Production of Soybean Plants for Hydroponic Cultivation from Seedling Cuttings in a Medium Containing Rhizobium Inoculum Depending on Various Concentrations of Nutrient Solution and Different Nitrogen Sources

Seeds of soybean cultivar Tambaguro were sown in 30 × 40 cm plastic trays filled with commercially available nursery soil (Type S; Yanmar, Japan). The trays were then covered with wet newspapers until the seedlings emerged. All the trays were placed in an incubator set at 25 °C, in darkness. After emergence (3 days after sowing), seedlings were transferred to a greenhouse at the Experimental Agricultural Facility of the University of Shiga Prefecture. At the primary leaf stage (8 days after sowing), uniformly growing seedlings were selected from the trays and their hypocotyls were cut 4 cm below the cotyledonary node.

The hypocotyledonous stems of the cuttings were individually inserted into polyvinyl chloride (PVC) tubes (45 mm tall and 56 mm internal diameter), containing 100 ml silica sand (No. 4F; Toyo Matelan, Japan) mixed with 200 mg commercial Rhizobium inoculant, which contained rhizobia mixed with peat (Mamezo; Tokachi Federation of Agricultural Cooperative Association, Japan). The bottom of each PVC tube was covered with root-resistant and water-permeable sheet (BKS9812; Toyobo, Japan) to enable capillary watering without collapsing the rooting medium (silica sand with a Rhizobium inoculant). Ten PVC tubes with cuttings were placed on one tray for subirrigation from the bottom side of each PVC tube with a capped 2 dm³ plastic bottle that refilled with a fresh solution as necessary. The plastic bottle had a small hole (8 × 8 mm) at the base about 2 mm from the bottom to maintain a relatively constant water level of about 1 cm depth in the tray (Fig. 1). Plants were subirrigated with tap water for the first week and then with the respective nutrient solutions for another 2 weeks.

Fig. 1

Seeds were sown as described above on May 27, 2016, and seedling stem cuttings were inserted into the rooting medium on June 4, 2016. Plants were cultivated for 3 weeks under natural sunlight without shading in a greenhouse, and irrigations with the nutrient solution were initiated after the first week. The treatments consisted of a nitrogen-free Enshi nutrient solution diluted to six concentrations (0, 10, 25, 50, 75, and 100% of full-strength).

The full-strength complete Enshi nutrient solution contained the following levels of salts per 1,000 dm³ tap water: 950 g Ca(NO₃)₂ · 4 H₂O, 810 g KNO₃, 500 g MgSO₄ · 7 H₂O, 155 g NH₄H₂PO₄, 3 g H₃BO₃, 2 g ZnSO₄ · 7 H₂O, 2 g MnSO₄ · 4 H₂O, 0.05 g CuSO₄ · 5 H₂O, 0.02 g Na₂MoO₄, and 25 g NaFe-EDTA. In the nitrogen-free solution, Ca(NO₃)₂ · 4 H₂O, KNO₃, and NH₄H₂PO₄ were substituted with CaCl₂ · 2 H₂O, K₂SO₄, and KH₂PO₄. Each solution was prepared by mixing the full-strength solution and tap water (the 0% solution consisted of only tap water). As normal growth control, additional seedling stem cuttings grown in rooting medium without the Rhizobium inoculant were similarly irrigated with half-strength complete Enshi nutrient solution. All the nutrient solutions were adjusted to pH 6.0 with H₂SO₄ before use.

The sowing and cutting dates were June 20 and 28, 2016, respectively. The cultural management of the cuttings was the same as in Experiment 1, except nitrogen-containing nutrient solutions were used (Table 1). Plants were irrigated with Enshi nutrient solutions at four concentrations (0, 10, 25, and 50% of full-strength), with nitrogen source as nitrate (NO₃-N), ammonium (NH₄-N), or urea (Urea-N). Half-strength nutrient solutions were prepared for each nitrogen source and diluted with tap water. Similar to Experiment 1, uninoculated plants cultivated with half-strength complete Enshi nutrient solution were served as normal growth control. All the nutrient solutions were adjusted to pH 6.0 with H₂SO₄ before use.

At 3 weeks after cutting, the following parameters were measured for 10 individuals per treatment: main stem length, number of trifoliate leaves, green leaf color intensity and number of nodules per root system. The green leaf color intensity of fully expanded second trifoliate leaves was measured with the SPAD-502 chlorophyll meter (Minolta Camera, Japan). After removing the rooting medium, the number of nodules ≥ 2 mm in diameter was determined. Roots and shoot were separated and dried in an oven at 60 °C, after which the constant dry weight was recorded. Differences in the values of the samples (n = 10) were analyzed with the Tukey-Kramer multiple comparison test following an ANOVA.

The rate of adventitious rooting of cuttings was as high as 100% without any auxin treatment. In all the rooting media with an inoculant, nodulated adventitious roots arose from the base of the cuttings (Fig. 2). Nodules on the adventitious roots were mainly distributed at the neighboring stem base, although some formed on the adventitious roots apart from the stem or the surface of the rooting medium.

Fig. 2

The growth of the rooted cuttings varied, but the highest values for the recorded shoot parameters were obtained for the normal growth control samples (Fig. 3 & 4A–D). The main stem length, number of trifoliate leaves, green leaf color intensity and shoot dry weight were significantly greater for the uninoculated plants supplied with nitrogen than for the inoculated plants not supplied with nitrogen. Regarding the nitrogen-free treatments, there were only small differences in the main stem length and number of trifoliate leaves among the treatments. The lowest and highest shoot dry weights resulted from the treatments with 0% and 25% dilutions, respectively, whereas they were not significantly different among plants of the other dilutions. However, the green leaf color intensity decreased substantially with increasing nutrient solution concentrations. Leaves turned yellow from the base towards the top of the plants, which was reflected in the green leaf color intensity.

Fig. 3

Fig. 4

In contrast to shoot growth, root growth was not significantly promoted by the application of the half-strength complete Enshi nutrient solution (Fig. 4E). Inoculated plants treated with the nitrogen-free nutrient solution, except the 0% dilution (i.e., tap water), tended to develop more adventitious roots than the uninoculated control plants, with the highest root dry weight for 25% dilution. Because semi-aseptic culture conditions were provided with the combination of silica sand and cuttings, uninoculated control plants never formed root nodules (Fig. 4F). When inoculated, the number of nodules per root system was higher for the 0% and 10% dilutions, and decreased with increasing nutrient solution concentrations.

The nutrient solution had a considerable effect on plant growth in a concentration-dependent manner (Fig. 5). Specifically, the nutrient solution promoted growth when nitrate or urea was used as the nitrogen source. The ammonium fertilization inhibited shoot and root growth compared with the effects of the nitrate, urea and tap water treatments. The leaves of the ammonium-fed plants became chlorotic and/or necrotic, which was more pronounced in the upper plant parts. The severity of these symptoms increased with increasing nutrient concentrations.

Fig. 5

The composition of the nutrient solution applied to the normal growth control was almost the same as that of the 50% dilution with nitrate as the sole nitrogen source, with the exception that the former contained a small amount of ammonium. Therefore, the shoot growth parameters were very similar for these two treatments, regardless of whether an inoculant was included in the rooting medium (Fig. 6A–D). The main stem length, number of trifoliate leaves, and shoot dry weight tended to increase with increasing concentrations from 0% to 50% for the plants treated with nitrate, whereas the stem length peaked with 25% dilution. These tendencies were also observed for the plants treated with urea, but there were no significant differences in the number of trifoliate leaves and shoot dry weight for the concentrations between 10% and 50%. For both the nitrate and urea treatments, the leaves from plants with the 0–25% dilutions were significantly less green than the leaves from the control plants, but the green leaf color intensity was the same for the plants treated with 50% dilution and the control plants at 5% significance level. At the same concentration, the shoot dry weight of the urea-fed plants was lower than that of the nitrate-fed plants, especially at higher concentrations. Regarding the ammonium treatment, there were no significant increases in the main stem length and number of trifoliate leaves for dilutions up to 50%. Furthermore, ammonium levels exceeding 25% significantly decreased the green leaf color intensity and shoot dry weight, with values lower than those of the plants treated with tap water.

Fig. 6

The root dry weights of the plants exposed to the no-nutrient treatment and the control plants were not significantly different (Fig. 6E). The positive effects of nitrate were observed for concentrations up to 25%, with a maximum weight of 0.81 g, which was approximately 28.5% higher than that resulting from the 0% treatment. The urea fertilization did not promote root growth, and the root dry weight was lower for the urea-fed plants than for the nitrate-fed plants at all concentrations. Both the nitrate- and urea-treated plants yielded more root dry matter than the ammonium-treated plants. Increases in the ammonium concentration of the nutrient solution resulted in a linear decrease in the root dry weight.

The number of root nodules on the adventitious roots was highest for the plants treated with the 10% nitrate-based solution, followed by the plants that underwent the 25% nitrate, 10% urea, and 25% urea treatments (Fig. 6F). Fewer root nodules were detected following the control (i.e., tap water) and 50% nitrate treatments. The 50% urea fertilization significantly inhibited root nodule formation. In addition to the uninoculated control plants, all ammonium-fed plants lacked root nodules, although they were inoculated similarly to the plants that underwent the nitrate and urea treatments.

The culture vessel used in this study, consisting of a PVC tube and a root-resistant and water-permeable sheet, has been used for growing tomato (Suzuki et al. 2011) and melon (Kawahara & Masuda 2012) seedlings in a subirrigation system. The results of the current study indicate that such a system can also be used to produce soybean transplants from seedling stem cuttings. Adventitious root formation was detected on all the cuttings within 7 days of cutting without any auxin treatment, suggesting that the adventitious rooting of soybean cuttings is promoted by large amounts of storage compounds in the cotyledons and not inhibited by the moisture status of silica sand under subirrigation system with a 1 cm depth.

The Rhizobium biofertilizer ‘Mamezo’ is usually applied as a pre-sowing seed dressing, but this study indicated that it can be incorporated in the rooting media under subirrigation as an inoculant. In a previous study on soybean plants under flooding conditions, the adventitious roots represented approximately 90% of the total root length, with root nodules on the adventitious roots, but not on the primary and lateral roots (Hattori et al. 2013). Accordingly, inoculating the adventitious roots of seedling stem cuttings may be better than inoculating seedling roots. Moreover, nodulated roots were not detected on uninoculated plants, implying that the silica sand used in this study, which was manufactured by crushing rocks, was nearly sterile, including rhizobial bacteria. Such conditions might provide Rhizobium species with an ecological niche suitable for root nodulation.

On the basis of our findings, we concluded that the subirrigation system may be used for the rooting and inoculation of soybean cuttings to produce transplants in a substrate-based hydroponic system.

According to Saito et al. (2014), the application of combined nitrogen, especially nitrate, inhibits the root nodule formation, growth, and nitrogen fixation activity of soybean plants. Therefore, we assumed that a nitrogen-free nutrient solution can provide other essential minerals without inhibiting nodulation, which can help induce the growth of inoculated plants that obtain nitrogen generated by symbiotic nitrogen fixation. However, we unexpectedly observed that the green leaf color intensity and the number of nodules per root system decreased with increasing concentrations of the nitrogen-free nutrient solution, implying the nitrogen-free nutrient solution did not promote the growth of inoculated plants (Fig. 3 & 4).

Soybean seeds are large and the storage compounds in the cotyledons can provide the nutrients required for the initial root and shoot growth (Ohyama et al. 2017). The relatively large amount of the storage compounds can partly explain why inoculated plants subirrigated with tap water were able to grow without any symptoms associated with a deficiency in essential minerals, including nitrogen, for 3 weeks (Fig. 3). However, when treated with a nitrogen-free nutrient solution at a concentration of 25% or more, the yellowing of leaves increased from the base to the top of the plants, which exhibited symptoms typically associated with nitrogen deficiency. These results suggest that the internal ratio of nitrogen to other essential minerals in plants following a nitrogen-free nutrient solution treatment might be lower than that resulting from tap water treatment. This decreased ratio may trigger stress responses to nitrogen deficiency.

The decrease in the number of root nodules was correlated with the decrease in the green leaf color intensity (Fig. 4). The yellowing of leaves indicates a loss of chlorophylls, which leads to a decrease in photosynthetic ability. Because nodule formation and activity require a considerable abundance of photosynthetic products provided by the host plant (Finn & Brun 1982), the number of root nodules and growth did not increase for the inoculated plants treated with high nitrogen-free nutrient solution concentrations. To avoid growth inhibition due to leaf yellowing accompanied by decreased nodule formation and activity, we propose that Rhizobium-inoculated plants should be subirrigated with a nutrient solution containing sufficient nitrogen to maintain an appropriate internal nitrogen ratio and to promote early growth up to the level of uninoculated control plants.

As expected, the green leaf color intensity of inoculated plants did not decrease with increasing nutrient solution concentrations when nitrogen was supplied as nitrate (Fig. 5 & 6C). Furthermore, urea also enhanced the green leaf color intensity. In both nitrate- and urea-fed plants, the internal nitrogen status, reflected by leaf greenness, may be enough to maintain photosynthetic ability. However, the chlorotic and/or necrotic lesions of the upper leaves of ammonium-fed plants increased in severity with increasing nutrient solution concentrations, likely because of ammonium toxicity (Tadano & Tanaka 1976; Ikeda & Osawa 1979; Yoneyama et al. 1985) rather than nitrogen deficiency. Although Xia et al. (2017) reported that the growth of soybean seedlings in a sand substrate was promoted by irrigation with a nutrient solution containing up to 100 ppm NH₄-N (7.14 mM), our results suggest that silica sand is nearly sterile, with minimal nitrifying bacteria that convert ammonium to nitrate, and the excess of ammonium remaining in the rhizosphere prior to absorption might result in ammonium toxicity, even when plants are subirrigated with a 25% treatment solution (4.33 mM NH₄-N).

Because the number of root nodules following the tap water treatment may have been underestimated more in Experiment 2 than in Experiment 1, it is unclear whether 10–25% nitrate fertilization will lead to more root nodules than the tap water treatment. However, our data indicate that nutrient solution concentrations up to 25% (4.33 mM NO₃-N) do not adversely influence nodulation (Fig. 6F). Although nitrate has long been known to strongly inhibit nodulation and the N₂ fixation activity in legumes, low concentrations of nitrate (1–2 mM) actually promote nodulation by ensuring early, rapid growth of the plant and the development of a healthy root system able to nodulate profusely (Giller & Wilson 1991; Ohyama et al. 2011). Furthermore, Xia et al. (2017) reported that the nodulation and N₂ fixation activity of soybean plants initially increase and then decrease with increasing nitrate concentrations, with the activity peaking at 50 ppm nitrate (3.57 mM). These results imply that the nitrate concentration can be increased to approximately 4 mM to promote the early plant growth and nodulation of inoculated soybean plants under sub-irrigation conditions. A treatment with 5 mM nitrate reportedly inhibits the growth and nitrogen fixation activity of soybean nodules by decreasing the amount of photosynthetic products supplied to the nodules (Ohyama et al. 2011; Saito et al. 2014). Thus, it is reasonable that the number of root nodules decreased when plants were treated with the 50% dilution (8.65 mM NO₃-N). Accordingly, nitrate concentrations exceeding 5 mM are unsuitable for cultivating inoculated soybean plants, even if they promote shoot growth (Fig. 6A–D).

According to Yoneyama et al. (1985) and Ohyama et al. (2013), the inhibitory effect of nitrate is stronger than that of ammonium or urea for the nodulation and nitrogen fixation activity of soybean plants. However, to our surprise, ammonium-fed plants never formed root nodules irrespective of the ammonium concentration (Fig. 6F), although soybean seedlings grown in a sand substrate can form many nodules, even when irrigated with 100 ppm NH₄-N (7.14 mM) (Xia et al. 2017). The excess ammonium in the rhizosphere due to the limited nitrification in semi-aseptic silica sand might have contributed to the plant injury observed as decrease in plant biomass (Fig. 6A–D) in addition to leaf chlorosis and/or necrosis. This is supported by the previous reports describing the severe injuries to hydroponically grown soybean plants caused by ammonium toxicity, even at low concentrations (2–3 mM NH₄-N) (Tadano & Tanaka 1976; Ikeda & Osawa 1979). Therefore, the decreased growth due to ammonium toxicity likely severely disrupts nodulation, even if the ammonium weakly inhibits nodulation.

Unlike the effects of ammonium, the urea treatment promoted the shoot growth (Fig. 6A–D) and nodulation (Fig. 6F) of inoculated plants, but less effectively than nitrate. The growth retardation of urea-fed plants may be partially explained by a previous report indicating that reduction of plant growth at the beginning of the growing cycle is likely related to the low use efficiency of urea in soybean seedlings (Paradiso et al. 2014b). Additionally, the long-term supply of urea as the sole nitrogen source results in a lower vegetative biomass and seed yield of hydroponically grown soybean plants compared with the effects of nitrate (Paradiso et al. 2014b, 2015). Regarding nodulation, several reports indicated that urea-fed plants formed more and larger root nodules than nitrate-fed plants (Yoneyama et al. 1985; Paradiso et al. 2014b, 2015), but these observations were following treatments with high nitrogen concentrations (7.5–20 mM), which are highly inhibitory to nodulation, especially when supplied as nitrate. Therefore, urea can be used as the nitrogen source for promoting the early growth of inoculated soybean plants, but may not be necessarily superior to nitrate for nodulation at relatively low nitrogen concentrations.

In this study, nitrate or urea fertilization at 25% dilution enhanced the growth and nodulation of inoculated plants, but the shoot growth at the transplanting stage was not comparable to the growth of uninoculated plants treated with the 50% nutrient solution (Fig. 6). The diversity in shoot growth may have been caused by differences in the supply of essential minerals other than nitrogen. Accordingly, the optimum ratio of the nitrogen content in 25% solution to the content of the other essential minerals should be determined. However, if the growth retardation is mainly due to a limited fixed nitrogen supply from the nodules during the early growth stage (Hamawaki & Kantartzi 2018; Cafaro La Menza et al. 2020), it may be restored in the later growth stages by increasing the amount of biologically-fixed nitrogen supplied to plants, and further improvements, except for enhanced nodulation, may not be required for the management of inoculated plants, at least during the early growth stage.