Phenotypic and Genotypic tests for identification of plant pathogens by SACHIN R KONDAGURI.pptx

Phenotypic and Genotypic tests for identification of plant pathogens

1. WELCOME

2. UNIVERSITY OF AGRICULTURAL SCIENCES, BANGALORE COLLEGE OF AGRICULTURE,V C FARM,MANDYA * Submitted by : SACHIN R KONDAGURI PAMM 2030 Jr M.Sc PAT 506 (0+2) 2 *Submitted to: Dr.N S PANKAJA ASSOCIATE PROFESSOR Dept. of Plant Pathology CoA, V C Farm, Mandya :Phenotypic and Genotypic tests for identification of plant pathogens:

3. 18-11-2023 PAMM2030 Dept. of Pathology 3 Phenotypic tests for identification of plant pathogens

4. 18-11-2023 PAMM2030 Dept. of Pathology 4 Phenotypic tests :   Phenotypic tests for identifying plant pathogens involve the observation of physical characteristics , growth patterns , and behaviors of the pathogen . These tests are often the initial steps in the identification process and can provide valuable information. Tests commonly used for this purpose: Symptom Observation test Cultural Characteristic test Morphological Feature test Serological Tests Pathogenicity Tests

5. Physiological and Biochemical Tests Host Range Studies 18-11-2023 PAMM2030 Dept. of Pathology 5

6. Description: Visual inspection of symptoms on the host plant, such as leaf spots , wilting , discoloration , and other abnormal growth patterns. Advantages: Quick and easy; can be done in the field. Limitations: Symptoms can be non-specific and may overlap between different pathogens or environmental stress conditions. 18-11-2023 PAMM2030 Dept. of Pathology 6 Symptom observation

7. SYMPTOMATOLOGY Small and diamond-shape Elongation of lesions as they mature Rectangular lesions

8. Description: Observation of the growth characteristics of the pathogen on various culture media, including color, texture, and rate of growth. Advantages: Can differentiate between species based on growth patterns. Limitations: Time-consuming; may not always provide definitive identification 18-11-2023 PAMM2030 Dept. of Pathology 8 Cultural characteristics

9. 3-day old 5-day old 10-day old 20-day old Pure culture of R. solani

10. 4 DAI 8 DAI 12 DAI Pure culture S.rolfsii on PDA Identification

11. Description: Examination of the physical characteristics of the pathogen, including spores, mycelium, and other structures under a microscope. Advantages: Provides detailed information for accurate identification. Limitations: Requires expertise; may not always be conclusive, especially for closely related species. 18-11-2023 PAMM2030 Dept. of Pathology 11 Morphological feature test

12. Pure culture of Rhizoctonia solani Microscopic view

13. Pure culture of B.maydis Speptate mycelia Conidia (septate) Bipolar germination of conidia

14. Description: Detection of specific pathogen proteins or antigens using antibodies. Advantages: High specificity; useful for lab studies. Limitations: Limited to known antibodies; may cross-react with closely related species. 18-11-2023 PAMM2030 Dept. of Pathology 14 Serological tests

15. Description: Inoculating healthy plants with the suspected pathogen and observing the development of disease symptoms. Advantages: Direct evidence of pathogenicity.It proves koch’s postulates Limitations: Requires host plants; not always practical for all pathogens. 18-11-2023 PAMM2030 Dept. of Pathology 15 Pathogenicity tests

16. Infected plant Pathogen culture Uninoculated plant     Symptoms expression Reisolation of pathogen Pathogenicity test of B. oryzae on PDA conidia Similar conidia

17. Description: Assessing physiological and biochemical characteristics, such as the ability to metabolize specific substrates or produce certain enzymes. Advantages: Can provide additional information for differentiation. Limitations: Requires specific reagents and conditions; may not be definitive. 18-11-2023 PAMM2030 Dept. of Pathology 17 Physiological and Biochemical Tests:

18. Description: Investigating the range of plant species that can be infected by the pathogen. Advantages: Provides information on host specificity. Limitations: Time-consuming; not always practical for routine identification. 18-11-2023 PAMM2030 Dept. of Pathology 18 Host range studies

19. 18-11-2023 PAMM2030 Dept. of Pathology 19 Ge notypic tests for identification of plant pathogens

20. 18-11-2023 PAMM2030 Dept. of Pathology 20 G enotypic tests Genotypic tests for the identification of plant pathogens involve the analysis of the genetic material of the pathogen. These tests provide a more precise and specific means of identification compared to phenotypic methods. Tests commonly used for this purpose: Polymerase Chain Reaction (PCR) Reverse Transcription PCR (RT-PCR) Molecular markers DNA Sequencing Restriction Fragment Length Polymorphism (RFLP)

21. Description: Amplification of specific DNA sequences using primers that target regions unique to the pathogen. Advantages: Highly sensitive and specific; rapid results. Limitations: Requires prior knowledge of target DNA sequences; may not differentiate closely related species 18-11-2023 PAMM2030 Dept. of Pathology 21 Polymerase Chain Reaction (PCR)

22. Description: Similar to PCR, but used to amplify RNA. Useful for RNA viruses. Advantages: Specific for RNA-based pathogens; can detect active infections. Limitations: Requires conversion of RNA to cDNA 18-11-2023 PAMM2030 Dept. of Pathology 22 Reverse Transcription PCR (RT-PCR):

23. Description: Using specific DNA markers associated with the pathogen, such as microsatellites or single nucleotide polymorphisms (SNPs). Advantages: Quick and specific; can be used for large-scale screening. Limitations: Requires prior knowledge of marker association. 18-11-2023 PAMM2030 Dept. of Pathology 23 Molecular markers

24. 18-11-2023 PAMM2030 Dept. of Pathology 24 940bp Ladder 1 2 3 4 5 6 7 8 9 10 Gel electrophoresis after amplification of isolates of B. maydis with NPS6 primer and 1 to 10 idicates the 10 isolates of B. maydis

25. Description: Determining the order of nucleotides in the pathogen's DNA, often the entire genome or specific regions. Advantages: Provides detailed genetic information; high resolution. Limitations: Expensive and time-consuming for routine use; requires bioinformatics expertise. 18-11-2023 PAMM2030 Dept. of Pathology 25 DNA Sequencing

26. Description: Digestion of amplified DNA with restriction enzymes , resulting in fragment patterns unique to different pathogens. Advantages: Provides genetic fingerprints; can differentiate between strains. Limitations: Requires large amounts of high-quality DNA; not as high throughput as other methods. 18-11-2023 PAMM2030 Dept. of Pathology 26 Restriction Fragment Length Polymorphism (RFLP):

27. 18-11-2023 PAMM2030 Dept. of Pathology 27

28. 18-11-2023 PAMM2030 Dept. of Pathology 28

29. 18-11-2023 PAMM2030 Dept. of Pathology 29