Alternaria - Science topic

Transcriptomics and comparative genomics are the most standard techniques in NGS, and I would say "easy to interpret" comparing to any other technique such as CHIP-seq, ATAC-seq and many others.

The order of elution alone is not a adequate description of the elution pattern. One should use the fractions of a column volume. As you know, small molecules are retained on a size exclusion column longer than the large ones. If, in your case, small molecules were eluted at approximately one column volume, then the column worked properly. Probably, your large molecules were very hydrophobic and were retained on the column by forces of different nature.

If the small molecules were eluted substantially earlier than one column volume, you have a problem either with the sorbent or the packing of your column.

I hope this helps.

Hi Jose, one thing you can try, and it always works for me is to grow the fungus on the same host of substrate that you isolate the fungus from. I should sporulate on it.

Good luck

The knowing of the fungus comes from the sayings in the New Testament Bible Gospel of Matthew that the fruits define the tree. Saccardo used that principle in his treatment of asexual or imperfect fungal species. Beside the spore morphology the trend is to look conidial development stages or conidial oontogeny. The study of the host ranges symptomatology and ecological habits are all important criteria. The analysis of genomic relationship is also of growing interest and concern. In the foto I see 5 septa the measurement should start in the conidial base foot cell and go to apical point the width measured at the widest diameter and the narrow point in that foot cell. Alternaria probably has a monographic treatment which needs be assessed. Genomic and pathological studies are suggested as well as ecological roles as many Alternaria have saprophytic roles.

Hi, Godfried

I think the problem lies in leaf samples, not methods. Are you sure if they were Alternaria spots or bacterial spots? And did you really use NaCl ???

Schrader, T. J., et al. "Further examination of the effects of nitrosylation on Alternaria alternata mycotoxin mutagenicity in vitro." Mutation Research/Genetic Toxicology and Environmental Mutagenesis 606.1-2 (2006): 61-71.

... To examine other targets for mutation, the metabolites Altertoxin I (ATX I), Altenuene (ALT), Alternariol (AOH), Alternariol monomethyl ether (AME), Tentoxin (TENT), Tenuazonic acid (TA) and Radicinin (RAD) were reexamined ± nitrosylation, using Ames Salmonella strain TA97, sensitive to frameshift mutations at a run of C's, as well as strains TA102 and TA104, reverted by base pair mutations at AT sites and more sensitive to oxidative damage. ... These results suggest ATX I, AME and AOH induce mutations at AT sites, possibly through oxidative damage, with nitrosylation enhancing ATX I frameshift mutagenicity at runs of C's. Nitrosylated ATX I was also directly mutagenic in mammalian test systems.

Yes, you will do well to choose a plant that is rich in saponins. They tend to confer antifungal effects. To identify a species with saponins, you can either do a literature search, or test them by mixing leaves with water and shaking to see if a soapy froth appears. The froth must persist for several hours for it to be caused by saponins.

I know it would be so late answered.

However, I set a method that supports maximum sporulation while do not decrease the infection percentage.

I move A. solani to new PDA media (pH 5.5) for five days at 12/12 light/dark and 28 C°.

After that I removed surface mysilium gently and keep the plate (without cover) under black light for 8 hours and 48 dark place.

Finally, I collected sporue by dissolving the plate in water.

Based on the two images on top, I also would go with Alternaria sp.. Those lesions, which do have a dark grey growth on them are most likely showing the presence of Alternaria sp. conidia (spores) on the leaf surface. Under high magnification using a dissecting microscope you should at least be able to see their club-shaped form.

Ascochyta spp. produced lesions do have small, spherical fruit bodies, which do contain the spores internally, until they are expelled from the pycnidia.

Some species of the genus Alternaria like A. solani can not produce conidia on the medium PDA. Instead, you can use V8 medium to obtain the conidia of the fungus.

To distinguish between Alternaria solani and Alternaria alternata which always isolated from the leaf spotting diseases in order to avoid the confusion between them, A. solani has a long peak reach to two third the conidium long or more, while A. alternata has a very short peak.

I do agree with Dr. Paul Reed Hepperly suggestions perfectly. Alternaria brassicae also grow on PDA media

You must grow the bacteria on Nutrient Agar medium and grow the fungus on PDA medium. Then you must take four discs from bacteria which grow on the Nutrient Agar and put them around the plate which contains PDA and incubate it under 28 C for 24 hours. After that, you must put 5 mm diameter disc of the fungus growing on PDA taken from the margin of seven days old colony in the center of the plate and incubate again for about 5-7 days with left a control treatment without any bacteria. Finally, you must measure the inhibition zone which has no contacting between the fungus and the bacterium.

Nina, that's what I would have done - I'm a long retired [pre-Brexit] scientist, but I would have contacted a colleague across the English Channel/La Manche; I would have used and disposed of the material responsibly. N.B. England is part of the UK - at the moment, and hopefully will remain so!

We are also trying for sporulation of the fungus under in vitro conditions.. But till date no success. Even though we have waited for 30 days on PDA medium.

Bikram Dhara thanks

ITS region along with Inter simple sequence repeat markers (ISSR) can be used to identify and differentiate between Alternaria sp. Just sequencing the ITS region and comparing from NCBI database (blast) can also be done for identification.

You can refer the following publications.

1. Molecular identification and genetic variation of Alternaria species isolated from tomatoes using ITS1 sequencing and inter simple sequence repeat methods (Mohammadi A and Seifollah B, 2019).

may be helpful to you sir..

There are several reasons for your observation

1. Mutation: The pathogen may have mutated due to long term storage thereby reducing its level of virulence when inoculated into a susceptible host.

Note: Try using a fresh isolate for the pathogenicity test

2. Method of inoculation: use abrasives for waxy or cutinized surfaces, or sharp objects to mechanically injure the plant prior to inoculation with the spores of the pathogen or spread around the root rhizosphere (for root infections of plants) etc.

Note: Whatever method or strategy you choose to adopt, the pathogen must be aseptically inoculated into the cortex of the host plant.

3. The Age of the host plant: Many pathogens are specific in their nutrient requirements

Example: a pathogen that can cause damping of disease in seedlings of a particular plant might not necessary cause infection in the matured stage of that same plant species.

Note: If your Alternaria spp was isolated from the fruit of matured rapeseed or mustard plant, then pathogenicity test should be conducted using a healthy plant of similar age range

It is equally possible for the fungicides to perform better when combined. The only way to find out is to carry out a pilot study

You can use a spore dilution method to get single-spore colonies, as Alex said above, or you can use the intriguingly weird method that we in the powdery mildew community use to get single spores: by attaching a human eyelash to a glass rod with wax, and, under a dissecting scope, catching a single spore on the tip of the eyelash. Alternaria has chains of spores just like powdery mildews, so it will be possible to catch the tip-most spore in a chain and deposit it on a fresh plate of agar.

I think this

help you.

Dear Samira, I am suggesting you to read this paper:

Characterisation of Alternaria species-groups associated with core rot of apples in South Africa.

Mycological Research

Volume 106, Issue 5, May 2002, Pages 561-569

First: Alex: I used to use leaf discs of different cucurbit cultivars to determine race in powdery mildews and it worked just fine. It was the right test for the right question. In your example, it's quite possible your results were correct: you got a 3:1 ratio for some cytoplasmic factor, but all your seedlings had, say, a waxy cuticle that masked the cytoplasmic factor.

Second: Arya: I will answer by giving you an idea of how complicated a simple-sounding question can be. I was once part of a study to determine the pathogenicity of Pythium isolates to geraniums. We used detached geranium stems as our assay. We made a wound in the stem and then placed over the wound: either a plug of CMA agar alone, a plug of agar colonized with a known pathogenic isolate, a plug with a known non-pathogenic isolate, and then all of our unknowns. We then measured the length of the lesions after a certain number of days. We found that we got lesions in the control agar-alone trials, but they were only a few mm long. Our non-pathogenic isolate gave lesions that were a little longer. Our known pathogen gave us lesions that were 10 centimeters or longer. And our unknowns were somewhere between the nonpathogen and the pathogen. We arbitrarily declared isolates that caused lesions over a certain length to be "strongly pathogenic"; ones that caused medium lesions to be "moderately pathogenic" and ones causing short lesions to be "weakly pathogenic". We understood that Pythiums are good saprophytes that, under the right conditions, could be pathogens, so we were using a wound to represent the "right condition". We understood that our assay was very artificial and used a detached organ, not a whole plant. On the other hand, we could test hundreds of isolates that way, and get a rough idea of how many of the pythiums present in greenhouses were possible pathogens. If we had gotten the pythiums from the plant potting mix, they tended to be highly pathogenic; if we got them from puddles on the greenhouse floor, they tended to be weak pathogens. We concluded, after sampling dozens of greenhouses, that the pathogenic pythiums were primarily coming in on the wholesale planting material itself, not from the surrounding greenhouse, and that was an important thing for the greenhouse owners to know! So it was the right test for the right question.

So all I can say about Alternaria and Fusarium is that they are good saprophytes and good secondary invaders, so you might want to go with healthy whole plants with no wounds.

You are most welcome dear Mohammad Imran with my highly appreciation.

Best greetings.

Dear colleague

I work with a similar genus, the curvularia, also isolated from leaves and are preserved for a long time in wedges of malt agar, they can also be preserved in 10% glycerol.

It is necessary to point out that there are various fungal preservation procedures, but periodic cultivation in agar wedges Under mineral oil, conservation of spores in soil, sand or silica gel, lyophilized strains, freezing in liquid nitrogen and in distilled water are the most used.

The choice of methods takes into account several factors, depending on the purpose of the collection, so a small teaching collection differs from those that are national repositories. If, on the contrary, the objective is to taxonomically classify the cultures, methods that guarantee the morphological stability of the microorganisms are required. Likewise, a collection of interest for the industry must emphasize the methods that maintain genetic stability.

In your case, I think you need to keep them in the long term and for that I suggest Cryo freezing or Lyophilization because they are the best where the growth of microbial cells is paralyzed, but they have not died. Thus, genetic stability is guaranteed to the maximum, because the appearance of successive generations is avoided.

I hope it has been helpful

regards

Thanks for answering this question, I want to test it by myself to versify this hypothesis.

There is quite a lot of confusion concernant the identification of the Alternaria isolates from lesions on cereals. Several saprophytic species colonize the tissues affected by other factors. In our experience on wheat, A. triticina is not easily differentiated from the other species based on morphological characteristics.

Thus first make sure you have isolated A. triticina. For inoculation of barley I would suggest to use the method we have used on wheat. See Mercado et al. 2006

Hello, by benzylchloride method you can extract both plant and fungi DNA by same procedure and same reaction mixture and you will receive both DNAs mixture. If you want to isolate only fungi DNA the better way is to isolate fungi culture from your material as pure culture and then perform DNA isolation.

Thank you @Laith. IN fact, the second article you sent me only studies association/correlation of sensitization to food and inhalant allergens in patients with rhinitis. Thus co-sensitization and subclinical sensitization can be the resaon. I have to thank also @Irene and @George for their inputs. As I really see, there is not much infomration about this topic

Alternaria solani sporulate very scarce in vitro. In carrot meal agar you may get few sporulation after a prolong incubation. I will suggest you to prepare a mass culture of this fungi in boiled sorghum or wheat or barley grain to get sufficient sporulation. You may also reinoculate on host tissue(tomato) for better sporulation.

Briefly:

1- Disease incidence: No. of infected plants x100 / Total no. of plant assessed

2- Disease severity is the percentage of relevant host tissues or organ covered by symptom or lesion or damaged by the disease. Severity results from the number and size of the lesions.

this link is useful

citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.841...

regards

I liked the publication which investigate the prediction capacity of ANN to obtain AUDPC values for tomato late blight using a lower number of evaluations to improve the process efficiency. Very informative

file:///C:/Users/Acer/Downloads/AUDPC-TomatoBlight-2016%20(3).pdf

Definitely not a TEM as a first approach. SEM, if coumpound of interest contains something behind C, O, H, especially if it is non-organing compound. For organic compound Raman could be a better technique.

Thank you. Will try these.

you are welcome

Houda

If you want to quantify fungal spores, why do you not count them with an optical microscope as real spores from a known volume and with a grid instead of with the more expensive and laborious technique of qPCR? DNA methods are not the answer to everything. Simple is best.

The protein molecule should be known first among various protein molecules and techniques for precipitation use ammonium sulphate or the certain solvent to give the effect. Furthermore preparation of separation that molecule. The target protein molecule can assessment with the biuret-spectrophotometry, barford or lowry. If total protein will be known then can use kjeldahl with various techniques like volumetric assay or spectrophotometry.    

Dear Djamel , 

In order to identify Alternaria solani and Alternaria alternata , you have to observe first of all  the conidia , there are some differences such as : seize of conidia and formation of conidia (in chain or not ) , and you have to check by molecular methods , because morphological characters are not enough to distinguish beteween them.

You should read this article :  Biodiversity and taxonomy of the pleomorphic genus Alternaria 

Best regards.

137

Hi Shuvrah

I think, The species is Alternaria alternate

Dear Laith

Thanks, the future with  the plant's immunity (cell talks), as important as in human immune system and cells communication (signaling and pathways)

regards

Houda

hi Tamas Toth

thanks a lot. I think your suggestion will help me to solve the final identity of my isolates

Hello Oadi Manty, I have done the pathogenicity test in case of plant pathogens. You can keep the barley leafs in moisture chamber and easily apply the 8 mm disc of Alternaria on the leaf. After this Incubate the moisture chamber in BOD incubator according to the growth condition.

I hope this method may help you.

Its look like toxicity effect

Hi, you can use either PDA or Malt Agar medium. You can also add streptomycin, to get rid of some bacteria etc, especially if you want to isolate Alternaria spp. from plant leaves. I found that sporulation work best with PDA and no streptomycin. You can also put your fungi under blacklight (2 weeks is a good start), which help a lot to obtain conidias. The color of the culture depends on the species, for example A. dauci produces some red pigments, A brassicicola or alternata produce black pigments...

Have a nice day

Dear Mrs Narimene 

this is your institutional adress : Avenue Hassan Badi - El Harrach - Alger 16040

Best regrads

Dear Alian,

Nowdays systematics and species delimitation in Alternaria is based primarily on molecular criteria because morphological characteristics proved not to correlate with phylogeny. Please see a paper attached, it's based on molecular study too but contains plenty of figures for different species conidia and sections morphological descriptions which may be helpful for identification.

Best wishes,

Elena

If you use the commercial fungicide formulation 'Bravo' as your source of chlorothalonil then you are likely to be dealing with a water-based suspension concentrate containing 50% chlorothalonil with inert dispersants.  The dispersants are added to the formulation to allow it to be diluted with water while still keeping the chlorothalonil in suspension (as its intended use is for spraying onto plants).  You won't be able to dissolve this in any solvent as the dispersants won't dissolve.  You would be best to make up your dilution series using sterile distilled water but be aware you will produce a suspension not a solution so you must be very careful to shake it well when making the dilutions and also when adding to your agar.  The alternative would be to purchase technical grade or 'pure' chlorothalonil from a chemical company then you could make up your dilution series as a true solution in a suitable solvent.  I agree with the others that you should add the fungicide to your media after autoclaving as I'm not sure about the heat stability of chlorothalnil (you could check this).

OGYA and Sabouraud chloramphenicol are better than PDA. they contains antibiotics ( Oxytetracyclin and Chloramphenicol) which inhibit the bacterial growth 

You should consider the virulence of iisolate and host resistance. In addition to this you could also try changing light exposure of the leaves to 16 h and try to do inoculation in the abaxial surface.

Hi MATI: You can use dilution methods for counting live spores .

Thank you. I am familiar with the book. But it does not describe any methods for measuring the parameters I am asking for.

Alex , 

It should !

Thank you ver much for that articles , it really helps me 

Both of these are not insect pathogens, why you have used them is need to be explained. There is no direct effect of them on Helicoverpa.

Dear Kamal Sadeghi,

1.

Fusarium cerealis (= Fusarium crookwellense L.W. Burgess, P.E. Nelson & Toussoun, Transactions of the British Mycological Society 79 (3): 498 (1982)  - A sister clade is F. culmorum,  similarly F. graminearum.

An outgroup is: Fusarium solani (Mart.) Sacc. 

2.

Fusarium delphinoides is a pathogen of the cactus-like African species Hoodia gordonii (Apocynaceae). Phylogenetic analyses based on combined sequences of the internal transcribed spacer region, LSU rDNA and partial sequences of the elongation factor 1-alpha and beta-tubulin genes identified a clade of several species producing predominately 2-septate macroconidia as the reciprocally monophyletic sister of F. dimerum. The basal sister group of the two aforementioned clades includes Fusarium lunatum .

An outgroup is: Fusarium tricinctum (Corda) Sacc. and Fusarium avenaceum (Fr.) Sacc.

3.

Alternaria alternata 

Alternaria arborescens and  Alternaria tenuissima formed monophyletic sister group to A. alternata

An outgroup is:  Alternaria brassicicola (Schwein.) Wiltshire, (1947)

It think 200 umol m-2s-1 it isn't enough. I won't recommend  less than 500 m-2s-1 to growth any plant, if it is possible. If the plants get tall and thin correct your pot size and the N available to the plant.

Ioannis, I agree with Alex. As you know, today we cannot talk about species, but complex species. Besides differences in virulence among the species which can infect potatoes, such as Alternaria alternata, A. grandis, and A. solani, we can find considerable differences among isolates of these species. Therefore, I suggest you choose a specific interaction to elucidade the infection process. Leaf age and nitrogen fertilization play an important role in disease establishment and evolution and might be considered, especially for a weak pathogen like A. alternata. 

Alternaria are not host specific. Tomato, Capsicum any Solanaceous crop plants leaf samples  you can use for bioassay.

Since Alternaria Spp are difficult to characterize morphologically, the best approach will be use of combinations of few genes like ITS, 1EF, btublin etc. Amplify genes using primers and later on sequence them. Use softwares like Paup, mega etc for phylogeny. By this way u will be able to differentiate different species of fungus. One marker can't help in this case, if u would have been dealing with oomycets then ITS is enough.

you can try the method provided by Liu et al 'Rapid Mini-Preparation of Fungal DNA for PCR'. it is good  for dematious fungi,

best,

Use the microculture method, take a slide put a drop of agar media, like corn meal agar or malt agar even PDA, on it when the media become solid transfer Alternaia inoculum to the borders of the drop, incubate in humid chamber and check undere microscope. You should able to see the chains of conidia

Plant parasitic nematodes, especially virus vectors obtained in Vicia faba will show in article( in press). Some aspects of fungi parasiting nematodes are discussed.. By the way, I ask in nematode faunistics surveys, in water plants from lakes and Baltic coast( Hirschmanniela sp) and nematodes Longidoridae and Trichodoridae as plant virus vectors  from Latvia.Greetings. Andrzej. Gdynia

One of the time tested methods is using short-wave UV source.

     

you can use ethanol to prepare the salicylic acid solution as 10 ml for 1g but add SA small amount gradually and completed to liter

PDA is best media for Alternaria..... but if not sporulating on PDA try giving physical or chemical stress.

Can any one provide me NahG tomato mutant lines with its wild type (cv. moneymaker). your help will be fruitful for my research. it is definitely acknowledged. thank you. you can even suggest where i can legally approach for buying it. waiting for valuable response .....