1. Pre- and post-harvest practical application of Pichia anomala strain K,  -1,3-glucans and calcium chloride on apples : Two years of monitoring and efficacy against post-harvest diseases M. H. Jijakli, D. De Clercq, C. Dickburt, P. Lepoivre Plant Pathology Unit University of Agricultural Sciences of Gembloux

2. Apple postharvest diseases l losses of 15-25% after harvest l Major fungal diseases : –wound parasites » Botrytis cinerea » Penicillium expansum –Latent infections » Gloeosporium

3. ? Biological control methods of postharvest diseases... Natural phenomenonEfficient and reliable control strategy Realistic approach ? Biological limits Technological limits Economical limits

4. Market definition Antagonistic strain isolation Selection Massive production Formulation Pilot trials Modes of action Practical trials Commercialization Registration procedure Registration Toxicology Monitoring Required steps for biopesticide development

5. Natural phenomenon Efficient and reliable control strategy ? Selection of antagonistic strain First step

6. Selection of one effective microbial strain Pichia anomala (strain K) A strain of yeast efficient against wound parasites (B. cinerea and P. expansum)

7. Natural phenomenon Selection of the antagonistic strain Efficient and reliable control strategy ? Mass production and formulation Second step

8. Standard formulation of P. anomala strain K l Quantity (>10 10 cells/g ) l Quality (biological activity preserved, identity, purity) Obtention of a wettable powder Production in fermentor l The technique needs to be transferred at an industrial scaleBut

9. Selection of the antagonistic strain Natural phenomenon Efficient and reliable control strategy ? Modes of action Third step

10. Modes of action l Colonisation related to protection suggesting competition for nutrients and/or space l No detectable antibiosis or stimulation of fruit defence Implication of exo-  -1,3-glucanases produced by strain K ? – In situ stimulation of exo-  -1,3-glucanase and protective activities of strain K against B. cinerea when applied with pathogen cell walls –The separate disruption of two genes coding for exo-  -1,3-glucanase had no detectable impact on protective level against B. cinerea

11. Selection of the antagonistic strain Natural phenomenon Efficient and reliable control strategy ? Biological formulation Fourth step

12. Biological formulation Goal : To stabilise the protection by increasing survival, efficacy of BCA and its persistance of efficacy Method l based on knowledge's on the mechanisms of action –Selection and patenting of molecules imitating pathogen cell walls and offering higher and longer efficacy (protective level) of strain K : YGT (71% of  -1,3-glucans) –Application of a composition containing strain K (10 5 ucf/ml) + YGT (0.2% w/v)+ Cacl 2 (2 % w/v) offers a higher and longer efficacy in comparison with the application of strain K alone (10 7 ucf/ml)

13. Selection of the antagonistic strain Natural phenomenon Efficient and reliable control strategy ? Monitoring Fith step

14. Monitoring of strain K l Specific identification –with a SCAR marker l Quantification –Yield of micro-organisms from fruit surface and plating on a semi- selective medium –Yield of micro-organisms from fruit surface, DNA extraction followed by a quantitative PCR (Q-PCR-ELOSA)

15. Selection of the antagonistic strain Natural phenomenon Efficient and reliable control strategy ? Semi-practical application of biological control Sixth step

16. Semi-practical application of strain K in combination with YGT and CaCl 2 in collaboration with the « Station Royale de Recherches Fruitières de Gorsem » (Belgium) l Application by spraying at low (300L/ha) and high (1000L/ha) volume 12, 5 or 2 days before harvest l Harvesting and artificial wounding of fruits l Application by dipping or drenching l Storage of the fruits at 1°C (15 days), at 15°C (1 month) and at 20°C (15 days) l Variance analysis on severity index l % of protection based on severity index l Pathogens inoculation ( B. cinerea and P. expansum )

17. Pre and post-harvest application of strain K powder (10 7 ufc/ml) + YGT (0.2%) + Cacl 2 (2 %) Year 2001 Biological treatments Contradictory results of efficacy between years 2000 and 2001 for biological and chemical treatments Year 2000 Biological treatments and B. cinerea

18. Climatic data Total Rain Mean t° Total Rain Time (days) Rain during 10h with a maximum of 1.4l/m 2 Short but heavy rains with a maximum of 4.4l/ m 2 Pre-harvest temperature : 18-25°C Pre-harvest temperature : 12-19°C Difference in climatic conditions between both trials could explain the lower efficacy observed for biological and chemical pre-harvest treatments during year 2001

19. Year 2000 % of protection for P. expansum 82.6 % B 65.7 % CDEFGH -4.0 % FGH 0.0 % H Monitoring of strain K by plating on semi-selective medium Year 2001 % of protection for P. expansum 26.9 EFGH 38.9 HI 69.0 BCD 0.0 GHI * Whatever the mode and the time of application, a density threshold of 10 4 cfu of strain K/cm 2 of apple surface seems to be required just after harvest to obtain an antagonistic activity against P. expansum Artificial inoculation B. cinerea and P. expansum

20. Conclusions l Concerning biological treatments during year 2000, the highest level of protection (83 %) against P. expansum was observed with the pre-harvest high volume spraying of strain K powder (including YGT and CaCl 2 ). This treatment was more efficient than post-harvest chemical treatment (74,71 %) but less efficient than pre-harvest chemical treatment (nearly 100 %) consisting in application of 4 different fungicides l During year 2001, strain K post-harvest treatments (dipping or drenching) offered the highest percentage of protection (aroudn 70 %) among the different biological treatments. This treatment was more efficient than pre-harvest chemical treatment (59,43%) but less efficient than post-harvest chemical treatment (84,91 %)

21. Conclusions l A minimal density threshold seems to be required just after harvest to obtain an antagonistic activity against P. expansum l Variations of climatic conditions between both trials are in accordance with population density and efficacy variations of pre-harvest treatments based on strain K l Need of a novel formulation taking into account –climatic detrimental factors for strain K during and after pre-harvest application –suspension stability and adherence during post-harvest application

22. l Cathy Grevesse l Sébastien Massart l Damien Friel l Flavia Nery l Deborah Declerq l Carol Kubjak l Stéphane Cognet l Catherine Dickburt l Eliane Hardy l Frédéric Dresen l Prof. Philippe Lepoivre Acknowledgements Walloon Region of Belgium Federal Ministry of Agriculture