Simultaneous plasmid profiling of phytopathogenic <i>Rhizobium</i> species (former <i>Agrobacterium </i>species) using multiplex colony-direct PCR.

Abstract

(2016). Simultaneous plasmid profiling of phytopathogenic Rhizobium species (former Agrobacterium species) using multiplex colony-direct PCR. Jpn. J. Among Rhizobium species, six species include plant pathogens as a member: R. radiobacter species complex (including R. nepotum and R. pusense), R. rhizogenes, R. vitis, R. rubi, R. larrymoorei, and R. skierniewicense. Based on their pathogenic states, the members belonging to the six species can be divided into three types, namely, crown gall bacteria carrying Ti plasmid, hairy root bacteria carrying Ri plasmid, and nonpathogenic bacteria carrying neither pathogenic plasmids. In this study, we developed a plasmid-profiling method using a multiplex colony-direct PCR to identify any pathogenic plasmid present in the respective strains and thus determine their pathogenic state. For that purpose, a universal primer set for both pathogenic plasmids and a specific primer set for the Ri plasmid were designed based on the sequences of virC1 and rolC, respectively. The universal primer set for 16S rRNA gene (16S rDNA), chosen as an internal control, was also added to the PCR mix to readily judge the success of the reaction and exclude false-negative reactions. Reliability of the method as a plasmid-profiling tool was then validated using the Rhizobium species containing plant pathogens and their relatives of 383 strains in total. As a result, two DNA fragments, one from virC1-specific amplification (278 bp) and the other (780-784 bp) from 16S rDNA as the internal control, were stably obtained from all crown gall bacteria used. For hairy root bacteria, three fragments derived from virC1 (278 bp), rolC (438 bp) and 16S rDNA were always observed. On the other hand, only a fragment derived from 16S rDNA was amplified from the other nonpathogenic bacteria. Therefore, this method is useful for rapid plasmid profiling of the Rhizobium species containing plant pathogens, which will improve the efficiency in areas such as surveillance and diagnosis of crown gall and hairy root diseases and epidemiological and ecological studies of the pathogens.

A simple method for creating transgenic pea hairy roots using a Japanese pea cultivar and a Japanese <i>Rhizobium radiobacter</i> strain

“…To date, the only reported case of crown gall (callus) being produced by inoculation of Agrobacterium with Ri-plasmid has been reported in opium poppy (Yoshimatsu and Shimomura 1992). Because R. radiobacter MAFF210266 contains the Ri-plasmid (Sawada et al 2016), the system used in this study may enable researchers to clarify the molecular mechanisms mediating hairy root and crown gall formation. Furthermore, it may be relevant for producing transgenic pea hairy roots and elucidating the (+)-pisatin biosynthesis mechanism.…”

The Presence of the Hairy-Root-Disease-Inducing (Ri) Plasmid in Wheat Endophytic Rhizobia Explains a Pathogen Reservoir Function of Healthy Resistant Plants

“…However, the strains are relatively variable in genomic DNA sequences and therefore are subclassified into more than nine genomic groups (or genomovars) (24)(25)(26)(27)(28). Several of the genomovar groups harbor most pathogenic strains (29,30), while several other genomovars have no identified phytopathogens but contain isolates from soil habitats. Genomovar G2 (Rhizobium pusense) harbors most isolates from human patients (31).…”