CN116333937B - 一株具有温室气体氧化亚氮减排功能的植物根际益生菌株及其应用 - Google Patents
一株具有温室气体氧化亚氮减排功能的植物根际益生菌株及其应用 Download PDFInfo
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Abstract
本发明公开了一株具有温室气体氧化亚氮减排功能的植物根际益生菌株及其应用。一株植物根际益生菌SQR9，该菌株属于Bacillusvelezensis，为革兰氏染色阳性菌，于2012年2月27日在中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏号CGMCCNO.5808。通过室内培养及盆栽试验测定SQR9的减排能力，发现该菌株减少土壤N2O排放的能力与其接种浓度相关，当浓度越高，减排能力越强。同时，菌株SQR9通过提高N2O还原速率实现土壤N2O减排。
Description
技术领域
本发明涉及一株植物根际益生菌，特别是涉及一株具有温室气体氧化亚氮减排功能的植物根际益生菌株及其应用。
背景技术
氧化亚氮(N2O)是一种重要的大气痕量气体，是三大温室气体之一，直接导致温室效应。氮肥施用导致土壤N2O排放增加，被认为是大气N2O浓度攀升的主要因素之一。据估计，在2007-2016年的十年间，人类活动引起的N2O排放占N2O总排放量(7.3TgN yr-1)的43％左右，其中，农业生产活动中大量氮肥施用导致的土壤N2O直接和间接排放约占52％。近年来，为追求农业高产和高经济效益，过量施肥引起土壤N2O排放量攀升，已成为全球氮循环研究和温室气体减排研究领域关注的热点。其中，我国菜地由于高氮肥施用、复种指数高等特点，成为农业土壤N2O排放关注的重点(Tian H,Xu R,Canadell J,et al.A comprehensivequantification of global nitrous oxide sources and sinks[J].Nature,2020,586(7828):248-256；Liu S,Lin F,Wu S,etal.A meta-analysis of fertilizer-inducedsoil NO and combined NO+N2O emissions[J].Global Change Biology,2017,23(6):2520-2532)。
在此背景下，优化可行策略来减少农田土壤N2O排放迫在眉睫。目前已经有很多减少农业土壤N2O排放的方法，包括调节水分管理、合理利用作物残渣以及施用特定化学物料等。近年来，生物质炭和硝化抑制剂等化学物质作为减少土壤N2O排放的有效手段被广泛报道。这些技术能够提高氮肥利用率、降低硝酸盐淋溶以及减少N2O排放。然而，这些调理剂普遍成本高且效果不一，还具有潜在的环境风险，限制了它们的广泛应用(Ji C,Li S,GengY,et al.Decreased N2O and NO emissions associated with stimulateddenitrification following biochar amendment in subtropical tea plantations[J].Geoderma,2020,365:114223；Lam S,Suter H,Mosier A,et al.Using nitrificationinhibitors to mitigate agricultural N2O emission:a double-edged sword[J].Global Change Biology,2017,23(2):485-489)。植物根际益生菌是一类能在植物根际存活定殖并具有促进植物生长或防控土传病害能力的有益微生物。目前国内外学者已在根际益生菌与植物促生方面取得了重要进展，发现其可通过活化养分、分泌植物激素、产生挥发性活性物质等方式促进植物生长。近年来，多项研究表明根际益生菌除具有提高作物产量的作用以外，还具有减少氮素损失和提高氮素利用率的功能(Wu S,Zhuang G,Bai Z,etal.Mitigation ofnitrous oxide emissions from acidic soils by Bacillusamyloliquefaciens,a plant growth-promoting bacterium[J].Global ChangeBiology,2018,24(6):2352-2365)。根际益生菌能提高作物的氮素养分吸收利用效率，降低土壤矿质氮积累；通过合作或竞争的关系与氮循环相关功能微生物发生互作，从而降低N2O排放。植物益生菌被广泛证实可定殖于植物根际，相较于传统减排技术，作为一种环境友好型的温室气体N2O减排措施，可在提高单产、改良土壤结构、构建健康高产土壤微生物区系的基础上同时实现减缓农业土壤N2O排放，减少农业源温室气体排放总量。
发明内容
本发明的目的在于提供一株植物根际益生菌SQR9，该菌株具有减少土壤氧化亚氮排放的功能。
本发明的目的可通过如下技术方案实现：
一株植物根际益生菌SQR9，该菌株属于Bacillus velezensis，为革兰氏染色阳性菌，于2012年2月27日在中国微生物菌种保藏管理委员会普通微生物中心保藏(北京市朝阳区北辰西路1号院3号)，保藏号CGMCC NO.5808。
通过室内培养及盆栽试验测定SQR9的减排能力，发现该菌株减少土壤N2O排放的能力与其接种浓度相关，当浓度越高，减排能力越强。同时，SQR9通过提高N2O还原速率，实现土壤N2O减排。
本发明所述的植物根际益生菌可应用于蔬菜种植田块，减少土壤N2O排放。
由权利要求1所述的贝莱斯芽孢杆菌(Bacillus velezensis)SQR9制备的菌剂。
本发明所述的菌剂在减少土壤N2O排放中的应用。
有益效果
本发明提供了一株可提高N2O还原速率，减少菜地土壤N2O排放的根际益生菌。
该菌株具有以下优点：
1)本发明提供的植物根际益生菌Bacillus velezensis SQR9，来源于植物根际，能作用于植物根际，改变土壤氮素转化过程；
2)本发明提供的植物根际益生菌接种浓度与土壤N2O减排量呈显著正相关。
3)本发明提供的植物根际益生菌可显著提高土壤N2O还原速率，减少土壤N2O排放通量。
附图说明
图1不同浓度梯度植物根际益生菌对土壤N2O的减排效果
图2植物根际益生菌对土壤N2O的减排效果图
图3植物根际益生菌提高土壤N2O还原速率效果图
生物材料保藏信息
SQR9，分类命名为贝莱斯芽孢杆菌Bacillus velezensis，于2012年2月27日在中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏地址北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏号CGMCC NO.5808。
具体实施方式
实施1不同浓度梯度植物根际益生菌的接种及土壤N2O排放速率
1、SQR9液体菌剂制备
将保藏号为CGMCC NO.5808的Bacillus velezensis SQR9接种到LB培养液中(蛋白胨10g，酵母粉5g，NaCl 10g，蒸馏水1L，121℃灭菌20min)，进行培养，培养条件为：培养温度30℃，摇床转速170转/分钟，时间12小时，制备菌液。将上述SQR9液体菌剂按照6000转/分钟离心10分钟，取沉淀溶于超纯水，按照8000转/分钟离心10分钟，重复三次；制得SQR9植物根际益生菌悬液；所述的菌悬液中功能菌含菌或孢子数量≥1×108CFU mL-1。
2、不同浓度植物根际益生菌接种及土壤N2O排放速率测定
将20g风干土壤样品(以干土计)置于250mL的锥形瓶中，用去离子水调节土壤水分至30％最大持水量(WHC)，于25℃黑暗条件预培养一周，稳定微生物活性。按照0、10、103、105、107和108CFU/g土的接种浓度梯度设置6个处理，每个处理三个重复。在锥形瓶中补充去离子水，以调节土壤水分含量至80％WHC，然后盖上带针孔的塑料薄膜，保持好氧条件并尽量减少水分蒸发。接种培养一周后，各处理均以0.15g N kg-1土施加尿素。锥形瓶立即用橡胶塞重新密封，在25℃黑暗条件下培养3天，然后采集瓶内气体，使用气象色谱测定N2O浓度，计算土壤N2O排放速率。
结果如附图1所示，在接种剂量试验中，接种菌株SQR9后各处理的N2O累积排放量(0.08～0.15mg kg-1)均低于对照处理(0.17mg kg-1)。N2O累积排放量与SQR9接种浓度呈现出显著负对数相关。在接种浓度最高时(108CFU g-1)，土壤N2O排放降低了53.4％，减排效果最显著。植物根际益生菌SQR9对土壤N2O的减排效果依赖于接种剂量。
实施2温室黄瓜盆栽试验
盆栽试验在南京农业大学牌楼科研基地温室进行，供试作物为黄瓜，共设置6个处理，分别为不施用SQR9且不种植黄瓜(Control)、仅施用活性SQR9不种植黄瓜(ActiveSQR9)、仅施用灭活SQR9不种植黄瓜(Inactive SQR9)、不施用SQR9仅种植黄瓜(Cucumber)、施用活性SQR9且种植黄瓜(Cucumber+active SQR9)和施用灭活SQR9且种植黄瓜(Cucumber+inactive SQR9)。每个处理包括3盆重复，每盆装14kg土。对于接种SQR9的处理，在黄瓜幼苗移栽前以108CFU g-1的终浓度分别施加活性和灭活SQR9菌株。黄瓜待其在育苗盘中长出两片真叶后，选取均匀的幼苗移栽至盆里，每盆4株苗。种植过程水分管理保持在80％WHC，分别在黄瓜幼苗移栽后第1、6和14天施肥，肥料与土充分混合均匀，全周期施肥总量为氮肥0.18g N/kg(尿素)、磷肥0.168g P2O5/kg(过磷酸钙)和钾肥0.05g K2O/kg(氯化钾)。N2O排放通量在施肥后加密采集，后期一周采集两次。土壤样品每周采集一次。
结果如附图2所示：盆栽实验中6个处理在黄瓜生育期内N2O排放通量的动态变化规律一致，所有处理的N2O排放通量均在每次施肥后的三天内达到峰值。各处理N2O累积排放量在差异显著，从Active SQR9处理的60.42mgN2O m-2到Cucumber处理的107.61mgN2O m-2。与Control和Cucumber处理相比，施用活性SQR9后，土壤N2O排放量分别显著降低了22.6％和33.5％。相较之下，接种灭活SQR9对土壤N2O排放无影响。说明活性植物根际益生菌SQR9发挥了显著的N2O减排作用。
SQR9对土壤理化性质的影响如表1所示：种植黄瓜的三个处理的土壤NH4 +-N和NO3 --N含量普遍低于不种植黄瓜的处理。在不种植黄瓜的处理中，与Control处理相比，活性SQR9显著提高了4.6％的土壤NH4 +-N，降低了13.1％的土壤NO3 --N。相较于Control处理，接种SQR9还促使土壤pH提高了0.11个单位，土壤溶解性有机碳提高了10.5％，而土壤电导率降低了23.7％。对于种植黄瓜的处理，接种SQR9后，这些理化性质没有显著变化。
表1不同处理对土壤理化性质的影响
实施例3N2O还原速率测定试验
运用直接氮气法，测定上述盆栽实验后期四个处理(Control、Active SQR9、Cucumber和Cucumber+active SQR9)的N2O还原速率。称取20g鲜土(以干土计)于250mL锥形瓶中，保持80％WHC在25℃厌氧条件下预培养24小时，以稳定微生物活性。在培养之前，用橡胶塞密封锥形瓶，抽出80mL气体后用等量的N2O替换，使得锥形瓶中N2O的终浓度为220ppm。土壤密封培养120小时。在此期间，使用注射器从锥形瓶顶空收集气体样品。用气相色谱仪分析样品中N2O浓度，并通过计算得到N2O还原速率。
结果如附图3所示：4个处理的N2O浓度表现出相似的动态下降趋势。其中，Cucumber处理的N2O浓度普遍高于其他处理，施用活性SQR9后N2O浓度下降最快。因此，N2O还原速率在不同处理中表现出显著差异，从Cucumber处理的0.011mg kg-1h-1到Active SQR9处理的0.019mg kg-1h-1。与Control和Cucumber处理相比，接种活性SQR9促使土壤N2O还原速率分别显著提高了46.8％和26.5％。说明活性植物根际益生菌SQR9通过提高土壤N2O还原速率，从而发挥了显著的N2O减排作用。
Claims (3)
1.贝莱斯芽孢杆菌（Bacillus velezensis）SQR9在提高N2O还原速率、减少土壤N2O排放中的应用，所述的贝莱斯芽孢杆菌（Bacillus velezensis）SQR9，于2012年2月27日在中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏号CGMCC NO.5808。
2.根据权利要求1所述的应用，其特征在于应用于蔬菜种植田块。
3.包含贝莱斯芽孢杆菌（Bacillus velezensis）SQR9的菌剂在提高N2O还原速率、减少土壤N2O排放中的应用，所述的贝莱斯芽孢杆菌（Bacillus velezensis）SQR9，于2012年2月27日在中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏号CGMCC NO.5808。
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