Surface sterilization of wheat plants - (Nov/27/2013 )
Well im doing a small study, to determine the efficiency of two different methods of surface sterillization methods on wheat plants, treated briefly with a M. anispoliae spore suspension. I so far found, that the methods i have been testing are insufficient at removing surface contaminats (or atleast all the spores from the suspension). Method 1 is subermerging the plantparts for 2 minutes in 70% ethanol, then 2-3 % sodium hypochlorite, then three waterbaths oh autoclaved ionized water. Method two is quite similar, but with significantly shorter times in each step (aproxematley 10-15 seconds), but with a 5-7,5 % sodiumhypochlorite concentration (this has been used for insect surface sterillization in the past). As i said, non of them seem to work, and thus im testing some tweaks to these methods. The first thing i tried, is adding 0,05% tween 20 to the hypochlorite, but i observed something peculiar. Ordinarily, when i submerge the plant parts into the hypochlorite, the part skipps across the liquids' surface, but when i submerge them in the dilution added tween, t does nothing. Also, the dilution has a strange, cloudy aperance. Im still waiting for the results of this test (incubating the sterillized plants on selective media, so it'll take a few weeks before i have anything conlcusive). with that said, here's my question. Do you think that the adittion of tween somehow ruins the hypochlorite?
Additional info:
I made the sodium hypochlorite dilutions twelve hours in advance (with the tween)
The goal of the study is to determine wheter the methods are sufficient at removing known surface contaminants (M. anisopliae)
I still would like someones' opionion on this problem... anyone?
Never worked with Metarhizium and its spores but aren't they quite susceptible to UV light?
Did you try different detergents and concentrations? i.e. as low concentration of tween as possible, or using SDS if the plant surface is not damaged by this treatment (and SDS offers some additional microbicidal potential too)
Well yeah, They are susceptible to UV, but in my experience, UV doesn't penetrate tissue that deeply, neither is it that reliable. What i have done so far (and im still waiting for the results of this) is adding 0,05 %tween to the chlorite, but so far, it doesn't realy seem to be significantly better... can't say for sure till the incubation is done though. I recently tried adding motion to the process, by doing the sterillisation steps (ethanol and hypochlorite) on magnetic stirers. The idea is to force liquid into grooves on the plant, and thus increase likelihood of succesfull sterillization. I tested the magnetic stirring on some plantmaterial first, to determine wheter the treament would be too rough, but it doesn't seem to cause any noticable injury to the plant (at least at 10 X magnifying it doesn't) I just recieved some PPM (plant Preservative mixture) from england. It interupts the krebs cycle in most single cell organisms, and thus works as a cheap alternative to broadspectrum antibiotics. The plan is to add a third sterillization step to the original methods, where the planttissue is submerged in a 4% solution of PPM. I have high hopes for this particular assay. Even so, im still open for sugestions, so please keep them comming
(sidenote, M. anisopliae is also know as M. bruneum... im just using it's old name out of habbit)
I wonder how many spores are needed for a successful infection? If it's only one you need of course a 100% clean surface, but if not, then perhaps a lower efficiency might be okay?
Another ideas would be:
Using a different wheat cultivar with a smoother leaf surface, to reduce adherence.
Using a kind of artificial insect surface, e.g. a chitin suspension to induce germination and therefore increase the vulnerability of the fungus. But this might need a longer incubation time then.
pH value of the solutions might have an influence on efficiency of the sterillization.
Well the ultimate goal of my particular study, is to use my findings in a paralel study, where they are examining M. anisopliae as an endophyte in specific plants. Thus, the plant that i use can't be changed. It is hoped, that i would be able to find a method with high enough efficiency to statistically determine wheter the metarhizium stems from residue spores or mycelia stuck to the surface of the host plant, or from within the plant tissue itself. Which again means, that the method im testing can't be too harsh either, since it is suposed to leave endophythes alive... or atleast some of the endophytes. I tried with prolonge suspension in higher concetrations of hypochlorite, but found (i kinda foresaw that) that the plant almost whitered away, and became pale and sligthly brittle.
okay what about a usual fungicide then which is not working systemically? There should be some.
Actually I've no idea about the pathway of the spores into the plant.
What spore concentration are you using? Maybe you are adding too many spores...
We used this on kiwifruit sprouts for tissue culture during some routine practices in the lab: 70% etOH 1 min, followed by sterile distilled water wash. Then 0,4% HClO plus a couple of drops of Tween 20 on the bench for 15 min (don't remember well this time) and then wash again with sterile distilled water in the laminar flow cabinet.
In case a sample would be too contaminated have you thought about about surfactant washes to remove excess spores on your samples. That could work not as sterilization step but as removal of microbes/spores or dirt that may have associated bugs on the surface, before trying to kill those that still are on the surface.
Also, ultrasound baths may be useful to increase the sterilising agent to the sample crevices and they aren't too aggresive on cells unless you leave them for very long times
Ultrasound seems like a good idea. The concentration of the sporesuspension i use, is aproxematley 107 / mL... i treat plants by submerging them in the suspension for 5 and 60 minutes (two different aproches). As for the fungicide, im currently awaiting the results of an assys, where i added a third step, containing plant preservative mixture, a biocide with documented effect on a broad spectrum of microbes. I have high hopes for that particular assay