参见作者原研究论文

本实验方案简略版
Mar 2017

本文章节


 

Quantification of the Composition Dynamics of a Maize Root-associated Simplified Bacterial Community and Evaluation of Its Biological Control Effect
玉米根系相关简化细菌群落组成动力学定量及其生物防治效果评价   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Besides analyzing the composition and dynamics of microbial communities, plant microbiome research aims to understanding the mechanism of plant microbiota assembly and their biological functions. Here, we describe procedures to investigate the role of bacterial interspecies interactions in root microbiome assembly and the beneficial effects of the root microbiota on hosts by using a maize root-associated simplified seven-species (Stenotrophomonas maltophilia, Ochrobactrum pituitosum, Curtobacterium pusillum, Enterobacter cloacae, Chryseobacterium indologenes, Herbaspirillum frisingense and Pseudomonas putida) synthetic bacterial community described in our previous work. Surface-sterilized maize seeds were grown in a gnotobiotic system based on double-tube growth chambers after being soaked in suspensions containing multiple species of bacteria. The dynamics of the composition of the bacterial communities colonized on maize roots were tracked by a culture-dependent method with a selective medium for each of the seven strains. The impact of bacterial interactions on the community assembly was evaluated by monitoring the changes of community structure. The plant-protection effects of the simplified seven-species community were assessed by quantifying (1) the growth of a fungal phytopathogen, Fusarium verticillioides on the surfaces of the seeds and (2) the severity of seedling blight disease the fungus causes, in the presence and absence of the bacterial community. Our protocol will serve as useful guidance for studying plant-microbial community interactions under the laboratory conditions.

Keywords: Maize (玉米), Synthetic community (合成群落), Selective medium (选择性培养基), Dynamics (动力学), Community assembly and biological control (群落构建和生物防治)

Background

In natural settings, plants are associated with myriad microorganisms of extremely high diversity. These microbes exploit the niches provided by plant hosts and form complex microbial communities (Bulgarelli et al., 2012; Ofek-Lalzar et al., 2014; Cardinale et al., 2015; Edwards et al., 2015; Beckers et al., 2016; de Souza et al., 2016; Niu et al., 2017). Such plant-associated microbiomes are able to affect the development and health of the hosts profoundly (Berendsen et al., 2012). Recently, huge amounts of data describing plant microbiome compositions and their dynamics have been obtained by using advanced DNA sequencing technologies and data analysis methods. Much has been learned about the community structure of plant microbiota (Ofek-Lalzar et al., 2014; Bai et al., 2015; Ritpitakphong et al., 2016). However, due to the great complexity, currently it is nearly impossible to directly define experimentally the mechanisms underlying the dynamics of plant microbiome assembly and their beneficial effects on hosts. The establishment of simplified plant-associated microbial communities under controlled laboratory conditions is an approach to overcome the challenges in analyzing the properties of plant microbiota (Bodenhausen et al., 2014; Bai et al., 2015; Lebeis et al., 2015). Testing of hypotheses by targeted manipulation in gnotobiotic systems with simplified synthetic communities become a lot easier (Vorholt et al., 2017).

Previously, through host-mediated selection, we assembled a greatly simplified, yet representative, synthetic bacterial community consisting of seven strains (Stenotrophomonas maltophilia, Ochrobactrum pituitosum, Curtobacterium pusillum, Enterobacter cloacae, Chryseobacterium indologenes, Herbaspirillum frisingense and Pseudomonas putida) (Niu et al., 2017). We found that the removal of E. cloacae caused dramatic changes of the community composition and that this seven-species community protects maize from colonization by a fungal pathogen, Fusarium verticillioides. These results suggest that this synthetic seven-species community has the potential to serve as a useful system to explore how bacterial interspecies interactions affect root microbiome assembly and to dissect the beneficial effects of the root microbiota on hosts under laboratory conditions (Niu et al., 2017). This protocol has been developed to set up a gnotobiotic system for cultivating maize seedlings colonized by the root-associated simplified communities, to track the dynamics of the composition of the simplified communities and to evaluate the in vivo biological control effects of the seven-species community against F. verticillioides.

Materials and Reagents

  1. Consumables
    1. Disposable Petri dishes (VWR, catalog number: 89022-320 )
    2. Pipette tips (Corning, Axygen®, catalog number: T1005WBCRS ; Biotix, catalog number: M-0200-1RCNS )
    3. Parafilm (VWR, catalog number: 52858-000)
      Manufacturer: Bemis, catalog number: PM996 .
    4. Inoculation loops (Globe Scientific, catalog number: 130118 )
    5. Centrifuge tubes 2.0 ml (Corning, Axygen®, catalog number: MCT-200-C-S )
    6. Centrifuge tubes 1.5 ml (VWR, catalog number: 20170-038 )
    7. Centrifuge tubes 50 ml (Corning, catalog number: 352098 )
    8. Scalpel blades (Integra LifeSciences, catalog number: 4-110 )
    9. Glass beads (Propper, catalog number: 03000600 )
    10. Paper wipers (KCWW, Kimberly-Clark, catalog number: 34155 )
    11. 96-well plates (Corning, catalog number: 351172 )
    12. Cell scrapers (VWR, catalog number: 89260-222 )
    13. Trays (Thermo Fisher Scientific, Nunc, catalog number: 242811 )

  2. Plants
    Zea mays cv. Sugar Buns F1 (se+) (Johnny’s Selected Seeds, catalog number: 267 )

  3. Bacterial strains
    Stenotrophomonas maltophilia ZK5342, Ochrobactrum pituitosum ZK5343,
    Curtobacterium pusillum ZK5344, Enterobacter cloacae ZK5345,
    Chryseobacterium indologenes ZK5346, Herbaspirillum frisingense ZK5347 and
    Pseudomonas putida ZK5348 (Niu and Kolter, 2017)
    These strains can be requested via e-mail: ben_niu@nefu.edu.cn or roberto_kolter@hms.harvard.edu

  4. Fungal strain
    Fusarium verticillioides MRC826 (Hinton and Bacon, 1995)

  5. Chemical reagents
    1. Ethanol (Decon Labs, catalog number: V1001 )
    2. Bleach (Janitorial Supplies, Clorox®, catalog number: CLO30966CT )
    3. BactoTM Tryptic Soy Broth without Dextrose (BD, catalog number: 286220 )
    4. Soyabean Casein Digest Agar (HiMedia Laboratories, catalog number: GM290-500G )
    5. Agar (BD, catalog number: 214010 )
    6. 10x Phosphate buffered saline (PBS) (Lonza, catalog number: 17-517Q )
    7. Murashige and Skoog Basal Salt Mixture (MS) (Sigma-Aldrich, catalog number: M5524-50L )
    8. Nalidixic acid (Sigma-Aldrich, catalog number: N8878-5G )
    9. Colistin (Sigma-Aldrich, catalog number: C4461-100MG )
    10. Lincomycin (Sigma-Aldrich, catalog number: 62143-1G )
    11. Chlortetracycline (Sigma-Aldrich, catalog number: C4881-5G )
    12. Erythromycin (Sigma-Aldrich, catalog number: E5389-1G )
    13. Vancomycin (Sigma-Aldrich, catalog number: 75423-5VL )
    14. Sodium chlorite (VWR, catalog number: BDH9286-500G )
    15. Novobiocin (Sigma-Aldrich, catalog number: N1628-1G )
    16. Tobramycin (Sigma-Aldrich, catalog number: T4014-100MG )
    17. Glucose (VWR, catalog number: BDH9230-500G )

  6. Media and buffers (see Recipes)
    1. Tryptone soya agar medium
    2. 0.1x Tryptone soya agar medium
    3. Tryptic soy broth medium
    4. 1x Phosphate buffered saline (PBS)
    5. ½ Murashige and Skoog (MS) agar medium
    6. Selective medium for S. maltophilia ZK5342
    7. Selective medium for O. pituitosum ZK5343
    8. Selective medium for C. pusillum ZK5344
    9. Selective medium for E. cloacae ZK5345
    10. Selective medium for C. indologenes ZK5346
    11. Selective medium for H. frisingense ZK5347
    12. Selective medium for P. putida ZK5348
    13. Potato dextrose agar medium
    14. Water agar medium

Equipment

  1. Forceps
  2. Pipettes (Gilson, models: P20, P200 and P1000, catalog numbers: F123600 , F123601 and F123602 ; Thermo Fisher Scientific, model: F1-ClipTipTM, catalog numbers: 4661140N and 4661130N )
  3. Class II biological safety cabinet (Thermo Fisher Scientific, model: HerasafeTM KS9 )
  4. Centrifuge (Eppendorf, model: 5424 )
  5. Vortex (Scientific Industries, model: Vortex-Genie 2, catalog number: G560 )
  6. Spectrophotometer (Beckman Coulter, model: DU 640 )
  7. Sonicator (Qsonica, model: Q125 , catalog number: Q125-110)
  8. Balance (Mettler-Toledo International, catalog number: AG135 )
  9. Hemacytometer (Hausser Scientific, catalog number: 1492 )
  10. Microscope (ZEISS, model: Axioscop 2 plus )
  11. Stereoscope (ZEISS, model: Stemi SV 6 )

Software

  1. RStudio (version 0.99.903)
  2. QIIME (version 1.6.0)
  3. PRISM (version 6.0c)

Procedure

  1. Surface sterilization and germination of maize seeds
    1. Pick ten intact maize seeds of no disease symptom and put them in a Petri dish (9-cm diameter) (Figure 1A) with tweezers.
    2. Immerse the seeds in 70% (v/v) ethanol for three minutes then remove the ethanol.
    3. Immerse the seeds in 5% (v/v) bleach for three minutes then remove the bleach.
    4. Rinse the seeds with sterile distilled water three times.
    5. Take 250 μl water from the third rinse and spread onto tryptone soya agar (TSA) plates in order to check for contamination.
    6. Incubate the TSA plates at 30 °C overnight.
    7. Continue with the following steps if there is no microbial colony presenting on the TSA plates, otherwise, discard the maize seeds and repeat Steps A1 to A6 until no colony is detected on the TSA plates.
    8. Remove the distilled water from the Petri dish containing maize seeds.
    9. Keep the embryos of the seeds up and fill the Petri dish with 7 ml sterile distilled water (Figure 1B).
    10. Put the Petri dish containing surface-sterilized seeds in the dark at 30 °C.
    11. After an incubation of 24 h, take 250 μl water from the Petri dish and spread onto TSA plates to check for contamination.
    12. Incubate the TSA plates at 30 °C overnight.
    13. Continue with the following steps if there is no microbial colony presenting on the TSA plates, otherwise, discard the maize seeds and repeat Steps A1 to A12 until no colony is detected on the TSA plates.
    14. Remove the water from the Petri dish and refill with 7 ml sterile distilled water.
    15. Put the Petri dish back in the dark at 30 °C.
    16. After an incubation of 50 to 55 h in total, choose the germinated maize seeds (Figure 1C) with a root of 1-2 cm for the following steps.
      Note: Perform Steps A2 to A17 in a biological safety cabinet.

  2. Inoculation of bacterial community on maize seedlings
    1. Streak the seven bacterial strains (Stenotrophomonas maltophilia ZK5342, Ochrobactrum pituitosum ZK5343, Curtobacterium pusillum ZK5344, Enterobacter cloacae ZK5345, Chryseobacterium indologenes ZK5346, Herbaspirillum frisingense ZK5347 and Pseudomonas putida ZK5348) on 0.1x TSA plates and incubate at 30 °C for 24-48 h.
    2. Inoculate a single colony of each strain in 5 ml of tryptic soy broth (TSB) and shake at 120 rpm at 30 °C overnight.
    3. Transfer 50 μl of overnight culture of each strain into 5 ml fresh TSB and shake at 30 °C for another 8 h.
    4. Collect the cells in 2.0-ml tubes by centrifuge at 2,940 x g for 10 min at 4 °C.
    5. Resuspend the cells in 1x phosphate buffered saline (PBS) and dilute the cell suspensions of each strain to ~108 cells per milliliter (Table 1).

      Table 1. The OD600 value for each strain corresponding to ~108 cells/ml


    6. Mix the cell suspension of each strain in a 50-ml Falcon tube in equal volume to prepare the multiple species (seven-species or six-species resulting from the removal of each of the seven species, respectively) bacterial suspensions.
    7. Soak no more than 30 surface-sterilized and germinated maize seeds with primary roots of 1-2 cm (Figure 1C) in 30 ml multiple species bacterial suspensions in a Petri dish (Figure 1D) without shaking at room temperature for 0.5-1.0 h. Move the seeds to make sure the roots are completely submerged in the suspensions. Soak another 10 surface-sterilized and germinated seeds in 1x PBS buffer for 0.5-1.0 h and use as a control.
    8. Transfer the maize seeds adhered by bacteria and sterile seeds onto 20 ml ½ Murashige and Skoog (MS) agar (0.8%) in glass tubes (16 x 150 mm) by sterile forceps. Press the seeds gently with the forceps to insert the primary roots into the agar (Figure 1E). Use the sterile empty glass tubes of the same size to close the tubes containing the seeds in a mouth-to-mouth way. Connect and fix the two tubes by parafilm (Figure 2A).


      Figure 1. Maize seeds. A. Dry seeds; B. The seeds after surface-sterilization; C. Surface-sterilized and germinated seeds; D. Surface-sterilized and germinated seeds soaked in suspensions of multiple bacterial species in a Petri dish; E. Surface-sterilized and germinated seeds with/without bacteria sitting in ½ MS agar. Scale bars = 1 cm.


      Figure 2. Growth of axenic maize seedlings. A. The double-tube growth chamber. The parafilm is used to hold the two glass tubes together. B. The axenic maize seedlings of different ages grown in the double-tube growth chambers.

    9. Place the maize seedlings in double-tube chambers under the following conditions: 16 h of light (day) and 8 h of dark (night), 4,000 lx, 25 °C and a relative humidity of 54%. Keep the maize seedlings under the above conditions for 15 days.
      Note: Perform the Steps B1 to B8 in a biological safety cabinet or close to a flame.

  3. Quantification of the compositions of the bacterial communities colonized on maize roots
    1. Sample three to five maize seedlings inoculated with each of the eight bacterial communities (one seven-species and seven six-species communities) at day 5, day 10 and day 15 after inoculation.
    2. Cut the root from each maize seedling with a sterile scalpel blade (Video 1).

      Video 1. Sampling root fragments from maize seedlings

    3. Rinse the root in sterile 1x PBS buffer quickly to remove the agar adheres to the root surface.
    4. Harvest a 1-cm-long primary root fragment below maize kernel by cutting the primary root with a sterile scalpel blade.
    5. Remove the lateral roots on the root fragment with the sterile scalpel blade.
    6. Transfer the root fragment into a 1.5-ml centrifuge tube with two sterile 200-μl pipette tips.
    7. Put six glass beads (diameter: 3 mm) in the tubes and add 1 ml sterile 1x PBS buffer.
    8. Dislodge the bacterial cells colonized on the root surfaces by sonicating (amplitude: 30%; pulse: on 01 sec, off 01 sec; time: 30 sec) for 1 min, then by vortexing for another 1 min.
    9. Repeat the Step C8 twice. Put the tube on ice for 1 min.
    10. Add 180 μl sterile 1x PBS buffer in one lane (8 wells) of a 96-well plate (Video 2).

      Video 2. Diluting bacterial suspension and plating

    11. Put 20 μl bacterial suspension obtained in Step C9 in the first well of the lane and mix by sucking and excluding the suspension with a pipette.
    12. Take the root fragment out with a pair of forceps, dry the fragment with paper wipers, then weigh it on a balance and record the weight of the root fragment.
    13. Transfer 20 μl well mixed bacterial suspension from the first to the eighth well sequentially to get 10 to 108 times dilutions of the bacterial suspension.
    14. Take 10 μl diluted bacterial suspension from each of the eight wells and spot on three selective 0.1x TSA plates (Table 2) for each strain with a multichannel pipette.

      Table 2. Supplements in the selective medium and incubation time for each strain


    15. Tilt the plates to make the bacterial suspension drops move toward one direction to spread the cells on agar surfaces.
    16. Air-dry the selective 0.1x TSA plates spread with the bacterial suspensions.
    17. Incubate the plates at 30 °C in the dark for 16 to 60 h (Table 2).
    18. Count and record the numbers of the Colony Formation Units (CFUs) (Figure 3A) on the selective plates.
    19. Use the CFU numbers between 10 and 200 (Figure 3B) to calculate the bacterial abundances:




      Figure 3. Colonies of the seven bacterial strains of the simplified community. A. Morphology of the colonies of each of the seven bacterial strains grown on the plain and selective 0.1x TSA plates. ‘P’ and ‘S’ designate the plain 0.1x TSA plates and the selective 0.1x TSA plates, respectively. B. Colonies of O. pituitosum grown on the selective plates. From left to right, the four lanes of colonies were formed through the growth of the cells in 104-, 103-, 102- and 10-times diluted bacterial suspensions, respectively. The CFUs (Colony Formation Units) numbers of the two lanes framed are fit for the quantification of bacteria.

    20. Calculate the relative abundance of each species in the communities.
      1. Perform the Steps C2 to C16 in a biological safety cabinet or close to a flame.
      2. Watch Video 1 for details of Steps C2 to C7.
      3. Watch Video 2 for details of Steps C10 to C16.

  4. In vivo assay for the inhibitory effect of bacterial community against F. verticillioides
    Note: Perform the Steps D1 to D10 in a biological safety cabinet or close to a flame.
    1. Place an F. verticillioides MRC826 mycelial disk of 0.5-cm diameter at the center of a potato dextrose agar (PDA) plate and incubate at 28 °C for 7 days until the mycelia cover the whole plate.
    2. Put 20 ml sterile 1x PBS buffer in the F. verticillioides colony grown on a PDA agar plate.
    3. Harvest the fresh spores with a cell scraper.
    4. Filter the F. verticillioides spore suspension through eight layers of sterile gauze.
    5. Dilute the spore suspension and count the spores using a hemacytometer under a microscope.
    6. Adjust the concentration of spores to ~108 spores per milliliter sterile 0.01% (vol/vol) Tween 20. Store the suspension at 4 °C.
    7. Dilute the suspension to ~106 spores per milliliter sterile 1x PBS buffer.
    8. Inoculate the spores by spreading on the trays (~103 CFU/cm2) containing 20 ml 2.25% (wt/vol) water agar.
    9. Soak 10 surface-sterilized maize seeds (Figure 1B) in a 30 ml suspension of the seven-species bacterial community, each single species of the community, or Escherichia coli DH5α in a Petri dish for 0.5-1.0 h. Soak another 10 surface-sterilized seeds in sterile 1x PBS for 0.5-1.0 h and use as a control.
    10. Put the seeds on the surfaces of water agar in trays with sterile forceps.
    11. Put the trays in the dark at 23 °C for 10 days.
    12. Count and record the number of seeds showing visible fungal mycelia (Figure 4A) in each treatment every day. Calculate the fungi colonization rate as:



    13. Take photographs of mycelial growth on the surface of each seed on day 4 and day 10 after incubation using a dissecting microscope (Figure 4B).


      Figure 4. The maize seedling co-inoculated with the seven-species simplified bacterial community and F. verticillioides. A. A ten-day-old maize seedling inoculated with the bacterial community grown on water agar spread with fungal spores. The white spots on the agar are the mycelia developed from the spores. B. The enlargement of the seed in (A) indicated by the two yellow dash lines. The white hairs on the seed are mycelia colonized on the surface of seed (scale bar = 2 mm).

    14. Evaluate the severities of maize seedling blight disease for each treatment on day 10 by calculating the disease severity indices based on the ranks described previously (Niu et al., 2017) following the formula (Sherwood and Hagedorn, 1958):



      The disease ranks were established as follows:
      rank 1 = no visible fungal mycelia grow on the surfaces of kernels, and tan lesions are present on the roots;
      rank 2 = the surfaces of kernels are partially covered by fungal mycelia, and tan lesions are present on the roots;
      rank 3 = the surfaces of kernels are partially covered by fungal mycelia, and tan to brown lesions are present on the roots;
      rank 4 = the surfaces of kernels are fully covered by fungal mycelia, and tan to brown lesions are present on the roots;
      rank 5 = the surfaces of kernels are fully covered by fungal mycelia, and reddish brown lesions are present on the roots (Niu et al., 2017).

Data analysis

Bray-Curtis (BC) dissimilarity indexes were calculated based on the relative abundance values of each species by the function of the package ‘Vegan’ of RStudio (version 0.99.903). The dissimilarity matrix was then used to generate corresponding cluster dendrograms by hierarchical clustering using the function ‘hclust’ of the R package ‘gplots.’ The BC distance between the community on the maize root inoculated with each six-species community and the community on the root inoculated with the seven-species model community was calculated using QIIME (version 1.6.0). The Fisher’s LSD test (PRISM, version 6.0c) was used to compare: 1). BC distances between each six-species community and the seven-species model community plant by plant, 2). fungi colonization rates and disease severity indices of maize seedlings treated with F. verticillioides alone, jointly with F. verticillioides and the seven-species model community, jointly with F. verticillioides and each of the seven species and jointly with F. verticillioides and E. coli DH5α, respectively.

Recipes

  1. Tryptone soya agar medium
    20 g Tryptone Soya Agar (HiMedia Laboratories)
    1,000 ml distilled water
    Autoclave at 121 °C for 20 min
  2. 0.1x tryptone soya agar medium
    1.38 g Tryptic Soy Broth without Dextrose (BD)
    7.5 g agar (BD)
    500 ml distilled water
    Autoclave at 121 °C for 20 min
  3. Tryptic soy broth medium
    13.75 g Tryptic Soy Broth without Dextrose (BD)
    500 ml distilled water 
  4. 1x phosphate buffered saline (PBS)
    100 ml PBS (10x) without calcium or magnesium
    900 ml distilled water
    Autoclave at 121 °C for 20 min
  5. ½ Murashige and Skoog (MS) agar medium
    2.15 g Murashige and Skoog Basal Salt Mixture (Sigma-Aldrich)
    4 g agar (BD)
    500 ml distilled water
    Autoclave at 121 °C for 20 min
  6. Selective medium for S. maltophilia ZK5342
    1.38 g Tryptic Soy Broth without Dextrose
    7.5 g agar
    500 ml distilled water
    Autoclave at 121 °C for 20 min. Cool down the sterilized medium to around 60 °C. Then add 300 μl novobiocin (Sigma-Aldrich) (100 mg/ml) and 12.8 μl tobramycin (Sigma-Aldrich) (39.1 mg/ml)
  7. Selective medium for O. pituitosum ZK5343
    1.38 g Tryptic Soy Broth without Dextrose
    7.5 g agar
    500 ml distilled water
    Autoclave at 121 °C for 20 min
    Cool down the sterilized medium to around 60 °C. Then add 200 μl colistin (Sigma-Aldrich) (10 mg/ml), 125 μl erythromycin (Sigma-Aldrich) (20 mg/ml) and 70 μl vancomycin (Sigma-Aldrich) (100 mg/ml)
  8. Selective medium for C. pusillum ZK5344
    1.38 g Tryptic Soy Broth without Dextrose
    7.5 g agar
    500 ml distilled water
    Autoclave at 121 °C for 20 min
    Cool down the sterilized medium to around 60 °C. Then add 1,132 μl nalidixic acid (Sigma-Aldrich) (5 mg/ml), 226.4 μl colistin (Sigma-Aldrich) (10 mg/ml) and 66 ml NaCl (VWR International) (30%, wt/vol)
  9. Selective medium for E. cloacae ZK5345
    1.38 g Tryptic Soy Broth without Dextrose
    7.5 g agar
    500 ml distilled water
    Autoclave at 121 °C for 20 min. Cool down the sterilized medium to around 60 °C. Then add 163.77 μl erythromycin (Sigma-Aldrich) (20 mg/ml) and 155.07 ml NaCl (VWR International) (30%, wt/vol)
  10. Selective medium for C. indologenes ZK5346
    1.38 g Tryptic Soy Broth without Dextrose
    7.5 g agar
    500 ml distilled water
    Autoclave at 121 °C for 20 min. Cool down the sterilized medium to around 60 °C. Then add 120.19 μl chlortetracycline (Sigma-Aldrich) (10 mg/ml)
  11. Selective medium for H. frisingense ZK5347
    1.38 g Tryptic Soy Broth without Dextrose
    7.5 g agar
    500 ml distilled water
    Autoclave at 121 °C for 20 min. Cool down the sterilized medium to around 60 °C. Then add 1000 μl nalidixic acid (Sigma-Aldrich) (5 mg/ml), 200 μl colistin (Sigma-Aldrich) (10 mg/ml) and 1,000 μl lincomycin (Sigma-Aldrich) (50 mg/ml)
  12. Selective medium for P. putida ZK5348
    1.38 g Tryptic Soy Broth without Dextrose
    7.5 g agar
    500 ml distilled water
    Autoclave at 121 °C for 20 min. Cool down the sterilized medium to around 60 °C. Then add 500 μl nalidixic acid (Sigma-Aldrich) (5 mg/ml) and 125 μl erythromycin (Sigma-Aldrich) (20 mg/ml)
  13. Potato dextrose agar medium
    Potato extracts from 200 g of potato tuber
    17 g agar (BD)
    20 g glucose (VWR International)
    1,000 ml distilled water
    Autoclave at 121 °C for 20 min
  14. Water agar medium
    11.25 g agar
    500 ml distilled water
    Autoclave at 121 °C for 20 min

Acknowledgments

We thank Yue Liu for helps in shooting the videos; Bo Shen for helps in culturing the maize seedlings; and members of the Kolter Laboratory for valuable advice. This work was supported by NIH Grant No. GM58213 (to R.K.) and Start-up Scientific Foundation of Northeast Forestry University JQ2017-02 (to B.N.). This protocol was adapted from Niu et al. (2017).

Competing interests

The authors have no conflict of interest or competing interests to declare.

References

  1. Bai, Y., Muller, D. B., Srinivas, G., Garrido-Oter, R., Potthoff, E., Rott, M., Dombrowski, N., Munch, P. C., Spaepen, S., Remus-Emsermann, M., Huttel, B., McHardy, A. C., Vorholt, J. A. and Schulze-Lefert, P. (2015). Functional overlap of the Arabidopsis leaf and root microbiota. Nature 528(7582): 364-369.
  2. Beckers, B., Op De Beeck, M., Weyens, N., Van Acker, R., Van Montagu, M., Boerjan, W. and Vangronsveld, J. (2016). Lignin engineering in field-grown poplar trees affects the endosphere bacterial microbiome. Proc Natl Acad Sci U S A 113(8): 2312-2317.
  3. Berendsen, R. L., Pieterse, C. M. and Bakker, P. A. (2012). The rhizosphere microbiome and plant health. Trends Plant Sci 17(8): 478-486.
  4. Bodenhausen, N., Bortfeld-Miller, M., Ackermann, M. and Vorholt, J. A. (2014). A synthetic community approach reveals plant genotypes affecting the phyllosphere microbiota. PLoS Genet 10(4): e1004283.
  5. Bulgarelli, D., Rott, M., Schlaeppi, K., Ver Loren van Themaat, E., Ahmadinejad, N., Assenza, F., Rauf, P., Huettel, B., Reinhardt, R., Schmelzer, E., Peplies, J., Gloeckner, F. O., Amann, R., Eickhorst, T. and Schulze-Lefert, P. (2012). Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota. Nature 488(7409): 91-95.
  6. Cardinale, M., Grube, M., Erlacher, A., Quehenberger, J. and Berg, G. (2015). Bacterial networks and co-occurrence relationships in the lettuce root microbiota. Environ Microbiol 17(1): 239-252.
  7. de Souza, R. S., Okura, V. K., Armanhi, J. S., Jorrin, B., Lozano, N., da Silva, M. J., Gonzalez-Guerrero, M., de Araujo, L. M., Verza, N. C., Bagheri, H. C., Imperial, J. and Arruda, P. (2016). Unlocking the bacterial and fungal communities assemblages of sugarcane microbiome. Sci Rep 6: 28774.
  8. Edwards, J., Johnson, C., Santos-Medellin, C., Lurie, E., Podishetty, N. K., Bhatnagar, S., Eisen, J. A. and Sundaresan, V. (2015). Structure, variation, and assembly of the root-associated microbiomes of rice. Proc Natl Acad Sci U S A 112(8): E911-920.
  9. Hinton, D. M. and Bacon, C. W. (1995). Enterobacter cloacae is an endophytic symbiont of corn. Mycopathologia 129(2): 117-125.
  10. Lebeis, S. L., Paredes, S. H., Lundberg, D. S., Breakfield, N., Gehring, J., McDonald, M., Malfatti, S., Glavina del Rio, T., Jones, C. D., Tringe, S. G. and Dangl, J. L. (2015). PLANT MICROBIOME. Salicylic acid modulates colonization of the root microbiome by specific bacterial taxa. Science 349(6250): 860-864.
  11. Niu, B. and Kolter, R. (2017). Complete genome sequences of seven strains composing a model bacterial community of maize roots. Genome Announc 5(36).
  12. Niu, B., Paulson, J. N., Zheng, X. and Kolter, R. (2017). Simplified and representative bacterial community of maize roots. Proc Natl Acad Sci U S A 114(12): E2450-E2459.
  13. Ofek-Lalzar, M., Sela, N., Goldman-Voronov, M., Green, S. J., Hadar, Y. and Minz, D. (2014). Niche and host-associated functional signatures of the root surface microbiome. Nat Commun 5: 4950.
  14. Ritpitakphong, U., Falquet, L., Vimoltust, A., Berger, A., Metraux, J. P. and L'Haridon, F. (2016). The microbiome of the leaf surface of Arabidopsis protects against a fungal pathogen. New Phytol 210(3): 1033-1043.
  15. Sherwood, R. and Hagedorn, D. (1958). Determining common root rot potential of pea fields. Agricultural Experiment Station, University of Wisconsin, Madison, WI.
  16. Vorholt, J. A., Vogel, C., Carlstrom, C. I. and Muller, D. B. (2017). Establishing causality: opportunities of synthetic communities for plant microbiome research. Cell Host Microbe 22(2): 142-155.

简介

除了分析微生物群落的组成和动态外,植物微生物群落研究的目的在于了解植物微生物群落组装的机制及其生物学功能。在这里,我们描述了通过使用与玉米根相关的简化的七种物种(嗜麦芽窄食单胞菌(Stenotrophomonas maltophilia),)来研究细菌种间相互作用在根微生物群组装中的作用以及根菌群对宿主的有益影响的程序。 Ochrobactrum pituitosum ,Curtobacterium pusillum ,阴沟肠杆菌, Chryseobacterium indologenes ,
【背景】在自然环境中,植物与无数极其多样化的微生物相关。这些微生物利用植物宿主提供的小生境并形成复杂的微生物群落(Bulgarelli等人,2012; Ofek-Lalzar等人,2014; Cardinale等人,2015; Edwards et al。,2015; Beckers et al。,2016; de Souza et。, 2016; Niu em et al。,2017)。这种与植物相关的微生物组能够深刻地影响宿主的发育和健康(Berendsen等人,2012年)。最近,通过使用先进的DNA测序技术和数据分析方法已经获得了大量描述植物微生物组成组成及其动力学的数据。关于植物微生物群落的群落结构已经有了许多了解(Ofek-Lalzar等人,2014; Bai等人,2015; Ritpitakphong等人, ,2016)。然而,由于复杂性大,目前几乎不可能直接定义植物微生物组装动力学及其对宿主的有益影响的机制。在受控实验室条件下建立简化的植物相关微生物群落是克服分析植物微生物群落特性方面的挑战的一种方法(Bodenhausen等人,2014; Bai等人, ,2015; Lebeis et。,2015)。通过在含有简化合成社区的假单胞菌系统中进行有针对性操作的假说检测变得容易得多(Vorholt et al。,2017)。

以前,通过宿主介导的选择,我们组装了一个大大简化但具有代表性的合成细菌群落,它由7个菌株组成(嗜麦芽窄食单胞菌(Stenotrophomonas maltophilia),青枯菌(Ochrobactrum pituitosum),压榨青枯菌阴沟肠杆菌,阴沟肠杆菌,

关键字:玉米, 合成群落, 选择性培养基, 动力学, 群落构建和生物防治

材料和试剂

  1. 耗材
    1. 一次性培养皿(VWR,目录号:89022-320)
    2. 移液器吸头(Corning,Axygen ,目录号:T1005WBCRS; Biotix,目录号:M-0200-1RCNS)
    3. Parafilm(VWR,目录号:52858-000)
      制造商:Bemis,目录号:PM996。
    4. 接种环(Globe Scientific,目录号:130118)
    5. 离心管2.0 ml(Corning,Axygen <\ sup>,目录号:MCT-200-C-S)
    6. 离心管1.5毫升(VWR,目录号:20170-038)
    7. 离心管50毫升(康宁,目录号:352098)
    8. 手术刀片(Integra LifeSciences,目录号:4-110)
    9. 玻璃珠(Propper,目录号:03000600)
    10. 刮纸器(KCWW,Kimberly-Clark,目录号:34155)
    11. 96孔板(Corning,目录号:351172)
    12. 细胞刮刀(VWR,目录号:89260-222)
    13. 托盘(Thermo Fisher Scientific,Nunc,目录号:242811)

  2. 植物
    Zea mays cv。糖包F1(se +)(约翰尼的精选种子,目录编号:267)

  3. 细菌菌株
    嗜麦芽窄食单胞菌ZK5342,垂体短肠杆菌 ZK5343,

    阴沟肠杆菌ZK5344,阴沟肠杆菌ZK5345, Chryseobacterium indologenes ZK5346, Herbaspirillum frisingense ZK5347和
    恶臭假单胞菌 ZK5348(牛和科尔特,2017)
    这些毒株可通过电子邮件申请: ben_niu@nefu.edu.cn 或 roberto_kolter@hms.harvard.edu

  4. 真菌菌株
    Fusarium verticillioides MRC826(Hinton and Bacon,1995)

  5. 化学试剂
    1. 乙醇(Decon Labs,目录号:V1001)
    2. 漂白剂(Janitorial Supplies,Clorox ,目录号:CLO30966CT)
    3. 没有葡萄糖的Bacto TM Tryptic大豆肉汤(BD,目录号:286220)
    4. Soyabean酪蛋白消化琼脂(HiMedia实验室,目录编号:GM290-500G)
    5. 琼脂(BD,目录号:214010)
    6. 10x磷酸盐缓冲盐水(PBS)(Lonza,目录号:17-517Q)
    7. Murashige和Skoog Basal盐混合物(MS)(Sigma-Aldrich,目录号:M5524-50L)
    8. 萘啶酮酸(Sigma-Aldrich,目录号:N8878-5G)
    9. 粘菌素(Sigma-Aldrich,目录号:C4461-100MG)
    10. 林可霉素(Sigma-Aldrich,目录号:62143-1G)
    11. 金霉素(Sigma-Aldrich,目录号:C4881-5G)
    12. 红霉素(Sigma-Aldrich,目录号:E5389-1G)
    13. 万古霉素(Sigma-Aldrich,目录号:75423-5VL)
    14. 亚氯酸钠(VWR,目录号:BDH9286-500G)
    15. Novobiocin(Sigma-Aldrich,目录号:N1628-1G)
    16. 妥布霉素(Sigma-Aldrich,目录号:T4014-100MG)
    17. 葡萄糖(VWR,目录号:BDH9230-500G)

  6. 媒体和缓冲区(请参阅食谱)
    1. 胰蛋白胨大豆琼脂培养基
    2. 0.1x胰蛋白胨大豆琼脂培养基
    3. 胰蛋白酶大豆肉汤培养基
    4. 1x磷酸盐缓冲盐水(PBS)
    5. ½Murashige和Skoog(MS)琼脂培养基
    6. 用于S的选择性媒体。麦芽糖杆菌 ZK5342
    7. 用于 O的选择性媒体。垂体瘤 ZK5343
    8. 用于 C的选择性媒体。 pusillum ZK5344
    9. E的选择性媒体。阴道菌 ZK5345
    10. 用于 C的选择性媒体。 indologenes ZK5346
    11. 用于 H的选择性媒体。 frisingense ZK5347
    12. 用于 P的选择性媒体。 putida ZK5348
    13. 马铃薯葡萄糖琼脂培养基
    14. 水琼脂培养基

设备

  1. 镊子
  2. 移液管(Gilson,型号:P20,P200和P1000,目录号:F123600,F123601和F123602; Thermo Fisher Scientific,型号:F1-ClipTip TM,目录号:4661140N和4661130N)
  3. II级生物安全柜(Thermo Fisher Scientific,型号:Herasafe TM KS9)
  4. 离心机(Eppendorf,型号:5424)
  5. 涡流(Scientific Industries,型号:Vortex-Genie 2,目录号:G560)
  6. 分光光度计(Beckman Coulter,型号:DU 640)
  7. Sonicator(Qsonica,型号:Q125,目录号:Q125-110)
  8. 余额(梅特勒 - 托利多国际,目录号:AG135)
  9. 血细胞计数器(Hausser Scientific,目录号:1492)
  10. 显微镜(ZEISS,型号:Axioscop 2 plus)
  11. 立体镜(蔡司,型号:Stemi SV 6)

软件

  1. RStudio(版本0.99.903)
  2. QIIME(版本1.6.0)
  3. PRISM(版本6.0c)

程序

  1. 玉米种子的表面灭菌和萌发
    1. 挑选10个完整无病症状的玉米种子,用镊子将它们放入培养皿(直径9厘米)(图1A)。
    2. 将种子浸入70%(v / v)乙醇中3分钟,然后除去乙醇。
    3. 将种子浸入5%(v / v)漂白剂中3分钟,然后除去漂白剂。

    4. 用无菌蒸馏水冲洗种子三次。
    5. 从第三次冲洗中取250μl水,涂在胰蛋白胨大豆琼脂(TSA)平板上,以检查是否有污染。

    6. 在30°C孵育TSA板过夜
    7. 如果TSA平板上不存在微生物菌落,则继续下列步骤,否则,丢弃玉米种子并重复步骤A1至A6,直到TSA平板上检测不到菌落。
    8. 从含有玉米种子的培养皿中取出蒸馏水。
    9. 保持种子的胚胎并用7ml无菌蒸馏水填充培养皿(图1B)。

    10. 将含有表面灭菌种子的培养皿置于30°C黑暗处。
    11. 孵育24小时后,从培养皿中取250μl水,铺在TSA平板上检查是否有污染。

    12. 在30°C孵育TSA板过夜
    13. 如果TSA平板上不存在微生物菌落,则继续下列步骤,否则,丢弃玉米种子并重复步骤A1至A12,直至TSA平板上未检测到菌落。
    14. 从培养皿中取出水并加入7 ml无菌蒸馏水。
    15. 在30°C下将培养皿放回黑暗中。
    16. 总共培养50至55小时后,选择1-2厘米根发芽的玉米种子(图1C),以进行以下步骤。
      注意:在生物安全柜中执行步骤A2至A17。

  2. 玉米幼苗接种细菌群落
    1. 将七种细菌菌株(嗜麦芽窄食单胞菌ZK5342,垂颈短杆菌ZK5343,革囊霉Cukobacterium pusillum ZK5344,阴沟肠杆菌ZK5345,阴性杆菌ZK5346,Herbaspirillum frisingense ZK5347和恶臭假单胞菌ZK5348),并在30℃下温育24-48小时。
    2. 接种5ml胰蛋白大豆肉汤(TSB)中的每种菌株的单菌落并在30℃以120rpm摇动过夜。
    3. 将每种菌株的50μl过夜培养物转移至5ml新鲜的TSB中,并在30℃再振荡8小时。

    4. 收集细胞在2.0毫升管中,通过在2,940×gg离心10分钟在4℃。
    5. 在1x磷酸盐缓冲盐水(PBS)中重悬细胞并将每种菌株的细胞悬液稀释至每毫升〜10 8个细胞(表1)。

      表1. 0 0 strong> 每个菌株对应t 0 > 8 cells / ml


    6. 将各菌株的细胞悬浮液以等体积混合在50ml Falcon管中以制备多种物种(分别由去除七种物种中的每一种产生的七种或六种)细菌悬浮液。
    7. 在培养皿中的30ml多种细菌悬浮液(图1D)中浸泡不超过30个具有1-2cm主根的表面灭菌和发芽的玉米种子(图1C),而不在室温下摇动0.5-1.0小时。移动种子以确保根部完全浸没在悬浮液中。将另外10个经表面灭菌和发芽的种子在1x PBS缓冲液中浸泡0.5-1.0h并用作对照。
    8. 将由细菌和无菌种子粘附的玉米种子通过无菌镊子转移到玻璃管(16×150mm)中的20ml 1/2 Murashige和Skoog(MS)琼脂(0.8%)上。用镊子轻轻按压种子,将主根插入琼脂中(图1E)。使用相同尺寸的无菌空玻璃管以口对口的方式关闭含种子的试管。通过石蜡膜连接并固定两个管(图2A)。


      图1.玉米种子A.干种子; B.表面消毒后的种子; C.表面灭菌和发芽的种子; D.在培养皿中浸泡在多种细菌物种的悬浮液中的表面灭菌和发芽的种子; E.表面灭菌和发芽的种子,有/无细菌,坐在半琼脂琼脂上。比例尺= 1厘米。


      图2.无菌玉米幼苗的生长A.双管生长室。封口膜用于将两个玻璃管固定在一起。 B.在双管生长室中生长的不同年龄的无菌玉米幼苗。

    9. 在下列条件下将玉米幼苗置于双管室中:16小时光照(天)和8小时黑暗(夜间),4000lx,25℃和54%的相对湿度。
      保持玉米苗在上述条件下15天。
      注意:在生物安全柜中或靠近火焰执行步骤B1至B8。

  3. 量化定殖在玉米根上的细菌群落的组成

    1. 在接种后的第5天,第10天和第15天,用八个细菌群落中的每一个(一个七种群和七个六种群落)接种三到五个玉米幼苗。

    2. 用无菌手术刀片切割每个玉米幼苗的根部(视频1)。

      视频1

    3. 在无菌1x PBS缓冲液中迅速冲洗根部以除去琼脂粘附在根表面。

    4. 用无菌手术刀切割主根,收获玉米籽粒下方1厘米长的主根碎片。

    5. 用无菌手术刀片取出根部的侧根
    6. 将根碎片转移到1.5毫升的离心管中,加入两个无菌200-μl移液器吸头。
    7. 将6个玻璃珠(直径:3 mm)放入试管中,并加入1 ml无菌1x PBS缓冲液。
    8. 通过超声处理(幅度:30%;脉冲:在01秒,关闭01秒;时间:30秒)下移动定殖在根表面上的细菌细胞1分钟,然后通过涡旋另外1分钟。
    9. 重复步骤C8两次。将试管放在冰上1分钟。

    10. 在96孔板(视频2)的一个泳道(8孔)中加入180μl无菌1x PBS缓冲液。

      视频2
    11. 将步骤C9中获得的20μl细菌悬浮液置于泳道的第一个孔中,并通过吸吮并用移液管排除混悬液进行混合。
    12. 用一把镊子取出根部碎片,用擦纸器擦干碎片,然后称重到天平上并记录碎片的重量。
    13. 从第一个到第八个孔顺序转移20μl混合好的细菌悬浮液,以获得细菌悬浮液的10至10 8倍稀释度。
    14. 从8个孔中的每一个中取10μl稀释的细菌悬液,用多通道移液管在每个菌株的3个选择性0.1x TSA平板(表2)上点出来。

      表2.每种菌株的选择培养基和培养时间的补充


    15. 倾斜平板使细菌悬液滴向一个方向移动,将细胞铺展在琼脂表面上。

    16. 空气干燥选择性0.1x TSA板与细菌悬浮液
    17. 将培养板在30°C黑暗中培养16至60小时(表2)。
    18. 计数并记录选择性平板上的菌落形成单位(CFU)数量(图3A)。
    19. 使用10到200之间的CFU数字(图3B)来计算细菌丰度:




      图3.简化群落中7种细菌菌落的菌落A.在普通和选择性0.1xTSA上生长的7种细菌菌株各自菌落的形态板。 'P'和'S'分别表示纯0.1x TSA板和选择性0.1x TSA板。 B. O的殖民地。在选择性平板上生长的pituito m 。从左至右,通过10 4 - ,10 - 3 - ,10 - 2 - 细胞的生长形成四条菌落, - 和10倍稀释的细菌悬液。
      两个泳道的CFU(菌落形成单位)数量适合细菌的定量分析。

    20. 计算社区中每个物种的相对丰度。

  4. 体内测定细菌群落对抗F的抑制作用。 verticillioide s
    注意:在生物安全柜内或靠近火焰处执行步骤D1至D10
    1. 放置一个 F。在马铃薯葡萄糖琼脂(PDA)平板的中心处直径为0.5厘米的MRC826菌丝盘,以及马铃薯葡萄糖琼脂(PDA)平板中心的直径为0.5厘米的MRC826菌丝盘和在28°C孵育7天,直到菌丝体覆盖整个平板。
    2. 将20ml无菌1x PBS缓冲液置于 F中。生长在PDA琼脂平板上的verticillioid s 菌落。

    3. 用细胞刮刀收获新鲜的孢子
    4. 过滤 F。 verticillioid e s 通过八层无菌纱布悬浮孢子。
    5. 稀释孢子悬浮液,用显微镜下的血细胞计数器计数孢子。
    6. 调整孢子浓度至每毫升无菌0.01%(vol / vol)Tween 20〜10 8孢子。将悬浮液储存在4°C。
    7. 将悬浮液稀释至每毫升无菌1x PBS缓冲液〜10 6孢子。
    8. 通过铺展在含有20ml 2.25%(wt / vol)水琼脂的托盘上(〜10 3 CFU / cm 2)接种孢子。
    9. 将10种表面灭菌的玉米种子(图1B)浸泡在7种细菌群落的30ml悬浮液中,每种单一种类的群落或埃希氏菌 c DH5α在培养皿中用于&lt; span style =“font-size:10pt;”> 0.5-1.0 h。将另外10个表面灭菌的种子在无菌1×PBS中浸泡0.5-1.0小时并用作对照。

    10. 用无菌镊子将种子放在托盘中的水琼脂表面上。
    11. 将托盘在23°C的黑暗中放置10天。
    12. 计数并记录每天每次治疗中显示可见真菌菌丝体的种子数(图4A)。计算真菌定殖率为:



    13. 使用解剖显微镜孵育后第4天和第10天,拍摄每个种子表面的菌丝体生长情况(图4B)。


      图4.与7种简化细菌群共同接种的玉米幼苗和 F。 A.一种十日龄的玉米幼苗,接种有在水琼脂上生长的细菌群落,并带有真菌孢子。琼脂上的白色斑点是由孢子发育而来的菌丝体。 B.由两条黄色虚线表示的(A)中种子的增大。
      种子上的白色毛发是种子表面的菌丝体(比例尺= 2毫米)。

    14. 通过基于先前描述的等级计算疾病严重程度指数(牛痘e抗体),在第10天评估每次治疗的玉米幼苗枯萎病的严重性。 (Sherwood and Hagedorn,1958):
      。,2017)


      这些疾病的排名如下:
      等级1 =在核仁表面没有可见的真菌菌丝体生长,并且根部存在褐色病变;
      等级2 =核仁表面被真菌菌丝体部分覆盖,并且根部存在褐色病斑;
      等级3 =内核的表面被真菌菌丝体部分覆盖,并且在根上存在棕褐色至棕色病变;
      等级4 =内核的表面被真菌菌丝体完全覆盖,并且在根上存在棕褐色至棕色病变;
      等级5 =内核的表面被真菌菌丝体完全覆盖,并且在根上存在红棕色病变(Ni-em-em-t a l 。,2017)。

数据分析

Bray-Curtis(BC)不相似性指数是根据每个物种的相对丰度值通过RStudio的包装素食(版本0.99.903)的函数计算的。然后使用差异矩阵通过使用R包'gplots'的函数'hclust'的等级聚类来生成相应的聚类树状图。在每个六种群落的玉米根上的社区与群落中的社区之间的BC距离使用QIIME(版本1.6.0)计算接种七种模型群落的根。 Fisher's LSD测试(PRISM,版本6.0c)用于比较:1)。每个六种群落与七种群落植物模型社区植物之间的BC距离,2)。用F em处理的玉米幼苗的真菌定植率和疾病严重性指数。 verticillioid e s 单独与 F 一起使用。 verticillioid e s 和七种模型社区,与 F 共同组成。 verticil l ioide s 以及七个物种中的每一个,并与F一起。 verticillioide s 和 。 col i DH5α。

食谱

  1. 胰蛋白胨大豆琼脂培养基
    20克胰蛋白胨大豆琼脂(HiMedia实验室)
    1000毫升蒸馏水
    在121°C高压灭菌20分钟

  2. 0.1x胰蛋白胨大豆琼脂培养基 1.38克不含葡萄糖(BD)的胰蛋白酶大豆肉汤
    7.5克琼脂(BD)
    500毫升蒸馏水
    在121°C高压灭菌20分钟
  3. 胰蛋白酶大豆肉汤培养基
    13.75克胰酶大豆肉汤没有葡萄糖(BD)
    500毫升蒸馏水&nbsp;
  4. 1x磷酸盐缓冲盐水(PBS)
    100毫升PBS(10倍)不含钙或镁
    900毫升蒸馏水
    在121°C高压灭菌20分钟
  5. ½Murashige和Skoog(MS)琼脂培养基
    2.15克Murashige和Skoog Basal盐混合物(Sigma-Aldrich)
    4克琼脂(BD)
    500毫升蒸馏水
    在121°C高压灭菌20分钟
  6. 用于S的选择性媒体。麦芽糖 i a ZK5342
    1.38克不含葡萄糖的胰蛋白酶大豆肉汤
    7.5克琼脂
    500毫升蒸馏水
    在121℃高压灭菌20分钟。将无菌培养基冷却至60°C左右。然后加入300μl新生霉素(Sigma-Aldrich)(100mg / ml)和12.8μl妥布霉素(Sigma-Aldrich)(39.1mg / ml)
  7. 用于 O的选择性媒体。垂体瘤 ZK5343
    1.38克不含葡萄糖的胰蛋白酶大豆肉汤
    7.5克琼脂
    500毫升蒸馏水
    在121°C高压灭菌20分钟
    将无菌培养基冷却至60°C左右。然后加入200μl粘菌素(Sigma-Aldrich)(10mg / ml),125μl红霉素(Sigma-Aldrich)(20mg / ml)和70μl万古霉素(Sigma-Aldrich)(100mg / ml)
  8. 用于 C的选择性媒体。 pusillu m ZK5344
    1.38克不含葡萄糖的胰蛋白酶大豆肉汤
    7.5克琼脂
    500毫升蒸馏水
    在121°C高压灭菌20分钟
    将无菌培养基冷却至60°C左右。然后加入1,132μl萘啶酮酸(Sigma-Aldrich)(5mg / ml),226.4μl粘菌素(Sigma-Aldrich)(10mg / ml)和66ml NaCl(VWR International)(30%,wt / vol) />
  9. E的选择性媒体。阴道菌 ZK5345
    1.38克不含葡萄糖的胰蛋白酶大豆肉汤
    7.5克琼脂
    500毫升蒸馏水
    在121℃高压灭菌20分钟。将无菌培养基冷却至60°C左右。然后加入163.77μl红霉素(Sigma-Aldrich)(20mg / ml)和155.07ml NaCl(VWR International)(30%,wt / vol)
  10. 用于 C的选择性媒体。 indologen e s ZK5346
    1.38克不含葡萄糖的胰蛋白酶大豆肉汤
    7.5克琼脂
    500毫升蒸馏水
    在121℃高压灭菌20分钟。将无菌培养基冷却至60°C左右。然后加入120.19μl金霉素(Sigma-Aldrich)(10 mg / ml)
  11. 用于 H 的选择性媒介。 frisingense ZK5347
    1.38克不含葡萄糖的胰蛋白酶大豆肉汤
    7.5克琼脂
    500毫升蒸馏水
    在121℃高压灭菌20分钟。将无菌培养基冷却至60°C左右。然后加入1000μl萘啶酮酸(Sigma-Aldrich)(5mg / ml),200μl粘菌素(Sigma-Aldrich)(10mg / ml)和1,000μl林可霉素(Sigma-Aldrich)(50mg / ml) >
  12. 用于 P的选择性媒体。 puti d a ZK5348
    1.38克不含葡萄糖的胰蛋白酶大豆肉汤
    7.5克琼脂
    500毫升蒸馏水
    在121℃高压灭菌20分钟。将无菌培养基冷却至60°C左右。然后加入500μl萘啶酸(Sigma-Aldrich)(5mg / ml)和125μl红霉素(Sigma-Aldrich)(20mg / ml)。
  13. 马铃薯葡萄糖琼脂培养基
    200克马铃薯块茎马铃薯提取物
    17克琼脂(BD)
    20克葡萄糖(VWR国际)
    1000毫升蒸馏水
    在121°C高压灭菌20分钟
  14. 水琼脂培养基

    11.25克琼脂 500毫升蒸馏水
    在121°C高压灭菌20分钟

致谢

我们感谢Yue Liu帮助拍摄视频;沉博帮助培育玉米幼苗;和Kolter实验室的成员提供宝贵的建议。这项工作得到了美国国立卫生研究院拨款号为GM58213(至R.K.)和东北林业大学启动科学基金JQ2017-02(至B.N.)的支持。这个协议是根据Niu em em t t em em一a em em em e l l em em e l em em e l em e l e em em e em em e l e(em)(2017)修改的。作者没有利益冲突或竞争利益声明。

参考

  1. Bai,Y.,Muller,DB,Srinivas,G.,Garrido-Oter,R.,Potthoff,E.,Rott,M.,Dombrowski,N.,Munch,PC,Spaepen,S.,Remus-Emsermann,M 。,Huttel,B.,McHardy,AC,Vorholt,JA和Schulze-Lefert,P.(2015)。 Arabidopsi s leaf的功能重叠和根微生物群。 528(7582):364-369。
  2. Beckers,B.,Op De Beeck,M.,Weyens,N.,Van Acker,R.,Van Montagu,M.,Boerjan,W。和Vangronsveld,J.(2016)。 田间种植的杨树中的木质素工程会影响内层细菌微生物群。 Proc Natl Acad Sci US A 113(8):2312-2317。
  3. Berendsen,R.L.,Pieterse,C.M。和Bakker,P.A。(2012)。 根际微生物组和植物健康。植物科学趋势 17(8):478-486。
  4. Bodenhausen,N.,Bortfeld-Miller,M.,Ackermann,M。和Vorholt,J.A。(2014)。 合成社区方法揭示植物基因型影响phyllosphere微生物群。 PLoS Genet 10(4):e1004283。
  5. Bulgarelli,D.,Rott,M.,Schlaeppi,K.,Ver Loren van Themaat,E.,Ahmadinejad,N.,Assenza,F.,Rauf,P.,Huettel,B.,Reinhardt,R.,Schmelzer, E.,Peplies,J.,Gloeckner,FO,Amann,R.,Eickhorst,T。和Schulze-Lefert,P.(2012)。 揭示 Arabidopsi s 的结构和组装线索>根源性细菌微生物群。 Natu r e 488(7409):91-95。
  6. Cardinale,M.,Grube,M.,Erlacher,A.,Quehenberger,J.和Berg,G。(2015)。 莴苣根微生物群中的细菌网络和共生关系 E nviron Microbiol 17(1):239-252。
  7. de Souza,RS,Okura,VK,Armanhi,JS,Jorrin,B.,Lozano,N.,da Silva,MJ,Gonzalez-Guerrero,M.,de Araujo,LM,Verza,NC,Bagheri,HC,Imperial, J.和Arruda,P。(2016)。 解开甘蔗微生物群落的细菌和真菌群落组合 科学杂志< 6:28774.
  8. Edwards,J.,Johnson,C.,Santos-Medellin,C.,Lurie,E.,Podishetty,N.K。,Bhatnagar,S.,Eisen,J.A。和Sundaresan,V.(2015)。 水稻根系相关微生物组的结构,变异和组装。 Proc Natl Acad Sci USA 112(8):E911-920。
  9. Hinton,D.M。和Bacon,C.W。(1995)。 肠杆菌阴道菌是一种内生共生菌玉米。 Mycopathologi a 129(2):117-125。
  10. Lebeis,SL,Paredes,SH,Lundberg,DS,Breakfield,N.,Gehring,J.,McDonald,M.,Malfatti,S.,Glavina del Rio,T.,Jones,CD,Tringe,SG和Dangl,JL (2015年)。 植物微生物。水杨酸通过特定的细菌分类群调节根微生物群落的定殖。科学 349(6250):860-864。
  11. Niu,B。和Kolter,R.(2017)。 构成玉米根系模型细菌群落的7个菌株的完整基因组序列 Genome Announc 5(36)。
  12. Niu,B.,Paulson,J.N.,Zheng,X。和Kolter,R。(2017)。 简化和具有代表性的玉米根细菌群落 P roc Natl Acad Sci USA 114(12):E2450-E2459。
  13. Ofek-Lalzar,M.,Sela,N.,Goldman-Voronov,M.,Green,S.J.,Hadar,Y。和Minz,D。(2014)。 根表微生物群的利基和宿主相关功能特征。 Nat COMMUN < 5:4950。
  14. Ritpitakphong,U.,Falquet,L.,Vimoltust,A.,Berger,A.,Metraux,J.P.和L'Haridon,F。(2016)。 噬菌体的叶表面的微生物群体 保护免受真菌病原体侵害 新Phytol 210(3):1033-1043。
  15. Sherwood,R.和Hagedorn,D.(1958)。 确定豌豆田的普通根腐病潜力 威斯康星大学农业实验站 ,麦迪逊,WI 。
  16. Vorholt,J.A.,Vogel,C.,Carlstrom,C.I和Muller,D.B。(2017)。 建立因果关系:植物微生物组研究的合成社区机会 Ce < ll Host Microbe 22(2):142-155。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:牛, 犇. and Kolter, R. (2018). Quantification of the Composition Dynamics of a Maize Root-associated Simplified Bacterial Community and Evaluation of Its Biological Control Effect. Bio-protocol 8(12): e2885. DOI: 10.21769/BioProtoc.2885.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。