Rhizobial diversity in shrub-tree legume-based silvopastoral systems

ABSTRACT

Silvopastoral systems based on tree legumes intercropped with forage grasses can harbor a high diversity of rhizobia, and these bacteria are good indicators of soil quality in several management systems. The objective of this work was to evaluate the morphophysiological, genetic and symbiotic diversity of cowpea [Vigna unguiculata (L.) Walp] rhizobia from soils under silvopastoral systems based on shrub-tree legumes. The experiment was performed in a randomized block design with three treatments and three replications, consisting of signalgrass (Urochloa decumbens Stapf.) intercropped with sabia (Mimosa caesalpiniaefolia); signalgrass intercropped with gliricidia (Gliricidia sepium) and single signalgrass. The samples were collected in the legume row (0 meter) and 4 and 8 meters away. Later, cowpea was used as a trap plant to capture the rhizobia. All strains were phenotypically characterized, authenticated, and genetically identified. Phenotypical characterization of the 431 isolates showed high diversity forming 69 groups at 100% similarity, of which 60 were able to nodulate cowpea during the authentication, and 36 presented relative efficiency superior or equal to the recommended bacteria for the crop. Most of the sequenced strains belonged to Bradyrhizobium (67%) and Methylobacterium (9%). Leifsonia (9%), Cohnella (6%), Rhizobium (3%), Burkholderia (3%), and Paenibacillus (3%) were also represented. Soils under silvopastoral systems harbor efficient rhizobia populations in cowpea with a high genetic diversity, which can be recommended for agronomic efficiency assays.

Key words
Methylobacterium ; Gliricidia sepium ; pasture; Mimosa caesalpiniaefolia ; Vigna unguiculata

Introduction

Silvopastoral systems integrate livestock, forage and shrub or tree species, with several economic benefits (^{Apolinário et al. 2015}). If the arboreal component includes legumes, they also promote an increase on soil fertility including nutrient input, mainly from nitrogen, into deep soil layers (^{Apolinário et al. 2015}) with significant effects up to 1 m depth (^{Lira Junior et al. 2020b}). This is particularly important since a large part of tropical subhumid pastures, typically based on pure grasses and not fertilized with N, are submitted to various stages of degradation with major environmental impacts (^{Lima et al. 2018}).

Gliricidia (Gliricidia sepium (Jacq.) Steud.) and sabia (Mimosa caesalpiniifolia Benth.) are tropical shrub-tree legumes that benefit grass pastures (^{Apolinário et al. 2015}) and provide nutritive support to ruminants. These legumes have fast growth, high regeneration capacity, drought resistance (^{Mendes et al. 2013}; ^{Paula et al. 2015}) and fix nitrogen when in symbiosis with diazotrophic microorganisms (^{Martins, J. C. R. et al. 2015}; ^{Martins, P. G. S. et al. 2015}) at around 200 kg N.ha^-1 yr^-1 (^{Apolinário et al. 2015}).

Soil microbial diversity can be affected by soil management and plant cover (^{Barros et al. 2018}), and the silvopastoral system based on shrub tree legumes determines the structure of the total bacterial and the nitrogen-cycle bacteria communities, as shown by ^{Barros et al. (2021)}. Soils under agriculture and agroforestry may harbor a high rhizobia genetic diversity due to increased nitrogen demand, stimulating the rhizobial population (^{Guimarães et al. 2012}), and this biodiversity may be both a means to evaluate management system impacts (^{Lima et al. 2009}) and a source of efficient rhizobial strains for use in inoculant production (^{Uzoh and Babalola 2018}).

The effects of silvopastoral systems on rhizobial diversity have not been published up to now, to the best of our knowledge, although general effects of land use systems and changes on rhizobial diversity are widely known (^{Berza et al. 2021}; ^{Gnangui et al. 2021}; ^{Souza and Procópio 2021}; ^{Wang et al. 2021}). This rhizobial diversity can be studied by either culture independent methods or by culture dependent ones, in this case typically using trap species. The selection of the trap species is a central point, due to the well-known specificity between legume and rhizobia.

While tropical legumes are generally considered promiscuous (^{Lira Junior et al. 2015}), cowpea [Vigna unguiculata (L.) Walp] is particularly so, and nodulates with a large rhizobial range to varying degrees of efficiency (^{Ndungu et al. 2018}), including both alpha and beta rhizobia (^{Castro et al. 2017}; ^{Lardi et al. 2017}; ^{Muindi et al. 2021}), so it is frequently used as a trap plant species when one of the goals is to obtain the largest cross section possible of rhizobial biodiversity with a single legume species (^{Jaramillo et al. 2013}).

The objective of this work was to determine phenotypical, genetic, and symbiotic diversity of cowpea [Vigna unguiculata (L.) Walp] rhizobia as an indicator of overall rhizobial diversity and silvopastoral systems effects upon it.

MATERIALS AND METHODS

Experimental area and design

Silvopastoral systems were established in 2011 at the experimental field of Instituto Agronômico de Pernambuco (IPA), Itambé, Pernambuco state, Brazil (7°25’S; 35°6’W, 190 m above average sea level), and the study station soil is classified as Ultisol Red-Yellow, according to ^{Jacomine et al. (1973)}. The climate is AS’ in the Köppen classification (i.e., hot and humid), with average rainfall and annual temperature of 1,300 mm.year^-1 and 24 ºC, respectively. The area was continuously grazed by crossbred bovines (Holstein-Frísia-Zebu), with grazing density variable according to the objectives of animal experiments carried out in the area (^{Santos et al. 2020}).

This experiment was conducted in a randomized block design with three treatments and three replications, in nine plots of 1 ha each (43.5 × 230 m), consisting of signalgrass (Urochloa decumbens Stapf.) intercropped with sabia (Mimosa caesalpiniaefolia) (B + S); signalgrass intercropped with gliricidia (Gliricidia sepium) (B + G) and single signalgrass (B). Legumes were planted in 14 double rows, spaced 15 × 1 × 0.5 m, with signalgrass in the rows among each double row.

Soil samples were collected in June 2016 to a 20-cm depth. Each intercropped plot was divided into three transects along, which samples were collected at a 0, 4 and 8-meter distance from the legume double row, to prepare a composite sample for each distance and each plot. Three samples were randomly collected in B treatment to form a composite soil sample at the same depth, totaling 21 samples. Soil chemical characterization is presented in Table 1.

Table 1
Soil chemical characteristics of the silvopastoral system in Itambé, Pernambuco, Brazil, 2016.

Distance (m)	pH Water 1:2.5		Ca	Mg	Al	Na	K	H + Al		P (mg·dm-3)
			(cmolc·dm-3)
			B + S
0	5.0		4.68	1.20	0.43	0.06	0.24	8.31		8
4	5.1		3.62	2.42	0.50	0.07	0.16	7.92		11
8	5.3		4.32	1.88	0.31	0.06	0.17	7.41		6
			B + G
0	5.2		4.43	1.17	0.41	0.06	0.23	7.60		9
4	5.5		4.68	1.90	0.15	0.08	0.30	6.75		7
8	5.6		4.60	2.43	0.20	0.05	0.24	6.48		6
			B
	5.6		3.70	2.00	0.17	0.04	0.19	6.48		9

• B + S: signalgrass intercropped with shrub-tree sabia; B + G: signalgrass intercropped with shrub-tree gliricidia; B: single signalgrass.

Rhizobia capture using cowpea as the trap species and morphophysiological characterization

Cowpea seeds (cultivar IPA-206) were superficially disinfected using 70% alcohol for 30 s, then submerged in 2.5% sodium hypochlorite and cleaned with ultrapure water. All seeds were placed in trays to germinate and incubated for two days at room temperature. Seedlings were moved to sterile 350-mL bottles filled with Hoagland solution without N (^{Hoagland and Arnon, 1950}) at 1/4 strength with two 1.8-cm wide germitest paper tapes placed inside each bottle. Each seedling was inoculated with 1 mL of each decimal serial dilution from 10^-1 to 10^-9 from a soil sample in a sterile saline solution of 0.85% NaCl.

A completely randomized block design in triplicate was used, with two positive controls inoculated with strains BR 3267 (Bradyrhizobium yuanmingense) or BR 3262 (Bradyrhizobium pachyrhizi) (^{Leite et al. 2018}) recommended for cowpea (^{Brasil 2011}), and two uninoculated controls, one without nitrogen and one with mineral N (52.5 mg.L^-1), totaling 513 experimental units. Both positive control strains are currently recommended for commercial rhizobial inoculant production, and according to Brazilian regulations, the usage of two or more strains for a given legume is recommended to increase inoculant effectiveness. After 35 days of inoculation, the plants were collected, the presence or absence of nodules was observed, and the rhizobia community density was estimated by the most probable number (^{Woomer et al. 1994}).

Later, nodules were detached, counted, and stored in silica gel tubes, with 20 nodules from each sample randomly selected for isolation. The nodules were superficially disinfected in 70% ethyl alcohol for 30 s, and subsequently in 2.5% sodium hypochlorite for 5 min, followed by 10 washes in sterile distilled water (^{Vincent 1970}). Nodules were macerated in Petri dishes with yeast-mannitol-agar (YMA) culture medium (^{Vincent 1970}), with Congo red (0.25% in 0.2 N KOH). The plates were incubated at room temperature, and, after bacterial growth, successive streaking was carried out until obtaining pure colonies.

Pure isolates were phenotypically characterized based on development of colonies (fast: up to three days; intermediate: four or five days; slow: six to 10 days; and very slow: more than 10 days); media pH change (acid, neutral, and alkaline); mucus amount (little, moderate or abundant); size (< 1, 1 to 2 and > 2 mm) and color of the colonies (yellow, white, pink, and cream). A binary matrix was created from the cultural characteristics, and the isolates were grouped by the unweighted pair group analysis (UPGMA) method using PAST 3.18 (^{Hammer et al. 2001}). For the diversity estimate, Shannon, Weaver diversity index (H), Simpson dominance index, and the Pielou equitability index (J) were also calculated using PAST 3.18 (^{Hammer et al. 2001}).

Strain authentication and symbiotic efficiency

A randomly selected representative from each of the phenotypical groups at 100% similarity was used for authentication under greenhouse conditions, as well as the same controls of the first phase, although the non-inoculated control with mineral nitrogen received the equivalent to 50 kg.ha^-1 N. The experiment consisted of 73 treatments in a completely randomized block design in triplicate, totaling 219 experimental units.

The planting was carried out in 200-mL disposable cups filled with a sand: vermiculite 2:1 (v:v) mix autoclaved. Three IPA-26 cultivar cowpea seeds were sown per cup, and later the plants were thinned to one plant per cup. The bacteria were grown under orbital shaking at 150 rpm for three days for fast growing and five days for slow growing strains. Inoculation was three days after planting using 2 mL of YMA medium per seed. Every three days, 30 mL of N-free Hoagland nutrient solution was applied. Irrigation was performed with sterile distilled water whenever necessary.

The plants were harvested 35 days after inoculation, and shoot dry mass (SDM), root dry mass (RDM), nodules number (NN) and nodule dry mass (NDM) were determined. Relative efficiency (RE) was calculated by shoot dry mass from inoculated treatments and shoot dry mass of the N-supplied control ratio. The mean values were organized by the Scott-Knott test at 5% probability, using the Sisvar 5.6 statistical program (^{Ferreira 2011}).

Genetic diversity of the 16S rRNA gene

From the authentication experiment, the isolates with relative efficiency not different or higher than the recommended strains were selected for characterization of genetic diversity by total bacteria gene sequencing.

During the extraction of the genomic bacterial DNA (bead beating method), a little part of the bacterial colony was introduced in TE buffer solution, and cell lysis was carried out in 10% sodium dodecyl sulfate solution (SDS). The compound was stirred with phenol:chloroform and centrifuged at 12,000 rpm to obtain a supernatant, which was also centrifuged in the presence of isopropanol. The pellet formed was washed with ethanol, centrifuged, and then eluted in 50 µL of sterile water and stored at -20°C. The DNA obtained was visualized on a 1% electrophoresis agarose gel (^{Araújo et al. 2020}).

The primers used during the amplification of the 16S rRNA gene were 27F (5’-AGAGTTTGACCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) (^{Lane 1991}). Amplification was performed in a solution containing 2 µL of DNA, 2.5 µL buffer of polymerase chain reaction (PCR) 10x, 1.5 mM of MgCl₂, 0.2 mM of dNTP, 0.4 mM of each primer, 1 U of Taq DNA polymerase and ultrapure water to a final volume of 50 µL. The entire amplification process was as follows: denaturation at 94°C for 5 min, 35 cycles of denaturation (94°C for 40 s), annealing (55°C for 40 s), extension (72°C for 1.5 min), and a final extension of 72°C, for 7 min. Then, the PCR products were sent to the Macrogen Laboratory in Korea. The 16S rRNA sequences identified were compared to those obtained within the EzBioCloud 16S-based ID (^{Yoon et al. 2017}). A Neighbor-Joining phylogenetic tree was constructed using the Kimura 2-parameter method to compare the obtained 16S rRNA sequences with those from type strains strived from the EzBioCloud service (https://www.ezbiocloud.net/identify), by the MEGA 11 program (^{Tamura et al. 2021}), applying a bootstrap with a minimum of 1,000 replications.

RESULTS AND DISCUSSION

Rhizobial communities’ density ranged from 5.7 × 10⁴ to 1.5 × 10⁶ cell.g soil^-1 without significant effect of intercropping or distance from the legume row.

We isolated 431 isolates, of which 35% showed rapid growth, 33% intermediate, 21% slow and 10% very slow; while 11% of the isolates alkalized the culture medium, 53% did not change the pH and 36% acidified the medium. Regarding colony color, cream (60%), yellow (24%), white (14%), pink (1%) and colorless (0.9%) bacteria were obtained. There was a predominance of bacteria capable of producing little to moderate mucus (44 and 32%) (Fig. 1). There was no significant effect of the silvopastoral system on the diversity (mean H = 2.3), dominance (mean D = 0.8) or equitability (mean J = 0.9) of the rhizobia.

Figure 1
Morphophysiological features of bacteria isolated in YMA culture medium under silvopastoral system soils in Itambé, Pernambuco, Brazil.

It was expected that the silvopastoral systems, based on shrub-tree legumes, would increase the density and diversity of rhizobia in relation to single signalgrass. In addition, it was expected that rhizobia diversity would be greater on the legume range soils (0 m) and it would decrease at an 8-m distance from the legume range. However, no significant differences were observed in the density and diversity of rhizobia, whose values were similar to those found in other studies (^{Lima et al. 2009}; ^{Castro et al. 2017}).

It is known that rhizobia in the soil respond quickly to changes in land use (^{Ormeño-Orrillo et al. 2012}). Hence, we assumed that five years of system implementation were sufficient to influence the soil rhizobia community. In addition, other research in the same experimental field and sampling year had already demonstrated that there was a significant increase in the abundance, spatial and temporal heterogeneity of diazotrophic microorganisms and ammonium-oxidizing bacteria (^{Barros et al. 2018}; ²⁰²¹), in the chemical attributes of the soil (^{Lima et al. 2018}), in soil C and N stocks (^{Lira Junior et al. 2020b}) and in soil organic matter quality (^{Lira Junior et al. 2020a}).

One possible reason for the lack of response observed for rhizobial diversity is the choice of cowpea as the trap plant, since it is able to nodulate with background rhizobial populations predating systems, and thus not differentiate the legume species. We must also consider that both studies of ^{Barros et al. (2018)} and ^{Barros et al. (2021)} were based on culture independent methods, which tend to be more sensitive to environmental effects, though not feasible when evaluation of the rhizobial strains as to their efficiency is desired.

Isolates were grouped into 69 phenotypical groups at 100% similarity. Among the 69 isolates evaluated in the authentication and in the symbiotic efficiency experiments, 60 produced nodules and fixed nitrogen in cowpea (Table 2), confirming cowpea’s promiscuity (^{Guimarães et al. 2012}; ^{Lira Junior et al. 2015}) and its efficiency as a trap plant for rhizobia capture. Cowpea is a promiscuous grain legume that is normally nodulated by Bradyrhizobium, which exhibits slow growing (^{Zhang et al. 2011}). It is known that sabia is preferentially associated with Burkholderia (^{Martins, P. G. S. et al. 2015}), a betaproteobacteria, and it is possible that cowpea was not effective in differentiating the silvopastoral system from single signalgrass due to its preference for association with Bradyrhizobium, an alphaproteobacteria. A possible alternative would be the use of other legumes as trap plants, such as sabia or gliricidia.

Table 2
Nodule number (NN), nodule dry mass (NDM), root dry mass (RDM), shoot dry mass (SDM), and relative efficiency (RE) of cowpea inoculated with rhizobia isolated from soils under silvopastoral system in Itambé, Pernambuco, Brazil.

Strain	Origin of isolates	NN	NDM (mg)	RDM (g)	SDM (g)	RE (%)
43 R1.1	B + G	86.00 a	70 a	0.24 b	1.20 a	338.31 a
35 R3.3	B + G	66.66 a	70 a	0.33 a	1.06 a	297.19 a
42 R2.1	B + G	64.66 a	280 a	0.37 a	1.04 a	291.59 a
44 R1.1	B + G	62.00 a	60 a	0.30 a	1.01 a	284.11 a
51 R2.2	B + G	49.00 a	50 a	0.16 b	0.96 a	271.03 a
57 R3.1	B + G	61.33 a	70 a	0.22 b	0.94 a	263.55 a
65A R1.1	B + G	78.33 a	70 a	0.42 a	0.91 a	257.01 a
1 R3.2	B + G	50.00 a	50 a	0.22 b	0.91 a	256.07 a
35 R3.1	B + G	52.66 a	50 a	0.27 a	0.86 a	241.12 a
17A R2.2	B + G	66.00 a	50 a	0.23 b	0.86 a	242.99 a
11 R3.2	B + G	56.33 a	70 a	0.28 a	0.86 a	242.05 a
58 R3.2	B + G	62.66 a	90 a	0.26 a	0.82 a	230.84 a
36 R1	B + G	54.00 a	80 a	0.25 b	0.81 a	227.10 a
10 R2.1	B + G	35.66 a	70 a	0.19 b	0.77 a	216.82 a
2B R2.2	B + G	39.66 a	40 a	0.14 b	0.55 b	211.33 a
37 R1.1	B + G	53.33 a	50 a	0.35 a	0.70 a	197.19 a
12 R2.1	B + G	31.00 b	30 a	0.18 b	0.69 a	194.39 a
33 R2.2	B + G	25.00 b	40 a	0.30 a	0.63 b	178.50 b
44A R1.1	B + G	49.33 a	40 a	0.13 b	0.60 b	170.09 b
18 R3.1	B + G	29.33 b	50 a	0.18 b	0.60 b	168.22 b
11 R2.1	B + G	0.00 c	0.00 c	0.27 a	0.48 b	135.51 b
25C R1	B + G	0.00 c	0.00 c	0.30 a	0.46 b	128.97 c
17A R2.1	B + G	0.00 c	0.00 c	0.19 b	0.44 b	125.23 c
49 R3.6	B + G	17.00 b	30 a	0.18 b	0.42 c	119.62 c
51 R2 1	B + G	16.00 b	20 a	0.33 a	0.41 c	113.08 c
65B R1.1	B + G	0.00 c	0.00 c	0.31 a	0.36 c	100.93 c
49B R1.1	B + G	0.00 c	0.00 c	0.33 a	0.27 c	77.57 c
49 R2.2	B + G	0.00 c	0.00 c	0.22 b	0.19 c	55.13 c
97A R2.1	B + S	57.33 a	70 a	0.30 a	1.15 a	323.36 a
114C R1	B + S	55.33 a	70 a	0.29 a	1.07 a	300 a
83 R1.1	B + S	58.66 a	70 a	0.32 a	1.06 a	299.06 a
113 R1.1	B + S	62.66 a	80 a	0.34 a	1.01 a	283.18 a
90A R1	B + S	63.66 a	60 a	0.19 b	1.00 a	280.37 a
91B R1.2	B + S	43.33 a	70 a	0.29 a	0.98 a	274.76 a
81A R2.2	B + S	63.00 a	80 a	0.29 a	0.95 a	267.29 a
124 R2.1	B + S	58.66 a	60 a	0.22 b	0.90 a	253.27 a
89 R3.1	B + S	55.33 a	60 a	0.27 a	0.89 a	251.40 a
115 R3.1	B + S	57.33 a	60 a	0.18 b	0.89 a	251.40 a
113 R2.2	B + S	45.66 a	70 a	0.22 b	0.89 a	251.40 a
138A R1.2	B + S	50.33 a	50 a	0.31 a	0.89 a	249.53 a
141 R3.2	B + S	51.66 a	60 a	0.23 b	0.89 a	249.53 a
90B R1	B + S	62.33 a	50 a	0.37 a	0.88 a	246.72 a
97 R3.3	B + S	51.33 a	50 a	0.22 b	0.86 a	242.05 a
138A R3.1	B + S	56.00 a	60 a	0.28 a	0.85 a	239.25 a
130 R3.1	B + S	46.33 a	70 a	0.32 a	0.84 a	236.45 a
75 R2.2	B + S	61.33 a	40 a	0.26 a	0.80 a	224.30 a
108 R3.4	B + S	41.66 a	40 a	0.28 a	0.64 b	180.37 b
107D R2.1	B + S	46.00 a	50 a	0.21 b	0.64 b	180.37 b
107B R2.2	B + S	21.66 b	50 a	0.19 b	0.60 b	169.16 b
105 R1.1	B + S	15.33 b	40 a	0.22 b	0.55 b	156.07 b
89A R1.2	B + S	30.33 b	30 a	0.15 b	0.53 b	149.53 b
122 R3.7	B + S	11.66 b	60 a	0.24 b	0.51 b	143.92 b
138B R1.2	B + S	0.00 c	0.00 c	0.30 a	0.45 b	126.16 c
92B R2.1	B + S	0.40 c	0.00 c	0.00 a	0.23 c	115.23 c
74A R1.2	B + S	0.00 c	0.00 c	0.16 b	0.37 c	104.67 c
75 R3.2	B + S	0.00 c	0.00 c	0.34 a	0.34 c	96.26 c
130 R2.2	B + S	0.00 c	0.00 c	0.35 a	0.33 c	92.52 c
92A R2.2	B + S	0.00 c	0.00 c	0.31 a	0.32 c	89.71 c
91A R1.2	B + S	12.50 b	40 a	0.26 a	0.46 b	86.91 c
113 R1.2	B + S	20.33 b	20 a	0.32 a	0.26 c	72.89 c
145 R3.1	B	48.00 a	50 a	0.33 a	1.08 a	303.73 a
162 R3.2	B	64.66 a	60 a	0.31 a	0.97 a	271.96 a
154 R2.2	B	66.00 a	70 a	0.30 a	0.90 a	253.27 a
155 R1.1	B	75.00 a	80 a	0.30 a	0.94 a	264.48 a
147 R3.6	B	64.33 a	80 a	0.27 a	0.93 a	262.61 a
148 R1	B	61.66 a	60 a	0.24 b	0.81 a	228.03 a
147 R3.3	B	54.00 a	50 a	0.29 a	1.06 a	199.06 a
155A R2.1	B	24.33 b	40 a	0.21 b	0.44 b	125.23 c
146 R2	B	14.66 b	0.00 c	0.17 b	0.31 c	86.91 c
BR3262		50.66 a	90 a	0.30 a	1.03 a	290.65 a
BR3267		42.33 a	60 a	0.20 b	0.79 a	221.49 a
TA		0.00 c	0.00 c	0.12 b	0.22 c	61.68 c
TN		0.00 c	0.00 c	0.34 a	0.35 c	100.00 c
Mean a		56.45	47.6	0.3	0.92	255.96
Mean b		20.72	-	0.19	0.53	163.17
Mean c		0.00	-	-	0.31	98.88
VC%		39.7	90.88	35.91	24.34	24.5

• B + S: signalgrass intercropped with shrub-tree sabia; B + G: signalgrass intercropped with shrub-tree gliricidia; B: single signalgrass.

Table 3
Identity of cowpea rhizobial isolates from silvopastoral system soil in Itambé, Pernambuco, Brazil, based on the most similar sequences in EzBioCloud.

Strain	Origin	Cultural characteristicsa	RE%b	Nucleotide number	Top-hit type strain on EzBioCloud	Similarity (%)
					Species
43 R1.1	B + G	S/AL/SC	338.31	1,191	Bradyrhizobium elkanii USDA 76	96.50
97A R2.1	B + S	I/A/M	323.36	1,172	Bradyrhizobium elkanii USDA 76	99.06
145 R3.1	B	S/AL/L	303.73	1,168	Bradyrhizobium huanghuaihaiense CGMCC 1.10948	99.91
114C R1	B + S	VS/AL/SC	300.00	874	Methylobacterium radiotolerans JCM 2831	99.77
83 R1.1	B + S	S/AL/L	299.06	1,171	Bradyrhizobium elkanii USDA 76	98.89
35 R3.3	B + G	F/A/L	297.19	1,181	Bradyrhizobium elkanii USDA 76	96.50
42 R2.1	B + G	I/N/L	291.59	1,251	Cohnella xylanilytica MX15-2	96.88
44 R1.1	B + G	S/N/SC	284.11	1,122	Rhizobium altiplani BR10423	98.31
113 R1.1	B + S	I/N/SC	283.18	1,173	Bradyrhizobium elkanii USDA 76	99.57
90A R1	B + S	S/A/L	280.37	1,035	Leifsonia shinshuensis JCM 10591	92.37
51 R2.2	B + G	I/A/M	271.03	1,167	Bradyrhizobium elkanii USDA 76	98.11
81A R2.2	B + G	F/AL/L	267.29	863	Methylobacterium longum 440	83.22
155 R1.1	B	VS/A/L	264.48	1,303	Cohnella plantaginis YN-83	93.92
57 R3.1	B + G	I/N/L	263.55	1,197	Bradyrhizobium elkanii USDA 76	96.24
147 R3.6	B	VS/AL/L	262.61	1,169	Bradyrhizobium elkanii USDA 76	98.89
65A R1.1	B + G	S/N/SC	257.01	1,188	Bradyrhizobium elkanii USDA 76	98.61
1 R3.2	B + G	I/AL/M	256.07	1,170	Bradyrhizobium elkanii USDA 76	99.83
154 R2.2	B	F/A/SC	253.27	1,079	Leifsonia shinshuensis JCM 10591	98.61
124 R2.1	B + S	VS/N/SC	253.27	1,173	Bradyrhizobium elkanii USDA 76	96.92
115 R3.1	B + S	S/N/M	251.40	1,171	Bradyrhizobium elkanii USDA 76	99.66
89 R3.1	B + S	F/AL/SC	251.40	850	Methylobacterium radiotolerans JCM 2831	94.35
138A R1.2	B + S	VS/N/M	249.53	1,173	Bradyrhizobium elkanii USDA 76	99.14
90B R1.1	B + S	VS/N/M	246.72	1,170	Bradyrhizobium elkanii USDA 76	99.40
17A R2.2	B + G	F/N/M	242.99	1,113	Burkholderia cepacia ATCC 25416	99.91
11 R3.2	B + G	S/AL/L	242.05	1,172	Bradyrhizobium elkanii USDA 76	99.06
35 R3.1	B + G	I/AL/SC	241.12	1,171	Bradyrhizobium elkanii USDA 76	98.89
130 R3.1	B + S	I/N/M	236.45	1,173	Bradyrhizobium elkanii USDA 76	99.57
58 R3.2	B + G	S/N/L	230.84	1,179	Bradyrhizobium elkanii USDA 76	97.61
148 R1	B	VS/N/SC	228.03	1,170	Bradyrhizobium elkanii USDA 76	99.74
36 R1	B + G	S/A/SC	227.10	1,085	Leifsonia shinshuensis JCM 10591	98.68
75 R2.2	B + S	F/A/M	224.30	1,245	Paenibacillus cineris LMG 18439	98.14
147 R3.3	B	S/AL/L	199.06	1,194	Bradyrhizobium elkanii USDA 76	96.93
162 R3.2	B	I/N/M	271.96	1,171	Bradyrhizobium elkanii USDA 76	99.57

• ^a
  F: fast growth; S: slow growth; I: intermediate growth; VS: very slow growth; AL: medium alkalination; A: medium acidification; N: none pH changed; SC: scarce production mucus; M: moderate mucus production; L: low mucus production; RE: relative efficiency; B + S: signalgrass intercropped with shrub-tree sabia; B + G: signalgrass intercropped with shrub-tree gliricidia; B: single signalgrass.

Figure 2
Neighbor-Joining phylogenetic tree of 16S rRNA sequences (950 nucleotides) of cowpea rhizobial isolates from silvopastoral system soil in Itambé, Pernambuco, Brazil. Bootstrap values (1,000 replicates) are shown next to the branches. The evolutionary distances were computed using the Kimura 2-parameter method.

Besides Bradyrhizobium sp., strains were also identified as Rhizobium and Burkholderia genera, which have already been reported to nodulate cowpea in different soil types and management systems, such as São Francisco Valley soils (^{Leite et al. 2009}), Amazonian soils under different land use systems (^{Guimarães et al. 2012}), and rehabilitated soils revegetated with grasses (^{Castro et al. 2017}), confirming the wide symbiotic compatibility spectrum of cowpea.

Three strains were identified as Methylobacterium, and this genus belongs to Methylobacteriaceae, a large Rhizobiales family. Methylobacterium nodulans, unlike other members of the genus, can induce nodule formation and fix atmospheric nitrogen in Crotalaria and Lotononis (^{Green and Ardley 2018}). Previously, ^{Leite et al. (2009)} found only one isolate in cowpea in soils of the São Francisco River Valley that had low similarity (36%) with strain BR 2006 of Methylobacterium nodulans.

Other genera identified were Leifsonia, Cohnella and Paenibacillus (Table 3). These bacteria are components of the nodule microbiome and can penetrate nodules through the infection cord, and the function of these bacteria is not fully known yet. Some non-rhizobial endophytic bacteria include plant growth promoting rhizobacteria, which can produce indole acetic acid, solubilize phosphate, siderophores producing, and it may exhibit pathogen antagonist activity (^{Velázquez et al. 2017}). The non-rhizobial endophytic bacteria identified in the present work should be characterized as to possible plant growth promotion mechanisms, because they can be used for new inoculants development.

CONCLUSION

Legume-based silvopastoral systems did not influence cowpea rhizobial population density and diversity, after five years of system implementation.

Silvopastoral system soils harbor populations of rhizobia efficient in biological nitrogen fixation in cowpea, which can be recommended for agronomic efficiency assays.

Cowpea nodulated with a high diversity of nitrogen-fixing bacterial genotypes obtained from silvopastoral systems soils. Most of the identified genotypes belong to different species of Bradyrhizobium.

This is the first report of Methylobacterium strains with high similarity to M. radiotolerans and M. longum nodulating cowpea in Brazil.

ACKNOWLEDGMENTS

The authors are indebted to Instituto Agronômico de Pernambuco for allowing us the use of the research area.

• How to cite: Santos, A. B., Fracetto, G. G. M., Fracetto, F. J. C. and Lira Junior, M. A. (2022). Rhizobial diversity in shrub-tree legume-based silvopastoral systems. Bragantia, 81, e2622. https://doi.org/10.1590/1678-4499.20210336

• DATA AVAILABILITY STATEMENT

  All dataset were generated and analyzed in the current study.

• FUNDING

  Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco
  http://dx.doi.org/10.13039/501100006162
  Grants Nos: BCT-0406-5.03/21 and APQ-0453-5.01/15
  Conselho Nacional de Desenvolvimento Científico e Tecnológico
  https://doi.org/10.13039/501100003593
  Grants Nos: 304107/2020-4, 401896/2013-7 and 483287/2013-0
  Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
  https://doi.org/10.13039/501100002322
  Finance Code: 001

REFERENCES

• ¹
  Apolinário, V. X., Dubeux Jr., J. C., Lira, M. A., Ferreira, R. L., Mello, A. C., Santos, M. V., Sampaio, E. V. S. B. and Muir, J. P. (2015). Tree legumes provide marketable wood and add nitrogen in warm‐climate silvopasture systems. Agronomy Journal, 107, 1915-1921. https://doi.org/10.2134/agronj14.0624
  » https://doi.org/10.2134/agronj14.0624
• ²
  Araújo, V. L. V. P., Lira Junior, M. A., Souza Júnior, V. S., Araújo Filho, J. C., Fracetto, F. J. C., Andreote, F. D., Pereira, A. P. A., Mendes Júnior, J. P., Barros, F. M. R. and Fracetto, G. G. M. (2020). Bacteria from tropical semiarid temporary ponds promote maize growth under hydric stress. Microbiological Research, 240, 126564. https://doi.org/10.1016/j.micres.2020.126564
  » https://doi.org/10.1016/j.micres.2020.126564
• ³
  Barros, F. M. R., Fracetto, F. J. C., Lira Junior, M. A., Bertini, S. C. B. and Fracetto, G. G. M. (2021). Spatial and seasonal responses of diazotrophs and ammonium-oxidizing bacteria to legume-based silvopastoral systems. Applied Soil Ecology, 158, 103797. https://doi.org/10.1016/j.apsoil.2020.103797
  » https://doi.org/10.1016/j.apsoil.2020.103797
• ⁴
  Barros, F. M. R., Fracetto, G. G. M., Fracetto, F. J. C., Mendes Júnior, J. P., Araújo, V. L. V. P. and Lira Junior, M. A. (2018). Silvopastoral systems drive the nitrogen-cycling bacterial community in soil. Ciência e Agrotecnologia, 42, 281-290. https://doi.org/10.1590/1413-70542018423031117
  » https://doi.org/10.1590/1413-70542018423031117
• ⁵
  Berza, B., Sekar, J., Ramalingam, P. V., Pagano, M. C. and Assefa, F. (2021). Genetically and functionally diverse symbiotic and non-symbiotic native bacteria colonized root nodules of Erythrina brucei growing in different land use types in Ethiopia. Rhizosphere, 17, 100301. https://doi.org/10.1016/j.rhisph.2020.100301
  » https://doi.org/10.1016/j.rhisph.2020.100301
• ⁶
  [Brasil]. 2011. Instrução Normativa SDA nº 13, de 24 de março de 2011. Aprova as normas sobre especificações, garantias, registro, embalagem e rotulagem dos inoculantes destinados à agricultura, bem como as relações dos micro-organismos autorizados e recomendados para produção de inoculantes no Brasil, na forma dos Anexos I, II e III, desta Instrução Normativa. Diário Oficial da União, Brasília, Seção 1, p. 3.
• ⁷
  Castro, J. L., Souza, M. G., Rufini, M., Guimarães, A. A., Rodrigues, T. L. and Moreira, F. M. S. (2017). Diversity and efficiency of rhizobia communities from iron mining areas using cowpea as a trap plant. Revista Brasileira de Ciência do Solo, 41, e0160525. https://doi.org/10.1590/18069657rbcs20160525
  » https://doi.org/10.1590/18069657rbcs20160525
• ⁸
  Costa, E. M., Carvalho, T. S., Guimarães, A. A., Leão, A. C. R., Cruz, L. M., Baura, V. A., Lebbe, L., Willems, A. and Moreira, F. M. S. (2019). Classification of the inoculant strain of cowpea UFLA03-84 and of other strains from soils of the Amazon region as Bradyrhizobium viridifuturi (symbiovar tropici). Brazilian Journal of Microbiology, 50, 335-345. https://doi.org/10.1007/s42770-019-00045-x
  » https://doi.org/10.1007/s42770-019-00045-x
• ⁹
  Ferreira, D. F. (2011). Sisvar: a computer statistical analysis system. Ciência e Agrotecnologia, 35, 1039-1042. https://doi.org/10.1590/S1413-70542011000600001
  » https://doi.org/10.1590/S1413-70542011000600001
• ¹⁰
  Gnangui, S. L. E., Fossou, R. K., Ebou, A., Amon, C. E. R., Koua, D. K., Kouadjo, C. G. Z., Cowan, D. A. and Zézé, A. (2021). The Rhizobial Microbiome from the Tropical Savannah Zones in Northern Côte d’Ivoire. Microorganisms, 9, 1842. https://doi.org/10.3390/microorganisms9091842
  » https://doi.org/10.3390/microorganisms9091842
• ¹¹
  Green, P. N. and Ardley, J. K. (2018). Review of the genus Methylobacterium and closely related organisms: a proposal that some Methylobacterium species be reclassified into a new genus, Methylorubrum gen. nov. International Journal of Systematic and Evolutionary Microbiology, 68, 2727-2748. https://doi.org/10.1099/ijsem.0.002856
  » https://doi.org/10.1099/ijsem.0.002856
• ¹²
  Guimarães, A. A., Jaramillo, P. M. D., Nóbrega, R. S. A., Florentino, L. A., Silva, K. B. and Moreira, F. M. S. (2012). Genetic and symbiotic diversity of nitrogen-fixing bacteria isolated from agricultural soils in the western Amazon by using cowpea as the trap plant. Applied and Environmental Microbiology, 78, 6726-6733. https://doi.org/10.1128/AEM.01303-12
  » https://doi.org/10.1128/AEM.01303-12
• ¹³
  Hammer, Ø., Harper, D. A. and Ryan, P. D. (2001). PAST: Paleontological statistics software package for education and data analysis. Palaeontologia Electronica, 4, 9. Available at: http://palaeo-electronica.org/2001_1/past/issue1_01.htm Accessed on: Jul. 3, 2017.
  » http://palaeo-electronica.org/2001_1/past/issue1_01.htm
• ¹⁴
  Hoagland, D. R. and Arnon, D. I. (1950). The water-culture method for growing plants without soil. 2. ed. Circular 347. California: California Agricultural Experiment Station.
• ¹⁵
  Jacomine, P. K., Cavalcanti, A. C., Burgos, N., Pessoa, S. C. P. and Silveira, C. O. (1973). Levantamento exploratório-reconhecimento de solos do estado de Pernambuco. Embrapa Solos-Séries Anteriores. Available at: http://www.infoteca.cnptia.embrapa.br/infoteca/handle/doc/331168 Accessed on: Jul. 3, 2017.
  » http://www.infoteca.cnptia.embrapa.br/infoteca/handle/doc/331168
• ¹⁶
  Jaramillo, P. M. D., Guimarães, A. A., Florentino, L. A., Silva, K. B., Nóbrega, R. S. A. and Moreira, F. M. S. (2013). Symbiotic nitrogen-fixing bacterial populations trapped from soils under agroforestry systems in the Western Amazon. Scientia Agricola, 70, 397-404. https://doi.org/10.1590/S0103-90162013000600004
  » https://doi.org/10.1590/S0103-90162013000600004
• ¹⁷
  Lane, D. J. (1991). 16S/23S rRNA sequencing. In E. Stackebrandt and M. Goodfellow (Eds.). Nucleic acid techniques in bacterial systematics (p. 115-147). West Sussex: John Wiley and Sons.
• ¹⁸
  Lardi, M., Campos, S. B., Purtschert, G., Eberl, L. and Pessi, G. (2017). Competition experiments for legume infection identify Burkholderia phymatum as a highly competitive -rhizobium. Frontiers in Microbiology, 8, 1527. https://doi.org/10.3389/fmicb.2017.01527
• ¹⁹
  Leite, J., Passos, S. R., Simões-Araújo, J. L., Rumjanek, N. G., Xavier, G. R. and Zilli, J. E. (2018). Genomic identification and characterization of the elite strains Bradyrhizobium yuanmingense BR 3267 and Bradyrhizobium pachyrhizi BR 3262 recommended for cowpea inoculation in Brazil. Brazilian Journal of Microbiology, 49, 703-713. https://doi.org/10.1016/j.bjm.2017.01.007
  » https://doi.org/10.1016/j.bjm.2017.01.007
• ²⁰
  Leite, J., Seido, S. L., Passos, S. R., Xavier, G. R., Rumjanek, N. G. and Martins, L. M. V. (2009). Biodiversity of rhizobia associated with cowpea cultivars in soils of the lower half of the São Francisco River Valley. Revista Brasileira de Ciência do Solo, 33, 1215-1226. https://doi.org/10.1590/S0100-06832009000500015
  » https://doi.org/10.1590/S0100-06832009000500015
• ²¹
  Lima, A. S., Nóbrega, R. S. A., Barberi, A., Silva, K., Ferreira, D. F. and Moreira, F. M. S. (2009). Nitrogen-fixing bacteria communities occurring in soils under different uses in the Western Amazon Region as indicated by nodulation of siratro (Macroptilium atropurpureum). Plant and Soil, 319, 127-145. https://doi.org/10.1007/s11104-008-9855-2
  » https://doi.org/10.1007/s11104-008-9855-2
• ²²
  Lima, H. N. B., Dubeux Jr., J. C. B., Santos, M. V. F., Mello, A. C. L., Lira, M. A. and Cunha, M. V. (2018). Soil attributes of a silvopastoral system in Pernambuco Forest Zone. Tropical Grasslands-Forrajes Tropicales, 6, 15-25. https://doi.org/10.17138/tgft(6)15-25
  » https://doi.org/10.17138/tgft(6)15-25
• ²³
  Lira Junior, M. A., Fracetto, F. J. C., Ferreira, J. D. S., Silva, M. B. and Fracetto, G. G. M. (2020a). Legume silvopastoral systems enhance soil organic matter quality in a subhumid tropical environment. Soil Science Society of America Journal, 84, 1209-1218. https://doi.org/10.1002/saj2.20106
  » https://doi.org/10.1002/saj2.20106
• ²⁴
  Lira Junior, M. A., Fracetto, F. J. C., Silva Ferreira, J., Silva, M. B. and Fracetto, G. G. M. (2020b). Legume-based silvopastoral systems drive C and N soil stocks in a subhumid tropical environment. Catena, 189, 104508. https://doi.org/10.1016/j.catena.2020.104508
  » https://doi.org/10.1016/j.catena.2020.104508
• ²⁵
  Lira Junior, M. A., Nascimento, L. R. S. and Fracetto, G. G. M. (2015). Legume-rhizobia signal exchange: promiscuity and environmental effects. Frontiers in Microbiology, 6, 945. https://doi.org/10.3389/fmicb.2015.00945
  » https://doi.org/10.3389/fmicb.2015.00945
• ²⁶
  Martins, J. C. R., Freitas, A. D. S., Menezes, R. S. C. and Sampaio, E. V. S. B. (2015). Nitrogen symbiotically fixed by cowpea and gliricidia in traditional and agroforestry systems under semiarid conditions. Pesquisa Agropecuária Brasileira, 50, 178-184. https://doi.org/10.1590/S0100-204X2015000200010
  » https://doi.org/10.1590/S0100-204X2015000200010
• ²⁷
  Martins, P. G. S., Lira Junior, M. A., Fracetto, G. G. M., Silva, M. L. R. B., Vincentin, R. P. and Lyra, M. C. C. P. (2015). Mimosa caesalpiniifolia rhizobial isolates from different origins of the Brazilian Northeast. Archives of Microbiology, 197, 459-469. https://doi.org/10.1007/s00203-014-1078-8
  » https://doi.org/10.1007/s00203-014-1078-8
• ²⁸
  Mendes, M. M. C., Chaves, L. F. C., Pontes Neto, T. P., Silva, J. A. A. and Figueiredo, M. V. B. (2013). Crescimento e sobrevivência de mudas de sabiá (Mimosa caesalpiniaefolia Benth.) inoculadas com micro-organismos simbiontes em condições de campo. Ciência Florestal, 23, 309-320. https://doi.org/10.5902/198050989277
  » https://doi.org/10.5902/198050989277
• ²⁹
  Muindi, M. M., Muthini, M., Njeru, E. M. and Maingi, J. (2021). Symbiotic efficiency and genetic characterization of rhizobia and non rhizobial endophytes associated with cowpea grown in semi-arid tropics of Kenya. Heliyon, 7, e06867. https://doi.org/10.1016/j.heliyon.2021.e06867
  » https://doi.org/10.1016/j.heliyon.2021.e06867
• ³⁰
  Ndungu, S. M., Messmer, M. M., Ziegler, D., Gamper, H. A., Mészáros, É., Thuita, M., Vanlauwe, B., Frossard, E. and Thonar, C. (2018). Cowpea (Vigna unguiculata L. Walp) hosts several widespread bradyrhizobial root nodule symbionts across contrasting agro-ecological production areas in Kenya. Agriculture, Ecosystems & Environment, 261, 161-171. https://doi.org/10.1016/j.agee.2017.12.014
  » https://doi.org/10.1016/j.agee.2017.12.014
• ³¹
  Ormeño-Orrillo, E., Rogel-Hernández, M. A., Lloret, L., López-López, A., Martínez, J., Barois, I. and Martínez-Romero, E. (2012). Change in land use alters the diversity and composition of Bradyrhizobium communities and led to the introduction of Rhizobium etli into the tropical rain forest of Los Tuxtlas (Mexico). Microbial Ecology, 63, 822-834. https://doi.org/10.1007/s00248-011-9974-9
  » https://doi.org/10.1007/s00248-011-9974-9
• ³²
  Paula, P. D., Campello, E. F. C., Guerra, J. G. M., Santos, G. A. and Resende, A. S. D. (2015). Decomposição das podas das leguminosas arbóreas Gliricidia sepium e Acacia angustissima em um sistema agroflorestal. Ciência Florestal, 25, 791-800. https://doi.org/10.5902/1980509819696
  » https://doi.org/10.5902/1980509819696
• ³³
  Pires, R. C., Reis Junior, F. B., Zilli, J. E., Fischer, D., Hofmann, A., James, E. K. and Simon, M. F. (2018). Soil characteristics determine the rhizobia in association with different species of Mimosa in central Brazil. Plant and Soil, 423, 411-428. https://doi.org/10.1007/s11104-017-3521-5
  » https://doi.org/10.1007/s11104-017-3521-5
• ³⁴
  Rocha, B. C. F., Santos, E. O. S., Santos, J. G. D., Takako, A. K. and Castro, F. J. (2017). Land use and vegetation cover on native symbionts and interactions with cowpea. Revista Brasileira de Engenharia Agrícola e Ambiental, 21, 116-121. https://doi.org/10.1590/1807-1929/agriambi.v21n2p116-121
  » https://doi.org/10.1590/1807-1929/agriambi.v21n2p116-121
• ³⁵
  Santos, A. M. G., Dubeux Junior, J. C. B., Santos, M. V. F., Lira, M. A., Apolinário, V. X. O., Costa, S. B. M., Coêlho, D. L., Peixôto, T. V. F. R. and Santos, E. R. S. (2020). Animal performance in grass monoculture or silvopastures using tree legumes. Agroforestry Systems, 94, 615-626. https://doi.org/10.1007/s10457-019-00431-2
  » https://doi.org/10.1007/s10457-019-00431-2
• ³⁶
  Souza, L. C. and Procópio, L. (2021). The profile of the soil microbiota in the Cerrado is influenced by land use. Applied Microbiology and Biotechnology, 105, 4791-4803. https://doi.org/10.1007/s00253-021-11377-w
  » https://doi.org/10.1007/s00253-021-11377-w
• ³⁷
  Tamura, K., Stecher, G., and Kumar, S. (2021). MEGA11: molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution, 38, 3022-3027. https://doi.org/10.1093/molbev/msab120
  » https://doi.org/10.1093/molbev/msab120
• ³⁸
  Uzoh, I. M. and Babalola, O. O. (2018). Rhizosphere biodiversity as a premise for application in bio-economy. Agriculture, Ecosystems & Environment, 265, 524-534. https://doi.org/10.1016/j.agee.2018.07.003
  » https://doi.org/10.1016/j.agee.2018.07.003
• ³⁹
  Velázquez, E., Carro, L., Flores-Félix, J. D., Martínez-Hidalgo, P., Menéndez, E., Ramírez-Bahena, M. H., Mulas, R., González-Andrés, F., Martínez-Molina, E. and Peix, A. (2017). The legume nodule microbiome: a source of plant growth-promoting bacteria. In V. Kumar, M. Kumar, S. Sharma and R. Prasad (Eds). Probiotics and plant health (p. 41-70). Singapore: Springer. https://doi.org/10.1007/978-981-10-3473-2_3
  » https://doi.org/10.1007/978-981-10-3473-2_3
• ⁴⁰
  Vincent, J. M. (1970). A manual for the practical study of root nodule bacteria. Oxford: Black well Scientific Publications. (IBP Handbook, 15.)
• ⁴¹
  Wakelin, S., Tillard, G., van Ham, R., Ballard, R., Farquharson, E., Gerard, E., Geurts, R., Brown, M., Ridgway, H. and O’Callaghan, M. (2018). High spatial variation in population size and symbiotic performance of Rhizobium leguminosarum bv. trifolii with white clover in New Zealand pasture soils. PLoS One, 13, e0192607. https://doi.org/10.1371/journal.pone.0192607
  » https://doi.org/10.1371/journal.pone.0192607
• ⁴²
  Wang, C., Zheng, M. M., Chen, J. and Shen, R. F. (2021). Land-use change has a greater effect on soil diazotrophic community structure than the plant rhizosphere in acidic ferralsols in southern China. Plant and Soil, 462, 445-458. https://doi.org/10.1007/s11104-021-04883-3
  » https://doi.org/10.1007/s11104-021-04883-3
• ⁴³
  Woomer, P. L. (1994). Most probable number counts. In P. W. Weaver, J. S. Angle and P. S. Bottomely (Ed). Methods of soil analysis (p. 59-79). Wisconsin: Soil Science Society of American Book.
• ⁴⁴
  Yoon, S. H., Ha, S. M., Kwon, S., Lim, J., Kim, Y., Seo, H. and Chun, J. (2017). Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. International Journal of Systematic and Evolutionary Microbiology, 67, 1613. https://doi.org/10.1099/ijsem.0.001755
  » https://doi.org/10.1099/ijsem.0.001755
• ⁴⁵
  Zhang, S., Xie, F., Yang, J. and Li, Y. (2011). Phylogeny of bradyrhizobia from Chinese cowpea miscellany inferred from 16S rRNA, atpD, glnII, and 16S–23S intergenic spacer sequences. Canadian Journal of Microbiology, 57, 316-327. https://doi.org/10.1139/w11-008
  » https://doi.org/10.1139/w11-008

Edited by

Section Editor: Hector Valenzuela

Publication Dates

• Publication in this collection
  15 June 2022
• Date of issue
  2022

History

• Received
  03 Dec 2021
• Accepted
  30 Mar 2022