Complete Genome Sequence Analysis of Bacillus subtilis MC4-2 Strain That against Tobacco Black Shank Disease.

The MC4-2 bacterium strain was isolated and purified from the Periplaneta americana intestine as a biocontrol agent with good antagonistic effect against the pathogens of a soil-borne disease called tobacco black shank. The MC4-2 strain was found to have good broad-spectrum inhibition by plate stand-off test. Based on 16S rRNA and gyrB genes, ANI analysis, and other comparative genomics methods, it was determined that the MC4-2 strain was Bacillus subtilis. The complete genome sequence showed that the genome size was 4,076,630 bp, the average GC content was 43.78%, and the total number of CDSs was 4,207. Genomic prediction analysis revealed that a total of 145 genes were annotated by the CAZy, containing mainly GH and CE enzymes that break down carbohydrates such as glucose, chitin, starch, and alginate, and a large number of enzymes involved in glycosylation were present. A total of ten secondary metabolite clusters were predicted, six clusters of which were annotated as surfactin, bacillaene, fengycin, bacillibactin, subtilosin A, and bacilysin. The present investigation found the biological control mechanism of B. subtilis MC4-2, which provides a strong theoretical basis for the best use of this strain in biological control methods and provides a reference for the subsequent development of agents of this bacterium.

Identification and expression analysis of heat shock protein family genes of gall fly (Procecidochares utilis) under temperature stress.

Heat shock proteins (HSP) are molecular chaperones involved in many normal cellular processes and environmental stresses. At the genome-wide level, there were no reports on the diversity and phylogeny of the heat shock protein family in Procecidochares utilis. In this study, 43 HSPs were identified from the genome of P. utilis, including 12 small heat shock proteins (sHSPs), 23 heat shock protein 40 (DNAJs), 6 heat shock protein 70 (HSP70s), and 2 heat shock protein 90 (HSP90s). The characteristics of these candidates HSP genes were analyzed by BLAST, and then phylogenetic analysis was carried out. Quantitative real-time PCR (qRT-PCR) was used to analyze the spatiotemporal expression patterns of sHSPs and HSP70s in P. utilis after temperature stress. Results showed that most sHSPs could be induced under heat stress during the adult stage of P. utilis, while a few HSP70s could be induced at the larval stage. This study provides an information framework for the HSP family of P. utilis. Moreover, it lays an important foundation for a better understanding of the role of HSP in the adaptability of P. utilis to various environments.

Bio-accumulation effects of heavy metals Pb, Zn and Cd on Procecidochares utilis parasitism to Eupatorium adenophorum at Suzu metal mines, Yunnan.

Procecidochares utilis is an obligatory parasitic insect to Eupatorium adenophorum. Both organisms have been spread to some metal mines areas. The objective of this study is to comprehend the trend of heavy metals transfer and the process of their bio-accumulation in the soil-E. adenophorum-P. utilis system and particularly their impact on the parasitic effect of P. utilis to E. adenophorum to reflect the impact of heavy metals on obligate parasitic insect and its host. Therefore, a detailed investigation was carried out at the Suzu Lead-Zinc Mine in Yunnan Province using the concentric circle's method. The results showed that the parasitic rate of P. utilis to a single plant and branch is positively correlated with distance. The metals content of the soil in E. adenophorum and P. utilis, decreased dramatically with an increase in distance away from the center of the mining area. From which is cleared that these metals could enter to E. adenophorum and P. utilis through the soil-E. adenophorum-P. utilis system which likely to affect its parasitic activities. In addition, the parasitic rate is impacted by per Zn content greatly, and the parasitic rate per plant is affected by Cd content enormously. This work could provide important basis of data for further understanding and clarifying the effects of bioaccumulation and heavy metals pollution on various aspects of the food chain. Simultaneously, it could clarify and simplify whether heavy metal contamination affects the parasitic behaviour of some obligatory parasitic insects.

Antifeedant Mechanism of Dodonaea viscosa Saponin A Isolated from the Seeds of Dodonaea viscosa.

Dodonaea viscosa is a medicinal plant which has been used to treat various diseases in humans. However, the anti-insect activity of extracts from D. viscosa has not been evaluated. Here, we found that the total saponins from D. viscosa (TSDV) had strong antifeedant and growth inhibition activities against 4th-instar larvae of Spodoptera litura. The median antifeeding concentration (AFC50) value of TSDV on larvae was 1621.81 µg/mL. TSDV affected the detoxification enzyme system of the larvae and also exerted antifeedant activity possibly through targeting the γ-aminobutyric acid (GABA) system. The AFC50 concentration, the carboxylesterase activity, glutathione S-transferases activity, and cytochrome P450 content increased to 258%, 205%, and 215%, respectively, and likewise the glutamate decarboxylase activity and GABA content to 195% and 230%, respectively, in larvae which fed on TSDV. However, D. viscosa saponin A (DVSA) showed better antifeedant activity and growth inhibition activity in larvae, compared to TSDV. DVSA also exerted their antifeedant activity possibly through targeting the GABA system and subsequently affected the detoxification enzyme system. Further, DVSA directly affected the medial sensillum and the lateral sensillum of the 4th-instar larvae. Stimulation of Spodoptera litura. with DVSA elicited clear, consistent, and robust excitatory responses in a single taste cell.

Phytotoxic Azaphilones From the Mangrove-Derived Fungus Penicillium sclerotiorum HY5.

Mangrove is a unique marine ecosystem growing in the intertidal zone of tropical and subtropical coast, with the characteristics of hypoxia tolerance, high salinity, and high humidity. In order to discover novel leading compounds with potent phytotoxicity, seven pairs of azaphilones E/Z isomers, isochromophilone H (1a/1b), sclerotiorins A and B (2a/2b and 3a/3b), ochlephilone (4a/4b), isochromophilone IV (5a/5b), isochromophilone J (6a/6b), and isochromophilone I (7a/7b), were isolated from the culture broth of the mangrove-derived fungus, the Penicillium sclerotiorum HY5, by various chromatographic methods. Among them, 1a, 1b, 2a, 3a, 4a, 5a, 6a, and 6b were new compounds. Their chemical structures and absolute configurations were elucidated based on high resolution electrospray ionization mass spectroscopy (HRESIMS), 1D/2D nuclear magnetic resonance (NMR) spectroscopic analysis, and comparisons of electronic circular dichroism (ECD) data. Compounds 3, 4, and 7 exhibited potent phytotoxicity against the growth of radicle and plumule on Amaranthus retroflexus L., with EC50 values ranging from 234.87 to 320.84 µM, compared to the positive control glufosinate-ammonium, with EC50 values of 555.11 µM for radicle, and 656.04 µM for plumule. Compounds 4 and 7 also showed inhibitory effects on the growth of velvetleaf (Abutilon theophrasti Medikus), with EC50 values ranging from 768.97 to 1,201.52 µM. This study provides new leading compounds for the research and development of marine-derived bioherbicides.

Nematicidal activity of tirotundin and parthenolide isolated from Tithonia diversifolia and Chrysanthemum parthenium.

Acetylcholinesterase (AChE) is an enzyme that catalyzes acetylcholine into choline and acetic acid. Conventional pesticides, including organophosphates and carbamates target and inhibit the activity of AChE. To obtain more pesticide precursors that meet the safety requirements, more than 200 compounds were screened. Tirotundin and parthenolide identified as potential neurotoxins to nematodes were isolated from Tithonia diversifolia and Chrysanthemum parthenium, respectively. Their IC50 values were 6.89 ± 0.30 and 5.51 ± 0.23 µg/mL, respectively against the AChE isolated from Caenorhabditis elegans. AChE was inhibited in a dose-dependent manner using the two compounds. And the Lineweaver-Burk and Dixon plots indicated that tirotundin and parthenolide were reversible inhibitors against AChE, both inhibiting AChE in a mixed-type competitive manner and demonstrating these compounds may possess dual binding site AChE inhibitors. LC50 values of tirotundin and parthenolide against C. elegans were 9.16 ± 0.21 and 7.23 ± 0.48 µg/mL, respectively. These results provide a certain theoretical basis for the development and utilization of novel pesticides.

Gross morphology and ultrastructure of the salivary glands of the stink bug predator Eocanthecona furcellata (Wolff).

Eocanthecona furcellata Wolff (Hemiptera: Pentatomidae) is a native generalist predator which attacks and kills its prey by first inserting its stylet into the prey's body and then injecting saliva into it. Here, we describe the histology and ultrastructure of its salivary glands. The study showed that the salivary glands were made up of pairs of principal and tubular accessory salivary glands. The principal salivary glands were bilobed and consisted of a smaller anterior lobe and a larger elongated posterior lobe. The ducts of the principal and accessory salivary glands were located in a narrow region between the anterior and posterior lobe known as the hilum. The principal salivary gland was lined with a single-layered epithelium. The cells cytoplasm was enriched with rough endoplasmic reticulum and secretory, and the nucleus showed a higher level of uncondensed chromatin. The basal region of the cell had plasma membrane infoldings. The cytoplasm of the accessory gland was rich in rough endoplasmic reticulum and many large cavities. The ducts of the principal salivary gland were made up of a single layer of flattened cells which had a thin cuticle lining the apical portion. Variation in the lumen content of the different lobes, which made up the principal gland suggested that their chemical products also varied. These results indicate that these two salivary glands produce the proteins found in the saliva.

Inhibitory activities of flavonoids from Eupatorium adenophorum against acetylcholinesterase.

Fifteen flavonoids isolated from the Eupatorium adenophorum showed inhibitory activities against acetylcholinesterase (AChE) isolated from Caenorhabditis elegans and Spodoptera litura. Their IC50 values ranged from 12.54 to 89.06µg/mL and 12.08 to 86.01µg/mL, respectively against the AChE isolated from the nematode and insect species. AChE was inhibited in a dose-dependent manner by all tested flavonoids, The isolated compound quercetagetin-7-O-(6-O-caffeoyl-ß-D-glucopyranoside) displayed the highest inhibitory effect against AChE from C. elegans and S. litura, with IC50 values of 12.54 µg/mL and 12.58 µg/mL, respectively. The structure-activity relationship of flavonoids on the inhibitory activities indicated that additional phenolic hydroxyl groups in the glucose were favorable for their inhibitory effects and the degree of increase in inhibitory activity also depended on the number of phenolic hydroxyl groups. The Lineweaver-Burk and Dixon plots indicated that quercetagetin-7-O-(6-O-caffeoyl-ß-d-glucopyranoside) is a reversible inhibitor against AChE. Quercetagetin-7-O-(6-O-caffeoyl-ß-d-glucopyranoside), 5,4'-Dihydroxytlavone and quercetin-3-O-ß-d-glucopyranoside inhibited AChE in a mixed-type competitive manner and these compounds might be the dual binding site AChE inhibitors. Further, nine compounds showed poisonous effects against C. elegans and inhibitory effects on the growth and development of S. litura.

Identification and preliminary characterization of chemosensory-related proteins in the gall fly, Procecidochares utilis by transcriptomic analysis.

Chemoreception is critical for insect behaviors such as foraging, host searching and oviposition. The process of chemoreception is mediated by a series of proteins, including odorant-binding proteins (OBPs), gustatory receptors (GRs), odorant receptors (ORs), ionotropic receptors (IRs), chemosensory proteins (CSPs) and sensory neuron membrane proteins (SNMPs). The tephritid stem gall fly, Procecidochares utilis Stone, is a type of egg parasitic insect, which is an effective biological control agent for the invasive weed Ageratina adenophora in many countries. However, the study of molecular components related to the olfactory system of P. utilis has not been investigated. Here, we conducted the developmental transcriptome (egg, first-third instar larva, pupa, female and male adult) of P. utilis using next-generation sequencing technology and identified a total of 133 chemosensory genes, including 40 OBPs, 29 GRs, 24 ORs, 28 IRs, 6 CSPs, and 6 SNMPs. The sequences of these candidate chemosensory genes were confirmed by BLAST, and phylogenetic analysis was performed. Quantitative real-time PCR (qRT-PCR) confirmed that the expression levels of the candidate OBPs varied at the different developmental stages of P. utilis with most OBPs expressed mainly in the pupae, female and male adults but scarcely in eggs and larvae, which was consistent with the differentially expressed genes (DEGs) analysis using the fragments per kilobase per million fragments (FPKM) value. Our results provide a significant contribution towards the knowledge of the set of chemosensory proteins and help advance the use of P. utilis as biological control agents.

De novo transcriptome analysis and identification of genes associated with immunity, detoxification and energy metabolism from the fat body of the tephritid gall fly, Procecidochares utilis.

The fat body, a multifunctional organ analogous to the liver and fat tissue of vertebrates, plays an important role in insect life cycles. The fat body is involved in protein storage, energy metabolism, elimination of xenobiotics, and production of immunity regulator-like proteins. However, the molecular mechanism of the fat body's physiological functions in the tephritid stem gall-forming fly, Procecidochares utilis, are still unknown. In this study, we performed transcriptome analysis of the fat body of P. utilis using Illumina sequencing technology. In total, 3.71 G of clean reads were obtained and assembled into 30,559 unigenes, with an average length of 539 bp. Among those unigenes, 21,439 (70.16%) were annotated based on sequence similarity to proteins in NCBI's non-redundant protein sequence database (Nr). Sequences were also compared to NCBI's non-redundant nucleotide sequence database (Nt), a manually curated and reviewed protein sequence database (SwissProt), and KEGG and gene ontology annotations were applied to better understand the functions of these unigenes. A comparative analysis was performed to identify unigenes related to detoxification, immunity and energy metabolism. Many unigenes involved in detoxification were identified, including 50 unigenes of putative cytochrome P450s (P450s), 18 of glutathione S-transferases (GSTs), 35 of carboxylesterases (CarEs) and 26 of ATP-binding cassette (ABC) transporters. Many unigenes related to immunity were identified, including 17 putative serpin genes, five peptidoglycan recognition proteins (PGRPs) and four lysozyme genes. In addition, unigenes potentially involved in energy metabolism, including 18 lipase genes, five fatty acid synthase (FAS) genes and six elongases of very long chain fatty acid (ELOVL) genes, were identified. This transcriptome improves our genetic understanding of P. utilis and the identification of a numerous transcripts in the fat body of P. utilis offer a series of valuable molecular resources for future studies on the functions of these genes.

RNA-seq analysis of the response of plant-pathogenic oomycete Phytophthora parasitica to the fungicide dimethomorph / Análisis RNA-seq de la respuesta del oomiceto fitopatógeno Phytophthora parasitica al fungicida dimetomorf

Phytophthora parasitica is an important oomycete that causes disease in a variety of plants, dimethomorph fungicides being specific for oomycetes. The aim of this study was to use RNA-seq to rapidly discover the mechanism by which dimethomorph acts in the treatment of P. parasitica. We found that the expression of 832 genes changed significantly after the dimethomorph treatment, including 365 up-regulated genes and 467 down-regulated genes. According to the Gene Ontology (GO) enrichment analysis, pathway enrichment and verification test results, the following conclusions are obtained: (i) the treatment of P. parasitica with dimethomorph causes changes in the expression levels of genes associated with the cell wall and cell wall synthesis; (ii) dimethomorph treatment results in reduced permeability of the cell membrane and changes in the expression of certain transport-related proteins; (iii) dimethomorph treatment increased reactive oxygen species and reduced the expression of genes related to the control of oxidative stress.

Phytophthora parasitica es un importante oomiceto que origina enfermedades en una variedad de plantas; el fungicida dimetomorf es específico contra oomicetos. El objetivo de este estudio fue utilizar la tecnología de RNA-seq para descubrir rápidamente el mecanismo por el que el dimetomorf actúa en el tratamiento de P. parasitica. Descubrimos que la expresión de 832 genes se modificaba significativamente tras el tratamiento con dimetomorf, incluyendo 365 genes que son sobrerregulados y 467 genes que son subrregulados. El análisis de enriquecimiento de ontología de genes (GO), análisis de enriquecimiento de las vías y pruebas de verificación permitieron extraer las conclusiones siguientes: 1) el tratamiento de P. parasitica con dimetomorf origina cambios en los niveles de expresión de los genes relacionados con la pared celular y su síntesis; 2) el tratamiento con dimetomorf origina una reducción de la permeabilidad de la membrana celular, así como cambios en la expresión de ciertas proteínas relacionadas con el transporte, y 3) el tratamiento con dimetomorf incrementó las especies reactivas del oxígeno y redujo la expresión de los genes relacionados con el control del estrés oxidativo.

RNA-seq analysis of the response of plant-pathogenic oomycete Phytophthora parasitica to the fungicide dimethomorph.

Phytophthora parasitica is an important oomycete that causes disease in a variety of plants, dimethomorph fungicides being specific for oomycetes. The aim of this study was to use RNA-seq to rapidly discover the mechanism by which dimethomorph acts in the treatment of P. parasitica. We found that the expression of 832 genes changed significantly after the dimethomorph treatment, including 365 up-regulated genes and 467 down-regulated genes. According to the Gene Ontology (GO) enrichment analysis, pathway enrichment and verification test results, the following conclusions are obtained: (i) the treatment of P. parasitica with dimethomorph causes changes in the expression levels of genes associated with the cell wall and cell wall synthesis; (ii) dimethomorph treatment results in reduced permeability of the cell membrane and changes in the expression of certain transport-related proteins; (iii) dimethomorph treatment increased reactive oxygen species and reduced the expression of genes related to the control of oxidative stress.

De novo transcriptomic analysis of the alimentary tract of the tephritid gall fly, Procecidochares utilis.

The tephritid gall fly, Procecidochares utilis, is an important obligate parasitic insect of the malignant weed Eupatorium adenophorum which biosynthesizes toxic secondary metabolites. Insect alimentary tracts secrete several enzymes that are used for detoxification, including cytochrome P450s, glutathione S-transferases, and carboxylesterases. To explore the adaptation of P. utilis to its toxic host plant, E. adenophorum at molecular level, we sequenced the transcriptome of the alimentary tract of P. utilis using Illumina sequencing. Sequencing and de novo assembly yielded 62,443 high-quality contigs with an average length of 604 bp that were further assembled into 45,985 unigenes with an average length of 674 bp and an N50 of 983 bp. Among the unigenes, 30,430 (66.17%) were annotated by alignment against the NCBI non-redundant protein (Nr) database, while 16,700 (36.32%), 16,267 (35.37%), and 11,530 (25.07%) were assigned functions using the Clusters of Orthologous Groups (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO) databases, respectively. Using the comprehensive transcriptome data set, we manually identified several important gene families likely to be involved in the detoxification of toxic compounds including 21 unigenes within the glutathione S-transferase (GST) family, 22 unigenes within the cytochrome P450 (P450) family, and 16 unigenes within the carboxylesterase (CarE) family. Quantitative PCR was used to verify eight, six, and two genes of GSTs, P450s, and CarEs, respectively, in different P. utilis tissues and at different developmental stages. The detoxification enzyme genes were mainly expressed in the foregut and midgut. Moreover, the unigenes were higher expressed in the larvae, pupae, and 3-day adults, while they were expressed at lower levels in eggs. These transcriptomic data provide a valuable molecular resource for better understanding the function of the P. utilis alimentary canal. These identified genes could be pinpoints to address the molecular mechanisms of P. utilis interacting with toxic plant host.