Yasufumi Urashima

Combined Analyses of Bacterial, Fungal and Nematode Communities in Andosolic Agricultural Soils in Japan

We simultaneously examined the bacteria, fungi and nematode communities in Andosols from four agro-geographical sites in Japan using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and statistical analyses to test the effects of environmental factors including soil properties on these communities depending on geographical sites. Statistical analyses such as Principal component analysis (PCA) and Redundancy analysis (RDA) revealed that the compositions of the three soil biota communities were strongly affected by geographical sites, which were in turn strongly associated with soil characteristics such as total C (TC), total N (TN), C/N ratio and annual mean soil temperature (ST). In particular, the TC, TN and C/N ratio had stronger effects on bacterial and fungal communities than on the nematode community. Additionally, two-way cluster analysis using the combined DGGE profile also indicated that all soil samples were classified into four clusters corresponding to the four sites, showing high site specificity of soil samples, and all DNA bands were classified into four clusters, showing the coexistence of specific DGGE bands of bacteria, fungi and nematodes in Andosol fields. The results of this study suggest that geography relative to soil properties has a simultaneous impact on soil microbial and nematode community compositions. This is the first combined profile analysis of bacteria, fungi and nematodes at different sites with agricultural Andosols.

Application of PCR-Denaturing-Gradient Gel Electrophoresis (DGGE) Method to Examine Microbial Community Structure in Asparagus Fields with Growth Inhibition due to Continuous Cropping

Growth inhibition due to continuous cropping of asparagus is a major problem; the yield of asparagus in replanted fields is low compared to that in new fields, and missing plants occur among young seedlings. Although soil-borne disease and allelochemicals are considered to be involved in this effect, this is still controversial. We aimed to develop a technique for the biological field diagnosis of growth inhibition due to continuous cropping. Therefore, in this study, fungal community structure and Fusarium community structure in continuously cropped fields of asparagus were analyzed by polymerase chain reaction/denaturing-gradient gel electrophoresis (PCR-DGGE). Soil samples were collected from the Aizu region of Fukushima Prefecture, Japan. Soil samples were taken from both continuously cropped fields of asparagus with growth inhibition and healthy neighboring fields of asparagus. The soil samples were collected from the fields of 5 sets in 2008 and 4 sets in 2009. We were able to distinguish between pathogenic and non-pathogenic Fusarium by using Alfie1 and Alfie2GC as the second PCR primers and PCR-DGGE. Fungal community structure was not greatly involved in the growth inhibition of asparagus due to continuous cropping. By contrast, the band ratios of Fusarium oxysporum f. sp. asparagi in growth-inhibited fields were higher than those in neighboring healthy fields. In addition, there was a positive correlation between the band ratios of Fusarium oxysporum f. sp. asparagi and the ratios of missing asparagus plants. We showed the potential of biological field diagnosis of growth inhibition due to continuous cropping of asparagus using PCR-DGGE.

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...

Multicolor staining of root systems in pot culture

Abstract We have developed a method for staining the root systems of neighboring plants distinguishably in pot culture to facilitate studies of the interactions between plants. Pot soil was desiccated until the plant wilted, and then the shoot was cut and a dye solution (Fantasy) was pressure-injected into the roots at 0.05 MPa (gauge). All the roots, including fine roots of double-planted tomato (Lycopersicon esculentum var. Momotaro), in a 10-cm-diameter pot were well stained, but the root hairs were not. The volume of dye and the time required for staining were 6.4 mL and 5 h, respectively (root length 179 m plant−1; root fresh weight 2.62 g plant−1 on average). The root distribution of double-planted tomato in a 40 L container was well determined. The volume of dye and the time required for staining were 100 mL and 40 h, respectively (root length 1,514 m plant−1; root dry weight 4.93 g plant−1 on average). Each plants roots accounted for 37–52% of the total root weight at an intermediate position between the two plants, whereas at the other positions, the roots of either plant were dominant (82–99% of total root weight). In principle, the proposed method is applicable to whole plant species with vascular systems.

Growth Promotion of Spinach by Fluorescent Pseudomonas Strains under Application of Organic Materials

We investigated whether the colonization of spinach roots by fluorescent Pseudomonas strains was promoted by the application of organic materials. Firstly, the bioluminescence (lux) gene was introduced into fluorescent Pseudomonas strains, and the colonization of on spinach roots by fluorescent Pseudomonas strains was observed using a CCD (charge-coupled device) camera. As a result, various organic materials were found to promote the colonization of spinach roots by fluorescent Pseudomonas strains. H3-4 strain colonized the roots when oat straw and betaine monohydrate, rice husks, Japanese pampas grass and horse feces compost (with rice straw) were applied. D23-2 strain colonized the roots when hippuric acid sodium salt, Japanese pampas grass, oat straw, horse feces compost (with rice straw) and trehalose dehydrate were applied. H23-1 strain colonized the roots when cattle feces compost (with rice straw), horse feces compost (with sawdust), Japanese pampas grass, betaine monohydrate, oat straw, horse feces compost (with rice straw) and hippuric acid sodium salt were applied. The organic materials (cattle feces compost (with rice straw), horse feces compost (with sawdust), Japanese pampas grass, betaine monohydrate or horse feces compost (with rice straw)) and fluorescent Pseudomonas (HS23 strain) were applied to soil, and spinach was cultivated. The growth of spinach was promoted when fluorescent Pseudomonas strains and organic materials except for Japanese pampas grass were applied. Since the growth of spinach was not promoted when only the organic materials were applied, this growth-promoting effect was considered to be due to the fluorescent Pseudomonas strains that colonized the roots more readily when the organic materials were applied. In conclusion, the growth-promoting effect of fluorescent Pseudomonas strains was demonstrated in soil culture by the application of fluorescent Pseudomonas strains and organic materials, which promoted root colonization by fluorescent Pseudomonas strains.

Gravitational water flow enhances the colonization of spinach roots in soil by plant growth-promoting Pseudomonas

Abstract Root colonization by a plant growth-promoting Pseudomonas (H3R strain) was examined to determine whether gravitational water flow affects the bacterial distribution on spinach roots. Firstly, the effect of the amount of irrigation water or soil moisture content on the distribution of the H3R strain resistant to three antibiotics was examined using a rhizobox, Pseudomonas When a large amount of water was irrigated from the top of the rhizobox, the strain inoculated onto the spinach seed was detected in the lower part of the rhizobox. However, when water was supplied from the side of the rhizobox with a sprayer, the Pseudomonas strain could not be detected in the lower part of the rhizobox. Secondly, to visualize root colonization by the inoculated bacteria, Pseudomonas strains were marked with the lux gene. Spinach seeds were inoculated with the lux-marked Pseudomonas strains and grown in a pot. A plastic tube with a hole at the upper, middle, or lower position was buried in a pot. Water was supplied from a hole of the tube. The bacterial distribution on roots was observed based on bioluminescence. Only when water was supplied from the upper hole, did the bacteria colonize the whole root. When water was supplied from the middle or lower hole, the bacteria remained around the seeds. These results indicated that the effect of the gravitational water flow by irrigation is important for the spread of colonization of spinach roots by Pseudomonas strains.

Trends of lettuce and carrot yields and soil enzyme activities during transition from conventional to organic farming in an Andosol

Abstract It has been reported that crop yields drop and then increase during the first few years of organic farming, and these yield recoveries have been attributed to gradual improvements in soil properties, such as soil microbial activities to mineralize nitrogen (N) or to suppress plant disease. To clarify whether yield increase during organic transition is caused by improvement of soil microbial activities, we compared identically managed organic and conventional plots of 1-year lettuce (Lactuca sativa L.)–carrot (Daucus carota L.) rotation for 3 years (organic plots: first 3 years after switching from conventional to organic management; conventional plots: managed in the same way as organic plots for 3 years but receiving chemical fertilizer, fungicide, insecticide and herbicide) in an Andosol field. During organic transition, yields of organic lettuce and carrots were lower than those of conventional lettuce and carrots for only the first year. Yield drop and recovery of lettuce were thought to be caused by changes in the amount of N uptake, though yield fluctuation of carrots was mainly caused by damage from insects. Although soil enzyme activities may be responsible for N mineralization, various soil enzyme activities promptly responded to organic amendment to become higher under organic management than under conventional management even after the first lettuce cropping (6 months after switching to organic management; much shorter than the period of organic transition). However, discriminant analysis using activities of six soil enzymes (dehydrogenase, β-glucosidase, β-galactosidase, α-glucosidase, cellulase and protease) indicated that 18–24 months (a period close to that of the organic transition) were needed for the pattern of various soil enzyme activities to be in a steady state after switching to organic management. The pattern of soil enzyme activities fluctuating to a plateau during the second lettuce cropping seemed to show a tendency similar to that of N uptake and yield of lettuce during organic transition. Soil available N in organic plots also became higher than that in conventional plots in the third year. These results suggested that improved N uptake and yield of lettuce during organic transition in an Andosol might be caused by either improvement in various soil enzyme activities or accumulation of soil available N. Yield response of carrots demanding less N was attributed not to N mineralization but to damage from insects.

Inoculum effect of arbuscular mycorrhizal fungi on soybeans grown in long-term bare-fallowed field with low phosphate availability

ABSTRACT Inoculation with arbuscular mycorrhizal fungi (AMF) can increase the growth of host plants, especially under condition of low phosphate (P) availability. Although this effect is shown relatively easily in simplified systems such as pot experiments, it is often hard to show in the field because of complicating factors such as competition with indigenous AMF. We conducted an AMF inoculation experiment with three Japanese soybean cultivars (Enrei, Misuzudaizu, and Akishirome) in an allophanic (Umbric Silandic) Andosol field under the long-term selective application of major nutrients (NPK and -P) and bare fallow. In the inoculation plots, introduced AMF were well colonized in soybean roots at flowering stage. In the -P plots, inoculation tended to increase the shoot dry weight of all the three soybean cultivars; this effect remained until harvest. Although a significant difference is not recognized, there was a tendency of residual effect on Enrei in the following year. In the NPK plots, inoculation did not significantly increase the shoot dry weight. We thought that in the -P plots, the long-term selective application of N and K and the long-term maintenance of bare fallow created the soil conditions of low P availability and poor native AMF. Thus, introduced AMF can benefit soybean growth and yield in the soil with low competitor AMF density and low phosphate availability.