假单胞菌YL11对扩展青霉的抑制作用及其机理初探
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摘要
【背景】苹果青霉病是由扩展青霉引起的一种重要的果实采后病害,影响果实品质导致苹果腐烂从而造成经济损失。【目的】研究假单胞菌YL11对扩展青霉的抑制作用和苹果采后青霉病的防治效果,并对抑菌机理进行初步探讨。【方法】以扩展青霉为供试菌株,研究不同浓度的假单胞菌YL11无菌发酵液对扩展青霉菌落直径、孢子萌发率、菌丝体干重、苹果损伤接种病斑直径扩展的影响,利用对电导率、核酸及蛋白释放量、AKP含量、SDH活性、ATP酶活性和ATP含量的影响对抑菌机理进行探究。【结果】假单胞菌YL11无菌发酵液能有效抑制扩展青霉生长,抑菌圈直径为22.33±0.27 mm,抑菌效价为71.67 mm/mL;能有效抑制孢子萌发,100%无菌发酵液对孢子萌发抑制率达到80.2%;对扩展青霉的生物量也有一定抑制作用,体积分数为100%时,菌丝体干重为4.7mg/mL,抑制率达到39.74%;无菌发酵液处理能有效抑制苹果青霉病病斑的扩展,3d时对病斑扩展的抑制率最大,达到47.1%;无菌发酵液处理均能引起电导率升高、胞内核酸和蛋白释放量增大、胞外AKP含量升高、SDH活性降低、ATP酶活性和ATP含量均降低,且随着发酵液浓度的增加效果越明显。【结论】假单胞菌YL11能显著抑制扩展青霉的生长,破坏细胞膜结构、降低能量代谢酶活性,从而扰乱扩展青霉的正常生长,对苹果青霉病有较好的生防效果,具有潜在的开发价值。
[Background] Apple blue mold rot is an important fruit post-harvest disease that caused by Penicillium expansum, which affects the quality of the fruit and leads to the rot of the apple resulting in economic losses. [Objective] To study the inhibitory effect and explore the inhibition mechanism of Pseudomonas sp. YL11 on P. expansum. [Methods] We studied the effects of cell-free fermentation filtrate of Pseudomonas sp. YL11 with different concentration on the growth colony diameter, spore germination, mycelial dry weight of P. expansum and the lesion diameter of wounded inoculated apple fruit; We also explored impact of the conductivity, nucleic acid and protein release, AKP content, SDH activity, ATPase activity and ATP content to reveal the inhibitory mechanism. [Results] Pseudomonas sp.YL11 sterile fermentation broth effectively inhibited the growth of P. expansum. The diameter of inhibition zone was 22.33±0.27 mm and the inhibition titer was 71.67 mm/mL. It also could effectively inhibit spore germination. The inhibition rate of 100% aseptic fermentation broth to germination reached80.2%. It had certain inhibition effect on the biomass of P. expansum. The dry weight of mycelium was4.7 mg/mL and the inhibition rate reached 39.74% when the volume fraction was 100%. Moreover, aseptic fermentation fluid effectively inhibited extension of lesion diameter of blue mold of apple. The maximum inhibitory rate reached 47.1% at 3 d. In addition, aseptic fermentation broth treatment increased electrical conductivity, intracellular nucleic acid, protein release, extracellular AKP content and decreased SDH activity, ATPase activity and ATP content. The effect was enhanced by the fermentation broth concentration. [Conclusion] Pseudomonas sp. YL11 disturbed the normal growth of P. expansum by significantly inhibited the growth of P. expansum, destroyed the structure of cell membrane and reduced the activity of energy metabolism enzyme. It had a good better biocontrol effect to against blue mold of apple and had a potential development value.
[Background] Apple blue mold rot is an important fruit post-harvest disease that caused by Penicillium expansum, which affects the quality of the fruit and leads to the rot of the apple resulting in economic losses. [Objective] To study the inhibitory effect and explore the inhibition mechanism of Pseudomonas sp. YL11 on P. expansum. [Methods] We studied the effects of cell-free fermentation filtrate of Pseudomonas sp. YL11 with different concentration on the growth colony diameter, spore germination, mycelial dry weight of P. expansum and the lesion diameter of wounded inoculated apple fruit; We also explored impact of the conductivity, nucleic acid and protein release, AKP content, SDH activity, ATPase activity and ATP content to reveal the inhibitory mechanism. [Results] Pseudomonas sp.YL11 sterile fermentation broth effectively inhibited the growth of P. expansum. The diameter of inhibition zone was 22.33±0.27 mm and the inhibition titer was 71.67 mm/mL. It also could effectively inhibit spore germination. The inhibition rate of 100% aseptic fermentation broth to germination reached80.2%. It had certain inhibition effect on the biomass of P. expansum. The dry weight of mycelium was4.7 mg/mL and the inhibition rate reached 39.74% when the volume fraction was 100%. Moreover, aseptic fermentation fluid effectively inhibited extension of lesion diameter of blue mold of apple. The maximum inhibitory rate reached 47.1% at 3 d. In addition, aseptic fermentation broth treatment increased electrical conductivity, intracellular nucleic acid, protein release, extracellular AKP content and decreased SDH activity, ATPase activity and ATP content. The effect was enhanced by the fermentation broth concentration. [Conclusion] Pseudomonas sp. YL11 disturbed the normal growth of P. expansum by significantly inhibited the growth of P. expansum, destroyed the structure of cell membrane and reduced the activity of energy metabolism enzyme. It had a good better biocontrol effect to against blue mold of apple and had a potential development value.
引文
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[4]Liu ZY,Zhang HY,Chen KP.Review on improving the biological control efficacity of postharvest fruits with antagonistic yeast cultivated with culture added elicitors[J].Science and Technology of Food Industry,2012,33(4):450-453,457(in Chinese)刘周阳,张红印,陈克平.激发子诱导培养提高拮抗酵母对水果采后病害生防效力的研究进展[J].食品工业科技,2012,33(4):450-453,457
[5]Zhao WY.Control effect of yeast antagonist on post-harvest blue mold of apple[J].Journal of Anhui Agricultural Sciences,2011,39(5):2676-2677(in Chinese)赵文亚.拮抗酵母菌对苹果采后青霉病的防治效果[J].安徽农业科学,2011,39(5):2676-2677
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[7]Fu RM,Xing WH,Zhang H,et al.Breeding of antagonistic bacteria against Penicillium expansum and study on its inhibition mechanism[J].Science and Technology of Food Industry,2016,37(5):154-158(in Chinese)付瑞敏,邢文会,张红,等.扩展青霉拮抗菌的选育及抑菌机制初探[J].食品工业科技,2016,37(5):154-158
[8]Li RP,Zhang HY,Liu WM,et al.Biocontrol of postharvest gray and blue mold decay of apples with Rhodotorula mucilaginosa and possible mechanisms of action[J].International Journal of Food Microbiology,2011,146(2):151-156
[9]Cai MX,Zhou YH,Zhang HY,et al.Effect and possible modes of action of Candida oleophila on controlling Penicillium expansum in apples[J].Food Science,2018,39(5):265-271(in Chinese)蔡孟轩,周雅涵,张鸿雁,等.橄榄假丝酵母控制苹果果实青霉病的效果及机制[J].食品科学,2018,39(5):265-271
[10]Zhang WW,Wang YL,Bi Y,et al.Screening and identification of an antagonistic strain against potato dry rot[J].Microbiology China,2018,45(8):1726-1736(in Chinese)张紊玮,王艳玲,毕阳,等.一株马铃薯干腐病拮抗菌的筛选、鉴定及其生物防效[J].微生物学通报,2018,45(8):1726-1736
[11]Niu LL,Bi Y,Zhang SG,et al.Comparison of inhibitory effect of sodium silicate at different concentrations and corresponding pHon Trichothecium roseum in vitro and in vivo[J].Science and Technology of Food Industry,2015,36(13):157-161(in Chinese)牛黎莉,毕阳,张盛贵,等.体内和体外条件下不同浓度硅酸钠和对应pH抑制粉红单端孢(Trichothecium roseum)效果比较[J].食品工业科技,2015,36(13):157-161
[12]Liu Q,Zhang XL,Fan SY.Preliminary research on antifungal effect of extracts from garland chrysanthemum on watermelon blight[J].Northern Horticulture,2016(14):114-118(in Chinese)刘琼,张新龙,范淑英.茼蒿提取物对西瓜枯萎病病菌抑制效果的初步研究[J].北方园艺,2016(14):114-118
[13]Bai XD,Niu LL,Bi Y,et al.18 kinds of salt on apple of core rot control and part of the mechanism[J].Science and Technology of Food Industry,2015,36(10):335-338(in Chinese)白小东,牛黎莉,毕阳,等.18种盐对苹果霉心病的控制及部分机理[J].食品工业科技,2015,36(10):335-338
[14]Gao XY.Studies on the mechanism of a new antibiotic Wanlongmycin against Phytophthora melonis[D].Guangzhou:Doctoral Dissertation of South China University of Technology,2005(in Chinese)高向阳.新抗生素万隆霉素对黄瓜疫病菌的作用机理研究[D].广州:华南理工大学博士学位论文,2005
[15]Chen CJ,Ning XB.Characteristics of the sublethal injured and resuscitative Vibrio parahaemolyticus[J].Science and Technology of Food Industry,2013,34(14):160-165(in Chinese)陈婵娟,宁喜斌.亚致死热损伤和复苏状态的副溶血性弧菌特性[J].食品工业科技,2013,34(14):160-165
[16]Huang WY,Cao W,Li J,et al.Determination of protein content in juice by coomassie brilliant blue[J].Food and Fermentation Industries,2009,35(5):160-162(in Chinese)黄婉玉,曹炜,李菁,等.考马斯亮蓝法测定果汁中蛋白质的含量[J].食品与发酵工业,2009,35(5):160-162
[17]Yang GF,Wei YL.The research status and application prospect of Pseudomonas[J].Biotechnology Bulletin,2011(1):37-39,49(in Chinese)杨光富,魏云林.假单胞菌研究现状及应用前景[J].生物技术通报,2011(1):37-39,49
[18]Zuo J,Liao XL.Study progress on plant fungal diseases prevention by Pseudomonas spp.[J].Modern Agricultural Science and Technology,2011(22):164-165,167(in Chinese)左静,廖晓兰.应用假单胞菌防治植物真菌性病害研究进展[J].现代农业科技,2011(22):164-165,167
[19]Zhang Y,Su P,Liu SQ,et al.Inhibition of antifungal Pseudomonas SU8 against several phytopathogenic fungi[J].Agrochemicals,2013,52(12):917-920(in Chinese)张亚,苏品,刘双清,等.拮抗假单胞菌SU8对几种植物病原真菌的抑制作用[J].农药,2013,52(12):917-920
[20]Ma LP,Hao BQ,Wang J,et al.Inhabitation of antagonistic bacterium B96-II to Fusarium oxysporum f.sp.asparagi[J].Chinese Journal of Applied&Environmental Biology,2009,15(6):861-865(in Chinese)马利平,郝变青,王静,等.拮抗菌B96-II对芦笋枯萎菌的抑菌作用[J].应用与环境生物学报,2009,15(6):861-865
[21]Huang XQ,Bie XM,LüFX,et al.Inhibitory effect of lipopeptides from Bacillus subtilis fmbJ on Penicillium notatum and its antisepsis to peach[J].Transactions of the CSAE,2008,24(1):263-267(in Chinese)黄现青,别小妹,吕凤霞,等.枯草芽孢杆菌fmbJ产脂肽抑制点青霉效果及其桃防腐试验[J].农业工程学报,2008,24(1):263-267
[22]Li CW,Yang LB,Deng X,et al.Inhibiting effects and mechanism of Trichoderma strains to Cytospora chrysosperma[J].Scientia Silvae Sinicae,2012,48(9):88-94(in Chinese)李冲伟,杨立斌,邓勋,等.木霉菌株对金黄壳囊孢菌的抑菌效应及机制[J].林业科学,2012,48(9):88-94
[2]Zhang YL,Zhang B,Tian SH,et al.Distribution detection of Penicillium sp.in apple tissues[J].Shandong Agricultural Sciences,2015,47(4):103-105(in Chinese)张悦丽,张博,田淑慧,等.苹果青霉病菌在苹果组织中的分布检测[J].山东农业科学,2015,47(4):103-105
[3]Fu RM,Xing WH,Gu YN,et al.Isolation and identification of antagonistic bacteria against postharvest blue mold decay of apple and its mutation by low energy N+implantation[J].Northern Horticulture,2015(21):116-120(in Chinese)付瑞敏,邢文会,谷亚楠,等.苹果采后青霉病拮抗菌株的分离、鉴定和低能N+注入诱变[J].北方园艺,2015(21):116-120
[4]Liu ZY,Zhang HY,Chen KP.Review on improving the biological control efficacity of postharvest fruits with antagonistic yeast cultivated with culture added elicitors[J].Science and Technology of Food Industry,2012,33(4):450-453,457(in Chinese)刘周阳,张红印,陈克平.激发子诱导培养提高拮抗酵母对水果采后病害生防效力的研究进展[J].食品工业科技,2012,33(4):450-453,457
[5]Zhao WY.Control effect of yeast antagonist on post-harvest blue mold of apple[J].Journal of Anhui Agricultural Sciences,2011,39(5):2676-2677(in Chinese)赵文亚.拮抗酵母菌对苹果采后青霉病的防治效果[J].安徽农业科学,2011,39(5):2676-2677
[6]Chen F,Yun JM,Ai DY,et al.Screening of bacterial strain against Penicillium expansum and isolating of its antibacterial compounds[J].Science and Technology of Food Industry,2014,35(9):160-164(in Chinese)陈芳,贠建民,艾对元,等.拮抗扩展青霉菌株的筛选及其抗菌活性物质分离[J].食品工业科技,2014,35(9):160-164
[7]Fu RM,Xing WH,Zhang H,et al.Breeding of antagonistic bacteria against Penicillium expansum and study on its inhibition mechanism[J].Science and Technology of Food Industry,2016,37(5):154-158(in Chinese)付瑞敏,邢文会,张红,等.扩展青霉拮抗菌的选育及抑菌机制初探[J].食品工业科技,2016,37(5):154-158
[8]Li RP,Zhang HY,Liu WM,et al.Biocontrol of postharvest gray and blue mold decay of apples with Rhodotorula mucilaginosa and possible mechanisms of action[J].International Journal of Food Microbiology,2011,146(2):151-156
[9]Cai MX,Zhou YH,Zhang HY,et al.Effect and possible modes of action of Candida oleophila on controlling Penicillium expansum in apples[J].Food Science,2018,39(5):265-271(in Chinese)蔡孟轩,周雅涵,张鸿雁,等.橄榄假丝酵母控制苹果果实青霉病的效果及机制[J].食品科学,2018,39(5):265-271
[10]Zhang WW,Wang YL,Bi Y,et al.Screening and identification of an antagonistic strain against potato dry rot[J].Microbiology China,2018,45(8):1726-1736(in Chinese)张紊玮,王艳玲,毕阳,等.一株马铃薯干腐病拮抗菌的筛选、鉴定及其生物防效[J].微生物学通报,2018,45(8):1726-1736
[11]Niu LL,Bi Y,Zhang SG,et al.Comparison of inhibitory effect of sodium silicate at different concentrations and corresponding pHon Trichothecium roseum in vitro and in vivo[J].Science and Technology of Food Industry,2015,36(13):157-161(in Chinese)牛黎莉,毕阳,张盛贵,等.体内和体外条件下不同浓度硅酸钠和对应pH抑制粉红单端孢(Trichothecium roseum)效果比较[J].食品工业科技,2015,36(13):157-161
[12]Liu Q,Zhang XL,Fan SY.Preliminary research on antifungal effect of extracts from garland chrysanthemum on watermelon blight[J].Northern Horticulture,2016(14):114-118(in Chinese)刘琼,张新龙,范淑英.茼蒿提取物对西瓜枯萎病病菌抑制效果的初步研究[J].北方园艺,2016(14):114-118
[13]Bai XD,Niu LL,Bi Y,et al.18 kinds of salt on apple of core rot control and part of the mechanism[J].Science and Technology of Food Industry,2015,36(10):335-338(in Chinese)白小东,牛黎莉,毕阳,等.18种盐对苹果霉心病的控制及部分机理[J].食品工业科技,2015,36(10):335-338
[14]Gao XY.Studies on the mechanism of a new antibiotic Wanlongmycin against Phytophthora melonis[D].Guangzhou:Doctoral Dissertation of South China University of Technology,2005(in Chinese)高向阳.新抗生素万隆霉素对黄瓜疫病菌的作用机理研究[D].广州:华南理工大学博士学位论文,2005
[15]Chen CJ,Ning XB.Characteristics of the sublethal injured and resuscitative Vibrio parahaemolyticus[J].Science and Technology of Food Industry,2013,34(14):160-165(in Chinese)陈婵娟,宁喜斌.亚致死热损伤和复苏状态的副溶血性弧菌特性[J].食品工业科技,2013,34(14):160-165
[16]Huang WY,Cao W,Li J,et al.Determination of protein content in juice by coomassie brilliant blue[J].Food and Fermentation Industries,2009,35(5):160-162(in Chinese)黄婉玉,曹炜,李菁,等.考马斯亮蓝法测定果汁中蛋白质的含量[J].食品与发酵工业,2009,35(5):160-162
[17]Yang GF,Wei YL.The research status and application prospect of Pseudomonas[J].Biotechnology Bulletin,2011(1):37-39,49(in Chinese)杨光富,魏云林.假单胞菌研究现状及应用前景[J].生物技术通报,2011(1):37-39,49
[18]Zuo J,Liao XL.Study progress on plant fungal diseases prevention by Pseudomonas spp.[J].Modern Agricultural Science and Technology,2011(22):164-165,167(in Chinese)左静,廖晓兰.应用假单胞菌防治植物真菌性病害研究进展[J].现代农业科技,2011(22):164-165,167
[19]Zhang Y,Su P,Liu SQ,et al.Inhibition of antifungal Pseudomonas SU8 against several phytopathogenic fungi[J].Agrochemicals,2013,52(12):917-920(in Chinese)张亚,苏品,刘双清,等.拮抗假单胞菌SU8对几种植物病原真菌的抑制作用[J].农药,2013,52(12):917-920
[20]Ma LP,Hao BQ,Wang J,et al.Inhabitation of antagonistic bacterium B96-II to Fusarium oxysporum f.sp.asparagi[J].Chinese Journal of Applied&Environmental Biology,2009,15(6):861-865(in Chinese)马利平,郝变青,王静,等.拮抗菌B96-II对芦笋枯萎菌的抑菌作用[J].应用与环境生物学报,2009,15(6):861-865
[21]Huang XQ,Bie XM,LüFX,et al.Inhibitory effect of lipopeptides from Bacillus subtilis fmbJ on Penicillium notatum and its antisepsis to peach[J].Transactions of the CSAE,2008,24(1):263-267(in Chinese)黄现青,别小妹,吕凤霞,等.枯草芽孢杆菌fmbJ产脂肽抑制点青霉效果及其桃防腐试验[J].农业工程学报,2008,24(1):263-267
[22]Li CW,Yang LB,Deng X,et al.Inhibiting effects and mechanism of Trichoderma strains to Cytospora chrysosperma[J].Scientia Silvae Sinicae,2012,48(9):88-94(in Chinese)李冲伟,杨立斌,邓勋,等.木霉菌株对金黄壳囊孢菌的抑菌效应及机制[J].林业科学,2012,48(9):88-94