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

Abstract: 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.

Abstract: A continuous set of low molecular weight chitosan (LMWC) products was successfully made for this study by coordinating three enzymes (chitinase, lysozyme and cellulase) and two different deacetylated chitosan substrates (80% and 92%). It was observed that the intrinsic viscosity molecular weight (MV) and SEC-HPLC-determined MW distribution of LMWC were directed by both the used enzyme and the degree of chitosan substrate acetylation. LMWC prepared using lysozyme and 92%-deacetylated chitosan had larger MW and, therefore, possessed higher antibacterial activity, as compared to other combinations. LMWC enzyme-directed properties suggest chitinase is more predictable and flexible to produce the specified MV of LMWC. LMWC’s solubility and antibacterial activity, determined as minimum inhibitory concentration (MIC), against Escherichia coli exhibited a negative linear relationship with log MV. E. coli strains showed much higher susceptibility to LMWCs than Staphylococcus aureus strains did. Both species also showed intra-species sensitivity diversity toward LMWC.

Abstract: 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

Abstract: 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

Abstract: Host Parasite Specificity and Pathogenesis R. Gupta, K.G. Mukerji. Yeast Species for Biocontrol of Apple Postharvest Diseases: An Encouraging Case of Study for Practical Use M. Haissam Jijakli, et al. Biological Control Agents of Canola and Rapeseed Diseases - Status and Practical Approaches S.M. Boyetchko. Innovative Applications of Microbial Agents for Biological Weed Control S.M. Boyetchko. Bacteria as Biocontrol Agents of Insects S. Kaur, K.G. Mukerji. Biocontrol-Plant Growth Promoting Rhizobacteria: Mechanism of Action K.V. B.R. Tilak, et al. Mycorrhiza in Control of Plant Pathogens: Molecular Approaches K.G. Mukerji. Biological Control of Bacterial Plant Diseases S. Kaur, K.G. Mukerji. Protoplast Fusion in Disease Control S. Mukerji, K.G. Mukerji. Role of Tissue Culture in Plant Disease Control P.S. Srivastava, et al. Genetic Manipulation of Antagonistic Fusarium spp Q. Migheli, et al. The Application of lux-Gene Technology in the Control of Soil-Borne Diseases D. White, et al. Index.

Abstract: A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.

Abstract: INTRODUCTION 21 ISOLATION OF PROTOPLASTS AND THEIR P ROPERTIES 22 Enzymatic Methods for Protoplast Isolation 22 Nonenzymatic Procedures for Protoplast Release 26 Properties of Protopiasts 27 WALL REGENERATION AND REVERSION OF PROTOPLASTS 28 Frequency of Protoplast Reversion 28 Morphology of Protoplast Reversion 29 Ultrastructure and Composition of the Regenerated Wall ..... 30 Biochemical Aspects oj Wall Biogenesis 31 PROTOPLAST FUSION AND GENETIC MANIPULATION 33 Protoplast Fusion and the Hybridization of Fungal Species 33 Transformation in Fungi by Using Protoplasts . .. ........ 36 CONCLUDING REMARKS 36

Abstract: Although protoplast fusion is of current interest because of its possibilities in pure and applied genetics, only a few reports exist on intraspecific fusion between fungal protoplasts and subsequent selection of heterokaryons. Naturally-occurring fusion between protoplasts of Geotrichum candidurn (Ferenczy, Kevei & Zsolt, I 974) and Aspergillz4s nidulans (Ferenczy, Kevei & Szegedi, I 975) has been reported. Binding & Weber (1974) described fusion between protoplasts of Phycomyces blakc~slceanus induced by seawater or calcium ions at high pH, but the frequency of heterokaryon formation was low in all cases. We here report heterokaryon formation, at high frequency, after induced intraspecific fusion between protoplasts of auxotrophs of Penicillium chryosogiwim, P . putulurn, P. roquejortii, A. nidulans, A . niger and Cephalosporium acrernonium, and fusion between protoplasts of P. chrysogenu~?z and P. rzotutunz auxotrophs, using solutions containing polyethylene glycol (PEG) mol.wt. 6000.

Abstract: The principles underlying the estimation of the viable proportion of cells in a suspension are discussed, viability being defined as the ability to multiply in a completely susceptible host. All estimates give equivocal results unless an ideal control system is available in which the inoculation of one cell always produces a tumour. The dose-response curves of such a system are defined. The results of four indirect in vitro methods of estimating the percentage viability of a cell suspension have been compared with the results of titrations in mice. The most reliable indirect index of viability was the ability to reduce triphenyltetrazolium chloride. All four in vitro methods gave similar results with a recently harvested cell suspension but three of them grossly overestimated the proportion of viable cells in a suspension which had been stored at 4 °C for 11 days.