Plant Pathology and Detection Methods

Plant Disease

• A disruption in normal plant physiology, negatively affecting survival or fitness.

• Includes infectious agents, nutrition, and air pollution (and nematodes, but not insects, mites, or genetic abnormalities unless caused by infectious agents).

• Plant pathologists primarily work with infectious agents.

How Pathogens Cause Disease

1. Enzymatic degradation:

   • Pathogens secrete enzymes that break down host tissues, similar to digestion in mammals.

2. Toxins:

   • Pathogens produce toxins to kill tissue before enzymatic degradation.

3. Growth regulators:

   • Pathogens produce growth regulators that cause:

     • Translocation of nutrients to host cells.

     • Enlargement or division of host cells near the pathogen, increasing food supply for the pathogen.

4. Genetic manipulations:

   • Viruses and some bacteria force the plant to produce pathogen gene products, starving plant cells and disrupting their function.

Common Pathogens

1. Bacteria

2. Fungi

3. Viruses

4. Nematodes

5. Parasitic higher plants

6. Insects

Bacteria

• Single-celled, no nucleus, one chromosome.

• Limited size, unlimited reproduction by fission (no chromosomal segregation), allowing faster reproduction than fungi and potentially quick epidemics.

• Absorptive nutrition, mostly saprophytic in nature.

• Cause blights (rapid, toxic killing of plant tissue), rots (mushy breakdown), wilts (plugging of vasculature), and galls (growth regulator-mediated enlarged areas on plants).

• Examples: fire blight on pear, crown gall on many woody plants, soft rot on many herbaceous plants.

Fungi

• Connected cells with nuclei, multiple chromosomes, mitochondria, and chitin for strength.

• Most can form differentiated structures, e.g., mushrooms, spores.

• Like bacteria, most are saprophytic.

• Plants infected with fungi exhibit many symptoms, including rot, blight, leaf spots, and wilts.

• Sensitive to light and dry conditions when growing but can make resistant structures to survive.

• Spread by wind, water, seed, and vectors.

• Examples: apple scab, powdery mildews, peach leaf curl.

Viruses

• Pieces of nucleic acid (RNA or DNA); those with a protein coat are viruses, those without are viroids.

• Nucleic acid codes for a few proteins and takes over a cell, upsetting normal metabolism and causing an excess or shortage of molecules used to make new cell components.

• Symptoms mimic genetic abnormalities and include mosaics, yellows, distortions, and death.

• Spread by mechanical means, seeds, or vectors (important for control method selection).

• Examples: squash mosaic on zucchini, yellow viruses on many plants, tobacco mosaic on tomato.

Nematodes

• Microscopic worms; the presence of a stylet (needle-like mouthpart) differentiates plant parasitic nematodes from saprophytes.

• Injection of nematode saliva upsets plant metabolism, causing nutrient or hormone imbalances; symptoms include tumors and death of affected parts.

• Examples: root knot nematode on many plants, beet cyst nematode on vegetables.

Parasitic Plants

• Rely on a host for water and minerals (green-colored leaves) and sometimes carbohydrates (non-green-colored leaves).

• Deleterious effects result from hormonal upset of the host rather than nutrient or water loss.

• Primarily occur in forestry, perennials, and poorly managed annual crops.

• Examples: mistletoe on trees, dodder on vegetables.

Examples of Plant Diseases and Disorders by Crop

• Field Crops:

  • Corn: Gibberella Stalk Rot (Fusarium graminearum), Gray Leaf Spot (Cercospora zeae-maydis)

  • Soybean: Frogeye Leaf Spot (Cercospora sojina), Fusarium Root Rot (Fusarium graminearum), Stem Canker (Phomopsis sp.)

• Fruits:

  • Raspberry: Anthracnose (Sphaceloma necator), Cane Blight (Coniothyrium fuckelii)

  • Strawberry: Root/Crown Rot (Pythium sp., Rhizoctonia sp., Fusarium sp., Cylindrocarpon sp.), Verticillium Wilt (Verticillium sp.), Bacterial Brown Spot (Pseudomonas syringae pv. syringae)

• Vegetables:

  • Bean (Kidney): Black Shoulder (Alternaria alternata)

  • Tomato: Verticillium Wilt (Verticillium sp.), Late Blight (Phytophthora infestans)

• Field Crops

  • Oats: Bacterial Streak (Xanthomonas campestris pv. translucens)

  • Wheat: Stripe Rust (Puccinia striiformis)

• Forage Crops

  • Alfalfa: Root Rot (Fusarium sp., Phytophthora sp.)

• Fruits

  • Blueberry: Phomopsis Canker (Phomopsis sp.)

• Vegetables

  • Carrot: Black Rot (Alternaria radicina)

  • Potato: Bacterial Soft Rot (Pectobacterium carotovorum), Black Heart (None)

  • Leek: Leak (Pythium sp.)

  • Tomato: Cucumber Mosaic (Cucumber mosaic virus)

Detection of Pesticide Residue

• A pesticide residue is any specified substance in food, agricultural commodities, or animal feed resulting from the use of a pesticide.

• Includes derivatives, conversion products, metabolites, reaction products, and impurities of toxicological significance.

• Detection methods include GC (Gas Chromatography), NMR (Nuclear Magnetic Resonance), HPLC (High-Performance Liquid Chromatography), and spectrophotometry.

Detection Methods of Disease-Causing Pathogens

• Important agricultural crops are threatened by plant diseases and pests, which can damage crops, lower quality, and wipe out harvests.

• Crop losses can be minimized by early and correct diagnoses.

• Two molecular diagnostic techniques:

  • ELISA (Enzyme-Linked Immunosorbent Assay)

  • PCR (Polymerase Chain Reaction)

ELISA (Enzyme-Linked Immunosorbent Assay)

• Biochemical technique to detect the presence of an antibody or antigen in a sample.

• Uses antibodies and color change to identify a substance.

• Used as a diagnostic tool in medicine and plant pathology, and as a quality-control check in various industries.

• Types:

  1. Indirect ELISA

  2. Sandwich ELISA (direct ELISA)

  3. Competitive ELISA

• Common enzymes used: alkaline phosphatase, horseradish peroxide, and $\beta$-galactosidase.

Indirect ELISA

• Serum containing primary antibody (Ab1) is added to an antigen-coated microtiter well and allowed to react.

• After washing away unbound Ab1, an enzyme-conjugated secondary antibody (Ab2) that binds to the primary antibody is added.

• After washing away unbound Ab2, a substrate for the enzyme is added.

• The amount of colored reaction product is measured using spectrophotometric plate readers.

Sandwich ELISA (Direct)

• The antibody is immobilized on a microtiter well.

• A sample containing antigen is added and allowed to react with the immobilized antibody.

• A second enzyme-linked antibody specific for a different epitope on the antigen is added and allowed to react.

• After washing, substrate is added, and the colored reaction product is measured.

Competitive ELISA

• Antibody is first incubated in solution with a sample containing antigen.

• The antigen-antibody mixture is added to an antigen-coated microtiter well.

• The more antigen in the sample, the less free antibody is available to bind to the antigen-coated well.

• An enzyme-conjugated secondary antibody (Ab2) specific for the isotype of the primary antibody is added.

ELISA Technique

• Antigens from the sample are attached to a surface.

• A specific antibody is applied to bind to the antigen.

• This antibody is linked to an enzyme, and a substance containing the enzyme's substrate is added.

• The reaction produces a detectable signal, such as a color change.

ELISA Applications

• Determining serum antibody concentrations (e.g., HIV test, West Nile Virus).

• Detecting food allergens (milk, peanuts, walnuts, almonds, eggs).

• Toxicology: rapid presumptive screen for certain classes of drugs.

• Detecting diseases and tracking outbreaks (HIV, bird flu, common colds, cholera, STD etc.).

• In vitro diagnostics in medical laboratories.

• Detection of antibodies in blood samples for past exposure to disease.

• Detection of antigens (pregnancy hormone, drug allergen, GMO, mad cow disease).

• Detection of Mycobacterium antibodies in tuberculosis, rotavirus in feces, hepatitis B markers in serum, enterotoxin of E. coli in feces, and HIV antibodies in blood samples.

PCR (Polymerase Chain Reaction)

• The most important and sensitive technique for detecting plant pathogens.

• Amplifies millions of copies of specific DNA sequences through repeated cycles of:

  • Denaturation

  • Annealing

  • Elongation

• Uses specific oligonucleotides (primers), deoxy-ribonucleotide triphosphates (dNTPs), and a thermostable Taq DNA polymerase in the appropriate buffer.

• Amplified DNA fragments are visualized by electrophoresis in agarose gel stained with EtBr, SYBR Green, or other DNA-intercalating molecules, or by colorimetric or fluorometric assays.

• The presence of a specific DNA band of the expected size indicates the presence of the target pathogen.

PCR Components and Reagents

1. DNA template: contains the DNA region (target) to be amplified.

2. Two primers: complementary to the 3’ ends of each strand of the DNA target.

3. Taq polymerase: a DNA polymerase with a temperature optimum around 70 °C.

4. Deoxynucleoside triphosphates (dNTPs): building blocks for synthesizing a new DNA strand.

5. Buffer solution: provides a suitable chemical environment for enzyme activity and stability.

6. Bivalent cation: magnesium or manganese ions (Mg2+ is generally used; Mn2+ can be used for PCR-mediated DNA mutagenesis).

7. Monovalent cation: potassium ions.

PCR-Based Diagnostic Methods

• Advances like real-time PCR allow fast, accurate detection and quantification of plant pathogens in an automated reaction.

• Advantages: high sensitivity, specificity, and reliability.

• It is not necessary to isolate the pathogen, reducing diagnosis time from weeks to hours.

• Allows detection and identification of non-culturable pathogens.

• Useful in the analysis of symptomless plants.

PCR Steps

1. DNA is unwound, and strands are separated by high temperatures.

2. As temperature lowers, primers bind to DNA strands at regions of homology, allowing Taq polymerase to make a new copy.

3. The cycle of denaturation-annealing-elongation is repeated 30-40 times, yielding millions of copies.

4. Primers in PCR diagnostic kits are very specific for the genes of a pathogen, and amplification only occurs in diseased plants.

PCR Applications

1. Selective DNA isolation: PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA.

2. Amplification and quantification of DNA: PCR can be used to analyze extremely small amounts of sample, which is critical for forensic analysis.

3. PCR in diagnosis of diseases: PCR permits early diagnosis of malignant and infectious diseases.

Other Versions of PCR

• Multiplex nested PCR

• Co-operative PCR

• Real-time monitoring of amplicons or quantitative PCR

DNA Microarray

• New method of detection.

DNA Microarray Assay of Gene Expression Levels

1. Isolate mRNA.

2. Make cDNA by reverse transcription, using fluorescently labeled nucleotides.

3. Apply the cDNA mixture to a microarray.

4. Rinse off excess cDNA; scan microarray for fluorescence.

DNA Microarray Making

1. Microscope glass slides coated with polylysine

2. RNA extraction

3. mRNA amplified by PCR

4. Reverse transcription

5. Spotting (deposit)

6. Hybridization

7. Scanning (lecture)

8. Results analysis

Characteristics for Pathogen and Contaminant Detection

• Accurate

• Fast

• Reliable

• Precise (for large number of samples)

• Easy to handle

• High sensitivity

Importance of Pathogen and Contaminant Detection

• Good quality of fruits

• Prevent disease transfer across countries borders

• Prevent transmission of disease to another plants

• Early detection may save other uninfected plant