Yasufumi Hikichi

In order to develop a method for discrimination of Corynespora cassiicola isolates pathogenic to sweet pepper among Japanese isolates, this study analysed pathogenic variations of 64 Japanese isolates of C. cassiicola on perilla, cucumber, tomato, aubergine and sweet pepper, and their multigene phylogeny. Japanese isolates were divided into seven pathogenicity groups (PG1–PG7). The virulence of isolates in PG1–PG5 was restricted to perilla, cucumber, tomato, aubergine and sweet pepper, respectively. Isolates in PG6 were virulent to sweet pepper, tomato and aubergine. Isolates in PG7 were avirulent to all tested plants. Multigene phylogenetic analysis of the isolates based on β‐tubulin, translation elongation factor 1‐α, calmodulin and actin genes showed three divergent clusters, MP‐A, MP‐B and MP‐C. These clusters included all isolates in PG1, PG2, PG8 and PG9 (MP‐A), PG3 and PG5 (MP‐B) and PG4 and PG6 (MP‐C). Isolates in PG7 were distributed amongst all clusters. Furthermore, random amplified polymorphic DNA (RAPD) analysis using universal primers, Q17 (5′‐GAAGCCCTTG‐3′) and Q13 (5′‐GGAGTGGACA‐3′), facilitated discrimination of isolates virulent on sweet pepper amongst isolates in MP‐B and MP‐C, respectively. Together, a combination of the multigene analysis and the RAPD technique allowed the discrimination of the isolates virulent to sweet pepper.

Pseudomonas cichorii causes necrotic lesions in eggplant and rot in lettuce. Through transposon insertion into P. cichorii strain SPC9018 we produced two mutants, 4-57 and 2-99, that lost virulence on eggplant but not lettuce. Analyses showed that a transposon was inserted into the hrpG gene in 4-57 and the hrcT gene in 2-99. Nucleotide sequences of the hrp genes of SPC9018 are homologous to those of Pseudomonas viridiflava BS group strains. The pathogenicity of 4-57 on eggplant was restored by transformation with an hrpF operon, originating from either SPC9018 or the BS group member P. viridiflava strain 9504 (Pv9504). These data suggested the involvement of hrp genes in the pathogenicity of SPC9018 on eggplant, and functional conservation of hrpF operons between SPC9018 and Pv9504. Both the hrpS mutant and the hrpL mutant were unable to cause necrotic lesions on eggplant leaves but retained their pathogenicity against lettuce. These results suggest that the pathogenicity of P. cichorii is hrp-dependent in eggplant, but not in lettuce.

Growth of Ralstonia solanacearum strain OE1-1 in roots after invasion is required for virulence. An Arg740Cys substitution in σ ⁷⁰ of OE1-1 resulted in loss of in planta growth and virulence. The negative dominance of mutant σ ⁷⁰ over the wild-type protein suggested that the amino acid substitution may affect the in planta growth of OE1-1, leading to a lack of virulence.

Tobamovirus resistance in Capsicum plants, which is mediated by L genes (L ¹ , L ² , L ³ or L ⁴ ), is known to be temperature sensitive. However, the L ^1a gene, a newly identified tobamovirus resistance gene that is mapped to the L locus, confers temperature-insensitive resistance against the tobamovirus P₀ pathotype. To identify the viral elicitor that activates the L ^1a -gene-mediated resistance, several chimeric viral genomes were constructed between tobacco mosaic virus-L (P₀ pathotype), paprika mild mottle virus-J (P₁ pathotype) and pepper mild mottle virus-J (P_1,2 pathotype). Infection patterns of these chimeric viruses in L ^1a -harboring plants revealed that the L ^1a -gene-mediated resistance was activated by the CP of a particular pathotype of tobamovirus, like other L-gene-mediated resistances, but the L ^1a -gene-mediated resistance differs from those conferred by other L genes in terms of temperature sensitivity.

Oxolinic acid (OA) resistance in field isolates ofBurkholderia glumae, a causal agent of bacterial grain rot, is dependent on an amino acid substitution at position 83 in GyrA (GyrA83). In the present study, among spontaneous in vitro mutants from the OA-sensitiveB. glumaestrain Pg-10, we selected OA-resistant mutants that emerged at a rate of 5.7 × 10⁻¹⁰. Nucleotide sequence analysis of the quinolone resistance-determining region in GyrA showed that Gly81Cys, Gly81Asp, Asp82Gly, Ser83Arg, Asp87Gly, and Asp87Asn are observed in these OA-resistant mutants. The introduction of each amino acid substitution into Pg-10 resulted in OA resistance, similar to what was observed for mutants with the responsible amino acid substitution. In vitro growth of recombinants with Asp82Gly was delayed significantly compared to that of Pg-10; however, that of the other recombinants did not differ significantly. The inoculation of each recombinant into rice spikelets did not result in disease. In inoculated rice spikelets, recombinants with Ser83Arg grew less than Pg-10 during flowering, and growth of the other recombinants was reduced significantly. On the other hand, the reduced growth of recombinants with Ser83Arg in spikelets was compensated for under OA treatment, resulting in disease. These results suggest that amino acid substitutions in GyrA ofB. glumaeare implicated in not only OA resistance but also fitness on rice plants. Therefore, GyrA83 substitution is thought to be responsible for OA resistance inB. glumaefield isolates.

In order to develop a detection method for the rice pathogens Burkholderia plantarii, Burkholderia glumae and Burkholderia gladioli, the phylogeny of six plant-pathogenic Burkholderia species was analysed using the combined nucleotide sequences of gyrB and rpoD. B. plantarii, B. glumae and B. gladioli formed tight monophyletic branches supported by high bootstrap probabilities. The high sequence similarity revealed a close phylogenetic relationship between B. glumae and B. plantarii. B. plantarii strains were divided into three subclusters comprising rice strains, whereas the single Vanda strain occupied a unique position in the phylogenetic tree. The gyrB and rpoD sequences of all B. glumae strains examined were highly conserved. In contrast, B. gladioli strains demonstrated a far greater sequence diversity, but this diversity did not correlate with pathovar, host plant or geographical origin of the strains. A multiplex-PCR protocol using specific primers from the gyrB sequences was designed that allowed the specific detection and identification of B. plantarii, B. glumae and B. gladioli in rice seeds infected with these pathogenic species.

The vigorous proliferation of Ralstonia solanacearum OE1-1 in host intercellular spaces after the invasion of host plants is necessary for the virulence of this bacterium. A folate auxotroph, RM, in which a mini-Tn 5 transposon was inserted into pabB encoding para -aminobenzoate synthase component I, lost its ability to vigorously proliferate in intercellular spaces along with its systemic infectivity and virulence after inoculation into roots and infiltration into leaves of tobacco plants. Complementation of RM with the pabB gene allowed the mutant to multiply in intercellular spaces and to cause disease. In tobacco plants that were pretreated with folate, RM was able to vigorously proliferate in the intercellular spaces and cause disease. Interestingly, when it was inoculated through cut stems, the mutant multiplied in the plants and was virulent. Moreover, the mutant multiplied well in stem fluids but not in intercellular fluids, suggesting that the folate concentration within intercellular spaces may be a limiting factor for bacterial proliferation. Therefore, folate biosynthesis contributes to the vigorous proliferation of bacteria in intercellular spaces and leads to systemic infectivity resulting in virulence.