A Highly Sensitive Method to Detect Avocado Sunblotch Viroid for the Maintenance of Infection-Free Avocado Germplasm Collections.

David N. Kuhn,Alan H. Chambers,Andrew Geering,Barbie Freeman

doi:10.3390/v11060512

Abstract

The United States Department of Agriculture (USDA) Agricultural Research Service (ARS) Subtropical Horticulture Research Station (SHRS) in Miami, FL holds a large germplasm collection of avocado (Persea americana). The recent threat of infection by laurel wilt has encouraged the creation of a backup collection at a disease-free site. Creating the backup collection is complicated by infection of some trees in the germplasm collection with avocado sunblotch viroid (ASBVd). Infected trees are frequently asymptomatic, necessitating the use of a molecular diagnostic assay. Although a reverse-transcription based assay already exists and has been used to assay all germplasm at the station, some trees showed inconsistent results. We have developed a more sensitive and specific assay involving pre-amplification of the entire viroid cDNA followed by detection using real-time PCR and a TaqMan assay. A second screening of all germplasm identified additional ASBVd -infected trees and allowed us to confidently remove these trees from the station. This method enables avocado germplasm curators to proceed with the creation of a viroid-free backup collection.

Highlights

Avocado sunblotch viroid (ASBVd) is a single-stranded circular RNA molecule of 247 nucleotides that is confined in nature to avocado (Persea americana) [1]
ASBVd is transmissible through seed and mechanical damage, but has no insect vectors
Precautions need to be taken during propagation to prevent infection as rates of seed transmission are high at 86%–100%, and ASBVd can be introduced through the use of infected budwood [3]

Summary

Introduction

Avocado sunblotch viroid (ASBVd) is a single-stranded circular RNA molecule of 247 nucleotides (reference sequence variant GenBank J02020.1) that is confined in nature to avocado (Persea americana) [1].Symptomatic and asymptomatic strains are recognized, even the apparently asymptomatic strains are associated with yield loss [2]. ASBVd is transmissible through seed and mechanical damage, but has no insect vectors. Precautions need to be taken during propagation to prevent infection as rates of seed transmission are high at 86%–100%, and ASBVd can be introduced through the use of infected budwood [3]. Rates of spread in the field are low and based on patterns of infection, it is suspected that ASBVd spreads in the field by root grafting. Pruning could potentially transmit the viroid but the efficiency of this mode of transmission is thought to be low [3]. But this only results in infection of the seed and not the mother plant [4]. Trees must be rogued to prevent infection of neighboring trees

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