Isolation, cloning and in silico analysis of β-tubulin gene from apple leaf blotch fungus Marssonina coronaria

Apple leaf blotch caused by Marssonina coronaria is an economically very important apple disease which leads to the loss in apple production. All the available commercial cultivars of apple are susceptible to this disease. Despite this, nothing much has been studied about the molecular biology of this fungus. Herein, we report the isolation of the beta-tubulin gene from DNA and cDNA of M. coronaria by PCR, then its cloning, sequencing and in silico analysis. The primer pairs were designed by utilizing the multiple sequence alignment based approach with already available β-tubulin sequences of other fungi in the GenBank database. The partial nucleotide sequences of 437 bp encoding 145 deduced amino acids were obtained with a theoretical isoelectric point and molecular weight of 10.10 and 16.50 kilodaltons, respectively. The BLASTn analysis of isolated gene sequences showed 90% identity with Marssonina brunnea whereas, BLASTp results of the deduced 145 aa sequences showed higher similarity to Melanomma pulvis - pyrius (100%) and Marssonina brunnea f. sp. ‘multigermtubi’ (99%). This gene was used for expression analysis using semi-quantitative RT-PCR at various stages and conditions of the fungal life cycle to ensure its use as an internal reference control for qRT-PCR studies. Further, the applicability of β-tubulin in the molecular detection of M. coronaria from infected apple tissues was also explored. This study will widen the molecular biological knowledge of this fungus and shed light on the apple - M. coronaria molecular interaction by studying the expression of various virulent genes during the infection process, detection, identification and differentiation of this pathogen.