玉米穗腐病致病禾谷镰孢复合种的遗传多样性、致病力与毒素化学型分析

doi:10.3864/j.issn.0578-1752.2020.23.005

摘要/Abstract

摘要：

【目的】明确中国玉米穗腐病致病禾谷镰孢复合种（Fusarium graminearum species complex,FGSC）的菌群遗传结构、致病力、毒素化学型及其相互关系,为玉米镰孢穗腐病防控提供参考信息。【方法】从中国玉米主产区采集玉米穗腐病样本,经单孢分离鉴定,选取代表性的禾谷镰孢复合种,利用22对SSR和10对VNTR引物,使用Popgen32、NTsys2.1、STRUCTURE2.3.4群体遗传学研究软件进行数据分析,结合TEF-1α、β-tubulin和RPB2基因测序构建系统发育树,分析7个地理区域禾谷镰孢复合种的遗传多样性,采用花丝通道注射接种法测定各镰孢菌的致病力,利用产毒基因特异性引物进行毒素化学型的检测。【结果】在禾谷镰孢复合种中共检测到等位位点数48个,多态性位点数39个,多态性条带比率为81.25%,每对引物扩增出多态性条带2—4条;禾谷镰孢复合种7个地理群体平均Shannon’s信息指数和Nei’s遗传多样性指数分别为0.41和0.29,7个地理区域遗传相似度集中在0.6677—0.8797,遗传距离为0.1282—0.4039,表明菌群间具有较丰富的遗传多样性。依据Nei’s遗传距离对7个地理种群进行UPGMA聚类分析,可划分为3个类群;STRUCTURE2.3.4群体结构分析表明,禾谷镰孢复合种菌群被分为2个大类群最合适,西北地区绝大部分属于类群A,华中地区和华南地区菌株均属于类群B,东北地区50%以上菌株属于类群B。TEF-1α、β-tubulin和RPB2基因序列分析表明禾谷镰孢复合种由禾谷镰孢（F. graminearum）、亚洲镰孢（F. asiaticum）、布氏镰孢（F. boothii）和南方镰孢（F. meridionale）构成,上述镰孢菌中存在142个单核苷酸多态性位点（SNP）,通过这些差异序列构建的聚类图能清楚显示出各个种内与种间的遗传分化情况,各镰孢种内遗传多样性十分丰富。禾谷镰孢致病力最强,平均发病面积百分比为20.79%;亚洲镰孢、布氏镰孢和南方镰孢的平均发病面积百分比分别为15.79%、11.77% 和 8.12%。统计分析表明,不同镰孢种所引起的穗腐病平均发病面积占比存在显著差异。禾谷镰孢毒素化学型为15ADON、3ADON、NIV和3ADON+15ADON+NIV 4种,以15ADON为主;布氏镰孢毒素化学型为15ADON、3ADON、NIV、3ADON+15ADON和3ADON+15ADON+NIV 5种,以15ADON为主;亚洲镰孢毒素化学型只有3ADON;南方镰孢毒素化学型为15ADON、3ADON、15ADON+NIV 3种,15ADON为优势类型。此外,15ADON、NIV和3ADON毒素类型菌株平均发病面积百分比分别为17.87%、17.20%和12.37%。【结论】我国不同地理区域尤其是相邻区域禾谷镰孢复合种菌群间存在较为频繁的基因交流与交换。本研究中禾谷镰孢复合种的主要毒素化学型为15ADON,致病力由强到弱依次为禾谷镰孢>亚洲镰孢>布氏镰孢>南方镰孢。整体看来,毒素化学型与菌种类型相关性不显著,致病力主要与菌种类型有关。

关键词: 玉米穗腐病, 禾谷镰孢复合种, 遗传多样性, 毒素化学型, 致病力

Abstract:

【Objective】The objective of this study is to clarify the genetic structure, pathogenicity, toxigenic chemotypes, and their relationship of Fusarium graminearum species complex (FGSC) causing maize ear rot in China, and to provide reference information for prevention and control of Fusarium ear rot of maize. 【Method】The samples were collected from the main maize producing areas in China. Twenty-two pairs of SSR and 10 pairs of VNTR primers, along with TEF-1α, β-tubulin and RPB2 gene sequences were used for genetic diversity and Popgen32, NTsys2.1, and STRUCTURE2.3.4 software were used to analyze the data and construct the phylogenetic tree. The pathogenicity of FGSC was determined using the silk channel injection inoculation method, and the specific primers were used to detect the toxigenic chemotypes. 【Result】A total of 48 alleles were detected by SSR and VNTR primers, 39 polymorphic sites were found, with a polymorphic band ratio of 81.25% among 45 strains, the polymorphic bands ranged from 2 to 4. The average Shannon’s information index and Nei’s genetic diversity index of the 7 FGSC geographic populations were 0.41 and 0.29, respectively, the genetic similarity in 7 regions was 0.6677-0.8797, and the genetic distance was 0.1282-0.4039, indicating that there existed rich genetic diversity among the flora. Based on the Nei’s genetic distance, 7 FGSC geographical populations were divided into 3 groups by UPGMA clustering. The population structure of FGSC stains could be divided into two different groups by STRUCTURE2.3.4. Most of the strains from Northwest China belonged to group A, those in Central China and South China belonged to group B, and more than 50% of the strains in Northeast China belonged to group B. Based on the sequences of TEF-1α, β-tubulin and RPB2, FGSC was composed of F. graminearum, F. asiaticum, F. boothii, and F. meridionale. There were 142 single nucleotide polymorphisms (SNPs) among these Fusarium strains. The dendrogram constructed by these differential sequences could clearly show the genetic differentiation within and between species. The genetic diversity within each Fusarium species was rich. Among four Fusarium species, the pathogenicity of F. graminearum was the strongest, with an average diseased ear area of 20.79%. The average diseased areas of F. asiaticum, F. boothii, and F. meridionale were 15.79%, 11.77%, and 8.12%, respectively. The difference in the percentage of average diseased area was significant between Fusarium species. The toxigenic chemotypes of F. graminearum were 15ADON, 3ADON, NIV, and 3ADON + 15ADON + NIV, F. boothii contained 15ADON, 3ADON, NIV, 3ADON + 15ADON, and 3ADON + 15ADON + NIV chemotypes, the toxigenic chemotypes of F. meridionale were 15ADON, 3ADON, and 15ADON+ NIV, the 15ADON chemotype was the most frequently detected, while F. asiaticum merely contained 3ADON chemotype. In addition, the average diseased ear areas of 15ADON, NIV and 3ADON type strains were 17.87%, 17.20%, and 12.37%, respectively. 【Conclusion】There existed frequent gene exchanges between different FGSC geographical populations, especially in adjacent regions. 15ADON type is the predominant toxigenic chemotype of FGSC in 7 geographic regions. The pathogenicity of F. graminearum is the strongest, followed by F. asiaticum, F. boothii, and F. meridionale according to the percentage of the diseased area caused by different Fusarium species. In this study, the correlation between toxigenic chemotypes and Fusarium species is not significant, and the pathogenicity is mainly related to Fusarium species.

Key words: maize ear rot, Fusarium graminearum species complex (FGSC), genetic diversity, toxigenic chemotype, pathogenicity

王宝宝,郭成,孙素丽,夏玉生,朱振东,段灿星. 玉米穗腐病致病禾谷镰孢复合种的遗传多样性、致病力与毒素化学型分析[J]. 中国农业科学, 2020, 53(23): 4777-4790.

WANG BaoBao,GUO Cheng,SUN SuLi,XIA YuSheng,ZHU ZhenDong,DUAN CanXing. The Genetic Diversity, Pathogenicity, and Toxigenic Chemotypes of Fusarium graminearum Species Complex Causing Maize Ear Rot[J]. Scientia Agricultura Sinica, 2020, 53(23): 4777-4790.

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图/表 10

表1

本试验所用引物"

引物 Primer	序列 Sequence (5′ to 3′)	退火温度 Tm (℃)	扩增片段大小 Product size (bp)	参考文献 Reference
SSR-L1	R: AGAAGAAGGGCAAATGG	57	378	[19]
	F: TGTCCGAGTCGTCTGAAT
SSR-L2	R: GGTGGTGGTTTCCGTGAG	60	214	[19]
	F: GCGTTGAGATACCCTTTCG
SSR-L3	R: CTGCTGCTATCACTTCCAC	58	344	[19]
	F: TTCACAACGACCGTATTTC
SSR-L4	R: GTTGTTGGGTTGCTTGTG	59	160	[19]
	F: GTCTCGTCCAGATTCCTATT
SSR-L5	R: GGTGGTGGTTTCCGTGAG	60	214	[19]
	F: GCGTTGAGATACCCTTTCG
SSR-L6	R: AATAGTGGCGGTGCGTAG	60	386	[19]
	F: GTCTCCCGAGTAGTTGGC
SSR-L7	R: TAGTGGCGGTGCGTAGTG	62	379	[19]
	F: GTCTCCCGAGTAGTTGGC
SSR-L8	R: GGTCCGCAACATAGAGGG	60	403	[19]
	F: CCGAAGAGGAGAACGAGC
SSR-G11	R: TTTTGGGTGTTGAAGAAGCC	56	200-300	[20]
	F: TTTTTGTTTGCGTCGTTCTG
SSR-G12	R: AATGCAGTCGATGGGAAACT	56	200-300	[20]
	F: GGGTTCCTTGTAAGTGGCTG
SSR-G13	R: AGTATCAGACAGGTTGGCCG	56	200-300	[20]
	F: GTATGCAAAGCAGCGTCGTA
SSR-G14	R: GCCCATTACGTTGAGCAAAT	56	200-300	[20]
	F: TTTTTCGGTCTGGCTATTGG
SSR-G15	R: CTCTAGTGGCAGACCCTTCG	56	200-300	[20]
	F: CTTGACATGTCGCGCTTTTA
SSR-G16	R: CTGATCTGCGGACATCTTCA	56	200-300	[20]
	F: GTTTTGGTTTGCCTGTCGTT
SSR-G17	R: TCTGTCGTTGACAAGCAAGC	56	200-300	[20]
	F: ACGGACCGACGACATAACTC
SSR-G18	R: GCGTCGACTAAAGGAACCAG	56	200-300	[20]
	F: TCCCGACGTTAGAGTGGAGT
SSR-G19	R: CCTTGTTCTTTCCACCCTGA	56	200-300	[20]
	F: ACGAGGACGAACTTGTTGCT
引物 Primer	序列 Sequence (5′ to 3′)	退火温度 Tm (℃)	扩增片段大小 Product size (bp)	参考文献 Reference
SSR-G20	R: AAGCTTGGGGGCTAACATCT	56	200-300
	F: CAGCTTTGGCGGACATTATT			[20]
SSR-G21	R: TCACATATTCAACCGACCCA	56	200-300	[20]
	F: GCTCCGTGTCCTTTCATTTC
SSR-G22	R: GACGGATCGTCGGATAGGTA	56	200-300	[20]
	F: CACTAAGCTGCTCCTCCACC
VNTR-F1	F: ACAGGCATCCAAGGACATTT	56	286	[21]
	R: GTTTGATGGCGCATTCAAAG
VNTR-F2	F: GCAGGACCTGGATGATGAA	56	234	[21]
	R: ATGTGTGCAGCCATGAGATT
VNTR-F3	F: ATCTCCCAAGCTGGCTAATT	56	234	[21]
	R: AGAACCGGCAAAGTTCGATT
VNTR-F4	F: TCCGAAGGTAGAAGCGTTGT	56	298
	R: TCAAGCCCATCTATGCTGTT			[21]
VNTR-F5	F: GAGATGGCAACATTATTTGCA	56	252
	R: ATTGGCAGCAGGGCTTGATT			[21]
VNTR-F6	F: AAGAGGGCGTGTCTCTGTTTT	56	215
	R: CGCTTCCTTCCTTTCAATTC			[21]
VNTR-F7	F: AAGACTGGTCAGCAGTAGGGA	56	248
	R: TGAGAGCGAGACTGAGCATGA			[21]
VNTR-F8	F: AAACGTAAACGGATCAACGG	56	210
	R: AGATTCGCAACTTTGTGCTG			[21]
VNTR-F9	F: TGGATATGGTTCCCCAGCT	56	239
	R: TACTGACCTTGAGGAGCACCATAC			[21]
VNTR-F10	F: TATGATGCAGCGAATGCAAC	56	221	[21]
	R: TAGAGACCTGGCCCATACCA
TEF-1α	R: ATGGGTAAGGAR-GACAAGAC	58	680	[22]
	F: GGAR-GTACCAGTSATCATGTT
β-tubulin	R: GGTAACCAAATCGGTGCTGCTTTC	55	380	[23]
	F: ACCCTCAGTGTAGTGACCCTTGGC
RPB2	R: CCCATRGCTTGYTTRCCCAT	55	900	[24]
	F: GGGGWGAYCAGAAGAAGGC
Tri13a/b	R: CTCSACCGCATCGAAGASTCTC	58	583, 644, 859	[25]
	F: GAASGTCGCARGACCTTGTTTC
Tri13F/R	R: GGTGTCCCAGGATCTGCG	57	415	[26]
	F: TACGTGAAACATTGTTGGC
Tri303F/R	R: GCCGGACTGCCCTATTG	52	586	[27]
	F: GATGGCCGCAAGTGGA
Tri315F/R	R: GTCTATGCTCTCAACGGACAAC	58	864	[27]
	F: CTCGCTGAAGTTGGACGTAA

表1

表2

表3

图1

图2

图3

图4

表4

禾谷镰孢复合种致病力与毒素化学型"

菌株 Strain	菌种 Species	发病面积 Diseased area (%)	毒素类型			采集地点 Collection location	地理区域 Geographical area
菌株 Strain	菌种 Species	发病面积 Diseased area (%)	3ADON	15ADON	NIV	采集地点 Collection location	地理区域 Geographical area
3HLJ	禾谷镰孢F. graminearum	10.70		√		黑龙江肇源Zhaoyuan, Heilongjiang	东北Northeast China
8HLJ	布氏镰孢F. boothii	4.00		√		黑龙江安达Anda, Heilongjiang	东北Northeast China
20HLJ	禾谷镰孢F. graminearum	10.09		√		黑龙江尚志Shangzhi, Heilongjiang	东北Northeast China
94HLJ	禾谷镰孢F. graminearum	42.63		√		黑龙江鹤岗Hegang, Heilongjiang	东北Northeast China
106HLJ	布氏镰孢F. boothii	6.88		√		黑龙江穆棱Muling, Heilongjiang	东北Northeast China
113HLJ	禾谷镰孢F. graminearum	27.58		√		黑龙江虎林Hulin, Heilongjiang	东北Northeast China
131HLJ	布氏镰孢F. boothii	11.63	√	√		黑龙江饶河Raohe, Heilongjiang	东北Northeast China
136HLJ	禾谷镰孢F. graminearum	23.36		√		黑龙江密山Mishan, Heilongjiang	东北Northeast China
162HLJ	布氏镰孢F. boothii	16.91		√		黑龙江依安Yian, Heilongjiang	东北Northeast China
180HLJ	禾谷镰孢F. graminearum	26.54		√		黑龙江嫩江Neijiang, Heilongjiang	东北Northeast China
184HLJ	禾谷镰孢F. graminearum	42.63		√		黑龙江龙江Longjiang, Heilongjiang	东北Northeast China
185HLJ	布氏镰孢F. boothii	22.54		√		黑龙江密山Mishan, Heilongjiang	东北Northeast China
195HLJ	禾谷镰孢F. graminearum	30.20		√		黑龙江宁安Ningan, Heilongjiang	东北Northeast China
200HLJ	禾谷镰孢F. graminearum	39.27		√		黑龙江穆棱Muling, Heilongjiang	东北Northeast China
212HLJ	禾谷镰孢F. graminearum	35.60		√		黑龙江五常Wuchang, Heilongjiang	东北Northeast China
231HLJ	禾谷镰孢F. graminearum	4.29		√		黑龙江哈尔滨Harbin, Heilongjiang	东北Northeast China
244HLJ	禾谷镰孢F. graminearum	13.70		√		黑龙江绥化Suihua, Heilongjiang	东北Northeast China
g1-CQ	南方镰孢F. meridionale	6.66		√		重庆合川Hechuan, Chongqing	西南Southwest China
g2-SXXA	禾谷镰孢F. graminearum	11.38	√			陕西西安Xian, Shaanxi	西北Northwest China
g3-SXSL	南方镰孢F. meridionale	9.67	√			陕西商洛Shangluo, Shaanxi	西北Northwest China
g4-SXSL	禾谷镰孢F. graminearum	25.33	√	√	√	陕西商洛Shangluo, Shaanxi	西北Northwest China
g5-YN	南方镰孢F. meridionale	6.56		√		云南弥勒Mile, Yunnan	西南Southwest China
g6-SD	禾谷镰孢F. graminearum	7.00		√		山东兖州Yanzhou, Shandong	华东East China
g7-HNZK	禾谷镰孢F. graminearum	12.44		√		河南周口Zhoukou, Henan	华中Central China
g8-HNSMX	禾谷镰孢F. graminearum	14.86		√		河南三门峡Sanmenxia, Henan	华中Central China
g9-SD	禾谷镰孢F. graminearum	29.00		√		山东微山Weishan, Shandong	华东East China
g10-SXYL	禾谷镰孢F. graminearum	2.75	√			陕西榆林Yulin, Shaanxi	西北Northwest China
g11-CQ	南方镰孢F. meridionale	4.71	√			重庆秀山Xiushan, Chongqing	西南Southwest China
g12-SXXY	亚洲镰孢F. asiaticum	12.36	√			陕西乾县Qianxian, Shaanxi	西北Northwest China
g14-BJ	布氏镰孢F. boothii	7.38	√	√	√	北京昌平Changping, Beijing	华北North China
g15-GZ	南方镰孢F. meridionale	6.00		√		贵州遵义Zunyi, Guizhou	西南Southwest China
g16-BJ	禾谷镰孢F. graminearum	20.10		√		北京顺义Shunyi, Beijing	华北North China
g17-LN	禾谷镰孢F. graminearum	17.60		√		辽宁锦州Jinzhou, Liaoning	东北Northeast China
g18-LN	禾谷镰孢F. graminearum	15.40		√		辽宁沈阳Shenyang Liaoning	东北Northeast China
g19-LN	禾谷镰孢F. graminearum	11.80	√	√		辽宁铁岭Tieling, Liaoning	东北Northeast China
g20-LN	禾谷镰孢F. graminearum	24.60			√	辽宁沈阳Shenyang, Liaoning	东北Northeast China
g23-GX	南方镰孢F. meridionale	13.11		√		广西德保Debao, Guangxi	华南South China
g24-GX	亚洲镰孢F. asiaticum	14.00	√			广西河池Hechi, Guangxi	华南South China
g25-GX	亚洲镰孢F. asiaticum	22.58	√			广西大新Daxin, Guangxi	华南South China
g26-GX	亚洲镰孢F. asiaticum	11.17	√			广西田林Tianlin, Guangxi	华南South China
g27-SC	亚洲镰孢F. asiaticum	16.64	√			四川巴中Bazhong, Sichuan	西南Southwest China
g28-SC	亚洲镰孢F. asiaticum	18.00	√			四川浦江Pujiang, Sichuan	西南Southwest China
g29-GS	布氏镰孢F. boothii	18.60			√	甘肃张掖Zhangye, Gansu	西北Northwest China
g30-GS	布氏镰孢F. boothii	6.20	√			甘肃张掖Zhangye, Gansu	西北Northwest China
g32-YN	南方镰孢F. meridionale	10.10		√	√	云南玉溪Yuxi, Yunnan	西南Southwest China

表4

表5

表6

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种群 Population	多态性位点数 Number of polymorphic loci	多态性位点百分比 Percentage of polymorphic loci (%)	等位基因数 Observed number of alleles	有效等位基因数 Effective number of alleles	Shannon’s信息指数 Shannon’s information index	Nei’s遗传多样性指数 Nei’s genetic diversity index
华北North China	18	46.15	1.46	1.46	0.32	0.23
华中Central China	10	25.64	1.26	1.26	0.18	0.13
华东East China	14	35.90	1.36	1.36	0.25	0.18
东北Northeast China	39	100.00	2.00	1.79	0.62	0.43
华南South China	26	66.67	1.67	1.46	0.40	0.27
西北Northwest China	37	94.87	1.95	1.65	0.55	0.37
西南Southwest China	36	92.31	1.92	1.71	0.56	0.39
平均Average	25.71	66.00	1.66	1.53	0.41	0.29

种群 Population	华北 North China	华中 Central China	华东 East China	东北 Northeast China	华南 South China	西北 Northwest China	西南 Southwest China
华北North China	—	0.6888	0.7100	0.7979	0.7011	0.8120	0.7910
华中Central China	0.3727	—	0.8489	0.8484	0.7067	0.6934	0.6677
华东East China	0.3424	0.1638	—	0.8572	0.7782	0.7556	0.7452
东北Northeast China	0.2257	0.1644	0.1540	—	0.8170	0.8797	0.8502
华南South China	0.3551	0.3471	0.2508	0.2021	—	0.8196	0.8609
西北Northwest China	0.2083	0.3662	0.2803	0.1282	0.1990	—	0.8775
西南Southwest China	0.2345	0.4039	0.2942	0.1623	0.1498	0.1307	—

菌种 Species	菌株数 Number of strains	发病面积和 Summary of diseased area (%)	平均发病面积 Percentage of average diseased area (%)	方差 Variance
禾谷镰孢F. graminearum	24	498.84	20.79±2.44A	137.3899
亚洲镰孢F. asiaticum	6	94.75	15.79±1.87B	17.6138
布氏镰孢F. boothii	8	94.12	11.77±2.56c	46.2450
南方镰孢F. meridionale	7	56.81	8.12±1.20d	8.6493

差异源 Source of difference	平方和 SS	自由度 df	平均平方和 MS	统计量 F	P值 P-value
组间Intergroup	1115.080	3	371.6934	4.206	0.011
组内Intragroup	3623.646	41	88.3816
总计Total	4738.727	44