【作者】 靳学慧；

【导师】 翟瑞常；

【作者基本信息】 黑龙江八一农垦大学 ， 作物栽培学与耕作学， 2015， 博士

【摘要】 氮素是马铃薯生长发育不可缺少的大量必需元素,要获得高产就必须供给充足的氮素营养,但高施氮量容易导致马铃薯晚疫病的严重发生而造成大幅度减产。从健身防病的理念出发,探索既能获得较高产量又能充分发挥植株抗性的施氮水平是非常必要的。本研究采用室内盆栽与田间小区控制实验相结合的研究方法,综合运用PCR-DGGE技术、生理生化检测技术、15N同位素示踪技术、微生物分离培养法等检测和研究手段,探讨了不同施氮水平对马铃薯根际土壤微生物数量及丰富度、土壤酶活性、植株体内抗性相关酶活性及抗性相关物质含量、晚疫病发生程度和产量的影响,确定出较为适宜的施纯氮水平范围为90～135 kg·hm-2,最佳施纯氮水平为135 kg·hm-2。具体研究结果如下：1.有46.4%～56.3%细菌、69.0%～82.1%真菌和53.6～61.2%放线菌分布于0～10cm土层,仅有17.2～21.8%细菌、4.6%～8.1%真菌和9.2～18.1%放线菌分布于20～30cm土层。土壤酶活性在土壤中垂直分布与微生物是一致的。在马铃薯生长时期,土壤细菌总量、芽孢菌和放线菌数量呈先升后降的趋势,播种后61d细菌数量最多,75d放线菌数量最多；真菌总量和木霉菌数量以播种后43d最多,后期逐渐减少；镰孢菌和立枯丝核菌数量呈缓慢增加趋势,但变化幅度不大。马铃薯根际土壤细菌群落的DGGE图谱显示,各生育期均有各自特定的DGGE图谱条带结构。土壤微生物数量的垂直分布规律及其时间变化动态趋势与施氮水平无关。2.适量施入氮素有利于土壤中有益微生物的增加而抑制植物病原菌的数量,并能增加土壤细菌的丰富度,提高土层中脲酶、蔗糖酶、磷酸酶和过氧化氢酶活性。施纯氮90～135kg·hm-2水平,马铃薯各生育期各耕层根际土壤中细菌总量、真菌总量、放线菌以及芽孢菌和木霉菌数量显著增加,而镰孢菌和立枯丝核菌数量显著减少,尤其是对自然状况下细菌总量和芽孢菌数量偏少的深层土壤、马铃薯生育前期与后期的促进效应更加明显,但不同生育时期有所波动。施纯氮157.5kg·hm-2以上时,细菌总量和芽孢菌数量的波动较大,真菌总量、镰孢菌和立枯丝核菌数量增加幅度较大,木霉菌增加量相对较少。各施氮处理之间根际土壤细菌群落DGGE图谱条带数目和亮度存在较大差异,以施纯氮135 kg·hm-2水平的谱带数目最多,多样性指数和均匀度指数最高,在播种后61d和75d显著高于其它施氮水平。施纯氮67.5～135.0 kg·hm-2水平有利于提高土壤脲酶的活性,施纯氮112.5～157.5kg·hm-2水平有利于提高各生育期土壤磷酸酶、过氧化氢酶和生育前期蔗糖酶活性。在马铃薯生长发育期间,各种土壤酶活性均呈先升后降的趋势。3.肥料15N在土壤中垂直分布随着时间推移呈动态变化。播种后45d时0～10cm土层中肥料氮素的含量(Nspf)和肥料氮素占土壤全氮的百分比(Ndffs)很大,播种后75d时Nspf和Ndffs很小；10-20cm土层中Nspf和Ndffs缓慢下降；20-30cm土层中Nspf和Ndffs呈先升后降趋势。Nspf和Ndffs大小对土壤细菌总量、芽孢菌数量、真菌总量、木霉菌数量及磷酸酶活性有一定影响,而对放线菌和其它土壤酶活性影响不大。土壤全氮含量对土壤磷酸酶和蔗糖酶活性有一定影响。4.在自然发病情况下,施入氮素能增加植株体内的多酚氧化酶(PPO)、苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)、β-1,3-葡聚糖酶和几丁质酶活性,并能提高总酚、绿原酸、类黄酮、可溶性蛋白和可溶性糖含量。以施纯氮135 kg·hm-2作用效应较强且较稳定,施纯氮180 kg·hm-2水平的促进效应也很强但不稳定。不同施氮水平对植物体内的过氧化氢酶(CAT)活性、超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量有一定的影响,但不稳定。人工接种病菌后,品种间存在差异。品种“大西洋”体内POD响应较快,施N区CAT活性显著高于不施氮,总酚含量响应慢但较稳定,可溶性蛋白含量增加值较高；品种“克新18号”体内POD响应较快,施N区CAT活性显著低于不施氮,总酚含量响应快且较稳定,可溶性蛋白含量增加值较高；而品种“克新1号”体内POD响应很慢,施N区CAT活性显著高于不施氮,总酚含量响应快但较不稳定,可溶性蛋白含量增加值较低。5.盆栽和小区实验均显示,施纯氮135 kg.hm-2水平发病程度轻,产量最高且薯块较大；施纯氮180kg·hm-2水平发病程度重,产量低且薯块较小。较为适宜的施纯氮水平范围为90～135kg·hm-2,最佳施纯氮水平为135 kg·hm-2。更多还原

【Abstract】 Nitrogen is one of the major elements essential for the growth and development of Potato (Solanum tuberosum), therefore adequate nitrogen nutrition must be supplied to achieve high yield, whereas high dosages of nitrogen application is easy to cause the serious occurrence of potato late blight, resulting in a substantial reduction of the yield. From the health and green plant protection point of view, it is necessary to explore the appropriate nitrogen level for both obtaining a higher yield and giving full play to the nitrogen fertilizer to plant resistance. In this study, indoor potted and field plots controlled experiments were combined, integrated research methods such as:PCR-DGGE technique, physiological and biochemical detection means,15N isotope tracer technique, microbial isolation and culture method were used, the effects of different nitrogen levels on potato rhizosphere microbial quantity and richness, soil enzyme activities, in vivo resistance related enzyme activities and the content of resistance related substances, the degree of Phytophthora infestans and its effect on yield were discussed, and the appropriate level of nitrogen application range of 90～135 kg · hm-2 and the optimal nitrogen application level of 135 kg · hm-2 were determined. The results of this study are as follows:1. There are 46.4%～56.3% bacteria,69.0%～82.1% fungi, and 53.6%～61.2% actinomycetes were distributed in 0～10cm soil layer, only 17.2%～21.8% bacteria,4.6%～8.1% fungi, and 9.2%～18.1% actinomycetes distributed in 20～30 cm soil layer. The vertical distribution of soil enzyme activity is consistent with the distribution of microorganisms in the soil. During the potato growth period, the amount of soil bacteria, Bacillus spp. and Actinomycetes increased first and then decreased, soil bacteria peaked at 61 d after sowing and Actinomycetes peaked at 75d after sowing, respectively; total number of fungi and Trichoderma spp. maximized on 43d after sowing, and then gradually reduced; the number of Fusarium spp. and Rhizoctonia solani was gradually increased, but the changes were not significant. The results of PCR-DGGE of potato rhizosphere soil bacterial communities showed that each different growth stages of potato had their own particular DGGE bands profiles. This vertical distribution of soil microorganisms and its dynamic changes over time were unrelated to nitrogen levels.2. The appropriate amount of nitrogen was helpful to increase beneficial soil microorganisms and inhibit the number of plant pathogens, to increase the richness of the soil bacteria, and to improve the activities of soil urease, invertase, phosphatase and catalase. When the supplied nitrogen level was 90～135 kg·hm-2, the total amount of bacteria, fungi, Actinomycetes, Bacillus spp and Trichoderma spp. was significantly increased in each potato rhizosphere soil tilth layers at different growth stages, while the number of Fusarium spp. and Rhizoctonia solani significantly reduced, especially for deep soil layer where total number of bacteria and Bacillus spp spores was smaller under natural conditions, and the improving effects on early and late growth stages of potato were more obvious, but fluctuated at different growth stages. When the nitrogen level was above 157.5 kg · hm-2, the total number of bacteria and Bacillus spp showed fluctuations, the total number of fungi, Fusarium spp. and R. solani increased greatly, and a relatively small increase in the amount of Trichoderma spp. was presented. There were big differences among band numbers and brightness of DGGE profiles of rhizosphere soil bacteria communities under different nitrogen treatments, the number of DGGE bands was at its maximum when the nitrogen level was at 135 kg · hm-2, the same as the richness, diversity and evenness index, and these parameters were significantly higher on 61 d and 75d after sowing than other nitrogen levels. Nitrogen level at 67.5～135.0 kg · hm-2 was helpful to improve the soil urease activity, nitrogen of 112.5～157.5 kg· hm-2 was helpful to improve the soil phosphatase and catalase activities at all growth stages and sucrase activity at early growth stage. During the potato growth and development, a variety of soil enzyme activities showed a trend of increased first and then decreased.3. The vertical distribution of 15N fertilizer in the soil showed dynamic changes over time. The content of nitrogen fertilizer in the soil (Nspf) and the percentage of nitrogen fertilizer in total soil nitrogen (Ndffs) was high in 0～10cm shallow soil layer on 45d after sowing, and smaller on 75d after sowing; Nspf and Ndffs decreased slowly in 10-20cm soil layer; while in 20-30cm soil layer, Nspf and Ndffs increased first and then decreased. The value of Nspf and Ndffs had influences on the total number of the soil bacterium, Bacillus spp quantity, total fungi, Trichoderma spp. quantity and phosphatase activity, while had little effects on Actinomycetes and other soil enzyme activities. Total nitrogen content of the soil had a certain impact on soil phosphatase and sucrase activities.4. Under the natural occurrence of diseases, nitrogen was applied to increase the plants PPO, PAL, POD,β-1,3- glucanase and chitinase activities, and to improve the total phenols, chlorogenic acid, flavonoids, soluble protein and soluble sugar content. The effect of nitrogen application of 135 kg·hm-2 was stronger and more stable, while the effect of nitrogen application of 180 kg·hm-2 was also very strong but unstable. The different levels of nitrogen fertilizer application had certain effects on CAT and SOD activities and MDA content, but not stable. After artificial inoculation of P. infestans, there were differences among the testing potato varieties. The potato variety Daxiyang had a rapid response in POD activity, and the activity of CAT was significantly higher than that without nitrogen application, the response of total phenol content was slower but stable, and the increase of soluble protein content was relatively high; the variety Kexin 18 also had a fast response in POD activity, the activity of CAT was significantly lower than that of no nitrogen, the response of total phenol content was fast and stable, and the increase of soluble protein content was significant; whereas for variety Kexin 1, the POD response was slow, the activity of CAT was significantly higher than that of no nitrogen, the response of total phenol content was fast and but less stable, and the increase of soluble protein content was relatively low.5. Potted plants and field experiments showed that the pure nitrogen application of 135 kg· hm-2 resulted in lower severity of disease incidence, the highest yield and larger tubers; nitrogen of 180 kg · hm-2 resulted in higher severity of disease incidence, lower yield and smaller tubers. More appropriate nitrogen application range was 90～135 kg·hm-2, and the optimum nitrogen application level was 135 kg· hm-2.更多还原