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Pre- and post-harvest practical application of
Pichia anomala strain K, b-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
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Apple postharvest diseases

• losses of 15-25 after harvest
• Major fungal diseases
• wound parasites
• Botrytis cinerea

• Penicillium expansum

• Latent infections
• Gloeosporium

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Biological control methods of postharvest
diseases...
?
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Required steps for biopesticide development
Antagonistic strain isolation
Massive production
Modes of action
Pilot trials
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?
Efficient and reliable control strategy
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Selection of one effective microbial strain
Pichia anomala (strain K)
A strain of yeast efficient against wound
parasites (B. cinerea and P. expansum)
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?
Efficient and reliable control strategy
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Standard formulation of P. anomala strain K
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Selection of the antagonistic strain
?
Efficient and reliable control strategy
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Modes of action

• Colonisation related to protection suggesting
  competition for nutrients and/or space
• No detectable antibiosis or stimulation of fruit
  defence
• Implication of exo-b-1,3-glucanases produced by
  strain K ?
• In situ stimulation of exo-b-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-b-1,3-glucanase had no detectable impact on
  protective level against B. cinerea

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Selection of the antagonistic strain
?
Efficient and reliable control strategy
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Biological formulation
Goal To stabilise the protection by increasing
survival, efficacy of BCA and its persistance of
efficacy

• Method
• 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 b-1,3-glucans)
• Application of a composition containing strain K
  (105ucf/ml) YGT (0.2 w/v) Cacl2 (2 w/v)
  offers a higher and longer efficacy in comparison
  with the application of strain K alone
  (107ucf/ml)

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Selection of the antagonistic strain
?
Efficient and reliable control strategy
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Monitoring of strain K

• Specific identification
• with a SCAR marker
• 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)

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Selection of the antagonistic strain
?
Efficient and reliable control strategy
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Semi-practical application of strain K in
combination with YGT and CaCl2
in collaboration with the  Station Royale de
Recherches Fruitières de Gorsem  (Belgium)

• Application by spraying at low (300L/ha) and high
  (1000L/ha) volume 12, 5 or 2 days before harvest
• Harvesting and artificial wounding of fruits
• Application by dipping or drenching

• Pathogens inoculation (B. cinerea and P.
  expansum)

• Storage of the fruits at 1C (15 days), at 15C
  (1 month) and at 20C (15 days)
• Variance analysis on severity index
• of protection based on severity index

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Pre and post-harvest application of strain K
powder (107ufc/ml) YGT (0.2) Cacl2 (2 )
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(No Transcript)
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Monitoring of strain K by plating on
semi-selective medium
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Conclusions

• 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 CaCl2 ). 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
• 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 )

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Conclusions

• A minimal density threshold seems to be required
  just after harvest to obtain an antagonistic
  activity against P. expansum
• Variations of climatic conditions between both
  trials are in accordance with population density
  and efficacy variations of pre-harvest treatments
  based on strain K
• 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

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Acknowledgements

• Cathy Grevesse
• Sébastien Massart
• Damien Friel
• Flavia Nery
• Deborah Declerq
• Carol Kubjak
• Stéphane Cognet
• Catherine Dickburt
• Eliane Hardy
• Frédéric Dresen
• Prof. Philippe Lepoivre

Walloon Region of Belgium Federal Ministry of
Agriculture