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TIAN Shi-Ping*, QIN Guo-Zheng, XU Yong, WANG You-Sheng. (Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany,.
Acta Botanica Sinica 植   物   学   报

2004, 46 (11): 1324-1330

http://www.chineseplantscience.com

Application of Antagonistic Yeasts Under Field Conditions and Their Biocontrol Ability Against Postharvest Diseases of Sweet Cherry TIAN Shi-Ping*, QIN Guo-Zheng, XU Yong, WANG You-Sheng (Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China)

Abstract: The yeasts Trichosporon pullulans (Lindner.) Diddens et Lodder, Cryptococcus laurentii (Kuffer.) Skinner and Rhodotorula glutinis (Fresenius) Harrison were sprayed at concentration of 1×108 CFU/mL onto sweet cherry (Prunus avivum L. cv. Hongdeng) fruit in two orchards prior to harvest. Survival of these species on fruit surfaces under field conditions was investigated. Also, their biocontrol efficacy against postharvest decay of cherry fruit stored under various conditions was assessed. All three yeasts colonized the surface of sweet cherry fruit. However, only C. laurentii and R. glutinis maintained populations at high and stable levels throughout the 4-d experimental period. C. laurentii was the most effective and promising of the three antagonists. It had strong survival ability on fruit surfaces under field conditions and adaptability to postharvest storage conditions of low temperature, low-O2 and high-CO2 concentrations. Key words: biocontrol efficacy; postharvest decay; preharvest application; sweet cherry fruit; yeast antagonists As use of high levels of chemical fungicide in fruit orchards results in the development of resistance of fungal pathogens to fungicides and the growing public concern over the health and environmental hazards, the need to develop other methods to control postharvest diseases has been encouraged in resent years (Wilson and Pusey, 1985; Wisniewski and Wilson, 1992; Lima et al., 1998). Biological control using microbial antagonists has emerged as one of the most promising alternatives, either alone or as part of an integrated control strategy to reduce synthetic fungicide inputs (Mercier and Wilson, 1994; Chand-Goyal and Spotts, 1996; Benbow and Sugar, 1999; Fan and Tian, 2000). The efficacy of several postharvest biocontrol agents has been evaluated in pilot tests under semi-commercial conditions (Chand-Goyal and Spotts, 1997; Janisiewicz et al., 1998; Tian et al., 2002a; Spotts et al., 2002). In the postharvest environments, yeasts appear to be particularly promising because production of antibiotics does not seem to be involved in their activity (Droby and Chalutz, 1994). Antagonistic yeasts have been selected mainly for their capability to rapidly colonize and grow in surface wounds and subsequently out compete pathogens for nutrients and space (Wisniewski et al., 1991; Arras, 1996; Spadaro et al., 2002) and parasitize postharvest pathogens directly through strong attachment to their hyphae (Droby et al., 2002; Wan and Tian, 2002). As the yeasts used in the experiment were originally isolated from fruit surfaces after

or near harvest, they might be tolerant of the field conditions and adversely affected by preharvest application of fungicides (Fan and Tian, 2001; Tian et al., 2002a). Some yeasts can colonize plant surfaces or wounds for long periods under dry conditions, and can produce extracellular polysaccharides that enhance their survival and restrict pathogen colonization sites (Wisniewski and Wilson, 1992; Chand-Goyal and Spotts, 1996). Infection of fruit by postharvest pathogens often occurs in the field prior to harvest (Roberts, 1994; Biggs, 1995). Therefore, it would be advantageous to apply antagonists before harvest. Preharvest application could reduce initial infection and the agents then remain active and suppress the pathogens in storage (Teixidóet al., 1998). Biocontrol activity of antagonists may also be influenced by the specific pathogen, host commodity and particularly by environmental conditions (Spotts et al., 1998; Tian et al., 2002a). The success of commercialization of an antagonist depends on its broad spectrum of action. When applied before harvest in the field, antagonistic yeasts need tolerance to environmental stresses, especially of high temperature, low water activity, low nutrient conditions, and UV light for effective establishment and disease control (Deacon, 1991; Conway et al., 1999). If an antagonist has potential for practical use, its ability to colonize the surface of fruit both in the field and in storage, and to persist for as long as possible is vitally important (Wisniewski and Wilson, 1992).

Received 19 Feb. 2004 Accepted 17 Jul. 2004 Supported by the National Science Fund for Distinguished Young Scholars of China (30225030) and the National Natural Science Foundation of China (30170663). * Author for correspondence. Fax: +86 (0)10 82594675; E-mail: .

TIAN Shi-Ping et al.: Application of Antagonistic Yeasts Under Field Conditions and Their Biocontrol Ability Against Postharvest Diseases of Sweet Cherry 1325

Although many studies have indicated the potential for biocontrol of postharvest decay when agents were applied after harvest (Wilson and Pusey, 1985; Chand-Goyal and Spotts, 1996; Lima et al., 1998; Tian et al., 2002b), relatively few studies have tried to improve the competence, survival and activity of antagonistic microbial agents in the field for optimizing subsequent disease control (Teixidóet al., 1998; Benbow and Sugar, 1999). Trichosporon pullulans, Cryptococcus laurentii and Rhodotorula glutinis were isolated selectively from the surface of apple fruit. We found previously that these yeasts had biocontrol ability against postharvest decay of apple and grape fruits when applied after harvest (Liu et al., 2002; Qin et al., 2003). The objectives of the study were (i) to investigate the survival of these antagonistic yeasts, (ii) to compare their establishment and population dynamics on the fruit surface under field conditions when applied 4 d prior to harvest, and (iii) to determine their biocontrol efficiency on sweet cherry fruit under different storage conditions

1 Materials and Methods 1.1 Antagonistic yeasts Trichosporon pullulans (Lindner.) Diddens et Lodder, Cryptococcus laurentii (Kuffer.) Skinner and Rhodotorula glutinis (Fresenius) Harrison were isolated selectively from the surface of apple fruit following the method of Wilson and Chalutz (1989). They were identified by CABI Bioscience Identification Services (International Mycological Institute, UK). The yeasts were cultured in 250 mL conical flasks containing 50 mL of nutrient yeast dextrose broth (NYDB: 8 g of nutrient broth, 5 g of yeast extract, and 10 g of dextrose in 1 000 mL water) on a rotary shaker at 200 r/ min for 48 h at 28 °C. Yeast cells were pelleted by centrifugation at 6 000 r/min (about 2 500g) for 10 min, resuspended in sterile distilled water, and adjusted to a concentration 1 ×108 CFU/mL with a haemocytometer. 1.2 Fruit and orchards Sweet cherry (Prunus avivum L. cv. Hongdeng) fruit were used in trials conducted in two orchards. One was the experiment orchard of the Institute of Forest and Fruits, Beijing Academy of Agricultural Sciences in Beijing. The other was a commercial orchard in the Jinzhou district of Liaoning Province, a major production area in China for sweet cherry. The trees in both orchards were 6-8 years old. They were grown under standard cultural practices. Application of fungicides (iprodione and thiabendazole) was stopped 1 month before the treatments.

1.3 Preharvest treatments The experiments were carried out on 21-25 May, 2002 in Beijing orchard, in June, 2002 in the Jinzhou orchard 4 d before harvest. The yeast suspensions of T. pullulans, C. laurentii and R. glutinis at 1×108 CFU/mL were sprayed uniformly onto fruit using a hand-held sprayer. Fruits sprayed with distilled water were used as the control (CK). There were three replicates of two trees per treatment in a completely randomized block design. Fruits were harvested carefully and put into different boxes according to the experimental design. Fruits harvested from the Beijing orchard were directly transported to our laboratory. Fruits from the Jinzhou orchard were immediately precooled after harvest and transported on the day of harvest to Beijing in a refrigerated van at 2-5 °C. Climatic data for each orchard were obtained during the field trial from nearby weather stations. 1.4 Storage conditions Storage conditions were room temperature (25 °C), and in air and under a controlled atmosphere (CA) condition of 10% O2 + 10 % CO2 at 0 °C, respectively. Fruit stored in air were put into plastic bag to maintain a relative humidity of about 95%. There were three boxes of 5 kg fruit in each treatment. Three kg fruits from each of three replicates in each treatment were used to determine disease incidence at various times of 10 and 20 d at 25 °C, 30 d in air at 0 °C and 60 d in CA at 0 °C. After 30 and 60 d, fruit stored at low temperature and under CA condition were raised to 25 °C for 3 d to monitor decay development under shelf life conditions. 1.5 Colonization of yeasts on cherry fruit surface Fifteen fruits from each treatment were taken at 0, 1, 2, 3, and 4 d after application to determine population numbers of the yeasts on the fruit surface by the method of Benbow and Sugar (1999). These fruits were placed in beakers containing 100 mL of sterile distilled water and then shaken at 200 r/min for 30 min. Serial (1:10) dilutions were made of the washing solutions. Four aliquots were taken from each dilution and plated on NYDA medium (NYDB plus 15 g of agar in 1 000 mL water). Colonies were counted after incubation at 28 °C for 48 h and expressed as Log10 CFU/fruit. The three yeasts used in this experiment could be visually distinguished because of their different colony color and morphology. Fruit treated with sterile water was used as the control and only those resembling the yeasts used in the treatments were counted. 1.6 Statistical analysis All data were analyzed by one-way analysis of variance (ANOVA). The treatment means were separated at the 5% significant level using Duncan’s multiple range test.

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Results

2.1 Population dynamics on fruit surface All three yeasts C. laurentii, R. glutinis and T. pullulans could colonize the surface of sweet cheery fruit under field conditions (Fig.1). Only C. laurentii and R. glutinis maintained populations at high and stable levels of 5.2×106 to 1.3×106 CFU per fruit throughout the experiment at both orchards. During the 4-d experimental period, populations of the yeasts on the surfaces of the sprayed fruit were significantly higher compared to the control (P = 0.05). C. laurentii and R. glutinis populations remained similar and high at 1.5-1.3 × 106 and 1.8-1.9 × 106 CFU per fruit throughout the 4 d, but T. pullulans populations rapidly dropped to 5.9×10 4-5.7 × 10 5 CUF per fruit on day 4 (Fig.1). There were no significant differences between population numbers of the same yeasts across the two orchards (P = 0.05). 2.2 Climatic data There were obvious differences during the 5 d prior to harvest in the minimum and maximum temperatures between the two orchards (Fig.2). Temperatures ranged from 10.815.8 °C to 28.7-32.2 °C in Beijing and from 18-19.3 °C to 23.1-26.6 °C in Jingzhou. The mean temperatures were not greatly different, being 22-24 °C in Beijing and 21-23 °C in Jingzhou during the experimental period. Temperature did

Fig.1. Populations of T. pullulans, C. laurentii, and R. glutinis on surface of cherry fruit in orchards in Beijing (A) and Jinzhou (B). Data are from 10 sample fruit for three replicates. Bars represent standard deviations of the means.

Fig.2. Temperatures and rainfall in sweet cheery orchards in Beijing (A) and Jinzhou (B) during the periods of the experiments.

not significantly (P > 0.05) affect populations of the yeasts on the surface of sweet cherry fruit, although there were differences in the field temperatures of the two orchards. 2.3 Storage decay control in different conditions C. laurentii was the most effective antagonist of the three yeasts for control of postharvest decay of sweet cherry over the different storage conditions (Figs.3-5). At 25 °C, fruit sprayed with C. laurentii before harvest had a significantly (P = 0.05) lower disease incidence than those treated with other yeasts or control (Fig.3). All fruit harvested from the Jingzhuo orchard appeared to have less decay compared to those from the Beijing orchard. Low temperature and controlled atmosphere storage limited decay development. Only 1.3%-3.3% of the fruit sprayed with C. laurentii decayed after 30 d at 0 °C (Fig.4). Treated fruit stored in 10% O2 + 10% CO2 CA showed only 0-5.1% decay after 60 d (Fig.5). Transferring fruit stored in either air or under CA at 0-25 °C for 5 d resulted in a significant increase in disease incidence (Figs.4,5). The pathogens causing sweet cherry decay were mainly Alternaria alternata, Monilinia fructicola, Penicillum expansum and Botrytis cinerea. A. alternata was the major pathogen during storage and shelf time.

4 Discussion Most pathogenic fungi inciting postharvest diseases of fruit attack wounds and cause decay immediately after

TIAN Shi-Ping et al.: Application of Antagonistic Yeasts Under Field Conditions and Their Biocontrol Ability Against Postharvest Diseases of Sweet Cherry 1327

Fig.3. Effects of preharvest application of Trichosporon pullulans, Cryptococcus laurentii, and Rhodotorula glutinis in orchards in Beijing (A, B) and Jinzhou (C, D) against postharvest diseases on sweet cherry fruit subsequently stored at 25 °C. There were three replications in each treatment and the experiment was repeated twice. Bars represented standard deviations of the means. Values followed by different letters were significantly different according to Duncan’s multiple range test at P = 0.05.

Fig.4. Effects of preharvest application of Trichosporon pullulans, Cryptococcus laurentii and Rhodotorula glutinis in orchards in Beijing (A, B) and Jinzhou (C, D) against postharvest diseases on sweet cherry fruit subsequently stored at 0 °C. There were three replications in each treatment and the experiment was repeated twice. Bars represented standard deviations of the means. Values followed by different letters were significantly different according to Duncan’s multiple range test at P = 0.05.

harvest (Sugar and Spotts, 1993; Spotts et al., 1998). Thus, application of biocontrol agents as close as possible to the time of wounding should provide the best opportunity to protect the fruit (Wilson and Pusey, 1985; Robberts, 1994).

Consequently, timing of applications of biocontrol agents is very important. Benbow and Sugar(1999)emphasized the important consideration in the context of preharvest application of the ability of antagonists to

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Fig.5. Effects of preharvest application of Trichosporon pullulans, Cryptococcus laurentii and Rhodotorula glutinis in orchards in Beijing (A, B) and Jinzhou (C, D) against postharvest diseases on sweet cherry fruit subsequently stored in controlled atmosphere (CA) condition at 0 °C. There were three replications in each treatment and the experiment was repeated twice. Bars represented standard deviations of the means. Values followed by different letters were significantly different according to Duncan’s multiple range test at P = 0.05.

survive at sufficient populations on the fruit surface after application. Good survival ability should allow antagonists to colonize wounds quickly. In the present study, C. laurentii was the most effective antagonist for control of postharvest decay of sweet cherry under different storage conditions and among the three yeasts (Figs.3-5). This antagonist could survive at high, relatively stable population levels on the surface of sweet cherry fruit under field conditions. In contrast, T. pullulans did not effectively control decay of sweet cherry under the different storage conditions. Its lower efficacy may be due to a rapid decrease in population numbers after the 4-d of application (Fig.1). These results agree with the view of Wilson and Wisniewki (1989), who considered that a major problem in biocontrol was maintaining a high population of an active biocontrol agent throughout the entire period of disease activity. Effective suppression of plant diseases by biocontrol agents is largely influenced by environmental conditions. Environment affects the establishment, survival and activity of the biocontrol agents (Benbow and Sugar, 1999). Both C. laurentii and R. glutinis remained at high and stable population levels on the surface of sweet cherry fruit under field conditions. However, C. laurentii more significantly reduced the fruit decay than R. glutinis (Figs.4, 5). The difference may relate to the adaptability of antagonists to postharvest storage environments (temperature, O2 and CO2

concentrations). C. laulentii apparently had better adaptability to storage conditions in comparison with R. glutinis. Antagonistic activity on fruit surfaces or in wounds depend well also on relative biocontrol ability against pathogenic fungi. Adaptability of the yeasts to storage environmental conditions influence the ability to control postharvest disease (Droby et al., 2002; Tian et al., 2002a). The positive characteristics of C. laulentii and R. glutinis coupled with the apparent evident broad range of activity and the ability to control decay at storage temperatures could pave the way for a practical use of these species. High populations on unwounded fruits under field conditions suggest that these isolates will perform well when applied before harvest onto fruit surfaces under commercial conditions. Moreover, the use of the yeasts prior to harvest might also reduce incidence of latent infections by pathogens that do not require wounds for infection (Biggs, 1995). The results of this study show that preharvest application of antagonistic yeasts should provide an alternative to chemical application. References: Arras G. 1996. Mode of action of an isolate of Candida famata in biological control of Penicillium digitatum in orange fruits. Postharvest Biol Tech, 8: 191-198. Benbow J M, Sugar D. 1999. Fruit surface colonization and biological control of postharvest diseases of pear by preharvest

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Qin G-Z , Tian S-P, Liu H-B , Xu Y. 2003. Biocontrol efficacy of

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