Growth and Yield Dynamics in Three Japanese Soybean Cultivars with Plant Growth-Promoting Pseudomonas spp. and Bradyrhizobium ottawaense Co-Inoculation.

Co-inoculation of soybeans with Bradyrhizobium and plant growth-promoting bacteria has displayed promise for enhancing plant growth, but concrete evidence of its impact on soybean yields is limited. Therefore, this study assessed the comparative efficacy of two 1-aminocyclopropane-1-carboxylate deaminase-producing Pseudomonas species (OFT2 and OFT5) co-inoculated with Bradyrhizobium ottawaense (SG09) on the growth, physiology, nodulation efficiency, and grain yield of three major Japanese soybean cultivars: Enrei, Fukuyutaka, and Satonohohoemi. The experiments were conducted in a warehouse under natural light conditions. The treatments included the inoculation of SG09, SG09 + OFT2, and SG09 + OFT5. Compared with Bradyrhizobium inoculation alone, co-inoculation led to significant improvements in nodulation efficiency, growth, and physiological performance in the Enrei and Fukuyutaka cultivars, but not in the Satonohohoemi cultivar. Furthermore, co-inoculation significantly boosted the total nitrogen content and ion uptake in the shoots, ultimately leading to a remarkable improvement in the grain yield in the Enrei and Fukuyutaka cultivars. These findings contribute to clarifying the interplay among Bradyrhizobium, Pseudomonas, and the plant host cultivar. Notably, Bradyrhizobium-Pseudomonas co-inoculation represents a potentially effective biofertilization strategy for soybean production, highlighting promising avenues for sustainable agricultural practices.

Synergistic N2-fixation and salt stress mitigation in soybean through dual inoculation of ACC deaminase-producing Pseudomonas and Bradyrhizobium.

We investigated the potential dual application of two Bradyrhizobium strains (B. diazoefficiens USDA110 and B. ottawaense SG09) and plant growth-promoting bacteria, PGPB (Pseudomonas spp.: OFT2 and OFT5), to improve nodulation and N2-fixation in soybean plants. The growth-promoting effects of dual inoculation were observed on plant growth, physiology, and nodulation of soybean under normal conditions compared with plants individually inoculated with either USDA110 or SG09. Both OFT2 and OFT5 promoted N2-fixation by 11% and 56%, respectively, when dual inoculation with USDA110 and by 76% and 81%, respectively, when dual inoculation with SG09. Salinity stress significantly reduces soybean growth, physiology, nutrient uptake, nodulation, and N2-fixation. However, these adverse effects were attenuated by the dual inoculation of PGPB and rhizobia depending on the combination of inoculants. In particular, dual inoculation of PGPB with SG09 was more effective in enhancing the salt tolerance of soybean by reducing salt-induced ethylene production and improving nutrient uptake. However, no such effect was observed with the combined inoculation of USDA110 and OFT5. An effective symbiotic association between SG09 and two Pseudomonas bacteria can be considered a beneficial approach to improving the symbiotic efficiency of nodulation and mitigating salinity stress in soybeans.

Clostridium folliculivorans sp. nov., isolated from soil samples of an organic paddy in Japan.

Two Gram-stain-negative, terminal endospore-forming, rod-shaped and aerotolerant bacterial strains designated D1-1T and B3 were isolated from soil samples of an organic paddy in Japan. Strain D1-1T grew at 15-37 °C, pH 5.0-7.3, and with up to 0.5 % (w/v) NaCl. Phylogenetic analysis of the 16S rRNA gene revealed that strain D1-1T belonged to the genus Clostridium and was closely related to Clostridium zeae CSC2T (99.7 % sequence similarity), Clostridium fungisolvens TW1T (99.7 %) and Clostridium manihotivorum CT4T (99.3 %). Strains D1-1T and B3 were whole-genome sequenced and indistinguishable, with an average nucleotide identity value of 99.7 %. The average nucleotide identity (below 91.1 %) and digital DNA-DNA hybridization (below 43.6 %) values between the two novel isolates and their corresponding relatives showed that strains D1-1T and B3 could be readily distinguished from their closely related species. A novel Clostridium species, Clostridium folliculivorans sp. nov., with type strain D1-1T (=MAFF 212477T=DSM 113523T), is proposed based on genotypic and phenotypic data.

Identification of Pseudomonas strains for the biological control of soybean red crown root rot.

Soybean red crown root rot (RCR), caused by the soil-borne fungal pathogen, Calonectria ilicicola, is the most destructive disease affecting soybean production in Japan. To date, no resistant cultivars or effective fungicides have been developed to control this disease. In this study, we evaluated 13 bacterial strains to determine their efficacy in controlling C. ilicicola. We first investigated whether the volatile organic compounds (VOCs) emitted by the bacterial strains exhibited any antifungal activity against C. ilicicola using the double-plate chamber method. The results showed that VOCs from three Pseudomonas bacterial strains, OFT2 (Pseudomonas sp.), OFT5 (Pseudomonas sp.), and Cab57 (Pseudomonas protegens), exhibited strong inhibitory activity against C. ilicicola mycelial growth. Some antifungal activity was also observed in the culture supernatants of these Pseudomonas strains. Greenhouse soil inoculation tests showed that application of OFT2, OFT5, and Cab57 cultures around soybean seeds after seed sowing significantly reduced the severity of RCR, as shown by up to 40% reduction in C. ilicicola fungal growth in the roots and 180-200% increase in shoot and root fresh weights compared to the water control. Our results suggest that OFT2, Cab57, and OFT5 produce potent antifungal compounds against C. ilicicola, thereby showing considerable potential for the biological control of C. ilicicola during soybean production.

Direct evidence for dynamics of cell heterogeneity in watercored apples: turgor-associated metabolic modifications and within-fruit water potential gradient unveiled by single-cell analyses.

Watercore is a physiological disorder in apple (Malus × domestica Borkh.) fruits that appears as water-soaked tissues adjacent to the vascular core, although there is little information on what exactly occurs at cell level in the watercored apples, particularly from the viewpoint of cell water relations. By combining picolitre pressure-probe electrospray-ionization mass spectrometry (picoPPESI-MS) with freezing point osmometry and vapor pressure osmometry, changes in cell water status and metabolisms were spatially assayed in the same fruit. In the watercored fruit, total soluble solid was lower in the watercore region than the normal outer parenchyma region, but there was no spatial difference in the osmotic potentials determined with freezing point osmometry. Importantly, a disagreement between the osmotic potentials determined with two methods has been observed in the watercore region, indicating the presence of significant volatile compounds in the cellular fluids collected. In the watercored fruit, cell turgor varied across flesh, and a steeper water potential gradient has been established from the normal outer parenchyma region to the watercore region, retaining the potential to transport water to the watercore region. Site-specific analysis using picoPPESI-MS revealed that together with a reduction in turgor, remarkable metabolic modifications through fermentation have occurred at the border, inducing greater production of watercore-related volatile compounds, such as alcohols and esters, compared with other regions. Because alcohols including ethanol have low reflection coefficients, it is very likely that these molecules would have rapidly penetrated membranes to accumulate in apoplast to fill. In addition to the water potential gradient detected here, this would physically contribute to the appearance with high tissue transparency and changes in colour differences. Therefore, it is concluded that these spatial changes in cell water relations are closely associated with watercore symptoms as well as with metabolic alterations.

Metabolic indices related to leaf marginal necrosis associated with potassium deficiency in tomato using GC/MS metabolite profiling.

To clarify the physiological factors associated with the development of tomato leaf marginal necrosis associated with potassium deficiency, tomato leaf blades prior to development of the symptoms were collected, and profiles of water-soluble metabolites were analyzed using gas chromatography-mass spectrometry. Multivariate analysis was conducted to screen for a component that was related to potassium deficiency-induced leaf necrosis among the 60 metabolites detected in tomato leaves. Polyamines, sugars, and branched amino acids were ranked highly. Putrescine was most strongly and negatively correlated with potassium concentration and exhibited an exponential response, regardless of the site or plant body. In addition to putrescine, glucose, xylose and l-isoleucine are known to play important roles in stress response and ion balance control, and it is thought that changes in metabolite profiles are linked to tomato leaf marginal necrosis associated with a decrease in endogenous potassium concentration. These findings will be useful for cultivation management to mitigate this physiological disorder of tomato.

Flavor and Texture Characteristics of 'Fuji' and Related Apple (Malus domestica L.) Cultivars, Focusing on the Rich Watercore.

Watercore is a so-called physiological disorder of apple (Malus domestica L.) that commonly occurs in several well-known cultivars. It is associated with a rapid softening of the flesh that causes a marked changed in flavor and texture. In Asia, apples with watercore are preferred and considered a delicacy because of their enhanced sweet flavor. The 'Fuji' cultivar, the first cultivar with rich watercore that is free from texture deterioration, has played a key role in the development of the market for desirable watercored apples. This review aimed to summarize and highlight recent studies related to the physiology of watercore in apples with special focus on 'Fuji' and related cultivars.

Volatile biomarkers for early-stage detection of insect-infested brown rice: Isopentenols and polysulfides.

To reduce food loss from stored products by insect attack, monitoring and early detection of insects are essential. Presently, monitoring with pheromone traps is the primary method for detection; however, traps are effective only after the insects propagate. Detection and identification of the early volatile biomarkers arising from insect-infested brown rice was performed in this study to develop an alternative detection strategy. Brown rice was infested with eggs of seven insect species, including Sitophilus zeamais and Plodia interpunctella. Infested rice emitted at least one of the volatile compounds prenol, isoprenol, dimethyl disulfide, and dimethyl trisulfide (DMTS). In particular, isopentenols were generated by moths within one week of infestation, whereas they were not released from non-infested rice. DMTS was detected from all insect-infested brown rice, especially S. zeamais and P. interpunctella. These volatiles are potential early biomarkers for the presence of insects in brown rice.

Point Analysis of Foods by Sheath-Flow Probe Electrospray Ionization/Mass Spectrometry (sfPESI/MS) Coupled with a Touch Sensor.

For quick, noninvasive, and high-sensitivity surface analysis of foods and agricultural products, a touch sensor was developed and applied to sheath-flow probe electrospray ionization/mass spectrometry (sfPESI/MS). Upon making contact with the sample, the probe stopped by detecting the current flowing through the circuit and analytes on the sample surface were extracted in the solvent preloaded in the plastic capillary. By lifting up the probe to the default position, an electrospray ionization mass spectrum of the sample was obtained. By scanning the sample stage using a programming tool, a point analysis of targeted positions of biological samples with a spot diameter of ≤0.3 mm was achieved. It took less than 10 s for one sample spot. This method was applied to various plants and animal tissues.

Component Profiling in Agricultural Applications Using an Adjustable Acupuncture Needle for Sheath-Flow Probe Electrospray Ionization/Mass Spectrometry.

In previous work, probe electrospray ionization/mass spectrometry (PESI/MS) and sheath-flow probe electrospray ionization/mass spectrometry (sfPESI/MS) were reported for the rapid and minimally invasive analysis of food. In this work, a modified version of sfPESI will be reported. The sample surface was pricked with an acupuncture needle inserted in the sfPESI probe that protruded from the terminus of the tip by 5 mm. The invasion depth of the needle into the sample was ∼1 mm. After sampling, the needle was retracted into the solvent-preloaded capillary with a protrusion length of 0.1-0.2 mm from the tip. A mass spectrum of the sample captured on the needle was obtained by applying a high voltage to the needle. This method could be applicable to profiling analyses of plants with the epicuticular wax covering on the surfaces that are difficult to analyze by sf-PESI. The on-site mass spectrometric analysis for a growing apricot in the field was performed to monitor the developing stage of the fruit.

Multiple strategies for heat adaptation to prevent chalkiness in the rice endosperm.

Heat-induced chalkiness of rice grains is a major concern for rice production, particularly with respect to climate change. Although the formation of chalkiness in the endosperm is suppressed by nitrogen, little is known about the cell-specific dynamics of this process. Here, using picolitre pressure-probe electrospray-ionization mass spectrometry together with transmission electron microscopy and turgor measurements, we examine heat-induced chalkiness in single endosperm cells of intact rice seeds produced under controlled environmental conditions. Exposure to heat stress decreased turgor pressure and increased the cytosolic accumulation of sugars, glutathione, and amino acids, particularly cysteine. Heat stress also led to a significant enlargement of the protein storage vacuoles but with little accumulation of storage proteins. Crucially, this heat-induced partial arrest of amyloplast development led to formation of chalkiness. Whilst increased nitrogen availability also resulted in increased accumulation of amino acids, there was no decrease in turgor pressure. The heat-induced accumulation of cysteine and glutathione was much less marked in the presence of nitrogen, and storage proteins were produced without chalkiness. These data provide important information on the cell dynamics of heat acclimation that underpin the formation of chalkiness in the rice endosperm. We conclude that rice seeds employ multiple strategies to mitigate the adverse effects of heat stress in a manner that is dependent on nitrogen availability, and that the regulation of protein synthesis may play a crucial role in optimizing organelle compartmentation during heat adaption.

Evaluation of the anti-hyperglycemic effect and safety of microorganism 1-deoxynojirimycin.

1-Deoxynojirimycin (DNJ) is a potent α-glucosidase inhibitor and thus beneficial for prevention of diabetes. While we have succeeded in obtaining the culture supernatant extract (CSE) rich in DNJ from microorganism source, information regarding its anti-hyperglycemic effect and safety were still limited. Therefore, this study was aimed to evaluate the anti-hyperglycemic effect and safety of microorganism DNJ. Oral sucrose tolerance test was performed, and the result showed that CSE was able to significantly suppress the blood glucose elevation and suggested DNJ as the main active compound. To determine its safety, the absorption and excretion of microorganism DNJ were evaluated using 15N labeling method. Our findings investigated the recovery rate of 15N from DNJ reached 80% up to 48 hours after oral administration, suggesting its rapid excretion, suggesting the safety of DNJ. This study verified the functional properties and safety of DNJ from microorganisms, suggesting its potential use for functional purpose.

The ACC deaminase expressing endophyte Pseudomonas spp. Enhances NaCl stress tolerance by reducing stress-related ethylene production, resulting in improved growth, photosynthetic performance, and ionic balance in tomato plants.

Plant growth promoting bacteria (PGPB) endophytes that express 1-aminocyclopropane-1-carboxylate (ACC) deaminase reportedly confer plant tolerance to abiotic stresses such as salinity by lowering stress-related ethylene levels. Two preselected ACC deaminase expressing endophytic Pseudomonas spp. strains, OFT2 and OFT5, were compared in terms of their potential to promote plant growth, leaf water contents, photosynthetic performance, and ionic balance of tomato plants under conditions of moderate NaCl stress (75 mM). Salinity stress strongly affected growth, leaf water contents, and photosynthetic performance of tomato seedlings, and inoculation with either OFT2 or OFT5 ameliorated these adverse effects. Decreases in plant biomass due to salinity stress were significant in both uninoculated control plants and in plants inoculated with OFT2 compared with plants without NaCl stress. However, no reductions in total biomass were observed in plants that were inoculated with the OFT5 strain. Strain OFT5 influenced growth, physiological status, and ionic balance of tomato plants more efficiently than strain OFT2 under NaCl stress. In particular, inoculated OFT5 reduced salt-induced ethylene production by tomato seedlings, and although it did not reduce shoot uptake of Na, it promoted shoot uptake of other macronutrients (P, K, and Mg) and micronutrients (Mn, Fe, Cu, and Zn). These nutrients may activate processes that alleviate the effects of salt, suggesting that OFT5 can be used to improve nutrient uptake and plant growth under moderate salt-affected conditions by reducing stress-related ethylene levels.

Turgor-responsive starch phosphorylation in Oryza sativa stems: A primary event of starch degradation associated with grain-filling ability.

Grain filling ability is mainly affected by the translocation of carbohydrates generated from temporarily stored stem starch in most field crops including rice (Oryza sativa L.). The partitioning of non-structural stem carbohydrates has been recognized as an important trait for raising the yield ceiling, yet we still do not fully understand how carbohydrate partitioning occurs in the stems. In this study, two rice subspecies that exhibit different patterns of non-structural stem carbohydrates partitioning, a japonica-dominant cultivar, Momiroman, and an indica-dominant cultivar, Hokuriku 193, were used as the model system to study the relationship between turgor pressure and metabolic regulation of non-structural stem carbohydrates, by combining the water status measurement with gene expression analysis and a dynamic prefixed 13C tracer analysis using a mass spectrometer. Here, we report a clear varietal difference in turgor-associated starch phosphorylation occurred at the initiation of non-structural carbohydrate partitioning. The data indicated that starch degradation in Hokuriku 193 stems occurred at full-heading, 5 days earlier than in Momiroman, contributing to greater sink filling. Gene expression analysis revealed that expression pattern of the gene encoding α-glucan, water dikinase (GWD1) was similar between two varieties, and the maximum expression level in Hokuriku 193, reached at full heading (4 DAH), was greater than in Momiroman, leading to an earlier increase in a series of amylase-related gene expression in Hokuriku 193. In both varieties, peaks in turgor pressure preceded the increases in GWD1 expression, and changes in GWD1 expression was correlated with turgor pressure. Additionally, a threshold is likely to exist for GWD1 expression to facilitate starch degradation. Taken together, these results raise the possibility that turgor-associated starch phosphorylation in cells is responsible for the metabolism that leads to starch degradation. Because the two cultivars exhibited remarkable varietal differences in the pattern of non-structural carbohydrate partitioning, our findings propose that the observed difference in grain-filling ability originated from turgor-associated regulation of starch phosphorylation in stem parenchyma cells. Further understanding of the molecular mechanism of turgor-regulation may provide a new selection criterion for breaking the yield barriers in crop production.

Sensory and metabolic profiles of "Fuji" apples (Malus domestica Borkh.) grown without synthetic agrochemicals: the role of ethylene production.

Flavors of "Fuji" apple cultivated with or without synthetic agrochemicals were compared using quantitative descriptive analyses (QDA) and metabolite profiling for 3 seasons. Experimental plots included conventional crops (with agrochemicals) and organic crops (without agrochemicals) at our institute and organic and conventional farms. Additionally, mass market samples were analyzed. Organic apples were weak in sweetness and floral characteristics and had enhanced green and sour flavors. Most esters and sugars were present in lower concentrations in organic than in conventional apples. Close relation of principal component 1 of QDA and metabolite profiles, to ethylene production suggested that ethylene is considerably involved in flavor synthesis. Reduced ethylene associated with immaturity accounted for insufficient flavor synthesis and weak aroma and flavor attributes of organic apples. Furthermore, organic apples from the farm were more flavorsome than those from the institute in 2012, suggesting possible recovery of ethylene production after a long organic cultivation period.

Metagenomic analysis of the bacterial community associated with the taproot of sugar beet.

We analyzed a metagenome of the bacterial community associated with the taproot of sugar beet (Beta vulgaris L.) in order to investigate the genes involved in plant growth-promoting traits (PGPTs), namely 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, indole acetic acid (IAA), N2 fixation, phosphate solubilization, pyrroloquinoline quinone, siderophores, and plant disease suppression as well as methanol, sucrose, and betaine utilization. The most frequently detected gene among the PGPT categories encoded ß-1,3-glucanase (18 per 10(5) reads), which plays a role in the suppression of plant diseases. Genes involved in phosphate solubilization (e.g., for quinoprotein glucose dehydrogenase), methanol utilization (e.g., for methanol dehydrogenase), siderophore production (e.g. isochorismate pyruvate lyase), and ACC deaminase were also abundant. These results suggested that such PGPTs are crucially involved in supporting the growth of sugar beet. In contrast, genes for IAA production (iaaM and ipdC) were less abundant (~1 per 10(5) reads). N2 fixation genes (nifHDK) were not detected; bacterial N2 -fixing activity was not observed in the (15)N2 -feeding experiment. An analysis of nitrogen metabolism suggested that the sugar beet microbiome mainly utilized ammonium and nitroalkane as nitrogen sources. Thus, N2 fixation and IAA production did not appear to contribute to sugar beet growth. Taxonomic assignment of this metagenome revealed the high abundance of Mesorhizobium, Bradyrhizobium, and Streptomyces, suggesting that these genera have ecologically important roles in the taproot of sugar beet. Bradyrhizobium-assigned reads in particular were found in almost all categories of dominant PGPTs with high abundance. The present study revealed the characteristic functional genes in the taproot-associated microbiome of sugar beet, and suggest the opportunity to select sugar beet growth-promoting bacteria.

A NMR-based, non-targeted multistep metabolic profiling revealed L-rhamnitol as a metabolite that characterised apples from different geographic origins.

This study utilises (1)H NMR-based metabolic profiling to characterise apples of five cultivars grown either in Japan (Fuji, Orin, and Jonagold) or New Zealand (Fuji, Jazz, and Envy). Principal component analysis (PCA) showed a clear separation between the Fuji-Orin-Jonagold class and the Jazz-Envy class, primarily corresponding to the differences in sugar signals, such as sucrose, glucose, and fructose. Multistep PCA removed the influence of dominant sugars and highlighted minor metabolites such as aspartic acid, 2-methylmalate, and an unidentified compound. These minor metabolites separated the apples into two classes according to different geographical areas. Subsequent partial least squares discriminant analysis (PLS-DA) indicated the importance of the unidentified metabolite. This metabolite was isolated using charcoal chromatography, and was identified as L-rhamnitol by 2D NMR and LC/MS analyses. The remarkable contribution of L-rhamnitol to geographic discrimination suggests that apples may be characterised according to various factors, including storage duration, cultivation method, and climate.

Rice chalky ring formation caused by temporal reduction in starch biosynthesis during osmotic adjustment under foehn-induced dry wind.

Foehn-like extreme hot and dry wind conditions (34°C, >2.5 kPa vapor pressure deficit, and 7 m s(-1)) strongly affect grain quality in rice (Oryza sativa L.). This is a current concern because of the increasing frequency and intensity of combined heat and water-deficit stress under climate change. Foehn-induced dry wind conditions during the grain-filling stage increase ring-shaped chalkiness as a result of spatiotemporal reduction in starch accumulation in the endosperm, but kernel growth is sometimes maintained by osmotic adjustment. Here, we assess the effects of dry wind on chalky ring formation in environmentally controlled growth chambers. Our results showed that hot and dry wind conditions that lasted for >24 h dramatically increased chalky ring formation. Hot and dry wind conditions temporarily reduced panicle water potential to -0.65 MPa; however, kernel growth was maintained by osmotic adjustment at control levels with increased transport of assimilate to the growing kernels. Dynamic tracer analysis with a nano-electrospray-ionization Orbitrap mass spectrometer and quantitative polymerase chain reaction analysis revealed that starch degradation was negligible in the short-term treatment. Overall expression of starch synthesis-related genes was found to be down-regulated at moderately low water potential. Because the events observed at low water potential preceded the packing of starch granules in cells, we concluded that reduced rates of starch biosynthesis play a central role in the events of cellular metabolism that are altered at osmotic adjustment, which leads to chalky ring formation under short-term hot and dry wind conditions.

Expressed nifH Genes of Endophytic Bacteria Detected in Field-Grown Sweet Potatoes (Ipomoea batatas L.).

We examined the nitrogenase reductase (nifH) genes of endophytic diazotrophic bacteria expressed in field-grown sweet potatoes (Ipomoea batatas L.) by reverse transcription (RT)-PCR. Gene fragments corresponding to nifH were amplified from mRNA obtained from the stems and storage roots of field-grown sweet potatoes several months after planting. Sequence analysis revealed that these clones were homologous to the nifH sequences of Bradyrhizobium, Pelomonas, and Bacillus sp. in the DNA database. Investigation of the nifH genes amplified from the genomic DNA extracted from these sweet potatoes also showed high similarity to various α-proteobacteria including Bradyrhizobium, ß-proteobacteria, and cyanobacteria. These results suggest that bradyrhizobia colonize and express nifH genes not only in the root nodules of leguminous plants but also in sweet potatoes as diazotrophic endophytes.