International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 2(9), 22-28, September (2013) Int. Res. J. Biological Sci. International Science Congress Association 22 Pectobacterium carotovorum Inhibition by Preservative agents in Sprouting Radish SeedsAtchara Chatkaew, Mukhamad Su’udi, Sooyeon Lim, Byung-Ho Hwang and Jongkee Kim* Department of Integrative Plant Science, Chung-Ang University, Anseong 456-756, KOREAAvailable online at: www.isca.in Received 15th May 2013, revised 30th June 2013, accepted 3rd August 2013Abstract In the present study, we report the effect of preservative agents against Pectobacterium carotovorum ssp. carotovorum (Pcc), the causal agent of soft rot disease, on sprouting radish seeds. Compounds were mixed with nutrient agar at concentrations of 0.002 M, 0.02 M and 0.2 M. In vitro assay showed that out of ten compounds, sodium metabisulfite and sodium sulfite were able to inhibit bacterial growth at all concentrations. In addition, the reduction of bacterial population was in agreement with increasing holding time and concentration of compounds. In vivo assay of both compounds also exhibited similar tendency, in which, high concentration shows more inhibition effects on bacterial growth. The population of Pcc on radish sprout after treated with sodium metabisulfite and sodium sulfite were 0.00 and 6.68 log10 CFU/ml, compared to the control (7.06 log10 CFU/ml). However, the sodium metabisulfite has stronger negative effects on seed germination compare to sodium sulfite at 0.2 M. High concentration of both compounds also interfere the seedling elongation and fresh weight. The results indicate that appropriate amount of both compounds might be used for controlling the Pcc growth. Keywords:Pcc, soft rot, Raphanus sativus LIntroduction Radish (Raphanus sativus ) is a root vegetable crop consumed by a majority of Asian people. Among all parts of mature radish plant, root was mostly used for daily food or side-dishes. In addition, seedling of radish (sprout) contain many kinds of secondary metabolites compounds such as glucosinolate, vitamin C, anti-oxidant and any other small molecules that contribute to human health. One of the small molecules found in radish is isothiocyanate, which has been reported to have beneficial effects against cancer cells in human. However, the radish production is restricted by several numbers of environmental conditions such as biotic and abiotic stresses. One example of biotic disturbance is caused by fungal or bacteria. Pectobacterium carotovorum ssp. carotovorum (Pcc) is one example of economically important bacteria infected the radish and other vegetable. This pathogen can reduce the vegetable yield, and therefore, has been included in the top ten bacterial pathogens infected plants. During the growing, harvesting, packaging and distribution processes, the radish might be contaminated with bacteria. Several sanitation methods have been applied to prevent and reduce the contamination problem. These methods including the physical treatment such as high pressure7,8, heat treatment, ozone exposure, electron beam and gamma irradiation10,11. Several chemical compounds were also used for sanitation purpose. These chemical usually have salt or acid properties such as chlorine dioxide12, sodium hypochlorite13, fumaric acid12 and acidic electrolyzed water14. One of commercial chemical used for sanitation is chlorine bleach. However, this compound might harmful for environments and generate very strong odor15. Recently, several preservative agents have been reported to have an inhibition effects against bacterial growth. In the previous report, the preservative agents can induce the stress responses in microorganism16,17. The preservative agents such as alum and lime have been reported to decrease the infection of soft rot in cabbages18. In addition, salt compounds were effective to reduce Pcc in potato tuber19. This study was conducted to observe the effect of preservative agents on Pcc growth. The effect of preservative agents against Pcc was also determined on radish seeds and sprouts. The side effects of preservative agents treatments on seed germination and sprouting is discussed. Material and Methods Plant Materials: The radish cultivar (cv.) Chungwoonplus was used in this study. Five hundred grams of radish seeds were sterilized through soaking in 1% of sodium hyperchlorite for 1 min, 70% ethanol for 20 sec, and then washed with deionized water as described20. The seeds were air dried inside the biosafety hood at 25ºC until uniformly dry. Bacterial Culture and Seed Inoculation: The Pectobacterium carotovorum ssp. carotovorum (Pcc) was streaked on Nutrient agar21,22 and was incubated at 28°C for 24 hours. A single colony was picked up and added into 10 ml of Nutrient broth (NB), and incubated overnight on shaking incubator at 28°C, International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(9), 22-28, September (2013) Int. Res. J. Biological Sci. International Science Congress Association 23 150 rpm. One ml of overnight bacterial seed culture was transferred to 100 ml of fresh NB medium and was incubated at 28°C for 24 hours.The cell suspension (109 CFU/ml) was mixed with the radish seeds at ratio of 5:3 (v/v) for 5 min. The cell suspension was removed from seeds with sterilized cheesecloth. The seeds were dried inside the biosafety hood at 25ºC for 30 min. The seeds (2 grams) were wrapped with sterilized cheesecloth and were prepared for next analysis according to the method described by Neetoo and Chen. Preservative Agent for Screening Analysis: The preservative agents (acetic acid, citric acid, oxalic acid, magnesium chloride, magnesium nitrate, potassium carbonate, potassium nitrate, sodium carbonate, sodium sulfite and sodium metabisulfite) were added to NA plate (pH 6.5) at three different concentrations, 0.002 M, 0.02M and 0.2 M. NA plate without any chemical compound was used as the control. The bacterial stock solution was grown in NB at 28°C for 24 hours, 1 ml of cell culture was added to 9 ml of 0.1% sterile peptone water to prepare 10-5 dilution. The aliquot (100 µl) of cell suspension was spread plated on NA using a sterile cell spreader. The plates were incubated at 28ºC for 3 days. Optimizing Time for Pcc Inactivation: The compounds and cell suspension were mixed in 9:1 (v/v) ratio. At 0 time incubation, 1 ml of mixture was directly diluted with sterile 0.1% peptone water for 10-1 - 10-7 serial dilution. For 5, 10, 15 and 20 min incubation time, the mixture were incubated at indicated times and was carried as mentioned above. The 100 µl of each diluted bacterial suspension was spread plated on NA using a sterile cell spreader and incubated at 28ºC for 3 days. Microbial Population on Seeds: The sodium metabisulfite concentration and incubation were 0.2 M for 5 min, 0.02 M for 20 min and 0.002 M for 20 min, while for sodium sulfite were 0.2 M for 5 min, 0.02 M for 10 min and 0.002 M for 20 min. Sterilized water was used as control. The infected seeds were randomly selected and spread on wet paper towels on the plastic rack and then placed in the chamber. Two grams of seeds were mixed with 18 ml of 0.1% peptone water for 2 min with agitation (260 rpm). 1 ml of mixture was directly diluted with sterile 0.1% peptone water and processed as described above. The plates were incubated in chamber at 28ºC for 3 days. Seed Germination, Length and Fresh Weight: From two grams infected seeds, 100 seeds were randomly selected and spread on the layers of wet paper towels on the plastic rack.The moisture condition on paper towels were maintained by spraying distilled water every 6 hours. The percentages of germinated were determined after 0, 1, 2 and 3 from the onset of germination. Each compound at each concentration was tested with three replications per experiment. The percentages of germination were calculated as the total number of germinating seeds to the total number of seeds. The lengths of sprouts were determined every day using digi-caliper (Cienceware®, U.S.A). The seeds (2 grams) were germinated on the layers of wet paper towels with maintained moisture condition on the bucket. The fresh weight was determined every day (0, 1, 2 and 3 days). The fresh weight was calculated as described previously23. Results and Discussion Growth Inhibition of Pcc in Media Containing Preservative Agents: The growth inhibition of Pectobacterium carotovorum ssp. carotovorum (Pcc) by several preservatives agents are summarized in table-1. At the concentration of 0.002 M, only sodium sulfite and sodium metabisulfite showed complete inhibition effects on bacterial growth. At moderate concentration (0.02 M), the acetic acid, potassium carbonate and sodium carbonate also inhibited Pcc growth. The effects of several preservative agents were significantly difference 0.05) at those both concentrations. However, at high concentration (0.2 M), all preservative agents were able to cease Pcc growth (0 log10 CFU/ml). In previous reports, low concentrations of organic acids were effectively inhibits the microbial growth. The application of acid condition in the food has been reported to have preventive effects to microbial growth24. At higher concentration, organic acids have been used for sweep over the microorganisms25. Similarly, Gurtler et al26reported that high concentration of organic acids can inhibit the activity of bacteria. In our experiment, at concentration of 0.02 M, only one acidic molecule (acetic acid) and four salt molecules (potassium carbonate, sodium carbonate, sodium sulfite and sodium metabisulfite) showed full bacterial growth inhibition. Optimizing Time for Pcc Inactivation: Since the sodium sulfite and sodium metabisulfite shown significant inhibition for Pcc growth, we conducted further experiment to determine the effect of incubation time. As shown in figure-1, there were no different of Pcc growth in media without preservative agents (control) and with sodium sulfite and sodium metabisulfite. However, at concentration of 0.2 M, the Pcc growth in media containing sodium metabisulfite was completely inhibited (0 log10 CFU/ml) at 5 minutes incubation time and further (figure-1A). In contrast, in the media containing the same concentration of sodium sulfite, the Pcc growth was slightly inhibited at all incubation times (figure-1B). This result is in agreement with previous reports for the positive correlation between preservative compound concentration and incubation (holding) times. Trinettaet al27 also observed that high concentration of preservative agents was able to inactivate food borne pathogens more effectively. Our results further confirmed the previous report of Mills et al19, in which the application of sodium metabisulfite can reduced the population of Erwinia sppFurthermore, Roberts and McWeeny28 reported that sulfur dioxide was effective against gram-negative rods bacteria such as E. coli and Pseudomonas, but less effective to the gram-positive rods bacteria such as Lactobacillus. In addition, Basaran-Akgul et al29 stated that sulfur dioxide was effective to reduce the bacterial population as low as 5-log by reaction with several essential macromolecules (protein carbonyl groups, FAD, RNA and DNA). International Research Journal of Biological Sciences ________________________________________________ Vol. 2(9), 22-28, September (2013) International Science Congress Association (A) Growth of Pcc in nutrient agar treated with sodium metabisulfite concentrations (0.2 M, 0.02 M and 0.002 M) and different inoculation times (0, 5, 10, 15 and 20 min) Pcc Inhibition by Preservative Agents on the next, we tested the effects of preservative agent in the radish seeds after pre-treated with Pcc. The results metabisulfite at concentration of 0.2, 0.02 and 0.002 M 0.00, 5.88 and 6.85 log10 CFU ml, respectively day). Complete Pcc growth inhibition caused by this chemical at concentration of 0.2 M was remains persistent up to 2 days. In contrast, for sodium sulfite, the colony forming units were 7.19 and 7.04 log10 CFUml at concentration of 0.2, 0.02 and 0.002 M, respectively (table-2). In both compounds, concentration of 0.2 for sodium metabisulfite population was increase at all concentrations after treatment day until the final day (3 days). The Pcc population in the final day in the control was 9.14 log10 CFU ml sodium metabisulfite and sodium sulfite were 7.91 and 9.26 log10 CFUml , respectively. In the present study, metabisulfite and sodium sulfite were effective Pcc growth in vivo . There are several possibilities for the mechanism of the sulfite ion to inhibit the bacterial cell growth. The sulfite ion could enter the cell membrane and normal metabolic activity of bacterial cells 30 decreasing pH caused by the presence of sulfite ion might inhibit bacterial growth and respiration31 . In the previous study using fungal pathogen, the sodium metabisulfite could increase the permeability of cell membrane through destruction of membrane lipids32. Similarly, Mecteau et al 3 sodium metabisulte has the ability to inhibit and spore germination completely and reduced the development of dry rot disease in potato tuber. Effect of Preservative Agents Treatmen t on Germination, Seedling Elongation and Fresh Weight 3 summarized the germination percentage of radish seeds after treated with sodium metabisulfite and sodium sulfite 24 hours (first day), at concentration of International Research Journal of Biological Sciences ________________________________________________ Association (B) Figure-1 in nutrient agar treated with sodium metabisulfite (A) and sodium sulfite (B) at three different concentrations (0.2 M, 0.02 M and 0.002 M) and different inoculation times (0, 5, 10, 15 and 20 min) on Radish Seeds: In the next, we tested the effects of preservative agent in the radish The results for sodium at concentration of 0.2, 0.02 and 0.002 M were ml, respectively , at initial day (0 growth inhibition caused by this chemical at concentration of 0.2 M was remains persistent up to 2 days. In contrast, for sodium sulfite, the colony forming units were 6.68, at concentration of 0.2, 0.02 and both compounds, except at sodium metabisulfite , bacterial after treatment at 0 population in the final ml , while at 0.2 M and sodium sulfite were 7.91 and 9.26 , respectively. In the present study, sodium effective to reduce the . There are several possibilities for the mechanism of the sulfite ion to inhibit the bacterial cell growth. ion could enter the cell membrane and disrupts the 30 . In addition, the decreasing pH caused by the presence of sulfite ion might . In the previous study using fungal pathogen, the sodium metabisulfite could increase the permeability of cell membrane through destruction of 3 3 reported that the has the ability to inhibit mycelial growth completely and reduced the development t on Radish Seed Germination, Seedling Elongation and Fresh Weight : Table- 3 summarized the germination percentage of radish seeds after sodium metabisulfite and sodium sulfite . In the first at concentration of 0.2 M sodium metabisulfite and sodium sulfite , the were 0.33% and 24.33% , respectively, the control. At the second day, the germination 54.67% for seeds treated with sodium metabisulfite and sulfite , respectively. Meanwhile, the mock treated seeds germination was 81.00%. At the final day, the germination percentage for seeds treated with and sodium sulfite were increased up to while in the control was 8 5.00%. germination percentages at the concentration of 0.002 M were approximately 90.00%. These indicate that high concentrations of both compounds germinatio n process. In contrast, compounds were not restrains the seed germination progression, as comparable with mock control ( t et al34 showed that low pH effectively inhibit bacterial growth. However, this condition also gave some negative ef germination. In addition, salt solution at high concentration might delay the seed germination due to osmotic pressure effect35 . Other researchers have also observed the effect of and osmotic stresses on seed germination, but not seedlin development36. This osmotic stresses uptake during imbibitions because of which probably evoked excessive uptake of ions The data for seedling elongation of radish seed after treated with sodium metabisulfite and sodium sulfite was summarized in table-4. At first day, all treatment at 0.2 M of sodium metabisulfite and sodium sulfite showed shorter other treatment. The lengths of seedling generated from after treated with sodium metabisulfite 4.68 and 6.95 mm. At second day, from sodium metabisulfite treatment was different with other treatment. A significant different ( 0.05) for all compounds except 0.2 M of International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Int. Res. J. Biological Sci. 24 (B) (A) and sodium sulfite (B) at three different concentrations (0.2 M, 0.02 M and 0.002 M) and different inoculation times (0, 5, 10, 15 and 20 min) , the germination percentage , respectively, compared to 68.00% in the germination was 8.00% and sodium metabisulfite and sodium , respectively. Meanwhile, the mock treated seeds At the final day, the germination for seeds treated with 0.2 M sodium metabisulfite increased up to 23.00% and 68.00%, 5.00%. On the other hand, the germination percentages at the concentration of 0.02 M and approximately 90.00%. These indicate that at high concentrations of both compounds somewhat interfere the n process. In contrast, low concentrations of these not restrains the seed germination progression, t able-3). Previous study by Li showed that low pH effectively inhibit bacterial growth. However, this condition also gave some negative ef fects to seed germination. In addition, salt solution at high concentration might delay the seed germination due to osmotic pressure . Other researchers have also observed the effect of salt on seed germination, but not seedlin g osmotic stresses might decrease the water because of high salt concentration excessive uptake of ions 37. of radish seed after treated with metabisulfite and sodium sulfite was summarized in first day, all treatment at 0.2 M of sodium metabisulfite and sodium sulfite showed shorter seedling than seedling generated from seed metabisulfite and sodium sulfite were 4.68 and 6.95 mm. At second day, the seedling length raised treatment was 4.88 mm, extremely different with other treatment. A t third day, there was no 0.05) for all compounds except 0.2 M of International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(9), 22-28, September (2013) Int. Res. J. Biological Sci. International Science Congress Association 25 sodium metabisulfite. The length of seedling was 8.12 mm, about 12 times shorter than control. Our study demonstrates that, all compounds have no effects for seedling elongation at all concentration, except 0.2 M of sodium metabisulfite (table-4). The fresh weight was significantly influenced (0.05) by preservative agents treatment, as shown in table-5. The fresh weight was increased gradually following the germination times. The lowest fresh weight was observed in 0.2 M sodium metabisulfite treatment at 3 days after germination. The fresh weight was 3.16 g, about 1.5 times lower than control. This study shows that the concentration of compounds govern both germination and growth rate. In accordance with previous study by Khan et al38, high salinity inhibits the seed germination at concentrations beyond the tolerance limits of the species. Kaya et al35 also mentioned that salt solution has effect on seedling growth rather than the germination from osmotic effect. In the case of different fresh weight between sodium metabisulfite and sodium sulfite treatments, it should be due to the different in the seed capability for absorbing water and minerals. The similar mechanism has been reported by Mao-Jun et al39. Table-1 Growth of Pcc in nutrient agar added with ten different preservative agents at three different concentrations (0.2 M, 0.02 M and 0.002 M) and incubated at 28°C for 3 days Compound Pcc (log10 CFU/ml) 0.002 M 0.02 M 0.2 M Control 8.14 8.14 8.14 Acetic acid 7.78 0.00 0.00 Citric acid 8.48 8.48 0.00 Magnesium chloride 8.15 7.81 0.00 Magnesium nitrate 8.48 8.18 0.00 Potassium carbonate 7.99 0.00 0.00 Potassium nitrate 8.15 7.72 0.00 Sodium carbonate 8.41 0.00 0.00 Sodium sulfite 0.00 0.00 0.00 Sodium metabisulfite 0.00 0.00 0.00 Oxalic acid 8.48 8.48 0.00 C.V. (%) 0.57 0.36 0.41 F-test ** ** ** Data represents the mean values of colony forming unit of Pcc on log survivors (CFU/ml). Within a column, different letters indicate significant (0.05). Table-2 Growth of inoculated Pcc on radish seeds treated with sodium metabisulfite (0.2 M for 5 min, 0.02 M for 20 min and 0.002 M for 20 min) and sodium sulfite (0.2 M for 5 min, 0.02 M for 10 min and 0.002 M for 20 min) Compound Time (min) Bacterial populationon seed (log10 CFU/ml) 0 Day 1 Day 2 Days 3 Days Control 0 7.06 8.46 9.25ab 9.14 0.2 M Na 5 0.00 0.00 0.00 7.91 0.02 M Na 20 5.88 7.95 9.06 9.17 0.002 M Na 20 6.85 8.17 9.23ab 9.26 0.2 M NaSO 5 6.68 8.24 9.30 9.26 0.02 M NaSO 10 7.19 8.46 9.20 9.25 0.002 M NaSO 20 7.04 8.28 8.88 9.24 C.V. (%) 1.21 0.43 0.57 0.77 F-test ** ** ** ** Data represents the mean values of colony forming unit of Pcc on log survivors (CFU/ml). Within a column, different letters indicate significant (0.05). International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(9), 22-28, September (2013) Int. Res. J. Biological Sci. International Science Congress Association 26 Table-3 Percentage of seed germination treated with sodium metabisulfite (0.2 M for 5 min, 0.02 M for 20 min and 0.002 M for 20 min) and sodium sulfite (0.2 M for 5 min, 0.02 M for 10 min and 0.002 M for 20 min) Compound Time (min) Percentage of radish seed germination (%) 0 Day 1 Day 2 Days 3 Days Control 0 0.00 68.00 81.00 85.00 0.2 M Na 5 0.00 0.33 8.00 23.00 0.02 M Na 20 0.00 70.67ab 86.67 92.00 0.002 M Na 20 0.00 75.00ab 86.00 91.67 0.2 M NaSO 5 0.00 24.33 54.67 68.00 0.02 M NaSO 10 0.00 77.00 85.33 91.00 0.002 M NaSO 20 0.00 78.67 85.33 90.67 C.V. (%) 0.00 8.09 7.41 5.19 F-test ns ** ** ** Data represents the mean value of percentage of seed germination. Within a column, different letters indicate significant (0.05). Table-4 Seeds elongation treated with sodium metabisulfite (0.2 M for 5 min, 0.02 M for 20 min and 0.002 M for 20 min) and sodium sulfite (0.2 M for 5 min, 0.02 M for 10 min and 0.002 M for 20 min) Compound Time (min) Seeds elongation (mm) 0 Day 1 Day 2 Days 3 Days Control 0 4.13 8.16 35.90 97.61 0.2 M Na 5 4.68 4.68 4.88 8.12 0.02 M Na 20 4.36ab 9.55 24.34 83.20 0.002 M Na 20 4.28ab 10.06 29.58ab 95.23 0.2 M NaSO 5 4.28ab 6.95 25.91 78.66 0.02 M NaSO 10 4.41ab 9.81 20.46 74.95 0.002 M NaSO 20 4.27ab 9.71 23.77 85.46 C.V. (%) 7.25 12.35 27.22 29.93 F-test ns ** ** ** Data represents the mean value of length of sprouting seeds. Within a column, different letters indicate significant (0.05Table-5 Fresh weight of sprouting seeds treated with sodium metabisulfite (0.2 M for 5 min, 0.02 M for 20 min and 0.002 M for 20 min) and sodium sulfite (0.2 M for 5 min, 0.02 M for 10 min and 0.002 M for 20 min) Compound Time (min) Fresh weight (g) 0 Day 1 Day 2 Days 3 Days Control 0 2.03 2.99 3.90 4.95 0.2 M Na 5 2.04 2.91 2.98 3.16 0.02 M Na 20 2.03 2.92 3.97 4.82 0.002 M Na 20 2.03 3.03 4.06 4.90ab 0.2 M NaSO 5 2.03 2.87 3.42 4.02 0.02 M NaSO 10 2.03 2.97 3.98 4.92 0.002 M NaSO 20 2.03 2.99 4.28 4.85bc C.V. (%) 0.41 0.62 0.53 0.88 F-test ns ** ** ** Data represents the mean value of fresh weight. Within a column, different letters indicate significant (0.05) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(9), 22-28, September (2013) Int. Res. J. Biological Sci. International Science Congress Association 27 Conclusion The present study shows that among ten preservative agents, sodium metabisulfite and sodium sulfite were effective to inhibit Pcc. Both compounds effectively reduce the cell population number at high concentration and longer incubation time. Although both compounds inhibit symptom development of radish seedling infected with Pcc, the sodium metabisulfites shows stronger inhibition effects. On the other hand, high concentration of sodium metabisulfite has negative effects on seed germination, seedling elongation and fresh weight. Since these compounds are not harmful for human consumption, the results of this study might be useful for application in the food processing, especially inhibition of microbial growth. Acknowledgment This work was supported by the GRRC program of Gyeonggi province [GRRC-CAU-2012-A01, Development of Baemoochae kimchi and postharvest technology]. 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