Isolation,Identification and Symbiotic Matching of Soybean Rhizobia from Shanxi Province

doi:10.13560/j.cnki.biotech.bull.1985.2021-1315

Abstract

Abstract:

As one of the origin of soybean,Shanxi is rich in rhizobia resources. In order to further excavate soybean rhizobia resources in Shanxi province and screen the rhizobia of having good symbiotic nitrogen fixation effect with the local main soybean varieties. In this experiment,root nodules of different soybean varieties were collected in Guangling of Datong,Qingxu of Taiyuan and Taigu of Jinzhong. After multiple streaking,the colony morphology,Congo red staining,BTB staining phenotype identification and 16S rDNA,nodA,nifH genotype identification of the isolated and purified single colonies were performed. BOX-PCR was used for electrophoresis detection of all rhizobia. The strains with completely consistent bands were excluded and the bacteria with inconsistent bands were clustered and analyzed in Bionumerics ^@ 7.5 software. Then the representative strains were selected and inoculated into two soybean materials Jinda 88 and Kefeng 1 to screen strains with good matching. A total of 203 strains were isolated,from 187 strains of them the nodA and nifH were amplified. While 172 fast-growing strains belonged to Sinorhizobium fredii and slowly-growing 15 strains belonged to Bradyrhizobium diazoefficiens and Bradyrhizobium daqingense,respectively. The 85 rhizobia strains with inconsistent bands were divided into 4 groups by BOX-PCR,which were divided into 16 subgroups with 70% as the limit. Rhizobium GL11 and GL43 were selected in better match with Jinda 88. Compared with USDA110 inoculation,the shoot dry weight increased by 38.5% and 30.1%,respectively;the root dry weight increased by 22.2% and 27.8%,respectively;the fresh weight of nodules increased by 24.3% and 41.5%,respectively;the dry weight of nodules increased by 36.6% and 31.0%,respectively. Rhizobium TG37 was selected as a good match with Kefeng 1 and its plant height,shoot dry weight,dry root weight,fresh nodule weight,dry nodule weight and number of nodules increased by 8.1%,15.0%,28.6%,27.3%,44.3% and 60.6%,respectively,compared with USDA110 inoculation. Shanxi province has rich resources of rhizobia. S. fredii is the dominant species. In addition,two types of slow-growing rhizobia B. diazoefficiens and B. daqingense were isolated,which enriches the rhizobia resources. The screening of rhizobia with good symbiosis and matching lays a material and theoretical basis for soybean production practice.

Key words: rhizobia, separation and identification, BOX-PCR, matching

WANG Xiao-li, QIN Jie, WANG Min, WANG Li-xiang, DU Wei-jun. Isolation,Identification and Symbiotic Matching of Soybean Rhizobia from Shanxi Province[J]. Biotechnology Bulletin, 2022, 38(3): 59-68.

Figures/Tables 13

References 27

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Primer name	Primer sequence（5'-3'）	Literature source
nifH-F	AAAGGYGGWATCGGYAARTCCACCAC	[12]
nifH-R	TTGTTSGCSGCRTACATSGCCATCAT	[12]
nodA-F	TGCRGTGGAATNTRNNCTGGGAAA	[13]
nodA-R	GGNCCGTCRTCRAAWGTCATGTA	[13]
16S rDNA-F	AGAGTTTGATCCTGGCTCAG	[14]
16S rDNA-R	GGTTACCTTGTTACGACTT	[14]

Primer name	Primer sequence（5'-3'）	Literature source
nifH-F	AAAGGYGGWATCGGYAARTCCACCAC	[12]
nifH-R	TTGTTSGCSGCRTACATSGCCATCAT	[12]
nodA-F	TGCRGTGGAATNTRNNCTGGGAAA	[13]
nodA-R	GGNCCGTCRTCRAAWGTCATGTA	[13]
16S rDNA-F	AGAGTTTGATCCTGGCTCAG	[14]
16S rDNA-R	GGTTACCTTGTTACGACTT	[14]

Rhizobium	Jinda 88			Kefeng 1
Rhizobium	Plant height/cm	Shoot dry weight/g	Root dry weight/g	Plant height/cm	Shoot dry weight/g	Root dry weight/g
CK	26.65±4.78 c	0.23±0.07 c	0.19±0.06 abc	19.55±1.93 abcd	0.18±0.02 bcde	0.15±0.01 abc
USDA110	31.40±2.91 abc	0.26±0.04 bc	0.18±0.02 abc	19.83±0.45 abcd	0.20±0.02 abcd	0.14±0.00 bc
TG1	30.15±4.16 abc	0.28±0.05 abc	0.19±0.01 abc	21.80±2.17 a	0.20±0.03 abc	0.16±0.02 ab
TG14	33.02±3.04 abc	0.26±0.05 bc	0.20±0.02 abc	19.30±1.06 abcd	0.16±0.03 cde	0.12±0.02 c
TG20	32.10±5.05 abc	0.25±0.04 c	0.19±0.02 abc	16.40±1.67 d	0.15±0.02 de	0.12±0.01 c
TG37	32.73±3.83 abc	0.31±0.01 abc	0.23±0.03 a	21.43±2.35 ab	0.23±0.01 a	0.18±0.02 a
TG53	34.45±4.74 abc	0.28±0.04 abc	0.21±0.03 abc	20.37±1.29 abc	0.17±0.02 bcde	0.13±0.02 bc
TG79	28.68±1.55 bc	0.26±0.06 bc	0.19±0.04 abc	20.13±1.96 abc	0.18±0.04 bcde	0.14±0.03 bc
GL11	30.63±5.23 abc	0.36±0.02 a	0.22±0.02 ab	17.20±1.57 cd	0.19±0.01 abcde	0.14±0.00 bc
GL16	31.77±2.96 abc	0.25±0.03 c	0.16±0.00 bc	19.37±0.85 abcd	0.18±0.01 bcde	0.12±0.01 c
GL17	32.33±5.72 abc	0.31±0.07 abc	0.19±0.04 abc	18.23±2.41 bcd	0.17±0.01 bcde	0.12±0.02 c
GL19	33.03±1.76 abc	0.28±0.03 abc	0.22±0.01 ab	20.93±0.71 ab	0.18±0.02 bcde	0.13±0.01 bc
GL28	34.60±2.25 ab	0.30±0.02 abc	0.21±0.03 ab	19.67±1.11 abcd	0.20±0.02 abc	0.14±0.02 bc
GL40	28.87±8.18 bc	0.31±0.05 abc	0.15±0.07 c	16.97±2.30 cd	0.16±0.03 cde	0.13±0.01 c
GL43	33.67±4.53 abc	0.34±0.09 ab	0.23±0.06 a	19.47±2.39 abcd	0.21±0.01 ab	0.15±0.01 abc
GL67	35.53±4.24 ab	0.29±0.04 abc	0.21±0.01 ab	17.93±2.57 bcd	0.16±0.00 cde	0.14±0.02 bc
GL75	36.97±0.47 a	0.32±0.03 abc	0.21±0.03 ab	18.10±1.76 bcd	0.15±0.02 e	0.12±0.02 c
QX13	29.83±2.42 abc	0.26±0.06 bc	0.21±0.04 ab	20.43±2.15 abc	0.19±0.06 abcde	0.15±0.03 abc

Rhizobium	Jinda 88			Kefeng 1
Rhizobium	Plant height/cm	Shoot dry weight/g	Root dry weight/g	Plant height/cm	Shoot dry weight/g	Root dry weight/g
CK	26.65±4.78 c	0.23±0.07 c	0.19±0.06 abc	19.55±1.93 abcd	0.18±0.02 bcde	0.15±0.01 abc
USDA110	31.40±2.91 abc	0.26±0.04 bc	0.18±0.02 abc	19.83±0.45 abcd	0.20±0.02 abcd	0.14±0.00 bc
TG1	30.15±4.16 abc	0.28±0.05 abc	0.19±0.01 abc	21.80±2.17 a	0.20±0.03 abc	0.16±0.02 ab
TG14	33.02±3.04 abc	0.26±0.05 bc	0.20±0.02 abc	19.30±1.06 abcd	0.16±0.03 cde	0.12±0.02 c
TG20	32.10±5.05 abc	0.25±0.04 c	0.19±0.02 abc	16.40±1.67 d	0.15±0.02 de	0.12±0.01 c
TG37	32.73±3.83 abc	0.31±0.01 abc	0.23±0.03 a	21.43±2.35 ab	0.23±0.01 a	0.18±0.02 a
TG53	34.45±4.74 abc	0.28±0.04 abc	0.21±0.03 abc	20.37±1.29 abc	0.17±0.02 bcde	0.13±0.02 bc
TG79	28.68±1.55 bc	0.26±0.06 bc	0.19±0.04 abc	20.13±1.96 abc	0.18±0.04 bcde	0.14±0.03 bc
GL11	30.63±5.23 abc	0.36±0.02 a	0.22±0.02 ab	17.20±1.57 cd	0.19±0.01 abcde	0.14±0.00 bc
GL16	31.77±2.96 abc	0.25±0.03 c	0.16±0.00 bc	19.37±0.85 abcd	0.18±0.01 bcde	0.12±0.01 c
GL17	32.33±5.72 abc	0.31±0.07 abc	0.19±0.04 abc	18.23±2.41 bcd	0.17±0.01 bcde	0.12±0.02 c
GL19	33.03±1.76 abc	0.28±0.03 abc	0.22±0.01 ab	20.93±0.71 ab	0.18±0.02 bcde	0.13±0.01 bc
GL28	34.60±2.25 ab	0.30±0.02 abc	0.21±0.03 ab	19.67±1.11 abcd	0.20±0.02 abc	0.14±0.02 bc
GL40	28.87±8.18 bc	0.31±0.05 abc	0.15±0.07 c	16.97±2.30 cd	0.16±0.03 cde	0.13±0.01 c
GL43	33.67±4.53 abc	0.34±0.09 ab	0.23±0.06 a	19.47±2.39 abcd	0.21±0.01 ab	0.15±0.01 abc
GL67	35.53±4.24 ab	0.29±0.04 abc	0.21±0.01 ab	17.93±2.57 bcd	0.16±0.00 cde	0.14±0.02 bc
GL75	36.97±0.47 a	0.32±0.03 abc	0.21±0.03 ab	18.10±1.76 bcd	0.15±0.02 e	0.12±0.02 c
QX13	29.83±2.42 abc	0.26±0.06 bc	0.21±0.04 ab	20.43±2.15 abc	0.19±0.06 abcde	0.15±0.03 abc

Rhizobium	Jinda 88			Kefeng 1
Rhizobium	Fresh nodule weight/mg	Dry nodule weight/mg	Nodule number per plant	Fresh nodule weight/mg	Dry nodule weight/mg	Nodule number per plant
USDA110	88.15±15.17 ab	19.18±4.20 ab	45.83±16.10 a	46.73±11.90 abc	10.00±2.14 abc	22.00±8.54 abcd
TG1	116.07±34.50 a	30.02±8.30 a	43.67±12.50 a	53.73±34.00 abc	10.77±6.90 abc	26.67±26.80 abc
TG14	101.15±17.40 ab	23.15±5.20 ab	36.33±9.29 ab	68.70±4.25 abc	17.57±4.70 a	25.67±16.10 abc
TG20	87.73±14.20 ab	18.30±3.44 b	42.33±5.86 a	69.77±13.50 ab	16.33±4.70 ab	41.67±18.00 a
TG37	98.13±4.66 ab	22.43±2.10 ab	34.67±12.80 ab	59.47±27.90 abc	14.43±7.00 abc	35.33±23.60 abc
TG53	77.65±30.64 ab	19.97±5.30 ab	40.33±7.39 a	70.83±19.20 a	15.27±6.00 abc	28.67±6.11 abc
TG79	93.38±40.84 ab	22.40±9.42 ab	36.50±18.69 ab	39.80±20.08 bc	7.80±2.95 c	12.00±1.00 cd
GL11	109.53±57.90 ab	26.20±16.20 ab	37.33±20.80 ab	54.17±6.70 abc	13.03±1.90 abc	37.67±8.08 ab
GL16	32.77±14.45 cd	7.73±3.55 cde	28.67±6.51 abc	46.83±7.92 abc	9.70±2.54 abc	35.33±9.50 abc
GL17	84.97±20.66 ab	19.97±3.00 ab	46.33±5.86 a	42.40±12.88 abc	10.13±3.40 abc	24.00±12.12 abc
GL19	81.40±14.60 ab	17.50±2.01 bc	36.33±10.70 ab	41.37±3.35 abc	7.70±1.51 c	22.67±6.11 abcd
GL28	66.27±21.01 bc	15.13±5.20 bcd	17.33±1.53 bcd	44.40±29.99 abc	11.50±8.92 abc	17.67±6.81 bcd
GL40	95.55±39.39 ab	22.60±7.07 ab	47.50±24.75 a	63.20±9.21 abc	14.27±2.10 abc	33.67±13.00 abc
GL43	124.73±39.90 a	25.13±7.10 ab	40.67±9.29 a	53.80±9.36 abc	12.83±2.50 abc	21.67±9.87 abcd
GL67	94.57±11.71 ab	21.60±3.31 ab	41.33±6.11 a	47.47±6.53 abc	9.37±1.39 abc	19.00±2.65 abcd
GL75	106.27±2.25 ab	24.33±2.20 ab	43.33±3.06 a	59.73±11.60 abc	13.33±2.80 abc	25.67±13.30 abc
QX13	29.30±16.95 cd	6.13±3.11 de	14.67±9.61 cd	38.97±1.85 c	7.83±1.37 c	27.33±10.00 abc