Kazuhira Yokoyama

Effect of Inorganic N Composition of Fertilizers on Nitrous Oxide Emission Associated with Nitrification and Denitrification

We studied the effect of repeated application (once every 2 d) of a fertilizer solution with different ratios of NH4 + - and NO3 −-N on N2O emission from soil. After the excess fertilizer solution was drained from soil, the water content of soil was adjusted to 50% of the maximum water-holding capacity by suction at 6 × 103 Pa. Repeated application of NH4 +- rich fertilizer solution stimulated nitrification in soil more than NO3 −-rich fertilizer. Although the evolution of N2O through nitrifier denitrification tended to increase with the repeated addition of a fertilizer solution rich in NH4 + rather than in NO3 −, the contribution of nitrifier denitrification remained at levels of 20 to 36% of the total emission regardless of the inorganic N composition. The total emission of N2O also tended to increase with the application of NH4 +- rather than NO3 −-rich fertilizer. It was suggested that the coupled process of nitrification and denitrification at micro-aerobic sites became important when fertilizer rich in NH4 + was applied to soil under relatively aerobic conditions.

Effect of peat on mycorrhizal colonization and effectiveness of the arbuscular mycorrhizal fungus Gigaspora margarita

Abstract The influence of the addition of Chinese peat and Canadian peat on arbuscular mycorrhizal colonization, mycorrhizal effectiveness and host-plant growth was investigated in a pot experiment. Chinese peat or Canadian peat was mixed with Masa soil (weathered granite soil) at different levels (0, 25, 50, 100, 150 or 200 g kg−1) into which an arbuscular mycorrhizal fungus (AMF) Gigaspora margarita Becker & Hall was inoculated, and seedlings of Miscanthus sinensis Anderess were planted. There was a significant increase in plant growth with increasing amounts of Chinese peat. The growth-promoting effect of the AMF on the host was enhanced when the addition of Chinese peat was increased from 25 to 100 g kg−1. Root colonization and the number of spores proliferating increased with increases at low levels of Chinese peat (from 25 to 100 g kg−1), and decreased gradually with higher Chinese peat increments. Although plant growth and root colonization with the addition of Canadian peat increased slightly, Canadian peat suppressed mycorrhizal effectiveness. In contrast to Canadian peat, the addition of Chinese peat improved considerably the physical and chemical properties of the soil, which might result in the promotion of AM formation and mycorrhizal effectiveness.

Stimulatory effect of peat on spore germination and hyphal growth of arbuscular mycorrhizal fungus Gigaspora margarita

Abstract The effects of peat extract solutions on spore germination and hyphal growth of the arbuscular mycorrhizal fungus Gigaspora margarita Becker & Hall were investigated. Spores were incubated in extract solutions of peat from China and peat moss from Canada, which were prepared at a weight ratio of 1:10, 1:20, 1:30, 1:50 and 1:100 for each substrate according to the weight ratio of water. Compared with peat and peat moss solutions, solutions of KH2PO4 and (NH4)2SO4 at various concentrations were used as media to investigate the effect of phosphorus (P) and nitrogen (N). Spores in the peat solutions showed the highest values for germination percentage, hyphal length and number of auxiliary cells, and these values were significantly higher than the values recorded for the other treatments. In contrast, in the peat moss extract solutions, which had a lower content of N and higher content of P than the peat solutions, the germination and hyphal growth of spores decreased. Spore growth was promoted with increased concentrations of the N solutions. However, P solutions exerted an inhibitory effect on spore germination and hyphal growth. Peat solutions showed a high N content and low P content, which might be related to the promotion of spore growth.

Estimation of mycorrhizal colonization of the roots of oak seedlings inoculated with an ectomycorrhizal fungus, Laccaria amethystea

Abstract Six-month-old seedlings of Quercus serrata and Quercus glauca in a nursery were inoculated with the ectomycorrhizal fungus Laccaria amethystea encapsulated in alginate gel and grown in the nursery. The seedlings were collected at 1, 3, and 5 months after the inoculation and examined for colonization of the root system with ectomycorrhizal fungi. The roots within 5 months after the inoculation showed rudimentary ectomycorrhizal colonization. The level of colonization of the root system was estimated based on the intensity of hyphal covering on the root tips by staining with a fluorescent dye and expressed as an index of mycorrhizal colonization (IMC). IMC increased with the time after inoculation and reached values of 4 and 12% in Q. serrata and Q. glauca, respectively at 5 months after the inoculation. The determination of IMC may enable to assess the development of mycorrhizal colonization of the root system that shows rudimentary ectomycorrhizas after the inoculation.

Promotion of host plant growth and infection of roots with arbuscular mycorrhizal fungus Gigaspora margarita by the application of peat

Abstract The influence of the addition of peat on arbuscular mycorrhizal formation and host plant growth was investigated using a pot experiment. Peat was mixed with Masa soil at different levels (0, 25, 50, 100, 150, 200 g kg−1) into which an arbuscular mycorrhizal fungus (AMF) Gigaspora margarita Becker & Hall was inoculated, and seedlings of Miscanthus sinensis Anderess were planted. There was a significant increase in plant growth with increasing levels of peat. Root colonization and the number of proliferating spores increased with increasing levels of peat. By decreasing the bulk density, increasing the maximum water-holding capacity and the content of total nitrogen, peat addition considerably improved the physical and chemical properties of the soil, which might result in the promotion of plant growth and AMF activity.

Multiple occupancy of nodules by nodulating rhizobia on field-grown soybeans with attendance of Sinorhizobium spp.

Abstract This study analyzed the phenotypic and genotypic characters of nodulating rhizobia isolated from two soybean cultivars, Kyushu 151 and Sachiyutaka, in the same field of the Yamaguchi Prefectural Technology Center of Agriculture and Forestry in Japan. The isolates were classified into groups using phenotypic characteristics, such as growth rate, color change on Bromothymol blue-containing yeast extract-mannitol agar (YMA) plates and colony morphology on YMA plates, and by genotypic characteristics, such as polymerase chain reaction–restriction fragment length polymorphism patterns of the 16S ribosomal RNA genes (16S rDNA) and the internal transcribed sequence (ITS) regions. In Kyushu 151, single phenotypic and genotypic groups were isolated from every nodule examined. In Sachiyutaka, plural strains belonging to distinct groups were obtained frequently from single nodules, indicating that multiple occupancy was established at high frequency. No fixed combination of the groups was found in the composition of multiple occupancy. An increase in the relative abundance of isolates belonging to Sinorhizobium fredii (Ensifer fredii) occurred concomitantly with the increase in the proportion of nodules with multiple occupancy. Nearly 60% of the isolates from Sachiyutaka belonged to S. fredii; 75% of them were obtained from nodules with multiple occupancy.

Application of a Molecular Method for the Identification of a Gigaspora margarita Isolate Released in a Field

A molecular technique for the identification of the Gigaspora margarita isolate CK based on the detection of a DNA sequence of 235 bp as its diagnostic marker was evaluated to investigate the survival and establishment of introduced arbuscular mycorrizal fungi (AMF) in a field ecosystem. In March 2001, roots and rhizosphere soil of Eragrostis curvula and Miscanthus sinensis were collected from the Mizunashi River at Mt. Fugendake (Nagasaki Prefecture, Japan), where plant seeds and AMF including G. margarita CK had been introduced for reforestation after the occurrence of repeated pyroclastic flows. We detected the marker sequence from DNA preparations of E. curvula roots and Gigaspora spores in the rhizosphere. This clearly showed that the isolate occurred at both hyphal and sporal stages. It was shown that the isolate survived and developed a life cycle in the revegetation area for 4 years. It was confirmed that the method was effective for tracing the isolate in samples collected from field ecosystems.

Characterization of denitrification and net N2O-reduction properties of novel aerobically N2O-reducing bacteria

ABSTRACT Nitrous oxide, one of the earth-warming and ozone-destructing gases, is produced through either nitrification or denitrification depending on the O2 availability in soil. Aerobically denitrifying bacteria express denitrification tract even under the gas phase containing O2 at the ambient air level. The net reduction of exogenous N2O by novel aerobically denitrifying bacteria were studied. We carried out two different isolation strategies in the primary screening. One was to select isolates of interest out of periplasmic nitrate reductase-dependent denitrifying bacteria in a eutrophic condition. The other was to use diluted nutrient agar to allow the formation of colonies of diverse bacteria. Among aerobically denitrifying bacteria, those which showed net aerobic N2O reduction were only minor populations. As a result, eight isolates belonging to Proteobacteria were obtained from soil and cow manure. The denitrification and net N2O reduction properties of the three representative isolates, Pseudomonas sp. CM1, Thauera sp. PM2 and Paracoccus denitrificans 96, were determined separately by the acetylene inhibition method after exposure to aerobic or low O2 conditions, a 24 h starvation prior to the determination of the aerobic activity and inoculation to a cow manure-amended sterile soil. The phenotype inversion from net N2O-reducing to N2O-emitting, and vice versa, attested to the fact that activity of the N2O-producing and -reducing steps changed in different intensities to each other. The activity values and the direction of activity changes varied among the isolates. When they were inoculated in a sterilized soil microcosm at 40% maximum water holding capacity, the denitrification and the N2O-reducing activities were comparable with or, in some cases, facilitated more than those determined under the low-O2 condition. It is possible that these isolates sensed the O2 deficiency even in such a relatively dry condition. Pseudomonas sp. CM1 was unique because it lacked nitrate reducing activity and acted as a net aerobic N2O reducer.

A method for isolation of soil microbial DNA that is suitable for analysis of microbial cellulase genes

ABSTRACT We developed a method for isolation of DNA of cellulolytic microbies from soil, so that DNA sample was free from soil organic matter (SOM). The method consists of three procedures: propagation of cellulolytic microbes, separation of microbial cells from SOM, and DNA extraction using cetyl trimethyl-ammonium bromide (CTAB). Differential centrifugation with Ficoll-PM400 successfully separated the cells from soil particles and SOM. From 1 g of soil, 0.12-0.51 μg of microbial DNA with an average size of 15-20 kbp was isolated. Denaturing Gradient Gel Electrophoresis showed that DNA from a variety of cellulolytic microbes was recovered.