Mitsuo Horita

Phylogenetic relationships of Ralstonia solanacearum species complex strains from Asia and other continents based on 16S rDNA, endoglucanase, and hrpB gene sequences

The 16S rDNA, endoglucanase, and hrpB genes were partially sequenced for Asian strains of Ralstonia solanacearum spp. complex, including 31 strains of R. solanacearum and two strains each of the blood disease bacterium (BDB) and Pseudomonas syzygii. Additional sequences homologous to these DNA regions, deposited at DDBJ/EMBL/GenBank databases were included in the analysis. Various levels of polymorphisms were observed in each of these DNA regions. The highest polymorphism (approximately 25%) was found in the endoglucanase gene sequence. The hrpB sequence had about 22% poly-morphism. The phylogenetic analysis consistently divided the strains into four clusters, as distinctly shown on the phylogenetic trees of 16S rDNA, hrpB gene, and endo-glucanase gene sequences. Cluster 1 contained all strains from Asia, which belong to biovars 3, 4, 5, and N2. Cluster 2 comprised the Asian strains of R. solanacearum (as biovars N2 and 1) isolated from potato and clove, as well as BDB and P. syzygii. Cluster 3 contained race 3 biovar 2 strains from potato, race 2 biovar 1 strains from banana, and race 1 biovar 1 strains isolated from America, Asia, and other parts of the world. Cluster 4 was exclusively composed of African strains. The results of the study showed the distribution and diversity of the Asian strains, which are present in three of the four clusters. The similarity of Asian strains to those in the other regions was also observed.

PCR-based specific detection of Ralstonia solanacearum race 4 strains

Two primer sets were designed based on the sequence of polymorphic bands that were derived from repetitive sequence-based polymerase chain reaction (rep-PCR) fingerprinting and specifically detected in Ralstonia solanacearum race 4 strains (ginger, mioga, and curcuma isolates). One primer set (AKIF-AKIR) amplified a single band (165 bp) from genomic DNA obtained from all mioga and curcuma and some ginger isolates; another set (21F-21R) amplified one band (125 bp) from the other ginger isolates. These primer sets did not amplify the bands from genomic DNA of other R. solanacearum strains or of other related bacteria. PCR detection limit for the pathogen was 2 × 102 cfu.

Spongospora subterranea soil contamination and its relationship to severity of powdery scab on potatoes

We investigated soil contamination by Spongospora subterranea f. sp. subterranea (Sss) and disease severity of powdery scab in 29 potato fields in Hokkaido, Japan, using a hydroponic culture method with tomato seedlings as bait plants. The quantity of Sss infection on the roots of bait plants was evaluated using the polymerase chain reaction (PCR) and expressed in terms of the infection potential in the soil. The infection potential was positively correlated with the disease severity of harvested tubers, whereas the spore ball density determined using PCR had an indistinct relationship with disease severity. The infection potential can be useful in evaluating soil contamination and in applying countermeasures against powdery scab.

Analysis of genetic and biological characters of japanese potato strains of ralstonia solanacearum

We assessed the geographic distribution, biovar, phylotype, DNA fingerprints (rep-PCR), and/or endoglucanase sequence of potato bacterial wilt pathogen, Ralstonia solanacearum (Rs), in Japan. Rs has been isolated from potato fields in southwestern, warm, temperate regions. Of the 188 isolates, 74 belonged to biovar N2 (39%), 44 to biovar 3 (24%), and 70 to biovar 4 (37%). Biovars N2 and 4 strains were widely distributed, from northern (Hokkaido) to southern (Okinawa) Japan. Based on the results of multiplex-PCR analysis, every potato strains belonged to either phylotype I or IV. Phylotype I comprised both biovars 3 and 4 strains. On the other hand, phylotype IV included biovar N2 strains. None of the strains belonged to phylotype II or III or biovar 1 or 2. Phylogenetic analysis based on DNA fingerprints and endoglucanase gene sequences clarified the genetic diversity of the Japanese potato strains and the close genetic relationship between the Japanese strains and the Asian strains in phylotypes I and IV.

Pathogenic characters of Japanese potato strains of Ralstonia solanacearum

Ralstonia solanacearum (Rs) strains in phylotypes I and IV isolated from potato in Japan were investigated for pathogenicity on potato, tomato, eggplant, Solanumintegrifolium, tobacco, groundnut, and pumpkin. The strains were divided into 17 types based on differences in their pathogenicity on the tested plants. Particularly, the pathogenicity of most phylotype I strains on eggplant was distinctly different from that of the phylotype IV strains. When nine potato varieties (included two breeding lines) were inoculated with several Rs strains, phylotype IV strains were highly virulent on the breeding lines that are regarded as resistant to phylotype I strains.

Comparative Analysis of Japanese and Foreign Strains of Ralstonia solanacearum Based on 16S Ribosomal RNA Gene Sequences

We determined nearly the complete sequences of the 16S ribosomal RNA gene (rDNA) for Japanese strains of R. solanacearum. The comparison of 1471 nucleotide positions separated the Japanese strains into two groups, group 1 with biovars 1, 2, 3 and 4 strains which belonged to race 1, and group 2 with biovar 2 strains corresponding to race 3. Group 1 strains all had identical sequences, and strains representing the four biovars within the group did not differ from each other. Group 2 strains had characteristic nucleotides which differed at seven positions from group 1 strains. Comparative analysis of Japanese and foreign strains based on 16S rDNA sequences showed that Japanese group 1 was closely related to Asian and Australian biovars 3, 4 and 5, and belonged to the known division 1. Japanese group 2 was homogeneous to Indonesian biovars 2 and N2 in subdivision 2b. Since the differences in the nucleotides corresponded to restriction sites for the AluI, RFLP analysis of PCR-amplified 16S rDNA efficiently differentiated not only Japanese group 1 from group 2, but also differentiated three types of foreign strains which differed in biovar and geographic origin.

DNA analysis of Ralstonia solanacearum and related bacteria based on 282-bp PCR-amplified fragment.

Strains of Ralstonia solanacearum, Pseudomonas syzygii, and the blood disease bacterium (BDB) from different countries were tested for polymerase chain reaction amplification of the 282-bp fragment using the primer pair 759 and 760. These 282-bp fragments from 49 strains of R. solanacearum, six strains of P. syzygii, and two strains of BDB were sequenced. A phylogenetic tree was generated based on the sequence alignment. The R. solanacearum strains were divided into three groups. Group I was composed of strains belonging to biovars 3, 4, 5, and biovar N2 from Japan. Most of the strains from this group were of Asian origin except for two strains from Australia and Guyana (GMI 1000), the type strain. Group II was composed of strains belonging to biovars 1 and 2 and biovar N2 from Brazil. Group III was composed of strains belonging to biovar N2 from Japan and the Philippines. All strains of P. syzygii and BDB clustered in group III. Based on nucleotide differences of the 282-bp fragments, restriction enzyme NlaIII was capable of differentiating R. solanacearum strains into the three groups. Restriction analysis of 165 R. solanacearum isolates from the Philippines using NlaIII showed that all biovar 3 and 4 (group 1) strains had restriction fragments of 116 and 166 bp, strains belonging to biovars 1 and 2 (group 2) showed no restriction, and one strain belonging to biovar 2 (group 3) showed restriction fragments of 54 and 228 bp in size. Thus, NlaIII could be used for rapid differentiation of R. solanacearum strains. Additionally, other restriction enzymes, such as McrI, BsiEI, and MnlI could be used to differentiate R. solanacearum strains from P. syzygii strains.

Current classification of Ralstonia solanacearum and genetic diversity of the strains in Japan

Ralstonia solanacearum is the causal organism of bacterial wilt of more than 200 species representing 50 families of plants in tropical, subtropical, and warm temperate regions in the world. Traditionally classified into five races based on differences in host range, R. solanacearum has also been grouped into six biovars on the basis of biochemical properties. With recent developments in molecular biology, various DNA-based analyses have been introduced and used to confirm that this binary system does not completely represent the diversity within R. solanacearum strains. Therefore, a new hierarchical classification scheme has been suggested, which defines R. solanacearum as a species complex and reorganized the concept of the species as a monophyletic cluster according to a phylogenetic analysis based on genomic sequence data. Here we discuss the current bacterial wilt situation and genetic relationships based on the recent classification system of Japanese R. solanacearum strains as well as worldwide strains. We also review the genetic, biochemical, and pathological characteristics of R. solanacearum strains, in particular, those affecting potato and Zingiberaceae plants as distinctly important pathogens in relation to continuously problematic and recent emergent diseases in Japan.

Relation of Japanese Xanthomonas campestris pv. vesicatoria with worldwide strains revealed with three specific monoclonal antibodies

Strains of Xanthomonas campestris pv. vesicatoria Dye 1978 (Xcv), the causal agent of bacterial spot, have been classified into two groups based on their ability to hydrolyze starch. Three monoclonal antibodies (MAbs), 7AH10, 5HB3, and 4AD2, were produced immunized against the living bacteria and were specific to and could distinguish Xcv strains able or unable to hydrolyze starch (Amy+ or Amy−). The MAb 7AH10, obtained against strain UPB141(Amy−) reacted in an enzyme-linked immunosorbent assay with all the Amy− strains (n = 19) and 1 of 11 Amy+ strains. Against Xcv 2625, an Amy− “unusual phenotype” strain, MAb 5HB3, recognized 97% of our worldwide collection of Xcvs (n = 30). Also against that strain, the MAb 4AD2 reacted with none of the homologous Amy− phenotypes and with 90% (n = 11) of the heterologous Amy+ phenotypes. For all the MAbs, cross reactions with other pathovars or species were less than 4% (n = 67). By assaying a Japanese collection of strains against the three MAbs, the Amy+ strains were distinguished from the Amy− strains, and their relation with other world strains could be demonstrated. All the MAbs reacted with the lipopolysaccharide fraction of the bacterial cell wall during immunoblotting.

Analysis of sources of oxolinic acid-resistant field strains of Burkholderia glumae based on rep-PCR analysis and nucleotide sequences of gyrB and rpoD

Repetitive sequence-based polymerase chain reaction (rep-PCR) analysis using BOX and ERIC as primers showed a highly divergent phylogeny among field strains of Burkholderia glumae. To elucidate the sources of oxolinic acid (OA) resistance in field strains of B. glumae isolated from rice seedlings cultivated in Mie, Toyama, and Iwate prefectures, Japan, the amino acid at position 83 of GyrA (GyrA83), which is involved in OA resistance, and the DNA patterns from the rep-PCR and the partial nucleotide sequences of gyrB and rpoD from various strains were analyzed. The ten Mie strains, in which GyrA83 was isoleucine (Ile), were divided into two groups based on the band patterns in rep-PCR analysis, although the nucleotide sequences of gyrB and rpoD were identical among the strains. Based on the band patterns in the rep-PCR analysis and the gyrB and rpoD sequences, two highly OA-resistant Toyama strains, Pg-13 and Pg-14, for which GyrA83 was serine (Ser) and Ile, respectively, were in the same lineage. This suggests that the bacteria might acquire OA resistance faster than phylogenic diversity as determined with the repetitive sequences BOX and ERIC and with gyrB and rpoD. Furthermore, three Iwate strains (H95, H101, and H104), isolated from seedlings of different cultivars grown in different years and having Ile at GyrA83, are probably in the same lineage, suggesting that OA-resistant bacteria might be transferred among different cultivars.