2.2.50
COMPARISON OF XANTHOMONAS ORYZAE PV. ORYZAE AND XANTHOMONAS ORYZAE PV. ORYZICOLA BASED ON PCR-RFLP AND SEQUENCES OF NUMERICAL GENES

H OCHIAI and H KAKU

Department of Genetic Resources I, National Institute of Agrobiological Resources, 2-1-2, Kannondai, Tsukuba 305, Japan

Background and objectives
Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) and bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola (Xocola) are two major bacterial diseases of rice. Phenotypic features of the two pathovars are very similar to each other [1,2], except for their mode of infection. In this study, therefore, they were compared to assess the genetic differences by molecular genetic analysis, such as PCR-RFLP, sequencing and southern hybridization.

Materials and methods
The pathotype strains or the field isolates of Xoo and Xocola were used in this study together with Xanthomonas campestris pv. campestris (Xcc) served as reference strains. The following analyses were used in this study. (1) PCR-RFLPs of rRNA genes, which were nearly full-length of 16S rRNA and about half-length of 23S rRNA, ITS regions and a gene-related EPS production, with 10 restriction enzymes. (2) Partial sequences of 5' end of 23S rRNA gene, ITS regions, and the hrpX gene regulated hrp gene cluster. (3) Southern hybridization with the avr gene as a probe.

Results and conclusions
No differences were observed in 16S and 23S rRNA genes among Xoo, Xocola and Xcc by PCR-RFLPs. In ITS regions, however, the difference was detected with restriction enzyme AluI between Xoo and Xocola. In addition, the results of RFLP analysis using the EPS-related gene as probe and four restriction enzymes showed that Xoo strains were clearly distinguishable from Xocola strains. Their RFLP patterns were different from those of Xcc with any of the four restriction enzymes. The results of direct sequencing of the PCR-amplified ITS regions showed that these were composed of 492 ;bp, and the differences in the sequence were in only four nucleotides between the type strains of Xoo and Xocola. These were all pointed on nearly 3' end of ITS regions, and one of them was detected on sequences recognized by restriction enzyme AluI. Partial sequencing of 544 ;bp of 5' end of 23S rRNA gene indicated that there was only one difference between them. Compared with partial sequences of 900 ;bp and the putative amino acid sequences of the hrpX gene, each homology value was as high as 98% and 97% between Xoo and Xocola, respectively; among strains of X. ;oryzae pathovars and Xcc, it contrasted with 82% and 88%, respectively. Based on southern hybridization with the avr gene probe, the RFLP patterns were also similar to each other between Xoo and Xocola strains. However, the cluster analysis showed that they were clearly distinguishable from each other.

The results obtained here indicated that Xoo and Xocola were closely related to each other in genetic features as well as phenotypic ones, except for slight differences in the analyses of certain genes.

References
1. Vera Cruz CM, Gossele F, Kersters K, Segers P, van den Mooter M, Swings J, De Ley J, 1984. Journal of General Microbiology 130, 2983-2999.
2. van den Mooter M, Swings J, 1990. International Journal of Systematic Bacteriology 40, 348-369.