Takeo Yamakawa

Phosphite (phosphorous acid): Fungicide, fertilizer or bio‐stimulator?

Abstract Phosphite (; Phi), a reduced form of phosphate (; Pi), is widely marketed as either a fungicide or fertilizer or sometimes as a biostimulant. This is confusing for both distributors and growers. The present paper explores data from various studies to clarify that Phi does not provide plant P nutrition and thus cannot complement or substitute Pi at any rate. In addition, Phi itself does not have any beneficial effect on the growth of healthy plants, regardless of whether it is applied alone or in combination with Pi at different ratios or different rates. The effect of Phi on plants is not consistent, but is strongly dependent on the Pi status of the plants. In most cases, the deleterious effect of Phi is evident in Pi-starved, but not Pi-sufficient, plants. Plants fertilized with Pi allowing for approximately 80–90% of its maximum growth might still be at risk of the effect. This negative effect becomes more pronounced under more seriously Pi-deficient conditions. Although a number of studies have shown positive crop responses to Phi, these responses are likely to be attributable to the suppression of plant diseases by Phi and/or to Pi formed from oxidation of Phi by microbes. In addition, indirectly providing P by Phi-to-Pi oxidation is not an effective means of supplying P to plants compared with Pi fertilizer. An understanding of these issues will aid the right selection of fertilizer as well as minimize the harmful effects of Phi use on crops.

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.

Effects of endophytic actinomycetes and Bradyrhizobium japonicum strains on growth, nodulation, nitrogen fixation and seed weight of different soybean varieties

Effects of endophytic actinomycetes and two Bradyrhizobium japonicum (Kirchner 1896) Jordan 1982 strains on growth, nodulation, nitrogen (N) fixation and seed weight of different soybean (Glycine max L. Merr.) varieties from Myanmar (Hinthada), Thailand (SJ5) and Cambodia (DT84) were studied in pot experiments in Thailand. The six treatments consisted of an uninoculated control (T1), a single inoculation with endophytic actinomycetes (Streptomyces sp. strain, P4) (T2), a single inoculation with Bradyrhizobium japonicum strains, USDA110 (T3) or THA7 (T4) and a dual inoculation of P4 with USDA110 (T5) or THA7 (T6). Sterile soil was used for cultivation of all treatments under open field conditions, using tap water for irrigation. N2 fixation activity of soybean was evaluated by the relative ureide method using xylem solute from the root bleeding sap at the early pod-fill stage. It was found that the single inoculation of P4 did not show significant effects on nodulation, N2 fixation, shoot dry weight and seed weight at harvest of all soybeans compared with the uninoculated control. In the Hinthada soybean, single inoculation of USDA110 or THA7 increased seed weight while USDA110 improved N2 fixation (P < 0.05). Dual inoculation of USDA110 and P4 showed the highest shoot N accumulation and seed weight among treatments. In the SJ5 soybean, similar trends to Hinthada were shown, although the THA7 and P4 combination showed the highest shoot N accumulation and seed weight. On the other hand, in the DT84 soybean, there were no significant differences of N2 fixation and seed weight between USDA110 and THA7 (both single and dual inoculation). In conclusion, dual inoculation of endophytic actinomycetes strain P4 may increase the nodulation and nitrogen fixation in some varieties; however, it is not effective for other varieties.

Diversity and distribution of indigenous soybean-nodulating rhizobia in the Okinawa islands, Japan

Abstract We investigated the genetic diversity and field distribution of indigenous soybean-nodulating rhizobia in the Okinawa islands, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of their 16S rDNA regions and 16S–23S rDNA internal transcribed spacer (ITS) regions. We collected rhizobia from four sites on Okinawa Island, one on Miyako Island and one on Ishigaki Island, isolating 360 strains from soybean cultivars with three Rj-genotypes, Akishirome or Bragg (non-Rj), CNS (Rj 2 Rj 3) and Hill or Fukuyutaka (Rj 4), planted with soil from each field site. Phylogenies of all isolates were analyzed by PCR-RFLP with Bradyrhizobium and Sinorhizobium USDA strains as reference strains. The PCR-RFLP analysis of 16S rDNA and the ITS regions revealed the dominance of Bradyrhizobium elkanii strains in acidic Okinawa soils, Miyako soil and Ishigaki soil, and the dominance of Sinorhizobium fredii strains in alkaline Okinawa soils. To detect phaAB genes, which are components of K+ efflux systems similar to Na+/H+ antiporters involved with alkaliphilic traits, we conducted Southern hybridization with a phaAB gene probe. Specific signals were observed in all representative S. fredii strains isolated from Okinawa alkaline soils, but not in any representative Bradyrhizobium strains.

Effect of Rj genotype and cultivation temperature on the community structure of soybean-nodulating bradyrhizobia

ABSTRACT The nodulation tendency and community structure of indigenous bradyrhizobia on Rj genotype soybean cultivars at cultivation temperatures of 33/28°C, 28/23°C, and 23/18°C for 16/8 h (day/night degrees, hours) were investigated using 780 bradyrhizobial DNA samples from an Andosol with 13 soybean cultivars of four Rj genotypes (non-Rj, Rj 2 Rj 3, Rj 4, and Rj 2 Rj 3 Rj 4). A dendrogram was constructed based on restriction fragment length polymorphism of the PCR products (PCR-RFLP) of the 16S-23S rRNA gene internal transcribed spacer region. Eleven Bradyrhizobium U.S. Department of Agriculture strains were used as a reference. The dendrogram indicated seven clusters based on similarities among the reference strains. The occupancy rate of the Bj123 cluster decreased with increasing cultivation temperature, whereas the occupancy rates of the Bj110 cluster, Be76 cluster, and Be94 cluster increased with increasing cultivation temperature. In particular, the Rj 2 Rj 3 Rj 4 genotype soybeans were infected with a number of Bj110 clusters, regardless of the increasing cultivation temperature, compared to other Rj genotype soybean cultivars. The ratio of beta diversity to gamma diversity (H′ β/H′ γ), which represents differences in the bradyrhizobial communities by pairwise comparison among cultivation temperature sets within the same soybean cultivar, indicated that the bradyrhizobial communities tended to be different among cultivation temperatures. Multidimensional scaling analysis indicated that the infection of the Bj110 cluster and the Bj123 cluster by host soybean genotype and the cultivation temperature affected the bradyrhizobial communities. These results suggested that the Rj genotypes and cultivation temperatures affected the nodulation tendency and community structures of soybean-nodulating bradyrhizobia.

Stimulation of dark carbon fixation in rice and tomato roots by application of ammonium nitrogen

Abstract Dark carbon fixation was investigated in roots of rice plants, an ammonium-tolerant species and tomato plants, an ammonium-sensitive one during periods of growth on nutrient media containing ammonium, ammonium nitrate, or nitrate as the nitrogen source. Application of ammonium nitrogen immediately stimulated the dark carbon fixation in the roots of both plants. The stimulation was greater in the plants grown on ammonium media than in those grown on ammonium nitrate media. The increase in the rates of dark carbon fixation continued in rice plants for 7 d whereas in tomato plants the rates reached the maximum value at day 1, and then gradually decreased. Most of the 14C fixed for 3 h remained in the roots: 75–90% in rice plants and 65–80% in tomato roots. Soluble fixation products were composed of amino acids and organic acids. Organic acids were more labelled than amino acids. Amino acids were more labelled in both plants grown on ammonium and ammonium nitrate media than in those grown on nitrate ...

Soybean preference for Bradyrhizobium japonicum for nodulation : Nodulation by Rj2Rj3Rj4-Genotypes isolated from the progenies of a cross between soybean cvs. IAC-2 (Rj2Rj3) and hill (Rj4)

Abstract For the increase of the occupation ratio of inoculum strain in the competition with indigenous rhizobia, the relationship between Rj-genotypes of soybean and the preference of Rj-cultivars for various types of rhizobia for nodulation was investigated by using the Rj 2 Rj 4-genotype of soybean isolated from the cross between the Rj 2 Rj 3-cultivar IAC-2 and Rj 4-one Hill (Ishizuka et al. 1993: Soil Sci. Plant Nutr., 39, 79-86). Firstly, these Rj 2 Rj 4-genotypes were found to harbor the Rj 3-gene. The Rj 2Rj3Rj4-genotypes of soybean were considered to exhibit a more narrow microsymbiont range for nodulation than the Rj 2 Rj 3-and Rj4-cultivars. Therefore, rhizobia were isolated from the nodules of various Rj-genotypes of soybeans grown in soils, and the preference of the Rj 2 Rj 3 Rj 4-genotype for indigenous rhizobia was examined. The nodule occupancy of serotype 110 was significantly higher in the bacteroids of the nodules from the Rj 2 Rj 3 Rj 4-rgenotypes than in those from the other genotypes...

Effects of nitrate application on growth, modulation, and nitrogen fixation of nitrate-tolerant mutants of soybean

Abstract Nitrate-tolerant mutants of soybean (Glycine max L. Merr.) cv. Bragg, nts1116 and nts1007, were cultured hydroponically to study the effects of nitrate on plant growth, nodulation, and nitrogen fixation compared to the parent cultivar Bragg. The vegetative growth of nts1116 was the most active. Absorption rate of nitrate increased with the duration of the treatment. The absorption rate was slightly higher in nts1116 compared to ntsl007 and Bragg. Nitrogen accumulation of the nitrate-treated plants through biological nitrogen fixation was larger in the order of ntsl007, nts1116, and Bragg. However, N accumulation from nitrate did not differ among the 3 lines. Nitrate reductase activities in the nodules were the highest in Bragg followed by nts1116 and nts1007, regardless of the nitrate treatment and assay time, and were negatively correlated with the nodule masses. Ureide concentration was the highest in nts1007 followed by nts1116 and Bragg, and was positively correlated with the nodule masses an...

Effects of phosphite, a reduced form of phosphate, on the growth and phosphorus nutrition of spinach (Spinacia oleracea L.)

Abstract Phosphite (Phi) may potentially supply phosphorus (P) nutrition to plants and is widely marketed as a super P fertilizer for many crops. This study investigated the effects of Phi on growth and P nutrition in spinach (Spinacia oleracea L.). High-rate foliar application experiments designed to evaluate the phytotoxicity and P nutritional potential of different Phi formulations by foliar application at two rates (0.15 and 0.3% P2O5) showed that all Phi formulations did not improve plant growth under different available P-soils, but rather significantly decreased shoot dry weight (DW) at the higher rate. In two other soil and hydroponic experiments, Phi was foliar applied at a low rate (0.05% P2O5) and root P treatments were combinations of Phi and phosphate (Pi) at different Pi : Phi ratios for a high P level (the soil experiment) or a low P level (the hydroponic experiment). In both experiments, shoot DW decreased markedly as the Pi : Phi ratios decreased from 100:0 to 0:100 and Phi foliar application did not improve plant growth. In the soil experiment, plants grew poorest at 0:100, but grew well when both Phi and Pi were applied at a high rate of 115 mg P pot−1, indicating that at this level Phi had a negative effect on only severely P-deficient plants. Root growth of no Pi-fertilized plants was strongly inhibited by Phi from either root or foliar application. In both experiments, P concentration drastically decreased with decreasing Pi : Phi ratios from 100:0 to 0:100, but increased substantially with foliar application of Phi compared with Pi, suggesting that Phi was absorbed poorly by the roots, but was well absorbed by the leaves compared to Pi. We conclude that Phi cannot be used as a P fertilizer for spinach via either root or foliar applications at low or high levels, and also that Phi has no beneficial effect on the growth of spinach. As Phi is now widely marketed as a P fertilizer for many crops, care should be taken in selecting Phi as a P fertilizer for a given crop.