Molecular Detection of the Crown Gall Disease Caused by an Agrobacterium Species in Tunisian Fruit Trees' Nurseries

Abstract

The importance of the crown gall disease caused by bacteria of the genus Agrobacterium was assessed for 13 isolates and 5 plant tissues belonging to 4 different species. Samples were collected from different growing areas (soil, plants and tumor tissues). The aim of this study was to isolate the bacterium from symptomatic plants, soils and for the first time from asymptomatic plant tissues and to identify new isolates of Agrobacterium sp. by sequence analysis of relevant marker genes. Two primers' pairs were used to test the incidence of crown gall disease in Tunisian nurseries. The first targeted the virulence region within the Ti plasmid and the second the polygalacturonase gene used for the identification of Allorhizobium vitis. Simple PCR and sequencing unambiguously demonstrated the unexpected contamination of the asymptomatic plant tissues by part of the Ti plasmid (virulence region). The samples were clustered according to the BLAST analyses of a sequenced vir gene region and phylogenetic trees allowed the distinction of two main groups, the first group carries vitopine type Ti plasmids, the second group carries nopaline, octopine, type Ti plasmids agropine, mikimopine, cucumopine type Ri plasmid and others Ri plasmid types. The BLAST analyses of a polygalacturonase encoding gene sequence from 6 isolates and 3 strains showed 95-99% identity to the sequences of Allorhizobium vitis previously deposited in NCBI GenBank database.

Keywords

• Agrobacterium
• crown gall
• fruit trees
• sequencing
• PCR