西藏沙棘根瘤内生假单胞菌的分离鉴定及促生性研究

马福林; 仁增卓玛; 王昌玲; 邓得坤; 冶贵生; 马玉花

西藏沙棘根瘤内生假单胞菌的分离鉴定及促生性研究

青海大学农牧学院农林系，青海　西宁　810016

基金项目: 国家自然科学基金(31660071)；青海省科技厅项目(2017-ZJ-734)；青海省“高端创新人才千人计划”项目（2020）

详细信息

作者简介:
马福林（1996−），男，硕士研究生，主要从事植物资源开发利用方面的研究（E-mail：qhdxmfl@163.com）

通讯作者:
马玉花（1978−），女，博士，教授，主要从事森林培育理论与技术、植物资源开发利用方面的研究（E-mail：qhxnmyh@163.com）

中图分类号: S154
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出版历程
- 收稿日期: 2022-10-09
- 修回日期: 2023-04-30
- 网络出版日期: 2023-05-09

Identification And Growth promotion Study of The Endophytic Pseudomonas sp. from Hippophae Thibetana root nodule

Department of Agriculture and Forestry, College of Agriculture and Animal Husbandry, Qinghai University, Xining　　810016, China

摘要

摘要: 目的获取西藏沙棘根瘤内生菌中具有多重生物学活性的假单胞菌属菌株，探究筛选所得菌株的促生作用，为研发高效生物菌肥提供基础材料。方法利用纯培养方法，从西藏沙棘根瘤中分离假单胞菌，通过形态、生理生化及16S rDNA序列比对鉴定菌株，测定菌株溶磷、产IAA、产铁载体及产降解纤维素酶的能力，接种宿主植物验证其促生效果。结果 4株根瘤内生菌与参考假单胞菌属同源性为99%以上，鉴定为假单胞菌属。溶磷和产IAA的定性及定量结果表明，4株菌均具有溶解无机磷和产IAA的能力，其中溶解无机磷能力较强的菌株是QY-X10和QY-X22，均达到400 mg·L⁻¹；菌株QY-X4产IAA能力较其他菌株强，达1.9 mg·L⁻¹；4株菌株具有产铁载体的能力，除QY-X10以外，均具产降解纤维素酶的能力；促生试验结果表明，QY-X6可有效促进种子的萌发；QY-X6、QY-X10处理组叶片数显著高于对照；QY-X6、QY-X10处理组苗长显著高于对照；QY-X10、QY-X22处理组最大叶片长显著高于对照。结论分离筛选出4株假单胞菌，均可溶解无机磷和产IAA，兼具产铁载体；3株兼具产降解纤维素的能力；接种试验发现，4株菌株处理组可有效促进植株的生长发育，分离菌株可为研发生物菌肥提供基础材料。
- 假单胞菌属 /
- 西藏沙棘 /
- 菌种鉴定 /
- 促生作用
Abstract: Objective The endogenous Pseudomonas sp. with biological activity in the root nodule of Hippophae thibetana was obtained, and the growth promotion effect of the selected strains was explored, which provided basic materials for the research and development of efficient biological fertilizers. Methods Pseudomonas sp. was isolated from the root nodule of Hippophae thibetana by pure culture method, and the strains were identified by morphology, physiological biochemistry and 16S rDNA sequence comparison, and the phosphorus-soluble, IAA-producing, iron-producing carrier and cellulase-producing capacity of the strains were determined, and the host plants were inoculated to verify their growth promotion effect. Results The results showed that 16S rDNA alignment of four endophytic strains is higher than 99% compared with the reference Pseudomonas sp., and can be identified as Pseudomonas sp. The qualitative and quantitative tests of phosphorus dissolving and IAA production showed that all the four strains had the ability to dissolve inorganic phosphorus and produce IAA, among which the strains with stronger ability to dissolve inorganic phosphorus were QY-X10 and QY-X22, both reaching 400 mg·L⁻¹. The QY-X4 showed stronger IAA production ability than other strains, reaching 1.9 mg·L⁻¹. Four strains have the ability to produce iron carriers, all three strains have the ability to degrade cellulose except QY-X10. Tthe growth promotion experiment showed that QY-X6 could effectively promote seed germination. The number of leaves in the QY-X6 and QY-X10 treatment groups was significantly higher than that of the control. The seedling lengths in the QY-X6 and QY-X10 treatment groups were significantly higher than those in the control. The maximum leaf length in the QY-X10 and QY-X22 treatment groups was significantly higher than that of the control. Conclusion The results showed that 4 strains of Pseudomonas were isolated and screened, all of which could dissolve inorganic phosphorus, produce IAA and excreting siderophore, and 3 of them had the ability to produce degradable cellulose. Inoculation tests found that the treatment group with 4 strains could effectively promote the growth and development of plants, and the isolated strains could provide basic materials for the development of biological fertilizers.
- Pseudomonas sp. /
- Hippophae thibetana /
- strain identification /
- growth-promoting effect

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图 1 镜检观察革兰氏染色的分离菌株

Figure 1. Microscopic observation of isolated strains after Gram staining

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图 2 基于16S rDNA构建系统发育树

Figure 2. Phylogenetic tree based on 16S rDNA

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图 3 不同pH培养对菌株生长量的影响

Figure 3. Effects of different pH cultures on strain growth

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图 4 不同氯化钠质量分数对菌株生长量的影响

Figure 4. Effects of different mass fractions of sodium chloride on the growth of strains

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图 5 菌株产IAA和溶磷能力

A: 产IAA，B: 溶解无机磷

Figure 5. IAA and Phosphate solubilization (inorganic) capacity of strains

A: IAA production, B: Phosphate solubilization (inorganic)

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图 6 菌株产铁载体及产降解纤维素酶能力效果图

A: 产铁载体, B: 产降解纤维素酶

Figure 6. Effect map of the ability of strain to produce siderophores and produce degradable cellulase

A: ferrous-producing carriers, B: degrading cellulase

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图 7 相关性分析

Figure 7. Correlation analysis

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表 1 分离菌株生化测定结果

Table 1. Biochemical identification test results of isolated strain

指标 Index	QY-X4	QY-X6	QY-X10	QY-X22	恶臭假单胞菌(Pseudomonas putida)A3菌株
半乳糖 Galactose	－	+	+	+	+
麦芽糖 Maltose	+	+	+	+	－
蔗糖生化管 Sucrose	－	－	+	+	－
葡萄糖 Glucose utilization	－	－	+	+	+
果糖 Fructose	+	+	+	+	+
明胶生化 Gelatin hydrolysis	－	－	－	－	N
硫化氢 H₂S	－	－	－	－	N
L-鼠李糖 L-Rhamnose	－	+	+	+	N
甲基红试验 Methyl red test	－	－	－	－	－
V-P反应 V-P Test	－	－	－	－	－
运动性 Motility	运动	运动	不运动	不运动	N
革兰氏染色 Gram stain	－	－	－	－	－
注：+表示阳性，－表示阴性。N表示不确定。 Note: +indicates positive, －indicates negative. N indicates Undetermined.

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表 2 菌株溶解磷、产IAA的定性测定

Table 2. Qualitative determination of Phosphate solubilization (inorganic) and IAA production in strains

菌株 strain	解无机磷 Phosphate solubilization (inorganic)/mm	产IAA IAA production
QY-X4	1.781±0.066 a	+
QY-X6	1.778±0.079 a	－
QY-X10	1.456±0.108 b	－
QY-X22	1.605±0.118 ab	+
注：同列不同小写字母表示处理间差异显著（P＜0.05），下同。“/”表示无效果。“+”表示浅红色，“－”表示无色。 Note: Lowercase letters are significantly at 0.05 probability level of the different treatments, the same below. “/” indicates no effect. “+” indicates light red and “－” indicates colorlessness.

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表 3 菌株溶解磷、产IAA的定量测定

Table 3. Quantitative determination of phosphorus dissolved and IAA produced by strains

菌株 strain	解无机磷 Phosphate solubilization (inorganic)/ （mg·L⁻¹）	产IAA IAA production/ （mg·L⁻¹）
QY-X4	230.232±3.482 b	1.919±0.243 a
QY-X6	207.335±26.067 b	1.019±0.141 bc
QY-X10	418.131±9.898 a	0.627±0.135 c
QY-X22	417.266±4.765 a	1.063±0.268 b

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表 4 菌株产铁载体及降解纤维素酶测定

Table 4. Determination of ferrous-producing carriers and degrading cellulase of strains

菌株 strain	产铁载体 ferrous-producing carriers/mm	产降解纤维素酶 degrading cellulase/mm
QY-X4	1.742±0.3232 a	1.671±0.3225 a
QY-X6	1.727±0.3556 a	2.177±0.2710 a
QY-X10	1.986±0.1822 a	/
QY-X22	1.830±0.1663 a	1.797±0.1597 a

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表 5 促生菌各项促生效果

Table 5. Various growth promoting effects of probiotic bacteria

菌株 Strain	叶片数 Leaf number	苗长 Seedling length/mm	根长 Root length/mm	最大叶片长 Maximum leaf length/mm	最大叶片宽 Maximum leaf width/mm	植株鲜重 fresh weight of plant/g	植株干重 Dry weight of plant/g	发芽率 germination percentage/%
CK	3.500±1.000 c	21.763±21.763 b	22.300±5.324 ab	5.803±0.616 c	4.125±0.530 ab	0.069±0.006 a	0.008±0.0004 a	3.3% b
QY-X4	4.000±0.816 bc	28.775±28.775 ab	18.460±2.761 b	6.290±1.521 bc	4.130±0.241ab	0.070±0.017 a	0.009±0.0032 a	3.3% b
QY-X6	4.750±0.500 ab	38.603±38.603 a	27.585±3.578 a	6.500±0.887 bc	4.380±1.076 ab	0.077±0.019 a	0.009±0.0006 a	13.0% a
QY-X10	5.250±0.500 a	34.778±34.778 a	27.635±6.579 a	8.418±0.910 a	4.823±0.597 a	0.090±0.009 a	0.009±0.0009 a	3.3% b
QY-X22	3.250±0.500 c	27.988±27.988 ab	20.708±3.412 ab	7.820±1.235 ab	3.628±0.364 b	0.073±0.014 a	0.009±0.0015 a	5.0% b

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