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Bacillus cereus AR156 induces resistance against Rhizopus rot through priming of defense responses in peach fruit
Food Chemistry 136 (2013) 400–406
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Bacillus cereus AR156 induces resistance against Rhizopus rot through priming of defense responses in peach fruit Xiaoli Wang, Feng Xu, Jing Wang, Peng Jin, Yonghua Zheng ⇑ College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China
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Article history: Received 5 May 2012 Received in revised form 26 July 2012 Accepted 4 September 2012 Available online 16 September 2012 Keywords: Peach fruit Bacillus cereus AR156 Rhizopus stolonifer Biocontrol Priming
a b s t r a c t The biocontrol effects of Bacillus cereus AR156 on Rhizopus rot caused by Rhizopus stolonifer in postharvest peach fruit and the possible mechanisms were investigated. The results showed that fruit treated with B. cereus AR156 had significantly lower disease incidence and smaller lesion diameter than the control fruit did. B. cereus AR156 treatment remarkably enhanced activities of chitinase and b-1,3-glucanase, promoted accumulation of H2O2, and improved total phenolic content and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity. Transcripts of four defense related genes were only significantly enhanced in fruit both treated with B. cereus AR156 and inoculated with R. stolonifer compared with those that were only treated with B. cereus AR156 or inoculated with R. stolonifer. These results suggest that B. cereus AR156 can effectively inhibit Rhizopus rot caused by R. stolonifer and enhance antioxidant activity in peach fruit through the priming of defense responses. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Peach fruit usually has a short postharvest life at room temperature due to its high susceptibility to pathogen infection. Rhizopus rot caused by Rhizopus stolonifer (Ehrenb.:Fr.) Vuill is one of the most common postharvest diseases of peach fruit. Disease control can be achieved through the use of fungicides that are commercially available for postharvest treatment to reduce decay and extend the shelf-life of peach fruit (Fan & Tian, 2000). Since the use of synthetic fungicides is becoming more restricted due to health and environmental concerns, non-chemical disease control methods are becoming increasingly important. Biological control has emerged as an effective strategy to combat major postharvest diseases of fruits because it is usually safe for humans and the environment. Debaryomyces hansenii (Mandal, Singh, & Sharma, 2007) and Cryptococcus laurentii (Zhang, Zheng, & Yu, 2007) have been reported as effective biocontrol agents against Rhizopus rot in peach fruit. Droby, Wisniewski, El Ghaouth, and Wilson (2003) reported that the biocontrol efficacy of Aspire was highly enhanced when using the yeast-based biocontrol product Aspire combined with 2% NaBi to control Rhizopus rot in peach fruit. Recently, some strains of Bacillus spp. have been evaluated as potential biocontrol agents against postharvest pathogens of peaches (Arrebola, Sivakumar, Bacigalupo, & Korsten, 2010; Zhou
⇑ Corresponding author. Tel.: +86 25 8439 9080; fax: +86 25 8439 5618. E-mail address: [email protected] (Y. Zheng). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.09.032
et al., 2011). How these biocontrol agents work, however, remains unclear. Upon treatment with necorotizing pathogens, many plants develop an enhanced capacity to activate defense responses, a phenomenon called ‘priming’. Priming is considered as a mechanism that is common to different types of induced resistances in plants (Conrath, 2009). Some chemical elicitors, beneficial microorganisms and biotic/abiotic stresses were found to have the ability of inducing priming in different crops (Beckers & Conrath, 2007; Conrath et al., 2006; Kohler, Schwindling, & Conrath, 2002). Recently, a rhizobacterium, Bacillus cereus AR156, was found to be able to significantly inhibit the leaf speck disease caused by Pseudomonas syringae pv. tomato, the bacterial wilt caused by Ralstonia solanacearum, the blight caused by Phytophthora capsici Leon., and the root-knot disease caused by Meloidogyne incognita in tomato and some other vegetables (Guo, Wei, & Li, 2007). This finding indicates that B. cereus AR156 can be a promising biocontrol agent against a broad spectrum of pathogens. The mechanism of B. cereus AR156 inducing disease resistance has been found to prime for potentiated expression of the cellular defense responses in plant leaves (Niu et al., 2011). However, no information is available regarding the biocontrol effects of B. cereus AR156 on disease incidence in postharvest fruits. The objectives of this work were first to evaluate the efficacy of B. cereus AR156 for the control of Rhizopus rot caused by R. stolonifer in postharvest peach fruit, and then to investigate whether the B. cereus AR156 induced disease resistance against Rhizopus rot is associated with priming of defense responses in peach fruit.
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2. Materials and methods 2.1. Biocontrol agent and pathogen The biocontrol agent, B. cereus AR156 was kindly supplied by Prof. Jianhua Guo of College of Plant Protection, Nanjing Agricultural University, China. The bacterial strain was cultured with LB medium in a 1 l conical flask at 30 °C and 200 rpm. A working volume of 500 ml of LB medium was used as a growth medium after inoculation with 1% (v/v) of an inoculum. Bacterial cells were harvested at the beginning of the stationary phase (24 h) by centrifugation at 5000g for 5 min at 20 °C in an Avanti-TMJ-25I centrifuge (Beckman, Palo Alto, CA, USA). The cell paste was resuspended in sterile distilled water and the cell concentration was adjusted to 1 108 CFU ml1. The challenging pathogen R. stolonifer was isolated from the surfaces of infected peach fruits and cultured on potato dextrose agar (PDA) medium (containing the extract of 200 g boiled potatoes, 20 g dextrose and 20 g agar in 1000 ml of distilled water). A conidiospore suspension was prepared from 2-week-old cultures incubated at 26 °C. Spores were removed from the surface of each Petri dish culture and suspended in 5 ml of sterile distilled water. The number of spores was calculated with a hemocytometer counting chamber, and the spore concentration was then adjusted to 1 105 spores ml1 with sterile distilled water. 2.2. Fruit material Peach [Prunus persica (L.) Batsch cv. Baifeng] fruits were handharvested at firm-mature stage from a commercial orchard in Nanjing, China, and transported to the laboratory on the day of collection. In the laboratory, the fruits were selected for uniform size and maturity and absence of visual defects. Fruits were surface-sterilized with 75% ethanol, and air dried prior to wounding. 2.3. Efficacy of B. cereus AR156 for control of Rhizopus rot Peaches were wounded with the tip of a sterile dissecting needle and two uniform 4 mm deep and 2 mm wide wounds made at two sides of each fruit around the fruit equator. 20 ll of washedcell suspension of B. cereus AR156 at 1 108 CFU ml1 or distilled water (as control) was pipetted onto each wound. The fruit were then air dried and put into 400 300 100 mm plastic trays wrapped with high density polyethylene sleeve. After keeping at 20 °C for 12 h, fruit pretreated with the biocontrol agent or distilled water were inoculated with 15 ll of a suspension of 1 105 spores of per ml R. stolonifer in each wound. The fruits were incubated at 20 °C with high humidity (about 95%) for 3 days. Disease incidence and lesion diameter on each fruit wound were observed at 1, 2 and 3 days postinoculation. Meanwhile, fruit samples were taken for enzyme assays and measurements of protein, total phenolic and H2O2 contents, DPPH radical-scavenging activity and quality parameters. There were three replicate of 10 fruit each per treatment, and the experiment was conducted three times. 2.4. Analysis of defense-related genes expression To further investigate whether the B. cereus AR156 induced disease resistance against Rhizopus rot is associated with priming of defense responses in peach fruit, RT-PCR was used to analyze the expression patterns of the defense-related genes PpNPR1-like, PpPR-like, chitinase (CHI), and b-1,3-glucanase (GNS) in peach fruit only inoculated with distilled water (Mock), R. stolonifer or B. cereus AR156, and in those both treated with B. cereus AR156 and inoculated with R. stolonifer. Total RNA was extracted from fruit accord-
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ing to the method described by Chang, Puryear, and John (1993) with some modifications. RT-PCR was performed according to the manufacturer’s instructions of PrimeScriptTM 16 1st Strand cDNA Synthesis Kit (TaKaRa, Japan). Short and conserved segments of PpNPR1-like (GenBank ID: DQ149935), PpPR-like (GenBank ID: AF362989), PpCHI (GenBank ID: AF206635), and PpGNS (GenBank ID: U49454) were cloned by degenerate primers. Independent PCR with 25 cycles was performed using aliquots (1 ll) of cDNA samples, and a constitutively expressed gene 18S-rRNA (GenBank ID: L28749.1) was used as a quantitative control in the RT-PCR analysis. The sequences of primers used were as follows: PpNPR1-like_forward: 50 -GACCCAAACATGCCAGCAGTG-30 , PpNPR1like_reverse: 50 -ATCCTTCGGCCTTGTCAACCT-30 ; PpPR1-like_forward: 50 -ATCAACTGGGACTTGCGTACT-30 , PpPR1-like_reverse: 50 TAGTCGCCACAGTCAACAAAG-30 ; PpCHI_forward: 50 -GTGGAAAAG CAATAGGGGAG-30 , PpCHI_reverse: 50 -TTCCAGCCCTTACCACAT-30 ; PpGNS_forward: 50 -ATTTCTCTTGCTGGTCTTG-30 , PpGNS_reverse: 50 -CTCTGGGGTCTTTCTATTCT-30 ; 18S-rRNA_forward: 50 -ATGGCCG TTCTTAGTTGGTG-30 , 18S-rRNA_reverse: 50 - GTACAAAGGGCAGGG ACGTA-30 . 2.5. Assay of enzyme activity Chitinase (EC 3.2.1.14) was extracted from 1 g of frozen tissue sample with 5 ml of 50 mM sodium acetate buffer (pH 5.0). Chitinase activity was measured by the release of N-acetyl-D-glucosamine (NAG) from colloidal chitin according to the method of Abeles, Bosshart, Forrence, and Habig (1971). A unit of chitinase activity is defined as the amount of enzyme required to catalyse the production of 1 lg NAG per hour at 37 °C. b-1,3-Glucanase (EC 3.2.1.58) activity was determined using the colorimetric assay based on the hydrolysis of laminarin. Frozen tissue sample (1 g) was ground with 5 ml of 50 mM sodium acetate buffer (pH 5.0). Enzyme preparation (1 ml) was incubated for 1 h at 37 °C with 1 ml of 4% laminarin (Aldrich, Chemical Co., Wilwaukee, WI, USA). The reaction was terminated by heating the sample in boiling water for 5 min and the amount of reducing sugars was measured spectrophotometrically at 540 nm after reaction with 250 ll 3,5-dinitrosalicyclic reagent (Aldrich). One unit is defined as the amount of enzyme catalyzing the formation of 1 lmol glucose equivalents in 1 h. Superoxide dismutase (SOD, EC 1.15.1.1) activity was determined by the method of Rao, Paliyath, and Ormrod (1996). Frozen tissue (1 g) was ground with 5 ml of 50 mM sodium phosphate buffer (pH 7.8). The reaction mixture contained 50 mM sodium phosphate buffer (pH 7.8), 14 mM methionine, 3 lM EDTA, 1 lM nitro blue tetrazolium (NBT), 60 lM riboflavin and 0.1 ml crude enzyme extract. One unit of SOD activity is defined as the amount of enzyme that caused a 50% inhibition of NBT. Catalase (CAT, EC 1.11.1.6) activity was measured according to the method of Chance and Maehly (1955). Frozen tissue (1 g) was ground with 5 ml of 50 mM sodium phosphate buffer (pH 7.0). The reaction mixture consisted of 50 mM sodium phosphate buffer (pH 7.0), 12.5 mM H2O2 and 20 ll of enzyme extract. One unit of CAT activity is defined as the amount of enzyme that decomposed 1 lmol H2O2 min1 at 30 °C. Ascorbate peroxidase (APX, EC 1.11.1.11) was extracted from 1 g frozen tissue ground with 5 ml of 50 mM sodium phosphate buffer (pH 7.0) that contained 0.1 mM EDTA, 1 mM ascorbic acid and 1% polyvinyl-pyrrolidone (PVP). The homogenate was centrifugated at 10,000g for 20 min at 4 °C and the supernatant was used to determine the APX activity. One unit of APX activity is defined as the amount of enzyme that oxidized 1 lmol ascorbate per minute at 30 °C. Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity was extracted with 0.2 M sodium borate buffer (pH 8.7) that contained
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Fig. 1. Changes in lesion diameter (A), and disease incidence (B) in peach fruit treated with B. cereus AR156 and inoculated with R. stolonifer and incubated at 20 °C. Each column represents the mean of triplicate samples. Vertical bars represent the standard errors of the means. Different letters above the bars indicate statistically significant differences at P < 0.05.
20 mM of b-mercaptoethanol. The assay medium contained 0.1 ml of enzyme extract and 1 ml of L-phenylalanine. After incubation at 40 °C for 1 h, the reaction was stopped by adding 0.2 ml of 6 M HCl. One unit of PAL activity is defined as the amount of enzyme that caused an increase in absorbance of 0.01 at 290 nm in 1 h under the assay conditions. Peroxidase (POD, EC 1.11.1.7) was extracted from 1 g of frozen tissue with 5 ml of 50 mM sodium phosphate buffer (pH 8.7). The extracts were then homogenized and centrifuged at 10,000g for 20 min at 4 °C. POD activity was assayed according to the method of Kochba, Lavee, and Spiege (1977) using guaiacol as donor and H2O2 as substrate. One unit of POD activity is defined as the amount of enzyme that caused an increase in absorbance of 0.01 at 470 nm per minute. For polyphenol oxidase (PPO, EC 1.10.3.1), the frozen tissue (1 g) was ground with 5 ml of 0.2 M sodium phosphate buffer (pH 6.5), together with 1% of polyvinylpolypyrrolidone (PVP). The crude PPO extraction was centrifuged at 10,000g for 20 min. Each 3 ml of assay medium contained 0.1 M catechol, 0.1 M sodium phosphate buffer (pH 6.5), and 0.1 ml enzyme extract. The increase in absor-
Fig. 3. Effect of B. cereus AR156 treatment on chitinase (A) and b-1,3-glucanase (B) activities in peach fruits inoculated with R. stolonifer and incubated at 20 °C. Data are expressed as the mean of triplicate samples. Vertical bars represent the standard errors of the means.
bance at 420 nm at 25 °C was recorded. One unit of PPO activity is defined as the amount of enzyme that caused an increase of 0.01 at 420 nm per minute. Protein content in the enzyme extracts was determined by the Bradford (1976) method, using bovine serum albumin as a standard. Specific activity of all the enzymes was expressed as units per milligram of protein. 2.6. Measurement of total phenolic, H2O2 contents and DPPH radicalscavenging activity Total phenolic content, expressed as milligram of gallic acid equivalent (GAE) per 100 g of fresh weight, was determined using
Fig. 2. Expression of representative defense related genes. Reverse transcription-polymerase chain reaction (RT-PCR) was carried out using 18S-rRNA as a standard.
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Fig. 4. Effect of B. cereus AR156 treatment on SOD (A), CAT (B) and APX (C) activities and H2O2 content (D) in peach fruits inoculated with R. stolonifer and incubated at 20 °C. Data are expressed as the mean of triplicate samples. Vertical bars represent the standard errors of the means.
the modified Folin–Ciocalteu procedure. Frozen tissue (1 g) were homogenized in 5 ml of 80% cold acetone and centrifuged at 10,000g for 20 min and the supernatant was used for analysis. The result was expressed as milligram of gallic acid equivalent (GAE) per 100 g of fresh weight. H2O2 content, expressed as lmol g1 FW, was determined using a method based on titanium oxidation. Frozen tissue (2 g) was ground and homogenized with 5 ml of chilled 100% acetone and then centrifuged at 10,000g for 20 min at 4 °C. Absorbance of the supernatant was measured at 412 nm. The DPPH radical-scavenging activity was estimated using the method of Larrauri, Sánchez-Moreno, and Saura-Calixto (1998). Half a gram of frozen sample was extracted with 50% ethanol and centrifuged at 10,000g for 20 min at 4 °C. An ethanolic solution of DPPH served as control. The DPPH radical-scavenging activity was calculated according to the following formula:
DPPH radical scavenging activityð%Þ ¼ 1 ðabsorbance of sample=absorbance of controlÞ 100%:
2.7. Statistical analysis Experiments were performed using a completely randomized design. All statistical analyses were performed with SPSS 11.0 (SPSS Inc., Chicago, IL, USA). The data were analyzed by one-way analysis of variance (ANOVA) to test the difference of the treatments. Mean separations were performed using Duncan’s multiple range tests. Significant tests were at level of P < 0.05. 3. Results
ease incidence, respectively, by 38.6% and 21.0% on the 2nd day of inoculation compared with the control (Fig. 1A and B). Although all the inoculated wounds in both B. cereus AR156 treated and control fruit developed decay symptoms after 3 days of inoculation, the lesion diameter in fruit treated with B. cereus AR156 was still significantly (P < 0.05) smaller than that in control fruit (Fig. 1A). 3.2. Effects of B. cereus AR156 treatment and R. stolonifer inoculation on defense-related genes expression in peach fruit Transcripts of the four defense related genes retained at very low level in fruits only inoculated with R. stolonifer or distilled water, while the transcripts were slightly enhanced in fruits only treated with B. cereus AR156 (Fig. 2). In fruit pretreated with AR156 and then inoculated with R. stolonifer, the transcripts of all four genes were significantly enhanced and attained at higher level in all the sampling time points compared with the other three treatments (Fig. 2), i.e. B. cereus AR156 treatment induced stronger expression of the four defense related genes in peach fruit upon challenged with the pathogen of R. stolonifer. 3.3. Effects of B. cereus AR156 treatment on chitinase and b-1,3glucanase activities in peach fruit Chitinase and b-1,3-glucanase are important defense related enzymes in peach fruit. The activities of both enzymes increased gradually during storage and maintained at significantly (P < 0.05) higher levels in fruit both treated with B. cereus AR156 and inoculated with R. stolonifer compared with the control. Fruit that both treated with B. cereus AR156 and inoculated with R. stolonifer showed 46.9% higher activity of chitinase and 31.8% higher activity of b-1,3-glucanase after 3 days of inoculation (Fig. 3A and B).
3.1. The biocontrol effects of B. cereus AR156 on Rhizopus rot of peach fruit The lesion diameter and disease incidence of Rhizopus rot in fruit treated with B. cereus AR156 were significantly (P < 0.05) lower than those in the control during the first 2 days of incubation at 20 °C (Fig. 1). B. cereus AR156 treatment reduced lesion diameter and dis-
3.4. Effects of B. cereus AR156 treatment on activities of SOD, CAT, APX and H2O2 content in peach fruit SOD activity increased on the first day of incubation and then decreased gradually during the following 2 days. B. cereus AR156
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treatment induced significantly (P < 0.05) higher SOD activity on the 1st and 2nd day compared with the control (Fig. 4A). The activity of CAT increased slightly during the first day of incubation and then decreased in the following 2 days. Fruit treated with B. cereus AR156 maintained significantly (P < 0.05) higher CAT activity on the 2nd and 3rd day of incubation (Fig. 4B). APX activity decreased during the incubation, B. cereus AR156 treatment significantly (P < 0.05) inhibited the activity of APX on the 1st and 3rd day of incubation (Fig. 4C). The level of H2O2 in both control and B. cereus AR156 treated fruit increased during the inoculation. B. cereus AR156 treatment promoted the accumulation of H2O2, and significantly (P < 0.05) higher H2O2 content was observed in fruit treated with B. cereus AR156 on the 2nd day of incubation (Fig. 4D). 3.5. Effects of B. cereus AR156 treatment on activities of PAL, PPO and POD in peach fruit PAL activity in control fruit increased slightly during storage. B. cereus AR156 treatment promoted the increase and maintained
significantly (P < 0.05) higher PAL activity during the whole storage period compared to the control fruit (Fig. 5A). POD and PPO activities increased with storage. B. cereus AR156 treatment significantly (P < 0.05) increased their activities (Fig. 5B and C). 3.6. Effects of B. cereus AR156 treatment on total phenolic content and DPPH radical-scavenging activity in peach fruit The level of total phenolic compounds in control fruit decreased gradually during storage. B. cereus AR156 treatment induced the accumulation of total phenolic content and resulted in significantly (P < 0.05) higher level of total phenolic content in the B. cereus AR156 treated fruits than that in control fruit during the whole storage (Fig. 6A). DPPH radical-scavenging activity in control fruit increased slightly during storage, while that in B. cereus AR156 treated fruit increased rapidly and reached a peak on the 1st day, and then decreased during the rest period of storage, but still kept significantly (P < 0.05) higher than that in the control fruit (Fig. 6B). 4. Discussion In this study, it was found that B. cereus AR156 treatment significantly reduced Rhizopus rot on peach fruit wounds inoculated with R. stolonifer. This result suggests that the disease resistance of peach fruit is enhanced by this biocontrol agent. Inducing resistance has been inferred to be one of the major mechanisms of biocontrol agents in controlling postharvest diseases of horticultural
Fig. 5. Effect of B. cereus AR156 treatment on PAL (A), POD (B) and PPO (C) activities in peach fruits inoculated with R. stolonifer and incubated at 20 °C. Data are expressed as the mean of triplicate samples. Vertical bars represent the standard errors of the means.
Fig. 6. Effect of B. cereus AR156 treatment on total phenolic content (A) and DPPH radical scavenging activity (B) in peach fruits inoculated with R. stolonifer and incubated at 20 °C. Data are expressed as the mean of triplicate samples. Vertical bars represent the standard errors of the means.
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crops (Terry & Joyce, 2004). The disease resistance in harvest fruit is associated with some inducible compounds including pathogenesis-related (PR) proteins. Among PR proteins, chitinase and b-1,3glucanase have been most extensively studied, and are thought to be involved in the plant defense mechanisms against fungal infection. Induction of the two defensive enzymes by Pichia mambranaefaciens was observed in harvested loquat and Chinese bayberry fruit, which was correlated to increased disease resistance and reduced disease severity (Cao, Zheng, Tang, Jin, & Wang, 2008; Wang, Jin, Cao, Rui, & Zheng, 2011). In this study, it was found that B. cereus AR156 significantly induced activities of chitinase and b-1,3glucanase and inhibited fruit decay in peach fruit (Figs. 1 and 3). These results indicate that the induced disease resistance is involved in the mechanisms by which B. cereus AR156 suppressed Rhizopus rot in peach fruit. The accumulation of reactive oxygen species (ROS) has the potential to serve not only as protectants against invading pathogen, but as signals activating further plant defense reactions (Lamb & Dixon, 1997). Generally, the metabolism of ROS is controlled by an array of enzymes including SOD, CAT, and APX. And H2O2 is destroyed predominantly by APX and CAT. It has been reported that a salicylic acid (SA)-induced increase in H2O2 content is mediated by an inhibition of CAT and APX in several plants (Dat, Lopez, Foyer, & Scott, 2000; Landberg & Greger, 2002). In the pulp of ‘Cara cara’ navel orange, treatment with SA accelerated the increase in SOD activity, but significantly inhibited CAT activity, which resulted in a higher level of H2O2 content during storage (Huang, Liu, Lu, & Xia, 2008). There is increasing evidence showing a close relationship between H2O2 accumulation and disease resistance in postharvest fruits. For example, higher level of H2O2 content was correlated with lower susceptibility to Penicillium expansum infection in apple fruit (Torres, Valentines, Usall, Vinas, & Larrigaudiere, 2003) and the enhanced resistance to anthracnose rot in methyl jasmonate-treated loquat fruit was correlated to higher H2O2 content (Cao et al., 2008). In this work, B. cereus AR156 treatment maintained higher activities of SOD and CAT, but lower APX activity, thus resulting in higher level of H2O2 in peach fruit. These results suggest that enhanced H2O2 generation may be one of the major factors that trigger the disease resistance in B. cereus AR156-treated peach fruit. The mechanisms of induced resistance appear to involve the direct induction of defenses by the inducing agent and/or the priming of defenses that are expressed following a challenge inoculation with a virulent strain of a pathogen (Hammerschmidt, 2009). A common feature of the resistance responses induced by beneficial microorganisms in plants is priming. For example, nonpathogenic rhizobacteria Pseudomonas fluorescens CHA0, P. fluorescens WCS417, P. putida WCS358, P. fluorescens Q2–87, and P. aeruginosa 7NSK2 were all effective in priming defense responses of grapevine against Botrytis cinerea (Verhagen, Trotel-Aziz, Couderchet, Höfte, & Aziz, 2010). A strain of rhizobacterium, P. putida strain LSW17S, protected tomato plants against biotrophic P. syringae pv. tomato strain DC3000 infection without direct accumulation of PR proteins. However, upon challenge with the pathogen, the LSW17S treated plants accumulated significantly more transcriptions of PR genes (Ahn et al., 2011). Challenge inoculation with the leaf pathogen P. syringae pv. lachrymans of cucumber plants that had been preinoculated with the plant growth-promoting fungus Trichoderma asperellum (strain T203) led to augmented PR gene expression (Conrath, 2009; Shoresh, Yedidia, & Chet, 2005). As a indicator of defense expression, the transcription of defense related genes measured in this study showed that only in fruit that had been pretreated with B. cereus AR156 and then challenged with R. stolonifer, was a significant increase in defense related genes expression observed (Fig. 2). This clearly suggests that the B. cereus AR156 induced disease resistance against Rhizopus rot in
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peach fruit is associated with priming of defense responses, rather than the direct induction. In conclusion, our results demonstrated that B. cereus AR156 treatment primed the expression of defense responses in peach fruit, which resulted in enhanced level of induced resistance against R. stolonifer infection. To our knowledge, this is the first report showing priming as an important mechanism of induced resistance phenomenon in postharvest fruits. Acknowledgments This study was supported by the National Natural Science Foundation of China (31172003). We thank Prof. Jianhua Guo of Nanjing Agricultural University for kindly providing the B. cereus AR156 strain and Dr. Chien Y. Wang of Food Quality Laboratory, US Department of Agriculture, for critical revising of this manuscript. References Abeles, F. B., Bosshart, R. P., Forrence, L. E., & Habig, W. H. (1971). Preparation and purification of glucanase and chitinase from bean leaves. Plant Physiology, 47, 129–134. Ahn, I. P., Lee, S. W., Kim, M. G., Park, S. R., Hwang, D. J., & Bae, S. C. (2011). Priming by rhizobacterium protects tomato plants from biotrophic and necrotrophic pathogen infections through multiple defense mechanisms. Molecules and Cells, 32, 7–14. Arrebola, E., Sivakumar, D., Bacigalupo, R., & Korsten, L. (2010). Combined application of antagonist Bacillus amyloliquefaciens and essential oils for the control of peach postharvest diseases. Crop Protection, 29, 369–377. Beckers, G. J. M., & Conrath, U. (2007). Priming for stress resistance: From lab to the field. Current Opinion in Plant Biology, 10, 425–431. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle-dye binding. Analytical Biochemistry, 72, 248–254. Cao, S. F., Zheng, Y. H., Tang, S. S., Jin, P., & Wang, K. T. (2008). Biological control of postharvest anthracnose rot of loquat fruit by Pichia membranaefaciens. The Journal of Horticultural Science and Biotechnology, 83, 816–820. Chance, B., & Maehly, A. C. (1955). Assay of catalases and peroxidase. Methods in Enzymology, 2, 764–775. Chang, S. J., Puryear, J., & John, C. (1993). A simple and efficient method for isolating RNA from pine trees. Plant molecular biology reporter, 11, 113–116. Conrath, U. (2009). Priming of induced plant defense responses. In L. C. Van Loon (Ed.), Plant innate immunity, advances in botanical research (Vol. 51, pp. 361– 395). Conrath, U., Beckers, G. J. M., Flors, V., Garcia-Agustin, P., Jakab, G., Mauch, F., et al. (2006). Priming: Getting ready for battle. Molecular Plant Microbe Interaction, 19, 1062–1071. Dat, J. F., Lopez, D. H., Foyer, C. H., & Scott, I. M. (2000). Effects of salicylic acid on oxidative stress and thermotolerance in tobacco. Journal of Plant Physiology, 156, 659–665. Droby, S., Wisniewski, M., El Ghaouth, A., & Wilson, C. (2003). Influence of food additives on the control of postharvest rots of apple and peach and efficacy of the yeast-based biocontrol product Aspire. Postharvest Biology and Technology, 27, 127–135. Fan, Q., & Tian, S. P. (2000). Postharvest biological control of Rhizopus rot of nectarine fruits by Pichia membranefaciens. Plant Disease, 84, 1212–1216. Guo, J. H., Wei, L. H., & Li, S. M. (2007). The strain for biocontrol root-knot disease of vegetable. US patent 200710021376.0. Hammerschmidt, R. (2009). Challenge inoculation reveals the benefits of resistance priming. Physiological and Molecular Plant Pathology, 73, 59–60. Huang, R. H., Liu, J. H., Lu, Y. M., & Xia, R. X. (2008). Effect of salicylic acid on the antioxidant system in the pulp of ‘Cara cara’ navel orange (Citrus sinensis L. Osbeck) at different storage temperatures. Postharvest Biology and Technology, 47, 168–175. Kochba, J., Lavee, S., & Spiege, R. P. (1977). Difference in peroxidase activity and isoenzymes in embryogenic and non-embryogenic ‘Shamouti’ orange ovular callus lines. Plant Cell Physiology, 18, 463–467. Kohler, A., Schwindling, S., & Conrath, U. (2002). Benzothiadiazole induced priming for potentiated responses to pathogen infection, wounding, and infiltration of water into leaves requires the NPR1/NIM1 gene in Arabidopsis. Plant Physiology, 128, 1046–1056. Lamb, C., & Dixon, R. A. (1997). The oxidative burst in plant disease resistance. Annual Review of Plant Physiology and Plant Molecular Biology, 48, 251–275. Landberg, T., & Greger, M. (2002). Differences in oxidative stress in heavy metal resistant and sensitive clones of Salix viminalis. Journal of Plant Physiology, 159, 69–75. Larrauri, J. A., Sánchez-Moreno, C., & Saura-Calixto, F. (1998). Effect of temperature on the free radical scavenging capacity of extracts from red and white grape pomace peels. Journal of agricultural and food chemistry, 46, 2694–2697.
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Bacillus cereus AR156 induces resistance against Rhizopus rot through priming of defense responses in peach fruit Xiaoli Wang, Feng Xu, Jing Wang, Peng Jin, Yonghua Zheng ⇑ College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China
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Article history: Received 5 May 2012 Received in revised form 26 July 2012 Accepted 4 September 2012 Available online 16 September 2012 Keywords: Peach fruit Bacillus cereus AR156 Rhizopus stolonifer Biocontrol Priming
a b s t r a c t The biocontrol effects of Bacillus cereus AR156 on Rhizopus rot caused by Rhizopus stolonifer in postharvest peach fruit and the possible mechanisms were investigated. The results showed that fruit treated with B. cereus AR156 had significantly lower disease incidence and smaller lesion diameter than the control fruit did. B. cereus AR156 treatment remarkably enhanced activities of chitinase and b-1,3-glucanase, promoted accumulation of H2O2, and improved total phenolic content and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity. Transcripts of four defense related genes were only significantly enhanced in fruit both treated with B. cereus AR156 and inoculated with R. stolonifer compared with those that were only treated with B. cereus AR156 or inoculated with R. stolonifer. These results suggest that B. cereus AR156 can effectively inhibit Rhizopus rot caused by R. stolonifer and enhance antioxidant activity in peach fruit through the priming of defense responses. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Peach fruit usually has a short postharvest life at room temperature due to its high susceptibility to pathogen infection. Rhizopus rot caused by Rhizopus stolonifer (Ehrenb.:Fr.) Vuill is one of the most common postharvest diseases of peach fruit. Disease control can be achieved through the use of fungicides that are commercially available for postharvest treatment to reduce decay and extend the shelf-life of peach fruit (Fan & Tian, 2000). Since the use of synthetic fungicides is becoming more restricted due to health and environmental concerns, non-chemical disease control methods are becoming increasingly important. Biological control has emerged as an effective strategy to combat major postharvest diseases of fruits because it is usually safe for humans and the environment. Debaryomyces hansenii (Mandal, Singh, & Sharma, 2007) and Cryptococcus laurentii (Zhang, Zheng, & Yu, 2007) have been reported as effective biocontrol agents against Rhizopus rot in peach fruit. Droby, Wisniewski, El Ghaouth, and Wilson (2003) reported that the biocontrol efficacy of Aspire was highly enhanced when using the yeast-based biocontrol product Aspire combined with 2% NaBi to control Rhizopus rot in peach fruit. Recently, some strains of Bacillus spp. have been evaluated as potential biocontrol agents against postharvest pathogens of peaches (Arrebola, Sivakumar, Bacigalupo, & Korsten, 2010; Zhou
⇑ Corresponding author. Tel.: +86 25 8439 9080; fax: +86 25 8439 5618. E-mail address: [email protected] (Y. Zheng). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.09.032
et al., 2011). How these biocontrol agents work, however, remains unclear. Upon treatment with necorotizing pathogens, many plants develop an enhanced capacity to activate defense responses, a phenomenon called ‘priming’. Priming is considered as a mechanism that is common to different types of induced resistances in plants (Conrath, 2009). Some chemical elicitors, beneficial microorganisms and biotic/abiotic stresses were found to have the ability of inducing priming in different crops (Beckers & Conrath, 2007; Conrath et al., 2006; Kohler, Schwindling, & Conrath, 2002). Recently, a rhizobacterium, Bacillus cereus AR156, was found to be able to significantly inhibit the leaf speck disease caused by Pseudomonas syringae pv. tomato, the bacterial wilt caused by Ralstonia solanacearum, the blight caused by Phytophthora capsici Leon., and the root-knot disease caused by Meloidogyne incognita in tomato and some other vegetables (Guo, Wei, & Li, 2007). This finding indicates that B. cereus AR156 can be a promising biocontrol agent against a broad spectrum of pathogens. The mechanism of B. cereus AR156 inducing disease resistance has been found to prime for potentiated expression of the cellular defense responses in plant leaves (Niu et al., 2011). However, no information is available regarding the biocontrol effects of B. cereus AR156 on disease incidence in postharvest fruits. The objectives of this work were first to evaluate the efficacy of B. cereus AR156 for the control of Rhizopus rot caused by R. stolonifer in postharvest peach fruit, and then to investigate whether the B. cereus AR156 induced disease resistance against Rhizopus rot is associated with priming of defense responses in peach fruit.
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2. Materials and methods 2.1. Biocontrol agent and pathogen The biocontrol agent, B. cereus AR156 was kindly supplied by Prof. Jianhua Guo of College of Plant Protection, Nanjing Agricultural University, China. The bacterial strain was cultured with LB medium in a 1 l conical flask at 30 °C and 200 rpm. A working volume of 500 ml of LB medium was used as a growth medium after inoculation with 1% (v/v) of an inoculum. Bacterial cells were harvested at the beginning of the stationary phase (24 h) by centrifugation at 5000g for 5 min at 20 °C in an Avanti-TMJ-25I centrifuge (Beckman, Palo Alto, CA, USA). The cell paste was resuspended in sterile distilled water and the cell concentration was adjusted to 1 108 CFU ml1. The challenging pathogen R. stolonifer was isolated from the surfaces of infected peach fruits and cultured on potato dextrose agar (PDA) medium (containing the extract of 200 g boiled potatoes, 20 g dextrose and 20 g agar in 1000 ml of distilled water). A conidiospore suspension was prepared from 2-week-old cultures incubated at 26 °C. Spores were removed from the surface of each Petri dish culture and suspended in 5 ml of sterile distilled water. The number of spores was calculated with a hemocytometer counting chamber, and the spore concentration was then adjusted to 1 105 spores ml1 with sterile distilled water. 2.2. Fruit material Peach [Prunus persica (L.) Batsch cv. Baifeng] fruits were handharvested at firm-mature stage from a commercial orchard in Nanjing, China, and transported to the laboratory on the day of collection. In the laboratory, the fruits were selected for uniform size and maturity and absence of visual defects. Fruits were surface-sterilized with 75% ethanol, and air dried prior to wounding. 2.3. Efficacy of B. cereus AR156 for control of Rhizopus rot Peaches were wounded with the tip of a sterile dissecting needle and two uniform 4 mm deep and 2 mm wide wounds made at two sides of each fruit around the fruit equator. 20 ll of washedcell suspension of B. cereus AR156 at 1 108 CFU ml1 or distilled water (as control) was pipetted onto each wound. The fruit were then air dried and put into 400 300 100 mm plastic trays wrapped with high density polyethylene sleeve. After keeping at 20 °C for 12 h, fruit pretreated with the biocontrol agent or distilled water were inoculated with 15 ll of a suspension of 1 105 spores of per ml R. stolonifer in each wound. The fruits were incubated at 20 °C with high humidity (about 95%) for 3 days. Disease incidence and lesion diameter on each fruit wound were observed at 1, 2 and 3 days postinoculation. Meanwhile, fruit samples were taken for enzyme assays and measurements of protein, total phenolic and H2O2 contents, DPPH radical-scavenging activity and quality parameters. There were three replicate of 10 fruit each per treatment, and the experiment was conducted three times. 2.4. Analysis of defense-related genes expression To further investigate whether the B. cereus AR156 induced disease resistance against Rhizopus rot is associated with priming of defense responses in peach fruit, RT-PCR was used to analyze the expression patterns of the defense-related genes PpNPR1-like, PpPR-like, chitinase (CHI), and b-1,3-glucanase (GNS) in peach fruit only inoculated with distilled water (Mock), R. stolonifer or B. cereus AR156, and in those both treated with B. cereus AR156 and inoculated with R. stolonifer. Total RNA was extracted from fruit accord-
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ing to the method described by Chang, Puryear, and John (1993) with some modifications. RT-PCR was performed according to the manufacturer’s instructions of PrimeScriptTM 16 1st Strand cDNA Synthesis Kit (TaKaRa, Japan). Short and conserved segments of PpNPR1-like (GenBank ID: DQ149935), PpPR-like (GenBank ID: AF362989), PpCHI (GenBank ID: AF206635), and PpGNS (GenBank ID: U49454) were cloned by degenerate primers. Independent PCR with 25 cycles was performed using aliquots (1 ll) of cDNA samples, and a constitutively expressed gene 18S-rRNA (GenBank ID: L28749.1) was used as a quantitative control in the RT-PCR analysis. The sequences of primers used were as follows: PpNPR1-like_forward: 50 -GACCCAAACATGCCAGCAGTG-30 , PpNPR1like_reverse: 50 -ATCCTTCGGCCTTGTCAACCT-30 ; PpPR1-like_forward: 50 -ATCAACTGGGACTTGCGTACT-30 , PpPR1-like_reverse: 50 TAGTCGCCACAGTCAACAAAG-30 ; PpCHI_forward: 50 -GTGGAAAAG CAATAGGGGAG-30 , PpCHI_reverse: 50 -TTCCAGCCCTTACCACAT-30 ; PpGNS_forward: 50 -ATTTCTCTTGCTGGTCTTG-30 , PpGNS_reverse: 50 -CTCTGGGGTCTTTCTATTCT-30 ; 18S-rRNA_forward: 50 -ATGGCCG TTCTTAGTTGGTG-30 , 18S-rRNA_reverse: 50 - GTACAAAGGGCAGGG ACGTA-30 . 2.5. Assay of enzyme activity Chitinase (EC 3.2.1.14) was extracted from 1 g of frozen tissue sample with 5 ml of 50 mM sodium acetate buffer (pH 5.0). Chitinase activity was measured by the release of N-acetyl-D-glucosamine (NAG) from colloidal chitin according to the method of Abeles, Bosshart, Forrence, and Habig (1971). A unit of chitinase activity is defined as the amount of enzyme required to catalyse the production of 1 lg NAG per hour at 37 °C. b-1,3-Glucanase (EC 3.2.1.58) activity was determined using the colorimetric assay based on the hydrolysis of laminarin. Frozen tissue sample (1 g) was ground with 5 ml of 50 mM sodium acetate buffer (pH 5.0). Enzyme preparation (1 ml) was incubated for 1 h at 37 °C with 1 ml of 4% laminarin (Aldrich, Chemical Co., Wilwaukee, WI, USA). The reaction was terminated by heating the sample in boiling water for 5 min and the amount of reducing sugars was measured spectrophotometrically at 540 nm after reaction with 250 ll 3,5-dinitrosalicyclic reagent (Aldrich). One unit is defined as the amount of enzyme catalyzing the formation of 1 lmol glucose equivalents in 1 h. Superoxide dismutase (SOD, EC 1.15.1.1) activity was determined by the method of Rao, Paliyath, and Ormrod (1996). Frozen tissue (1 g) was ground with 5 ml of 50 mM sodium phosphate buffer (pH 7.8). The reaction mixture contained 50 mM sodium phosphate buffer (pH 7.8), 14 mM methionine, 3 lM EDTA, 1 lM nitro blue tetrazolium (NBT), 60 lM riboflavin and 0.1 ml crude enzyme extract. One unit of SOD activity is defined as the amount of enzyme that caused a 50% inhibition of NBT. Catalase (CAT, EC 1.11.1.6) activity was measured according to the method of Chance and Maehly (1955). Frozen tissue (1 g) was ground with 5 ml of 50 mM sodium phosphate buffer (pH 7.0). The reaction mixture consisted of 50 mM sodium phosphate buffer (pH 7.0), 12.5 mM H2O2 and 20 ll of enzyme extract. One unit of CAT activity is defined as the amount of enzyme that decomposed 1 lmol H2O2 min1 at 30 °C. Ascorbate peroxidase (APX, EC 1.11.1.11) was extracted from 1 g frozen tissue ground with 5 ml of 50 mM sodium phosphate buffer (pH 7.0) that contained 0.1 mM EDTA, 1 mM ascorbic acid and 1% polyvinyl-pyrrolidone (PVP). The homogenate was centrifugated at 10,000g for 20 min at 4 °C and the supernatant was used to determine the APX activity. One unit of APX activity is defined as the amount of enzyme that oxidized 1 lmol ascorbate per minute at 30 °C. Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity was extracted with 0.2 M sodium borate buffer (pH 8.7) that contained
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Fig. 1. Changes in lesion diameter (A), and disease incidence (B) in peach fruit treated with B. cereus AR156 and inoculated with R. stolonifer and incubated at 20 °C. Each column represents the mean of triplicate samples. Vertical bars represent the standard errors of the means. Different letters above the bars indicate statistically significant differences at P < 0.05.
20 mM of b-mercaptoethanol. The assay medium contained 0.1 ml of enzyme extract and 1 ml of L-phenylalanine. After incubation at 40 °C for 1 h, the reaction was stopped by adding 0.2 ml of 6 M HCl. One unit of PAL activity is defined as the amount of enzyme that caused an increase in absorbance of 0.01 at 290 nm in 1 h under the assay conditions. Peroxidase (POD, EC 1.11.1.7) was extracted from 1 g of frozen tissue with 5 ml of 50 mM sodium phosphate buffer (pH 8.7). The extracts were then homogenized and centrifuged at 10,000g for 20 min at 4 °C. POD activity was assayed according to the method of Kochba, Lavee, and Spiege (1977) using guaiacol as donor and H2O2 as substrate. One unit of POD activity is defined as the amount of enzyme that caused an increase in absorbance of 0.01 at 470 nm per minute. For polyphenol oxidase (PPO, EC 1.10.3.1), the frozen tissue (1 g) was ground with 5 ml of 0.2 M sodium phosphate buffer (pH 6.5), together with 1% of polyvinylpolypyrrolidone (PVP). The crude PPO extraction was centrifuged at 10,000g for 20 min. Each 3 ml of assay medium contained 0.1 M catechol, 0.1 M sodium phosphate buffer (pH 6.5), and 0.1 ml enzyme extract. The increase in absor-
Fig. 3. Effect of B. cereus AR156 treatment on chitinase (A) and b-1,3-glucanase (B) activities in peach fruits inoculated with R. stolonifer and incubated at 20 °C. Data are expressed as the mean of triplicate samples. Vertical bars represent the standard errors of the means.
bance at 420 nm at 25 °C was recorded. One unit of PPO activity is defined as the amount of enzyme that caused an increase of 0.01 at 420 nm per minute. Protein content in the enzyme extracts was determined by the Bradford (1976) method, using bovine serum albumin as a standard. Specific activity of all the enzymes was expressed as units per milligram of protein. 2.6. Measurement of total phenolic, H2O2 contents and DPPH radicalscavenging activity Total phenolic content, expressed as milligram of gallic acid equivalent (GAE) per 100 g of fresh weight, was determined using
Fig. 2. Expression of representative defense related genes. Reverse transcription-polymerase chain reaction (RT-PCR) was carried out using 18S-rRNA as a standard.
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Fig. 4. Effect of B. cereus AR156 treatment on SOD (A), CAT (B) and APX (C) activities and H2O2 content (D) in peach fruits inoculated with R. stolonifer and incubated at 20 °C. Data are expressed as the mean of triplicate samples. Vertical bars represent the standard errors of the means.
the modified Folin–Ciocalteu procedure. Frozen tissue (1 g) were homogenized in 5 ml of 80% cold acetone and centrifuged at 10,000g for 20 min and the supernatant was used for analysis. The result was expressed as milligram of gallic acid equivalent (GAE) per 100 g of fresh weight. H2O2 content, expressed as lmol g1 FW, was determined using a method based on titanium oxidation. Frozen tissue (2 g) was ground and homogenized with 5 ml of chilled 100% acetone and then centrifuged at 10,000g for 20 min at 4 °C. Absorbance of the supernatant was measured at 412 nm. The DPPH radical-scavenging activity was estimated using the method of Larrauri, Sánchez-Moreno, and Saura-Calixto (1998). Half a gram of frozen sample was extracted with 50% ethanol and centrifuged at 10,000g for 20 min at 4 °C. An ethanolic solution of DPPH served as control. The DPPH radical-scavenging activity was calculated according to the following formula:
DPPH radical scavenging activityð%Þ ¼ 1 ðabsorbance of sample=absorbance of controlÞ 100%:
2.7. Statistical analysis Experiments were performed using a completely randomized design. All statistical analyses were performed with SPSS 11.0 (SPSS Inc., Chicago, IL, USA). The data were analyzed by one-way analysis of variance (ANOVA) to test the difference of the treatments. Mean separations were performed using Duncan’s multiple range tests. Significant tests were at level of P < 0.05. 3. Results
ease incidence, respectively, by 38.6% and 21.0% on the 2nd day of inoculation compared with the control (Fig. 1A and B). Although all the inoculated wounds in both B. cereus AR156 treated and control fruit developed decay symptoms after 3 days of inoculation, the lesion diameter in fruit treated with B. cereus AR156 was still significantly (P < 0.05) smaller than that in control fruit (Fig. 1A). 3.2. Effects of B. cereus AR156 treatment and R. stolonifer inoculation on defense-related genes expression in peach fruit Transcripts of the four defense related genes retained at very low level in fruits only inoculated with R. stolonifer or distilled water, while the transcripts were slightly enhanced in fruits only treated with B. cereus AR156 (Fig. 2). In fruit pretreated with AR156 and then inoculated with R. stolonifer, the transcripts of all four genes were significantly enhanced and attained at higher level in all the sampling time points compared with the other three treatments (Fig. 2), i.e. B. cereus AR156 treatment induced stronger expression of the four defense related genes in peach fruit upon challenged with the pathogen of R. stolonifer. 3.3. Effects of B. cereus AR156 treatment on chitinase and b-1,3glucanase activities in peach fruit Chitinase and b-1,3-glucanase are important defense related enzymes in peach fruit. The activities of both enzymes increased gradually during storage and maintained at significantly (P < 0.05) higher levels in fruit both treated with B. cereus AR156 and inoculated with R. stolonifer compared with the control. Fruit that both treated with B. cereus AR156 and inoculated with R. stolonifer showed 46.9% higher activity of chitinase and 31.8% higher activity of b-1,3-glucanase after 3 days of inoculation (Fig. 3A and B).
3.1. The biocontrol effects of B. cereus AR156 on Rhizopus rot of peach fruit The lesion diameter and disease incidence of Rhizopus rot in fruit treated with B. cereus AR156 were significantly (P < 0.05) lower than those in the control during the first 2 days of incubation at 20 °C (Fig. 1). B. cereus AR156 treatment reduced lesion diameter and dis-
3.4. Effects of B. cereus AR156 treatment on activities of SOD, CAT, APX and H2O2 content in peach fruit SOD activity increased on the first day of incubation and then decreased gradually during the following 2 days. B. cereus AR156
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treatment induced significantly (P < 0.05) higher SOD activity on the 1st and 2nd day compared with the control (Fig. 4A). The activity of CAT increased slightly during the first day of incubation and then decreased in the following 2 days. Fruit treated with B. cereus AR156 maintained significantly (P < 0.05) higher CAT activity on the 2nd and 3rd day of incubation (Fig. 4B). APX activity decreased during the incubation, B. cereus AR156 treatment significantly (P < 0.05) inhibited the activity of APX on the 1st and 3rd day of incubation (Fig. 4C). The level of H2O2 in both control and B. cereus AR156 treated fruit increased during the inoculation. B. cereus AR156 treatment promoted the accumulation of H2O2, and significantly (P < 0.05) higher H2O2 content was observed in fruit treated with B. cereus AR156 on the 2nd day of incubation (Fig. 4D). 3.5. Effects of B. cereus AR156 treatment on activities of PAL, PPO and POD in peach fruit PAL activity in control fruit increased slightly during storage. B. cereus AR156 treatment promoted the increase and maintained
significantly (P < 0.05) higher PAL activity during the whole storage period compared to the control fruit (Fig. 5A). POD and PPO activities increased with storage. B. cereus AR156 treatment significantly (P < 0.05) increased their activities (Fig. 5B and C). 3.6. Effects of B. cereus AR156 treatment on total phenolic content and DPPH radical-scavenging activity in peach fruit The level of total phenolic compounds in control fruit decreased gradually during storage. B. cereus AR156 treatment induced the accumulation of total phenolic content and resulted in significantly (P < 0.05) higher level of total phenolic content in the B. cereus AR156 treated fruits than that in control fruit during the whole storage (Fig. 6A). DPPH radical-scavenging activity in control fruit increased slightly during storage, while that in B. cereus AR156 treated fruit increased rapidly and reached a peak on the 1st day, and then decreased during the rest period of storage, but still kept significantly (P < 0.05) higher than that in the control fruit (Fig. 6B). 4. Discussion In this study, it was found that B. cereus AR156 treatment significantly reduced Rhizopus rot on peach fruit wounds inoculated with R. stolonifer. This result suggests that the disease resistance of peach fruit is enhanced by this biocontrol agent. Inducing resistance has been inferred to be one of the major mechanisms of biocontrol agents in controlling postharvest diseases of horticultural
Fig. 5. Effect of B. cereus AR156 treatment on PAL (A), POD (B) and PPO (C) activities in peach fruits inoculated with R. stolonifer and incubated at 20 °C. Data are expressed as the mean of triplicate samples. Vertical bars represent the standard errors of the means.
Fig. 6. Effect of B. cereus AR156 treatment on total phenolic content (A) and DPPH radical scavenging activity (B) in peach fruits inoculated with R. stolonifer and incubated at 20 °C. Data are expressed as the mean of triplicate samples. Vertical bars represent the standard errors of the means.
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crops (Terry & Joyce, 2004). The disease resistance in harvest fruit is associated with some inducible compounds including pathogenesis-related (PR) proteins. Among PR proteins, chitinase and b-1,3glucanase have been most extensively studied, and are thought to be involved in the plant defense mechanisms against fungal infection. Induction of the two defensive enzymes by Pichia mambranaefaciens was observed in harvested loquat and Chinese bayberry fruit, which was correlated to increased disease resistance and reduced disease severity (Cao, Zheng, Tang, Jin, & Wang, 2008; Wang, Jin, Cao, Rui, & Zheng, 2011). In this study, it was found that B. cereus AR156 significantly induced activities of chitinase and b-1,3glucanase and inhibited fruit decay in peach fruit (Figs. 1 and 3). These results indicate that the induced disease resistance is involved in the mechanisms by which B. cereus AR156 suppressed Rhizopus rot in peach fruit. The accumulation of reactive oxygen species (ROS) has the potential to serve not only as protectants against invading pathogen, but as signals activating further plant defense reactions (Lamb & Dixon, 1997). Generally, the metabolism of ROS is controlled by an array of enzymes including SOD, CAT, and APX. And H2O2 is destroyed predominantly by APX and CAT. It has been reported that a salicylic acid (SA)-induced increase in H2O2 content is mediated by an inhibition of CAT and APX in several plants (Dat, Lopez, Foyer, & Scott, 2000; Landberg & Greger, 2002). In the pulp of ‘Cara cara’ navel orange, treatment with SA accelerated the increase in SOD activity, but significantly inhibited CAT activity, which resulted in a higher level of H2O2 content during storage (Huang, Liu, Lu, & Xia, 2008). There is increasing evidence showing a close relationship between H2O2 accumulation and disease resistance in postharvest fruits. For example, higher level of H2O2 content was correlated with lower susceptibility to Penicillium expansum infection in apple fruit (Torres, Valentines, Usall, Vinas, & Larrigaudiere, 2003) and the enhanced resistance to anthracnose rot in methyl jasmonate-treated loquat fruit was correlated to higher H2O2 content (Cao et al., 2008). In this work, B. cereus AR156 treatment maintained higher activities of SOD and CAT, but lower APX activity, thus resulting in higher level of H2O2 in peach fruit. These results suggest that enhanced H2O2 generation may be one of the major factors that trigger the disease resistance in B. cereus AR156-treated peach fruit. The mechanisms of induced resistance appear to involve the direct induction of defenses by the inducing agent and/or the priming of defenses that are expressed following a challenge inoculation with a virulent strain of a pathogen (Hammerschmidt, 2009). A common feature of the resistance responses induced by beneficial microorganisms in plants is priming. For example, nonpathogenic rhizobacteria Pseudomonas fluorescens CHA0, P. fluorescens WCS417, P. putida WCS358, P. fluorescens Q2–87, and P. aeruginosa 7NSK2 were all effective in priming defense responses of grapevine against Botrytis cinerea (Verhagen, Trotel-Aziz, Couderchet, Höfte, & Aziz, 2010). A strain of rhizobacterium, P. putida strain LSW17S, protected tomato plants against biotrophic P. syringae pv. tomato strain DC3000 infection without direct accumulation of PR proteins. However, upon challenge with the pathogen, the LSW17S treated plants accumulated significantly more transcriptions of PR genes (Ahn et al., 2011). Challenge inoculation with the leaf pathogen P. syringae pv. lachrymans of cucumber plants that had been preinoculated with the plant growth-promoting fungus Trichoderma asperellum (strain T203) led to augmented PR gene expression (Conrath, 2009; Shoresh, Yedidia, & Chet, 2005). As a indicator of defense expression, the transcription of defense related genes measured in this study showed that only in fruit that had been pretreated with B. cereus AR156 and then challenged with R. stolonifer, was a significant increase in defense related genes expression observed (Fig. 2). This clearly suggests that the B. cereus AR156 induced disease resistance against Rhizopus rot in
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peach fruit is associated with priming of defense responses, rather than the direct induction. In conclusion, our results demonstrated that B. cereus AR156 treatment primed the expression of defense responses in peach fruit, which resulted in enhanced level of induced resistance against R. stolonifer infection. To our knowledge, this is the first report showing priming as an important mechanism of induced resistance phenomenon in postharvest fruits. Acknowledgments This study was supported by the National Natural Science Foundation of China (31172003). We thank Prof. Jianhua Guo of Nanjing Agricultural University for kindly providing the B. cereus AR156 strain and Dr. Chien Y. Wang of Food Quality Laboratory, US Department of Agriculture, for critical revising of this manuscript. References Abeles, F. B., Bosshart, R. P., Forrence, L. E., & Habig, W. H. (1971). Preparation and purification of glucanase and chitinase from bean leaves. Plant Physiology, 47, 129–134. Ahn, I. P., Lee, S. W., Kim, M. G., Park, S. R., Hwang, D. J., & Bae, S. C. (2011). Priming by rhizobacterium protects tomato plants from biotrophic and necrotrophic pathogen infections through multiple defense mechanisms. Molecules and Cells, 32, 7–14. Arrebola, E., Sivakumar, D., Bacigalupo, R., & Korsten, L. (2010). Combined application of antagonist Bacillus amyloliquefaciens and essential oils for the control of peach postharvest diseases. Crop Protection, 29, 369–377. Beckers, G. J. M., & Conrath, U. (2007). Priming for stress resistance: From lab to the field. Current Opinion in Plant Biology, 10, 425–431. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle-dye binding. Analytical Biochemistry, 72, 248–254. Cao, S. F., Zheng, Y. H., Tang, S. S., Jin, P., & Wang, K. T. (2008). Biological control of postharvest anthracnose rot of loquat fruit by Pichia membranaefaciens. The Journal of Horticultural Science and Biotechnology, 83, 816–820. Chance, B., & Maehly, A. C. (1955). Assay of catalases and peroxidase. Methods in Enzymology, 2, 764–775. Chang, S. J., Puryear, J., & John, C. (1993). A simple and efficient method for isolating RNA from pine trees. Plant molecular biology reporter, 11, 113–116. Conrath, U. (2009). Priming of induced plant defense responses. In L. C. Van Loon (Ed.), Plant innate immunity, advances in botanical research (Vol. 51, pp. 361– 395). Conrath, U., Beckers, G. J. M., Flors, V., Garcia-Agustin, P., Jakab, G., Mauch, F., et al. (2006). Priming: Getting ready for battle. Molecular Plant Microbe Interaction, 19, 1062–1071. Dat, J. F., Lopez, D. H., Foyer, C. H., & Scott, I. M. (2000). Effects of salicylic acid on oxidative stress and thermotolerance in tobacco. Journal of Plant Physiology, 156, 659–665. Droby, S., Wisniewski, M., El Ghaouth, A., & Wilson, C. (2003). Influence of food additives on the control of postharvest rots of apple and peach and efficacy of the yeast-based biocontrol product Aspire. Postharvest Biology and Technology, 27, 127–135. Fan, Q., & Tian, S. P. (2000). Postharvest biological control of Rhizopus rot of nectarine fruits by Pichia membranefaciens. Plant Disease, 84, 1212–1216. Guo, J. H., Wei, L. H., & Li, S. M. (2007). The strain for biocontrol root-knot disease of vegetable. US patent 200710021376.0. Hammerschmidt, R. (2009). Challenge inoculation reveals the benefits of resistance priming. Physiological and Molecular Plant Pathology, 73, 59–60. Huang, R. H., Liu, J. H., Lu, Y. M., & Xia, R. X. (2008). Effect of salicylic acid on the antioxidant system in the pulp of ‘Cara cara’ navel orange (Citrus sinensis L. Osbeck) at different storage temperatures. Postharvest Biology and Technology, 47, 168–175. Kochba, J., Lavee, S., & Spiege, R. P. (1977). Difference in peroxidase activity and isoenzymes in embryogenic and non-embryogenic ‘Shamouti’ orange ovular callus lines. Plant Cell Physiology, 18, 463–467. Kohler, A., Schwindling, S., & Conrath, U. (2002). Benzothiadiazole induced priming for potentiated responses to pathogen infection, wounding, and infiltration of water into leaves requires the NPR1/NIM1 gene in Arabidopsis. Plant Physiology, 128, 1046–1056. Lamb, C., & Dixon, R. A. (1997). The oxidative burst in plant disease resistance. Annual Review of Plant Physiology and Plant Molecular Biology, 48, 251–275. Landberg, T., & Greger, M. (2002). Differences in oxidative stress in heavy metal resistant and sensitive clones of Salix viminalis. Journal of Plant Physiology, 159, 69–75. Larrauri, J. A., Sánchez-Moreno, C., & Saura-Calixto, F. (1998). Effect of temperature on the free radical scavenging capacity of extracts from red and white grape pomace peels. Journal of agricultural and food chemistry, 46, 2694–2697.
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