Identification and Biology of Leaf Spot Pathogen on Chimonanthus salicifolius
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摘要:目的 为明确柳叶蜡梅（Chimonanthus Salicifolius）的叶部病害的病原种类，进行病叶病原分离鉴定并研究病原菌株生物学特性。方法 对福建省寿宁县福瑞泰生物技术有限责任公司种植基地的柳叶腊梅叶斑病病叶采用离体组织分离法进行分离，对分离的病原菌采用形态学结合分子方法（rDNA-ITS和TUB序列）进行鉴定，并对该致病菌菌丝体在不同温度、pH值、光照、碳源、氮源及致死温度等条件下的生物学特性进行研究。结果 病原菌分离株经形态特征观测及rDNA-ITS和TUB序列在NCBI数据库的分析比对，将寿宁县柳叶腊梅叶斑病病原菌鉴定为子囊菌门，格孢腔目，附球真菌属，高粱附球菌（Epicoccum Sorghinum）。生物学特性结果表明：在 5～30 ℃均可生长，最适生长温度 25 ℃；菌丝体生长最适pH值范围为5～9，在供试的11种碳源上均可生长，最佳碳源为蔗糖；在8种氮源培养基中最适菌丝生长的氮源为蛋白胨；光暗交替不影响菌丝生长，菌丝体致死温度为 52 ℃。结论 分离鉴定了福建省寿宁县柳叶腊梅叶斑病病害的病原菌为高粱附球菌(E. sorghinum)，生物学测定结果显示其对环境适应性强。Abstract:Objective Pathogen that caused the leaf spot disease on Chimonanthus salicifolius was isolated and classified, and its biological characteristics determined.Method Specimens of C. salicifolius plants infected by leaf spot disease at Shouning county in Fujian were collected for in vitro tissue culture in the laboratory. Morphological and molecular examinations were performed on the isolates. Biological properties and culture conditions including temperature, pH, light, carbon source, nitrogen source, and lethal temperature were scrutinized for the pathogenic identification and characterization.Result Based on the morphology as well as the rDNA ITS and TUB sequences, the pathogen responsible for the disease was identified to be Ascomycetes Pleosporales Epicoccum sorghinum. It could grow at 5-30 ℃ with an optimum temperature at 25 ℃ and pH ranging from 5 to 9 in media of 11 various carbon sources including sucrose and 8 different nitrogen source including peptone under no specific light/dark conditions. The lethal temperature of the fungal mycelia was 52 ℃.Conclusion E. sorghinum was identified to be the major pathogen that caused the leaf spot disease on C. salicifolius in Shouning county. It had strong ability to grow on, resist to, and survive under adverse conditions.
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图 1 菌落形态和致病性鉴定
注：a为1013-1正面菌落形态；b为1013-1背面菌落形态；c为孢子；d 为10 min NaOH处理；e 为2 h NaOH处理；f为分生孢子器；h为病叶；h为CK(对照)；i为1013-1的致病性测定
Figure 1. Colony morphology and pathogenic assay
Note: a is the colony morphology; b is the back of colony morphology; c is the spore morphology; d the strain on medium 10 min after NaOH treatment; e the strain on medium 2 h after NaOH treatment; f is pycnidium; g is the disease spot of C. salicifolius; h is CK; i is the pathogenic assay of 1013-1strain
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图 2 PCR结果
注：M：为DNA marker；1：为1013-1菌株ITS，2：为1013-1菌株的β-Tub
Figure 2. Result of PCR
Note: M was DNA marker; 1 is 1013-1 stain’s ITS single band, 2 is 1013-1 stain’s β-Tub single band
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图 3 基于ITS+TUB序列联合分析构建NJ-系统树
Figure 3. NJ-system tree derived from ITS and β-Tub sequence analysis
表 1 ITS和β-Tub PCR扩增反应程序
Table 1 Primer ITS and β-Tub PCR amplification procedures
成分 Ingredient 浓度 Concentration 用量 Dosage/μL 扩增程序 Procedure 反应液 2xTaq PCR Master Mix — 10 94 ℃ 3 min；94 ℃ 30 s， 上下游引物 F/R primer 10 μmol·L−1 1 57 ℃ 30 s， 模板 DNA 60 ng·μL−1 2 72 ℃ 1 min，30 cycle； 水 ddH2O — 12 72 ℃ 7 min，end 16 ℃ 
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表 2 不同温度和pH处理病原菌的菌落直径
Table 2 Diameters of pathogen colonies at different temperatures and pHs
处理
Treatments菌落直径
Colony diameter/cm处理
Treatments菌落直径
Colony diameter/cm5 ℃ 0.11±0.10 D pH 3 3.98±0.13 D 10 ℃ 1.12±0.10 C pH 4 7.63±0.12 B 15 ℃ 5.82±0.10 B pH 5 8.00±0.00 A 20 ℃ 5.71±0.23 B pH 6 8.00±0.00 A 25 ℃ 6.89±0.35 A pH 7 8.00±0.00 A 30 ℃ 6.68±0.17 A pH 8 8.00±0.00 A 35 ℃ 0.82±0.03 C pH 9 8.00±0.00 A 40 ℃ 0 pH 10 7.63±0.12 B pH 11 7.37±0.15 B 注：不同大写字母表示0.01水平上的差异极显著。下同。
Note: Different uppercase letters indicate highly significant difference at 0.01 level .The same as below.
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表 3 光照对生长影响
Table 3 Effect of light on mycelial growth
光照处理
Lighting condition菌落直径
Colony diameter/cm全光照 Full light 8.00±0.0 A 半光照 12 h light and dark 7.90±0.10 A 全黑暗 Full dark 8.00±0.00 A
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表 4 病原菌在不同碳源和氮源处理下的菌落直径
Table 4 Diameters of pathogen colonies on media of different carbon and nitrogen sources
不同碳源处理
Different carbon source treatments菌落直径
Colony diameter/cm不同氮源处理
Different nitrogensource treatments菌落直径
Colony diameter/cm葡萄糖 Glucose 7.65±0.05 B 硝酸钾
Potassium nitrate8.00±0.00 A 果糖 Fructose 6.97±0.20 D 酵母粉 Yeast powder 8.00±0.00 A 甘露醇 Mannitol 7.31±0.05 C 蛋白胨 Peptone 8.00±0.00 A 木糖 Xylose 7.40±0.13 C 硝酸钠 Sodium nitrate 8.00±0.00 A 蔗糖 Sucrose 8.00±0.00 A 尿素 The urea 0 乳糖 Lactose 6.59±0.08 E 氯化氨
Ammonia chloride4.25±0.09 C 麦芽糖 Maltose 8.00±0.00 A 甘氨酸 Glycine 8.00±0.0 A 可溶性淀粉
Soluble starch6.02±0.10 F 硫酸铵
Ammonium sulfate5.94±0.31 B 山梨醇 Sorbitol 7.02±0.08 D 肌醇 Inositol 6.88±0.08 D 木糖醇 Xylitol 6.96±0.10 D
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