For antimicrobial activity, you can used both Bacteria and the fungus, but you test the antimicrobial activity against phytopathogens, so in that case we select fungus for antimicrobial activity. The best fungus for that case is the Penicillium sp.,Fusarium sp.,and Aspergillus sp.

You need to use a strain which almost susceptible to all antimicrobials. Try using S.epidermidis ATCC12228, which is already use in microbiology laboratorie to performe RAA test (residual Antimicorbial Activity test). I'd be very careful in the use of a fungi like Aspergillus, Candida or Fusarium most of them are quire resistant!

We tested over 19 desert plants and divided the plant parts into flower, leaf, stem and root (seed if we had it). We used 3 extracts - water, methanol and hexane for each plant part. We chose 11 microbes which were mainly human pathogens - including one fungus Candida albicans. We had varying results depending on the plant and the microorganism. We did have a lot of success with Staphyloccoccus aureus, as well as Bacillus cereus. We had some success with the fungus Candida albicans. Hope this helps.

First of all one gram positive (e.g Staphylococcus aureus) one gram negative (E.coli) standart sensitive ATCC strain could be used, then you choose other bacteria or fungi.

if you want to go and start with an easy one to prepare and also test, first try with Fungus . i worked with F.Solani , F.Oxyspurum and F.Culmurom and they are very easy to prepare. But bacteria is a little tricky :-s

Economically important Mollicutes (e.g.Spiroplasmas) would be nice ! You if you need collaboration please get in touch !

Fungus: Colletotrichum spp., Phytophtora infestans, Fusarium oxysporum, Fusarium roseum, Botrytis cinerea, Aspergillus niger...

Bacteria: Xanthomonas campestris, Pseudomonas syringae...

I agree with the answer given by many researchers .

Fungus: Colletotrichum spp., Phytophtora infestans, Fusarium oxysporum, Fusarium roseum, Botrytis cinerea, Aspergillus niger...

Bacteria: Xanthomonas campestris, Pseudomonas syringae...

Dear Mr. Amal,

You want to test with human pathogens or vegetable? Fungi or bacteria?

Human pathogens:

BACTERIA

Gram-positive

Bacillus subtilis (ATCC 6633)

Enterococcus faecalis (ATCC 12953)

Staphylococcus aureus (ATCC29213)

Streptococcus pyogenes (ATCC19615)

Gram-negative

Escherichia coli (ATCC8739)

Klebsiella pneumoniae (ATCC13885)

Proteus mirabilis (ATCC25933)

Pseudomonas aeruginosa (ATCC15442)

Salmonella typhimurium (ATCC14028)

FUNGI

Aspergillus fumigatus (ATCC 46645)

Blastomyces dermatitidis ( ATCC 18188)

Candida albicans (atcc 10231)

Cryptococcus neoformans (208821)

Histoplasma capsulatum (ATCC 11408)

Sporothrix schenckii (ATCC 22433)

One option is to buy a phytopathogenic strain ATCC any widespread. (Erwinia carotovora, Pseudomonas syringae)

Another is to isolate and identify the phytopathogenic affecting the crop of interest. Then test the effect against the isolated

Great thanks for all

you can test against Plant pathogens for get good result

Erwinia amylovora, Pseudomonas syringae.

I agree with Gabriele protocol....either procure from collection of any ICAR institute or isolate from infected plants.

happy to test them against the TB pathogen. get in touch for collaboration.

You can follow different approaches:

1. testing your extracts against plant pathogens important in your region or Country (I agree with Gabriela Feresin)

2. testing your extracts against a set of phytopathogenic fungi or fungus-like organisms belonging to different Division (Oomycota, Zygomycota, Basidiomycota and Ascomycota) possibly including, for each Division, organisms belonging to different Orders or Families. This is a "systematic" approach.

There are a lot of studies have been done on the antimicrobial activity of the plant extracts against the plant pathogens, fungi and bacteria in order to use in the control of plant diseases. you can survey the literature in this respect. You can obtain fungal and bacterial plant pathogens from colleagues in the agriculture research center in Egypt or from faculties of agriculture and science.

I routinely assay extracts and purified compounds for their antifungal activity against Rhizoctonia solani, which is particularly suitable since it does not sporulate in culture, and radial growth can be measured without interference of secondary colonies

I agree with Giovanni Vannacci

From Agriculture point of view Alternaria spp, Fusarium spp., Magnaporthe grisea are the most common plant pathogens. you can try your extract on these.

You may also use the bacterium Ralstonia solanacearum which is a soilborne root pathogen, in addition to leaf pathogens that have been proposed (Pseudomonas syringae, Xanthomonas campestris or Erwinia amylovora...).

Thanks for all

can anyone provide me with protocol to isolate mentioned microorganisms?

Actually different microorganisms have different susceptibility to different antimicrobial agents. So first of all check microorganisms associated with your plants and then use microorganisms differ from them.

The selection of microorganism depends upon the type of phytopathogen you are targeting. So first thing that you have to decide is whether you are targeting single pathogen or broad range of pathogen. I think testing of extract against human pathogenic bacteria/ candida albicans are perview of your study since you are targeting phyto-pathogens. Specifically select plant-system and pathogen affecting that plant would be your target therefore use them for testing.

There are several plant pathogens sensitive to several plant exracts in literatures. lef spot pathogens and even soil born pathogens can be used

I will test on abroad range of phytopathogens

how i can identify isolated phyto-pathogens?i isolated from different hosts.but i need protocl to identify them scientifically

I think you better to test on the pathogen(s) that you have targeted for your product. The effects of extract on different strain of one fungus could be different. However, if you want to have something very general then I will go for Fusarium spp. they are very common and economically important..

Well if u are interested to work out the broad spectrum effect of your product, then i believe among fungi u should chose one member of lower fungi i. e phytophthora at least one member of ascomycetes fungi like fusarium and one basediomycetes fungi like rhizoctonia. similarly among bacteria you can chose common widespread bacteria like xanthomonas compesrtis, Erwinia carotovora/ amylovora. and any phytopathogenic Pseudomonas using these Plant pathogens the in-vitro tests will give the idea of efficacy of your product.

Because you worked on citrus diseases, I sugest you to continue on this area and achived their activity against phytophthora citrophthora (postharvest fruit disease): their activity of growth, germination, sporulation and diseases. You could used microscopy for helping you to assess all of these tasks.

Dr Rachid Lahlali

In the beginning you should to define the problem of disease and would rather be a crop of vegetables that are grown and easier to place the seeds in pot experiments in greenhouse or lab easily. There are two types of fungi, foliar fungi first and second soil-borne fungi and fungal treatment is easier in the wind, but you can test both types at the same time by planting a larger number of pots. BUT, the most important and easily to you in this field, POSTHARVEST DISEASES, we can used the aqueous extract and essential oils against pathogenic fungi and decreased the fungicides treatments and pollution of environment. Best wishes and good luck.

Dr.

Khaled Arafat

Essential oils/plant extracts are usually incorporated into sprayable formulations that may be used for foliar application or postharvest applications. In addition, when screening fungi, one should cover the different fungal groups - asco, basidio, Zygo, etc. With this in mind, pathogens of interest may be P. infestans, Alternaria spp., Botrytis cinerea, Pennicillium spp., Aspergilus niger, R. stolonifer. Yeasts may be Rhodotorula or aerobasidium/schytalidium. Bacteria such as P. caratovora is also a good option.

Good luck.

Dear Amal, regarding your questions about how to identify phytopathogens. What I would do is have isolates from the crops of your interest, with symptoms of disease. They might be fungi, bacteria and even viruses. For fungi ID, you can use traditional morphological identification based on fruiting bodies. There is a book you can use (it is really basic though) The Identification of Fungi: An Illustrated Introduction With Keys, Glossary, And Guide to Literature by Frank M. Dugan. In addition, you should sequence the ITS region using specific primers (this paper explains how to do it: Bellemain (2010) BMC microbiology 10:189). Also, once you know which pathogens you have, it would be convenient to do bioassays to check the level of virulence.

There is other options are there to work with human pathogenic fungi

You may evaluate the extracts for its nematicidal efficacy against root knot nematodes in petridishes (effect on egg hatching and juvenile mortality).

for antifungal activity of plant extract you can use the fungus is Fusarium species, colletotrichum sps drascelaria sps

Use Alterneria as test organism. Alterneria is a foliar disease and the spores are coloured as well as fast growing.

As several colleagues have alrready told you, it is important that before starting to evaluate the effect on several pathogens, to define the problem that you want to address. I thonk it´s better to focus on one or two pathogens and do not forget to work with different isolates (monosporic) or your results will be unreliable.

For your choice it may help you to consider how you might apply the extract in the plant. If you do it by soraying plants you should better to evaluate fungi or bacteria affecting the aerial part of the plant so that, in future, you´ll have a potential use in agriculture. Take also into account that methanol extracts may be toxic to the fungus by methanol bot by extract per se.

Dear Amal, there is a good literature on the use of plant extracts to control plant diseases. While in the field there is a strong competition with the use of chemical fungicides, that usually are much more effective, you could try to control postharvest decay. You can go on the market, choose the fruit you like, leave them in a high humidity environment and in few days you will have fungal decay. You can isolate causal organism, identify, so wound fruits and apply your plant extracts before or after pathogen inoculation, to check for preventive or curative effects. You can find the procedure in some of my papers. Good luck.

Gianfranco Romanazzi

Dear Amal:

In order you results will be sound for your country, I recommend to you to select a pair of plant pathogens that attack an important crop at your country. I prefer to work with cultivated phytopathogenic fungi with has a well known biology, because you must design first, the lab test, an later the field tests. Also it will be very important that the fungus sporulated well at lab, so that you can test for example, the extracts effects on fungus spore production and viability.

Evaluate the effficacy against any plant pathogen of interest and of high economic importance in your country. This is will be practical in term of application later.

You can isolate any phytopathogen from infected tissue of plants, it is better if you go for fungi as phytopathogens. You may select Phytophthora sp, Alternaria sp, Fusarium sp, Helminthosporium sp...

Many phytopathogenic fungi and bacteria could be isolated from diseased and apparent healthy plants. Fusarium spp. ( wilt and root rot ), Rhizoctonia ( root rot ) Pyrenophora ( foliar disease) are well known to cause serious problems to many crpos and trees. You can evaluate the plant extracts In Vivo as foliar spray, seedling treatment and seed soaking. In Vitro studies could be applied under controlled conditions to test the antimicrobial activity of the plant extracts on the pathogens ( colony diameter, spores viability, germination .......etc.)

It all depends on your objectives. If your target is for aerial borne pathogens like in our case as we are targeting for post harvest treatment , we are using Colletotrichum spp.. It is a very common post harvest pathogen. For soil borne we normally use Fusarium as our test pathogen.

you can also use either soil-borne or air-borne bacterial or fungal plant pathogens to be tested with the plant extract. Such as Bacillus sp. orFusarium sp. (soil-borne plant pathogen) or Xanthomonas sp. or Botrytis cinerea (air-borne plant pathogen).

I recommend you to test this extract against Phytophthora species as they are spreaded worldwide. I specially work with P. capsici which is an agressive pathogen of hortyicultural crops.

I think you shoud use the pathogen of the plant of which extract you are using as antibiotic effect. Then try it on other phytopathogens of both bacterial and fungul origin in the fertile soil.

Hi,

I suggest you to evaluate the efficacy of plant extract against a pathogen that is most important in perspective of your country and that causes great damage to your high value crops. Alternatively, you can use few pathogens covering fungal and bacterial groups.

Hi Dear Amal,

form your question, this suggestion comes out: in my opinion you may first look for recent studies published about biocontrol of phytopathogenic system.

You start, first, by omiting all the 'already done antimicrobial tests' 'depending on the extract used and compare these results with your own extract material

Then see the efficacity of tests: check is it well powerfull activity -medium or does not give any result in these publications even that may lead you to an ideal protocol after selecting some good articles related or near to what you work on like those already done studies (publications/ PhD thesis) may let you choose one of the pathogens or you may look after its pathogenesis (pathways and signaling)

my proposal is to work on most known pathogenic fungus (since it is a antimicrobial activity test) that attact important economical crops in your country

subject that are focuced on Crops biocontrol (SAR, biotic/abiotic stress resistance, beyond biocontrol agents/elicitors or receptors identification)

I think once you do your check list that will allow to organize more your ideas

best of luck

We use Phytophthora palmivora isolate from cocoa to test purified constiuents of methanol and ethyl acetate extracts of medicinal herbs

What is the genus and species of the plant you are testing.? That would be a good place to start as certain plants are susceptible to certain pathogens. Let us know the plant you are using and we can give you a more thoughtful answer. Also, which country is this plant growing in - certain areas have higher incidences of certain plant pathogens. Could you provide more information in order to give you an informed answer?

You should also consider using dilutions of your extract as you do not want to use all of the stock which is often tedious to get and costly. Also, very often your extract is composed of a mixture of various molecules that are not all effective against your pathogen. It is important to know which one is controlling the pathogen.

Some extracts have volatile effects too and you can test them in Petri dish having compartments. In one compartment you would grow the funguson the medium and in another compartment you can put a drop of the extract. I have had interesting results where the fungus would not grow at all. Use water as a control in another Petri dish.

Hi

I would prefer to Choose a specific disease to work with, then start my test with the causal agent

Hi Amal,

This may help you to take into consideration some aspects worth mentioning here. Do you know the actual AI (active ingradient) of your extract? If so, how much of it do you have in the extract (concentration)? Also, consider the fact that some of the microbes suggested by our colleagues here may have developed resistance against some groups of antimcrobial components. These factors may give conflicting results to your research.

Best of luck

Thanks for all.

Hai,

There are lots of plant pathogens are causing various diseases in plants, the most common plant pathegenic bacteria were following

Burkholderia, Xanthomonas, Pseudomonas and fungi Fusarium spp., Verticillium spp. etc.

I agree with the idea of ​​Carmen. However, a cosmopolitan fungus Fusarium can use.

We do such tests on 8-10 strains of bacteria including Xanthomonas, Pseudomonas, Pectobacterium, Erwinia, Pantoea, Dickeya, Clavibacter, Agrobacterium, 3 strains of fungi: Alternaria, Fusarium, Septoria, and Phytophthora. They cover >75% of reaction variation across other pathogens.

Fusarium, Pythium, Phytopthora and other soil borne fungi are best to study the effect of plant extract against phytopathogens. Because these are common problems in agricultural lands throughout the world.

I am for dear Khurshid: You had better to test your extracts against representatives from different taxonomic groups, monilaceous Fusarium sp. and/ dematiaceous Alternaria sp. (Ascomycetes), Rhizoctonia solani (Basidiomycetes), Pythium sp. (Oomycetes). I prefer Pythium to Phytophthora because of its more saprophytic-necrotrophic wild and robust growth. With bacteria, I am again for dear Khurshid but plus a gram positive bacterium like a Clavibacter sp. You can use Petri dish based tests as mentioned by dear Yvan but use solvent in controls: water for water, and alcohol for alcohol. Consider the sterilizing impact of alcohol: use little amounts and let alcohol evaporate under laminar flow if your active ingredient is not volatile. You can use TLC to separate materials in your extract sample, then to spray the spores of fungi to find the number of bands of toxicity against fungi. You can determine their range of antifungal activities, and more.

Plant Pathogens like Pyricularia oryzae, Rhizotonia solani, Xanthomonas oryzae etc.

Well there are many option but I will suggest for Alternaria, which is very common.

I recommend Penicillium expansum, the causal agent of postharvest fruit rots. You can test various isolates (races) of this taxon. On the other hands, this is more applied than soil-borne pathogens. If you use Dimethysulfuxide, DMSO, as solvent, please note that in some cases it has microbial inhibitory effect....

You may use this effective method on this article (A new bioassay using Chlorella vulgaris cell density for detecting mycotoxins M. B. Aboul-Nasr1*, Abdel-Naser A. Zohri2 and Enas Mahmoud Amer2,.African Journal of Microbiology ResearchVol. 7(50), pp. 5709-5712, 18 December, 2013

You must test it against Bacterial Soft Rots of Potato

It depennds on which microorganismyou want to use. Generally, you should use the type member of the family of that organism. OR, you could choose the most important disease in your area, so your research would be more applicable. You can check the articles below:

1. "Efficiency of Thuja orientalis and Artimisia campestris extracts to control of Potato leaf roll virus (PLRV) in potato plants." Rakib A, NH Diwan, MA Adhab. Agriculture & Biology Journal of North America 1 (4)

2. Systemic resistance induced in potato plants against Potato virus Y common strain (PVYO) by plant extracts in Iraq. RA Al-Ani, MA Adhab, SNH Diwan. Advances in Environmental Biology 5 (1), 209-215

3. Antiviral activity of Vit-org and 2-nitromethyl phenol and Thuja extract against Eggplant blister mottled virus (EBMV). RA Al-Ani, MA Adhab, KA Hassan. African Journal of Microbiology Research 5 (21), 3555-3558.

4. Tomato yellow leaf curl virus (TYLCV), identification, virus vector relationship, strains characterization and a suggestion for its control with plant extracts in Iraq. A Al-ani, MA Adhab, SAH Hamad, SNH Diwan. African Journal of Agricultural Research 6 (22), 5149-5155.

5. Antibacterial activity of clove, cinnamon, and datura extracts against Erwinia carotovora subsp. atroseptica causative agent of black stem and soft rot on potato

RA Al-Ani, MA Adhab, HH Nawar. J Med Plants Res 6, 1891-1895.

I hope they help you to get an idea.

Good luck,

Mustafa

Your selection depends on what you want to do in the end.

If you are interested in an academic study and in isolating the antimicrobial compounds responsible for the activity you should use pathogens that are easy to grow and where methods have been well established.

We have frequently found that acetone is by far the best extractant to use to get extracts with high activity. (ELOFF, J N 1998 Which extractant should be used for the screening and isolation of antimicrobial components from plants? Journal of Ethnopharmacology 60, 1-8.)

If you want the results to have a practical application you should select important pathogens.

On this basis we have selected the following fungi: Aspergillus niger, A. parasiticus, Colletotrichum gloeosporioides, Penicillium janthinellum, P. expansum, Trichoderma harzianum and Fusarium oxysporum

Methods have been developed to determine the MIC and also bioautography of these organisms that makes it easy to isolate antifungal compounds:.

For methods see MAHLO SM, et al. (2010) Some tree leaf extracts have good activity against plant fungal pathogens. Crop Protection 29, 1529-1533.s

I think E.coli K12 is very useful for assessment of plant extract antibacterial activity that you can rely on.

Sclerotium rolfsii or Macrophomina phaseolina would be the best choice as these two are fast growing, easy to culture, less contaminating and sometime called omnivorous causing diseases of wide range of plant hosts.

You can use E.coli K12 for testing the active substance in your preparation utilizing MIC techniques.

Bacteria:

gram positive (Clavibacter spp.), and

gram negative (Pseudomonas spp.);

True Fungi:

Ascomycetes (Fusarium oxysporum, Macrophomina phaseolina, Alternaria sp.),

Basidiomycetes (Rhizoctonia solani)

Oomycetes: (Pythium spp.)

These are good members to be included in such tests. You may study the effect of extracts on spore germination, fungal growth, and sporulation.

Try out the anti microbial activity on some Gram positive, some Gram negative bacteria and some pathogenic fungi. While choosing see that all these are phyto-pathogens.

As above, Penicillium sp.,Fusarium sp.,and Aspergillus sp. are the most appropriate ones.

Clavibacter michiganensis ssp. michiganensis

Erwinia amylovora

Rhizoctonia solani

Fusarium oxysporum f. sp. lycopersici

Use OD of Chlorella vulgaris

use nematode (meloidoyne). it can be found on tomato and brinjals plant

well , you have not given specification of phytopathogens. whether you want to see antimicrobial activity against fungal phytopathogens or bacterial or viral. then you can select the microorganism to test the antimicrobial activity against that particular pathogen group. good luck

If you want to determine the broad spectrum of antimicrobial activity of your sample, you can select different fungi as mentioned before: fungal endophytes as Fusarium or Rhizoctonia; the most common for postharvest diseases are Botrytis or Penicillium; and some Gram negative (Pseudomonas or Erwinia) and Gram positive bacteria (Bacillus or Clavibacter).

But it is also interested to select a relevant target with an impact in agriculture in your country or worldwide, so it could have a commercial interest.   

You can try with Pyricularia oryzae, Rhizoctonia solani, Colletotrichum falcatum . All are plant pathogens .

I think Fusarium oxysporum is good to evaluate the anti fungal property of the given plant extract.

You choose your pathosystem with relevant support, then work on its causal agent, afterwards, you should demonstrate that you are controlling the disease.

You can choice a wedspread plant pathogens for serious disease. For example, nemthode, fungi or bacteria

it must be tested on the best known phytopathogens in your area

I agree with previous comments, in you I would choose a widespread pathogen easy to grow and measure, Botrytis cinerea affect a long list of plants both in the field and postharvest, so it can be an excellent candidate. Good luck!