Method for analyzing grass seed zone microorganisms by using molecular ecological network analysis platform

Abstract

The invention discloses a method for analyzing grass seed zone microorganisms by utilizing a molecular ecological network analysis platform, which comprises the following steps of: obtaining six glume microorganism samples of EI achnatherum inebrians caryopses infected by # imgabs0 # endophytic fungi and EF achnatherum inebrians caryopses not infected by # imgabs1 # endophytic fungi, treating seed epiphytic microorganism samples and in-seed microorganism samples, extracting DNA (Deoxyribonucleic Acid) and sequencing; submitting OTU and / or ASV data, obtained by sequencing, of all the microorganisms to a molecular ecological network analysis MENA platform for analysis; and then an obtained data result is uploaded to Cytoscape and / or Gephi software for analysis and drawing. According to the application, the knowledge of the network structure of the achnatherum inebrians seed zone microorganisms and the interaction relationship between the microorganisms is enhanced through network analysis, a network analysis thought is developed for the subsequent research on the microorganisms related to each part of the achnatherum inebrians, and the cognition on the microorganisms is deepened.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant and microorganism interaction, and in particular relates to a method for analyzing grass seed-borne microorganisms using a molecular ecological network analysis platform. Background Art

[0002] Microorganisms, especially bacteria or fungi, are essential to the functioning of all ecosystems because of their huge abundance (nearly 5 × 10 30 bacteria), with a large cumulative mass and more diversity than other organisms. Microbial interactions can drive ecosystem functions such as plant productivity, variation, and even biodiversity, which is essential to maintain stable productivity in ecosystems. Biodiversity includes not only species but also the interactions between species.

[0003] Ceratonia Endophytes grow in cool-season grasses such as Lolium, Festuca and Achnatherum inebrians in arid and semi-arid grasslands of China. Almost 100% of Achnatherum inebrians plants were infected by E. gansuensis or E. inebrians. The presence of endophytic fungi enhances the resistance of Drunken Horse Grass plants to heavy metals, insect pests, low temperature, pathogenic fungi, salt and drought stress. The presence of endophytes affects the structure and diversity of wine grape rhizosphere microbial communities in soil, roots, leaves and seeds, but the interactions between these microbes have never been analyzed by network analysis. Summary of the invention

[0004] The purpose of the present invention is to provide a method for analyzing grass seed-borne microorganisms using a molecular ecological network analysis platform to solve the problem that the prior art has never analyzed the interactions between these microorganisms through network analysis.

[0005] The embodiment of the present application is implemented by providing a method for analyzing grass seed-borne microorganisms using a molecular ecological network analysis platform, including:

[0006] Provide Endophytic fungi infect caryopsis and uninoculated Endophytic fungi infected the caryopsis of EF intoxicating horse grass;

[0007] Obtained respectively Endophytic fungi infect caryopsis and uninoculated There are 6 treatments in total, including samples of lemma microorganisms, epiphytic microorganisms and microorganisms in seeds of EF euphorbia caryopsis infected by endophytic fungi;

[0008] DNA was extracted from the above six treated microbial samples and sequenced;

[0009] Submit the OTU and / or ASV data of all microorganisms obtained by sequencing to the molecular ecological network analysis MENA platform for analysis;

[0010] After analysis, the obtained data results are uploaded to Cytoscape and / or Gephi software for analysis and drawing.

[0011] In some embodiments, Endophytic fungi infect caryopsis and uninoculated The method for obtaining the caryopsis of EF intoxicated grass infected by endophytic fungi comprises: randomly selecting eight plots of intoxicated grass with endophytic fungi EI and eight plots of intoxicated grass without endophytic fungi EF to collect caryopsis, selecting six mature plants in each plot to collect caryopsis, and then mixing the caryopsis of intoxicated grass with endophytic fungi EI and without endophytic fungi EF, respectively, to obtain the following: Endophytic fungi infect caryopsis and uninoculated The caryopsis of EF sphaerocephala infected by endophytic fungi.

[0012] In some embodiments, the obtained Endophytic fungi infect caryopsis and uninoculated The caryopsis of EF euphorbia infected by endophytic fungi were stored in a refrigerator at 4°C.

[0013] In some embodiments, the EI scutellaria scutellaria caryopsis is separated into the EI scutellaria scutellaria husk and the EI scutellaria scutellaria seeds, the EI scutellaria scutellaria seeds are placed in a first conical flask, and a first sterile PB buffer solution is added for 10 -1 , seal the mouth of the first conical flask with a sealing film, shake and elute for the first time, let it stand for the first time, take the washing solution to be the EI intoxicated horse grass seed epiphytic microorganism sample; add the EI intoxicated horse grass seeds after shaking and elution into the first sterile PBS buffer solution for 10 -1 After grinding, the filtrate obtained by filtration is the microbial sample in the seeds of EI intoxicated horse grass; the husk of EI intoxicated horse grass is placed in a second conical flask, and a second sterile PB buffer solution is added for 10 -1 , seal the mouth of the second conical flask with a sealing film, shake and elute for the second time, let it stand for the second time, and take the washing and elution liquid to obtain the EI glume hull microbial sample.

[0014] In some embodiments, the amount of EI scutellaria baicalensis caryopsis is 40 g to 60 g; and / or

[0015] The volume of the first conical flask is 50 ml, and the volume of the second conical flask is 50 ml; and / or

[0016] The rotation speed of the first shaking elution is 140r / min to 160r / min; and / or

[0017] The rotation speed of the second oscillation elution is 140r / min to 160r / min; and / or

[0018] The first standing time is 4 to 6 minutes; and / or

[0019] The second standing time is 4 to 6 minutes; and / or

[0020] First sterile PB buffer 10 -1 The volume is 20ml to 40ml; and / or

[0021] Second sterile PB buffer 10 -1 The volume is 20ml to 40ml; and / or

[0022] First sterile PBS buffer 10 -1 The volume is 20ml~40ml.

[0023] In some embodiments, the EF scutellaria baicalensis caryopsis is separated into the EF scutellaria baicalensis glumes and the EF scutellaria baicalensis seeds, and the EF scutellaria baicalensis seeds are placed in a third conical flask, and a third sterile PB buffer solution is added for 10 -1 , seal the mouth of the third conical flask with a sealing film, shake and elute for the third time, let it stand for the third time, take the washing solution to be the epiphytic microorganism sample of EF scutellaria seed; add the EF scutellaria seed after shaking and elution into the second sterile PBS buffer for 10 -1 After grinding, the filtrate obtained by filtration is the microbial sample in the seeds of EF scutellaria baicalensis. The husk of EF scutellaria baicalensis is placed in a fourth conical flask, and 10% of the fourth sterile PB buffer is added. -1 , seal the mouth of the fourth conical flask with a sealing film, shake and elute for the fourth time, let it stand for the fourth time, and take the washing and elution liquid to obtain the EF glume microbial sample.

[0024] In some embodiments, the amount of EF physalis caryopsis is 40 g to 60 g; and / or

[0025] The volume of the third conical flask is 50 ml, and the volume of the fourth conical flask is 50 ml; and / or

[0026] The rotation speed of the third oscillation elution is 140r / min to 160r / min; and / or

[0027] The rotation speed of the fourth oscillation elution is 140r / min to 160r / min; and / or

[0028] The third standing time is 4 to 6 minutes; and / or

[0029] The fourth standing time is 4 to 6 minutes; and / or

[0030] Third sterile PB buffer 10 -1 The volume is 20ml to 40ml; and / or

[0031] Fourth sterile PB buffer 10 -1 The volume is 20ml to 40ml; and / or

[0032] Second sterile PBS buffer 10 -1 The volume is 20ml~40ml.

[0033] In some embodiments, the MN NucleoSpin 96 Soil DNA Kit was used to extract fungal and / or bacterial DNA from the microbial samples of the above 6 treatments, with 8 replicates in each treatment.

[0034] In some embodiments, the bacterial amplification primers are: 335F (5'-CADACTCCTACGGGGAGGGC-3') and 769R (5'-ATCCTGTTTGMTMCCCCRC-3');

[0035] The primers for fungal amplification were: ITS1F (5'-CTTGGTCATAGGAAGAAGTAA-3') and ITS2 (5'-GCTGCGTTCTTCATCGATGC-3').

[0036] In some embodiments, the PCR product is purified using an OMEGA DNA kit, and the purified sample is subjected to high-throughput sequencing analysis of bacterial and / or fungal rDNA genes using the Illumina Hiseq 2500 platform of Beijing Biomike Biotechnology Co., Ltd. (BMK), China, to obtain OTU and / or ASV data of all microorganisms, and the obtained OTU and / or ASV data of all microorganisms are submitted to the molecular ecological network analysis MENA platform for data analysis.

[0037] In this application:

[0038] The full name of EI is endophyte-infected;

[0039] EF stands for endophyte-free;

[0040] OTU stands for Operational Taxonomic Units;

[0041] ASV stands for Amplicon Sequence Variants;

[0042] MENA stands for molecular ecological network analysis;

[0043] PB buffer is phosphate buffer;

[0044] PBS buffer is phosphate buffered saline.

[0045] PBS buffer 10 -1 The stock solution in PBS buffer was diluted 10 times;

[0046] PB Buffer 10 -1 The stock solution of PB buffer was diluted 10 times.

[0047] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0048] This application has been approved by Endophytic fungi infect caryopsis and uninoculated A total of 6 treatments were conducted for the microbial samples of the glumes, epiphytic microbial samples, and microbial samples in the seeds of EF euphorbia caryopsis infected by endophytic fungi. DNA was extracted and sequenced. The OTU and / or ASV data of all microorganisms obtained by sequencing were submitted to the molecular ecological network analysis MENA platform for analysis. After the analysis, the obtained data results were uploaded to Cytoscape and / or Gephi software for analysis and drawing. The presence of endophytic fungi has an effect on the complexity of the bacterial and / or fungal network in the seeds of Drunken Horse Grass. The endophytic fungi increase the complexity of the endophytic bacterial network and the epiphytic bacterial network of the glume of Drunken Horse Grass, while reducing the complexity of the endophytic fungal network and the epiphytic fungal network of the glume. Endophytic fungi reduce the complexity of the seed-attached bacterial network, while increasing the complexity of the seed-attached bacterial network. In general, the interactions between fungi are mostly positive in these fungal co-occurrence networks. The key species of the microbial networks of the three parts of the caryopsis, seeds and seeds are mainly Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes and Acidobacteria, and the key species of the seed-transmitted fungal network are mainly Ascomycota, Basidiomycota and Mortierella. This application enhances the understanding of the microbial network structure of the seed belt of Drunken Horse Grass and the interaction relationship between microorganisms through network analysis, opens up the idea of ​​network analysis for the subsequent research on microorganisms related to various parts of Drunken Horse Grass, and deepens the understanding of microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 A technical roadmap for analyzing grass seed-borne microorganisms using a molecular ecological network analysis platform provided in an embodiment of the present invention;

[0050] Figure 2The embodiment of the present invention uses Cystocape software to analyze the bacterial community network structure diagram of the species of the drunk horse grass;

[0051] Figure 3 The Cystocape software is used to analyze the fungal community network structure diagram of the species of the drunk horse grass in the embodiment of the present invention;

[0052] Figure 4 The embodiment of the present invention uses Gephi software to analyze the bacterial community network structure diagram of the drunk horse grass species;

[0053] Figure 5 The Gephi software was used to analyze the fungal community network structure diagram of the species of Drunken Horse Grass in the embodiment of the present invention. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0055] The technical solution of this application is as follows:

[0056] See also Figure 1 The present application embodiment provides a method for analyzing grass seed-borne microorganisms using a molecular ecological network analysis platform, including:

[0057] S01. Provide Endophytic fungi infect caryopsis and uninoculated Endophytic fungi infected the caryopsis of EF intoxicating horse grass;

[0058] S02, respectively obtain Endophytic fungi infect caryopsis and uninoculated There are 6 treatments in total, including samples of lemma microorganisms, epiphytic microorganisms and microorganisms in seeds of EF euphorbia caryopsis infected by endophytic fungi;

[0059] S03, extracting DNA from the above 6 treated microbial samples and sequencing them;

[0060] S04. Submit the OTU and / or ASV data of all microorganisms obtained by sequencing to the molecular ecological network analysis MENA platform for analysis;

[0061] S05. After analysis, the obtained data results are uploaded to Cytoscape and / or Gephi software for analysis and drawing.

[0062] In this application, the Endophytic fungi infect caryopsis and uninoculated A total of 6 treatments were conducted for the microbial samples of the glumes, epiphytic microbial samples, and microbial samples in the seeds of EF euphorbia caryopsis infected by endophytic fungi. DNA was extracted and sequenced. The OTU and / or ASV data of all microorganisms obtained by sequencing were submitted to the molecular ecological network analysis MENA platform for analysis. After the analysis, the obtained data results were uploaded to Cytoscape and / or Gephi software for analysis and drawing. The presence of endophytic fungi has an effect on the complexity of the bacterial and / or fungal network in the seeds of Drunken Horse Grass. The endophytic fungi increase the complexity of the endophytic bacterial network and the epiphytic bacterial network of the glume of Drunken Horse Grass, while reducing the complexity of the endophytic fungal network and the epiphytic fungal network of the glume. Endophytic fungi reduce the complexity of the seed-attached bacterial network, while increasing the complexity of the seed-attached bacterial network. In general, the interactions between fungi are mostly positive in these fungal co-occurrence networks. The key species of the microbial networks of the three parts of the caryopsis, seeds and seeds are mainly Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes and Acidobacteria, and the key species of the seed-transmitted fungal network are mainly Ascomycota, Basidiomycota and Mortierella. This application enhances the understanding of the microbial network structure of the seed belt of Drunken Horse Grass and the interaction relationship between microorganisms through network analysis, opens up the idea of ​​network analysis for the subsequent research on microorganisms related to various parts of Drunken Horse Grass, and deepens the understanding of microorganisms.

[0063] In the S01:

[0064] In some implementations, Endophytic fungi infect caryopsis and uninoculated The method for obtaining the caryopsis of EF intoxicated grass infected by endophytic fungi comprises: randomly selecting eight plots of intoxicated grass with endophytic fungi EI and eight plots of intoxicated grass without endophytic fungi EF to collect caryopsis, selecting six mature plants in each plot to collect caryopsis, and then mixing the caryopsis of intoxicated grass with endophytic fungi EI and without endophytic fungi EF, respectively, to obtain the following: Endophytic fungi infect caryopsis and uninoculated The caryopsis of EF sphaerocephala infected by endophytic fungi.

[0065] Furthermore, the obtained Endophytic fungi infect caryopsis and uninoculated The caryopsis of EF euphorbia infected by endophytic fungi were stored in a refrigerator at 4°C.

[0066] In the S02:

[0067] In some embodiments, the EI scutellaria scutellaria caryopsis is separated into the EI scutellaria scutellaria husk and the EI scutellaria scutellaria seeds, the EI scutellaria scutellaria seeds are placed in a first conical flask, and a first sterile PB buffer solution is added for 10 -1, seal the mouth of the first conical flask with a sealing film, shake and elute for the first time, let it stand for the first time, take the washing solution to be the EI intoxicated horse grass seed epiphytic microorganism sample; add the EI intoxicated horse grass seeds after shaking and elution into the first sterile PBS buffer solution for 10 -1 After grinding, the filtrate obtained by filtration is the microbial sample in the seeds of EI intoxicated horse grass; the husk of EI intoxicated horse grass is placed in a second conical flask, and a second sterile PB buffer solution is added for 10 -1 , seal the mouth of the second conical flask with a sealing film, shake and elute for the second time, let it stand for the second time, and take the washing and elution liquid to obtain the EI glume hull microbial sample.

[0068] Further, the amount of EI scutellaria baicalensis caryopsis is 40 g to 60 g, for example, 40 g, 45 g, 50 g, 55 g, 60 g, etc.;

[0069] The volume of the first conical flask is 50 ml, and the volume of the second conical flask is 50 ml;

[0070] The speed of the first oscillation elution is 140 r / min to 160 r / min, for example, 140 r / min, 145 r / min, 150 r / min, 155 r / min, 160 r / min, etc., and the time is 2 h to 4 h, for example, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, etc. Within the speed and time range, the microorganisms on the surface of the EI drunk horse grass seeds can be completely eluted;

[0071] The rotation speed of the second oscillation elution is 140r / min to 160r / min, for example, it can be 140r / min, 145r / min, 150r / min, 155r / min, 160r / min, etc., and the time is 2h to 4h, for example, it can be 2h, 2.5h, 3h, 3.5h, 4h, etc.; within the said rotation speed and time range, the microorganisms on the surface of EI drunk horse grass seeds can be completely eluted.

[0072] The first standing time is 4 to 6 minutes, for example, 4 minutes, 4.5 minutes, 5 minutes, 5.5 minutes, 6 minutes, etc.;

[0073] The second standing time is 4 to 6 minutes, for example, 4 minutes, 4.5 minutes, 5 minutes, 5.5 minutes, 6 minutes, etc.;

[0074] First sterile PB buffer 10 -1 The volume is 20ml to 40ml, for example, 20ml, 25ml, 30ml, 35ml, 40ml, etc.;

[0075] Second sterile PB buffer 10 -1The volume is 20ml to 40ml, for example, 20ml, 25ml, 30ml, 35ml, 40ml, etc.;

[0076] First sterile PBS buffer 10 -1 The volume is 20ml to 40ml, for example, it can be 20ml, 25ml, 30ml, 35ml, 40ml, etc.

[0077] It will be appreciated that the grinding is carried out in a mortar.

[0078] For example, 50 g of EI scutellaria caryopsis was separated into EI scutellaria glume and EI scutellaria seeds, and the separated EI scutellaria seeds were placed in a 50 ml first conical flask, and 30 ml of a first sterile PB buffer solution was added for 10 min. -1 , seal the mouth of the first conical flask with a sealing film, shake and elute at 150r / min for 3h, let it stand for 5min, take the washing solution to be the epiphytic microorganism sample of EI intoxicated horse grass seeds; add 30ml of the first sterile PBS buffer solution for 10 -1 After grinding, the filtrate obtained by filtration is the microbial sample in the seeds of EI intoxicated horse grass; the separated EI intoxicated horse grass husk is placed in a 50 ml second conical flask, and 30 ml of the second sterile PB buffer solution is added for 10 -1 , seal the mouth of the second conical flask with a sealing film, shake and elute at 150r / min for 3h, let it stand for 5min, and take the washing elution to obtain the EI glume hull microbial sample.

[0079] In some embodiments, the EF scutellaria baicalensis caryopsis is separated into the EF scutellaria baicalensis glumes and the EF scutellaria baicalensis seeds, and the EF scutellaria baicalensis seeds are placed in a third conical flask, and a third sterile PB buffer solution is added for 10 -1 , seal the mouth of the third conical flask with a sealing film, shake and elute for the third time, let it stand for the third time, take the washing solution to be the epiphytic microorganism sample of EF scutellaria seed; add the EF scutellaria seed after shaking and elution into the second sterile PBS buffer for 10 -1 After grinding, the filtrate obtained by filtration is the microbial sample in the seeds of EF scutellaria baicalensis. The husk of EF scutellaria baicalensis is placed in a fourth conical flask, and 10% of the fourth sterile PB buffer is added. -1 , seal the mouth of the fourth conical flask with a sealing film, shake and elute for the fourth time, let it stand for the fourth time, and take the washing and elution liquid to obtain the EF glume microbial sample.

[0080] Furthermore, the amount of EF physalis caryopsis is 40 g to 60 g, for example, 40 g, 45 g, 50 g, 55 g, 60 g, etc.;

[0081] The volume of the third conical flask is 50 ml, and the volume of the fourth conical flask is 50 ml;

[0082] The rotation speed of the third oscillation elution is 140 r / min to 160 r / min, for example, 140 r / min, 145 r / min, 150 r / min, 155 r / min, 160 r / min, etc., and the time is 2 h to 4 h, for example, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, etc. Within the rotation speed and time range, the microorganisms on the surface of EF drunk horse grass seeds can be completely eluted;

[0083] The rotation speed of the fourth shock elution is 140r / min~160r / min, for example, it can be 140r / min, 145r / min, 150r / min, 155r / min, 160r / min, etc., and the time is 2h~4h, for example, it can be 2h, 2.5h, 3h, 3.5h, 4h, etc.; within the said rotation speed and time range, the microorganisms on the surface of EF drunken horse grass seeds can be completely eluted.

[0084] The third standing time is 4 to 6 minutes, for example, 4 minutes, 4.5 minutes, 5 minutes, 5.5 minutes, 6 minutes, etc.;

[0085] The fourth standing time is 4 to 6 minutes, for example, 4 minutes, 4.5 minutes, 5 minutes, 5.5 minutes, 6 minutes, etc.;

[0086] Third sterile PB buffer 10 -1 The volume is 20ml to 40ml, for example, 20ml, 25ml, 30ml, 35ml, 40ml, etc.;

[0087] Fourth sterile PB buffer 10 -1 The volume is 20ml to 40ml, for example, 20ml, 25ml, 30ml, 35ml, 40ml, etc.;

[0088] Second sterile PBS buffer 10 -1 The volume is 20ml to 40ml, for example, it can be 20ml, 25ml, 30ml, 35ml, 40ml, etc.

[0089] It will be appreciated that the grinding is carried out in a mortar.

[0090] For example, 50 g of EF spatholobus caryopsis was separated into EF spatholobus husk and EF spatholobus seeds, and the separated EF spatholobus seeds were placed in a 50 ml third conical flask, and 30 ml of the third sterile PB buffer solution was added. -1, seal the mouth of the third conical flask with a sealing film, shake and elute at 150r / min for 3h, let it stand for 5min, take the washing solution to be the epiphytic microorganism sample of EF scutellaria seed; add 30ml of the second sterile PBS buffer solution for 10 -1 After grinding, the filtrate obtained by filtration is the microbial sample in the seeds of EF euphorbia pulex. The separated EF euphorbia pulex husk is placed in a 50 ml fourth conical flask, and 30 ml of the fourth sterile PB buffer solution is added for 10 -1 , seal the mouth of the fourth conical flask with a sealing film, shake and elute at 150r / min for 3 hours, let it stand for 5 minutes, and take the washing liquid to obtain the EF glume microbial sample.

[0091] Furthermore, there were a total of 6 treatments including EI infamyssin seeds epiphytic microorganism samples, EI infamyssin seeds microorganism samples, EI infamyssin glume microorganism samples, EF infamyssin seeds epiphytic microorganism samples, EF infamyssin seeds microorganism samples and EF infamyssin glume microorganism samples, and each treatment had 8 replications.

[0092] In some embodiments, the microorganisms include fungi and / or bacteria.

[0093] In said S03:

[0094] In some embodiments, the fungal and / or bacterial DNA in the microbial samples of the above 6 treatments, with 8 replicates in each treatment, was extracted using the MN NucleoSpin 96 Soil DNA Kit.

[0095] Further, the primers for bacterial amplification were: 335F (5′-CADACTCCTACGGGGAGGGC-3′) and 769R (5′-ATCCTGTTTGMTMCCCCRC-3′);

[0096] The primers for fungal amplification were: ITS1F (5'-CTTGGTCATAGGAAGAAGTAA-3') and ITS2 (5'-GCTGCGTTCTTCATCGATGC-3').

[0097] Furthermore, the PCR amplification system 50 μL: 10 μL TE buffer (pH 8.0), 0.2 μL Q5 high-fidelity DNA polymerase, 10 μL high GC enhancer, 1 μL dNTP, 1 μL upstream primer (10 μM) of fungi or bacteria, 1 μL downstream primer (10 μM) of fungi or bacteria, 60 ng genomic DNA; supplemented with dd H2O to 50 μL.

[0098] PCR amplification program: pre-denaturation at 98°C, 30s; denaturation at 98°C, 10s, annealing at 65°C, 30s, extension at 72°C, 30s, 10 cycles; extension at 72°C, 300s.

[0099] The PCR products were electrophoresed on agarose gel.

[0100] The PCR products were purified using the OMEGA DNA kit, and the purified samples were subjected to high-throughput sequencing analysis of bacterial and / or fungal rDNA genes using the Illumina Hiseq 2500 platform (2x250 paired ends) of Beijing Biomike Biotechnology Co., Ltd., China, to obtain the OTU and / or ASV data of all microorganisms, which were then submitted to the molecular ecological network analysis MENA platform for data analysis.

[0101] It can be understood that the website of the MENA platform for molecular ecological network analysis is http: / / ieg2.ou.edu / MENA / .

[0102] Understandably, the version number of Cytoscape is v.3.9.1 and the version number of Gephie is v.0.9.5.

[0103] In said S05:

[0104] It can be understood that the data exported by the MENA platform is a file saved with delimiters and must be saved as a .lsx file that Gephi can recognize. These operations can be completed in Excel.

[0105] Experimental example

[0106] The caryopsis of E. ilex with endophytic fungus (EI) and E. ilex without endophytic fungus (EF) were each given three treatments (seed attachment, seed endophytic and glume attachment). The purified samples were subjected to high-throughput sequencing analysis of bacterial and / or fungal rDNA genes using the Illumina Hiseq2500 platform (2x250 paired end) of Beijing Biomike Biotechnology Co., Ltd. (BMK). The results are shown in Tables 1 and 2.

[0107] Table 1 Bacterial composition of the three parts of EI and EF intoxicated horse grass, namely, epiphytic, endophytic and husk epiphytic

[0108]

[0109]

[0110] In Table 1, SEpI1-SEpI8 are 8 replicates of EI Drunkard species with epiphytic sites (seed epiphytic); SEpF1-SEpF8 are 8 replicates of EF Drunkard species with epiphytic sites (seed epiphytic); SEnI1-SEnI8 are 8 replicates of EI Drunkard species with endophytic sites (seed endophytic); SEnF1-SEnF8 are 8 replicates of EF Drunkard species with endophytic sites (seed endophytic); GEpI1-GEpI8 are 8 replicates of EI Drunkard glume epiphytic sites (glume epiphytic); GEpF1-GEpF8 are 8 replicates of EF Drunkard glume epiphytic sites (glume epiphytic), OUT (Optical Transmission Unit) is the classification operation unit, referring to each different bacterial species.

[0111] Table 2 Fungal composition of the three parts of EI and EF intoxicating horse grass, namely, epiphytic, endophytic and glume epiphytic parts

[0112]

[0113]

[0114]

[0115] In Table 2, SEpI1-SEpI8 are 8 replicates of EI Drunkard species with epiphytic sites (seed epiphytic); SEpF1-SEpF8 are 8 replicates of EF Drunkard species with epiphytic sites (seed epiphytic); SEnI1-SEnI8 are 8 replicates of EI Drunkard species with endophytic sites (seed endophytic); SEnF1-SEnF8 are 8 replicates of EF Drunkard species with endophytic sites (seed endophytic); GEpI1-GEpI8 are 8 replicates of EI Drunkard husk epiphytic sites (husk epiphytic); GEpF1-GEpF8 are 8 replicates of EF Drunkard husk epiphytic sites (husk epiphytic), OUT (Optical Transmission Unit) is the classification operation unit, referring to each different fungal species.

[0116] The tables in Table 1 and Table 2 were uploaded to the Molecular Ecological Network Analysis (MENA) platform (http: / / ieg2.ou.edu / MENA / ) for data analysis. All the data analyzed by the platform were downloaded. The results of the downloaded network points and edge files are shown in Table 3 and Table 4:

[0117] Table 3. Point files in network analysis data

[0118]

[0119]

[0120] In Table 3 , OTU+ numbers refer to individual species.

[0121] Table 4. Edge files in network analysis data

[0122]

[0123]

[0124] In Table 4, OUT+numbers refer to individual species.

[0125] The network graph features constructed by analyzing the point and edge table data obtained in Tables 3 and 4 above are shown in Tables 5 and 6:

[0126] Table 5 Topological characteristics of the phylogenetic molecular ecological network of bacterial communities under the action of SEpF, SEpI, SEnF, SEnI, GEpF, GEpI and their related random networks in the six treatments of the scutellaria baicalensis seed zone

[0127]

[0128] Note: Random networks: 100 networks were generated by rewiring all links of a pMEN with the same number of nodes and links of the corresponding empirical pMEN.

[0129] Table 6 Topological characteristics of the phylogenetic molecular ecological network of fungal communities under the influence of SEpF, SEpI, SEnF, SEnI, GEpF, GEpI and their related random networks in the six treatments of the scutellaria baicalensis seed zone

[0130]

[0131] From Tables 5 and 6, we can see that for the bacterial network (Table 5), the thresholds (St) of SEpF (0.9300), SEpI (0.9200), SEnF (0.9300), SEnI (0.9300), GEpF (0.9200) and GEpI (0.9300) are very similar. The connectivity of all networks conforms to the power law model, with R2 values ​​ranging from 0.85 to 0.91. At the same time, the average geodesic distance and average clustering coefficient of the empirical networks are higher than those of the corresponding random networks. The modularity values ​​of the six empirical networks are all greater than those of the random networks, all greater than 0.4 (Table 5), while the St values ​​of the fungal networks are between 0.89 and 0.95, and the power law R2 values ​​are between 0.66 and 0.87 (Table 6). For the average geodesic distance, the average clustering coefficient and modularity values ​​of the empirical networks are different from those of the 100 random networks (Table 6). These indicators indicate that the empirical networks are non-random co-occurrence patterns. These constructed networks are all small-world, scale-free, and modular.

[0132] In the present application: SEpF is the epiphytic part of EF spatholobus seeds, SepI is the epiphytic part of EI spatholobus seeds, SEnF is the endogenous part of EF spatholobus seeds, SenI is the endogenous part of EI spatholobus seeds, GEpF is the epiphytic part of EF spatholobus glume, and GEpI is the epiphytic part of EI spatholobus glume.

[0133] The point and edge tables obtained above were uploaded to the Cystoscap software for drawing. The obtained network diagram of bacteria and / or fungi with the species of the scutellaria baicalensis was as follows: Figure 2 and Figure 3 The results show that about 70% of the nodes in the six bacterial networks belong to Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Acidobacteria ( Figure 2 It is worth mentioning that the relative number of Proteobacteria in the EI network is less than that in the EF network, while the relative number of Actinobacteria in the EI network is more than that in the EF network ( Figure 2 Nearly 90% of the nodes in the six fungal networks belonged to the phyla Ascomycota, Basidiomycota, and Mortierellomycota ( Figure 3 )

[0134] Figure 2 In the figure, Cystocape software was used to analyze the network structure of bacterial communities in the seed belt of the sedum. A: SEpI with endophytic fungus sedum seeds; B: SEpF without endophytic fungus sedum seeds; C: SEnI with endophytic fungus sedum seeds; D: SEnF without endophytic fungus sedum seeds; E: GEpI with endophytic fungus sedum hull; F: GEpF without endophytic fungus sedum hull. The color of the node indicates different bacterial phyla, and the pie chart indicates the module composition with 10 nodes. The red line indicates a positive relationship between two nodes, while the blue link indicates a negative relationship. The node in the middle of the module is the module hub, the node in the black box is the connector, and there is no network hub.

[0135] Figure 2Among them, Acidobacteria, Gemmatimonadetes, Actinobacteria, Nitrospirae, Armatimonadetes, Patescibacteria, Bacteroidetes, Chloroflexi, Planctomycetes, Proteobacteria, Cyanobacteria, Rokubacteria, Deferribacteres, Deinococcus-Thermus, Entotheonollaeota, Epsilonombacteraeota, Spirochates, Synergistetes, Tenericutes, unclassified, Firmicutes, Verrucomicrobia, Fusobacteria.

[0136] Figure 3 In the figure, Cystocape software was used to analyze the network structure of the fungal community with the seeds of Sophora japonica. A: SEpI with endophytic fungi on the seeds of Sophora japonica; B: SEpF without endophytic fungi on the seeds of Sophora japonica; C: SEnI with endophytic fungi on the seeds of Sophora japonica; D: SEnF without endophytic fungi on the seeds of Sophora japonica; E: GEpI with endophytic fungi on the glumes of Sophora japonica; F: GEpF without endophytic fungi on the glumes of Sophora japonica. The colors of the nodes represent different fungal phyla, and the pie chart represents the module composition with 10 nodes. The red line represents a positive relationship between two nodes, while the blue link represents a negative relationship. The node in the middle of the module is the module hub, the nodes in the black box are connectors, and there is no network hub.

[0137] The point and edge tables obtained above are uploaded to Gephi software for drawing. The obtained network diagram of bacteria and / or fungi with the species of Drunken Horse Grass is as follows Figure 4 and Figure 5 The results show that: in bacterial networks, EI makes the network process more connected. In addition to SEnI having fewer modules, EI has a larger number of modules than EF ( Figure 4 ). Overall, taxa tend to co-exclude (negative correlation, blue line) rather than co-occur (positive correlation, red line), with negative correlation accounting for 40%-63% and greater than positive correlation (37%-60%) ( Figure 4 ). In the fungal network, the number of modules of GEPI showed a consistent decreasing trend. ( Figure 5 ). SEnI has more nodes and modules than SEnF, but fewer links. SEpI has more nodes and links than SEpF. Unlike bacterial networks, fungal network taxa tend to co-occur (72%-98% positive correlation) rather than co-exclude ( Figure 5 ).

[0138] Figure 3 Among them, Ascomycota, Basidiomycota, Unclassified, Mortierellomycota, Chytridiomycota, Mucoromycota, Olpidiomycota, Rozellomycota, Zoopagomycota.

[0139] Figure 2 and Figure 3 In the above figure, modules are modules, node is node, link is connection, positive is positive correlation, and negative is negative correlation.

[0140] Figure 4 In the figure, Gephi software was used to analyze the network structure of bacterial communities in the seed belt of the sedum. A: SEpI with endophytic fungus sedum seeds; B: SEnI with endophytic fungus sedum seeds; C: GEpI with endophytic fungus sedum husk; D: SEpF with no endophytic fungus sedum seeds; E: SEnF with no endophytic fungus sedum seeds; F: GepF with no endophytic fungus sedum husk.

[0141] Figure 5 In the figure, Gephi software was used to analyze the fungal community network structure of the species of the sedum. A: SEpI with endophytic fungi on the seeds of the sedum; B: SEnI with endophytic fungi on the seeds of the sedum; C: GEpI with endophytic fungi on the hulls of the sedum; D: SEpF with no endophytic fungi on the seeds of the sedum; E: SEnF with no endophytic fungi on the seeds of the sedum; F: GepF with no endophytic fungi on the hulls of the sedum.

[0142] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for analyzing grass seed-borne microorganisms using a molecular ecological network analysis platform, characterized in that: include: Provide Endophytic fungi infect caryopsis and uninoculated Endophytic fungi infected the caryopsis of EF intoxicating horse grass; Obtained respectively Endophytic fungi infect caryopsis and uninoculated There are 6 treatments in total, including samples of lemma microorganisms, epiphytic microorganisms and microorganisms in seeds of EF euphorbia caryopsis infected by endophytic fungi; DNA was extracted from the above six treated microbial samples and sequenced; Submit the OTU and / or ASV data of all microorganisms obtained by sequencing to the molecular ecological network analysis MENA platform for analysis; After analysis, the obtained data results are uploaded to Cytoscape and / or Gephi software for analysis and drawing.

2. The method for analyzing grass seed-borne microorganisms using a molecular ecological network analysis platform according to claim 1, characterized in that: by Endophytic fungi infect caryopsis and uninoculated The method for obtaining the caryopsis of EF intoxicated grass infected by endophytic fungi comprises: randomly selecting eight plots of intoxicated grass with endophytic fungi EI and eight plots of intoxicated grass without endophytic fungi EF to collect caryopsis, selecting six mature plants in each plot to collect caryopsis, and then mixing the caryopsis of intoxicated grass with endophytic fungi EI and without endophytic fungi EF, respectively, to obtain the following: Endophytic fungi infect caryopsis and uninoculated The caryopsis of EF sphaerocephala infected by endophytic fungi.

3. The method for analyzing grass seed-borne microorganisms using a molecular ecological network analysis platform according to claim 1, characterized in that: Obtained by Endophytic fungi infect caryopsis and uninoculated The caryopsis of EF euphorbia infected by endophytic fungi were stored in a refrigerator at 4°C.

4. The method for analyzing grass seed-borne microorganisms using a molecular ecological network analysis platform according to claim 1, characterized in that: Separate the EI scutellaria scutellariae caryopsis into EI scutellaria scutellariae glumes and EI scutellaria scutellariae seeds, place the EI scutellaria scutellariae seeds in a first conical flask, add 10% of the first sterile PB buffer solution, and -1 , seal the mouth of the first conical flask with a sealing film, shake and elute for the first time, let it stand for the first time, take the washing solution to be the EI intoxicated horse grass seed epiphytic microorganism sample; add the EI intoxicated horse grass seeds after shaking and elution into the first sterile PBS buffer solution for 10 -1 After grinding, the filtrate obtained by filtration is the microbial sample in the seeds of EI intoxicated horse grass; the husk of EI intoxicated horse grass is placed in a second conical flask, and a second sterile PB buffer solution is added for 10 -1 , seal the mouth of the second conical flask with a sealing film, shake and elute for the second time, let it stand for the second time, and take the washing and elution liquid to obtain the EI glume hull microbial sample.

5. The method for analyzing grass seed-borne microorganisms using a molecular ecological network analysis platform according to claim 4, characterized in that: The amount of EI caryopsis of the herb is 40 g to 60 g; and / or The volume of the first conical flask is 50 ml, and the volume of the second conical flask is 50 ml; and / or The rotation speed of the first shaking elution is 140r / min to 160r / min; and / or The rotation speed of the second oscillation elution is 140r / min to 160r / min; and / or The first standing time is 4 to 6 minutes; and / or The second standing time is 4 to 6 minutes; and / or First sterile PB buffer 10 -1 The volume is 20ml to 40ml; and / or Second sterile PB buffer 10 -1 The volume is 20ml to 40ml; and / or First sterile PBS buffer 10 -1 The volume is 20ml~40ml.

6. The method for analyzing grass seed-borne microorganisms using a molecular ecological network analysis platform according to claim 1, characterized in that: Separate the EF scutellaria baicalensis caryopsis into EF scutellaria baicalensis glumes and EF scutellaria baicalensis seeds, place the EF scutellaria baicalensis seeds in a third conical flask, add 10% of the third sterile PB buffer solution, and -1 , seal the mouth of the third conical flask with a sealing film, shake and elute for the third time, let it stand for the third time, take the washing solution to be the epiphytic microorganism sample of EF scutellaria seed; add the EF scutellaria seed after shaking and elution into the second sterile PBS buffer for 10 -1 After grinding, the filtrate obtained by filtration is the microbial sample in the seeds of EF scutellaria baicalensis. The husk of EF scutellaria baicalensis is placed in a fourth conical flask, and 10% of the fourth sterile PB buffer is added. -1 , seal the mouth of the fourth conical flask with a sealing film, shake and elute for the fourth time, let it stand for the fourth time, and take the washing and elution liquid to obtain the EF glume microbial sample.

7. The method for analyzing grass seed-borne microorganisms using a molecular ecological network analysis platform according to claim 6, characterized in that: The amount of EF physalis caryopsis is 40 g to 60 g; and / or The volume of the third conical flask is 50 ml, and the volume of the fourth conical flask is 50 ml; and / or The rotation speed of the third oscillation elution is 140r / min to 160r / min; and / or The rotation speed of the fourth oscillation elution is 140r / min to 160r / min; and / or The third standing time is 4 to 6 minutes; and / or The fourth standing time is 4 to 6 minutes; and / or Third sterile PB buffer 10 -1 The volume is 20ml to 40ml; and / or Fourth sterile PB buffer 10 -1 The volume is 20ml to 40ml; and / or Second sterile PBS buffer 10 -1 The volume is 20ml~40ml.

8. The method for analyzing grass seed-borne microorganisms using a molecular ecological network analysis platform according to claim 1, characterized in that: The MN NucleoSpin 96 soil DNA kit was used to extract fungal and / or bacterial DNA from the microbial samples of the above six treatments, with eight replicates in each treatment.

9. The method for analyzing grass seed-borne microorganisms using a molecular ecological network analysis platform according to claim 8, characterized in that: The primers for bacterial amplification were: 335F (5′-CADACTCCTACGGGGAGGGC-3′) and 769R (5′-ATCCTGTTTGMTMCCCCRC-3′); The primers for fungal amplification were: ITS1F (5'-CTTGGTCATAGGAAGAAGTAA-3') and ITS2 (5'-GCTGCGTTCTTCATCGATGC-3').

10. The method for analyzing grass seed-borne microorganisms using a molecular ecological network analysis platform according to claim 1, characterized in that: The PCR products were purified using the OMEGA DNA kit, and the purified samples were subjected to high-throughput sequencing analysis of bacterial and / or fungal rDNA genes using the Illumina Hiseq 2500 platform of Beijing Biomike Biotechnology Co., Ltd. (BMK), China, to obtain the OTU and / or ASV data of all microorganisms, which were then submitted to the molecular ecological network analysis MENA platform for data analysis.

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