生物技术通报 ›› 2022, Vol. 38 ›› Issue (3): 103-112.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0619
收稿日期:
2021-05-12
出版日期:
2022-03-26
发布日期:
2022-04-06
作者简介:
高亚慧,女,硕士研究生,研究方向:微生物与植物的互作;E-mail: 基金资助:
GAO Ya-hui(), JIANG Ming-guo, FENG Jing(), ZHOU Gui
Received:
2021-05-12
Published:
2022-03-26
Online:
2022-04-06
摘要:
旨在筛选出能产生促进植物生长的挥发性有机化合物(volatile organic compounds,VOCs)的潜在植物根际促生菌(plant growth promoting rhizobacteria,PGPR),考察其VOCs对植物的促生作用及其它促生功能,为研发微生物肥料提供新的思路及可靠的材料。以本实验室从海洋样品中分离保存的48株功能菌为供试菌株,通过二分隔平板实验、VOCs盆栽实验进行筛选,结合菌株的16S rRNA进行鉴定,通过气相色谱-质谱联用的方法(Gas Chromatography-Mass,GC-MS)对菌株所产VOCs成分进行分析。通过平板活性实验对细菌的固氮,溶磷及产IAA进行活性检测。研究结果显示:筛选得到1株潜在PGPR - GX14001,其所产生的VOCs对本氏烟草(Nicotiana benthamiana)和上海青(Brassica chinensis L.)均表现出明显的促生作用,菌株经16S rRNA鉴定为橙色微杆菌(Microbacterium aurantiacum)。经GC-MS对其VOCs成分分析,共得到7种特异性化合物。经平板活性检测,GX14001具有较强的溶有机/无机磷活性和一般固氮活性,而其产IAA能力较弱,为1.737 μg/mL。PGPR可通过不同的促生机制对植物表现出促生作用,研究结果表明GX14001所产VOCs对植物有明显促生作用,其他方面的促生活性并不高,两者之间不存在较高的正相关性,说明其最主要的促生作用是通过所产生的VOCs获得的。
高亚慧, 姜明国, 丰景, 周桂. 产生促生挥发性物质的潜在PGPR菌株筛选及其促生特性研究[J]. 生物技术通报, 2022, 38(3): 103-112.
GAO Ya-hui, JIANG Ming-guo, FENG Jing, ZHOU Gui. Screening of Potential PGPR Strains Producting Growth-promoting Volatile Compounds and Study on Their Growth-promoting Characteristics[J]. Biotechnology Bulletin, 2022, 38(3): 103-112.
菌株编号Strain No. | 菌株名称Name of strain |
---|---|
GX13159 | Bacillus cereus |
GX13529 | Staphylococcus edaphicus |
GX13571 | Asticcacaulis excentricus |
GX13580 | Micrococcus aloeverae |
GX13585 | Microbacterium hydrothermale |
GX13594 | Microbacterium laevaniformans |
GX13656 | Bacillus acidiceler |
GX13667 | Bacillus aryabhattai |
GX13668 | Micrococcus aloeverae |
GX13676 | Streptomyces diastaticus subsp. ardesiacus |
GX13678 | Streptomyces sanglieri |
GX13679 | Falsibacillus pallidus |
GX13685 | Sinomonas humi |
GX13699 | Cellulosimicrobium funkei |
GX13717 | Kocuria indica |
GX13747 | Paenibacillus silvae |
GX13767 | Bacillus mangrovi |
GX13774 | Bacillus marisflavi |
GX13777 | Halobacillus marinus |
GX13785 | Kosakonia oryziphila |
GX13790 | Serratia oryzae |
GX14001 | Microbacterium aurantiacum |
GX14008 | Microbacterium maritypicum |
GX14011 | Exiguobacterium aestuarii |
GX14016 | Staphylococcus haemolyticus |
GX14017 | Staphylococcus hominis subsp. novobiosepticus |
GX14022 | Staphylococcus nepalensis |
GX14028 | Kytococcus sedentarius |
GX14031 | Sporosarcina koreensis |
GX14032 | Macrococcus canis |
GX14046 | Glutamicibacter creatinolyticus |
GX14059 | Pontibacter chinhatensis |
GX14066 | Kocuria tytonicola |
GX14070 | Micrococcus luteus |
GX14080 | Macrococcus caseolyticus subsp. Caseolyticus |
GX14105 | Bacillus altitudinis |
GX14108 | Brachybacterium squillarum |
GX14201 | Staphylococcus sciuri |
GX14203 | Bacillus haikouensis |
GX14062 | Agromyces indicus |
GX14082 | Psychrobacter pulmonis |
GX14096 | Pseudomonas deceptionensis |
GX13112 | Klebsiella Trevisan |
GX13213 | Klebsiella variicola subsp. Variicola |
GX14104 | Streptomyces griseus subsp. griseus |
GX14111 | Streptomyces koyangensis |
GX14119 | Streptomyces gramineus |
GX14125 | Streptomyces flavovirens |
表1 菌株信息
Table 1 Strain information
菌株编号Strain No. | 菌株名称Name of strain |
---|---|
GX13159 | Bacillus cereus |
GX13529 | Staphylococcus edaphicus |
GX13571 | Asticcacaulis excentricus |
GX13580 | Micrococcus aloeverae |
GX13585 | Microbacterium hydrothermale |
GX13594 | Microbacterium laevaniformans |
GX13656 | Bacillus acidiceler |
GX13667 | Bacillus aryabhattai |
GX13668 | Micrococcus aloeverae |
GX13676 | Streptomyces diastaticus subsp. ardesiacus |
GX13678 | Streptomyces sanglieri |
GX13679 | Falsibacillus pallidus |
GX13685 | Sinomonas humi |
GX13699 | Cellulosimicrobium funkei |
GX13717 | Kocuria indica |
GX13747 | Paenibacillus silvae |
GX13767 | Bacillus mangrovi |
GX13774 | Bacillus marisflavi |
GX13777 | Halobacillus marinus |
GX13785 | Kosakonia oryziphila |
GX13790 | Serratia oryzae |
GX14001 | Microbacterium aurantiacum |
GX14008 | Microbacterium maritypicum |
GX14011 | Exiguobacterium aestuarii |
GX14016 | Staphylococcus haemolyticus |
GX14017 | Staphylococcus hominis subsp. novobiosepticus |
GX14022 | Staphylococcus nepalensis |
GX14028 | Kytococcus sedentarius |
GX14031 | Sporosarcina koreensis |
GX14032 | Macrococcus canis |
GX14046 | Glutamicibacter creatinolyticus |
GX14059 | Pontibacter chinhatensis |
GX14066 | Kocuria tytonicola |
GX14070 | Micrococcus luteus |
GX14080 | Macrococcus caseolyticus subsp. Caseolyticus |
GX14105 | Bacillus altitudinis |
GX14108 | Brachybacterium squillarum |
GX14201 | Staphylococcus sciuri |
GX14203 | Bacillus haikouensis |
GX14062 | Agromyces indicus |
GX14082 | Psychrobacter pulmonis |
GX14096 | Pseudomonas deceptionensis |
GX13112 | Klebsiella Trevisan |
GX13213 | Klebsiella variicola subsp. Variicola |
GX14104 | Streptomyces griseus subsp. griseus |
GX14111 | Streptomyces koyangensis |
GX14119 | Streptomyces gramineus |
GX14125 | Streptomyces flavovirens |
组别 Group | 鲜重 Fresh weight/g | 根长 Root lengt/cm | 侧根数 Number of lateral root/roots | 叶片长 Leaf length/cm | 叶片宽 Leaf width/cm |
---|---|---|---|---|---|
对照组Control | 0.081±0.015 | 2.855±0.656 | 1.887±0.726 | 0.551±0.093 | 0.443±0.089 |
GX13594 | 0.503±0.214** | 4.627±1.481** | 4.400±1.183** | 0.990±0.217** | 0.730±0.158** |
GX13747 | 0.387±0.078** | 5.187±1.351** | 4.267±0.799** | 1.023±0.145** | 0.772±0.157** |
GX14001 | 0.703±0.300** | 4.530±1.129** | 4.267±1.870** | 1.250±0.260** | 0.880±0.190** |
GX14016 | 0.487±0.106** | 4.853±1.255** | 5.333±1.589** | 1.133±0.247** | 0.775±0.191** |
GX14066 | 0.620±0.100** | 4.243±0.918** | 4.533±1.552** | 1.285±0.244** | 0.958±0.260** |
GX14070 | 0.300±0.183** | 4.600±0.858** | 3.867±1.125** | 0.950±0.163** | 0.742±0.129** |
GX14201 | 0.457±0.093** | 4.486±1.143** | 4.643±1.082** | 1.107±0.289** | 0.764±0.209** |
表2 部分菌株产生的VOCs对烟草作用效果
Table 2 Effect of VOCs produced by some strains on tobacco
组别 Group | 鲜重 Fresh weight/g | 根长 Root lengt/cm | 侧根数 Number of lateral root/roots | 叶片长 Leaf length/cm | 叶片宽 Leaf width/cm |
---|---|---|---|---|---|
对照组Control | 0.081±0.015 | 2.855±0.656 | 1.887±0.726 | 0.551±0.093 | 0.443±0.089 |
GX13594 | 0.503±0.214** | 4.627±1.481** | 4.400±1.183** | 0.990±0.217** | 0.730±0.158** |
GX13747 | 0.387±0.078** | 5.187±1.351** | 4.267±0.799** | 1.023±0.145** | 0.772±0.157** |
GX14001 | 0.703±0.300** | 4.530±1.129** | 4.267±1.870** | 1.250±0.260** | 0.880±0.190** |
GX14016 | 0.487±0.106** | 4.853±1.255** | 5.333±1.589** | 1.133±0.247** | 0.775±0.191** |
GX14066 | 0.620±0.100** | 4.243±0.918** | 4.533±1.552** | 1.285±0.244** | 0.958±0.260** |
GX14070 | 0.300±0.183** | 4.600±0.858** | 3.867±1.125** | 0.950±0.163** | 0.742±0.129** |
GX14201 | 0.457±0.093** | 4.486±1.143** | 4.643±1.082** | 1.107±0.289** | 0.764±0.209** |
图1 菌株产生的VOCs对烟草的促生作用 A:空白对照;B:菌株GX14001处理后对烟草的影响
Fig. 1 Growth-promoting effects of VOCs produced by the strains on tobacco A:Control. B:Effect of strain GX14001 on tobacco
图2 菌株GX14001产生的VOCs对烟草的促生结果分析 A:菌株GX14001处理后对烟草鲜重的影响;B:菌株GX14001处理后对烟草叶片长和叶片宽的影响;C:菌株GX14001处理后对烟草根长的影响;D:菌株GX14001处理后对烟草侧根数的影响. “*”表示处理间差异显著P<0.05,“**”表示P<0.01,下同
Fig. 2 Growth-promoting effects of VOCs produced by strain GX14001 on tobacco A:Effect of strain GX14001 on the fresh weight of tobacco. B:Effects of strain GX14001 on the tobacco leaf length and width. C:Effect of strain GX14001 on the root length of tobacco. D:Effect of strain GX14001 on the lateral root number of tobacco.“*” refers to significant difference between treatments,P < 0.05,“**” refers to P < 0.01,The same below
图3 根据16S rRNA基因序列构建菌株GX14001系统发育树 分支上的数值表示采用Neighbor-Joining方法构建系统树时,1 000次计算时形成该节点的百分比;括号内为菌株16S rRNA基因序列在GenBank中的登录号;标尺表示碱基置换频率
Fig. 3 Constructed phylogenetic tree of strain GX14001 based on 16S rRNA gene sequence The value on the branch represents the percentage of the node formed in 1 000 calculations when the Neighbor-Joining method is used to construct the system tree. The registration number of 16S rRNA gene sequence in GenBank is shown in brackets. The ruler represents the base substitution frequency
图4 菌株GX14001产生的VOCs对上海青的促生作用 A:GX14001-VOCs盆栽结果图;B:GX14001-VOCs盆栽根部结果图;C:GX14001-VOCs处理后对上海青叶片长和叶片宽的影响;D:GX14001-VOCs处理后对上海青根长的影响;E:GX14001-VOCs处理后对上海青鲜重的影响;F:GX14001-VOCs处理后对上海青干重的影响
Fig. 4 Growth promoting effect of VOCs produced by strain GX14001 on rape A:Results of GX14001-VOCs pot culture. B:Results of GX14001-VOCs potted roots. C:The effects of GX14001-VOCs treatment on the leaf length and width of rape. D:Effect of GX14001-VOCs treatment on root length of rape. E:Effect of GX14001-VOCs on the fresh weight of rape. F:Effect of GX14001-VOCs on the dry weight of rape
图5 空白对照TSA及菌株GX14001产生的VOCs成分SPME-GC-MS分析总离子流图
Fig. 5 SPME-GC-MS analysis of total ion flow diagram of blank control TSA and VOCs produced by strain GX14001
编号No. | 保留时间Retaining time/ min | 化学名称Chemical name | 峰面积Peak area |
---|---|---|---|
1 | 9.3353 | 邻乙基甲苯(1-ethyl-2-methyl- Benzene) | 1335701 |
2 | 9.9132 | 1,2,3-三甲苯(1,2,3-trimethyl- Benzene) | 2357671 |
3 | 11.1091 | 1,2,4-三甲苯(1,2,4-trimethyl- Benzene) | 294469 |
4 | 11.298 | 4-甲基-癸烷(4-methyl-Decane) | 917363 |
5 | 12.8658 | 十二烷(Dodecane) | 3497194 |
6 | 32.2232 | 二十五烷(Pentacosane) | 671059 |
7 | 34.1057 | 9-辛基-十七烷(9-octyl-Heptadecane) | 387135 |
表3 菌株GX14001产生的VOCs成分
Table 3 VOCs produced by strain GX14001
编号No. | 保留时间Retaining time/ min | 化学名称Chemical name | 峰面积Peak area |
---|---|---|---|
1 | 9.3353 | 邻乙基甲苯(1-ethyl-2-methyl- Benzene) | 1335701 |
2 | 9.9132 | 1,2,3-三甲苯(1,2,3-trimethyl- Benzene) | 2357671 |
3 | 11.1091 | 1,2,4-三甲苯(1,2,4-trimethyl- Benzene) | 294469 |
4 | 11.298 | 4-甲基-癸烷(4-methyl-Decane) | 917363 |
5 | 12.8658 | 十二烷(Dodecane) | 3497194 |
6 | 32.2232 | 二十五烷(Pentacosane) | 671059 |
7 | 34.1057 | 9-辛基-十七烷(9-octyl-Heptadecane) | 387135 |
菌株编号 Strain No. | 菌株名称 Name of a strain | 溶无机磷 Inorganic phosphorus solubilization | 溶有机磷 Organic phosphorus solubilization | 固氮 Nitrogen fixation | IAA/(μg·mL-1) |
---|---|---|---|---|---|
GX14001 | Microbacterium aurantiacum | ++ | ++ | + | 1.737 |
表4 菌株溶磷固氮及IAA能力测定汇总
Table 4 Summary of determining strain's IAA and ability of fixing nitrogen and solubilizing phosphorus
菌株编号 Strain No. | 菌株名称 Name of a strain | 溶无机磷 Inorganic phosphorus solubilization | 溶有机磷 Organic phosphorus solubilization | 固氮 Nitrogen fixation | IAA/(μg·mL-1) |
---|---|---|---|---|---|
GX14001 | Microbacterium aurantiacum | ++ | ++ | + | 1.737 |
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