Isolation of a New Mexican Strain of Bacillus subtilis with Antifungal and Antibacterial Activities

Although several strains of B. subtilis with antifungal activity have been isolated worldwide, to date there are no published reports regarding the isolation of a native B. subtilis strain from strawberry plants in Mexico. A native bacterium (Bacillus subtilis 21) demonstrated in vitro antagonistic activity against different plant pathogenic fungi. Under greenhouse conditions, it was shown that plants infected with Rhizoctonia solani and Fusarium verticillioides and treated with B. subtilis 21 produced augment in the number of leaves per plant and an increment in the length of healthy leaves in comparison with untreated plants. In addition, B. subtilis 21 showed activity against pathogenic bacteria. Secreted proteins by B. subtilis 21 were studied, detecting the presence of proteases and bacteriocin-like inhibitor substances that could be implicated in its antagonistic activity. Chitinases and zwittermicin production could not be detected. Then, B. subtilis 21 could potentially be used to control phytopathogenic fungi that infect strawberry plants.


Introduction
Data published by the Food and Agriculture Organization [1] indicated that Mexico is among the tenth largest producers of strawberries worldwide. In Mexico, the State of Guanajuato is one of the major producers of this crop, but during the past few years different factors, including the "secadera" disease, have led to an ∼50% decline in productivity [2]. The etiologic agents of "secadera" are fungi, primarily Rhizoctonia sp., Fusarium sp., Verticillium sp., and Phytophthora sp. [3].
In Mexico, chemical pesticides are used to control agricultural pests, but their long-term effects on animals and the environment have stimulated much concern [4]. To circumvent known and proposed detrimental effects related to widespread use of these pesticides, much interest is focused in identifying and developing biological agents to control insects and phytopathogenic microbes [5]. In particular, the use of selected nonpathogenic microorganisms which are ubiquitous in the soil and has potential applied use if they naturally produce antagonistic metabolites that kill bacterial or fungal pathogens or significantly inhibit their proliferation. For example, it has been demonstrated that an antibiotic isolated from Bacillus cereus inhibited mycelial growth of Fusarium roseum, and chitinases synthesized by Serratia marcescens, Streptomyces spp. Bacillus circulans, and Trichoderma harzianum are involved in the degradation of the host fungus cellular walls [6].
Interestingly, wild type and recombinant strains of Bacillus subtilis are known to not only promote plant growth but also synthesize different metabolites with antibacterial and antifungal activities [9]. As such, natural strains of probiotic B. subtilis could be beneficial as biocontrol agent in the strawberry industry. However, though many different environmental isolates and strains of B. subtilis have been described, to date there is no report regarding the identification of native B. subtilis isolates from strawberries in Mexico that elicit significant antagonistic activity against fungi that cause the "secadera" disease [10].
In this study, we isolated and cultured a new antifungal isolate of B. subtilis (isolate 21) from strawberries grown in Irapuato Guanajuato, Mexico, that showed antagonistic effect in vitro and under greenhouse conditions to pathogenic fungi.

Microbial
Strains. Bacillus thuringiensis subsp. morrisoni (LBIT 269) was obtained from a native bacterial stock collection held at CINVESTAV, Campus Guanajuato, Mexico. This strain synthesizes the bacteriocin called morricin 269 [11]. Bacillus cereus 183 was obtained from a collection of Bacillus strains maintained in the International Entomopathogenic Bacillus Centre, Institute Pasteur, Paris, France, and was employed as the indicator bacterium for determining, by a fluorogenic method, the time of the highest bacteriocin production by B. subtilis 21 [12]. Antibacterial activity of morricin 269 and bacteriocin-like inhibitor substances (BLISs) produced by B. subtilis 21

Isolation and Bacterial
Identification. Soil samples collected from the rhizosphere of healthy strawberry plants grown in different localities in Irapuato Guanajuato, Mexico, were homogenized to obtain uniform particle size. Crown and roots of the same plants were cut into 1 cm pieces, washed with 1.5% (v/v) sodium hypochlorite, and rinsed several times with distilled water. Homogenized soil was inoculated onto PDA (potato dextrose agar, Bioxon) using a small cylindrical sponge. Crown and roots pieces were also grown on the same media and incubated at room temperature (25-28 • C). Axenic bacterial cultures were maintained on PDA slants. Bacterial identification was performed by general and specific biochemical tests to determine the genus and the species [14].

Isolation and Pathogenic Fungi Identification.
Crown and roots of strawberry plants with the "secadera" disease were washed with distilled water to eliminate soil and cut into ∼1 cm pieces. Subsequently, pieces were disinfected with 1.5% (v/v) sodium hypochlorite, rinsed twice with distilled water, and placed in plates with PDA supplemented with 10% (w/v) lactic acid. Plates were incubated at 22 • C or 28 • C for Rhizoctonia species and other fungi, respectively. Fungal identification was performed as previously described [15,16].

In Vitro Antifungal Activity.
Agar disks from axenic fungus culture were inoculated on the middle of Petri dishes and cultivated at 28 • C. When mycelium reached about 2 cm of radial grow, B. subtilis 21 was inoculated at approximately 4.5 cm from the fungal margin and incubated at the same temperature to determine the formation of an inhibition zone. Bacillus subtilis 1.2.2 was used for comparison [17].

Antifungal Activity of Bacillus subtilis 21 in Greenhouse
Environment. Antagonism assays with both R. solani and F. verticillioides were carried out for a period of 90 days under greenhouse conditions [18]. To evaluate antagonism activity, the number of strawberry leaves without "secadera" disease symptoms and their length in centimeters were recorded. Treatments were untreated plants; strawberry plants infected with R. solani or F. verticillioides and treated separately with B. subtilis 21, B. subtilis1.2.2, and the fungicide Busan 30WB (2-tiocianometiltiobenzotiazol) (TCMTB). Roots plants were submerged for 1 min in suspensions of B. subtilis 21 or B. subtilis 1.2.2 containing ∼ 1 × 10 7 cells/mL or in 50 ppm of the fungicide TCMTB before planted. All assays were carried out in triplicate (three pods with three strawberry plants/pot) and a mix of vermiculate, pro-moss, and organic soil (1 : 1 : 2) as substrate was used [10]. Analysis of variance (ANOVA) was performed using the statistical model for a completely randomized block design. Treatment means were compared using the Tukey's test at significance level of 0.05%.

], and 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide [4-MU-GlcNAc]
(Sigma) to detect endochitinases, exochitinases, and N-acetylglucosaminidases, respectively. Reaction mixtures were prepared as described previously, diluted appropriately, and incubated at 37 • C for at least 15 min. Chitinase activity was measured spectrophotofluorometrically (excitation 340 nm, emission 415 nm). One unit of enzyme activity was defined as the amount of enzyme required to release 1 μmol of 4methylumbelliferone (MU) in 1 h [19]. Additionally, 72 h cultures were centrifuged, and ammonium sulfate was added to supernatant at 80% saturation to concentrated extracellular proteins with constant stirring overnight. Concentrated proteins were resuspended in 2 mL of 100 mM phosphate buffer and dialyzed overnight against the same buffer using membranes of 10 kDa cut-off (Spectrum Laboratories Company). Chitinase activity was determined as described above using fluorogenic substrates [19,20].

Proteolytic
Activity. Bacteria were cultivated in 100 mL of liquid Castañeda medium supplemented with 1% (w/v) casein under the same conditions described above for chitinolytic activity determination. One hundred microliters of cultures were collected at 24, 48, and 72 h, centrifuged and supernatants mixed with 0.4 mL of double-distilled water, 1 mL of 1% (w/v) casein in 200 mM glycine-NaOH buffer (pH 9), and incubated at 37 • C for 30 min. Then, reactions were stopped by adding 3.5 mL of 4% (w/v) trichloroacetic acid, centrifuged, and the absorbance was measured at 280 nm. A standard curve of tyrosine (0-300 μM/mL) was carried out to determine the protease units (PU). One PU was defined as the amount of enzyme required to release 1 μg of tyrosine/min [21].

Antibacterial Activity.
To study the kinetics of bacteriocin-like inhibitor substances produced by B. subtilis 21 (Bs21-BLIS), bacterium was cultured in TSB (tryptic soy broth) at 28 • C and samples were assayed in duplicate at different times over a 72 h period. One of the samples was used for monitoring cell growth spectrophotometrically at 600 nm, and the other was employed for bacteriocin detection using a rapid fluorogenic method [12], using B. cereus 183 as indicator bacterium. After determining the time that yielded the highest level of bacteriocin production, the bacterium was cultivated in fresh TSB and culture supernatant was concentrated with ammonium sulfate as described previously [12,13]. Precipitated proteins were pelleted by centrifugation at 16.000× g for 30 min at 4 • C, resuspended in 100 mM phosphate buffer (pH 7.0), and dialyzed overnight against the same buffer using a minidialysis kit with a 1 kDa cut-off (Amersham Biosciences). Antibacterial activity of concentrated proteins was tested using the modified well-diffusion method [12]. Briefly, twenty-five ml of TSB with soft agar 0.7% (w/v) was mixed with 50 μL (∼ 1 × 10 9 cells/mL) of indicator bacteria and plated. Wells, 7 mm in diameter, were dug into the agar and stored for 2 h at 37 • C to dry the humidity. Then 100 μL of the concentrated proteins were added to each well and incubated for 12 h at 4 • C to allow the diffusion of the samples followed by an additional incubation at 28 • C for 1 day before diameters of zones of inhibition were measured. The minimum detectable zone measured for analytic purposes was 1 mm beyond the well diameter. Each point of activity was repeated in triplicate and the average was recorded. For our purposes, we define one unit (U) of bacteriocin activity as equal to 1 mm 2 of the zone of inhibition of growth of the indicator bacterium [12,13].

Pathogenic Fungi Isolation and Identification.
Two hundred and twenty-six morphologically distinct fungal colonies were isolated from strawberry with the "secadera" disease. From those isolates, 63 were selected based on their morphology and microscopic characteristics and placed into three groups. Group 1 was composed of 33 strains (52.4%) of Fusarium verticillioides (Sacc.) Nuremberg and had purple cottony aerial mycelia and sickle-shaped thinwalled macro conidia. In the second group, 21 isolates (33.3%) of Rhizoctonia solani Kühn were included as they showed aerial mycelia in radial layers and segmented hyphae with "T" joints. Finally, 9 isolates (14.2%) that could not be identified comprised Group 3 [10].  The intensity of the antagonistic activity was recorded on basis of the size of growth inhibition from the place where bacterium was inoculated to the edge of the spreading fungal mycelium as follows: strong antagonism (+++), middle antagonism (++), and light antagonism (+) if the fungal growth was, respectively, ≤3.5 cm, 3.5 to 4.5 cm, or >4.5 cm. and then subjected to the treatment with B. subtilis 21 and B. subtilis 1.1.2 and the TCMTB fungicide. The results of this study were analyzed after 90 days. When infected plants were exposed to B. subtilis, they developed more leaves than those exposed to the TCMTB fungicide and also they were larger (see below). It has been previously reported that some strains of B. subtilis secrete compounds that could act as growth promoters [9]

Chitinolytic and Proteolytic Activity Determination.
In order to test whether B. subtilis 21 was able to synthesize chitinase, the bacterium was grown in two media containing salts and varying the carbon and nitrogen sources. The media were supplemented with either colloidal chitin or dry mycelium of Rhizoctonia solani. No chitinase production was detected in both media. In addition, B. subtilis 21 synthesized proteases during growth but the maximum production (∼ 450 UP/mL/min) was detected during the middle of the logarithmic phase ∼20 h (data not shown).

Bactericidal
Activity. Antibacterial activity was evaluated using a fluorogenic rapid method [12], employing B. cereus 183 as indicator bacterium (Figure 1). In this assay, bacteriocin activity was observed in samples collected at the middle of the logarithmic phase and achieved the highest levels at the start of the stationary period ( Figure 1). Subsequently, Bs21-BLIS was concentrated at the time corresponding to the highest level of bacteriocin production. Samples were evaluated against different Gram-positive and Gram-negative bacteria known to be etiologic agents of human diseases with the well-diffusion method and activities were compared to that of morricin 269 (Table 3). This bacteriocin showed a wide spectrum of antibacterial inhibitory effect whereas Bs21-BLIS had a narrow inhibitory effect obtaining the highest activity against B. cereus 183 (276 U) and Staphylococcus aureus (276 U), followed by Streptococcus pyogenes (25 U) and Enterobacter cloacae (25 U).

Discussion
One  Figure 1: Correlation between growth and the appearance of Bs21-BLIS in the culture medium. Bacterium was grown in tryptic soy broth, and duplicate samples were collected at different times. One sample was used for measuring the optical density at 600 nm ( ) and the other for evaluating the Bs21-BLIS activity ( ) against Bacillus cereus employing a fluorogenic rapid method.  Antimycotic activity is known to be induced by chitinases produced by a variety of microorganisms including Enterobacter agglomerans [25] and Trichoderma harzianum [6]. It appears that the antifungal activity of B. subtilis 21 is not due to constitutive or induced chitinase as such activity was not detected in our assays using preparations of the bacterium obtained from growth media supplemented with chitin or Rhizoctonia solani mycelia or without these inducers. Additionally, the role of subtilisin-like proteases with activity against Alternaria alternata, Fusarium oxysporum, Rhizoctonia solani, Sclerotinia sclerotiorum, and Cytospora chrysosperma has been demonstrated with Trichoderma harzianum T88 [26]. Our results showed that antifungal protease production occurred during all phases of B. subtilis 21 growth which suggests that those organic molecules could play an important role in its antifungal activity.
Other compounds that confer antimycotic activity include zwittermicin A, an aminopolyol antibiotic encoded by the zwittermicin A gene in Bacillus species such as B. cereus UW85 [23,24]. Our inability to produce an amplicon corresponding to the B. cereus UW85 zwittermicin A gene in B. subtilis 21 suggests that a closely related homolog is absent in this isolate. However, it is possible that a more diverged gene not amplifiable with the primers used in this study could be present in the isolate. Therefore we are unable to conclude that the antifungal activity of B. subtilis 21 is not due to a zwittermicin-A-like antibiotic.
Studies on the kinetics of bacteriocins production by B. subtilis 21 were very similar to those observed for B. thuringiensis subsp. kenyae (LBIT 404), B. thuringiensis subsp. entomocidus (LBIT 420), and B. thuringiensis subsp. tolworthi (LBIT 524), where bactericidal activity was present in samples collected at middle of the logarithmic phase of growth and achieved the highest levels at the start of the stationary period Barboza-Corona et al. [11]. The Bs21-BLIS activity against Staphylococcus aureus, Streptococcus pyogenes, and Enterobacter cloacae, potent agents of a number of community acquired and nosocomial diseases, including emesis, diarrhea, sore throat and scarlet fever, and urinary tract infections [27], suggests that these substances could be useful as a preservative in stored consumable products.
In conclusion, we cultured an isolate B. subtilis 21 that showed inhibitory activity in vitro and under greenhouse conditions against several fungi of economic importance including Verticillium sp. and agent of "secadera disease" and also showed that it produces extracellular compounds, such as proteases and bacteriocin-like inhibitor which could be implicated in the antagonistic activity against fungi and food-borne pathogenic bacteria.
Future efforts will focus on two important items: (a) identifying specific substance produced by B. subtilis 21, including volatile substances, that could play an important role in its antifungal activity and could be important in controlling fungal agents of "secadera disease" and (b) performing antagonistic assays against fungi under field conditions.