Topic of Doctoral Seminar-I Fluorescence In Situ Hybridization (FISH) Technique in Detection of Plant Pathogens Presented by Prashant Waghrulkar, 200136006 Ph.D. Agri. (Plant Pathology), 2nd Year, 3rd Semester College of Agriculture, JNKVV, Jabalpur Academic Year: 2021-22

1. Fluorescence In Situ Hybridization (FISH)
Technique
in Detection of Plant Pathogens
Topic of Doctoral Seminar-I
Presented by
Prashant Waghrulkar,
200136006
Ph.D. Agri. (Plant Pathology),
2nd Year, 3rd Semester
College of Agriculture,
JNKVV, Jabalpur Academic Year: 2021-22

2. Introduction
• It has been estimated that out of 36.5% average total crop losses . . .
• 70-80% of these losses were caused by Fungi
Crop losses (%) Causes
14.1 Diseases
10.2 Insects
12.2 Weeds
• More than 40% of crop loss is due to viral infections

3. Current Methods for Crop Disease Detection
Direct Detection Methods
• Polymerase Chain Reaction (PCR)
• Enzyme-Linked Immunosorbent Assay (ELISA)
• Immunofluorescence (IF)
• Flow Cytometry (FCM)
• Fluorescence in-situ Hybridization (FISH)
Indirect Detection Methods
• Thermography
• Fluorescence Imaging
• Hyperspectral Techniques
• Gas Chromatography
Detection of Plant Diseases Using Portable Sensors
• Biosensor Platforms Based on Nanomaterials
• Affinity Biosensors
- Antibody-Based Biosensors
- DNA/RNA-Based Affinity Biosensor
• Enzymatic Electrochemical Biosensors
• Bacteriophage-Based Biosensors

4. History of ISH
1953:
Watson & Crick demonstrated how the 2 strands of DNA are hold by
hydrogen bonding.
1969:
Gall & Pardue performed the first in situ hybridization experiment.
1991:
First application of FISH to plant cytogenetics by Leitch et al.
Joseph Gall Mary-Lou Pardue

5. Fluorescence In Situ Hybridization (FISH)
• It is a molecular detection technique which include hybridization of
DNA probes & target gene followed by microscopy.
• In this technique fluorescent probes bind to particular parts of
a nucleic acid sequence with a high degree of
sequence complementarity.

6. Probe: Single strand of DNA or RNA that is complementary to a
nucleotide sequence of interest.
• It is approx. 20-50 oligonucleotide pairs & covering a space of 40–50
bp.
• A wide range of probes, extending from whole genomes to small
cloned probes (1–10 kb), can be used.
There are basically 3 types of probes
• Whole chromosome painting probes
• Repetitive sequence probes
• locus specific probes
Probe Labelling (Tagging by Nick translation)
• DNase1 nick the one strand of DNA & break the phosphodieaster
bond then some of the nucleotides of a DNA sequence replaced with
label.
• It can also be used for Radiolabeling.

7. • Nonradioactive probe label: Haptens (biotin, fluorescein,
rhodamine, & coumarin etc.)
• Radioactive probe label: Radioisotopes (32P,35S etc.)

8. Applications
• To detect & localize the presence or absence of
specific DNA sequences on chromosomes.
• To detect chromosomal aberrations (additions, deletions, insertions,
inversions, copy number, translocations).
• Used for gene mapping.
• Karyotyping by Chromosome painting
Types of chromosome painting
Whole chromosome
painting probe
(WPP)
Chromosome terminal
band painting probes
(TPP)
Chromosome arm
painting probes
(APP)
Chromosome
enumeration probes
(CEP)
Cont...

9. Identification of human chromosomes through chromosome painting
Applications
Cont...

10. • To detect & localize specific RNA targets (mRNA: messenger
RNA, lncRNA: Long non-coding RNA & miRNA: micro RNA) in cells,
circulating tumor cells, & tissue samples.
• It help to define the spatial-temporal patterns of gene
expression within cells and tissues.
Central DOGMA
DNA to RNA to Protein
rRNA
Large subunit
Small subunit
tRNA
mRNA
RNA
Ribosomal RNA
Messenger RNA
Transfer RNA
Amino acid delivered by tRNA form a
Chain, then fold & form a protein
Applications
Cont...

11. • Used in prenatal diagnosis of diseases like Downy syndrome, cancer
etc.
• Helps in genetic counseling, medicine, & species identification.
• Due to the presence of pathogen-specific ribosomal RNA (rRNA)
sequences in plants, recognizing this specific information by FISH can
help detect the pathogen infections.
• In addition to bacterial pathogens, FISH could also be used to detect
fungi & viruses and other endosymbiotic bacteria that infect the
plant.
Applications

12. Fluorescence Microscopy
Fluorescence: The phenomenon in which a molecule absorbs light of higher frequency
& emits light of lower frequency.

13. Transmitted light Actin in Green
Actin is a microfilamentous protein of cytoskeleton
& important for shape of the cell
Mitochondria in Orange Nucleus in Blue

14. Advantages
• High Affinity & Specificity of DNA probes provide high single-cell sensitivity
because the probe will bind to each of the ribosomes in the sample.
• Can be detect culturable & yet-to-be cultured (so called unculturable)
organisms in order to investigate complex microbial communities.
Limitations
Detection Limit
Lies in the range of around 103 CFU/mL.
Accuracy & reliability
Highly dependent on specificity of nucleotide probes. Probe designing is
challenging.
Cause of false negative results
Insufficient penetration, higher order structure of target or probe (e.g., 3D
rRNA, loop & hairpin formation), low rRNA content, & photobleaching.

15. Limitations
Photobleaching
Fluroscence Loss in Photobleaching (FLIP)
It proves the motility of phospholipid
molecules in cell membrane

16. Limitations
Autofluorescence (false positive results)
Natural fluorescence
• Lipofuscins: A native autofluorescent material in certain large neurons in the
CNS.
• Elastin: It found in blood vessel walls & contain a similar fluorophore that
found in collagen.
• Collagen: Extracellular material between the smooth muscle and in the
adventitia layer.
Fixative-induced Fluorescence: Aldehyde, Glutaraldehyde
Cont...

17. Tomato yellow leaf curl virus
(TYLCV)
Whitefly
(Bemisia tabaci)

18. Chrysanthemum × morifolium Puccinia horiana
Gladiolus × hortulanus Uromyces transversalis
Glycine max Phakopsora pachyrhizi

19. Downy mildew of
Impatiens walleriana caused by
Plasmopara obducens

20. Plasmopara obducens sporangiophores & sporangia hybridized to probes labeled with
Alexa594 (red, a–f) or Alexa350 (blue, g–i) fluorophores. Scale bar = 20 µm