ISSN: 0973-7510

E-ISSN: 2581-690X

Mohammad Shahid, Mukesh Srivastava, Sonika Pandey , Vipul Kumar, Anuradha Singh, Shubha Trivedi and Y.K. Srivastava
Biocontrol Laboratory, Department of Plant Pathology, CSA University of Agriculture & Technology, Kanpur – 208002, India.
J. Pure Appl. Microbiol., 2016, 10 (1): 713-723
© The Author(s). 2016
Received: 09/09/2015 | Accepted: 01/11/2015 | Published: 31/03/2016
Abstract

Strain identification in situ is an important factor in the monitoring of microorganisms used in the field. In this study, we demonstrated the use of sequence-characterized amplified region (SCAR) markers to detect genomic DNA from Trichoderma harzianum Th azad from soil. Two primers (SCAR A1/SCAR A1c) were tested against DNA of 49 isolates of Trichoderma spp. and amplified a 900-bp fragment from T. atroviride 11 and a 1.5-kb fragment from T. harzianum Th azad, using an annealing temperature of 68°C. These fragments showed no significant homology to any sequence deposited in the databases. The primer pair, BR1 and BR2, was designed to the 1.5-kb fragment amplified from T. harzianum Th azad, generating a SCAR marker. To test the specificity of these primers, experiments  were conducted  using  the  DNA from  49 Trichoderma spp. strains and  22 field soil samples obtained from different agro-geographical condition of UP. PCR results showed that BR1 and BR2 amplified an 830-bp fragment unique to T. harzianum Th azad. Assays in which total DNA was extracted from sterile and nonsterile soil samples, inoculated with spore or mycelium combinations of Trichoderma spp.  strains,  indicated  that  the  BR1 and  BR2 primers could specifically detect T. harzianum Th azad in a pool  of  mixed  DNA.  No other soil-microorganisms containing these sequences were amplified using these primers. To test whether the 830-bp SCAR marker of T. harzianum Th azad could be used in real-time PCR experiments, new primers  (CSA-Th azadf and  CSA-Th azad)  conjugated with a TaqMan fluorogenic probe were designed. Real- time PCR assays were applied using DNA from sterile and nonsterile soil samples inoculated with a  known quantity of spores of Trichoderma spp. strains.

Keywords

RT-PCR, Trichoderma, Genomic DNA, SCAR marker.

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© The Author(s) 2016. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.