枣树抗枣疯病生理机制的研究
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摘要
枣树是我国重要的经济林树种之一,在我国大部分地区广泛种植。枣疯病是由植原体引起的枣树上一种毁灭性系统侵染病害,给我国的枣树生产造成巨大的经济损失。本研究采用嫁接病皮的方法接种病原,以嫁接病原后的不同枣树品种和单株为试材,观察其田间生长状况,嫁接情况并对体内的病原进行PCR检测鉴定;病理生理学方面,生长季节内对嫁接枣树不同品系抗病植株和感病植株枣吊内过氧化物酶(POD)、多酚氧化酶(PPO)、吲哚乙酸氧化酶(IAAO)、苯丙氨酸解氨酶(PAL)的活性及POD、IAAO同工酶进行了测定和比较;同时,对不同枣树品系叶片的酚类物质、绿原酸、蛋白质、氨基酸含量进行了比较和研究,旨在进一步从生理生化的角度探讨枣疯病问题,为枣疯病的研究提供一些基础数据。主要试验结果如下:
     1.枣树通过嫁接病皮可以传病,用聚合酶链反应(PCR)技术对枣树体内的病原进行检测,结果发现,在所有嫁接枣树植株内都能检测到植原体的存在,抗病婆枣单株、蛤蟆枣内病原浓度相对较低,婆枣感病株和酸枣病株病原浓度较高,胡瓶枣内病原浓度也比较高。病菌的存在与否和枣树的发病症状间没有直接关系,一些抗病性强的品种和单株尽管体内含有植原体,但不表现症状。
     2.用分光光度法对枣树枣吊内POD、PPO、IAAO、PAL酶活性进行测定,结果表明同一枣树品种不同单株间四种酶活性的变化趋势在生长季节中没有太大的差异,但是婆枣中感病单株的POD、PPO、IAAO酶活性比抗病单株高,PAL酶活性比抗病株低;胡瓶枣中感病株POD、IAAO酶活性比抗病株高,PPO、PAL酶活性比抗病株低。这表明不同枣树品种对植原体的侵染反应是有差异的。
     3.用不连续聚丙烯酰胺凝胶电泳(PAGE)法对枣树叶片的POD、IAAO同工酶进行了研究和比较,结果表明婆枣抗病感病单株叶片的POD和IAAO同工酶酶谱在8月份的测定中差别都比较大,在7月和9月份的测定中差别不大;胡瓶枣不同单株间两种同工酶谱在3次测定中差别都不明显。
     4.同一枣树品种不同单株叶片内酚类物质和绿原酸含量有一定差异。在7、8、9月3次测定中,婆枣抗病单株叶片总酚和绿原酸含量都比感病单株高。胡瓶枣中抗病株叶片的总酚和绿原酸含量也比感病株高。不同枣树品种叶片内总酚和绿原酸含量进行比较,结果表明抗病品系叶片内酚类物质和绿原酸含量也比感病品系高,因此酚类物质和绿原酸可以作为研究枣树抗病生理的一个重要指标。
     5.同一枣树品种不同单株叶片内蛋白质含量有一定差异。在7、8、9月3次测定中,婆枣感病单株叶片内蛋白质含量都比抗病株高;胡瓶枣感病病株叶片蛋白质含量也比抗病单株高。不同枣树品种叶片蛋白质含量比较表明,感病品种叶片蛋白质含量均比抗病品系高。而氨基酸含量的变化在不同品种和同一品种内的变化都没有明显的规律性。
Zizyphus jujube, as one of the important economic trees, is planted widely in china. Jujube witches' broom (JWB) caused by phytoplasma, is becoming the most destructive systematic disease of Chinese jujube and has brought much economic damage to our country. In this paper, the pathogen detection and growth condition in the field of different ziziphus jujube cultivars were studied after grafted infected barks. In addition, on the physio-pathological aspect, the POD,PPO,PAL,IAAO enzyme activity, POD and IAAO isozyme in different zizyphus jujube cultivars bearing branch and the content of phenolic compounds , protein, amino acids, chlorogenic acids in leaves were also analyzed respectively, the main result were as follows:
    1. Zizuphus jujube may be infected by phytoplasma through graft transmission using disease barks. After grafted infected barks, almost all the jujube cultivars and single tree have phytoplasma through PCR detection. However, in Pozao and Hamazao healthy single tree leaves, the content of phytoplasma is relative lower, in Pozao and Suanzao disease single tree, higher; while in Hupingzao cultivars, both healthy trees and disease trees have high phytoplasma content. It can be concluded that there is no direct relationship between phytoplasma content and sympotom, what's more, different jujube cultivars may have different reaction to phytoplasma.
    2. Using ultraviolet spectrophotometry determining the enzyme activity of POD,PPO,IAAO,PAL in jujube bearing branch, the result showed that in the same cultivar, there is no significant difference among all the single tree on the enzyme change tendency. But in Pozao cultivar, the POD,PPO,IAAO enzyme activity of susceptible single tree are a little higher than resistant ones, the PAL enzyme activity lower than resistant ones. While in Hupingzao cultivar, the POD,IAAO activity of susceptible tree are higher than resistant ones, the PPO,PAL activity lower than resistant ones. This also indicated that different jujube cultivars may have different reaction to phytoplasma.
    
    
    3. The POD and IAAO isozyme of different jujube cultivars bearing branch were determined by using incontinous polyaerylamicle electrophoretic (PAGE) method. It showed that in Pozao cultivar, the PODJAAO isozyme band have obvious difference between susceptible trees and resistant ones in August, while in Hupingzao cultivar, there is no significant difference between susceptible trees and resistant trees.
    4. The phenolic content and chlorogenic acid content in different jujube cultivars leaves were studied , the result suggested that in the same strain , the phenolic content and chlorogenic acid content of resistant single tree are usually higher than susceptible ones. Comparing different cultivars also indicated the content of phenolic compound and chlorogenic acid in resistant cultivars and single tree are shortly higher than susceptible ones. Therefore, these two indexes may be used to study JWB.
    5. The content of protein and amino acid in different cultivars leaves were also analyzed. It indicated that in the same strain, the protein and amino acid content of susceptible trees are a little higher than resistant ones. Furthermore, all diseased cultivars protein content are higher than resistant ones. But the amino content has no obvious regularity.
引文
[1]Doi Y, Teranaka K., Yora K,et al. Mycoplasma-or PLA-group-like micro-organisms found in the phloem element of plant infected with mulberry drawf, potato Witche's-broom[J]. Ann Phytopath Soc Japan, 1976,33:259~266.
    [2]田国忠,朱永芳,张成良.植原体分类和鉴定新动向[M].植物病理学研究,北京:中国农业出版社.1997,9~13.
    [3]刘仲健,罗换亮,张景宁.植原体病理学[M].北京:中国林业出版社,1999,26.
    [4]Sinclair W A, Griffiths H M. Ash yellow and its relationship to dieback and decline of ash[J]. Ann. Rev. Phytopathol, 1994,32:49~60.
    [5]田国忠,张锡津,熊耀国.泡桐筛管内胼胝质积累与抗丛枝病的关系[J].植物病理学报,1994,24(4):
    [6]田国忠.泡桐丛枝病植原体与泡桐的生化和分子互作研究[D]北京:中国农业大学,1999.
    [7]田国忠.植物菌原体的检测和鉴定研究新动态[J].植物检疫,1992,(增刊):70~73.
    [8]林木兰,杨继红,陈捷,等.泡桐丛枝病类菌原体单克隆抗体的研制及初步应用[J].植物学报,1993,35(9):710~715.
    [9]金开璇,田国忠,汪跃.组织化学技术快速检测泡桐丛枝病研究[J].植物病理学报,1989,19:185~188.
    [10]兰平,李文凤.甘薯丛枝病植原体的PCR检测[J].植物学通报,2001,18(2):210~215.
    [11]张春立,林木兰,胡勤学,等.泡桐丛枝病类菌原体DNA的分子克隆与序列分析[J].植物学报,1994,36(4):278~282.
    [12]林木兰,张春立,杨继红,等.用核酸杂交技术检测泡桐丛枝病类菌原体[J].科学通报,1994,39(4):376~380.
    [13]李江山,金开璇,汪跃,等.用PCR扩增16SrDNA检测泡桐组培苗丛枝病类菌原体[J],林业科学,1996,32(6):569~573.
    [14]邱并生,李横红,史春霖,等.从我国20种感病植物中扩增植原体16SrDNA片断及其RFLP分析[J].微生物学报,1998,34(6):67~74.
    [15]朱澄,徐丽云,金开璇,等.用DAPI荧光显微技术检测泡桐丛枝病[J].植物学报,1991,33(7):495~499.
    [16]Huiruki c, Dijkstra J, Light and electron microscopy of Vinca plant infected with mycoplasma lide bodies ofsandspike disease [J]. Neth J PI Path,1973,79:207~217.
    [17]Kirkpatrick B C, Stenger D C, Morris T J, et al. Cloning and detection of DNA from a nonculturable plant pathogenic mycoplasma-like organism[J]. Science, 1987,238:197~200.
    
    
    [18]Bak W C, La Y J. Purification of mulberry dwarf and jujube witches'broom mycoplasmas-like organisms and their serological relationship determined by enzyme-linked immunosorbent assay (ELISE)[J]. Korean J Plant Pathol,1992,8(2):149~153.
    [19]Davis R E, Lee I M, Douglas S M, et al. Molecular cloning and detection of chromosomal and extra chromosomal DNA of mycoplasma like organism associated with little leaf disease in Periwinkle(catharanthus roseus)[J]. Phytopathology,1990,80:233~237.
    [20]Harrison N A, Bourne C M, Cox R L, et al. DNA probes for detection of mycoplamas like organism associated with lethal yellowing disease of palms in Florida[J]. Phytopathology, 1992,82:216~224.
    [21]Lee I M, Hammond R W, Davis R E, et al. Universal amplification and analysis of Pathogen 16SrDNA for classification and identification of mycoplasma like organisms[J]. Phytopathology, 1993,83:834~842.
    [22]Ahrens U. Detection of DNA of plant pathologenic mycoplasma like organism by a polymerase chain reaction that amplifies a sequence of the 16SrRNA gene[J]. Phytopathology, 1992,82:828~832.
    [23]Kuske C R, Kirkpatrick B C, Seemuller E, et al. Differentiation of Viresence MLOs using western aster yellows mycoplasmas like organism chromosomal DNA probes and restriction fragment length polymophism analysis[J]. J.Gen.Microbiol, 1991,137:153~159.
    [24]蔡红,祖旭宁,陈海如,等.植原体分类研究进展[J].植物保护,2002,28(3):39~42.
    [25]赵锦.枣疯病病原分布消长规律及其病害生理研究[D].保定:河北农业大学,2003.
    [26]何放亭,武红巾,陈子文,等.C/A值与甘薯丛枝病症状发生的关系[J].植物病理学报,1997,27(1):43~46.
    [27]田国忠,黄钦才,袁巧平,等.感染MLO泡桐组培苗代谢变化和致病机理的关系[J].中国科学B辑,1994,24(5):484~490.
    [28]金红.枣疯病丛枝症状形成的生理病理学基础[D].北京:中国农业科学院植物保护研究所,1990.
    [29]田国忠,张锡津.泡桐丛枝病新研究进展[J].世界林业研究,1996,(2):32~38.
    [30]任国兰.泡桐丛枝病的研究现状与展望[J].河南农业大学学报,1996,30(4):358~364.
    [31]周俊义,刘孟军,侯宝林.枣疯病研究进展[J].果树科学,1998,15(4):354~359.
    [32]陈子文,陈永萱,陈泽安.枣疯病研究的进展[J].南京农业大学学报,1991,14(4):49~55.
    [33]徐绍华.枣疯病痫枝超薄切片中类菌原体的电镜观察[J].微生物学报,1980,20(2):219~220.
    [34]陈作义,沈菊英,龚祖,等.枣疯病病原的电子显微镜研究[J].科学通报,1978,(12):751.
    [35]王秀伶,刘孟军,刘丽娟.荧光显微技术在枣疯病病原鉴定中的作用[J].河北农业大学学报,1999,22(4):46~49.
    [36]韩国安,郭永红,陈永萱.用单克隆抗体检测枣疯病类菌原体[J].南京农业大学学
    
    报,1990,13(1):123.
    [37]陈子文,张凤舞,田旭东,等.枣疯病传病途径的研究[J].植物病理学报,1984,14(3):141~146.
    [38]La Y. J., Woo K. S. Transmission of jujube witches's-broom mycoplasma by the leafhopper(Hishimonus sellatus Uhler)[J]. J Korean For Soc,1980,48:29~39.
    [39]王焯.枣疯病几个有关问题[J].落叶果树,1995,(4):8~10.
    [40]莽克强,李德葆,王小凤,等.枣疯叶内游离氨基酸纸层析的研究[J].微生物学报,1974,14(2):224~229.
    [41]Kim Y H.Variation of phenolic substances in the leaves ofjujube tree(zizyphus jujuba Miller var. inermis Rehder) infected with witches'-broom diaease(1)[C].韩国全北大学校农大论文集,1973,4:24~29.
    [42]孙朝晖,温秀军,孙士学.枣疯病研究现状[J].河北林业科技,1998,(2):50~54.
    [43]温秀军,孙朝晖,孙士学,等.抗枣疯病枣树的品种及品系的选择[J].林业科学,2001,17(5):87~92.
    [44]任国兰,郑铁民,陈功友,等.枣疯病发病因子和防治技术研究[J].河南农业大学学报,1993,27(1):67~70.
    [45]尔吉辉.枣疯病的发生及综合防治技术[J].河北林果研究,2000,15(1):61~64.
    [46]焦荣斌,李亚.太行山区枣疯病发生规律及防治对策[J].中国果树,2001,(6):40~41.
    [47]田国忠.枣疯病的预防和治疗策略研究[J].林业科技通讯,1998(2):14~17.
    [48]潘青华.枣疯病研究进展及防治措施[J]。北京农业科学,2002,(3):4~8.
    [49]王焯,刘秀芳,周佩珍,等.枣疯病综合防治技术研究[J].中国果树,1988,(2):40~41.
    [50]江顺庆,吕明,江向勇.枣疯病综合防治技术[J].江西园艺,2002,(2):24~25.
    [51]侯保林,齐秋锁,赵善香,等.手术治疗枣疯病的初步探索[J].河北农业大学学报,1987,10(4):11~17.
    [52]王焯,于保文,德全,等.四环素族等药物对枣疯病的初步治疗试验[J].中国农业科学,1980,(4):65-69.
    [53]王焯,周佩珍,于保文,等.枣疯病媒介昆虫-中华拟菱纹叶蝉生物学防治的研究[J].植物保护学报,1984,11(4):247~252.
    [54]田旭东,张凤舞,孙淑梅.凹缘菱纹叶蝉的越冬习性与传播枣疯病的关系[J].植物病理学报,1988,18(2):103~106.
    [55]田砚亭,王红艳,牛晨,等.枣树脱除类菌原体技术的研究[J].北京林业大学学报,1993,15(2):16~26.
    [56]戴洪义,沈德绪,林佰年.枣疯病热处理脱毒的初步研究[J].落叶果树,1988,(10):1~2.
    [57]朱文勇,杜学海,郭黄萍,等.骏枣茎尖培养脱除枣疯病MLO[J].园艺学报,1996,23(2):197~198.
    [58]李和生,孙群,赵世杰,等.植物生理生化实验原理和技术[M].北京:高等教育出版社,2002.
    
    
    [59]胡能书.同工酶技术及应用[M].长沙:湖南科学技术出版社,1985.
    [60]汪跃,金开璇,张锐,等.山楂丛枝病过氧化物同工酶的研究[J].林业科学研究,1994,7(2):203~205.
    [61]荣向东,王静,张景宁.木麻黄丛枝病病健株过氧化物酶同工酶的初步研究[J].广东林业科学通讯,1984,(5):1~4.
    [62]鞠志国,朱广廉,曹宗馔.莱阳茌梨果实褐变与多酚氧化酶及酚类物质区域化分布的关系[J].植物生理学报,1988,14(4):356~361.
    [63]杨家书,吴畏,吴友三,等.植物苯丙酸类代谢与小麦对白粉病抗性关系[J].植物病理学报,1986,16(3):169~173.
    [64]田国忠,李怀方,裘维蕃.植物过氧化物酶研究进展[J].武汉植物学研究,2001,19(4):332~344.
    [65]Lagrimin L M, Rothstein S. Tissue specificity of tobacco peroxidase isozymes and their induction by wounding and tobacco mosaic virus infection [J].Plant Physiol,1987,84:438~442.
    [66]Quiroga M,Guerrero C,Botella M A,et al.A tomato peroxidase involved in the synthesis of lignin and suberin[J] .Plant Physiol,2000,122:1119~1129.
    [67]Goy P A,Feria G,Metraux J P, et al. Resistance to disease in hybrid Nicotiana glutinosa ×Nicotiana debneyi is associated with high constitutive levels of β-1,3-glucanase,chitinase,peroxidase and polyphenoloxidase[J].Physiol Mol Plant Pathol, 1992,41: 11~21.
    [68]Reimers P J,Guo A,Leadi J E. Incresed activity of a cationic peroxidase associated with an incompatible interaction between Xanthomonas oryzae pv.oryzac, and rice (oryza sativa)[J].Ptant Physiol,1992,99: 1044~1050.
    [69]任国兰,刘延忠,时向阳,等.泡桐丛枝病诱导抗性机制的的研究[J].河南农业大学学报,1995,29(4):378~381.
    [70]蒋建平,朱建军,刘延志,等.泡桐丛枝病与过氧化物酶含量关系的研究初报[J].河南农业大学学报,1993,27(4):301~304.
    [71]秦国夫,祁和艾,赵明光,等.泡桐丛枝病带毒苗过氧化物酶同工酶分析[J].森林病虫通讯,1993,(2):13~14.
    [72]张会梅,胡喜米,方立红,等.枣树过氧化物酶同工酶研究初探[J].河南农业科学,1997,(7):38~40.
    [73]王生荣.不同黄瓜品种感染黑星病菌后一些酶含量的变化[J].山东农业大学学报,1999,30(增刊):179~182.
    [74]叶建仁,黄素红,李传道,等.抗松针褐斑病湿地松体内氧化酶的变化[J].南京林业大学学薄,1994,19(1):8~14.
    [75]古月景江,文建雷,景耀,等.过氧化物酶和多酚氧化酶与杨树溃疡病抗性的关系[J].西北林学院学报,1990,5(1):46~51。
    
    
    [76]Miyazawa J,Kawabata T, Ogasawara A, Induction of an acidic isozyme of peroxidase and acquired resistance to wilt disease in response to treatment of tomato roots with 2-furoic acid ;4-hydroxybenoic hydrazide,or salicylic hydrazide [J].Physiol Mol Plant Pathol,1998,52:115~126.
    [77]Calderon A A ,Zapata J M,Redreno M A,et al. Levels of 4-hdroxystilbene-oxidizing isoperoxidase related to constitutive disease resistance in in vitro-cultured grapevine[J].Plant Cell Tiss Organ Culture, 1992,239:63~67.
    [78]Martinez C,Montillet S L,Bresson F, et al. Apoplastic peroxidase generates superoxide anions in cells of cotton cotyledons undergoing the hypersensitive reaction to Xanthomonas campestris pv. Malvacearum race 18.Mol Plant-Microbe Interact[J]. 1998, 11:1038~1047.
    [79]宋淑梅,宋东辉,史建伟,等.同工酶与枣疯病病树病理变化的关系[J].林业科学研究,2000,13(专):106~113.
    [80]蒋建平,刘延忠,王政堂.泡桐丛技病过氧化物酶季节性变化规律的研究[J].河南农业大学学报,1996,30(2):123~126.
    [81]郑翠明,高凤兰.感染SMV后大豆种皮超氧物歧化酶过氧化物和多酚氧化酶的变化[J].中国农业科学,1999,32(1):99~101.
    [82]Gentile L A, Ferrais L,Matta. Variation of phenoloxidase activities as a consequence of stress that indude resistance to Fusariumwilt of tomato[J]. J Phytopathol, 1988,122:45~51.
    [83]薛俊杰,王永琳,张淑改,等.泡桐丛枝病过氧化物酶和多酚氧化酶研究[J].山西农业科学,2000,28(1):62~64.
    [84]胡勤学,周咏芝,马修理,等.泡桐丛枝病感病指示植物的病理变化[J].中南林学院学报,1992,12(1):57~62.
    [85]周桂元,梁炫强.花生种子苯丙氨酸解氨酶活性与抗黄曲霉侵染的关系[J].花生学报,2002,31(1):14~17.
    [86]Bolwell G P, Bell J N,Cramer C L,et al..L-Phenylalanine ammonia-lyase from Phaseolus vulgaris characterization and differetial induction of multiple from elicitor-treated cell suspension cultures[J].Eur J biochem, 1985,149:411.
    [87]胡景江,文建雷,景耀,等.杨树体内苯丙烷代谢与其对溃疡病抗性的关系[J].植物病理学报,1992,22(2):185-188.
    [88]阳传和,杨旺,周仲铭.树皮内酚类物质的含量及苯丙氨酸解氨酶的活性与杨树抗溃疡病的关系[J].林业科学,1989,25(4):311~316.
    [89]樊自红,沈瑞祥,周仲铭,等.过氧化物酶和苯丙氨酸解氨酶与毛白杨抗锈性的关系[J].植物病理学报,1989,19(2):95~100.
    [90]刘亚光,李海英,杨庆凯.大豆品种的抗病性与叶片内苯丙氨酸解氨酶活性关系的研究[J].东北农
    
    业大学学报,1999,13(3):43~48.
    [91]叶建仁,吴小芹.树木抗病的生理生化学研究进展[J].林业科学研究,1996,9(3):311~317.
    [92]翟晓巧,王国周,毕会涛.泡桐丛枝病发生叶片酚和氨基酸变化关系研究[].河南科学,2000,18(3):276~278.
    [93]何学友,黄衍庆.木麻黄酚类、单宁及基本营养化学物质与抗病性的关系[J].林业科学研究,2000,13(专):125-129.
    [94]Pirie A,Mullins MG.Changes in amthocyanin and phenolic content of grapevine leaf and abscisic acid [J].Plant Physiol,1976,58:468~472.
    [95]Ralph L,Nicholson. Phenlic compounds and their role in disease resistance[J].Ann.Rev.Phytopathol., 1992,30:369~389.
    [96]Cole ,R.A,Relationship between the concentration of chlorogenic acid in carrotroots and the incidence of carrot fly larval damage Ann[J].Applied Biology, 1985,106(2):211~217.07.
    [97]郑建伟,毕会涛,寇首道.泡桐丛枝病发生过程中物质变化研究现状[J].河南科学,2000,18(4):405~407.
    [98]La Y J,Park W C,Park W M, et al. Electrophoresis of proteins in tissue from witches'broom infected jujube tree[J]. Korean For Soc,1984,66:79~81.
    [99]Saho H.On the amino acid of Paulownia tomentosa (Thumb) Staud affected by witches'broom Part Ⅰ The amino acid of leaves and twigs[J].Jap.For.Soc,1957,39(1):447~451.
    [100]景耀,朱玮.杨树树皮中化学成分与溃疡病关系的初步研究Ⅰ.常量营养元素与溃疡病的关系[J].林业科学,1989,25(4):304~310.
    [101]Entry J A, Martin N E, Kelsey R, et al. Chemical constituents in root bark offive species of western conifer saplings and infection by Armillaria ostoyae[J]. Phytopathology, 1972,82(4):393~397.
    [102]范国强,蒋建平.泡桐丛枝病发生与叶片蛋白质和氨基酸变化关系的研究[J].林业科学研究,1997,10(6):570~573.