再植枸杞根际真菌群落对长期连作的响应研究

Effects of Long-term Monocropping of Lycium barbarum L. on Function and Composition of Fungal Community in Rhizosphere of Replanted Lycium barbarum L.

受枸杞自身种植特点和道地产区土地资源限制,连作障碍已成为制约宁夏枸杞产业可持续发展的主要因素之一,导致严重的经济损失和生态问题。前期研究表明,连作能够显著影响再植枸杞根际土壤细菌的群落结构和多样性,但就连作对真菌群落结构和功能的影响目前仍不清楚。本文利用实时荧光定量PCR和高通量测序技术,研究连作对再植枸杞根际真菌群落丰度及多样性的影响。实时荧光定量PCR分析表明,与对照样地相比,连作显著促进再植枸杞根际及非根际土壤中细菌和真菌的丰度。但连作对真菌的促进作用明显高于细菌,导致细菌/真菌比例失衡,使再植枸杞根际及非根际土壤微生物环境偏向于真菌型。对测序结果的分析发现,所测定样地中枸杞根际及非根际土壤中的优势真菌门分别为子囊菌门、担子菌门、接合菌门、壶菌门及球囊菌门,其中连作地再植枸杞根际子囊菌门的相对丰度较对照样地显著降低,而接合菌门的相对丰度则显著增加(P<0.05)。FUNGuild真菌功能预测也证实连作显著抑制再植枸杞根际土壤丛枝菌根真菌的相对丰度(P<0.05)。基于距离的冗余分析(db-RDA)结果表明,土壤pH、电导率、硝态氮和有效磷含量是影响枸杞根际土壤真菌群落变化的主要因子(P<0.05),而土壤硝态氮和有效磷含量则是解释非根际土壤真菌群落变化的主要因子(P<0.05)。这些结果说明长期施用化肥可能是改变连作地再植枸杞根际土壤真菌群落结构及枸杞-真菌互作关系的主要因素之一。这一研究结果为理解枸杞连作障碍的形成机制提供理论基础。

【Objective】Owing to the growth characteristics of wolfberry(Lycium barbarum L.)per se and the limitation of soil resources in the traditional wolfberry plantation regions,long-term monocropping of the plants has become one of the main factors hindering sustainable development of the industry of wolfberry production and causing serious economic loss and potential eco-problems.Previous studies found that monocropping could significantly affect diversity and composition of the bacterial communities in the rhizosphere of replanted L.barbarum,however,they did not deal much with responses of the fungal communities therein to the long-term monocropping.【Method】Soil samples were collected from the topsoil layer(0~20 cm)near the plants in the longterm monocropping wolfberry fields andrandomly in the control(no wolfberry cultivation history)with a soil auger(n=3).In order to haveenough rhizosphere soils,root segments from at least ten individual plants were collected.Rhizosphere soil adhered to the roots were collected by washing with 10 mmol·L-1 NaCl solution and centrifuging.The genomic DNA was extracted from 0.1 g wet rhizosphere and bulk soil sample using the PowerSoil DNA isolation Kit,separately.The ITS2 region was amplified and sequenced with the Illumina HiSeq technique.Then metrics ofαdiversity(Shannon index and OTU richness)and weighted and unweighted UniFrac distances were calculated with the aid of QIIME.And real time PCR was used to quantify abundance of the bacterial and fungal populations in each DNA samples.【Result】Real time PCR found that monocropping significantly increased abundances of the bacterial and fungal communities in both bulk soil and rhizosphere of L.barbarum as compared with what was found in the control fields(P<0.05).However,the effect was much higher on fungi than on bacteria,and hence disturbed the balance between bacteria and fungi in ratio,thus making the microbial environments in the rhizosphere and bulk soil tend to be fungal dominant.Sequencing analysis discovered that Ascomycota,Basidiomycota,Zygomycota,Chytridiomycota,and Glomeromycota were the dominant phyla in all the samples,and that the relative abundance of Zygomycota in the rhizosphere of replanted wolfberry was significantly lower than that in the control,whereas an opposite trend was found with Glomeromycota(P<0.05).And FUNGuild predication of fungal functions demonstrated that the relative of arbuscular mycorrhizal fungi(AMF)in the rhizosphere of replanted wolfberry in the monocropping fields decreased significantly relative to what in the control fields(P<0.05).Non-metric multidimensional scaling analysis(NMDS)revealed that the fungal communities in bulk soil and rhizosphere of replanted wolfberry were quite similar to each other in the monocropping fields,but not so in the control,although they did not very much in diversity from those in the control.Furthermore,distance based redundancy analysis indicated that soil pH,electrical conductivity,and contents of nitrate nitrogen and readily available phosphorous were the main factors affecting soil fungal communities in the rhizosphere of wolfberry,while contents of nitrate nitrogen and readily available phosphorous were the ones explaining changes in soil fungal communities in the bulk soils(P<0.05).【Conclusion】All the findings in this study suggest that long-term fertilization in the monocropping wolfberry fields might be one of the major causes driving the succession of soil fungal communities in the rhizosphere of replanted wolfberry and altering the interaction between wolfberry and soil fungal community.Therefore,the study may shed some new light on mechanism of the forming of the hazard of long-term monocropping of wolfberry to sustainable development of the industry of wolfberry production.