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Isolation and characterization of Bacillus amyloliquefaciens PG12 for the biological control of apple ring rot
Postharvest Biology and Technology 115 (2016) 113–121
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Isolation and characterization of Bacillus amyloliquefaciens PG12 for the biological control of apple ring rot Xinyi Chen, Yuanyuan Zhang, Xuechi Fu, Yan Li, Qi Wang* Key Laboratory of Plant Pathology, Ministry of Agriculture, Department of Plant Pathology, China Agricultural University, No. 2 Yuanmingyuan West Road, Haidian District, Beijing 100193, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 March 2015 Received in revised form 14 December 2015 Accepted 15 December 2015 Available online xxx
Bacillus spp. are promising candidates for biological control of postharvest diseases. Bacillus amyloliquefaciens PG12 was isolated from apple fruit and exhibited broad-spectrum antifungal activity. Botryosphaeria dothidea was significantly suppressed by PG12 in in vitro and in vivo. Lumpy appearance and abnormal structure of the mycelia from the edge of inhibition zone were observed using scanning electron microscopy (SEM) in in vitro assays. Furthermore, the lipopeptide crude extracts from cell-free supernatant of PG12 had remarkable antifungal activity against B. dothidea, indicating that lipopeptides played a major role in the biological control ability of PG12. PCR detection revealed that PG12 harbored the gene clusters required for the biosynthesis of the two main families of lipopeptide, including iturin and fengycin. One iturin-like compound (Rf 0.4) showed inhibitory activity against B. dothidea using thin layer chromatography (TLC)-bioautography analysis and were further fractionated by semipreparative high performance liquid chromatography (HPLC). The fraction with a molecular weight of 1043.55 m/z was identified as iturin A by electrospray ionization quadrupole-time-of-flight mass spectrometry/mass spectrometry (ESI-Q-TOF MS). Taken together, B. amyloliquefaciens PG12 was an effective biocontrol agent against apple ring rot caused by B. dothidea and iturin A was an important factor in its activity. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Biological control Bacillus amyloliquefaciens Botryosphaeria dothidea Postharvest diseases Iturin A
1. Introduction Apple ring rot disease, caused by the latent pathogen Botryosphaeria dothidea, is one of the most significant diseases affecting apple production in Asia (Xu et al., 2015). This disease is characterized by wart-like symptoms on twigs and slightly sunken lesions with alternating tan and brown rings on the fruit. With the widespread planting of the Fuji apple cultivar in eastern China, apple ring rot disease has resulted in significant economic losses of apple production during the postharvest period (Tang et al., 2012). Currently in China, the apple trees are sprayed with fungicides ten or more times during each season to control this disease. Due to the increased fungicide resistance, ecological balance and food safety as well as new sustainable strategies are required (Kexiang et al., 2002). Biological control with microbial antagonists has emerged as a promising alternative treatment for reducing the use of synthetic fungicides with a low environmental impact. In recent decades,
* Corresponding author. E-mail addresses: [email protected] (X. Chen), [email protected] (Y. Zhang), [email protected] (X. Fu), [email protected] (Y. Li), [email protected] (Q. Wang). http://dx.doi.org/10.1016/j.postharvbio.2015.12.021 0925-5214/ ã 2015 Elsevier B.V. All rights reserved.
some antagonistic microorganisms have been identified as biological control agents (BCAs) for postharvest diseases, such as Bacillus spp., Pseudomonas spp., Trichoderma spp., Pichia spp. and Candida spp. (Liu et al., 2013; Talibi et al., 2014). Potential BCAs against apple ring rot disease include B. licheniformis, T. harzianum and T. atroviride (Kexiang et al., 2002; Ji et al., 2008). Among the most promising BCAs, Bacillus spp. are safe microorganisms which can be found ubiquitously in the environment. They exhibit versatility in protecting plants from pathogen infection, and have excellent abilities to colonize and to sporulate (Romero et al., 2007; Arrebola et al., 2010). The biocontrol mechanisms of BCAs involve the production of antifungal compounds, colonization, nutrient competition and the induced systemic resistance phenomenon (ISR) in the host plants (Santoyo et al., 2012). Previous studies revealed that antibiosis was one of the primary mechanisms of Bacillus spp. against plant pathogens. Various antibiotics are produced by Bacillus spp. (Kilian et al., 2000). Bacillus amyloliquefaciens and its close relative B. subtilis have been reported to be effective in controlling plant pathogens through the production of non-ribosomally synthesized antibiotics, mainly cyclic lipopeptides (Romero et al., 2007; Xu et al., 2013). Based on their structure, cyclic lipopeptides can be generally classified into three families: iturin, fengycin, and surfactin (Ongena and Jacques,
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2008). The members of the iturin family, represented by iturin A and C, bacillomycin D, F, L and LC and mycosubtilin, have been widely studied for their strong antifungal activity against a wide range of plant pathogens (Stein, 2005). Iturins are heptapeptides linked to a b-amino fatty acid chain with a length of 14–17 carbons. The fungitoxicity of iturins is almost certainly due to their membrane permeabilization properties, which are based on osmotic perturbations resulting from the formation of ionconducting pores (Ongena and Jacques, 2008). It has been reported that members of the iturin family, particularly iturin A, play an active role in the biological control of postharvest pathogens by Bacillus spp. (Kim et al., 2010; Gong et al., 2015; Waewthongrak et al., 2015). However, the role of iturin A in controlling apple ring rot disease has not been reported. A few BCAs that can effectively control apple ring rot by antifungal compounds are available. Although Li et al. (2013) has showed that B. amyloliquefaciens 9001 effectively suppressed apple ring rot disease, the biocontrol mechanisms of B. amyloliquefaciens 9001 were not well known. In this study, PG12 exhibited stronger antifungal activity than 9001. The objectives of this study were (i) to identify and characterize the selected bacterial isolate PG12 with antagonistic activity against B. dothidea, (ii) to optimize its biocontrol activity against B. dothidea on apples in the postharvest period and (iii) to identify the main antimicrobial compounds involved in its antifungal activity. Collectively, we aimed to identify a new promising candidate for use as BCA against apple ring rot and further investigate its biocontrol mechanisms. 2. Materials and methods 2.1. Fungal pathogens B. dothidea YL1, a particularly virulent strain, was supplied by Prof. Liyun Guo of College of Agronomy and Biotechnology, China Agricultural University, China. B. dothidea YL1 was cultured on potato dextrose agar (PDA) at 25 C in the dark and then subcultured on 2% malt extract agar with a 12-h photoperiod using near UV light to induce sporulation. The conidial suspension was obtained according to the method used by Tang et al. (2012). The plant pathogens listed in Table 1 were kindly provided by the Department of Plant Pathology, College of Agronomy and Biotechnology, China Agricultural University, China. 2.2. Isolation of bacterial strains Bacterial strains used in this study were originally isolated from healthy apples (Malus domestica cv. Fuji) collected at commercial maturity from several orchards in Northern China (Beiliu village, Beijing; Sanmenxia, Henan Province; Qixia city, Shandong Province; Tianshui city, Gansu Province; Xi’an city, Shaanxi Province and Yuncheng city, Shanxi Province) that have been affected by severe apple ring rot disease. For bacterial isolation, fruit samples were processed using surface sterilization and serial dilution methods according to protocols described previously by Lai et al. (2012). One gram samples were collected close to the fruit skin and ground in 3 mL of sterilized water and sterilized quartz sands using a mortar and pestle. The homogenates were serially diluted, incubated for 30 min at 80 C in a water bath, and plated on NA. The plates were then incubated at 30 C for one day (Urrea et al., 2011). Individual bacterial colonies were isolated and purified.
Table 1 Antagonist activity against various fungal plant pathogens by strain PG12 in vitro. Target fungal pathogens
Inhibitory activitya
Botryosphaeria dothidea Botryosphaeria ribis Valsa mali Monilinia fructicola Botrytis cinerea Penicillium expansum Botryosphaeria berengeriana Colletotrichum gloeosporioides Rhizoctonia cerealis Phytophthora infestans Phytophthora capsici Fusarium oxysporum Trichothecium roseum Alternaria alternata
+++ ++ ++ +++ + + + + ++ ++ ++ ++ +++ +
a + ++ and +++ represent relative inhibition rates against mycelia growth of each fungal colony on the PDA medium to the extent of 31–50%, 51–70% and 71%, respectively.
2.3. Screening of antagonistic bacteria The dual culture technique was used to detect the antagonistic activities of bacterial isolates towards fungi. A 5-mm-diameter plug from a 5-day-old mycelial culture of B. dothidea was placed in the center of fresh PDA plates (90 mm). A fresh colony was patched around the fungal plug at a distance of 3 cm using a sterile toothpick. PDA only inoculated with the pathogen was used as a control. The plates were incubated at 28 C for five days. The antagonistic effect was assessed by measuring the inhibition zones (mm) and the colony diameters. The percentage of growth inhibition was calculated using the formula n = [(a b)/a] 100, where n is the percentage of growth inhibition, a is the colony area of uninhibited fungi, and b is the colony area of treated fungi (Etebarian et al., 2005). The values were recorded as the means of three replicates. Experiments were repeated twice, with B. amyloliquefaciens 9001 mentioned previously used as the control isolate. 2.4. In vivo test Fuji apples were used to evaluate the biocontrol activity of strain PG12 on apple ring rot in the postharvest period (Li et al., 2013). Healthy apples were sampled from an orchard containing 10-year-old trees badly infected with B. dothidea and situated in the Beiliu village of Changping District in Beijing, China. Twelve trees were chosen for the biocontrol trial. Routine chemical control was applied in the experimental field. The bacterial inoculum was prepared in nutrient broth (NB) in a shaker at 160 rpm for 2 days at 30 C. The bacterial cells were collected by centrifugation at 6000 g for 5 min and diluted to the desired concentration using a PBS buffer (pH 7.0). Cell concentration was determined by a hemocytometer. After fruit maturation, healthy samples were harvested and soaked in bacterial suspensions of PG12 at a concentration of 108 CFU mL1 for 2 min, and then dried in a transfer hood. Fruit soaked in sterile distilled water were used as the controls. The treated apples were placed in plastic trays at room temperature (20 C). The sample numbers of rotted fruit were recorded weekly, and the percentage of disease incidence (the numbers of rotted apples divided by the numbers of total apples 100) was calculated. Ten fruit samples were used for each replicate, and the values were recorded as the means of three replicates for each treatment. The experiments were repeated in two years, and data from the three replicates were combined.
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2.5. Identification of PG12
2.6. Scanning electron microscopy analysis
PG12 was preliminarily characterized through means of conventional bacteriological methods including morphological, physiological, and biochemical characteristics (Gerhardt et al., 1981). PG12 was further identified through a phylogenetic analysis of its 16 S rDNA and partial gyrase gene sequences (gyrA). The genomic DNA of PG12 was extracted using standard procedures (Sambrook et al., 2001). The 16 S rDNA was amplified by PCR using primers 63 F (50 -CAGGCCTAACACATGCAAGTC-30 ) and 1387R (50 -GGGCGGWGTGTACAAGGC-30 ) (Beijing Sunbiotech Co., Ltd) (Marchesi et al., 1998). Part of the gyrA sequence was amplified with the primers p-gyrA-f (50 -CAGTCAGGAAATGCGTACGTCCTT-30 ) and p-gyrA-r (50 -CAAGGTAATGCTCCAGGCATTGCT-30 ) (Beijing Sunbiotech Co., Ltd.) (Chun and Bae, 2000). The concentrations of the primers, polymerase, and DNA used in the PCR were 10 mM, 5.0 U mL1, and 50 ng mL1, respectively. PCR amplification (in a total volume of 50 mL, including 2 mL of each primer, 5 mL of 10 buffer, 4 mL of 2.5 nM dNTP, 0.5 mL of Taq polymerase, and 2 mL of DNA template) was achieved using a Taq DNA polymerase kit (Beijing TransGen Biotech Co., Ltd.) with a modified thermocycler protocol that included an initial denaturation at 95 C for 5 min followed by 35 cycles of denaturation at 95 C for 1 min, annealing at 56 C for 40 s, extension at 72 C for 1 min, and a final extension at 72 C for 10 min (Li et al., 2012). The PCR products were ligated into pMD19-T vectors (Takara Co., Ltd.) to generate a recombinant plasmid. The plasmid was then mini-prepped from an overnight Luria–Bertani broth (LB broth) culture using the StarPrep Plasmid Miniprep Kit (Bioteke Biosolutions Co., Ltd.), and sent to Zhongkebaiao Biotechnologies Co., Ltd., (China) for sequencing. Multiple alignments with sequences of closely related Bacillus strains and sequence similarity calculations were performed using CLUSTAL X (Thompson et al., 1997). The phylogenetic trees of strain PG12 based on 16 S rDNA and the gyrA sequences were constructed using the neighbor-joining method and MEGA 4.0 software (Kim et al., 1993). The topology of the phylogenetic tree was evaluated by 1000 bootstrap resampling replicates (Felsenstein, 1985).
The morphology of the mycelia was further investigated by scanning electron microscopy (SEM, S-3400N, HITACHI, Japan). Five mycelial plugs (5 mm in diameter) were cut from PDA at outer edges of the inhibition zone as well as the corresponding position on the control. The mycelia were fixed with 2.5% (v/v) glutaraldehyde at 4 C for 2 h, rinsed three times with 0.1 mL of phosphate buffer (0.02 M) and subsequently fixed with 1% osmium tetroxide for 2 h at 20 C. Dehydration was performed three times with a graded series of ethanol (30%, 50%, 70%, 80%, 90%, 100%, v/v). The samples were dried with a desiccator (LEICA EM CPD), affixed to stubs using carbon tape and coated with a thin coating of gold (EIKO IB-3) for viewing under a SEM. The mycelia were observed by SEM and all of the images were processed computationally. The experiments were repeated twice, and the means of the three replicates were recorded. 2.7. Antifungal activity of metabolites from B. amyloliquefaciens PG12 against B. dothidea The antifungal activity of cell-free supernatant and lipopeptide crude extracts from PG12 against B. dothidea was explored using a technique similar to dual culture analysis (Yoshida et al., 2001). The pre-culture was grown in LB for 6 h to an OD600 of approximately 3.0. Briefly, 500 mL of culture was transferred to 50 mL of Landy medium and incubated at 30 C and 200 rpm for approximately 64 h (Landy et al., 1948). The cell-free supernatant and lipopeptide crude extracts were obtained following Niu et al. (2013). Briefly, cell-free supernatant was collected by centrifugation at 6000 g for 10 min, followed by filtration through 0.22 mm cellulose nitrate filters. To obtain the lipopeptide extract, six grams of XAD16 (Sigma) adsorbent resin held in a column (Sigma Amberlite) were washed with 50 mL of deionized water to remove salts. The cellfree supernatant was loaded onto the XAD16 resin column, washed with deionized water and eluted with 14 mL of 100% methanol. After drying with a rotary evaporator, the lipopeptide crude extracts were further dissolved in 1 mL of methanol. One hundred-microliter aliquots of cell-free supernatant, lipopeptide crude extracts or methanol (control) were spread on the PDA. A 7-mm diameter agar plug from an actively growing fungal mycelia of the B. dothidea was inoculated in the center of PDA. The diameters of the fungal colonies were measured after five
Fig. 1. Antagonistic activity of isolate PG12 and control isolate 9001 of B. amyloliquefaciens against B. dothidea in a dual culture test. (A) The mycelia growth of B. dothidea in absence of selected isolates. (B) The mycelia growth of B. dothidea in presence of the control isolate 9001 and PG12. Images were taken after seven days.
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Fig. 2. Disease incidence of ring rot in apples treated with bacterial suspensions of strain PG12 (PG12) or water (Control) after harvest. The vertical error bars represent the standard errors of the means calculated from three replicates for each treatment at each time point. Different letters indicate significant differences between different treatments according to Student’s t-test (P < 0.05).
days of incubation at 28 C, and the mean diameters from three replicates were calculated. The inhibition of fungal growth was calculated by measuring the diameter of the fungal colony and was expressed as the percentage of the inhibition of fungal growth divided by the growth obtained in the control plates. The values were recorded as the means of three replicates. The experiments were repeated twice. 2.8. PCR detection of antibiotic biosynthesis genes
chloroform/methanol/water (65:25:4, v/v/v) as mobile phase. Thereafter, bioautography was used to show the inhibitive activity of the methanolic fractions on a thin agar layer containing the fungus as an indicator organism. Bioautography was performed as described by Niu et al. (2013). In short, the TLC plates were stuck on the surface of the 1% PDA agar containing fungal spores with the final concentration of 4 107 CFU/mL at room temperature for 2 h and then removed. The PDA plates were incubated at 28 C for five days. Afterwards, the inhibition zones were documented the positions of the antifungal compounds separated by TLC. Their corresponding retention factor (Rf) values were estimated and the experiments were repeated three times. Matrix material of the TLC plates from the positions at which the antifungal compounds were located was scraped from the silica gel and then extracted with methanol. The extracts were further purified by semipreparative reverse phase HPLC (RP-HPLC). RP-HPLC analysis was carried out using a 5-mm Waters Symmetry C18 column (150 4.6 mm) at room temperature. The mobile phase consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile and was applied at a flow rate of 15 mL min1. The elution pattern was monitored by determining the absorbance at 215 nm. The peaks were collected and the substances dissolved in methanol were deposited in 7 mmdiameter wells made in the PDA plates at a distance of 2 cm from both the fungal plug and the control well containing methanol (M). The antagonistic activity against B. dothidea was observed after incubation at 28 C for 5 d. The eluted fractions with antifungal activity were analyzed by ESI-Q-TOF MS. Both the molecular weight and the structure of the purified lipopeptide were determined by ESI-Q-TOF MS using a Q-TOF instrument (Micromass, Bruker Daltonics). A mass spectrum was acquired over the range of 50–2200 m/z in the positive mode, and the capillary voltage was 4000 V. The precursor mass was set to 1043.55 m/z and the isolation window width was 10 Da. The collision gas was nitrogen and the fragment energy was 55 eV, and a ramping factor ranging from 100% to 150% (i.e., the real fragment energy ranged from 55 eV to 82.5 eV) was used to achieve improved fragmentation. The MS data were processed with Data Analysis 4.1 SP3, and the de novo sequencing analysis was performed using BioTools 3.2 (both from Bruker Daltonics). The amino acid sequence tag could be directly assigned by BioTools, but there was still a 225-Da gap that could not be interpreted. This gap was characterized using the Bruker Mol Weight to Formula software tools, and its formula was determined as C14H27NO, as determined directly from its accurate mass of 225.2087 Da (no other possibility in the mass error tolerance of 5 ppm).
Genomic DNA extraction and amplification of 19 antibiotic biosynthesis genes were performed using specific primers, which were listed in the supplementary information (Table S1). PCR amplifications were achieved in 50-ml of reaction mixtures containing 10 EasyTaq buffer, 2.5 nM of dNTP, 5.0 U mL1 of EasyTaq DNA polymerase (Beijing TransGen Biotech Co., Ltd.), 10 mM of each primer and 2 mL of template DNA (approximately 100 ng of bacterial genomic DNA). The amplifications were performed using a DNA thermal cycler (Model No. 9700, PerkinElmer Co., Wellesley, USA) with the following cycle conditions: an initial denaturation at 95 C for 5 min; 30 cycles of denaturation of 95 C for 1 min, an annealing step of 40 s (annealing temperature was listed in Table S 1), and extension at 72 C for 1 min; and a final extension at 72 C for 10 min (Chung et al., 2008). Expected PCR products were gel-purified and then ligated into pMD19-T vectors (Takara Co., Ltd.), generating a recombinant plasmid. Plasmids were isolated from the transformed cells using the StarPrep Plasmid Miniprep Kit (Bioteke Biosolutions Co., Ltd.) and sent to Zhongkebaiao Biotechnologies Co., Ltd., (China) for sequencing. The PCR sequences were compared using the BLAST program in the GenBank nucleotide database from the National Center for Biotechnology Information (Bethesda, MD, USA; http://www. ncbi.nlm.nih.gov/).
The data were subjected to variance analysis (ANOVA) using the SPSS software (SPSS Statistics 20.0). The statistical differences between means of different treatments was assessed by Student’s t-test at P < 0.05.
2.9. Isolation and characterization of antifungal lipopeptides
3. Results
The presence of antifungal lipopeptides in the supernatant of B. amyloliquefaciens PG12 was determined first by thin layer chromatography (TLC) (Razafindralambo et al., 1993). The control isolate FZB42 was kindly supplied by Dr. Rainer Borriss from Humboldt Universität Berlin, Germany. The methanolic fractions containing lipopeptide crude extracts were obtained as described above. Twenty microliters of each methanol extract were spotted onto TLC aluminum sheets coated with silica gel 60 F254 (20 cm 20 cm, Merck, Germany) and separated by TLC plates using
3.1. Screening of antagonistic bacteria
2.10. Statistical analysis
Eighty-three bacterial strains from 254 isolates were screened for potential antagonistic activity towards B. dothidea in the dual culture test (data not shown). Among these isolates, PG12 (CGMCC Accession No. 7132; China General Microbiology Culture Collection Center), which was isolated from apple fruit harvested from an orchard of Beiliu village, showed strongest antagonistic activity against B. dothidea. The inhibition zone of PG12 was 5.3 mm, and
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the percentage of growth inhibition after five days of incubation was approximately 87.5%. The antagonistic activity of PG12 was compared with that of 9001, which was previously identified as a potential biocontrol agent against apple ring rot (Li et al., 2013). There was significant (P < 0.05) difference in the inhibition zone and the percentage of fungal growth inhibition between isolate PG12 and the control isolate 9001. The inhibition ratio of isolate PG12 was higher than 9001 against B. dothidea (87.5% for PG12 versus 45.2% for 9001), indicating that antagonist PG12 was more effective than the control isolate 9001 in suppressing B. dothidea (Fig. 1). Moreover, PG12 exhibited broad-spectrum in vitro antagonistic activity against 13 other pathogens causing
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postharvest diseases in apple, pear, peach and other plants (Table 1). 3.2. In vivo test To investigate the effect of PG12 in the postharvest period, apples were treated with a PG12 suspension after harvest. The disease incidence of the PG12-treated apples was noticeably lower than that of the water-treated control apples for a period of 56 days after inoculation (Fig. 2). Similar results were obtained from the experiment conducted in the subsequent year (data not shown), suggesting PG12 effectively suppressed fungal diseases in the postharvest period.
Fig. 3. Phylogenetic tree based on the partial nucleotide sequence of 16 S rDNA (A) and gyrA (B). A neighbor-joining phylogenetic tree of strain PG12 was constructed using MEGA 4.0. The numbers at the nodes indicate the levels of bootstrap support (%) based on a neighbor-joining analysis of 1000 resampled datasets; only values greater than 50% are provided. Bars = 0.005 (A) and 0.05 (B) nucleotide substitutions per site (T = type strain).
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3.3. Identification of PG12
inhibited fungal growth by approximately 89%. No significant difference was found between these two treatments, suggesting that lipopeptides were responsible for the inhibitory activity against B. dothidea.
The physiological and biochemical characteristics of strain PG12 are summarized in Table S2. Strain PG12 is a Gram-positive and spore-forming bacterium with an optimal growth temperature of 28–37 C. A partial 16 S rDNA sequence analysis demonstrated that strain PG12 was most likely B. amyloliquefaciens subsp. plantarum CAU B946 (99%), indicating that strain PG12 belongs to the species B. amyloliquefaciens (Fig. 3A). This result was confirmed by a partial gyrA sequence analysis, which has been shown to be effective in distinguishing these closely related taxa of the B. subtilis group (Chun and Bae, 2000) (Fig. 3B). Therefore, PG12 was identified as B. amyloliquefaciens (B. amyloliquefaciens subsp. plantarum). The 16 S rDNA sequence and partial gyrA nucleotide sequence of the test strain PG12 were deposited in the GenBank database with the accession numbers KC797583 and KC797584, respectively.
PCR detection and nucleotide BLAST analysis showed that PG12 had antibiotic biosynthesis genes: ituD (GeneBank Accession No. KT342867), ituC (GeneBank Accession No. KT342866), bamD (GeneBank Accession No. KT342867), bamC (GeneBank Accession No. KT342868), bacAB (GeneBank Accession No. KT342869), bacD (GeneBank Accession No. KT342865), fenB (GeneBank Accession No. KT342870), fenC (GeneBank Accession No. KT342871) and fenF (GeneBank Accession No. KT342867) (Table 2). However, eriB,eriSa, mrsA, mrsM, spaB, spaC, albF, albA, mycC and sunT were not detected in B. amyloliquefaciens PG12.
3.4. Scanning electron microscopy analysis
3.7. Identification of lipopeptides from B. amyloliquefaciens PG12
SEM analysis revealed that mycelia of B. dothidea collected from the edge of inhibition zone were morphologically different from that of the control on PDA. Normal mycelia of B. dothidea were smooth and presented a uniform in thickness (Fig. 4A). Whereas, mycelia collected from the inhibition zone were thick and abnormal, irregular swelling, curling of the hyphal tips, cell wall collapse and many hollows (Fig. 4B–D).
To identify the compounds that suppress the growth of B. dothidea, the methanol extracts of lipopeptides obtained from PG12 were subjected to TLC in combination with bioautography. It was found that two active fractions of PG12 inhibited the conidial germination and mycelial growth of B. dothidea. One of the active fractions was found in both B. amyloliquefaciens FZB42 and PG12, showing Rf values typical for fengycin-like lipopeptides (Rf 0.1 to 0.2). However, another active fraction (Rf 0.4) was found in PG12, but not in B. amyloliquefaciens FZB42 (Fig. 5). The active compounds (Rf 0.4) were extracted from silica gel of TLC plates for further purification by HPLC (Fig. 6A). Three fractions were obtained and only fraction 1 showed inhibitory activity against B. dothiedea (Fig. 6B). The identification of fraction 1 by LCMS showed that the molecular mass of the active compound was 1043.55 m/z at a retention time of 3.45 min, resembling C 14 iturin,
3.5. Effect of B. amyloliquefaciens PG12 cell-free supernatant and lipopeptide crude extracts on B. dothidea The dual culture assay showed that cell-free supernatant of B. amyloliquefaciens PG12 significantly inhibited the growth of the fungal pathogen by approximately 86%. The methanol extracts of lipopeptide from cell-free supernatant of PG12 significantly
3.6. Antibiotic biosynthesis genes in B. amyloliquefaciens PG12
Fig. 4. Morphological analysis of mycelia by scanning electron microscopy. Mycelia of B. dothidea in the absence (control) (A) or presence of bacterial isolate PG12 (B–D). The arrows indicate B. dothidea mycelia damaged by strain PG12.
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Table 2 PCR detection of antibiotic biosynthesis genes from B. amyloliquefaciens PG12. Antibiotic
Genes
Detection in PG12a
Iturin A
ituD ituC bam D bam C bacAB bacD eriB eriSa fenB fenC mrsA mrsM fenF mycC sunT spaB spaC albF albA
+ + + + + + + + +
Bacillomycin D Bacilysin Ericin Fengycin Mersacidin Mycosubtilin Sublancin Subtilin Subtilosin
a
+ and represent presence or absence of each gene, respectively.
as previously reported (Chen et al., 2008). ESI-Q-TOF MS analysis further revealed the amino acid sequence of fraction 1. The resulting fragmentation spectrum was analyzed, revealing a partial series of a-type and b-type ions covered the entire amino acid sequence of the peptide. The sequences indicated that Ser and Asn were connected to a fatty acid moiety, and the molecule was deduced to be a cyclic lipopeptide based on data obtained through tandem mass spectrometry regulation. This sequence was found to be N-Ser-Asn-Pro-Gln-Asn-Tyr-Asn-C. Based on an analysis of its cyclic structure, the lipopeptide was determined to be iturin A (Fig. 7). The molecular mass and complete peptide sequence were similar to those of iturin A, produced by B. amyloliquefaciens TF28 (Zhang et al., 2012). Altogether, iturin A was identified as an inhibitor of B. dothidea. 4. Discussion Many species in the Bacillus genus are considered as potential biocontrol agents for reduction of postharvest phytopathogens such as Penicilium spp., Botrytis cinerea and Monilinia spp. (Sharma et al., 2009; Pretorius et al., 2015). Few studies have reported Bacillus spp. for biocontrol of apple ring rot caused by B. dothidea which is an important disease for apple production (Li et al., 2013). So far, the mechanisms of Bacillus strains against B. dothidea have not been extensively investigated. In this study, B. amyloliquefaciens PG12 originally isolated from apple fruit proved its biocontrol potential against apple ring rot. The antifungal activity of B. amyloliquefaciens PG12 against B. dothidea was attributed to the action of lipopeptides. The results of this study demonstrated for the first time the biological potential of a B. amyloliquefaciens strain to control apple ring rot disease by the production of iturin A. In the present study, B. amyloliquefaciens PG12 with broadspectrum of antagonistic activity efficiently controlled apple ring rot in in vitro and in vivo. These results confirmed previous findings on the potential of B. amyloliquefaciens species as biocontrol agent of apple ring rot (Li et al., 2013). A few of other antagonistic microorganisms were tested on fruit to verify the antifungal activity against apple ring rot, such as B. licheniformis, T. harzianum and T. atroviride. For example, biocontrol field trials suggested that apple ring rot on apple shoots, stems and fruit had been efficiently controlled by the application of T. harzianum T88 and T. atroviride T95 (Kexiang et al., 2002). Ji et al. (2008) reported that B.
Fig. 5. Thin layer chromatography (TLC)-bioautography analysis against B. dothidea. Developed chromatograms were covered with a PDA medium inoculated with a fungal spore suspension at concentration of 4 107 CFU mL1. Pictures were taken after five days of incubation. The retention factor (Rf) values of the different lipopeptides iturin A (Rf 0.4) and fengycin (Rf 0.1–0.2) are indicated on the right. The fungal growth inhibition zones produced by iturin A-like activity from B. amyloliquefaciens PG12 are indicated by an arrow.
licheniformis W10 could suppress necrosis spread of apple ring rot on fruit. Therefore, the postharvest application of biocontrol agents is a practical and useful method for controlling apple ring rot. Previous studies have reported that B. amyloliquefaciens strains showed strong antifungal properties against plant pathogenic fungi based on the production of antimicrobial compounds such as cyclic lipopeptides (Yu et al., 2012; Yuan et al., 2014; Ben Abdallah et al., 2015). However, the mechanism of biocontrol of apple ring rot by B. amyloliquefaciens is not clear. In this study, B. amyloliquefaciens PG12 reduced mycelial growth of B. dothidea in vitro tests. The large inhibition zones could be due to the effect of antimicrobial compounds produced by PG12. The mycelia from the edge of inhibition zone exhibited lumpy appearance and abnormal structure observed by SEM. Antimicrobial compounds not only inhibited the growth of mycelia but also damaged the morphology. These effects were very similar to the morphological damage of apple ring rot treated with B. licheniformis W10 (Ji et al., 2008). Additional experiments were carried out to evaluate whether the efficacy was associated with lipopeptides. It was found that cellfree supernatant of PG12 and its lipopeptide crude extracts had similar efficacy against B. dothidea in vitro. These results suggested that the mechanism for PG12 to suppress apple ring rot may mainly include antimicrobial lipopeptides. This finding was consistent with previous reports in which lipopeptides in cellfree supernatant were responsible for the antifungal activity of B. subtilis CPA-8 against peach brown rot (Yánez-Mendizábal et al., 2012). The lipopeptide antibiotics produced by Bacillus spp. have been generally assumed to be responsible for antifungal activity. B. amyloliquefaciens strains produce various antimicrobial
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Fig. 6. The lipopeptide mixture was isolated and purified by semipreparative reverse phase HPLC (RP-HPLC) and antagonism assay against B. dothidea in vitro. Three peaks were detected at 215 nm and collected (A), and the antagonistic activity against B. dothidea was assayed (B).
lipopeptides including iturins surfactins and fengycins against phytopathogens (Falardeau et al., 2013; Cawoy et al., 2015). Based on PCR-assays, B. amyloliquefaciens PG12 harbored the gene clusters involved in the synthesis of iturin A, bacillomycin D and fengycin. In many cases, it was possible to associate the presence of lipopeptides of B. amyloliquefaciens strains with the corresponding biosynthetic genes. However, further bioautography analysis of lipopeptide crude extracts revealed that B. amyloliquefaciens PG12 obviously inhibited mycelial growth of B. dothidea by two active fractions (iturin A [Rf 0.4] and fengycin [Rf 0.1–0.2]). As shown in this study, the production of iturin A is a very important factor conferring antifungal activity to B. amyloliquefaciens strains against phytopathogens (Arrebola et al., 2010; Gong et al., 2015; Han et al., 2015). Fengycin-like families of B. amyloliquefaciens PG12 also exhibited antifungal activity against B. dothidea according to control isolate FZB42. Iturin A and fengycin were well known for its broad-spectrum of antifungal activity (Stein, 2005). The strong antifungal activity of PG12 against 13 other postharvest pathogens may be due to its production of iturin A and fengycin, but further evidence is needed to confirm this finding. Furthermore, Koumoutsi et al. (2004) reported that iturins and fengycins of B. amyloliquefaciens FZB42 exhibited synergistic actions against F. oxysporum. In contrast, iturins and fengycins of
B. amyloliquefaciens SQR9 did not show synergistic actions against F. oxysporum (Xu et al., 2013). Whereas, as evidenced for isolate BO7 of this bacterial species, the strong biocontrol activity is only due to new identified surfactins (Vitullo et al., 2012). Further research will be needed to identify and characterize the relationship between iturins and fengycins against B. dothidea in our isolate PG12. Joshi and McSpadden Gardener (2006) has demonstrated that the presence of a particular gene is not a reliable predictor of the biocontrol capacity in a Bacillus strain. In this study, although B. amyloliquefaciens PG12 harbored the gene clusters required for bacillomycin D, no active fraction (Rf 0.3) corresponding to bacillomycin D on TLC plate showed obvious antifungal activity against B. dothidea. This result might be due to no bacillomycin D production or a low concentration of bacillomycin D from PG12. Our result was similar to those previously reported for B. amyloliquefaciens ARP23. The peptide synthetase genes of B. amyloliquefaciens ARP23 involved in the production of bacillomycin were detected by PCR, but bacillomycin were not found in the culture supernatants of ARP23 (Alvarez et al., 2012). The production of lipopeptides may be related to the growth medium, culture condition or genes related to the biosynthesis of lipopeptides. Future work involving gene expression studies and molecular analysis by mutagenesis will be useful for establishing the involvement of the lipopeptide synthetase genes to biological control. Moreover, lipopeptides production is not the only factor that needs to be considered and more work is required to also explore other possible biocontrol mechanisms. 5. Conclusions In conclusion, B. amyloliquefaciens PG12 efficiently controlled apple ring rot disease caused by B. dothidea in vitro and in vivo. The antifungal activity of B. amyloliquefaciens PG12 against B. dothidea was attributed to the action of lipopeptides. This is the first report revealing the role of iturin A produced by B. amyloliquefaciens PG12 in the reduction of apple ring rot disease caused by B. dothidea. In brief, B. amyloliquefaciens PG12 has a great potential as a biocontrol agent against apple ring rot. Acknowledgments
Fig. 7. The a, b and y ions obtained in the ESI-Q-TOF MS are indicated in the spectrum at m/z 1043.551.
This work was financially supported by the National Natural Science Foundation of China (Grant No. 31371977), the Earmarked Fund for Modern Agro-industry Technology Research System (Grant No. CARS-28) and the Specialized Research Fund for the
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Doctoral Program of Higher Education (SRFDP) (Grant No. 20110008120013). The authors are grateful to Ryan Mcnaughton (Boston University, USA) for English revision of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. postharvbio.2015.12.021. References Alvarez, F., Castro, M., Principe, A., Borioli, G., Fischer, S., Mori, G., Jofre, E., 2012. The plant-associated Bacillus amyloliquefaciens strains MEP218 and ARP23 capable of producing the cyclic lipopeptides iturin or surfactin and fengycin are effective in biocontrol of sclerotinia stem rot disease. J. Appl. Microbiol. 112, 159–174. Arrebola, E., Jacobs, R., Korsten, L., 2010. Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens. J. Appl. Microbiol. 108, 386–395. Ben Abdallah, D., Frikha-Gargouri, O., Tounsi, S., 2015. 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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Isolation and characterization of Bacillus amyloliquefaciens PG12 for the biological control of apple ring rot Xinyi Chen, Yuanyuan Zhang, Xuechi Fu, Yan Li, Qi Wang* Key Laboratory of Plant Pathology, Ministry of Agriculture, Department of Plant Pathology, China Agricultural University, No. 2 Yuanmingyuan West Road, Haidian District, Beijing 100193, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 March 2015 Received in revised form 14 December 2015 Accepted 15 December 2015 Available online xxx
Bacillus spp. are promising candidates for biological control of postharvest diseases. Bacillus amyloliquefaciens PG12 was isolated from apple fruit and exhibited broad-spectrum antifungal activity. Botryosphaeria dothidea was significantly suppressed by PG12 in in vitro and in vivo. Lumpy appearance and abnormal structure of the mycelia from the edge of inhibition zone were observed using scanning electron microscopy (SEM) in in vitro assays. Furthermore, the lipopeptide crude extracts from cell-free supernatant of PG12 had remarkable antifungal activity against B. dothidea, indicating that lipopeptides played a major role in the biological control ability of PG12. PCR detection revealed that PG12 harbored the gene clusters required for the biosynthesis of the two main families of lipopeptide, including iturin and fengycin. One iturin-like compound (Rf 0.4) showed inhibitory activity against B. dothidea using thin layer chromatography (TLC)-bioautography analysis and were further fractionated by semipreparative high performance liquid chromatography (HPLC). The fraction with a molecular weight of 1043.55 m/z was identified as iturin A by electrospray ionization quadrupole-time-of-flight mass spectrometry/mass spectrometry (ESI-Q-TOF MS). Taken together, B. amyloliquefaciens PG12 was an effective biocontrol agent against apple ring rot caused by B. dothidea and iturin A was an important factor in its activity. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Biological control Bacillus amyloliquefaciens Botryosphaeria dothidea Postharvest diseases Iturin A
1. Introduction Apple ring rot disease, caused by the latent pathogen Botryosphaeria dothidea, is one of the most significant diseases affecting apple production in Asia (Xu et al., 2015). This disease is characterized by wart-like symptoms on twigs and slightly sunken lesions with alternating tan and brown rings on the fruit. With the widespread planting of the Fuji apple cultivar in eastern China, apple ring rot disease has resulted in significant economic losses of apple production during the postharvest period (Tang et al., 2012). Currently in China, the apple trees are sprayed with fungicides ten or more times during each season to control this disease. Due to the increased fungicide resistance, ecological balance and food safety as well as new sustainable strategies are required (Kexiang et al., 2002). Biological control with microbial antagonists has emerged as a promising alternative treatment for reducing the use of synthetic fungicides with a low environmental impact. In recent decades,
* Corresponding author. E-mail addresses: [email protected] (X. Chen), [email protected] (Y. Zhang), [email protected] (X. Fu), [email protected] (Y. Li), [email protected] (Q. Wang). http://dx.doi.org/10.1016/j.postharvbio.2015.12.021 0925-5214/ ã 2015 Elsevier B.V. All rights reserved.
some antagonistic microorganisms have been identified as biological control agents (BCAs) for postharvest diseases, such as Bacillus spp., Pseudomonas spp., Trichoderma spp., Pichia spp. and Candida spp. (Liu et al., 2013; Talibi et al., 2014). Potential BCAs against apple ring rot disease include B. licheniformis, T. harzianum and T. atroviride (Kexiang et al., 2002; Ji et al., 2008). Among the most promising BCAs, Bacillus spp. are safe microorganisms which can be found ubiquitously in the environment. They exhibit versatility in protecting plants from pathogen infection, and have excellent abilities to colonize and to sporulate (Romero et al., 2007; Arrebola et al., 2010). The biocontrol mechanisms of BCAs involve the production of antifungal compounds, colonization, nutrient competition and the induced systemic resistance phenomenon (ISR) in the host plants (Santoyo et al., 2012). Previous studies revealed that antibiosis was one of the primary mechanisms of Bacillus spp. against plant pathogens. Various antibiotics are produced by Bacillus spp. (Kilian et al., 2000). Bacillus amyloliquefaciens and its close relative B. subtilis have been reported to be effective in controlling plant pathogens through the production of non-ribosomally synthesized antibiotics, mainly cyclic lipopeptides (Romero et al., 2007; Xu et al., 2013). Based on their structure, cyclic lipopeptides can be generally classified into three families: iturin, fengycin, and surfactin (Ongena and Jacques,
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2008). The members of the iturin family, represented by iturin A and C, bacillomycin D, F, L and LC and mycosubtilin, have been widely studied for their strong antifungal activity against a wide range of plant pathogens (Stein, 2005). Iturins are heptapeptides linked to a b-amino fatty acid chain with a length of 14–17 carbons. The fungitoxicity of iturins is almost certainly due to their membrane permeabilization properties, which are based on osmotic perturbations resulting from the formation of ionconducting pores (Ongena and Jacques, 2008). It has been reported that members of the iturin family, particularly iturin A, play an active role in the biological control of postharvest pathogens by Bacillus spp. (Kim et al., 2010; Gong et al., 2015; Waewthongrak et al., 2015). However, the role of iturin A in controlling apple ring rot disease has not been reported. A few BCAs that can effectively control apple ring rot by antifungal compounds are available. Although Li et al. (2013) has showed that B. amyloliquefaciens 9001 effectively suppressed apple ring rot disease, the biocontrol mechanisms of B. amyloliquefaciens 9001 were not well known. In this study, PG12 exhibited stronger antifungal activity than 9001. The objectives of this study were (i) to identify and characterize the selected bacterial isolate PG12 with antagonistic activity against B. dothidea, (ii) to optimize its biocontrol activity against B. dothidea on apples in the postharvest period and (iii) to identify the main antimicrobial compounds involved in its antifungal activity. Collectively, we aimed to identify a new promising candidate for use as BCA against apple ring rot and further investigate its biocontrol mechanisms. 2. Materials and methods 2.1. Fungal pathogens B. dothidea YL1, a particularly virulent strain, was supplied by Prof. Liyun Guo of College of Agronomy and Biotechnology, China Agricultural University, China. B. dothidea YL1 was cultured on potato dextrose agar (PDA) at 25 C in the dark and then subcultured on 2% malt extract agar with a 12-h photoperiod using near UV light to induce sporulation. The conidial suspension was obtained according to the method used by Tang et al. (2012). The plant pathogens listed in Table 1 were kindly provided by the Department of Plant Pathology, College of Agronomy and Biotechnology, China Agricultural University, China. 2.2. Isolation of bacterial strains Bacterial strains used in this study were originally isolated from healthy apples (Malus domestica cv. Fuji) collected at commercial maturity from several orchards in Northern China (Beiliu village, Beijing; Sanmenxia, Henan Province; Qixia city, Shandong Province; Tianshui city, Gansu Province; Xi’an city, Shaanxi Province and Yuncheng city, Shanxi Province) that have been affected by severe apple ring rot disease. For bacterial isolation, fruit samples were processed using surface sterilization and serial dilution methods according to protocols described previously by Lai et al. (2012). One gram samples were collected close to the fruit skin and ground in 3 mL of sterilized water and sterilized quartz sands using a mortar and pestle. The homogenates were serially diluted, incubated for 30 min at 80 C in a water bath, and plated on NA. The plates were then incubated at 30 C for one day (Urrea et al., 2011). Individual bacterial colonies were isolated and purified.
Table 1 Antagonist activity against various fungal plant pathogens by strain PG12 in vitro. Target fungal pathogens
Inhibitory activitya
Botryosphaeria dothidea Botryosphaeria ribis Valsa mali Monilinia fructicola Botrytis cinerea Penicillium expansum Botryosphaeria berengeriana Colletotrichum gloeosporioides Rhizoctonia cerealis Phytophthora infestans Phytophthora capsici Fusarium oxysporum Trichothecium roseum Alternaria alternata
+++ ++ ++ +++ + + + + ++ ++ ++ ++ +++ +
a + ++ and +++ represent relative inhibition rates against mycelia growth of each fungal colony on the PDA medium to the extent of 31–50%, 51–70% and 71%, respectively.
2.3. Screening of antagonistic bacteria The dual culture technique was used to detect the antagonistic activities of bacterial isolates towards fungi. A 5-mm-diameter plug from a 5-day-old mycelial culture of B. dothidea was placed in the center of fresh PDA plates (90 mm). A fresh colony was patched around the fungal plug at a distance of 3 cm using a sterile toothpick. PDA only inoculated with the pathogen was used as a control. The plates were incubated at 28 C for five days. The antagonistic effect was assessed by measuring the inhibition zones (mm) and the colony diameters. The percentage of growth inhibition was calculated using the formula n = [(a b)/a] 100, where n is the percentage of growth inhibition, a is the colony area of uninhibited fungi, and b is the colony area of treated fungi (Etebarian et al., 2005). The values were recorded as the means of three replicates. Experiments were repeated twice, with B. amyloliquefaciens 9001 mentioned previously used as the control isolate. 2.4. In vivo test Fuji apples were used to evaluate the biocontrol activity of strain PG12 on apple ring rot in the postharvest period (Li et al., 2013). Healthy apples were sampled from an orchard containing 10-year-old trees badly infected with B. dothidea and situated in the Beiliu village of Changping District in Beijing, China. Twelve trees were chosen for the biocontrol trial. Routine chemical control was applied in the experimental field. The bacterial inoculum was prepared in nutrient broth (NB) in a shaker at 160 rpm for 2 days at 30 C. The bacterial cells were collected by centrifugation at 6000 g for 5 min and diluted to the desired concentration using a PBS buffer (pH 7.0). Cell concentration was determined by a hemocytometer. After fruit maturation, healthy samples were harvested and soaked in bacterial suspensions of PG12 at a concentration of 108 CFU mL1 for 2 min, and then dried in a transfer hood. Fruit soaked in sterile distilled water were used as the controls. The treated apples were placed in plastic trays at room temperature (20 C). The sample numbers of rotted fruit were recorded weekly, and the percentage of disease incidence (the numbers of rotted apples divided by the numbers of total apples 100) was calculated. Ten fruit samples were used for each replicate, and the values were recorded as the means of three replicates for each treatment. The experiments were repeated in two years, and data from the three replicates were combined.
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2.5. Identification of PG12
2.6. Scanning electron microscopy analysis
PG12 was preliminarily characterized through means of conventional bacteriological methods including morphological, physiological, and biochemical characteristics (Gerhardt et al., 1981). PG12 was further identified through a phylogenetic analysis of its 16 S rDNA and partial gyrase gene sequences (gyrA). The genomic DNA of PG12 was extracted using standard procedures (Sambrook et al., 2001). The 16 S rDNA was amplified by PCR using primers 63 F (50 -CAGGCCTAACACATGCAAGTC-30 ) and 1387R (50 -GGGCGGWGTGTACAAGGC-30 ) (Beijing Sunbiotech Co., Ltd) (Marchesi et al., 1998). Part of the gyrA sequence was amplified with the primers p-gyrA-f (50 -CAGTCAGGAAATGCGTACGTCCTT-30 ) and p-gyrA-r (50 -CAAGGTAATGCTCCAGGCATTGCT-30 ) (Beijing Sunbiotech Co., Ltd.) (Chun and Bae, 2000). The concentrations of the primers, polymerase, and DNA used in the PCR were 10 mM, 5.0 U mL1, and 50 ng mL1, respectively. PCR amplification (in a total volume of 50 mL, including 2 mL of each primer, 5 mL of 10 buffer, 4 mL of 2.5 nM dNTP, 0.5 mL of Taq polymerase, and 2 mL of DNA template) was achieved using a Taq DNA polymerase kit (Beijing TransGen Biotech Co., Ltd.) with a modified thermocycler protocol that included an initial denaturation at 95 C for 5 min followed by 35 cycles of denaturation at 95 C for 1 min, annealing at 56 C for 40 s, extension at 72 C for 1 min, and a final extension at 72 C for 10 min (Li et al., 2012). The PCR products were ligated into pMD19-T vectors (Takara Co., Ltd.) to generate a recombinant plasmid. The plasmid was then mini-prepped from an overnight Luria–Bertani broth (LB broth) culture using the StarPrep Plasmid Miniprep Kit (Bioteke Biosolutions Co., Ltd.), and sent to Zhongkebaiao Biotechnologies Co., Ltd., (China) for sequencing. Multiple alignments with sequences of closely related Bacillus strains and sequence similarity calculations were performed using CLUSTAL X (Thompson et al., 1997). The phylogenetic trees of strain PG12 based on 16 S rDNA and the gyrA sequences were constructed using the neighbor-joining method and MEGA 4.0 software (Kim et al., 1993). The topology of the phylogenetic tree was evaluated by 1000 bootstrap resampling replicates (Felsenstein, 1985).
The morphology of the mycelia was further investigated by scanning electron microscopy (SEM, S-3400N, HITACHI, Japan). Five mycelial plugs (5 mm in diameter) were cut from PDA at outer edges of the inhibition zone as well as the corresponding position on the control. The mycelia were fixed with 2.5% (v/v) glutaraldehyde at 4 C for 2 h, rinsed three times with 0.1 mL of phosphate buffer (0.02 M) and subsequently fixed with 1% osmium tetroxide for 2 h at 20 C. Dehydration was performed three times with a graded series of ethanol (30%, 50%, 70%, 80%, 90%, 100%, v/v). The samples were dried with a desiccator (LEICA EM CPD), affixed to stubs using carbon tape and coated with a thin coating of gold (EIKO IB-3) for viewing under a SEM. The mycelia were observed by SEM and all of the images were processed computationally. The experiments were repeated twice, and the means of the three replicates were recorded. 2.7. Antifungal activity of metabolites from B. amyloliquefaciens PG12 against B. dothidea The antifungal activity of cell-free supernatant and lipopeptide crude extracts from PG12 against B. dothidea was explored using a technique similar to dual culture analysis (Yoshida et al., 2001). The pre-culture was grown in LB for 6 h to an OD600 of approximately 3.0. Briefly, 500 mL of culture was transferred to 50 mL of Landy medium and incubated at 30 C and 200 rpm for approximately 64 h (Landy et al., 1948). The cell-free supernatant and lipopeptide crude extracts were obtained following Niu et al. (2013). Briefly, cell-free supernatant was collected by centrifugation at 6000 g for 10 min, followed by filtration through 0.22 mm cellulose nitrate filters. To obtain the lipopeptide extract, six grams of XAD16 (Sigma) adsorbent resin held in a column (Sigma Amberlite) were washed with 50 mL of deionized water to remove salts. The cellfree supernatant was loaded onto the XAD16 resin column, washed with deionized water and eluted with 14 mL of 100% methanol. After drying with a rotary evaporator, the lipopeptide crude extracts were further dissolved in 1 mL of methanol. One hundred-microliter aliquots of cell-free supernatant, lipopeptide crude extracts or methanol (control) were spread on the PDA. A 7-mm diameter agar plug from an actively growing fungal mycelia of the B. dothidea was inoculated in the center of PDA. The diameters of the fungal colonies were measured after five
Fig. 1. Antagonistic activity of isolate PG12 and control isolate 9001 of B. amyloliquefaciens against B. dothidea in a dual culture test. (A) The mycelia growth of B. dothidea in absence of selected isolates. (B) The mycelia growth of B. dothidea in presence of the control isolate 9001 and PG12. Images were taken after seven days.
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Fig. 2. Disease incidence of ring rot in apples treated with bacterial suspensions of strain PG12 (PG12) or water (Control) after harvest. The vertical error bars represent the standard errors of the means calculated from three replicates for each treatment at each time point. Different letters indicate significant differences between different treatments according to Student’s t-test (P < 0.05).
days of incubation at 28 C, and the mean diameters from three replicates were calculated. The inhibition of fungal growth was calculated by measuring the diameter of the fungal colony and was expressed as the percentage of the inhibition of fungal growth divided by the growth obtained in the control plates. The values were recorded as the means of three replicates. The experiments were repeated twice. 2.8. PCR detection of antibiotic biosynthesis genes
chloroform/methanol/water (65:25:4, v/v/v) as mobile phase. Thereafter, bioautography was used to show the inhibitive activity of the methanolic fractions on a thin agar layer containing the fungus as an indicator organism. Bioautography was performed as described by Niu et al. (2013). In short, the TLC plates were stuck on the surface of the 1% PDA agar containing fungal spores with the final concentration of 4 107 CFU/mL at room temperature for 2 h and then removed. The PDA plates were incubated at 28 C for five days. Afterwards, the inhibition zones were documented the positions of the antifungal compounds separated by TLC. Their corresponding retention factor (Rf) values were estimated and the experiments were repeated three times. Matrix material of the TLC plates from the positions at which the antifungal compounds were located was scraped from the silica gel and then extracted with methanol. The extracts were further purified by semipreparative reverse phase HPLC (RP-HPLC). RP-HPLC analysis was carried out using a 5-mm Waters Symmetry C18 column (150 4.6 mm) at room temperature. The mobile phase consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile and was applied at a flow rate of 15 mL min1. The elution pattern was monitored by determining the absorbance at 215 nm. The peaks were collected and the substances dissolved in methanol were deposited in 7 mmdiameter wells made in the PDA plates at a distance of 2 cm from both the fungal plug and the control well containing methanol (M). The antagonistic activity against B. dothidea was observed after incubation at 28 C for 5 d. The eluted fractions with antifungal activity were analyzed by ESI-Q-TOF MS. Both the molecular weight and the structure of the purified lipopeptide were determined by ESI-Q-TOF MS using a Q-TOF instrument (Micromass, Bruker Daltonics). A mass spectrum was acquired over the range of 50–2200 m/z in the positive mode, and the capillary voltage was 4000 V. The precursor mass was set to 1043.55 m/z and the isolation window width was 10 Da. The collision gas was nitrogen and the fragment energy was 55 eV, and a ramping factor ranging from 100% to 150% (i.e., the real fragment energy ranged from 55 eV to 82.5 eV) was used to achieve improved fragmentation. The MS data were processed with Data Analysis 4.1 SP3, and the de novo sequencing analysis was performed using BioTools 3.2 (both from Bruker Daltonics). The amino acid sequence tag could be directly assigned by BioTools, but there was still a 225-Da gap that could not be interpreted. This gap was characterized using the Bruker Mol Weight to Formula software tools, and its formula was determined as C14H27NO, as determined directly from its accurate mass of 225.2087 Da (no other possibility in the mass error tolerance of 5 ppm).
Genomic DNA extraction and amplification of 19 antibiotic biosynthesis genes were performed using specific primers, which were listed in the supplementary information (Table S1). PCR amplifications were achieved in 50-ml of reaction mixtures containing 10 EasyTaq buffer, 2.5 nM of dNTP, 5.0 U mL1 of EasyTaq DNA polymerase (Beijing TransGen Biotech Co., Ltd.), 10 mM of each primer and 2 mL of template DNA (approximately 100 ng of bacterial genomic DNA). The amplifications were performed using a DNA thermal cycler (Model No. 9700, PerkinElmer Co., Wellesley, USA) with the following cycle conditions: an initial denaturation at 95 C for 5 min; 30 cycles of denaturation of 95 C for 1 min, an annealing step of 40 s (annealing temperature was listed in Table S 1), and extension at 72 C for 1 min; and a final extension at 72 C for 10 min (Chung et al., 2008). Expected PCR products were gel-purified and then ligated into pMD19-T vectors (Takara Co., Ltd.), generating a recombinant plasmid. Plasmids were isolated from the transformed cells using the StarPrep Plasmid Miniprep Kit (Bioteke Biosolutions Co., Ltd.) and sent to Zhongkebaiao Biotechnologies Co., Ltd., (China) for sequencing. The PCR sequences were compared using the BLAST program in the GenBank nucleotide database from the National Center for Biotechnology Information (Bethesda, MD, USA; http://www. ncbi.nlm.nih.gov/).
The data were subjected to variance analysis (ANOVA) using the SPSS software (SPSS Statistics 20.0). The statistical differences between means of different treatments was assessed by Student’s t-test at P < 0.05.
2.9. Isolation and characterization of antifungal lipopeptides
3. Results
The presence of antifungal lipopeptides in the supernatant of B. amyloliquefaciens PG12 was determined first by thin layer chromatography (TLC) (Razafindralambo et al., 1993). The control isolate FZB42 was kindly supplied by Dr. Rainer Borriss from Humboldt Universität Berlin, Germany. The methanolic fractions containing lipopeptide crude extracts were obtained as described above. Twenty microliters of each methanol extract were spotted onto TLC aluminum sheets coated with silica gel 60 F254 (20 cm 20 cm, Merck, Germany) and separated by TLC plates using
3.1. Screening of antagonistic bacteria
2.10. Statistical analysis
Eighty-three bacterial strains from 254 isolates were screened for potential antagonistic activity towards B. dothidea in the dual culture test (data not shown). Among these isolates, PG12 (CGMCC Accession No. 7132; China General Microbiology Culture Collection Center), which was isolated from apple fruit harvested from an orchard of Beiliu village, showed strongest antagonistic activity against B. dothidea. The inhibition zone of PG12 was 5.3 mm, and
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the percentage of growth inhibition after five days of incubation was approximately 87.5%. The antagonistic activity of PG12 was compared with that of 9001, which was previously identified as a potential biocontrol agent against apple ring rot (Li et al., 2013). There was significant (P < 0.05) difference in the inhibition zone and the percentage of fungal growth inhibition between isolate PG12 and the control isolate 9001. The inhibition ratio of isolate PG12 was higher than 9001 against B. dothidea (87.5% for PG12 versus 45.2% for 9001), indicating that antagonist PG12 was more effective than the control isolate 9001 in suppressing B. dothidea (Fig. 1). Moreover, PG12 exhibited broad-spectrum in vitro antagonistic activity against 13 other pathogens causing
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postharvest diseases in apple, pear, peach and other plants (Table 1). 3.2. In vivo test To investigate the effect of PG12 in the postharvest period, apples were treated with a PG12 suspension after harvest. The disease incidence of the PG12-treated apples was noticeably lower than that of the water-treated control apples for a period of 56 days after inoculation (Fig. 2). Similar results were obtained from the experiment conducted in the subsequent year (data not shown), suggesting PG12 effectively suppressed fungal diseases in the postharvest period.
Fig. 3. Phylogenetic tree based on the partial nucleotide sequence of 16 S rDNA (A) and gyrA (B). A neighbor-joining phylogenetic tree of strain PG12 was constructed using MEGA 4.0. The numbers at the nodes indicate the levels of bootstrap support (%) based on a neighbor-joining analysis of 1000 resampled datasets; only values greater than 50% are provided. Bars = 0.005 (A) and 0.05 (B) nucleotide substitutions per site (T = type strain).
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3.3. Identification of PG12
inhibited fungal growth by approximately 89%. No significant difference was found between these two treatments, suggesting that lipopeptides were responsible for the inhibitory activity against B. dothidea.
The physiological and biochemical characteristics of strain PG12 are summarized in Table S2. Strain PG12 is a Gram-positive and spore-forming bacterium with an optimal growth temperature of 28–37 C. A partial 16 S rDNA sequence analysis demonstrated that strain PG12 was most likely B. amyloliquefaciens subsp. plantarum CAU B946 (99%), indicating that strain PG12 belongs to the species B. amyloliquefaciens (Fig. 3A). This result was confirmed by a partial gyrA sequence analysis, which has been shown to be effective in distinguishing these closely related taxa of the B. subtilis group (Chun and Bae, 2000) (Fig. 3B). Therefore, PG12 was identified as B. amyloliquefaciens (B. amyloliquefaciens subsp. plantarum). The 16 S rDNA sequence and partial gyrA nucleotide sequence of the test strain PG12 were deposited in the GenBank database with the accession numbers KC797583 and KC797584, respectively.
PCR detection and nucleotide BLAST analysis showed that PG12 had antibiotic biosynthesis genes: ituD (GeneBank Accession No. KT342867), ituC (GeneBank Accession No. KT342866), bamD (GeneBank Accession No. KT342867), bamC (GeneBank Accession No. KT342868), bacAB (GeneBank Accession No. KT342869), bacD (GeneBank Accession No. KT342865), fenB (GeneBank Accession No. KT342870), fenC (GeneBank Accession No. KT342871) and fenF (GeneBank Accession No. KT342867) (Table 2). However, eriB,eriSa, mrsA, mrsM, spaB, spaC, albF, albA, mycC and sunT were not detected in B. amyloliquefaciens PG12.
3.4. Scanning electron microscopy analysis
3.7. Identification of lipopeptides from B. amyloliquefaciens PG12
SEM analysis revealed that mycelia of B. dothidea collected from the edge of inhibition zone were morphologically different from that of the control on PDA. Normal mycelia of B. dothidea were smooth and presented a uniform in thickness (Fig. 4A). Whereas, mycelia collected from the inhibition zone were thick and abnormal, irregular swelling, curling of the hyphal tips, cell wall collapse and many hollows (Fig. 4B–D).
To identify the compounds that suppress the growth of B. dothidea, the methanol extracts of lipopeptides obtained from PG12 were subjected to TLC in combination with bioautography. It was found that two active fractions of PG12 inhibited the conidial germination and mycelial growth of B. dothidea. One of the active fractions was found in both B. amyloliquefaciens FZB42 and PG12, showing Rf values typical for fengycin-like lipopeptides (Rf 0.1 to 0.2). However, another active fraction (Rf 0.4) was found in PG12, but not in B. amyloliquefaciens FZB42 (Fig. 5). The active compounds (Rf 0.4) were extracted from silica gel of TLC plates for further purification by HPLC (Fig. 6A). Three fractions were obtained and only fraction 1 showed inhibitory activity against B. dothiedea (Fig. 6B). The identification of fraction 1 by LCMS showed that the molecular mass of the active compound was 1043.55 m/z at a retention time of 3.45 min, resembling C 14 iturin,
3.5. Effect of B. amyloliquefaciens PG12 cell-free supernatant and lipopeptide crude extracts on B. dothidea The dual culture assay showed that cell-free supernatant of B. amyloliquefaciens PG12 significantly inhibited the growth of the fungal pathogen by approximately 86%. The methanol extracts of lipopeptide from cell-free supernatant of PG12 significantly
3.6. Antibiotic biosynthesis genes in B. amyloliquefaciens PG12
Fig. 4. Morphological analysis of mycelia by scanning electron microscopy. Mycelia of B. dothidea in the absence (control) (A) or presence of bacterial isolate PG12 (B–D). The arrows indicate B. dothidea mycelia damaged by strain PG12.
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Table 2 PCR detection of antibiotic biosynthesis genes from B. amyloliquefaciens PG12. Antibiotic
Genes
Detection in PG12a
Iturin A
ituD ituC bam D bam C bacAB bacD eriB eriSa fenB fenC mrsA mrsM fenF mycC sunT spaB spaC albF albA
+ + + + + + + + +
Bacillomycin D Bacilysin Ericin Fengycin Mersacidin Mycosubtilin Sublancin Subtilin Subtilosin
a
+ and represent presence or absence of each gene, respectively.
as previously reported (Chen et al., 2008). ESI-Q-TOF MS analysis further revealed the amino acid sequence of fraction 1. The resulting fragmentation spectrum was analyzed, revealing a partial series of a-type and b-type ions covered the entire amino acid sequence of the peptide. The sequences indicated that Ser and Asn were connected to a fatty acid moiety, and the molecule was deduced to be a cyclic lipopeptide based on data obtained through tandem mass spectrometry regulation. This sequence was found to be N-Ser-Asn-Pro-Gln-Asn-Tyr-Asn-C. Based on an analysis of its cyclic structure, the lipopeptide was determined to be iturin A (Fig. 7). The molecular mass and complete peptide sequence were similar to those of iturin A, produced by B. amyloliquefaciens TF28 (Zhang et al., 2012). Altogether, iturin A was identified as an inhibitor of B. dothidea. 4. Discussion Many species in the Bacillus genus are considered as potential biocontrol agents for reduction of postharvest phytopathogens such as Penicilium spp., Botrytis cinerea and Monilinia spp. (Sharma et al., 2009; Pretorius et al., 2015). Few studies have reported Bacillus spp. for biocontrol of apple ring rot caused by B. dothidea which is an important disease for apple production (Li et al., 2013). So far, the mechanisms of Bacillus strains against B. dothidea have not been extensively investigated. In this study, B. amyloliquefaciens PG12 originally isolated from apple fruit proved its biocontrol potential against apple ring rot. The antifungal activity of B. amyloliquefaciens PG12 against B. dothidea was attributed to the action of lipopeptides. The results of this study demonstrated for the first time the biological potential of a B. amyloliquefaciens strain to control apple ring rot disease by the production of iturin A. In the present study, B. amyloliquefaciens PG12 with broadspectrum of antagonistic activity efficiently controlled apple ring rot in in vitro and in vivo. These results confirmed previous findings on the potential of B. amyloliquefaciens species as biocontrol agent of apple ring rot (Li et al., 2013). A few of other antagonistic microorganisms were tested on fruit to verify the antifungal activity against apple ring rot, such as B. licheniformis, T. harzianum and T. atroviride. For example, biocontrol field trials suggested that apple ring rot on apple shoots, stems and fruit had been efficiently controlled by the application of T. harzianum T88 and T. atroviride T95 (Kexiang et al., 2002). Ji et al. (2008) reported that B.
Fig. 5. Thin layer chromatography (TLC)-bioautography analysis against B. dothidea. Developed chromatograms were covered with a PDA medium inoculated with a fungal spore suspension at concentration of 4 107 CFU mL1. Pictures were taken after five days of incubation. The retention factor (Rf) values of the different lipopeptides iturin A (Rf 0.4) and fengycin (Rf 0.1–0.2) are indicated on the right. The fungal growth inhibition zones produced by iturin A-like activity from B. amyloliquefaciens PG12 are indicated by an arrow.
licheniformis W10 could suppress necrosis spread of apple ring rot on fruit. Therefore, the postharvest application of biocontrol agents is a practical and useful method for controlling apple ring rot. Previous studies have reported that B. amyloliquefaciens strains showed strong antifungal properties against plant pathogenic fungi based on the production of antimicrobial compounds such as cyclic lipopeptides (Yu et al., 2012; Yuan et al., 2014; Ben Abdallah et al., 2015). However, the mechanism of biocontrol of apple ring rot by B. amyloliquefaciens is not clear. In this study, B. amyloliquefaciens PG12 reduced mycelial growth of B. dothidea in vitro tests. The large inhibition zones could be due to the effect of antimicrobial compounds produced by PG12. The mycelia from the edge of inhibition zone exhibited lumpy appearance and abnormal structure observed by SEM. Antimicrobial compounds not only inhibited the growth of mycelia but also damaged the morphology. These effects were very similar to the morphological damage of apple ring rot treated with B. licheniformis W10 (Ji et al., 2008). Additional experiments were carried out to evaluate whether the efficacy was associated with lipopeptides. It was found that cellfree supernatant of PG12 and its lipopeptide crude extracts had similar efficacy against B. dothidea in vitro. These results suggested that the mechanism for PG12 to suppress apple ring rot may mainly include antimicrobial lipopeptides. This finding was consistent with previous reports in which lipopeptides in cellfree supernatant were responsible for the antifungal activity of B. subtilis CPA-8 against peach brown rot (Yánez-Mendizábal et al., 2012). The lipopeptide antibiotics produced by Bacillus spp. have been generally assumed to be responsible for antifungal activity. B. amyloliquefaciens strains produce various antimicrobial
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Fig. 6. The lipopeptide mixture was isolated and purified by semipreparative reverse phase HPLC (RP-HPLC) and antagonism assay against B. dothidea in vitro. Three peaks were detected at 215 nm and collected (A), and the antagonistic activity against B. dothidea was assayed (B).
lipopeptides including iturins surfactins and fengycins against phytopathogens (Falardeau et al., 2013; Cawoy et al., 2015). Based on PCR-assays, B. amyloliquefaciens PG12 harbored the gene clusters involved in the synthesis of iturin A, bacillomycin D and fengycin. In many cases, it was possible to associate the presence of lipopeptides of B. amyloliquefaciens strains with the corresponding biosynthetic genes. However, further bioautography analysis of lipopeptide crude extracts revealed that B. amyloliquefaciens PG12 obviously inhibited mycelial growth of B. dothidea by two active fractions (iturin A [Rf 0.4] and fengycin [Rf 0.1–0.2]). As shown in this study, the production of iturin A is a very important factor conferring antifungal activity to B. amyloliquefaciens strains against phytopathogens (Arrebola et al., 2010; Gong et al., 2015; Han et al., 2015). Fengycin-like families of B. amyloliquefaciens PG12 also exhibited antifungal activity against B. dothidea according to control isolate FZB42. Iturin A and fengycin were well known for its broad-spectrum of antifungal activity (Stein, 2005). The strong antifungal activity of PG12 against 13 other postharvest pathogens may be due to its production of iturin A and fengycin, but further evidence is needed to confirm this finding. Furthermore, Koumoutsi et al. (2004) reported that iturins and fengycins of B. amyloliquefaciens FZB42 exhibited synergistic actions against F. oxysporum. In contrast, iturins and fengycins of
B. amyloliquefaciens SQR9 did not show synergistic actions against F. oxysporum (Xu et al., 2013). Whereas, as evidenced for isolate BO7 of this bacterial species, the strong biocontrol activity is only due to new identified surfactins (Vitullo et al., 2012). Further research will be needed to identify and characterize the relationship between iturins and fengycins against B. dothidea in our isolate PG12. Joshi and McSpadden Gardener (2006) has demonstrated that the presence of a particular gene is not a reliable predictor of the biocontrol capacity in a Bacillus strain. In this study, although B. amyloliquefaciens PG12 harbored the gene clusters required for bacillomycin D, no active fraction (Rf 0.3) corresponding to bacillomycin D on TLC plate showed obvious antifungal activity against B. dothidea. This result might be due to no bacillomycin D production or a low concentration of bacillomycin D from PG12. Our result was similar to those previously reported for B. amyloliquefaciens ARP23. The peptide synthetase genes of B. amyloliquefaciens ARP23 involved in the production of bacillomycin were detected by PCR, but bacillomycin were not found in the culture supernatants of ARP23 (Alvarez et al., 2012). The production of lipopeptides may be related to the growth medium, culture condition or genes related to the biosynthesis of lipopeptides. Future work involving gene expression studies and molecular analysis by mutagenesis will be useful for establishing the involvement of the lipopeptide synthetase genes to biological control. Moreover, lipopeptides production is not the only factor that needs to be considered and more work is required to also explore other possible biocontrol mechanisms. 5. Conclusions In conclusion, B. amyloliquefaciens PG12 efficiently controlled apple ring rot disease caused by B. dothidea in vitro and in vivo. The antifungal activity of B. amyloliquefaciens PG12 against B. dothidea was attributed to the action of lipopeptides. This is the first report revealing the role of iturin A produced by B. amyloliquefaciens PG12 in the reduction of apple ring rot disease caused by B. dothidea. In brief, B. amyloliquefaciens PG12 has a great potential as a biocontrol agent against apple ring rot. Acknowledgments
Fig. 7. The a, b and y ions obtained in the ESI-Q-TOF MS are indicated in the spectrum at m/z 1043.551.
This work was financially supported by the National Natural Science Foundation of China (Grant No. 31371977), the Earmarked Fund for Modern Agro-industry Technology Research System (Grant No. CARS-28) and the Specialized Research Fund for the
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