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Interaction Between Cryptococcus laurentii, Monilinia fructicola, and Sweet Cherry Fruit at Different Temperatures
Available online at www.sciencedirect.com '#
Agricultural Sciences in China 2008, 7(1): 48-57
*8b
.1. * ScienceDirect
January 2008
Interaction Between Cryptococcus laurentii, Monilinia fructicola, and Sweet Cherry Fruit at Different Temperatures WANG You-shengl-2and TIAN Shi-ping2 1
Beijing Key Laboratory of Plant Resources Research and Development, Beijing Technology and Business University, Beijing 100037, P.R.China Key Laboratory of' Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, P.R.China
Abstract The present study was performed mainly to investigate the antagonist-pathogen-host interaction in wounds of the sweet cherry fruits. The antagonistic yeast Cryptococcus laurentii could significantly reduce the brown rot of the sweet cherry fruit caused by Monilinia fructicola at 25 and 1°C. The populations of yeast increased faster in the presence of the pathogen initially, but then decreased rapidly. In the fruits inoculated with M. fructicola alone or combined with C. laurentii, an induction of lipid peroxidation as well as activities of the antioxidant enzymes, such as, superoxide dismutases (SOD), catalase (CAT), and peroxidase (POD), was observed. The isoenzyme pattern of polyphenol oxidase (PPO) changed greatly after the symptoms appeared, with new PPO isoforms being induced. By contrast, the induction of lipid peroxidation and activities of SOD, CAT, and POD were low, although no significant changes were found in the PPO isoenzyms in the fruits inoculated with antagonist C. laurentii alone. The inhibition of brown rot during the antagonistpathogen-host interaction in wounds of the sweet cherry fruits was mainly on account of the stimulated growth of C. laurentii as well as the induction of antioxidant enzymes of the fruits by M. fructicola. Key words: Cryptococcus laurentii, Monilinia fructicola, sweet cherry, interaction
INTRODUCTION It is essential to investigate the interactions between host, pathogen, and antagonist, so as to improve the biocontrol ability of antagonistic yeast, because wounds are the main approach through which pathogens attack fruits, whereas, the antagonistic yeasts show their biocontrol ability during postharvest periods (Droby and Chalutz 1994; Filonow 1998). Currently, most studies focus on the mode of action of biocontrol yeast, and it is confirmed that the antagonistic yeast can attach to the pathogen along with
the secretion of extracellular lytic enzymes, and induce the fruit to produce some hydrolyzed enzymes, such as, chitinase and p- 1,3-glucanase, besides which they reproduce rapidly in wounds of fruits to compete for nutrition and space with pathogens (Ippolito et al. 2000; Wan and Tian 2002). Of late, Castoria et al. (2003) has reported that the oxidative stress in wounds of the fruit also shows a significant influence on the biocontrol ability of the antagonistic yeast. Thus, further detailed information is needed with regard to the response of pro- and anti-oxidant enzymes in the fruits to pathogen and antagonistic yeast. The sweet cherry fruits were susceptible to spoil-
This paper is translated from its Chinese version in Scienria Agricultura Sinica WANG You-sheng, Ph 0.Tel: 46-10-68984940, E-mail: [email protected]; Correspondence TIAN Shi-ping, Tel: +86-10-62836559, Fax: +86- 10.82594675, E-mail: [email protected]
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Interaction Between Cryptococcus laurentii, Monilinia fructicola, and Sweet Cherry Fruit at Different
ing and Monilinia fructicola was the major pathogen (Tian et al. 2001). Meanwhile, the yeast CryptoCOCCUS inurentiis was isolated from the surface of apples and had proved to be an effective biological control agent against the major pathogens of different fruits (Qin et al. 2003a, 2004). However, no detailed work have been done with regard to the biocontrol ability evaluation of C. laurentii against M. fructicolu on sweet cherry fruit, as well as on the investigation of its mechanism. The present work was mainly to investigate the antagonist-pathogen-host interactions in wounds of the sweet cherry fruits at different temperatures, especially the influence of pathogen and/or antagonist inoculation on pro- and anti-oxidant enzyme in fruit, which could provide more detailed information for the biocontrol of brown rot disease of sweet cherry fruit.
MATERIALS AND METHODS Fruit The sweet cherry fruits (Prunus avivum L. cv. Van) were harvested from an orchard in the Institute of Forestry and Pomology, Beijing Academy of Agriculture and Forestry Sciences, China, and immediately transported to the laboratory. The fruit firmness was 3.6 newton (N), soluble solid content (SSC) was 14.8%, and the pH value was 3.45 at the beginning of the experiment. The fruits were selected according to the uniformities of color, maturity, size and weight. The surface was disinfected with 2% sodium hypochlorite solution for 5 min, and then washed with tap water and dried in air for the experiment.
Pathogen M . fructicola was isolated from the infected sweet cherry fruit and cultured on potato dextrose agar (PDA). A spore suspension was obtained by flooding one-weekold PDA cultures of M. fructicola with sterile distilled water containing 0.05% (v/v) Tween 80. Spore concentration of the pathogen was determined with a hemacytometer and adjusted with sterile distilled water to a concentration of 5 x lo4 spores mL-'.
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Antagonistic yeast The antagonistic yeast, C. laurentii, was isolated from the surface of apple fruit and identified by the CAB1 Bioscience Identification Services (International Mycological Institute, UK). The yeast was grown in 250mL conical flasks containing 50 mL nutrient yeast dextrose broth (NYDB: 8 g nutrient broth, 5 g yeast extract, and 10 g dextrose in 1 000 mL water), on a rotary shaker at 200 r/min for 48 h at 25°C. The yeast cells were harvested by centrifugation at 4 000 x g for 10 min, resuspended in sterile distilled water, and adjusted to a c o n c e n t r a t i o n of 1 x l o * c e l l s m L - ' u s i n g a hemocytometer.
Biocontrol assays on the sweet cherry fruits A uniform wound of 4 mm deep and 3 mm in diameter was made in the equatorial zone (one wound per fruit) with a flame-sterilized needle, The wounds were inoculated with 20 pL of C. laurentii at lo8cells mL-', 20 yL of M . fructicola suspension at 5 x lo4 spore mL-', or 20 yL of C . laurentii at lo8cells mL-I plus 20 yL of M . fructicola suspension at 5 x lo4 spores mL-'. The fruits inoculated with 20 pL of distilled water served as the control. The treated fruits were arranged separately on 200 mm x 150 mm x 50 mm plastic trays. The tray was placed in a plastic bag to maintain relative humidity at about 95% and stored at 25 and 1 ° C respectively. Infection rate, lesion diameter, and some enzymes were measured at regular times. There were 10 fruits in each treatment with three replicates arranged in complete randomization.
Population dynamics of the yeasts The fruits were inoculated with 20 yL of C. laurentii suspension at 1 x lo8cells mL-' or 20 yL of C . lnurentii suspension at 1 x l o 8 cells m L - ' plus 20 V L of M . fructicola at 5 x lo4 spores mL-'. The treated fruits were arranged separately on 200 mm x I50 mm x 50 mm plastic trays. The tray was placed in a plastic bag to maintain relative humidity at about 95% and stored at 25 and l"C, respectively. The wounded tissue of five fruits was removed with a cork borer (1 cm diameter and 1 cm deep), ground with a mortar and pestle in
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1 mL of 0.05 M phosphate buffer at pH 7.0. Serial tenfold dilutions were made in phosphate buffer. Each dilution of 0.1 mL was spread on NYDA medium in Petri plates. The plates were inoculated at 26°C for 48 h and the colonies were counted. Colony counts were expressed as log,,CFU/wound. There were five fruits in each treatment with three replicates arranged in complete randomization.
Preparation of extracts and determination of antioxidant enzymes Enzyme extraction The fruit flesh (3 g) was obtained from the area 2 mm away from the wounds and homogenized in 20 mL ice-cold extraction buffer phosphate (100 mM, pH 7.8, 0.5 g of polyvinyl polypyrrolidone). The homogenate was centrifuged at 18 000 x g for 50 min at 4°C and the resulting supernatants were used for enzyme activity assay and isoenzyme analysis. SOD activity The SOD activity was analyzed by monitoring the inhibition of the photochemical reduction of nitro blue tetrazolium (NBT) according to the method of Constantine and Stanley (1977). The reaction mixture (3 mL) contained 50 mM sodium phosphate buffer (pH 7.8), 13 mM methionine, 75 pM nitroblue tetrazolium (NBT), 10 pM EDTA, 2 pM riboflavin, and 0.1 mL enzyme extract. The mixtures were illuminated by a fluorescent lamp (60 pmol m-2s-') for 10 min and then the absorbance was determined at 560 nm. Identical solutions held in the dark were served as blanks. One unit of SOD was defined as the amount of enzyme that caused a 50% decrease in the SOD-inhibitable NBT reduction. CAT activity Determination of CAT activity was performed according to the method of Beers and Sizer (1 952) with slight modifications. The standard assay mixture consisted of 2 mL sodium phosphate buffer (50 mM, pH 7.0), 0.5 mL H,O, (40 mM), and 0.5 mL enzyme in a total volume of 3.0 mL. The decomposition of H 2 0 2was measured by the decline in absorbance at 240 nm. The specific activity was expressed in units per g fresh weight, where one unit of catalase converted one pmol of H20, per rnin E = 36 mM-' cm-I). POD activity POD activity was assayed according to
WANC You-sheng et al.
the method of Chance and Maehly (1959, with slight modifications. 0.5 mL enzyme in 2 mL buffered substrate (100 mM sodium phosphate, pH 6.4 and 8 mM guaiacol) was incubated for 5 rnin at 30°C and the increase were measured in absorbance at 460 nm for 120 s after adding 1 mL H20, (24 mM). The specific activity was expressed in units per g fresh weight, where one unit of POD converted one pmol of guaiacol per rnin (E = 26.6 mM-I cm-I). PPO activity PPO activity was determined according to the method of Waite (1976) with slight modifications. The reaction was initiated by incubating 0.5 mL enzyme preparation in 3 mL buffered substrate ( 1 O O m M sodium phosphate, pH 6.4, and 500 mM catechol) and monitoring the change of absorbance at 398 nm for 10 s. The specific activity was expressed in units per one g fresh weight, where one unit of PPO converted one pmol of catechol per rnin (E = 1.4 mM-' cm-I). Isoenzyme analysis Isoenzymes were separated by native polyacrylamide gel electrophoresis (PAGE) using 10% separating and 4% stacking gels. An equal amount of protein was loaded in each lane and electrophoresis was carried out at 4°C under nondenaturing conditions. Following electrophoretic separation, the gels were stained for PPO isoenzymes by the procedure described by Mohammadi and Kazemi (2002). The gel was incubated in 100 mM citrate-200 mM Na phosphate buffer containing 15 mM catechol and 0.05% phenylenediamine, till the PPO activity-containing brown band was visualized carefully. Then the gel was washed in 1 mM ascorbic acid for 5 min to stop the reaction.
Preparationof extracts and determination of lipid peroxides The fruit flesh was collected from the area 2 mm far from the wounds and homogenized in 15 mL 10% ice-cold extraction. The homogenate was centrifuged at 18OOO x g for 50 rnin at 4"C, and the supernatants were used directly for assay, according to the method of Buege and Aust (1978). The determination was conducted by adding 0.8 mL 0.6% thibarbituric acid (TBA) in 15% trichloroacetic acid to 1.0 mL of the crude enzyme sample. The solution was heated at 90°C for 20 min, quickly cooled in ice bath for 5 min, and then centri-
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Interaction Between Cryprococcus laurenfii, Monilinia fructicola, and Sweet Cherry Fruit at Different
fuged at 12 000 x g for 10 min to clarify the solution. Absorbance at 532 nm was measured and subtracted from the nonspecific absorbance at 600 nm. The amount of lipid peroxide was calculated with an extinction coefficient of 155 mM cm-'.
Data analysis To test the significance of the effect of treatments, mean separations were performed by a paired sample T-test or Duncan's multiple range test with SPSS. The differences at P < 0.05 were considered significant.
RESULTS Biocontrol assays on the sweet cherry fruits The sweet cherry fruits inoculated with M. fructicola had an incidence of infection of 100% with lesion diameters of 13.4mm after 48 h at 25°C and the lesion diameter enlarged to 22.1 mm after 96 h (Fig.1). In contrast, no disease symptom was found in the fruits inoculated by C. laurentii +M. fructicola within 48 h,
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and only 13% of the fruits were infected with lesion diameters of 2.2 mm after 96 h. In all treatments, when stored at 1°C for 21 d, no fruit got brown rot disease, indicating that low temperature delayed the decay of sweet cherry fruits caused by M. fructicola. All fruits inoculated with M.fructicola alone got the disease after 40 d, and their lesion diameters expanded from 12.6 to 32.7mm during the subsequent shelf-life of 25°C for 3 d. However, no disease incidence was observed in fruits inoculated with C . laurentii+M. fructicola after 40 d, and the rate of disease incidence was only 35% with lesion diameters of 5.9 mm when transferred to 25°C for 3 d.
Population dynamics of antagonistic yeast The antagonistic yeast C. laurentii grew rapidly in the wound of the sweet cherry at both 25 and 1 "C (Fig.2). The yeast growth was stimulated significantly in the fruits inoculated with C. laurentii+M. fructicola at an early stage, whereas, it was inhibited after 108 h and 21 d, respectively, at 25 and 1°C. The results indicated that the influence of the pathogen on the growth of the antagonist was time-dependent, showing a promotion
Fig. 1 Disease incidence (top) and lesion diameter (bottom) in the sweet cherry fruits inoculated with M. fructicola alone and M . frucficola + C . laurentii at 25 and 1°C.
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WANG You-sheng et al.
at an earlier stage while an inhibition at a later stage.
Lipidperoxidation When stored at 25"C, lipid peroxide was rapidly induced in the fruits inoculated with only M . fructicola or combined with C. laurentii, and the fruits inoculated with only M . fructicola showed significantly higher severity of lipid peroxidation than those inoculated by C. lauentii+M. fructicola at 60 h (Fig.3). In contrast, the induction of lipid peroxidation by C. laurentii alone was not evident until 36 h after inoculation and the intensity was much lesser when compared with that of M . fructicola. The content of fruit lipid peroxide increased during the first seven days of storage at 1"C, but no e v i d e n t d i f f e r e n c e b e t w e e n t r e a t m e n t s was observed. Similarly, clear elevation of lipid peroxide levels appeared at 21 d in the fruits inoculated with M . fructicola, whereas, there was no significant difference between the yeast-inoculated fruit and the control during the whole storage time. The inoculation of C . lauentii + M . fructicola also could not induce evident lipid peroxidation in the sweet cherry fruits during the whole storage period at l "C, but lipid peroxidation was significantly evaluated when the C. lauentii + M . fructicola-inoculated fruit was stored at 1°C for 40 d and then transferred to 25°C.
Fig. 2 Population dynamics of C. laurentii in wounds of the sweet cherry fruits with and without M. fructicola at 25 and 1°C. At each point of time, data followed by different letters were significantly different according to paired sample T-test at P c 0.05.
SOD activity SOD activity increased at first and then decreased in the control fruit during the storage at 25°C (Fig.4). SOD activity in the M .fructicola-inoculated fruit was induced during the first 12 h of postinoculation,but was inhibited before the fruit disease appeared (at 36 h), and then induced after the fruit disease appeared (at 60 h). In comparison, no evident difference in SOD activity was observed between the C. lauentii + M .fructicola-inoculated fruit and the control during the first 12 h, and induction was also observed at the end of the experiment. In the C. lauentii-inoculated fruit, SOD activity was inhibited at 12 h, whereas, induced thereafter. SOD activity in all the treated fruits maintained at a steady level in the first seven days and there was no
Fig. 3 Changes of lipid peroxidation production in the sweet cherry fruits inoculated with C. laurentii, C. laurentii + M . fructicola, M . fructicola, and the control at 25 and 1°C. At each point of time, data followed by different letters were significantly different according to Duncan's multiple range test at PcO.05. The same as below.
significant difference between treatments, however, it increased to become 2.8-3.3 times higher than the initial level after 21 d. SOD activity was significantly evaluated by C. laurentii or M . fructicola as compared to the
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Interaction Between Cryptococcus laurentii, Monilinia fructicola, and Sweet Cherry Fruit at Different
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Fig. 4 Changes of SOD activity in the sweet cherry fruits inoculated with C. laurentii, C. laurentii + M.fructicola, M.fructicola, and the control at 25 and 1°C.
Fig. 5 Changes of CAT activity in the sweet cherry fruits inoculated with C. laurentii, C. laurentii + M . fructicola, M. fructicola, and the control at 25 and 1°C.
control, and the induction of C. lauentii + M . fructicola showed a coordinated effect of C . laurentii and M . fructicola. When transferred to 25°C after storage at 1°C for 40 d, SOD activity in the C . lauentii+ M . fructicola and M . fructicola-inoculated fruits were significantly higher than that of the control, whereas, no significant difference was found between the C. laurentii-inoculated fruit and the control.
POD activity Activity of POD in M . fructicola-inoculated fruit increased during the first 12 h of postinoculation, however, it was inhibited at 36 h, before which the disease appeared and then elevated to about 2.6-3.4 0
CAT activity When stored at 25"C, CAT activities increased both in the M . fructicola and C. lauentii + M . fructicola-inoculated fruits within 12 h, and the evaluations were markedly higher in the fruits inoculated by M . fhcticolu alone (Fig.5). CAT activity in the fruits inoculated by M . fructicola was inhibited before the symptoms appeared (36 h), however, elevated rapidly after the disease appeared (60 h). In contrast, the CAT activity in the C . laurentii-inoculated fruits showed no significant difference as compared with the control, during the whole experiment. CAT activity in the fruits inoculated by M.fructicola alone and C. lauentii + M. fructicola was significantly higher than in the control, whereas, the inoculation of C. laurentii alone showed minor effect during the storage at 1°C.
Fig. 6 Changes of POD activity in the sweet cherry fruits inoculated with C. laurentii, C . laurentii+M.fructicola, M.fructicola, and the control at 25 and 1°C.
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WANG You-sheng et uI.
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times higher than that of the other treatments, at 60 h, when the fruit disease appeared (Fig.6). In comparison, no evident difference in POD activity was observed between the C . lauentii-inoculated fruits and the control during the first 12 h, and POD activity in the C. lauentii-inoculated fruits subsequently increased, and the induction effect increased with time. In the C. lauentii + M . fructicola-inoculated fruits, the POD activity was induced during the whole storage time and the induction effect was gradually strengthened with time. POD activity in the control fruit kept steady during the whole storage at 1°C. In contrast, the induction effect of C . laurentii, M . fructicola, and C . lauentii + M.fructicola inoculation on POD activity evaluated with time, and increased as high as 2.3, 1.9, and 1.7 times of the control at the end of low temperature storage.
PPO activity PPO activity in all treatments elevated to about 1.9-3.3 times as compared with the initial level at 12 h at 25°C (Fig.7). However, in both C. lauentii and M . fructicolatreated fruit, the PPO activity was significantly lower than the control during the whole experiment. While
the PPO activity in the C. lauentii+M. fructicoln-inoculated fruit was stimulated during the first 36 h, but inhibited thereafter. Activity of PPO in all treatments elevated 2.4-2.7 times within seven days when stored at 1"C, and decreased thereafter, and then increased evidently at 40 d. However, no evident difference was observed in PPO activity of treated fruit and the control at 1°C.
lsoenzyme pattern for PPO PPO isoenzymes pattern in the sweet cherry fruits varied greatly during the course of disease development of M . fructicola at 25°C (Fig.8-A). Isoenzyme bands of PPO in the b region disappeared at 36 h, before whch the disease symptoms occurred, whereas, five new isoenzyme bands in a and c regions appeared at 60 and 108 h after the symptoms occurred. It could be deduced that the five new isoenzymes bands were induced by the pathogen M . fructicola, as they were not observed in the fruits inoculated by C . laurentii alone or C. laurentii+M.fructicola at the same time. New isoenzymes appeared in the d region in all treatments when stored at 1°C as compared to 25"C, which indicated that these new isoenzymes were induced by low temperature (Fig.8-B). Isoenzyme bands of PPO in the b region disappeared, whereas, additional bands in the c region were observed in the M . fructicolainoculated fruits at 48 d when disease symptoms in all fruits appeared, suggesting that M . fructicola inhibited the expression of isoenzymes in the b region, whereas, induced those in the c region. In contrast, the intensity of PPO isoenzymes in the b region increased considerably with time in the other treatments, but became weak when the fruits were transferred to 25°C. Therefore, it appeared that the increased intensity of PPO isoenzymes in the b region correlated with increased PPO activity at a later stage.
DISCUSSION
Fig. 7 Changes of PPO activity in sweet cherry fruit inoculated with C. laurentii, C. laurentii+M.fructicola, M .fructicola, and the control at 25 and 1°C.
Data from the present experiment showed that when the fruits were inoculated by M . fructicola alone, disease could be found in the sweet cherry at both 1 and 25°C. The antagonistic yeast C. Zuurentii could delay
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Interaction Between Cryprococcus luurenrii, Monilinia frucricola, and Sweet Cherry Fruit at Different
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Fig. 8 Active staining of polyphenol oxidase in sweet cherry fruit at 25°C (A) and 1°C (B). Y, C. luurenrii-inoculated; YP, C. luurentii M. fructicolu co-inoculated; P, M . fructicolu-inoculated; C, control.
the incidence and reduce the severity of brown rot in the sweet cherry fruits, but disease was found partly in the C. Zauentii + M. fructicola-inoculated fruit at the end of the experiment, showing that C. Zaurentii inhibited rather than killed the pathogen M. fructicola, which was in correspondence with the characterization of antagonistic yeast (Janisiewicz and Korsten 2002). In addition, it was obvious that the growth of C. laurentii was stimulated at an early stage and inhibited at a later stage in fruit treated with C. luuentii+M. fructicola as compared with that inoculated with C. lauentii alone. Similar results were also found in the authors' previous studies (Fan and Tian 2001). It remains to be further investigated as to how M.fructicola stimulated C. laurentii to grow rapidly at the earlier stage. The accumulation of reactive oxygen species (ROS) could cause lipid peroxidation, and SOD, CAT, as well as POD, are the major enzymes in metabolizing ROS (Rice-Evans et al. 1991; Bolwell et al. 1995; Deighton et aZ. 1999). Thus, the formation of lipid peroxidation, along with the induction of antioxidant enzymes, indicated an excessive production of ROS and oxidative stress. The present results showed that M.fructicola promoted SOD, CAT, and POD activities within 12 h in fruits stored at 25"C, indicating that M . fructicola in-
+
duced fruit to produce excess ROS and thus led to oxidative stress (Baker and Orlandi 1995). Although the activities of antioxidant enzymes were significantly inhibited before disease incidence (36 h), the sustained increasing of lipid peroxidation indicated that excess ROS was indeed found. Similarly, the infected rate of brown rot was reduced in fruit stored at 1"C, but the elevation of lipid peroxidation and antioxidant enzyme activity were also observed at the earlier stage of inoculation (21 d), as well as when symptoms appeared (40 d). The results showed that the fruits inoculated with M . fructicola produced excess ROS and thus led to oxidative stress at two temperatures. In comparison, SOD activity was significantly inhibited within the first 12 h, in the fruits inoculated with C. laurntii alone, whereas, no significant difference was found in CAT and POD activities as well as in lipid peroxidation, showing that the antagonist alone could not lead to oxidative stress during this period. It could be speculated that C. laurntii mainly competed for nutrition and space in fruit wounds at the early stage and the number of C. laurntii was not large enough to induce the antioxidant enzymes of the host. At 108 h, the increased levels of SOD and POD activities as well as lipid peroxide content, combined with the decline of
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CAT activity in the fruits inoculated with C. laurntii, could be because of the strong elevation in yeast number and the promotion of the influence of yeast, but it could not be excluded that the antagonist might secrete something that acted as an elicitor when the nutrition was used up. This could be further confirmed by the results that POD activity in fruit inoculated C. laurntii also increased greatly as compared with the control after 40 d of storage at 1°C. However, the antagonist C. laurntii showed less influence on fruit when compared with the pathogen M . fructicola. From the fact that SOD, CAT, and.POD activities as well as the rate of lipid peroxidation significantly increased in fruit inoculated by C. lauentii+M. fructicola, together with the results that the C'laurntii alone could not lead to the oxidative stress of fruit, it can be deduced that the oxidative stress stimulated by C. lauentii + M . fructicola mainly contributed to the pathogen M . fructicola at the early stage. It was probable that M . fructicola not only stimulated the growth of C. laurntii, but also induced ROS production of fruit at the earlier stage of inoculation, and that the influence of M . fructicola on the fruit still existed in wounds inoculated by C. lauenrii + M . fructicola. In contrast, the activities of antioxidant enzymes and the degree of lipid peroxidation in the C. lauentii +M. fructicolainoculated fruit indicated that serious oxidative stress occurred around the wound in agreement with the coordinated induction of both C. lauentii and M .fructicola at 108 h, which might be one of the reasons that the number of C. laurntii became less and at the same time the disease incidence was found in part of the fruits. Thus, the M. fructicola might attack the sweet cherry fruit by making the host accumulate excess ROS, which killed the host and antagonist cells. The results supported the view of Tiedemann (1997), who stated that Botrytis cinerea could cause disease by inducing the host to produce a large amount of ROS. PPO was capable of oxidizing a range of phenolic acids in the fruit to quinine, which had two aspects to its functions. On one hand, quinine accumulated and cross linked with amino acid and proteins and thus led to flesh browning (Martinez and Whitaker 1995). On the other hand, quinine was also implicated in pathogen resistance as it showed a much stronger killing ability
to fungi than phenolic acids (Mayer and Hare1 1979; Bashan et al. 1987). Previous investigations had demonstrated that both R. stolonifer and P. membranefaciens +R. stolonifer could elevate PPO activity in peach fruit (Qin et al. 2003b), whereas, the present study has demonstrated that both C. laurntii and M. fructicola inhibited the PPO activity in the sweet cherry. In addition, the disappearance of the PPO isoenzyme band in the b region along with the occurrence of the fruit disease at both temperatures revealed the relationship between PPO isoenzyme of the b region and the fruit-induced resistance. In contrast, isoenzymes in the a and c regions might be just a passive reaction to the pathogen M .fructicola, as they appeared only after fruit incidence. There were different kinds of polyphenols in the sweet cherry fruit and each isoenzyme of PPO had its specific substrate. Thus, the variation of PPO isoenzyme pattern during pathogenesis, in the fruit inoculated with M. fructicola, represented the change of metabolic pathway of polyphenol in sweet cherry (Lanzarini et al. 1972). Future studies will need to address the relationship between PPO isoenzyme pattern and the fruit resistance in different conditions.
CONCLUSION In the interaction between C. laurentii, M . fructicola, and sweet cherry, C. laurentii could effectively inhibit brown rot disease incidence infected by M . fructicola at 25 and 1°C. M .fructicola could stimulate the growth of C. laurentii at the earlier stage, but inhibit it at a later stage. Inoculated M. fructicola alone or combined with C. laurentii could promote SOD, CAT, and POD activities, and accelerate lipid peroxidation. Therefore, the inhibition of brown rot during the antagonist-pathogen-host interaction in wounds of sweet cherry fruit was mainly because of the stimulated growth of C. laurentii, as well as the induction of antioxidant enzymes of fruit by M. fructicola.
Acknowledgements This work was supported by the Knowledge Innovation Program of the Chinese Academy of Sciences, China (KSCX2-YW-G-010), and the National Natural Science Foundation of China (30671473).
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Interaction Between Cryptococcus laurentii, Monilinia fructicola, and Sweet Cherry Fruit at Different
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11, 89-94. Janisiewicz W J, Korsten L. 2002. Biological control of postharvest diseases of fruits. Annual Review of Phytopathology, 40,411-441. Martinez M V, Whitaker J R. 1995. The biochemistry and control of enzymatic browning. Trends in Food Science and Technology, 6, 195-200. Mayer A M, Hare1 E. 1979. Polyphenol oxidases in plants. Phytochemistry, 18,193-215. Mohammadi M, Kazemi H. 2002. Changes in peroxidase and polyphenol oxidase activities in susceptible and resistant wheat heads inoculated with Fusarum graminearurn and induced resistance. Plant Science, 162,491-498. Qin G 2,Tian S P, Liu H B, Xu Y. 2003a. Biocontrol efficacy of three antagonistic yeasts against Penicillium expansum in harvested apple fruits, Acra Botanic Sinica, 45,417-421. Qin G 2,Tian S P, Liu H B, Xu Y. 2003b. Polyphenol oxidase, peroxidase and phenylalanine ammonium lyase in postharvest peach fruits induced by inoculation with Pichia membranefaciens or Rhizopus stolonifer. Scientia Agricultura Sinica, 36, 89-93. (in Chinese) Qin G Z, Tian S P, Xu Y.2004. Biocontrol of postharvest diseases on sweet cherries by four antagonistic yeasts in different storage conditions. Postharvest Biology and Technology, 31, 51-58. Rice-Evans C A, Diplock A T, Symons M C R. 1991. Investigations of the consequences of free radicl attack on lipids. In: Burdon R H, Knippenberg P H, eds, Techniques in Free Radical Research. Elsevier, Amsterdam. pp. 125183. Tiedemann A V. 1997. Evidence for a primary role of active oxygen species in induction of host cell death during infection of bean leaves with Botrytis cinerea. Physiological and Molecular Plant Pathology, 50, 151-166. Tian S P, Fan Q, Xu Y, Wang Y, Jian A L. 2001. Evaluation of the use of high CO, concentrations and cold storage to control Monilinia fructicola on sweet cherries. Postharvest Biology and Technology, 22,53-60. Waite J H. 1976. Calculating extinction coefficients for enzymatically produced o-quinones. Analytical Biochemistry, 75, 211-218. Wan Y K, Tian S P. 2002. Antagonistical mode of Pichia membranefaciens to Rhizopus stolonifer in wounds of peach fruit. Acta Botanic Sinica, 44, 1384-1386. (Edited by ZHANG Juan)
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Agricultural Sciences in China 2008, 7(1): 48-57
*8b
.1. * ScienceDirect
January 2008
Interaction Between Cryptococcus laurentii, Monilinia fructicola, and Sweet Cherry Fruit at Different Temperatures WANG You-shengl-2and TIAN Shi-ping2 1
Beijing Key Laboratory of Plant Resources Research and Development, Beijing Technology and Business University, Beijing 100037, P.R.China Key Laboratory of' Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, P.R.China
Abstract The present study was performed mainly to investigate the antagonist-pathogen-host interaction in wounds of the sweet cherry fruits. The antagonistic yeast Cryptococcus laurentii could significantly reduce the brown rot of the sweet cherry fruit caused by Monilinia fructicola at 25 and 1°C. The populations of yeast increased faster in the presence of the pathogen initially, but then decreased rapidly. In the fruits inoculated with M. fructicola alone or combined with C. laurentii, an induction of lipid peroxidation as well as activities of the antioxidant enzymes, such as, superoxide dismutases (SOD), catalase (CAT), and peroxidase (POD), was observed. The isoenzyme pattern of polyphenol oxidase (PPO) changed greatly after the symptoms appeared, with new PPO isoforms being induced. By contrast, the induction of lipid peroxidation and activities of SOD, CAT, and POD were low, although no significant changes were found in the PPO isoenzyms in the fruits inoculated with antagonist C. laurentii alone. The inhibition of brown rot during the antagonistpathogen-host interaction in wounds of the sweet cherry fruits was mainly on account of the stimulated growth of C. laurentii as well as the induction of antioxidant enzymes of the fruits by M. fructicola. Key words: Cryptococcus laurentii, Monilinia fructicola, sweet cherry, interaction
INTRODUCTION It is essential to investigate the interactions between host, pathogen, and antagonist, so as to improve the biocontrol ability of antagonistic yeast, because wounds are the main approach through which pathogens attack fruits, whereas, the antagonistic yeasts show their biocontrol ability during postharvest periods (Droby and Chalutz 1994; Filonow 1998). Currently, most studies focus on the mode of action of biocontrol yeast, and it is confirmed that the antagonistic yeast can attach to the pathogen along with
the secretion of extracellular lytic enzymes, and induce the fruit to produce some hydrolyzed enzymes, such as, chitinase and p- 1,3-glucanase, besides which they reproduce rapidly in wounds of fruits to compete for nutrition and space with pathogens (Ippolito et al. 2000; Wan and Tian 2002). Of late, Castoria et al. (2003) has reported that the oxidative stress in wounds of the fruit also shows a significant influence on the biocontrol ability of the antagonistic yeast. Thus, further detailed information is needed with regard to the response of pro- and anti-oxidant enzymes in the fruits to pathogen and antagonistic yeast. The sweet cherry fruits were susceptible to spoil-
This paper is translated from its Chinese version in Scienria Agricultura Sinica WANG You-sheng, Ph 0.Tel: 46-10-68984940, E-mail: [email protected]; Correspondence TIAN Shi-ping, Tel: +86-10-62836559, Fax: +86- 10.82594675, E-mail: [email protected]
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Interaction Between Cryptococcus laurentii, Monilinia fructicola, and Sweet Cherry Fruit at Different
ing and Monilinia fructicola was the major pathogen (Tian et al. 2001). Meanwhile, the yeast CryptoCOCCUS inurentiis was isolated from the surface of apples and had proved to be an effective biological control agent against the major pathogens of different fruits (Qin et al. 2003a, 2004). However, no detailed work have been done with regard to the biocontrol ability evaluation of C. laurentii against M. fructicolu on sweet cherry fruit, as well as on the investigation of its mechanism. The present work was mainly to investigate the antagonist-pathogen-host interactions in wounds of the sweet cherry fruits at different temperatures, especially the influence of pathogen and/or antagonist inoculation on pro- and anti-oxidant enzyme in fruit, which could provide more detailed information for the biocontrol of brown rot disease of sweet cherry fruit.
MATERIALS AND METHODS Fruit The sweet cherry fruits (Prunus avivum L. cv. Van) were harvested from an orchard in the Institute of Forestry and Pomology, Beijing Academy of Agriculture and Forestry Sciences, China, and immediately transported to the laboratory. The fruit firmness was 3.6 newton (N), soluble solid content (SSC) was 14.8%, and the pH value was 3.45 at the beginning of the experiment. The fruits were selected according to the uniformities of color, maturity, size and weight. The surface was disinfected with 2% sodium hypochlorite solution for 5 min, and then washed with tap water and dried in air for the experiment.
Pathogen M . fructicola was isolated from the infected sweet cherry fruit and cultured on potato dextrose agar (PDA). A spore suspension was obtained by flooding one-weekold PDA cultures of M. fructicola with sterile distilled water containing 0.05% (v/v) Tween 80. Spore concentration of the pathogen was determined with a hemacytometer and adjusted with sterile distilled water to a concentration of 5 x lo4 spores mL-'.
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Antagonistic yeast The antagonistic yeast, C. laurentii, was isolated from the surface of apple fruit and identified by the CAB1 Bioscience Identification Services (International Mycological Institute, UK). The yeast was grown in 250mL conical flasks containing 50 mL nutrient yeast dextrose broth (NYDB: 8 g nutrient broth, 5 g yeast extract, and 10 g dextrose in 1 000 mL water), on a rotary shaker at 200 r/min for 48 h at 25°C. The yeast cells were harvested by centrifugation at 4 000 x g for 10 min, resuspended in sterile distilled water, and adjusted to a c o n c e n t r a t i o n of 1 x l o * c e l l s m L - ' u s i n g a hemocytometer.
Biocontrol assays on the sweet cherry fruits A uniform wound of 4 mm deep and 3 mm in diameter was made in the equatorial zone (one wound per fruit) with a flame-sterilized needle, The wounds were inoculated with 20 pL of C. laurentii at lo8cells mL-', 20 yL of M . fructicola suspension at 5 x lo4 spore mL-', or 20 yL of C . laurentii at lo8cells mL-I plus 20 yL of M . fructicola suspension at 5 x lo4 spores mL-'. The fruits inoculated with 20 pL of distilled water served as the control. The treated fruits were arranged separately on 200 mm x 150 mm x 50 mm plastic trays. The tray was placed in a plastic bag to maintain relative humidity at about 95% and stored at 25 and 1 ° C respectively. Infection rate, lesion diameter, and some enzymes were measured at regular times. There were 10 fruits in each treatment with three replicates arranged in complete randomization.
Population dynamics of the yeasts The fruits were inoculated with 20 yL of C. laurentii suspension at 1 x lo8cells mL-' or 20 yL of C . lnurentii suspension at 1 x l o 8 cells m L - ' plus 20 V L of M . fructicola at 5 x lo4 spores mL-'. The treated fruits were arranged separately on 200 mm x I50 mm x 50 mm plastic trays. The tray was placed in a plastic bag to maintain relative humidity at about 95% and stored at 25 and l"C, respectively. The wounded tissue of five fruits was removed with a cork borer (1 cm diameter and 1 cm deep), ground with a mortar and pestle in
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1 mL of 0.05 M phosphate buffer at pH 7.0. Serial tenfold dilutions were made in phosphate buffer. Each dilution of 0.1 mL was spread on NYDA medium in Petri plates. The plates were inoculated at 26°C for 48 h and the colonies were counted. Colony counts were expressed as log,,CFU/wound. There were five fruits in each treatment with three replicates arranged in complete randomization.
Preparation of extracts and determination of antioxidant enzymes Enzyme extraction The fruit flesh (3 g) was obtained from the area 2 mm away from the wounds and homogenized in 20 mL ice-cold extraction buffer phosphate (100 mM, pH 7.8, 0.5 g of polyvinyl polypyrrolidone). The homogenate was centrifuged at 18 000 x g for 50 min at 4°C and the resulting supernatants were used for enzyme activity assay and isoenzyme analysis. SOD activity The SOD activity was analyzed by monitoring the inhibition of the photochemical reduction of nitro blue tetrazolium (NBT) according to the method of Constantine and Stanley (1977). The reaction mixture (3 mL) contained 50 mM sodium phosphate buffer (pH 7.8), 13 mM methionine, 75 pM nitroblue tetrazolium (NBT), 10 pM EDTA, 2 pM riboflavin, and 0.1 mL enzyme extract. The mixtures were illuminated by a fluorescent lamp (60 pmol m-2s-') for 10 min and then the absorbance was determined at 560 nm. Identical solutions held in the dark were served as blanks. One unit of SOD was defined as the amount of enzyme that caused a 50% decrease in the SOD-inhibitable NBT reduction. CAT activity Determination of CAT activity was performed according to the method of Beers and Sizer (1 952) with slight modifications. The standard assay mixture consisted of 2 mL sodium phosphate buffer (50 mM, pH 7.0), 0.5 mL H,O, (40 mM), and 0.5 mL enzyme in a total volume of 3.0 mL. The decomposition of H 2 0 2was measured by the decline in absorbance at 240 nm. The specific activity was expressed in units per g fresh weight, where one unit of catalase converted one pmol of H20, per rnin E = 36 mM-' cm-I). POD activity POD activity was assayed according to
WANC You-sheng et al.
the method of Chance and Maehly (1959, with slight modifications. 0.5 mL enzyme in 2 mL buffered substrate (100 mM sodium phosphate, pH 6.4 and 8 mM guaiacol) was incubated for 5 rnin at 30°C and the increase were measured in absorbance at 460 nm for 120 s after adding 1 mL H20, (24 mM). The specific activity was expressed in units per g fresh weight, where one unit of POD converted one pmol of guaiacol per rnin (E = 26.6 mM-I cm-I). PPO activity PPO activity was determined according to the method of Waite (1976) with slight modifications. The reaction was initiated by incubating 0.5 mL enzyme preparation in 3 mL buffered substrate ( 1 O O m M sodium phosphate, pH 6.4, and 500 mM catechol) and monitoring the change of absorbance at 398 nm for 10 s. The specific activity was expressed in units per one g fresh weight, where one unit of PPO converted one pmol of catechol per rnin (E = 1.4 mM-' cm-I). Isoenzyme analysis Isoenzymes were separated by native polyacrylamide gel electrophoresis (PAGE) using 10% separating and 4% stacking gels. An equal amount of protein was loaded in each lane and electrophoresis was carried out at 4°C under nondenaturing conditions. Following electrophoretic separation, the gels were stained for PPO isoenzymes by the procedure described by Mohammadi and Kazemi (2002). The gel was incubated in 100 mM citrate-200 mM Na phosphate buffer containing 15 mM catechol and 0.05% phenylenediamine, till the PPO activity-containing brown band was visualized carefully. Then the gel was washed in 1 mM ascorbic acid for 5 min to stop the reaction.
Preparationof extracts and determination of lipid peroxides The fruit flesh was collected from the area 2 mm far from the wounds and homogenized in 15 mL 10% ice-cold extraction. The homogenate was centrifuged at 18OOO x g for 50 rnin at 4"C, and the supernatants were used directly for assay, according to the method of Buege and Aust (1978). The determination was conducted by adding 0.8 mL 0.6% thibarbituric acid (TBA) in 15% trichloroacetic acid to 1.0 mL of the crude enzyme sample. The solution was heated at 90°C for 20 min, quickly cooled in ice bath for 5 min, and then centri-
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Interaction Between Cryprococcus laurenfii, Monilinia fructicola, and Sweet Cherry Fruit at Different
fuged at 12 000 x g for 10 min to clarify the solution. Absorbance at 532 nm was measured and subtracted from the nonspecific absorbance at 600 nm. The amount of lipid peroxide was calculated with an extinction coefficient of 155 mM cm-'.
Data analysis To test the significance of the effect of treatments, mean separations were performed by a paired sample T-test or Duncan's multiple range test with SPSS. The differences at P < 0.05 were considered significant.
RESULTS Biocontrol assays on the sweet cherry fruits The sweet cherry fruits inoculated with M. fructicola had an incidence of infection of 100% with lesion diameters of 13.4mm after 48 h at 25°C and the lesion diameter enlarged to 22.1 mm after 96 h (Fig.1). In contrast, no disease symptom was found in the fruits inoculated by C. laurentii +M. fructicola within 48 h,
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and only 13% of the fruits were infected with lesion diameters of 2.2 mm after 96 h. In all treatments, when stored at 1°C for 21 d, no fruit got brown rot disease, indicating that low temperature delayed the decay of sweet cherry fruits caused by M. fructicola. All fruits inoculated with M.fructicola alone got the disease after 40 d, and their lesion diameters expanded from 12.6 to 32.7mm during the subsequent shelf-life of 25°C for 3 d. However, no disease incidence was observed in fruits inoculated with C . laurentii+M. fructicola after 40 d, and the rate of disease incidence was only 35% with lesion diameters of 5.9 mm when transferred to 25°C for 3 d.
Population dynamics of antagonistic yeast The antagonistic yeast C. laurentii grew rapidly in the wound of the sweet cherry at both 25 and 1 "C (Fig.2). The yeast growth was stimulated significantly in the fruits inoculated with C. laurentii+M. fructicola at an early stage, whereas, it was inhibited after 108 h and 21 d, respectively, at 25 and 1°C. The results indicated that the influence of the pathogen on the growth of the antagonist was time-dependent, showing a promotion
Fig. 1 Disease incidence (top) and lesion diameter (bottom) in the sweet cherry fruits inoculated with M. fructicola alone and M . frucficola + C . laurentii at 25 and 1°C.
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WANG You-sheng et al.
at an earlier stage while an inhibition at a later stage.
Lipidperoxidation When stored at 25"C, lipid peroxide was rapidly induced in the fruits inoculated with only M . fructicola or combined with C. laurentii, and the fruits inoculated with only M . fructicola showed significantly higher severity of lipid peroxidation than those inoculated by C. lauentii+M. fructicola at 60 h (Fig.3). In contrast, the induction of lipid peroxidation by C. laurentii alone was not evident until 36 h after inoculation and the intensity was much lesser when compared with that of M . fructicola. The content of fruit lipid peroxide increased during the first seven days of storage at 1"C, but no e v i d e n t d i f f e r e n c e b e t w e e n t r e a t m e n t s was observed. Similarly, clear elevation of lipid peroxide levels appeared at 21 d in the fruits inoculated with M . fructicola, whereas, there was no significant difference between the yeast-inoculated fruit and the control during the whole storage time. The inoculation of C . lauentii + M . fructicola also could not induce evident lipid peroxidation in the sweet cherry fruits during the whole storage period at l "C, but lipid peroxidation was significantly evaluated when the C. lauentii + M . fructicola-inoculated fruit was stored at 1°C for 40 d and then transferred to 25°C.
Fig. 2 Population dynamics of C. laurentii in wounds of the sweet cherry fruits with and without M. fructicola at 25 and 1°C. At each point of time, data followed by different letters were significantly different according to paired sample T-test at P c 0.05.
SOD activity SOD activity increased at first and then decreased in the control fruit during the storage at 25°C (Fig.4). SOD activity in the M .fructicola-inoculated fruit was induced during the first 12 h of postinoculation,but was inhibited before the fruit disease appeared (at 36 h), and then induced after the fruit disease appeared (at 60 h). In comparison, no evident difference in SOD activity was observed between the C. lauentii + M .fructicola-inoculated fruit and the control during the first 12 h, and induction was also observed at the end of the experiment. In the C. lauentii-inoculated fruit, SOD activity was inhibited at 12 h, whereas, induced thereafter. SOD activity in all the treated fruits maintained at a steady level in the first seven days and there was no
Fig. 3 Changes of lipid peroxidation production in the sweet cherry fruits inoculated with C. laurentii, C. laurentii + M . fructicola, M . fructicola, and the control at 25 and 1°C. At each point of time, data followed by different letters were significantly different according to Duncan's multiple range test at PcO.05. The same as below.
significant difference between treatments, however, it increased to become 2.8-3.3 times higher than the initial level after 21 d. SOD activity was significantly evaluated by C. laurentii or M . fructicola as compared to the
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Interaction Between Cryptococcus laurentii, Monilinia fructicola, and Sweet Cherry Fruit at Different
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Fig. 4 Changes of SOD activity in the sweet cherry fruits inoculated with C. laurentii, C. laurentii + M.fructicola, M.fructicola, and the control at 25 and 1°C.
Fig. 5 Changes of CAT activity in the sweet cherry fruits inoculated with C. laurentii, C. laurentii + M . fructicola, M. fructicola, and the control at 25 and 1°C.
control, and the induction of C. lauentii + M . fructicola showed a coordinated effect of C . laurentii and M . fructicola. When transferred to 25°C after storage at 1°C for 40 d, SOD activity in the C . lauentii+ M . fructicola and M . fructicola-inoculated fruits were significantly higher than that of the control, whereas, no significant difference was found between the C. laurentii-inoculated fruit and the control.
POD activity Activity of POD in M . fructicola-inoculated fruit increased during the first 12 h of postinoculation, however, it was inhibited at 36 h, before which the disease appeared and then elevated to about 2.6-3.4 0
CAT activity When stored at 25"C, CAT activities increased both in the M . fructicola and C. lauentii + M . fructicola-inoculated fruits within 12 h, and the evaluations were markedly higher in the fruits inoculated by M . fhcticolu alone (Fig.5). CAT activity in the fruits inoculated by M . fructicola was inhibited before the symptoms appeared (36 h), however, elevated rapidly after the disease appeared (60 h). In contrast, the CAT activity in the C . laurentii-inoculated fruits showed no significant difference as compared with the control, during the whole experiment. CAT activity in the fruits inoculated by M.fructicola alone and C. lauentii + M. fructicola was significantly higher than in the control, whereas, the inoculation of C. laurentii alone showed minor effect during the storage at 1°C.
Fig. 6 Changes of POD activity in the sweet cherry fruits inoculated with C. laurentii, C . laurentii+M.fructicola, M.fructicola, and the control at 25 and 1°C.
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WANG You-sheng et uI.
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times higher than that of the other treatments, at 60 h, when the fruit disease appeared (Fig.6). In comparison, no evident difference in POD activity was observed between the C . lauentii-inoculated fruits and the control during the first 12 h, and POD activity in the C. lauentii-inoculated fruits subsequently increased, and the induction effect increased with time. In the C. lauentii + M . fructicola-inoculated fruits, the POD activity was induced during the whole storage time and the induction effect was gradually strengthened with time. POD activity in the control fruit kept steady during the whole storage at 1°C. In contrast, the induction effect of C . laurentii, M . fructicola, and C . lauentii + M.fructicola inoculation on POD activity evaluated with time, and increased as high as 2.3, 1.9, and 1.7 times of the control at the end of low temperature storage.
PPO activity PPO activity in all treatments elevated to about 1.9-3.3 times as compared with the initial level at 12 h at 25°C (Fig.7). However, in both C. lauentii and M . fructicolatreated fruit, the PPO activity was significantly lower than the control during the whole experiment. While
the PPO activity in the C. lauentii+M. fructicoln-inoculated fruit was stimulated during the first 36 h, but inhibited thereafter. Activity of PPO in all treatments elevated 2.4-2.7 times within seven days when stored at 1"C, and decreased thereafter, and then increased evidently at 40 d. However, no evident difference was observed in PPO activity of treated fruit and the control at 1°C.
lsoenzyme pattern for PPO PPO isoenzymes pattern in the sweet cherry fruits varied greatly during the course of disease development of M . fructicola at 25°C (Fig.8-A). Isoenzyme bands of PPO in the b region disappeared at 36 h, before whch the disease symptoms occurred, whereas, five new isoenzyme bands in a and c regions appeared at 60 and 108 h after the symptoms occurred. It could be deduced that the five new isoenzymes bands were induced by the pathogen M . fructicola, as they were not observed in the fruits inoculated by C . laurentii alone or C. laurentii+M.fructicola at the same time. New isoenzymes appeared in the d region in all treatments when stored at 1°C as compared to 25"C, which indicated that these new isoenzymes were induced by low temperature (Fig.8-B). Isoenzyme bands of PPO in the b region disappeared, whereas, additional bands in the c region were observed in the M . fructicolainoculated fruits at 48 d when disease symptoms in all fruits appeared, suggesting that M . fructicola inhibited the expression of isoenzymes in the b region, whereas, induced those in the c region. In contrast, the intensity of PPO isoenzymes in the b region increased considerably with time in the other treatments, but became weak when the fruits were transferred to 25°C. Therefore, it appeared that the increased intensity of PPO isoenzymes in the b region correlated with increased PPO activity at a later stage.
DISCUSSION
Fig. 7 Changes of PPO activity in sweet cherry fruit inoculated with C. laurentii, C. laurentii+M.fructicola, M .fructicola, and the control at 25 and 1°C.
Data from the present experiment showed that when the fruits were inoculated by M . fructicola alone, disease could be found in the sweet cherry at both 1 and 25°C. The antagonistic yeast C. Zuurentii could delay
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Interaction Between Cryprococcus luurenrii, Monilinia frucricola, and Sweet Cherry Fruit at Different
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Fig. 8 Active staining of polyphenol oxidase in sweet cherry fruit at 25°C (A) and 1°C (B). Y, C. luurenrii-inoculated; YP, C. luurentii M. fructicolu co-inoculated; P, M . fructicolu-inoculated; C, control.
the incidence and reduce the severity of brown rot in the sweet cherry fruits, but disease was found partly in the C. Zauentii + M. fructicola-inoculated fruit at the end of the experiment, showing that C. Zaurentii inhibited rather than killed the pathogen M. fructicola, which was in correspondence with the characterization of antagonistic yeast (Janisiewicz and Korsten 2002). In addition, it was obvious that the growth of C. laurentii was stimulated at an early stage and inhibited at a later stage in fruit treated with C. luuentii+M. fructicola as compared with that inoculated with C. lauentii alone. Similar results were also found in the authors' previous studies (Fan and Tian 2001). It remains to be further investigated as to how M.fructicola stimulated C. laurentii to grow rapidly at the earlier stage. The accumulation of reactive oxygen species (ROS) could cause lipid peroxidation, and SOD, CAT, as well as POD, are the major enzymes in metabolizing ROS (Rice-Evans et al. 1991; Bolwell et al. 1995; Deighton et aZ. 1999). Thus, the formation of lipid peroxidation, along with the induction of antioxidant enzymes, indicated an excessive production of ROS and oxidative stress. The present results showed that M.fructicola promoted SOD, CAT, and POD activities within 12 h in fruits stored at 25"C, indicating that M . fructicola in-
+
duced fruit to produce excess ROS and thus led to oxidative stress (Baker and Orlandi 1995). Although the activities of antioxidant enzymes were significantly inhibited before disease incidence (36 h), the sustained increasing of lipid peroxidation indicated that excess ROS was indeed found. Similarly, the infected rate of brown rot was reduced in fruit stored at 1"C, but the elevation of lipid peroxidation and antioxidant enzyme activity were also observed at the earlier stage of inoculation (21 d), as well as when symptoms appeared (40 d). The results showed that the fruits inoculated with M . fructicola produced excess ROS and thus led to oxidative stress at two temperatures. In comparison, SOD activity was significantly inhibited within the first 12 h, in the fruits inoculated with C. laurntii alone, whereas, no significant difference was found in CAT and POD activities as well as in lipid peroxidation, showing that the antagonist alone could not lead to oxidative stress during this period. It could be speculated that C. laurntii mainly competed for nutrition and space in fruit wounds at the early stage and the number of C. laurntii was not large enough to induce the antioxidant enzymes of the host. At 108 h, the increased levels of SOD and POD activities as well as lipid peroxide content, combined with the decline of
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CAT activity in the fruits inoculated with C. laurntii, could be because of the strong elevation in yeast number and the promotion of the influence of yeast, but it could not be excluded that the antagonist might secrete something that acted as an elicitor when the nutrition was used up. This could be further confirmed by the results that POD activity in fruit inoculated C. laurntii also increased greatly as compared with the control after 40 d of storage at 1°C. However, the antagonist C. laurntii showed less influence on fruit when compared with the pathogen M . fructicola. From the fact that SOD, CAT, and.POD activities as well as the rate of lipid peroxidation significantly increased in fruit inoculated by C. lauentii+M. fructicola, together with the results that the C'laurntii alone could not lead to the oxidative stress of fruit, it can be deduced that the oxidative stress stimulated by C. lauentii + M . fructicola mainly contributed to the pathogen M . fructicola at the early stage. It was probable that M . fructicola not only stimulated the growth of C. laurntii, but also induced ROS production of fruit at the earlier stage of inoculation, and that the influence of M . fructicola on the fruit still existed in wounds inoculated by C. lauenrii + M . fructicola. In contrast, the activities of antioxidant enzymes and the degree of lipid peroxidation in the C. lauentii +M. fructicolainoculated fruit indicated that serious oxidative stress occurred around the wound in agreement with the coordinated induction of both C. lauentii and M .fructicola at 108 h, which might be one of the reasons that the number of C. laurntii became less and at the same time the disease incidence was found in part of the fruits. Thus, the M. fructicola might attack the sweet cherry fruit by making the host accumulate excess ROS, which killed the host and antagonist cells. The results supported the view of Tiedemann (1997), who stated that Botrytis cinerea could cause disease by inducing the host to produce a large amount of ROS. PPO was capable of oxidizing a range of phenolic acids in the fruit to quinine, which had two aspects to its functions. On one hand, quinine accumulated and cross linked with amino acid and proteins and thus led to flesh browning (Martinez and Whitaker 1995). On the other hand, quinine was also implicated in pathogen resistance as it showed a much stronger killing ability
to fungi than phenolic acids (Mayer and Hare1 1979; Bashan et al. 1987). Previous investigations had demonstrated that both R. stolonifer and P. membranefaciens +R. stolonifer could elevate PPO activity in peach fruit (Qin et al. 2003b), whereas, the present study has demonstrated that both C. laurntii and M. fructicola inhibited the PPO activity in the sweet cherry. In addition, the disappearance of the PPO isoenzyme band in the b region along with the occurrence of the fruit disease at both temperatures revealed the relationship between PPO isoenzyme of the b region and the fruit-induced resistance. In contrast, isoenzymes in the a and c regions might be just a passive reaction to the pathogen M .fructicola, as they appeared only after fruit incidence. There were different kinds of polyphenols in the sweet cherry fruit and each isoenzyme of PPO had its specific substrate. Thus, the variation of PPO isoenzyme pattern during pathogenesis, in the fruit inoculated with M. fructicola, represented the change of metabolic pathway of polyphenol in sweet cherry (Lanzarini et al. 1972). Future studies will need to address the relationship between PPO isoenzyme pattern and the fruit resistance in different conditions.
CONCLUSION In the interaction between C. laurentii, M . fructicola, and sweet cherry, C. laurentii could effectively inhibit brown rot disease incidence infected by M . fructicola at 25 and 1°C. M .fructicola could stimulate the growth of C. laurentii at the earlier stage, but inhibit it at a later stage. Inoculated M. fructicola alone or combined with C. laurentii could promote SOD, CAT, and POD activities, and accelerate lipid peroxidation. Therefore, the inhibition of brown rot during the antagonist-pathogen-host interaction in wounds of sweet cherry fruit was mainly because of the stimulated growth of C. laurentii, as well as the induction of antioxidant enzymes of fruit by M. fructicola.
Acknowledgements This work was supported by the Knowledge Innovation Program of the Chinese Academy of Sciences, China (KSCX2-YW-G-010), and the National Natural Science Foundation of China (30671473).
02008,CAAS. All rights reserved.Published by ElsevierLtd.
Interaction Between Cryptococcus laurentii, Monilinia fructicola, and Sweet Cherry Fruit at Different
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