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西藏沙棘根瘤内生假单胞菌的分离鉴定及促生性研究

马福林 仁增卓玛 王昌玲 邓得坤 冶贵生 马玉花

马福林,仁增卓玛,王昌玲,等. 西藏沙棘根瘤内生假单胞菌的分离鉴定及促生性研究 [J]. 福建农业学报,2023,38(5):624−631 doi: 10.19303/j.issn.1008-0384.2023.05.014
引用本文: 马福林,仁增卓玛,王昌玲,等. 西藏沙棘根瘤内生假单胞菌的分离鉴定及促生性研究 [J]. 福建农业学报,2023,38(5):624−631 doi: 10.19303/j.issn.1008-0384.2023.05.014
MA F L, RENZENG Z M, WANG C L, et al. Identification and Growth-promoting Effects of Endophytic Pseudomonas sp. from Hippophae thibetana Root Nodules [J]. Fujian Journal of Agricultural Sciences,2023,38(5):624−631 doi: 10.19303/j.issn.1008-0384.2023.05.014
Citation: MA F L, RENZENG Z M, WANG C L, et al. Identification and Growth-promoting Effects of Endophytic Pseudomonas sp. from Hippophae thibetana Root Nodules [J]. Fujian Journal of Agricultural Sciences,2023,38(5):624−631 doi: 10.19303/j.issn.1008-0384.2023.05.014

西藏沙棘根瘤内生假单胞菌的分离鉴定及促生性研究

doi: 10.19303/j.issn.1008-0384.2023.05.014
基金项目: 国家自然科学基金(31660071);青海省科技计划项目(2017-ZJ-734);青海省“高端创新人才千人计划”项目(2020)
详细信息
    作者简介:

    马福林(1996−),男,硕士研究生,主要从事植物资源开发利用方面的研究(E-mail:qhdxmfl@163.com

    通讯作者:

    马玉花(1978−),女,博士,教授,主要从事森林培育理论与技术、植物资源开发利用方面的研究(E-mail:qhxnmyh@163.com

  • 中图分类号: S154

Identification and Growth-promoting Effects of Endophytic Pseudomonas sp. from Hippophae thibetana Root Nodules

  • 摘要:   目的  获取西藏沙棘根瘤内生菌中具有多重生物学活性的假单胞菌属菌株,探究筛选所得菌株的促生作用,为研发高效生物菌肥提供基础材料。  方法  利用纯培养方法,从西藏沙棘根瘤中分离假单胞菌,通过形态、生理生化及16S rDNA序列比对鉴定菌株,测定菌株溶磷、产IAA、产铁载体及产降解纤维素酶的能力,接种宿主植物验证其促生效果。  结果  4株根瘤内生菌与参考假单胞菌属同源性为99%以上,鉴定为假单胞菌属。溶磷和产IAA的定性及定量结果表明,4株菌均具有溶解无机磷和产IAA的能力,其中溶解无机磷能力较强的菌株是QY-X10和QY-X22,均达到400 mg·L−1;菌株QY-X4产IAA能力较其他菌株强,达1.9 mg·L−1;4株菌株具有产铁载体的能力,除QY-X10以外,均具产降解纤维素酶的能力;促生试验结果表明,QY-X6可有效促进种子的萌发;QY-X6、QY-X10处理组叶片数显著高于对照;QY-X6、QY-X10处理组苗长显著高于对照;QY-X10、QY-X22处理组最大叶片长显著高于对照。  结论  分离筛选出4株假单胞菌,均可溶解无机磷和产IAA,兼具产铁载体;3株兼具产降解纤维素的能力;接种试验发现,4株菌株处理组可有效促进植株的生长发育,分离菌株可为研发生物菌肥提供基础材料。
  • 图  1  镜检观察革兰氏染色的分离菌株

    Figure  1.  Gram-stained isolated Pseudomonas sp. under microscope

    图  2  基于16S rDNA构建系统发育树

    Figure  2.  Phylogenetic tree based on 16S rDNA

    图  3  不同pH培养对菌株生长量的影响

    Figure  3.  Effect of medium pH on growth of isolated strains

    图  4  不同氯化钠用量对菌株生长量的影响

    Figure  4.  Effect of NaCl concentration in medium on growth of isolated strains

    图  5  菌株产IAA和溶磷能力

    A: 产IAA;B: 溶解无机磷

    Figure  5.  IAA-producing and phosphorus-dissolving abilities of isolated Pseudomonas sp.

    A: IAA-producing ability; B: phosphorus-dissolving ability.

    图  6  菌株产铁载体及产降解纤维素酶能力

    A: 产铁载体; B: 产降解纤维素酶

    Figure  6.  Effect of iron-producing siderophores and cellulase in isolatedPseudomonas sp.

    A: iron-producing siderophore; B: cellulase.

    图  7  菌株促生各指标间的相关性分析

    Figure  7.  Correlation analysis of growth-promoting indexes of Pseudomonas sp.

    表  1  分离菌株生化测定结果

    Table  1.   Biochemical identification of isolated Pseudomonas sp.

    指标
    Index
    QY-X4QY-X6QY-X10QY-X22恶臭假单胞菌A3菌株Pseudomonas putida A3 strain
    半乳糖
    Galactose
    ++++
    麦芽糖
    Maltose
    ++++
    蔗糖生化管
    Sucrose biochemical tube
    ++
    葡萄糖
    Glucose utilization
    +++
    果糖
    Fructose
    +++++
    明胶生化
    Gelatin hydrolysis
    N
    硫化氢
    H2S
    N
    L-鼠李糖
    L-Rhamnose
    +++N
    甲基红试验
    Methyl red test
    V-P反应
    V-P Test
    运动性
    Motility
    运动运动不运动不运动N
    革兰氏染色
    Gram stain
    +表示阳性,-表示阴性。N表示不确定。
    +indicates positive; -indicates negative; N indicates undetermined.
    下载: 导出CSV

    表  2  菌株溶解磷、产IAA的定性测定

    Table  2.   Qualitative determination of phosphorus-dissolving and IAA-producing abilities of isolated Pseudomonas sp.

    菌株
    Strain
    溶解无机磷能力SI
    Phosphate solubilization (inorganic) SI/mm
    产IAA能力
    IAA production
    QY-X41.781±0.066 a++
    QY-X61.778±0.079 a+
    QY-X101.456±0.108 b+
    QY-X221.605±0.118 ab++
    同列不同小写字母表示处理间差异显著(P<0.05),下同。/表示无效果。+表示浅红色,++表示粉红。
    Datas with different lowercase letters on the same column indicate significant difference at 0.05 level. Same for below. /indicates no effect; + indicates light red; and ++ indicates pink.
    下载: 导出CSV

    表  3  菌株溶解磷、产IAA的定量测定

    Table  3.   Quantitative determination of phosphorus-dissolving and IAA-producing abilities of isolated Pseudomonas sp.

    菌株
    Strain
    溶解无机磷量
    Phosphate solubilization (inorganic)/
    (mg·L−1
    IAA含量
    IAA production/
    (mg·L−1
    QY-X4230.232±3.482 b1.919±0.243 a
    QY-X6207.335±26.067 b1.019±0.141 bc
    QY-X10418.131±9.898 a0.627±0.135 c
    QY-X22417.266±4.765 a1.063±0.268 b
    下载: 导出CSV

    表  4  菌株产铁载体及降解纤维素酶测定

    Table  4.   Determination of iron-producing and cellulose-degrading abilities of isolated Pseudomonas sp.

    菌株
    strain
    产铁载体能力SI
    iron-producing siderophore SI/mm
    产降解纤维素酶能力SI
    Degrading cellulase SI/mm
    QY-X41.742±0.3232 a1.671±0.3225 a
    QY-X61.727±0.3556 a2.177±0.2710 a
    QY-X101.986±0.1822 a/
    QY-X221.830±0.1663 a1.797±0.1597 a
    下载: 导出CSV

    表  5  菌株对西藏沙棘幼苗的促生效果

    Table  5.   Growth promoting effects of Pseudomonas sp. on Hippophae thibetana seedlings

    菌株
    Strain
    叶片数
    Leaf number
    苗长
    Seedling length/mm
    根长
    Root length/mm
    最大叶片长
    Maximum leaf length/mm
    最大叶片宽
    Maximum leaf width/mm
    植株鲜重
    fresh weight of plant/g
    植株干重
    Dry weight of plant/g
    发芽率
    germination percentage/%
    CK3.500±1.000 c21.763±21.763 b22.300±5.324 ab5.803±0.616 c4.125±0.530 ab0.069±0.006 a0.008±0.0004 a3.3 b
    QY-X44.000±0.816 bc28.775±28.775 ab18.460±2.761 b6.290±1.521 bc4.130±0.241ab0.070±0.017 a0.009±0.0032 a3.3 b
    QY-X64.750±0.500 ab38.603±38.603 a27.585±3.578 a6.500±0.887 bc4.380±1.076 ab0.077±0.019 a0.009±0.0006 a13.0 a
    QY-X105.250±0.500 a34.778±34.778 a27.635±6.579 a8.418±0.910 a4.823±0.597 a0.090±0.009 a0.009±0.0009 a3.3 b
    QY-X223.250±0.500 c27.988±27.988 ab20.708±3.412 ab7.820±1.235 ab3.628±0.364 b0.073±0.014 a0.009±0.0015 a5.0 b
    下载: 导出CSV
  • [1] 林美珍, 张吉科, 张国伟, 等. 论沙棘根系与功能Ⅲ: 共生固氮机理、条件与初级氮代谢 [J]. 沙棘, 2006, 19(3):7−14.

    LIN M Z, ZHANG J K, ZHANG G W, et al. Root systems of seabuckthorn and their functions: Ⅲ. Mechanism of symbiotic nitrogen fixation, conditions, and primary nitrogen metabolism [J]. Hippophae, 2006, 19(3): 7−14.(in Chinese)
    [2] 邱并生. 根瘤内生细菌 [J]. 微生物学通报, 2013, 40(3):544.

    QIU B S. Nodule endophytic bacteria [J]. Microbiology China, 2013, 40(3): 544.(in Chinese)
    [3] LUGTENBERG B, KAMILOVA F. Plant-growth-promoting rhizobacteria [J]. Annual Review of Microbiology, 2009, 63: 541−556. doi: 10.1146/annurev.micro.62.081307.162918
    [4] COOMBS J T, FRANCO C M M. Visualization of an endophytic Streptomyces species in wheat seed [J]. Applied and Environmental Microbiology, 2003, 69(7): 4260−4262. doi: 10.1128/AEM.69.7.4260-4262.2003
    [5] 张爱梅, 韩雪英, 王嘉, 等. 马衔山中国沙棘根瘤内共生细菌多样性研究 [J]. 生态学报, 2019, 39(1):294−301.

    ZHANG A M, HAN X Y, WANG J, et al. Diversity of endophytic bacteria in root nodules of Hippophae rhamnoides in the Maxian Mountains [J]. Acta Ecologica Sinica, 2019, 39(1): 294−301.(in Chinese)
    [6] 郭振华, 陈立红. 阿尔山不同时期落叶松根际土壤固氮菌的多样性研究 [J]. 西北植物学报, 2019, 39(4):729−739. doi: 10.7606/j.issn.1000-4025.2019.04.0729

    GUO Z H, CHEN L H. Community structure and diversity of culturable nitrogen fixing bacteria from Larix gmelinii rhizosphere soil at different stages in Arxan [J]. Acta Botanica Boreali-Occidentalia Sinica, 2019, 39(4): 729−739.(in Chinese) doi: 10.7606/j.issn.1000-4025.2019.04.0729
    [7] 池景良, 郝敏, 王志学, 等. 解磷微生物研究及应用进展 [J]. 微生物学杂志, 2021, 41(1):1−7. doi: 10.3969/j.issn.1005-7021.2021.01.001

    CHI J L, HAO M, WANG Z X, et al. Advances in research and application of phosphorus-solubilizing microorganism [J]. Journal of Microbiology, 2021, 41(1): 1−7.(in Chinese) doi: 10.3969/j.issn.1005-7021.2021.01.001
    [8] 雷海英, 赵青松, 杨潇, 等. 苦参根际高效固氮菌的分离及复合菌肥对幼苗的促生效应 [J]. 生物技术通报, 2020, 36(9):157−166.

    LEI H Y, ZHAO Q S, YANG X, et al. Isolation of efficient nitrogen-fixing bacteria from the rhizosphere of Sophora flavescens and the growth-promoting effect of compound microbial fertilizer on seedlings [J]. Biotechnology Bulletin, 2020, 36(9): 157−166.(in Chinese)
    [9] 闫双堆, 刘利军, 曹燕篆, 等. 3株多环芳烃高效降解菌株的分离鉴定及降解特性 [J]. 应用生态学报, 2021, 32(12):4439−4446.

    YAN S D, LIU L J, CAO Y Z, et al. Isolation, identification, and degradation characteristics of three effective PAHs degradation strains [J]. Chinese Journal of Applied Ecology, 2021, 32(12): 4439−4446.(in Chinese)
    [10] 杨顺, 杨婷, 林斌, 等. 两株溶磷真菌的筛选、鉴定及溶磷效果的评价 [J]. 微生物学报, 2018, 58(2):264−273.

    YANG S, YANG T, LIN B, et al. Isolation and evaluation of two phosphate-dissolving fungi [J]. Acta Microbiologica Sinica, 2018, 58(2): 264−273.(in Chinese)
    [11] 罗兴, 邹兰, 吴清山, 等. 乌头产吲哚乙酸内生细菌遗传多样性、抗逆性及其对水稻幼苗生长的影响 [J]. 微生物学报, 2022, 62(4):1485−1500.

    LUO X, ZOU L, WU Q S, et al. Genetic diversity, stress resistance, and effect on rice seedling growth of indoleacetic acid-producing endophytic bacteria isolated from Aconitum carmichaelii Debeaux [J]. Acta Microbiologica Sinica, 2022, 62(4): 1485−1500.(in Chinese)
    [12] 东秀珠, 蔡妙英. 常见细菌系统鉴定手册[M]. 北京: 科学出版社, 2001.
    [13] 刘艳萍, 滕松山, 赵蕾. 高产嗜铁素恶臭假单胞菌A3菌株的鉴定及其对黄瓜的促生作用 [J]. 植物营养与肥料学报, 2011, 17(6):1507−1514.

    LIU Y P, TENG S S, ZHAO L. Identification of a siderophore-producing bacterium Pseudomonas putida A3 and its growth-promoting effects on cucumber seedlings [J]. Plant Nutrition and Fertilizer Science, 2011, 17(6): 1507−1514.(in Chinese)
    [14] GLICKMANN E, DESSAUX Y. A critical examination of the specificity of the salkowski reagent for indolic compounds produced by phytopathogenic bacteria [J]. Applied and Environmental Microbiology, 1995, 61(2): 793−796. doi: 10.1128/aem.61.2.793-796.1995
    [15] 樊梦颖, 张明明, 张情, 等. 不同月份沙棘根瘤细菌群落结构特征的分析对比 [J]. 西北林学院学报, 2020, 35(6):160−167.

    FAN M Y, ZHANG M M, ZHANG Q, et al. Comparison of the characteristics of bacterial community in root nodules of Hippophae rhamnoides in different months [J]. Journal of Northwest Forestry University, 2020, 35(6): 160−167.(in Chinese)
    [16] 田国杰, 王晗, 陈立红. 内蒙古赤峰地区主要树种根际固氮菌的分离和鉴定 [J]. 内蒙古林业科技, 2016, 42(1):21−26. doi: 10.3969/j.issn.1007-4066.2016.01.006

    TIAN G J, WANG H, CHEN L H. Isolation and determination of nitrogen-fixing bacteria in rhizosphere of main tree species in Chifeng, Inner Mongolia [J]. Journal of Inner Mongolia Forestry Science and Technology, 2016, 42(1): 21−26.(in Chinese) doi: 10.3969/j.issn.1007-4066.2016.01.006
    [17] 朱培淼, 杨兴明, 徐阳春, 等. 高效解磷细菌的筛选及其对玉米苗期生长的促进作用 [J]. 应用生态学报, 2007, 18(1):107−112. doi: 10.3321/j.issn:1001-9332.2007.01.018

    ZHU P M, YANG X M, XU Y C, et al. High effective phosphate-solubilizing bacteria: Their isolation and promoting effect on corn seedling growth [J]. Chinese Journal of Applied Ecology, 2007, 18(1): 107−112.(in Chinese) doi: 10.3321/j.issn:1001-9332.2007.01.018
    [18] 雷平, 黄军, 黄彬彬, 等. 1株产铁载体辣椒内生细菌的分离鉴定及其促生长作用 [J]. 激光生物学报, 2020, 29(4):379−384. doi: 10.3969/j.issn.1007-7146.2020.04.012

    LEI P, HUANG J, HUANG B B, et al. Isolation, identification and growth promoting effect of a siderophore-producing endophytic bacterium from capscium [J]. Acta Laser Biology Sinica, 2020, 29(4): 379−384.(in Chinese) doi: 10.3969/j.issn.1007-7146.2020.04.012
    [19] 苑博华, 廖祥儒, 郑晓洁, 等. 吲哚乙酸在植物细胞中的代谢及其作用 [J]. 生物学通报, 2005, 40(4):21−23. doi: 10.3969/j.issn.0006-3193.2005.04.010

    YUAN B H, LIAO X R, ZHENG X J, et al. Metabolism and function of 1AA in plant cell [J]. Bulletin of Biology, 2005, 40(4): 21−23.(in Chinese) doi: 10.3969/j.issn.0006-3193.2005.04.010
    [20] 吴婧, 聂彩娥, 朱媛媛, 等. 一株兼具产IAA能力纤维素降解菌的筛选、鉴定及条件优化 [J]. 生物技术通报, 2020, 36(12):54−63.

    WU J, NIE C E, ZHU Y Y, et al. Isolation, identification of a cellulose-degrading bacterium with IAA-producing ability and optimization of its culture conditions [J]. Biotechnology Bulletin, 2020, 36(12): 54−63.(in Chinese)
    [21] 刘丹丹, 李敏, 刘润进. 我国植物根围促生细菌研究进展 [J]. 生态学杂志, 2016, 35(3):815−824. doi: 10.13292/j.1000-4890.201603.033

    LIU D D, LI M, LIU R J. Recent advances in the study of plant growth-promoting rhizobacteria in China [J]. Chinese Journal of Ecology, 2016, 35(3): 815−824.(in Chinese) doi: 10.13292/j.1000-4890.201603.033
    [22] SARWAR M, FRANKENBERGER W T Jr. Influence of L-tryptophan and auxins applied to the rhizosphere on the vegetative growth of Zea mays L [J]. Plant and Soil, 1994, 160(1): 97−104. doi: 10.1007/BF00150350
    [23] 刘梦洁, 尹清强, 常娟, 等. 植物乳杆菌和纤维素酶对玉米秸秆降解及糖化效果的影响 [J]. 中国饲料, 2020(23):34−39.

    LIU M J, YIN Q Q, CHANG J, et al. Effects of Lactobacillus plantarum and cellulase on the degradation and saccharification of corn straw [J]. China Feed, 2020(23): 34−39.(in Chinese)
    [24] 张爱梅, 殷一然, 齐汝楠. 产纤维素酶沙棘根瘤内生放线菌的筛选、鉴定及其酶活性测定 [J]. 西北师范大学学报(自然科学版), 2019, 55(5):71−76. doi: 10.16783/j.cnki.nwnuz.2019.05.013

    ZHANG A M, YIN Y R, QI R N. Screening, identification and enzyme activity of endophytic actinomycetes producing cellulase from root nodules of Hippophae rhamnoides [J]. Journal of Northwest Normal University (Natural Science), 2019, 55(5): 71−76.(in Chinese) doi: 10.16783/j.cnki.nwnuz.2019.05.013
    [25] 朱凤, 陈夕军, 童蕴慧, 等. 水稻内生细菌的分离及其拮抗性与潜在致病性测定 [J]. 中国生物防治, 2007, 23(1):68−72.

    ZHU F, CHEN X J, TONG Y H, et al. Isolation of rice endophytic bacteria and their antagonism and potential pathogenicity [J]. Chinese Journal of Biological Control, 2007, 23(1): 68−72.(in Chinese)
    [26] 金玲, 巴峰, 计平生, 等. 小麦内生有害细菌的发现和作用研究 [J]. 植物病理学报, 1996, 26(2):123−126.

    JIN L, BA F, JI P S, et al. Discovery of deleterious endobacteria and their effects on growth of wheat [J]. Acta Phytopathologica Sinica, 1996, 26(2): 123−126.(in Chinese)
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  • 收稿日期:  2022-10-09
  • 修回日期:  2023-04-30
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