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Journal of Environmental Biology

pISSN: 0254-8704 ; eISSN: 2394-0379 ; CODEN: JEBIDP
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    Abstract - Issue Jan 2017, 38 (1)                                     Back


nstantaneous and historical temperature effects on a-pinene

Rapid identification of endophytic fungi of sugarcane

(Saccharum spp. hybrid) using PCR-RFLP of rDNA

 

Sangeeta Srivastava1*, Prashant Shekhar Gupta1, Sunita Lal1 and O.K. Sinha2

1Division of Crop Improvement, ICAR-Indian Institute of Sugarcane Research, Lucknow-226 002, India

2AICRP(S), ICAR-Indian Institute of Sugarcane Research, Lucknow- 226 002, India

*Corresponding Author E-mail: sangeeta_iisr@yahoo.co.in

 

 

Key words

Banding pattern,

Endophytic fungi,

Internal transcribed spacer,

PCR-RFLP assay,

ribosomal DNA

 

 

 

Publication Data

Paper received : 03.12.2015

Revised received : 03.03.2016

Accepted : 04.05.2016

 

Abstract

Aim : Restriction fragment length polymorphism analysis of PCR-amplified internal transcribed spacer (ITS) regions of rDNA is a cheap and convenient molecular tool used in fungal taxonomy. This tool has been used for rapid identification of the endophytic fungi of sugarcane belonging to different genera for their further potential utilization.

 

Methodology : Eight isolates belonging to three endophytic fungi viz. Colletotrichum falcatum, Fusarium moniliforme and Trichoderma viride isolated from sugarcane plant were used for differentiation using PCR-RFLP pattern of rDNA. For this, the ITS1-5.8S-ITS2 region of rDNA was amplified with the help of universal primers and the PCR products were digested with various restriction enzymes.

 

Results : The size variation of amplified products ranged from 637 bp (C. falcatum isolates), 592-614 bp (F. moniliforme isolates) to 631-698 bp (T.viride isolates). Five restriction enzymes viz. Alu I, EcoR I, Bam H I, MSe I and Sma I used for PCR-RFLP of ITS fragments?? yielded a total of 54 bands that were arranged in three RFLP patterns.

 

Interpretation : Banding pattern obtained with the combination of Alu I and Mse I enzymes presented the best profile to identify the endophytic fungi and allowed distinction at molecular level. This reflected the potential of PCR-ITS-RFLP using a combination of restriction endonucleases as a rapid and convenient differentiation tool to distinguish these endophytic fungi.

 

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