Expression patterns of chitinase produced from Paenibacillus chitinolyticus with different two culture media

To investigate the expression patterns of chitinase isozymes on native-PAGE and SDS-PAGE gels Paenibacillus chitinolyticus MP-306 was cultured on culture media with and without chitin substrate. P. chitinolyticus MP-306 had a strong chitinolytic activity on colloidal chitin medium. Chitinase isozymes of MP-306 were expressed as six bands (CN1-CN6) on native-PAGE gels and thirteen bands (CS1-CS13) on SDS-PAGE gels after incubation in chitin medium. Three bands (CN1, CN2, and CN3) of chitinase isozymes of MP-306 on native-PAGE gels were expressed as nine bands (CS1, CS2, CS3, CS4, CS5, CS6, CS8, CS10, and CS13) of chitinase isozymes on SDS-PAGE gels. Three bands (CN4, CN5, and CN6) of chitinase isozymes of MP-306 were strongly inhibited by metal ions on native-PAGE and SDS-PAGE gels.     

Benzothiadiazole as an Inducer of β-1,3-Glucanase and Chitinase Isozymes in Sugar Beet

The effect of benzothiadiazole (BTH) on protein synthesis was studied in sugar beet plants. Extracellular proteins induced by 0.025 % BTH were examined and their pattern was compared with that induced by sodium salicylate, chitosan, paraquat, AgNO₃, and by tobacco necrosis virus. BTH induced synthesis of at least 9 acidic and 6 basic proteins; three of them appeared as acidic chitinase isozymes, three as acidic β-1,3-glucanase isozymes, three as basic chitinase isozymes, and one as a basic β-1,3-glucanase isozyme. One of the basic chitinase isozymes was found also in control plants. The most of the newly formed proteins was also induced by the other inducers under study regardless of the necrotic or symptomless reaction of plants. The benzothiadiazole proved to be an efficient inducer of proteins in sugar beet. This revised version was published online in July 2006 with corrections to the Cover Date.     

High-level expression of a specific β-1,3-1,4-glucanase from the thermophilic fungus Paecilomyces thermophila in Pichia pastoris

In this study, a novel β-1,3-1,4-glucanase gene (designated as PtLic16A) from Paecilomyces thermophila was cloned and sequenced. PtLic16A has an open reading frame of 945 bp, encoding 314 amino acids. The deduced amino acid sequence shares the highest identity (61%) with the putative endo-1,3(4)-β-glucanase from Neosartorya fischeri NRRL 181. PtLic16A was cloned into a vector pPIC9K and was expressed successfully in Pichia pastoris as active extracellular β-1,3-1,4-glucanase. The recombinant β-1,3-1,4-glucanase (PtLic16A) was secreted predominantly into the medium which comprised up to 85% of the total extracellular proteins and reached a protein concentration of 9.1 g l⁻¹ with an activity of 55,300 U ml⁻¹ in 5-l fermentor culture. The enzyme was then purified using two steps, ion exchange chromatography, and gel filtration chromatography. The purified enzyme had a molecular mass of 38.5 kDa on SDS–PAGE. It was optimally active at pH 7.0 and a temperature of 70°C. Furthermore, the enzyme exhibited strict specificity for β-1,3-1,4-d-glucans. This is the first report on the cloning and expression of a β-1,3-1,4-glucanase gene from Paecilomyces sp.     

Purification and characterization of a β-1,3-glucanase from the novel mycoparasite <Emphasis Type="Italic">Periconia byssoides</Emphasis>

An extracellular β-1,3-glucanase with antifungal properties was secreted by the novel mycoparasite, Periconia byssoides. The glucanase has a molecular mass of 35 kDa estimated by SDS-PAGE. Its optimum activity was at pH 6.0 and 50°C (over 2 h). The purified β-1,3-glucanase was capable of degrading cell walls, and inhibiting mycelia growth and spore germination of plant pathogenic fungi including Fulvia fulva, Fusarium sp. and Rhizoctonia solani. The N-terminal amino acid residues of the purified β-1,3-glucanase are LKNGGPSFGA, which do not have any homology with previously described glucanases, suggesting it may be a novel member of the fungal β-1,3-glucanases. Chao Lin and Jinkui Yang contributed equally to this work.     

Purification,characterization and differential hormonal regulation of a β-1,3-glucanase and two chitinases from chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) cell-suspension cultures were used to isolate one -1,3-glucanase (EC 3.2.1.29) and two chitinases (EC 3.2.1.14). The -1,3-glucanase (M_r = 36 kDa) and one of the chitinases (M_r = 32 kDa) belong to class I hydrolases with basic isoelectric points (10.5 and 8.5, respectively) and were located intracellularly. The basic chitinase (BC) was also found in the culture medium. The second chitinase (M_r = 28 kDa), with an acidic isoelectric point of 5.7, showed homology to N-terminal sequences of class III chitinases and represented the main protein accumulating in the culture medium. Polyclonal antibodies raised against the basic -1,3-glucanase (BG) and the acidic chitinase (AC) were shown to be monospecific. The anti-AC antiserum failed to recognize the BC on immune blots, confirming the structural diversity between class I and class III chitinases. Neither chitinase exhibitied lysozyme activity. All hydrolases were endo in action on appropriate substrates. The BC inhibited the hyphal growth of several test fungi, whereas the AC failed to show any inhibitory activity. Expression of BG activity appeared to be regulated by auxin in the cell culture and in the intact plant. In contrast, the expression of neither chitinase was apparently influenced by auxin, indicating a differential hormonal regulation of -1,3-glucanase and chitinase activities in chickpea. After elicitation of cell cultures or infection of chickpea plants with Ascochyta rabiei, both system were found to have hydrolase patterns which were qualitatively and quantitatively comparable. Finally, resitant (ILC 3279) and susceptible (ILC 1929) cultivars of chickpea showed no appreciable differences with regard to the time and amount of hydrolase accumulation after inoculation with spores of A. rabiei.Abbreviations AC acidic chitinase - BC basic chitinase - BG = basic -1,3-glucanase - CM-Chitin-RBV carboxymethylated-chitin-remazol brilliant violet - 2,4-D 2,4-dichlorophenoxyacetic acid - ILC international legume chickpea - M_r relative molecular mass - pI isoelectric point - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis We thank the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie for financial support and ICARDA, Aleppo, Syria, for the provision of seed material. We also thank Dr. B. Fritig (Institut de Biologie Moléculaire des Plantes, CNRS, Straßbourg, France) and Dr. F. Meins, Jr. (Friedrich-Miescher-Institut, Basel, Switzerland) for their kind gifts of antibodies.     

β-1,3-Glucanase and dimorphism in Paracoccidioides brasiliensis

Mycelial and yeast forms of P. brasiliensis were tested for several glucohydrolases. In addition to high levels of -blucanases, low amounts of -glucanase, chitinase and maltase were found. Tests for invertase, amylase and lactase were negative. The levels of -1,3-glucanase were higher in the mycelial form. The shift to the mycelial phase correlated with an increase in the levels of -1,3-glucanase. The enzyme was present in the cytoplasm, cell wall and culture medium. The extracellular enzyme was purified 42 fold by ammonium sulphate precipitation and gel filtration. Maximal activity was obtained at 60°C and pH of 5.0 acetate buffer or pH 6.0 (phosphate buffer). Its K _m was 0.205 mg/ml. The cell wall-bound enzyme showed a higher temperature optimum. Optimum pH and K _m were also slightly different. Following treatment of the cell walls with chitinase, -1,3-glucanase was released into the medium.     

Variability in antifungal proteins in the grains of maize, sorghum and wheat

Crude protein extracts were made from kernels of 12 cultivars each of maize, sorghum and wheat. These preparations were fractionated on sodium dodecyl sulfate (SDS)-polyacrylamide gels and subjected to Western blot analyses. Bands corresponding to chitinases and β-glucanases were identified immunologically (Western blots) and on activity gels. Ribosome Inactivating Protein(s) (RIP) and permatins were identified immunologically. In maize, two chitinase bands (25–29 kDa) were seen in all cultivars, and a third band of about 23 kDa was detected in 7 of the 12 cultivars. Two or three β-glucanase bands of sizes between 24 and 36 kDa (probably a mixture of 1,3–β- and 1,3–1,4-β-glucanases) were detected in blots of SDS gels, and one band was detected in activity gels (1,3-β-glucanase). In sorghum, one chitnase band of approximately 29 kDa, and two or three additional bands ranging in size from 21–24 kDa were observed. Only one β-glucanase band was identified, with an estimated molecular weight of 30 kDa. All bands that appeared on Western blots of SDS gels corresponded to bands detected on activity gels. In wheat, one chitmase band of around 20 kDa, one β-glucanase band of about 30 kDa and one RIP band of about 30 kDa were identified. Permatins (molecular weight about 22 kDa) were identified in maize, sorghum and wheat, with the different cultivars having varying amounts of permatins.     

Induction of pathogenesis-related proteins during biocontrol of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> with <Emphasis Type="Italic">Pseudomonas aureofaciens</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merr.) plants

To investigate the biocontrol effectiveness of the antibiotic producing bacterium, Pseudomonas aureofaciens 63–28 against the phytopathogen Rhizoctonia solani AG-4 on Petri plates and in soybean roots, growth response and induction of PR-proteins were estimated after inoculation with P. aureofaciens 63–28 (P), with R. solani AG-4 (R), or with P. aureofaciens 63–28 + R. solani AG-4 (P + R). P. aureofaciens 63–28 showed strong antifungal activity against R. solani AG-4 pathogens in Petri plates. Treatment with P. aureofaciens 63–28 alone increased the emergence rate, shoot fresh weight, shoot dry weight and root fresh weight at 7 days after inoculation, when compared to R. solani AG-4; P + R treatment showed similar effects. Peroxidase (POD) and β-1,3-glucanase activity of P. aureofaciens 63–28 treated roots increased by 41.1 and 49.9%, respectively, compared to control roots. POD was 26% greater in P + R treated roots than R. solani treated roots. Two POD isozymes (59 and 27 kDa) were strongly induced in P + R treated roots. The apparent molecular weight of chitinase from treated roots, as determined through SDS-PAGE separation and comparison with standards, was about 29 kDa. Five β-1,3-glucanase isozymes (80, 70, 50, 46 and 19 kDa) were observed in all treatments. These results suggest that inoculation of soybean plants with P. aureofaciens 63–28 elevates plant growth inhibition by R. solani AG-4 and activates PR-proteins, potentially through induction of systemic resistance mechanisms.     

Purification of exo-1,3-beta-glucanase, a new extracellular glucanolytic enzyme from Talaromyces emersonii

The moderately thermophilic aerobic ascomycete Talaromyces emersonii secretes, under selected growth conditions, several β-glucan hydrolases including an exo-1,3-β-glucanase. This enzyme was purified to apparent homogeneity in order to characterise its biochemical properties and investigate hydrolysis of different β-glucans, including laminaran, a 1,3-β-glucan from brown algae. The native enzyme is monomeric with a molecular mass of ~40 kDa and a pI value of 4.3, and is active over broad ranges of pH and temperature, with optimum activity observed at pH 5.4 and 65 °C. At pH 5.0, the enzyme displays strict specificity for laminaran (apparent K _m 1.66 mg mL⁻¹; V _max 7.69 IU mL⁻¹) and laminari-oligosaccharides and did not yield activity against 1,4-β-glucans, 1,3;1,4-β-glucans or 4-nitrophenyl- and methylumbelliferyl-β-d-glucopyranosides. Analysis of hydrolysis products formed during time-course hydrolysis of laminaran by high-performance anion exchange chromatography with pulsed amperometric detection revealed a strict exo mode of action, with glucose being the sole reaction product even at the initial stages of hydrolysis. The T. emersonii exo-1,3-β-glucanase was inhibited by glucono-δ-lactone (K _i 1.25 mM) but at significantly higher concentrations than typically inhibitory for exo-glycosidases such as β-glucosidase. ‘De novo’ sequence analysis of the purified enzyme suggests that it belongs to family GH5 of the glycosyl hydrolase superfamily. The results clearly show that the exo-1,3-β-glucanase is yet another novel enzyme present in the β-glucanolytic enzyme system of T. emersonii.     

Induction of pathogenesis-related proteins in sugarcane leaves and cell-cultures by a glycoprotein elicitor isolated from <Emphasis Type="Italic">Colletotrichum falcatum</Emphasis>

The induction of pathogenesis-related (PR) proteins in sugarcane (Saccharum officinarum L.) leaves and suspension-cultured cells in response to treatment with a glycoprotein elicitor isolated from Colletotrichum falcatum (the red rot pathogen) was investigated. Treatment of leaves and cells with the elicitor resulted in a much marked increase in the activities of chitinase and β-1,3-glucanase in red rot resistant (BO 91) than susceptible (CoC 671) sugarcane cultivar. SDS-PAGE analysis revealed that C. falcatum elicitor induced the accumulation of several proteins in suspension-cultured cells of resistant cultivar (BO 91); among them the 35 kDa protein was predominant. Whereas, a 27 kDa protein was induced predominantly in the cells of susceptible cultivar upon treatment with the elicitor. When sugarcane leaves were treated with C. falcatum elicitor, two proteins with apparent molecular masses of 25 and 27 kDa were induced both in the resistant and susceptible cultivars. However, the induction was stronger in the resistant than the susceptible cultivar. Immunoblot analysis for chitinase indicated that a protein with an apparent molecular mass of 37 kDa cross-reacting with barley chitinase antiserum was strongly induced in the suspension cultured cells of both the cultivars. The induction of 37 kDa chitinase was more in the cells of resistant cultivar than in the susceptible cultivar. Western blot analysis revealed that a 25 kDa thaumatin-like protein (TLP) cross-reacting with bean TLP antiserum was strongly induced in leaves and cultured cells of both resistant and susceptible cultivars due to elicitor treatment.     

Local and Systemic Induction of β-1,3-Glucanase and Chitinase in Coffee Leaves Protected Against Hemileia vastatrix by Bacillus thuringiensis

β-1,3-glucanase and chitinase activities were induced locally and systemically 4–25 and 11–25 days, respectively, after spraying the surface of the third pair of coffee leaves from the apex of 8-month-old plants with a 50 mg/ml aqueous suspension of Bacillus thuringiensis in a commercial formulation (Thuricide HP-Sandoz). The treatment also induced local and systemic resistance against Hemileia vastatrix after the application of the inducer. Within 14–18 days of application of the Thuricide inducer, the β-1,3-glucanase activity in the locally and systemically-protected unchallenged leaves reached maximum levels of 226% and 279% higher levels respectively, than in control plants. The chitinase activity reached maximum levels of 224% and 181% respectively, within 18–21 days after treatment with the inducer. Two β-1,3-glucanase bands were detected by native PAGE electrophoresis in extracts from locally-and systemicallyprotected unchallenged coffee leaves.     

Ethylene-induced chitinase and β-1,3-glucanase accumulate specifically in the lower epidermis and along vascular strands of bean leaves

We have studied the spatial pattern of accumulation of chitinase (EC 3.2.1.14) and -1,3-glucanase (EC 3.2.1.39) in ethylene-treated leaves of bean (Phaseolus vulgaris L.). Electron-microscopical examination of chemically fixed tissue demonstrated the presence of large electron-dense aggregates in the vacuoles of ethylene-treated leaf cells. No such vacuolar structures were observed in untreated control cells. Immunogold labelling with antisera directed against the basic forms of chitinase and -1,3-glucanase indicated that the vacuolar aggregates were the major site of accumulation of chitinase and -1,3-glucanase. The chitinase- and -1,3-glucanase-containing vacuolar aggregates were not randomly distributed within the leaf tissue but were restricted to the lower epidermal cells and to parenchyma cells adjacent to vascular strands. In addition, heavy -1,3-glucanase labelling was observed over spongy plugs of expanded middle-lamella material that appear to occlude the transition regions between the airspaces underlying the stomata and those throughout the rest of the leaf. Some labelling was also seen to extend along the surface layer of the cell walls lining all of the airspaces. Protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting as well as enzyme-activity measurements showed that the peeled lower epidermis of the ethylene-treated leaves contained on a protein and on a per-weight basis several times more chitinase and -1,3-glucanase than the remainder of the leaf.Abbreviation in Text SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Abbreviations in Micrographs AS air space - C chloroplast - EP (epidermal) cell - G guard cell - P parenchyma cell - S stoma - V vacuole - VE] vein - VP vascular parenchyma cell - W cell wall - X xylem We thank Dr. L.A. Hadwiger, Pullman, Wash., and Dr. U. Vögeli, Lexington, Ky., for their kind gifts of antibodies. This work was supported by the National Science Foundation grant DCB-8615763 to L.A.S.     

Co-ordinated regulation of chitinase and β-1,3-glucanase in bean leaves

Ethylene induced chitinase (EC 3.2.1.14) and -1,3-glucanase (EC 3.2.1.29) to a similar extent in primary leaves of bean seedlings (Phaseolus vulgaris cv. Saxa). Both enzymes were purified from ethylene-treated leaves, and monospecific antibodies were raised aginst them. Ethylene treatments strongly increased the amount of immunore-active chitinase and -1,3-glucanase. Ethylene enhanced synthesis of chitinase in vivo, as tested by immunoprecipitation after pulse-labelling with [³⁵S]methionine. RNA was isolated from bean leaves and translated in a rabbit reticulocyte lysate system in vitro. The chitinase and the -1,3-glucanase antiserum each precipitated a single polypeptide from the translation products. The precipitated polypeptides were 1500 and 4000 daltons larger, respectively, than native chitinase and native -1,3-glucanase, indicating that the two enzymes were synthesized as precursors in vitro. The translatable mRNAs for both enzymes increased at least tenfold within 2 h in response to a treatment with ethylene. When ethylene was withdrawn after 8 h of incubation, the translatable mRNAs for both enzymes decreased somewhat more slowly, reaching the basal level about 25 h later. In all cases, there was a close correlation between the levels of translatable mRNA for chitinase and -1,3-glucanase. A putative -1,3-glucanase cDNA clone, pCH16, was isolated by hybrid-selected translation. The amount of -1,3-glucanase mRNA, as measured by RNA blot analysis using pCH16 as a probe, increased rapidly in response to ethylene and decreased again after withdrawal of ethylene, indicating that the amount of hybridizable RNA and of translatable mRNA for -1,3-glucanase were correlated. In conclusion, the results indicate that chitinase and -1,3-glucanase are regulated co-ordinately at the level of mRNA.Abbreviations poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Purification and partial characterization of a 36-kDa chitinase from Bacillus thuringiensis subsp. colmeri,and its biocontrol potential

Chitinase A (ChiA) produced by Bacillus thuringiensis subsp. colmeri 15A3 (Bt. 15A3) was expressed in Escherichia coli XL-Blue. The ChiA was purified using Sephadex G-200 and its molecular mass was estimated to be 36 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Detection of chitinase activity on SDS-PAGE after protein renaturation indicated that the molecular mass of the protein band expressing chitinase activity was approximately 72 kDa. This suggests that the dimeric form of ChiA is the enzymatically active form when glycol chitin is used as a substrate. ChiA has optimal activity at 50 °C and retains most of its activity between 20 and 60 °C. The optimum pH for ChiA activity is pH 5.0, and the enzyme is active between pH 4.0 and 8.0. The enzyme activity was significantly inhibited by Ag⁺ and Zn²⁺. ChiA significantly inhibited the spore germination of four species of fungi. The median inhibitory concentrations (IC₅₀) of ChiA on the spore germination of Penicillium glaucum and Sclerotinia fuckelian were 11.27 and 10.57 μg/ml, respectively. In surface contamination bioassays, the crude ChiA protein (12.6 mU) reduced the LC₅₀ (50% lethal concentration) of the crystal protein of Bt. 15A3 against the larvae of Spodoptera exigua and Helicoverpa armigera.     

Purification and partial characterization of β-1,3-glucanase from <Emphasis Type="Italic">Chaetomium thermophilum</Emphasis>

A thermostable extracellular β-1,3-glucanase from Chaetomium thermophilum was purified to homogeneity by fractional ammonium sulfate precipitation, Pheny1-Sepharose hydrophobic interaction chromatography, ion exchange chromatography on DEAE-Sepharose and gel filtration on Sephacryl S-100. SDS-PAGE of the purified enzyme showed a single protein band of molecular weight 76.3 kDa. The enzyme exhibited optimum catalytic activity at pH 6.0 and 60 °C. It was thermostable at 50 °C, and retained 90% activity after 60 min at 60 °C. The half-life at 65 °C, 70 °C and 80 °C was 55 min, 21.5 min, and 5 min, respectively. The N-terminal amino acid sequence (8 residues) of the enzyme was HWLGDIPH. The HPLC analysis showed that the only enzymatic product formed from laminarin by the purified β-1,3-glucanase was glucose, indicating that the enzyme is an exo-β-1,3-glucanase (EC 3.2.1.58).     

Characterization of chitinases and β-1,3-glucanases in grapefruit flavedo during fruit development

Chitinase (EC 3.2.1.14) and β-1,3-glucanase (EC 3.2.1.39) activities in the flavedo of grapefruit ( Citrus paradisi cv. Marsh) were determined at 17 times during the course of fruit development. Chitinase activity is initially high in flavedo, but drops rapidly and is low, although fairly constant throughout the remainder of fruit development. In contrast to chitinase, β-1,3-glucanase activity is lowest in young fruit and increases during development. Western blots of crude flavedo extracts following SDS-PAGE were probed with antibodies raised against purified citrus chitinase and β-1,3-glucanase. Results of immunostaining revealed that changes in the activities of chitinase and β-1,3-glucanase were reflected in the amount of chitinase and glucanase protein present in the extracts. Only a single chitinase band was detected on western blots of crude flavedo extracts, whereas one glucanase band was present in young fruit and a second one appeared later in older fruit. Partial purification of flavedo chitinases and glucanases was performed using extracts prepared from immature and mature fruit for the two enzymes, respectively. Acidic and basic forms of both enzymes were present in the extracts; acidic and basic forms of chitinase were present in nearly equal amounts whereas basic glucanases predominated (91% of total activity). Acidic and basic chitinases differed in substrate specificity as well as products of degradation indicating the heterogeneous nature of the enzymes. Both acidic and basic glucanases required the presence of β-1,3 linkages for activity, were active against both soluble and insoluble β-1,3 glucans and generated similar products.     

Evidence for secretion of vacuolar α-mannosidase, class I chitinase, and class I β-1,3-glucanase in suspension cultures of tobacco cells

We have investigated the possibility that vacuolar proteins can be secreted into the medium of cultured cells of Nicotiana tabacum L. Time-course and balance-sheet experiments showed that a large fraction, up to ca. 19%, of vacuolar α-mannosidase (EC 3.2.1.24) and vacuolar class I chitinase (EC 3.2.1.14) in suspension cultures accumulated in the medium within one week after subculturing. This effect was most pronounced in media containing 2,4-dichlorophenoxyacetic acid (2,4-D). Under comparable conditions only a small fraction, 1.8–5.1% of the total protein and ca. 1% of malate dehydrogenase (EC 1.1.1.37), which is localized primarily in the mitochondria and cytoplasm, accumulated in the medium. Pulse-chase experiments showed that newly synthesized vacuolar class I isoforms of chitinase and β-1,3-glucanase (EC 3.2.1.39) were released into the medium. Post-translational processing, but not the release of these proteins, was delayed by the secretion inhibitor brefeldin A. Only forms of the proteins present in the vacuole, i.e. mature chitinase and pro-β-1,3-glucanase and mature β-1,3-glucanase, were chased into the medium of tobacco cell-suspension cultures. Our results provide strong evidence that vacuolar α-mannosidase, chitinase and β-1,3-glucanase can be secreted into the medium. They also suggest that secretion of chitinase and β-1,3-glucanase might be via a novel pathway in which the proteins pass through the vacuolar compartment. Received: 3 September 1997 / Accepted: 30 October 1997     

Selection and differentiation of Bacillus spp. Antagonistic to Fusarium oxysporum f.sp. lycopersici and Alternaria solani infecting Tomato

Antagonistic Bacillus spp. displaying in vitro production of siderophore, chitinase, and β-1,3-glucanase were identified from dual culture assays. In independent greenhouse studies, seed bacterization and soil application of Bacillus atrophaeus S2BC-2 challenge inoculated with Fusarium oxysporum f.sp. lycopersici (FOL) and Alternaria solani (AS) recorded low percent disease index of 25.3 and 28.7, respectively, over nonbacterised pathogen control (44.3 and 56.4). The low disease incidence corroborated with tomato growth promotion with high vigor index (8,041.2) and fresh plant weight (82.5 g) on challenge inoculation with FOL. Analysis of root and leaf samples in rhizobacterial treatment challenged with FOL and AS revealed maximum induction of chitinase (1.9 and 1.7 U/mg of protein, respectively) and β-1,3-glucanase (23.5 and 19.2 U/mg of protein, respectively). In native gel activity assays, the rhizobacterial treatment on challenge inoculation strongly expressed three high intensity PO isoforms along with one low intensity isoform. In studies on genetic diversity of the Bacillus strains by repetitive extragenomic palindromic-polymerase chain reaction (REP-PCR) and amplified rDNA restriction analysis (ARDRA) patterns, ARDRA was more highly discriminant than REP-PCR and allowed grouping of the strains and differentiation of the antagonistic strains from other isolates.     

聚丙烯酰胺凝胶电泳配合荧光增白剂显色检测木霉几丁质酶同工酶谱

为提高木霉几丁质酶检测方法的准确性和灵敏度,建立一种快速检测几丁质酶同工酶的方法。采用活性凝胶电泳、变性凝胶电泳、原位显色凝胶电泳结合荧光增白剂（Calcofluor white M2R）显色从绿色木霉LTR-2发酵产物中检测几丁质酶同工酶。活性凝胶电泳在粗酶液浓缩5倍时显示两条活性谱带,变性凝胶电泳在浓缩10倍时显示一条活性谱带,原位显色凝胶电泳在浓缩20倍时显示两条不清晰的活性谱带,SDS-PAGE显示这两条活性谱带的分子量分别为65kDa和42kDa。结果表明活性聚丙烯酰胺凝胶电泳和Calcofluor white M2R显色相结合的方法在几丁质酶上样量为0.47U时具有较好的分辨能力,是检测木霉几丁质酶同工酶的有效的方法。