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Overuse of pesticides can result in the development of resistance, secondary pest outbreaks, and pest resurgence due to a reduction in natural enemies. The present study compares the residual toxicity of lambda-cyhalothrin, a relatively nonselective insecticide, with abamectin, indoxacarb, and spinosad, compounds which have been reported to be less harmful to arthropod natural enemies. Two key cosmopolitan pests of crucifer crops, (Plutella xylostella) and (Myzus persicae), and two of their respective hymenopteran parasitoids, (Cotesia vestalis) and (Aphidius colemani) were used as representative pests and natural enemies. The pyrethroid lambda-cyhalothrin was found to be the most persistent toxicant against both pest and both parasitoid species tested, while the lactones abamectin and spinosad were the least persistent toxicants. A leaf wax stripping technique was used to compare the contact toxicity of insecticide residues against adult C. vestalis and A. colemani in the epicuticular wax layer. For each compound, removal of epicuticular wax reduced the 24 h residual toxicity (LC50) of fresh deposits (day 0) by about an order of magnitude against C. vestalis. A second residual toxicity experiment showed that removal of epicuticular wax significantly reduced the residual toxicity of each compound against A. colemani at 0, 7, and 14 d after application, with little or no detectable residual activity for the oxadiazine indoxacarb or abamectin/spinosad respectively after 14 d. The present data supports the view that in addition to the intrinsic toxicity of insecticides to natural enemies, differences in their persistence as foliar residues should also be considered in IPM systems., (© The Author(s) 2022. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We examined differences in the physiology and life history between dimorphs of the common pistachio psyllid, Agonoscena pistaciae (Burckhardt and Lauterer) (Hemiptera: Aphalaridae), and how they differ in elicitating host plant production of key metabolites and volatile compounds involved in the recruitment of herbivores and natural enemies. Summer morphs had higher activities of glutathione S-transferase, carboxylesterase, acetylcholinesterase, and cytochrome P450 monooxygenase, superoxide dismutase, catalase, peroxidase, phenoloxidase, and a higher total protein content compared to winter morphs, whereas the latter had higher amounts of lipid, carbohydrate, and glycogen. Winter morphs were heavier, with a higher chitin content and longer preoviposition period, but greater fecundity and longevity than summer morphs. A lower LC50 to thiamethoxam for winter morphs resulted in higher mortality following exposure to the recommended rate of this insecticide in a greenhouse trial. Feeding by winter morphs elicited more strongly the release of volatile compounds known to be attractive to other herbivores, whereas feeding by summer morphs elicited more strongly the release of volatiles implicated in the attraction of natural enemies. Feeding by psyllids increased the concentrations of nitrogenous compounds, carbohydrates, vitamins, and amino acids in plants, the winter morph eliciting larger changes and more improved host plant quality. We conclude that winter morphs are more vulnerable targets for chemical control in early spring, whereas management of summer morphs could rely more on conservation biological control., (© The Author(s) 2022. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The spore-forming bacterium Bacillus thuringiensis (Bt) of the Bacillus cereus group uses toxin-opened breaches at the insect midgut epithelium to infest the hemolymph, where it can rapidly propagate despite antimicrobial host defenses and induce host death by acute septicemia. The response of Bt to host hemolymph and the latter's role in bacterial pathogenesis is an area that needs clarification. Here, we report a proteomic analysis of the Bt kurstaki strain HD73 (Btk) hemolymph stimulon showing significant changes in 60 (34 up- and 26 downregulated) differentially accumulated proteins (DAPs). Gene ontology (GO) enrichment analysis revealed that DAPs were mainly related to glutamate metabolism, transketolase activity, and ATP-dependent transmembrane transport. KEGG analysis disclosed that DAPs were highly enriched in the biosynthesis of bacterial secondary metabolites, ansamycins. Interestingly, about 30% of all DAPs were in silico predicted as putative virulence factors. Further characterization of hemolymph effects on Btk showed enhanced autoaggregation in liquid cultures and biofilm formation in microtiter polystyrene plates. Hemolymph-exposed Btk cells were less immunogenic in mice, suggesting epitope masking of selected surface proteins. Bioassays with intrahemocoelically infected Bombyx mori larvae showed that hemolymph preexposure significantly increased Btk toxicity and reproduction within the insect (spore count per cadaver) at low inoculum doses, possibly due to 'virulence priming'. Collectively, our findings suggest that the Btk hemolymph stimulon could be partially responsible for bacterial survival and propagation within the hemolymph of infected insects, contributing to its remarkable success as an entomopathogen. All mass spectrometry data are available via ProteomeXchange with identifier PXD021830. IMPORTANCE After ingestion by a susceptible insect and damaging its midgut epithelium, the bacterium Bacillus thuringiensis (Bt) reaches the insect blood (hemolymph), where it propagates despite the host's antimicrobial defenses and induces insect death by acute septicemia. Although the hemolymph stage of the Bt toxic pathway is determinant for the infested insects' fate, the response of Bt to hemolymph and the latter's role in bacterial pathogenesis has been poorly explored. In this study, we identified the bacterial proteins differentially expressed by Bt after hemolymph exposure. We found that about 30% of hemolymph-regulated Bt proteins were potential virulence factors, including manganese superoxide dismutase, a described inhibitor of hemocyte respiratory burst. Additionally, contact with hemolymph enhanced Bt virulence phenotypes, such as cell aggregation and biofilm formation, altered bacterial immunogenicity, and increased Bt toxicity to intrahemocoelically injected insects.

Background: The Plutella xylostella PxSDF2L1 gene was previously reported to enhance insect resistance to pathogen at high basal transcription rate. PxSDF2L1 shows similitude with the stromal cell-derived factor 2 (SDF2), an ER stress-induced chaperon protein that is highly conserved throughout animals and plants. The precise biological function of SDF2 is not clear, but its expression is required for innate immunity in plants. Here, we investigate whether a continuous expression of PxSDF2L1 in Nicotiana benthamiana can similarly confer resistance to plant pathogen, particularly, the black shank Phytophthora parasitica var. nicotianae., Results: The N. benthamiana plants were inoculated with agrobacteria transformed with a PVX-based binary vector carrying the PxSDF2L1 gene; similar agroinoculation experiments with a PVX vector carrying the GFP gene were used for controls. In pot trials, agroinfected N. benthamiana plants constitutively expressing PxSDF2L1 showed a significant reduction of stem disease symptoms caused by the inoculation with P. parasitica, compared with controls., Conclusions: We confirm a role of PxSDF2L1 in resistance to black shank, with a potential application to engineering active resistance against this oomycete in the commercial N. tabacum species and propose its evaluation in other crop families and plant pathogens.

The Vip3Aa insecticidal protein from Bacillus thuringiensis (Bt) is produced by specific transgenic corn and cotton varieties for efficient control of target lepidopteran pests. The main threat to this technology is the evolution of resistance in targeted insect pests and understanding the mechanistic basis of resistance is crucial to deploy the most appropriate strategies for resistance management. In this work, we tested whether alteration of membrane receptors in the insect midgut might explain the >2000-fold Vip3Aa resistance phenotype in a laboratory-selected colony of Heliothis virescens (Vip-Sel). Binding of 125 I-labeled Vip3Aa to brush border membrane vesicles (BBMV) from 3rd instar larvae from Vip-Sel was not significantly different from binding in the reference susceptible colony. Interestingly, BBMV from Vip-Sel larvae showed dramatically reduced levels of membrane-bound alkaline phosphatase (mALP) activity, which was further confirmed by a strong downregulation of the membrane-bound alkaline phosphatase 1 ( HvmALP1 ) gene. However, the involvement of HvmALP1 as a receptor for the Vip3Aa protein was not supported by results from ligand blotting and viability assays with insect cells expressing HvmALP1 .

Multitoxin Bt-crops expressing insecticidal toxins with different modes of action, for example, Cry and Vip, are expected to improve resistance management in target pests. While Cry1A resistance has been relatively well characterized in some insect species, this is not the case for Vip3A, for which no mechanism of resistance has yet been identified. Here we applied HT-SuperSAGE to analyze the transcriptome of the gut tissue of tobacco budworm Heliothis virescens (F.) laboratory-selected for Vip3Aa resistance. From a total of 1 324 252 sequence reads, 5 895 126-bp tags were obtained representing 17 751 nonsingleton unique transcripts (UniTags) from genetically similar Vip3Aa-resistant (Vip-Sel) and susceptible control (Vip-Unsel) strains. Differential expression was significant (≥2.5 fold or ≤0.4; P < 0.05) for 1989 sequences (11.2% of total UniTags), where 420 represented overexpressed (OE) and 1569 underexpressed (UE) genes in Vip-Sel. BLASTN searches mapped 419 UniTags to H. virescens sequence contigs, of which, 416 (106 OE and 310 UE) were unambiguously annotated to proteins in NCBI nonredundant protein databases. Gene Ontology distributed 345 of annotated UniTags in 14 functional categories with metabolism (including serine-type hydrolases) and translation/ribosome biogenesis being the most prevalent. A UniTag homologous to a particular member of the REsponse to PAThogen (REPAT) family was found among most overexpressed, while UniTags related to the putative Vip3Aa-binding ribosomal protein S2 (RpS2) were underexpressed. qRT-PCR of a subset of UniTags validated the HT-SuperSAGE data. This study is the first providing lepidopteran gut transcriptome associated with Vip3Aa resistance and a foundation for future attempts to elucidate the resistance mechanism., (© 2017 The Authors. Insect Science published by John Wiley & Sons Australia, Ltd on behalf of Institute of Zoology, Chinese Academy of Sciences.)

Peritrophins are associated with structural and functional integrity of peritrophic membranes (PM), structures composed of chitin and proteins. PM lines the insect midgut and has roles in digestion and protection from toxins. We report the full-length cDNA cloning, molecular characterization and functional analysis of SfPER, a novel PM peritrophin A protein, in Spodoptera frugiperda. The predicted amino acid sequence indicated SfPER's domain structure as a CMCMC-type, consisting of a signal peptide and three chitin-binding (C) domains with two intervening mucin-like (M) domains. Phylogenetic analysis determined a close relationship between SfPER and another S. frugiperda PM peritrophin partial sequence. SfPER transcripts were found in larvae and adults but were absent from eggs and pupae. Chitin affinity studies with a recombinant SfPER-C1 peritrophin A-type domain fused to SUMO/His-tag confirmed that SfPER binds to chitin. Western blots of S. frugiperda larval proteins detected different sized variants of SfPER along the PM, with larger variants found towards the posterior PM. In vivo suppression of SfPER expression did not affect susceptibility of larvae to Bacillus thuringiensis toxin, but significantly decreased pupal weight and adult emergence, possibly due to PM structural alterations impairing digestion. Our results suggest SfPER could be a novel target for insect control.

Laboratory selection with Vip3Aa of a field-derived population of Heliothis virescens produced >2,040-fold resistance in 12 generations of selection. The Vip3Aa-selected (Vip-Sel)-resistant population showed little cross-resistance to Cry1Ab and no cross-resistance to Cry1Ac. Resistance was unstable after 15 generations without exposure to the toxin. F 1 reciprocal crosses between Vip3Aa-unselected (Vip-Unsel) and Vip-Sel insects indicated a strong paternal influence on the inheritance of resistance. Resistance ranged from almost completely recessive (mean degree of dominance [ h ] = 0.04 if the resistant parent was female) to incompletely dominant (mean h = 0.53 if the resistant parent was male). Results from bioassays on the offspring from backcrosses of the F 1 progeny with Vip-Sel insects indicated that resistance was due to more than one locus. The results described in this article provide useful information for the insecticide resistance management strategies designed to overcome the evolution of resistance to Vip3Aa in insect pests. IMPORTANCE Heliothis virescens is an important pest that has the ability to feed on many plant species. The extensive use of Bacillus thuringiensis (Bt) crops or spray has already led to the evolution of insect resistance in the field for some species of Lepidoptera and Coleoptera. The development of resistance in insect pests is the main threat to Bt crops. The effective resistance management strategies are very important to prolong the life of Bt plants. Lab selection is the key step to test the assumption and predictions of management strategies prior to field evaluation. Resistant insects offer useful information to determine the inheritance of resistance and the frequency of resistance alleles and to study the mechanism of resistance to insecticides., (Copyright © 2017 American Society for Microbiology.)

The assessment of sub-lethal effects is important to interpret the overall insecticide efficacy in controlling insect pest populations. In addition to the lethal effect, sub-lethal effects may also occur in exposed insects. Vegetative insecticidal proteins (Vips) have shown a broad spectrum of insecticidal activity against many insect pest species. In this study the sub-lethal effects of the Bacillus thuringiensis vegetative insecticidal toxin Vip3A on the development and reproduction of Heliothis virescens F. and Plutella xylostella L. were evaluated in the laboratory. The results indicated that the sub-lethal concentration of Vip3A increased the duration of the larval and pupal stages as compared with the control treatment for both species. The percent pupation and percent adult emergence were significantly lower for Vip3A-treated insects. The proportion of pairs that produced eggs and the longevity of adults were not significantly different between treatments. H. virescens and P. xylostella treated with Vip3A showed an 11 and 17 % decrease in their intrinsic rate of increase (rm) respectively compared with untreated insects. The results from this study will be helpful to develop the strategy to incorporate Vip 3A containing crops in an integrated pest management programme.

Herbivory is an important modulator of plant biodiversity and productivity in grasslands, but our understanding of herbivore-induced changes on below-ground processes and communities is limited. Using a long-term (17 years) experimental site, we evaluated impacts of rabbit and invertebrate grazers on some soil functions involved in carbon cycling, microbial diversity, structure and functional composition. Both rabbit and invertebrate grazing impacted soil functions and microbial community structure. All functional community measures (functions, biogeochemical cycling genes, network association between different taxa) were more strongly affected by invertebrate grazers than rabbits. Furthermore, our results suggest that exclusion of invertebrate grazers decreases both microbial biomass and abundance of genes associated with key biogeochemical cycles, and could thus have long-term consequences for ecosystem functions. The mechanism behind these impacts are likely to be driven by both direct effects of grazing altering the pattern of nutrient inputs and by indirect effects through changes in plant species composition. However, we could not entirely discount that the pesticide used to exclude invertebrates may have affected some microbial community measures. Nevertheless, our work illustrates that human activity that affects grazing intensity may affect ecosystem functioning and sustainability, as regulated by multi-trophic interactions between above- and below-ground communities., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Gaining a better understanding of climate and atmospheric change effects on species interactions is one of the great challenges facing modern ecology. Here, we review the literature concerning the responses of insect herbivores and their natural enemies to atmospheric and climate change, focusing specifically on elevated concentrations of atmospheric CO 2 and air temperatures. We recommend that future work on the responses of systems to climate change incorporates as far as possible the trophic complexity inherent in ecosystems, and where feasible, considers the effects of interrelated climate factors in tandem. Such studies will produce more realistic insights into how species interactions may respond under future climates., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Transgenic crops expressing Bt toxins have substantial benefits for growers in terms of reduced synthetic insecticide inputs, area-wide pest management and yield. This valuable technology depends upon delaying the evolution of resistance. The 'high dose/refuge strategy', in which a refuge of non-Bt plants is planted in close proximity to the Bt crop, is the foundation of most existing resistance management. Most theoretical analyses of the high dose/refuge strategy assume random oviposition across refugia and Bt crops., Results: In this study we examined oviposition and survival of Spodoptera frugiperda across conventional and Bt maize and explored the impact of oviposition behavior on the evolution of resistance in simulation models. Over six growing seasons oviposition rates per plant were higher in Bt crops than in refugia. The Cry1F Bt maize variety retained largely undamaged leaves, and oviposition preference was correlated with the level of feeding damage in the refuge. In simulation models, damage-avoiding oviposition accelerated the evolution of resistance and either led to requirements for larger refugia or undermined resistance management altogether. Since larval densities affected oviposition preferences, pest population dynamics affected resistance evolution: larger refugia were weakly beneficial for resistance management if they increased pest population sizes and the concomitant degree of leaf damage., Conclusions: Damaged host plants have reduced attractiveness to many insect pests, and crops expressing Bt toxins are generally less damaged than conventional counterparts. Resistance management strategies should take account of this behavior, as it has the potential to undermine the effectiveness of existing practice, especially in the tropics where many pests are polyvoltinous. Efforts to bring down total pest population sizes and/or increase the attractiveness of damaged conventional plants will have substantial benefits for slowing the evolution of resistance.

Pesticide mixtures can reduce the rate at which insects evolve pesticide resistance. However, with live biopesticides such as the naturally abundant pathogen Bacillus thuringiensis (Bt), a range of additional biological considerations might affect the evolution of resistance. These can include ecological interactions in mixed infections, the different rates of transmission post-application and the impact of the native biodiversity on the frequency of mixed infections. Using multi-generation selection experiments, we tested how applications of single and mixed strains of Bt from diverse sources (natural isolates and biopesticides) affected the evolution of resistance in the diamondback moth, Plutella xylostella, to a focal strain. There was no significant difference in the rate of evolution of resistance between single and mixed-strain applications although the latter did result in lower insect populations. The relative survivorship of Bt-resistant genotypes was higher in the mixed-strain treatment, in part owing to elevated mortality of susceptible larvae in mixtures. Resistance evolved more quickly with treatments that contained natural isolates, and biological differences in transmission rate may have contributed to this. Our data indicate that the use of mixtures can have unexpected consequences on the fitness of resistant and susceptible insects.

Drought can alter plant quality and the strength of trophic interactions between herbivore groups, and is likely to increase in occurrence and severity under climate change. We hypothesized that changes in plant chemistry due to root herbivory and drought stress would affect the performance of a generalist and a specialist aphid species feeding on a Brassica plant. High drought stress increased the negative effect of root herbivory on the performance of both aphid species (30% decrease in fecundity and 15% reduction in intrinsic rate of increase). Aphid performance was greatest at moderate drought stress, though the two species differed in which treatment combination maximized performance. Nitrogen concentration was greatest in high and moderately drought-stressed plants without root herbivores and moderately drought-stressed plants under low root herbivore density, and correlated positively with aphid fecundity for both species. Glucosinolate concentrations increased 62% under combined drought stress and root herbivory, and were positively correlated with extended aphid development time. Root herbivory did not influence relative water content and foliar biomass under normal water regimes but they decreased 24 and 63%, respectively, under high drought stress. This study shows that drought can alter the strength of interactions between foliar and root herbivores, and that plant chemistry is key in mediating such interactions. The two aphid species responded in a broadly similar way to root herbivore and drought-stress treatments, which suggests that generalized predictions of the effects of abiotic factors on interactions between above- and below-ground species may be possible.

Multitrophic interactions are likely to be altered by climate change but there is little empirical evidence relating the responses of herbivores and parasitoids to abiotic factors. Here we investigated the effects of drought on an above/below-ground system comprising a generalist and a specialist aphid species (foliar herbivores), their parasitoids, and a dipteran species (root herbivore).We tested the hypotheses that: (1) high levels of drought stress and below-ground herbivory interact to reduce the performance of parasitoids developing in aphids; (2) drought stress and root herbivory change the profile of volatile organic chemicals (VOCs) emitted by the host plant; (3) parasitoids avoid ovipositing in aphids feeding on plants under drought stress and root herbivory. We examined the effect of drought, with and without root herbivory, on the olfactory response of parasitoids (preference), plant volatile emissions, parasitism success (performance), and the effect of drought on root herbivory. Under drought, percentage parasitism of aphids was reduced by about 40-55% compared with well watered plants. There was a significant interaction between drought and root herbivory on the efficacy of the two parasitoid species, drought stress partially reversing the negative effect of root herbivory on percent parasitism. In the absence of drought, root herbivory significantly reduced the performance (e.g. fecundity) of both parasitoid species developing in foliar herbivores. Plant emissions of VOCs were reduced by drought and root herbivores, and in olfactometer experiments parasitoids preferred the odour from well-watered plants compared with other treatments. The present work demonstrates that drought stress can change the outcome of interactions between herbivores feeding above- and below-ground and their parasitoids, mediated by changes in the chemical signals from plants to parasitoids. This provides a new insight into how the structure of terrestrial communities may be affected by drought.

Agricultural intensification and greater production of Brassica vegetable and oilseed crops over the past two decades have increased the pest status of the diamondback moth (DBM), Plutella xylostella L., and it is now estimated to cost the world economy US$4-5 billion annually. Our understanding of some fundamental aspects of DBM biology and ecology, particularly host plant relationships, tritrophic interactions, and migration, has improved considerably but knowledge of other aspects, e.g., its global distribution and relative abundance, remains surprisingly limited. Biological control still focuses almost exclusively on a few species of hymenopteran parasitoids. Although these can be remarkably effective, insecticides continue to form the basis of management; their inappropriate use disrupts parasitoids and has resulted in field resistance to all available products. Improved ecological understanding and the availability of a series of highly effective selective insecticides throughout the 1990s provided the basis for sustainable and economically viable integrated pest management (IPM) approaches. However, repeated reversion to scheduled insecticide applications has resulted in resistance to these and more recently introduced compounds and the breakdown of IPM programs. Proven technologies for the sustainable management of DBM currently exist, but overcoming the barriers to their sustained adoption remains an enormous challenge.

Microbial insecticides derived from the soil bacterium Bacillus thuringiensis (Bt) have become increasingly important for pest management. In addition to crystal (Cry) insecticidal protein toxins formed during sporulation, vegetative insecticidal protein (Vip) toxins can be produced during the vegetative phase. Resistance to Cry toxins has been reported in laboratory- and field-selected populations of various Lepidoptera species and several studies have identified fitness costs associated with Cry toxin resistance. Here, fitness costs are examined in the first insect population to be reported with resistance to a Vip toxin, a laboratory-selected Vip3A-resistant subpopulation of the tobacco budworm, Heliothis virescens (L.) (Vip-Sel). The Vip-Sel population showed reduced survival to adult eclosion compared with an unselected subpopulation at all test temperatures, including the culture temperature (25 degrees C). Vip3A resistance was also associated with reduced egg viability and mating success and a lower intrinsic rate of population increase (r(m)) at temperatures below (20 degrees C) and above (30 degrees C) the optimal laboratory culture temperature. The latter findings agree with previous studies, that fitness costs associated with resistance are usually greater under stressful conditions. Such data can help predict the impact of fitness costs on the rate of development of resistance in the field and in the development of resistance management strategies that more fully exploit fitness costs.

Plants produce volatile organic compounds (VOCs) in response to herbivore attack, and these VOCs can be used by parasitoids of the herbivore as host location cues. We investigated the behavioural responses of the parasitoid Cotesia vestalis to VOCs from a plant-herbivore complex consisting of cabbage plants (Brassica oleracea) and the parasitoids host caterpillar, Plutella xylostella. A Y-tube olfactometer was used to compare the parasitoids' responses to VOCs produced as a result of different levels of attack by the caterpillar and equivalent levels of mechanical damage. Headspace VOC production by these plant treatments was examined using gas chromatography-mass spectrometry. Cotesia vestalis were able to exploit quantitative and qualitative differences in volatile emissions, from the plant-herbivore complex, produced as a result of different numbers of herbivores feeding. Cotesia vestalis showed a preference for plants with more herbivores and herbivore damage, but did not distinguish between different levels of mechanical damage. Volatile profiles of plants with different levels of herbivores/herbivore damage could also be separated by canonical discriminant analyses. Analyses revealed a number of compounds whose emission increased significantly with herbivore load, and these VOCs may be particularly good indicators of herbivore number, as the parasitoid processes cues from its external environment.

Classical models of the evolution of virulence predict that multiple infections should select for elevated virulence, if increased competitiveness arises from faster growth. However, diverse modes of parasite competition (resource-based, antagonism, immunity manipulation) can lead to adaptations with different implications for virulence. Using an experimental evolution approach we investigated the hypothesis that selection in mixed-strain infections will lead to increased antagonism that trades off against investment in virulence. Selection in mixed infections led to improved suppression of competitors in the bacterial insect pathogen Bacillus thuringiensis. Increased antagonism was associated with decreased virulence in three out of four selected lines. Moreover, mixed infections were less virulent than single-strain infections, and between-strain competition tended to decrease pathogen growth in vivo and in vitro. Spiteful interactions among these bacteria may be favoured because of the high metabolic costs of virulence factors and the high risk of mixed infections., (© 2011 Blackwell Publishing Ltd/CNRS.)

Indirect competition is often mediated by plant responses to herbivore feeding damage and is common among phytophagous insect species. Plant-mediated responses may be altered by abiotic conditions such as nutrient supply, which can affect plant growth, morphology, and the concentration of primary and secondary metabolites. Nutrient supply can be manipulated by the type and amount of fertilizer applied to a plant. Brassica oleracea plants were grown in several types of fertilizer, including those commonly used in sustainable and conventional agricultural systems. The occurrence of indirect competition between two phytophagous species from different feeding guilds (a phloem-feeder and leaf-chewer) was assessed. The leaf-chewer reduced aphid populations on plants growing in most fertilizer treatments, but not on those in the ammonium nitrate fertilizer treatment, which caused the highest concentration of foliar nitrogen. The potential consequences of our findings are discussed for phytophagous species in conventional and sustainable agricultural systems.

SfT6 has been identified in a subtracted cDNA library of Spodoptera frugiperda larval midgut transcripts as a serine-protease gene downregulated within 24 h of intoxication with Bacillus thuringiensis Cry1Ca1 protein. In the present study, the specific role of SfT6 during Cry1Ca1 intoxication was investigated by RT-PCR and in vivo RNA interference. Quantitative real-time RT-PCR analysis showed SfT6 mRNA levels in the midgut tissue were significantly reduced after injecting or feeding 4th-instar larvae with specific long-size dsRNA. Gut juice-mediated in vitro protoxin activation and susceptibility for Cry1Ca1 were investigated in Sft6-knockdown larvae and compared with control treated with nonspecific dsRNA. Our results demonstrate SfT6 plays a determinant role in Cry1Ca1 toxicity against S. frugiperda since a decreased expression caused a reduced protoxin activation by larval gut juice and reduced susceptibility of insects to toxin in bioassays. We propose SfT6 downregulation occurring at the early stages of Cry1Ca1 intoxication is part of a complex and multifaceted defensive mechanism triggered in the insect gut to withstand B. thuringiensis pathogenesis., (© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

The hypothesis that plants supplied with organic fertilizers are better defended against insect herbivores than those supplied with synthetic fertilizers was tested over two field seasons. Organic and synthetic fertilizer treatments at two nitrogen concentrations were supplied to Brassica plants, and their effects on the abundance of herbivore species and plant chemistry were assessed. The organic treatments also differed in fertilizer type: a green manure was used for the low-nitrogen treatment, while the high-nitrogen treatment contained green and animal manures. Two aphid species showed different responses to fertilizers: the Brassica specialist Brevicoryne brassicae was more abundant on organically fertilized plants, while the generalist Myzus persicae had higher populations on synthetically fertilized plants. The diamondback moth Plutella xylostella (a crucifer specialist) was more abundant on synthetically fertilized plants and preferred to oviposit on these plants. Glucosinolate concentrations were up to three times greater on plants grown in the organic treatments, while foliar nitrogen was maximized on plants under the higher of the synthetic fertilizer treatments. The varying response of herbivore species to these strong differences in plant chemistry demonstrates that hypotheses on defence in organically grown crops have over-simplified the response of phytophagous insects.

The mode of action of the entomopathogenic bacterium Bacillus thuringiensis (Bt) remains a matter of debate. Recent reports have claimed that aseptic lepidopteran hosts were not susceptible to Bt and that inoculation with mid-gut bacteria restores pathogenicity. These claims are controversial because larvae were rendered aseptic by consuming antibiotics, although the effect of these antibiotics on Bt was not examined. We tested the generality of the mid-gut bacteria hypothesis in the diamondback moth, Plutella xylostella using properly controlled experiments that investigated the effect of antibiotic consumption and absence of gut microbiota separately. We found that purified Bt toxin and spore/toxin mixtures were fully pathogenic to larvae reared aseptically. Persistence of antibiotics in larval tissues was implicated in reducing host mortality because larval consumption of the antibiotic rifampicin reduced the pathogenicity of rifampicin-sensitive Bt strains but not rifampicin-resistant strains. Inoculating larvae with Enterobacter sp. Mn2 reduced the mortality of larvae feeding on Bt HD-1 and the presence of a culturable gut microbiota also reduced the pathogenicity of the Bt toxin Cry1Ac, in agreement with other studies indicating that an intestinal microbiota can protect taxonomically diverse hosts from pathogen attack. As ingestion of antibiotics suppresses host mortality the vegetative growth of Bt in the host must be important for its pathogenicity. Furthermore, claims that aseptic larvae are not susceptible to Bt must be supported by experiments that control for the effect of administering antibiotics.

Background: Bacillus thuringiensis Berliner (Bt) crystal (Cry) toxins are expressed in various transgenic crops and are also used as sprays in integrated pest management and organic agricultural systems. The diamondback moth (Plutella xylostella L.) is a major worldwide pest of crucifer crops and one that has readily acquired field resistance to a broad range of insecticides., Results: Selection of a subpopulation of the P. xylostella SERD4 population with the pyrethroid deltamethrin increased resistance to both deltamethrin and Cry1Ac relative to an unselected subpopulation. Selection of a second subpopulation with the Bt toxin Cry1Ac also increased resistance to both Cry1Ac and deltamethrin. A complementation test between the Cry1Ac-selected and deltamethrin-selected subpopulations suggested the presence of a common genetic locus or loci that control resistance to both insecticides. A piperonyl butoxide analogue with potent inhibitory activity against insect esterases significantly increased the toxicity of Cry1Ac and deltamethrin against the respective resistant subpopulations, but showed no such synergism with the unselected subpopulation of SERD4., Conclusion: Selection of one resistance phenotype resulted in the simultaneous selection of the other. This phenomenon could be due to a single mechanism acting against both classes of insecticide or to genetically linked, but separate, mechanisms.

The use of Bacillus thuringiensis Cry delta-endotoxins as bioinsecticides is threatened by the possibility of pest resistance. Determining transcriptional profiles of midgut cells early in Cry toxin poisoning is crucial for understanding the biochemical and molecular aspects of insect detoxification and for sustained use of such toxins. In this study, transcriptional responses of midgut cells from Spodoptera frugiperda third-instar larvae following treatment with Cry1Ca were investigated. Suppression subtractive hybridization (SSH) on insect midguts dissected at different time intervals during the first 24h of exposure to a sublethal concentration of Cry1Ca was used to isolate and identify S. frugiperda gut genes that change in expression on intoxication. After differential screening by membrane-based hybridization, 86 cDNA fragments were selected, sequenced, and analyzed in databases using BLASTN/BLASTX. The cDNA collection comprised a repertoire of genes mainly associated with metabolism, defence and oxidative stress. The expression of a subset of these genes was further investigated. Northern blot analysis confirmed the differential expression patterns between intoxicated and control larvae. The transcript accumulation rate at six different times taken after the initiation of the intoxication point was also examined. Differential expression of most genes examined was detected within 15 min after toxin challenge, where defence and oxidative stress-related genes were transcriptionally enhanced and metabolic-related genes were repressed.

Novel adaptations often cause pleiotropic reductions in fitness. Under optimal conditions individual organisms may be able to compensate for, or reduce, these fitness costs. Declining environmental quality may therefore lead to larger costs. We investigated whether reduced plant quality would increase the fitness costs associated with resistance to Bacillus thuringiensis in two populations of the diamondback moth Plutella xylostella. We also measured the rate of decline in resistance on two host-plant (Brassica) species for one insect population (Karak). Population X plant species interactions determined the fitness costs in this study. Poor plant quality increased the fitness costs in terms of development time for both populations. However, fitness costs seen in larval survival did not always increase as plant quality declined. Both the fitness and the stability experiment indicated that fitness costs were higher on the most suitable plant for one population. Theoretically, if the fitness cost of a mutation interacts additively with environmental factors, the relative fitness of resistant insects will decrease with environmental quality. However, multiplicative costs do not necessarily increase with declining quality and may be harder to detect when fitness parameters are more subject to variation in poorer environments.

Bioassays (at generation G2) with a newly collected field population (designated CH3) of Plutella xylostella L. from farmers' fields in the Cameron Highlands, Malaysia, indicated resistance ratios of 813-, 79-, 171-, 498- and 1285-fold for indoxacarb, fipronil, spinosad, deltamethrin and Bacillus thuringiensis toxin Cry1Ac respectively compared with a laboratory susceptible population (Lab-UK). At G2 the field-derived population was divided into two subpopulations: one was selected (G2 to G7) with indoxacarb (indoxa-SEL), while the second was left unselected (UNSEL). A significant reduction in the resistance ratio for each compound was observed in UNSEL at G8. For indoxa-SEL, bioassays at G8 found that selection with indoxacarb gave a resistance ratio of 2594 compared with Lab-UK and of 90 compared with UNSEL. The toxicity of fipronil, spinosad and deltamethrin was not significantly different in indoxa-SEL at G8 compared with G2 but was significantly greater than UNSEL at G8. The toxicity of Cry1Ac was significantly reduced in indoxa-SEL at G8 compared with G2 but was also significantly greater than UNSEL at G8. This suggests that indoxacarb selection maintained resistance to these compounds in the indoxa-SEL population. Synergist studies indicated that resistance to indoxacarb in indoxa-SEL was esterase associated. Logit regression analysis of F1 reciprocal crosses between indoxa-SEL and Lab-UK indicated that resistance to indoxacarb was inherited as an autosomal, incompletely recessive (D(LC) = 0.35) trait. Tests of monogenic inheritance suggested that resistance to indoxacarb was controlled by a single locus., (Copyright (c) 2006 Society of Chemical Industry.)

A field collected population of Plutella xylostella (SERD4) was selected in the laboratory with Bacillus thuringiensis endotoxins Cry1Ac (Cry1Ac-SEL) and Cry1Ab (Cry1Ab-SEL). Both subpopulations showed similar phenotypes: high resistance to the Cry1A toxins and little cross-resistance to Cry1Ca or Cry1D. A previous analysis of the Cry1Ac-SEL showed incompletely dominant resistance to Cry1Ac with more than one factor, at least one of which was sex influenced. In the present study reciprocal mass crosses between Cry1Ab-SEL and a laboratory susceptible population (ROTH) provided evidence that Cry1Ab resistance was also inherited as incompletely dominant trait with more than one factor, and at least one of the factors was sex influenced. Analysis of single pair mating indicated that Cry1Ab-SEL was still heterogeneous for Cry1Ab resistance genes, showing genes with different degrees of dominance. Binding studies showed a large reduction of specific binding of Cry1Ab and Cry1Ac to midgut membrane vesicles of the Cry1Ab-SEL subpopulation. Cry1Ab-SEL was found to be more susceptible to trypsin-activated Cry1Ab toxin than protoxin, although no defect in toxin activation was found. Present and previous results indicate a common basis of resistance to both Cry1Ab and Cry1Ac in selected subpopulations and suggest that a similar set of resistance genes are responsible for resistance to Cry1Ab and Cry1Ac and are selected whichever toxin was used. The possibility of an incompletely dominant trait of resistant to these toxins should be taken into account when considering refuge resistance management strategies.

Genes which provide resistance to novel challenges such as pesticides, toxins or pathogens often impose fitness costs on individuals with a resistant phenotype. Studies of resistance to Bacillus thuringiensis and its insecticidal Cry toxins indicate that fitness costs may be variable and cryptic. Using two field populations (Karak and Serd4) of the diamondback moth, Plutella xylostella, we tested the hypothesis that the costs associated with resistance to the B. thuringiensis toxin Cry1Ac would be evident when insects were grown under poor environmental conditions, namely limited or poor quality resources. On a poor quality resource, a cultivar of Brassica oleracea var. capitata with varietal resistance to P. xylostella, only one resistant population, Karak, showed reduced fitness. Conversely, when we limited a high quality resource, Brassica pekinensis, by imposing larval competition, only resistant Serd4 insects had reduced survival at high larval densities. Furthermore, Cry1Ac resistance in Serd4 insects declined when reared at high larval densities while resistance at low densities fluctuated but did not decline significantly. These results confirm the hypothesis that resistance costs can appear under stressful conditions and demonstrate that the fitness cost of resistance to Bacillus thuringiensis can depend on the particular interaction between genes and the environment.

A field population of Plutella xylostella (L) from Pakistan was found to be highly resistant to deltamethrin (>500-fold) but had little or no resistance to spinosad, fipronil, indoxacarb, abamectin, Bacillus thuringiensis (Bt) var kurstaki, Bt var aizawai or Cry1Ac when compared with a susceptible laboratory population, Lab-UK. A sub-population was selected for six generations (laboratory G3-G8) with deltamethrin (delta-SEL), while a second sub-population was left unselected (UNSEL). Bioassays at G9 found that selection with deltamethrin gave resistance ratios of >230 compared with UNSEL (>6730 compared with Lab-UK). The delta-selected population showed no apparent cross-resistance to spinosad, fipronil or indoxacarb. Logit regression analysis of F1 reciprocal crosses between delta-SEL and Lab-UK indicated that resistance to deltamethrin in the delta-SEL population was inherited as an autosomal, incompletely dominant (D(LC) = 0.67) trait. A direct test of monogenic inheritance based on a back-cross of F1 progeny with delta-SEL suggested that resistance to deltamethrin was controlled by more than one factor., (Copyright 2005 Society of Chemical Industry)

The long-term usefulness of Bacillus thuringiensis Cry toxins, either in sprays or in transgenic crops, may be compromised by the evolution of resistance in target insects. Managing the evolution of resistance to B. thuringiensis toxins requires extensive knowledge about the mechanisms, genetics, and ecology of resistance genes. To date, laboratory-selected populations have provided information on the diverse genetics and mechanisms of resistance to B. thuringiensis, highly resistant field populations being rare. However, the selection pressures on field and laboratory populations are very different and may produce resistance genes with distinct characteristics. In order to better understand the genetics, biochemical mechanisms, and ecology of field-evolved resistance, a diamondback moth (Plutella xylostella) field population (Karak) which had been exposed to intensive spraying with B. thuringiensis subsp. kurstaki was collected from Malaysia. We detected a very high level of resistance to Cry1Ac; high levels of resistance to B. thuringiensis subsp. kurstaki Cry1Aa, Cry1Ab, and Cry1Fa; and a moderate level of resistance to Cry1Ca. The toxicity of Cry1Ja to the Karak population was not significantly different from that to a standard laboratory population (LAB-UK). Notable features of the Karak population were that field-selected resistance to B. thuringiensis subsp. kurstaki did not decline at all in unselected populations over 11 generations in laboratory microcosm experiments and that resistance to Cry1Ac declined only threefold over the same period. This finding may be due to a lack of fitness costs expressed by resistance strains, since such costs can be environmentally dependent and may not occur under ordinary laboratory culture conditions. Alternatively, resistance in the Karak population may have been near fixation, leading to a very slow increase in heterozygosity. Reciprocal genetic crosses between Karak and LAB-UK populations indicated that resistance was autosomal and recessive. At the highest dose of Cry1Ac tested, resistance was completely recessive, while at the lowest dose, it was incompletely dominant. A direct test of monogenic inheritance based on a backcross of F1 progeny with the Karak population suggested that resistance to Cry1Ac was controlled by a single locus. Binding studies with 125I-labeled Cry1Ab and Cry1Ac revealed greatly reduced binding to brush border membrane vesicles prepared from this field population.

Bioassays (at generation 1, G1) using fipronil, spinosad, indoxacarb, and Bacillus thuringiensis toxins Cry1Ac and Cry1Ca with a newly collected field population of Plutella xylostella (L.) from farmers fields in the Cameron Highlands, Malaysia, indicated a resistance ratio of approximately 400-, 1,170-, 330-, 2,840-, and 1,410-fold, respectively, compared with a laboratory-susceptible population of P. xylostella (ROTH). At G3, the field-derived population was divided into two subpopulations, one was selected (G3 to G7) with fipronil (fip-SEL), whereas the second was left unselected (UNSEL). Bioassays at G8 found that selection with fipronil gave a resistance ratio of approximately 490 compared with UNSEL and approximately 770 compared with ROTH. The resistance ratio for fipronil, spinosad, indoxacarb, Cry1Ac, and Cry1Ca in the UNSEL population declined significantly by G8. Logit regression analysis of F1 reciprocal crosses between fip-SEL (at G8) and UNSEL indicated that resistance to fipronil in the fip-SEL population was inherited as an autosomal, incompletely recessive (D(LC) = 0.37) trait. At the highest dose of fipronil tested, resistance was completely recessive, whereas at the lowest dose it was incompletely recessive. A direct test of monogenic inheritance based on a backcross of F1 progeny with fip-SEL suggested that resistance to fipronil was controlled by a single locus. The fip-SEL population at G8 showed little change in its response to spinosad and indoxacarb compared with G1, whereas its susceptibility to Cry1Ac and Cry1Ca increased markedly over the selection period. This suggests that there may be some low level of cross-resistance between fipronil, spinosad, and indoxacarb.

Resistance to the bacteria-derived insecticides spinosad (Conserve), abamectin (Vertimec), Bacillus thuringiensis var kurstaki (Btk) (Dipel), B thuringiensis var aizawai (Bta) (Xentari), B thuringiensis crystal endotoxins Cry1Ac and Cry1Ca, and to the synthetic insecticide fipronil was estimated in a freshly-collected field population (CH1 strain) of Plutella xylostella (L) from the Cameron Highlands, Malaysia. Laboratory bioassays at G1 indicated significant levels of resistance to spinosad, abamectin, Cry1Ac, Btk, Cry1Ca, fipronil and Bta when compared with a laboratory insecticide-susceptible population. Logit regression analysis of F1 reciprocal crosses indicated that resistance to spinosad in the CH1 population was inherited as a co-dominant trait. At the highest dose of spinosad tested, resistance was close to completely recessive, while at the lowest dose it was incompletely dominant. A direct test of monogenic inheritance based on a back-cross of F1 progeny with CH1 suggested that resistance to spinosad was controlled by a single locus.

Six chlorpyrifos-degrading bacteria were isolated from an Australian soil and compared by biochemical and molecular methods. The isolates were indistinguishable, and one (strain B-14) was selected for further analysis. This strain showed greatest similarity to members of the order Enterobacteriales and was closest to members of the Enterobacter asburiae group. The ability of the strain to mineralize chlorpyrifos was investigated under different culture conditions, and the strain utilized chlorpyrifos as the sole source of carbon and phosphorus. Studies with ring or uniformly labeled [(14)C]chlorpyrifos in liquid culture demonstrated that the isolate hydrolyzed chlorpyrifos to diethylthiophospshate (DETP) and 3, 5, 6-trichloro-2-pyridinol, and utilized DETP for growth and energy. The isolate was found to possess mono- and diphosphatase activities along with a phosphotriesterase activity. Addition of other sources of carbon (glucose and succinate) resulted in slowing down of the initial rate of degradation of chlorpyrifos. The isolate degraded the DETP-containing organophosphates parathion, diazinon, coumaphos, and isazofos when provided as the sole source of carbon and phosphorus, but not fenamiphos, fonofos, ethoprop, and cadusafos, which have different side chains. Studies of the molecular basis of degradation suggested that the degrading ability could be polygenic and chromosome based. Further studies revealed that the strain possessed a novel phosphotriesterase enzyme system, as the gene coding for this enzyme had a different sequence from the widely studied organophosphate-degrading gene (opd). The addition of strain B-14 (10(6) cells g(-1)) to soil with a low indigenous population of chlorpyrifos-degrading bacteria treated with 35 mg of chlorpyrifos kg(-1) resulted in a higher degradation rate than was observed in noninoculated soils. These results highlight the potential of this bacterium to be used in the cleanup of contaminated pesticide waste in the environment.

Repeated treatment with fenamiphos (ethyl 4-methylthio-m-tolyl isopropylphosphoramidate) resulted in enhanced biodegradation of this nematicide in two United Kingdom soils with a high pH (>/= 7.7). In contrast, degradation of fenamiphos was slow in three acidic United Kingdom soils (pH 4.7 to 6.7), and repeated treatments did not result in enhanced biodegradation. Rapid degradation of fenamiphos was observed in two Australian soils (pH 6.7 to 6.8) in which it was no longer biologically active against plant nematodes. Enhanced degrading capability was readily transferred from Australian soil to United Kingdom soils, but only those with a high pH were able to maintain this capability for extended periods of time. This result was confirmed by fingerprinting bacterial communities by 16S rRNA gene profiling of extracted DNA. Only United Kingdom soils with a high pH retained bacterial DNA bands originating from the fenamiphos-degrading Australian soil. A degrading consortium was enriched from the Australian soil that utilized fenamiphos as a sole source of carbon. The 16S rRNA banding pattern (determined by denaturing gradient gel electrophoresis) from the isolated consortium migrated to the same position as the bands from the Australian soil and those from the enhanced United Kingdom soils in which the Australian soil had been added. When the bands from the consortium and the soil were sequenced and compared they showed between 97 and 100% sequence identity, confirming that these groups of bacteria were involved in degrading fenamiphos in the soils. The sequences obtained showed similarity to those from the genera Pseudomonas, Flavobacterium, and CAULOBACTER: In the Australian soils, two different degradative pathways operated simultaneously: fenamiphos was converted to fenamiphos sulfoxide (FSO), which was hydrolyzed to the corresponding phenol (FSO-OH) or was hydrolyzed directly to fenamiphos phenol. In the United Kingdom soils in which enhanced degradation had been induced, fenamiphos was oxidized to FSO and then hydrolyzed to FSO-OH, but direct conversion to fenamiphos phenol did not occur.

This paper describes the assessment of nematode spray application methods and details the redesign of a spinning disc applicator for the improved application of the infective juveniles of entomopathogenic nematodes to foliage. The assessment highlighted fundamental design features that prevent efficient and accurate nematode application and these are dealt with in turn specifically to enable improved nematode application. The redesigned or prototype spinning disc was shown to improve nematode carriage in droplets, leading to greater deposition of nematodes per unit area. There is a trade-off between spray droplet coverage of the target surface and the production of a suitable droplet spectrum for nematode carriage.

Crops expressing Bacillus thuringiensis (Bt) insecticidal Cry proteins are grown on millions of hectares. Recommendations to delay resistance are based on a high expression/refugia strategy that aims to kill resistant heterozygotes and enable some susceptible insects to survive. Leaf-dip bioassays on F1 crosses of Malaysian populations of diamondback moth (Plutella xylostella (L)) showed that CrylAc resistance was not fully recessive. The survival of ca 50% of heterozygotes on Bt canola (Brassica napus L) leaves expressing low concentrations of CrylAc agreed with a non-fully-recessive model for resistance. Extrapolations based on log dose-logit mortality regressions for heterozygotes using leaf-dip bioassays showed that a relatively high level of expression, of ca 2000 ng CrylAc mg(-1) total leaf protein, would be required to give 90% mortality to heterozygotes. If high enough levels of expression of Bt toxin to kill heterozygotes cannot be achieved and maintained under field conditions, the effectiveness of the high-dose/refugia strategy would be reduced.

We examined the role of microorganisms in the degradation of the organophosphate insecticide chlorpyrifos in soils from the United Kingdom and Australia. The kinetics of degradation in five United Kingdom soils varying in pH from 4.7 to 8.4 suggested that dissipation of chlorpyrifos was mediated by the cometabolic activities of the soil microorganisms. Repeated application of chlorpyrifos to these soils did not result in the development of a microbial population with an enhanced ability to degrade the pesticide. A robust bacterial population that utilized chlorpyrifos as a source of carbon was detected in an Australian soil. The enhanced ability to degrade chlorpyrifos in the Australian soil was successfully transferred to the five United Kingdom soils. Only soils with a pH of >/=6.7 were able to maintain this degrading ability 90 days after inoculation. Transfer and proliferation of degrading microorganisms from the Australian soil to the United Kingdom soils was monitored by molecular fingerprinting of bacterial 16S rRNA genes by PCR-denaturing gradient gel electrophoresis (DGGE). Two bands were found to be associated with enhanced degradation of chlorpyrifos. Band 1 had sequence similarity to enterics and their relatives, while band 2 had sequence similarity to strains of Pseudomonas. Liquid enrichment culture using the Australian soil as the source of the inoculum led to the isolation of a chlorpyrifos-degrading bacterium. This strain had a 16S rRNA gene with a sequence identical to that of band 1 in the DGGE profile of the Australian soil. DNA probing indicated that genes similar to known organophosphate-degrading (opd) genes were present in the United Kingdom soils. However, no DNA hybridization signal was detected for the Australian soil or the isolated degrader. This indicates that unrelated genes were present in both the Australian soil and the chlorpyrifos-degrading isolate. These results are consistent with our observations that degradation of chlorpyrifos in these systems was unusual, as it was growth linked and involved complete mineralization. As the 16S rRNA gene of the isolate matched a visible DGGE band from the Australian soil, the isolate is likely to be both prominent and involved in the degradation of chlorpyrifos in this soil.

The effects of repeated application and of combinations of pesticides on their degradation rates in soil and on some soil microbial properties were studied. Repeated application of chlorpyrifos did not modify its degradation rate, whereas repeated applications of fenamiphos and chlorothalonil suppressed their own rates of degradation. When applied in combination, the presence of chlorothalonil reduced the degradation rate of both chlorpyrifos and fenamiphos, and the half-life of chlorothalonil was extended in the presence of chlorpyrifos. The dynamics of residues of the major metabolites of the different compounds were also affected by the pesticide combinations and, particularly, by the presence of chlorothalonil. The measured soil microbial parameters (enzyme activities and total microbial biomass) were stable in the pesticide-free control soils throughout the 90-d incubation period, but they were all adversely affected by the presence of chlorothalonil in the soil. The effects from fenamiphos or chlorpyrifos on the soil microbial characteristics were either very small or insignificant.