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Molecular characterization of an endophytic Phomopsis liquidambaris CBR-15 from Cryptolepis buchanani Roem. and impact of culture media on biosynthesis of antimicrobial metabolites

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ABSTRACT

An endophytic fungus Phomopsis liquidambaris CBR-15, was isolated from Cryptolepis buchanani Roem. (Asclepiadaceae) and identified by its characteristic culture morphology and molecular analysis of the ITS region of rDNA and intervening 5.8S rRNA gene. The impact of different culture media on biosynthesis of antimicrobial metabolites was tested by disc diffusion assay. Polyketide synthase gene (PKS) of the endophytic fungus was investigated using three pairs of degenerate primers LC1–LC2c, LC3–LC5c and KS3–KS4c by PCR. TLC-bioautography method was employed to detect the antimicrobial metabolites. Antimicrobial metabolites fractionated with ethyl acetate extract showed significant antimicrobial activity against the test bacteria and fungi. Biosynthesis of antimicrobial metabolites was optimum as depicted by zone of inhibition from ethyl acetate extract cultured in potato dextrose broth. Strain CBR-15 was identified as Phomopsisliquidambaris and PKS genes of the fungus were amplified with LC3–LC5c and KS3–KS4c sets of degenerate primers. These findings suggest that endophytic P.liquidambaris CBR-15 harbor iterative type I fungal PKS gene domain which indicates the biosynthetic potential of endophytic fungi as producers of natural antimicrobial metabolites. The study also demonstrates the utilization and optimization of different culture media which best supports for the biosynthesis of the antimicrobial metabolites from P.liquidambaris.

Electronic supplementary material: The online version of this article (doi:10.1007/s13205-014-0204-2) contains supplementary material, which is available to authorized users.

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ITS sequence-based Neighbor Joining tree of Phomopsis sp. isolates. A consensus NJ dendrogram with bootstrap values (1,000 replications) based on multiple sequence alignment. Scale bar indicated nucleotide substitutions per nucleotide position. * denotes the isolate obtained in the present study (accession no. KF032029)
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Fig3: ITS sequence-based Neighbor Joining tree of Phomopsis sp. isolates. A consensus NJ dendrogram with bootstrap values (1,000 replications) based on multiple sequence alignment. Scale bar indicated nucleotide substitutions per nucleotide position. * denotes the isolate obtained in the present study (accession no. KF032029)

Mentions: The amplified ITS region of rDNA was sequenced and aligned with the ITS sequences of the different organisms retrieved from NCBI databases, using CLUSTAL W (Thompson et al. 1994). Dendrogram was generated using neighbor joining (NJ) plot and the boot strapping was carried out using 1,000 replications. The acquisition of ITS1-5.8S-ITS2 sequence data and NJ plot showed that the isolate belongs to P. liquidambaris CJBB25-20, KC895530 (Fig. 3) which is also an endophytic fungus isolated from Saraca asoca (http://www.ncbi.nlm.gov/nuccore/KC895530). The partial ITS sequence data of this fungus was deposited in GenBank, under accession no. KF032029.Fig. 3


Molecular characterization of an endophytic Phomopsis liquidambaris CBR-15 from Cryptolepis buchanani Roem. and impact of culture media on biosynthesis of antimicrobial metabolites
ITS sequence-based Neighbor Joining tree of Phomopsis sp. isolates. A consensus NJ dendrogram with bootstrap values (1,000 replications) based on multiple sequence alignment. Scale bar indicated nucleotide substitutions per nucleotide position. * denotes the isolate obtained in the present study (accession no. KF032029)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4362740&req=5

Fig3: ITS sequence-based Neighbor Joining tree of Phomopsis sp. isolates. A consensus NJ dendrogram with bootstrap values (1,000 replications) based on multiple sequence alignment. Scale bar indicated nucleotide substitutions per nucleotide position. * denotes the isolate obtained in the present study (accession no. KF032029)
Mentions: The amplified ITS region of rDNA was sequenced and aligned with the ITS sequences of the different organisms retrieved from NCBI databases, using CLUSTAL W (Thompson et al. 1994). Dendrogram was generated using neighbor joining (NJ) plot and the boot strapping was carried out using 1,000 replications. The acquisition of ITS1-5.8S-ITS2 sequence data and NJ plot showed that the isolate belongs to P. liquidambaris CJBB25-20, KC895530 (Fig. 3) which is also an endophytic fungus isolated from Saraca asoca (http://www.ncbi.nlm.gov/nuccore/KC895530). The partial ITS sequence data of this fungus was deposited in GenBank, under accession no. KF032029.Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

An endophytic fungus Phomopsis liquidambaris CBR-15, was isolated from Cryptolepis buchanani Roem. (Asclepiadaceae) and identified by its characteristic culture morphology and molecular analysis of the ITS region of rDNA and intervening 5.8S rRNA gene. The impact of different culture media on biosynthesis of antimicrobial metabolites was tested by disc diffusion assay. Polyketide synthase gene (PKS) of the endophytic fungus was investigated using three pairs of degenerate primers LC1–LC2c, LC3–LC5c and KS3–KS4c by PCR. TLC-bioautography method was employed to detect the antimicrobial metabolites. Antimicrobial metabolites fractionated with ethyl acetate extract showed significant antimicrobial activity against the test bacteria and fungi. Biosynthesis of antimicrobial metabolites was optimum as depicted by zone of inhibition from ethyl acetate extract cultured in potato dextrose broth. Strain CBR-15 was identified as Phomopsisliquidambaris and PKS genes of the fungus were amplified with LC3–LC5c and KS3–KS4c sets of degenerate primers. These findings suggest that endophytic P.liquidambaris CBR-15 harbor iterative type I fungal PKS gene domain which indicates the biosynthetic potential of endophytic fungi as producers of natural antimicrobial metabolites. The study also demonstrates the utilization and optimization of different culture media which best supports for the biosynthesis of the antimicrobial metabolites from P.liquidambaris.

Electronic supplementary material: The online version of this article (doi:10.1007/s13205-014-0204-2) contains supplementary material, which is available to authorized users.

No MeSH data available.