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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 - PowerPoint PPT Presentation
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Pre- and post-harvest practical Pre- and post-harvest practical application of Pichia anomala strain K, application of Pichia anomala strain K, --
1,3-glucans and calcium chloride on 1,3-glucans and calcium chloride on apples : Two years of monitoring and apples : Two years of monitoring and efficacy against post-harvest diseasesefficacy against post-harvest diseases
M. H. Jijakli, D. De Clercq, C. Dickburt, P. Lepoivre
Plant Pathology Unit
University of Agricultural Sciences of Gembloux
Apple postharvest Apple postharvest diseasesdiseases losses of 15-25% after harvest
Major fungal diseases :– wound parasites
» Botrytis cinerea
» Penicillium expansum
– Latent infections » Gloeosporium
?
Biological control methods Biological control methods of postharvest diseases...of postharvest diseases...
Natural phenomenon
Efficient and reliable
control strategy
Realistic approach ?
•Biological limits
•Technological limits
•Economical limits
Market definition
Antagonistic strain isolation
SelectionMassive production
Formulation
Pilot trialsModes of action
Practical trials
Commercialization
Registration procedure
Registration
Toxicology
Monitoring
Required steps for biopesticide developmentRequired steps for biopesticide development
Natural phenomenon
Efficient and reliable
control strategy
?
Selection ofantagonistic strain
First step
Selection of one effective Selection of one effective microbial strainmicrobial strain
Pichia anomala (strain K)
A strain of yeast efficient against wound parasites (B. cinerea and P. expansum)
Natural phenomenon Selection of the antagonistic strain
Efficient and reliable
control strategy
? Mass production and formulation
Second step
Standard formulation of P. anomala Standard formulation of P. anomala strain Kstrain K
Quantity (>1010cells/g ) Quality (biological
activity preserved, identity, purity)
Obtention of a wettable Obtention of a wettable powderpowder
Production in fermentorProduction in fermentor
The technique needs to be transferred at an industrial scale
ButBut
Selection of the antagonistic strain
Natural phenomenon
Efficient and reliable
control strategy
?
Modes of action
Third step
Modes of actionModes of action
Colonisation related to protection suggesting competition for nutrients and/or space
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
Selection of the antagonistic strain
Natural phenomenon
Efficient and reliable
control strategy
?
Biological formulation
Fourth step
Biological formulationBiological formulation
GoalGoal : To stabilise the protection by : To stabilise the protection by increasing survival, efficacy of BCA and its increasing survival, efficacy of BCA and its persistance of efficacy persistance of efficacy
MethodMethod 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 (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)
Selection of the antagonistic strain
Natural phenomenon
Efficient and reliable
control strategy
?
Monitoring
Fith step
Monitoring of strain KMonitoring 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)
Selection of the antagonistic strain
Natural phenomenon
Efficient and reliable
control strategy
?Semi-practical application of
biological control
Sixth step
Semi-practical application of strain K in Semi-practical application of strain K in combination with YGT and CaClcombination with YGT and CaCl22
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
Storage of the fruits at 1°C (15 days), at 15°C (1 month) and at 20°C (15 days)
Variance analysis on severity index % of protection based on severity index
Pathogens inoculation (B. cinerea and P. expansum)
Pre and post-harvest application of strain K powder Pre and post-harvest application of strain K powder (10(1077ufc/ml)ufc/ml) + + YGT YGT (0.2%)(0.2%) + Cacl + Cacl2 2 (2 %)(2 %)
GHI
EFGHI
EFGH
FGHI
HI
HI
DEFGH
BCDEF
BCD
BCDE
AB
-20 0 20 40 60 80 100
Post-harvest chemical treatment (Sumico)
Post-harvest drenching
Post-harvest dipping
Pre-harvest strandard treatement with chemical
Pre-harvest high volume spray (3 days)
Pre-harvest low volume spray (3 days)
Pre-harvest high volume spray (7 days)
Pre-harvest low volume spray (7 days)
Pre-harvest high volume spray (15 days)
Pre-harvest low volume spray (15 days)
Untreated but inoculated control
% of protection against P. expansum
Year 2001
Biological treatments
Biological treatments
Contradictory results of efficacy between years 2000 and 2001 for biological and chemical treatments
H
BCDEF
B
CDEFGH
CDEFGH
CDEFGH
CDEFGH
A
A
FGH
FGH
BCDE
-20 0 20 40 60 80 100
Post-harvest chemical treatment (Sumico)
Post-harvest drenching
Post-harvest dipping
Pre-harvest strandard treatement with chemical
Pre-harvest high volume spray (2 days)
Pre-harvest low volume spray (2 days)
Pre-harvest high volume spray (5 days)
Pre-harvest low volume spray (5 days)
Pre-harvest high volume spray (12 days)
Pre-harvest low volume spray (12 days)
Untreated but inoculated control
% of protection against P. expansum
Year 2000
Biological treatments
Biological treatments
and B. cinerea
Climatic data Climatic data Données climatiques
2000 2001Revel és c li mat iques
0
5
1 0
1 5
2 0
-1 5 -1 2 - 9 - 6 - 3 0 3
t emp s (j ou rs avant récolte )
tem
pé
ratu
re (
°C)
0
2
4
6
8
1 0
1 2
temp mo y
pluie tot
Pluie to t l /m 2
Re velé s c li ma tiques
0
5
1 0
1 5
2 0
te mp s -1 1 - 9 - 7 - 5 - 3 - 1 1
te m ps (jours )
tem
pé
ratu
re (
°C
)
0
2
4
6
8
1 0
1 2
Pl u iet ot( l/ m2 )
tem p m o y
P luie to t
Pluie pendant 10 h avec un maximum de 1.4 l / m2 Forte
pluiejusqu’à
2.8 l / m2
Fortepluie
jusqu’à4.4 l / m2
Climatic data
Total Rain
Total Rain
Mean t° Mean t°
Total Rain
Total Rain
Time (days)
Time (days)
•Rain during 10h with a maximum of 1.4l/m2
•Short but heavy rains with a maximum of 4.4l/ m2
• 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
1,E+00
1,E+01
1,E+02
1,E+03
1,E+04
1,E+05
-15 0 15 30 45Time before harvest (days)
Log
(cf
u/c
m2)
Spraying 12 days before harvest Spraying 2 days before harvest
Dipping after harvest No treatment
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 Monitoring of strain K by plating on semi-selective mediummedium
1,E+00
1,E+01
1,E+02
1,E+03
1,E+04
1,E+05
-15 0 15 30 45Time before harvest (days)
Log
(cf
u/c
m2)
Spraying 12 days before harvest Spraying 2 days before harvest
Dipping after harvest No treatment
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 104 cfu of strain K/cm2 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
ConclusionsConclusions
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 %)
ConclusionsConclusions
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
Cathy Grevesse Sébastien Massart Damien Friel Flavia Nery
Deborah Declerq Carol Kubjak Stéphane Cognet
Catherine Dickburt Eliane Hardy Frédéric Dresen
Prof. Philippe Lepoivre
AcknowledgementsAcknowledgements
Walloon Region of BelgiumFederal Ministry of Agriculture