柿叶枯病病原菌鉴定、生物学特性及杀菌剂毒力测定

doi:10.16420/j.issn.0513-353x.2024-0337

摘要/Abstract

摘要：

为明确引起柿（Diospyros kaki L.）叶枯病的病原菌，采集具有典型叶枯病症状的柿叶片，通过组织分离法和柯赫氏法致病性验证，获得柿叶枯病致病菌。通过形态学观察、ITS分析和TEF-CAL-HIS-TUB多基因序列串联系统发育分析，将柿叶枯病病原菌鉴定为间座壳属真菌Diaporthe eres。生物学特性测定表明，菌株的最适生长碳源为葡萄糖和麦芽糖，最适氮源为酵母粉，最适生长温度为28 ℃，最适pH 6.0。杀菌剂室内毒力测定结果表明，供试的9种杀菌剂对柿叶枯病菌的菌丝生长均表现出不同程度的抑制作用，其中95%咯菌腈的抑制作用最强，EC₅₀值为0.046 mg · L^-1；其次是95%己唑醇、80%多菌灵和97%苯醚甲环唑，EC₅₀值分别为0.217、0.224和0.705 mg · L^-1。

关键词: 柿, 叶枯病, 病原菌, 分离鉴定, 间座壳属, 杀菌剂筛选, 多基因序列分析

Abstract:

To determine the primary pathogen responsible for persimmon leaf blight disease，leaves exhibiting typical symptoms of the blight were collected. The fungus was isolated and purified using the tissue separation method，and its pathogenicity was confirmed through Koch’s postulation. Based on morphological characteristics，ITS analysis，and the combined gene sequence phylogenetic analysis of TEF-CAL-HIS-TUB，the pathogen was identified as Diaporthe eres. The biological characteristics of the isolated D. eres strain DHB1 were investigated. The results showed that a culture medium containing glucose or maltose as the carbon source，along with yeast extract as the nitrogen source，significantly promoted rapid mycelium growth. The optimal conditions for mycelial growth were found to be a temperature of 28 ℃ and a pH of 6.0. In vitro toxicity tests were conducted to evaluate the effects of nine fungicides on the mycelial growth of D. eres DHB1. The results indicated that all tested fungicides had varying degrees of inhibitory effects. Among them，95% Fludioxonil had the strongest inhibitory effect with an EC₅₀value of 0.046 mg · L^-1，followed by 95% Hexaconazole，80% Carbendazim WP and 97% Difenoconazole，with EC₅₀values of 0.217，0.224 and 0.705 mg · L^-1，respectively.

Key words: Diospyros kaki, leaf blight, pathogen, Diaporthe, fungicide screening, multi-locus phylogeny

王洁, 董晓旭, 陈金霄, 余贤美, 高瑞, 艾呈祥, 沈广宁. 柿叶枯病病原菌鉴定、生物学特性及杀菌剂毒力测定[J]. 园艺学报, 2025, 52(3): 737-748.

WANG Jie, DONG Xiaoxu, CHEN Jinxiao, YU Xianmei, GAO Rui, AI Chengxiang, SHEN Guangning. Identification and Biological Characteristics of the Pathogen Causing Persimmon Leaf Blight and Toxicity Test of Different Fungicides[J]. Acta Horticulturae Sinica, 2025, 52(3): 737-748.

图/表 9

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片段 Target	引物 Primer	引物序列（5′-3′） Sequence	退火温度/℃ T_m	参考文献 Reference
ITS	ITS-4	TCCTCCGCTTATTGATATGC	53	White et al.，1990
ITS	ITS-5	GGAAGTAAAAGTCGTAACAAGG	53	White et al.，1990
TEF	EF1-728F	CATCGAGAAGTTCGAGAAGG	53	Carbone & Kohn，1999
TEF	EF1-986a	TACTTGAAGGAACCCTTACC	53	Carbone & Kohn，1999
CAL	CAL-228F	GAGTTCAAGGAGGCCTTCTCCC	58	Carbone & Kohn，1999
CAL	CAL-737R	CATCTTCTGGCCATCATGG	58	Carbone & Kohn，1999
HIS	CYLH3F	AGGTCCACTGGTGGCAAG	63	Crous et al.，2006
HIS	H3-1b	GCGGGCGAGCTGGATGTCCTT	63	Glass & Donaldson，1995
TUB	Bt-2a	GGTAACCAAATCGGTGCTGCTTTC	59	Glass & Donaldson，1995
TUB	Bt2b	ACCCTCAGTGTAGTGACCCTTGGC	59	Glass & Donaldson，1995

片段 Target	引物 Primer	引物序列（5′-3′） Sequence	退火温度/℃ T_m	参考文献 Reference
ITS	ITS-4	TCCTCCGCTTATTGATATGC	53	White et al.，1990
ITS	ITS-5	GGAAGTAAAAGTCGTAACAAGG	53	White et al.，1990
TEF	EF1-728F	CATCGAGAAGTTCGAGAAGG	53	Carbone & Kohn，1999
TEF	EF1-986a	TACTTGAAGGAACCCTTACC	53	Carbone & Kohn，1999
CAL	CAL-228F	GAGTTCAAGGAGGCCTTCTCCC	58	Carbone & Kohn，1999
CAL	CAL-737R	CATCTTCTGGCCATCATGG	58	Carbone & Kohn，1999
HIS	CYLH3F	AGGTCCACTGGTGGCAAG	63	Crous et al.，2006
HIS	H3-1b	GCGGGCGAGCTGGATGTCCTT	63	Glass & Donaldson，1995
TUB	Bt-2a	GGTAACCAAATCGGTGCTGCTTTC	59	Glass & Donaldson，1995
TUB	Bt2b	ACCCTCAGTGTAGTGACCCTTGGC	59	Glass & Donaldson，1995

种类名称 Species name	菌株 Strain	GenBank登录号GenBank accession number
种类名称 Species name	菌株 Strain	ITS	CAL	HIS	TEF	TUB
D. alnea	CBS146.46	KC343008	KC343250	KC343492	KC343734	KC343976
D. betulina	CFCC 52562	MH121497	MH121421	MH121457	MH121539	MH121579
D. bicincta	CBS 121004	KC343134	KC343376	KC343618	KC343860	KC344102
D. celastrina	CBS 139.27	KC343047	KC343289	KC343531	KC343773	KC344015
D. celeris	CBS 143349	MG281017	MG281712	MG281363	MG281538	MG281190
D. citri	CBS 135422	KC843311	KC843157	MF418281	KC843071	KC843187
D. eres	CBS 101742	KC343073	KC343315	KC343557	KC343799	KC344041
	FAU506	KJ210526	KJ435012	KJ420842	JQ807403	KJ420792
	CPC 30073	MG281052	MG281749	MG281400	MG281573	MG28122
	NFFT-3-8	MN816419	MN894369	MN894402	MN894440	MN894479
	DHB1	PP464122	PP481215	PP481216	PP481217	PP471558
	CPC 29677	MG281039	MG281736	MG281387	MG281560	MG281212
	DLR12A	KJ210518	KJ434996	KJ420833	KJ210542	KJ420783
	ACCC35482	OP963195	-	OP968955	OP968958	OP978141
	PSCG 041	MK626880	MK691144	MK726219	MK654840	MK691265
D. eres (D. biguttusis)	CGMCC3.17081	KF576282	-	-	KF576257	KF576306
D. eres (D. camptothecicola)	CFCC 51632	KY203726	KY228877	KY228881	KY228887	KY228893
D. eres (D. castaneae-mollissimae)	DNP128	JF957786	KJ435040	KJ420852	KJ210561	KJ420801
D. eres (D. cotoneastri)	CBS 439.82	FJ889450	JX197429	-	GQ250341	JX275437
D. eres (D. ellipicola)	CGMCC3.17084	KF576270	-	-	KF576245	KF576291
D. eres (D. henanensis)	CGMCC3.17639	KC898258	-	KF600609	-	KF600608
D. eres (D. longicicola)	CGMCC3.17089	KF576267	-	-	KF576242	KF576291
D. eres (D. lonicerae)	MFLUCC 17-0963	KY964190	KY964116	-	KY964146	KY964073
D. eres (D. mahothocarpus)	CGMCC3.15181	KC153096	-	-	KC153087	KF576312
D. eres (D. momicola)	CGMCC3.17466	KU557563	KU557611	-	KU557631	KU557587
D. eres (D. nobilis)	CBS113470	KC343146	KC343388	KC343630	KC343872	KC344114
D. eres (D. rosicola)	MFLU17-0646	MG828895	-	-	MG829270	MG843877
D. eres (Phomopsis fukushii)	MAFF625033	JQ807468	KJ435017	KJ420865	JQ807417	KJ420814
D. helicis	CBS138596	KJ210538	KJ435043	KJ420875	KJ210559	KJ420828
D. neilliae	CBS 144.27	KC343144	KC343386	KC343628	KC343870	KC344112
D. padina	CFCC52590	MH121525	MH121443	MH121483	MH121567	MH121604
D. phragmitis	CBS138897	KP004445	-	KP004503	-	KP004507
D. pulla	CBS338.89	KC343152	KC343394	KC343636	KC343878	KC344120

种类名称 Species name	菌株 Strain	GenBank登录号GenBank accession number
种类名称 Species name	菌株 Strain	ITS	CAL	HIS	TEF	TUB
D. alnea	CBS146.46	KC343008	KC343250	KC343492	KC343734	KC343976
D. betulina	CFCC 52562	MH121497	MH121421	MH121457	MH121539	MH121579
D. bicincta	CBS 121004	KC343134	KC343376	KC343618	KC343860	KC344102
D. celastrina	CBS 139.27	KC343047	KC343289	KC343531	KC343773	KC344015
D. celeris	CBS 143349	MG281017	MG281712	MG281363	MG281538	MG281190
D. citri	CBS 135422	KC843311	KC843157	MF418281	KC843071	KC843187
D. eres	CBS 101742	KC343073	KC343315	KC343557	KC343799	KC344041
	FAU506	KJ210526	KJ435012	KJ420842	JQ807403	KJ420792
	CPC 30073	MG281052	MG281749	MG281400	MG281573	MG28122
	NFFT-3-8	MN816419	MN894369	MN894402	MN894440	MN894479
	DHB1	PP464122	PP481215	PP481216	PP481217	PP471558
	CPC 29677	MG281039	MG281736	MG281387	MG281560	MG281212
	DLR12A	KJ210518	KJ434996	KJ420833	KJ210542	KJ420783
	ACCC35482	OP963195	-	OP968955	OP968958	OP978141
	PSCG 041	MK626880	MK691144	MK726219	MK654840	MK691265
D. eres (D. biguttusis)	CGMCC3.17081	KF576282	-	-	KF576257	KF576306
D. eres (D. camptothecicola)	CFCC 51632	KY203726	KY228877	KY228881	KY228887	KY228893
D. eres (D. castaneae-mollissimae)	DNP128	JF957786	KJ435040	KJ420852	KJ210561	KJ420801
D. eres (D. cotoneastri)	CBS 439.82	FJ889450	JX197429	-	GQ250341	JX275437
D. eres (D. ellipicola)	CGMCC3.17084	KF576270	-	-	KF576245	KF576291
D. eres (D. henanensis)	CGMCC3.17639	KC898258	-	KF600609	-	KF600608
D. eres (D. longicicola)	CGMCC3.17089	KF576267	-	-	KF576242	KF576291
D. eres (D. lonicerae)	MFLUCC 17-0963	KY964190	KY964116	-	KY964146	KY964073
D. eres (D. mahothocarpus)	CGMCC3.15181	KC153096	-	-	KC153087	KF576312
D. eres (D. momicola)	CGMCC3.17466	KU557563	KU557611	-	KU557631	KU557587
D. eres (D. nobilis)	CBS113470	KC343146	KC343388	KC343630	KC343872	KC344114
D. eres (D. rosicola)	MFLU17-0646	MG828895	-	-	MG829270	MG843877
D. eres (Phomopsis fukushii)	MAFF625033	JQ807468	KJ435017	KJ420865	JQ807417	KJ420814
D. helicis	CBS138596	KJ210538	KJ435043	KJ420875	KJ210559	KJ420828
D. neilliae	CBS 144.27	KC343144	KC343386	KC343628	KC343870	KC344112
D. padina	CFCC52590	MH121525	MH121443	MH121483	MH121567	MH121604
D. phragmitis	CBS138897	KP004445	-	KP004503	-	KP004507
D. pulla	CBS338.89	KC343152	KC343394	KC343636	KC343878	KC344120

杀菌剂 Fungicides	毒力回归方程 Regress equation	相关系数 Correlation coefficient		抑制中浓度/（mg · L^-1） EC₅₀
95% 咯菌腈 Fludioxonil	y = 1.153x + 1.542	0.983		0.046
95% 己唑醇 Hexaconazole	y = 1.407x + 0.933	0.999		0.217
80% 多菌灵 Carbendazim WP	y = 1.518x + 0.986	0.993		0.224
97% 苯醚甲环唑 Difenoconazole	y = 0.860x + 0.130	0.998		0.705
98% 吡唑醚菌酯 Pyraclostrobin	y = 1.068x - 0.130	0.998		1.323
97% 戊唑醇 Tebuconazole	y = 1.569x - 0.305	0.993		1.565
80% 代森锰锌 Mancozeb WP	y = 1.395x - 0.911	0.972		4.496
94% 腈菌唑 Myclobutanil	y = 1.265x - 1.162		0.982	8.289
98% 肟菌酯 Trifloxystrobin	y = 0.987x - 1.038		0.995	11.245