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Biocontrol of Aspergillus flavus on peanut kernels by use of a strain of marine Bacillus megaterium
International Journal of Food Microbiology 139 (2010) 31–35
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Biocontrol of Aspergillus flavus on peanut kernels by use of a strain of marine Bacillus megaterium Qing Kong a,⁎, Shihua Shan b, Qizheng Liu a, Xiudan Wang a, Fangtang Yu c a b c
School of Food Science and Engineering, Ocean University of China, Qingdao, Shandong 266003, China Shandong Peanut Research Institute, Qingdao, Shandong 266110, China College of Marine Life Sciences, Ocean University of China, Qingdao, Shandong 266003, China
a r t i c l e
i n f o
Article history: Received 17 September 2009 Received in revised form 15 December 2009 Accepted 25 January 2010 Keywords: Peanut kernels Biological control Aspergillus flavus Bacillus megaterium Marine microbiology
a b s t r a c t A strain of marine Bacillus megaterium isolated from the Yellow Sea of East China was evaluated for its activity in reducing postharvest decay of peanut kernels caused by Aspergillus flavus in in vitro and in vivo tests. The results showed that the concentrations of antagonist had a significant effect on biocontrol effectiveness in vivo: when the concentration of the washed bacteria cell suspension was used at 1 × 109 CFU/ml, the percentage rate of rot of peanut kernels was 31.67% ± 2.89%, which was markedly lower than that treated with water (the control) after 7 days of incubation at 28 °C. The results also showed that unwashed cell culture of B. megaterium was as effective as the washed cell suspension, and better biocontrol was obtained when longer incubation time of B. megaterium was applied. When the incubation time of B. megaterium was 60-h, the rate of decay declined to 41.67% ± 2.89%. Furthermore, relative to the expression of 18S rRNA, the mRNA abundances of aflR gene and aflS gene in the experiment group were 0.28 ± 0.03 and 0.024 ± 0.005 respectively, indicating that this strain of B. megaterium could significantly reduce the biosynthesis of aflatoxins and expression of aflR gene and aflS gene (p < 0.01). To the best of our knowledge, this is a first report demonstrating that the marine bacterium B. megaterium could be used as a biocontrol agent against postharvest fungal disease caused by A. flavus. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Peanuts (Arachis hypogaea) are one of the most important food and oilseed crops cultivated and utilized in most parts of the world. They are widely accepted as an excellent source of nutrition to both man and poultry due to their high protein content. Several investigations have listed a large number of fungi which could be isolated from peanuts during storage (Mukherjee and Nandi, 2001; Hedayati et al., 2007). Aspergillus flavus is the dominant storage fungus colonizing peanuts, capable of causing seed rots, moulding of seeds, pre- and post-emergence damping off, and reducing seed viability and seedling growth in peanuts (Kumar et al., 2008; Horn and Dorner, 2009). Colonization of peanuts with this mould is of importance because of its potential to produce aflatoxins, which are potent toxic, carcinogenic, mutagenic, immunosuppressive, and teratogenic agents (Calvo et al., 2002; Krishnamurthy et al., 2008). Aflatoxin biosynthesis has been proposed to involve at least 23 enzymatic reactions and 25 genes or open reading frames (ORFs) representing a well-defined aflatoxin pathway gene cluster (Yu et al., 2004). Among the aflatoxin pathway
⁎ Corresponding author. Tel.: +86 532 8203 1851; fax: +86 532 8203 2272. E-mail address: [email protected] (Q. Kong). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.01.036
genes, aflR is involved in transcription activation and aflS (originally named aflJ) is involved in regulation of aflatoxin biosynthesis, so aflR and aflS are the most important genes involved in pathway regulation (Meyers et al., 1998; Fox and Howlett, 2008). The need for protection of food and feedstuffs against A. flavus is universally recognized and several approaches such as treatment of peanuts with fungicides, fumigants, and plant and earth products have been suggested. Although the use of synthetic fungicides is a most effective decay control treatment, there is an urgent need to find effective and safe non-fungicide means of controlling postharvest pathogens mainly because of the toxicity of the synthetic fungicide residues to human health and the environment (Droby, 2006). In recent years, some antagonists have been applied in biocontrol of postharvest diseases of agricultural products. For example, a new strain of Bacillus pumilus isolated from Korean soybean sauce showed strong antifungal activity against the aflatoxin-producing fungi A. flavus and A. parasiticus (Cho et al., 2009). Serratia plymuthica 5-6, isolated from the rhizosphere of pea, is reported to reduce dry rot of potato caused by Fusarium sambucinum (Gould et al., 2008). Generally, most of these antagonistic microorganisms were isolated from fruit surface, fruit plant, and soil (Wilson et al., 1993; Janisiewicz, 1996; Janisiewicz and Korsten, 2002). Marine microorganisms are capable of producing many unique bioactive substances, and therefore could be a rich resource for
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Q. Kong et al. / International Journal of Food Microbiology 139 (2010) 31–35
antagonists (Blunt et al., 2008). We have screened a marine strain of Bacillus megaterium which has not been reported previously as a biological control agent. The aim of this study was to investigate the efficacy of B. megaterium in inhibiting the growth of A. flavus in vitro and in vivo. The effect of B. megaterium on the biosynthesis of aflatoxins and expression of aflR gene and aflS gene was also studied. 2. Materials and methods 2.1. Peanut kernels and microorganisms Peanut kernels with a commercial level of maturity were used immediately after harvest, or stored at 4 °C for no longer than 48 h before using (Brown et al., 2000). Before treatments, the peanut kernels were washed with tap water, then surfaced-disinfected with 0.1% sodium hypochlorite for 1 min, cleaned with tap water, and air dried prior to wounding. The marine antagonist B. megaterium used in this study was isolated from the Yellow Sea of East China. It was identified by morphological, physiological, and chemotaxonomic features of the organism and confirmed by 16S rRNA based phylogenetic analysis (Sneath, 1986; Olofsson and Molin, 2007). Bacteria cells were prepared by growing cultures in beef extract peptone broth (BEPB: beef extract, 3 g/l; peptone, 10 g/l; and sodium chloride, 5 g/l) for 24 h at 37 °C with shaking at 200 rpm. The cells were harvested by centrifuging (Refrigerated Centrifuge 3-18R, New Jersey TOMOS) at 8000 rpm (10,000 g) for 45 s and were washed twice with sterile distilled water to remove the growth medium. The cells were counted by the plate count method and then were diluted with sterile distilled water as required. The aflatoxigenic strain of A. flavus 2810 was obtained from Fujian Agriculture and Forestry University (Fuzhou, China) and maintained on potato dextrose agar (PDA) medium (containing the extract of 200 g boiled potato, 20 g glucose and 20 g agar in 1 l of distilled water) at 4 °C. Spore suspensions were prepared by flooding 7-day-old sporulating cultures of A. flavus with sterile distilled water. Spores were counted with a haemocytometer and then were diluted with sterile distilled water as required. 2.2. In vitro antifungal assay To evaluate the interactions between the antagonist and the pathogen in culture, 6-mm diameter plugs were cut from 7-day-old PDA cultures of A. flavus and then placed on another PDA plate (15 ml/ plate) seeded with 1.0 ml of different treatments of B. megaterium. The treatments were as follows: (A) autoclaved culture; (B) culture filtrate; (C) 1 × 108 CFU/ml unwashed cell culture mixture; and (D) 1 × 108 CFU/ml washed cell suspension and sterile distilled water as a control. Autoclaved cultures were prepared by autoclaving a sample containing B. megaterium in culture broth for 20 min at 121 °C. Culture filtrates were prepared by filtering the supernatant of centrifuged culture of the antagonist through a 0.2-μm polycarbonate membrane filter (Wang et al., 2008). Unwashed cells from 24-h cultures were adjusted to 1 × 108 CFU/ml by adding additional culture filtrate. Plates were incubated for 7 days at 37 °C. Fungal growth was recorded after 7 days. Growth inhibition was calculated as the percentage of inhibition of radial growth relative to the control (Bouchra et al., 2003). Three replicates were used per treatment and the experiments were repeated twice. The effect of B. megaterium on spore germination and germ tube elongation of pathogen was tested in potato dextrose broth (PDB). The washed cell suspension of B. megaterium (concentration in PDB was finally adjusted to 1 × 108 CFU/ml) or sterile distilled water as a control was added into the 100-ml beaker flask containing 15 ml PDB, respectively. Then, 100 μl of spore suspension (5 × 106 spores/ml) of A. flavus were added into each beaker flask. 100–150 spores per
replicate were observed microscopically following 20 h of incubation at 37 °C on a rotary shaker (QYC 2102, Shanghai FUMA) at 200 rpm. The treatment was replicated three times and the experiment was conducted twice (Yu et al., 2007). 2.3. Efficacy of B. megaterium on control of A. flavus in vivo Peanut kernels were wounded (6 mm diameter and approximately 3 mm deep) using a sterile borer and then treated with 20 μl of one of the following: (A) autoclaved culture; (B) culture filtrate; (C) 1 × 108 CFU/ml unwashed cell culture mixture; and (D) 1 × 108 CFU/ ml washed cell suspension; and sterile distilled water as a control. Two hours later, 10 μl of A. flavus spores at 5 × 104 spores/ml was inoculated into each wound. The kernels were placed in artificial weather chamber (QHX-300BS-III, Shanghai Xinmiao) to maintain high humidity (85%) and incubated at 28 °C. The number of kernel wounds that were infected was recorded after 7 days inoculation. There were three replicates of 20 kernels per treatment and the experiment was conducted twice. 2.4. Effect of different incubation times of B. megaterium on control of A. flavus in vivo The peanut kernels were wounded as described above. 20 μl of 1 × 108 CFU/ml washed cell suspension of B. megaterium that had been grown in BEPB for 18, 24, 36, 48 and 60 h was respectively inoculated into the wounds of the peanut kernels, and sterile distilled water as a control. After 2 h, 10 μl of 5 × 104 spores/ml suspension of A. flavus was inoculated into each wound. The kernels were placed in an artificial weather chamber as described above. 2.5. Effect of different concentrations of B. megaterium on control of A. flavus in vivo The peanut kernels were wounded as described above. The suspensions of washed cells of B. megaterium were adjusted to concentrations of 1 × 106, 1 × 107, 1 × 108 and 1 × 109 CFU/ml with sterile distilled water, respectively (Fan and Tian, 2000). 20 μl of 1 × 106, 1 × 107, 1 × 108 and 1 × 109 CFU/ml washed cell suspension respectively was inoculated into each wound, and sterile distilled water as a control. 10 μl of 5 × 104 spores/ml suspension of A. flavus was inoculated to each wound 2 h later. Treated kernels were stored in an artificial weather chamber as described above. 2.6. Effect of B. megaterium on aflR and aflS expression A. flavus and B. megaterium were mixed in 50-ml beaker flasks containing 10 ml PDB and concentrations were adjusted to 1 × 106 spores/ml of A. flavus and 1 × 108 CFU/ml of B. megaterium. 1 × 106 spores/ml of A. flavus in PDB was the control. After 48 h of cultivation in rotary shaker (28 °C, 200 rpm), the amount of aflatoxins was detected by aflatoxin plate kit (Beacon Analytical Systems Inc., Portland, Maine, USA). Quantitative detection of aflR and aflS expression was carried out using an ABI PRISM 7500 Fast real-time PCR system (Applied Biosystems Inc., Foster City, CA, USA). The mycelia of A. flavus were collected by centrifugation (Refrigerated Centrifuge 3-18R, New Jersey TOMOS) at 10,000 g for 30 s and total RNA was prepared from mycelia using a UNIQ-10 Total RNA Kit (Sangon Biological Co. Ltd., Shanghai, China) according to the manufacturer's instructions. Quality and quantity of RNA was assessed by measuring the A260/ A280 ratio and by analysis on ethidium bromide stained 1% agar gels (Marino et al., 2003). The quantitative real-time PCR was performed using a One Step SYBR® Prime Script™ RT-PCR Kit (TaKaRa Bio Inc., Otsu, Shiga, Japan). Each PCR reaction (20 μl) was incubated at 42 °C for 5 min followed by incubation at 95 °C for 10 s. Then the tubes were
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cycled for 40 repeats at 95 °C for 5 s, and at 60 °C for 34 s. A melting curve was generated at the end of every run to ensure product uniformity (Ririe et al., 1997). Primers for PCR: 18S rRNA forward: 5′GGCTCAAGCCGATGGAAGT-3′; 18S rRNA reverse: 5′-AGCACGACAGGGTTTAACAAGA-3′; aflR gene forward: 5′- GGGAACAAGAGGGCTACCGA-3′; aflR gene reverse: 5′- TGCCAGCACCTTGAGAACG-3′; aflS gene forward: 5′- GGTCGTGCATGTGCGAATC-3′; aflS gene reverse: 5′- GAGGGCAACAACCAGTGAGG-3′. The 18S rRNA, aflR gene and aflS gene primers were synthesized by SBS Genetech (Beijing, China). Each PCR reaction was replicated three times and the experiment was repeated twice.
the treatment with the unwashed bacteria cell suspension at 1 × 108 CFU/ml was 36.67% ± 5.77%, which was significantly lower than the treatments with the autoclaved culture, culture filtrate and water control. Meanwhile, the average percentage of the disease incidence in the treatment with the washed cell culture mixture was 41.67% ± 2.89%, which also showed remarkable decay control compared with the treatments of water control, autoclaved culture and culture filtrate (p < 0.05).
2.7. Data analysis
Different incubation times of B. megaterium significantly influenced the effectiveness of the disease control in peanut kernels at 28 °C (p < 0.05). The results showed that better biocontrol was obtained when longer incubation time of B. megaterium was applied (Fig. 2). When the incubation time of B. megaterium was 60-h, the rate of decay declined to 41.67% ± 2.89%. So the best control effect was obtained by the washed cell suspension of B. megaterium when the incubation time was 48–60 h.
In this study, all experiments were repeated twice and the data from two experiments were combined. All data were analyzed by one way analysis of variance (ANOVA) in the statistical software SAS v. 8.0. Mean separations were performed by Duncan's multiple range tests. Differences at P = 0.05 were considered as significant.
3.3. Effect of different incubation times of B. megaterium on control of rot caused by A. flavus in vivo
3. Results 3.1. Effects of B. megaterium on germination and growth of A. flavus in vitro In the tests on PDA plates, different treatments exhibited different inhibitory effects on A. flavus. The percentage of inhibition of culture filtrate, washed cell suspension (1 × 108 CFU/ml) and unwashed cell suspension (1 × 108 CFU/ml) reached 30.6 ± 4.5, 35.1 ± 4.8 and 21.7 ± 5.2, respectively. In contrast, the treatments of B. megaterium including autoclaved culture and sterile distilled water as a control did not inhibit the growth of A. flavus, because the growth reached the edge of the plates. In the assay of spore germination, the washed cell suspension of B. megaterium at 108 CFU/ml significantly suppressed germination of A. flavus spores compared with the control (p < 0.05) in PDB. 3.2. Efficacy of B. megaterium on control of decay caused by A. flavus in vivo As shown in Fig. 1, the unwashed cell suspension of B. megaterium was the most effective treatment on control of the rots in peanut kernels caused by A. flavus. After 7 days at 28 °C, the disease incidence of the water control and autoclaved culture were both 100%, whereas
Fig. 1. Effect of B. megaterium on control of A. flavus. (A) autoclaved culture; (B) culture filtrate; (C) 1 × 108 CFU/ml unwashed cell mixture; and (D) 1 × 108 CFU/ml washed cell suspension. The control is treated with sterile distilled water. Values followed by different letters were significantly different according to Duncan's multiple range test p = 0.05.
3.4. Effect of different concentrations of B. megaterium on control of rot caused by A. flavus in vivo The disease incidence on all antagonist treated kernels (treated with 20 μl of 1 × 106, 1 × 107, 1 × 108 and 1 × 109 CFU/ml washed cell suspension, respectively) was significantly lower than those on the control (p < 0.05). The concentrations of B. megaterium significantly influenced disease incidence on peanut kernels (p < 0.05, Fig. 3). The results showed that the higher the concentrations of the antagonist, the lower the disease incidence exhibited. When the concentration of B. megaterium at 1 × 109 CFU/ml and spore suspension of A. flavus was at 5 × 104 cells/ml, the rate of decay declined to 31.67% ± 2.89%. 3.5. Effect of B. megaterium on aflR and aflS expression After 48 h of cultivation, the aflatoxin concentration of the control was 112.36 ± 5.17 ppb, whereas in the treatment with the B. megaterium (1× 108 CFU/ml) was 25.05 ± 2.10 ppb, which was significantly lower than the control (p < 0.01). Moreover, as shown in Fig. 4, relative to the expression of 18S rRNA, the mRNA abundances of aflR and aflS genes in control were 1.11± 0.24 and 0.18 ± 0.05 respectively, while in experiment group were 0.28 ± 0.03 and 0.024 ± 0.005 respectively, indicating that B. megaterium (1 × 108 CFU/ml) could significantly suppress the expression of these two genes (p < 0.01). The concentration of aflatoxins and the mRNA abundances of these two genes in the
Fig. 2. Effect of different incubation times of B. megaterium on control of A. flavus. The control is treated with sterile distilled water. Values followed by different letters were significantly different according to Duncan's multiple range test p = 0.05.
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Fig. 3. Effect of different concentrations of B. megaterium on control of A. flavus. (A) 1 × 106 CFU/ml B. megaterium; (B) 1 × 107 CFU/ml B. megaterium; (C) 1 × 108 CFU/ ml B. megaterium; and (D) 1 × 109 CFU/ml B. megaterium. The control is treated with sterile distilled water. Values followed by different letters were significantly different according to Duncan's multiple range test p = 0.05.
control experiment corresponded: the lower the expression of aflR gene and aflS gene, the lower the aflatoxin concentration detected. 4. Discussion The results of this study demonstrated for the first time that the marine bacteria B. megaterium had great potential in controlling postharvest disease caused by A. flavus on peanut kernels. In the in vitro tests, the washed cell suspension of B. megaterium at 1 × 108 CFU/ ml significantly suppressed spore germination of A. flavus in PDB. Moreover, the percentage of inhibition of culture filtrate, washed and unwashed cell suspension (1 × 108 CFU/ml) could influence the growth of the pathogen on PDA plates. Thus, we concluded the cells of B. megaterium had some inhibitory effect on A. flavus, but the metabolites of B. megaterium were more strongly inhibitory to A. flavus (p < 0.05) on PDA plates. This finding suggested that B. megaterium could inhibit the pathogens due to some toxic compounds accumulated in the culture medium or antibiotic production. This result was in agreement with that reported from other antagonists such as B. subtilis (Pusey and Wilson, 1984) and B. pumilus (Cho et al., 2009). The results from in vivo test showed that the unwashed cell culture of B. megaterium was as effective as the washed cell suspension, and better control was obtained with longer incubation time. The concentrations of antagonist had significant effects on biocontrol
Fig. 4. Real-time qRT-PCR analysis of changes in aflR and aflS genes expression. Each mRNA was assayed in triplicate in two independent experiments. Representative results (mean ± SD) from control (106 spores/ml of A. flavus in PDB) and experiment (106 spores/ml of A. flavus and 108 CFU/ml of B. megaterium in PDB) are plotted.
effectiveness: the higher the concentration of B. megaterium, the better biocontrol activity. The best biocontrol was obtained with B. megaterium at 1 × 109 CFU/ml, because the disease incidence was the lowest compared with other concentration treatments (Fig. 3). These results indicate that, apart from the production of antimicrobial substances by B. megaterium, bacterial competition for space and nutrition is perhaps another mode of action (Fan and Tian, 2001; Wang et al., 2008). Our findings also showed the biosynthesis of aflatoxins was related to the expression of aflatoxin pathway genes. This strain of B. megaterium was able to significantly inhibit the biosynthesis of aflatoxins in PDB (p < 0.01) and the primary inhibitory mechanism is die to inhibition of the expression of the genes relating to aflatoxin biosynthesis (such as aflR gene and aflS gene). But our finding raise the question of whether B. megaterium could inhibit the expression of all aflatoxin pathway genes or only some of them. The use of DNA microarrays and real-time qPCR could answer this question (Hooper et al., 2001). In conclusion, our results showed that the marine bacterium B. megaterium has potential biocontrol activity against the decay and aflatoxin biosynthesis caused by A. flavus. This potential may extend to direct use in the market to prolong shelf life, provided the antagonist and its metabolites are safe for human consumption. Acknowledgements This research was supported by the research grant from Qingdao Municipal Science and Technology Commission (08-1-3-24-jch), Shandong Province, People's Republic of China; the National Natural Science Foundation of China (30771361); and Undergraduates' Innovation Experimental Program, Ministry of Education of China (081042309). References Blunt, J.W., Copp, B.R., Hu, W.P., Munro, M.H.G., Northcote, P.T., Prinsep, M.R., 2008. Marine natural products. Natural Product Reports 25, 35–94. Bouchra, C., Achouri, M., Hassani, L.M.I., Hmamouchi, M., 2003. Chemical composition and antifungal activity of essential oils of seven Moroccan Labiatae against Botrytis cinerea Pers: Fr. Journal of Ethnopharmacology 89, 165–169. Brown, G.E., Davis, C., Chambers, M., 2000. Control of citrus green mold with Aspire is impacted by the type of injury. Postharvest Biology and Technology 18, 57–65. Calvo, A.M., Wilson, R.A., Bok, J.W., Keller, N.P., 2002. Relationship between secondary metabolism and fungal development. Microbiology and Molecular Biology Reviews 66, 447–459. Cho, K.M., Math, R.K., Hong, S.Y., Islam, S.M.A., Mandanna, D.K., Cho, J.J., Yun, M.G., Kim, J.M., Yun, H.D., 2009. Iturin produced by Bacillus pumilus HY1 from Korean soybean sauce (kanjang) inhibits growth of aflatoxin producing fungi. Food Control 20, 402–406. Droby, S., 2006. Improving quality and safety of fresh fruits and vegetables after harvest by the use of biocontrol agents and natural materials. Acta Horticulturae 709, 45–51. Fan, Q., Tian, S.P., 2000. Postharvest biological control of Rhizopus rot of nectarine fruits by Pichia membranefaciens. Plant Disease 84, 1212–1216. Fan, Q., Tian, S.P., 2001. Postharvest biological control of grey mold and blue mold on apple by Cryptococcus albidus (Saito) Skinner. Postharvest Biology and Technology 21, 341–350. Fox, E.M., Howlett, B.J., 2008. Secondary metabolism: regulation and role in fungal biology. Current Opinion in Microbiology 11, 481–487. Gould, M., Nelson, L.M., Waterer, D., Hynes, R.K., 2008. Biocontrol of Fusarium sambucinum, dry rot of potato, by Serratia plymuthica 5-6. Biocontrol Science and Technology 18, 1005–1016. Hedayati, M.T., Pasqualotto, A.C., Warn, P.A., Bowyer, P., Denning, D.W., 2007. Aspergillus flavus: human pathogen, allergen and mycotoxin producer. Microbiology 153, 1677–1692. Hooper, L.V., Wong, M.H., Thelin, A., Hansson, L., Falk, P.G., Gordon, J.I., 2001. Molecular analysis of commensal host-microbial relationships in the intestine. Science 291, 881–884. Horn, B.W., Dorner, J.W., 2009. Effect of nontoxigenic Aspergillus flavus and A. parasiticus on aflatoxin contamination of wounded peanut seeds inoculated with agricultural soil containing natural fungal populations. Biocontrol Science and Technology 19, 249–262. Janisiewicz, W.J., 1996. Ecological diversity, niche, overlap, and coexistence of antagonists used in developing diseases of apples. Phytopathology 86, 473–479. Janisiewicz, W.J., Korsten, L., 2002. Biological control of postharvest diseases of fruits. Annual Review of Phytopathology 40, 411–441. Krishnamurthy, Y.L., Shashikala, J., Naik, B.S., 2008. Antifungal potential of some natural products against Aspergillus flavus in soybean seeds during storage. Journal of Stored Products Research 44, 305–309.
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Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Biocontrol of Aspergillus flavus on peanut kernels by use of a strain of marine Bacillus megaterium Qing Kong a,⁎, Shihua Shan b, Qizheng Liu a, Xiudan Wang a, Fangtang Yu c a b c
School of Food Science and Engineering, Ocean University of China, Qingdao, Shandong 266003, China Shandong Peanut Research Institute, Qingdao, Shandong 266110, China College of Marine Life Sciences, Ocean University of China, Qingdao, Shandong 266003, China
a r t i c l e
i n f o
Article history: Received 17 September 2009 Received in revised form 15 December 2009 Accepted 25 January 2010 Keywords: Peanut kernels Biological control Aspergillus flavus Bacillus megaterium Marine microbiology
a b s t r a c t A strain of marine Bacillus megaterium isolated from the Yellow Sea of East China was evaluated for its activity in reducing postharvest decay of peanut kernels caused by Aspergillus flavus in in vitro and in vivo tests. The results showed that the concentrations of antagonist had a significant effect on biocontrol effectiveness in vivo: when the concentration of the washed bacteria cell suspension was used at 1 × 109 CFU/ml, the percentage rate of rot of peanut kernels was 31.67% ± 2.89%, which was markedly lower than that treated with water (the control) after 7 days of incubation at 28 °C. The results also showed that unwashed cell culture of B. megaterium was as effective as the washed cell suspension, and better biocontrol was obtained when longer incubation time of B. megaterium was applied. When the incubation time of B. megaterium was 60-h, the rate of decay declined to 41.67% ± 2.89%. Furthermore, relative to the expression of 18S rRNA, the mRNA abundances of aflR gene and aflS gene in the experiment group were 0.28 ± 0.03 and 0.024 ± 0.005 respectively, indicating that this strain of B. megaterium could significantly reduce the biosynthesis of aflatoxins and expression of aflR gene and aflS gene (p < 0.01). To the best of our knowledge, this is a first report demonstrating that the marine bacterium B. megaterium could be used as a biocontrol agent against postharvest fungal disease caused by A. flavus. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Peanuts (Arachis hypogaea) are one of the most important food and oilseed crops cultivated and utilized in most parts of the world. They are widely accepted as an excellent source of nutrition to both man and poultry due to their high protein content. Several investigations have listed a large number of fungi which could be isolated from peanuts during storage (Mukherjee and Nandi, 2001; Hedayati et al., 2007). Aspergillus flavus is the dominant storage fungus colonizing peanuts, capable of causing seed rots, moulding of seeds, pre- and post-emergence damping off, and reducing seed viability and seedling growth in peanuts (Kumar et al., 2008; Horn and Dorner, 2009). Colonization of peanuts with this mould is of importance because of its potential to produce aflatoxins, which are potent toxic, carcinogenic, mutagenic, immunosuppressive, and teratogenic agents (Calvo et al., 2002; Krishnamurthy et al., 2008). Aflatoxin biosynthesis has been proposed to involve at least 23 enzymatic reactions and 25 genes or open reading frames (ORFs) representing a well-defined aflatoxin pathway gene cluster (Yu et al., 2004). Among the aflatoxin pathway
⁎ Corresponding author. Tel.: +86 532 8203 1851; fax: +86 532 8203 2272. E-mail address: [email protected] (Q. Kong). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.01.036
genes, aflR is involved in transcription activation and aflS (originally named aflJ) is involved in regulation of aflatoxin biosynthesis, so aflR and aflS are the most important genes involved in pathway regulation (Meyers et al., 1998; Fox and Howlett, 2008). The need for protection of food and feedstuffs against A. flavus is universally recognized and several approaches such as treatment of peanuts with fungicides, fumigants, and plant and earth products have been suggested. Although the use of synthetic fungicides is a most effective decay control treatment, there is an urgent need to find effective and safe non-fungicide means of controlling postharvest pathogens mainly because of the toxicity of the synthetic fungicide residues to human health and the environment (Droby, 2006). In recent years, some antagonists have been applied in biocontrol of postharvest diseases of agricultural products. For example, a new strain of Bacillus pumilus isolated from Korean soybean sauce showed strong antifungal activity against the aflatoxin-producing fungi A. flavus and A. parasiticus (Cho et al., 2009). Serratia plymuthica 5-6, isolated from the rhizosphere of pea, is reported to reduce dry rot of potato caused by Fusarium sambucinum (Gould et al., 2008). Generally, most of these antagonistic microorganisms were isolated from fruit surface, fruit plant, and soil (Wilson et al., 1993; Janisiewicz, 1996; Janisiewicz and Korsten, 2002). Marine microorganisms are capable of producing many unique bioactive substances, and therefore could be a rich resource for
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antagonists (Blunt et al., 2008). We have screened a marine strain of Bacillus megaterium which has not been reported previously as a biological control agent. The aim of this study was to investigate the efficacy of B. megaterium in inhibiting the growth of A. flavus in vitro and in vivo. The effect of B. megaterium on the biosynthesis of aflatoxins and expression of aflR gene and aflS gene was also studied. 2. Materials and methods 2.1. Peanut kernels and microorganisms Peanut kernels with a commercial level of maturity were used immediately after harvest, or stored at 4 °C for no longer than 48 h before using (Brown et al., 2000). Before treatments, the peanut kernels were washed with tap water, then surfaced-disinfected with 0.1% sodium hypochlorite for 1 min, cleaned with tap water, and air dried prior to wounding. The marine antagonist B. megaterium used in this study was isolated from the Yellow Sea of East China. It was identified by morphological, physiological, and chemotaxonomic features of the organism and confirmed by 16S rRNA based phylogenetic analysis (Sneath, 1986; Olofsson and Molin, 2007). Bacteria cells were prepared by growing cultures in beef extract peptone broth (BEPB: beef extract, 3 g/l; peptone, 10 g/l; and sodium chloride, 5 g/l) for 24 h at 37 °C with shaking at 200 rpm. The cells were harvested by centrifuging (Refrigerated Centrifuge 3-18R, New Jersey TOMOS) at 8000 rpm (10,000 g) for 45 s and were washed twice with sterile distilled water to remove the growth medium. The cells were counted by the plate count method and then were diluted with sterile distilled water as required. The aflatoxigenic strain of A. flavus 2810 was obtained from Fujian Agriculture and Forestry University (Fuzhou, China) and maintained on potato dextrose agar (PDA) medium (containing the extract of 200 g boiled potato, 20 g glucose and 20 g agar in 1 l of distilled water) at 4 °C. Spore suspensions were prepared by flooding 7-day-old sporulating cultures of A. flavus with sterile distilled water. Spores were counted with a haemocytometer and then were diluted with sterile distilled water as required. 2.2. In vitro antifungal assay To evaluate the interactions between the antagonist and the pathogen in culture, 6-mm diameter plugs were cut from 7-day-old PDA cultures of A. flavus and then placed on another PDA plate (15 ml/ plate) seeded with 1.0 ml of different treatments of B. megaterium. The treatments were as follows: (A) autoclaved culture; (B) culture filtrate; (C) 1 × 108 CFU/ml unwashed cell culture mixture; and (D) 1 × 108 CFU/ml washed cell suspension and sterile distilled water as a control. Autoclaved cultures were prepared by autoclaving a sample containing B. megaterium in culture broth for 20 min at 121 °C. Culture filtrates were prepared by filtering the supernatant of centrifuged culture of the antagonist through a 0.2-μm polycarbonate membrane filter (Wang et al., 2008). Unwashed cells from 24-h cultures were adjusted to 1 × 108 CFU/ml by adding additional culture filtrate. Plates were incubated for 7 days at 37 °C. Fungal growth was recorded after 7 days. Growth inhibition was calculated as the percentage of inhibition of radial growth relative to the control (Bouchra et al., 2003). Three replicates were used per treatment and the experiments were repeated twice. The effect of B. megaterium on spore germination and germ tube elongation of pathogen was tested in potato dextrose broth (PDB). The washed cell suspension of B. megaterium (concentration in PDB was finally adjusted to 1 × 108 CFU/ml) or sterile distilled water as a control was added into the 100-ml beaker flask containing 15 ml PDB, respectively. Then, 100 μl of spore suspension (5 × 106 spores/ml) of A. flavus were added into each beaker flask. 100–150 spores per
replicate were observed microscopically following 20 h of incubation at 37 °C on a rotary shaker (QYC 2102, Shanghai FUMA) at 200 rpm. The treatment was replicated three times and the experiment was conducted twice (Yu et al., 2007). 2.3. Efficacy of B. megaterium on control of A. flavus in vivo Peanut kernels were wounded (6 mm diameter and approximately 3 mm deep) using a sterile borer and then treated with 20 μl of one of the following: (A) autoclaved culture; (B) culture filtrate; (C) 1 × 108 CFU/ml unwashed cell culture mixture; and (D) 1 × 108 CFU/ ml washed cell suspension; and sterile distilled water as a control. Two hours later, 10 μl of A. flavus spores at 5 × 104 spores/ml was inoculated into each wound. The kernels were placed in artificial weather chamber (QHX-300BS-III, Shanghai Xinmiao) to maintain high humidity (85%) and incubated at 28 °C. The number of kernel wounds that were infected was recorded after 7 days inoculation. There were three replicates of 20 kernels per treatment and the experiment was conducted twice. 2.4. Effect of different incubation times of B. megaterium on control of A. flavus in vivo The peanut kernels were wounded as described above. 20 μl of 1 × 108 CFU/ml washed cell suspension of B. megaterium that had been grown in BEPB for 18, 24, 36, 48 and 60 h was respectively inoculated into the wounds of the peanut kernels, and sterile distilled water as a control. After 2 h, 10 μl of 5 × 104 spores/ml suspension of A. flavus was inoculated into each wound. The kernels were placed in an artificial weather chamber as described above. 2.5. Effect of different concentrations of B. megaterium on control of A. flavus in vivo The peanut kernels were wounded as described above. The suspensions of washed cells of B. megaterium were adjusted to concentrations of 1 × 106, 1 × 107, 1 × 108 and 1 × 109 CFU/ml with sterile distilled water, respectively (Fan and Tian, 2000). 20 μl of 1 × 106, 1 × 107, 1 × 108 and 1 × 109 CFU/ml washed cell suspension respectively was inoculated into each wound, and sterile distilled water as a control. 10 μl of 5 × 104 spores/ml suspension of A. flavus was inoculated to each wound 2 h later. Treated kernels were stored in an artificial weather chamber as described above. 2.6. Effect of B. megaterium on aflR and aflS expression A. flavus and B. megaterium were mixed in 50-ml beaker flasks containing 10 ml PDB and concentrations were adjusted to 1 × 106 spores/ml of A. flavus and 1 × 108 CFU/ml of B. megaterium. 1 × 106 spores/ml of A. flavus in PDB was the control. After 48 h of cultivation in rotary shaker (28 °C, 200 rpm), the amount of aflatoxins was detected by aflatoxin plate kit (Beacon Analytical Systems Inc., Portland, Maine, USA). Quantitative detection of aflR and aflS expression was carried out using an ABI PRISM 7500 Fast real-time PCR system (Applied Biosystems Inc., Foster City, CA, USA). The mycelia of A. flavus were collected by centrifugation (Refrigerated Centrifuge 3-18R, New Jersey TOMOS) at 10,000 g for 30 s and total RNA was prepared from mycelia using a UNIQ-10 Total RNA Kit (Sangon Biological Co. Ltd., Shanghai, China) according to the manufacturer's instructions. Quality and quantity of RNA was assessed by measuring the A260/ A280 ratio and by analysis on ethidium bromide stained 1% agar gels (Marino et al., 2003). The quantitative real-time PCR was performed using a One Step SYBR® Prime Script™ RT-PCR Kit (TaKaRa Bio Inc., Otsu, Shiga, Japan). Each PCR reaction (20 μl) was incubated at 42 °C for 5 min followed by incubation at 95 °C for 10 s. Then the tubes were
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cycled for 40 repeats at 95 °C for 5 s, and at 60 °C for 34 s. A melting curve was generated at the end of every run to ensure product uniformity (Ririe et al., 1997). Primers for PCR: 18S rRNA forward: 5′GGCTCAAGCCGATGGAAGT-3′; 18S rRNA reverse: 5′-AGCACGACAGGGTTTAACAAGA-3′; aflR gene forward: 5′- GGGAACAAGAGGGCTACCGA-3′; aflR gene reverse: 5′- TGCCAGCACCTTGAGAACG-3′; aflS gene forward: 5′- GGTCGTGCATGTGCGAATC-3′; aflS gene reverse: 5′- GAGGGCAACAACCAGTGAGG-3′. The 18S rRNA, aflR gene and aflS gene primers were synthesized by SBS Genetech (Beijing, China). Each PCR reaction was replicated three times and the experiment was repeated twice.
the treatment with the unwashed bacteria cell suspension at 1 × 108 CFU/ml was 36.67% ± 5.77%, which was significantly lower than the treatments with the autoclaved culture, culture filtrate and water control. Meanwhile, the average percentage of the disease incidence in the treatment with the washed cell culture mixture was 41.67% ± 2.89%, which also showed remarkable decay control compared with the treatments of water control, autoclaved culture and culture filtrate (p < 0.05).
2.7. Data analysis
Different incubation times of B. megaterium significantly influenced the effectiveness of the disease control in peanut kernels at 28 °C (p < 0.05). The results showed that better biocontrol was obtained when longer incubation time of B. megaterium was applied (Fig. 2). When the incubation time of B. megaterium was 60-h, the rate of decay declined to 41.67% ± 2.89%. So the best control effect was obtained by the washed cell suspension of B. megaterium when the incubation time was 48–60 h.
In this study, all experiments were repeated twice and the data from two experiments were combined. All data were analyzed by one way analysis of variance (ANOVA) in the statistical software SAS v. 8.0. Mean separations were performed by Duncan's multiple range tests. Differences at P = 0.05 were considered as significant.
3.3. Effect of different incubation times of B. megaterium on control of rot caused by A. flavus in vivo
3. Results 3.1. Effects of B. megaterium on germination and growth of A. flavus in vitro In the tests on PDA plates, different treatments exhibited different inhibitory effects on A. flavus. The percentage of inhibition of culture filtrate, washed cell suspension (1 × 108 CFU/ml) and unwashed cell suspension (1 × 108 CFU/ml) reached 30.6 ± 4.5, 35.1 ± 4.8 and 21.7 ± 5.2, respectively. In contrast, the treatments of B. megaterium including autoclaved culture and sterile distilled water as a control did not inhibit the growth of A. flavus, because the growth reached the edge of the plates. In the assay of spore germination, the washed cell suspension of B. megaterium at 108 CFU/ml significantly suppressed germination of A. flavus spores compared with the control (p < 0.05) in PDB. 3.2. Efficacy of B. megaterium on control of decay caused by A. flavus in vivo As shown in Fig. 1, the unwashed cell suspension of B. megaterium was the most effective treatment on control of the rots in peanut kernels caused by A. flavus. After 7 days at 28 °C, the disease incidence of the water control and autoclaved culture were both 100%, whereas
Fig. 1. Effect of B. megaterium on control of A. flavus. (A) autoclaved culture; (B) culture filtrate; (C) 1 × 108 CFU/ml unwashed cell mixture; and (D) 1 × 108 CFU/ml washed cell suspension. The control is treated with sterile distilled water. Values followed by different letters were significantly different according to Duncan's multiple range test p = 0.05.
3.4. Effect of different concentrations of B. megaterium on control of rot caused by A. flavus in vivo The disease incidence on all antagonist treated kernels (treated with 20 μl of 1 × 106, 1 × 107, 1 × 108 and 1 × 109 CFU/ml washed cell suspension, respectively) was significantly lower than those on the control (p < 0.05). The concentrations of B. megaterium significantly influenced disease incidence on peanut kernels (p < 0.05, Fig. 3). The results showed that the higher the concentrations of the antagonist, the lower the disease incidence exhibited. When the concentration of B. megaterium at 1 × 109 CFU/ml and spore suspension of A. flavus was at 5 × 104 cells/ml, the rate of decay declined to 31.67% ± 2.89%. 3.5. Effect of B. megaterium on aflR and aflS expression After 48 h of cultivation, the aflatoxin concentration of the control was 112.36 ± 5.17 ppb, whereas in the treatment with the B. megaterium (1× 108 CFU/ml) was 25.05 ± 2.10 ppb, which was significantly lower than the control (p < 0.01). Moreover, as shown in Fig. 4, relative to the expression of 18S rRNA, the mRNA abundances of aflR and aflS genes in control were 1.11± 0.24 and 0.18 ± 0.05 respectively, while in experiment group were 0.28 ± 0.03 and 0.024 ± 0.005 respectively, indicating that B. megaterium (1 × 108 CFU/ml) could significantly suppress the expression of these two genes (p < 0.01). The concentration of aflatoxins and the mRNA abundances of these two genes in the
Fig. 2. Effect of different incubation times of B. megaterium on control of A. flavus. The control is treated with sterile distilled water. Values followed by different letters were significantly different according to Duncan's multiple range test p = 0.05.
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Fig. 3. Effect of different concentrations of B. megaterium on control of A. flavus. (A) 1 × 106 CFU/ml B. megaterium; (B) 1 × 107 CFU/ml B. megaterium; (C) 1 × 108 CFU/ ml B. megaterium; and (D) 1 × 109 CFU/ml B. megaterium. The control is treated with sterile distilled water. Values followed by different letters were significantly different according to Duncan's multiple range test p = 0.05.
control experiment corresponded: the lower the expression of aflR gene and aflS gene, the lower the aflatoxin concentration detected. 4. Discussion The results of this study demonstrated for the first time that the marine bacteria B. megaterium had great potential in controlling postharvest disease caused by A. flavus on peanut kernels. In the in vitro tests, the washed cell suspension of B. megaterium at 1 × 108 CFU/ ml significantly suppressed spore germination of A. flavus in PDB. Moreover, the percentage of inhibition of culture filtrate, washed and unwashed cell suspension (1 × 108 CFU/ml) could influence the growth of the pathogen on PDA plates. Thus, we concluded the cells of B. megaterium had some inhibitory effect on A. flavus, but the metabolites of B. megaterium were more strongly inhibitory to A. flavus (p < 0.05) on PDA plates. This finding suggested that B. megaterium could inhibit the pathogens due to some toxic compounds accumulated in the culture medium or antibiotic production. This result was in agreement with that reported from other antagonists such as B. subtilis (Pusey and Wilson, 1984) and B. pumilus (Cho et al., 2009). The results from in vivo test showed that the unwashed cell culture of B. megaterium was as effective as the washed cell suspension, and better control was obtained with longer incubation time. The concentrations of antagonist had significant effects on biocontrol
Fig. 4. Real-time qRT-PCR analysis of changes in aflR and aflS genes expression. Each mRNA was assayed in triplicate in two independent experiments. Representative results (mean ± SD) from control (106 spores/ml of A. flavus in PDB) and experiment (106 spores/ml of A. flavus and 108 CFU/ml of B. megaterium in PDB) are plotted.
effectiveness: the higher the concentration of B. megaterium, the better biocontrol activity. The best biocontrol was obtained with B. megaterium at 1 × 109 CFU/ml, because the disease incidence was the lowest compared with other concentration treatments (Fig. 3). These results indicate that, apart from the production of antimicrobial substances by B. megaterium, bacterial competition for space and nutrition is perhaps another mode of action (Fan and Tian, 2001; Wang et al., 2008). Our findings also showed the biosynthesis of aflatoxins was related to the expression of aflatoxin pathway genes. This strain of B. megaterium was able to significantly inhibit the biosynthesis of aflatoxins in PDB (p < 0.01) and the primary inhibitory mechanism is die to inhibition of the expression of the genes relating to aflatoxin biosynthesis (such as aflR gene and aflS gene). But our finding raise the question of whether B. megaterium could inhibit the expression of all aflatoxin pathway genes or only some of them. The use of DNA microarrays and real-time qPCR could answer this question (Hooper et al., 2001). In conclusion, our results showed that the marine bacterium B. megaterium has potential biocontrol activity against the decay and aflatoxin biosynthesis caused by A. flavus. This potential may extend to direct use in the market to prolong shelf life, provided the antagonist and its metabolites are safe for human consumption. Acknowledgements This research was supported by the research grant from Qingdao Municipal Science and Technology Commission (08-1-3-24-jch), Shandong Province, People's Republic of China; the National Natural Science Foundation of China (30771361); and Undergraduates' Innovation Experimental Program, Ministry of Education of China (081042309). References Blunt, J.W., Copp, B.R., Hu, W.P., Munro, M.H.G., Northcote, P.T., Prinsep, M.R., 2008. Marine natural products. Natural Product Reports 25, 35–94. Bouchra, C., Achouri, M., Hassani, L.M.I., Hmamouchi, M., 2003. Chemical composition and antifungal activity of essential oils of seven Moroccan Labiatae against Botrytis cinerea Pers: Fr. Journal of Ethnopharmacology 89, 165–169. Brown, G.E., Davis, C., Chambers, M., 2000. Control of citrus green mold with Aspire is impacted by the type of injury. Postharvest Biology and Technology 18, 57–65. Calvo, A.M., Wilson, R.A., Bok, J.W., Keller, N.P., 2002. Relationship between secondary metabolism and fungal development. Microbiology and Molecular Biology Reviews 66, 447–459. Cho, K.M., Math, R.K., Hong, S.Y., Islam, S.M.A., Mandanna, D.K., Cho, J.J., Yun, M.G., Kim, J.M., Yun, H.D., 2009. Iturin produced by Bacillus pumilus HY1 from Korean soybean sauce (kanjang) inhibits growth of aflatoxin producing fungi. Food Control 20, 402–406. Droby, S., 2006. Improving quality and safety of fresh fruits and vegetables after harvest by the use of biocontrol agents and natural materials. Acta Horticulturae 709, 45–51. Fan, Q., Tian, S.P., 2000. Postharvest biological control of Rhizopus rot of nectarine fruits by Pichia membranefaciens. Plant Disease 84, 1212–1216. Fan, Q., Tian, S.P., 2001. Postharvest biological control of grey mold and blue mold on apple by Cryptococcus albidus (Saito) Skinner. Postharvest Biology and Technology 21, 341–350. Fox, E.M., Howlett, B.J., 2008. Secondary metabolism: regulation and role in fungal biology. Current Opinion in Microbiology 11, 481–487. Gould, M., Nelson, L.M., Waterer, D., Hynes, R.K., 2008. Biocontrol of Fusarium sambucinum, dry rot of potato, by Serratia plymuthica 5-6. Biocontrol Science and Technology 18, 1005–1016. Hedayati, M.T., Pasqualotto, A.C., Warn, P.A., Bowyer, P., Denning, D.W., 2007. Aspergillus flavus: human pathogen, allergen and mycotoxin producer. Microbiology 153, 1677–1692. Hooper, L.V., Wong, M.H., Thelin, A., Hansson, L., Falk, P.G., Gordon, J.I., 2001. Molecular analysis of commensal host-microbial relationships in the intestine. Science 291, 881–884. Horn, B.W., Dorner, J.W., 2009. Effect of nontoxigenic Aspergillus flavus and A. parasiticus on aflatoxin contamination of wounded peanut seeds inoculated with agricultural soil containing natural fungal populations. Biocontrol Science and Technology 19, 249–262. Janisiewicz, W.J., 1996. Ecological diversity, niche, overlap, and coexistence of antagonists used in developing diseases of apples. Phytopathology 86, 473–479. Janisiewicz, W.J., Korsten, L., 2002. Biological control of postharvest diseases of fruits. Annual Review of Phytopathology 40, 411–441. Krishnamurthy, Y.L., Shashikala, J., Naik, B.S., 2008. Antifungal potential of some natural products against Aspergillus flavus in soybean seeds during storage. Journal of Stored Products Research 44, 305–309.
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