Immunological detection of the Weligama coconut leaf wilt disease associated phytoplasma: Development and validation of a polyclonal antibody based indirect ELISA.

Chamini Kanatiwela-De Silva,Matthew Dickinson,Malini Damayanthi,Nalin De Silva,Devaka Weerakoon,Preethi Udagama,Rohana Wijesekera

doi:10.1371/journal.pone.0214983

Abstract

Weligama coconut leaf wilt disease (WCLWD) causes heavy losses in the coconut cultivations of southern Sri Lanka. The in-house developed and validated indirect ELISA was based on specific polyclonal antibodies raised in female New Zealand White rabbits, against partially purified WCLWD associated phytoplasma. This ELISA has the potential to distinguish secA PCR confirmed, WCLWD associated phytoplasma positive palms from phytoplasma free palms at high accuracy (93%) and sensitivity (92.7%), but with marginal specificity (79%). The calculated ELISA cross reactivity index (CRI) values were low for sugarcane white leaf (7%) and sugarcane grassy shoot (8%) infected leaves, but with marked highCRIfor both Bermuda grass white leaf (69%) and areca nut yellow leaf (70%) infected leaves. SecA gene based phylogenetic relationships of the WCLWD associated phytoplasma with these other locally prevalent phytoplasma strains elucidated this immunological cross reactivity, which was further reiterated by virtual restriction fragment length polymorphism analysis. Based on scanning electron microscopy, this study provides additional visual evidence, for the presence of phytoplasmas in WCLWD infected tissues.

Highlights

Coconut (Cocos nucifera L.), an important tropical perennial crop, is a major source of export revenue to Sri Lanka that provides livelihood to more than 0.8 million people of the island [1]
This study aimed to develop and validate an indirect ELISA based on specific polyclonal antibodies raised in rabbits to the partially purified Weligama coconut leaf wilt disease (WCLWD) associated phytoplasma, to be subsequently developed as a rapid diagnostic method
Nucleotide BLAST of secA sequences confirmed the association of phytoplasma with WCLWD, closely related to phytoplasma strains of AYLD (KM978910.1) and Bermuda Grass White Leaf Disease (BGWLD) (KT369127.1- KT369134.1)

Summary

Introduction

Coconut (Cocos nucifera L.), an important tropical perennial crop, is a major source of export revenue to Sri Lanka that provides livelihood to more than 0.8 million people of the island [1]. It is a key food crop in the country responsible for about 22% of the per capita caloric intake in the diet, being second only to rice, the staple food of Sri Lankans [2]. One of the oldest agrobased industries of Sri Lanka, coconut cultivation, spans approximately 402,649 ha and accounts for 21% of agricultural lands of the island [2].

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Conclusion