Indexing Agrobacterium vitis in Asymptomatic Grapevine Propagation Material by Two Nested PCR Assays

Abstract

Agrobacterium vitis, the causal agent of grapevine crown gall, may systemically infest nursery propagating material, which remains asymptomatic until conditions conducive for tumor induction are met. Two sensitive and specific diagnostic tools capable of detecting A. vitis in the total DNA extracted from asymptomatic grapevine tissues were set up. Starting from two preexisting standard PCR assays targeting the chromosomal gene pehA and the Ti plasmid borne gene virA, two nested PCR assays were developed. These assays showed increased sensitivity when combined with a simple but effective DNA extraction. Regardless of the type of tissue examined (rootstock canes or trunks, roots, green shoots, leaves), the physiological state of the tissues (dormant propagating material or vegetating plants), or the composition of the bacterial community naturally associated to them, results indicated that the nested PCR method is two to five times more effective at detecting A. vitis DNA in asymptomatic grapevine tissues than the standard PCR analysis. The overall results indicate that one out of two samples in which the presence of the pathogen was revealed appears to be infected with tumorigenic strains of A. vitis, highlighting the risk of disease dissemination.