2023, Vol. 63
表1(Table 1)
醋酸钙不动杆菌对盐碱胁迫下达乌里胡枝子促生效应研究
http://dx.doi.org/10.13343/j.cnki.wsxb.20220911
中国科学院微生物研究所，中国微生物学会
中国科学院微生物研究所，中国微生物学会
文章信息
- 黄臣, 蒋霖, 梁银萍, 韩玲娟, 杨凯元, 秦云梦, 高鹏, 赵祥. 2023
- HUANG Chen, JIANG Lin, LIANG Yinping, HAN Lingjuan, YANG Kaiyuan, QIN Yunmeng, GAO Peng, ZHAO Xiang.
- 醋酸钙不动杆菌对盐碱胁迫下达乌里胡枝子促生效应研究
- Acinetobacter calcoaceticus promotes the seedling growth of Lespedeza daurica under saline-alkaline stress
- 微生物学报, 63(8): 3264-3278
- Acta Microbiologica Sinica, 63(8): 3264-3278
-
文章历史
- 收稿日期：2022-12-08
- 网络出版日期：2023-03-21
引用本文 |
黄臣, 蒋霖, 梁银萍, 韩玲娟, 杨凯元, 秦云梦, 高鹏, 赵祥. 醋酸钙不动杆菌对盐碱胁迫下达乌里胡枝子促生效应研究[J]. 微生物学报, 2023, 63(8): 3264-3278.
Huang Chen, Jiang Lin, Liang Yinping, Han Lingjuan, Yang Kaiyuan, Qin Yunmeng, Gao Peng, Zhao Xiang. Acinetobacter calcoaceticus promotes the seedling growth of Lespedeza daurica under saline-alkaline stress[J]. Acta Microbiologica Sinica, 2023, 63(8): 3264-3278.
醋酸钙不动杆菌对盐碱胁迫下达乌里胡枝子促生效应研究
山西农业大学草业学院, 山西 晋中 030600
摘要：达乌里胡枝子(Lespedeza davurica)是黄土高原地区重要的生态修复草种，但土壤盐碱化极大影响了达乌里胡枝子的生长，制约了其经济和生态价值。接种根际促生菌剂是提高植物对盐碱胁迫抗性的重要途径。[目的] 验证醋酸钙不动杆菌(Acinetobacter calcoaceticus)对盐碱胁迫下达乌里胡枝子幼苗和成株的促生效应，初步揭示其促进豆科植物生长的潜在机理，为田间应用提供理论依据。[方法] 采用解钾培养基对2株达乌里胡枝子根际分离的醋酸钙不动杆菌的解钾能力进行测定；采用液体发酵法对菌株分泌铁载体、吲哚乙酸(indole-3-acetic acid, IAA)的能力进行定量测定；分别在培养皿和盆栽条件下，验证菌株的促生能力及其机制。[结果] 菌株DP25和DP27具有解钾和分泌铁载体、IAA的能力，其中相对铁载体表达量值(siderophore, SU)分别为53.13%和86.67%，IAA含量分别为1.01 mg/L和17.31 mg/L；菌株DP25显著提高了培养皿中达乌里胡枝子幼苗的茎长和根长；菌株DP25和DP27对盐碱胁迫下达乌里胡枝子成株的光合色素合成及其光合作用产生积极影响；菌株DP25和DP27显著提高了盆栽条件下达乌里胡枝子成株的株高、茎粗、根表面积、总根长、分叉数；菌株DP27还显著提高了地上和地下生物量、根体积、总根尖数、根系活力。[结论] 接种醋酸钙不动杆菌对达乌里胡枝子幼苗和成株均表现出良好的促生作用，其中分泌IAA是促进幼苗生长的主要因素，而提高光合能力和根系形态建成在促进成株生长中发挥关键作用。醋酸钙不动杆菌可用于开发黄土高原盐碱地区建植达乌里胡枝子的促生菌剂。
关键词：黄土高原 盐碱胁迫 达乌里胡枝子 醋酸钙不动杆菌
Acinetobacter calcoaceticus promotes the seedling growth of Lespedeza daurica under saline-alkaline stress
College of Grassland Science, Shanxi Agricultural University, Jinzhong 030600, Shanxi, China
Abstract: Lespedeza davurica is a grass for ecological restoration in the Loess Plateau. The saline-alkaline soil in the Loess Plateau greatly affects the growth and restricted the economic and ecological values of L. davurica. Inoculation of growth-promoting rhizobacteria is an important way to improve plant tolerance to saline-alkaline stress. [Objective] This study aims to reveal the effect and mechanism of Acinetobacter calcoaceticus in promoting the seedling growth of L. davurica under saline-alkaline stress, preliminarily disclose the potential growth-promoting mechanism of legumes, and provide a theoretical basis for the field application of this bacterium. [Methods] The potassium-containing medium was used to measure the potassium-solubilizing ability of two A. calcoaceticus strains isolated from L. davurica rhizosphere. The liquid fermentation method was employed to determine the production of siderophore and indole-3-acetic acid (IAA) by the two strains. Furthermore, we carried out petri dish and pot experiments to determine the growth-promoting effects of A. calcoaceticus on L. davurica seedlings under saline-alkaline stress. [Results] Strains DP25 and DP27 had the abilities to solubilize potassium and showed the siderophore production of 53.13% and 86.67% and the IAA production of 1.01 mg/L and 17.31 mg/L, respectively. Compared with the control, the inoculation of DP25 significantly improved the stem length and root length of L. davurica seedlings in petri dishes. The pot experiments showed that strains DP25 and DP27 had positive effects on the photosynthetic pigment synthesis and photosynthesis of the plants under saline-alkaline stress. Compared with the control, the inoculation with strains DP25 and DP27 increased the plant height, stem diameter, root surface area, total root length, and bifurcation number of L. davurica plants in pots. In addition, strain DP27 increased the aboveground biomass, underground biomass, root volume, total root tips, and root activity. [Conclusion] A. calcoaceticus demonstrates growth-promoting effect on the seedlings and adults of L. davurica. It secretes IAA to promote the growth of seedlings and improves the photosynthetic capacity and root morphogenesis to facilitate the growth of the adults under saline-alkaline stress. A. calcoaceticus can be used as a growth-promoting bacterium for planting L. daurica in the saline-alkaline area of Loess Plateau.
Keywords:
Loess Plateau saline-alkaline stress Lespedeza daurica Acinetobacter calcoaceticus
达乌里胡枝子(Lespedeza daurica)为豆科(Leguminosae)胡枝子属(Lespedeza)的多年生草本状半灌木，是黄土高原水土流失区和土壤贫瘠地块形成次生群落的重要组分[1-2]，在尾矿区修复中也发挥着重要作用[3]。由于农业灌溉、水利工程和土地管理方案不当等因素导致黄土高原地区约有30%耕地面积受到土壤盐碱化的影响，且盐碱化程度及盐碱化面积呈逐年加剧趋势[4]，其中晋北地区盐碱地总面积约为1.88×106 hm2[5]。研究表明，土壤中高浓度钠离子会抑制植物的生长、发芽、蒸腾作用、光合作用等，造成严重的毒害作用[6]，每年约10%的作物因土地盐碱化导致减产[7]。因此，极大限制了达乌里胡枝子的经济和生态价值。
此外，高盐浓度会改变土壤渗透和基质潜力，降低土壤微生物活性，进而导致土壤退化[8]。微生物是土壤生物多样性的关键组成部分，同时土壤微生物和植物之间存在独特的共生关系，包括互作和空间上相邻[9]。微生物可通过多种途径改善植物适应性和土壤质量，从而提高农作物生产力和土壤稳定性[10-11]。根际是一个独特的生态位，蕴含丰富的微生物类群，其中一类具有直接或间接促进植物生长、提高植物抗性功能的有益细菌称为根际促生菌(plant growth-promoting rhizobacteria, PGPR)[12]，其作用为：(1) 可通过溶磷、解钾、产有机酸和铁载体、产植物激素如吲哚乙酸、赤霉素等促进植物的生长发育[13]；(2) 分泌铁载体、1-氨基环丙烷-1-羧酸(1-aminocyclopropane-1-carboxylate, ACC)脱氨酶等，增强植物对盐碱、重金属等逆境的抗性及抗病性[14-15]。
盐碱化是植物生存面临的主要非生物胁迫，也是制约农业生产力的关键因素[16]，利用耐盐促生菌能有效缓解盐碱胁迫对作物生长造成的毒害。不动杆菌属(Acinetobacter)属于假单胞目(Pseudomonadales)莫拉菌科(Moraxellaceae)，是一类在土壤中广泛分布且多兼具耐盐和促生功能的微生物[17-18]。Han等[19]从海边土壤中分离到1株在7% NaCl胁迫下可产生乳糖酸(lactobionic acid, LBA)的不动杆菌(Acinetobacter halotolerans) KRICT-1；赵廷伟等[20]从甘肃地区盐碱土中分离出1株可在盐碱环境下(pH为9.0，盐浓度为20%、10%，pH为12.0)生长且兼具产IAA、解磷能力的不动杆菌属菌株21；张巍等[21]从羊草(Leymus chinensis)根际盐碱土分离出1株可在pH值为10或盐浓度为7.5%条件下生长，且兼具解磷功能的醋酸钙不动杆菌C111。耐盐的根际促生菌可降低盐胁迫对植物的毒害，提高植物生产性能[22]。目前，关于醋酸钙不动杆菌的研究对象多为番茄(Solanum lycopersicum)[23]、燕麦(Avena sativa)[24]、苜蓿(Medicago sativa)[18]、黄瓜(Cucumis sativus)[24]等经济作物。接种醋酸钙不动杆菌SJ4后显著促进番茄幼苗根和芽的伸长，促进植物鲜重的增加，并显著提高了番茄幼苗对灰霉病菌(Bacillus cinerea)的抗病性。复合菌剂接种普罗威登斯菌(Providencia rettgeri) P2、克什米尔小陌生菌(Advenella kashmirensis) P4、醋酸钙不动杆菌(Acinetobacter calcoaceticus) P19、腐败沙雷菌(Serratia plymuthica) P35显著提高了燕麦、苜蓿、黄瓜幼苗的株高、根长、根平均直径、根表面积、根体积、叶绿素含量和抗氧化酶活性[24]。此外，醋酸钙不动杆菌在柴油降解[25]、土壤重金属修复[26]和石油烃降解[27]等方面也发挥着重要作用，但鲜有研究关注盐碱条件下醋酸钙不动杆菌与黄土高原地区生态修复植物的互作效应。
本团队前期从盐碱地达乌里胡枝子根际分离筛选得到2株具有解磷能力的中度耐盐醋酸钙不动杆菌DP25、DP27[28]。在此基础上，本研究结合黄土高原地区盐碱化程度和醋酸钙不动杆菌促生特性，开展醋酸钙不动杆菌与达乌里胡枝子互作研究，进一步分析盐碱条件下醋酸钙不动杆菌对达乌里胡枝子的促生效应。以期初步揭示其在盐胁迫下对达乌里胡枝子的促生机理，为微生物提高植物适应盐胁迫提供理论依据，同时对黄土高原地区盐碱地修复利用及醋酸钙不动杆菌促生机制研究具有重要意义。
1 材料与方法 1.1 供试菌株及达乌里胡枝子种子A. calcoaceticus菌株DP25 (GenBank登录号：ON077030)和DP27 (GenBank登录号：ON077031)由本团队筛选，分离自达乌里胡枝子根际[28]。
‘晋农1号’达乌里胡枝子种子由本团队选育，2014年获国家草品种审定委员会审定的育成品种。
1.2 主要试剂和仪器振荡培养箱，上海知楚仪器有限公司；酶标仪，博藤仪器有限公司；涡旋仪，大龙兴创实验仪器有限公司；光合仪，思爱迪(北京)生态科学仪器有限公司；根系扫描仪，中国中晶科技有限公司。
1.3 培养基采用漫静等[29]的方法使用LB培养基进行促生菌的常规培养；解钾培养基(g/L)：葡萄糖10.0，钾长石2.5，NaH2PO4 0.2，MgSO4·7H2O 0.2，NaCl 0.2，CaSO4·7H2O 0.1，CaCO3 5.0，琼脂18.0，蒸馏水1 000 mL，pH 7.0–7.5[30]。改良金氏(modified King B, MKB)液体培养基(g/L)：酪蛋白5.0，甘油15.0 mL，K2HPO4·7H2O 2.5，MgSO4·7H2O 2.5，pH 7.2[31]；金氏培养基B (King’s B medium, KMB, g/L)：胰蛋白胨20.0，K2HPO4·7H2O 1.5，MgSO4·7H2O 1.5，甘油10 mL，色氨酸0.5，pH 7.0[32]。上述培养基均在121 ℃高压灭菌锅中灭菌20 min。
1.4 醋酸钙不动杆菌功能特性测定 1.4.1 菌悬液制备供试菌株在LB液体培养基中活化培养12 h后，室温10 000 r/min离心5 min，收集菌体，反复重悬3次，菌体重新悬浮于无菌水中，通过无菌水稀释至OD600值约为0.1、0.5和0.8。
1.4.2 解钾能力测定将菌株点接种于解钾固体培养基上，每株菌重复3次，28 ℃、恒温培养5 d，观察菌株周围是否出现透明圈，有透明圈则表明具有解钾能力。
1.4.3 产铁载体能力测定将0.5 mL菌悬浮液(OD600约为0.5)接种于盛有50 mL MKB液体培养基的锥形瓶中，每组重复3次，28 ℃、180 r/min振荡培养箱培养2 d。培养结束后，取适量发酵液10 000 r/min离心10 min，取3 mL上清液与等体积CAS检测液充分混匀，避光静置30 min，测定OD630值(As)，测定未接种的MKB液体培养基OD630值(Ar)，用相对铁载体表达量单位表示[33]。
1.4.4 产IAA能力测定将分离纯化后的菌株接种于0.5 g/L的KMB (0.5 g/L l-色氨酸)液体培养基中，30 ℃、180 r/min振荡培养48 h，10 000 r/min离心10 min，取3 mL上清液与等体积Salkowski比色液充分混合，未接菌培养基为对照，避光静置30 min，颜色变粉红为具有产IAA能力菌株，并在OD530测定发酵液中IAA含量[34]。
1.5 醋酸钙不动杆菌对达乌里胡枝子苗期生长影响为了验证菌株分泌的激素对种子促生效应，挑选成熟饱满的达乌里胡枝子，用10%次氯酸钠溶液浸泡5 min，无菌水冲洗至无味。设置2个处理，A处理：种子置于盛有待测菌菌悬液(OD600约为0.5)的无菌培养皿中浸泡12 h；B处理：将种子置于盛有无菌水的无菌培养皿中浸泡12 h。取出晾干种子表面水分，置于装有2层无菌滤纸的培养皿中，每皿50粒种子，每个处理4次重复，置于光照培养箱中，培养条件为25 ℃ (16 h，光照，12 000 lx)/20 ℃ (8 h，暗培养)。14 d后统计达乌里胡枝子根长、茎长、苗长。
1.6 醋酸钙不动杆菌对达乌里胡枝子成株期生长的影响 1.6.1 盆栽土壤制备供试土壤取自山西省晋中市人工栽培达乌里胡枝子种植田(112°60′E, 37°44′N)，理化性质如表 1所示。土壤过2 mm筛，备用。试验用盆为直径15 cm、高20 cm的PVC管。
表 1. 盆栽土壤基本理化性质
Table 1. Basic characteristics of soil for experiment
| Item | Value |
| pH | 8.60 |
| Water soluble salt (g/kg) | 5.33 |
| Total phosphorus (g/kg) | 0.57 |
| Total nitrogen (g/kg) | 1.20 |
| Total potassium (g/kg) | 18.71 |
| Available phosphorus (mg/kg) | 33.40 |
| Alkaline hydrolysis nitrogen (mg/kg) | 10.00 |
| Available potassium (mg/kg) | 275 |
| Total iron (g/kg) | 31.13 |
| Saturation capacity (%) | 24.01 |
1.6.2 试验设计
盆栽试验在2022年9–11月于山西农业大学草业学院草地微生物实验室的植物培养间进行。共设置3个处理，A处理：等量无菌水(control)；B处理：接种菌株DP25；C处理：接种菌株DP27。每个处理4次重复。培养条件：白天温度为23– 25 ℃，夜间温度18–20 ℃，光照强度8 000 lx，光照周期为16 h/8 h (光照/黑暗)，相对湿度为40%–50%。步骤：试验前将达乌里胡枝子种子进行消毒处理：用10% NaClO溶液表面消毒5 min后，无菌水冲洗至无味。无纺布于121 ℃高压灭菌锅中灭菌20 min，75%酒精溶液擦拭PVC管内外壁，装填3.5 kg过筛的土壤于PVC管中，加水至最大田间持水量的75%左右，平衡1 d后。每盆3穴，每穴播种达乌里胡枝子种子10颗。待生长7 d后，间苗至每穴1株。待植株生长至第10天，处理施入菌悬液(108 CFU/mL)，对照施入无菌水，间隔7 d施入1次，每次施入量均为35 mL，共计3次。
管理培养60 d后，进行取样，测定植物光合指标、形态指标、生物量指标和土壤有效磷指标。试验期间，每隔3 d补充各PVC管缺失质量等量的无菌水。
1.6.3 测定指标形态指标：采用直尺测定植株株高、根长；使用游标卡尺测定茎粗；量筒测定根系体积；采用MRS-9600TFU2L根系扫描仪测定并计算单株根系长度(cm)、表面积(cm2)、根平均直径(mm)、根尖数。
光合指标：参考乙醇提取分光光度法[35]测定叶绿素a (chlorophyll a, Chl a)、叶绿素b (chlorophyll b, Chl b)、叶绿素总量[Chl (a+b)]、叶绿素a/叶绿素b (Chl a/b)和类胡萝卜素(carotenoid, Car)含量；在9:00–11:30，使用便携式光合仪Cl-340测定叶片净光合速率(Pn)、蒸腾速率(Tr)、胞间CO2浓度(Ci)、气孔导度(Gs)等参数。使用PAM 2500荧光仪测定叶片叶绿素荧光参数，叶片暗适应30 min后，测定暗适应后光系统Ⅱ (photo system Ⅱ, PSII)的最大光化学效率(Fv/Fm)和PSⅡ潜在活性(Fv/Fo)。
生物量指标：将地上部植株和地下部根系分开并清洗干净，分析天平测量并计算单株鲜重；根系活力采用2, 3, 5-三苯基氯化四氮唑(2, 3, 5-triphenyltetrazolium chloride, TTC)法；将地上部植株和地下部根系装入档案袋，105 ℃杀青30 min，75 ℃烘至恒重，分析天平测量并计算单株干重。
土壤指标：采用凯氏法测定土壤全氮；采用原子吸收光谱法测定土壤全铁；土壤有效磷测定采用钼锑抗比色法；采用火焰光度计法测定土壤有效钾；根际土壤pH采用pH计测定。
1.7 数据分析利用Microsoft Excel 2019整理数据，使用SPSS 20.0进行数据分析，对达乌里胡枝子干鲜重、株高、根长、茎粗、Chl a、Chl b、Chl (a+b)、Chl a/b和Car等进行单因素方差分析和Duncan法多重比较(P < 0.05)，采用Origin 2021统计软件绘制达乌里胡枝子幼苗株高、根长和苗长柱状图和达乌里胡枝子土壤养分含量柱状图。
2 结果与分析 2.1 醋酸钙不动杆菌功能特性测定定性测定结果表明菌株DP25和DP27均具备产IAA能力。进一步测定菌株DP25和DP27产IAA的含量分别为17.31 mg/L和1.01 mg/L (表 2)，铁载体的相对浓度分别为53.13%、86.67%。此外，菌株DP25、DP27可在解钾培养基上形成清晰的透明圈，表明菌株DP25、DP27均具备解钾能力(图 1)。
表 2. 菌株DP25、DP27产IAA、铁载体、解钾能力测定结果
Table 2. Determination results of IAA, siderophore and potassium releasing ability of Acinetobacter calcoaceticus DP25 and DP27
| Strain | IAA (mg/L) | Siderophore (%) | Potassium release |
| DP25 | 17.31±1.01 | 53.13±3.55 | + |
| DP27 | 1.01±0.80 | 86.67±0.98 | + |
|
| 图 1 菌株DP25和DP27解钾能力 Figure 1 Potassium-dissolving ability of strains DP25 and DP27. |
2.2 醋酸钙不动杆菌对达乌里胡枝子幼苗的促生作用
如图 2所示，菌株DP25能够显著促进达乌里胡枝子幼苗的茎长、根长生长，较无菌水处理分别提高了19.21%、22.67% (P < 0.05)。
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| 图 2 接种醋酸钙不动杆菌DP25和DP27对达乌里胡枝子幼苗生长指标的影响 Figure 2 The growth indexes of Lespedeza daurica seedlings was affected by Acinetobacter calcoaceticus DP25 and DP27. Different letters represent significant differences in different treatment at 0.05 level. |
2.3 醋酸钙不动杆菌对盐碱胁迫下达乌里胡枝子成株期生长影响 2.3.1 对盐碱胁迫下达乌里胡枝子光合作用的影响
如表 3所示，与无菌水对照组相比，菌株DP25处理的达乌里胡枝子叶片Chl a、Chl b、Chl (a+b)、Car含量、Fv/Fm值和Fv/Fo值分别显著增加43.97%、61.29%、47.62%、38.10%、2.67%和10.47% (P < 0.05)；菌株DP27处理的达乌里胡枝子叶片Chl a、Chl b、Chl (a+b)、Car含量、Fv/Fm值和Fv/Fo值分别显著增加44.83%、58.06%、47.62%、35.71%、2.67%和12.50% (P < 0.05)。
表 3. 接种醋酸钙不动杆菌DP25和DP27对达乌里胡枝子叶片光合色素含量及其叶绿素荧光参数的影响
Table 3. Effects on photosynthetic pigment content and chlorophyll fluorescence parameters of Lespedeza davurica leaves by Acinetobacter calcoaceticus DP25 and DP27
| Treatment | Chlorophyll content | Determination of fluorescent chlorophyll | ||||||
| Chl a (mg/kg) | Chl b (mg/kg) | Chl (a+b) (mg/kg) | Chl a/b | Car (mg/kg) | Fv/Fm | Fv/Fo | ||
| Control | 1.16+0.06b | 0.31+0.02b | 1.47+0.08b | 3.81+0.05a | 0.42+0.02b | 0.75+0.01b | 2.96+0.15b | |
| DP25 | 1.67+0.10a | 0.50+0.09a | 2.17+0.19a | 3.42+0.36a | 0.58+0.05a | 0.77+0.01a | 3.27+0.18a | |
| DP27 | 1.68+0.10a | 0.49+0.11a | 2.17+0.14a | 3.44+0.16a | 0.57+0.04a | 0.77+0.01a | 3.33+0.13a | |
| Different letters represent significant differences in different treatment at 0.05 level. | ||||||||
与无菌水对照组相比(表 4)，菌株DP25、DP27处理的达乌里胡枝子叶片Pn值分别增加12.77%和14.74% (P < 0.05)；菌株DP25、DP27处理的达乌里胡枝子叶片Ci值分别降低65.20%和42.44% (P < 0.05)。
表 4. 接种醋酸钙不动杆菌DP25和DP27对达乌里胡枝子Pn、Tr、Gs和Ci的影响
Table 4. Effects of inoculation with Acinetobacter calcoaceticus DP25 and DP27 on Pn, Tr, Gs and Ci of Lespedeza davurica
| Treatment | Pn | Tr | Gs | Ci |
| Control | 5.89+1.31b | 1.18+0.23a | 74.67+4.34a | 159.00+14.98b |
| DP25 | 9.73+1.78a | 1.28+0.16a | 79.60+6.54a | 138.69+8.21ab |
| DP27 | 8.39+0.90a | 1.39+0.49a | 70.13+25.52a | 135.56+16.34a |
| Different letters represent significant differences in different treatment at 0.05 level. | ||||
2.3.2 对盐碱胁迫下达乌里胡枝子生长的影响
盐碱环境下，接种菌株DP25后达乌里胡枝子株高、茎粗均显著高于无菌水对照组(表 5)，分别增加了31.65%和25.53% (P < 0.05)；接种菌株DP27后达乌里胡枝子株高提高了35.35%，根长提升了16.33%，茎粗提高了21.99% (P < 0.05)。
表 5. 接种醋酸钙不动杆菌DP25和DP27对达乌里胡枝子生长指标的影响
Table 5. Effects of Acinetobacter calcoaceticus DP25 and DP27 inoculation on growth indexes of Lespedeza davurica
| Item | Treatment | ||
| Control | DP25 | DP27 | |
| Plant acid height (cm/plant) | 14.06±1.18b | 18.51±1.58a | 19.03±2.80a |
| Root length (cm/plant) | 23.33±1.25b | 25.53±1.25ab | 27.14±2.63a |
| Whole plant length (cm/plant) | 37.38±1.41b | 44.04±0.88a | 46.17±5.21a |
| Stem thick (cm/plant) | 46.17±5.21a | 1.77±0.09a | 1.72±0.11a |
| Root top ratio | 0.13±0.02a | 0.13±0.01a | 0.13±0.01a |
| Aboveground fresh weight (g/plant) | 2.33±0.36b | 2.59±0.32ab | 3.17±0.68a |
| Underground fresh weight (g/plant) | 0.60±0.06b | 0.57±0.11b | 0.87±0.33a |
| Terrestrial dry matter (g/plant) | 0.55±0.05b | 0.65±0.09b | 0.81±0.15a |
| Underground weights (g/plant) | 0.07±0.01b | 0.08±0.01b | 0.12±0.02a |
| Root volume (m3/plant) | 0.69±0.07b | 0.84±0.09b | 1.21±0.28a |
| Root surface (cm2/plant) | 60.76±9.45c | 84.39±8.02b | 115.57±11.45a |
| Total root length (cm/plant) | 285.89±46.04c | 395.48±28.06b | 485.69±68.71a |
| Mean diameter (cm/plant) | 0.90±0.22a | 0.77±0.03a | 0.91±0.07a |
| Root tips number | 331.75±14.92b | 577.00±138.57b | 1075.5±257.33a |
| Bifurcation number | 407.42±80.68b | 596.17±64.73a | 715.92±119.71a |
| Root activity (μg/(g·h)) | 93.23±9.15b | 83.64±4.79b | 133.35±8.33a |
| Different letters represent significant differences in different treatment at 0.05 level. | |||
接种菌株DP25后达乌里胡枝子根表面积、总根长及分叉数显著高于无菌水对照组(图 3和表 5)，分别增加了38.89%、38.33%、46.43% (P < 0.05)；菌株DP27对达乌里胡枝子根系促生效果最为显著，根体积、根表面积、总根尖数、分叉数和根系活力分别提高了75.36%、90.21%、224.19%、75.72%和43.03% (P < 0.05)。
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| 图 3 接种醋酸钙不动杆菌DP25和DP27对达乌里胡枝子植株地上部分及其地下根系结构的影响 Figure 3 The Structure of aboveground parts and underground roots of Lespedeza davurica was affected by Acinetobacter calcoaceticus DP25 and DP27. A, a: The structure of aboveground parts and underground roots of L. davurica in control. B, b: The structure of aboveground parts and underground roots of Lespedeza davurica inoculated with strain DP25. C, c: The structure of aboveground parts and underground roots of L. davurica inoculated with strain DP24. White line segment for 10 cm ruler. |
从不同水平根系指标总量及其占比情况分析，与对照组相比(表 6)，菌株DP25和DP27均显著提高植株L < 0.50 cm、2.00≤L < 3.00 cm、TL < 2.00 cm、SA < 0.50 cm2和2.00≤SA < 3.00 cm2总量，菌株DP27还可促进0.50≤L < 2.00 cm、3.00 cm≤L、0.50≤SA < 2.00 cm2、3.00 cm2≤SA积累(P < 0.05)。此外，接种菌株DP25较对照2.00≤L < 3.00 cm、0.50≤TL < 2.00 cm和2.00≤SA < 3.00 cm2占比显著提高，接种菌株DP27较对照0.50≤TL < 2.00 cm占比显著提高，而L < 0.50 cm、TL < 0.50 cm和SA < 0.50 cm2占比水平较对照显著降低(P < 0.05)。
表 6. 接种醋酸钙不动杆菌DP25和DP27对达乌里胡枝子不同水平根系长度(L, cm)、根尖长度(TL, cm)、表面积(SA, cm2)总量的影响
Table 6. Effects of Acinetobacter calcoaceticus DP25 and DP27 on root length (L), root tip length (TL) and surface area (SA) of Lespedeza davurica at different levels
| Treatment | L | TL | SA | ||||||||||
| L1 | L2 | L3 | L4 | TL1 | TL2 | TL3 | SA1 | SA2 | SA3 | SA4 | |||
| Control | 490.83±74.15b | 320.06±82.84b | 21.87±16.85b | 24.91±19.30b | 244.81±25.07c | 51.84±7.75c | 13.12±12.53a | 15.81±2.53b | 28.23±6.58b | 5.07±3.87b | 8.91±6.77b | ||
| Prencent (%) | 51.34±2.85a | 36.91±4.33a | 2.76±2.35b | 2.98±2.22a | 78.96±3.21a | 16.67±1.26c | 4.36±4.32a | 27.56±4.94a | 48.98±11.20a | 8.69±6.38b | 14.76±10.34a | ||
| DP25 | 656.81±55.90a | 446.32±70.63b | 69.31±11.09a | 13.99±4.13b | 335.01±35.44b | 93.70±11.48b | 5.84±2.04a | 21.01±1.91a | 37.92±5.88b | 16.74±2.41a | 4.92±1.39b | ||
| Prencent | 55.46±4.47a | 37.52±4.08a | 5.83±0.70a | 1.19±0.36a | 77.10±1.55a | 21.55±1.18b | 1.35±0.48a | 26.20±2.83ab | 46.89±3.02a | 20.72±1.41a | 6.19±2.06a | ||
| DP27 | 733.45±105.13a | 618.46±105.66a | 58.85±4.88a | 46.31±4.86a | 431.55±75.48a | 184.90±14.73a | 16.60±2.02a | 24.17±3.20a | 55.11±7.36a | 15.01±5.92a | 16.07±1.76a | ||
| Prencent (%) | 50.33±1.59b | 42.36±1.87a | 4.07±0.37ab | 3.25±0.67a | 67.93±2.59b | 29.40±2.04a | 2.67±0.57a | 21.85±1.18b | 49.85±2.40a | 13.62±0.66b | 14.68±2.14a | ||
| L, TL, SA: Root length, root tip length and surface area of L. davurica; L1, L2, L3 and L4: L < 0.50 cm, 0.50≤L < 2.00 cm, 2.00≤L < 3.00 cm and 3.00 cm≤L; TL1, TL2 and TL3: TL < 0.50 cm, 0.50≤TL < 2.00 cm and 2.00 cm≤TL; SA1, SA2, SA3 and SA4: SA < 0.50 cm2, 0.50≤SA < 2.00 cm2, 2.00≤SA < 3.00 cm2 and 3.00 cm≤SA2. Prencent (%) indicates the proportion. Different letters represent significant differences in different treatment at 0.05 level. | |||||||||||||
2.3.3 对盐碱胁迫下达乌里胡枝子生物量的影响
植株地上部分鲜重提高了36.05%，地下部分鲜重提高了45.00%，地上部分干重提高了47.27%，地下部分干重提高了71.43% (P < 0.05)。
2.3.4 对土壤养分的影响盐碱环境下，接种菌株DP25后，土壤有效磷含量较对照降低了6.50% (图 4C)；土壤有效钾含量显著上升，较对照提高了3.06% (图 4D)；此外，接种菌株DP25后土壤pH较对照下降了0.89% (P < 0.05) (图 4E)。
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| 图 4 接种醋酸钙不动杆菌DP25和DP27对土壤养分的影响 Figure 4 Effects of Acinetobacter calcoaceticus DP25 and DP27 inoculation on soil nutrient. Different letters represent significant differences in different treatment at 0.05 level. |
3 讨论与结论
醋酸钙不动杆菌是一类在土壤中广泛分布且多具有促生功能的微生物[17]。醋酸钙不动杆菌类促生菌具有或兼具固氮、解磷、解钾、产IAA、产铁载体等能力[36-37]。本文研究对象醋酸钙不动杆菌DP25和DP27同时具备产IAA、解钾能力和产铁载体等能力。IAA作为植物中最重要的生长素之一，在调节植物生长发育等方面发挥着重要的作用[38]。从美国龙舌兰(Agave americana)根际分离出的醋酸钙不动杆菌ACO-40产IAA含量为8.5 mg/L[37]，远低于菌株DP25产IAA含量，远高于菌株DP27产IAA含量，盆栽实验表明菌株ACO-40显著促进美国龙舌兰植株干重和叶片数的积累，分别增加了61.84%和125.00%。而Li等[24]从三叶草(Trifolium)根际分离的醋酸钙不动杆菌P19不具备产IAA能力，这可能是由于菌株自身遗传特性所引起的促生功能差异。产铁载体能力的根际促生菌可直接供给作物铁营养，有效抑制作物病害的发生，促进作物生长[39-40]；此外，铁载体还可以通过螯合金属提高作物对重金属的耐受性[41-42]。但醋酸钙不动杆菌DP25和DP27分泌的铁载体是否可提高达乌里胡枝子对抗病性、重金属耐受等能力，还需要进一步试验论证。总之，上述结果表明醋酸钙不动杆菌是具有良好应用潜力的PGPR菌种。
通过纸培养基法研究醋酸钙不动杆菌对达乌里胡枝子幼苗促生作用，结果表明菌株DP25可显著促进达乌里胡枝子幼苗根长、茎长，菌株DP27对达乌里胡枝子无显著促生作用，究其原因可能是由于纸培养基中不存在外源性养分，植物激素在植物生长中发挥着主导作用，菌株DP25较菌株DP27具有更优的产IAA能力，而IAA可促进植物根系新陈代谢及根系的发育，进而增加植物对水分及养分的吸收，从而促进植物生长。因此，纸培养基法可作为初步验证微生物促生能力及其部分促生机制的手段。
光合作用能够为植物生长发育提供必需的物质与能量，是植物最主要的代谢活动之一[43]。光合色素包括叶绿素和类胡萝卜素，其中叶绿素是植物光合光能捕获的关键物质，叶绿素含量在某种程度上可反映植物光合作用及其抗逆性能力强弱[44]。本研究发现在盐碱条件下，接种菌株DP25和DP27的达乌里胡枝子叶片叶绿素含量显著高于对照，这与王艳宇等[45]的研究一致，这可能是由于盐碱胁迫会造成叶绿素含量的下降[46]，而促生菌对受盐碱胁迫的叶绿体有保护作用，减缓了叶绿素的降解，进而缓解盐碱胁迫对植物的毒害。一定程度的盐碱胁迫会促使植物叶片中净光合速率下降[47]。杨丽娟等[48]发现盐碱胁迫下施用产酸克雷伯氏菌(Klebsiella oxytoca)显著提高玉米幼苗光合速率、蒸腾速率、气孔导度，与本研究结论存在差异。本研究中，接种菌株DP25和DP27的达乌里胡枝子植株的净光合速率显著升高，胞间CO2浓度显著降低，而气孔导度、蒸腾速率无显著差异，造成这一差异的原因可能是由于促生菌影响植物光合机制的途径不同。此外，接种菌株DP25和DP27后植物光合速率的显著提高，表明促生菌缓解了盐碱胁迫对达乌里胡枝子光合的限制[49]。这是由于叶绿素含量在光合作用中起特殊作用，光合色素的减少会降低植物生长的光合作用速率[46]，接种菌株DP25和DP27后减缓了达乌里胡枝子叶绿素的降解，叶肉细胞的光合活性增强，从而提高植物光合速率，进而降低植物胞间CO2浓度。
叶绿素荧光参数可作为光合诱导的探针，能够快速无损地检测PSⅡ光系统对逆境的响应机制，还可间接反映光合作用的光能吸收传递情况和光合诱导能量的利用耗散过程[50]。盐碱胁迫会对植物叶绿体造成损伤，因此掌握叶绿素荧光参数的变化规律有助于明确盐碱胁迫对植物光合结构的损伤情况，同时有利于探究盐碱条件下植物光合作用对接种促生菌的响应。PSⅡ的最大光能转化效率(Fv/Fm值)表示PSⅡ原初光化学效率，主要用来反映PSⅡ复合物的光抑制是否受到伤害，若Fv/Fm值值降低，则表明发生了光抑制现象，进而导致植物光合活性降低[51]。本研究中，盐碱胁迫下达乌里胡枝子植株叶片的Fv/Fm值和Fv/Fo值较低，接种菌株DP25和DP27后显著缓解了盐碱胁迫对达乌里胡枝子植株叶片叶绿素荧光参数的抑制，说明盐碱胁迫下达乌里胡枝子植株PSⅡ结构及其生理状态受到破坏，使得光合电子由PSⅡ反应中心向下游电子传递受到抑制，极大地降低了原初光能捕获效率[51]。而接种菌株DP25和DP27可提高光能转化效率，从而提高达乌里胡枝子植株的光合能力，缓解叶绿体损伤，促进植物干物质积累和根系发育。
根系是连接植物地上与地下部分的唯一枢纽，当植物暴露在盐碱胁迫下时，根系最先感知到胁迫信息，并逐步传递到地上部分[52]。长期的盐碱胁迫最终会导致植物产生生理干旱[53]，而根系形态结构和生理特征是影响植物对水分的吸收与运输的主要因素。因此，良好的根系结构和生理状态在缓解植物盐碱胁迫中发挥着重要作用[54]。盐碱胁迫下，接种菌株DP25和DP27后达乌里胡枝子总根长、根表面积、根体积、根尖数和分枝数均显著升高，表明菌株DP25和DP27在盐碱条件下对达乌里胡枝子根系形态建成仍具有较好的促生效果。这可能是与微生物分泌的激素、土壤磷养分供给充足存在着潜在联系，低浓度的IAA可促进植物根尖细胞的伸长[55]；同时菌株DP25和DP27具备较好的解磷能力，土壤有效磷供给较为充足，促进了植物根系的形成和生长，进而提高植物对逆境的适应能力。但接种菌株DP25和DP27后对植物根系形态指标促生效应存在差异，与纸培养基试验结果不一致。与Zhang等[56]的研究一致，醋酸钙不动杆菌P19仅对无盐胁迫条件下燕麦(Avena sativa)表现出显著的促生作用，盐胁迫条件下对燕麦无促生效应，这可能是由于盐碱胁迫抑制菌株的促生特性，进而导致菌株DP25促生能力减弱。细根是根系中生理活性最高的植物成分，主要负责吸收大量的水分和养分[57]，发达的根系构型有利于在土壤多方位、多空间的吸收水分和养分[54]。接种菌株DP25和DP27均显著促进盐碱胁迫达乌里胡枝子根系发育，表明微生物在植物根系发育中发挥着积极作用，促使植物根系在胁迫环境中扩展，通过植物丰富的根系从盐碱土壤中捕获最大限度的水源[54]。由此可见，菌株DP25和DP27有助于达乌里胡枝子缓解盐碱胁迫，促进植物生长发育，表明菌株DP25和DP27在促进植株适应盐碱胁迫方面具有良好的应用潜力。
盐碱胁迫会限制植物地上部分生长和发育[58]，而本研究中发现接种醋酸钙不动杆菌显著促进植物地上部分生长及生物量积累，这可能是由于接种菌株DP25和DP27促进了植物根系生长和植物的光合作用，缓解植物的水分胁迫，确保为植物光合作用、生长和维持提供充足的水和养分[59]。接种菌株DP25植株的地下鲜重、地上和地下生物量显著低于接种菌株DP27达乌里胡枝子植株，这可能是由于接种菌株DP25的植物根系活力不佳，限制了地下部分对地上部分的水分及矿物质养分的供给，进而影响植物的生长发育。此外，菌株DP25和DP27还需进一步在不同程度盐碱环境下进行大田试验验证其促生稳定性。
菌株DP25和DP27对达乌里胡枝子在幼苗期和成株期均具有促生作用。其中，菌株DP25显著促进达乌里胡枝子幼苗生长；盆栽试验中接种菌株DP25和DP27可提高达乌里胡枝子成株耐盐碱能力，显著促进达乌里胡枝子光合能力和根形态的建成。综上所述，醋酸钙不动杆菌作为土壤中常见的耐盐微生物，在耐盐碱促生菌剂的开发和应用方面具有巨大的潜力，可为黄土高原地区盐碱地建植达乌里胡枝子提供菌种资源。
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