酵母菌的分离、筛选以及对柑橘保鲜的机理研究

Isolation,Screening of Yeasts with Antagonistic Effect to Green and Blue Mold of Citrus,and the Preliminary Study of the Mechanism

作者: 专业:果树学 导师:邓伯勋;邓秀新 年度:2005 学位:博士 

关键词
酵母菌 柑橘 青霉病 绿霉病 生物防治 抑菌机理

Keywords
Yeast, Citrus, Penicillium italicum, Penicillium digitatum, Biological control, Control mechanism
        柑橘果品安全(无毒)防腐保鲜是国内外关注的问题,它关系到人体的健康及果品贸易、柑橘产业和农业经济的发展。多年来,国内外科技工作者非常重视无毒防腐保鲜柑橘的研究。由于研究手段和方法等问题,至今没有理想的成果投产。本研究旨在利用柑橘果实表面及柑橘根际土壤中的有益酵母菌,研究生物防腐保鲜,以菌杀菌,以菌抑菌,以生物制剂代替化学防腐保鲜剂,克服化学药剂对人体的毒害作用,提高柑橘果品防腐保鲜效果,主要结果如下: 1.酵母菌的分离、筛选和鉴定 从果园及柑橘果实伤口处中共分离和筛选了381株酵母菌(yeast)。经过离体和活体实验,筛选出一株能够有效防治柑橘采后青、绿霉病的拮抗菌株——34-9。通过形态学观察和生理生化实验,在确定属之后,根据属的具体特征选择与本属的鉴定有关的实验,进一步鉴定表明该菌株属于真菌门半知菌纲丛梗孢目,隐球酵母科,克勒克酵母属(Kloeckera)的柠檬形克勒克酵母(Kloeckera apiculata)。 2.菌株(34-9)在活体上对柑橘青、绿霉病的抑制效果评价 在25℃下,2×10~8 CFU/ml的菌株(34-9)的悬浮液能够完全抑制病菌孢子浓度为2×10~4个/ml时柑橘果实青霉病(Penicillium italicum Wehmer.)的发生,对柑橘果实绿霉病(Penicillium digitatum Sacc.)也有极好的抑制效果;当菌株(34-9)的浓度降低为2×10~7CFU/ml时,对柑橘青、绿霉病的抑制效果相对较差,但可以延缓病害的发生。 3.菌株(34-9)在柑橘果实伤口处的生长情况 菌株(34-9)在有无柑橘青、绿霉病存在的情况下均能迅速在柑橘果实伤口定殖,接种48h后,数量可增加20倍以上;在有病菌存在的情况下,24h后菌株(34-9)的数量就增加66倍。菌株(34-9)和病菌孢子的接种时间与防治的效果有关,先接种菌株(34-9)的抑菌效果显著地好于同时或后于病菌接种的效果。 4.菌株(34-9)与常规的果实采后处理措施的研究 菌株(34-9)能够与常规的果实采后处理措施如化学杀菌剂、低温冷藏条件相结合。菌株(34-9)与低浓度的杀菌剂如多菌灵等混合使用,可以达到高浓度杀菌剂的
    Biological control of postharvest diseases in citrus has been affected the affection, it relate human health, fruit trade and the citrus industry development. In recent decades, scientists have studied on biological control of postharvest diseases in citrus. Since there was a series of problems on method and ways, progress is not great. With increasing consumer concern and the loss of registration of many fungicides, alternatives are necessary to provide decay control. The efficacy of several treatments of biological control, rapid colonization, and common postharvest practice was evaluated for decay control on citrus. Some conclusions were drawn as following:1. Screening and identification of yeastsThree hundred and eighty-one yeast isolates were selected from different fruits and the epiphytes of citrus roots. A yeast isolate named 34-9, from the rhizosphere of citrus, was tested for antagonistic activity against blue mold (Penicillium italicum) and green mold (Penicillium digitatum) of citrus fruit in bean sprout juice agar. In order to further study its characters, the strain (34-9) was needed to identified to genus. According to morphological and physiological experiments, it belonged to Kloeckera apiculata, Kloeckera.2. Inhibition effect on blue mold and green mold of citrus in vivo of strain (34-9) The strain (34-9) at 2× 108 CFU/ml of washed-cell suspension completely inhibitedblue mold (P. italicum) and mostly inhibited green mold (P. digitatum) in citrus wounds artificially inoculated with 2 × 104 spores/ml at 25 . After challenge with 2 × 104 spores/ml of postharvest pathogens of blue mold and green mold in citrus wounds, the strain (34-9) at 2 ×10 CFU/ml of washed-cell suspension did not inhibit decay in citrus fruits at 25℃. But it could delay the decay of the postharvest pathogens in citrus fruits.3. Rapid colonization of the strain (34-9) in citrus woundsRapid colonization of the strain (34-9) in citrus wounds was observed either when inoculated alone or co-inoculated with the fungal pathogen. The population increased more than 20 folds after 48 h inoculation. And when co-inoculated with Penicillium rot, the population increased 66 folds after 24 h. Efficacy of the strain (34-9) againstpostharvest diseases in citrus was maintained when applied before pathogens, but when applied simultaneously with or after spores, biological control efficacy was significantly reduced.4. Study on strain (34-9) with common postharvest practiceThe strain (34-9) was compatible with common postharvest practice such as fungicide, low-temperature storage. The strain (34-9) could be applied with low dose of fungicide such as carbendazim, and efficacy had the same as carbendazim; furthermore, Temperature had litter effect on biological control efficacy of the strain (34-9). The control efficacy under low temperature was the same as that at room temperature. Therefore, the strain (34-9) has a great potential in the future..5. Study on mechanism of the strain (34-9)Although the effect have been preferably obtained on biological control of the strain (34-9) against P. italicum) and P. digitatum of citrus, the study on mechanism of the strain (34-9) activity was still lagged. According to latest researches of some antagonists, the following studies were performed subsequently.1) The mechanism of the strain (34-9) activity did not involve in the production of antifungal substances, since supernatant solution and treatment liquid did not inhibit the pathogens, but limited spore germination in vivo.2) The strain (34-9) inhibited Penicillium rot by producing directly antagonist such as attachment of the yeast cells to the hypha of the pathogen and metamorphosed hypha of Penicillium rot3) Nutrient competition played a major role in controlling postharvest diseases in citrus. The populations of the strain (34-9) in drop-inoculated wounds in fruits increased rapidly. The populations of the antagonist in wounds were more than 20 times greater than inoculation after 48h.Finally, the possible mechanism of the strain (34-9) against P. italicum and P. digitatum of citrus were also discussed in the paper.
        

酵母菌的分离、筛选以及对柑橘保鲜的机理研究

摘要8-10
Abstract10
缩略词12-13
第一章 文献综述13-31
    1 生物保鲜研究的必要性13-14
        1.1 大力发展柑橘生产的需要13
        1.2 提高柑橘产品市场竞争力的需要13
        1.3 柑橘产业化生产的需要13-14
        1.4 减少用于柑橘防腐保鲜药剂毒性的需要14
    2 生物防治的概念及研究现状14-17
        2.1 生物防治的概念14-15
        2.2 生物防治的研究现状15-17
    3 采后病害生物防治的特点17
    4 目前采后病害生物防治的研究进展及防治状况17-19
    5 当前采后病害生物防治的研究热点19-29
        5.1 拮抗菌的分离与筛选19-20
        5.2 菌种的复壮与保藏20-25
            5.2.1 尽量减少传代21-22
            5.2.2 菌种经常纯化22
            5.2.3 采用有效的菌种保藏方法22-25
        5.3 拮抗菌的抑菌机理25-29
            5.3.1 依靠产生抗菌素抑制病菌的生长26
            5.3.2 与病菌竞争营养和空间26-27
            5.3.3 重寄生现象(真菌寄生现象)27-28
            5.3.4 影响寄主抗性28
            5.3.5 其它物质对拮抗效果的影响28-29
    6 采后病害生物防治商业开发的可能性以及发展前景29-31
        6.1 拮抗菌的商品化应用29-30
        6.2 发展前景30-31
第二章 酵母菌的分离与筛选31-45
    1 引言31
    2 材料与方法31-37
        2.1 从果蔬表面或根际土壤自然生长的微生物中分离31-34
            2.1.1 从柑橘果实表面分离酵母菌31-33
            2.1.2 从柑橘根际土壤中分离酵母菌33-34
        2.2 从柑橘果实伤口分离酵母菌34
        2.3 “in vitro”筛选实验34-36
        2.4 从现有的酵母菌中筛选36-37
        2.5 所有数据均采用SAS软件进行方差分析37
    3 结果与分析37-43
        3.1 酵母菌分离结果37
        3.2 “in vitro”筛选实验37-40
            3.2.1 初选37-38
            3.2.2 复选38-39
            3.2.3 第二次复选39
            3.2.4 酵母与病菌的活菌数39-40
        3.3 酵母菌与青、绿霉菌的混合培养结果40-43
        3.4 酵母拮抗菌的筛选43
    4 讨论43-45
第三章 酵母菌对柑橘采后病害的抑制效果45-50
    1 引言45-46
    2 材料与方法46-47
        2.1 材料46
        2.2 方法46-47
            2.2.1 “in vivo”实验46
            2.2.2 菌株(34-9)抑菌效果的分析46-47
            2.2.3 拮抗菌株抑菌浓度的筛选47
            2.2.4 菌株(34-9)发挥效益的分析47
    3 结果与分析47-49
        3.1 菌株(34-9)不同处理对柑橘青、绿霉病的抑制效果47
        3.2 菌株(34-9)的不同浓度对柑橘采后青、绿霉病的防治47-48
        3.3 菌株(34-9)的接种时间对柑橘青、绿霉病防治效果的影响48-49
    4 讨论49-50
第四章 菌株(34-9)的鉴定及部分生理指标的测定50-63
    1 引言50
    2 材料与方法50-57
        2.1 无丝状营养细胞的培养形态50
        2.2 丝状酵母菌的镜检50-51
        2.3 掷孢子(Ballistoconidia)的镜检51
        2.4 子囊孢子的镜检51
        2.5 碳源同化实验-生长谱法51-52
        2.6 氮源同化实验52
        2.7 糖类发酵鉴定52
        2.8 维生素需要量鉴定52-53
        2.9 类淀粉化合物形成测定53
        2.10 对高浓度D-葡萄糖生长的鉴定53
        2.11 不同的培养基配方53-56
        2.12 部分生理指标的测定56-57
            2.12.1 耐乙醇能力测试56
            2.12.2 致死温度测试56
            2.12.3 生长曲线测试56
            2.12.4 比生长速率的计算56-57
    3 结果与分析57-61
        3.1 形态学特征57-58
        3.2 生理生化特征58-59
        3.3 部分生理指标的测定59-61
            3.3.1 耐乙醇能力59
            3.3.2 致死温度59-60
            3.3.3 生长曲线60
            3.3.4 比生长速率60-61
    4 讨论61-63
第五章 K.apiculata在柑橘果实上的生长动态63-67
    1 引言63
    2 材料与方法63-64
        2.1 材料63
        2.2 方法63-64
            2.2.1 K.apiculata在柑橘果实伤口的繁殖速度63
            2.2.2 不同处理的K.apiculata在果实伤口的生长速度63-64
    3 结果与分析64-65
        3.1 K.apiculata在柑橘果实伤口的繁殖速度64
        3.2 不同处理的K.apiculata在果实伤口的生长速度64-65
    4 讨论65-67
第六章 K.apiculata抑菌机理的初步研究67-73
    1 引言67
    2 材料与方法67-68
        2.1 K.apiculata产生抗菌素的研究67-68
        2.2 K.apiculata与病菌竞争营养的检测68
            2.2.1 K.apiculata在PDA上抑菌实验68
            2.2.2 K.apiculata在PDB上的抑菌实验68
        2.3 K.apiculata对病菌的直接作用68
    3 结果与分析68-71
        3.1 拮抗菌产生抗生素的情况69
        3.2 K.apiculata与病菌竞争营养的情况69-70
            3.2.1 在PDA上对病菌的作用69
            3.2.2 在PDB上对病菌的作用69-70
        3.3 K.apiculata对柑橘青、绿霉病菌的直接作用70-71
    4 讨论71-73
第七章 K.apiculata在柑橘贮藏中的应用73-79
    1 引言73
    2 材料与方法73-75
        2.1 实验材料73
        2.2 实验方法73-75
            2.2.1 2002年—2003年实验73-74
            2.2.2 2003年—2004年实验74-75
    3 结果与分析75-77
        3.1 2002年—2003年实验结果75-76
        3.2 2003年-2004年实验结果76-77
    4 讨论77-79
第八章 总讨论79-85
    1.拮抗酵母菌对采后病害生物防治的应用79-82
        1.1 应用于采后病害生物防治研究的酵母菌特点及种类79
            1.1.1 拮抗酵母菌的特点79
            1.1.2 拮抗酵母菌的种类79
        1.2 拮抗酵母菌的作用机制79-80
            1.2.1 竞争作用(营养、空间等的竞争)79-80
            1.2.2 寄生作用80
            1.2.3 诱导寄主抗性80
            1.2.4 抗生作用80
        1.3 拮抗酵母菌的商品化应用及发展前景80-82
            1.3.1 拮抗酵母菌的商品化应用80-81
            1.3.2 发展前景81-82
    2.生物保鲜的安全性82
    3.应用的方法82-83
    4.预期的风险分析和效益分析83-85
        4.1 风险分析83
        4.2 效益分析83-85
            4.2.1 经济效益分析83
            4.2.2 社会、环境效益分析83-85
创新点与下一步研究打算85-86
参考文献86-100
照片及说明100-102
附录Ⅰ 酵母各属检索表102-105
附录Ⅱ 酵母菌(34-9)急性检测报告105-109
附录Ⅲ 论文及成果109-110
致谢110


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