Many parasitoids lack adult fat accumulation, despite fatty acid synthesis: A discussion of concepts and considerations for future research.

Fat reserves, specifically the accumulation of triacylglycerols, are a major energy source and play a key role for life histories. Fat accumulation is a conserved metabolic pattern across most insects, yet in most parasitoid species adults do not gain fat mass, even when nutrients are readily available and provided ad libitum. This extraordinary physiological phenotype has evolved repeatedly in phylogenetically dispersed parasitoid species. This poses a conundrum because it could lead to significant constraints on energy allocation toward key adult functions such as survival and reproduction. Recent work on the underlying genetic and biochemical mechanisms has spurred a debate on fat accumulation versus fat production, because of incongruent interpretation of results obtained using different methodologies. This debate is in part due to semantics, highlighting the need for a synthetic perspective on fat accumulation that reconciles previous debates and provides new insights and terminology. In this paper, we propose updated, unambiguous terminology for future research in the field, including "fatty acid synthesis" and "lack of adult fat accumulation", and describe the distinct metabolic pathways involved in the complex process of lipogenesis. We then discuss the benefits and drawbacks of the main methods available to measure fatty acid synthesis and adult fat accumulation. Most importantly, gravimetric/colorimetric and isotope tracking methods give complementary information, provided that they are applied with appropriate controls and interpreted correctly. We also compiled a comprehensive list of fat accumulation studies performed during the last 25 years. We present avenues for future research that combine chemistry, ecology, and evolution into an integrative approach, which we think is needed to understand the dynamics of fat accumulation in parasitoids.

Deadly scents: Exposure to plant volatiles increases mortality of entomopathogenic nematodes during infection.

Plants attacked by insects commonly mobilize various defense mechanisms, including the biosynthesis and release of so-called herbivore-induced plant volatiles (HIPVs). Entomopathogenic nematodes (EPNs) can be attracted to these belowground HIPVs, which can enhance biocontrol services from EPNs. However, recent research has also demonstrated that HIPVs can induce and initiate insect immune responses, decreasing the insect's susceptibility to pathogens and parasites. Therefore, experiments were conducted to test the impact of HIPVs on insects and EPNs during the initial stage of EPN infection. Compounds that can impact EPN attraction and infectivity such as pregeijerene, ß-caryophyllene, and α-pinene, and compounds that have been determined to increase or decrease susceptibility of insects to pathogens, such as (Z)-3-hexenyl acetate, linalool, and ß-ocimene, were selected. Exposure of Galleria mellonella larvae to pregeijerene, linalool, ß-ocimene and α-pinene during invasion significantly increased mortality of Steinernema diaprepesi and Heterorhabditis bacteriophora after 48 h. Larval treatment with ß-caryophyllene only increased mortality for Heterorhabditis bacteriophora. (Z)-3-hexenyl acetate did not cause differential mortality from the controls for either nematode species. In additional experiments, we found that EPNs exposed to α-pinene and linalool were more readily recognized by the insects' immune cells compared to the control treatment, thus the observed increased mortality was likely due to HIPVs-EPN interactions with the insect's immune system. These results show that the presence of HIPVs can impact EPN survival in the model host, G. mellonella.

Production of the Green Leaf Volatile (Z)-3-Hexenal by a Zea mays Hydroperoxide Lyase.

Plant-produced volatile compounds play important roles in plant signaling and in the communication of plants with other organisms. Many plants emit green leaf volatiles (GLVs) in response to damage or attack, which serve to warn neighboring plants or attract predatory or parasitic insects to help defend against insect pests. GLVs include aldehydes, esters, and alcohols of 6-carbon compounds that are released rapidly following wounding. One GLV produced by maize (Zea mays) is the volatile (Z)-3-hexenal; this volatile is produced from the cleavage of (9Z,11E,15Z)-octadecatrienoic acid by hydroperoxide lyases (HPLs) of the cytochrome P450 CYP74B family. The specific HPL in maize involved in (Z)-3-hexenal production had not been determined. In this study, we used phylogenetics with known HPLs from other species to identify a candidate HPL from maize (ZmHPL). To test the ability of the putative HPL to produce (Z)-3-hexenal, we constitutively expressed the gene in Arabidopsis thaliana ecotype Columbia-0 that contains a natural loss-of-function mutant in AtHPL and examined the transgenic plants for restored (Z)-3-hexenal production. Volatile analysis of leaves from these transgenic plants showed that they did produce (Z)-3-hexenal, confirming that ZmHPL can produce (Z)-3-hexenal in vivo. Furthermore, we used gene expression analysis to show that expression of ZmHPL is induced in maize in response to both wounding and the insect pests Spodoptera frugiperda and Spodoptera exigua. Our study demonstrates that ZmHPL can produce GLVs and highlights its likely role in (Z)-3-hexenal production in response to mechanical damage and herbivory in maize.

Detecting the Conspecific: Herbivory-Induced Olfactory Cues in the Fall Armyworm (Lepidoptera: Noctuidae).

The fall armyworm (FAW), Spodoptera frugiperda (Smith), is a polyphagous pest whose larval feeding threatens several economically important crops worldwide with especially severe damage to corn (Zea mays L.). Field-derived resistance to several conventional pesticides and Bt toxins have threatened the efficacy of current management strategies, necessitating the development of alternative pest management methods and technologies. One possible avenue is the use of volatile organic compounds (VOCs) and other secondary metabolites that are produced and sequestered by plants as a response to larval feeding. The effects of conspecific larval feeding on fall armyworm oviposition preferences and larval fitness were examined using two-choice oviposition experiments, larval feeding trials, targeted metabolomics, and VOC analyses. There was a significant preference for oviposition on corn plants that lacked larval feeding damage, and larvae fed tissue from damaged plants exhibited reduced weights and head capsule widths. All larval feeding promoted significantly increased metabolite and VOC concentrations compared to corn plants without any feeding. Metabolite differences were driven primarily by linoleic acid (which is directly toxic to fall armyworm) and tricarboxylic acids. Several VOCs with significantly increased concentrations in damaged corn plants were known oviposition deterrents that warrant further investigation in an integrated pest management context.

Sampling of Volatiles in Closed Systems: A Controlled Comparison of Three Solventless Volatile Collection Methods.

Complex inter-organismal communication among plants, insects, and microbes in natural and agricultural ecological systems is typically governed by emitted and perceived semiochemicals. To understand and ultimately utilize the role of volatile semiochemicals in these interactions, headspace volatiles are routinely collected and analyzed. Numerous collection systems are available (e.g., static or dynamic; adsorption or absorption) where the choice of technique should be dependent upon the plant, insect, or microbial ecological system studied, the information sought, and the limitations of each method. Within these constraints, it remains necessary that each method detects and provides the accurate in situ, or in vitro, volatile profile of the studied system. Herein, we analyzed and compared the pros and cons of three solventless, thermal desorption systems (SPME, Tenax/cold trap, SPDE) using a synthetic standard blend of compounds mimicking a simple natural blend (benzaldehyde, b-caryophyllene, (Z)-3-hexenol, 6-methyl-5-hepten-2-one, and limonene). Direct splitless injection and Super Q collections of the standard blend were used as controls. The results indicated that related qualitative, as well as quantitative differences, could be correlated with adsorbent sampling capacity and structural bias. The results for Tenax/cold trap and SPDE also were affected by sampled headspace volumes. All solventless techniques exhibited high analytical reproducibility, with SPME and SPDE providing ease of use, low cost, and minimal instrument modifications. The more complex Tenax/cold trap technique provided higher collection efficiency. Using these results, we provide guidance for technique selection for chemical communication applications.

Using Chemical Ecology to Enhance Weed Biological Control.

In agricultural systems, chemical ecology and the use of semiochemicals have become critical components of integrated pest management. The categories of semiochemicals that have been used include sex pheromones, aggregation pheromones, and plant volatile compounds used as attractants as well as repellents. In contrast, semiochemicals are rarely utilized for management of insects used in weed biological control. Here, we advocate for the benefit of chemical ecology principles in the implementation of weed biocontrol by describing successful utilization of semiochemicals for release, monitoring and manipulation of weed biocontrol agent populations. The potential for more widespread adoption and successful implementation of semiochemicals justifies multidisciplinary collaborations and increased research on how semiochemicals and chemical ecology can enhance weed biocontrol programs.

Phenotypic plasticity explains apparent reverse evolution of fat synthesis in parasitic wasps.

Numerous cases of evolutionary trait loss and regain have been reported over the years. Here, we argue that such reverse evolution can also become apparent when trait expression is plastic in response to the environment. We tested this idea for the loss and regain of fat synthesis in parasitic wasps. We first show experimentally that the wasp Leptopilina heterotoma switches lipogenesis on in a fat-poor environment, and completely off in a fat-rich environment. Plasticity suggests that this species did not regain fat synthesis, but that it can be switched off in some environmental settings. We then compared DNA sequence variation and protein domains of several more distantly related parasitoid species thought to have lost lipogenesis, and found no evidence for non-functionality of key lipogenesis genes. This suggests that other parasitoids may also show plasticity of fat synthesis. Last, we used individual-based simulations to show that a switch for plastic expression can remain functional in the genome for thousands of generations, even if it is only used sporadically. The evolution of plasticity could thus also explain other examples of apparent reverse evolution.

Desorption Temperature, Solid-Phase Microextraction (SPME), and Natural Product Analyses, how Low Can we Go?

Solid phase microextraction (SPME) has become a common technique for volatile sampling due to its ease of use and limited technical requirements. The solvent-free nature of SPME is also exceptionally attractive for gas chromatography mass spectrometry (GC/MS) analysis. To ensure efficient transfer of the sample to the GC, the manufacturer recommend injector desorption temperatures in the range of 200 to 320 °C. A high desorption temperature can, however, have unwanted effects on analyses of plant and insect produced semiochemicals. By investigating the quantitative and qualitative chromatographic responses at varying inlet temperatures for a component blend of seven plant produced volatile compounds, we found the thermally labile plant-nematode signaling compound, pregeijerene to degrade to geijerene at all tested temperatures within the recommended range (200, 240, and 280 °C), but that it did not break down with an inlet temperature below 200 °C (100 °C and 150 °C). Degradation was also detected for the sesquiterpene germacrene D, but only at the highest inlet temperature tested (280 °C). Surprisingly, an inlet temperature of 200 °C gave the highest sample recovery, measured as total peak area while an inlet temperature of 100 °C as well as 280 °C gave the lowest total area values. An increase in desorption time from 3 to 5 min. Resulted in a recovery at 100 °C close to that obtained at 200 °C. Peak broadening was minimal, and only observed at the 100 °C inlet temperature. Based on these results, we highly recommend that SPME users include desorption temperature as one variable when developing sampling procedures for novel biological systems to ensure that potentially present thermally labile compounds are not degraded.

Metabolomics by UHPLC-HRMS reveals the impact of heat stress on pathogen-elicited immunity in maize.

INTRODUCTION: Studies investigating crop resistance to abiotic and biotic stress have largely focused on plant responses to singular forms of stress and individual biochemical pathways that only partially represent stress responses. Thus, combined abiotic and biotic stress treatments and the global assessment of their elicited metabolic expression remains largely unexplored. In this study, we employed targeted and untargeted metabolomics to investigate the molecular responses of maize (Zea mays) to abiotic, biotic, and combinatorial stress. OBJECTIVE: We compared the inducible metabolomes of heat-stressed (abiotic) and C. heterostrophus-infected (biotic) maize and examined the effects of heat stress on the ability of maize to defend itself against C. heterostrophus. METHODS: Ultra-high-performance liquid chromatography-high-resolution mass spectrometry was performed on plants grown under control conditions (28 °C), heat stress (38 °C), Cochliobolus heterostrophus infection, or combinatorial stress [heat (38 °C) + C. heterostrophus infection]. RESULTS: Multivariate analyses revealed differential metabolite expression between heat stress, C. heterostrophus infection, and their respective controls. In combinatorial experiments, treatment with heat stress prior to fungal inoculation negatively impacted maize disease resistance against C. heterostrophus, and distinct metabolome separation between combinatorial stressed plants and the non-heat-stressed infected controls was observed. Targeted analysis revealed inducible primary and secondary metabolite responses to abiotic/biotic stress, and combinatorial experiments indicated that deficiency in the hydroxycinnamic acid, p-coumaric acid, may contribute to the heat-induced susceptibility of maize to C. heterostrophus. CONCLUSION: These findings demonstrate that abiotic stress can predispose crops to more severe disease symptoms, underlining the increasing need to investigate defense chemistry in plants under combinatorial stress.

Setaria viridis as a model for translational genetic studies of jasmonic acid-related insect defenses in Zea mays.

Little is known regarding insect defense pathways in Setaria viridis (setaria), a model system for panicoid grasses, including Zea mays (maize). It is thus of interest to compare insect herbivory responses of setaria and maize. Here we use metabolic, phylogenetic, and gene expression analyses to measure a subset of jasmonic acid (JA)-related defense responses to leaf-chewing caterpillars. Phylogenetic comparisons of known defense-related maize genes were used to identify putative orthologs in setaria, and candidates were tested by quantitative PCR to determine transcriptional responses to insect challenge. Our findings show that while much of the core JA-related metabolic and genetic responses appear conserved between setaria and maize, production of downstream secondary metabolites such as benzoxazinoids and herbivore-induced plant volatiles are dissimilar. This diversity of chemical defenses and gene families involved in secondary metabolism among grasses presents new opportunities for cross species engineering. The high degree of genetic similarity and ease of orthologous gene identification between setaria and maize make setaria an excellent species for translational genetic studies, but the species specificity of downstream insect defense chemistry makes some pathways unamenable to cross-species comparisons.

Preferential infectivity of entomopathogenic nematodes in an envenomed host.

Entomopathogenic nematodes and parasitoid wasps are used as biological control agents for management of insect pests such as the Indian meal moth, Plodia interpunctella. The parasitoid wasp Habrobracon hebetor injects a paralytic venom into P. interpunctella larvae before laying eggs. A previous study reported that the entomopathogenic nematode Heterorhabditis indica preferentially infects P. interpunctella that have been envenomed by H. hebetor while results in this study showed a similar preference by the entomopathogenic nematode, Steinernema glaseri. We therefore tested four hypotheses for why nematode infection rates are higher in envenomed hosts: (1) elevated CO2 emission from envenomed hosts attracts nematodes, (2) paralysis prevents hosts from escaping nematodes, (3) volatile chemicals emitted from envenomed hosts attract nematodes and increase infection, and (4) reduced immune defenses in envenomed hosts increase nematode survival. Results showed that envenomed P. interpunctella larvae emitted lower amounts of CO2 than non-envenomed larvae. Physical immobilization of P. interpunctella larvae did not increase infection rates by S. glaseri but did increase infection rates by H. indica. Emissions from envenomed hosts were collected and analyzed by thermal desorption gas chromatography/mass spectrometry. The most abundant compound, 3-methyl-3-buten-1-ol, was found to be an effective cue for S. glaseri attraction and infection but was not an effective stimulus for H. indica. Envenomed P. interpunctella exhibited a stronger immune response toward nematodes than non-envenomed hosts. Altogether, we conclude that different mechanisms underlie preferential infection in the two nematode species: host immobilization for H. indica and chemical cues for S. glaseri.

Pheromone extracts act as boosters for entomopathogenic nematodes efficacy.

Inconsistency in entomopathogenic nematode (EPN) efficacy is still one of the biggest challenges for the wider adoption of EPNs as biocontrol agents. Previous studies demonstrated that extracts from EPN-infected hosts enhance dispersal and efficacy, two key factors in success of EPNs. Some active components in the insect host cadavers responsible for dispersal, ascarosides, have been identified as nematode pheromones. We hypothesized that pheromone extracts increase dispersal of EPN infective juveniles (IJs) leading to increased efficacy. First, we determined whether pheromone extracts improved IJ movement/dispersal in soil columns baited with Tenebrio molitor larvae. We found that pheromone extracts induced higher numbers of Steinernema carpocapsae and Steinernema feltiae IJs to move towards T. molitor larvae in the bottom of the column compared to IJs treated with infected cadaver macerate and water, positive and negative controls, respectively. Furthermore, the number of S. carpocapsae IJs that invaded T. molitor larvae was higher for the pheromone extract treatment than the controls. S. feltiae IJs that were pretreated with pheromone extracts and macerate (positive control) infected T. molitor at the same rate but invasion was superior to IJs that were treated with water. Consistent with the soil column tests, both S. carpocapsae and S. feltiae IJs treated with pheromone extracts performed better in killing larvae of two economically important insect larvae, pecan weevil, Curculio caryae, and black soldier fly, Hermetia illucens, in greenhouse tests compared to IJs treated with water. We demonstrated pheromone-mediated behavioral manipulation of a biological control agent to enhance pest control potential. Conceivably, nematodes can be exposed to efficacy-enhancing pheromones prior to field application.

Electrophysiological and Behavioral Responses of the Kudzu Bug, Megacopta cribraria (Hemiptera: Plataspidae), to Volatile Compounds from Kudzu and Soybean Plants.

The kudzu bug, Megacopta cribraria, is a key pest of soybean in the United States. Electrophysiological and behavioral responses of adult M. cribraria to kudzu and soybean volatile compounds were examined to identify semiochemicals used for host location. Headspace volatiles collected from undamaged potted plants were analyzed by gas chromatography with electroantennographic detection (GC-EAD). Subsequently, six GC-EAD-active compounds were identified by gas chromatography-mass spectrometry (GC-MS). These six compounds, along with some previously reported insect attractants, were selected for electroantennogram (EAG) assays. The four chemicals that elicited the strongest EAG responses, 1-octen-3-ol, nonanal, benzaldehyde, and ocimene, were selected for evaluation in olfactometer bioassays. Both benzaldehyde and 1-octen-3-ol exhibited dose-dependent responses at higher concentration. Our results provide insight into host location compounds used by adult M. cribraria. These results may be evaluated in future field tests and ultimately useful to develop a semiochemical-based monitoring technique and integrated pest management program for M. cribraria.

Infected host macerate enhances entomopathogenic nematode movement towards hosts and infectivity in a soil profile.

Vertical dispersal and infectivity of the infective juveniles (IJs) of three entomopathogenic nematodes (EPNs), Steinernema carpocapsae, S. feltiae, and Heterorhabditis bacteriophora, were tested in the presence or absence of cadaver macerate of nematode-infected Galleria mellonella. Infected host macerate applied on the top of column surface induced higher numbers of IJs to move to the bottom of the column for all three species, indicating a dispersal-enhancing effect of host cadaver on IJs. Among the three EPNs, H. bacteriophora was the most responsive to host macerate, followed by S. feltiae, and S. carpocapsae was the least. Also, more IJs of H. bacteriophora invaded Tenebrio molitor hosts at the bottom of soil columns in the presence of host macerate compared with the treatment without cadaver macerate. These findings suggest enhanced dispersal and/or infectivity of all three EPNs may be leveraged toward superior biocontrol efficacy.

Risk taking of educated nematodes.

Nematode parasites rely on successful host infection to perpetuate their species. Infection by individual nematode parasites can be risky, however; any one individual could be killed by the host's immune response. Here we use a model system to show that environmental cues and parasite past experience can be used by entomopathogenic nematodes to reduce individual risk of infection. Past parasite experience can more than double the infective virulence (number of host invaders) of a given cohort of entomopathogenic nematode parasites. This plasticity in individual parasite risk-taking and associated infection can be used to manage infection of parasitic nematodes: enhancing biological control with entomopathogenic nematodes and developing behavioral and chemical strategies to reduce infection by vertebrate and plant parasitic nematodes.

Interactions Among Plants, Insects, and Microbes: Elucidation of Inter-Organismal Chemical Communications in Agricultural Ecology.

The last 2 decades have witnessed a sustained increase in the study of plant-emitted volatiles and their role in plant-insect, plant-microbe, and plant-plant interactions. While each of these binary systems involves complex chemical and biochemical processes between two organisms, the progression of increasing complexity of a ternary system (i.e., plant-insect-microbe), and the study of a ternary system requires nontrivial planning. This planning can include an experimental design that factors in potential overarching ecological interactions regarding the binary or ternary system, correctly identifying and understanding unexpected observations that may occur during the experiment and thorough interpretation of the resultant data. This challenge of planning, performing, and interpreting a plant's defensive response to multiple biotic stressors will be even greater when abiotic stressors (i.e., temperature or water) are factored into the system. To fully understand the system, we need to not only continue to investigate and understand the volatile profiles but also include and understand the biochemistry of the plant's response to these stressors. In this review, we provide examples and discuss interaction considerations with respect to how readers and future authors of the Journal of Agricultural and Food Chemistry can contribute their expertise toward the extraction and interpretation of chemical information exchanged between agricultural commodities and their associated pests. This holistic, multidisciplinary, and thoughtful approach to interactions of plants, insects, and microbes, and the resultant response of the plants can lead to a better understanding of agricultural ecology, in turn leading to practical and viable solutions to agricultural problems.

Role of Plant Volatiles in Host Plant Recognition by Listronotus maculicollis (Coleoptera: Curculionidae).

The annual bluegrass weevil (ABW), Listronotus maculicollis Kirby, is an economically important pest of short cut turfgrass. Annual bluegrass, Poa annua L., is the most preferred and suitable host for ABW oviposition, larval survival and development. We investigated the involvement of grass volatiles in ABW host plant preference under laboratory and field conditions. First, ovipositional and feeding preferences of ABW adults were studied in a sensory deprivation experiment. Clear evidence of involvement of olfaction in host recognition by ABW was demonstrated. Poa annua was preferred for oviposition over three bentgrasses, Agrostis spp., but weevils with blocked antennae did not exhibit significant preferences. ABW behavioral responses to volatiles emitted by Agrostis spp. and P. annua were examined in Y-tube olfactometer assays. Poa annua was attractive to ABW females and preferred to Agrostis spp. cultivars in Y-tube assays. Headspace volatiles emitted by P. annua and four cultivars of Agrostis stolonifera L. and two each of A. capillaris L. and A. canina L. were extracted, identified and compared. No P. annua specific volatiles were found, but Agrostis spp. tended to have larger quantities of terpenoids than P. annua. (Z)-3-hexenyl acetate, phenyl ethyl alcohol and their combination were the most attractive compounds to ABW females in laboratory Y-tube assays. The combination of these compounds as a trap bait in field experiments attracted adults during the spring migration, but was ineffective once the adults were on the short-mown turfgrass. Hence, their usefulness for monitoring weevil populations needs further investigation.

Herbivore-derived fatty-acid amides elicit reactive oxygen species burst in plants.

Reactive oxygen species (ROS) can be elicited by many forms of stress, including pathogen attack, abiotic stress, damage and insect infestation. Perception of microbe- or damage-associated elicitors triggers an ROS burst in many plant species; however, the impact of herbivore fatty-acid amides on ROS elicitation remains largely unexplored. In this study we show that the lepidopteran-derived fatty-acid amide elicitor N-linolenoyl-L-glutamine (GLN18:3) can induce a ROS burst in multiple plant species. Furthermore, in Arabidopsis this ROS burst is partially dependent on the plasma membrane localized NADPH oxidases RBOHD and RBOHF, and an Arabidopsis rbohD/F double mutant produces enhanced GLN18:3-induced jasmonic acid. Quantification of GLN18:3-induced ROS in phytohormone-deficient lines revealed that in Arabidopsis reduced levels of jasmonic acid resulted in a larger elicitor-induced ROS burst, while in tomato reduction of either jasmonic acid or salicylic acid led to higher induced ROS production. These data indicate that GLN18:3-induced ROS is antagonistic to jasmonic acid production in these species. In biological assays, rbohD/F mutant plants were more resistant to the generalist herbivores Spodoptera exigua and Trichoplusia ni but not to the specialist Plutella xylostella. Collectively, these results demonstrate that in Arabidopsis herbivore-induced ROS may negatively regulate plant defense responses to herbivory.

A Technique for Thermal Desorption Analyses Suitable for Thermally-Labile, Volatile Compounds.

Many plant and insect interactions are governed by odors released by the plants or insects and there exists a continual need for new or improved methods to collect and identify these odors. Our group has for some time studied below-ground, plant-produced volatile signals affecting nematode and insect behavior. The research requires repeated sampling of volatiles of intact plant/soil systems in the laboratory as well as the field with the help of probes to minimize unwanted effects on the systems we are studying. After evaluating solid adsorbent filters with solvent extraction or solid phase micro extraction fiber sample collection, we found dynamic sampling of small air volumes on Tenax TA filters followed by thermal desorption sample introduction to be the most suitable analytical technique for our applications. Here we present the development and evaluation of a low-cost and relatively simple thermal desorption technique where a cold trap cooled with liquid carbon dioxide is added as an integral part of a splitless injector. Temperature gradient-based focusing and low thermal mass minimizes aerosol formation and eliminates the need for flash heating, resulting in low sample degradation comparable to solvent-based on-column injections. Additionally, since the presence of the cold trap does not affect normal splitless injections, on-the-fly switching between splitless and thermal desorption modes can be used for external standard quantification.

Commercial hybrids and mutant genotypes reveal complex protective roles for inducible terpenoid defenses in maize.

Plant defense research is facilitated by the use of genome-sequenced inbred lines; however, a foundational knowledge of interactions in commercial hybrids remains relevant to understanding mechanisms present in crops. Using an array of commercial maize hybrids, we quantified the accumulation patterns of defense-related metabolites and phytohormones in tissues challenged with diverse fungal pathogens. Across hybrids, Southern leaf blight (Cochliobolus heterostrophus) strongly elicited specific sesqui- and diterpenoid defenses, namely zealexin A4 (ZA4) and kauralexin diacids, compared with the stalk-rotting agents Fusarium graminearum and Colletotrichum graminicola. With respect to biological activity, ZA4 and kauralexin diacids demonstrated potent antimicrobial action against F. graminearum. Unexpectedly, ZA4 displayed an opposite effect on C. graminicola by promoting growth. Overall, a negative correlation was observed between total analyzed terpenoids and fungal growth. Statistical analyses highlighted kauralexin A3 and abscisic acid as metabolites most associated with fungal suppression. As an empirical test, mutants of the ent-copalyl diphosphate synthase Anther ear 2 (An2) lacking kauralexin biosynthetic capacity displayed increased susceptibility to C. heterostrophus and Fusarium verticillioides. Our results highlight a widely occurring defensive function of acidic terpenoids in commercial hybrids and the complex nature of elicited pathway products that display selective activities on fungal pathogen species.