Dear collegues,
Can we use protocols of DNA extraction from plant tissues (from leaves) to extract DNA of fungal pathogen which lives inside of tissue? If yes, will the quantity of the pathogen's DNA will be sufficient for further amplification with specific primers?
Or we have to use special protocols to extract fungal DNA from plant leaves? In this case please send a link.
The pathogen is Taphrina deformans causing leaf curl disease in Peah tree.
Thanks a lot in advance,
Looking forward to your reply,
Lidia
Maybe you can get problems by solving the chitinwall of the fungal cells.
Do you have an insect-DNA kit?
We dont have insect-DNA kit.
we extracted DNA from peach leaves using standard protocol for plant tissues. Then we tried to make PCR with primers for this pathogen , but we have not seen amplification products on the gel, even in the samples with symptomatic leaves (curly leaves).
try something that destroys the chitin in the cells
Good luck :-)
you can first culture the fungus in agar plates and then extract DNA from the fungi directly. but initially might be difficult if you don't know how your fungus look like in invitro cultures. because many fungus will come in 1 st plate and the have to screen by re culturing several times till end up in your desired fungi mono culture plate.
Good luck
I think that you should firstly isolate the fungus from the leaves ant do the DNA extraction.
Do you have a positive control to ensure that your primers are working under the PCR conditions you are testing?
Is is possible that the concentration of the fungus is too low in the leaves you are testing? You might consider disrupting the leaf tissue and culturing the fungus on plates first. Then extract DNA directly from the fungus for PCR.
Dear collegues,
Many thanks to you,
Yes, now we are working to get pure culture of the fungus to have positive control.
150
Hi Lidia
You can read some references in attachment 1
https://bmcresnotes.biomedcentral.com/articles/10.1186/1756-0500-5-266
2
http://cdn.intechopen.com/pdfs/34844/intech-molecular_tools_for_detection_of_plant_pathogenic_fungi_and_fungicide_resistance.pdf
3
https://onlinelibrary.wiley.com/doi/10.1111/j.1472-765X.2010.02867.x/full
4
https://onlinelibrary.wiley.com/doi/10.1111/j.1472-765X.2010.02867.x/full
5
https://onlinelibrary.wiley.com/doi/10.1111/j.1472-765X.2010.02867.x/full
7
https://www.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132010000200001
8
https://www.academicjournals.org/journal/AJB/article-full-text-pdf/956CF8231201
10
http://www.nbpgr.ernet.in/Portals/6/DMX/GENOMIC_RESOURCES/DNA%20extraction-Comparison%20of%20methodologies.pdf
12
https://pubag.nal.usda.gov/pubag/downloadPDF.xhtml?id=20257&content=PDF
13
http://www.sigmaaldrich.com/technical-documents/articles/biology/isolation-of-plant-and-fungal-rna.html
14
https://www.qiagen.com/us/shop/sample-technologies/dna/genomic-dna/dneasy-plant-mini-kit/
19
https://www.biotechniques.com/multimedia/archive/00010/97223rr01_10666a.pdf
Dear Lidia Samarina, did you find any protocol for fungal DNA isolation avoiding too much plant DNA? Thanks
Hi Lidia Samarina , have you found the way to extract fungal DNA from plant tissues? I hope you can kindly share your findings.
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