福建无花果细菌性叶斑病的症状及病原菌鉴定

doi:10.13304/j.nykjdb.2023.0546

摘要/Abstract

摘要：

为明确2020年以来在福州市郊区种植无花果上新发生的细菌性叶斑病的病因，在果园内采集病样进行病原菌分离纯化，利用烟草过敏性反应测定、喷雾接种无花果叶片以及柯赫氏法则验证，明确其致病性；通过对病原菌的形态特征观察、生理生化测定，结合16S rDNA、gyrB和rpoD基因序列分析，确定病原菌的分类地位。结果表明，从病样中分离纯化获得10株细菌菌株；烟草过敏性反应阳性；人工接种结果显示，供试细菌可侵染健康的无花果叶片，并产生与田间相似的症状，且重新分离到与原菌落形态相同的细菌，柯赫氏法则证明这些菌株为该病害的病原菌。该病菌在NA培养基上培养2 d后，菌落为淡黄色、圆形、扁平；菌体形状为短杆状，革兰氏染色阴性；病菌在KB培养基上能产生绿色水溶性荧光色素；供试菌株的16S rDNA、gyrB和rpoD基因序列分析结果显示，供试菌株与菊苣假单胞菌（Pseudomonas cichorii）聚为一支；其生理生化和Biolog表型测定结果以及菊苣假单胞菌特异性引物检测结果均与菊苣假单胞菌的模式菌株5707一致。因此将该致病菌鉴定为菊苣假单胞菌（P. cichorii）。这是首次发现菊苣假单胞菌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, 菊苣假单胞菌, 管家基因

Abstract:

To clarify the taxonomy of the pathogen causing a new bacterial leaf spot disease on fig （Ficus carica） in Fuzhou in 2020， diseased fig leaves were collected as test materials. The bacterial strains were isolated from the diseased leaves by streak plate method， and then the pathogenicity of the strains was determined by tobacco allergic reaction， spray inoculated fig leaves and Koch’s rule. The pathogen was identified based on morphological characteristics， physiological and biochemical determination， and phylogenetic tree analysis using partial sequences of 16S rDNA， gyrB and rpoD. The results showed that 10 bacterial strains were isolated and purified from the diseased leaves， healthyfig leaves demonstrated similar symptoms as the specimen collected from fields， and the identical morphological bacterial strains were re-isolated from diseased fig leaves. Thus， all of the bacterial strains were identified as the causal pathogens of bacterial leaf spot disease by Koch’s rule. The colony morphology was light yellow， round， and flat on the NA medium after 2 d of culture， and the gram staining reaction was negative. The bacterial strains could produce green water-soluble fluorescent on KB medium. The sequencing analysis based on 16S rDNA， gyrB and rpoD showed that the test strains were clustered with Pseudomonas cichorii； and the results of physiological and biochemical characteristics， Biolog phenotypes instrument， and specific primers were similar to the P. cichorii strain 5707， therefore the 10 isolated bacterial strains were identified as P. cichorii. It was the first report that P. cichorii could naturally cause the leaf spot disease in F. carica.

Key words: Ficus carica bacterial leaf spot disease, pathogen identification, Pseudomonas chicorii, housekeeping gene

中图分类号:

S663.3

季梦婷, 朱玲, 罗晓华, 郑钰婷, 肖顺, 胡方平, 蔡学清. 福建无花果细菌性叶斑病的症状及病原菌鉴定[J]. 中国农业科技导报, 2025, 27(1): 147-154.

Mengting JI, Ling ZHU, Xiaohua LUO, Yuting ZHENG, Shun XIAO, Fangping HU, Xueqing CAI. Symptoms and Pathogen Identification of Ficus carica Bacterial Leaf Spot Disease in Fujian[J]. Journal of Agricultural Science and Technology, 2025, 27(1): 147-154.

图/表 7

图1 无花果细菌性叶斑病的田间症状A：整株发病植株症状；B：初期症状；C：中期症状（黄色晕圈）；D：后期症状

Fig. 1 Symptoms of bacterial leaf spot disease on Ficus carica in the fieldA： Symptom of whole plant； B： Early symptom； C： Middle symptom （yellow halo）； D： Late symptom

图2 接种W10和未接种的无花果叶片正面和背面A：接种W10的叶片正面；B：接种W10的叶片背面；C：CK的叶片正面；D：CK的叶片背面

Fig. 2 Front and back of fig leaf inoculated with bacterial strain W10 and uninoculated bacterial strainA： Front of leaf inoculated with W10； B： Back of leaf inoculated with W10； C： Front of leaf inoculated with water； D： Back of leaf inoculated with water

图3 供试细菌菌株在NA培养基上培养24 h的菌落形态和电子显微镜下的鞭毛特征A：平板菌落；B：单菌落；C：鞭毛

Fig. 3 Colony morphology characteristics of test strain on NA medium after culture 24 h and the flagellum under electron microscopyA： Plate colony； B： Single colony； C： Flagellum

图4 系统发育树A：基于16S rDNA；B：基于gyrB和rpoD基因

Fig.4 Phylogenetic treeA： Based on 16S rDNA； B： Based on gyrB and rpoD genes

图5 供试菌株的特异性引物Hrp1a/Hrp2a扩增结果注：M—DL2000 marker；1~10—菌株W1~W10；11—5707；12—Pc-GD-1。

Fig. 5 PCR amplification results of specific primer Hrp1a/Hrp2a of the test strainsNote：M—DL2000 marker；1~10—Strain W1~W10；11—Strain 5707； 12—strain Pc-GD-1.

表1 供试菌株生理生化测定结果

Table 1 Results of physiological and biochemical characteristics of the test strains

项目Item	供试菌株（W1~W10） Test strain（W1~W10）	菌株 5707	项目Item	供试菌株（W1~W10） Test strains（W1~W10）	菌株 5707
KB荧光 Produce fluorescence on KB	+	+	丙二酸纳 sodium malonate	+	+
革兰氏染色 Gram stain	-	-	麦芽糖 Maltose	-	-
精氨酸双水解 Arginine hydrolysis	-	-	纤维二糖 Cellobiose	-	-
过氧化氢反应 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 Reduction of potassium nitrate	-	-	鼠李糖 L-rhamnose monohydrate	-	-
吲哚的产生 Indole production	-	-	海藻糖 D-trehalose anhydrous	-	-
溶解果胶活性 Hydrolysis of pectinase	-	-	棉籽糖 Raffinose	-	-
果聚糖的产生 Fructan production	-	-	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	+	+

表 2 供试细菌菌株的 Biolog 测定结果

Table 2 Biolog test results of the test bacterial strain

菌株编号 Strain number	最佳匹配种类 Best match species	可能性 Probability	相似性 Similarity	位距值 Bit distance value
W1	P. cichorii	0.970	0.801	2.432
W2	P. cichorii	0.963	0.800	2.393
W3	P. cichorii	0.961	0.786	2.611
W5	P. cichorii	0.960	0793	2.394
W10	P. cichorii	0.907	0.721	2.969

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