【作者】 马旭；

【导师】 安莲英； 曹坳程；

【作者基本信息】 成都理工大学 ， 分析化学， 2010， 硕士

【摘要】 本文以北京市通州区大棚蔬菜试验田土壤为材料,运用现代生物化学手段,采用BIOLOG微平板与磷脂脂肪酸分析相结合的方法,探讨了熏蒸剂氯化苦和硫酰氟对农田土壤微生物群落结构和功能多样性的影响,对群落系统受到熏蒸剂扰动后的恢复能力进行了初步评价。本研究所获主要结论如下:1.采用BIOLOG方法系统研究了熏蒸剂氯化苦施用对土壤微生物功能多样性的影响以及氯化苦胁迫下土壤微生物功能恢复情况。研究结果表明,氯化苦处理后,土壤微生物的丰富度、均匀性和多样性均受到一定程度地影响。第一次取样,三个浓度氯化苦处理土壤的McIntosh指数与对照相比差异显著,分别降低72.31%、95.73%、97.33%,表明物种均匀性受到不同程度地影响,并且随浓度递增而加大;Simpson指数分别降低39.36%、45.93%、21.96%,说明氯化苦的添加降低了土壤微生物种群的生物多样性;而从Shannon指数来看,800mg/kg浓度处理过的土壤微生物的该指数值与对照相比增加6.66%,而80mg/kg浓度处理的该指数值与对照相比无显著差异,表明随着施药浓度的增大开始出现一定的耐受能力,生物量有所增大,丰富度指数有所增加。7d后取样结果表明,随着时间的推移,三指数都出现了波动,40mg/kg浓度处理的土壤其McIntosh指数值下降,但变化不显著,而80mg/kg和800mg/kg处理土壤的McIntosh值有小幅度的上升;高、中、低浓度处理土壤的Simpson多样性指数值均呈下降趋势,且减幅不显著;而Shannon丰富度指数经浓度梯度处理后均下降,其幅度不明显。AWCD值、Simpson指数、McIntosh指数和Shannon指数的变化能够较好的表征氯化苦胁迫下土壤微生物的功能抗性。均一性指数McIntosh能有效地指示污染环境的微生物学变化,在整个处理期间,该指数变化趋势与AWCD值变化值基本一致,表明AWCD值和McIntosh指数的表征效果较好。2.土壤微生物在原始状态下以糖类和氨基酸类为基础碳源进行代谢。氯化苦施用初期,对土壤微生物群落利用碳源的能力产生抑制作用。随着土壤培养时间的增加,土壤微生物逐渐适应了氯化苦,导致氯化苦对土壤微生物群落碳源利用能力的抑制程度减弱,土壤中优势种群大量繁殖,微生物对碳源的利用类型也逐渐增多。培养后期,随着土壤中内源性碳源的消耗,土壤中微生物种群出现功能性衰减,多样性降低,对BIOLOG中碳源的利用能力也减弱。80mg/kg和800mg/kg氯化苦对氨基酸、羧酸、胺类碳源的利用影响较大。4种样品中微生物生理代谢活性和碳代谢类群变现出明显差异,形成各自独特的微生物类群。3.在BIOLOG研究基础上,采用磷脂脂肪酸(PLFAs)方法,对熏蒸剂氯化苦和硫酰氟施用后土壤微生物群落结构变化进行了研究。研究发现,土壤中检测到PLFAs从C13到C20共计16种,包括饱和脂肪酸(16:0,18:0,20:0)、支链脂肪酸(i13:0,a14:0,i15:0,i16:0,a16:0,9Me18:0)、环丙烷脂肪酸(cy18:0)、单不饱和脂肪酸(16:1ω7c,18:1ω9c)和多不饱和脂肪酸(18:2ω6,9、22:4、20:3)。根据每种脂肪酸占总脂肪酸含量的百分数得出,偶数碳饱和脂肪酸的相对含量为41.97%,是土壤中含量最丰富的脂肪酸种类,其次是单不饱和脂肪酸和支链脂肪酸,相对含量分别为29.18%和13.22%。其中,饱和脂肪酸16:0和18:0、单不饱和脂肪酸18:1ω9c和16:1ω7c、支链脂肪酸a14:0为优势脂肪酸。多不饱和脂肪酸的相对含量较低,仅占到总PLFA的9.76%,而环丙基脂肪酸的相对含量最低,不到总PLFA的5%。通过对特征脂肪酸的分析表明,在所研究的农田生态系统中,真菌在该土壤基质中占有优势,并且在5～20cm土壤中好氧细菌为优势菌种。4.两种熏蒸剂氯化苦和硫酰氟对土壤微生物群落结构会产生不同程度的影响。研究发现,熏蒸剂处理过的土壤中脂肪酸a14:0、i15:0、i16:0(革兰氏阳性菌),16:1ω7c(革兰氏阴性菌),18:2ω6,9(真菌)的相对含量均有所下降,544mg/kg(SF64)的硫酰氟与212.48mg/kg(CP64)的氯化苦使i15:0/16:0下降幅度分别达到73.4%和59.8%,可以推断高浓度的气态熏蒸剂硫酰氟对细菌的杀灭能力更强。同时,硫酰氟和氯化苦施药后,16:1ω7c(革兰氏阴性菌)的降幅分别为79%和84%,a14:0、i15:0、i16:0(革兰氏阳性菌)的平均降幅分别为51.7%和68.4%。以上结果表明,在熏蒸剂的胁迫下,革兰氏阳性菌的生存能力较革兰氏阴性菌强。更多还原

【Abstract】 As a sort of wide-spectrum fungicides,application of fumigants to agricultural soils before planting of high-value cash crops has been widely accepted by agriculture-related practitioners for decades due to its high efficiency in controlling soilborne pathogens,nematodes,rodents and weeds,but recently concerns have been raised over its pollution to the environment and potential hazards to the human health that might in turn influence the whole balance of ecosystem. To assess the influence of fumigants on soil quality,studies were conducted concerning its impact on soil microbial community function and structure.The results were summarized as follows:1. Soils were sampled from experimental plots of Chinese Academy of Agricultural Sciences,Tongzhou District ,Beijing.Microbial functional diversity and functional resilience after chloropicrin application were estimated with BIOLOG Eco-microplates.The results showed that the richness,evenness and diversity of soil microorganisms ,to some extent,were affected after chloropicrin treatment.The fact that the McIntosh index after chloropicrin application decreased by 72.31%,95.73% and 97.33% respectively in contrast to control soils indicated that species evenness were influenced while the magnified effects were observed as concentration increased.The Simpson index decreased by 39.36%,45.93% and 21.96% respectively showed the reduction of biodiversity after chloropicrin application.Soil Shannon index increased by 6.66% after application with 80mg/kg chloropicrin in contrast to control soils while the index differed less significantly with controls after treatment with 800mg/kg chloropicrin,which demonstrated that microbial functional diversity recovered,biomass and richness of microorganisms rising as concentration increased.Sampling after 7 days showed that all three indics fluctuated as time passed.McIntosh index decreased slightly after treatment with 40mg/kg chloropicrin,but figures rised after treatment with 80mg/kg and 800mg/kg.Simpson diversity indices of soil treated with three concentration gradients dropped less significantly.Shannon richness indics decreased in all treatments.AWCD and diversity index,such as Shannon,McIntosh and Simpson indics can be used to indicate the functional resistance to stress of chloropicrin.The fact that McIntosh evenness index whose tendency is in agreement with AWCD during the application can indicate the microbiology change of polluted environment showed that the best results can be obtained by using AWCD and McIntosh.2. Soil microorganisms metabolized mainly using carbonhydrates and amino acids as basic carbon sources.An obvious inhibitory effect was observed in microbial utilization of carbon substrates after the first introduction of chloropicrin.As increasing of incubation time,the decreased inhibitory effect of chloropicrin to microbial utilization of carbon substrates was observed due to the gradual adaptation of soil microbial community to chloropicrin.Soil dominant species multiplied substantially while the utilization type of microorganisms to carbon substrates increasing gradually.As comsuption of endogenous carbon source in soil,microbial species dropped functionally that resulted in the decrease of diversity and utilization of carbon substrates existing in Biolog wells.80mg/kg and 800mg/kg chloropicrin exerted strongly influence on uitilization of amino acids,carboxylic acids and amides. microbial physiological metabolism activity and carbon-metabolic species varied significantly and unique microbial communities were shaped in respective four soil samples.3. The effects of the applications of chloropicrin and sulfuryl fluoride on the change of microbial community structure exposed to two fumigants was studied under microcosm conditions using phospholipid fatty acid analysis(PLFA).The results showed that in control soils PLFAs within carbon number from 13 to 20 were detected,including saturated fatty acids(16:0,18:0,20:0),branched chain fatty acids(i13:0,a14:0,i15:0,i16:0,a16:0,9Me18:0),cyclopropane fatty acid(cy18:0),monounsaturated fatty acids(16:1ω7c,18:1ω9c) and polyunsaturated fatty acids(18:2ω6,9、22:4、20:3).The results based on the mole percentage of gross fatty acids showed that saturated fatty acids within even carbon number,which were the most abundant fatty acids type in soils,accounted for 41.97%.The contents of monounsaturated fatty acids and branched chain fatty acids which accounted for 29.18% and 13.22% respectively were relatively small in amount.Of those,saturated fatty acids(16:0,18:0),monounsaturated fatty acids(18:1ω9c,16:1ω7c) and brached chain fatty acid(a14:0) were dominant fatty acids.Polyunsaturated fatty acids were less popular accounting for merely 9.76%;and cyclopropane fatty acid accounting for less than 5% was the lowest proportion of gross fatty acids.Analysing the specific fatty acids indicated that in our study areas of agroecosystem, fungus were in large proportion and the dominant species were aerobic bacteria at depths from 5 to 20cm in the subsurface soil.4. Influene of chloropicrin and sulfuryl fluoride on soil microbial community structure was different to the extent.The study found that in soils treated with fumigants the relative contents of the fatty acids a14:0,i15:0 and i16:0 generally referred to as the biomarkers of Gram-positive bacteria, 16:1ω7c referred to the biomarkers of Gram-negative bacteria and the biomarkers of fungi, 18:2ω6,9 had decreased,while the reduction of the ratio of i15:0/16:0 in soils treated with 544mg/kg sulfuryl fluoride and chloropicrin,by 73.4% and 59.8% respectively,had been observed as to assume that gaseous sulfuryl fluoride had stronger lethality to bacteria than liquid chloropicrin. Meanwhile, after application of sulfuryl fluoride and chloropicrin, 16:1ω7c(Gram-negative bacteria) had decreased by 79% and 84% respectively, a14:0,i15:0 and i16:0(Gram-positive bacteria) had decreased by 51.7% and 68.4% respectively.The results showed that under stress of fumigants the survival rate of Gram-positive bacteria was higher than Gram-negative bacteria.更多还原