红肉火龙果采后病原菌分离鉴定与植物精油抑菌研究
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摘要
分离、纯化并鉴定了浙江产区红肉火龙果贮藏期病原菌,筛选出对抑制病原菌效果较好的精油,并验证了该精油抑菌效果。通过分离、纯化及致病性检测,得到了病原菌菌株HS3和HS6。以病原菌18S r DNA序列进行进化树分析,确定两种病原菌为层生镰刀菌和平头炭疽菌。选择6种植物精油进行抑菌性试验,结果发现香芹酚精油、肉桂精油及百里香精油在2 000μL/L时对两种病原菌生长抑制率均为100%。采用体外直接接触法和体外熏蒸法,研究了香芹酚精油、肉桂精油和百里香精油对两种病原菌的抑制效果,发现香芹酚精油表现出较强的抑菌性。无论直接接触还是熏蒸方式,香芹酚精油对层生镰刀菌和平头炭疽菌的抑菌效果均优于肉桂精油和百里香精油。精油熏蒸处理使接种层生镰刀菌和平头炭疽菌火龙果腐烂率下降,香芹酚精油的效果最优,可作为火龙果贮藏前的熏蒸剂。研究结果可为浙江地区火龙果贮藏期腐烂病害的预防和控制提供一定的参考依据。
Red pitaya fruit is highly susceptible to fungal infection during postharvest,causing quality deterioration and huge economic losses. This research was intended to isolate,purify and identify the pathogens which cause rotting of red pitaya during storage in Zhejiang Province,screen essential oils for controlling the pathogenic isolates, and test their effectiveness. HS3 and HS6 were identified as pathogens through isolation,purification and pathogenicity tests. These two strains were proved as F. proliferatum and C. fructicola by morphological identification and r DNA-ITS molecular identification.Six essential oils were investigated for their activity to control these pathogens. The result indicated that carvacrol,cinnamon and thyme oil showed complete inhibitory effect on all pathogens at a concentration of 2 000 μL/L. Then,the inhibitory effects of carcanol,cinnamon and thyme oil on these two pathogens were studied by direct contact in vitro and in vitro fumigation. It was found that carcanol showed higher antifungal activity against F. proliferatum and C. fructicola than cinnamon and thyme in the drug sensitivity test. Moreover,essential oil fumigation inhibited the decay incidence of pitaya fruit inoculated with F. proliferatum and C. fructicola during storage at 4℃ and carcanol showed the best effect.Carcanol oil seemed to be a promising potential fumigant. The results can provide reference for disease control during storage of red pitaya in Zhejiang Province.
Red pitaya fruit is highly susceptible to fungal infection during postharvest,causing quality deterioration and huge economic losses. This research was intended to isolate,purify and identify the pathogens which cause rotting of red pitaya during storage in Zhejiang Province,screen essential oils for controlling the pathogenic isolates, and test their effectiveness. HS3 and HS6 were identified as pathogens through isolation,purification and pathogenicity tests. These two strains were proved as F. proliferatum and C. fructicola by morphological identification and r DNA-ITS molecular identification.Six essential oils were investigated for their activity to control these pathogens. The result indicated that carvacrol,cinnamon and thyme oil showed complete inhibitory effect on all pathogens at a concentration of 2 000 μL/L. Then,the inhibitory effects of carcanol,cinnamon and thyme oil on these two pathogens were studied by direct contact in vitro and in vitro fumigation. It was found that carcanol showed higher antifungal activity against F. proliferatum and C. fructicola than cinnamon and thyme in the drug sensitivity test. Moreover,essential oil fumigation inhibited the decay incidence of pitaya fruit inoculated with F. proliferatum and C. fructicola during storage at 4℃ and carcanol showed the best effect.Carcanol oil seemed to be a promising potential fumigant. The results can provide reference for disease control during storage of red pitaya in Zhejiang Province.
引文
1 WICHIENCHOT S,JATUPORNPIPAT M,RASTALL R A.Oligosaccharides of pitaya(dragon fruit)flesh and their prebiotic properties[J].Food Chemistry,2010,120(3):850-857.
2 WU L,HSU H,CHEN Y,et al.Antioxidant and antiproliferative activities of red pitaya[J].Food Chemistry,2006,95(2):319-327.
3 DEMBITSKY V M,POOVARODOM S,LEONTOWICZ H,et al.The multiple nutrition properties of some exotic fruits:biological activity and active metabolites[J].Food Research International,2011,44(7):1671-1701.
4 SONG H,CHU Q,YAN F,et al.Red pitaya betacyanins protects from diet-induced obesity,liver steatosis and insulin resistance in association with modulation of gut microbiota in mice[J].Journal of Gastroenterology and Hepatology,2016,31(8):1462-1469.
5 STINTZING F C,SCHIEBER A,CARLE R.Betacyanins in fruits from red-purple pitaya:Hylocereus polyrhizus(Weber)Britton&Rose[J].Food Chemistry,2002,77:101-106.
6朱迎迎,陈亮,祝庆刚,等.火龙果采后病害与防控技术研究进展[J].中国热带农业,2014(4):55-58.ZHU Y Y,CHEN L,ZHU Q G,et al.Progress in post-harvest disease and preservative technology of pitaya[J].China Tropical Agriculture,2014(4):55-58.(in Chinese)
7 MA T,YANG B,YU Y,et al.Market disease pathogens detection of imported fruits in Shanghai[J].Agricultural Sciences in China,2009(8):1087-1096.
8 GUO L,WU Y,HO H,et al.First report of dragon fruit(Hylocereus undatus)anthracnose caused by Colletotrichum truncatum in China[J].Journal of Phytopathology,2014,162(4):272-275.
9姚昇华,范诗睿,邢云莱,等.越南‘红心’火龙果黑腐病病原真菌鉴定及环境因素影响分析[J].植物生理学报,2015,51(9):1419-1424.YAO S H,FAN S R,XING Y L,et al.Identification of black fungus black rot in red pitaya and effects of environmental factors[J].Journal of Plant Physiology,2015,51(9):1419-1424.(in Chinese)
10王生有,陈于陇,徐玉娟,等.火龙果采后生理和保鲜技术的研究进展[J].食品工业科技,2014,35(13):396-400.WANG S Y,CHEN Y L,XU Y J,et al.Progress in postharvest physiology and preservative technology of pitaya[J].Science and Technology of Food Industry,2014,35(13):396-400.(in Chinese)
11王彬,郑伟,何绪晓,等.1-MCP对火龙果果实采后生理特性的影响[J].西南农业学报,2011,24(6):2127-2131.WANG B,ZHENG W,HE X X,et al.Effects of 1-Methylcyclopropene fumigation treatment on physiological characteristics of pitaya fruits during storage period[J].Southwest China Journal of Agricultural Sciences,2011,24(6):2127-2131.(in Chinese)
12王彬,郑伟,何绪晓,等.Ca Cl2处理对火龙果低温贮藏期品质的影响[J].西南农业学报,2010,23(3):836-840.WANG B,ZHENG W,HE X X,et al.Effect of Ca Cl2on quality of pitaya fruits under cold storage[J].Southwest China Journal of Agricultural Sciences,2010,23(3):836-840.(in Chinese)
13张绿萍,金吉林,邓仁菊.保鲜剂及保鲜方式对火龙果贮藏时间的影响[J].贵州农业科学,2011,39(5):215-217.ZHANG L P,JIN J L,DENG R J.Effect of antistaling agent and preservation method on pitaya during storage[J].Guizhou Agricultural Sciences,2011,39(5):215-217.(in Chinese)
14冯武.植物精油对果蔬采后病害的防治及其防治机理研究[D].杭州:浙江大学,2006.FENG W.Study on prevention of postharvest diseases of fruits and vegetables with plant essential oil and its control mechanism[D].Hangzhou:Zhejiang University,2006.(in Chinese)
15 BURT S.Essential oils:their ant in foods-a review[J].International Journal of Food Microbiology,2004,94:223-253.
16 SIVAKUMAR D,BAUTISTA-BAOS S.A review on the use of essential oils for postharvest decay control and maintenance of fruit quality during storage[J].Crop Protection,2014,64(3):27-37.
17吴新,金鹏,孔繁渊.植物精油对草莓果实腐烂和品质的影响[J].食品科学,2011,32(14):323-326.WU X,JIN P,KONG F Y.Effects of plant essential oils on strawberry rotting and quality[J].Food Science,2011,32(14):323-326.(in Chinese)
18魏景超.真菌鉴定手册[M].上海:上海科学技术出版社,1979.
19郜海燕,肖尚月,陈杭君,等.蓝莓采后主要病原真菌的分离鉴定与生物学特性研究[J/OL].农业机械学报,2017,48(5):327-334.http:∥www.j-csam.org/jcsam/ch/reader/view_abstract.aspx?file_no=20170541&flag=1.DOI:10.6041/j.issn.1000-1298.2017.05.041.GAO H Y,XIAO S Y,CHEN H J,et al.Identification and biological characteristics of fungal pathogens in blueberry during storage[J/OL].Transactions of the Chinese Society for Agricultural Machinery,2017,48(5):327-334.(in Chinese)
20 LUO G,MITCHELL T G.Rapid identification of pathogenic fungi directly from cultures by using multiplex PCR[J].Journal of Clinical Microbiology,2002,40:2860-2865.
21 TAMURA K,DUDLEY J,NEI M,et al.MEGA4:molecular evolutionary genetics analysis(MEGA)software version 4.0[J].Molecular Biology and Evolution,2007,24:1596-1599.
22杨帅,王健生,马振川,等.引致大豆根腐病的层出镰孢菌分离鉴定及其特性[J].植物保护学报,2012,39(2):187-188.YANG S,WANG J S,MA Z C,et al.Isolation and identification of Fusarium proliferatum causing soybean root rot and its biological characterization[J].Acta Phytophylacica Sinica,2012,39(2):187-188.(in Chinese)
23吴婧波,詹儒林,柳凤,等.芒果畸形病病原菌Fusarium mangiferae的快速分子检测[J].菌物学报,2016,35(3):298-308.WU J B,ZHAN R L,LIU F,et al.Rapid molecular detection of mango malformation Fusarium mangiferae[J].Mycosystema,2016,35(3):298-308.(in Chinese)
24崔志婧,王奕文,于岳.上海市进口火龙果软腐病病害分析[J].微生物学通报,2011,38(10):1499-1506.CUI Z J,WANG Y W,YU Y.Pathogens analysis of soft rot disease of imported pitaya in Shanghai[J].Microbiology,2011,38(10):1499-1506.(in Chinese)
25朱迎迎,高兆银,李敏,等.火龙果镰刀菌果腐病病原菌鉴定及生物学特性研究[J].热带作物学报,2016,37(1):164-171.ZHU Y Y,GAO Z Y,LI M,et al.Identification and biological characteristics of dragon fruit(Hylocereus undatus Britt)fusarium rot pathogen[J].Journal of Tropical Crops,2016,37(1):164-171.(in Chinese)
26朱迎迎,李敏,高兆银,等.火龙果炭疽病病原菌的鉴定及生物学特性研究[J].南方农业学报,2016,47(1):59-66.ZHU Y Y,LI M,GAO Z Y,et al.Identification and biological characteristics of anthracnose pathogen from pitaya(Hylocereus undulatus)[J].Journal of Southern Agriculture,2016,47(1):59-66.(in Chinese)
27周丹丹,王卓,邢梦珂,等.植物精油抑制炭疽菌及对枇杷采后炭疽病与品质的影响[J].食品科学,2017,38(19):212-217.ZHOU D D,WANG Z,XING M K,et al.Inhibitory effect of plant essential oils on colletorichum acutatum and postharvest anthracnose and quality of loquat fruits[J].Food Science,2017,38(19):212-217.(in Chinese)
28李素清,何靖柳,秦文.几种植物精油对红阳猕猴桃中两种致病菌抑制效果的研究[J].四川农业大学学报,2014,32(3):315-320.LI S Q,HE J L,QIN W.Inhibitory effects of several plant essential oils against two pathogens of‘red sun’kiwifruit[J].Journal of Sichuan Agricultural University,2014,32(3):315-320.(in Chinese)
29刘继,颜静,何靖柳,等.生姜贮藏期病原菌分离鉴定及精油抑菌效果[J].食品科学,2014,35(18):172-177.LIU J,YAN J,HE J L,et al.Isolation,identification and inhibition of pathogens from mature ginger during storage[J].Food Science,2014,35(18):172-177.(in Chinese)
30 TEPE B,DAFERERA D,SOKMEN A,et al.Antimicrobial and antioxidant activities of the essential oil and various extracts of Salvia tomentosa Miller(Lamiaceae)[J].Food Chemistry,2005,90(3):333-340.
31 MIGUEL M G.Antioxidant and anti-inflammatory activities of essential oils:a short review[J].Molecules,2010,15(12):9252-9287.
2 WU L,HSU H,CHEN Y,et al.Antioxidant and antiproliferative activities of red pitaya[J].Food Chemistry,2006,95(2):319-327.
3 DEMBITSKY V M,POOVARODOM S,LEONTOWICZ H,et al.The multiple nutrition properties of some exotic fruits:biological activity and active metabolites[J].Food Research International,2011,44(7):1671-1701.
4 SONG H,CHU Q,YAN F,et al.Red pitaya betacyanins protects from diet-induced obesity,liver steatosis and insulin resistance in association with modulation of gut microbiota in mice[J].Journal of Gastroenterology and Hepatology,2016,31(8):1462-1469.
5 STINTZING F C,SCHIEBER A,CARLE R.Betacyanins in fruits from red-purple pitaya:Hylocereus polyrhizus(Weber)Britton&Rose[J].Food Chemistry,2002,77:101-106.
6朱迎迎,陈亮,祝庆刚,等.火龙果采后病害与防控技术研究进展[J].中国热带农业,2014(4):55-58.ZHU Y Y,CHEN L,ZHU Q G,et al.Progress in post-harvest disease and preservative technology of pitaya[J].China Tropical Agriculture,2014(4):55-58.(in Chinese)
7 MA T,YANG B,YU Y,et al.Market disease pathogens detection of imported fruits in Shanghai[J].Agricultural Sciences in China,2009(8):1087-1096.
8 GUO L,WU Y,HO H,et al.First report of dragon fruit(Hylocereus undatus)anthracnose caused by Colletotrichum truncatum in China[J].Journal of Phytopathology,2014,162(4):272-275.
9姚昇华,范诗睿,邢云莱,等.越南‘红心’火龙果黑腐病病原真菌鉴定及环境因素影响分析[J].植物生理学报,2015,51(9):1419-1424.YAO S H,FAN S R,XING Y L,et al.Identification of black fungus black rot in red pitaya and effects of environmental factors[J].Journal of Plant Physiology,2015,51(9):1419-1424.(in Chinese)
10王生有,陈于陇,徐玉娟,等.火龙果采后生理和保鲜技术的研究进展[J].食品工业科技,2014,35(13):396-400.WANG S Y,CHEN Y L,XU Y J,et al.Progress in postharvest physiology and preservative technology of pitaya[J].Science and Technology of Food Industry,2014,35(13):396-400.(in Chinese)
11王彬,郑伟,何绪晓,等.1-MCP对火龙果果实采后生理特性的影响[J].西南农业学报,2011,24(6):2127-2131.WANG B,ZHENG W,HE X X,et al.Effects of 1-Methylcyclopropene fumigation treatment on physiological characteristics of pitaya fruits during storage period[J].Southwest China Journal of Agricultural Sciences,2011,24(6):2127-2131.(in Chinese)
12王彬,郑伟,何绪晓,等.Ca Cl2处理对火龙果低温贮藏期品质的影响[J].西南农业学报,2010,23(3):836-840.WANG B,ZHENG W,HE X X,et al.Effect of Ca Cl2on quality of pitaya fruits under cold storage[J].Southwest China Journal of Agricultural Sciences,2010,23(3):836-840.(in Chinese)
13张绿萍,金吉林,邓仁菊.保鲜剂及保鲜方式对火龙果贮藏时间的影响[J].贵州农业科学,2011,39(5):215-217.ZHANG L P,JIN J L,DENG R J.Effect of antistaling agent and preservation method on pitaya during storage[J].Guizhou Agricultural Sciences,2011,39(5):215-217.(in Chinese)
14冯武.植物精油对果蔬采后病害的防治及其防治机理研究[D].杭州:浙江大学,2006.FENG W.Study on prevention of postharvest diseases of fruits and vegetables with plant essential oil and its control mechanism[D].Hangzhou:Zhejiang University,2006.(in Chinese)
15 BURT S.Essential oils:their ant in foods-a review[J].International Journal of Food Microbiology,2004,94:223-253.
16 SIVAKUMAR D,BAUTISTA-BAOS S.A review on the use of essential oils for postharvest decay control and maintenance of fruit quality during storage[J].Crop Protection,2014,64(3):27-37.
17吴新,金鹏,孔繁渊.植物精油对草莓果实腐烂和品质的影响[J].食品科学,2011,32(14):323-326.WU X,JIN P,KONG F Y.Effects of plant essential oils on strawberry rotting and quality[J].Food Science,2011,32(14):323-326.(in Chinese)
18魏景超.真菌鉴定手册[M].上海:上海科学技术出版社,1979.
19郜海燕,肖尚月,陈杭君,等.蓝莓采后主要病原真菌的分离鉴定与生物学特性研究[J/OL].农业机械学报,2017,48(5):327-334.http:∥www.j-csam.org/jcsam/ch/reader/view_abstract.aspx?file_no=20170541&flag=1.DOI:10.6041/j.issn.1000-1298.2017.05.041.GAO H Y,XIAO S Y,CHEN H J,et al.Identification and biological characteristics of fungal pathogens in blueberry during storage[J/OL].Transactions of the Chinese Society for Agricultural Machinery,2017,48(5):327-334.(in Chinese)
20 LUO G,MITCHELL T G.Rapid identification of pathogenic fungi directly from cultures by using multiplex PCR[J].Journal of Clinical Microbiology,2002,40:2860-2865.
21 TAMURA K,DUDLEY J,NEI M,et al.MEGA4:molecular evolutionary genetics analysis(MEGA)software version 4.0[J].Molecular Biology and Evolution,2007,24:1596-1599.
22杨帅,王健生,马振川,等.引致大豆根腐病的层出镰孢菌分离鉴定及其特性[J].植物保护学报,2012,39(2):187-188.YANG S,WANG J S,MA Z C,et al.Isolation and identification of Fusarium proliferatum causing soybean root rot and its biological characterization[J].Acta Phytophylacica Sinica,2012,39(2):187-188.(in Chinese)
23吴婧波,詹儒林,柳凤,等.芒果畸形病病原菌Fusarium mangiferae的快速分子检测[J].菌物学报,2016,35(3):298-308.WU J B,ZHAN R L,LIU F,et al.Rapid molecular detection of mango malformation Fusarium mangiferae[J].Mycosystema,2016,35(3):298-308.(in Chinese)
24崔志婧,王奕文,于岳.上海市进口火龙果软腐病病害分析[J].微生物学通报,2011,38(10):1499-1506.CUI Z J,WANG Y W,YU Y.Pathogens analysis of soft rot disease of imported pitaya in Shanghai[J].Microbiology,2011,38(10):1499-1506.(in Chinese)
25朱迎迎,高兆银,李敏,等.火龙果镰刀菌果腐病病原菌鉴定及生物学特性研究[J].热带作物学报,2016,37(1):164-171.ZHU Y Y,GAO Z Y,LI M,et al.Identification and biological characteristics of dragon fruit(Hylocereus undatus Britt)fusarium rot pathogen[J].Journal of Tropical Crops,2016,37(1):164-171.(in Chinese)
26朱迎迎,李敏,高兆银,等.火龙果炭疽病病原菌的鉴定及生物学特性研究[J].南方农业学报,2016,47(1):59-66.ZHU Y Y,LI M,GAO Z Y,et al.Identification and biological characteristics of anthracnose pathogen from pitaya(Hylocereus undulatus)[J].Journal of Southern Agriculture,2016,47(1):59-66.(in Chinese)
27周丹丹,王卓,邢梦珂,等.植物精油抑制炭疽菌及对枇杷采后炭疽病与品质的影响[J].食品科学,2017,38(19):212-217.ZHOU D D,WANG Z,XING M K,et al.Inhibitory effect of plant essential oils on colletorichum acutatum and postharvest anthracnose and quality of loquat fruits[J].Food Science,2017,38(19):212-217.(in Chinese)
28李素清,何靖柳,秦文.几种植物精油对红阳猕猴桃中两种致病菌抑制效果的研究[J].四川农业大学学报,2014,32(3):315-320.LI S Q,HE J L,QIN W.Inhibitory effects of several plant essential oils against two pathogens of‘red sun’kiwifruit[J].Journal of Sichuan Agricultural University,2014,32(3):315-320.(in Chinese)
29刘继,颜静,何靖柳,等.生姜贮藏期病原菌分离鉴定及精油抑菌效果[J].食品科学,2014,35(18):172-177.LIU J,YAN J,HE J L,et al.Isolation,identification and inhibition of pathogens from mature ginger during storage[J].Food Science,2014,35(18):172-177.(in Chinese)
30 TEPE B,DAFERERA D,SOKMEN A,et al.Antimicrobial and antioxidant activities of the essential oil and various extracts of Salvia tomentosa Miller(Lamiaceae)[J].Food Chemistry,2005,90(3):333-340.
31 MIGUEL M G.Antioxidant and anti-inflammatory activities of essential oils:a short review[J].Molecules,2010,15(12):9252-9287.