PGPR微胶囊菌剂研发及其对玉米的促生效果评价

刘文佳; 刘天一; 张俐敏; 徐畅; 莫继先

PGPR微胶囊菌剂研发及其对玉米的促生效果评价

1.
齐齐哈尔大学生命科学与农林学院，黑龙江　齐齐哈尔　161006

基金项目: 黑龙江省省属高等学校基本科研业务费项目（135409420）；黑龙江省大学生创新创业训练计划项目（S202310232017）

详细信息

作者简介:
刘文佳（1999 —），女，硕士研究生，研究方向：资源环境微生物学（E-mail：1740203063@qq.com）

通讯作者:
莫继先（1982 —），男，博士，教授，研究方向：资源环境微生物学（E-mail：mojixian8208@sina.com）

中图分类号: S541
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出版历程
- 收稿日期: 2023-12-22
- 修回日期: 2024-02-07
- 网络出版日期: 2024-03-28

Development of microencapsulated rhizosphere growth promoting bacteria and evaluation of the growth promoting effects on corn

1.
College of Life Science and Agriculture and Forestry, Qiqihar University, Qiqihar, Heilongjiang　161006, China

摘要

摘要: 目的为了提高PGPR对作物促生作用的稳定性，研制出以PGPR复合菌液为原料的微胶囊菌剂，探究其对玉米的促生效果。方法从玉米根际土壤中分离得到2株PGPR菌株并制备成复合液体菌液。分别用海藻酸钠（SA）和CaCl₂作为包埋剂和交联剂，对复合菌液进行包埋以制备微胶囊菌剂。以复合微胶囊菌剂制备上的难易程度和菌株包埋率、增殖前活菌数及增殖后活菌数为评价指标，确定微胶囊菌剂制备的最优条件。通过分析微胶囊菌剂在不同聚乙二醇浓度、温度、pH、存储时间以及盐浓度下的菌株生长及其对玉米生长和根系形态的影响确定PGPR微胶囊菌剂的特性。结果研究分离得到了2株PGPR菌株，具有多种植物促生功能。使用1%SA-3%CaCl₂制备的微胶囊菌剂稳定性较好，包埋率达到91.27%，增殖后的微胶囊活菌数达到8.73×10⁹ cfu·g⁻¹，增殖7.79倍。微胶囊菌剂在不同干旱、温度、pH、存储时间和盐浓度条件下对PGPR菌株具有较好的保护作用。PGPR微胶囊菌剂对玉米有显著的促生作用，施用微胶囊菌剂使玉米株高、苗干重和根干重分别增加91.83%、81.82%和29.57%。同时微胶囊菌剂也显著提高了玉米总根长、根表面积、根平均直径、根体积和根尖数。结论确定了一种PGPR微胶囊菌剂的制备方法，施用该PGPR微胶囊剂能够显著促进玉米生长、改善玉米根系形态，对玉米的促生效果优于液体菌剂。
- 玉米 /
- 根际促生菌 /
- 促生效果 /
- 微胶囊菌剂
Abstract: Objective In order to improve the stability of PGPR in promoting crop growth, microcapsule microbial agents were developed using PGPR composite bacteria as raw materials, and its growth-promoting effects on corn were investigated. Method Isolation of PGPR strains from corn rhizosphere soil and preparation of composite liquid microbial agents. Sodium alginate (SA) and CaCl₂ were used as embedding and crosslinking agents to embed the composite bacterial solution and prepare microcapsule microbial agents. Based on the difficulty level, strain embedding rate, and pre-proliferative and post-proliferative viable bacterial count in the preparation of composite microcapsule microbial agents, the optimal conditions for preparing microcapsule microbial agents were determined. The excellent characteristics of PGPR microcapsules were determined by analyzing the strains growth in PGPR microcapsules under different concentrations of PEG, temperature, pH, storage time, and salt concentration, as well as their effects on corn growth and root morphology. Result This study isolated two PGPR strains with various plant growth-promoting functions. The microcapsule microbial agents made with 1% SA-3% CaCl₂ had good stability, with an embedding rate of 91.27%. After proliferation, the number of live bacteria in the microcapsule reached 8.73 × 10⁹ cfu·g⁻¹, with a proliferation rate of 7.79 times. Under different concentrations of PEG, temperature, pH, storage time, and salt concentration, the concentration of microencapsulated bacterial solution was higher than that of liquid bacterial solution. PGPR microcapsule microbial agents had significant promoting effects on corn growth. Applying microcapsule microbial agents increased corn plant height, seedling dry weight, and root dry weight by 91.83%, 81.82%, and 29.57%, respectively. At the same time, microencapsulated microbial agents significantly increased the total root length, root surface area, average root diameter, root volume, and root tip number of corn. Conclusion This study determined a preparation method for PGPR microcapsule microbial agents. The application of the PGPR microcapsule agents significantly promoted corn growth, improved corn root morphology, and had better growth-promoting effects on corn than liquid microbial agents.
- Corn /
- Rhizosphere growth promoting bacteria /
- Promoting growth effects /
- Microcapsule microbial agents

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图 1 菌株P1和K5的菌间拮抗实验

Figure 1. Interbacterial antagonism assay of strains P1 and K5

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图 2 菌株 P1（a）和K5（b）的显微形态图

Figure 2. Microscopic morphology of P1 (a) and K5 (b) strains

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图 3 菌株P1的系统进化树

Figure 3. Phylogenetic tree of strain P1

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图 4 菌株K5的系统进化树

Figure 4. Phylogenetic tree of strain K5

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图 5 菌株P1和K5的生长曲线

Figure 5. Growth curves of strains P1 and K5

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图 6 SA1%-CaCl₂3%微胶囊颗粒的形态图

Figure 6. Morphological diagram of SA1%-CaCl₂3% microcapsule particles

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图 7 不同环境条件对两种剂型菌株生长的影响

＊表示两组之间差异显著（P＜0.05）

Figure 7. Effects of different environmental conditions on the growth of two dosage form strains

＊indicated a significant difference between the two groups (P＜0.05)

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图 8 不同剂型PGPR对玉米生长的影响

a：各处理组对玉米株高的影响；b：各处理组对玉米苗干重的影响；c：各处理组对玉米根干重的影响；不同小写字母表示不同组别间差异显著（P＜0.05）

Figure 8. Effects of different dosage forms of PGPR on the growth of corn

a: The effects of each treatment group on the plant height of corn; b: The effects of each treatment group on the dry weight of corn seedlings; c: The effects of each treatment group on the dry weight of corn roots; Different letters indicated significant differences between different groups (P＜0.05)

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图 9 不同剂型PGPR对玉米根系形态的影响

a：各处理组对玉米根长的影响；b：各处理组对玉米根总表面积的影响；c：各处理组对玉米根平均直径的影响；d：各处理组对玉米根尖数的影响；e：各处理组对玉米根体积的影响；不同字母表示不同组别间差异显著（P＜0.05）

Figure 9. Effects of different dosage forms of PGPR on corn root morphology

a: The effects of each treatment group on the corn root length; b: The effects of each treatment group on the total surface area of corn roots; c: The effects of each treatment group on the average diameter of corn roots; d: The effects of each treatment group on the number of corn root tips; e: The effects of each treatment group on the volume of corn roots; Different letters indicated significant differences between different groups (P＜0.05)

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表 1 玉米根际土壤中筛选的具有促生特性的菌株及促生特性分析

Table 1. Analysis of strains with growth-promoting characteristics and growth-promoting characteristics screened in corn rhizosphere soil

菌株 Strains	解磷量 Phosphate solubilizing/ (μg·mL⁻¹)		解钾量 Potassium releasing/ (μg·mL⁻¹)	固氮量 Nitrogen fixation/ (μg·mL⁻¹)	吲哚乙酸 Indoleacetic acid/ (μg·mL⁻¹)	铁载体活性 Siderophore activity/(%)	ACC含量 ACC content/ (μg·mL⁻¹)
P1	33.50±0.37a	—	1.44±0.16b	78.06±0.51a	55.72±0.67a	0.19±0.07b
P2	31.49±0.51b	—	—	65.74±1.61b	44.19±0.94d	0.30±0.07e
P3	31.34±0.58b	—	—	—	49.12±0.35b	0.27±0.05d
P4	30.85±0.59b	—	—	—	46.47±0.27c	0.22±0.14c
P5	30.12±0.03c	—	—	46.23±3.25d	40.48±0.44e	0.14±0.07a
P6	31.13±0.04b	—	—	—	39.65±0.38e	0.20±0.06b
N1	—	—	1.96±0.35a	—	33.5±0.527f	0.27±0.11d
N2	—	—	1.27±0.21b	—	—	0.14±0.02a
N3	—	—	1.23±0.10b	—	—	0.22±0.03c
K1	—	1.22±0.09b	—	55.63±0.54c	31.52±0.39g	0.14±0.02a
K2	—	1.17±0.07b	—	—	—	0.30±0.06e
K3	—	1.26±0.07b	—	—	29.34±0.35g	0.25±0.13cd
K4	—	1.35±0.28b	—	—	—	0.18±0.07b
K5	—	2.26±0.07a	—	63.43±2.9b	50.46±0.71b	0.34±0.05f
同列不同行有不同字母表示差异显著（P＜0.05）；—表示无此项促生功能 Different letters in different rows indicated significant differences (P＜0.05); —indicated no growth-promoting function

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表 2 菌株P1和K5的生理生化特性

Table 2. Physiological and biochemical characteristics of strains P1 and K5

实验项目 Items	菌株 Strains
实验项目 Items	P1	K5
淀粉水解实验 Starch hydrolysis	−	−
蔗糖发酵实验 Sucrose fermentation	+	−
乳糖发酵实验 Lactose fermentation	−	+
葡萄糖发酵实验 Glucose fermentation	+	−
甲基红实验 Methyl red experiment	−	−
柠檬酸盐实验 Citrate experiment	−	−
吲哚实验 Indole experiment	+	+
革兰氏染色实验 Gram staining experiment	+	+
触酶实验 Catalase experiment	+	+
伏-普实验 Volt-P experiment	+	−
+：阳性；−：阴性； +: positive; −: negative

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表 3 SA-CaCl₂不同浓度配比下微胶囊的操作性和成球性

Table 3. Operability and spherical properties of microcapsules under different concentration ratios of SA-CaCl₂

SA浓度/% Concentration of SA/ (%)	CaCl₂浓度/% Concentration of CaCl₂/ (%)	操作性 Operation	成球性 Sphericity
1	2	易 Easy	较好，拖尾，无粘连 Better, trailing, no sticking
1	3	易 Easy	较好，拖尾，少量粘连 Better, trailing, few sticks
1	4	易 Easy	较好，拖尾，少量粘连 Better, trailing, few sticks
2	2	易 Easy	较好，拖尾，无粘连 Better, trailing, no sticking
2	3	易 Easy	较好，拖尾，少量粘连 Better, trailing, few sticks
2	4	易 Easy	较好，拖尾，少量粘连 Better, trailing, few sticks
3	2	难 Difficult	较差，无拖尾，少量破碎 Bad, no tailing, few fragile
3	3	难 Difficult	较差，拖尾，少量粘连 Bad, trailing, few sticks
3	4	难 Difficult	差，拖尾，少量破碎，易粘连 Poor, trailing, few fragile, sticks

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表 4 微胶囊菌剂颗粒的理化性质

Table 4. Physicochemical properties of microcapsule particles

指标 Index	SA1%-CaCl₂2%	SA1%-CaCl₂3%	SA2%-CaCl₂2%
直径（mm） Diameter (mm)	3.14±0.13b	3.11±0.07c	3.22±0.04a
机械强度（g） Mechanical Strength (g)	3.29±0.16b	3.21±0.13c	3.35±0.03a
增殖前胶囊活菌数（10⁹ cfu·g⁻¹） Number of viable cells before proliferation (10⁹ cfu·g⁻¹)	1.01±0.11b	1.12±0.03c	1.20±0.03a
包埋率（%） Embedding rate (%)	85.60±2.25b	91.27±1.05c	81.17±1.91a
增殖后胶囊活菌数（10⁹ cfu·g⁻¹） Number of viable cells in after proliferation (10⁹ cfu·g⁻¹)	5.68±0.37b	8.73±0.50c	6.91±0.49a
增殖倍数 Multiplication ratio	5.62	7.79	5.76
同行不同列有不同字母表示差异显著（P＜0.05） Different letters in the different column indicated significant differences (P＜0.05)

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