I am trying to extract tightly attached microorganism from apple tree roots (rhizoplane and endorhizosphere) without grinding the roots in liquid N2 due to the size of roots (larger than 1cm in diameter). Afterwards I am extracting the DNA of the entire sample and want to avoid having large amounts of plant DNA (16s amplification).
I have quite a large sample number and thus was hoping to avoid cutting the outer parts of the root off and then grind it. I was thinking to maybe use beads?
Any ideas?
Thanks in advance.
Max
Have you considered sonicating the whole root slice in a suitable. Buffer. The sonication should hopefully dislodge tightly attached bacteria without disrupting the hardy root tissue
Not a plant biologist so please excuse if answer seems tOo simplistic
Thanks for the reply. Yes I have thought about and will try it out. Only thing I am not sure about is whether I will get the mycorrhiza (which I want to include in my studies) out of the roots as well.
Flores-Vargas, R.D. and O'Hara, G.W. (2006) Isolation and characterization of rhizosphere bacteria with potential for biological control of weeds in vineyards. Journal of Applied Microbiology, 100 (5). pp. 946-954.
Aims: Deleterious rhizosphere inhabiting bacteria (DRB) have potential to suppress plant growth. This project focuses on the isolation of DRB with potential for development as commercial products for weed control.
Methods and Results: Bacteria were isolated from the rhizosphere, rhizoplane, and endorhizosphere of seedlings and mature plants of wild radish (Raphanus raphanistrum), annual ryegrass (Lolium rigidum) and capeweed (Arctotheca calendula) growing in vineyards in the Swan Valley, Western Australia. A majority (81·5%) of the 442 strains was obtained from either rhizospheres or rhizoplanes. Rapid screening techniques were developed to evaluate in the laboratory and glasshouse the effects of bacteria on plants. Strains were screened in the glasshouse for deleterious effects on annual ryegrass, wild radish, grapevine rootlings (Vitis vinifera) and the legume cover crop subterranean clover (Trifolium subterraneum). Three strains were identified using the Biolog system and 16S rRNA gene sequencing as two strains of Pseudomonas fluorescens (WSM3455 and WSM3456) and one strain of Alcaligenes xylosoxidans (WSM3457). One of the P. fluorescens (WSM3455) strain produced hydrogen cyanide, an inhibitor of plant roots and a broad-spectrum antimicrobial compound.
If you were planning to do culture independent methods to analyse the microbiome composition such as DGGE or metabarcoding you may use 799F primer that can avoid plastid amplification and use 1393R that amplify V5-V7 region of 16S rRNA gene
refer to this paper
Article Bodenhausen N, Horton MW, Bergelson J.. Bacterial communitie...
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