Fungal Biotechnology

Fungal Biotechnology

  • What are your sampling and DNA extraction methods for fungal communities?
    I have been trying to get a sample with a high concentration of fungal DNA. I tried using dry sterile swabs, swabs moistened with saline solution, sodium chloride and Tween 20 solution and water. However I wasn't able to get a concentration good enough for amplification (my kit calls for 5-20 ng/ul).
    I was hoping to get any advice from those who has already done that.
    Thank you!
    Mohammed saleem Ali-shtayeh · Biodiversity and Environmental Research Center

    You might like to see the attached file.

    Good luck

  • 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 · Research Center for Food and Development A.C.

    Thank you so much, Dr. Baseia! I'll look for it.

  • Sangeetha Siva Sangu added an answer:
    What is the air humidity inside of a Petri dish?
    What is the air humidity inside of a Petri dish (94x16) with fresh PDA medium, inoculated with 0,5 ml of spore suspension, at 25 degrees Celsius, during a 32 hours incubation? It depends on the incubator humidity? I need saturated atmosphere for 32 hours. Thank you very much in advance!
    Sangeetha Siva Sangu · University Malaysia Sarawak

    thank you very much Mr. Vijay Kumar Sharma

  • Nhật Minh Võ added an answer:
    is there any method to isolated and purified ganoderic acids from Ganoderma lucidum without HPLC ?
    I only have this file that ganoderic acids from Ganoderma lucidum isolated by HPLC.
    Nhật Minh Võ · Can Tho University

    Thank you so much. i will try.

  • Mansoor Hussain added an answer:
    What is the treatment condition for KT5720, PKA inhibitor for fibroblast cells?

    I would like to know the treatment condition for KT 5720 PKA inhibitor for fibroblast cells. Earlier i have used 5um concentration for 16 hours. 

    Mansoor Hussain · National Institute of Immunology

    thats informative Mr.Kaushik.. my only concern is the limitation of inhibitor that i have. i've used 5um concentration. i cant do time course expt as i mentioned above. But i think i should do that. And yeah i will also use the positive controls when i do my experiment next time. Thanks for your valuable suggestions!!! 

  • Helmut Brandl added an answer:
    What else can cause by-product formation in Aspergillus terreus fermentations besides low pH conditions?

    I am using a strain of Aspergillus terreus which mainly produces oxalic acid and a little of other organic acids, but it doesn't produce much of itaconic acid which is the metabolite of interest, even at pH 2. Considering the fact that low pH is one factor that minimizes undesirable by-product formation, what else can i do to ensure the strain produces more itaconic acid. The medium contains sufficient amounts of glucose and N-source

    Helmut Brandl · University of Zurich

    Perhaps this publication helps: Hevekert et al. (2014). Applied Microbiology and Biotechnology 98:6983.  I do not have experience with A. terreus, but with A. niger regarding the formation of citrate and gluconate (see Bosshard et al. (1996) Environmental Science and Technology 30:3066). In the absence of manganese citrate is formed, whereas in its presence gluconate is formed. Perhaps there is a similar regulation in A. terreus.

  • Michaela Conrad added an answer:
    Does the accumulating CO2 levels become toxic to yeast during fermentation?

    What will happen to the yeasts if the vessel carrying out yeast fermentation is tightly sealed? Will the rising level of CO2 inside the vessel be toxic to the growing yeasts or the yeasts have a strategy to overcome this situation?

    Michaela Conrad · Vlaams Instituut voor Biotechnologie

    You will also need a very stable vessel otherwise it might just blow up. Not a direct problem for your yeast, but not nice for your bench neighbours ;)

  • Martua Manullang added an answer:
    How to prepare samples for fungal hypahe/spores for environmental scanning electron microscopy?
    For Alternaria alternata. Any suggestions?
    Martua Manullang · Universitas Riau

    How to prepare dry mycelium for SEM analysis ??
    I want to SEM (Scanning Electron Microscop ) analyzes on samples that had been dried mycelium with a freeze drier.. Thanks

  • Dhinakarasamy Inbakandan added an answer:
    Can someone explain the effects of nanoparticles on plant or fungi gene expression, please?

    Manganese peroxidase is a ligninolytic enzyme encoded by MnP genes in fungi. Expression of this gene is increased by Mn ions as substrate of MnP enzyme. Do manganese nanoparticles have any effect on mnp gene expression as well?

    Dhinakarasamy Inbakandan · Sathyabama University

    Dear Mojtaba Lotfi,

    Normally Metal Nanoparticles will have impact on oxidative stress genes, in particular metallothionein genes. In your case, hope Manganese peroxidase kinetics in the presence of manganese nanoparticles itself is a good work. Further you can confirm it in your fungal model with enzyme production followed by gene expression studies in the presence and absence of NP.

  • Ervín Jankura added an answer:
    How to control fusarium in microbiology QC lab?
    There are number of fungicide which can be used to control different type of fungi but when you test a particular product for presence of fungi you got to avoid such fungicide, what you will do in this situation if you are having problem of fusarium contamination in your media you used for yeast and mold analysis?
    Ervín Jankura · Plant Production Research Center Piešťany
    Formalin alternative:

    https://www.researchgate.net/publication/230182896_Evaluation_of_a_Peroxyacetic_Acid_Disinfectant_in_Hotwater_Treatment_for_the_Control_of_Basal_Rot_%28Fusarium_oxysporum_f._sp._narcissi%29_and_Stem_Nematode_%28Ditylenchus_dipsaci%29_in_Narcissus
  • Deepti Agrawal added an answer:
    How can I determine inulinase activity?
    I am working on inulinase production from fungal sources. I followed DNS method for inulinase assay. However, I have a problem. How can I calculate inulinase activity in Units per mL/min?
    Deepti Agrawal · Indian Institute of Petroleum (IIP)
    You are most welcome Aftab
  • Ali Vaiz Garipoglu asked a question:
    How can I find or obtain Lentinula edodes and Bacillus pumilus cultures to be used in my study?
    How can I find or obtain Lentinula edodes and Bacillus pumilus cultures to be used in my study?
  • Senthil Sankar added an answer:
    What is the best way to induce zoospores in a Phytophthora palmivora sample?
    I have a culture of P palmivore on V8 and agar oats, the culture is 10 days old, but the zoospores production is to low only 2 or 3 zoosporores. Could anyone help me with a better protocol?
    Senthil Sankar · Aix-Marseille Université
    V8 is used for zoospore production in wide Phytophtora species but i did not get enough zoospores of P. palmivora in V8 agar media. Even the host material can also be tried.
  • Andrea Lessing asked a question:
    Has a complete pathway for methoxypyrazine production in either plants or microorganisms specifically fungi been described?
    I have recently read a publication which describes the last enzyme in the pathway but I need information regarding enzymes which function in earlier steps.
  • Muhammad Faraz Bhatti added an answer:
    Does someone have a reliable protocol for transformation of Botrytis cinerea (or other fungi with melanised cell wall)?
    I have tried a couple of published protoplast/PEG mediated methods and tried a homebrew electroporation (similar to a method which has been very successful for Neurospora), none of which have been successful.
    I know that my protoplasting is working as the protoplasts look big and round and recover well before the antibiotic is added. I have a tried and tested positive control for insert DNA so it must be my transformation step that is not working.
    I heard from other Botrytis workers that transformation is tricky and needs optimizing, any hot tips would be appreciated.
    Muhammad Faraz Bhatti · National University of Science and Technology
    I have adapted this method of protoplast fusion / fungal transformation (Please see attached file). It worked really well for me. Please find the paper as an attachment. I hope that will help. Regards
  • Ayat AL-Laaeiby added an answer:
    What is the best protocol for transforming fungal cell?
    Is electroporation of conidia more efficient than peg mediated transformation of protoplasts? Does the protoplast suspension in peg plated on large sized petri plate have a layer of soft agar? How long does it take for regeneration of the protoplast?
    Ayat AL-Laaeiby · University of Exeter
    I want to knock out gene, I used protoplast it is succeed for one gene and I want to KO another gene with electroporation. Can someone share protocol for KO gene with electroporation technique?
  • Narong Chamkasem added an answer:
    HPLC determination of Ochratoxin A.
    I have isolated Aspergillus ochraceus strains. I would like to quantify the ochratoxin-A produced by those cultures. I am growing the cultures in 250ml of broth medium. Now I would like to quantify the Ochratoxin-A of those cultures. How can I extract Ochratoxin from those liquid cultures?How can I test the presence of OTA through TLC? Later how can I quantify OTA through HPLC? What is the best detector (UV/Florescent) for detection of OTA? What are safe levels of OTA in foods and feeds especially in Poultry feeds.
    Narong Chamkasem · U.S. Food and Drug Administration

    I got an old paper here.

  • Matheus Sanita Lima asked a question:
    Has anyone seen a endoxylanase with an optimum pH at 3.5? And a b-xylodase at 2.5/3.0? I think I got some nice stuff here!
    I am working with the xylanolytic system of Aspergillus niger and I have found these two enzymes (endoxylanases and beta-xylodases) with very low optimum pHs!

    I have checked many times if there was something wrong with my methods but it really seems those enzymes are very acid resistant.

    Has anyone ever seen very low pHs like those?

    Thanks!
  • Ruchi Singh 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?
    Ruchi Singh · Indian Council of Medical Research
    a correction


    read

    even if LAMP reaction has not taken place.
  • Binnur Tüzün added an answer:
    How to elute the adsorbed metals from the cell wall on mycelia? Can I do AAS from the elute?
    I'm doing the metal adsorption by different fungi and got stuck on this step
    Binnur Tüzün · International kolan hospital
    ıf you reverse them water soluble you can elute them. For example copper acetate or sulphate is water soluble but copper insoluble. 1:1000 acetic acid may be added
  • Vladimir Ostry added an answer:
    How can I identify Fusarium culmorum species?
    I want to identify (confirmed) Fusarium culmorum by molecular tech. Can anyone tell me the specific species or ITS primer sequence for this species or any other tech. to confirm the identity of this species?
    Vladimir Ostry · Státní Zdravotní Ústav
    Dear Vipin Panwar,
    I recommend the use of primers from an article published in International Journal of Food Microbiology "Verification of the effectiveness of SCAR (sequence characterized amplified region) primers for the identification of Polish strains of Fusarium culmorum and their potential ability to produce B-trichothecenes and zearalenone.
    Authors: Anna Baturo-Ciesniewska and Michalina Suchorzynska.
    My best regards
    Vladimir
  • Hima Ranjith asked a question:
    What is the best protocol for trichoderma protoplast fusion?
    Please suggest some article.
  • Mª Victoria Durantez asked a question:
    Does anyone know what tag is used to purify proteins by immunoaffinity column method in Botrytis?
    I want to isolate certain proteins in Botrytis and want to know which tag is used
  • Subash Nachimuthu added an answer:
    Can anyone suggest for me a known biofungicide?
    Fungicide
    Subash Nachimuthu · Nehru Memorial College
    Dear
    Trichoderma harzianum has been proved to be a fungicide to control Rhizoctonia solani and damping-off in chilli.

    Please see this article
  • Kalyanaraman Rajagopal added an answer:
    How to identify and characterize fungi growing on PDA?
    How to identify and characterize fungi growing on PDA?
    Kalyanaraman Rajagopal · Vels University
    For identification best way is slide culture. Remove block of mycelium growing from margin of the colony. Keep it in the moisture chamber. Keep the agar block in the middle of glass slide and keep sterile cover slip over block. Incubate for 72 to 96 hrs. Remove the cover slip stain if required and observe through the microscope.
  • Beatriz elena Guerra added an answer:
    Why does Cd inhibit sporulation in fungi?
    What is the mechanism by which Cd inhibits sporulation of fungi? I\m working in the laboratory and I have some strains of Aspergillus that do not sporulate in the presence of Cd in the culture medium.
    Beatriz elena Guerra · Universidad de Santander
    Thanks Alex
  • Jyoti S added an answer:
    Can anyone please help me to give your suggestions about a biochemical test for fungal isolates isolated from water and soil?
    I want to know is their hope for differential media based biochemical tests?
    Jyoti S · Babasaheb Bhimrao Ambedkar University
    Thanks a lot Anna...
  • Can someone advise on extracting fungal DNA with Promega Kit?
    I try to extract DNA from Septoria tritici with Wizard genomic DNA purification kit from Promega Madison WI. the manufacturer's manual does not give any indications for fungus but only for yeast, blood cells and plant tissues. Could anybody point me to a reliable protcole for my fungus, Septoria tritici
    thank you in advance
    Graham Robert David McGrann · Scotland's Rural College
    Hi Meamiche,

    I've extracted good quality genomic DNA from Mycosphaerella graminicola cultures using the Nucelon Phytopure kit:

    http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences/25006720

    If you make a spore suspension with sterile water, then spin it down in an eppendorf to pellet the cells and remove the supernatent you can freeze the pellet at -80C for storage and/or proceed with the DNA extraction following the Phytopure kits instructions. I've isolates good quality, high yielding genomic DNA this way for use in qPCR reactions and other downstream processes. IThis kit has been particularly good for extracting genomic DNA from Mycosphaerella species that are difficult to obtain high yields good quality DNA extraction from (e.g. Ramularia collo-cygni).

    Hope it helps?

    G
  • Mukesh Dubey added an answer:
    Why does Agrobacterium tumefaciens develop visible clumps (agglutination) in liquid LB medium?
    What is the solution to stop this? And is it okay to use clumped cells for transformation?
    Mukesh Dubey · Swedish University of Agricultural Sciences
    Thank you Alex Ignatov for suggestion.
  • Trung Trieu added an answer:
    Which is the most effective way to isolate the homokaryotic transformants in filamentous fungi?
    I have several transformants of Mucor circinelloides which are heterokaryons. I often isolate the homokaryotic strains by growing spores on the selective media.
    However, this method requires a long time for selection, and it is not effective sometimes.

    Can you give me some other protocols that could be more effective than that?
    Thank you in advanced.
    Trung Trieu · University of Murcia
    Thanks a lot for everyone for your kind suggestions.
    I have tried using the filter with the pore-size is 5 micro meter to select the small spores (these spores could have one single nucleus).
    However, the percentage of positive transformant nuclei was significantly reduced after filtering, and I also could not isolate the homokaryon strains.
    No, I am trying to isolate homokaryon strains by subculture without filter. I will check the results after 9th vegetative cycle.

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