【作者】 张初署；

【导师】 刘阳；

【作者基本信息】 中国农业科学院 ， 农产品质量与食物安全， 2013， 博士

【摘要】 黄曲霉毒素污染是制约我国花生产业发展的重要限制因子。黄曲霉毒素主要是黄曲霉菌和寄生曲霉菌的次级代谢产物，具有高致癌性和高毒性。花生作为地上开花地下结果的作物，很容易受到土壤中黄曲霉菌的侵染，研究表明土壤是花生黄曲霉菌的主要来源，花生荚果中的黄曲霉菌与土壤中的黄曲霉菌有直接联系，因此本文首次对四个生态区同时也是四大花生主产区（东南沿海、长江流域、黄河流域和东北地区）的花生土壤中黄曲霉菌分布和产毒特征进行系统研究，揭示花生土壤中黄曲霉菌的分布和产毒规律，探讨其与生态环境的关系；研究四个生态区黄曲霉菌的遗传多样性，掌握黄曲霉菌的遗传变异情况，揭示遗传变异与地理来源的关系，为花生黄曲霉毒素污染的综合防治提供理论依据和技术支持。主要研究结论如下：1.筛选出适宜黄曲霉菌分离的培养基。通过比较7种常用于霉菌分离的培养基对10个土壤样品的黄曲霉菌分离效果，发现7种培养基对黄曲霉菌培养效果差别很大，培养效果最好的是DG18，培养的黄曲霉数是PDA的7.3倍。同时DG18能很好的抑制毛霉的生长，与DRBC培养基相比，能抑制81%的毛霉菌生长。DG18培养基能最大限度的保证黄曲霉的生长分离，同时也能更好的抑制毛霉的生长，因此选择DG18培养基作为黄曲霉菌分离培养基。2.分离出324株黄曲霉菌和20株寄生曲霉菌。对东南沿海、长江流域、黄河流域和东北地区的20个采样地600份花生土壤样品进行黄曲霉菌的分离纯化，共分离出344株黄曲霉毒素产生菌，其中黄曲霉菌为324株，寄生曲霉菌为20株。黄曲霉菌所占比例为94.2%，寄生曲霉菌为5.8%。3.掌握了中国花生土壤中黄曲霉菌在四个生态区的分布特征。黄曲霉菌的分布呈现明显的地域特征，不同生态区土壤中黄曲霉菌的菌落数和检出率均存在显著差异。数量最多、分布最广泛的为长江流域(菌落数为1039.3cfu·g^-1土壤，检出率为80.7%)；其次是东南沿海，再次是黄河流域，最少的为东北地区(菌落数只有2.4cfu·g^-1土壤，检出率仅为6.6%)。土壤中黄曲霉菌的菌落数与平均气温成正相关，检出率与平均气温成极显著的正相关，且检出率与经度成极显著的负相关。中国花生土壤中NS型菌株所占的比例（41.59%）>L型菌株（31.75%）>S型菌株（26.67%）。4.掌握了中国花生土壤中黄曲霉菌在四个生态区的产毒特征。对分离的344个菌株通过液体发酵，用HPLC法测定发酵液中AFB1的量，研究菌株的产毒量。中国花生土壤中黄曲霉菌产毒菌所占的比例为69.19%，不产毒菌所占的比例为30.81%。研究表明黄曲霉菌的产毒能力存在明显的地域特征。四个生态区菌株平均产毒量差异很大，最高的是东南沿海地区(平均产毒量为5836ng·mL^-1)，其次是黄河流域(其平均产毒量为5453ng·mL^-1)，再次是东北地区(1886ng·mL^-1)，最小的为长江流域(平均产毒量为1566ng·mL^-1)。不同产毒能力的菌株所占的比例在四个生态区间均存在显著性差异。四个生态区中，东南沿海高产毒菌(>1000ng·mL^-1)所占的比例最高，为55.4%，其次是黄河流域为31.8%，最少的是长江流域为10.2%。东南沿海高产毒菌所占的比例是长江流域的5.4倍。不产毒菌中长江流域所占比例最高为40.2%，其次是东北地区为36.4%，最少的东南沿海为15.7%。5.评估了四个生态区花生受黄曲霉毒素污染的潜在风险。四个生态区每克土壤中产AFB1的量也存在显著差异，最多的为长江流域，为1627544ng·mL^-1；其次为东南沿海，每克土壤中黄曲霉产AFB1的量为1117594ng·mL^-1；然后是黄河流域，每克土壤中黄曲霉产AFB1的量为18602ng·mL^-1；数量最少的是东北地区，每克土壤中AFB1的量为4478ng·mL^-1。长江流域每克土壤中的潜在产毒量是东北地区的363倍。因此可以看出花生黄曲霉毒素的污染潜在风险最大的地区为长江流域，其次是东南沿海，最小的生态区为东北地区。6.构建了ISSR标记用于黄曲霉菌遗传多样性的方法。建立并优化了黄曲霉菌ISSR-PCR的体系。在100个ISSR引物中，通过系统的引物筛选（初筛和精筛），确定引物UBC809、UBC817、UBC834、UBC895和UBC899作为黄曲霉菌ISSR遗传多样性分析的引物。对供试的菌株进行ISSR聚类分析，菌株的遗传相似系数为0.59-0.90。表明ISSR标记在黄曲霉菌遗传多样性研究上是一个十分有效的方法。7.掌握了中国花生土壤中黄曲霉菌在四个生态区的遗传多样性。对分离自四个生态区的343株黄曲霉菌株进行基于ISSR的遗传多样性研究，聚类分析表明黄曲霉菌ISSR群与地理来源密切相关。供试菌株划分成四个大的类群，东北地区的菌株全部在类群Ⅰ中，黄河流域的菌株全部在类群Ⅳ中，长江流域88.9%的株菌存在于类群Ⅱ中，东南沿海除8株菌外，其他均存在于类群Ⅲ中。因此来自于四个生态区的菌株，除极少部分菌株在ISSR类群上有交叉外，四个生态区的菌株均分布在不同的类群中。黄曲霉的ISSR群与黄曲霉的产毒情况和菌核类型没有明显的相关性。更多还原

【Abstract】 Aflatoxin contamination is an important limiting factor for the growth of China’s peanutindustry. Aflatoxins are the secondary metabolites formed mainly from the Aspergillus flavus andA. parasiticus. Soil is the main source of inoculum for A. flavus and since peanut developunderground, pods are in direct contact with the soil fungal populations. The study was firstly,systematically researched on the distribution and production of aflatoxin of A. flavus in soils ofpeanut fields in four agroecological zones （Southeast coast、Yangtze River Basin、Yellow RiverBasin and Northeast area） of China and revealed the distribution and aflatoxin production of A.flavus. And the genetic diversity of A. flavus from the four agroecological zones was studied inorder to grasp genetic variation and it revealed the relationship of genetic variation and geographicorigin. The study could provide a theoretical basis and technical support for the prevention ofaflatoxin contamination in peanut.The results are listed as follows:1. Screened the suitable medium for the isolation of A. flavus. Seven agar media used to isolatemildew were compared for utility in isolating fungi in the A. flavus from soils of peanut fields.The seven agar media were Dicloran18%glycerol medium、High salt Czapek medium、Patatodextrose agar medium、Modified rose Bengal agar medium、YES medium、Bengal medium、Dicloran Bengal agar medium. The results showed that there was a big difference in seven mediain isolating A. flavus. DG18was the most useful for studying the population biology of this groupbecause it permitted both identification of the greatest number of A. flavus group strains andgrowth of the fewest competing fungi. DG-18supported about7.3times more A. flavus coloniesthan the PDA while reducing the number of mucorales colonies by81%than the DRBC. So DG18should be the most useful for studies on the population biology of the A. flavus group.2. Isolated324strains of A. flavus and20strains of Aspergillus parasiticus.344strains ofaflatoxin producing Aspergillus species were isolated from600soil samples of20peanut fields infour agroecological zones.323（94.2%） were A. flavus and20（5.8%） were A. parasiticus. Inpeanut soil population of aflatoxin producing Aspergillus species, A. flavus was found to be thedominant species.3. Mastered the distribution of A. flavus in soils of peanut fields in four agroecologicalzones of China. The distribution of A. flavus isolates significantly varied among the districts andagroecological zones of China. The largest number and the most widely distribution of A. flavuswas Yangtze River Basin (the densities of A. flavus in soils was1039.3cfu·g^-1 and the positiverate of A. flavus in soils was80.7%). The second was Southeast coast, and the third was YellowRiver Basin. The last was Northeast area (The densities of A. flavus in soils was only2.4cfu·g^-1and the positive rate was6.6%). The positive rate of A. flavus in soils and the temperature had thenotable positive correlation. There was the notable positive correlation between the densities of A.flavus in soils and the temperature. And the densities of A. flavus and the longitude had negative correlation. The A. flavus NS-strain was the most commonly isolated member of A. flavus（41.59%） across the four examined agroecological zones, and the second was A. flavus S-strain.The last was L-strain.4. Mastered the aflatoxin production of A. flavus in soils of peanut fields in fouragroecological zones of China. The aflatoxin producing ability was quantified for343isolatedstrains by liquid fermentation with HPLC.69.19%of the tested isolates were toxigenic A. flavusand the percent of atoxigenic A. flavus was30.81%. The aflatoxin producing potential of A. flavusisolates significantly varied among the districts and agroecological zones of China. A. flavusisolate of the Southeast coast zones had the largest aflatoxin producing potential (averagedaflatoxin production was5836ng·mL^-1), the second was Yellow River Basin, the third wasNortheast area and the last was Yangtze River Basin. Incidence of toxigenic A. flavusisolates(>1000ng·mL^-1) was largest in the Southeast area （54.4%）, the second was Yellow RiverBasin,（31.8%）, the last was Yangtze River Basin （10.2%）.The distribution of the toxigenic andatoxigenic A. flavus isolates varied among the districts and agroecological zones of China.Incidence of atoxigenic A. flavus isolates was largest in the Yangtze River Basin （40.2%）, thesecond was Northeast area （36.4%）. The last was Southeast coast zones （15.7%）.5. Estimated the risk of aflatoxin contamination of peanut. Yangtze River Basin had thelargest aflatoxin producing potential in1g soils (1627544ng·mL^-1in1g soils), the second wasNortheast area (1117594ng·mL^-1), the third was Yellow River Basin (18602ng·mL^-1). The lastwas Northeast area (4478ng·mL^-1). So highest risk of aflatoxin contamination of peanut was inYangtze River Basin, the lowest was Northeast area.6. Established ISSR-PCR method for genetic diversity of A. flavus. The optimal reactionsystem of ISSR-PCR amplification was optimized. Of the100primers, the primers UBC834,UBC809, UBC817, UBC895, and UBC899were selected for for genetic diversity of A. flavus.The range of genetic similarity coefficients was from0.59to0.90. The study showed that theISSR technology is an effective molecular approach for studying diversity of A. flavus.7. Mastered the genetic diversity of A. flavus in soils of peanut fields in four agroecologicalzones of China. The genetic diversity of343A. flavus isolates was researched with ISSR. Thecluster analysis showed that the ISSR groups were closely related with geography origin. Theisolates were classified into four large groups. All the strains of Northeast area were in group Ⅰ,and all the strains Yellow River Basin were in group IV.88.9%strains of Yangtze River Basinwere in group Ⅱ. All the strains of Yellow River Basin excepted for8strains were in group Ⅲ.The ISSR groups were not notable correlation with sclerotia character and aflatoxin production.更多还原