Fungal Biotechnology

Fungal Biotechnology

  • Rosa Caiazzo added an answer:
    How can I get rid of non-specific amplification in a LAMP assay?
    I am trying to develop a LAMP assay for a fungal plant pathogen using HNB as the indicator. However, I am getting amplification even in negative controls without any DNA template added. I have changed reagents and rooms where I set up the assay, varied reagent concentrations and also checked all the reagents for contamination. I have also used different primer sets (all HPLC purified) for the LAMP assay but I still get this non-specific amplification in the negative controls. Is there anything else that I am supposed to look out for to prevent non-specific amplification in a LAMP assay?
    Rosa Caiazzo

    I think my problem is in the buffer, but still I can't have a color detection, they all look violet! how much HNB do you guys use? 


    I send you a prv message Andrea 


  • Eduardo Hernández-Navarro added an answer:
    Which is the best Fungal DNA extraction Kit in your opinion?

    I need to extract DNA from spores from herbaria material. I've used the Phenol-Chloroform method with success, but I'm interested in a Kit.

    Eduardo Hernández-Navarro

    Thank you so much, Dr. Vlassov!

  • Asma Shahzad added an answer:
    If we are making nanoparticles by fungi.what should be the concentration of cell free filtrate of fungus and silver nitrate solution?

    what volume of silver nitrate and cell free filtrate of fungi should be mixed to get smaller size nanoparticles?

    Asma Shahzad

    Thank you

  • Maryam Moudi added an answer:
    Which factors can have an affect on the mineral properties of edible mushrooms?

    Dear All, Hi, i have question about edible mushrooms especially Pleurotus florida. Actually i wanted to know that some factors like storage time, preserving methods, freezing and.... can be effected on mineral properties especially heavy metal concentration? Although the mushrooms are not wild and are cultivated.indeed they store in the freezer -19c for maybe one or two months.

    Maryam Moudi

    Dear All, Thank yo so much. Warmest Regards

  • Mehdi Nikkhah added an answer:
    Can I use the broth microdilution method for the antifungal effects (MIC , MFC and FIC ) of essential oils (Thyme, Rosemary, Oreganum )?

    Fungal strains are Botrytis cinerea، Penicillium expansum and Alternaria alternate

    Is there any protocol?

    Mehdi Nikkhah

    Thank you very much for your comments and pdf

  • Florence Obani added an answer:
    How is Aflasafe applied to groundnut fields? and at what rate?

    Aflasafe is a biological control which reduces aflatoxin contamination.

    Florence Obani

    By broadcasting and at 10kg/hectare

  • Brigitte Sthepani Orozco Colonia added an answer:
    Does T. harzianum produce cellulase?

    I've done the solid state fermentation on a plant sample. The crude fibre for the plant sample treated with the T. harzianum decreased much more compared to the plant sample treated with the T. reesei. T. reesei is a well-known fungi strains that producing cellulase enzyme. However, in my study it showed that the T. reesei decreased the crude fibre content less than the T. harzianum. Please help me.

    Brigitte Sthepani Orozco Colonia

    Yes, I suggested you that performed a test for detection and selection of microorganisms that produce cellulases.

    • Source
      [Show abstract] [Hide abstract]
      ABSTRACT: The screening plate method is commonly used for previous detection of cellulases produced by micro-organisms with biotechnological potential. In this manuscript, the authors aim to evaluate the hydrolytic ability of different fungi isolated from soil for the production of cellulolytic enzymes for cellulose degradation and determining the enzymatic index (EI) in relation to the growth of fungal colony and halo. The fungi were grown in carboxymethyl cellulose medium (CMC 1% w/v) and Avicel medium (Cellulose microcrystalline 1% w/v) for the determination of endo-glucanases and exo-glucanases respectively at 28°C for 48 h. Four chromogenic dyes were used: Congo Red, Phenol Red, Trypan Blue and Gram’s Iodine. Also, another screening method was compared using carboxymethyl cellulose medium (CMC 1% w/v) at 28°C for 96 h and exposed with Congo Red dye in buffer Tris HCl 0.1 M, pH 8.0. The results obtained allowed to find significant differences between the tested fungi, the growth time and chromogenic dyes. The strains with higher Enzymatic Index (EI) were JCO1, UFT1, UFT2 and UFT3 for endo-glucanases and JCO2, UFT1, UFT2 and UFT3 for exo-glucanases.
      AFRICAN JOURNAL OF BIOTECHNOLOGY 12/2014; 13(52):4694-4701. DOI:10.5897/AJB2014.14221
  • Konstantinos Kesanopoulos added an answer:
    What should the primer concentration be in a "multiplex PCR"?

    I am using a mixture of 2 forward primers and 4 reverse primers in a single pcr-mix for the detection of Arbuscular Mycorrhiza Fungi and I was wondering in what concentration should I use them. The reference article I am using is Kruger et al. (2009) where they describe the mixture of 2-4 primers respectively as "one primer" and the concentration they use of this "each primer" is 0.5 uM. I doubt whether the concentration of the 2 or 4 primers together would be 0.5 uM or each single primer's concentration should be 0.5 uM.

    Konstantinos Kesanopoulos

    Testing of different concentration is the key, as Luis mentioned before! Important role is also playing the ''efficiency'' (or quality if you prefer), influencing the necessary concentration. Be systematic and methodical and you will find the perfect primer mixture !

  • Christian Q. Scheckhuber added an answer:
    Does anyone have a protocol for isolating IMM and OMM in fungus?

    Ultimately, I'm attempting to find a giant (~1900 aa) protein encoded in the mt genome. I'll be isolating the mites via a sucrose flotation gradient protocol and then plan on using BN-PAGE and the 100k centrifugation method of isolating membrane bound proteins (from Schagger 1991). 

    Currently, all we know via the sequence data, is that it has 6 TM domains, so this is seemingly the logical way to go about finding it. My PI has expressed the concern that the sheer number of nuclear proteins in the mitochondria will cause too much noise for identification with simple Coomassie. He suggests separation of the inner and outer membrane fractions before isolation of membrane bound proteins.

    I've come across a protocol that uses digitonin with rat mitochondria, but have also seen issues associated with that protocol in other animal models. So I'm wondering if anyone has established a protocol that is optimized for fungi.

    Also - I've found alternative protocols for isolation of membrane bound proteins via treatment with ice cold sodium carbonate. If anyone could speak to the efficacy of this vs centrifugation, that would be swell.

    Christian Q. Scheckhuber

    A while ago I was working on the characterization of protein complexes in the inner mitochondrial membrane from the fungus Podospora anserina. The attached publication by Frank Krause et al. might be interesting for you because it contains BN-PAGE and CN-PAGE protocols optimized for the use with fungal mitochondria.

    Regards, Christian

    • Source
      [Show abstract] [Hide abstract]
      ABSTRACT: To elucidate the molecular basis of the link between respiration and longevity, we have studied the organization of the respiratory chain of a wild-type strain and of two long-lived mutants of the filamentous fungus Podospora anserina. This established aging model is able to respire by either the standard or the alternative pathway. In the latter pathway, electrons are directly transferred from ubiquinol to the alternative oxidase and thus bypass complexes III and IV. We show that the cytochrome c oxidase pathway is organized according to the mammalian "respirasome" model (Schägger, H., and Pfeiffer, K. (2000) EMBO J. 19, 1777-1783). In contrast, the alternative pathway is composed of distinct supercomplexes of complexes I and III (i.e. I(2) and I(2)III(2)), which have not been described so far. Enzymatic analysis reveals distinct functional properties of complexes I and III belonging to either cytochrome c oxidase- or alternative oxidase-dependent pathways. By a gentle colorless-native PAGE, almost all of the ATP synthases from mitochondria respiring by either pathway were preserved in the dimeric state. Our data are of significance for the understanding of both respiratory pathways as well as lifespan control and aging.
      Journal of Biological Chemistry 07/2004; 279(25):26453-61. DOI:10.1074/jbc.M402756200
  • Mehrdad Khatami added an answer:
    Can anybody suggest the simple procedure to isolate the nanoparticles from fungal fruit body at low experimental cost?

    Can anybody suggest me the simple procedure to isolate the nano-particles from fungal fruit body at low experimental cost?



    Mehrdad Khatami


    In the first cells of fungus should be disrupted by ultrasonication at 100W for 5min (cell degradation). Then the suspension then centrifuged at 10,000 × g for 30 min to separate disrupted cells (pellet) from NPs(supernatant).

    Have good time.


  • Boris Michaylovich Sharga added an answer:
    What is the best way to initially screen fungal isolates with potential biocontrol properties?

    I have almost a hundred fungal isolates. It wont be economical if assay all of them. Are there any set of fungal characters that I can use or look for to initially screen them to limit the number of isolates that I will be testing?

    Boris Michaylovich Sharga

    OK. It is not too much. You can evaluate them in vitro first, then in fild or greenhouse conditions. Actually, about 6% of them can show activity in field to different extent.

  • El-Sayed Ramadan El-Sayed added an answer:
    I am working on endophytic fungi, but the yield of ethyl acetate extracts is very low. Can someone suggest extraction method to increase yield ?

    Yield of  mass culture is good. But after extraction of fungal mass with  ethyl alcohol or other solvent  like methanol, yield is very low.

    El-Sayed Ramadan El-Sayed

    You can try methylene chloride and methanol (9:1).

  • Cheng Zhou added an answer:
    How to check the fungal spore germination?

    Currently I am working on the penicillium fungus strain and facing problem in spore germination. When I inoculate the production medium with spores 1X107 spores/ml, I did not get the desired concentration of cellulase protein but when I inoculated  by  disk of 1 cm diameter from plate I got the desired concentration of protein. I want to check the viability and germination of spores. Is it possible to see the germinating spores in microscope? If yes, Please do let me know. Please let me know any other relevant information regarding this. I am enclosing SDS gel pic for your convenience.

    Cheng Zhou

    the hanging-drop culture method with concavity slides can be use to observe the process of Spore germination. in addition,  Serial dilution and plating can be used to investigae viability of spores.

  • Sandip Chakraborty added an answer:
    How to extract DNA from Aspergillus spp. grown on solid medium?
    Protocol for DNA extraction
    Sandip Chakraborty

    You can please refer to the links below to obtain certain valuable informations:-



  • Sufi Hanna added an answer:
    How long can a sample for mycology (infected plant) prior to screening be stored under 4 degree Celsius?

    I took sample from infected stem of tropical tree in the forest. I read from some sources that screening should be done as soon as possible to get accurate result. It is optional to store sample under 4 degree but the duration limit of fungi viability or potential of contamination is not stated.Is there any other way to lengthen the viability of fungi in the infected sampel?

    Sufi Hanna

    Alphus Dan Wilson,

    Thank you for your answer but I have question.

    To induce sporulation besides having the sample under high humidity environment, does the sample require certain lighting mode  incubation method?

    The tissue sample may have more than one pathogen and  spores may mixed.  I am thinking of diluted the collected spores, streaking on plate, and subculture of each different colony appear. Please correct me if my method is wrong.

    Thank you

  • Olívio Fernandes Galão added an answer:
    What cell disruption method is recommended for freeze-dried fungal mycelium for the subsequent solvent extraction and fatty acid analysis by GC-FID?

    I have to determine total fatty acid content (mg/g dry biomass) and fatty acid composition from oleaginous filamentous fungi samples. 

    I have several freeze-dried fungal mycelium sample (low amount: 2-3 extraction possible with 50mg/extraction). I have tried so far grinding in morter with the addition of solvent. I did for at least 1 minute until complete homogenization, but still after extraction and transesterification (direct transesterification method by Lewis) I saw some intact hypha and lipid bodies inside the hypha. I'm working with Mucor, Mortierella and Penicillium at the moment. Does bed beating work without the addition of water? I need dry biomass for the extraction..

    Olívio Fernandes Galão

    Extraction of Lipids in Solution by the Method of Bligh & Dyer
    (Bligh,E.G. and Dyer,W.J. 1959. A rapid method for total lipid extraction and purification.
    Can.J.Biochem.Physiol. 37:911-917.)

  • Alexander Vlassov added an answer:
    Fungal DNA extraction kit
    Does anyone have good recommendations for kits for fungal DNA extraction from solid medium? I have many colonies to identify.
    Alexander Vlassov

    Try PureLink Microbiome DNA Purification Kit –

  • Tim Alexander Dahlmann added an answer:
    How can I purify fungal isolate from unknown contaminant?

    I have a culture seems to be Alternaria alternata . Culture is looking pure having single type but very very less spores. In sequencing showing mixed peaks, what reason could be if it is contaminated how can i purify it?  

    Tim Alexander Dahlmann

    I'm glad to hear that you solved your problem. Good luck with your studies.

  • Damion Neath added an answer:
    Can Hemileia vastatrix grow on agar?

    I want to know if Hamileia vastatrix can grow in the lab on artificial media. And if yes what type of agar?

    Damion Neath

    Thank you all for your responses. 

  • Jegan Sekar added an answer:
    Any advice on the microbes (apart from human pathogen) involved in rotting of pulp and paper?

    name of the species responsible for rotting of pulp and paper specially bacteria and fungi.

    Jegan Sekar

    Fungal community will be best source, it can produce laccase, Mnp and Lip which degrade the complex material easily. In bacteria Clostridium, pseudomonas and bacillus

  • Agu K C added an answer:
    Does anyone know the protocol for investigation of fungal induced deterioration of mineral pigments?

    I wish to test the ability of wall painting fungal isolates to degrade mineral pigments (eg. Red Ochre, Green Earth...) often used during the painting of murals, but I am having problem finding the suitable method.

    Any idea or protocol on how to prove that degradation took place, or even better quantify the amount of degraded pigment, would be really helpful.

    Thank you in advance.

    Agu K C

    The vapour-Phase transfer method of Okpokwasili et al., will be helpful with subsequent mathematical models of Freudlich, Lagmuir etc

  • Tim Jenkins added an answer:
    Does anyone know what is the name of the Enzyme involved in Alcoholic sugar biosynthesis in Candida albicans?

    Candida albicans groups Does anyone know what is the name of the Enzyme involved in Alcoholic sugar biosynthesis in Candida albicans?

    Tim Jenkins

    Sugar Alcohol Biosynthesis: Potential enzymes can include xylose reductase for xylitol synthesis and aldose reductase for L-arabitol synthesis - see attached link. Mannose-6-phosphate isomerase has been reported in Candida albicans (this could be followed by mannose reductase as identified in high mannitol producing Candida magnoliae HH-01 but not sure if it is present in C. albicans).

  • Juraj Medo added an answer:
    How can I design a primer with GC-clamp attached to ITS ?

    I have read the articel that is suggested by Kuldeep Patel.Thank you so much.

    I carried out DGGE to study microflora on 18S RNA using primer (NS1 and GC fung) based on Technical report from National Institute for Agro-Environmental Science japan (NIAES).

    Currently,I would like to identify spesies from DNA band of DGGE

    I would like to use ITS1 and ITS4 for DGGE to study microflora of fungi, I would like to ask how to attach GC clamp on the primer, and how long  the GC sequence?

    Anybody could help me please?

    Thank you

    Juraj Medo

    Generally, yes. But you will need amplify or to re-amplifiy the sliced band using DGGE primers i.e. one of them should have GC-clamp.  For  100bp you should use 8% acrylamide gel. For first time use high denaturant range, 30-70% and according to bands position you can use narow range in next run.

  • Ephrem John Ora Agorob added an answer:
    Good Day! What is the ratio for the amount of fungi to be included in the waste-water to the BOD/COD of the untreated waste-water?

    Good Day! I'm a chemical engineering student and I am doing my final year research on waste-water treatment using fungal strain (A. oryzeae). I just want to ask what is the ratio for the fungi to be placed in the waste-water (COB*:fungi or BOD*:fungi)

    *data fron the untreated waste-water to be used in the study.

    Ephrem John Ora Agorob

    Thank You sir C.P. Chin-pao Huang and David N Ogbonna.

  • Prithvi Shirahatti added an answer:
    How to prepare the spore suspension of Magnaporthe oryzae?

    Instead of the spore spread all over, I see lumps while using the haemocytometer for spore counting. 

    Prithvi Shirahatti

    The simplest way is to overlay tween 20/tween 80 to the culture and tease the ooze a bit with the needle and then collect the liquid using a pipette. Once you have this suspension, you can filter it to remove the impurities and then you are good to set your spore concentration.

  • Heba Shawky Mohamed added an answer:
    I want to know how to calculate the diameter of fungal spores and the length of the hyphae through scanning electron microscope?

    I make analysis using scanning electron microscope on fungal spores and I want to measure their diameters.

    Heba Shawky Mohamed

    I install it but can not use it

  • Virgil Tudor added an answer:
    How to extract ahcc from shiitake mushroom?

    I am trying to find some information on how the extraction of Active Hexose Correlated Compound (AHCC) from Shiitake mushroom can be done. I can't seem to find anything online. 

    Thank you for the help. 

    Virgil Tudor

    Hi Woo-Sik Jo,

    Thank you for your troubles.

  • Oadi Matny added an answer:
    How do I extract spore of Rhizoctonia solani?

    I want to extract spore of Rhizoctonia solani, so please anyone suggest me how can i extract the same and is their any specific stain for spore staining...?


    Oadi Matny

    you can grow Rhizoctonia on millet sorghum seed after soaking in water for 3-6 h after that pour out the water and sterilized in autoclave. after the seed be could inoculated with 3-4 disc of Rhizoctonia and incubated in 25 C for 10 day with shaking every 3 days. You can use this seed as inoculation to apply the soil with the fungus, 

  • Hero M. Ismael added an answer:
    How to exract fungul DNA from grains?

    I am trying to exract DNA of fungi out of grains. How do I extract DNA of fungi which are on a plant, grains and silage, since the DNA is dameged from enzymes of the host? How can I exract the DNA without damage?


    Hero M. Ismael

    as mentioned above CTAB is the best, also you can use kits as bioneer kit or GenAid kit

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