野生中国沙棘根际假单胞属菌株的筛选鉴定及其对空心菜促生效果的影响

高佩; 徐淑琴; 贺曦; 三鸿源; 马玉花; 冶贵生

野生中国沙棘根际假单胞属菌株的筛选鉴定及其对空心菜促生效果的影响

高佩^{1, 2,},
徐淑琴²,
贺曦²,
三鸿源²,
马玉花^2, ,,
冶贵生²

1.
青海大学畜牧兽医科学院，青海　西宁　810016
2.
青海大学农牧学院，青海　西宁　810016

基金项目: 青海省“高端创新人才千人计划”项目（2022）

详细信息

作者简介:
高佩（1996 —），女，博士生，主要从事植物资源开发与利用研究，E-mail：1639245486@qq.com

通讯作者:
马玉花（1978 —），女，博士，教授，硕士生导师，主要从事森林培育理论与技术、植物资源开发利用研究，E-mail:qhxnmy@163.com

中图分类号: S759;S714.6
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出版历程
- 收稿日期: 2024-01-15
- 修回日期: 2024-05-17
- 网络出版日期: 2024-08-15

Pseudomonas in Hippophae rhamnoides Rhizosphere Affecting Growth of Ipomoea aquatica

GAO Pei^{1, 2
,},
XU Shuqin²,
HE Xi²,
SAN Hongyuan²,
MA Yuhua^{2
, ,},
YE Guisheng²

1.
College of Animal Science and Veterinary Science, Qinghai University, Xining, Qinghai　810016, China
2.
College of Agriculture and Animal Husbandry, Qinghai University, Xining, Qinghai　810016, China

摘要

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。以空心菜为试验材料，检测各假单胞菌属菌株促进空心菜种子萌发以及空心菜幼苗生长能力。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，假单胞菌可提高空心菜种子发芽率，并且可显著提高空心菜幼苗生长。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显著增加了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空心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。
- 中国沙棘 /
- 空心菜 /
- 假单胞菌 /
- 根际促生菌 /
- 促生能力
Abstract: Objective Pseudomonas sp. in the rhizosphere of Hippophae rhamnoides were isolated and studied for potential application as a biofertilizer. Method Microorganisms 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. Result On different media of specific formulations, the diameters of the turbid circles born by the 7 isolated Pseudomonas strains cultured for 3d ranged 4.28-13.71mm with 5.15-25.41 μg·mL^-1 of dissolved organic phosphorus, those of clear circles 3.51-7.62mm with 2.15-22.26 μg·mL⁻¹ of dissolved inorganic phosphorus, those of halos 11.12-21.85mm 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. Conclusion Presence 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.
- Hippophae rhamnoides sinensis subsp. /
- Ipomoea aquatica Forssk. /
- Pseudomonas sp. /
- rhizosphere growth-promoting bacteria /
- growth-promoting capacity

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图 1 菌落形态特征图

Figure 1. Colony morphology

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图 2 普通光学显微镜镜检结果（显微镜倍数，×100）

Figure 2. Optical microscope images of bacterial colonies, ×100

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图 3 菌株系统发育树

不同小写字母表示不同处理间差异显著（P＜0.05）。

Figure 3. Phylogenetic tree of isolates

Different lowercase letters indicate significant difference in different treatment (P＜0.05).

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图 4 不同菌株菌液中K⁺含量

Figure 4. K⁺ content in broths of isolates

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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).

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图 6 菌株降解纤维素功能测定结果

Figure 6. Cellulose-degradation by isolates

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表 1 菌株生理生化特征鉴定结果

Table 1. Physio-biochemical characteristics of isolates

试验项目 Pilot project	菌株 Strain							试验项目 Pilot project	菌株 Strain
试验项目 Pilot project	ZGSJ-1	ZGSJ-2	ZGSJ-3	ZGSJ-4	ZGSJ-5	ZGSJ-6	ZGSJ-7	试验项目 Pilot project	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 redMethyl 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 OFGlucose 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 Strain	1 d后的浑浊圈直径 Diameter of turbid circle after 1 d/mm	3 d后的浑浊圈直径 Diameter of turbid circle after 3 d/mm	1 d后的透明圈直径 Diameter of transparent ring after 1 d/mm	3 d后的透明圈直径 Diameter of transparent ring after 3 d/mm
ZGSI-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表达显著水平（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 Strain	1 d后的黄色光圈直径 Diameter of yellow aperture after 1 d /mm	3 d后的黄色光圈直径 Diameter of yellow aperture after 3 d /mm
ZGSI-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 菌株对空心菜发芽率的影响

Table 5. Effect of isolates on I. aquatica seed germination rate

菌株 Strain	第3 天/%	第5 天/%
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 菌液处理对空心菜生长的影响

Table 6. Effect of bacterial broth treatment on I. aquatica growth and development

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 hairs
CK	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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