Impact of non‐pathogenic bacteria on insect disease resistance: importance of ecological context

1. The aerial surface of plants is a habitat for large and diverse microbial communities; termed the phyllosphere. These microbes are unavoidably consumed by herbivores, and while the entomopathogens are well studied, the impact of non‐pathogenic bacteria on herbivore life history is less clear. 2. Previous work has suggested that consumption of non‐entomopathogenic bacteria induces a costly immune response that might decrease the risk of infection. However, we hypothesised that insect herbivores should be selective in how they respond to commonly encountered non‐pathogenic bacteria on their host plants to avoid unnecessary and costly immune responses. 3. An ecologically realistic scenario was used in which we fed cabbage looper, Trichoplusia ni Hübner, larvae on cabbage or cucumber leaves treated with the common non‐entomopathogenic phyllosphere bacteria, Pseudomonas fluorescens and P. syringae. Their constitutive immunity and resistance to a pathogenic bacterium (Bacillus thuringiensis; Bt) and a baculovirus (T. ni single nucleopolyhedrovirus) were then examined. 4. While feeding on bacteria‐treated leaves reduced the growth rate and condition of T. ni, there was no effect on immunity (haemolymph antibacterial and phenoloxidase activities and haemocyte numbers). Phyllosphere bacteria weakly affected the resistance of T. ni to Bt but the direction of this effect was concentration dependent; resistance to the virus was unaffected. Host plant had an impact, with cucumber‐fed larvae being more susceptible to Bt. 5. The lack of evidence for a costly immune response to non‐entomopathogenic bacteria suggests that T. ni are probably adapted to consuming common phyllosphere bacteria, and highlights the importance of the evolutionary history of participants in multi‐trophic interactions.     

Host plant species affects the quality of the generalist Trichoplusia ni as a host for the polyembryonic parasitoid Copidosoma floridanum

Diet of herbivorous insects can influence both the herbivores and their natural enemies. We examined the direct and indirect effects of diet on the interactions between the polyphagous herbivore Trichoplusia ni Hübner (Lepidoptera: Noctuidae) and its polyembryonic parasitoid Copidosoma floridanum Ashmead (Hymenoptera: Encyrtidae). To determine how host plant species and host plant iridoid glycoside content affect host caterpillars and their parasitoids, parasitized and unparasitized T. ni were given leaves of either Plantago lanceolata L., which contains the iridoid glycosides aucubin and catalpol, Plantago major L. (Plantaginaceae), which contains only aucubin, or Taraxacum officinale F.H. Wigg (Asteraceae), which contains neither. Survival of unparasitized T. ni was much lower when fed P. major compared with the other two host plants, whereas pupae were smallest when fed T. officinale and developed most slowly when fed P. lanceolata as larvae. Neither aucubin nor catalpol were detected in intact Plantago‐fed T. ni larvae or their hemolymph, and only trace amounts of aucubin were detected in frass, suggesting that these compounds are mostly metabolized in the midgut and are not encountered by the parasitoid. Copidosoma floridanum clutch size was almost doubled when reared from P. lanceolata‐fed T. ni compared with T. officinale‐fed larvae and tripled compared with P. major‐fed larvae, although the percent of parasitoids surviving to adulthood was uniformly high regardless of host diet. The observed variation in C. floridanum fitness among host diets is likely mediated by the effect of the diets on host quality, which in turn may be influenced more by other factors in the host plants than their iridoid glycoside profiles. Interactions between plant metabolites, generalist herbivores like T. ni, and their parasitoids may be predominantly indirect.     

Lepidopteran pest populations and crop yields in row intercropped broccoli

Abstract

• 1 To determine how the presence of non‐host plants impact populations of lepidopteran pests and yield of broccoli, Brassica oleracea var. italica L., broccoli was intercropped with tomato, Lycopersicon esculentum Miller, or yellow sweetclover (YSC), Melilotus officinalis L., and compared with broccoli monoculture grown at two densities (high and low).
• 2 During a 1997 study, numbers of early instar imported cabbageworm, Artogeia rapae L., were greater in broccoli monoculture compared with intercropped plantings. In contrast, late instar A. rapae numbers were significantly higher in intercropped broccoli during most of the season, indicating greater survival of early instar stages in intercropped habitats.
• 3 Cabbage looper, Trichoplusia ni Hübner, eggs and larvae were more abundant in broccoli monoculture than intercropped broccoli in 1997. In addition, T. ni populations were found at significantly lower levels in broccoli‐YSC compared with broccoli‐tomato habitat throughout the season.
• 4 During a 1998 study, numbers of early and late A. rapae instars were greater in intercropped broccoli on most sampling dates.
• 5 The mean percentages of harvested broccoli heads infested with insects and associated frass were higher in monocultures than intercropped treatments in 1997. Additionally, a significantly lower number of contaminants were found in the crowns of broccoli harvested from broccoli‐YSC (YSC) compared with broccoli‐tomato (tomato) habitat. However, broccoli heads were significantly smaller in intercropped habitats.
• 6 Variations in insect responses and challenges to using crop diversification as a management tool are discussed.

     

Effects of crop type on Bacillus thuringiensis toxicity and residual activity against Trichoplusia ni in greenhouses

We assessed the efficacy and persistence of a Bacillus thuringiensiskurstaki (Btk) formulation (Dipel) against Trichoplusia ni (Hubner) (Lep., Noctuidae), the cabbage looper, on three greenhouse vegetable crops (tomato, bell pepper and cucumber). First, T. ni larvae were fed leaf discs treated with Btk to assess how Btk toxicity varies with host plant. Secondly, T. ni larvae were fed leaf discs harvested from plants that had been sprayed with Btk 1, 5 and 9 days previously to assess the residual activity of Btk toxicity in greenhouse environments. Mortality of T. ni larvae fed tomato leaf discs was significantly higher than T. ni fed cucumber or pepper leaf discs. The toxicity of Btk had declined by less than 50% after 9 days, which suggests that Btk persistence is lengthy in greenhouse environments. No crop effects on the residual activity of Btk were found. These results demonstrate that the greenhouse environment and the crop should be considered when using Btk for insect management on greenhouse crops.     

A plant pathogen modulates the effects of secondary metabolites on the performance and immune function of an insect herbivore

Host plant chemical composition critically shapes the performance of insect herbivores feeding on them. Some insects have become specialized on plant secondary metabolites, and even use them to their own advantage such as defense against predators. However, infection by plant pathogens can seriously alter the interaction between herbivores and their host plants. We tested whether the effects of the plant secondary metabolites, iridoid glycosides (IGs), on the performance and immune response of an insect herbivore are modulated by a plant pathogen. We used the IG‐specialized Glanville fritillary butterfly Melitaea cinxia, its host plant Plantago lanceolata, and the naturally occurring plant pathogen, powdery mildew Podosphaera plantaginis, as model system. Pre‐diapause larvae were fed on P. lanceolata host plants selected to contain either high or low IGs, in the presence or absence of powdery mildew. Larval performance was measured by growth rate, survival until diapause, and by investment in immunity. We assessed immunity after a bacterial challenge in terms of phenoloxidase (PO) activity and the expression of seven pre‐selected insect immune genes (qPCR). We found that the beneficial effects of constitutive leaf IGs, that improved larval growth, were significantly reduced by mildew infection. Moreover, mildew presence downregulated one component of larval immune response (PO activity), suggesting a physiological cost of investment in immunity under suboptimal conditions. Yet, feeding on mildew‐infected leaves caused an upregulation of two immune genes, lysozyme and prophenoloxidase. Our findings indicate that a plant pathogen can significantly modulate the effects of secondary metabolites on the growth of an insect herbivore. Furthermore, we show that a plant pathogen can induce contrasting effects on insect immune function. We suspect that the activation of the immune system toward a plant pathogen infection may be maladaptive, but the actual infectivity on the larvae should be tested.     

Linking belowground and aboveground phenology in two boreal forests in Northeast China

Plants are frequently attacked by both pathogens and insects, and an attack from one can induce plant responses that affect resistance to the other. However, we currently lack a predictive framework for understanding how pathogens, their vectors, and other herbivores interact. To address this gap, we have investigated the effects of a viral infection in the host plant on both its aphid vector and non-vector herbivores. We tested whether the infection by three different strains of Potato virus Y (PVY^NTN, PVY^NO and PVY^O) on tomato plants affected: (1) the induced plant defense pathways; (2) the abundance and fecundity of the aphid vector (Macrosiphum euphorbiae); and (3) the performance of two non-vector species: a caterpillar (Trichoplusia ni) and a beetle (Leptinotarsa decemlineata). While infection by all three strains of PVY induced the salicylate pathway, PVY^NTN induced a stronger and longer response. Fecundity and density of aphids increased on all PVY-infected plants, suggesting that the aphid response is not negatively associated with salicylate induction. In contrast, the performance of non-vector herbivores positively correlated with the strength of salicylate induction. PVY^NTN infection decreased plant resistance to both non-vector herbivores, increasing their growth rates. We also demonstrated that the impact of host plant viral infection on the caterpillar results from host plant responses and not the effects of aphid vector feeding. We propose that pathogens chemically mediate insect–plant interactions by activating the salicylate pathway and decreasing plant resistance to chewing insects, which has implications for both disease transmission and insect community structure.     

Serological relationships among plusiinae baculoviruses

Antisera were produced against nucleocapsids, NP-40 detergent soluble proteins, or polyhedral protein of the multiply embedded nuclear polyhedrosis virus (MNPV) of Autographa californica, nucleocapsids of Trichoplusia ni singly embedded virus (SNPV), and polyhedral protein of Lymantria dispar MNPV. Antigens consisting of nucleocapsids, NP-40 soluble proteins, and polyhedral protein were prepared from A. californica MNPV, T. ni MNPV, L. dispar MNPV, Rachiplusia ou MNPV, T. ni SNPV, and Pseudoplusia includens SNPV. Radial immunodiffusion patterns formed with Plusiinae nucleocapsid antigens and antiserum to nucleocapsids of A. californica MNPV or T. ni SNPV revealed a distinction between multiply and singly embedded viruses. The same alignment of Plusiinae viruses was observed in reactions between A. californica NP-40 soluble protein antiserum and the NP-40 soluble protein fractions from the Plusiinae NPVs. There were no reactions between the Plusiinae SNPV nucleocapsid antigens and the A. californica MNPV nucleocapsid antiserum. However, there were faint precipitin bands between MNPV nucleocapsid antigens and T. ni SNPV nucleocapsid antiserum. Each of the polyhedral protein fractions from the Plusiinae formed a single precipitin band with the antiserum to polyhedral protein of either A. californica or L. dispar. The precipitin bands formed with the A. californica antiserum by polyhedral proteins of T. ni SNPV, P. includens SNPV, and R. ou MNPV were confluent, and shared partial identity with those formed by A. californica MNPV and T. ni MNPV. All precipitin bands formed by Plusiinae polyhedral proteins against antiserum to L. dispar polyhedral protein were confluent, and shared partial identity with that formed by L. dispar polyhedral protein.     

Behavior of a Recombinant Baculovirus in Lepidopteran Hosts with Different Susceptibilities

Insect pathogens, such as baculoviruses, that are used as microbial insecticides have been genetically modified to increase their speed of action. Nontarget species will often be exposed to these pathogens, and it is important to know the consequences of infection in hosts across the whole spectrum of susceptibility. Two key parameters, speed of kill and pathogen yield, are compared here for two baculoviruses, a wild-type Autographa californica nucleopolyhedrovirus (AcNPV), AcNPV clone C6, and a genetically modified AcNPV which expresses an insect-selective toxin, AcNPV-ST3, for two lepidopteran hosts which differ in susceptibility. The pathogenicity of the two viruses was equal in the less-susceptible host, Mamestra brassicae, but the recombinant was more pathogenic than the wild-type virus in the susceptible species, Trichoplusia ni. Both viruses took longer to kill the larvae of M. brassicae than to kill those of T. ni. However, whereas the larvae of T. ni were killed more quickly by the recombinant virus, the reverse was found to be true for the larvae of M. brassicae. Both viruses produced a greater yield in M. brassicae, and the yield of the recombinant was significantly lower than that of the wild type in both species. The virus yield increased linearly with the time taken for the insects to die. However, despite the more rapid speed of kill of the wild-type AcNPV in M. brassicae, the yield was significantly lower for the recombinant virus at any given time to death. A lower yield for the recombinant virus could be the result of a reduction in replication rate. This was investigated by comparing determinations of the virus yield per unit of weight of insect cadaver. The response of the two species (to both viruses) was very different: the yield per unit of weight decreased over time for M. brassicae but increased for T. ni. The implications of these data for risk assessment of wild-type and genetically modified baculoviruses are discussed.     

A high‐quality chromosome‐level genome assembly of a generalist herbivore,Trichoplusia ni

The cabbage looper, Trichoplusia ni, is a globally distributed highly polyphagous herbivore and an important agricultural pest. T. ni has evolved resistance to various chemical insecticides, and is one of the only two insect species that have evolved resistance to the biopesticide Bacillus thuringiensis (Bt) in agricultural systems and has been selected for resistance to baculovirus infections. We report a 333‐Mb high‐quality T. ni genome assembly, which has N50 lengths of scaffolds and contigs of 4.6 Mb and 140 Kb, respectively, and contains 14,384 protein‐coding genes. High‐density genetic maps were constructed to anchor 305 Mb (91.7%) of the assembly to 31 chromosomes. Comparative genomic analysis of T. ni with Bombyx mori showed enrichment of tandemly duplicated genes in T. ni in families involved in detoxification and digestion, consistent with the broad host range of T. ni. High levels of genome synteny were found between T. ni and other sequenced lepidopterans. However, genome synteny analysis of T. ni and the T. ni derived cell line High Five (Hi5) indicated extensive genome rearrangements in the cell line. These results provided the first genomic evidence revealing the high instability of chromosomes in lepidopteran cell lines known from karyotypic observations. The high‐quality T. ni genome sequence provides a valuable resource for research in a broad range of areas including fundamental insect biology, insect‐plant interactions and co‐evolution, mechanisms and evolution of insect resistance to chemical and biological pesticides, and technology development for insect pest management.     

Examining the relationship between hemolymph phenoloxidase and resistance to a DNA virus, Plodia interpunctella granulosis virus (PiGV)

We have a detailed understanding of invertebrate immune responses to bacteria and fungal pathogens, but we know less about how insects respond to virus challenge. Phenoloxidase (PO) functions as an important immune response against many parasites and pathogens and is routinely used as a measure of immune competance. We examine the role of haemolymph PO activity in Plodia interpuncetella's response to its natural granulosis virus (PiGV). Larvae were challenged with virus by both oral inoculation of occluded virus (the natural infection route) and direct intrahaemocoelic injection of budded virus. Haemolymph was collected at time points post-viral challenge using a novel method that allows the volume of haemolymph to be quanitified. The haemolmyph was collected without killing the larvae so that haemolymph samples from individuals that developed viral disease could be distinguished from samples collected from those that fought off infection. The level of haemolymph PO activity in resistant larvae did not differ from control larvae. Therefore we have no evidence that PO is involved in resistance to virus in the haemocoel whether larvae are challenged naturally by oral innoculation or directly by intraheamocoelic injection. Phenoloxidase may therefore not be a relevant metric of immunocompetence for viral infection.     

Composition of the Spruce Budworm (Choristoneura fumiferana) Midgut Microbiota as Affected by Rearing Conditions

The eastern spruce budworm (Choristoneura fumiferana) is one of the most destructive forest insect pests in Canada. Little is known about its intestinal microbiota, which could play a role in digestion, immune protection, communication and/or development. The present study was designed to provide a first characterization of the effects of rearing conditions on the taxonomic diversity and structure of the C. fumiferana midgut microbiota, using a culture-independent approach. Three diets and insect sources were examined: larvae from a laboratory colony reared on a synthetic diet and field-collected larvae reared on balsam fir or black spruce foliage. Bacterial DNA from the larval midguts was extracted to amplify and sequence the V6-V8 region of the 16S rRNA gene, using the Roche 454 GS-FLX technology. Our results showed a dominance of Proteobacteria, mainly Pseudomonas spp., in the spruce budworm midgut, irrespective of treatment group. Taxonomic diversity of the midgut microbiota was greater for larvae reared on synthetic diet than for those collected and reared on host plants, a difference that is likely accounted for by several factors. A greater proportion of bacteria from the phylum Bacteroidetes in insects fed artificial diet constituted the main difference between this group and those reared on foliage; within the phylum Proteobacteria, the presence of the genus Bradyrhizobium was also unique to insects reared on artificial diet. Strikingly, a Bray-Curtis analysis showed important differences in microbial diversity among the treatment groups, pointing to the importance of diet and environment in defining the spruce budworm midgut microbiota.     

The effect of host plant species on performance and movement behaviour of the cabbage looper Trichoplusia ni and their potential influences on infection by Autographa californica multiple nucleopolyhedrovirus

• 1 Cabbage loopers Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) are serious pests in greenhouses growing tomatoes, cucumbers and bell peppers. A potential microbial control, now in development, is the broad host‐range virus Autographa californica multiple nucleopolyhedrovirus (AcMNPV).
• 2 The relationships between the three host plants and the feeding behaviour, larval movement and performance of cabbage looper larvae that might relate to their interaction with AcMNPV applications were investigated.
• 3 Larvae reared on cucumber plants consumed approximately ten‐fold more leaf area than larvae reared on pepper plants and almost five‐fold more than larvae reared on tomato plants. This could influence the amount of AcMNPV consumed if it were used as a microbial spray because increased consumption can be associated with increased probability of infection. Survival from neonate to pupa also varied, with the greatest being on cucumber, followed by tomato and pepper plants. Larvae fed cucumber were approximately four‐fold heavier than larvae fed tomato and over 15‐fold heavier than larvae fed pepper plants.
• 4 The distribution of larvae on plants in commercial greenhouses where a single crop was being grown also varied with food plant with 73% being found on the bottom and middle portions of tomato plants and 87% occurring in the top portions of pepper plants. Larvae tended to be distributed on the middle portion of cucumber plants, the lower portion of tomato plants and the top portion of pepper plants. Larval movement did not vary between AcMNPV‐infected and uninfected controls.
• 5 It is predicted that the higher leaf area consumption and location of larvae in the middle portion of cucumber plants may make them more susceptible to viral sprays. Furthermore, given their greater survival than larvae fed tomato and pepper, there may be a greater need for virus applications.

     

Biochemical comparison of polyhedral protein from five nuclear polyhedrosis viruses infecting plusiine larvae (Lepidoptera: Noctuidae)

Polyhedral protein preparations from five nuclear polyhedrosis viruses isolated from four closely related host insects of the noctuid subfamily Plusiinae were characterized by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE), high voltage paper electrophoresis, and amino acid analysis. The viruses were Autographa california multiple-embedded virion type (MEV), Pseudoplusia includens singly embedded virion type (SEV), Rachiplusia ou MEV, Trichoplusia ni MEV, and T. ni SEV. Each was produced in its own host; A. californica MEV was also produced in T. ni larvae to determine possible host influence on polyhedral protein chemistry. Each test revealed minor, reproducible differences among most isolates. In SDS-PAGE, the major protein component ranged from 26,700 to 28,300 MW among the isolates. Differences were confined to minor protein bands or to band intensity. Peptide maps showed differences among most isolates in numbers of acidic and basic peptide spots, but all had an identical number of neutral spots. Migration patterns also differed among most isolates. The amino acid compositions of the six polyhedral inclusions were very similar, with aspartic and glutamic acids being the predominant residues. The greatest differences were found between the MEV and SEV groups, with lesser differences within each group. In all analyses, A. californica MEV produced in A. californica was indistinguishable from virus produced in T. ni.     

Occurrence and accumulation of viruses of Trichoplusia ni in treated field plots

Concentrations of the nuclear-polyhedrous virus (T. ni NPV) and the granulosis virus (T. ni GV) of the cabbage looper, Trichoplusia ni, in soil and on foliage were monitored up to 4 years after treatment.A single application of T. ni NPV to soil in August or 5 foliar applications of the virus at 10-day intervals in August and early September maintained substantial concentrations of the virus on foliage and high concentrations of the virus accumulated in soil. With development of natural epizootics of the virus disease in populations of the host larvae in September and October, substantial concentrations of the virus accumulated in soil and on foliage in nontreated plots, eventually becoming equal in amount with the virus in virus-treated plots. The virus accumulated more slowly in plots treated with chemical insecticides or Bacillus thuringiensis because few host larvae survived to support late-season epizootics of the disease. Small quantities of T. ni NPV were detected in heads of cabbage harvested from the plots in October.Long-term studies in which nontreated plots and plots treated with T. ni NPV or T. ni GV were replanted for up to 4 years after treatment showed that concentrations of T. ni NPV in surface soil remained constant during the winter but were reduced by dilution during cultivation preparatory to planting in the spring. T. ni NPV accumulated during the late summer and autumn with development of epizootics of the disease in populations of host larvae. Increased concentrations of the virus in soil coincided with increased concentrations on leaves in each year. T. ni GV did not persist on leaves or in soil following application and only small amounts were found 2 years after application.T. ni NPV disease was prevalent in September and October in populations of host larvae in plots in which substantial residues of the virus were found. These epizootics contributed substantially to late-season control of the looper after completion of spraying.     

Sequence variation and differential splicing of the midgut cadherin gene in Trichoplusia ni

The insect midgut cadherin serves as an important receptor for the Cry toxins from Bacillus thuringiensis (Bt). Variation of the cadherin in insect populations provides a genetic potential for development of cadherin-based Bt resistance in insect populations. Sequence analysis of the cadherin from the cabbage looper, Trichoplusia ni, together with cadherins from 18 other lepidopterans showed a similar phylogenetic relationship of the cadherins to the phylogeny of Lepidoptera. The midgut cadherin in three laboratory populations of T. ni exhibited high variability, although the resistance to Bt toxin Cry1Ac in the T. ni strain is not genetically associated with cadherin gene mutations. A total of 142 single nucleotide polymorphisms (SNPs) were identified in the cadherin cDNAs from the T. ni strains, including 20 missense mutations. In addition, insertion and deletion polymorphisms (indels) were also identified in the cadherin alleles in T. ni. More interestingly, the results from this study reveal that differential splicing of mRNA also occurs in the cadherin gene expression. Therefore, variation of the midgut cadherin in insects may not only be caused by cadherin gene mutations, but could also result from alternative splicing of its mRNA regulated by factors acting in trans. Analysis of cadherin gene alleles in F2, F3 and F4 progenies from the cross between the Cry1Ac resistant and the susceptible strain after consecutive selections with Cry1Ac for three generations showed that selection with Cry1Ac did not result in an increase of frequencies of the cadherin alleles originated from the resistant strain.     

Effect of Bt broccoli and resistant genotype of Plutella xylostella (Lepidoptera: Plutellidae) on life history and prey acceptance of the predator Coleomegilla maculata (Coleoptera: Coccinellidae)

The ecological implications on biological control of insecticidal transgenic plants, which produce crystal (Cry) proteins derived from the soil bacterium Bacillus thuringiensis (Bt), remains a contentious issue and affects risk assessment decisions. In this study, we used a unique system of resistant insects, Bt plants and a predator to critically evaluate this issue. The effects of broccoli type (normal or expressing Cry1Ac protein) and insect genotype (susceptible or Cry1Ac-resistant) of Plutella xylostella L. (Lepidoptera: Plutellidae) were examined for their effects on the life history of the predator, Coleomegilla maculata DeGeer (Coleoptera: Coccinellidae) over two generations. Additional behavioral studies were conducted on prey choice. C. maculata could not discriminate between Bt-resistant and susceptible genotypes of P. xylostella, nor between Bt and normal broccoli plants with resistant genotypes of P. xylostella feeding on them. The larval and pupal period, adult weight and fecundity of each female were not significantly different when C. maculata larvae fed on different genotypes (Bt-resistant or susceptible) of insect prey larvae reared on Bt or non-Bt broccoli plants. The life-history parameters of the subsequent generation of C. maculata fed on Bt broccoli-reared resistant P. xylostella were also not significantly different from those on non-Bt broccoli. These results indicated that Cry1Ac did not harm the life history or prey acceptance of an important predator after two generations of exposure. Plants expressing Cry1Ac are unlikely to affect this important predator in the field.     

Generalized plant defense: effects on multiple species

Summary Two species of lepidopteran herbivores, Manduca sexta (Sphingidae) and Trichoplusia ni (Noctuidae), were reared on synthetic diet containing either the alkaloid nicotine or the flavonoid rutin. Survival and pupal weight of the specialist M. sexta did not differ when larvae were reared on diet containing nicotine or rutin. In contrast, the generalist T. ni did not survive on diet containing 0.125% nicotine or greater, whereas larvae survived on all concentrations of rutin. These data demonstrate that the alkaloid nicotine is inhibitory toward generalist, but not specialist herbivores, whereas the flavonoid rutin has no effect on specialist herbivores and limited effects on generalist herbivores. Five species of Pseudomonas bacterial pathogens: P. syringae, P. syringae pv. angulata, P. syringae pv. tabaci, P. fluorescens, and P. solanacearum were grown on nutrient agar containing nicotine or rutin at concentrations ranging from 0.0 to 1.0% wet weight in 0.1% intervals. No species of Pseudomonas grew at concentrations greater than 0.5% nicotine when 10⁶ colony forming units (cfu) were used, but growth occurred at all concentrations of rutin when 10² cfu were used. These data indicate that nicotine was inhibitory to growth of both herbivores and pathogens, suggesting that certain plant secondary chemicals with high toxicity are of a generalized nature and affect multiple species. Differences in the sensitivity of organisms to allelochemicals such as generalist or specialist can make it appear that specific allelochemicals affect specific organisms, when in fact it is the tolerance of the organism to the plant chemical that is responsible. In four separate studies, the growth of M. sexta, T. ni and Helicoverpa zea was significantly lower on plants inoculated with P. solanacearum. Alteration in leaf quality by P. solanacearum was due to either reductions in leaf nutrients or increases in allelochemicals. We speculate that localized or systemic induction by both herbivores and pathogens can cause changes in leaf quality, effecting each other's subsequent colonization. The generalized nature of plant secondary compounds and potential reciprocal effects on induction by both species suggests that herbivores and pathogens may affect plant quality through induction and diffuse interactions of disparate species can alter the community of organisms colonizing a plant.     

Potential of Lecanicillium spp. for management of insects, nematodes and plant diseases

Fungi in the genus Lecanicillium (formerly classified as the single species Verticillium lecanii) are important pathogens of insects and some have been developed as commercial biopesticides. Some isolates are also active against phytoparasitic nematodes or fungi. Lecanicillium spp. use both mechanical forces and hydrolytic enzymes to directly penetrate the insect integument and the cell wall of the fungal plant pathogen. In addition to mycoparasitism of the plant pathogen, the mode of action is linked to colonization of host plant tissues, triggering an induced systemic resistance. Recently it was demonstrated that development of Lecanicillium hybrids through protoplast fusion may result in strains that inherit parental attributes, thereby allowing development of hybrid strains with broader host range and other increased benefits, such as increased viability. Such hybrids have demonstrated increased virulence against aphids, whiteflies and the soybean cyst nematode. Three naturally occurring species of Lecanicillium, L. attenuatum, L. longisporum, and an isolate that could not be linked to any presently described species based on rDNA sequences have been shown to have potential to control aphids as well as suppress the growth and spore production of Sphaerotheca fuliginea, the causal agent of cucumber powdery mildew. These results suggest that strains of Lecanicillium spp. may have potential for development as a single microbial control agent effective against several plant diseases, pest insects and plant parasitic nematodes due to its antagonistic, parasitic and disease resistance inducing characteristics. However, to our knowledge, no Lecanicillium spp. have been developed for control of phytopathogens or phytoparasitic nematodes.     

Microbial Symbionts in Insects Influence Down-Regulation of Defense Genes in Maize

Diabrotica virgifera virgifera larvae are root-feeding insects and significant pests to maize in North America and Europe. Little is known regarding how plants respond to insect attack of roots, thus complicating the selection for plant defense targets. Diabrotica virgifera virgifera is the most successful species in its genus and is the only Diabrotica beetle harboring an almost species-wide Wolbachia infection. Diabrotica virgifera virgifera are infected with Wolbachia and the typical gut flora found in soil-living, phytophagous insects. Diabrotica virgifera virgifera larvae cannot be reared aseptically and thus, it is not possible to observe the response of maize to effects of insect gut flora or other transient microbes. Because Wolbachia are heritable, it is possible to investigate whether Wolbachia infection affects the regulation of maize defenses. To answer if the success of Diabrotica virgifera virgifera is the result of microbial infection, Diabrotica virgifera virgifera were treated with antibiotics to eliminate Wolbachia and a microarray experiment was performed. Direct comparisons made between the response of maize root tissue to the feeding of antibiotic treated and untreated Diabrotica virgifera virgifera show down-regulation of plant defenses in the untreated insects compared to the antibiotic treated and control treatments. Results were confirmed via QRT-PCR. Biological and behavioral assays indicate that microbes have integrated into Diabrotica virgifera virgifera physiology without inducing negative effects and that antibiotic treatment did not affect the behavior or biology of the insect. The expression data and suggest that the pressure of microbes, which are most likely Wolbachia, mediate the down-regulation of many maize defenses via their insect hosts. This is the first report of a potential link between a microbial symbiont of an insect and a silencing effect in the insect host plant. This is also the first expression profile for a plant attacked by a root-feeding insect.     

Heritability of immune function in the caterpillar Spodoptera littoralis

Phenoloxidase (PO) is believed to be a key mediator of immune function in insects and has been implicated both in non-self recognition and in resistance to a variety of parasites and pathogens, including baculoviruses and parasitoids. Using larvae of the Egyptian cotton leafworm, Spodoptera littoralis, we found that despite its apparent importance, haemolymph PO activity varied markedly between individuals, even amongst insects reared under apparently identical conditions. Sib-analysis methods were used to determine whether individuals varied genetically in their PO activity, and hence in one aspect of immune function. The heritability estimate of haemolymph PO activity was high (h(2) = 0.690 +/- 0.069), and PO activity in the haemolymph was strongly correlated with PO activity in both the cuticle and midgut; the sites of entry for most parasites and pathogens. Haemolymph PO activity was also strongly correlated with the degree to which a synthetic parasite (a small piece of nylon monofilament) was encapsulated and melanized (r = 0.622 +/- 0.142), suggesting that the encapsulation response is also heritable. The mechanism maintaining this genetic variation has yet to be elucidated.