Growth-promoting effects of Bradyrhizobium soybean symbionts in black oats, white oats, and ryegrass

Abstract

Although inoculating soybean with rhizobia for biological nitrogen fixation is a common practice in agriculture, rhizobia are also known to associate with grasses. In this study, we evaluate the potential utility of the rhizobial strains SEMIA 587 and 5019 (Bradyrhizobium elkanii), 5079 (Bradyrhizobium japonicum), and 5080 (Bradyrhizobium diazoefficiens), recommended for Brazilian soybean inoculation, in colonizing black oat plants and promoting growth in black and white oats, and ryegrass. Inoculation of white oats with SEMIA 587 increase the seed germination (SG) by 32.09%, whereas the SG of black oats inoculated with SEMIA 587 and 5019 increased by 40.38% and 37.85%, respectively. Similarly, inoculation of ryegrass with all strains increased SG values between 24.63 and 27.59%. In addition, white oats with SEMIA 587 and 5080 had root areas significantly superior to those in other treatments, whereas inoculation with SEMIA 5079 and 5080 resulted in the highest volume of roots. Likewise, SEMIA 5079 and 5080 significantly increased the length, volume, and area of black oats roots, whereas SEMIA 587 increased the volume, area, and dry mass of roots and shoot. Inoculation in ryegrass with SEMIA 587 significantly increased the root volume. Moreover, most strains transformed with gfp and gus were observed to colonize the roots of black oats. Collectively, the findings of this study indicate that rhizobial strains recommended for inoculation of soybean can also be used to promote the growth of the three assessed grass species, and are able to colonize the roots of black oats.

Introduction

White oats (Avena sativa L.) and black oats (Avena strigosa Schreb) are cereal species of considerable agricultural importance, given their strong exploration potential, and are used both for grain production and pasture [1]. The advantages associated with cultivation of these crops can also extend to the subsequently grown crops, by reducing the infestation of unwanted plants due to an allelopathic suppressive effect, and thereby reducing the costs of manual or chemical weeding [2]. In the southern Brazilian state of Rio Grande do Sul (RS), oats are among the main winter-cultivated crops, accounting for more than 265.8 thousand hectares, with an average yield of 2.293 kg/ha, both in mono-cultivated areas and those under crop–livestock rotations, in which they are intercropped with soybean [3]. In addition to oats, ryegrass (Lolium multiflorum Lam.), an annual winter grass [4] grown throughout RS, is an excellent source of forage, producing up to 25 tons of green mass per hectare, with good palatability and nutritional value [5].

Agriculturally, RS is notable with respect to the favorable combination of climatic factors, investment in technology, and cultivable land area. However, productivity is limited to a certain extent by the nutritionally poor soils, particularly low nitrogen levels, and consequently, a high reliance on nitrogen fertilizers accounts for a high proportion of agricultural expenditure in this region [6]. An alternative to the application of expensive nitrogen fertilizers is the exploitation of biological nitrogen fixation, which is widely used for soybean production in Brazil. In this regard, a number of programs have led to the identification rhizobia strains and development of inoculants that could be used to increase crop plant nitrogen fixation [7], and the selection and recommendation of strains were carried out during the 1960s and 1970s [8]. In 1992, four strains of Bradyrhizobium (SEMIA 587, 5019, 5079, and 5080) were recommended for soybean cultivation [9] and are still widely used today [10].

In addition to forming symbiotic associations with legumes, rhizobia can also interact with other plants, including grasses, and the beneficial effects of these association have already been observed in rice [11–16], maize [17–19], white oats [19, 20], wheat [18, 21, 22], ryegrass [23], aries grass and sorghum forage [24], and millet and Sudan grass [24, 25]. In grasses, rhizobia can stimulate plant growth through the production of phytohormones and 1-aminocyclopropane-1-carboxylate deaminase [13, 26], phosphate solubilization [27], and protection against pathogens [14, 28]. After penetrating the root system of grasses, rhizobia undergo an upward migration in plants [29], and have been detected in the emergent lateral roots, stalk, and leaves of rice plants [15, 30]. Moreover, the ability of rhizobia to survive and multiply in the rhizosphere has been explored as an alternative approach for enhancing soil populations to desirable levels, via the inoculation of winter cereal seeds prior to sowing soybeans [31]. However, in addition to simply increasing soil populations, inoculation of non-legumes can result in an intense rhizobial colonization of roots [32]. Given the benefits regarding the development of grasses and legumes, rhizobia have been studied as a potential alternative for the management of these plants grown in succession and/or intercropping [23, 29, 33–36]. The objective of the present study was to evaluate the potential of the rhizobial strains SEMIA 587 and 5019 (Bradyrhizobium elkanii), 5079 (Bradyrhizobium japonicum), and 5080 (Bradyrhizobium diazoefficiens), which are recommended for soybean inoculation in Brazil, for the purposes of promoting the growth of black oats, white oats, and ryegrass, In addition, we sought to evaluate the colonization capacity of Bradyrhizobium strains in inoculated black oat plants.

Material and methods

Biological material source

The following Bradyrhizobium strains used in the present study were obtained from the SEMIA Rhizobia Collection of the Department of Agricultural Research and Diagnosis of the Secretariat of Agriculture, Livestock, and Rural Development of Rio Grande do Sul: SEMIA 587 (Bradyrhizobium elkanii), SEMIA 5019 (29W, B. elkanii ), SEMIA 5079 (CPAC15, B. japonicum), and SEMIA 5080 (CPAC7, B. diazoefficiens). As controls, we used strains of Azospirillum sp. (Ab-v5 and Ab-v6) obtained as a commercial liquid inoculant (Simbiose, lot 2018). All bacterial strains were maintained at the Soil Microbiology Laboratory of the Federal University of Rio Grande do Sul (UFRGS). The seeds of black oats, white oats, and ryegrass used were respectively those of the varieties Embrapa 139, URS Altiva, and LE-284.

Inoculation with rhizobial strains and evaluation of white oat, black oat, and ryegrass seed germination

To assess the effects of rhizobial inoculation on grass seed germination, we applied the following of five treatments: plants inoculated with SEMIA 587, SEMIA 5019, SEMIA 5079, and SEMIA 5080, and a control treatment without inoculation. The seeds were washed and disinfected with 70% alcohol for 30 s, followed by 1% hypochlorite for 30 s, and ten washes with sterile distilled water. The strains of Bradyrhizobium were cultured in yeast mannitol broth with agitation (120 rpm) at 28°C for 7 days. Thirty seeds of each plant (black oats, white oats, and ryegrass) were inoculated with 1.1 × 10⁹ CFU·mL⁻¹ of each strain, placed on 28 × 38-cm filter paper rolls, and incubated for 7 days in plant growth chambers (Model 290L; Mangelsdorf) at 20°C and relative humidity 90%, under a 12-h light:12-h dark photoperiod. Daily evaluations of germination were performed to determine the seed germination (% SG) and average germination time (AGT), according to Maguire [37]. The design was completely randomized with three replicates of each treatment, giving 90 seeds. The index values obtained were evaluated statistically using the Scott–Knott test (p < 0.05).

Growth promotion of seedlings inoculated with rhizobial strains

Seeds of black and white oat were disinfected as described in the previous section, pre-germinated on filter paper, and then individually transferred to tubes containing sterile nutrient solution [38] and 2.5% agar, giving 80 replicates of seeds inoculated with strains SEMIA 587, 5019, 5079, and 5080, or without inoculation (control). The seedlings were incubated at 20°C under a 12-h light:12-h dark photoperiod for 15 days. Owing to the fragility of ryegrass seedling roots, which are readily broken by filter paper, we did not perform similar evaluations for this plant. Following incubation, the roots were stained with 1% methylene blue and scanned on a printer (HP 1610 all in one) according to the methodology adapted from Moraes [39]. The length, volume, and area of seedlings roots were analyzed using the Safira 2.0 program [40], after which, the seedlings were dried in an oven at 60°C for 8 days until the dry mass was constant. The design was completely randomized, and all data were evaluated using the Scott–Knott test (p < 0.01).

Growth promotion in inoculated greenhouse-grown white oats, black oats, and ryegrass plants

To assess the growth characteristics of inoculated plants, we raised plants in greenhouses in 3-kg pots containing a 2:1 mixture of sterile vermiculite and sand. As treatments, the different plants were inoculated separately with 1 mL of 1.1 × 10⁹ CFU·mL⁻¹ SEMIA 587, 5019, 5079, 5080, or Azospirillum sp. (Ab-v5 and Ab-v6), with control plants remaining uninoculated. For each treatment, five disinfected and pre-germinated seeds were sown, as described above. The plants were irrigated with sterile nutrient solution [38] without added nitrogen, and all treated plant received the same dose of nitrogen (NH₄NO₃) fertilizer equivalent to 40 kg ha⁻¹. In total, nutrient solution and nitrogen were applied 14 times over the course of 50 days growth. In the end of the experiment, each plant received 1020 mg of NH₄NO₃. The roots of the grown plants were subsequently washed and total root volume was determined as described above. The shoot and roots were after that dried in an oven at 60°C for 10 days until dry mass was constant. To determinate dry mass and the total nitrogen, content of shoots was determined using the methodology described by Tedesco et al. [41]. The experimental design was completely randomized, and the results were analyzed using the Scott–Knott test (p < 0.05 and p < 0.10).

Transformation of Bradyrhizobium strains with the plasmid pHRGFPGUS

The transformation of the Bradyrhizobium strains SEMIA 587, 5019, 5079, and 5080 was performed based on triparental conjugation using the DH5α strain of Escherichia coli with two distinct plasmids, namely, pHRGFPGUS [42], harboring gfp (green fluorescent protein) [43] and the gusA gene, and pRK2013 [44], which contains genes that promote transfer and resistance to kanamycin. Conjugation was performed according to the methodology of O’Hara et al. [45], and the selection of transformed colonies was carried out on Luria–Bertani (LB) medium containing X-gluc 40 µg mL⁻¹ and ampicillin (150 µg mL⁻¹). The antibiotics used to select SEMIA strains 587, 5019, and 5079 were ampicillin (150 µg mL⁻¹) and nalidixic acid (50 µg mL⁻¹) and for SEMIA 5080 were ampicillin (150 µg mL⁻¹) and vancomycin (50 µg mL⁻¹). To quantify gfp expression in transformed Bradyrhizobium strains, we performed fluorimeter readings at a wavelength 510 nm, together with untransformed bacteria (control) cultured in the same medium.

Colonization of black oat plants by transformed Bradyrhizobium strains

Black oat seeds were initially disinfected and pre-germinated under the same conditions described above, and were after that placed in test tubes containing sterile nutrient solution [38]. The experiment had four replications of the following treatments: inoculation with 1.3 × 10⁹ CFU·mL⁻¹ of the transformed Bradyrhizobium strains, a control without inoculation, a control with inoculation of the same untransformed strains, and a “positive” control of soybean (Glycine max) inoculated with each of the transformed Bradyrhizobium strains. The plants were maintained for 15 day at 23°C in a plant growth chamber under a 12-h light:12-h dark photoperiod. Following incubation, evaluations were carried out under a fluorescence microscope (Imager. D2; Zeiss) with an attached camera (AxioCam MRc Zeiss) to visualize GFP and a phase-contrast microscope for the visualization of GUS.

Results and discussion

Inoculation with rhizobial strains and evaluation of white oat, black oat, and ryegrass seed germination

Values obtained for the seed germination (SG) and average germination time (AGT) of inoculated white oat, black oat, and ryegrass seeds are shown in Table 1. White oat seeds inoculated with the SEMIA 587 strain showed a higher SG (32.09%) than the other treatments, as did black oat seeds inoculated with SEMIA strains 587 (40.38%) and 5019 (37.85%). In the case of ryegrass, seeds inoculated with SEMIA strains 587, 5079, and 5080 showed higher SG values of approximately 24.63%, 27.59%, and 26.20%, respectively (Table 1). Contrastingly, we detected no significant differences among treatments with respect to the AGT of inoculated white oat, black oat, and ryegrass seed (Table 1).

Table 1.

Seed germination (SG) and average germination time (AGT) of white oat, black oat, and ryegrass seeds inoculated with the strains of Bradyrhizobium SEMIA 587, 5019, 5079, and 5080

Treatments	White oat		Black oat		Ryegrass
	SG (%) **	AGT (days)	SG (%) **	AGT (days)	SG (%) *	AGT (days)
SEMIA 587	32.09 a ± 3.59	3.08 ± 0.86	40.38 a ± 1.6	2.02 ± 0.24	24.63 a ± 4.84	4.49 ± 1.6
SEMIA 5019	21.29 c ± 5.75	5.38 ± 0.69	37.85 a ± 3.3	1.96 ± 0.23	19.16 b ± 2.8	5.96 ± 3.5
SEMIA 5079	27.79 b ± 3.23	2.45 ± 0.9	31.08 c ± 0.83	2.07 ± 0.69	27.59 a ± 7.7	2.96 ± 0.7
SEMIA 5080	29.49 b ± 5.53	4.84 ±1.87	36.15 b ± 3.5	1.97 ± 0.44	26.20 a ± 4.14	3.29 ± 0.96
Control	23.64 b ± 1.51	3.52 ± 0.63	36.40 b ± 5.13	2.19 ± 0.67	18.24 b ± 3.51	4.8 ± 1.13

Means three replicates (90 seeds of each plant) grouped by the same letter in the column do not differ for the Scott–Knott test

^*Significant for p <0.1

^**Significant for p <0.05

Osório Filho et al. [35] obtained similar results for rice seeds inoculated with rhizobia isolated from Trifolium vesiculosum, Trifolium repens, Lotus glaber, Lotus corniculatus, and Lotus uliginosus, whereas Silva et al. [19], who examined the effects of inoculating rice and wheat with rhizobia isolated from Desmodium incanum, observed significant effects on the germination of rice plants. Similarly, Machado [46], who evaluated the germination of ryegrass plants, observed that inoculation with the rhizobial strains SEMIA 816 (Mesorhizobium sp.), UFRGS L524 (Mesorhizobium sp.), and UFRGS Lc111 accelerated germination from the second day after inoculation, compared to the control group. Furthermore, in a study conducted by Hahn et al. [16], inoculation with the strain UFRGS Lc348 (Mesorhizobium sp.), isolated from birdsfoot trefoil, was found to accelerate germination of the rice cultivar IRGA 422CL by more than 38%, compared with the control, whereas the strains UFRGS Vp16 (Burkholderia sp.) and UFRGS Lg111 (Mesorhizobium sp.) stimulated rice seed germination by 21% and 20%, respectively. Stroschein et al. [12] also observed that rhizobia isolated from alfalfa enhanced the germination speed of rice seeds, notably UFRGS Ms72 (Rhizobium sp.), which promoted an increase of approximately 25%.

Growth promotion in white oat and black oat seedlings inoculated with rhizobial strains

Evaluation of selected root parameters of white oat plants inoculated with the strains SEMIA 587 and 5080 revealed that these strains promoted an increase in root area that was superior to that obtained for plants inoculated with the other assessed strains; however, changes in root length did not differ significantly from those observed in control plants (Table 2). Nevertheless, the roots of white oat plants inoculated with strains SEMIA 5079 and 5080 were found to have a greater volume compared with those inoculated with other strains (Table 2).

Table 2.

Length, volume, area, and root dry mass (RDM) of white oat and black oat inoculated with Bradyrhizobium SEMIA 587, 5019, 5079, and 5080 strains after fifteen days of growth

Treatments	White oat				Black oat
	Length (mm)*	Volume (mm3)*	Area (mm2)*	RDM (mg)	Length (mm)*	Volume (mm3)*	Area (mm2)*	RDM (mg)*
SEMIA 587	137.08a ± 26.29	33.84b ± 3.01	203.14a ± 8.99	59.60 ± 6.05	73.90b ± 3.96	21.82a ± 4.31	115.51a ± 15.30	39.30 a ± 16.64
SEMIA 5019	86.24b ± 1.56	31.06b ± 11.53	157.21b ± 34.28	39.60 ± 4.60	29.30c ± 10.17	9.62b ± 3.00	45.92b ± 13.98	17.10b ± 4.92
SEMIA 5079	101.30b ± 49.05	38.14a ± 18.70	192.21b ± 94.69	53.17 ± 1.79	91.61a ± 37.79	24.04a ± 8.00	140.62a ± 54.18	16.87b ± 0.67
SEMIA 5080	130.58a ± 36.47	40.59a ± 16.33	223.56a ± 74.42	52.23 ± 15.53	78.14a ± 7.59	22.29a ± 1.49	122.87a ± 7.49	27.53b ± 2.28
Control	116.46a ± 12.65	31.72b ± 6.12	186.59b ± 26.58	52.13 ± 8.79	58.43b ± 25.14	12.91b ± 4.43	80.32b ± 32.48	20.67b ± 7.84

^*Means (80 repetitions), values followed by the same letter in the column do not differ for the Scott–Knott test (p <0.01)

The SEMIA 5079 and 5080 strains were also found to significantly increase the length, volume, and area of black oat roots (Table 2), whereas inoculation of seeds with SEMIA 587 also increased root volume and area, as well as root dry mass (RDM) (Table 2). Similarly, Machado et al. [47] also noted increases in the RDM of pensacola grass following inoculation with the rhizobia UFRGS Lc 323 and Lc 348 (Mesorhizobium sp.), as well as in Tanzania grass inoculated with the rhizobia SEMIA 816, UFRGS Lc134, 323, 336, 348, 510, and 524.

Growth promotion in inoculated white oat, black oat, and ryegrass plants grown in a greenhouse

White oat plants inoculated with Bradyrhizobium sp. SEMIA 587, SEMIA 5019, SEMIA 5079, and SEMIA 5080 strains and grown in a greenhouse were found to show no significant differences with respect to shoot dry mass (SDM), RDM, or shoot nitrogen content, whereas those inoculated with Azospirillum sp. were characterized by a significantly higher SDM and RDM (Table 3), although no significant differences in shoot nitrogen or root volume were detected (Table 3). These observations accordingly indicate that white oat plants respond positively to inoculation with Azospirillum sp., which is a well-established promoter of grass growth, not only due to its capacity to fix nitrogen but also with respect to the production of phytohormones [48, 49]. Consistently, studies that have examined the effects of Azospirillum inoculation have revealed a significant influence on the productivity of important agricultural crops [50]. The Bradyrhizobium genus also can produce IAA (indole acetic acid). This capacity has been demonstrated in vitro and in planta after soybean seed inoculation [51, 52]. Nodules caused by an IAA-overproducing strain of Bradyrhizobium japonicum contained more IAA than nodules induced by a wild strain. This indicates that the bacteroid in the nodule is able to synthesize the phytohormone [53, 54]. Torres et al. [55] showed the presence of several genes responsible for IAA biosynthesis in Bradyrhizobium japonicum E109, but IAA is not accumulated in the culture medium in significant amounts. Santos [18] working with maize plants observed that inoculation with the rhizobia UFRGS Vp16 (Burkholderia sp.) and UFRGS Lc348 (Mesorhizobium sp.), either alone or in combination with Azospirillum, maintains the production of maize grains fertilized with 50% of the recommended nitrogen. This beneficial effect is assumed to be attributable to the production of auxins, gibberellins, and cytokinins, which promote an increase in root growth [56]. Gupta et al. [57] evaluated the effects of inoculating white oats with bacterial isolates from the rhizosphere of wheat and maize, and demonstrated that the auxins, siderophores, and solubilized phosphates produced by these isolates accelerated germination and subsequently stimulated root growth when compared with an uninoculated control.

Table 3.

Shoot dry mass (SDM), root dry mass (RDM), shoot nitrogen content (N), and root volume of white oat, black oat, and ryegrass inoculated with Bradyrhizobium SEMIA 587, 5019, 5079, and 5080 strains, and grown in a greenhouse

Treatments	White oak				Black oak				Ryegrass
	SDM* (g)	RDM** (g)	N (mg)	Root volume (cm3)	SDM* (g)	RDM (g)	N (mg)	Root volume (cm3)	SDM* (g)	RDM (g)	N** (mg)	Root volume (cm3)
SEMIA 587	4.01 b ± 0.72	2.52 b ± 0.29	69.62 ± 8.08	46.5 ± 25.1	5.01 a ± 0.19	2.38 ± 0.43	94.0 ± 17.6	36.49 ± 11.99	3.52 b ± 0.39	3.68 ± 0.32	75.4 b ± 7.6	83.86 a ± 27.87
SEMIA 5019	3.85 b ± 0.44	2.50 b ± 0.37	67.43 ± 12.44	61.3 ± 34.7	4.77 b ± 0.49	3.10 ± 0.46	84.4 ± 11.4	51.26 ± 9.92	3.47 b ± 0.68	2.19 ± 0.57	66.1 b ± 27.9	55.87 b ± 49.97
SEMIA 5079	4.04 b ± 0.53	2.61 b ± 0.53	70.16 ± 11.4	72.5 ± 45.9	4.59 b ± 0.66	2.61 ± 0.4	75.7 ± 8.4	48.72 ±15.34	3.88 b ± 1.18	3.58 ± 0.88	71.1 b ± 11.4	58.95 b ± 16.01
SEMIA 5080	3.68 b ± 0.36	2.30 b ± 0.20	67.2 ± 12.0	57.8 ± 22.4	4.52 b ± 0.45	2.16 ± 0.55	88.8 ± 12.8	52.36 ± 24.00	3.61 b ± 0.26	3.13 ± 0.3	78.0 b ± 8.2	43.52 b ± 9.07
Azospirillum sp. Ab-v5/Ab-v6	4.54 a ± 0.57	3.31 a ± 0.39	79.4 ± 10.8	54.8 ± 25.8	4.64 b ± 1.08	2.80 ± 0.44	88.9 ± 23.4	30.88 ±17.08	4.50 a ± 0.38	2.85 ± 0.58	97.4 a ± 8.8	58.01 b ± 24.27
Control	3.88 b ± 0.59	2.59 b ± 0.24	69.6 ± 11.8	44.2 ± 24.8	4.49 b ± 0.57	2.24 ± 0.52	83.0 ± 3.9	40.71 ± 24.28	3.57 b ± 0.27	3.46 ± 0.27	80.0 b ± 9.4	72.58 b ±27.92

Values followed by the same letter in the column do not differ for the Scott–Knott test

^*Significant means for p <0.10

^**Significant means for p <0.05

With respect to greenhouse-grown black oat plants, we recorded a significant increase in the SDM of plants inoculated with the SEMIA 587 strain (Table 3), although no significant difference was detected for the parameters RDM, shoot nitrogen, or root volume (Table 3). The SEMIA 587 strain of Bradyrhizobium elkanii was originally isolated in 1968 from soybeans in the Santa Rosa/RS region, and on the basis of its high symbiotic efficiency and competitiveness, this strain has since been recommended as an effective inoculant [8, 10]. Silveira [58] has pointed out the ability of SEMIA 587 to produce siderophores, which are secondary metabolites with a high affinity for Fe³⁺ ions that are produced by several microorganisms under conditions of environmental Fe deficiency. Stajkovic-Srbinovic et al. [20], working with Enterococcus, Bacillus, and Pseudomonas in white oats, verified an increase in the shoot of plants grown with half the recommended nitrogen dose. Silva et al. [19] similarly observed that white oat plants inoculated with rhizobia isolated from Desmodium incanum showed increases in SDM, compared with the control treatment in which plants received 100% of the recommended nitrogen dose, whereas Machado [46], who evaluated the effects of the Bradyrhizobium sp. strain UFRGS Lc 394 co-inoculated with Trichoderma in black oat plants, found that SDM was higher in plants inoculated with rhizobia alone and in combination with the fungus than in the control with twice the normal nitrogen application. Similar increases in SDM have been found by Osório Filho et al. [35] in rice plants, in which the UFRGS Vp16 isolate of Burkholderia sp. was noted as being particularly effect in stimulating the growth of aerial parts of plants receiving either 21% of the normal nitrogen application or no applied nitrogen. Hahn et al. [16] found that the same strain increased the SDM of the rice cultivar IRGA 422CL by 11%, and also reported that inoculation with the UFRGS Lc336 strain of Mesorhizobium sp., isolated from birdsfoot trefoil, increased the SDM of the rice cultivar IRGA 422CL by 8%. Furthermore, in Sudan grass, inoculation with the UFRGS Vp16 strain enriched with tryptophan increased the SDM of mature plants [25], whereas Machado et al. [47] found that the dry mass of Pensacola grass inoculated with the rhizobial strains UFRGS Lc134, Lc336, and Lc394 and supplied with 50% of the recommended nitrogen dose was superior to that of control plants, although comparable in plants receiving a 100% nitrogen dose. Similar observations were made for Tanzania grass plants in the same study.

We also evaluated the efficacy of rhizobia inoculation in greenhouse-cultivated ryegrass plants. With respect SDM, we found that the average values in plants inoculated with Azospirillum sp. were significantly higher than those obtained using the other assessed strains, whereas in the case of root volume, the SEMIA 587 strain was observed to be significantly more effective (Table 3). However, we detected no significant differences among treatments with regard to the RDM and shoot nitrogen contents of inoculated ryegrass plants (Table 3). Machado [46], who examined the effects of inoculating ryegrass plants with different rhizobia from the UFRGS strain bank, found that there were no significant increases in SDM when compared with a treatment in which plants were provided same nitrogen dosage as used in the present study. However, in plants inoculated with the UFRGS Lc323 strain, a larger root volume was observed compared with the control treatment, although this increase did not promote a greater accumulation of total nitrogen in the aerial parts. Furthermore, Bhattacharjee et al. [13] demonstrated that Rhizobium leguminosarum bv. trifolii SN10 promotes the growth of four rice varieties in terms of total biomass, root ramification, and nitrogen content.

Colonization of black oat plants by strains of Bradyrhizobium

On the basis of results obtained in the seedling and greenhouse assessments, which indicated an acceleration of seed germination, increases in root volume, area, and dry mass, and increases in SDM in black oat plants inoculated with SEMIA 587, we selected black oats for inoculation with transformed bacteria, in order to verify whether the inoculated bacteria colonize host plants.

As shown in Fig. 1a–c, plants respectively inoculated with the SEMIA 587, SEMIA 5079, and SEMIA 5080 strains transformed with pHRGFPGUS expressed gfp gene, although no colonization was detected in plants inoculated with SEMIA 5019. All strains expressed GUS and colonized the roots of black oat plants (Fig. 2). Moreover, we found that all the transformed Bradyrhizobium strains retained the capacity to nodulate soybean plants.

Fig. 1.

Black oat roots inoculated with Bradyrhizobium strains expressing the gfp gene: (a) SEMIA 587; (b) SEMIA 5079; (c) SEMIA 5080; (d) control (without inoculation). The red arrows indicate bacterial cells expressing GFP

Fig. 2.

Black oat roots inoculated with Bradyrhizobium strains expressing the gus gene: (a) SEMIA 587; (b) SEMIA 5019; (c) SEMIA 5079; (d) SEMIA 5080. The red arrows indicate bacterial cells expressing GUS

In India, Bhatia et al. [59] transformed lentil symbiont strains of Bradyrhizobium with the gfp gene via biparental conjugation with E. coli S17-1 carrying the EDS 15 plasmid, and verified the efficacy of rhizobia transformation in inoculated host plants, based on nodule counts and wet and dry weight determinations performed 45 days post-inoculation, as well as the respective re-isolation of nodule-forming bacteria. Previous analysis of the stability of gfp fluorescence in transformed Bradyrhizobium diazoefficiens strain USDA 110 has indicated established that fluorescence was weak when inserted in this strain Ledermann et al. [60], and on the basis of the observed fluorescence intensity, plasmid susceptibility to degradation, and plant tissue autofluorescence, these authors constructed a synthetic gene expressing mCherry (mChe-1); (mChe-4) gene, found in plasmids that express red autofluorescence from jellyfish. Using this approach, it was possible to observe fluorescence in soybean roots as early as the 4th day after inoculation with the transformed rhizobia [55]. However, Hahn et al. [16] confirmed the presence of rhizobia inoculated and labeled with gfp (UFRGS Vp16 and UFRGS Lc348) and Azospirillum brasilense colonizing only the intercellular spaces of the epidermis and root surface of rice seedlings, primarily in the root hairs and lateral roots.

Conclusions

In this study, we found that inoculation with Bradyrhizobium strains recommended as inoculants for soybean cultivation in Brazil (SEMIA 587, 5019, 5079, and 5080) promoted the growth of white oat, black oat, and ryegrass plants, by increasing parameters such as the seed germination, thereby yielding plants with superior root area, and significantly increased root length and volume, and enhanced shoot and root dry mass. SEMIA 587, SEMIA 5079, and SEMIA 5080 bacteria transformed and labeled with the gfp gene, as well as all strains transformed and tagged with the gus gene, colonized the roots of the black oat plants, and retained their ability to nodulate soybean. Accordingly, we have established that the rhizobial strains recommended for inoculation of Brazilian soybean can also be used to promote the growth of selected grass species (black oat and ryegrass), although we highlight the need for a field experiment to confirm those positive interactions.

Author contribution

Material and methods preparation, and analysis were performed by Carolina Leal de Castilho, Camila Gazolla Volpiano, Lucas Zulpo, Adriana Ambrosini, and Anelise Beneduzi. Grant and fellowships acquisition were provided by Enílson Luiz Saccol de Sá, and Luciane Passaglia. The manuscript was written by Carolina Leal de Castilho, Enílson Luiz Saccol de Sá, and Anelise Beneduzi. All authors commented on previous versions of the manuscript, and approved the final manuscript.

Funding

This work was financed by a grant and fellowships from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/Brazil).

Declarations

Conflict of interest

The authors declare no competing interests.