Enzymatic comparison and mortality of Beauveria bassiana against cabbage caterpillar Pieris brassicae LINN

Abstract Beauveria bassiana, an entomopathogenic fungus, is the alternative biocontrol agent exploited against major economic crop pests. Pieris brassicae L. is an emerging pest of the Brassicaceae family. Therefore, in the present study, fungal isolates of Beauveria bassiana, viz. MTCC 2028, MTCC 4495, MTCC 6291, and NBAII-11, were evaluated for their virulence against third instar larvae of P. brassicae. Among all these fungal isolates, maximum mortality (86.66%) was recorded in B. bassiana MTCC 4495 at higher concentration of spores (109 conidia/ml), and the minimum mortality (30.00%) was recorded in B. bassiana MTCC 6291 at a lower concentration (107 conidia/ml) after ten days of treatment. The extracellular cuticle-degrading enzyme activities of fungal isolates were measured. Variability was observed both in the pattern of enzyme secretion and the level of enzyme activities among various fungal isolates. B. bassiana MTCC 4495 recorded the maximum mean chitinase (0.51 U/ml), protease (1.12 U/ml), and lipase activities (1.36 U/ml). The minimum mean chitinase and protease activities (0.37 and 0.91 U/ml, respectively) were recorded in B. bassiana MTCC 6291. The minimum mean lipase activity (1.04 U/ml) was recorded in B. bassiana NBAII-11. Our studies revealed B. bassiana MTCC 4495 as the most pathogenic isolate against P. brassicae, which also recorded maximum extracellular enzyme activities, suggesting the possible roles of extracellular enzymes in the pathogenicity of B. bassiana against P. brassicae.

Thermostable chitinase from Cohnella sp. A01: isolation and product optimization

Abstract Twelve bacterial strains isolated from shrimp farming ponds were screened for their growth activity on chitin as the sole carbon source. The highly chitinolytic bacterial strain was detected by qualitative cup plate assay and tentatively identified to be Cohnella sp. A01 based on 16S rDNA sequencing and by matching the key morphological, physiological, and biochemical characteristics. The cultivation of Cohnella sp. A01 in the suitable liquid medium resulted in the production of high levels of enzyme. The colloidal chitin, peptone, and K2HPO4 represented the best carbon, nitrogen, and phosphorus sources, respectively. Enzyme production by Cohnella sp. A01 was optimized by the Taguchi method. Our results demonstrated that inoculation amount and temperature of incubation were the most significant factors influencing chitinase production. From the tested values, the best pH/temperature was obtained at pH 5 and 70 °C, with Km and V max values of chitinase to be 5.6 mg/mL and 0.87 µmol/min, respectively. Ag+, Co2+, iodoacetamide, and iodoacetic acid inhibited the enzyme activity, whereas Mn2+, Cu2+, Tweens (20 and 80), Triton X-100, and EDTA increased the same. In addition, the study of the morphological alteration of chitin treated by enzyme by SEM revealed cracks and pores on the chitin surface, indicating a potential application of this enzyme in several industries.

Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan

Abstract This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract.

Biological control of the grapevine diseases ‘grey mold’ and ‘powdery mildew’ by Bacillus B27 and B29 strains.

Uncinula necator and Botrytis cinerea are the most destructive pathogens of the grapevine in Tunisia and elsewhere. We used two strains of Bacillus subtilis group, B27 and B29 to control powdery mildew and the grey mold disease of the grapevine. Green house experiments showed that B29 and B27 strains of the bacteria efficiently reduced the severity of powdery mildew up to 50% and 60%, respectively. Further, they decreased Botrytis cinerea development on grape leaf by 77% and 99%, respectively. The mode of action has been shown to be chitinolytic. These two bacteria showed significant production of total proteins discharged into the culture medium. Determination of some chitinolytic enzymes revealed the involvement of N-acetyl glucosaminidase (Nagase), the chitin-1,4-chitobiosidase (Biase) and endochitinase in degrading the mycelium of B. cinerea.

Production, purification and properties of fungal chitinases—A review.

After cellulose, chitin is the second most abundant organic and renewable polysaccharide in nature. This polymer is degraded by enzymes called chitinases which are a part of the glycoside hydrolase family. Chitinases have many important biophysiological functions and immense potential applications especially in control of phytopathogens, production of chito-oligosaccharides with numerous uses and in treatment and degradation of chitinous biowaste. At present many microbial sources are being explored and tapped for chitinase production which includes potential fungal cultures. With advancement in molecular biology and gene cloning techniques, research on fungal chitinases have made fast progress. The present review focuses on recent advances in fungal chitinases, containing a short introduction to types of chitinases, their fermentative production, purification and characterization and molecular cloning and expression.

Distinct chitinases are expressed during various growth phases of the human pathogen Paracoccidioides brasiliensis

The aim of this work was the partial purification and subsequent evaluation of chitinase expression during the various growth phases of Paracoccidioides brasiliensis. Initially, PbCTS1r was expressed as a recombinant protein and displayed enzymatic activity against 4-MU-[N-acetylglucosamine (GlcNAc)]3 and 4-MU-(GlcNAc)2. Two proteins, 45 kDa and 39 kDa in size, were partially purified from P. brasiliensis yeast crude extract using cation-exchange chromatography coupled with HPLC and were characterised as PbCTS1 and PbCTS2, respectively. Anti-PbCTS1r antibody recognised two proteins in the crude extracts of yeast and the transitional stage between mycelial and yeast phases. In crude extracts of mycelium, only the 45 kDa protein was detected. However, quantitative real-time polymerase chain reaction led to the detection of small quantities of Pbcts2 transcript in the mycelial phase. In the yeast cell wall extract, only the 39 kDa protein was detected. Moreover, both proteins were secreted by the yeast parasitic phase, suggesting that these proteins participate in the modulation of the fungal environment. Phylogenetic analysis of the predicted PbCTS1 and PbCTS2 proteins indicated that they code for distinct chitinases in P. brasiliensis. During evolution, P. brasiliensis could have acquired the paralogues Pbcts1 and Pbcts2 for growth and survival in diverse environments in both saprophytic and parasitic phases.

Induction of resistance in host against the infection of leaf blight pathogen (Alternaria palandui) in onion (Allium cepa var aggregatum).

The Pseudomonas fluorescens isolate Pfl was found to inhibit the growth of pathogen Alternaria palandui, in vitro. In the present study, foliar application of a talc-based formulation of Pfl significantly reduced the incidence of leaf blight of onion, caused by A. palandui. Induction of defense-related proteins viz., chitinase, beta-1,3 glucanase, peroxidase (PO) and polyphenol oxidase (PPO) by application of Pfl, was studied against A. palandui infection in resistant (IHR 56) and susceptible (MDUI) onion cultivars. Chitinase in both cultivars, with or without challenge-inoculation of A. palandui revealed changes in the isoform pattern. The Native-PAGE of PO showed induction of PO2 isoform in both the cultivars, in response to inoculation of pathogen. Isoform analysis of PPO also exhibited induction in the Pfl-treated plants challenged with pathogen. Similarly, the activity of beta-1,3-glucanase was greatly induced in Pfl-treated plants, challenged with pathogen as compared to controls. Thus, the P. fluorescens-treated plants showed significant increase in the levels of the defense enzymes, in comparison to the plants challenged with the pathogen.

Formulation of medium constituents by multiresponse analysis of central composite design to enhance chitinase production in Pantoea dispersa.

In the present study, a high chitinase producing strain Pantoea dispersa was isolated from the sea dumps at Bhavnagar, India. Chitin, urea, CaCl2 and MgSO4 x 7H2O were variables used in central composite design for chitinase production. Chitinase, biomass and pH were the responses used in different models to evaluate individually fit ones. Quadratic model was found to be fit for chitinase response whereas in the case of biomass and pH, linear model was found to be fit without the effect of others. Chitinase production was optimized with respect to other responses such as biomass and pH in multiresponse analysis of response surface design by using desirability approach. In multiresponse analysis, following medium formulation (g/l), chitin, 15; urea, 0.32; CaCl2, 0.10 and MgSO4 x 7H2O, 0.08 was found to predict optimum chitinase production of 482.77 units/ml with overall highest desirability of 0.854 as compared to other formulations. The selection of model was done on the basis of high Adjusted R-squared value and lowered p-value for each model in individual analysis of each response. In multiresponse experiment, it was found that for response chitinase quadratic model and for responses pH and biomass linear models were well fit. Through desirability analysis, it was found that in the chitinase production, pH was essential as compared to biomass in P. dispersa. Endochitinase and chitobiase actvities were also studied.

Chitinolytic activity in viable spores of encephalitozoon species

By employing 4-methylumbelliferyl-beta-D-NN',N"-triacetylchitotriose substrate in a semi quantitative assay, chitinolytic activity in viable spores of Encephalitozoon cuniculi and E. intestinalis was detected and dependence on reaction time, spore concentration, concentration of substrate and temperature were demonstrated. It was possible to block the chitinolytic activity by chitin hydrolysate. By incubation at 80§C for 10 min or at 55§C for 20 min the spores were loosing the chitinolytic activity. Incubation of the spores in trypsin reduced the chitinolytic activity. Cellulase activity could not be detected.