Application of biological and single-strand conformation polymorphism assays for characterizing potential mild isolates of Citrus tristeza virus for cross protection

Sagheer Atta,Abdul Hannan,Ummad Ud Din Umar,Ateeq Ur Rehman,Syed Atif Hasan Naqvi,Muhammad Amjad Bashir,Changyong Zhou

doi:10.1186/s13568-019-0903-5

Abstract

Citrus tristeza virus (CTV) by killing millions of citrus cultivars grown on sour orange rootstock worldwide has become one of the most dangerous viral pathogen. Characterization of 12 CTV isolates was analyzed by biological indexing. Infected samples of citrus were collected from citrus growing areas of Pakistan and CTV was detected by symptoms on indicator plants and confirmed by direct tissue blot immunoassay (DTBIA). CTV positive samples were graft inoculated on six biological indicator hosts in the study. A standardized protocol was deployed to study biological characteristics of these isolates. All biological indicators induced mild and from mild to moderate reactions against all of the CTV isolates tested. About two isolates produced stem-pitting symptoms from moderate to severe on Mexican lime. CTV strains were further characterized and confirmed by the analysis of p25 gene of CTV isolates using single-strand conformation polymorphism (SSCP) assay. SSCP analysis revealed that most isolates confined only one predominant sequence variant. SSCP profiles of PCR amplified products from CTV isolates showed bands patterns corresponding to mild and sever strain. Three isolates (4MF, 8KBS and 10GS) from different regions and cultivars were identified as potential source of mild strains for cross protection. These results are the best base for mild strain cross protection (MSCP) in the country.

Highlights

Pakistan is always considered among the top citrus producers of the world, and its production is consumed locally and mainly exported to fresh fruit markets
Conformation thorough direct tissue blot immunoassay (DTBIA) All 12 isolates were inoculated on six indicators plants collected from different citrus growing areas or districts of Punjab province of Pakistan and confirmed by DTBIA to know Citrus tristeza virus (CTV) infection
All 12 samples were found infected with CTV by DTBIA suggesting that CTV isolates could be inoculated successfully on all indicators plant (Table 1)

Summary

Introduction

Pakistan is always considered among the top citrus producers of the world, and its production is consumed locally and mainly exported to fresh fruit markets. Citrus tristeza virus (CTV), a member of family Closteroviridae genus clotserovirus, is causal agent of economically most important disease of citrus which has caused death of about 100 million citrus trees around the globe (Moreno et al 2008) and damage is increasing (Ferretti et al 2014). It causes different disease outbreaks in the form of stem-pitting, quick decline, and stunting of tress are important one (Bar-Joseph et al 1989). Symptoms produced by the different CTV strains range from invisible to visible severe symptoms which include pits in the stems, branches and trunks, stunted growth, quick decline, seedling yellows, vein clearing and death of whole tree

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Results

Conclusion