Endophytic Fungi - Science topic

Leonardo Brandão I am afraid that only plant reaction can differenciate endophytic (probably beneficial for host) and pathogenic (quiescent / latent infection, thus harmful) fungi. I remember that somebody estimated that >60% of plant infection is present in latent stage a (symptomless) .

Kumbar Manjunath 1) Follow the recommendations of Ricardo Julian Licea-Moreno

2) Meristem culture helps to eliminate virus, endophytic bacteria, and fungi.

See:

Cassells, A.C. (1991). Problems in tissue culture: culture contamination. In: Debergh, P.C., Zimmerman, R.H. (eds) Micropropagation. Springer, Dordrecht. https://doi.org/10.1007/978-94-009-2075-0_3

You will have to perform GCMS or LCMS. Find the probable compounds for 0the highest peaks. For preparing the sample, you let the extracted potion air dry until the ethyl acetate portion evaporates. You need to mix the remains in a solvent (eg. methanol) which depends on your GC configurations.

As of inputs by Francis Mwaura Mwaura based on the studies, fungi are able to degrade the Carbendazim if presence during the experimental conditions or if the isolates have gained resistance at its natural habitite. If so during the detection the derviative or degraded products of Carbendazium is expected.

Secondly, in order to confirm, repeation of experiments is needed considering different factors: nature of the media, sample preparation process, and importantly using a righ tool for analysis and identification of componds in different avaiable liberaries meant for fungal metabolities.

Another way is using standard Carbendazium during the sample preparation and analysis.

A good response by @Shasha Hu.

In addition, I feel that the primary purpose of a small scale production is the verification of laboratory procedures and their modification before engaging in large scale. Some of the benefits include minimize manual handling and economic on scale. They also help the researcher to adjust key variable before a move to large scale

Large scale fermentations are utilized to produce massive quantities of secondary metabolites by selected fungi. Large scale experimentation are time consuming and expensive, which is why the small scale is necessary (especially, in the case of novel study).

In summary, the small scale endophytic fungi culture allows for the preliminary screening of numerous fungal extracts for desired bioactivities or products. From which the best performers can be selected for large scale to further the study, increase the scope and characterize the compounds produced.

Good luck !

Six different isolation media: plain PDA, plain MEA, PDA supplemented with host plant powder (PHP), PDA supplemented with host plant water extract (PPE), MEA supplemented with host plant powder (MHP) and MEA supplemented with host plant water extract (MPE) were studied and the results showed that the isolated

The lyophilization conditions depend on the quantity of dissolved matter, the solvent that use in the sample and the type of the freeze drying requirements. The deep freeze degree must be -196 ⁰C.

Hi! According to a study (Peteros and Uy, 2010), we also used the same concentrations of the experimental control (plant or fungal extract) for the Potassium Dichromate, for comparison.

Potassium Dichromate is also mixed with DMSO with the Artificial Sea Water (ASW), just like what we did for the plant extracts.

Hope this helps!

Ref: Peteros & Uy, 2010. Antioxidant and cytotoxic activities and phytochemical screening of four Philippine medicinal plants. Journal of Medicinal Plant Research.

A method for the quantitative determination of gibberellic acid in fermentation media, using descending paper chromatography in butylacetate-water.

Using only colony morphology is impossible. Light microscopy could help identifying some isolates at higher taxonomic levels, but you will still need to at least sequence the ITS region. For some genera you need to sequence more regions.

It seems that an another fungus is appearing in the plate.

The genes you mentioned of are more reserve and stable

We have a paper in press about endophytic fungus Saccharomyces cerevisiae in medicinal plant and see that this yeast idinduces the plant to produce phenol, quinines and other importnat secondary metabolites.

Check https://bmccomplementmedtherapies.biomedcentral.com/articles/10.1186/s12906-021-03269-3

Endophytic microorganisms may control growth and development of host plants. Importantly, endophytic microorganisms living inside their host can often be cultured and grown after isolation from their host plants.

Are these fungal growths present in the dormant seed prior to hydroponic growth? What is the relative abundance of the sequences from the fungal growth relative to other sequences present in the dormant seed? What is the species of these fungal growths, are they endophytes?

My thought would be that the semi-hydroponic environment would be creating perhaps a greater selective pressure for some fungal species over others, this pressure maybe greater than that created by the host plant species genotype?

thank you so much for your reply

ppm

With the continual improvement in high‐throughput sequencing technology and constant updates to fungal reference databases, the use of amplicon‐based DNA markers as a tool to reveal fungal diversity and composition in various ecosystems has become feasible. However, both the primer selection and the experimental procedure require meticulous verification.

however, with the continual improvement of sequencing technology and constant updating of fungal reference databases, it is necessary to re‐evaluate existing primers and design new sets continuously. Trade‐offs among coverage, specificity, and amplification length will inevitably alter community detection. This will lead to more accurate estimations, especially for soil and other highly diverse ecosystems.

Jack Silver and Francisco Kuhar many thanks for the suggestions and the links. I will have a look of this. Best wishes

Within the ergot like fungi there is an apparent evolutionary series which show how a parasitic plant parasitic pathogen can evolve into a mutualistic endophyte.

In tbe dance of adaptation this can be seen in the way a toxic principle like curcurbitin can be something that the cucumber beetles can actually key into.

Bact to the fungi in the case of ergot fungi there can be the reduction of the seed by the fungi.

As the fungal alkaloids the compounds of the fungus become something that is positive for the host plant allowing that drought and insect attack can be better tolerated the parasitic and pathogenic relationship transforms into mutualistic symbiosis. .

The disease fungus which eliminated the seed of the grass cereal becomes a e

accommodating mutualist symbiont co inhabitant that becomes transmitted with the seed rather than killing it. When a systemic fungicide frees the seed the resulting seedling are disadvantaged and readily attacked by insect and drought.

In this way a negative host parasitic relationship leading to disease becomes converted into a symbiosis where the host and parasite are both favored by co evolving for their mutual benefit.

Dear colleague,

I have always put the roots in the water for one hour, then I have cleaned them with a brush, then I have washed them with distilled water. Then I have always put the roots of annual plants in NaOCl 1% for one hour, especially for thick roots. After that, I have washed them with distilled water three times, and then I have cut them with a sterile scalpel, and finally, I have put them in alcohol 70% for one minute and have washed with distilled water three times. I have moved them to PDA plates when they got dried. I have always added 200 ppm/liter of antibiotics include Penicillin G, and Streptomycin when I prepare the PDA medium.

For leaves, you can put them in NaOCl 1% for two or three minutes.

All the steps have to do in the laminar hood!

this article is good for your question

https://www.ijcmas.com/6-3-2017/Swapan%20K.%20Tripathy,%20et%20al.pdf

Thanks Dear Rajesh Dev Sarkar , Mubarak Ali Khan and Abhijeet Singh for your valuable information.

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

Hi,

The important part of endophytic fungi isolation is disinfecting of plant organs.

You have to do the washing with distilled-water and disinfecting with hypochlorite-sodium and also ethanol, because it is necessary to eliminate of epiphytic fungi. So, if you do this step better, you will be sure that the isolated fungi are endophytes but not 100 percent! Because maybe a resistant spore such as chlamydospores could survive.

The endophytic fungi are in part of plant host, and you can find them everywhere.

I am agree with Rohit. Please find out the attached document. A fascinating article reported the significance of sold media on production of interesting secondary metabolites (SMs). they investigated the impact of different media types, liquid media and solid media, on the SMs production.

Following

Good question........

Guba, E. F. 1961. Monograph of Pestalotia and Monochaetia. Harvard University Press, Cambridge, MA.

Hepperly, P. R., and J. B. Sinclair. 1977. Aqueous polyethylene glycol solutions for treating soybean seeds. Seed Science and Technology 5:727-733.

PEG 6000 molecular weight is nontoxic and forms aqueous solutions when water is add. Adding the proper amount of PEG can produce a swelling of the seeds without the seed coats being rupture. If these aerated solutions are rinsed off and dried the dried seeds will germinated more uniformly and faster.

I believe the technique can be widely adapted to various grain seeds.

I would suggest the colonization of endophytic fungi be done by growing the inoculum on vermiculite perlite with nutrients and then applying the inoculum with the seeds at planting. The endophyte will enter the germinating seedling roots and colonization of plants will spread alone with the root systems.

For this type of inoculation Pirisporoforma indica and acremonium endophytes which produce plant stimulating alkaloids would be good candidates. The first fungus would be a good selection based on its good ability to grow on media and soils.

In relation to the inoculum one volume of perlite is mixed with one volume vermiculite then a potato carrot broth could be the nutrient source.

Grow the inoculum in stoppered Erlenmeyer flask for mason jars using aseptic techniques.

Dear Sir. Concerning your issue about the determination of bio/neurological activity of purified metabolites obtained from endophytic fungi . Endophytic fungi are capable of producing plant associated metabolites and their analogs with therapeutic value. In order to identify the potential endophytic isolates producing bioactive compounds, one need to screen all isolated endophytes, which may run into hundreds. Isolation of endophytic fungi is relatively a simple process; but screening of the isolated fungi for required metabolite production is a cumbersome process. Endophytic fungi producing plant associated metabolites may contain genes involved in the entire biosynthetic pathway(s). Therefore, ascertaining the presence of key enzymes of a particular biosynthetic pathway could serve as a molecular marker for screening of these endophytes to produce that metabolite. In absence of entire biosynthetic pathways in endophytic fungi, plant genes associated with that metabolic pathway could serve as markers. This review focuses on the impact of molecular approaches to screen the endophytic fungi for the production of bioactive compounds. An attempt has been made on screening of anticancer compounds like taxol (paclitaxel), podophyllotoxin, and camptothecin using molecular markers. The advantages of molecular approaches over conventional methods to screen endophytic fungi and also identification of endophytic fungi are discussed. I think the following below links may help you in your analysis:

https://www.frontiersin.org/articles/10.3389/fmicb.2016.01774/full https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5042905/pdf/13205_2016_Article_518.pdf https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5270730/pdf/MBT2-10-175.pdf

Thanks

Elizabeth Kimbrough,

I got it, like this you have to measure growth rate daily till the fungus cover the plate (control) then you have to take the measurement for the last day and dived by total days.

As an example, I want to measure Phytophthora growth rate, usually the fungus covers the plate within 7 days. I take the measurement every day but to calculate, I will take the measurement for day 7 only then dived by 7.

Note: you have to -0.5 (Agar disk).

Yes, you can surface sterilize after the explants cut, but it will certainly affect the explants growth. However, you cannot eliminate endophytes in this way. You have to follow different approaches for the endophytes like selection & types of explants, culture medium, sub-culturing, use of antimicrobial solution in the medium etc.

I am very much on board with using sequence variants (i.e. ESVs, ASVs, ZOTUs are all terms used) as opposed to clustering sequences by similarity. My rationale is based on both biology and technical concerns.

1. As you mentioned, the similarity threshold one chooses is largely arbitrary and determined via consensus among practitioners. In some cases, taxa that are >97% similar ARE functionally different and have been labeled different things by taxonomists, and in other cases the 97% threshold works ok for delineating "species". There is not a whole lot of guidance out there for when the threshold works and when it doesn't. If you are working in a remote system and sequencing things nobody has sequenced before, then your guess is as good as anyone's regarding how well the 97% threshold captures variation at some specific level of the biological hierarchy. To sum up, the arbitrary nature of the 97% threshold is unsatisfying.

2. Depending upon the length of your amplicon, a percentage based threshold will require more or fewer bases to generate a new OTU. E.g. for a 100 bp long amplicon 4 bases need to differ between two sequences for them to be >3% different, but for a 200bp long amplicon 7 bases do. The denominator changes in the percentage calculation. For fungi in particular I think this is an issue because the ITS region is variable in length, so the 97% threshold (or any other threshold) likely means different things across the fungal phylogeny.

3. If you use a similarity threshold then you can inflate richness estimates and community membership slightly within a sample. Say for instance you have two 100bp sequences in a sample (call it sample 1) that are identical except one has AAAA and the other has TTTA at the same locus. If you processed this sample by itself these two sequences would probably get assigned to the same OTU because they are 97% similar. However, further suppose that you are processing several samples together and in one of these other samples you call an OTU that has the sequence TTTT at our focal locus. Now, if we processed sample 1 with this extra OTU sequence in our consensus OTU database we would find that two OTUs are in sample one! To sum up, the similarity threshold can lead to inflated OTU estimates in some cases when processing more samples together (which we all do) than if you processed those samples independently.

4. When using similarity thresholds one throws away biologically meaningful variation. One can use relative abundance of exact sequence variants to learn about genetic variation within a taxon. In my mind, the goal is to learn about biological variation, what led to that variation, and what are the consequences of that variation to communities. Therefore we should retain all our information. This is the Bayesian in me....

You could check out a paper I just published for an example of analyzing ESVs within a focal taxon to link genetic variation to differences in the production of a focal metabolite (Harrison et al. 2018, J of Ecology). I found the ESV approach very rewarding here as it let me learn something regarding the consequences of genetic variation within a fungal taxon that I would have missed if I had only used the standard 97% similarity threshold

5. Using exact sequence variants better facilitates cobbling data together from disparate labs, methods, etc. because one has the exact sequences instead of a nebulous OTU that hides biologically meaningful variation. For a cool example of how ESVs can be useful see:

Thompson, L. R., Sanders, J. G., McDonald, D., Amir, A., Ladau, J., Locey, K. J., ... & Navas-Molina, J. A. (2017). A communal catalogue reveals Earth’s multiscale microbial diversity. Nature, 551(7681).

6. A criticism of ESVs is that if you look at the genome with enough resolution everything becomes different. However, we must remember that by using standard primer pairs we limit the portion of the genome under consideration. Therefore, I do not think this criticism holds water for amplicon based work.

For all these reasons I do not think it is relevant for us to continue using similarity thresholds for OTU delineation.

See Callahan et al. for more thoughts on this: Callahan, B. J., McMurdie, P. J., & Holmes, S. P. (2017). Exact sequence variants should replace operational taxonomic units in marker-gene data analysis. The ISME journal, 11(12), 2639.

I am keen to hear counterarguments from folks!

Dear Aya,

Tukey’s Honest Significant Difference (HSD) test, is a post-hoc test based on the studentized range distribution. An ANOVA test can tell you if your results are significant overall, but it won’t tell you where exactly those differences lie. After you have run an ANOVA and found significant results, then you can run Tukey’s HSD to find out which specific groups’s means (compared with each other) are different. The test compares all possible pairs of means.

I hope this helps.

Hello Nischitha

Use a clearance solution ((0.15% trichloroacetic acid [w ⁄ v] in ethanol: chloroform [4:1; v ⁄ v]) for 48 h, with the solution being changed once during this time. Then wash 2 *15 min with 50% ethanol, 2 * 15 min with 50 mM NaOH and 3 * 10 min with MilliQ H2O, subsequently followed by 30 min of incubation in 0.1 M Tris⁄ HCl (pH 8.5). Samples were then either stained immediately or stored in 50% (v ⁄ v) glycerol se mentioned in the following references. Good luck

Hi

It is not so clear but it seems to be Fusarium sp. and Alternaria sp. conidia.

Good luck

Follow the articles

Some other useful PDFs...

Pelease se:

You can also try adding a vitamin solution to your broth, sometimes it helps . You can make a stock solution and freeze it. Then filter sterilize it before adding it to your broth.

You could tease out the mycelium in LPCB and examine under a microscope to view the distinct microscopic char. Then upload the images.

i am very interesting with, so please i will follow answers.

thanks

agree with dr Arizaldo Castro

No, one some of the fungal species can harbor more than one bacterial species. The attached files may provide you some information.

Hi Genese: The easy way to identify fungus by using sequencing the ITS region.

You are right Subhpriya, I too feel so since I feel endophytes are simply the better version of rhizosphere microbes

Congratulation for transforming fungi with GFP. I think you should first check direct sections of target plant tissue under confocal with GFP filter. it will only recognize GFP location. It may work well.

Inoculate the growing medium . let endophytes find entry into the roots, dpending upon their competence to act as endophytes within certain tissues of host plant.....

Thank you very much Oussama Ali Bensaci. I have followed the method as mentioned in Li et al., 2014. The papers you have attached are really very helpful and hopefully I will contact Prof. Pirttilä, Anna Maria for further information. Thanks again...

You should check data on Gliocladium.

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Thank you dear  Barkat and Dhurgham for responding me!

Dear Tiolo:

I went through several papers concerning biodegradation of polyurathane and other olastics. From the papers above it is not clear that  Pestalotiopsis microspora is the best degrader. It is stated there that this fungus has "robust activity" to degrade PUR. According to my opinion it would be very usefull to perform some screening using various microorganisms before you start your further research.

Best regards

Vit

By using PCR you can detect the presence of DNA, nothing else. Although there are some papers indicating the feasability to use PCR to detect mycotxigenic fungi, by no way you can use PCR for  the determination of the mycotoxin. You have to use any analytical method in your matirx of interest for derterminig  the presence of the mycotoxin. You may have a postive PCR result with no production of myxcotoxin and the other way around.

Thank you sir for providing useful information  

The use of EtOAc is under the assumption that it is polar/non-polar enough to extract all metabolites (both non-polar n polar). Additionally caustic enough to break cell membranes. You can use other solvents if you so desire (chloroform, butanol) or just soak the cells in methanol etc with mechanical agitation.

Good Luck

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/ 

Methanol in my opinion is adecuate

Dear Prof.  Gibbs, I really appreciate your time and efforts dedicated  to get me the answer, your data is so valuable , I am going to test the potivirid primers to detect ipomoviruses....

Many thanks

thank for your answers Oadi, but it seems like the only site that has an actual identification key is Discoverlife.org, and i'm not sure how much it can be trusted. 

thank you all  for the answers

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Thanks Laith Al-Ani

Kinds regards from Mexico

Porfirio

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Dear Mohammad,

You should check any literature on tropical lichens, e.g.

Also some links:

Best wishes,

Andrei.

Dear all,

Thank you very much for your helpful answers.

Best wishes,

Estefanía.

Dear Vikas,

I wish you a great journey in a Mycology field! learning studying researching. YOu are obviously at the beggining. enjoy that all! Go and start from the fundamentals (the best taxonomy books) and learn as much as possible from the Grand Masters in Mycology such as Gams, Domsch,  Ellis, Samson, De Hoog, Seifert, Simmons, Pitt etc. etc  (go and study phenotypic traits (not only molecular ;-D then you will never ask where is the differences between acremonias and fusarias and other allied related genera.. YOu will master them all based on their morphology in pure cultures in vitro, Im sure! it need only several years of skills, and You are there, I am sure..  :-)  Welcome in traditional mycology!

With all my respect

Yours Roman

You never know, which line  could invite which pest or disease inadvertantly , thereby , ruin the ruling variety..?

1. Only a small fraction of microbes occurring in nature have been isolated.

2. Only a fraction of isolated microbes have had their genomes sequenced.

3. With any newly found microbes, the only way to determine if it has already been isolated and characterized is to compare its 16S RNA or entire genome to those already published in a database.

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.

Dear Farrukh Baig

Even though NCBI is a great database collecting a lot of information useful for scientists, it has the problem that too many data are registered with wrong species determination, bad sequence quality etc.

Therefore we use some other databases for the molecular idenfication of taxa, which are managed by curators. That means curators ensure the quality of (i) DNA sequences and of (ii) species determination.

First we check databases with curators, then we compare with NCBI...

- UNITE: https://unite.ut.ee/ (go to "run analysis", use field "blastn"). Check the taxon/taxa with the best score bits. Below the list of taxa you will find the nucleotide sequences displayed and they are linked to the corresponding NCBI entry. If your sequence has gaps or differs in some nucleotide, check your raw data. Are you sure your sequence quality is "good"?

- BOLD: http://www.boldsystems.org/index.php/IDS_OpenIdEngine (go to "ITS", use "all barcodes"). You can check the best match on the right column: press on "published". If sequences are depostited in BOLD but still not published, you will not be able to open this file ("early release").

I hope this helps you!

Carolina

Thank you so much Laith sir as well as Jawad sir for your valuable suggestion. I am an undergrad student and apprentice in this field. So any further suggestions and experiences will be very appreciable and greatness.