禾谷镰孢复合种毒素化学型及遗传多样性分析

doi:10.3864/j.issn.0578-1752.2018.01.008

摘要/Abstract

摘要： 【目的】明确不同省（自治区）引起玉米穗腐病的禾谷镰孢复合种（Fusarium graminearum species complex，FGSC）的毒素化学型和它们之间的遗传差异以及亲缘关系。【方法】选用根据基因Tri13和Tri3序列设计的特异性引物分析来自11个省（自治区）的92株禾谷镰孢复合种菌株的毒素化学型；从100条哥伦比亚大学（UBC）开发的通用引物中筛选出13 条扩增条带丰富、重复性好、信号强、背景清晰的引物，利用筛选出的引物对供试菌株进行ISSR-PCR扩增，使用Popgen32软件计算多态性位点百分率、Shannon’s多样性指数、群体间的遗传距离和遗传相似性；依据Nei’s 遗传距离，利用NTsys2.10e软件进行UPGMA聚类分析，并构建供试菌株的聚类图。【结果】供试的92株禾谷镰孢复合种菌株的产毒素化学型有4种：DON、15-ADON、DON+15-ADON和NIV+15-ADON。其中产生DON和15-ADON的菌株分别为1株和20株；同时产生15-ADON和NIV的有1株；同时产生15-ADON和DON的有55株。筛选出的13条引物对所有供试菌株进行PCR扩增，共获得102条带，其中多态性条带101条，多态性比率为99.02%，平均每条引物产生条带为7.85条。在群体平均水平上，基因多样性指数（H）为0.3129，Shannon’s的信息指数（I）为0.4774，表明禾谷镰孢复合种存在较高的遗传多样性水平；不同地理种群间，各群体的遗传多样性水平具有一定差异，河北、山西、黑龙江和吉林种群遗传多样性最高，安徽和河南种群最低。地理种群间的基因分化系数（Gst）为0.2722，说明不同地理种群间存在一定的遗传变异，但大部分遗传变异（72.78%）发生在种群内。遗传分化系数估算的基因流值Nm=1.3372（＞1），表明不同地理种群间存在一定的基因流动；遗传关系结果表明河北、山西、黑龙江、吉林、辽宁、内蒙古和甘肃菌株群体间亲缘关系较近，山东、江苏、河南和安徽菌株间亲缘关系较近，安徽和内蒙古菌株间的亲缘关系最远。聚类分析显示所有菌株相似系数为0.43—0.95。在相似系数为0.43时，所有菌株分成2大类群，Group 1包括4株北方春播区菌株（吉林、山西和河北张家口），不产生NIV和DON；Group 2由余下的88株菌株构成，产生的毒素类型主要为DON和15-ADON，其来源于北方春播区（山西、黑龙江、吉林、辽宁、甘肃和河北张家口、唐山）和黄淮海夏播区（河北石家庄、河南、山东、江苏和安徽），在相似系数为0.664时，各类群分成不同的亚群，亚群的结果与菌株来源有一定的相关性。聚类分析结果和毒素化学型分析显示，同一地理种群的菌株多数聚在一起，个别菌株分散到不同的分支上。【结论】北方春播区和黄淮海夏播区的禾谷镰孢复合种主要的毒素化学型为DON和15-ADON。引起玉米穗腐病的禾谷镰孢复合种菌株群体内存在丰富的遗传变异，不同地理种群间存在一定的基因交流，遗传多样性与地理来源有关。

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

Abstract: 【Objective】The objective of this study is to clarify the toxigenic chemotype and genetic diversity of Fusarium graminearum species complex (FGSC) causing maize ear rot from different provinces (autonomous region).【Method】The toxigenic chemotypes of 92 FGSC isolates collected from 11 provinces (autonomous region) were analyzed by using the specific primer designed based on Tri13 and Tri3 sequences. Thirteen primers with abundant bands, good repeatability, strong signal and clear background were screened from 100 universal primers developed by Columbia University (UBC) and used for ISSR-PCR amplification of these isolates. The Popgen32 software was used to calculate the percentage of polymorphic loci, Shannon’s diversity index, genetic distance and genetic similarity among populations. According to Nei’s genetic distance, UPGMA cluster analysis was carried out using NTsys2.10e software, and the dendrogram of different geographical populations was constructed. 【Result】 Four different toxigenic chemotypes including DON, 15-ADON, DON+15-ADON and NIV+15-ADON were detected among 92 FGSC isolates. One and 20 isolates represented the DON and 15-ADON chemotypes, respectively. One isolate produced both 15-ADON and NIV, and 55 isolates carried both 15-ADON and DON producing segments. The PCR amplification of all FGSC isolates was performed with 13 ISSR primers and a total of 102 fragments were obtained, of which 101 fragments displayed polymorphic and accounted for 99.02% in the total amplified fragments. The average number of fragments amplified per primer was 7.85. At population average level, the Nei’s gene diversity index (H) was 0.3129 and the Shannon’s information index (I) was 0.4774, which indicated that there was a high genetic diversity in FGSC isolates. The diversity existed among fungal populations from different geographic regions. Hebei, Shanxi, Heilongjiang and Jilin had the highest genetic diversity, while Anhui and Henan had the lowest genetic diversity. The genetic differentiation coefficient (Gst) of geographical populations was 0.2722, indicating that there were some genetic variations among different geographical populations, but most of the genetic variation (72.78%) occurred in the population. The Nm=1.3372 (＞1) indicated that there was a certain gene flow among different geographical populations. The results of genetic relationship showed that the populations of Hebei, Shanxi, Heilongjiang, Jilin, Liaoning, Inner Mongolia and Gansu were relatively close, and the populations of Shandong, Jiangsu, Henan and Anhui also displayed a close relationship, whereas the population isolated from Anhui had the lowest similarity with population isolated from Inner Mongolia. Cluster analysis showed that the similarity coefficient of all isolates was 0.43-0.95. All the isolates were divided into two groups with the similarity coefficient of 0.43. Group 1 included 4 isolates of northern spring sowing region (Jilin, Shanxi and Zhangjiakou, Hebei), which did not produce NIV and DON. Group 2 consisted of the remaining 88 isolates which originated from northern spring sowing region (Shanxi, Heilongjiang, Jilin, Liaoning, Gansu and Hebei (Zhangjiakou and Tangshan)) and Huang-Huai-Hai summer sowing region (Henan, Shandong, Jiangsu, Anhui and Shijiazhuang, Hebei), and the toxins produced by these isolates were mainly DON and 15-ADON. When the similarity coefficient was 0.664, each group was divided into different subgroups, and the results of subgroups were related to the source of the isolates.【Conclusion】The main toxigenic chemotypes of FGSC from northern spring sowing region and Huang-Huai-Hai summer sowing region are DON and 15-ADON. There are abundant genetic variations in the populations of FGSC causing maize ear rot. There are certain gene exchanges among different geographical populations. The genetic diversity was related to the geographical origin.

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

马红霞，孙华，郭宁，张海剑，石洁，常佳迎. 禾谷镰孢复合种毒素化学型及遗传多样性分析[J]. 中国农业科学, 2018, 51(1): 82-95.

MA HongXia, SUN Hua, GUO Ning, ZHANG HaiJian, SHI Jie, CHANG JiaYing. Analysis of Toxigenic Chemotype and Genetic Diversity of the Fusarium graminearum Species Complex[J]. Scientia Agricultura Sinica, 2018, 51(1): 82-95.

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参考文献

[1] 秦子惠, 任旭, 江凯, 武小菲, 杨知还, 王晓鸣. 我国玉米穗腐病致病镰孢种群及禾谷镰孢复合种的鉴定. 植物保护学报, 2014, 41(5): 589-596.

QIN Z H, REN X, JIANG K, WU X F, YANG Z H, WANG X M. Identification of Fusarium species and F. graminearum species complex causing maize ear rot in China. Journal of Plant Protection, 2014, 41(5): 589-596. (in Chinese)

[2] 石洁. 玉米镰刀菌型茎腐、穗腐、苗期根腐病的相互关系及防治[D]. 保定: 河北农业大学, 2002.

SHI J. Relationship and control of Fusarium stalk rot, ear rot and seeding root rot in maize[D]. Baoding: Hebei Agricultural University, 2002. (in Chinese)

[3] 任金平, 吴新兰, 孙秀华. 吉林省玉米镰刀菌穗腐病和茎腐病病原菌传染循环研究. 玉米科学, 1995, 3(增刊1): 25-28.

Ren J P, Wu X L, Sun X H. A study on infection cycle of pathogen of corn ear rot and stalk rot in Jilin Province. Journal of Maize Sciences, 1995, 3(suppl. 1): 25-28. (in Chinese)

[4] 邹庆道, 许远, 王立, 陈捷. 玉米镰孢菌穗腐病和茎腐病侵染规律相互关系的研究. 沈阳农业大学学报, 2000, 31(5): 487-489.

ZOU Q D, XU Y, WANG L, CHEN J. Relationship of infection cycle between ear rot and stalk rot in maize caused by Fusarium. Journal of Shenyang Agricultural University, 2000, 31(5): 487-489. (in Chinese)

[5] 朱维芳. 玉米籽粒中镰孢菌的分离及相互作用对产毒的影响[D]. 保定: 河北农业大学, 2014.

ZHU W F. The isolation of Fusarium species and the effect of interaction between Fusarium species for mycotoxin accumulation[D]. Baoding: Hebei Agricultural University, 2014. (in Chinese)

[6] 蔡静平, 刘新影, 翟焕趁. 禾谷镰刀菌DON毒素生物合成调控研究进展. 河南工业大学学报(自然科学版), 2016, 37(1): 114-119.

CAI J P, LIU X Y, ZHAI H C. Progress on the regulation of DON toxin produced by Fusarium graminearum. Journal of Henan University of Technology (Natural Science Edition), 2016, 37(1): 114-119. (in Chinese)

[7] Champeil A, Doré T, Fourbet J F. Fusarium head blight: epidemiological origin of the effects of cultural practices on head blight attacks and the production of mycotoxins by Fusarium in wheat grains. Plant Science, 2004, 166(6): 1389-1415.

[8] Nicholson P, Simpson D R, Wilson A H, Chandler E, Thomsett M. Detection and differentiation of trichothecene and enniatin-producing Fusarium species on small-grain cereals. European Journal of Plant Pathology, 2004, 110(5/6): 503-514.

[9] Albayrak G, Yörük E, Gazda?li A, Sharifnabi B. Genetic diversity among Fusarium graminearum and F. culmorum isolates based on ISSR markers. Archives of Biological Sciences, 2016, 68(2): 333-343.

[10] 邢锦城. 禾谷镰刀菌致病相关基因的鉴定及其毒素DON特异亲和肽段的淘选[D]. 南京: 南京农业大学, 2010.

XING J C. Identification of genes related to virulence of Fusarium graminearum and biopanning of DON toxin specific affinity peptides[D]. Nanjing: Nanjing Agricultural University, 2010. (in Chinese)

[11] Ortega L M, Dinolfo M I, Astoreca A L, Alberione E J, Stenglein S A, Alconada T M. Molecular and mycotoxin characterization of Fusarium graminearum isolates obtained from wheat at a single field in Argentina. Mycological Progress, 2016, 15: 1.

[12] Miedaner T, Schilling A G, Geiger H H. Molecular genetic diversity and variation for aggressiveness in populations of Fusarium graminearum and Fusarium culmorum sampled from wheat fields in different countries. Journal of Phytopathology, 2010, 149(11): 641-648.

[13] 孙华, 张海剑, 马红霞, 石洁, 郭宁, 陈丹. 春玉米区穗腐病病原菌组成、分布及禾谷镰孢复合种的鉴定. 植物病理学报, doi:10.13926/j.cnki.apps.000161.

SUN H, ZHANG H J, MA H X, SHI J, GUO N, CHEN D. Composition and distribution of pathogens causing ear rot in spring maize region and identification of Fusarium graminearum species complex. Acta Phytopathologica Sinica, doi:10.13926/j.cnki.apps. 000161. (in Chinese)

[14] Lee T, Han Y K, Kim K H, Yun S H, Lee Y W. Tri13 and Tri7 determine deoxynivalenol- and nivalenol-producing chemotypes of Gibberella zeae. Applied and Environmental Microbiology, 2002, 68(5): 2148-2154.

[15] Brown D W, McCormick S P, Alexander N J, Proctor R H, Desjardins A E. Inactivation of a cytochrome P-450 is a determinant of trichothecene diversity in Fusarium species. Fungal Genetics and Biology, 2002, 36(3): 224-233.

[16] Chandler E A, Simpson D R, Thomsett M A, Nicholson P. Development of PCR assays to Tri7 and Tri13 trichothecene biosynthetic genes, and characterisation of chemotypes of Fusarium graminearum, Fusarium culmorum and Fusarium cerealis. Physiological and Molecular Plant Pathology, 2003, 62(6): 355-367.

[17] 董怀玉, 徐婧, 王丽娟, 刘可杰, 姜钰, 胡兰, 张明会, 徐秀德. 我国北方玉米子粒禾谷镰孢菌群产毒素化学型检测分析. 玉米科学, 2014, 22(6): 126-130.

DONG H Y, XU J, WANG L J, LIU K J, JIANG Y, HU L, ZHANG M H, XU X D. Molecular detection of mycotoxin chemotypes of seeds borned Fusarium graminearum clade on maize. Journal of maize sciences, 2014, 22(6): 126-130. (in Chinese)

[18] Jennings P, Coates M E, Turner J A, Chandler E A, Nicholson P. Determination of deoxynivalenol and nivalenol chemotypes of Fusarium culmorum isolates from England and Wales by PCR assay. Plant Pathology, 2010, 53(2): 182-190.

[19] 李伟, 胡迎春, 陈莹, 张爱香, 陈怀谷. 长江流域禾谷镰孢菌群部分菌株系统发育学、产毒素化学型及致病力研究. 菌物学报, 2010, 29(1): 51-58.

LI W, HU Y C, CHEN Y, ZHANG A X, CHEN H G. Phylogenetic analysis, chemotype diversity, and pathogenicity of the Fusarium graminearum clade in the Yangtze basin. Mycosystema, 2010, 29(1): 51-58. (in Chinese)

[20] 纪莉景. 中国不同生态地区禾谷镰刀菌种群分化及遗传多样性分析[D]. 保定: 河北农业大学, 2007.

JI L J. Population and genetic diversity of Fusarium graminearum clade from different ecological regions of China[D]. Baoding: Hebei Agricultural University, 2007. (in Chinese)

[21] 邹庆道. 玉米穗腐病和茎腐病镰孢菌病原学相互关系及遗传多态性研究. 植物病理学报, 2003, 33(1): 93-94.

ZOU Q D. Studies on mutual relationship in etiology and genetic polymorphism of Fusarium from stalk rot and ear rot in maize. Acta Phytopathologica Sinica, 2003, 33(1): 93-94. (in Chinese)

[22] 孙枫. 禾谷镰孢菌群体遗传结构研究[D]. 南京: 南京农业大学, 2004.

SUN F. Population genetic structure of Fusarium graminearum species complex[D]. Nanjing: Nanjing Agricultural University, 2004. (in Chinese)

[23] 刘恒, 侯丽娟, 马红娜, 白耀博, 李强, 王保通. 陕西省小麦禾谷镰刀菌的遗传多样性研究. 植物病理学报, 2010, 40(6): 615-621.

LIU H, HOU L J, MA H N, BAI Y B, LI Q, WANG B T. Genetic diversity of Fusarium graminearum from wheat in Shaanxi Province. Acta Phytopathologica Sinica, 2010, 40(6): 615-621. (in Chinese)

[24] 洪旭. 四川省小麦赤霉病菌致病性分化及遗传多样性分析[D]. 雅安: 四川农业大学, 2012.

HONG X. Studies of Fusarium spp. pathogenicity differentiation and analysis of Fusarium graminearum genetic diversity in Sichuan[D]. Yaan: Sichuan Agricultural University, 2012. (in Chinese)

[25] 董怀玉, 王大为, 王丽娟, 刘可杰, 姜钰, 徐秀德, 左驰. 北方春玉米区玉米穗腐病禾谷镰孢菌复合种SCAR类型测定. 东北农业大学学报, 2015, 46(10): 23-28.

DONG H Y, WANG D W, WANG L J, LIU K J, JIANG Y, XU X D, ZUO C. SCAR type detection of Fusarium graminearum clade species complex on spring maize producing areas of north China. Journal of Northeast Agricultural University, 2015, 46(10): 23-28. (in Chinese)

[26] 任旭. 我国玉米穗腐病主要致病镰孢菌多样性研究[D]. 北京: 中国农业科学院, 2011.

REN X. Diversity analyses of Fusarium spp., the main causal agents of maize ear rot in China[D]. Beijing: Chinese Academy of Agricultural Sciences, 2011. (in Chinese)

[27] 李蕊倩, 何瑞, 张跃兵, 徐玉梅, 王建明. 镰刀菌ISSR标记体系的建立及遗传多样性分析. 中国农业科学, 2009, 42(9): 3139-3146.

LI R Q, HE R, ZHANG Y B, XU Y M, WANG J M. Establishment of ISSR reaction system of Fusarium and its analysis of genetic diversity. Scientia Agricultura Sinica, 2009, 42(9): 3139-3146. (in Chinese)

[28] 王建明, 李蕊倩, 畅引东, 何瑞, 李新风, 徐玉梅, 高俊明. 尖孢镰刀菌及芬芳镰刀菌遗传多样性的ISSR分析. 植物病理学报, 2011, 41(4): 337-344.

WANG J M, LI R Q, CHANG Y D, HE R, LI X F, XU Y M, GAO J M. ISSR analysis of genetic diversity of Fusarium oxysporum and F. redolens. Acta Phytopathologica Sinica, 2011, 41(4): 337-344. (in Chinese)

[29] 李新凤. 山西镰刀菌种类鉴定及遗传多样性分析研究[D]. 太谷: 山西农业大学, 2013.

LI X F. The morphological identification and genetic diversity of Fusarium in Shanxi province[D]. Taigu: Shanxi Agriculture University, 2013. (in Chinese)

[30] Bayraktar H, Dolar F S, Maden S. Use of RAPD and ISSR markers in detection of genetic variation and population structure among Fusarium oxysporum f. sp. ciceris isolates on chickpea in Turkey. Journal of Phytopathology, 2008, 156(3): 146-154.

[31] Waalwijk C, Kastelein P, Vries I D, Kerényi Z, Lee T V D, Hesselink T, Köhl J, Kema G. Major changes in Fusarium spp. in wheat in the Netherlands. European Journal of Plant Pathology, 2003, 109: 743-754.

[32] Nei M. Genetic distance between populations. The American Naturalist, 1972, 106(949): 283-292.

[33] QU B, LI H P, ZHANG J B, XU Y B, HUANG T, WU A B, ZHAO C S, CARTER J, NICHOLSON P, LIAO Y C. Geographic distribution and genetic diversity of Fusarium graminearum and F. asiaticum on wheat spikes throughout China. Plant Pathology, 2008, 57(1): 15-24.

[34] 何婧, 郭庆元, 王晓鸣, 宋利宁, 张维娜, 武小菲. 利用ISSR技术分析禾谷镰孢菌群体遗传多样性的研究. 玉米科学, 2011, 19(2): 129-134.

HE J, GUO Q Y, WANG X M, SONG L N, ZHANG W N, WU X F. Study on genetic diversity of Fusarium graminearum populations causing maize stalk rot by ISSR analysis. Journal of maize sciences, 2011, 19(2): 129-134. (in Chinese)

[35] 瞿波. 中国禾谷镰刀菌(Fusarium graminearum)的遗传多样性及其与尼泊尔、欧美菌系的比较[D]. 武汉: 华中农业大学, 2003.

QU B. Genetic diversity of Fusarium gaminearum in China and its comparison with the isolates from of Nepal, Europe and USA[D]. Wuhan: Huazhong Agricultural University, 2003. (in Chinese)