【作者】 孙萍；

【导师】 郑晓冬；

【作者基本信息】 浙江大学 ， 食品科学与工程， 2003， 硕士

【摘要】 果蔬采后病害造成的损失是巨大的。据报道，发达国家有10％-30％的新鲜果蔬损失于采后腐烂，而在发展中国家，由于缺乏储运冷藏设备，果蔬腐烂损失率竟高达40％-50％。防治果蔬腐烂的传统有效方法是使用化学杀菌剂，但长期使用化学杀菌剂，会造成环境污染、农药残留、危害人类身体健康、病原菌对其逐渐产生抗性等一系列问题。因此迫切需要寻找一种有效而对人类无害的防治方法。生物防治作为一项新兴技术，已引起了人们的注意。本研究以浙江特产水果柑橘为试验材料，研究了粘红酵母(Rhodotorula glutinis)对引起柑橘采后病害的主要病原菌指状青霉(Penicillium digitatum)的生物防治作用。探讨了粘红酵母菌种特性及处理形式、活化条件对抑菌效果的影响以及粘红酵母与病原菌接种间隔、与佐剂、化学杀菌剂结合使用、贮藏温度、贮前热处理、添加营养物质(碳源和氮源)、与其它生防菌结合使用、采前使用等对生物防治效果的影响。并确定了最佳的处理方法。同时还探讨了粘红酵母对柑橘采后病害生物防治的机理。主要研究结果如下： 1 筛选出一株具有良好抑菌效果的粘红酵母菌株。在PDB试管中，粘红酵母菌悬液能抑制病原菌孢子萌发和芽管伸长。且菌悬液浓度越大，对孢子和芽管抑制作用越强。培养原液对孢子萌发率有很强抑制作用，对芽管伸长的抑制作用不很明显，滤液对两者均没有抑制作用；在柑橘果实上，研究了其培养原液、培养液滤液、菌悬液和热杀死液的抑菌效果，发现菌悬液对病原菌有良好的抑制作用，且菌悬液浓度越大，抑菌效果越好。相同细胞浓度的培养原液的抑菌效果不如菌悬液的好，而滤液和热杀死液均没有抑菌效果。 在菌悬液中添加不同的营养物(碳源、氮源)对抑菌效果有不同影响。适量的有机碳源和有机氮源可以提高粘红酵母的抑菌效果，它可能是通过促进粘红酵母生长而起作用。而添加无机氮源(硝基氮、氨基氮)会降低其粘红酵母抑菌效果。 粘红酵母和病原菌接种间隔时间长短对抑菌效果有很大影响。一般来说，越早接种粘红酵母，效果越好。本实验中，在接种病原菌之前4小时或36小时接种粘红酵母，抑菌效果最佳。提示在采前使用或采后立即使用该菌将会有更好的效果。 粘红酵母和其他生防菌如枯草芽孢杆菌培养液结合使用可提高粘红酵母抑菌效果。枯草芽孢杆菌通过产生抗生素来发挥作用。将粘红酵母菌悬液和37℃下培养20小时的细菌滤液结合使用可得到最好的效果。 2 粘红酵母的抑菌机理主要是与病原菌之间对营养物质和空间的竞争有关，而不是通过产生抗生素达到目的的。粘红酵母能快速利用柑橘伤口上的营养物质生长存活，占据生存空间，排斥病原菌生长，生长速度较病原菌快。粘红酵母还浙江大学硕士论文可能分泌降解病原菌细胞壁的酶来发挥生防作用。此外通过显微镜观察，粘红酵母与病原菌丝之间没有紧密粘附和溶解作用。 3无论果蔬伤口上是否存在病原菌，在室温20℃和低温4℃下，粘红酵母都能快速增长。 4用响应面分析方法对粘红酵母活化条件进行了优化，借用SAS软件对实验数据进行了处理，发现麦芽汁培养基浓度对抑菌效果几乎没有影响。培养时间对其抑菌效果影响最大，装液量影响次之。活化培养时间为20.4小时，装液量38.0mL(250mL三角瓶)时为最佳条件。 5研究了金属离子对粘红酵母抑菌效果的影响，发现:培养基中Fe2+浓度对粘红酵母抑菌效果有较显著影响，当在培养基中添加5卿ol/L的Fe2+时，能够提高抑菌效果。菌悬液中添加NaZCO3和NaHCO3对粘红酵母抑菌效果影响不大;二阶阳离子MgZ+(o.Zmol几)和eaZ+(o一mol/L)都能提高粘红酵母抑菌效果，但与ca2+相比，Mg2+的效果不够明显，c扩+可能通过多种作用机制来提高粘红酵母的抑菌效果。 6探讨了粘红酵母与杀菌剂结合使用的效果，发现不同杀菌剂有不同效果。粘红酵母与多菌灵一起使用时，低浓度如250mg/L多菌灵结合使用时有较好效果，而多菌灵浓度越高，抑菌效果并没有相应地随着提高，因为高浓度的多菌灵对粘红酵母细胞有毒害作用，而粘红酵母对低浓度多菌灵有一定的抗性。当粘红酵母与扑海因结合使用时，不能降低腐烂率，可见扑海因对粘红酵母细胞有较大毒害作用，且其本身也不能有效地防治柑橘病害。 7柑橘的贮藏温度和贮前热处理(37℃、4天)会影响粘红酵母抑菌效果和果蔬的腐烂率。贮藏温度越低，贮藏效果越好。该菌株在低温下仍能生长并发挥其拮抗作用，同时低温能抑制病原菌的生长。贮前热处理可以杀死部分病原菌或者使病原菌失活，推迟果蔬上病原菌抱子的萌发，延长保质期;贮藏前热处理可使果实表面干燥，不利于病原菌生长;受伤后热处理还可治疗愈合伤口。有人则认为:热处理可诱导果实的抗病性。 7在最后确定最佳生防方法时，采用正交试验方法得出最佳组合即:菌体经Zoh活化培养、培养基中添加5啤01/L FesO4、菌悬液中添加0.lmol/L eaCI:，细胞浓度为10sCFU/mL，生防菌和致病菌接种间隔时间为10h时可达到最佳生防效果。 8采前喷施粘红酵母菌悬液，除创伤的象山红没有降低腐烂率外，其余均获得了更多还原

【Abstract】 Losses of fresh vegetables and fruits after postharvest are very serious in the world, almost 10-30% of those got rot postharvest every year. It is particularly high in undeveloped countries (almost 40-50%) for being short of refrigerate equipment.In general, fungicides still provide a primary means of control. However, due to the development of fungicides-resistant isolate of pathogen, the possible deregistration of some of more effective postharvest fungicides, concern for public safety, and the residue of fungicide and so on, the fungicides is forbidden to use. The microbial antagonist for biocontrol of postharvest disease of fruits and vegetables has been made actively in the world as an alternative method to fungicidal treatment in recent years. Furthermore, the biocontrol had achieved a success. Several isolates of bacteria and yeasts had been shown to be effective against postharvest pathogens on a range of crops. Some had been patented and commercialized as biocontrol agents.In the present study, we evaluated the biocontrol activity of Rhodotorula glutinis against the major postharvest pathogen (Penicillium digitatuni) on citrus fruits and studied the biocontrol measures. In the study, we discussed the effect of some factors on the inhibiting activity of R.glutinis. The factors are as below: the characteristic and different treatments of R.glutinis; the condition of culture; different interval between antagonist and pathogen inoculated; combination of R.glutinis with assistant substance such as chemical compound, fungicides; the store temperature of citrus fruits; heat treatment before store; adding nutrients (carbohydrate and nitrogen); combination with other strains. In the same time, we determined the most effective treatment of R. glutinis, discussed the mechanism of the biocontrol of R.glutinis and explored the effect in vitro. The main results were as following:1. One strain (R. glutinis 3) was selected for its good biocontrol activity from the strains preserved in laboratory. In PDB, the cell suspension could inhibit the germination of pathogen spores and the length of the germ tube, and the biocontrol activity was inverse proportion with cell concentration. However, the culture filtrate and culture broth hadn’t the same activity. The large of cell concentration of R.glutinis, the stronger of inhibition on the pathogen spores. The culture of R.glutinis had stronger inhibition activity on the germination of pathogen and not on the length of germ tube. The filtrate had no effect on the pathogen spores. We study the effect of the culture on citrus, the filtrate, the cells suspension, and theautoclaved culture. The cells suspension of R.glutinis had the best biocontrol activity; the effect of the culture broth containing the same concentration of yeast cells had the lower activity. However, culture filtrate and autoclaved culture weren’t had inhibition activity. The higher concentration of cell suspension was, the better biocontrol effect achieved.Nutrients (carbohydrate and nitrogen) added to the cell suspension had different influence on the activity of R.glutinis. Moderate dose of organic carbohydrate and nitrogen could promote the inhibition activity of it, maybe it exhibit its action by promoting the growth of R.glutinis. The inorganic nitrogen added to cell suspension would decrease the biocontrol activity of the antagonist by promoting the growth of pathogen.The interval between R.glutinis and pathogen inoculation influence the activity of R.glutinis greatly. The earlier of antagonist inoculation, the better of biocontrol achieved. When R.glutinis was inoculated 4h or 36h before pathogen, the best biocontrol activity of it was obtained. So we forecast that the better inhibition activity of R.glutinis if we used it preharvest or immediately after harvest.The culture filtrate of Bacillus subtilis could enhance the biocontrol when combined with R.glutinis. It was considered B.subtilis exhibit its activity by producing the antibiotic (iturin). The best activity could be achi更多还原