海南三亚和黄淮海地区玉米小斑病菌致病性及遗传多样性分析

doi:10.3864/j.issn.0578-1752.2020.06.007

摘要/Abstract

摘要：

【目的】明确海南三亚玉米南繁基地和黄淮海玉米主产区的玉米小斑病菌（Bipolaria maydis）菌株的致病性差异,以及遗传多样性和亲缘关系的远近,为海南三亚南繁基地育种过程中玉米品种的抗病性筛选提供理论依据,并对玉米品种在黄淮海地区的推广种植提供参考。【方法】对采集自海南三亚和黄淮海地区的玉米小斑病病叶进行病原菌分离,经过形态学和分子生物学方法对分离到的菌株进行鉴定,共获得61株玉米小斑病菌菌株。采用人工喷雾接种法对61株小斑病菌菌株的致病力进行鉴定;并选取16条扩增条带清晰、重复性好、多态性高的引物进行ISSR-PCR扩增,利用Popgen32生物软件计算群体间的遗传距离和遗传相似性,并利用NTsys2.10e软件进行聚类分析,构建海南三亚与黄淮海地区小斑病菌的聚类图。【结果】致病性测定结果表明,海南三亚地区未分离到弱致病力菌株,强致病力菌株个数占三亚地区总菌株的57.1%;黄淮海地区分离到的弱致病力菌株占黄淮海地区总菌株的47.5%,强致病力菌株占黄淮海地区总菌株的17.5%。遗传多样性分析结果表明,在群体平均水平上,Nei’s基因多样性指数（H）为0.2820,Shannon’s信息指数（I）为0.4197,表明玉米小斑病菌具有一定的遗传变异;不同地理种群间的遗传相似系数为0.9028—0.9618,遗传距离为0.0390—0.1023,表明整体遗传相似系数较高,遗传距离较近;但不同地理种群间仍存在不同程度的遗传分化,其中,河南与河北的菌株群体遗传相似系数最高（GS=0.9618）,亲缘关系最近;河南和三亚地区的菌株群体遗传相似系数最低（GS=0.9028）,亲缘关系相对较远。聚类分析结果表明,三亚地区菌株和黄淮海地区菌株在相似系数为0.722时,明显被分为两大类群。【结论】黄淮海玉米主产区与海南三亚玉米南繁基地的玉米小斑病菌在致病力上存在一定差异,三亚地区强致病力菌株的分离频率高于黄淮海地区。黄淮海地区与三亚地区的菌株群体具有丰富的遗传多样性,且遗传多样性与地理来源有一定关系,地域相邻的菌株遗传关系较近;但三亚地区与黄淮海地区的菌株遗传相似系数相对较高,亲缘关系较近,且两种群间存在一定基因交流。因此,三亚地区作为玉米的南繁基地,其自然发病条件下筛选出对小斑病具有抗性的玉米品种或抗性亲本材料,对黄淮海地区小斑病抗性的逐年提高具有一定的影响,有利于减轻黄淮海地区病害的发生。

关键词: 玉米小斑病菌, 玉米小斑病, 南繁基地, 海南三亚, 黄淮海地区, 致病性, 遗传多样性

Abstract:

【Objective】The objective of this study is to clarify the pathogenicity differences, genetic diversity and relationship among the Bipolaria maydis strains from southern maize breeding base of Sanya, Hainan and Huang-Huai-Hai region, and to provide a theoretical basis for screening resistance of maize varieties under natural disease conditions in the breeding process. It also provides a reference for the popularization and utilization of maize varieties in Huang-Huai-Hai region.【Method】The pathogens were isolated from diseased leaves collected from Sanya, Hainan and Huang-Huai-Hai region, and a total of 61 strains were obtained by identification of morphology and molecular biology. The pathogenicity of 61 strains was identified by an artificial spray inoculation method, and 16 primers with clear, repetitive and polymorphic bands were used for ISSR-PCR. Popgen32 software was used to calculate the genetic distance and genetic similarity among the populations, and NTsys2.10e software was used for cluster analysis and constructing a cluster map of the pathogens in Sanya, Hainan and Huang-Huai-Hai region.【Result】The results of pathogenicity test showed that there was no weak pathogenic strain and 57.1% strong pathogenic strains isolated from Sanya, Hainan, while that of Huang-Huai-Hai region accounted for 47.5% and 17.5%, respectively. The results of genetic diversity analysis showed that the Nei’s gene diversity index (H) was 0.2820 and Shannon’s information index (I) was 0.4197 on the average level of population, indicating that there was a certain genetic variation in the pathogen. The genetic similarity coefficient was 0.9028-0.9618 and the genetic distance was 0.0390-0.1023 among different geographical populations, which indicated that the overall genetic similarity coefficient was relatively high and the genetic distance was close, but there were still different degrees of genetic differentiation among different geographical populations. Among which, the genetic similarity coefficient of strains in Henan and Hebei was the highest (GS=0.9618), and the genetic relationship was the closest; the genetic similarity coefficient of strains in Henan and Sanya was the lowest (GS=0.9028), and the genetic relationship was relatively far. The cluster analysis showed that the strains in Sanya and Huang-Huai-Hai region were obviously divided into two groups when the similarity coefficient was 0.722.【Conclusion】There are some differences in B. maydis pathogenicity between the main maize producing area of Huang-Huai-Hai region and the southern maize breeding base of Sanya, Hainan. The isolation frequency of the strong pathogenic strains in Sanya was higher than that in Huang-Huai-Hai region. The strain populations in Huang-Huai-Hai region and Sanya region have rich genetic diversity, and genetic diversity has a certain relationship with geographical origin. The genetic relationship of the strains in the adjacent regions was relatively close. However, the genetic similarity coefficient of strains between Sanya region and Huang-Huai-Hai region is relatively high, the genetic relationship is close, and also there are some gene exchanges between the two populations. Therefore, selection of resistant maize varieties or resistant parent materials under natural conditions in southern maize breeding base of Sanya has a certain effect on the yearly increase of resistance to southern corn leaf blight in Huang-Huai-Hai region, which is helpful to reduce the occurrence of disease in Huang-Huai-Hai region.

Key words: Bipolaria maydis, southern corn leaf blight, southern maize breeding base, Sanya,Hainan, Huang-Huai-Hai region, pathogenicity, genetic diversity

常佳迎,刘树森,石洁,郭宁,张海剑,马红霞,杨春凤. 海南三亚和黄淮海地区玉米小斑病菌致病性及遗传多样性分析[J]. 中国农业科学, 2020, 53(6): 1154-1165.

JiaYing CHANG,ShuSen LIU,Jie SHI,Ning GUO,HaiJian ZHANG,HongXia MA,ChunFeng YANG. Pathogenicity and Genetic Diversity of Bipolaria maydis in Sanya, Hainan and Huang-Huai-Hai Region[J]. Scientia Agricultura Sinica, 2020, 53(6): 1154-1165.

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图/表 5

表1

黄淮海地区和海南三亚小斑病菌的致病性测定"

菌株编号 Strain number	采样地点 Collection site	平均发病面积Average disease area (%)	病斑大小 Lesion size (mm)	致病力 Pathogenicity	病斑描述 Lesion description
菌株编号 Strain number	采样地点 Collection site	平均发病面积Average disease area (%)	病斑大小 Lesion size (mm)	致病力 Pathogenicity	病斑颜色 Lesion color	形状 Shape	褐色边缘 Brown edge
HB1802-1	河北省保定市满城区 Mancheng, Baoding, Hebei	7.50	(2-8) × (1-2)	弱 Weak	浅黄色 Light yellow	长方形Rectangle	-
HB1802-2	河北省保定市满城区 Mancheng, Baoding, Hebei	16.75	(1-7) × (1-1.5)	中 Moderate	深褐色 Dark brown	椭圆形 Oval	+
HB1803-1	河北省邯郸市邯郸县 Handan, Handan, Hebei	7.50	(2-7) × (1-2)	弱 Weak	黄褐色 Yellow brown	长方形Rectangle	-
HB1803-2	河北省邯郸市肥乡县 Feixiang, Handan, Hebei	27.75	(2-8) × (1-2)	中 Moderate	浅黄色 Light yellow	长方形Rectangle	+
HB1803-3	河北省邯郸市肥乡县 Feixiang, Handan, Hebei	5.00	(3-8) × (1-1.5)	弱 Weak	黄褐色 Yellow brown	长方形Rectangle	+
HB1804-1	河北省廊坊市香河县 Xianghe, Langfang, Hebei	1.75	(2-9) × (1-3)	弱 Weak	浅黄色 Light yellow	长方形Rectangle	-
HB1804-2	河北省廊坊市固安县 Gu^’an, Langfang, Hebei	76.75	(4-7) × (1-2)	强 Strong	浅黄色 Light yellow	长方形Rectangle	-
HB1804-3	河北省廊坊市固安县 Gu^’an, Langfang, Hebei	4.25	(5-8) × (1-2)	弱 Weak	黄褐色 Yellow brown	长方形Rectangle	-
HB1804-4	河北省廊坊市香河县 Xianghe, Langfang, Hebei	8.75	(1-3) × (1-1)	弱 Weak	黄褐色 Yellow brown	长方形Rectangle	+
HB1804-5	河北省廊坊市固安县 Gu^’an, Langfang, Hebei	7.75	(4-6) × (1-1.5)	弱 Weak	浅黄色 Light yellow	椭圆形 Oval	-
HB1804-6	河北省廊坊市固安县 Gu^’an, Langfang, Hebei	10.25	(5-7) × (1-2)	中 Moderate	黄褐色 Yellow brown	长方形Rectangle	+
HB1807-1	河北省唐山市滦南县 Luannan, Tangshan, Hebei	1.25	(3-9) × (1.5-2)	弱 Weak	浅黄色 Light yellow	椭圆形 Oval	-
HB1807-2	河北省唐山市乐亭县 Laoting, Tangshan, Hebei	8.25	(2-9) × (1-2)	弱 Weak	黄褐色 Yellow brown	长方形Rectangle	+
HB1807-3	河北省唐山市滦南县 Luannan, Tangshan, Hebei	20.00	(3-8) × (1-2)	中 Moderate	深褐色 Dark brown	椭圆形 Oval	+
HB1807-4	河北省唐山市开发区 Development Zone, Tangshan, Hebei	9.50	(3-8) × (1-2)	弱 Weak	深褐色 Dark brown	长方形Rectangle	+
HB1807-5	河北省唐山市开发区 Development Zone, Tangshan, Hebei	85.50	(3-5) × (1-2)	强 Strong	浅黄色 Light yellow	长方形Rectangle	-
HB1807-6	河北省唐山市乐亭县 Laoting, Tangshan, Hebei	81.25	(1-5)×(1-2)	强 Strong	黄褐色 Yellow brown	椭圆形 Oval	+
HB1807-7	河北省唐山市乐亭县 Laoting, Tangshan, Hebei	72.00	(3-7) × (1-2)	强 Strong	黄褐色 Yellow brown	椭圆形 Oval	+
HB1807-8	河北省唐山市迁安 Qian^’an, Tangshan, Hebei	13.25	(2-6) × (1-2)	中 Moderate	浅黄色 Light yellow	椭圆形 Oval	-
HB1807-9	河北省唐山市迁安 Qian^’an, Tangshan, Hebei	24.50	(1-4) × (1-2)	中 Moderate	黄褐色 Yellow brown	椭圆形 Oval	+
HB1807-10	河北省唐山市玉田县 Yutian, Tangshan, Hebei	9.50	(2-8) × (1-1.5)	弱 Weak	浅黄色 Light yellow	长方形Rectangle	-
HB1807-11	河北省唐山市开发区 Development Zone, Tangshan, Hebei	58.00	(2-6) × (1-2)	强 Strong	浅黄色 Light yellow	椭圆形 Oval	+
HB1807-12	河北省唐山市滦南县 Luannan, Tangshan, Hebei	2.75	(4-8) × (1-2)	弱 Weak	浅黄色 Light yellow	长方形Rectangle	-
HB1808-1	河北省衡水市深州市 Shenzhou, Hengshui, Hebei	27.50	(3-9) × (1-1.5)	中 Moderate	深褐色 Dark brown	长方形Rectangle	+
HB1808-2	河北省衡水市深州市 Shenzhou, Hengshui, Hebei	13.50	(2-9) × (1-2)	中 Moderate	深褐色 Dark brown	长方形Rectangle	+
HB1808-3	河北省衡水市深州市 Shenzhou, Hengshui, Hebei	17.50	(3-8) × (1-2)	中 Moderate	浅黄色 Light yellow	长方形Rectangle	-
HB1808-4	河北省衡水市深州市 Shenzhou, Hengshui, Hebei	9.50	(2-5) × (1-2)	弱 Weak	浅黄色 Light yellow	椭圆形 Oval	-
HN1802-1	河南省鹤壁市浚县 Xunxian, Hebi, Henan	12.50	(4-9) × (1-2)	中 Moderate	深褐色 Dark brown	长方形Rectangle	-
HN1802-2	河南省鹤壁市浚县 Xunxian, Hebi, Henan	3.25	(1-8) × (1-1.5)	弱 Weak	浅黄色 Light yellow	长方形Rectangle	-
HN1802-3	河南省鹤壁市浚县 Xunxian, Hebi, Henan	12.50	(4-6) × (1-1.5)	中 Moderate	深褐色 Dark brown	椭圆形 Oval	-
HN1802-4	河南省鹤壁市浚县 Xunxian, Hebi, Henan	9.50	(4-7) × (1-1.5)	弱 Weak	深褐色 Dark brown	长方形Rectangle	-
HN1803-1	河南省安阳市滑县 Huaxian, Anyang, Henan	13.25	(1-7) × (1-1.5)	中 Moderate	深褐色 Dark brown	长方形Rectangle	+
HN1804-1	河南省新乡市辉县 Huixian, Xinxiang, Henan	15.00	(5-8) × (1-1.5)	中 Moderate	深褐色 Dark brown	长方形Rectangle	+
HN1807-1	河南省漯河市召陵区 Zhaoling, Luohe, Henan	77.50	(1-8) × (1-2)	强 Strong	黄褐色 Yellow brown	长方形Rectangle	+
HN1807-2	河南省漯河市源汇区 Yuanhui, Luohe, Henan	3.25	(3-7) × (1-2)	弱 Weak	浅黄色 Light yellow	长方形Rectangle	-
HN1807-3	河南省漯河市临颍县 Linying, Luohe, Henan	4.00	(3-8) × (1-2)	弱 Weak	深褐色 Dark brown	长方形Rectangle	+
HN1810-1	河南省周口市西华县 Xihua, Zhoukou, Henan	13.50	(4-6) × (1-1.5)	中 Moderate	深褐色 Dark brown	椭圆形 Oval	+
SD1811-1	山东青岛市平度市 Pingdu, Qingdao, Shandong	6.50	(3-8) × (1-1.5)	弱 Weak	浅黄色 Light yellow	长方形Rectangle	+
SD1811-2	山东省青岛市平度市 Pingdu, Qingdao, Shandong	3.50	(2-8) × (1-1.5)	弱 Weak	黄褐色 Yellow brown	长方形Rectangle	+
SD1812-1	山东省东营市利津县 Lijin, Dongying, Shandong	36.25	(2-9) × (1-2)	强 Strong	深褐色 Dark brown	椭圆形 Oval	-
SY-1	海南省乐东黎族自治县九所镇 Jiusuo, Ledong Li Autonomous County, Hainan	20.00	(3-7) × (1-1.5)	中 Moderate	深褐色 Dark brown	长方形Rectangle	+
SY-2	海南省乐东黎族自治县九所镇 Jiusuo, Ledong Li Autonomous County, Hainan	18.75	(1-8) × (1-2)	中 Moderate	黄褐色 Yellow brown	长方形Rectangle	+
SY-3	海南省乐东黎族自治县九所镇 Jiusuo, Ledong Li Autonomous County, Hainan	21.25	(1-9) × (1-2)	中 Moderate	黄褐色 Yellow brown	椭圆形 Oval	-
SY-4	海南省乐东黎族自治县九所镇 Jiusuo, Ledong Li Autonomous County, Hainan	18.25	(1-7) × (0.5-2)	中 Moderate	浅黄色 Light yellow	长方形Rectangle	-
SY-5	海南省乐东黎族自治县九所镇 Jiusuo, Ledong Li Autonomous County, Hainan	70.25	(2-8) × (1-1.5)	强 Strong	黄褐色 Yellow brown	长方形Rectangle	-
SY-6	海南省三亚市吉阳区 Jiyang, Sanya, Hainan	57.00	(2-8) × (1-1.5)	强 Strong	深褐色 Dark brown	长方形Rectangle	-
SY-7	海南省三亚市吉阳区 Jiyang, Sanya, Hainan	52.00	(2-8) × (1-2)	强 Strong	深褐色 Dark brown	长方形Rectangle	+
SY-8	海南省三亚市吉阳区 Jiyang, Sanya, Hainan	25.00	(4-7) × (1-2)	中 Moderate	黄褐色 Yellow brown	长方形Rectangle	-
SY-9	海南省三亚市吉阳区 Jiyang, Sanya, Hainan	10.25	(1-8) × (1-2)	中 Moderate	黄褐色 Yellow brown	椭圆形 Oval	-
SY-10	海南省三亚市天涯区 Tianya, Sanya, Hainan	33.75	(1-7) × (0.5-2)	强 Strong	黄褐色 Yellow brown	长方形Rectangle	-
SY-11	海南省三亚市天涯区 Tianya, Sanya, Hainan	52.50	(1-9)×(1-1.5)	强 Strong	浅黄色 Light yellow	长方形Rectangle	-
SY-12	海南省乐东黎族自治县九所镇 Jiusuo, Ledong Li Autonomous County, Hainan	54.50	(2-8) × (1-1.5)	强 Strong	浅黄色 Light yellow	长方形Rectangle	-
SY-13	海南省三亚市海棠区 Haitang, Sanya, Hainan	24.50	(2-9) × (1-1.5)	中 Moderate	浅黄色 Light yellow	椭圆形 Oval	-
SY-14	海南省三亚市海棠区 Haitang, Sanya, Hainan	67.50	(5-8) × (1-2)	强 Strong	深褐色 Dark brown	长方形Rectangle	+
SY-15	海南省三亚市海棠区 Haitang, Sanya, Hainan	23.25	(5-8) × (1-1.5)	中 Moderate	黄褐色Yellow brown	椭圆形 Oval	+
SY-16	海南省三亚市海棠区 Haitang, Sanya, Hainan	86.25	(1-7) × (0.5-1.5)	强 Strong	浅黄色 Light yellow	椭圆形 Oval	-
SY-17	海南省三亚市海棠区 Haitang, Sanya, Hainan	92.50	(1-8) × (1-2)	强 Strong	浅黄色 Light yellow	长方形Rectangle	-
SY-18	海南省乐东黎族自治县九所镇 Jiusuo, Ledong Li Autonomous County, Hainan	64.00	(4-8) × (1-2)	强 Strong	浅黄色 Light yellow	长方形Rectangle	-
SY-19	海南省三亚市陵水县 Lingshui, Sanya, Hainan	12.25	(3-8) × (1-1.5)	中 Moderate	浅黄色 Light yellow	长方形Rectangle	-
SY-20	海南省三亚市陵水县 Lingshui, Sanya, Hainan	38.75	(2-8) × (1-1.5)	强 Strong	黄褐色 Yellow brown	长方形Rectangle	+
SY-21	海南省三亚市陵水县 Lingshui, Sanya, Hainan	38.25	(3-8) × (1-1.5)	强 Strong	深褐色 Dark brown	椭圆形Oval	+

表1

表2

表3

表4

图1

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引物 Primer	序列 Sequence (5′-3′)	退火温度 Tm (℃)	扩增条带数 Number of amplified bands	多态性条带 Number of polymorphic bands	多态性比率 Polymorphism percent (%)
807	(AG)₈T	46	10	8	80.00
808	(AG)₈C	46	17	14	82.35
812	(GA)₈A	45	7	7	100.00
825	(AC)₈T	50	8	5	62.50
834	(AG)₈YT	48	14	14	100.00
835	(AG)₈YC	50	12	11	91.67
841	(GA)₈YC	48	16	14	87.50
856	(AC)₈YA	50	8	7	87.50
857	(AC)₈YG	51	12	8	66.67
880	(GGAGA)₃	47	12	11	91.67
884	HBHA(GA)₆G	46	10	8	80.00
885	BHB(GA)₇	46	11	8	72.73
887	DVD(TC)₇	45	12	6	50.00
888	BDB(CA)₇	49	9	4	44.44
889	DBD(AC)₇	49	9	8	88.89
891	HVH(TG)₇	50	10	7	70.00
总计 Total			177	140	79.10

	多态性位点 Number of polymorphic loci	多态性比率 Polymorphism percent (%)	观察等位基因数 Observed number of alleles (Na)	有效等位基因 Effective number of alleles (Ne)	基因多样性指数Nei’s gene diversity index (H)	Shannon’s信息指数 Shannon’s information index (I)
河北HB	127	71.75	1.7175	1.4930	0.2783	0.4072
河南HN	100	56.50	1.5650	1.3822	0.2137	0.3134
山东SD	75	42.37	1.4237	1.3060	0.1717	0.2505
三亚SY	121	68.36	1.6836	1.4186	0.2437	0.3637
群体水平 Population level	140	79.10	1.7910	1.4904	0.2820	0.4197

种群 Group	河北HB	山东SD	河南HN	三亚SY
河北HB	****	0.9455	0.9618	0.9404
山东SD	0.0560	****	0.9351	0.9116
河南HN	0.0390	0.0671	****	0.9028
三亚SY	0.0614	0.0925	0.1023	****