Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (19): 3393-3403.doi: 10.3864/j.issn.0578-1752.2019.19.010

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

Isolation and Application of Effective Rhizobium Strains in Peanut on Acidic Soils

LIU Peng,TIAN YingZhe,ZHONG YongJia,LIAO Hong()   

  1. Root Biology Center, College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002
  • Received:2019-03-25 Accepted:2019-06-04 Online:2019-10-01 Published:2019-10-11
  • Contact: Hong LIAO E-mail:hliao@fafu.edu.cn

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

Table 1

Basic physiochemical properties of soils from different sampling sites"

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

Table 2

PCR primers used for rhizobia identification"

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
nodA-F TGCRGTGGARDCTRYGCTGGGAAA 10 750 56 [18]
nodA-R GNCCGTCRTCRAASGTCARGTA
16S 27-F AGAGTTTGATCMTGGCTCAG 25 1470 60 [19-20]
16S 1492-R AGAGTTTGATCMTGGCTCAG

Fig. 1

Gel electrophoresis of nodA (A) and nifH (B) genes from PCR products of the bacterial strains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 represent AXLQ1-6, AXLQ1-7, YZHO5-5, FAMB12-6, YZMB13-3, YZHO13-7, FAMB13-9, YZLH14-3, YZMO10-8, YZMO14-4, CK"

Table 3

Effects of inoculation with different Rhizobium stains on peanut nodulation and growth in hydroponic experiments"

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*

Fig. 3

Comparative analysis on nitrogenase activity of different rhizobia in the hydroponic experiment The data in the figure were the mean ± standard error (n=3). ***: Significant at P<0.001 level through One-way ANOVA. Different letters indicated significantly different among different rhizobium strain at 0.05 level"

Table 4

Effects of inoculation with different rhizobium stains on peanut nodulation and growth in field trails"

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

Fig. 4

Comparative analysis on nitrogenase activity of different rhizobia in the field trials Each bar was the mean ± standard error (n=10). ***: significant at P<0.001 level through One-way ANOVA. Different letters indicated significantly different among different rhizobium strain at 0.05 level"

Fig. 5

Effects of inoculation with YZMB13-3 rhizobium stains on peanut growth on acidic soils"

Fig. 2

Phylogenetic tree of the rhizobium strains ①, ② and ③ represent Bradyrhizobium, Rhizobium and Staphylococcus, respectively. The number in the bracket was the 16S rRNA accession number of each reference stain in GenBank"

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