Fungal Biotechnology - Science topic

You can use cellophane overlaid media plates. Once the fungus growth is completed on cellophane sheet, you can directly use to extract gDNA using liquid nitrogen and phenol chloroform extraction.

Please check it.

V-8 Agar is a medium consists of 8 types of vegetables including carrot, spinach, tomato, celery, parsley, beets, watercresses and lettuce. They are mixing together with known percent and preparing a juice from them and adding CaCO3 (3gm). It's a good medium for isolating Pythium spp.

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.

I am also looking for this, don’t see an attachment.

This compound may be useful in covid-19: https://www.physiciansweekly.com/possible-therapeutic-role-of-a-highly-standardized-mixture-of-active-compounds-derived-from-cultured-lentinula-edodes-mycelia-ahcc-in-patients-infected-with-2019-novel-coronavirus/

You can follow the Gerdemann and Nicolson (1963) method, see the

Baccharis incarum and fungus Arbuscular Mycorrhizal symbiotic relationship for land fallow in the Bolivian highland article (CienciAgro (2014) 3(1): 51- 58)

@Mattias Anderson. Sorry to say than orsellinic acid derivatives are colorless unless those are conjugated with other aromatic systems (dipole/quadrupoles preferably)

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

wheat germ agglutinin (WGA) staining

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

hi andrej did you ever find a source for Monascus purpreus . I've been looking for either live cultures or even spores just alittle harder to clean but not to hard.

thx steve

You can use PDA or SDA with chloramphenicol to recover common environmental fungi like Aspergillus, Fusarium, Chaetomium, Mucor, Rhizomucor, Alternaria, Rhizopus etc.If you wish to isolate dermatophytes such as Microsporum gypseum, you should use SDA with chloramphenicol and cycloheximide. However, for isolation of Cryptococcus neoformans, Candida, Geotrichum, Rhototorula from soil, avian droppings, bat guano, air, fruits and vegetables, we employed Pal sunflower seed medium with chloramphenicol.This medium was developed by us in 1980 and is used in many laboratories.We developed one more medium designated as "APRM" in 2015 and is helpful to recover common saprophytic fungi from soil, avian excreta,water, and air.I have uploaded several papers on Research Gate and Academia on the efficacy of Pal sunflower seed medium to isolate many fungi from environmental and clinical specimens.

Prof.Dr.Mahendra Pal

Founder Director of Narayan Consultancy on Veterinary Public Health and Microbiology, Anand, India

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.

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For obtained sufficient fungal filtates is necessary temperature, aireation etc in optimal points whenyou selecting ^{shake flasks in submerged fermentations.}

Dear Khalil

there are vats specified for that you can make yourself like cheese vats with double jacket in order to keep the temperature of your mix in the same time these vats made from stateless so the feed will be safe from metal contamination. in order to do your work it so easy to sterilize the vats, the feed mix the continua your experimental

shenana 

Hi!

It seems to be an Aspergillus but you must to look for species identification as Yehia told you. Colony morphology depends on culture media...useful for identification, also (i don't know what culture media you have used). 

I find this articles usefuls:

Best wishes!

Beatrice

Hi 

you can see this link its useful for you 

good luck

V

Some bacteria live in close association with fungal colonies, and use hyphae as corridors.

In order to separate the bacteria from the fungus, take a sterile inoculating loop and touch over your Penicillium culture where you see colour (spores). Then perform a "streak" inoculation. Draw an s-shaped pattern with the loop (the streak). This technique allows for your sample to be progressively diluted accross the streaked surface. After culture, you should find individual Penicillium colonies, and probably, some bacterial colonies. Allow the single Penicillium colonues to spore and subculture from those.

If the contamination persissts, conduct the same experiment in agar containing chloramphenicol.

Hello

Yes, you can use . It is standard method

The effect of the type of incubation just in the amount of metabolites not in the type of it.

Hi

You can read this thesis on attachment

Are you extracting the DNA or protein?

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Dear & Simon, Saida and Łukasz

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

Hi Dr,

Hope you are doing well.

Please see the latest molecular ID procedures in  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4260807/ 

We use the calmodulin (CaM) primers in this article as a DNA barcode.  Though ITS is also recommended, it rarely IDs aspergilli below the species group level.  To ID, blast the CaM sequence against the curated Aspergillus & Penicllium Database at http://www.westerdijkinstitute.nl/Aspergillus/DefaultInfo.aspx?Page=Home.

Other good articles include:  https://link.springer.com/article/10.1007/s00253-012-4165-2/fulltext.html and https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4255536/

See also:  http://www.aspergillus.org.uk/ 

Dear Nur,

In the following link you can find a proposed methodology to store R. solani:

Which contains the following protocol and a link to the original research paper:

A Method for Long-time Culture Storage of Rhizoctonia solani. Edward E. Butler, Professor, Department of Plant Pathology, University of California, Davis 95616; Phytopathology 70:820-821. Accepted for publication 22 February 1980. Copyright 1980 The American Phytopathological Society. DOI: 10.1094/Phyto-70-820.

A method is described for long-time preservation of cultures of Rhizoctonia solani in tubes in a dry soil containing 4% (w/w) wheat bran. The moisture level of the mixture at the time of inoculation with R. solani was 0.23 g water per gram (dry weight) of soil. About 1 mo at 23–27 C was required to allow the colonized mix to air dry; then it was stored at –25 C. Isolates from diverse sources survived 55 mo at –25 C. Virulence, morphology, and cultural characteristics of R. solani were not changed.

I guess it shouldn´t be difficult to find alternative protocols at the NCBI database.

Good luck!

If your protein is very dilute, use a binding technique like ion exchange first to concentrate. The flavine has an absorbance maximum at 450 nm, so you are looking for something that absorbs there and at 280 nm (the aromatic aa in the protein). Since you have done 2DE, you know the pI of your protein, designing an IEC experiment should be straightforward.

Thanks Oadi Matny thats very kind of you

Methanol in my opinion is adecuate

I have never seen it growing like that. Which strain is it? Cultivation time and temp?

Mohammed Fayyadh; Oadi Matny:

thank you for your help!!!

Thank you very much Mr. Oadi and Mr. Laith.

Good Luck

You welcome 

thank you all  for the answers

You most welcome

Houda

@Laith Al-Ani but sir i cannot open these links....please guide me to open these.thanks sir.

Hi

you can disinfect with hypochlorite sodium %1 for 30 second and wash with autoclaved  distilled water two times and then dry samples with autoclaved paper and culture it on PDA or MEA or PCA

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I think you can use the following primers, and  then sequencing and bioinformatics:

forward primer ITS1 (5’ TCC  GTA  GGT  GAA  CCT  GCG G 3’) and reverse primer ITS4 (5’ TCC  TCC  GCT  TAT  TGA  TAT  GC 3’)  (White et al., 1990)

Dear Yehya Salih, 

Thank you for your response and attached

documents.

Mr.  Abbas,  I  suggested that you can recheck the procedure and be highly aseptic in the operations especially during subculturing the isolates. 

Another "Reference36"

Several good references here on ResearchGate.

Check this discussion on a 5x10(4) spore concentration:

How to find the find the concentration of fungal spores? - ResearchGate. https://www.researchgate.net/post/How_to_find_the_find_the_concentration_of_fungal_spores2 [accessed Feb 7, 2017].

Also:

Also "Reference22"

You are welcome.

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Sometimes problem:

1) DNA concentration may be very little

2) Primer concentration is very high

Dear Karthik

Hi it's a TRY (It's worked for me)

Anyway 

Divide the entire cell culture in to small portions and observe under microscope for the fungal contamination: (there is a chance to get a portion of cells without fungi)

Gentle centrifugation  can also settles the mesh of fungi+cells

TRY 

Best of Luck

clean the filter with a 2 % formaldehyde and then autoclave by packing 

you can add 5ml formaldehyde to the fermenter containing water or remaining fungal culture and sterilize for 10mins keep a mask to your nose while sterilizing

and then clean thoroughly

Dear Dr. Yehya,

I hope you find the attached document useful.

Ajay:

Have a look at this link for insights:

Best

Syed

@ali

As far as i know, i haven't saw anyone using ETBR with PDA so it was on trial basis just to make sure that strain is susceptible to the carcinogenic media or not, and now as per your comment i am going to increase the amount of ETBR and so to check its Growth rate if enhanced over time or not. Secondly i ll be surely in contact with because this whole thing is my very own idea and i have pretty much no idea what parameters could effect the growth rate other then the ETBR concentrations and TEMP....

Looking Forward to talk with you 

It has been Solicorynespora litchii since1990.

Both genera are valid but some species of Corynespora were transferred to Solicorynespora when this was created.

For this kind of questions check Mycobank and Index Fungorum

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Hi Kuan

You can see this paper "Entomopathogenic fungi in soils from Alicante province"

Good Luck

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Hi Dhiraj Gautam

You can read this paper "Sodium Thiosulfate"

Hi Dr Jawid it is known that Penicillium dangeadii(Telemorphic:Talaromyces flavus) reduce Verticillium wilt infection on potato, olive and eggplant.the mechanism by which P.dangeardii may be due to glucose oxidase releases from  Penicillium ,glucose oxidase metabolize glucose released from root plants and form hydrogen peroxide which is toxic to Verticillium(Fravel &Robert ,1991)

Fravel,D,Rand Roberts,D.P.(1991).In suito evidence for thr role of glucose oxidase in the biocontrol of verticillium wilt by Talaromyces flavus .Biocontrol Sci Tecnol,1:90-99

Dear Liqaa,

Looking at the pics, it looks like you have Fusarium sp., definitely not T. rubrum.

Regards

Mohammed

You most welcome

Houda

Dear Yehya A. Salih

No I did not test pathogenicity on termites. Till now I am completing isolation protocol. Yes I will upload plate culture.

Thanks.

Shuvrah

Collect your blue extract. Filter it. Reduce it to dryness in vacuo under mild conditions to keep it under safer conditions for storage in the refrigerator than in solution.Take a small portion of your blue extract and give it on several TLC plates (but use enough material). Then develop it in small TLC chambers with pure solvents as eluents. These solvents should have different polarities, e.g. hexane, chloroform, ether or MTBE, ethyl acetate, methanol, acetone and others. But do not use mixtures. After the TLC ist developed have a look where the BLUE is. Upstairs, in the middle, downstairs?This will allow you to get an intention on the polarity of your compound and the qualitiy of your extract.

Then, it is possible to find a suitable eluent for column chromatography (CC) by mixing two of the solvents in an appropriate ratio. All of this can be done only by trial and error. But with small amounts it is not difficult to find a solution for the separation. I would recommend to try CC for separation of the blue.

Caution: Real dyestuffs with a high epsilon value may cause a colour already with tiny amounts of material. However, it cannot be seen at the begin if this is the case in your case or not.

Hi Jawad: There are a master thesis about formalization and spore production of Trichoderma  in the department of plant protection - University of Baghdad. I dont remember the title of the thesis but the name of the MS student is Mohammad Al-Hittar, his supervisor is Dr Farkad Al-Rawi. I think there is an electronic copy of the thesis, if you have any contact with any one in plant protection department - university of Baghdad, ask him to provide it to you. Unfortunately i am now in sabbatical leave, other whys I will send it to you.

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Hi John,

I think this pictorial overview maybe can help you "Wood Decay in Living and Dead Trees:, A Pictorial Overview"

Good Luck

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Dear Sergey

I coincide with Prof. Antoun. AMFspores can be present inside roots (if the product consist in a traditional inocula of spores reproduced at the host roots) or free in the proper product´s substrate. AMF can be extracted by wet sieving and decanting method, followed by centrifugation in sucrose solution (50%w/v) (Walker et al., 1982), be grouped initially by morphotypes, and finally identified the species under a microscope. By molecular methods, DNA extraction from the isolated can be performed using UltraCleanTM soil DNA kit (MO BIO Laboratories, Solana beach, CA, USA) following the manufacturer’s specifications. DNA extracted is checked by agarose gel electrophoresis (0.8%), stained with ethidium bromide and visualized under UV light. Then you can use use a nested-PCR DGGE analysis of the 18S rDNA followed by sequencing of excised bands.The products are analyzed by DGGE and the bands are excised for sequencing.

The fatty acid composition of lipids in AM fungi diferentiates them from other organisms. In most AM fungi, a large proportion of the total fatty acids is found as 16:1ω5c, 18:1ω7c, 20:3, 20:4 and 20:5. Furthermore, AM fungi have a rather high content of polyunsaturated 20-carbon fatty acids. The 16:1ω5c has been used as a marker fatty acid for AM fungi in controlled environments and the 18:1ω9c, 20:1ω9c, 20:2ω6c and 22:1ω9c could be used as possible markers for the detection of G. margarita, for example. You  can check also Madan et al. (2002).

Madan RC, Pankhurst BH, Smith H (2002) Use of fatty acids for identification of AM fungi and estimation of the biomass of AM spores in soil. Soil Biol Biochem 34:125–128. A big hug.

Sure sir

The best option is to develop nested pcr model please refer this article Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts

Dear Nizzal Syafiq

May be different requirement of temperature, Usually any slow growing rate in fungal we consider it as suspected viral infected fungal (mycoviruses), it may your isolate get infected with viruses

Pls. find the attached files

Page 206 in: Saul L. Neidleman . 1989. Advances in Applied Microbiology, Academic Press,Pp.320. ISBN 0080564488, 9780080564487

Ursula Kües, Reinhard Fischer. 2006. Growth, Differentiation and Sexuality Springer Science & Business Media,Pp.449. ISBN 3540281355, 9783540281351

Marcel Borgers, Roderick Hay, Michael G. Rinaldi. 2012. Current Topics in Medical Mycology, Springer Science & Business Media,Pp.274 ISBN 1461227623, 9781461227625

Hoping this will be helpful

Regards

Prof. Houda Kawas

Thank you so much you all to share your valuable suggestions and good wishes for you.

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)

Hi Alaa

for isolation of total cellular DNA from plant cells and tissues, or fungi (or plant pathogen infected plants) I have good experience using the DNeasy Plant Mini Kit. Good luck