黄瓜抗白粉病突变体筛选与鉴定

doi:10.3864/j.issn.0578-1752.2020.01.016

摘要/Abstract

摘要：

【目的】白粉病是危害黄瓜产量、品质的最严重的病害之一,抗白粉病材料的发掘与研究,可以实现从根本上解决病害问题。对获得的‘长春密刺’突变体材料进行分析,旨在筛选出黄瓜抗白粉病突变体材料,丰富育种群体。【方法】对400份‘长春密刺’突变体材料进行苗期接种白粉病菌试验,通过叶片病斑观察结合病情指数分析,初步筛选出抗病材料,将筛选出的抗病材料在大田环境下自然发病进一步观察表型。对初步筛选出的抗病材料进行苗期生理鉴定,测定并分析超氧化物歧化酶、过氧化物酶、过氧化氢酶活性以及叶绿素a、叶绿素b、类胡萝卜素、可溶性蛋白含量等生理指标,通过田间自然发病进一步筛选抗病材料,并测定其蒸腾速率、胞间二氧化碳浓度、气孔导度、净光合速率等光合作用指标及乙烯、茉莉酸、水杨酸激素含量,并通过荧光定量PCR分析叶片中乙烯、茉莉酸、水杨酸、木质素、病程相关蛋白等防御信号途径中相关基因的表达。【结果】与感病材料相比,抗病材料的表面菌斑少,净光合速率、气孔导度都优于对照‘长春密刺’,胞间CO₂浓度低于‘长春密刺’。在防御激素方面,抗病材料的乙烯、茉莉酸、水杨酸含量同样优于感病材料,而在成熟期叶片的防御信号途径相关基因表达上,抗病材料的表达高于感病材料。通过苗期接种白粉病菌和田间自然发病综合筛选出了两份抗白粉病突变体材料：Mu-86-2、Mu-58-9。【结论】通过对突变体库的筛选可以获得抗白粉病新材料,这些材料的获得对黄瓜抗白粉病的遗传研究和新品种培育具有重要价值。

关键词: 黄瓜, 突变体, 抗白粉病, 筛选鉴定

Abstract:

【Objective】Powdery mildew is one of the most serious diseases that effects cucumber yield and quality. The discovery and research of materials resistant to powdery mildew can fundamentally solve the disease problem. To screen out materials of cucumber resistant to powdery mildew and enrich the breeding population, the Changchunmici mutants were investigated and analyzed in the study.【Method】Totally, 400 Changchunmici mutant lines were inoculated with powdery mildew at seedling stage, and resistant materials were screened preliminarily based on leaf lesion observation combined with disease index analysis. The selected resistant materials were further observed in the natural environment in the field environment. Physiological indexes of resistant materials in the seedling stages were analyzed, including the activities of superoxide dismutase, peroxidase and catalase activity, and physiological indicators such as the contents of chlorophyll a, chlorophyll b, carotenoids and soluble protein. The resistant materials were screened by natural pathogenesis in the field and were further analyzed for the photosynthetic indexes such as transpiration rate, intercellular carbon dioxide concentration, stomatal conductance and net photosynthetic rate. And the contents of ethylene, jasmonic acid and salicylate were determined, and expression of related genes in defense signaling pathways such as ethylene, jasmonic acid, salicylic acid, lignin, and disease-related proteins in leaves were analyzed by real-time PCR. 【Result】Compared with susceptible materials, the resistant materials had less plaque. The net photosynthetic rate and stomatal conductance of resistant materials were higher than those of wild-type Changchunmici, and the intercellular CO₂ concentration of them was lower than that of Changchunmici. In terms of defensive hormones, the contents of ethylene, jasmonic acid and salicylate of resistant materials were higher than those in control. The expression of defense signal related genes in mature leaves of resistant materials was higher compared with susceptible materials. Two powdery mildew resistance materials, Mu-86-2 and Mu-58-9 were screened by inoculation of powdery mildew in seedling stage and natural disease. 【Conclusion】The new materials resistant to powdery mildew could be obtained by screening mutant libraries. The acquisition of these materials had the important value for the genetic research and new varieties breeding of cucumber resistance to powdery mildew.

Key words: cucumber, mutant, resistance to powdery mildew, screening and identification

亓飞,林姝,宋蒙飞,张孟茹,陈姝延,张乃心,陈劲枫,娄群峰. 黄瓜抗白粉病突变体筛选与鉴定[J]. 中国农业科学, 2020, 53(1): 172-182.

Fei QI,Shu LIN,MengFei SONG,MengRu ZHANG,ShuYan CHEN,NaiXin ZHANG,JinFeng CHEN,QunFeng LOU. Screening and Identification of Cucumber Mutant Resistant to Powdery Mildew[J]. Scientia Agricultura Sinica, 2020, 53(1): 172-182.

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参考文献 36

[1]	BERG J A, APPIANO M, BIJSTERBOSCH G, VISSER R G F, SCHOUTEN H J, BAI Y L . Functional characterization of cucumber (Cucumis sativus L.) clade vmlogenes. BMC Plant Biology, 2017,17:80.
[2]	冯东昕, 李宝栋 . 主要瓜类作物抗白粉病育种研究进展. 中国蔬菜, 1996(1):55-59.
	FENG D Y, LI B D . Research progress on breeding of main melon crops against powdery mildew. Chinese Vegetables, 1996(1):55-59. (in Chinese)
[3]	曲丽, 秦智伟 . 黄瓜白粉病病原菌及抗病性研究进展. 东北农业大学学报, 2007,38(6):835-841.
	QU L, QIN Z W . Advance on cucumber resistance to powdery mildew. Journal of Northeast Agricultural University, 2007,38(6):835-841. (in Chinese)
[4]	刘秀波, 崔琦, 崔崇士 . 瓜类白粉病抗性育种研究进展. 东北农业大学学报, 2005,36(6):794-798.
	LIU X B, CUI Q, CUI C S . Advance on the resistance to powdery mildew in cucurbit. Journal of Northeast Agricultural University, 2005,36(6):794-798. (in Chinese)
[5]	NIE J T, WANG Y L, HE H L, GUO C L, ZHU W Y, PAN J, LI D D, LIAN H L, PAN J S, CAI R . Loss-of-function mutations incsmlo1confer durable powdery mildew resistance in cucumber (Cucumis sativus L.). Frontiers in Plant Science, 2015,6:612.
[6]	THOMAS C E . Influence of dew on downy mildew of cantaloups in South Texas. Phytopathology, 1977,77(11):1368-1369.
[7]	JAHN M, MUNGER H M, MCCREIGHT J D. Breeding cucurbit crops for powdery mildew resistance//The Powdery Mildews: A Comprehensive Treatise. American Phytopathological Society (APS Press), 2002: 239-248.
[8]	周凤珍, 王永健 . 黄瓜白粉病抗病鉴定方法的研究. 蔬菜, 1987(1):10-11.
	ZHOU F Z, WANG Y J . Study on identification method of resistance to cucumber powdery mildew. Vegetables, 1987(1):10-11. (in Chinese)
[9]	阚光锋, 张广民, 房保海, 刘萍 . 烟草野火病菌(Pseudomonas syringae pv. tabaci)对烟草细胞内5种防御酶系统的影响. 山东农业大学学报(自然科学版), 2002,33(1):28-31.
	ZHAI G F, ZHANG G M, FANG B H, LIU P . The effects of Pseudomonas syringae pv. tabaci on the defendant enzymes in tobacco cells. Journal of Shandong Agricultural University (Natural Science Edition), 2002,33(1):28-31. (in Chinese)
[10]	邱金龙, 金巧玲, 王钧 . 活性氧与植物抗病反应. 植物生理学通讯, 1998,34(1):56-63.
	QIU J L, JIN Q L, WANG J . Active oxygen and plant defense responses. Plant Physiology Communications, 1998,34(1):56-63. (in Chinese)
[11]	陈少裕 . 膜脂过氧化对植物细胞的伤害. 植物生理学通讯, 1991,27(2):84-90.
	CHEN S Y . Injury of membrane lipid peroxidation to plant cell. Plant Physiology Communications, 1991,27(2):84-90. (in Chinese)
[12]	梁芳芳 . 霜霉病胁迫对黄瓜幼苗叶片光合作用的影响[D]. 郑州: 河南农业大学, 2010.
	LIANG F F . Effects of downy mildew stress on photosynthesis in cucumber seedling leaves[D]. Zhengzhou: Henan Agricultural University, 2010. (in Chinese)
[13]	NEJAT N, ROOKES J, MANTRI N L, CAHILL D M . Plant-pathogen interactions: toward development of next-generation disease-resistant plants. Critical Reviews in Biotechnology, 2017,37(2):229-237.
[14]	THOMMA B P H J, EGGERMONT K, PENNINCKX I A M A, MAUCH-MANI B, VOGELSANG R, CAMMUE B P A, BROEKAERT W F . Separate jasmonate-dependent and salicylate- dependent defense-response pathways in arabidopsis are essential for resistance to distinct microbial pathogens. Proceedings of the National Academy of Sciences of the United States of America, 1998,95:15107-15111.
[15]	MCGRATH M T . Fungicide resistance in cucurbit powdery mildew: Experiences and challenges. Plant Disease, 2001,85:236-245.
[16]	SAKATA Y, KUBO N, MORISHITA M, KITADANI E, SUGIYAMA M, HIRAI M . QTL analysis of powdery mildew resistance in cucumber (Cucumis sativus L.). Theoretical and Applied Genetics, 2006,112:986.
[17]	ZHANG K J, WANG X, ZHU W W, QIN X D, XU J, CHENG C Y, KOU Q F LI J, CHEN J X, . Complete resistance to powdery mildew and partial resistance to downy mildew in a Cucumis hystrix introgression line of cucumber were controlled by a co-localized locus. Theoretical & Applied Genetics, 2018,131(10):1-15.
[18]	康振生 . 植物病原真菌超微结构. 北京:中国科学技术出版社, 1996.
	KANG Z S. Ultrastructure of Phytopathogenic Fungi. Beijing: China Science and Technology Press, 1996. (in Chinese)
[19]	李宗霆, 周燮 . 植物激素及其免疫检测技术. 南京: 江苏科学技术出版社, 1996.
	LI Z T, ZHOU X. Plant Hormones and Their Immunoassay Technology. Nanjing: Jiangsu Science and Technology Press, 1996. (in Chinese)
[20]	马华 . 黄瓜—酸黄瓜渐渗系抗蔓枯病鉴定及相关抗性机制研究[D]. 南京: 南京农业大学, 2015.
	MA H . Identification of cucumber-sour cucumber introgression resistance and related resistance mechanism[D]. Nanjing: Nanjing Agricultural University, 2015. (in Chinese)
[21]	周梦韩, 汪静, 孟新刚, 王俊刚 . 感染白粉病菌初期黄瓜叶片生理指标的变化. 北方园艺, 2013(3):133-135.
	ZHOU M H, WANG J, MENG X G, WANG J G . Physiological indexes changes of cucumber after infected bySphaerotheca fuliginea at the beginning period. Northern Horticulture, 2013(3):133-135. (in Chinese).
[22]	沈喜, 李红玉, 文雅, 孟雪琴, 周功克, 张立新, 梁厚果 . 白粉病菌侵染对黄瓜叶片光合电子传递及其psⅡ功能蛋白d1表达的影响. 植物病理学报, 2003,33(6):546-549.
	SHEN X, LI H Y, WEN Y, MENG X Q, ZHOU G K, ZHANG L X, LIANG H G . Influence of cucumber powdery mildew (Sphaerotheca fuliginea) infection on photosynthetic electron transport and protein d1 expression of cucumber(Cucumis sativus) leaf. Chinese Journal of Plant Pathology, 2003,33(6):546-549. (in Chinese)
[23]	张子山, 张立涛, 高辉远, 贾裕娇, 部建雯, 孟庆伟 . 不同光强与低温交叉胁迫下黄瓜psⅠ与psⅡ的光抑制研究. 中国农业科学, 2009,42(12):4288-4293.
	ZHANG Z S, ZHANG L T, GAO H Y, JIA Y J, BU J W, MENG Q W . Research of photoinhibition of psI and psII in leaves of cucumber under chilling stress combined with different light intensities. Scientia Agricultura Sinica, 2009,42(12):4288-4293. (in Chinese).
[24]	HAO Y H, LIN C M, FAN H Y, YU Y, LI N, CHEN S L . Proteomic analysis of Cucumis sativus cotyledons after glucohexaose treatment as a part of ros accumulation related resistance mechanism. Proteome Science, 2014,12:34-43.
[25]	张梅, 刘瑶, 丛慧芳, 孙治军, 王红艳, 王洪磊 . 草莓抗白粉病生理生化指标研究. 中国农学通报, 2011,27(28):249-253.
	ZHANG M, LIU Y, CONG H F, SUN Z J, WANG H Y, WANG H L . Study on index of physiological and biochemical in resistance to strawberry powdery mildew. Chinese Agricultural Science Bulletin, 2011,27(28):249-253. (in Chinese)
[26]	VANACKER H, HARBINSON J, RUISCH J, CARVER T L W, FOYER C H . Antioxidant defences of the apoplast. Protoplasma, 1998,205:129-140.
[27]	GILL S S, TUTEJA N . Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry, 2010,48:909-930.
[28]	BLOKHINA O, VIROLAINEN E, FAGERSTEDT K V . Antioxidants, oxidative damage and oxygen deprivation stress: A review. Annals of Botany (London), 2003,91:179-194.
[29]	GLAZEBROOK J . Genes controlling expression of defense responses in Arabidopsis-2001 status. Current Opinion in Plant Biology, 2001,4:301-308.
[30]	COOPER W, BOUZAYEN M, HAMILTON A, BARRY C, ROSSALL S, GRIERSON D . Use of transgenic plants to study the role of ethylene and polygalacturonase during infection of tomato fruit by Colletotrichum gloeosporioides. Plant Pathology, 2010,47(3):308-316.
[31]	MAYERS C N, LEE K C, MOORE C A, WONG S M, CARR J P . Salicylic acid-induced resistance to cucumber mosaic virus in squash and Arabidopsis thaliana: Contrasting mechanisms of induction and antiviral action. Molecular Plant-Microbe Interactions, 2007,18:428-434.
[32]	石延霞, 于洋, 傅俊范, 李宝聚 . 病原菌诱导后黄瓜叶片中脂氧合酶活性与茉莉酸积累的关系. 植物保护学报, 2008,35(6):486-490.
	SHI Y X, YU Y, FU J F, LI B J . Relationship between LOX activity and JA accumulation in cucumber leaves induced by pathogen. Acta Phytophylacica Sinica, 2008,35(6):486-490. (in Chinese)
[33]	LIEBERMAN M . Biosynthesis and action of ethylene. Annual Review of Plant Physiology, 1985,30:533-591.
[34]	武健东, 洪玲, 江海洋, 朱苏文 . 玉米抗病相关基因NPR1的同源克隆及表达分析. 安徽农业大学学报, 2015,42(5):651-656.
	WU J D, HONG L, JIANG H Y, ZHU S W . Cloning and expression analysis of the disease-related gene NPR1 in maize. Journal of Anhui Agricultural University, 2015,42(5):651-656. (in Chinese)
[35]	鲁秀梅, 张宁, 夏美玲, 钱春桃, 陈劲枫 . 不同抗性甜瓜接种蔓枯病菌后内源激素含量变化及其相关基因的表达分析. 南京农业大学学报, 2018,41(2):248-255.
	LU X M, ZHANG N, XIA M L, QIAN C T, CHEN J F . Changes of endogenous hormone contents and expression analysis of related genes in melon with different resistance after inoculated with Didymella bryoniae. Journal of Nanjing Agricultural University, 2018,41(2):248-255. (in Chinese)
[36]	高庆华, 曾祥然, 贾霖, 牛东东, 李雪姣, 关明俐, 贾盟, 兰金苹, 窦世娟, 李莉云, 刘丽娟, 刘国振 . 水稻病程相关蛋白质在逆境胁迫下的表达研究. 生物化学与生物物理进展, 2013,40(11):1140-1147.
	GAO Q H, ZENG X R, JIA L, NIU D D, LI X J, GUAN M X, JIA M, LAN J P, DOU S J, LI L Y, LIU L J, LIU G Z . The expression profiling of rice pathogenesis-related proteins in seedling stage under environmental stresses. Progress in Biochemistry and Biophysics, 2013,40(11):1140-1147. (in Chinese)

编号Code	病情指数Disease index	抗性类型Disease resistance	编号Code	病情指数Disease index	抗性类型Disease resistance
Mu-86-2	2.22	抗R	Mu-104-2	20.20	中抗MR
Mu-58-9	3.33	抗R	Mu-106-3	22.22	中抗MR
Mu-50-10	4.18	抗R	Mu-59-8	22.22	中抗MR
Mu-9-9	9.00	中抗MR	Mu-150-3	23.23	中抗MR
Mu-60-5	10.00	中抗MR	Mu-118-2	24.24	中抗MR
Mu-84-8	15.56	中抗MR

编号 Code	叶绿素a含量 Chlorophyll a content	叶绿素b含量 Chlorophyll b content	类胡萝卜素 Carotenoids content
CK	13.556±1.089abcd	4.713±1.081b	2.420±0.987b
Mu-86-2	14.869±0.521abc	5.536±0.179ab	2.946±0.195ab
Mu-58-9	16.723± 0.536a	6.298±0.504ab	2.887±0.484ab
Mu-50-10	11.825±3.908d	4.566±0.756ab	2.501±0.942b
Mu-9-9	11.597±2.778bcd	4.291±0.886b	2.526±0.450b
Mu-60-5	14.317±2.577abc	5.342±0.709ab	2.853±0.356ab
Mu-84-8	14.921±2.864abc	5.635±1.691ab	2.950±0.296ab
Mu-104-2	11.845±1.490cd	4.557±0.526b	2.609±0.475b
Mu-106-3	14.333±2.758abc	5.599±0.923ab	2.993±0.292ab
Mu-59-8	15.372±3.327abc	5.930±1.075ab	3.042±0.938a
Mu-150-3	14.749±0.876abc	5.594±0.886ab	2.858±0.466ab
Mu-118-2	15.839±1.838ab	6.100±0.822a	2.783±0.185ab

编号Code	POD活性POD activity (U·g^-1·min^-1)	SOD活性SOD activity (U·g^-1)	CAT活性CAT activity (U·g^-1·min^-1)
CK	0.876±0.011bc	0.331±0.041bcde	13.104±0.381c
Mu-86-2	2.143±0.651a	0.450±0.108b	16.906±0.565ab
Mu-58-9	2.186±0.311a	0.415±0.028bc	18.800±2.630a
Mu-50-10	1.836±0.526b	0.588±0.024a	14.960±0.876bc
Mu-9-9	1.588±0.503b	0.309±0.003cde	13.120±0.155c
Mu-60-5	1.208±0.520b	0.358±0.085bcde	14.040±0.501bc
Mu-84-8	1.245±0.497b	0.302±0.115bcde	18.506±0.966a
Mu-104-2	0.832±0.119bc	0.327±0.049bcde	11.920±0.969d
Mu-106-3	0.589±0.237c	0.441±0.091bc	15.093±1.927ab
Mu-59-8	2.272±0.404a	0.247±0.046e	13.040±0.855c
Mu-150-3	0.977±0.228bc	0.268±0.087de	14.640±1.222bc
Mu-118-2	0.964±0.152bc	0.380±0.031bcd	14.340±0.650bc

编号 Code	净光合速率 Net photosynthetic rate (μmol·m^-2·s^-1)	气孔导度 Stomatal conductance (mmol·m^-2·s^-1)	胞间CO₂浓度 Intercellular CO₂ concentration (μmol·mol^-1)	蒸腾速率 Transpiration rate (mmol·m^-2·s^-1)
CK	16.500±1.252b	0.569±0.042b	302.666±9.865a	10.866±0.351a
Mu-58-9	22.866±1.625a	1.173±0.188a	286.000±1.000b	10.666±0.650a
Mu-86-2	22.866±2.281a	0.824±0.155b	278.000±10.000b	10.123±0.849a
IL52	23.600±0.435a	0.789±0.074 b	295.666±3.780b	11.133±0.416a

编号 Code	茉莉酸含量 Jasmonic acid content (pmol·L^-1)	水杨酸含量 Salicylic acid content (pmol·L^-1)	乙烯含量 Ethylene content (ng·L^-1)
CK	1346.912±25.758b	960.066±23.283d	159.509±3.868d
Mu-58-9	1696.515±31.178a	1114.259±19.026b	189.744±5.398b
Mu-86-2	1752.818±51.115a	1063.894±29.433a	194.694±6.823b
IL52	1692.587±47.625a	1009.656±10.652c	222.705±5.158a