Genetic enhancement of Trichoderma viride to overproduce different hydrolytic enzymes and their biocontrol potentiality against root rot and white mold diseases in bean plants

Abstract

A major part of Trichoderma antifungal system consists of a number of genes encoding for an astonishing variety of secreted lytic enzymes including; e.g. chitinase, cellulases and β-1,3glucanases. The present work aimed to apply the mutagenesis technique in genetically breeding program of the bioagent Trichoderma viride to enhance three effective hydrolytic enzymes in their biocontrol abilities against two of the important plant fungal pathogens; Sclerotia rolfesii and Sclerotinia sclerotiorum the causal fungi of rot-root and white-rot diseases, respectively. Subjecting the spores of the wild type (T. viride) to the first UV-exposure time induced highly decrease in survival reached to 99.85%. Also, subjecting the spores of the two selected mutants (TvM1-UV1 and TvM9-UV1) to the second UV-exposure time induced highly decrease in their survival reached to 99.81 and 99.52%, respectively. The first and the second UV-treatments induced mutants with high growth and better sporulation measurements as well as superiority in biological control activities against some rot pathogens formed sclerotia than their parental wild type, Trichoderma viride. Four UV-induced mutants (TvM1-UV1, TvM9-UV1, TvM1-UV2 and TvM9-UV2) that obtained after UV-exposure of the parental wild type, Trichoderma viride were in vitro assessed for their biological control activities (Dual culture, filtrate inhibition, inhibition of sclerotia viability and inhibition of Polygalacuronase (PG) and Pectin methyl esterase (PME) activities against both pathogens. Through in vitro inhibitory activity by culture filtrates of the selected mutants (TvM1-UV1, TvM9-UV1, TvM1-UV2 and TvM9-UV2) and their parental wild type, T. viride, the growth rates, sclerotia viability as well as PG and PME activities were decreased the pathogens compared with the wild type. The filtrate inhibition activities of the selected mutants were considerably increased than that of their parental wild type. The pathogen S. sclerotiorum was more senstive to the inhibition effects of the culture filtrates of the tested T. viride mutants than the other pathogen S. rolfsii. Spectrophotometer was used to assay the hydrolytic enzymes; chitinase, β-1,3-glacturoranase and cellulases activities from the selected mutants (TvM1-UV1, TvM9-UV1, TvM1-UV2 and TvM9-UV2) and their parental wild type, T. viride. The selected mutants showed higher hydrolytic enzyme activities than their parental wild type. The greatest enzyme production by the tested T. viride mutants was cellulases followed by β-1,3-glucanase then chitinase. Total protein extraction and banding patterns SDS-polyacrylamid gel electrophoresis of mutants showed fold increased in bands numbers compared with wild type. In biological control experiments against rot-root and white-rot diseases caused by S. rolfesii and S. sclerotiorum, respectively in bean plants under artificial and natural infested soil, complete control of the disease was achieved, in which even the higher producer-hydrolytic enzymes of T. viride mutants treatment failed to provide significant control compared with their parental wild type. Treatment of the bean seeds with T. viride mutants resulted in reducing colonization of S. rolfesii and S. sclerotiorum in bean rhizosphere compared with treatment with their parental wild type and increased plant yield. keywords: Biological control , Mutation, hydrolytic enzymes, T. viride, sclerotia. INTRODUCTION Plant diseases are limiting factors in the production of most crops. Rot-root and white-rot cause by Sclerotia rolfesii and Sclerotinia sclerotiorum ,respectively are among the most devastating diseases in different crops causing thoughtful economic outlay. These diseases are troublesome because of the high plant Agric. Biol. J. N. Am., 2010, 1(3): 273-284 274 densities and auspicious ecosystem for their development (Rafael et al. 2007 and Ender and Kelly 2005). We know that a fungicide program is required to manage fungal diseases. But fungicide application caused many negative effects on environment. As a consequence been directed primarily towards identified new control methods that could be effective, reliable and safe for the environment. Biological control of several plant pathogens has shown promise as an option diseases management strategy (Jensen 2000). Antagonistic microorganisms represent the most diverse group of organisms on the plant. Even through the natural microflora, antagonistic fungi included Trichoderma species are surmised of special share as biological control agents against numerous phytopathogenic fungi (Jensen 2000). Trichoderma species are known as cogent producer of many antifungal metabolites including enzymes and others (Ghisalberti, 2002 and Harman et al., 2004). Trichoderma classifcationally is a genus of fungi within the division Deutromycetes, which are a heterogeneous group of fungi in which sexual stages (perfect stages) are not known or rarely found and reproduction is limited to the production of conidia (Hunter and Barnett 1974 and Harman et al. 2004). Unfortunately, many of the microorganisms that have gained industrial importance do not have a clearly defined sexual cycle (e.g. Fungi imerfacti). This has meant that the only way to change the genome with a view to enhancing productivity has been indulge in massive mutational programs, followed by screening and selection to detect the new variants that might arise (Smith, 1996 ; Brunner et al. 2005 and Ximena et al. 2007). Mutagenesis of the microorganisms including those used as biocontrol agents were applied to improve of the antifungal production and antagonistic potential over a broad spectrum of phytopathogens, survival, longevity and activity (Ximena et al. 2007). Several successful endeavors had been made to rectify the biocontrol potential of Trichoderma or Gliocladum species (which were bankrupted to generate new biotypes) by exposing the spores to chemical or physical mutagens. Physical mutagen like UV-ray (Melo et al. 1997 and Brunner et al. 2005). In this respect, Haggag and Mohamed (2002) found that mutagenesis of three Trichoderma species by gamma irradiation exhibited high capabilities to produce efficient antibiotics, enzymes and phenols, corresponded to better onion white rot disease control in overall biocontrol ability. The present work aimed to apply the mutagenesis technique in genetically breeding program of the bioagent Trichoderma viride to enhancement three effective hydrolytic enzymes in their biocontrol abilities against two of the important plant fungal pathogens, Sclerotia rolfesii and Sclerotinia sclerotiorum, the causal fungi of root-rot and damping-off diseases. MATERIALS AND METHODS Fungal cultures : The bioagent fungi, Trichoderma viride and the pathogens; Sclerotia rolfsii and Sclerotinia sclerotiorum, the causal fungi of White mold diseases, respectively were isolated from bean plants, collected from El-Bohira (Nobaria districts) and identified in Plant Pathology Department, National Research Center, Dokki, Cairo, Egypt. Cultures were maintained on Bean dextrose agar (PDA) medium. Dextrose broth (PDB) medium or Czapek Dox salt solution (CDS) were used to obtain the fungal culture