LABORATORY EFFECT OF Boni Protect CONTAINING Aureobasidium pullulans ( de Bary ) Arnoud IN THE CONTROL OF SOME FUNGAL DISEASES OF APPLE FRUIT

The efficacy of Aureobasidium pullulans (in the biopreparation Boni Protect) against different pathogens of apples (Botrytis cinerea, Monilinia fructigena, Penicillium expansum, and Pezicula malicorticis) was evaluated under laboratory conditions. The biocontrol product was applied at concentrations of 0.05%, 0.1%, and 0.5%. Fruits of apple cultivars ‘Jonagold Decosta’ and ‘Pinova’ were used. Boni Protect was very effective against B. cinerea on cv. ‘Jonagold Decosta’, reducing disease incidence by 55–83.8%. On ‘Pinova’ apples, this biological control product was the most efficient at earlier stages of the experiment. It inhibited grey mold by 65% after 5 days from inoculation and only by 14% after 20 days. On cv. ‘Jonagold Decosta’, Boni Protect at a concentration of 0.1% was also effective against M. fructigena, reducing brown rot by 31.4–74.5%, but its efficiency on cv. ‘Pinova’ was not significant. Blue mold caused by P. expansum was inhibited only slightly by the biocontrol product, while P. malicorticis proved to be the most resistant to its antagonistic abilities.


INTRODUCTION
Fungal diseases cause major losses in apple production.The most important pathogens of apple fruits are Pezicula alba Gunthrie, Pezicula malicorticis (Jacks.)Nannf., Botrytis cinerea Pers., Monilinia fructigena (Aderh.et Ruhl.)Honey, and Penicillium expansum Link.The importance of each pathogen depends on climatic and storage conditions as well as on the growing system.The protection of apples is dif-ficult because many fungi are resistant to fungicides (R o s s l e n b r o i c h and S t u e b l e r , 2000) and customers are afraid of chemical residues in fruits.Recently, more attention has been paid to microorganisms occurring on fruits under natural conditions and to their abilities to protect fruits against pathogens (V i n a s et al. 1998; W a g n e r et al. 2006).One of these microorganisms is a yeast-like fungus Aureobasidium pullulans (De Bary) Arnoud.The fungus is a widespread epiphyte well adapted to the phyllosphere and carposphere.Some A. pullulans isolates exhibit antagonistic activity against a number of phytopathogenic fungi (S c h e n a et al. 1999, 2003), competing for nutrients and space and producing numerous compounds such as pectolytic enzymes (P o u l i o t et al. 2005), antimicrobial metabolites (T a k e s a k o et al. 1991), and high-molecular-weight polysaccharides (pullulan) (P r a s o n g s u k et al. 2005).The fungus colonizes the surface of fruits and induces their resistance to fungal pathogens, especially those involved in postharvest diseases.Therefore, it can be used as a biocontrol agent against pathogens (e.g.Botrytis cinerea and Penicillium expansum) (S c h e n a et al. 1999; P o u l i o t et al. 2005).Based on this fungus, the biopreparation Boni Protect was developed and it is now produced by Bio-Protect GmbH.The active ingredients are two strains of A. pullulans isolated from apples at LS Phytopathology of Konstanz University.This biocontrol product contains living blastospores of A. pullulans in natural medium.
The aim of the study was to estimate the effect of A. pullulans in Boni Protect on the development of some fungal diseases of apple fruit.

MATERIALS AND METHODS
Aureobasidium pullulans was used in the biocontrol product Boni Protect (produced by Bio-Protect Gmbh, Konstanz, Germany).This biopreparation was tested at concentrations of 0.05%, 0.1%, and 0.5%.Four species of fungi (Botrytis cinerea, Monilinia fructigena, Penicillium expansum, Pezicula malicorticis) isolated from apples were used in the experiment.The study was carried out on fruits of cvs 'Jonagold Decosta' and 'Pinova'.The surface of fruits was sterilized with 70% ethanol.With a sterilized cork borer, four 2 mm deep holes were made in each fruit at the same distance from each other.5 μl of Boni Protect suspension at each concentration was introduced into each hole.In the control combinations, 5 ml of sterilized water was used.After 24 hrs 3mm disks of mycelium of each pathogen were placed in the holes.Before that, the fungi were grown for 10 days on PDA medium (Difco) in Petri dishes.6 fruits were tested for Boni Protect in one of the concentrations, one of the pathogens, and one of the cultivars.The apples were placed in plastic containers disinfected with ethanol and stored in a chamber at 22 o C and relative humidity of 80%.The diameters of necrotic spots were measured after 5, 10, 15 and 20 days of incubation.
The fungistatic activity of the biocontrol product was estimated as the percentage of inhibition of rot development on apples compared to the control according to Abbott's formula (B o r e c k i , 1984): C -T I (%) = × 100% C where: I -inhibition index (%); C -necrosis diameter in the control treatment; -necrosis diameter in the treatment containing the biopreparation.The data were analyzed by analysis of variance (Duncan's test) at a significance level of 5% using the SAS statistical system, ANOVA (SAS Version 9.1, SAS Inst., Cary, N.C., U.S.A.).

RESULTS
The results showed that the efficacy of Aureobasidium pullulans in Boni Protect depends on pathogen species, product concentration, and apple cultivar.The biopreparation was the most effective in controlling fruit rot caused by B. cinerea and M. fructigena.Aureobasidium pullulans strongly inhibited the development of diseases already after 5 days of experiment.The size of necrotic spots caused by B. cinerea on 'Jonagold Decosta' fruits was significantly smaller in the treatments with Boni Protect than in the control combination.The biopreparation was effective at all concentrations but the strongest effect was achieved at the concentration of 0.5% after 10 days (83.8%).Aureobasidium pullulans was less effective on apples of 'Pinova', although in most combinations the development of disease was significantly slower than in the control combination.The best results were achieved after 5 days of experiment but after 20 days the necrotic spots were significantly smaller only when 0.1% concentration of the product was used (Tables 1-2, Figs 1-2, 9).
Brown rot caused by M. fructigena can also be controlled by A. pullulans.The biocontrol product in 0.1% concentration proved to be very efficient on cv.'Jonagold Decosta' fruits, inhibiting the development of disease by 31.4-78.4%.The strongest effect was noticed after 15 days after the inoculation.The other concentrations were not effective.The concentration of 0.1% was the most efficient also on cv.'Pinova' apples but the diameters of spots did not differ significantly from the control combination (Tables 1-2,  Figs 3-4, 10).
The efficacy of A. pullulans to control rots caused by P. expansum and P. malicorticis was much lower.On 'Jonagold Decosta' apples, the concentration of 0.1% inhibited significantly P. expansum rot after 5, 15 and 20 days, the concentration of 0.5% after 10, 15 and 20 days, whereas the concentration of 0.05% only after 5 days.The differences between the treatments and controls were much smaller than in the combinations with B. cinerea and M. fructigena.On cv.'Pinova' fruits, only the development of Penicillium rot was slightly inhibited and only at the beginning of the experiment.The rot caused by P. malicorticis was not inhibited by Boni Protect (Tables 1-2, [11][12].

DISCUSSION
Losses of fruits and vegetables during harvest, handling and storage are high.In Poland there are no registered fungicides for controlling postharvest diseases caused by B. cinerea, M. fructigena, and P. expansum.There are four products registered for P. malicorticis but some strains of this fungus are resistant to synthetic fungicides.In addition, consumers' concerns and restrictions about fungicide residues have made more ecological alternatives necessary.
The efficacy of biocontrol agents to control diseases of fruit and vegetables has been investigated by many researchers and several commercial products are now available in many countries  2007) observed the first symptoms of blue mold on apples only after 13 days since A. pullulans application.In our study the effect of Boni Protect on blue mold was rather poor even if in some combinations the lesions were significantly smaller than those in the control.
The most difficult to control with Boni Protect was P. malicorticis.Perhaps the concentration of the antagonist was too low, since L e i b i n g e r et al. (1997) proved that bull's-eye rot could be controlled only with high concentrations of A. pullulans.
The differences in Boni Protect efficacy between the cultivars, especially noticeable in the combinations with B. cinerea, are difficult to interpret.This cannot be explained by cultivar susceptibility.A uw e r k e r k e n et al. (2007) proved that late-ripening cultivars are more resistant to B. cinerea than earlyripening ones.Both cultivars tested in our study are late-ripening, so this feature cannot have any effect on the results.Further studies are required to investigate the effect of cultivar on the efficiency of the biocontrol product in question.

CONCLUSIONS
1. Boni Protect (based on Aureobasidium pullulans) can be recommended for the protection of apples against Botrytis cinerea at all concentrations.2. The biopreparation proved effective in the protection of apples against Monilinia fructigena only at a concentration of 0.1%.3. Boni Procect cannot be recommended for the protection of apples against Penicillium expansum and Pezicula malicorticis.
Fig. 1.Effect of Boni Protect on the inhibition of necrosis caused by B. cinerea on 'Pinova'.
(D r o b y et al. 1998; S u g a r and S p o t t s , 1999; L i m a , 2003).Recently, yeasts and the yeast-like fungus A. pullulans are of special interest as antagonists.Similarly to yeasts, A. pullulans is an effective competitor for nutrients, especially for amino acids, iron and nitrogen (V e r o et al. 2002; B e n c h e q r o u n et al. 2006; C h i et al. 2009).It also produces antifungal compounds such as pullulan, protease, amylase, lipase, cellulase, xylanase and single cell protein (C h i et al. 2009).The fungus is also known for inducing plant defense mechanisms against fungi.It increases the activity of 1,3-glucanase, peroxidase and chitinase enzymes in wounds, which stimulates wound healing (I p p o l i t o et al. 2000).In the present research, A. pullulans in Boni Protect has proved quite effective to inhibit grey mold on apples.Similar results have been obtained by many researchers applying A. pullulans (F a l c o n i and M e n d g e n 1994; L i m a 1997).A c h b a n i et al. (2005) observed complete control of B. cinerea on apples using selected strains of this fungus.The efficiency of A. pullulans in grey mold control seems to be similar to that of yeasts (S a r a v a n a k u m a r et al. 2008; S c h a r m a et al. 2009).Although some authors have presented the ability of the fungus to protect fruits against brown rot (Z h a n g et al. 2010a, b), the biocontrol product was not always efficient in our study.Boni Protect inhibited the development of brown rot only in the concentration of 0.1% applied to 'Jonagold Decosta' apples.On 'Pinova' apples, the lesions in the treatments with the product were not significantly smaller than those in the control treatment.Many reports show the abilities of A. pullulans to control P. expansum (E l -G h a o u t et al. 2000; B e n c h e q r o u n et al. 2006; Z h a n g et al. 2010a).M o u n i r et al. (

Table 1
Effect of Boni Protect on necrosis diameter on 'Pinova' apples B1 -0.05% concentration of Boni Protect; B2 -0.1% concentration of Boni Protect; B3 -0.5% concentration of Boni Protect *values designated with the same letters (a,b,c…) within columns do not significantly differ at 5% error (Duncan's test) B1 -0.05% concentration of Boni Protect; B2 -0.1% concentration of Boni Protect; B3 -0.5% concentration of Boni Protect *values designated with the same letters (a,b,c…) within columns do not significantly differ at 5% error (Duncan's test)