Ascomycetes - Science topic

Kouadri Mohamed El Amine Thank you for your reply. Yes. I tried slide culture method by putting a tiny agar media on the slide with spores, and let it grow for a while. I also prepare another version of this by putting the slide on a heat block around 60°C for a while to melt agar thereby flatten the slide. This works good sometimes.

Also, you can use the fungicides Benlate, Pentachloronitrobenzen (PCNB), Hexachlorobenzen (HCB), Vitavax and Bayletan to control basididiomycetes.

With best regards!

The genes you mentioned of are more reserve and stable

Benomyl is a good option but it does not inhibit fast-growing Zygomycetes. You can incubate plates at a low temperature (4-10-15ºC) to slow them down and give basidiomycetes better chances to grow.

I am in agreement with Cristian Riquelme.

Yes you can use the medium YMA which consists of Yeast extract (3 gm), Malt extract (3 gm), Peptone (5 gm), Glucose (10 gm) and Agar (20 gm) per one Liter. Its suitable medium for growing Peziza spp.

I have now added more photos to this comment of the photobiont, ascii, paraphyses, and the ascospores, which are much clearer than I was able to attain before. The ascospores are 1-4 septate, and appear to either come in two types/maturities (one of which seems to have very distinct jigsaw-puzzle shaped septa, as shown in the photos), or the ascocarp I squashed may have had another species present. Average spore sizes of the two types (n=8 each) are 15.7 x 3.8 μm (jigsaw septa) and 17.9 x 4.4 μm (opaque spores) for the two types.

I still don't have access to Iodine to stain the ascus tips, but I am working on it!

Dear Cameron,

You can find useful information about ribosomal DNA (which includes ITS) copy number in fungal genomes in this recent paper:

Best,

vincent

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There is essentially no worthwhile documentation for making fungal mounts, and it mostly boils down to trial and error with a full sharps bin full of failures before you start making progress.

Do not use Scotch tape, it decreases the amount of light which makes it to your camera and reduces the dynamic range. Also it barely picks up anything worth photographing and rips apart relevant structures (e.g. you pick up a conidiophore of an Aspergillus but the stipe got town and there is no foot cell)

What I typically do is slice out a 1 cm^2 piece of agar with a sterile scalpel. Make 2 series of parallel cuts, perpendicular to each other (essentially make a # pattern). Gently remove the square (containing the leading edge of a colony) and place onto a microscope slide (with the side containing growth face up).

I use DIC as well, and I just place a drop of sterile water on top of the agar square, place a cover slip down, and mount onto the microscope stage.

Now I typically work with Fusarium species which spread out on a clear water agar surface, and the philiades are dispersed enough that this method works very conveniently. If you are working with Aspergillus or Penicillia the above method will probably result in a large, opaque, knotted mess under the microscope. The nice thing about DIC is that the depth of field is super small so you may be able to focus in on a conidiophore while the hyphae are mostly out of focus.

The alternative is to make 3 cuts at the leading edge of a colony. Make one cut perpendicular to the leading edge behind where you see sporulation (think cutting the crust off a pizza) like ---------. Then make 2 cuts around 1 cm apart perpendicular to the first into the agar like --|---------|--. Then you need to make another cut parallel to the first one (--------) at like a 45 degree angle towards the colony. Instead of a square like above you are extracting a triangle with the least amount of agar as possible.

From there you extract the agar piece containing the colony and place onto a microscope slide. Add a couple drops and use needles to "tease" out the knotted mess that the hyphae is in. Do not poke repeatedly, do not rip to oplivion, just try to loosen the mess a bit and try to flatten everything.

If possible, cut off any excess agar. Then place a cover slip and try to crush the hyphae the best you can. If you picked up too big of a piece the cover slip may snap and break. You can try shearing the slide a bit to spread out the hyphae, but too much will ruin everything worse than Scotch tape.

The goal is to have as flat a layer of hyphae as possible before you begin looking for structures under the microscope. Do not waste your time looking around the center of the knotted mess, instead look around the edges of the clumps you eased apart. If all goes well you will see some conidiophores free from the mess and suspended loosely enough to photograph.

If you end up with a slide which is essentially an ocean of spores, my recommendation is to hold the agar piece you just extracted onto the microscope slide with a teasing needle and rinse the colony with sterile water before teasing to remove the bulk of the spores. There will still be plenty left, but hopefully you could now see if that Aspergillus isolate has metulae or if that Penicillium isolate is biverticillate.

-Best, someone who has had to make microscopic mounts of fungal food contaminants for the past 5 years

James Bonitati Thanks for your suggestion. Will try it for sure

Dear Faiza

Indeed, the picture is not clear, but it seems that You have genomic DNA band on the top near the bright cloud. The "smearing" looks like a degraded RNA. I am not sure about light spots on the bottom. How many samples do you analize in this run? I have had such results (similar, as I think) in gel after CTAB. Be carefull during "washing".

https://nt.ars-grin.gov/fungaldatabases/fungushost/fungushost.cfm

Check this BUSCO software = Opposite Polarity Monospore Genome De Novo Sequencing

Both mating types in the heterothallic fungus Ophiostoma quercus contain MAT1- 1 MAT1 - 2 genes........ type 1 idiomorph, and MAT1 - 2 - 1 ( of the mating type 2 idiomorph ) genes in these isolates. Reults obtained showed that all isolates contained the full gene sequence for the MAT1 - 2 - 1 gene. In addition fragments of the MAT1 - 1 and MAT1 - 1 - 3 genes were sequenced from all isolates. These results were unexpected, as each isolate from a heterothallic species would typically contain only one of the two possible MAT idiomorphs. For more details consult https://www.ncbi.nlm.gov - pubmed.

you're welcome

fred

Hi, These are sclerotia , The fungus produces highly resistant sclerotia as survival structures in older cultures.

Try less concentration of the primer (0,2 µM) because more time the higher concentration of the primer can be produced second products or artefact. Which thermoprofile did you use?

Try this from the paper Korabecna 2007: 94°C for 3 min, 35 cycles of 94°C for 1 min, 55,5°C for 2 min, 72°C for 2 min, followed 72°C for 30 min.

hi see this link please 

https://www.ncbi.nlm.nih.gov › NCBI ›

Reference: Ancient material from museum collections exhibit dramatic DNA degradation

Hi, I´m working with plants and we also purified mitochondria and performed MS analysis we made good experience with a urea based buffer, we also performed sequential extractions with different urea based buffers. Here you can find the composition:

doi: 10.1007/s00299-010-0935-4

Further, heating of protein sampples is absolutely not recommended if you want to run MS analysis because it will lead in artificial carbamoylation of aminogroups. this will mask proteins from detection.  also if thiourea is contained in the buffer it will hydrolyse  your proteins.

Additionally, we have good experience in combining ruea based protein extraction with methanol/chloroform preciptation as we have to get rid of the chlorophyl. this works very satisfying.

Anyway your buffer composition does not (at least not for me) appear unsuitable but maybe its worth to include methanol/chloroform precipitation additionally.

Please let me know if this is helpful or if you have further questions.

cheers

Sebastian

Hi Matthias,

I was looking for yeast specific FISH probes. I came across this information which I think may be suitable for you.

Fluorescent In situ hybridization allows rapid identification of microorganisms in blood cultures. Kempf VA, Trebesius K, Autenrieth IB. Journal of clinical microbiology. 2000.

Probes Sequence

5'- CTC TGG CTT CAC CCT ATT C -3'

Please go through the publication and find out if it is helpful.

Regards

Riyaz

There is a publication in which cytoplasmic ROS levels in the filamentous ascomycete Podospora anserina (which is closely related to N. crassa) are studied by using the HyPer protein (please see attachment). You could think about constructing a strain that expresses a gene encoding the HyPer protein with a mitochondrial targeting signal. There is no guarantee that it would work, though. When I was working with Podospora anserina I was not able to find a fluorescent dye that reliably reports on mitochondrial ROS levels (usually the background levels were much too high). But you could consider testing MitoSOX which seems to be a good probe for measuring mitochondrial oxidative stress in mammalian cells (I didn't test this dye for use with Podospora anserina so I can't give you any hints).

Regards, Christian  

Dear Mrs Narimene 

this is your institutional adress : Avenue Hassan Badi - El Harrach - Alger 16040

Best regrads

Dear  Carlos Barco,

Thank you very much indeed for your detail explanation. I think Your Answer will help me a lot.

Are you sure these are ascospores?  The spore shape is typical of Pithomyces chartarum, which make them asexual conidia and not sexual ascospores.  I must say that the spores are arranged in rows like ascospores, but I think that is just by accident.  There are suppose to be 8 spores arranged together in a sac-like structure called an ascus....which is not the case here. Hope it helps.

The proper  (current)species name now is:

Mycosphaerella nubilosa (Cooke) Hansf.

You can check latin names of fungi on web page below.

Regards, Franci

Dear Otto,

from the literature it seems, that Cenococcum is to be expected to be very common on dry and sandy soils. See for instance LoBuglio KF (1999, Cenococcum. In: Cairney J, Chambers S (eds) Ectomycorrhizal Fungi Key Genera in Profile. Springer Berlin Heidelberg, pp 287-309) or Ferdinandsen (1925, abstract: http://www.cabdirect.org/abstracts/19261100585.html;jsessionid=6FC06E1B9AD1348AA2A011D1DB9F0D04;jsessionid=3F68F02FC3B1E653FD79508E21864FA8?freeview=true).

Best wishes

Frank

Dear Hajar,

you can make a 500 µg/ml stock solution of benomyl in 70% ethanol, which you can then dilute to your working concentration in water. Some examples are given here and in the attached file:

The final concentration of ethanol will be very low.

Best Regards,

Christoph Kumann

dried filter paper or silicon gel would be ideal, especially for ascocarps that may sampled for DNAs. Glycerol would be good for cultures.

Hydrophobins are a family of small relatively hydrophobic fungal proteins with

interesting properties. These secreted proteins have an ability to convert

hydrophobic surfaces to hydrophilic and hydrophilic surfaces to hydrophobic by

self-assembly into an amphipathic protein membrane. They also

belong to the most surface active molecules known. The surface activity of hydrophobins is in the level of the commercial synthetic surfactants and other biosurfactants. In nature, these exceptional characteristics serve different functions. The hydrophobin membrane covers emergent fungal structures making the cell wall hydrophobic which facilitates attachment of hyphae to hydrophobic surfaces, aerial growth of the hyphae, spore dispersal, and proper gas exchange in fungal air channels

. The high surface activity of hydrophobins enables the fungal hyphae to escape from liquid media to the air .

There are several possible applications for hydrophobins including use of

hydrophobin membrane in immobilization of cells or proteins to surfaces like in

biosensors, changing surface hydrophobicity e.g. in order to increase

biocompatibility in tissue engineering, and acting as oil dispersing agents in

different branches of industry.

Hydrophobins can be extrcated from wet mycelium of T. reesei VTT-D-98692 (2.15 kg

wet weight, 19.5% dw, from 15 l of culture) three times with

4190 ml of 100 mM Tris/HCl buffer, pH 9.0, containing 1% SDS at room

temperature for 1 h with occasional mixing. The solution can be chosen according

to the preliminary trials in which different pH values, detergents (0.6% Triton

X-100, 0.6% Tween 20, and SDS), and SDS concentrations were tested. The

diluting effect of the moisture in the mycelium should be taken into account in the first

extraction by using 1.7x buffer. The mycelium was separated by centrifugation

(8000 g, 25 min, 6o C; Heraeus Sepatech Cryofuge 8000). SDS was precipitated

form the first extract as water insoluble potassium dodecyl sulfate by adding 0.4

sample volumes of 2 M KCl and discarded after centrifugation. The SDS content

is determined as described by Hayashi (Hayashi 1975) using dichloromethane

instead of chloroform. Ammonium sulfate concentration and pH of the first

extract are adjusted to 0.6 M and pH 7.5, respectively, and the solution

(5110 ml) is applied to the column of high substituted Phenyl Sepharose 6 FF

(45 x 10 cm, Pharmacia Biotech) equilibrated with 100 mM Tris/HCl, pH 7.5,

containing 2 M ammonium sulfate. Most of the HFBI is eluted with water

after a linear gradient from the equilibrium buffer to 20 mM Tris/HCl pH 7.5.

HFBI-containing fractions are pooled (4590 ml). Part of the pool (3x200 ml)

is further purified by anion exchange fast performance liquid chromatography

(ResourceTM Q, 6 ml, Pharmacia Biotech) to separate different form

Ascomycetes and Basidiomycetes produce characteristic spores after the

processes of plasmogamy, karyogamy, and meiosis. Heterothallism, which requires two

mating types, is more common than homothallism; therefore, zygospores, ascospores, and basidiospores are not commonly seen in the clinical laboratory because only one mating type is usually recovered.Ascomycetes are characterized by the development of sac-like cells called asci, which usually contain eight ascospores. Asci may form in a specialized fruiting body called an ascocarp. The development of the centrum is very important in distinguishing ascocarp types.Casein agar

casein, 10% acid hydrolyzed 25.0 ml

glucose 40.0 gm

Mg S04 0.1 gm

KH2

PO4 1.8 gm

agar 20.0 gm

distilled H O q.s. 1000 ml 2

adjust to pH 6.8

 Ammonium nitrate agar can be used- Same as casein agar except substitute 1.5 gm NH N0 for 4 3 casein, 10% acid hydrolzyed

The principal host of E. fawcettii is sour oranges (Citrus aurantium), but it is also

important on grapefruits (C. paradisi), lemons (C. limon), mandarins (C. reticulata) and

some cultivars of oranges (C. sinensis) and tangelos (C. paradisi x C. reticulata). Many other species and hybrids of Rutaceae include susceptible or moderately susceptible cultivars or clones, e. g. calamondins (C. madurensis), C. hystrix, C. limonia, C. nobilis, Poncirus trifoliata, rough lemons (C. jambhiri) and satsumas (C. unshiu). The potential host range extends to other ornamental Citrus and rootstocks. Most cultivars of C. latifolia,Fortunella margarita, oranges and pummelos (C. maxima) are rarely attacked. cultivars of citrons (C. medica), kumquats (Fortunella), limes (C. aurantiifolia) and oranges are highly resistant. Inoculum for new infections consists of conidia, and presumably ascospores, from scabs formed on leaves, twigs and fruits. Conidia are formed abundantly on wet scabs, in a nearly saturated atmosphere, between 20 and 28°C.Germination of conidia and infection do not require liquid water, both processes being possible with dew, fog or under high moisture conditions. A wet period of 2.5-3.5 h is needed for conidial infection. The temperature range required for germination of conidia is 13-32°C, but infection does not take place below 14°C or above 25°C. The incubation period is at least 5 days. The optimal temperature for disease development is 20-21°C. Leaves, shoots and fruits are infected when young, i.e. when leaves are up to 15 mm wide and fruits are not more than 20 mm across.

The pathogen is able to survive in scab pustules on fruits remaining on the tree and on other plant organs, providing the inoculum for the next season. Even in resistant cultivars, the fungus can survive on diseased shoots from susceptible rootstocks (Whiteside, 1988). For more information, see Yamada (1961), Whiteside (1975; 1988).

Biotypes of the fungus virulent on certain hosts are known (Whiteside, 1978). 

Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. nature biotechnology,2014,doi:10.1038/nbt.2969

Dear Raquel,

I upload a paper on gas diffusion in porous media and a PPT on gas diffusion in liquid that lead to convection for your reference.  You need to combine both concepts in one set up in FLUENT, and add reaction, but remove g force to stop convection.  You can estimate the time steps from the spreading rate of Altenaria sp.  kk

CDH gene family is responsible for the lignocellutytic degradation in wood.The GH61 represents the most enigmatic Glycoside Hydrolase family (GH) regarding enzymatic activity and importance in cellulose degradation. Heterobasidion irregulare is a necrotizing pathogen and white-rot fungus that causes enormous damages in conifer forests. Important genes in ascomyectes are HiGH61 members divided into two groups; one that show up regulation on lignocellulosic substrates (HiGH61A, HiGH61B, HiGH61D, HiGH61G, HiGH61H, and HiGH61I) and a second showing either down-regulation or constitutive expression (HiGH61C, HiGH61E, HiGH61F, and HiGH61J). 

Basidiomycetes have the capacity to degrade lignocellulose using a variety of enzymes

We successfully used  Malt Extract Agar (MEA) media for Periconia spp. cultivation.

In addition to thee public collection centers, you can simply look at who works on this pathogen and request some strains

It's not too complicated, and please follow Zheng Wang's advice. Use a good dictionary or a good text book in mycology.

In short:

1. The term heterokaryon describes a mycelium containing more than one genotype. It does, in this case, not matter how many genetically different karyotypes are harboured by this mycelium.

2. Consequently, a homokaryon contains only a single karyotype.

3. The term dikaryon is used exclusively in sexual context. Dikaryon means that a given mycelium contains both nuclei with complementary mating type. Thus, a dikaryon is always heterokaryotic, because it contains two different genotypes that differ at the mating type locus. However, you may not switch that sentence: A heterokaryon is not necessarily a dikaryon, the latter word being used only for nuclei differing in mating type.

4. The term  monokaryon, too, is used if talking about sex and means a mycelium containing only one of the two mating types.

5. Polykaryotic or multikaryotic do not normally refer to the genetic System in a given fungus and just state thar there are many nuclei in a given compartment of the mycelium or, if you wish, in a cell. These terms are normally used for describing the cytological situation in a fungus.

Cheer up!

If you do some bibliographic research you will rapidly see that there is no media of choice.

Il you look to Bode et al. litterature (OSMAC approach) you will see that they are all good !

PDA is best media for Alternaria..... but if not sporulating on PDA try giving physical or chemical stress.

Dear Matthias,

 Mostly soil isolated Trichoderma sp. are resistant to fungicides so you may enriched your culture media with some of the fungicides such as captan, penta-chloro nitrobenzene , metaloxyl etc. but place to place the concentration of the fungicidal quantity may vary little  in the media preparation. But I didnot know what are the soil you are looking for. But fungicidal enriched media are best for the isolation of Trichoderma sp. from the soil. You may try the below given media for your purpose. But if you use this media it took about 4 to 5 days to enumerate fungal colony on it.

 Trichoderma specific medium: MgSO47H2O, 0.2; K2HPO4,0.9;KCl,0.3;NH4NO3,3.0; glucose, 3.0; chloromophenicol, 0.25; penta-chloro nitrobenzene, 0.2; Rose Bengal, 1.5; captan,0.2; metaloxyl, 1.6 (g/1000ml)  and pH adjusted to 5.5

 Hope it may find your solutions.

Depend on the size of your sample and what you look for. Based on my experience,  rotary microtome with a freezing stage works very well with sample smaller than 2-3mm in diameter, and this should be good for most microscopy work in ascomycetes.

Many persons across globe are working on apple scab like Xu (East malling) patocchi (Switzerland) and V. Bus(NewZealand). By morphology it is easy to identify apple scab fungus. You can also use realtime CPR for detection. 

You are lucky that only few areas are infected in China however, in our place (Kashmir, India) it is a catastrophic disease and needs 12 fungicidal sprays from pink bud to harvest.

Thank you very much Andrew