 4NO 3), or glucose plus nitrogen were added with the biomass. The C:N ratios of the other Bt plants (including two other hybrids of Bt corn) and their near-isogenic non- Bt counterparts were also not related to their relative biodegradation. Bt corn had a significantly higher lignin content than near-isogenic non- Bt corn. However, the lignin content of the other Bt plants, which was significantly lower than that of both Bt and non- Bt corn, was generally not statistically significantly different, although 10–66% higher, from that of their respective non- Bt near-isolines. The numbers of culturable bacteria and fungi and the activity of representative enzymes involved in the degradation of plant biomass were not significantly different between soil amended with biomass of Bt or non- Bt corn. The degradation of the biomass of all Bt plants in the absence of soil but inoculated with a microbial suspension from the same soil was also significantly less than that of their respective inoculated non- Bt plants. The addition of streptomycin, cycloheximide, or both to the soil suspension did not alter the relative degradation of Bt+ and Bt− biomass, suggesting that differences in the soil microbiota were not responsible for the differential decomposition of Bt+ and Bt− biomass. All samples of soil amended with biomass of Bt plants were immunologically positive for the respective Cry proteins and toxic to the larvae of the tobacco hornworm ( Manduca sexta), which was used as a representative lepidopteran in insect bioassays (no insecticidal assay was done for the Cry3A protein from potato). The ecological and environmental relevance of these findings is not clear.

Extrações de óleos de sementes de citros e suas atividades sobre a formiga cortadeira Atta sexdens e seu fungo simbionte

Seeds Citrus oils (C. sinensis, C. limon and C. reticulata) extraction with hexane in a soxhlet apparatus and through supercritical fluid (CO2) were done. Besides triglycerides, the oils obtained with hexane comprised volatile compounds such as terpenes and fatty alcohols, esters, and aldehydes. However, the oils obtained by extraction with supercritical fluid presented only triglycerides. These results indicate that the extraction using supercritical fluid presents better selectivity. The activity of the oils on the development of the ant symbiotic fungus, Leucoagaricus gongylophorus, showed week activity and the topic insecticide assay showed better activity for the tangerine seed oil.

Recombinant scorpion insectotoxin AaIT kills specifically insect cells but not human cells

The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LC50 value of 18.4 microM and 0.70 microM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 microM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3 microM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells.

A venom protein from the endoparasitoid wasp Pimpla hypochondriaca is similar to snake venom reprolysin-type metalloproteases.

Parasitoid species regulate the nutritional and physiological states of their hosts to ensure their eggs and larvae successfully develop inside the host. Due to the insecticidal and paralyzing properties of venoms from idiobiont parasitoids, parasitic wasps are important natural enemies of several insect pests. For this purpose, in this report, we investigated the oxidative, genotoxic, and mutagenic potential of venom from Pimpla turionellae L. (Hymenoptera: Ichneumonidae) on the laboratory host Galleria mellonella L. (Lepidoptera: Pyralidae). The insecticidal assay showed the LC50 and LC99 values of endoparasitoid venom for G. mellonella pupal stage to be 0.44 µg and 1.17 µg in 2 µl insect saline buffer, respectively. According to the mechanical stimulus–response (MSR) assay, pupal paralysis increased significantly within four hours post-injection at LC50 and higher venom concentrations. Envenomation by LC10, LC30, and LC50 concentrations of endoparasitoid venom and parasitism reduced the activities of superoxide dismutase, catalase, and glutathione-S-transferase enzymes and the level of malondialdehyde in the hemolymph of host pupae 1, 2 and 4 h post-treatment. On the other hand, comet and micronucleus assays displayed that, envenomation by the effective concentrations of P. turionellae venom-induced concentration and time-dependent DNA and chromosomal damages in host hemocytes. Therefore, these data indicated that envenomation by P. turionellae induces anti-oxidative stress, genotoxicity, and mutagenicity which alter the hosts’ physiological state to create a suitable host environment for the successful development of their progeny. In conclusion, we suggest that this study could provide useful information for bioinsecticidal potential of idiobiont wasp venom for sustainable integrated pest management programs.

Inheritance of resistance to pyrethroid insecticides in Tribolium castaneum (Herbst)

A strain of Tribolium castaneum, highly resistant to pyrethroids, was isolated (single-pair method) from a pyrethroid-resistant field strain and made isogenic with a susceptible laboratory strain. Insecticide assays were used to determine the mode of inheritance and linkage of pyrethroid resistance. Resistance to deltamethrin could not be measured directly but at a 2% mortality response was about 1600-fold. The responses of SR and RS hybrids to deltamethrin were intermediate between the resistant and susceptible parent strains; there was no significant difference between the SR and RS hybrids ( P>0.05). Chi-square analysis of the observed responses of the F 1–backcross and F 2 progenies rejected the null hypothesis that a single gene was responsible for resistance. Successive backcrosses with selection indicated the involvement of more than one gene. Test crosses with visible mutant markers suggested that resistance was associated with linkage groups VIII and IX. Assays with deltamethrin applied to grain indicated that this resistance will be rapidly selected in the field resulting in control failures.

Toxicological and Biochemical Characterization of Insecticide Resistance in a Field-Collected Strain of Blattella germanica (Dictyoptera: Blattellidae)

A combination of insecticidal, biochemical, and electrophoretic assays were conducted on adult male and 1st instars of a field-collected strain of German cockroach, Blattella germanica (L.) (Muncie‘86), to characterize its insecticide resistance. In topical applications to adult males, 11 technical grade insecticides were tested over 3 yr. These included representatives of 4 chemical classes: (1) pyrethroids (5 compounds), (2) oregano phosphates (3 compounds), (3) carbamates (2 compounds), and (4) cyclodienes (1 compound). The highest levels of resistance were to chlordane and bendiocarb with a resistance ratio at 95% response of 21.9 and 15.7, respectively. To examine the stability of resistance, the responses of adult males to chlorpyrifos and cypermethrin were tested repeatedly over 3 yr and results were compared by chi-square analysis. Resistance levels showed significant yearly variation in each of the 50 and 95% resistance ratios, but only significant insecticidal variation at the 50% level. Treatment with piperonyl butoxide before insecticides restored susceptibility to only 1 of 4 insecticides tested on adult males, suggesting that a monooxygenase resistance mechanism coexists with other mechanisms in this strain. Biochemical assays confirmed that this strain possessed both elevated levels of cytochrome P450 monooxygenase (in the adult male stage) and est erase activity (in 1st instars), compared with a standard susceptible strain of German cockroach. Native est erases from 1st instars in the field-collected strain differed electrophoretically from those in the susceptible strain.

New Destruxins from the Entomopathogenic Fungus Aschersonia sp.

Two new insecticidal cyclic depsipeptides, destruxins A4 (1) and A5 (2), were isolated by bioassay-guided fractionation of organic extracts from an undescribed species of entomopathogenic fungus from the genus Aschersonia. Another previously identified but related compound, homodestruxin B (3), was also isolated as a product of this fungus. The chemical structures of 1−3 were determined based on analysis of NMR and mass spectral data. In common with homodestruxin B (3), the novel compounds destruxins A4 (1) and A5 (2) have a MeIle moiety in place of the MeVal residue more typically found in destruxins. In addition, destruxin A5 has a β-MePro unit in place of the Pro residue found in most destruxins. LC50 values for 1 and 2 in an insecticidal assay against Drosophila melanogaster were estimated at 41 and 52 ppm, respectively. Homodestruxin B (3) showed no activity at 400 ppm in the same assay. Destruxins A4 (1) and A5 (2) are the first biologically active secondary metabolites reported from the fungal genu...

Effects of pesticide residues on ground-dwelling lycosid and micryphantid spiders in laboratory tests

The effects of residues of three commercial pesticides in common use in Europe against cereal pests, on two dominant spider species, a lycosid and a micryphantid, were evaluated in the laboratory. Insecticide assay methods for juvenile and adult hunting and adult web-building, ground-dwelling spiders are described. Results varied with different sprayed substrates and species. Fenvalerate was more toxic than other pesticides and had a longer residual effect on all spiders when tested on moistened sand than on filter paper. Endosulfan was of high initial toxicity on sand, but was more toxic on filter paper to both adults and juveniles ofPardosa spp. Pyrazophos was non-toxic to the spiders tested, regardless of substrate. In general, the lycosids were more affected by pesticides than web-building spiders because of their different life strategies. A sprayed sand substrate matched field conditions better as a method to test the effects of pesticides on ground-dwelling spiders.

Genetic Analysis of Fenitrothion Resistance in the Sawtoothed Grain Beetle, Oryzaephilus surinamensis (Coleoptera: Cucujidae)

Insecticide assays were used to analyze the inheritance of fenitrothion resistance (145-fold) in a strain of the sawtoothed grain beetle, Oryzaephilus surinamensis (L.), from Australia. At the LC50, the response of the RS hybrid to fenitrothion was closer to its resistant than to its susceptible parent strain; there was no significant difference between the RS and SR hybrids (P < 0.05). X2 analyses of the observed responses of the F2 progeny and the F1-backcross progenies rejected the null hypothesis that a single gene was responsible for resistance. Fenitrothion resistance appears to be controlled by two or more autosomal factors and the major gene(s) involved is incompletely dominant.

A rapid chitin synthase preparation for the assay of potential fungicides and insecticides

A rapid technique for the preparation of large quantities of solubilized chitin synthase is described. The enzyme derived is very stable and has high specific and total activities. It has been used to screen compounds with potential fungicidal and insecticidal properties.