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

doi:10.19303/j.issn.1008-0384.2023.05.014

西藏沙棘根瘤内生假单胞菌的分离鉴定及促生性研究

doi: 10.19303/j.issn.1008-0384.2023.05.014

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）；青海省科技计划项目（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
- 刊出日期: 2023-05-28

Identification and Growth-promoting Effects of Endophytic Pseudomonas sp. from Hippophae thibetana Root Nodules

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 EndogenousPseudomonas sp. in the root nodules of Hippophae thibetana that displayed a biological activity were isolated for toxicological identification and growth-promoting effect determination for the development of a biological agent for crop fertilization. Methods Strains of Pseudomonas sp. were isolated from the root nodules of H. thibetana by pure culture method. They were identified by morphology, physiological biochemistry, and 16S rDNA sequence, as well as examined for the growth-promoting effect on the host plant by artificial inoculation for evidence of phosphorus-dissolving and IAA-, iron- and cellulase-producing capacities. Results Out of the isolated endophytic strains, 4 showed greater than 99% 16S rDNA alignment with the reference Pseudomonas. Among them, QY-X10 and QY-X22 produced 400 mg·L⁻¹ of solubilized phosphorus; QY-X4 yielded the highest IAA at 1.9 mg·L⁻¹; all 4 strains contained iron-generating carriers; except QY-X10, all could degrade cellulose; QY-X6 promoted seed germination; and QY-X6 and QY-X10 helped the host plant to grow significantly more leaves and longer seedlings, while QY-X10 and QY-X22 did significantly to make the maximum-length leaves longer than did control. Conclusion There were 4 Pseudomonas sp. identified in this study that exhibited significant abilities to dissolve inorganic phosphorus and produce IAA and iron. Three of them could also degrade cellulose. By inoculating these strains on the host plants, the seed germination and seedling growth were effectively promoted. It appeared that they could be applied for crop fertilization.
- 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. Gram-stained isolated Pseudomonas sp. under microscope

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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. Effect of medium pH on growth of isolated strains

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图 4 不同氯化钠用量对菌株生长量的影响

Figure 4. Effect of NaCl concentration in medium on growth of isolated 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-producing and phosphorus-dissolving abilities of isolated Pseudomonas sp.

A: IAA-producing ability; B: phosphorus-dissolving ability.

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图 6 菌株产铁载体及产降解纤维素酶能力

A: 产铁载体； B: 产降解纤维素酶

Figure 6. Effect of iron-producing siderophores and cellulase in isolatedPseudomonas sp.

A: iron-producing siderophore; B: cellulase.

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图 7 菌株促生各指标间的相关性分析

Figure 7. Correlation analysis of growth-promoting indexes of Pseudomonas sp.

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表 1 分离菌株生化测定结果

Table 1. Biochemical identification of isolated Pseudomonas sp.

指标 Index	QY-X4	QY-X6	QY-X10	QY-X22	恶臭假单胞菌A3菌株Pseudomonas putida A3 strain
半乳糖 Galactose	－	+	+	+	+
麦芽糖 Maltose	+	+	+	+	－
蔗糖生化管 Sucrose biochemical tube	－	－	+	+	－
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	－	－	－	－	－
+表示阳性，－表示阴性。N表示不确定。 +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 phosphorus-dissolving and IAA-producing abilities of isolated Pseudomonas sp.

菌株 Strain	溶解无机磷能力SI Phosphate solubilization (inorganic) SI/mm	产IAA能力 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	++
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，++表示粉红。 Datas with different lowercase letters on the same column indicate significant difference at 0.05 level. Same for below. /indicates no effect; + indicates light red; and ++ indicates pink.

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表 3 菌株溶解磷、产IAA的定量测定

Table 3. Quantitative determination of phosphorus-dissolving and IAA-producing abilities of isolated Pseudomonas sp.

菌株 Strain	溶解无机磷量 Phosphate solubilization (inorganic)/ （mg·L⁻¹）	IAA含量 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 iron-producing and cellulose-degrading abilities of isolated Pseudomonas sp.

菌株 strain	产铁载体能力SI iron-producing siderophore SI/mm	产降解纤维素酶能力SI Degrading cellulase SI/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 菌株对西藏沙棘幼苗的促生效果

Table 5. Growth promoting effects of Pseudomonas sp. on Hippophae thibetana seedlings

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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