Fungal methods (e.g., Fusarium oxysporum): theory and protocols

1. Theory: How Fungal Induction Works

  • Mechanism
    • Agarwood resin is a tree defense response to injury or infection.
    • Fusarium oxysporum acts as a biotic elicitor, simulating natural infection.
    • Infection triggers the tree to produce secondary metabolites (sesquiterpenes, chromones) → resin formation.
  • Advantages of Using Fungi
    • Mimics natural resin formation → potentially high-quality aroma.
    • Can be applied to young or plantation-grown trees.
    • Faster than waiting for natural infections.
  • Biological Principles
    • Infection & Wounding: Fungal hyphae enter via wounds or drilled holes.
    • Defense Response: Tree cells detect pathogen → produce reactive oxygen species (ROS).
    • Resin Synthesis: Enzymatic pathways are activated → accumulation of sesquiterpenes and chromones.
    • Localization: Resin forms around the infection site, gradually spreading over months.

2. Materials Required

  • Pure culture of Fusarium oxysporum (verified strain for agarwood induction)
  • Potato Dextrose Agar (PDA) or other suitable fungal media
  • Autoclave & laminar flow hood (for aseptic culture)
  • Sterile syringes / inoculation tools
  • Drill or chisel for creating inoculation sites
  • Protective gloves, masks, and safety tools

3. Laboratory Protocol (Culture Preparation)

  1. Isolation & Maintenance
    • Grow F. oxysporum on PDA plates at 25–28°C for 5–7 days.
    • Subculture every 2–3 weeks to maintain viability.
  2. Spore Suspension Preparation
    • Add sterile distilled water to the fungal culture plate.
    • Gently scrape to collect spores.
    • Filter through sterile gauze to remove mycelial clumps.
    • Adjust spore concentration: 1 × 10⁶ to 1 × 10⁸ spores/mL.
  3. Optional: Fungal Carrier
    • Mix spores with a carrier (e.g., sawdust, rice bran) to allow slow release inside the tree.

4. Field Protocol (Tree Inoculation)

  • Step 1: Site Selection
    • Choose healthy, mature trees (typically 3–5+ years old).
    • Avoid trees under severe stress or disease.
  • Step 2: Wound Creation
    • Drill small holes (1–2 cm diameter, 5–10 cm deep) at multiple sites around the trunk.
    • Space holes 30–50 cm apart vertically and horizontally to avoid excessive stress.
  • Step 3: Inoculation
    • Inject spore suspension (or carrier mixture) into each wound.
    • Seal holes with paraffin wax or sterile cotton to prevent contamination and retain moisture.
  • Step 4: Post-Inoculation Care
    • Water the tree and maintain soil moisture.
    • Apply organic fertilizers to support tree recovery.
    • Monitor for resin formation every 3–6 months.

5. Monitoring and Harvest

  • Initial resin detection: 6–12 months after inoculation.
  • Harvesting: Typically 12–36 months post-inoculation depending on tree age, species, and infection response.
  • Signs of success: Darkened wood around inoculation site, resin accumulation, distinct fragrance upon scraping.

6. Safety and Risk Management

  • Always use protective gloves and masks; Fusarium can be pathogenic to humans in immunocompromised cases.
  • Avoid over-inoculation; excessive fungal load can kill the tree.
  • Rotate inoculation sites and allow tree recovery between applications.

7. Notes on Resin Quality

  • Fungal-induced resin can be comparable to natural agarwood, but aroma depends on:
    • Tree species (e.g., A. malaccensis)
    • Age of tree
    • Fungal strain
    • Site and depth of inoculation
  • Combining mechanical wounding + fungal inoculation generally produces higher yield and more uniform resin.