连翘褐斑病病原菌鉴定及其对杀菌剂敏感性研究

doi:10.16420/j.issn.0513-353x.2021-0335

摘要/Abstract

摘要：

采用组织分离法分离纯化得到连翘褐斑病病原菌,依据科赫氏法则验证其致病性,结合病原菌形态学特征及其ITS、LSU和SSU序列的多基因联合分析,确定病原菌为链格孢Alternaria alternata。测定了培养基、温度、pH、光照、碳源和氮源等处理对该病原菌生长、产孢和孢子萌发的影响,并采用菌丝生长速率法测定其对8种杀菌剂的敏感性。结果表明,菌丝生长最适条件为25 ~ 30 ℃,pH 7.0,碳源为木糖醇和甘露醇,氮源为蛋白胨;产孢最适条件为25 ℃,pH 7.0,碳源木糖醇,氮源为尿素;孢子萌发最适条件为15 ~ 35 ℃,pH 5 ~ 9,碳源为木糖醇,氮源为牛肉浸粉。在测试的8种杀菌剂中,嘧菌环胺（EC₅₀ = 0.032 mg · L^-1）和苯醚甲环唑（EC₅₀ = 0.054 mg · L^-1）对其具有较强的抑制作用。

关键词: 连翘, 褐斑病, 链格孢, 生物学特性, 病原菌, 鉴定, 药剂筛选

Abstract:

In order to clarify the species and biological characteristics of the pathogen of the brown spot of Forsythia suspensa（Thunb.）Vahl,the pathogen was isolated by tissue separation method and was tested for its pathogenicity following Koch’s postulation. Combing morphological feature with molecular identification of the ITS,LSU and SSU sequences,the pathogen was identified as Alternaria alternata. Growth response of the pathogen to different medium,light treatments,temperature,pH,carbon sources,and nitrogen sources were also studied. The toxicities of eight fungicides against the pathogen were tested using mycelial growth rate method. The results showed that the optimal conditions for mycelial growth were 25-30 ℃ at pH 7.0 with xylitol and mannitol as the carbon sources and peptone as the nitrogen source. The optimal sporulation conditions were 25 ℃ at pH 7.0 with xylitol as the carbon source and urea as nitrogen source. The optimal conditions for conidia germination were 15-35 ℃ at pH 5-9 with xylitol as the carbon source and beef extract powder as the nitro gen source. Among eight fungicides tested in toxicity assays,Cyprodinil（EC₅₀= 0.032 mg · L^-1）and Difenoconazole（EC₅₀= 0.054 mg · L^-1） had stronger inhibitory effect on A. alternata.

Key words: Forsythia suspensa, brown spot, Alternaria alternata, biological characteristics, pathogen, identification, fungicide screening.

中图分类号:

S685.24

刘鹏, 李钦, 张维瑞, 贺社起, 张素平, 马晓旭, 袁王俊. 连翘褐斑病病原菌鉴定及其对杀菌剂敏感性研究[J]. 园艺学报, 2022, 49(8): 1805-1814.

LIU Peng, LI Qin, ZHANG Weirui, HE Sheqi, ZHANG Suping, MA Xiaoxu, YUAN Wangjun. Identification,Biological Characteristics and Fungicide Sensitivity of the Pathogen Causing Brown Spot Disease on Forsythia[J]. Acta Horticulturae Sinica, 2022, 49(8): 1805-1814.

图/表 7

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碳源 Carbon source	萌发率 Germination rate	氮源 Nitrogen source	萌发率 Germination rate
木糖醇 Xylitol	78.01 ± 5.25 a	氯化铵 Ammonium chloride	0 c
葡萄糖 Glucose	40.14 ± 4.28 d	草酸铵 Ammonium oxalate monohydrate	0 c
蔗糖 Sucrose	74.83 ± 5.38 ab	甘氨酸 Glycine	69.23 ± 6.51 a
山梨醇 Sorbitol	74.15 ± 5.59 ab	尿素 Urea	62.77 ± 3.41 a
乳糖 Lactose	56.40 ± 7.44 c	牛肉浸粉 Beef Powder	66.35 ± 5.94 a
甘露醇 Mannitol	74.14 ± 2.28 ab	硝酸钠 Sodium nitrate	62.44 ± 7.42 a
果糖 Fructose	68.19 ± 5.10 b	蛋白胨 Peptone bacteriological	43.80 ± 3.30 b
对照 Control	67.29 ± 4.38 b	对照 Control	67.29 ± 4.38 a

碳源 Carbon source	萌发率 Germination rate	氮源 Nitrogen source	萌发率 Germination rate
木糖醇 Xylitol	78.01 ± 5.25 a	氯化铵 Ammonium chloride	0 c
葡萄糖 Glucose	40.14 ± 4.28 d	草酸铵 Ammonium oxalate monohydrate	0 c
蔗糖 Sucrose	74.83 ± 5.38 ab	甘氨酸 Glycine	69.23 ± 6.51 a
山梨醇 Sorbitol	74.15 ± 5.59 ab	尿素 Urea	62.77 ± 3.41 a
乳糖 Lactose	56.40 ± 7.44 c	牛肉浸粉 Beef Powder	66.35 ± 5.94 a
甘露醇 Mannitol	74.14 ± 2.28 ab	硝酸钠 Sodium nitrate	62.44 ± 7.42 a
果糖 Fructose	68.19 ± 5.10 b	蛋白胨 Peptone bacteriological	43.80 ± 3.30 b
对照 Control	67.29 ± 4.38 b	对照 Control	67.29 ± 4.38 a

温度/℃ Tempearture	萌发率 Germination rate	pH	萌发率 Germination rate	光照条件 Photoperiod	萌发率 Germination rate
5	0 c	4	64.29 ± 4.15 b	黑暗 Dark	63.23 ± 5.60 a
10	22.45 ± 9.36 b	5	76.36 ± 7.01 a	半光 Half light	64.16 ± 2.21 a
15	62.22 ± 9.26 a	6	72.16 ± 4.36 a	全光 Full light	71.08 ± 6.57 a
20	61.32 ± 6.29 a	7	75.03 ± 1.25 a
25	67.29 ± 4.38 a	8	76.69 ± 8.03 a
30	55.48 ± 11.72 a	9	76.24 ± 4.09 a
35	58.47 ± 4.48 a	10	65.21 ± 2.63 b

温度/℃ Tempearture	萌发率 Germination rate	pH	萌发率 Germination rate	光照条件 Photoperiod	萌发率 Germination rate
5	0 c	4	64.29 ± 4.15 b	黑暗 Dark	63.23 ± 5.60 a
10	22.45 ± 9.36 b	5	76.36 ± 7.01 a	半光 Half light	64.16 ± 2.21 a
15	62.22 ± 9.26 a	6	72.16 ± 4.36 a	全光 Full light	71.08 ± 6.57 a
20	61.32 ± 6.29 a	7	75.03 ± 1.25 a
25	67.29 ± 4.38 a	8	76.69 ± 8.03 a
30	55.48 ± 11.72 a	9	76.24 ± 4.09 a
35	58.47 ± 4.48 a	10	65.21 ± 2.63 b

药剂 Fungicide	毒力回归方程 Regression equation	决定系数 R² Coefficient of determination	抑制中浓度/（mg · L^-1） EC₅₀
嘧菌环胺 Cyprodinil	y = 0.415x + 6.8651	0.9614	0.032
苯醚甲环唑 Difenoconazole	y = 0.2601x + 6.11	0.9513	0.054
吡唑醚菌酯 Pyraclostrobine	y = 0.2014x + 5.4204	0.9138	8.2
百菌清 Chlorothalonil	y = 0.2481x + 5.279	0.9435	75.1
春雷霉素 Kasugamycin	y = 1.8876x + 6.0587	0.9941	274.9
甲基硫菌灵 Thiophanate-methyl	y = 1.2704x + 4.1356	0.9629	4 790.9
枯草芽孢杆菌 Bacillus subtilis	y = -0.7751x + 6.0136	0.9499	20 309.6
嘧菌酯 Azoxystrobin	y = 0.4271x + 3.5136	0.9428	3 021 450