Specific herbivore-induced volatiles defend plants and determine insect community composition in the field

In response to insect attack, plants release complex blends of volatile compounds. These volatiles serve as foraging cues for herbivores, predators and parasitoids, leading to plant-mediated interactions within and between trophic levels. Hence, plant volatiles may be important determinants of insect community composition. To test this, we created rice lines that are impaired in the emission of two major signals, S-linalool and (E)-β-caryophyllene. We found that inducible S-linalool attracted predators and parasitoids as well as chewing herbivores, but repelled the rice brown planthopper Nilaparvata lugens, a major pest. The constitutively produced (E)-β-caryophyllene on the other hand attracted both parasitoids and planthoppers, resulting in an increased herbivore load. Thus, silencing either signal resulted in specific insect assemblages in the field, highlighting the importance of plant volatiles in determining insect community structures. Moreover, the results imply that the manipulation of volatile emissions in crops has great potential for the control of pest populations.

3-β-d-Glucopyranosyl-6-methoxy-2-benzoxazolinone (MBOA-N-Glc) is an insect detoxification product of maize 1,4-benzoxazin-3-ones

In order to defend themselves against arthropod herbivores, maize plants produce 1,4-benzoxazin-3-ones (BXs), which are stored as weakly active glucosides in the vacuole. Upon tissue disruption, BXs come into contact with β-glucosidases, resulting in the release of active aglycones and their breakdown products. While some aglycones can be reglucosylated by specialist herbivores, little is known about how they detoxify BX breakdown products. Here we report on the structure of an N-glucoside, 3-β-d-glucopyranosyl-6-methoxy-2-benzoxazolinone (MBOA-N-Glc), purified from Spodoptera frugiperda faeces. In vitro assays showed that MBOA-N-Glc is formed enzymatically in the insect gut using the BX breakdown product 6-methoxy-2-benzoxazolinone (MBOA) as precursor. While Spodoptera littoralis and S. frugiperda caterpillars readily glucosylated MBOA, larvae of the European corn borer Ostrinia nubilalis were hardly able to process the molecule. Accordingly, Spodoptera caterpillar growth was unaffected by the presence of MBOA, while O. nubilalis growth was reduced. We conclude that glucosylation of MBOA is an important detoxification mechanism that helps insects tolerate maize BXs.

Synergies and trade-offs between insect and pathogen resistance in maize leaves and roots

Determining links between plant defence strategies is important to understand plant evolution and to optimize crop breeding strategies. Although several examples of synergies and trade-offs between defence traits are known for plants that are under attack by multiple organisms, few studies have attempted to measure correlations of defensive strategies using specific single attackers. Such links are hard to detect in natural populations because they are inherently confounded by the evolutionary history of different ecotypes. We therefore used a range of 20 maize inbred lines with considerable differences in resistance traits to determine if correlations exist between leaf and root resistance against pathogens and insects. Aboveground resistance against insects was positively correlated with the plant's capacity to produce volatiles in response to insect attack. Resistance to herbivores and resistance to a pathogen, on the other hand, were negatively correlated. Our results also give first insights into the intraspecific variability of root volatiles release in maize and its positive correlation with leaf volatile production. We show that the breeding history of the different genotypes (dent versus flint) has influenced several defensive parameters. Taken together, our study demonstrates the importance of genetically determined synergies and trade-offs for plant resistance against insects and pathogens.

Induction and detoxification of maize 1,4-benzoxazin-3-ones by insect herbivores

In monocotyledonous plants, 1,4-benzoxazin-3-ones, also referred to as benzoxazinoids or hydroxamic acids, are one of the most important chemical barriers against herbivores. However, knowledge about their behavior after attack, mode of action and potential detoxification by specialized insects remains limited. We chose an innovative analytical approach to understand the role of maize 1,4-benzoxazin-3-ones in plant–insect interactions. By combining unbiased metabolomics screening and simultaneous measurements of living and digested plant tissue, we created a quantitative dynamic map of 1,4-benzoxazin-3-ones at the plant–insect interface. Hypotheses derived from this map were tested by specifically developed in vitro assays using purified 1,4-benzoxazin-3-ones and active extracts from mutant plants lacking 1,4-benzoxazin-3-ones. Our data show that maize plants possess a two-step defensive system that effectively fends off both the generalist Spodoptera littoralis and the specialist Spodoptera frugiperda. In the first step, upon insect attack, large quantities of 2-β-d-glucopyranosyloxy-4,7-dimethoxy-1,4-benzoxazin-3-one (HDMBOA-Glc) are formed. In the second step, after tissue disruption by the herbivores, highly unstable 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one (HDMBOA) is released by plant-derived β-glucosidases. HDMBOA acts as a strong deterrent to both S. littoralis and S. frugiperda. Although constitutively produced 1,4-benzoxazin-3-ones such as 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) are detoxified via glycosylation by the insects, no conjugation of HDMBOA in the insect gut was found, which may explain why even the specialist S. frugiperda has not evolved immunity against this plant defense. Taken together, our results show the benefit of using a plant–insect interface approach to elucidate plant defensive processes and unravel a potent resistance mechanism in maize.

The Role of Roots in Plant Defence

Roots play an important role for plant defence and resistance against pathogens and insect herbivores: They act as environmental sensors for space, nutrients and water, they are important biosynthetic sites of plant toxins, they can store assimilates for future regrowth, and they possess themselves a potent defensive system to fend off belowground attackers. Although roots are often seen as passive tissue that only delivers services to the rest of the plant, it is becoming increasingly evident that roots actively respond to environmental conditions and are a vital part of the plant’s signaling and perception machinery. This chapter summarizes what is known about roots as constituents of plant resistance and defense mechanisms, with a particular emphasis on signaling aspects. It also discusses how the increasing knowledge about roots can be used to help protect plants from harmful pests.

Effects of biotechnology on biodiversity: herbicide-tolerant and insect-resistant GM crops

Biodiversity is threatened by agriculture as a whole, and particularly also by traditional methods of agriculture. Knowledge-based agriculture, including GM crops, can reduce this threat in the future. The introduction of no-tillage practices, which are beneficial for soil fertility, has been encouraged by the rapid spread of herbicide-tolerant soybeans in the USA. The replacement of pesticides through Bt crops is advantageous for the non-target insect fauna in test-fields. The results of the British Farm Scale experiment are discussed. Biodiversity differences can mainly be referred to as differences in herbicide application management.

Sequestration of plant secondary metabolites by insect herbivores: molecular mechanisms and ecological consequences

Numerous insect herbivores can take up and store plant toxins as self-defense against their own natural enemies. Plant toxin sequestration is tightly linked with tolerance strategies that keep the toxins functional. Specific transporters have been identified that likely allow the herbivore to control the spatiotemporal dynamics of toxin accumulation. Certain herbivores furthermore possess specific enzymes to boost the bioactivity of the sequestered toxins. Ecologists have studied plant toxin sequestration for decades. The recently uncovered molecular mechanisms in combination with transient, non-transgenic systems to manipulate insect gene expression will help to understand the importance of toxin sequestration for food-web dynamics in nature.

Roots under attack: contrasting plant responses to below- and aboveground insect herbivory

The distinctive ecology of root herbivores, the complexity and diversity of root–microbe interactions, and the physical nature of the soil matrix mean that plant responses to root herbivory extrapolate poorly from our understanding of responses to aboveground herbivores. For example, root attack induces different changes in phytohormones to those in damaged leaves, including a lower but more potent burst of jasmonates in several plant species. Root secondary metabolite responses also differ markedly, although patterns between roots and shoots are harder to discern. Root defences must therefore be investigated in their own ecophysiological and evolutionary context, specifically one which incorporates root microbial symbionts and antagonists, if we are to better understand the battle between plants and their hidden herbivores.

Effects of genetic diversity of grass on insect species diversity at higher trophic levels are not due to cascading diversity effects

Genetic diversity in plant populations has been shown to affect the species diversity of insects. In grasses, infection with fungal endophytes can also have strong effects on insects, potentially modifying the effects of plant genetic diversity. We manipulated the genetic diversity and endophyte infection of a grass in a field experiment. We show that diversity of primary parasitoids (3rd trophic level) and, especially, secondary parasitoids (4th trophic level) increases with grass genetic diversity while there was no effect of endophyte infection. The increase in insect diversity appeared to be due to a complementarity effect rather than a sampling effect. The higher parasitoid diversity could not be explained by a cascading diversity effect because herbivore diversity was not affected and the same herbivore species were present in all treatments. The effects on the higher trophic levels must therefore be due to a direct response to plant traits or mediated by effects on traits at intermediate trophic levels.

Ecotoxicology of pesticides on natural enemies of olive groves. Potential of ecdysone agonists for controlling Bactrocera oleae (Rossi) (Diptera: Tephritidae)

Pesticide applications are still one of the most common control methods against the main olive grove pests and diseases: the olive fruit fly, Bactrocera oleae (Rossi), the olive moth, Prays oleae (Bernard), the black scale, Saissetia oleae (Olivier), and the olive leaf spot, caused by the fungus Spilocaea oleagina Fries. However, and because the new pesticide legislation is aimed at an integrated pest and disease management, it is still important to evaluate and to know the ecotoxicology of pesticides on the natural enemies of the different agrosystems. A part of this work has been focusses on evaluating the direct and indirect effects of kaolin particle films and two copper-based products (Bordeaux mixture and copper oxychloride) through different laboratory, extended laboratory and semi-field experiments. Two natural enemies have been chosen: Psyttalia concolor (Szèpligeti), a parasitoid of the olive fruit fly, and Chilocorus nigritus (F.), predator of Diaspididae. This predator has been used instead of C. bipustulatus (L.), which is the species found in olive orchards. Kaolin mainly acts as a repellent of insects and/or as an oviposition deterrent. It is used in olive groves to control the olive fruit fly and the olive moth. Copper is applied against fungal and bacterial diseases. In olive groves it is used against the olive leaf spot and other diseases. No statistical differences were found in any of the experiments performed, compared to the controls, except when the oral toxicity of the products was evaluated on P. concolor females. In this case, kaolin and copper oxychloride caused a higher mortality 72 hours after the treatments, and both kaolin and the two copper formulations decreased females’ life span. Reproductive parameters were only negatively affected when kaolin was ingested. Apart from these experiments, due to the uncommon mode of action of kaolin, two extra experiments were carried out: a dual choice and a no-choice experiment. In this case, both P. concolor females and C. nigritus adults showed a clear preference for the untreated surfaces when they had the possibility of choosing between a treated surface and an untreated one. When there was no choice, no statistical differences were found between the treatments and the controls. Furthermore, the efficacy and the selectivity of three insect growth regulators (methoxyfenozide, tebufenozide and RH-5849) on B. oleae and P. concolor, respectively, have also been evaluated. In addition to laboratory experiments to evaluate the toxicity of the insecticides, also molecular approaches were used. RNA of both insects was isolated. cDNA was subsequently synthesized and the complete sequences of the ligand biding domain (LBD) of the ecdysone receptor of each insect were then determined. Afterwards the three dimensional structures of both LBDs were constructed. Finally, the docking of the insecticide molecules in the cavity delineated by the 12 α-helix that composed the LBD was performed. Both toxicity assays and molecular docking approaches showed that either methoxyfenozide or tebufenozide had no negative effects nor on B. oleae nor on P. concolor. In contrast, RH-5849 had no deleterious effect to the parasitoid but decreased olive fruit fly adults’ life span, especially when they were in contact with the fresh residue of the insecticide applied on a glass surface. The docking study of RH-5849 molecule has shown a very light hindrance with the wall of the LBD pocket. This means that this molecule could more or less adjust in the cavity. Thus, searching of new insecticides for controlling the olive fruit fly could be based on the basic lead structure of RH-5849 molecule.

Spatio-temporal dynamics of viruses are differentially affected by parasitoids depending on the mode of transmission by their insect vectors.

Relationships between agents in multitrophic systems are complex and very specific. Insect-transmitted plant viruses are completely dependent on the behaviour and distribution patterns of their vectors. The presence of natural enemies may directly affect aphid behaviour and spread of plant viruses, as the escape response of aphids might cause a potential risk for virus dispersal. The spatio-temporal dynamics of Cucumber mosaic virus (CMV) and Cucurbit aphid-borne yellows virus (CABYV), transmitted by Aphis gossypii in a non-persistent and persistent manner, respectively, were evaluated at short and long term in the presence and absence of the aphid parasitoid, Aphidius colemani. SADIE methodology was used to study the distribution patterns of both the virus and its vector, and their degree of association. Results suggested that parasitoids promoted aphid dispersion at short term, which enhanced CMV spread, though consequences of parasitism suggest potential benefits for disease control at long term. Furthermore, A. colemani significantly limited the spread and incidence of the persistent virus CABYV at long term. The impact of aphid parasitoids on the dispersal of plant viruses with different transmission modes is discussed.

Insect growth regulators as potential insecticides to control olive fruit fly (Bactrocera oleae Rossi): insect toxicity bioassays and molecular docking approach

Olive fruit fly, Bactrocera oleae (Rossi), is a key pest in olive orchards, causing serious economic damage. To date, the pest has already developed resistance to the insecticides commonly applied to control it. Thus, in searching for new products for an accurate resistance management programme, targeting the ecdysone receptor (EcR)might provide alternative compounds for use in such programmes. RESULTS: Residual contact and oral exposure in the laboratory of B. oleae adults to the dibenzoylhydrazine-based compounds methoxyfenozide, tebufenozide and RH-5849 showed different results. Methoxyfenozide and tebufenozide did not provoke anynegative effectsontheadults,but RH-5849 killed98-100%of the treated insects15 days after treatment. Theligand-binding domain (LBD) of the EcR of B. oleae (BoEcR-LBD) was sequenced, and a homology protein model was constructed. Owing to a restricted extent of the ligand-binding cavity of the BoEcR-LBD, docking experiments with the three tested insecticides showed a severe steric clash in the case of methoxyfenozide and tebufenozide, while this was not the case with RH-5849. CONCLUSION: IGR molecules similar to the RH-5849 molecule, and different from methoxyfenozide and tebufenozide, might have potential in controlling this pest.

Control of insect vectors and plant viruses in protected crops by novel pyrethroid-treated nets

Long-lasting insecticide-treated nets (LLITNs) constitute a novel alternative that combines physical and chemical tactics to prevent insect access and the spread of insect-transmitted plant viruses in protected enclosures. This approach is based on a slow-release insecticide-treated net with large hole sizes that allow improved ventilation of greenhouses. The efficacy of a wide range of LLITNs was tested under laboratory conditions against Myzus persicae, Aphis gossypii and Bemisia tabaci. Two nets were selected for field tests under a high insect infestation pressure in the presence of plants infected with Cucumber mosaic virus and Cucurbit aphid-borne yellows virus. The efficacy of Aphidius colemani, a parasitoid commonly used for biological control of aphids, was studied in parallel field experiments.

Effects of a bifenthrin-treated net on the natural enemies Aphidius colemani (Haliday) and Adalia bipunctata L. in a cucumber crop in Central Spain. Semi-field experiments

Pest management practices that rely on pesticides are growing increasingly less effective and environmentally inappropriate in many cases and the search of alternatives is under focus nowadays. Exclusion of pests from the crop by means of pesticide-treated screens can be an eco-friendly method to protect crops, especially if pests are vectors of important diseases. The mesh size of nets is crucial to determine if insects can eventually cross the barrier or exclude them because there is a great variation in insect size depending on the species. Long-lasting insecticide-treated (LLITN) nets, factory pre-treated, have been used since years to fight against mosquitoes vector of malaria and are able to retain their biological efficacy under field for 3 years. In agriculture, treated nets with different insecticides have shown efficacy in controlling some insects and mites, so they seem to be a good tool in helping to solve some pest problems. However, treated nets must be carefully evaluated because can diminish air flow, increase temperature and humidity and decrease light transmission, which may affect plant growth, pests and natural enemies. As biological control is considered a key factor in IPM nowadays, the potential negative effects of treated nets on natural enemies need to be studied carefully. In this work, the effects of a bifentrhin-treated net (3 g/Kg) (supplied by the company Intelligent Insect Control, IIC) on natural enemies of aphids were tested on a cucumber crop in Central Spain in autumn 2011. The crop was sown in 8x6.5 m tunnels divided in 2 sealed compartments with control or treated nets, which were simple yellow netting with 25 mesh (10 x 10 threads/cm2; 1 x 1 mm hole size). Pieces of 2 m high of the treated-net were placed along the lateral sides of one of the two tunnel compartments in each of the 3 available tunnels (replicates); the rest was covered by a commercial untreated net of a similar mesh. The pest, Aphis gossypii Glover (Aphidae), the parasitoid Aphidius colemani (Haliday) (Braconidae) and the predator Adalia bipunctata L. (Coccinellidae) were artificially introduced in the crop. Weekly sampling was done determining the presence or absence of the pest and the natural enemies (NE) in the 42 plants/compartment as well as the number of insects in 11 marked plants. Environmental conditions (temperature, relative humidity, UV and PAR radiation) were recorded. Results show that when aphids were artificially released inside the tunnels, neither its number/plant nor their distribution was affected by the treated net. A lack of negative effect of the insecticide-treated net on natural enemies was also observed. Adalia bipunctata did not establish in the crop and only a short term control of aphids was observed one week after release. On the other hand, A. colemani did establish in the crop and a more long-term effect on the numbers of aphids/plant was detected irrespective of the type of net. KEY WORDS: bifenthrin-treated net, Adalia bipunctata, Aphidius colemani, Aphis gossypii, semi-field