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Characterization and identification of hot pepper-associated endospore-forming bacteria with potential applications as biofertilizers and in biocontrol of pepper wilt pathogens

BMC Microbiology volume 25, Article number: 198 (2025) Cite this article

Abstract

Background

Although hot pepper contributes significantly to Ethiopia’s national economy, its production is hindered by devastating outbreaks of phytopathogens such as Fusarium wilt and Meloidogyne incognita disease complexes. It is known that bacteria in the pepper rhizosphere can promote plant growth by suppressing soil-borne pathogens and producing growth-promoting substances. Therefore, hot pepper-associated endospore-forming bacteria were evaluated for plant growth-promoting traits and in vitro antagonism to pepper wilt-causing pathogens, revealing some potentially valuable isolates.

Results

One hundred and forty-seven heat-resistant endospore-forming rhizobacteria were recovered from 48 rhizosphere samples. Thirty-five of these isolates solubilized phosphate efficiently with solubilization index values of 2.8–10, and produced indole acetic acid (27. 31–59.16 µg/ml). Moreover, 20 isolates hydrolyzed chitin effectively, 21 of them reduced the radial growth of three pathogenic Fusarium oxysporum strains by between 26.7% and 79.2%, and cell-free supernatants of 12 isolates reduced the hatching of M. incognita eggs by 51–96.4% while also increasing juvenile mortality by 45–98.7%. After 16S rRNA gene sequence analysis, 31 of the isolates were identified as Bacillus spp. (B. siamensis, B. velezensis, and B. cereus; n = 26) and Paenibacillus polymyxa (n = 5).

Conclusions

The bacterial strains JUBC7 (B. cereus) and JUBC12 (B. siamensis) have multiple phytobeneficial traits that make them promising microbial inoculants for protecting high value crops against phytopathogens.

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Background

Hot pepper (Capsicum spp.) cultivation in Ethiopia is hindered by the increasingly devastating impact of abiotic and biotic factors [1,2,3,4], causing pepper production to decline from 12.5 t/ha in 2010/11 to 6.5 t/ha in 2021/22 [5]. The primary factors limiting Ethiopian pepper production are diseases and pests such as Fusarium wilt and Meloidogyne incognita, which are particularly prevalent in southwest Ethiopia due to the local cropping system, intensive cultivation, and edaphic factors [6,7,8].

Such problems are not limited to Ethiopia; the emergence and wide distribution of virulent phytopathogens and formae speciales such as Fusarium wilt strains and root-knot nematodes (RKN) is a global problem. Of special concern is the formation of disease complexes in which one pathogen facilitates the invasion of another for example, infestation by RKN species such as M. incognita predisposes plants to Fusarium infection. This problem is especially severe in the tropics where diverse soil-borne diseases are found [9,10,11].

The synergy of Fusarium wilt and RKN during co-infection was first recognized in cotton [12, 13]. Previous studies showed that approximately 7.7 x 104Fusarium conidia (g soil)−1 were required to cause visible foliar wilt symptoms on cotton but just 6.5 x 102 conidia (g soil)−1 were sufficient to induce disease development in the presence of 50 M. incognita individuals [14]. Moreover, combined exposure to M. incognita and Fusarium spp. increased inka nut plant mortality by 25–30% when compared to M. incognita alone (5–10% mortality) [15], and several researchers have noted that pepper cultivation in Ethiopia is increasingly threatened by multiple pathogens, particularly those causing wilt complex diseases [1, 16,17,18,19].

Previous studies have demonstrated the high prevalence of M. incognita on pepper and other vegetables in Ethiopia as follows; this pest’s reported prevalence is 58% in the country’s central and southwestern regions [20], 90% in the eastern region [21], 55.3–57.7% in the central and western regions [22], and 38.5% in the Oromia, SNNP, Tigray, and Amhara regions [23]. In addition, a recent analysis of the Nad5 gene revealed that all available Meloidogyne spp. samples from the pepper-growing region of the southwestern (Jimma Zone) could be assigned to the species M. incognita, whose prevalence in this region ranges from 73 to 89% [8]. Fusarium wilt is also a major pathogen of hot pepper in Ethiopia, with disease incidence rates of 50% in the Benishangul-Gumuz region (northwestern) [24], 18–37% in Wallaga (western) [18], 40–65% in Gojam (northwestern) [16, 25], and 58.3–93% in the central region [26]. Our recent analysis of the ITS region revealed F. oxysporum to be highly abundant in pepper fields within the Jimma Zone [7], which includes some of Ethiopia’s main pepper-growing areas. Yield losses caused by Fusarium wilt are reportedly as high as 68–71% in Ethiopia’s Central Rift Valley [27], demonstrating a clear need for reliable methods of managing soil-borne pathogens in order to sustainably increase Ethiopian pepper production.

Agrochemicals are widely used to control soil-borne pathogens but several studies have shown that these substances can be disappointingly ineffective in addition to causing significant ecological harm [28]. It is therefore notable that recent publications indicate that agricultural production can be sustainably increased by inoculation with plant growth-promoting rhizobacteria (PGPR), which help maintain biodiversity in ecosystems [29, 30]. In particular, endospore-forming rhizobacteria such as Bacillus subtilis, B. velezensis, B. siamensis, B. amyloliquefaciens, B. cereus, and Paenibacillus spp. have drawn attention because they can produce diverse plant growth-promoting substances and antimicrobial agents that combat a wide range of phytopathogens [29, 31,32,33,34,35,36].

The protective effects of rhizobacteria in the pepper rhizosphere have been demonstrated in several studies: rhizobacteria were found to inhibit bacterial wilt colony growth by 61.5% to 72%, and radial growth of Fusarium wilt by 18.6-43.7%, while also reducing M. incognita survival by 16.6-65.8% [29, 34, 37, 38]. Moreover, rhizobacteria have been shown to perform diverse plant growth-promoting functions including phosphate solubilization, nitrogen fixation, biosynthesis of antimicrobials and phytohormones such as indole-3-acetic acid, and induction of systematic resistance in pepper via overexpression of genes encoding enzymes involved in pathogen defense such as peroxidases, polyphenol oxidase, and superoxide dismutase [39,40,41].

Chitinolytic bacteria reduced the occurrence of visible Fusarium wilt symptoms in hot pepper by 29–61% while also promoting increases in shoot height and biomass of 6.5–14.4% and 17.4–87%, respectively [42]. Moreover, P. polymyxa inhibited the radial growth of Fusarium wilt in vitro and reduced the disease severity of F. oxysporum f.sp. capsici on pepper by 67.7% [43], and culture filtrates of the same species (25–100%) inhibited M. incognita egg hatching by 84–91% and increased J2 mortality by 100% in vitro [44]. In addition to producing phytohormones, B. velezensis induced systemic resistance in chili peppers and reduced Fusarium wilt disease severity by 60% [40]. Although the rhizosphere of hot pepper plants from Ethiopia could harbor diverse plant growth promoting rhizobacteria (PGPR), no bacterial species from this environment had previously been shown to exhibit the full range of advantageous traits, which include effective biofertilization and biopesticide activity.

This study aims to address two major challenges in Ethiopian pepper cultivation, namely nutrient depletion and the spread of phytopathogens, both of which severely affect the production of this high-value crop. The growing threat of soil-borne diseases such as Fusarium wilt and M. incognita has created an urgent need for sustainable solutions to protect pepper crops. Endospore-forming bacteria from the pepper rhizosphere with PGPR traits and antagonism towards pathogens could provide such a solution, acting as both biofertilizers and biocontrol agents with the potential to enhance plant growth, improve nutrient uptake, and suppress soil-borne pathogens. By isolating, characterizing, and evaluating hot pepper rhizobacteria for beneficial traits and pathogen antagonism, this study lays a foundation for mass production of biofertilizers and the application of biocontrol agents in Ethiopian agriculture.

Results

Physiological assays and identification of pepper-associated endospore-forming bacteria

A total of 147 bacteria that can withstand heat treatment and dissolve phosphate were collected from 48 soil samples. Thirty-five of these isolates were highly effective at dissolving tricalcium phosphate, with phosphate solubilization index (PSI) values between 2.8 and 10; the isolates with the highest PSI values were JUBC11, JUBC24, and JUBC26 (Table 1 and Fig. 1A). In addition, 29 isolates hydrolyzed chitin, forming clear zones with diameters of 1 to 9 mm, corresponding to chitinolytic index (CI) values of 0.3 to 7.5 (Table 1). Twenty isolates had CI values above 1; those with the strongest chitinase activity were JUBC7 (B. cereus), JUBC12 (B. siamensis), and JUBC13 (B. siamensis) (Fig. 1B). Additionally, 27 isolates (77%) produced hydrogen cyanide (HCN) when grown on a glycine-supplemented tryptic soya agar medium, with JUBC7 (B. cereus) exhibiting the highest HCN production (Fig. 1C). All 35 isolates that could solubilize phosphate also produced indole-3-acetic acid (IAA) at concentrations between 27.31 and 59.16 (µg/ml). The strains JUBC5 and JUBC11 (P. polymyxa), JUBC8 (B. velezensis), and JUBC18 (B. siamensis) produced the largest quantities of IAA (Table 1 and Fig. 1D). The bacterial isolates tended to form white, rough, large, and irregular colonies on growth media. Microscopically, they appeared as Gram-positive endospore-forming rods (Fig. 1E).

Table 1 Taxonomic identity and physiological traits of selected hot pepper rhizosphere-associated bacteria
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Fig. 1
figure 1

Morphological and physiological traits of hot pepper-associated endospore-forming rhizobacterial isolates. A Phosphate solubilization, B Chitinase activity C HCN production, D Indole-3-acetic acid (IAA) production, and E Micrograph showing the rod-like shape of the most active isolates

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Antagonistic effects of bacterial isolates against Fusarium oxysporum

Dual culture assays using three Fusarium wilt isolates were performed to evaluate the rhizobacterial isolates’ ability to inhibit the pathogen’s growth (Fig. 2). In total, 60% (n = 21 isolates) of the rhizobacterial isolates effectively inhibited Fusarium growth.

Fig. 2
figure 2

Antagonistic effects of endospore-forming rhizobacteria against Fusarium wilt. Control plates (left-most column) and treatment plates using three antagonistic rhizobacterial isolates. JUBC7; Bacillus cereus, JUBC12 and JUBC16; B. siamensis. **FI1, FI4, FI5; Fusarium oxysporum isolates 1, 4, and 5

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The antagonistic bacteria demonstrated statistically significant (F = 7.9–19.7, P < 0.0001) levels of inhibition, with inhibition percentages between 26.7 and 79.2% (Table 2). Of the 21 tested bacterial isolates, over 95% markedly inhibited Fusarium radial growth, achieving significant (> 50%) inhibition percentages against FI4 and up to 52.4% inhibition of FI1 but only 28.6% inhibition of FI5 (Table 2). JUBC12 (B. siamensis) and JUBC7 (B. cereus) were the most efficient inhibitors, while JUBC22 (B. velezensis) was less effective against the tested Fusarium isolates (Table 2).

Table 2 Antagonistic effects of pepper-associated rhizobacteria against mycelial growth of Fusarium pepper wilt isolates. Data are presented as means ± SD
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Effect of cell-free supernatant on M. incognita

Cell-free supernatants of 12 rhizobacterial isolates were tested for their ability to suppress egg hatching and the J2 activity of M. incognita (Fig. 3). All of the isolates significantly (P < 0.0001, LSD = 1.3–4.6, CV = 0.9–4.5) suppressed the hatchability of the eggs (> 50%) and increased J2 mortality by over 45% (P < 0.0001, LSD = 1.7–3.3, CV = 1.2–4.1) for all supernatant concentrations) compared to the control group (Fig. 3). The best-performing isolate was JUBC7 (B. cereus), which had the highest egg-hatching inhibition percentage and J2 mortality rate. Increasing the concentration of JUBC7 supernatant from 25 to 100% caused a corresponding increase in egg hatchability inhibition (76–96%) and J2 mortality (73–99%). Similarly, increasing the applied concentration of JUBC12 (B. siamensis) increased inhibition of egg hatching (75–96%) and J2 mortality (64–97%).

Fig. 3
figure 3

Effects of cell-free supernatants of 12 pepper-associated endospore-forming rhizobacterial isolates on egg hatching (A) and J2 mortality of Meloidogyne incognita (B) in vitro. DW; distilled sterile water. NB; nutrient broth. SM; solid medium. JUBC1, JUBC6, JUBC12, JUBC13, JUBC14, JUBC16, JUBC18; Bacillus siamensis. JUBC5 and JUBC11; Paenibacillus polymyxa. JUBC7 and JUBC19; B. cereus. JUBC8; B. velezensis. Line graphs with error bars generated using Origin Pro 2024 version 10.10.178

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Two of the tested bacterial strains, JUBC7 and JUBC12, significantly outperformed the other bacterial isolates in terms of their effects against M. incognita, even at the lowest tested supernatant concentration (25%). For example, JUBC7 and JUBC12 induced 76% and 75% inhibition of egg hatching, respectively, whereas the corresponding values for JUBC11 and JUBC16 were 51% and 55%, respectively. Similar outcomes were observed for J2 mortality (73% for JUBC7 and 64.2% for JUBC12, compared to 45.1% and 45.3% for JUBC13 and JUBC18, respectively). JUBC19 (B. cereus) and JUBC11 (P. polymyxa) also effectively reduced egg hatchability and caused substantial J2 mortality at concentrations of 75% and 100%, respectively (Fig. 3).

Molecular analysis of endospore-forming bacterial isolates

The 16S rRNA gene sequences of 31 bacterial isolates were analyzed, revealing that they belonged to the genera Bacillus and Paenibacillus (Table 3). Each isolate was assigned a unique GenBank accession number starting with OM766257-OM766283, although four were excluded from subsequent analysis due to poor sequence quality. We compared the newly identified isolates to the NCBI database and found that they were all highly similar to known strains, with similarities of 99% to 100% (Table 3).

Table 3 Similarity of the identified isolates to strains retrieved from databases
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The new isolates were closely related to bacteria from diverse sources, as shown by the topology of a Maximum Likelihood tree with bootstrapping values > 50 (Fig. 4). The majority of the isolates (26, corresponding to 83.9% of the total) were assigned to the genus Bacillus, while the remaining 5 (16.1% of the total) belonged to Paenibacillus. Within the Bacillus genus, three different species were identified: B. siamensis (50%), B. velezensis (34.6%), and B. cereus (15.4%). All Paenibacillus isolates belonged to the species P. polymixa (Fig. 4).

Fig. 4
figure 4

Maximum Likelihood phylogenetic tree based on 16S rRNA gene sequences showing the relationship between the potential PGPR isolates from the hot pepper rhizosphere and reference strains from the GenBank database including Pseudomonas protegens (MT355569), which was used as an outgroup taxon. Newly identified species are enclosed in diamond-shaped blue boxes. The numbers on the tree indicate the bootstrap percentages after 1000 iterations

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Discussion

Heat treatment-resistance rhizobacteria were successfully isolated from the rhizosphere of hot peppers (Table 1). Thirty-five of these isolates displayed multiple plant growth-promoting traits including phosphate solubilization, chitin hydrolysis, and production of IAA and HCN, all of which are (along with production of ammonia, siderophores, and antimicrobials) valuable for promoting plant growth [34]. Additionally, 21 isolates effectively suppressed the mycelial growth of pepper wilt fusarium, while 12 strongly inhibited egg hatchability and J2 survival in the disease-causing nematode parasite M. incognita. These findings suggest that the pepper rhizosphere is a rich source of heat-tolerant bacterial strains with remarkable plant growth-promoting properties and strongly antagonistic activity against phytopathogens. This is consistent with previous reports showing that pepper-associated rhizobacteria exhibit biopesticidal and biofertilizing activity in vitro resulting from production of plant growth substances and antagonistic activity against bacterial wilt (R. solanacearum) [29, 34]. In particular, the endospore-forming pepper rhizobacteria B. subtilis, B. amyloliquefaciens, and B. cereus reportedly inhibited bacterial wilt colony growth by 66–68.4%, 64.5%, and 61.5%, respectively [29]. In another study, the inhibition of fungal mycelial growth of Sorghum anthracnose (Colletotrichum graminicola) due to Trichoderma spp. in the range of 60.4- 76.5% was observed on a dual culture plate [45]. The use of beneficial rhizobacteria as biofertilizers and biocontrol agents could thus offer substantial environmental and economic benefits, providing sustainable and eco-friendly alternatives to chemical treatments and reducing reliance on agrochemicals that harm both ecosystems and human health [41, 45, 46]. By suppressing pathogens such as Fusarium wilt and parasitic nematodes, these microbial inoculants enhance the health of soil and crops, which in turn fosters biodiversity and minimizes long-term environmental damage caused by synthetic pesticides and fertilizers. Another important advantage of adopting strategies based on inoculation with beneficial rhizobacteria is that it could significantly reduce input costs for farmers. This is particularly important in tropical agricultural regions such as Ethiopia, where chemical pesticides and fertilizers are expensive and difficult to apply, and farming is predominantly done on smallholdings with high turnover of pathogens/pests. The strains identified in this work could potentially be incorporated into integrated pest management (IPM) systems to improve vegetable production and increase yields [47, 48]. For instance, combined use of biocontrol agents and fungicides increased pepper yields by over 22% in one study [49]. The bacterial strains identified here could further enhance crop production after validation under varied field conditions, offering a cost-effective way to improve both productivity and profitability for pepper growers. Such a shift towards biological control would increase agricultural sustainability and support long-term food security while reducing environmental risks.

Phosphate solubilization activity was assayed using Pikovskaya’s medium. Several isolates produced clear zones indicating varying levels of organic acid production and phosphate mobilization [34, 50], with PSI values ranging from 2.8–10. Three stood out as being especially suitable for use in the acidic soils of Ethiopia’s Jimma areas, where complexation by Fe2+ and Al3+ ions greatly limits phosphate availability to crops [51]: JUBC11 (P. polymyxa), JUBC24 (B. cereus), and JUBC26 (B. velezensis). The genera Bacillus and Pseudomonas thus appear to be the best phosphate solubilizers and suppliers of available phosphate to plants [52]. This is notable because both this work and previous studies have shown that Bacillus spp. are also effective producers of plant growth-promoting substances and antagonists of soil-borne phytopathogens [41].

All 35 of the bacterial isolates produced the phytohormone indole-3-acetic acid (IAA), in concentrations ranging from 27.3 to 59.16 (µg/ml). The highest concentrations were generated by the strains JUBC5 (P. polymyxa), JUBC8 (B. velezensis), JUBC11 (P. polymyxa), and JUBC18 (B. siamensis). Other studies have similarly demonstrated effective IAA production by pepper-associated rhizobacteria [34, 53]. Exogenous IAA can enhance root growth and improve nutrient absorption by plants, especially in nutrient-deficient soils, making IAA production a valuable plant growth-promoting trait. Additionally, 27 of the bacterial isolates produced HCN, with JUBC7 (B. cereus) producing the most. HCN is a rhizobacterial secondary metabolite that can suppress pathogen activity by inhibiting energy metabolism while also promoting plant growth by sequestering metal ions and thus increasing phosphate availability [54].

Twenty-nine of the rhizobacterial isolates demonstrated at least some chitinase activity, forming distinct clear zones on the assay plates. This is desirable for pest control because microbial chitinases can degrade fungal cell walls as well as nematode eggshells and cuticles [37]. Formerly, chitinase-producing bacteria isolated from rhizosphere soil were shown to effectively inhibit the growth of phytopathogenic fungi [55], and treatment with the metabolite of pepper rhizobacteria was recently shown to induce disintegration of the bacterial wilt cell wall and deformation of bacterial wilt cells [29]. The isolates with the highest chitinolytic activity were Bacillus species, namely JUBC7 (B. cereus), JUBC12 (B. siamensis), and JUBC13 (B. siamensis). The CI values of these isolates were all above 1, indicating that they surpass the useful activity threshold for potential biocontrol agents [56]. B. cereus was previously confirmed to produce chitinase [57].

Previous studies have identified the production of diverse secondary metabolites and tolerance of harsh environments as characteristic traits of endospore-forming bacteria [31, 58]. The isolates identified in this work displayed both traits. For example, B. velezensis can combat diverse fungal pathogens including F. oxysporum, F. moniliforme, and C. falcatum due to its production of metabolites that effectively suppress pathogen growth, including antibiotics, lipopeptides, hydrolytic enzymes, and HCN [29, 46, 59].

Supernatants from twelve of the isolates identified in this work also significantly reduced the hatchability of nematode eggs and caused severe J2 mortality in M. incognita. This is consistent with an earlier report noting that a cell-free supernatant from a B. cereus strain isolated from the rhizosphere of healthy tomato plants grown on Chinese farmland infested with M. incognita increased J2 mortality by 92.8% and reduced egg hatching by 33.8% [60]. In another study, supernatants from P. polymyxa reduced M. incognita egg hatchability by 91% and caused 100% J2 mortality in vitro [44]. These nematicidal effects may be due to the production of metabolites such as HCN, chitinase, and other toxic compounds that would both increase J2 mortality and reduce egg hatchability by degrading the cuticle and changing the behavior of nematodes [61,62,63]. The high efficacy of Bacillus strains in this context is supported by the literature: multiple studies have shown that Bacillus spp. increase M. incognita mortality more than other bacterial genera [41, 64]. This may be due to their ability to produce diverse bioactive substances and to form endospores while also competing effectively with phytopathogens.

Several of the isolated bacterial strains thus exhibited both potential PGPR features and strong antagonism towards the phytopathogens Fusarium and M. incognita. Strains exhibiting particularly good activity in both senses included JUBC7 (B. cereus), JUBC12 (B. siamensis), JUBC11 (P. polymyxa), JUBC13 (B. siamensis), JUBC16 (B. siamensis), and JUBC8 (B. velezensis). The results of greenhouse experiments also indicated that JUBC7 (B. cereus) and JUBC12 (B. siamensis) effectively suppressed Fusarium wilt infection and M. incognita infestation while increasing the growth of pepper plants (data to be presented in a forthcoming publication). Previous studies have shown that several of these bacterial species possess diverse phytobeneficial properties that can increase crop production and productivity [29, 40, 43, 44, 65].

The 16S rRNA gene sequence was used to identify 35 phytobeneficial bacterial isolates (four isolates excluded from analysis due to their bad sequences), which were assigned to different species of Bacillus such as Bacillus cereus, B. siamensis, B. velezensis, and Paenibacillus polymyxa. Similarly, earlier workers indicated the presence of those bacteria in the plant rhizosphere, but in this study considerable number and effective strains of P. polymyxa were obtained [29, 31, 34, 38, 66, 67]. The earlier studies also found that endospore-forming bacteria from the pepper rhizosphere produce multiple plant growth-promoting substances and are antagonistic towards various soil-borne pathogens [37, 38] as well as suppressing bacterial wilt [34].

The plant growth promoting activity and phytopathogen antagonism observed among the rhizobacterial isolates identified here is consistent with previous reports. For example, B. velezensis was shown to contain genes promoting plant growth and biocontrol activity, making it a preferred species for developing inoculants [56], and another study showed that B. cereus has typical PGPR characteristics and can combat pathogenic fungi due to its abundance of gene clusters for producing plant growth promoting and antifungal compounds [66]. Additionally, Paenibacillus spp. from plant roots can have multiple growth-promoting traits and a wide range of antimicrobial activities [68], and a B. siamensis strain was found to be antagonistic towards F. oxysporum both in vitro and in vivo [69]. Our results confirm these reported activities and demonstrate the high diversity of endospore-forming bacteria in the pepper rhizosphere.

Conclusions

The results presented here show that the hot pepper rhizosphere harbors diverse microbial inoculants with beneficial plant growth promoting activity and antagonism towards important phytopathogens. It was particularly rich in Bacillus species, with B. siamensis being dominant, and many of the identified strains could potentially be used to manage pepper wilt caused by Fusarium and M. incognita in the major pepper growing areas of Ethiopia’s Jimma Zone. Our analyses suggest that the isolates JUBC12 (B. siamensis) and JUBC7 (B. cereus) have particularly high potential for development as biofertilizers and biocontrol agents against Fusarium wilt and nematode infection. Thus, to exploit these microbial inoculums efficiently, in future, their genes and or metabolites that are responsible for phytopathogen antagonism and producing of plant growth-promoting substances should be determined.

Materials and methods

Origin of pathogens and biocontrol agents

Endospore-forming rhizobacteria were isolated from healthy pepper rhizospheres, while Fusarium isolates were collected from infected pepper plants and M. incognita was collected randomly from nematode-infested pepper fields in the Jimma Zone, where the crop is widely cultivated [7, 8]. Characterization of the Fusarium wilt and rhizobacterial isolates as well as in vitro antagonism tests were performed at Jimma University. DNA extraction and sequencing of Fusarium wilt and rhizobacterial isolates were performed at the Swedish University of Agricultural Sciences (Sweden). Molecular identification of Meloidogyne spp. was conducted at Ghent University (Belgium).

Sample collection and isolation of endospore-forming bacteria from pepper rhizosphere

During the 2018 pepper growing season, 48 pepper plant samples were collected from major pepper cultivating districts in the Jimma Zone. These samples included 24 from the Omo Nada district and 12 each from Sekoru and Kersa districts. In all cases, the sampling sites were similar to those from which M. incognita and Fusarium wilt samples were collected. Healthy plant samples were uprooted along with non-rhizosphere soil from different sites within the study areas. Intact roots and the adhered soil were transferred to a sterile polythene bag with a proper label and transported to laboratories at the Biology Department of Jimma University. After removing loosely adhered soil by gently shaking, the roots were cut into 2 cm pieces along with firmly adhered soil using sterile scissors.

From each rhizosphere sample, 10 g of soil was suspended in a flask containing 90 ml standard saline solution (0.85% NaCl) and kept on a rotary shaker at 125 rpm for 30 min. Upon preparing appropriate dilutions (10–1−10–6 ml−1), the serially diluted samples were heat treated at 80 °C for 10 min to eliminate non-endospore-forming rhizobacteria.

Screening of phosphate solubilizing bacteria

Aliquots (0.1 ml) of each heat-treated diluted sample were spread onto pre-solidified Pikovskaya’s agar plates (10 g glucose, 5 g Ca3(PO4)2, 0.5 g NH4(SO4)2, 0.5 g yeast extract, 0.1 g MgSO4.7H2O, 0.2 g NaCl, 0.2 g KCl, 0.002 g MnSO4, 0.002 g FeSO4, and 15 g Agar in 1L distilled water) and incubated at 32 °C for 2–5 days [70]. Pure cultures were then obtained by separately transferring 10–15 bacterial colonies that formed clear zones on each Pikovskaya’s agar plate into screw cap test tubes containing nutrient broth (i.e., one colony per test tube). One loopful of each of the resulting overnight cultures was re-streaked onto nutrient agar medium (Oxoid, 1.5 g beef extract, 5 g peptone, 1.5 g yeast extract, 5 g NaCl, 15 g agar in 1L distilled water) for verification of purity and further study.

Evaluation of isolates for phosphate solubilization

The phosphate solubilization activity of the rhizobacterial cultures was evaluated by spotting them on a Pikovskaya agar plate that was then incubated for 2 to 5 days at 32 °C. The sizes of the resulting colonies and halo zones were measured with a transparent ruler and the phosphate solubilization index (PSI) was calculated using the following equation [71]:

$$PSI=\frac{Colony\ diameter + Halo\ zone\ diameter}{Colony\ diameter}$$

Assays of plant growth-promoting traits

HCN production assay

A 24 h old culture was spread on pre-solidified Tryptic soya agar medium (5 g peptic digest of soybean meal, 5 g sodium chloride, 15 g agar in 1L distilled water) supplemented with glycine (4.4 g/l). A sterilized Whatman No.1 filter paper was soaked with 2 ml of picric acid solution (2.5 g picric acid and 12.5 g Na2CO3 in 1L distilled water) and placed on the plate’s lid, which was then sealed with parafilm, after which the plate was incubated at 32 °C for seven days. HCN production was evaluated based on the development of orange to red coloration as described previously [54].

IAA production assay

The IAA production of the bacterial isolates was determined using an established method [72]. Briefly, a loopful of a young bacterial culture was inoculated into nutrient broth amended with 5 mg/ml L-tryptophan and incubated at 32 °C for 48 h. After incubation, the culture was centrifuged at 5000 rpm for 20 min, then 2 ml of the supernatant was mixed with two drops of orthophosphoric acid and 4 ml of Solawaski's reagent (50 ml, 35% perchloric acid, 1 ml of 0.5 M FeCl3) in a test tube that was then kept at room temperature for 20 min. The development of pink coloration indicated the production of IAA, which was quantified by measuring absorbance at 530 nm using a V-600 UV spectrophotometer (Japan Spectroscopic Company, JASCO) and the level of IAA production was determined using a standard IAA curve.

Chitinase activity assay

Chitinolytic activity was evaluated using a modification of a published method [56]. A loopful of a young bacterial culture was spotted on pre-solidified minimal salt medium (Na2HPO4 0.65 g, 12.25 g NaCl, 1.5 g KH2PO4, 0.12 g MgSO4, 0.5 g NH4Cl, 0.005 g CaCl2 and 5 g colloidal chitin dissolved in 1L distilled water) in two locations and incubated at 32 °C for seven days, then soaked with Congo red dye 0.1% (w/v) for ten min and washed with distilled water. The diameters of the bacterial colonies and the clear zones formed around them were measured using a transparent ruler, then the chitinolytic index was calculated as follows:

$$Chitinolytic\ index=\frac{Clear\ zone\ diameter- Colony\ diameter}{Colony\ diameter}$$

Characterization of the endospore-forming bacterial isolates

Cultural and microscopic characteristics of isolates

Bacterial isolates considered effective were characterized based on their colonial morphology (i.e., colony color, shape, size, and texture). Additionally, gram and endospore staining was performed for microscopic observation after smear preparation [73].

In vitro assay for antagonism of isolates towards Fusarium and nematodes

Source of Fusarium and nematode pests

Fusarium oxysporum (FI1, FI4, and FI5) isolates were selected based on a preliminary pathogenicity test using the Mareko Fana pepper variety [7]. M. incognita is known to be a major parasite of hot peppers and was collected from infested fields in the Jimma Zone, where its activity is a significant problem [8].

Antagonism towards Fusarium isolates

Thirty-five rhizobacterial isolates (JUBC1-35) displaying PGPR traits were identified using the assays described in the preceding section. Their activity against Fusarium wilt fungi was evaluated using a dual culture technique in which the positions of the pathogen and antagonistic microbes are interchanged on the same medium. For primary screening, a fungal suspension (107 spores/ml) was seeded on a pre-solidified 1:1 mix of PDA (potato dextrose agar) and NA (nutrient agar). Then, 48 h-old bacterial agar blocks (1 × 1.5 cm2) were placed at two positions on the pathogen-inoculated medium and incubated at 28 °C for 5–7 days [74].

Under similar conditions to the preliminary screening test, the degree of fungal radial growth inhibition caused by the bacterial isolates was quantified by placing a 5-day-old Fusarium mycelial disc (1 × 2 cm2) at the center of a 90 mm Petri dish containing a pre-solidified 1:1 mixture of PDA and NA. A loopful of overnight bacterial culture (109 CFU/ml) was then streaked in a broadband fashion about 3 cm away from the mycelial block on two opposite sides of the plate and incubated at 28 °C for 5–7 days until complete plate coverage was achieved by the mycelium of a Fusarium disc on a control plate without antagonistic microbes. These experiments were performed in triplicate. The antagonistic effect of the bacterial strains against the fungal pathogen was quantified by computing the degree of fungal radial growth inhibition relative to the control using the equation shown below [45], where P is the growth inhibition percentage (%), R1 is the diameter of the Fusarium mycelium in the absence of antagonistic bacteria (mm), and R2 is the diameter of the Fusarium mycelium in the presence of antagonistic bacteria.

$$P=\frac{{R}_{1}-{R}_{2}}{{R}_{1}}\times 100$$

Assay of activity against Meloidogyne incognita

Twelve of the rhizobacterial isolates that inhibited Fusarium wilt growth were tested for effects on nematode egg hatching and second-stage juvenile (J2) mortality.

Preparation of cell-free extract

A single colony from each antagonistic bacterium was inoculated into a screw-cap test tube containing 10 ml sterilized nutrient broth and incubated at 32 °C for 48 h. An aliquot of the bacterial culture was then placed in a 2 ml Eppendorf tube and centrifuged at 12,000 rpm for 10 min. The supernatant was passed through a 0.22 µ Millipore filter and used directly in the intended assay while the cell pellet was discarded [75].

Meloidogyne incognita egg hatching and J2 mortality assays

Effects on egg-hatching and J2 mortality were evaluated separately, as described previously [75]. For the egg-hatching assay, the cell-free rhizobacterial supernatants were used as stock solutions (100%) and also diluted with distilled sterile water to obtain dilutions at 75, 50, and 25% of the initial supernatant concentration. For each concentration, 1 ml of supernatant from each of the 12 bacterial isolates was separately placed in one well of a 12-well plate. M. incognita eggs harvested from a susceptible tomato rootstock using 0.5% NaOCl solution with a mean density of 50 eggs per 100 µl) were then placed in the wells and incubated at room temperature. The number of hatched eggs in each well was counted under a stereomicroscope at 24 h intervals for 120 h. The trial was repeated twice and each treatment was tested in triplicate in both cases. Distilled sterile water and autoclaved nutrient broth were used as controls. The egg hatching inhibition rate was calculated using a previously reported equation [75]: I (%) = (C-T)/C × 100, where I represents the egg hatching inhibition percentage, T is the hatchability of the eggs in the treatment groups and C is the hatchability of the eggs in the control group.

To evaluate effects on J2 mortality, a freshly hatched 30/100 µl J2 suspension of M. incognita was placed in each well of a 12-well plate containing 1 ml of cell-free extract (separate multi-well plate were prepared for extract concentrations of 25, 50, 75, and 100%) and in a control plate containing sterile nutrient agar and distilled sterile water in place of the bacterial extract. The plates were then covered with parafilm and incubated at room temperature for 120 h. J2 survival was determined by counting under a stereomicroscope at 24 h intervals for 120 h. Nematodes were considered dead if they did not move when probed with a fine needle. The experiment was repeated twice, with three replicates of each treatment (including controls) in both runs. J2 mortality was calculated using the equation JM (%) = (T/C) × 100, where JM is the J2 mortality, T is the number of dead individuals, and C is the total number of J2 individuals used in the test [75].

Molecular identification of endospore-forming bacterial isolates

Thirty-five (35) endospore-forming bacterial isolates that showed multiple plant growth traits and antagonism towards phytopathogens were selected for molecular identification. Pure cultures of the bacterial colonies were inoculated into Luria–Bertani (LB) broth (Duchefa Biochemie, Netherlands) and incubated at 28 °C for 12 h. Genomic DNA was then extracted from each isolate using a Quick-DNA Fungal/Bacterial Microprep Kit (Zymo Research, Irvine, CA, USA). DNA yield was measured using a NanoDrop microphotometer (NanoDrop Technologies, South San Francisco, CA, USA) and its integrity was checked by electrophoresis on a 1% agarose gel.

The 16S rRNA gene of each isolate was amplified with the following primer pair: 27F (5′-AGA GTT TGA TCM TGG CTC AG-3′) and 907R (5′-CCG TCA ATT CMT TTR AGT TT-3′) [76]. PCR was performed using 1 × PCR buffer, 1.5 mM MgCl2, 200 µM dNTPs, 10 pmol of each primer, 1 U HotStar Taq polymerase (Qiagen Valencia, CA), and 10 ng of genomic DNA as the template set, with the following thermal profile: heating at 94 °C for 3 min, followed by 35 cycles of 94 °C for 45 s, 50 °C for 30 s, and 72 °C for 30 s with a final incubation for 5 min at 72 °C.

The PCR products were purified using the Qiagen PCR purification kit (Qiagen, UK) and sequenced at the Eurofins sequencing facility (Konstanz, Germany). BioEdit version 7.0.5 was used to visualize raw sequences and edit low-quality sequences. The newly obtained sequences were used to perform BLAST similarity searches against the GenBank NCBI database [77]. Search hits for sequences from records in the database were analyzed for sequence coverage and identity, and the best-matched NCBI accession was recorded.

After aligning and trimming the unaligned sequences using Seaview version 5, the newly obtained 16S rRNA gene sequences together with the sequences retrieved from GenBank and a sequence from the outgroup taxon Pseudomonas protegens (MT355569) were used to reconstruct phylogenetic trees using the maximum likelihood method. The support for each branch was estimated using the bootstrap method with a heuristics search and 1000 replicates.

Data analysis

Data on bacterial physiology, antagonistic activity against Fusarium wilt, and inhibition of egg hatching and J2 mortality in M. incognita were analyzed using SAS statistical software version 9.3 (SAS Institute, 211). Duncan’s multiple range test (DMRT) was used to compare the means for each treatment at P < 0. 05. Graphs showing the results of the nematode antagonism assays were generated using Origin Pro 2024 version 10.10.178.

Data availability

The nucleotide sequences of the 16S rDNA gene and accession numbers of bacterial isolates deposited in the GenBank database under accession numbers OM766257- OM766283(https://www.ncbi.nlm.nih.gov/Genbank/update.html) and also ITS region sequences and accession numbers of the Fusarium isolates; OQ771938- OQ771941 (https://submit.ncbi.nlm.nih.gov/subs/?search=SUB13039407). For the rest of the experiments, their analyzed data and material are available in the manuscript.

Abbreviations

CI:

Chitinolytic index

HCN:

Hydrogen cyanide

IAA:

Indole-3-acetic acid

IPM:

Integrated pest management

JM:

J2 mortality

J2:

Second-stage juvenile

LB:

Luria-Bertani broth

NA:

Nutrient agar

PDA:

Potato dextrose agar

PGPR:

Plant growth-promoting rhizobacteria

PSI:

Phosphate solubilization index

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Acknowledgements

The authors would like to express their gratitude to Jimma University for providing financial support and access to facilities. RRV acknowledges support from FORMAS (2019–01316), the Carl Tryggers Foundation for Scientific Research (CTS 20:464), the Crafoord Foundation (20200818), the Novo Nordisk Foundation (0074727), and the SLU Centre for Biological Control.

Funding

This work was supported by Jimma University.

Author information

Authors and Affiliations

  1. College of Agriculture and Veterinary Medicine, Jimma University, P.O. Box 307, Jimma, Ethiopia

    Shiferaw Demissie Tola & Beira Hailu Meressa

  2. Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia

    Diriba Muleta & Fassil Assefa

  3. Swedish University of Agricultural Sciences (SLU), Box 190, Lomma, SE-234 22, Sweden

    Farideh Ghadamgahi & Ramesh Raju Vetukuri

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  2. Diriba Muleta
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  3. Fassil Assefa
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  4. Farideh Ghadamgahi
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  5. Ramesh Raju Vetukuri
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  6. Beira Hailu Meressa
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Contributions

SDT and BHM designed and executed the experiments and carried out the study. SDT wrote the draft manuscript. RRV and FG carried the molecular part. SDT analyzed the data including the bioinformatics aspect. SDT, BHM, DM, FA, and RRV read and approved the final manuscript.

Corresponding authors

Correspondence to Shiferaw Demissie Tola, Ramesh Raju Vetukuri or Beira Hailu Meressa.

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Tola, S.D., Muleta, D., Assefa, F. et al. Characterization and identification of hot pepper-associated endospore-forming bacteria with potential applications as biofertilizers and in biocontrol of pepper wilt pathogens. BMC Microbiol 25, 198 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12866-025-03896-7

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12866-025-03896-7

Keywords

BMC Microbiology

ISSN: 1471-2180

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