Determination of tumorigenic Agrobacterium density in soil by real-time PCR assay and its effect on crown gall disease severity

Abstract

Tumorigenic Agrobacterium-induced crown gall disease significantly limits the production of peach, cherry, and other fruit trees in China. Thus, the rapid detection of pathogens is necessary for disease prediction and management. In this study, we designed the primer pair ipt 3F/ipt 3R on the basis of the ipt gene. This primer pair effectively amplified a 247 bp DNA fragment from the transferred DNA (T-DNA) sequences of tumorigenic Agrobacterium. By using this primer pair, we developed a sensitive real-time PCR method for quantifying tumorigenic Agrobacterium in soil at a level as low as 102 cfu/g. We used this assay to detect tumorigenic Agrobacterium in 19 soil samples. Levels were ≤102 cfu/g in maize or uncultivated soil and 103 to106 cfu/g in soil planting with peach or cherry. The relationship between the disease severity and the tumorigenic Agrobacterium density in soil was verified by artificial inoculation test. The disease incidence was 30 to 50 %, and the disease index was 20 to 30 in soil inoculated with A. tumefaciens at 103 cfu/g. The disease incidence increased to >80 % when the inoculation density of A. tumefaciens was increased to >105 cfu/g soil. These results indicated that tumorigenic Agrobacterium density in soil severely affected the incidence of crown gall formation on peach seedlings, and 103 bacterial cells in 1 g of soil were inappropriate for peach planting. Determination of pathogen density for gall formation can improve the prediction of crown gall disease outbreaks as well as aid in the management of this disease.