超声波处理对稻谷表面微生物的影响
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摘要
【目的】考察超声波处理对种谷表面真菌与细菌的影响,比较不同处理方式的杀菌效果,分析影响超声波杀菌作用的因素以及超声波促进稻谷萌发与壮秧的原因。【方法】以水稻品种象牙香占的种谷为材料,设25～50 kHz变频超声波湿法(T_(wet)),50 kHz超声波干法(T_(dry1))和25 kHz超声波干法(T_(dry2))3个处理,以常规湿法(CK1)与干法(CK2)为对照,3次重复。采用PDA平板涂布法分离种谷表面微生物,结合菌落形态及菌体显微结构,以及ITS序列同源性分析,鉴定真菌与细菌。【结果】超声波处理对种谷有杀菌作用,其中超声波湿法处理(T_(wet))优于干法处理,而50 kHz超声波干法处理(T_(dry1))优于25 kHz超声波干法处理(T_(dry2))。检测到的15种种谷表面细菌中,超声波对甘蔗黄单胞菌Xanthomonas sacchari、假单胞菌Pseudomonas aeruginosa、粘金黄杆菌Chryseobacterium gleum、成团泛菌Pantoea agglomerans、嗜麦芽窄食单胞菌Stenotrophomonas maltophilia等丰度较低的细菌杀菌效果较好,但对阴沟肠杆菌Enterobacter cloacae和肺炎克雷伯菌Klebsiella pneumoniae等高丰度细菌杀菌效果较差。从稻谷表面检测到11种真菌,超声波对层生镰刀菌Fusarium proliferatum、尖孢镰刀菌F. oxysporum、串珠镰刀菌F. moniliforme,木贼镰刀菌F. equiseti与茎点霉菌Phoma sp.等杀菌效果较好,而对稻叶鞘腐败病菌Sarocladium oryzae及草酸青霉菌Penicillium oxalicum等杀菌效果不显著,可能是由于后者丰度太高所致杀菌效果偏低。【结论】超声波处理对水稻种谷表面微生物具有杀菌作用,但其效果与超声波处理方式、微生物种类及其丰度有关,超声波湿法处理杀菌效果比干法处理更好,超声波对低丰度的镰孢菌类具有显著杀菌效果,可能是促进水稻萌发与壮秧的原因之一。
【Objective】Ultrasound wave(USW) treatment of rice seeds can promote seed germination and seedling growth, but its bactericidal effect is not clear. In this study, we intend to investigate the effects of USW on fungi and bacteria on the surface of rice seeds, and to evaluate the antimicrobial efficacy of different USW treatments and the influencing factors. We also try to analyze the reasons of the promotion of rice germination and seedling growth by USW. 【Methods】Using the rice cultivar, Xiangyaxiangzhan, as material, three treatments, 25-50 kHz USW _(wet) method(T_(wet)), 50 kHz USW drying method(T_(dry1)) and 25 kHz USW drying method(T_(dry2)), were set up. The conventional _(wet) method(CK1) and dry method(CK2) were taken as controls. The experiments were repeated three times. Microorganisms on the seed surfaces were separated by PDA plates. The fungi and bacteria were identified by combining colony features, microscopic structures and ITS sequences analysis. 【Results】USW had substantial bactericidal and fungicidal effects on seed surfaces. _(Wet) treatment of USW(T_(wet)) was better than dry treatment(T_(dry1), T_(dry2)), and the 50 kHz USW(T_(dry1)) was better than 25-kHz USW(T_(dry2)). Among the 15 species of bacteria detected on seed surfaces, USW had the significant bactericidal effects on low abundance bacterial species such as Xanthomonas sacchari, Pseudomonas aeruginosa, Chryseobacterium gleum, Pantoea agglomerans and Stenotrophomo maltophilia, but USW had not effects on the higher abundance bacteria such as Enterobacter cloacae and Klebsiella pneumoniae. Eleven fungi were detected on the seed surfaces, USW had better fungicidal effects on Fusarium proliferatum, F. oxysporum, F. moniliforme, F. equiseti and Phoma sp. But USW had not apparent effects on Sarocladium oryzae and Penicillium oxalicum with the relatively higher richness. 【Conclusion】USW has antimicrobial effects on rice seed surfaces, but the effects are related to the treatment ways of USW, microorganism species and their abundance. USW treatment on _(wet) seeds has better bactericidal and fungicidal effects than those of USW treatment on dry seeds. USW has significant fungicidal effects on Fusarium spp with low abundance, which may be one of the reasons for promoting rice germination and seedling growth.
【Objective】Ultrasound wave(USW) treatment of rice seeds can promote seed germination and seedling growth, but its bactericidal effect is not clear. In this study, we intend to investigate the effects of USW on fungi and bacteria on the surface of rice seeds, and to evaluate the antimicrobial efficacy of different USW treatments and the influencing factors. We also try to analyze the reasons of the promotion of rice germination and seedling growth by USW. 【Methods】Using the rice cultivar, Xiangyaxiangzhan, as material, three treatments, 25-50 kHz USW _(wet) method(T_(wet)), 50 kHz USW drying method(T_(dry1)) and 25 kHz USW drying method(T_(dry2)), were set up. The conventional _(wet) method(CK1) and dry method(CK2) were taken as controls. The experiments were repeated three times. Microorganisms on the seed surfaces were separated by PDA plates. The fungi and bacteria were identified by combining colony features, microscopic structures and ITS sequences analysis. 【Results】USW had substantial bactericidal and fungicidal effects on seed surfaces. _(Wet) treatment of USW(T_(wet)) was better than dry treatment(T_(dry1), T_(dry2)), and the 50 kHz USW(T_(dry1)) was better than 25-kHz USW(T_(dry2)). Among the 15 species of bacteria detected on seed surfaces, USW had the significant bactericidal effects on low abundance bacterial species such as Xanthomonas sacchari, Pseudomonas aeruginosa, Chryseobacterium gleum, Pantoea agglomerans and Stenotrophomo maltophilia, but USW had not effects on the higher abundance bacteria such as Enterobacter cloacae and Klebsiella pneumoniae. Eleven fungi were detected on the seed surfaces, USW had better fungicidal effects on Fusarium proliferatum, F. oxysporum, F. moniliforme, F. equiseti and Phoma sp. But USW had not apparent effects on Sarocladium oryzae and Penicillium oxalicum with the relatively higher richness. 【Conclusion】USW has antimicrobial effects on rice seed surfaces, but the effects are related to the treatment ways of USW, microorganism species and their abundance. USW treatment on _(wet) seeds has better bactericidal and fungicidal effects than those of USW treatment on dry seeds. USW has significant fungicidal effects on Fusarium spp with low abundance, which may be one of the reasons for promoting rice germination and seedling growth.
引文
[1]闵红,李好海,赵利民.河南省种子处理技术的发展及展望[J].中国植保导刊,2019,39(2):92-94.MING H,LI H H,ZHAO L M.Development and prospect of seed processing technology in Henan Province[J].China Plant Protection,2019,39(2):92-94.
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[3]肖宜安,李化茂,冯若.超声辐照对苏铁种子萌发的影响[J].植物生理学通讯,1999,35(4):293.doi:10.13592/j.cnki.ppj.1999.04.009.XIAO Y A,LI H M,FENG R.Effect of ultrasound irradiation on cycad seed germination[J].Plant Physiological Communication,1999,35(4):293.doi:10.13592/j.cnki.ppj.1999.04.009.
[4]袁畹兰.超声处理对冬小麦生长发育和增产的研究总结[J].陕西师大学报(自然科学版),1978(1):83-93.doi:10.15983/j.cnki.jsnu.1978.01.007.YUAN W L.Summary of research on growth and yield increase of winter wheat by ultrasound treatment[J].Journal of Shaanxi Normal University(Natural Science Edition),1978(1):83-93.doi:10.15983/j.cnki.jsnu.1978.01.007.
[5]武睿,郭晔红,萧明明,郭云云,袁佩举.超声波处理对锁阳种子萌发特性影响研究[J].甘肃农业大学学报,2010,45(6):122-124.doi:10.39669/j.issn.1003-4315.2010.06.017.WU R,GUO Y H,XIAO M M,GUO Y Y,YUAN P J.Study on the effect of ultrasound treatment on germination characteristics of Cynomorium songaricum seeds[J].Journal of Gansu Agricultural University,2010,45(6):122-124.doi:10.39669/j.issn.1003-4315.2010.06.017.
[6]赵燕,杨青松,王莹,陈毅坚,罗万玲,刘云华.不同时间超声波处理对油菜种子萌发的影响[J].种子,2012,31(10):90-92.doi:10.3969/j.issn.1001-4705.2012.10.025.ZHAO Y,YANG Q S,WANG Y,CHEN Y J,LUO W L,LIU Y H.Effects of different time ultrasound treatment on seed germination of rapeseed[J].Seeds,2012,31(10):90-92.doi:10.3969/j.issn.1001-4705.2012.10.025.
[7]林海.国槐种子超声波处理效应研究[J].北方园艺,2011(20):51-54.LIN,H..Study on the effect of Sophora japonica seeds treated by ultrasound[J].Northern Horticulture,2011(20):51-54.
[8]杨葵.超声波技术试验增产效果明显[J].现代农业装备,2017(4):75.YANG K.The effect of increasing production by ultrasonic technology test is obvious[J].Modern Agricultural Equipment,2017(4):75.
[9]张思聪,孔雷蕾,唐湘如.超声波预处理对作物种子及幼苗的影响综述[J].安徽农业科学,2017,45(21):11-12.doi:10.13989/j.cnki.0517-6611.2017.21.005.ZHANG S C,KONG L L,TANG X R.Summary of the effects of ultrasound pretreatment on crop seeds and seedlings[J].Journal of Anhui Agricultural Sciences,2017,45(21):11-12.doi:10.13989/j.cnki.0517-6611.2017.21.005.
[10]刘西莉,李健强,朱春雨,张国珍,张龙,罗军,李小林,朱建军,房双龙.不同水稻品种种子带菌检测及药剂消毒处理效果[J].中国农业大学学报,2000(5):42-47.doi:10.3321/j.issn:1007-4333.2000.05.008.LIU X L,LI J Q,ZHU C Y,ZHANG G Z,ZHANG L,LUO J,LI XL,ZHU J J,FANG S L.Detection of bacteria in seeds of different rice varieties and effect of disinfection with drugs[J].Journal of China Agricultural University,2000(5):42-47.doi:10.3321/j.issn:1007-4333.2000.05.008.
[11]刘烨,赵兴红,韩雨桐,张琳淋,张俊华.黑龙江省水稻立枯病病原鉴定及致病性研究[J].吉林农业科学,2015,40(5):75-78,84.doi:10.16423/j.cnki.1003-8701.2015.05.019.LIU Y,ZHAO X H,HAN Y T,ZHANG L L,ZHANG J H.Identification and pathogenicity of rice standing blight in Heilongjiang Province[J].Jilin Agricultural Science,2015,40(5):75-78,84.doi:10.16423/j.cnki.1003-8701.2015.05.019.
[12]尚毅,方伟,廖万清.中国多变根毛霉感染性皮肤接合菌病13例回顾性分析[J].中国真菌学杂志,2013,8(1):30-34.doi:10.3969/j.issn.1673-3827.2013.01.007.SHANG Y,FANG W,LIAO W Q.Retrospective analysis of 13 cases of infectious dermatoconjunctivitis caused by Rhizopus multiformis in China[J].Chinese Journal of Mycology,2013,8(1):30-34.doi:10.3969/j.issn.1673-3827.2013.01.007.
[13]罗宽,廖小兰,陈寊.水稻褐鞘病研究Ⅴ.稻褐鞘病菌与其它病原的关系[J].湖南农学院学报,1991(3):471-476.doi:10.13331/j.cnki.jhau.1991.03.008.LUO K,LIAO X L,CHEN Z.Studies on rice brown sheath v.relationship between brown sheath fungi and other pathogens[J].Journal of Hunan Agricultural College,1991(3):471-476.doi:10.13331/j.cnki.jhau.1991.03.008.
[14]ZHANG R Y,SHAN H L,LI W F,WANG X Y,YIN J,LUO Z M,HUANG Y K.First report of sugarcane leaf chlorotic streak disease caused by Xanthomonas sacchari in Guangxi,China[J].Plant Disease,2017,101(8):1541-1542.doi:10.1094/PDIS-12-16-1774-PDN.
[15]MAROON L C J,SCHNEIDER K L,TURNER R.S,PRESTING G G,ALVAREZ A M.Xanthomonas sacchari-a pathogen or an endophyte[J].Phytopathology,2010,100(6):S78.
[16]FANG X Y,LIN H Y,WU L W,REN D Y,YE W J,DONG G J,ZHU L,GUO L B.Genome sequence of Xanthomonas sacchari R1,a biocontrol bacterium isolated from the rice seed[J].Journal of Biotechnology,2015,206:77-78.doi:10.1016/j.jbiotec.2015.04.014.
[17]唐敏,李丽,肖蓉.嗜麦芽寡养单胞菌胞外蛋白酶功能研究进展[J].微生物学报,2016,56(5):731-739.doi:10.13343/j.cnki.wsxb.20150289.TANG M,LI L,XIAO R.Research progress on extracellular protease function of Stenotrophomonas maltophilia[J].Journal of Microbiology,2016,56(5):731-739.doi:10.13343/j.cnki.wsxb.20150289.
[18]NEI T,SONOBE K,ONODERA A,ITABASHI T,YAMAGUCHI H,MAEDA M,SAITO R.Two cases with bacteremia suspected to be due to relatively rare Pseudomonas(Flavimonas)oryzihabitans[J].Journal of Infection and Chemotherapy,2015,21(10):751-755.doi:10.1016/j.jiac.2015.06.005.
[19]TENA D,FEMANDEZ C.Pseudomonas oryzihabitans:an unusual cause of skin and soft tissue infection[J].Infectious Diseases,2015,47(11):820-824.doi:10.3109/2374423.2015.1034170.
[20]冯婷婷,王佳贺.阴沟肠杆菌感染与耐药机制的研究进展[J].中国人兽共患病学报,2017,33(10):933-937.doi:10.3969/j.issn.1002-2694.2017.10.018.FENG T T,WANG J H.Advances in the study of Enterobacter cloacae infection and drug resistance mechanisms[J].Chinese Journal of Zoonoses,2017,33(10):933-937.doi:10.3969/j.issn.1002-2694.2017.10.018.
[2]袁经天,严锦璇,汪德锋,曹明,柯用春.超声波处理对水稻种子萌发,产量及产量构成的影响[J].中国稻米,2014(2):53-55.YUAN J T,YAN J X,WANG D F,CAO M,KE Y C.Effects of ultrasound treatment on germination,yield and yield composition of rice seeds[J].China Rice,2014(2):53-55.
[3]肖宜安,李化茂,冯若.超声辐照对苏铁种子萌发的影响[J].植物生理学通讯,1999,35(4):293.doi:10.13592/j.cnki.ppj.1999.04.009.XIAO Y A,LI H M,FENG R.Effect of ultrasound irradiation on cycad seed germination[J].Plant Physiological Communication,1999,35(4):293.doi:10.13592/j.cnki.ppj.1999.04.009.
[4]袁畹兰.超声处理对冬小麦生长发育和增产的研究总结[J].陕西师大学报(自然科学版),1978(1):83-93.doi:10.15983/j.cnki.jsnu.1978.01.007.YUAN W L.Summary of research on growth and yield increase of winter wheat by ultrasound treatment[J].Journal of Shaanxi Normal University(Natural Science Edition),1978(1):83-93.doi:10.15983/j.cnki.jsnu.1978.01.007.
[5]武睿,郭晔红,萧明明,郭云云,袁佩举.超声波处理对锁阳种子萌发特性影响研究[J].甘肃农业大学学报,2010,45(6):122-124.doi:10.39669/j.issn.1003-4315.2010.06.017.WU R,GUO Y H,XIAO M M,GUO Y Y,YUAN P J.Study on the effect of ultrasound treatment on germination characteristics of Cynomorium songaricum seeds[J].Journal of Gansu Agricultural University,2010,45(6):122-124.doi:10.39669/j.issn.1003-4315.2010.06.017.
[6]赵燕,杨青松,王莹,陈毅坚,罗万玲,刘云华.不同时间超声波处理对油菜种子萌发的影响[J].种子,2012,31(10):90-92.doi:10.3969/j.issn.1001-4705.2012.10.025.ZHAO Y,YANG Q S,WANG Y,CHEN Y J,LUO W L,LIU Y H.Effects of different time ultrasound treatment on seed germination of rapeseed[J].Seeds,2012,31(10):90-92.doi:10.3969/j.issn.1001-4705.2012.10.025.
[7]林海.国槐种子超声波处理效应研究[J].北方园艺,2011(20):51-54.LIN,H..Study on the effect of Sophora japonica seeds treated by ultrasound[J].Northern Horticulture,2011(20):51-54.
[8]杨葵.超声波技术试验增产效果明显[J].现代农业装备,2017(4):75.YANG K.The effect of increasing production by ultrasonic technology test is obvious[J].Modern Agricultural Equipment,2017(4):75.
[9]张思聪,孔雷蕾,唐湘如.超声波预处理对作物种子及幼苗的影响综述[J].安徽农业科学,2017,45(21):11-12.doi:10.13989/j.cnki.0517-6611.2017.21.005.ZHANG S C,KONG L L,TANG X R.Summary of the effects of ultrasound pretreatment on crop seeds and seedlings[J].Journal of Anhui Agricultural Sciences,2017,45(21):11-12.doi:10.13989/j.cnki.0517-6611.2017.21.005.
[10]刘西莉,李健强,朱春雨,张国珍,张龙,罗军,李小林,朱建军,房双龙.不同水稻品种种子带菌检测及药剂消毒处理效果[J].中国农业大学学报,2000(5):42-47.doi:10.3321/j.issn:1007-4333.2000.05.008.LIU X L,LI J Q,ZHU C Y,ZHANG G Z,ZHANG L,LUO J,LI XL,ZHU J J,FANG S L.Detection of bacteria in seeds of different rice varieties and effect of disinfection with drugs[J].Journal of China Agricultural University,2000(5):42-47.doi:10.3321/j.issn:1007-4333.2000.05.008.
[11]刘烨,赵兴红,韩雨桐,张琳淋,张俊华.黑龙江省水稻立枯病病原鉴定及致病性研究[J].吉林农业科学,2015,40(5):75-78,84.doi:10.16423/j.cnki.1003-8701.2015.05.019.LIU Y,ZHAO X H,HAN Y T,ZHANG L L,ZHANG J H.Identification and pathogenicity of rice standing blight in Heilongjiang Province[J].Jilin Agricultural Science,2015,40(5):75-78,84.doi:10.16423/j.cnki.1003-8701.2015.05.019.
[12]尚毅,方伟,廖万清.中国多变根毛霉感染性皮肤接合菌病13例回顾性分析[J].中国真菌学杂志,2013,8(1):30-34.doi:10.3969/j.issn.1673-3827.2013.01.007.SHANG Y,FANG W,LIAO W Q.Retrospective analysis of 13 cases of infectious dermatoconjunctivitis caused by Rhizopus multiformis in China[J].Chinese Journal of Mycology,2013,8(1):30-34.doi:10.3969/j.issn.1673-3827.2013.01.007.
[13]罗宽,廖小兰,陈寊.水稻褐鞘病研究Ⅴ.稻褐鞘病菌与其它病原的关系[J].湖南农学院学报,1991(3):471-476.doi:10.13331/j.cnki.jhau.1991.03.008.LUO K,LIAO X L,CHEN Z.Studies on rice brown sheath v.relationship between brown sheath fungi and other pathogens[J].Journal of Hunan Agricultural College,1991(3):471-476.doi:10.13331/j.cnki.jhau.1991.03.008.
[14]ZHANG R Y,SHAN H L,LI W F,WANG X Y,YIN J,LUO Z M,HUANG Y K.First report of sugarcane leaf chlorotic streak disease caused by Xanthomonas sacchari in Guangxi,China[J].Plant Disease,2017,101(8):1541-1542.doi:10.1094/PDIS-12-16-1774-PDN.
[15]MAROON L C J,SCHNEIDER K L,TURNER R.S,PRESTING G G,ALVAREZ A M.Xanthomonas sacchari-a pathogen or an endophyte[J].Phytopathology,2010,100(6):S78.
[16]FANG X Y,LIN H Y,WU L W,REN D Y,YE W J,DONG G J,ZHU L,GUO L B.Genome sequence of Xanthomonas sacchari R1,a biocontrol bacterium isolated from the rice seed[J].Journal of Biotechnology,2015,206:77-78.doi:10.1016/j.jbiotec.2015.04.014.
[17]唐敏,李丽,肖蓉.嗜麦芽寡养单胞菌胞外蛋白酶功能研究进展[J].微生物学报,2016,56(5):731-739.doi:10.13343/j.cnki.wsxb.20150289.TANG M,LI L,XIAO R.Research progress on extracellular protease function of Stenotrophomonas maltophilia[J].Journal of Microbiology,2016,56(5):731-739.doi:10.13343/j.cnki.wsxb.20150289.
[18]NEI T,SONOBE K,ONODERA A,ITABASHI T,YAMAGUCHI H,MAEDA M,SAITO R.Two cases with bacteremia suspected to be due to relatively rare Pseudomonas(Flavimonas)oryzihabitans[J].Journal of Infection and Chemotherapy,2015,21(10):751-755.doi:10.1016/j.jiac.2015.06.005.
[19]TENA D,FEMANDEZ C.Pseudomonas oryzihabitans:an unusual cause of skin and soft tissue infection[J].Infectious Diseases,2015,47(11):820-824.doi:10.3109/2374423.2015.1034170.
[20]冯婷婷,王佳贺.阴沟肠杆菌感染与耐药机制的研究进展[J].中国人兽共患病学报,2017,33(10):933-937.doi:10.3969/j.issn.1002-2694.2017.10.018.FENG T T,WANG J H.Advances in the study of Enterobacter cloacae infection and drug resistance mechanisms[J].Chinese Journal of Zoonoses,2017,33(10):933-937.doi:10.3969/j.issn.1002-2694.2017.10.018.