| 杨秀超1, 俞文静1, 段作营1, 夏森玉2, 沈志松2, 金 坚3, 李华钟1*.土壤中盾壳霉双标记平板计数法的建立及应用[J].植物保护,2017,43(5):134-142. |
| 土壤中盾壳霉双标记平板计数法的建立及应用 |
| Establishment and application of double-labeling plate counting method for detection of Coniothyrium minitans in soil |
| 投稿时间：2017-01-19 修订日期：2017-03-30 |
| DOI： |
| 中文关键词: 生物防治 盾壳霉 农杆菌介导转化 平板计数 |
| 英文关键词:biological control Coniothyrium minitans Agrobacterium-mediated transformation plate count |
| 基金项目:2015江苏省重点研发计划(现代农业)重点项目(BE2015309) |
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| 中文摘要: |
| 盾壳霉是一种重要的生防菌，其在土壤中的存活数量直接关系到防治病害的效果。然而目前没有对土壤中盾壳霉直接计数的方法，构建一种简单易行的土壤中盾壳霉计数方法对研究盾壳霉在土壤中的存活动态具有重要意义。本研究利用农杆菌转化法构建了潮霉素基因和绿色荧光蛋白基因标记的双标记盾壳霉菌株，并测定转化子的生长速度、产孢量和菌核致腐能力，初步分析了该方法计数土壤盾壳霉的有效性和可行性。结果显示，潮霉素基因和绿色荧光蛋白基因可以稳定地遗传和表达，并且部分转化子生长速度、产孢量和菌核致腐能力与出发盾壳霉菌株JN-CM没有显著差异。加入土壤中的盾壳霉转化子可以在含潮霉素(50 μg/mL)、氯霉素(100 μg/mL)和链霉素(100 μg/mL)的PDA平板培养，杂菌得到充分抑制，呈现绿色荧光的盾壳霉转化子被有效检出，检出限达到2×103个/g土。本研究所构建的计数方法能有效检出施入土壤中的盾壳霉并进行活菌计数，可以用于盾壳霉JN-CM产品在土壤中的定殖、生长、繁殖和存活情况的研究。应用双标记平板计数法研究了不同温度、湿度、接种量和添加菌核等条件下盾壳霉JN-CM在土壤中的存活规律。结果显示，在含有核盘菌菌核的土壤中，盾壳霉JN-CM可以通过重寄生维持一段时间(12周)的数量增长，在长达半年左右(24周)的时间里其存活率仍然可以维持在65%左右。在不含菌核的土壤中，在一般土壤温度(10~20℃)范围内，无论土壤水分含量高低，其半年存活率也可以维持在50%左右。因此，可以预测，连续施用盾壳霉JN-CM生防制剂，可以使其数量在土壤中长期维持在一定的水平范围，达到长效防治效果。 |
| 英文摘要: |
| Coniothyrium minitans is an important biocontrol fungus. The quantity of the fungus surviving in the soil affects control efficacy directly. At present, there is still no direct way to count the quantity of C.minitans in the soil, therefore, a simple and easy method for counting C.minitans in the soil will be very important. In this study, a double-labeling C.minitans strain with hygromycin resistance gene and enhanced green fluorescent protein gene was constructed by Agrobacterium-mediated transformation. Subsequently, the stability, growth rate, reproduction capacity and sclerotium-parasitizing ability of the transformants were measured. The effectiveness and feasibility of the method were also analyzed. The results showed that the hygromycin resistance gene and enhanced green fluorescent gene could inherited and expressed stably, and there was no significant difference between C.minitans strain JN-CM and the transformants in the ability of growth rate, reproduction and parasitizing on sclerotia of Sclerotinia sclerotiorum. PDA plates containing hygromycin (50 μg/mL), chloramphenicol (100 μg/mL) and streptomycin (100 μg/mL) could inhibit most of the microbes in the soil, while the transformants with green fluorescence could be detected and counted. The detection limit was 2 × 103 spores per gram soil. Thus, the counting method can be used to study the colonization, growth, reproduction and survival of C.minitans JN-CM in soil. The effects of temperature, soil humidity, inoculation amount, sterilization and adding of sclerotia on the survival of C.minitans in soil were further investigated by the double-labeling plate counting method. The results showed that in the soil with sclerotia, the number of C.minitans JN-CM increased in a period of time (12 weeks) and its survival rate can be maintained at about 65% in about half a year (24 weeks). While, in the soil without sclerotia, regardless of the soil moisture, the half-year survival rate can still be maintained at about 50% under the soil temperature of 10-20℃. Therefore, it can be predicted that continuous application of C.minitans JN-CM biocontrol agent can keep the number of C.minitans in the soil within a certain range for a long time, indicating that the long-term control effect can be achieved. |
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