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Dynamics of symbiotic relationship of soybean with Bradyrhizobium diazoefficiens and involvements of root-secreted daidzein behind the continuous cropping
European Journal of Soil Biology 93 (2019) 103098
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European Journal of Soil Biology journal homepage: www.elsevier.com/locate/ejsobi
Dynamics of symbiotic relationship of soybean with Bradyrhizobium diazoefficiens and involvements of root-secreted daidzein behind the continuous cropping
T
Clarissien Ramongolalainaa,b,∗ a
Department of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto, 606-8502, Japan Department of Life Science Frontiers, Center for iPS Cell Research and Application, Graduate School of Medicine, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
b
A R T I C LE I N FO
A B S T R A C T
Handling editor: Bryan Griffiths
Symbiosis with a selectively nodulating rhizobium strain, such as Bradyrhizobium diazoefficiens USDA110, improves soybean productivity. In continuous cropping, however, the concentrations of root exudates – of which daidzein is one of the major secretions – might influence nodulating rhizobium communities and cause ineffective nitrogen fixation in soybean. The dynamics of indigenous rhizobia that form nodules with the soybean cultivars Tamahomare, Enrei and Tamabaguro at three cycles of a continuous cropping plot under different experimental conditions were examined. PCR-RFLP analysis targeted to the 16S-23S rRNA gene internal transcribed spacer (ITS) region of a bacterium, confirmed by sequencing analysis, was used to determine the bacterial type for each root nodule. The results showed that as the number of cycles increased, the proportion of B. diazoefficiens USDA110 of all cultivars decreased. Under all experimental conditions, the average differences between the proportion of B. diazoefficiens USDA110 of the first and third cycles in Tamahomare, Enrei and Tamabaguro were 67%, 70% and 44%, respectively. After the HPLC analysis of the root-secreted isoflavonoids, the results showed that the root-secreted daidzein from Tambaguro was excreted in the highest levels among the three cultivars. Increase of the proportion of B. diazoefficiens USDA110 among seedlings treated with daidzein was also noted. Therefore, the present study reports that the symbiotic relationship of soybean with B. diazoefficiens USDA110 decreases in continuous cropping and suggests that root-secreted daidzein helps to maintain populations of this rhizobial strain in rhizosphere of some cultivars.
Keywords: Symbiotic relationship Soil bacteria Bradyrhizobium diazoefficiens USDA110 Soybean continuous cropping Root-secreted daidzein
1. Introduction Soybean is cultivated in many regions around the world because of its great health and economic benefits. For climatic and other technical reasons, soybean is commonly grown in a continuous monoculture, resulting in yield decline and quality deterioration [1]. The reduced yield of soybean in continuous cultivation is mainly attributed to intraspecific allelopathy and the buildup of pathogens and other pests [1,2]. Notwithstanding, alterations in microbial communities, especially beneficial microbes, may also be important and require further investigation. In continuous cropping systems, microbial communities are continuously exposed to the roots of the same crop that selects and enriches certain groups of microorganisms – including yield-debilitating populations (i.e., soilborne pathogens) of that crop [3]. In the field of soybean crop, the most important legume crop in the world, soil
rhizobia are one of the main actors improve soybean productivity and soil fertility [4–7]. So far, scarcely ever are articles focused on soil rhizobia under continuous cropping of soybean. At this point, it might be still wondering, is this alteration of soil rhizobia worth attention in term of soybean cultivation? But, Sugiyama and colleagues [8] demonstrated that bacterial communities – including rhizobial community – of the soybean rhizosphere alter even during growth in the field. Taking into account these few previous reports, it is only appropriate next to discover the effects of the continuous cropping of soybean on nodulating rhizobia. The concentrations of root exudates in the soybean rhizosphere include phenolic acids, and isoflavonoids (daidzein and genistein) might be critical factors influencing changes in microbial communities [9–11]. Daidzein and genistein are known to be the inducers of nod gene expression; the inducers initiate the establishment of legume-
∗ Corresponding author. Department of Life Science Frontiers, Center for iPS Cell Research and Application, Graduate School of Medicine, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan. E-mail address: [email protected].
https://doi.org/10.1016/j.ejsobi.2019.103098 Received 12 March 2019; Received in revised form 11 June 2019; Accepted 12 June 2019 Available online 28 June 2019 1164-5563/ © 2019 Elsevier Masson SAS. All rights reserved.
European Journal of Soil Biology 93 (2019) 103098
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2
Water Tm, Enr, Tb 12 nodules
Number of cycles Treatment of soil medium after each cycle Watering supplement Used genotypes Harvested organs per seedling
Exp-2
Table. 1 Particularities of each independent experiment
2.1.1. First and second sets of experiments (Exp-1 and Exp-2) Exp-1 and Exp-2, primarily aimed to investigate dynamics of symbiotic relationship between soil rhizobia and soybean plants successively cultivated in real field under different temperature conditions, were carried out in incubators set at 28 °C with an 18/6 h photoperiod, and automatically set – the settings were being unable to adjust – at 30 °C with a 20/4 h photoperiod, respectively. Surface-sterilized soybean seeds were sown in a pot filled with field soil (90%) mixed, to aerate the soil medium, with S-sized pumice Kanuma soil (10%); this Kanuma soil was purchased from Tachikawa-heiwa Nouen Co., LTD., Japan. The field soil samples were taken from a Kyoto University experimental farm field – rotated between soybean field and paddy field every year – after either rice or soybean harvest. The seedlings were watered every 2 days. The seedlings were harvested at the third to fourth trifoliolate stage (V3 – V4; 22–23 days after sowing) and 12 very young nodules per plant were sampled from four seedlings. Once the seedlings of the first cycle were harvested, the soil substrate was mixed and put back – without further treatments – to the same pot, and the soybean seeds of the same cultivar were grown in this substrate 3–4 days later. After harvesting the seedlings from the second cycle at the fourth trifoliolate stage, the same approach was undertaken for the third cycle.
Tm, Enr, Tb: Tamahomare, Enrei, Tambaguro Tm, Enr, Tb + 7 others: Tamahomare, Enrei, Tambaguro, Peking, Shintambaguro, Fukuyutaka, William 82, Norin 2, Tachinagaha and Iyodaizu
Water Tm, Enr, Tb 12 nodules
Rice field soil (90%) + Kanuma soil (10%) 3 Mixing
2.1. Successive cultivation of soybean
Water Tm, Enr, Tb 12 nodules
Rice field soil (90%) + Kanuma soil (10%) 3 Mixing Soybean field soil (90%) + Kanuma soil (10%) 3 Mixing
28°C and 18/6 h photoperiod
Conditions (incubator settings) Original soil medium
2. Method
Three sets of experiments were performed simultaneously to simulate successive cultivation using three soybean cultivars well-known in Japan: Tamahomare, Tambaguro, and Enrei. Using culture pots (Ø = 15 cm, H = 12 cm), the experiments were performed in three different incubators; the particularities of each experiment were summarized in Table 1. Of note, in all experiments, the textural and chemical properties of the used soil samples were analyzed before their use (Table. A1).
Water Tm, Enr, Tb 12 nodules
Rice field soil (2 g) + Vermiculite soil 3 Addition of vermiculite soil and mixing N-free nutrient solution Tm, Enr, Tb 12 nodules and shoot
30°C and 20/4 h photoperiod
Exp-1 Expriments
Soybean field soil (90%) + Kanuma soil (10%) 3 Mixing
30°C and 20/4 h photoperiod
Exp-3
Tambaguro field soil (2 g) + Vermiculite soil 4 Addition of vermiculite soil and mixing N-free nutrient solution Tm, Enr, Tb + 7 others 12 nodules
rhizobium symbiotic relationships in Bradyrhizobium [12–14]. They induce the secretion of nod factors and of other molecular signals from type III secretion systems (T3SSs) of rhizobia [15]. Genistein is related to the genetic factors controlling soybean-rhizobium compatibility [16]. Daidzein, on the other hand, is the main root exudate of soybean seedlings [9,17], and has been considered to be a type of signaling molecule associated with competitiveness [18]. Even so, Guo and coworkers [10] reveal that both isoflavonoids were easily degraded in soil. To date, it is unclear whether the role of these isoflavonoids on nodulating rhizobia is retained in continuous cropping or not. In that sense, if the microbial communities – inclusive of Bradyrhizobium – change under the continuous cropping of soybean, what could be the most important factors related to their changes? In our previous study investigating the efficient compatibility of indigenous Bradyrhizobium strains with soybean in different fields, it was noted that the proportion of the compatible rhizobium strain B. diazoefficiens – formerly named B. japonicum – USDA110 with the Tamahomare-cultivated soybean field was different from that of the non-cultivated field [16]. This result suggests the influence of cropping system on symbiotic relationship between soybean and indigenous rhizobia. Therefore, the goals of the current research are to assess the effects of successive soybean cultivation on shifting symbiotic relationship with Bradyrhizobium strains in the field and to speculate the factors involve in this modification. For these purposes, the evolution of nodulating rhizobia of Tamahomare, Enrei and Tambaguro was surveyed in three cycles of continuous cropping. HPLC analysis of the rootsecreted isoflavonoids of these cultivars and assessment of the effects of a daidzein solution applied in the medium on the proportion of the B. diazoefficiens strain were independently carried out.
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polymerase – provided by Takara Bio, Japan – was used and mixed in accordance with the manufacturer's protocol. PCR conditions were 94 °C for 2 min, 35 cycles of amplification at 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 90 s, followed by a 10-min extension period at 72 °C. PCR products were electrophoresed on an agarose gel, and target DNA fragments were extracted from the agarose gel and purified using a DNA purification kit (Takara Bio Inc., Tokyo, Japan), ligated with the pGEMT Easy (Promega, Madison, WI, USA) cloning vector as described by the manufacturer, and then used to transform E. coli DH5α-competent cells. The cloned inserts underwent amplification by PCR again, electrophoresis and purification with a DNA purification kit. The purified DNA fragments were sent to FASMAC sequencing service (Fasmac, Japan) for sequence analysis. The sequenced DNA clones were analyzed using a standard nucleotide BLAST search of GenBank for homology with known bacterial 16S rRNA sequences. In this study, 16S rRNA sequences with > 99% identity with known bacterial strains were considered as homologous with that strains.
2.1.2. Third set of experiments (Exp-3) These experiments were also performed in an incubator set at 30 °C with a 20/4 h photoperiod. Using a modified version of a model approach for isolation of indigenous bradyrhizobia from a field soil [16,19,20], Exp-3 was mainly implemented to efficiently track the dynamics of soybean-bradyrhizobium symbiotic relationship in different field soil media. Surface-sterilized soybean seeds were sown in culture pots filled with autoclaved (121 °C for 20 min) vermiculite. Soybean seeds along with 2 g of air-dried soil were placed in the vermiculite soil at a depth of 1–1.5 cm at the 1st cycle of successive cultivation. The soil samples were taken either from a Kyoto University experimental farm field after harvest of rice or from the Tambaguro soybean field. The latter was provided by the Biotechnology Research Department, Kyoto Prefectural Agriculture, Forestry, and Fisheries Technology Research Center. The seedlings were watered daily with sterile distilled water for the first week and with sterile N-free nutrient solution subsequently. The N-free solution contains the following (concentrations in g L−1): K2SO4, 10.9 × 10−2; MgSO4, 12.3 × 10−2; K2HPO4, 0.85 × 10−2; CaCl2, 18.3 × 10−2; EDTANa2, 1.86 × 10−2; FeSO4, 1.39 × 10−2; KCl, 9.4 × 10−4; H3BO3, 3.7 × 10−4; and micronutrient solution (CuSO4, 3.2 × 10−5; MnSO4, 1.89 × 10−4; ZnSO4, 1.44 × 10−4; (NH4)6Mo7O24, 4 × 10−6; NiSO4, 3.5 × 10−6; and CoCl2, 0.28 × 10−6). From each plant at stage V3 – V4, 12 nodules were harvested; and seedlings from the medium using rice field soil sample were cut at the stem base and oven-dried at 75 °C for two days to measure shoot dry weight. After harvesting, the soil substrate of each previous cycle was added with a small quantity of autoclaved vermiculite soil (to adjust the volume), mixed, put back to the same pot, and 3–4 days later, sown with the soybean seeds of the same cultivar. For the Exp-3 performed on Tambaguro field soil samples, the same method was conducted using seven other cultivars (Peking, Norin 2, Shintambaguro, Fukuyutaka, William 82, Tachinagaha and Iyodaizu).
2.4. Analysis of root-secreted isoflavonoids Surface sterilized seeds of Tamahomare, Tambaguro, Enrei and seven other cultivars were grown in vermiculite-containing water for 7 days at 28 °C in the dark. Seedlings were transferred into a hydroponic culture system and grown in an incubator at 28 °C with a 16/8 h photoperiod for 48h. Regarding the hydroponic culture, the roots of each seedling were immersed into a test tube (one seedling per tube) filled with N-free solution (similar to the above-mentioned N-free solution). Isoflavonoids secreted into the hydroponic medium during these last 48 h were collected, filtered through Omnipore membrane filters (Millipore, Darmstadt, Germany), passed through a Sep-pak C18 Plus short cartridge (Waters, Milford, MA, USA) after adjusting the pH to 3.0 using HCl, washed with 3 mL water and eluted with 2 mL MeOH [17]. The root-secreted daidzein and genistein were analyzed using HPLC machine (LC-10A, Shimadzu, Kyoto, Japan), as described previously with some modifications [17]. The detection of the mobile phase consisted of 0.1% (v/v) acetic acid in filtered distilled water (solvent A) and 0.1% (v/v) formic acid in acetonitrile (solvent B), and the detection wavelength was 264 nm. The system was run at 0.8 mL/min with the solvent A and B with a linear gradient elution program from 15 to 22% B in 45 min, followed by a linear gradient from 22 to 35% B in 55 min, and a linear gradient from 35 to 70% B in 5 min. 500 μL of root-secretion (eluted with MeOH) of each plant were transferred into a tube and, immediately afterwards, loaded into the machine. For quantification of isoflavonoids, 500 μL of daidzein and genistein solutions – prepared by dissolution of 1 g pure isoflavonoids (daidzein and genistein standards) in 60 mL of MeOH – are separately analyzed at first. The peak locations of these standards are used to identify the corresponding compounds and their peak areas are used as a standard to calculate the root-secreted daidzein and genistein from each seedling.
2.2. PCR-RFLP analysis of bacterial DNA from nodules DNA of bradyrhizobia were extracted from nodules according to the method adopted by Ramongolalaina and colleagues [16]. Briefly, the nodules were washed thoroughly to remove soil, surface-sterilized with 70% ethanol for 1 min and 50% sodium hypochlorite for 3 min and rinsed three times with sterilized distilled water. They were crushed into a microplate (1 nodule/well) containing 5–30 μL of TE buffer (60%), Proteinase K (7.5%), RNase (2.5%), and bacterial lyse buffer (30%) depending on nodule size, and boiled at 99 °C for 60 min. The samples were diluted 5 to 10 times with TE buffer for DNA templates. For PCR analysis, 0.5 μL of DNA templates were mixed with 5 μL of EmeralAmp Max PCR Master (Takara Bio, Japan), 0.5 μL of DMSO, 2.5 μL of sterilized distilled water and 1.5 μL of the 10-μL internal transcribed spaces (ITS) primer set [21]. The ITS primer set (BraITS-F: 5′-GACTGGGGTGAAGTCGTAAC-3′, BraITS-R: 5′-ACGTCCTTCATCGC CTC -3′) was used for the amplification of the 16S−23S rDNA ITS region of rhizobia. The PCR cycle consisted of a pre-run at 96 °C for 5 min, 30 cycles of denaturation at 96 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 1 min, followed by a final post-run at 72 °C for 10 min. For PCR-RFLP analysis of the 16S−23S rDNA ITS region, 5 μL of each PCR product were digested with the restriction enzyme HaeIII at 37 °C for 6 h in a 10 μL reaction. The HaeIII is supplied by TaKaRa Bio, Japan.
2.5. Treatment of soybean with daidzein In Exp-3, the soil medium of the third cycle was returned once more to the same pot, after finishing the identical preparations carried out on substrates of precedent cycles. In this fourth cycle's medium, were sown daidzein-treated soybean seeds. Eight surface-sterilized soybean seeds of each cultivar Tamahomare, Enrei and Tambaguro were soaked for 20–30 min in 8-mL solution of daidzein. The daidzein solution was prepared by dissolving 0.2 g/L daidzein standard (with 254.24 molecular weight and purity ≥ 98%) – bought from Fujicco Co, Japan – in DMSO. The seeds were directly sown, and the remaining daidzein solution was also applied to the soil medium.
2.3. Bacterial DNA sequencing and cloning Prior to bacterial DNA sequencing, 16S−23S rRNA genes were cloned in order to avoid overlapping genome DNA fragments. Fragments of 16S−23S rRNA genes were amplified using general bacterial primers (ITS-F: 5′-CTGGGGTGAAGTCGTAACAAGG-3′, ITS-R: 5′-ACGTCCTTCATCGCCTCTCAG-3′). For each PCR, Taq DNA 3
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shown in Table. A.2, are: B. diazoefficiens USDA110 (USDA110-type), B. elkanii USDA94 (USDA94-type) and Rhizobium sp. (U-type). Their proportions for Tamahomare, Enrei and Tambaguro cultivars at three cycles of successive cropping were examined. Generally, the proportions of USDA110-type and U-type significantly decreased inversely with the proportions of USDA94-type as the number of cycles increased (Fig. 2). Overall, the proportions of USDA110-type, USDA94-type and U-type were different among all experimental conditions. They were also significantly different between cycles and experiments (p < 0.001). Only the proportion of USDA94type was significantly different between the genotypes (p = 0.0046). USDA110-type, USDA94-type and U-type levels appeared to be slightly higher with the soil samples taken from the rice field, but, conversely to the expectation, they were not significantly different among soil samples. Correspondingly, the proportions of U-type were lower in Exp-3 (Fig. 2e and f) than those in Exp-1 and Exp-2, because the medium in Exp-3 was vermiculite soil mixed with 2 g of field soil, while that of Exp-1 and Exp-2 was 90% field soil mixed with 10% kanuma soil. Therefore, this difference must be a result of the initial density of U-type in the first cycle and the soil properties. Because the intensity of decreases in USDA110-type appeared to be different among soybean genotypes, experiments, and soil types, the differences between the proportion of USDA110-type of the 1st cycles and the 3rd cycles were calculated. As predicted, the results (Fig. 3) showed that the decreases of proportions of USDA110-type were statistically different in genotypes (p = 0.023) and in experiments (p < 0.001). The proportions of USDA110-type of Tambaguro moderately decrease compared to those of Tamahomare and Enrei in all experimental conditions. Interestingly, these declines of USDA110-type
2.6. Calculation and statistical analysis The differences between the proportion of a bacterial type of the 1st cycles and the 3rd cycles were calculated by the following formula:
Δ B=
(BC3 − BC1) x100 BC1
where ΔB is the differences between the proportion of a bacterial type of two cycles; BC1 is the proportion of this bacterial type of the 1st cycles, and BC3 of the 3rd cycles. Statistical analysis of the six independently performed experiments was conducted using R programming (RStudio version 1.1.463 - Mac OS X 10.6) and IBM SPSS Statistics (SPSS v23.0 MacOS, SPSS Inc., Chicago, IL, USA) with significance level of 0.05. A one-way ANOVA was used to assess differences in the proportion of each identified bacterial type between themselves, treatments, cycles, and soybean genotypes. 3. Results 3.1. Decrease of the density of B. diazoefficiens strains in successive cultivation Similar to the results of our previous study [16], the PCR-RFLP analysis using the 16S−23S rRNA ITS region of rhizobia in each nodule identified three groups of indigenous rhizobium strains (members of the indigenous soil microbial community) that could form nodules with soybean in the field (Fig. 1). The sequencing analysis performed on current research clearly confirmed that these three types of rhizobia, as
Fig. 1. PCR-RFLP patterns of the three nodule bacterial types identified in 48 nodules (12 nodules per plant) from each cultivar at 1st cycle of Exp-1 (rice field soil sample): a) Tamahomare, b) Enrei and c) Tambaguro. d) Schematic representation of amplicon patterns based on PCR-RFLP analysis of the l6S-23S rDNA internal transcribed spacer (ITS) region of B. diazoefficiens USDA110 (USDA110-type), B. elkanii USDA94 (USDA94-type), and Rrhizobium sp. (U-type) when digested with restriction enzyme HaeIII. 4
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Fig. 2. Proportions of the three nodule bacterial types found in each nodule of Tamahomare, Enrei and Tambaguro throughout the three continuous cropping cycles. a) represents results from Exp-1, using rice field soil sample; b) from Exp-1, using soybean field soil sample; c) from Exp-2, using rice field soil sample; d) from Exp-2, using soybean field soil sample; e) from Exp-3, using rice field soil sample; and f) from Exp-3, using Tambaguro field soil sample. Values are means across four replicates.
3.3. Increase in competitiveness of B. Diazoefficiencs treated with isoflavonoids
nodules were significantly greater in Exp-3. Based on this approach, therefore, besides the experimental conditions, soybean genotypes contribute the intensity of decreases in USDA110-type that can form nodules with plants.
It may be reasonable to assume that the root-secreted daidzein might contribute to the alteration of USDA110-type that can nodulate soybean plants in continuous cropping, because Tambaguro secreted a large amount of daidzein from the root compared to Tamahomare and Enrei, also its decrease in proportion of USDA110-type nodules in successive cropping was moderate compared with those of two other cultivars. To strengthen this assumption, the effect of treating soybean seeds with daidzein on the proportions of USDA110-type was evaluated in the fourth cycle of Exp-3. As can be seen in Fig. 5, the proportion of USDA110-type of Tamahomare, Enrei and Tambaguro in the fourth cycle were further decreased compared to those in the third cycle. This result is consistent with those previously obtained on this study, confirming the further decrease in the population of USDA110-type under continuous cropping conditions. The proportions of USDA110-type among all seedlings increased from 10% to 38% in response to daidzein treatment. The proportion of USDA110-type of Tambaguro was higher in response to daidzein treatment compared with those of Tamahomare and Enrei.
3.2. Amount of root-secreted isoflavonoids An HPLC analysis of root-secreted isoflavonoids from Tamahomare, Enrei, Tambaguro and other genotypes (Peking, Shintambaguro, Fukuyutaka, William 82, Norin 2, Tachinagaha and Iyodaizu) was performed. The average amount of root-secreted daidzein from oneweek old Tamahomare, Enrei and Tambaguro seedlings in 48 h were 20.28, 14.19, and 127.31 μg/seedling, respectively (Fig. 4). The amount of root-secreted daidzein was significantly different among genotypes (p < 0.001). All the genotypes can be classified in two groups: one group with high amount of root-secreted daidzein (Tambaguro, Peking, William 82 and Shintambaguro) and another group with low amount (Tamahomare, Enrei, Norin 2, Fufuyutaka, Tachinagaha and Iyodaizu). For the root-secreted genistein, trace amounts of secretion in all genotypes, which ranged from 0.61 μg to 1.29 μg, were found. Therefore, the current results were almost similar to those gotten in our previous study, and confirm that daidzein is the main root-secreted isoflavonoid of soybean – as mentioned in the preceding studies of Cesco et al. [9] and Sugiyama et al. [17].
4. Discussion Prior work on soybean-rhizobia symbiotic relationship has never focused on the evolution of rhizobial communities in the field. None 5
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Fig. 3. Decreases of the proportion of USDA110-type of Tamahomare, Enrei and Tamabaguro from the first cycle to the third cycle (%) under all experiment conditions. Values are means across four replicates.
Fig. 5. Effect of soybean seed treatment with daidzein on the proportion of nodule bacterial type for Tamahomare, Enrei, and Tambaguro. 3rd cycle, 4th cycle and 4th cycle (with daidzein) represent proportions of the nodule USDA110-type of the seedlings at third cycle, at fourth cycle without and with daidzein treatment, respectively (Exp-3, using Tambaguro soil sample). Values are means across four replicates.
and co-workers [22] demonstrated that the addition of root exudates collected from liquid medium to soils was found to modify the microbial composition of these soils, depending on the fractions of root exudates. Root exudates are composed of sugars, amino acids, flavonoids, proteins, and fatty acids [23]. These substances, documented Bais and colleagues [24], can serve as growth substrates or signals for suitable microbial partners and as antimicrobials or growth deterrents for other microbes. Bradyrhizobium has been shown to be abundant in rhizosphere soil – soil around the roots – because of its chemotaxis with the root exudates of soybean [8,25]. Additionally, root exudate contents and concentrations are correlate with the metabolic activities of bacterial communities in rhizospheres [26,27].
Fig. 4. Boxplot representation of root-secreted daidzein and genistein from oneweek-old Tamahomare, Enrei, Tambaguro, Peking, Shintambaguro, Fukuyutaka, William 82, Norin 2, Tachinagaha and Iyodaizu seedlings. The dots represent the total amount of root-secreted isoflavonoid of each plant in 48 h (4 replicates).
previous report is, therefore, comparable to the current investigation. Herein, it was showed – in all experimental conditions – that the proportion of USDA110-type decreases significantly during successive soybean cropping. This decrease in USDA110-type must be a result of accumulation of root exudates in the soil and other environmental conditions. It is, of course possible to object those reasons, but Badri 6
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continuous cropping (Figs. 3 and 4 and Fig. A2). This suggests that the high accumulation of daidzein increases the competitiveness of USDA110. Consequently, the treatment of seeds with daidzein increased the proportion of USDA110 (Fig. 5). These findings extend those of Guan et al. [44], confirming that when treated with daidzein, a large proportion of proteins associated with nodulation and the metabolism of energy and material – the reason for its good symbiotic matching for nodulation – are upregulated in B. diazoefficiens USDA110-A, while nodulation-related proteins and defensive proteins are downregulated in B. diazoefficiens 2178. Supporting these finding also, a comparison of the competitiveness of two B. diazoefficiens strains for nodulation investigated by Li and co-workers [45] proved that a large proportion of proteins associated with nodulation are upregulated in the highly competitive B. diazoefficiens 4534 treated with daidzein. Daidzein regulates the expression of hopanoid synthesis genes in rhizobia [46]. Hopanoids are pentacyclic triterpenoid lipids that are widely occurring in bacteria, which act as membrane reinforcers, conferring resistance to different environmental stresses. In bradyrhizobia, these compounds can account for nearly half of the total cell lipid fraction [47]. Daidzein also has been considered to be a type of signaling molecule that is associated with competitiveness [18], although the maximal response of B. diazoefficiens USDA110 to daidzein is 50% or less compared with that of genistein [48]. In conclusion, though the results could be partly influenced by the experimental design, the present study discovered that the population of the efficient nodulating strain B. diazoefficiens USDA110 decreases in successive cropping, which is a factor in the decrease in shoot dry weight. There is a strong possibility that this alteration of nodulating rhizobial community is due to the accumulation and decomposition of root exudates. The accumulation of daidzein might be able to rescue the ability of B. diazoefficiens USDA110. Some limitations are worth noting. Although the hypotheses were supported statistically, the evolution of Bradyrhizobum and daidzein in soil was not assessed. Future work should therefore include follow-up work designed to evaluate how isoflavonoids are degraded and how much exactly nodulating rhizobial population is altered in the field soil in long-term cultivation of soybean. Therefore, further investigation is needed to determine the amounts of root exudate residues, including daidzein and genistein, in soybean fields.
After having discussed the root exudates, let us look at the environmental conditions. The results (Fig. 3) displayed that the intensities of the decrease in USDA110-type from the first cycle to the third cycle were statistically different among experiments. The decrease in the USDA110-type was more intense in the vermiculite medium (Exp-3) compared to a mixed soil medium (Exp-1 and Exp-2), because growing soybean plants under laboratory conditions using soil samples diluted with vermiculite might undermine the possible effects of soil properties on the rhizobial nodulating properties [28]. This decrease in USDA110-type was also even more intense in high-temperature conditions (Exp-2) than in low-temperature conditions (Exp-1). B. diazoefficiens is dominant in the soils of cooler environments in Japan and Nepal [29–31]. Moreover, relationships exist between the geographic distribution of indigenous soybean-nodulating rhizobia, soil temperature (and its variations because of latitude and altitude), and soil properties, such as soil pH [32,33]. B. elkanii strains showed growth stability at varying temperatures and pH levels [32,34], and widely distribution in acid soil [35]. It is also necessary to discuss further the importance of B. diazoefficiens USDA110 in symbiotic relationship with soybean. B. diazoefficiens USDA110 has greater N2 fixation potential than the other species, resulting in greater biomass production than with unselected strains [36]. In addition, Rj2, Rj3, and Rj4 – the loci that control the formation of symbiotic root nodules in soybean plants [37] – conferred improved nodulation when inoculated with B. diazoefficiens USDA110 [38]. B. diazoefficiens USDA110 is used in many countries as an inoculant to increase soybean yield [39]. Furthermore, B. diazoefficiens can form nodules faster than B. elkanii [34], but B. elkanii strains produce rhizobitoxine, a compound that induces chlorosis in the host plant, and the strains are relatively inefficient symbionts for soybean [40–42]. It was noted that the shoot dry weights of Tamahomare, Enrei and Tambaguro were highly decreased as the number of times of cultivation increased (Fig. A3) and that the seedlings in the third cycle of cropping were susceptible to chlorosis (Fig. A4b). Rhizobitoxine increases nodule formation via the inhibition of endogenous ethylene synthesis in host plants because ethylene restricts nodulation in many legumes [41,43]. Rhizobitoxine production by B. elkanii USDA94 gave the bacterium a nodulation competitiveness approximately 10 times higher than that of a non-rhizobitoxine-producing mutant strain on Macroptilium atropurpureum [41,43]. Rhizobitoxine production correlates with the amount of rtxC transcript in B. elkanii USDA94 [42]. Bolzan de Campos et al. [15] and Ramongolalaina et al. [16] reported that the Peking cultivar can efficiently establish a symbiotic relationship with B. elkanii because the transcriptional regulator TtsI used to activate the type III secretion system of B. elkanii displays host-dependent characteristics [15]. Up to this point, the discussion has been focusing on B. diazoefficiens USDA110, but what does it have to do daidzein? The results provide compelling evidence of the involvement of daidzein in alteration of B. diazoefficiens USDA110. Genotypes (Tambaguro, Peking, William 82 and Shintambaguro) with a high amount of root-secreted daidzein corresponded to the moderate decrease in the USDA110-type in
Funding This research was conducted using no grant from funding agencies in the public, commercial, or not-for-profit sectors. Acknowledgement I am very grateful to my previous advisors, Dr. Okumoto Yutaka and Dr. Teraishi Masayoshi (Plant Breeding laboratory, Department of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University), for allowing me to carry my experiments in their laboratory and, most importantly, for granting me to publish this work.
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejsobi.2019.103098.
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Appendices
Fig. A.1. Proportions of the three nodule bacterial types found in each nodule Peking, Shintambaguro, Fukuyutaka, William 82, Norin 2, Tachinagaha and Iyodaizu throughout the three continuous cropping cycles in Exp-3 (Tambaguro field soil samples).
Fig. A.2. Decreases of the proportion of USDA110-type of Tamahomare, Enrei, Tambaguro, Peking, Shintambaguro, Fukuyutaka, William 82, Norin 2, Tachinagaha and Iyodaizu from to 1st cycles to the 3rd cycles (%) in Exp-3 (Tambaguro field soil samples).
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Fig. A.3. Shoot dry weight (mg) of Tamahomare, Enrei and Tambaguro throughout the three continuous cropping cycles.
Fig. A.4. a) Harvested seedlings of Tamahomare, Enrei, Peking and Tambaguro at the second cycle in Exp-3 (Tambaguro field soil samples). b) Patterns of the leaves of Tamahomare seedlings at the three continuous cropping. c) Seedlings in hydroponic culture system, ready for root-secreted isoflavonoids (right after their growth in an incubator).
Supplementary data and tables Table. A.1 Textural and chemical properties of the soil samples. Table. A.2 Results of sequencing analysis of each bacterial type. Table. A.3 Increases in the proportion of USDA94-type from the thirst to the third cycles Reference Data Whole data.xlsx - 3_cycles: Proportions of the three nodule bacterial types found in each nodule of Tamahomare, Enrei and Tambaguro throughout the three continuous cropping cycles, and root dry weight (mg) of Peking, Tamahomare, Enrei and Tambaguro throughout the three continuous cropping cycles. - Daidzein_in_soil: Proportions of the nodule bacterial type without incubation and incubation of seeds in daidzein solution of the seedlings at fourth cycle of Exp-3 (using Tambaguro soil sample). - Other_genotypes: Proportions of the three nodule bacterial types found in each nodule Peking, Shintambaguro, Fukuyutaka, William 82, Norin 2, Tachinagaha and Iyodaizu throughout the three continuous cropping cycles in Exp-3 (Tambaguro field soil samples). - Statistic test: oneway ANOVA analysis of differences of the proportion of each identified bacterial type between themselves, treatments, cycles, 9
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and soybean genotypes - Root-secreted daidzein and genistein.xlsx: Method to calculate the amounts of root-secreted daidzein and genistein and their values from oneweek-old Tamahomare, Enrei, Tambaguro, Peking, Shintambaguro, Fukuyutaka, William 82, Norin 2, Tachinagaha and Iyodaizu seedlings.
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Abbreviations DMSO: Dimethyl sulfoxide HPLC: High-Performance Liquid Chromatography ITS: Internal Transcribed Spacer PCR: Polymerase Chain Reaction rRNA: ribosomal Ribonucleic Acid RFLP: Restriction Fragment Length Polymorphism T3SS: Type III Secretion System
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Dynamics of symbiotic relationship of soybean with Bradyrhizobium diazoefficiens and involvements of root-secreted daidzein behind the continuous cropping
T
Clarissien Ramongolalainaa,b,∗ a
Department of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto, 606-8502, Japan Department of Life Science Frontiers, Center for iPS Cell Research and Application, Graduate School of Medicine, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
b
A R T I C LE I N FO
A B S T R A C T
Handling editor: Bryan Griffiths
Symbiosis with a selectively nodulating rhizobium strain, such as Bradyrhizobium diazoefficiens USDA110, improves soybean productivity. In continuous cropping, however, the concentrations of root exudates – of which daidzein is one of the major secretions – might influence nodulating rhizobium communities and cause ineffective nitrogen fixation in soybean. The dynamics of indigenous rhizobia that form nodules with the soybean cultivars Tamahomare, Enrei and Tamabaguro at three cycles of a continuous cropping plot under different experimental conditions were examined. PCR-RFLP analysis targeted to the 16S-23S rRNA gene internal transcribed spacer (ITS) region of a bacterium, confirmed by sequencing analysis, was used to determine the bacterial type for each root nodule. The results showed that as the number of cycles increased, the proportion of B. diazoefficiens USDA110 of all cultivars decreased. Under all experimental conditions, the average differences between the proportion of B. diazoefficiens USDA110 of the first and third cycles in Tamahomare, Enrei and Tamabaguro were 67%, 70% and 44%, respectively. After the HPLC analysis of the root-secreted isoflavonoids, the results showed that the root-secreted daidzein from Tambaguro was excreted in the highest levels among the three cultivars. Increase of the proportion of B. diazoefficiens USDA110 among seedlings treated with daidzein was also noted. Therefore, the present study reports that the symbiotic relationship of soybean with B. diazoefficiens USDA110 decreases in continuous cropping and suggests that root-secreted daidzein helps to maintain populations of this rhizobial strain in rhizosphere of some cultivars.
Keywords: Symbiotic relationship Soil bacteria Bradyrhizobium diazoefficiens USDA110 Soybean continuous cropping Root-secreted daidzein
1. Introduction Soybean is cultivated in many regions around the world because of its great health and economic benefits. For climatic and other technical reasons, soybean is commonly grown in a continuous monoculture, resulting in yield decline and quality deterioration [1]. The reduced yield of soybean in continuous cultivation is mainly attributed to intraspecific allelopathy and the buildup of pathogens and other pests [1,2]. Notwithstanding, alterations in microbial communities, especially beneficial microbes, may also be important and require further investigation. In continuous cropping systems, microbial communities are continuously exposed to the roots of the same crop that selects and enriches certain groups of microorganisms – including yield-debilitating populations (i.e., soilborne pathogens) of that crop [3]. In the field of soybean crop, the most important legume crop in the world, soil
rhizobia are one of the main actors improve soybean productivity and soil fertility [4–7]. So far, scarcely ever are articles focused on soil rhizobia under continuous cropping of soybean. At this point, it might be still wondering, is this alteration of soil rhizobia worth attention in term of soybean cultivation? But, Sugiyama and colleagues [8] demonstrated that bacterial communities – including rhizobial community – of the soybean rhizosphere alter even during growth in the field. Taking into account these few previous reports, it is only appropriate next to discover the effects of the continuous cropping of soybean on nodulating rhizobia. The concentrations of root exudates in the soybean rhizosphere include phenolic acids, and isoflavonoids (daidzein and genistein) might be critical factors influencing changes in microbial communities [9–11]. Daidzein and genistein are known to be the inducers of nod gene expression; the inducers initiate the establishment of legume-
∗ Corresponding author. Department of Life Science Frontiers, Center for iPS Cell Research and Application, Graduate School of Medicine, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan. E-mail address: [email protected].
https://doi.org/10.1016/j.ejsobi.2019.103098 Received 12 March 2019; Received in revised form 11 June 2019; Accepted 12 June 2019 Available online 28 June 2019 1164-5563/ © 2019 Elsevier Masson SAS. All rights reserved.
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Water Tm, Enr, Tb 12 nodules
Number of cycles Treatment of soil medium after each cycle Watering supplement Used genotypes Harvested organs per seedling
Exp-2
Table. 1 Particularities of each independent experiment
2.1.1. First and second sets of experiments (Exp-1 and Exp-2) Exp-1 and Exp-2, primarily aimed to investigate dynamics of symbiotic relationship between soil rhizobia and soybean plants successively cultivated in real field under different temperature conditions, were carried out in incubators set at 28 °C with an 18/6 h photoperiod, and automatically set – the settings were being unable to adjust – at 30 °C with a 20/4 h photoperiod, respectively. Surface-sterilized soybean seeds were sown in a pot filled with field soil (90%) mixed, to aerate the soil medium, with S-sized pumice Kanuma soil (10%); this Kanuma soil was purchased from Tachikawa-heiwa Nouen Co., LTD., Japan. The field soil samples were taken from a Kyoto University experimental farm field – rotated between soybean field and paddy field every year – after either rice or soybean harvest. The seedlings were watered every 2 days. The seedlings were harvested at the third to fourth trifoliolate stage (V3 – V4; 22–23 days after sowing) and 12 very young nodules per plant were sampled from four seedlings. Once the seedlings of the first cycle were harvested, the soil substrate was mixed and put back – without further treatments – to the same pot, and the soybean seeds of the same cultivar were grown in this substrate 3–4 days later. After harvesting the seedlings from the second cycle at the fourth trifoliolate stage, the same approach was undertaken for the third cycle.
Tm, Enr, Tb: Tamahomare, Enrei, Tambaguro Tm, Enr, Tb + 7 others: Tamahomare, Enrei, Tambaguro, Peking, Shintambaguro, Fukuyutaka, William 82, Norin 2, Tachinagaha and Iyodaizu
Water Tm, Enr, Tb 12 nodules
Rice field soil (90%) + Kanuma soil (10%) 3 Mixing
2.1. Successive cultivation of soybean
Water Tm, Enr, Tb 12 nodules
Rice field soil (90%) + Kanuma soil (10%) 3 Mixing Soybean field soil (90%) + Kanuma soil (10%) 3 Mixing
28°C and 18/6 h photoperiod
Conditions (incubator settings) Original soil medium
2. Method
Three sets of experiments were performed simultaneously to simulate successive cultivation using three soybean cultivars well-known in Japan: Tamahomare, Tambaguro, and Enrei. Using culture pots (Ø = 15 cm, H = 12 cm), the experiments were performed in three different incubators; the particularities of each experiment were summarized in Table 1. Of note, in all experiments, the textural and chemical properties of the used soil samples were analyzed before their use (Table. A1).
Water Tm, Enr, Tb 12 nodules
Rice field soil (2 g) + Vermiculite soil 3 Addition of vermiculite soil and mixing N-free nutrient solution Tm, Enr, Tb 12 nodules and shoot
30°C and 20/4 h photoperiod
Exp-1 Expriments
Soybean field soil (90%) + Kanuma soil (10%) 3 Mixing
30°C and 20/4 h photoperiod
Exp-3
Tambaguro field soil (2 g) + Vermiculite soil 4 Addition of vermiculite soil and mixing N-free nutrient solution Tm, Enr, Tb + 7 others 12 nodules
rhizobium symbiotic relationships in Bradyrhizobium [12–14]. They induce the secretion of nod factors and of other molecular signals from type III secretion systems (T3SSs) of rhizobia [15]. Genistein is related to the genetic factors controlling soybean-rhizobium compatibility [16]. Daidzein, on the other hand, is the main root exudate of soybean seedlings [9,17], and has been considered to be a type of signaling molecule associated with competitiveness [18]. Even so, Guo and coworkers [10] reveal that both isoflavonoids were easily degraded in soil. To date, it is unclear whether the role of these isoflavonoids on nodulating rhizobia is retained in continuous cropping or not. In that sense, if the microbial communities – inclusive of Bradyrhizobium – change under the continuous cropping of soybean, what could be the most important factors related to their changes? In our previous study investigating the efficient compatibility of indigenous Bradyrhizobium strains with soybean in different fields, it was noted that the proportion of the compatible rhizobium strain B. diazoefficiens – formerly named B. japonicum – USDA110 with the Tamahomare-cultivated soybean field was different from that of the non-cultivated field [16]. This result suggests the influence of cropping system on symbiotic relationship between soybean and indigenous rhizobia. Therefore, the goals of the current research are to assess the effects of successive soybean cultivation on shifting symbiotic relationship with Bradyrhizobium strains in the field and to speculate the factors involve in this modification. For these purposes, the evolution of nodulating rhizobia of Tamahomare, Enrei and Tambaguro was surveyed in three cycles of continuous cropping. HPLC analysis of the rootsecreted isoflavonoids of these cultivars and assessment of the effects of a daidzein solution applied in the medium on the proportion of the B. diazoefficiens strain were independently carried out.
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polymerase – provided by Takara Bio, Japan – was used and mixed in accordance with the manufacturer's protocol. PCR conditions were 94 °C for 2 min, 35 cycles of amplification at 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 90 s, followed by a 10-min extension period at 72 °C. PCR products were electrophoresed on an agarose gel, and target DNA fragments were extracted from the agarose gel and purified using a DNA purification kit (Takara Bio Inc., Tokyo, Japan), ligated with the pGEMT Easy (Promega, Madison, WI, USA) cloning vector as described by the manufacturer, and then used to transform E. coli DH5α-competent cells. The cloned inserts underwent amplification by PCR again, electrophoresis and purification with a DNA purification kit. The purified DNA fragments were sent to FASMAC sequencing service (Fasmac, Japan) for sequence analysis. The sequenced DNA clones were analyzed using a standard nucleotide BLAST search of GenBank for homology with known bacterial 16S rRNA sequences. In this study, 16S rRNA sequences with > 99% identity with known bacterial strains were considered as homologous with that strains.
2.1.2. Third set of experiments (Exp-3) These experiments were also performed in an incubator set at 30 °C with a 20/4 h photoperiod. Using a modified version of a model approach for isolation of indigenous bradyrhizobia from a field soil [16,19,20], Exp-3 was mainly implemented to efficiently track the dynamics of soybean-bradyrhizobium symbiotic relationship in different field soil media. Surface-sterilized soybean seeds were sown in culture pots filled with autoclaved (121 °C for 20 min) vermiculite. Soybean seeds along with 2 g of air-dried soil were placed in the vermiculite soil at a depth of 1–1.5 cm at the 1st cycle of successive cultivation. The soil samples were taken either from a Kyoto University experimental farm field after harvest of rice or from the Tambaguro soybean field. The latter was provided by the Biotechnology Research Department, Kyoto Prefectural Agriculture, Forestry, and Fisheries Technology Research Center. The seedlings were watered daily with sterile distilled water for the first week and with sterile N-free nutrient solution subsequently. The N-free solution contains the following (concentrations in g L−1): K2SO4, 10.9 × 10−2; MgSO4, 12.3 × 10−2; K2HPO4, 0.85 × 10−2; CaCl2, 18.3 × 10−2; EDTANa2, 1.86 × 10−2; FeSO4, 1.39 × 10−2; KCl, 9.4 × 10−4; H3BO3, 3.7 × 10−4; and micronutrient solution (CuSO4, 3.2 × 10−5; MnSO4, 1.89 × 10−4; ZnSO4, 1.44 × 10−4; (NH4)6Mo7O24, 4 × 10−6; NiSO4, 3.5 × 10−6; and CoCl2, 0.28 × 10−6). From each plant at stage V3 – V4, 12 nodules were harvested; and seedlings from the medium using rice field soil sample were cut at the stem base and oven-dried at 75 °C for two days to measure shoot dry weight. After harvesting, the soil substrate of each previous cycle was added with a small quantity of autoclaved vermiculite soil (to adjust the volume), mixed, put back to the same pot, and 3–4 days later, sown with the soybean seeds of the same cultivar. For the Exp-3 performed on Tambaguro field soil samples, the same method was conducted using seven other cultivars (Peking, Norin 2, Shintambaguro, Fukuyutaka, William 82, Tachinagaha and Iyodaizu).
2.4. Analysis of root-secreted isoflavonoids Surface sterilized seeds of Tamahomare, Tambaguro, Enrei and seven other cultivars were grown in vermiculite-containing water for 7 days at 28 °C in the dark. Seedlings were transferred into a hydroponic culture system and grown in an incubator at 28 °C with a 16/8 h photoperiod for 48h. Regarding the hydroponic culture, the roots of each seedling were immersed into a test tube (one seedling per tube) filled with N-free solution (similar to the above-mentioned N-free solution). Isoflavonoids secreted into the hydroponic medium during these last 48 h were collected, filtered through Omnipore membrane filters (Millipore, Darmstadt, Germany), passed through a Sep-pak C18 Plus short cartridge (Waters, Milford, MA, USA) after adjusting the pH to 3.0 using HCl, washed with 3 mL water and eluted with 2 mL MeOH [17]. The root-secreted daidzein and genistein were analyzed using HPLC machine (LC-10A, Shimadzu, Kyoto, Japan), as described previously with some modifications [17]. The detection of the mobile phase consisted of 0.1% (v/v) acetic acid in filtered distilled water (solvent A) and 0.1% (v/v) formic acid in acetonitrile (solvent B), and the detection wavelength was 264 nm. The system was run at 0.8 mL/min with the solvent A and B with a linear gradient elution program from 15 to 22% B in 45 min, followed by a linear gradient from 22 to 35% B in 55 min, and a linear gradient from 35 to 70% B in 5 min. 500 μL of root-secretion (eluted with MeOH) of each plant were transferred into a tube and, immediately afterwards, loaded into the machine. For quantification of isoflavonoids, 500 μL of daidzein and genistein solutions – prepared by dissolution of 1 g pure isoflavonoids (daidzein and genistein standards) in 60 mL of MeOH – are separately analyzed at first. The peak locations of these standards are used to identify the corresponding compounds and their peak areas are used as a standard to calculate the root-secreted daidzein and genistein from each seedling.
2.2. PCR-RFLP analysis of bacterial DNA from nodules DNA of bradyrhizobia were extracted from nodules according to the method adopted by Ramongolalaina and colleagues [16]. Briefly, the nodules were washed thoroughly to remove soil, surface-sterilized with 70% ethanol for 1 min and 50% sodium hypochlorite for 3 min and rinsed three times with sterilized distilled water. They were crushed into a microplate (1 nodule/well) containing 5–30 μL of TE buffer (60%), Proteinase K (7.5%), RNase (2.5%), and bacterial lyse buffer (30%) depending on nodule size, and boiled at 99 °C for 60 min. The samples were diluted 5 to 10 times with TE buffer for DNA templates. For PCR analysis, 0.5 μL of DNA templates were mixed with 5 μL of EmeralAmp Max PCR Master (Takara Bio, Japan), 0.5 μL of DMSO, 2.5 μL of sterilized distilled water and 1.5 μL of the 10-μL internal transcribed spaces (ITS) primer set [21]. The ITS primer set (BraITS-F: 5′-GACTGGGGTGAAGTCGTAAC-3′, BraITS-R: 5′-ACGTCCTTCATCGC CTC -3′) was used for the amplification of the 16S−23S rDNA ITS region of rhizobia. The PCR cycle consisted of a pre-run at 96 °C for 5 min, 30 cycles of denaturation at 96 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 1 min, followed by a final post-run at 72 °C for 10 min. For PCR-RFLP analysis of the 16S−23S rDNA ITS region, 5 μL of each PCR product were digested with the restriction enzyme HaeIII at 37 °C for 6 h in a 10 μL reaction. The HaeIII is supplied by TaKaRa Bio, Japan.
2.5. Treatment of soybean with daidzein In Exp-3, the soil medium of the third cycle was returned once more to the same pot, after finishing the identical preparations carried out on substrates of precedent cycles. In this fourth cycle's medium, were sown daidzein-treated soybean seeds. Eight surface-sterilized soybean seeds of each cultivar Tamahomare, Enrei and Tambaguro were soaked for 20–30 min in 8-mL solution of daidzein. The daidzein solution was prepared by dissolving 0.2 g/L daidzein standard (with 254.24 molecular weight and purity ≥ 98%) – bought from Fujicco Co, Japan – in DMSO. The seeds were directly sown, and the remaining daidzein solution was also applied to the soil medium.
2.3. Bacterial DNA sequencing and cloning Prior to bacterial DNA sequencing, 16S−23S rRNA genes were cloned in order to avoid overlapping genome DNA fragments. Fragments of 16S−23S rRNA genes were amplified using general bacterial primers (ITS-F: 5′-CTGGGGTGAAGTCGTAACAAGG-3′, ITS-R: 5′-ACGTCCTTCATCGCCTCTCAG-3′). For each PCR, Taq DNA 3
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shown in Table. A.2, are: B. diazoefficiens USDA110 (USDA110-type), B. elkanii USDA94 (USDA94-type) and Rhizobium sp. (U-type). Their proportions for Tamahomare, Enrei and Tambaguro cultivars at three cycles of successive cropping were examined. Generally, the proportions of USDA110-type and U-type significantly decreased inversely with the proportions of USDA94-type as the number of cycles increased (Fig. 2). Overall, the proportions of USDA110-type, USDA94-type and U-type were different among all experimental conditions. They were also significantly different between cycles and experiments (p < 0.001). Only the proportion of USDA94type was significantly different between the genotypes (p = 0.0046). USDA110-type, USDA94-type and U-type levels appeared to be slightly higher with the soil samples taken from the rice field, but, conversely to the expectation, they were not significantly different among soil samples. Correspondingly, the proportions of U-type were lower in Exp-3 (Fig. 2e and f) than those in Exp-1 and Exp-2, because the medium in Exp-3 was vermiculite soil mixed with 2 g of field soil, while that of Exp-1 and Exp-2 was 90% field soil mixed with 10% kanuma soil. Therefore, this difference must be a result of the initial density of U-type in the first cycle and the soil properties. Because the intensity of decreases in USDA110-type appeared to be different among soybean genotypes, experiments, and soil types, the differences between the proportion of USDA110-type of the 1st cycles and the 3rd cycles were calculated. As predicted, the results (Fig. 3) showed that the decreases of proportions of USDA110-type were statistically different in genotypes (p = 0.023) and in experiments (p < 0.001). The proportions of USDA110-type of Tambaguro moderately decrease compared to those of Tamahomare and Enrei in all experimental conditions. Interestingly, these declines of USDA110-type
2.6. Calculation and statistical analysis The differences between the proportion of a bacterial type of the 1st cycles and the 3rd cycles were calculated by the following formula:
Δ B=
(BC3 − BC1) x100 BC1
where ΔB is the differences between the proportion of a bacterial type of two cycles; BC1 is the proportion of this bacterial type of the 1st cycles, and BC3 of the 3rd cycles. Statistical analysis of the six independently performed experiments was conducted using R programming (RStudio version 1.1.463 - Mac OS X 10.6) and IBM SPSS Statistics (SPSS v23.0 MacOS, SPSS Inc., Chicago, IL, USA) with significance level of 0.05. A one-way ANOVA was used to assess differences in the proportion of each identified bacterial type between themselves, treatments, cycles, and soybean genotypes. 3. Results 3.1. Decrease of the density of B. diazoefficiens strains in successive cultivation Similar to the results of our previous study [16], the PCR-RFLP analysis using the 16S−23S rRNA ITS region of rhizobia in each nodule identified three groups of indigenous rhizobium strains (members of the indigenous soil microbial community) that could form nodules with soybean in the field (Fig. 1). The sequencing analysis performed on current research clearly confirmed that these three types of rhizobia, as
Fig. 1. PCR-RFLP patterns of the three nodule bacterial types identified in 48 nodules (12 nodules per plant) from each cultivar at 1st cycle of Exp-1 (rice field soil sample): a) Tamahomare, b) Enrei and c) Tambaguro. d) Schematic representation of amplicon patterns based on PCR-RFLP analysis of the l6S-23S rDNA internal transcribed spacer (ITS) region of B. diazoefficiens USDA110 (USDA110-type), B. elkanii USDA94 (USDA94-type), and Rrhizobium sp. (U-type) when digested with restriction enzyme HaeIII. 4
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Fig. 2. Proportions of the three nodule bacterial types found in each nodule of Tamahomare, Enrei and Tambaguro throughout the three continuous cropping cycles. a) represents results from Exp-1, using rice field soil sample; b) from Exp-1, using soybean field soil sample; c) from Exp-2, using rice field soil sample; d) from Exp-2, using soybean field soil sample; e) from Exp-3, using rice field soil sample; and f) from Exp-3, using Tambaguro field soil sample. Values are means across four replicates.
3.3. Increase in competitiveness of B. Diazoefficiencs treated with isoflavonoids
nodules were significantly greater in Exp-3. Based on this approach, therefore, besides the experimental conditions, soybean genotypes contribute the intensity of decreases in USDA110-type that can form nodules with plants.
It may be reasonable to assume that the root-secreted daidzein might contribute to the alteration of USDA110-type that can nodulate soybean plants in continuous cropping, because Tambaguro secreted a large amount of daidzein from the root compared to Tamahomare and Enrei, also its decrease in proportion of USDA110-type nodules in successive cropping was moderate compared with those of two other cultivars. To strengthen this assumption, the effect of treating soybean seeds with daidzein on the proportions of USDA110-type was evaluated in the fourth cycle of Exp-3. As can be seen in Fig. 5, the proportion of USDA110-type of Tamahomare, Enrei and Tambaguro in the fourth cycle were further decreased compared to those in the third cycle. This result is consistent with those previously obtained on this study, confirming the further decrease in the population of USDA110-type under continuous cropping conditions. The proportions of USDA110-type among all seedlings increased from 10% to 38% in response to daidzein treatment. The proportion of USDA110-type of Tambaguro was higher in response to daidzein treatment compared with those of Tamahomare and Enrei.
3.2. Amount of root-secreted isoflavonoids An HPLC analysis of root-secreted isoflavonoids from Tamahomare, Enrei, Tambaguro and other genotypes (Peking, Shintambaguro, Fukuyutaka, William 82, Norin 2, Tachinagaha and Iyodaizu) was performed. The average amount of root-secreted daidzein from oneweek old Tamahomare, Enrei and Tambaguro seedlings in 48 h were 20.28, 14.19, and 127.31 μg/seedling, respectively (Fig. 4). The amount of root-secreted daidzein was significantly different among genotypes (p < 0.001). All the genotypes can be classified in two groups: one group with high amount of root-secreted daidzein (Tambaguro, Peking, William 82 and Shintambaguro) and another group with low amount (Tamahomare, Enrei, Norin 2, Fufuyutaka, Tachinagaha and Iyodaizu). For the root-secreted genistein, trace amounts of secretion in all genotypes, which ranged from 0.61 μg to 1.29 μg, were found. Therefore, the current results were almost similar to those gotten in our previous study, and confirm that daidzein is the main root-secreted isoflavonoid of soybean – as mentioned in the preceding studies of Cesco et al. [9] and Sugiyama et al. [17].
4. Discussion Prior work on soybean-rhizobia symbiotic relationship has never focused on the evolution of rhizobial communities in the field. None 5
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Fig. 3. Decreases of the proportion of USDA110-type of Tamahomare, Enrei and Tamabaguro from the first cycle to the third cycle (%) under all experiment conditions. Values are means across four replicates.
Fig. 5. Effect of soybean seed treatment with daidzein on the proportion of nodule bacterial type for Tamahomare, Enrei, and Tambaguro. 3rd cycle, 4th cycle and 4th cycle (with daidzein) represent proportions of the nodule USDA110-type of the seedlings at third cycle, at fourth cycle without and with daidzein treatment, respectively (Exp-3, using Tambaguro soil sample). Values are means across four replicates.
and co-workers [22] demonstrated that the addition of root exudates collected from liquid medium to soils was found to modify the microbial composition of these soils, depending on the fractions of root exudates. Root exudates are composed of sugars, amino acids, flavonoids, proteins, and fatty acids [23]. These substances, documented Bais and colleagues [24], can serve as growth substrates or signals for suitable microbial partners and as antimicrobials or growth deterrents for other microbes. Bradyrhizobium has been shown to be abundant in rhizosphere soil – soil around the roots – because of its chemotaxis with the root exudates of soybean [8,25]. Additionally, root exudate contents and concentrations are correlate with the metabolic activities of bacterial communities in rhizospheres [26,27].
Fig. 4. Boxplot representation of root-secreted daidzein and genistein from oneweek-old Tamahomare, Enrei, Tambaguro, Peking, Shintambaguro, Fukuyutaka, William 82, Norin 2, Tachinagaha and Iyodaizu seedlings. The dots represent the total amount of root-secreted isoflavonoid of each plant in 48 h (4 replicates).
previous report is, therefore, comparable to the current investigation. Herein, it was showed – in all experimental conditions – that the proportion of USDA110-type decreases significantly during successive soybean cropping. This decrease in USDA110-type must be a result of accumulation of root exudates in the soil and other environmental conditions. It is, of course possible to object those reasons, but Badri 6
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continuous cropping (Figs. 3 and 4 and Fig. A2). This suggests that the high accumulation of daidzein increases the competitiveness of USDA110. Consequently, the treatment of seeds with daidzein increased the proportion of USDA110 (Fig. 5). These findings extend those of Guan et al. [44], confirming that when treated with daidzein, a large proportion of proteins associated with nodulation and the metabolism of energy and material – the reason for its good symbiotic matching for nodulation – are upregulated in B. diazoefficiens USDA110-A, while nodulation-related proteins and defensive proteins are downregulated in B. diazoefficiens 2178. Supporting these finding also, a comparison of the competitiveness of two B. diazoefficiens strains for nodulation investigated by Li and co-workers [45] proved that a large proportion of proteins associated with nodulation are upregulated in the highly competitive B. diazoefficiens 4534 treated with daidzein. Daidzein regulates the expression of hopanoid synthesis genes in rhizobia [46]. Hopanoids are pentacyclic triterpenoid lipids that are widely occurring in bacteria, which act as membrane reinforcers, conferring resistance to different environmental stresses. In bradyrhizobia, these compounds can account for nearly half of the total cell lipid fraction [47]. Daidzein also has been considered to be a type of signaling molecule that is associated with competitiveness [18], although the maximal response of B. diazoefficiens USDA110 to daidzein is 50% or less compared with that of genistein [48]. In conclusion, though the results could be partly influenced by the experimental design, the present study discovered that the population of the efficient nodulating strain B. diazoefficiens USDA110 decreases in successive cropping, which is a factor in the decrease in shoot dry weight. There is a strong possibility that this alteration of nodulating rhizobial community is due to the accumulation and decomposition of root exudates. The accumulation of daidzein might be able to rescue the ability of B. diazoefficiens USDA110. Some limitations are worth noting. Although the hypotheses were supported statistically, the evolution of Bradyrhizobum and daidzein in soil was not assessed. Future work should therefore include follow-up work designed to evaluate how isoflavonoids are degraded and how much exactly nodulating rhizobial population is altered in the field soil in long-term cultivation of soybean. Therefore, further investigation is needed to determine the amounts of root exudate residues, including daidzein and genistein, in soybean fields.
After having discussed the root exudates, let us look at the environmental conditions. The results (Fig. 3) displayed that the intensities of the decrease in USDA110-type from the first cycle to the third cycle were statistically different among experiments. The decrease in the USDA110-type was more intense in the vermiculite medium (Exp-3) compared to a mixed soil medium (Exp-1 and Exp-2), because growing soybean plants under laboratory conditions using soil samples diluted with vermiculite might undermine the possible effects of soil properties on the rhizobial nodulating properties [28]. This decrease in USDA110-type was also even more intense in high-temperature conditions (Exp-2) than in low-temperature conditions (Exp-1). B. diazoefficiens is dominant in the soils of cooler environments in Japan and Nepal [29–31]. Moreover, relationships exist between the geographic distribution of indigenous soybean-nodulating rhizobia, soil temperature (and its variations because of latitude and altitude), and soil properties, such as soil pH [32,33]. B. elkanii strains showed growth stability at varying temperatures and pH levels [32,34], and widely distribution in acid soil [35]. It is also necessary to discuss further the importance of B. diazoefficiens USDA110 in symbiotic relationship with soybean. B. diazoefficiens USDA110 has greater N2 fixation potential than the other species, resulting in greater biomass production than with unselected strains [36]. In addition, Rj2, Rj3, and Rj4 – the loci that control the formation of symbiotic root nodules in soybean plants [37] – conferred improved nodulation when inoculated with B. diazoefficiens USDA110 [38]. B. diazoefficiens USDA110 is used in many countries as an inoculant to increase soybean yield [39]. Furthermore, B. diazoefficiens can form nodules faster than B. elkanii [34], but B. elkanii strains produce rhizobitoxine, a compound that induces chlorosis in the host plant, and the strains are relatively inefficient symbionts for soybean [40–42]. It was noted that the shoot dry weights of Tamahomare, Enrei and Tambaguro were highly decreased as the number of times of cultivation increased (Fig. A3) and that the seedlings in the third cycle of cropping were susceptible to chlorosis (Fig. A4b). Rhizobitoxine increases nodule formation via the inhibition of endogenous ethylene synthesis in host plants because ethylene restricts nodulation in many legumes [41,43]. Rhizobitoxine production by B. elkanii USDA94 gave the bacterium a nodulation competitiveness approximately 10 times higher than that of a non-rhizobitoxine-producing mutant strain on Macroptilium atropurpureum [41,43]. Rhizobitoxine production correlates with the amount of rtxC transcript in B. elkanii USDA94 [42]. Bolzan de Campos et al. [15] and Ramongolalaina et al. [16] reported that the Peking cultivar can efficiently establish a symbiotic relationship with B. elkanii because the transcriptional regulator TtsI used to activate the type III secretion system of B. elkanii displays host-dependent characteristics [15]. Up to this point, the discussion has been focusing on B. diazoefficiens USDA110, but what does it have to do daidzein? The results provide compelling evidence of the involvement of daidzein in alteration of B. diazoefficiens USDA110. Genotypes (Tambaguro, Peking, William 82 and Shintambaguro) with a high amount of root-secreted daidzein corresponded to the moderate decrease in the USDA110-type in
Funding This research was conducted using no grant from funding agencies in the public, commercial, or not-for-profit sectors. Acknowledgement I am very grateful to my previous advisors, Dr. Okumoto Yutaka and Dr. Teraishi Masayoshi (Plant Breeding laboratory, Department of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University), for allowing me to carry my experiments in their laboratory and, most importantly, for granting me to publish this work.
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejsobi.2019.103098.
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Appendices
Fig. A.1. Proportions of the three nodule bacterial types found in each nodule Peking, Shintambaguro, Fukuyutaka, William 82, Norin 2, Tachinagaha and Iyodaizu throughout the three continuous cropping cycles in Exp-3 (Tambaguro field soil samples).
Fig. A.2. Decreases of the proportion of USDA110-type of Tamahomare, Enrei, Tambaguro, Peking, Shintambaguro, Fukuyutaka, William 82, Norin 2, Tachinagaha and Iyodaizu from to 1st cycles to the 3rd cycles (%) in Exp-3 (Tambaguro field soil samples).
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Fig. A.3. Shoot dry weight (mg) of Tamahomare, Enrei and Tambaguro throughout the three continuous cropping cycles.
Fig. A.4. a) Harvested seedlings of Tamahomare, Enrei, Peking and Tambaguro at the second cycle in Exp-3 (Tambaguro field soil samples). b) Patterns of the leaves of Tamahomare seedlings at the three continuous cropping. c) Seedlings in hydroponic culture system, ready for root-secreted isoflavonoids (right after their growth in an incubator).
Supplementary data and tables Table. A.1 Textural and chemical properties of the soil samples. Table. A.2 Results of sequencing analysis of each bacterial type. Table. A.3 Increases in the proportion of USDA94-type from the thirst to the third cycles Reference Data Whole data.xlsx - 3_cycles: Proportions of the three nodule bacterial types found in each nodule of Tamahomare, Enrei and Tambaguro throughout the three continuous cropping cycles, and root dry weight (mg) of Peking, Tamahomare, Enrei and Tambaguro throughout the three continuous cropping cycles. - Daidzein_in_soil: Proportions of the nodule bacterial type without incubation and incubation of seeds in daidzein solution of the seedlings at fourth cycle of Exp-3 (using Tambaguro soil sample). - Other_genotypes: Proportions of the three nodule bacterial types found in each nodule Peking, Shintambaguro, Fukuyutaka, William 82, Norin 2, Tachinagaha and Iyodaizu throughout the three continuous cropping cycles in Exp-3 (Tambaguro field soil samples). - Statistic test: oneway ANOVA analysis of differences of the proportion of each identified bacterial type between themselves, treatments, cycles, 9
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and soybean genotypes - Root-secreted daidzein and genistein.xlsx: Method to calculate the amounts of root-secreted daidzein and genistein and their values from oneweek-old Tamahomare, Enrei, Tambaguro, Peking, Shintambaguro, Fukuyutaka, William 82, Norin 2, Tachinagaha and Iyodaizu seedlings.
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Abbreviations DMSO: Dimethyl sulfoxide HPLC: High-Performance Liquid Chromatography ITS: Internal Transcribed Spacer PCR: Polymerase Chain Reaction rRNA: ribosomal Ribonucleic Acid RFLP: Restriction Fragment Length Polymorphism T3SS: Type III Secretion System
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