Songqing Wu

Pretreatment of spent mushroom substrate for enhancing the conversion of fermentable sugar.

To develop a cost-effective biopesticide, spent mushroom substrate (SMS) extract was studied as a potential carbon source for cultivating Bacillus thuringiensis (Bt). Several pretreatments were compared to determine the optimal method for degrading cellulose to produce reducing sugars, including dilute sulfuric acid (0.5-2.0% v/v, 50-121°C, 1h), sodium hydroxide (0.5-2% w/v, 50-121°C, 1h), calcium hydroxide (0.2-4% w/v, 50-121°C, 1h), and hot water (50-121°C, 1h). Pretreatment was followed by standard enzymatic hydrolysis and fermentation. Results showed that the highest cellulose degradation was obtained using 2% dilute sulfuric acid pretreatment at 121°C for 1h, resulting in a high yield of reducing sugar (284.24 g/kg SMS). Sporulation was also highest using the same pretreatment. Use of SMS is not only an alternative way to commercialize Bt-based biopesticide, but also a potential solution for the environmental pollution associated with accumulation of the spent substrate of the mushroom industry.

Transcriptional profiling analysis of Spodoptera litura larvae challenged with Vip3Aa toxin and possible involvement of trypsin in the toxin activation.

Vip proteins, a new group of insecticidal toxins produced by Bacillus thuringiensis, are effective against specific pests including Spodoptera litura. Here, we report construction of a transcriptome database of S. litura by de novo assembly along with detection of the transcriptional response of S. litura larvae to Vip3Aa toxin. In total, 56,498 unigenes with an N50 value of 1,853 bp were obtained. Results of transcriptome abundance showed that Vip3Aa toxin provoked a wide transcriptional response of the S. litura midgut. The differentially expressed genes were enriched for immunity-related, metabolic-related and Bt-related genes. Twenty-nine immunity-related genes, 102 metabolic-related genes and 62 Bt-related genes with differential expression were found. On the basis of transcriptional profiling analysis, we focus on the functional validation of trypsin which potentially participated in the activation of Vip3Aa protoxin. Zymogram analysis indicated that the presence of many proteases, including trypsin, in S. litura larvae midgut. Results of enzymolysis in vitro of Vip3Aa by trypsin, and bioassay and histopathology of the trypsin-digested Vip3Aa toxin showed that trypsin was possibly involved in the Vip3Aa activation. This study provides a transcriptome foundation for the identification and functional validation of the differentially expressed genes in an agricultural important pest, S. litura.

The potential of the novel mosquitocidal Bacillus thuringiensis strain LLP29 for use in practice

1Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, 350002 Fuzhou, Fujian, People’s Republic of China 2Fujian International Travel Health Care Center, 350001 Fuzhou, Fujian, People’s Republic of China 3Biological Centre of the Academy of Sciences of the Czech Republic, Institute of Entomology, Branisovska 31, 37005 Ceske Budějovice, Czech Republic, gelbic@entu.cas.cz

Loop replacements with gut-binding peptides in Cry1Ab domain II enhanced toxicity against the brown planthopper, Nilaparvata lugens (Stål)

Bacillus thuringiensis (Bt) Cry toxins have been used widely in pest managements. However, Cry toxins are not effective against sap-sucking insects (Hemiptera), which limits the application of Bt for pest management. In order to extend the insecticidal spectrum of Bt toxins to the rice brown planthopper (BPH), Nilaparvata lugens, we modified Cry1Ab putative receptor binding domains with selected BPH gut-binding peptides (GBPs). Three surface exposed loops in the domain II of Cry1Ab were replaced with two GBPs (P2S and P1Z) respectively. Bioassay results showed that toxicity of modified toxin L2-P2S increased significantly (~9 folds) against BPH nymphs. In addition, damage of midgut cells was observed from the nymphs fed with L2-P2S. Our results indicate that modifying Cry toxins based on the toxin-gut interactions can broaden the insecticidal spectrum of Bt toxin. This method provides another approach for the development of transgenic crops with novel insecticidal activity against hemipteran insects and insect populations resistant to current Bt transgenic crops.

Use of Spent Mushroom Substrate for Production of Bacillus thuringiensis by Solid-State Fermentation

ABSTRACT The aim of this study was to explore a cost-effective method for the mass production of Bacillus thuringiensis (Bt) by solid-state fermentation. As a locally available agroindustrial byproduct, spent mushroom substrate (SMS) was used as raw material for Bt cultivation, and four combinations of SMS-based media were designed. Fermentation conditions were optimized on the best medium and the optimal conditions were determined as follows: temperature 32°C, initial pH value 6, moisture content 50%, the ratio of sieved material to initial material 1:3, and inoculum volume 0.5 ml. Large scale production of B. thuringiensis subsp. israelensis (Bti) LLP29 was conducted on the optimal medium at optimal conditions. High toxicity (1,487 international toxic units/milligram) and long larvicidal persistence of the product were observed in the study, which illustrated that SMS-based solid-state fermentation medium was efficient and economical for large scale industrial production of Bt-based biopesticides. The cost of production of 1 kg of Bt was approximately US

Identification of Genes Relevant to Pesticides and Biology from Global Transcriptome Data of Monochamus alternatus Hope (Coleoptera: Cerambycidae) Larvae

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Isolation and characterization of a novel native Bacillus thuringiensis strain BRC-HZM2 capable of degrading chlorpyrifos

Monochamus alternatus Hope is the main vector in China of the Pine Wilt Disease caused by the pine wood nematode Bursaphelenchus xylophilus. Although chemical control is traditionally used to prevent pine wilt disease, new strategies based in biological control are promising ways for the management of the disease. However, there is no deep sequence analysis of Monochamus alternatus Hope that describes the transcriptome and no information is available about gene function of this insect vector. We used next generation sequencing technology to sequence the whole fourth instar larva transcriptome of Monochamus alternatus Hope and successfully built a Monochamus alternatus Hope transcriptome database. In total, 105,612 unigenes were assigned for Gene Ontology (GO) terms, information for 16,730 classified unigenes was obtained in the Clusters of Orthologous Groups (COGs) database, and 13,024 unigenes matched with 224 predicted pathways in the Kyoto Encyclopedia of Genes and Genome (KEGG). In addition, genes related to putative insecticide resistance-related genes, RNAi, the Bt receptor, intestinal digestive enzymes, possible future insect control targets and immune-related molecules are described. This study provides valuable basic information that can be used as a gateway to develop new molecular tools for Monochamus alternatus Hope control strategies.

Pretreatment of wheat straw leads to structural changes and improved enzymatic hydrolysis

Studies were carried out to isolate chlorpyrifos degrading Bacillus thuringiensis (Bt) strains from chlorpyrifos‐contaminated samples. Six Bt strains (isolation rate 2.7%) were isolated by modified sodium acetate antibiotic heat treatment, and one novel strain (BRC‐HZM2) was selected for further analysis. Phenotype and phylogeny analysis of this strain was conducted on the basis of biochemical reactions, antibiotic sensitivity, 16s rRNA genes, plasmid profile, insecticidal crystal protein profiles, and PCR–RFLP for cry and cyt genes. The degradation rate of chlorpyrifos in liquid culture was estimated during 48 h of incubation for the isolate BRC‐HZM2. More than 50% of the initial chlorpyrifos concentration degraded within 12 h, 88.9% after 48 h. These results highlight the potential of the Bt strain for biological control and the bioremediation of environments contaminated with chlorpyrifos.

Identification of transcripts involved in digestion, detoxification and immune response from transcriptome of Empoasca vitis (Hemiptera: Cicadellidae) nymphs

Wheat straw (WS) is a potential biomass for production of monomeric sugars. However, the enzymatic hydrolysis ratio of cellulose in WS is relatively low due to the presence of lignin and hemicellulose. To enhance the enzymatic conversion of WS, we tested the impact of three different pretreatments, e.g. sulfuric acid (H2SO4), sodium hydroxide (NaOH), and hot water pretreatments to the enzymatic digestions. Among the three pretreatments, the highest cellulose conversion rate was obtained with the 4% NaOH pretreatment at 121 °C (87.2%). In addition, NaOH pretreatment was mainly effective in removing lignin, whereas the H2SO4 pretreatment efficiently removed hemicellulose. To investigate results of pretreated process for enhancement of enzyme-hydolysis to the WS, we used scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy to analyze structural changes of raw and treated materials. The structural analysis indicated that after H2SO4 and NaOH pretreatments, most of the amorphous cellulose and partial crystalline cellulose were hydrolyzed during enzymatic hydrolysis. The findings of the present study indicate that WS could be ideal materials for production of monomeric sugars with proper pretreatments and effective enzymatic base hydrolysis.

Physiological and biochemical response of Aedes aegypti tolerance to Bacillus thuringiensis

Tea production has been significantly impacted by the false-eye leafhopper, Empoasca vitis (Göthe), around Asia. To identify the key genes which are responsible for nutrition absorption, xenobiotic metabolism and immune response, the transcriptome of either alimentary tracts or bodies minus alimentary tract of E. vitis was sequenced and analyzed. Over 31 million reads were obtained from Illumina sequencing. De novo sequence assembly resulted in 52,182 unigenes with a mean size of 848nt. The assembled unigenes were then annotated using various databases. Transcripts of at least 566 digestion-, 224 detoxification-, and 288 immune-related putative genes in E. vitis were identified. In addition, relative expression of highly abundant transcripts was verified through quantitative real-time PCR. Results from this investigation provide genomic information about E. vitis, which will be helpful in further study of E. vitis biology and in the development of novel strategies to control this devastating pest.