Combination of microbial strains and the promotion effect on beneficial soil microbial genes and maize growth
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摘要:目的
微生物肥料一般需要采用复合菌种提高有益菌在作物根际的定殖和促生效果。我们对本实验室前期分离鉴定的26个具有不同功能的玉米根际菌株进行复配,并研究复合菌株在土壤中的有益作用及其促生效果。
方法按照功能共构建了4组合成菌群:C1,将铁载体分泌量最高的解磷菌、解钾菌、自生固氮菌、联合固氮菌各1株进行组合;C2,将吲哚乙酸 (IAA) 分泌量最高的解磷菌、解钾菌、自生固氮菌、联合固氮菌各1株进行组合;C3,每个菌属选择1株综合促生能力最强的菌株进行组合;C4,由C1与C2所有菌株组合。用以上4组菌群在灭菌土壤和未灭菌土壤上分别进行玉米接种菌剂和土壤浇灌菌剂盆栽试验,包括无菌剂对照在内共10个处理,灭菌土壤处理分别记为MCK、MC1、MC2、MC3、MC4,未灭菌土壤处理分别记为WCK、WC1、WC2、WC3和WC4。玉米接菌试验是将初生胚根长1~2 cm的玉米种子浸泡在菌群悬浮液中1 h,然后种植在灭菌土壤和未灭菌土壤中,9天后测定株高、鲜重、根长、根尖数和分叉数。土壤浇灌试验采用种子发芽方法,将种子播在灭菌和未灭菌土壤中,每5天浇灌1次菌悬液,待玉米生长15天后,收集玉米根际土壤用于宏基因组学测序,揭示菌群对玉米根际微生物的影响及其促生机制。
结果4个复合菌剂相比,C4表现出最好的综合促生能力。玉米接种C4后,幼苗株高、地上鲜重、根鲜重、根长、根尖数和分叉数在灭菌组中分别比对照提高了28.97%、46.00%、59.99%、61.99%、62.27%和46.70%,在未灭菌组中分别提高了27.49%、25.86%、50.13%、40.11%、62.26%和82.55%。复合菌群C4提高了根际土壤中有益菌属Pseudomonas、Klebsiella、Duganella、Cellvibrio、Pantoea、Pseudoxanthomonas的丰度,在浇灌菌群C4的玉米根际土壤中,有益菌属Enterobacter和Klebsiella的注释丰度与对照组相比显著提高。菌群C4提高了根际土壤中磷和氮的转运、IAA的合成、铁载体的转运与调控系统和碳循环相关的功能基因的丰度,部分丰度上升的功能基因的微生物溯源分析结果显示,多数有益微生物的菌属丰度与功能基因丰度呈正相关,同时在ugqB、phnW的基因集中注释到了菌群C4的成员Klebsiella。
结论菌群C4改变了玉米根际细菌群落组成,提高了有益菌属丰度,富集了促生相关功能基因,具备微生物复合菌肥开发的潜能。
Abstract:ObjectiveBiofertilizers containing composite strains are generally more effective to improve the colonization of beneficial bacteria in crop rhizosphere. We identified 26 strains in maize rhizosphere with different functions in our laboratory and constructed four composite strains. In this research, we investigated their effects in assembling beneficial genus in rhizosphere soil and growth-promotion on crops.
MethodsThe 26 strains belong to four functional groups: phosphate solubilizing bacteria, potassium solubilizing bacteria, autochthonous nitrogen-fixing bacteria and combined nitrogen-fixing bacteria. The composite microbial agents (SynComs) were constructed by combining one strain from each of the four functioanl groups. C1 contains the four strains with the highest secretion of iron carriers; C2 contains the four strains with the highest secretion of indole acetic acid (IAA); C3 contains the four strains with the strongest growth promotion; C4 contains all the strains in C1 and C2. Maize pot experiment was conducted using the four SynComs under sterilized soil (MCK, MC1, MC2, MC3, MC4), and unsterilized soil (WCK, WC1, WC2, WC3, and WC4). Maize seeds with primary radicle length of 1−2 cm were soaked in the suspension of SymComs for 1 h, and then planted in sterilized and unsterilized soil. After 9 days, the seedlings were harvested for the measurement of plant height, fresh weight, root length, number of root tips and number of forks. The same maize seeds were planted in sterilized and unsterilized soils, and watered with the composite strain suspension every 5 days. And the maize was harvested after 15 days growth for collecting the rhizosphere soil for metagenomics sequencing.
ResultsThe SynComs C4 exhibited the best integrative ability among the four SynComs. Compared with the corresponding CK, C4 significantly promoted plant height, aboveground fresh weight, root fresh weight, root length, number of root tips and number of forks of maize seedlings by 28.97%, 46.00%, 59.99%, 61.99%, 62.27%, and 46.70% in sterilized group, and by 27.49%, 25.86%, 50.13%, 40.11%, 62.26% and 82.55% in unsterilized group, respectively. SynComs C4 increased the abundance of beneficial bacterial genera such as Pseudomonas, Klebsiella, Duganella, Cellvibrio, Pantoea, and Pseudoxanthomonas. Genera Enterobacter and Klebsiella in maize rhizosphere water with SynComs C4 had significantly higher annotated abundance than the control. SynComs C4 increased the abundance of functional genes related to phosphorus and nitrogen transport, IAA synthesis, iron carrier transport and carbon cycling in the rhizosphere soil, microbial traceability analyses of some of the functional genes with increased abundance showed a positive correlation between genus abundance and functional gene abundance for most beneficial microorganisms, while the members of SynComs C4 (Klebsiella) were annotated in the gene sets of ugqB, phnW.
ConclusionsSynComs C4 changed the rhizosphere soil bacterial community composition, increased the abundance of beneficial bacterial genera and enriched growth-promoting related functional genes, and possessed the potential for the development of microbial composite fertilizers.
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玉米是我国最重要的农作物之一,全国玉米种植面积占农作物播种面积的37%,其年产量高达27255.2万t[1]。玉米是高产喜肥作物[2],然而,大量使用化肥破坏土壤结构,同时加剧环境负担[3−4]。植物根际促生细菌 (PGPR) 是一类定殖于植物根际并能对植物生长产生促进作用的有益菌,在微生物肥料领域具有重要的应用价值[5]。PGPR是最常用的微生物肥料的菌种来源[6]。研究发现,PGPR菌肥与半量化肥共同处理黑青稞,供试的PGPR菌肥大多数能显著促进黑青稞生长、提高产量和改善营养品质[7]。然而,单一菌株施入土壤后,难以与土著微生物竞争[8],不能在植物根系上很好地定殖,达不到预期的效果[9−10]。复合菌剂具有协同作用,通过分泌不同生理活性物质,影响植株的生理代谢活动,促进植物生长。以梭梭根际促生菌为主要菌源制备的复合菌肥,与单株菌肥相比,显著提高了番茄产量且优化了番茄根际土壤特性[11]。由4株促生菌组成的细菌联盟,通过增加植物激素、土壤养分的有效性以及有益菌群的丰度,间接促进了植物的生长[12]。玉米施用生物菌肥能够减少化肥用量并提高玉米产量[13],但近年来,如何制备玉米根际促生合成菌群并研究其促生效应的相关报道较少。
本研究以前期从玉米根际筛选出的26株优质PGPR,根据菌株的解磷、解钾、固氮、分泌吲哚乙酸 (IAA) 和产铁载体的特性,构建多个合成菌群。通过宏基因组测序,揭示玉米根际促生菌群对玉米的促生机制,为玉米根际促生菌肥的开发提供菌种资源和理论依据。
1. 材料与方法
1.1 试验材料
供试玉米为垦粘1号,购自齐齐哈尔市胜利种子商店。
供试土壤采自黑龙江省齐齐哈尔市梅里斯区,土壤基本理化性质如下:有机质22.85 g/kg,全氮2.19 g/kg,有效磷4.12 mg/kg,速效钾136 mg/kg,pH为7.58。
供试菌株由齐齐哈尔大学微生物学实验室提供。依据菌株具有的解磷、解钾、固氮、分泌IAA和产铁载体能力,构建合成菌群。具体信息如表1所示。
表 1 菌群构建原则及组合方式Table 1. Principles of synthetic microbial community construction and combination合成菌群
Synthetic microbial communities (SynComs)构建原则
Construction principle菌株组成
Strain compositionC1 每个菌属选择一株铁载体分泌量最高的菌株进行组合
One strain with the highest secretion of iron carriers from ezch genus was selected, including one phosphate solubilizing bacteria strain, one potassium solubilizing bacteria strain, one autochthonous N-fixing bacteria strain and one combined N-fixing bacteria strainEnterobacter huaxiensis LN1, Bacillus velezensis P33, Enterobacter huaxiensis K6, Klebsiella pneumoniae ZN17 C2 每个菌属选择一株吲哚乙酸 (IAA) 分泌量最高的菌株进行组合
One strain with the highest secretion of indole acetic acid (IAA) from each genus was selectedLelliottia jeotgali P43, Klebsiella granulomatis K11, Klebsiella granulomatis LN3, Klebsiella pneumoniae ZN18 C3 每个菌属选择一株促生综合能力最强的菌株进行组合
One strain from each genus with the strongest growth promotion effect was selectedLelliottia jeotgali P23, Enterobacter hormaechei P24, Priestia filamentosa P37, Pseudomonas hunanensis P45, Staphylococcus epidermidis P46, Agrobacterium arsenijevicii ZN4, Klebsiella granulomatis K5, Bacillus zanthoxyli LN2 C4 将合成菌群C1与合成菌群C2的所有菌株进行合并
Amalgamation of all strains in SynComs C1 and SynComs C2Lelliottia jeotgali P43, Klebsiella granulomatis K11, Klebsiella granulomatis LN3, Klebsiella pneumoniae ZN18, Bacillus velezensis P33, Enterobacter huaxiensis K6, Enterobacter huaxiensis LN1, Klebsiella pneumoniae ZN17 1.2 复合菌剂促生效果盆栽试验
挑选健康、完整的玉米种子,放置于5% (v/v) 次氯酸钠中浸泡3 min,然后在70% (v/v) 乙醇中浸泡3 min,最后用无菌蒸馏水冲洗3次。种子在光照培养箱 (Thermo Scientific,美国) 中培养至芽长1~2 cm。将各功能菌株分别活化于牛肉膏蛋白胨培养基中,30°C、200 r/min振荡过夜,吸取适量菌液稀释100或1000倍并在旋涡混合仪上混合均匀,用细菌计数板统计菌数,并计算原始菌液浓度,将菌液离心并收集菌体至10 mL离心管中。将收集的菌体悬浮于1×磷酸盐缓冲液 (PBS) 中,并将各菌株的悬浮液稀释至108 CFU/mL。选择无拮抗作用的菌株,根据表1的原则进行组合构建,将各菌株的悬浮液等体积混合于50 mL离心管中,制备成多菌株悬浮液。
此外,将表面灭菌并发芽的玉米种子浸泡在悬浮液中1 h,并用等量的灭菌PBS缓冲溶液浸泡作为对照。将接种菌悬浮液的玉米种子用镊子分别播种在灭菌土壤和未灭菌土壤中,进行盆栽试验。盆栽试验共设置10个处理:MCK (灭菌土+灭菌水)、WCK (未灭菌土+灭菌水)、MC1 (灭菌土+合成菌群C1)、WC1 (未灭菌土+合成菌群C1)、MC2 (灭菌土+合成菌群C2)、WC2 (未灭菌土+合成菌群C2)、MC3 (灭菌土+合成菌群C3)、WC3 (未灭菌土+合成菌群WC3)、MC4 (灭菌土+合成菌群C4) 和WC4 (未灭菌土+合成菌群C4) 。在恒温恒湿培养箱中培养9天。轻轻将玉米幼苗从土壤中拔出,用流动水将根部清洗干净后,测量其株高和鲜重,并用根系扫描仪分析其根长、根尖数和分叉数。
1.3 土壤施用复合菌剂试验
采用同样方法处理玉米种子,播种在灭菌和未灭菌土壤中。每5天浇灌1次菌株悬浮液(×108 CFU/mL,5 mL),待玉米幼苗生长至第15天,将玉米幼苗轻轻拔出,刷去大块土壤,摇晃去除多余土壤,仅保留紧贴根部约1 mm的土壤,经离心、超声振荡后收集根际土壤[14]。收集促生效果最佳的菌群处理组及对照组玉米幼苗的根际土壤,用于宏基因组学测序。每个处理设置3个重复。使用FastDNA®SPIN试剂盒 (MP Biomedicals,Solon,USA),从0.5 g土壤中提取总DNA[15]。使用Illumina NovaSeq测序平台进行宏基因组测序 (上海美吉生物医药科技有限公司)。使用SOAPaligner软件,将样品中高质量reads与非冗余基因集比对,统计基因的丰度信息。使用Diamond将非冗余基因集的氨基酸序列与NR数据库和KEGG数据库比对,并通过分类学信息数据库获得对应的物种注释,同时获得基因对应的KEGG功能信息,使用KO、Pathway对应的基因丰度总和计算对应功能类别的丰度。
1.4 统计分析
利用托普根系分析仪对根系数据进行分析,用Excel进行数据整理。利用SPSS 26软件的Student’s T 检验方法在0.05水平进行差异显著性分析,采用Origin 2021作图。
2. 结果与分析
2.1 复合菌群的促生效果分析
对C1、C2、C3和C4菌群的促生效果进行盆栽验证,结果(图1)表明,C4处理后的灭菌组(MC4) 与对照组 (MCK) 相比,玉米幼苗的株高、地上鲜重、根鲜重、根长、根尖数和分叉数分别提高了28.97%、46.00%、59.99%、61.99%、62.27%和46.70%。在未灭菌组 (WC4) 分别提高了27.49%、25.86%、50.13%、40.11%、62.26%和82.55%。对根鲜重进行测定后发现,菌群C1在未灭菌组中的促生效果最好,根鲜重提高了72.67%,而在灭菌组中C2促生效果最好,根鲜重提高了81.23%。对4个菌群的综合能力进行分析,发现无论在灭菌组还是未灭菌组,菌群C4都具有最强的综合能力,其次是菌群C2、C1和C3。
图 1 合成菌群对玉米幼苗生长的影响及菌群综合能力评价注:M—灭菌土处理,W—未灭菌土处理。柱旁不同小写字母表示不同处理之间玉米幼苗指标差异显著 (P<0.05)。Figure 1. Effects of SynComs on the growth of maize seedlings and evaluation of the integrated capacity of the SynComsNote: M—Sterilized soil treatment; W—Unsterilized soil treatment. Different small letters beside the bars indicate significant differences in maize seedling indexes among treatments (P<0.05).2.2 菌群C4对根际细菌群落多样性的影响
接种菌群C4后玉米根际细菌属水平的α多样性分析和β多样性分析结果(图2)显示,在灭菌组中,MC4的Chao指数显著高于MCK;在未灭菌组处理中,WC4的Simpson指数显著低于WCK。这说明接种菌群C4改变了土壤微生物α多样性及丰富度。
采用非度量多维尺度NMDS进行微生物群落的距离分析,表征β多样性。在属水平上,在灭菌与未灭菌处理中,应力函数值均为0.000,小于0.05 (图2),能较好地揭示不同处理间细菌群落组成的差异,初步判断复合菌群的接种影响了细菌群落的组成。
2.3 菌群处理对玉米根际微生物群落的影响
对相对丰度大于1%的菌属进行注释,将相对丰度小于1%的菌属合并。图3A和3C显示,不同处理在属水平上的土壤细菌群落组成存在差异。在灭菌组处理中,相对丰度较高的菌属包括Massilia、Kosakonia、Duganella、Pseudomonas、Exiguobacterium、Stenotrophomonas等。而未灭菌组中相对丰度较高的菌属包括Actinbacteria、ChlorofLexi、Rubrobacter、Nocardioides、Arthrobacter等。
图 3 不同处理组的属水平细菌群落组成及两组显著差异菌属比较分析注:MCK、MC4为灭菌土对照、C4菌群组,WCK、WC4分别为未灭菌土对照、C4菌群组。*,**,***分别表示不同处理间在P<0.05、P<0.01和P<0.001水平差异显著。Figure 3. Bacterial community composition at genus level in rhizosphere soil of C4 group treatment and comparison of genera showing significant difference with controlNote: MCK and MC4 represent the sterilized soil control and C4 group; WCK and WC4 represent the unsterilized soil control and C4 group. *, ** and *** indicate significant difference between treatments at P<0.05, P<0.01 and P<0.001 levels, respectively.对丰度前25的菌属进行了两组比较分析,图3B和3D显示,在MC4处理中Duganella、Pseudomonas、Klebsiella、Cellvibrio、Azoarcus、Fluviicola、Streptomyces、Delftia、Pseudoduganella、Dyadobacter和Sphingobium相对丰度与对照相比显著上升,分别上升了37.53%、12.53%、46.24%、50.89%、109.63%、34.83%、101.67%、27.06%、26.09%、95.23%和118.18%。在WC4处理中,Lysobacter、Azospirillum、Pseudoxanthomonas、Pantoea、Pseudomonas和Klebsiella的相对丰度与对照相比显著上升,分别上升了36.17%、45.45%、102.27%、17.9%、104.9%和38.7%。
2.4 菌群C4的成员在玉米根际中丰度变化分析
菌群C4中包含2株Enterobacter,4株Klebsiella,1株Bacillus和1株Lelliolttia,将这8株菌所属的4个菌属与NR数据库进行对比,获得了样本物种的分类学注释信息,注释结果如图4所示。Enterobacter和Klebsiella在MC4和WC4处理中的注释丰度与对照相比均显著上升,其中Enterobacter注释到的丰度最高。而Bacillus和Lelliolttia的注释丰度与对照组相比虽略有上升,但未有显著差异。
图 4 菌群C4中各菌属在灭菌和非灭菌根际土壤中的丰度注:MCK、MC4分别为灭菌土对照、C4菌群组,WCK、WC4分别为未灭菌土对照、C4菌群组。*表示CK与C4菌群组间微生物丰度差异显著 (P<0.05),ns表示差异不显著。Figure 4. The abundance of genus of the syncoms C4 in rhizosphere of sterilized and unsterilized soilNote: MCK and MC4 represent the sterilized soil control and C4 group; WCK and WC4 represent the unsterilized soil control and C4 group. * indicates significant difference in microbial abundance between CK and C4 group (P<0.05), and ns indicates no significant difference.2.5 玉米根际土壤中促生相关基因的注释结果及分析
基于KEGG数据库对宏基因组数据中的相关功能基因进行注释,分别注释了磷转化相关基因 (图5A)、固氮相关基因 (图5B)、IAA合成、铁载体转运调控、碳循环及微生物对植物的影响相关基因 (图6) 的丰度。将注释到的相关基因以Heatmap图的方式进行聚类分析。如图5A所示,在灭菌处理共筛选到34个与有机酸合成、磷的溶解和转运有关的基因。在未灭菌处理中共筛选到33个相关基因,结果显示,接种菌群C4后,pho、gcd、phn、ugp、ppx等基因都在MC4和WC4处理组显著富集。说明菌群C4处理后的土壤中解磷相关功能基因的数量增多,可提升土壤中的磷转化效率。图5B显示,接种菌群C4后,nif、vnf、nrt、nar等与固氮及氮代谢相关的基因,在MC4和WC4处理组丰度显著上升。说明接种菌群C4后,可通过提升土壤中固氮基因的丰度来提高土壤的固氮活性。
对不同处理组中与IAA合成、铁载体转运调控、碳循环及微生物对植物的影响的相关基因各10个进行了聚类分析(图6),发现iaaH、iaaA、iro、bas、fhu等基因都在菌群C4处理后富集。因此,菌群C4可以通过增加促生相关功能基因的丰度来丰富土壤功能,进而达到促生的效果。
2.6 玉米根际土壤中促生相关基因的溯源分析
为探索功能基因及功能菌株间的联系,图7分别筛选了结果2.5中在MC4和WC4处理组丰度均上升的部分功能基因 (nifK, nasA, nifH, norB, ugqB, phnW, phnJ),并对其进行微生物溯源分析,构建单个基因的基因集后进行物种注释、功能注释,得到携带相同功能基因但在分类上不同的微生物菌属分类信息。并将追溯到的微生物 (属水平) 丰度与功能基因丰度进行线性回归方程拟合,得到相关性系数。结果表明,丰度上升的Pseudomonas在7个基因集中均被注释到,在ugqB、phnW的基因集中注释到了与菌群C4的部分成员相同的菌属Klebsiella。相关系数的分析结果显示,多数有益微生物菌属丰度与功能基因丰度呈正相关。说明菌群C4通过提高玉米根际土壤的有益菌属丰度来富集促生相关功能基因,进而发挥其促生功能。
3. 讨论
合成菌群中包括多个微生物物种,功能不同的微生物菌株通过协同作用,为植物寄主提供多种利益,使植物更好地适应新环境,以改善植物生长并最终提高作物产量[16−17]。物种间可通过营养互补和代谢分工等不同的代谢作用,影响彼此之间的相互作用,进而影响菌群的作用效果[18−19]。
接种菌群C4后,未灭菌与灭菌土壤中的细菌群落存在差异,这可能是由于在灭菌土壤中加入促生菌群后重塑了土壤微生物群落。Pseudomonas被证明具有溶磷的作用[20]。Klebsiella也被证明可在无氮环境中生长,溶解无机磷和钾,并产生IAA和铁载体[21]。Duganella、Pseudoduganella、Delftia、Azoarcus都被证明具有高效的固氮作用,并可以改善氮代谢[22−24]。Streptomyces在土壤中无处不在,以产生次生代谢物而闻名,研究表明它会被特异性地招募到植物的根际和内圈[25]。以上菌属均在本研究MC4处理中被注释到且丰度与对照相比显著上升。Pantoea和Azospirillum可以提高固氮酶的活性,提高植物的产量[26];Lysobacter具有杀线虫活性、产ACC脱氨酶和促进植物生长的能力[27];Enterobacter可通过固氮、产IAA等作用促进玉米生长[28]。在WC4处理组中,Pantoea、Azospirillum、Lysobacter、Enterobacter等有益细菌的丰度与对照相比显著上升。其中Pseudomonas和Klebsiella在MC4和WC4处理组中的相对丰度均显著增加。显著增加的菌属大多是潜在的植物生长促进细菌,证明加入菌群可提高促生相关的有益菌的丰度,以此促进植物生长[29]。物种注释的结果表明,菌群C4所包含的4个菌属均可在根际土壤中被注释到。其中,Enterobacter和Klebsiella在MC4和WC4处理中的丰度均显著增加,而Bacillus和Lelliolttia虽然注释到但丰度变化不大,它们可以分泌植物生长激素和提高植物营养元素吸收来促进植物生长[30]。
本研究筛选了一些功能基因进而揭示菌群的促生机制。gcd、ppa和ppx是与磷增溶有关的基因,phoA和phoD与有机磷的矿化有关,ugpQ、phoB和phoR与磷酸盐转运蛋白有关[31−33],pqq、gdh与编码膜结合的葡萄糖脱氢酶相关[34],由PPX基因编码的外聚磷酸酶被证明,其在无机聚磷酸盐降解成磷酸盐的过程中发挥重要作用[35]。本研究基于宏基因组数据筛选了与解磷相关的基因,结果显示,上述与解磷相关基因的丰度都在MC4和WC4处理中升高,说明菌群可能通过提升磷转运相关基因的丰度,有效地获取和竞争磷元素,利用微生物群落来提高可持续农业中的磷有效性。固氮基因家族nif相关的基因都是固氮所必需的基因[36],nir、nor和nos等基因都是参与氮循环的基因[37]。上述基因在接种菌群C4后的丰度都得到提升,说明菌群C4可能通过提升土壤中固氮基因的丰度来改善土壤的固氮活性,从而提高植物对氮元素的利用率。碳循环、微生物对植物的影响、IAA合成及铁载体的转运与调控均是与植物促生密切相关的因素。fab、gdh、ppc是参与碳循环和转运的相关基因[37]。vir基因家族与膜转运、细菌分泌系统相关,tur是植物−病原体相互作用相关途径的重要基因,hcp是能量代谢相关途径的重要基因,imp、vgr和vas是与生物膜形成相关的基因,以上基因都是与微生物对植物的影响相关的基因[37]。iaaH、iaaA等基因与IAA的合成有关[38];iro、bas、fhu等基因与铁载体合成、运输及调节相关[39];本研究结果表明上述基因在MC4和WC4处理中的丰度升高,证明菌群C4还可能通过提高碳循环、微生物对植物的影响、IAA的合成及铁载体的转运与调控相关功能基因的丰度,来实现对植物促生和保护植物健康的作用。
4. 结论
复合菌群C4为本研究中促生能力最强的菌群,菌群C4显著提高玉米幼苗的株高、鲜重、根长、根尖数和分叉数,影响玉米根际微生物的组成和功能,提高微生物群落的多样性及丰富度,亦可以提升有益菌及相关功能基因丰度以促进玉米生长。因此,菌群C4具有良好的促生能力,具备微生物复合菌肥开发的潜能。
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图 1 合成菌群对玉米幼苗生长的影响及菌群综合能力评价
注:M—灭菌土处理,W—未灭菌土处理。柱旁不同小写字母表示不同处理之间玉米幼苗指标差异显著 (P<0.05)。
Figure 1. Effects of SynComs on the growth of maize seedlings and evaluation of the integrated capacity of the SynComs
Note: M—Sterilized soil treatment; W—Unsterilized soil treatment. Different small letters beside the bars indicate significant differences in maize seedling indexes among treatments (P<0.05).
图 3 不同处理组的属水平细菌群落组成及两组显著差异菌属比较分析
注:MCK、MC4为灭菌土对照、C4菌群组,WCK、WC4分别为未灭菌土对照、C4菌群组。*,**,***分别表示不同处理间在P<0.05、P<0.01和P<0.001水平差异显著。
Figure 3. Bacterial community composition at genus level in rhizosphere soil of C4 group treatment and comparison of genera showing significant difference with control
Note: MCK and MC4 represent the sterilized soil control and C4 group; WCK and WC4 represent the unsterilized soil control and C4 group. *, ** and *** indicate significant difference between treatments at P<0.05, P<0.01 and P<0.001 levels, respectively.
图 4 菌群C4中各菌属在灭菌和非灭菌根际土壤中的丰度
注:MCK、MC4分别为灭菌土对照、C4菌群组,WCK、WC4分别为未灭菌土对照、C4菌群组。*表示CK与C4菌群组间微生物丰度差异显著 (P<0.05),ns表示差异不显著。
Figure 4. The abundance of genus of the syncoms C4 in rhizosphere of sterilized and unsterilized soil
Note: MCK and MC4 represent the sterilized soil control and C4 group; WCK and WC4 represent the unsterilized soil control and C4 group. * indicates significant difference in microbial abundance between CK and C4 group (P<0.05), and ns indicates no significant difference.
表 1 菌群构建原则及组合方式
Table 1 Principles of synthetic microbial community construction and combination
合成菌群
Synthetic microbial communities (SynComs)构建原则
Construction principle菌株组成
Strain compositionC1 每个菌属选择一株铁载体分泌量最高的菌株进行组合
One strain with the highest secretion of iron carriers from ezch genus was selected, including one phosphate solubilizing bacteria strain, one potassium solubilizing bacteria strain, one autochthonous N-fixing bacteria strain and one combined N-fixing bacteria strainEnterobacter huaxiensis LN1, Bacillus velezensis P33, Enterobacter huaxiensis K6, Klebsiella pneumoniae ZN17 C2 每个菌属选择一株吲哚乙酸 (IAA) 分泌量最高的菌株进行组合
One strain with the highest secretion of indole acetic acid (IAA) from each genus was selectedLelliottia jeotgali P43, Klebsiella granulomatis K11, Klebsiella granulomatis LN3, Klebsiella pneumoniae ZN18 C3 每个菌属选择一株促生综合能力最强的菌株进行组合
One strain from each genus with the strongest growth promotion effect was selectedLelliottia jeotgali P23, Enterobacter hormaechei P24, Priestia filamentosa P37, Pseudomonas hunanensis P45, Staphylococcus epidermidis P46, Agrobacterium arsenijevicii ZN4, Klebsiella granulomatis K5, Bacillus zanthoxyli LN2 C4 将合成菌群C1与合成菌群C2的所有菌株进行合并
Amalgamation of all strains in SynComs C1 and SynComs C2Lelliottia jeotgali P43, Klebsiella granulomatis K11, Klebsiella granulomatis LN3, Klebsiella pneumoniae ZN18, Bacillus velezensis P33, Enterobacter huaxiensis K6, Enterobacter huaxiensis LN1, Klebsiella pneumoniae ZN17 -
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