Predicted structure of odorant-binding protein 12 from Monochamus alternatus (Hope) suggests a mechanism of flexible odorant-binding.

Odorant-binding proteins (OBPs) are thought to bind and deliver hydrophobic odorants from the environment to receptors on insect sensory neurons, and have been used to screen behaviorally active compounds of insects. In order to screen behaviorally active compounds for Monochamus alternatus by OBPs, we cloned full length of Obp12 coding sequence from M. alternatus and proved secretion property of MaltOBP12, then tested binding affinities of recombinant MaltOBP12 to 12 pine volatiles in vitro. We confirmed MaltOBP12 has binding affinities to 9 pine volatiles. The structure of MaltOBP12 and protein-ligand interactions were further analyzed by homology modeling, molecular docking, site-directed mutagenesis, and ligand-binding assays. These results demonstrated that the binding pocket of MaltOBP12 consists of several large aromatic and hydrophobic residues, and four aromatic residues (Tyr50, Phe109, Tyr112, Phe122) are essential for odorant-binding; ligands adopt extensive hydrophobic interactions with an overlapping subset of residues in the binding pocket. Finally, based on non-directional hydrophobic interactions, MaltOBP12 binds odorants flexibly. These findings will not only help us understand how OBPs flexibly bind odorants but also promote to screen of behaviourally active compounds by computer methods to prevent M. alternatus in the future.

A Highly Expressed Antennae Odorant-Binding Protein Involved in Recognition of Herbivore-Induced Plant Volatiles in Dastarcus helophoroides.

Natural enemies such as parasitoids and parasites depend on sensitive olfactory to search for their specific hosts. Herbivore-induced plant volatiles (HIPVs) are vital components in providing host information for many natural enemies of herbivores. However, the olfactory-related proteins involved in the recognition of HIPVs are rarely reported. In this study, we established an exhaustive tissue and developmental expression profile of odorant-binding proteins (OBPs) from Dastarcus helophoroides, an essential natural enemy in the forestry ecosystem. Twenty DhelOBPs displayed various expression patterns in different organs and adult physiological states, suggesting a potential involvement in olfactory perception. In silico AlphaFold2-based modeling and molecular docking showed similar binding energies between six DhelOBPs (DhelOBP4, 5, 6, 14, 18, and 20) and HIPVs from Pinus massoniana. While in vitro fluorescence competitive binding assays showed only recombinant DhelOBP4, the most highly expressed in the antennae of emerging adults could bind to HIPVs with high binding affinities. RNAi-mediated behavioral assays indicated that DhelOBP4 was an essential functional protein for D. helophoroides adults recognizing two behaviorally attractive substances: p-cymene and γ-terpinene. Further binding conformation analyses revealed that Phe 54, Val 56, and Phe 71 might be the key binding sites for DhelOBP4 interacting with HIPVs. In conclusion, our results provide an essential molecular basis for the olfactory perception of D. helophoroides and reliable evidence for recognizing the HIPVs of natural enemies from insect OBPs' perspective.

BarH1 regulates odorant-binding proteins expression and olfactory perception of Monochamus alternatus Hope.

Insect odorant-binding proteins (OBPs) are a class of small soluble proteins that can be found in various tissues wherein binding and transport of small molecules are required. Thus, OBPs are not only involved in typical olfactory function by specific activities with odorants but also participate in other physiological processes in non-chemosensory tissues. To better understand the complex biological functions of OBPs, it is necessary to study the transcriptional regulation of their expression patterns. In this paper, an apparent gradient expression pattern of Obp19, that was highly and specifically expressed in antennae and played an essential role in the detection of camphene, was defined in the antennae of the Japanese pine sawyer. Further, the transcription factor BarH1, that also presented gradient expression pattern in antennae, was found to regulate expression of Obp19 directly through binding to its upstream DNA sequence. The condition of BarH1 gene silence, the gene expression levels of Obp19 significantly decreased. At the same time, additional olfactory genes also were regulated and thus influence camphene reception. These findings provide us an opportunity to incorporate Obps in the gene regulatory networks of insects, which contribute to a better understanding of the multiplicity and diversity of OBPs and the olfactory mediated behaviors.

Phytopathogenic infection alters rice-pest-parasitoid tri-trophic interactions.

BACKGROUND: Plant pathogens and pests often occur together, causing damage while interfering with plant growth. The effects of phytopathogenic infections on plant-herbivore-natural enemy tri-trophic interactions (TTIs) have been extensively investigated, but little is known about how the interval of infection influences such relationships. Here, the effect of rice plants infected by the phytopathogen Rhizoctonia solani on the herbivorous rice brown planthopper (BPH) and associated egg parasitoid Anagrus nilaparvatae over a temporal scale was examined. RESULTS: Our results showed that rice plants infected by R. solani showed increased volatile profiles and significantly attracted BPH and A. nilaparvatae at 5-15 days post infection (DPI) and 5-10 DPI, respectively, when compared with healthy plants. Jasmonic acid and salicylic acid content decreased significantly in BPH-damaged plants after 15 DPI, whereas oxalic acid accumulated soon after 5 DPI when compared with healthy plants. To adapt to adverse environment, BPH laid more eggs and developed into macropterous adults. Under field conditions, R. solani infection had no substantial effect on the arthropod community when compared with healthy plants. CONCLUSION: Taken together, R. solani infection altered rice-pest-parasitoid TTIs over a temporal scale. This result will shed more light on our understanding of plant pathogen-insect cross-talk essential for developing novel pest management strategies. © 2021 Society of Chemical Industry.

Correction: Dihydroxyacetone of wheat root exudates serves as an attractant for Heterodera avenae.

[This corrects the article DOI: 10.1371/journal.pone.0236317.].

An Odorant Binding Protein (SaveOBP9) Involved in Chemoreception of the Wheat Aphid Sitobion avenae.

Odorant binding proteins play a key role in the olfactory system and are involved in the odor perception and discrimination of insects. To investigate the potential physiological functions of SaveOBP9 in Sitobion avenae, fluorescence ligand binding experiments, molecular docking, RNA interference, and behavioral tests were performed. Fluorescence binding assay results showed that SaveOBP9 had broad and high (Ki < 10 µM) binding abilities with most of the wheat volatiles, but was more obvious at pH 7.4 than pH 5.0. The binding sites of SaveOBP9 to the volatiles were predicted well by three-dimensional docking structure modeling and molecular docking. Moreover, S. avenae showed a strong behavioral response with the four compounds of wheat. The reduction in mRNA transcript levels after the RNA interference significantly reduced the expression level of SaveOBP9 and induced the non-significant response of S. avenae to the tetradecane, octanal, decanal, and hexadecane. This study provides evidence that SaveOBP9 might be involved in the chemoreception of wheat volatile organic compounds and can successfully contribute in the integrated management programs of S. avenae.

Dihydroxyacetone of wheat root exudates serves as an attractant for Heterodera avenae.

Heterodera avenae, as an obligate endoparasite, causes severe yield loss in wheat (Triticum aestivum). Investigation on the mechanisms how H. avenae perceives wheat roots is limited. Here, the attractiveness of root exudates from eight plant genotypes to H. avenae were evaluated on agar plates. Results showed that the attraction of H. avenae to the root exudates from the non-host Brachypodium distachyon variety Bd21-3 was the highest, approximately 50 infective second-stage juveniles (J2s) per plate, followed by that from three H. avenae-susceptible wheat varieties, Zhengmai9023, Yanmai84 and Xiangmai25, as well as the resistant one of Xinyuan958, whereas the lowest attractive activity was observed in the two H. avenae-resistant wheat varieties, Xianmai20 (approximately 12 J2s/plate) and Liangxing66 (approximately 11 J2s/plate). Then Bd21-3, Zhengmai9023 and Heng4399 were selected for further assays as their different attractiveness and resistance to H. avenae, and attractants for H. avenae in their root exudates were characterized to be heat-labile and low-molecular compounds (LM) by behavioral bioassay. Based on these properties of the attractants, a principle of identifying attractants for H. avenae was set up. Then LM of six root exudates from the three plants with and without heating were separated and analyzed by HPLC-MS. Finally, dihydroxyacetone (DHA), methylprednisolone succinate, embelin and diethylpropionin in the root exudates were identified to be putative attractants for H. avenae according to the principle, and the attraction of DHA to H. avenae was validated by behavioral bioassay on agar. Our study enhances the recognition to the orientation mechanism of H. avenae towards wheat roots.

A chemosensory protein MsepCSP5 involved in chemoreception of oriental armyworm Mythimna separata.

Chemosensory proteins (CSPs) have been suggested to perform several functions in insects, including chemoreception. To find out whether MsepCSP5 identified from Mythimna separata shows potential physiological functions in olfaction, gene expression profiles, ligand-binding experiments, molecular docking, RNA interference, and behavioral test were performed. Results showed that MsepCSP5 was highly expressed in female antennae. MsepCSP5 showed high binding affinities to a wide range of host-related semiochemicals, and displayed that 26 out of 35 candidate volatiles were highly bound (Ki < 10 µM) at pH 5.0 rather than pH 7.4. The binding sites of MsepCSP5 to candidate volatiles were well predicted by three-dimensional structure modeling and molecular docking experiments. Pursuing further, biological activities of M. separata to highly bound compounds elicited strong behavioral responses, such as alcoholic compounds displayed strong attractiveness whereas terpenes showed repellency to M. separata. The transcript expression level of MsepCSP5 gene significantly decreased after injecting target dsRNAs, and resulted in non-significant preference responses of M. separata to semiochemicals, such as 3-pentanol and 1-octene-3-ol. In conclusion, MsepCSP5 may involve in semiochemical reception of M. separata.

Isolation, Characterization, and Application of Bacteriophage LPSE1 Against Salmonella enterica in Ready to Eat (RTE) Foods.

Salmonella infection is an important foodborne consumer health concern that can be mitigated during food processing. Bacteriophage therapy imparts many advantages over conventional chemical preservatives including pathogen specificity, natural derivation, potency, and providing a high degree of safety. The objective of this study aimed to isolate and characterize a phage that effectively control Salmonella food contamination. Out of 35 isolated phages, LPSE1 demonstrated a broad Salmonella host range, robust lytic ability, extensive pH tolerance, and prolonged thermal stability. The capacity for phage LPSE1 to control Salmonella Enteritidis-ATCC13076 in milk, sausage, and lettuce was established. Incubation of LPSE1 at 28°C in milk reduced recoverable Salmonella by approximately 1.44 log10 CFU/mL and 2.37 log10 CFU/mL at MOI of 1 and 100, respectively, as relative to the phage-excluded control. Upon administration of LPSE1 at an MOI of 1 in sausage, Salmonella count decreased 0.52 log10 at 28°C. At MOI of 100, the count decreased 0.49 log10 at 4°C. Incubation of LPSE1 on lettuce reduced recoverable Salmonella by 2.02 log10, 1.71 log10, and 1.45 log10 CFU/mL at an MOI of 1, 10, and 100, respectively, as relative to the negative control. Taken together, these findings establish LPSE1 as an effective weapon against human pathogenic Salmonella in various ready to eat foods.

Silencing of Chemosensory Protein Gene NlugCSP8 by RNAi Induces Declining Behavioral Responses of Nilaparvata lugens.

Chemosensory proteins (CSPs) play imperative functions in chemical and biochemical signaling of insects, as they distinguish and transfer ecological chemical indications to a sensory system in order to initiate behavioral responses. The brown planthopper (BPH), Nilaparvata lugens Stål (Hemiptera: Delphacidae), has emerged as the most destructive pest, causing serious damage to rice in extensive areas throughout Asia. Biotic characteristics like monophagy, dual wing forms, and annual long-distance migration imply a critical role of chemoreception in N. lugens. In this study, we cloned the full-length CSP8 gene from N. lugens. Protein sequence analysis indicated that NlugCSP8 shared high sequence resemblance with the CSPs of other insect family members and had the typical four-cysteine signature. Analysis of gene expression indicated that NlugCSP8 mRNA was specifically expressed in the wings of mated 3-day brachypterous females with a 175-fold difference compare to unmated 3-day brachypterous females. The NlugCSP8 mRNA was also highly expressed in the abdomen of unmated 5-day brachypterous males and correlated to the age, gender, adult wing form, and mating status. A competitive ligand-binding assay demonstrated that ligands with long chain carbon atoms, nerolidol, hexanal, and trans-2-hexenal were able to bind to NlugCSP8 in declining order of affinity. By using bioinformatics techniques, three-dimensional protein structure modeling and molecular docking, the binding sites of NlugCSP8 to the volatiles which had high binding affinity were predicted. In addition, behavioral experiments using the compounds displaying the high binding affinity for the NlugCSP8, revealed four compounds able to elicit significant behavioral responses from N. lugens. The in vivo functions of NlugCSP8 were further confirmed through the testing of RNAi and post-RNAi behavioral experiments. The results revealed that reduction in NlugCSP8 transcript abundance caused a decrease in behavioral response to representative attractants. An enhanced understanding of the NlugCSP8 is expected to contribute in the improvement of more effective and eco-friendly control strategies of BPH.

Screening behaviorally active compounds based on fluorescence quenching in combination with binding mechanism analyses of SspOBP7, an odorant binding protein from Sclerodermus sp.

Reverse chemical ecology approaches based on the recognition and transport function of odorant binding proteins (OBPs) have been used to screen behaviorally active compounds of insects. In the first place, behaviorally active compounds from Sclerodermus sp., an important ectoparasite of Monochamus alternatus Hope, were screened by SspOBP7. The Fluorescence quenching assays revealed that only six of 19 ligands that had binding affinities in fluorescence competition-binding assays formed complexes with SspOBP7. Pursuing this further, two non-polar ligands, terpinolene and (+)-α-longipinene showed strong attractant activities for Sclerodermus sp. The pH change could lead to conformational transition of SspOBP7 from one state to another, which results in low binding affinities at low pH. Finally, a mutational analysis of the SspOBP7 binding cavity proved that changing the cavity had a greater effect on non-polar ligands, and the specific recognition of ligands by SspOBP7 might depend mainly on the appropriate shapes of the cavity and ligands. The most obvious finding to emerge from this work is that the use of fluorescence quenching to study the binding mechanism of OBPs could aid reverse chemical ecology approaches by narrowing the scope of candidate behaviorally active compounds.

Complete Genome Sequence of Salmonella enterica Lytic Bacteriophage LPST10, Isolated in China.

Bacteriophage LPST10 was isolated from Wuhan, China. Lytic activity was demonstrated against multiple Salmonella enterica serovars, including Salmonella enterica serovar Typhimurium strains. This bacteriophage has a 47,657-bp double-stranded DNA genome encoding 87 putative coding sequences.

Impact of direct and indirect application of rising furfural concentrations on viability, infectivity and reproduction of the root-knot nematode, Meloidogyne incognita in Pisum sativum.

The gradual withdraw of several broadly used nematicides from market has enhanced the need to develop sustainable and eco-friendly alternatives with nematicidal properties. Furfural is one of the promising alternatives to fill this need. Baseline information about the impact of furfural on egg hatch, penetration potential and ultrastructure of nematode is lacking. In this study, the reagent-grade (purity ≥ 99.0%) of furfural was applied against Meloidogyne incognita. In vitro tests showed gradual reduction in either the rate of egg hatch or second stage juvenile (J2) viability of M. incognita when immersed in concentrations ranging from 0 to 10.0 µl/ml furfural. The mean EC50 for J2 and egg hatch was 0.37 and 0.27 µl/ml furfural, respectively. Furfural, even at low concentrations, resulted in a considerable suppression in egg hatch. Hatch was <5% after 8 days at 0.63 µl/ml furfural. The same furfural concentrations after 12 h caused 57.25% loss of viability in J2. Moreover, the penetration rate of juveniles to pea roots was suppressed when furfural was even applied at low rates. In pot experiments, furfural was applied as liquid (direct) or vapor (indirect) treatments at rates of 0-1.5 ml/kg soil. Significant reduction in galling, egg production and population density of M. incognita observed when furfural was applied at rates >0.2 ml/kg soil. No adverse effect was detected on plants or free-living nematodes as a result of furfural application. Liquid furfural proved to have superior juvenile-suppressive effect whereas its vapor has such superiority against eggs. Scanning electron microscope (SEM) study showed irregular appearance of the body surface accompanied with some cuticle disfigurement of furfural-treated juveniles. These results indicated that furfural can adversely affect egg hatch, juvenile viability, penetration potential and ultrastructure of M. incognita. Furfural may therefore be of a considerable potential as an appropriate alternative for class I nematicides.

Assessment of DAPG-producing Pseudomonas fluorescens for Management of Meloidogyne incognita and Fusarium oxysporum on Watermelon.

Pseudomonas fluorescens isolates Clinto 1R, Wayne 1R, and Wood 1R, which produce the antibiotic 2,4-diacetylphloroglucinol (DAPG), can suppress soilborne diseases and promote plant growth. Consequently, these beneficial bacterial isolates were tested on watermelon plants for suppression of Meloidogyne incognita (root-knot nematode: RKN) and Fusarium oxysporum f. sp. niveum (Fon). In a greenhouse trial, Wayne 1R root dip suppressed numbers of RKN eggs per gram root on 'Charleston Gray' watermelon by 28.9%. However, in studies focused on 'Sugar Baby' watermelon, which is commercially grown in Maryland, a Wayne 1R root dip did not inhibit RKN reproduction or plant death caused by Fon. When all three isolates were applied as seed coats, plant stand in the greenhouse was reduced up to 60% in treatments that included Fon ± P. fluorescens, and eggs per gram root did not differ among treatments. In a microplot trial with Clinto 1R and Wayne 1R root dips, inoculation with P. fluorescens and/or Fon resulted in shorter vine lengths than treatment with either P. fluorescens isolate plus RKN. Root weights, galling indices, eggs per gram root, and second-stage juvenile (J2) numbers in soil were similar among all RKN-inoculated treatments, and fruit production was not affected by treatment. Plant death was high in all treatments. These studies demonstrated that the tested P. fluorescens isolates resulted in some inhibition of vine growth in the field, and were not effective for enhancing plant vigor or suppressing RKN or Fon on watermelon.

A mutant of the nematophagous fungus Paecilomyces lilacinus (Thom) is a novel biocontrol agent for Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum causes severe stem rot and yield loss in oilseed rape (Brassica napus L.) and other crops worldwide. Extensive studies have been conducted on Paecilomyces lilacinus as a nematophagous bioagent. However, no reports stated the effect of P. lilacinus as a biocontrol agent against oilseed rape rot S. sclerotiorum. This study describes such effect in lab and field trials using the new transformant pt361 derived from the wild strain P. lilacinus 36-1. Unlike the wild-type strain, the mutant pt361 showed high antagonistic effect against S. Sclerotiorum A. Under lab conditions, the pt361 inhibited (65%) radial mycelial growth of S. sclerotiorum in dual culture test producing 5.9 mm inhibition zone IZ in front of the S. sclerotiorum colony. Moreover, the cell-free filtrate of pt361 culture showed strong inhibitory effects (60.3-100%) on mycelial growth of S. sclerotiorum. In leaf detached assay, pt361 significantly (p < 0.05) inhibited (40.4-97.9%) the extension of the leaf spots caused by S. sclerotiorum A at all tested concentrations. The genomic DNA sequences of the inserted T-DNA flanking obtained from pt361 strain was cloned, verified as a glycoside hydrolase 31 family by homologous analysis with other fungal strains, and named PGH31 (2556bp). Secondary structure prediction showed a domain (Glycoside hydrolase31). Three years field trial confirmed that the cell-free filtrates or spores suspension of pt361 achieved significant (p < 0.05) suppression of oilseed rape stem rot, promoted growth and increased yield compared to the control and exceeded, at dose 100%, the action of the fungicide procymidone(®). In conclusion, the mutant pt361 of P. lilacinus is a novel and promising biocontrol agent against oilseed rape Sclerotinia stem rot.

The role of a phospholipase (PLD) in virulence of Purpureocillium lilacinum (Paecilomyces lilacinum).

Phospholipases are key enzymes in pathogenic fungi that cleave host phospholipids, resulting in membrane destabilization and host cell penetration. However, understanding the role of phospholipases on the virulence of the filamentous fungus Purpureocillium lilacinum has been still rather limited. In this study, pld gene was characterized. It encodes the protein phospholipase D (PLD) in P. lilacinum. This gene, 3303 bp open reading frame fragment (ORF), encodes a protein of 1100 amino acids with high similarity to the same gene from Penicillium oxalicum and Aspergillus fumigatus. Secondary structure prediction showed two PLD phosphodiesterase domains (437-464 bp and 885-912 bp). The pld gene was significantly regulated during infection of Meloidogyne incognita eggs by P. lilacinum. The expression of pld gene using RT-PCR was the highest at 36 and 48 h, which introduce evidence that the presence of M. incognita may induce the expression of the pld gene in P. lilacinum. In addition, maltose and l-alanine were found to increase the expression of pld gene. An acidic environment (pH 3.0-4.0) and moderate temperatures (27-29 °C) are favorable for pld expression in P. lilacinum.

Toxicity of 2,4-diacetylphloroglucinol (DAPG) to plant-parasitic and bacterial-feeding nematodes.

The antibiotic 2,4-diacetylphloroglucinol (DAPG) is produced by some isolates of the beneficial bacterium Pseudomonas fluorescens. DAPG is toxic to many organisms, and crop yield increases have been reported after application of DAPG-producing P. fluorescens. This study was conducted to determine whether DAPG is toxic to selected nematodes. The plant-parasitic nematodes Heterodera glycines, Meloidogyne incognita, Pratylenchus scribneri and Xiphinema americanum, and the bacterial-feeding nematodes Caenorhabditis elegans, Pristionchus pacificus, and Rhabditis rainai, were immersed in concentrations ranging from 0 to 100 µg/ml DAPG. Egg hatch and viability of juveniles and adults were determined. DAPG was toxic to X. americanum adults, with an LD50 of 8.3 µg/ml DAPG. DAPG decreased M. incognita egg hatch, but stimulated C. elegans hatch during the first hours of incubation. Viability of M. incognita J2 and of C. elegans J1 and adults was not affected. There were no observed effects on the other nematodes. The study indicated that DAPG is not toxic to all nematodes, and did not affect the tested species of beneficial bacterial-feeding nematodes. Augmentation of DAPG-producing P. fluorescens populations for nematode biocontrol could be targeted to specific nematode species known to be affected by this compound and by other antibiotics produced by the bacteria, or these bacteria could be used for other possible effects, such as induced plant resistance.