The bacterial strain 5-18, isolated from field soil, showed strong antifungal activity against some plant pathogens in vitro. Based on 16s rRNA and tetB-yyaO / yyaR genes analysis, the strain was identified as Bacillus amyloliquefaciens. In this study, crude lipopeptides were extracted with methanol from the precipitate by adding concentrated HCl to the bacillus-free culture broth. Both the culture filtrate and the crude lipopeptides showed strong growth inhibition activity in vitro against the plant disease phytopathogens Rhizoctonia solani, Fusarium oxysPorium f.sp. cucumerinum, Sclerotinia sclerotiorum. The antifungal substance in the culture filtrate was relatively stable to temperature and pH. They still remained strong antifungal activity after treatment at 100°C for 30 min or at pH values ranging from 2 to 10 for 24 h. The active components were isolated by high-performance liquid chromatography (HPLC). A component of a molecular weight of 1042 Da was identified as iturin A2 after electrospray ionization quadrupole time of flight tandem mass spectrometry analysis (ESI-Q-TOF-MS/MS). This result was confirmed by polymerase chain reaction (PCR) detection of the ituA gene of the iturin operon.
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Key words: B. amyloliquefaciens, antifungal activity, lipopeptide
Plant diseases caused by viruses, bacteria and fungi are responsible for significant losses and decrease the quality of agricultural products (Benitez et al., 2010). For example, Sclerotinia sclerotiorum (Lib.) de Bary is a soil-borne plant pathogen that infects over 400 plant species at all stages of growth, development and harvested products (Abdullah, 2008). Chemical fungicides have been widely used to control those pathogens, but at the same time abuse of chemical pesticides has contributed to the development of resistant pathogens and many of these synthetic chemicals are gradually becoming ineffective (Ge et al., 2004; Kim et al., 2010). Moreover, chemicals pesticides can be lethal to useful insects of the environment and beneficial microorganisms in the rhizosphere, eventually, they may enter the food chain and accumulate in the human body as undesirable chemical residues (Bartlett et al., 2002). Therefore, more effective and safer alternative control methods are necessary to be developed, especially those with novel modes of action with disease resistance-breaking properties.
Biological methods using antagonistic microorganisms or their active metabolites to control plant disease have attracted increasing attention for their rapid bactericidal and/or fungicidal activity over a broad spectrum and the low propensity of microorganisms to develop resistance against them (Benitez et al., 2010). Several species of the Bacillus have been reported to be effective against plant pathfungal (Chen et al., 2012; Zhang et al., 2012). Through competing for nutrients, inducing defensive capacities of the host plant and producing antifungal compounds, they can strongly suppress pathfungal (Arrebola et al., 2009; Hanene et al., 2012). Among these antifungal compounds, lipopeptides such as iturin, surfactin and fengycin families play an important role in suppressing the plant disease. Besides showed strong antifungal activity, they have low toxicity, high biodegradability and are environmentally friendly at the same time. Many strains of Bacillus subtilis and Bacillus amyloliquefaciens have been reported to suppress fungal growth in vitro by the production of several antifungal compounds and their role in biocontrol has been studied (Yu et al., 2002; Velho et al., 2011; Li et al., 2012).
The aim of this study was to screen antagonistic bacteria, which have the strong activity to inhibition of the growth of several commonly phytopathogens. A potential antagonist, Bacillus amyloliquefaciens 5-18 was isolated by an in vitro screening technique and was characterized by 16S rDNA sequence analysis and tetB-yyaO / yyaR genes analysis. It is effective against the growth of several phytopathogenic fungi such as Rhizoctonia solani, Fusarium oxysPorium f.sp. cucumerinum, Sclerotinia sclerotiorum. Furthermore, the antifungal compounds from the culture filtrate of the strain 5-18 were isolated and characterized, and found to be a member of the iturin family.
2.1 Isolation and cultivation of the bacteria
The bacteria isolation from the root zone of the rice plant located in huazhong agriculture university. The principal for screening various Bacillus strains were based mainly on the resistance of their endospores to elevated temperatures (Sadfi et al., 2001). We collected these soil samples and they were treated in a water-bath at 80°C for 10 min so that endospores would be separated from other microorganism (Walker et al., 1998).Then a decimal dilution series was prepared in distilled water and 100ul aliquots were spread onto TSA plates for each dilution. The plates were incubated at 28°C for several days. The distinct single colonies were picked and streaked several times to obtain pure cultures. The strains were maintained on LB plates at 4°C.
2.2 In vitro screening of isolates for antagonism
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Bacillus isolates were screened in vitro against different phyto-pathogenic fungi by applying a dual culture technique in Petri dishes on PDA medium. Bacillus isolates were streaked in a straight line at one side of the Petri dish (3 cm from the center). Simultaneously, a 0.5cm mycelia plug cut from the edge of a five day-old culture of the fungal strain was placed at the center of the plate. After four days at 28°C the inhibition of fungal growth was assessed by measuring the inhibition zone (mm) using the method described by Sadfi-Zouaoui et al. (2008). All in vitro antagonism assays were made in triplicate.
2.3 Molecular identification of the bacterial strains
Total genomic DNA for PCR amplification of 16S rDNA, tetB- yyaO / yyaR sequences were extracted from strain 5-18 according to the described method (Zhao et al., 2010). The specific primers used for PCR amplification 16S rRNA and the sequences between tetB and yyaO / yyaR are show in table 1 respectively. The following cycling conditions were used: Initial denaturation at 94°C for 5 min; 30 cycles of 94°C for 1 min, 58°C for 1 min and 72°C for 40s ; and final extension at 72°C for 10 min. The amplification products were purified from agarose gels using the PCR purification kit, and then sequenced. The sequences were compared with similar sequences retrieved from the DNA databases by using the BLAST search program in the National Center for Biotechnology Information (NCBI) and aligned with BioEdit and Mega 4 software.
2.3 Microorganisms and culture conditions
A loop of 5-18 cell was incubated in a 100 ml Erlenmeyer flask with 10 ml seed medium (1% tryptone, 0.5% yeast extract and 1%NaCl) for 24h at 28°C on a rotary shaker (180r/min), and then 2 ml of culture liquid was added to a 250 ml Erlenmeyer flask containing 100 ml Landy medium medium (20 g glucose, 5 g L-glutamic acid, 0.5 g MgS04, 0.5 g KCl, 1 g KH2P04, 0.15 mg Fe2(SO4)3.6H2O, 5.0 mg MnS04.H20, 0.16 mg CuS04.5H20 and 1000 ml distilled water; final pH is 7.0)(Landy et al. 1948). The culture was incubated for 60 h at 28°C under shaking condition (180 r/min).
The phytopathogenic fungi listed in Table 1 were maintained on potato dextrose agar (PDA) at 4°C ready for use.
2.4 Extraction of crude lipopeptides
After fermentation for 60 h at 28°C, 500 milliliter of the culture broth was centrifuged at 8,000 g for 15 min to remove the pellet. The cell-free culture broth was precipitated by adjusting pH to 2.0 with concentrated HCl and was stored overnight at 4°C. The precipitates were collected by centrifugation at 8,000 g for 20 min and then extracted twice with methanol. The solvent was removed under reduced pressure to give an brown yellow solid. Further purification was achieved by recrystallization. The methane extract was dissolved in distilled water containing sufficient NaOH to produce a pH of 8. This solution was filtered through a 0.45 μm pore size polytetrafluoroethylene membrane (Millipore, USA) and titrated to pH 2 with concentrated HCl. The brown yellow solid was collected as a pellet after centrifugation and stored at -20°C, for further analysis.
2.5 Antifungal activity of strain 5-18
The culture filtrate of strain 5-18 obtained at 60 h after inoculation was evaluated for its in vitro antifungal activity against the phytopathogens. 1 mL of the culture filtrate mixed with 19 mL of the PSA medium was poured into a Petri dish (9 cm in diameter). Once the medium had cooled, discs (5 mmin diameter) of the target fungi, taken from the fresh margin of the mycelium, were spaced equally on the Petri dish, and the dish was incubated at 28°C for two days. The inhibitory activity of the filtrate against fungal growth was recorded as the percentage reduction of mycelia growth in comparison with that of the control plates: Growth inhibition (%) = [(length of mycelia in the negative control plate-length of mycelia in the treated plate) / (length of mycelia in the negative control plate)] -100.
The antifungal activity of crude lipopeptides was tested against six phytopathogenic fungi with mycelial growth inhibition method. 50 μl of lipopeptide (2.0 μg/μl) was spread on PDA plates. Sterile methanol was used to replace lipopeptides as a negative control. Fermentation liquid was used as a positive control. The mycelial plug of each fungus was deposited in the centre of plates. After incubation for three days at 28°C, the diameter of mycelium growth was measured. Growth inhibition effect was evaluated by comparing the percentage of reduction of mycelium expansion with control plates without lipopeptides. The experiment was performed twice (Zhang et al., 2012).
2.6 Effects of pH and temperature on the stability of antifungal metabolites
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In the test for pH stability, samples of the filtered and sterilized culture broth of strain 5-18 were adjusted to various pH values on 2, 4, 6, 7, 8, 10, 12, 14 using 2 M HCl or 2 M NaOH and maintained at 4 °C for 24 h. Antifungal activity was assayed after the samples were readjusted to pH 7.0. A similar procedure was used to assess thermal stability of the antifungal metabolites produced by strain 5-18. The culture filtrate was held at temperatures of room temperature, 60, 80, 100 and 121 °C for 30 min, and was tested for their antifungal activity after being cooled to room temperature.
Antifungal activity of the pH and temperatures treated samples against Rhizoctonia solani, Fusarium oxysPorium f.sp. cucumerinum and Sclerotinia sclerotiorum was determined using the procedure described above. The relative remaining activity was measured by comparison with that of the samples held at pH 7.0 and room temperature. The data are presented as the mean values of the three replications ±SEM (standard error of the mean).
2.7 Purification of antifungal compound
The isolated brown yellow solid was dissolved in 1 mL of methanol followed by passed through a 0.22 um pore filter. The filtrate was subjected to HPLC on a reversed-phase column (RP-18, 5 um, 4.6-250 mm; Agilent). The column was eluted at a flow rate of 1.0 mL/min with acetonitrile-water (40:60, v/v) in the presence of 0.1% trifluoroacetic acid (TFA) and monitored at 214 nm. The peaks were collected manually, evaporated to dryness by speed vacuum concentrator and assayed for their antifungal activity. The eluted fractions with antifungal activity were analyzed by ESI-Q-TOF -MS/MS.
2.8 Structure identification of antifungal compound
Electrospray ionization quadrupole time of flight tandem mass spectrometry analysis was performed on Q-TOF2 instrument (Waters Micromass) to determine the molecular weight and structure of the purified lipopeptide. The electrospray source was operated at a capillary voltage of 32 V, a spray voltage of 5 kV and a capillary temperature of 320°C Positive ionization mode was used.
2.9 PCR analysis
DNA was extracted from overnight cultures of B. amyloliquefacies 5-18 and B. subtilis 168 according to the described method (Zhao et al., 2010). Specific primers for the functional genes of the bacteriocins iturin A and surfactin are listed in Table 2. PCR was conducted under the following parameters: denaturation for 1 min at 94°C, annealing for 1 min at 50°C, and elongation for 1.5 min at 72°C for a total of 30 cycles for iturin A and denaturation for 1 min at 94°C, annealing for 30 sec at 46°C, and elongation for 1 min at 72°C for a total of 25 cycles for surfactin.
In vitro screening of isolates for antagonism
For the selection of a potential antagonist to inhibit the test phytopathogens, over 100 bacterial strains were isolated from the soil and eight of them showed a broad and strong antifungal activity against the test phytopathogens in vitro. Among these eight isolates, strain 5-18 showing the strongest antifungal activity than others through comparing the size of the inhibition zone.
Identification of strain 5-18
Analysis of the gene encoding 16S rDNA, the strain 5-18 was identified as belonging to the genus Bacillus. 16S rDNA sequence analysis revealed that this Bacillus strain had high similarity with B. subtilus and B. amyloliquefaciens. Obviously, it is impossible to classify the strain 5-18 in the species level only dependent on sequence analysis of 16S rRNA gene.
It has been studied that B.amyloliquefaciens is very close to B. subtilis. In order to prove the strain 5-18 belonging to B. amyloliquefaciens, we used two pairs of primers such as tetB_R / yyaR_F (specific for B. amyloliquefaciens) and tetB_R / yyaO_F (specific for B. subtilis) amplification the tetL gene that encode antiporter accomplish with tetB gene in B. subtilis and B. amyloliquefaciens. Results revealed that the chromosome DNA of strain 5-18 could be amplified with tetB_R / yyaR_F primers but not tetB_R/yyaO_F, which proved that the strain 5-18 belong to B. amyloliquefaciens. The 16S rDNA sequence obtained for B. amyloliquefaciens 5-18 (1510 bp) has been submitted to GenBank under the accession number KC480058.
Antifungal activity of strain 5-18
Results in Table 4 showed that the culture filtrate significantly inhibited the growth of the test fungi, especially on Sclerotinia sclerotiorum. To characterize the antifungal compounds in the culture filtrate of strain 5-18, the crude lipopeptides was obtained. The in vitro antifungal activity of the crude lipopeptides revealed significant growth inhibition of the test fungi which was consistent with the culture filtrate.
Stability of the antifungal culture filtrate
Results shown in Fig. 2 demonstrated that the antifungal activity of the culture filtrate of strain 5-18 against Rhizoctonia solani, Fusarium oxysPorium f.sp. cucumerinum, Sclerotinia sclerotiorum was significantly reduced after it had been exposed to basic conditions, but remained almost unchanged when the filtrate was exposed to conditions in the pH range 2-8 (Fig. 2a). The antifungal culture filtrate was relatively thermostable with more than 50% of its activity being retained even after the sample had been held at 100°C for 30 min (Fig. 2b).
Isolation and structure elucidation of the antifungal Compounds
The HPLC analysis appeared five major peaks (Figure 5A). Samples from each peak were condensed and assayed for antifungal activity against Fusarium oxysPorium f.sp. cucumerinum,Only fraction 1 showed antifungal activity and the other four peaks' antifungal activity couldn't be detected. (Figure 5B).
ESI-Q-TOF-MS was chosen to measure the mass of peak 1. The results showed that the predominant ion mass peaks in the positive ion mode were that of m/z 1043.55, 1065.53 and 1081.50 (Figure 6). M/z 1043.55 was (M+H)+ ion peaks. M/z 1065.53 and 1081.50 were (M+Na)+ ion peaks and (M+K)+ ion peaks, respectively. The molecular mass of purified substance was 1 042, which was identical to that of iturin A2 by comparison with reference data (Chen et al., 2008; Yu et al., 2002).
For further identification, the amino acid sequences of m/z 1043.55 were analyzed by ESI-Q-TOF-MS/MS. The result contained some typical b- and y-type fragments (including b2-b7 and y2-y6) (Fig. 5-B) other collision induced dissociation (CID) fragment ions (data not shown). The amino acid sequences were confirmed according to debris ions mass of b type: m/z 916, 802, 638, 524, 299 and 212. The amino acid sequences were Ser, Asn, Pro, Gln, Asn, Tyr and Asn. According to the spectrum, the molecular ion of βAA was b4-b3=225 and the peptide sequences was Pro-Asn-Ser-βAA-Asn-Tyr-Asn-Gln which was consistent with that of iturin A2.
Identification of genes related to antimicrobial peptides
PCR analysis of the B. amyloliquefaciens5-18 showed that the strain exhibited potential for the functional gene encoding iturinA synthetase (ituA) but not for to putative transcription terminator gene (sfp) (Fig. 1). The result revealed that this strain might harbor the gene cluster required for iturinA biosynthesis. The agreement between PCR and HPLC results suggest that the PCR method can be used as a reliable and quick screening tool to isolate iturinA producing Bacillus strains.
There have been reported that microorganism can be beneficial to the host plant directly through the production of bioactive compounds that suppress the growth of phytopathogenic fungi (Van Loon et al. 1998). Meanwhile, the production of bioactive compounds also can be regulated by strain selection, genetic manipulation, metabolic regulation, large-scale fermentation and other techniques (Koji et al., 2010). Moreover, microbe derived pesticides are more easily degradable with lower residue and higher environmental compatibility compared with pesticides (Makkar et al., 2002). In this study, we isolated six strains to suppress the growth of fungal pathogen, among them the strain 5-18 showed the strongest activity against many fungal plant pathogens in vitro.
Bacillus amyloliquefaciens is a closely related species to B. subtilis (Arguelles-Arias et al. 2009). By analysing 16S rRNA gene sequence and tetB-yyaO / yyaR genes, we were able to confirm the strain 5-18 as B. amyloliquefaciens (Reva et al., 2004). To our knowledge, few reports were found to use B. amyloliquefaciens to control sclerotinia stem rot of colza caused by Sclerotinia sclerotiorum. Therefore, the strain is potential for agricultural application.
The potential of some strains of Bacillus spp. to synthesize a wide variety of metabolites with antifungal activity has been described (Yu et al. 2002; Souto et al., 2004; Chen et al., 2008). It can produce anti-microbial active compounds include predominantly peptides that are either ribosomally synthesized and post-translationally modified or non-ribosomally generated (Stein, 2005). Among these antimicrobial compounds, lipopeptides through non-ribosomally generated such as surfactin, fengycin and the members of iturin family (iturin, mycosubtilin, bacilomycin) plays an important role in control of the growth of fungal pathogens (Steller and Vater, 2000). In this study, we extracted crude lipopeptides from culture filtrate of strain 5-18 using HCl precipitation. Both the culture filtrate and crude lipopeptides exhibited a wide-spectrum of antifungal activity. The antifungal activity of the culture filtrate of strain 5-18 is very stable to heat and pH which is an extremely interesting feature in view of its potential use in agro-industries, resistant to the action of many hydrolytic enzymes. Therefore, the crude lipopeptides in the culture filtrate may be a potent candidate in controlling pathfungal of crops. However, the yield of crude lipopeptides is very low. Further research is needed to improve the yield of crude lipopeptides through changing culture condition and medium components. And also the efficiency of in vivo disease control provided by the broth filtrate, the crude extract, and iturin itself obtained from strain 5-18, should be confirmed in field trials.
Chromatographic analysis showed that crude lipopeptides mainly included five compounds. Only one compound (peak 1) showed antifungal activity. The compound was isolated and was identified as iturin A2 based on the molecular weight by ESI-Q-TOF-MS/MS analytical techniques. Iturin A has been reported to be produced by several Bacillus strains and other close phylogenetic relationship of several species of bacteria in the soil and to show strong inhibitory activity against various fungi in vitro (Yu et al. 2002; Cho et al. 2003; Marc and Philippe, 2007; Benitez et al., 2010; Ye et al., 2012; Zhang et al., 2012). Iturins are a group of antifungal, cyclic lipopeptodes, consisting of iturin A-E, bacillomycin D, F and L, and mycosubtilin. There are isomers for each of iturins because their fatty acid chain can be in the n-, iso-, or anteiso-form. Iturin A has a cyclic structure consisting of β-amino fatty acid integrated into a peptide moiety [Asn-Tyr-Asn-Gln-Pro-Asn-Ser]. In nature, iturin A is produced as a mixture of up to eight isomers (Zhang et al., 2012). It has been reported that some of B. subtilis and B. amyloliquefaciens strain could co-produce iturin A, surfactin and fengycin and their anifungal activity is higher than only iturin A production (Arrebola et al., 2010; Chen et al., 2008). However, the co-production of lipopeptides makes it difficult to isolate and purification. In our study B. amyloliquefaciens strain 5-18 only produces iturin A, which makes the purification easier than co-production of lipopeptides. The easy purification reinforces the potential application of this strain. Moreover, besides iturinA, no other antifungal compound was detected, suggesting that these last might play a main part in the biocontrol of the phytopathogenic fungus.
PCR amplification of biosynthetic gene corresponding to iturin A confirms the HPLC and the EST-TOF- MS concerning the iturinA production by B. amyloliquefaciens 5-18. Both peptides are synthesized by the action of large multienzyme complex, and these genes are essential to their respective production (Schneider et al., 1998; Stein, 2005). In fact the detection of a particular antibiotic biosynthetic operon in bacterial strain would signify the function of the operon and the production of the antibiotics (Rajesh et al., 2007).
It has been reported that the biological activity of iturins produced by B. subtilis, depended on the chain length of their fatty acids and the composition of the amino acids in their peptide rings (Peypoux et al., 1978; Quentin et al., 1982). However, about the mechanisms such as direct antagonism or induced systemic resistance employed by these potential biocontrol bacteria needs further research.
In conclusion, a bacteria B. amyloliquefaciens 5-18, isolated from the field soil. The culture filtrate and the crude extract showed strong in vitro inhibition activity against pepper replant disease causing pathogens such as Rhizoctonia solani, Fusarium oxysPorium f.sp. cucumerinum, Sclerotinia sclerotiorum. Further study disclosed that the compound responsible for the antifungal activity in the crude extract of culture filtrate of strain 5-18 was iturin A2. The bacteria B. amyloliquefaciens 5-18 and its bioactive component may provide an alternative resource for the biocontrol of replant diseases
Acknowledgements This work was cofinanced by grants from the Ministry of Education of the People's Republic of China ( ). We are also grateful to jibin Zhang, for providing the pathogen fungi, who is at the National Engineering Research Center of Microbe Pesticides, China; and erning Yang at the State Key Laboraty of Agricultural Microbiology, China, for helpful discussions regarding the ESI-MS/MS techniques.