Gut Microbiota Mediate Plutella xylostella Susceptibility to Bt Cry1Ac Protoxin and Exopolysaccharides.

Exopolysaccharides (EPSs) are carbohydrate polymers that are synthesized and secreted into the extracellular during the growth of microorganisms. Bacillus thuringiensis (Bt) is a type of entomopathogenic bacterium, that produces various insecticidal proteins and EPSs. In our previous study, the EPSs produced by Bt strains were first found to enhance the toxicity of insecticidal crystal proteins against Plutella xylostella. However, the response of the intestinal bacterial communities of P. xylostella under the action of EPSs is still unelucidated. In this study, 16S rRNA amplicon sequencing was used to characterize the intestinal bacterial communities in P. xylostella treated with EPSs alone, Cry1Ac protoxin alone, and both the Cry1Ac protoxin and EPSs. Compared with the control group, alpha diversity indices, the Chao1 and ACE indices were significantly altered after treatment with EPSs alone, and no significant difference was observed between the groups treated with Cry1Ac protoxin alone and Cry1Ac protoxin + EPSs. However, compared with the gut bacterial community feeding on Cry1Ac protoxin alone, the relative abundance of 31 genera was significantly changed in the group treated with Cry1Ac protoxin and EPSs. The intestinal bacteria, through the oral of Cry1Ac protoxin and EPSs, significantly enhanced the toxicity of the Cry1Ac protoxin towards the axenic P. xylostella. In addition, the relative abundance of the 16S rRNA gene in the chloroplasts of Brassica campestris decreased after adding EPSs. Taken together, these results show the vital contribution of the gut microbiota to the Bt strain-killing activity, providing new insights into the mechanism of the synergistic insecticidal activity of Bt proteins and EPSs.

Identification of a biomarker for Bacillus thuringiensis strains with high toxicity against Spodoptera frugiperda based on insecticidal gene linkage analysis.

BACKGROUND: Bacillus thuringiensis (Bt) is a Gram-positive bacterium that produces various insecticidal proteins used to control insect pests. Spodoptera frugiperda is a global insect pest which causes serious damage to crops, but bio-insecticides currently available to control this pest have limited activity and so new ones are always being sought. In this study we have tested the hypothesis that a biomarker for strain toxicity could be found that would greatly facilitate the identification of new potential products. RESULTS: Using genomic sequencing data we constructed a linkage network of insecticidal genes from 1957 Bt genomes and found that four gene families, namely cry1A, cry1I, cry2A and vip3A, showed strong linkage. For 95 strains isolated from soil samples we assayed them for toxicity towards S. frugiperda and for the presence of the above gene families. All of the strains that showed high toxicity also contained a member of the vip3A gene family. Two of them were more toxic than a commercially available strain and genomic sequencing identified a number of potentially novel toxin-encoding genes. CONCLUSIONS: The presence of a vip3A gene in the genome of a Bt strain proved to be a strong indicator of toxicity towards S. frugiperda validating this biomarker approach as a strategy for future discovery programs. © 2024 Society of Chemical Industry.

Characterization and evaluation of actinomycete from the Protaetia brevitarsis Larva Frass.

Protaetia brevitarsis larvae (PBL) are soil insects important for the soil organic carbon cycle, and PBL frass not only contains a large amount of humic acid but also affects the diversity, novelty, and potential functions of actinomycetes. Here, we characterized and assessed the actinomycete. The operational taxonomic unit (OTU) data showed that 90% of the actinomycetes cannot be annotated to species, and pure culture and genome analysis showed that 35% of the strains had the potential to be new species, indicating the novelty of PBL frass actinomycetes. Additionally, genome annotation showed that many gene clusters related to antifungal, antibacterial and insecticidal compound synthesis were identified, and confrontation culture confirmed the antifungal activities of the actinomycetes against soil-borne plant pathogenic fungi. The incubation experiment results showed that all isolates were able to thrive on media composed of straw powder and alkaline lignin. These results indicated that PBL hindgut-enriched actinomycetes could survive in soil by using the residual lignocellulose organic matter from plant residues, and the antibiotics produced not only give them a competitive advantage among soil microflora but also have a certain inhibitory effect on plant diseases and pests. This study suggests that the application of PBL frass can not only supplement soil humic acid but also potentially affect the soil microbiota of cultivated land, which is beneficial for the healthy growth of crops.

In Vivo and In Vitro Interactions between Exopolysaccharides from Bacillus thuringensis HD270 and Vip3Aa11 Protein.

Bacillus thuringiensis (Bt) secretes the nutritional insecticidal protein Vip3Aa11, which exhibits high toxicity against the fall armyworm (Spodoptera frugiperda). The Bt HD270 extracellular polysaccharide (EPS) enhances the toxicity of Vip3Aa11 protoxin against S. frugiperda by enhancing the attachment of brush border membrane vesicles (BBMVs). However, how EPS-HD270 interacts with Vip3Aa11 protoxin in vivo and the effect of EPS-HD270 on the toxicity of activated Vip3Aa11 toxin are not yet clear. Our results indicated that there is an interaction between mannose, a monosaccharide that composes EPS-HD270, and Vip3Aa11 protoxin, with a dissociation constant of Kd = 16.75 ± 0.95 mmol/L. When EPS-HD270 and Vip3Aa11 protoxin were simultaneously fed to third-instar larvae, laser confocal microscopy observations revealed the co-localization of the two compounds near the midgut wall, which aggravated the damage to BBMVs. EPS-HD270 did not have a synergistic insecticidal effect on the activated Vip3Aa11 protein against S. frugiperda. The activated Vip3Aa11 toxin demonstrated a significantly reduced binding capacity (548.73 ± 82.87 nmol/L) towards EPS-HD270 in comparison to the protoxin (34.96 ± 9.00 nmol/L). Furthermore, this activation diminished the affinity of EPS-HD270 for BBMVs. This study provides important evidence for further elucidating the synergistic insecticidal mechanism between extracellular polysaccharides and Vip3Aa11 protein both in vivo and in vitro.

Plants recruit insecticidal bacteria to defend against herbivore attacks.

Pest feeding affects the rhizobacteria community. The rhizomicrobiota activates salicylic acid and jasmonic acid signaling pathways to help plants deal with pest infestation. However, whether plants can recruit special pesticidal microorganisms to deal with attack from herbivores is unclear. A system composed of peanuts and first-instar larvae of Holotrichia parallela were used to analyze whether peanuts truly enrich the insecticidal bacteria after feeding by larvae, and whether inoculation of the enriched bacteria promotes the resistance of plants to herbivore. In this study, high-throughput sequencing of 16 S rRNA gene amplicons was used to demonstrate that infestation of the subterranean pest H. parallela quickly changed the rhizosphere bacterial community structure within 24 h, and the abundance of Enterobacteriaceae, especially Enterobacter, was manifestly enriched. Root feeding induced rhizobacteria to form a more complex co-occurrence network than the control. Rhizosphere bacteria were isolated, and 4 isolates with high toxicity against H. parallela larvae were obtained by random forest analysis. In a back-inoculation experiment using a split-root system, green fluorescent protein (GFP)-labeled Enterobacter sp. IPPBiotE33 was observed to be enriched in uneaten peanut roots. Additionally, supplementation with IPPBiotE33 alleviated the adverse effects of H. parallela on peanuts. Our findings indicated that herbivore infestation could induce plants to assemble bacteria with specific larvicidal activity to address threats.

Analysis of Synergism between Extracellular Polysaccharide from Bacillus thuringensis subsp. kurstaki HD270 and Insecticidal Proteins.

Bacillus thuringiensis (Bt) is the most widely used biopesticide worldwide and can produce several insecticidal crystal proteins and vegetative insecticidal proteins (Vips) at different growth stages. In our previous study, extracellular polysaccharides (EPSs) of Bt strain HD270 were found to enhance the insecticidal activity of Cry1Ac protoxin against Plutella xylostella (L.) and promote the binding of Cry1Ac to the intestinal brush border membrane vesicles (BBMVs). Whether the synergistic activity of Bt EPSs is common to other Cry1-type or Vip proteins is unclear, as is the potential synergistic mechanism. In this study, crude EPS-HD270 was found to increase the toxicity of Cry1-type toxins and Vip3Aa11 against different lepidopteran pests by approximately 2-fold. The purified EPS-HD270 also possessed synergistic activity against the toxicity of Cry1Ac and Vip3Aa11 against Spodoptera frugiperda (J.E. Smith) and Helicoverpa armigera (Hübner). Furthermore, we found that EPS-HD270 had a strong binding ability with Vip3Aa11 and promoted the binding of Vip3Aa11 to the BBMVs of H. armigera and S. frugiperda. Bt EPS-HD270 also protected Vip3Aa11 from proteolytic processing in larval midgut juice. Bt EPSs had universal synergistic effects on Cry1-type or Vip toxins against S. frugiperda and H. armigera. Bt EPS-HD270 exhibited synergistic activity with Vip3Aa through promotion of binding to BBMVs and protection from digestion by midgut protease. The results indicated that synergistic activity with Bt toxins was an important function of Bt EPSs, which was very different from other Bacillus spp.

Enterobacter Strain IPPBiotE33 Displays a Synergistic Effect with Bacillus thuringiensis Bt185.

The discovery and isolation of new non-Bt insecticidal bacteria and genes are significant for the development of new biopesticides against coleopteran pests. In this study, we evaluated the insecticidal activity of non-Bt insecticidal bacteria, PPBiotE33, IPPBiotC41, IPPBiotA42 and IPPBiotC43, isolated from the peanut rhizosphere. All these strains showed insecticidal activity against first- and third-instar larvae of Holotrichia parallela, Holotrichia oblita, Anomala corpulenta and Potosia brevitarsis. IPPBiotE33 showed the highest toxicity among the four strains and exhibited virulence against Colaphellus bowringi. The genome of IPPBiotE33 was sequenced, and a new protein, 03673, with growth inhibition effects on C. bowringi was obtained. In addition, IPPBiotE33 had a synergistic effect with Bacillus thuringiensis Bt185 against H. parallela in bioassays and back-inoculation experiments with peanut seedlings. IPPBiotE33 induced a decrease in hemocytes and an increase in phenol oxidase activity in H. parallela hemolymph, known as the immunosuppressive effect, which mediated synergistic activity with Bt185. This study increased our knowledge of the new insecticidal strain IPPBiotE33 and shed new light on the research on new insecticidal coaction mechanisms and new blended pesticides.

Identification and functional characterization of eight novel tpp family genes from Bacillus thuringiensis.

BACKGROUND: Bacillus thuringiensis (Bt) and its transgenic crops are widely used as biological control agents for agricultural pests. The tpp family is a branch of Bt insecticidal genes and consists of a few members. Research on the Tpp family proteins has focused on the binary toxins Gpp34Ab/Tpp35Ab and Tpp1/Tpp2, which need to function together to achieve insecticidal activity. However, only a few tpp family genes have been reported to exert insecticidal activity independently. This study aimed to identify and characterize tpp family genes that independently perform insecticidal functions. RESULTS: A total of 162 nucleotide sequences homologous to the single component Bt insecticidal gene tpp78Aa were obtained from the genome data of 1368 wild-type Bt strains, and 25 new full-length tpp family genes were identified. Eight new tpp family genes were successfully cloned and expressed, and bioassays of the expressed products were performed against five different pests. Bioassay results showed that these proteins exerted high insecticidal activity only against Laodelphax striatellus, a globally important rice pest, and were named Tpp78Ab1, Tpp78Bb1, Tpp78Ca1, Tpp78Da1, Tpp80Aa3, Tpp80Ac1, Tpp80Ad1, and Tpp80Ae1. The LC50 values of Tpp78Ab1, Tpp78Bb1, Tpp78Ca1, and Tpp80Ae1 against L. striatum were 8.1, 8.6, 10.1, and 9.6 µg mL-1 , respectively. The phylogenetic tree and conserved motifs indicated that the Tpp family had a common evolutionary ancestor. During evolution, the C-terminal pore-forming domain of the Tpp family adopted a similar arrangement; however, the N-terminal conserved motif showed high variability. CONCLUSION: Twenty-five full-length tpp family genes were identified. Eight new tpp family genes were cloned successfully, which could independently achieve insecticidal activity against L. striatellus. This provides abundant genetic resources for the biological control of important rice pests. In this study, we found that the relative conservation of the Tpp family proteins in the lengthy evolutionary process and the diversity generated for adapting to the environment can lay a theoretical foundation for an in-depth analysis of the function and evolution of the Tpp family. © 2023 Society of Chemical Industry.

Development of an Online Genome Sequence Comparison Resource for Bacillus cereus sensu lato Strains Using the Efficient Composition Vector Method.

An automated method was developed for differentiating closely related B. cereus sensu lato (s.l.) species, especially biopesticide Bacillus thuringiensis, from other human pathogens, B. anthracis and B. cereus sensu stricto (s.s.). In the current research, four typing methods were initially compared, including multi-locus sequence typing (MLST), single-copy core genes phylogenetic analysis (SCCGPA), dispensable genes content pattern analysis (DGCPA) and composition vector tree (CVTree), to analyze the genomic variability of 23 B. thuringiensis strains from aizawai, kurstaki, israelensis, thuringiensis and morrisoni serovars. The CVTree method was the best option to be used for typing B. thuringiensis strains since it proved to be the fastest method, whilst giving high-resolution data about the strains. In addition, CVTree agrees well with ANI-based method, revealing the relationship between B. thuringiensis and other B. cereus s.l. species. Based on these data, an online genome sequence comparison resource was built for Bacillus strains called the Bacillus Typing Bioinformatics Database to facilitate strain identification and characterization.

Prediction and bioactivity of small-molecule antimicrobial peptides from Protaetia brevitarsis Lewis larvae.

Antimicrobial peptides (AMPs) are widely recognized as promising natural antimicrobial agents. Insects, as the group of animals with the largest population, have great potential as a source of AMPs. Thus, it is worthwhile to investigate potential novel AMPs from Protaetia brevitarsis Lewis larvae, which is a saprophagous pest prevalent in China. In this study, comparing the whole-genome sequence of Protaetia brevitarsis Lewis larvae with the Antimicrobial Peptide Database (APD3) led to the identification of nine peptide templates that were potentially AMPs. Next, based on the peptide templates, 16 truncated sequences were predicted to the AMPs by bioinformatics software and then underwent structural and physicochemical property analysis. Thereafter, candidate small-molecule AMPs were artificially synthesized and their minimal inhibitory concentration (MIC) values were assessed. A candidate peptide, designated FD10, exhibited strong antimicrobial activity against both bacteria and fungi comprising Escherichia coli (MIC: 8 µg/mL), Pseudomonas aeruginosa (MIC: 8 µg/mL), Bacillus thuringiensis (MIC: 8 µg/mL), Staphylococcus aureus (MIC: 16 µg/mL), and Candida albicans (MIC: 16 µg/mL). Additionally, two other candidate peptides, designated FD12 and FD15, exhibited antimicrobial activity against both E. coli (MIC: both 32 µg/mL) and S. aureus (MIC: both 16 µg/mL). Moreover, FD10, FD12, and FD15 killed almost all E. coli and S. aureus cells within 1 h, and the hemolytic effect of FD10 (0.31%) and FD12 (0.40%) was lower than that of ampicillin (0.52%). These findings indicate that FD12, FD15, and especially FD10 are promising AMPs for therapeutic application. This study promoted the development of antibacterial drugs and provided a theoretical basis for promoting the practical application of antimicrobial peptides in the Protaetia brevitarsis Lewis larvae.

A New Method for Discovering Plant Biostimulants.

Structurally well-defined compounds have advantages for quality control in plant biostimulant production and application processes. Humic acid (HA) is a biostimulant that significantly affects plant growth, and soil-dwelling Protaetia brevitarsis larva (PBLs) can rapidly convert agricultural waste into HA. In this study, we use PBLs as a model to investigate HA formation and screen for structurally well-defined HA-related plant biostimulant compounds. Dephasing magic angle spinning nuclear magnetic resonance (13C DD-MAS NMR) analysis indicated HA structural changes during PBL digestion; metabolic profiling detected seven HA-related aromatic ring-containing compounds. A total of six compounds that significantly stimulate plant growth were identified through plant experiments, and all six compounds demonstrate the ability to enhance seed germination. It is noteworthy that piperic acid exhibits a remarkable promotion of root growth in plants, a finding reported for the first time in this study. Thus, this study not only provides insights into the insect-mediated transformation of HA but also illustrates a new method for discovering structurally well-defined plant biostimulant compounds.

Microflora for improving the Auricularia auricula spent mushroom substrate for Protaetia brevitarsis production.

Mushroom cultivation is a sustainable agricultural waste utilization method, but the lack of high-value utilization of the produced spent mushroom substrate (SMS) has hindered the development of mushroom cultivation-based circular agricultural systems. Conversion and utilization of SMS via Protaetia brevitarsis larvae (PBL) have proven to be a high-value AASMS utilization strategy. However, Auricularia auricula SMS (AASMS), which contains woodchips, is less palatable and digestible for PBL. To solve this problem, in this investigation, we screened out microflora (MF) for AASMS fermentation by comparing the fermentation performance as well as the effect on PBL feed intake, weight gain, and AASMS phytotoxic compound removal efficiency. In addition, by bacterial community analysis, the genera Luteimonas, Moheibacter, and Pseudoxanthomonas were predicted to be functional bacteria for AASMS fermentation and contribute to palatability and digestibility improvement.

The crystal structure of Cry78Aa from Bacillus thuringiensis provides insights into its insecticidal activity.

Genetically modified plants with insecticidal proteins from Bacillus thuringiensis (Bt) have been successfully utilized to control various kinds of pests in crop production and reduce the abuse of pesticides. However, a limited number of genes are available for the protection of crops from rice planthopper. Recently, Cry78Aa protein from Bt strain C9F1 has been found to have high insecticidal activity against Laodelphax striatellus and Nilaparvata lugens. It is the first reported single-component protein in the world to combat rice planthoppers, making it very promising for use in transgenic crops. The ambiguous mechanism of Cry78Aa functions prevented further engineering or application. Here, we report the crystal structure of Cry78Aa, which consists of two domains: a C-terminal ß-pore forming domain belonging to the aerolysin family and an N-terminal trefoil domain resembling the S-type ricin B lectin. Thus, Cry78Aa could represent a distinctive type of ß-pore forming toxin. We also found that Cry78Aa binds carbohydrates such as galactose derivatives and is essential for insecticidal activity against Laodelphax striatellus. Our results suggest a mechanism underlying the function of Cry78Aa against rice planthoppers and pave the way to maximizing the usage of the toxin.

Alteration of Manure Antibiotic Resistance Genes via Soil Fauna Is Associated with the Intestinal Microbiome.

Livestock wastes contain high levels of antibiotic resistance genes (ARGs) and a variety of human-related pathogens. Bioconversion of livestock manure using larvae of the beetle Protaetia brevitarsis is an effective technique for waste reduction and value creation; however, the fate of manure ARGs during gut passage and interaction with the gut microbiome of P. brevitarsis remains unclear. To investigate this, we fed P. brevitarsis with dry chicken manure for 6 days and measured bacterial community dynamics and ARG abundance and diversity along the P. brevitarsis gut tract using high-throughput quantitative PCR and metagenomics approaches. The diversity of ARGs was significantly lower in larval midgut, hindgut, and frass than in raw chicken manure, and around 80% of pathogenicity-related genes (PRGs) exhibited reduced abundance. Network analysis demonstrated that Bacteroidetes and Firmicutes were the key bacterial phyla associated with ARG reduction. Metagenomic analysis further indicated that ARGs, mobile genetic elements (MGEs), and PRGs were simultaneously attenuated in the hindgut, implicating a decreased likelihood for horizontal gene transfer (HGT) of ARGs among bacteria and pathogens during manure bioconversion. Our findings demonstrated that the attenuation of ARGs is strongly associated with the variation of the gut microbiome of P. brevitarsis, providing insights into mechanisms of risk mitigation of ARG dissemination during manure bioconversion. IMPORTANCE Saprophagous fauna like the oriental edible beetle (P. brevitarsis) plays a fundamental role in converting organic wastes into biofertilizer. Accumulating evidence has shown that soil fauna can reduce the abundance of ARGs, although the underlying mechanism of ARG reduction is still unclear. In our previous research, we found a large reduction of ARGs in vegetable roots and leaves from frass compared with raw manure, providing a promising biofertilizer for soil-vegetable systems. Therefore, in this study, temporal dynamic changes in the microbiomes of the donor (chicken manure) and host (P. brevitarsis) were investigated, and we found a close association between the gut microbiome and the alteration of ARGs. These results shed new light on how the insect gut microbiome can mitigate manure-borne ARGs and provide insights into the bioconversion process via a typical member of the saprophagous fauna, P. brevitarsis.

Lignocellulose degradation in Protaetia brevitarsis larvae digestive tract: refining on a tightly designed microbial fermentation production line.

BACKGROUND: The Scarabaeidae insect Protaetia brevitarsis (PB) has recently gained increasing research interest as a resource insect because its larvae can effectively convert decaying organic matter to plant growth-promoting frass with a high humic acid content and produce healthy, nutritional insect protein sources. Lignocellulose is the main component of PB larvae (PBL) feed, but PB genome annotation shows that PBL carbohydrate-active enzymes are not able to complete the lignocellulose degradation process. Thus, the mechanism by which PBL efficiently degrade lignocellulose is worthy of further study. RESULTS: Herein, we used combined host genomic and gut metagenomic datasets to investigate the lignocellulose degradation activity of PBL, and a comprehensive reference catalog of gut microbial genes and host gut transcriptomic genes was first established. We characterized a gene repertoire comprising highly abundant and diversified lignocellulose-degrading enzymes and demonstrated that there was unique teamwork between PBL and their gut bacterial microbiota for efficient lignocellulose degradation. PBL selectively enriched lignocellulose-degrading microbial species, mainly from Firmicutes and Bacteroidetes, which are capable of producing a broad array of cellulases and hemicellulases, thus playing a major role in lignocellulosic biomass degradation. In addition, most of the lignocellulose degradation-related module sequences in the PBL microbiome were novel. PBL provide organic functional complementarity for lignocellulose degradation via their evolved strong mouthparts, alkaline midgut, and mild stable hindgut microenvironment to facilitate lignocellulosic biomass grinding, dissolving, and symbiotic microbial fermentation, respectively. CONCLUSIONS: This work shows that PBL are a promising model to study lignocellulose degradation, which can provide highly abundant novel enzymes and relevant lignocellulose-degrading bacterial strains for biotechnological biomass conversion industries. The unique teamwork between PBL and their gut symbiotic bacterial microbiota for efficient lignocellulose degradation will expand the knowledge of holobionts and open a new beginning in the theory of holobionts. Video Abstract.

Characterization of Microorganisms from Protaetia brevitarsis Larva Frass.

Decomposers play an important role in the biogeochemical cycle. Protaetia brevitarsis larvae (PBLs) can transform wastes into frass rich in humic acid (HA) and microorganisms, which may increase the disease resistance of plants and promote plant growth. Beyond HA, the microorganisms may also contribute to the biostimulant activity. To address this hypothesis, we investigated the potential microbial community in the PBL frass samples and elucidated their functions of disease resistance and plant growth promotion. High-throughput sequencing analysis of four PBL-relevant samples showed that their frass can influence the microbial community of the surrounding environment. Further analysis showed that there were many microorganisms beneficial to agriculture, such as Bacillus. Therefore, culturable Bacillus microbes were isolated from frass, and 16S rDNA gene analysis showed that Bacillus subtilis was the dominant species. In addition, some Bacillus microorganisms isolated from the PBL frass had antibacterial activities against pathogenic fungi. The plant growth promotion pot experiment also proved that some strains promote plant growth. In conclusion, this study demonstrated that the microorganisms in the PBL frass are conducive to colonizing the surrounding organic matrix, which will help beneficial microbes to increase the disease resistance of plants and promote plant growth.

Attenuation of antibiotic resistance genes in livestock manure through vermicomposting via Protaetia brevitarsis and its fate in a soil-vegetable system.

Scarab larvae (Protaetia brevitarsis) could transform large quantities of agricultural waste into compost, providing a promising bio-fertilizer for soil management. There is an urgent need to assess the risk of antibiotic resistance genes (ARGs) in soil-vegetable system with application of compost derived from P. brevitarsis larvae. We conducted a pot experiment to compare the changes of ARGs in the soil and lettuce by adding four types of manure, livestock manure (chicken and swine manure) and the corresponding larval frass. Significantly low numbers of ARGs and mobile genetic elements (MGEs) were detected in both larval frass compared with the corresponding livestock manure. Pot experiment showed that the detected numbers of ARGs and MGEs in bulk soil, rhizosphere soil, and root endophytes were significantly lower in the frass-amended treatments than the raw manure-amended treatments. Furthermore, the relative abundance of ARGs and MGEs with application of chicken-frass was significant lower in rhizosphere soil and leaf endophyte. Using non-metric multidimensional scaling analysis, the patterns of soil ARGs and MGEs with chicken-frass application were more close to those from the bulk soil in the control. Structural equation models indicated that livestock manure addition was the main driver shaping soil ARGs with raw manure application, while MGEs were the key drivers in frass-amended treatments. These findings demonstrated that application of livestock manure vermicomposting via scarab larvae (P. brevitarsis) may be at low risk in spreading manure-borne ARGs through soil-plant system, providing an alternative technique for reducing ARGs in organic waste.

Structure characteristics and function of a novel extracellular polysaccharide from Bacillus thuringiensis strain 4D19.

Bacillus thuringiensis (Bt) are entomopathogenic bacteria that produce different kinds of insecticidal proteins. However, studies on Bt exopolysaccharides are lacking. Here, we aimed to explore the characteristics and insecticidal synergism of EPS, an exopolysaccharide from Bt strain 4D19. The molecular weight of EPS-2 was 58.0 kDa, which consisted of mannose (44.2%), GlcN (35.5%), D-GalN (8.0%), glucose (5.5%), arabinose (5.1%), galactose (0.9%), Man-UA (0.3%) and Glc-UA (0.2%). The toxicity of insecticidal proteins against Plutella xylostella was increased by adding EPS. EPS-2 bound to Cry1Ac protoxin and promoted the binding of Cry1Ac protoxin to the gut membrane of P. xylostella, but did not bind to activated toxins. These results suggested that EPS-2 may bind to the protoxin C-terminal region to enhance insecticidal activity. Our findings indicated that Bt strains produce exopolysaccharide to enhance the toxicity of insecticidal crystal proteins, which could be applied in biopesticide research and product development.

Plant Polysaccharides Modulate Biofilm Formation and Insecticidal Activities of Bacillus thuringiensis Strains.

After the biological pesticide Bacillus thuringiensis (Bt) is applied to the field, it has to remain on the surface of plants to have the insecticidal activities against insect pests. Bt can form biofilms on the surface of vegetable leaves, which were rich in polysaccharides. However, the relationship between polysaccharides of the leaves and the biofilm formation as well as the insecticidal activities of Bt is still unknown. Herein, this study focused on the effects of plant polysaccharides pectin and xylan on biofilm formation and the insecticidal activities of Bt strains. By adding pectin, there were 88 Bt strains with strong biofilm formation, 69 strains with weak biofilm formation, and 13 strains without biofilm formation. When xylan was added, 13 Bt strains formed strong biofilms, 98 strains formed weak biofilms, and 59 strains did not form biofilms. This indicated that two plant polysaccharides, especially pectin, modulate the biofilm formation of Bt strains. The ability of pectin to induce biofilm formation was not related to Bt serotypes. Pectin promoted the biofilms formed by Bt cells in the logarithmic growth phase and lysis phase at the air-liquid interface, while it inhibited the biofilms formed by Bt cells in the sporangial phase at the air-liquid interface. The dosage of pectin was positively correlated with the yield of biofilms formed by Bt cells in the logarithmic growth phase or lysis phase at the solid-liquid interfaces. Pectin did not change the free-living growth and the cell motility of Bt strains. Pectin can improve the biocontrol activities of the spore-insecticidal crystal protein mixture of Bt and BtK commercial insecticides, as well as the biofilms formed by the logarithmic growth phase or lysis phase of Bt cells. Our findings confirmed that plant polysaccharides modulate biofilm formation and insecticidal activities of Bt strains and built a foundation for the construction of biofilm-type Bt biopesticides.

Dominant egg surface bacteria of Holotrichia oblita (Coleoptera: Scarabaeidae) inhibit the multiplication of Bacillus thuringiensis and Beauveria bassiana.

Holotrichia oblita (Coleoptera: Scarabaeidae) and some other scarab beetles are the main soil-dwelling pests in China. Bacillus thuringiensis (Bt) and Beauveria bassiana (Bb) are entomopathogens that have been used as biocontrol agents of various pests. However, scarab larvae especially H. oblita exhibited strong adaptability to these pathogens. Compared to other scarabs, H. oblita could form a specific soil egg case (SEC) structure surrounding its eggs, and young larvae complete the initial development process inside this structure. In this study, we investigated the role of SEC structure and microorganisms from SEC and egg surface in pathogen adaptability. 16S rRNA gene analysis revealed low bacterial richness and high community unevenness in egg surface, with Proteobacteria, Firmicutes, Bacteroidetes and Fusobacteria dominating. In terms of OTUs composition analysis, the data show that the egg surface contains a large number of unique bacteria, indicating that the egg bacterial community may be derived from maternal transmission. Furthermore, we found that all culturable bacteria isolated from egg surface possessed antimicrobial activity against both Bt and Bb. The Pseudomonas bacteria with a significantly higher abundance in egg surface showed strong Bt- and Bb antagonistic ability. In conclusion, this study demonstrated a unique and antimicrobial bacterial community of H. oblita egg surface, which may contribute to its adaptability. Furthermore, the specific SEC structure surrounding the H. oblita eggs will provide a stable microenvironment for the eggs and egg surface bacteria, which probably provides more advantages for H. oblita adaptation ability.