Plant Pathology exam 3

How do pathogens attack plants

1. enzymatic degradation

2. toxins

3. growth regulators

4. polyssacharides

Enzymatic Degradation of Attack Mechanism

 Target: Phospholipids and glycolipids in the plant cell membrane.

· Enzymes: Lipases/Phospholipases (PLs).

· Action: Degrade lipids, disrupting cell membrane integrity.

· Consequences:

  · For the Pathogen: The degradation products can be utilized as a food source.

  · For the Plant:

    · Plants also have their own PLs (e.g., PLC, PLA).

    · The products of lipid degradation can act as signal molecules to induce plant defense responses.

· Visual Model (from slide): Pathogen-derived PLs (PILP, PLP-PLC) act on the plasma membrane, potentially interacting with plant transcription factors like TGA5 and TGA3.

Toxins

· General Function: Damage plant cells.

· Producers: Necrotrophic and some hemibiotrophic pathogens.

· Modes of Action:

  · Affect cell membrane permeability.

  · Inactivate or inhibit plant enzymes.

  · Mimic plant hormones and disrupt hormonal balance.

  · Kill cells directly (leading to necrosis).

· Classification:

  · General (Non-Host Specific) Toxins

  · Host-Specific Toxins (HSTs)

General (Non-Host Specific) Toxins

· Nature: Toxic to a wide range of plant species, not specific to one host.

· Role: Typically function as virulence factors (enhance disease severity).

· Effects:

  · Inhibit host enzymes.

  · Affect cellular transport systems.

  · Disrupt cell membranes.

  · Suppress the plant immune system.

· Symptoms: Chlorosis (yellowing) followed by Necrosis (cell death).

· Examples: Tabtoxin, Phaseolotoxin, Tentoxin, Cercosporin.

Host-Specific Toxins (HSTs)

 Nature: Induce toxicity and promote disease only in specific host species or genotypes.

· Role: Can be pathogenicity factors (essential for causing disease) or virulence factors.

· Key Examples:

  · Victorin (HV Toxin): From Cochliobolus victoriae (necrotroph). Causes Victoria blight in oats. It was the first discovered HST and is a pathogenicity factor.

  · T-Toxin: Associated with the Southern Corn Leaf Blight epidemic (Race T).

  · HC Toxin: From Cochliobolus carbonum, affects specific maize lines. Its exact mode of action is unknown, but it is a virulence factor.

Gene-for-Gene Interaction in Toxin Compatibility

· Example: HC Toxin from Cochliobolus carbonum and maize.

· Mechanism:

  · The maize Hm1 gene confers resistance to the fungus.

  · Hm1 encodes a reductase enzyme that detoxifies the HC-toxin.

· Compatibility Table:

  Pathogen Genotype Host Plant Genotype Interaction Outcome

  HC-TOX (produces toxin) hm1 (susceptible, no resistance gene) Compatible (Disease)

  HC-TOX (produces toxin) Hm1 (resistant) Incompatible (Resistance)

  hc-tox (no toxin) hm1 or Hm1 Incompatible (Resistance)

 Growth Regulators (Hormones)

Pathogens manipulate plant hormones to cause imbalances, leading to disease symptoms.

· Agrobacterium tumefaciens (Case Study):

  · Transfers a segment of DNA (T-DNA) from its Ti plasmid into the plant genome.

  · The T-DNA contains genes for the synthesis of auxin and cytokinin.

  · This hormone overproduction leads to uncontrolled cell division and growth, forming crown galls (tumors).

  · The T-DNA also includes genes for opine synthesis, which the bacterium uses as a food source.

· Specific Hormones and Pathogenic Effects:

  · Auxin: Required for cell elongation and differentiation. Nematodes can manipulate auxin pathways.

  · Cytokinins: Promote cell division, growth, and differentiation. Pathogens may alter their levels.

  · Gibberellins: Have growth-promoting effects and can induce auxin production. Some viruses can inhibit gibberellin, leading to stunting.

  · Ethylene: Induces senescence and fruit ripening. Can be produced by both plants and pathogenic fungi/bacteria. Fungal infection often triggers ethylene production.

Polysaccharides

Producers: Fungi, bacteria, and nematodes produce Extracellular Polymeric Substances (EPS).

· Functions in Pathogenesis:

  · Vascular Blockage: EPS can clog the plant's vascular system (xylem), wilting.

  · Role in Bacteria: EPS and Lipopolysaccharides (LPS) are key virulence factors.

  · Biofilm Formation: Polysaccharides help form protective biofilms.

  · Survival: Protect survival structures like nematode eggs.

  · Adhesion: Help pathogens and their infectious structures (e.g., spores) adhere to the plant surface.

How Do Plants Recognize Pathogens? (Sensing Elicitors)

Plants recognize "elicitors"—molecular signals from pathogens or damaged self.

· Elicitors can be:

  · Non-Host Specific: Common to many microbes.

    · E.g., Chitin (fungi), Flagellin (bacteria, specifically flg22 epitope), Lipopolysaccharide - LPS (bacteria), Pathogen Enzymes (Cell Wall Degrading Enzymes), General Toxins.

  · Host Specific: Specific to a particular pathogen-host interaction.

    · E.g., Host-Specific Toxins (HSTs), Avirulence (Avr) proteins (e.g., AvrPto from P. syringae).

Recognition of Conserved Microbial Patterns (MAMPs/PAMPs/NAMPs)

· MAMP/PAMP/NAMP: Conserved, essential molecular motifs unique to microbes.

  · MAMP: Microbe-Associated Molecular Pattern.

  · PAMP: Pathogen-Associated Molecular Pattern (term used specifically for pathogens).

  · NAMP: Nematode-Associated Molecular Pattern.

· Examples:

  · Bacterium: Flagellin (flg22), Elongation factor Tu (EF-Tu), LPS, Peptidoglycan (PGN).

  · Fungus: Chitin, β-glucan.

  · Oomycete: β-glucan, Pep13 (oligopeptide), Elicitins (lipid-transfer proteins).

  · Nematodes: Ascarosides.

· Recognition & Response (PTI/MTI):

  · Plants have pattern recognition receptors (PRRs) on their cell surfaces (e.g., FLS2 for flg22).

  · Upon binding a MAMP, a signaling cascade is activated (e.g., involving BAK1, MAPK cascades).

  · This leads to MAMP-Triggered Immunity (MTI) or PAMP-Triggered Immunity (PTI).

  · Defense Outputs: Production of Reactive Oxygen Species (ROS), activation of PR genes, ethylene production.

Recognition of Effectors

· Effectors: Molecules secreted and delivered by pathogens into the plant apoplast or cytoplasm.

· They can be:

  · Non-Host Specific: e.g., Cell wall degrading enzymes, growth regulators, proteases, lipases, immunity suppressors, general toxins.

  · Host Specific: e.g., Some HSTs, Avirulence (Avr) proteins.

Recognition of Damage/Danger Signals (DAMPs)

· DAMPs: Damage-Associated Molecular Patterns.

· Origin: These are plant's own molecules that are produced, released, or exposed due to pathogen activity (e.g., by pathogen enzymes).

· Function: Act as "alarm signals" to activate plant defenses.

· Examples:

  · Cell wall fragments (e.g., Oligogalacturonides - OGs)

  · Cutin monomers

  · Short plant peptides: Peps, Systemins

· DAMPs are recognized by specific plant receptors, amplifying the defense response.

 Summary of Plant Defense Activation

1. Pathogen Exposure occurs.

2. Plant recognizes:

   · Microbial Patterns (MAMPs/PAMPs/NAMPs) via PRRs, leading to PTI/MTI.

   · Damage Signals (DAMPs) via their receptors.

3. This recognition triggers two levels of response:

   · Local Response: At the site of infection.

   · Systemic Response: Throughout the plant, triggered by a systemic signal.

Introduction to Plant Defense Responses - Plants employ a multi-layered defense system against pathogens, categorized into two main types:

1. A. Passive (Constitutive) Defenses: Pre-existing defenses present before any contact with a pathogen.

   1. Structural Defenses: Physical barriers.

   2. Biochemical Defenses: Pre-formed antimicrobial compounds.

2. Active (Inducible) Defenses: Defenses activated after pathogen recognition.

   1. Triggered by pathogen attack.

   2. Involves recognition, signal transduction, and the activation of resistance responses.

   3. Includes both local and systemic signals.

Pre-formed (Constitutive) Structural Defenses - These are physical barriers that prevent pathogen entry and establishment.

1. Waxy Cuticle:

   1. A hydrophobic layer covering the epidermal cells.

   2. Function: Prevents direct contact and penetration by microbes.

2. Cell Wall:

   1. A rigid structural barrier.

   2. Function: Varies in thickness and toughness; physically inhibits the advance of pathogens attempting direct penetration.

3. Stomata:

   1. Pores used for gas exchange, but also a common entry point for pathogens.

   2. Defense Role: Stomatal opening and closing is a regulated defense. Plants can close stomata in response to pathogens.

   3. Stomatal regulation is influenced by: Light, Stress, CO₂, Circadian clock, Humidity, and hormones like Abscisic Acid (ABA).

   4. Some pathogens can manipulate stomatal opening to gain entry.

4. Trichomes:

   1. Leaf hairs (specialized epidermal cells).

   2. Can be glandular (secrete compounds) or non-glandular.

   3. Function: Create a physical barrier that deters insects (making feeding and egg-laying difficult) and pathogens.

5. Bark:

   1. The outermost layer of stems and roots of woody plants.

   2. Function: Contains high amounts of suberin and lignin, making it highly water-resistant and tough, providing a formidable barrier.

6. Thorns:

   1. Function: Primarily deter herbivores, which can be vectors for pathogens.

Pre-formed (Constitutive) Biochemical Defenses - These are antimicrobial compounds present in the plant before infection.

1. Inhibitors Released into the Environment:

   1. Fungitoxic Exudates: Compounds released by plants that can inhibit fungal spore germination on the plant surface.

   2. Examples:

      1. Phenolic compounds: e.g., Catechol in onion bulbs, which confers resistance to onion smudge disease (Collectotrichum circinans). White onions lack catechol and are susceptible.

      2. Terpenoids: Volatile Organic Compounds (VOCs) with antimicrobial properties.

2. Inhibitors Present inside Plant Cells:

   1. Phenolic Compounds: Can have protein-degrading, corrosive, and antiseptic effects. Many inhibit pathogen-derived hydrolytic enzymes.

   2. Saponins: Soap-like compounds that disrupt the cell membranes of invading microbes.

   3. Proteins:

      1. Inhibitors of pathogen proteinases.

      2. Some plants maintain a basal level of antimicrobial enzymes like glucanases and chitinases.

Induced Structural Defenses - Reinforcements formed at the site of infection after pathogen attack.

1. Cell Wall Reinforcement:

   1. Lignin Accumulation: Deposition of lignin strengthens the cell wall, making it more difficult to penetrate. (e.g., Increased lignin in wheat leaves infected with Puccinia graminis tritici).

   2. Papillae Formation: Localized cell wall appositions (reinforcements) formed directly beneath the site of pathogen penetration (e.g., beneath a fungal appressorium).

      1. Composition: Complex, containing phenolics, Reactive Oxygen Species (ROS), proteins, and polymers.

      2. Callose is the most abundant polymer in papillae.

   3. Callose Deposition: Callose is a polysaccharide (polymer of glucose with β-1,3-linkages) produced in response to stress or damage. It is a key component of papillae and other defensive structures.

2. Tyloses:

   1. Definition: Outgrowths from parenchyma cells that balloon into xylem vessels in response to vascular pathogens.

   2. Function: Block the spread of the pathogen by physically clogging the xylem.

   3. Trade-off: While defensive, tyloses also contribute to disease symptoms (e.g., wilting) by impeding water flow.

Induced Biochemical Responses - A cascade of chemical defenses activated upon pathogen recognition. - Early Induced Responses (Immediate):

1. Ion Fluxes: Rapid changes in ion flow across the plasma membrane, particularly an influx of Calcium (Ca²⁺), which acts as a second messenger to activate downstream defenses.

2. Reactive Oxygen Species (ROS) Burst:

   1. The rapid production of chemically reactive molecules containing oxygen (e.g., Superoxide O₂⁻, Hydrogen Peroxide H₂O₂, Hydroxyl radical •OH).

3. Functions:

   1. Direct Toxicity: Kills microbes directly.

   2. Signaling: Acts as a second messenger for plant immunity.

   3. Cell Wall Defense: Contributes to cross-linking and strengthening (penetration resistance).

   4. Inducing Cell Death: Can trigger the Hypersensitive Response (HR).

   5. Hormone Synthesis: Required for the production of some defense hormones.

Induced Biochemical Responses - A cascade of chemical defenses activated upon pathogen recognition. - Late Induced Responses (slower and sustained)

1. Pathogenesis-Related (PR) Proteins:

   1. Small, acidic/basic proteins produced after pathogen attack.

   2. Function: Many have direct antimicrobial activity (e.g., Chitinases and Glucanases that degrade fungal cell walls).

   3. Can accumulate both locally and systemically.

2. Phytoalexins:

   1. Definition: Low molecular weight, antimicrobial compounds that are "plant protectors."

   2. Characteristics: Chemically diverse secondary metabolites (e.g., Stilbenes) that are toxic to pathogens.

   3. pathogen produce detoxification enzymes in response

3. Terpenoids:

   1. The largest and most diverse group of plant volatiles.

   2. Function: Primarily toxic to insects and pathogens; also act as alarm signals to prime other plants or parts of the plant.

4. Glucosinolates:

   1. Plant natural chemicals involved in defense against pathogens and insects.

   2. Key Role: Required for callose deposition and papillae formation at penetration sites.

Induced Biochemical Responses - A cascade of chemical defenses activated upon pathogen recognition. - Phytohormones in Defense Regulation

1. Plant hormones form a complex signaling network to regulate defense.

2. Key Hormones:

   1. Salicylic Acid (SA): Defense against biotrophic and hemibiotrophic pathogens.

   2. Jasmonic Acid (JA) & Ethylene (ET): Defenses against necrotrophic pathogens, insects, and some nematodes. They often work synergistically.

   3. Abscisic Acid (ABA): Involved in stomatal closure and stress responses.

3. Hormone Crosstalk:

   1. Synergistic: JA and ET pathways reinforce each other.

   2. Antagonistic: SA and JA pathways often suppress each other. This allows the plant to fine-tune its response to the specific threat.

4. Pathogen Manipulation: Pathogens often target these hormone pathways. A key example is Pseudomonas syringae, which produces the toxin Coronatine (COR). COR mimics JA, activating the JA pathway and suppressing the protective SA pathway, leading to increased susceptibility.

Systemic Resistance - Whole-plant resistance induced by a localized infection or beneficial microbes.

1. Systemic Acquired Resistance (SAR):

   1. Trigger: Localized infection by a pathogen.

   2. Signaling Pathway: Dependent on Salicylic Acid (SA).

   3. Characteristics:

      1. Persistent and broad-spectrum resistance throughout the plant.

      2. Associated with the accumulation of PR proteins.

   4. Classic Experiment (Ross, 1961): A local infection with Tobacco Mosaic Virus (TMV) on a lower leaf led to fewer TMV lesions on upper leaves challenged one week later, demonstrating SAR.

2. Induced Systemic Resistance (ISR):

   1. Trigger: Colonization of roots by beneficial microbes (e.g., Rhizobacteria, Mycorrhizal fungi).

   2. Signaling Pathway: Dependent on Jasmonic Acid (JA) and Ethylene (ET).

   3. Characteristics:

      1. Does not typically involve major PR protein accumulation.

      2. Often primes the plant for a faster and stronger defense response upon subsequent attack ("primed state").

Genetic Interactions & Resistance Mechanisms - Plant resistance is governed by the genetic interplay between plant and pathogen. - Types of Resistance:

1. Non-host Resistance: A plant species is completely resistant to all isolates of a pathogen that is not adapted to it.

2. Qualitative Resistance:

   1. "All-or-nothing" response (complete resistance or severe disease).

   2. Controlled by a single R gene.

   3. Race-specific (effective only against pathogen strains with a corresponding Avr gene).

3. Quantitative Resistance:

   1. "Partial" resistance; plants get diseased but symptoms develop more slowly and are less severe.

   2. Controlled by multiple genes (polygenic), often referred to as Quantitative Trait Loci (QTL).

   3. Race-non-specific and broad-spectrum.

   4. Considered more durable because it is harder for a pathogen to simultaneously evade multiple plant resistance mechanisms.

Genetic Interactions & Resistance Mechanisms - Plant resistance is governed by the genetic interplay between plant and pathogen. - Effector-Triggered Immunity (ETI) - The Basis of Qualitative Resistance:

1. Gene-for-Gene Model: For every pathogen Avirulence (Avr) gene (encoding an effector), there is a corresponding plant Resistance (R) gene.

2. Mechanism: An R protein directly or indirectly recognizes a specific pathogen effector. This recognition triggers a strong defense response.

3. Hypersensitive Response (HR): A key feature of ETI. It is a programmed cell death at the infection site.

   1. Function: "Sacrifices" a few cells to confine and starve the pathogen, preventing its spread.

   2. Effective Against: Biotrophic and hemibiotrophic pathogens (which require living tissue). It is not effective against necrotrophs (which kill tissue and feed on the dead matter).

Genetic Interactions & Resistance Mechanisms - Plant resistance is governed by the genetic interplay between plant and pathogen. - The Plant-Pathogen Arms Race (Zig-Zag Model):

1. PTI (PAMP-Triggered Immunity): Plants use Pattern Recognition Receptors (PRRs) to detect general Pathogen/Microbe-Associated Molecular Patterns (PAMPs/MAMPs). This provides a basal defense.

2. ETS (Effector-Triggered Susceptibility): Pathogens evolve effectors to suppress PTI, leading to disease.

3. ETI (Effector-Triggered Immunity): Plants evolve R genes to recognize specific effectors, triggering a strong HR and resistance.

4. The cycle continues as pathogens evolve new effectors that are not recognized, and plants evolve new R genes.

Importance & Application

1. Food Security: Understanding plant defense mechanisms is crucial for developing new strategies to protect crops, ensuring global food security in the face of constant pathogen evolution and the threat of famine.

2. Biotechnology Example: The Arabidopsis PAMP receptor gene EFR was expressed in tobacco and tomato. These transgenic plants showed enhanced resistance to bacterial pathogens like Ralstonia solanacearum, demonstrating that transferring key immune components can boost disease resistance across species.

Introduction to Disease Control Principles

1. Plant disease management is a multi-faceted approach focused on disrupting the disease triangle (susceptible host, virulent pathogen, favorable environment). Control strategies are broadly categorized based on which component of the triangle they target.

Strategy 1: Avoid the Pathogen

1. This strategy involves manipulating planting practices to prevent the host and pathogen from coinciding in space and time.

2. Key Tactics:

   1. Site Selection: Choosing a field or geographic area where the pathogen is not present or where environmental conditions are unfavorable for the disease.

   2. Planting Time: Adjusting planting dates so that the most susceptible stage of crop development does not coincide with periods of high pathogen inoculum or favorable environmental conditions (e.g., planting after the primary spore release period).

Strategy 2: Exclude the Pathogen

1. This strategy aims to prevent the introduction and establishment of a pathogen into a new area or a clean crop.

2. Key Tactics:

   1. A. Quarantines:

      1. Definition: Legal restrictions on the movement of plant material, soil, seeds, machinery, or any item that could introduce a dangerous pathogen into a new area.

      2. Goal: To prevent the introduction of exotic or high-consequence pathogens.

   2. B. Pathogen-Free Propagative Material:

      1. Using certified pathogen-free seeds, tubers, and transplants.

      2. Production Methods:

         1. Growing crops in isolated regions that are free of the pathogen.

         2. Growing in areas where the environment is unsuitable for the pathogen or its vector.

         3. Testing seeds and plant material for the presence of pathogens, especially hard-to-detect ones like viruses and viroids.

Strategy 3: Eradicate or Reduce the Pathogen

1. This strategy focuses on reducing, eliminating, or destroying the pathogen population from a field, plant, or piece of equipment.

2. Cultural Practices:

   1. Crop Rotation:

      1. Practice: Rotating a susceptible crop with a non-host crop

      2. Mechanism: "Starves" the pathogen by removing its host, reducing inoculum levels over time

      3. Limitations:

         1. Primarily effective for annual crops and pathogens with a narrow host range.

         2. Not effective against pathogens with wide host ranges or those that can survive as saprophytes (living on dead organic matter).The economic value of the non-host crop may be lower.

1. Fallowing:

   1. Practice: Leaving a field free of any crops for a season or more.

   2. Limitation: Similar to crop rotation, it is not effective against long-lived saprophytes or pathogens with resilient survival structures.

2. Flooding:

   1. Practice: Flooding fields after a season to create anaerobic conditions.

   2. Mechanism: Depletes oxygen, killing many soil-borne pathogens.

   3. Trade-off: Also reduces populations of beneficial soil organisms.

3. Sanitation:

   1. Practice: Removing and destroying infected plant debris (e.g., burning, deep burial).

   2. Practice: Cleaning and decontaminating tools, machinery, and clothing (e.g., with disinfectants like bleach).

   3. Practice: Treating irrigation water (for pathogens like Pythium and Phytophthora) with chlorine, filtration, ozone, or UV radiation.

4. Heat Treatments:

   1. Burning: Directly burning infected plant debris to destroy pathogens.

   2. Steam Heat:

      1. Used to disinfect soil, pots, and tools

      2. Steam at 60-70°C kills most pathogens while preserving soil structure.

   3. Solarization:

      1. Practice: Covering moist soil with clear plastic to trap solar radiation, heating the soil to levels lethal to many pathogens.

      2. Mechanism: The "greenhouse effect" under the plastic raises soil temperatures, effectively reducing the primary inoculum.

   4. Hot Water Treatment: Used to kill pathogens on or in seeds, fruits, and propagative parts (e.g., bulbs, cuttings).

   5. Thermotherapy: Treating entire plants or plant parts with mild heat to eradicate viruses, often used before tissue culture propagation.

Biological Control:

1. Using other organisms to reduce the pathogen population.

2. Mechanisms:

   1. Parasitism: A parasite attacks the pathogen (e.g., the fungus Trichoderma parasitizing other fungi).

3. Predation: An organism consumes the pathogen (e.g., some nematodes that feed on fungal spores and mycelium).

4. Hypovirulence: A pathogen is infected by a virus or other parasite that reduces its virulence.

5. Antagonism: An organism produces antibiotics or other compounds that inhibit the pathogen.

6. Competition: Beneficial microbes outcompete the pathogen for space and nutrients. Timing is critical ("first come, first served").

Chemical Treatment:

1. Applying pesticides that are toxic to pathogens.

2. Herbicides:

   1. Used to kill parasitic plants and weed hosts that can harbor fungal pathogens or serve as alternative hosts.

3. Insecticides:

   1. Used to control insect vectors that transmit pathogens (e.g., aphids spreading viruses).

4. Nematicides:

   1. Target nematodes. They are not selective (they affect the nervous system of many animals) and are highly regulated due to human toxicity.

5. Soil Fumigants:

   1. Broad-spectrum biocides (e.g., methyl bromide) that eradicate most soil-borne pathogens, weeds, and pests.

   2. Drawbacks: Expensive, harmful to humans and the environment, and strictly regulated.

6. Bactericides (Antibiotics):

   1. Used to control bacterial diseases.

   2. Drawbacks: Expensive, and there is a high risk of bacteria developing resistance. Sometimes injected directly into trees.

Fungicides:

1. Chemicals toxic to fungi and oomycetes.

2. Toxicity Measurement: LD₅₀ (Lethal Dose 50)

   1. The dose required to kill 50% of a test population.

   2. A relative measurement used to compare the inherent toxicity of different chemicals to humans and animals. A high LD₅₀ indicates low mammalian toxicity.

1. Efficacy Measurement: EC₅₀ (Effective Concentration 50)

   1. The concentration of a chemical required to inhibit 50% of mycelial growth (or another biological process) in vitro.

   2. A lower EC₅₀ indicates a more effective/potent fungicide.

   3. Example: Oxathiapiprolin (EC₅₀ = 0.0002-0.0007 µg/ml) is much more potent than Mefenoxam (EC₅₀ = 0.023-0.138 µg/ml).

2. Modes of Action:

   1. Contact (Protectant): Remains on the plant surface. Must be applied before infection to form a protective barrier.

3. Systemic (Penetrant): Absorbed by the plant and translocated internally. Can act curatively after infection has begun. Often has a specific biochemical target in the pathogen (e.g., a specific enzyme).

Strategy 4: Immunize or Improve Host Resistance - This strategy focuses on enhancing the plant's own ability to resist infection.

1. Cross-Protection:

   1. Definition: Deliberately infecting a plant with a mild (less virulent) strain of a virus to protect it from infection by a more severe, related virus.

   2. Application: Used almost exclusively for viral diseases.

2. Induced Resistance:

   1. Systemic Acquired Resistance (SAR):

      1. Trigger: A localized infection by a pathogen.

      2. Signaling: Salicylic Acid (SA) pathway.

      3. Markers: Accumulation of PR (Pathogenesis-Related) proteins.

   2. Induced Systemic Resistance (ISR):

      1. Trigger: Colonization of roots by beneficial microbes (e.g., rhizobacteria).

      2. Signaling: Jasmonic Acid (JA) and Ethylene (ET) pathways.

      3. Mechanism: Often primes the plant for a faster, stronger defense response upon subsequent attack, rather than directly activating defenses.

3. Application of Plant Defense Activators:

   1. Spraying plants with compounds that "switch on" defense pathways.

   2. Examples: Plant hormones (SA, JA) or their chemical analogs, or elements like Silicon, which strengthens cell walls and primes defense responses.

4. Genetic Resistance:

   1. Using plant varieties that are genetically resistant to a pathogen.

   2. Advantages: Inherited, permanent, environmentally friendly, and compatible with other management practices.

   3. Deployment Strategies (to manage resistance durability and avoid pathogen adaptation):

      1. Geographic Deployment: Using different resistant varieties in different regions.

      2. Temporal Deployment: Rotating resistant varieties with different resistance genes over time.

      3. Pyramiding: Breeding multiple resistance (R) genes into a single plant variety. This makes it harder for the pathogen to evolve virulence against all genes simultaneously.

      4. Multilines and Mixtures:

         1. Multilines: A mixture of genetically similar plant lines, each carrying a different R gene.

         2. Mixtures: A mixture of genetically different cultivars, each carrying different R genes.

         3. Mechanism: Creates a heterogeneous host population that acts as a "speed bump" for an epidemic, preventing a single pathogen race from devastating the entire crop.

Integration and Importance

1. Integrated Pest Management (IPM): integrates multiple strategies (avoidance, exclusion, eradication, protection) to manage the disease triangle effectively.

2. Epidemiological Principles: The choice and timing of control measures are guided by understanding the pathogen's life cycle, sources of inoculum, environmental conditions that favor disease, and host susceptibility.

3. Goal: The ultimate goal is sustainable and economically viable disease control to ensure food security and agricultural productivity.

Introduction to Biotechnology and OMICS

• Biotechnology is defined as the development of genetically modified or gene edited organism using modern technology and processes. The field is haveily supported by “Omics” technologies, which provide a comprehensive, systems-level understanding of biological system.

• Genomics - the study of the entire set of genes (the genome) in an organism.

• Transcriptomics - the study of all the RNA molecules ( the transcriptome), inculding mRNA, produced in a cell. This shows which genes are being actively expressed

• Proteomics - the study of the entire set of proteins (the proteome) expressed by a cell or organism

• Metabolomics - the study of the complete of small molecule metabolites (the metabolome) present in a cell, which represents he functional outcome of cellular processes

• epigenetics - the study of changes in gene expression that do not involve changes to the underlying DNA sequence itself (the epigenome). These can be influenced by the environment

• Phenomics - the study of the physical and biochemical traits ( the phenotypes) of an organism, which results from the interaction of its genetics with the environment.

• These Omics field are interconnected: DNA is transcribed to mRNA, which is translated into proteins, which then catalyze metabolic reactions, all influenced by the environment and epigenetic factors, ultimately producing the observable phenotype.

Genetic Engineering vs. Gene Editing

1. Genetic Engineering:

   1. Definition: The transfer of specific DNA fragments between organisms using laboratory techniques, rather than traditional breeding.

   2. GMO (Genetically Modified Organism): An organism that possesses a genomic region from another organism, created through genetic engineering.

      1. Transgenic: The introduced gene comes from a different, genetically unrelated species (e.g., a bacterial gene inserted into a plant).

      2. Cisgenic: The introduced gene comes from the same species or a closely related species that could be crossed by conventional breeding.

2. Gene Editing (Genome Editing):

   1. Definition: A more precise technique where specific DNA within the genome of a living organism is directly inserted, deleted, or replaced in situ (in its original place) using engineered nucleases, often called "molecular scissors."

   2. This does not necessarily involve inserting foreign DNA but can simply edit the existing DNA sequence.

Plant Transformation Methods - To create genetically engineered plants, foreign DNA must be introduced into plant cells, which are then regenerated into whole plants.

1. Biolistic Transformation (gene g u n )

   1. process - plant cells are bombarded with microscopic tungsten or gold particles that are coated with the foreign DNA.

   2. The "gene gun" literally shoots the DNA into the cells.

   3. Regeneration: The bombarded cells are then placed on a special growth medium (e.g., Murashige and Skoog (MS) media) and treated with specific hormones to regenerate into whole plants via tissue culture.

2. Agrobacterium tumefaciens-Mediated Transformation:

   1. Process: Utilizes the natural genetic engineer, the soil bacterium Agrobacterium tumefaciens.

   2. The bacterium naturally transfers a segment of its DNA (T-DNA) from its Ti (Tumor-inducing) plasmid into the plant genome, causing crown gall disease.

   3. In the lab, the disease-causing genes are removed from the T-DNA and replaced with the gene of interest.

   4. The recombinant Agrobacterium then infects plant cells in culture, transferring the new gene into the plant's chromosomes.

   5. Regeneration: Transformed cells are selected and regenerated into whole plants using tissue culture and specific hormone treatments (e.g., balancing auxin and cytokinin ratios to induce root and shoot formation).

Generations of Genetically Engineered Crops

1. 1st Generation (1994~): Focus on agronomic input traits.

   1. Herbicide-Resistant Crops (e.g., Roundup Ready Soybean): Engineered to tolerate specific herbicides (like glyphosate), allowing farmers to control weeds without harming the crop.

   2. Insect-Resistant Crops (e.g., Bt Corn): Engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). These "delta-endotoxins" are toxic to specific insect pests (e.g., caterpillars) but safe for humans and other animals.

   3. Virus-Resistant Crops (e.g., Rainbow Papaya): Engineered to resist devastating viruses, such as the papaya ringspot virus, which saved the Hawaiian papaya industry.

   4. Disease Tolerance & Delayed Ripening.

2. 2nd Generation (1995~): Focus on output traits and consumer benefits.

   1. Improved Nutrition:

      1. Golden Rice: Engineered to produce beta-carotene (a precursor to Vitamin A) in the grain to combat vitamin A deficiency.

      2. Purple Tomato: Engineered to produce high levels of anthocyanins (powerful antioxidants) by expressing transcription factors from snapdragon flowers.

   2.  Improved Quality: Non-browning apples and potatoes (e.g., Arctic Apple, Innate Potato), modified oils.

3. 3rd Generation (1998~): Plant-Made Pharmaceuticals (PMPs) and Industrial Products.

   1. Using plants as bio-factories to produce valuable proteins.

   2. Edible Vaccines: Plants engineered to produce pathogen antigens (e.g., rabies antigen in tomatoes, cholera antigen in potatoes). The plant tissue can be eaten to deliver the vaccine.

   3. Other Pharmaceuticals: Production of antibodies and other therapeutic proteins in plants (e.g., Ebola vaccine produced in tobacco).

4. 4th Generation: Gene-Edited Crops.

   1. Crops developed using precise genome editing tools (ZFNs, TALENs, CRISPR-Cas9) to make targeted changes (knock-outs, point mutations, insertions) without necessarily adding foreign DNA.

Genome Editing Tools - These tools create double-strand breaks in DNA at specific locations, which the cell then repairs, allowing for precise genetic modifications.

1. Zinc Finger Nucleases (ZFNs) (mid-2000s): Artificial enzymes created by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain.

2. TALENs (Transcription Activator-Like Effector Nucleases) (early 2010s): More flexible and easier to design than ZFNs. Created by fusing a TAL effector DNA-binding domain to a DNA-cleavage domain.

   1. Application Example: Used to create disease-resistant rice against bacterial blight.

   2. recognize dna based on - Repeat-variable diresidues (RVDs) in protein repeats

3. CRISPR-Cas9 (2013~): The most versatile and widely adopted system.

   1. Origin: An acquired adaptive immune system in bacteria that defends against viruses.

   2. Mechanism: The system uses a guide RNA (gRNA) to recognize and bind to a specific DNA sequence, and the Cas9 nuclease to cut the DNA at that location.

   3. Advantages: Simpler, cheaper, and more easily programmable than ZFNs or TALENs.

   4. Applications: Rapidly being adopted by many companies (e.g., Corteva, Syngenta, Pairwise) for crop improvement, focusing on traits like higher yield, disease resistance, and improved quality (e.g., non-browning mushrooms, decaffeinated coffee).

Introduction to Plant Disease Diagnosis

• Diagnosis is the process of identifying the cause of a plant disease. Accurate diagnosis is critical for implementing effective management strategies. The process is a multi-step investigation that moves from the field to the laboratory, using increasingly specific tools.

• Key Concepts for Diagnostic Tests:

  • Sensitivity: The test's ability to correctly identify the presence of a pathogen (low rate of false negatives). A highly sensitive test can detect very low levels of the pathogen.

  • Specificity: The test's ability to correctly identify the absence of a pathogen (low rate of false positives). A highly specific test will not react with non-target organisms.

The Diagnostic Process: A Stepwise Approach

1. Field Observation (Symptoms & Signs):

   1. Symptoms: The plant's visible reaction to the pathogen (e.g., wilting, spots, cankers, stunting).

   2. Signs: Physical evidence of the pathogen itself (e.g., fungal mycelium, bacterial ooze, nematode cysts, insect vectors). This is the most direct evidence.

2. Pathogen Isolation: The process of separating the pathogen from plant tissue and other microbes to obtain a pure culture.

   1. For Bacteria:

      1. Small pieces of tissue from the margin of the lesion (where the pathogen is most active) are surface-sterilized (e.g., with 10% bleach).

      2. The tissue is macerated in sterile water.

      3.  A serial dilution is performed to reduce microbial density.

      4. Dilutions are plated on a nutrient agar medium. After incubation, single bacterial colonies appear and can be subcultured for further analysis.

   2. For Fungi/Oomycetes:

      1. Surface-sterilized tissue sections from the lesion margin are placed directly onto a nutrient agar medium.

      2. After incubation, the pathogen grows out from the tissue. A pure culture is obtained by transferring a hyphal tip or single spore from the leading edge of the growth to a new plate

3. Pathogen Identification: Using various laboratory techniques to identify the isolated pathogen.

Laboratory Identification Techniques - Culturing on Specific Media

1. Differential Media: Allows distinction between different microbes based on their colony morphology (color, shape, texture) on the medium.

   1. YDC Medium (Yeast extract-Dextrose-CaCO₃): Differentiates Xanthomonas (yellow colonies) from Clavibacter (orange colonies).

   2. King’s B Medium: An iron-deficient medium that promotes the production of siderophores (iron-chelating compounds) by fluorescent Pseudomonas species (e.g., P. syringae), causing them to fluoresce under UV light

2. Selective Media: Contains additives (antibiotics, specific nutrients) that inhibit the growth of most microbes, selectively allowing only the target pathogen to grow.

Laboratory Identification Techniques - Biochemical Tests - These tests identify pathogens based on their metabolic capabilities.

1. Example: Urease Test

   1. The bacterium is cultured in a medium containing urea and a pH indicator (phenol red).

   2. If the bacterium produces the urease enzyme, it hydrolyzes urea into ammonia and CO₂.

   3. The release of ammonia raises the pH, turning the indicator pink-red, indicating a positive test.

2. Commercial kits (e.g., API strips) test for the use of various sugars, amino acids, and enzyme production.

Laboratory Identification Techniques - Protein-Based Detection (Immunoassays) - These methods use antibodies that bind specifically to pathogen proteins (antigens).

1. ELISA (Enzyme-Linked Immunosorbent Assay):

   1. A plate-based technique where a primary antibody captures the pathogen antigen.

   2. A secondary antibody with an enzyme conjugate is added. When a substrate is added, the enzyme produces a color change, indicating a positive result.

   3. Can be quantitative.

2. ImmunoStrips (Lateral Flow Devices):

   1. A rapid, field-deployable version of ELISA.

   2. The plant extract is applied to a strip. Capillary action moves it along, and if the pathogen antigen is present, it binds to antibodies, creating a visible test line.

   3. Advantages: Very fast (minutes), simple, and portable.

Laboratory Identification Techniques- Nucleic Acid-Based Detection - These are the most sensitive and specific methods, based on detecting the pathogen's unique DNA or RNA.

1. PCR (Polymerase Chain Reaction):

   1. Process: Amplifies a specific target DNA sequence through repeated cycles of:

   2. Denaturation: Heating to separate DNA strands.

   3. Annealing: Cooling to allow specific primers to bind to the target sequence.

   4. Extension: A heat-stable DNA polymerase (Taq) synthesizes new DNA strands.

   5. Visualization: The amplified DNA (amplicon) is visualized using gel electrophoresis, which separates DNA fragments by size. A band of the expected size indicates a positive detection.

2. Primer Specificity:

   1. General Primers: Target conserved genes like 16S rRNA (bacteria) or ITS (fungi) for broad identification.

   2. Specific Primers: Target genes unique to a specific pathogen species or strain.

3. qPCR (Quantitative PCR or Real-Time PCR):

   1. An advanced form of PCR that monitors DNA amplification in real-time using fluorescent reporters.

   2. More sensitive than conventional PCR and provides quantitative data (how much pathogen DNA is present).

   3. Fluorescent Reporters:

      1. SYBR Green: Binds to any double-stranded DNA.

      2. TaqMan Probes: Specific probes that bind only to the target sequence, providing higher specificity.

4. RPA (Recombinase Polymerase Amplification):

   1. An isothermal nucleic acid amplification technique (works at a constant low temperature).

   2. Advantages: Extremely fast, portable, and does not require a thermocycler. Ideal for field diagnosis.

5. RFLP (Restriction Fragment Length Polymorphism):

   1. A technique to distinguish between closely related pathogens.

   2. Process: PCR products are digested with restriction enzymes that cut DNA at specific sequences. The resulting DNA fragments are separated by gel electrophoresis.

   3. Different pathogens will have different cutting patterns (restriction patterns), producing a unique "fingerprint" of band sizes.

6. Next-Generation Sequencing (NGS):

   1. A powerful tool for studying complex microbial communities (microbiomes).

   2. Marker Gene Sequencing (e.g., 16S, ITS): Amplifies and sequences a specific gene from all organisms in a sample to identify community members.

   3. Shotgun Metagenomics: Sequences all the DNA in a sample, providing a comprehensive view of the genes and organisms present.

Emerging and Future Diagnostic Technologies

• Multispectral/Hyperspectral Sensors: Cameras and sensors that detect light reflectance beyond the visible spectrum. Healthy and diseased plants have unique spectral "signatures," allowing for early, pre-symptomatic disease detection from drones, planes, or satellites. A key tool for Precision Agriculture.

• Metabolite-Based Detection: Identifying diseases by detecting unique volatile organic compounds (VOCs) or other metabolites that plants release when stressed or infected.

• Smartphone-Enabled Diagnostics: Using smartphone cameras and attachments to perform colorimetric assays (like ELISA strips) or even basic microscopy, bringing lab-quality diagnostics to the field.

Measuring Disease Impact

• Disease Intensity: The amount of disease present on a plant or in a field, often expressed as a percentage (e.g., % leaf area covered in lesions).

• AUDPC (Area Under the Disease Progress Curve): A single numerical value that summarizes the severity and speed of a disease epidemic over time. A larger AUDPC indicates a more severe and rapidly developing epidemic.