Eco-friendly Management of Rhizoctonia solani, the Cause of Tomato Root Rot Disease

The tomato (Solanum lycopersicum L.) holds considerable importance as a food source that is cultivated and exchanged on a global scale, this species belongs to the Solanaceae family, which encompasses approximately 3000 species (Padmanabhan et al., 2016). The tomato crop stands out among all major food crops for experiencing the highest yield losses (Roux et al., 2014). Soil-borne pathogens, especially R. solani, are the leading pathogenic agents that cause disease in tomato crops (Hussein, 2023). The decline in tomato production in Iraq can be linked to various factors, such as the contamination of the soil with pathogenic fungi such as R. solani (Hussein et al., 2022). R. solani, a soil-borne pathogen that causes seedling damping-off and foot rot in tomatoes, was found by Gondal  et al. (2019). R. solani, the main species within the genus Rhizoctonia, is a soil-dwelling plant pathogen (Bhamra et al., 2022), it shows considerable differences in cultural morphology, host range and levels of aggressiveness. It has a documented history of inflicting losses on economically important crops worldwide (Ajayi-Oyetunde and Bradley, 2022). R. solani affects plants during various growth stages, resulting in considerable reductions in crop yield, the pathogen has the capability to infect seeds present in the soil, seedlings at any stage of emergence, roots and multiple aerial parts of plants such as pods, fruits, leaves and stems (Agrios, 2005). The implementation of biocontrol methods in agricultural production presents a promising solution (Lavanya et al., 2023), as it decreases the reliance on harmful fungicides, thereby lessening pollution within the ecosystem (Madbouly, 2018). Biological control agents function as either outcompeting pests or directly eliminating them (Qaisy et al., 2016; Hussin et al., 2018). In contrast to chemical pesticides, the application of biocontrol agents presents numerous benefits, the outcomes encompass a reduced environmental impact, minimized harm to non-target species and a lower likelihood of target pests developing resistance to pesticides (Alwan, 2018; Lahlali et al., 2022). This study focused on assessing rhizobacterial isolates as a sustainable biological alternative to chemical fungicides and fertilizers for the protection of tomato plants.
 

Antagonistic rhizobacterial isolates used
 
Four rhizobacterial isolates of L. adecarboxylata DKS3, B. halotolerans DMC8, B. subtilis NAS1 and P. polymyxa TRS4 were obtained from a prior study (Hussein et al., 2024).
 
Assessment of antagonistic activity
 
The fungal pathogen R. solani was isolated from infected tomato roots in a previous study (Hussein et al., 2022).  The dual culture method was utilized to evaluate the antifungal efficacy of four rhizobacterial isolates in vitro, adhering to the protocol established by Hussein  et al. (2024). The experiment comprised three replicates and was performed twice. The inhibition zone was measured using the following formula:
 

 

Characterization of rhizobacteria
 
The morphological characteristics of the 4 rhizobacterial isolates, such as their colony boundary, color and form, as well as their Gram staining, were examined according to Kloepper  et al. (1992). The durability of bacterial isolates at several temperatures was assessed in Nutrient Broth injected with 50 µl of bacterial cultures (109 cfu ml^-1). The cultures were incubated at 15^oC, 25^oC, 35^oC, 45^oC and 50^oC. The optical density (O.D.) was determined at 420 nm using a UV-Vis spectrophotometer (Shimadzu, Japan) after 48 hours (Hussein, 2019). To assess the growth of bacteria at different pH levels, 50 ml of bacterial cultures (109 cfu ml^-1) were used to inoculate nutritive broth maintained at different pH values (pH 4, 5, 6, 7, 8, 9) using either HCl or 1N NaOH. O.D. value was measured at 420 nm in a UV-Vis spectrophotometer 48 hours later. The experiment conducted with three replicates and repeated twice (Hussein et al., 2024).
 
Greenhouse experiment
 
Four rhizobacterial isolates were evaluated in greenhouse studies for their efficacy in controlling root rot disease in tomato plants caused by R. solani, as well as their capacity to enhance plant growth. All bacterial isolates were grown in Nutrient broth for 48 hours at 27^oC with 150 rpm shaking. The bacterial cells were pelleted by centrifugation at 6000 rpm for 15 minutes and the cell pellet was resuspended in 2 ml of sterile distilled water. The bacterial suspension was adjusted to 109 cfu ml^-1 in sterile 1% carboxymethyl cellulose (CMC) solution. The fungal inoculum was generated by cultivating R. solani on sterilized millet grains for 20 days at 28^oC, after which the colonized grains were dried, crushed and combined with sterilized soil at a ratio of 10 g per kg of soil, following the procedure of Etebarian  et al. (2000). The seeds of tomato (cv. Super Marmande) were subjected to surface sterilization using a 2% sodium hypochlorite solution. The seeds were submerged in a specialized bacterial solution containing 1% CMC and subsequently air-dried, resulting in a bacterial cell population of 10^-9 to 10^-11 colonies per seed. Plastic pots (1 Kg) filled with sterile soil and peat moss in a 2:1 volume ratio. Ten grams of fungal inoculum were introduced seven days before seed sowing for the treatments involving pathogenic fungus. Each pot included five coated seeds, except for the negative control treatment, which was exclusively treated with a 1% CMC solution and excluded any bacterial inoculum. The pots were kept in a greenhouse using a fully randomized design (CRD) with four replications. At 0.05% level of significance, means were compared using LSD. The treatments included negative and positive control and rhizobacterial isolates individually and against R. solani (Rs).
       
Percentage of seed germination was calculated after 15 days of seed sowing as follows (Hussein, 2024):

 

The disease incidence (DI) was determined and expressed as a percentage of diseased plants (Hussein and Al Zubidy, 2019) as follows:

 

The disease severity index (DSI) was assessed with a six-class scale described by Castro  et al. (2017) and the disease severity index (%) was calculated (Hussein and Ibrahim, 2019) as follows:

 

 
Further, the growth parameters comprising of fresh and dry weight of shoot and root systems were measured and recorded.
 
Assay of enzyme activity in tomato plants
 
Peroxidase
 
For each treatment, 0.5 g of leaf tissues were utilized, which was subsequently macerated in 2 ml of a phosphate buffer solution composed of Na₂HPO₄ and NaH₂PO₄ at a concentration of 0.1 M and a pH of 6, maintained at 4^oC. The enzyme extract was acquired by filtering the solution through a linen cloth, facilitating the measurement of the peroxidase enzyme. The tubes were centrifuged for 15 minutes at 6000 rpm and 4^oC. The peroxidase enzyme activity was assessed using a UV-Vis spectrophotometer (Infitek Co., China) by mixing 1 ml of the supernatant with 1 ml of 20% hydrogen peroxide and 1 ml of 1% catechol. The mean of four repetitions was calculated to assess the variation in light absorption at 470 nm (Hussein et al., 2024).
 
Phenylalanine ammonium lyase (PAL)
 
To assess the activity of the phenylalanine ammonium lipase (PAL) enzyme, 2.0 g of leaf tissues from each treatment were homogenized and subsequently diluted 1:2 with phosphate buffer at pH 7. The test tubes were filled with the leaf mixture and the phosphate buffer solution. The tubes were centrifuged for fifteen minutes at a speed of 4,000 rpm. The activity of the PAL enzyme was assessed using a UV-Vis spectrophotometer (Infitek Co., China) by combining 0.2 ml of the filtrate, 0.5 ml of a 0.5 M Tris-HCl buffer solution at pH 8 and 0.5 ml of phenylalanine at a concentration of 6 micromoles in test tubes. Following 60 minutes of incubation at 37^oC. The hue shift at 290 nm was subsequently recorded by averaging four repeats (Hussein  et al., 2022; Hussein et al., 2024).

Antagonistic assay
 
The inhibition rates of four rhizobacterial isolates against R. solani varied from 66.67% to 100.00% (Table 1). The isolate Bacillus halotolerans DMC8 demonstrated superior performance, exhibiting 100% antagonistic activity (Fig 1). Rhizobacteria are recognized for their ability to produce various secondary metabolites with antifungal properties (Wani et al., 2022). The variation in antifungal efficacy may be attributed to disparities in the ability of rhizobacterial isolates to produce and release these bioactive compounds (Hussein et al., 2024). Isolates like DMC8, TRS4, DKS3 and NAS1 may possess unique metabolic pathways or genetic attributes that enable them to synthesize potent antifungal agents (Wani et al., 2022).
 
 



Morphological and physiological evaluation of rhizobacteria
 
Three of the four rhizobacterial isolates were Gram-positive, except L. adecarboxylata DKS3, which was negative (Table 2). Furthermore, the results of the phenotypic examination also revealed that all of the bacterial cells were rod-shaped and the bacterial colonies were all circular, but they differed in terms of elevation and margin, the optimum temperature and pH of all the isolates were 35^oC and 7 respectively (Table 2).


 
Greenhouse experiment
 
The result indicateds (Table 3) that the rhizobacterial isolates, in the absence of the pathogen, did not substantially vary from the negative control treatment (plant alone) in attaining a tomato seed germination rate of 100%. In treatments contaminated with the pathogen, the B. halotolerans DMC8 treatment significantly increased the seed germination rate to 70%, compared to the positive control treatment (pathogenic fungus alone), which was 60%. The remaining treatments did not exhibit significant differences compared to the positive control treatment (Table 3). The findings align with previous research. Bhatt and Manuel (2014) demonstrated that rhizobacterial isolates greatly enhanced seed germination and seedling vigour in mung bean (Vigna radiate) plants subjected to pathogen stress. Research by Olanrewaju  et al. (2017) has shown that rhizobacteria enhance plant growth and seed germination by counteracting the effects of pathogens through several physiological and biochemical mechanisms.


       
The data (Table 3) indicated that the treatments with B. halotolerans DMC8, B. subtilis NAS1 and P. polymyxa TRS4 resulted in a significant reduction in disease incidence rates of 50%, 55% and 65%, respectively, compared to the positive control treatment, which recorded an 85% incidence (Fig 2). In contrast, the treatment with L. adecarboxylata DKS3 did not demonstrate any significant difference. All therapies, however, resulted in a notable decrease in disease severity, with reductions ranging from 30% to 49%, in contrast to the positive control therapy, which yielded a reduction of 56% (Table 3). The treatments of B. halotolerans DMC8 and B. subtilis NAS1 demonstrated superiority by attaining a disease severity of 30%. These data illustrated the isolates’ have the capacity to function as autonomous biocontrol agents for integrated pest management. These results conform the previous research on the topic. Kloepper  et al. (2004) revealed that rhizobacteria may successfully protect plants from phytopathogens while enhancing their growth and general health. This is accomplished through many mechanisms, including induced systemic resistance (ISR) and nutritional competition. Additionally, a research by Glick (2012) showed the efficacy of rhizobacteria in controlling soil-borne diseases through antibiotic production and other antagonistic mechanisms. Various biocontrol treatments have demonstrated efficacy in managing R. solani across diverse crops. The most efficacious bacterial treatments for managing root rot disease predominantly belong to the Bacillus species (Szczech and Shoda, 2004; Hussein et al., 2025). El-Kazzaz  et al. (2022) demonstrated that P. polymyxa reduced both the prevalence and severity of root rot and wilt diseases in pepper plants induced by R. solani. The B. halotolerans strain effectively mitigated the strawberry gray mold produced by Botrytis cinerea (Wang et al., 2021). The results indicated that treatments with rhizobacteria, in the absence of the pathogen, significantly enhanced growth parameters, including plant length and both fresh and dry weight, compared to the negative control treatment, thereby demonstrating the efficacy of these isolates as biofertilizer (Table 4). The treatments contaminated with R. solani yielded varied results, the isolates B. halotolerans DMC8 and B. subtilis NAS1 demonstrated a notable enhancement in the average lengths of the vegetative and root systems (Fig 2), measuring 18.1 cm, 18.5 cm and 9.0 cm, 8.6 cm, respectively, in contrast to the positive control treatment, which measured 12.9 cm and 5.7 cm, respectively (Table 4). The treatments of B. halotolerans DMC8, B. subtilis NAS1 and P. polymyxa TRS4 resulted in a notable enhancement in the average fresh weight of the vegetative and root systems, ranging from 2.226 to 3.450 g and 0.197 to 0.301 g, respectively, in contrast to the positive control treatment, which yielded 2.159 g and 0.175 g, respectively (Table 4). The dry weight for the three isolates DMC8, NAS1 and TRS4 exhibited substantial increases, ranging from 0.445 to 0.690 g and 0.039 to 0.060 g, respectively, in comparison to the positive control treatment, which recorded 0.432 g and 0.035 g, respectively (Table 4). The treatment with L. adecarboxylata DKS3 bacterium did not result in any notable enhancement under biotic stress conditions. The results shown in Table 4 conform the previous research findings demonstrating the beneficial effects of rhizobacteria on plant growth. Abdeljalil  et al. (2016) discovered that rhizobacterial isolates of B. thuringiensis and B. subtilis significantly enhanced the growth of tomato plants infected with R. solani, resulting in a 62-76% increase in plant height, a 53-86% increase in root fresh weight and a 34-67% increase in the fresh weight of aerial parts. Kang  et al. (2021) found that isolates of L. adecarboxylata displayed significant differences in plant growth traits when cucumber (Cucumis sativus L) seeds were infected, resulting in a marked increase in shoot length, root length and shoot fresh weight of the plants. Zhang  et al. (2015) discovered that P. polymyxa and B. subtilis significantly enhanced the growth of tomato plants in greenhouse pot experiments, resulting in increased plant height, root length and total fresh and dry biomass compared to an untreated control group.




 
Assay enzyme activity in tomato plants
 
The research revealed peroxidase enzyme activity significantly changed in tomato plants subjected to several treatments, including rhizobacterial isolates both independently and in conjunction with R. solani. The peroxidase enzyme is crucial for the plant’s defense mechanisms, particularly by facilitating the neutralization of reactive oxygen species and fortifying cell walls to inhibit pathogen invasion (Hiraga et al., 2001). The results (Table 5) demonstrated that the rhizobacterial isolates, when exposed to the pathogen, led to a significant enhancement in peroxidase enzyme activity, in contrast to treatments utilizing rhizobacteria only. The enzyme activity in pathogen-contaminated treatments varied between 18.11 and 20.21 D470.min^-1.g-1.F.wt^-1 (Table 5), significantly exceeding the enzyme activities observed in the positive and negative control treatments, which were 7.10 D470.min^-1.g^-1.F.wt^-1 and 4.82 D470.min^-1.g^-1.F.wt^-1, respectively. The elevated peroxidase activity seen in the presence of pathogens signifies that the rhizobacterial isolates effectively induce systemic resistance in tomato plants, hence enhancing their defensive responses. The findings align with previous studies indicating that some beneficial bacteria may activate induced systemic resistance (ISR) in plants, hence enhancing the synthesis of defense-related enzymes like peroxidase (Van Loon  et al., 1998).


               
Phenylalanine ammonium lyase (PAL) is a crucial enzyme in plant defense mechanisms. It activates the phenylpropanoid pathway, which generates defense-related compounds such as phytoalexins and lignin (Gho et al., 2020). The findings (Table 5) indicateds that all rhizobacterial treatments, irrespective of pathogen presence, significantly increased PAL activity compared to the control group. The treatment of rhizobacterial isolates against R. solani resulted in an increase in PAL activity ranging from 19.20 to 20.70 mg Cinnamic Acid.h^-1.g^-1.F.wt. The increases were substantial relative to the positive control (13.96 mg Cinnamic Acid.h^-1.g^-1.F.wt.) and the negative control (7.82 mg Cinnamic Acid.h^-1.g^-1.F.wt.). This indicateds that the rhizobacterial isolates significantly enhanced PAL activity and maintained elevated enzyme levels even in the absence of pathogens, suggesting a strong priming effect. This corresponds with the principle of priming, wherein plants pre-conditioned by advantageous microbes exhibit a more robust and rapid defensive response to subsequent pathogen assaults (Goellner et al., 2008). The results demonstrated the effectiveness of rhizobacterial treatments in promoting the production of essential defense enzymes in tomato plants. The notable increase in peroxidase and phenylalanine ammonia lyase activity in pathogen-contaminated treatments suggests that these bacteria may induce a heightened state of readiness in plants, allowing for a more effective response to pathogen attacks. Yasmin  et al. (2016) identified a robust correlation between the activity of the antioxidant enzymes peroxidase (PO), phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO) in plants and disease suppression, proposing that these enzymes may function as elicitors of induced systemic resistance (ISR). Al-Himiry (2013) conducted an experiment demonstrating that the application of a mixture of rhizobacterial isolates of P. putida and E. cloacae as an inoculum significantly improved the resistance of tomato plants against F. oxysporum f. sp. lycopersici, correlating with increased levels of peroxidase, phenylalanine ammonia lyase and phenolic compounds. Jayaraj  et al. (2004) demonstrated that the application of B. subtilis strain AUBS1 significantly reduced the occurrence of sheath blight disease in rice during greenhouse trials.

Three isolates of B. halotolerans DMC8, B. subtilis NAS1 and P. polymyxa TRS4, reduced the root rot disease incidence caused by R. solani in tomato plants. All of the four isolates, however, resulted in a considerable decrease in the disease severity. The isolates showed significant abilities in enhancing plant growth, including height and both fresh and dry biomass of the plants. The results demonstrate the effectiveness of rhizobacterial treatments in augmenting the production of essential defensive enzymes, namely peroxidase (PO) and phenylalanine ammonia lyase (PAL), in tomato plants. This study shows that rhizobacterial isolates may promote plant development in sustainable agriculture as biocontrol agents and biofertilizer. These results provide the groundwork for future field studies on crop productivity and soil health in different environments.

The authors would like to extend their sincere appreciation to Mustansiriyah University in Iraq and Tarbiat Modares University in Iran for their invaluable assistance and contributions to the field of plant pathology and scientific development.
 
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