An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Podsumowanie

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Streszczenie

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD₆₀₀ = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Wprowadzenie

Chemotaxis is important for colonization of plant growth-promoting rhizobacterial (PGPR) on roots and for understanding plant-microbe interactions¹. A class of low molecular weight compounds (chemoattractants) in plant root exudates induce the chemotactic movement of PGPR to the rhizosphere². Malic acid, citric acid, and other components in the root exudates stimulate chemotaxis of Bacillus strains³. For example, glucose, citric acid, and fumaric acid in maize root exudates recruit bacteria to the root surface⁴. D-galactose, which is derived from....

Protokół

1. Materials and equipment

NOTE: Bacillus altitudinis LZP02 (CP075052) was isolated from the rhizosphere of rice in northeast China^12,13 for this study.

1. Culture B. altitudinis LZP02 in Luria-Bertani (LB) medium (peptone, 10 g L^-1; NaCl, 8 g L^-1 and yeast extract, 5 g L^-1) for 10 h. Collect cells by centrifugation at 9,569 x g for 2 min at 4 °C. and store with 15% glycerol at -80 °C.
   NOTE: For this experiment, rice seeds (Oryza sativa Longgeng 46) were provided ....

Wyniki

A total of 584 and 937 known metabolites were detected in the positive and negative ion indices, respectively. Previous studies have shown that chemoattractants are typically organic acids, amino acids, and carbohydrates^17,18.

In this study, 16 kinds of chemoattractants from the LC-MS studies in the rice rhizosphere exudates were selected for subsequent experiments (Table 1). Using the swimming plate method, we screene.......

Dyskusje

Increasing research indicates that plant-bacteria interactions mainly occur in the rhizosphere and are influenced by root exudates^{20,21,22,23,24}. Plant root exudates include a diverse array of primary metabolites, including phenolic acids, organic acids and amino acids as well as more complex secondary compounds^25,

Materiały

Name	Company	Catalog Number	Comments
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