Increased Chitinase Activity Research Articles

English

An incubation study investigated the decomposition patterns, microbial activities, soil aggregates distribution, and soil organic carbon (SOC) contents, using four crop residues; wheat (Triticum aestivum L.), rice (Oryza sativa L.), cotton (Gossypium hirsutum L.), and maize (Zea mays L.), at low and high rates (10 and 20 g kg–1). An average increase of about 35% in cumulative C-CO2 emissions and 26% in soil microbial biomass carbon (MBC) was observed at high residue rates as compared to low rates. A positive and strong correlation (0.96) was found between C-CO2 emissions and MBC, and the effect of residue quality on decomposition was found to be less at high rates. The relative reduction in C-CO2 emissions by adding rice residues may be due to their high silica content. Differences in extracellular enzyme activities (EEA) with different residues indicated the effect of residue quality on microbial activities. Cotton residues increased β-glucosidase and chitinase activities by 41 and 38%, respectively, at high rates than at low rates. High maize residues also markedly increased chitinase activity by 41%, implying higher N cycling and fungal prevalence. Acid phosphatase activity was found to increase by about 25 to 32% with high rice and wheat residues, respectively. Higher EEA at high residues input likely reflected the microbial nutritional limitations. Crop residues raised SOC content from 0.7 under control to 1.0% at low residues input but not at high input. Increased macro-aggregates fraction at high residues input may be attributed to higher microbial activities. In summary, high residues input can minimize residue biochemical quality’s effect on decomposition, with no further increase in SOC content. While a positive effect on the soil MBC, but not on the SOC content, was observed at high residue input, suggesting moderation in the additions of organic amendments is key for SOC buildup. Crop residues, added at 10 g kg–1, may help maintain a positive SOC balance in the arid agroecosystems by moderating higher microbial activities and soil respiration.

Defense related enzymes implicated in resistance and susceptibility to Rhizoctonia solani, the causal agent of sheath blight of rice

Plants have evolved various defense mechanisms to defend themselves from phytopathogens. Pathogenesis-related proteins are produced as part of the active defenses. In this work, changes in the activity of defense-related enzymes in rice in response to Rhizoctonia solani were examined at different time intervals viz., 0, 24, 48, and 72 h post inoculation (hpi), in late tillering tolerant and susceptible cultivars of rice. When the pathogen was induced, the activities of defense-related enzymes like chitinase, b-1,3-glucanase, peroxidase (PO), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL), and total phenol content significantly increased in the tolerant cultivar (Tetep) compared to susceptible (KRH-4). Tetep showed increased chitinase activity up to 72 hpi, whereas KRH-4 showed no significant change up to 48 hpi but then increased activity at that time. When compared to the susceptible cultivar, tolerant Tetep had the maximum b-1-3 glucanase activity at 48 hpi. Tetep had an early induction of PAL with the highest activity at 24 hours compared to the control, but no proper trend was seen in susceptible cultivar. The early PO activity culminates in Tetep at 48 hpi. Tetep and KRH-4 both showed higher PPO enzyme activity at 48 hpi. In both the tolerant and susceptible cultivars, SOD activity steadily rose to 24 hpi and subsequently increased gradually up to 72 hpi. Tetep reported a higher total phenol concentration in the infected sheath samples than KRH-4 at 72 hpi. In this work, we looked at the biochemical methods for describing the defense-related enzymes against the sheath blight pathogen.

Alkaline phosphatase LapA regulates quorum sensing-mediated virulence and biofilm formation in Pseudomonas aeruginosa PAO1 under phosphate depletion stress.

Among various environmental stresses, the depletion of inorganic phosphate, which is an important signaling molecule that modulates virulence production, is notable. In our previous study, we demonstrated that lapA, encoding alkaline phosphatase, plays a key role in Pseudomonas aeruginosa biofilm formation in porcine skin explants, an ex vivo chronic wound model. However, the mechanism by which lapA participates in the virulence and biofilm formation of P. aeruginosa remains unclear. In the present study, the lapA deletion mutant was constructed in P. aeruginosa PAO1, and the phenotypic characterization was assessed. Our data demonstrated that phosphate depletion stress significantly enhanced elastase activity, hemolysis, rhamnolipid production, swarming and swimming motilities, and 3-oxo-C12-homoserine lactone (HSL) production in P. aeruginosa. Moreover, phosphate starvation evidently induced the virulence of P. aeruginosa in Caenorhabditis elegans (C. elegans) through fast-kill and slow-kill infections. Deletion of the lapA gene led to broad phenotypic changes, including reduced elastase activity, swimming motility, C4-HSL, and 3-oxo-C12-HSL production, as well as increased chitinase activity and rhamnolipid production under phosphate depletion stress, which was unrelated to C4-HSL. Deletion of the lapA gene also significantly inhibited PAO1 biofilm formation in porcine skin explants by reducing the expression levels of las and rhl quorum sensing systems and extracellular polymeric substance synthesis. Finally, lapA deletion also reduced the virulence of PAO1 in C. elegans in fast-kill and slow-kill infection assays. Thus, the findings suggest that targeting lapA can help control biofilm formation and reduce virulence in nonhealing-infected wounds, where P. aeruginosa is a persistent problem. IMPORTANCE Our previous study demonstrated that the expression of lapA was induced under phosphate depletion conditions, but its roles in virulence and biofilm formation by Pseudomonas aeruginosa remain largely unknown. This study presents a systematic investigation of the roles of lapA in virulence induction and biofilm formation by constructing a lapA-deficient strain with P. aeruginosa PAO1. The results showed that deletion of the lapA gene evidently reduced elastase activity, swimming motility, C4-HSL, and 3-oxo-C12-HSL production, and increased rhamnolipid production under phosphate depletion stress. Moreover, lapA gene deletion inhibited PAO1 biofilm formation in porcine skin explants by reducing the expression levels of las and rhl quorum sensing systems and extracellular polymeric substance synthesis. Finally, lapA gene deletion also reduced the virulence of PAO1 in Caenorhabditis elegans in fast-kill and slow-kill infection assays. This study provides insights into the roles of lapA in modulating P. aeruginosa virulence and biofilm formation under phosphate depletion stress.

Enzymatically hydrolyzed fluorescence-based chemical probe enables in situ mapping of chitinase activity in the rhizosphere

Chitin is an insoluble and ubiquitous soil biopolymer, estimated to be the second most abundant organic soil biopolymer on Earth. Despite its abundance, role as a source of C and N in soil, and importance to ecosystem function, further research is required to elucidate key controls on chitin breakdown under varying environmental conditions. Previous work highlights the important role rhizosphere microbiomes and root exudates can play in chitin catabolism. To enable mapping of chitinase activity within the highly heterogeneous and spatially organized rhizosphere, we designed and synthesized an enzymatically activated fluorogenic substrate, chitotriose-TokyoGreen (chitotriose-TG), by incorporating a fluorescein derivative (TG) onto the trimeric unit of chitin. This non-fluorescent substrate is selectively hydrolyzed by chitinase to release TG and yield a fluorescence signal, which can be used to spatially image and measure chitinase activity in the rhizosphere. To demonstrate the application of this technique, we grew switchgrass (Panicum virgatum) in rhizoboxes amended with a horizontal layer supplemented with chitin. We extracted mobile proteins from the rhizobox using a nitrocellulose membrane blotting technique which offers non-destructive enzyme extraction while preserving the 2D spatial position of the enzymes. We then subjected these membranes to the synthesized chitotriose-TG stain to spatially visualize the distribution of chitinase activity within the rhizosphere. We observed increased chitinase activity near switchgrass roots and higher activity within the soil zone enriched in chitin, showing an adaptive response of chitinase production with spatial focusing in areas of higher chitin abundance. Thus, the enzyme extraction and visualization strategy we describe here can enhance efforts to better understand spatial controls on chitin breakdown in rhizosphere, further elucidating the role of chitin as a C and N source in these systems.

Gene Expression and Metabolomics Profiling of the Common Wheat Obtaining Leaf Rust Resistance by Salicylic or Jasmonic Acid through a Novel Detached Leaf Rust Assay

Wheat leaf rust caused by Puccinia triticina is a destructive fungal disease causing considerable grain yield loss. In this study, we developed a novel assay to test the rust resistance of detached wheat leaves on defined media with retarded senescence. We observed that salicylic and jasmonic acid confer leaf rust resistance to a susceptible Keumkang wheat (Triticum aestivium L.). Transcription analysis revealed that atchi8 was highly expressed with an increased chitinase activity in the salicylic acid-treated leaves, while expression of PR-9, atpodL, and PR-5 increased in the jasmonic acid-treated leaves. Additionally, the metabolic profile suggested that the phenylalanine pathway might link flavonoid production to leaf rust resistance in the salicylic acid-treated leaves, while the alanine, aspartate, and glutamate metabolism might control the production of other amino acids to enhance pathogen stress response in the jasmonic acid-treated leaves. Finally, all identified genes and metabolites could be potential targets for screening chemical compounds for leaf rust resistance. Future studies on the underlying mechanisms of leaf rust resistance obtained by exogenous treatment of salicylic and jasmonic acids remain necessary.

A Na2CO3-Responsive Chitinase Gene From Leymus chinensis Improve Pathogen Resistance and Saline-Alkali Stress Tolerance in Transgenic Tobacco and Maize.

Salinity and microbial pathogens are the major limiting factors for crop production. Although the manipulation of many genes could improve plant performance under either of these stresses, few genes have reported that could improve both pathogen resistance and saline-alkali stress tolerance. In this study, we identified a new chitinase gene CHITINASE 2 (LcCHI2) that encodes a class II chitinase from Leymus chinensis, which grows naturally on alkaline-sodic soil. Overexpression of LcCHI2 increased chitinase activity in transgenic plants. The transgenic tobacco and maize exhibited improved pathogen resistance and enhanced both neutral salt and alkaline salt stress tolerance. Overexpression of LcCHI2 reduced sodium (Na+) accumulation, malondialdehyde content and relative electrical conductivity in transgenic tobacco under salt stress. In addition, the transgenic tobacco showed diminished lesion against bacterial and fungal pathogen challenge, suggesting an improved disease resistance. Similar improved performance was also observed in LcCHI2-overexpressed maize under both pathogen and salt stresses. It is worth noting that this genetic manipulation does not impair the growth and yield of transgenic tobacco and maize under normal cultivation condition. Apparently, application of LcCHI2 provides a new train of thought for genetically engineering saline-alkali and pathogen resistant crops of both dicots and monocots.

Purification, physicochemical and thermodynamic studies of antifungal chitinase with production of bioactive chitosan-oligosaccharide from newly isolated Aspergillus griseoaurantiacus KX010988

In our search for chitinase and chitosanase producer from unconventional sources, the marine-derived fungus Aspergillus griseoaurantiacus KX010988 was obviously the best producer of the highest chitinase and chitosanase activities by solid state fermentation of potato shells. Chitinase was purified in three steps involving ammonium sulphate precipitation, DEAE-cellulose ion-exchange chromatography and Sephacryl S-300 gel chromatography. 12.55 fold increase in purity with a recovery of 17.6 was obtained. The molecular mass of the purified chitinase was found to be 130kDa. It was optimally active at pH 4.5 and 40°C. Km and Vmax values were 0.22mgmL−1 and 19.6μmolemin−1mg−1 respectively. Mn2+ and Zn2+ ions lead to increased chitinase activity. While Fe2+and Cu2+ions strongly inhibited the chitinase activity. The thermodynamics of pure chitinase including activation energy for thermal denaturation (Ea,d), change of free energy (ΔGd), enthalpy(ΔHd), entropy(ΔSd) and half life values (T1/2) at 40, 50 and 60°C were determined. Chitinase showed antifungal activity against pathogenic fungus Fusarium solani. Chitosanase was partially purified by acetone precipitation (50–75%) v/v concentration. The hydrolytic products of moderate molecular weight of chitosan by chitosanase were analyzed by thin layer chromatography (TLC) after 12 and 24h respectively. Chitosan-oligosaccharides showed good antibacterial and antioxidant activities.

On the ecosystemic network of saliva in healthy young adults

A dysbiotic state is believed to be a key factor in the onset of oral disease. Although oral diseases have been studied for decades, our understanding of oral health, the boundaries of a healthy oral ecosystem and ecological shift toward dysbiosis is still limited. Here, we present the ecobiological heterogeneity of the salivary ecosystem and relations between the salivary microbiome, salivary metabolome and host-related biochemical salivary parameters in 268 healthy adults after overnight fasting. Gender-specific differences in the microbiome and metabolome were observed and were associated with salivary pH and dietary protein intake. Our analysis grouped the individuals into five microbiome and four metabolome-based clusters that significantly related to biochemical parameters of saliva. Low salivary pH and high lysozyme activity were associated with high proportions of streptococcal phylotypes and increased membrane-lipid degradation products. Samples with high salivary pH displayed increased chitinase activity, higher abundance of Veillonella and Prevotella species and higher levels of amino acid fermentation products, suggesting proteolytic adaptation. An over-specialization toward either a proteolytic or a saccharolytic ecotype may indicate a shift toward a dysbiotic state. Their prognostic value and the degree to which these ecotypes are related to increased disease risk remains to be determined.

Characterization of extracellular chitinase produced from Streptomyces rubiginosus isolated from rhizosphere of Gossypium sp.

AbstractIn the present study, 28 chitinase producing Actinomycetes were isolated from rhizoshperic soil samples of Gossypium sp. (Cotton) growing in Dastan farm, Naroda, Gujarat. Amongst them, eleven of these isolates produced significantly higher levels of chitinase and were further studied quantitatively. Results indicated that SP 24 was found to be the most prominent chitinase producer, giving 2.790 U/ml chitinase activity by using 1% colloidal chitin as substrate concentration, amongst the isolates under study. In case of isolate SP 24, maximum chitinase production was obtained after 72 h of incubation period and in culture medium having pH 7 at temperature 30°C. Amendment of peptone as a sole nitrogen source to colloidal chitin broth (CCB) enhanced chitinase activity. Furthermore, xylose as a sole carbon source added to CCB medium also increased chitinase activity. Effective production method of chitinase by Actinomycetes has captured the attention due to its varied applications in industries as well...

The components of rice and watermelon root exudates and their effects on pathogenic fungus and watermelon defense

ABSTRACTWatermelon (Citrullus lanatus) is susceptible to wilt disease caused by the fungus Fusarium oxysporum f. sp niveum (FON). Intercropping management of watermelon/aerobic rice (Oryza sativa) alleviates watermelon wilt disease, because some unidentified component(s) in rice root exudates suppress FON sporulation and spore germination. Here, we show that the phenolic acid p-coumaric acid is present in rice root exudates only, and it inhibits FON spore germination and sporulation. We found that exogenously applied p-coumaric acid up-regulated the expression of ClPR3 in roots, as well as increased chitinase activity in leaves. Furthermore, exogenously applied p-coumaric acid increased β-1,3-glucanase activity in watermelon roots. By contrast, we found that ferulic acid was secreted by watermelon roots, but not by rice roots, and that it stimulated spore germination and sporulation of FON. Exogenous application of ferulic acid down-regulated ClPR3 expression and inhibited chitinase activity in watermelon leaves. Salicylic acid was detected in both watermelon and rice root exudates, which stimulated FON spore germination at low concentrations and suppressed spore germination at high concentrations. Exogenously applied salicylic acid did not alter ClPR3 expression, but did increase chitinase and β-1,3-glucanase activities in watermelon leaves. Together, our results show that the root exudates of phenolic acids were different between rice and watermelon, which lead to their special ecological roles on pathogenic fungus and watermelon defense.

Purification, characterization and antimicrobial activity of chitinase from marine-derived Aspergillus terreus

Chitinase (EC 3.2.1.14) was produced from the culture filtrate of marine-derived Aspergillus terreus and purified by 65% ammonium sulphate precipitation, followed by gel filtration on Sephadex G-100 and DEAE-Sephadex A-50 ion exchange chromatography, with 5.16-fold of purification and specific activity of 182.08U/mg protein. The molecular weight of the purified chitinase was 60kDa, determined by a sodium dodecyl sulphate polyacrylamide gel electrophoresis. The optimum pH and temperature of purified chitinase were 5.6 and 50°C, respectively. The chitinase enzyme was stable from pH 5 to 7.5 and stable up to 70°C. The effect of activators and inhibitors was studied, Hg+, pb, EDTA, ethanol, methanol and acetone strongly inhibited the enzyme activity, while, metal ions such as Ca2+, Mn2+ and Na2+ highly increased chitinase activity. The purified chitinase produced by A. terreus inhibited the growth of Aspergillus niger, Aspergillus oryzae, Penicillum oxysporium, Rhizocotonia solani, Candida albicans and Fusarium solani, while did not inhibit the growth of Rhizopus oryzae. Moreover, the purified enzyme had antibacterial effects against some pathogenic bacteria such as; Staphylococcus aureus, Salmonella typhi and Pseudomonas aeruginosa, while, it had not any activity against Escherichia coli, Aeromonas hydrophila and Photobacterium damsela.

A highly conserved metalloprotease effector enhances virulence in the maize anthracnose fungus Colletotrichum graminicola.

Colletotrichum graminicola causes maize anthracnose, an agronomically important disease with a worldwide distribution. We have identified a fungalysin metalloprotease (Cgfl) with a role in virulence. Transcriptional profiling experiments and live cell imaging show that Cgfl is specifically expressed during the biotrophic stage of infection. To determine whether Cgfl has a role in virulence, we obtained null mutants lacking Cgfl and performed pathogenicity and live microscopy assays. The appressorium morphology of the null mutants is normal, but they exhibit delayed development during the infection process on maize leaves and roots, showing that Cgfl has a role in virulence. In vitro chitinase activity assays of leaves infected with wild-type and null mutant strains show that, in the absence of Cgfl, maize leaves exhibit increased chitinase activity. Phylogenetic analyses show that Cgfl is highly conserved in fungi. Similarity searches, phylogenetic analysis and transcriptional profiling show that C. graminicola encodes two LysM domain-containing homologues of Ecp6, suggesting that this fungus employs both Cgfl-mediated and LysM protein-mediated strategies to control chitin signalling.

Seed treatment with benzothiadiazole induces resistance against powdery mildew disease caused by Sphaerotheca fuliginea and increases the activities of pathogenesis-related enzymes in cucumber plants

SummaryApplications of acibenzolar S-methyl (ASM), commercially known as Bion® WG 50 [benzothiadiazole (benzo 1, 2, 3) thiadiazole-7-carbothioic acid S-methyl ester], or Milsana® by seed imbibition were evaluated for their efficacy in protecting cucumber plants against powdery mildew caused by the biotrophic fungus, Sphaerotheca fuliginea.Among the different concentrations of ASM (0, 25, 50, 75, or 100 ?g a.i. ml_1) or Milsana® (10 or 20 ml l_1) tested, soaking seed in 75 ?g a.i. ml_1 ASM for 12 h was the optimum concentration and duration to induce systemic acquired resistance (SAR) without adverse effects on seed germination or seedling vigour. Higher concentrations of ASM delayed the germination of cucumber seed, and longer durations of seed treatment negatively affected the seedling vigour index. Seed treatment with ASM increased the activities of pathogenesis-related (PR) proteins, but not treatment with Milsana®.The activity of chitinase in cotyledons from ASM-treated seed increased six-fold compared to its activity in untreated (control) plant tissue. Longer durations of soaking with ASM significantly increased chitinase activity in shoot tissues, reaching a peak at 12 h before declining.The enhanced levels of chitinase and peroxidase activity elicited by seed treatment with ASM were similar to those triggered by foliar application of ASM alone, or by seed treatment followed by foliar application of ASM. The application of ASM by seed imbibition significantly reduced the severity of powdery mildew in cucumber cotyledon-disc assays. Increased resistance to powdery mildew in ASM seed-treated cucumber plants was positively correlated with the enhanced activities of PR proteins that are known to be associated with pathogen resistance in cucumber leaves.This research indicates that ASM may be applied as a seed treatment to protect cucumber seedlings against powdery mildew.

Isolation and identification of a virulent Ralstonia solanacearum by fliC gene amplification and induction of chitinase by 2-amino butyric acid for control of bacterial wilt in tomato plants

Ralstonia solanacearum is a devastating, soil borne bacterial pathogen of tomato. The pathogen is nonmotile in planta but highly motile in culture. On the basis of physiological and biochemical characteristics 26 isolates have been purified and identified as Ralstonia solanacearum. The flic gene is responsible for the movement of bacteria. Ralstonia specific fliC gene amplification is the indication of virulence of the pathogen. In the present study one R. solanacearum isolate has been identified by PCR amplification of the fliC gene using fliC gene specific primer. Following isolation and identification of the virulent isolate, fresh tomato plants were induced by application of 2- amino butyric acid (ABA). The defense enzyme, chitinase was estimated in treated plants. Treated inoculated plants did not show any visible symptoms of wilt even after 14 days of inoculation. Significantly it was observed that chitinase was increased in the 2-ABA-treated plants and also in the treated-inoculated plants. The increased chitinase activity in the treated plants showed that 2-ABA has the resistance inducing capacity in tomato plants against Ralstonia solanacearum.

Optimization of protease and chitinase production by Bacillus cereus SV1 on shrimp shell waste using statistical experimental design. Biochemical and molecular characterization of the chitinase

The current increase in the amount of shell waste produced by the shrimp industries has led to the need to find new methods for its disposal. In this study, statistical methodologies were used for chitinase and protease co-production optimization by Bacillus cereus SV1 in media containing shrimp shell powder. Medium composition and culture conditions were optimized using two statistical methods: Plackett–Burman design was applied to find the key ingredients and conditions for the best yield of enzymes production, and central composite design was used to optimize the levels of the five significant variables: shrimp shell powder (SSP), NH4Cl, CaCl2, K2HPO4 and speed of agitation. The medium optimization resulted in protease and chitinase activities of 8,445.8 U ml−1 and 82.8 mU ml−1, respectively. The crude chitinase exhibited maximal activity at 55°C and pH 7.0 using colloidal chitin as substrate. The metal ions Fe2+ and Mg2+ increased chitinase activity, while Hg2+ strongly inhibited this activity. The nucleotide sequencing of SV1 chitinase revealed an open reading frame containing 2,025 bp and encoding 642 amino acids.

Increased chitinase expression and fungal‐specific antibodies in the bronchoalveolar lavage fluid of asthmatic children

Increasing evidence highlights the contribution of chitinases and fungal infection to the development of asthma. The purpose of this study was to characterize chitinase expression and serological markers of fungal infection in children with severe asthma. Bronchoalveolar lavage fluid (BALF) was collected from children undergoing clinically indicated flexible bronchoscopy. A diagnosis of asthma was confirmed by pulmonary function testing. BALF was tested for chitinase activity and YKL-40 (an enzymatically inactive chitinase) concentrations. Specimens were cultured for fungal organisms and tested for cryptococcal antigen by ELISA. IgG and IgA reactivity to whole extract fungal (Aspergillus fumigatus, Alternaria alternata, Cryptococcus neoformans and Candida albicans) proteins were determined by immunoblot assay. Among the 37 patients studied, 30 were asthmatic and 7 were non-asthmatic. Asthmatics exhibited elevated serum IgE levels (median: 748IU/mL, IQR: 219-1765IU/mL). Chitinase activity was greater in the BALF of asthmatics (mean, 0.85±1.2U/mL) compared with non-asthmatics (mean: 0.23±0.21U/mL, P=0.012). Likewise YKL-40 concentrations were higher in the BALF of asthmatics and correlated with chitinase activity. There was a trend towards increased fungal-specific IgG in the BALF of asthmatics compared with non-asthmatics and for C. albicans this difference reached statistical significance. IgA reactivity to C. neoformans and A. fumigatus was greater in the BALF of asthmatics compared with non-asthmatics. Compared with non-asthmatics, asthmatic children exhibited increased chitinase activity and increased YKL-40 levels in BALF. Increased IgG and IgA reactivity to fungal proteins in the BALF of asthmatics may reflect a local response to fungal infection. Our findings are consistent with and suggest a role for chitinases in asthma pathogenesis among Bronx children and provide serological evidence of an association between fungal infection and severe asthma.

A single application of Milsana® followed by Bion® assists in the control of powdery mildew in cucumber and helps overcome yield losses

SummaryThe efficacy of candidate activators of systemic acquired resistance (SAR), Bion® (Syngenta, Basel, Switzerland), Rezist® (Stoller Enterprises, Houston, Texas, USA), and Milsana® (P. J. Margo Pvt. Ltd., Bangalore, India), was evaluated for the control of powdery mildew (Podosphaera xanthii) on cucumber and zucchini. Each candidate activator significantly reduced the incidence of disease on both crops in a glasshouse, where spore inoculum was applied to treated leaves. Both Bion® and Rezist® induced increased chitinase activity in plants, confirming their action as activators of SAR. However, Milsana® did not have this effect, suggesting that its mode of action was as a direct inhibitor of infection by spores. In the field, two or three applications of Bion®, at 14-day intervals, gave consistently better results in protecting cucumber plants from powdery mildew. Several applications of Milsana® alone were less effective than a single application of Bion® alone. However, several applications of Bion® adversely affected plant growth, resulting in lower yields. The application of Milsana® before or after Bion® provided effective control of powdery mildew and minimised the adverse effects of Bion® on yield. Application of all three candidate activators alone, on field-grown zucchini did not control powdery mildew, although a systemic increase in chitinase activity occurred in response to Bion® or Rezist®. A greater understanding of the mode of action of Milsana®, and of the interaction between disease load and the timing of activator application(s) may improve the control of powdery mildew.

Indução de resistência em tomateiro por extratos aquosos de Lentinula edodes e Agaricus blazei contra Ralstonia solanacearum

O presente trabalho foi conduzido com o objetivo de avaliar o efeito dos extratos aquosos dos cogumelos Lentinula edodes e Agaricus blazei e do indutor de resistência acibenzolar-S-metil (ASM) no controle da murcha bacteriana em tomate (Lycopersicon esculentum Mill.), bem como investigar o modo de ação destes produtos na atividade protetora, através de alterações na atividade de determinadas enzimas. Extratos aquosos dos cogumelos e o ASM foram testados na inibição do crescimento da bactéria in vitro. Em casa-de-vegetação, plantas foram tratadas com diferentes doses dos extratos e com ASM (0,05 g/L), dois dias antes da inoculação. As avaliações foram efetuadas com base na severidade da doença e determinação da atividade de algumas enzimas. Os isolados de A. blazei e L. edodes, bem como o ASM, não exerceram efeito inibitório direto no crescimento da bactéria. Quanto à indução de resistência, o isolado Le-96/17 de L. edodes na concentração de 10 % (v/v) e o ASM reduziram significativamente a ocorrência de murcha. As atividades de quitinase, fenilalanina amônia-liase e polifenoloxidase não foram alteradas para a maioria dos tratamentos, porém foi constatado um aumento na atividade de quitinase nas plantas tratadas com ASM. Para a peroxidase, os tratamentos ASM, Abl-26 e Le-96/17 ocasionaram aumentos na atividade da enzima e, com base nos resultados, o cogumelo L. edodes e o ASM apresentam potencial para reduzir a murcha bacteriana provavelmente através da indução de resistência.

Directed evolution for increased chitinase activity

Directed evolution through DNA shuffling and screening was used to enhance the catalytic ability of a fungal, Beauveria bassiana, chitinase, Bbchit1. The Bbchit gene was first linked to various prokaryotic signal sequences and expressed in Escherichia coli. The signal peptide, PelB, from Erwinia carotovora resulted in greatest chitinase secretion into broth. The nucleotide sequence expressing PelB signal peptide was then incorporated into an E. coli vector to express Bbchit1 variants generated by three rounds of DNA shuffling. A Bbchit1 library with 150,000 variants was constructed with a nucleotide point mutation frequency of 0.6% and screened for chitinolytic activity. Two Bbchit1 variants (SHU-1 and SHU-2) were selected that showed increased chitinolytic activity compared to the wild type. Sequence analysis of these variants revealed mutations in amino acid residues that would not normally be considered for rational design of improved chitinase activity. The amino acid substitutions occurred outside of the two putative substrate-binding sites and the catalytic region.

Agrobacterium-mediated transformation of indica rice with chitinase gene for enhanced sheath blight resistance

Four rice indica genotypes of local importance were transformed with RC7, rice chitinase cDNA clone through Agrobacterium-mediated gene transfer method using mature seed derived calli as explants. The putative hygromycin resistant calli showed varied level of regeneration efficiency ranging from 2.0 to 7.6 %. The stable integration and expression of RC7 was confirmed through polymerase chain reaction (PCR) and Western analysis. Transformation efficiency ranged from 0.9 to 5.2 %. The expression of RC7 (35 kDa chitinase) in different tissues of transgenic plant (root, sheath and leaf) was proved through Western analysis and in terms of increased chitinase activity. The inheritance of transgene was studied through PCR and Western analysis in transgenic plants of Pusa Basmati 1. Bioassays with transgenic plants of local cultivars exhibited enhanced resistance up to 33.3 % to rice sheath blight pathogen Rhizoctonia solani under glasshouse conditions. Enhanced expression or 3-to 4-fold increased activity of chitinase in transgenic plants was correlated with sheath blight resistance.