一种用于快速鉴定根系分泌物中根细菌化学引诱剂的改进趋化性测定
Summary
在这里，我们提出了一种改进的趋化性测定方案。该协议的目标是减少传统细菌趋化性方法的步骤和成本，并作为理解植物 – 微生物相互作用的宝贵资源。
Abstract
趋化性鉴定对于根际生长促进菌的研究与应用具有重要意义。我们建立了一种简单的方法来快速鉴定 可以通过简单的步骤 在无菌载玻片上诱导根际生长促进细菌的趋化运动的化学引诱剂。细菌溶液（OD600 = 0.5）和无菌化学引诱剂水溶液以1cm的间隔滴加在载玻片上。使用接种回路将化学引诱剂水溶液连接到细菌溶液。将载玻片在室温下在干净的工作台上保持20分钟。最后，收集化学引诱剂水溶液进行细菌计数和显微镜观察。本研究通过对实验结果的多重比较，克服了传统细菌趋化性方法的多重缺点。该方法降低了计板误差，缩短了实验周期。对于化学引诱物质的鉴定，与传统方法相比，这种新方法可以节省2-3天。此外，这种方法允许任何研究人员在1-2天内系统地完成细菌趋化性实验。该协议可以被认为是理解植物 – 微生物相互作用的宝贵资源。
Introduction
趋化性对于植物根系上促进生长的根际细菌（PGPR）的定植和理解植物 – 微生物相互作用非常重要1。植物根系分泌物中的一类低分子量化合物（化学引诱剂）诱导PGPR向根际的趋化运动2。根系分泌物中的苹果酸、柠檬酸和其他成分刺激芽孢杆菌菌株的趋化性3。例如，玉米根系分泌物中的葡萄糖、柠檬酸和富马酸将细菌招募到根部表面4。D-半乳糖来源于根系分泌物，诱导黄芽孢杆菌SQR95的趋化性。有机酸，包括富马酸盐、苹果酸和琥珀酸盐，会影响Cajanus cajan – Zea mays间作系统中各种PGPR的趋化性和定植6。水稻根系分泌物中的齐墩果酸可作为菌株FP357的化学引诱剂。其他植物渗出物（包括组氨酸、精氨酸和天冬氨酸）可在细菌的趋化反应中起至关重要的作用8。植物分泌物作为引导细菌运动的信号，这是根际定植的第一步。PGPR的植物定植是一个具有巨大相关性的过程，因为PGPR对植物宿主有益。
许多方法已被用于分析细菌趋化性。游泳板法是前面描述的方法之一9。在这种方法中，用半固体介质制成板。将含有琼脂（1.0%，w / v）的趋化缓冲液加入平板中。将缓冲液加热，然后与化学吸引剂混合。然后，将8μL细菌悬浮液滴入板的中间，并将板置于28°C的培养箱中。 该板材定期被观察和拍照。然而，游泳板法的实验周期非常长。在毛细管样方法10中，移液器吸头用作容纳100μL细菌悬浮液的腔室。1 mL注射器针头用作毛细管。将含有具有不同浓度梯度的化学吸引剂的注射器针头插入100μL移液器尖端。在室温下孵育3小时后，取出注射器针头，稀释内容物并镀在培养基上。注射器中的细菌积累由板中的集落形成单元（CFU）表示。然而，类似毛细管的方法的重复实验误差很大。另一种方法使用微流体滑片装置11。简而言之，将牛血清白蛋白（BSA）溶液注射到所有通道中并使用真空除去。将含有不同化学引诱剂（仅定性检测的1 mM浓度）、悬浮在磷酸盐缓冲盐水和磷酸盐缓冲液中的细菌细胞（阴性对照）的溶液分别加入到顶部、中间和底部微孔中。然后在室温下的黑暗环境中孵育30分钟。然后在微孔中检测到细菌细胞。然而，微流体SlipChip设备很昂贵。因此，上述每种方法都有优点和缺点。
我们建立了一种改进的趋化性测定法，用于使用无菌载玻片快速鉴定根系分泌物中的根际细菌化学引诱剂，而无需复杂的步骤。本研究通过对实验结果的多重比较，克服了传统细菌趋化性方法的多重缺点。该方法降低了计板误差，缩短了实验周期。因此，如果用于鉴定化学引诱剂物质，这种新方法可以节省2-3天并降低实验材料的成本。
Protocol
1. 材料和设备 注：从东北水稻根际中分离出 高山芽孢杆菌 LZP02（CP075052）12，13 。 文化 B. 山茱萸 LZP02在卢里亚 – 贝尔塔尼（LB）培养基（蛋白胨，10克L-1;NaCl，8克L-1 和酵母提取物，5克L-1）10小时。通过在4°C下以9，569× g 离心2分钟收集细胞，并在-80°C下用15%甘?…
Representative Results
在阳性和阴离子指数中分别检测到584种和937种已知代谢物。先前的研究表明，化学引诱剂通常是有机酸，氨基酸和碳水化合物17，18。 本研究选取了水稻根际渗出物中LC-MS研究中的16种化学引诱剂进行后续实验（表1）。使用游泳板法，我们筛选了根分泌的低分子量化合物，以寻找根际 B. altitudinis LZP02的引诱剂。为?…
Discussion
越来越多的研究表明，植物与细菌的相互作用主要发生在根际，并受根系分泌物的影响20，21，22，23，24。植物根系分泌物包括多种初级代谢物，包括酚酸、有机酸和氨基酸以及更复杂的二次化合物25、26、27</s…
Divulgations
The authors have nothing to disclose.
Acknowledgements
本研究由国家自然科学基金（第31870493号）、黑龙江重点研发项目（GA21B007）和黑龙江省高校基础研究费用（第135409103号）资助。
Materials
| 2,5-dihydroxybenzoic acid | Beijing InnoChem Science & Technology C.,Ltd. | 490-79-9 | |
| Acetonitrile | CNW Technologies | 75-05-8 | |
| Ammonium acetate | CNW Technologies | 631-61-8 | |
| Caffeic acid | Beijing InnoChem Science & Technology C.,Ltd. | 331-39-5 | |
| Centrifuge | Thermo Fisher Scientific | Heraeus Fresco17 | |
| Citric acid | Beijing InnoChem Science & Technology C.,Ltd. | 77-92-9 | |
| Clean bench | Shanghai Boxun Industrial Co., Ltd. | BJ-CD | |
| Ferulic acid | Beijing InnoChem Science & Technology C.,Ltd. | 1135-24-6 | |
| Formic acid | CNW Technologies | 64-18-6 | |
| Fructose | Beijing InnoChem Science & Technology C.,Ltd. | 57-48-7 | |
| Galactose | Beijing InnoChem Science & Technology C.,Ltd. | 59-23-4 | |
| Glycine | Beijing InnoChem Science & Technology C.,Ltd. | 56-40-6 | |
| Grinding Mill | Shanghai Jingxin Industrial Development Co., Ltd. |
JXFSTPRP-24 | |
| Histidine | Beijing InnoChem Science & Technology C.,Ltd. | 71-00-1 | |
| Internal standard: 2-Chloro-L-phenylalanine | Shanghai Hengbai Biotech C.,Ltd. | 103616-89-3 | |
| Leucine | Beijing InnoChem Science & Technology C.,Ltd. | 61-90-5 | |
| Malic acid | Beijing InnoChem Science & Technology C.,Ltd. | 6915-15-7 | |
| Mannose | Beijing InnoChem Science & Technology C.,Ltd. | 3458-28-4 | |
| Mass Spectrometer | Thermo Fisher Scientific | Q Exactive Focus | |
| Methanol | CNW Technologies | 67-56-1 | |
| Optical Microscope | Olympus | BX43 | |
| Phenylalanine | Beijing InnoChem Science & Technology C.,Ltd. | 63-91-2 | |
| Proline | Beijing InnoChem Science & Technology C.,Ltd. | 147-85-3 | |
| Scales | Sartorius | BSA124S-CW | |
| Serine | Beijing InnoChem Science & Technology C.,Ltd. | 56-45-1 | |
| Threonine | Beijing InnoChem Science & Technology C.,Ltd. | 72-19-5 | |
| UHPLC | Agilent | 1290 UHPLC | |
| Ultrasound Instrument | Shenzhen Leidebang Electronics Co., Ltd. |
PS-60AL | |
| Valine | Beijing InnoChem Science & Technology C.,Ltd. | 7004-03-7 |
References
- Belas, R. Biofilms, flagella, and mechanosensing of surfaces by bacteria. Trends in Microbiology. 22 (9), 517-527 (2014).
- Haichar, Z., Santaella, C., Heulin, T., Achouak, W. Root exudates mediated interactions belowground. Soil Biology and Biochemistry. 77 (7), 69-80 (2014).
- Zhang, N., et al. Effects of different plant root exudates and their organic acid components on chemotaxis, biofilm formation and colonization by beneficial rhizosphere-associated bacterial strains. Plant and Soil. 374 (1-2), 689-700 (2014).
- Zhang, N., et al. Whole transcriptomic analysis of the plant-beneficial rhizobacterium Bacillus amyloliquefaciens SQR9 during enhanced biofilm formation regulated by maize root exudates. BMC Genomics. 16 (1), 685 (2015).
- Lui, Y., et al. Induced root-secreted D-galactose functions as a chemoattractant and enhances the biofilm formation of Bacillus velezensis SQR9 in an mcpa-dependent manner. Applied Microbiology and Biotechnology. 104 (17), 785-797 (2020).
- Vora, S. M., Joshi, P., Belwalkar, M., Archana, G. Root exudates influence chemotaxis and colonization of diverse plant growth-promoting rhizobacteria in the Cajanus cajan – Zea mays intercropping system. Rhizosphere. 18 (12), 100331 (2021).
- Sampedro, I., et al. Effects of halophyte root exudates and their components on chemotaxis, biofilm formation and colonization of the halophilic bacterium halomonas anticariensis FP35T. Microorganisms. 8 (4), 575 (2020).
- Liu, X. L., Raza, W., Ma, J. H., Huang, Q. W., Shen, Q. R. A dual role amino acid from sesbania rostrata seed exudates in the chemotaxis response of Azorhizobium caulinodans ORS571. Molecular Plant-Microbe Interactions. 32 (9), 1134-1147 (2019).
- Ling, N., Raza, W., Ma, J. H., Huang, Q. W., Shen, Q. R. Identification and role of organic acids in watermelon root exudates for recruiting Paenibacillus polymyxa SQR-21 in the rhizosphere. European Journal of Soil Biology. 47 (6), 374-379 (2011).
- Rudrappa, T., Czymmek, K. J., Pare, P. W., Bais, H. P. Root-secreted malic acid recruits beneficial soil bacteria. Plant Physiology. 148 (3), 1547-1556 (2008).
- Shen, C., et al. Bacterial chemotaxis on Slipchip. Lab on a Chip. 14 (16), 3074-3080 (2014).
- Liu, H., et al. Bacillus pumilus LZP02 promotes rice root growth by improving carbohydrate metabolism and phenylpropanoid biosynthesis. Molecular Plant-Microbe Interactions. 33 (10), 1222-1231 (2020).
- Goswami, M., Deka, S. Isolation of a novel rhizobacteria having multiple plant growth promoting traits and antifungal activity against certain phytopathogens. Microbiological Research. 240, 126516 (2020).
- Kaiira, M., Chemining’Wa, G., Ayuke, F., Baguma, Y., Nganga, F. Profiles of compounds in root exudates of rice, cymbopogon, desmodium, mucuna and maize. Journal of Agricultural Sciences Belgrade. 64 (4), 399-412 (2019).
- Shi, Y., et al. Effect of rice root exudates and strain combination on biofilm formation of Paenibacillus polymyxa and Paenibacillus macerans. African Journal of Microbiology Research. 6 (13), 3343-3347 (2012).
- Lee, H. W., Ghimire, S. R., Shin, D. H., Lee, I. J., Kim, K. U. Allelopathic effect of the root exudates of K21, a potent allelopathic rice. Weed Biology and Management. 8 (2), 85-90 (2008).
- Belimov, A. A., et al. Rhizobacteria that produce auxins and contain 1-amino-cyclopropane-1-carboxylic acid deaminase decrease amino acid concentrations in the rhizosphere and improve growth and yield of well-watered and water-limited potato (Solanum tuberosum). Annals of Applied Biology. 167 (1), 11-25 (2015).
- Ankati, S., Podile, A. R. Metabolites in the root exudates of groundnut change during interaction with plant growth promoting rhizobacteria in a strain-specific manner. Journal of Plant Physiology. 243, 153057 (2019).
- Gordillo, F., Chavez, F., Jerez, C. A. Motility and chemotaxis of Pseudomonas sp. B4 towards polychlorobiphenyls and chlorobenzoates. FEMS Microbiology Ecology. 60 (2), 322-328 (2007).
- Bais, H. P., Weir, T. L., Perry, L. G., Gilroy, S., Vivanco, J. M. The role of root exudates in rhizosphere interactions with plants and other organisms. Annual Review of Plant Biology. 57, 233-266 (2006).
- Badri, D. V., Vivanco, J. M. Regulation and function of root exudates. Plant Cell Environment. 32 (6), 666-681 (2009).
- Badri, D. V., Weir, T. L., Lelie, D., Vivanco, J. M. Rhizosphere chemical dialogues: plant-microbe interactions. Curr Opin Biotech. Current Opinion in Biotechnology. 20 (6), 642-650 (2009).
- Kamilova, F., Kravchenko, L. V., Shaposhnikov, A. I., Makarova, N., Lugtenberg, B. Effects of the tomato pathogen Fusarium oxysporum f. sp. radicis-lycopersici and of the biocontrol bacterium Pseudomonas fluorescens WCS365. Molecular Plant-Microbe Interactions. 19 (10), 1121-1126 (2006).
- Kamilova, F., et al. Organic acids, sugars, and L-tryptophane in exudates of vegetables growing on stonewool and their effects on activities of rhizosphere bacteria. Molecular Plant-Microbe Interactions. 19 (3), 250-256 (2006).
- Badri, D. V., Vivanco, J. M. Regulation and function of root exudates. Plant Cell Environment. 32 (6), 666-681 (2009).
- Hao, W. Y., Ren, L. X., Ran, W., Shen, Q. R. Allelopathic effects of root exudates from watermelon and rice plants on Fusarium oxysporum f.sp. Niveum. Plant and Soil. 336 (1-2), 485-497 (2010).
- Hao, Z. P., Wang, Q., Christie, P., Li, X. L. Allelopathic potential of watermelon tissues and root exudates. Scientia Horticulturae. 112 (3), 315-320 (2007).
Citer Cet Article
Jiao, H., Lyu, C., Xu, W., Chen, W., Hu, Y., Wang, Z. An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates. J. Vis. Exp. (181), e63249, doi:10.3791/63249 (2022).