Hello, we are currently using an enzyme solution containing macerozyme R10: 0.2g/l, cellulase R10 : 1g/l, driselase: 0.5g/l, and we are trying to digest roots to remove microbes, which will then be cultured. The solution is able to digest leaf material without any issue, but has no effect on roots. We have used several species of plant roots (leek, maize, wheat, etc.), tried adjusting the pH between 5 and 7, tried both a liquid media buffer and a phosphate buffer, tried temperatures that range from 20C to 55C, tried very small quantities of roots, and tried vacuum infiltration, but unfortunately, nothing seems to work.
The method is based on Strullu and Romand (1987), and we have tried it word for word and with the adaptations noted above.
Could anyone provide some advice? It was suggested to us that there may be exudates in the roots that are preventing the enzymes from working, and perhaps adding PVPP could help, but I thought it best to see if anyone has specific experience using enzymes to digest roots.
One key aspect is that we are looking to culture microbes from the resulting solution after digestion, so we can't use any steps that would kill them.
Many thanks in advance - any thoughts are much appreciated!
Why do you have to do "complete" digestion for obtaining endophytic microbes ? Cant´t you culativate those, that are obtained after mazeration and partly digestion ?
I think i don't know the actual answer of your question but i think you can choose alternate options to culture root endophytes by properly surface sterilizing the root (use 0.5% NaClO for 2minutes followed by dipping them in 20% Tween 80 solution then wash with 98% ethanol solution and finally rinse them with sterilized RO water) and then crushing them in normal saline then spread 0.1ml of crushed roots mixed with normal saline to priorly sterilized media plates then incubate them under required temprature based on the type of microbes you are targeting to isolate and also the media you are using. Hope this may help you.
Thanks for the answers. We would like to use enzyme digestion instead of maceration and surface sterilisation, as we would like to isolate individual fungal structures, whereas even a small section of the roots contain many, and are a community (not pure). Thanks again.
Dear Peter, I do not know if this is of any help, but some few hints maybe:
1- I would do a (very) soft surface sterilization, in order to get rid of ectophytes contamination during process when keeping focus on endophytes.
2- Maybe you need to consider to add plant extract in your culture medium: Article Optimization of isolation and cultivation of bacterial endop...
3- Did you see also this (I am aware it is OMF; but there are probably some ideas to catch there): Article Isolation and Identification of Endophytic Bacteria from Myc...
4- Dr Paola Bonfante & team is one of the rare research group working on bacterial endosymbiont in endomycorrhizal fungi, it might be they have some advices.
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