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Acid adaptation and biocontrol efficacy of antagonistic marine yeast Rhodosporidium paludigenum

Acid adaptation and biocontrol efficacy of antagonistic marine yeast Rhodosporidium paludigenum Ann Microbiol (2014) 64:503–508 DOI 10.1007/s13213-013-0681-2 ORIGINAL ARTICLE Acid adaptation and biocontrol efficacy of antagonistic marine yeast Rhodosporidium paludigenum Yifei Wang & Shoukui He & Jindan Xia & Ting Yu & Xiaodong Zheng Received: 21 September 2012 /Accepted: 24 June 2013 /Published online: 11 July 2013 # Springer-Verlag Berlin Heidelberg and the University of Milan 2013 Abstract The objectives of this work were to assess the Introduction optimum conditions for induction of acid tolerance in the marine yeast Rhodosporidium paludigenum and evaluate the The marine yeast Rhodosporidium paludigenum was previ- biocontrol activity of non-adapted and acid-adapted yeasts in ously isolated from the East China Sea and exhibited its controlling apple blue mold caused by Penicillium expansum. antimicrobial activity against Alternaria alternata and Bo- R. paludigenum grown in malic and lactic acid treatments trytis cinerea, which are two major postharvest pathogens of were stimulated after 12 h incubation. Moreover, medium cherry tomatoes and Chinese winter jujubes in China (Wang modified with malic and lactic acid significantly enhanced et al. 2010a, 2011). Its biocontrol efficacy was also found to the acid tolerance of R. paludigenum (p<0.05). In acid toler- be improved in combination with calcium chloride (Wang ance response test, the highest viability of R. paludigenum et al. 2010b). Previous studies showed that competition for was obtained at initial pH of 5.5 in the NYDB medium nutrients is an important antagonist mechanism of R. modified with malic acid (91.6 %). In addition, all R. paludigenum (Wang et al. 2008). paludigenum treatments significantly reduced the disease in- Before applying to fruits, antagonists can be exposed to cidences and lesion diameters of blue mold in apples. Further- variable environments. A number of biotic or abiotic factors more, there was no significant difference between acid- reduce the efficacy and stability of biocontrol agents adapted and unadapted yeasts in the apple wounds after 48 h (BCAs). Recently, several researchers have shown that salt dynamics. Acid stress improved R. paludigenum viability adaptation of yeasts (Lahlali and Jijakli 2009) and bacteria under acidic conditions. However, there was no significant (Teixidó et al. 2005, 2006) may have promising potentials to difference between acid-adapted and unadapted yeasts in con- develop industrial formulation of antagonists. Hence, to be trolling P. expansum on apple fruit (p<0.05). These results successful in the food industry, stress tolerance of BCA must indicate the potential for maintaining the survival level of be enhanced in order to extend shelf life and maintain bio- biocontrol agents by physiological inducement strategy. control activity (Lahlali and Jijakli 2009). Acid tolerance response (ATR) can be defined as the resis- . . Keywords Acid tolerance response Biocontrol tance of microbes to lethal low pH when they are exposed to . . Antagonistic yeast Postharvest Penicillium expansum mild acid conditions, or grown at moderately low pH. ATR can enhance the antagonist viability after a lethal acid shock. Cañamás et al. (2009) demonstrated that acid tolerance response induced the ability to survive under acidic conditions with a BCA Pantoea agglomerans. On the other hand, the BCAs are also exposed to acidic conditions, including acidic carbon Y. Wang S. He sources (Arneborg et al. 1995), fruit wound surface (Costa School of Perfume and Aroma Technology, Shanghai Institute of et al. 2002), and additives in formulations (Kren et al. 2003). Technology, Shanghai 201418, People’s Republic of China Furthermore, our group previously found salt-adapted R. : : : Y. Wang J. Xia T. Yu (*) X. Zheng (*) paludigenum had better survival and biocontrol acitivity Department of Food Science and Nutrition, Zhejiang University, after cold treatment (Wang et al. 2010c). To the best of our Hangzhou 310029, People’s Republic of China knowledge, there is no literature about mild acid stress on e-mail: yuting@zju.edu.cn antagonistic yeast survival under acidic conditions and its e-mail: xdzheng@zju.edu.cn 504 Ann Microbiol (2014) 64:503–508 effect on biocontrol efficacy. Consequently, the objectives of citric acids, respectively. The non-acidified NYDB medium this work were to assess the optimum conditions for acidic (pH 6.5) was used as a control. Each treatment (50 ml NYDB induction in the marine yeast R. paludigenum and the effec- in 250-ml flasks) was inoculated with 1 ml of R. tiveness of acid-adapted yeast in controlling Penicillium paludigenum suspensions at 1×10 cells/ml and incubated expansum on apple fruit. at 28 °C and 200 rpm for 48 h. Cultures were taken at 12, 24, 36, and 48 h, respectively, and recorded the absorbance at 600 nm and 25 °C (Spectra max plus 384; Molecular De- Materials and methods vices, USA). The experiments were replicated twice and each treatment was carried out in triplicate. Fruit material Acid tolerance response of R. paludigenum after a lethal acid Apples (Fushi cultivar), purchased from a local fruit market- shock place, were selected according to their uniformity of maturity and size. The fruit were stored until use (1 month at maxi- Rhodosporidium paludigenum was prepared as described mum) at −1 °C. Samples (without decay or apparent injuries) above. A 1-ml aliquot of the yeast cell suspension was were washed with running tap water, surface-sterilized in inoculated into a 250-ml flask containing 50 ml NYDB 0.1 % (v/v) sodium hypochlorite for 60 s, scoured under (pH 4.0 or 5.5 modified with malic and lactic acid, respec- tap water, and then air-dried. tively). The non-acidified medium was used as a control. After incubating at 28 °C for 36 h with shaking at 200 rpm, Pathogen acid-adapted or non-adapted R. paludigenum cultures (1 ml) were centrifuged at 7,000g for 10 min. For the lethal acid Penicillium expansum was obtained from the Institute of shock test, R. paludigenum cells were resuspended in deion- Microbiology, Chinese Academy of Sciences (Beijing, PR ized water (50 ml) with a low pH of 1.0, which was previ- China) and maintained at 4 °C on potato dextrose agar ously acidified with 1 M sulfuric acid. After 3 h exposure and (PDA) containing 200 ml extract of boiled potatoes, 20 g 200 rpm agitation, yeast suspensions diluted to an appropriate dextrose, and 20 g agar in 800 ml of distilled water. Conidial concentration were plating onto NYDA. R. paludigenum colo- suspensions (1-week-old mono-conidial grown at 28 °C on nies were counted after approximately 48 h incubation at 28 °C. PDA Petri dishes) were prepared by flooding and suspending The following formula was used to calculate the viability level in the sterile distilled water (SDW). The concentration of spore of R. paludigenum: viable cell counts after acid treatment/viable suspensions was determined by using a haemocytometer and cell counts without acid treatment×100 %. The experiments adjusted to about 1×10 spores/ml. were performed twice over time in three biological replicates (three plates/replicate). Yeast Effect of acid adaptation on biocontrol efficacy The yeast antagonist R. paludigenum (IMI 394084) was pre- of R. paludigenum viously isolated from marine water and identified by CABI Bioscience Identification Services (Wang et al. 2008). The In order to evaluate the inhibition of acid-adapted or non- strain was maintained at 4 °C on nutrient yeast dextrose agar adapted R. paludigenum against P. expansum, yeast sus- (NYDA) containing 8 g nutrient broth, 5 g yeast extract, 10 g pensions were prepared as described above. Two wounds dextrose, and 20 g agar in 1,000 ml distilled water. R. were made on the opposite sides of each apple with a sterile paludigenum were sub-cultured in 50 ml (in 250-ml flasks) cork-borer (5 mm×5 mm). Sample wounds were dipped with nutrient yeast dextrose broth (NYDB) and incubated at 28 °C aliquots (30 μl) of non-adapted (control) or malic or lactic and 200 rpm for 24 h. After centrifuging (KA1000, Shanghai acid-adapted R. paludigenum suspensions at 1×10 cells/ml, Anke) at 7,000 g for 10 min, R. paludigenum cells were respectively. Four hours later, 30 μlof a 1×10 spores/ml suspended in SDW and washed twice to remove nutritional suspension of P. expansum were inoculated into each wound. compounds from the initial medium. Yeast suspension with The fruit were sealed up in enclosed plastic boxes (approxi- expected concentration was obtained by adjusting with SDW mately 90 % relative humidity) at 25 °C after being air-dried. and a haemocytometer prior to use. The disease incidences (percentages of infected fruit wounds) and corresponding lesion diameters (the mean Screening of acidic media of the diameter perpendicular) were examined after 3 days postharvest inoculation. The experiments were performed The initial pH levels of 4.0, 4.5, 5.0, 5.5, and 6.0 was twice over time in three biological replicates (20 modified with proper amounts of acetic, lactic, malic, and samples/replicate). Ann Microbiol (2014) 64:503–508 505 Effect of acid adaptation on R. paludigenum population conducted by using the software Statistical Analysis Systems dynamics in the apple wounds (SAS v.8.0, SAS Institute, Cary, NC, USA). Homogeneity of variances was improved by log-transforming microbial pop- Apples and R. paludigenum suspensions were prepared as ulations of antagonist (cells/wound) and the viable counts described above. An aliquot of 30 μl non-adapted or acid- before being analyzed. adapted yeast suspensions (1×10 cells/ml) was inoculated into each wound. After being air-dried, fruits were stored at 25 °C in plastic trays lined with polythene. Samples were Results taken after 0, 24, and 48 h incubation. The wounded tissues were removed by using another sterile punch (10 mm Screening of acidic media diameter×10 mm deep), ground with a mortar, and then suspended in 10 ml of SDW. R. paludigenum populations The effects of different acidic solutes on the R. paludigenum were counted with a hemacytometer. The experiments were survival are shown in Fig. 1. In the acetic acid treatments, the performed twice over time in three biological replicates (6 initial pH of 4.0 and 4.5 limited the growth of yeast during samples/replicate). the first 36 h of incubation (Fig. 1a). The growths of R. paludigenum in all citric acidified medium were similar with Data analysis that of control (Fig. 1b). As shown in Fig. 1c, d, the growth of R. paludigenum was stimulated in malic and lactic acidi- One-way ANOVA was performed to analyze all data and the fied NYDB with the pH value of 4.0 and 5.5. |n addition, the significance of differences between means was tested by stimulating effect was more obvious at the initial pH value of Duncan’s multiple range tests at p<0.05. The analysis was 4.0 at 36 h. Thus, the NYDB media containing malic and 0.8 0.8 (a) (b) 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0.0 0.0 0 1224364860 012 24 36 48 0.8 0.8 (c) (d) 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0.0 0.0 012 24 36 48 012 24 36 48 Time (h) Time (h) Control pH 4.0 pH 4.5 pH 5.0 pH 5.5 pH 6.0 Fig. 1 Growth curves of R. paludigenum in medium at different initial pH level modified by using: a acetic acid, b citric acid, c malic acid, and d lactic acid. Vertical bars SEM OD OD OD OD 600 600 506 Ann Microbiol (2014) 64:503–508 lactic acid (pH=4.0 and 5.5), respectively, were selected for and non-adapted treatments during the storage. After 48 h the subsequent tests. incubation, yeast populations in all wounds reached an ap- proximately 150-fold increase compared with the initial Acid tolerance response of R. paludigenum after a lethal acid number. shock The viability levels of non-adapted and acid-adapted R. Discussion paludigenum were presented in Fig. 2. Both malic and lactic acid treatments significantly enhanced the acid tolerance in A key factor in the commercialization of antagonist is the R. paludigenum compared with the control (p<0.05). Studies stress tolerance under variable conditions (Teixidó et al. after 3 h lethal shock showed that the viability of non- 1998). To evaluate the effect of initial pH level, R. induced R. paludigenum declined rapidly from 53.2 to paludigenum was cultured in medium containing different 26.3 %, either through combined 4.0 pH and lactic acid organic acids, since these materials have shown to induce treatment or combined 4.0 pH and malic acid treatment. acid tolerance response of bacteria (Chorianopoulos et al. The best growth of R. paludigenum (91.6 %) was obtained 2011). In the current work, acid tolerance response of R. at pH of 5.5 when malic acid was used. However, at the pH paludigenum depended on the acid solutes. The medium value of 5.5, there was no significant survival difference modified with both lactic and malic acid were efficient at between malic and lactic acid treatments. inducing acid tolerance in R. paludigenum. Interestingly, in another case of antagonistic bacteria P. agglomerans CPA-2, Effect of acid adaptation on biocontrol efficacy acid tolerance was induced with citric and malic acid at of R. paludigenum initial pH of 5.0 (Cañamás et al. 2009). These results sug- gested that the differences of acid tolerance response As shown Fig. 3, all R. paludigenum treatments significantly depended on a factor as medium. Also, medium modified reduced the disease incidences and lesion diameters after with acetic acid reduced R. paludigenum, which might be 5 days incubation at 25 °C. Furthermore, acidified medium related to its effect on the intracellular pH of yeast cells had no negative influence on the biocontrol activity of R. (Stratford 1999). paludigenum. Furthermore, the influences of initial pH value on acid tolerance response have been reported (Wong et al. 1998; Effect of acid adaptation on population dynamics Ravishankar and Harrison 1999; Greenacre et al. 2003). It of R. paludigenum in apple wounds was found that a pH value from 4.0 to 5.5 in the medium improved P. agglomerans survival rates (Cañamás et al. As presented in Fig. 4, there was no statistical difference in 2009). Chorianopoulos et al. (2011) reported that acid- R. paludigenum growth in wounds between acid-adapted adapted Listeria monocytogenes had higher log-reductions compared to non-acid-adapted ones exposured to strong acid shock (pH 2.0) with either hydrocholric or lactic acid. A 100 a similar result was found in this work that acid-adapted R. paludigenum at initial pH levels of 4.0 or 5.5 showed higher viabilities than non-acid-adapted ones after 3 h exposure to strong acid challenges. And a pH value of 5.5 was the best induction condition. This result implied that the initial pH was 40 another important factor in acid tolerance response. Addition- ally, our previously work found that R. paludigenum showed little bicontrol activity in controlling natural decay of some Chinese traditional fruit, such as waxberry (Myrica rubra)and apricot (Armeniaca vulgaris) (data not shown), which might NYDB NMA4 NMA5 NLA4 NLA5 be due to its high total acid content (Huang et al. 2012). The Treatment effects of acid-adapted R. paludigenum in controlling Fig. 2 Viability of nonacid- and acid-adapted R. paludigenum at 28 °C postharvest disease of waxberry are underway. for 36 h. NMA4 or NMA5 medium acidified with malic acid at pH of 4.0 or 5.5. NLA4 or NLA5 medium acidified with lactic acid at pH of 4.0 or The influence of acid tolerance response of yeast on the 5.5. Data are mean values. The bars labeled with at least one common efficacy of a biological control agent is also required after letter were not significantly different according to Duncan’s Multiple application in the fruit (Wang et al. 2010c). In the second part Range Test at p<0.05. The results from individual experiments are in of this work, results obtained in the in vivo test demonstrated high accordance; hence, data shown are from one representative experiment that there was no significant difference among malic and Viability (%) Ann Microbiol (2014) 64:503–508 507 (a) Control NYDB NMA4 NMA5 Control NYDB NMA4 NMA5 Treatment Treatment (b) 4 a 20 b 2 Control NYDB NLA4 NLA5 Control NYDB NLA4 NLA5 Treatment Treatment Fig. 3 Biocontrol effects of nonacid- and acid-adapted R. paludigenum 5.5. Data are mean values. The bars labeled at least one common letter on disease incidence (a) and lesion diameter (b) against P. expansum on were not significantly different according to Duncan’s Multiple Range apples after 5 days incubation at 25 °C. The control was treated with Test at p<0.05. The results from individual experiments are in high SDW. NMA4 or NMA5 medium acidified with malic acid at pH of 4.0 or accordance; hence, data shown are from one representative experiment 5.5. NLA4 or NLA5 medium acidified with lactic acid at pH of 4.0 or NYDB lactic acid-adapted and non-adapted yeast-treated apples in NMA4 disease incidence and lesion diameter against P. expansum. NMA5 Furthermore, the population growth in non-acid treatment NLA4 a a was also not influenced by the pH level in the apple wounds. NLA5 a a a Similar results were reported by Cañamás et al. (2009) that a a acid adaptation only protected Pantoea agglomerans against lethal acid shocks, suggesting that acid tolerance only pro- tects antagonist cells against lethal pH conditions. These results could also be explained by the complex environment in the fruit wounds. On the other hand, orange, mandarin, apple, and pear fruit exhibit different pH levels, from 3.5 to 4.6. BCAs suffer other environmental circumstances during the formulation, transport, and fruit storage period. Further 024 48 research will need to be conducted to establish the survival Time (h) level of non-adapted and acid-adapted yeast in these adverse Fig. 4 Population dynamics of non- and acid-adapted R. paludigenum in conditions. the apple wounds. NMA4 or NMA5 medium acidified with malic acid at pH of 4.0 or 5.5. NLA4 or NLA5 medium acidified with lactic acid at pH of 4.0 Antagonistic yeasts are more acceptable from the food or 5.5. Data are mean values. The bars labeled at least one common letter safety point of view. Moreover, the results reported here were not significantly different according to Duncan’s Multiple Range Test imply the potential for improving the stress tolerance of at p<0.05. The results from individual experiments are in high accordance; BCAs by physiological inducement strategy. hence, data shown are from one representative experiment Population (log cells per wound) Disease incidence (%) Disease incidence (%) Lesion diameters (mm) Lesion diameter (mm) 508 Ann Microbiol (2014) 64:503–508 Acknowledgments This research was supported by grants from the Ravishankar S, Harrison MA (1999) Acid adaptation ofListeria National Natural Science Foundation of China (31271962) and Ph.D. Pro- monocytogenes strains does not offer cross-protectionagainst an grams Foundation of Ministry of Education of China (20100101110087) activated lactoperoxidase system. JFood Protect 62:670–673 and Program for Key Innovative Research Team of Zhejiang Province Stratford M (1999) Traditional preservatives – organic acids. In: Rob- (2009R50036) and the Special Fund for Agro-scientific Research in the inson RK, Batt CA, Patel PD (eds) Encyclopedia of Food Micro- Public Interest of China (200903044). biology vol 3. Academic, London, pp 1729–1737 Teixidó N, Vinas I, Usall J, Magan N (1998) Improving ecological fitness and environmental stress tolerance of the biocontrol yeast Candida sake by manipulation of intracellular sugar alcohol and References sugar content. Mycol Res 102:1409–1417 Teixidó N, Canamas TP, Usall J, Torres R, Magan N, Vinas I (2005) Accumulation of the compatible solutes, glycine-betaine and Arneborg H, Moos N, Jakobsen M (1995) The effect of acetic acid and ectoine, in osmotic stress adaptation and heat shock cross- specific growth rate on acetic acid tolerance and trehalose content protection in the biocontrol agent Pantoea agglomerans CPA-2. of Saccharomyces cerevisiae. Biotechnol Lett 17:1299–1304 Lett Appl Microbiol 41:248–252 Cañamás PT, Viñas I, Abadias M, Usall J, Torres R, Teixidó N (2009) Teixidó N, Canamas TP, Abadiasm M, Usall J, Solsona C, Casals C, Acid tolerance response induced in the biocontrol agent Pantoea Vinas I (2006) Improving low water activity and desiccation agglomerans CPA-2 and effect on its survival ability in acidic tolerance of the biocontrol agent Pantoea agglomerans CPA-2 environments. Microbiol Res 164:438–450 by osmotic treatments. J Appl Microbiol 101:927–937 Chorianopoulos N, Giaouris E, Grigoraki I, Skandamis P, Nychas G Wang Y, Bao Y, Shen D, Feng W, Yu T, Zhang J, Zheng X (2008) (2011) Effect of acid tolerance response (ATR) on attachment of Biocontrol of Alternaria alternata on cherry tomato fruit by use of Listeria monocytogenes Scott A to stainless steel under extended marine yeast Rhodosporidium paludigenum Fell & Tallman. Int J exposure to acid or/and salt stress and resistance of sessile cells to Food Microbiol 123:234–239 subsequent strong acid challenge. Int J Food Microbiol 145:400–406 Wang Y, Yu T, Xia J, Yu D, Wang J, Zheng X (2010a) Biocontrol of Costa E, Usall J, Teixidó N, Delgado J, Viñas I (2002) Water activity, postharvest gray mold of cherry tomatoes with the marine yeast temperature, and pH effects on growth of the biocontrol agent Rhodosporidium paludigenum. Biol Control 53:178–182 Pantoea agglomerans CPA-2. Can J Microbiol 48:1082–1088 Wang Y, Ren X, Song X, Yu T, Lu H, Wang P, Wang J, Zheng X (2010b) Greenacre EJ, Brocklehurst TF, Waspe CR, Wilson DR, Wilson PDG (2003) Control of postharvest decay on cherry tomatoes by marine yeast Salmonella enterica serovar Typhimuriumand Listeria monocytogenes Rhodosporidium paludigenum and calcium chloride. J Appl acid toleranceresponse induced by organic acids at 20 °C: optimiza- Microbiol 109:651–656 tionand modeling. Appl Environ Microbiol 69:3945–3951 Wang Y, Wang P, Xia J, Yu T, Lou B, Wang J, Zheng X (2010c) Effect Huang H, Fu G, Yang Z, Zhang J, Jin Z (2012) Effect of protected of water activity on stress tolerance and biocontrol activity in cultivation to the development and quality of Myrica rubra.J antagonistic yeast Rhodosporidium paludigenum. Int J Food Nanjing For Univ 36:47–52 Microbiol 143:103–108 Kren A, Mamnun YM, Bauer BE, Schüller C, Wolfger H, Hatzixanthis Wang Y, Tang F, Xia J, Yu T, Wang J, Azhati R, Zheng X (2011) A K, Mollapour M, Gregori C, Piper P, Kuchler K (2003) War1p, a combination of marine yeast and food additive enhances preven- novel transcription factor controlling weak acid stress response in tive effects on postharvest decay of jujubes (Zizyphus jujuba). yeast. Mol Cell Biol 23:1775–1785 Food Chem 125:835–840 Lahlali R, Jijakli M (2009) Enhancement of the biocontrol agent Can- Wong H-C, Peng P-Y, Han J-M, Chang C-Y, Lan S-L (1998) Effect of dida olephila (strain O) survival and control efficiency under mild acid treatment onthe survival, enteropathogenicity, and protein extreme conditions of water activity and relative humidity. Biol productionin Vibrio parahaemolyticus. Infect Immun 66:3066–3071 Control 51:403–408 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Acid adaptation and biocontrol efficacy of antagonistic marine yeast Rhodosporidium paludigenum

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Springer Journals
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Copyright © 2013 by Springer-Verlag Berlin Heidelberg and the University of Milan
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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Abstract

Ann Microbiol (2014) 64:503–508 DOI 10.1007/s13213-013-0681-2 ORIGINAL ARTICLE Acid adaptation and biocontrol efficacy of antagonistic marine yeast Rhodosporidium paludigenum Yifei Wang & Shoukui He & Jindan Xia & Ting Yu & Xiaodong Zheng Received: 21 September 2012 /Accepted: 24 June 2013 /Published online: 11 July 2013 # Springer-Verlag Berlin Heidelberg and the University of Milan 2013 Abstract The objectives of this work were to assess the Introduction optimum conditions for induction of acid tolerance in the marine yeast Rhodosporidium paludigenum and evaluate the The marine yeast Rhodosporidium paludigenum was previ- biocontrol activity of non-adapted and acid-adapted yeasts in ously isolated from the East China Sea and exhibited its controlling apple blue mold caused by Penicillium expansum. antimicrobial activity against Alternaria alternata and Bo- R. paludigenum grown in malic and lactic acid treatments trytis cinerea, which are two major postharvest pathogens of were stimulated after 12 h incubation. Moreover, medium cherry tomatoes and Chinese winter jujubes in China (Wang modified with malic and lactic acid significantly enhanced et al. 2010a, 2011). Its biocontrol efficacy was also found to the acid tolerance of R. paludigenum (p<0.05). In acid toler- be improved in combination with calcium chloride (Wang ance response test, the highest viability of R. paludigenum et al. 2010b). Previous studies showed that competition for was obtained at initial pH of 5.5 in the NYDB medium nutrients is an important antagonist mechanism of R. modified with malic acid (91.6 %). In addition, all R. paludigenum (Wang et al. 2008). paludigenum treatments significantly reduced the disease in- Before applying to fruits, antagonists can be exposed to cidences and lesion diameters of blue mold in apples. Further- variable environments. A number of biotic or abiotic factors more, there was no significant difference between acid- reduce the efficacy and stability of biocontrol agents adapted and unadapted yeasts in the apple wounds after 48 h (BCAs). Recently, several researchers have shown that salt dynamics. Acid stress improved R. paludigenum viability adaptation of yeasts (Lahlali and Jijakli 2009) and bacteria under acidic conditions. However, there was no significant (Teixidó et al. 2005, 2006) may have promising potentials to difference between acid-adapted and unadapted yeasts in con- develop industrial formulation of antagonists. Hence, to be trolling P. expansum on apple fruit (p<0.05). These results successful in the food industry, stress tolerance of BCA must indicate the potential for maintaining the survival level of be enhanced in order to extend shelf life and maintain bio- biocontrol agents by physiological inducement strategy. control activity (Lahlali and Jijakli 2009). Acid tolerance response (ATR) can be defined as the resis- . . Keywords Acid tolerance response Biocontrol tance of microbes to lethal low pH when they are exposed to . . Antagonistic yeast Postharvest Penicillium expansum mild acid conditions, or grown at moderately low pH. ATR can enhance the antagonist viability after a lethal acid shock. Cañamás et al. (2009) demonstrated that acid tolerance response induced the ability to survive under acidic conditions with a BCA Pantoea agglomerans. On the other hand, the BCAs are also exposed to acidic conditions, including acidic carbon Y. Wang S. He sources (Arneborg et al. 1995), fruit wound surface (Costa School of Perfume and Aroma Technology, Shanghai Institute of et al. 2002), and additives in formulations (Kren et al. 2003). Technology, Shanghai 201418, People’s Republic of China Furthermore, our group previously found salt-adapted R. : : : Y. Wang J. Xia T. Yu (*) X. Zheng (*) paludigenum had better survival and biocontrol acitivity Department of Food Science and Nutrition, Zhejiang University, after cold treatment (Wang et al. 2010c). To the best of our Hangzhou 310029, People’s Republic of China knowledge, there is no literature about mild acid stress on e-mail: yuting@zju.edu.cn antagonistic yeast survival under acidic conditions and its e-mail: xdzheng@zju.edu.cn 504 Ann Microbiol (2014) 64:503–508 effect on biocontrol efficacy. Consequently, the objectives of citric acids, respectively. The non-acidified NYDB medium this work were to assess the optimum conditions for acidic (pH 6.5) was used as a control. Each treatment (50 ml NYDB induction in the marine yeast R. paludigenum and the effec- in 250-ml flasks) was inoculated with 1 ml of R. tiveness of acid-adapted yeast in controlling Penicillium paludigenum suspensions at 1×10 cells/ml and incubated expansum on apple fruit. at 28 °C and 200 rpm for 48 h. Cultures were taken at 12, 24, 36, and 48 h, respectively, and recorded the absorbance at 600 nm and 25 °C (Spectra max plus 384; Molecular De- Materials and methods vices, USA). The experiments were replicated twice and each treatment was carried out in triplicate. Fruit material Acid tolerance response of R. paludigenum after a lethal acid Apples (Fushi cultivar), purchased from a local fruit market- shock place, were selected according to their uniformity of maturity and size. The fruit were stored until use (1 month at maxi- Rhodosporidium paludigenum was prepared as described mum) at −1 °C. Samples (without decay or apparent injuries) above. A 1-ml aliquot of the yeast cell suspension was were washed with running tap water, surface-sterilized in inoculated into a 250-ml flask containing 50 ml NYDB 0.1 % (v/v) sodium hypochlorite for 60 s, scoured under (pH 4.0 or 5.5 modified with malic and lactic acid, respec- tap water, and then air-dried. tively). The non-acidified medium was used as a control. After incubating at 28 °C for 36 h with shaking at 200 rpm, Pathogen acid-adapted or non-adapted R. paludigenum cultures (1 ml) were centrifuged at 7,000g for 10 min. For the lethal acid Penicillium expansum was obtained from the Institute of shock test, R. paludigenum cells were resuspended in deion- Microbiology, Chinese Academy of Sciences (Beijing, PR ized water (50 ml) with a low pH of 1.0, which was previ- China) and maintained at 4 °C on potato dextrose agar ously acidified with 1 M sulfuric acid. After 3 h exposure and (PDA) containing 200 ml extract of boiled potatoes, 20 g 200 rpm agitation, yeast suspensions diluted to an appropriate dextrose, and 20 g agar in 800 ml of distilled water. Conidial concentration were plating onto NYDA. R. paludigenum colo- suspensions (1-week-old mono-conidial grown at 28 °C on nies were counted after approximately 48 h incubation at 28 °C. PDA Petri dishes) were prepared by flooding and suspending The following formula was used to calculate the viability level in the sterile distilled water (SDW). The concentration of spore of R. paludigenum: viable cell counts after acid treatment/viable suspensions was determined by using a haemocytometer and cell counts without acid treatment×100 %. The experiments adjusted to about 1×10 spores/ml. were performed twice over time in three biological replicates (three plates/replicate). Yeast Effect of acid adaptation on biocontrol efficacy The yeast antagonist R. paludigenum (IMI 394084) was pre- of R. paludigenum viously isolated from marine water and identified by CABI Bioscience Identification Services (Wang et al. 2008). The In order to evaluate the inhibition of acid-adapted or non- strain was maintained at 4 °C on nutrient yeast dextrose agar adapted R. paludigenum against P. expansum, yeast sus- (NYDA) containing 8 g nutrient broth, 5 g yeast extract, 10 g pensions were prepared as described above. Two wounds dextrose, and 20 g agar in 1,000 ml distilled water. R. were made on the opposite sides of each apple with a sterile paludigenum were sub-cultured in 50 ml (in 250-ml flasks) cork-borer (5 mm×5 mm). Sample wounds were dipped with nutrient yeast dextrose broth (NYDB) and incubated at 28 °C aliquots (30 μl) of non-adapted (control) or malic or lactic and 200 rpm for 24 h. After centrifuging (KA1000, Shanghai acid-adapted R. paludigenum suspensions at 1×10 cells/ml, Anke) at 7,000 g for 10 min, R. paludigenum cells were respectively. Four hours later, 30 μlof a 1×10 spores/ml suspended in SDW and washed twice to remove nutritional suspension of P. expansum were inoculated into each wound. compounds from the initial medium. Yeast suspension with The fruit were sealed up in enclosed plastic boxes (approxi- expected concentration was obtained by adjusting with SDW mately 90 % relative humidity) at 25 °C after being air-dried. and a haemocytometer prior to use. The disease incidences (percentages of infected fruit wounds) and corresponding lesion diameters (the mean Screening of acidic media of the diameter perpendicular) were examined after 3 days postharvest inoculation. The experiments were performed The initial pH levels of 4.0, 4.5, 5.0, 5.5, and 6.0 was twice over time in three biological replicates (20 modified with proper amounts of acetic, lactic, malic, and samples/replicate). Ann Microbiol (2014) 64:503–508 505 Effect of acid adaptation on R. paludigenum population conducted by using the software Statistical Analysis Systems dynamics in the apple wounds (SAS v.8.0, SAS Institute, Cary, NC, USA). Homogeneity of variances was improved by log-transforming microbial pop- Apples and R. paludigenum suspensions were prepared as ulations of antagonist (cells/wound) and the viable counts described above. An aliquot of 30 μl non-adapted or acid- before being analyzed. adapted yeast suspensions (1×10 cells/ml) was inoculated into each wound. After being air-dried, fruits were stored at 25 °C in plastic trays lined with polythene. Samples were Results taken after 0, 24, and 48 h incubation. The wounded tissues were removed by using another sterile punch (10 mm Screening of acidic media diameter×10 mm deep), ground with a mortar, and then suspended in 10 ml of SDW. R. paludigenum populations The effects of different acidic solutes on the R. paludigenum were counted with a hemacytometer. The experiments were survival are shown in Fig. 1. In the acetic acid treatments, the performed twice over time in three biological replicates (6 initial pH of 4.0 and 4.5 limited the growth of yeast during samples/replicate). the first 36 h of incubation (Fig. 1a). The growths of R. paludigenum in all citric acidified medium were similar with Data analysis that of control (Fig. 1b). As shown in Fig. 1c, d, the growth of R. paludigenum was stimulated in malic and lactic acidi- One-way ANOVA was performed to analyze all data and the fied NYDB with the pH value of 4.0 and 5.5. |n addition, the significance of differences between means was tested by stimulating effect was more obvious at the initial pH value of Duncan’s multiple range tests at p<0.05. The analysis was 4.0 at 36 h. Thus, the NYDB media containing malic and 0.8 0.8 (a) (b) 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0.0 0.0 0 1224364860 012 24 36 48 0.8 0.8 (c) (d) 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0.0 0.0 012 24 36 48 012 24 36 48 Time (h) Time (h) Control pH 4.0 pH 4.5 pH 5.0 pH 5.5 pH 6.0 Fig. 1 Growth curves of R. paludigenum in medium at different initial pH level modified by using: a acetic acid, b citric acid, c malic acid, and d lactic acid. Vertical bars SEM OD OD OD OD 600 600 506 Ann Microbiol (2014) 64:503–508 lactic acid (pH=4.0 and 5.5), respectively, were selected for and non-adapted treatments during the storage. After 48 h the subsequent tests. incubation, yeast populations in all wounds reached an ap- proximately 150-fold increase compared with the initial Acid tolerance response of R. paludigenum after a lethal acid number. shock The viability levels of non-adapted and acid-adapted R. Discussion paludigenum were presented in Fig. 2. Both malic and lactic acid treatments significantly enhanced the acid tolerance in A key factor in the commercialization of antagonist is the R. paludigenum compared with the control (p<0.05). Studies stress tolerance under variable conditions (Teixidó et al. after 3 h lethal shock showed that the viability of non- 1998). To evaluate the effect of initial pH level, R. induced R. paludigenum declined rapidly from 53.2 to paludigenum was cultured in medium containing different 26.3 %, either through combined 4.0 pH and lactic acid organic acids, since these materials have shown to induce treatment or combined 4.0 pH and malic acid treatment. acid tolerance response of bacteria (Chorianopoulos et al. The best growth of R. paludigenum (91.6 %) was obtained 2011). In the current work, acid tolerance response of R. at pH of 5.5 when malic acid was used. However, at the pH paludigenum depended on the acid solutes. The medium value of 5.5, there was no significant survival difference modified with both lactic and malic acid were efficient at between malic and lactic acid treatments. inducing acid tolerance in R. paludigenum. Interestingly, in another case of antagonistic bacteria P. agglomerans CPA-2, Effect of acid adaptation on biocontrol efficacy acid tolerance was induced with citric and malic acid at of R. paludigenum initial pH of 5.0 (Cañamás et al. 2009). These results sug- gested that the differences of acid tolerance response As shown Fig. 3, all R. paludigenum treatments significantly depended on a factor as medium. Also, medium modified reduced the disease incidences and lesion diameters after with acetic acid reduced R. paludigenum, which might be 5 days incubation at 25 °C. Furthermore, acidified medium related to its effect on the intracellular pH of yeast cells had no negative influence on the biocontrol activity of R. (Stratford 1999). paludigenum. Furthermore, the influences of initial pH value on acid tolerance response have been reported (Wong et al. 1998; Effect of acid adaptation on population dynamics Ravishankar and Harrison 1999; Greenacre et al. 2003). It of R. paludigenum in apple wounds was found that a pH value from 4.0 to 5.5 in the medium improved P. agglomerans survival rates (Cañamás et al. As presented in Fig. 4, there was no statistical difference in 2009). Chorianopoulos et al. (2011) reported that acid- R. paludigenum growth in wounds between acid-adapted adapted Listeria monocytogenes had higher log-reductions compared to non-acid-adapted ones exposured to strong acid shock (pH 2.0) with either hydrocholric or lactic acid. A 100 a similar result was found in this work that acid-adapted R. paludigenum at initial pH levels of 4.0 or 5.5 showed higher viabilities than non-acid-adapted ones after 3 h exposure to strong acid challenges. And a pH value of 5.5 was the best induction condition. This result implied that the initial pH was 40 another important factor in acid tolerance response. Addition- ally, our previously work found that R. paludigenum showed little bicontrol activity in controlling natural decay of some Chinese traditional fruit, such as waxberry (Myrica rubra)and apricot (Armeniaca vulgaris) (data not shown), which might NYDB NMA4 NMA5 NLA4 NLA5 be due to its high total acid content (Huang et al. 2012). The Treatment effects of acid-adapted R. paludigenum in controlling Fig. 2 Viability of nonacid- and acid-adapted R. paludigenum at 28 °C postharvest disease of waxberry are underway. for 36 h. NMA4 or NMA5 medium acidified with malic acid at pH of 4.0 or 5.5. NLA4 or NLA5 medium acidified with lactic acid at pH of 4.0 or The influence of acid tolerance response of yeast on the 5.5. Data are mean values. The bars labeled with at least one common efficacy of a biological control agent is also required after letter were not significantly different according to Duncan’s Multiple application in the fruit (Wang et al. 2010c). In the second part Range Test at p<0.05. The results from individual experiments are in of this work, results obtained in the in vivo test demonstrated high accordance; hence, data shown are from one representative experiment that there was no significant difference among malic and Viability (%) Ann Microbiol (2014) 64:503–508 507 (a) Control NYDB NMA4 NMA5 Control NYDB NMA4 NMA5 Treatment Treatment (b) 4 a 20 b 2 Control NYDB NLA4 NLA5 Control NYDB NLA4 NLA5 Treatment Treatment Fig. 3 Biocontrol effects of nonacid- and acid-adapted R. paludigenum 5.5. Data are mean values. The bars labeled at least one common letter on disease incidence (a) and lesion diameter (b) against P. expansum on were not significantly different according to Duncan’s Multiple Range apples after 5 days incubation at 25 °C. The control was treated with Test at p<0.05. The results from individual experiments are in high SDW. NMA4 or NMA5 medium acidified with malic acid at pH of 4.0 or accordance; hence, data shown are from one representative experiment 5.5. NLA4 or NLA5 medium acidified with lactic acid at pH of 4.0 or NYDB lactic acid-adapted and non-adapted yeast-treated apples in NMA4 disease incidence and lesion diameter against P. expansum. NMA5 Furthermore, the population growth in non-acid treatment NLA4 a a was also not influenced by the pH level in the apple wounds. NLA5 a a a Similar results were reported by Cañamás et al. (2009) that a a acid adaptation only protected Pantoea agglomerans against lethal acid shocks, suggesting that acid tolerance only pro- tects antagonist cells against lethal pH conditions. These results could also be explained by the complex environment in the fruit wounds. On the other hand, orange, mandarin, apple, and pear fruit exhibit different pH levels, from 3.5 to 4.6. BCAs suffer other environmental circumstances during the formulation, transport, and fruit storage period. Further 024 48 research will need to be conducted to establish the survival Time (h) level of non-adapted and acid-adapted yeast in these adverse Fig. 4 Population dynamics of non- and acid-adapted R. paludigenum in conditions. the apple wounds. NMA4 or NMA5 medium acidified with malic acid at pH of 4.0 or 5.5. NLA4 or NLA5 medium acidified with lactic acid at pH of 4.0 Antagonistic yeasts are more acceptable from the food or 5.5. Data are mean values. The bars labeled at least one common letter safety point of view. Moreover, the results reported here were not significantly different according to Duncan’s Multiple Range Test imply the potential for improving the stress tolerance of at p<0.05. The results from individual experiments are in high accordance; BCAs by physiological inducement strategy. hence, data shown are from one representative experiment Population (log cells per wound) Disease incidence (%) Disease incidence (%) Lesion diameters (mm) Lesion diameter (mm) 508 Ann Microbiol (2014) 64:503–508 Acknowledgments This research was supported by grants from the Ravishankar S, Harrison MA (1999) Acid adaptation ofListeria National Natural Science Foundation of China (31271962) and Ph.D. Pro- monocytogenes strains does not offer cross-protectionagainst an grams Foundation of Ministry of Education of China (20100101110087) activated lactoperoxidase system. 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Annals of Microbiology – Springer Journals

Published: Jul 11, 2013

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