Yoshitaka Nagatomo

Phylogenetic Analysis of Soybean‐Nodulating Rhizobia Isolated from Alkaline Soils in Vietnam

In order to analyze the phylogeny of soybean-nodulating bacteria in alkaline soils in Vietnam, indigenous soybean-nodulating bacteria were isolated from root nodules by cultivating three kinds of Rj-soybean cultivars on two alkaline soils in Vietnam. The 120 isolates were classified into two major genera of soybean-nodulating rhizobia, namely Bradyrhizobium and Sinorhizobium genera, based on a growth analysis on medium and PCR-RFLP analyses of 16S rDNA and of the 16S–23S rDNA internal transcribed spacer (ITS) region. Most of the isolates of B. japonicum were extra-slow-growing and their ITS types were similar to that of B. japonicum USDA 135. They were not isolated from the soybean cultivar CNS used as Rj2Rj3 genotype. Isolates of Sinorhizobium were divided into two groups, S. fredii and S. sp., based on a PCR-RFLP analysis of 16S rDNA. Furthermore, PCR-RFLP analysis of the 16S–23S rDNA ITS region enabled to separate them into five types, three ITS types associated with S. fredii and two with S. sp. Sinorhizobium was frequently isolated from the three soybean cultivars on two soils. From the isolate ratio, it was suggested that B. japonicum strains similar to B. japonicum USDA 135 and S. fredii predominated in the alkaline soils of Vietnam. Additionally, our findings indicated that the Rj-genotypes affected not only the compatibility, but also the preference for nodulation between the host soybean and rhizobia.

Diversity and geographical distribution of indigenous soybean-nodulating bradyrhizobia in Japan

Abstract Genetic diversity and distribution of indigenous soybean-nodulating bradyrhizobia in Japan were investigated based on restriction fragment length polymorphism analysis of PCR product (PCR-RFLP) analysis of the 16S−23S rDNA internal transcribed spacer (ITS) region using Bradyrhizobium USDA strains as reference strains. Soil samples were collected from five field sites in Hokkaido, Fukushima, Kyoto, Miyazaki and Okinawa in Japan. A total of 300 isolates were derived from three Rj-genotype soybean cultivars, Akishirome (non-Rj), CNS (Rj 2 Rj 3) and Fukuyutaka (Rj 4), and five field site combinations. The PCR products of the ITS region were digested with HaeIII, HhaI, MspI and XspI. Electrophoresed patterns were analyzed for phylogenetic relationship using Bradyrhizobium reference strains. Results revealed 22 RFLP patterns and 11 clusters. The RFLP patterns of the seven clusters were similar or identical to Bradyrhizobium japonicum USDA 6, 38, 110, 115, 123 and Bradyrhizobium elkanii USDA 76 and 94. Four minor clusters were independent from the clusters of the reference strains. The isolation ratio revealed the major clusters at each field site. These results suggested that major clusters of indigenous bradyrhizobia might be in the order Bj123, Bj38, Bj110, Bj6 and Be76 from the northern to southern regions in Japan.

Diversity of indigenous Bradyrhizobium strains isolated from three different Rj-soybean cultivars in terms of randomly amplified polymorphic DNA and intrinsic antibiotic resistance

Abstract Diversity of indigenous Bradyrhizobium strains isolated from three different Rj-genotypes of soybean (Glycine max L. Merrill) cultivars which was investigated based on randomly amplified polymorphic DNA (RAPD) fingerprinting, intrinsic antibiotic resistance (JAR) and indole-3-aceticacid (IAA) production, was correlated with preference for and compatibility with Rj-genotypes of soybean cultivars for nodulation. Seventeen strains from Akishirome (nonRj), 22 strains from CNS (Rj 2 Rj 3) and 19 strains from Fukuyutaka (Rj 4) were isolated. All of the isolates from Akishirome and Fukuyutaka and 8 isolates from CNS formed effective nodules on each Rj-soybean cultivar, while 14 isolates from CNS did not form effective nodules on Fukuyutaka after inoculation under sterile conditions. The 14 isolates from CNS produced IAA. Cluster analysis based on RAPD fingerprinting using 5 primers revealed the presence of tw.o clusters, A and B. Cluster B contained B. elkanii USDA 61, USDA 76 and indigenous bradyrhizobia of 14 isolates from CNS showing IAA production and cluster A contained B. japonicum USDA 110, AI017 and indigenous bradyrhizobia not showing IAA production. Indigenous bradyrhizobia strains in cluster A were further divided into 3 subclusters, A-I, A-2, and A-3 different from the cluster of B. japonicum USDA 110 and AI017. Identity of subclusters between RAPD fingerprinting and IAR phenotype was not completely obtained. Sub clusters A-I, A-2, and A-3 consisted mainly of isolates from Akishirome, CNS, and Fukuyutaka, respectively. Preferences of bradyrhizobia for Rj-soybean cultivars for nodulation may be estimated by genetic diversity at the strain level.

Distribution of zinc, manganese, copper, cobalt, and nickel in andosols profiles

Abstract The distribution of zinc, manganese, copper, cobalt, and nickel in Andosols was investigated. Sixty nine soil samples were collected from different horizons of an Andosols profile in Miyakonojo Basin in south Kyushu, Japan, The total contents of heavy metals were determined by digestion and four extraction solutions, 1 M NH4Ac (ammonium acetate) pH 4.5, 0.1 M HCl, 0.01 M EDTA (ethylenediaminetetraacetic acid) pH 6.5, and 0.005 M DTPA (diethylenetri-aminepentaacetic acid) pH 7.3 were used to determine the contents of available Zn, Mn, Cu, Co, and Ni in Andosols in relation to the organic carbon content. The results of the extraction analysis showed that by the use of 0.1 M H Cl high value of extracted heavy metals in the upper layers of the humus horizons were obtained while EDTA extraction yielded a large amount of the above mentioned metals in the high humus horizons. The extractable heavy metals contents were high and these metals closely related to the organic carbon content mostly in the humu...

Detection of plant parasitic nematodes, Meloidogyne incognita and Pratylenchus coffeae by multiplex PCR using specific primers

Abstract We investigated the internal transcribed spacer (ITS) region of plant parasitic nematodes, namely the root-knot nematode, Meloidogyne incognita and root-lesion nematode, Pratylenchus coffeae that hamper the development of sustainable cropping systems in the southern region of Kyushu, Japan. In the current study, we attempted to detect M. incognita and P. coffeae using multiplex PCR using specific primer sets. M. incognita specific primer set (Mi1-Mi2) was designed based on the sequence of the ITS region of M. incognita. P. coffeae specific primer set (Pc1-Pc2) was designed according to both the report of Uehara et al. (Nematoiogica, 44: 357–368, 1998) and the sequence of P. coffeae obtained in this study. The use of multiplex PCR enable to detect two distinctive bands derived from the two kinds of plant parasitic nematodes. Furthermore, these two specific bands were detectable, when the genome of other nematodes was mixed in the template, with at least one nematode of the plant parasitic nematodes, M. incognita and P. coffeae being present with hundreds of other soil nematodes.

Free-tryptophan and indoleacetic acid in zinc-deficient radish shoots

Abstract Free-tryptophan was detected In higher amounts in zinc-deficient radish shoots than in the control shoots with the use of HPLC. Two-dimensional TLC and GC-MS analysis revealed the presence of IAA in zinc-deficient radish shoots. Quantitative estimation of IAA by gas chromatography showed that zinc-deficient and control radish shoots contained 0.32 and 0.36 μg/100 g f.w., respectively. It could be deduced from these results that zinc nutrition did not affect significantly the amount of IAA in radish shoots.

Zinc nutrition and levels of endogenous lndole‐3‐acetic acid in radish shoots

Abstract Radish (Raphanus sativus cv. Akamaru‐Hatsukadaikon) was grown for several experiments in a glasshouse with zinc (Zn) supply in the nutrient solution. Lack of Zn resulted in stunted growth and reduced leaf of radish shoots were observed. Two‐dimensional thin layer chromatography (TLC) and gas chromatography‐mass spectrometry (GC‐MS) analysis revealed the presence of endogenous indole‐3‐acetic acid (IAA) in Zn‐deficient radish shoots. An estimate has been made of alkali‐labile (1 and 7N NaOH) IAA in Zn‐deficient radish shoots with the use of gas chromatography (GC). The level of 7N NaOH‐labile IAA (peptidic + ester + free) and IN NaOH‐labile IAA (ester + free) in Zn‐deficient radish shoots was almost the same as that of control radish shoots. These results suggest that Zn nutrition did not affect the level of endogenous IAA in radish shoots.

Distinctive Expression of a Zinc-Binding Protein in Rice Callus Grown in Medium with High Zinc Concentration

Abstract We investigated the growth, contents of water-soluble protein and free amino acids of the callus of japonica rice (Oryza sativa L. cv. Nipponbare) cultured in liquid N6 medium containing a high concentration of zinc. Furthermore, we determined the N-terminal amino acid sequence of a Zn-binding protein expressed in large quantities in the callus. The addition of Zn stimulated the growth of the callus and increased the Zn concentration. The callus subjected to the high-Zn treatment (hereafter referred to as CHZn) contained a larger amount of soluble proteins and a smaller amount of free amino acids than the control callus. Zinc-binding proteins were separated by affinity chromatography. The SDS-PAGE pattern of these proteins showed a distinctive protein band of about 29 kDa. Especially, CHZn contained larger quantities of 29 kDa protein than the control callus. Twenty-seven N-terminal amino acids of the protein were sequenced as DYAPMTLTIVNNCPYPVWPGIQANSGH. Results of homology search to the amino acid sequences from the nr-aa database and the dbEST database suggested that this 29 kDa protein may be a novel zinc-binding protein and that the protein may regulate the concentration of free zinc in the cytoplasm of callus cells through its binding to zinc ions.

Indole carboxylic acid in zinc-deficient radish shoots

Abstract van Urk-Salkowskys reactive spots were detected on a two dimensional thin layer chromatogram of the acidic fraction from zinc deficient radish shoots. A yellowish and dominant spot gave the same R f value and color reaction as those of the authentic carboxylic acid (ICA). For further identification of ICA in the zinc-deficient radish shoots, GC-MS was employed. The fraction of the methylated final extract, which appeared as a peak on the gas chromatogram at the same retention time as that of the methylated authentic ICA showed high peaks at 175, 144, and 116 m/e. Mass spectra of the methylated authentic ICA revealed the presence of peaks at 175, 144, and 116 m/e. These results suggest that ICA was present in the zinc deficient radish shoots. The amount of ICA in the zinc deficient and control radish shoots were 32 and 1.4 μg/100 g F.W., respectively.

Distribution of micro elements in an andisol profile in Takatoge, Kagoshima, Japan

Abstract The distribution of micro-elements (Zn, Mn, Cu, Co, and Ni) in an Andisol profile of Takatoge in Osumi Peninsula, Kagoshima, Japan was investigated and the factors that influence their distribution during the Andisol forming process were discussed. The upper part of the soil profile consisted of a new ash layer which was deposited from 1946 to 1995 A.D. and originated from Sakurajima volcano. Thirty soil samples were collected from the soil profile for the determination of total and extractable contents of the micro-elements. Total contents of Zn and Mn were almost uniform, but the distribution of total Cu, Co, and Ni was irregular throughout the profile. The ratio of the content of extractable Zn and Mn to that of the total content was very low. The most of Zn and Mn occurred in a non-extractable form (total minus extractable) which was related to the presence of pyroxenese in the fine sand fraction. The ratio of the amount of extractable Cu, Co, and Ni to that of their total was very high compa...