Post-harvest non-chemical treatments consists of a large range of different approaches,

including strengthening of the commodity’s natural defence mechanisms, thermotherapy,

application of antagonistic microorganisms and natural antimicrobial substances.

NEX0101 is a promising antagonistic biocontrol agent containing the yeast Candida

oleophila as the active ingredient. NEX0101 was developed by Bionext, a spin-off from the

laboratory of Dr. H. Jijakli, and is currently evaluated for commercial use. The product

contains a yeast strain isolated from apple fruit and was originally developed for the control

of post-harvest diseases on apple. The antifungal effectiveness of this antagonist can be

increased by addition of calcium salts. As the mode of action of this yeast is based on the

colonisation of wounds, the mean targets of NEX0101 are blue mould, caused by

Penicillium expansum, and grey mould, caused by Botrytis cinerea. Facing possible latent

infections, thermotherapy by using hot water treatments could provide an advanced control

towards lenticelrot (Gloeosporium spp.). A combination of both physical and biological

treatment techniques could broaden the spectrum to all key pathogens on apple and pear.

The use of NEX0101 for the control of P. expansum was examined in combination with

calcium gluconate. According to the results the combination NEX0101 with calcium

gluconate provides an advanced mould control towards P. expansum The best results

were achieved using NEX0101 in combination with post-harvest dipping by thermotherapy.

The hot water treatment alone was clearly inefficient towards wound parasite P.

expansum, on the contrary thermotherapy stimulates the decay caused by this postharvest

pathogen. For the future a combination of biological and physical treatments could offer a

worthy non-chemical alternative for organic and integrated fruit growers towards fruit rot

decay, although more research is necessary to implement these methods in practice

Creemers, P.

Jijakli, H.

Pujos, P.

Van Laer, S.

Vorstermans, B.

English

Organic Eprints

172Improving control of storage diseases on apple by combining biological and physical post-harvest methodsB. Vorstermans1, S. Van Laer1, P. Creemers1, H. Jijakli2, P. Pujos3AbstractPost-harvest non-chemical treatments consists of a large range of different approaches, including strengthening of the commodity’s natural defence mechanisms, thermotherapy, application  of  antagonistic  microorganisms  and  natural  antimicrobial  substances. NEX0101  is  a  promising  antagonistic  biocontrol  agent  containing  the  yeast  Candida oleophila as the active ingredient. NEX0101 was developed by Bionext, a spin-off from the laboratory of Dr. H. Jijakli, and is currently evaluated for commercial use. The product contains a yeast strain isolated from apple fruit and was originally developed for the control of post-harvest diseases on apple. The antifungal effectiveness of this antagonist can be increased by addition of calcium salts. As the mode of action of this yeast is based on the colonisation  of  wounds,  the  mean  targets  of  NEX0101  are  blue  mould,  caused  by Penicillium expansum, and grey mould, caused by Botrytis cinerea. Facing possible latent infections, thermotherapy by using hot water treatments could provide an advanced control towards  lenticelrot  (Gloeosporium  spp.).  A  combination  of  both  physical  and  biological treatment techniques could broaden the spectrum to all key pathogens on apple and pear. The use of NEX0101 for the control of P. expansum was examined in combination with calcium  gluconate.  According  to  the  results  the  combination  NEX0101  with  calcium gluconate  provides  an  advanced  mould  control  towards  P.  expansum  The  best  results were achieved using NEX0101 in combination with post-harvest dipping by thermotherapy. The  hot  water  treatment  alone  was  clearly  inefficient  towards  wound  parasite  P.expansum, on the contrary thermotherapy stimulates the decay caused by this postharvest pathogen. For the future a combination of biological and physical treatments could offer a worthy non-chemical alternative for organic and integrated fruit growers towards fruit rot decay, although more research is necessary to implement these methods in practice. Keywords: Botrytis, Penicillium, storage IntroductionThe purpose of this trial was to compare biological and physical post-harvest methods of storage  disease  control  on  apple,  and  potentially  to  improve  it  by  combining  them. Concerning the biological control strategy, NEX0101 is included in the trial setup. This promising antagonistic biocontrol agent contains the yeast Candida oleophila strain O as the  active  ingredient  and  is  currently  evaluated  for  commercial  use.  The  antifungal effectiveness of this antagonist can be increased by addition of a specific additive. As the mode of action of this yeast is based on the colonisation of wounds, the mean targets of NEX0101 are blue mould, caused by Penicillium expansum, and grey mould, caused by Botrytis cinerea.                                                 1 Proefcentrum Fruitteelt* Mycology, De Brede Akker 13, B-3800 Sint-Truiden, Belgium,    E-mail: ben.vorstermans@pcfruit.be, stijn.vanlaer@pcfruit.be, piet.creemers@pcfruit.be; 2 Unité de Phytopathologie, Faculté Universitaire des Sciences Agronomiques, Passage des déportés, 2, 5030 Gembloux, Belgium, E-mail : jijakli.h@fsagx.ac.be  3 Bionext, Passage des déportés, 2, 5030 Gembloux, Belgium pps@bionext.be Archived at http://orgprints.org/13667/173Improving control of storage diseases on apple by combining biological and physical post-harvest methodsB. Vorstermans1, S. Van Laer1, P. Creemers1, H. Jijakli2, P. Pujos3AbstractPost-harvest non-chemical treatments consists of a large range of different approaches, including strengthening of the commodity’s natural defence mechanisms, thermotherapy, application  of  antagonistic  microorganisms  and  natural  antimicrobial  substances. NEX0101  is  a  promising  antagonistic  biocontrol  agent  containing  the  yeast  Candida oleophila as the active ingredient. NEX0101 was developed by Bionext, a spin-off from the laboratory of Dr. H. Jijakli, and is currently evaluated for commercial use. The product contains a yeast strain isolated from apple fruit and was originally developed for the control of post-harvest diseases on apple. The antifungal effectiveness of this antagonist can be increased by addition of calcium salts. As the mode of action of this yeast is based on the colonisation  of  wounds,  the  mean  targets  of  NEX0101  are  blue  mould,  caused  by Penicillium expansum, and grey mould, caused by Botrytis cinerea. Facing possible latent infections, thermotherapy by using hot water treatments could provide an advanced control towards  lenticelrot  (Gloeosporium  spp.).  A  combination  of  both  physical  and  biological treatment techniques could broaden the spectrum to all key pathogens on apple and pear. The use of NEX0101 for the control of P. expansum was examined in combination with calcium  gluconate.  According  to  the  results  the  combination  NEX0101  with  calcium gluconate  provides  an  advanced  mould  control  towards  P.  expansum  The  best  results were achieved using NEX0101 in combination with post-harvest dipping by thermotherapy. The  hot  water  treatment  alone  was  clearly  inefficient  towards  wound  parasite  P.expansum, on the contrary thermotherapy stimulates the decay caused by this postharvest pathogen. For the future a combination of biological and physical treatments could offer a worthy non-chemical alternative for organic and integrated fruit growers towards fruit rot decay, although more research is necessary to implement these methods in practice. Keywords: Botrytis, Penicillium, storage IntroductionThe purpose of this trial was to compare biological and physical post-harvest methods of storage  disease  control  on  apple,  and  potentially  to  improve  it  by  combining  them. Concerning the biological control strategy, NEX0101 is included in the trial setup. This promising antagonistic biocontrol agent contains the yeast Candida oleophila strain O as the  active  ingredient  and  is  currently  evaluated  for  commercial  use.  The  antifungal effectiveness of this antagonist can be increased by addition of a specific additive. As the mode of action of this yeast is based on the colonisation of wounds, the mean targets of NEX0101 are blue mould, caused by Penicillium expansum, and grey mould, caused by Botrytis cinerea.                                                 1 Proefcentrum Fruitteelt* Mycology, De Brede Akker 13, B-3800 Sint-Truiden, Belgium,    E-mail: ben.vorstermans@pcfruit.be, stijn.vanlaer@pcfruit.be, piet.creemers@pcfruit.be; 2 Unité de Phytopathologie, Faculté Universitaire des Sciences Agronomiques, Passage des déportés, 2, 5030 Gembloux, Belgium, E-mail : jijakli.h@fsagx.ac.be  3 Bionext, Passage des déportés, 2, 5030 Gembloux, Belgium pps@bionext.be As for the physical control strategy, thermotherapy using hot water treatment was included in  this  trial.  Facing  potent  latent  infections,  thermotherapy  could  provide  an  advanced control  towards  lenticelrot  (Gloeosporium  spp.).  A  combination  of  both  physical  and biological treatment techniques could broaden the spectrum to all key pathogens on apple and pear. Material and Methods The trial was executed on apple variety Golden delicious. The fruits originated from one orchard and were stored till the start of the trial, under standard storage conditions (1°C) at the Research Station of Gorsem. The fruits were sorted on fruit size and injured apples were removed. Apples with a fruit size of 75-85 mm were intended to be used in the trial. Artificial woundingThe fruits were infected with the storage disease Penicillium expansum. This fungus is a wound parasite, for infection the fruits need to be wounded in advance. In concrete the apples were punctured at 4 places, divided equally over the fruit, using a sterilized nail, mounted in a cork plug, creating a 1-mm wide by 2-mm deep cylindrical wound. As the fruits  were  dipped  before  the  artificial  infection,  the  trial  was  set  up  to  evaluate  the preventive efficiency of the test objects. Post-harvest dipping/drenching treatmentOn day one, the wounded fruits were dipped in the treatment solution containing the test product NEX0101. To prepare the test solution the biomass granules of NEX0101 have been reactivated in a small volume of water (13.33g in 0.5l). The suspension was smoothly agitated  for  one  hour  to  homogenize.  As  the  antifungal  effectiveness  of  this  biocontrol agent can be increased by addition of a specific additive, the trial was set up using the biomass  in  combination  with  the  additive,  at  a  dose  rate  of  2g/l  with  the  target concentration of the biomass (0.033%). In this trial each test object contains 4 replicates of 50 fruits. Next to the untreated dry check, one water dipped check object was included and dipped for 2 minutes, just as was done for NEX0101. Also one object was included facing a submersion time of 30 seconds. Dipping was done in plastic baskets with a content of 60 l and filled up with 40 l of the test solution. Perforated baskets containing 50 fruits were dipped in the test solution. Four dips were needed for the four replicates of 50 fruits per test object. In the trial setup also one object was included using post-harvest drenching. In concrete a spray equipment was used. The construction consists of a spray tower and a moving table. In the spray tower, 3 different nozzles are available, one above the fruits (spray tip: Twin Jet TJ-60 8004 VS) and two sideways (spray tip: Teejet UB8502 SS) guaranteeing a high flow rate (drenching). The fruits were placed on the moving table (0.045 m/s) and treated with the test solution (spray pressure 3 bar). Next to the biological control of Penicillium expansum also a physical control strategy was evaluated towards blue mould. In the trial setup, two objects were included facing thermotherapy (a hot water treatment for 2 minutes at 50°C) respectively alone or in combination with a biological dip treatment with NEX0101. An incubation time of about one hour was respected between both post-harvest dip treatments. After all objects were treated, the fruits were placed in small  wooden  boxes  (50  fruits/box  =  1  replicate),  marked  and  wrapped  in  plastic  foil leaving holes to insure a high level of humidity. The boxes were placed at random on pallets in a 100% humidified room (with a mist defensor) and incubated for 24 hours at about 20°C. Archived at http://orgprints.org/13667/174Artificial infectionOn  day  two,  four  replicates  of  50  fruits  per  object  were  inoculated  with  a  conidial suspension of Penicillium expansum. The conidial suspension was made from a culture plated on PDA-agar (Potato dextrose agar 39g/l, Merck, Germany). The spores of 10 days old Penicillium cultures, sporulated under normal light, were used to inoculate the fruits. The sporulating plates were rinsed using sterilized demineralised water, containing 0.05% of  Tween  20  (Fluka  Chemie,  Switserland).  After  counting  in  a  Burker  cel,  the  conidial suspension was assessed, by controlling the germination of the conidia on a PDA-agar. In this trial the germination level was more than 90%. To infect the fruits, a spray equipment was used. The construction (spray tower and a moving table) was also used for the object using post-harvest drenching. Only some preset parameters have been adjusted: in the spray tower 3 flat spray tips (TeeJet 650050, spraying angle 65°) are installed and the speed of the mobile table amounts to 0.19 m/s. The fruits were placed on the moving table and infected with the conidial suspension (spray pressure: 2 bar). When they were passed the  nozzles,  the  fruits  were  turned  and  infected  in  the  same  way  on  the  other  side. Afterwards the fruits were collected again in wooden boxes and wrapped in plastic foil leaving holes to insure a high level of humidity. The boxes were stored on pallets in a 100%  humidified  room  (with  a  mist  defensor)  and  incubated  for  48  hours  at  room temperature (20°C). The wooden boxes were arranged at random on the pallets. On  day  four,  the  pallets  were  stored  in  a  cold  storage  room  at  1°C  for  one  week. Afterwards the pallets were placed at 20°C and regularly moistened till wet to accelerate the development of the disease.Assessment on fruit rot decayAbout  18  days  later  the  degree  of  decay  of the  apple  fruits  was  evaluated.  A  second assessment of Penicillium took place one week later. To assess the wounded fruits on decay, per fruit the diameter of the lesion area is measured for each of the 4 wounds (mm). The total diameter of the lesions is expressed per fruit and when the whole fruit was infected with the disease, a total maximum diameter of 200mm was noted. The efficacy (Abbott value) of the test substances is calculated on the total diameter of lesions on 50 fruits.  The  number  of  infected  fruits  and  the  percentage  of  infected  fruit  were  also calculated.ResultsThe infection with Penicillium expansum was successful, about 43.5% of all apple fruits were infected in the untreated dry check. In the untreated dry check, the total average diameter per object of all infected fruits amounted to 1393.8mm. NEX0101 provided an advanced  mould  control  towards  P.  expansum.  All  NEX0101  treated  objects  gave  a significant reduction in decay. All objects testing NEX0101 gave a statistically identical result,  whatever  the  dipping  time  (2’  or  30’’),  the  application  technique  (dipping  or drenching), or the addition of thermotherapy to NEX0101. The best results were achieved using  NEX0101  in  combination  with  post-harvest  dipping  by  thermotherapy  (89.4% efficacy). The hot water treatment alone was clearly inefficient towards wound parasite P. expansum,  on  the  contrary  thermotherapy  stimulates  the  decay  caused  by  this  post-harvest pathogen. In this object 63.0% of all fruits were infected compared to 43.5% in the non-treated dry check object.Archived at http://orgprints.org/13667/175Discussion The results obtained are consistent with other results published by the lab of H. Jijakli on the use of the Candida oleophila strain O. The effect of hot water dipping on P. expansumis in agreement with was is seen in practice. However this negative effect is not always visible. Probably the physiological condition of the fruit at harvest and the interval between harvest and dipping play a major role in determining the outcome of hot water dipping towards the infection of P. expansum. For the future NEX0101 appears to be a flexible biological solution able to be used with different dipping times, application techniques, and alone or after thermotherapy.Figure 1. Percentage of P. expansum infected fruits per object (Statistics: Anova, Duncan p<0.05) Table 1. Total evaluation of Penicillium fruit rot infestation Penicillium expansum % infection  Diameter total lesionsFungicide  Dose (%)Mean    Mean   Efficacy ABB1  Check dry    43.5  bc  1393.8  b   2  Check water treated    29.5  b  891.3  b  36.1 3  NEX0101 dipping 2’ Biomass + Additive 0.033+ 0.200 8.5  a  201.3  a  85.6 4  NEX0101 dipping 30’’ Biomass + Additive 0.033+ 0.200 12.5  a  312.5  a  77.6 5  NEX0101 drenching Biomass + Additive 0.033+ 0.200 9.0  a  213.8  a  84.7 6  Thermotherapy 2’ in hot water (50°C)   63.0  c  1923.8  c  -38.0 7  Thermotherapy 2’ + dipping NEX0101 (2’) (Biomass+Additive)0.033+ 0.200 7.5  a  147.5  a  89.4 (Statistics: Anova, Duncan p<0.05) 0%10%20%30%40%50%60%70%dry checkwet checkNEX 2'dipNEX 30''dipNEX drenchTT 2' 50°CTT2'+NEX 2'dipBCBCA AAA% PenArchived at http://orgprints.org/13667/176Acknowledgement Research centre subsidized by the Ministry of the Flemish Community References Creemers P., Van Laer S., Dickburt C., Jijakli H. (Mars 2004). Biological control of storage rot on apple  by  postharvest  treatments  with  NEX0101.  International  Workshop:  Development  of Biocontrol  Agents  of  Diseases  for  Commercial  Applications  in  Food  Production  Systems, Sevilla, Spain. Jijakli H., De Clercq D., Dickburt C., Lepoivre P. (2002). 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. IOBC/WPRS Bulletin: Biological Control of Foliar and Post-Harvest Diseases, Vol 25, 29-32. Massart S., Sens-Espel R., Lepoivre P. Jijakli H (2002). Application of RAP-PCR and cDNA-AFLP to isolate genes of Candida oleophila (strain O) induced by the presence of galacturonic acid. IOBC/WPRS Bulletin: Biological Control of Foliar and Post-HarvestDiseases, 25, 399-402. Unistat 5.5. Statistical Package for Windows. User’s Guide, 930 pp. Version 5.5 November 2002. Unistat House, Shirland Mews, 4, London W9 3DY, England. Zar  J.H.  1999.  Biostatistical  analysis.  Fourth  Edition,  Prentice-Hall,  Upper  Saddle  River,  New Jersey. 663 pp. plus Appendices. ISBN: 0-13-081542-X. Archived at http://orgprints.org/13667/

Improving control of storage diseases on apple by combining biological and physical post-harvest methods

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