Molecular Tools for Detection of Plant Pathogenic Fungi ByMAZIN.S.SELMAN

I believe that many of us heard when he was a small child a story that says at one days one bird love fish and they had agreed to the marriage, but there was one obstacle ! which is where they will build their own house? In the river where the fish live or in the air where the bird live ?

We Learn from this story important thing is that there are genetic barriers separating species races and genus,this undebatable and clear fact and No one can said this fact is not true

So genetic convergence and divergence regulates all relations and even the relationship between pathogens and plants

Plant pathogenic fungi are the causal agents of the most detrimental diseases in plants, including economically important crops, provoking considerable yield losses worldwide. Fungal pathogens can infect a wide range of plant species or be restricted to one or few host species.

Therefore, we must focus on two things 1- Fungal pathogen genetic diversity comes from Mutation, Recombination and Gene Flow.

2-Genetic polymorphism in plant may be make plant more resistant or more Susceptible to certain fungi. In next lecture I will explain the genetics polymorphism in plants

Fungal pathogen genetic diversity This genetic diversity may be lead to increase or decrease the fugal pathogenicity ( virulence ). This genetic diversity may be lead to increase or decrease the fugal pathogenicity ( virulence ). So for example we can detect some of genetics features that present in pathogenic fungi :- So for example we can detect some of genetics features that present in pathogenic fungi :- A- Genome size variation The genomes of several, but not all, filamentous plant pathogens are larger than those of their free-living relatives and represent remarkable cases of genome

B-Genome architecture Filamentous plant pathogen genomes are typically rich in non-coding DNA and display an irregular architecture, with an uneven distribution of genes and repetitive elements across and between chromosomes. Here, we can discuss the different types of genomic regions that harbor virulence and effector genes in pathogenic fungi Gene clusters. In plant pathogenic fungi, genes encoding metabolic enzymes often occur in co-expressed clusters. Gene-sparse regions. (also known as transposon islands ) Gene-sparse regions. (also known as transposon islands ) regions (structure) rich in certain repeats (CA repeats ) OR (GC repeats) for examples Isochore-like regions. A genome regions (structure) rich in certain repeats (CA repeats ) OR (GC repeats) for examples

Subtelomeric regions. Effector genes in plant pathogenic fungi are sometimes located near telomeres, which tend to evolve at higher rates than the rest of the genome. Conditionally dispensable chromosomes. Conditionally dispensable chromosomes (CDCs) that differ from the remainder of the genome in several structural features, such as the number of repeats, gene density and GC content. CDCs often carry virulence and effector genes, but they are accessory chromosomes: their loss does not affect fungus viability.

Molecular methods for detection of plant pathogenic fungi 1-Polymerase Chain Reaction (PCR) Selection of target DNA to amplify conserved known genes with enough sequence variation are selected Internal transcribed spacer regions (ITS) Single nucleotide polymorphisms (SNPs)

PCR-based methods : Conventional PCR

2- Nested-PCR and Cooperational-PCR (Co-PCR) Nested PCR approach is used when an improvement of the sensitivity and/or specificity of detection is necessary. This method consists in two consecutive rounds of amplification in which two external primers amplify a large amplicon that is then used as a target for a second round of amplification using two internal primers.

3-Multiplex PCR :- Multiplex PCR is based on the use of several PCR primers in the same reaction allowing the simultaneous and sensitive detection of different DNA targets, reducing time and cost. This method is useful in plant pathology since plants are usually infected by more than one pathogen.

4-in situ PCR :- This technique allows the amplification of specific gene sequences within intact cells or tissues combining two technologies: PCR and in situ hybridization (ISH).

5-PCR-DGGE :- Small samples of DNA (or RNA) are added to an electrophoresis gel that contains a denaturing agent. The denaturing gel induces melting of the DNA at various stages. As a result of this melting, the DNA spreads through the gel and can be analyzed for single components.DNARNAelectrophoresis

6-Fingerprinting Fingerprinting approaches allow the screening of random regions of the fungal genome for identifying species-specific sequences when conserved genes have not enough variation to successfully identify species. Fingerprinting analyses are generally used to study the phylogenetic structure of fungal populations. fingerprinting include three techniques Restriction fragment length polymorphism (RFLP) Random Amplified Polymorphic DNA (RAPD) Amplified fragment length polymorphism (AFLP)

7-Microsatellites :- Microsatellites, also known as simple sequence repeats (SSRs) or short tandem repeats (STRs), are motifs of one to six nucleotides repeated several times in all eukaryotic genomes (generally in non- coding regions). These nucleotide units can differ in repeat number among individuals and their distribution in the genome is almost random. Using primers flanking such variable regions PCR products of different lengths can be obtained. So, the microsatellites are highly versatile genetic markers that have been widely exploited for DNA fingerprinting. The advantages of SSRs are that they are multiallelic, codominant, highly polymorphic and several thousand potentially polymorphic markers are available