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Characterization of Native Isolates of Trichoderma Spp. and Cloning of Endochitinase

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Title Characterization of Native Isolates of Trichoderma Spp. and Cloning of Endochitinase
 
Creator Upendra Mahato
 
Contributor Sumangala Bhat
 
Subject Plant Bio-Technology
 
Description In the present study, 23 Trichoderma isolates belonging to different Trichoderma
species viz., T. viride, T. virens, T. polysporum, T. harzianum, T. pseudokoningii, and T.
koningii were isolated from 51 soil samples collected from different parts of India. Sixteen
Trichoderma isolates were screened for antagonistic action against Sclerotium rolfsii,
Rhizoctonia bataticola and Fusarium solani. Four T. virens isolates (IABT-1002, 1005, 1006
and 1007) were highly antagonistic against S. rolfsii and all the isolates of Trichoderma
except T. viride (IABT-1027), T.harzianum (IABT-1033) and T. polysporum (IABT-1011)
were capable of overlapping the R. bataticola colony. None of the Trichoderma isolates were
able to inhibit the growth of F. solani completely. When these isolates were tested for their
chitinolytic activity, T. koningii (IABT-1030) were found to be better followed by four T.
virens (IABT-1002, 1005, 1006 and 1007) isolates. Same sixteen isolates were subjected to
Randomly Amplified Polymorphic DNA (RAPD) analysis using nine oligodeoxynucleotide
primers (I-14, AK-10, AK-07, I-18, L-16, N-04, N-05, AB-11 and AB-07) to study the
genetic variability, high inter and intraspecific variation was observed.
The endochitinase gene was cloned from four T. virens isolates (IABT-1002, 1005,
1006 and 1007) using specific primers. All the cloned endochitinase genes were identical at
nucleotide and amino acid levels for the sequenced region except endochitinase gene from T.
virens IABT-1002 which was fully sequenced and found to code for 430 amino acids with a
molecular weight of 46559.9 Da and theoretical pI of 5.90. It has conserved domain of
Glycosyl hydrolases family 18, conserved catalytic and potential chitin binding domain.
Endochitinase gene from T. virens IABT-1002 in recombinant plasmid pSUM1C was
transferred to a plant transformation vector pHS100 to facilitate transformation of crop
plants. The recombinant clones of pHS100; pUSK1C was mobilized into Agrobacterium
tumefaciens LBA4404 by triparental mating which was used to transform tobacco and the
putative transformants were confirmed by PCR with gene specific primers.
 
Date 2016-10-17T13:35:36Z
2016-10-17T13:35:36Z
2005
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/80704
 
Format application/pdf
 
Publisher UAS, Dharwad