Journal of Applied Microbiology ISSN 1364-5072
ORIGINAL ARTICLE
Effects of trehalose on stress tolerance and biocontrol efficacy of Cryptococcus laurentii B.Q. Li1,2 and S.P. Tian1 1 Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China 2 Graduate School of Chinese Academy of Sciences, Beijing, China
Keywords apple fruit, biocontrol, stress tolerance, trehalose, yeast. Correspondence Shi Ping Tian, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Xiangshan Nanxincun 20, Haidian District, Beijing 100093, China. E-mail:
[email protected]
2005/0665: received 12 June 2005, revised 20 July 2005 and accepted 20 July 2005 doi:10.1111/j.1365-2672.2006.02852.x
ABSTRACT Aims: To investigate the effects of internal trehalose on viability and biocontrol efficacy of antagonistic yeast Cryptococcus laurentii under stresses of low temperature (LT), controlled atmosphere (CA) and freeze drying. Methods and Results: The content of trehalose in C. laurentii was increased by culturing the yeast in trehalose-containing medium. Compared with yeast cells with low trehalose level, the yeast cells with high level of internal trehalose not only obtained higher viability, but also showed higher population and better biocontrol efficacy against Penicillium expansum on apple fruit both at 1C and in CA condition (5% O2, 5% CO2, 1C). After freeze drying, survival of the yeast with high trehalose level was markedly increased when stored at 25C for 0, 15 and 30 days. Meanwhile, high integrity of plasma membrane was detected in the freeze-dried yeast with high trehalose level by propidium iodide staining. Conclusions: Induced accumulation of internal trehalose could improve viability and biocontrol efficacy of C. laurentii under stresses of LT and CA. Moreover, survival of the yeast was also increased as internal trehalose accumulation after freeze drying, and one of the reasons might be that trehalose gave an effective protection to plasma membrane. Significance and Impact of the Study: The results of this experiment show a promising way to improve the biocontrol performance of antagonistic yeasts under the commercial conditions.
Introduction Postharvest diseases caused by fungal pathogens and spoilage micro-organisms result in the major losses of fruit (Eckert and Ogawa 1988). When permitted, synthetic fungicides are the primary means to control postharvest diseases (Spadaro and Gullino 2004). However, fungicide efficacy has been frequently decreased by the development of resistant strains of pathogens. In addition, public concern and regulatory restrictions about the presence of fungicide residues on plants have emphasized the need to find alternative methods for disease control (Wilson and Wisniewski 1994). Biological control using antagonists has been proved to be one of the most promising alternatives, either alone or as part of an integrated pest management policy to reduce pesticide use (Wilson 854
and Wisniewski 1994; Fan and Tian 2000). By comparison with antagonistic bacteria, yeasts have been pursued actively in recent years since producing of antibiotics or other toxic secondary metabolites were not involved in their activities against postharvest pathogens, generally. Many researches have shown that several yeast antagonists could effectively inhibit the development of postharvest pathogens on various fruits (Droby et al. 1989; McLaughlin et al. 1992; Qin et al. 2004). However, biological control is only effective when applying high concentrations of antagonistic yeasts (Fan and Tian 2001). Moreover, environmental stresses during formulation, distribution and application of biocontrol agents can decrease the viability of yeast cells (Tian et al. 2002b). In order to apply biocontrol microbial agents successfully in commercial treatment, combining biocontrol yeasts with some chemical
ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 100 (2006) 854–861
B.Q. Li and S.P. Tian
compounds, such as Ca (Tian et al. 2002a), salicylic acid (Qin et al. 2003) and sodium bicarbonate (Yao et al. 2004), has been proved to be an effective way in enhancing their biocontrol ability against pathogenic fungi. On the other hand, enhancing tolerance of biocontrol agents to stress conditions may be another useful approach. Trehalose, a nonreducing disaccharide and a major reserve carbohydrate, has been generally accepted as a protection metabolite against various stresses, such as heat, dehydration, freezing and hyperosmotic stress (Kwon et al. 2003). Based on the cell protection and viability improvement, trehalose has been used in cryogenic preservation of yeast strains, as well as in brewing, winemaking and dry yeast production (Aranda et al. 2004). But, there was little information about the application of trehalose in agriculture, especially in biological control of postharvest diseases on fruits. The objectives of this study were to evaluate the relationship between internal trehalose content of Cryptococcus laurentii and its tolerance to stress conditions such as freezing drying, low temperature (LT) and controlled atmosphere (CA) with low O2 + high CO2 concentrations and to determine the influence of trehalose on biocontrol efficiency of C. laurentii against Penicillium expansum in apple fruit under different storage conditions.
Effects of trehalose on antagonism
Fruit ‘Fuji’ apples (Malus domestica Borkh.) were harvested when commercially ripe from an orchard of the Institute of Forest and Fruit, the Beijing Academy of Agricultural Sciences. Fruits were classified according to uniformity of size and maturity without wounds or rot, and then surfaced-disinfected with 2% sodium hypochlorite prior to use according to the method of Yao et al. (2004). Determination of trehalose contents After 48 h, yeast cells were harvested by centrifugation at 7660 g for 3 min and washed three times with cold distilled water in order to remove residual medium. Trehalose was extracted from 10 mg yeast cells (dry weight) with water according to the method of Lee and Goldberg (1998) and determined using high-pressure liquid chromatography (HPLC, DIONEX-P680; Dionex, Sunnyvale, CA, USA). The HPLC was fitted with a CARBOSep COREGEL 87C column (300 · 7Æ8 mm), CARBOSep COREGEL 87C Cartridge as guard column (TRANSGENOMIC, San Jose, CA, USA) and a refractive index detector (RI-101; Shodex, Japan). The mobile phase was water at 0Æ5 ml min)1. There were three replicates in each treatment, the experiment was repeated twice.
Materials and Methods Stress tolerance Yeast Cryptococcus laurentii was isolated from the surfaces of apple fruit in the previous experiment (Liu et al. 2002) and identified by CABI Bioscience Identification Services (International Mycological Institute, Egham, UK). Yeast was cultured with nutrient yeast dextrose broth (NYDB: 1 g of beef extract, 5 g of soybean peptone, 5 g of NaCl, 7 g of yeast extract, and 10 g of glucose within 1000 ml distilled water). To induce accumulation of internal trehalose, yeast was cultured with nutrient yeast trehalose broth (NYTB: replacing glucose in NYDB with trehalose at the same concentration). The yeast was cultured in 50 ml conical flasks containing 20 ml of medium on a rotary shaker at 200 r.p.m. at 25C. Pathogen Penicillium expansum was isolated from decayed apple fruit. The pathogen was maintained on potato dextrose agar (PDA) at 4C, and inoculated and re-isolated on apples before the experiment. Spore suspension of P. expansum was obtained from 2-week-old cultures and adjusted to 1 · 104 spores ml)1 with sterile distilled water by a hemacytometer.
Yeast cells cultured with or without trehalose for 48 h were obtained by centrifugation. Paste of each treatment was washed for three times with sterile distilled water, then resuspended and divided into two parts. One part of the sample was adjusted to the concentration of 1 · 106 cells ml)1 with a hemacytometer. A 50 ll yeast sample was spread on NYDA plate (NYDB with agar) and cultured at 25 ± 1C, 1 ± 1C and CA (5% O2, 5% CO2) at 1C, respectively. In CA treatment, plates were placed in a CA cabinet, which was linked with an atmosphere analyser (Fruit slr control FC-710; N. Copernico, Milan, Italy). After 12 h and 7, 15 days, plates were observed with a Zeiss Axioskop 40 microscope (Carl Zeiss, Oberkochen, Germany) and the numbers of viable cells and nonviable cells were counted, respectively. At least 400 cells were counted and the viable percentage was calculated. Survival rates under LT or CA stresses were expressed as viable percentage at 1C or under CA condition relative to that at 25C. There were three replicates in each treatment, the experiment was repeated twice. Another part of the yeast sample was adjusted to the concentration of 2 · 108 cells ml)1. Tenfold dilutions of this suspension were prepared and spread onto plates in order to calculate the initial concentration. Plates were
ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 100 (2006) 854–861
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incubated at 25C for 72 h and the initial number (CFU ml)1) was measured. A 250 ll sample was added to 1Æ5-ml autoclaved Eppendorf tube and frozen at )20C overnight. These frozen samples were freeze-dried for 6 h with a freeze-drier ()50C, 1 Pa) (Labconco Corporation, Kansas City, MO, USA) and then stored at 25C. After 0, 15 and 30 days, freeze-dried sample was rehydrated to its original volume with sterile distilled water for 10 min at 25C and CFU ml)1 was determined as described above. Survival rates were expressed as percentages of the number of colonies after freeze drying relative to the number of colonies in the untreated controls. There were three replicates in each treatment, the experiment was repeated twice. Membrane integrity assay and microscopy Freeze-dried cells of C. laurentii were rehydrated with 500 ll pH 7Æ4 phosphate buffer solution (PBS) for 10 min, and stained with 10 lg ml)1 propidium iodide (PI) for 5 min at 30C. Then yeast cells were collected by centrifugation, and washed twice with PBS (pH 7Æ4) to remove residual dye. The cells were observed on a Zeiss Axioskop 40 microscope (Carl Zeiss) equipped with individual fluorescein rhodamine filter set (Zeiss no. 15: excitation BP 546/12 nm, emission LP 590 nm), and imaged using an Axiocam MRc digital camera (Carl Zeiss). Pictures were then processed by the Adobe Photoshop 7.0 (Adobe, San Jose, CA, USA). Three fields of view from each cover slip were randomly chosen, and the number of cells in brightfield was defined as total number. Membrane integrity (MI) was calculated with the following formula: MI ¼[1 ) (number of stained cells/number of total cells)] · 100%, and relative MI was defined as the rate of MI after and before freeze drying. Biocontrol assays of yeasts under different storage conditions Three wounds (uniform 4 mm deep · 3 mm wide) were made on the cheek of each apple fruit with a sterile nail. Yeast cells of C. laurentii were cultured for 48 h before inoculation. These fruits were inoculated with aliquots of 20 ll washed cell suspension of the yeast growing in NYDB or NYTB at 1 · 107 cells ml)1, respectively. The same volume of sterile distilled water was pipetted into each wound site as the control. Yeast cells freeze-dried as above procedure were rehydrated with sterile distilled water for 10 min, adjusted to the concentration of 1 · 107 cells ml)1, and pipetted into the wounds with aliquots of 20 ll. After 2 h, a 20 ll suspension of P. expansum at 1 · 104 spores ml)1 was inoculated into each wound. These treated fruits were put in 400 · 300 · 100 mm plastic boxes and stored at 25C, 1C and CA condi856
tion at 1C, respectively. Fruit treated with freeze-dried yeast cells were stored only at 25C. Plastic trays, kept at 25C and 1C, were put in high density polyethylene bags in order to retain high humidity (about 95% relative humidity). Disease incidence and severity (lesion diameter) caused by P. expansum were determined at regular times (each day at 25C, each week at 1C and each 15 days under CA condition, respectively). Disease severity was divided to five levels according to lesion diameter: 0 ¼ no infection, 1 ¼ lesion diameter
