MOLECULAR TOOLS TO STUDY DIVERSITY OF ANTAGONISTIC FUNGI

A.BHARANIDEEPAN 13-513-301 DEPARTMENT OF PLANT PATHOLOGY

Detection of fungi on the basis of visual examination of morphology is highly selective and species - specific identification of fungi and spore is difficult.

based on arrangements, forms, sizes and ornamentations of mature sexual and asexual structures

 Morphological features occasionally overlap and standardization of incubation conditions and the use of chemically defined media have been proposed to avoid these problems. Image analysis DNA sequencing Micro array technology Expressed sequencing tag (EST)

TYPES OF MOLECULAR MARKERS

Molecular markers for antagonist fungi can be derived from both variable and conserved region of nucleic and mitochondrial genome . Markers have potential for the detection of specific genomic DNA sequence from initial plant samples. Probes and primers are developed from variety of DNA sequences.

SELECTION OF MOLECULAR MARKERS

1. 2. Taxonomic rank Life cycle of fungus

HYBRIDIZATION BASED METHODS

Direct hybridization techniques used for detection are in situ hybridization and colony or dot blot hybridization. DNA probes and peptide nucleic acid are used. Recent development - DNA micro array.

PCR BASED METHODS

Any DNA or RNA sequence that is specific to particular antagonist fungi can be used for PCR identification of that antagonist. Sensitivity, speed, versatility of PCR are primary factors. To increase the specificity of PCR reactions, post-PCR hybridization, PCR-ELISA reactions are used.

RIBOSOMAL DNA GENE CLUSTER

DNA sequence that encode for RNA have been extensively used to study taxonomic relationships and genetic variations in fungi. rRNA gene cluster found in nuclei and mitochondria consists of highly conserved and variable regions.

INTERGENIC TRANSCRIBED SPACE (ITS)

ITS mainly used as a target for species - specific detection of fungi. Its mainly used for molecular characterization studies of fungi.

ITS 1
rDNA Subunits

ITS 2

RANDOM AMPLIFIED P O LY M O R P H I C D N A ( R A P D )
RAPD used for developing suitable species or strain specific probe for detection. On contrast to PCR, low annealing temperature is maintained (35C). SCAR Sequenced characterized amplified region. Band patterns are used to differentiate both within and between individual species.

 ISSR Inter simple sequence repeat SSR Simple sequence repeat

Amplify the microsatellite regions of fungal chromosomal DNA.

A M P L I F I E D F R AG M E N T L E N G T H P O LY M O R P H I S M ( A F L P )
Good combination of PCR and RFLP. DNA is cut with two restriction enzymes and known sequence is ligated. Primers accord to the known base sequence is designed. PCR is run to amplify fragments.

M O L E C U L A R TA XO N O M Y A N D GENOMICS OF Trichoder ma species

Internal transcribed sequences (IST) are used to identify T.longibrachiatum, T.harzianum, T.viride, T.aureoviride.

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