I am working with pathogenic oomycetes in grasses which are asymptomatic and rarely produce sporangia growing out from the host tissue. The grass samples were fixed in 5% Glutaraldehyde in PBS (pH 7.5) and thus difficult to proceed with oomycete-specific probes for FISH (GA creates a very strong fluorescent background that even my negative controls reacts with the different filters). Apparently, I tried using multiple fluorescent stains and combinations (Aniline Blue, Trypan Blue, and Uvitex2b) although these stains also binds to chitin. The problem is the host plant may have both fungal and oomycete pathogens together. Likewise, it is already sure that my target parasite is present since specific primers were designed (sequences obtained were 99-100% identical and forms a clade with the same species with >95% support by RaxML) and the samples for microscopy where derived from the same batch. I had also considered morphological aspects like the presence and absence of septation, but reports of septation in the hyphae of oomycetes had been widely observed in different genera (Plasmopara, Peronospora, etc). Trying chitin degradation had been an option too, but the procedure is too tedious, likewise the chances of degrading cellulose and glucans of oomycete cell walls are highly possible. Considering my little amount of samples, this risk is also quite the least option for me. Sorry for the long detail, I hope someone can offer some suggestions. Thanks!
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Hi Bismark
You can see this document "Lab Practical study Guide"
https://www.science.oregonstate.edu/bpp/bot350/Lab%20Practical%20Study%20Guide.doc
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You can read this "Reference"
http://apsjournals.apsnet.org/doi/abs/10.1094/PHYTO-96-0212
And "Vital fluorescent stains for detection of stress in Pythium ultimum"
https://onlinelibrary.wiley.com/doi/10.1111/j.1574-6941.1999.tb00631.x/full
Also "F-actin and integrin like proteins in Phytophthora cinnamomi"
https://ir.canterbury.ac.nz/bitstream/handle/10092/1895/thesis_fulltext.pdf?sequence=1
And "Rapid staining method to detect and identify downy mildew"
https://academicworks.cuny.edu/cgi/viewcontent.cgi?article=1012&context=bm_pubs
Also "The rhizosphere: a playground and battlefield for soilborne pathogen"
http://edepot.wur.nl/18591
And "Fungi in lake ecosystems"
https://www.int-res.com/articles/ame_oa/a059p125.pdf
Also "Studies on Saprolegnia ferax suggest the general importance"
https://www.usask.ca/biology/kaminskyj/pub/K%26HmycologiaRev.pdf
Also "oomycete infections in fish - Cabi"
https://www.cabi.org/isc/datasheet/71941
Also "GFP-tagging of cell components reveals the dynamics of subcellular"
https://www.ibt.unam.mx/computo/pdfs/ubiquita/gfptagging.pdf
Also "Pythium insidiosum Keratitis"
http://journals.lww.com/claojournal/Abstract/2011/03000/Pythium_insidiosum_Keratitis_in_Israel.10.aspx
And "Oomycete Genetics and Genomics"
https://elibrary.com.ng/UploadFiles/file0_10095.pdf
ohhh. Thanks a lot. sorry for my delayed response. But these are really helpful. I will check them! Thanks again
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