CN109752510B - Phospholipid fatty acid biomarker of rhizobium meliloti and biomass measurement and calculation method - Google Patents
Phospholipid fatty acid biomarker of rhizobium meliloti and biomass measurement and calculation method Download PDFInfo
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Abstract
The invention discloses a phospholipid fatty acid biomarker of Rhizobium meliloti and a biological measurement method, which comprises the following steps: 1) collecting a soil sample to extract phospholipid fatty acid; 2) analyzing the composition of phospholipid fatty acid by a gas chromatography-mass spectrometer; 3) classifying the phospholipid fatty acids and finding out phospholipid fatty acid biomarkers of the rhizobium meliloti; 4) and calculating the biomass of the rhizobium meliloti according to the content of the phospholipid fatty acid marker. The method comprises the steps of screening specific phospholipid fatty acid of a pure culture strain of the rhizobium meliloti, determining the specific phospholipid fatty acid as a specific biomarker, and confirming the specific phospholipid fatty acid biomarker by soil sampling analysis. The invention relates to a specific phospholipid fatty acid biomarker screened from Rhizobium meliloti, which can be used for measuring and calculating the biomass of the Rhizobium meliloti in soil.
Description
Technical Field
The invention relates to the field of soil microorganism research, in particular to a phospholipid fatty acid biomarker of alfalfa rhizobia and a biomass measurement and calculation method, which are suitable for measuring and calculating the biomass of alfalfa rhizobia in alfalfa soil.
Background
Phospholipid Fatty acids (PLFAs) are important components of the cellular structure of living microorganisms, differ in PLFA synthesis pathways and composition status of different kinds of microorganisms, and their PLFAs are rapidly degraded once the cells die. Therefore, the content and composition status of soil PLFA can indicate the biomass and community structure status of different kinds of microorganisms in soil more accurately (Zelles, 1999). The PLFA method has the advantages of being able to estimate soil microbial biomass, easy to separate bacterial and fungal populations, relatively low analysis cost, etc. In recent 20 years, soil PLFA analysis has become an important analytical tool in the field of soil microbial biomass and community structure research (Haack et al, 1994; Zhouyika and butyl Ming 25035, 2007; Wang et al, 2019). The PLFA method is commonly used for differentiating soilThe amount of bacterial and fungal biomass in the soil, and also for the indication of groups such as gram-positive bacteria, gram-negative bacteria, actinomycetes, protozoa and green algae (
et al, 1993; ruess and chamberland, 2010). The uses of the PLFA assay are mainly two: one is to analyze the PLFA composition of pure cultured strains for enrichment of microbial PLFA databases by comparing PLFA differences of different groups of microorganisms and for commercial PLFA databases; the second is for analyzing the PLFA composition of environmental soil samples or laboratory culture samples, for comparing differences and changes in microbial community composition, etc. (Zelles, 1999). However, most of the current research focuses on the use of the PLFA analysis, and the application of the PLFA analysis method to the comparison of PLFA analysis of pure culture strains and the development and expansion of PLFA databases is severely deficient. Therefore, it is necessary to analyze the PLFA composition of various microorganisms cultured in pure form to improve the effect of PLFA in soil microorganism classification.
Disclosure of Invention
The invention aims to provide a phospholipid fatty acid biomarker screening and biomass measuring and calculating method for rhizobium meliloti, which is easy to implement and simple and convenient to operate, wherein the biomarker is screened after phospholipid fatty acid component analysis and comparison are carried out on pure cultured rhizobium meliloti, and can be used for indicating the rhizobium meliloti and estimating the biomass of the rhizobium meliloti according to the amount of the phospholipid fatty acid. The method is provided based on PLFA analysis, and has the advantages of low requirements on sample collection, storage and transportation, low cost, rapid analysis, accurate result and the like.
In order to achieve the purpose, the invention adopts the following technical measures:
phospholipid fatty acid composition analysis was performed on pure culture strains of Rhizobium meliloti, and the obtained phospholipid fatty acids were compared with phospholipid fatty acids in the existing phospholipid fatty acid biomarker database, and a total of 2 unused phospholipid fatty acid biomarkers were found to be a17:1 ω 9 and 18:1 ω 5, respectively. After further analysis of phospholipid fatty acid composition of the alfalfa-planted soil, only 18:1 omega 5 phospholipid fatty acid is detected, and the content of the fatty acid in the alfalfa-planted soil is obviously higher than that in the alfalfa-unplanted soil. Therefore, the phospholipid fatty acid 18:1 ω 5 can be used as a biomarker for Rhizobium meliloti and can be used for estimating the biomass of Rhizobium meliloti.
A phospholipid fatty acid biomarker of Rhizobium meliloti and a biomass estimation method comprise the following steps:
A. collecting soil samples for cultivating alfalfa, and extracting soil Phospholipid Fatty Acid (PLFA);
B. analyzing the composition of phospholipid fatty acid by a gas chromatography-mass spectrometer (GC-MS) instrument (such as Agilent 7890), and determining the types of the phospholipid fatty acid by utilizing a Sherlock microorganism identification system (PLFAD2 or RTSBA6) of MIDI company in the United states;
C. the phospholipid fatty acid is classified into bacteria, fungi, actinomycetes, protozoa, green algae, rhizobia and the like, wherein the phospholipid fatty acid of 18:1 omega 5 is used as a biomarker of the rhizobia;
D. response data of each phospholipid fatty acid was obtained from the output of the Sherlock microorganism identification system, and the mass of each phospholipid fatty acid was calculated and the content of 18: 1. omega.5 was used as the biomass of rhizobia.
The biomass calculation formula B ═ (Re)PLFA/Re19)×(C19×A/W)
Wherein: b is the biomass of phospholipid fatty acid (ng/g dry soil), RePLFAResponse, Re for unknown phospholipid fatty acids19Response, C of 19:0 (internal standard phospholipid fatty acid)19The concentration of the solution is 19:0 (5 ng/ul in the method), A is the sample amount of the gas chromatograph-mass spectrometer (200 ul in the method), and W is the dry weight of the soil for analysis (8 g in the method);
E. taking the difference of the 18:1 omega 5 content of the cultivated alfalfa soil and the uncultured alfalfa soil as the biomass of the alfalfa rhizobium.
The biomass of the rhizobium meliloti can be estimated (measured) by the technical measures of the five steps, wherein the phospholipid fatty acid biomarker 18:1 omega 5 in the fifth step (E) is a specific marker aiming at the rhizobium meliloti, and the phospholipid fatty acid marker aiming at the rhizobium is not available in the prior art, so that the technical problem that the qualitative and quantitative evaluation of the rhizobium can not be carried out by the prior phospholipid fatty acid technology is solved. Through the method, the biomass of various groups of microorganisms in the alfalfa soil is measured by the applicant, and the biomass of the alfalfa rhizobia is measured, so that the result shows that the alfalfa rhizobia is contained in the alfalfa soil at 0.27ng (the content of the alfalfa rhizobia per gram of dry soil) and is higher than the content of fungi and protozoa, the method is used for directly analyzing and calculating the biomass of the alfalfa rhizobia by finding out the biomarker aiming at the alfalfa rhizobia without changing the extraction and analysis process and the related cost of the existing phospholipid fatty acid technology, and the method is a supplement to the existing phospholipid fatty acid technology.
Compared with the prior art, the invention has the following advantages and effects:
according to the phospholipid fatty acid composition of the pure culture strain of the rhizobium meliloti, a phospholipid fatty acid biomarker which can indicate the rhizobium meliloti is screened by comparing the phospholipid fatty acid composition with the existing phospholipid fatty acid biomarker database and verifying a field soil sample, and the biomarker is the only phospholipid fatty acid marker of the rhizobium meliloti at present. Compared with the conventional common DNA mapping technology, the method for analyzing the biomass of the alfalfa rhizobia by using the phospholipid fatty acid method can quantify the biomass of the alfalfa rhizobia, has relatively low requirements on collection and storage of soil samples, and is simpler in analysis method and lower in cost.
Compared with the difference between the prior art method and the method in soil microorganism classification and biomass measurement, the result shows that only the contents of bacteria, fungi, actinomycetes, protozoa and green algae in the soil can be obtained in the prior art, the method takes the phosphatide fatty acid 18:1 omega 5 as the biomarker of the alfalfa rhizobia, not only can the biomass of each microorganism group be obtained, but also the content of the alfalfa rhizobia in the cultivated alfalfa soil can be obtained (figure 4), wherein the contents of the bacteria, the fungi, the actinomycetes, the protozoa and the green algae measured by the two methods are not different, and the contents of the bacteria, the fungi, the actinomycetes, the protozoa, the green algae and the alfalfa rhizobia in the soil are respectively 6.98, 023, 3.16, 0.11, 1.18 and 0.27ng (the content in each gram of dry soil) (figure 4).
Drawings
FIG. 1 is a diagram of the phospholipid fatty acids of a pure culture strain of Rhizobium meliloti.
The figure totals 27 phospholipid fatty acids, the y-axis is the phospholipid fatty acid name and the x-axis is the percentage of phospholipid fatty acids.
FIG. 2 is a graph of the relationship of potential biomarkers for Rhizobium meliloti to Rhizobium meliloti biomass.
In the figure, the total content of 18 types of alfalfa rhizobium phospholipid fatty acids has a significant positive correlation with the total content of alfalfa rhizobium phospholipid fatty acids (alfalfa rhizobium biomass), and the content of the other 9 types of alfalfa rhizobium phospholipid fatty acids has no significant positive correlation with the total content of alfalfa rhizobium phospholipid fatty acids (not shown in the figure). Only when the content of single phospholipid fatty acid has a correlation with the total phospholipid fatty acid content of the rhizobium meliloti can be used as a potential marker of the rhizobium meliloti; if no obvious positive correlation exists, the biomass change of the rhizobium meliloti cannot be reflected, and the rhizobium meliloti cannot be used as a biomarker. Indicates a very significant linear correlation.
FIG. 3 is a graph showing the phospholipid fatty acid profile of alfalfa soil grown and alfalfa soil not grown.
In the figure, the alfalfa cultivation soil is the soil for planting alfalfa for 2 years, the uncultivated alfalfa soil (control) is the soil for planting paspalum latifolium for 2 years, and management measures such as fertilization and weeding of the two kinds of soil are completely consistent except for different types of planted plants. Indicates that phospholipid fatty acids 18:1 ω 5 and i18:0 differ significantly between the two soils, and that only 18:1 ω 5 can be used as a biomarker for rhizobia since i18:0 is not among the phospholipid fatty acids of 27 species of S.meliloti.
FIG. 4 is a biological quantity chart of various groups of microorganisms in alfalfa cultivation soil
The figures list the bacterial, fungal, actinomycete, protozoan, green algae and alfalfa rhizobia populations of the alfalfa soil, where the alfalfa rhizobia biomass is significantly different from the populations of other species except fungal biomass. The letters in the figure indicate whether there was a significant difference between microbial biomass for each group, and no significant difference between groups labeled with the same letter.
Detailed Description
The invention is described in detail below with reference to the attached drawing figures: schematic diagrams of the implementation of the principles of the present invention are shown in fig. 1-3.
Example 1:
a phospholipid fatty acid biomarker of Rhizobium meliloti and a biomass measurement method (taking the comparison of alfalfa cultivation soil and uncultured soil as an example) comprise the following steps:
1) 500g of soil sample was sampled by random sampling using a soil auger, and after picking up roots and stones, the soil was sieved through a 10-mesh soil sieve, and fresh soil equivalent to 8g of dry soil was weighed to extract phospholipid fatty acids (Bossio and Scow, 1998).
2) Analyzing PLFA composition by gas chromatography-mass spectrometer (GC-MS), and determining the types of phospholipid fatty acids by using Sherlock microorganism identification system (RTSBA6) of MIDI company in America;
3) the PLFAs are classified as bacteria, fungi, actinomycetes, protozoa, green algae and rhizobia, wherein i15:0, a15:0,15:0, i16:0,16:1x7, i17:0, a17:0,17:0, cy17: 0and cy19:0 are used as bacterial markers and the bacterial biomass is calculated by the sum of their responses, taking 18:2 omega 6,9 as a fungal marker and calculating fungal biomass by using the Response thereof, the actinomycete biomass was calculated as the sum of their responses with 10Me 16:0,10Me17: 0and 10Me18:0 as actinomycete markers, protozoan biomass was calculated as the sum of their responses using 20:0and 20:4 ω 6,9,12,15 as protozoan markers, taking 18:1 omega 9 as a green algae marker and calculating the green algae biomass by using the Response thereof, taking 18:1 omega 5 as a rhizobium marker and calculating rhizobium biomass by using Response thereof;
D. response data of each phospholipid fatty acid was obtained from the output of the Sherlock microorganism identification system, and formula B ═ Re (Re) was calculated from the biomassPLFA/Re19)×(C19xA/W) calculating the content of each phospholipid fatty acid;
wherein: b is the biomass of phospholipid fatty acid (ng/g dry soil), RePLFAResponse, Re for unknown phospholipid fatty acids19Response, C of 19:0 (internal standard phospholipid fatty acid)19The concentration of the solution is 19:0 (5 ng/ul in the method), A is the sample amount of the gas chromatograph-mass spectrometer (200 ul in the method), W is the dry weight of the soil for analysis (8 g in the method), and the biomass of bacteria, fungi, actinomycetes, protozoa, green algae and rhizobia is determined;
E. taking the difference between the 18:1 omega 5 content in the alfalfa cultivation soil and the 18:1 omega 5 content in the alfalfa cultivation soil as the biomass of the alfalfa rhizobia (figure 4), wherein the 18:1 omega 5 is a phospholipid fatty acid with a carbon chain length of 18 carbons, a double bond of 1 carbon and a double bond at the 5 th carbon of the molecular terminal, and the molecular formula is CH3(CH2)3CH＝CH(CH2)11CO2CH3。
The method can directly obtain corresponding data along with the analysis of the fatty acid components of the soil phospholipid, does not need to independently analyze the phospholipid fatty acid, and is more convenient and faster.
Through the specific implementation process, the biomass of bacteria, fungi, actinomycetes, protozoa and green algae can be obtained through the analysis of the existing phospholipid fatty acid technology, and meanwhile, the quantitative analysis result of the rhizobium meliloti can be obtained; according to the existing phospholipid fatty acid analysis technology, the contents of bacteria, fungi, actinomycetes, protozoa and green algae in the alfalfa cultivation soil are respectively 6.98, 023, 3.16, 0.11 and 1.18ng (the content in each gram of dry soil) (figure 4), and by using the phospholipid fatty acid 18:1 omega 5 as the biomarker of the alfalfa rhizobia, the biomass of the microorganism groups can be obtained, and the content of the alfalfa rhizobia in the alfalfa cultivation soil can be 0.27ng (the content in each gram of dry soil) (figure 4).
Claims (1)
1. A method for measuring and calculating biomass of rhizobium meliloti comprises the following steps:
1) collecting a soil sample for cultivating alfalfa, and extracting soil phospholipid fatty acid;
2) analyzing the composition of the phospholipid fatty acid by a gas chromatography-mass spectrometer, and determining the types of the phospholipid fatty acids by using a Sherlock microorganism identification system;
3) the phospholipid fatty acid 18:1 omega 5 is used as a biomarker of Rhizobium meliloti;
4) extracting the Response data of each phospholipid fatty acid, calculating the molar mass of each phospholipid fatty acid, and calculating the biomass of the rhizobium meliloti according to the content of the phospholipid fatty acid of 18:1 omega 5;
the biomass calculation formula B ═ (Re)PLFA/Re19)×(C19×A/W)；
Wherein: b is the biomass of phospholipid fatty acids, RePLFAResponse, Re for unknown phospholipid fatty acids19Response, C of 19:019The concentration of the solution was 19:0, A represents the amount of sample taken by the GC, and W represents the dry weight of the soil used for analysis.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1588048A (en) * | 2004-07-09 | 2005-03-02 | 中国科学院生态环境研究中心 | Analyzing soil microbial community diversity using lipoid fatty acid atlas technology |
| CN102033114A (en) * | 2010-10-12 | 2011-04-27 | 四川大学 | Method for quickly and simply analyzing microbial community structure of yeast for traditional brewage |
| CN102382874A (en) * | 2010-08-30 | 2012-03-21 | 中国食品发酵工业研究院 | Biochemistry method for researching white spirit omagari microbial community structure |
| CN104611271A (en) * | 2015-02-09 | 2015-05-13 | 河北大学 | Efficient nitrogen-fixing alfalfa rhizobium strain and method for screening molecular marker |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1588048A (en) * | 2004-07-09 | 2005-03-02 | 中国科学院生态环境研究中心 | Analyzing soil microbial community diversity using lipoid fatty acid atlas technology |
| CN102382874A (en) * | 2010-08-30 | 2012-03-21 | 中国食品发酵工业研究院 | Biochemistry method for researching white spirit omagari microbial community structure |
| CN102033114A (en) * | 2010-10-12 | 2011-04-27 | 四川大学 | Method for quickly and simply analyzing microbial community structure of yeast for traditional brewage |
| CN104611271A (en) * | 2015-02-09 | 2015-05-13 | 河北大学 | Efficient nitrogen-fixing alfalfa rhizobium strain and method for screening molecular marker |
Non-Patent Citations (3)
| Title |
|---|
| Effects of different chloroform stabilizers on the extraction efficiencies of phospholipid fatty acids from soils;Andrea Fuhrmann 等;《Soil Biology & Biochemistry》;20081206(第41期);428–430 * |
| Soil microbial community dynamics and phenanthrene degradation as affected by rape oil application;Maren Mellendorf 等;《Applied Soil Ecology》;20101231(第46期);329–334 * |
| 基于磷脂脂肪酸生物标记法的松茸生长土壤微生物测定;马大龙 等;《贵州农业科学》;20150315;第43卷(第3期);106-109 * |
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