Multiplex PCR for the identification of Agrobacterium biovar 3 strains

Abstract

A biovar 3-specific primer set Ab3-F3/Ab3-R4 was designed based on the comparison of sequences of the 16S rDNA region of agrobacteria and related rhizobia for rapid identification of Agrobacterium biovar 3 strains. A 570-bp 16S rDNA fragment was amplified from cell lysates of Agrobacterium biovar 3 strains by polymerase chain reaction (PCR) using Ab3-F3/Ab3-R4 primers. Discrimination of Agrobacterium tumefaciens biovar 3 from Agrobacterium radiobacter biovar 3 and of Agrobacterium biovar 3 strains from other Agrobacterium strains was done simultaneously using multiplex PCR with a mixture of two primer sets (Ab3-F3/Ab3-R4 and VCF3/VCR3) previously designed for the virC region of Ti-plasmid and Ri-plasmid.

Inhibition of crown gall formation by Agrobacterium radiobacter biovar 3 strains isolated from grapevine

“…DNA fragments 414 bp from the virC operon are expected to be amplified from the cell lysate of tumorigenic or rhizogenic Agrobacterium strains by PCR with VCF3 and VCR3 primers (Sawada 2005;Sawada and Tsuchiya 2003) and 570-bp fragments from 16S rDNA are expected to be amplified from Agrobacterium biovar 3 strains by PCR with Ab3-F3 and Ab3-R4 primers (Kawaguchi et al 2005).…”

“…The multiplex PCR was performed using a mixture of two primer sets -Ab3-F3 (5¢-ATG ACG GTA GTC GGA GAA GAA GCC-3¢)/Ab3-R4 (5¢-CTG TCT CTG TGT CCC CGA AAG G-3¢) and VCF3 (5¢-GGC GGG CGY GCY GAA AGR AAR ACY T-3¢)/VCR3 (5¢-AAG AAC GYG GNA TGT TGC ATC TYA C-3¢) -to identify A. tumefaciens biovar 3 or A. radiobacter biovar 3 (Kawaguchi et al 2005). DNA fragments 414 bp from the virC operon are expected to be amplified from the cell lysate of tumorigenic or rhizogenic Agrobacterium strains by PCR with VCF3 and VCR3 primers (Sawada 2005;Sawada and Tsuchiya 2003) and 570-bp fragments from 16S rDNA are expected to be amplified from Agrobacterium biovar 3 strains by PCR with Ab3-F3 and Ab3-R4 primers (Kawaguchi et al 2005).…”

Grapevine crown gall caused by Rhizobium radiobacter (Ti) in Japan

“…DNA fragments (414 bp) from a partial sequence of the virC1 and virC2 genes encoded in the virC operon are expected to be amplified from the cell lysate of tumorigenic or rhizogenic Rhizobium strains by PCR with VCF3 and VCR3 primers (Kawaguchi et al 2005a;Sawada and Tsuchiya 2003), and 570 bp fragments from 16S rDNA are expected to be amplified from R. vitis strains by PCR with Ab3-F3 and Ab3-R4 primers (Kawaguchi et al 2005a). …”

“…The multiplex polymerase chain reaction (PCR) was performed using a mixture of two primer sets Ab3-F3 (5 0 -ATGACGGTAGTCGGAGAAGAAGCC-3 0 )/Ab3-R4 (5 0 -CTG TCTCTGTGTCCCCGAAAGG-3 0 ) and VCF3 (5 0 -GGCGGGCGYGCYGAAAGRAARACYT-3 0 )/VCR3 (5 0 -AAGAACGYGGNATGTTGCATCTYAC-3 0 ) to identify tumorigenic and nonpathogenic strains of R. vitis according to the procedure of Kawaguchi et al (2005a). DNA fragments (414 bp) from a partial sequence of the virC1 and virC2 genes encoded in the virC operon are expected to be amplified from the cell lysate of tumorigenic or rhizogenic Rhizobium strains by PCR with VCF3 and VCR3 primers (Kawaguchi et al 2005a;Sawada and Tsuchiya 2003), and 570 bp fragments from 16S rDNA are expected to be amplified from R. vitis strains by PCR with Ab3-F3 and Ab3-R4 primers (Kawaguchi et al 2005a).…”

Comparative study of diagnostic methods used for monitoring of common grape vine (Vitis vinifera L.) crown gall (Agrobacterium vitis Ophel & Kerr) in Slovenia

“…Also, the interpretation can be doubtful when the PCR shows the absence of pTi, but the test plants develop tumors. Although PCR techniques for simultaneous identification of pathogenic and nonpathogenic A. vitis are available (Kawaguchi et al, 2005), the traditional pathogenicity test is still a standard technique in strain pathogenicity determination. …”