Biological Control: Competitive Exclusion

1. Biological Control:Competitive Exclusion 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

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

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

17. Cotty, P.J. USDA-ARS. Univ. of Arizona, 1997 biopesticide.ucr.edu/abstracts/assets/Cotty_abstract.pdf BCA AF36 Timeline 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: MaryblytTM • 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 N2 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. BlightBanTM • 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 • BeforeE. 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 -20oC • Ice needs something to form crystals on… • Water full of ice nucleation minerals, bacteria, etc. which lowers the freezing temp to 0oC • 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 -5 C • 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.

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 BlightBan & Strep. BlightBan & Strep. Copper Start here Dormant pruning Strep. every 7 days* pruning pruning 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. • Non-toxic A. flavus reduces aflatoxin contamination in cotton and corn. • BlightBan A506 prevents E. amylovora from quorum sensing in apple & pear flowers. • A506 (Ice-) also reduces frost damage on many fruits and veggies by reducing populations of ice-nucleating bacteria.

36. Questions?