Fungal Biology - Science topic

Hi Jakob, As we work with fungi from the technology side, our deep learning algorithms track spores from very dormancy through swelling. As soon as spores develop a germtube our algorithm let's go and thereby we quantify how many spores are present in sample, when do they germinate under given conditions and how many within a population germinates. It is exciting work in fungi and you can see more if of interest on biosensesolutions.dk

Best wishes, Søren

Hello, mainly storage in clinical laboratories in glycerol stocks, but for interesting fungi you can grow in reach media, than you can put in freezer for sporulation after you can collect spores and keep in dried conditions as long as you want.

Having worked in microbiology for years, including providing accredition sampling to meat testing. I found there was a limit to how far I could go. Having a life long love of learning I decided to go back to school and pursue a masters in counselling psychology.

Since opening a private practice www.mindsetsolutionscounselling.ca I have found my passion and can changed direction as my interests and knowledge increase.

Dear Pablo Reguera

If you are having issues with fungal contamination you maintain the aseptic condition. Sterilize everything that is being used in the experiment, like filter paper, Petri plates, and distilled water, and carry out your experiment in a laminar. Sterilization of seeds with a 30-45 seconds dip in 1% NaOCl and 3 successive 30 seconds of wash on sterilized distilled water is a must to prevent any contamination.

Kind regards

Jiwan

The picture is useless.

"fungus spread in rock music treatment is vigorous on the planting media compared to the ballad music treatment. The germination is completely zero for both treatment."

What do you mean by vigorous and germination?

Hi,

See the first report of Hf from Spain:

When it is moist and not too hot, the fungus will thrive well. The rather warm and dry conditions on the Iberian penninsula will likely limit the pathogen and thus disease intensity.

F. angustifolia is likely as susceptible as F. excelsior, but in Southern Europe the impact of ash dieback may be less, due to climatic conditions.

I hope that this helps.

With kind regards,

Thomas Kirisits

In the picture above, the grayish brown mycelium represents the fungus Rhizopus. While the white and greenish blue colonies represent the fungus Penicillium.

The above micrograph shows penicillium spp.

The colony which limited with yellow marker with white mycelium may be belong to the fungus Beauveria bassiana, but not exactly. It needs more images to clearly identified.

Decoloration est dur aux rzyons solaire ou n'importe quels paramètres qui peuvent dégrader les couleurs

This may help you https://www.nature.com/articles/s41598-019-49284-w#Sec9

Mohamed S Ahmed yes when we are talking about fungal contaminations specially like aspergillus and penicillum which are the common source causing breakdown of the compounds such as gelatine and most of the fungi found in the environment cause infections. And the ways by which these can be avoided or reduced is specially the spores from the culture can contaminate the other cultures and we need to remove them and after removing the contaminated cultures clean the entire incubation centre with a cloth soaked in hypochlorite and then with ethanolto reomvoe the fungal contaminations

I figured it out. It was some freaking copepods contaminated in my samples from Carolina

Dear Abhik.

Most fungi can be kept alive on many of the agar media for 6 months or 1 year before transfers are necessary. Sporulating agar cultures may be frozen at -20 C. in the deep freeze and maintained for considerable periods of time. Another good method for keeping cultures for a number of years is by pouring sterilized mineral oil in the test tube until the culture is completely submerged . The best method for preservation of most fungi that sporulate is by the lyophil process or the freeze dry method . Cultures have been maintained for at least 20 years by this method.(Alexopoulos and Beneke, 1962). you can also use sterilized sand for preserve of F.oxysporum.

The sequences should be the reverse complement to each other, so you will need to convert one of your sequences to a reverse complement so you can combine them to form a consensus sequence.

If you did the above and it was still a problem, it is possible the sequence is long and there was a breakdown towards the end of sequencing, meaning there may be no clear sections of overlap. If this is the problem, you can rerun the PCR and sequencing using primers closer together to crease a shorter contig.

That being said, since both the forward and reverse sequences agree, you probably have identified the organism.

Which are best Methods to extract Metarhizum toxins?

I can provide you a simple protocol for fungal DNA extraction.

Take 2-3 colonies directly from your culture plate using a pipette tip and dip it in a tube containing 100 microliters of nuclease free water. Vortex for a few seconds and heat in a water bath or incubator at 96 degree C for 10-15 min. Centrifuge at 13000 RPM and use 1 or 2 microliters of the supernatant for PCR.

Hello,yes you can extract mycotoxin from the substrate. You may culture the fung in a specific media preferred by this fungi .

Typically, superoxide (O2*-) is the first ROS being produced in most cells under aerobic conditions.

For bacterial cells, while it is true that a fraction of molecular oxygen consumed during respiration and ATP production is converted to superoxide, this amount of superoxide cannot be compared to the level of the host cells (which contains multiple mitochondria as a source of superoxide at resting state and during inflammation).

In modeling infection, typically host cell densities used are 1x10^4 to 1x10^5 cells /mL. In realistic terms, clinical infections involve infecting pathogens at densities at or below 10^7 microbes / mL at most (because the nutrient requirements including aeration in vivo won't allow them to grow to supraphysiological levels like 10^8 / mL) .

Also, typically, infection experiments use MOI (multiplicity of infection) ranging from 1 to 10. MOI like 100 (equivalent to 10^7/mL or more) again seems unrealistic for the reasons stated above.

Most bacteria possess multiple enzymatic and non-enzymatic antioxidant defenses to deal with superxide -- first by dismutation by SOD, then catalase or peroxiredoxins to terminate the ROS reactions. So, effectively, what you can expect to detect or see in resting bacteria for endogenous superoxide / ROS is very very low (we have some unpublished imaging data). Certainly not to be compared with a resting host cell.

***

Having said that, it means endogenous bacterial superoxide (or ROS) in host-pathogen interactions may not be a key concern in measuring the overall ROS turnover. It is expected to be a small contribution.

***

If you have an appropriately selective chemical probe for ROS (say superoxide), then what you do is to use enzyme inhibitors (e.g. NOX inhbitors) or antioxidants (say mito-TEMOL for mitochondrial superoxide) to measure the effects of the intervention, compared to un-intervened controls.

***

Also, if you are absolutely interested in finding about ROS in the bacteria/microbe, fluorescent protein based H2O2 reporting has been established (the HyPer series). Those can be integrated into your model strains by transgenesis. Then, you can image or quantify H2O2 changes during infection.

Currently with the known tools, precise investigation on ROS kinetics and dynamics remain quite challenging.

Good luck on your experiments!

Hi Nasim

You can remove making a shaker for the broth that contain mycelium and spores, then you can do a filtration for it

Dear colleagues,

Thank you for your comments,

we just used traditional CTAB method and our experience is that it works always much better than any kind of commercial kits.

Yes, we have lots od plant DNA together with the fungal DNA in the result. But if we have a fungal-specific primers PCR always wrks good. We just use higher concentration of DNA in PCR-mix:

DNA sample - 2 mkl (from the solution of 200-300 ng/mkl)

PCR mix volume - 15 mkl.

The last two decades have shown an increase incidence of nosocomial fungal infections in hospital environment.The important fungi that are implicated in nosocomial infections are Candida albicans, Non-Candida albicans, Aspergillus fumigatus and Non-Aspergillus fumigatus, Fusarium species and others. We have isolated several fungal pathogens from burn wounds of patients admitted in burn ward of the hospital. Certain measures, such as personal protective wear, proper hand hygiene, respiratory hygiene, thorough cleaning and disinfection, safe injection practice, avoidance of sharp needle and scalpel injury, and waste disposal besides prompt medical attention to skin injury, and appropriate treatment with anti-fungal drugs (fluconazole, itraconazole, Amphotericin B, posaconazole etc) can be effective to prevent nosocomial mycoses.

One see the following paper on nosocomial mycotic infections.

Alangaden GJ. Nosocomial Fungal Infections: Epidemiology, Infection Control, and Prevention. Infectious Disease Clinics of North America 2011;25:201-25.

Michael Dare Asemoloye Factors which greatly limits the generation of fungal vaccines are,

1. Human commensal nature of fungi,

2. Capacity of fungi to establish clinical latency (Candida, Cryptococcus etc.),

3. Potential high costs in preparing the vaccines,

4. Mostly immunocompromised individuals are susceptible to fungal infections,

5. Vaccine against commensal organisms ( Candida, etc.) becomes a challenge as autoimmunity against the organism develops.

Despite these factors, many fungal vaccines are in development stages.

Please go through the following RG links.

Could you please tell the name of this mushroom??

@ Jose, you have to inoculate separately as Tricoderma is growing very fast and they are not compatible with Glomus species. I think if you are very eager to see the combined effect you must inoculate first with Glomus and wait atleast for 2 weeks then go for Tricoderma inoculation.

It is preferable to spray chemical pesticides at the beginning of flowering or adding biological fungi to the soil to increase the defenses of the host

While doing leaf litter decomposition experiment following Olsen (1963) we got k value (per year) 0.18 and half life 3.85. However, 54.01% weight has already lost at the end of first year. How can we interprete the value of half life and weight loss?

Please have a look at the following RG link.

I face the same problem in growing fusarium oxysporum >> after one year of continuous sub- culturing on PDA medium the growth became transparent without apparent mycelium or spores . would you please tell me any solution for this case

thanks

Generally, this kind of contaminants are found when the culture plate is too old (≥6 months) or handled inappropiately. Normally yeast are found in our body and a part of healthy mix of normal flora. If I am not mistaken, it looks like yeast contamination; avoid unwanted movements, speaking while plating. Keep your work bench clean and wipe with 70% ethanol.

Hello, can't be evidence of ascorbate peroxidase in fungi. Peroxidase structures have significantly increased our understanding of the evolutionary and functional relationships within the plant peroxidase superfamily.

Three distantly related structural classes have emerged:

1. mitochondrial yeast cytochrome c peroxidase, chloroplast and cytosol ascorbate peroxidases, and gene duplicated bacterial peroxidase class I

2.secretory fungal peroxidases classII

3. classical, secretory plant peroxidases (class III).

So ascorbate peroxidase is class I peroxidase enzyme and catalyse the H2O2-dependent oxidation of ascorbate in plants, algae and certain cyanobacteria but not fungi. For this type of peroxidase your culture shuld be photosynthetic. My be we will see this activity in future in Radiotrophic fungus.

Good luck!

The Mycology Unit of Universitat Rovira i Virgili (Reus, Catalonia, Spain) is an excellent research group working on systematics of clinical and environmental fungi. Some outstanding scientists who have worked (or currently work) there are Drs. Josep Guarro, Javier Pastor, Josepa Gené, Josep Cano, Alberto Stchigel and Maria José Figueras, among others.

Yes, it is possible. When fungus grow on different media or different environment exposure, their morphology could be change because of different substrate, environment parameters and medium condition.

Other titles of peer-reviewed journals indexed in Scopus and Clarivate:

1-Current Research in Environmental and Applied Mycology

2-Sabouraudia Journal of Medical and Veterinary Mycology

3-Medical Mycology Case Report

Regards

Thank you.

Hi Francisco. Not really clear about your question, as the true coprophils are already in the dung when it is excreted - they require a passage through the herbivore gut to stimulate spore germination. If dung is dry when collected it can be kept in paper bags for months, even years, and will produce the normal growth of fungi when rehydrated and incubated. Coprophilous fungi alone are not responsible for decomposition - they will be interacting with the bacteria and fauna in the dung. I suppose you could do something along the lines of Webster and his students, e.g sterilise dung and then inoculate with different species, alone and in combination, and record the decomposition rate by weighing at intervals.

It seems to be Coprinus comatus

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

It is fluorescent, but can be made more or less fluorescent depending on the protein it binds to. Here is a link to the absorbance spectrum and other info: http://www.rsc.org/suppdata/cc/b8/b821687h/b821687h.pdf Hopefully after 3 years this helps someone :-)

It seems that there are at least three issues of importance besides dx. and tx.:

(1) prevention,

(1.1) who acquires them,

(1.2) do certain individuals/groups have a propensity to acquire them,

(1.3) do certain individuals/groups use successful preventative strategies already and, if so, what are the strategies and success rates.

Also, any RCTs out there and what are the results.

Dennis

Dennis Mazur

Kinda easy. Have been using it in my fungus for 2years now and it works perfectly well.

Hola Cristian

VCG is a classification at the subspecies level. You are not going to be sure if two isolates, belonging to the same VCG, are the same, albeit they are more closely related than two isolates of two different VCGs. However, it is a well-known (and relatively easy) tool that is going to give you useful ecologic information about your population; for example, in general, and depending on your species, you population could sexually reproduce if you have isolates of different MAT within a given VCG.

Un saludo

Any co-dominant marker can be used

CAPS markers are easy to develop. Three markers linked to MAT are interesting: (1) PR6 used in the paper cited above by Aydin Hassanzadeh, (2) markers based on polymorphism in RPB2 (also linked to MAT), or (3) in gene mip (tightly linked to MAT). For the latter, the couple of primers used for A. subrufescens (marker mip) in the paper indicated below has been also successfully used with A. bisporus.

Evidence for amphithallism and broad geographical hybridization potential among Agaricus subrufescens isolates from Brazil, France and Thailand. Fungal Biology 118:1013-1023, DOI 10.1016/j.funbio.2014.10.004

While pouring hot agar medium, stack petri plates in size of 10 or even more. It will prevent condensation in all plates except the top one.

20

The optimal media depend on the fungus being grown.  A good reference for several of the general media for fungal growth is Singleton et al. 1992. Methods for Research on Soilborne Phytopathogenic Fungi.  This mentions several of the types that have been widely used, such as malt extract agar, cornmeal agar, oatmeal  agar, etc.

Yes, V8 juice is most commonly clarified for use in media (e.g. Miller 1955, Ayers and Lumsden 1975, etc.) and then autoclaved.

how long have you been staining the media? because in my experience, you need to flood it with congo red for about 15 minutes then wash it with aquadest (flood it also for 15 minutes) 2-3 times, then the clear zone that indicated enzyme activity will be shown

 

Dear & Simon, Saida and Łukasz

I attached new photos related to the blight reed fungus, Thank you

You need to find the ascospores and photograph them. 

Hei,

To obtain a sensitive and specific assay, I would recommend the use of a primer/probe -based real-time PCR assay. It is probably very challenging to use the ITS rDNA gene cluster to design a specific assay for Botrytis cinerea. One option is to use betatubulin -see e.g. New Phytol (2005) 168: 465-474.

Dear Yosep,

Not because I work on protist pathogens, but to me looks like trophozoites of some amoeba. Try to grow the swab assisted wound material on sabouraud's medium to rule out fungus. Could be Acanthamoebal trophozoites, deep eosinophilic cells??

Best,

Mannan

Thank you Jean sir

You are welcome.

Hi Abbas, I used autoclaved mineral oil to preserve  Sclerotia at room temperature. it works good up to six month.

I hope it will help you.

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Hi Shuvrah,

You can also see this book "Seifert K, Morgan-Jones G, Gams W, Kendrick B. 2011. The Genera of Hyphomycetes. CBS Biodiversity Series no. 9: 1–997. CBS-KNAW Fungal Biodiversity Centre, Utrecht, Netherlands ".

Dear colleague Mostakim

There are not an exact time for find that structures in roots. It depends of diverse causes, among then, physiological processes, soil nutrient levels. For example it has been demonstrated that late embryogenesis abundant (LEA) proteins also accumulate in vegetative plant tissues during periods of water deficit, which reinforced a role for these proteins as desiccation protectant. It seems that during cellular dehydration LEA proteins play an important role in maintenance of the structure of other proteins, vesicles or endomembrane structures in the sequestration of ions such as calcium, in binding or replacement of water, and functioning as molecular chaperones. Any problem with that proteins could be one of the causes of the ausence of that vesicles.On the other hand, such vesicles, frequently hard to distinguish from intraradical spores, are more frequently formed on the most polluted locations and are seen as a part of the mycorrhizal survival strategy on metal-polluted sites (Pawlowska et al. 1996; Turnau et al. 1996; Regvar et al. 2006). Finally, with a management of nutrient solutions to the plant we can induce the formation of such structures also.

I agree with Christoph Ottenheim  and all answers above ...good luck.

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Hi Duarte

I think, you can identify the races dependence on VCG, after you determine the race

you can do molecular biology

First, things do not necessary have a function in Nature. Structures that do not impact the fitness of a genotype (neutral traits) will not be eliminated by natural selection and may thus get fixed and transmitted. 

This being said, the function of cystidia, whether cheilo or pleuro, is still unclear. They may play a role in maintaining a higher degree humidity around the gills (boundary layer effect). They may also play a defensive role in protecting  against gills feeding animals. See for instance:

HI, Elena

We published a small paper on Thuemenidium (http://www.mycologia.org/content/102/5/1089.full), and observed shared habitat between the fungus and crawberry. We have not collected any molecular evidence for any associations between earth tongues and other plants. However, I did observed tiny apothecia of Trichoglossum associated with mosses' rhizomes. Below was what we addressed about earth tongue ecology in that paper, hope this is helpful for your research.

"...The ecology of earth tongue fungi in Geoglossum, Trichoglossum, Microglossum and Thuemenidium was once considered homogenous, not only because all were found commonly in more or less damp lawns or pastureland (Nannfeldt 1942) but also because the ecology of these fungi, indeed of most Leotiomycetes, has been both understudied and overlooked for a long time. Although there is no available hard evidence many species of Geoglossum and Trichoglossum are believed to be associated in some way with bryophytes. The ecology of T. arenarium is unique because it grows in sand dunes near the seacoast. Of note it often grows with Clavaria argillacea (Ohenoja 1995, 2000) and has been confirmed to form mycorrhizae with the black crowberry Empetrum nigrum (Nitare 1982). In contrast T. atropurpureum usually is collected from acidic grasslands where diverse mosses are common. So far no relationships between T. atropurpureum and specific mosses have been proposed. Lumbsch and Huhndorf kept T. arenarium in Geoglossum following Nitrare (1982) and assigned only T. atropurpureum to Microglossum on the basis of molecular evidence (http://www8.umu.se/myconet)."

Hi Shridhar

You can follow this paper "Effect of root feeding by striped cucumber beetle larvae on the incidence and severity of Fusarium wilt of muskmelon"

Good Luck

You can also write to Prof. Ning Zhang at Rutgers Un. She works with Magnaporthe.

Dear Fatia, 

As other colleagues have mentioned, not all conidial fungi have the ability to produce a sexual stage. Furthermore, those which do produce a sexual stage, can be either homothallic or heterothallic.

Homothallic fungi can produce sexual reproductive structures in the absence of a sexually compatible strain, but mating compatible strains is required to obtain a sexual phase in heterothallic species.

In general terms, it is easier to obtain a sexual stage on poor media which resemble the conditions in which the fungus grows in nature. For example, keratinophilic Onygenales commonly produce sexual stages on keratin-rich substrates such as sterilized feathers or hairs. If you have a fungus which is associated with decaying plant material, you can try water agar with sterilized fragments of straw, carnation leaves, etc., and poor media such as oatmeal agar or potato-carrot agar.

Success!

Hugo 

i am working on it now. At our location Central Kalimantan Indonesia we have many of C, militaris infected on lavae and pupa of Setora nitens (Lepidoptera: Limacodidae)

first make 5 to 6 sets of saw dust soaked with double distilled water ( including a control set without any inoculation). then add the microbes aseptically to each set after serial dilution with .01% MgSO₄ solution ( upto 10^{-5/-6 level}) and take your desired data after certain period of inoculation. 

Hi,

I aggree that you shouldn't base oyour decisions in molecular ID only. Some kind of preliminary phenotypic identification can sometimes help you define what type of fungus you are dealing with, and what other genes you should use for molecular ID.

Even though ITS is the current fungal barcode region, it is not fully discriminatory for numerous fungal genera of Ascomycota. βT1, TEF, calmodulin and other genes are not as standardised, but are in some cases more useful than ITS. You should have a multilocus approach.

I would advise you to go through publications of Studies in Mycology or others describing newly discovered species to have a clearer picture of the steps you should take when novelty is suspected. 

Good luck,

Paula Rodrigues

I agree with Franci Aco Celar.  It's classical method where in control plate must be only one pathogen inoculum. In addition for Trichoderma strains with active growth is necessary to use the method of "deferred antagonism". In our studies, 7 days was too much. Trichoderma covers the surface of petri dish for 3-4 days.

yes culture media, nutrients, pH variation affect spore morphology and well as cultural characteristics.  

I don't know the aim of your study but  I red about some methods used in metagenomics  (study of genetic material recovered directly from environmental sample like soil). You can extract microbiome DNA from soil and then use AMF specific primers and NGS to explore diversity in AMF community. So maybe you should address your question to research groups that have used this new approach for AMF.