B1 Sequenzierung

Begonnen von Ahriman, 25.Feb.09 um 21:20 Uhr

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Ahriman

Hello!

Unfortunately our first attempt to identify B1 totally failed due to PCR problems.

I will receive new cultures from Claus soon, then we'll see if we can solve the mistery.

Regards,
Christian

fred

Probably a useless question, but ...

Is it possible that our B1 culture isn't pure ? I.e. it contains Tulasnella + Ceratobasidium ?

These are the reasons I'm asking:

  • - when starting a new culture on a strong medium (20g/l and more), I always see a (sparse) fluffy white ring growing out of the innoculation. This only lasts a few days and then collapses into the appressed culture we know
  • - This is what's really bugging me: what's the chance that I accidentally contaminated the culture with Ceratobasidium, a mycorrhizal genus ? In a microelectronics clean room ?
  • - There is no data on the isolation of B1 apart that it was done from Dactylorhiza fuchsii roots by someone in the HOS. Ceratobasidium can also be found in roots of this species.

My "theory": B1 = Tulasnella (germinator) + Ceratobasidium (not a germinator)

Claus

Fred,
that's possible. I got a fungus, let's call it D1, which contains at minimum three individual species, a mould, a symbiotic fungus and a third one with only hyphae in the air. Despite there is a mould in the mixture it germinates quite a lot of orchid seeds.

Besides my trials to grow asymbiotic cultures I try to separate these three individuals. Maybe that I was already successful with the air hyphae, because I picked one very tiny part directly from the air, and the new culture is now, only four days later, already again "in the air". Maybe I also separated the symbiotic part, as it is appressed to the surface, and I got a part from that area short after infecting the agar.

I also know the fluffy structure form the B1 when the concentration of oat exceeds about 10 g/l, but I thought this occurs when the fungus becomes parasitic.

Well, what to do? There is a possibility to pick up very tiny parts of the agar from the front of the hyphae short after infection (about one day). But when two individuals run with the same speed the a separation is not possible.

Do you have other ideas?

Greetings Claus
Wer Chemiker werden will, muss Chemie studieren; wer Jurist oder Arzt werden will, muss Jura oder Medizin studieren. Aber um Politiker zu werden, ist lediglich das Studium der eigenen Interessen notwendig. (Max O'Rell)

winwen

The thought of B1 consisting of two fungi is quite an interesting theory, Claus, BUT even if this is the case, fred should at least have been able to germinate Anacamptis morio. Instead of that he got a total failure in his trial-germinations. If the fungus, that fred received is composed of Tulasnella AND Ceratobasidium, he should have had success in germinating some of the mentioned species. Therefore this is currently not an acceptable theory for me (from a logical point of view) and we should first try to find the answer why there were no germinations.
Fred, what kind of medium did you use for germinating the mentioned species with the B1?

Kind regards
Erwin

fred

Winwen,

It's not me who is having problems germinating these orchids, it's Michele who wanted to do the identification/sequencing. My B1 comes from Roman, I took a sample from which my cultures are grown and I sent the vial to Michele. He isolated a fungus, sequenced it and got Ceratobasidium. He tested it as a germinator and it failed. That's the problem: my sample works, he isolated something which doesn't work. Sorry for the confusion.

Claus,

very interesting info you've given. Now you mention air hyphae, my old cultures have hyphae which run to the top of the jar and don't form these coils/clusters like at the bottom - I think the fast aerial one is Ceratobasidium. I've just taken a sample from these "aerial" hyphae and I'm interested to see what'll come out. It's in a 30g/l OMA so we'll know this weekend. I also have jars with other media (rye agar, with and without dextrins) so maybe it's possible to provide a condition where the Tulasnella grows faster than the other and isolate it.

Ahriman

Hello!

Just some more thoughts:
Usually if you try to sequence a mixture of fungi with universal primers you get some unreadable mess in the chromatogram.
Since Michele obviously got some results (using which PCR primers?) this would be a point against the mixture-hypothesis.
However, fungal standard-primers often don't work well with Tulasnellales so it may happen that you don't find them even if they are there and detect some contamination instead.

If somenthing similar happens in our 2nd attempt I'll use specific primers for Tulasnellales and Ceratobasidiales, this should shed some light oh this issue.

Claus:
In order to seperate such mixtures I'd try to grow them on stress-inducing media, varying pH, salinity and content of essential nutrients.
If you can create conditions that one component can tolerate but not the others you should be successful.

Anyway I find it astonishing that such associations remain stable. Usualy I would expect fungi to compete with or even attack each other if conditions are less favorable.
Unless they have formed some symbiotic relationship among themselves?

Regards,
Christian

fred

Hi Christian,

could you shed some light on how exactly you process the culture for sequencing? If I understand correctly they're grown in a floating culture, then washed and processed for sequencing. But when you receive a culture on agar, do you first isolate single hyphae under the microscope or do you cut out a piece of the agar and assume it's pure? I've sent a link of this discussion to Michele, maybe he will join and answer some questions.

Claus,

In addition to the tips given by Christian, some literature reports that different fungi have a different optimum growing speed in relation to the temperature.  Example (Perfect states of Rhizoctonias associated with orchids, Warcup & Talbot 1967): Ceratobasidium obscurum grows optimal at 15-20°C, C. cornigerum at 25°C. It's a simple variable to experiment with.

As a last question, would anybody be interested in running the same experiment as I am for verification? I took a sample from the B1 hyphae growing near the top of a jar (near the lid) in an old culture and put it on 30g/l OMA. Even if I get something different than "normal" B1 it could still be caused by contamination from me. If it's as simple as I think it is we could solve this puzzle quite quickly.

regards,
Fred

Claus

Hi Fred and Christian,
separating different species of fungus seems to be not my main work. I tried this, because I was curious which species of Dieters D1-mixture will be the "good" one or is the mixture essential? Such a mixture is obvious in natural environment.

If you put a fresh sample of Dieters mixture on a fresh agar, then you get different kinds of surfaces, parts where the surface becomes hydrophobic in the first days, parts where you can see hyphae appressed to the surface and finally some first white, later blue green spots.

Finally one of these species dominates the surface. As I'm in an advanced stage, I infect new vessels normally not with pieces of agar but with germinated protocorms, and you could see different developments on the surface dependent of the single protocorm. It's my opinion that the protocorm, grown on an infected agar, contains at minimum the symbiotic fungus, so the chance to get the right one seems to be higher.

For the time being I have different vessels with different stages of development and with the chance, I already have the right one.

It is obvious that I'm not able with my limited resources to vary also the conditions of temperature and components in the agar, as I'm concentrated on the propagation of rare orchid species mainly on asymbiotic media. For my work the symbiotic propagation is only interesting because the development of seedlings is much faster – if it works.

The next time I prepare fresh oat agar I will cook also 30 g/l and infect it with a single hyphae from an appropriate location.

Greetings
Claus
Wer Chemiker werden will, muss Chemie studieren; wer Jurist oder Arzt werden will, muss Jura oder Medizin studieren. Aber um Politiker zu werden, ist lediglich das Studium der eigenen Interessen notwendig. (Max O'Rell)

Berthold

Zitat von: Ahriman am 24.Jun.09 um 03:48 Uhr
Claus:
In order to seperate such mixtures I'd try to grow them on stress-inducing media, varying pH, salinity and content of essential nutrients.
If you can create conditions that one component can tolerate but not the others you should be successful.

Regards,
Christian

Christian that's a fine idea but is doesn't sound easy to find the conditions to stress the right fungus only
Weniger gelobt ist genug kritisiert (frei nach Peter Altmaier)

Ahriman

Berthold, I assume you want to grow the right fungus only and stress the others. grins
Anyway, I guess it's just trial & error and much, much work.

Claus, I know what you mean - it's not my main work either, I never found time to really experiment with symbiotic germination at all...

Fred, to make it clear I am neither Mycologist nor an exprt for sequencing procedures but a student of chemical ecology. I just do this because I find it an interesting idea...
The B1 samples were not processed but directly sequenced out of agar as there were visibly sclerotia present that could easily be removed.
But I did't do this part myself so I cannot tell you more - I will do all steps myself mext time, so if it goes wrong again at least I know it's my fault. :whistle

Regards,
Christian

fred

Christian,

I forwarded some of the questions to Michele:

Regarding the possibility of B1 = mix of fungi:

ZitatThat may be possible, but I think it is quite remote since
usually fungi do not grow together without taking over,
even on oat meal agar.
In my opinion if a mix up happened, the Ceratobasidium is
most likely to lose the battle since usually it does not
cover the agar surface as quickly as the Tulasnella
(usually, then I have seen very aggressive Ceratobasidium
but usually have dark hyphae).
If two fungi were present in the culture that I used to
make the DNA extraction, I would have not been able to
have a clear DNA sequence, so, if there is a mix up I may
only have the one fungus left (strangely the
Ceratobasidium).
I have to admit that the general morphology of the colony
that I have is more Tulasnella than Ceratobasidium-like.
Still in the recent past I have seen many Ceratobasidium
looking Tulasnella and many Tulasnella looking
Ceratobasidium.
To be absolutely sure that no further mix up happened in
my laboratory I will re-perform sequencing on the culture.

Regarding O. morio germination failure:

ZitatI only tried a germination
experiment with B1 once, and I may need to re-test it,
maybe with different orchid species.

Regarding rich OMA vs malt agar:

ZitatI never used so rich oatmeal, so I cannot make comparisons
with my culture.
I will try to re-inoculate it on fresh oatmeal and see
what happens.
Generally speaking Ceratobasidium develop quite woolly
colonies, either whitish yellow or brown. Tulasnella
instead appear very flat on the medium, often mucid and
yellowish only. The picture that you sent me looks like a
Ceratobasidium to me.

Regarding Christian's question on the PCR primers:

ZitatI am aware that many primers commonly used for ITS
amplification of fungi do not work well on the Tulasnella
calospora group.
Till a few years ago all isolates (and all direct root
amplifications) in my lab were PCR amplified using ITS1F-
ITS4, but with poor results on some strains (likely of T.
calospora). Other primers used were ITS1Tul and ITS4B, but
again with poor results.
At the moment we are using ITS1OF A+B--ITS4OF for direct
amplification from root and ITS1-ITS4 for isolates.
Sometimes, when the chromatogram is unreadable we clone in
PgemT, but this is a rare occasion. It is more likely that
the need for cloning is due to the dicaryon in the fungus
itself than a mix of two fungi.

Did you [Christian] get contrasting results from the other sequencing
of B1?

regards,
Fred

Ahriman

Hello!

I agree with Michele - as far as I understand  :-D
Morphology and genetic identity are two very diffrent things in fungi. Not as weird as in bacteria but still very confusing.
I did and will not attempt any cloning as that's too much work for me, I'd rather grow orchids invitro in my spare time...

But what does he mean with contrasting results?
I only did one sequencing attempt which failed in all samples, possibly DNA Polymerase had turned bad.

It's funny that by now at least 3 people are working on sequencing B1 but so far noone seems to have been successful :whistle

Regards,
Christian

Claus

Zitat von: Ahriman am 27.Jun.09 um 03:18 Uhr
It's funny that by now at least 3 people are working on sequencing B1 but so far noone seems to have been successful :whistle
Regards,
Christian
Yes, but unfortunately Rafael successfully sequenzed the T&M and not the B1. And what is worse, we don't hear anything of him since weeks.

Claus
Wer Chemiker werden will, muss Chemie studieren; wer Jurist oder Arzt werden will, muss Jura oder Medizin studieren. Aber um Politiker zu werden, ist lediglich das Studium der eigenen Interessen notwendig. (Max O'Rell)

fred

As promissed, a culture of the aerial hyphae ...



Some explanation:
To sample the 'aerial' hyphae I used a little piece of agar and pushed it against the top of an old jar - it's a trick to get hold of those few hyphae. That's why you see that small chunk in the center.

The fungus clearly develops a woolly growth, which becomes appressed later on. The ring is about 4 cm in diameter. This is different from my typical B1 cultures which only shows this behaviour during the first few days and then only grows as a mucus. The second thing I noticed is that it's completely white ... my B1 is normally yellowish at the appressed stage.

The piece of dust is because I removed the lid and photographed directly above the culture - the glass blurred my other photographs. I have 2 other cultures running which I keep closed. I'll let them develop to see if they also grow the 'coils' at the edge of the jar.

Berthold

Fred the woolly hyphae are from a fungus that does not germinate any orchid. I am very familiar with this species of fungus and I throw it away immediately when I detect it.
Weniger gelobt ist genug kritisiert (frei nach Peter Altmaier)