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大豆连作土壤线虫群落结构的影响

DOI: 10.11674/zwyf.2015.0422, PP. 1022-1031

Keywords: 连作,大豆,线虫群落,T-RFLP,定量PCR

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Abstract:

【目的】由根系活动引起的根际微生态系统的改变,特别是病原生物数量的增加是导致作物产生连作障碍的主要因素。其中,植生性病原线虫的危害是大豆连作障碍产生的重要原因之一。由于植生性病原线虫的存在往往受到其它营养类型线虫的影响,因而从线虫群落结构进行分析,不仅可以更好地反映不同营养类型的线虫之间的相互关系,而且能全面了解土壤的健康状况。本文利用末端限制性片段长度多态性分析(T-RFLP)和实时荧光定量PCR(qPCR)等分子生物学的方法,比较短期连作和长期连作线虫群落的差异,揭示长期连作大豆土壤线虫群落的变化规律,理解线虫群落与植物健康的关系,阐明线虫群落的变化在大豆连作障碍中的作用。【方法】首先,基于16srDNA的T-RFLP指纹图谱,分析土壤中线虫的物种丰富度和不同大小的末端限制性片段(T-RF)的相对丰度。然后,通过构建克隆文库和系统发育树,鉴定T-RF片段对应的线虫种类。最后,利用qPCR,采用绝对定量的方法确定线虫群落的大小。【结果】线虫的物种丰富度随着连作年限的增加呈逐渐降低的趋势。第1年物种丰富度最高,第3年的丰富度显著低于第1年,之后逐渐降低,9年之后保持不变。大豆根际土中共检测到16个T-RF,且大多数T-RF能从克隆文库中鉴定。其中,食细菌线虫(Acrobeloides)是最为丰富的线虫种类。在连作2~3年后,植物寄生线虫相对丰度增加,而在连作后期,植物寄生线虫相对丰度减少。非度量多维尺度分析(NMDS)示,第1年线虫群落与其余年限分开,而第2和第3年聚集较近,而连作9、11和13年后聚集较近。另外,线虫群落结构与pH、土壤有机质(SOM)、速效磷(AP)、细菌数量和真菌数量相关。线虫群落总丰度呈先增后降的趋势,最高值出现在第6年。线虫的基因拷贝数与土壤NH+4和染料木因浓度呈显著正相关,而与NO-3和细菌的基因拷贝数呈显著负相关。【结论】大豆根际土壤中,线虫群落丰度在连作第2~3年下降最为明显,到第6~9年有一定的恢复,但不能完全修复。大豆种植为第一,基线虫属(556bp)丰度最高。土壤功能正常,连作第2~3年后,摄食性线虫(555bp、558bp、560bp等)丰度增加,线虫浸染机会增加。

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