Development of a PCR based assay for the detection of Rigidoporus microporus (causative organism of white root disease of rubber)

Abstract

Introduction: Rigidoporus microporus, one of the most harmful fungal pathogens which cause white root disease, affects Para rubber (Hevea brasiliensis) plantation, the second largest plantation in Sri Lanka. R. Microporus, a member of basidiomycetes division, affects the plants starting from its root to other parts. The damage is caused due to certain enzymes produced by the fungus and this occurs stage by stage. Firstly, a formation of white rhizomorphs grows and firmly adheres to the root’s debris. Next, the collar region of trees gets infected and basidiocarps occur. The primary justification for conducting this study is because the roots of plants are initially affected making it difficult to detect the fungus. Hence initial identification of the fungus is important so that the pathogen can be detected at an early stage which helps to halt the progression of the disease. Accordingly, the objective of the study was to develop a polymerase chain reaction (PCR) based assay for the detection of R. microporus. Methods: R. microporus isolates’ DNA was extracted using two methods and PCR amplification was performed. For PCR amplification, β-tubulin primers were designed using the Bioedit software and Primer3 Input (version 0.4.0). With the chosen primer, temperature, MgCl2 and dNTPs were optimised. Sensitivity and specificity assay was performed using prior PCR conditions.