Suppression of Bacterial Wilt of Tomato by Bioorganic Fertilizer Made

Article pubs.acs.org/JAFC

Suppression of Bacterial Wilt of Tomato by Bioorganic Fertilizer Made from the Antibacterial Compound Producing Strain Bacillus amyloliquefaciens HR62 Jianfeng Huang, Zhong Wei, Shiyong Tan, Xinlan Mei, Qirong Shen, and Yangchun Xu* National Engineering Research Center for Organic-Based Fertilizers and Jiangsu Collaborative Innovation Center for Solid Organic Waste Utilization, Nanjing Agricultural University, Nanjing, 210095, China S Supporting Information *

ABSTRACT: Ralstonia solanacearum (Smith) is an important soil-borne pathogen worldwide. We investigated the effects of a new bioorganic fertilizer, BIO62, which was made from organic fertilizer and antagonist Bacillus amyloliquefaciens HR62, on the control of bacterial wilt of tomato in greenhouse condition. The results showed that the application of BIO62 significantly decreased disease incidence by 65% and strongly reduced R. solanacearum populations both in the rhizosphere soil (8.04 log cfu g−1 dry soil) and crown sections (5.63 log cfu g−1 fresh plant section) at 28 days after pathogen challenge. Antibacterial compounds produced by HR62 were purified by silica gel, Sephadex LH-20, and HPLC and then identified using HPLC/ electrospray ionization mass spectrometry analysis. Macrolactin A and 7-O-malonyl macrolactin A (molecular weights of 402 and 488 Da, respectively), along with surfactin B (molecular weights of 994, 1008, 1022, and 1036 Da), were observed to inhibit the growth of R. solanacearum. KEYWORDS: bacterial wilt of tomato, antibacterial compounds, bioorganic fertilizer, biological control, Ralstonia solanacearum

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INTRODUCTION

Antibacterial compounds are suppression factors and play a major role in biocontrol of soil-borne diseases. Many studies have reported that B. amyloliquefaciens, one of the Bacillus spp., can produce a broad range of peptide metabolites with antibacterial and/or antifungal activities.13−16 B. amyloliquefaciens forms highly resistant endospores to both chemical and physical stresses. These properties, combined with the ability of these bacteria to inhabit the soil, make them well-suited for biocontrol applications. Lipopeptides are one type of antimicrobial substances isolated from B. amyloliquefaciens thatmare not produced by ribosomes and include surfactins, fengycins, and iturins.13,17,18 B. amyloliquefaciens can also produce polyketides, a large family of secondary metabolites that include macrolactin, difficidin, and oxidifficidin.16,19 Dipeptides and siderophores are two additional low-molecular compounds purified from B. amyloliquefaciens.19,20 Furthermore, high-molecular compounds, such as antibacterial proteins, can also be produced by B. amyloliquefaciens.21,22 However, there are few reports concerning antibacterial compounds secreted by B. amyloliquefaciens against R. solanacearum. We have reported on the biocontrol efficacy of B. amyloliquefaciens HR62 against bacterial wilt of tomato in a previous study; this strain was not a satisfactory biocontrol agent due to its weak root-colonizing capacity.23 In the present study, the application of the bioorganic fertilizer BIO62 may improve the colonization capacity of HR62 in the tomato rhizosphere. The identification of the antibacterial compounds produced by

Bacterial wilt caused by the soil-borne pathogen Ralstonia solanacearum (Smith 1896) is one of the most devastating diseases in the tropical and subtropical regions of the world. It constantly threatens tomato production in southern China and causes increasing economic loss over time.1 Biological control is the proper way to suppress disease, and its aim is to attack, repel, or otherwise antagonize disease-causing pathogens and suppress disease in soil in an environmentally friendly way.2 Beneficial microorganisms, known as biocontrol agents, have been used to effectively control R. solanacearum under laboratory and/or greenhouse conditions.1,3−5 However, inoculation into soils with antagonistic microorganisms without a suitable organic substrate will not be successful due to the absence of nutrients.6,7 It is believed that a combination of antagonistic microbes with organic amendments may be more efficient in inhibiting disease than the use of a single antagonistic microbial strain or the organic amendment alone.8,9 In the present study, the combination of the antagonist Bacillus amyloliquefaciens HR62 and organic fertilizer (e.g., compost, manure, and plant waste) for the control of bacterial wilt of tomato was studied. This type of product can minimize organic waste and reduce fertilizer and fungicide use in crop production.9 Luo et al.10 found that the application of bioorganic fertilizer significantly affected fungal diversity in the soil, resulting in a reduction in cotton verticillium wilt. Wei et al.11 applied a bacillus-fortified organic fertilizer to control bacterial wilt of tomato both in greenhouse and field conditions; R. solanacearum disease incidence was effectively reduced. Yuan et al.12 evaluated the successful use of bacillusfortified organic fertilizer for the control of tobacco bacterial wilt in the greenhouse and the field. © 2014 American Chemical Society

Received: Revised: Accepted: Published: 10708

July 14, 2014 October 15, 2014 October 16, 2014 October 16, 2014 dx.doi.org/10.1021/jf503136a | J. Agric. Food Chem. 2014, 62, 10708−10716

Journal of Agricultural and Food Chemistry

Article

DI = [∑(number of diseased plants in the index × di)/(total number of plants investigated × highest di)] × 100%. To evaluate the R. solanacearum populations in the rhizosphere soils and crown sections, three tomato plants were uprooted from each treatment at 1, 7, 14, 21, and 28 days after pathogen challenge. Ten grams of rhizosphere soils or tomato crown sections was added to 90 mL of sterile water (crown sections were ground beforehand). The suspensions were shaken for 30 min on a rotary shaker at 200 rpm. Several 10-fold dilutions were made, and 0.1 mL aliquots were spread on the surface of M-SMSA medium.27 After 2 days of incubation at 30 °C, colonies typical of R. solanacearum were counted. Colony forming units (cfu) were calculated per gram (dry weight) of soil and the values were log-transformed before statistical analysis was performed. Amplification of HR62 Antagonistic Genes. The primers used to amplify the genes encoding bioactive compounds in HR62 were either used as previously described by Arguelles-Arias et al.19 and Joshi and Gardener28 or designed using Primer 5 software from Premier Biosoft based on consensus sequences of known Bacillus subtilis lipopeptide antibiotic genes that were deposited in GenBank (Table S1, Supporting Information).29,30 PCR reactions were carried out in a 25 μL reaction volume containing 1 μL of genomic DNA, 2.5 μL of 10× PCR buffer, 20 mM MgCl2, 0.2 mM of each dNTP, 0.5 μM of each primer, and 1.25 U of Taq DNA polymerase (Takara, Dalian). Amplification was performed using a PCR System DNA thermal cycler (Bio-Rad) programmed for one cycle of 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, annealing for 30 s at 51 °C, extension for 1 min and 30 s, and a final extension at 72 °C for 10 min. A negative control without DNA was included in each PCR run. Each PCR reaction had three replicates. The amplified products were visualized by gel electrophoresis in 1% agarose gels; gels were stained with ethidium bromide. Expected PCR fragments were extracted using a gel extraction kit (Axygen, China). The obtained fragments were sequenced by Majorbio Co. (Shanghai, China). Nucleotide sequences were compared with those from GenBank using the BlastN software provided online by the National Center for Biotechnology Information. Isolation and Purification of Antibacterial Compounds from B. amyloliquefaciens HR62. To select the best extractant, HR62 was incubated in an optimized culture (100 mL) for 36 h and extracted twice (for 1 h each) in an equal volume for five the following extractants: toluene, hexane, chloroform, ethyl acetate, and n-butyl alcohol. Among these, n-butyl alcohol was found to be the best extractant and was therefore used in subsequent experiments. Then, 5 mL of an overnight optimized culture of HR62 was used to inoculate 500 mL of fresh liquid optimized culture. The culture was then placed in an incubator shaker (at 170 rpm and 30 °C) for 36 h. The liquid culture was centrifuged at 10 000g for 10 min, and the supernatants were pooled together. Active compounds were extracted twice (for 1 h each) with an equal volume of n-butyl alcohol. The extract was concentrated using a rotary evaporator, and the residues were dissolved in 100 mL of distilled water. The sample was adjusted to pH 2.0 with 6 mol L−1 HCl and stored overnight at 4 °C. The precipitate was recovered by centrifugation at 8000 g for 20 min. The pellet was washed twice with acidic deionized waster (pH 2.0) and extracted twice with methanol.31 The solution was dried in a rotary vacuum evaporator, and the residue was dissolved in 10 mL of 0.01 mol L−1 phosphate buffer solution (PBS) at pH 7.4. A chloroform and methanol gradient (e.g., 100 mL of chloroform, 90 mL of chloroform + 10 mL of methanol, 80 mL of chloroform + 20 mL of methanol, and so on, up to 100 mL of methanol) was used to elute active compounds from a silica gel column (2 × 24 cm). Twenty-eight fractions (10 50-mL fractions and 18 30-mL fractions) were collected. The fractions were dried using a rotary evaporator and dissolved separately in 2 mL of PBS buffer. Active fractions were identified by an agar diffusion assay against R. solanacearum on KB plates; these fractions were then pooled and eluted further using a water and methanol gradient (e.g., 100 mL of water, 90 mL of water + 10 mL of methanol, 80 mL of water + 20 mL of methanol, and so on, up to 100 mL of methanol) in a Sephadex LH-20 column (2 × 24 cm). Active fractions were found using a bioassay and were then pooled. The pooled fractions were purified further using an HPLC device (1200 series, Agilent, Santa Clara, CA).

HR62 allows more comprehensive understanding of the mechanisms involved in this biocontrol system. The objectives of this work were as follows: (1) to assess the capability of a new bioorganic fertilizer containing B. amyloliquefaciens HR62 to control bacterial wilt of tomato under greenhouse conditions and (2) to purify and to identify the antibacterial compounds produced by B. amyloliquefaciens HR62 that are responsible for the in vitro antagonism of R. solanacearum.

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MATERIALS AND METHODS

Bacterial Strains and Culture Conditions. HR62 was isolated from the rhizosphere soil of healthy tomato plants from a heavily wilt diseased field using dilution plate techniques. The strain was identified as B. amyloliquefaciens based on its biochemical characteristics and partial 16s rRNA.23 B. amyloliquefaciens HR62 was cultured on Luria−Bertani medium (LB) plates and stored at −70 °C in a mixture of LB liquid broth and 30% glycerol for further use. R. solanacearum QL-Rs1115 was provided by the Soil-Microbe-Interaction Laboratory at Nanjing Agricultural University in Nanjing, China.11 The strain was maintained on KB plates supplemented with 0.005% triphenyltetrazolium chloride (m/v) for 2 days at 30 °C. A single colony was transferred into sterilized water for storage at room temperature. For the production of antibiotics, B. amyloliquefaciens HR62 was grown in an optimized culture.24 Preparation of Bioorganic Fertilizers. The preparation of the bioorganic fertilizer (BIO62) followed the procedure described by Zhang et al.36 The organic fertilizers (OF) consisted of a compost of pig manure and rice straw containing 33.7% OM (organic material), 2.51% N, 2.4% P2O5, 1.13% K2O, and 22.3% H2O (Jiangsu Tianniang Agritechnology Ltd., Jiangsu, China) and an amino acid organic fertilizer containing a biologically hydrolyzed rapeseed cake with 44.2% OM, 8.0% amino acids, 4.4% N, 3.5% P2O5, 0.67% K2O, and 28.5% H2O36 provided by Jiangsu Xintiandi Ltd. (Jiangsu, China). The two organic fertilizers were evenly mixed (1:1, w/w). B. amyloliquefaciens HR62 was incubated in 1 L of optimized culture in a 5 L fermentation tank on a shaker at 170 rpm at 30 °C for 36 h. The bacterial culture was centrifuged (at 10 000g for 10 min at 4 °C), and the collected cell pellets were resuspended in sterile water. The suspension of HR62 (5.0 × 109 cfu mL−1) was mixed with the OF mixture at a 1:5 (v/w) ratio. The final concentration of HR62 in the fortified organic fertilizer was approximately 109 cfu g−1 dry soil (ds). Design of the Pot Experiment under Greenhouse Conditions. Tomato seedlings were grown in nursery cups (8.5 × 11 cm) containing 300 g of healthy nursery soil (i.e., soil that had been previously planted with tomato). One seedling was maintained in each cup. The seedlings were grown in a greenhouse, with temperatures ranging from 26 to 35 °C, until the seedlings had four true leaves. The seedlings were then transplanted to pots with 5 kg of R. solanacearum-infested soil. The following three treatments were included in the pot experiment: (1) CK treatment, where OF was applied to the soil; (2) HR62 treatment, where OF and a cell-free fermentation of HR62 was applied to the soil; and (3) BIO62 treatment, where BIO62 was added in the pot soil. The rate of the OF and BIO applications was 0.5% (dry weight basis). The OF and BIO applications were mixed separately into the soil prior to the transplantation of the tomato seedlings. Seedlings with four leaves were transplanted into the ceramic pots (5 kg soil per pot). One week after the transplantation, a 1 mL suspension of the R. solanacearum strain QLRs1115 (5 × 107 cfu mL−1) was poured into the soil to give a final bacterial concentration of 104 cfu g−1 ds. A total of 45 pots (15 pots with 30 plants for each treatment) were placed on a glasshouse bench in a completely randomized design and watered regularly. The experiment was repeated twice. Disease development, expressed as disease incidence (DI), was based on a disease index (di) scale of 0−4,25 where 0 = no wilting, 1 = 1−25% wilting, 2 = 26−50% wilting, 3 = 51−75% wilting, and 4 = 76−100% wilting or dead. The di was recorded daily for 28 days after pathogen challenge. The DI was calculated according to the following equation:26 10709

dx.doi.org/10.1021/jf503136a | J. Agric. Food Chem. 2014, 62, 10708−10716


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A 20 μL of sample was injected into an HPLC column (Eclipse XDBC18, 4.6 × 250 mm, 5 μm, Agilent, Santa Clara, CA). Two conditions were applied. In condition 1, the temperature of the column was maintained at 30 °C throughout the experiment. The purification was performed using a solvent containing 40% A (CH3CN) and 60% B [0.1% (v/v) CH3COOH], with a flow rate of 0.6 mL min−1 for 30 min. In condition 2, the temperature of the column was maintained at 35 °C throughout the experiment. The sample was eluted in 60−93% A (CH3CN) and 40−7% B [0.1% (v/v) CH3COOH] over 9 min. The ratio was then kept stable until the end of the experiment, with a flow rate of 0.84 mL min−1 for 25 min. A standard sample of surfactin was also tested using condition 2. An ultraviolet (UV) detector was used to detect peaks at 210 nm. A fraction collector (Analyt FC, G1364C, Agilent, Santa Clara, CA) was used to collect the pure compounds; the fractions were collected using the time and peaks mode. The injections were performed repeatedly to allow for the collection of a sufficient quantity of antibiotics. The fractions were lyophilized, and the residues were dissolved in 500 μL of methanol. After an agar diffusion assay against R. solanacearum on KB plates, the active peaks were located. Mass Spectrometry Analysis. The antibacterial material was subjected to a liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) system (1200 series, Agilent, Santa Clara, CA, and ESI-MS, 6410 Triple Quad LC/MS, Agilent, Santa Clara, CA) with a C18 column (4.6 × 250 mm, 5 μm). Two conditions were used; these conditions were the same as those described for the purification in the HPLC section. For the MS analysis, the electrospray needle was operated at a spray voltage of 4.5 kV. The capillary temperature was 300 °C. The fractions eluted in condition 1 were measured in the both positive and negative ion modes to determine the accurate molecular weights of the antibiotics. The fractions eluted in condition 2 were measured in positive ion mode. The mass spectra were acquired in an m/ z range of 100−2000 at a scan rate of 500 atomic mass units (amu)/s. Sensitivity of the Antibacterial Compounds to Temperature, pH, Enzymes, and Organic Solvents. For the thermal stability test, the antibacterial compounds were exposed to temperatures of 30, 40, 50, 60, 70, 80, 90, and 100 °C for 1 h; 115 °C for 30 min; or 121 °C for 20 min. For the pH stability test, the antibacterial compounds were solubilized in a buffer containing 100 mM citrate and 200 mM phosphate buffer (pH 1−8), a buffer containing 100 mM phosphate and 100 mM HCl (pH 9−11), or a buffer containing 200 mM KCl and 200 mM NaOH (pH 12−14), followed by an incubation at 30 °C for 12 h. The residual antibacterial activity was assessed after neutralizing the samples to pH 7.0 with an appropriate weak base or weak acid. The sensitivity of the antibacterial compounds to various enzymes was tested. The enzymes used were as follows: pepsin (800−2500 U mg−1; P8160, Solarbio), recombinant proteinase K (600−100 U mg−1; 03115828001, Roche Applied Science), albumin fraction V (≥95% protein; A8020, Solarbio), and trypsin from bovine pancreas (≥2500 U mg−1; 64008834, SCRS). All of the enzyme solutions were prepared in 25 mM phosphate buffer (pH 7.0). Each solution contained 1 mg mL−1 of its respective enzyme. Solutions containing the antibacterial compounds were prepared by centrifuging the HR62 fermentation. The stock solutions of the antibacterial compounds and the enzymes were mixed at a 1:1 ratio (v/v) and incubated at 37 °C for 1 h before the residual antibacterial activity was measured using an agar diffusion assay against R. solanacearum. The sensitivity of the antibacterial compounds to the following organic solvents was tested: acetone, methanol, isopropyl alcohol, and ethanol. A 100 mL aliquot of cultured HR62 was centrifuged at 10 000g for 10 min, and the supernatants were added to the organic solvents in equal volumes. The mixture was then placed at room temperature for 4 h. The mixtures were dried using a rotary evaporator, and the residues were dissolved in 100 mL of PBS buffer. The resistance activity of the PBS solutions to R. solanacearum was determined using KB plates. Statistical Analysis. The arcsin conversion was performed before the data were analyzed for percentages. Differences were assessed by one-way ANOVA. Duncan’s multiple-range test was applied when oneway ANOVA revealed significant differences (P < 0.05). All statistical analyses were performed with SPSS 16.0 (SPSS Inc.) and SigmaPlot 11.0 (Systat Software Inc.).

Article

RESULTS Biological Control Assays against Bacterial Wilt of Tomato. Treating plants with BIO62 resulted in reduced disease incidence (Figure 1). Disease symptoms were apparent 7 days

Figure 1. Effect of different treatments on bacterial wilt disease incidences at 1, 7, 14, 21, and 28 days after challenge. All values are means of three blocks with ten replicates per block. Bars show standard errors of the mean. An asterisk on the top of the bar represents significance at P < 0.05 (Duncan’s multiple range test).

after the pathogen challenge in all treatments. The BIO62treated plants had significantly lower disease incidence compared with the control at 14, 21, and 28 days after the pathogen challenge. At the end of the experiment, the disease incidence in HR62- and BIO62-treated plants was reduced by 53% and 65%, respectively. The disease incidence in the control was 85%. Population Dynamics of R. solanacearum in the Rhizosphere Soil and Crown Sections of Tomato. Populations of R. solanacearum in the rhizosphere soil of CKtreated tomatoes were significantly higher than those observed in HR62 and BIO62 treatments (Figure 2A) throughout the challenge period. The rhizosphere soil of BIO62-treated plants had the lowest population density at 8.04 log cfu g−1 ds. For the CK treatment, the observed population density was 9.30 log cfu g−1 ds at the end of experiment. The same trend was observed in the dynamics of R. solanacearum among three treatments in the crown sections of tomatoes (Figure 2B). BIO62-treated plants had the lowest population density at 5.63 log cfu g−1 fresh plant section (fps). For the CK treatment, the observed population density was 8.08 log cfu g−1 fps at the end of experiment. Identification of Antibiotic Biosynthesis Genes from B. amyloliquefaciens HR62. PCR was used to detect the genes involved in the biosynthesis of the six antibiotics reported to be produced by B. amyloliquefaciens. A total of 10 gene fragments of expected sizes correlated with biological control activities were efficiently amplified from HR62 (Figure S2, Supporting Information). The DNA sequences obtained from these amplifications confirmed the identity of these genes (Table 1). FenBF/FenBR and FNDF/FNDR primers amplified fragments with 99% and 96% homology, respectively, to the gene that encodes fengycin (synonymous with plipastatin) synthetase. The analysis of sequences amplified by the primers ituAF/ituAR, bamB1F/bamB1R, ituCF/ituCR, and ituD2F/ituD2R showed 10710



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Figure 2. Populations of R. solanacearum in the rhizosphere soil (A) and crown section (B) of tomato plants were determined using the plate count method at 1, 7, 14, 21, and 28 days after challenge with R. solanacearum QL-Rs1115. Bars show standard errors of the mean. An asterisk on the top of the bar represents significance (Duncan’s multiple range test, P < 0.05).

Table 1. Genes Showing the Highest Similarity to the Sequenced Products Obtained from PCR Amplification of Biosynthetic Genes primer names

isolate

BacF/BacR ituAF/ituAR bamB1F/bamB1R

B. subtilis 916 B. subtilis MH25 B. subtilis SQR9

ituCF/ituCR ituD2F/ituD2R

B. subtilis B. subtilis SQR9

FNDF/FNDR fenBF/fenBR BACDF1/BACDR1 110F/110R mln1F/mln1R

B. amyloliquefaciens Q-426 B. amyloliquefaciens Lx-11 B. subtilis ME488 B. subtilis B006 B. amyloliquefaciens FZB42

match with proper genes by BlastN

GenBank accession numbers of matching genes

bacillorin synthetase A iturin A operon iturin A synthetase B (ituB) gene iturin A synthetase C malonyl-CoA transacylase (ituD) fengycin synthetase D fengycin synthetase B BacD gene product srfAB gene macrolactin synthesis

e value of BlastN

% identity of corresponding gene by BlastN

FJ194462.1 EU263005.1 JN093026.1

0.0 0.0 0.0

98 99 97

AB050629.1 JN093033.1

0.0 0.0

99 97

JQ271536.1 JN086144.1 EU334357.1 GU062711.1 CP000560.1

4e-108 0.0 0.0 8e-115 0.0

96 99 99 95 98

− OH − H2O]− in negative ion mode (Figure 4D) and 511.2 [M + Na]+ and 367.2 [M − malonic acid − 2H2O + H]+ in positive ion mode (Figure 4C). This fraction was identified as containing 7-O-malonyl macrolactin A, with a molecular weight of 488 Da.16 In addition, five fractions collected from sample HR62 showed antagonism to R. solanacearum QL-Rs1115. These fractions were collected at almost the same retention time as the standard surfactin sample (Figure 3B,C). The collection times were 14.385, 15.563, 17.476, 18.303, and 20.248 min (Figure 3B); the fractions were named HR62-1′, HR62-2′, HR62-3′, HR62-4′, and HR62-5′, respectively. The mass spectra of HR62-1′, HR622′, HR62-3′, HR62-4′, and HR62-5′ exhibited m/z ratios of 516.8, 523.7, 530.9, 530.9, and 537.8, respectively. According to the already available information about surfactin, the molecular weight of surfactin is approximately 1000 Da. We deduced that the sample took two charges after ionization and analyzed the m/ z results as 516.8 [M + K + H]2+, 523.7 [M + K + H]2+, 530.9 [M + K + H]2+, 530.9 [M + K + H]2+, and 537.8 [M + K + H]2+ in positive ion mode; these fractions were identified as surfactin with molecular weights of 994 (C13), 1008 (C14), 1022 (C15), 1022 (C15), and 1036 (C16), respectively. The prominent masses from the HPLC/ESI-MS spectra and the molecular weights are listed in Table 2. The Effect of Temperature, pH, Organic Solvents and Enzymes on Antibacterial Activity. The antibacterial

97−99% identity with regions of genes encoding iturin synthetase or enzymes synthesizing closely related compounds in the iturin family. A sequence with 95% (e value = 8e-115) homology to the surfactin synthetase gene was detected in HR62 by the primer pair 110F/110R. BacF/BacR primers amplified a fragment similar to the Bacillorin operon with 98% homology. The Macrolactin gene cluster was amplified by mLn1F/mLn1R primers with 98% homology. Moreover, the analysis of sequences amplified by BACDF1/BACDR1 primers revealed 99% identity with the bacilysin synthetase gene cluster. Characterization of the Antibacterial Compounds from B. amyloliquefaciens HR62. After the final purification with RP-HPLC, the fractions eluted at 4.740 and 5.808 min (Figure 3A) in condition 1 showed pathogen antagonism; these fractions were recorded as HR62-1 and HR62-2, respectively. To determine the molecular weight and molecular formula of the HPLC-purified antibiotics, the samples were subjected to HPLC analysis coupled with ESI-Q-trap MS. The data from the HPLC/ ESI-MS analyses are shown in Figure 4. The spectra of HR62-1 revealed molecular masses of 401.3 [M − H]− in negative ion mode (Figure 4B) and 425.2 [M + Na]+, 441.2 [M + K]+, 385.2 [M + H − H2O]+, 367.2 [M + 2K − 2H2O]2+, and 349.1 [M + 2K − 3H2O]3+ in positive ion mode (Figure 4A). Therefore, HR62-1 contained macrolactin A, with a molecular weight of 402 Da.16 The spectra of HR62-2 exhibited molecular masses of 435.5 [M 10711



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Figure 3. Reversed-phase HPLC chromatograms of antibiotics produced by B. amyloliquefaciens HR62 by condition 1 (A) and condition 2 [B and C (standard sample)]. Antibacterial activities were found in peaks located at 4.740 and 5.808 min (A) and at 14.385, 15.563, 17.476, 18.303, and 20.248 min (B).

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DISCUSSION Bacterial wilt of tomato was significantly suppressed by both of the treatments tested in the present study. The application of organic fertilizer and antagonists separately (treatment HR62) reduced the incidence of bacterial wilt disease by 53% (Figure 1), in agreement with the established effect of organic amendments and biocontrol agents such as Bacillus on soilborne diseases.32−34 The application of BIO62 conferred a biocontrol efficacy of 65% (Figure 1). The combination of antagonists with a suitable organic fertilizer may provide sufficient nutrients to the antagonists to ensure high inoculum to the plants at the time of treatment, promote the root-colonization capacity of the antagonist, and prevent the invasion of the pathogen.35,36

compounds were stable at different temperatures (40, 50, and 60 °C) (Figure 5A) and at a pH range of 5−7 (Figure 5B). Antibacterial activities were decreased at 100, 115, and 121 °C. In addition, the antibacterial compounds were stable in methanol, ethanol, acetone, and isopropyl alcohol (Figure 5C). The enzymes pepsin (800−2500 U mg−1; P8160, Solarbio), recombinant proteinase K (600−100 U mg−1; 03115828001, Roche Applied Science), albumin fraction V (≥95% protein; A8020, Solarbio), and trypsin from bovine pancreas (≥2500 U mg−1; 64008834, SCRS) were unable to degrade the antibacterial compounds (Figure 5D). 10712



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Figure 4. Mass spectroscopic analysis of antibacterial compounds observed in peaks eluted at 4.740 min (A and B), 5.808 min (C and D), 14.385 min (E), 15.563 min (F), 17.476 min (G), 18.303 min (H), and 20.248 min (I) by reversed-phase HPLC.

Table 2. Active Compound Production by B. amyloliquefaciens HR62 Detected by HPLC/ESI-MS in Positive and (or) Negative Modes m/z name of sample retention time (min) negative HR62-1

4.740

401.3

HR62-2

5.808

435.5

HR62-1′ HR62-2′ HR62-3′ HR62-4′ HR62-5′

14.385 15.563 17.476 18.303 20.248

ND ND ND ND ND

product ions

positive 425.2 441.2 385.2 367.2 349.1 511.2 367.2 516.8 523.7 530.9 530.9 537.8

negative [M − H]−

[M − OH − H2O]− ND ND ND ND ND

positive

mol wt

[M + Na]+ [M + K]+ [M + H − H2O]+ [M + 2K − 2H2O]2+ [M + 2K − 3H2O]3+ [M + Na]+ [M − malonic acid − 2H2O + H]+ [M + K + H]2+ [M + K + H]2+ [M + K + H]2+ [M + K + H]2+ [M + K + H]2+

402

macrolactin A

488

7-O-malonyl macrolactin A

994 1008 1022 1022 1036

compd

C13 surfactin B C14 surfactin B C15 surfactin B C15 surfactin B C16 surfactin B

al.,11 which indicated that the application of bioorganic fertilizers significantly reduced the incidence of bacterial wilt disease both in the greenhouse and in field conditions. Several B. amyloliquefaciens strains have been used as biological control agents of plant diseases.21,38,39 The most convincing antagonism properties are the broad-spectrum antibiotic and antimicrobial compounds synthesized by these microorgan-

Biomass yields of plants treated with HR62 and BIO62 were significantly higher than that of the control (Figure S1, Supporting Information). This result demonstrates that BIO62 not only reduced bacterial wilt of tomato but also promoted plant growth. This was most likely due to the beneficial contribution of the enriched microorganism community in the compost.37 These results were in agreement with the report published by Wei et 10713



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Figure 5. Sensitivities of antibacterial compounds produced by B. amyloliquefaciens HR62 to temperature (A), pH (B), organic solvents (C), and proteinases (D).

isms.20 Antibiotics from Bacillus protect plants either by hampering the pathogens directly or by inducing systemic resistance in the host.40,41 In the present study, B. amyloliquefaciens HR62 showed antagonism against seven soil-borne fungi pathogens (Table S2, Supporting Information). This result suggests that a large range of antibiotics may be secreted by HR62. Furthermore, PCR analysis of the HR62 genome revealed the existence of lipopeptide-synthetic genes encoding for surfactin, fengycin, iturin, and bacillicon; polyketide-synthetic genes coding for macrolactin; and dipeptide-synthetic genes encoding for bacilysin synthetase (Table 1). Therefore, B. amyloliquefaciens HR62 is an effective biocontrol agent. We focused on its antagonism to R. solanacearum, a causative agent of bacterial wilt of tomato, both in vitro and in vivo. In this study, we tried to isolate the antibiotics produced by HR62 to explain another biocontrol mechanism. After purification and characterization using extraction, precipitation, and purification using silica gel columns (Sephadex LH-20, RP-HPLC and LC−MS), two types of polyketides were identified, macrolactin A and 7-Omalonyl macrolactin A. One type of lipopeptide, surfactin B (C13, C14, C15, C15, and C16), was observed and identified according to previous studies.16,19,42 Macrolactin, which consists of a 24-membered lactone ring, was first isolated from a deep-sea bacterium. Macrolactin was used in clinical antibiosis because of its selective antibacterial activities, its cytotoxicity against murine melanoma cancer cells, and its antiviral activities against Herpes simplex virus (HSV) and human immunodeficiency virus (HIV).43,45 7-O-Malonyl macrolactin A (MMA) has the same configuration as its parent

molecule macrolactin A. MMA is a bacteriostatic antibiotic that inhibits a number of multidrug-resistant Gram-positive pathogens, such as B. subtilis, Staphylococcus aureus, and Burkholderia cepacia.44,45 Dunlap et al.46 identified and confirmed the presence of the PKS cluster that encodes the synthesis of macrolactin from antagonist B. amyloliquefaciens AS 43.3, which showed antagonism to Fusarium head blight. In this present study, we observed the inhibition activities of these compounds to the phytopathogen R. solanacearum; this activity was supported by the results published by Yuan et al.16 In the present study, the antibiotic surfactin was also shown to act as an antagonist to R. solanacearum QL-Rs1115. This compound is applied in an extremely wide variety of industrial processes involving emulsification, foaming, detergency, wetting, dispersing, and solubilization. Kim et al.42 reported that surfactin isolated from Bacillus polyfermenticus KJS-2 showed antimicrobial activity to several Gram-positive and Gram-negative bacteria. Zhu et al.47 tested the antagonism of lipopeptides produced by B. amyloliquefaciens XZ-173 to R. solanacearum QL-Rs1115; one of these lipopeptides was surfactin. Bacon et al.48 reported that Bacillus mojavensis produce a considerable amount of surfactin A to inhibit of the maize mycotoxic fungus Fusarium verticillioides. Surfactin resists soil-borne pathogens by stimulating plant growth, increasing the adhesion of biocontrol agents to plant surfaces, inhibiting the formation of biofilms by pathogenic organisms, and activating systemic resistance in a wide range of plants.24,49,50 In this work, the combination of organic fertilizers with B. amyloliquefaciens HR62 significantly reduced the disease 10714



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asperellum strain T-34 suppress Rhizoctonia solani in cucumber seedlings. Biol. Control 2006, 39, 32−38. (10) Luo, J.; Ran, W.; Hu, J.; Yang, X.; Xu, Y.; Shen, Q. Application of bio-organic fertilizer significantly affected fungal diversity of soils. Soil Sci. Soc. Am. J. 2010, 74, 2039−2048. (11) Wei, Z.; Yang, X. M.; Yin, S. X.; Shen, Q. R.; Ran, W.; Xu, Y. C. Efficacy of bacillus-fortified organic fertiliser in controlling bacterial wilt of tomato in the field. Appl. Soil Ecol. 2011, 48, 152−159. (12) Yuan, S. F.; Wang, L. L.; Wu, K.; Shi, J. X.; Wang, M. S.; Yang, X. M.; Shen, Q. R.; Shen, B. Evaluation of bacillus-fortified organic fertilizer for controlling tobacco bacterial wilt in greenhouse and field experiments. Appl. Soil Ecol. 2014, 75, 86−94. (13) Lee, S. C.; Kim, S. H.; Park, I. H.; Chung, S. Y.; Chandra, M. S.; Choi, Y. L. Isolation, purification and characterization of novel fengycin S from Bacillus amyloliquefaciens LSC04 degrading-crude oil. Biotechnol. Bioproc. E 2010, 15, 246−253. (14) Scholz, R.; Molohon, K. J.; Nachtigall, J.; Vater, J.; Markley, A. L.; Süssmuth, R. D.; Mitchell, D. A.; Borriss, R. Plantazolicin, a novel microcin B17/streptolysin S-like natural product from Bacillus amyloliquefaciens FZB42. J. Bacteriol. 2011, 193, 215−224. (15) Wang, X. X.; Huang, L. L.; Kang, Z. S.; Buchenauer, H.; Gao, X. N. Optimization of the fermentation process of Actinomycete strain Hhs.015T. J. Biomed. Biotechnol. 2010, 2010, 141876. (16) Yuan, J.; Li, B.; Zhang, N.; Waseem, R.; Shen, Q. R.; Huang, Q. W. Production of bacillomycin- and macrolactin-type antibiotics by Bacillus amyloliquefaciens NJN-6 for suppressing soilborne plant pathogens. J. Agric. Food Chem. 2012, 60, 2976−2981. (17) Lee, S. C.; Kim, S. H.; Park, I. H.; Chung, S. Y.; Choi, Y. L. Isolation and structural analysis of bamylocin A, novel lipopeptide from Bacillus amyloliquefaciens LP03 having antagonistic and crude oilemulsifying activity. Arch. Microbiol. 2007, 188, 307−312. (18) Yu, G. Y.; Sinclair, J. B.; Hartman, G. L.; Bertagnolli, B. L. Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani. Soil Biol. Biochem. 2002, 34, 955−963. (19) Arguelles-Arias, A.; Ongena, M.; Halimi, B.; Lara, Y.; Brans, A.; Joris, B.; Fickers, P. Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens. Microb. Cell Fact. 2009, 8, 63. (20) Chen, X. H.; Koumoutsi, A.; Scholz, R.; Schneider, K.; Vater, J.; Süssmuth, R.; Piel, J.; Borriss, R. Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens. J. Biotechnol. 2009, 140, 27−37. (21) Kim, P. I.; Chung, K.-C. Production of an antifungal protein for control of Colletotrichum lagenarium by Bacillus amyloliquefaciens MET0908. FEMS Microbiol Lett. 2004, 234, 177−83. (22) Rapp, C.; Jung, G.; Katzer, W.; Loeffler, W. Chlorotetain from Bacillus subtilis, an antifungal dipeptide with an unusual chlorinecontaining amino acid. Angew. Chem. Int. Ed. 1988, 27, 1733−1734. (23) Huang, J. F.; Wei, Z.; Tan, S. Y.; Mei, X. L.; Yin, S. X.; Shen, Q. R.; Xu, Y. C. The rhizosphere soil of diseased tomato plants as a source for novel microorganisms to control bacterial wilt. Appl. Soil Ecol. 2013, 72, 79−84. (24) Jourdan, E.; Henry, G.; Duby, F.; Dommes, J.; Barthélemy, J. P.; Thonart, P.; Ongena, M. Insights into the defense-related events occurring in plant cells following perception of surfactin-type lipopeptide from Bacillus subtilis. Mol. Plant−Microbe Interact. 2009, 22, 456−468. (25) Tans-Kersten, J.; Brown, D.; Allen, C. Swimming motility, a virulence trait of Ralstonia solanacearum, is regulated by FlhDC and the plant host environment. Mol. Plant−Microbe Interact. 2004, 17, 686. (26) Guo, J. H.; Qi, H. Y.; Guo, Y. H.; Ge, H. L.; Gong, L. Y.; Zhang, L. X.; Sun, P. H. Biocontrol of tomato wilt by plant growth-promoting rhizobacteria. Biol. Control 2004, 29, 66−72. (27) French, E. B.; Elphinstone, J. Culture media for Ralstonia solanacearum isolation, identification and maintenance. Fitopatologia 1995, 30, 126−130. (28) Joshi, R.; Gardener, B. B. M. Identification and characterization of novel genetic markers associated with biological control activities in Bacillus subtilis. Phytopathology 2006, 96, 145−54.

incidence of bacterial wilt of tomato. Macrolactin and surfactin analogs were detected in an HR62 culture filtrate. Although these two compounds have been previously reported in the literature, here they were found in one strain that could inhibit R. solanacearum for the first time. Polyketide and lipopeptide production were pursued as another possible mechanism underlying the biocontrol of bacterial wilt disease by HR62. These data provide a theoretical basis for the development of commercial bioorganic fertilizers with different functional biocontrol agents.

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ASSOCIATED CONTENT

* Supporting Information S

Figures S1 and S2 and Tables S1 and S2, as noted in the text. This material is available free of charge via the Internet at http://pubs. acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 25 84396824. Fax: +86 25 84396824. E-mail: ycxu@ njau.edu.cn. Funding

This research was financially supported by the National Natural Science Foundation of China (41301262), the Jiangsu Province Science Foundation for Youths (BK20130677), the Innovative Research Team Development Plan of the Ministry of Education of China (IRT1256), the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and the 111 project (B12009). Notes

The authors declare no competing financial interest.

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Suppression of Bacterial Wilt of Tomato by Bioorganic Fertilizer Made

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