Pectobacterium araliae sp. nov., a pathogen causing bacterial soft rot of Japanese angelica tree in Japan.

Phytopathogenic bacteria (MAFF 302110T and MAFF 302107) were isolated from lesions on Japanese angelica trees affected by bacterial soft rot in Yamanashi Prefecture, Japan. The strains were Gram-reaction-negative, facultatively anaerobic, motile with peritrichous flagella, rod-shaped, and non-spore-forming. The genomic DNA G+C content was 51.1 mol % and the predominant cellular fatty acids included summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0, summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), summed feature 2 (comprising any combination of C12 : 0 aldehyde, an unknown fatty acid with an equivalent chain length of 10.928, C16 : 1 iso I, and C14 : 0 3OH), and C12 : 0. Phylogenetic analyses based on 16S rRNA and gyrB gene sequences, along with phylogenomic analysis utilizing whole-genome sequences, consistently placed these strains within the genus Pectobacterium. However, their phylogenetic positions did not align with any known species within the genus. Comparative studies involving average nucleotide identity and digital DNA-DNA hybridization with the closely related species indicated values below the thresholds employed for the prokaryotic species delineation (95-96 % and 70 %, respectively), with the highest values observed for Pectobacterium polonicum DPMP315T (92.10 and 47.1 %, respectively). Phenotypic characteristics, cellular fatty acid composition, and a repertoire of secretion systems could differentiate the strains from their closest relatives. The phenotypic, chemotaxonomic, and genotypic data obtained in this study show that MAFF 302110T/MAFF 302107 represent a novel species of the genus Pectobacterium, for which we propose the name Pectobacterium araliae sp. nov., designating MAFF 302110T (=ICMP 25161T) as the type strain.

Pseudomonas solani sp. nov. isolated from the rhizosphere of eggplant in Japan.

The search for bacteria that can be used as biocontrol agents to control crop diseases yielded a promising candidate, Sm006T, which was isolated from the rhizosphere of eggplant (Solanum melongena) growing in a field in Aichi Prefecture, Japan, in 2006. The cells were Gram-stain-negative, aerobic, non-spore-forming, rod-shaped and motile with one polar flagellum. The results of homology searches and phylogenetic analyses based on the 16S rRNA gene sequence indicated that Sm006T represents a member of the genus Pseudomonas. The genomic DNA G+C content was 66.3 mol% and the major cellular fatty acids (more than 5 % of the total fatty acids) were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and C12 : 0. Phylogenetic analyses using the rpoD gene sequence and phylogenomic analysis of the whole genome sequence revealed that Sm006T represents a member of the Pseudomonas resinovorans group; however, its phylogenetic position does not match that of any known species of the genus Pseudomonas. The average nucleotide identity and digital DNA-DNA hybridisation values between the strain and closely related species were lower than the thresholds for prokaryotic species delineation (95-96 and 70 %, respectively), with the highest values observed for Pseudomonas tohonis TUM18999T (92.05 and 46.3 %, respectively). Phenotypic characteristics, cellular fatty acid composition and possession of 2,4-diacetylphloroglucinol biosynthetic gene cluster could be used to differentiate the strain from its closest relatives. The phenotypic, chemotaxonomic and genotypic data obtained during this study indicated that Sm006T represents a novel species of the genus Pseudomonas, for which we propose the name Pseudomonas solani sp. nov., with Sm006T (= MAFF 212523T = ICMP 24689T) as the type strain.

Pseudomonas petroselini sp. nov., a pathogen causing bacterial rot of parsley in Japan.

Phytopathogenic bacterial strains (MAFF 311094T, MAFF 311095, MAFF 311096 and MAFF 311097), which were isolated from rot lesions of parsley (Petroselinum crispum) sampled in Miyagi, Japan, were subjected to polyphasic characterization to determine their taxonomic position. The cells were Gram-reaction-negative, aerobic, non-spore-forming, motile with one or two polar flagella and rod-shaped. The 16S rRNA gene sequences analyses revealed that the strains belong to the genus Pseudomonas, exhibiting the highest sequence similarity to Pseudomonas sivasensis P7T (99.93% sequence similarity), Pseudomonas cyclaminis MAFF 301449T (99.93 %), Pseudomonas extremaustralis 14-3T (99.86 %), Pseudomonas kitaguniensis MAFF 212408T (99.86 %) and Pseudomonas antarctica CMS 35T (99.79 %). The genomic DNA G+C content was 60.1 mol%, and the major cellular fatty acids (>5 % of the total fatty acids) were C16:0, summed feature 3 (C16:1 ω7c/C16:1 ω6c), summed feature 8 (C18:1 ω7c/C18:1 ω6c) and C17:0 cyclo. The rpoD sequence-based phylogenetic and whole genome-based phylogenomic analyses demonstrated that the strains are a member of the Pseudomonas fluorescens subgroup, but their phylogenetic position does not match those of any members of this subgroup. The average nucleotide identity and digital DNA-DNA hybridization values between the strains and their closely related species were ≤90.64% and ≤41.9 %, respectively, which were below the thresholds for prokaryotic species delineation (95-96 and 70%, respectively). Phenotypic characteristics, pathogenicity toward parsley and cellular fatty acid composition could differentiate the strains from their closest relatives. The phenotypic, chemotaxonomic and genotypic data presented in this study revealed that the strains constitute a novel Pseudomonas species, for which we propose the name Pseudomonas petroselini sp. nov., with MAFF 311094T (=ICMP 24279T) being the type strain.

Pseudomonas lactucae sp. nov., a pathogen causing bacterial rot of lettuce in Japan.

Two phytopathogenic bacteria, MAFF 301380T and MAFF 301381, isolated from rot lesions of lettuce (Lactuca sativa L. var. capitata L.) in Japan, were characterized using a polyphasic approach. The cells were Gram-reaction-negative, aerobic, non-spore-forming, rod-shaped and motile with one to three polar flagella. Analysis of the 16S rRNA gene sequences showed that the strains belong to the genus Pseudomonas and are closely related to Pseudomonas cedrina subsp. cedrina CFML 96-198T (99.72 %), Pseudomonas cedrina subsp. fulgida P515/12T (99.65 %), Pseudomonas gessardii DSM 17152T (99.51 %), Pseudomonas synxantha DSM 18928T (99.44 %), Pseudomonas libanensis CIP 105460T (99.44 %) and Pseudomonas lactis DSM 29167T (99.44 %). The genomic DNA G+C content was 60.4 mol% and the major fatty acids consisted of summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c), C16 : 0 and summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c). Phylogenetic analysis using the rpoD gene sequences and phylogenomic analyses based on the whole genome sequences demonstrated that the strains are members of the Pseudomonas fluorescens subgroup but formed a monophyletic and robust clade separated from their closest relatives. The average nucleotide identity and digital DNA-DNA hybridization values between the strains and their closely related species were 88.65 % or less and 36.3 % or less, respectively. The strains could be distinguished from their closest relatives by phenotypic characteristics, pathogenicity towards lettuce and whole-cell MALDI-TOF MS profiles. The evidence presented in this study supports the classification of the strains as representing a novel Pseudomonas species, for which we propose the name Pseudomonas lactucae sp. nov., with the type strain MAFF 301380T (=ICMP 23838T).

Pseudomonas allii sp. nov., a pathogen causing soft rot of onion in Japan.

Six phytopathogenic bacterial strains, MAFF 301512, MAFF 301513, MAFF 301514T, MAFF 301515, MAFF 301516 and MAFF 301517, were isolated from soft rot lesions of onion (Allium cepa L.) in Japan. The cells were Gram-reaction-negative, aerobic, non-spore-forming, motile with one or two polar flagella and rod-shaped. Analysis of their 16S rRNA gene sequences showed that they belong to the genus Pseudomonas, with the highest similarities to Pseudomonas poae DSM 14936T (99.86 %), Pseudomonas simiae OLiT (99.85 %), Pseudomonas trivialis DSM 14937T (99.79 %) and Pseudomonas extremorientalis KMM 3447T (99.79 %). Their genomic DNA G+C content was 60.9 mol% and the major fatty acids (>5 % of the total fatty acids) present were C16 : 0, summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c), summed feature 8 (C18 : 1 ω7c /C18 : 1 ω6c) and C17 : 0 cyclo. Phylogenetic and phylogenomic analyses based on the rpoD gene and whole genome sequences, respectively, demonstrated that the strains belong to the Pseudomonas fluorescens subgroup, but form a monophyletic and robust clade, with Pseudomonas azotoformans as their neighbour. Between the strains and P. azotoformans, the average nucleotide identity scores were 95.63-95.70 %, whereas the digital DNA-DNA hybridization scores of the strains against their closest relatives, including P. azotoformans, were 65.4 % or less, which are lower than the 70 % cut-off for prokaryotic species delineation. The strains were differentiated from their closest relatives by phenotypic characteristics, pathogenicity in onion and cellular fatty acid composition. The phenotypic, chemotaxonomic and genotypic data showed that the strains represent a novel Pseudomonas species, proposed to be named Pseudomonas allii sp. nov., with MAFF 301514T (=ICMP 23680T) being the type strain.

Pseudomonas cyclaminis sp. nov., a pathogen causing bacterial bud blight of cyclamen in Japan.

Five phytopathogenic bacterial strains, MAFF 301449T, MAFF 301450, MAFF 301451, MAFF 301452, and MAFF 301453, which were isolated from bud blight lesions of cyclamen (Cyclamen persicum Mill.) in Miyagi, Japan, were subjected to polyphasic taxonomic characterisation. The cells were Gram-reaction-negative, aerobic, non-spore-forming, motile with one to five polar flagella, and rod-shaped. Analysis of 16S rRNA gene sequences showed that they belong to the genus Pseudomonas, with Pseudomonas extremaustralis 14-3T (99.79 % sequence similarity), Pseudomonas trivialis DSM 14937T (99.79 %), Pseudomonas poae DSM 14936T (99.72 %), and Pseudomonas antarctica CMS 35T (99.72 %) as their relatives. The genomic DNA G+C content was 60.3 mol% and the major fatty acids (>5 % of the total fatty acids) were C16 : 0, summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c), summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c), and C17 : 0 cyclo. Phylogenetic analysis using the rpoD gene sequences and phylogenomic analyses based on the whole genome sequences demonstrated that the strains are members of the Pseudomonas fluorescens subgroup, but form a monophyletic and robust clade separated from their relatives. Average nucleotide identity and digital DNA-DNA hybridisation analyses with the closely related Pseudomonas species corroborated their novel species status. The strains were differentiated from their relatives by phenotypic characteristics, pathogenicity towards cyclamen, cellular fatty acid composition, and whole-cell MALDI-TOF mass spectrometry profiles. Based on the phenotypic, chemotaxonomic, and genotypic data obtained, we conclude that the strains represent a novel Pseudomonas species, for which we propose the name Pseudomonas cyclaminis sp. nov.; the type strain is MAFF 301449T (=ICMP 23720T).

Use of LAMP Detection to Identify Potential Contamination Sources of Plant-Pathogenic Pythium Species in Hydroponic Culture Systems of Tomato and Eustoma.

Hydroponic culture systems are subject to high risks of diseases caused by zoosporic plant pathogens. Control is generally difficult because of the rapid spread of zoospores in the nutrient solutions. In Japan, tomato and eustoma, which are cultivated using the D-tray and nutrient film techniques, respectively, are susceptible to diseases caused by Pythium aphanidermatum and P. irregulare. We used loop-mediated isothermal amplification to identify potential contamination sources of these two pathogens by monitoring their presence in the water supply wells, seedling terraces, nutrient solutions, diseased plants, and ground soils of a tomato greenhouse complex and a eustoma greenhouse complex. The results indicated that the pathogens may enter the culture systems from the soils around the greenhouses. Entry most likely occurs when seedlings are moved from the seedling terraces to the greenhouses, and sterilization of the hydroponic systems may not be sufficient. Therefore, monitoring pathogens in the culture systems and ground soils is very important for the management and prevention of these diseases.

[A Case of Descending Colon Adenocarcinoma with Neuroendocrine Differentiation Successfully Treated with FOLFIRI plus Bevacizumab].

We report a case of descending colon adenocarcinoma with neuroendocrine differentiation that was effectively treated with FOLFIRI plus bevacizumab. A 70-year-old man underwent a colonoscopy and was found to have a type 2 tumor of the descending colon. A well-to-moderately differentiated adenocarcinoma was diagnosed by biopsy. A preoperative CT scan showed paraaortic lymph node and liver metastasis, and that the tumor was directly invading the spleen. The patient underwent a left hemicolectomy with resection of the pancreas tail, spleen, and diaphragm. The pathological diagnosis was adenocarcinoma with less than 30% neuroendocrine differentiation in the primary tumor and almost 100% neuroendocrine differentiation in the metastatic lymph nodes. After surgery, FOLFIRI plus bevacizumab treatment was initiated. After 33 treatment cycles, the paraaortic lymph node and liver metastasis disappeared and the patient has remained progression-free to date. Adenocarcinoma with neuroendocrine differentiation of the colon is rare and an effective chemotherapy has not yet been established. We report this case with a review of the literature.

Enhanced sensitivity to cis-diamminedichloroplatinum(II) of a human carcinoma cell line with mutated p53 gene by cyclin-dependent kinase inhibitor p21(WAF1) expression.

p53-mediated induction of p21(WAF1), a cyclin-dependent protein kinase inhibitor, is known to protect cancer cells from the cytotoxic effects of anti-cancer drugs or gamma-irradiation. Since the p53 gene is frequently inactivated in cancer cells, we examined whether p21(WAF1) expression may alter the sensitivity of cancer cells with mutated p53 gene to anti-cancer drugs. Cells of a colon cancer cell line DLD-1 were transfected with p21(WAF1) expression vector controlled by a tetracycline-repressable promoter and transfectants were cloned (Dp21-1). p21(WAF1) expression induced by removal of tetracycline from culture media repressed cell proliferation and resulted in altered cell shape, suggesting induction of differentiation. Dp21-1 cells with p21(WAF1) expression were more sensitive to cis-diamminedichloroplatinum(II) (CDDP) (IC(50) value, 10 microM) than those without p21(WAF1) expression (IC(50), 22 microM). Sensitivity to doxorubicin was not different between Dp21-1 cells with and without p21(WAF1) expression. DNA ladder formation was observed in Dp21-1 cells treated with CDDP, indicating that the enhanced sensitivity to CDDP involves apoptosis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytosolic protein revealed that subunit protein bands with M(r) 55 kDa and 44 kDa were markedly increased in cells with p21(WAF1) expression. By immunoblotting, these proteins were identified as c-Jun N-terminal kinase (JNK) 2 and p38 mitogen-activated protein kinase (MAPK) delta, respectively, both of which are believed to be involved in apoptosis induction by CDDP. These results suggest that p21(WAF1) may enhance the sensitivity of colon cancer cells with mutated p53 gene to CDDP, possibly through the JNK and p38 MAPK pathways.