吉林省玉米穗腐病致病镰孢菌的鉴定与部分菌株对杀菌剂的敏感性

doi:10.3864/j.issn.0578-1752.2023.01.005

摘要/Abstract

摘要：

【目的】明确吉林省玉米穗腐病主要致病镰孢种群分布及杀菌剂对镰孢菌菌丝生长的抑制效果, 为针对性地开展玉米镰孢穗腐病的防治提供依据。【方法】通过组织分离法和分子生物学方法对2020年采自吉林省36个市（县）的149份玉米穗腐病样品进行病原菌分离鉴定，利用禾谷镰孢复合种（Fusarium graminearum species complex，FGSC）毒素合成相关基因特异性引物检测其产生毒素的化学型，对部分禾谷镰孢复合种进行致病力测定；采用菌丝生长速率法测定7种杀菌剂对禾谷镰孢复合种的抑制效果。【结果】分离获得233株镰孢菌，隶属4个镰孢复合种，含9种镰孢菌，包括拟轮枝镰孢（F. verticillioides）、布氏镰孢（F. boothii）、禾谷镰孢（F. graminearum）、层出镰孢（F. proliferatum）、亚洲镰孢（F. asiaticum）、厚垣镰孢（F. chlamydosporum）、藤仓镰孢（F.fujikuroi）、木贼镰孢（F.equiseti）和亚黏团镰孢（F. subglutinans），分离频率依次为33.05%、26.18%、25.32%、12.45%、0.86%、0.86%、0.43%、0.43%和0.43%，其中禾谷镰孢复合种分离频率最高，为52.36%，是吉林省玉米穗腐病的优势致病镰孢菌。布氏镰孢、禾谷镰孢和亚洲镰孢在禾谷镰孢复合种中的占比分别为50.00%、48.36%和1.46%。系统发育树结果表明禾谷镰孢复合种种间、种内遗传多样性均较为丰富。致病力测定结果表明52.73%的禾谷镰孢复合种为中致病型菌株，东部玉米主产区分离的禾谷镰孢致病力最强。毒素化学型检测表明，亚洲镰孢为雪腐镰刀烯醇（NIV）化学型，禾谷镰孢与布氏镰孢为15-乙酰基-脱氧雪腐镰刀烯醇（15-AcDON）化学型。7种杀菌剂抑制禾谷镰孢复合种菌丝生长的EC₅₀介于0.02—19.45 μg·mL^-1，其中咯菌腈（FS）、抑霉唑（FS）、氟硅唑（EC）、戊唑醇（TC）和腈菌唑（EW）对禾谷镰孢复合种抑制效果较好且差异不大，EC₅₀小于1.20 μg·mL^-1，EC₉₀小于100 μg·mL^-1；30%吡唑醚菌酯抑制禾谷镰孢和布氏镰孢生长的EC₅₀差异明显，抑制禾谷镰孢的EC₅₀是布氏镰孢的10.24倍。【结论】吉林省不同玉米产区的玉米穗腐病的优势致病镰孢不同，东部和西部为禾谷镰孢和布氏镰孢，中部为拟轮枝镰孢。禾谷镰孢复合种种间、种内遗传多样性均较丰富。咯菌腈、抑霉唑、氟硅唑、戊唑醇和腈菌唑对禾谷镰孢复合种的抑菌效果较好且在禾谷镰孢复合种间的药剂敏感性差别不明显。

关键词: 玉米, 穗腐病, 镰孢菌, 禾谷镰孢复合种, 杀菌剂

Abstract:

【Objective】The objective of this study is to clarify the population distribution of Fusarium spp. of maize ear rot in Jilin Province and the inhibitory effect of fungicides on the growth of Fusarium mycelium, and to provide a basis for the targeted control of maize ear rot. 【Method】149 samples of maize ear rot collected from 36 cities and counties in Jilin Province in 2020 were isolated and identified by tissue isolation and molecular biology methods. The specific toxin synthesis primers of related genes were synthesized using Fusarium graminearum species complex (FGSC) toxin. The toxigenic chemotypes were detected, and the pathogenicity of some FGSC was determined. The inhibitory effect of 7 fungicides on FGSC was determined by the mycelial growth rate method. 【Result】A total of 233 Fusarium strains were isolated, belonging to 4 Fusarium complex species, including 9 Fusarium species, which were F. verticillioides, F. boothii, F. graminearum, F. proliferatum, F. asiaticum, F. chlamydosporum, F. fujikuroi, F. equiseti and F. subglutinans. The isolation frequencies were 33.05%, 26.18%, 25.32%, 12.45%, 0.86%, 0.86%, 0.43%, 0.43% and 0.43%, respectively. The isolate frequency of FGSC was the highest, which was 52.36%, and it was the dominant pathogen of maize ear rot in Jilin Province. The proportions of F. boothii, F. graminearum and F. asiaticum in FGSC were 50.00%, 48.36% and 1.46%, respectively. The phylogenetic tree showed that the interspecific and intraspecific genetic diversity of FGSC was rich. The results of pathogenicity assay showed that 52.73% of FGSC were medium pathogenic strains. F. graminearum isolated from the main maize producing areas in the east had the strongest pathogenicity. Toxigenic chemotype detection showed that F. asiaticum produced nivalenol (NIV) chemotype, F. graminearum and F. boothii produced 15-acetyl-deoxynivalenol (15-AcDON) chemotype. The EC₅₀ of the 7 fungicides for inhibiting the growth of FGSC ranged from 0.02 to 19.45 μg·mL^-1. Fludioxonil (FS), imazalil (FS), flusilazole (EC), tebuconazole (TC) and myclobutanil (EW) had good inhibitory effects on FGSC and the difference was not significant. The EC₅₀ of FGSC was less than 1.20 μg·mL^-1 and EC₉₀ was less than 100 μg·mL^-1. The difference of EC₅₀ between F. graminearum and F. boothii was significant under 30% pyraclostrobin treatment. The EC₅₀ of F. graminearum was 10.24 times higher than that of F. boothii. 【Conclusion】The dominant pathogenic Fusarium of maize ear rot in different maize producing areas of Jilin Province is different. F. graminearum and F. boothii are dominant species in the east and west, and F. verticillioides is dominant species in the middle. The interspecific and intraspecific genetic diversity of FGSC is rich. Fludioxonil, imazalil, flusilazole, tebuconazole and myclobutanil have better antifungal effect on FGSC. There is no significant difference in the fungicides susceptibility among FGSC.

Key words: maize, ear rot, Fusarium spp., Fusarium graminearum species complex (FGSC), fungicide

柴海燕,贾娇,白雪,孟玲敏,张伟,金嵘,吴宏斌,苏前富. 吉林省玉米穗腐病致病镰孢菌的鉴定与部分菌株对杀菌剂的敏感性[J]. 中国农业科学, 2023, 56(1): 64-78.

CHAI HaiYan,JIA Jiao,BAI Xue,MENG LingMin,ZHANG Wei,JIN Rong,WU HongBin,SU QianFu. Identification of Pathogenic Fusarium spp. Causing Maize Ear Rot and Susceptibility of Some Strains to Fungicides in Jilin Province[J]. Scientia Agricultura Sinica, 2023, 56(1): 64-78.

图/表 9

表1

图1

表2

图2

图3

表3

表4

禾谷镰孢复合种致病力与毒素化学型分析"

菌株 Strain	禾谷镰孢复合种 FGSC	来源 Source	平均病情级别 Average disease grade	致病类型 Pathogenic type	毒素化学型 Toxigenic chemotype
DASJ-1	F. b	大安Daan	1.48	LV	15-AcDON
DASJ-2	F. b	大安Daan	3.51	MV	15-AcDON
DASJ-4	F. b	大安Daan	4.00	MV	15-AcDON
DASJ-5	F. b	大安Daan	3.29	SL	15-AcDON
DATSZ-1	F. b	大安Daan	3.68	MV	15-AcDON
DATSZ-2	F. b	大安Daan	4.26	MV	15-AcDON
DHXST-1	F. a	德惠Dehui	4.43	MV	NIV
DHXST-2	F. g	德惠Dehui	4.28	MV	15-AcDON
DHXST-3	F. g	德惠Dehui	5.29	SH	15-AcDON
DHXST-4	F. g	德惠Dehui	3.86	MV	15-AcDON
DHXCZ-2	F. g	德惠Dehui	6.06	MV	15-AcDON
DHXCZ-3	F. b	德惠Dehui	2.95	SL	15-AcDON
DF-1	F. g	东丰Dongfeng	3.69	MV	15-AcDON
DFHH-3	F. b	东丰Dongfeng	3.97	MV	15-AcDON
DL-1	F. g	东辽Dongliao	2.95	SL	15-AcDON
DL-2	F. g	东辽Dongliao	5.55	SH	15-AcDON
FYYP-1	F. b	扶余Fuyu	4.48	MV	15-AcDON
SJZ-1	F. b	扶余Fuyu	6.77	SH	15-AcDON
SJZ-2	F. b	扶余Fuyu	3.60	MV	15-AcDON
SJZ-3	F. b	扶余Fuyu	5.59	SH	15-AcDON
SJZ-4	F. g	扶余Fuyu	4.22	MV	15-AcDON
SJZ-5	F. b	扶余Fuyu	3.05	SL	15-AcDON
SJZ-6	F. b	扶余Fuyu	3.34	SL	15-AcDON
HLZ-11	F. g	公主岭Gongzhuling	5.12	MV	15-AcDON
HLZ-12	F. a	公主岭Gongzhuling	2.52	SL	NIV
HLZ-25	F. g	公主岭Gongzhuling	4.28	MV	15-AcDON
HLZ-26	F. g	公主岭Gongzhuling	6.42	SH	15-AcDON
HLZ-35	F. b	公主岭Gongzhuling	5.42	MV	15-AcDON
JHXZWHC-2	F. g	蛟河Jiaohe	5.00	MV	15-AcDON
JHXZWHC-3	F. g	蛟河Jiaohe	5.98	SH	15-AcDON
JYXJ-1	F. g	靖宇Jingyu	4.62	MV	15-AcDON
LY-1	F. g	辽源市区Liaoyuan	4.39	MV	15-AcDON
LYDT-2	F. b	辽源市区Liaoyuan	5.63	SH	15-AcDON
NAXQKJ-1	F. g	农安Nongan	6.57	SH	15-AcDON
NAXQKJ-2	F. g	农安Nongan	8.58	HV	15-AcDON
QG-1	F. b	前郭Qianguo	5.04	SH	15-AcDON
QG-2	F. b	前郭Qianguo	5.72	SH	15-AcDON
QG-4	F. g	前郭Qianguo	4.05	MV	15-AcDON
QG-5	F. b	前郭Qianguo	6.35	SH	15-AcDON
1SL-1	F. g	舒兰Shulan	3.27	SL	15-AcDON
SLSYZ-1	F. g	舒兰Shulan	1.70	SL	15-AcDON
SL-1	F. b	双辽Shuangliao	2.79	SL	15-AcDON
SYSNJQ-1	F. b	松原市区Songyuan	4.36	MV	15-AcDON
SYSNJQ-2	F. b	松原市区Songyuan	5.15	MV	15-AcDON
SYSNJQ-3	F. b	松原市区Songyuan	3.76	MV	15-AcDON
TNHS-1	F. g	洮南Taonan	3.61	MV	15-AcDON
TNHS-2	F. g	洮南Taonan	3.74	MV	15-AcDON
TYNLB-1	F. g	洮南Taonan	3.85	MV	15-AcDON
WQ-1	F. b	汪清Wangqing	2.76	SL	15-AcDON
WQ-3	F. b	汪清Wangqing	4.09	MV	15-AcDON
YJ-1	F. g	永吉Yongji	2.00	MV	15-AcDON
TJT-2	F. b	榆树Yushu	3.33	SL	15-AcDON
CL-2	F. b	长岭Changling	3.10	SL	15-AcDON
CL-3	F. b	长岭Changling	3.69	MV	15-AcDON
CL-8	F. b	长岭Changling	1.67	SL	15-AcDON

表4

图4

表5

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真菌 Fungi	引物 Primer	引物序列 Primer sequence (5′-3′)	扩增片段 Target fragment (bp)	退火温度 Tm (℃)
镰孢菌属 Fusarium spp.	ItsF	AACTCCCAAACCCCTGTGAACATA	431	58
镰孢菌属 Fusarium spp.	ItsR	TTTAACGGCGTGGCCGC	431	58
禾谷镰孢复合种 F. graminearum species complex	Fg16NF	ACAGATGACAAGATTCAGGCACA	280	57
禾谷镰孢复合种 F. graminearum species complex	Fg16NR	TTCTTTGACATCTGTTCAACCCA	280	57
拟轮枝镰孢 F. verticillioides	VER1	CTTCCTGCGATGTTTCTCC	578	56
拟轮枝镰孢 F. verticillioides	VER2	AATTGGCCATTGGTATTATATATCTA	578	56
层出镰孢 F. proliferatum	PRO1	CTTTCCGCCAAGTTTCTTC	585	56
层出镰孢 F. proliferatum	PRO2	TGTCAGTAACTCGACGTTGTTG	585	56
亚黏团镰孢 F. subglutinans	SUB1	CTGTCGCTAACCTCTTTATCCA	340	58
亚黏团镰孢 F. subglutinans	SUB2	CAGTATGGACGTTGGTATTATATCTAA	340	58

镰孢菌 Fusarium spp．	总分离频率 Total isolation frequency (%)	分离频率<BOLD>I</BOLD>solation frequency (%)
镰孢菌 Fusarium spp．	总分离频率 Total isolation frequency (%)	东部East	中部Central	西部West
禾谷镰孢 F. graminearum	25.32	33.85	19.86	36.36
亚洲镰孢 F. asiaticum	0.86	0	1.37	0
布氏镰孢 F. boothii	26.18	30.77	13.01	54.55
拟轮枝镰孢 F. verticillioides	33.05	27.69	40.41	0
层出镰孢 F. proliferatum	12.45	6.15	17.12	0
厚垣镰孢 F. chlamydosporum	0.86	1.54	0.68	4.55
藤仓镰孢 F. fujikuroi	0.43	0	0	0
木贼镰孢F. equiseti	0.43	0	0.68	0
亚黏团镰孢 F. subglutinans	0.43	0	0	4.55

区域 Area	禾谷镰孢 F. graminearum		亚洲镰孢 F. asiaticum		布氏镰孢 F. boothii
区域 Area	菌株数 Number of strains	平均病情级别 Average disease grade	菌株数 Number of strains	平均病情级别 Average disease grade	菌株数 Number of strains	平均病情级别 Average disease grade
东部East	3	5.23	-	-	2	3.42
中部Middle	18	4.47	2	3.47	18	4.48
西部West	3	3.73	-	-	9	3.29
合计Total	24	4.55	2	3.47	29	4.13

药剂 Fungicide	禾谷镰孢复合种 FGSC	毒力回归方程 Toxic regression equation (y=)	相关系数 Correlation coefficient (r)	P值 P value	EC₅₀(μg·mL^-1)	EC₉₀ (μg·mL^-1)
1%咯菌腈 1% Fludioxonil (FS)	F. g	6.7459+1.1782x	0.9867	0.0003	0.03	0.40
	F. a	7.0660+1.3816x	0.9864	0.0003	0.03	0.27
	F. b	6.5108+0.8802x	0.9956	0	0.02	0.55
97%戊唑醇 97% Tebuconazole (TC)	F. g	5.1913+0.6573x	0.9810	0.0001	0.51	45.57
	F. a	5.2057+0.6911x	0.9901	0	0.50	36.05
	F. b	5.1031+0.6849x	0.9817	0.0001	0.71	52.55
400 g·L^-1氟硅唑 400 g·L^-1 Flusilazole (EC)	F. g	5.5130+0.7766x	0.9944	0.0000	0.63	28.36
	F. a	5.0412+0.7940x	0.9755	0.0009	0.89	36.48
	F. b	5.2173+0.6697x	0.9963	0	0.47	38.84
10%抑霉唑 10% Imazalil (FS)	F. g	5.4567+2.4924x	0.9642	0.0019	0.66	2.14
	F. a	5.2131+2.1992x	0.9397	0.0053	0.80	3.06
	F. b	6.0591+2.1246x	0.9525	0.0033	0.32	1.27
12.5%腈菌唑 12.5% Myclobutanil (EW)	F. g	5.0197+0.8285x	0.9829	0.0004	0.95	33.35
	F. a	4.9353+0.9961x	0.9821	0.0005	1.16	22.46
	F. b	5.1448+0.9618x	0.9961	0	0.71	15.20
80%福美双 80% Famous Double (WG)	F. g	4.5324+0.4878x	0.9697	0.0003	9.09	3855.64
	F. a	4.5260+0.5899x	0.9820	0.0001	6.36	946.20
	F. b	4.4917+0.7141x	0.9739	0.0002	5.15	320.84
30%吡唑醚菌酯 30% Pyraclostrobin (SC)	F. g	4.6753+0.2519x	0.9619	0.0021	19.45	2378665.18
	F. a	4.7013+0.2785x	0.9661	0.0017	11.81	471493.34
	F. b	4.9428+0.2048x	0.9601	0.0024	1.90	3434875.02