Selection of type A and type B strains for improving symbiotic effectiveness on non-Rj and Rj₄ genotype soybean varieties

ABSTRACT

The selection of effective rhizobia for higher efficiency nitrogen fixation is one of the most important steps for inoculant production. Therefore, this experiment was conducted to select the most effective type A and type B strains for specific Rj-gene harboring soybean varieties and to test the symbiotic effectiveness of selected strains on different Rj-gene harboring soybean varieties. Screening experiments using the specific soybean varieties were done with a completely randomized design and three replications in this study. Evaluation of the effective Myanmar Bradyrhizobium strains for plant growth, nodulation and N₂ fixation were studied in pot experiments using sterilized vermiculite in the Phytotron (controlled-environmental condition). Then, a pot experiment was conducted using Futsukaichi soil in the screen house (natural environmental condition). The N₂ fixation ability of soybean was evaluated by acetylene reduction activity (ARA) and the relative ureide index method. In the first screening experiment, type A and type B strains with higher nitrogen fixation and proper nodulation on their respective soybean cultivars were selected for the next screening. In the second screening, Bradyrhizobium elkanii AHY3-1 (type A), Bradyrhizobium japonicum SAY3-7 (type A), B. elkanii BLY3-8 (type B) and B. japonicum SAY3-10 (type B) isolates, which showed higher nitrogen fixation and nodulation in Yezin-3 (Rj₄) and Yezin-6 (non-Rj), were selected for the next experiment. In the third screening experiment, SAY3-7 and BLY3-8, which had higher nitrogen fixing potential and proper nodulation, were selected as effective isolates. These two isolates were compatible with non-Rj and Rj₄ soybean varieties for nodulation and nitrogen fixation. Based on the results of the screening experiment, these two strains were tested for their symbiotic efficacy in Futsukaichi soil. This study shows that inoculation treatment of SAY3-7 and BLY3-8 significantly increased plant growth, nodulation, and N₂ fixation at the V6, R3.5 and R8 stages in Yezin-3 (Rj₄) and/or Yezin-6 (non-Rj), and the seed yield at R8 stage, in Yezin-3 (Rj₄) and Yezin-6 (non-Rj) soybean varieties compared with the control treatment. It can be concluded that SAY3-7 and BLY3-8 are suitable for inoculant production because of their higher nitrogen fixation ability, proper nodulation and better productivity of Myanmar soybean cultivars.

KEY WORDS:

• Nodulation type
• nodulation regulatory gene (Rj gene)
• nitrogen fixation
• nodulation
• seed yield

1. Introduction

Soybean seeds are a major source of protein, oil, carbohydrate, and minerals for human and animal nutrition. Proteins, oil, carbohydrates, and minerals constitute 36%, 19%, 35%, and 5%, respectively, of the total soybean dry weight (Liu 1997). In addition to providing nutritious food, the soybean increases soil fertility (FAO 1984). The yield of soybeans (1.51 tons ha⁻¹) in Myanmar is still low compared to the world average yield (2.52 tons ha⁻¹; MOAI 2015). This reduction of yield is related to inadequate nitrogen application and lack of inoculation of an alternative source of nitrogen fertilizer.

Inoculation is the treatment of the seed or soil with inoculants of specific bacterial strains (NifTAL 1990). The aim of inoculation is to provide sufficient numbers of viable, effective rhizobia to enhance rapid colonization in the rhizosphere to form nodules as soon as possible after germination and thereby promote optimal crop yields (Catroux 1991). In many soils, the strain, number, quality, or virulence of indigenous nodulating bacteria is not adequate for nitrogen fixation (FAO 1984). Therefore, inoculation against seeds or soils with effective strains is necessary for successful nodulation because biological nitrogen fixation (BNF) depends on effective nodules.

Soybeans, in symbiosis with Bradyrhizobium, have the ability to fix nitrogen at a rate of up to 300 kg N ha⁻¹ under favorable conditions (Smith and Hume 1987). However, the symbiosis is dependent on host specificity. This specificity might also be related to the nodulation regulatory genes of soybean cultivars and nodulation types of rhizobia, especially in the soybean. Ishizuka et al. (1991a); (1991b) tested the compatibility and preference of Rj-genotype soybean cultivars with specific Bradyrhizobium strains. The Bradyrhizobium strains are classified into nodulation types A, B, and C, based on their compatibility with Rj cultivars. Type A strains are preferred by the non-Rj genotype soybean cultivars and nodulate with all the Rj-genotype soybean cultivars. Type B strains are preferred by Rj₄ genotype soybean cultivars and inhibit nodulation with the Rj₂Rj₃ genotype soybean cultivars. Type C strains are preferred by the Rj₂Rj₃ cultivars and restrict effective nodule formation with the Rj₄ genotype soybean cultivars.

The responses of soybean cultivars vary with the Rhizobium strains. While some cultivars are fully compatible with other Rhizobium, others cannot form nodules, although the Rhizobium belongs to same serogroups (Van et al. 2007). This might be due to nodulation regulatory genes called Rj genes found in the soybean cultivars. The Rj-genotypes (non-Rj, rj₁, Rj₂, Rj₃, and Rj₄) have been found to exist in nature (Devine and Kuykendall 1996). In Myanmar, non-Rj, Rj₂Rj₃, and Rj₄ genotypes soybean cultivars were reported in a previous study (Htwe et al. 2015b). Among them, Rj₄ genotype soybean cultivars are widely grown in Myanmar and account for 60% of all the cultivars. Devine and Kuykendall (1996) reported that more than 60% of the soybean cultivars in Southeast Asia have the Rj₄ gene. In Myanmar, Soe et al. (2013) reported that six cultivars, such as Hinthada, Southern Shan local, Northern Shan local, Yezin-3, Yezin-11, and Daewoncong, were Rj₄ genotype soybean cultivars, but the remaining eight cultivars, such as Shan Sein, Shan Wha, Yezin-6, Yezin-8, Yezin-14, Daepung, Cheongga-3, and Zhongpin-661, were non-Rj genotype soybean cultivars among the 14 soybean cultivars. From their results, non-Rj and Rj₄ genotype soybean cultivars are widely grown in Myanmar. Therefore, selection of the type A strain, which prefers non-Rj genotype soybean cultivar, and type B strains, which prefer to Rj₄ genotype soybean cultivars, are important steps for increasing nodulation and nitrogen fixation. Moreover, selected type A and type B strains can be useful as effective inoculants for soybean cultivation in Myanmar, where non-Rj and Rj₄-gene harboring soybean cultivars are widely grown.

In Myanmar, the use of Rhizobium for soybean production is still limited. To promote soybean productivity, it is necessary to select strains compatible with cultivars. Therefore, the goal of the present study was to select the most effective type A and type B strains for non-Rj and Rj₄-gene harboring soybean cultivars and to evaluate the symbiotic effectiveness of selected Bradyrhizobium type A and type B isolates on non-Rj and Rj₄-gene soybean cultivars.

2. Materials and methods

2.1. Inoculant preparation

Bradyrhizobium diazoefficiens USDA110 was obtained from the Laboratory of Plant Nutrition, Kyushu University. Indigenous Bradyrhizobium strains were obtained from a previous experiment (Htwe et al. 2015a). The origins of Myanmar indigenous Bradyrhizobium strains are presented in supplementary Table 1. Bradyrhizobium strains were cultured in A1E liquid media (Kuykendall 1987) on a rotary shaker (100 rpm) at 30°C for 7 days.

2.2. Soybean varieties

Myanmar soybean varieties [Yezin-3 (Rj₄), Yezin-6 (non-Rj), Yezin-11 (Rj₄), and Yezin-14 (non-Rj)] were collected from the Food Legumes Section, Department of Agricultural Research, Yezin, Myanmar. Their Rj genes were identified by (Soe et al. 2013; Htwe et al. 2015b). Rj₄ and non-Rj in the parenthesis refer to the nodulation regulatory genes (Devine and Kuykendall 1996).

2.3. Cultivation, crop management, plant sampling, and analysis of screening experiments under controlled environmental conditions

The 1 L pots were filled with vermiculite and 0.6 L of half-strength modified Hoagland nutrient (MHN) solution (Nakano et al. 1997). The pots were autoclaved at 120°C for 20 min. Surface sterilization of the seeds was done by soaking them in 2.5% sodium hypochlorite solution for 5 min, rinsing five times with 10 mL of 99.5% ethanol, and washing five times with sterilized MHN solution. Five surface sterilized seeds were sown in the pots. The liquid bacterial cultures were diluted with sterilized MHN solution to 10⁷ cells mL⁻¹. Each seed was inoculated with 5 mL of bacterial suspension. The plants were cultivated in an environmentally controlled room (25°C and 75% relative humidity) for 30 days. In Myanmar, soybean is cultivated from November to February with residual moisture after rice cultivation. From November to February, the average monthly temperature is between 20°C and 25°C. Therefore, we set the temperature at 25°C for screening experiments to obtain better soybean growth. Completely randomized design was used with three replications. Watering was done as necessary using autoclaved deionized water. At the harvest time, N₂ fixation was analyzed using an acetylene reduction assay (ARA) as described by Haider et al. (1991). After the assay, nodules were counted. Shoots, roots and nodules were collected separately and oven dried at 70°C for 24 h to record dry weights. This experiment was conducted from January 2017 to June 2017.

2.4. Cultivation, crop management, plant sampling and analysis of pot experiment under natural environmental conditions

Futsukaichi soil was collected from Kamigoka, Chikushino city, Fukuoka prefecture, Japan. Before cultivation, the indigenous rhizobial population of the Futsukaichi soil was evaluated by the most probable number (MPN) method (Vincent 1970) using Yezin-3 (Rj₄) as the host plant. The estimated rhizobial population was 2.9 × 10³ rhizobia per 1 g dried soil. For pot preparation, the a/5000 Wagner pot was filled with 3.7 kg (oven dry basis) of Futsukaichi soil. Futsukaichi soil was collected from Kamigoka, Chikushino city, Fukuoka prefecture, Japan. The physiochemical properties of Futsukaichi soil were analyzed by Myint et al. (2011). This soil had a sandy loam with pH 6.11 (soil: water, 1:2.5). The soil had a total nitrogen (N) content of 0.68 g Kg⁻¹; and total phosphorus (P) content of 0.37 g Kg⁻¹. The cation exchange capacity (CEC) of the soil was 9.75 cmol_c kg⁻¹. Exchangeable potassium (K), calcium (Ca), magnesium (Mg), sodium (Na), and Iron (Fe) contained 0.32, 10.76, 0.89, 0.14, and 0.10 cmol_c kg⁻¹, respectively. We adjusted the soil from pH 6.1 to pH 6.5 by using CaMg(CO₃)₂ powder. Then, compound fertilizer (Kumiai Mame-kasei 300, Ryoto Fertilizer Co., Ltd., Ooita, Japan) containing 3% N, 10% P₂O₅, and 10% K₂O, was applied at a rate of 1.6 g pot⁻¹ at the time of pot preparation. The water content was maintained at 60% of the water holding capacity at the time of sowing. In this experiment, the seed inoculation method was used prior to seed sowing. A total of 100 soybean seeds were mixed thoroughly with 10 g peat soil, 7 mL 20% liquid solution of gum arabic, and 0.1 mL 1 × 10⁸ cell mL⁻¹ Bradyrhizobium to obtain the required inoculation density (1 × 10⁵ cells seed⁻¹). Four inoculated seeds were planted in one pot and covered with soil just after seed sowing. At 20 days after sowing, thinning was done to maintain one plant per pot. Pesticides were sprayed as necessary. Plant samples were collected from three growing stages: V6 (six unfolded trifoliate leaves), R3.5 (early pod-fill stage), and R8 (maturity stage). The growth stages referred to Fehr et al. (1971).

At the V6 stage, the plants were uprooted and washed to collect data. ARA measurements and collected data were the same as described above. Shoots, roots, and nodules were oven-dried at 70°C for 72 h to record their dry weights.

At the R3.5 stage, the plants were cut just under the cotyledonary nodes, inserted into a silicon tube, and the xylem sap was collected within 1 h after cutting. The sap samples were stored at −30°C before analysis. Then, the amino acid (Moore and Stein 1954), nitrate (Cataldo et al. 1975), and ureide (Young and Conway 1942) concentrations were analyzed from the root bleeding saps. The relative ureide index (RUI) of the root bleeding sap was calculated using the following formula (Peoples et al. 1989);

The percentage of N derived from N fixation (%Ndfa), was calculated using the following formula (Herridge and Peoples 1990):

where y is RUI (%) and x is %Ndfa, respectively.

At R8, the plants were cut at the cotyledon nodes to determine yield components such as the number of pods per plant, number of seeds per pod, 100-seed weight, and seed yield. This experiment was conducted in the screen house under open environmental conditions from July 2017 to November 2017.

3. Results

3.1. Screening of effective Bradyrhizobium type A strains

In the first screening, Yezin-6 (non-Rj) was used as a host plant for the determination of the nitrogen fixing potential and nodulation ability of 30 bradyrhizobial type A strains. USDA110 was used as the positive control. There were no significant differences in the shoot and root dry weights (supplementary Table 2). AHY3-1, AHY6-1, BLY6-5, MMY3-1, MMY3-2, SAY3-7, SAY6-2, SMY3-5, SMY6-1 and USDA110 significantly enhanced nitrogen fixation compared to the other tested strain. Therefore, these strains with higher nitrogen fixation and proper nodulation were selected for the next screening.

In the second screening experiment, the strains were compared for their ability to fix nitrogen, in terms of the ARA per plant using Yezin-3 (Rj₄), and Yezin-6 (non-Rj). The results of the plant growth and nodulation are shown in Table 1. The results of nitrogen fixation are shown in Fig. 1B. In both soybean varieties, shoot biomass and root biomass were not significantly different among inoculated strains (Table 1). Nodule numbers of Yezin-3 were significantly different among inoculated bacteria, but the nodule dry weights were not significantly different (Table 1). B. elkanii AHY3-1 and SAY6-2 produced significantly higher nodule numbers than other tested isolates except BLY6-5 in Yezin-3. In Yezin-6 soybean cultivars, nodule number and nodule mass were significantly different among inoculated bacteria (Table 1). AHY3-1, SAY6-2, and SMY3-5 produced significantly higher nodule numbers, but they were not different from USDA110. Moreover, AHY3-1, SAY6-2, and SMY3-5 produced higher nodule dry weights than other tested strains, except SAY3-7. Nitrogen fixation of B. japonicum SAY3-7 was significantly higher than that of other tested isolates and USDA110 (Fig. 1B) in Yezin-3 (Rj₄). In Yezin-6 (non-Rj), the nitrogen fixation of B. japonicum strains SAY3-7 and AHY3-1 was significantly higher than that of other tested isolates and USDA110 (Table 1). It was interesting that SAY3-7 consistently showed higher nitrogen fixation in both soybean varieties, whereas AHY3-1 fixed higher nitrogen in Yezin-6. Therefore, AHY3-1 and SAY3-7 was selected for the next screening.

Table 1. Effect of inoculation of selected Bradyrhizobium type A strains on NN, NDW, RDW and SDW of Yezin-3 and Yezin-6 soybean varieties at 30 days after sowing.

Cultivar	Strain	NN (No. plant^−1)	NDW (mg plant^−1)	RDW (g plant^−1)	SDW (g plant^−1)
Yezin-3	AHY3-1	20.3 ± 3.1 a	29.57 ± 1.42 a	0.23 ± 0.04 a	0.58 ± 0.04 a
	AHY3-6	11.7 ± 1.9 bc	21.80 ± 0.66 a	0.25 ± 0.05 a	0.52 ± 0.09 a
	AHY6-1	12.7 ± 1.2 bc	23.63 ± 2.98 a	0.22 ± 0.03 a	0.52 ± 0.08 a
	BLY6-5	18.3 ± 2.4 ab	32.60 ± 3.40 a	0.31 ± 0.03 a	0.55 ± 0.02 a
	MMY3-1	10.7 ± 0.9 c	29.83 ± 2.78 a	0.28 ± 0.03 a	0.54 ± 0.04 a
	MMY3-2	13.3 ± 1.2 bc	27.73 ± 3.21 a	0.27 ± 0.03 a	0.51 ± 0.04 a
	SAY3-7	12.3 ± 0.5 bc	31.23 ± 2.86 a	0.22 ± 0.04 a	0.55 ± 0.04 a
	SAY6-2	20.7 ± 3.7 a	30.70 ± 4.48 a	0.21 ± 0.02 a	0.49 ± 0.02 a
	SMY3-5	9.7 ± 0.5 c	33.23 ± 4.87 a	0.23 ± 0.01 a	0.50 ± 0.03 a
	SMY6-1	12.0 ± 1.4 bc	27.37 ± 3.55 a	0.23 ± 0.02 a	0.48 ± 0.02 a
	USDA110	13.3 ± 0.9 bc	25.37 ± 4.32 a	0.22 ± 0.03 a	0.51 ± 0.04 a
Yezin-6	AHY3-1	20.0 ± 1.6 ab	63.13 ± 3.31 a	0.43 ± 0.08 a	0.72 ± 0.06 a
	AHY3-6	10.0 ± 1.2 c	33.67 ± 5.68 cd	0.41 ± 0.06 a	0.61 ± 0.07 a
	AHY6-1	15.0 ± 0.8 bc	38.90 ± 0.67 bcd	0.34 ± 0.02 a	0.54 ± 0.09 a
	BLY6-5	15.3 ± 1.2 bc	40.63 ± 3.84 bcd	0.42 ± 0.04 a	0.58 ± 0.02 a
	MMY3-1	11.7 ± 1.9 c	30.37 ± 4.91 cd	0.39 ± 0.04 a	0.63 ± 0.00 a
	MMY3-2	11.7 ± 2.4 c	36.77 ± 2.42 cd	0.34 ± 0.01 a	0.66 ± 0.02 a
	SAY3-7	11.7 ± 2.4 c	63.97 ± 4.68 a	0.43 ± 0.06 a	0.65 ± 0.05 a
	SAY6-2	24.0 ± 2.9 a	52.57 ± 1.93 ab	0.45 ± 0.08 a	0.60 ± 0.06 a
	SMY3-5	19.0 ± 2.2 ab	61.07 ± 5.59 a	0.39 ± 0.02 a	0.60 ± 0.06 a
	SMY6-1	11.7 ± 1.9 c	28.6 ± 3.82 d	0.35 ± 0.03 a	0.61 ± 0.05 a
	USDA110	21.0 ± 2.2 ab	46.43 ± 2.85 bc	0.41 ± 0.02 a	0.71 ± 0.02 a

Mean value ± SD in each column at each variety followed by the same letter is not significantly different at P < 0.05 (Tukey’s test).

Figure 1. Effect of inoculation of Bradyrhizobium type A strains on acetylene reduction activity (ARA) of Yezin-6 in the first screening (A), and Yezin-3 and Yezin-6 in the second screening (B) at 30 days after sowing. The histograms with the same letter at each variety are not significantly different at P < 0.05 (Tukey’s test). The bar on each histogram indicates standard deviation (SD).

3.2. Screening of effective Bradyrhizobium type B strains

In the first screening, Yezin-3 (Rj₄) was inoculated with 26 bradyrhizobial type B strains. USDA110 was used as a positive control. In the first screening experiment, shoot biomass and root biomass were not significantly different among the inoculated bacteria (supplementary Table 3). However, the nodule number, nodule mass and nitrogen activities of the inoculated bacteria were significantly different (supplementary Table 3 and Fig. 2A). The isolates showed the proper nodulation and higher nitrogen fixing potential were selected for the next screening experiment. They were B. elkanii strains BLY3-8 and BLY6-1, B. japonicum strains SAY3-3 and SAY3-10, and Bradyrhizobium spp. strains SHY3-1, SHY3-3, SHY6-1, SHY6-5, SHY6-6, and SHY6-10.

Figure 2. Effect of inoculation of Bradyrhizobium type B strains on ARA of Yezin-3 in the first screening (A), and Yezin-3 and Yezin-6 in the second screening (B) at 30 days after sowing. The histograms with the same letter at each variety are not significantly different at P < 0.05 (Tukey’s test). The bar on each histogram indicates SD.

In the second screening experiment, the strains were compared for their nitrogen fixation ability in terms of the ARA per plant using Yezin-3 (Rj₄) and Yezin-6 (non-Rj). The results of the plant growth and nodulation are shown in Table 2. The nitrogen fixation results are shown in Fig. 2B. In both soybean varieties, the shoot biomass and root biomass were not significantly different among the inoculated strains (Table 2). However, the nodule number, nodule mass, and nitrogen fixing potential were significantly different among the inoculated bacteria (Table 2 and Fig. 2B). Nodule number, nodule mass and nitrogen fixation of B. elkanii BLY3-8, and B. japonicum strains SAY3-10 were significantly higher than those of the other tested isolates, but these strains were not different from Bradyrhizobium spp. SHY3-1, and USDA110 (Table 2) in both varieties. Therefore, BLY3-8 and SAY3-10 were selected for the next screening.

Table 2. Effect of inoculation of selected Bradyrhizobium type B strains on NN, NDW, RDW, and SDW of Yezin-3 and Yezin-6 soybean varieties at 30 days after sowing.

Cultivar	Strain	NN (No. plant^−1)	NDW (mg plant^−1)	RDW (g plant^−1)	SDW (g plant^−1)
Yezin-3	BLY3-8	27.0 ± 1.6 a	53.50 ± 0.85 a	0.32 ± 0.02 a	1.17 ± 0.08 a
	BLY6-1	16.3 ± 0.5 bc	39.03 ± 3.97 c	0.36 ± 0.02 a	1.25 ± 0.03 a
	SAY3-3	15.3 ± 1.7 c	38.90 ± 3.65 c	0.33 ± 0.04 a	1.15 ± 0.11 a
	SAY3-10	24.7 ± 1.2 a	54.73 ± 1.39 a	0.32 ± 0.04 a	1.07 ± 0.12 a
	SHY3-1	24.7 ± 1.7 a	56.23 ± 3.49 a	0.32 ± 0.07 a	0.94 ± 0.14 a
	SHY3-3	18.7 ± 2.6 bc	44.00 ± 1.50 bc	0.27 ± 0.01 a	1.00 ± 0.15 a
	SHY6-1	18.3 ± 0.9 bc	41.13 ± 0.24 c	0.32 ± 0.01 a	0.98 ± 0.07 a
	SHY6-5	10.0 ± 0.8 c	45.07 ± 2.60 bc	0.34 ± 0.02 a	0.95 ± 0.10 a
	SHY6-6	18.7 ± 0.5 bc	42.57 ± 1.79 bc	0.37 ± 0.04 a	1.07 ± 0.08 a
	SHY6-10	15.0 ± 0.8 c	45.07 ± 0.73 bc	0.28 ± 0.02 a	0.90 ± 0.08 a
	USDA110	21.7 ± 2.4 ab	50.67 ± 1.59 ab	0.26 ± 0.04 a	0.91 ± 0.11 a
Yezin-6	BLY3-8	33.0 ± 2.2 a	75.80 ± 1.14 a	0.40 ± 0.05 a	1.37 ± 0.18 a
	BLY6-1	14.7 ± 3.7 d	45.73 ± 2.29 c	0.40 ± 0.04 a	1.07 ± 0.08 a
	SAY3-3	25.0 ± 0.8 bc	56.80 ± 4.24 b	0.46 ± 0.05 a	1.18 ± 0.16 a
	SAY3-10	32.7 ± 1.9 ab	73.67 ± 0.45 a	0.45 ± 0.05 a	1.11 ± 0.08 a
	SHY3-1	33.0 ± 2.2 a	74.37 ± 0.69 a	0.33 ± 0.04 a	1.05 ± 0.03 a
	SHY3-3	22.0 ± 2.2 cd	62.50 ± 2.55 b	0.44 ± 0.12 a	1.17 ± 0.13 a
	SHY6-1	14.7 ± 2.9 d	60.13 ± 2.87 b	0.39 ± 0.09 a	1.17 ± 0.17 a
	SHY6-5	25.0 ± 0.8 bc	62.47 ± 3.73 b	0.30 ± 0.03 a	0.94 ± 0.02 a
	SHY6-6	22.0 ± 2.2 cd	59.03 ± 1.60 b	0.43 ± 0.04 a	1.17 ± 0.05 a
	SHY6-10	17.0 ± 1.6 d	63.10 ± 4.82 b	0.36 ± 0.03 a	1.02 ± 0.08 a
	USDA110	35.3 ± 2.5 a	75.07 ± 1.33 a	0.36 ± 0.07 a	1.04 ± 0.09 a

Mean value ± SD in each column at each variety followed by the same letter is not significantly different at P < 0.05 (Tukey’s test).

3.3. Effects selected type A and type B strains on symbiotic effectiveness of various soybean varieties

n this experiment, the selected Bradyrhizobium type A and type B strains were compared for their nitrogen fixation potential using Yezin-3 (Rj₄), Yezin-6 (non-Rj), Yezin-11 (Rj₄) and Yezin-14 (non-Rj). The plant growth and nodulation results are shown in Table 3. The nitrogen fixation results are shown in Fig. 3. Shoot and root dry weights were not significant in all tested soybean varieties. Nodule numbers were significantly different among inoculated bacteria, especially in Yezin-3 varieties, but not in other tested varieties (Table 3). BLY3-8 produced significantly higher nodule numbers compared with SAY3-10 and USDA110, but it was not different from AHY3-1 and SAY3-7. Nodule dry weights were significantly different among inoculated bacteria, especially in Yezin-3, Yezin-6, and Yezin-14 varieties, but not in Yezin-11 variety. SAY3-7 produced significantly higher nodule dry weights in Yezin-3, Yezin-6, and Yezin-14 varieties, but it was not different from BLY3-8 in Yezin-3 and Yezin-14 varieties. Nitrogen fixation rates differed significantly in the Yezin-3, Yezin-6 and Yezin-14 varieties, but not in the Yezin-11 variety (Fig. 3). AHY3-1 and USDA110 showed significantly lower nitrogen fixation than the other tested isolates in Yezin-3, Yezin-6, and Yezin-14 varieties. However, BLY3-8 and SAY3-7 showed higher nitrogen fixing potential than other tested isolates in Yezin-3, Yezin-6, and Yezin-14 varieties, but nitrogen fixation of BLY3-8 and SAY3-7 was not significantly different compared to SAY3-10, Yezin-6, and Yezin-14 varieties, respectively. Therefore, BLY3-8 and SAY3-7 were more compatible in all the tested varieties.

Table 3. Effect of inoculation of selected B. elkanii AHY3-1 (type A), B. elkanii BLY3-8 (type B), B. japnicum SAY3-7 (type A) and B. japnicum SAY3-10 (type B) strains on NN, NDW, RDW, and SDW of Yezin-3, Yezin-6, Yezin-11 and Yezin-14 soybean varieties at 30 days after sowing.

Variety	Strain	NN (No. plant^−1)	NDW (mg plant^−1)	RDW (g plant^−1)	SDW (g plant^−1)
Yezin-3	AHY3-1	18.0 ± 1.4 ab	39.70 ± 0.50 b	0.41 ± 0.03 a	0.89 ± 0.12 a
	BLY3-8	23.0 ± 2.5 a	55.50 ± 4.70 a	0.40 ± 0.07 a	0.96 ± 0.06 a
	SAY3-7	19.7 ± 1.3 ab	49.50 ± 1.02 a	0.32 ± 0.02 a	0.91 ± 0.04 a
	SAY3-10	12.0 ± 1.6 c	34.83 ± 2.65 b	0.37 ± 0.09 a	1.10 ± 0.30 a
	USDA110	15.0 ± 1.6 bc	36.87 ± 0.90 b	0.38 ± 0.02 a	0.87 ± 0.10 a
Yezin-6	AHY3-1	17.3 ± 3.3 a	45.07 ± 1.16 b	0.43 ± 0.03 a	0.98 ± 0.03 a
	BLY3-8	14.3 ± 1.3 a	46.23 ± 6.06 b	0.42 ± 0.01 a	0.97 ± 0.02 a
	SAY3-7	14.3 ± 1.3 a	70.37 ± 3.03 a	0.48 ± 0.08 a	1.03 ± 0.04 a
	SAY3-10	17.3 ± 0.5 a	56.20 ± 2.76 b	0.43 ± 0.03 a	1.08 ± 0.06 a
	USDA110	19.0 ± 0.8 a	45.57 ± 3.74 b	0.46 ± 0.01 a	0.97 ± 0.13 a
Yezin-11	AHY3-1	19.0 ± 2.8 a	33.53 ± 4.52 a	0.46 ± 0.07 a	0.88 ± 0.03 a
	BLY3-8	18.7 ± 0.5 a	32.80 ± 2.61 a	0.44 ± 0.06 a	0.91 ± 0.03 a
	SAY3-7	15.3 ± 2.5 a	37.00 ± 4.24 a	0.38 ± 0.06 a	0.88 ± 0.01 a
	SAY3-10	16.0 ± 1.4 a	40.00 ± 1.50 a	0.55 ± 0.06 a	1.00 ± 0.07 a
	USDA110	18.3 ± 2.1 a	33.13 ± 0.92 a	0.42 ± 0.05 a	0.89 ± 0.04 a
Yezin-14	AHY3-1	19.0 ± 1.6 a	41.70 ± 3.10 c	0.38 ± 0.05 a	0.97 ± 0.05 a
	BLY3-8	15.3 ± 3.9 a	57.10 ± 4.29 ab	0.51 ± 0.01 a	1.21 ± 0.05 a
	SAY3-7	18.7 ± 2.5 a	61.60 ± 8.42 a	0.42 ± 0.05 a	1.03 ± 0.17 a
	SAY3-10	22.7 ± 4.2 a	45.50 ± 0.82 bc	0.42 ± 0.09 a	1.10 ± 0.14 a
	USDA110	20.3 ± 0.9 a	43.73 ± 2.80 bc	0.39 ± 0.03 a	1.01 ± 0.15 a

Mean value ± SD in each column at each variety followed by the same letter is not significantly different at P < 0.05 (Tukey’s test).

Figure 3. Effect of inoculation of selected B. elkanii AHY3-1 (type A), B. elkanii BLY3-8 (type B), B. japnicum SAY3-7 (type A) and B. japnicum SAY3-10 (type B) strains on ARA of Yezin-3, Yezin-6, Yezin-11 and Yezin-14 soybean varieties at 30 days after sowing. The histograms with the same letter at each variety are not significantly different at P < 0.05 (Tukey’s test). The bar on each histogram indicates SD.

3.4. Effects of selected type A and type B strains on symbiotic effectiveness of Yezin-3 (Rj₄) and Yezin-6 (non-Rj) soybean varieties at different growth stages

The plant growth and nodulation results of Yezin-3 and Yezin-6 at different growth stages are shown in Table 4. The N₂ fixation results for both varieties are shown in Fig. 4. At the V6 growth stage, except nitrogen fixation, other parameters such as nodule numbers, nodule dry weight, root dry weight, and shoot dry weight were not significantly different among the treatments in Yezin-3 variety (Table 4), whereas the nodule dry weight, root dry weight, shoot dry weight, and nitrogen fixation were significantly different by inoculation in Yezin-6 (Table 4). These results show that the inoculation of SAY3-7 induced more symbiotic effectiveness at the early growth stage (V6) in the Yezin-6 variety. Moreover, inoculation of SAY-7 and BLY3-8 significantly enhanced N₂ fixation in terms of the C₂H₄ production compared with the control treatment (Fig. 4A) in both varieties.

Table 4. Effect of inoculation of B. japonicum SAY3-7 (type A) and B. elkanii BLY3-8 (type B) on NN, NDW, RDW, and SDW of Yezin-3 and Yezin-6 soybean varieties at different growth stages.

Growth Stage	Variety	Treatment	NN (No. plant^−1)	NDW (mg plant^−1)	RDW (g plant^−1)	SDW (g plant^−1)
V6	Yezin-3	Control	9.7 ± 0.5 a	24.70 ± 0.71 a	0.49 ± 0.11 a	2.01 ± 0.31 a
		SAY3-7	11.3 ± 1.9 a	31.0 ± 0.73 a	0.51 ± 0.04 a	1.91 ± 0.05 a
		BLY3-8	11.3 ± 1.9 a	27.73 ± 4.53 a	0.49 ± 0.13 a	2.13 ± 0.32 a
	Yezin-6	Control	10.3 ± 0.5 a	22.40 ± 0.96 b	0.41 ± 0.01 b	1.51 ± 0.05 b
		SAY3-7	14.0 ± 1.4 a	34.83 ± 2.22 a	0.51 ± 0.04 a	1.68 ± 0.03 a
		BLY3-8	12.7 ± 1.7 a	30.63 ± 0.75 a	0.53 ± 0.02 a	1.73 ± 0.00 a
R3.5	Yezin-3	Control	55.3 ± 10.7 b	413.33 ± 17.00 b	1.38 ± 0.11 a	7.38 ± 0.15 b
		SAY3-7	63.3 ± 2.4 ab	476.67 ± 20.55 ab	1.69 ± 0.24 a	7.75 ± 0.07 ab
		BLY3-8	86.0 ± 8.5 a	523.33 ± 44.97 a	1.64 ± 0.06 a	8.38 ± 0.34 a
	Yezin-6	Control	72.7 ± 5.3 b	376.67 ± 4.71 b	1.85 ± 0.04 b	7.22 ± 0.14 b
		SAY3-7	93.7 ± 4.5 a	456.67 ± 17.00 a	2.07 ± 0.06 a	7.80 ± 0.03 a
		BLY3-8	92.0 ± 2.2 a	473.33 ± 17.00 a	2.07 ± 0.06 a	7.80 ± 0.03a
R8	Yezin-3	Control	–	–	–	9.21 ± 0.51 b
		SAY3-7	–	–	–	11.45 ± 0.40 a
		BLY3-8	–	–	–	11.51 ± 0.52 a
	Yezin-6	Control	–	–	–	11.0 ± 0.84 b
		SAY3-7	–	–	–	13.37 ± 0.41 a
		BLY3-8	–	–	–	13.31 ± 0.45 a

Mean value ± SD in each column at each growth stage at each variety followed by the same letter is not significantly different at P < 0.05 (Tukey’s test). Control means un-inoculated treatment.

Figure 4. Effect of inoculation of B. japonicum SAY3-7 (type A) and B. elkanii BLY3-8 (type B) on (A) ARA at V6 stage, (B) relative ureide index (%) at R3.5 stage, (C) N derived from N fixation (%Ndfa) at R3.5 of Yezin-3 and Yezin-6 soybean varieties. The histograms with the same letter at each variety are not significantly different at P < 0.05 (Tukey’s test). The bar on each histogram indicates SD.

At the R3.5 growth stage, the nodule number, nodule dry weight, and shoot dry weight were significantly different among the treatments in both varieties, but the root dry weight was not significant in Yezin-3. In the Yezin-3 variety, inoculation of BLY3-8 produced significantly higher nodule number, nodule dry weight, shoot dry weight and nitrogen fixation in terms of RUI (%) and %Ndfa compared with the control, but not SAY3-7 (Fig. 4B and Fig. 4C). In the Yezin-6 variety, inoculation of SAY3-7 and BLY3-8 produced a higher nodule number, nodule dry weight, root dry weight, and shoot dry weight compared with the control. At R3.5 stage, nodule number of inoculated plants with BLY3-8 (86.0 plant⁻¹) increased 35.7% compared with non-inoculated plant (55.3 plant⁻¹) in Yezin-3. Nodule number of inoculated plants with SAY3-7 (93.7 plant⁻¹) and BLY3-8 (92.0 plant⁻¹) and increased 23.2% and 21.7%, respectively, compared with non-inoculated plant (72.0 plant⁻¹) in Yezin-6. These results suggested that competitiveness of SAY3-7 and BLY3-8 strains for nodulation with indigenous rhizobia existing in the soil.

At the R8 growth stage, shoot biomass was significantly different in both the soybean varieties (Table 4). Inoculation of SAY3-7 and BLY3-8 produced at higher shoot biomass than the control treatment.

The yield component results are shown in Table 5. Yield components, such as the number of pods per plant, number of seeds per pod, and 100-seed weight, were not significantly different among the treatments in both varieties. However, the seed yields were significantly different. Inoculation of SAY3-7 and BLY3-8 resulted in significantly higher seed yields than the control treatment in both varieties. This result suggests that SAY3-7 and BLY3-8 are effective bacteria, which are compatible with Yezin-6 (non-Rj) and Yezin-3 (Rj₄) because of the higher production of the seed yield.

Table 5. Effect of inoculation of B. japonicum SAY3-7 (type A) and B. elkanii BLY3-8 (type B) on yield components and seed yield of Yezin-3 and Yezin-6 soybean varieties at maturity stage.

Variety	Treatment	Pods (No. plant^−1)	Seeds per pod (No. pod^−1)	100 seed weight (g)	Seed yield (g plant^−1)
Yezin-3	Control	18.0 ± 1.6 a	1.97 ± 0.2 a	14.08 ± 0.24 a	4.92 ± 0.14 b
	SAY3-7	19.0 ± 1.4 a	1.88 ± 0.2 a	14.08 ± 0.31 a	5.65 ± 0.16 a
	BLY3-8	20.0 ± 2.2 a	2.20 ± 0.2 a	13.70 ± 0.55 a	5.86 ± 0.24 a
Yezin-6	Control	18.7 ± 1.9 a	2.01 ± 0.1 a	14.31 ± 0.93 a	5.31 ± 0.25 b
	SAY3-7	22.0 ± 1.4 a	1.97 ± 0.1 a	14.71 ± 0.80 a	6.36 ± 0.30 a
	BLY3-8	21.0 ± 1.4 a	1.99 ± 0.1 a	15.33 ± 0.17 a	6.38 ± 0.29 a

Mean value ± SD in each column at each variety followed by the same letter is not significantly different at P < 0.05 (Tukey’s test). Control means un-inoculated treatment.

4. Discussion

Rhizobia have been used extensively in agricultural systems to enhance the ability of legumes to fix atmospheric N₂ (Elkan 1992). Therefore, the isolation and selection of indigenous bacteria for their respective soybean cultivars is necessary. Recently, Soe et al. (2013) and Htwe et al. (2015b) identified Myanmar soybean cultivars with non-Rj and Rj₄ genotypes. Soe et al. (2013) and Htwe et al. (2015c) noted that the cultivars Yezin-6, harboring the non-Rj gene, and Yezin-3, harboring the Rj₄ gene, have enhanced nodulation and nitrogen fixation compared to indigenous strains. However, their initial screening was based on using Yezin-6 (non-Rj) soybean cultivars. As a consequence, type A strains, which prefer non-Rj soybean cultivars, have more potential for selection. In this study, the type A and type B groups were separately screened using the respective cultivars, Yezin-6 (non-Rj) and Yezin-3 (Rj₄), using the USDA110, which is used in many countries as an inoculant. In the first screening, the type A and type B strains that showed proper nodulation and higher nitrogen fixing potential were selected for the next screening experiment (supplementary Table 2 and Fig. 1A for type A) and (supplementary Table 3 and Fig. 2A for type B). According to the results of this study, the isolates belonging to the same species showed different responses for nitrogen fixation. This was in line with the previous findings of our group (Soe and Yamakawa 2013; Htwe et al. 2015c).

Isolation and selection were aimed at producing rhizobial inoculants. The first step in manufacturing rhizobial inoculants is to obtain the most effective strain, since rhizobial strains within a species vary in their effectiveness (Boonkerd et al. 1978). Therefore, the second screening was done to determine the symbiotic effectiveness of the selected isolates on Yezin-3 (Rj₄) and Yezin-6 (non-Rj) because the selected isolates were isolated from Yezin-3 (Rj₄), and Yezin-6 (non-Rj). Among the selected type A strains, SAY3-7 consistently showed higher nodulation and nitrogen fixation in both soybean varieties compared with the other type A strains (Table 1 and Fig. 1B). However, AHY3-1 fixed higher nitrogen and produced proper nodulation in the Yezin-6 soybean cultivar, but not in Yezin-3 (Table 1 and Fig. 1B). Among the selected type B strains, the nodulation and nitrogen fixation of BLY3-8 and SAY3-10 were shown to be significantly higher than those of the other tested isolates, in both the Yezin-3 and Yezin-6 soybean cultivars (Table 2 and Fig. 2B). To confirm the results of the second screening and to select the most effective strains, a third screening was done. In the third screening, BLY3-8 and SAY3-7 showed higher nitrogen fixing potential than the other tested isolates in the Yezin-3, Yezin-6 and Yezin-14 varieties, but nitrogen fixation of BLY3-8 and SAY3-7 was not significant from SAY3-10 in both the Yezin-6 and Yezin-14 varieties (Fig. 3). Therefore, BLY3-8 and SAY3-7 seemed to be more compatible for proper nodulation and nitrogen fixation in all the tested varieties (Table 3). This result supports the finding of others in which BNF in soybean production can be increased by the selection of effective Bradyrhizobium japonicum strains and efficient soybean cultivars as cultivar-bacterial strain pairs (Israel et al. 1986; Soe and Yamakawa  2013; Htwe et al. 2015c). However, plant growth was not significant among the inoculated treatments. This was in agreement with (Sarr et al. 2016) in which N₂ fixation was increased significantly by Bradyrhizobium strains, but it was not accompanied by a significant increase in plant growth.

It has been reported that soybean growth parameters and yield were significantly increased due to the inoculation of bradyrhizobial isolates (Purcino et al. 2000; Pant and Prasad 2004). In this study, inoculation of SAY3-7 and BLY3-8 significantly increased N₂ fixation at the V6 and R3.5 stages; plant growth, nodulation, N₂ fixation and N uptake at the V6, R3.5 and R8 stages (Table 4 and Fig. 4), and the seed yield at the R8 stage (Table 5), in Yezin-3 (Rj₄) and Yezin-6 (non-Rj) soybean varieties were compared with the control treatment. These results support the findings of our group in which inoculation of bradyrhizobial strains increased the plant growth, nodulation, N₂ fixation, and seed yield of soybeans cultivated in the soil condition (Soe et al. 2012; Yamakawa and Fukushima 2014).

A successful legume-Rhizobium symbiosis mainly depends on the presence of a compatible strain in the soil for a particular legume. Therefore, inoculation with the compatible strain is important for successful legume-Rhizobium symbiosis. However, one of the major problems in the inoculation technology with soybeans is the establishment of an inoculated strain from populations of indigenous bradyrhizobia existing in the soil (Tang 1979). Therefore, the occupancy of inoculated strains in nodules is relatively low compared with indigenous rhizobia because of their competition to occupy root nodules (Weaver and Frederick 1974; Kvien et al. 1981). However, in this study, the nodule number and nodule dry weight in inoculation treatment was significantly higher than those of the control at R3.5 stage. The abundance of nodulation in the plants inoculated with SAY3-7 and BLY3-8 shows that the inoculated strain might have a competitive nodulation ability with indigenous rhizobia. These results were observed because of the use of proper variety, compatible strain and suitable inoculation methods with proper inoculation density as described in (Yamakawa et al. 2003) and (Yamakawa and Fukushima 2014). Nodule occupancy of inoculated strain can be increased by the use of reciprocal relationship between Rj-genotype soybean cultivars and rhizobia (Yamakawa et al. 2003). Better competitiveness for nodulation with indigenous rhizobia can be promoted by using suitable inoculation methods such as inoculation of seeds with 1000-fold higher density than that in the soil (Yamakawa and Fukushima 2014).

Soybean-Bradyrhizobium symbiosis provides the required nitrogen to soybean plants through the symbiotic nitrogen fixation of Bradyrhizobium. To optimize this symbiosis, selection of compatible strains for currently used soybean cultivars is considered to be one of the major factors in improving the nodulation and symbiotic effectiveness. Moreover, Rj genes of soybean varieties and the nodulation types of inoculated strains need to be considered because many scientists reported that the indigenous Bradyrhizobium strains in the soil exhibit preferences for nodulation on compatible Rj-genotypes (Saeki et al. 2008; Minami et al. 2009). The Rj-genotypes play a role in controlling the plant’s compatibility with specific rhizobial strains, and the preference for indigenous soybean-nodulating rhizobia (Ishizuka et al. 1991a, 1991b). In addition, non-Rj genotype soybean cultivars are compatible with all types of bradyrhizobial strains, but Rj₄ has unique features that restrict nodulation with specific strains of Bradyrhizobium (Vest and Caldwell 1972). In the present study, the symbiotic effectiveness on plant growth, nodulation, nitrogen fixation and productivity were observed in SAY3-7 (type A strain) and BLY3-8 (type B strain) compared with the control, in both Yezin-3 (Rj₄) and Yezin-6 (non-Rj) soybean cultivars. The improvement of the symbiotic efficacy in inoculation treatments is due to the preferable use of Rj-genotype soybean cultivars by inoculated Bradyrhizobium strains.

According to the results of this study, it can be concluded that B. japonicum SAY3-7 (type A strain) and B. elkanii BLY3-8 (type B strain) were the most effective isolates with proper nodulation and higher nitrogen fixation in all the Rj₄ and non-Rj soybean cultivars cultivated in rhizobial free vermiculite conditions. Moreover, these two effective isolates showed significantly higher plant growth, nodulation, N₂ fixation, and seed yield of soybeans cultivated in Futsukaichi soil, where rhizobia existed at a density of 2.9 × 10³ cells g⁻¹ compared with the control treatment. In this study, the symbiotic effectiveness of inoculated isolates was observed in soil containing low level of indigenous rhizobial population (2.9 × 10³ cells g⁻¹). As further experiment, these two isolates should be tested in soil containing not only low density of rhizobia but also high density of native rhizobia to know their competitiveness to occupancy in nodules by inoculum and to evaluate their symbiotic effectiveness on soybean.

Acknowledgments

We are grateful to Ministry of Education, Culture, Sports, Sciences and Technology (MEXT) of Japan for financial support of this study.

Supplementary materials

Supplementary data for this article are accessed here

Additional information

Funding

This work was supported by the Ministry of Education, Culture, Sports, Science and Technology [130287].