The role of protease of Trichoderma harzianum in the biocontrol of Botrytis cinerea was examined. Two isolates of T. harzianum were compared for their ability to produce protease in liquid culture medium and on the surface of bean leaves. The biocontrol agent T. harziaum T39 produced 58 mU/ml of protease and T. harzianum NCIM1185 produced 54 mU/ml on the 5th day of growth in liquid culture medium. On bean leaves, combinations of B. cinerea and T. harzianum isolates were examined for the synthesis of protease. The protease activities were 0.9 and 0.6 mU/ml for T. harzianum T39 and NCIM1185, respectively, and 0.5 mU/ml for B. cinerea alone after 48 h of incubation. In the presence of T. harzianum T39 culture liquid containing protease, a 55% reduction in B. cinerea germination and a 80% reduction in the germ tube length were observed after 17 h of incubation in vitro. When T. harzianum isolates were added to B. cinerea on bean leaves, increased synthesis of protease was observed (1.0 and 1.2 mU/ml for T39 and NCIM1185, respectively). In the presence of T. harzianum NCIM1185 protease, although the rate of germination was reduced, B. cinerea attained 98% germination after 17 h of incubation. The hydrolytic enzymes produced by B. cinerea, endo-polygalacturonase (PG) and exoPG were partially deactivated by protease from the T. harzianum isolates. Carboxymethyl cellulase was deactivated only by protease of NCIM1185. On the surface of bean leaves, the protease (obtained from liquid culture medium of T. harzianum isolates) resulted in 56–100% reduction of disease severity. The culture liquid containing protease synthesized on the surface of bean leaves treated with B. cinerea and with T. harzianum was collected and added to fresh leaves infected by B. cinerea. There was 56–100% and 30–75% reduction of disease severity with liquid droplet collected from the leaves treated with T. harzianum T39 and NCIM1185, respectively. Increased control of disease was obtained by combining the conidia of T. harzianum isolates with protease obtained from culture media. Protease inhibitors, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64), antipain hydrochloride, and a mixture of inhibitors, but not pepstatin A, fully or partially nullified the biocontrol effect of T39. T39 was found to be a poor producer of chitinase and β-1,3-glucanase in vitro. These enzymes were not detected on leaves treated with T39. Involvement of protease in biocontrol of B. cinerea is suggested.

The role of trichoderma harzianum protease in the biocontrol of botrytis cinerea

Low molecular weight chitosan prepared with the aid of cellulase, lysozyme and chitinase: Characterisation and antibacterial activity

A range of chitinase genes from microorganisms have been cloned and the potential uses of these genetically manipulated organisms are being investigated by various researchers Fungi and yeast are better producers of chitinase than bacteria Since fungi grow at a slower rate, there have been efforts to clone the fungal chitinase genes into fast-growing bacteria This review gives a brief survey of recent progress in the regulation and cloning of microbial chitinase genes Emphasis is placed on the post-translational modification and localization of the recombinant protein in the host Various amino acid domains are present in this protein The mode of catalytic activity of the recombinant protein in comparison to the wild-type protein is discussed in the available literature The different mechanisms involved in the regulation of chitinase genes from various microorganisms is discussed by the researchers The scope of future research and conclusions yet to be obtained in this particular area are also outlined in this review

Regulation and cloning of microbial chitinase genes.

Nine isolates of Trichoderma were collected from Assiut Governorate,Egypt, as leaf surface and endophytic fungi associated with onion ﬂorastalks. Four isolates were identiﬁed as Trichoderma harzianum, while ﬁveisolates were belonging to Trichoderma longibrachiatum. The antagonisticactivity of these isolates against onion purple blotch pathogen Alternariaporri was studied in vitro using dual culture assay. All tested Trichodermaisolates showed mycoparasitic activity and competitive capability againstthe mycelial growth of A. porri. Mycoparastic activity of Trichoderma wasmanifested morphologically by the overgrowth upon the mycelial growthof the pathogen and microscopically by production of coiling hyphaearound pathogen hyphae. Isolates of T. harzianum exhibited high abilityto compete on potato dextrose agar (PDA) medium causing the maximumrate of pathogen inhibition (73.12%), while isolates of T. longibrachiatumshowed inhibition rate equalling 70.3%. Chitinase activity of Trichodermawas assayed, and T. harzianum Th-3013 showed the maximum value con-tributing 2.69 U/min. Application of T. harzianum Th-3013 to control pur-ple blotch disease in vivo under greenhouse conditions caused diseasereduction up to 52.3 and 79.9% before and after 48 h of pathogen inocu-lation, respectively, while the fungicide Ridomil Gold Plus caused diseasereduction comprising 56.5 and 71.7%, respectively. This study proved thatT. harzianum Th-3013 as a biocontrol agent showed signiﬁcant reductionin onion purple blotch disease compared with the tested fungicide.IntroductionPurple blotch disease, caused by Alternaria porri (Ellis)Cif., is one of the most serious diseases that wide-spread in many parts of the world, restricted to Alliumspp. and more prevalent in warm and humid environ-ments (Cramer 2000; Suheri and Price 2000). Purpleblotch disease caused signiﬁcant reduction in bulband seed yield of the onion crop (Gupta and Pathak1988). The disease is more severe on seed crop ascompared to bulb crop causing sometimes 100% loos-ing of the seed production (Singh et al. 1992;Schwartz 2004). Although chemical control of onionblotch had been practised and its success depends lar-gely on high frequency of spraying, but today, thereare strict regulations on chemical fungicide use due tocarcinogenic effects, residual toxicity problems, envi-ronmental pollution and development of fungicide-resistant strains (Benitez et al. 2004; Rial-Otero et al.2005). Therefore, there are a large number of studiesthat have been devoted to apply a biological control asnature-friendly alternative method (Siameto et al.2010; Soria et al. 2012). The potential of Trichodermaspecies as biocontrol agents of plant diseases was ﬁrstrecognized in the early 1930s (Weindling 1932), andsince then, there have been extensive efforts in thecommercial production of them for disease control ina number of crops (Harman 1996; Gardener and Frav-el 2002). Recently, several investigation proved thatTrichoderma have been used to control many foliar

Isolation of Trichoderma and Evaluation of their Antagonistic Potential against Alternaria porri

BACKGROUND: Chito-oligosaccharide (COS) is generally known to possess many specific biological functions, especially antibacterial activity, depending on its size. To prepare a specific size range of COS, however, has proved difficult. The aim of this study was to establish a method for preparing a specific size range of antibacterially active COS by adjusting the degree of deacetylation (DD) of β-chitosan in a Trichoderma harzianum chitinase-hydrolysing process.

RESULTS: The molecular weight spectrum, elucidated by viscosity-average molecular weight, high-performance liquid chromatography and thin layer chromatography, of COS in chitosan hydrolysate was significantly related to the DD of its original chitosan. Compared with the original form, COS produced at 90% DD showed superior activity against most Gram-negative bacteria tested, with a minimum inhibition concentration (MIC) ranging from 55 ± 27 to 200 ± 122 µ g mL−1. Conversely, most Gram-positive strains tested were less sensitive to COS (MIC > 880 ± 438 µ g mL−1) than to its original form. Among the Gram-positive strains, Staphylococcus xylosus was the only exception in that it showed a high susceptibility to COS and had an MIC as low as 45 ± 11 µ g mL−1.

CONCLUSION: The results indicate that the production of a specific size range of COS product is possible by altering the DD of chitosan in the chitinase-catalysed process. To produce various sizes of COS for versatile biological functions, as seen in this study to inhibit various types of bacteria, is made possible in this established process. Copyright © 2008 Society of Chemical Industry

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Preparation of antibacterial chito‐oligosaccharide by altering the degree of deacetylation of β‐chitosan in a Trichoderma harzianum chitinase‐hydrolysing process

Studies on critical analysis of factors influencing improved production of protoplasts from Trichoderma reesei mycelium

Intergeneric hybridization of Trichoderma reesei QM9414 and Saccharomyces cerevisiae NCIM 3288 by protoplast fusion

The regeneration of Saccharomyces cerevisiae, NCIM 3288, cells from its sphaeroplasts were found to be influenced by a number of factors. The most suitable conditions of regeneration were also dependent on growth medium, that is, using malt-extractglucose-yeast extract-peptone (MGYP) medium: mannitol 0.7 M, pH 6.5, 30 °C and using yeast extract-peptone-glucose (YPG) medium: sucrose 0.7 M, pH 5.0 and 30 °C. The maximum regeneration frequency was observed in YPG medium.

Analysis of factors responsible for the regeneration to intact cells from sphaeroplasts of Saccharomyces cerevisiae

Important parameters in the regeneration of protoplasts are viability, the capacity to synthesize cell walls and the retention of properties of the parent cell. Mycelial protoplasts of Trichoderma longibrachiatum (Trichoderma reesei) have been regenerated. Factors influencing the regeneration of protoplasts of T. longibrachiatum QM 9414 were found to be, the nature of osmotic stabilizer, the concentration of osmotic stabilizer, pH, temperature, and the composition of regeneration medium. With glucose-mineral regeneration medium, the optimum conditions for protoplasts regeneration were 0.5 M KCl, pH 6.0 and temperature 30°C. With Czapek-Dox medium, the optimum conditions were 0.7 M mannitol, pH 6.0 and temperature 30°C. Maximum regeneration frequency of T. longibrachiatum protoplasts were obtained using glucose-mineral medium.

Improved technique on reversion of mycelial protoplast of Trichoderma reesei into mycelia

The propagation of animal cells in artificial media may be the basis for large scale biosynthesis of commercial animal proteins. This is equally applicable for the in-vitro development of chick-embryo. The major problems of in-vitro growth of chick-embryo are artificial environment for the growaills, suitable reactor configuration for cultivation and the mode of growth. In-vitro growth of chick-embryo can be better understod in stationary f1sk, agitated flask, continuous oscillatory glass reactor, and intermittent oscillation in glass reactor and continuous rotary reactor cf the reactor and methods developed for general cell growth. A survey of its advantages and disadvantages are summarized.

J. Anjani Kumari

Papers

Studies on critical analysis of factors influencing improved production of protoplasts from Trichoderma reesei mycelium

Intergeneric hybridization of Trichoderma reesei QM9414 and Saccharomyces cerevisiae NCIM 3288 by protoplast fusion

Analysis of factors responsible for the regeneration to intact cells from sphaeroplasts of Saccharomyces cerevisiae

Improved technique on reversion of mycelial protoplast of Trichoderma reesei into mycelia

Critical Analysis of Process Development on in-Vitro Growth of Chick-Embryo