酸性土壤上花生高效根瘤菌的分离及应用

doi:10.3864/j.issn.0578-1752.2019.19.010

摘要/Abstract

摘要：

【背景】 花生（Arachis hypogaea L.）是世界上重要的油料和经济作物。在我国南方产区,大部分花生种植地土壤为酸性。酸性土壤不仅pH值低、瘦瘠,而且还有低磷、铝毒等诸多障碍因素,严重限制了花生的生物固氮及产量。【目的】 本文旨在分离及应用适应酸性土壤的高效固氮根瘤菌,提高花生固氮效率及产量,改良酸性土壤。【方法】 利用平板划线结合镜检的方法,从田间采集的新鲜花生根瘤中分离纯化单菌落;通过PCR技术检测分离物中是否含有结瘤基因nodA和固氮基因nifH,进行根瘤菌的初步分子鉴定;再通过16S rRNA基因序列比对,对根瘤菌进行进一步分子鉴定。候选根瘤菌通过水培回接,检测其与花生的共生结瘤及固氮能力;再通过田间试验评价筛选出来的候选根瘤菌在酸性土壤上的应用效果。【结果】 本研究首先从不同酸性土壤种植区域的花生根瘤中分离、纯化得到256个分离物;其中10株含有nodA和nifH,初步确定为根瘤菌。经16S rRNA基因全长序列测定,发现8株为慢生型根瘤菌（Bradyrhizobium）,2株为根瘤菌属根瘤菌（Rhizobium）。水培回接试验发现,这10株根瘤菌均能够与花生共生、形成有效根瘤,证实是花生根瘤菌。在此基础上,选取4株固氮效率较高的根瘤菌,在酸性土壤上应用。结果表明,4株根瘤菌均能与花生在酸性土壤上形成根瘤,而未接种的花生根部不能形成根瘤。并且接种根瘤菌后显著改善了花生氮营养,提高了花生的生物量和产量。与不接种的对照相比,接种根瘤菌后,花生生物量、产量和氮含量分别提高了27.1%—38.0%、24.7%—104.2%和73.9%—151.3%。【结论】 本研究分离鉴定的花生根瘤菌能够高效固氮和适应酸性土壤,具有重要的应用前景。

关键词: 酸性土壤, 花生, 根瘤菌, 生物固氮

Abstract:

【Background】 Peanut (Arachis hypogaea L.) is an important oil and economic crop in the world. Most of the peanut cultivation soils in South China are acidic. Since low pH value, nutrition deficiencies and aluminium (Al) toxicity on acidic soils severely limit yield and biological nitrogen fixation (BNF) capacity in peanut. 【Objective】 The purpose of this paper is to isolate acidic soil adaptive rhizobium stains to improve BNF and yield of peanut, as well as to remediate acidic soils.【Method】 Firstly, strains were isolated and purified from fresh peanut nodules by collecting from the field by streak plate method combined with microscope observation, the nodulation gene nodA and nitrogen fixing gene nifH were detected by PCR to preliminary identification of potential Rhizobia strains, and then 16S rRNA sequencing was used for further taxonomy identification of the isolate strains. The symbiotic nodulation and nitrogen fixation ability of potential Rhizobium strains were validated by inoculation to the peanut under hydroponics conditions. Further, the candidate rhizobium strains in the field trial on acidic soils were evaluated. 【Result】 A total of 256 microbe strains were isolated and purified from nodules of peanuts grown in different sites on acidic soils. Based on the results of 16S rRNA sequencing analysis, ten of them contained nodA and nifH, and were initially identified as potential rhizobium. Of which, 8 strains belonged to Bradyrhizobium sp. and 2 belonged to Rhizobium sp. Hydroponic experiments further confirmed that the 10 strains could form functional nodules with peanuts. Subsequently, 4 strains with higher BNF capacity were selected for field evaluation on acidic soils. The results showed that all the 4 strains successfully formed functional nodules with peanuts in the field. On contrary, none of nodules could be formed in peanuts without rhizobium inoculation. Besides, Rhizobia inoculation significantly improved N nutrition, and increased biomass and yield of peanut. By comparison with CK plants, biomass, yield and N content of peanuts inoculated with rhizobium strains were increased by 27.1%-38.0%, 24.7%-104.2% and 73.9%-151.3%, respectively. 【Conclusion】 Taken together, the peanut Rhizobia isolated and identified in this study could fix N₂ effectively and adapted well to acidic soils, and therefore which had great application potentials.

Key words: acidic soils, peanut, rhizobia, biological nitrogen fixation

刘鹏,田颖哲,钟永嘉,廖红. 酸性土壤上花生高效根瘤菌的分离及应用[J]. 中国农业科学, 2019, 52(19): 3393-3403.

LIU Peng,TIAN YingZhe,ZHONG YongJia,LIAO Hong. Isolation and Application of Effective Rhizobium Strains in Peanut on Acidic Soils[J]. Scientia Agricultura Sinica, 2019, 52(19): 3393-3403.

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试验点 Site	pH	碱解氮Alkali-hydrolyzable nitrogen (mg·kg^-1)	速效磷 Available phosphorus (mg·kg^-1)	速效钾 Available potassium (mg·kg^-1)	有机质 Organic matter (g·kg^-1)
安溪 Anxi	5.10±0.16	128.98±30.33	5.05±0.16	50.61±9.38	13.2±1.1
福安 Fuan	4.22±0.17	86.93±12.50	105.49±32.37	106.33±14.78	19.6±6.0
洋中 Yangzhong	4.73±0.13	105.84±20.22	85.23±10.96	128.88±2.99	14.6±2.1
安溪茶园 Anxi tea garden	4.58±0.02	35.81±1.70	4.90±0.74	100.15±8.83	19.4±0.8

引物 Primer	引物序列 Primer sequence (5′-3′)	PCR反应体积 Reaction volume of PCR (μL)	产物大小 Product size (bp)	退火温度 Annealing temperature (℃)	参考文献 Reference
nifH-F	GTCATGTCYTCSAGYTCNTCCA	10	450	58	[17]
nifH-R	GCTTCCATGGTGATCGGGGT	10	450	58	[17]
nodA-F	TGCRGTGGARDCTRYGCTGGGAAA	10	750	56	[18]
nodA-R	GNCCGTCRTCRAASGTCARGTA	10	750	56	[18]
16S 27-F	AGAGTTTGATCMTGGCTCAG	25	1470	60	[19-20]
16S 1492-R	AGAGTTTGATCMTGGCTCAG	25	1470	60	[19-20]

根瘤菌 Strain of rhizobia	根瘤数 Nodule number (NO./plant)	生物量 Biomass (g/plant)	SPAD值 SPAD value	含氮量 N content (mg/plant)
CK	0	0.62±0.06	18.83±3.88	8.45±0.74
AXLQ1-6	23±6	0.67±0.18	26.53±1.96**	51.35±1.37***
AXLQ1-7	39±8	0.65±0.24	25.93±2.89*	14.11±0.53*
YZHO5-5	18±3	0.59±0.00	24.07±1.43*	25.99±2.74**
YZMO10-8	21±6	0.63±0.17	27.80±2.26**	12.10±1.90*
FAMB12-6	11±1	0.60±0.30	22.73±1.95*	17.35±1.22**
YZMB13-3	18±4	0.67±0.05	25.43±5.06*	25.06±6.43**
YZHO13-7	19±7	0.54±0.13	20.87±3.55	20.33±3.33**
FAMB13-9	28±6	0.51±0.11	19.60±4.44	19.89±5.30**
YZLH14-3	16±8	0.61±0.15	24.53±4.65	28.16±3.24**
YZMO14-4	22±5	0.69±0.19	23.60±4.16	13.40±0.98*

根瘤菌 Species of rhizobia	根瘤数 Nodule number (NO./plant)	生物量 Biomass (g/plant)	SPAD值 SPAD value	植株含氮量 Nitrogen content (g/plant)	产量 Yield (g/plant)	单株荚数 Pod number (NO./plant)
CK	0b	24.12±1.40b	37.97±2.71b	0.27±0.03c	19.02±1.48d	15.14±0.81c
AXLQ1-6	98.20±47.66a	32.23±1.03a	47.10±3.78a	0.47±0.03b	31.50±1.99b	22.50±1.00b
FAMB12-6	146.80±40.39a	33.21±2.61a	46.38±1.23a	0.58±0.08b	38.84±3.57a	28.71±1.91a
YZMB13-3	194.50±35.40a	33.28±2.10a	48.88±1.56a	0.70±0.07a	29.41±0.84b	26.38±1.11a
YZMO14-4	68.70±17.57a	30.66±1.72a	46.24±2.41a	0.48±0.04b	25.25±1.23c	27.25±1.01a