Identification and Evaluation of Strain B37 of Bacillus subtilis Antagonistic to Sapstain Fungi on Poplar Wood

Devaluation of poplar products by sapstain accounts for huge and unpredictable losses each year in China. We had isolated four poplar sapstain fungi, Ceratocystis adiposa Hz91, Lasiodiplodia theobromae YM0737, L. theobromae Fx46, and Fusarium sp. YM05, from five poplar varieties and 13 antagonistic bacteria from nine diverse varieties. After being experimented with agar plates, wood chips, and enzyme activities, strain B37 was identified as the best poplar sapstain biocontrol bacterium. The strain B37 was identified as Bacillus subtilis using sequences of the 16S rRNA gene, physiological biochemical, and morphological characteristics.

Sapstain and mold growth on lumber were the serious problems in the wood process industry [7]. Biodiscoloration caused by molds in the initial stage of wood decay was one of the most serious discolorations. It caused serious damage to wood, which not only existed on the surface of the wood, but also penetrated wood into its deep interior [8,9]. Chapman and Scheffer [10] proved that blue stain fungi (Ceratostomella pilifera (Fr.) G. Winter, C. pini Münch, C. ips Rumbold, Graphium rigidum (Pers.) Sacc.) could discolor Pinus echinata Mill., P. ponderosa Dougl. ex Laws., and P. taeda L. The toughness reduced from 9% to 75%; specific gravity losses reached from 1.4% to 3.8%, respectively. The discoloration was objectionable to buyers and highly detrimental to the pulp and paper industry.
Because toxic chemicals may cause secondary pollution in the environment and accumulation in animal tissues, many researchers used various biological methods to inhibit  the growth of sapstain [22].  [23,24]. Morin et al. [25] discovered that a spontaneous albino strain, Ceratocystis resinifera Kasper, has the ability to prevent discoloration of spruce sapwood caused by wild-type sapstain fungi. Some genera of bacteria (i.e., Bacillus, Pseudomonas, Lactobacillus, and Streptomyces) also have been investigated in laboratory experiments or field trails to control sapstain fungi [24,[26][27][28][29][30][31][32]. Forty-five fungal isolates were tested for the sensitivity to Bacillus subtilis C186.
Only Phialophora richardsiae 616E appeared unaffected by the Bacillus [30]. The results showed that Bacillus subtilis C186 had a wide variety of inhibitory effects on sapstain fungi. However, the report about biocontrol of sapstain fungi in poplar is seldom. The objectives of our study were to screen bacterial strains for their antagonistic activity against the discoloration of poplar wood and to compare the efficiency of different bacteria in preventing sapstain under the same conditions. from P. deltoides cv. "harvard" × P. deltoides cv. "Lux" cv. "Nanlin351" at Jurong City, were isolated by the Center of Biotechnology in Jiangsu Polytechnic College of Agriculture and Forestry, Jiangsu Province, China.

Screening for Bacteria for Biocontrol against Poplar
Discoloration Fungi. The method used to select and identify bacteria for biocontrol against poplar discoloration fungi was shown in Figure 1.

Screening for Antifungal
In Vitro Activity. The method of screening for antifungal in vitro activity was shown in Figure 2. Four species of sapstain fungi were cultivated with PDA plates for 5 days at 28 ± 1 ∘ C and cut into 0.8-cm diameter plugs using a cork borer, respectively. Each plug was  inoculated to the center of PDA plates. Different bacterial strains were dibbled at four corners 2 centimeters from the center. All treatments were conducted in quintuplicate. Different fungi (Hz91, YM05, YM0737, and Fx46) were used for control treatment. The cultures were incubated for 48 hours in dark at 28 ± 1 ∘ C and RH 70% in a PRC-250A intelligent artificial climate incubator. The radius of colony (R1) and width of bacteriostatic band (R2) were measured. The ratio of R2 to R1 represented bacteriostatic intensity.

Biocontrol Experiment of Chip.
Thin chips (5 × 2 × 0.6 cm in size) of Populus × euramericana (Dode) Guineir cv. "San Martino" (I-72/58) were sterilized for 30 min at 121 ∘ C. LB liquid cultures of 13 bacteria were cultured for 60 hours at 120 rpm/min and 28 ± 1 ∘ C. The chips were dipped in bacterial cultures for 1 min, removed, and then air dried. The chip for control experiment was dipped in LB liquid medium for 1 min. Plugs of 0.8 cm diameter of four species of sapstain fungi that were grown on PDA plates for 5 days were used to inoculate the center of chips, respectively. Sterilized chips were put on a piece of filter paper over vermiculite in Petri dishes. The treated chips were cultivated separately for 4, 8, and 12 days at 28 ± 1 ∘ C for observing results.

Screening for Enzyme Activity.
Thirteen bacteria were inoculated on casein, chitin, or beta-glucan extract agar in Petri dishes according to the method of Cota et al. [33]. All treatments were conducted in quintuplicate and all plates were incubated at 28 ± 1 ∘ C. The color change of the medium was observed after 2 days. The width of color change band represents protease, chitinase, and beta-glucanase activity.

Detailed Identification for Candidate of the Biocontrol
Agents. For identification for candidates of the biocontrol agents, the methods of morphological, physiological, and biochemical characteristics as well as molecular biological technology were used. The detailed procedure and methods of physiological and biochemical tests of the candidate were according to the Manual of Systematic and Determinative Bacteriology [34] and Bergey's Manual of Determinative Bacteriology, 9th edition [35]. Gram-stain of the candidate was observed under an optical compound microscope ZEISS Imager A1 (Carl Zeiss, Jena, Germany) ( = 30). The morphology of cells was examined by transmission electron microscopy (Hitachi, Tokyo, Japan). Bacterial cells cultured for 10 hours for electron microscopy were picked up using an inoculating loop and dipped in a drop of water to make suspension. The suspension was transferred to carbon-coated copper grids with a capillary pipet. After a short time, excess liquid was absorbed with a piece of filter paper from the edge of the copper grids. Cells were negatively stained with 2% phosphotungstic acid for 90 seconds. A piece of filter paper was used to absorb extra dye. The sample was air dried and examined.

PCR Amplification and
Sequencing of 16s rRNA. Total DNA was extracted and purified according to the procedure described by Devereux and Willis [36]. Agarose 4 The Scientific World Journal (0.7%) gel electrophoresis was used to verify DNA quality.
The following universal primers were used to amplify the nearly complete 16s rRNA. The forward primer was 5 -AGAGTTTGATCCTGGCTCAG-3 , and the reverse primer was 5 -GCCTTGTACACACCGCCC-3 [37]. The total volume ( Table 3. The strains of B4, B9, B33, B123, and B1 had no antagonistic effect on the 4 species of poplar sapstain fungi. The strains of B37 and B82 expressed antagonistic effect. FX46, YM05, and Hz91 were inhibited for 12 days by B37, whereas YM0737 was inhibited only for 8 days. The strain of B82 had antagonistic effect on FX46 and YM05 for 12 days, but on Hz91 for 8 days and on YM0737 for 4 days.

Results and Analysis
The enzymes activity of 13 bacteria was showed in Table 3. The results showed that all 13 bacteria did not produce chitinase. Strain B37 was the best bacterium to produceglucanase; the width of color change band is 4.25 cm. There was only a little amount of -glucanase produced by strains B33 and B13; the width of color change band is 0.29 cm and 0.38 cm, respectively. Strains B82, B8, and B9 showed a better activity to produce protease than those of strains of B47, B27, B4, B30, and B37. The width of color change band is 4.13 cm by strain B82. Other bacteria did not produce protease. Analysis of enzymes activity showed a significant difference ( < 0.05) between strain B82 and strain B37.

Morphological Characteristics of Strain B37. Strain B37
was Gram-positive rod-shaped with blunt ends (Figure 3(a)). On LB medium, colony was larger, milky white, of 3∼5 mm diameter, after training 24 hours, with the raised surface being dry and wrinkled. The size was 1.83∼2.41 × 0.56-1.02 m, with an average of 2.1 × 0.74 m with peritrichous flagella (Figure 3(b)). Endospore was located in the middle of the cell and the size was 0.62∼1.08 × 0.44∼0.66 m with an average of 0.86 × 0.51 m.

Physiological and Biochemical Characteristics of Strain
B37. According to the Manual of Systematic and Determinative Bacteriology [34] and Bergey's Manual of Determinative Bacteriology, 9th edition [35], results of tests on physiological and biochemical characteristics of strain B37 identified strain B37 as Bacillus subtilis (Ehreberg) Cohn (Table 4). The results showed that treatment of indole reaction, gas production, and lactose have no effect, and the other 18 treatments have positive effect.

The 16S rRNA of Strain B37 Sequencing and Phylogenetic
Tree. The results of the 16S rRNA of strain B37 sequencing and aligning with published sequences from the GenBank database demonstrated that strain B37 had a 99.3% similarity to Bacillus subtilis NCIB 3610 T (GenBank accession number ABQL01000001) (CP000560) (Figure 4). According to morphology, physiological and biochemical characteristics,

Discussion
Sapstain fungi are economically important and have been studied for many years in different countries. The major sapstaining groups belong to the Aureobasidium, Ceratocystis, Ophiostoma, Sporothrix, Graphium, or Hyalorhynocladiella and Leptographium genera [4,38].
In our work, we used four sapstain fungi (C. adiposa Hz91, L. theobromae YM0737 and FX46, and F. lateritium YM05) isolated from discolored poplar as pathogen. Lasiodiplodia theobromae which can cause death of the kumquat (Fortunella margarita (Lour.) Swingle) tree was isolated from tissue taken from the margin of discolored kumquat bark and wood on symptomatic branches in orchards in Taiwan [2]. Various studies confirmed that L. theobromae was the causal agents of dieback and canker of grapevines in northern Mexico and Pinus caribaea var. hondurensis and Citrus aurantifolia in Venezuela [3,39]. It also caused discoloration of rubberwood and heveawood in tropical countries [40,41]. The ascomycete Ceratocystis resinifera is a pathogen of Picea and Pinus tree 8 The Scientific World Journal or freshly felled spruce and pine logs in Continental Europe, North America, Norway, and Canada [4,42,43]. Ceratocystis coerulescens (Münch) B. K. Bakshi can stain Jack pine, white spruce, lodgepole pine, and Corsican pine in Canada [44]. Fusarium spp. is one kind of Sapstain fungi from Pinus radiata D. Don in Korea [45]. However, the study on stain to poplar was scarce. Strain B37 of Bacillus subtilis was the best biocontrol bacterium on poplar sapstain (C. adiposa Hz91, L. theobromae YM0737, L. theobromae Fx46, and Fusarium sp., YM05) of the 13 bacteria in our work. It was isolated from normal leaf of Pinus thunbergii. These observations agree with Feio et al. [46], who reported that the growth of Fusarium oxysporum was inhibited by B. subtilis 335 on yeast extract glucose broth (YGB) medium on days 11 and 14. Growth of F. oxysporum was significantly inhibited by 50% solution of YGB inoculated with 11-day-old B. subtilis 335 culture in blocks of maritime pine (Pinus pinaster). The 12-h yeast malt (YM) broth medium of B. subtilis C186 can prevent the growth of several fungi, isolated from Pinus spp., Aureobasidium sp., Ceratocystis sp., Cytospora pini Desm., Ophiostoma sp., Penicillium sp., and Trichocladium sp. [30]. However, very few studies were biocontrol poplar sapstain by B. subtilis.
Chitinase and -l,3-glucanase that degrade the fungal cell wall components, chitin and -l,3-glucan, are important enzymes to control fungi (Fusarium solani (Mart.) Sacc., Neurospora crassa Shear & B. O. Dodge, Rhizoctonia solani J. G. Kühn, Sclerotium rolfsii Sacc., and Pythium ultimum Trow) [47,48]. Cultivated for 2 days, strain B37 produced a significant amount of -1,3-glucanase (the width of color change band is 4.25 cm), effectively inhibiting the growth of four poplar sapstain fungi. It can produce a small amount of proteases (the width of color change band is 0.76 cm), but chitinase was not found. -1,3-Glucanase can catalyze the hydrolysis of -1,3-dextran polymers, which inhibit the growth and reproduction of fungi [49]. -1,3-Glucanase genes clearly resulted in resistance to infection by Fusarium [50]. Therefore, -1,3-glucanase is one of the important inhibitions of the plant fungal disease, so the metabolic mechanism of strain B37 will be the direction of our further study.