月季病原真菌鉴定及青岛假单胞菌BYX7生防潜力研究

doi:10.13560/j.cnki.biotech.bull.1985.2026-0073

摘要/Abstract

摘要：

目的探究南阳月季产区病原真菌种类及青岛假单胞菌BYX7的生防潜力与应用价值，为月季真菌病害绿色防控提供菌株资源与技术支撑。方法采用组织分离法从南阳月季病叶及茎段中分离病原真菌，结合形态学特征与ITS分子生物学技术进行鉴定；通过平板对峙法筛选拮抗菌并分析其抑菌谱；采用透明圈法、比色法、无氮培养基接种法、CAS培养基法分别检测目标菌株的产酶、解磷、固氮、产铁载体及IAA合成能力；运用单因素试验与响应面法优化菌株高密度发酵条件；通过载体与保护剂筛选制备微生物菌粉；采用盆栽试验评价菌剂对月季褐斑病的防控效果。结果分离鉴定出8种对月季具有致病性的真菌，其中隐秘刺盘孢菌为优势病原菌；青岛假单胞菌BYX7对8种病原菌的抑制率为50%‒99%，兼具尿素酶、纤维素酶活性，可产12.40 mg/L IAA，且具有产铁载体、溶磷（将1 g/L无机磷降至10.1 mg/L）和固氮能力；优化后的发酵条件为木糖20 g/L、鱼蛋白胨20 g/L、K₂HPO₄ 1 g/L、初始pH 7.5、33 ℃、179 r/min，菌体浓度达2.71×10¹⁰ CFU/mL（较优化前提高22倍）；以沸石粉为载体、海藻糖为保护剂制备的菌粉活菌数1.60×10⁸ CFU/g，存活率66.46%，对月季褐斑病的盆栽防治指数达38.61%。结论青岛假单胞菌BYX7兼具广谱抑菌活性与多重促生功能，其优化后的高密度发酵工艺及微生物菌剂制备技术可行，对月季褐斑病具有良好防控效果，可作为月季真菌病害绿色防控的优质菌株资源。

关键词: 月季褐斑病, 微生物防治, 青岛假单胞菌BYX7, 拮抗作用, 促生特性, 高密度发酵, 菌剂制备, 盆栽防效

Abstract:

Objective This study aimed to identify the pathogenic fungi associated with rose production areas in Nanyang and to evaluate the biocontrol potential and application value of Pseudomonas qingdaonensis BYX7, thereby providing strain resources and technical support for the green control of rose fungal diseases. Method Pathogenic fungi were isolated from diseased leaves and stems of roses in Nanyang using the tissue isolation method and were identified based on morphological characteristics and ITS molecular analysis. Antagonistic bacteria were screened by plate confrontation assays, and their inhibition spectra were determined. The enzyme production, phosphate solubilization, nitrogen fixation, siderophore production, and IAA synthesis ability of the target strain were assessed using the transparent zone method, colorimetric assays, nitrogen-free medium inoculation, and the CAS medium method, respectively. High-density fermentation conditions were optimized through single-factor experiments followed by response surface methodology. Microbial powder was prepared by screening suitable carriers and protectants. The control efficacy of the microbial agent against rose brown spot disease was evaluated in pot experiments. Result Eight pathogenic fungi were isolated and identified, among which Colletotrichum aenigma was the dominant pathogen. Pseudomonas qingdaonensis BYX7 exhibited inhibition rates of 50%–99% against the eight pathogens. The strain possessed urease and cellulase activities, produced 12.40 mg/L IAA and siderophores, and showed phosphate-solubilizing capacity (reducing 1 g/L inorganic phosphorus to 10.1 mg/L) as well as nitrogen-fixing ability. Under optimized fermentation conditions (xylose 20 g/L, fish peptone 20 g/L, K₂HPO₄ 1 g/L, initial pH 7.5, 33 ℃, 179 r/min), the cell concentration reached 2.71×10¹⁰ CFU/mL, representing a 22-fold increase compared to pre-optimization levels. The microbial powder prepared with zeolite powder as carrier and trehalose as protectant had a viable cell count of 1.60×10⁸ CFU/g and a survival rate of 66.46%, and achieved a control efficacy of 38.61% against rose brown spot disease in pot experiments. Conclusion Pseudomonas qingdaonensis BYX7 possesses broad-spectrum antifungal activity and multiple plant growth-promoting traits. The optimized high-density fermentation process and microbial agent preparation technology are feasible, and the strain shows promising control efficacy against rose brown spot disease. Therefore, it represents a valuable strain resource for the green control of rose fungal diseases.

Key words: Rose brown spot disease, microbial control, Pseudomonas qingdaonensis BYX7, antagonistic activity, growth-promoting characteristics, high-density fermentation, microbial agent preparation, pot control efficacy

张愈甜, 王怡, 王子阳, 付曼青, 吴鹏宇. 月季病原真菌鉴定及青岛假单胞菌BYX7生防潜力研究[J]. 生物技术通报, 2026, 42(11): 240-251.

ZHANG Yu-tian, WANG Yi, WANG Zi-yang, FU Man-qing, WU Peng-yu. Identification of Pathogenic Fungi on Rosa chinensis and Biocontrol Potential of Pseudomonas qingdaonensis BYX7[J]. Biotechnology Bulletin, 2026, 42(11): 240-251.

图/表 16

表1 拮抗微生物种类

Table 1 Antagonistic microbial species

编号 Number	名称 Name	保藏编号 Preservation number
BYXC5	日本假黄单胞菌 Pseudoxanthomonas japonensis	2021060102
BYX7	青岛假单胞菌 P. qingdaonensis	2021060105
BYX1E	边缘假单胞菌 P. marginalis	2021060112
BYX5	空气芽胞杆菌 Bacillus aerius	2021060123
BYXB	海口芽胞杆菌 B. haikouensis	2021060124
BYXF	托拉氏假单胞菌 P. tolaasii	2021060502
BYX1J	摩氏假单胞菌 P. mosselii	2021060504
BYXD2	克氏假单胞菌 P. knackmussii	2021060522

表2 Box-Behnken试验设计

Table 2 Box-Behnken experimental design

编号 Symbol	因素 Factor	编码水平 Coded levels
编号 Symbol	因素 Factor	-1	0	+1
A	温度 Temperature （℃）	31	33	35
B	转速 Speed （r/min）	160	180	200
C	pH	7	7.5	8

表3 不同载体的初始添加比例及干燥参数

Table 3 Initial addition ratios of different carriers and their corresponding drying parameters

载体种类 Type of adsorption carrier	载体质量 Carrier quality （g）	生理盐水 Sodium chloride solution （mL）	干燥时间 Drying time （min）	干燥前质量 The mass before drying （g）	干燥后质量 The mass after drying （g）
活性炭 Activated carbon	5	4.7	60	1.79	1
木屑 Sawdust	8	6.0	65	1.57	1
硅藻土 Diatomaceous earth	4	5.0	50	2.00	1
玉米芯粉 Corn cob powder	2	7.0	90	4.13	1
沸石粉 Zeolite powder	9	4.0	45	1.32	1
麸皮 Bran	3	5.0	65	2.52	1

图1 病原真菌的菌落形态（A）及基于ITS序列构建的系统发育树（B）

Fig. 1 Colony morphology (A) of the pathogenic fungi and phylogenetic tree (B) based on ITS sequences

表4 病原真菌鉴定结果

Table 4 Identification of pathogenic fungi

编号 Number	菌株名称 Strain name	序列相似度 Similarity （%）	GenBank 登录号 Accession number
BYZ1	Colletotrichum aenigma	100.00	NR 120140.1
BYZ2	Botryosphaeria wangensis	100.00	NR 159555.1
BYZ3	Cladosporium oxysporum	100.00	NR 152267.1
BYZ4	Fusarium boothii	99.81	NR 121203.1
BYZ5	Alternaria alstroemeriae	99.81	NR 163686.1
BYZ6	Fusarium longifundum	99.81	NR 171059.1
BYZ7	Colletotrichum cobbittiense	100.00	NR 163538.1
BYZ8	Fusarium fujikuroi	99.81	NR 111889.1

图2 8种真菌的致病性测定

Fig. 2 Pathogenicity test of the eight fungi

图3 8种拮抗菌对隐秘刺盘孢菌的抑菌效果（A）及抑菌率（B）不同字母表示差异显著（P<0.05），下同

Fig. 3 Antifungal effects (A) and inhibition rates (B) of eight antagonistic bacteria against C. aenigmaDifferent letters indicate significant differences(P<0.05). The same below

图4 青岛假单胞菌BYX7对8种病原菌的抑菌效果（A）及抑菌率（B）

Fig. 4 Antifungal effects (A) and inhibition rates (B) of P. qingdaonensis BYX7 against eight pathogenic fungi

图5 青岛假单胞菌BYX7生物酶活性A：尿素酶活性；B：纤维素酶活性

Fig. 5 The enzymatic activity of P. qingdaonensis BYX7A: Urease activity. B: Cellulase activity

图6 青岛假单胞菌BYX7促生能力测定A：解磷实验；B：产铁载体测定；C：固氮实验；D：产IAA测定

Fig. 6 Determination of the growth-promoting ability of P. qingdaonensis BYX7A: Phosphorus dissolution experiment. B: Determination of siderophores. C: Nitrogen fixation experiment. D: Determination of IAA production

图7 发酵培养基成分优化A：碳源种类；B：碳源添加量；C：氮源种类；D：氮源添加量；E：无机盐种类

Fig. 7 Optimization of fermentation medium compositionA: Types of carbon sources. B: Addition amount of carbon source. C: Type of nitrogen source. D: Nitrogen source addition amount. E: Types of inorganic salts

图8 发酵条件的优化

Fig. 8 Optimization of fermentation conditions

图9 发酵条件影响青岛假单胞菌BYX7活菌数的响应曲面（A、C、E）和等高线（B、D、F）

Fig. 9 Response surface (A, C, E) and contour plots (B, D, F) illustrating the effect of fermentation conditions on the viable cell count of P. qingdaonensis BYX7

图10 菌粉吸附载体和保护剂种类的优化A：吸附载体种类；B：保护剂种类

Fig. 10 Optimization of the types of adsorption carriers and protective agents for bacterial powderA: Types of adsorption carriers. B: Types of protective agents

图11 BYX7对月季盆栽苗的回接试验（A）及对褐斑病的防治效果（B）A1：回接试验；A2：对照组；A3：实验组；B1‒B3：健康叶片；B4‒B6：病害叶片

Fig. 11 The re-inoculation experiment of BYX7 on rose potted seedlings (A) and its efficacy in preventing brown spot disease (B)A1: Re-inoculation group. A2: Control group. A3: Experimental group. B1-B3: Healthy leaves. B4-B6: Diseased leaves

表5 盆栽实验发病率、病情指数及防治指数

Table 5 Incidence rate, disease index, and prevention and control index of the pot experiment

组别

Group

发病率

Incidence rate （%）

病情指数

Disease index （%）

防治指数

Prevention and control index （%）

对照组

Control group

72.29±17.28a

90.50±10.14a

—

实验组

Experimental group

32.40±22.08b

55.56±15.72a

38.61

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