1株白三叶根际产铁载体菌的功能特性及培养条件
为充分利用贵州岩溶山区牧草根际促生菌资源，以野生白三叶(Trifolium repens)根际铁载体产生菌TSQA26-3为研究对象，结合形态观察、VITEK-2全自动微生物鉴定系统和16S rRNA基因序列分析对菌株进行分类鉴定，开展菌株促生特性研究，筛选适宜菌株合成铁载体的发酵基础培养基，通过单因素试验和正交试验相结合，确定最佳培养基成分和培养条件，促进菌株合成铁载体能力的提升。结果表明，菌株TSQA26-3为恶臭假单胞菌(Pseudomonas putida)；菌株同时还具有溶解无机磷、产植物生长素(IAA)和固氮能力；优化后的培养基(MG培养基)配方为，甘露醇25.0 g·L−1，L-谷氨酰胺2.5 g·L−1，K2HPO4 1.0 g·L−1，MgSO4·7H2O 0.25 g·L−1；最佳培养条件为，培养基初始pH 7.0，摇床转速170 r·min−1，培养温度30 ℃，培养基装液量为每250 mL瓶中装70 mL。
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参考文献
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图 1 可溶性指数和噬铁圈计算方法
A：晕圈直径；B：菌落直径；可溶性指数 = A/B；噬铁圈 = (A − B)/2。
Figure 1. The calculation method for soluble index and zone radius
A: halo diameter; B: colony diameter; Soluble index = A/B; Zone radius = (A − B)/2.
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图 2 TSQA26-3菌落形态及革兰氏染色结果
A为菌株TSQA26-3在CAS培养基上的形态；B为菌株在NB固体培养基上的形态；C为革兰氏染色镜检结果。
Figure 2. The morphology and Gram staining result of TSQA26-3
A is the morphology of the strain TSQA26-3 on CAS medium; B is the morphology of the strain TSQA26-3 on NB medium; C is the result of Gram-stain microscopy.
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图 3 菌株基于16S rRNA基因序列的系统发育树
Figure 3. Phylogenetic tree of the strain TSQA26-3 based on its 16S rRNA gene sequences
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图 4 不同培养基对菌株铁载体合成的影响
不同小写字母表示不同处理之间差异显著(P < 0.05)；下同。
Figure 4. Effects of different media on the siderophores produced by TSQA26-3
Different lowercase letters indicate significant differences between treatments at the 0.05 level. This is applicable for the following figures and table as well.
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图 5 不同碳源对菌株铁载体合成的影响
Glu、Suc、Mal、Man、Amb、Sol分别代表葡萄糖、蔗糖、麦芽糖、甘露醇、琥珀酸和可溶性淀粉。
Figure 5. Effects of different carbon sources on the siderophores produced by TSQA26-3
Glu, Suc, Mal, Man, Amb, Sol indicate glucose, sucrose, maltose, mannitol, amber acid and soluble starch, respectively.
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图 6 不同氮源对菌株铁载体合成的影响
Asp、Pro、Arg、Glu、Gly、Amm分别表示L-天冬酰胺、L-脯氨酸、L-精氨酸、L-谷氨酰胺、甘氨酸和硫酸铵。
Figure 6. Effects of different nitrogen sources on the siderophores produced by TSQA26-3
Asp, Pro, Arg, Glu, Gly, Amm indicate L-Asparagine, L-Proline, L-Arginine, L-Glutamine, Glycine and (NH4)2SO4.
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图 7 不同培养条件对菌株合成铁载体的影响
Figure 7. Effects of different culture conditions on the siderophores produced by TSQA26-3
表 1 培养基成分正交试验设计
Table 1 The orthogonal test of factors of the medium
水平
Level因素 Factor 碳源
Carbon
source/(g·L−1)氮源
Nitrogen
source/(g·L−1)K2HPO4/
(g·L−1)MgSO4·7H2O/
(g·L−1)1 15.0 1.5 0.5 0.25 2 20.0 2.0 1.0 0.50 3 25.0 2.5 1.5 0.75 
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表 2 分离菌株产铁载体能力分析
Table 2 Analysis of siderophores produced by the isolated strains
序号
No.土壤编号
Soil No.菌株编号
Strains No.可溶性指数
Soluble index噬铁圈
Zone radius/mm1 TSQA26 TSQA26-3 5.14 ± 0.08aA 8.42 ± 0.22aA 2 TSTS36 TSTS36-3 5.07 ± 0.09aA 6.53 ± 0.46bB 3 TSTP39 TSTP39-1 4.96 ± 0.06aA 6.31 ± 0.31bB 同列不同大写字母和小写字母分别表示差异极显著(P < 0.01)和差异显著(P < 0.05)。
The capital letters indicate significant differences at the 0.01 level, and the lowercase letters indicate significant differences at the 0.05 level.
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表 3 TSQA26-3促生特性检测
Table 3 Detection of the plant-promoting ability of TSQA26-3
促生特性检测 Plant-promoting ability 定量分析 Quantitative analysis 指标
Parameter活性
Activity指标
Parameter值
Value溶解无机磷能力
Phosphate solubilization (inorganic)+ 无机磷溶磷量
Inorganic phosphorus solubilizing amount/(mg·L−1)30.31 ± 5.29 溶解有机磷能力
Phosphate solubilization (organic)− 有机磷溶磷量
Organic phosphorus solubilizing amount/(mg·L−1)0 固氮能力 Nitrogen fixation + 固氮酶活性 Nitrogenase activity/(IU·L−1) 88.24 ± 3.04 产IAA IAA production + 产IAA能力 Capacity of IAA secretion/(mg·L−1) 9.71 ± 0.31 解钾能力 Potassium decomposition − 解钾能力 Soluble potassium content/(mg·L−1) 0
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表 4 培养基中各营养成分正交试验结果
Table 4 Results of the orthogonal test with different nutritional facts in the medium
序号
No.甘露醇
MannitolL-谷氨酰胺
L-GlutamineK2HPO4 MgSO4·7H2O su/% 1 1 1 1 1 69.19 ± 3.47b 2 1 2 2 2 69.85 ± 1.58b 3 1 3 3 3 76.56 ± 1.11b 4 2 1 2 3 46.79 ± 5.07d 5 2 2 3 1 88.79 ± 0.67a 6 2 3 1 2 57.75 ± 4.31c 7 3 1 3 2 68.88 ± 4.48b 8 3 2 1 3 45.07 ± 4.98d 9 3 3 2 1 89.44 ± 0.53a K1 71.87 61.62 57.34 82.47 K2 64.44 67.90 68.69 65.49 K3 67.80 74.58 78.07 56.14 R 7.43 12.96 20.74 26.33 碳氮源，K2HPO4和MgSO4·7H2O下的1，2，3与表1相同。R为极差。
1, 2, and 3 in the carbon, nitrogen sources, K2HPO4, and MgSO4·7H2O column are the same as in Table 1. R is a range.
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