Postharvest losses refer to the losses that occur along the food supply chain due to pathogens infection, handling, storage, transportation and processing, thereby resulting in the reduction in quality, quantity and market value of agricultural commodities [1
]. Food and Agriculture Organization reported that global average loss due to the food postharvest losses in North America, Europe and Oceania was about 29%, compared to an average of about 38% in industrialized Asia, Africa, Latin America and South East Asia [3
]. Among all the factors for reducing the losses on food supply, postharvest diseases of fruits present a major factor that causes the postharvest losses and limits the duration of storage [4
]. In addition, postharvest diseases are often the major concern in influencing consumer prices, requirements and mode of transportation [6
]. China is the largest producer of peaches with a production of 13.5 million metric tons (MMT), and exporter to North Korea, Russia, Singapore, USA, Philippines and Malaysia, with a very different climate (from tropical, continental or oceanic climatic climates), but the postharvest diseases of peach fruits have been considered one of the most severe factors that results in the loss of production [7
]. Additionally, the diseases caused by fungal pathogens in harvested fresh fruits are considered as one of the most serious losses of production at the postharvest and consumption levels [8
]. Some research showed that the main worldwide postharvest diseases caused by fungi in peach fruits are brown rot caused by Monilinia fructicola
or M. laxa
; rot caused by R. stolonifer
; grey mold caused by Botrytis cinerea
], and other economically important fungal diseases such as those of stone fruits caused by Penicillium
spp. and Aspergillus
]. However, little is known about the species of main fungal pathogens that cause the postharvest disease of peaches in China.
A number of strategies have been adapted to manage of postharvest diseases worldwide [15
]. Chemical control (synthetic fungicides) is known to be highly effective and widely applied method in orchard after harvesting [17
]. However, some fungicides have toxicological risks, such as dangerous to human health and causing environmental pollution, even in some cases their use is prohibited by law in postharvest phase [19
]. Particularly, the increased level of fungicide use in fruit orchards has led to the growing public concern over the health and environmental hazards associated with fungicides [22
]. Therefore, development of alternative safe and natural methods in controlling postharvest diseases have become urgent in recent years worldwide [23
]. In particular, there has been extensive research to reduce synthetic fungicide usage based on microbial antagonists to biologically control postharvest pathogens in the past years with higher control efficiency [10
]. In recent years, the bacterium Bacillus
spp. has been widely studied as a potential biological agent against various plant diseases, increases plant systemic resistance and improves rhizosphere microbial community structure [26
]. It is common in nature and nontoxic and harmless to humans and other animals, and nonpathogenic to plants [30
]. However, there is little information on the bio-control activity of the bacterial antagonist B. subtilis
JK-14 and its mechanisms involved in the postharvest disease management of peaches.
Therefore, the objectives of the present study were to (i) isolate and identify the main species of fungal pathogens causing postharvest disease on peaches, (ii) explore the antifungal potential and controlling efficiency of B. subtilis JK-14 against the main postharvest fungal infection, and (iii) determine the possible mechanisms involved in the strain of B. subtilis JK-14 in controlling postharvest fruit diseases on peaches.
3. Discussion and Conclusions
Peach is one of the most ancient and world-popular fruits due to its high marketing value with favorable taste and abundant phytonutrients [31
]. However, postharvest fungal diseases limit the storage period and marketing life of peaches, and result in serious economic losses worldwide. Recently, application of bio-control agents for the management of postharvest fruit decay has been explored as an alternative method instead of synthetic fungicides worldwide [15
spp. has been considered as the bio-control agent in controlling number of plant diseases with a high efficacy [32
]. However, there is little information available regarding the identification the fungal pathogen species that cause the peach postharvest diseases, and explore the potential and mechanisms of Bacillus subtilis
JK-14 in controlling postharvest peach diseases. Our present study showed that a total of six fungal isolates were isolated from the mature peaches, and in particular the species of Alternaria tenuis
and Botrytis cinerea
have been identified as the main pathogens for causing the host of mature peach decay. Interestingly, the strain of B. subtilis
JK-14 has been found and exhibited a potent activity in inhibiting the growth of A. tenuis
and B. cinerea
, and controlling peaches fruits fungal disease in the present study. The possible mechanisms for the strain of B. subtilis
JK-14 in inhibiting and controlling postharvest peaches fungal disease were due to the direct effect by inhibiting the pathogens infection, and the indirect effect by activating the host defense response to pathogens infection. To the best of our knowledge, the present study is the first to discover the role of the antagonistic B. subtilis
JK-14 in controlling peach fungal disease that are caused by the pathogens of A. tenuis
and B. cinerea
. In view of the high control efficacy in comparison to the control, the strain of B. subtilis
JK-14 can be considered as an environmentally-safe biological control agent instead of chemical fungicides for the management of postharvest disease.
Some previous studies found and identified numerous postharvest pathogens which can cause the decay of stone fruits and belong to the genera of Monilinia
] and Aspergillus
]. Interestingly, six fungal isolates were isolated from the mature peach fruits in the present study, including A. tenuis
, B. cinerea
, P. digitatum
, T. roseum
, R. nigricans
and A. niger
. Our results confirm for the first time that these species are pathogenic to peach fruit and cause decay on wounded peach fruits. However, we have discovered that the isolate of T. roseum
was not pathogenic to the intact peach fruits. The reason may due to the lack of wounds that prevent the T. roseum
invasion. A similar study demonstrated that the wounds can provide the pathways for the pathogens invasion [25
]. In addition, some previous studies revealed that the gray mold decay, blue mold decay and Rhizopus
decay caused by the fungi of B. cinerea
, P. expansum
and R. stolonifer
were the most economically significant and destructive postharvest diseases of peaches [5
]. However, our results found that the isolates of A. tenuis
and B. cinerea
presented the highest pathogenicity and virulence on the host of mature peaches, and also considered as the main pathogens that cause the postharvest disease of peach fruits. The average disease incidences of A. tenuis
and B. cinerea
were 100% and 96.17% after inoculation onto the wounded and intact fruits, respectively. The difference from the previous studies may due to the relationship between the pathogenicity of microbial isolates and the ripening index of peach fruits at harvest [39
In view of the need for reducing environmental pollution due to fungicide over-use in controlling plant diseases in previous years, recently, biological control has emerged as an effective strategy to combat major postharvest decay of fruits [25
]. It is well-known that B. subtilis
is an effective antagonistic bacterium and has been applied in controlling plant fungal diseases such as root diseases [42
], foliar diseases [43
] and postharvest diseases [15
]. A significant advancement from the present study is the finding that B. subtilis
JK-14 provided a significant inhibitory effect on the peach fruits pathogens of A. tenuis
and B. cinerea
, and also different formulations of B. subtilis
JK-14 exhibited significant controlling effect on the peach fruits decay after inoculation with pathogens of A. tenuis
and B. cinerea.
Our findings suggest that the strain of B. subtilis
JK-14 can be considered as a bio-control agent in the effort of developing alternative approaches to control postharvest diseases of fruits.
A previous study showed that Bacillus
sp. C06 suppressed the disease incidences of the postharvest disease brown rot by 92% and decreased the lesion diameters by 88% compared to the pathogen-only, and Bacillus
sp.T03-c reduced disease incidences and lesion diameters by 40% and 62%, respectively [44
]. Similarly, Xu et al. reported that the treatment with Pichia caribbica
significantly reduced the disease incidences and lesion diameters of Rhizopus
decay of peaches compared with the control fruits in a dose dependent manner [7
]. However, our results revealed that the greatest mean percent reduction of disease incidences and lesion diameters of peach postharvest fungal disease by 82.40% and 72.46% after the application of B. subtilis
JK-14 at 1 × 107
among all the different concentrations from 1 × 105
to 1 × 109
. Such differences may be related to the effect of the species of pathogens and different conditions of ripening index of peach fruits (pH value) at harvest on the inhibitory effect of B. subtilis
To further understand the mechanisms of B. subtilis
JK-14 in controlling postharvest diseases of peaches, we explored the effects of B. subtilis
JK-14 on the activities of defense-related enzymes after inoculation with the pathogens in the present study, and found that the treatment of peach fruits with B. subtilis
JK-14 effectively enhanced the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) after inoculation with the pathogen of A. tenuis
or B. cinerea
. Our results indicate that the enhanced activities of defense-related enzymes may play a significant role in the resistance of peaches to the pathogens infection and the induced activity of defense-related enzymes to be part of the mechanism of B. subtilis
JK-14 in controlling postharvest diseases of peach fruits. Some previous studies revealed that one of the important mechanisms for the genus Bacillus
in controlling plant diseases is by increasing and activating the plant systemic resistance [45
]. In addition, the enhanced activities of antioxidant enzymes (SOD, POD, CAT and ascorbate peroxidase, APX) and their coordinated action have been reported to be a part of the mechanism implicated in the alleviation of lipid peroxidation and delay of senescence in peach fruits [48
]. Similarly, Xu et al. [7
] have demonstrated that peach fruits inoculated with P. caribbica
exhibited higher level of POD, CAT and phenylalanine aminolase (PAL) activities than the untreated fruits during the storage period.
In summary, a total of six isolates were isolated from the peach fruits, and the isolates of A. tenuis and B. cinerea were considered as the main pathogens with the highest pathogenicity and virulence on the host of mature peaches. The strain of B. subtilis JK-14 exhibits a high efficacy in controlling postharvest decay of peaches, and may be considered as an environmentally-safe biological control agent for the management of postharvest decay diseases. The possible mechanisms of B. subtilis JK-14 for the management of peach postharvest disease were due to (i) the direct effect by inhibiting the postharvest fungal pathogens growth and infection, and (ii) the indirect effect by activating the defense-related enzymes to enhance the resistance of peaches response to the postharvest fungal pathogens infection during the storage period.
4. Materials and Methods
Experiments were carried out at the Gansu Provincial Biocontrol Engineering Laboratory of Crop Diseases and Pests. The peach (Prunus persica L., cultivar Baifeng) fruits were collected from the stone fruit orchards in Gansu, China. Gansu is located in the northwest of China, at the longitude of 103.8264470 E and latitude of 36.0595610 N, with a dry and strong continental temperate monsoon climate. The average temperature, precipitation and relative humidity of the air were about 8 °C, 300 mm and 30% in 2011–2012.
4.1. Fungal Pathogens Isolation and Identification
During 2011–2012, the mature peach (cultivar Baifeng) fruits were collected from the stone fruit orchards in Gansu, China. The ripening index of peach fruits at harvest: pH 3.75–3.98, organic acid 2.28–2.64 mg g−1, ethylene production 16.23–21.46 µL kg−1 h−1, soluble solids content 12.24–13.16%, total sugar 90.85–110.90 mg g−1, pectic substances 9.2–13.8 mg g−1. Thereafter, fruits were moist-incubated by placing in plastic containers with lids, lined with moist paper towels to maintain high relative humidity, and incubated at room temperature (20 °C) for 1–2 weeks to promote the pathogens growth and development. Small fruits sections (2 cm) were surface sterilized with 2% sodium hypochlorite (NaClO) for 3 min, and followed by 3 min rinses in sterile water. Fruits were then cut lengthwise along the lesion (1 cm) and placed individually onto PDA for 5 days at 25 °C. The spores and mycelium were transferred with a sterile needle from the colony to fresh Petri dishes containing PDA medium at Day 5. These cultures were grown for 5 days in an incubator at 25 °C, and then identified according to the colony and spores characteristics. Finally, all isolates were maintained and stored in 20% glycerol at −80 °C until use.
4.2. Spore Suspensions of Fungal Pathogen Preparation
The identified pathogens of peaches were cultured on PDA medium for 5 days, and then suspended in 5 mL of sterile water containing 0.05% (v
) Tween-80. Thereafter, the spore suspensions were filtered through 0.22 mm Millipore membranes to remove any adhering mycelia. The concentration of the spore suspension was determined using a hemacytometer, and then, the final concentration was adjusted to 1 × 106
4.3. Fruit Preparation
For inoculum production, the experiments were conducted with the peach (Prunus persica
L.) fruit cultivars Baifeng. The fresh fruits (pH = 3.53–3.64) were collected one week before commercial harvest during the 2012 production season, and the mature fruits (pH = 3.75–3.98) were collected and harvested at the mature stage, and sorted based on the size and the absence of physical injuries or disease infection. Before treatments, fruits were disinfected on the surface with 2% (v
) NaClO for 3 min, and then rinsed with sterile water and air-dried for approximately 30 min at room temperature (20 °C) prior to use [50
] and inoculation.
4.4. Pathogenicity of the Isolates on Peach Fruits
All the isolates in the present study were tested for pathogenicity on the mature peach fruits. Two groups of treatments were designed in this experiment, (i) one group of the sterile fruits were wounded once to a depth of 3 mm with a sterilized needle in the equatorial zone (wounded fruits) and (ii) another group of the sterile fruits were non-wounded with a sterilized needle (intact fruits). A 5-mm-diameter plug from a 5-day-old mycelial culture of isolates was inoculated onto intact and wounded peach fruits. Additionally, a 5-mm-diameter PDA plug was used as the untreated control treatment. Thereafter, all the treatments fruits were moist-incubated by placing in plastic containers with lids, lined with moist paper towels to maintain high relative humidity and incubated at room temperature (20 °C). Pathogenicity was determined as the ability to cause the typical decay symptom, and the number of fruit infected. The parameter of disease incidences was measured at 5 days after inoculation. Each experiment had three replications and each replication had three fruits, and all the experiments were repeated twice. The highest pathogenic pathogens were used to determine the antagonistic activity of Bacillus subtilis JK-14 in later experiments.
4.5. Formulations of Bacillus Subtilis JK-14 Preparation
The strain of B. subtilis JK-14 used in the present study was obtained from the College of Plant Protection, Gansu Agricultural University, isolated from the surface of peach fruits from an orchard in Gansu, China, and tested for its antifungal potential against the highest pathogenicity of the isolates on mature peach fruits. The active colony was then prepared by culturing on nutrient agar (NA, pH = 7.0) in Petri dishes for 3 days at 28 °C. A culture of B. subtilis JK-14 was obtained by transferring a colony from the activated culture plate into a 150 mL flask containing 30 mL liquid broth (peptone 0.3 g, yeast extract 0.3 g, NaCl 0.05 g) and shaking in an orbital shaker (200 rpm min−1) at 28 °C for 48 h. A formulation of fermentation liquid with bacterial cells (FLBC) was made by incubating the bacterial culture under the same conditions, and then dissolved with the sterile water to prepare the final concentrations of FLBC from 1 × 105 to 1 × 109 CFU mL−1. A formulation of bacterial cell suspension (BCS) was prepared by centrifuging the fermentation liquid at 12, 000 rpm min−1 at 4 °C for 20 min, and filtered by 0.22 µm biofilter to collect the bacterial sediment. Thereafter, the bacterial sediment was washed with an equal volume of saline (0.85% NaCl), and then dissolved with the sterile water to prepare the final concentrations of BCS from 1 × 105 to 1 × 109 CFU mL−1. The two formulations were stored at 4 °C for later use.
4.6. In Vitro and in Vivo Antagonistic Activity Determination
In vitro experiments, the antagonistic activity of B. subtilis
JK-14 against the main pathogens, were conducted following dual culture plate technique [51
]. The inhibitory effects of B. subtilis
JK-14 on the isolates with the highest pathogenicity were done by examining the growth rates inhibition using the paper–disc method on PDA [52
]. Each experiment had six replications and was repeated twice.
For the confirmation of the antagonistic activity of B. subtilis JK-14 (BCS formulation) in controlling A. tenuis and B. cinerea decay in fresh peach wounds, the fruits experiments were conducted to determine the controlling effects in vivo. A uniform wound (3 mm diameter and 3 mm deep) was made at the equator of each peach fruit using sterilized needle. An aliquot (30 µL) of B. subtilis JK-14 at 1 × 108 CFU mL−1 was pipetted into each wound site, and 30 µL of sterile water in place of the B. subtilis JK-14 was used as the control. Two hours later, 15 µL spores suspension of A. tenuis and B. cinerea (1 × 106 CFU mL−1) were inoculated into each wound, respectively. After air drying, the peaches were stored in enclosed plastic containers to maintain a high relative humidity (RH 85%) at 20 °C. Disease incidences and lesion diameters, and the symptoms of the treated peach fruits were measured and observed at 5 days after inoculation. All treatments were carried out with three replicates and three fruits for each treatment, and the experiment was conducted twice.
4.7. Efficacy of Bacillus subtilis JK-14 in Controlling of Peach Postharvest Disease
For the fruits inoculation, peach fruit samples were treated as described above to determine the antagonistic activity of B. subtilis JK-14 formulations (FLBC and BCS) in inhibiting A. tenuis and B. cinerea decay in mature peach wounds in vivo. An aliquot (30 µL) of different formulations of B. subtilis JK-14 at 1 × 105, 1 × 106, 1 × 107, 1 × 108, and 1 × 109 CFU mL−1 was pipetted into each wound site, and 30 µL of sterile water in place of the B. subtilis JK-14 formulations was used as the control. Two hours later, 15 µL spores suspension of A. tenuis and B. cinerea (1 × 106 CFU mL−1) were inoculated into each wound, respectively. After air drying, the incubation condition of treated peaches as described above. Disease incidences and lesion diameters, and the symptoms of the treated mature peach fruits were measured and observed at 5 days after inoculation. All treatments were carried out with three replicates and three fruits for each treatment, and the experiment was conducted twice.
4.8. Effects of Bacillus subtilis JK-14 on the Activities of Defense-Related Enzymes of Peaches
Peach fruit samples were treated as described above to test the efficacy of B. subtilis JK-14 in inhibiting A. tenuis and B. cinerea decay in mature peach wounds. The wounds were then treated with 30 µL of BCS of B. subtilis JK-14 at 1 × 107 CFU mL−1, and 30 µL of sterile water in place of the BCS formulation of B. subtilis JK-14 was used as the control. Two hours later, 15 µL spores suspension of the highest pathogenicity of the isolates of A. tenuis and B. cinerea (1 × 106 CFU mL−1) were inoculated into each wound. The treatments of sterile water and B. subtilis JK-14 alone were considered as the controls. The peach fruits were stored in enclosed plastic containers to maintain a high relative humidity (RH 85%) and incubated at 20 °C after air drying. In order to measure the activities of defense-related enzymes of peaches after the treatment of B. subtilis JK-14, the tissue surrounding each wound of fruit was collected at Day 4 after treatment. Three replicates consistent of three fruits were sampled in both inoculated group and control group, and the experiments were conducted twice.
4.9. Determination and Analysis of Defense-Related Enzyme Activities of Peaches
The extraction procedures of the enzyme extract from the collected samples were conducted following the method of Xu et al. [7
]. The tissue surrounding each wound of fruits (2 g) were collected and homogenized with 4 mL of ice-cold sodium phosphate buffer (50 mM, pH 7.8) containing 1.33 mM EDTA and 1% PVP. Thereafter, the homogenates were then centrifuged at 12,000× g
for 15 min at 4 °C, and the supernatants were collected and used as enzyme extract to assay the activity of POD, SOD and CAT of peaches after extraction using the spectrophotometer (AOE (UV1900), Shanghai, China).
POD activity was assayed following the method of Meng et al., with some minor modifications [54
]. The reaction mixture containing 0.2 mL of the enzyme extract and 2.2 mL of 0.3% guaiacol was incubated for 5 min at 30 °C, and then the reaction was initiated immediately by adding 0.6 mL of 0.3% H2
. The activity of POD was determined by measuring absorbance at 470 nm, and expressed as U per g fresh weight (U g−1
SOD activity was measured following the method of Giannopolitis and Ries, and determined by assaying the ability to inhibit the photochemical reduction of nitroblue tetrazolium chloride (NBT) [55
]. The reaction mixture (1.5 mL) contained 50 mM phosphate buffer (pH 7.8), 0.1 µM EDTA, 13 mM methionine, 75 µM NBT, 2 µM riboflavin and 50 µL enzyme extracts. One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of the NBT photo reduction rate, and the results were expressed as U g−1
CAT activity was measured according to the method described by Wang et al. [56
], with some modifications. The reaction mixtures contained 1.4 mL buffered substrate (50 mM sodium phosphate, pH 7.8, and 30 mM H2
) and 100 µL of enzyme extracts. The decomposition of H2
was measured by the decline in absorbance at 240 nm. One unit of the CAT activity was defined as the amount of the H2
decomposing, and the activity was expressed as U g−1
4.10. Statistical Analysis
Data presented in the present paper were pooled across two independent repeated experiments. All statistical analyses were performed with SPSS version 16.0 (SPSS Inc., Chicago, IL, USA, 2007). Data were analyzed by multi-way ANOVA. Duncan’s multiple range test were computed using standard error and T values of adjusted degrees of freedom. Differences at p < 0.05 were considered significant.