Fumihiko Suzuki

Phylogenetic analysis of Pseudomonas syringae pathovars suggests the horizontal gene transfer of argK and the evolutionary stability of hrp gene cluster.

Abstract.Pseudomonas syringae are differentiated into approximately 50 pathovars with different plant pathogenicities and host specificities. To understand its pathogenicity differentiation and the evolutionary mechanisms of pathogenicity-related genes, phylogenetic analyses were conducted using 56 strains belonging to 19 pathovars. gyrB and rpoD were adopted as the index genes to determine the course of bacterial genome evolution, and hrpL and hrpS were selected as the representatives of the pathogenicity-related genes located on the genome (chromosome). Based on these data, NJ, MP, and ML phylogenetic trees were constructed, and thus 3 trees for each gene and 12 gene trees in total were obtained, all of which showed three distinct monophyletic groups: Groups 1, 2 and 3. The observation that the same set of OTUs constitute each group in all four genes suggests that these genes had not experienced any intergroup horizontal gene transfer within P. syringae but have been stable on and evolved along with the P. syringae genome. These four index genes were then compared with another pathogenicity-related gene, argK (the phaseolotoxin-resistant ornithine carbamoyltransferase gene, which exists within the argK–tox gene cluster). All 13 strains of pv. phaseolicola and pv. actinidiae used had been confirmed to produce phaseolotoxin and to have argK, whose sequences were completely identical, without a single synonymous substitution among the strains used (Sawada et al. 1997a). On the other hand, argK were not present on the genomes of the other 43 strains used other than pv. actinidiae and pv. phaseolicola. Thus, the productivity of phaseolotoxin and the possession of the argK gene were shown at only two points on the phylogenetic tree: Group 1 (pv. actinidiae) and Group 3 (pv. phaseolicola). A t test between these two pathovars for the synonymous distances of argK and the tandemly combined sequence of the four index genes showed a high significance, suggesting that the argK gene (or argK–tox gene cluster) experienced horizontal gene transfer and expanded its distribution over two pathovars after the pathovars had separated, thus showing a base substitution pattern extremely different from that of the noncluster region of the genome.

A phylogenomic study of the OCTase genes in Pseudomonas syringae pathovars: the horizontal transfer of the argK-tox cluster and the evolutionary history of OCTase genes on their genomes.

Abstract. Phytopathogenic Pseudomonas syringae is subdivided into about 50 pathovars due to their conspicuous differentiation with regard to pathogenicity. Based on the results of a phylogenetic analysis of four genes (gyrB, rpoD, hrpL, and hrpS), Sawada et al. (1999) showed that the ancestor of P. syringae had diverged into at least three monophyletic groups during its evolution. Physical maps of the genomes of representative strains of these three groups were constructed, which revealed that each strain had five rrn operons which existed on one circular genome. The fact that the structure and size of genomes vary greatly depending on the pathovar shows that P. syringae genomes are quite rich in plasticity and that they have undergone large-scale genomic rearrangements. Analyses of the codon usage and the GC content at the codon third position, in conjunction with phylogenomic analyses, showed that the gene cluster involved in phaseolotoxin synthesis (argK–tox cluster) expanded its distribution by conducting horizontal transfer onto the genomes of two P. syringae pathovars (pv. actinidiae and pv. phaseolicola) from bacterial species distantly related to P. syringae and that its acquisition was quite recent (i.e., after the ancestor of P. syringae diverged into the respective pathovars). Furthermore, the results of a detailed analysis of argK [an anabolic ornithine carbamoyltransferase (anabolic OCTase) gene], which is present within the argK–tox cluster, revealed the plausible process of generation of an unusual composition of the OCTase genes on the genomes of these two phaseolotoxin-producing pathovars: a catabolic OCTase gene (equivalent to the orthologue of arcB of P. aeruginosa) and an anabolic OCTase gene (argF), which must have been formed by gene duplication, have first been present on the genome of the ancestor of P. syringae; the catabolic OCTase gene has been deleted; the ancestor has diverged into the respective pathovars; the foreign-originated argK–tox cluster has horizontally transferred onto the genomes of pv. actinidiae and pv. phaseolicola; and hence two copies of only the anabolic OCTase genes (argK and argF) came to exist on the genomes of these two pathovars. Thus, the horizontal gene transfer and the genomic rearrangement were proven to have played an important role in the pathogenic differentiation and diversification of P. syringae.

Molecular characterization of the tox operon involved in toxoflavin biosynthesis of Burkholderia glumae

Two toxoflavin biosynthesis-related proteins (TRP-1, TRP-2) from wild strains of the phytopathogen Burkholderia glumae were previously identified, and toxA was determined to encode TRP-1, which has characteristics of a methyltransferase. An 8.2-kb region in the chromosomal DNA of B. glumae that contains the tox operon (toxABCDE) and an upstream regulatory gene (toxR) involved in phytotoxin toxoflavin biosynthesis was cloned and sequenced in this study. The sequence downstream of toxA contains four open reading frames – toxB, toxC, toxD and toxE – which encode polypeptides with calculated mole-cular masses of 23.3, 61.6, 34.9, and 38.3u2009kDa, respectively, all having the same transcriptional orientation as toxA. Mutants disrupted in the tox operon lost their ability to produce toxoflavin and did not induce typical chlorosis on infected rice panicles. Based on results from reverse transcription-polymerase chain reaction experiments, the message encoded by the tox operon may be polycistronic for all five genes. Also, the toxR gene was located upstream of this operon. The toxR gene product, with a calculated molecular mass of 37.5u2009kDa, might be a member of the LysR family of regulatory molecules and an activator that allows transcription of the tox operon. Furthermore, the deduced proteins of ToxB and ToxE had significant similarity to the GTP cyclohydrolase II and the deaminase, respectively, involved in riboflavin synthesis in several organisms. These results suggest that toxoflavin is synthesized in part through a biosynthetic pathway common to the synthesis of riboflavin, starting with GTP as the precursor.

Effect of the Timing of Fungicide Application on Fusarium Head Blight and Mycotoxin Accumulation in Closed-Flowering Barley

Fungicide application is one measure available to reduce the risk of Fusarium head blight (FHB) and mycotoxin contamination in barley. The stage at or near anthesis, or at full head emergence, is generally thought to be optimal for fungicide application, regardless of cultivar. However, we have previously found that the most critical time for Fusarium graminearum infection and mycotoxin accumulation in barley differs among cultivars. Whereas chasmogamous (open-flowering) cultivars were most susceptible at anthesis, cleistogamous (closed-flowering) cultivars were considerably resistant at anthesis but became susceptible after spent anther extrusion. Therefore, this study evaluated the effect of the timing of fungicide application on FHB and mycotoxin (deoxynivalenol and nivalenol) accumulation in cleistogamous barley. Thiophanate-methyl fungicide was applied at different developmental stages, from before anthesis to 30 days after anthesis (DAA), under artificial inoculation conditions in the field in which inoculum spores were provided throughout the testing period. As expected, the optimal timing for chemical control of FHB and mycotoxin accumulation was the time around the beginning of spent anther extrusion rather than at anthesis. Later application, as late as 30 DAA, was also effective in controlling mycotoxin accumulation, although it was not effective in controlling disease levels.

Changes in Fungicide Resistance Frequency and Population Structure of Pyricularia oryzae after Discontinuance of MBI-D Fungicides

The changes in fungicide resistance frequency and population structure of the rice blast fungus Pyricularia oryzae were monitored after the discontinuance of melanin biosynthesis inhibitor targeting scytalone dehydratase (MBI-D) fungicides use in Saga Prefecture, Japan. After discontinuance in 2003, the frequency of resistant isolates decreased from 71.8% in 2002 to 25% in 2003, and became undetectable in 2007. The initial marked decrease was due to a decline of isolates possessing the predominant haplotype, although the haplotypic diversity among resistant isolates remained high from 2003 to 2005. These results revealed that resistant isolates were less fit in comparison with sensitive isolates in the absence of MBI-D fungicide pressure under field conditions. Pairwise FST values indicated that the change in population structure after MBI-D discontinuance was explainable by a rapid change in the proportions of resistant and sensitive subpopulations. Depending upon the existence of fitness cost and rapid changes in population structure, it may be possible to reintroduce MBI-D fungicides in areas where resistance has already developed, although we speculate that fitness cost related to MBI-D resistance may be small based on our present results and previous findings.

DNA fingerprinting of Pyricularia grisea by rep-PCR using a single primer based on the terminal inverted repeat from either of the transposable elements Pot2 and MGR586

We investigated the use of single primers complementary to sequences in the terminal inverted repeat (TIR) of either Pot2 or MGR586, transposable elements found in Pyricularia grisea, for DNA fingerprinting by repetitive-element-based polymerase chain reaction (rep-PCR). Under standard amplification conditions, rep-PCR with each single primer generated distinct fingerprint patterns among rice-infecting P. grisea isolates collected in Japan. With the Pot2-TIR primer, bands ranging in size from 0.2 to 8u2009kb and in number from 8 to 13 per isolate were amplified. Although fewer bands were amplified with the MGR586-TIR primer, this molecular technique should be more reliable to identify and classify P. grisea isolates by combining the data of fingerprint patterns from each TIR primer. In a cluster analysis based on DNA fingerprints from this rep-PCR with the Pot2-TIR primer, 10 reference isolates and 12 field isolates from Saga Prefecture in 2002 were separated into six clonal lineages. We also demonstrated that the 12 field isolates belonged to one clonal lineage. Thus, this rep-PCR method using the single primer Pot2-TIR will be useful for the analysis of the population structure of rice blast pathogens.

Genetic Analysis of Pyricularia grisea Population by rep-PCR During Development of Resistance to Scytalone Dehydratase Inhibitors of Melanin Biosynthesis

In 2001, field isolates of Pyricularia grisea resistant to scytalone dehydratase inhibitors of melanin biosynthesis (MBI-D) were reported in Saga prefecture, Kyushu. Among 1,175 isolates collected from six prefectures of Kyushu in 2002 and 2003, 647 were resistant to MBI-D fungicides, each due to a single point mutation of the scytalone dehydratase (SDH) gene. On the basis of repetitive element-based polymerase chain reaction (rep-PCR) fingerprint data, the haplotypes of the resistant isolates showed high genetic diversity, indicating that the resistance existed in a multigenetic background. Three predominant haplotypes mainly contributed to the widespread resistance in Kyushu; haplotype Sa4 was observed frequently in Saga, Sa18 was predominant in Oita and Miyazaki, and Sa5 was widely distributed among all four prefectures. Also, phylogenetic analysis showed that both the resistant and sensitive isolates were clustered together in a closely related group. These results suggest that isolates possessing the SDH mutation would have been selected and then multiplied rapidly in each region of Kyushu as a result of the widespread introduction of MBI-D fungicides in a short period.

Simultaneous identification of species and trichothecene chemotypes of Fusarium asiaticum and F. graminearum sensu stricto by multiplex PCR

A multiplex PCR assay was developed for simultaneous identification of the species and trichothecene chemotypes for Fusarium asiaticum and F. graminearum sensu stricto based on the genes related to trichothecene biosynthesis. PCR was carried out in a single reaction with three pairs of primers designed for the tri6 region and one pair of primers designed for tri3. We confirmed that the multiplex PCR was able to identify species and chemotypes for all tested strains of F. asiaticum and F. graminearum s. str. isolated in Japan. This technique would be a useful and rapid tool for diagnosis, epidemiology, and population structure studies of the F. graminearum complex in Japan.

Population structure of rice blast isolates resistant to scytalone dehydratase inhibitors in Mie Prefecture and implications for their origin

In Mie Prefecture in Japan, rice blast isolates resistant to melanin biosynthesis inhibitors targeting scytalone dehydratase (SDH) were first observed in 2005. To analyze the distribution of the resistant isolates, 527 isolates were collected from wide areas in this prefecture during 2006 and 2007. Almost half of the isolates collected (233 of 527 isolates) carried a point mutation in the SDH gene conferring the resistant phenotype. To compare population structures of resistant and sensitive isolates, we analyzed the isolates with repetitive-element-based PCR DNA fingerprinting using a single primer complementary to a sequence in the terminal inverted repeat of transposable element Pot2. A majority of the resistant isolates were classified into a single DNA fingerprint haplotype, Mie1. Despite its prevalence in the resistant isolates, Mie1 was not found in the sensitive isolates. Furthermore, in a dendrogram constructed from the DNA fingerprint data, Mie1 and six other haplotypes formed a cluster composed of resistant isolates alone. These results suggest that the resistant isolates that belonged to the Mie1 haplotype had migrated from regions outside Mie Prefecture and selectively propagated in a short period in this prefecture.