Ryuichi Uegaki

Genotypic and phenotypic characterization of lactic acid bacteria isolated from Italian ryegrass silage

Twenty-three lactic acid bacteria (LAB) isolated from three cultivars (Akiaoba, Nagahahikari and Tachiwase) of Italian ryegrass (Lolium multiflorum Lam.) silage were precisely characterized by a combination of phenotypic tests, genotypic 16S ribosomal DNA sequencing and rapid PCR-based analyses, focusing on their useful phenotypes for silage preparation as inoculants. We successfully identified both at the species and subspecies levels: phenotypically novel Lactococcus lactis subsp. lactis, Lactobacillus brevis, Lactobacillus coryniformis subsp. torquens, Lactobacillus curvatus, Lactobacillus plantarum subsp. plantarum, Lactobacillus sakei subsp. carnosus, Leuconostoc mesenteroides subsp. dextranicum and Pediococcus parvulus. This is the first report to elucidate the presence of Lactobacillus coryniformis ssp. torquens and Leuconostoc mesenteroides subsp. dextranicum in Italian ryegrass silages. Physiological and biochemical tests revealed that phenotypic characteristics are different among the different strains of the same species and subspecies, and that the isolates show unique and diverse phenotypes related to fermentation factors, such as available carbohydrates, optimal growth pH and temperature. These results suggest that, for various well-preserved silage preparations, the isolates may be useful in producing novel inoculants corresponding to their optimally climatic and ecological niches.

Identification and characterization of lactic acid bacteria isolated from mixed pasture of timothy and orchardgrass, and its badly preserved silage.

In order to understand the relationship between lactic acid bacteria (LAB) species and silage fermentation, a total of 65 LAB strains isolated from mixed pasture of timothy (Phleum pratense L.) and orchardgrass (Dactylis glomerata L.), and its badly preserved silages were subjected to phenotypic and genetic analysis. According to these analyses, the isolates were divided into 13 groups, including Enterococcus gallinarum, Lactobacillus acidipiscis, L. coryniformis subsp. coryniformis, L. coryniformis subsp. torquens, L. curvatus, L. paraplantarum, L. plantarum subsp. argentoratensis, L. plantarum subsp. plantarum, L. sakei subsp. carnosus, Lactococcus garvieae, Lactococcus lactis subsp. cremoris, Leuconostoc pseudomesenteroides, Pediococcus acidilactici, Pediococcus pentosaceus, Weissella hellenica, Weissella paramesenteroides and Carnobacterium divergens. This is the first report to document that C. divergens, L. acidipiscis, L. sakei subsp. carnosus, L. garvieae, phenotypically novel L. lactis subsp. cremoris, E. gallinarum and W. hellenica are present in vegetative forage crops. L. plantarum group strains were most frequently isolated from the badly preserved silages. Some isolates showed a wide range of growth preferences for carbohydrate utilization, optimal growth pH and temperature in vitro, indicating that they have a high growth potential. These results are useful in understanding the diversity of LAB associated with decayed silage of timothy and orchardgrass.

Weissella oryzae sp. nov., isolated from fermented rice grains.

A taxonomic study was conducted on two Gram-reaction-positive, catalase-negative, irregular short-rod-shaped or coccoid lactic acid bacteria, designated strains SG25(T) and SG23, that were isolated from grains of fermented Japanese rice (Oryza sativa L. subsp. japonica). A phylogenetic analysis based on 16S rRNA gene sequence data clearly showed that the strains belonged to the genus Weissella and were most closely related to Weissella soli LMG 20113(T) (with a sequence similarity of 96.9 % for each novel strain). The peptidoglycan of each strain contained the amino acids glutamic acid, lysine, serine and alanine in a molar ratio of 1.0 : 1.2 : 0.5 : 3.0, respectively. On the basis of the unusual phenotypic characteristics of the novel strains and the low levels of DNA-DNA relatedness recorded between each novel strain and Weissella soli JCM 12536(T), strains SG25(T) and SG23 represent a single novel species in the genus Weissella, for which the name Weissella oryzae sp. nov. is proposed. The type strain is SG25(T) ( = JCM 18191(T)  = DSM 25784(T)).

Lactobacillus hokkaidonensis sp. nov., isolated from subarctic timothy grass (Phleum pratense L.) silage.

Four strains of Gram-positive, non-spore-forming, rod-shaped, catalase-negative and non-motile lactic acid bacteria, LOOC260(T), LOOC253, LOOC273 and LOOC279, were isolated from timothy grass (Phleum pratense L.) silage produced in Hokkaido, a subarctic region of Japan. These isolates grew at 4-37 °C, indicating the psychrotolerant nature of these strains. Phylogenetic analysis on the basis of 16S rRNA and pheS gene sequences, as well as biochemical and physiological characteristics, indicated that these four strains were members of the genus Lactobacillus. 16S rRNA gene sequence analysis of strain LOOC260(T) demonstrated that the closest neighbours were the type strains of Lactobacillus suebicus (97.7 %), Lactobacillus oligofermentans (96.7 %) and Lactobacillus vaccinostercus (96.7 %). Strain LOOC260(T) showed low levels of DNA-DNA association with Lactobacillus suebicus JCM 9504(T) (14.7 ± 3.5 %), Lactobacillus oligofermentans JCM 16175(T) (15.1 ± 4.8 %) and Lactobacillus vaccinostercus JCM 1716(T) (10.7 ± 3.0 %). The cell wall contained meso-diaminopimelic acid and the major fatty acids were C18 : 1ω9c and C19 : 1 cyclo 9,10. On the basis of phenotypic, physiological and phylogenetic evidence, these isolates represent a novel species of the genus Lactobacillus, for which the name Lactobacillus hokkaidonensis sp. nov. is proposed. The type strain is LOOC260(T) ( = JCM 18461(T) = DSM 26202(T)).

Strain-dependent effects of inoculation of Lactobacillus plantarum subsp. plantarum on fermentation quality of paddy rice (Oryza sativa L. subsp. japonica) silage.

Paddy rice has been of particular interest as a forage crop in Japan. In this study, the isolated strains TO1000, TO1001, TO1002, and TO1003 were characterized by phenotypic and genotypic approaches. These strains were identified as Lactobacillus plantarum subsp. plantarum by species-specific PCR. Phenotypic characteristics varied among different strains of the same subspecies, and the strains represented unique and diverse phenotypes related to fermentation factors, such as carbohydrate assimilation and range of pH and temperature allowing growth. PCR analysis revealed that the patterns of presence/absence of known plantaricin genes differed in a strain-specific manner. Using these strains as inoculants for preparation of whole crop paddy rice silage, fermentation quality was significantly improved, as shown by lower pH, higher lactic acid content, and inhibition of the growth of undesirable microorganisms such as molds, coliform bacteria, and clostridia, after 30 and 60 days of storage, with effectiveness differing from strain to strain. These observations suggest that suitable candidates for bacterial inoculants in silage preparation should be screened at the strain level. Strain TO1002 may be useful for producing silage inoculants for the production of well-preserved whole crop paddy rice silage.

Lactobacillus oryzae sp. nov., isolated from fermented rice grain (Oryza sativa L. subsp. japonica).

The taxonomic position of three Lactobacillus-like micro-organisms (strains SG293(T), SG296 and SG310) isolated from fermented rice grain (Oryza sativa L. subsp. japonica) in Japan was investigated. These heterofermentative lactic acid bacteria were Gram-stain-positive, rod-shaped, facultatively anaerobic, non-motile, non-spore-forming and did not show catalase activity. 16S rRNA gene sequence analysis of strain SG293(T) revealed that the type strains of Lactobacillus malefermentans (98.3 %), Lactobacillus odoratitofui (96.2 %), Lactobacillus similis (96.1 %), Lactobacillus kimchicus (96.1 %), Lactobacillus paracollinoides (95.9 %) and Lactobacillus collinoides (95.7 %) were the closest neighbours. Additional phylogenetic analysis on the basis of pheS and rpoA gene sequences, as well as biochemical and physiological characteristics, indicated that these three strains were members of the genus Lactobacillus and that the novel isolates had a unique taxonomic position. The predominant cellular fatty acids were C18 : 1ω9c and C19 : 1 cyclo 9,10. Because low DNA-DNA hybridization values among the isolates and Lactobacillus malefermentans JCM 12497(T) were observed, it is proposed that these unidentified isolates be classified as a novel species of the genus Lactobacillus, Lactobacillus oryzae sp. nov. The type strain is SG293(T) (= JCM 18671(T) = DSM 26518(T)).

Changes in the concentrations of fumonisin, deoxynivalenol and zearalenone in corn silage during ensilage.

We assessed the production of the mycotoxins fumonisin, deoxynivalenol and zearalenone during the ensiling of corn. Corn was harvested at yellow-ripe or full-ripe stage and separated into the stem and leaf parts and the ear parts, including bracts. Each material was ensiled under five conditions: (1) no fungus added, anaerobic conditions; (2) no fungus added, aerobic conditions; (3) mycotoxin-producing fungus added, anaerobic conditions; (4) mycotoxin-producing fungus added, aerobic conditions; and (5) mycotoxin-producing fungus added to autoclaved material, aerobic conditions. After 40 days of ensilage, we analyzed the silage fermentative quality and mycotoxin concentration. The fermentative quality of all materials was good in treatments (1) and (3), because the pH < 4 increased the lactic acid content preventing mycotoxin levels from increasing. In treatments (2) and (4), fermentative quality of all materials was poor, and mycotoxin levels were slightly increased. In treatment (5), fermentative quality was poor, and mycotoxin levels were increased remarkably. These results indicate that mycotoxins are not produced under anaerobic conditions and are hardly produced under aerobic condition during the ensiling of corn. Our findings suggest that almost all mycotoxins in corn silage are produced pre-harvest.

Changes of fumonisin production in rice grain during ensiling

We assessed fumonisin production during the ensiling of rice grain. Rice grain was harvested at the full-ripe stage and prepared as rough rice, crushed rough rice, brown rice or crushed brown rice. Each material was ensiled under six conditions: (1) no fungus, anaerobic; (2) no fungus, aerobic; (3) water added, anaerobic; (4) water and fumonisin-producing fungus added, anaerobic; (5) water and fumonisin-producing fungus added, aerobic; or (6) fumonisin-producing fungus added to autoclaved material, aerobic. After 40 days of ensilage, we analyzed the silage fermentative quality and fumonisin concentration. The fermentative quality of all materials was good in treatments (3) and (4) (pH < 4), reasonable in treatment (5) (pH = 5∼6) and unacceptable in treatments (1) and (2) (pH > 6.5). The fumonisin concentration was low in all materials in treatments (1) to (4), slightly increased in the three materials other than rough rice in treatment (5), and enormously increased in all materials in treatment (6). The results indicate that the fumonisin-producing fungus does not produce fumonisin in anaerobic conditions. It is important that an anaerobic condition be maintained during ensiling in order to reduce the fumonisin content in rice grain silage.

A single nucleotide polymorphism in the translation elongation factor 1α gene correlates with the ability to produce fumonisin in Japanese Fusarium fujikuroi.

PCR-RFLP based on the translation elongation factor 1α (TEF) gene was developed to identify Fusarium fujikuroi in the Fusarium (Gibberella) fujikuroi species complex. Ninety-three strains, most of which were obtained from various sources in Japan, were identified as F. fujikuroi and their capability to produce fumonisin was investigated using an in vitro assay. Fumonisin production was detected in 50 strains isolated from maize, strawberry, wheat, and rice, whereas it was undetectable in 43 strains derived from rice seeds and rice seedlings carrying the bakanae disease, and from unknown sources. A single nucleotide polymorphism in the TEF gene (T618G) correlated with the ability to synthesize fumonisin.

Lactobacillus silagei sp. nov., isolated from orchardgrass silage.

A Gram-reaction-positive, facultatively anaerobic, non-spore-forming and catalase-negative rod-shaped bacterial strain, designated IWT126(T), was isolated from orchardgrass (Dactylis glomerata L.) silage preserved in Hachimantai, Iwate, Japan. The isolate showed growth at 15-45 °C, pH 3.5-7.5 and with 4.0 % (w/v) NaCl. The cell wall peptidoglycan did not contain meso-diaminopimelic acid, and the DNA G+C content was 45.6 mol%. The major cellular fatty acids were C16 : 0 and C19 : 1 cyclo 9,10. Based on 16S rRNA gene sequence similarity, strain IWT126(T) was classified as a member of the genus Lactobacillus and was most closely related to Lactobacillus odoratitofui YIT 11304(T) (98.7 %), Lactobacillus similis JCM 2765(T) (98.5 %), Lactobacillus collinoides JCM 1123(T) (97.6 %), Lactobacillus paracollinoides DSM 15502(T) (97.6 %) and Lactobacillus kimchicus DCY51(T) (96.9 %). Based on sequence analysis of the phenylalanyl-tRNA synthase α-subunit (pheS) gene, strain IWT126(T) was well separated from its phylogenetic neighbours in the genus Lactobacillus. Based on physiological, biochemical and genotypic results, as well as low DNA-DNA relatedness to recognized phylogenetic relatives in the genus Lactobacillus, classification of strain IWT126(T) as a representive of a novel species named Lactobacillus silagei sp. nov. is proposed. The type strain is IWT126(T) ( = JCM 19001(T) = DSM 27022(T)).