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PGPR菌剂配合化肥减施对植烟土壤nosZ型细菌群落的影响

来源:用户上传      作者:余伟 闫芳芳 冯文龙 陈强 张映杰 张宗锦 辜运富

  摘  要:研究化肥減施情况下PGPR(plant growth promoting rhizobacteria)菌剂对植烟土壤反硝化作用的微生物调控机制,为植烟土壤科学施肥、培肥地力提供理论依据。本试验在四川攀枝花米易县的传统烤烟种植区通过化学分析和末端限制性长度多态性分析手段(T-RFLP)分别对PGPR菌剂配合化肥减肥处理下的植烟土壤理化性质及土壤nosZ型细菌群落组成和多样性进行研究。结果表明,与常规施肥相比,PGPR菌剂配合化肥减施的处理土壤pH和碱解氮含量显著提高,有机质、全氮含量提高但未达显著水平,部分处理速效磷、速效钾含量显著提高。PGPR菌剂配合化肥减施改变了nosZ型细菌的物种组成,Rhodobacter (红杆菌属)和Bacterium(杆菌属)为5个施肥处理共有的优势菌属,而Bradyrhizobium(慢生根瘤菌属)和Azospirillum(固氮螺菌属)仅为施用PGPR菌剂处理的优势菌。全量化肥配施PGPR菌剂处理下的Shannon多样性指数和均匀度显著低于其他处理,其他各处理细菌群落多样性之间无显著差异。冗余分析表明,土壤pH、有机质和速效钾是影响植烟土壤nosZ型细菌群落结构变化的主要因子。综上所述,PGPR菌剂配合化肥减施影响了土壤理化性质进而导致土壤nosZ型细菌群落结构组成发生改变。
  关键词:PGPR菌剂;化肥减施;T-RFLP;nosZ细菌群落
  Abstract: The nosZ-type bacterial community variation in flue-cured tobacco cultivation soils under the condition of plant growth promoting rhizobacteria (PGPR) application plus reduction of chemical fertilizer (RCF) was investigated so as to reveal the microbial mediated mechanisms of the denitrifying process in these specific soils, and to establish reasonable fertilizer regimes and provide theoretical foundations for maintaining soil quality. The study was carried out in a traditional flue-cured tobacco cultivation field in Miyi County, Sichuan Province. Chemical analysis and terminal restriction fragment length polymorphism (T-RFLP) were conducted to study the variation of soil physicochemical parameters and nosZ-type bacterial community composition under the condition of PGPR+RCF fertilizer. The results showed that the soil pH and available nitrogen were significantly increased under the treatment of PGPR+RCF fertilizer as compared with the conventional fertilization (CK) (p<0.05). Meanwhile, the soil organic carbon and total nitrogen were also increased although not significantly, and available phosphorus and available potassium were also increased under some fertilizer treatments (30% RCF). The T-RFLP experiment showed that using PGPR in combination with fertilizers increased the composition and diversity of the nosZ-type bacterial community. Rhodobacter and Bacterium were the dominant genera in the soils under the five different fertilization treatments, while Bradyrhizobium and Azospirillum were dominant in the soil treated with only PGPR inocula. The Shannon diversity index and Evenness in the soil under total amount chemical fertilizer plus PGPR inoculant were the lowest among the five different fertilizer treatments, while those diversity indexes in the soil under the other four fertilizer treatments showed no significantly difference. Redundancy Analysis (RDA) showed that soil pH, soil organic matter and available potassium were the most important factors in shaping nosZ-type bacterial community in the flue-cured tobacco cultivation soil. Taken together, the using of PGPR in combination with reduced chemical fertilizers would change the soil physicochemical properties thus modify the composition of nosZ-type bacterial community composition, and increase their diversity.   1.5  数据分析
  土壤理化性质及nosZ细菌群落多样性指数等基础数据的处理和绘图利用Excel 2013进行,单因素方差分析利用SPSS 21.0完成。利用CANOCO 5.0软件对土壤环境参数和nosZ细菌群落相关性进行冗余分析(Redundancy Analysis, RDA)。
  2  结  果
  2.1  PGPR菌剂配合化肥减施对植烟土壤理化性质的影响
  由表1可知,与不施PGPR菌剂处理(T1)相比,在全量化肥基础上加施菌肥(T2),土壤各理化指标均无显著变化。而与T1相比,PGPR菌剂配合化肥减施的处理(T3、T4、T5)土壤pH均显著提高,T3处理最高,但各减肥处理之间差异不显著;土壤有机质、全氮含量略有提高,但未达到显著差异;土壤碱解氮含量显著提高;除T4外,有效磷和有效钾含量均较T1、T2提高,其中减少30 %化肥用量的处理土壤有效磷含量最高,减施化肥10%的处理土壤有效钾含量最高,与T1、T2处理达到显著差异。此可见,施用PGPR菌剂同时减施10%~30%的化肥对提高植烟区土壤肥力、改善土壤质量具有一定促进作用。
  2.2   PGPR菌剂配合化肥减施对植烟土壤nosZ细菌群落组成的影响
  图1示出了nosZ细菌群落T-RFLP分析中丰度排名前10的优势菌。结果表明,PGPR菌剂配合化肥减施明显改变土壤nosZ土壤细菌的群落组成。各处理中共有的优势菌属为Polymorphum(多形菌属),Rhodobacter(红杆菌属)和Bacterium(杆菌属)。其中,Polymorphum(多形菌属)随着化肥施用量减少呈先增加后降低的趋势,在减肥10%~20%的处理中占主导;Bacterium(杆菌属)在施用PGPR菌剂处理中丰度均有所提高;Rhodobacter 则在不施PGPR菌剂的处理中占优势,配施菌剂之后随化肥施用量减少有逐渐减少趋势;而Bradyrhizobium(慢生根瘤菌属)和Azospirillum(固氮螺菌属)仅为施用PGPR菌剂处理的优势菌。总体而言,PGPR菌剂配合化肥减施会对土壤中的nosZ细菌群落组成产生明显影响。
  2.3  PGPR菌剂配合化肥减施对植烟土壤nosZ细菌群落多样性的影响
  由表2可知,除T2处理Shannon多样性指数和均匀度显著低于其他处理外,其他处理之间Shannon多样性指数、丰富度和均匀度均未见显著差异。说明在全量化肥的条件下加施菌剂(Streptomycete sp.),会对部分nosZ细菌造成竞争,导致其群落多样性的下降,而化肥减施后则不产生影响。
  2.4  土壤环境因子与nosZ细菌群落组成的相关性分析
  运用冗余分析(RDA),对植烟土壤nosZ细菌群落组成与土壤理化性质相关性进行分析(图2)。从RDA排序图中可以看出,第 1 排序轴解释了土壤理化性质对nosZ细菌群落变异贡献率的44.62%,第2排序轴解释了土壤理化性质对nosZ细菌群落变异贡献率的21.47%,累计贡献率为66.09%。nosZ细菌群落与土壤pH、有机质和速效钾含量显著相关(p<0.05),可见,土壤pH、有机质(SOM)和速效钾(AK)含量是影响nosZ细菌群落的主要环境因子。图2显示,不施用PGPR菌剂(T1)处理与施用菌剂的T2、T4和T5处理相距较远,这表明,PGPR菌剂对nosZ细菌群落影响较大。施用菌剂的T2、T4、T5处理相对较为集中,表明施用PGPR菌剂及减施化肥20%~30%的土壤中nosZ细菌群落的差异性较小,而当PGPR菌剂配合减少10%的化肥施用量(T3)时,植烟土壤的nosZ细菌群落与其他处理差异较大。
  3  讨  论
  目前PGPR菌剂已广泛应用在不同作物的生产中,其中许多报道指出外源PGPR可以改变根际土壤中微生物结构和种群数量,且总体变化表现为细菌数量增加,真菌数量减少,放线菌数量变化不一致[20-21]。但关于PGPR菌剂结合化肥减施对土壤肥力的影响的报道尚不多见。本试验表明,PGPR菌剂配合化肥减量施用对提高土壤肥力具有重要影响。PGPR菌剂配合化肥减量施用显著增加土壤pH、碱解氮含量,一定程度上提高速效磷和速效钾含量,而土壤有机质、全氮也有不显著的增加,表明施用PGPR菌剂同时减施化肥对提高土壤肥力、改善土
  壤状况具有积极影响。主要原因可能是本试验中所用PGPR菌剂为具有溶磷溶钾能力的抗病促生型菌株,有利于活化土壤中的磷钾元素,促进植物吸收,进而为土壤反馈更多的细胞脱落物及有机代谢物,从而提高土壤有机质[12]。
  已有研究證实施用PGPR菌剂能增加土壤微生物菌群种类和数量,如施用PGPR菌剂配合有机肥,增加了玉米收获期土壤中的固氮菌、溶磷菌及纤维素分解菌数量[22],而施用PGPR菌剂和适量的氮肥能增加土壤纤维素分解菌、硝化细菌及固氮菌数量[23]。关于nosZ型反硝化细菌的研究已有大量报道,HARTER等[24]研究发现,Bradyrhizobium(慢生根瘤菌属)是nosZ反硝化细菌中最丰富的物种。Azoarcus(固氮弓菌属)和Bradyrhizobium(慢生根瘤菌属)能够在自由生活状态下进行脱氮作用,并能诱导豆科植物大量结瘤,与豆科植物形成共生关系,刺激植物生长。此外,Bradyrhizobium(慢生根瘤菌属)对土壤具有生物修复功能[24]。ITELIMA等[25]认为Azospirillum(固氮螺菌属)能够分泌赤霉素、乙烯和生长素,还可以刺激植物中黄酮类化合物的分泌,进而刺激根瘤菌中结瘤基因(nod)的表达。本试验中,施用PGPR菌剂均增加了土壤中Bradyrhizobium和Bacterium的丰度,并使Bradyrhizobium(慢生根瘤菌属)和Azospirillum(固氮螺菌属)丰度增加成为优势菌属,这表明施用PGPR改变了土壤含nosZ反硝化细菌群落组成且增加了有益微生物的数量。   研究表明,在煙草上应用PGPR菌剂能够增加土壤微生物群落多样性,促进烟株生长[26]。PGPR菌剂配合化肥减施对植烟土壤nosZ细菌群落多样性无显著影响,而全量化肥配施PGPR菌剂则降低了nosZ细菌群落多样性。这可能是由于化肥的施入调节土壤中的C/N从而影响了不同菌群的定殖能力,使PGPR菌剂中的菌群与nosZ菌群造成竞争导致。适当降低化肥用量有助于保持土壤中nosZ群落的多样性。这也再次证实了nosZ型反硝化细菌群落对施肥的响应较为敏感[4]。
  环境因子与土壤微生物群落相互作用,密切相关[27]。大量研究表明土壤pH、有机碳、氮是影响反硝化细菌群落组成的重要环境因子[28-29],也有研究指出,nosZ菌群与土壤水分含量和有效磷相关性最强[30]。这都证实了土壤环境质量与功能微生物的生存息息相关。本试验中,RDA相关性分析表明土壤pH、有机质和有效钾是影响植烟土壤nosZ细菌群落组成的主要环境因子,其次是速效钾、全氮及碱解氮。这与前人的研究结果[31-32]一致。
  4  结  论
  研究表明PGPR菌剂结合化肥减施改变了土壤理化性质,不同程度地提高了土壤pH、碱解氮、速效磷和速效钾等养分含量。PGPR菌剂配合化肥减施对植烟土壤nosZ细菌群落多样性无显著影响,却明显改变了其结构组成;而全量化肥配施PGPR菌剂则降低细菌群落多样性。红杆菌属 (Rhodobacter)和杆菌属(Bacterium)是该地区植烟土壤主要nosZ型反硝化细菌,而慢生根瘤菌属(Bradyrhizobium)和固氮螺菌属(Azospirillum)等nosZ基因型反硝化细菌对施用PGPR菌剂处理响应最敏感。土壤pH、有机质及有效钾是影响植烟土壤nosZ型细菌群落的主要环境因子。相关研究可为在攀枝花烟区植烟土壤上建立合理的施肥制度、保护土壤质量提供理论依据。
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