引起乙烯褪绿蜜橘果实腐烂的主要致病真菌分离及鉴定
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摘要
为明确贮藏过程中乙烯褪绿蜜橘果实腐烂的主要致病真菌,以重庆市北碚产区无核蜜橘为研究材料,利用乙烯利对其果实进行褪绿处理,观察贮藏期间褪绿蜜橘果实的发病症状,确定病害种类,统计发病率;并从发病果实中分离主要致病真菌,采用形态学特征观察和ITS序列分子生物学分析对其进行鉴定。结果表明,乙烯褪绿加剧贮藏蜜橘果实病害的发生。从发病果实中共分离出5株主要致病真菌菌株,分别编号为A、B、C、D、E;结合形态学特征和5株致病真菌的ITS序列分析结果,A、B菌株均被鉴定为胶孢炭疽菌Colletotrichum gloeosporioides,但这2株菌株具有不同的菌落形态及致病特性;C菌株被鉴定为焦腐病菌Lasiodiplodia theobromae;D菌株被鉴定为交链格孢菌Alternaria alternata;E菌株被鉴定为棘孢曲霉Aspergillus aculeatus。A、B菌株引起的果实腐烂占乙烯褪绿蜜橘总腐烂果实的81.96%,是引起贮藏过程中乙烯褪绿蜜橘果实腐烂的主要致病真菌,其次为C菌株,D、E菌株为次要致病真菌。
To identify the main postharvest pathogens of ethylene-degreened satsuma mandarin fruit during storage, satsuma mandarin fruits harvested in Beibei District of Chongqing City were degreened with ethephon, and then the diseases symptoms were investigated, and the disease types and the incidence rates of different diseases were determined. The main pathogenic fungi were isolated from disease-infected fruits, and the fungi were identified according to the morphology and r DNA-ITS sequences of the fungi. The results indicated that degreening treatment aggravated the occurrence of the postharvest pathogens in satsuma mandarin fruits. Five major pathogenic fungi were isolated from the infected mandarin fruit and numbered as A, B, C, D, E, respectively. The strains A and B were identified as Colletotrichum gloeosporioides, but they had different colony morphology and pathogenic characteristics;strains C, D and E were identified as Lasiodiplodia theobromae, Alternaria alternata and Aspergillus aculeatus, respectively. Strains A and B were the main postharvest fungi in ethephon-degreened satsuma mandarin fruits, and the disease caused by them accounted for 81.96% of the total disease, followed by strain C, and strains D and E were the minor fungi.
To identify the main postharvest pathogens of ethylene-degreened satsuma mandarin fruit during storage, satsuma mandarin fruits harvested in Beibei District of Chongqing City were degreened with ethephon, and then the diseases symptoms were investigated, and the disease types and the incidence rates of different diseases were determined. The main pathogenic fungi were isolated from disease-infected fruits, and the fungi were identified according to the morphology and r DNA-ITS sequences of the fungi. The results indicated that degreening treatment aggravated the occurrence of the postharvest pathogens in satsuma mandarin fruits. Five major pathogenic fungi were isolated from the infected mandarin fruit and numbered as A, B, C, D, E, respectively. The strains A and B were identified as Colletotrichum gloeosporioides, but they had different colony morphology and pathogenic characteristics;strains C, D and E were identified as Lasiodiplodia theobromae, Alternaria alternata and Aspergillus aculeatus, respectively. Strains A and B were the main postharvest fungi in ethephon-degreened satsuma mandarin fruits, and the disease caused by them accounted for 81.96% of the total disease, followed by strain C, and strains D and E were the minor fungi.
引文
Brown GE.1973.Development of green mold in degreened oranges.Phytopathology,63(9):1104-1107
Brown GE.1975.Anthracnose,a serious decay of degreened‘Robinson’tangerines.Proceedings of the Florida State Horticultural Society,88:308-311
Brown GE,Burns JK.1998.Enhanced activity of abscission enzymes predisposes oranges to invasion by Diplodia natalensis,during ethylene degreening.Postharvest Biology and Technology,14(2):217-227
Brown GE,Lee HS.1993.Interactions of ethylene with citrus stemend rot caused by Diplodia natalensis.Phytopathology,83(11):1204-1208
Cronje PJR,Barry GH,Huysamer M.2011.Postharvest rind breakdown of‘Nules Clementine’mandarin is influenced by ethylene application,storage temperature and storage duration.Postharvest Biology and Technology,60(3):192-201
Cronje PJR,Crouch EM,Huysamer M.2005.Postharvest calyx retention of citrus fruit.Acta Horticulturae,682:369-376
Deng LL,Yuan ZY,Yin BF,Ren SY,Li SP,Yao SX,Zeng KF 2017.Effect of LED light treatment on coloration of ethephon-degreened mandarin fruit.Food&Machinery,33(3):127-133(in Chinese)[邓丽莉,袁梓洢,尹保凤,任书颖,李四平,姚世响,曾凯芳.2017.LED光照处理对乙烯褪绿蜜橘果实着色的影响.食品与机械,33(3):127-133]
Deng WP,Yang M,Du F,Yang JZ,Zhu SS.2013.Identification of the pathogen causing grape anthracnose in Yunnan and its pathogenicity.Journal of Plant Protection,40(1):61-67(in Chinese)[邓维萍,杨敏,杜飞,杨积忠,朱书生.2013.云南葡萄产区葡萄炭疽病病原鉴定及致病力分析.植物保护学报,40(1):61-67]
Fang ZD.1998.Research methods of plant pathology(3rd edition).Beijing:China Agriculture Press(in Chinese)[方中达.1998.植病研究方法(第3版).北京:中国农业出版社]
He K,Li ZL,Yang SY,Li XR.2013.Biological characteristics of citrus Colletotrichum gloeosporioides and inhibitory effect of boron on the fungus.Journal of Plant Protection,40(6):571-572(in Chinese)[何凯,李振轮,杨水英,李晓蕊.2013.柑橘炭疽菌的生物学特性及硼对其抑制作用.植物保护学报,40(6):571-572]
Ismail M,Zhang JX.2004.Post-harvest citrus diseases and their control.Outlooks on Pest Management,15(1):29-35
Li HY,Mei XF,Fu YS,Huang F,Chen ZL,Yang XP,Wang YG,Huang HH.2015.Alternaria brown spot of citrus:the risk and management strategy.Journal of Fruit Science,32(5):969-976(in Chinese)[李红叶,梅秀凤,符雨诗,黄峰,陈再廖,杨小平,王一光,黄海华.2015.柑橘链格孢褐斑病的发生危害风险和治理对策.果树学报,32(5):969-976]
Liu CL,Wen JZ,Yang MX,Li YG.2007.Application of rDNA-ITS in molecular test of phytopathogenic fungi.Journal of Northeast Agricultural University,38(1):101-106(in Chinese)[刘春来,文景芝,杨明秀,李永刚.2007.rDNA-ITS在植物病原真菌分子检测中的应用.东北农业大学学报,38(1):101-106]
Liu WX,Miao HX,Jin ZQ,Liu JH,Jia CH,Sun PG,Xu BY.2013.Effects of ethylene and 1-MCP on pigment content and degreening of banana during ripening.Journal of Fruit Science,30(6):1051-1055(in Chinese)[刘伟鑫,苗红霞,金志强,刘菊华,贾彩红,孙佩光,徐碧玉.2013.乙烯和1-MCP处理对香蕉采后果皮色素含量及褪绿变黄的调控.果树学报,30(6):1051-1055]
Lu LM,Du DC,Cheng BP,Hu XR,Chen GQ.2014.Isolation and identification of marine actinomyces A3202 and its control efficacy against postharvest citrus diseases.Journal of Plant Protection,41(3):327-334(in Chinese)[鹿连明,杜丹超,程保平,胡秀荣,陈国庆.2014.海洋放线菌A3202的分离鉴定及其对柑橘采后病害的防效.植物保护学报,41(3):327-334]
Mayuoni L,Tietel Z,Patil BS,Porat R.2011.Does ethylene degreening affect internal quality of citrus fruit?Postharvest Biology and Technology,62(1):50-58
Moscoso-Ramírez PA,Palou L.2014.Effect of ethylene degreening on the development of postharvest penicillium molds and fruit quality of early season citrus fruit.Postharvest Biology and Technology,91:1-8
Plaza P,Sanbruno A,Usall J,Lamarca N,Torres R,Pons J,Vi?as I.2004.Integration of curing treatments with degreening to control the main postharvest diseases of clementine mandarins.Postharvest Biology and Technology,34(1):29-37
Poole ND,Gray K.2002.Quality in citrus fruit:to degreen or not degreen?British Food Journal,104(7):492-505
Porat R,Weiss B,Cohen L,Daus A,Goren R,Droby S.1999.Effects of ethylene and 1-methylcyclopropene on the postharvest qualities of‘Shamouti’oranges.Postharvest Biology and Technology,15(2):155-163
Rodrigo MJ,Alquézar B,Alós E,Lado J,Zacarías L.2013.Biochemical bases and molecular regulation of pigmentation in the peel of citrus fruit.Scientia Horticulturae,163:46-62
Sdiri S,Navarro P,Benabda J,Monterde A,Salvador A.2012b.Antioxidant activity and vitamin C are not affected by degreening treatment of clementine mandarins.Acta Horticulturae,934:893-900
Sdiri S,Navarro P,Monterde A,Benabda J,Salvador A.2012a.Effect of postharvest degreening followed by a cold-quarantine treatment on vitamin C,phenolic compounds and antioxidant activity of early-season citrus fruit.Postharvest Biology and Technology,65(3):13-21
Sdiri S,Navarro P,Monterdea,Benabda J,Salvador A.2012c.New degreening treatments to improve the quality of citrus fruit combining different periods with and without ethylene exposure.Postharvest Biology and Technology,63(1):25-32
Sdiri S,Navarro P,Salvador A.2013.Postharvest application of auxins to control calyx senescence in clementines submitted to degreening treatment.Postharvest Biology and Technology,75:68-74
Tanaka A,Tanaka R.2006.Chlorophyll metabolism.Current Opinion in Plant Biology,9(3):248-255
Tang XH.2008.Development strategy of Chongqing citrus industry based on“Diamond Model”.Master Thesis.Chongqing:Southwest University(in Chinese)[唐晓华.2008.基于“钻石模型”的重庆柑橘产业发展研究.硕士学位论文.重庆:西南大学]
Wei JC.1979.Fungal identification manual.Shanghai:Shanghai Scientific&Technical Publishers(in Chinese)[魏景超.1979.真菌鉴定手册.上海:上海科学技术出版社]
Yi RH,Gan LJ,Yan DH,Wu ZJ,Tong YT,Wu FF.2013.Identification and biological characteristics of Neoscytalidium dimidiatum causing pitaya canker.Journal of Plant Protection,40(2):102-108(in Chinese)[易润华,甘罗军,晏冬华,吴泽菁,童依婷,吴凤发.2013.火龙果溃疡病病原菌鉴定及生物学特性.植物保护学报,40(2):102-108]
Yi RH,Zhu XR,Zhou EX.2003.Simplified CTAB method to extract DNA of filamentous fungi.Journal of Zhanjiang Ocean University,23(6):72-73(in Chinese)[易润华,朱西儒,周而勋.2003.简化CTAB法快速微量提取丝状真菌DNA.湛江海洋大学学报,23(6):72-73]
Yin BF,Zeng KF,Zhang ZQ,Deng LL.2015.Advances in ethylene degreening treatment of citrus fruits.Food Science,36(3):245-249(in Chinese)[尹保凤,曾凯芳,张昭其,邓丽莉.2015.柑橘果实乙烯褪绿技术研究进展.食品科学,36(3):245-249]
Zeng DX,Qi PK,Jiang ZD.2003.Analysis of intraspecific variation in the species Colletotrichum gloeosporiodies.Mycosystema,22(1):50-55(in Chinese)[曾大兴,戚佩坤,姜子德.2003.胶孢炭疽菌的种内遗传多样性研究.菌物系统,22(1):50-55]
Zhang C.2014.Screening of Aspergillus aculeatus ZC-1005 based on citrus mesocarp degradation and basic research for application.Master Thesis.Changsha:Central South University(in Chinese)[张闯.2014.降解柑橘囊衣Aspergillus aculeatus ZC-1005的筛选及其应用基础研究.硕士学位论文.长沙:中南大学]
Zhang JX.2004.Effect of ethylene on natural resistance of citrus fruit to stem-end rot caused by Diplodia natalensis and its relation to harvest control of this decay.Proceedings of the Florida State Horticultural Society,117:364-367
Zhou JY,Sun CD,Zhang LL,Dai X,Xu CJ,Chen KS.2010.Preferential accumulation of orange-colored carotenoids in Ponkan(Citrus reticulata)fruit peel following postharvest application of ethylene or ethephon.Scientia Horticulturae,126(2):229-235
Zhou Y,Chen HM,Wang XF,Tang J,Jin X,Zhou CY.2017.Development and application of a duplex RT-PCR protocol for detection of two new citrus viruses in China.Journal of Plant Protection,44(4):699-700(in Chinese)[周彦,陈洪明,王雪峰,唐杰,金鑫,周常勇.2017.两种柑橘新病毒双重RT-PCR检测体系的建立及应用.植物保护学报,44(4):699-700]
Brown GE.1975.Anthracnose,a serious decay of degreened‘Robinson’tangerines.Proceedings of the Florida State Horticultural Society,88:308-311
Brown GE,Burns JK.1998.Enhanced activity of abscission enzymes predisposes oranges to invasion by Diplodia natalensis,during ethylene degreening.Postharvest Biology and Technology,14(2):217-227
Brown GE,Lee HS.1993.Interactions of ethylene with citrus stemend rot caused by Diplodia natalensis.Phytopathology,83(11):1204-1208
Cronje PJR,Barry GH,Huysamer M.2011.Postharvest rind breakdown of‘Nules Clementine’mandarin is influenced by ethylene application,storage temperature and storage duration.Postharvest Biology and Technology,60(3):192-201
Cronje PJR,Crouch EM,Huysamer M.2005.Postharvest calyx retention of citrus fruit.Acta Horticulturae,682:369-376
Deng LL,Yuan ZY,Yin BF,Ren SY,Li SP,Yao SX,Zeng KF 2017.Effect of LED light treatment on coloration of ethephon-degreened mandarin fruit.Food&Machinery,33(3):127-133(in Chinese)[邓丽莉,袁梓洢,尹保凤,任书颖,李四平,姚世响,曾凯芳.2017.LED光照处理对乙烯褪绿蜜橘果实着色的影响.食品与机械,33(3):127-133]
Deng WP,Yang M,Du F,Yang JZ,Zhu SS.2013.Identification of the pathogen causing grape anthracnose in Yunnan and its pathogenicity.Journal of Plant Protection,40(1):61-67(in Chinese)[邓维萍,杨敏,杜飞,杨积忠,朱书生.2013.云南葡萄产区葡萄炭疽病病原鉴定及致病力分析.植物保护学报,40(1):61-67]
Fang ZD.1998.Research methods of plant pathology(3rd edition).Beijing:China Agriculture Press(in Chinese)[方中达.1998.植病研究方法(第3版).北京:中国农业出版社]
He K,Li ZL,Yang SY,Li XR.2013.Biological characteristics of citrus Colletotrichum gloeosporioides and inhibitory effect of boron on the fungus.Journal of Plant Protection,40(6):571-572(in Chinese)[何凯,李振轮,杨水英,李晓蕊.2013.柑橘炭疽菌的生物学特性及硼对其抑制作用.植物保护学报,40(6):571-572]
Ismail M,Zhang JX.2004.Post-harvest citrus diseases and their control.Outlooks on Pest Management,15(1):29-35
Li HY,Mei XF,Fu YS,Huang F,Chen ZL,Yang XP,Wang YG,Huang HH.2015.Alternaria brown spot of citrus:the risk and management strategy.Journal of Fruit Science,32(5):969-976(in Chinese)[李红叶,梅秀凤,符雨诗,黄峰,陈再廖,杨小平,王一光,黄海华.2015.柑橘链格孢褐斑病的发生危害风险和治理对策.果树学报,32(5):969-976]
Liu CL,Wen JZ,Yang MX,Li YG.2007.Application of rDNA-ITS in molecular test of phytopathogenic fungi.Journal of Northeast Agricultural University,38(1):101-106(in Chinese)[刘春来,文景芝,杨明秀,李永刚.2007.rDNA-ITS在植物病原真菌分子检测中的应用.东北农业大学学报,38(1):101-106]
Liu WX,Miao HX,Jin ZQ,Liu JH,Jia CH,Sun PG,Xu BY.2013.Effects of ethylene and 1-MCP on pigment content and degreening of banana during ripening.Journal of Fruit Science,30(6):1051-1055(in Chinese)[刘伟鑫,苗红霞,金志强,刘菊华,贾彩红,孙佩光,徐碧玉.2013.乙烯和1-MCP处理对香蕉采后果皮色素含量及褪绿变黄的调控.果树学报,30(6):1051-1055]
Lu LM,Du DC,Cheng BP,Hu XR,Chen GQ.2014.Isolation and identification of marine actinomyces A3202 and its control efficacy against postharvest citrus diseases.Journal of Plant Protection,41(3):327-334(in Chinese)[鹿连明,杜丹超,程保平,胡秀荣,陈国庆.2014.海洋放线菌A3202的分离鉴定及其对柑橘采后病害的防效.植物保护学报,41(3):327-334]
Mayuoni L,Tietel Z,Patil BS,Porat R.2011.Does ethylene degreening affect internal quality of citrus fruit?Postharvest Biology and Technology,62(1):50-58
Moscoso-Ramírez PA,Palou L.2014.Effect of ethylene degreening on the development of postharvest penicillium molds and fruit quality of early season citrus fruit.Postharvest Biology and Technology,91:1-8
Plaza P,Sanbruno A,Usall J,Lamarca N,Torres R,Pons J,Vi?as I.2004.Integration of curing treatments with degreening to control the main postharvest diseases of clementine mandarins.Postharvest Biology and Technology,34(1):29-37
Poole ND,Gray K.2002.Quality in citrus fruit:to degreen or not degreen?British Food Journal,104(7):492-505
Porat R,Weiss B,Cohen L,Daus A,Goren R,Droby S.1999.Effects of ethylene and 1-methylcyclopropene on the postharvest qualities of‘Shamouti’oranges.Postharvest Biology and Technology,15(2):155-163
Rodrigo MJ,Alquézar B,Alós E,Lado J,Zacarías L.2013.Biochemical bases and molecular regulation of pigmentation in the peel of citrus fruit.Scientia Horticulturae,163:46-62
Sdiri S,Navarro P,Benabda J,Monterde A,Salvador A.2012b.Antioxidant activity and vitamin C are not affected by degreening treatment of clementine mandarins.Acta Horticulturae,934:893-900
Sdiri S,Navarro P,Monterde A,Benabda J,Salvador A.2012a.Effect of postharvest degreening followed by a cold-quarantine treatment on vitamin C,phenolic compounds and antioxidant activity of early-season citrus fruit.Postharvest Biology and Technology,65(3):13-21
Sdiri S,Navarro P,Monterdea,Benabda J,Salvador A.2012c.New degreening treatments to improve the quality of citrus fruit combining different periods with and without ethylene exposure.Postharvest Biology and Technology,63(1):25-32
Sdiri S,Navarro P,Salvador A.2013.Postharvest application of auxins to control calyx senescence in clementines submitted to degreening treatment.Postharvest Biology and Technology,75:68-74
Tanaka A,Tanaka R.2006.Chlorophyll metabolism.Current Opinion in Plant Biology,9(3):248-255
Tang XH.2008.Development strategy of Chongqing citrus industry based on“Diamond Model”.Master Thesis.Chongqing:Southwest University(in Chinese)[唐晓华.2008.基于“钻石模型”的重庆柑橘产业发展研究.硕士学位论文.重庆:西南大学]
Wei JC.1979.Fungal identification manual.Shanghai:Shanghai Scientific&Technical Publishers(in Chinese)[魏景超.1979.真菌鉴定手册.上海:上海科学技术出版社]
Yi RH,Gan LJ,Yan DH,Wu ZJ,Tong YT,Wu FF.2013.Identification and biological characteristics of Neoscytalidium dimidiatum causing pitaya canker.Journal of Plant Protection,40(2):102-108(in Chinese)[易润华,甘罗军,晏冬华,吴泽菁,童依婷,吴凤发.2013.火龙果溃疡病病原菌鉴定及生物学特性.植物保护学报,40(2):102-108]
Yi RH,Zhu XR,Zhou EX.2003.Simplified CTAB method to extract DNA of filamentous fungi.Journal of Zhanjiang Ocean University,23(6):72-73(in Chinese)[易润华,朱西儒,周而勋.2003.简化CTAB法快速微量提取丝状真菌DNA.湛江海洋大学学报,23(6):72-73]
Yin BF,Zeng KF,Zhang ZQ,Deng LL.2015.Advances in ethylene degreening treatment of citrus fruits.Food Science,36(3):245-249(in Chinese)[尹保凤,曾凯芳,张昭其,邓丽莉.2015.柑橘果实乙烯褪绿技术研究进展.食品科学,36(3):245-249]
Zeng DX,Qi PK,Jiang ZD.2003.Analysis of intraspecific variation in the species Colletotrichum gloeosporiodies.Mycosystema,22(1):50-55(in Chinese)[曾大兴,戚佩坤,姜子德.2003.胶孢炭疽菌的种内遗传多样性研究.菌物系统,22(1):50-55]
Zhang C.2014.Screening of Aspergillus aculeatus ZC-1005 based on citrus mesocarp degradation and basic research for application.Master Thesis.Changsha:Central South University(in Chinese)[张闯.2014.降解柑橘囊衣Aspergillus aculeatus ZC-1005的筛选及其应用基础研究.硕士学位论文.长沙:中南大学]
Zhang JX.2004.Effect of ethylene on natural resistance of citrus fruit to stem-end rot caused by Diplodia natalensis and its relation to harvest control of this decay.Proceedings of the Florida State Horticultural Society,117:364-367
Zhou JY,Sun CD,Zhang LL,Dai X,Xu CJ,Chen KS.2010.Preferential accumulation of orange-colored carotenoids in Ponkan(Citrus reticulata)fruit peel following postharvest application of ethylene or ethephon.Scientia Horticulturae,126(2):229-235
Zhou Y,Chen HM,Wang XF,Tang J,Jin X,Zhou CY.2017.Development and application of a duplex RT-PCR protocol for detection of two new citrus viruses in China.Journal of Plant Protection,44(4):699-700(in Chinese)[周彦,陈洪明,王雪峰,唐杰,金鑫,周常勇.2017.两种柑橘新病毒双重RT-PCR检测体系的建立及应用.植物保护学报,44(4):699-700]