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.

Rhizosphere frame system enables nondestructive live-imaging of legume-rhizobium interactions in the soil.

Most plants interact with various soil microorganisms as they grow through the soil. Root nodule symbiosis by legumes and rhizobia is a well-known phenomenon of plant-microbe interactions in the soil. Although microscopic observations are useful for understanding the infection processes of rhizobia, nondestructive observation methods have not been established for monitoring interactions between rhizobia and soil-grown roots. In this study, we constructed Bradyrhizobium diazoefficiens strains that constitutively express different fluorescent proteins, which allows identification of tagged rhizobia by the type of fluorophores. In addition, we constructed a plant cultivation device, Rhizosphere Frame (RhizoFrame), which is a soil-filled container made of transparent acrylic plates that allows observation of roots growing along the acrylic plates. Combining fluorescent rhizobia with RhizoFrame, we established a live imaging system, RhizoFrame system, that enabled us to track the nodulation processes with fluorescence stereomicroscope while retaining spatial information about roots, rhizobia, and soil. Mixed inoculation with different fluorescent rhizobia using RhizoFrame enabled the visualization of mixed infection of a single nodule with two strains. In addition, observation of transgenic Lotus japonicus expressing auxin-responsive reporter genes indicated that RhizoFrame system could be used for a real-time and nondestructive reporter assay. Thus, the use of RhizoFrame system is expected to enhance the study of the spatiotemporal dynamics of plant-microbe interactions in the soil.

Seed phytic acid concentration affects rice seedling vigor irrespective of soil phosphorus bioavailability.

Rice with a black-colored pericarp (hereafter, black rice) is well-known as an antioxidant-rich food, but a high grain phytic acid (PA) concentration affects its nutritional quality. However, phytic acid helps improve seedling vigor, which is crucial for enhancing subsequent plant growth. This study investigated the effect of seed phytic acid concentration in black rice on seedling vigor compared to the effects on white rice. In the first experiment, three phytic acid concentrations in the seeds of black rice, low (LPA, 15.5 mg g-1 per seed), medium (MPA, 24.7 mg g-1 per seed), and high (HPA, 35.4 mg g-1 per seed) were tested for seedling vigor in phosphorus-deficient soils. The HPA seedlings showed substantially increased seedling vigor and shoot P uptake due to early root development and enhanced physiological processes. LPA grown seedlings showed increased ethylene production in response to P stress, which is the main physiological mechanism modulating seedling growth under P stress conditions. In the second experiment, the three phytic acid concentrations in black and white rice seeds were tested under low and high soil P conditions. Again, LPA seedlings showed significantly reduced seedling vigor in both rice varieties in P-deficient soils. Interestingly, seed phytic acid and external P application had an additive effect on seedling vigor, suggesting that the combined effect further improved seedling growth. Our results reveal that black rice seeds with a HPA concentration can be used as a seed source for planting in P-deficient ecosystems for rice plants as they can increase seedling vigor and subsequent growth, thus reducing dependence on finite P resources.

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.

A fresh weight-based method for evaluating soybean resistance to red crown rot.

Soybean red crown rot (RCR) caused by Calonectria ilicicola is a serious soil-borne disease affecting soybean production and quality. The current visual necrosis-based method for the measurement of RCR severity is prone to subjectivity as well as time consuming and laborious as it requires digging out and washing the roots to remove adhering soil prior to the visual scoring. Using cultivar Enrei, we show that, upon C. ilicicola infection, relative fresh weights (RFW; fresh weights relative to non-inoculated control plants) showed a significant negative correlation with visual RCR severity in apical shoot (trifoliate and above, R2 = 0.96), shoot (unifoliate and above, R2 = 0.82) and roots (R2 = 0.89). Furthermore, apical shoot RFW efficiently correlated with varying levels of C. ilicicola resistance in two test sets containing 37 soybean cultivars and three wild soybean accessions, exhibiting a significant correlation with visual severity (R2 = 0.72 and 0.79, p < 0.01). Taken together, our results suggest that RFW can serve as an index of soybean RCR severity, providing a simple, rapid, consistent, and cost-effective method for evaluating C. ilicicola resistance in soybeans.

Soybean Hypocotyls Prevent Calonectria ilicicola Invasion by Multi-Layered Defenses.

In plants, many pathogens infect a specific set of host organs to cause disease, yet the underlying mechanisms remain unclear. Here, we show that inoculation of soybean plants with Calonectria ilicicola, the soil-borne causal agent of soybean red crown rot, caused typical disease symptoms of root rot and leaf chlorosis and necrosis. However, the pathogen DNA was only detected in the roots and stem (hypocotyl) base but not other aerial parts of the plants. As we observed vigorous fungal growth in all culture media made of extracts from roots, stems, and leaves, differences in key components including available nutrients did not determine organ-specific infection and reproduction by C. ilicicola. Furthermore, inoculation of stems both with and without a surface wound showed that the stems resisted C. ilicicola infection via both the pre- and post-invasion defense layers. Transcriptomic comparison of roots and stems using RNA-seq analysis further revealed that upon C. ilicicola inoculation, a greater expression of genes involved in stress response was induced in the plant stems, including receptor-like kinase, AP2/ERF, MYB, and WRKY. In addition, pathways related to amino acid metabolism were also more upregulated in the stems in response to C. ilicicola infection. These results suggest that soybean stems provide C. ilicicola resistance, at least in part, by activating an organ-specific defense response.

Influence of rice-husk biochar and Bacillus pumilus strain TUAT-1 on yield, biomass production, and nutrient uptake in two forage rice genotypes.

Biochar is widely used as a soil amendment to increase crop yields. However, the impact of the interaction between the biochar and microbial inoculants (e.g., biofertilizer) on plant nutrient uptake and yield in forage rice is not fully understood. A greenhouse study was conducted to evaluate the synergistic effects of rice-husk biochar and Bacillus pumilus strain TUAT-1 biofertilizer application on growth, yield, and nutrient uptake in two forage rice genotypes; Fukuhibiki and the newly bred line, LTAT-29. Positive effects of biochar and biofertilizer, alone or in a combination, on growth traits, nutrient uptake, and yield components were dependent on the rice genotypes. Biochar and TUAT-1 biofertilizer influenced the overall growth of plants positively and increased straw and above-ground biomass in both genotypes. However, although biochar application significantly increased grain yield in LTAT-29, this was not the case in Fukuhibiki. Biochar and TUAT-1 biofertilizer, either alone or combined, significantly affected plant nutrient uptake but the effect largely depended on rice genotype. Results of this study indicate that biochar amendment and TUAT-1 biofertilizer can enhance forage rice productivity depending on genotypes, and therefore, there is a need to consider plant genetic composition when evaluating the potential for crop response to these soil amendments before application on a commercial scale.

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.

Influence of pruning waste biochar and oyster shell on N2O and CO2 emissions from Japanese pear orchard soil.

Two incubation experiments were conducted under controlled moisture and temperature conditions to determine the effects of soil amendment treatments based on pruning waste biochar and oyster shell, on N2O and CO2 emissions from an orchard soil. In experiment 1, four treatments were tested including, control (CK), pruning waste biochar at 2% (B2%), at 10% (B10%), and oyster shell (OS), mixed with soil from two different depths, namely, from the 0-5 cm and the 0-10 cm layers. In experiment 2, only the 0-10 cm soil layer was used to study the effect of surface application of pruning waste biochar (B2% and B10%) on soil N2O and CO2 emissions. The results showed that soil pH, total C and C: N ratio increased with biochar amendment treatments. Significant reduction in soil NO3- content was observed for the B10% treatment. Although OS application increased soil pH, no effect was observed on soil mineral N content, total C or C: N ratio. The rate of N2O emissions from the 0-5 cm soil layer after B2% and B10% addition, significantly declined by 12.5% and 26.3%, respectively. However, only the B10% treatment caused significant reduction in N2O emissions from the 0-10 cm soil layer and from surface soil, by 15.1% and 13.8%, respectively. Oyster shell application had no effect on either soil N2O or CO2 emissions from either soil layer tested. Our results suggest that the addition of pruning waste biochar at a high rate has the potential to mitigate N2O emissions from orchard soils; while, oyster shell can be used for liming without altering soil N2O nor CO2 emissions.

Effect of dolomite and biochar addition on N2O and CO2 emissions from acidic tea field soil.

A laboratory study was conducted to study the effects of liming and different biochar amendments on N2O and CO2 emissions from acidic tea field soil. The first experiment was done with three different rates of N treatment; N 300 (300 kg N ha-1), N 600 (600 kg N ha-1) and N 900 (900 kg N ha-1) and four different rates of bamboo biochar amendment; 0%, 0.5%, 1% and 2% biochar. The second experiment was done with three different biochars at a rate of 2% (rice husk, sawdust, and bamboo) and a control and lime treatment (dolomite) and control at two moisture levels (50% and 90% water filled pore space (WFPS)). The results showed that dolomite and biochar amendment significantly increased soil pH. However, only biochar amendment showed a significant increase in total carbon (C), C/N (the ratio of total carbon and total nitrogen), and C/IN ratio (the ratio of total carbon and inorganic nitrogen) at the end of incubation. Reduction in soil NO3--N concentration was observed under different biochar amendments. Bamboo biochar with the rates of 0.5, 1 and 2% reduced cumulative N2O emission by 38%, 48% and 61%, respectively, compare to the control soil in experiment 1. Dolomite and biochar, either alone or combined significantly reduced cumulative N2O emission by 4.6% to 32.7% in experiment 2. Reduction in N2O production under biochar amendment was due to increases in soil pH and decreases in the magnitude of mineral-N in soil. Although, both dolomite and biochar increased cumulative CO2 emission, only biochar amendment had a significant effect. The present study suggests that application of dolomite and biochar to acidic tea field soil can mitigate N2O emissions.

Influence of soil types and osmotic pressure on growth and 137Cs accumulation in blackgram (Vigna mungo L.).

A pot experiment was conducted to study the effects of soil types and osmotic levels on growth and 137Cs accumulation in two blackgram varieties differing in salinity tolerance grown in Fukushima contaminated soils. The contamination levels of the sandy clay loam and clay soil were 1084 and 2046 Bq kg-1 DW, respectively. The 137Cs activity was higher in both plants grown on the sandy clay loam than on the clay soil regardless of soil 137Cs activity concentration. No significant differences were observed in all measured growth parameters between the two varieties under optimal water conditions for both types of soil. However, the growth, leaf water contents and 137Cs activity concentrations in both plants were lower in both soil types when there was water stress induced by addition of polyethylene glycol. Water stress-induced reduction in total leaf area and total biomass, in addition to leaf relative water content, were higher in salt sensitive 'Mut Pe Khaing To' than in salt tolerant 'U-Taung-2' plants for both soil types. Varietal difference in decreased 137Cs uptake under water stress was statically significant in the sandy clay loam soil, however, it was not in the clay soil. The transfer of 137Cs from soil to plants (i.e., root, stem and leaf) was higher for the sandy clay loam for both plants when compared with those of the clay soil. The decreased activity of 137Cs in the above ground samples (leaf and stem) in both plants in response to osmotic stress suggested that plant available 137Cs decreased when soil water is limited by osmotic stress.

Genotypic difference in (137)Cs accumulation and transfer from the contaminated field in Fukushima to azuki bean (Vigna angularis).

The screening of mini-core collection of azuki bean accessions (Vigna angularis (Willd.) Ohwi & Ohashi) for comparative uptake of (137)Cs in their edible portions was done in field trials on land contaminated by the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident. Ninety seven azuki bean accessions including their wild relatives from a Japanese gene bank, were grown in a field in the Fukushima prefecture, which is located approximately 51 km north of FDNPP. The contamination level of the soil was 3665 ± 480 Bq kg(-1) dry weight ((137)Cs, average ± SD). The soil type comprised clay loam, where the sand: silt: clay proportion was 42:21:37. There was a significant varietal difference in the biomass production, radiocaesium accumulation and transfer factor (TF) of radiocaesium from the soil to edible portion. Under identical agricultural practice, the extent of (137)Cs accumulation by seeds differed between the accessions by as much as 10-fold. Inter-varietal variation was expressed at the ratio of the maximum to minimum observed (137)Cs transfer factor for seeds ranged from 0.092 to 0.009. The total biomass, time to flowering and maturity, and seed yield had negative relationship to (137)Cs activity concentration in seeds. The results suggest that certain variety/varieties of azuki bean which accumulated less (137)Cs in edible portion with preferable agronomic traits are suitable to reduce the (137)Cs accumulation in food chain on contaminated land.

Changes in hydraulic conductance cause the difference in growth response to short-term salt stress between salt-tolerant and -sensitive black gram (Vigna mungo) varieties.

Black gram (Vigna mungo) is an important crop in Asia, However, most black gram varieties are salt-sensitive. The causes of varietal differences in salt-induced growth reduction between two black gram varieties, 'U-Taung-2' (salt-tolerant; BT) and 'Mut Pe Khaing To' (salt-sensitive; BS), were examined the potential for the first step toward the genetic improvement of salt tolerance. Seedlings grown in vermiculite irrigated with full-strength Hoagland solution were treated with 0mM NaCl (control) or 225 mM NaCl for up to 10 days. In the 225 mM NaCl treatment, plant growth rate, net assimilation rate, mean leaf area, leaf water potential, and leaf photosynthesis were reduced more in BS than in BT plants. Leaf water potential was closely related to leaf photosynthesis, net assimilation rate, and increase in leaf area. In response to salinity stress, hydraulic conductance of the root, stem, and petiole decreased more strongly in BS than in BT plants. The reduction in stem and petiole hydraulic conductance was caused by cavitation, whereas the reduction in root hydraulic conductance in BS plants was caused by a reduction in root surface area and hydraulic conductivity. We conclude that the different reduction in hydraulic conductance is a cause of the differences in the growth response between the two black gram varieties under short-term salt stress.

Within field spatial variation in methane emissions from lowland rice in Myanmar.

An assessment of within field spatial variations in grain yield and methane (CH4) emission was conducted in lowland rice fields of Myanmar. Two successive rice fields (1(st) field and 2(nd) field) were divided into fertilized and non-fertilized parts and CH4 measurements were conducted at the inlet, middle and outlet positions of each field. The results showed that CH4 emissions at non-fertilized parts were higher than those at fertilized part in both rice fields. The average CH4 emissions ranged from 8.7 to 26.6 mg m(-2) h(-1) in all positions in both rice fields. The spatial variation in CH4 emission among the positions was high in both rice fields with the highest emissions in the outlet of the 1(st) field and the inlet of the 2(nd) field. The CH4 emissions at these two positions showed 2 - 2.5 times higher than those at other positions in both rice fields. Stepwise regression analysis indicates that soil total carbon content is the primary factor for CH4 emission. The average CH4 emissions during rice growing season were 13.5 mg m(-2) h(-1) for the 1(st) field and 15.7 mg m(-2) h(-1) for the 2(nd) field. Spearman rank order correlation analysis showed that CH4 emission was significantly and positively correlated with soil temperature, surface water depth and negatively correlated with soil redox potential. The result indicated that high within field spatial variation in CH4 emissions required different site specific management practices to mitigate CH4 emissions in lowland paddy rice soil.