The biological control agent for bacterial canker of kiwifruit, in Aureo® Gold, is a strain of Aureobasidium pullulans identifiable by novel SCAR marker primers

Abstract

The yeast-like fungus strain CG163, also known as YBCA5, is the active ingredient in Aureo® Gold, a biological control agent sold for control of bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae. Analysis of the whole genome sequence of CG163 as well as a multi locus sequence analysis based on the ITS, ELO, EF-1α and β-tubulin genes indicate that CG163 is a strain of Aureobasidium pullulans. None of the published PCR primers developed for the identification of other strains of A. pullulans led to an amplicon when using CG163 total DNA. Following PCR using 14 random amplified polymorphic DNA primers by themselves or in combination, we identified a Sequence Characterised Amplified Region (SCAR) marker specific to CG163. One set of primers designed on this SCAR marker gave a unique amplicon only with CG163 and not with any of the 164 strains of A. pullulans tested. In addition, an in silico analysis revealed that none of the 75 strains of A. pullulans for which the whole genome sequence is available had the same sequence as either one of those primers. To prevent the detection of false negatives, we developed a duplex polymerase chain reaction (PCR) based on those primers and primers which amplify a segment of the ITS. The limit of detection of this duplex PCR assay was 1 pg. This assay can be used for quality control; however, for detection and quantification of CG163 we developed a quantitative polymerase chain reaction (qPCR) assay. Using this assay, we positively identified CG163 and followed its establishment on kiwifruit leaves for seven days post treatment with Aureo Gold. Therefore, the primers designed in this project can be used to study the establishment and colonisation of plant tissues by CG163 and allow the development of a robust set of recommendations on how and when to best use Aureo Gold.