Pseudomonas in Hippophae rhamnoides Rhizosphere Affecting Growth of Ipomoea aquatica
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摘要:目的
从青海野生中国沙棘根际土中筛选出具有多重功能的假单胞属菌株，为生物菌肥的研发创造条件。
方法利用筛选培养基对沙棘根际土中的微生物进行分离，利用平板划线法对菌株进行纯化。通过形态、生理生化及16S rDNA序列比对鉴定菌株，并测定菌株解有机磷、解无机磷、解钾、固氮和降解纤维素能力。以雍菜为试验材料，检测各假单胞菌属菌株促进雍菜种子萌发以及雍菜幼苗生长能力。
结果从中国沙棘根际土壤中分离出7株假单胞菌，培养3 d，7株假单胞菌溶解有机磷浑浊圈直径为4.28～13.71 mm，溶解无机磷透明圈直径为3.51～7.62 mm，解有机磷菌液中磷的质量浓度为5.15～25.41 μg·mL−1，解无磷菌液中磷的质量浓度为2.15～22.26 μg·mL−1，解钾黄色光圈直径为11.12～21.85 mm，解钾菌液中K+质量浓度为5.07～14.33 μg·mL−1，固氮透明圈直径（D）和菌落生长直径（d）的比值（D/d）为1.33～1.86，降解纤维素透明圈直径为4.61～10.22 mm。平板促生试验结果表明，假单胞菌可提高雍菜种子发芽率，并且可显著提高雍菜幼苗生长。其中菌株ZGSJ-3促生效果最好，其叶宽和茎长分别为3.69 mm和50.25 mm，较CK显著增加35.2%和41.2%。
结论经综合评价后得出，不同假单胞菌菌液处理下雍菜的生长状况均得到一定程度改善，发芽率得到显著提高，其中ZGSJ-3和ZGSJ-7效果较好。
Abstract:ObjectivePseudomonas sp. in the rhizosphere of Hippophae rhamnoides were isolated and studied for potential application as a biofertilizer.
MethodMicroorganisms in the rhizosphere soil of Hippophae rhamnoides sinensis subsp. in the wild in Qinghai Province were isolated by using selected media and purified by plate streaking. Candidate Pseudomonas strains were morphologically, physiologically, and biochemically identified as well as 16S rDNA sequenced. Abilities of the isolates to degrade organic and inorganic phosphorus, potassium, cellulose and/or to fix nitrogen were examined. Effects of spraying the bacterial culture broth of the individual isolates on the seed germination and seedling growth of Ipomoea aquatica Forssk were observed.
ResultOn different media of specific formulations, the diameters of the turbid circles born by the 7 isolated Pseudomonas strains cultured for 3 d ranged 4.28–13.71 mm with 5.15–25.41 μg·mL-1 of dissolved organic phosphorus, those of clear circles 3.51–7.62 mm with 2.15–22.26 μg·mL−1 of dissolved inorganic phosphorus, those of halos 11.12–21.85 mm with 5.07–14.33 μg·mL-1 of dissolved potassium, those of transparent circles 4.61–10.22 mm of cellulose-degradation, and the ratios of the nitrogen-fixing clear circle diameter (D) to the colony growth circle diameter (d) 1.33–1.86. The isolated Pseudomonas strains significantly improved the I. aquatica seed germination rate and seedling growth. Among them, ZGSJ-3 showed the largest increases of 35.2% on the 3.69 mm leaf width and of 41.2% on the 50.25 mm stem length over control.
ConclusionPresence of the Pseudomonas sp., especially ZGSJ-3 and ZGSJ-7, isolated in this study significantly improved the seed germination and seedling growth of I. aquatica.
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图 5 菌株固氮透明圈直径与菌落生长直径的比值
不同小写字母表示同一时期不同处理间差异显著（P＜0.05）。
Figure 5. Colony D/d ratios of isolates on media
Different lowercase letters indicate significant difference in different treatment on the same period (P＜0.05).
表 1 菌株生理生化特征鉴定结果
Table 1 Physio-biochemical characteristics of isolates
项目
Item菌株
Strain
项目
Item菌株
StrainZGSJ-1 ZGSJ-2 ZGSJ-3 ZGSJ-4 ZGSJ-5 ZGSJ-6 ZGSJ-7 ZGSJ-1 ZGSJ-2 ZGSJ-3 ZGSJ-4 ZGSJ-5 ZGSJ-6 ZGSJ-7 接触酶
Catalase+ + + + + + + 7%氯化钠
7%sodium
chloride+ − − + + + + 氧化酶
Oxidase− + − − + − − 木糖
Xylose+ − − + − − + 甲基红
Methyl
red− − − − − − − pH5.7 + + + + + + + 明胶液化
Gelatin liquefaction+ − − − − − + 水杨苷
Salicin− − − − − − − 淀粉水解
Starch
hydrolysis− − − − − − − 吲哚
Indole− − − − − − − 硝酸盐
还原
Nitrate
reduction+ − − − − − + L-精氨酸
双水解
L-arginine dihydrolyze− − − + − + − 葡萄糖
Glucose− + + − + + − 葡萄糖OF
Glucose
OF产碱
Alkali production产碱
Alkali production氧化
Oxidation发酵
Fermentation发酵
Fermentation氧化
Oxidation氧化
Oxidation
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表 2 菌株解磷能力的定性测定结果
Table 2 Phosphate-degrading ability of isolates
菌株
Strain1 d后的浑浊圈直径
Diameter of turbid circle
after 1 d/mm3 d后的浑浊圈直径
Diameter of turbid circle
after 3 d/mm1 d后的透明圈直径
Diameter of transparent ring
after 1 d/mm3 d后的透明圈直径
Diameter of transparent ring
after 3 d/mmZGSI-1 7.79±1.10b 13.71±0.49a 1.66±0.16c 4.27±1.06b ZGSI-2 2.91±2.91d 4.28±0.70c 0.82±0.51d 5.13±0.78b ZGSI-3 3.75±0.74cd 8.05±0.49b 0.63±0.35d 5.45±0.8ab ZGSI-4 7.56±0.98b 11.65±2.48a 3.62±0.96ab 7.62±0.78a ZGSI-5 4.92±0.52c 9.07±0.36b 4.30±0.11a 4.31±0.85b ZGSI-6 9.75±0.80a 12.05±0.67a 1.93±0.07c 4.82±2.75b ZGSI-7 4.12±0.00cd 7.95±0.29b 2.94±0.24b 3.51±0.01b 同列不同小写字母表示差异达显著水平（P＜0.05）。下同。
Data with different lowercase letters on same column indicate significant differences at P＜0.05. Same for below.
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表 3 菌株的解磷量
Table 3 Phosphorus-degrading capacity of isolates
菌株
Strain解有机磷量
Organophosphorus
hydrolysis
/(μg·mL−1)解无机磷量
Hydrolysis of
inorganic phosphorus
/(μg·mL−1)ZGSJ-1 8.08±0.41e 2.15±0.13f ZGSJ-2 7.41±0.39e 2.49±0.43f ZGSJ-3 18.77±0.14b 16.62±0.36b ZGSJ-4 17.79±0.47c 9.46±0.41d ZGSJ-5 25.41±0.77a 22.26±0.68a ZGSJ-6 5.15±0.14f 13.01±0.07c ZGSJ-7 9.76±0.19d 3.06±0.51e
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表 4 菌株解钾能力的定性测定结果
Table 4 Potassium-degrading ability of isolates
菌株
Strain1 d后的黄色光圈直径
Diameter of yellow
aperture after
1 d /mm3 d后的黄色光圈直径
Diameter of yellow
aperture after
3 d /mmZGSI-1 9.18±1.39c 13.70±1.94bc ZGSI-2 3.01±0.42e 11.12±1.85c ZGSI-3 21.66±0.40a 21.85±0.60a ZGSI-4 12.17±2.29b 16.98±0.19b ZGSI-5 5.99±1.73d 14.00±3.10bc ZGSI-6 8.25±1.34cd 17.61±3.58b ZGSI-7 3.48±0.67e 11.49±1.39c
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表 5 菌株对雍菜发芽率的影响
Table 5 Effect of isolates on I. aquatica seed germination rate
菌株 Strain 第3 天 The 3rd day/% 第5 天 The 5th day/% CK 53.3±5.77d 63.3±5.77d ZGSI-1 63.3±23.09c 66.7±11.55d ZGSI-2 80.0±20.00a 80.0±20.00ab ZGSI-3 80.0±10.37a 80.0±10.00b ZGSI-4 70.0±10.00b 90.0±0.00ab ZGSI-5 76.7±20.81ab 96.7±5.80a ZGSI-6 66.7±5.77bc 76.7±15.3bc ZGSI-7 73.3±11.54b 86.7±11.5a
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表 6 菌液处理对雍菜生长的影响
Table 6 Effect of bacterial broth treatment on I. aquatica growth
处理
Treatment茎长
Stem length/mm鲜重
Fresh weight/g叶长
Leaf length/mm叶宽
Blade width/mm主根长
Taproot length/mm须根数
Number of hairsCK 35.60±4.34c 0.21±0.04a 15.57±3.05bc 2.73±0.43c 13.47±3.03de 15.00±8.58d ZGSI-1 38.48±9.44bc 0.18±0.04b 15.79±2.94bc 2.48±0.47e 14.88±3.81d 24.83±7.08b ZGSI-2 48.39±5.52ab 0.20±0.03a 17.01±2.00b 3.08±0.63b 15.92±3.73c 25.00±7.32b ZGSI-3 50.25±7.15a 0.22±0.04a 19.88±3.05a 3.69±0.62a 18.23±3.15a 25.33±4.41ab ZGSI-4 39.23±6.49bc 0.21±0.04a 17.86±3.65ab 3.11±0.63b 14.30±2.76d 31.50±7.61a ZGSI-5 42.87±6.36b 0.17±0.04b 18.99±2.34a 3.67±0.79a 14.32±3.11d 23.17±10.19b ZGSI-6 44.92±3.07b 0.22±0.03a 14.60±3.51c 2.44±0.51de 17.05±4.90b 21.67±7.76c ZGSI-7 46.54±10.76ab 0.22±0.03a 20.23±2.25a 3.25±0.44b 18.36±7.48a 17.50±6.47d
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