【作者】 杨帆；

【导师】 胡滨； 张丽萍； 董超；

【作者基本信息】 河北工业大学 ， 生物工程（专业学位）， 2019， 硕士

【摘要】 随着马铃薯主粮化战略的实施,马铃薯现已成为我国第四大粮食作物。作为马铃薯主产区之一的河北省张家口具有悠久的马铃薯种植传统,马铃薯一直是该地区重要的粮食作物和经济作物。近年来由于轮作倒茬困难,不合理的施肥用药及常年种植单一品种致使田间马铃薯真菌性病害普遍发生,品质下降,增施复合肥产量难再提高。以生防菌及其代谢产物为核心的生物防治是解决这一问题的有效途径,本文以实验室前期筛选到的一株高效拮抗菌株BA-26为依托,以16SrDNA序列分析结合形态特征和生理生化特征对其进行分类鉴定,通过室内抑菌活性测定、代谢产物稳定性研究、离体防病实验以及盆栽实验明确其田间应用的可行性。从不同时期的土壤理化指标、酶活、微生物群落组成及马铃薯农艺性状和品质指标等方面对BA-26田间应用效果进行了系统性研究。取得的主要研究结果如下:1.通过16SrDNA序列分析结合形态特征和生理生化特征将菌株BA-26鉴定为解淀粉芽孢杆菌Bacillus amyloliquefaciens,该菌株马铃薯病原真菌抑菌谱广,对所试8种马铃薯病原真菌均表现出较强的拮抗作用,其中对茄链格孢菌、茄病镰刀菌、燕麦镰刀菌的抑制率大于80%;胞外代谢产物稳定性强,经100℃处理30 min、紫外照射8 h、pH2~10酸碱处理后抑菌活性维持80%以上;室内防病实验表明BA-26无菌滤液对茄病镰刀菌抑制率为91.3%;每盆施用20 mL浓度为4.0×109 cfu/mL的BA-26发酵液可使马铃薯根长增加12.52%,须根数增加2.1倍,株高提高48%,叶片中叶绿素含量提升19.7%。2.马铃薯连作和轮作种植模式相比,马铃薯商品性、土壤理化和生物学指标均具有极显著差异。连作导致土壤酸化,土壤孔隙度降低,不同时期土壤有机质和碱解氮含量变少,而土壤速效磷和速效钾含量变高,土壤酸性磷酸酶和蔗糖酶活性显著降低,土壤细菌多样性和丰度下降,真菌多样性降低,丰度变高,链格孢属和镰刀菌属均是两种种植模式的优势真菌属,连作导致它们的相对丰度变高,生长期放线菌门、酸杆菌门、绿弯菌门和疣微菌门丰度下降,变形杆菌门和不明菌门丰度变高,马铃薯连作果实薯皮粗糙,病斑较多,芽眼较深,商品性变差。3.马铃薯连作实验田,BA-26和有机肥联用及单施有机肥均能起到改善土壤结构活化土壤养分的作用,BA-26和有机肥联用效果更佳,和对照相比,BA-26和有机肥联用使单株产量提高6.67%,果实中可溶性糖和Vc含量分别增加50%和23.08%。幼苗期土壤EC值降低65.37%,盛花期土壤速效磷、速效钾和有机质分别提高52.24%、56.4%和9.58%,提高盛花期土壤真菌多样性,降低真菌丰度。土壤排名前十优势真菌属相对丰度分析结果表明幼苗期、盛花期和收获后链格孢属的相对丰度分别降低39.01%、60.8%和93.1%,幼苗期和收获后土壤镰刀菌属相对丰度分别降低31.65%和32.44%。4.马铃薯轮作实验田,BA-26和有机肥联用替代10%化肥使产量提高了7.96%,果实中可溶性糖和Vc含量分别提升了15.58%和28.9%,替代15%化肥用量在不影响作物产量的前提下,使果实中可溶性糖含量提升6.49%,两处理显著提高了生长期土壤有机质和碱解氮含量,并提高了马铃薯生长期土壤脲酶活性。土壤中可培养微生物统计结果表明,替代10%和15%化肥均使幼苗期和盛花期土壤真菌和放线菌数量降低,成熟期和收获期土壤真菌和放线菌数量增加。土壤真菌α多样性和Top10优势真菌属相对丰度研究表明替代10%化肥处理对幼苗期土壤真菌影响最剧烈,使幼苗期真菌丰度降低,排名前十的优势真菌属中8个真菌属的丰度均较对照显著降低,从而起到了调整土壤微生物群落结构的作用,可见BA-26和有机肥联用替代10%和15%化肥均为可行的施肥方案。更多还原

【Abstract】 With the implementation of the strategy of potato staple food normalization,potatoes have become the fourth largest food crop in China.Zhangjiakou,Hebei Province,one of the main potato producing areas,has a long tradition of potato planting.In recent years,because of the difficulty of rotational cropping,unreasonable fertilization and medication,and the single planting of the whole year have led to the widespread occurrence of fungal diseases in the field,which has led to a decline in the quality of the potato and an increase in the yield of compound fertilizer.Biocontrol technology with biocontrol bacteria and its metabolites as the core is an effective way to solve this problem.Based on a highly effective Antagonistic Bacterial Strain BA-26 screened in the laboratory,16 SrDNA sequence analysis combined with morphological and physiological and biochemical characteristics was used to classify and identify it.The feasibility of its application in the field was confirmed by indoor bacteriostasis test,metabolite stability study,in vitro disease prevention experiment and pot experiment.The physical and chemical indicators,enzyme activity,microbial community composition and potato agronomic characteristics and quality indicators of soils in different periods were systematically studied in order to clarify the field application effect.The main results are as follows:1.The strain BA-26 was identified as Bacillus amyloliquefaciens by 16 SrDNA sequence analysis,morphological characteristics and physiological and biochemical characteristics.The strain had a broad spectrum of inhibition against pathogenic fungus of potato,and showed strong antagonistic effects on the eight pathogenic fungus of potato tested.The inhibition rate of Alternaria solani,Fusarium solani,Fusarium avenaceum was more than 80%.The stability of extracellular metabolites was strong,and the antibacterial activity is maintained above 80% after treatment at 100 °C for 30 min,UV irradiation for 8 h,and pH 2-10 acid-base treatment.The indoor disease prevention experiment showed that the inhibition rate of BA-26 sterile filtrate against Fusarium solani was 91.3%.The potato root length,fibrous root number,plant height and chlorophyll content in leaves were increased by 12.52%,2.1 times,48% and 19.7% respectively by applying BA-26 fermentation broth with 20 mL concentration of 4.0×109 cfu/mL per pot.2.Comparing the continuous cropping and rotation cropping planting patterns of potato,it was found that there were significant differences in potato commerciality,soil physical and chemical indicators and biological indicators.Continuous cropping resulted in soil acidification and soil porosity decreased.Soil organic matter and alkaline hydrolysis N content decreased in different periods,while soil available phosphorus and available potassium increased,soil acid phosphatase and invertase activities decreased significantly,soil bacterial diversity and abundance decreased,fungal diversity decreased,and abundance increased.Both Alternaria and Fusarium were the dominant fungal genus of two planting patterns,and their relative abundance is increased by continuous cropping.The abundance of Actinomycetes,Acidobacteria,Chloroflexi and Verrucomicrobia decreased during growth period,while the abundance of Proteobacteria and Unknown Microflora increased.Continuous cropping of potatoes has rough skin,more spots,deeper bud eyes and worse commodity.3.In the continuous cropping experiment field of potato,BA-26 combined with organic fertilizer and single application of organic fertilizer could improve the soil structure and activate soil nutrients,but the combination of BA-26 and organic fertilizer had better effect.Compared with the control,BA-26 combined with organic fertilizer could increase yield per plant by 6.67%,soluble sugar and Vc content in fruit by 50% and 23.08%,respectively.The EC value of soil decreased by 65.37% in seedling stage,and the soil available phosphorus,available potassium and organic matter increased by 52.24%,56.4% and 9.58% in florescence stage,which increased soil fungal diversity and reduced fungal abundance in florescence stage.Relative abundance of dominant fungal genus in the top 10 of the soil rankings showed that the relative abundance of Alternaria decreased by 39.01%,60.8% and 93.1% at seedling stage,blooming stage and post-harvest stage,respectively,and that of Fusarium decreased by 31.65% and 32.44% at seedling stage and post-harvest stage,respectively.4.In the rotation cropping experiment field of potato,BA-26 and organic fertilizer were used to replace 10% chemical fertilizer,which increased the yield by 7.96%,and the soluble sugar and Vc content in the fruit increased by 15.58% and 28.9%,respectively.Under the premise of not affecting crop yield,BA-26 and organic fertilizer were used to replace 15% chemical fertilizer,which increased the soluble sugar content of fruits by 6.49%.Both treatments significantly increased Soil organic matter and alkaline hydrolysis N content in growing period,and increased soil urease activity in potato growing period.The statistic results of culturable microorganisms in soil showed that substitution of 10% and 15% chemical fertilizer reduced the number of soil fungi and Actinomycetes at seedling and blooming stage,and increased the number of soil fungi and Actinomycetes at maturity and harvest stages.The relative abundance of soil fungal α diversity and Top10 dominant fungal genus showed that the substitution of 10% chemical fertilizer had the most severe effect on soil fungi at seedling stage,which reduced the abundance of fungi in seedling stage.The abundance of 8 dominant fungi genera in the Top10 dominant fungal genus was significantly lower than that of the control group,which played a role in adjusting the soil microbial community structure.It can be seen that the combination of BA-26 and organic fertilizers to replace 10% and 15% fertilizers is a feasible fertilization scheme.更多还原