Pepper growth promotion and biocontrol against Xanthomonas euvesicatoria by Bacillus cereus and Bacillus thuringiensis formulations

Samples collection

Soil samples were collected from 30 chili pepper commercial lots from pepper producers in Meoqui, Chihuahua, MX (28°23′23″N, 105°37′25″W), who are current and former members of the Fundación Produce Chihuahua, A.C. Sampling was performed by selecting five diagonal points per lot, and soil was collected at a depth of 20 cm at each point (Ha, 2014). Samples were then placed in new zip-lock plastic bags, labeled, placed in a cooler, and transported to the Applied Microbiology, Plant Pathology, and Postharvest Physiology Laboratory (MAFFP) of the Autonomous University of Chihuahua (UACH), where they were kept at 4 °C, until use.

Bacillus strains isolation and storage

Bacillus strains isolation was performed according to the method adapted by Astorga-Quirós et al. (2014). In brief, one gram of soil from each sample was placed in a test tube containing nine mL of sterile saline solution (0.85%) and covered with a lid. Tubes were then shaken for three minutes with a vortex (Daigger Vortex-Genie 2; Scientific Industries Inc., Bohemia, NY, USA) set at speed 3. Next, tubes were placed in a water bath at 90 °C for 10 min, after which serial microdilutions were prepared in microplates, according to Chen, Nace & Irwin (2003) and Lugo et al. (2012). For this, 100 µL of soil suspension were taken from each tube and placed in the first series of microplate wells (stock sample), and subsequent wells were filled with 180 µL of sterile saline solution (0.85%). Next, 20 µL of the stock sample were transferred into one well of the second series, shaking in circles with the micropipette tip to have the first dilution. From this, 20 µL of the sample were transferred into the third series of wells, repeating the previous step, until obtaining a dilution of 1 × 10⁶ (Chen, Nace & Irwin, 2003). Dilutions were then transferred to Petri dishes containing nutrient agar medium (NA; BD Difco Laboratories, Sparks, Maryland, MD, USA) using a replicator (Lugo et al., 2012) and incubated for 24 h at 28 °C in a Lab-Line Imperial III incubator (Fisher Scientific, Dallas, TX, USA). Developing colonies were characterized as typical Bacillus genus based on the size, shape, elevation, and texture (Calvo & Zúñiga, 2010), and were isolated in NA medium and stored at −20 °C in a 40% glycerol solution in distilled water, until use.

Phenotypic identification

Presumed Bacillus isolates were subjected to standard biochemical and physiological tests (Shaad, Jones & Chun, 2001). Gram stain (Hycel^®, Zapopan, Jalisco, Mexico), KOH tests, and Schaeffer-Fulton stain were also performed to determine endospores presence (Mormak & Casida, 1985). The hanging drop motility test (Vázquez et al., 2011) was used to determine the motility of an isolate.

Molecular identification

PCR was implemented to identify Bacillus species, using specific primer pairs for B. anthracis, B. cereus, B. licheniformis, B. mycoides, B. subtilis, and B. thuringiensis (Table 1) (Park et al., 2007; Sadeghi et al., 2012). DNA extraction was performed using the modified phenol-chloroform method (Bardakci & Skibinski, 1994). For this, 1.5 mL of each bacterial isolate after 24 h growth in nutrient broth (NB) (BD Difco Laboratories) was centrifuged at 12,000 rpm for 5 min at 4 °C, and the supernatant was discarded. Next, 100 µL of 10% sodium dodecyl sulfate, 100 µL of 5 M NaCl, and 100 µL of 10% cetyltrimethylammonium bromide were added, after which the mixture was homogenized in a vortex at 3,400 rpm for two minutes (VX-200, Labnet International, Inc., Edison, NJ, USA). This mixture was then incubated at 65 °C in a water bath for 10 min, followed by the addition of 750 µL of a phenol:chloroform:isoamyl alcohol (25:24:1) mixture, stirred in a vortex at 3,400 rpm, and centrifuged at 12,000 rpm for 5 min at 4 °C. The upper aqueous phase was carefully collected with a pipette in a new tube. For DNA precipitation, 500 µL of isopropanol were added to the supernatant, shaken at 2,000 rpm, and placed in the freezer at 20 °C for 24 h. This solution was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was discarded. Next, the precipitate was washed twice with 300 µL of 70% ethanol, centrifuged for 10 min, dried at 25 °C for 30 min, suspended in 50 µL of Tris-EDTA (TE) buffer (10 mM Tris–HCl and 1 mM disodium ethylene diaminetetra-acetic acid (EDTA) solution at pH 8), and stored at −20 °C, until use.

Samples DNA concentration was adjusted to 5 ng/µL with a basic Eppendorf BioSpectrometer (Eppendorf do Brasil Ltda., Mexico City, Mexico). We used 25 µL samples in the PCR reaction mix, including 15.5 µL of sterile water, 5 µL of 5X PCR buffer, 1.5 µL of 25 mM MgCl₂, 1 µL of 25 mM dNTP, 1 µL of each primer (10 µM stock concentration), 1.5 µL of DNA at 5 ng/µL, and 0.2 µL of 5 U/µL Taq DNA polymerase. PCR reactions were performed using a thermocycler (Mastercycler model 5333; Eppendorf AG, Hamburg, Germany). The amplification program consisted of an initial denaturation cycle at 94 °C for 5 min, 30 denaturation cycles at 94 °C for 30 s, and 63 °C for 30 s, an extension at 72 °C for 30 s, and a final extension cycle at 72 °C for 5 min, followed by an initial denaturation cycle at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 5 min, annealing at 64 °C for 1 min, extension at 72 °C for 1 min, and a final extension cycle at 72 °C for 7 min (Park et al., 2007; Sadeghi et al., 2012). Results were analyzed by 1.5% agarose gel electrophoresis, using 1 X ethidium bromide as intercalating agent for 120 min and visualized in a GenLogic 200 photodocumenter (Kodak, New York, NY, USA).

Pepper plants growth promotion and formulations

Bacillus activity as PGPB in pepper plants was evaluated in two stages, under greenhouse conditions (27 °C ± 2 °C and 75% relative humidity (RH)). In the first experiment, we evaluated pepper seedlings growth promotion (seedling emergence) by 22 Bacillus strains. In a second experiment, three formulations were prepared with selected isolated identified and coded as F-BC26 for B. cereus strain CBC26, F-BC08 for B. cereus strain CBC08, and F-BT24 for B. thuringiensis strain CBT24. They were grown in 100 mL of tryptone soy broth (TSB) at room temperature for 72 h, under continuous shaking at 120 rpm. After incubation and sporulation, cultures were placed in a water bath at 90 °C for 10 min to eliminate other non-forming spore bacteria, present as contaminants. Next, the culture was centrifuged at 9,000 rpm and suspended in 10 mL of 1X phosphate buffered saline (PBS) solution (Fisher Scientific), which was inoculated in one liter of TSB and incubated at room temperature for 24 h at 120 rpm.

As previously reported by Tamez-Guerra et al. (2000), formulations were prepared for microgranules production by spray drying (Niro Mobile Minor 2000; GEA, Munich, Germany), using the composite formula. Bacterial cultures were used to inoculate 4 L of TSB in a 7 L flask and incubated at room temperature for 72 h at 120 rpm. Cultures were then mixed with one kilogram of nixtamalized corn flour (Maseca, Molinos Azteca, S.A. de C.V., Guadalupe NL, México), previously dissolved in 5 L of purified water, and kept under stirring. Next, 100 mL of cottonseed oil, 200 mL of Inex A, and 5 L of culture were added, followed by 40 g of CaCl₂ and 300 mL of isopropyl alcohol. The final volume was processed in the spray dryer, with a turbine pressure of 1.5 bar, an inlet flow of 55 mL/min at 200 °C, and an outlet temperature of 80 °C ± 3 °C, after which microcapsules were prepared by spray-drying (Tamez-Guerra et al., 2000). Formulations were then stored in plastic bags at 4 °C, and CFU was determined.

Formulations were evaluated in pepper seedlings, seedbeds, and potted plants (seedling emergence stage and vegetative stage). The bacterial mixture as consortium was not included as treatment because, in previous experiments, they did not show differences from their individual effect (data not shown).

In vitro antagonism of Bacillus spp. versus Xanthomonas spp.

In vitro Xanthomonas growth inhibition by Bacillus isolates was determined in microplates, using 22 Bacillus strain isolates as treatments (antagonists). In addition, B. subtilis QST 713 (Serenade^®) was evaluated as the control antagonist, X. euvesicatoria Xp47 and Xe65 (Cepario Lab MAFFP, UACH) as pathogens, and X. campestris ATTC1395 as the negative control. Bacterial suspensions were prepared from 24 h growth cultures at 28 °C and 120 rpm in Luria Bertani (LB) liquid medium at pH 7.0. Bacterial density was adjusted to 1 ×10⁸ CFU/mL, corresponding to 0.4 at 600 nm OD by UV-visible spectrophotometry (Evolution 60 S; Thermo Fisher Scientific Inc., Waltham, MA, USA) for Xanthomonas and Bacillus spp. (Rabbee, Ali & Baek, 2019). Bacterial suspensions were placed in a microplate, adding 75 µL of LB-cultured Xanthomonas per well plus 75 µL of Bacillus suspensions. We used 150 µL of LB as an absolute control, whereas as a positive control, we tested 75 µL of LB plus 75 µL of each phytopathogenic bacterium. Microplates were sealed with parafilm^® and incubated at 28 °C for 72 h. Xanthomonas growth was confirmed every 24 h by reseeding, using the streak plate technique in NA and incubating at 28 °C for 48 h. Xanthomonas growth inhibition by Bacillus was qualitatively determined, considering, as a positive result, the presence of the antagonistic effect of Bacillus. For this, the following arbitrary scale was used: +, regular; ++, good; and +++, intense, when no pathogen colonies were observed in the reseeding in NA at 24 h, 48 h, and 72 h of confrontation. It was considered a negative result (absence of Bacillus antagonistic effect) when the growth of at least one colony of the pathogen was detected in the reseeding, during the evaluation period (the “-” sign indicates no growth inhibition).

Resistance induction in pepper against Xanthomonas

We evaluated plant resistance to Xanthomonas by F-BC26, F-BC08, and F-BT24 strains. This test was established in a plant growth room with a photoperiod of 16 h light at 28 °C and 8 h dark at 18 °C and 80% to 90% RH. In this assay, we used 35-d-old jalapeño pepper plants variety M. Seedlings were cultivated in 10 cm diameter pots with sterile peat moss and irrigated every third day with the nutrient solution proposed by Otazu (2010). Plants were inoculated during transplantation time at stem base level, using 10 mL of F-BC26, F-BC08, or F-BT24 ( 1 × 10⁸ CFU/mL) suspensions, whereas 10 mL of Serenade^® (1 × 10⁸ CFU/mL) or nutrient solution (FitoFort^® (15.9% P, 21.5% K, 1.5% Zn, 1.7% sulfur, 1.6% Mn, and 58.7% plant extracts))

(Fruverint Comercializadora S.A de C.V., Mexico City, Mexico). Plants without any inducing treatment were used as controls. After seven days, plants foliage (two points on two leaves per plant) was inoculated through infiltration with one milliliter Plastipak SFP syringes (Becton-Dickinson, Brooklyn, NY), using 10 µL of X. euvesicatoria Xp47 and Xe65 suspensions at 1 × 10⁸ CFU/mL or 0.85% sterile saline solution (control). Before and after pathogens inoculation, plants were conditioned for two days in darkness, with an RH greater than 90%. Disease inhibition by inducing resistance was determined after 15 d of pathogens inoculation, using the Nutter, Esker & Netto (2006) modified formula as follows: ${\displaystyle \text{Disease inhibition}\left(\text{\%}\right)=\frac{1-\text{Disease severity in leaf infiltrated phytobacterial formulation}}{\text{Disease severity in control}}\times 100}$

Where: $\text{disease severity}\left(\text{\%}\right)=\frac{\text{disease leaf area}}{\text{total leaf area}}\times 100$

Foliar biocontrol of Xanthomonas

In this assay, we used 35-d-old jalapeño pepper plants variety M. Seedlings were cultivated under the same conditions as for the resistance bioassay mentioned above. For biocontrol testing, three milliliters of treatment suspensions were applied by foliage spraying. Treatments consisted of F-BC26, F-BC08, or F-BT24 strain suspensions (1 × 10⁸ CFU/mL) plus 0.005% Tween 20, Serenade^® (as a positive control at 1 × 10⁸ CFU/mL), 10 mL of 50% Anglosan CL^® (didecyldimethylammonium chloride (DDAC); American Pharma S.A. de C.V.), 10 mL of FitoFort^® applied to the stem base (as resistance inducer), or 10 mL of sterile distilled water. After 48 h, approximately three milliliters of X. euvesicatoria Xp47 and Xe65 suspensions at 1 ×10⁸ CFU/mL or sterile saline solution at 0.85% were sprayed on foliage as negative controls. Before and after pathogens inoculation, plants were conditioned for two days in darkness, with a RH higher than 90%. Xanthomonas spp. biocontrol by Bacillus spp. was evaluated after 15 d of pathogen inoculation and Bacillus positive antagonistic effect was recorded if disease symptoms were not observed in leaves, whereas Bacillus absence of antagonistic effect was recorded when disease symptoms on the foliage were observed. In addition, pathogen presence in leaf tissue was determined by taking samples, grinding them with a pestle, and taking microdilutions to inoculate on MacConkey Agar plates (Lugo et al., 2012).

Statistical analysis

Pepper growth promotion by Bacillus spp. test in the seedlings emergence was established under a completely randomized design, where 22 isolated strains from soil were tested as treatments, Serenade^® as a positive control, and seedlings without PGPB as control. All treatments had five replicate determinations. Pepper growth promotion by formulated Bacillus spp. tests in the seedlings emergence assay and the vegetative stage were established under a completely randomized design, where F-BC26, F-BC08, and F-BT24 formulations were tested as treatments, BactoRacine-B^® as positive control, and seedlings or plants without formulations as controls. Seedlings emergence assay and vegetative stage assays had four and five replicate determinations per treatment, respectively.

In vitro antagonism of Bacillus spp. versus Xanthomonas spp. the test was established under a completely randomized design. The 22 Bacillus isolated strains from soil were evaluated as treatments, Serenade^® as a positive control, and X. euvesicatoria Xp47, X. euvesicatoria Xe65, and X. campestris ATTC1395 as the control group. All treatments had three replicate determinations.

Resistance induction in pepper against Xanthomonas spp. and foliar biocontrol of Xanthomonas spp. by formulated Bacillus spp. tests were established under a completely randomized design, where F-BC26, F-BC08, and F-BT24 formulations were tested as treatments, Serenade^®, FitoFort^®, and Anglosan^® CL (only foliar biocontrol test) as a positive control group, and X. euvesicatoria Xp47 and X. euvesicatoria Xe65 as the negative controls, and plants without formulations as controls. Resistance induction assays were performed five times, whereas foliar biocontrol experiments had four replicate determinations per treatment.

Data from Bacillus- induced pepper plants growth with unformulated and formulated Bacillus spp. strains were analyzed by ANOVAs, and the Scott-Knott’s (α = 0.05) and the Tukey’s (α = 0.05) mean separation tests, respectively. Data from Xanthomonas spp. biocontrol assays were analyzed by ANOVAs and Tukey’s (α = 0.05) mean separation test. All analyzes were performed with the InfoStat software (InfoStat version 2009; InfoStat Group, Cordoba, Argentina).

Isolation and identification of Bacillus spp.

Samples exposure to 90 °C is recommended for selecting mostly Bacillus spp. isolates. This step is recommended since these temperature range does not affect spore survival, but helps to eliminate other undesirable bacteria. After exposing samples at 90 °C by 10 min, mostly viable Bacillus spp. endospores will develop in the culture medium (Cohan, Roberts & King, 1991). Processing soil samples at such conditions, allowed the isolation and purification of 22 bacterial strains with morphological characteristics expected for the genus Bacillus. Macroscopic analysis showed that bacteria grew in large, circular shapes, wavy edges, opaque and smooth textures, and low elevation colonies, which also presented colors with variations between light gray or creamy to whitish. Microscopically, bacteria showed bacillary form, were Gram-positive, and positive to Schaeffer-Fulton stain, thus indicating endospores presence. Strains were positive for motility and negative to the KOH test. Most Bacillus spp. isolates were molecularly identified by multiplex PCR as B. cereus (21 isolates) and only one as B. thuringiensis. Isolates identification was confirmed with the 400 bp and 299 bp DNA fragments amplification of one isolate, using B. thuringiensis-specific primers, and 400 bp and 475 bp fragments amplification in isolates testing B. cereus-specific primers (Fig. 1). No amplification was observed for negative controls and other primer pairs specific for B. anthrasis, B. licheniformis, B. mycoides, or B. subtilis.

Bacillus spp. as pepper PGPB

Pepper seedlings growth under controlled conditions significantly (P < 0.05) improved after Bacillus spp. application (seedling emergence assay) (Table 2). After individual application of 22 isolates in seedlings, seven isolates increased leaves area by 46.8%, and 12 increased leaves number by 11.4%, as compared with the absolute control. In addition, seedling height improved between 14.2% and 28.8%, as compared with the absolute control seedlings, after applying 20 of the strains. After application, 21 out of 22 Bacillus spp. strains, improved pepper stem diameter between 9.9% and 27.8%, as compared with the control seedlings. Furthermore, 11 out of 22 strains, showed 8.8% higher root length, compared with the control (Table 2).

The evaluated strains (3, 7, and 13 of 22) increased the dry weight of roots, stems, and leaves by 46.1%, 45.4%, and 37.8%, respectively, as compared with the absolute control seedlings and commercial product. Regarding seedlings quality, expressed in terms of the Dickson Index, CBC08 and CBC26 strains improved plant quality by 60.7%, as compared with the absolute control seedlings (Table 3). Overall, 15 of all tested strains induced the same quality of seedlings as the commercial product treatment (Table 3).

Pepper seedlings grown under greenhouse conditions (seedlings emergence assay), significantly (P < 0.05) improved after applying formulated B. cereus and B. thuringiensis (Table 4). Formulated F-BT24 strain improved the stem diameter and root length by 16.2% and 10.2%, respectively, as compared with the un-inoculated control seedlings and commercial product. Both formulations separately applied, improved seedling height, leaf area, and stem and leaves dry weights by 46.1%, 36.9%, 38.1% and 39.1%, respectively, as compared with the absolute control seedlings. Formulated F-BC08 strain only improved height by 25.5% and induced comparable results as the commercial treatment and the control in the remaining assayed parameters.

Potted pepper plants development under greenhouse conditions (vegetative stage) was significantly higher (P <  0.05) after applying formulated B. cereus and B. thuringiensis strains, as compared with fertilized plants (Table 5 and Fig. 2). The three tested formulations and the commercial product significantly (P < 0.05) improved leaf area, stem diameter, and leaf and stem dry weights by 82.0%, 20.6%, 62.9%, and 72.7%, respectively, as compared with the absolute control. F-BC26, F-BT24, and commercial product treatments, increased height and root dry weight by 48% and 141%, respectively, as compared with the absolute control. Furthermore, F-BC26 formulation and the commercial product improved the number of leaves by 41.1%, whereas only the F-BT24 formulation enhanced root length by 30.6%. Similarly, F-BC08 formulation improved chlorophyll “b” and carotenoid content by 157.7% and 33.0%, respectively, as compared with the commercial product. Regarding flowering stimulation, formulated F-BC26 strain significantly increased the number of flowers to 140.7%, as compared with fertilized plants (Table 5 and Fig. 2).

In vitro Bacillus spp. antagonism versus Xanthomonas spp

All Bacillus isolates tested inhibited in vitro X. euvesicatoria Xp47 and Xe65 growth after 24 h of confrontation (Table 6). However, only X. campestris ATTC1395 was inhibited by Bacillus spp. after 24 h of confrontation (63% inhibition), but at 48 h, its growth was completely inhibited (Table 6).

Resistance induction in pepper against Xanthomonas

Formulated B. cereus and B. thuringiensis were applied as bacterial spot resistance promoters in pepper plants under controlled conditions, showing a significant disease inhibition (P < 0.05) (Fig. 3). Similarly, the bacterial spot was reduced by 44.7%, after applying the commercial product Serenade^®, whereas FitoFort^® induced the highest disease inhibition (61.2% disease inhibition) (Fig. 3A).

Xp47 strain in interaction with Bacillus formulations or commercial products showed characteristic disease signs in treated plants but lower severity depending on the treatment. Necrotic spots with chlorotic edges presence characterized them, leaves with chlorosis or perforation areas with leaf blades, and necrotic spots with chlorosis (Figs. 3A and 3B).

Foliar Xanthomonas biocontrol

Bacterial spot development on pepper plants was inhibited after applying Bacillus spp. formulations and Serenade^® directly to foliage. Although it was intended for the root system, FitoFort^® inhibited disease development. Xp47 treated plants showed small necrotic spots on leaf blade. However, pathogen presence in foliage was found among all biological treatments, but as expected, it was not shown in the chemical treatment (Anglosan^® CL50%). Overall, maximum pathogen population reduction by biocontrol agents was 8.6% (Table 7).