Comparative transcriptomic analysis of chemoreceptors in two sympatric scarab beetles, Hylamorpha elegans and Brachysternus prasinus.

Chemoreception through odorant receptors (ORs), ionotropic receptors (IRs) and gustatory receptors (GRs) represents the functions of key proteins in the chemical ecology of insects. Recent studies have identified chemoreceptors in coleopterans, facilitating the evolutionary analysis of not only ORs but also IRs and GRs. Thus, Cerambycidae, Tenebrionidae and Curculionidae have received increased attention. However, knowledge of the chemoreceptors from Scarabaeidae is still limited, particularly for those that are sympatric. Considering the roles of chemoreceptors, this analysis could shed light on evolutionary processes in the context of sympatry. Therefore, the aim of this study was to identify and compare the repertoires of ORs, GRs and IRs between two sympatric scarab beetles, Hylamorpha elegans and Brachysternus prasinus. Here, construction of the antennal transcriptomes of both scarab beetle species and analyses of their phylogeny, molecular evolution and relative expression were performed. Thus, 119 new candidate chemoreceptors were identified for the first time, including 17 transcripts for B. prasinus (1 GR, 3 IRs and 13 ORs) and 102 for H. elegans (22 GRs, 14 IRs and 66 ORs). Orthologs between the two scarab beetle species were found, revealing specific expansions as well as absence in some clades. Purifying selection appears to have occurred on H. elegans and B. prasinus ORs. Further efforts will be focused on target identification to characterize kairomone and/or pheromone receptors.

A highly conserved plant volatile odorant receptor detects a sex pheromone component of the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae).

Odorant receptors (ORs) are key specialized units for mate and host finding in moths of the Ditrysia clade, to which 98% of the lepidopteran species belong. Moth ORs have evolved to respond to long unsaturated acetates, alcohols, or aldehydes (Type I sex pheromones), falling into conserved clades of pheromone receptors (PRs). These PRs might have evolved from old lineages of non-Ditrysian moths that use plant volatile-like pheromones. However, a Ditrysian moth called the greater wax moth, Galleria mellonella (a worldwide-distributed pest of beehives), uses C9-C11 saturated aldehydes as the main sex pheromone components (i.e., nonanal and undecanal). Thus, these aldehydes represent unusual components compared with the majority of moth species that use, for instance, Type I sex pheromones. Current evidence shows a lack of consensus in the amount of ORs for G. mellonella, although consistent in that the moth does not have conserved PRs. Using genomic data, 62 OR candidates were identified, 16 being new genes. Phylogeny showed no presence of ORs in conserved PR clades. However, an OR with the highest transcript abundance, GmelOR4, appeared in a conserved plant volatile-detecting clade. Functional findings from the HEK system showed the OR as sensitive to nonanal and 2-phenylacetaldehyde, but not to undecanal. It is believed that to date GmelOR4 represents the first, but likely not unique, OR with a stable function in detecting aldehydes that help maintain the life cycle of G. mellonella around honey bee colonies.

Identification and ligand binding of a chemosensory protein from sea louse Caligus rogercresseyi (Crustacea: Copepoda).

Caligus rogercresseyi is an ectoparasitic copepod that negatively affects the salmon farming industry, causing economic losses. To use phytochemicals as feed additives, or other chemicals that could elicit behavioral responses in C. rogercresseyi, the chemosensory recognition process is crucial. Therefore, to establish how C. rogercresseyi recognizes glucosinolates and their derivates isothiocyanates, a chemosensory protein (CSP) described as specific carrier of these chemicals in sea louse (CrogCSP) was identified in this study. The recombinant CSP and its selectivity against different chemical compounds was tested by fluorescence binding assays. Phylogenetic analysis revealed a close relationship among CrogCSP and other reported CSPs. Our results indicate that phenyl isothiocyanate and isophorone exhibited dissociation constants of 4.17 and 4.28 µM of Ki, respectively, indicating affinity over other chemicals, such as fatty acids and sinigrin. Structural findings suggest a unique binding site capable of accept several types of chemicals, similar to what has been reported for crystallized insect CSPs. Finally, this study lays the foundation for a deeper understanding of CSPs in crustaceans and especially in C. rogercresseyi. Likewise, the identification of chemosensory proteins could serve as the first step towards novel semiochemicals discovery to being applied in the sea louse controlling.

Arbuscular mycorrhizal fungi enhance monoterpene production in red clover (Trifolium pratense L.): a potential tool for pest control.

The possibility of modifying terpene production in plants is a defensive strategy that has been studied in conjunction with their biosynthetic pathways. A biotic factor such as Arbuscular Mycorrhizal Fungi (AMF) could modify terpene production in Trifolium pratense L. In this work, the enzymatic production of monoterpenes in Superqueli INIA cultivar with two AMF was evaluated via HeadSpace-Gas Chromatography (HS-GC). A significant increase of (S)-limonene was found in plants inoculated with Claroideoglomus claroideum as well as with the AMF mix (genera Scutellospora, Acaulospora and Glomus). Moreover, significant increases in other monoterpenes such as (-)-ß-pinene, myrcene, linalool, were observed. Results showed higher monoterpene production capacities in the Superqueli-INIA cultivar, suggesting the participation of monoterpene synthases (MTS). The significant rise of (S)-limonene in red clover plants inoculated with AMF suggests this strategy could be implemented in an agronomical manage for controlling the H. obscurus, the primary pest.

Characterization of Two Aldehyde Oxidases from the Greater Wax Moth, Galleria mellonella Linnaeus. (Lepidoptera: Pyralidae) with Potential Role as Odorant-Degrading Enzymes.

Odorant-degrading enzymes (ODEs) are proposed to degrade/inactivate volatile organic compounds (VOCs) on a millisecond timescale. Thus, ODEs play an important role in the insect olfactory system as a reset mechanism. The inhibition of these enzymes could incapacitate the olfactory system and, consequently, disrupt chemical communication, promoting and complementing the integrated pest management strategies. Here, we report two novel aldehyde oxidases, AOX-encoding genes GmelAOX2 and GmelAOX3, though transcriptomic analysis in the greater wax moth, Galleria mellonella. GmelAOX2 was clustered in a clade with ODE function, according to phylogenetic analysis. Likewise, to unravel the profile of volatiles that G. mellonella might face besides the sex pheromone blend, VOCs were trapped from honeycombs and the identification was made by gas chromatography-mass spectrometry. Semi-quantitative RT-PCR showed that GmelAXO2 has a sex-biased expression, and qRT-PCR indicated that both GmelAOX2 and GmelAOX3 have a higher relative expression in male antennae rather than female antennae. A functional assay revealed that antennal extracts had the strongest enzymatic activity against undecanal (4-fold) compared to benzaldehyde (control). Our data suggest that these enzymes have a crucial role in metabolizing sex pheromone compounds as well as plant-derived aldehydes, which are related to honeycombs and the life cycle of G. mellonella.

Analysis of glutathione-S-transferases from larvae of Galleria mellonella (Lepidoptera, Pyralidae) with potential alkaloid detoxification function.

The greater wax moth, Galleria mellonella, is a global pest for beehives, doing damage in the larval stage. Although a significant number of studies have reported on larvae and adults, to date no effective pest control has been implemented. In this study, we tested larval resistance to alkaloids from Berberis microphylla, and the objective was to identify enzymes that participate in alkaloid detoxification through enzymatic assays, bioinformatics analysis and qRT-PCR. Findings suggest glutathione-S-transferases (GSTs), from an increased metabolic mechanism, are responsible for alkaloid detoxification rather than cytochrome P450 (CYP), carboxylesterases (CarE). A bioinformatics analysis from transcriptome data revealed 22 GSTs present in both G. mellonella larvae and adults. The qRT-PCR experiments corroborated the presence of the 22 GSTs in larvae, where GST8 and GST20 stood out with the highest expression after berberine treatment. Structural information around GST8 and GST20 suggests that GST8 could bind berberine stronger than GST20. These findings represent an important advance in the study of detoxification enzymes in G. mellonella, expanding the role of delta-class GSTs towards alkaloids. Likewise, GST inhibition by alkaloid analogs is proposed in the framework of integrated pest management strategies.

Insights Into Chemosensory Proteins From Non-Model Insects: Advances and Perspectives in the Context of Pest Management.

Nowadays, insect chemosensation represents a key aspect of integrated pest management in the Anthropocene epoch. Olfaction-related proteins have been the focus of studies due to their function in vital processes, such ashost finding and reproduction behavior. Hence, most research has been based on the study of model insects, namely Drosophila melanogaster, Bombyx mori or Tribolium castaneum. Over the passage of time and the advance of new molecular techniques, insects considered non-models have been studied, contributing greatly to the knowledge of insect olfactory systems and enhanced pest control methods. In this review, a reference point for non-model insects is proposed and the concept of model and non-model insects is discussed. Likewise, it summarizes and discusses the progress and contribution in the olfaction field of both model and non-model insects considered pests in agriculture.

Optimization of enzymatic parameters for the production of formononetin from red clover (Trifolium pratense L.) through a response surface methodology.

Trifolium pratense, is a forage found worldwide, but it is negatively impacted by the clover root borer, Hylastinus obscurus. Methanolic extraction has been reported for isolating formononetin from vegetal tissues, with an antifeeding effect on H. obscurus. However, this methodology is time-consuming and also extracts other secondary metabolites, whereas enzymatic assays can provide higher specificity. Hence, the objective of this work was to determine the optimal conditions in pH, temperature, and incubation time for the activity of isoflavone synthase via a response surface model. Once these parameters were optimized, the concentration of formononetin in cultivars and experimental lines of T. pratense was evaluated enzymatically. The results showed that the best condition for developing the enzymatic assay was pH 9.1 with an incubation at 34.5 °C for 155 min. The formononetin content fluctuated between 0.74 and 1.96 mg/g of fresh weight, where Precoz-3, Precoz-1, and Superqueli-INIA presented the highest production.

Acetylcholinesterase inhibitory activity from Amaryllis belladonna growing in Chile: enzymatic and molecular docking studies.

Alkaloid profiles from Amaryllis belladonna plants collected in Chile were examined by GC-MS to assess their inhibitory activity on acetylcholinesterase (AChE) using in vitro and in silico methodologies. The alkaloid extract was roughly separated by column chromatography on silica gel. AChE inhibitory activities from extracts and purified alkaloids were tested by the Ellman method and a molecular docking study was performed to assess the interaction between AChE and purified alkaloids. Sixteen alkaloids were found from hexane and chloroform extracts, and three were isolated and identified as buphanidrine, acetylcaranine and lycorine. Chloroform extract showed the greatest AChE inhibitory activity with IC50 value 8.89 µg/mL, whereas buphanidrine exhibited the highest inhibitory activity, with IC50 value 17.56 µg/mL. Inhibition kinetics showed that buphanidrine acts as a mixed inhibitor and molecular docking supports this inhibition mechanism. Overall, our study supports the potential use of A. belladonna as an alkaloid source with AChE inhibitory activity.[Formula: see text].

An Overview of Antennal Esterases in Lepidoptera.

Lepidoptera are used as a model for the study of insect olfactory proteins. Among them, odorant degrading enzymes (ODEs), that degrade odorant molecules to maintain the sensitivity of antennae, have received less attention. In particular, antennal esterases (AEs; responsible for ester degradation) are crucial for intraspecific communication in Lepidoptera. Currently, transcriptomic and genomic studies have provided AEs in several species. However, efforts in gene annotation, classification, and functional assignment are still lacking. Therefore, we propose to combine evidence at evolutionary, structural, and functional level to update ODEs as well as key information into an easier classification, particularly of AEs. Finally, the kinetic parameters for putative inhibition of ODEs are discussed in terms of its role in future integrated pest management (IPM) strategies.

Galanthamine and other Amaryllidaceae related alkaloids are inhibitors of α7, α4β2 and α3β4 nicotinic acetylcholine receptors

Abstract Galanthamine is an Amaryllidaceae-derived acetylcholinesterase inhibitor used to treat memory impairment in Alzheimer's disease and vascular dementia. There is evidence that galanthamine, in addition to its effects on acetylcholinesterase, may enhance or inhibit brain nicotinic acetylcholine receptors, which could increase or decrease the therapeutic efficacy of galanthamine, respectively. Here, we evaluated the effects of galanthamine and two others Amaryllidaceae acetylcholinesterase inhibitors (haemanthamine and tazettine) analyzed by gas chromatography-mass spectrometry and identified by comparing their mass fragmentation patterns with literature and database NIST vs.2.0 on the agonist responses of brain nicotinic acetylcholine receptors α7, α3β4, (α4)2(β2)3 and (α4)3(β2)2. Using nicotinic acetylcholine receptors expressed heterologously in Xenopus oocytes, in conjunction with two-electrode voltage clamping, we found that galanthamine inhibits the function of nicotinic acetylcholine receptors assayed through a mix competitive and non-competitevely. Nicotinic acetylcholine receptor α7 were significantly more sensitive to inhibition (17 ± 0.6 µM) than the heteromeric receptor, α3β4 (90 ± 3.4 µM). Neither haemanthamine nor tazettine were more potent than galanthamine.

Structural investigation of selective binding dynamics for the pheromone-binding protein 1 of the grapevine moth, Lobesia botrana.

The European grapevine moth, Lobesia botrana (Denis & Schiffermüller), is a serious pest in vineyards in North and South America. Mating disruption techniques have been used to control and monitor L. botrana on the basis of its sexual communication. This needs a well-tuned olfactory system, in which it is believed that pheromone-binding proteins (PBPs) are key players that transport pheromones in the antennae of moths. In this study, the selectivity of a PBP, named as LbotPBP1, was tested by fluorescence binding assays against 11 sex pheromone components and 6 host plant volatiles. In addition, its binding mechanism was predicted on the basis of structural analyses by molecular docking and complex and steered molecular dynamics (SMD). Our results indicate that LbotPBP1 binds selectively to sex pheromone components over certain host plant volatiles, according to both in vitro and in silico tests. Thus, chain length (14 carbon atoms) and functional groups (i.e., alcohol and ester) appear to be key features for stable binding. Likewise, residues such as Phe12, Phe36, and Phe118 could participate in unspecific binding processes, whilst Ser9, Ser56, and Trp114 could participate in the specific recognition and stabilization of sex pheromones instead of host plant volatiles. Moreover, our SMD approach supported 11-dodecenyl acetate as the best ligand for LbotPBP1. Overall, the dynamics simulations, contact frequency analysis and SMD shed light on the binding mechanism of LbotPBP1 and could overcome the imprecision of molecular docking, supporting the in vitro binding assays. Finally, the role of LbotPBP1 in the chemical ecology of L. botrana is discussed.

Differential formononetin content in cultivars and experimental lines of red clover (Trifolium pratense L. ) plants affect the feeding behaviour of Hylastinus obscurus (Coleoptera: Curculionidae) / Contenido diferencial de formononetina en cultivares y líneas experimentales de plantas de trébol rosado (Trifolium pratense L. ) afecta la conducta alimenticia de Hylastinus obscurus (Coleoptera: Curculionidae)

Red clover (Trifolium pratense L.) is a perennial plant widely used as a forage resource for several animals. This plant is the exclusive host of Hylastinus obscurus (Marsham) which causes irreparable damages to the root system affecting their persistence. It has been reported that the presence of the isoflavonoid formononetin in roots of red clover could act as an antifeedant on H. obscurus. There are not studies related to the formononetin content in red clover roots to the antifeedant effect elicited by experimental lines and cultivar of red clover. Six red clover genotypes were investigated in both formononetin content and their respective antifeedant action. The results showed to Sabtoron High and Superqueli-INIA with both the highest formononetin content in red clover roots and antifeedant effect, allowing to suggest that this secondary metabolites could be used as a chemical factor for red clover plants. Moreover, a rapid methodology for searching red clover genotypes with high formononetin content is reported.

El trébol rosado (Trifolium pratense L.) es una planta perenne ampliamente utilizada como fuente de forraje de variados animales. Esta planta es el exclusivo hospedero de Hylastinus obscurus (Marsham) el cual causa irreparables daños al sistema radical afectando seriamente su persistencia. Se ha reportado que la presencia del isoflavonoide formononetina en raíces del trébol rosado podría actuar como antialimentario sobre H. obscurus. Actualmente no existen estudios que relacionen el contenido de formononetina en raíces de trébol rosado con el efecto antialimentario elicitado por líneas experimentales y cultivares de trébol rosado. Seis genotipos de esta leguminosa fueron evaluados en cuanto a su contenido de formononetina y actividad antialimentaria. Los resultados mostraron que los cultivares Sabtoron High y Superqueli-INIA presentaron altos niveles de formononetina en sus raíces y efecto antialimentario sobre H. obscurus, lo que permite sugerir que este metabolito secundario podría ser usado como factor químico para incrementar la persistencia de plantas de trébol rosado. Además, se informa una metodología rápida para la búsqueda de genotipos con altos contenidos de formononetina.

Analysis of the grapevine moth Lobesia botrana antennal transcriptome and expression of odorant-binding and chemosensory proteins.

The grapevine moth, Lobesia botrana, is considered a harmful pest for vineyards in Chile as well as in North America and Europe. Currently, monitoring and control methods of L. botrana are based on its main sex pheromone component, being effective for low population densities. In order to improve control methods, antennal olfactory proteins in moths, such as odorant-binding proteins (OBPs) and odorant receptors (ORs) have been studied as promising targets for the discovery of new potent semiochemicals, which have not been reported for L. botrana. Therefore, the objective of this study was to identify the repertoire of proteins related to chemoreception in L. botrana by antennal transcriptome and analyze the relative expression of OBPs and CSPs in male and female antennae. Through next-generation sequencing of the antennal transcriptome by Ilumina HiSeq2500 we identified a total of 118 chemoreceptors, from which 61, 42 and 15 transcripts are related to ORs, ionotropic receptors (IRs) and gustatory receptors (GRs), respectively. Furthermore, RNA-Seq data revealed 35 transcripts for OBPs and 18 for chemosensory proteins (CSPs). Analysis by qRT-PCR showed 20 OBPs significantly expressed in female antennae, while 5 were more expressed in males. Similarly, most of the CSPs were significantly expressed in female than male antennae. All the olfactory-related sequences were compared with homologs and their phylogenetic relationships elucidated. Finally, our findings in relation to the improvement of L. botrana management are discussed.

Rhodolirium andicola: a new renewable source of alkaloids with acetylcholinesterase inhibitory activity, a study from nature to molecular docking

ABSTRACT Acetylcholinesterase is an important target for control of neurodegenerative diseases causing cholinergic signaling deficit. Traditionally, galanthamine has been used as an Amaryllidaceae-derived acetylcholinesterase inhibitor, although new Amaryllidaceae plants could serve as source for better acetylcholinesterase inhibitors. Therefore, the objective of this study was to characterize the alkaloid composition from bulbs of Rhodolirium andicola (Poepp.) Traub, a native Chilean Amaryllidaceae specie, and assess their inhibitory activity on acetylcholinesterase by in vitro and in silico methodologies. Alkaloidal extracts from R. andicola exhibited an inhibitory activity with IC50 values between 11.25 ± 0.04 and 57.78 ± 1.92 µg/ml that included isolated alkaloid, galanthamine (2.3 ± 0.18 µg/ml), Additionally, 12 alkaloids were detected using gas chromatography-mass spectrometry and identified by comparing their mass fragmentation patterns with literature and database NIST vs.2.0. To better understand the bioactivity of isolated compounds and alkaloidal extracts against acetylcholinesterase, a molecular docking approach was performed. Results suggested that alkaloids such as lycoramine, norpluvine diacetate and 6α-deoxy-tazettine expand the list of potential acetylcholinesterase inhibitors to not only galanthamine. The role of R. andicola as a source for acetylcholinesterase inhibitors is further discussed in this study.

Copper immobilization by biochar and microbial community abundance in metal-contaminated soils.

Biochar (BC) is gaining attention as a soil amendment that can remediate metal polluted soils. The simultaneous effects of BC on copper (Cu) mobility, microbial activities in soil using metallophytes have scarcely been addressed. The objective of this study was to evaluate the effects of biochar BCs on Cu immobilization and over soil microbial communities in a Cu-contaminated soil evaluated over a two-year trial. A Cu-contaminated soil (338mgkg-1) was incubated with chicken manure biochar (CMB) or oat hull biochar (OHB) at rates of 1 and 5% w/w. Metallophyte Oenothera picensis was grown over one season (six months). The above process was repeated for 3 more consecutive seasons using the same soils. The BCs increased the soil pH and decreased the Cu exchangeable fraction Cu by 5 and 10 times (for OHB and CMB, respectively) by increasing the Cu bound in organic matter and residual fractions, and its effects were consistent across all seasons evaluated. BCs provided favorable habitat for microorganisms that was evident in increased microbial activity. The DHA activity was increased in all BC treatments, reaching a maximum of 7 and 6 times higher than control soils in CMB and OHB. Similar results were observed in microbial respiration, which increased 53% in OHB and 61% in CMB with respect to control. The BCs produced changes in microbial communities in all seasons evaluated. The fungal and bacterial richness were increased by CMB and OHB treatments; however, no clear effects were observed in the microbial diversity estimators. The physiochemical and microbiological effects produced by BC result in an increase of plant biomass production, which was on average 3 times higher than control treatments. However, despite being a metallophyte, O. picensis did not uptake Cu efficiently. Root and shoot Cu concentrations decreased or changed insignificantly in most BC treatments.

Combined effect of enzyme inducers and nitrate on selective lignin degradation in wheat straw by Ganoderma lobatum.

Lignin is one of the main barriers to obtaining added-value products from cellulosic fraction of lignocellulosic biomass due to its random aromatic structure and strong association with cellulose and hemicellulose. Inorganic and organic compounds have been used as enzyme inducers to increase the ligninolytic potential of white-rot fungi, without considering their effect on the selectivity of degradation. In this study, the selective lignin degradation in wheat straw by Ganoderma lobatum was optimized using a central composite design to evaluate the combined effect of Fe2+ and Mn2+ as inducers of ligninolytic enzymes and NO3- as an additional nitrogen source. Selective lignin degradation was promoted to maximize lignin degradation and minimize weight losses. The optimal conditions were 0.18 M NO3-, 0.73 mM Fe2+, and 1 mM Mn2+, which resulted in 50.0% lignin degradation and 18.5% weight loss after 40 days of fungal treatment. A decrease in absorbance at 1505 and 900 cm-1 in fungal-treated samples was observed in the FTIR spectra, indicating lignin and cellulose degradation in fungal-treated wheat straw, respectively. The main ligninolytic enzymes detected during lignin degradation were manganese-dependent and manganese-independent peroxidases. Additionally, confocal laser scanning microscopy revealed that lignin degradation in wheat straw by G. lobatum resulted in higher cellulose accessibility. We concluded that the addition of enzyme inducers and NO3- promotes selective lignin degradation in wheat straw by G. lobatum.

Volatiles emitted by Bacillus sp. BCT9 act as growth modulating agents on Lactuca sativa seedlings.

Chemical products are applied during horticulture to increase food production, but the environmental problems resulting from these applications have led to a search for more sustainable products. Volatile organic compounds (VOCs) demonstrating plant growth promoter (PGP) activity released by bacterial species have emerged as alternatives, but their effects on Lactuca sativa growth are unknown. In this study, VOCs released by Bacillus sp. BCT9 cultures grown in different media (Methyl Red & Voges Proskauer, Murashige & Skoog and nutrient media) at concentrations of 0.1, 0.2, 0.5 and 0.7 (measured as the absorbance, λ=600nm) were tested to evaluate their activity as growth inducers of L. sativa after 10days of exposure. Lower concentrations of BCT9 increased root length, and higher concentrations induced shoot length and lateral root length. The dry weight and number of lateral roots increased similarly, independent of concentration, for VOCs produced in all culture media. BCT9 cultures grown in Methyl Red & Voges Proskauer medium as bioactive compounds with or without lanolin. These VOCs increased shoot length, root length and dry weight at low concentrations, independent of the presence of lanolin. Lateral root length increased with the application of 2-nonanone (50ppm) and 2-undecanone (0.05ppm). Based on these results, the use of bioactive volatiles as growth inducers of horticultural species represents an alternative or complementary strategy.

Growth promotion of Lactuca sativa in response to volatile organic compounds emitted from diverse bacterial species.

Agrochemicals are currently used in horticulture to increase crop production. Nevertheless, their indiscriminate use is a relevant issue for environmental and legal aspects. Alternative tools for reducing fertilizers and synthetic phytohormones are being investigated, such as the use of volatile organic compounds (VOCs) as growth inducers. Some soil bacteria, such as Pseudomonas and Bacillus, stimulate Arabidopsis and tobacco growth by releasing VOCs, but their effects on vegetables have not been investigated. Lactuca sativa was used as model vegetable to investigate bacterial VOCs as growth inducers. We selected 10 bacteria strains, belonging to Bacillus, Staphylococcus and Serratia genera that are able to produce 3-hydroxy-2-butanone (acetoin), a compound with proven growth promoting activity. Two-day old-seedlings of L. sativa were exposed to VOCs emitted by the selected bacteria grown in different media cultures for 7 days. The results showed that the VOCs released from the bacteria elicited an increase in the number of lateral roots, dry weight, root growth and shoot length, depending on the media used. Three Bacillus strains, BCT53, BCT9 and BCT4, were selected according to its their growth inducing capacity. The BCT9 strain elicited the greatest increases in dry weight and primary root length when L. sativa seedlings were subjected to a 10-day experiment. Finally, because acetoin only stimulated root growth, we suggest that other volatiles could be responsible for the growth promotion of L. sativa. In conclusion, our results strongly suggest that bacteria volatiles can be used as growth-inducers as alternative or complementary strategies for application in horticulture species.

Virtual Screening of Plant Volatile Compounds Reveals a High Affinity of Hylamorpha elegans (Coleoptera: Scarabaeidae) Odorant-Binding Proteins for Sesquiterpenes From Its Native Host.

Hylamorpha elegans(Burmeister) is a native Chilean scarab beetle considered to be a relevant agricultural pest to pasture and cereal and small fruit crops. Because of their cryptic habits, control with conventional methods is difficult; therefore, alternative and environmentally friendly control strategies are highly desirable. The study of proteins that participate in the recognition of odorants, such as odorant-binding proteins (OBPs), offers interesting opportunities to identify new compounds with the potential to modify pest behavior and computational screening of compounds, which is commonly used in drug discovery, may help to accelerate the discovery of new semiochemicals. Here, we report the discovery of four OBPs inH. elegans as well as six new volatiles released by its native host Nothofagus obliqua(Mirbel). Molecular docking performed between OBPs and new and previously reported volatiles from N. oblique revealed the best binding energy values for sesquiterpenic compounds. Despite remarkable divergence at the amino acid level, three of the four OBPs evaluated exhibited the best interaction energy for the same ligands. Molecular dynamics investigation reinforced the importance of sesquiterpenes, showing that hydrophobic residues of the OBPs interacted most frequently with the tested ligands, and binding free energy calculations demonstrated van der Waals and hydrophobic interactions to be the most important. Altogether, the results suggest that sesquiterpenes are interesting candidates for in vitro and in vivo assays to assess their potential application in pest management strategies.