1. December 5, 2008

2. Outline Introduction to competition How? Where? Two biocontrol examples Aspergillus in cotton/corn Fire Blight How do they work? How effective?

3. What is a Niche? The totality of environmental factors that influence growth, survival, and reproduction of a species. Factors can be biotic, abiotic, resources, etc. The relational position of a species in an ecosystem. Temperature Moisture

4. Competitive Exclusion Principle Gause’s Law: Two species occupying the identical niche cannot co- exist indefinitely. One species (with slight advantage) will out-compete the other for resources. Inferior species will shift towards another niche. Extinction in extreme cases

5. Modes of Competition Competition Niche Exclusion Take away pathogen’s home Nutrient Competition Starve Pathogen

6. Agro-ecosystem Microbes: Where does competition take place? Phylloplane: On the leaf surface Dynamic environment (exposed to the elements) Food source: Endogenous –leached from leaf Exogenous – pollen, dead epiphytes, aphid honeydew Rhizoplane/Rhizosphere: Root surface & specialized area of soil, immediately surrounding the roots More stable environment Food source: root exudates, decaying material

7. How do Plant Pathologists take advantage of this? Introduce new species (Biocontrol Agent) to farm ecosystem NOT a (virulent) pathogen! Competes with pathogen for same niche Keeps pathogen population in check by being better competitor. Pathogen must find another “home” or resources Disease remains at a minimum. Niche displacement: shifting one species out of its niche by competition from another species.

8. Two Examples Ear Rot of Corn/Cotton Pathogen: Aspergillus flavus BCA: avirulent Aspergillus flavus Fire Blight of Apple Pathogen: Erwinia amylovora BCA: Pseudomonas fluorescens

9. Aspergillus Diseases Aspergillus flavus Asexual fungus Soil saprophyte Infects oil-rich seed crops Corn, Cotton, Peanut Through silks, wounds Not a strong/aggressive pathogen Affects crops already suffering from insect or environmental damage

10. A. Flavus Life Cycle Conidia Asexual Conidia Stressed/wounded plants Crop debris/soil

11. A. flavus Produces Aflatoxins Toxic to humans and animals in small doses Turkey X disease (1962) 2006: Dog food contaminated Associated with liver cancer/mutagenic Kenya, Africa Found in contaminated grain and feed (silage, hulls). Can pass to milk FDA regulations limit aflatoxin in food and feed 0.5 ppb in milk; 20 ppb for dairy cows and peanut butter

12. Aflatoxin Management Prevention Early harvest of corn and cottonseed Minimize insect damage Dry storage post-harvest Often aflatoxin problem not known until time of sale Biocontrol with non-toxigenic A. flavus strain Avirulent pathogen

13. Biocontrol of Aflatoxin A. flavus strains differ in their toxin-production ability. A. flavus 36 (AF36) does not produce toxin Defective polyketide synthase gene Essential to aflatoxin synthesis pathway Otherwise, exhibits wild-type growth. Occupies same niche as toxigenic A. flavus COMPETITION! Peter Cotty, USDA-ARS, Arizona

14. Effectiveness of AF36 on cotton In vitro studies showed AF36 out-competes toxigenic stains Would the same be true for in vivo/ in the field? Entire AZ cotton fields treated with AF36 Colonized wheat seed spread in field Measured resident Af and AF36 populations (in soil) for 2 years Measured aflatoxin in cottonseeds Cotty, P. J., 1994. Phytopathology. 84: 1270-1277.

15. Results AF36 is an efficient colonizer & competitor and persists in the field. Cotty, P.J. USDA-ARS. Univ. of Arizona, 1997 biopesticide.ucr.edu/abstracts/assets/Cotty_abstract.pdf Cotty, P.J. USDA-ARS. Univ. of Arizona, 1997 biopesticide.ucr.edu/abstracts/assets/Cotty_abstract.pdf S strain: highly toxic

16. Results As the population of AF36 increases in the field, aflatoxin contamination of cottonseed decreases. Cotty, P.J. USDA-ARS. Univ. of Arizona, 1997 biopesticide.ucr.edu/abstracts/assets/Cotty_abstract.pdf Cotty, P.J. USDA-ARS. Univ. of Arizona, 1997 biopesticide.ucr.edu/abstracts/assets/Cotty_abstract.pdf

17. BCA AF36 Timeline Cotty, P.J. USDA-ARS. Univ. of Arizona, 1997 biopesticide.ucr.edu/abstracts/assets/Cotty_abstract.pdf Cotty, P.J. USDA-ARS. Univ. of Arizona, 1997 biopesticide.ucr.edu/abstracts/assets/Cotty_abstract.pdf EUP: Experimental Use Permit (for biopesticide)

18. Commercial Production of AF36 Arizona Cotton Research and Protection Council Phoenix, Arizona.

19. Summary: AF36 for Competitive Exclusion Out-competes toxigenic strains for niche in cotton Decreases aflatoxin contamination Persists in soil Registration of Biopesticide is time consuming Approved for use in cotton, in few states Pending approval for use in pistachio and corn…

20. Disadvantages to AF36 AF36 is endemic to southwestern U.S. Not as effective/adapted to other regions of the world? Regulatory concerns Need to find more nontoxic A. flavus isolates native to other regions Found atoxigenic isolates in Nigeria (La3279 and La3303) effective at reducing aflatoxin in corn! Atehnkeng, J. et al., 2008.

21. Two Examples Ear Rot of Corn/Cotton Pathogen: Aspergillus flavus BCA: avirulent Aspergillus flavus Fire Blight of Apple Pathogen: Erwinia amylovora BCA: Pseudomonas fluorescens

22. Fire Blight: Review Erwinia amylovora Main mode of entry: Through flowers (nectarthodes) Wounds Conventional control: Pruning (cultural) Copper Antibiotics: streptomycin

23. Challenges of Controlling FB Disease is sporadic Forecasting models: Maryblyt TM Modern orchards: Increasing tree density 100-200 trees/acre (historically) to nearly 10x that density Demand for dwarf trees But popular rootstocks (M.26 and M.9) highly susceptible. New trees encouraged (through N 2 applications) to produce in 3 years

24. More Challenges New varieties very susceptible (Gala, Fuji, Granny Smith) As well as “old favorites” (Rome, Jonathan, Ida Red) Copper inhibits bacteria colonization efficacy of stems and buds Phytotoxic Streptomycin only very effective against floral infections Emerging antibiotic resistance! Timeliness of pruning (during dormancy) Thoroughness and monitoring Adding biocontrol option to IPM?

25. BlightBan TM Contains live bacterium: Pseudomonas fluorescens A506 Isolated from CA pear tree (S. Lindow) A506 is an excellent colonist of flower tissues. Competes with E. amylovora for niche (blossom resources) Prevents E. amylovora from obtaining high epiphytic populations No quorum sensing! Why is this good for control? No amylovorin: Prevents blossom infections Stockwell and Stack. 2007. Phytopathology. 97: 244-249.

26. How to use BlightBan A506 Preventative: must be sprayed at blossom onset Before E. amylovora emergence Shows no effect when co-inoculated E. amylovora is superior colonist to A506 Applied 1-2x per season (during early to full bloom) Designed for IPM: Used in combination with streptomycin and pruning Does not prevent branch or trunk infection

27. Other Benefits of BlightBan Prevents fruit russeting (from other bacteria) Protects grape from sour rot Prevents frost damage

28. Ice & Frost Damage Pure water forms ice at -15 to -20 o C Ice needs something to form crystals on… Water full of ice nucleation minerals, bacteria, etc. which lowers the freezing temp to 0 o C Provide initiation point for ice crystals Ice crystals puncture cell membrane of plant Destroy membrane integrity Cell dehydrates – plant necrosis and wilt Wounded tissue provides entry for bacteria and fungi

29. Frost Damage of Apple M. Longstroth, Michigan State Univ.

30. Ice Nucleating Bacteria Pseudomonas: naturally occurring epiphytes Ice + bacteria: Ice Nucleation Active (INA + ) Cell membrane has proteins that serve as ice nucleation centers Allow ice crystals to form at warmer temperature Ice - mutants Lack this protein Provide less favorable environment for ice formation. Must be colder for ice to form. Ice + Ice - -5 C

31. BlightBan Combats Frost P. fluorescens A506 is Ice - mutant: Competes with Ice + Pseudomonas species (i.e. P. syringae) Reduces accumulation of ice nuclei on leaves, flowers, young fruit. Prevents frost damage, by keeping the freezing point lower (i.e. colder) Provides frost protection for apple, pear, cherry, peach, tomato, almond, strawberry, potato

32. Effectiveness of BlightBan When used alone… Incidence of fire blight on A506-treated trees reduced by 60 – 80% (compared to un-treated control) Reduced fruit russet 25-40% Reduced frost injury by >50% Designed for IPM!!! A506 and Streptomycin can be tank-mixed -S. E. Lindow - Stockwell and Stack. 2007. Phytopathology. 97: 244-249. -S. E. Lindow - Stockwell and Stack. 2007. Phytopathology. 97: 244-249.

33. Challenges with BlightBan Pseudomonas does not form spores More susceptible to stress Less stable during storage, formulation High-titer biocontrol product required for effective establishment in field. Cells rapidly lose viability in storage Lyophilized cells require freezer storage Refrigerated, high-titer formulation of A506 shows good shelf-life. Silver Bullet

34. Fire Blight Management Copper Dormant pruning BlightBan & Strep. Strep. every 7 days* pruning Start here Strep. every 7 days* *very aggressive. Especially needed after hail/heavy storm

35. Summary Biocontrol via Competitive Exclusion Utilizes BCA competitor of the pathogen. Pathogen populations are reduced due to lack of resources (niche displacement). Reduces disease. 1. Non-toxic A. flavus reduces aflatoxin contamination in cotton and corn. 2. BlightBan A506 prevents E. amylovora from quorum sensing in apple & pear flowers. 3. A506 (Ice - ) also reduces frost damage on many fruits and veggies by reducing populations of ice-nucleating bacteria.

36. Questions?