Wheat root exudate harvesting - Revision history

Louisa perry jic at 17:15, 15 February 2022

2022-02-15T17:15:23Z

← Older revision		Revision as of 17:15, 15 February 2022
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	Plant roots are nutrient rich environments for soil microbes which form root-associated microbial communities. These communities make up the root microbiome consisting of the endosphere (the space inside of the root), the rhizosphere (the soil that is directly influenced by the root) and the bulk soil (any soil exterior to the root rhizosphere).		Plant roots are nutrient rich environments for soil microbes which form root-associated microbial communities. These communities make up the root microbiome consisting of the endosphere (the space inside of the root), the rhizosphere (the soil that is directly influenced by the root) and the bulk soil (any soil exterior to the root rhizosphere).

	16S rRNA gene sequencing has shown us that there is lower bacterial species diversity in the endosphere of roots compared to the rhizosphere and bulk soil (Lundberg et al, 2012; Prudence et al, 2012). This demonstrates that some bacteria have developed adaptations that allows them to acquire this niche; it is hypothesised that this is either through outcompeting other bacteria or a mechanism of protrusion. Some initial studies have found that plants grown side by side in the same environment recruit different microbes which suggest that plants can select for specific microbes which benefit them (Reinhold-Hurek et al., 2015). One factor which is being explored is the role of plant exudates on this selection.		16S rRNA gene sequencing has shown us that there is lower bacterial species diversity in the endosphere of roots compared to the rhizosphere and bulk soil (Lundberg et al, 2012; Prudence et al, 2012). This demonstrates that some bacteria have developed adaptations that allows them to acquire this niche; it is hypothesised that this is either through outcompeting other bacteria or a mechanism of protrusion. Some initial studies have found that plants grown side by side in the same environment recruit different microbes which suggest that plants can select for specific microbes which benefit them (Smalla, et al. 2001; Reinhold-Hurek et al., 2015). One factor which is being explored is the role of plant exudates on this selection.

	Exudates are metabolites excreted by plants into the rhizosphere which allows plants to support healthy growth, for example to break down nutrients in a more readily accessible form and to signal PGP soil microbes (Vives-Peris, 2019). This protocol outlines how to extract root exudates.		Exudates are metabolites excreted by plants into the rhizosphere which allows plants to support healthy growth, for example to break down nutrients in a more readily accessible form and to signal PGP soil microbes (Vives-Peris, 2019). This protocol outlines how to extract root exudates.
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	* Electric hand drill		* Electric hand drill
	* 10mm drill bit		* 10mm drill bit
	* 2ml ~~microcentifuge~~ tubes		* 2ml microcentrifuge tubes
	* Dog nail cutters		* Dog nail cutters
	* Vermiculite (washed and autoclaved)		* Vermiculite (washed and autoclaved)
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	# Attach a sterile 0.22 micron Büchner filter to another sterile Duran bottle and fix to a vacuum pump.		# Attach a sterile 0.22 micron Büchner filter to another sterile Duran bottle and fix to a vacuum pump.
	# Pour the exudate solution into the Büchner filter and turn on the vacuum pump.		# Pour the exudate solution into the Büchner filter and turn on the vacuum pump.
	# ~~Seperate~~ the filtered exudate solution into clean 50ml falcon tubes (about 25-30ml each) and freeze at -80°C.		# Separate the filtered exudate solution into clean 50ml falcon tubes (about 25-30ml each) and freeze at -80°C.

	<h2>Week 5, day 5</h2>		<h2>Week 5, day 5</h2>
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	Reinhold-Hurek, B., Bünger, W., Burbano, C., Sabale, M. and Hurek, T., 2015. Roots Shaping Their Microbiome: Global Hotspots for Microbial Activity. Annual Review of Phytopathology, 53(1), pp.403-424.		Reinhold-Hurek, B., Bünger, W., Burbano, C., Sabale, M. and Hurek, T., 2015. Roots Shaping Their Microbiome: Global Hotspots for Microbial Activity. Annual Review of Phytopathology, 53(1), pp.403-424.

			References
			Lundberg, D., Lebeis, S., Paredes, S., Yourstone, S., Gehring, J., Malfatti, S., Tremblay, J., Engelbrektson, A., Kunin, V., Rio, T., Edgar, R., Eickhorst, T., Ley, R., Hugenholtz, P., Tringe, S. and Dangl, J., 2012. Defining the core Arabidopsis thaliana root microbiome. Nature, 488(7409), pp.86-90.

			Prudence, S., Newitt†, J., Worsley, S., Macey, M., Murrell, J., Lehtovirta-Morley, L. and Hutchings, M., 2021. Soil, senescence and exudate utilisation: characterisation of the Paragon var. spring bread wheat root microbiome. Environmental Microbiome, 16(1).

			Reinhold-Hurek, B., Bünger, W., Burbano, C., Sabale, M. and Hurek, T., 2015. Roots Shaping Their Microbiome: Global Hotspots for Microbial Activity. Annual Review of Phytopathology, 53(1), pp.403-424.

			Smalla, K., Wieland, G., Buchner, A., Zock, A., Parzy, J., Kaiser, S., Roskot, N., Heuer, H. and Berg, G., 2001. Bulk and Rhizosphere Soil Bacterial Communities Studied by Denaturing Gradient Gel Electrophoresis: Plant-Dependent Enrichment and Seasonal Shifts Revealed. Applied and Environmental Microbiology, 67(10), pp.4742-4751.

	Vives-Peris, V., de Ollas, C., Gómez-Cadenas, A. and Pérez-Clemente, R., 2019. Root exudates: from plant to rhizosphere and beyond. Plant Cell Reports, 39(1), pp.3-17.		Vives-Peris, V., de Ollas, C., Gómez-Cadenas, A. and Pérez-Clemente, R., 2019. Root exudates: from plant to rhizosphere and beyond. Plant Cell Reports, 39(1), pp.3-17.

Louisa perry jic at 17:06, 15 February 2022

2022-02-15T17:06:24Z

Louisa perry jic at 16:55, 15 February 2022

2022-02-15T16:55:37Z

← Older revision		Revision as of 16:55, 15 February 2022
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	# Dry down your sample again (on a freeze drier or alternatively a Genevac) and weigh.		# Dry down your sample again (on a freeze drier or alternatively a Genevac) and weigh.
	# Your sample can be resuspended at a known concentration. It is now ready for use.		# Your sample can be resuspended at a known concentration. It is now ready for use.

			<h1>References:</h1>
			Lundberg, D., Lebeis, S., Paredes, S., Yourstone, S., Gehring, J., Malfatti, S., Tremblay, J., Engelbrektson, A., Kunin, V., Rio, T., Edgar, R., Eickhorst, T., Ley, R., Hugenholtz, P., Tringe, S. and Dangl, J., 2012. Defining the core Arabidopsis thaliana root microbiome. Nature, 488(7409), pp.86-90.

			Prudence, S., Newitt†, J., Worsley, S., Macey, M., Murrell, J., Lehtovirta-Morley, L. and Hutchings, M., 2021. Soil, senescence and exudate utilisation: characterisation of the Paragon var. spring bread wheat root microbiome. Environmental Microbiome, 16(1).

			Reinhold-Hurek, B., Bünger, W., Burbano, C., Sabale, M. and Hurek, T., 2015. Roots Shaping Their Microbiome: Global Hotspots for Microbial Activity. Annual Review of Phytopathology, 53(1), pp.403-424.

			Vives-Peris, V., de Ollas, C., Gómez-Cadenas, A. and Pérez-Clemente, R., 2019. Root exudates: from plant to rhizosphere and beyond. Plant Cell Reports, 39(1), pp.3-17.

Louisa perry jic at 16:49, 15 February 2022

2022-02-15T16:49:24Z

← Older revision		Revision as of 16:49, 15 February 2022
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	Exudates are metabolites excreted by plants into the rhizosphere which allows plants to support healthy growth, for example to break down nutrients in a more readily accessible form and to signal PGP soil microbes (Vives-Peris, 2019).		Exudates are metabolites excreted by plants into the rhizosphere which allows plants to support healthy growth, for example to break down nutrients in a more readily accessible form and to signal PGP soil microbes (Vives-Peris, 2019).


	<h1>You will need:</h1>		<h1>You will need:</h1>

Louisa perry jic at 16:48, 15 February 2022

2022-02-15T16:48:19Z

← Older revision		Revision as of 16:48, 15 February 2022
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	<h1>Wheat root exudates and the recruitment of root microbes</h1>		<h1>Wheat root exudates and the recruitment of root microbes</h1>
	...		Plant roots are nutrient rich environments for soil microbes which form root-associated microbial communities. These communities make up the root microbiome consisting of the endosphere (the space inside of the root), the rhizosphere (the soil that is directly influenced by the root) and the bulk soil (any soil exterior to the root rhizosphere).

			16S rRNA gene sequencing has shown us that there is lower bacterial species diversity in the endosphere of roots compared to the rhizosphere and bulk soil (Lundberg et al, 2012; Prudence et al, 2012). This demonstrates that some bacteria have developed adaptations that allows them to acquire this niche; it is hypothesised that this is either through outcompeting other bacteria or a mechanism of protrusion. Some initial studies have found that plants grown side by side in the same environment recruit different microbes which suggest that plants can select for different microbes (Reinhold-Hurek et al., 2015). One factor which is being explored is the role of plant exudates on this selection.

			Exudates are metabolites excreted by plants into the rhizosphere which allows plants to support healthy growth, for example to break down nutrients in a more readily accessible form and to signal PGP soil microbes (Vives-Peris, 2019).


	<h1>You will need:</h1>		<h1>You will need:</h1>

Louisa perry jic at 14:33, 23 November 2021

2021-11-23T14:33:46Z

← Older revision		Revision as of 14:33, 23 November 2021
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	<h1>You will need:</h1>		<h1>You will need:</h1>
	Preparation of growth tubes		Preparation of growth tubes
	* 50ml falcon/centrifuge tubes		* 50ml falcon/centrifuge tubes (1 tube = 1 plant)
	* Electric hand drill		* Electric hand drill
	* 10mm drill bit		* 10mm drill bit

Louisa perry jic at 14:30, 23 November 2021

2021-11-23T14:30:26Z

← Older revision		Revision as of 14:30, 23 November 2021
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	# Pour away the 1/2 MS0 and rinse the roots and tube with water (to remove any remaining salts).		# Pour away the 1/2 MS0 and rinse the roots and tube with water (to remove any remaining salts).
	# Fill the growth tubes with ~45ml fresh water (this will be the media which the exudates will be extracted from).		# Fill the growth tubes with ~45ml fresh water (this will be the media which the exudates will be extracted from).
	# Keep the ~~grow~~ tubes in a controlled environment room.		# Keep the growth tubes in a controlled environment room.

	<h2>Week 5</h2>		<h2>Week 5</h2>

Louisa perry jic at 14:29, 23 November 2021

2021-11-23T14:29:23Z

← Older revision		Revision as of 14:29, 23 November 2021
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	# Fill the falcon tubes with ~45ml 1/2 MS0 media.		# Fill the falcon tubes with ~45ml 1/2 MS0 media.
	# When the seeds have sprouted two roots of ~0.5-1cm long, transfer the seed to the vermiculite tube so that the roots are pointing downwards into the vermiculite.		# When the seeds have sprouted two roots of ~0.5-1cm long, transfer the seed to the vermiculite tube so that the roots are pointing downwards into the vermiculite.
	# Keep the ~~grow~~ tubes in a controlled environment room (...)		# Keep the growth tubes in a controlled environment room (...)

	<h2>Week 2-4</h2>		<h2>Week 2-4</h2>

Louisa perry jic at 12:36, 29 October 2021

2021-10-29T12:36:34Z

← Older revision		Revision as of 12:36, 29 October 2021
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	{{DISPLAYTITLE: ~~Harvesting~~ root ~~exudates~~}}		{{DISPLAYTITLE: Wheat root exudate harvesting}}

	<h1>Wheat root exudates and the recruitment of root microbes</h1>		<h1>Wheat root exudates and the recruitment of root microbes</h1>

Louisa perry jic at 12:35, 29 October 2021

2021-10-29T12:35:34Z

← Older revision		Revision as of 12:35, 29 October 2021
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	{{DISPLAYTITLE: ~~Wheat~~ root ~~exudate harvesting~~}}		{{DISPLAYTITLE: Harvesting root exudates}}

	<h1>Wheat root exudates and the recruitment of root microbes</h1>		<h1>Wheat root exudates and the recruitment of root microbes</h1>

← Older revision		Revision as of 17:06, 15 February 2022
Line 4:		Line 4:
	Plant roots are nutrient rich environments for soil microbes which form root-associated microbial communities. These communities make up the root microbiome consisting of the endosphere (the space inside of the root), the rhizosphere (the soil that is directly influenced by the root) and the bulk soil (any soil exterior to the root rhizosphere).		Plant roots are nutrient rich environments for soil microbes which form root-associated microbial communities. These communities make up the root microbiome consisting of the endosphere (the space inside of the root), the rhizosphere (the soil that is directly influenced by the root) and the bulk soil (any soil exterior to the root rhizosphere).

	16S rRNA gene sequencing has shown us that there is lower bacterial species diversity in the endosphere of roots compared to the rhizosphere and bulk soil (Lundberg et al, 2012; Prudence et al, 2012). This demonstrates that some bacteria have developed adaptations that allows them to acquire this niche; it is hypothesised that this is either through outcompeting other bacteria or a mechanism of protrusion. Some initial studies have found that plants grown side by side in the same environment recruit different microbes which suggest that plants can select for ~~different~~ microbes (Reinhold-Hurek et al., 2015). One factor which is being explored is the role of plant exudates on this selection.		16S rRNA gene sequencing has shown us that there is lower bacterial species diversity in the endosphere of roots compared to the rhizosphere and bulk soil (Lundberg et al, 2012; Prudence et al, 2012). This demonstrates that some bacteria have developed adaptations that allows them to acquire this niche; it is hypothesised that this is either through outcompeting other bacteria or a mechanism of protrusion. Some initial studies have found that plants grown side by side in the same environment recruit different microbes which suggest that plants can select for specific microbes which benefit them (Reinhold-Hurek et al., 2015). One factor which is being explored is the role of plant exudates on this selection.

	Exudates are metabolites excreted by plants into the rhizosphere which allows plants to support healthy growth, for example to break down nutrients in a more readily accessible form and to signal PGP soil microbes (Vives-Peris, 2019).		Exudates are metabolites excreted by plants into the rhizosphere which allows plants to support healthy growth, for example to break down nutrients in a more readily accessible form and to signal PGP soil microbes (Vives-Peris, 2019). This protocol outlines how to extract root exudates.

	<h1>You will need:</h1>		<h1>You will need:</h1>
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	<h4>Preparation of growth tubes</h4>		<h4>Preparation of growth tubes</h4>
	# Prepare as many tubes as plants required (1 batch of 50 tubes ~ ~~...~~ mg final exudate).		# Prepare as many tubes as plants required (1 batch of 50 tubes ~ 500 mg final exudate).
	# Drill a 10mm hole in each tube and rinse with water to remove any plastic debris.		# Drill a 10mm hole in each tube and rinse with water to remove any plastic debris.
	# Using the dog nail cutters, cut the end of a 2ml microcentrifuge tube to create a ~0.5cm opening. Loosely fill the tube with the prepared vermiculite and close the lid.		# Using the dog nail cutters, cut the end of a 2ml microcentrifuge tube to create a ~0.5cm opening. Loosely fill the tube with the prepared vermiculite and close the lid.
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	# Fill the falcon tubes with ~45ml 1/2 MS0 media.		# Fill the falcon tubes with ~45ml 1/2 MS0 media.
	# When the seeds have sprouted two roots of ~0.5-1cm long, transfer the seed to the vermiculite tube so that the roots are pointing downwards into the vermiculite.		# When the seeds have sprouted two roots of ~0.5-1cm long, transfer the seed to the vermiculite tube so that the roots are pointing downwards into the vermiculite.
	# Keep the growth tubes in a controlled environment room (~~...~~)		# Keep the growth tubes in a controlled environment room (temp 24C, light 18h)

	<h2>Week 2-4</h2>		<h2>Week 2-4</h2>