番木瓜感染畸形花叶病毒后的生理变化及扫描电镜观察
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摘要
【目的】阐明番木瓜畸形花叶病毒(Papaya leaf-distortion mosaic virus,PLDMV)的侵染机理及侵染后番木瓜自身防御体系对抗病毒的作用机制,为番木瓜病毒病防治提供参考。【方法】以台农二号番木瓜为材料,通过RTPCR检测接种PLDMV处理组(T)与健康空白对照组(CK)的病毒感染情况,采用分光光度法测定PLDMV侵染对番木瓜相关生理指标的影响,运用扫描电镜法观察两组番木瓜叶片的微观结构差异。【结果】通过RT-PCR检测,证明T番木瓜植株已感染PLDMV。T与CK番木瓜叶片在表征、微观解剖结构及生理指标等方面均存在明显差异,与CK番木瓜叶片相比,T番木瓜叶片纵切面栅海比及背部绒毛密度明显小于CK番木瓜,气孔密度大于CK番木瓜;T番木瓜的过氧化物酶(POD)、多酚氧化酶(PPO)、苯丙氨酸解氨酶(PAL)活性和脯氨酸(Pro)、丙二醛(MDA)、超氧阴离子(O)含量均显著高于CK番木瓜(P<0.05,下同),过氧化氢酶(CAT)活性和叶绿素(Chl)含量显著低于CK番木瓜,而超氧化物歧化酶(SOD)和几丁质酶活性两组间无显著差异(P>0.05)。【结论】番木瓜受PLDMV侵染后,其叶片结构被破坏,细胞毒害损伤严重,体内代谢紊乱,多种代谢调节平衡被打破,番木瓜的正常生长发育受阻。
【Objective】The infection mechanism of Papaya leaf-distortion mosaic virus(PLDMV)and the mechanism of Carica papaya L. defense system against virus after infection were elucidated to provide reference for papaya virus disease.【Method】C. papaya variety Tainong 2 was used as material. The infection situations of C. papaya in treatment group with PLDMV inoculation(T)and healthy blank control group(CK)were confirmed by RT-PCR detection. The influence of PLDMV infection on C. papaya related physiological indexes was determined by spectrophotometry. Scanning electron microscopy was used to observe the difference in microstructure between the two groups.【Result】RT-PCR test showed that T group was infected with PLDMV. There were obvious differences in the characterization,microstructure and physiological indexes between leaves in T and CK. Palisade tissue to cavernous tissue ratio of vertical section leaves and density of back tomentum in T were smaller than those in group CK,and the stomatal density in group T was larger than that in group CK. In physiological indexes,the activities of peroxidase(POD),polyphenol oxidase(PPO)and phenylalanine ammonia lyase(PAL),and contents of proline(Pro),malondialdehyde(MDA)and superoxide anion(O)in T group were significantly higher than those in CK group(P<0.05,the same below). Catalase(CAT)activity and chlorophyll(Chl)content in T group was significantly lower than those in CK group. Superoxide dismutase(SOD)and chitinase were not significantly different from each other(P>0.05).【Conclusion】After PLDMV infection,C. papaya leaf structure is damaged,the cells are seriously poisoned,metabolic disturbance occurs,multiple metabolic regulation are imbalanced,therefore,the development and growth of papaya is blocked.
【Objective】The infection mechanism of Papaya leaf-distortion mosaic virus(PLDMV)and the mechanism of Carica papaya L. defense system against virus after infection were elucidated to provide reference for papaya virus disease.【Method】C. papaya variety Tainong 2 was used as material. The infection situations of C. papaya in treatment group with PLDMV inoculation(T)and healthy blank control group(CK)were confirmed by RT-PCR detection. The influence of PLDMV infection on C. papaya related physiological indexes was determined by spectrophotometry. Scanning electron microscopy was used to observe the difference in microstructure between the two groups.【Result】RT-PCR test showed that T group was infected with PLDMV. There were obvious differences in the characterization,microstructure and physiological indexes between leaves in T and CK. Palisade tissue to cavernous tissue ratio of vertical section leaves and density of back tomentum in T were smaller than those in group CK,and the stomatal density in group T was larger than that in group CK. In physiological indexes,the activities of peroxidase(POD),polyphenol oxidase(PPO)and phenylalanine ammonia lyase(PAL),and contents of proline(Pro),malondialdehyde(MDA)and superoxide anion(O)in T group were significantly higher than those in CK group(P<0.05,the same below). Catalase(CAT)activity and chlorophyll(Chl)content in T group was significantly lower than those in CK group. Superoxide dismutase(SOD)and chitinase were not significantly different from each other(P>0.05).【Conclusion】After PLDMV infection,C. papaya leaf structure is damaged,the cells are seriously poisoned,metabolic disturbance occurs,multiple metabolic regulation are imbalanced,therefore,the development and growth of papaya is blocked.
引文
蔡庆生.2013.植物生理学实验[M].北京:中国农业大学出版社.[Cai Q S.2013.Experiment of Plant Physiology[M].Beijing:China Agricultural University Press.]
陈建勋,王晓峰.2015.植物生理学实验指导[M].广州:华南理工大学出版社.[Chen J X,Wang X F.2015.Experimental Guidance for Plant Physiology[M].Guangzhou:South China University of Technology Press.]
陈银萍,蘧苗苗,苏向楠,Ngabire M,杨志娟,杨述文,摆玉林.2015.外源一氧化氮对镉胁迫下紫花苜蓿幼苗活性氧代谢和镉积累的影响[J].农业环境科学学报,34(12):2261-2271.[Chen Y P,Qu M M,Su X N,Ngabire M,Yang Z J,Yang S W,Bai Y L.2015.Effect of exogenous nitric oxide on active oxygen metabolism and cadmium accumulation in alfalfa seedlings under cadmium stress[J].Journal of Agro-Environment Science,34(12):2261-2271.]
董炜博,严敦余,郭兴启,竺晓平.1997.感染花生条纹病毒(Ps TV)后花生生理生化性状变化的研究[J].植物病理学报,27(3):281-285.[Dong W B,Yan D Y,Guo X Q,Zhu X P.1997.Effect of Ps TV-infection on physiological and biochemical properties of peanut[J].Acta Phyto Pathologica Sinica,27(3):281-285.]
付春霞.2014.锌对苹果叶片结构发育、光合及荧光特性的影响[D].泰安:山东农业大学.[Fu C X.2014.Effect of Zinc on structure development,photosynthetic and fluorescence characteristics of apple tree leaves[D].Tai’an:Shandong Agricultural University.]
洪健,徐颖,徐正.2001.植物病毒感染寄主叶绿体的形态结构变化[J].电子显微学报,19(3):335-336.[Hong J,Xu Y,Xu Z.2001.Morphological structure changes of host chloroplasts infected by plant viruses[J].Journal of Chinese Electron Microscopy Society,19(3):335-336.]
霍鹏.2015.利用多重荧光定量PCR鉴定番木瓜病毒及番木瓜e IF4E家族基因干扰效应研究[D].海口:海南大学.[Huo P.2015.Papaya viruses identification using multiplex real-time rt-pcr and the interference effect research of papaya e IF4E family genes[D].Haikou:Hainan University.]
李和平.2009.植物显微技术[M].第2版.北京:科学出版社.[Li H P.2009.Plant Micro Technology[M].The 2ndEdition.Beijing:Science Press.]
李琴,马庆华,王贵禧,张日清,梁丽松.2010.冬枣锈病发生与叶龄及其解剖结构关系的研究[J].中国农学通报,26(6):231-235.[Li Q,Ma Q H,Wang G X,Zhang R Q,Liang L S.2010.Correlation of rust occurrence to leaf age and leaf anatomical structure in Zizyphus jujuba Mill.cv.‘Dongzao’[J].Chinese Agricultural Science Bulletin,26(6):231-235.]
毛健民,郑爱珍,白岩,白宝璋.2002.烟草叶片感染花叶病毒时的某些生理生化变化[J].吉林农业大学学报,24(4):19-21.[Mao J M,Zheng A Z,Bai Y,Bai B Z.2002.Some physiological-biochemical changes of tobacco leaves when infected by mosaic virus disease[J].Journal of Jilin Agricultural University,24(4):19-21.]
倪国仕,章新军,毕庆文,王瑞.2010.受烟草花叶病毒侵染程度不同的烤烟叶片光合特性的变化[J].中国烟草科学,31(5):58-61.[Ni G S,Zhang X J,Bi Q W,Wang R.2010.Changes in photosynthesis characteristics of fluecured tobacco infected with tobacco mosaic virus[J].Chinese Tobacco Science,31(5):58-61.]
孙玉娟,张雨良,黄启星,王红星,龚殿,刘志昕.2011.番木瓜的PRSV接种及其组培苗的RT-PCR检测[J].广东农业科学,38(21):132-134.[Sun Y J,Zhang Y L,Huang Q X,Wang H X,Gong D,Liu Z X.2011.PRSV vaccination and RT-PCR detection of Carica papaya[J].Guangdong Agricultural Sciences,38(21):132-134.]
庹德财.2015.番木瓜畸形花叶病毒检测鉴定及侵染性克隆构建与应用[D].海口:海南大学.[Tuo D C.2015.Identification,generation of infectious full-length c DNA clones of Papaya leaf distortion mosaic virus from Hainan(PLD-MV-DF)and its use for GFP expression[D].Haikou:Hainan University.]
吴自林,李华平.2016.广东省番木瓜畸形花叶病毒的发现与鉴定[C]//中国植物病理学会.中国植物病理学会2016年学术年会论文集.北京:267.[Wu Z L,Li H P.2016.Discovery and identification of papaya malformed mosaic virus in Guangdong[C]//Chinese Society for Plant Pathology.Proceedings of the Annual Meeting of Chinese Society for Plant Pathology(2016).Beijing:267.]
徐恩静,闫小燕,王志学,周志良.2011.低温处理对小麦几丁质酶活性的影响[J].安徽农业科学,39(22):13324-13325.[Xu E J,Yan X Y,Wang Z X,Zhou Z L.2011.Influences of low temperature treatment on the chitinase activity of wheat[J].Journal of Anhui Agricultural Sciences,39(22):13324-13325.]
杨勇,庹德财,沈文涛,言普,黎小瑛,周鹏.2013.海南地区番木瓜畸形花叶病毒的发现与鉴定[J].热带作物学报,34(12):2442-2445.[Yang Y,Tuo D C,Shen W T,Yan P,Li X Y,Zhou P.2013.Papaya leaf distortion mosaic virus was identified in Hainan[J].Chinese Journal of Tropical Crops,34(12):2442-2445.]
杨志敏,蒋立科.2010.生物化学[M].第2版.北京:高等教育出版社.[Yang Z M,Jiang L K.2010.Biochemistry.[M].The 2ndEdition.Beijing:Higher Education Press.]
于力,阎君,郭世荣,黑银秀,许爽,朱为民.2012.番茄黄化曲叶病毒侵染对番茄叶片光合及荧光特性的影响[J].上海农业学报,28(3):22-25.[Yu L,Yan J,Guo S R,Hei Y X,Xu S,Zhu W M.2012.Effects of tomato yellow leaf curl virus(TYLCV)infection on photosynthetic and fluorescent characteristics of tomato leaves[J].Acta Agriculturae Shanghai,28(3):22-25.]
张乐华,周广,孙宝腾,李晓花,王书胜,单文.2011.高温胁迫对两种常绿杜鹃亚属植物幼苗生理生化特性的影响[J].植物科学学报,29(3):362-369.[Zhang L H,Zhou G,Sun B T,Li X H,Wang S S,Shan W.2011.Physiological and biochemical effects of high temperature stress on the seedlings of two rhododendron species of subgenus Hymenanthes[J].Plant Science Journal,29(3):362-369.]
张梦如,杨玉梅,成蕴秀,周滔,段晓艳,龚明,邹竹荣.2014.植物活性氧的产生及其作用和危害[J].西北植物学报,34(9):1916-1926.[Zhang M R,Yang Y M,Cheng Y X,Zhou T,Duan X Y,Gong M,Zou Z R.2014.Generation of reactive oxygen species and their functions and deleterious effects in plants[J].Acta Botanica Boreali-occidentalia Sinica,34(9):1916-1926.]
张琦,刘千,张智,罗锟,陈萍.2016.硝普钠对香蕉采后品质及生理指标的影响[J].南方农业学报,47(7):1198-1202.[Zhang Q,Liu Q,Zhang Z,Luo K,Chen P.2016.Effects of sodium nitroprusside on postharvest quality and physiological indexes of banana[J].Journal of Southern Agriculture,47(7):1198-1202.]
张荣萍,樊俊华,陈慧娟,鄢兴祥,陈萍.2017.3种生物农药对番木瓜环斑病的防效研究[J].中国南方果树,46(3):79-82.[Zhang R P,Fan J H,Chen H J,Yan X X,Chen P.2017.Study on the control effects of 3 biological pesticides on Papaya ringspot virus[J].South China Fruits,46(3):79-82.]
赵天宏,孙加伟,付宇.2008.逆境胁迫下植物活性氧代谢及外源调控机理的研究进展[J].作物杂志,(3):10-13.[Zhao T H,Sun J W,Fu Y.2008.Advances of researchon metabolism of plant reactive oxygen species and exogenous regulation under abiotic stresses[J].Crops,(3):10-13.]
Yin L H,Wang P,Li M J,Ke X W,Li C Y,Liang D,Wu S,Ma X L,Li C,Zou Y J,Ma F W.2013.Exogenous melatonin improves malus resistance to marssonina apple blotch[J].Journal of Pineal Research,54(4):426-434.
陈建勋,王晓峰.2015.植物生理学实验指导[M].广州:华南理工大学出版社.[Chen J X,Wang X F.2015.Experimental Guidance for Plant Physiology[M].Guangzhou:South China University of Technology Press.]
陈银萍,蘧苗苗,苏向楠,Ngabire M,杨志娟,杨述文,摆玉林.2015.外源一氧化氮对镉胁迫下紫花苜蓿幼苗活性氧代谢和镉积累的影响[J].农业环境科学学报,34(12):2261-2271.[Chen Y P,Qu M M,Su X N,Ngabire M,Yang Z J,Yang S W,Bai Y L.2015.Effect of exogenous nitric oxide on active oxygen metabolism and cadmium accumulation in alfalfa seedlings under cadmium stress[J].Journal of Agro-Environment Science,34(12):2261-2271.]
董炜博,严敦余,郭兴启,竺晓平.1997.感染花生条纹病毒(Ps TV)后花生生理生化性状变化的研究[J].植物病理学报,27(3):281-285.[Dong W B,Yan D Y,Guo X Q,Zhu X P.1997.Effect of Ps TV-infection on physiological and biochemical properties of peanut[J].Acta Phyto Pathologica Sinica,27(3):281-285.]
付春霞.2014.锌对苹果叶片结构发育、光合及荧光特性的影响[D].泰安:山东农业大学.[Fu C X.2014.Effect of Zinc on structure development,photosynthetic and fluorescence characteristics of apple tree leaves[D].Tai’an:Shandong Agricultural University.]
洪健,徐颖,徐正.2001.植物病毒感染寄主叶绿体的形态结构变化[J].电子显微学报,19(3):335-336.[Hong J,Xu Y,Xu Z.2001.Morphological structure changes of host chloroplasts infected by plant viruses[J].Journal of Chinese Electron Microscopy Society,19(3):335-336.]
霍鹏.2015.利用多重荧光定量PCR鉴定番木瓜病毒及番木瓜e IF4E家族基因干扰效应研究[D].海口:海南大学.[Huo P.2015.Papaya viruses identification using multiplex real-time rt-pcr and the interference effect research of papaya e IF4E family genes[D].Haikou:Hainan University.]
李和平.2009.植物显微技术[M].第2版.北京:科学出版社.[Li H P.2009.Plant Micro Technology[M].The 2ndEdition.Beijing:Science Press.]
李琴,马庆华,王贵禧,张日清,梁丽松.2010.冬枣锈病发生与叶龄及其解剖结构关系的研究[J].中国农学通报,26(6):231-235.[Li Q,Ma Q H,Wang G X,Zhang R Q,Liang L S.2010.Correlation of rust occurrence to leaf age and leaf anatomical structure in Zizyphus jujuba Mill.cv.‘Dongzao’[J].Chinese Agricultural Science Bulletin,26(6):231-235.]
毛健民,郑爱珍,白岩,白宝璋.2002.烟草叶片感染花叶病毒时的某些生理生化变化[J].吉林农业大学学报,24(4):19-21.[Mao J M,Zheng A Z,Bai Y,Bai B Z.2002.Some physiological-biochemical changes of tobacco leaves when infected by mosaic virus disease[J].Journal of Jilin Agricultural University,24(4):19-21.]
倪国仕,章新军,毕庆文,王瑞.2010.受烟草花叶病毒侵染程度不同的烤烟叶片光合特性的变化[J].中国烟草科学,31(5):58-61.[Ni G S,Zhang X J,Bi Q W,Wang R.2010.Changes in photosynthesis characteristics of fluecured tobacco infected with tobacco mosaic virus[J].Chinese Tobacco Science,31(5):58-61.]
孙玉娟,张雨良,黄启星,王红星,龚殿,刘志昕.2011.番木瓜的PRSV接种及其组培苗的RT-PCR检测[J].广东农业科学,38(21):132-134.[Sun Y J,Zhang Y L,Huang Q X,Wang H X,Gong D,Liu Z X.2011.PRSV vaccination and RT-PCR detection of Carica papaya[J].Guangdong Agricultural Sciences,38(21):132-134.]
庹德财.2015.番木瓜畸形花叶病毒检测鉴定及侵染性克隆构建与应用[D].海口:海南大学.[Tuo D C.2015.Identification,generation of infectious full-length c DNA clones of Papaya leaf distortion mosaic virus from Hainan(PLD-MV-DF)and its use for GFP expression[D].Haikou:Hainan University.]
吴自林,李华平.2016.广东省番木瓜畸形花叶病毒的发现与鉴定[C]//中国植物病理学会.中国植物病理学会2016年学术年会论文集.北京:267.[Wu Z L,Li H P.2016.Discovery and identification of papaya malformed mosaic virus in Guangdong[C]//Chinese Society for Plant Pathology.Proceedings of the Annual Meeting of Chinese Society for Plant Pathology(2016).Beijing:267.]
徐恩静,闫小燕,王志学,周志良.2011.低温处理对小麦几丁质酶活性的影响[J].安徽农业科学,39(22):13324-13325.[Xu E J,Yan X Y,Wang Z X,Zhou Z L.2011.Influences of low temperature treatment on the chitinase activity of wheat[J].Journal of Anhui Agricultural Sciences,39(22):13324-13325.]
杨勇,庹德财,沈文涛,言普,黎小瑛,周鹏.2013.海南地区番木瓜畸形花叶病毒的发现与鉴定[J].热带作物学报,34(12):2442-2445.[Yang Y,Tuo D C,Shen W T,Yan P,Li X Y,Zhou P.2013.Papaya leaf distortion mosaic virus was identified in Hainan[J].Chinese Journal of Tropical Crops,34(12):2442-2445.]
杨志敏,蒋立科.2010.生物化学[M].第2版.北京:高等教育出版社.[Yang Z M,Jiang L K.2010.Biochemistry.[M].The 2ndEdition.Beijing:Higher Education Press.]
于力,阎君,郭世荣,黑银秀,许爽,朱为民.2012.番茄黄化曲叶病毒侵染对番茄叶片光合及荧光特性的影响[J].上海农业学报,28(3):22-25.[Yu L,Yan J,Guo S R,Hei Y X,Xu S,Zhu W M.2012.Effects of tomato yellow leaf curl virus(TYLCV)infection on photosynthetic and fluorescent characteristics of tomato leaves[J].Acta Agriculturae Shanghai,28(3):22-25.]
张乐华,周广,孙宝腾,李晓花,王书胜,单文.2011.高温胁迫对两种常绿杜鹃亚属植物幼苗生理生化特性的影响[J].植物科学学报,29(3):362-369.[Zhang L H,Zhou G,Sun B T,Li X H,Wang S S,Shan W.2011.Physiological and biochemical effects of high temperature stress on the seedlings of two rhododendron species of subgenus Hymenanthes[J].Plant Science Journal,29(3):362-369.]
张梦如,杨玉梅,成蕴秀,周滔,段晓艳,龚明,邹竹荣.2014.植物活性氧的产生及其作用和危害[J].西北植物学报,34(9):1916-1926.[Zhang M R,Yang Y M,Cheng Y X,Zhou T,Duan X Y,Gong M,Zou Z R.2014.Generation of reactive oxygen species and their functions and deleterious effects in plants[J].Acta Botanica Boreali-occidentalia Sinica,34(9):1916-1926.]
张琦,刘千,张智,罗锟,陈萍.2016.硝普钠对香蕉采后品质及生理指标的影响[J].南方农业学报,47(7):1198-1202.[Zhang Q,Liu Q,Zhang Z,Luo K,Chen P.2016.Effects of sodium nitroprusside on postharvest quality and physiological indexes of banana[J].Journal of Southern Agriculture,47(7):1198-1202.]
张荣萍,樊俊华,陈慧娟,鄢兴祥,陈萍.2017.3种生物农药对番木瓜环斑病的防效研究[J].中国南方果树,46(3):79-82.[Zhang R P,Fan J H,Chen H J,Yan X X,Chen P.2017.Study on the control effects of 3 biological pesticides on Papaya ringspot virus[J].South China Fruits,46(3):79-82.]
赵天宏,孙加伟,付宇.2008.逆境胁迫下植物活性氧代谢及外源调控机理的研究进展[J].作物杂志,(3):10-13.[Zhao T H,Sun J W,Fu Y.2008.Advances of researchon metabolism of plant reactive oxygen species and exogenous regulation under abiotic stresses[J].Crops,(3):10-13.]
Yin L H,Wang P,Li M J,Ke X W,Li C Y,Liang D,Wu S,Ma X L,Li C,Zou Y J,Ma F W.2013.Exogenous melatonin improves malus resistance to marssonina apple blotch[J].Journal of Pineal Research,54(4):426-434.