View source for Isolating Streptomyces from plant roots

{{DISPLAYTITLE: Isolating ''Streptomyces'' from plant roots}}

<h2><strong>Sampling of plant roots for isolation of ''Streptomyces'' and other microorganisms from the plant rhizosphere and endosphere</h2></strong>

This method can be used to sample plant roots for total or selective bacterial isolation, as well as nucleic extraction for various experiments.

Depending on soil type and number of plants being sampled, this protocol can take up to a full day.

<h2><strong>Preparation and reagents needed</h2></strong>
*Phosphate buffer saline (PBS)
**6.33g NaH<sub>2</sub>PO<sub>4</sub>.H<sub>2</sub>O
**16.5g Na<sub>2</sub>HPO<sub>4</sub>.H<sub>2</sub>O
**1L dH<sub>2</sub>O
**Autoclave then add 200µl Silwett L-77
*Ethanol 100%
*Ethanol 70%
*50ml falcon tubes
*Sterile petri dishes
*Sterile micro centrifuge tubes (MCT)
*Sterile 10% glycerol
*Tweezers
*Sonicating water bath
*Disposable gloves

<h2><strong>Root sampling protocol</h2></strong>
#Set out foil and wipe down with 70% Ethanol
#Take plants from pots: shake off large soil particles in a gloved hand (make sure gloves have been disinfected with ethanol), cut off the stems aseptically.
#Put sampled roots into a 50ml tube containing 25ml of sterile PBS buffer. Wash on a shaker (30mins 180rpm) to remove particles. Alternatively, vortex vigorously for 20 seconds.
#Transfer the roots to a fresh tube of PBS, repeat step 3 (wash or vortex). 
#If sampling rhizosphere, take the previous tube and centrifuge soil/PBS mixture at 3200g for 15 minutes to form a pellet. The resulting pellet can be resuspended in a smaller volume of PBS and diluted for spread plating, if the end goal is to isolate live bacteria. Alternatively, if the sampling is for nucleic acid isolation, snap freeze pellet in liquid nitrogen.
#Repeat steps 3 and 4 with the root samples until no further particles are removed by the washing.
#If there are still soil particles attached to the root, transfer to a sterile petri dish and remove as much soil debris as possible with ethanol flame sterilised tweezers.
#Transfer to a new falcon with PBS (25ml) and sonicate for 15-20 minutes in a sonicating water bath.
#If you are sampling from ''Arabidopsis'' roots, continue onto step 10 or 11. If you are sampling from thicker roots like ''Triticum aestivum'', see root sterilisation section below before continuing.
#For culturing endophytic bacteria: crush the roots in 1-2ml (depending on root size) in sterile 1G with a sterile pestle and mortar, transfer to an MCT. Make serial dilutions (0,-1,-2) and plate out onto replicate agar plates. It is possible to partially select for filamentous actinomycetes by plating on [[SFM]] with nystatin, cyclohexamide and naladixic acid.
#For DNA extraction from endophytic community, remove roots from PBS and separate into 2 samples for each plant. Snap freeze in liquid nitrogen and crush with a sterile mortar and pestle. Store at -80&deg; until sample is extracted. Use MPBio fast spin kit for soil to extract from crushed roots.

<h2><strong>Wheat root sterilisation protocol</h2></strong>
#For sterilisation of wheat (''Triticum aestivum''), transfer the PBS washed roots into a falcon containing 10-20ml 70% ethonol and incubate at room temperature for 30 seconds.
#Transfer the roots to a fresh falcon tube containing 10-20ml 3% bleach solution and incubate at room temperature for 10 minutes.
#Pour off the bleach and wash with 10-20ml sterile dH<sub>2</sub>O at least 5 times. Continue to wash until the smell of bleach mostly dissipates.
#Continue with steps 10 or 11 of root sampling protocol above.

<h2><strong>Uses</h2></strong>
*[[Nucleic acid isolation from wheat roots]] for microbial community analysis
*General isolation of novel root associated microorganisms
*[[Root colonization assay – colony forming unit (CFU) count method]]

<h2><strong>References</h2></strong>

Root sampling protocol adapted from:-
*Lebeis, S.L., Paredes, S.H., Lundberg, D.S., Breakfield, N., Gehring, J., McDonald, M., Malfatti, S., Del Rio, T.G., Jones, C.D., Tringe, S.G. and Dangl, J.L., 2015. Salicylic acid modulates colonization of the root microbiome by specific bacterial taxa. Science, 349(6250), pp.860-864.
*Lundberg, D.S., Lebeis, S.L., Paredes, S.H., Yourstone, S., Gehring, J., Malfatti, S., Tremblay, J., Engelbrektson, A., Kunin, V., Del Rio, T.G. and Edgar, R.C., 2012. Defining the core Arabidopsis thaliana root microbiome. Nature, 488(7409), p.86.
*Bulgarelli, D., Rott, M., Schlaeppi, K., van Themaat, E.V.L., Ahmadinejad, N., Assenza, F., Rauf, P., Huettel, B., Reinhardt, R., Schmelzer, E. and Peplies, J., 2012. Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota. Nature, 488(7409), p.91.
Wheat root sterilisation adapted from:-
*Larran, S., Perello, A., Simon, M.R. and Moreno, V., 2002. Isolation and analysis of endophytic microorganisms in wheat (Triticum aestivum L.) leaves. World Journal of Microbiology and Biotechnology, 18(7), pp.683-686.

Protocol adapted for ActinoBase by Jake T. Newitt from the University of East Anglia.