Shoichiro Akao

Root, Root Hair, and Symbiotic Mutants of the Model Legume Lotus japonicus

To gain an overview of plant factors controlling nodule number and organogenesis, an extensive screening using model legume Lotus japonicus was carried out. This screening involved 40,000 M2 seeds, and 32 stable mutant lines were isolated. From these, 16 mutant lines maintaining the phenotypic variation were selected and genetically analyzed. With respect to nodule number, four loci were identified, Ljsym77, Ljsym78, slippery root (slp), and radial organization1 (rdo1). The former two mutants have an increased number of nodules, while the latter two have a decreased number. Ljsym78-1 and Ljsym78-2 are hypernodulating mutants with a branched root system and were found to be allelic to Ljsym16. The phenotype of the Ljsym77 mutant was highly pleiotropic, being deficient in light and gravity responses. The slp mutant was isolated as a low-nodulating mutant lacking root hairs. Concerning nodule organogenesis, nine symbiotic loci were identified, including the two loci alb1 and fen1. Mutants affecting the developmental process of nodule organogenesis were placed in three phenotypic categories: Nod- (Ljsym70 to Ljsym73), Hist- (alb1-1, alb1-2, and Ljsym79), and Fix- (fen1, Ljsym75, and Ljsym81).

A Supernodulating Mutant Isolated from Soybean Cultivar Enrei

Abstract Symbiotic nitrogen fixation in nodules of legumes depends on the complex interaction between the legume plant and (Brady)Rhizobium bacteria. Nodule formation and nitrogen fixation are closely regulated by both the host plant and the microsymbiont. Plant mutants with altered symbiotic performance are considered to be useful to gain a better understanding of the plant—microbe interactions in the legume—(Brady)Rhizobium symbiosis (Jacobsen 1984; Carroll et al 1985a, b; Park and Buttery 1988; Duc and Messager 1989; Gremaud and Harper 1989). Recently, Carroll et al. (1985a, b) have isolated the supernodulating mutants of the soybean cv. “Bragg,” which display a very large number of nodules and “nitrate-tolerant-symbiotic” (nts) characteristics. More recently, Gremaud and Harper (1989) have also isolated similar mutants from the soybean cv. “Williams.” These mutants not only provide materials that are useful for investigatings on the interaction in the nodule formation processes but also for agricultur...

Rhizobitoxine production by Bradyrhizobium elkanii enhances nodulation and competitiveness on Macroptilium atropurpureum

ABSTRACT Application of 1-aminoocyclopropane-1-carboxylic acid, an ethylene precursor, decreased nodulation of Macroptilium atropurpureum by Bradyrhizobium elkanii. B. elkaniiproduces rhizobitoxine, an ethylene synthesis inhibitor. Elimination of rhizobitoxine production in B. elkanii increased ethylene evolution and decreased nodulation and competitiveness on M. atropurpureum. These results suggest that rhizobitoxine enhances nodulation and competitiveness of B. elkanii on M. atropurpureum.

Characterization of Mycorrhizas Formed by Glomus sp. on Roots of Hypernodulating Mutants of Lotus japonicus

Lotusjaponicus hypernodulating mutants, Ljsym78-1 and Ljsym78-2, by the arbuscular mycorrhizal fungus Glomus sp. was characterized. The mutants are defective in systemic autoregulation of nodulation and nitrate inhibition, and form an excess of nodules and lateral roots. The percent root length colonized by the arbuscular mycorrhizal fungi was significantly higher for the mutant than wild-type roots. Detailed assessment of the colonization indicated that the percentage of colonization by arbuscules was increased, but that by external hyphae, internal hyphae and vesicles was decreased, in the mutant roots compared with the wild-type. The succinate dehydrogenase activity of arbuscules, external hyphae and internal hyphae showed similar trends. In addition, the majority of individual arbuscules that formed on the mutant roots had a well-developed and seemingly tough morphology. The results suggest that mutation at the Ljsym78 locus positively stimulates the growth and activity of arbuscules, but leads to reduced growth and activity of hyphae. We report the first identification of Lotus japonicus mutants that show significantly increased arbuscule formation and termed these mutants Arb++.

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.

Determination of leghemoglobin components and xylem sap composition by capillary electrophoresis in hypernodulation soybean mutants cultivated in the field

Abstract The hypernodulation soybean mutant lines (NOD1-3, NOD2-4, NOD3-7) and their parent Williams, and the mutant En6500 and its parent Enrei were cultivated in a sandy dune field in Niigata, and the nodules and root bleeding xylem sap were sampled at 50, 70, 90, and 120 d after planting (DAP). The nodule size distribution patterns and concentration of leghemoglobin components were determined. The number of nodules of the hypernodulation mutant lines was about two to three times higher than that of the parent lines irrespective of the sampling date. At 50 DAP the nodule size was relatively smaller in the hypernodulation mutant lines, and the total dry weight of the nodules was almost the same in the mutant lines and their parents. At 70 DAP and 90 DAP, the size distribution of the hypernodulation mutant nodules became .almost the same as that of the parent lines, and both the number and total dry weight of the nodules were higher than those of the parent lines. The concentration of four Lb components w...

The use of GUS-reporter gene to study the effect of Azospirillum-Rhizobium coinoculation on nodulation of white clover

Abstract The gusA-marked Azospirillum lipoferum T1371, constructed by inserting transposon mTn5SSgusA20 from Escherichia coli S17-1 λ-pir into the genome of Azospirillum lipoferum 137, was used to evaluate its effect on nodulation of white clover with and without Rhizobium inoculation. When inoculated alone, Azospirillum colonized the tap roots, secondary roots and root hairs. The combined inoculation of white clover with Rhizobium leguminosarum bv trifolii and A. lipoferum enhanced the number of nodules by 2–3 times from 5 to 20 days after inoculation (DAI). The combined inoculation also enhanced acetylene reduction activity by 2.3–2.7 times at 20 DAI. Moreover, Azospirillum was observed colonizing the tap root, root hairs and sites near or on the nodules. These results suggest that the formation of additional infection sites by A. lipoferum, with a combined inoculation, may be the mechanism that will enhance nodulation and nitrogen fixation of white clover.

Isolation and partial characterization of endophytic diazotrophs associated with Japanese sugarcane cultivar

Abstract Endophytic bacteria were isolated from the juice of a 6 month-old sugarcane cv. NiF-8, which was collected from Miyako, Okinawa and planted in a field lysimeter in Tsukuba, Ibaraki, Japan. The most probable number of N2-fixing endophytes was 4.5 × 105 cells per gram of fresh weight sample and 21 isolates have a positive reaction for ARA in an N2-free semi-solid medium with 10% crystallized sugarcane sugar and 0.5% sugarcane juice. Analyses of some of the biochemical properties of the N2-fixing isolates indicated that 13 isolates were putative strains of Acetobacter diazotrophicus, 4 isolates showed similar characteristics to those of Herbaspirillum seropedicae, and 4 isolates consisted of Herbaspirillum rubribalbicans-like strains. This study confirmed the existence of N2-fixing endophytic bacteria in the Japanese sugarcane cv. NiF-8.

Infection and colonization of aseptically micropropagated sugarcane seedlings by nitrogen-fixing endophytic bacterium, Herbaspirillum sp. B501gfp1

In this study, we used Herbaspirillum sp. B501gfp1 (B501gfp1), an isolate from wild rice, to investigate the interaction between a non-host nitrogen-fixing endophytic bacterium and micropropagated sugarcane plants under aseptic condition. Two Japanese sugarcane plants (Saccharum sp.) cultivars (cvs) NiF8 and Ni15 were inoculated using B501gfp1 in two inoculum doses of 108 and 102 bacterial-cells-per-milliliter suspension. The results showed that bacterial cells colonized both the root and stem tissues, and colonization was apparent in the intercellular spaces. Higher bacterial numbers were detected in plant tissues inoculated with the higher inoculum concentration treatment. Bacterial numbers also varied between the two cultivars, with the higher values determined in cv Ni15. This study provides evidence that Herbaspirillum sp. B501gfp1, a rice isolate, could colonize sugarcane tissues, suggesting non-specificity of host plant among endophytes.