Chili peppers (Capsicum spp.) are among the most important crops worldwide, and in Mexico, where in 2024 production reached 3.2 million tons of green chili peppers, ranking fourth in global production and representing the second highest production value nationwide (FAO, 2023; SIAP, 2024). Phytophthora root rot, caused by the oomycete Phytophthora capsici, is the main limiting factor in pepper production, causing severe economic losses (Silva-Rojas et al., 2009). Its presence has reduced the cultivated area in important regions and displaced susceptible crops. This pathogen causes damping-off, wilting, leaf blight and the rotting of roots, stems, and fruits. The control methods for P. capsici are based on integrated management practices, including cultural practices, fungicide applications, and the cultivation of resistant varieties. However, fungicide use remains predominant, leading to considerable environmental and human health concerns and contributing to pathogen resistance to fungicides (Hausbeck and Lamour, 2004; Sánchez-Gurrola et al., 2019). Together, the high impact of the disease on pepper production and the limitations and risks associated with current control practices highlight the need for alternative and more sustainable management strategies.

Mexico is the center of origin and domestication of Capsicum annuum, the most economically important of all five domesticated species of the genus Capsicum (C. annuum, C. chinense, C. frutescens, C. baccatum and C. pubescens). A large diversity of peppers is cultivated across Mexico; therefore, the country harbors a high genetic diversity of P. capsici, which is associated with its high host-pathogen coevolutionary rate, rapid spread, and adaptability to new hosts and environments. These factors make this pathogen economically significant and a potential global threat to food security (Retes-Manjarrez et al., 2020; Reyes-Tena et al., 2020; Sánchez-Gurrola et al., 2019). The sporangia and zoospores of P. capsici provide high reproductive potential under wet conditions, facilitating rapid spread via water, root penetration, and successive infection cycles within a single growing season (Granke et al., 2012). Thus, it is essential to explore alternative approaches for the control of Phytophthora blight and root rot, such as biological control.

Various bacterial genera have been assessed in vitro and in vivo as antagonists of P. capsici, demonstrating promising potential (Akgül and Mirik, 2008; Lee et al., 2008; Ley-López et al., 2018; Xu et al., 2020). However, due to the high aggressiveness of this pathogen and the variability in reported outcomes, it is important to continue screening for new antagonists and to further assess their interactions (Lee et al., 2013; Xu and Kim, 2016).

The genus Bacillus comprises ubiquitous bacteria inhabiting diverse environments, including soil, water, air, the rhizosphere, and many extreme environments. This genus is among the most commonly used bacterial groups for the biological control of phytopathogenic fungi and oomycetes, such as P. capsici, mainly due to its ability to produce a wide array of secondary metabolites with antimicrobial activity and plant growth-promoting properties (Ferreira et al., 1991; Fira et al., 2018; Zhang et al., 2010).

First described in 2016 after being isolated from shrimp farm water in Zhangzhou, China (Ruginescu et al., 2018), Bacillus zhangzhouensis has attracted increasing attention in recent years as a promising halophyte plant growth-promoting bacterium (Alp et al., 2024; Bezerra et al., 2022; Emami et al., 2019). Regarding biological control traits, several studies have reported the antimicrobial potential of B. zhangzhouensis strains against animal, human, and plant pathogens, underscoring their broad-spectrum antagonistic activity (Anil et al., 2019; Aytenov et al., 2024; Molina-Garza et al., 2021; Soliman et al., 2022). However, to date, no studies have evaluated the biocontrol performance of Bacillus zhangzhouensis against Mexican isolates of P. capsici, nor its effectiveness in pepper plants, which is particularly relevant given the high variability of this pathogen. The objective of this study was to evaluate the biocontrol capacity of a bacterial isolate as a biocontrol agent against six P. capsici and one P. cinnamomi isolates. Biocontrol effects were assessed based on mycelial growth, sporangia and zoospore release, light and electron microscopy, as well as severity of Phytophthora root rot in pepper plants.

Microbial culture conditions. Bacterial isolate JJR198 was provided by the Laboratory of Bioremediation, Universidad Autónoma de Aguascalientes. The isolate was cultured by streaking on LB agar plates and incubated for 24-48 h at 30 °C. A single colony was transferred to liquid LB medium and grown at 30 °C for 24 h on a rotary shaker at 160 rpm. After incubation, the bacterial suspension was adjusted with sterile distilled water (SDW) to final concentrations of 1.0 × 10⁴, 1.0 × 10⁶, and 1.0 × 10⁸ CFU mL⁻¹ for subsequent evaluations, with the highest concentration corresponding to an optical density of 0.1 at 600 nm (Kim et al., 2009; Xu et al., 2020).

Six isolates of Phytophthora capsici (Pc1, Pc2aIT, Pc43M, PcAR-2, Pc57, and PcAR-14) and one isolate of P. cinnamomi (Pcn5-1) were kindly provided by the Plant Pathology Laboratory, Instituto de Investigaciones Agropecuarias y Forestales, Universidad Michoacana de San Nicolás de Hidalgo. Information on host, mating type, and geographic origin was available for P. capsici isolates Pc2aIT, Pc43M, PcAR-2, Pc57, PcAR-14 and Pcn5-1. Specifically, PcAR-2 was isolated from poblano pepper mating type (MT) A1 in Copándaro de Galeana, Michoacán, Mexico; PcAR-14 from serrano pepper (MT A1) in Cuitzillo el Grande, Tarímbaro, Michoacán, Mexico; Pc57 from pepper (MT A2) in Guanajuato, Mexico; Pc43M from pepper in Guanajuato, Mexico; and Pc2aIT from poblano pepper (MT A2) in Yurécuaro, Michoacán, Mexico and Pcn5-1 from avocado in Michoacán, Mexico. All isolates were grown on PDA, and medium disks containing 7-day-old mycelium were transferred to 15 mL tubes containing sterile distilled water (SDW) and stored at 4 °C until use.

Identification of the bacterial isolate. Phenotypic tests, including cell morphology, Gram staining, and catalase and oxidase assays, were performed. The bacterial isolate was identified through 16S rRNA gene sequencing carried out by the Laboratory of Genomic Services at CINVESTAV (Irapuato, Mexico). The axenic isolate was shipped, and the service consisted of DNA extraction and PCR amplification of the 16S rRNA gene were performed using universal primers, according to the provider’s standard protocol. The exact length of the amplified fragment and primer sequences were not specified by the service provider. Sequencing was performed using the Sanger terminal di-deoxy method (Sanger et al., 1977). Homologous sequences of the amplified regions were searched using the Basic Local Alignment Search Tool (BLASTn) from the National Center for Biotechnology Information (NCBI) database. Multiple sequence alignment was performed using the MUSCLE tool. Phylogenetic analysis was conducted using MEGA 11 (Molecular Evolutionary Genetics Analysis version 11), applying the average-distance method with 1,000 bootstrap replicates (Tamura et al., 2021)

Inhibition of mycelial growth. The inhibitory effect of JJR198 was evaluated using a dual-culture method (Chávez-Díaz et al., 2016). Six of the Phytophthora isolates described above were included in this assay and cultured on PDA at 25 °C for four days. Mycelial plugs (8 mm in diameter) of each isolate were excised and placed at the center of PDA plates. A sterile cotton swab was used to inoculate the bacterial suspension (1.0 × 10⁸ CFU mL-1) at four equidistant edges, 2.7 cm from the center, toward the outer margin, forming an uninoculated square area around the mycelial plug. Plates of each isolate without bacterial inoculation served as controls. After five days of incubation at 25 °C, the mycelial growth diameter was measured, and the inhibition rate was calculated based on mycelial growth of the control. The experiment followed a completely randomized design with two treatments (with and without bacterial inoculation), six replicates per Phytophthora isolate, and individual Petri dishes as experimental units.

Effect of bacteria inoculum concentrations on sporangia formation. To evaluate the inhibitory effect of the bacterial isolate on sporangia formation, two P. capsici isolates: PcAR-2 and Pc57 selected for their high and stable sporangia production, were cultured on V8 juice agar (200 mL L-1 V8 juice, 800 mL L-1 distilled water, 3 g L-1 CaCO₃, 15 g L-1 agar, pH 7.2) at 25 °C for four days. Three mycelial plugs (8 mm in diameter) were placed in each 80 mm x 10 mm Petri dish and flooded with 25 mL of bacterial suspensions at three concentrations (1.0 × 10⁴, 1.0 × 10⁶, and 1.0 × 10⁸ CFU mL-1). The plates were incubated at 25 °C for 48 hours to induce sporangia formation. Petri plates with only sterile distilled water (SDW) served as a control. Sporangia formed on the mycelial plugs were observed in situ for each concentration by placing a Petri dish on a Zeiss Primo Star light microscope with a 4x objective lens; for each plug, sporangia were counted within 2 mm² observation areas using ImageJ software. The experiment was performed with four replicates (Petri plates) per concentration treatment.

Scanning electron microscopy. Mycelial plugs of PcAR-2 with sporangia, either untreated or treated with JJR198 (1.0 × 10⁸ CFU mL-1), were fixed by immersion in 2.5% (v/v) glutaraldehyde and washed several times with 1X PBS. The samples were dehydrated through a graded ethanol series (60, 70, 80, 90, 96, and 100%), for 20 minutes at each concentration. After dehydration in 100% ethanol, the samples were critical-point dried using a Samdri critical point dryer (Tousimis Research Corporation), mounted on stubs, gold-coated for 3 minutes using a Denton Vacuum Desk II sputter coater, and examined with a JEOL JSM-5900LV scanning electron microscope (Hayat, 1981, 1989; JEOL, 2000).

Effect of bacteria inoculum concentrations on zoospore release. To assess the effect of JJR198 on zoospore release, three V8 agar mycelial plugs (8 mm in diameter) with four-day-old mycelium of the Pc57 isolate (the most prolific sporangia producer) were placed in the center of a Petri dish (80 mm x 10 mm). The mycelial plugs were flooded with 25 mL of SDW and incubated for 48 h at 25 °C to induce sporangia formation. Afterward, the water was removed with a pipette and replaced with 25 mL of bacterial suspensions at three concentrations (1.0 × 104, 1.0 × 106 and 1.0 × 108 CFU mL-1). To induce zoospore release, the Petri dishes were incubated at 10 °C for 30 minutes followed by 30 min at 25 °C. After incubation, the number of zoospores per mL were estimated for each Petri plate using a hemocytometer. Each concentration treatment was replicated four times, with SDW serving as a control.

Evaluation of disease suppression in potted pepper plants. Pepper seedlings of a susceptible Guajillo landrace were produced in a pest and disease-free commercial greenhouse. After eight weeks individual seedlings were transplanted into plastic trays with six-cell cavities of 195 cm3 each. Cell cavities were prefilled with sterilized Berger BM2 substrate (70-80% peat moss, perlite, vermiculite, and wetting agent) and supplemented with 13 beads of controlled-release fertilizer (Osmocote 14-14-14). All seedlings were maintained in a greenhouse for two weeks at 11 to 30 °C; irrigation was provided every two days or as needed. To evaluate the protective effect of bacterial isolate JJR198 against P. capsici, plants were preinoculated with JJR198 at 7, 3, or 0 days before the oomycete inoculation All bacterial treatments were performed by applying 5 mL (1.0 × 108 CFU mL-1) suspensions to the substrate at the plant base above the roots. Plants free of bacteria and P. capsici, plants infected only with the oomycete, and plants treated only with bacteria were used as controls. Each of the four treatments consisted of six plants and was replicated twice. Prior to inoculate P. capsici isolate AR-14 all plant trays were placed in water-filled containers to ensure root saturation. A zoospore suspension was prepared and adjusted as described above for P. capsici inoculations. Inoculated plants with the oomycete, received 10 000 zoospores per cell. All plant trays were maintained under flooded conditions for 48 hours; afterward excess water was removed. The severity of root rot per plant was based on aerial parts of the plant at 7 and 14 days after inoculation (DAI) with P. capsici using a modified scale of severity (0 to 5) based on Ristaino (1990), where: 0 = no visible symptoms; 1 = slight leaf wilting with initial brown lesions on the stem; 2 = 30-50% of the plant showing disease symptoms; 3 = 50-70% of the plant affected; 4 = 70-90% of the plant affected; 5 = dead plant.

Salt Tolerance Assay. Following molecular identification of the bacterial isolate and considering the physiological characteristics previously reported in the literature for phylogenetically related species, its salt resistance was evaluated by assessing growth on nutrient agar (NA) medium with increasing NaCl concentrations (5, 7.5, 10, and 15%). Plates without NaCl served as controls. All assays were performed in triplicate, and plates were incubated at 30 °C for 48-72 h before measuring bacterial colony growth.

Presence of bacterial inoculum in pepper roots. Bacterial isolate JJR198 was recovered from the rhizoplane and rhizosphere of pepper plants two weeks after inoculation using the dilution plating technique (Shankar et al., 2011; Shahid et al., 2012). Samples of bacterial inoculated pepper root balls were shaken to remove excess substrate. For the rhizosphere bacterial population, one gram of adhering substrate was suspended in 9 mL of SDW in 25 mL glass tubes. For the rhizoplane bacterial population, roots were washed ten times with SDW, and then one gram of roots was vigorously shaken with glass beads in 9 mL of SDW, also in 25 mL glass tubes, to dislodge root-adhering bacteria. All tubes were subsequently heated in a water bath at 80 °C for 15 minutes to eliminate vegetative cells. Serial 10-fold dilutions were prepared up to 1.0 × 10⁴, and 0.1 mL of each dilution was plated onto NA plates supplemented with 6% NaCl. Colony-forming units (CFU) were counted, and Gram staining was performed after 48 hours of incubation at 30 °C. Uninoculated plants served as controls. Three replicates were performed in this assay.

Statistical analysis. Data of each experiment was organized in a specific database and analyzed with STATISTICA software 8.0 (StatSoft Inc. USA). For each experiment, statistical analyses included analysis of variance (ANOVA), followed by mean separation using Tukey’s test at p ≤ 0.05. Assumptions of normality and homogeneity of variances were also evaluated.

Identification of the bacterial isolate. The JJR198 strain was Gram-positive, rod shaped catalase and oxidase positive. JJR198 sequence was 1513 nt. The search for homologous genes in NCBI showed 99.6% similarity with Bacillus zhangzhouensis strain MCCC 1A08372 and Bacillus pumilus. The phylogenetic tree showed that JJR198 is in the clade together with B. zhangzhouensis and B. pumilus (Figure 1). The 16S rRNA gene sequence was deposited in the GeneBank under accession number PV826766.

Figure 1

Inhibition of mycelial growth. The JJR198 bacterial strain (1.0 × 108 CFU mL-1) exhibited similar inhibitory activity against the mycelial growth of the five P. capsici isolates and one isolate of P. cinnamomi (Figures 2, and 3) Inhibition rates ranged from 42. to 52.7%. The highest inhibition was observed in isolate PcAR-2 (52.7%), which differed significantly from Pc43M (42.1%). Pc2aIT (51.6%) also showed higher inhibition than Pc43M, while Pc1 (42.0%), Pc57 (47.0%), and Pcn5-1 (46.6%) exhibited intermediate values without significant differences among them (Tukey, p ≤ 0.05).

Figure 2

Figure 3

Effect of bacteria inoculum concentrations on sporangium formation. Under the sporangia-inducing conditions (48 h at 25 °C), the JJR198 bacterial strain did not exhibit a significant antagonistic effect against P. capsici isolates PcAR-2 and Pc57 at low and medium concentrations (1.0 × 10⁴ and 1.0 × 10⁶ CFU mL-1). However, sporangia formation was completely inhibited at 1.0 × 10⁸ CFU mL-1 (Table 1). At the highest bacterial inoculum, the morphology of the P. capsici isolates appeared altered, suggesting modifications of the mycelium and hyphae, along with the presence of bacterial aggregates (Figure 4).

Figure 4

Effect of bacteria inoculum concentrations on zoospore release. Zoospore release from Pc57 sporangia was not inhibited by 1.0 × 104 or 1.0 × 106 CFU mL-1 suspensions of strain JJR198, showing no significant difference (p > 0.05) compared to the control. However, a significant reduction was observed at a concentration of 1.0 × 108 CFU mL-1, with almost no spore release detected (Table 1).

Table 1

JJR198 inoculum density (CFU mL-1)	No. of sporangia/VA*		No. of zoospores (1.0 × 103 mL-1)
	PcAR-2	Pc57	Pc57
SDW¤ (control)	60.1 ± 6.2 a	234.2 ± 12.7 a	25.9 ± 4.9 a
1.0 × 104	41.0 ± 4.9 a	207.2 ± 15.3 a	26.1 ± 2.6 a
1.0 × 106	50.1 ± 10.7 a	205.5 ± 31.4 a	20.6 ± 3.3 a
1.0 × 108	0.0 ± 0.0 b	0.0 ± 0.0 b	0.2 ± 0.1 b

*VA, viewing area of 2 mm² under light microscope. ^¤SDW, sterile distilled water. Results are means ± standard errors of four replications. Same letters in each column denote no significant difference (Tukey, p ≤ 0.05).

Evaluation of disease suppression in potted pepper plants. The bacterial isolate JJR198 did not significantly reduced root rot severity in treated plants regardless of bacterial application at 7, 3 or 0 days prior to P. capsici inoculation. Root rot severity increased gradually with DAI in all four treatments. No statistically significant differences were observed between the bacterial treatments and the uninoculated control (Figure 5).

Figure 5

Salt Tolerance Assay. JJR198 was assessed for its capacity to grow at high NaCl concentrations. JJR198 was able to grow in NA media after 48 hours of incubation at 30

°C with 7.5% NaCl.

Presence of bacterial inoculum in pepper roots. In rhizosphere and rhizoplane samples from two-week inoculated pepper roots, bacterial colonies with the same macro- and micro-morphological characteristics as the JJR198 isolate were observed, including irregular, medium-sized colonies with irregular texture, cream coloration, and Gram-positive rods. No other colonies grew on NA medium supplemented with 6% NaCl. The bacterial count was 1.6 × 10⁶ CFU mL-1 in rhizosphere and 0.19 × 10⁶ CFU mL-1 in rhizoplane samples.

In previous studies, Liu et al. (2016) isolated a strain from a shrimp aquaculture farm in Zhangzhou City, China and they called as DW5-4. The Molecular identification of DW5-4 was performed using 16S and gyr genes, and the search for homologous genes showed an identity with Bacillus pumilus. However, a detailed bioinformatic analysis demonstrated that it was a different strain, which was named Bacillus zhangzhouensis. Thus, JJR198 has a very high similarity to both strains, as demonstrated in the phylogenetic tree. The molecular identification of JJR198 using 16S is consistent with other strains of B. zhangzhouensis and its close similarity with B. pumilus (Anil et al., 2019; Liu et al., 2016; Soliman et al., 2022).

Strain JJR198 exhibited in vitro antagonistic activity against mycelial growth of five P. capsici isolates and one P. cinnamomi isolate. Although JJR198 achieved a maximum mycelial inhibition of 52.7%, it showed a comparable effect across all Phytophthora isolates, suggesting a relevant potential broad-spectrum protective role. This is particularly relevant considering the biological differences in growth rate, sporangia production, and pathogenicity between P. capsici isolates (Li et al., 2007). A similar effect of B. zhangzhouensis on mycelial growth was reported by Anil et al. (2019), with 62% inhibition of mulberry (Morus spp.) leaf spot. Likewise, Aytenov et al. (2024) found that 47 strains of B. zhangzhouensis obtained from the rhizosphere and phyllosphere of plants in a hypersaline environment displayed broad-spectrum inhibition of fungal mycelial growth against 13 pathogenic fungi.

In the evaluation against sporangia and zoospores of P. capsici, JJR198 completely restricted sporangia formation and halted zoospore release at a bacterial concentration of

1.0 × 10⁸ CFU mL-1 but showed no effect on either at 1.0 × 10⁴ and 1.0 × 10⁶ CFU mL-1. Similar results were described by Kim et al. (2009) and Kim et al. (2010), who studied various Paenibacillus polymyxa strains, observing little to no effect on sporangia formation and zoospore release at low bacterial inoculum concentrations, but complete inhibition at

1.0 × 10⁷ and 1.0 × 10⁸ CFU mL-1, where higher inoculum levels also altered sporangial morphology.

Soliman et al. (2022) determined through mass spectrometric analysis that phenol, 2,4-bis(1,1-dimethylethyl), is the bioactive compound responsible for the antimicrobial activity of B. zhangzhouensis, which they also evaluated as a broad-spectrum biological control agent exhibiting high activity against different pathogens, including fungi, Gram-positive, and Gram-negative bacteria. This compound has been previously reported for its anti-oomycete properties against P. capsici and P. cinnamomi (Rangel-Sánchez et al., 2014; Romero-Correa et al., 2014; Sang et al., 2011; Sang and Kim, 2012).

In vivo evaluations of JJR198 comprised two complementary approaches. First, the bacterium was successfully re-isolated from the rhizoplane and rhizosphere of inoculated pepper plants, confirming its ability to persist in the root environment. Second, and critically, its antagonistic performance against P. capsici was assessed in potted pepper plants, where no reduction in disease severity was observed. This lack of disease suppression, despite effective root colonization, underscores an important limitation in the in planta performance of JJR198 and highlights the discrepancy between root colonization, in vitro antagonistic traits, and effective disease control. Although other B. zhangzhouensis isolates have been reported as rhizosphere-associated or endophytic bacteria with promising plant growth-promoting and biocontrol traits in potted plant and field studies (Bhutani et al., 2021; Carreiras et al., 2023; Karagoz et al., 2018; Maheshwari et al., 2022). Strain JJR198 was found to tolerate up to 7.5% NaCl. Several studies have described B. zhangzhouensis strains as halotolerant bacteria with promising agricultural potential due to their plant growth-promoting and antagonistic properties in saline soils (Aytenov et al., 2024). This is particularly relevant given that soil salinization is a major and increasingly prevalent problem in agricultural systems (Kramer and Mau, 2023). Primarily isolated from marine samples, hypersaline coastal lakes, and the rhizosphere and phyllosphere of plants in saline environments, this species has also been reported to exhibit significant enzymatic activities, including cellulolytic, protease and keratinase (Moridshahi et al., 2020; Niu-Niu et al., 2023; Ruginescu et al., 2018), with proteases being among the main enzymes involved in the degradation of the cell walls of fungal and oomycete pathogens (Latijnhouwers et al., 2003; Carmona-Hernandez et al., 2019).

The results of this study demonstrate that, under in vitro conditions, B. zhangzhouensis JJR198 exhibits potential as a biocontrol agent against P. capsici due to its inhibitory effects on mycelial growth, sporangia formation, and zoospore release. Nevertheless, since it did not show antagonistic activity against Phytophthora root rot in pepper plants under greenhouse conditions, factors limiting its in planta performance, including environmental conditions, rhizosphere dynamics, and host-mediated responses, should be addressed in future studies, while its in vitro activity suggests that this strain could be further explored as a source of antimicrobial bioactive compounds.

This study demonstrated that Bacillus zhangzhouensis JJR198 exhibited in vitro antagonistic activity against several Phytophthora capsici isolates. JJR198 bacterial isolate inhibited mycelial growth by 42.0 to 52.7% across five P. capsici isolates and one P. cinnamomi isolate. The biocontrol response was strongly dependent on the initial bacterial concentration, as sporangia formation and zoospore release were completely suppressed at

1.0 × 10⁸ CFU mL-1, whereas no inhibitory effect was detected at lower concentrations.

Microscopic observations revealed that hyphae and mycelia were covered by bacterial cells, suggesting a possible association between bacterial aggregation and alterations in sporangial development. Additionally, the presence of the bacterial inoculum was confirmed in the rhizosphere and rhizoplane of inoculated pepper plants. However, pretreatment of pepper plants with JJR198 at different times prior to the oomycete inoculation (7, 3, or 0 days) did not reduce disease severity caused by P. capsici under the conditions evaluated.

These findings highlight the in vitro biocontrol potential of Bacillus zhangzhouensis JJR198 against multiple Phytophthora isolates; however, its effectiveness as a live inoculant in pepper plants remained limited under the conditions evaluated. Consequently, further studies are needed to identify the factors restricting the in planta performance of JJR198.

Conflict of interest. The authors declare that they have no conflict of interest in the publication.

Funding. This research received funding from SECIHTI, grant number 2022-000002-01NACF-04856, and from the Universidad Autónoma de Aguascalientes through project PIBT23-3.

Contribution of authors. D.F.-R., N.A.C.-V. and J.J.L.-R. designed the work.: D.F.-R. conducted the experiments and wrote the manuscript. N.A.C.-V. provided partial funding and resources, N.A.C.-V., J.J.L.-R. and J.F.M.-D. corrected and approved the final manuscript. J.F.M.-D. validated and completed the molecular identification data analysis. S.P.F.-P. provided the Phytophthora isolates. S.D.T.-Z. refined the laboratory methodology.