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Postharvest biological control of gray mold decay of strawberry with Rhodotorula glutinis
Biological Control 40 (2007) 287–292 www.elsevier.com/locate/ybcon
Postharvest biological control of gray mold decay of strawberry with Rhodotorula glutinis Hongyin Zhang ¤, Lei Wang, Ying Dong, Song Jiang, Jian Cao, Rujie Meng College of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, People’s Republic of China Received 22 July 2006; accepted 18 October 2006 Available online 10 November 2006
Abstract Rhodotorula glutinis was evaluated for its activity in reducing postharvest gray mold decay of strawberry caused by Botrytis cinerea in vitro and in vivo. In the test on PDA plates, R. glutinis signiWcantly inhibit the growth of B. cinerea. Spore germination of pathogens in PDB was greatly controlled in the presence of living cell suspensions. Rapid colonization of the yeast in wounds was observed during the Wrst 3 days at 20 °C, and then the populations stabilized for the remaining storage period. On strawberry wounds kept at 4 °C, the increase in population density of R. glutinis was lower than those kept at 20 °C, but continued over 8 days after application of the antagonist until it reached a high level. Number of inoculated strawberry fruit treated with 1 £ 108 CFU/ml washed cell suspension of R. glutinis was 10% after 2 days at 20 °C, compared to 100%, respectively, in the control. Washed cell suspensions of yeast controlled gray mold better than yeast in culture broth. Treatment of wounds with autoclaved cell cultures or cell-free culture Wltrate did not prevent decay. The concentrations of antagonist had signiWcant eVects on biocontrol eVectiveness: the higher the concentrations of the antagonist, the lower the disease incidence regardless of whether the fruit was stored at 20 °C for 2 days or 4 °C for 7 days. At concentrations of R. glutinis 1 £ 109 CFU/ml, the incidence of gray mold was reduced by 94.7 or 95%, respectively, compared with control, after storage at 20 °C for 2 days or 4 °C for 7 days, respectively. R. glutinis signiWcantly reduced the natural development of decay of fruit following storage at 20 °C for 3 days or 4 °C for 5 days followed by 20 °C for 3 days. © 2006 Elsevier Inc. All rights reserved. Keywords: Strawberry; Gray mold; Postharvest decay; Biocontrol; Rhodotorula glutinis
1. Introduction Strawberry fruit have a very short postharvest life, due in part to gray mold caused by Botrytis cinerea Pers.:Fr. (Wszelaki and Mitcham, 2003). Infection may occur in the Xower, remain quiescent until fruits mature, and then develop abundantly, causing fruit decay accompanied by profuse sporulation of the pathogen (Kovach et al., 2000). B. cinerea also causes signiWcant losses during shipping and marketing (Ceponis et al., 1987), making it one of the most economically important pathogens of strawberry (Mertely et al., 2002; Romanazzi et al., 2001). Control of B. cinerea is normally carried out by the application of
*
Corresponding author. Fax: +86 511 86971139. E-mail address: [email protected] (H. Zhang).
1049-9644/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2006.10.008
fungicides. Although fungicides use reduces yield losses due to Botrytis in strawberries, there are studies that show that commercially available fungicides can reduce pollination and cause fruit malformation (Kovach et al., 2000). Fungicide eYcacy is frequently decreased by the development of resistant strains of pathogens. In addition, public concern and regulatory restrictions about the presence of fungicide residues on crops have emphasized the need to Wnd alternative methods for disease control (Smilanick, 1994). Microbial biocontrol agents have shown great potential as an alternative to synthetic fungicides for the control of postharvest decay of fruits and vegetables (Wisniewski and Wilson, 1992). Several biological control agents are eVective in reducing decay caused by gray mold on strawberry (Peng and Sutton, 1991; Swadling and JeVries, 1996; Lima et al., 1997; Guinebretiere et al., 2000).
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H. Zhang et al. / Biological Control 40 (2007) 287–292
However, few of these have been tested under postharvest storage conditions. The objective of the present study was to study the postharvest control of gray mold of strawberry by Rhodotorula glutinis and determine: (1) biocontrol in vitro; (2) the population dynamics of R. glutinis in wounds; (3) eYcacy of R. glutinis in controlling of gray mold decay of strawberry; (4) eVect of concentration of R. glutinis on biocontrol eYcacy at ambient temperatures and cold storage condition; (5) eYcacy of R. glutinis in controlling of natural decay development of strawberry. 2. Materials and methods 2.1. Pathogen inoculum Botrytis cinerea Pers.: Fr. was isolated from infected strawberry fruits. The culture was maintained on potatodextrose agar medium (PDA: extract of boiled potatoes, 200 ml; dextrose, 20 g; agar, 20 g and distilled water, 800 ml) at 4 °C; fresh cultures were grown on PDA plates at 25 °C before use. Spore suspensions were prepared by removing the spores from the sporulating edges of a 10-day-old culture with a bacteriological loop, and suspending them in 5 ml of sterile distilled water. Suspensions were Wltered through four layers of cheesecloth to remove fungal mycelium and spore concentrations were determined with a hemocytometer with the concentration being adjusted as required for diVerent experiments by adding sterile distilled water. 2.2. Antagonist The yeast antagonist R. glutinis (Fresenins) Harrison was isolated from the surfaces of strawberries harvested in unsprayed orchards and identiWed by VITEK 32 Automicrobic system (bioMérieux Company, Marcy l’Etoile, France). R. glutinis isolates were maintained at 4 °C on Nutrient Yeast Dextrose Agar (NYDA) medium containing 8 g nutrient broth, 5 g yeast extract, 10 g glucose, and 20 g agar, in 1 L of distilled water. Liquid cultures of the yeast were grown in 250-ml Erlenmeyer Xasks containing 50 ml of NYD Broth (NYDB) which had been inoculated with a loop of the culture. Flasks were incubated on a rotary shaker at 28 °C for 20 h. Following incubation, cells were centrifuged at 5000g for 10 min and washed twice with sterile distilled water in order to remove the growth medium. Cell pellets were re-suspended in sterile distilled water and adjusted to an initial concentration of 2 £ 109 to 5 £ 109 CFU/ml (CFU, colony-forming units) before being adjusted to the concentrations required for diVerent experiments. Culture Wltrates were prepared by Wltering the supernatant of centrifuged cultures of the antagonist through a 0.2 m polycarbonate membrane Wlter. Autoclaved cultures were prepared by autoclaving a sample containing yeast in culture broth for 20 min at 121 °C. Unwashed cells
from 20-h cultures were adjusted to 1 £ 108 CFU/ml by adding additional culture Wltrate. Treatments were as follows: A, 1 £ 108 CFU/ml unwashed cell culture mixture; B, culture Wltrate; C, autoclaved culture; D, 1 £ 108 CFU/ml washed cell suspension; and E, sterile distilled water as control. 2.3. Fruits Strawberries (Fragaria ananassa Duch.) cultivars ‘chunxing’ were harvested from randomized blocks and rapidly transferred to the laboratory. Berries were sorted on the basis of size, color (75% full red color), and absence of physical damage, and were randomly divided into lots of ten fruit. 2.4. In vitro antagonism To evaluate the interactions between the antagonist and the pathogens in culture, we cut 5-mm-diameter disks from potato-dextrose agar (PDA) plates, then, a 300-l quantity of 1 £ 108 CFU/ml washed cell suspension of R. glutinis or sterile distilled water as a control was, respectively, added into each wound site of PDA plates. After these, 100 l of 1 £ 104 spores/ml suspension of B. cinerea was inoculated onto each wound. The plates were incubated at 28 °C for 5 days after which the colony diameter of B. cinerea were recorded. There were three replicate trials of three plates in each test. The test was repeated three times. The eVect of R. glutinis on spore germination and germ tube elongation of pathogen was tested in potato-dextrose broth (PDB). A 100-l quantity of 1 £ 108 CFU/ml washed cell suspension of R. glutinis or sterile distilled water as a control was added into 10 ml glass tube containing 5 ml PDB, respectively. At the same time, aliquots (100 l) of spore suspensions (1 £ 107 spores/ml) of B. cinerea were added into each tube. After 20 h incubation at 25 °C on a rotary shaker (50 rpm), at least 100 spores per replicate were observed microscopically to determine germination rate and germ tube length. All treatments consisted of three replicates, and experiments were repeated three times (Droby et al., 1997). 2.5. Population studies of R. glutinis in fruit wounds Strawberries were rinsed in fresh water and air dried. A uniform 3 mm deep by 3 mm wide wound was made at the equator of each fruit (put on its side) using the tip of a sterile dissecting needle. Twenty l of 1 £ 108 CFU/ml washed cell suspension of R. glutinis was applied to wounds on fruit. Fruits were incubated at 20 °C (90% relative humidity) or at 4 °C (98% relative humidity). R. glutinis was recovered from the wounds after incubation at 20 °C for 0 (just prior to storage), 1, 2, 3, and 4 days, and at 4 °C, for 0 (just prior to storage), 2, 4, 6, and 8 days, respectively. Wounded tissue was removed with an ethanol-Xamed,
H. Zhang et al. / Biological Control 40 (2007) 287–292
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7 mm (internal diameter) cork borer and ground with a autoclaved mortar and pestle in 10 ml of sterile 0.05 M phosphate buVer (pH 7.0). The slurry (15 ml) was plated on Petri plates (9 ml wide) with NYDA medium. Serial tenfold dilutions were made and 0.1 ml of each dilution was spread in NYDA. The plates were incubated at 28 °C for 2 days and the colonies were counted. Population densities of R. glutinis were expressed as log10 CFU per wound. There were three single fruit replicates per treatment, and the experiments were repeated twice (Viñas et al., 1998).
2.9. Statistical analysis
2.6. EYcacy of R. glutinis in controlling of gray mold decay
In the test on PDA plates, R. glutinis signiWcantly inhibit the growth of B. cinerea (Fig. 1). By the end of 5 days incubation at 25 °C, the colony diameter of B. cinerea on PDA plates incubated with R. glutinis was reduced by 68.1% compared with control. Spore germination and germ tube elongation of B. cinerea in PDB was greatly controlled by the living cell of R. glutinis (Table 1). By the end of 20 h incubation at 25 °C, the percentage of spore germination and the length of germ tube of B. cinerea incubated with R. glutinis was 20.5% and 9.7 m, respectively; while the percentage of spore germination and the length of germ tube of the control was 66.7% and 28.4 m, respectively.
A uniform 3 mm deep by 3 mm wide wound was made at the equator of each fruit (put on its side) using the tip of a sterile dissecting needle. Aliquots (30 l) of the above-mentioned suspensions of A, B, C, D, and E were, respectively, pipetted into each wound site. Two hours later, 15 l of 1 £ 105 spores/ml suspension of B. cinerea was inoculated onto each wound. After air drying, the fruits were sealed in 5-l capacity plastic boxes lined with polythene and incubated at 20 °C for 2 days after which the number of infected fruit were recorded. There were three replicate trials of 10 fruits with a complete randomization in each test. The test was repeated three times.
Data were subjected to analysis of variance (ANOVA) using SAS Software (SAS Institute, version 6.08, Cary, NC). Statistical signiWcance was assessed at p < 0.05 and Tukey’s HSD Test was used to separate means. 3. Results 3.1. Biocontrol activity of R. glutinis in vitro
9
2.7. EVect of concentration of yeast on eVectiveness of control at ambient temperatures and under cold storage condition
Lesion diameter(mm)
8
Aliquot (30 l) of 1 £ 106, 1 £ 107, 1 £ 108, 1 £ 109, and 0 (sterile distilled water alone) CFU/ml washed cell suspension were pipetted onto wounds. Two hours later, 15 l of 1 £ 105 spores/ml suspension of B. cinerea were inoculated onto each wound. Treated fruits were stored at 20 °C for 2 days or 4 °C for 7 days. Infection rate was recorded afterwards. There were three replicate trials of 10 fruits per treatment with complete randomization. The experiment was repeated three times. 2.8. EYcacy of R. glutinis for reducing natural decay development The suspensions of washed cells were adjusted to concentrations of 5 £ 108 CFU/ml with sterile distilled water by a hemocytometer, and sterile distilled water as a control. Intact fruits were inoculated by dipping them in the suspension of washed cells or sterile distilled water, respectively, for 30 s, and air dried. The treated fruits were sealed in polyethylene-lined plastic boxes to retain high humidity. Fruits were stored at 20 °C for 3 days or 4 °C for 5 days followed by 20 °C for 3 days in order to determine disease development under normal shelf-life conditions. Infection rate was recorded afterwards. There were three replicate trials of 10 fruits with a complete randomization in each test and experiments were repeated three time.
7 6
a
5 4 3 2
b
1 0
Control
Antagonist Treatments
Fig. 1. EVect of R. glutinis on the growth of B. cinerea on PDA plates. Disease incidences was measured after 2 days incubation at 20 °C. Bars represent standard error of the mean. Data in columns with the diVerent letters are signiWcantly diVerent according to Tukey’s HSD Test at p D 0.05.
Table 1 EVect of washed cell suspensions of R. glutinis on spore germination and germ tube elongation of B. cinerea Treatments
Spore germinationa(%)
Germ tube length (m)
R. glutinis control
20.5 § 1.80 bb 66.7 § 4.30aa
9.7 § 0.89 b 28.4 § 1.64a
a
Germination rate and germ tube length were measured microscopically after 20 h incubation at 25 °C in PDB. b Means are averaged values of three trials § the standard error. Values of each column followed by the same letter are not signiWcantly diVerent at p D 0.05 according to Tukey’s HSD Test.
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3.2. Population studies of R. glutinis in fruit wounds Population density of R. glutinis in wounds increased rapidly with incubation time at all temperatures (Fig. 2). At the beginning of the experiment (time 0), the yeast population was 1.26 £ 104 CFU per wound and increased to 9.77 £ 104 CFU per wound at 20 °C after 1 day, and 6.92 £ 104 CFU per wound at 4 °C after 2 days. Temperature and incubation time also aVected population of the R. glutinis in strawberry wounds. Regression analysis showed the relationship of the log of the mean number of cells per wound (Y) and days of incubation (X) was: at 4 °C, Y D 4.1669 ¡ 0.0084X2 + 0.2636X, R2 D 0.9654; at 20 °C, Y D 4.0546 ¡ 0.1507X2 + 1.2219X, R2 D 0.9903. Repeated trials gave similar results. Rapid colonization of the yeast in wounds was observed during the Wrst 3 days at 20 °C, and then the populations stabilized for the remaining storage period. On strawberry wounds kept at 4 °C, the increase in population density of R. glutinis was lower than those kept at 20 °C, but continued over 8 days after application of the
antagonist until it reached a high level. Repeated trials gave similar results. 3.3. EYcacy of R. glutinis in controlling of gray mold rot of strawberry Best control of gray mold was achieved with suspensions of washed yeast cell (Fig. 3). Number of infected strawberry fruit treated with 1 £ 108 CFU/ml washed cell suspension of R. glutinis was 10% after 2 days at 20 °C, compared to 100%, respectively, in the control. Unwashed cell suspensions also gave some control (p < 0.05), but lower than achieved with washed cell suspensions at the same concentration. Autoclaved cultures and culture Wltrate gave no control of gray mold compared to the water control (p < 0.05). 3.4. EVect of concentration of R. glutinis on biocontrol eYcacy at ambient temperatures and cold storage condition Disease incidence on all treated fruits after storage at 20 °C for 2 days or 4 °C for 7 days were signiWcantly lower than those on the untreated control (p < 0.05). The concentrations of R. glutinis signiWcantly aVected disease incidence of gray mold in strawberries (Fig. 4): in general, the higher the concentrations of the antagonist, the lower the disease incidence. At concentrations of R. glutinis 1 £ 109 CFU/ml, the incidence of gray mold was reduced by 94.7% or 95%, respectively, compared with control, after storage at 20 °C for 2 days or 4 °C for 7 days, respectively. The relationship of the percentage of strawberry developing lesions (Y) and concentration of R. glutinis applied to the wounds (X) could be described by the following equations: Stored at 20 °C for 2 days, Y D 71.625 ¡ 6 £ 10¡7X + 5 £ 10¡16X2, R2 D 0.7356; Stored
Fig. 2. Population dynamics of R. glutinis in wounds of strawberries and incubated at 4 and 20 °C. Data are from one trial with each point representing the mean colony counts from three replicate fruits, each plated in triplicate at each sampling time for each temperature. Y means log10 CFU per wound.
Fig. 3. Incidence of gray mold rot on inoculated strawberry fruits after various treatments with the antagonistic yeast R. glutinis. (A) 1 £ 108 CFU/ml unwashed cell culture mixture; (B) culture Wltrate; (C) autoclaved culture; (D) 1 £ 108 CFU/ml washed cell suspension, (E) sterile distilled water. Each value is pooled from three experiments. Disease incidences was measured after 2 days incubation at 20 °C. Bars represent standard error of the mean. Data in columns with diVerent letters are signiWcantly diVerent according to Tukey’s HSD Test at p D 0.05.
H. Zhang et al. / Biological Control 40 (2007) 287–292
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4. Discussion
Fig. 4. EVect of concentration of R. glutinis on biocontrol eYcacy at ambient temperatures and cold storage condition. Each value is the mean of three experiments. Disease incidences were measured after 2 days at 20 or 4 °C for 7 days. Bars represent standard error of the mean. Data in columns with diVerent letters within the same Wgure are statistically diVerent according to Tukey’s HSD Test at p D 0.05.
Fig. 5. EYcacy of R. glutinis for reducing natural decay development. Each value is the mean of three experiments. Disease incidences were measured after 4 °C for 5 days followed by 20 °C for 3 days. Bars represent standard error of the mean. Data in columns with diVerent letters within the same Wgure are statistically diVerent according to Tukey’s HSD Test at p D 0.05.
at 4 °C for 7 days, Y D 81.398 ¡ 7 £ 10¡7X + 7 £ 10¡16X2, R2 D 0.9148. 3.5. EYcacy of R. glutinis for reducing natural decay development Our experiments evaluated the eYcacy of yeast antagonist in reducing the natural development of decay following storage at 20 °C for 3 days or 4 °C for 5 days followed by 20 °C for 3 days. The result presented in Fig. 5 indicated that the application of R. glutinis resulted in low average decay incidence in fruit 25%, compared with 100% in the water-treated control fruit after storage at 20 °C for 3 days. Similarly, after storage at 4 °C for 5 days followed by 20 °C for 3 days, the decay incidence of the strawberries treated with R. glutinis was 15%, while the control fruit had 95% decay incidence.
On PDA plates, R. glutinis signiWcantly inhibit the growth of B. cinerea. Under in vitro conditions, spore germination of B. cinerea was signiWcantly inhibited by the living cells of R. glutinis. Furthermore, on the experiment of eVects of yeast antagonist on gray mold decay in artiWcially inoculated and wounded fruits, the percentage of infected wounds in fruits treated with R. glutinis were signiWcantly lower than those of the control fruit. These results suggested that R. glutinis can control postharvest gray mold decay of strawberries, and limiting spore germination of pathogens may be one of the mechanisms of action of R. glutinis. The ability of R. glutinis to rapidly colonize the wounded fruits (the major points of entry for pathogens) at ambient temperature (20 °C) and low temperature (4 °C) may indicate biocontrol by nutrient competition and/or site exclusion. The wounds, which may inevitably occur during harvest, transport, and handling, not only damage the harvested fruit (Miller, 2003), but also provide pathways for pathogen invasion, especially for the wound-invading necrotrophic fungi (Janisiewicz and Korsten, 2002). The nutritional environment at the wound site may be favourable to antagonistic R. glutinis, which rapidly colonizes the fruit tissues and will be competing with the pathogen for nutrients. When wounds on strawberries were pretreated with R. glutinis, development of lesions of B. cinerea was eVectively reduced or prevented. Best control was achieved with washed cell suspensions of R. glutinis, much better than unwashed cell cultures. Culture Wltrate and autoclaved cell cultrate did not give any control. Furthermore, the higher the concentration of R. glutinis, the better biocontrol activity it had. These results suggest that the main mode of action of R. glutinis is competition for nutrients. An antagonist is very promising if it can be combined with routine postharvest treatments. Cold storage treatment is a routine practice to prolong the storage period of fruits. The demonstrated biocontrol eVect of R. glutinis at low temperature (4 °C) implied that R. glutinis can be used combined with cold storage to enhance the control eYcacy. R. glutinis signiWcantly reduced the natural development of decay of strawberry following storage at 20 °C for 3 days or 4 °C for 5 days followed by 20 °C for 3 days. These suggested that R. glutinis may have a control eYcacy to other pathogens of strawberry such as Rhizopus stolonifer. In conclusion, our results show that R. glutinis can be used as a non-chemical alternative treatment against postharvest diseases of strawberries. Future research will be aimed at developing the technology to be used under largescale operations and investigating about the mode of action of R. glutinis to control postharvest diseases of fruits. Acknowledgments This research was supported by China Postdoctoral Science Foundation (2005038016) and Nature Science
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Foundation of Jiangsu Provincial Education Department (06KJB210013). References Ceponis, M.J., Cappellini, R.A., Lightner, G.W., 1987. Disorders in sweet cherry and strawberry shipments in the New York market, 1972–1984. Phytopathology 71, 472–475. Droby, S., Wisniewski, M.E., Cohen, L., Weiss, B., Touitou, D., Eilam, Y., Chalutz, E., 1997. InXuence of CaCl2 on Penicillium digitatum, grapefruit peel tissue, and biocontrol activity of Pichia guilliermondii. Phytopathology 87, 310–315. Guinebretiere, M.H., Nguyen-The, C., Morrison, N., Reich, M., Nicot, P., 2000. Isolation and characterization of antagonists for the biocontrol of the postharvest wound pathogen Botrytis cinerea on strawberry fruits. J. Food Prot. 63, 386–394. Janisiewicz, W.J., Korsten, L., 2002. Biological control of postharvest diseases of fruits. Annu. Rev. Phytopathol. 40, 411–441. Kovach, J., Petzoldt, R., Harman, G.E., 2000. Use of honey bees and bumble bees to disseminate Trichoderma harzianum 1295-22 to strawberries for botrytis control. Biol. Control 18, 235–242. Lima, G., Ippolito, A., Nigro, F., Salerno, M., 1997. EVectiveness of Aureobasidium pullulans and Candida oleophila against postharvest strawberry rots. Postharvest Biol. Technol. 10, 169–178.
Mertely, J.C., MacKenzie, S.J., Legard, D.E., 2002. Timing of fungicide applications for Botrytis cinerea based on development stage of strawberry Xowers and fruit. Plant Dis. 86, 1019–1024. Miller, A.R., 2003. Harvest and handing injury: physiology, biochemistry, and deletion. In: Bartz, J., Brecht, J. (Eds.), Postharvest Physiology and Pathology of Vegetables. Marcel Dekker, New York, pp. 177–208. Peng, G., Sutton, J.C., 1991. Evaluation of microorganisms for biocontrol of Botrytis cinerea in strawberry. Can. J. Plant Pathol. 13, 247–257. Romanazzi, G., Nigro, F., Ippolito, A., Salerno, M., 2001. EVect of short hypobaric treatments on postharvest rots of sweet cherries, strawberries and table grapes. Postharvest Biol. Technol. 22, 1–6. Smilanick, J.L., 1994. Strategies for the isolation and testing of biocontrol agents. In: Wilson, C.L., Wisniewski, M.E. (Eds.), Biological Control of Postharvest Diseases, Theory and Practice. CRC Press, Boca Raton, FL. Swadling, I.R., JeVries, P., 1996. Isolation of microbial antagonists for biocontrol of grey mould disease of strawberries. Biocontrol Sci. Technol. 6, 125–136. Viñas, I., Usall, J., Teixidó, N., Sanchis, V., 1998. Biological control of major postharvest pathogens on apple with Candida sake. Int. J. Food Microbiol. 40, 9–16. Wisniewski, M.E., Wilson, C.L., 1992. Biological control of postharvest diseases of fruits and vegetables: recent advance. HortScience 27, 94–98. Wszelaki, A.L., Mitcham, E.J., 2003. EVect of combinations of hot water dips, biological control and controlled atmospheres for control of gray mold on harvested strawberries. Postharvest Biol. Technol. 27, 255–264.
Postharvest biological control of gray mold decay of strawberry with Rhodotorula glutinis Hongyin Zhang ¤, Lei Wang, Ying Dong, Song Jiang, Jian Cao, Rujie Meng College of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, People’s Republic of China Received 22 July 2006; accepted 18 October 2006 Available online 10 November 2006
Abstract Rhodotorula glutinis was evaluated for its activity in reducing postharvest gray mold decay of strawberry caused by Botrytis cinerea in vitro and in vivo. In the test on PDA plates, R. glutinis signiWcantly inhibit the growth of B. cinerea. Spore germination of pathogens in PDB was greatly controlled in the presence of living cell suspensions. Rapid colonization of the yeast in wounds was observed during the Wrst 3 days at 20 °C, and then the populations stabilized for the remaining storage period. On strawberry wounds kept at 4 °C, the increase in population density of R. glutinis was lower than those kept at 20 °C, but continued over 8 days after application of the antagonist until it reached a high level. Number of inoculated strawberry fruit treated with 1 £ 108 CFU/ml washed cell suspension of R. glutinis was 10% after 2 days at 20 °C, compared to 100%, respectively, in the control. Washed cell suspensions of yeast controlled gray mold better than yeast in culture broth. Treatment of wounds with autoclaved cell cultures or cell-free culture Wltrate did not prevent decay. The concentrations of antagonist had signiWcant eVects on biocontrol eVectiveness: the higher the concentrations of the antagonist, the lower the disease incidence regardless of whether the fruit was stored at 20 °C for 2 days or 4 °C for 7 days. At concentrations of R. glutinis 1 £ 109 CFU/ml, the incidence of gray mold was reduced by 94.7 or 95%, respectively, compared with control, after storage at 20 °C for 2 days or 4 °C for 7 days, respectively. R. glutinis signiWcantly reduced the natural development of decay of fruit following storage at 20 °C for 3 days or 4 °C for 5 days followed by 20 °C for 3 days. © 2006 Elsevier Inc. All rights reserved. Keywords: Strawberry; Gray mold; Postharvest decay; Biocontrol; Rhodotorula glutinis
1. Introduction Strawberry fruit have a very short postharvest life, due in part to gray mold caused by Botrytis cinerea Pers.:Fr. (Wszelaki and Mitcham, 2003). Infection may occur in the Xower, remain quiescent until fruits mature, and then develop abundantly, causing fruit decay accompanied by profuse sporulation of the pathogen (Kovach et al., 2000). B. cinerea also causes signiWcant losses during shipping and marketing (Ceponis et al., 1987), making it one of the most economically important pathogens of strawberry (Mertely et al., 2002; Romanazzi et al., 2001). Control of B. cinerea is normally carried out by the application of
*
Corresponding author. Fax: +86 511 86971139. E-mail address: [email protected] (H. Zhang).
1049-9644/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2006.10.008
fungicides. Although fungicides use reduces yield losses due to Botrytis in strawberries, there are studies that show that commercially available fungicides can reduce pollination and cause fruit malformation (Kovach et al., 2000). Fungicide eYcacy is frequently decreased by the development of resistant strains of pathogens. In addition, public concern and regulatory restrictions about the presence of fungicide residues on crops have emphasized the need to Wnd alternative methods for disease control (Smilanick, 1994). Microbial biocontrol agents have shown great potential as an alternative to synthetic fungicides for the control of postharvest decay of fruits and vegetables (Wisniewski and Wilson, 1992). Several biological control agents are eVective in reducing decay caused by gray mold on strawberry (Peng and Sutton, 1991; Swadling and JeVries, 1996; Lima et al., 1997; Guinebretiere et al., 2000).
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However, few of these have been tested under postharvest storage conditions. The objective of the present study was to study the postharvest control of gray mold of strawberry by Rhodotorula glutinis and determine: (1) biocontrol in vitro; (2) the population dynamics of R. glutinis in wounds; (3) eYcacy of R. glutinis in controlling of gray mold decay of strawberry; (4) eVect of concentration of R. glutinis on biocontrol eYcacy at ambient temperatures and cold storage condition; (5) eYcacy of R. glutinis in controlling of natural decay development of strawberry. 2. Materials and methods 2.1. Pathogen inoculum Botrytis cinerea Pers.: Fr. was isolated from infected strawberry fruits. The culture was maintained on potatodextrose agar medium (PDA: extract of boiled potatoes, 200 ml; dextrose, 20 g; agar, 20 g and distilled water, 800 ml) at 4 °C; fresh cultures were grown on PDA plates at 25 °C before use. Spore suspensions were prepared by removing the spores from the sporulating edges of a 10-day-old culture with a bacteriological loop, and suspending them in 5 ml of sterile distilled water. Suspensions were Wltered through four layers of cheesecloth to remove fungal mycelium and spore concentrations were determined with a hemocytometer with the concentration being adjusted as required for diVerent experiments by adding sterile distilled water. 2.2. Antagonist The yeast antagonist R. glutinis (Fresenins) Harrison was isolated from the surfaces of strawberries harvested in unsprayed orchards and identiWed by VITEK 32 Automicrobic system (bioMérieux Company, Marcy l’Etoile, France). R. glutinis isolates were maintained at 4 °C on Nutrient Yeast Dextrose Agar (NYDA) medium containing 8 g nutrient broth, 5 g yeast extract, 10 g glucose, and 20 g agar, in 1 L of distilled water. Liquid cultures of the yeast were grown in 250-ml Erlenmeyer Xasks containing 50 ml of NYD Broth (NYDB) which had been inoculated with a loop of the culture. Flasks were incubated on a rotary shaker at 28 °C for 20 h. Following incubation, cells were centrifuged at 5000g for 10 min and washed twice with sterile distilled water in order to remove the growth medium. Cell pellets were re-suspended in sterile distilled water and adjusted to an initial concentration of 2 £ 109 to 5 £ 109 CFU/ml (CFU, colony-forming units) before being adjusted to the concentrations required for diVerent experiments. Culture Wltrates were prepared by Wltering the supernatant of centrifuged cultures of the antagonist through a 0.2 m polycarbonate membrane Wlter. Autoclaved cultures were prepared by autoclaving a sample containing yeast in culture broth for 20 min at 121 °C. Unwashed cells
from 20-h cultures were adjusted to 1 £ 108 CFU/ml by adding additional culture Wltrate. Treatments were as follows: A, 1 £ 108 CFU/ml unwashed cell culture mixture; B, culture Wltrate; C, autoclaved culture; D, 1 £ 108 CFU/ml washed cell suspension; and E, sterile distilled water as control. 2.3. Fruits Strawberries (Fragaria ananassa Duch.) cultivars ‘chunxing’ were harvested from randomized blocks and rapidly transferred to the laboratory. Berries were sorted on the basis of size, color (75% full red color), and absence of physical damage, and were randomly divided into lots of ten fruit. 2.4. In vitro antagonism To evaluate the interactions between the antagonist and the pathogens in culture, we cut 5-mm-diameter disks from potato-dextrose agar (PDA) plates, then, a 300-l quantity of 1 £ 108 CFU/ml washed cell suspension of R. glutinis or sterile distilled water as a control was, respectively, added into each wound site of PDA plates. After these, 100 l of 1 £ 104 spores/ml suspension of B. cinerea was inoculated onto each wound. The plates were incubated at 28 °C for 5 days after which the colony diameter of B. cinerea were recorded. There were three replicate trials of three plates in each test. The test was repeated three times. The eVect of R. glutinis on spore germination and germ tube elongation of pathogen was tested in potato-dextrose broth (PDB). A 100-l quantity of 1 £ 108 CFU/ml washed cell suspension of R. glutinis or sterile distilled water as a control was added into 10 ml glass tube containing 5 ml PDB, respectively. At the same time, aliquots (100 l) of spore suspensions (1 £ 107 spores/ml) of B. cinerea were added into each tube. After 20 h incubation at 25 °C on a rotary shaker (50 rpm), at least 100 spores per replicate were observed microscopically to determine germination rate and germ tube length. All treatments consisted of three replicates, and experiments were repeated three times (Droby et al., 1997). 2.5. Population studies of R. glutinis in fruit wounds Strawberries were rinsed in fresh water and air dried. A uniform 3 mm deep by 3 mm wide wound was made at the equator of each fruit (put on its side) using the tip of a sterile dissecting needle. Twenty l of 1 £ 108 CFU/ml washed cell suspension of R. glutinis was applied to wounds on fruit. Fruits were incubated at 20 °C (90% relative humidity) or at 4 °C (98% relative humidity). R. glutinis was recovered from the wounds after incubation at 20 °C for 0 (just prior to storage), 1, 2, 3, and 4 days, and at 4 °C, for 0 (just prior to storage), 2, 4, 6, and 8 days, respectively. Wounded tissue was removed with an ethanol-Xamed,
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7 mm (internal diameter) cork borer and ground with a autoclaved mortar and pestle in 10 ml of sterile 0.05 M phosphate buVer (pH 7.0). The slurry (15 ml) was plated on Petri plates (9 ml wide) with NYDA medium. Serial tenfold dilutions were made and 0.1 ml of each dilution was spread in NYDA. The plates were incubated at 28 °C for 2 days and the colonies were counted. Population densities of R. glutinis were expressed as log10 CFU per wound. There were three single fruit replicates per treatment, and the experiments were repeated twice (Viñas et al., 1998).
2.9. Statistical analysis
2.6. EYcacy of R. glutinis in controlling of gray mold decay
In the test on PDA plates, R. glutinis signiWcantly inhibit the growth of B. cinerea (Fig. 1). By the end of 5 days incubation at 25 °C, the colony diameter of B. cinerea on PDA plates incubated with R. glutinis was reduced by 68.1% compared with control. Spore germination and germ tube elongation of B. cinerea in PDB was greatly controlled by the living cell of R. glutinis (Table 1). By the end of 20 h incubation at 25 °C, the percentage of spore germination and the length of germ tube of B. cinerea incubated with R. glutinis was 20.5% and 9.7 m, respectively; while the percentage of spore germination and the length of germ tube of the control was 66.7% and 28.4 m, respectively.
A uniform 3 mm deep by 3 mm wide wound was made at the equator of each fruit (put on its side) using the tip of a sterile dissecting needle. Aliquots (30 l) of the above-mentioned suspensions of A, B, C, D, and E were, respectively, pipetted into each wound site. Two hours later, 15 l of 1 £ 105 spores/ml suspension of B. cinerea was inoculated onto each wound. After air drying, the fruits were sealed in 5-l capacity plastic boxes lined with polythene and incubated at 20 °C for 2 days after which the number of infected fruit were recorded. There were three replicate trials of 10 fruits with a complete randomization in each test. The test was repeated three times.
Data were subjected to analysis of variance (ANOVA) using SAS Software (SAS Institute, version 6.08, Cary, NC). Statistical signiWcance was assessed at p < 0.05 and Tukey’s HSD Test was used to separate means. 3. Results 3.1. Biocontrol activity of R. glutinis in vitro
9
2.7. EVect of concentration of yeast on eVectiveness of control at ambient temperatures and under cold storage condition
Lesion diameter(mm)
8
Aliquot (30 l) of 1 £ 106, 1 £ 107, 1 £ 108, 1 £ 109, and 0 (sterile distilled water alone) CFU/ml washed cell suspension were pipetted onto wounds. Two hours later, 15 l of 1 £ 105 spores/ml suspension of B. cinerea were inoculated onto each wound. Treated fruits were stored at 20 °C for 2 days or 4 °C for 7 days. Infection rate was recorded afterwards. There were three replicate trials of 10 fruits per treatment with complete randomization. The experiment was repeated three times. 2.8. EYcacy of R. glutinis for reducing natural decay development The suspensions of washed cells were adjusted to concentrations of 5 £ 108 CFU/ml with sterile distilled water by a hemocytometer, and sterile distilled water as a control. Intact fruits were inoculated by dipping them in the suspension of washed cells or sterile distilled water, respectively, for 30 s, and air dried. The treated fruits were sealed in polyethylene-lined plastic boxes to retain high humidity. Fruits were stored at 20 °C for 3 days or 4 °C for 5 days followed by 20 °C for 3 days in order to determine disease development under normal shelf-life conditions. Infection rate was recorded afterwards. There were three replicate trials of 10 fruits with a complete randomization in each test and experiments were repeated three time.
7 6
a
5 4 3 2
b
1 0
Control
Antagonist Treatments
Fig. 1. EVect of R. glutinis on the growth of B. cinerea on PDA plates. Disease incidences was measured after 2 days incubation at 20 °C. Bars represent standard error of the mean. Data in columns with the diVerent letters are signiWcantly diVerent according to Tukey’s HSD Test at p D 0.05.
Table 1 EVect of washed cell suspensions of R. glutinis on spore germination and germ tube elongation of B. cinerea Treatments
Spore germinationa(%)
Germ tube length (m)
R. glutinis control
20.5 § 1.80 bb 66.7 § 4.30aa
9.7 § 0.89 b 28.4 § 1.64a
a
Germination rate and germ tube length were measured microscopically after 20 h incubation at 25 °C in PDB. b Means are averaged values of three trials § the standard error. Values of each column followed by the same letter are not signiWcantly diVerent at p D 0.05 according to Tukey’s HSD Test.
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3.2. Population studies of R. glutinis in fruit wounds Population density of R. glutinis in wounds increased rapidly with incubation time at all temperatures (Fig. 2). At the beginning of the experiment (time 0), the yeast population was 1.26 £ 104 CFU per wound and increased to 9.77 £ 104 CFU per wound at 20 °C after 1 day, and 6.92 £ 104 CFU per wound at 4 °C after 2 days. Temperature and incubation time also aVected population of the R. glutinis in strawberry wounds. Regression analysis showed the relationship of the log of the mean number of cells per wound (Y) and days of incubation (X) was: at 4 °C, Y D 4.1669 ¡ 0.0084X2 + 0.2636X, R2 D 0.9654; at 20 °C, Y D 4.0546 ¡ 0.1507X2 + 1.2219X, R2 D 0.9903. Repeated trials gave similar results. Rapid colonization of the yeast in wounds was observed during the Wrst 3 days at 20 °C, and then the populations stabilized for the remaining storage period. On strawberry wounds kept at 4 °C, the increase in population density of R. glutinis was lower than those kept at 20 °C, but continued over 8 days after application of the
antagonist until it reached a high level. Repeated trials gave similar results. 3.3. EYcacy of R. glutinis in controlling of gray mold rot of strawberry Best control of gray mold was achieved with suspensions of washed yeast cell (Fig. 3). Number of infected strawberry fruit treated with 1 £ 108 CFU/ml washed cell suspension of R. glutinis was 10% after 2 days at 20 °C, compared to 100%, respectively, in the control. Unwashed cell suspensions also gave some control (p < 0.05), but lower than achieved with washed cell suspensions at the same concentration. Autoclaved cultures and culture Wltrate gave no control of gray mold compared to the water control (p < 0.05). 3.4. EVect of concentration of R. glutinis on biocontrol eYcacy at ambient temperatures and cold storage condition Disease incidence on all treated fruits after storage at 20 °C for 2 days or 4 °C for 7 days were signiWcantly lower than those on the untreated control (p < 0.05). The concentrations of R. glutinis signiWcantly aVected disease incidence of gray mold in strawberries (Fig. 4): in general, the higher the concentrations of the antagonist, the lower the disease incidence. At concentrations of R. glutinis 1 £ 109 CFU/ml, the incidence of gray mold was reduced by 94.7% or 95%, respectively, compared with control, after storage at 20 °C for 2 days or 4 °C for 7 days, respectively. The relationship of the percentage of strawberry developing lesions (Y) and concentration of R. glutinis applied to the wounds (X) could be described by the following equations: Stored at 20 °C for 2 days, Y D 71.625 ¡ 6 £ 10¡7X + 5 £ 10¡16X2, R2 D 0.7356; Stored
Fig. 2. Population dynamics of R. glutinis in wounds of strawberries and incubated at 4 and 20 °C. Data are from one trial with each point representing the mean colony counts from three replicate fruits, each plated in triplicate at each sampling time for each temperature. Y means log10 CFU per wound.
Fig. 3. Incidence of gray mold rot on inoculated strawberry fruits after various treatments with the antagonistic yeast R. glutinis. (A) 1 £ 108 CFU/ml unwashed cell culture mixture; (B) culture Wltrate; (C) autoclaved culture; (D) 1 £ 108 CFU/ml washed cell suspension, (E) sterile distilled water. Each value is pooled from three experiments. Disease incidences was measured after 2 days incubation at 20 °C. Bars represent standard error of the mean. Data in columns with diVerent letters are signiWcantly diVerent according to Tukey’s HSD Test at p D 0.05.
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4. Discussion
Fig. 4. EVect of concentration of R. glutinis on biocontrol eYcacy at ambient temperatures and cold storage condition. Each value is the mean of three experiments. Disease incidences were measured after 2 days at 20 or 4 °C for 7 days. Bars represent standard error of the mean. Data in columns with diVerent letters within the same Wgure are statistically diVerent according to Tukey’s HSD Test at p D 0.05.
Fig. 5. EYcacy of R. glutinis for reducing natural decay development. Each value is the mean of three experiments. Disease incidences were measured after 4 °C for 5 days followed by 20 °C for 3 days. Bars represent standard error of the mean. Data in columns with diVerent letters within the same Wgure are statistically diVerent according to Tukey’s HSD Test at p D 0.05.
at 4 °C for 7 days, Y D 81.398 ¡ 7 £ 10¡7X + 7 £ 10¡16X2, R2 D 0.9148. 3.5. EYcacy of R. glutinis for reducing natural decay development Our experiments evaluated the eYcacy of yeast antagonist in reducing the natural development of decay following storage at 20 °C for 3 days or 4 °C for 5 days followed by 20 °C for 3 days. The result presented in Fig. 5 indicated that the application of R. glutinis resulted in low average decay incidence in fruit 25%, compared with 100% in the water-treated control fruit after storage at 20 °C for 3 days. Similarly, after storage at 4 °C for 5 days followed by 20 °C for 3 days, the decay incidence of the strawberries treated with R. glutinis was 15%, while the control fruit had 95% decay incidence.
On PDA plates, R. glutinis signiWcantly inhibit the growth of B. cinerea. Under in vitro conditions, spore germination of B. cinerea was signiWcantly inhibited by the living cells of R. glutinis. Furthermore, on the experiment of eVects of yeast antagonist on gray mold decay in artiWcially inoculated and wounded fruits, the percentage of infected wounds in fruits treated with R. glutinis were signiWcantly lower than those of the control fruit. These results suggested that R. glutinis can control postharvest gray mold decay of strawberries, and limiting spore germination of pathogens may be one of the mechanisms of action of R. glutinis. The ability of R. glutinis to rapidly colonize the wounded fruits (the major points of entry for pathogens) at ambient temperature (20 °C) and low temperature (4 °C) may indicate biocontrol by nutrient competition and/or site exclusion. The wounds, which may inevitably occur during harvest, transport, and handling, not only damage the harvested fruit (Miller, 2003), but also provide pathways for pathogen invasion, especially for the wound-invading necrotrophic fungi (Janisiewicz and Korsten, 2002). The nutritional environment at the wound site may be favourable to antagonistic R. glutinis, which rapidly colonizes the fruit tissues and will be competing with the pathogen for nutrients. When wounds on strawberries were pretreated with R. glutinis, development of lesions of B. cinerea was eVectively reduced or prevented. Best control was achieved with washed cell suspensions of R. glutinis, much better than unwashed cell cultures. Culture Wltrate and autoclaved cell cultrate did not give any control. Furthermore, the higher the concentration of R. glutinis, the better biocontrol activity it had. These results suggest that the main mode of action of R. glutinis is competition for nutrients. An antagonist is very promising if it can be combined with routine postharvest treatments. Cold storage treatment is a routine practice to prolong the storage period of fruits. The demonstrated biocontrol eVect of R. glutinis at low temperature (4 °C) implied that R. glutinis can be used combined with cold storage to enhance the control eYcacy. R. glutinis signiWcantly reduced the natural development of decay of strawberry following storage at 20 °C for 3 days or 4 °C for 5 days followed by 20 °C for 3 days. These suggested that R. glutinis may have a control eYcacy to other pathogens of strawberry such as Rhizopus stolonifer. In conclusion, our results show that R. glutinis can be used as a non-chemical alternative treatment against postharvest diseases of strawberries. Future research will be aimed at developing the technology to be used under largescale operations and investigating about the mode of action of R. glutinis to control postharvest diseases of fruits. Acknowledgments This research was supported by China Postdoctoral Science Foundation (2005038016) and Nature Science
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