Nested PCR-Based Rapid Detection of Phytoplasma Leaf Wilt Disease of Coconut in Sri Lanka and Systemic Movement of the Pathogen

Abstract

Phytoplasmas are associated with many plant diseases. In palms, lethal bronzing disease, Texas Phoenix palm decline, and coconut lethal yellowing decline are some of them. In Sri Lanka, coconut leaf wilt decline has been reported in the Weligama area of the Southern province, and the disease is called Weligama coconut leaf wilt disease (WCLWD). Unlike other phytoplasma diseases of palms, WCLWD shows slow disease progress. Pathogen detection entirely relies on nested polymerase chain reaction (PCR). However, inconsistencies in pathogen detection have been experienced, i.e., symptomatic plants often produce negative results. The objectives of this study were to reconsider the choice of primers and to determine the best sampling tissue types for consistent detection of the pathogen. Among the six universal primer combinations tested, P1/Tint nested with fU5/rU3 produced consistent results. BLASTn searches of the sequences showed 99–100% similarity to sugarcane white leaf disease (SWL) or grassy shoot (SGS) disease-causing phytoplasma. The optimized nested PCR protocol was successful, with the minimum success rating of 88% and 100% specificity. Midribs of milky white bud leaf samples were the best tissue type for rapid detection. Systemic movement of the pathogen and a tentative latent period were also reported. The findings are helpful in the early detection of the disease.

Keywords

• phytoplasma
• coconut
• leaf wilt disease
• nested PCR
• 16S rRNA