Fungal Biotechnology

Fungal Biotechnology

  • Fatma F. Abdel-Motaal added an answer:
    1
    What is the best method to visualize Trichoderma hyphae in the cortical region of the young roots of a tree?

    I have tried aniline blue and cotton blue staining for root sections. They're not effective, in this case.

    Fatma F. Abdel-Motaal

    Hi Benji

    Please try trypan blue, I think it is a good stain as I used to use it in fungal hyphae staining in plant tissue

  • Rama Krishnan Sankar Ganesh added an answer:
    7
    How to prepare a fungal spore suspension from a culture tube?

    I need Concentration like 106 or 107.

    Rama Krishnan Sankar Ganesh

     Viviana V Rivera thanking you for your timely help.

  • Laith Al-Ani added an answer:
    7
    What are the best primers to analyse fungal community by Illumina MiSeq: ITS1/ITS2 or ITS3 / ITS4 ?

    I'm going to send some soil samples to analyse the fungal community by Illumina MiSeq. I can choose between two pairs of primers: ITS1F-ITS2aR and ITS3F-ITS4R. I've been reading about this and I've found some studies that use ITS3 / ITS4. I have to use the region that I can amplify by PCR but I don't know if I have to consider anything else before choosing one or another pair with the aim to get the best results.

    Laith Al-Ani

    Hi Tatiana

    The primer ITS is a universal primers, and Forward ITS1 - reverse ITS4

  • Thiyam General added an answer:
    4
    Is there any water soluble purple pigment?

    Is there any report for purple pigment from fungal culture?

    -It is extracellular

    -water soluble and soluble in n-butanol.

    Your suggestion and advice will be helpful.

    Thank you

    general 

    + 1 more attachment

    Thiyam General

    Dear all 

    First of all thank you all for your kind reply.

    In think Violacein is insoluble in water. Whereas, this pigment is an extracellular pigment.

    I have checked the morphology of the fungal culture, and it has spores structure similar to that of Fusarium spp. Literature suggested Fusarium spp  producing purple pigment.

    Thank you everyone for your kind suggestion. 

  • Sattar Shah added an answer:
    5
    Which method is best for fungal isolation, Rhizpus rot (Rhizopus stolonifer) in stone fruits?

    Which method is best for fungal isolation, Rhizpus rot (Rhizopus stolonifer) in stone fruits?

    Sattar Shah

    Thanks to all

  • Aleksa Mijuca added an answer:
    3
    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. 

    Aleksa Mijuca

    I cant find attachment, could you please leave it here. Thank you!

  • Shaoxuan Qu added an answer:
    5
    Do you know the scientific name of this Basidiomycetes?

    This wood rotting fungi produce basidiocarp in cluster, having free gills stipe without ring

    + 1 more attachment

    Shaoxuan Qu

    I mean this fungi.

  • Feng Jin Liew added an answer:
    12
    How do I homogenise fungal mycelium from plates or liquid cultures to make an inoculum?

    Often in literature it is only mentioned that fungal cultures are homogenized but rarely it mentions the  time, type of homogenizer etc. There surely must be optimal choices as some bad choices as different homogenizers have different rpm power, blades can be of different quality and can be blunt of sharp. How long to homogenize to get best  propagules (mucelial fragments, blastospores, other spores..)  and the least of dead shattered mycelial fragments. I am working with basidiomycetes (wood decay fungi) . Any suggestions and personal experimences?

    Feng Jin Liew

     Hi, I am interested in homogenize my fungal biomass but is there a way to tell if the mycelium is from the aerial hyphae or the submerged hyphae? 

  • Xiao Jiang added an answer:
    15
    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?
    Xiao Jiang

    Hi Rosa,

    Thank you for sharing your experience. I use 120uM HNB from sigma, though I have no betaine in the buffer. However, when I try to use 4mM MgSO4 with the neb buffer, the HNB stays sky blue (both negative and positive, before and after incubation). So I assume the PH in the buffer is right. It is possible that something in that buffer is preventing the Mg ion to precipitate out with pyrophosphate ion. I have not tried other buffer yet but the neb buffer might be the cause if you are also experiencing it. They do suggest HNB for colorimetric detection in their website so I was thinking that there is something I have done is wrong to cause the problem. 

    Maybe you can try the neb buffer or whatever buffer you have problem with and try to adjust its Mg concentration by adding MgSO4. For neb I am able to get from 2mM all the way to above. And add HNB, if the HNB changes its color (from sky blue to violet) from the lower concentration to the higher, that would mean at lest the PH and HNB is right. 

  • Camilla Maciel Rabelo Pereira added an answer:
    4
    Why did the primers AML1/AML2 and NS31/AML2 amplify so many Dikarya Ascomycota?

    We tried to amplify  AM fungal DNA from root or soil samples from Tibetan Plateau with the primer combination AML1/AML2 or NS31/AML2. But the result is not good. There are so many sequences belonging to Dikarya ,Ascomycota. Only 20% was AM fungi. It was like winning the lottery. I do not know whether you had similar results. How to solve it ?

    Camilla Maciel Rabelo Pereira

    You could try the combination NS31-AML2 

  • Abid Aziz Ajaz added an answer:
    4
    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?

    Abid Aziz Ajaz

     As said above, the enzyme name will be after the name of the carbohydrate sugar involved in the process. So first determine  / name the sugar you are going to react enzymatically. 

  • Gururaja G M added an answer:
    4
    What is the best technique for polysaccharide extraction in Dictyophora indusiata?

    Optimum extraction for polysaccharides  What is the best technique for polysaccharide extraction in Dictyophora indusiata?

    Gururaja G M

    Dear Researcher,

    Please follow our publication.

    http://www.phcogres.com/article.asp?issn=0974-8490;year=2013;volume=5;issue=2;spage=71;epage=79;aulast=Chandrasekaran

  • Rafik Karaman added an answer:
    1
    Is it possible to perform anti-denaturation assay using heat treated BSA with fungal extracts?

    How can I perform anti-denaturation assay " screening assay for detecting anti-inflammatory compounds" using heat treated BSA with fungal extracts?

    Rafik Karaman

    Dear Skanda,

    The attached publication covers the answer to your question.

    Phytochemical Screening, Antimicrobial and in vitro Anti-inflammatory Activity of Endophytic Extracts from Loranthus sp.

    ARTICLE in PHARMACOGNOSY JOURNAL 3(25):82–90

    ABSTRACT

    Four different endophytes were isolated from different parts of Loranthus sp. Methanol and water extracts of all the endophytes was assessed for its antimicrobial and anti-inflammatory activity and phytochemical screening. Phytochemical analysis revealed the presence of tannins, flavonoids, terpenoids, steroids, alkaloids, phenols and saponins. The antimicrobial efficacy was determined using paper disc diffusion method against different fungi and bacteria. Sensitivity in terms of zones of inhibition and phytochemical composition of the all endophytic extracts were also determined. The results show that, A. niger, Penicillium sp., Alternaria alternata extracts effective against all the bacteria and fungi tested, whereas A. flavus extract was failure in inhibiting the growth of all bacteria and bacteria. In vitro anti-inflammatory activity was evaluated using albumin denaturation, membrane stabilization assay and proteinase inhibitory assay. Aspirin was used as a standard drug for the study of anti-inflammatory activity. A. niger, Penicillium sp. and Alternaria alternata methanol fractions showed in vitro anti-inflammatory activity by inhibiting the heat induced albumin denaturation (87.88, 86.89, 87.03 g/ml) and red blood cells membrane stabilization with 78.42, 77.61, 77.98 g/ml respectively. Proteinase activity was also significantly inhibited by the A. niger (85.21 g/ml), Alternaria alternata (84.09 g/ml) and Penicillium sp. (79.17 g/ml). BSA anti-denaturation and HRBC membrane stabilization assay indicated that the methanol extracts of A. niger, Penicillium sp., Alternaria alternata possess constituents with anti-inflammatory properties. From the result, it is concluded that phytochemicals (tannins, flavonoids, terpenoids, phenols, steroids, alkaloids and saponins) present in the A. niger, Penicillium sp., Alternaria alternata extract may be responsible for the antimicrobial and anti-inflammatory activity.

    Hoping this will be helpful,

    Rafik

  • Nadeem Ramadan added an answer:
    5
    Does anybody have an idea about isolating a single spore of Beauveria bassiana from a suspension?

    I am using an electrofusion technique to develop a hybrid isolate. After staining two types of spores by two different fluorescent dyes and applying the electric shock, I am able to see some hybrid spores under the fluorescent microscope. My goal is to isolate the hybrids from a mix suspension that contains parental spores and hybrid ones. Any idea or method for this?

    Nadeem Ramadan

    Single spore technique method ( suspension and distributed on water agar)

  • Enas Elfadly added an answer:
    1
    Could anyone help me understanding why CAPPED Ag NPs with tri sodium citrate don't have any antifungal activity?

    All references reported that Ag NPs have an anti-fungal activity, but after I'd synthesised it using tri sodium citrate as a capping agent it didn't have any activity at any concentration. So, Does tri sodium citrate have any effect on the nanopartecles nature?   

    Enas Elfadly
    @Thalij thanks alot for your quick response. So, how could I synthesis Ag with a small size without capping agent?
  • Eduardo Hernández-Navarro added an answer:
    13
    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:
    2
    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:
    10
    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:
    3
    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

  • Vladimir Ostry added an answer:
    1
    How is Aflasafe applied to groundnut fields? and at what rate?

    Aflasafe is a biological control which reduces aflatoxin contamination.

    Vladimir Ostry

    Dear Ephrem Guchi,

    an answer to your request is in attached article pp. 39-41

    I send you a contact to foremost expert in this area:

    Contact information: Dr Ranajit Bandyopadhyay, plant pathologist, r.bandyopadhyay@cgiar.org

    + 1 more attachment

  • Brigitte Sthepani Orozco Colonia added an answer:
    10
    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.
      Full-text · Article · Dec 2014 · AFRICAN JOURNAL OF BIOTECHNOLOGY
  • Konstantinos Kesanopoulos added an answer:
    4
    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:
    3
    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.
      Full-text · Article · Jul 2004 · Journal of Biological Chemistry
  • Mehrdad Khatami added an answer:
    3
    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?

    regards

    DR.KiranRanadive

    Mehrdad Khatami

    regardDR.KiranRanadive

    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.

    regard

  • Boris Michaylovich Sharga added an answer:
    3
    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:
    5
    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:
    5
    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:
    7
    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:-

    1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC516345/

    2. http://www.fgsc.net/fgn50/fgn50punekar.htm

  • Sufi Hanna added an answer:
    4
    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

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