Effects of disease severity stage and quality of extracted DNA on molecular detection of the Weligama coconut leaf wilt disease agent in Sri Lanka

Weligama coconut leaf wilt disease (WCLWD) is one of the devastating diseases in the Southern province of Sri Lanka, causing drastic yield reductions in coconut. The agent associated with WCLWD is a phytoplasma, which is an intracellular bacterium. Accurate molecular detection of phytoplasmas is important for effective disease management, especially due to the long latent period, during which the disease could be spread from non-symptomatic coconut palms. Two sampling rounds were conducted using 30 selected WCLWD palms, with 10 each from mild, moderate and severe disease categories and 10 asymptomatic coconut palms as control. DNA was extracted from the midrib of milky white emerging bud leaf tissues using a CTAB method, and nested PCR was performed to detect phytoplasmas. Extracted DNA was purified using Qiagen DNA purification kit to evaluate the effect of DNA purification on PCR detection. PCR results were assessed and compared between pre-purified and purified DNA samples. The highest PCR positivity was observed in the moderate disease severity stage after the DNA purification. However, upon DNA purification PCR positivity increased to 30%, 60% and 30% respectively for mild, moderate and severe palm categories. None of the asymptomatic control samples showed PCR positivity. In conclusion, DNA purification improved the quality of extracted DNA and efficiency of PCR detection, and the moderate disease severity stage was identified to be the best stage for detection of the WCLWD phytoplasma. The findings would be useful in WCLWD management programs.