Biological Control of Diseases Caused by Rhizoctonia solani AG-4 in Bean (Phaseolus vulgaris L.) using Rhizobacteria and Endophytic Bacteria

Beans (Phaseolus vulgaris L.) are nutritionally valuable crops consumed fresh, dried, or canned. However, various pathogens, especially the soil-borne Rhizoctonia solani Kühn, which causes root rot, significantly limit production (Meziadi et al., 2016). This widespread disease leads to 20-40% global yield losses (Vural and Soylu, 2012; Bhamra et al., 2022; Sulaiman and Bello, 2024) and is difficult to control due to the long-term survival of its reproductive structures in the soil. Control strategies include crop rotation, resistant cultivars and primarily fungicides. Although resistant varieties are most effective, their development is time-consuming and new pathogen strains may overcome resistance genes (Palacıoğlu et al., 2019).
       
The primary strategy against these diseases is the use of agricultural pesticides. Although effective in reducing R. solani-related symptoms, they do not eliminate sclerotia. Due to concerns about environmental and human health impacts and resistance development, many active ingredients have been banned or increasingly restricted worldwide. Microbial activity also influences the spread of soil-borne diseases. However, natural beneficial microorganisms may not exist in sufficient quantities. Therefore, microbial fertilizers and biopreparations are used to boost disease-suppressing microbes in the soil. Consequently, interest in biological control as an alternative approach has grown (Patel and Singh, 2021).
       
Biological methods, particularly those utilizing antagonistic interactions of soil- or plant-associated microor-ganisms, are increasingly favored in plant disease prevention. Plant growth-promoting (PGP) bacteria have proven effective against soil-borne diseases (Bozic et al., 2014). These bacteria colonize both the rhizosphere and internal plant tissues as endophytes, residing within sterilized tissues without harming the host. They inhabit various organs such as roots, stems, leaves, seeds, fruits and xylem vessels. Rhizospheric and endophytic bacteria offer eco-friendly disease control while supporting sustainable plant growth (Adeleke et al., 2021; Abbas et al., 2024). In recent years, Pseudomonas, Bacillus and Trichoderma species have been widely used as biocontrol agents, leading to the development of numerous biopreparations.
       
Biological control studies highlight the potential of endophyte-containing weeds, especially Datura stramonium (Jimsonweed) from the Solanaceae family. Despite its harmful effects on crops, D. stramonium is a strong competitor. Its rising interest stems from its resistance mechanisms and endophytic bacteria that may suppress or eliminate pathogens while promoting beneficial plant processes. Endophytic antagonistic bacteria isolated from bean fields and Datura species have demonstrated effectiveness against various root diseases (Ben Abdallah et al., 2016; Sendi et al., 2020).
       
Beyond their biocontrol effects, certain bacterial species produce hydrogen cyanide (HCN), siderophores, cyclic lipopeptides, indole-3-acetic acid (IAA) and solubilize phosphate (Keshavarz-Tohid  et al., 2017). Investigating the traits of plant growth-promoting microorganisms aids in developing more sustainable biological control strategies.
This study aimed to identify bacterial biocontrol agents from indigenous plant and soil samples as an alternative method to control Rhizoctonia solani-induced root rot in beans (Phaseolus vulgaris L.), particularly in the Konya region where the disease is prevalent.

This study was conducted at the laboratories of the Department of Plant Protection, Faculty of Agriculture, Selçuk University, Konya, Turkey, during the research period 2021-2023.
 
Source of the pathogen and inoculum preparation
 
Rhizoctonia solani (GenBank accession no. OQ526209.1) was obtained from the Mycology Laboratory, Faculty of Agriculture, Selçuk University, Konya. It was isolated from infected bean roots showing typical symptoms in a Konya field (Salman and Boyraz, 2023). For inoculum preparation, fungal isolates from PDA stock cultures were incubated at 28±2^oC for 7 days to obtain fresh cultures.
 
Isolation of endophytic and rhizospheric bacteria
 
Healthy bean (Phaseolus vulgaris L.) and Datura spp. plants were collected from the same fields for bacterial isolation. Soil samples from their rhizospheres were taken to study rhizospheric bacteria. Endophytes were isolated from plant tissues (leaves, flowers, roots, stems) following Küsek (2007) for soil bioagents and Sendi et al., (2020) for plant tissues. Plant materials were washed, surface-sterilized in 1% NaOCl for 1 minute, rinsed thrice with sterile distilled water, dried on sterile paper and placed on nutrient agar. After 24-48 hours incubation, bacterial colonies were purified by streaking on fresh plates. Pure rhizospheric and endophytic cultures were stored at -20^oC in 30% glycerol.
 
Characterization of bacteria
 
The Gram reaction of endophytic and rhizospheric bacteria cultured for 48 hours was assessed using the potassium hydroxide (KOH) test. For hypersensitive response (HR) testing, bacterial isolates were infiltrated into tobacco leaves and incubated for 48 hours; HR-inducing samples were removed (Schaad et al., 2001). Isolates were stored at -20^oC in 30% glycerol. Subsequently, their antagonistic effects against plant diseases were evaluated. Bacteria showing antagonism were tested for phosphate solubilization, hydrogen cyanide (HCN) production, indole-3-acetic acid (IAA) production, siderophore production and ACC deaminase activity. Effective bioagents were selected for in vivo trials based on a weighted scoring system.
 
Determination of antagonistic effects of bacteria
 
Antifungal activity and antibiosis-based control of R. solani by bioagents were evaluated using the dual culture test. A 5 mm R. solani disc was placed at the center of PDA plates (antibiotic-free) and bacterial cultures were streaked circularly 3 cm from the center. After 7 days incubation at 27^oC, fungal growth in treated plates was measured and compared to controls. The experiment used a randomized complete block design (RCBD) with three replicates per treatment. Inhibition percentage was calculated as follows (Tariq et al., 2010).
 

% inhibition = [1 - (Treatment growth/Control growth)] x 100

 
Phosphate solubilization
 
Bacterial colonies were inoculated on Pikovskaya (PVK) medium containing Tri-Calcium Phosphate (TCP) (Pikovskaya, 1948). After 7 days incubation at 28±2^oC, colonies with clear halos were considered phosphate-solubilizing (positive).
 
Indole acetic acid (Iaa) production
 
IAA synthesis was detected following modified methods of Ambrosini and Passaglia (2017) and Aktan and Soylu (2020). Bacteria (108 cells/ml) were cultured in LB medium with 1 g/l L-tryptophan at 30^oC, 200 rpm for 3 days. After centrifugation (5000 rpm, 30 min), 1 ml supernatant was mixed with 4 ml Salkowski reagent and two drops of orthophosphoric acid. A pink color after 30 minutes indicated IAA production.
 
ACC deaminase production
 
The presence of ACC deaminase was inferred from the ability of isolates to utilize ACC as the sole nitrogen source, following Ambrosini and Passaglia (2017). Bacteria were streaked onto DF medium with and without 50 µl of 0.5 M ACC. After 48-72 hours of incubation at 27^oC, growth on ACC-supplemented plates compared to ACC-free controls indicated positive ACC deaminase activity.
 
Siderophores production
 
Siderophore activity was assessed using the method of Schwyn and Neilands (1987). Bacterial cultures (10⁸ cells/ml) were streaked four times onto Blue-CAS agar. Orange-yellow halos around colonies indicated siderophore production.
 
Hydrogen cyanide (HCN) production
 
Bacteria were streaked on Tryptic Soy Agar (4.4 g/l) and incubated at 28^oC for 3 days. A single colony was then restreaked onto fresh medium. A filter paper soaked in 0.5% picric acid-2% sodium carbonate was placed on the lid and plates were incubated inverted at 28^oC for 3-5 days. A yellow to brownish-orange color change indicated HCN production (Bakker and Schippers, 1987).
 
Selection of bacteria according to the weighted rating system
 
Candidate bacterial isolates were evaluated using a weighted rating system (WRS) based on predefined coefficients and their performance in characterization tests. Isolates with the highest scores were considered the most effective (Koçak and Salman, 2023).
 
Molecular characterization
 
Isolates identified as effective via the weighted rating system were analyzed through 16S rRNA gene sequencing. PCR amplification was performed using universal primers 27F (5'-AGA GTT TGA TCM TGG CTC AG-3') and 1492R (5'-ACG GCT ACC TTG TTA CGA CTT-3') (Weisburg et al., 1991) with a PTC 100 thermal cycler (M.J. Research, USA). Sequences from 10 isolates were compared with NCBI database entries. Partial 16S rDNA sequences were submitted to GenBank under accession numbers PP125781.1–PP125790.1.
 
Determination of in vivo biocontrol activity
 
Bacterial isolates effective in vitro were further tested in vivo against R. solani. Bean seeds were surface-sterilized with 2% sodium hypochlorite, rinsed with sterile water and pre-germinated on diluted water agar. Upon sprouting, three seeds were sown per pot containing 2 kg sterilized peat-perlite mix. Bacterial suspensions (10⁸ cells/ml) were prepared from 48-hour cultures and seeds were bioprimed by immersion in 15 ml of the suspension for 10 minutes. Barley grains inoculated with R. solani and treated seeds were placed in pots, followed by the addition of the remaining bacterial suspension. Root lesions were evaluated using the scale of Muyolo et al., (1993): healthy seedlings (0 points); very small, superficial brown lesions on roots or stems (1 point); deep and wide lesions on roots or stems with retardation in root development (2 points); severe root rot with deep lesions surrounding the main root or stem and significantly reduced root length (3 points); and dead plants (4 points). All experiments were performed in triplicate. Biocontrol efficacy was calculated using Abbott’s formula:
  

  

 
Statistical analysis
 
Disease scale values were analyzed using Minitab 17 and mean differences were evaluated with Fisher’s LSD test at P = 0.05.

Identification of selected antagonistic bacteria
 
Survey studies in eight bean fields yielded 56 samples from soil and healthy plants (Phaseolus vulgaris L. and Datura spp.), resulting in 175 bacterial isolates 140 from plant tissues and 35 from rhizospheric soil. Among the 10 most bioactive isolates, 9 were endophytic and only one (DP143.6) was rhizospheric.
       
Dual culture and biochemical test results were evaluated using a weighted rating system with a base score of 70. The top 10 isolates were identified by 16S rRNA gene sequencing and sequences were submitted to NCBI GenBank (Table 1).


       
BLAST comparisons with GenBank reference sequences confirmed ~100% similarity to Bacillus spp. (Fig 1). Phylogenetic analysis revealed that Bacillus altitudinis DP26 formed an early-diverging branch, suggesting distinct evolutionary history. B. pumilus DP25, B. aerophilus DP45 and B. altitudinis DP88 clustered in a strongly supported clade (100% bootstrap), indicating close genetic relatedness. Similarly, B. amyloliquefaciens DP76 and B. velezensis DP98 shared a common ancestor. B. halotolerans DP47 and DP48 grouped with B. subtilis DP143.6, also with 100% bootstrap support, indicating close genetic affiliation.


 
In vitro dual culture antagonism
 
Fifty-three bacterial isolates showed efficacy against Rhizoctonia solani. Some isolates completely inhibited mycelial growth, while others partially controlled it. Among these, 29 exhibited over 70% antagonism and four (DP25, DP64, DP81, DP98) achieved 100% inhibition. The remaining isolates showed 62–70% inhibition. Forty-two isolates were endophytic and the rest were rhizospheric. Dual culture assessed antagonistic activity, complemented by biochemical (PGP) traits. The top candidates were selected via a weighted grading system and tested on host plants.
       
The weighted evaluation results in Fig 2 and Table 2 reveal significant differences among the 10 bacterial isolates in suppressing R. solani.




 
Biochemical properties of bacterial antagonists
 
Growth-promoting potential (PGP) of 53 biocontrol isolates was assessed using a standard curve, revealing that 81% produced IAA between 2.2-13.2 ppm; all but DP76 and DP78 showed strong IAA activity. Except for 13 isolates, all tested positive for siderophore production (2-32 mm). While 30 isolates solubilized phosphorus, only DP78, also selected in vivo, was negative. Among four ACC-positive isolates, DP48 used in the next study phase tested positive. Only seven isolates produced HCN, including DP78. Ten isolates with the highest activity were selected for in vivo tests (Table 3). Biochemical and dual culture results were scored via a weighted rating system with a base of 70 points.


 
Ability of bacterial isolates to control the pathogen in vivo
 
Under climate chamber conditions, the ability of bacterial isolates to control Rhizoctonia solani induced root rot in beans was evaluated. All isolates significantly (P<0.05) reduced disease severity compared to the negative control (Table 4, Fig 3).




       
Biocontrol efficacy was calculated by comparing treated plants to the positive control and is presented in Table 4. Except for Bacillus amyloliquefaciens DP88, nine isolates showed over 50% efficacy in vivo. Bacillus pumilus DP25 showed the highest control (87%), followed by B. halotolerans DP48 (82%) and B. amyloliquefaciens DP76 (80%). Bacillus amyloliquefaciens DP78, B. altitudinis DP26 and B. subtilis DP143.6 exhibited 73-76% efficacy, while B. halotolerans DP47 and B. velezensis DP98 had similar inhibition (~78%). Except for B. aerophilus DP45 (53%) and B. amyloliquefaciens DP88 (40%), eight isolates demonstrated strong antagonistic potential as biocontrol agents against bean root rot. Fig 3 shows the effectiveness of bacterial bioagents at a high efficacy against R. solani in vivo.
       
R. solani survives in soil for years due to its sclerotia, making control difficult; thus, chemical methods are often preferred (Liu et al., 2021). However, ecological alternatives using chemical-free products are increasingly recommended to avoid harmful effects on human health and the environment. Many countries market microorganisms with biocontrol activity, known as biopesticides (Abbas et al., 2019). This study evaluated the traits and biocontrol potential of naturally occurring endophytic and rhizospheric bacteria against R. solani induced bean root rot. Unlike previous research focusing solely on rhizobacteria, isolating both bacterial types from the same ecosystem allows comparative analysis of their biocontrol mechanisms.
       
The Bacillus genus, widely used as beneficial biopesticides, included many species isolated in this study. Previous research has demonstrated Bacillus potential as biological control agents against R. solani. In the dual culture test, 32 isolates significantly (over 70%) inhibited R. solani mycelial growth, consistent with earlier studies using bacterial biocontrol agents against this pathogen (Aydın, 2022; Lan et al., 2024).
       
Bacillus species exhibit diverse antifungal mechanisms, producing low molecular weight antifungal compounds, especially lipopeptide antibiotics (bacillomycin, fengycin, surfactin) and bacteriocins critical for biocontrol (Radhakrishnan et al., 2017). Our study indicates that B. subtilis isolates with high inhibition likely produce these lipopeptides, as supported by Mnif and Ghribi (2015). Biocontrol agents with lipopeptide genes have been commercialized due to their enhanced fungal suppression and antibiotic production (Joshi and McSpadden Gardener, 2006). Recent genomics reveal that gene copy number variations, such as ituD, affect lipopeptide synthesis and biocontrol efficacy (Wang et al., 2024; Bai et al., 2025). Genetic analysis of our top isolates could clarify their mechanisms.
       
Endophytic Bacillus species have gained attention for their positive impact on plant health. Fall et al., (2004) reported root colonization by B. subtilis. In this study, endophytic B. subtilis strains showing 73% inhibition underscore their dual biocontrol potential in both rhizosphere and plant tissues.
       
Martins et al., (2018) similarly confirmed that 76-80% of isolated B. amyloliquefaciens strains function as effective biocontrol agents. Our findings further support the hypothesis that the tested isolates suppress pathogens primarily through antibiosis.
       
Endophytic bacteria appear more prominent in the plant microbiome than rhizospheric ones, as 99% of the isolates effective against R. solani in this study were endophytes. They offer stronger protection against environmental stress (Miliute et al., 2015). Unlike rhizobacteria, which mainly promote growth, endophytes form stable, direct interactions with plants, ensuring more consistent and lasting protection.
       
Bacillus species are among the most important plant growth-promoting (PGP) bacteria, capable of producing phytohormone-like compounds that stimulate plant growth. Various species, including B. megaterium, B. thuringiensis, B. pumilus, B. weihenstephanensis, B. cereus, B. toyonensis and B. subtilis, have been isolated from rhizospheric and endophytic environments and reported to produce indole-3-acetic acid (IAA) (Martins et al., 2018; Shabana and Ambreen, 2019). Studies suggest that approximately 80% of plant-associated bacteria are capable of IAA production; for instance, 81% of 61 isolates from the potato rhizosphere were shown to produce IAA (Calvo et al., 2010). However, the amount of IAA produced varies among isolates. For example, endophytic and rhizospheric bacteria isolated from wheat have been reported to produce IAA in the range of 0.27 to 77.98 ppm (Majeed et al., 2015). In our study, 64.2% of the tested isolates produced IAA within the range of 1.4 to 13.2 ppm. These findings align with those of Elsoud et al., (2023), who reported significant in vitro IAA production by Bacillus species. However, it is important to note that in vitro IAA production levels (1.4-13.2 ppm) may not directly reflect field conditions. Boonmahome and Mongkolthanaruk (2023) demonstrated that IAA stability decreases by up to 70% when soil pH falls below 6.5 and temperature drops.
       
Positive phosphate-solubilizing activity was observed in 45% of the tested isolates. Phosphate-solubilizing bacteria enhance the availability of phosphorus to plants by mineralizing organic phosphorus compounds and converting inorganic phosphorus into more usable forms. The acids produced by these bacteria lower the pH, releasing phosphorus trapped in calcareous soils and contributing to biological disease control (Rasul et al., 2019). Phosphatase activity was detected in 15% of the 499 potential PGP bacterial isolates obtained from 39 different soil samples (Çetinkaya Yıldız and Aysan, 2014). Furthermore, many studies have shown that endophytic and rhizospheric bacteria possess phosphate-solubilizing capabilities (Ahmad et al., 2008; Özaktan et al., 2015).
       
Hydrogen cyanide (HCN), a broad-spectrum antibacterial agent, is used in biocontrol of root diseases by inhibiting cytochrome oxidase in the respiratory chain, thereby disrupting pathogen energy synthesis. Our study found that the tested bacteria produced notable levels of HCN. While HCN is recognized as a key secondary metabolite in biocontrol (Nandi et al., 2017), its role in suppressing pathogenic fungi remains debated, likely due to differing experimental conditions (Michelsen and Stougaard, 2012).
       
Siderophore production is an effective strategy employed by bacterial biocontrol agents (Kesaulya et al., 2018). In particular, Bacillus species synthesize siderophores that chelate and solubilize iron, thereby promoting plant growth. Additionally, they inhibit the growth of phytopathogenic bacteria and fungi, while supporting water movement and ionic balance in plant tissues (Joshi and McSpadden Gardener, 2006). In this study, 85% of the isolates were capable of producing siderophores, forming halos ranging from 2 to 32 mm in diameter. Bacillus amyloliquefaciens DP78 exhibited a high antagonistic potential with a 25 mm halo. Priyanka Agrawal  et al. (2017) reported a strong correlation between siderophore production and antifungal activity.
       
Furthermore, plant growth-promoting (PGP) bacteria are known to utilize 1-aminocyclopropane-1-carboxylate (ACC) as a nitrogen source by converting it into ACC deaminase (ACC-d), which enhances plant growth (Gupta and Pandey, 2019). However, only 7.6% of the isolates in this study demonstrated ACC-d activity.
       
The top ten isolates were identified by 16S rRNA sequencing following characterization and dual-culture tests. These included B. pumilus DP25, B. halotolerans DP48, B. amyloliquefaciens DP76, DP78 and others. Numerous studies have demonstrated the effectiveness of Bacillus species as biocontrol agents against R. solani in vitro and in vivo (Sunkad et al., 2023; Sirivella et al., 2025).
       
In vivo biocontrol tests provided a clearer evaluation of the isolates’ antagonistic potential, highlighting that in vitro and in vivo results do not always correlate. Notably, B. amyloliquefaciens DP88 showed strong in vitro antagonism but only 40% disease inhibition in vivo. Three isolates B. pumilus DP25, B. halotolerans DP48 and B. amyloliquefaciens DP76 exceeded 80% efficacy. Others (B. halotolerans DP47, B. velezensis DP98, B. amyloliquefaciens DP78, B. altitudinis DP26, B. subtilis DP143.6 and B. aerophilus DP45) inhibited R. solani between 53% and 78%.
       
Our study identified B. amyloliquefaciens DP76, B. halotolerans DP48 and B. pumilus DP25 as the most effective biocontrol agents against R. solani. These findings support previous research (Feng et al., 2022;  Abdelaziz  et al., 2023; Kumar et al., 2023; Resmi et al., 2024) and offer new strategies for sustainable agriculture.

The results indicate that both plant tissues and soil may serve as reservoirs for bacteria with diverse PGP traits and antagonistic activity. Due to their endospore-forming ability, Bacillus isolates can persist under varying environmental conditions. Therefore, the tested isolates hold potential as antagonistic PGP agents against R. solani in bean cultivation. However, field trials are needed to validate their biocontrol and growth-promoting effects. Most endophytic and rhizospheric bacteria identified in this study show promise as biocontrol agents in commercial bean production and may also enhance soil health, nutrient availability and microbial balance, contributing to improved crop yield.

The present study was supported by Selcuk University Scientific Research Projects Coordinatorship (Project number: BAP-21401059).
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.
 

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.