Comm. Appl. Biol. Sci, Ghent University, 71/3b, 2006 973 AUREOBASIDIUM PULLULANS AS A BIOCONTROL AGENT OF BLUE MOLD IN ROCHA PEAR M.M. FERREIRA-PINTO 1, M.C. MOURA-GUEDES 1, M.G. BARREIRO 2, I. PAIS 2, M.R. SANTOS 1 & M.J. SILVA 1 1 ECO-BIO, Instituto de Investigação Científica Tropical (IICT) Apartado 3041, PT-1301-901 Lisboa, Portugal 2 Departamento de Fisiologia Vegetal, EAN-INIAP Quinta do Marquês, PT-2745-505 Oeiras, Portugal SUMMARY The blue mold of Rocha pear caused by Penicillium expansum is an important postharvest disease which is adequately controlled by application of synthetic fungicides. In recent years, strategies like biological control have been considered a desirable alternative to chemicals. Several studies have demonstrated the potential of the yeastlike fungus Aureobasidium pullulans for control of postharvest decay of pear. A Portuguese isolate of Aureobasidium pullulans was characterized and evaluated for its activity in reducing postharvest blue mold decay of Rocha pear caused by Penicillium expansum. Study of optimal conditions for antagonist growth was carried out in six different culture media. The effect of four maturity stages of fruits in the development of A. pullulans was also studied. Biocontrol studies were performed with two concentrations of the antagonist (3 10 8 and 4 10 9 CFU/ml). A. pullulans growth was significantly different (P 0.001) according to the various media and time of incubation. Best results were obtained in Corn Meal Agar (CMA) and Potato Dextrose Agar (PDA) media which contains the higher concentration of glucose (20 mg/l). Medium resulted from fruits of the first harvest date presented lower colony diameter. Inoculation of A. pullulans at 3 10 8 and 4 10 9 CFU/ml reduced the incidence of the disease by 23 and 63%, and reduced the lesion diameter by 36 and 46%, respectively. Key words: Aureobasidium pullulans, Penicillium expansum, Rocha pear, culture media, biocontrol INTRODUCTION The blue mold of Rocha pear caused by Penicillium expansum (Link) is an important postharvest disease which is adequately controlled by application of synthetic fungicides such as imazalil and thiabendazole. However, its use has become increasingly more restricted due to human health risks, environmental concerns and development of fungicide resistance by pathogens (Vero et al., 2002). In recent years, strategies like biological control have been considered a desirable alternative to chemical fungicides. Several species of bacteria and yeasts have been reported to reduce postharvest decay of pome fruits and some of them are already commercially available in some countries (Lima et al., 2003; Spadaro & Gullino, 2004). Several studies have also demonstrated the potential of the yeast-like fungus Aureobasidium pullulans (de Bary) Arnaud for control of postharvest decay of pear. This antagonist operates mainly through an efficient competition essentially based on a preventive colonization of fruit wounds (Ippolito et al., 2000; Lima et al., 2003).
974 In previous work the natural epiphytic microorganisms from surfaces of Rocha pear fruits were isolated and tested as antagonists of P. expansum. One isolate was selected for its potential as biocontrol agent due to its strong inhibitory activity against this fungus (Borges et al., 2004). The objectives of this work were to evaluate the influence of different synthetic culture media and natural media made with fruits at different maturity stages on the development of a Portuguese isolate of A. pullulans and to compare the biocontrol efficacy of two concentrations of the yeast against P. expansum in Rocha pear. MATERIALS AND METHODS Antagonist strain A Portuguese isolate of A. pullulans (isolate Rocha) was previously obtained from leaves and fruits of Rocha pear trees grown in Alcobaça, Portugal (Borges et al., 2004). The yeast was maintained on Potato Dextrose Agar (PDA, Difco) at 5ºC. Fresh cultures were grown on PDA for 7 days at 25ºC, before use. To obtain more rapidly higher cell concentrations the yeast was grown on 50 ml of Nutrient Yeast Dextrose Broth (NYDB: 8 g of nutrient broth (Merck), 5 g of yeast extract and 10 g of dextrose per litre) in Erlenmeyer flask cultures. Each flask was inoculated with a loop of a fresh yeast culture and incubated in a rotary shaker at 25ºC for 48 h. Following incubation, yeast cells were centrifuged at 5000 rpm for 10 m, washed with sterile distilled water to remove the growth medium and filtered through sterilized cheesecloth. The concentrations of the antagonist were adjusted to 3 10 8 and 4 10 9 CFU/ml respectively, using a haemocytometer. Pathogen inoculum Penicillium expansum was isolated from naturally infected Rocha pears collected from orchards located in Alcobaça, Portugal (Borges et al., 2004). The culture was maintained on PDA at 5ºC. Inoculum suspension was prepared by removing the spores with a bacteriological loop from a 7-day-culture grown on PDA at 25ºC. The spores were suspended in sterile distilled water and spore concentration was determined with a haemocytometer and adjusted to a concentration of 1 10 4 spores/ml. Influence of culture media on growth of A. pullulans Growth of the yeast was compared in six synthetic culture media: Corn Meal Agar (CMA, Difco), Czapeck-Dox agar (CZD, Merck), Nutrient Agar (NA, Oxoid), Nutrient Yeast Dextrose Agar (NYDA: 8 g of nutrient broth, 5 g of yeast extract, 10 g of dextrose and 15 g agar per litre), Plate Count Agar (PCA, Difco) and PDA. The different media (10 dishs/medium) were inoculated in the centre of each Petri dish containing 15 ml of medium with a 4 mm-diameter agar disk collected with a cork borer from the edges of fresh cultures and incubated at 25ºC. Colonies diameters were measured over a period of 9 days. Influence of fruit maturity stage in development of A. pullulans Development of the yeast was evaluated in pear juice agar (pear juice 50% v/v and 9 g agar per litre) made with Rocha pears weekly collected at four harvest dates: earlier harvests (DC1 and DC2), commercial harvest (DC3) and late harvest (DC4). The media were inoculated in the centre of each dish with
Comm. Appl. Biol. Sci, Ghent University, 71/3b, 2006 975 a 4 mm-diameter agar disk of A. pullulans cultures and incubated at 25ºC. Development of yeast was evaluated by measuring the colony diameter during 6 days. Biocontrol assays on pear fruits Biocontrol studies were performed on Rocha pear fruits surface-disinfected by immersion for 2 min in a 1% sodium hypochlorite solution, rinsed with distilled water and air-dried at room temperature before wounding. Two artificial wounds of 5 mm diameter and 5 mm in depth (each wound distant 15 mm from the equatorial region) were made on each fruit, followed by inoculation of 25 µl of each antagonist suspension. Three h later, 20 µl of the aqueous suspension of P. expansum was pipetted into all wounds. Fifteen fruits per treatment were used (thirty inoculation sites) and inoculated control fruit were treated with sterile distilled water. After inoculation, pears from different treatments were placed in tray-boxes, covered with polyethylene bags and transferred to a cabinet at 24ºC and high relative humidity conditions. Fruits were evaluated for symptoms over a period of 5 days and disease incidence (percentage of inoculated wounds that developed lesions) and severity (lesion diameter of diseased wounds sites) were recorded. Statistical analysis Data of all experiments were subjected to analysis of variance (ANOVA) and the mean values were compared using Tukey test. RESULTS AND DISCUSSION The results of A. pullulans development in the different media culture showed that yeast growth was significantly different (P 0.001) according to the various media and time of incubation. After 9 days of incubation at 25ºC, the best results were obtained with CMA and PDA with colonies diameter of 3.71 and 4.59 cm, respectively (Figure 1). Since mycelial growth presented some differences it is probable that nutritional composition of media influences the development of yeast. Our results showed that better growth of A. pullulans was obtained in media with higher concentration of glucose (20 mg/l). The experiments of Punnapayak et al. (2003) also concluded that the greatest dry weights of all A. pullulans isolates studied were obtained in media supplemented with glucose as only carbon source. The morphological characteristics of A. pullulans in the different media were similar: color, white and gradually turned pink; colonies flat, moist and shiny in appearance; conidia hyaline, one-celled, 4-6 2-3 µm in size and oval to cylindrical in shape. The growth of A. pullulans in pear juice agar was significantly different (P 0.05) depending on the maturity stage of fruits and time of incubation (Figure 2). In general, colony diameter values of A. pullulans in pear juice agar were similar to those obtain on PDA medium with a lower value for the medium resulted from fruits of the first harvest date (DC1) in comparison with the three others harvest dates, DC2, DC3 and DC4. Our results showed that pear juice agar made with fruits collected on an earlier stage of maturity resulted in less radial growth of A. pullulans. This fact may be related to
976 fruit composition namely less quantities of simple and complex sugars as suggested by Spadaro et al. (2002). PDA PCA Culture media NYDA NA 9 days 7 days 3 days 1 day CZD CMA 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Colony diameter (cm) Figure 1. Development of A. pullutans in different synthetic culture media 4 3.5 3 2.5 2 1.5 DC1 DC2 DC3 DC4 1 0.5 0 2 3 6 Figure 2. Development of A. pullutans in pear juice agar made with fruits collected at four harvest dates (DC1, DC2, DC3 and DC4) All treatments reduced decay significantly (P 0,001) compared with control inoculated only with sterile distilled water. Inoculum concentration of A. pullulans also significantly influenced control efficacy of the antagonist. The inoculation of the yeast at 3 10 8 and 4 10 9 CFU/ml reduced the incidence of the disease by 23 and 63%, and reduced the lesion diameter by 36 and 46%, respectively (Figure 3). The results showed that the higher the concentration of the antagonist the lower the disease incidence and smaller the lesion diameter. Recently, Zheng et al. (2004) and Zhang et al. (2005) reported that inoculation of high concentration of the yeast Cryptococcus laurentii (1x10 9
Comm. Appl. Biol. Sci, Ghent University, 71/3b, 2006 977 CFU/ml) gave best control of gray mold rot of pear, delaying the onset of decay and prolonging the storage period. These results suggest that competition for space and nutrition may play a major role in the biocontrol capability of these yeasts against pathogens. However, the mechanism by which the biocontrol activity of A. pullulans works is not clearly understood. Further investigations are in progress to optimize the antagonistic activity of A. pullulans against P. expansum. Figure 3. Biocontrol efficacy of two inoculum concentrations of A. pullulans on decay of Rocha pear after 5 days of inoculation with: a. control, b. 3 10 8 CFU/ml and c. 4 109 CFU/ml ACKNOWLEDGEMENTS This work was financially supported by project POCI/AGR/59270/2004 from FCT/MCES (Portugal) and FEDER (UE). REFERENCES BORGES M., PEDRO J.M. & BARREIRO G. (2004). Biological control of postharvest decay with epiphytic microorganisms of Rocha pear. In: Barreiro, G. (Ed.), livro de Actas do IV Simpósio Ibérico de Maturação e Pós-colheita 2004, EAN, Oeiras, pp. 513-517. IPPOLITO A., GHAOUTH A.EL, WILSON C.L. & WISNIEWSKI M. (2000). Control of postharvest decay of apple fruit by Aureobasidium pullulans and induction of defense responses. Postharvest Biology and Technology, 19:265-272. LIMA G., DE CURTIS F., CASTORIA R. & DE CICCO V. (2003). Integrated control of apple postharvest pathogens and survival of biocontrol yeasts in semi-commercial conditions. European Journal of Plant Pathology, 109:341-349. PUNNAPAYAK H., SUDHADHAM M., PRASONGSUK S. & PICHAYANGKURA S. (2003). Characterization of Aureobasidium pullulans isolated from airborne spores in Thailand. J. Ind Microbiol Biotechnol, 30:89-94. SPADARO D., GARIBALDI A. & GULLINO M. L. (2004). Control of Penicillium expansum and Botrytis cinerea on apple combining a biocontrol agent with hot water dipping and acibenzolar-s-methyl, baking soda or ethanol application. Postharvest Biology and Technology, 33:141-151.
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