Fluorescent Quantitative PCR of nifH Gene and Diversity Analysis of Nitrogen-fixing Bacteria in the Rhizosphere Soil of Caragana spp. of Desert Grassland

doi:10.13560/j.cnki.biotech.bull.1985.2022-0211

Abstract

Abstract:

Exploring the abundance information of nifH gene and the composition and diversity of nitrogen-fixing bacterial community in the rhizosphere soil of the desert sand-fixing plant may provide basic information for enriching the microbial resource pool in desert areas. Real-time quantitative PCR and high-throughput sequencing were applied to analyze the abundance of nifH gene and the community composition of nitrogen-fixing bacteria in the rhizosphere soil of 5 shrublands of Caragana spp. growing in the desert grassland of Ningxia. The method of isolation and culture were to screen nitrogen-fixing bacteria in the rhizosphere soil of Caragana spp. and to study their diversities. The results showed that the copy number of nifH gene in the 5 rhizosphere soil samples were significantly different, the highest in Luoshan（LS）and the lowest in Shapotou（SPT）. A total of 7 phyla, 12 classes, 26 families and 30 genera of nitrogen-fixing bacteria were detected in the rhizosphere soil of Caragana spp. At the family and genus level, the community composition of nitrogen-fixing bacteria in Shapotou sample was quite different. Unclassified Proteobacteria and Phyllobacteriaceae accounted for 25.54% and 22.77%, respectively, Rhodospirillaceae accounted for 17.43%, and Mesorhizobium were dominant genus, with a relative abundance of 22.77%; the other four soil samples were dominated by Rhodospirillaceae and Skermanella, with relative abundances of 23.71%-73.45% and 23.19%-71.14%, respectively, and it accounted for the highest proportion in Luoshan sample. The isolated nitrogen-fixing bacteria belong to 15 genera, in addition to the common nitrogen-fixing groups such as Pseudomonas, Rhizobium and Enterobacter, also including Leclercia, Chryseobacterium, Stenotrophomonas, etc., the diversity is abundant.

Key words: Caragana spp., nifH gene, nitrogen-fixing bacteria community, diversity

LIU Shuang, YAO Jia-ni, SHEN Cong, DAI Jin-xia. Fluorescent Quantitative PCR of nifH Gene and Diversity Analysis of Nitrogen-fixing Bacteria in the Rhizosphere Soil of Caragana spp. of Desert Grassland[J]. Biotechnology Bulletin, 2022, 38(12): 252-262.

Figures/Tables 9

References 37

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样地Sampling site	Ct值Ct value	拷贝数Number of copies/（copies·μL^-1 DNA）	称重Weight/g	拷贝数Number of copies/（copies·g^-1 ）
LS	22.76	3.7298E+03	0.27	1.5922E+06a
HSP	25.67	6.3276E+02	0.38	1.7188E+05b
SPT	25.36	7.4268E+02	0.48	1.5550E+05b
ZQ	25.41	7.1586E+02	0.28	2.6036E+05b
TSH	24.47	1.4555E+03	0.30	5.2318E+05b

样地Sampling site	Ct值Ct value	拷贝数Number of copies/（copies·μL^-1 DNA）	称重Weight/g	拷贝数Number of copies/（copies·g^-1 ）
LS	22.76	3.7298E+03	0.27	1.5922E+06a
HSP	25.67	6.3276E+02	0.38	1.7188E+05b
SPT	25.36	7.4268E+02	0.48	1.5550E+05b
ZQ	25.41	7.1586E+02	0.28	2.6036E+05b
TSH	24.47	1.4555E+03	0.30	5.2318E+05b

样地 Sampling site	有效序列数No. of valid sequences	操作分类单元数目 No. of OTU	覆盖度Coverage	香农指数 Shannon index	辛普森指数 Simpson index	Ace 指数 Ace index	Sobs指数 Sobs index	Chao1指数 Chao1 index
LS	32 736	546	0.98864	3.2507b	0.076655a	546.53b	320.00b	462.98b
SPT	56 020	2 320	0.98389	4.9285a	0.031739b	1 459.70a	1 244.70a	1 415.70a
ZQ	62 271	2 703	0.98551	4.6876a	0.047986ab	1 468.50a	1 266.70a	1 454.50a
TSH	52 969	1 546	0.98491	4.0688ab	0.040206b	981.44ab	710.67ab	942.75ab
HSP	60 767	2 462	0.98696	4.3394a	0.049855ab	1 281.00a	1 077.70a	1 249.90a

样地 Sampling site	有效序列数No. of valid sequences	操作分类单元数目 No. of OTU	覆盖度Coverage	香农指数 Shannon index	辛普森指数 Simpson index	Ace 指数 Ace index	Sobs指数 Sobs index	Chao1指数 Chao1 index
LS	32 736	546	0.98864	3.2507b	0.076655a	546.53b	320.00b	462.98b
SPT	56 020	2 320	0.98389	4.9285a	0.031739b	1 459.70a	1 244.70a	1 415.70a
ZQ	62 271	2 703	0.98551	4.6876a	0.047986ab	1 468.50a	1 266.70a	1 454.50a
TSH	52 969	1 546	0.98491	4.0688ab	0.040206b	981.44ab	710.67ab	942.75ab
HSP	60 767	2 462	0.98696	4.3394a	0.049855ab	1 281.00a	1 077.70a	1 249.90a

菌株编号 Strain No.	革兰氏染色 Gram stain	H₂O₂酶实验 Catalase test	淀粉水解 Starch hydrolysis	V.P实验 V.P test	甲基红实验 Methyl red test	吲哚实验 Indole test	硝酸盐还原 Nitrate reduction
LG-H2	-	+	-	-	-	-	-
LG-H3	+	-	-	-	-	-	-
LG-H4	+	+	-	-	+	+	+
LG-H5	+	+	-	-	+	+	+
LG-L3	-	+	-	+	-	-	-
LG-T2	-	+	-	-	-	-	+
LG-T3	-	+	-	-	-	-	+
LG-T6	-	+	-	-	+	-	+
LG-Z1	-	+	-	-	-	-	+
LG-Z3	-	+	-	-	-	-	-
LG-Z5	-	+	-	+	-	-	+
LG-Z7	+	+	+	-	-	-	+
GY-T1	-	+	-	-	-	-	+
GY-T4	-	+	-	-	-	-	+
GY-T5	+	+	-	-	+	-	+
GY-L1	-	+	-	-	-	-	+
GY-L5	+	+	-	+	-	-	+
GY-H3	-	+	-	+	-	-	-
GY-H8	+	+	-	-	+	+	-
GY-Z2	-	+	-	-	-	-	+
WD-L1	-	-	-	-	-	-	-
WD-L2	-	+	-	-	-	-	-
WD-Z1	-	-	-	-	-	-	-
WD-T3	-	+	-	-	-	+	-
WD-H1	-	-	-	-	-	-	-
YMA-T2	-	+	-	-	-	-	+