Effects of co-inoculation of Bradyrhizobium elkanii BLY3-8 and Streptomyces griseoflavus P4 on Rj₄ soybean varieties

ABSTRACT

Co-inoculation of selected nitrogen-fixing bacteria with plant growth-promoting bacteria is the promising way for the improvement of soybean production through enhancing plant growth, nodulation, and N₂ fixation. Therefore, this experiment was conducted to study the effects of co-inoculation of Bradyrhizobium elkanii BLY3-8 with Streptomyces griseoflavus P4 on plant growth, nodulation, N₂ fixation, N uptake, and seed yield of Rj₄ soybean varieties. Two experiments with completely randomized design and three replicates were done in this study. N₂-fixation ability of soybean was evaluated by acetylene reduction activity (ARA) and relative ureide method. In the first experiment, synergetic effect in N₂ fixation and nodulation was occurred in co-inoculation treatment (BLY3-8 + P4) in Yezin-3 and Fukuyutaka. Based on these results, co-inoculation effect of BLY3-8 and P4 was assessed on Yezin-3 and Fukuyutaka varieties at three different growth stages, using Futsukaichi soil under natural environmental conditions. This study shows that co-inoculation of BLY3-8 and P4 significantly increased N₂ fixation at V6 stage; plant growth, nodulation, N₂ fixation, and N uptake at R3.5 stage; and shoot growth, N uptake, and seed yield at R8 stage, in Rj₄ soybean varieties compared with the control. Significant difference in plant growth, nodulation, N₂ fixation, N uptake, and yield between co-inoculation and control, not between single inoculation and control, suggests that there is a synergetic effect due to co-inoculation of BLY3-8 and P4.

KEY WORDS:

• Co-inoculation
• N₂ fixation
• nodulation
• plant growth
• seed yield

1. Introduction

Soybean, in symbiosis with Bradyrhizobium, has the ability to fix nitrogen up to 300 kg N ha⁻¹ under favorable conditions (Smith and Hume 1987). However, the symbiosis depends on the host specificity. This specificity might be also related to nodulation regulatory genes of soybean cultivars and nodulation types of rhizobia, especially in soybean. Ishizuka et al. (1991a); (1991b)) tested the compatibility and preference of Rj genotypes of 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 cultivars and nodulate with all Rj genotype cultivars. Type B strains are preferred by Rj₄ cultivars and inhibit nodulation with the Rj₂Rj₃ gene harboring cultivars. Type C strains are preferred by Rj₂Rj₃ cultivars and restrict effective nodule formation with the Rj₄ genotype cultivars. Therefore, selection of type A, B, and C for their specific soybean cultivars is a novel phenomenon because their positive effects depend on the combination of nodulation type of rhizobia and the host soybean genotype.

Different soybean cultivars possess different nodulation regulatory genes known as Rj genes. These Rj (rj) genes involved in the formation of nitrogen-fixing root nodule in soybean (Hayashi et al. 2012). In soybean, the Rj genes Rj₂, Rj₃, and Rj₄ restrict the nodule formation by certain Bradyrhizobium strains (Caldwell 1966; Caldwell et al. 1966; Vest 1970; Vest and Caldwell 1972). The regulatory gene Rfg1 inhibits nodulation by the fast-growing strain Sinorhizobium fredii USDA257 (Trese 1995). The compatibility of Bradyrhizobium and Ensifer/Sinorhizobium species is dependent on the Rj (rj) gene (Hayashi et al. 2012). Therefore, inoculation of specific soybean cultivars with compatible strain is necessary to form functional nodules, which can fix the atmospheric nitrogen.

Soybean cultivars in Myanmar harboring non-Rj, Rj₂Rj₃, Rj₃, or Rj₄ genes were reported in a previous study (Htwe et al. 2015). Among them, Rj₄ gene harboring soybean cultivars are widely grown in Myanmar and account for 60% of all cultivars. Devine and Kuykendall (1996) reported that >60% of soybean in Southeast Asia have the Rj₄ gene. Therefore, inoculation of type B strains, which prefer Rj₄ genotype cultivars, is necessary to increase nodulation and N₂ fixation in Myanmar where 60% of Rj₄ gene harboring soybean cultivars are widely grown.

Streptomyces is a beneficial actinomycetes and a plant growth-promoting rhizobacterium with many effects on plant growth (Ardakani and Mafakheri 2011). Streptomyces griseoflavus P4 used in this experiment can induce production of the plant growth hormone indole acetic acid (Soe 2013). Moreover, P4 used in this study was reported that it is compatible with some bradyrhizobial strains, and combined use of P4 and compatible bradyrhizobial strains had a synergetic effect for plant growth, nodulation, and N₂ fixation (Soe et al. 2012). Those facts are attractive to use this endophytic strain as a co-inoculant with indigenous Bradyrhizobium to increase plant growth and N₂-fixation rates. Therefore, the present study was aimed to evaluate co-inoculation effects of B. elkanii type B strain with P4 on plant growth, nodulation, N₂ fixation, N uptake, and seed yield of Rj₄ soybean varieties.

2. Materials and methods

2.1. Inoculant preparation

B. elkanii BLY3-8 (type B) strain was obtained from a previous experiment (Htwe et al. 2015). B. diazoefficiens strain USDA110 (Type strain) was obtained from the Laboratory of Plant Nutrition, Kyushu University, Japan. Bradyrhizobium strains and S. griseoflavus P4 were cultured in A1E liquid media (Kuykendall 1987) and in IMA-2 medium (Shimizu et al. 2000), respectively, on a rotary shaker (100 rpm) at 30°C for 7 days.

2.2. Soybean varieties

Myanmar soybean varieties, Yezin-3 (Rj₄) and Yezin-6 (non-Rj), were collected from Food Legumes Section, Department of Agricultural Research, Yezin, Myanmar. Their Rj genes were identified by Soe et al. (2013). Yezin-3 and Yezin-6 are semideterminate types. Japanese soybean varieties, Fukuyutaka (Rj₄) and Akisirome (non-Rj), were obtained from the Laboratory of Plant Nutrition, Kyushu University, Japan. Their Rj genes were described in Ishizuka et al. (1991a). Fukuyutaka and Akisirome are determinate types.

2.3. Cultivation, crop management, plant sampling, and analysis of pot experiment 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 (Nakano et al. 1997) to 10⁵ 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 1 month. 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 acetylene reduction activity (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 May 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 rhizobial population of Futsukaichi soil was evaluated by the most probable number method (Vincent 1970) using Yezin-3 (Rj₄) as the host plant. The estimated rhizobial population was 0.29 × 10⁴ 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. 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) and with low content of total nitrogen (0.68 g kg⁻¹). CaMg(CO₃)₂ was added to adjust soil pH (pH 6.1 to pH 6.5). Then, compound fertilizer (Kumiai Mame-kasei 300, Ryoto Fertilizer Co., Ltd., Ooita, Japan) containing 3% N, 10% P₂O₅, and 10% K₂O was applied at the rate of 1.6 g pot⁻¹ at the time of pot preparation. Water content was kept at 60% of the water-holding capacity at the time of sowing. In this experiment, seed inoculation method was used prior to seed sowing as shown in Htwe et al. (2018). Four inoculated seeds were planted in one pot and covered with soil just after seed sowing. After 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 refer to Fehr et al. (1971).

At V6 stage, the plants were uprooted and washed to collect data. ARA measurement 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. Dried shoot was grounded into a powder using a Cyclotec 1093 sample mill (100–120 mesh, Tecator AB, Hoedanaes, Sweden). Total nitrogen accumulation in the shoot was measured by using indophenol method (Cataldo et al. 1974) after extracting the nutrient contents using H₂SO₄–H₂O digestion method (Ohyama et al. 1991).

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

The percentage of N derived from N₂ fixation was calculated using the following formula (Herridge and Peoples 1990);

in which y is RUI (%) and x is the percentage of N derived from N₂ fixation (% Ndfa), respectively. Determination of N accumulation and other data were collected as described above.

At R8, the plants were cut at the cotyledon nodes to determine seed yield and yield component parameters such as number of pods per plant, number of seeds per pod, hundred-seed weight, and seed yield. Determination of N accumulation was the same as described above. This experiment was conducted from July 2017 to November 2017.

2.5. Statistical analysis

Data were analyzed using the STATISTIX 8 software package (Analytical Software, Tallahassee, FL, USA) and the means were compared by Tukey’s HSD test with a P value < 0.05 taken to indicate statistical significance.

3. Results

3.1. Effects of single or co-inoculation on symbiotic effectiveness of different soybean varieties

The results of plant growth, nodulation, and N₂ fixation are shown in Table 1. Shoot dry weight was significantly affected by single or dual inoculation of bradyrhizobial strains compared with control in Yezin-3, Yezin-6, and Akisirome, except Fukuyutaka variety. However, root dry weight was not significantly increased among the treatments in all tested varieties. Nodule number, nodule dry weight, and N₂ fixation were significantly different among single or dual inoculation of N₂-fixation bacteria in Yezin-3 and Fukuyutaka, but not in Yezin-6 and Akisirome soybean varieties. In both Rj₄ soybean varieties (Yezin-3 and Fukuyutaka), inoculation with USDA110, USDA110 + P4, and BLY3-8 + P4 showed the higher nodulation and nitrogenase activity compared with single inoculation of BLY3-8. Moreover, co-inoculation treatment (BLY3-8 + P4) shows the higher nodulation and N₂ fixation compared with single inoculation with BLY3-8, although USDA110 did not show the synergetic effect due to co-inoculation with P4. Considering all the varieties, synergetic effect in nodulation and N₂ fixation were occurred due to co-inoculation compared with single inoculation of BLY3-8 in Rj₄ soybean varieties (Yezin-3 and Fukuyutaka), but not in non-Rj soybean varieties (Yezin-6 and Akisirome). Positive effect in shoot growth was formed by single or co-inoculation in some varieties except Fukuyutaka soybean variety.

Table 1. Effect of single or dual inoculation of B. elkanii BLY3-8 strain and B. diazoefficiens USDA110 on nodule number (NN), nodule dry weight (NDW), shoot dry weight (SDW), root dry weight (RDW), and the acetylene reduction activity (ARA) of Yezin-3, Yezin-6, Fukuyutaka, and Akisirome soybean varieties at 30 days after sowing.

Variety	Treatment	NN (number of plant^−1)	NDW (mg plant^−1)	RDW (g plant^−1)	SDW (g plant^−1)	ARA (µmol C2H4 h^−1 plant^−1)
Yezin-3	Control	-	-	0.33 ± 0.04 a	0.61 ± 0.03 b	-
	P4	-	-	0.33 ± 0.01 a	0.64 ± 0.02 b	-
	BLY3-8	16.7 ± 5.7 b	46.54 ± 3.60 b	0.30 ± 0.01 a	0.99 ± 0.12 a	0.83 ± 0.01 b
	BLY3-8 + P4	35.7 ± 7.8 a	59.07 ± 5.08 a	0.35 ± 0.04 a	0.92 ± 0.06 a	1.04 ± 0.04 a
	USDA110	38.7 ± 5.7 a	62.37 ± 2.98 a	0.34 ± 0.03 a	0.93 ± 0.04 a	0.98 ± 0.03 ab
	USDA110 + P4	50.0 ± 2.2 a	66.93 ± 2.17 a	0.38 ± 0.05 a	1.06 ± 0.03 a	1.09 ± 0.05 a
Yezin-6	Control	-	-	0.42 ± 0.03 a	0.69 ± 0.05 b	-
	P4	-	-	0.47 ± 0.04 a	0.92 ± 0.03 a	-
	BLY3-8	12.3 ± 1.9 a	51.23 ± 4.41 a	0.51 ± 0.07 a	1.11 ± 0.09 a	1.48 ± 0.01 a
	BLY3-8 + P4	18.7 ± 4.2 a	54.40 ± 3.06 a	0.44 ± 0.02 a	1.04 ± 0.04 a	1.66 ± 0.03 a
	USDA110	16.0 ± 4.5 a	62.97 ± 9.86 a	0.44 ± 0.06 a	0.93 ± 0.10 a	1.55 ± 0.14 a
	USDA110 + P4	16.3 ± 1.2 a	51.37 ± 5.55 a	0.43 ± 0.06 a	0.99 ± 0.02 a	1.55 ± 0.26 a
Fukuyutaka	Control	-	-	0.46 ± 0.06 a	0.82 ± 0.04 a	-
	P4	-	-	0.54 ± 0.06 a	0.99 ± 0.02 a	-
	BLY3-8	10.0 ± 0.8 c	22.27 ± 0.76 c	0.51 ± 0.04 a	0.97 ± 0.03 a	0.64 ± 0.04 c
	BLY3-8 + P4	21.3 ± 2.4 b	35.07 ± 0.86 b	0.51 ± 0.07 a	0.92 ± 0.04 a	1.03 ± 0.16 a
	USDA110	28.3 ± 1.2 ab	51.13 ± 3.94 a	0.42 ± 0.05 a	0.94 ± 0.09 a	0.71 ± 0.06 bc
	USDA110 + P4	30.3 ± 4.2 a	57.16 ± 6.39 a	0.45 ± 0.02 a	1.04 ± 0.12 a	0.95 ± 0.04 ab
Akisirome	Control	-	-	0.46 ± 0.04 a	0.84 ± 0.01 b	-
	P4	-	-	0.51 ± 0.05 a	0.95 ± 0.01 b	-
	BLY3-8	18.0 ± 3.7 a	38.87 ± 1.74 a	0.50 ± 0.03 a	1.21 ± 0.07 a	1.02 ± 0.04 a
	BLY3-8 + P4	17.7 ± 1.9 a	41.23 ± 4.67 a	0.44 ± 0.02 a	1.20 ± 0.08 a	0.77 ± 0.02 a
	USDA110	19.0 ± 9.5 a	55.03 ± 1.18 a	0.47 ± 0.02 a	1.28 ± 0.08 a	0.78 ± 0.03 a
	USDA110 + P4	20.0 ± 7.3 a	53.73 ± 0.62 a	0.46 ± 0.06 a	1.26 ± 0.05 a	0.93 ± 0.06 a

Mean values ± standard deviation (SD) in each column at each growth stage followed by the same letters are not significantly different at P < 0.05 (Tukey’s test).

3.2. Effects of single or co-inoculation on symbiotic effectiveness of Yezin-3 (Rj₄) and Fukuyutaka (Rj₄) soybean varieties at different growth stages

The results of plant growth, nodulation, and N uptake at different growth stages are shown in Table 2 for Yezin-3 and Table 3 for Fukuyutaka. The results of N₂ fixation for both varieties are shown in Fig. 1. At V6 growth stage, root dry weight, shoot dry weight, and N uptake were not significantly different among inoculated treatments in both varieties (Tables 2 and 3). Nodule numbers and nodule dry weight were significantly differed by inoculation in Fukuyutaka (Table 3), but not in Yezin-3 (Table 2). In Fukuyutaka soybean variety, co-inoculation BLY3-8 and P4 produced relatively higher nodule numbers and nodule dry weight than control, although it was not significantly different from single inoculation of BLY3-8 and P4 in nodule number (Table 3). N₂ fixation in terms of C₂H₄ production was significantly different among treatments (Fig. 1A) in both varieties. Co-inoculation of BLY3-8 + P4 showed the highest nitrogenase activity compared with single inoculation P4 and control. Besides, single inoculation of BLY3-8 significantly enhanced N₂ fixation compared with control in both varieties. These results indicate that single inoculation of BLY3-8 also promoted N₂ fixation. Consequently, co-inoculation of both bacteria occurred the synergetic effect in N₂ fixation.

Table 2. Effect of co-inoculation of B. elkanii BLY3-8 and S. griseoflavus P4 nodule number (NN), nodule dry weight (NDW), shoot dry weight (SDW), root dry weight (RDW), and shoot N uptake of Yezin-3 soybean variety at different growth stages.

Growth stage	Treatment	NN (number of plant^−1)	NDW (mg plant^−1)	RDW (g plant^−1)	SDW (g plant^−1)	N uptake (mg plant^−1)
V6	Control	10.7 ± 1.7 a	22.00 ± 2.29 a	0.40 ± 0.08 a	1.75 ± 0.34 a	18.34 ± 1.39 a
	P4	8.3 ± 1.7 a	25.27 ± 3.41 a	0.59 ± 0.04 a	2.33 ± 0.13 a	23.33 ± 2.36 a
	BLY3-8	10.0 ± 2.8 a	33.30 ± 5.91 a	0.65 ± 0.12 a	2.53 ± 0.45 a	22.70 ± 3.60 a
	BLY3-8 + P4	7.7 ± 1.7 a	27.73 ± 4.53 a	0.49 ± 0.13 a	2.13 ± 0.32 a	18.15 ± 3.90 a
R3.5	Control	56.0 ± 11.6 b	403.33 ± 18.86 b	1.31 ± 0.05 b	7.34 ± 0.20 b	161.92 ± 2.92 b
	P4	73.3 ± 3.4 ab	460.00 ± 14.14 ab	1.59 ± 0.05 ab	7.87 ± 0.20 ab	173.04 ± 7.75 ab
	BLY3-8	78.0 ± 1.6 ab	500.00 ± 35.59 ab	1.60 ± 0.07 ab	8.08 ± 0.21 ab	171.86 ± 5.75 ab
	BLY3-8 + P4	89.3 ± 7.4 a	526.67 ± 47.14 a	1.78 ± 0.16 a	8.40 ± 0.33 a	187.98 ± 4.51 a
R8	Control	-	-	-	9.21 ± 0.51 b	158.98 ± 8.87 b
	P4	-	-	-	11.25 ± 0.44 a	187.94 ± 7.46 ab
	BLY3-8	-	-	-	11.34 ± 0.83 a	205.29 ± 27.69 ab
	BLY3-8 + P4	-	-	-	11.51 ± 0.52 a	227.95 ± 10.79 a

Mean values ± standard deviation (SD) in each column at each growth stage followed by the same letters are not significantly different at P < 0.05 (Tukey’s test).

Table 3. Effect of co-inoculation of B. elkanii BLY3-8 and S. griseoflavus P4 nodule number (NN), nodule dry weight (NDW), shoot dry weight (SDW), root dry weight (RDW), and shoot N uptake of Fukuyutaka soybean variety at different growth stages.

Growth stage	Treatment	NN (number of plant^−1)	NDW (mg plant^−1)	RDW (g plant^−1)	SDW (g plant^−1)	N uptake (mg plant^−1)
V6	Control	10.0 ± 0.8 b	7.05 ± 0.07 b	0.65 ± 0.08 a	2.07 ± 0.30 a	24.47 ± 0.59 a
	P4	14.0 ± 1.4 ab	6.01 ± 1.40 b	0.69 ± 0.12 a	2.18 ± 0.50 a	26.20 ± 2.32 a
	BLY3-8	20.3 ± 0.5 ab	9.40 ± 5.07 b	0.78 ± 0.12 a	2.77 ± 0.40 a	32.71 ± 9.37 a
	BLY3-8 + P4	21.0 ± 5.0 a	19.23 ± 0.99 a	0.79 ± 0.01 a	2.74 ± 0.02 a	32.05 ± 4.29 a
R3.5	Control	101.7 ± 8.2 b	480.00 ± 49.67 b	4.07 ± 0.01 b	8.35 ± 0.14 b	179.31 ± 35.34 b
	P4	120.0 ± 4.1 ab	580.00 ± 8.16 ab	4.65 ± 0.13 ab	8.88 ± 0.40 ab	204.44 ± 3.30 ab
	BLY3-8	122.7 ± 7.7 ab	566.67 ± 47.14 ab	5.03 ± 0.41 ab	9.68 ± 0.85 ab	230.90 ± 17.23 ab
	BLY3-8 + P4	126.7 ± 3.4 a	653.33 ± 12.47 a	5.54 ± 0.42 a	10.69 ± 0.80 a	248.07 ± 15.22 a
R8	Control	-	-	-	10.46 ± 0.57 b	225.64 ± 8.25 b
	P4	-	-	-	12.37 ± 0.34 a	284.40 ± 20.78 a
	BLY3-8	-	-	-	12.36 ± 0.24 a	271.38 ± 9.84 a
	BLY3-8 + P4	-	-	-	12.38 ± 0.33 a	280.25 ± 7.34 a

Mean values ± standard deviation (SD) in each column at each growth stage followed by the same letters are not significantly different at P < 0.05 (Tukey’s test).

Figure 1. Effect of co-inoculation of B. elkanii BLY3-8 and S. griseoflavus on (A) acetylene reduction activity (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 Fukuyutaka 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 standard deviation (SD).

At R3.5 growth stage, nodule number, nodule dry weight, root dry weight, shoot dry weight, and N uptake were significantly different among treatments in both varieties (Table 2 and Table 3). Co-inoculation treatment produced higher nodule number, nodule dry weight, root dry weight, and shoot dry weight compared with control, but not single inoculation treatments. These results highlight that synergetic effects for plant growth and nodulation was formed by co-inoculation of BLY3-8 and P4 in both varieties. RUI (%) and %Ndfa were significantly different among treatments (Fig. 1B and 1C) in Yezin-3 variety, but not in Fukuyutaka. Inoculation with BLY3-8 + P4 or BLY3-8 showed the higher RUI (%) and %Ndfa compared with single inoculation of P4 and control in Yezin-3. Moreover, single inoculation of P4 showed the significantly higher RUI (%) and %Ndfa than control. These results highlight that sole inoculation or dual inoculation of BLY3-8 and P4 increased plant growth, nodulation, and N₂ fixation in Yezin-3 soybean variety.

At R8 growth stage, shoot biomass and shoot N uptake were significantly different in both soybean varieties (Table 2 and Table 3). Inoculation of BLY3-8, P4, and BLY3-8 + P4 produced the higher shoot biomass than control. Moreover, BLY3-8 + P4 increased N uptake of shoot compared with control but not single inoculation treatments in both varieties.

The yield and yield component results are shown in Table 4. Yield component parameters, such as the number of pods per plant, number of seeds per pod, and 100-seed weight, were not significantly different among treatments in both varieties. However, seed yields were significantly different. Co-inoculation of BLY3-8 with P4 resulted in significantly higher seed yield than control in Yezin-3, but not single inoculation treatments. Significant difference in yield between co-inoculation and control, not between single inoculation and control, indicates that a synergetic effect is formed by combined application of both bacteria in Yezin-3. In Fukuyutaka soybean variety, single inoculation of BLY3-8, P4, and co-inoculation of BLY3-8 with P4 resulted in significantly higher seed yield than control. This result suggests that BLY3-8 and P4 are the effective bacteria, which are suitable for increasing soybean productivity. Although co-inoculation of both bacteria did not significantly increase seed yield, its co-inoculation did not show any detrimental effect in Fukuyutaka.

Table 4. Effect of co-inoculation of B. elkanii BLY3-8 and S. griseoflavus on seed yield and yield components of Yezin-3 and Fukuyutaka soybean varieties at maturity stage.

Variety	Treatment	Pods (number of plant^−1)	Seeds per pod (number of pod^−1)	100 seed weight (g)	Seed yield (g plant^−1)
Yezin-3	Control	17.7 ± 1.7 a	2.0 ± 0.2 a	13.62 ± 0.68 a	4.63 ± 0.32 b
	P4	24.7 ± 4.7 a	1.7 ± 0.2 a	13.19 ± 0.85 a	5.29 ± 0.25 ab
	BLY3-8	24.3 ± 1.2 a	1.9 ± 0.1 a	11.82 ± 1.33 a	5.41 ± 0.43 ab
	BLY3-8 + P4	20.0 ± 2.2 a	2.2 ± 0.2 a	13.85 ± 0.58 a	6.04 ± 0.14 a
Fukuyutaka	Control	13.3 ± 0.5 a	1.7 ± 0.1 a	24.17 ± 1.20 a	5.46 ± 0.17 b
	P4	17.3 ± 0.9 a	1.6 ± 0.2 a	22.86 ± 0.99 a	6.32 ± 0.09 a
	BLY3-8	16.7 ± 0.5 a	1.7 ± 0.1a	23.04 ± 2.34 a	6.40 ± 0.19 a
	BLY3-8 + P4	16.3 ± 0.9 a	1.8 ± 0.1 a	23.53 ± 0.31 a	6.74 ± 0.20 a

Mean values ± standard deviation (SD) in each column followed by the same letters are not significantly different at P < 0.05 (Tukey’s test).

4. Discussion

Co-inoculation of rhizobia with plant growth-promoting bacteria rather than a single inoculation of rhizobia has become popular because co-inoculation improves soybean yield and contributes to sustainable agriculture (Hungria et al. 2015). Therefore, co-inoculation effects of BLY3-8 with P4 for N₂ fixation were studied in pot experiments using sterilized vermiculite and standard Hoagland nutrient solution in the Phytotron under environmentally controlled condition. In this study, synergetic effect in nodulation and N₂ fixation was occurred in co-inoculation treatment (BLY3-8 + P4) in Rj₄ soybean varieties (Yezin-3 and Fukuyutaka), but not in non-Rj soybean varieties (Table 1). This supports the results of our previous study (Soe et al. 2012; Htwe and Yamakawa 2016), in which the dual inoculation of P4 with bradyrhizobial strains increased the nodulation and N₂ fixation in some soybean varieties but not in other varieties.

Based on the results of the first experiment, a pot experiment was conducted to assess co-inoculation effects of BLY3-8 and P4, in Yezin-3 and Fukuyutaka varieties using Futsukaichi soil in screen house (natural environmental conditions). The combination of P4 with BLY3-8 resulted in significant increases in plant biomasses at R3.5 and R8 stages (Table 2 and Table 3), number of nodules and nodule dry weight at V6 and/or R3.5 stages (Table 2 and Table 3), N₂ fixation at V6 and/or R3.5 stage (Fig. 1A, B and C), N uptake at R3.5 and R8 stages (Table 2 and Table 3), and seed yield (Table 4) in both Yezin-3 and Fukuyutaka soybean varieties. These results support the findings of our group in which dual inoculation of bradyrhizobial strains and P4 increased plant growth, nodulation, N₂ fixation, N uptake, and seed yield of soybeans (Soe et al. 2012; Soe and Yamakawa 2013; Htwe et al. 2018).

We previously reported that co-inoculation of Streptomyces griseoflavus P4 with a type A Bradyrhizobium strain, SAY3-7, had synergetic effects on the plant growth, nodulation, N₂ fixation, N uptake, and seed yield of non-Rj soybean variety (Htwe et al. 2018). The present study showed that co-inoculation of a type B Bradyrhizobium strain with P4 strain synergistically enhanced the plant growth, nodulation, N₂ fixation, N uptake, and seed yield of Rj₄ soybean varieties. Differences between the present and previous studies were that the different types of Bradyrhizobium strain with P4 were investigated using different varieties in a field in the previous study and in a pot in the present study, under natural environmental conditions. However, our previous study (Htwe et al. 2018) and present study suggests that there is a synergetic effect due to co-inoculation of Bradyrhizobium strain and P4 because of the significant difference in plant growth, nodulation, N₂ fixation, N uptake, and yield between co-inoculation and control, not between single inoculation of Bradyrhizobium strain and control.

According to the results of this study, it can be concluded that B. elkanii BLY3-8 (type B strain) together with P4 can be useful as effective inoculants for soybean cultivation of Myanmar where Rj₄ gene harboring soybean cultivars are widely grown.

Acknowledgments

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

Additional information

Funding

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