Xingming Yang

A new bioorganic fertilizer can effectively control banana wilt by strong colonization with Bacillus subtilis N11

Fusarium wilt is one of the most serious diseases caused by a soil-borne pathogen affecting banana production. The goal of this study was to evaluate the capability of a novel bio-organic fertilizer (BIO2) that integrated the biocontrol agent Bacillus subtilis N11, and mature composts to control Fusarium wilt of banana in pot experiments. The results showed that the application of the BIO2 significantly decreased the incidence rate of Fusarium wilt compared to the control. To determine the antagonistic mechanism of the strain, we also studied the colonization of the natural biocontrol agent on banana roots using a GFP marker. The studies were performed in a hydroponic culture system, a sand system and a natural soil system. The results indicated that the bacteria colonized predominantly by forming biofilms along the elongation and differentiation zones of the roots. The fact that similar observations were obtained in all three systems suggests that colonization by N11 can be studied in a defined system. The population of B. subtilis N11 in the rhizosphere and on banana roots was also monitored. We speculate that the colonization pattern of B.subtilis N11 can be linked to the mechanism of protection of plants from fungal infection.

Biocontrol of Rhizoctonia solani damping-off disease in cucumber with Bacillus pumilus SQR-N43.

Biological control is an efficient and environmentally friendly way to prevent damping-off disease. Micrographs were used to investigate the ability of Bacillus pumilus (B. pumilus) SQR-N43 to control Rhizoctonia solani (R. solani) Q1 in cucumbers. The root colonization ability of B. pumilus SQR-N43 was analyzed in vivo with a green fluorescent protein (GFP) tag. A pot experiment was performed to assess the in vivo disease-control efficiency of B. pumilus SQR-N43 and its bio-organic fertilizer. Results indicate that B. pumilus SQR-N43 induced hyphal deformation, enlargement of cytoplasmic vacuoles and cytoplasmic leakage in R. solani Q1 mycelia. A biofilm on the root surface was formed when the roots were inoculated with 10(7)-10(8)cells g(-1) of soil of GFP-tagged B. pumilus SQR-N43. In the pot experiment, the biocontrol reduced the concentration of R. solani. In contrast to applications of only B. pumilus SQR-N43 (N treatment), which produced control efficiencies of 23%, control efficiencies of 68% were obtained with applications of a fermented organic fertilizer inoculated with B. pumilus SQR-N43 (BIO treatment). After twenty days of incubation, significant differences in the number of CFUs and the percentage of spores of B. pumilus SQR-N43 were recorded between the N treatment (2.20×10(7)CFU g(-1) of soil and 79%, respectively) and the BIO treatment (1.67×10(8)CFU g(-1) of soil and 52%, respectively). The results indicate that B. pumilus SQR-N43 is a potent antagonist against R. solani Q1. The BIO treatment was more effective than the N treatment because it stabilized the population and increased the active form of the antagonist.

Characterization of a thermostable β-glucosidase from Aspergillus fumigatus Z5, and its functional expression in Pichia pastoris X33

BackgroundRecently, the increased demand of energy has strongly stimulated the research on the conversion of lignocellulosic biomass into reducing sugars for the subsequent production, and β-glucosidases have been the focus because of their important roles in a variety fundamental biological processes and the synthesis of useful β-glucosides. Although the β-glucosidases of different sources have been investigated, the amount of β-glucosidases are insufficient for effective conversion of cellulose. The goal of this work was to search for new resources of β-glucosidases, which was thermostable and with high catalytic efficiency.ResultsIn this study, a thermostable native β-glucosidase (nBgl3), which is secreted by the lignocellulose-decomposing fungus Aspergillus fumigatus Z5, was purified to electrophoretic homogeneity. Internal sequences of nBgl3 were obtained by LC-MS/MS, and its encoding gene, bgl3, was cloned based on the peptide sequences obtained from the LC-MS/MS results. bgl 3 contains an open reading frame (ORF) of 2622 bp and encodes a protein with a predicted molecular weight of 91.47 kDa; amino acid sequence analysis of the deduced protein indicated that nBgl3 is a member of the glycoside hydrolase family 3. A recombinant β-glucosidase (rBgl3) was obtained by the functional expression of bgl 3 in Pichia pastoris X33. Several biochemical properties of purified nBgl3 and rBgl3 were determined - both enzymes showed optimal activity at pH 6.0 and 60°C, and they were stable for a pH range of 4-7 and a temperature range of 50 to 70°C. Of the substrates tested, nBgl3 and rBgl3 displayed the highest activity toward 4-Nitrophenyl-β-D-glucopyranoside (pNPG), with specific activities of 103.5 ± 7.1 and 101.7 ± 5.2 U mg-1, respectively. However, these enzymes were inactive toward carboxymethyl cellulose, lactose and xylan.ConclusionsAn native β-glucosidase nBgl3 was purified to electrophoretic homogeneity from the crude extract of A. fumigatus Z5. The gene bgl 3 was cloned based on the internal sequences of nBgl3 obtained from the LC-MS/MS results, and the gene bgl3 was expressed in Pichia pastoris X33. The results of various biochemical properties of two enzymes including specific activity, pH stability, thermostability, and kinetic properties (Km and Vmax) indicated that they had no significant differences.

PARAFAC modeling of fluorescence excitation−emission spectra for rapid assessment of compost maturity

Assessment of compost maturity is crucial for achieving high quality compost in order to guarantee its marketability. In this context, a novel technique that combines fluorescence excitation-emission matrices (EEMs) with parallel factor (PARAFAC) analysis to assess compost maturity is presented. A total of 60 fluorescence EEMs of composts were successfully decomposed into a three-factor model using PARAFAC analysis. Components 1 [excitation/emission (Ex/Em) wavelengths=(230, 330)/410] and 2 [Ex/Em wavelengths=(250, 350)/450] were attributable to humic-like and fulvic-like substances, whereas component 3 [Ex/Em wavelengths=(220, 280)/340] belonged to protein-like substances. Pearson correlation analysis between the common maturity indices and log scores of three components demonstrated that components 1 and 3 are more suitable to assess compost maturity than component 2. These results reveal that EEM-PARAFAC could be applied as a valuable tool for assessing compost maturity, given its high sensitivity and selectivity.

Solid-state fermentation of agro-industrial wastes to produce bioorganic fertilizer for the biocontrol of Fusarium wilt of cucumber in continuously cropped soil.

Agro-industrial wastes of cattle dung, vinegar-production residue and rice straw were solid-state fermented by inoculation with Trichoderma harzianum SQR-T037 (SQR-T037) for production of bioorganic fertilizers containing SQR-T037 and 6-pentyl-α-pyrone (6PAP) to control Fusarium wilt of cucumber in a continuously cropped soil. Fermentation days, temperature, inoculum and vinegar-production residue demonstrated significant effects on the SQR-T037 biomass and the yield of 6PAP, based on fractional factorial design. Three optimum conditions for producing the maximum SQR-T037 biomass and 6PAP yield were predicted by central composite design and validated. Bioorganic fertilizer containing 8.46 log(10) ITS copies g(-1) dry weight of SQR-T037 and 1291.73 mg kg(-1) dry weight of 6PAP, and having the highest (p<0.05) biocontrol efficacy, was achieved at 36.7 fermentation days, 25.9°C temperature, 7.6% inoculum content, 41.0% vinegar-production residue, 20.0% rice straw and 39.0% cattle dung. This is a way to offer a high value-added use for agro-industrial wastes.

Expression, purification and characterization of two thermostable endoglucanases cloned from a lignocellulosic decomposing fungi Aspergillus fumigatus Z5 isolated from compost

Two genes encoding endoglucanase, designated as egl2 and egl3, were cloned from a lignocellulosic decomposing fungus Aspergillus fumigatus Z5 and were successfully expressed in Pichia pastoris X33. The deduced amino acid sequences encoded by egl2 and egl3 showed strong similarity with the sequence of glycoside hydrolase family 5. SDS-PAGE and western blot assays indicated that the recombinant enzymes were secreted into the culture medium and the zymogram analysis confirmed that both recombinant enzymes had endoglucanase activity. Several biochemical properties of the two recombinant enzymes were studied: Egl2 and Egl3 showed optimal activity at pH 5.0 and 4.0, respectively, and at 50 and 60°C, respectively. Egl2 and Egl3 showed good pH stability in the range of 4-7, and both enzymes demonstrated good thermostability ranging from 30 to 60°C. The K(m) and V(max) values using carboxymethyl cellulose (CMC, soluble cellulose, polymerized by β-1, 4-linked glucose residues) as the substrate at optimal conditions were determined. The activities of the enzymes on a variety of cello-oligosaccharide substrates were investigated, and Egl2 can hydrolyze cellotetraose and cellopentaose but not cellobiose and cellotriose, whereas Egl3 can hydrolyze all cello-oligosaccharides, except cellobiose.

Application of Trichoderma harzianum SQR-T037 bio-organic fertiliser significantly controls Fusarium wilt and affects the microbial communities of continuously cropped soil of cucumber

BACKGROUND The reduction in diversity of the soil microbial community causes the disorder of continuous cropping. The aim of this study was to determine the effects of applying Trichoderma harzianum SQR-T037 bio-organic fertiliser (BIO) on the microbial community in continuously cropped cucumber soil. Four treatments were set: (1) control, where neither seedling nursery soil (N) nor transplanted soil (T) was amended with BIO; (2) N treatment, where nursery soil was amended with BIO (1% w/w) but transplanted soil was not; (3) N + T treatment, where BIO was added to both nursery soil (1% w/w) and transplanted soil (0.5% w/w); (4) uncropped soil, where soil was left uncropped consistently. RESULTS A disease index of 72.2% was found for the control treatment, while the N and N + T treatments had disease indices of only 25 and 15% respectively. Analysis of the denaturing gradient gel electrophoresis (DGGE) profiles showed that the bacterial communities of the N and N + T treatments were similar to those of the uncropped soil but distinct from those of the control soil. The fungal communities of the N and N + T treatments differed from those of both the uncropped soil and the control. CONCLUSION Addition of BIO to both the nursery soil and the transplanted soil can diversify the microbial community in continuously cropped cucumber soil and thus effectively control Fusarium wilt of cucumber plants.

Optimization of the production of poly-γ-glutamic acid by Bacillus amyloliquefaciens C1 in solid-state fermentation using dairy manure compost and monosodium glutamate production residues as basic substrates.

Poly-γ-glutamic acid (γ-PGA) is a polymer with uses in foods, cosmetics, medicine and agriculture. The medium for the production of γ-PGA by Bacillusamyloliquefaciens C1 was optimized by response surface methodology using agro-industrial wastes in solid-state fermentation (SSF). The optimal SSF medium (20g substrates with 50% initial moisture) for producing γ-PGA was determined to contain 5.51g dairy manure compost, 1.91g soybean cake, 0.57g corn flour, 2.15g monosodium glutamate production residues, 1.5g wheat bran, 0.5g rapeseed cake, 0.1g citric acid, 0.05g MgSO(4)·7H(2)O and 0.03g MnSO(4)·H(2)O. In this medium the strain produced up to 0.0437g γ-PGA per gram of substrates when cultured for 48h at 37°C. SDS-PAGE showed that the molecular weight of the γ-PGA was more than 130kDa. Due to the high-yields observed and the low-cost nature of the optimal medium, this study indicates a possibility to establish economical large-scale production of γ-PGA.

Fluorescence excitation―emission spectroscopy with regional integration analysis for assessment of compost maturity

Composting of animal manures is believed as an alternative way for directly recycling them in farms, and therefore assessment of compost maturity is crucial for achieving high quality compost. Fluorescence excitation-emission matrices (EEMs) combined with regional integration analysis is presented to assess compost maturity. The results showed that the EEM contours of water-extract organic matter (WEOM) from immature composts exhibited four peaks at excitation/emission (Ex/Em) of 220/340nm, 280/340nm, 220/410nm, and 330/410nm, whereas EEM contour of WEOM from mature composts had only two peaks at Ex/Em of 230/420nm and 330/420nm. Pearson correlation demonstrated that peaks intensity rather than their ratios had a significantly correlation with the common indices assessing compost maturity, whereas the normalized excitation-emission area volumes (Φ(i,n)s) from regional integration analysis had a stronger correlation with the common indices assessing compost maturity than peaks intensity. It is concluded that the Φ(i,n)s from regional integration analysis are more suitable to assess the maturity of compost than the intensities of peaks. Therefore, the fluorescence spectroscopy combined with regional integration analysis can be used as a valuable industrial and research tool for assessing compost maturity, given its high sensitivity and selectivity.

Evaluation of metal ions (Zn2+, Fe3+ and Mg2+) effect on the production of fusaricidin-type antifungal compounds by Paenibacillus polymyxa SQR-21

Effect of metal ions (Mg(2+), Zn(2+) and Fe(3+)) on the production of fusaricidin-type antifungal compounds by Paenibacillus polymyxa SQR-21 was studied in liquid culture. First, one-factor; three-level experiments were conducted to find out optimal concentrations of each metal ion for maximum production of fusaricidins. Later, three-factor; five-level experiments were performed and a quadratic predictive model was developed using response surface methodology (RSM). The results indicated that Fe(3+) and Mg(2+) positively affected the growth of P. polymyxa as determined by measuring the OD(600) of the liquid culture. The production of fusaricidin-type antifungal compounds was significantly inhibited by Zn(2+) (P=0.0114) and increased by Mg(2+) (P=0.0051) but the effect of Fe(3+) (P=0.2157) was non-significant. However, a synergistic positive effect of Mg(2+) and Fe(3+) on the production of antifungal compounds was observed. This study sheds lights on the pertinent effects of the individual and combined metal ions on the production of fusaricidins in P. polymyxa. It provides the key information for optimization of the metal ions in the fermentation media to achieve the maximum antibiotic production in this strain.