The bacterial strain 5-18, stray from field dirt, showed strong fungicidal activity against some works pathogens in vitro. Based on 16s rRNA and tetB-yyaO / yyaR cistrons analysis, the strain was identified as Bacillus amyloliquefaciens. In this survey, crude lipopeptides were extracted with methyl alcohol from the precipitate by adding concentrated HCl to the bacillus-free civilization stock. Both the civilization filtrate and the petroleum lipopeptides showed strong growing suppression activity in vitro against the works disease phytopathogens Rhizoctonia solani, Fusarium oxysPorium f.sp. cucumerinum, Sclerotinia sclerotiorum. The fungicidal substance in the civilization filtrate was comparatively stable to temperature and pH. They still remained strong fungicidal activity after intervention at 100 & A ; deg ; C for 30 min or at pH values runing from 2 to 10 for 24 h. The active constituents were isolated by high-performance liquid chromatography ( HPLC ) . A constituent of a molecular weight of 1042 Da was identified as iturin A2 after electrospray ionisation quadrupole clip of flight tandem mass spectroscopy analysis ( ESI-Q-TOF-MS/MS ) . This consequence was confirmed by polymerase concatenation reaction ( PCR ) sensing of the ituA cistron of the iturin operon.
Cardinal words: B. amyloliquefaciens, fungicidal activity, lipopeptide
Plant diseases caused by viruses, bacteriums and Fungis are responsible for important losingss and diminish the quality of agricultural merchandises ( Benitez et al. , 2010 ) . For illustration, Sclerotinia sclerotiorum ( Lib. ) de Bary is a soil-borne works pathogen that infects over 400 works species at all phases of growing, development and harvested merchandises ( Abdullah, 2008 ) . Chemical antifungals have been widely used to command those pathogens, but at the same clip maltreatment of chemical pesticides has contributed to the development of immune pathogens and many of these man-made chemicals are bit by bit going uneffective ( Ge et al. , 2004 ; Kim et al. , 2010 ) . Furthermore, chemicals pesticides can be deadly to utile insects of the environment and good micro-organisms in the rhizosphere, finally, they may come in the nutrient concatenation and accumulate in the human organic structure as unwanted chemical residues ( Bartlett et al. , 2002 ) . Therefore, more effectual and safer alternate control methods are necessary to be developed, particularly those with fresh manners of action with disease resistance-breaking belongingss.
Biological methods utilizing counter micro-organisms or their active metabolites to command works disease have attracted increasing attending for their rapid bactericidal and/or antifungal activity over a wide spectrum and the low leaning of micro-organisms to develop opposition against them ( Benitez et al. , 2010 ) . Several species of the Bacillus have been reported to be effectual against works pathfungal ( Chen et al. , 2012 ; Zhang et al. , 2012 ) . Through viing for foods, bring oning defensive capacities of the host works and bring forthing fungicidal compounds, they can strongly stamp down pathfungal ( Arrebola et al. , 2009 ; Hanene et al. , 2012 ) . Among these fungicidal compounds, lipopeptides such as iturin, surfactin and fengycin households play an of import function in stamp downing the works disease. Besides showed strong fungicidal activity, they have low toxicity, high biodegradability and are environmentally friendly at the same clip. Many strains of Bacillus subtilis and Bacillus amyloliquefaciens have been reported to stamp down fungous growing in vitro by the production of several fungicidal compounds and their function in biocontrol has been studied ( Yu et al. , 2002 ; Velho et al. , 2011 ; Li et al. , 2012 ) .
The purpose of this survey was to screen counter bacteriums, which have the strong activity to suppression of the growing of several normally phytopathogens. A possible adversary, Bacillus amyloliquefaciens 5-18 was isolated by an in vitro showing technique and was characterized by 16S rDNA sequence analysis and tetB-yyaO / yyaR cistrons analysis. It is effectual against the growing of several phytopathogenic Fungis such as Rhizoctonia solani, Fusarium oxysPorium f.sp. cucumerinum, Sclerotinia sclerotiorum. Furthermore, the fungicidal compounds from the civilization filtrate of the strain 5-18 were isolated and characterized, and found to be a member of the iturin household.
2 Methods
2.1 Isolation and cultivation of the bacteriums
The bacteriums isolation from the root zone of the rice works located in huazhong agribusiness university. The principal for testing assorted Bacillus strains were based chiefly on the opposition of their endospores to elevated temperatures ( Sadfi et al. , 2001 ) . We collected these dirt samples and they were treated in a water-bath at 80 & A ; deg ; C for 10 min so that endospores would be separated from other micro-organism ( Walker et al. , 1998 ) .Then a denary dilution series was prepared in distilled H2O and 100ul aliquots were spread onto TSA home bases for each dilution. The home bases were incubated at 28 & A ; deg ; C for several yearss. The distinguishable individual settlements were picked and streaked several times to obtain pure civilizations. The strains were maintained on LB home bases at 4 & A ; deg ; C.
2.2 In vitro showing of isolates for hostility
Bacillus isolates were screened in vitro against different phyto-pathogenic Fungi by using a double civilization technique in Petri dishes on PDA medium. Bacillus isolates were streaked in a consecutive line at one side of the Petri dish ( 3 centimeter from the centre ) . Simultaneously, a 0.5cm mycelia stopper cut from the border of a five day-old civilization of the fungous strain was placed at the centre of the home base. After four yearss at 28 & A ; deg ; C the suppression of fungous growing was assessed by mensurating the suppression zone ( millimeter ) utilizing the method described by Sadfi-Zouaoui et Al. ( 2008 ) . All in vitro hostility checks were made in triplicate.
2.3 Molecular designation of the bacterial strains
Entire genomic Deoxyribonucleic acid for PCR elaboration of 16S rDNA, tetB- yyaO / yyaR sequences were extracted from strain 5-18 harmonizing to the described method ( Zhao et al. , 2010 ) . The specific primers used for PCR elaboration 16S rRNA and the sequences between tetB and yyaO / yyaR are show in table 1 severally. The following cycling conditions were used: Initial denaturation at 94 & A ; deg ; C for 5 min ; 30 rhythms of 94 & A ; deg ; C for 1 min, 58 & A ; deg ; C for 1 min and 72 & A ; deg ; C for 40s ; and concluding extension at 72 & A ; deg ; C for 10 min. The elaboration merchandises were purified from agarose gels utilizing the PCR purification kit, and so sequenced. The sequences were compared with similar sequences retrieved from the Deoxyribonucleic acid databases by utilizing the BLAST hunt plan in the National Center for Biotechnology Information ( NCBI ) and aligned with BioEdit and Mega 4 package.
2.3 Microorganisms and civilization conditions
A cringle of 5-18 cell was incubated in a 100 milliliter Erlenmeyer flask with 10 milliliters seed medium ( 1 % tryptone, 0.5 % yeast infusion and 1 % NaCl ) for 24h at 28 & A ; deg ; C on a rotary shaker ( 180r/min ) , and so 2 milliliter of civilization liquid was added to a 250 milliliter Erlenmeyer flask incorporating 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 milligram Fe2 ( SO4 ) 3.6H2O, 5.0 milligram MnS04.H20, 0.16 milligram CuS04.5H20 and 1000 milliliter distilled H2O ; concluding pH is 7.0 ) ( Landy et al. 1948 ) . The civilization was incubated for 60 H at 28 & A ; deg ; C under agitating status ( 180 r/min ) .
The phytopathogenic Fungi listed in Table 1 were maintained on murphy dextroglucose agar ( PDA ) at 4 & A ; deg ; C ready for usage.
2.4 Extraction of petroleum lipopeptides
After agitation for 60 H at 28 & A ; deg ; C, 500 millilitre of the civilization stock was centrifuged at 8,000 g for 15 min to take the pellet. The cell-free civilization stock was precipitated by seting pH to 2.0 with concentrated HCl and was stored overnight at 4 & A ; deg ; C. The precipitates were collected by centrifugation at 8,000 g for 20 min and so extracted twice with methyl alcohol. The dissolver was removed under decreased force per unit area to give an brown xanthous solid. Further purification was achieved by recrystallization. The methane infusion was dissolved in distilled H2O incorporating sufficient NaOH to bring forth a pH of 8. This solution was filtered through a 0.45 ?m pore size Teflon membrane ( Millipore, USA ) and titrated to pH 2 with concentrated HCl. The brown xanthous solid was collected as a pellet after centrifugation and stored at -20 & A ; deg ; C, for farther analysis.
2.5 Antifungal activity of strain 5-18
The civilization filtrate of strain 5-18 obtained at 60 H after vaccination was evaluated for its in vitro fungicidal activity against the phytopathogens. 1 milliliter of the civilization filtrate mixed with 19 milliliter of the PSA medium was poured into a Petri dish ( 9 centimeter in diameter ) . Once the medium had cooled, phonograph record ( 5 mmin diameter ) of the mark Fungi, taken from the fresh border of the mycelium, were spaced every bit on the Petri dish, and the dish was incubated at 28 & A ; deg ; C for two yearss. The repressive activity of the filtrate against fungous growing was recorded as the per centum decrease of mycelia growing in comparing with that of the control plates: Growth suppression ( % ) = [ ( length of mycelia in the negative control plate-length of mycelia in the treated home base ) / ( length of mycelia in the negative control home base ) ] -100.
The fungicidal activity of petroleum lipopeptides was tested against six phytopathogenic Fungis with mycelial growing suppression method. 50 ?l of lipopeptide ( 2.0 ?g/?l ) was spread on PDA home bases. Sterile methyl alcohol was used to replace lipopeptides as a negative control. Fermentation liquid was used as a positive control. The mycelial stopper of each fungus was deposited in the Centre of home bases. After incubation for three yearss at 28 & A ; deg ; C, the diameter of mycelium growing was measured. Growth suppression consequence was evaluated by comparing the per centum of decrease of mycelium enlargement with control home bases without lipopeptides. The experiment was performed twice ( Zhang et al. , 2012 ) .
2.6 Effectss of pH and temperature on the stableness of fungicidal metabolites
In the trial for pH stableness, samples of the filtered and sterilised civilization stock of strain 5-18 were adjusted to assorted pH values on 2, 4, 6, 7, 8, 10, 12, 14 utilizing 2 M HCl or 2 M NaOH and maintained at 4 & A ; deg ; C for 24 h. Antifungal activity was assayed after the samples were readjusted to pH 7.0. A similar process was used to measure thermic stableness of the fungicidal metabolites produced by strain 5-18. The civilization filtrate was held at temperatures of room temperature, 60, 80, 100 and 121 & A ; deg ; C for 30 min, and was tested for their fungicidal 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 utilizing the process described above. The comparative remaining activity was measured by comparing with that of the samples held at pH 7.0 and room temperature. The informations are presented as the average values of the three reproductions ±SEM ( standard mistake of the mean ) .
2.7 Purification of fungicidal compound
The stray brown xanthous solid was dissolved in 1 milliliter of methyl alcohol 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 millimeter ; 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 nanometer. The extremums were collected manually, evaporated to dryness by velocity vacuity concentrator and assayed for their fungicidal activity. The eluted fractions with fungicidal activity were analyzed by ESI-Q-TOF -MS/MS.
2.8 Structure designation of fungicidal compound
Electrospray ionisation quadrupole clip of flight tandem mass spectroscopy analysis was performed on Q-TOF2 instrument ( Waters Micromass ) to find the molecular weight and construction of the purified lipopeptide. The electrospray beginning was operated at a capillary electromotive force of 32 V, a spray electromotive force of 5 kilovolts and a capillary temperature of 320 & A ; deg ; C Positive ionisation manner was used.
2.9 PCR analysis
Deoxyribonucleic acid was extracted from nightlong civilizations of B. amyloliquefacies 5-18 and B. subtilis 168 harmonizing to the described method ( Zhao et al. , 2010 ) . Specific primers for the functional cistrons of the bacteriocins iturin A and surfactin are listed in Table 2. PCR was conducted under the undermentioned parametric quantities: denaturation for 1 min at 94 & A ; deg ; C, tempering for 1 min at 50 & A ; deg ; C, and elongation for 1.5 min at 72 & A ; deg ; C for a sum of 30 rhythms for iturin A and denaturation for 1 min at 94 & A ; deg ; C, tempering for 30 sec at 46 & A ; deg ; C, and elongation for 1 min at 72 & A ; deg ; C for a sum of 25 rhythms for surfactin.
Consequences
In vitro showing of isolates for hostility
For the choice of a possible adversary to suppress the trial phytopathogens, over 100 bacterial strains were isolated from the dirt and eight of them showed a wide and strong fungicidal activity against the trial phytopathogens in vitro. Among these eight isolates, strain 5-18 demoing the strongest fungicide activity than others through comparing the size of the suppression zone.
Designation of strain 5-18
Analysis of the cistron 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 sort the strain 5-18 in the species level merely dependent on sequence analysis of 16S rRNA cistron.
It has been studied that B.amyloliquefaciens is really close to B. subtilis. In order to turn out the strain 5-18 belonging to B. amyloliquefaciens, we used two braces of primers such as tetB_R / yyaR_F ( specific for B. amyloliquefaciens ) and tetB_R / yyaO_F ( specific for B. subtilis ) amplification the tetL cistron that encode antiporter accomplish with tetB cistron in B. subtilis and B. amyloliquefaciens. Consequences revealed that the chromosome DNA of strain 5-18 could be amplified with tetB_R / yyaR_F primers but non 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 figure KC480058.
Antifungal activity of strain 5-18
Consequences in Table 4 showed that the civilization filtrate significantly inhibited the growing of the trial Fungi, particularly on Sclerotinia sclerotiorum. To qualify the fungicidal compounds in the civilization filtrate of strain 5-18, the petroleum lipopeptides was obtained. The in vitro fungicidal activity of the petroleum lipopeptides revealed important growing suppression of the trial Fungi which was consistent with the civilization filtrate.
Stability of the fungicidal civilization filtrate
Consequences shown in Fig. 2 demonstrated that the fungicidal activity of the civilization 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 about unchanged when the filtrate was exposed to conditions in the pH scope 2-8 ( Fig. 2a ) . The fungicidal civilization filtrate was comparatively thermostable with more than 50 % of its activity being retained even after the sample had been held at 100 & A ; deg ; C for 30 min ( Fig. 2b ) .
Isolation and construction elucidation of the fungicidal Compounds
The HPLC analysis appeared five major extremums ( Figure 5A ) . Samples from each extremum were condensed and assayed for fungicidal activity against Fusarium oxysPorium f.sp. cucumerinum, Merely fraction 1 showed fungicidal activity and the other four extremums ‘ fungicidal activity could n’t be detected. ( Figure 5B ) .
ESI-Q-TOF-MS was chosen to mensurate the mass of extremum 1. The consequences showed that the prevailing ion mass extremums in the positive ion manner were that of m/z 1043.55, 1065.53 and 1081.50 ( Figure 6 ) . M/z 1043.55 was ( M+H ) + ion extremums. M/z 1065.53 and 1081.50 were ( M+Na ) + ion extremums and ( M+K ) + ion extremums, severally. The molecular mass of purified substance was 1 042, which was indistinguishable to that of iturin A2 by comparing with mention informations ( Chen et al. , 2008 ; Yu et al. , 2002 ) .
For farther designation, the amino acerb sequences of m/z 1043.55 were analyzed by ESI-Q-TOF-MS/MS. The consequence contained some typical b- and y-type fragments ( including b2-b7 and y2-y6 ) ( Fig. 5-B ) other hit induced dissociation ( CID ) fragment ions ( informations non shown ) . The amino acid sequences were confirmed harmonizing 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. Harmonizing to the spectrum, the molecular ion of ?AA was b4-b3=225 and the peptide sequences was Pro-Asn-Ser-?AA-Asn-Tyr-Asn-Gln which was consistent with that of iturin A2.
Designation of cistrons related to antimicrobic peptides
PCR analysis of the B. amyloliquefaciens5-18 showed that the strain exhibited possible for the functional cistron encoding iturinA synthetase ( ituA ) but non for to putative written text eradicator cistron ( sfp ) ( Fig. 1 ) . The consequence revealed that this strain might harbour the cistron bunch required for iturinA biogenesis. The understanding between PCR and HPLC consequences suggest that the PCR method can be used as a dependable and speedy testing tool to insulate iturinA bring forthing Bacillus strains.
Discussion
There have been reported that micro-organism can be good to the host works straight through the production of bioactive compounds that suppress the growing of phytopathogenic Fungis ( Van Loon et Al. 1998 ) . Meanwhile, the production of bioactive compounds besides can be regulated by strain choice, familial use, metabolic ordinance, large-scale agitation and other techniques ( Koji et al. , 2010 ) . Furthermore, bug derived pesticides are more easy degradable with lower residue and higher environmental compatibility compared with pesticides ( Makkar et al. , 2002 ) . In this survey, we isolated six strains to stamp down the growing of fungous pathogen, among them the strain 5-18 showed the strongest activity against many fungous works pathogens in vitro.
Bacillus amyloliquefaciens is a closely related species to B. subtilis ( Arguelles-Arias et al. 2009 ) . By analyzing 16S rRNA cistron sequence and tetB-yyaO / yyaR cistrons, we were able to corroborate the strain 5-18 as B. amyloliquefaciens ( Reva et al. , 2004 ) . To our cognition, few studies were found to utilize B. amyloliquefaciens to command sclerotinia root putrefaction of rape caused by Sclerotinia sclerotiorum. Therefore, the strain is possible for agricultural application.
The potency of some strains of Bacillus spp. to synthesise a broad assortment of metabolites with fungicidal activity has been described ( Yu et al. 2002 ; Souto et al. , 2004 ; Chen et al. , 2008 ) . It can bring forth anti-microbial active compounds include preponderantly peptides that are either ribosomally synthesized and post-translationally modified or non-ribosomally generated ( Stein, 2005 ) . Among these antimicrobic compounds, lipopeptides through non-ribosomally generated such as surfactin, fengycin and the members of iturin household ( iturin, mycosubtilin, bacilomycin ) plays an of import function in control of the growing of fungous pathogens ( Steller and Vater, 2000 ) . In this survey, we extracted rough lipopeptides from civilization filtrate of strain 5-18 utilizing HCl precipitation. Both the civilization filtrate and petroleum lipopeptides exhibited a wide-spectrum of fungicidal activity. The fungicidal activity of the civilization filtrate of strain 5-18 is really stable to heat and pH which is an highly interesting characteristic in position of its possible usage in agro-industries, resistant to the action of many hydrolytic enzymes. Therefore, the petroleum lipopeptides in the civilization filtrate may be a powerful campaigner in commanding pathfungal of harvests. However, the output of petroleum lipopeptides is really low. Further research is needed to better the output of petroleum lipopeptides through altering civilization status and medium constituents. And besides the efficiency of in vivo disease control provided by the stock filtrate, the petroleum infusion, and iturin itself obtained from strain 5-18, should be confirmed in field tests.
Chromatographic analysis showed that petroleum lipopeptides chiefly included five compounds. Merely one compound ( peak 1 ) showed fungicidal 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 phyletic relationship of several species of bacteriums in the dirt and to demo strong repressive activity against assorted Fungis 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 fungicide, cyclic lipopeptodes, dwelling of iturin A-E, bacillomycin D, F and L, and mycosubtilin. There are isomers for each of iturins because their fatty acid concatenation can be in the n- , iso- , or anteiso-form. Iturin A has a cyclic construction dwelling of ?-amino fatty acid integrated into a peptide mediety [ 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 merely iturin A production ( Arrebola et al. , 2010 ; Chen et al. , 2008 ) . However, the co-production of lipopeptides makes it hard to insulate and purification. In our survey B. amyloliquefaciens strive 5-18 merely produces iturin A, which makes the purification easier than co-production of lipopeptides. The easy purification reinforces the possible application of this strain. Furthermore, besides iturinA, no other fungicidal compound was detected, proposing that these last might play a chief portion in the biocontrol of the phytopathogenic fungus.
PCR elaboration of biosynthetic cistron matching to iturin A confirms the HPLC and the EST-TOF- MS refering the iturinA production by B. amyloliquefaciens 5-18. Both peptides are synthesized by the action of big multienzyme composite, and these cistrons are indispensable to their several production ( Schneider et al. , 1998 ; Stein, 2005 ) . In fact the sensing of a peculiar antibiotic biosynthetic operon in bacterial strain would mean the map 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 concatenation length of their fatty acids and the composing of the amino acids in their peptide rings ( Peypoux et al. , 1978 ; Quentin et al. , 1982 ) . However, about the mechanisms such as direct hostility or induced systemic opposition employed by these possible biocontrol bacterium needs farther research.
In decision, a bacterium B. amyloliquefaciens 5-18, isolated from the field dirt. The civilization filtrate and the petroleum infusion showed strong in vitro suppression activity against Piper nigrum replant disease doing pathogens such as Rhizoctonia solani, Fusarium oxysPorium f.sp. cucumerinum, Sclerotinia sclerotiorum. Further survey disclosed that the compound responsible for the fungicidal activity in the petroleum infusion of civilization filtrate of strain 5-18 was iturin A2. The bacterium B. amyloliquefaciens 5-18 and its bioactive constituent may supply an alternate resource for the biocontrol of replant diseases
Recognitions This work was cofinanced by grants from the Ministry of Education of the People ‘s Republic of China ( ) . We are besides thankful to jibin Zhang, for supplying 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 treatments sing the ESI-MS/MS techniques.
