陆地棉种质黄萎病抗性生理鉴定分析

doi:10.6048/j.issn.1001-4330.2023.04.025

新疆农业科学 ›› 2023, Vol. 60 ›› Issue (4): 992-1002.DOI: 10.6048/j.issn.1001-4330.2023.04.025

• 草业·植物保护·畜牧兽医·农业经济 • 上一篇下一篇

陆地棉种质黄萎病抗性生理鉴定分析

程利华¹^,²(), 杨红兰²(), 马清倩¹, 史莹³, 张大伟⁴, Alisher A. Abdullaev⁵, 张道远²()

1.新疆农业大学农学院/棉花教育部工程研究中心,乌鲁木齐 830052
2.新疆抗逆植物基因资源保育与利用重点实验室/中国科学院新疆生态与地理研究所,乌鲁木齐 830011
3.石河子大学生命科学学院,新疆石河子 832003
4.新疆农业科学院经济作物研究所,乌鲁木齐 830091
5.Center for Advanced Technologies under Ministry of Innovative Development of Uzbekistan,Uzbekistan 100174

收稿日期:2022-08-19 出版日期:2023-04-20 发布日期:2023-05-06
通信作者: 张道远(1973-),女,安徽合肥人,研究员,研究方向为干旱区资源保育与分子生物学,(E-mail)zhangdy@ms.xjb.ac.cn;杨红兰(1982-),女,四川丰都人,副研究员,研究方向为棉花抗逆分子育种,(E-mail)yanghonglan@ms.xjb.ac.cn
作者简介:程利华(1995-),女,河南商丘人,硕士研究生,研究方向为植物分子育种,(E-mail)chenglh5258@163.com
基金资助:
新疆维吾尔自治区优秀青年科技人才培养项目(2019Q035);国家自然科学基金青年科学项目(31700289)

Physiological identification and analysis of Verticillium wilt resistance of 10 foreign cotton germplasm resources

CHENG Lihua¹^,²(), YANG Honglan²(), MA Qingqian¹, SHI Ying³, ZHANG Dawei⁴, Alisher A. Abdullaev⁵, ZHANG Daoyuan²()

1. Cotton Engineering Research Center, Ministry of Education of the P.R.C., Xinjiang Agricultural University, Urumqi 830052, China
2. Xinjiang Key Lab of Conservation and Utilization of Gene Resources, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences,Urumqi 830011, China
3. College of Life Sciences, Shihezi University, Shihezi Xinjiang 832003, China
4. Institute of Economic Crops, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China
5. The Advanced Technology Center of the Ministry of National Innovation and Development of Uzbekistan, Tashkent 100174, Uzbekistan

Received:2022-08-19 Online:2023-04-20 Published:2023-05-06
Correspondence author: ZHANG Daoyuan(1973-), female, researcher. Her research interest is resource conservation and molecular biology in arid region, (E-mail)zhangdy@ms.xjb.ac.cn;YANG Honglan(1982-), female, associate professor, research interest: molecular breeding of cotton under stress, (E-mail)yanghonglan@ms.xjb.ac.cn
Supported by:
Outstanding Young Scientific and Technological Talents Training Program Project of Xinjiang Uygur Autonomous Region(2019Q035);National Natural Science Foundation of China(31700289)

摘要/Abstract

摘要：

【目的】研究陆地棉种质黄萎病抗性及生理鉴定,为棉花黄萎病抗性品种选育提供数据支持。【方法】以10份国外棉花种质(Gossypium hirsutum)为材料对室内盆栽棉花接种大丽轮枝菌(Verticillium dahliae Kleb),菌液浓度为10⁷ CFU/mL,接菌14 d后,观察并记录表型发病情况;采用荧光定量PCR检测叶片的菌含量;测定棉花叶片的木质素、过氧化氢(H₂O₂)、苯丙氨酸解氨酶(PAL)、丙二醛(MDA)和脯氨酸(PRO)等生理生化指标,结合相关性、主成分、隶属函数和聚类分析等方法,综合评价棉花的5个生理指标抗性。【结果】03804、A-6、04841和05189发病较轻;03804和A-6病菌DNA含量最少;在病菌侵染过程中,这些品种对PAL、PRO、木质素等的依赖程度不同,呈现出品种间差异;当棉花受到V. dahliae侵染后,MDA与H₂O₂呈极显著正相关,与PAL和PRO呈极显著负相关;五项抗病指标通过简化得到2个主成分,贡献率分别为64.951%,20.386%;10份材料根据棉花抗病综合指标可划分为1份抗病(03804)、5份耐病(05160、A-6、A-688、A-91、A-924)和4份易感病(04219、04841、A-1139、05189)材料。【结论】筛选出1份抗病、5份耐病和4份易感病的陆地棉种质。

关键词: 棉花; 黄萎病; 生理指标; 主成分; 种质资源

Abstract:

【Objective】Physiological identification and analysis of Verticillium wilt resistance of 10 foreign cotton germplasm resources, and provide data support for the breeding of Verticillium wilt resistance in China.【Methods】To study the Verticillium wilt resistance of 10 foreign cotton varieties (Gossypium hirsutum),The concentration of Verticillium dahliae Kleb infecting cotton seedling was 10⁷ spores /mL. After 14 days of inoculation, the morphology and phenotype were observed and photographed. Real-time PCR was used to determine the pathogene content in cotton leaves. The physiological and biochemical indexes (lignin, hydrogen peroxide (H₂O₂), phenylalanine ammonia lyase (PAL), malondialdehyde (MDA) and proline (PRO) in cotton leaves were determined. Correlation analysis, principal component analysis, subordinate function analysis and cluster analysis were conducted to evaluate the cotton resistance. 【Results】Phenotypic results showed that 03804, A-6, 04841 and 05189 had mild incidence. The results of qPCR showed that the 03804 and A-6 contained the least pathogene. In the course of pathogen infection, these varieties had different dependence on PAL, Proline, lignin, etc., indicating different characteristics of varieties. Correlation analysis showed that MDA was significantly positively correlated with H₂O₂ and negatively correlated with PAL and Proline after V. dahliae infection in cotton. Principal component analysis showed that two principal components were obtained from the five resistance indexes, and their contribution rates covered 64.951% and 20.38%, respectively. Analyzed by subordinate function and hierarchical cluster of comprehensive index of cotton disease resistance, 10 cotton cultivars could be divided into three categories, including one resistance, five tolerance and four susceptible cultivars. 【Conclusion】H₂O₂, PAL, MDA, PRO and lignin content can be used as physiological indexes for the identification of Verticillium wilt resistance in cotton.In this study, one disease-resistant, five disease-tolerant and four susceptible foreign land cotton germplasm are identified by morphological and physiological index after Verticillium dahliae Kleb infection.

Key words: cotton; Verticillium wilt; physiological indexes; principal component; germplasm resources

中图分类号:

S562

程利华, 杨红兰, 马清倩, 史莹, 张大伟, Alisher A. Abdullaev, 张道远. 陆地棉种质黄萎病抗性生理鉴定分析[J]. 新疆农业科学, 2023, 60(4): 992-1002.

CHENG Lihua, YANG Honglan, MA Qingqian, SHI Ying, ZHANG Dawei, Alisher A. Abdullaev, ZHANG Daoyuan. Physiological identification and analysis of Verticillium wilt resistance of 10 foreign cotton germplasm resources[J]. Xinjiang Agricultural Sciences, 2023, 60(4): 992-1002.

图/表 11

表1 棉花种质资源信息

Tab.1 Information of cotton germplasm resources

编号 Code	原名称 Original name	来源国家 Original country
03804	Ryad 285	土库曼斯坦
04219	Acal Shafter P18C	美国
04841	Qalla lot 361	澳大利亚
05160	Lightning express	阿塞拜疆
05189	Acala India	阿塞拜疆
A-1139	C-1211	乌兹别克斯坦
A-6	no name	秘鲁
A-688	Chimbayski 1086	乌兹别克斯坦
A-91	#4F Upland	美国
A-924	C-1472	乌兹别克斯坦

表2 实时荧光定量PCR体系

Tab.2 Real time PCR system

试剂 Reagent	使用量 Usage(μL)
SYBR PremixEx TAq^TMⅡ(TliRNAseHPlus)	10
PCR-ForwArd Primer(10 μM)	0.5
PCR-Reverse Primer(10 μM)	0.5
DNA溶液	2.0
灭菌水	7.0
Total	20

图1 棉苗在大丽轮枝菌(菌液107 个/mL)接菌后0、14 d的表型动态

Fig.1 Dynamic phenotype observation of cotton seedlings at 0 d and 14 d after inoculation with Verticillium dahliae Kleb(bacterial solution 107CFU/mL)

图2 室内和病田棉花黄萎病病情指数注:(a)室内棉苗接大丽轮枝菌14 d病情指数,(b)大田病情指数;CK:对照组,T:处理组;不同字母(a,b…)代表各种质组间在0.05水平上差异显著,P<0.05,下同

Fig.2 Verticillium wilt disease index of indoor and diseased fields Note: (a) Verticillium wilt disease index of indoor cotton seedlings exposed to R. dahliae for 14 days, (b) Disease index of Daejeon;CK:the control group, T: the treatment group; Different letters (a, b...) It means that there were significant differences among all quality groups at 0.05 level, P<0.05, the same as below

图3 棉花叶片含菌量qPCR分析注: (a) 接大丽轮枝菌菌液14d叶片含菌量qPCR分析 (b)qPCR标曲

Fig.3 Real-time PCR analysis of bacterial content in leaves of cotton Note: (a)Real-time PCR analysis of V. dahliae content in leaves after 14 days post inoculation of Verticillium dahliae Kleb (b) Real-time PCR scale-up

图4 棉苗接大丽轮枝菌菌液14 d后的生理生化指标注:CK:对照组,T:处理组;‘*’‘ **’:分别表示种质组内P<0.05和P<0.01,下同

Fig.4 Physiological and biochemical indexes of 10 cotton seedlings at 14 days after inoculation of Verticillium dahliae Kleb Note: CK:the control group, T: the treatment group; "*" "**" respectively represent P < 0.05 and P < 0.01 within germplasm group, the same as below

表3 棉花黄萎病抗性指标的相关系数矩阵

Tab.3 Correlation coefficient matrix of cotton Verticillium wilt resistance index

项目 Item	丙二醛含量 MDA content	过氧化氢含量 H₂O₂ content	苯丙氨酸解氨酶活性 PAL activity	木质素含量 Lignin content	游离脯氨酸含量 PRO content	发病率 Incidencet
过氧化氢含量 H₂O₂content	0.833^**
苯丙氨酸解氨酶活性 PAL activity	0.782^**	0.954^**
木质素含量 Lignin content	-0.454	-0.638^*	-0.615
游离脯氨酸含量 PRO content	0.383	0.134	0.17	-0.129
发病率 Incidence	0.639^*	0.813^**	0.839^**	-0.829^**	0.367
含菌量 Bacterial content	-0.322	0.109	0.013	-0.333	-0.25	0.283

表4 棉花黄萎病各综合指标的特征值、贡献率及累计贡献率

Tab.4 Characteristic values, contribution andcumulative contribution of each comprehensive index of cotton after Verticillium wilt pathogen infection

成分 Component	特征值 Eigen value	贡献率 Contribution rate (%)	累计贡献率 Cumulative contribution rate(%)
1	4.457	64.951	64.951
2	1.427	20.386	85.337
3	0.56	8.005	93.342
4	0.381	5.442	98.784
5	0.054	0.765	99.550
6	0.025	0.357	99.906
7	0.007	0.094	100

表5 棉花黄萎病抗性各生理指标的主成分矩阵

Tab.5 Principal component coefficients of each physiological index in cotton after Verticillium wilt pathogen infection

主成分 Princial component	丙二醛含量 MDA content	过氧化氢含量 H₂O₂ content	苯丙氨酸解氨酶活性 PAL Activity	木质素含量 Lignin content	游离脯氨酸含量 PRO content	发病率 Incidence	含菌量 Bacterial content
第一主成分 PC₁	0.936	0.985	-0.867	0.879	-0.344	0.978	0.317
第二主成分 PC₂	-0.265	0.065	-0.041	0.139	0.791	0.032	0.8

表6 棉花各品种黄萎病抗性的综合指标值Y、权重、U(Xj)、D值及综合评价

Tab.6 Value of each comprehensive index(CI), index weight, U(Xj)and comprehensive evaluation value (D value) of cotton after Verticillium wilt pathogen infection

品种(系) Variety(line)	Y₁	Y₂	U(X₁)	U(X₂)	D值 D value	综合评价 Comprehensive evaluation
03804	5.330	0.020	1.000	0.437	0.866	抗
A-6	0.630	1.860	0.340	1.000	0.498	耐
05160	1.420	-1.410	0.451	0.000	0.343	耐
A-91	-0.580	1.240	0.170	0.810	0.323	耐
A-924	-1.490	1.560	0.042	0.908	0.249	耐
A-688	-0.140	-0.510	0.232	0.275	0.242	耐
04219	-0.590	-0.720	0.169	0.211	0.179	敏
04841	-1.530	0.160	0.037	0.480	0.143	敏
A-1139	-1.260	-0.830	0.074	0.177	0.099	敏
05189	-1.790	-1.380	0.000	0.009	0.002	敏
权重Weight			0.761	0.239

图5 10份棉花种质资源黄萎病抗性聚类

Fig.5 Cluster diagram of Verticillium wilt resistance of 10 cotton germplasm resources

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陆地棉种质黄萎病抗性生理鉴定分析

Physiological identification and analysis of Verticillium wilt resistance of 10 foreign cotton germplasm resources

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