广西玉米穗腐病致病镰孢种群构成与毒素化学型分析

doi:10.3864/j.issn.0578-1752.2019.11.005

中国农业科学 ›› 2019, Vol. 52 ›› Issue (11): 1895-1907.doi: 10.3864/j.issn.0578-1752.2019.11.005

广西玉米穗腐病致病镰孢种群构成与毒素化学型分析

杜青¹,唐照磊¹,李石初¹,上官玲玲²,李华娇²,段灿星³()

¹ 广西壮族自治区农业科学院玉米研究所,南宁 530007
² 广西农业职业技术学院,南宁 530007
³ 中国农业科学院作物科学研究所/ 国家农作物基因资源与基因改良重大科学工程,北京100081

收稿日期:2019-02-19 接受日期:2019-03-21 出版日期:2019-06-01 发布日期:2019-06-11
通讯作者: 段灿星
作者简介:杜青,E-mail：duq82@163.com。
基金资助:
国家重点研发计划(2018YFD0200705);农作物种质资源保护与利用专项(2018NWB036-12);广西科技重大专项桂科(AA17204050-2);广西农业科学院玉米研究所科技发展基金桂玉科(2017003)

Composition of Fusarium Species Causing Maize Ear Rot and Analysis of Toxigenic Chemotype in Guangxi

DU Qing¹,TANG ZhaoLei¹,LI ShiChu¹,SHANGGUAN LingLing²,LI HuaJiao²,DUAN CanXing³()

¹ Institute of Maize Research, Guangxi Academy of Agricultural Sciences, Nanning 530007
² Guangxi Agricultural Vocational and Technical College, Nanning 530007
³ Institute of Crop Sciences, Chinese Academy of Agricultural Sciences/National Key Facility of Crop Gene Resources and Genetic Improvement, Beijing 100081

Received:2019-02-19 Accepted:2019-03-21 Online:2019-06-01 Published:2019-06-11
Contact: CanXing DUAN

摘要/Abstract

摘要：

目的明确广西玉米穗腐病致病镰孢的种群构成及毒素化学型,为玉米穗腐病的综合防治、品种的合理布局和抗病育种提供重要指导和理论依据。方法从广西玉米主产区采集玉米穗腐病样本,经组织分离和单孢纯化共获得138个镰孢菌分离物,分别来自21个县（区）。利用形态学和分子生物学相结合的方法进行镰孢菌种的鉴定,构建TEF-1α系统发育树,利用产毒基因特异性引物进行毒素化学型检测。结果在138个镰孢菌分离物中,鉴定出10种镰孢菌,包括拟轮枝镰孢（Fusarium verticillioides）、层出镰孢（F. proliferatum）、九州镰孢（F. kyushuense）、南方镰孢（F. meridionale）、甘蔗镰孢（F. sacchari）、藤仓镰孢（F. fujikuroi）、亚洲镰孢（F. asiaticum）、轮纹镰孢（F. concentricum）、变红镰孢（F. incarnatum）和禾谷镰孢（F. graminearum）,分离频率依次为50.72%、12.32%、10.87%、8.70%、6.52%、3.62%、3.62%、1.45%、1.45%和0.72%。禾谷镰孢复合种（F. graminearum species complex,FGSC）由南方镰孢、亚洲镰孢和禾谷镰孢3个独立种组成。拟轮枝镰孢为优势致病菌,禾谷镰孢复合种、层出镰孢和九州镰孢为次优势致病菌,而甘蔗镰孢和轮纹镰孢则是国内首次报道为玉米穗腐病致病菌。拟轮枝镰孢、层出镰孢、甘蔗镰孢和藤仓镰孢检测到伏马毒素关键合成基因FUM1的菌株数分别为67、13、5、3,具有潜在的产伏马毒素能力,轮纹镰孢则未检测到FUM1。供试禾谷镰孢复合种、九州镰孢和变红镰孢菌株的毒素化学型有4种：NIV、15-ADON、NIV+15-ADON和DON+15-ADON。8个九州镰孢菌株、2个亚洲镰孢菌株、2个南方镰孢菌株和1个变红镰孢菌株携带NIV毒素化学型;2个南方镰孢菌株携带15-ADON毒素化学型;8个南方镰孢菌株、2个九州镰孢菌株、1个亚洲镰孢菌株和1个变红镰孢菌株携带NIV+15-ADON毒素化学型;只有1个禾谷镰孢菌株携带DON+15-ADON毒素化学型。未检测到3-ADON毒素化学型菌株。结论拟轮枝镰孢为广西玉米穗腐病优势致病菌,禾谷镰孢复合种、层出镰孢和九州镰孢为次优势致病菌。拟轮枝镰孢、层出镰孢、甘蔗镰孢、藤仓镰孢均检测到FUM1,广西禾谷镰孢复合种的主要毒素化学型为NIV和15-ADON,变红镰孢和部分九州镰孢的主要毒素化学型为NIV。广西玉米穗腐病镰孢菌种群构成与我国温带玉米相关研究结果存在差异,原因可能为镰孢菌种群适应广西热带和亚热带高温、高湿的玉米生长环境并因此导致毒素化学型的不同。

关键词: 玉米, 穗腐病, 镰孢菌, 分离频率, 毒素化学型

Abstract:

【Objective】 The objective of this study is to clarify the composition and toxigenic chemotype of Fusarium species causing maize ear rot in Guangxi, and to provide important guidance and theoretical basis for comprehensive control of maize ear rot and reasonable distribution of varieties and resistance breeding.【Method】 The diseased ear samples were collected from main maize producing areas in Guangxi between 2016 and 2018, a total of 138 isolates from 21 counties (districts) were obtained by tissue separation and single-spore purification. Fusarium species were identified and determined according to morphological characteristics and molecular methods. The phylogenetic tree was constructed based on TEF-1α gene sequences, and specific primers were used to detect toxigenic chemotype.【Result】 A total of 10 Fusarium species were identified and confirmed among 138 isolates, including F. verticillioides, F. proliferatum, F. kyushuense, F. meridionale, F. sacchari, F. fujikuroi, F. asiaticum, F. concentricum, F. incarnatum and F. graminearum, with the isolation frequencies of 50.72%, 12.32%, 10.87%, 8.70%, 6.52%, 3.62%, 3.62%, 1.45%, 1.45% and 0.72%, respectively. F. graminearum species complex (FGSC) contained three independent species, i.e. F. meridionale, F. asiaticum and F. graminearum. F. verticillioides was the predominant pathogen, FGSC, F. proliferatum and F. kyushuense were the secondary predominant pathogens. F. sacchari and F. concentricum were the first reported to be the pathogen of maize ear rot in China. The key gene FUM1 responsible for the biosynthesis of fumonisins was detected among 67 F. verticillioides, 13 F. proliferatum, 5 F. sacchari and 3 F. fujikuroi strains, respectively, which indicated the potential ability of producing fumonisins. FUM1 was not detected in the F. concentricum strains. Four toxigenic chemotypes including NIV, 15-ADON, NIV+15-ADON and DON+15-ADON were detected among FGSC, F. kyushuense and F. incarnatum strains. Strains containing NIV chemotype included 8 F. kyushuense, 2 F. asiaticum, 2 F. meridionale, and 1 F. incarnatum. Strains containing 15-ADON chemotype included 2 F. meridionale. The NIV+15-ADON chemotype was detected among 8 F. meridionale, 2 F. kyushuense, 1 F. asiaticum and 1 F. incarnatum strains. Only 1 F. graminearum strain carried DON+15-ADON chemotype. The 3-ADON chemotype was not detected among these strains.【Conclusion】 F. verticillioides is the predominant pathogen of maize ear rot in Guangxi. FGSC, F. proliferatum and F. kyushuense are the secondary predominant species. FUM1 was detected among F. verticillioides, F. proliferatum, F. sacchari and F. fujikuroi. The main toxigenic chemtype of FGSC in Guangxi is NIV+15-ADON, while NIV is the main toxigenic chemotype of F. incarnatum and some F. kyushuense. The population composition of Fusarium species causing maize ear rot in Guangxi is different from equivalent study of temperate zone in China, which may be due to the fact that Fusarium species adapt to the high temperature and humidity growth environment in tropical and subtropical Guangxi and thus leads to the differences of toxigenic chemotypes.

Key words: maize, ear rot, Fusarium spp., isolation frequency, toxigenic chemotype

杜青, 唐照磊, 李石初, 上官玲玲, 李华娇, 段灿星. 广西玉米穗腐病致病镰孢种群构成与毒素化学型分析[J]. 中国农业科学, 2019, 52(11): 1895-1907.

DU Qing, TANG ZhaoLei, LI ShiChu, SHANGGUAN LingLing, LI HuaJiao, DUAN CanXing. Composition of Fusarium Species Causing Maize Ear Rot and Analysis of Toxigenic Chemotype in Guangxi[J]. Scientia Agricultura Sinica, 2019, 52(11): 1895-1907.

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检测真菌 Detected fungus	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	ItsF	AACTCCCAAACCCCTGTGAACATA	431	58
Fusarium spp.	ItsR	TTTAACGGCGTGGCCGC
禾谷镰孢复合种	Fg16NF	ACAGATGACAAGATTCAGGCACA	280	57
F. graminearum	Fg16NR	TTCTTTGACATCTGTTCAACCCA
species complex
拟轮枝镰孢	VER1	CTTCCTGCGATGTTTCTCC	578	56
F. verticillioides	VER2	AATTGGCCATTGGTATTATATATCTA
层出镰孢	PRO1	CTTTCCGCCAAGTTTCTTC	585	56
F. proliferatum	PRO2	TGTCAGTAACTCGACGTTGTTG

引物 Primer	引物序列 Primer sequence (5′-3′)	毒素化学型 Toxigenic chemotype	扩增片段 Product size (bp)	退火温度 Tm (℃)
Tri13F	TACGTGAAACATTGTTGGC	DON, NIV	234, 415	57
Tri13R	GGTGTCCCAGGATCTGCG
Tri303F	GATGGCCGCAAGTGGA	3-DON	586	56
Tri303R	GCCGGACTGCCCTATTG
Tri315F	CTCGCTGAAGTTGGACGTAA	15-ADON	864	58
Tri315R	GTCTATGCTCTCAACGGACAAC

镰孢菌 Fusarium spp.	拟轮枝镰孢 F. v	层出镰孢 F. p	甘蔗镰孢 F. s	藤仓镰孢 F. f	轮纹镰孢 F. c	亚洲镰孢 F. a	禾谷镰孢 F. g	南方镰孢 F. m	九州镰孢 F. k	变红镰孢 F. i	总计 Total
巴马 Bama	2	4	-	-	-	1	-	-	3	-	10
大化 Dahua	2	-	1	1	-	-	-	-	-	-	4
东兰 Donglan	9	-	-	-	-	-	-	-	-	-	9
都安 Duan	4	-	1	-	-	1	-	2	3	-	11
凤山Fengshan	5	-	-	-	-	-	-	3	-	-	8
金城江Jinchengjiang	-	-	2	-	-	-	-	-	-	-	2
乐业 Leye	3	-	-	-	-	2	-	-	-	-	5
凌云 Lingyun	9	-	-	2	-	-	-	-	-	-	11
平果 Pingguo	1	1	-	-	-	-	1	1	3	1	8
靖西 Jingxi	-	6	-	-	2	-	-	-	-	-	8
田东Tiandong	6	-	-	-	-	-	-	1	-	-	7
田林 Tianlin	2	-	-	-	-	-	-	-	-	-	2
田阳 Tianyang	2	-	-	-	-	-	-	-	-	-	2
德保 Debao	1	2	-	1	-	-	-	2	2	-	8
西林 Xilin	-	1	-	-	-	-	-	-	-	-	1
隆安 Longan	-	-	-	-	-	-	-	-	-	1	1
江南 Jiangnan	7	-	-	-	-	-	-	-	-	-	7
马山 Mashan	7	-	-	-	-	-	-	-	-	-	7
武宣 Wuxuan	-	2	2	1	-	-	-	-	2	-	7
大新 Daxin	3	-	1	-	-	1	-	-	1	-	6
天等 Tiandeng	7	1	2	-	-	-	-	3	1	-	14
总计 Total	70	17	9	5	2	5	1	12	15	2	138

菌株编号 Strain number	镰孢菌 Fusarium spp.	毒素化学型 Toxigenic chemotype
菌株编号 Strain number	镰孢菌 Fusarium spp.	NIV	DON	3-ADON	15-ADON
Bama7	F. asiaticum	-	-	-	-
Daxin1	F. asiaticum	+	-	-	-
Duan1	F. asiaticum	-	-	-	-
Leye2	F. asiaticum	+	-	-	-
Leye4	F. asiaticum	+	-	-	+
Pingguo4	F. graminearum	-	+	-	+
Longan1	F. incarnatum	+	-	-	-
Pingguo8	F. incarnatum	+	-	-	+
Bama2	F. kyushuense	+	-	-	+
Bama3	F. kyushuense	+	-	-	+
Bama9	F. kyushuense	-	-	-	-
Daxin5	F. kyushuense	-	-	-	-
Debao3	F. kyushuense	+	-	-	-
Debao5	F. kyushuense	-	-	-	-
Duan3	F. kyushuense	+	-	-	-
Duan4	F. kyushuense	+	-	-	-
Duan5	F. kyushuense	+	-	-	-
Pingguo3	F. kyushuense	+	-	-	-
Pingguo5	F. kyushuense	-	-	-	-
Pingguo6	F. kyushuense	+	-	-	-
Tiandeng12	F. kyushuense	+	-	-	-
Wuxuan6	F. kyushuense	+	-	-	-
Wuxuan7	F. kyushuense	-	-	-	-
Debao6	F. meridionale	+	-	-	+
Debao7	F. meridionale	+	-	-	-
Duan2	F. meridionale	-	-	-	+
Duan8	F. meridionale	+	-	-	+
FengShan2	F. meridionale	-	-	-	+
FengShan3	F. meridionale	+	-	-	+
FengShan7	F. meridionale	+	-	-	+
Pingguo2	F. meridionale	+	-	-	+
Tiandeng1	F. meridionale	+	-	-	+
Tiandeng2	F. meridionale	+	-	-	+
Tiandeng8	F. meridionale	+	-	-	+
Tiandong4	F. meridionale	+	-	-	-

广西玉米穗腐病致病镰孢种群构成与毒素化学型分析

Composition of Fusarium Species Causing Maize Ear Rot and Analysis of Toxigenic Chemotype in Guangxi

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