Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (14): 2751-2760.doi: 10.3864/j.issn.0578-1752.2023.14.010

• SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT • Previous Articles     Next Articles

Rapid Identification for Bradyrhizobium japonicum 5873 by PCR and Its Evaluation in Application

MA MingChao(), JIANG Xin(), WANG PengHui, GUAN DaWei, LI Jun   

  1. Institute of Agricultural Resources and Agricultural Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2022-11-13 Accepted:2022-12-21 Online:2023-07-16 Published:2023-07-21
  • Contact: JIANG Xin

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Abstract:

【Objective】Bradyrhizobium japonicum is one of the important functional bacteria in microbial fertilizer products. As a commercialized strain, B. japonicum 5873 was wildly used in agricultural production and played important roles in symbiotic nitrogen fixation. Therefore, it is necessary to establish a rapid identification method at the strain level by screening and identifying specific primers for rhizobia, and is of great importance in the aspects of microbial fertilizer product quality inspection, strain identification and function evaluation.【Method】According to the whole genome sequencing of B. japonicum 5873 and other strain related sequences from NCBI, as well as the differential fragments of B. japonicum USDA 6T which is highly homologous to B. japonicum 5873 (the ANI value of the genome is greater than 99.95%), specific PCR primers were designed and obtained. After optimization of PCR reaction conditions and the detection of sensitivity and specificity, a rapid detection method for B. japonicum 5873 was established. Then, using pot experiment, B. japonicum 5873 was mixed with other rhizobium strains and inoculated in the soybean rhizosphere, and the above method was used to evaluate the competitive nodulation ability of B. japonicum 5873.【Result】A set of species-specific primers 4-4 and Q1 (4-4-F 5′-GATAAGGCCACGGGTGAACA-3′/4-4-R 5′-CACTCGATAAGCTCCGCTGT-3′ and Q1-F 5′-CCGGTCGTGACTGGAATGAT-3′/Q1-R 5′-TCGAGGCCTACAAGAACGTC -3′), were successfully designed to selectively amplify 355 and 218 bp amplicon from B. japonicum 5873. Well specificity was demonstrated by reference strains, from which only the targeted bands of B. japonicum 5873 were observed. Amplifications were performed in a 25 μL reaction mixture, which contained Premix TaqTM 12.5 μL, template DNA (15 ng of genomic DNA), primers 4-4 and Q1 (1.0 μL, respectively). The PCR cycling consisted of one cycle of 5 min at 95 ℃ and 30 cycles of 45 s at 94 ℃, 45 s at 61 ℃, and 60 s at 72 ℃. A final extension step was run for 10 min at 72 ℃. The sensitivity was 1 850 CFU/µL. In addition, the method mentioned above was successfully used to evaluate the competitive nodulation ability of B. japonicum 5873, which was consistent with the results of traditional BOX-PCR identification.【Conclusion】The established rapid detection method can directly use the bacterial solution or squeezed nodules as templates, which will eliminate laborious tasks, such as processes of nodule isolation, purification, culture, DNA extraction, sequencing and sequence comparison. It only takes a few hours to accurately detect B. japonicum 5873, which provides a reference for product quality detection and competitive nodulation ability evaluation of microbial fertilizers.

Key words: Bradyrhizobium japonicum, strain-specific primer pair, PCR amplification

Table 1

The strains used in this study"

种Species 菌株编号No. of strain
大豆根瘤菌
Bradyrhizobium japonicum		 USDA 6T, 5821, 5841, 5876, 5547, 5588, 5665, 5129, 4892, 5452, 5599, 5016, 5437, 4129, 4470, 4530, 4438, 5248, 4784, 5528, 5412, 5260, 4503, 5039, 4248, 5240, 5190, 5742, 4855, 4794, 4291, 4698, 5128, 4770, 4296, 4207, 4543
有效慢生根瘤菌Bradyrhizobium diazoefficiens USDA 110T, 5119
埃氏慢生根瘤菌Bradyrhizobium elkanii 5136, 5161, 5109
弗氏中华根瘤菌Sinorhizobium fredii USDA 205T, 4475
奥氏根瘤菌Rhizobium alamii 4370

Fig. 1

Comparison of sequence diversity of the two strains"

Fig. 2

Schematic diagram for the design of specific sequence primers"

Table 2

Design results of specific sequence primers for B. japonicum 5873 in primer-BLAST"

引物Primer 序列Sequence (5′-3′) 长度Length (bp) Tm GC (%)
4-4 Forward primer GATAAGGCCACGGGTGAACA 20 60.04 55.00
4-4 Reverse primer CACTCGATAAGCTCCGCTGT 20 59.90 55.00
Q1 Forward primer CCGGTCGTGACTGGAATGAT 20 59.82 55.00
Q1 Reverse primer TCGAGGCCTACAAGAACGTC 20 59.48 55.00

Fig. 3

PCR results of No.4-4 primer specificity (355 bp) A: M: 2-Log ladder Marker. 1: B. japonicum 5873; 2: B. japonicum 5841; 3: B. elkanii 5136; 4: B. diazoefficiens 5119; 5-17: B. japonicum 5821, 5129, 4892, 5452, 5599, 5016, 5437, 4129, 4470, 4530, 4438, 5547, 5248; 18: S. fredii 4475; 19: R. alamii 4370; 20-24: B. japonicum 5876, 4784, 5528, 5412, 5260. B: M: 2-Log ladder Marker. 1: B. japonicum 5873; 25: B. diazoefficiens USDA 110T; 26: B. japonicum 5665; 27: S. fredii USDA 205T; 28-33: B. japonicum 5588, 4503, 5039, 4248, 5240, 5190; 34-35: B. elkanii 5109, 5161; 36-45: B. japonicum 5742, 4855, 4794, 4291, 4698, 5128, 4770, 4296, 4207, 4543; 46: B. japonicum USDA 6T"

Fig. 4

PCR results of different primers"

Fig. 5

Amplification products of B. japonicum 5873 under different conditions and the optimized amplification procedure"

Table 3

The nodule occupancy of B. japonicum 5873"

复合接种
Co-inoculation		 占瘤率Nodule occupancy (%)
快速检测技术
Rapid identification		 BOX-PCR
B. diazoefficiens USDA 110T+
B. japonicum 5873		 78.3±1.2 76.9±2.2
B. japonicum 5136+B. japonicum 5873 73.7±1.6 76.0±1.7
B. japonicum 5665+B. japonicum 5873 84.2±0.9 82.1±2.5
B. japonicum 5841+B. japonicum 5873 87.0±1.6 85.5±2.6
B. japonicum 5821+B. japonicum 5873 95.2±1.9 92.9±0.9
B. japonicum 5119+B. japonicum 5873 87.5±1.3 85.8±1.9
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