CONTROL OF PENICILLIUM DIGITATUM ON ORANGE FRUIT COMBINING PANTOEA AGGLOMERANS WITH HOT SODIUM BICARBONATE DIPPING

020_JPP410Zamani_437 25-06-2009 12:27 Pagina 437 Journal of Plant Pathology (2009), 91 (2), 437-442 Edizioni ETS Pisa, 2009 437 CONTROL OF PENICILLIUM DIGITATUM ON ORANGE FRUIT COMBINING PANTOEA AGGLOMERANS WITH HOT SODIUM BICARBONATE DIPPING M. Zamani 1, A. Sharifi Tehrani 1, M. Ahmadzadeh 1, V. Hosseininaveh 1 and Y. Mostofy 2 1 Department of Plant Protection, and 2 Department of Horticulture, Faculty of Horticultural Sciences and Plant Protection, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran SUMMARY The antagonistic bacterium, Pantoea agglomerans HR, was evaluated for controlling citrus green mould caused by Penicillium digitatum Sacc. at 20 C (room temperature) and 4 C (cold storage). This isolate was also assessed in combination with dipping in 3% sodium bicarbonate solution at 24 C and 45 C on artificially inoculated Thomson navel oranges. Application of the antagonist alone reduced green mould by more than 75% at both temperatures, but was not as effective as Imazalil (more than 87% decay reduction). The antagonistic bacterium was completely tolerant to sodium bicarbonate up to a concentration of 3%. In addition, its efficacy for controlling green mould was improved at least by 5% and 11% when combined with 3% sodium bicarbonate at 24 C and 45 C, respectively. Key words: Citrus green mould, Pantoea agglomerans, sodium bicarbonate, heat treatment, biological control. INTRODUCTION Postharvest fungal decay may cause significant losses to the citrus industry. Injuries on citrus fruit caused during harvest, provide entries to wound pathogens, including Penicillium digitatum Sacc. and P. italicum Wehmer, causal agents of green and blue mould, respectively. These pathogens occur in almost all citrus growing regions of the world (Palou et al., 2001). The synthetic fungicide Imazalil, has been routinely used to control postharvest diseases including green and blue moulds (Eckert, 1990). Attempts to find alternatives to chemical control have been ongoing for some time, and indeed many fungi have become resistant to commonly used fungicides. In the mean time, there is increasing consumer concern in respect to chemical residues on fruit products regarding health and environmental issues. However, alternative measures are generally less effective than fungicides. Corresponding author: M. Zamani Fax: +98.261.2238529 E-mail: mzamany@ut.ac.ir Biological control is becoming increasingly effective in replacing chemicals used to control plant diseases (Conway et al., 1999; El-Ghouth et al., 2000). This method has lower environmental impact than fungicides either alone or as part of integrated pest management in reducing synthetic fungicide application (Wisniewski and Wilson, 1992). Several strains of Pantoea agglomerans have been reported as effective in suppressing diseases of fruit crops, such as fire blight of apple and pear (Beer et al., 1984; Kearns and Hale, 1995), brown spot of pear (Montensinos et al., 1996), and cranberry cotton ball caused by Monilinia oxycocci (Voland et al., 1999). P. agglomerans was reported as an antagonist of P. expansum, Botrytis cinerea and Rhizopus stolonifer on apple cv. Golden Delicious and on pear (Bryk et al., 1998; Nunes et al., 2001, 2002; Usall et al., 2001). However, biocontrol agents do not generally posses a broad spectrum of activity and they are not as effective as fungicides. Simultaneous application of several physical and chemical methods could provide more effective means of control and consistent results than that of one approach alone. Some physical treatments and exogenous substances, such as chitosan, amino acids, antibiotics, calcium salts, and carbohydrates have also been used to enhance biocontrol of antagonists against fungal pathogens (Conway et al., 1999; El-Ghouth et al., 2000). Pre storage hot water dips of fruit at temperatures above 40ºC have been shown to be effective in controlling storage decay, not only by reducing the pathogen but also by enhancing the resistance of fruit tissue, influencing host metabolism and ripening (Barkai-Golan and Philips, 1991). Postharvest dips are applied for a few minutes at high temperatures, because fungal spores and latent infections of the pathogen are either on the surface or in the first few cell layers under the peel of the fruit (Lurie, 1998). Their beneficial effects in controlling Penicillium rots on citrus fruit have been documented (Porat et al., 2000; Auret, 2001). Hot water treatment may eliminate incipient infections by removing spores from wounds and acting directly on spore viability and/or inducing defence mechanisms in the outer layers of the epicarp which inhibit pathogen growth (Schirra et al., 2000). Heat treatment can also provide further advantage of enhancing fruit coloration, but

020_JPP410Zamani_437 25-06-2009 12:27 Pagina 438 438 Control of Penicillium digitatum Journal of Plant Pathology (2009), 91 (2), 437-442 does not lead to softening. It inhibits the activities of cell wall hydrolytic enzymes in apple fruit and reduces ethylene production (Lurie, 1998). Sodium bicarbonate (NaHCO 3 ), commonly known as baking soda, was selected for integration with the biocontrol agent. It is a common food additive for phadjustment, taste, texture modification, and spoilage control, and has been shown to have antimicrobial activity against P. digitatum on citrus fruit (Smilanick et al., 1999; Zamani et al., 2008). It is inexpensive, readily available, and could be used with minimal risk of injury to the fruit. However, it is a poor eradicant unable to kill spores and its inhibitory effect is not very persistent. Its inhibitory activity depends on the presence of salt residues within the wound infection sites occupied by the fungus and on interactions between this residue and constituents of the peel (Palou et al., 2001). Biocontrol agents which can persist for long periods may protect fruit from post-treatment infection (Teixidó et al., 2001). Combining heat treatment and chemical compounds with an antagonist, could possibly be synergistic (Conway et al., 1999). The antagonist is applied after hot water treatment as it cannot survive at 50ºC. Thus the problem with application of the biocontrol agent before hot water treatment is that it must be heat tolerant (Leverentz et al., 2000). The objective of this study was to determine if green mould on oranges could be reduced by combination of the biocontrol agent P. agglomerans along with sodium bicarbonate and hot water treatment. The experiments were designed to develop an integrated strategy to control postharvest decay on oranges caused by P. digitatum that would be as effective as chemical control. MATERIALS AND METHODS Fruit. Commercially harvested Thomson Navel oranges, [Citrus sinensis L. (Osbeck)], with healthy appearance were used within 2 weeks of storage at 4ºC. Source of pathogen. A highly aggressive isolate of P. digitatum (M121) originally isolated from rotten grapefruit, was used. This isolate was grown on potato dextrose agar (PDA) at 25ºC for 7 days. Spores were harvested by adding 5 ml of water containing 0.05% (v/v) of Triton X-100 to the Petri dish, rubbing the surface with a sterile glass rod, and passing through two layers of cheesecloth. The suspension was diluted with water to an optical density (OD) of 0.1 at 425 nm, equivalent to roughly 10 6 spores ml -1 (Smilanick and Denis-Arrue, 1992). Antagonist isolate. The antagonistic bacterium, P. agglomerans strain HR, was obtained from the Department of Plant Protection, University of Mazandaran, Iran. It was originally isolated from leaf and fruit surfaces of Mexican lime in 2005. Preparation of aqueous antagonist suspension. To prepare antagonist suspension, cells were grown for 24-48 h on liquid nutrient yeast dextrose broth (NYDB) medium at 25±1ºC while shaken at 150 rpm. The culture was centrifuged at 8315g for 10 min and cells were resuspended in deionized water. Using a haemocytometer, the cell density was adjusted to 10 8 cells ml -1 (Nunes et al., 2001). Compatibility of P. agglomerans HR with sodium bicarbonate solution. To test for compatibility with sodium bicarbonate (SB), P. agglomerans HR cells were suspended in 400 µl of 1.0, 3.0, and 5.0% SB for 1, 12, and 24 h in microtitre plate wells. Subsequently, 100 µl was plated on fresh nutrient agar (NA). Plates were incubated at 27ºC for 24 h and compared with normal growth in the absence of SB. Serial dilutions were prepared from antagonist-salt suspensions at the end of each time interval and colony forming units (fu) determined using the spread plate technique (Obagwu and Korsten, 2003). Effect of sodium bicarbonate on P. digitatum spore germination. To assess the influence of SB on germination of P. digitatum spores, 100 µ1 of P. digitatum spore suspension (about 10 6 spores ml -1 ) along with 1.0, 3.0 or 5.0% SB were added to tubes containing 5.0 ml potato dextrose broth (PDB), These were incubated on a rotary shaker at 110 rpm at 25 ºC. After 13 h incubation, germination of approximately 200 spores was screened by measuring germ tube length. Spores were considered germinated when germ tube length was equal to or greater than spore length. Effect of P. agglomerans HR on P. digitatum spore germination. To assess the influence of P. agglomerans HR on germination of P. digitatum spores, 100 µl of P. digitatum spore suspension (about 10 6 spores ml -1 ) and 100 µl of P. agglomerans HR 10 8 fu ml -1 with or without 3% SB were added to tubes containing 5.0 ml of PDB. These were incubated on a rotary shaker as above. After 13 h incubation, germination rate and germ tube elongation of approximately 200 spores were screened as described above. Effect of sodium bicarbonate on citrus peel. Sodium bicarbonate treatment was performed by immersing the oranges in 1, 3 and 5 % SB for 2 min. Treated fruit were air-dried, and stored at 10±1ºC, and 90-95% relative humidity (RH) for 4 weeks following which they were screened for signs of peel damage. Immersion in sterile distilled water was used as negative control.

020_JPP410Zamani_437 25-06-2009 12:28 Pagina 439 Journal of Plant Pathology (2009), 91 (2), 437-442 Zamani et al. 439 Effect of antagonist on pathogen control. Fruits were surface-sterilized in 70% ethanol, and wound-inoculated with P. digitatum (10 6 spores ml -1 ) using sterile needles. One wound, approximately 1 mm wide and 2 mm deep, was made in the middle of each fruit. About 5-6 h post-inoculation, fruit were dipped in a suspension of antagonist (10 8 cells ml -1 ) for 2 min. Treated fruit were stored in cardboard boxes at 4ºC (for 35 days) and 20ºC (for 20 days), and 80-85% RH. Disease was rated as 0 or 1 for healthy and diseased fruit respectively. A fruit was considered diseased if decay was visible at the inoculation point regardless of lesion diameter. The controls consisted of infected fruit immersed in sterile distilled water (positive control), non-infected fruit immersed in sterile distilled water (negative control) and infected fruit treated with Imazalil (1-[2-(2, 4- dichlorophenyl)-2-(2-propenyloxy) ethyl]-1h-imidazole; technical, 97.5% a.i.) at a concentration of 1 g l -1. Effect of heat treatment along with sodium bicarbonate and P. agglomerans HR on disease control. Fruit inoculated as described previously were immersed in 3% SB solution at 45ºC, for 2 min, and then immersed in antagonist suspension for a further 2 min. Their surface was then air dried, and they were stored at 20ºC for 5 weeks. The control groups consisted of fruit immersed in aqueous antagonist suspension (10 8 fu l -1 ), 3% SB solution at 24ºC (room temperature), sterile distilled water at 24ºC, sterile distilled water at 45ºC and Imazalil at the concentration of 1 g l -1. Experimental design and statistical analysis. All experiments were conducted on the basis of completely randomized designs (CRD). Each treatment was replicated at least three times. In some cases the whole experiment was duplicated. For statistical analysis, data were subjected to the analysis of variance (ANOVA) using the GLM procedure of the SAS program (SAS Institute, 2004). Statistical comparisons among means were performed using Duncan s multiple range test using the same procedure. decreased spore germination of P. digitatum from 11% to 83% (Fig. 2). Effect of P. agglomerans HR on P. digitatum spore germination. P. agglomerans HR inhibited spore germination of P. digitatum (P<0.05), which was completely inhibited when P. agglomerans HR was combined with 3% SB at 20ºC (Table 1). Effect of sodium bicarbonate on citrus peel. After 4 weeks of storage at 10±1ºC, and 80-85% RH, none of the fruit immersed in 1, 3, and 5% SB showed any visi- Fig. 1. Effect of different concentrations (%), and duration of cell suspensions in sodium bicarbonate (SB) solution, on the growth of Pantoea agglomerans HR grown on NA for 24h at 27ºC. Values of columns with different letters differ significantly at P<0.05 RESULTS Effect of sodium bicarbonate on growth of P. agglomerans HR. Growth of this isolate following cell suspension in SB solution was affected by both concentration and duration of the cell suspension and duration of the treatment (Fig. 1). Cells grew normally in 1 and 3% solution for all incubation times. P. agglomerans HR showed noticeable reduction (up to 60%) in growth following 1, 12 and 24 h suspension in 5% bicarbonate. Effect of sodium bicarbonate on P. digitatum spore germination. Different concentrations of SB solution Fig. 2. Effect of different concentrations of sodium bicarbonate (SB) on Penicillium digitatum spore germination. Values of columns with different letters differ significantly at P<0.05

020_JPP410Zamani_437 25-06-2009 12:28 Pagina 440 440 Control of Penicillium digitatum Journal of Plant Pathology (2009), 91 (2), 437-442 Table 1. Effect of Pantoea agglomerans HR with and without sodium bicarbonate on spore germination a and germ tube length of Penicillium digitatum in PDB at 20º C after 13 h. Treatments P. digitatum Spore germination (%) Germ tube length (µm) Control 96.60a 121.45a P. agglomerans HR 47.5b 52b SB 18.3c 31.21c P. agglomerans HR + SB 0d 0d a Germination and germ tube length were measured with a light microscope after 13 h incubation at 25º C in potato dextrose broth. Figures in a column followed by a different letter differ significantly at P<0.05 by Duncan ' s multiple range tests. 80% at 4 ºC and more than 70% at 20ºC) (Fig. 3). Effect of heat treatment along with sodium bicarbonate and P. agglomerans HR on disease control. Results (Fig. 4) showed significant differences among the controls. P. agglomerans HR used alone, was significantly more efficient in controlling green mould than the positive control (water). However, the treatment was not efficient enough compared to the fungicide treatment, which provided approximately complete disease control. A remarkable improvement in the performance of the biocontrol agent was observed when it was applied following hot SB treatment (Fig. 4). No peel damage was observed following the treatment. ble damage. No symptoms of internal damage or weight loss were observed between the control and the treated fruit. Effect of the antagonist on pathogen control. Significant differences were observed among the treatments and positive control (infected with pathogen but no antagonist) at both temperatures (P<0.01).The antagonist, P. agglomerans HR, controlled development of the pathogen, P. digitatum, as effectively as Imazalil (about Fig. 4. Influence of Pantoea agglomerans HR (Pan) alone, sodium bicarbonate (SB) dip alone at ambient temperature (24ºC; cold) or at 45ºC (hot), or hot SB followed by Pan on the incidence of postharvest green mould of oranges subsequently held in cold storage. Treatments are significantly different at the 5% level. P. agglomerans HR (10 8 fu ml -1 ); SB (3% w/v); Imazalil (0.1%). Values of columns with different letters differ significantly at P<0.05. DISCUSSION Fig. 3. Percentage of fruit remaining uninfected after inoculation with Penicillium digitatum at 10 6 spore ml -1, followed by Pantoea agglomerans HR at 10 8 fu ml -1 in comparison with Imazalil treatment. Fruits were stored at 20 and 4ºC. There is significant difference between treatments, using Duncan s multiple range test (P<0.01) at both temperatures. A: Imazalil (1 g l -1 ), B: P. agglomerans HR (10 8 fu ml -1 ), C: negative control and D: positive control. Values of columns with different letters differ significantly at P<0.05 The present study demonstrated that P. agglomerans HR is an effective biocontrol agent against P. digitatum on oranges, and could protect the fruit from green mould decay under cold storage. The effectiveness of P. agglomerans HR was comparable to that of Imazalil at commercial doses, indicating that this biocontrol agent could be used as a substitute for chemicals to control P. digitatum. P. agglomerans HR is tolerant and compatible with sodium bicarbonate (SB) solution at 3%. Other biocontrol agents, such as P. agglomerans CPA-2, is also totally tolerant to 2% SB solution (Teixidó et al., 2001). Differ-

020_JPP410Zamani_437 25-06-2009 12:28 Pagina 441 Journal of Plant Pathology (2009), 91 (2), 437-442 Zamani et al. 441 ences of ph and ionic strength of the SB solutions may be the reason for different effect of them on the antagonist. Our results indicated that control of fruit decay using a mixture of the antagonist bacterium and SB was effective in two ways. First, SB could directly inhibit the spore germination of the fungal pathogen (Aharoni et al., 1997). The presence of SB in antagonist suspension enhanced the inhibition of spore germination and germ tube growth of P. digitatum (Zamani et al., 2008). Secondly, biocontrol bacteria have been selected mainly for their capacity to rapidly colonize and multiply in surface wounds, and subsequently to compete with the pathogen for nutrients and space (Nunes et al., 2002). In our tests, oranges treated with different concentration of SB were not injured. Heating SB solution to 45 C improved its efficacy compared to treatment at room temperature. Many authors have observed synergic effects of heat and chemicals applied to control post harvest decays (Teixidó et al., 2001; Palou et al., 2001; Smilanik et al., 1999). Heat treatment and sodium bicarbonate are not curative when used alone; their effects in vivo are primarily fungistatic and not very persistent. Simultaneous application of chemical and heat treatment with an antagonist, in some cases, could complement their effectiveness with the residual activity of the biocontrol agent (Conway et al., 1999). How P. agglomerans controls postharvest diseases is not clearly understood. In our previous experiments we have observed decay suppression by this isolate to be correlated with the presence of bacterial cells, while their filter sterilized, cell-free culture fluids did not influence decay, and the biocontrol activity of this isolate is probably related to the number of viable cells, so to achieve effective control it is sometimes necessary to use high concentrations of antagonist (Zamani et al., 2009). It has been speculated that P. agglomerans inhibits plant pathogens by colonizing the fruit and competing for nutrients, and that physical contact between pathogen and antagonist is important for effective control (Kempf and Wolf, 1989). This may induce plant defences (Slade and Tiffin, 1984), perhaps by the production of antibiotics such as Pantocin A and B (Beer et al., 1984; Bonaterra et al., 2003; Poppe et al., 2003; Wright et al., 2006). The isolate P. agglomerans HR used in this study has shown a high tolerance to SB treatment at commonly used concentrations. SB is a routine food product, and hot water treatment is also a normal practice in many citrus packing houses. The present approach in simultaneous application of P. agglomerans HR along with SB or warm SB solution is therefore compatible with already practiced routines in citrus packing houses, and therefore could easily be adopted. Further confirmation and comprehensive studies in the exact mode of action and any non-target effects will have to be carried out before commercializing this protocol. ACKNOWLEDGEMENTS This work was supported through a grant from the Higher Council for Researchers, University of Tehran. We also gratefully acknowledge the Ramsar Citrus Research Institute for providing the fresh fruit. We thank Professor H. Rahimian, Department of Plant Protection, University of Mazandaran for the antagonistic bacterial isolate. 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