Zhipeng Huang

Detection and Isolate Differentiation of Citrus tristeza virus in Infected Field Trees Based on Reverse Transcription-Polymerase Chain Reaction

Reverse transcription-polymerase chain reaction (RT-PCR) was compared with enzyme-linked immunosorbent assay (ELISA) and direct tissue blot immunoassay (DTBIA) for detection of non-decline-inducing and decline-inducing isolates of Citrus tristeza virus (CTV) in 21 field sweet orange and grapefruit plants on sour orange rootstock in Fort Pierce, FL. Among these samples, seven, six, and eight were infected with decline-inducing, non-decline-inducing, and both decline-inducing and non-decline-inducing isolates of CTV, respectively. However, there was not a good correlation between field symptoms and detection of the decline-inducing isolate. The results confirmed that RT-PCR is not only able to detect and differentiate decline-inducing and non-decline-inducing isolates of CTV in Florida, but also can detect both isolate types in a single field sweet orange or grapefruit tree. For most samples, results from RT-PCR, ELISA, and DTBIA were the same. However, the 320-bp fragments produced only from decline-inducing isolates were amplified from two sweet orange and two grapefruit samples that did not react with decline-inducing CTV-specific monoclonal antibody MCA13 in ELISA or DTBIA, indicating that RT-PCR has a higher sensitivity than these immunological tests for field sweet orange or grapefruit samples. Thus, RT-PCR is a simple, rapid, and specific procedure for CTV identification applicable to both research and diagnostic needs.

Expression and characterization of aiiA gene from Bacillus subtilis BS-1.

AHL-lactonase (AiiA), a metallo-beta-lactamase produced by Bacillus thuringiensis, Bacillus cereus and Bacillus anthracis, specifically hydrolyzes N-acyl-homoserine lactones (AHLs) secreted by Gram-negative bacteria and thereby attenuates the symptoms caused by plant pathogens. In this study, an aiiA gene was cloned from Bacillus subtilis BS-1 by PCR with a pair of degenerate primers. The deduced 250 amino acid sequence contained two small conserved regions, 103SHLHFDH109 and 166TPGHTPGH173, which are characteristic of the metallo-beta-lactamase family. Homology comparison revealed that the deduced amino acid sequence had a high degree of similarity with those of the known AiiA proteins in the B. cereus group. Additionally, the aiiA gene was expressed in Escherichia coli BL21 (DE3) pLysS and the expressed AiiA protein could attenuate the soft rot symptoms caused by Erwinia carotovora var. carotovora.

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.

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.

Insecticidal activity and histopathological effects of Vip3Aa protein from Bacillus thuringiensis on Spodoptera litura.

Vegetative insecticidal proteins (Vips) are insecticidal proteins synthesized by Bacillus thuringiensis during the vegetative stage of growth. In this study, Vip3Aa protein, obtained by in vitro expression of the vip3Aa gene from B. thuringiensis WB5, displayed high insecticidal activity against Spodoptera litura aside from Spodoptera exigua and Helicoverpa armigera. Bioassay results showed that the toxicity of Vip3Aa protein against S. litura larvae statistically decreased along with the increase of the age of the larvae, with LC50 = 2.609 ng/cm2 for neonatal larvae, LC50 = 28.778 ng/cm2 for first instar larvae, LC50 = 70.460 ng/cm2 for second instar larvae, and LC50 = 200.627 ng/cm2 for third instar larvae. The accumulative mortality of 100% larvae appeared at 72 h for all instars of S. litura larvae, when feeding respectively with 83.22, 213.04, 341.40, and 613.20 ng/cm2 of Vip3Aa toxin to the neonatal and first to third instar larvae. The histopathological effects of Vip3Aa toxin on the midgut epithelial cells of S. litura larvae was also investigated. The TEM observations showed wide damage of the epithelial cell in the midgut of S. litura larvae fed with Vip3Aa toxin.

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

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

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Biological Activity of Bacillus thuringiensis (Bacillales: Bacillaceae) Chitinase Against Caenorhabditis elegans (Rhabditida: Rhabditidae)

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.

Physiological and biochemical response of Aedes aegypti tolerance to Bacillus thuringiensis

ABSTRACT In addition to being used increasingly as a model system in modern molecular biology studies, the free-living nematode Caenorhabditis elegans (Maupas, 1900) is an important pathogen in fungi and straw mushrooms. In this study, Bacillus thuringiensis strain 010 was found to have significantly detrimental activity against C. elegans. To further characterize this activity, the toxicological mechanism was elucidated at molecular level. Genes encoding for crystal protein and chitinase were isolated, cloned, and sequenced. However, the toxicity was detected only in the chitinase. Under transmission electron microscopy, change in the body wall and gut structures of C. elegans was observed, and thus degeneration of body wall and gut in the worms was also investigated. Further bioassay also confirmed the mortality of C. elegans fed with Escherichia coli TB1 strain. These observations suggest great potential for B. thuringiensis 010 as a biocontrol agent against C. elegans and other nematodes.