Isolation and identification of phosphate solubilizing bacteria from rice rhizosphere soil, and promoting effect on rice plant growth
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摘要:目的
为明确水稻根际溶磷菌株溶磷能力及其对水稻植株及根际土壤磷含量的影响。
方法利用溶磷圈法从水稻根际土壤中分离获得具有较强溶解Ca3(PO4)2能力的2株细菌NDW1和NDW3，根据16S rDNA对菌株进行鉴定。以NBRIP为基础培养基，通过正交试验对2株菌株利用氮源、碳源及初始pH进行优化，鉴定菌株的吲哚乙酸（IAA）分泌量，研究溶磷菌对水稻的促生作用，以及对土壤速效磷和水稻幼苗全磷含量的影响。
结果菌株NDW1和菌株NDW3分别被鉴定为Enterobacter sp.和Serratia sp.，2株菌株溶磷的最佳条件组合均为葡萄糖、蛋白胨及初始pH 6。2株菌株24 h内最高溶磷量分别为294.95和312.93 μg·mL-1，且都可分泌IAA。土培和沙培条件下，溶磷菌NDW3对水稻株高、根长、最大叶长及地上干质量都有明显的促进作用，并且NDW3在2种种植条件下均显著增加了根际土壤速效磷及水稻植株全磷含量。
结论从水稻根际土壤分离获得2株溶磷能力较好的细菌菌株Enterobacter sp. NDW1和Serratia sp. NDW3，菌株NDW3对水稻的促生作用强于菌株NDW1。
Abstract:ObjectiveTo determine the ability of phosphate dissolving of phosphate solubilizing bacteria(PSB)in rice rhizosphere, and study the effect of PSB on soluble phosphorus contents in rice plants and rhizosphere soil.
MethodTwo bacteria strains NDW1 and NDW3 with strong ability of dissolving Ca3(PO4)2 were isolated from rice rhizosphere soil based on the halo zone formation on media due to phosphate solubilization, and identified by 16S rDNA method. Phosphorus dissolving conditions of two strains were optimized by orthogonal test with NBRIP as the basic medium. The production of indole-3-acetic acid(IAA) were determined. The effects of NDW1 and NDW3 on promotion of rice growth, the soil available phosphorus contents and plant total phosphorus contents were studied.
ResultNDW1 and NDW3 were identified as Enterobacter sp. and Serratia sp.. The best combination of carbon, nitrogen source and pH for phosphate solubilition by NDW1 and NDW3 were both the combination of glucose, peptone and pH 6. The highest phosphorus contents dissolved by NDW1 and NDW3 were 294.95 and 312.93 μg·mL-1 after 24 h incubation, respectively. Both PSB could produce IAA. NDW3 significantly increased the shoot height, root length, maximum leaf length and above-ground dry mass of rice plant in both soil culture and sand culture. NDW3 also significantly increased the contents of soil available phosphorus and rice total phosphorus under both culture conditions.
ConclusionEnterobacter sp. NDW1 and Serratia sp. NDW3 isolated from rice rhizosphere soil have strong ability of phosphate dissolving. The growth promoting effect of NDW3 for rice plant is better than that of NDW1.
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Keywords:
- soil phosphate solubilizing bacteria /
- IAA /
- growth promoting effect /
- phosphorus content /
- rice /
- rhizosphere
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图 1 菌株NDW1和NDW3溶磷效果
Figure 1. Phosphate solubilizing effects of bacteria NDW1 and NDW3
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图 2 基于菌株NDW1、NDW3和其他溶磷菌16S rDNA序列构建的系统发育树
Figure 2. Phylogeny tree of 16S rDNA sequences from NDW1, NDW3 and other phosphoate solubilizing strains
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图 3 不同碳源、氮源对菌株溶磷量和生长的影响
柱状图为菌株溶磷量，折线图为菌液光密度。
Figure 3. Effect of different carbon sources and nitrogen sources on phosphorus contents dissolved by bacteria strains and bacterial growth
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图 4 不同pH对菌株溶磷量和生长的影响
柱状图为菌株溶磷量，折线图为菌液光密度。
Figure 4. Effect of different pH values on phosporus contents dissolved by bacteria strains and bacterial growth
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图 5 不同处理对水稻根际土壤速效磷含量及幼苗全磷含量的影响
沙培条件下，CK1：加全磷营养液，CK2：加Ca3(PO4)2和无磷营养液；土培条件下，CK1:不加Ca3(PO4)2，CK2：加Ca3(PO4)2; 2种种植条件下，NDW1和NDW3处理分别在CK2处理的基础上接种相应菌株。相同种植条件不同柱子上相同小写字母表示不同处理间差异不显著(P>0.05, Duncan’s法)。
Figure 5. Effects of different treatments on soil available phosphorus contents and total phosphorus contents of rice seedlings
表 1 菌株溶磷的正交试验结果1）
Table 1 Results of the orthogonal test of phosphate solubilizing by bacteria strains
序号 NDW1 序号 NDW3 碳源 氮源 pH 溶磷量/(μg·mL-1) 碳源 氮源 pH 溶磷量/(μg·mL-1) 1 1 1 1 281.60 1 1 1 1 278.12 2 1 2 2 294.95 2 1 2 2 312.93 3 1 3 3 207.35 3 1 3 3 245.02 4 2 1 2 2.07 4 2 1 2 5.40 5 2 2 3 4.55 5 2 2 3 4.55 6 2 3 1 39.34 6 2 3 1 37.60 7 3 1 3 7.76 7 3 1 3 6.94 8 3 2 1 5.34 8 3 2 1 19.05 9 3 3 2 32.50 9 3 3 2 30.50 k1 261.30 97.14 108.76 k1 278.69 96.82 111.59 k2 15.32 101.61 109.84 k2 15.85 112.18 116.28 k3 15.20 93.06 73.22 k3 18.83 104.37 85.50 R 246.10 8.55 36.62 R 262.84 15.36 30.77 最优组合 1 2 2 最优组合 1 2 2 主次顺序 碳源>pH >氮源 主次顺序 碳源>pH >氮源 1）碳源1、2、3分别表示葡萄糖、麦芽糖、蔗糖；氮源1、2、3分别表示牛肉膏、蛋白胨、硝酸铵；pH 1、2、3分别表示pH为5、6、7。 
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表 2 溶磷菌产IAA的测定
Table 2 Measurements of IAA production of two phosphorus solubilizing strains
King培养液 菌株 ρ定量（IAA）/(μg·mL-1) 定性 加色氨酸 NDW1 11.37± 0.36 粉色 NDW3 15.62± 0.83 粉色 不加色氨酸 NDW1 5.41± 0.71 浅粉色 NDW3 5.60± 0.24 浅粉色
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表 3 不同处理对水稻幼苗性状的影响1）
Table 3 Effects of different treatments on traits of rice seedlings
种植条件 处理 株高/cm 根长/cm 最大叶长/cm 地上干质量/mg 地下干质量/mg w(叶绿素)/(μg·g-1) 沙培 CK1 31.67±4.58b 9.50±0.10b 21.57±0.72b 29.00±7.21ab 8.33±0.58b 81.64±7.43b CK2 32.03±0.80b 9.46±0.23ab 19.27±0.83c 27.67±4.04b 8.67±1.53ab 79.27±0.17ab NDW1 34.20±1.91ab 9.73±0.11ab 20.93±1.00b 30.67±3.79ab 9.67±1.15ab 88.90±9.53ab NDW3 37.53±1.00a 9.83±0.25a 23.47±0.58a 39.00±5.57a 11.00±2.00a 103.57±9.37a 土培 CK1 31.20±1.11b 9.73±0.31b 19.73±0.23c 44.67±4.04b 17.67±5.86b 37.82±2.36b CK2 31.80±0.87b 9.83±0.25b 21.40±1.60bc 47.67±2.08b 18.33±3.51ab 54.82±0.39a NDW1 32.93±0.42ab 10.03±0.21ab 22.47±0.31ab 49.33±2.08b 26.67±5.50ab 56.67±0.18a NDW3 34.87±1.70a 10.37±0.20a 23.93±1.92a 58.00±5.29a 18.00±3.00a 59.72±2.81a 1) 沙培条件下，CK1：加全磷营养液，CK2：加Ca3(PO4)2和无磷营养液；土培条件下，CK1:不加Ca3(PO4)2，CK2：加Ca3(PO4)2; 2种种植条件下，NDW1和NDW3处理分别在CK2处理的基础上接种相应菌株。相同种植条件同列数据后相同小写字母表示不同处理间差异不显著(P>0.05, Duncan’s法)。
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