Identification and Evaluation of the Salt and Acid—Alkaline Resistance of Rhizobium Species of Desmodium

Abstract

Inoculation with Rhizobium can improve not only host yield and quality but also host adaptability to stress. The match between inoculated strains and hosts and resistance to stress are the key factors determining the inoculation effect. Four types of Desmodium, Pleurolobus gangeticus, Puhuaea sequax, Grona heterocarpos, and Polhillides velutina, were used to capture Rhizobium strains in soil, and the phylogenetic classification and salt and acid—alkaline resistances of the strains were determined through isolation, identification, and different salt and pH treatments. The phylogenetic classification of the 62 rhizobial strains was determined by 16S rDNA analysis. After comparison, the strains were grouped into 10 groups with known strains of rhizobia, including 14 strains of fast-growing rhizobia grouped with Rhizobium tropici and Rhizobium oryzicola and 48 strains of slow-growing rhizobia. The salt tolerance and acid—base adaptability of 52 strains were qualitatively and quantitatively determined in this study, and salt stress was found to have varying effects on the growth of different strains. Some strains grew normally on media with NaCl concentrations of 0.4 and 0.35 mol/L and had strong salt tolerance. The plants grew normally on media with pH 5–9. This study provides theoretical support for the classification of Desmodium and provides efficient Rhizobium strains for the cultivation and application of Desmodium.

1. Introduction

Desmodium Desv., a butterfly flower subfamily of the leguminous family, is an annual or perennial herb or shrub plant widely distributed in tropical and subtropical regions, with approximately 350–450 species worldwide [1,2]. Desmodium Desv. can be used for embankment protection, artificial grassland construction, or natural grassland improvement, and it has high medicinal value [3,4]. Desmodium Desv. is rich in nutrients, with an average crude protein content of greater than 8.40% and a relatively low crude fibre content, and the best time for its use is from the vegetative stage to the initial flowering stage [5,6]. In China, the Desmodium Desv. varieties used as forage grasses and in green fertiliser applications research include Pleurolobus gangeticus, Puhuaea sequax, Grona heterocarpos, and Polhillides velutina [7,8,9]. Among them, Reyan No. 16 Pleurolobus gangeticus is the first grass variety approved by the National Grass Variety Approval Committee in China.

The phylogenetic relationship of modern rhizobia is based mainly on the 16S rRNA gene sequence, and the phylogenetic status of rhizobia can be accurately determined by gene comparison after sequencing [6,10]. There are few studies on rhizobia of the genus Desmodium in China, and the nodules of the four species of Desmodium are all spherical or nearly spherical in size (2~4 mm) [11,12,13]. Most of the root nodules of this genus are slow-growing rhizobia (strains can be grown within 5 to 7 d). Although the nodules are very strong and the host range is wide, they often form ineffective nodules [14,15]. Sequence analysis of the nodC gene in Desmodium revealed that it was diverse, among which the slow-growing rhizobia formed independent phylogenetic branches, whereas some Rhizobium pods were able to establish symbiotic relationships with different species of Desmodium [16,17]. Terrill et al. determined the abundance of indigenous Rhizobium in Desmodium in different regions of China, analysed the results of 16S rDNA sequencing to establish phylogenetic trees, and initially established the classification and status of the Rhizobium of Desmodium in China [18]. Among the 35 strains of rhizobia, 5 strains that were highly effective were selected [19].

Soil salinization restricts the rapid development of agriculture. Inoculation with rhizobia can alleviate salt damage, but different types of rhizobia have different effects on salt damage [20]. Rhizobia may play an important role in plant tolerance to salt stress [21,22]. After inoculation with rhizobia, Reyan No. 5 Desmodium protected its roots by increasing the number of nodules. This inhibited the upwards transport of Na+, increased the absorption of K+, improved the availability of soil phosphorus, and improved the salt tolerance of the strain [23,24]. After inoculation with rhizobia, the composition of the rhizosphere soil microbial community was affected, the availability of soil nutrients improved, and the salt tolerance of the plants increased [25,26]. Sun et al. studied the genetic and phenotypic diversity of rhizobia from several leguminous plants of Mimosa and Desmodium in eight regions of Yunnan Province and reported that most Rhizobium strains could tolerate high temperatures of 60 °C, high salt concentrations (4–5 mol/L), and high concentrations of antibiotics (300 μg/mL) [27]. After fast- and slow-growing rhizobia were inoculated into Puhuaea sequax, the results indicated that fast-growing rhizobia (strains could be grown within 3 to 5 d) could still grow normally at pH values of 9~12, whereas slow-growing rhizobia could not grow when the pH was greater than 9 [17,28]. In our study, the salt tolerance and acid—alkaline resistance of rhizobia in soil captured by different types of Desmodium were analysed through identification and classification to provide theoretical support for the classification of the Rhizobium of Desmodium and to provide efficient Rhizobium strains for the cultivation and application of four types of Desmodium.

2. Materials and Methods

2.1. Materials

The Desmodium used in the experiment were Pleurolobus gangeticus, Puhuaea sequax, Grona heterocarpos, and Polhillides velutina, which were stored in the National Tropical Pasture Interim Backup Bank Velutina. The tested soil was collected from the 10th team base of the Grass Industry Research Center, Tropical Crop Variety Resources Research Institute, Chinese Academy of Tropical Agricultural Sciences.

The soil physical and chemical properties were as follows: pH, 4.70; total phosphorus content, 0.27 g/kg; available potassium content, 72.93 mg/kg; available phosphorus content, 5.32 mg/kg; available nitrogen content, 151.90 mg/kg; and organic matter content, 39.40 g/kg.

2.2. Seed Treatment and Culture of Desmodium

The Desmodium seeds were soaked in hot water at 80 °C for 5 min, the water was absorbed, the seeds were sterilised in 95% ethanol for 5 min, and the seeds were rinsed with sterile water 5 to 6 times after the ethanol solution was absorbed. Then, the seeds were sprayed in a presterilised Petri dish, and sterile 0.05 mmol/L calcium sulphate solution was poured onto the sprouting paper. When the seeds were white, they were transplanted into a spare pot and watered daily with half of the field water capacity. Four pots were planted per type of Desmodium. The planted Desmodium was placed in the greenhouse for 2 months, the root system was removed, and the nodules were collected [20,21].

2.3. Isolation and Purification of Desmodium Rhizobia

The collected Desmodium roots were cleaned with water, and the root nodules were picked and placed in a Petri dish. After the root nodules were removed from the Petri dish with ddH₂O, they were soaked in ddH₂O for 5 min and transferred to a sterile bench. The samples were treated with 95% ethanol solution for 6 min and 30% H₂O₂ for 6 min, and then rinsed with sterile water 5–10 times. The washed Desmodium root nodule was transferred to a sterile plate for use. Then, sterilised tweezers were used to break the surface of the root nodule to break out the bacterial mixture, and the bacterial mixture was dipped into YMA solid medium with an inoculation ring. The sterile plate was placed in a storage bag and cultured in an incubator (28 ± 1 °C, dark) for 7 days, after which the colony morphology of the strain was observed. Until the colonies on the YMA solid medium were uniform, different numbers of bacteria were examined via microscopy, and the numbers of bacteria with the same morphology were combined for DNA extraction [23,24].

2.4. Total DNA Extraction, 16S rDNA Amplification, and Sequencing of Rhizobia

After the above isolated and purified strains were cultured in YMA medium to the logarithmic growth stage, the total DNA of the rhizobia was extracted with a kit. Using total DNA as a template, the upstream primer 27F (5-AGAGTTTGATCCTGGCTCAG-3) was used, and the downstream primer 1492R (5-CTACGGCTACCTTGTTACGA-3) was used. The reaction system was as follows: 10× buffer (including 2.5 mM Mg²⁺), 5.0 μL; dNTP (10 mM), 1.0 μL; template DNA (20 ng/μL), 1.0 μL; Taq DNA polymerase (5 μ/μL), 1.0 μL; 1492R primer (10 µM), 1.5 μL; 27F primer (10 µM), 1.5 μL; ddH₂O, 39.0 μL; and total volume, 50.0 μL. The reaction conditions were as follows: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s; annealing at 58 °C for 30 s; extension at 72 °C for 1 min 30 s; and final elongation at 72 °C for 7 min. The amplified products were subjected to agarose gel electrophoresis, purified, and sent to the Shenzhen Micomeng Technology Group Co., Ltd. (Shenzhen, China), for sequencing [25,26].

2.5. Multiple Sequence Alignment and Establishment of a Phylogenetic Tree

The sequencing results were analysed and compared via BLAST on the website of the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/), and 13 known strains with the highest similarity were obtained from the NCBI GenBank as reference strains. Multiple sequences of the 16S rRNA gene were compared between the test strain and the reference strain via BioEdit 7 software. Mega7 software (v7.0.26) was used to perform neighbour-joining analysis via the Kimura-2 parameter, and 1000 repeats were performed to generate the phylogenetic tree [27,28].

2.6. Salt Tolerance of Desmodium Rhizobia

The concentrations of YMA solid media prepared with NaCl were 0.1, 0.2, 0.3, 0.35, and 0.4 mol/L. The Desmodium Rhizobium strains were cultured on YMA media with single colonies at different concentrations (three repetitions per strain) on a super clean workbench. After being cultured at 28 °C for 5~7 days, the growth of 4 species of Rhizobium colonies at different salt concentrations was observed. The growth was recorded as not long (−), weak (+), good (++), or the same as the control growth (+++). The 52 strains to be tested were picked out from YMA slant medium, activated in YMA solid medium, and cultured in an incubator at 28 °C for 5 d. The colonies were picked out with sterilised toothpick segments (1 cm) and inoculated into liquid medium with different NaCl concentrations, respectively, and cultured at 180 r.min⁻¹ on a shaker at 28 °C, timed, for 12 h. A portion of bacterial liquid was taken by pipette gun, and the absorbance value of the bacterial liquid OD₆₀₀ nm was measured spectrophotometrically. Three replicates were measured for each strain, and the culture was continued after the bacterial liquid was taken; then, it was measured once every 12 h until 168 h, with a total of 14 measurements [1,2].

2.7. Acid–Alkaline Resistance of Desmodium Rhizobia

YMA medium with different pH values was prepared as follows: 10 mL/L HCl and 10 g/L NaOH solutions prepared in advance were added to YMA media, and the pH values were adjusted to 5.0, 6.0, 7.0, 8.0, and 9.0. After inoculation with Desmodium rhizobia for 5 days, the growth of 4 species of Rhizobium colonies under different acidic and alkaline conditions was observed [3,4].

2.8. Statistical Analysis

The datasets were replicated more than three times, and the resulting data are presented as the means ± standard deviations. Statistical analysis was performed via SPSS 26 software, while processing and mapping were conducted via Origin 2021 software.

3. Results

3.1. PCR Gene Amplification and 16S rDNA Identification Results for Desmodium Rhizobia

The results of PCR amplification of the 16S rDNA of some strains are shown in Figure 1, with 1500 bp indicated by the arrow. As shown in Figure 1, the amplified band was bright and undrag-free, and the amplified 16S rDNA was relatively pure 16S rDNA with a fragment size of approximately 1500 bp. The PCR products purified from each strain can be used for sequencing.

A total of 104 amplified products were sequenced, and the sequencing results were compared with the 16S rDNA genes of other reference strains of Desmodium rhizobia listed in the NCBI. The results revealed that the homology between the 78 isolates and reference strains was greater than 99%, which confirmed that the isolates were rhizobia and that 15 of them were rapidly growing rhizobia. The rapidly growing rhizobia were NM1, BY3, RM4, RM5, RM6, DY1, DY2, DY3, DY7, DY8, DY9, DY15, DY22, DY23, and RM20, and the other 63 strains were slow-growing rhizobia.

3.2. Phylogenetic Tree Analysis of Desmodium Rhizobia

The 13 known Rhizobium strains with the highest similarity were searched in the NCBI GenBank, and the 16S rDNA sequences of the 78 Desmodium Rhizobium strains isolated from the root nodules were compared via MEGA7.0 software. A phylogenetic tree was constructed. The relationships between the isolated strains and other strains were determined.

As shown in Figure 2, among the 78 strains of rhizobia isolated from Desmodium, DY7, DY8, and DY23 were fast-growing rhizobia, but the NCBI ratio could not be grouped into the genus of fast-growing rhizobia. DY7 was more closely related to NR112928, and DY23 was more closely related to NR137225. DY8 could not be grouped with the reference strains selected in GenBank.

The Rhizobium strains in the evolutionary tree can be divided into eight groups. LY2, LY3, ZF4, DY21, JD7, RM11, JD2, DY16, RM10, RM12, JD9, and DY7 were closely related to known strains NR117945 and NR112928 and were categorised into group I; DY14, RM19, RM3, RM15, DY19, DY20, RM13, and RM21 were closely related to known strain NR145861 and were clustered into group II; DY1, DY3, NM1, BY3, RM4, and DY23 were closely related to known strains NR137225 and NR102511 and were clustered into group III; DY12, LY4, RM1, DY5, RM7, FZ2, JD4, ZF5, and ZF6 were closely related to known strains NR112927, NR133706, and NR145925 and clustered into group IV; MR5, MR6, MR20, DY2, DY15, DY9, and DY22 were closely related to the known strain NR117850 and clustered into group V; JD6, JD13, JD3, JD1, JD5, JD8, LY1, ZF1, ZF3, and XY1 were closely related to known strains NR114138 and NR149804 and clustered into group VI; and RM14, DY10, DY18, DY13, JD12, JD14, JD10, LY5, JD11, DY4, DY6, RM8, RM2, RM9, BY4, NM2, RM16, BY2, and DY17 were closely related to known strains NR043037 and NR036953 and clustered into group VII. BY1, MR18, XY3, XY2, DY11, and RM17, however, failed to cluster with the reference strains selected in GenBank and were categorised into group VIII separately.

3.3. Resistance of Desmodium Rhizobia in Media with Different NaCl Concentrations

The growth of the rhizobia of Desmodium strains in YMA media with different NaCl concentrations is shown in

Table 1. Effects of different NaCl concentrations on the growth of Desmodium rhizobia.

Strain/Salt Concentration	0.1 mol/L	0.2 mol/L	0.3 mol/L	0.35 mol/L	0.4 mol/L	Strain Type
BY1	+++	+++	+++	+++	−	slow-growing
BY2	+	−	−	−	−	slow-growing
BY3	+++	+++	++	+	−	fast-growing
BY4	+	−	−	−	−	slow-growing
ZF3	+++	++	++	++	−	slow-growing
RM1	+	−	−	−	−	slow-growing
RM2	+	−	−	−	−	slow-growing
RM3	+	−	−	−	−	slow-growing
RM4	+++	+++	−	−	−	fast-growing
RM5	+++	+++	+	−	−	fast-growing
RM6	+++	+++	−	−	−	fast-growing
RM7	+	+	−	−	−	slow-growing
RM8	+++	+++	++	−	−	slow-growing
RM9	+	−	−	−	−	slow-growing
RM13	+	−	−	−	−	slow-growing
RM14	+	+	−	−	−	slow-growing
RM15	+	+	−	−	−	slow-growing
RM16	+++	+++	+++	+++	+	slow-growing
RM17	−	+	−	−	−	slow-growing
RM18	+	−	−	−	−	slow-growing
RM19	+++	+++	+++	+++	−	slow-growing
RM20	+++	+++	+++	++	−	fast-growing
RM21	−	−	−	−	−	slow-growing
DY1	+++	+++	++	++	−	fast-growing
DY2	+++	+++	++	++	−	fast-growing
DY3	+++	+++	+	+	−	fast-growing
DY4	+	−	−	−	−	slow-growing
DY6	+	−	−	−	−	slow-growing
DY7	+++	+++	++	++	−	fast-growing
DY8	+++	+++	+++	++	+	fast-growing
DY9	+++	+++	+++	+++	+	fast-growing
DY10	+++	+++	++	++	+	slow-growing
DY11	+++	+++	++	+	−	slow-growing
DY12	++	−	−	−	−	slow-growing
DY13	+++	+++	++	++	−	slow-growing
DY14	++	−	−	−	−	slow-growing
DY15	+++	+++	++	++	-	fast-growing
DY16	−	−	−	−	−	slow-growing
DY17	+	−	−	−	−	slow-growing
DY18	++	−	−	−	−	slow-growing
DY19	++	−	−	−	−	slow-growing
DY20	+++	−	−	−	−	slow-growing
DY21	+	−	−	−	−	slow-growing
DY23	+++	+++	++	++	+	fast-growing
JD10	+	+	+	+	-	slow-growing
JD11	−	−	−	−	−	slow-growing
JD12	+	−	−	−	−	slow-growing
JD13	+++	+++	++	++	-	slow-growing
JD14	−	−	−	−	−	slow-growing
JD1	+	+	+	+	−	slow-growing
LY4	+++	++	++	++	+	slow-growing
LY5	++	+	+	−	−	slow-growing

Note: (+++) indicates strong salt tolerance, (++) indicates some salt tolerance, (+) indicates weak salt tolerance, and (−) indicates no salt tolerance.

and Figure 3. Strains RM16, DY8, DY9, DY10, DY23, and LY4 still grew weakly under 0.4 mol/L NaCl, indicating strong salt tolerance. Among them, RM16, DY10, and LY4 are slow-growing Rhizobium, whereas DY8, DY9, and DY23 are fast-growing rhizobia. Of the 13 fast-growing rhizobial strains, 10 were able to grow on 0.35 mol/L NaCl solid medium, and only 10 of the 39 slow-growing rhizobial strains were able to grow on 0.35 mol/L NaCl solid medium, which indicated that the salt tolerance of fast-growing rhizobia was greater than that of slow-growing rhizobia. The growth of most slow-growing Rhizobium strains was completely inhibited when the NaCl concentration was 0.2 mol/L, and some slow-growing Rhizobium strains also presented strong salt tolerance, with BY1, ZF3, MR19, DY10, DY11, DY13, DJ1, DJ10, and DJ13 growing at 0.35 mol/L. The growth was not completely inhibited until 0.4 mol/L, while all the fast-growing Rhizobium strains still grew at 0.35 mol/L. However, some fast-growing Rhizobium stains were completely inhibited at 0.4 mol/L.

3.4. Growth Curves of Desmodium Strains in Media with Different NaCl Concentrations

The OD₆₀₀ values of the Desmodium Rhizobium strains in the stable stage at different NaCl concentrations are shown in Figure 4. The OD₆₀₀ values of the different strains under different salt (acid) treatments for different times are included in

Table 2. Effect of different salt treatments on the growth of Rhizobium strains.

Strain	NaCl (mol/L)	Incubation Time (h)
		12	36	60	84	108	132	156	168
BY1	0	0.130 ± 0.007	0.790 ± 0.014	1.561 ± 0.025	1.809 ± 0.038	1.920 ± 0.046	1.899 ± 0.040	1.918 ± 0.063	1.939 ± 0.049
	0.2	0.097 ± 0.002	0.330 ± 0.023	0.919 ± 0.025	1.319 ± 0.020	1.699 ± 0.042	1.700 ± 0.020	1.694 ± 0.025	1.752 ± 0.042
	0.4	0.113 ± 0.010	0.115 ± 0.006	0.131 ± 0.017	0.122 ± 0.020	0.144 ± 0.024	0.108 ± 0.007	0.101 ± 0.012	0.108 ± 0.012
BY2	0	0.233 ± 0.016	0.746 ± 0.046	1.311 ± 0.028	1.572 ± 0.017	1.708 ± 0.060	1.981 ± 0.006	2.122 ± 0.038	2.158 ± 0.052
	0.2	0.121 ± 0.016	0.121 ± 0.009	0.111 ± 0.011	0.100 ± 0.012	0.105 ± 0.007	0.108 ± 0.005	0.105 ± 0.004	0.105 ± 0.004
	0.4	0.122 ± 0.013	0.157 ± 0.008	0.157 ± 0.011	0.149 ± 0.006	0.121 ± 0.024	0.118 ± 0.028	0.138 ± 0.007	0.134 ± 0.005
BY3	0	1.570 ± 0.043	2.323 ± 0.013	2.400 ± 0.011	2.284 ± 0.020	2.209 ± 0.009	2.145 ± 0.010	2.195 ± 0.005	2.201 ± 0.020
	0.2	0.651 ± 0.017	1.237 ± 0.041	1.671 ± 0.020	2.017 ± 0.016	2.244 ± 0.044	2.292 ± 0.018	2.339 ± 0.008	2.335 ± 0.023
	0.4	0.169 ± 0.020	0.148 ± 0.012	0.151 ± 0.016	0.123 ± 0.013	0.120 ± 0.011	0.124 ± 0.003	0.125 ± 0.002	0.122 ± 0.002
BY4	0	0.340 ± 0.015	0.831 ± 0.028	1.277 ± 0.020	1.865 ± 0.019	2.099 ± 0.019	2.054 ± 0.047	2.217 ± 0.025	2.254 ± 0.023
	0.2	0.106 ± 0.010	0.091 ± 0.010	0.129 ± 0.007	0.131 ± 0.003	0.124 ± 0.000	0.111 ± 0.011	0.108 ± 0.004	0.109 ± 0.002
	0.4	0.100 ± 0.003	0.088 ± 0.004	0.057 ± 0.018	0.085 ± 0.018	0.068 ± 0.005	0.084 ± 0.011	0.077 ± 0.002	0.087 ± 0.006
ZF3	0	0.147 ± 0.042	0.432 ± 0.062	0.674 ± 0.129	0.812 ± 0.105	0.957 ± 0.098	1.228 ± 0.067	1.463 ± 0.113	1.429 ± 0.061
	0.2	0.079 ± 0.001	0.119 ± 0.024	0.561 ± 0.084	0.771 ± 0.083	0.958 ± 0.061	1.168 ± 0.051	1.295 ± 0.010	1.328 ± 0.018
	0.4	0.090 ± 0.013	0.042 ± 0.019	0.022 ± 0.016	0.024 ± 0.007	0.026 ± 0.007	0.034 ± 0.004	0.046 ± 0.013	0.049 ± 0.014
RM1	0	0.212 ± 0.009	0.883 ± 0.020	1.186 ± 0.015	1.354 ± 0.061	1.673 ± 0.072	2.114 ± 0.088	2.264 ± 0.043	2.299 ± 0.013
	0.2	0.192 ± 0.005	0.109 ± 0.011	0.157 ± 0.008	0.165 ± 0.004	0.139 ± 0.010	0.152 ± 0.018	0.175 ± 0.003	0.171 ± 0.001
	0.4	0.147 ± 0.004	0.105 ± 0.005	0.129 ± 0.004	0.109 ± 0.008	0.122 ± 0.011	0.123 ± 0.004	0.104 ± 0.003	0.098 ± 0.003
RM2	0	0.273 ± 0.034	0.703 ± 0.024	1.098 ± 0.013	1.450 ± 0.094	1.661 ± 0.080	1.914 ± 0.049	2.152 ± 0.062	2.242 ± 0.064
	0.2	0.190 ± 0.004	0.161 ± 0.009	0.143 ± 0.014	0.127 ± 0.009	0.142 ± 0.013	0.157 ± 0.013	0.164 ± 0.011	0.172 ± 0.013
	0.4	0.181 ± 0.006	0.103 ± 0.005	0.093 ± 0.005	0.106 ± 0.006	0.103 ± 0.009	0.119 ± 0.011	0.121 ± 0.005	0.116 ± 0.008
RM3	0	0.277 ± 0.004	0.731 ± 0.006	1.162 ± 0.013	1.453 ± 0.042	1.671 ± 0.026	1.963 ± 0.029	2.095 ± 0.051	2.164 ± 0.021
	0.2	0.136 ± 0.035	0.118 ± 0.022	0.120 ± 0.033	0.111 ± 0.031	0.121 ± 0.019	0.106 ± 0.013	0.110 ± 0.020	0.106 ± 0.024
	0.4	0.215 ± 0.010	0.148 ± 0.022	0.144 ± 0.019	0.155 ± 0.011	0.165 ± 0.014	0.144 ± 0.004	0.157 ± 0.012	0.161 ± 0.015
RM4	0	1.589 ± 0.014	2.122 ± 0.027	2.371 ± 0.017	2.194 ± 0.023	2.088 ± 0.028	1.997 ± 0.026	1.922 ± 0.021	1.887 ± 0.008
	0.2	1.353 ± 0.007	2.093 ± 0.014	2.395 ± 0.007	2.270 ± 0.021	2.225 ± 0.042	2.061 ± 0.036	1.942 ± 0.029	1.925 ± 0.042
	0.4	0.341 ± 0.008	0.509 ± 0.004	0.526 ± 0.041	0.468 ± 0.019	0.471 ± 0.037	0.467 ± 0.065	0.447 ± 0.032	0.434 ± 0.028
RM5	0	1.589 ± 0.016	2.138 ± 0.061	2.346 ± 0.034	2.150 ± 0.034	2.062 ± 0.061	1.996 ± 0.058	1.913 ± 0.051	1.866 ± 0.049
	0.2	1.361 ± 0.007	2.096 ± 0.016	2.384 ± 0.011	2.261 ± 0.012	2.245 ± 0.029	2.239 ± 0.008	1.933 ± 0.064	1.922 ± 0.048
	0.4	0.387 ± 0.006	0.510 ± 0.008	0.529 ± 0.043	0.474 ± 0.016	0.484 ± 0.049	0.467 ± 0.063	0.448 ± 0.033	0.440 ± 0.029
RM6	0	1.641 ± 0.064	2.099 ± 0.081	2.317 ± 0.026	2.139 ± 0.013	2.080 ± 0.040	2.027 ± 0.033	1.974 ± 0.043	1.938 ± 0.032
	0.2	1.306 ± 0.010	2.145 ± 0.023	2.384 ± 0.011	2.303 ± 0.007	2.259 ± 0.011	2.213 ± 0.026	2.117 ± 0.023	2.071 ± 0.029
	0.4	0.358 ± 0.006	0.488 ± 0.002	0.519 ± 0.014	0.476 ± 0.006	0.479 ± 0.018	0.458 ± 0.010	0.450 ± 0.004	0.446 ± 0.006
RM7	0	0.070 ± 0.033	0.503 ± 0.162	1.663 ± 0.076	1.895 ± 0.040	2.028 ± 0.073	2.053 ± 0.085	2.086 ± 0.093	2.125 ± 0.084
	0.2	0.066 ± 0.001	0.048 ± 0.001	0.063 ± 0.010	0.074 ± 0.020	0.063 ± 0.005	0.049 ± 0.018	0.048 ± 0.019	0.047 ± 0.021
	0.4	0.067 ± 0.028	0.039 ± 0.036	0.046 ± 0.033	0.042 ± 0.023	0.048 ± 0.029	0.067 ± 0.041	0.031 ± 0.017	0.034 ± 0.020
RM8	0	0.194 ± 0.006	0.710 ± 0.009	1.283 ± 0.041	1.469 ± 0.064	1.492 ± 0.023	1.802 ± 0.043	2.099 ± 0.062	2.158 ± 0.072
	0.2	0.105 ± 0.009	0.259 ± 0.091	0.508 ± 0.104	0.937 ± 0.118	1.271 ± 0.097	1.493 ± 0.165	1.714 ± 0.210	1.603 ± 0.193
	0.4	0.093 ± 0.007	0.071 ± 0.011	0.030 ± 0.004	0.064 ± 0.007	0.062 ± 0.011	0.058 ± 0.018	0.051 ± 0.013	0.056 ± 0.013
RM9	0	0.453 ± 0.025	0.909 ± 0.004	1.276 ± 0.042	1.711 ± 0.014	1.978 ± 0.028	2.203 ± 0.030	2.277 ± 0.029	2.299 ± 0.030
	0.2	0.355 ± 0.028	0.280 ± 0.018	0.278 ± 0.024	0.281 ± 0.011	0.262 ± 0.023	0.244 ± 0.030	0.249 ± 0.033	0.267 ± 0.011
	0.4	0.333 ± 0.008	0.281 ± 0.004	0.294 ± 0.081	0.275 ± 0.082	0.266 ± 0.051	0.247 ± 0.020	0.258 ± 0.015	0.234 ± 0.011
RM13	0	0.477 ± 0.027	1.218 ± 0.012	1.541 ± 0.042	1.841 ± 0.033	1.958 ± 0.013	1.949 ± 0.030	1.926 ± 0.012	1.926 ± 0.007
	0.2	0.374 ± 0.031	0.322 ± 0.033	0.268 ± 0.017	0.274 ± 0.024	0.273 ± 0.017	0.258 ± 0.004	0.235 ± 0.019	0.267 ± 0.011
	0.4	0.354 ± 0.017	0.315 ± 0.030	0.309 ± 0.012	0.237 ± 0.024	0.341 ± 0.019	0.310 ± 0.017	0.299 ± 0.011	0.289 ± 0.017
RM14	0	0.332 ± 0.012	1.321 ± 0.044	1.801 ± 0.021	1.907 ± 0.022	2.001 ± 0.010	2.144 ± 0.039	2.193 ± 0.079	2.171 ± 0.051
	0.2	0.140 ± 0.016	0.123 ± 0.020	0.128 ± 0.010	0.131 ± 0.001	0.136 ± 0.005	0.147 ± 0.019	0.138 ± 0.016	0.136 ± 0.024
	0.4	0.135 ± 0.013	0.157 ± 0.009	0.178 ± 0.014	0.174 ± 0.017	0.104 ± 0.007	0.105 ± 0.011	0.107 ± 0.006	0.110 ± 0.002
RM15	0	0.261 ± 0.007	0.767 ± 0.035	1.314 ± 0.064	1.671 ± 0.051	1.852 ± 0.046	1.920 ± 0.032	2.024 ± 0.030	2.065 ± 0.044
	0.2	0.153 ± 0.009	0.153 ± 0.007	0.186 ± 0.006	0.165 ± 0.010	0.146 ± 0.022	0.151 ± 0.009	0.141 ± 0.006	0.147 ± 0.007
	0.4	0.137 ± 0.007	0.089 ± 0.012	0.080 ± 0.015	0.100 ± 0.007	0.100 ± 0.003	0.099 ± 0.019	0.097 ± 0.018	0.107 ± 0.014
RM16	0	0.276 ± 0.031	1.452 ± 0.044	1.735 ± 0.032	1.782 ± 0.018	1.846 ± 0.010	1.850 ± 0.010	1.813 ± 0.025	1.793 ± 0.023
	0.2	0.234 ± 0.028	1.417 ± 0.028	1.655 ± 0.036	1.734 ± 0.031	1.773 ± 0.020	1.775 ± 0.017	1.744 ± 0.018	1.740 ± 0.019
	0.4	0.240 ± 0.016	1.070 ± 0.020	1.571 ± 0.008	1.635 ± 0.019	1.731 ± 0.004	1.767 ± 0.009	1.740 ± 0.009	1.737 ± 0.008
RM17	0	0.101 ± 0.017	0.485 ± 0.023	1.092 ± 0.012	1.328 ± 0.065	1.438 ± 0.059	1.511 ± 0.073	1.575 ± 0.060	1.630 ± 0.063
	0.2	0.167 ± 0.005	0.177 ± 0.003	0.194 ± 0.006	0.199 ± 0.020	0.194 ± 0.007	0.158 ± 0.012	0.150 ± 0.012	0.146 ± 0.013
	0.4	0.098 ± 0.003	0.029 ± 0.003	0.042 ± 0.003	0.040 ± 0.002	0.039 ± 0.005	0.033 ± 0.004	0.030 ± 0.006	0.027 ± 0.007
RM18	0	0.417 ± 0.013	1.171 ± 0.051	1.597 ± 0.033	1.821 ± 0.024	1.956 ± 0.021	1.977 ± 0.038	2.086 ± 0.020	2.220 ± 0.099
	0.2	0.257 ± 0.033	0.298 ± 0.018	0.239 ± 0.028	0.229 ± 0.010	0.211 ± 0.004	0.193 ± 0.008	0.176 ± 0.006	0.178 ± 0.003
	0.4	0.245 ± 0.010	0.205 ± 0.015	0.199 ± 0.021	0.336 ± 0.025	0.276 ± 0.020	0.319 ± 0.048	0.305 ± 0.022	0.318 ± 0.021
RM19	0	0.073 ± 0.027	0.535 ± 0.048	1.355 ± 0.037	1.719 ± 0.036	1.930 ± 0.053	1.935 ± 0.084	1.924 ± 0.053	1.952 ± 0.046
	0.2	0.232 ± 0.007	0.236 ± 0.008	0.234 ± 0.009	0.235 ± 0.012	0.227 ± 0.012	0.207 ± 0.010	0.200 ± 0.008	0.200 ± 0.004
	0.4	0.177 ± 0.010	0.137 ± 0.015	0.121 ± 0.029	0.172 ± 0.037	0.145 ± 0.008	0.127 ± 0.017	0.120 ± 0.005	0.112 ± 0.011
RM20	0	1.617 ± 0.070	2.238 ± 0.019	2.218 ± 0.106	2.050 ± 0.062	1.987 ± 0.057	1.933 ± 0.057	1.999 ± 0.050	2.016 ± 0.062
	0.2	1.144 ± 0.041	2.070 ± 0.009	2.301 ± 0.011	2.300 ± 0.018	2.212 ± 0.004	2.141 ± 0.010	2.142 ± 0.013	2.122 ± 0.023
	0.4	0.229 ± 0.010	0.276 ± 0.012	0.245 ± 0.003	0.226 ± 0.005	0.221 ± 0.003	0.156 ± 0.003	0.204 ± 0.004	0.211 ± 0.003
RM21	0	0.199 ± 0.004	0.715 ± 0.008	1.273 ± 0.037	1.487 ± 0.040	1.656 ± 0.042	1.944 ± 0.036	2.102 ± 0.061	2.164 ± 0.082
	0.2	0.098 ± 0.007	0.113 ± 0.006	0.115 ± 0.010	0.124 ± 0.012	0.141 ± 0.010	0.111 ± 0.012	0.106 ± 0.009	0.114 ± 0.007
	0.4	0.088 ± 0.006	0.069 ± 0.010	0.030 ± 0.003	0.059 ± 0.004	0.066 ± 0.009	0.058 ± 0.017	0.051 ± 0.013	0.057 ± 0.012
DY1	0	1.039 ± 0.109	1.911 ± 0.051	2.167 ± 0.107	2.087 ± 0.013	1.882 ± 0.083	1.764 ± 0.061	1.723 ± 0.099	1.699 ± 0.096
	0.2	0.956 ± 0.004	2.091 ± 0.011	2.310 ± 0.012	2.258 ± 0.003	2.174 ± 0.009	2.114 ± 0.026	2.047 ± 0.014	2.008 ± 0.006
	0.4	0.166 ± 0.015	0.265 ± 0.019	0.319 ± 0.037	0.296 ± 0.017	0.380 ± 0.037	0.480 ± 0.067	0.447 ± 0.034	0.432 ± 0.035
DY2	0	1.395 ± 0.022	1.699 ± 0.052	2.026 ± 0.033	1.823 ± 0.081	1.633 ± 0.043	1.547 ± 0.066	1.621 ± 0.058	1.656 ± 0.098
	0.2	0.908 ± 0.036	1.942 ± 0.050	2.232 ± 0.069	2.290 ± 0.022	2.207 ± 0.027	2.083 ± 0.015	2.066 ± 0.024	2.051 ± 0.028
	0.4	0.154 ± 0.003	0.235 ± 0.014	0.297 ± 0.007	0.207 ± 0.010	0.227 ± 0.020	0.255 ± 0.009	0.223 ± 0.014	0.219 ± 0.003
DY3	0	1.312 ± 0.043	2.040 ± 0.061	2.225 ± 0.015	2.040 ± 0.023	1.896 ± 0.052	1.840 ± 0.047	1.769 ± 0.013	1.738 ± 0.019
	0.2	0.935 ± 0.036	2.031 ± 0.021	2.306 ± 0.013	2.264 ± 0.008	2.179 ± 0.025	2.101 ± 0.008	2.024 ± 0.012	1.990 ± 0.004
	0.4	0.154 ± 0.009	0.273 ± 0.009	0.272 ± 0.056	0.285 ± 0.010	0.301 ± 0.001	0.272 ± 0.003	0.264 ± 0.003	0.263 ± 0.006
DY4	0	0.348 ± 0.064	1.081 ± 0.128	1.615 ± 0.070	1.866 ± 0.044	1.985 ± 0.019	2.081 ± 0.042	2.181 ± 0.029	2.182 ± 0.015
	0.2	0.224 ± 0.034	0.153 ± 0.036	0.153 ± 0.005	0.115 ± 0.026	0.107 ± 0.024	0.114 ± 0.007	0.080 ± 0.034	0.136 ± 0.031
	0.4	0.188 ± 0.002	0.137 ± 0.005	0.152 ± 0.012	0.183 ± 0.014	0.165 ± 0.022	0.173 ± 0.027	0.167 ± 0.026	0.171 ± 0.025
DY6	0	0.151 ± 0.006	0.763 ± 0.011	1.608 ± 0.012	1.815 ± 0.048	1.943 ± 0.006	2.069 ± 0.021	2.145 ± 0.028	2.204 ± 0.010
	0.2	0.159 ± 0.025	0.130 ± 0.028	0.094 ± 0.013	0.103 ± 0.009	0.099 ± 0.011	0.119 ± 0.010	0.116 ± 0.026	0.110 ± 0.021
	0.4	0.112 ± 0.008	0.084 ± 0.004	0.047 ± 0.005	0.058 ± 0.004	0.072 ± 0.007	0.063 ± 0.006	0.073 ± 0.007	0.075 ± 0.009
DY7	0	1.569 ± 0.023	2.253 ± 0.040	2.212 ± 0.022	2.077 ± 0.036	1.942 ± 0.053	1.894 ± 0.011	1.973 ± 0.056	2.090 ± 0.085
	0.2	1.333 ± 0.049	2.006 ± 0.021	1.577 ± 0.012	1.621 ± 0.019	1.591 ± 0.065	1.459 ± 0.053	1.546 ± 0.034	1.566 ± 0.068
	0.4	0.141 ± 0.003	0.255 ± 0.010	0.312 ± 0.010	0.261 ± 0.006	0.233 ± 0.011	0.253 ± 0.012	0.223 ± 0.014	0.219 ± 0.003
DY8	0	1.625 ± 0.033	2.253 ± 0.006	2.240 ± 0.026	2.061 ± 0.033	1.995 ± 0.013	1.933 ± 0.009	1.913 ± 0.012	1.905 ± 0.007
	0.2	1.395 ± 0.006	2.033 ± 0.005	1.944 ± 0.006	1.641 ± 0.004	1.550 ± 0.027	1.495 ± 0.017	1.497 ± 0.016	1.477 ± 0.018
	0.4	0.357 ± 0.004	0.346 ± 0.008	0.296 ± 0.006	0.262 ± 0.008	0.244 ± 0.011	0.254 ± 0.009	0.232 ± 0.007	0.209 ± 0.003
DY9	0	1.709 ± 0.025	2.398 ± 0.005	2.346 ± 0.047	2.208 ± 0.038	2.082 ± 0.042	1.942 ± 0.004	1.936 ± 0.026	1.917 ± 0.036
	0.2	1.469 ± 0.019	2.208 ± 0.008	2.307 ± 0.025	2.193 ± 0.042	2.189 ± 0.055	2.110 ± 0.056	2.194 ± 0.066	2.192 ± 0.066
	0.4	0.509 ± 0.032	0.482 ± 0.041	0.430 ± 0.029	0.368 ± 0.018	0.341 ± 0.020	0.297 ± 0.019	0.352 ± 0.013	0.326 ± 0.016
DY10	0	0.095 ± 0.012	1.161 ± 0.009	1.309 ± 0.010	1.347 ± 0.023	1.346 ± 0.029	1.353 ± 0.041	1.366 ± 0.038	1.409 ± 0.042
	0.2	0.077 ± 0.004	0.025 ± 0.004	0.027 ± 0.005	0.032 ± 0.008	0.052 ± 0.016	0.051 ± 0.014	0.303 ± 0.321	0.069 ± 0.005
	0.4	0.098 ± 0.002	0.003 ± 0.004	0.026 ± 0.008	0.023 ± 0.006	0.016 ± 0.003	0.020 ± 0.006	0.022 ± 0.003	0.025 ± 0.004
DY11	0	0.166 ± 0.016	0.835 ± 0.048	1.588 ± 0.048	1.846 ± 0.020	1.970 ± 0.041	2.041 ± 0.039	2.099 ± 0.052	2.140 ± 0.038
	0.2	0.278 ± 0.013	0.232 ± 0.015	0.297 ± 0.018	0.255 ± 0.004	0.210 ± 0.003	0.194 ± 0.006	0.177 ± 0.005	0.182 ± 0.005
	0.4	0.138 ± 0.020	0.274 ± 0.300	0.090 ± 0.022	0.091 ± 0.035	0.080 ± 0.033	0.078 ± 0.029	0.070 ± 0.015	0.075 ± 0.012
DY12	0	0.126 ± 0.031	0.721 ± 0.080	1.412 ± 0.006	1.770 ± 0.013	1.983 ± 0.018	2.087 ± 0.014	2.146 ± 0.011	2.234 ± 0.001
	0.2	0.133 ± 0.026	0.077 ± 0.022	0.076 ± 0.018	0.062 ± 0.020	0.071 ± 0.019	0.053 ± 0.006	0.053 ± 0.006	0.052 ± 0.004
	0.4	0.033 ± 0.001	0.006 ± 0.010	0.010 ± 0.004	0.007 ± 0.005	0.016 ± 0.003	0.008 ± 0.003	0.006 ± 0.005	0.008 ± 0.006
DY13	0	0.093 ± 0.015	0.328 ± 0.009	0.724 ± 0.022	1.124 ± 0.027	1.339 ± 0.028	1.429 ± 0.022	1.617 ± 0.014	1.666 ± 0.026
	0.2	0.071 ± 0.015	0.031 ± 0.014	0.013 ± 0.005	0.018 ± 0.001	0.031 ± 0.006	0.025 ± 0.009	0.027 ± 0.002	0.033 ± 0.008
	0.4	0.065 ± 0.012	0.018 ± 0.015	0.013 ± 0.016	0.015 ± 0.003	0.031 ± 0.007	0.026 ± 0.006	0.024 ± 0.002	0.027 ± 0.006
DY14	0	0.275 ± 0.004	0.785 ± 0.040	1.317 ± 0.060	1.669 ± 0.041	1.848 ± 0.050	1.912 ± 0.041	1.923 ± 0.073	2.074 ± 0.061
	0.2	0.173 ± 0.009	0.180 ± 0.009	0.199 ± 0.015	0.193 ± 0.007	0.193 ± 0.002	0.154 ± 0.013	0.163 ± 0.002	0.169 ± 0.008
	0.4	0.161 ± 0.014	0.105 ± 0.017	0.095 ± 0.009	0.111 ± 0.004	0.134 ± 0.002	0.130 ± 0.008	0.131 ± 0.005	0.135 ± 0.006
DY15	0	1.590 ± 0.014	2.246 ± 0.004	2.297 ± 0.021	2.145 ± 0.065	2.078 ± 0.065	1.994 ± 0.007	1.945 ± 0.038	1.875 ± 0.019
	0.2	1.073 ± 0.052	2.039 ± 0.063	2.311 ± 0.015	2.274 ± 0.027	2.188 ± 0.030	2.104 ± 0.034	1.921 ± 0.029	1.888 ± 0.043
	0.4	0.211 ± 0.010	0.276 ± 0.011	0.246 ± 0.004	0.227 ± 0.004	0.222 ± 0.004	0.161 ± 0.011	0.146 ± 0.004	0.146 ± 0.002
DY16	0	0.119 ± 0.006	0.548 ± 0.023	1.174 ± 0.021	1.497 ± 0.012	1.674 ± 0.021	1.732 ± 0.032	1.742 ± 0.032	1.751 ± 0.016
	0.2	0.032 ± 0.007	0.030 ± 0.005	0.073 ± 0.042	0.064 ± 0.020	0.046 ± 0.004	0.036 ± 0.008	0.037 ± 0.007	0.037 ± 0.007
	0.4	0.013 ± 0.005	0.011 ± 0.004	0.051 ± 0.028	0.025 ± 0.006	0.016 ± 0.006	0.019 ± 0.009	0.023 ± 0.005	0.025 ± 0.007
DY17	0	0.201 ± 0.015	0.685 ± 0.027	1.153 ± 0.058	1.315 ± 0.080	1.476 ± 0.196	1.673 ± 0.040	1.943 ± 0.005	1.986 ± 0.015
	0.2	0.388 ± 0.513	0.129 ± 0.007	0.119 ± 0.018	0.100 ± 0.005	0.113 ± 0.006	0.099 ± 0.006	0.107 ± 0.018	0.108 ± 0.011
	0.4	0.093 ± 0.013	0.123 ± 0.006	0.114 ± 0.005	0.103 ± 0.008	0.091 ± 0.004	0.088 ± 0.005	0.095 ± 0.006	0.092 ± 0.005
DY18	0	0.195 ± 0.017	0.870 ± 0.051	1.422 ± 0.084	1.667 ± 0.073	1.827 ± 0.004	1.982 ± 0.061	2.138 ± 0.049	2.124 ± 0.100
	0.2	0.133 ± 0.012	0.218 ± 0.009	0.218 ± 0.018	0.167 ± 0.006	0.142 ± 0.011	0.147 ± 0.005	0.174 ± 0.006	0.171 ± 0.008
	0.4	0.128 ± 0.014	0.182 ± 0.004	0.169 ± 0.004	0.139 ± 0.005	0.128 ± 0.009	0.135 ± 0.012	0.158 ± 0.016	0.151 ± 0.022
DY19	0	0.232 ± 0.032	0.900 ± 0.055	1.340 ± 0.029	1.602 ± 0.018	1.753 ± 0.062	1.941 ± 0.022	1.993 ± 0.004	2.069 ± 0.049
	0.2	0.129 ± 0.010	0.235 ± 0.008	0.264 ± 0.007	0.267 ± 0.017	0.266 ± 0.013	0.274 ± 0.024	0.257 ± 0.032	0.255 ± 0.036
	0.4	0.129 ± 0.019	0.186 ± 0.007	0.192 ± 0.014	0.179 ± 0.007	0.126 ± 0.007	0.122 ± 0.002	0.137 ± 0.005	0.144 ± 0.007
DY20	0	0.184 ± 0.019	0.899 ± 0.056	1.521 ± 0.034	1.775 ± 0.038	1.922 ± 0.019	1.927 ± 0.052	2.040 ± 0.062	2.105 ± 0.065
	0.2	0.150 ± 0.020	0.204 ± 0.007	0.230 ± 0.012	0.202 ± 0.003	0.213 ± 0.018	0.189 ± 0.017	0.174 ± 0.010	0.177 ± 0.024
	0.4	0.141 ± 0.016	0.139 ± 0.047	0.176 ± 0.016	0.125 ± 0.096	0.183 ± 0.009	0.186 ± 0.004	0.160 ± 0.002	0.155 ± 0.004
DY21	0	0.241 ± 0.034	0.684 ± 0.047	1.182 ± 0.026	1.430 ± 0.028	1.536 ± 0.003	1.622 ± 0.039	1.638 ± 0.003	1.675 ± 0.008
	0.2	0.159 ± 0.018	0.139 ± 0.006	0.115 ± 0.011	0.098 ± 0.008	0.112 ± 0.007	0.100 ± 0.006	0.110 ± 0.014	0.116 ± 0.009
	0.4	0.144 ± 0.010	0.092 ± 0.016	0.065 ± 0.005	0.082 ± 0.014	0.088 ± 0.016	0.087 ± 0.009	0.083 ± 0.006	0.086 ± 0.008
DY23	0	1.645 ± 0.033	2.357 ± 0.002	2.296 ± 0.022	2.152 ± 0.028	1.997 ± 0.042	1.827 ± 0.066	1.854 ± 0.019	1.817 ± 0.027
	0.2	1.419 ± 0.026	2.029 ± 0.010	1.688 ± 0.014	1.668 ± 0.032	1.631 ± 0.033	1.527 ± 0.036	1.572 ± 0.022	1.554 ± 0.029
	0.4	0.274 ± 0.069	0.338 ± 0.005	0.274 ± 0.013	0.256 ± 0.003	0.200 ± 0.005	0.149 ± 0.009	0.178 ± 0.006	0.187 ± 0.004
JD10	0	0.184 ± 0.007	0.603 ± 0.006	1.053 ± 0.017	1.234 ± 0.024	1.573 ± 0.206	1.771 ± 0.213	2.059 ± 0.075	2.110 ± 0.068
	0.2	0.237 ± 0.013	0.183 ± 0.010	0.171 ± 0.018	0.157 ± 0.006	0.164 ± 0.009	0.171 ± 0.009	0.170 ± 0.008	0.173 ± 0.006
	0.4	0.040 ± 0.003	0.019 ± 0.008	0.018 ± 0.005	0.024 ± 0.009	0.024 ± 0.014	0.032 ± 0.013	0.022 ± 0.004	0.019 ± 0.004
JD11	0	0.253 ± 0.024	0.721 ± 0.062	1.082 ± 0.057	1.387 ± 0.035	1.772 ± 0.089	1.952 ± 0.077	2.034 ± 0.011	2.139 ± 0.040
	0.2	0.136 ± 0.006	0.102 ± 0.019	0.119 ± 0.012	0.115 ± 0.006	0.140 ± 0.004	0.140 ± 0.004	0.135 ± 0.006	0.129 ± 0.007
	0.4	0.137 ± 0.012	0.113 ± 0.019	0.115 ± 0.019	0.100 ± 0.008	0.117 ± 0.010	0.130 ± 0.012	0.112 ± 0.019	0.116 ± 0.014
JD12	0	0.233 ± 0.039	0.639 ± 0.016	0.777 ± 0.584	1.379 ± 0.038	1.572 ± 0.044	1.771 ± 0.061	2.083 ± 0.105	2.182 ± 0.044
	0.2	0.175 ± 0.020	0.178 ± 0.003	0.127 ± 0.012	0.088 ± 0.016	0.099 ± 0.006	0.105 ± 0.023	0.108 ± 0.024	0.111 ± 0.024
	0.4	0.121 ± 0.012	0.174 ± 0.008	0.188 ± 0.037	0.196 ± 0.027	0.166 ± 0.001	0.134 ± 0.012	0.139 ± 0.013	0.129 ± 0.005
JD13	0	0.279 ± 0.011	0.860 ± 0.053	1.383 ± 0.017	1.793 ± 0.064	1.891 ± 0.014	1.998 ± 0.081	2.236 ± 0.038	2.274 ± 0.025
	0.2	0.177 ± 0.022	0.133 ± 0.019	0.113 ± 0.010	0.138 ± 0.025	0.153 ± 0.026	0.165 ± 0.018	0.161 ± 0.007	0.163 ± 0.011
	0.4	0.089 ± 0.019	0.302 ± 0.458	0.054 ± 0.027	0.063 ± 0.034	0.072 ± 0.038	0.075 ± 0.048	0.069 ± 0.034	0.071 ± 0.039
JD14	0	0.181 ± 0.004	0.778 ± 0.044	1.421 ± 0.052	1.633 ± 0.052	1.782 ± 0.106	1.795 ± 0.091	1.892 ± 0.167	1.947 ± 0.171
	0.2	0.045 ± 0.003	0.035 ± 0.007	0.047 ± 0.007	0.044 ± 0.005	0.041 ± 0.010	0.055 ± 0.009	0.047 ± 0.003	0.050 ± 0.007
	0.4	0.127 ± 0.022	0.079 ± 0.015	0.127 ± 0.012	0.118 ± 0.012	0.118 ± 0.007	0.105 ± 0.008	0.105 ± 0.005	0.108 ± 0.002
JD1	0	0.555 ± 0.062	0.889 ± 0.431	1.953 ± 0.022	2.281 ± 0.011	2.404 ± 0.012	2.406 ± 0.023	2.419 ± 0.018	2.411 ± 0.019
	0.2	0.411 ± 0.009	0.424 ± 0.027	0.456 ± 0.025	0.453 ± 0.017	0.472 ± 0.041	0.415 ± 0.065	0.439 ± 0.029	0.477 ± 0.050
	0.4	0.369 ± 0.012	0.276 ± 0.008	0.206 ± 0.001	0.184 ± 0.007	0.185 ± 0.007	0.167 ± 0.000	0.148 ± 0.019	0.152 ± 0.013
LY4	0	0.162 ± 0.004	0.674 ± 0.009	1.021 ± 0.007	1.634 ± 0.054	1.930 ± 0.035	1.913 ± 0.011	1.893 ± 0.010	1.854 ± 0.021
	0.2	0.098 ± 0.004	0.112 ± 0.007	0.137 ± 0.006	0.098 ± 0.006	0.093 ± 0.004	0.122 ± 0.005	0.110 ± 0.002	0.107 ± 0.003
	0.4	0.004 ± 0.003	0.000 ± 0.002	0.011 ± 0.002	0.013 ± 0.002	0.014 ± 0.002	0.007 ± 0.002	0.038 ± 0.053	0.010 ± 0.003
LY5	0	0.277 ± 0.009	0.793 ± 0.004	1.282 ± 0.014	1.687 ± 0.037	2.059 ± 0.071	1.930 ± 0.041	1.871 ± 0.035	1.829 ± 0.036
	0.2	−0.001 ± 0.005	0.010 ± 0.011	0.148 ± 0.015	0.158 ± 0.023	0.142 ± 0.018	0.108 ± 0.034	0.112 ± 0.042	0.115 ± 0.039
	0.4	0.001 ± 0.002	−0.001 ± 0.003	0.010 ± 0.004	0.011 ± 0.002	0.014 ± 0.004	0.008 ± 0.004	0.016 ± 0.010	0.012 ± 0.003

. The growth of 51 of the 52 Rhizobium strains was inhibited at 0.4 mol/L, whereas that of MR16 was not inhibited. Most of the strains were inhibited at 0.2 mol/L, while all of the strains grew normally at 0 mol/L.

3.5. The Growth Curves of Desmodium Rhizobium Strains in Media at Different pH Values

The OD₆₀₀ values of the Desmodium Rhizobium strains grown at different pH values changed with time. As shown in Figure 5 and

Table 3. Effect of different pH liquid media on the growth of Rhizobium strains.

Strain	pH	Incubation Time (h)
		12	36	60	84	108	132	156	168
BY1	4.5	0.067 ± 0.009	0.078 ± 0.011	0.091 ± 0.011	0.210 ± 0.028	1.442 ± 0.123	1.857 ± 0.153	2.023 ± 0.122	2.064 ± 0.115
	7.0	0.029 ± 0.007	0.683 ± 0.006	1.467 ± 0.026	1.781 ± 0.020	1.936 ± 0.037	1.894 ± 0.011	1.945 ± 0.045	1.967 ± 0.021
	9.0	0.089 ± 0.011	0.123 ± 0.016	0.120 ± 0.009	0.119 ± 0.005	0.117 ± 0.002	0.090 ± 0.004	0.108 ± 0.012	0.102 ± 0.010
BY2	4.5	0.051 ± 0.011	0.136 ± 0.014	0.548 ± 0.035	1.285 ± 0.109	1.725 ± 0.166	1.932 ± 0.163	1.959 ± 0.135	1.990 ± 0.145
	7.0	0.128 ± 0.007	0.547 ± 0.009	1.183 ± 0.020	1.406 ± 0.023	1.607 ± 0.040	1.674 ± 0.018	1.740 ± 0.037	1.773 ± 0.034
	9.0	0.061 ± 0.014	0.438 ± 0.008	0.896 ± 0.021	1.010 ± 0.002	1.093 ± 0.011	1.164 ± 0.010	1.136 ± 0.017	1.134 ± 0.015
BY3	4.5	0.125 ± 0.004	0.967 ± 0.057	1.983 ± 0.041	2.098 ± 0.027	1.984 ± 0.027	1.934 ± 0.077	1.850 ± 0.043	1.819 ± 0.042
	7.0	0.550 ± 0.008	1.183 ± 0.020	2.059 ± 0.014	2.126 ± 0.027	2.034 ± 0.015	1.955 ± 0.018	1.859 ± 0.043	1.832 ± 0.040
	9.0	0.430 ± 0.019	1.341 ± 0.024	2.116 ± 0.012	2.167 ± 0.033	2.078 ± 0.069	1.995 ± 0.031	1.895 ± 0.034	1.864 ± 0.036
BY4	4.5	0.053 ± 0.007	0.121 ± 0.005	0.548 ± 0.035	1.436 ± 0.236	1.754 ± 0.260	1.847 ± 0.168	1.906 ± 0.150	1.937 ± 0.155
	7.0	0.132 ± 0.010	0.555 ± 0.008	1.183 ± 0.020	1.452 ± 0.026	1.604 ± 0.033	1.674 ± 0.022	1.735 ± 0.031	1.765 ± 0.035
	9.0	0.097 ± 0.002	0.435 ± 0.020	0.896 ± 0.021	1.008 ± 0.008	1.093 ± 0.011	1.148 ± 0.017	1.130 ± 0.002	1.125 ± 0.003
ZF3	4.5	0.143 ± 0.022	0.021 ± 0.005	0.025 ± 0.006	0.024 ± 0.004	0.023 ± 0.007	0.048 ± 0.009	0.052 ± 0.011	0.017 ± 0.007
	7.0	0.132 ± 0.007	0.363 ± 0.040	0.651 ± 0.084	0.931 ± 0.074	1.009 ± 0.012	1.192 ± 0.047	1.424 ± 0.069	1.456 ± 0.051
	9.0	0.126 ± 0.017	0.139 ± 0.007	0.100 ± 0.004	0.097 ± 0.001	0.090 ± 0.002	0.103 ± 0.004	0.102 ± 0.003	0.096 ± 0.002
RM1	4.5	0.065 ± 0.003	0.069 ± 0.003	0.095 ± 0.008	0.213 ± 0.027	1.499 ± 0.115	1.997 ± 0.102	2.112 ± 0.087	2.165 ± 0.088
	7.0	0.121 ± 0.020	0.141 ± 0.015	1.115 ± 0.046	1.801 ± 0.038	1.966 ± 0.029	2.079 ± 0.030	2.181 ± 0.027	2.244 ± 0.023
	9.0	0.108 ± 0.023	0.113 ± 0.021	0.132 ± 0.018	0.134 ± 0.018	0.138 ± 0.026	0.130 ± 0.023	0.124 ± 0.020	0.121 ± 0.021
RM2	4.5	0.060 ± 0.006	0.061 ± 0.011	0.087 ± 0.012	0.201 ± 0.003	0.783 ± 0.013	1.493 ± 0.115	1.935 ± 0.075	2.023 ± 0.020
	7.0	0.099 ± 0.010	0.122 ± 0.021	0.554 ± 0.021	1.114 ± 0.019	1.538 ± 0.022	1.870 ± 0.012	2.018 ± 0.013	2.069 ± 0.011
	9.0	0.105 ± 0.005	0.110 ± 0.008	0.118 ± 0.009	0.136 ± 0.011	0.141 ± 0.010	0.141 ± 0.008	0.137 ± 0.008	0.136 ± 0.008
RM3	4.5	0.064 ± 0.007	0.054 ± 0.006	0.073 ± 0.015	0.215 ± 0.035	1.394 ± 0.218	1.960 ± 0.161	2.072 ± 0.164	2.125 ± 0.149
	7.0	0.089 ± 0.013	0.159 ± 0.018	1.120 ± 0.058	1.836 ± 0.032	2.038 ± 0.012	2.175 ± 0.006	2.213 ± 0.024	2.239 ± 0.036
	9.0	0.072 ± 0.018	0.063 ± 0.005	0.275 ± 0.334	0.094 ± 0.025	0.125 ± 0.037	0.117 ± 0.022	0.101 ± 0.032	0.102 ± 0.048
RM4	4.5	0.536 ± 0.058	1.879 ± 0.091	2.124 ± 0.121	2.118 ± 0.090	1.996 ± 0.013	1.853 ± 0.017	1.744 ± 0.028	1.687 ± 0.021
	7.0	1.234 ± 0.038	2.107 ± 0.020	2.364 ± 0.017	2.189 ± 0.026	2.088 ± 0.033	1.991 ± 0.028	1.937 ± 0.033	1.892 ± 0.019
	9.0	1.101 ± 0.005	2.006 ± 0.020	2.350 ± 0.032	2.265 ± 0.034	2.164 ± 0.035	2.069 ± 0.038	1.983 ± 0.040	1.950 ± 0.044
RM5	4.5	0.527 ± 0.026	1.818 ± 0.062	2.083 ± 0.014	1.980 ± 0.016	1.900 ± 0.016	1.841 ± 0.012	1.783 ± 0.009	1.739 ± 0.018
	7.0	1.239 ± 0.151	2.088 ± 0.074	2.252 ± 0.053	2.257 ± 0.004	2.163 ± 0.008	2.021 ± 0.020	1.928 ± 0.015	1.905 ± 0.010
	9.0	1.161 ± 0.040	2.035 ± 0.033	2.247 ± 0.023	2.252 ± 0.023	2.172 ± 0.078	2.016 ± 0.045	1.918 ± 0.041	1.868 ± 0.036
RM6	4.5	0.420 ± 0.073	1.604 ± 0.034	1.981 ± 0.018	1.758 ± 0.052	1.680 ± 0.047	1.633 ± 0.048	1.607 ± 0.034	1.592 ± 0.024
	7.0	0.829 ± 0.636	1.999 ± 0.012	2.287 ± 0.007	2.239 ± 0.019	2.095 ± 0.009	2.041 ± 0.012	1.989 ± 0.018	1.961 ± 0.027
	9.0	1.220 ± 0.109	2.045 ± 0.053	2.252 ± 0.048	2.260 ± 0.047	2.094 ± 0.045	1.959 ± 0.037	1.896 ± 0.030	1.877 ± 0.020
RM7	4.5	0.133 ± 0.007	0.386 ± 0.023	1.302 ± 0.058	1.793 ± 0.023	1.920 ± 0.034	2.054 ± 0.009	2.011 ± 0.007	1.989 ± 0.008
	7.0	0.200 ± 0.012	0.987 ± 0.024	1.604 ± 0.033	1.712 ± 0.033	1.723 ± 0.030	1.759 ± 0.037	1.882 ± 0.033	1.930 ± 0.016
	9.0	0.140 ± 0.012	0.547 ± 0.148	1.120 ± 0.110	1.209 ± 0.032	1.261 ± 0.055	1.279 ± 0.058	1.170 ± 0.047	1.124 ± 0.043
RM8	4.5	0.197 ± 0.033	0.276 ± 0.012	1.066 ± 0.048	1.777 ± 0.016	1.977 ± 0.021	2.187 ± 0.025	2.209 ± 0.027	2.330 ± 0.027
	7.0	0.478 ± 0.035	1.178 ± 0.001	1.619 ± 0.026	1.809 ± 0.018	1.921 ± 0.010	2.009 ± 0.008	2.083 ± 0.008	2.149 ± 0.025
	9.0	0.345 ± 0.032	0.896 ± 0.010	1.171 ± 0.066	1.184 ± 0.069	1.301 ± 0.083	1.513 ± 0.098	1.613 ± 0.101	1.746 ± 0.098
RM9	4.5	0.069 ± 0.005	0.112 ± 0.005	0.621 ± 0.025	1.632 ± 0.022	1.974 ± 0.041	2.149 ± 0.015	2.219 ± 0.016	2.243 ± 0.029
	7.0	0.090 ± 0.005	0.327 ± 0.011	1.164 ± 0.034	1.726 ± 0.023	1.912 ± 0.040	2.051 ± 0.009	2.127 ± 0.013	2.174 ± 0.013
	9.0	0.055 ± 0.006	0.108 ± 0.005	0.327 ± 0.010	0.626 ± 0.034	0.762 ± 0.050	0.935 ± 0.047	0.942 ± 0.059	0.947 ± 0.070
RM13	4.5	0.180 ± 0.023	0.202 ± 0.013	0.977 ± 0.073	1.462 ± 0.012	1.671 ± 0.015	1.767 ± 0.014	1.837 ± 0.020	1.878 ± 0.033
	7.0	0.319 ± 0.030	0.851 ± 0.004	1.348 ± 0.006	1.528 ± 0.024	1.636 ± 0.014	1.713 ± 0.018	1.765 ± 0.007	1.796 ± 0.032
	9.0	0.217 ± 0.011	0.374 ± 0.022	0.847 ± 0.024	0.997 ± 0.010	1.065 ± 0.014	1.111 ± 0.016	1.138 ± 0.012	1.145 ± 0.030
RM14	4.5	0.170 ± 0.008	0.505 ± 0.019	1.646 ± 0.013	1.927 ± 0.007	1.954 ± 0.016	2.042 ± 0.005	2.042 ± 0.005	2.027 ± 0.005
	7.0	0.205 ± 0.017	1.058 ± 0.036	1.684 ± 0.026	1.750 ± 0.012	1.689 ± 0.012	1.741 ± 0.000	1.700 ± 0.002	1.681 ± 0.002
	9.0	0.179 ± 0.006	0.552 ± 0.010	1.179 ± 0.052	1.288 ± 0.056	1.232 ± 0.122	1.133 ± 0.065	1.100 ± 0.038	1.072 ± 0.036
RM15	4.5	0.068 ± 0.011	0.056 ± 0.002	0.061 ± 0.007	0.107 ± 0.013	0.622 ± 0.186	1.814 ± 0.153	2.032 ± 0.053	2.106 ± 0.065
	7.0	0.078 ± 0.003	0.099 ± 0.007	0.830 ± 0.099	1.772 ± 0.044	2.022 ± 0.014	2.218 ± 0.118	2.278 ± 0.100	2.299 ± 0.096
	9.0	0.057 ± 0.005	0.054 ± 0.004	0.060 ± 0.004	0.061 ± 0.013	0.078 ± 0.002	0.090 ± 0.006	0.142 ± 0.014	0.175 ± 0.006
RM16	4.5	0.003 ± 0.002	0.012 ± 0.002	0.083 ± 0.111	0.020 ± 0.008	0.028 ± 0.004	0.032 ± 0.012	0.025 ± 0.006	0.028 ± 0.004
	7.0	0.023 ± 0.007	0.292 ± 0.003	0.666 ± 0.004	1.326 ± 0.008	1.773 ± 0.026	1.845 ± 0.011	1.848 ± 0.012	1.827 ± 0.019
	9.0	0.014 ± 0.006	0.152 ± 0.012	0.379 ± 0.004	0.974 ± 0.014	1.546 ± 0.011	1.746 ± 0.009	1.732 ± 0.010	1.756 ± 0.019
RM17	4.5	0.016 ± 0.006	0.027 ± 0.015	0.016 ± 0.007	0.014 ± 0.006	0.031 ± 0.004	0.035 ± 0.003	0.017 ± 0.008	0.012 ± 0.008
	7.0	0.045 ± 0.008	0.075 ± 0.010	0.369 ± 0.014	0.937 ± 0.051	1.150 ± 0.059	1.175 ± 0.014	1.237 ± 0.009	1.296 ± 0.010
	9.0	0.012 ± 0.006	0.019 ± 0.004	0.056 ± 0.013	0.101 ± 0.036	0.078 ± 0.009	0.077 ± 0.011	0.070 ± 0.010	0.069 ± 0.010
RM18	4.5	0.085 ± 0.018	0.383 ± 0.044	1.239 ± 0.091	1.779 ± 0.009	1.960 ± 0.010	2.016 ± 0.018	1.721 ± 0.039	2.153 ± 0.035
	7.0	0.278 ± 0.017	1.046 ± 0.030	1.668 ± 0.022	1.748 ± 0.025	1.795 ± 0.021	1.838 ± 0.014	1.824 ± 0.002	1.890 ± 0.026
	9.0	0.195 ± 0.005	0.719 ± 0.023	1.180 ± 0.055	1.217 ± 0.063	1.260 ± 0.087	1.195 ± 0.094	1.111 ± 0.081	1.182 ± 0.085
RM19	4.5	0.021 ± 0.004	0.099 ± 0.021	0.426 ± 0.033	1.236 ± 0.089	1.776 ± 0.010	1.954 ± 0.010	2.012 ± 0.018	1.887 ± 0.010
	7.0	0.080 ± 0.020	0.431 ± 0.030	1.165 ± 0.023	1.715 ± 0.037	1.922 ± 0.064	1.934 ± 0.078	1.921 ± 0.053	1.948 ± 0.044
	9.0	0.061 ± 0.007	0.242 ± 0.010	0.717 ± 0.028	1.175 ± 0.056	1.207 ± 0.054	1.257 ± 0.089	1.189 ± 0.095	1.119 ± 0.124
RM20	4.5	0.067 ± 0.002	0.240 ± 0.007	0.721 ± 0.028	1.176 ± 0.050	1.203 ± 0.054	1.258 ± 0.082	1.184 ± 0.095	1.119 ± 0.115
	7.0	0.129 ± 0.014	1.605 ± 0.079	2.234 ± 0.018	2.213 ± 0.105	2.051 ± 0.057	1.982 ± 0.055	1.930 ± 0.058	1.980 ± 0.046
	9.0	0.102 ± 0.005	0.808 ± 0.010	1.749 ± 0.083	1.917 ± 0.059	2.099 ± 0.034	2.129 ± 0.028	1.980 ± 0.008	1.941 ± 0.036
RM21	4.5	0.138 ± 0.009	0.357 ± 0.012	1.384 ± 0.048	1.850 ± 0.019	1.807 ± 0.170	2.055 ± 0.011	2.002 ± 0.006	1.987 ± 0.006
	7.0	0.188 ± 0.010	0.847 ± 0.235	1.703 ± 0.030	1.849 ± 0.011	1.918 ± 0.008	1.988 ± 0.007	1.997 ± 0.003	1.976 ± 0.004
	9.0	0.143 ± 0.009	0.527 ± 0.096	1.247 ± 0.187	1.409 ± 0.146	1.295 ± 0.077	1.279 ± 0.168	1.233 ± 0.154	1.217 ± 0.147
DY1	4.5	0.147 ± 0.043	1.754 ± 0.153	1.823 ± 0.202	1.829 ± 0.206	1.858 ± 0.134	1.804 ± 0.164	1.750 ± 0.168	1.675 ± 0.109
	7.0	0.285 ± 0.038	1.591 ± 0.138	2.065 ± 0.022	1.676 ± 0.082	1.628 ± 0.063	1.594 ± 0.064	1.653 ± 0.066	1.692 ± 0.044
	9.0	0.344 ± 0.049	1.696 ± 0.141	1.813 ± 0.035	1.879 ± 0.011	2.163 ± 0.016	2.016 ± 0.025	1.946 ± 0.031	1.907 ± 0.022
DY2	4.5	0.562 ± 0.076	1.882 ± 0.018	2.019 ± 0.080	1.975 ± 0.020	1.804 ± 0.086	1.695 ± 0.058	1.632 ± 0.038	1.598 ± 0.017
	7.0	1.156 ± 0.037	1.818 ± 0.129	2.051 ± 0.098	1.985 ± 0.145	1.838 ± 0.033	1.722 ± 0.128	1.701 ± 0.098	1.695 ± 0.088
	9.0	1.169 ± 0.036	1.938 ± 0.096	2.250 ± 0.035	2.086 ± 0.044	2.087 ± 0.073	2.007 ± 0.021	1.949 ± 0.035	1.918 ± 0.029
DY3	4.5	0.149 ± 0.035	1.525 ± 0.163	1.962 ± 0.104	2.061 ± 0.037	1.839 ± 0.140	1.661 ± 0.095	1.634 ± 0.047	1.608 ± 0.007
	7.0	0.287 ± 0.038	1.377 ± 0.244	1.951 ± 0.127	2.061 ± 0.018	1.867 ± 0.050	1.749 ± 0.065	1.716 ± 0.063	1.687 ± 0.056
	9.0	0.347 ± 0.039	1.731 ± 0.111	2.152 ± 0.052	2.163 ± 0.040	2.130 ± 0.029	2.012 ± 0.021	1.934 ± 0.026	1.900 ± 0.021
DY4	4.5	0.053 ± 0.009	0.081 ± 0.012	0.198 ± 0.035	0.903 ± 0.102	1.685 ± 0.038	1.925 ± 0.011	1.976 ± 0.024	2.088 ± 0.013
	7.0	0.211 ± 0.003	0.950 ± 0.028	1.561 ± 0.017	1.728 ± 0.071	1.796 ± 0.043	1.927 ± 0.050	1.938 ± 0.043	2.022 ± 0.026
	9.0	0.115 ± 0.010	0.454 ± 0.037	0.960 ± 0.025	1.095 ± 0.124	1.105 ± 0.062	1.110 ± 0.024	1.056 ± 0.047	1.128 ± 0.082
DY6	4.5	0.070 ± 0.006	0.147 ± 0.009	0.713 ± 0.061	1.436 ± 0.042	1.618 ± 0.015	1.916 ± 0.020	1.991 ± 0.035	2.037 ± 0.061
	7.0	0.139 ± 0.003	0.826 ± 0.011	1.361 ± 0.027	1.532 ± 0.021	1.549 ± 0.022	1.817 ± 0.023	2.027 ± 0.015	2.099 ± 0.009
	9.0	0.120 ± 0.007	0.579 ± 0.020	0.782 ± 0.099	1.021 ± 0.048	1.071 ± 0.048	1.136 ± 0.058	1.111 ± 0.028	1.100 ± 0.018
DY7	4.5	0.212 ± 0.027	1.726 ± 0.127	1.964 ± 0.062	1.872 ± 0.031	1.752 ± 0.032	1.625 ± 0.067	1.590 ± 0.061	1.573 ± 0.042
	7.0	0.631 ± 0.029	1.567 ± 0.020	2.100 ± 0.140	2.163 ± 0.043	2.007 ± 0.034	1.950 ± 0.018	1.856 ± 0.065	1.845 ± 0.069
	9.0	0.467 ± 0.041	1.426 ± 0.015	1.830 ± 0.038	2.128 ± 0.028	2.005 ± 0.008	1.930 ± 0.020	1.864 ± 0.014	1.842 ± 0.014
DY8	4.5	0.215 ± 0.013	1.549 ± 0.025	2.001 ± 0.008	1.823 ± 0.045	1.684 ± 0.052	1.593 ± 0.052	1.578 ± 0.045	1.571 ± 0.042
	7.0	0.524 ± 0.011	1.475 ± 0.008	2.124 ± 0.054	2.079 ± 0.032	1.988 ± 0.022	1.857 ± 0.013	1.830 ± 0.068	1.825 ± 0.070
	9.0	0.325 ± 0.010	1.323 ± 0.011	2.056 ± 0.030	2.065 ± 0.021	1.981 ± 0.029	1.881 ± 0.017	1.824 ± 0.029	1.805 ± 0.021
DY9	4.5	0.192 ± 0.010	1.554 ± 0.030	1.998 ± 0.016	1.808 ± 0.036	1.656 ± 0.054	1.589 ± 0.064	1.570 ± 0.059	1.562 ± 0.060
	7.0	0.245 ± 0.010	1.377 ± 0.010	2.110 ± 0.051	2.087 ± 0.027	1.983 ± 0.010	1.881 ± 0.012	1.836 ± 0.058	1.828 ± 0.067
	9.0	0.234 ± 0.020	1.221 ± 0.013	2.043 ± 0.024	2.046 ± 0.034	1.956 ± 0.033	1.888 ± 0.016	1.828 ± 0.008	1.815 ± 0.008
DY10	4.5	0.169 ± 0.016	0.192 ± 0.011	0.830 ± 0.010	1.105 ± 0.044	1.185 ± 0.042	1.195 ± 0.048	1.164 ± 0.037	1.148 ± 0.042
	7.0	0.093 ± 0.017	0.860 ± 0.040	1.228 ± 0.009	1.359 ± 0.017	1.339 ± 0.014	1.330 ± 0.030	1.316 ± 0.008	1.351 ± 0.032
	9.0	0.063 ± 0.027	1.130 ± 0.031	1.279 ± 0.053	1.334 ± 0.052	1.360 ± 0.049	1.370 ± 0.029	1.361 ± 0.025	1.352 ± 0.020
DY11	4.5	0.078 ± 0.009	0.535 ± 0.026	1.071 ± 0.055	1.151 ± 0.055	1.210 ± 0.047	1.321 ± 0.084	1.401 ± 0.087	1.470 ± 0.127
	7.0	0.151 ± 0.015	0.812 ± 0.068	1.593 ± 0.040	1.842 ± 0.018	1.965 ± 0.057	2.031 ± 0.031	2.105 ± 0.040	2.145 ± 0.027
	9.0	0.242 ± 0.008	0.871 ± 0.016	1.702 ± 0.086	1.964 ± 0.084	2.043 ± 0.056	2.103 ± 0.062	2.122 ± 0.059	2.116 ± 0.046
DY12	4.5	0.146 ± 0.018	0.030 ± 0.006	0.062 ± 0.004	0.259 ± 0.005	0.668 ± 0.007	1.214 ± 0.007	1.433 ± 0.057	1.550 ± 0.043
	7.0	0.155 ± 0.005	0.356 ± 0.014	0.681 ± 0.018	0.952 ± 0.015	1.098 ± 0.034	1.300 ± 0.026	1.326 ± 0.032	1.380 ± 0.048
	9.0	0.147 ± 0.012	0.168 ± 0.013	0.556 ± 0.025	0.756 ± 0.024	0.854 ± 0.030	0.935 ± 0.034	0.892 ± 0.022	0.984 ± 0.086
DY13	4.5	0.145 ± 0.025	0.030 ± 0.011	0.058 ± 0.007	0.257 ± 0.005	0.672 ± 0.006	1.212 ± 0.004	1.436 ± 0.056	1.552 ± 0.039
	7.0	0.160 ± 0.005	0.362 ± 0.014	0.688 ± 0.020	0.956 ± 0.016	1.102 ± 0.034	1.290 ± 0.037	1.329 ± 0.036	1.380 ± 0.052
	9.0	0.151 ± 0.014	0.163 ± 0.029	0.557 ± 0.023	0.758 ± 0.027	0.854 ± 0.034	0.933 ± 0.036	0.885 ± 0.027	0.983 ± 0.093
DY14	4.5	0.182 ± 0.010	0.220 ± 0.026	0.803 ± 0.076	1.498 ± 0.077	1.850 ± 0.040	2.005 ± 0.022	2.123 ± 0.038	2.207 ± 0.007
	7.0	0.432 ± 0.014	1.035 ± 0.010	1.573 ± 0.004	1.856 ± 0.012	1.991 ± 0.022	2.002 ± 0.034	2.174 ± 0.011	2.213 ± 0.004
	9.0	0.276 ± 0.041	0.720 ± 0.079	1.217 ± 0.045	1.513 ± 0.040	1.626 ± 0.044	1.661 ± 0.027	1.709 ± 0.041	1.681 ± 0.048
DY15	4.5	0.295 ± 0.040	0.807 ± 0.062	1.501 ± 0.077	1.846 ± 0.039	1.918 ± 0.040	1.851 ± 0.036	1.804 ± 0.027	1.785 ± 0.024
	7.0	0.577 ± 0.018	1.586 ± 0.015	2.246 ± 0.005	2.296 ± 0.020	2.145 ± 0.064	2.080 ± 0.061	1.984 ± 0.020	1.964 ± 0.008
	9.0	0.731 ± 0.016	2.124 ± 0.012	2.269 ± 0.016	2.251 ± 0.006	2.134 ± 0.012	2.047 ± 0.035	1.973 ± 0.019	1.953 ± 0.012
DY16	4.5	0.144 ± 0.006	0.030 ± 0.004	0.027 ± 0.008	0.035 ± 0.008	0.038 ± 0.010	0.216 ± 0.272	0.294 ± 0.404	0.214 ± 0.291
	7.0	0.231 ± 0.017	0.502 ± 0.011	1.045 ± 0.020	1.402 ± 0.006	1.638 ± 0.028	1.696 ± 0.019	1.772 ± 0.018	1.762 ± 0.021
	9.0	0.186 ± 0.028	0.148 ± 0.005	0.174 ± 0.010	0.192 ± 0.028	0.156 ± 0.025	0.183 ± 0.012	0.133 ± 0.001	0.165 ± 0.012
DY17	4.5	0.086 ± 0.007	0.122 ± 0.007	0.154 ± 0.017	0.459 ± 0.068	0.776 ± 0.035	1.543 ± 0.051	1.718 ± 0.016	1.743 ± 0.010
	7.0	0.163 ± 0.007	1.156 ± 0.564	1.449 ± 0.002	1.574 ± 0.022	1.525 ± 0.031	1.708 ± 0.017	1.907 ± 0.012	1.953 ± 0.014
	9.0	0.123 ± 0.009	0.662 ± 0.016	1.140 ± 0.018	1.230 ± 0.008	1.227 ± 0.006	1.206 ± 0.012	1.189 ± 0.011	1.182 ± 0.019
DY18	4.5	0.164 ± 0.033	0.782 ± 0.022	1.418 ± 0.085	1.639 ± 0.117	1.821 ± 0.003	1.983 ± 0.063	2.128 ± 0.035	2.138 ± 0.033
	7.0	0.184 ± 0.014	0.812 ± 0.029	1.419 ± 0.081	1.676 ± 0.082	1.824 ± 0.009	1.981 ± 0.062	2.129 ± 0.045	2.119 ± 0.026
	9.0	0.075 ± 0.002	0.134 ± 0.008	0.222 ± 0.015	0.323 ± 0.011	0.383 ± 0.029	0.370 ± 0.031	0.357 ± 0.031	0.354 ± 0.031
DY19	4.5	0.134 ± 0.005	0.781 ± 0.015	1.419 ± 0.085	1.675 ± 0.069	1.832 ± 0.005	1.980 ± 0.061	2.144 ± 0.059	2.180 ± 0.029
	7.0	0.192 ± 0.021	0.886 ± 0.026	1.511 ± 0.045	1.780 ± 0.030	1.924 ± 0.011	1.925 ± 0.052	2.041 ± 0.058	2.085 ± 0.071
	9.0	0.070 ± 0.004	0.184 ± 0.062	0.225 ± 0.059	0.296 ± 0.033	0.376 ± 0.024	0.405 ± 0.022	0.396 ± 0.010	0.391 ± 0.014
DY20	4.5	0.172 ± 0.016	0.778 ± 0.011	1.326 ± 0.002	1.667 ± 0.082	1.833 ± 0.007	1.977 ± 0.048	2.070 ± 0.053	2.075 ± 0.061
	7.0	0.170 ± 0.008	0.885 ± 0.014	1.346 ± 0.027	1.609 ± 0.015	1.758 ± 0.062	1.943 ± 0.022	1.991 ± 0.005	2.032 ± 0.025
	9.0	0.060 ± 0.006	0.164 ± 0.010	0.254 ± 0.019	0.331 ± 0.027	0.443 ± 0.021	0.428 ± 0.003	0.412 ± 0.012	0.403 ± 0.014
DY21	4.5	0.040 ± 0.003	0.245 ± 0.012	0.979 ± 0.036	1.382 ± 0.004	1.527 ± 0.011	1.654 ± 0.019	1.682 ± 0.017	1.688 ± 0.028
	7.0	0.194 ± 0.003	0.836 ± 0.014	1.251 ± 0.009	1.380 ± 0.007	1.411 ± 0.028	1.458 ± 0.024	1.456 ± 0.014	1.470 ± 0.021
	9.0	0.098 ± 0.043	0.332 ± 0.028	0.784 ± 0.005	0.909 ± 0.046	0.909 ± 0.032	0.891 ± 0.032	0.886 ± 0.035	0.911 ± 0.037
DY23	4.5	0.042 ± 0.004	0.391 ± 0.084	1.345 ± 0.042	1.257 ± 0.041	1.191 ± 0.068	1.062 ± 0.290	1.015 ± 0.237	0.985 ± 0.235
	7.0	0.412 ± 0.155	0.922 ± 0.054	1.837 ± 0.130	2.283 ± 0.129	1.816 ± 0.536	2.148 ± 0.034	2.154 ± 0.021	2.145 ± 0.024
	9.0	0.360 ± 0.121	0.738 ± 0.029	1.962 ± 0.015	1.791 ± 0.012	1.722 ± 0.028	1.723 ± 0.018	1.712 ± 0.014	1.731 ± 0.045
JD10	4.5	0.021 ± 0.003	0.313 ± 0.019	0.370 ± 0.018	0.403 ± 0.016	0.454 ± 0.005	0.395 ± 0.009	0.305 ± 0.010	0.301 ± 0.004
	7.0	0.184 ± 0.007	0.600 ± 0.014	1.048 ± 0.016	1.219 ± 0.016	1.567 ± 0.205	1.770 ± 0.205	2.042 ± 0.054	2.098 ± 0.063
	9.0	0.006 ± 0.004	0.012 ± 0.004	0.058 ± 0.011	0.039 ± 0.006	0.022 ± 0.003	0.020 ± 0.008	0.128 ± 0.104	0.019 ± 0.005
JD11	4.5	0.130 ± 0.029	0.532 ± 0.007	0.998 ± 0.011	1.254 ± 0.016	1.689 ± 0.005	1.821 ± 0.129	1.868 ± 0.207	1.853 ± 0.218
	7.0	0.253 ± 0.024	0.698 ± 0.070	1.079 ± 0.054	1.383 ± 0.035	1.776 ± 0.091	1.944 ± 0.074	2.034 ± 0.007	2.063 ± 0.011
	9.0	0.161 ± 0.004	0.229 ± 0.014	0.853 ± 0.009	1.279 ± 0.051	1.543 ± 0.018	1.780 ± 0.023	1.962 ± 0.012	2.009 ± 0.019
JD12	4.5	0.093 ± 0.018	0.380 ± 0.014	1.074 ± 0.047	1.383 ± 0.021	1.474 ± 0.009	1.508 ± 0.010	1.422 ± 0.008	1.397 ± 0.010
	7.0	0.223 ± 0.024	0.633 ± 0.016	1.105 ± 0.035	1.377 ± 0.042	1.566 ± 0.043	1.766 ± 0.058	2.025 ± 0.056	2.075 ± 0.039
	9.0	0.122 ± 0.011	0.199 ± 0.004	0.351 ± 0.007	0.526 ± 0.013	0.490 ± 0.013	0.445 ± 0.025	0.376 ± 0.012	0.363 ± 0.011
JD13	4.5	0.175 ± 0.036	0.452 ± 0.014	1.114 ± 0.008	1.612 ± 0.079	1.618 ± 0.070	1.624 ± 0.056	1.634 ± 0.068	1.653 ± 0.057
	7.0	0.280 ± 0.018	0.843 ± 0.063	1.377 ± 0.019	1.786 ± 0.065	1.879 ± 0.022	1.972 ± 0.065	2.120 ± 0.062	2.189 ± 0.077
	9.0	0.075 ± 0.022	0.135 ± 0.021	0.274 ± 0.019	0.472 ± 0.012	0.450 ± 0.028	0.406 ± 0.020	0.355 ± 0.012	0.338 ± 0.003
JD14	4.5	0.054 ± 0.020	0.354 ± 0.025	1.147 ± 0.017	1.502 ± 0.058	1.752 ± 0.043	1.787 ± 0.046	1.619 ± 0.030	1.564 ± 0.031
	7.0	0.170 ± 0.013	0.770 ± 0.039	1.418 ± 0.053	1.623 ± 0.061	1.775 ± 0.102	1.795 ± 0.091	1.921 ± 0.133	1.947 ± 0.151
	9.0	0.081 ± 0.008	0.130 ± 0.009	0.248 ± 0.005	0.381 ± 0.014	0.362 ± 0.008	0.327 ± 0.006	0.307 ± 0.012	0.295 ± 0.017
JD1	4.5	0.056 ± 0.007	0.056 ± 0.003	0.062 ± 0.009	0.080 ± 0.014	0.083 ± 0.022	0.099 ± 0.017	0.092 ± 0.022	0.102 ± 0.017
	7.0	0.159 ± 0.010	0.639 ± 0.019	0.927 ± 0.014	1.580 ± 0.042	1.844 ± 0.034	1.983 ± 0.009	2.052 ± 0.020	2.072 ± 0.035
	9.0	0.083 ± 0.005	0.143 ± 0.032	0.135 ± 0.006	0.134 ± 0.006	0.148 ± 0.003	0.127 ± 0.003	0.138 ± 0.016	0.136 ± 0.006
LY4	4.5	0.113 ± 0.009	0.319 ± 0.008	0.444 ± 0.010	0.601 ± 0.021	0.565 ± 0.011	0.469 ± 0.060	0.434 ± 0.039	0.419 ± 0.037
	7.0	0.161 ± 0.024	0.672 ± 0.021	1.020 ± 0.018	1.668 ± 0.015	1.906 ± 0.081	1.895 ± 0.053	1.865 ± 0.044	1.828 ± 0.022
	9.0	0.136 ± 0.004	0.466 ± 0.012	0.555 ± 0.032	0.662 ± 0.035	0.619 ± 0.017	0.572 ± 0.028	0.532 ± 0.023	0.508 ± 0.022
LY5	4.5	0.051 ± 0.007	0.268 ± 0.015	0.467 ± 0.052	0.709 ± 0.044	0.605 ± 0.035	0.558 ± 0.035	0.514 ± 0.024	0.503 ± 0.026
	7.0	0.139 ± 0.007	0.522 ± 0.013	1.022 ± 0.006	1.412 ± 0.023	1.937 ± 0.058	1.996 ± 0.011	1.873 ± 0.058	1.857 ± 0.033
	9.0	0.126 ± 0.006	0.346 ± 0.032	0.523 ± 0.075	0.649 ± 0.063	0.612 ± 0.080	0.598 ± 0.067	0.558 ± 0.061	0.542 ± 0.058

, RM17, JD1, RM16, ZF3, and DY16 were inhibited and unable to grow at a pH of 4.5, whereas growth of JD10, LY4, and LY5 was significantly inhibited. The OD₆₀₀ values of these strains were all less than one. The acid resistance of these strains significantly differed from those of the other strains. All the strains grew normally at a pH of 7.0, but RM4, RM5, RM6, DY15, DY2, RM20, DY8, DY1, BY3, DY3, and DY9 reached the logarithmic growth stage at 60 h. At a pH of 9.0, JD10, RM17, RM15, JD1, JD16, RM3, RM2, RM1, and BY1 were inhibited and could not grow. The growth of RM16, JD11, DY14, RM8, DY23, RM20, DY1, DY11, DY7, DY9, DY8, BY3, DY3, DY15, RM4, DY2, RM6, and RM5 was not inhibited, and that of the remaining strains was inhibited to varying degrees.

4. Discussion

4.1. Determination of the Taxonomic Status of Desmodium Rhizobia

With the development of sequencing technology, researchers usually use 16S rRNA gene sequence alignment to initially determine the taxonomic status of rhizobia [5,7]. In this study, 62 strains with large differences were selected for 16S rDNA gene sequence determination on the basis of the apparent traits of the Rhizobium culture and Gram staining results and then compared with the NCBI results, and the classification locations of the isolated strains were preliminarily determined. Among the 62 strains, 48 were slow-growing Rhizobium strains, and 14 were fast-growing Rhizobium strains. The fast-growing Rhizobium strains were mainly isolates of Pleurolobus gangeticus and Polhillides velutina. Grona heterocarpos did not isolate with the fast-growing Rhizobium strains, which is consistent with the results obtained in previous studies in China, in which most maechium Rhizobium strains were slow-growing, and only a few strains were located in the genus of fast-growing Rhizobium [8,9]. By means of phenotypic cluster analysis (including numerical classification and SDS—PAGE whole-cell protein analysis) and analysis on the basis of genetic information, Jin et al. classified the locations of 50 Rhizobium strains of 11 species of Desmodium from 9 provinces, including Anhui and Jiangxi. Among these strains, 29 Rhizobium strains were classified as slow-growing, whereas the remaining 18 were identified as fast-growing [7]. These fast-growing bacteria are distributed mainly in the Rhizobium, Sinorhizobium, Agrobacterium, and Mesorhizobium genera. In addition, D. caudatum, D. heterocarpon, D. racemosum, D. sequax, D. elegans, D. microphyllum, D. ovalophyllum, D. racemosum, and D. fallax were isolated via BOX-PCR. A total of 25 strains belonged to 3 different lineages of Bradyrhizobia, which had close genetic relationships with Bradyrhizobia Erdzei, soybean Bradyrhizobia, and Yuanmingyuan Bradyrhizobia. Chang et al. numerically classified rhizobia isolated from a variety of tropical legumes in Hainan Province, China, and reported a new population of rhizobia isolated from several species of Desmodium, such as D. riquetrum, D. yroides, and D. eterophylum [3]. The rhizobia of Desmodium species obtained in our study can be divided into 10 groups on the evolutionary tree. The strains were Bradyrhizobium elkanii, Rhizobium oryzicola, Bradyrhizobium huanghuaihaiense, Rhizobium tropici, Rhizobium oryzicola, Bradyrhizobium ganzhouense, Bradyrhizobium centrosematis, Bradyrhizobium pachyrhizi, Bradyrhizobium yuanmingense, and Bradyrhizobium iriomotense. These results were consistent with the results of the classification of Desmodium Rhizobium strains at home and abroad, and no new strains appeared [5,7]. According to the evolutionary tree, with the exception of Bradyrhizobium sp. isolated from Grona heterocarpos, the other bacterial groups were derived from two or more hosts. The results indicated that the Bradyrhizobium sp. strain had strong fixation specificity of Rhizobium in four types of Desmodium.

4.2. Determination of the Salt Tolerance and Acid—Alkaline Resistance of Desmodium Rhizobia

The nitrogen fixation ability of root nodules is jointly dependent on the strain, host, and environmental conditions [6,10]. Desmodium are found mainly in tropical and subtropical areas, where acidic soil is dominant. Therefore, the adaptability of a strain to acid and alkali becomes the key factor for its symbiosis with the host and nitrogen fixation ability [11,12]. Moreover, large areas of saline land also exist in tropical and subtropical coastal areas [13,18]. Studies have shown that inoculation with salt-tolerant rhizobia can significantly improve the salt tolerance of the host. Therefore, screening Rhizobium strains with strong salt-tolerant capacities can increase the adaptability of Desmodium in saline land and provide excellent materials for the utilization and improvement of saline land [8,19]. The results of this study revealed that the strains with strong salt tolerance were mainly isolated from Grona heterocarpos and Polhillides velutina and could grow on YMA media supplemented with NaCl at a concentration of 0.4 mol/L. In terms of the phylogenetic classification of the strains, 9 of the 11 strains with high pH adaptability were slow-growing Rhizobium strains, and 2 were fast-growing Rhizobium strains. The alkali-tolerant strain RM20 is also a strain of the Rhizobium genus. Among the 15 strains with strong acid resistance, 14 were slow-growing Rhizobium strains, and 1 was a fast-growing Rhizobium strain. Salazar et al. reported that rhizobia grown in liquid media secrete acid or alkali, which affects the pH of the culture medium. In the numerical classification of rhizobia, acid production and alkali production are used as indices to distinguish between rapidly growing rhizobia and slow-growing rhizobia [12]. In our study, slow-growing bacteria with strong acid resistance were dominant, and fast-growing bacteria with strong alkali resistance were dominant, which may be due to the difference in tolerance of the different types of strains due to the acid and alkali production characteristics.

In the next step of this study, the following research steps will be carried out using the isolated strains. The obtained strains will be inoculated to screen for strains that can efficiently symbiose with the host under adverse conditions (acid or salt) and to study their promoting mechanisms. Then, the adaptation mechanisms of strains with strong tolerance (such as RM16) to adverse conditions will be studied, the key metabolic pathways will be analysed, and the key genes will be explored, providing strains and a foundation for enhancing the stress resistance of the rhizobia–host symbiosis and offering a gene source for the stress-resistance modification of other rhizobia.

5. Conclusions

A total of 152 cultures were isolated from the root nodules of the Desmodium genus, including Pleurolobus gangeticus, Puhuaea sequax, Grona heterocarpos, and Polhillides velutina. The phylogenetic classification of 62 strains of Rhizobium was determined by 16S rDNA. After comparison, the known Rhizobium strains were grouped into 10 groups, among which 14 strains were fast-growing Rhizobium strains. Two types of fast-growing Rhizobium strains were identified: Rhizobium tropici and Rhizobium oryzicola. Forty-eight Rhizobium strains were slow-growing. The strains with strong salt tolerance ability were isolated mainly from Pleurolobus gangeticus. The isolates from slow-growing Rhizobium strains presented strong acid resistance, whereas those from fast-growing Rhizobium strains presented strong alkali resistance. The salt tolerance and acid—base adaptability of 49 strains were qualitatively and quantitatively determined. Some strains grew normally on media supplemented with 0.4 mol/L or 0.35 mol/L NaCl and presented strong salt tolerance. The strains that grew normally on media with pH 5–9 with high pH adaptability were BY3, RM4, RM5, RM6, RM16, RM19, DY2, DY3, DY7, DY8, and DY9. RM16 had good salt, acid, and alkali resistance.