Hi Fatemah

for identification of fungi, you have to go through with different parameters, like mycelium type, spore type, colony appearance, production of conidia, shape size of conidia or other sopores. and i must say that is a very lengthy, tedious and skilled job.

So i would suggest you to go for PCR based identification.

Extract DNA from the pathogenic fungi and run PCR with Universal Fungal primers. Extract-PCR product (DNA) from gel, sequence it and further go for BLAST (phylogentic analysis). your fungal pathogen will show maximum similarity/homology with some already reported group and hence you will able to identify it easily.

Good Luck

Hi Fatemah

for identification of fungi, you have to go through with different parameters, like mycelium type, spore type, colony appearance, production of conidia, shape size of conidia or other sopores. and i must say that is a very lengthy, tedious and skilled job.

So i would suggest you to go for PCR based identification.

Extract DNA from the pathogenic fungi and run PCR with Universal Fungal primers. Extract-PCR product (DNA) from gel, sequence it and further go for BLAST (phylogentic analysis). your fungal pathogen will show maximum similarity/homology with some already reported group and hence you will able to identify it easily.

Good Luck

To complete the excellent Swati answer: 

The most frequently used primers for filamentous fungi are ITS4 and ITS5.  You should find a good database to compare your results against in GeneBank. Using frequently-used primers can be advantageous due to the size of the databank already built by other researchers. Good luck!

I think you should explore the literature first or post the symptom picture on Research Gate, so that any one can give you a clue about the pathogen. 

there are two approaches to the identification of fungal pathogens on plants:

1. based on disease symptoms appear, because every disease showing different symptoms

2. The use of common methods for identification of fungi. you can apply the Koch's postulates

Hello Fatemah,

check out e.g.: McTaggart, Alistair R., et al. "Rust (Puccinia psidii) recorded in Indonesia poses a threat to forests and forestry in South-East Asia." Australasian Plant Pathology (2015): 1-7. and Tan, Mui-Keng, et al. "A brief overview of the size and composition of the myrtle rust genome and its taxonomic status." Mycology 5.2 (2014): 52-63.

Both discuss methods like morphological analysis, molecular barcode comparisons and

molecular phylogenetic approaches to identify a rust. Still there are uncertainties about the taxonomic classification about this rust e.g. because of high morphological plasticity. I am not an taxonomist but identifying a rust seems to be a tricky task.

Cheers,

Rene

Koch's postulates established:

1- The plant pathogen must be or have been always associated with the disease and conversely, this should not manifest itself if the organism is absent.

2- The plant pathogen to be isolated in pure culture medium to study morphological, physiological and cultural characteristics.

3-The host plant must reproduce the characteristic symptoms of the disease to be inoculated with the isolated agent, on favorable terms.

4- The plant pathogen must be re-isolated in pure culture and identified with the isolated first.

Subsequently with the isolate should be developed to molecular or taxonomic study. 

It is not ease. First we need to examinated carefully the diseased  plants looking for signs and also  to known symtoms on death plants and on plants where the disease is beggining. Also is important  to observe the infect tissues with  mycroscope to  obtain information about the possible pathogen. It permit confirm if the causal agent  is  fungus and select  the appropiated  isolation medium. Later is neccesary  apply Koch´s postulates for correct identification  of the pathogen. Traditional plant pathology is based on morphometric features of the reproductive structures. 

You must isolate from the diseased  plants and then test the pathogenic fungi. Later  apply Koch´s postulates.

Fatemah,

If your 'unknown isolate' is on an artificial medium, you have several options of identifying the fungus. If it is still maintained on live plants (obligate pathogen), you have just a limited scope. 

You can use  these books also:   The Fungi: An Advanced Treatise [Ainsworth, G.C., Sparrow, F.K., Sussman, A.S. (eds.)]: 1-4 Vol. -1965–1973.– Academic Press, New York.

Whichever morphological, microscopic or genetic methods help in narrowing down to the fungus. Isolates can only be verified with Koch's postulates.

FIRST OBSERVES CAREFULLY  SYMPTOMS AND SIGNS OF THE DISEASE AND REGISTER THEM. IT IS IMPORTANT TO OBSERVE, NOT JUST LOOK. THUS YOU ALLOW CONCER EXACTLY WHICH IS THE PHYSIOLOGICAL FUNCTION THAT IS BEING ALTERED OR INTERRUPTED. PROCEEDS TO EXAMINE THE TISSUES OF SYMPTOMS AND SIGNS IN THE MICROSCOPE. SO YOU'LL HAVE AN IDEA OF THE NATURE OF THE PATHOGEN (FUNGUS, BACTERIA, OOMYCOTA, NEMATODE). THEN CHOOSE THE CULTURE  MEDIA OF CROPS MORE APPROPRIATE AND MAKES THE CORRESPONDING ISOLATES. BEFORE IDENTIFYING THE PATHOGEN PERFORMS PRELIMINARY INOCULATION TESTS TO SAVE TIME AND DISCARD ISOLATES. SELECT IDENTIFICATION KEYS.

As stated above, the first thing to determine is whether the isolate is indeed a pathogen.  Once that is done, for accurate identification of a fungus, a combination of morphological and molecular data is the best method.  Care needs to be taken with what identification keys and which genetic region(s) and databases are used for identification.  For example, the "Universal Fungal primers" do not provide reliable information for species identification in a number of important genera like Fusarium (e.g. Leslie and Summerell 2006), etc.  In addition, many of the public databases have a high error rate, particularly for material that is not curated (e.g. Bridge et al. 2003, Nilsson et al. 2006) and may not include ITS sequences for some important fungi (e.g.Brock et al. 2008, Romanelli et al 2010).  Thus relying only on a maximum similarity to something deposited in a general database is problematic.  Similarly, keys can vary in accuracy and may be outdated.  Using a combination allows morphological cues to help make informed decisions on the appropriate database(s) and genetic regions to use for DNA-based tests and molecular indications to determine appropriate keys and descriptions of a fungus, appropriate media, etc. for morphological observations.

First, you could focus on disease symptoms and selected pathogens based on plant host. Used this information for plate morphological identification. If this does not help or the pathogen cannot be grown on artificial media (for example Puccinia) using ITS primers could be helpfull. At least It will narrow down the search range. However then it comes to Fusarium genera, ITS primers give sometimes misleading data in comparison to dedicated barcoding methods we developed.