Masao Sakai

Isolation and Characterization of Agar-degrading Paenibacillus spp. Associated with the Rhizosphere of Spinach

Agar-degrading bacteria in spinach plant roots cultivated in five soils were screened, and four strains of Paenibacillus sp. were isolated from roots cultivated in three soils. The agar-degrading bacteria accounted for 1.3% to 2.5% of the total bacteria on the roots. In contrast, no agar-degrading colony was detected in any soil (non-rhizosphere soil samples) by the plate dilution method, and thus these agar-degrading bacteria may specifically inhabit plant roots. All isolates produced extracellular agarase, and could grow using agar in the culture medium as the sole carbon source. Zymogram analyses of agarase showed that all four isolates extracellularly secreted multiple agarases (75-160 kDa). In addition, the isolates degraded not only agar but also various plant polysaccharides, i.e., cellulose, pectin, starch, and xylan.

Influence of soil salinity on the populations and composition of fluorescent pseudomonads in plant rhizosphere

Abstract Effect of soil salinity associated with intensive cultivation including horticulture on microbial communities in the soil-root system was investigated in a growth chamber experiment. Spinach plants were grown on three soil samples of the same origin but with low, medium, and high levels of salinity. Microbial populations, including fluorescent pseudomonads, in three sites of the soil-root system, namely root-free soil, rhizosphere soil, and rhizoplane, of 3-week-old spinach plants were compared in relation to the levels of soil salinity. Effect of soil salinity on the microbial populations differed widely among the three sites of the soil-root system, as well as among the microbial groups. In the root-free soil, populations of total bacteria (TB) and gram-negative bacteria (GN) did not change significantly through salinity, while the populations of total fluorescent pseudomonads (FP) apparently increased. In the rhizosphere soil, however, soil salinity induced changes in the populations depending...

Tn5 mutants of Bradyrhizobium japonicum Is-1 with altered compatibility with Rj 2-soybean cultivars

Abstract Bradyrhizobium japonicum Is-1 is incompatible with soybean (Glycine max L. Merr.) cv. CNS (Rj 2 Rj 3) and compatible with cv. Hill (Rj 4) and is, therefore, classified into nodulation-type B. Tn5 mutants (1C1, 1C2, 5C1, 6C1, 7C1, 7C2, 10C1 and 10C2) were isolated from eight nodules produced on CNS that had been inoculated with the kanamycin-resistant (Kmr) transconjugants of strain Is-1. To investigate the shift of host range, Tn5 mutants were inoculated to soybean cvs. CNS, D-51 (Rj 3), Hardee (Rj 2 Rj 3), Hill and IAC-2 (Rj 2 Rj 3). They acquired the ability to nodulate Rj 2 Rj 3 soybeans without losing the ability to nodulate both Rj 3 and Rj 4. These results suggest that the Tn5 mutation in B. japonicum strain Is-1 is only related to the ability to nodulate Rj 2 soybean. Although the host ranges were common to Tn5 mutants and all sections of nodules produced by Tn5 mutants indicate a red color, the nodule numbers and nitrogen fixation activities were different for each mutant. These results suggest that Tn5 insertion sites are different from each other. Tn5-flanking sequences in these mutants were specifically amplified. The electrophoresis analyses of the polymerase chain reaction (PCR) products showed that Tn5 mutants contained a single copy of Tn5, except for 10C1. These PCR products were cloned and sequenced. The sequence analysis of Tn5-flanking sequences revealed that these Tn5 insertion sites were different from each other and all homologous sequences to them were found in the complete genome sequence of B. japonicum USDA 110. These results are in good accordance with our expectation that Tn5 insertion sites are different for each mutant. Most Tn5-inserted gene products relate to the cell membrane structure. This suggests that the change in the cell surface structure in Tn5 mutants may overcome nodulation restriction conditioned by Rj 2 soybean.

Application of DGGE analysis to the study of bacterial community structure in plant roots and in nonrhizosphere soil

Abstract To analyze the structure of bacterial communities in spinach roots and in the nonrhizosphere soil, we used PeR-amplified 16S rRNA gene fragments separated by denaturing gradient gel electrophoresis (DGGE). DGGE revealed a large number of band patterns, which were ascribed to various bacterial species composing each of the bacterial communities. The pattern from the roots was less complex than that from the soil. It is considered that DGGE analysis is suitable for studies of bacterial community structure in soil-plant ecosystems.

Effect of soil salinity on population structure of fluorescent pseudomonads in spinach rhizosphere

Abstract Use of PGPR (plant growth-promoting rhizobacteria) for stimulating plant growth and for the biological control of soil-borne diseases is necessary mainly in fields with intensive cropping. The intensive cultivation systems in horticulture have generally led to soil salinity due tp the high rates of fertilizer application. Therefore, the high concentration of inorganic salts accumulated in the plant rhizosphere is likely to be a factor of salinity stress affecting both roots and root-associated rhizobacteria. In a previous report (Matsuguchi and Sakai 1995), we observed that high salinity of soil had induced a significant decrease in the populations of fluorescent pseudomonads in spinach roots. Furthermore, the adverse effect of high salinity of soil was more pronounced on Pseudomonas fluorescens strains than on Pseudomonas putida strains, resulting in an alteration of the composition of fluorescent pseudomonads in the roots where vP. putida} predominated. The objective of the present study was to...

Effect of cations on the growth of fluorescent pseudomonad isolates from spinach roots grown in soils with different salinity levels

Abstract Effect of Inorganic ion concentrations in culture solution on the growth of Pseudomonas putida and P. fluorescens Isolates from spinach roots grown in soils with high-salinity (H-soil) and low-salinity (L-soil) levels was investigated. Both H- and L-soils were taken from the topsoil of a greenhouse for spinach cultivation and a nearby fallow field, respectively. In the H-soil inorganic ions had accumulated due to the high rates of application of fertilizer. Among the ions, the concentrations of Ca2+, NO3 -, and SO4 2- in the H-soil were significantly higher than those in the L-soil. Among the inorganic ions tested (K+, Na+, Ca2+, Mg2+, Cl-, NO3 -, and SO4 2-), Ca2+ was found to be the most inhibitory on the growth of isolates. In addition, the isolates from the H-soil were more Ca2+-tolerant than those from the L-soil. Consequents the growth tolerance to Ca2+ stress of the total 80 isolates, 40 each from the H-soil and L-soil, was compared at 150 mM Ca2+. Most isolates from the H-soil showed a si...

Isolation of Asticcacaulis sp. SA7, a novel agar-degrading alphaproteobacterium.

An agar-degrading bacterium, strain SA7, was isolated from plant roots cultivated in soil. Analysis of the 16S rDNA sequence showed that strain SA7 is affiliated with the genus Asticcacaulis. Strain SA7 produced extracellular agarase, and grew utilizing agar in the culture medium as sole carbon source. Zymogram analysis showed that strain SA7 extracellularly secreted single agarase protein (about 70 kDa).

Chemotactic response to amino acids of fluorescent pseudomonads isolated from spinach roots grown in soils with different salinity levels

Abstract Chemotactic response to amino acids of fluorescent pseudomonads isolated from spinach roots grown in a low-salinity soil (LR-group) and its corresponding high-salinity soil (HR-group) was investigated. Furthermore, the amount and composition of amino acids in the root exudate under low- and high-salinity conditions were investigated. All the isolates examined showed a chemotactic response exclusively to the amino acid fraction, while most of them did not respond to the sugar and the organic acid fractions. Based on these results, the chemotactic response to 20 amino acids of randomly selected 48 isolates, of which 12 isolates each belongs to the LR- and HR-groups of both Pseudomonas putida and P. fluorescens, was investigated. The results showed that the spectrum of chemotaxis to 20 amino acids of the H-group of each species was markedly different from that of the corresponding L-group. Also, the amino acid composition of the root exudate in the high-salinity culture solution significantly differ...

Use of Bacterial Bioluminescence for Monitoring the Behavior of Rhizobacteria Introduced to Plant Rhizosphere

Abstract Bioluminescence (lux) genes from Vibrio harveyi were introduced into an artificial transposon vector pJFF350 as a constitutively expressing lux-genes set. The resultant recombinant plasmid pSOL2 was then transposed into the chromosome of fluorescent pseudomonad strains, in which the lux genes were stably integrated. Root colonization by the lux-marked strains was visually monitored by autophotography, and it was found that the bioluminescence intensities were highly correlated with the populations of the strains colonizing roots. A strain of Pseudomonas putida thus lux-marked was inoculated to spinach seedlings in nursery pots, and the root colonization was assayed. In sterile soil, the strain extensively colonized the entire part of the developing roots and the population densities at any sites were high. In non-sterile soil, only the inoculated site of the roots was weakly colonized. These results suggest that the presence of the indigenous microflora is an environmental constraint on root colo...

Application of T-RFLP analysis to the study of bacterial community structure in the rhizosphere

Abstract To analyze the structure of the bacterial communities in the spinach rhizosphere, we used a terminal-restriction fragment length polymorphism (TRFLP) analysis. The T-RFLP electropherograms revealed several large peaks and smaller peaks, which were ascribed to various bacterial species composing each of the bacterial communities. It is considered that T-RFLP analysis is a convenient method for assessing the microbial diversity or community structure in the rhizosphere. Bacterial DNA from colonies on d~lution plates and directly extracted bacterial DNA of rhizosphere samples were analyzed by TRFLP in order to determine whether agar-grown colonies are representative of the dominant bacterial groups in the rhizosphere. The T-RFLP patterns obtained by using bacterial colonies from dilution plates as the source of target DNA were different from those found with the direct DNA extracts. The results indicated that many bacterial groups grown on agar media selective for the rhizosphere are not likely to be dominant in the rhizosphere.