新疆冬枣贮藏期拮抗酵母菌筛选及鉴定的研究

发布时间：2019-04-17 05:18

【摘要】：本课题通过从冬枣患病部分的病健交界处取样进行分离纯化,将分离的菌种对冬枣进行回接,确定冬枣采后主要的病原菌,所得到的主要病原菌通过平板观察菌落形态特征及显微观察菌种形态对其进行初步鉴定,并通过对冬枣创伤及无伤接种判断病原菌侵染途径。筛选得到三株新疆北疆地区冬枣采后贮藏期病理性病害的主要侵染病原菌,分别为B64A,G和Z。通过离体及活体筛选,从土壤及冬枣表面筛选得到一株拮抗酵母11-2,该酵母对三种病原菌有良好的控制作用。对该酵母11-2进行26S r DNA D1/D2区基因扩增进行鉴定,研究该酵母的生物学特性,确定了该酵母菌悬液最佳抑制效果的浓度,探讨接种时间对该拮抗酵母效力的影响,通过不同酵母处理液对病原菌的作用以及被拮抗酵母悬浮液处理后冬枣生理指标的变化来推测该酵母可能的生防方式,拮抗酵母菌细胞悬液处理对冬枣采后贮藏品质的影响,最后,初步研究了外源物质对于该酵母的生理影响以及外源物质与该拮抗酵母联合使用对冬枣采后主要病原菌的控制作用。测定结果利用Oringin 9.0和Spss18.0软件分析各组数据,得出主要结论如下:(1)从冬枣患病部分的病健交界处取样进行分离纯化,通过回接实验得到的三株病原菌,经过平板菌落形态及显微观察,初步鉴定表明北疆冬枣采后贮藏期病理病害病原菌中B64A为青霉属(Penicillium)、G为交链孢属(Alternaria)和Z为灰霉葡萄孢(Botrytis cinerea),灰霉、交链孢霉和青霉在枣果有伤及无伤时均可让枣果病变,灰霉和交链孢霉在枣果果柄处有伤及无伤时会引起病害,青霉只有在枣果果柄有伤时才会使枣果患病;(2)从土壤及枣果等来源筛选得一株对北疆冬枣采后贮藏期主要侵染病原菌有良好控制作用的拮抗酵母11-2,经过26S r DNA D1/D2区基因扩增进行鉴定为假丝酵母属Candida metapsilosis。该拮抗酵母的拮抗效果随着菌悬液浓度的增加而增强,1×109cells/m L下该酵母对冬枣采后青霉抑制率可达31.11%,对交链孢霉抑制率可达30.00%,灰霉的抑制率可达24.44%。不同接种时间的试验表明,拮抗酵母先于病原菌2h接种对病原菌控制较好,酵母不同处理液以及冬枣生理实验推测营养与空间竞争是该拮抗酵母主要的抑制病原菌方式,拮抗酵母菌细胞悬液的处理能影响冬枣果实采后主要品质指标如可滴定酸、光泽、硬度、抗坏血酸和可溶性固形物含量的变化;(3)外源物质对酵母影响的试验表明,一定浓度的壳聚糖溶液和一定浓度的羧甲基纤维素钠溶液不会影响该拮抗酵母的生长,0.5%壳聚溶液和0.3%羧甲基纤维素钠溶液都可以有效的减缓酵母菌悬液静置而出现的沉降现象,0.5%壳聚糖溶液与酵母菌联合使用时对于北疆冬枣贮藏期病原菌有较好的抑制作用,在用0.5%壳聚糖溶液配制的酵母悬浮液处理下5d保存的冬枣青霉抑制率为35.55%、交链孢霉30.00%以及灰霉的21.11%。
[Abstract]:The main pathogen of Dongzao jujube postharvest was determined by sampling and purification from the junction of disease and health of Dongzao jujube, and then the isolated strains were rejoined to Dongzao jujube, and the main pathogenic bacteria were determined after harvest. The main pathogenic bacteria were identified by plate observation of colony morphology and microscopic observation of the morphology of bacteria, and the pathogeny of pathogen infection was determined by inoculation of Dongzao jujube with wound and without injury. Three strains of pathogenic bacteria, B64A, G and Z., were isolated from winter jujube (Zizyphus jujuba L.) during postharvest storage period in northern Xinjiang. Through in vitro and in vivo screening, an antagonistic yeast 11m2 was obtained from soil and surface of winter jujube. This yeast has good control effect on three pathogenic bacteria. The gene amplification of the 26s r DNA D1/D2 region was carried out to study the biological characteristics of the yeast, to determine the concentration of the optimal inhibition effect of the yeast suspension, and to explore the effect of inoculation time on the efficacy of the antagonistic yeast. The effects of different yeast treatments on pathogenic bacteria and physiological indexes of winter jujube treated with antagonistic yeast suspension were studied to predict the possible biocontrol mode of the yeast and to antagonize the effect of yeast cell suspension treatment on postharvest storage quality of Dongzao jujube (Zizyphus jujuba L.) after being treated with antagonistic yeast suspension. Finally, the physiological effects of exogenous substances on the yeast and the control effects of exogenous substances combined with this antagonistic yeast on the main pathogens of postharvest winter jujube were studied. The results were analyzed by Oringin 9.0and Spss18.0 software. The main conclusions were as follows: (1) three pathogenic bacteria were isolated and purified from the junction of the diseased part of Dongzao jujube. The morphology of plate colony and microscopic observation showed that B64A was Penicillium (Penicillium), G, (Alternaria) and Z (Botrytis cinerea), Botrytis graminearum in postharvest storage stage of Dongzao jujube in North Xinjiang, and the results showed that B64A belonged to Penicillium sp., (Alternaria) and Z was Botrytis cinerea. Alternaria and Penicillium can cause the disease of jujube fruit when there is injury and no injury. Gray mold and Alternaria can cause disease when there is injury and no injury at the stem of jujube fruit, Penicillium can only make jujube fruit sick when there is injury to the fruit stem of jujube fruit. (2) from the soil and jujube fruit, a strain of antagonistic yeast 11 脳 2, which has good control effect on the main infectious pathogenic bacteria in postharvest storage period of Dongzao jujube in North Xinjiang, was screened and identified as Candida metapsilosis. by gene amplification of 26s r DNA D1/D2 region. The antagonistic effect of the antagonistic yeast increased with the increase of suspension concentration. The inhibition rate of this yeast on postharvest Penicillium, Alternaria and Botrytis cinerea was 31.11%, 30.00% and 24.44% at 1 脳 109cells/m / L, respectively. The experiment of different inoculation time showed that antagonistic yeast could control the pathogen better than 2 hours before pathogen inoculation. The physiological experiments of different treatments of yeast and winter jujube suggested that nutrition and spatial competition were the main ways to inhibit the pathogen of the antagonistic yeast. The treatment of antagonistic yeast cell suspension could affect the changes of main quality indexes such as titratable acid, luster, hardness, ascorbic acid and soluble solid content after harvest. (3) the effects of exogenous substances on yeast showed that chitosan solution and sodium carboxymethyl cellulose solution had no effect on the growth of the antagonistic yeast. 0.5% chitosan solution and 0.3% carboxymethyl cellulose sodium solution can effectively reduce the sedimentation of yeast suspension. 0.5% chitosan solution combined with yeast had a good inhibitory effect on the pathogen in storage period of Dongzao jujube in Northern Xinjiang. The inhibition rate of Penicillium jujube preserved for 5 days was 35.55% in yeast suspension prepared with 0.5% chitosan solution, 30.00% in Alternaria and 21.11% in Botrytis cinerea.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S436.65;S476.1

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