Preliminary Research On The Diversity Based On ITs Molecular Maker And Cloning Of β-1,3-glucanase Gene Fragment From Trichoderma

Species of the genus Trichoderma are ubiquitous and relatively easy to isolate andculture. They are the most important coenosis of soil microorganisms. There is a specialgroup in Trichoderma which can restrain the growth of pathogen that called antagonisticTrichoderma. Twenty two Trichoderma strains with disease resistance were screened in2007, in this study, the information of disease resistance were completed based on duelingculture for their antagonisms against pathogens. In this paper, eighty six Trichodermastrains conserved in our laboratory, including twenty two Trichoderma strains with diseaseresistance, were used as experiment materials. The diversity of Trichoderma and theirdisease resistance were investigated by ITS (Internal Transcribed Spacer) PCR-RFLPmethod and β-1,3-glucanase gene framgment was cloned and analyzed. The main resultsare as following.1) There were six of twenty two Trichoderma strains assayed uncompletely by theprevious study, which were used as experiment materials based on duel culture for theirantagonisms against pathogens. Results showed that72.22%Trichoderma had a inhibitionratio of over50%against pathogens, suggesting that the good disease resistance againstnine pathogens was detected. Synthesizing the information of disease resistance, we foundthat there were difference in some extent on inhibitory action, which was from differentTrichoderma strains against a certain pathogen or from a certain Trichoderma strain againstdifferent pathogens.2) PCR-RFLP method was used to study the systematic relationships of Trichodermaincluding eighty six strains representing fifteen species. The ITS region was amplified byPCR with primers ITS1/ITS4and then cleaved with two restriction enzymes HaeIII andHinfI. Results showed that, in total, nineteen distinct and polymorphic DNA bands wereobtained after digested by two restriction enzymes respectively. There were eightpolymorphic DNA bands and six restriction patterns by the use of HaeIII, elevenpolymorphic DNA bands and nine restriction patterns by the use of HinfI. These resultsshowed that HinfI enzyme has more discriminatory power than HaeIII. Both of the tworestriction enzymes digested maps of ACCC30371(T. harzianum) were special for itsunique among the eighty six strains tested, suggesting that ACCC30371may paly a specialrole in the evolution of Trichoderma.3) Code “1” and “0” represented a certain band of the whole nineteen polymorphicDNA bands which appeared and disappeared, respectively. All of the codes were collected to generate a matrix, which was assayed by NTSYS-PC. The dendrogram showed extensivepolymorphism and the eighty six strains could be clustered into twenty one groups. Theseresults were basically consistent with the classification of Gams and Bissett, whoseclassification mainly based on morphological characters.4) Twenty two strains with high level disease resistance (classes A) and thirteen strainswith low level disease resistance (classes B) were selected as materials, degenerate primerswere designed and Touch-down PCR were preformed to obtain the target fragment. Thefragments from nineteen of classes A and four of classes B strains were identified asβ-1,3-glucanase gene fragments by NCBI BALSTx. The alignment of the deduced aminoacid sequences shared sequence similarity between40.7%and100%among twenty threestrains, and the predicted amino acid sequences locus from sixty to eighty and from eightynine to one hundred were the most conservative, which provided the basic information forfurther cloning the full length of β-1,3-glucanase gene and screening genotype with highenzyme activity.