1Research Institute for Bioresources, Okayama University, Kurashiki 710-0046, Japan; 2Hokkaido Central Agricultural Experiment Station, Naganuma, Hokkaido 069-1300, Japan

Background and objectives
Beet necrotic yellow vein virus (BNYVV), which causes rhizomania of sugar beet and is transmitted by the soil-inhabiting fungus Polymyxa betae, consists generally of four RNA components; RNA 1 and RNA 2 are required for viral RNA replication, assembly and virus movement, whereas RNA 3 and RNA 4 are needed for disease development and spread in nature. Some Japanese [1] and European isolates [2] contain the fifth RNA species RNA 5, which is present together with RNA 3 and RNA 4. RNA 5 is 1342-1347 nucleotides in length and contains a single ORF encoding a 26 kDa protein (P26) [1]. When RNA 5 is present, the root symptoms are more intense. BNYVV has now been distributed in many sugar beet-growing countries in the world, since it was first recorded in Italy in the early 1950s. We have described geographical distribution and sequence variation of RNA 5 molecules which were detected in Japanese, Chinese and French isolates.

Materials and methods
All BNYVV isolates were obtained by inoculating sugar beet seedlings from rhizomania-infested soil samples which were collected from different areas of Japan and China. Two French isolates were generously supplied by M. Fattori. BNYVV infection in sugar beet roots was determined by ELISA. Detection of RNA 5 was made by Northern blotting or RT-PCR. For CDNA synthesis, total nucleic acids were extracted by direct phenol methods from inoculated leaves of Tetragonia expansa. RNA 5- specific CDNA products within positions 308-1192 in RNA 5 from D5 isolate were determined by RT-PCR as described previously [1]. Two specific primers RT5F (5'-GTTTTTC CGCTCGCAGCACAAG-3', representing nucleotides 308 to 326) and RT5R (5'-CGAGCCCGTAAA CACCGCATA-3', complementary to nucleotides 1172-1192) were used. Nucleotide sequences were determined by dideoxynucleotide chain termination.

Results and conclusions
Out of 194 Japanese BNYVV isolates, 88 (46%) contained RNA 5. The result shows that the RNA 5 is widely present in Hokkaido, although there is a slight difference in their geographical distribution. Two isolates derived from Nei Menggu and Ningia in China were also found to contain RNA 5. As described by Koenig et al. [2], RNA 5 was detected from two French isolates. Nucleotide sequences of RNA 5 molecules from 13 Japanese, two Chinese and two French isolates were compared with each other and those from six isolates reported previously [1, 2]. The differences between individual sequences ranged from 0.1 to 7.9% at the nucleotide level, and from 0 to 17 amino acid changes within the P26 ORF. RNA 5 sequences were divided into three groups: group I contains 17 isolates from Japan and China, group II contains two Japanese isolates (from Shari), and group III contains four French isolates. The sequence differences of P26 gene between the groups were 10 to 17 amino acid changes and within each group were 0 to 7 amino acid changes. In addition, a few or several sequences were deleted or inserted at an A-rich region downstream from the nucleotide position 356 in the 5' end-noncoding region. Based on the differences at this region, the RNA 5 sequences belonging to group I fell into further three clusters. These comparisons indicate that sequence differences between groups and clusters are associated with geographical origin, and are probably due to independent evolution of recently separated populations.

1. Kiguchi T, Saito M, Tamada T, 1996. Journal of General Virology 77, 575-580
2. Koenig R, Haeberle AM, Commandeur U, 1997. Archives of Virology 142, 1499-1504.