基于Illumina MiSeq测序平台分析长期不同施肥处理对黑土真菌群落的影响

Effects of long-term different fertilization on soil fungal communities in black soil based on the Illumina Mi Seq platform

【目的】不合理施肥所引发的土壤环境问题逐渐成为制约我国农业可持续发展的重要因素之一,土壤真菌作为一类重要的土壤微生物,研究长期施肥对土壤真菌多样性及群落分布格局,探讨其理化因子对真菌群落结构的影响具有一定意义。【方法】本研究以东北黑土玉米田长期定位施肥试验(1984–2017)为基础,通过常规分析和Illumina Mi Seq高通量测序技术,分析长期施肥对黑土玉米田土壤养分含量和真菌群落结构变化的影响。【结果】长期施用氮肥明显降低土壤p H,却增加了玉米产量,秸秆与化肥配施可以增加土壤有机质和全氮的含量。稀释曲线结果表明长期施肥降低了土壤真菌序列的丰度和均匀度,并且在秸秆与化肥配施中序列数最低;在优势菌群中,共检测出5个已知真菌门,分别是子囊菌门(Ascomycota)、担子菌门(Basidiomycota)、接合菌门(Zygomycota)、球囊菌门(Glomeromycota)和壶菌门(Chytridiomycota),子囊菌门占总序列平均值的57.0%,并且在氮磷钾配施高量秸秆有机肥(NPK+S0.5)的土壤中,子囊菌门丰度高达70.35%。在土壤真菌属水平的物种丰度分析中,共检测出109个已知真菌属,Humicola、Fusarium、Verticillium、Mortierella这4个菌属为优势菌属;Chaetomium、Trichocladium、Podospora、Preussia 4个菌属在秸秆与化肥配施处理中丰度较高,并同属一个分支聚类。从多样性指数分析得出,秸秆与化肥配施可以增加物种丰度和群落多样性;从热图分析可知,施用氮肥和不施用氮肥处理间真菌群落组成存在明显差异。RDA分析中,土壤理化性质影响着土壤真菌群落结构,尤其是土壤的p H、全量氮磷钾(T-N、T-P、T-K)、有效磷钾(A-P、A-K)和铵态氮(NH4+-N)浓度是重要环境因素。【结论】因此,施用氮肥虽然增加了产量,但也造成土壤酸化,真菌数量增加,其丰富度和多样性明显降低。而秸秆与化肥配施可以维持土壤健康生态环境和真菌群落多样性。

[Objective] Soil environmental problems caused by irrational fertilization are gradually becoming one of the important factors that restrict the sustainable development of agriculture in China.Soil fungi is a kind of important microorganisms in soil.Hence,it is significant to study the diversity and community distribution patterns of soil fungi under long-term fertilization and investigate the physical and chemical factors on the fungal community structure.[Methods] The black soil under the long-term fertilization experiment（1984–2017） in the northeast of China was adopted to be analyzed by routine analysis and Illumina Mi Seq high-throughput sequencing.And then,the effects of long-term fertilization on soil nutrient contents and fungal community structures in black soil corn fields were studied.[Results] The results showed that the long-term application of nitrogen fertilizer significantly decreased p H in soil,but increased the yield of maize.The combined application of straw and fertilizer can increase the content of soil organic matter and total nitrogen.The results of operational taxonomic unites（OTU） curves showed that the long-term fertilization reduced the abundance and evenness of soil fungal sequences,and there was the lowest sequence number in the combined application of straw and fertilizer.Among the dominant communities,it has been found a total of five known fungal Phylum,Ascomycota,Basidiomycota,Zygomycota,Glomeromycota,Chytridiomycota.Among of them,Ascomycota accounted for not only 57.0% in the average of the total sequence,but also 70.35% in the soils with Fertilizer add high amount of straw treatment（NPK＋S0.5）.In the analysis of species abundance in soil fungal genus level,it has detected 109 known fungi.Humicola,Fusarium,Verticillium and Mortierella were the dominant genus.In addition to that,it was found that the abundance of Chaetomium,Trichocladium,Podospora and Preussia were high in the combined application of straw and fertilizer,and it all belong to a branch cluster.According to the analysis of diversity index,it can suggest that the combined application of straw and fertilizer can increase species abundance and community diversity.According to the Heatmap analysis,it can indicate that there were obviously different in the fungal community between the nitrogenous and non-nitrogenous fertilizers.In Redundancy analysis（RDA）,the soil physical and chemical properties affected the community structure of soil fungi,especially soil p H,total nitrogen,total phosphorus and total potassium（TN,TP,TK）,available phosphorus and available potassium（AP,AK） and ammonium nitrogen（NH4＋-N） is an important environmental factor.[Conclusion] The application of nitrogen fertilizer increased the yield.however,it caused soil acidification.Meanwhile,the application of nitrogen fertilizer increase the number of fungi,but the richness and diversity of fungi was decreased.Nevertheless,the combined application of straw and fertilizer can maintain soil healthy ecological environment and fungal community diversity.