Antagonistic Action of Bacillus subtilis Strain SG6 on Fusarium graminearum

Fusarium graminearum causes Fusarium head blight (FHB), a devastating disease that leads to extensive yield and quality loss of wheat and barley. Bacteria isolated from wheat kernels and plant anthers were screened for antagonistic activity against F. graminearum. Based on its in vitro effectiveness, strain SG6 was selected for characterization and identified as Bacillus subtilis. B. subtilis SG6 exhibited a high antifungal effect on the mycelium growth, sporulation and DON production of F. graminearum with the inhibition rate of 87.9%, 95.6% and 100%, respectively. In order to gain insight into biological control effect in situ, we applied B. subtilis SG6 at anthesis through the soft dough stage of kernel development in field test. It was revealed that B. subtilis SG6 significantly reduced disease incidence (DI), FHB index and DON (P≤0.05). Further, ultrastructural examination shows that B. subtilis SG6 strain induced stripping of F. graminearum hyphal surface by destroying the cellular structure. When hypha cell wall was damaged, the organelles and cytoplasm inside cell would exude, leading to cell death. The antifungal activity of SG6 could be associated with the coproduction of chitinase, fengycins and surfactins.


Introduction
Fusarium graminearum causes Fusarium head blight (FHB), a widespread destructive disease of small grained cereals, resulting in yield loss [1][2][3]. Also FHB causes the reduction of grain quality, by producing a range of toxic metabolites, especially deoxynivalenol (DON) which poses a serious threat to animal health and food safety [4,5].
Though some success in controlling FHB can be expected by plowing fields to remove or bury crop residues infected with F. graminearum after harvest, minimal tillage practices render this method unacceptable [6]. The use of host resistance is an economically and environmentally effective strategy for controlling FHB. Till date, only a few highly resistant wheat cultivars have been identified from different geographic regions, including Asia, South and North America, and Europe [7][8][9][10]. Foliar fungicides applied at anthesis can be useful in reducing scab [8]. Due to the growing cost of chemical pesticides and increasing awareness about their negative effect, the farmers are looking for alternative substitutes for these products to fulfill the consumers demand on pesticide-free food while maintaining environmental safety.
Biological control of F. graminearum has shown promise in previous studies due to their low enviromental impact, and their ability to help reduce growers' dependency on chemicals, thereby slowing the development of fungicide resistance in pathogen populations [11,12]. Several bacteria or fungal strains have been reported to have antagonistic effects against F. graminearum [13]. Among them, Bacillus strains are well-known antibiotic producers, which have advantage over other biocontrol microorganisms due to their inherent property to form endospores and resistance to extreme conditions. The antagonistic effects of Bacillus strains have been shown by in vitro antibiosis [14] and in situ disruption of spikelet infection leading to reduced disease severities [15][16][17][18][19][20], and identifying the lipopeptides [11,21]. Regarding antimicrobial mechanism study, production of antifungal compounds is thought to be the main mode of action by the antagonistic bacteria.
In an attempt to develop biological control of FHB and DON contamination using antagonistic microorganism, we isolated a B. subtilis strain SG6 displaying a strong inhibitory effect on F. graminearum. The objective of the present study was to (1) evaluate inhibitory effect of B. subtilis strain SG6 on F. graminearum mycelial growth, sporulation and DON production; (2) determine the antagonistic efficacy of B. subtilis strain SG6 in controlling FHB in field condition; (3) examine the ultrastructural alterations occurring in hypha cells of F. graminearum during interaction with B. subtilis SG6 by transmission electron microscopy (TEM) and scanning electron microscope (SEM); (4) analyze antifungal peptides to investigate the putative biocontrol mechanism.

Isolation and Screening of Bacteria
Totally 136 isolates were obtained from wheat kernels and plant anthers. Of these, 24 isolates showed a wide range of apparent antagonistic activity against F. graminearum. Notably, isolate SG6 showed the highest apparent antagonistic activity (Table 1), and was selected for further characterization and investigation.

Characterization and Identification of Isolate SG6
The morphological, biochemical and physiological characteristics of strain SG6 were determined. The cells are Grampositive, endospore-forming, aerobic, rods. Oxidase reactions, catalase reactions, Voges-Proskauer test, methyl red reaction and nitrite reduction are positive. It was capable of utilizing citrate and hydrolyzed starch and casein. It was able to grow at 50uC or at pH 5.7. According to 16S rRNA gene sequence analysis, it was found that the closest relatives of strain SG6 were B. subtilis subsp. subtilis NCIB 3610 (99.72%) and B. siamensis KCTC 13613 (99.72%). Based on gyrB gene sequence analysis, strain SG6 displayed the highest sequence similarity (99%) to several Bacillus subtilis strains, such as strain PY79, 6051-HGW and BEST7003. Strain SG6 was finally identified as B. subtilis SG6. The partial 16S rRNA gene and gyrB gene sequences of strain SG6 were submitted to the database of DNA Data Bank of Japan, and the accession numbers are AB858386 and AB909427, respectively.
In vitro Studies on the Effect of B. subtilis SG6 Strain against F. graminearum B. subtilis SG6 showed a high level of antifungal activity. Hyphal growth of F. graminearum was inhibited (Fig. 1). Then the mycelial growth was analyzed with different concentration of B. subtilis SG6 (Table 2). It showed that the mycelium diameter of F. graminearum was significantly decreased with increase in concentration of B. subtilis SG6 in PDA plate, resulting to a gradual increase in inhibition ratio of F. graminearum. The inhibition ratio of F. graminearum could reach the highest as 87.9% at 10 8 CFU ml 21 concentrations of B. subtilis SG6.
Further, inhibition of sporulation in F. graminearum by B. subtilis SG6 strain was significant (Table 3). Stain SG6 at a concentration of 10 4 CFU ml 21 could reduce the spore number of F. graminearum by 83.7% compared with the untreated control. With the increase in concentration of B. subtilis SG6, the inhibition ratio of sporulation gradually increased. No obvious differences in inhibitory effects between different concentrations were found.

Effects of B. subtilis SG6 Strain on Deoxynivalenol Production of F. graminearum
When co-cultured with B. subtilis SG6, growth of F. graminearum D187 was greatly inhibited and ergosterol extracted decreased by 87.18% when compared with the control ( B. subtilis SG6 significantly reduced DI, FHB index and DON in the field trials. Strain SG6 reduced DI by 72.6%, and FHB index by 77.5% compared with the untreated controls. These effects were more pronounced than those of carbendazim, which reduce DI by 8.8% and FHB by 40.2%. Based on the data under field conditions, strain SG6 was more effective than the chemical fungicide Carbendazim widely used in China in reducing DI and FHB index (Table 5). While yield is a widely used parameter for evaluating the efficacy of control of FHB [19], strain SG6 did not show a significant decrease in 100-kernel weight (P.0.05) when compared to the untreated controls. Strain SG6 could reduce DON by 69.1% when compared with the untreated controls. Similar DON reduction effects were found for carbendazim, which reduce DON by 73.2%.

Effects of B. subtilis SG6 Strain on Ultrastructure of F. graminearum
Inhibition of F. graminearum growth in vitro was further complemented by SEM investigations (Fig. 2). Healthy looking hyphae of F. graminearum cultured without B. subtilis SG6 strain were regular in shape and their surfaces were smooth ( Fig. 2A, B and C). Noticeable morphological changes were found in the hyphae of F. graminearum in the presence of the antagonistic bacteria. One of the most striking features was a marked hyphal surface flaking (Fig. 2D, E and F). Strain SG6 induced stripping of hyphae surface, leading to debris accumulation or dispersion.
TEM analysis further elucidated morphological changes of hyphal ultrastructure of F. graminearum induced by B. subtilis SG6.
TEM observation showed that the structure of F. graminearum cell remained intact, the enclosing cell wall was well defined and all cell components arranged in order in the untreated controls. However, most treated F. graminearum had more or less degradation in cell walls (Fig. 3B, D, F, G and H). The organelles and cytoplasms in the hyphae cell were irregular and degenerated even appeared empty holes (Fig. 3F, G and H). These results indicated that B. subtilis SG6 initially break down the cell walls of F. graminearum, leading to release of cell contents. Further, chitinase activity of SG6 was detected on chitin-amended media, and clearance halos around and beneath the growth were observed.

B. subtilis SG6 Strain AMP Genes and AMP Profile Analysis
AMP biosynthetic genes were reported to be related to biocontrol of plant pathogen in several Bacillus [22][23][24]. The presence of five AMP genes markers were checked by PCR (Fig. 4). Amplification of these gene markers showed that each gene had one specific band with the right size.
To further characterize the AMP profiles of strain SG6, lipopeptides mixture was precipitated with 6 N HCl and extracted by methanol, the assignment of lipopeptides was on the basis of molecular weight using ESI-MS/CID. A summary of the accumulated lipopeptides is reported in Table 6. The results show stain SG6 mainly produce surfactins and fengycins. The mass spectra of several typical lipopeptides are shown in Figure 5. The masses of the [M+Na] + molecular ions at m/z 1030.6, 1058.7, 1072.7 and 1086.7 differed by 14 Da, suggesting that they are homologous molecules.   [25,26,27]. Among those, several B. subtilis strains had the potential for biocontrol against F. graminearum [15][16][17][18][19][20]. Understanding the mode of action between B. subtilis and F. graminearum is important for developing B. subtilis as a successful biological control agent.
It seems that inhibition of hyphal growth is the main pattern of inhibition of Bacillus stains against Fusarium pathogens. For example, B. subtilis EU07 strain could inhibit F. oxysporum f. sp. radicis-lycopersici growth by 64% [28]. B. subtilis strains reduced mycelial growth of F. solani by 34.4% [29]. Studies by Chan et al. [14] showed that B. subtilis D1/2 showed the inhibition against F. graminearum, F. subglutinans, and F. verticilliodes with the wider target spectrum. Besides, inhibition of pathogen fungi sporulation could also be observed in some Bacillus strains. Dihazi et al. [30] showed in his study on the 5 th day B. amyloliquefaciens inhibited the sporulation of F. oxysporum to 86%. In our study, B. subtilis SG6 could effectively inhibit both growth and sporulation of F. graminearum.
Selection of antagonists that not only inhibit of pathogen growth and sporulation but also reduce DON production is critical to biocontrol of FHB [31]. More and more studies have considered the importance of reduction in DON production by antagonistic stains. A concurrent selection method for microbial suppression of F. graminearum, Fusarium head blight and deoxynivalenol in wheat was established by He et al. [32]. 9 isolates screened in Argentina were able to reduce the growth of F. graminearum and the production of DON on irradiated wheat grains by 60-100%, and in greenhouse conditions they could significantly reduce DON content in spikes by 32-100% compared to the control treatment [31]. Similar results have been observed in our study. SG6 could significantly reduce DON production in wheat DON assay in lab, but also reduce DON by 69.1% when compared with the untreated control in field test.
B. subtilis strains produce a broad spectrum of antimicrobial compounds, including predominantly peptides as well as a couple of non-peptidic compounds such as polyketides, an aminosugar, and a phospholipid [33]. The antifungal effects might have been due to one or more antifungal compounds produced by this biocontrol agent. Chitin is a common constituent of fungal cell walls [34]. SG6 could induce cell wall degradation of F. graminearum D187 based on the ultrastructural analysis (Fig. 3D). SG6 could produce chitinase on chitin-amended media. It indicates that SG6 could break down cell wall of F. graminearum D187 by producing chitinase. The cell wall of fungi provides both protective and aggressive functions. If removed or weakened, the fungi die unless they are osmotically protected [35]. Secretion of chitinase could be involved in biocontrol of F. graminearum in SG6.
Antimicrobial peptides produced by Bacillus spp. have been implicated in the biocontrol of several plant pathogens [33,36,37]. The presence of AMP biosynthetic genes has been linked to biocontrol of plant pathogens in several Bacillus strains [38][39][40]. Presence of five AMP genes (bmyB, fenD, ituC, srfAA and bacA ) in strain SG6 was checked by PCR. The result of electrophoresis showed that all the five genes exist in SG6. It indicates the presence of the five genes in strain SG6 could be due to the benefit provided by complementary mechanisms of action among the gene products [41].
Lipopeptides profile of strain SG6 had been analyzed. Fengycins and surfactins are the prominent products of strain SG6 when it is cultured in NB for 72 h. Fengycins are cyclic lipodecapeptides which specifically inhibits against filamentous fungi [42]. Fengycins have also been identified as the prominent lipopeptides in other B. subtilis strains acting against F. graminearum [43,44]. Possibly fengycins affect the cell membrane of F. graminearum to alter its permeability, resulting in release of cell contents (Fig. 3G, H). Surfactins could synergistically impact the anti-fungal activity of other lipopeptides [45]. The simultaneous production of fengycins and surfactins would be important for the efficiency of F. graminearum control by strain SG6.
As known, the effectiveness of biological control in the field tests depends on the antagonist dose, the carbon to nitrogen ratio of the antagonist production medium and the wheat cultivar utilized [19]. Further systematic field study is necessary to study under different conditions to compare and evaluate the efficacy of strain SG6 at a larger level.

Ethics Statement
Specific permission was not needed for our field studies. The strains used in our field study were isolated from natural environment. B. subtilis strain SG6 was isolated for anthers of luffa grown in yard in Beijing, while F. graminearum D187 was isolated from Fusarium-infected wheat kernel collected from Hebei Province (the place of our further field test). No transgenic or created mutant microbes have been used in our study. Also we confirm that the field studies did not involve endangered or protected species.

Strains, Culture Media and Conditions
F. graminearum D187, from the culture collection of our lab, was primarily grown on PDA at 28uC under white fluorescent light. Nutrient broth (NB; Disco) was used at 3 ml per 176100 mm tube for preparing bacterial cultures on an incubator shaker at 28uC and 200 rpm. Agar (Aobox, Beijing, China) at 15 g/L was added to solidify the liquid media. Bacteria strains were recovered from storage in 10% glycerol at 280uC by briefly warming the vial at room temperature and streaked onto Nutrient Agar (NA) plate. To obtain a large amount of bacterial culture supernatant for cyclic lipopeptides, the bacterium was grown in 2 L conical flasks each containing 0.5 L of NB. The culture was started with a 1% inoculum and incubated at 28uC and 200 rpm for 48 h. After the cells were separated by centrifugation, the supernatant was filter sterilized by using a 500-mL Stericup TM fitted with a 0.22 mM GP Express membrane (Millipore Corp., Bedford, Mass.).   (Table S1). Anthers of luffa, bean, Chinese Rose and pumpkin were collected in summer of 2011 in Beijing (Table S1). Bacteria from wheat kernels and plant anthers were isolated according to Hartnett et al. and Khan et al. [15,46]. Thereafter, 1 ml of cell suspension was serially diluted. Dilutions were plated onto NA plates.

Morphological and Physiological Properties of Strain SG6
General physiological and biochemical tests were carried out using previously described methods, including Gram-type, morphology, growth properties, catalase and oxidase activities, methyl red reaction, Voges-Proskauer reaction, nitrate reduction and anaerobic growth [47].

Phylogenetic Identification of Strain SG6 based on 16S rRNA Gene and gyrB Gene Sequence Analysis
Genomic DNA of strain SG6 was extracted using the method described previously [48]. Universal primer sets (27F and 1492R; UP1 and UP2r) were used to amplify the 16S rRNA gene and gyrB gene [49,50]. The nucleotide sequences were determined by direct sequencing and compared with available 16S rRNA gene sequences in EZTAXON (http://eztaxon-e.ezbiocloud.net/) and gyrB gene in the GenBank database using the BLAST program [51]. Strain SG6 (CGMCC No. 7621) was registered by the China Committee for Culture Collection of Microorganisms.

Antagonism Assay
Antagonistic effect of isolates was evaluated by a dual-culture assay using relative growth of F. graminearum [14,52]. A 5-mm agar plug cut from an agar culture of D187 was seeded at the center of the PDA plate, and isolates were inoculated at 4 equidistance sites 2.5 cm from the centre. Other plates were inoculated with same size plug of F. graminearum colony in the absence of test stain as the control. All treatments were replicated triplicates and the plates were incubated at 28uC for 5 days. And then the antagonistic effect of test strains on F. graminearum D187 was observed.

Preparations of Bacteria and Pathogen Spore Suspensions
Cells of B. subtilis strain SG6 were obtained by rolling a sterile cotton swab on the 36 to 48 h culture and suspended in isotonic saline water (0.85% NaCl). Then the cell suspension was diluted from 10 9 to l0 5 CFU/ml as a stock suspension with isotonic saline water (0.85% NaCl) [21,52].
F. graminearum D187was grown in a 250 ml flask with 100 ml of CMC medium (1.5 g of CMC, 0.05 g of NH 4 NO 3 ?7H 2 O, 0.1 g of yeast extract and 100 ml of H 2 O ) on a rotary shaker at 200 rev min 21 at 25uC for 3 to 5 days to produce macroconidia [53]. A macroconidia suspension was prepared by filtering the culture through sterile filter paper to remove mycelia. The concentration of the suspension was adjusted to 10 5 conidia/ml.

Effect of B. subtilis Strain SG6 on Mycelial Growth of F. graminearum
PDA (1/9 diluted) medium was combined with B. subtilis SG6 cells at concentrations of 10 8 , 10 7 , 10 6 , 10 5 and 10 4 CFU ml 21 and isotonic saline water (0.85% NaCl) was used as a control in 9:1 proportions. A 5-mm agar plug from an actively growing mycelium of F. graminearum D187 was placed on the center of the test plate [54]. All the plates were incubated for 5 d at 28uC Experiment was performed in triplicate. The diameters of fungal colonies were measured. The inhibition ratios of mycelium growth of F. graminearum D187 were calculated with the following formula [52].
Inhibition ratio (%) = (C 2 E)/C6100%, where C is the diameter of the control colony and E is the diameter of the treatment colonies.
Effect of B. subtilis Strain SG6 on Sporulation of F. graminearum 5-mm agar plugs of F. graminearum D187 were placed on the center of SNA plates that contained 5 concentrations of B. subtilis SG6 cells (10 8 CFU ml 21 , 10 7 CFU ml 21 , 10 6 CFU ml 21 , l0 5 CFU ml 21 and 10 4 CFU ml 21 ) and isotonic saline water (0.85% NaCl) as a control. All treated sets were incubated at 28uC for 21 d. At the 22 th day, 5 ml of isotonic saline water (0.85% NaCl) containing 0.01% (v/v) Tween 80 was added to the plate and the mature spores were harvested with a pipette. The volume of spore suspension was adjusted to 5 ml with 0.01% Tween 80 and dispersed by vortexes, and the spore concentration was determined by a haemocytometer. The inhibition ratios of sporulation of F. graminearum were calculated with the following formula: Inhibition ratio (%) = (C 2 E)/C6100%, where C is the numbers of sporulation of the control groups and E is the numbers of sporulation of the experimental group [52]. All treatments were replicated 4 times.

Effect of B. subtilis Strain SG6 on DON Production of F. graminearum
A 50 g aliquot of healthy wheat kernels was sterilized and inoculated with 1 ml spore suspension (10 5 spores/ml) of F. graminearum D187 and 1ml B. subtilis strain SG6 suspension (10 8 CFU/ml). As control, 1 ml spore suspension (10 5 spores/ml) of F. graminearum D187 and 1 ml NB were inoculated. After incubation at 25uC for 20 days, subsequent DON extraction and analysis was performed as described by Bluhm et al. [55]. Ergosterol levels were used to normalize DON content per fungal mass. All treatments were replicated 3 times.

Hyphal Cell Preparation for Ultrastructural Study
One hundred microliters of spore suspensions of F. graminearum D187 (about 10 5 conidia ml 21 ) were spread uniformly on the surface of each PDA plate contained B. subtilis SG6 cells at the concentration of 10 8 CFU ml 21 and 0.85% NaCl as a control. Plates were placed at 28uC After 5 d, F. graminearum D187 hyphae were harvested for SEM and TEM.

Transmission Electron Microscopy (TEM)
Similar procedure (in section 'Scanning electron microscopy') was used until hyphae dehydrated. After dehydration, the samples  were embedded in Epon812 and ultrafine sections (80 nm) of tissue were prepared at room temperature using an ultramicrotome LeicaUC6 and a glass knife. Once the tissue had been mounted on a copper grid, poststaining was carried out (uranyl acetate for 30 min and lead citrate for 20 min) [52,57]. Samples were kept in a desiccator until examination with a TEM (HITACHI, H-7500, Japan) operated at 80 kV.

Field Disease Management Trials
Field experiment was conducted at Lianjiazhuang Village, Songlindian Town, Zhuozhou, Hebei Province, P. R. China (E39u239, N115u569), in 2012. The FHB intermediately resistant wheat ''Shixin 838'' was used to test the efficacy of B. subtilis SG6 and compared with carbendazim. Experiments were arranged as one-factor factorial design with triplicates. Plants were grown in 10-row plots, 2 m long with 10 cm row spacing. Plots were fertilized based on soil test recommendations. Appropriate herbicides for efficient weed control were applied [58].
At anthesis (Zadoks growth stage 65), wheat spikes were sprayed with 300 ml suspension of strain SG6 at a concentration of 10 8 CFU ml 21 , 0.9 g/L carbendazim, or sterile distilled water immediately before treating heads with 300 ml suspension containing 2610 5 CFU ml 21 conidia of F. graminearum D187. At each application, the suspension was sprayed evenly on to the spikes in each plot using a polyethylene compressed air sprayer (Yuanhua Sprayer Inc., Taizhou, Zhejiang Province, P.R. China). The treatments were applied in late afternoon approximately 2h before sunset [13,58]. At the soft dough stage, plot disease severity for a population of approximately 300 spikes per plot was estimated for both incidence (percentage of infected spikes) and severity (percentage of infected spikelets of the diseased spikes) [16]. An FHB index (incidence6severity/100) was derived to give an assessment of plot disease severity [59]. 100-kernel weight was determined after harvest [13,16].
For each plot, 30 g seed sample was taken and ground to a fine powder and stored in paper bags at room temperature. From each ground sample, a 5 g subsample was used for DON analysis. The concentration of DON was determined according to the method reported by Maragos [60] and Liu [61] with few modifications, and toxin determination was quantified by HPLC/UV.
PCR was carried out in a total volume of 50 ml containing 25 ml 16 Go Tag H Colorless Master Mix (Promega), 0.4 mM of each primer, and 5 ml of genomic DNA. The cycling conditions for the amplification of all targets were as follows: 95uC for 4 min, 40 cycles of 94uC for 1 min, annealing temperature for 1 min, and 70uC for 1 min. A final extension step at 70uC for 10 min was followed by a 4uC soak. The annealing temperature was set to 58uC for fenD, ituC, srfAA and bacA, to 55uC for bmyB [41].
AMP was collected and ESI-MS/CID analysis using methods similar to those previously described [21,62]. Briefly, lipopeptides were precipitated from cell-free supernatants with 6 N HCl, and extracted with dichloromethane. After evaporation, the recovered materials were re-dissolved in methanol. AMP extract was subjected to the analyzed HPLC and ESI-MS/CID spectrometric analysis.

Production of Cell Wall Degrading Enzyme
The qualitative assay for chitinase production was performed according to the method described by Marten et al. [25]. Strain SG6 was inoculated as single streak on the chitin containing medium, the plates were incubated at 28uC and clearance halos around and beneath the growth indicating the enzymatic degradation was observed and measured after 5-10 days.

Statistical Analyses
The mycelia radius and the number of spores were subjected to analysis of variance without transformation. Variance was stabilized using the arcsine square root transformation for DS, IS and FHB index and logarithmic transformation of DON values when needed [63]. Means were separated at P#0.05 using Fisher's protected least significance difference test (SPSS Statistics, ver. 17.0, IBM).