Enhanced plant growth and / or nitrogen fixation by leguminous and non-leguminous crops after single or dual inoculation of Streptomyces griseoflavus P 4 with Bradyhizobium strains

1 Laboratory of Plant Nutrition, Graduate School of Bioresource and Bioenvironmental Sciences, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. 2 Laboratory of Plant Nutrition, Division of Molecular Biosciences, Department of Bioresource and Bioenvironmental Sciences, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.

The symbiosis between the nodulating bacteria and legumes results in the fixation of atmospheric nitrogen in the nodules.Bio-fertilizer promotes nodulation efficiency and increases yield by 16-60% (DAR, 2004).Moreover, symbiotic nitrogen fixation by soybean provides 65 to greater than 160 kg fixed N ha −1 (Klubeck et al., 1988), accounting for 40-70% of the total nitrogen requirement.Therefore, maintaining this significant nitrogen input is important for economical and sustainable soybean yields, particularly in soils with low available soil nitrogen (Zablotowicz and Reddy, 2004).Therefore, the symbiosis between leguminuous plants and rhizobia is of considerable importance for the environment and agriculture (Ogutcu et al., 2009).
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).Co-inoculation of Bradyrhizobium japonicum with an Azospirillum brasilense strain improved nitrogen fixation in soybean (14.1%) as compared to that of the control (Hungria et al., 2013).Hungria et al. (2015) demonstrated that co-inoculation of Bradyrhizobium spp. with an A. brasilense strain increases soybean yield without additional nitrogen application to soils with an indigenous rhizobial population.
The endophytic actinomycete strain, P4, was isolated from sweet pea root at Kurima, Tsu-City, Japan by Thapanapongworakul (2003).Tang-um and Niamsup (2012b) reported that the P4 16S rRNA sequence has 99.7% sequence similarity with that of Streptomyces griseoflavus (Accession number: EU741217).The production of plant growth-promoting hydrolytic enzymes, such as chitinase, amylase (Tang-um and Niamsup, 2012a) and IAA (Soe, 2013) from S. griseoflavus P4 has been reported.N uptake by adzuki beans and Thai sweet pea occurred due to co-inoculation of Streptomyces spp.P4 with nitrogen-fixing rhizobia (Thapanapongworakul, 2003).A P4 symbiotic interaction similar to that with Myanmar rhizobia has been reported by Soe et al. (2012) and Soe andYamakawa (2013a, 2013b) who stated that the synergistic effect of P4 with soybean-nodulating rhizobia increases nodulation and nitrogen fixation in Myanmar soybean cultivars.Moreover, Soe (2013) reported that S. griseoflavus P4 increases growth performance in leguminous and non-leguminous cereal and vegetable crops.
Using this endophytic strain as a single inoculant in non-leguminous crops or as a co-inoculant with indigenous rhizobial strains to increase plant growth and nitrogen fixation rates of leguminous crops would be of interest.Therefore, this study was conducted to test the effect of a single inoculation of S. griseoflavus P4 on various crops and to evaluate the co-inoculation effects of S. griseoflavus P4 with various Bradyrhizobium strains on plant growth, nodulation and nitrogen fixation in soybean cultivars.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License through a 4-mm sieve to obtain fine growth medium for the crops.S. griseoflavus P4 was inoculated at 2 mL per seed.The plants were cultivated in a Phytotron (25°C and 75% relative humidity) for 2 weeks.Autoclaved deionized water was used for watering.This experiment was conducted during July of 2015.Five replicate plants were used from pots of the various crops to measure the growth parameters.

Symbiosis of the Bradyrhizobium strains and S. griseoflavus P4
Growth conditions occurred under nitrogen-free conditions.The treatments were single inoculations of P4, B. japonicum SAY3-7, B. elkanii AHY3-1 and B. liaoningense SMY3-1 and co-inoculation of P4 with each of the Bradyrhizobium strains.Six seeds per pot were grown in prepared culture pots (1 L volume) filled with 1 L vermiculite and 0.6 L MHN solution and autoclaved at 120°C for 20 min.A control un-inoculated treatment was also provided.S. griseoflavus P4 was inoculated at 2 mL per seed.Bradyrhizobium strain was inoculated at 5 mL per seed.The effectiveness of the single and co-inoculations was determined using the Rj cultivars Yezin-6 (non-Rj) and .Rj genes of these two varieties and their symbiotic effectiveness with B. japonicum USDA110 were described in Htwe et al. (2015b).This experiment was conducted from July to August of 2015.Three replicate plants of the two soybean varieties were used to measure acetylene reduction activity (ARA).
ARA was measured by flame ionization gas chromatography (GC-14A; Shimadzu, Kyoto, Japan) after 4 weeks of cultivation as described by Soe and Yamakawa (2013a).Nodules were removed from the roots after the assay, and the number of nodules was recorded.Shoots, roots and nodules were collected separately and oven dried at 70°C for 24 h to obtain dry weights.

Data analysis
The data were analyzed statistically using STATISTIX 8 analytical software (Tallahassee, FL, USA) and means were compared using Tukey's HSD test at P < 0.05.

Symbiosis of S. griseoflavus P4 with various crops under nitrogen-free condition
The growth performance of the leguminous plants is presented in Tables 1 and 2. Soybean growth parameters, such as shoot and root length and shoot and root dry weight, differed significantly between the plants inoculated with S. griseoflavus P4 and the control plants (Table 1).Shoot length in common bean was affected significantly by inoculation with S. griseoflavus P4 (Table 2) although none of the other growth parameters differed from the control.
The growth performance of the cereal crops is presented in Tables 3 and 4. Maize shoot length and shoot and root biomass were significantly higher than those of the un-inoculated control (Table 3).In rice, no differences were observed in any of the growth parameters between the inoculated treatment and the control (Table 4).The growth performance of the vegetable crops is presented in Tables 5 and 6.Spinach biomass did not increase after inoculation with S. griseoflavus P4 (Table 5); however, root and shoot lengths differed significantly from the control.Radish root biomass was affected significantly by inoculation with S. griseoflavus P4 (Table 6), although none of the other growth parameters differed from the control.
Taken together, the bacterial inoculation treatments increased shoot length, root length, shoot biomass and root biomass when compared with the control.

Symbiosis of three selected Bradyrhizobium strains and S. griseoflavus P4
The performance of the Bradyrhizobium strains and S. griseoflavus P4 on two different soybean varieties is shown in Tables 7 and 8.The numbers of nodules and the nodule dry weights in the Yezin-6 (non-Rj) cultivar differed significantly among treatments but were not significantly different between single and dual inoculations of each strain.Shoot dry weight did not increase among the treatments; however, root dry weight differed significantly among treatments.A single inoculation of B. japonicum SAY3-7 resulted in the highest root dry weight but the difference was not significant from those of a single inoculation of P4, single and dual inoculation of B. liaoningense SMY3-1, or a dual inoculation of B. elkanii AHY3-1.Co-inoculation of B. elkanii AHY3-1 with P4 resulted in significantly higher root dry weight when compared with a single inoculation of this strain.Nitrogen fixation as indicated by the ARA value differed significantly between the P4 co-inoculation and single inoculation treatments, and the control.Coinoculation of P4 with either B. japonicm SAY3-7 or B. liaoningense SMY3-1 did not increase the nitrogen fixation rate when compared with a single inoculation of these strains.Nitrogenase activity increased approximately 24% over the control after co-inoculation of P4 with B. elkanii AHY3-1, although no difference was found between single and dual inoculation.These results show that co-inoculation of B. elkanii AHY3-1 with P4 promotes nitrogenase activity and root growth in the Yezin-6 (non-Rj) cultivar.The nodule dry weight of the Yezin-11 (Rj 4 ) cultivar did not differ between the co-inoculation and single inoculation treatments with the Bradyrhizobium strains.The number of nodules differed significantly among treatments but it was not significantly different between single and dual inoculations of each strain.Root dry weight did not change among treatments.However, shoot dry weight was significantly different among the treatments.Both single and dual inoculation of the tested strains resulted in a significant increase in shoot growth when compared with the control and P4 alone.A significant difference in the nitrogenase activity of the Yezin-11 (Rj 4 ) cultivar was observed between the single and dual inoculation treatments (Table 8).The highest ARA per plant was found after dual inoculation of B. japonicum SAY3-7, followed by dual inoculation of B. elkanii AHY3-1, but these ARA values were not significantly different from those of single or dual inoculation of B. liaoningense SMY3-1.These results show that co-inoculation of B. japonicum SAY3-7 and B. elkanii AHY3-1 with P4 increased nitrogen fixation significantly by approximately 45 and 31%, respectively, when compared with single inoculation of these strains.

DISCUSSION
In this study, the growth and plant biomass of soybean was enhanced after inoculation with S. griseoflavus P4, which agrees with the results of Soe (2013) who reported that root and shoot biomass was significantly higher in plants inoculated with S. griseoflavus P4 than in uninoculated controls.Soe et al. (2010) reported improved shoot growth of the SJ5 Thailand soybean cultivar after inoculation with P4.Moreover, maize shoot and root biomass amounts were significantly higher than those of un-inoculated controls.These findings agree with those of Soe (2013) who reported that maize shoot and root dry weights increased significantly after inoculation with S. griseoflavus P4 when compared with the control.Radish root biomass increased significantly after inoculation with S. griseoflavus P4, although other growth parameters did change relative to the un-inoculated control.These results support the findings of Soe (2009Soe ( , 2013)), who found that the root biomass of sweet pea inoculated with P4 was significantly higher than that of the un-inoculated control, although the shoot biomass did not differ significantly (Soe, 2013).Moreover, inoculation of S. griseoflavus P4 increased the root dry weight of Myanmar soybean, Hinthada by approximately 63% over the control (Soe, 2009).
Soybean, common bean, maize and spinach shoots and roots elongated significantly after inoculation with the P4 strain.The plant growth-promoting effects of S. griseoflavus P4 on leguminous and non-leguminous crops may be due to the production of plant growthpromoting compounds, such as IAA.Soe (2013) reported that S. griseoflavus P4 can produce IAA.Meguro et al. (2006) reported that an endophytic strain of Streptomyces spp.MBR-52 caused pronounced enhancement of emergence and elongation of plant adventitious roots.IAA-producing microorganisms are known to stimulate root elongation and enhance plant growth (Patten and Glick, 2002).
In this study, the symbiotic interaction between P4 and B. elkanii AHY3-1 improved root growth significantly in the Yezin-6 (non-Rj) cultivar as compared to that of a single inoculation of a Bradyrhizobium strain and S. griseoflavus P4.Co-inoculating P4 with the selected indigenous bradyrhizobial strains B. japonicum SAY3-7 and B. elkanii AHY3-1 in the Yezin-11 (Rj 4 ) cultivar significantly increased shoot dry weight as compared to a single inoculation of a Bradyrhizobium strain.Bai et al. (2002) reported that the endophytic Bacillus subtilis strains NEB4 and NEB5 and Bacillus thuringiensis strain NEB17 have excellent potential as plant growth promoters in soybean.They stated that the weight of soybean increased after co-inoculation of B. japonicum with another strain under nitrogen-free conditions when compared with plants inoculated with B. japonicum alone.The symbiotic interaction between P4 and B. elkanii AHY3-1 improved nitrogen fixation significantly as compared to a single inoculation of a Bradyrhizobium strain and S. griseoflavus P4 in the Yezin-6 (non-Rj) cultivar.The symbiotic interaction between P4 and the selected indigenous bradyrhizobial strains B. japonicum SAY3-7 and B. elkanii AHY3-1 increased nitrogen fixation significantly in the Yezin-11 (Rj 4 ) cultivar as compared to a single inoculation of a Bradyrhizobium strain.This result was supported by findings of others that dual inoculation of a bradyrhizobial strain and an endophytic actinomycete (Streptomyces spp.P4) increases nodulation and the nitrogen fixation rate in various soybean varieties (Soe et al., 2012;Soe and Yamakaw, 2013a, b).Thapanapongworakul (2003) reported that dual inoculation of P4 S. griseoflavus and the B. japonicum USDA 110 bradyrhizobial strain increases N uptake by SJ5 (Thailand soybean cultivar) with approximately 44.3% as compared to single inoculation of the same strain.
The Rj genes play roles in adaptation and preference for specific rhizobial strains (Ishizuka et al., 1991;Saeki et al., 2000).In addition, non-Rj is compatible with all bradyrhizobial strains, but Rj 4 has unique features that restrict nodulation with specific Bradyrhizobium strains (Vest and Caldwell, 1972).Soe and Yamakawa (2013a) reported that symbioses were observed after dual inoculation of P4 and MAS34 and dual inoculation of P4 and MAS23 in Yezin-3 (Rj 4 ) and Yezin-6 (non-Rj), respectively.The MAS34 and MAS23 strains have been isolated from Rj 4 -and non-Rj-genotype cultivars, respectively (Soe et al., 2013b).In this study, dual inoculation of P4 with B. japonicum SAY3-7 and B. elkanii AHY3-1 isolated from Rj 4 cultivars increased the symbiotic nitrogen fixation rates in Yezin-11 (Rj 4 ) cultivars.No difference was found for the Yezin-6 (non-Rj) cultivar.
Streptomyces strains are frequently reported to be plant growth-promoting microbes (Nassar et al., 2003;El-Tarabily, 2008).Plant growth-promoting traits such as IAA production and ACC deaminase, cellulase and nitrogenase activities by Bacillus megaterium LNL6 and Methylobatcterium oryzae CBMB20, contribute to the improvement of the overall symbiosis of nitrogen fixation (Subramanian et al., 2015).Plant growth-promoting traits, including the activity of hydrolytic enzymes such as chitinase and amylase (Tang-um and Niamsup, 2012a and 2012b) and IAA production (Soe, 2013), have been documented for S. griseoflavus P4.Therefore, the enhancement of nitrogen fixation observed in this study may be related to the induction of plant growth hormones by S. griseoflavus P4.Sturz et al. (2003) and Sarr et al. (2010) reported that beneficial, detrimental and neutral effects of endophytic bacteria depend on the coinoculated strains of nitrogen-fixing bacteria.In this study, co-inoculation of P4 with either B. japonicum SAY3-7 or B. elkanii AHY3-1 had a synergistic effect on plant growth and nitrogen fixation in the Yezin-11 cultivar; however, co-inoculation of P4 with B. liaoningense SMY3-1 was not beneficial or detrimental to growth or nitrogen fixation rates in either tested cultivar.Therefore, co-inoculation of this endophytic bacterium with B. japonicum SAY3-7 or B. elkanii AHY3-1 may be useful for producing biofertilizer and reduce the need for nitrogen fertilizer by enhancing growth and nitrogen fixation rates in soybean.

Conclusion
A beneficial effect of S. griseoflavus P4 on the growth of leguminous and non-leguminous crops was observed in this study.It was shown that S. griseoflavus P4 was effective in both leguminous and non-leguminous crops.The root dry weight of the Yezin-6 (non-Rj) cultivar increased significantly after co-inoculation of B. elkanii AHY3-1 with P4 when compared with a single inoculation of this strain.Moreover, nitrogenase activity increased (24%) after inoculating B. elkanii AHY3-1 with P4 when compared with a single inoculation of this strain, although no difference among treatments was observed.Coinoculation of B. japonicum SAY3-7 and B. elkanii AHY3-1 with P4 increased nitrogen fixation in the Yezin-11 (Rj 4 ) cultivar by approximately 45 and 31%, respectively, when compared with single inoculation of these strains.The results demonstrate that S. griseoflavus P4 promotes growth and nitrogen fixation in soybean after coinoculation with the Bradyrhizobium strains B. japonicum SAY3-7 or B. elkanii AHY3-1.This study was conducted in pots under controlled conditions.Further studies should be conducted under open field conditions.

Table 1 .
The effect of P4 on soybean growth at two weeks after sowing.

Table 2 .
The effect of P4 on common bean growth at two weeks after sowing.
Means in each column followed by the same letters are not significantly different at P < 0.05 (Tukey's test).SL, RL, SDW, and RDW are shoot length, root length, shoot dry weight, and root dry weight, respectively.

Table 3 .
The effect of P4 on rice growth at two weeks after sowing.

Table 4 .
The effect of P4 on maize growth at two weeks after sowing.Means in each column followed by the same letters are not significantly different at P < 0.05 (Tukey's test).SL, RL, SDW, and RDW are shoot length, root length, shoot dry weight, and root dry weight, respectively.

Table 5 .
The effect of P4 on spinach growth at two weeks after sowing.
Means in each column followed by the same letters are not significantly different at P < 0.05 (Tukey's test).SL, RL, SDW, and RDW are shoot length, root length, shoot dry weight, and root dry weight, respectively.

Table 6 .
The effect of P4 on radish growth at two weeks after sowing.
Means in each column followed by the same letters are not significantly different at P < 0.05 (Tukey's test).SL, RL, SDW, and RDW are shoot length, root length, shoot dry weight, and root dry weight, respectively.

Table 7 .
Effect of single and coinoculation of bradyrhizobial strains on plant growth, nodulation and acetylene reduction activity of Yezin-6 soybean cultivar at 28 DAS.Mean in each column followed by the same letters are not significantly different at P < 0.05 (Tukey's test).NN, NDW, SDW, and RDW indicate nodule number, nodule dry weight, shoot dry weight, and root dry weight, respectively.DAS means days after sowing.

Table 8 .
Effect of single and coinoculation of bradyrhizobial strains on plant growth, nodulation and acetylene reduction activity of Yezin-11 soybean cultivar at 28 DAS.
Mean in each column followed by the same letters are not significantly different at P < 0.05 (Tukey's test).NN, NDW, SDW, and RDW indicate nodule number, nodule dry weight, shoot dry weight, and root dry weight, respectively.DAS means days after sowing.