Comparative analysis of defensive secondary metabolites in wild teosinte and cultivated maize under flooding and herbivory stress.

Climate change is driving an alarming increase in the frequency and intensity of abiotic and biotic stress factors, negatively impacting plant development and agricultural productivity. To survive, plants respond by inducing changes in below and aboveground metabolism with concomitant alterations in defensive secondary metabolites. While plant responses to the isolated stresses of flooding and insect herbivory have been extensively studied, much less is known about their response in combination. Wild relatives of cultivated plants with robust stress tolerance traits provide an excellent system for comparing how diverse plant species respond to combinatorial stress, and provide insight into potential germplasms for stress-tolerant hybrids. In this study, we compared the below and aboveground changes in the secondary metabolites of maize (Zea mays) and a flood-tolerant wild relative Nicaraguan teosinte (Zea nicaraguensis) in response to flooding, insect herbivory, and their combination. Root tissue was analyzed for changes in belowground metabolism. Leaf total phenolic content and headspace volatile organic compound emission were analyzed for changes in aboveground secondary metabolism. Results revealed significant differences in the root metabolome profiles of teosinte and maize. Notably, the accumulation of the flavonoids apigenin, naringenin, and luteolin during flooding and herbivory differentiated teosinte from maize. Aboveground, terpenes, including trans-α-bergamotene and (E)-4,8-dimethylnona-1,3,7-triene, shaped compositional differences in their volatile profiles between flooding, herbivory, and their combination. Taken together, these results suggest teosinte may be more tolerant than maize due to dynamic metabolic changes during flooding and herbivory that help relieve stress and influence plant-insect interactions.

Plant Variety, Mycorrhization, and Herbivory Influence Induced Volatile Emissions and Plant Growth Characteristics in Tomato.

Plants produce a range of volatile organic compounds (VOCs) that mediate vital ecological interactions between herbivorous insects, their natural enemies, plants, and soil dwelling organisms including arbuscular mycorrhizal fungi (AMF). The composition, quantity, and quality of the emitted VOCs can vary and is influenced by numerous factors such as plant species, variety (cultivar), plant developmental stage, root colonization by soil microbes, as well as the insect developmental stage, and level of specialization of the attacking herbivore. Understanding factors shaping VOC emissions is important and can be leveraged to enhance plant health and pest resistance. In this greenhouse study, we evaluated the influence of plant variety, mycorrhizal colonization, herbivory, and their interactions on the composition of emitted volatiles in tomato plants (Solanum lycopersicum L.). Four tomato varieties from two breeding histories (two heirlooms and two hybrids), were used. Tomato plants were inoculated with a commercial inoculum blend consisting of four species of AMF. Plants were also subjected to herbivory by Manduca sexta (Lepidoptera: Sphingidae L.) five weeks after transplanting. Headspace volatiles were collected from inoculated and non-inoculated plants with and without herbivores using solid phase-microextraction. Volatile profiles consisted of 21 different volatiles in detectable quantities. These included monoterpenes, sesquiterpenes, and alkane hydrocarbons. We documented a strong plant variety effect on VOC emissions. AMF colonization and herbivory suppressed VOC emissions. Plant biomass was improved by colonization of AMF. Our results show that mycorrhization, herbivory and plant variety can alter tomato plant VOC emissions and further shape volatile-mediated insect and plant interactions.

Deciphering Plant-Insect-Microorganism Signals for Sustainable Crop Production.

Agricultural crop productivity relies on the application of chemical pesticides to reduce pest and pathogen damage. However, chemical pesticides also pose a range of ecological, environmental and economic penalties. This includes the development of pesticide resistance by insect pests and pathogens, rendering pesticides less effective. Alternative sustainable crop protection tools should therefore be considered. Semiochemicals are signalling molecules produced by organisms, including plants, microbes, and animals, which cause behavioural or developmental changes in receiving organisms. Manipulating semiochemicals could provide a more sustainable approach to the management of insect pests and pathogens across crops. Here, we review the role of semiochemicals in the interaction between plants, insects and microbes, including examples of how they have been applied to agricultural systems. We highlight future research priorities to be considered for semiochemicals to be credible alternatives to the application of chemical pesticides.

Flooding and herbivory: the effect of concurrent stress factors on plant volatile emissions and gene expression in two heirloom tomato varieties.

BACKGROUND: In nature and in cultivated fields, plants encounter multiple stress factors. Nonetheless, our understanding of how plants actively respond to combinatorial stress remains limited. Among the least studied stress combination is that of flooding and herbivory, despite the growing importance of these stressors in the context of climate change. We investigated plant chemistry and gene expression changes in two heirloom tomato varieties: Cherokee Purple (CP) and Striped German (SG) in response to flooding, herbivory by Spodoptera exigua, and their combination. RESULTS: Volatile organic compounds (VOCs) identified in tomato plants subjected to flooding and/or herbivory included several mono- and sesquiterpenes. Flooding was the main factor altering VOCs emission rates, and impacting plant biomass accumulation, while different varieties had quantitative differences in their VOC emissions. At the gene expression levels, there were 335 differentially expressed genes between the two tomato plant varieties, these included genes encoding for phenylalanine ammonia-lyase (PAL), cinnamoyl-CoA-reductase-like, and phytoene synthase (Psy1). Flooding and variety effects together influenced abscisic acid (ABA) signaling genes with the SG variety showing higher levels of ABA production and ABA-dependent signaling upon flooding. Flooding downregulated genes associated with cytokinin catabolism and general defense response and upregulated genes associated with ethylene biosynthesis, anthocyanin biosynthesis, and gibberellin biosynthesis. Combining flooding and herbivory induced the upregulation of genes including chalcone synthase (CHS), PAL, and genes encoding BAHD acyltransferase and UDP-glucose iridoid glucosyltransferase-like genes in one of the tomato varieties (CP) and a disproportionate number of heat-shock proteins in SG. Only the SG variety had measurable changes in gene expression due to herbivory alone, upregulating zeatin, and O-glucosyltransferase and thioredoxin among others. CONCLUSION: Our results suggest that both heirloom tomato plant varieties differ in their production of secondary metabolites including phenylpropanoids and terpenoids and their regulation and activation of ABA signaling upon stress associated with flooding. Herbivory and flooding together had interacting effects that were evident at the level of plant chemistry (VOCs production), gene expression and biomass markers. Results from our study highlight the complex nature of plant responses to combinatorial stresses and point at specific genes and pathways that are affected by flooding and herbivory combined.

Herbivory Protection via Volatile Organic Compounds Is Influenced by Maize Genotype, Not Bacillus altitudinis-Enriched Bacterial Communities.

Belowground, plants interact with beneficial soil microbes such as plant growth-promoting rhizobacteria (PGPR). PGPR are rhizosphere bacteria that colonize roots and elicit beneficial effects in plants such as improved plant growth, pathogen resistance, abiotic stress tolerance, and herbivore protection. Treatment of plants with PGPR has been shown to trigger the emission of volatile organic compounds (VOCs). Volatile emissions can also be triggered by herbivory, termed herbivore-induced plant volatiles (HIPV), with important ramifications for chemical-mediated plant and insect interactions. Much of our current understanding of PGPR and herbivore-induced volatiles is based on studies using one plant genotype, yet domestication and modern breeding has led to the development of diverse germplasm with altered phenotypes and chemistry. In this study, we investigated if volatile emissions triggered by PGPR colonization and herbivory varies by maize genotype and microbial community assemblages. Six maize genotypes representing three decades of crop breeding and two heterotic groups were used, with four microbiome treatments: live or sterilized soil, with or without a Bacillus inoculant. Soil sterilization was used to delay microbiome establishment, resulting in low-diversity treatments. At planting, maize seeds were inoculated with PGPR Bacillus altitudinis AP-283 and grown under greenhouse conditions. Four weeks post planting, plants were subjected to feeding by third instar Helicoverpa zea (Lepidoptera: Noctuidae) larvae. Volatiles were collected using solid phase microextraction and analyzed with gas chromatography-mass spectrometry. Illumina NovaSeq 16S rRNA amplicon sequencing was carried out to characterize the rhizosphere microbiome. Maize genotype significantly influenced total volatile emissions, and relative abundance of volatile classes. We did not document a strong influence of microbe treatment on plant VOC emissions. However, inoculating plants with PGPR improved plant growth under sterile conditions. Taken together, our results suggest that genotypic variation is the dominant driver in HIPV composition and individual HIPV abundances, and any bacterial-mediated benefit is genotype and HIPV-specific. Therefore, understanding the interplay of these factors is necessary to fully harness microbially-mediated benefits and improve agricultural sustainability.

Flooding and Herbivory Interact to Alter Volatile Organic Compound Emissions in Two Maize Hybrids.

Flooding is a major plant abiotic stress factor that is frequently experienced by plants simultaneously with other biotic stresses, including herbivory. How plant volatile emissions, which mediate interactions with a wide range of organisms, are influenced by flooding and by multiple co-occurring stress factors remains largely unexplored. Using Spodoptera frugiperda (Lepidoptera: Noctuidae) (fall armyworm) as the insect pest and two maize (Zea mays, L. Poaceae) hybrids differentially marketed for conventional and organic production, we assessed the effects of flooding, herbivory, and both stress factors on the composition of blends of emitted volatiles. Headspace volatiles were collected from all treatment combinations seven days after flooding. We documented metrics indicative of biomass allocation to determine the effects of individual and combined stressors on plant growth. We also evaluated relationships between volatile emissions and indicators of soil chemical characteristics as influenced by treatment factors. Flooding and herbivory induced the emission of volatile organic compounds (VOCs) in similar ways on both maize hybrids, but the interaction of both stress factors produced significantly larger quantities of emitted volatiles. Thirty-eight volatile compounds were identified, including green leaf volatiles, monoterpenes, an aldehyde, a benzoate ester, sesquiterpenes, a diterpene alcohol, and alkane hydrocarbons. The hybrid marketed for organic production was a stronger VOC emitter. As expected, plant biomass was detrimentally affected by flooding. Soil chemical properties were less responsive to the treatment factors. Taken together, the results suggest that flooding stress and the interactions of flooding and insect attack can shape the emission of plant volatiles and further influence insect-plant interactions.

Differential regulation of cytochrome P450 genes associated with biosynthesis and detoxification in bifenthrin-resistant populations of navel orangewom (Amyelois transitella).

Pyrethroid resistance was first reported in 2013 for the navel orangeworm, Amyelois transitella, but the genetic underpinnings of pyrethroid resistance are unknown. We investigated the role of cytochrome P450 monooxygenases (P450s) belonging to the CYP3 and CYP4 clans using colonies derived from individuals collected in 2016 from almond orchards in two counties. One colony (ALM) originated from an almond orchard in Madera County with no reported pyrethroid resistance and the second colony (R347) originated from the same Kern County orchard where pyrethroid resistance was first reported. We used high-throughput quantitative real-time PCR (qRT-PCR) analyses of 65 P450s in the CYP3 and CYP4 clans of A. transitella to identify P450s induced by bifenthrin and associated with pyrethroid resistance. Nine P450s were constitutively overexpressed in R347 compared to ALM, including CYP6AE54 (11.7-fold), belonging to a subfamily associated with metabolic pesticide detoxification in Lepidoptera and CYP4G89 (33-fold) belonging to a subfamily associated with cuticular hydrocarbon (CHC) synthesis and resistance via reduced pesticide penetrance. Cuticular hydrocarbons analysis revealed that R347 produced twice as many total CHCs in the egg and adult stages as ALM. Topical toxicity bioassays for R347 determined that egg mortality was reduced at low bifenthrin concentrations and larval mortality was reduced at high concentrations of bifenthrin compared to ALM. Our discovery of both changes in metabolism and production of CHCs for R347 have implications for the possible decreased efficacy of other classes of insecticide used to control this insect. The threat of widespread pyrethroid resistance combined with the potential for cross-resistance to develop through the mechanism of reduced penetrance warrants developing management strategies that facilitate insecticide passage across the cuticle.

Author Correction: Factors Associated with Variation in Cuticular Hydrocarbon Profiles in the Navel Orangeworm, Amyelois transitella (Lepidoptera: Pyralidae).

The original version of this article unfortunately contained a mistake. When filing the final publication details, we failed to include the following statement in our Acknowledgments paragraph: We thank the Almond Board of California for research funding.

Factors Associated with Variation in Cuticular Hydrocarbon Profiles in the Navel Orangeworm, Amyelois transitella (Lepidoptera: Pyralidae).

Cuticular hydrocarbons (CHCs) are the main components of the epicuticular wax layer that in many insects functions as a barrier against desiccation. CHCs also play many other roles, including serving as sex pheromones, kairomones, primer pheromones, and colony-, caste-, species- and sex-recognition signals. In insects, CHC profiles can vary depending upon age, species, sex, and strain. Understanding factors associated with variation in hydrocarbon profiles is important for identifying potential vulnerabilities relating to pest ecology and life histories and for developing tools for pest monitoring and management strategies. In this study, we assessed potential sources of variation in CHC profiles in the navel orangeworm Amyelois transitella (Walker) (Lepidoptera: Pyralidae), an economically important pest of nut crops in California. Using coupled gas chromatography-mass spectrometry, we characterized and compared CHC profiles between adults of pyrethroid-resistant (R347) and susceptible (ALMOND) strains. We further compared CHC profiles from adults differing in age (1, 3, 5, and 7 d post-eclosion) and sex. Hydrocarbon profiles comprised 47 different CHCs in detectable quantities that ranged from C17 to C43 in chain length and included straight-chain alkanes and a variety of mono-, di-, and tri-methylalkanes. Adults from resistant populations had greater quantities of CHCs in total than those from susceptible strains, but relative quantities of individual components were similar. The six most abundant compounds were n-pentacosane, n-heptacosane, n-nonacosane, n-hentriacontane, 11,25 + 13,23 + 15,21-dimethylpentatriacontane, and 13,23 + 11,25 + 9,17-dimethylheptatriacontane. Post-eclosion, total CHCs increased with adult age, with males producing greater quantities than females at all ages. Our results show that CHC profiles vary depending on age, sex, and strain and suggest that CHC profiles may be useful as biomarkers to differentiate between insecticide- resistant and susceptible populations.

Topical and vapor toxicity of saturated fatty acids to the German cockroach (Dictyoptera: Blattellidae).

Topical and fumigant toxicity of saturated aliphatic fatty acids with chain lengths of C1 through C14 were determined against the German cockroach, Blattella germanica (L.). In the C1 to C11 series, topical toxicity (LD50 in milligram per adult male) ranged from 0.145 (C1) to 0.322 mg (C2). Toxicity declined dramatically with C12 and C14 acids whose LD50 values could not be calculated. The relative fumigation toxicity (LC50 in microliter per liter) of C1 through C5 acids was positively correlated with topical toxicity with values ranging from 6.159 (C3) to 12.302 microl/liter (C2). Fumigant toxicity decreased sharply with C6 (LC50 = 37.691 microl/liter) and there was no mortality of cockroaches exposed to vapors from C7 to C14 acids. The low fumigant toxicity of the C6 to C11 acids was correlated with their relatively low vapor pressure, but differences in diffusion of the vapors into the spiracles and subsequent passage to the target sites may have also been involved.

Electroantennogram (EAG) responses of Microplitis croceipes and Cotesia marginiventris and their lepidopteran hosts to a wide array of odor stimuli: correlation between EAG response and degree of host specificity?

In order to test whether the electroantennogram (EAG) response spectrum of an insect correlates to its degree of host specificity, we recorded EAG responses of two parasitoid species with different degrees of host specificity, Microplitis croceipes (specialist) and Cotesia marginiventris (generalist), to a wide array of odor stimuli including compounds representing green leaf volatiles (GLVs), herbivore-induced plant volatiles (HIPV), ecologically irrelevant (not used by the parasitoid species and their hosts for host location) plant volatiles, and host-specific odor stimuli (host sex pheromones, and extracts of host caterpillar body and frass). We also tested the EAG responses of female moths of the caterpillar hosts of the parasitoids, Heliothis virescens and Spodoptera exigua, to some of the odor stimuli. We hypothesized that the specialist parasitoid will have a narrower EAG response spectrum than the generalist, and that the two lepidopteran species, which are similar in their host plant use, will show similar EAG response spectra to plant volatiles. As predicted, the specialist parasitoid showed greater EAG responses than the generalist to host-specific odor and one HIPV (cis-3-hexenyl butyrate), whereas the generalist showed relatively greater EAG responses to the GLVs and unrelated plant volatiles. We detected no differences in the EAG responses of H. virescens and S. exigua to any of the tested odor.

Comparative GC-EAD responses of a specialist (Microplitis croceipes) and a generalist (Cotesia marginiventris) parasitoid to cotton volatiles induced by two caterpillar species.

Plants emit volatile blends that may be quantitatively and/or qualitatively different in response to attack by different herbivores. These differences may convey herbivore-specific information to parasitoids, and are predicted to play a role in mediating host specificity in specialist parasitoids. Here, we tested the above prediction by using as models two parasitoids (Hymenoptera: Braconidae) of cotton caterpillars with different degree of host specificity: Microplitis croceipes, a specialist parasitoid of Heliothis spp., and Cotesia marginiventris, a generalist parasitoid of caterpillars of several genera including Heliothis spp. and Spodoptera spp. We compared GC-EAD (coupled gas chromatography electroantennogram detection) responses of both parasitoid species to headspace volatiles of cotton plants damaged by H. virescens (a host species for both parasitoids) vs. S. exigua (a host species for C. marginiventris). Based on a recent study in which we reported differences in the EAG responses of both parasitoids to different types of host related volatiles, we hypothesized that M. croceipes (specialist) would show relatively greater GC-EAD responses to the herbivore-induced plant volatile (HIPV) components of cotton headspace, whereas C. marginiventris (generalist) would show greater response to the green leaf volatile (GLV) components. Thirty volatile components were emitted by cotton plants in response to feeding by either of the two caterpillars, however, 18 components were significantly elevated in the headspace of H. virescens damaged plants. Sixteen consistently elicited GC-EAD responses in both parasitoids. As predicted, C. marginiventris showed significantly greater GC-EAD responses than M. croceipes to most GLV components, whereas several HIPV components elicited comparatively greater responses in M. croceipes. These results suggest that differences in the ratios of identical volatile compounds between similar volatile blends may be used by specialist parasitoids to discriminate between host-plant and non-host-plant complexes.

Rhopalosiphum padi (Hemiptera: Aphididae) responses to volatile cues from Barley yellow dwarf virus-infected wheat.

In choice bioassays, Rhopalosiphum padi L. nonviruliferous apterae preferentially locate near volatile organic compounds (VOCs) emitted from Barley yellow dwarf virus (BYDV)-infected wheat plants compared with VOCs from noninfected plants. However, the specific VOCs responsible for R. padi responses are unknown. It is unclear also if R. padi responses to BYDV-infected wheat are caused by arrestment or attraction. Additionally, the responses of viruliferous apterae and nonviruliferous alate to BYDV-infected wheat have not been examined. R. padi responses were studied through emigration, immigration, and settling laboratory bioassays using BYDV-infected and noninfected wheat plants. Two wheat genotypes, virus-susceptible Lambert and virus-resistant Lambert-derived transgenic 103.1J expressing the BYDV-PAV coat protein gene, were evaluated. In a settling bioassay, alates preferentially settled on noninfected 103.1J. Responses of viruliferous and nonviruliferous R. padi to virus-infected, noninfected, and sham-inoculated (exposed to nonviruliferous aphids) Lambert and 103.1J were examined in separate bioassays. A paper leaf model served as a control. Immigration by viruliferous apterae was significantly lower toward the paper leaf model, but no significant differences were observed among plant treatments. Nonviruliferous apterae exhibited no significant differences in emigration among treatments, suggesting no arrestment occurred. Nonviruliferous apterae significantly preferred to immigrate toward BYDV-infected Lambert. Immigration toward the paper leaf model was significantly lower compared with plant treatments. Responses of R. padi to VOCs were tested by applying compounds to paper leaf models at concentrations designed to mimic those present in headspace of wheat plants. Nonviruliferous apterae immigrated in significantly greater numbers toward paper leaf models individually treated with nonanal, (Z)-3-hexenyl acetate, decanal, caryophyllene, and undecane than toward paper leaf models that served as controls and toward leaf models treated with synthetic blends made to mimic headspace of BYDV-infected compared with blends made to mimic headspace of noninfected wheat plants. Results suggest responses of R. padi to BYDV-infected plants are caused by attraction rather than arrestment.

Myzus persicae is arrested more by blends than by individual compounds elevated in headspace of PLRV-infected potato.

Volatiles from potato plants (Solanum tuberosum L.) infected with Potato leaf roll virus (PLRV) attract and arrest the principal vector of PLRV, the green peach aphid, Myzus persicae (Sulzer), more strongly than volatiles from non-infected plants. The total concentration of volatiles detectable in the headspace of PLRV-infected plants is greater than that in the headspace of non-infected controls, and the relative composition is altered. To determine the basis of the aphid response to PLRV-infection-induced volatiles from potato, behavioral bioassays were conducted. We measured arrestment of aphids by individual components, by synthetic blends of these, and by a naturally occurring blend by using an emigration rate bioassay, and quantified observations of the behavior of individual aphids. The components tested were those elevated at least twofold in response to PLRV infection. Before conducting the behavioral bioassays, electroantennograms confirmed the electrophysiological responses of aphids to the components of the blend. For bioassays, individual compounds or blends were tested by applying them in solution to paper strips at concentrations designed to mimic those present in the headspace of the plants. All bioassays were conducted by placing aphids on fine-mesh screening positioned above treated paper strips. Arrestment was measured by placing groups of 30 aphids directly over the treated strips and counting the number moving away at 10-min intervals for 50 min. Among the individual compounds tested, only beta-pinene was a mild arrestant. The other compounds did not elicit significant changes in arrestment or behavior at a range of physiologically relevant concentrations. In contrast, synthetic blends that mimicked the concentration and composition present in headspace of PLRV-infected potato plants arrested aphids significantly more strongly than blends mimicking volatiles from the headspace of non-infected plants. The naturally occurring blend collected from headspace of PLRV-infected plants also arrested M. persicae more strongly than the blend collected from headspace of non-infected plants. Aphid behavior was quantified by directly observing individual aphids and recording their activities during a 5-min period on screening above strips treated with test materials. Few differences in time budgets were observed among aphids exposed to individual components, but synthetic blends and trapped headspace volatiles from PLRV-infected plants resulted in significantly less time spent walking by aphids than synthetic blends and trapped headspace from non-infected controls. Our results indicate that arrestment of M. persicae by PLRV-infected plants requires the blend of volatile organic compounds released by these plants and is not produced in response to a single compound.