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