Phenotypic and genotypic test for identification of plant by akhil dhawane.pptx

This presentation provides an in-depth overview of phenotypic and genotypic methods used for the accurate identification of plant pathogens, a core topic in the M.Sc. Plant Pathology curriculum.

1. DR panjabrao Deshmukh Krishi vidyapeth , akola college of agriculture Nagpur department of plant pathology topic – phenotypic and genotypic test for identification of plant pathogen course no – pl. path 506 course titlie –technique for detection and diagnosis of plant diseases presented by - Akhil g. Dhawane ( msc plant pathology) submitted to – dr. r w ingle sir (professor and head of department)

2. Phenotypic and genotypic test for identification of plant pathogen Identification of plant pathogens is crucial for effective disease management in agriculture. Both phenotypic and genotypic tests are used to identify these pathogens. Pathogen can include a wide range of organism such as bacteria, fungi and viruses each with their own unique characteristics and symptoms. Accurate identification is essential for developing effective treatment and prevention strategies.

3. Phenotypic test 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. Phenotypic tests are as follows – 1) symptoms observation test 2) microscopic examination test 3) cultural characteristics test 4) biochemical test 5) pathogenicity test 6) serological test

4. 1) Symptom observation test Identifying a plant pathogen involves careful observation of various symptoms exhibited by the plant. 1) General symptoms – Wilting : water loss in leaves and stems. Stunting : Reduced growth and development compared to healthy plants. Yellowing (Chlorosis) : Leaves may turn yellow due to nutrient deficiencies or caused by the pathogen. Necrosis : Death of tissue in leaves, stem. Abnormal Growths : Galls, tumors, or other unusual growths. Fusarium oxysporium f.sp . ciceris Rice grassy stunt virus Rice yellow dwarf virus Plasmodiophora brassicae

5. 2) Specific symptoms – Leaf Spots : Circular, irregular, or angular spots on leaves, often with a yellow halo. May be caused by bacteria or fungi. Blights ( burnt like appearance ) : Rapid and extensive browning and death of plant tissues. Cankers : Sunken, necrotic lesions on stems, branches, leaves or fruits. Rusts : Powdery pustules or blisters on leaves, typically orange, red, brown, or black, indicating fungal infection. Powdery Mildew : White, powdery growth on leaves and stems. Downy Mildew : Yellow patches on upper leaf surface leaves. Rots : Soft, discolored, and often foul-smelling decay of fruits. Root Rot : Black, mushy, and decaying roots often caused by fungal or bacterial pathogens. Colletotrichum capsici Uncinula necator Xanthomonas citri subsp. citri Pectobacterium carotovorum Puccinia triticina Plasmopara viticola

6. 3) Viral Symptoms - Mosaic Patterns : Mottled, patchy discoloration of leaves. Leaf Curl : Distorted, curled leaves. Streaking : Stripes or streaks on leaves , stems and fruits. Tobacco mosaic virus (TMV) Chilli leaf curl virus Tomato streak virus

7. Advantages : - Quick and easy It can be done in the field. Limitations : - Symptoms can be non-specific and may overlap between different pathogens or environmental stress conditions.

8. 2) Microscopic examination test – Microscopic examination is a crucial step in identifying plant pathogens. For fungi - Collect Sample - Obtain a small section of the infected plant tissue, such as leaves, stems, or roots. Preparation - Place the sample on a glass slide. Add a drop of water or a staining solution like lactophenol cotton blue, which helps highlight fungal structures. Cover with a coverslip. Microscopic Examination - Hyphae : Look for filamentous structures. Hyphae are the branching threads of fungal mycelium. Spores : Identify different types of spores (conidia, sporangia, zoospores). Their shape, size, and arrangement can help in identifying the fungal species. Fruit Bodies : Observe structures like sporangia, pycnidia, and perithecia, which produce spores. Bipolaris oryzae (brown spot of rice ) Podosphaera aphanis (powdery mildew of strawberry )

9. For bacteria – Collect Sample - Obtain a sample from the infected area, such as ooze from cankers or discolored vascular tissues. Preparation: - Smear the sample onto a glass slide. - Stain with Gram stain or another bacterial staining technique. Microscopic Examination - Cell Shape and Arrangement : Identify the shape (rod-shaped, spherical) and arrangement (chains, clusters). Gram Stain : Determine if bacteria are Gram-positive (purple) or Gram-negative (pink/red). Xanthomonas (gram -negative ) Clavibacter (gram –positive )

10. For viruses - Viruses are too small to be seen with electron microscope. Collect Sample: - Use symptomatic plant tissue, such as leaves showing mosaic patterns or curling. Electron Microscopy : - Prepare ultra-thin sections of the infected tissue. Stain with heavy metals like uranyl acetate and lead citrate. Examine under an electron microscope to observe virus particles. Tobacco mosaic virus (under electron microscope)

11. 3) Cultural characteristics test – Cultural characteristics are the observable traits of microorganisms when they are grown on nutrient media. These traits can be very helpful in identifying plant pathogens For Fungi - Growth Medium : - Potato Dextrose Agar (PDA)

12. Steps for Culturing Pathogens – 1) Sample Collection: - Collect plant tissues showing symptoms of disease. 2) Isolation : - Surface sterilize the plant tissue, cut into small pieces, and place on the appropriate growth medium. 3) Incubation - Incubate the cultures under suitable conditions (temperature, light) for a specified period. 4) Observation: - Regularly observe and record the growth characteristics of the colonies. 5) Sub-culturing: - If necessary, sub-culture individual colonies to obtain pure cultures for more detailed observation and testing

13. For Bacteria – Growth Medium: - Nutrient Agar (NA)

14. Culture of Xanthomonas axonopodis pv . citri

15. For virus Viruses cannot be cultured on nutrient media as they require living host cells to replicate. Identification is usually based on symptomatology, serological tests (e.g., ELISA), and molecular techniques (e.g., PCR). Advantages :- It Can differentiate genus of plant pathogen based on growth patterns. Limitations : - Time-consuming It may not always provide definitive identification

16. 4) Biochemical test – Enzyme Activity Tests - Pathogens are tested for specific enzymatic activities. For example, bacteria may be tested for catalase, oxidase, and urease activities. 1) Catalase Test: - Purpose:- Detects the presence of catalase, an enzyme that breaks down hydrogen peroxide into water and oxygen. - Procedure: - A small amount of the bacterial culture is placed on a slide, and a drop of hydrogen peroxide is added. The presence of bubbles indicates a positive result. - Application:- Commonly used to differentiate between catalase-positive bacteria (e.g., Pseudomonas spp.) and catalase-negative bacteria (e.g., some species of Streptococcus).

17. 2) Oxidase Test: - Purpose: - Identifies bacteria that produce cytochrome c oxidase, an enzyme involved in the electron transport chain. Procedure: - A sample of the bacterial culture is placed on filter paper saturated with an oxidase reagent. A color change to dark blue or purple within a few seconds indicates a positive result. Application: - Used to distinguish oxidase-positive bacteria (e.g., Pseudomonas spp., Xanthomonas spp.) from oxidase-negative bacteria (e.g., Enterobacteriaceae).

18. 3) Urease Test: - Purpose: - Detects the presence of urease, an enzyme that hydrolyzes urea into ammonia and carbon dioxide. - Procedure:-The bacterium is inoculated into a urea broth or agar containing phenol red as a pH indicator. A color change to pink indicates a positive result due to the alkaline ammonia produced. Application: - Helps identify urease-positive pathogens like some species of Rhodococcus and certain fungi. .

19. 4) Gelatinase Test : - Purpose: - Assesses the ability of the pathogen to hydrolyze gelatin using gelatinase enzymes. Procedure:- The organism is inoculated into a gelatin medium. Liquefaction of the medium indicates a positive result. Application:- Used to identify gelatinase-positive bacteria (e.g., some species of Bacillus) and differentiate them from gelatinase-negative bacteria

20. 5) Amylase Test: - Purpose:- Detects the production of amylase, an enzyme that breaks down starch into simpler sugars. Procedure:- The pathogen is grown on a starch agar plate. After incubation, the plate is flooded with iodine solution. Clear zones around colonies indicate starch hydrolysis and a positive result. Application:- Helps identify amylase-producing pathogens like certain species of Xanthomonas.

21. 6) Pectinase Test: - Purpose: - Detects pectinase enzymes that degrade pectin, a component of plant cell walls. Procedure: - The pathogen is inoculated onto a pectin-containing medium. Clear zones around colonies indicate pectin degradation. Application: - Important for identifying pectinase-producing pathogens like some species of Erwinia, which cause soft rot.

22. 7) Cellulase Test: - Purpose:- Identifies the production of cellulase, an enzyme that breaks down cellulose. Procedure: - The organism is grown on a cellulose-containing medium. Clear zones around colonies after staining with Congo red indicate cellulase activity. Application: - Used for identifying cellulase-producing pathogens like certain fungi (e.g., Trichoderma spp.). Advantages: Can provide additional information for differentiation. Limitations: Requires specific reagents conditions It may not be definitive.

23. Pathogenicity tests – Inoculating healthy plants with the suspected pathogen and observing the development of disease symptoms . Procedure -Host Plant Selection: The first step is to choose a suitable host plant that is known to be susceptible to the suspected pathogen. This host plant should exhibit characteristic symptoms. Inoculum Preparation : The pathogen is cultured and grown under appropriate conditions to produce a concentrated inoculum. This inoculum may consist of spores, mycelium or bacterial cells. Inoculation: The inoculum is then applied to the host plant through various methods such as spraying, injecting, rubbing, or soil drenching, depending on the nature of the pathogen and the host plant. Observation of Symptoms: The inoculated plants are monitored over a period of time for the development of disease symptoms. These symptoms may include leaf spots, wilting, necrosis, chlorosis, stunting, cankers, or other characteristic signs of disease caused by the pathogen. Control Tests: Control plants, which are not inoculated with the suspected pathogen, are included in the experiment to ensure that any observed symptoms are indeed caused by the inoculated organism and not by other factors such as environmental stress or unrelated pathogens. Re-isolation: After observing disease symptoms, the pathogen is often re-isolated from the infected tissues and cultured again to confirm its identity. This helps ensure that the observed symptoms were caused by the inoculated organism and not by any other opportunistic pathogens present in the plant tissue.

24. Advantages: Direct evidence of pathogenicity. It proves koch's postulates (recognition , isolation , inoculation , reisolation ) Limitations: Requires host plants it not be always practical for all pathogens.

25. Serological Tests: - ELISA (Enzyme-Linked Immunosorbent Assay):- Uses antibodies specific to the pathogen’s antigens to detect its presence in plant tissues. Principle of ELISA :- ELISA relies on antibodies to detect the presence of specific antigens associated with a pathogen. There are several types of ELISA, but the most common in plant pathology is the indirect ELISA. microtitre plate ELISA reader

26. Steps in ELISA for Plant Pathogen Detection:- Sample Collection: -Collect plant tissue or extract sap from the plant suspected of infection. Processing : - Homogenize the tissue or sap in a suitable buffer to release the pathogen antigens. Microplate Coating : - Coat the wells of a microplate with the antigen (pathogen extract). Incubate to allow binding. Blocking : - Add a blocking solution (e.g., BSA-bovine serum albumin or non-fat dry milk) to block non-specific binding sites on the microplate. Primary Antibody Binding : - Add a primary antibody specific to the pathogen antigen. This antibody binds to the antigen if it is present. Enzyme-Linked Secondary Antibody: - Add a secondary antibody that binds to the primary antibody. This secondary antibody is conjugated to an enzyme (e.g., horseradish peroxidase). Substrate Addition : - Add a substrate that the enzyme can convert to a detectable product, usually a color change. The intensity of the color is proportional to the amount of antigen present. Detection and reading : - Measure the color change using a spectrophotometer or ELISA reader. Higher absorbance indicates a higher concentration of the pathogen antigen

27. Applications in Plant Pathology Virus Detection : - ELISA is commonly used to detect plant viruses such as Tomato spotted wilt virus (TSWV), Tobacco mosaic virus (TMV), and Potato virus Y (PVY). Bacterial Pathogen Detection : - ELISA can identify bacterial pathogens like Xanthomonas spp. and Pseudomonas syringae . Fungal Pathogen Detection : - Fungi such as Fusarium spp. and Phytophthora spp. can also be detected using ELISA. Advantages of ELISA in Plant Pathology- High specificity. Useful for lab studies. Limitations- Limited to known antibodies. It may cross react with closely related species.

28. Genotypic 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. 1) Polymerase chain reaction ( PCR ) 2 ) DNA sequencing 3) DNA barcoding 4) Molecular markers

29. 1) Polymerase chain reaction ( PCR ) – Amplification of specific DNA sequences using primers that target regions unique to the pathogen. PCR step by step- Sample Collection :- Obtain samples from the plant tissue showing symptoms of disease (e.g., leaves, stems, roots) and Use sterile tools and containers to avoid contamination. Isolate DNA: - Extract DNA from the plant tissue using a suitable plant DNA extraction kit or protocol. This often involves grinding the tissue in a buffer, followed by a series of purification steps. Purify DNA: Ensure the DNA is free from contaminants that could inhibit the PCR reaction. Primers Target Pathogen DNA: - Select primers specific to the DNA sequences of the pathogen. Primers are short sequences of nucleotides that bind to specific regions of the pathogen’s DNA.Specificity : - Ensure that the primers are specific to the pathogen and do not amplify plant DNA

30. Analysis of PCR Products Agarose Gel Electrophoresis: Load the PCR products onto an agarose gel and run electrophoresis to separate DNA fragments by size. Staining and Visualization: Stain the gel with a DNA-binding dye (e.g., ethidium bromide) and visualize under UV light. The presence of a band at the expected size indicates the presence of the pathogen. Interpretation of Results Positive Control: Ensure a positive control (known pathogen DNA) was included to confirm the PCR worked correctly. Negative Control: Ensure a negative control (no DNA template) was included to check for contamination.

31. Type of PCR techniques Reverse transcription PCR :- similar to PCR but used to amplify RNA and useful for RNA viruses in plant Conventional PCR: - Detects specific DNA sequences of the pathogen. Requires pathogen-specific primers. Multiplex PCR: - Detects multiple pathogens in a single reaction using different sets of primers. Real time PCR Digital PCR

32. Advantages : Highly sensitive and specific. rapid results. Limitations : Requires prior knowledge of target DNA sequences It may not differentiate closely related species

33. 2) DNA Sequencing DNA sequencing is a powerful tool for identifying plant pathogens. The process involves several steps : Sample Collection and Preparation: Samples from infected plant tissues (leaves, stems, roots, etc.) are collected and prepared for DNA extraction. This preparation might involve cleaning, grinding, and homogenizing the tissues to release DNA. DNA Extraction: The DNA is extracted from the plant tissue using various methods, including commercial kits that use buffers and enzymes to break down cell walls and membranes, releasing the DNA into solution. PCR Amplification: Specific regions of the pathogen's DNA are amplified using polymerase chain reaction (PCR). Primers designed to match sequences of known pathogens or conserved regions across related pathogens are used in this step. This ensures that even small amounts of pathogen DNA can be detected and analyzed.

34. Sequencing: The amplified DNA is then sequenced. There are several sequencing technologies available, including Sanger sequencing for smaller, targeted regions and next-generation sequencing (NGS) for larger-scale sequencing efforts. Bioinformatics Analysis: The raw sequence data is processed and analyzed using bioinformatics tools. Sequences are compared to databases of known pathogen sequences (like GenBank) to identify the pathogen. Identification and Confirmation: Based on the sequence data and bioinformatics analysis, the pathogen is identified. This identification can be further confirmed by comparing the symptoms, environmental conditions, and other characteristics observed in the field

35. Advantages : Provide detail genetic information High resolution Limitation : Expensive Time consuming Require bioinformatics expertise

36. 3) DNA Barcoding DNA barcoding is indeed a powerful tool for identifying plant pathogens. It involves using short standardized DNA sequences from a specific region of the genome to identify species. For plant pathogens, this can be particularly useful in distinguishing between closely related species or strains that may have similar morphological characteristics but differ genetically.

37. Steps of DNA barcoding – Selection of Barcode Region: The first step is to select a suitable region of the pathogen's genome to serve as the barcode. This region should ideally have enough genetic variation to distinguish between different species or strains but also be conserved enough within species. DNA Extraction and Amplification: DNA is extracted from the pathogen samples, which can include infected plant tissues or pure cultures of the pathogen. The selected barcode region is then amplified using polymerase chain reaction (PCR) techniques. Sequencing: The amplified DNA is then sequenced using high-throughput sequencing technologies, such as Sanger sequencing or next-generation sequencing (NGS). This generates a DNA sequence for each sample. Sequence Analysis: The generated sequences are compared to a reference database of known sequences using bioinformatics tools. By comparing the sequences, researchers can determine the identity of the pathogen present in the sample. Identification: Based on the similarity of the sequences to those in the reference database, the pathogen present in the sample can be identified at the species or strain level

38. 4) Molecular markers – RFLP : - Restriction Fragment Length Polymorphism (RFLP) is a molecular marker technique that has been widely used for the identification and characterization of plant pathogens Principle of RFLP : RFLP is based on the detection of variations in DNA sequences among different individuals or strains. It relies on the fact that DNA sequences are cut (digested) by specific restriction enzymes into fragments of varying lengths. Variations in the lengths of these DNA fragments, resulting from differences in DNA sequences (polymorphisms), can be visualized using gel electrophoresis. Procedure: DNA Extraction: The first step in RFLP analysis involves extracting DNA from the pathogen of interest, typically from infected plant tissues or pure cultures. Digestion : The extracted DNA is then digested with one or more restriction enzymes, which cleave the DNA at specific recognition sites. The choice of restriction enzymes depends on the target DNA sequence and the desired resolution of the RFLP analysis. Gel Electrophoresis : The digested DNA fragments are separated by size using gel electrophoresis, where they migrate through a gel matrix in response to an electric field. Smaller fragments move faster through the gel, while larger fragments move more slowly. Visualization : After electrophoresis, the DNA fragments are visualized using staining techniques or by leaser and hybridizing them with labeled probes specific to the target DNA sequence.