茉莉酸甲酯诱导采后脐橙抗青霉病的机制研究

doi:10.16420/j.issn.0513-353x.2023-0564

摘要/Abstract

摘要：

以‘纽荷尔’脐橙果实为试验材料，采用50 μmol · L^-1茉莉酸甲酯（MeJA）熏蒸处理24 h后接种青霉病病原菌意大利青霉（Penicillium italicum）孢子悬浮液，探究MeJA诱导脐橙果实抗青霉病的效应和机理。结果表明，MeJA处理的青霉病病斑直径在发病后1 ~ 7 d显著小于对照；过氧化物酶（POD）、多酚氧化酶（PPO）、漆酶（LAC）、苯丙氨酸解氨酶（PAL）、肉桂醇脱氢酶（CAD）、4-香豆酸辅酶A连接酶（4CL）、肉桂酸-4-羟化酶（C4H）活性及相关基因（CsPPO除外）表达量的最高值均显著高于对照；促进了木质素、总酚和类黄酮含量显著增加，而苯丙氨酸和色氨酸含量明显减少。上述结果暗示MeJA处理可能通过调控芳香族氨基酸含量并激发苯丙烷代谢途径相关酶活性和基因表达，促进木质素、总酚和类黄酮含量的积累，诱导脐橙果实增强对青霉病的抗性。

关键词: 脐橙, 茉莉酸甲酯, 青霉病, 诱导抗性, 苯丙烷代谢途径

Abstract:

In this study，‘Newhall’navel orange fruits were used as experimental material and fumigated with 50 μmol · L^-1 MeJA for 24 h，then 20 µL 1.25 × 10⁶ spores · mL^-1 blue mold pathogen Penicillium italicum spore suspension were injected，which to investigate the induced role of methyl jasmonate（MeJA）treatment on the resistance of postharvest navel orange fruit against blue mold. The results showed the blue mold lesion diameters of MeJA treatment group was significantly smaller than that of the control at 1-7 d after disease onset. The highest activities of POD，PPO，LAC，PAL，CAD，4CL and C4H and their relative gene expression levels except CsPPO in MeJA treatment group were higher than those of the control，respectively. At the same time，MeJA treatment promoted the accumulation of lignin，total phenolics，and flavonoids in navel orange fruits. Moreover，the content of phenylalanine and tryptophan in MeJA treatment group was significantly reduced. The above results indicated MeJA treatment might promote the accumulation of lignin，total phenolics and flavonoids content through regulating aromatic amino acids as well as stimulating the activities and gene expression of phenylpropanoid metabolic pathway-related enzymes，thereby inducing an enhancement in resistance of navel orange fruits against postharvest blue mold.

Key words: navel orange, methyl jasmonate, blue mold, induced resistance, phenylpropanoid metabolic pathway

陈明, 张洁茹, 杨航云, 王印宝, 郑致远, 曾教科, 陈金印, 付永琦, 向妙莲. 茉莉酸甲酯诱导采后脐橙抗青霉病的机制研究[J]. 园艺学报, 2024, 51(9): 2183-2194.

CHEN Ming, ZHANG Jieru, YANG Hangyun, WANG Yinbao, ZHENG Zhiyuan, ZENG Jiaoke, CHEN Jinyin, FU Yongqi, XIANG Miaolian. Induction Mechanism of Methyl Jasmonate on Postharvest Navel Orange Fruit Against Blue Mold[J]. Acta Horticulturae Sinica, 2024, 51(9): 2183-2194.

图/表 6

表1 实时荧光定量PCR引物

Table 1 Primers for qRT-PCR analysis

甜橙基因 Orange gene	引物序列（5′-3′） Primer sequence	片段大小/bp Size	NCBI号 NCBI No.
CsPOD	F-GACATTCGCTATGCCGTTCT；R-TCGGTCCCGTTGTAATCG	94	XM_006492612.2
CsPPO	F-ATGAGATGCCAATAGGGCCG；R-AATGCGAGCTAGACCCTTCC	253	XM_006468155.2
CsPAL	F-GATTACGGATTCAAGGGTGC；R-TTGGTGACAGGTATTGGCGAG	80	XM_006485585.3
CsCAD	F-CCTTTGGTGCCTGGACATGAG；R-GTGTAAACTGACCGAGCACAA	170	XM_006476176.3
Cs4CL	F-ACCGGCGACATTGGTTACAT；R-TGGAGTGCGAGAGAAGCAAG	130	XM_006494749.3
CsC4H	F-GTACTTGCCTTTTGGCGTGG；R-TGCAATCACAAGCCCCAAGA	79	NM_001288840.1
CsLAC	F-CGCAATGTCCAATTCAGCC；R-GCTTTCCACCATTCACCGA	208	XM_006486310.3
CsActin	F-CATCCCTCAGCACCTTCC；R-CCAACCTTAGCACTTCTCC	195	XM_006464503.3

图1 茉莉酸甲酯（MeJA）及其合成抑制剂（SHAM）预处理对接种青霉病菌脐橙果实抗病性的影响不同小写字母代表各处理间在0.05水平存在显著性差异。下同。

Fig. 1 Effects of methyl jasmonate（MeJA）and its synthesis inhibitor（SHAM）pretreatments on the disease resistance of navel orange inoculated with Penicillium italicum The different lowercase letters indicate significant differences among treatments at 0.05 level. The same below.

图2 茉莉酸甲酯（MeJA）及其合成抑制剂（SHAM）对接种青霉病菌脐橙相关酶活性的影响 *和**分别表示MeJA处理和对照之间差异显著（P < 0.05）和极显著（P < 0.01）。下同。

Fig. 2 Effects of methyl jasmonate（MeJA）and its synthesis inhibitor（SHAM）on the activities of related enzyme. in navel orange inoculated with Penicillium italicum * indicates significant differences between MeJA treatment and the control（P < 0.05），** indicate extremely significant differences between MeJA treatment and the control（P < 0.01）. The same below.

图3 茉莉酸甲酯（MeJA）及其合成抑制剂（SHAM）预处理对接种青霉病菌脐橙苯丙烷代谢酶基因相对表达量的影响

Fig. 3 Effects of methyl jasmonate（MeJA）and its synthesis inhibitor（SHAM）pretreatments on phenylpropane metabolizing enzyme genes expression in navel orange inoculated with Penicillium italicum

图4 茉莉酸甲酯（MeJA）及其合成抑制剂（SHAM）预处理对接种青霉病菌脐橙木质素等含量的影响

Fig. 4 Effects of methyl jasmonate（MeJA）and its synthesis inhibitor（SHAM）pretreatments on the content of lignin etc. in navel orange inoculated with Penicillium italicum

图5 茉莉酸甲酯处理对脐橙果实木质素生物合成通路的影响示意图白色框表示未检测，红色框表示含量显著增加，蓝色框表示含量显著下降。虚线箭头表示多个催化步骤和中间产物，实线箭头表示进一步催化；向上红色箭头表示上调；问号表示未知。

Fig. 5 The sketch map for effects of methyl jasmonate（MeJA）treatment on lignin biosynthesis pathway in navel orange fruits The white box indicates no detection，the red box indicates a significant increase in content，and the blue box indicates a significant decrease in content. The dotted arrow indicates multiple catalytic steps and intermediate products，and the solid arrow indicates further catalysis；the upward red arrow indicates upregulation；the question mark indicates unknown.

参考文献 45

[1]	Bhatta U K. 2022. Alternative management approaches of citrus diseases caused by Penicillium digitatum(Green Mold)and Penicillium italicum(Blue Mold). Frontiers in Plant Science,12:833328.
[2]	Chen Yanru. 2019. Effect of postharvest sodium nitroprusside treatment on the quality,shikimate and phenylpropanoid pathway of apple fruit[M. D. Dissertation]. Jinzhou: Bohai University. (in Chinese)
	陈延儒. 2019. 采后硝普钠处理对苹果果实品质、莽草酸和苯丙烷代谢途径的影响[硕士论文]. 锦州: 渤海大学.
[3]	Cheong J J, Choi Y D. 2003. Methyl jasmonate as a vital substance in plants. Trends in Genetics, 19 (7):409-413.
[4]	Cui Xixi, Li Fujun, Zhang Xinhua, Guo Yanyin, Li Xiaoan. 2019. Recent progress in mechanism of action and application of methyl jasmonate in postharvest quality regulation of fruits and vegetables. Food Science, 40 (13):304-311. (in Chinese) doi: 10.7506/spkx1002-6630-20180805-036
	崔席席, 李富军, 张新华, 郭衍银, 李晓安. 2019. 茉莉酸甲酯调控果蔬采后品质的机制及应用研究进展. 食品科学, 40 (13):304-311. doi: 10.7506/spkx1002-6630-20180805-036
[5]	Deng Chengfeng, Li Suping, Zhang Ruixuan, Han Leng, Li Yong. 2023. Control effect of Lysobacter enzymogenes LE 16 on rot disease in post-harvest citrus fruit caused by Penicillium digitatum and P. italicum. Acta Horticulturae Sinica, 50 (7):1574-1586. (in Chinese)
	邓成凤, 李素平, 张瑞轩, 韩冷, 李勇. 2023. 产酶溶杆菌LE16对采后柑橘青霉绿霉病的防治作用. 园艺学报, 50 (7):1574-1586.
[6]	Dubouzet J G, Matsuda F, Ishihara A, Miyagawa H, Wakasa K. 2013. Production of indole alkaloids by metabolic engineering of the tryptophan pathway in rice. Plant Biotechnology Journal, 11 (9):1103-1111. doi: 10.1111/pbi.12105 pmid: 23980801
[7]	Ge Y H, Chen Y R, Li C Y, Wei M L, Li X, Tang Q, Duan B. 2019a. Effect of trisodium phosphate treatment on black spot of apple fruit and the roles of anti-oxidative enzymes. Physiological and Molecular Plant Pathology,106:226-231.
[8]	Ge Y H, Li X, Li C Y, Tang Q, Duan B, Cheng Y, Hou J B, Li J R. 2019b. Effect of sodium nitroprusside on antioxidative enzymes and the phenylpropanoid pathway in blueberry fruit. Food Chemistry,295:607-612.
[9]	Guo J, Fang W W, Lu H P, Zhu R Y, Lu L F, Zheng X D, Yu T. 2014. Inhibition of green mold disease in mandarins by preventive applications of methyl jasmonate and antagonistic yeast Cryptococcus laurentii. Postharvest Biology and Technology,88:72-78.
[10]	Hayat K, Zhang X M, Farooq U, Abbas S, Xia S Q, Jia C S, Zhong F, Zhang J. 2010. Effect of microwave treatment on phenolic content and antioxidant activity of citrus mandarin pomace. Food Chemistry, 123 (2):423-429.
[11]	He F T, Zhao L N, Zheng X F, Abdelhai M H, Boateng N S, Zhang X H, Zhang H Y. 2020. Investigating the effect of methyl jasmonate on the biocontrol activity of Meyerozyma guilliermondii against blue mold decay of apples and the possible mechanisms involved. Physiological and Molecular Plant Pathology,109:101454.
[12]	Jiang L L, Jin P, Wang L, Yu X, Wang H Y, Zheng Y H. 2015. Methyl jasmonate primes defense responses against Botrytis cinerea and reduces disease development in harvested table grapes. Scientia Horticulturae,192:218-223.
[13]	Jing X, Wang H, Gong B, Liu S Q, Wei M, Ai X Z, Li Y, Shi Q H. 2018. Secondary and sucrose metabolism regulated by different light quality combinations involved in melon tolerance to powdery mildew. Plant Physiology and Biochemistry,124:77-87.
[14]	Kanashiro A M, Akiyama D Y, Kupper K C, Fill T P. 2020. Penicillium italicum:an underexplored postharvest pathogen. Frontiers in Microbiology,11:606852.
[15]	Li Dong. 2021. The regulation of elevated CO₂ on anthocyanin synthesis in postharvest strawberry fruit[Ph. D. Dissertation]. Hangzhou: Zhejiang University. (in Chinese)
	李栋. 2021. 高浓度CO₂处理调控采后草莓花色苷合成机制研究[博士论文]. 杭州: 浙江大学.
[16]	Li H, Suo J T, Han Y, Liang C Q, Jin M J, Zhang Z K, Rao J P. 2017. The effect of 1-methylcyclopropene,methyl jasmonate and methyl salicylate on lignin accumulation and gene expression in postharvest‘Xuxiang’kiwifruit during cold storage. Postharvest Biology and Technology,124:107-118.
[17]	Li Xue. 2020. Partial mechanism of applying acibenzolar-s-methyl after harvest inducing disease resistance against black spot in docteur jules Guyot pears[M. D. Dissertation]. Jinzhou: Bohai University. (in Chinese)
	李雪. 2020. 采后苯并噻重氮处理诱导三季梨果实抗黑斑病的部分机理[硕士论文]. 锦州: 渤海大学.
[18]	Liang Wei, Zhu Yatong, Chai Xiuwei, Kong Rui, Li Binshan, Li Yongcai, Bi Yang, Prusky D. 2021. p-Coumaric acid promoted wound healing of potato tubers by accelerating the deposition of suberin poly phenolic and lignin at wound sites. Scientia Agricultura Sinica, 54 (20):4434-4445. (in Chinese)
	梁伟, 朱亚同, 柴秀伟, 孔蕊, 李斌山, 李永才, 毕阳, Prusky Dov. 2021. P-香豆酸通过加速伤口处聚酚软木脂和木质素的沉积促进马铃薯块茎愈伤. 中国农业科学, 54 (20):4434-4445. doi: 10.3864/j.issn.0578-1752.2021.20.016
[19]	Liu Lian, Tang Zhipeng, Li Feifei, Xiong Jiang, Lü Biwen, Ma Xiaochuan, Tang Chaolan, Li Zehang, Zhou Tie, Sheng Ling, Lu Xiaopeng. 2021. Fruit quality in storage,storability and peel transcriptome analysis of Rong’an kumquat,Huapi kumquat and Cuimi kumquat. Scientia Agricultura Sinica, 54 (20):4421-4433. (in Chinese) doi: 10.3864/j.issn.0578-1752.2021.20.015
	刘恋, 唐志鹏, 李菲菲, 熊江, 吕壁纹, 马小川, 唐超兰, 李泽航, 周铁, 盛玲, 卢晓鹏. 2021. ‘融安金柑’‘滑皮金柑’及‘脆蜜金柑’贮藏期品质、贮藏特性及果皮转录组分析. 中国农业科学, 54 (20):4421-4433. doi: 10.3864/j.issn.0578-1752.2021.20.015
[20]	Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCt method. Methods, 25 (4):402-408. doi: 10.1006/meth.2001.1262 pmid: 11846609
[21]	Lü Xinning, Wang Yue, Jia Runpu, Wang Shengnan, Yao Yuxin. 2022. Effects of melatonin treatment on quality of stored shine muscat grapes under different storage temperatures. Scientia Agricultura Sinica, 55 (7):1411-1422. (in Chinese) doi: 10.3864/j.issn.0578-1752.2022.07.012
	吕馨宁, 王玥, 贾润普, 王胜男, 姚玉新. 2022. 不同温度下褪黑素处理对‘阳光玫瑰’葡萄采后品质的影响. 中国农业科学, 55 (7):1411-1422. doi: 10.3864/j.issn.0578-1752.2022.07.012
[22]	Ma Jie, Hu Wenzhong, Bi Yang, Jiang Aili, Sa Sa. 2013. Effect of MeJA treatments on benzene propane metabolism in tissues of fresh-cut lettuce and cabbage. Science and Technology of Food Industry, 34 (7):333-335. (in Chinese)
	马杰, 胡文忠, 毕阳, 姜爱丽, 萨仁高娃. 2013. 茉莉酸甲酯处理对鲜切莴苣和甘蓝苯丙烷代谢的影响. 食品工业科技, 34 (7):333-335.
[23]	Ma Qiaoli. 2014. Mechanism underlying 2,4-dichlorophenoxyacetic acid maintaining citrus fruit postharvest quality[Ph. D. Dissertation]. Wuhan: Huazhong Agricultural University. (in Chinese)
	马巧利. 2014. 2,4-二氯苯氧乙酸保鲜柑橘果实采后品质机理[博士论文]. 武汉: 华中农业大学.
[24]	Maeda H, Dudareva N. 2012. The shikimate pathway and aromatic amino acid biosynthesis in plants. Annual Review of Plant Biology,63:73-105.
[25]	Miao Yuhuan. 2019. Signalling and regulatory roles of tryptophan metabolites in cotton immune system to Verticillium dahliae[Ph. D. Dissertation]. Wuhan: Huazhong Agricultural University.
	苗玉焕. 2019. 色氨酸代谢与棉花抗黄萎病免疫调控[博士论文]. 武汉: 华中农业大学. (in Chinese)
[26]	Min D D, Li Z L, Fu X D, Li F J, Li X A, Zuo J H, Zhang X H. 2022. Autophagy is involved in methyl jasmonate-mediated resistance against Botrytis cinerea in postharvest tomato fruit by regulating jasmonate signaling and reactive oxygen species homeostasis. Scientia Horticulturae,305:111361.
[27]	Morrison I M. 1972. A semi-micro method for the determination of lignin and its use in predicting the digestibility of forage crops. Journal of the Science of Food and Agriculture, 23 (4):455-463. pmid: 5029974
[28]	Pan L Y, Chen X R, Xu W, Fan S S, Wan T, Zhang J, Cai Y L. 2022. Methyl jasmonate induces postharvest disease resistance to decay caused by Alternaria alternata in sweet cherry fruit. Scientia Horticulturae,292:110624.
[29]	Pan L Y, Zhao X Y, Chen M, Fu Y Q, Xiang M L, Chen J Y. 2020. Effect of exogenous methyl jasmonate treatment on disease resistance of postharvest kiwifruit. Food Chemistry,305:125483.
[30]	Pascual M B, Jorge E A, De La Torre F N, Cañas R A, Concepción A, Cánovas F M. 2016. Biosynthesis and metabolic fate of phenylalanine in conifers. Frontiers in Plant Science,7:1030-1042.
[31]	Pérez A G, Olías R, Luaces P, Sanz C. 2002. Biosynthesis of strawberry aroma compounds through amino acid metabolism. Journal of Agricultural and Food Chemistry, 50 (14):4037-4042. pmid: 12083879
[32]	Tao X Y, Wu Q, Li J Y, Huang S Q, Cai L Y, Mao L C, Luo Z S, Li L, Ying T J. 2022. Exogenous methyl jasmonate regulates phenolic compounds biosynthesis during postharvest tomato ripening. Postharvest Biology and Technology,184:111760.
[33]	Valenzuela-Riffo F, Zúñiga P E, Morales-Quintana L, Lolas M, Cáceres M, Figueroa C R. 2020. Priming of defense systems and upregulation of MYC2 and JAZ1 genes after Botrytis cinerea inoculation in methyl jasmonate-treated strawberry fruits. Plants, 9 (4):447.
[34]	Wang H B, Kou X H, Wu C E, Fan G J, Li T T. 2020. Methyl jasmonate induces the resistance of postharvest blueberry to gray mold caused by Botrytis cinerea. Journal of the Science of Food and Agriculture, 100 (11):4272-4281.
[35]	Wang K T, Jin P, Han L, Shang H T, Tang S S, Rui H J, Duan Y F, Kong F Y, Kai X, Zheng Y H. 2014. Methyl jasmonate induces resistance against Penicillium citrinum in Chinese bayberry by priming of defense responses. Postharvest Biology and Technology,98:90-97.
[36]	Wang S Y, Shi X C, Liu F Q, Laborda P. 2021. Effects of exogenous methyl jasmonate on quality and preservation of postharvest fruits:a review. Food Chemistry,353:129482.
[37]	Wang Y C, Chuang Y C, Hsu H W. 2008. The flavonoid,carotenoid and pectin content in peels of citrus cultivated in Taiwan. Food Chemistry, 106 (1):277-284.
[38]	Wang Yinbao, Wu Fan, Xiao Liuhua, Peng Wenwen, Chen Ming, Chen Jinyin, Xiang Miaolian. 2022. Effects of methyl jasmonate treatment on blue mold and defense enzymes activity of navel orange fruit. Acta Agriculturae Universitatis Jiangxiensis, 44 (3):560-568. (in Chinese)
	王印宝, 吴帆, 肖刘华, 彭文文, 陈明, 陈金印, 向妙莲. 2022. 茉莉酸甲酯处理对脐橙果实青霉病及防御酶活性的影响. 江西农业大学学报, 44 (3):560-568.
[39]	Wei X B, Guan W L, Yang Y J, Shao Y L, Mao L C. 2021. Methyl jasmonate promotes wound healing by activation of phenylpropanoid metabolism in harvested kiwifruit. Postharvest Biology and Technology,175:111472.
[40]	Xu J, Zhang Z, Li X P, Wei J, Wu B. 2019a. Effect of nitrous oxide against Botrytis cinerea and phenylpropanoid pathway metabolism in table grapes. Scientia Horticulturae,254:99-105.
[41]	Xu X Y, Zhou Y P, Wang B, Ding L, Wang Y, Luo L, Zhang Y L, Kong W W. 2019b. Genome-wide identification and characterization of laccase gene family in Citrus sinensis. Gene, 20 (689):114-123
[42]	Zhang H Y, Ma L C, Turner M, Xu H X, Dong Y, Jiang S. 2009. Methyl jasmonate enhances biocontrol efficacy of Rhodotorula glutinis to postharvest blue mold decay of pears. Food Chemistry, 117 (4):621-626.
[43]	Zhang Ji, Li Junjie, Wan Lianjie, Yang Jiangbo, Zheng Yongqiang, Lü Qiang, Xie Rangjin, Ma Yanyan, Deng Lie, Yi Shilai. 2020. Effects of potassium application levels on nutrient,yield and quality of Newhall navel orange. Scientia Agricultura Sinica, 53 (20):4271-4286. (in Chinese)
	张绩, 李俊杰, 万连杰, 杨江波, 郑永强, 吕强, 谢让金, 马岩岩, 邓烈, 易时来. 2020. 施钾水平对纽荷尔脐橙养分、产量和品质的影响. 中国农业科学, 53 (20):4271-4286. doi: 10.3864/j.issn.0578-1752.2020.20.015
[44]	Zhang Zhao, Lü Zefang, Wu Hongmei, Zhou Zhiqin, Yu Jie. 2015. Phenolic contents of ten famous Chongqing pummelo(Citrus grandis)cultivars and their antioxidant capacities. Journal of Southwest University(Natural Science Edition), 37 (5):58-65. (in Chinese)
	张昭, 吕泽芳, 吴洪梅, 周志钦, 于杰. 2015. 重庆市10大名柚果实酚类物质质量分数及抗氧化活性研究. 西南大学学报(自然科学版), 37 (5):58-65.
[45]	Zhou Z, Zhi T T, Liu Y, Chen Y C, Ren C M. 2014. Tyrosine induces anthocyanin biosynthesis in Arabidopsis thaliana. American Journal of Plant Sciences, 5 (3):328-331.