Identification and detection of Macrophomina phaseolina by using species-specific oligonucleotide primers and probe.
A study was conducted to develop specific primers and oligonucleotide probe (within the ITS region) and subsequently evaluate their efficiency for identification/detection of Macrophomina phaseolina under in vitro conditions. The genomic DNA amplification of ITS region of different isolates yielded a fragment of approximately 650 bp. The restriction enzymes EcoR I, Sau3A I, Alu I and Cla I gave identical pattern for all isolates and produced two fragments of different sizes at single cut. The enzymes Hap II, Msp I and Taq I have multiple cleavage sites in ITS amplified product and produced fragments less than 100 bp in size. The enzymes Apa I, Hind III and Sac I failed to cleave the ITS region. Sequence comparison of ITS region encompassing ITS-1, 5.8 S rRNA gene and ITS-2 of M. phaseolina and other related fungal species revealed three regions that were conserved among the M. phaseolina isolates. After editing and rearrangement of aligned sequences and comparison with the sequences of closely related genera of fungi, regions 1 and 5 were selected for the development of species-specific primers for M. phaseolina. Two primers MpKFI and MpKRI were designed from the specific nucleotide areas and one oligonucleotide probe MpKHI (19 mer) was designed from the conserved region, adjacent to 5.8 S gene showed in the region 3. The designed primers yielded single amplified product of 350 bp with all the M. phaseolina isolates tested. Dot blot assay initially conducted with variable quantities of PCR product from different strains of M. phaseolina did not show any appreciable differences in signal intensity. In the later dot blot assay conducted with 5 µl of PCR product irrespective of their concentrations allowed the detection of all the spots of PCR products from M. phaseolina strains. Probe MpKHI selectively hybridized with strains of M. phaseolina but failed to do so with all representative strains of different groups of fungi.