无花果细菌性叶斑病病原鉴定

doi:10.3969/j.issn.1004-1524.20240149

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (5): 1097-1106.DOI: 10.3969/j.issn.1004-1524.20240149

无花果细菌性叶斑病病原鉴定

季梦婷¹(), 陈长江², 朱玲³, 詹梦琳¹, 肖顺¹, 蔡学清¹^,^*()

1.福建农林大学植物保护学院,福建福州 350002
2.福建农林大学计算机与信息学院,福建福州 350002
3.福建天禾绿宝农资有限公司,福建福州 350193

收稿日期:2024-02-18 出版日期:2025-05-25 发布日期:2025-06-11
作者简介:季梦婷(1998—),女,江苏灌南人,硕士,主要从事植物病害综合防治研究。E-mail: 1249169841@qq.com
通讯作者: *蔡学清,E-mail:caixq90@163.com
基金资助:
福建省现代农业(水果)产业技术体系植保与土肥岗位(闽农科教〔2023〕16号);福建省科技特派员工作经费(KTP19128A);福建省科技特派员工作经费(KTP21267A)

Identification of etiological bacterium causing leaf spot disease on Ficus carica

JI Mengting¹(), CHEN Changjiang², ZHU Ling³, ZHAN Menglin¹, XIAO Shun¹, CAI Xueqing¹^,^*()

1. College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, China
2. College of Computer and Information Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China
3. Fujian Tianhe Lyubao Agricultural Materials Co., Ltd., Fuzhou 350193, China

Received:2024-02-18 Online:2025-05-25 Published:2025-06-11

摘要/Abstract

摘要： 2021—2023年,在福建省福州市的无花果果园发现细菌性叶斑病,田间株发病率达50%以上。为明确病因,采集病叶进行实验室菌株分离,经柯赫氏法则验证其致病性,并综合细菌的生物学特征、生理生化反应、多基因序列分析明确其分类地位。结果表明,从采集的病叶中分离纯化获得6株细菌菌株,经柯赫氏法则验证其为病原菌。菌株在NA培养基平板上的菌落为乳白色,圆形,微隆起。细菌菌体呈短杆状,极生1~6根鞭毛,革兰氏染色阴性。对菌株开展的27项生理生化测定结果与对照菌株——丁香假单胞菌(Pseudomonas syringae)DC3000菌株一致,在金氏B(KB)培养基平板上可产生绿色水溶性荧光色素,过氧化氢酶反应和淀粉水解的测试结果为阳性,精氨酸双水解、硝酸钾还原反应、吲哚反应、果聚糖利用和氧化酶反应的测试结果为阴性,可利用的碳水化合物为葡萄糖、甘露醇、丙二酸钠和丙三醇。基于16S rDNA序列,及gyrB、rpoD和cts的基因构建系统发育树,供试菌株与丁香假单胞菌丁香致病变种(P. syringae pv. syringae)聚为一支。综上,将分离到的细菌菌株鉴定为丁香假单胞菌丁香致病变种。这是国内首次发现丁香假单胞菌丁香致病变种在自然状态下侵染无花果并引起叶斑病。

关键词: 无花果细菌性叶斑病, 病原菌鉴定, 丁香假单胞菌丁香致病变种

Abstract:

From 2021 to 2023, a new bacterial leaf spot disease on Ficus carica was found in Fuzhou, Fujian Province, China, with an incidence above 50%. To clarify the cause of the disease, symptomatic leaf samples were collected and taken back to the laboratory to isolate the bacteria. The pathogenicity of the isolated strains were determined via Koch's rule, and its taxonomic status was confirmed based on the bacterial biological characteristics, physiological and biochemical characters, and multi-gene phylogenetic analysis. The results showed that 6 bacterial strains were obtained by isolating and purifying from the diseased leaves, and all the bacterial strains could cause the same disease on Ficus carica. The bacterial colonies were round and bulged with milk white. The bacterial cells were rod-shaped and had 1-6 flagella on polar. The Gram staining reaction was negative. The test results regarding 27 biological and biochemical characters of the isolated strains were consistent with Pseudomonas syringae strain DC3000. The bacterial cells could produce green water-soluble fluorescent on King's B medium. They were capable of hydrolyzing starch and had hydrogen peroxide activities. Meanwhile, they were not capable of potassium nitrate reduction, arginine hydrolysis, indole production or fructan utilization, and had no oxidase activities. They could utilize glucose, mannitol, sodium malonate, glycerol as carbon sources. As revealed by the constructed phylogenetic trees based on the sequences of 16S rDNA, and gyrB, rpoD, cts genes, the isolated strains were clustered with P. syringae pv. syringae. Above all, the isolated bacterial strains were identified as P. syringae pv. syringae. This was the first report that P. syringae pv. syringae could naturally cause leaf spot disease on Ficus carica in China.

Key words: Ficus carica bacterial leaf spot disease, pathogen identification, Pseudomonas syringae pv. Syringae

中图分类号:

S432.42

季梦婷, 陈长江, 朱玲, 詹梦琳, 肖顺, 蔡学清. 无花果细菌性叶斑病病原鉴定[J]. 浙江农业学报, 2025, 37(5): 1097-1106.

JI Mengting, CHEN Changjiang, ZHU Ling, ZHAN Menglin, XIAO Shun, CAI Xueqing. Identification of etiological bacterium causing leaf spot disease on Ficus carica[J]. Acta Agriculturae Zhejiangensis, 2025, 37(5): 1097-1106.

图/表 9

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测定项目 Test	各菌株的测定结果 Result of strains		测定项目 Test	各菌株的测定结果 Result of strains
测定项目 Test	WP1~WP6	DC3000	测定项目 Test	WP1~WP6	DC3000
KB荧光Produce fluorescence on KB	+	+	麦芽糖Maltose	-	-
精氨酸双水解Arginine Hydrolysis	-	-	纤维二糖Cellobiose	-	-
过氧化氢酶反应Reaction with hydrogen peroxide	+	+	蔗糖Sucrose	-	-
氧化酶反应Oxidase test	-	-	阿拉伯糖L-Arabinose	-	-
硝酸钾还原反应Potassium nitrate reduction	-	-	鼠李糖L-Rhamnose monohydrate	-	-
吲哚反应Indole production	-	-	海藻糖D-Trehalose anhydrous	-	-
果胶酶溶解活性Hydrolysis of pectinase	-	-	棉籽糖Raffinose	-	-
果聚糖利用Fructan utilization	-	-	山梨醇Sorbitol	-	-
淀粉水解Starch hydrolysis	+	+	酒石酸钠Sodium tartaric	-	-
甲基红试验Methyl red test	-	-	甜醇Dulcitol	-	-
V-P试验V-P test	-	-	丙三醇Glycerol	+	+
葡萄糖Glucose	+	+	香叶醇Geraniol	-	-
柠檬酸钠Trisodium citrate dihydrate	-	-	甘露醇Mannitol	+	+
丙二酸纳Sodium malonate	+	+

测定项目 Test	各菌株的测定结果 Result of strains		测定项目 Test	各菌株的测定结果 Result of strains
测定项目 Test	WP1~WP6	DC3000	测定项目 Test	WP1~WP6	DC3000
KB荧光Produce fluorescence on KB	+	+	麦芽糖Maltose	-	-
精氨酸双水解Arginine Hydrolysis	-	-	纤维二糖Cellobiose	-	-
过氧化氢酶反应Reaction with hydrogen peroxide	+	+	蔗糖Sucrose	-	-
氧化酶反应Oxidase test	-	-	阿拉伯糖L-Arabinose	-	-
硝酸钾还原反应Potassium nitrate reduction	-	-	鼠李糖L-Rhamnose monohydrate	-	-
吲哚反应Indole production	-	-	海藻糖D-Trehalose anhydrous	-	-
果胶酶溶解活性Hydrolysis of pectinase	-	-	棉籽糖Raffinose	-	-
果聚糖利用Fructan utilization	-	-	山梨醇Sorbitol	-	-
淀粉水解Starch hydrolysis	+	+	酒石酸钠Sodium tartaric	-	-
甲基红试验Methyl red test	-	-	甜醇Dulcitol	-	-
V-P试验V-P test	-	-	丙三醇Glycerol	+	+
葡萄糖Glucose	+	+	香叶醇Geraniol	-	-
柠檬酸钠Trisodium citrate dihydrate	-	-	甘露醇Mannitol	+	+
丙二酸纳Sodium malonate	+	+

项目Items	Wp3	Wp6	项目Items	Wp3	Wp6	项目Items	Wp3	Wp6
糊精Dextrin	−	−	梭链孢酸Fusidic acid	−	−	黏酸Mucic acid	+	+
D-麦芽糖D-Maltose	−	−	D-丝氨酸D-Serine	−	−	奎宁酸QuINIC Acid	+	+
D-海藻糖D-Trehalose	−	−	D-山梨醇D-Sorbitol	−	B	糖质酸D-Saccharic acid	+	+
D-纤维二糖D-Cellobiose	−	−	D-甘露醇D-Mannitol	+	B	万古霉素Vancomycin	+	+
龙胆二糖Gentiobiose	−	−	D-阿拉伯醇D-Arabitol	+	+	四唑紫Tetrazolium violet	+	+
蔗糖Sucrose	−	B	肌醇Inositol	+	B	四唑蓝Tetrazolium blue	+	+
D-松二糖D-Turanose	−	−	丙三醇Glycerol	+	B	p-羟基-苯乙酸p-Hydroxy phenylacetic acid	−	−
水苏糖Stachyose	−	−	D-葡糖-6-磷酸D-Glucose-6-phosphate	−	−	丙酮酸甲酯Methyl pyruvate	B	B
pH=6	+	+	D-果糖-6-磷酸D-Fructose-6-phosphate	B	B	D-乳酸甲酯D-Lactic acid methyl ester	−	−
pH=5	−	−	D-天冬氨酸D-Aspartic acid	B	B	L-乳酸L-Lactic acid	+	+
棉子糖D-Raffinose	−	−	D-Serine D-丝氨酸	B	B	柠檬酸Citric acid	+	+
α-D-乳糖α-D-Lactose	−	−	醋竹桃霉素Troleandomycin	B	−	α-酮-戊二酸α-Keto-glutaric acid	+	+
蜜二糖D-Melibiose	−	−	利福霉素SV Rifamycin SV	+	+	D-苹果酸D-Malic acid	+	+
β-甲酰-D-葡糖苷β-Methyl-D-glucoside	−	−	二甲胺四环素Minocycline	-	−	L-苹果酸L-Malic acid	+	+
D-水杨苷D-Salicin	−	−	明胶Gelatin	−	−	溴-丁二酸Bromo-succinic acid	B	B
N-乙酰-D-葡糖胺N-Acetyl-D-Glucosamine	−	−	氨基乙酰-L-脯氨酸Glycyl-L-proline	B	B	萘啶酮酸Nalidixic acid	−	−
N-乙酰-β-D-甘露糖胺N-Acetyl-β-D-mannosamine	−	−	L-丙氨酸L-Alanine	+	+	氯化锂Lithium chloride	−	−
N-乙酰-D-半乳糖胺N-Acetyl-D-Gal actosamine	−	−	L-Arginine L-精氨酸	B	B	亚碲酸钾Potassium tellurite	+	+
N-乙酰神经氨酸N-Acetyl Neuraminic Acid	−	−	L-天冬氨酸L-Aspartic acid	+	+	吐温40 Tween 40	B	B
1% NaCl	+	+	L-谷氨酸L-Glutamic acid	+	+	γ-氨基-丁酸γ-Amino-butryric acid	+	+
4% NaCl	B	B	L-组胺L-Histidine	B	B	α-羟基-丁酸α-Hydroxy butyric acid	B	B
8% NaCl	−	−	L-焦谷氨酸L-Pyroglutamic acid	B	B	β-羟基-D,L丁酸β-Hydroxy-D,L butyric acid	B	B
α-D-Glucoseα-D-葡萄糖	+	+	L-丝氨酸L-Serine	+	+	α-酮-丁酸α-Keto-butyric acid	B	−
D-甘露糖D-Mannose	+	+	林肯霉素Lincomycin	+	+	乙酰乙酸Acetoacetic acid	B	B
D-果糖D-Fructose	B	B	盐酸胍Guanidine hydrochloride	B	B	丙酸Propionic acid	B	−
D-半乳糖D-Galactose	+	+	硫酸四癸钠Tetradecyl sodium sulfate	+	+	乙酸Acetic acid	+	+
3-甲酰葡糖3-Methyl Glucose	−	−	果胶Pectin	B	B	甲酸Formic acid	+	+
D-果糖D-Fucose	+	+	D-半乳糖醛酸D-Galacturonic Acid	+	+	氨曲南Aztreonam	−	−
L-果糖L-Fucose	+	+	L-半乳糖醛酸内酯L-Galactonic acid lactone	+	+	丁酸钠Sodium butyrate	-	−
L-鼠李糖L-Rhamnose	−	−	D-葡糖酸D-Gluconic acid	+	+	溴酸钠Sodium bromate	-	−
肌苷Inosine	+	+	D-葡糖醛酸D-Glucuronic acid	+	+
1%乳酸钠1%Sodium Lactate	+	+	葡糖醛酰胺Glucuronamide	+	+

项目Items	Wp3	Wp6	项目Items	Wp3	Wp6	项目Items	Wp3	Wp6
糊精Dextrin	−	−	梭链孢酸Fusidic acid	−	−	黏酸Mucic acid	+	+
D-麦芽糖D-Maltose	−	−	D-丝氨酸D-Serine	−	−	奎宁酸QuINIC Acid	+	+
D-海藻糖D-Trehalose	−	−	D-山梨醇D-Sorbitol	−	B	糖质酸D-Saccharic acid	+	+
D-纤维二糖D-Cellobiose	−	−	D-甘露醇D-Mannitol	+	B	万古霉素Vancomycin	+	+
龙胆二糖Gentiobiose	−	−	D-阿拉伯醇D-Arabitol	+	+	四唑紫Tetrazolium violet	+	+
蔗糖Sucrose	−	B	肌醇Inositol	+	B	四唑蓝Tetrazolium blue	+	+
D-松二糖D-Turanose	−	−	丙三醇Glycerol	+	B	p-羟基-苯乙酸p-Hydroxy phenylacetic acid	−	−
水苏糖Stachyose	−	−	D-葡糖-6-磷酸D-Glucose-6-phosphate	−	−	丙酮酸甲酯Methyl pyruvate	B	B
pH=6	+	+	D-果糖-6-磷酸D-Fructose-6-phosphate	B	B	D-乳酸甲酯D-Lactic acid methyl ester	−	−
pH=5	−	−	D-天冬氨酸D-Aspartic acid	B	B	L-乳酸L-Lactic acid	+	+
棉子糖D-Raffinose	−	−	D-Serine D-丝氨酸	B	B	柠檬酸Citric acid	+	+
α-D-乳糖α-D-Lactose	−	−	醋竹桃霉素Troleandomycin	B	−	α-酮-戊二酸α-Keto-glutaric acid	+	+
蜜二糖D-Melibiose	−	−	利福霉素SV Rifamycin SV	+	+	D-苹果酸D-Malic acid	+	+
β-甲酰-D-葡糖苷β-Methyl-D-glucoside	−	−	二甲胺四环素Minocycline	-	−	L-苹果酸L-Malic acid	+	+
D-水杨苷D-Salicin	−	−	明胶Gelatin	−	−	溴-丁二酸Bromo-succinic acid	B	B
N-乙酰-D-葡糖胺N-Acetyl-D-Glucosamine	−	−	氨基乙酰-L-脯氨酸Glycyl-L-proline	B	B	萘啶酮酸Nalidixic acid	−	−
N-乙酰-β-D-甘露糖胺N-Acetyl-β-D-mannosamine	−	−	L-丙氨酸L-Alanine	+	+	氯化锂Lithium chloride	−	−
N-乙酰-D-半乳糖胺N-Acetyl-D-Gal actosamine	−	−	L-Arginine L-精氨酸	B	B	亚碲酸钾Potassium tellurite	+	+
N-乙酰神经氨酸N-Acetyl Neuraminic Acid	−	−	L-天冬氨酸L-Aspartic acid	+	+	吐温40 Tween 40	B	B
1% NaCl	+	+	L-谷氨酸L-Glutamic acid	+	+	γ-氨基-丁酸γ-Amino-butryric acid	+	+
4% NaCl	B	B	L-组胺L-Histidine	B	B	α-羟基-丁酸α-Hydroxy butyric acid	B	B
8% NaCl	−	−	L-焦谷氨酸L-Pyroglutamic acid	B	B	β-羟基-D,L丁酸β-Hydroxy-D,L butyric acid	B	B
α-D-Glucoseα-D-葡萄糖	+	+	L-丝氨酸L-Serine	+	+	α-酮-丁酸α-Keto-butyric acid	B	−
D-甘露糖D-Mannose	+	+	林肯霉素Lincomycin	+	+	乙酰乙酸Acetoacetic acid	B	B
D-果糖D-Fructose	B	B	盐酸胍Guanidine hydrochloride	B	B	丙酸Propionic acid	B	−
D-半乳糖D-Galactose	+	+	硫酸四癸钠Tetradecyl sodium sulfate	+	+	乙酸Acetic acid	+	+
3-甲酰葡糖3-Methyl Glucose	−	−	果胶Pectin	B	B	甲酸Formic acid	+	+
D-果糖D-Fucose	+	+	D-半乳糖醛酸D-Galacturonic Acid	+	+	氨曲南Aztreonam	−	−
L-果糖L-Fucose	+	+	L-半乳糖醛酸内酯L-Galactonic acid lactone	+	+	丁酸钠Sodium butyrate	-	−
L-鼠李糖L-Rhamnose	−	−	D-葡糖酸D-Gluconic acid	+	+	溴酸钠Sodium bromate	-	−
肌苷Inosine	+	+	D-葡糖醛酸D-Glucuronic acid	+	+
1%乳酸钠1%Sodium Lactate	+	+	葡糖醛酰胺Glucuronamide	+	+

菌株 Strain	最佳匹配种类 Best match species	可能性值 Probable value	相似性值 Similarity value	位距值 Bit distance value
Wp3	P. syringae	0.653	0.570	3.769
Wp6	P. syringae	0.624	0.540	3.260

无花果细菌性叶斑病病原鉴定

Identification of etiological bacterium causing leaf spot disease on Ficus carica

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