Biological Control of Tomato Root Rot Disease by Indigenous Rhizobacteria in Greenhouse Setting

Rhizobacteria are soil-resident bacteria that inhabit plant roots and significantly influence their growth and health (de Andrade et al., 2023). Rhizobacteria are considered effective alternatives to chemical pesticides and fertilizers because of their ability to address several issues and constraints linked to these traditional approaches (de Andrade et al., 2023). Tomato (Solanum lycopersicum L.) playing an essential role in the Iraq’s agricultural sector and dietary practices, according to FAOSTAT (2024), its annual production has attained 630,180 tons. Root rot infections are highly significant and devastating plant ailments with a worldwide impact (Williamson-Benavides and Dhingr, 2021). They may result in a reduction in agricultural yield and quality between 10% and 90%, dependent upon the particular crop and pathogen implicated. Moreover, they lead to significant economic detriments for both agricultural producers and consumers (Agrios, 2005). Root rot diseases are caused by several fungal infections, including R. solani, P. aphanidermatum and M. phaseolina (Hussein et al., 2022). Bonaterra et al., (2022) identified multiple bacterial genera, including Alcaligenes, Arthrobacter, Bacillus, Enterobacter, Erwinia, Pseudomonas, Rhizobium, Serratia, Stenotrophomonas, Streptomyces and Xanthomonas, that demonstrate protective properties against fungal and bacterial pathogens responsible for plant diseases. The mechanisms employed by rhizobacteria include the enhancement of plant resistance, the secretion of potent antimicrobial agents such as antibiotics or cell wall-degrading enzymes to combat pathogens and the occupation of infection sites to outcompete the pathogens (Hussein et al., 2024). Rana et al., (2024) demonstrated that the soil bacterium Bacillus halotolerans NYG5 serves as an effective biocontrol agent against various phytopathogenic fungi, including M. phaseolina, R. solani, P. aphanidermatum and S. sclerotiorum, owing to its capacity to produce volatile organic compounds (VOCs). Anak et al., 2021, revealed that strains of Bacillus thuringiensis HV43, Bacillus subtilis HV34, Bacillus cereus GC and Bacillus subtilis HT30 efficiently inhibited root rot disease in bean (Vigna radiate) plants induced by R. solani, attaining a 100% success rate. The primary hypothesis and aim of this study is to assess the antifungal efficacy of local rhizobacterial isolates against the pathogens R. solani, P. aphanidermatum and M. phaseolina, which cause tomato root rot disease, in a greenhouse environment and to subsequently demonstrate its effectiveness in improving various facets of plant growth.

Preparing of rhizobacterial isolates inoculum
 
Twelve rhizobacterial isolates were derived from a prior research (Hussein et al., 2024), extracted from the roots of several plant species across Iraqi governorates (Table 1). The bacterial isolates were generated by inoculating 1 ml of fresh individual bacterial cultures, cultivated overnight in 500 ml flasks containing 250 ml of nutrient broth, at 27°C for 48 hours with shaking at 150 rpm.  Bacterial cells were pelleted via centrifugation at 6000 rpm for 15 minutes at the room temperature and the supernatant was discarded. The cell pellet was resuspended in sterile distilled water and subjected to centrifugation under identical conditions. The supernatant was discarded and the bacterial cells were washed and resuspended in 2 ml of sterile distilled water. The concentration of cells in the suspension was adjusted to 10-9 cfu ml-1 in sterile 1% carboxymethyl cellulose (CMC) solution (Hussein, 2024).


 
Preparing of fungal pathogens inoculum
 
The fungal pathogens of R. solani (AG4) isolate (Rs-12), P. aphanidermatum isolate (Pa-m7) and M. phaseolina isolate (Mp-03) were previously obtained from the infected roots of diseased tomato plants and have shown a high level of virulence in pathogenicity tests (Hussein et al., 2022). Fungal inoculums were prepared individually by cultivating them on millet grains (Panicum miliaceum) in 250 ml flasks. The grains were soaked in distilled water and autoclaved twice on two successive days at 121°C for 60 minutes. They were then inoculated with five agar discs (5 mm diameter) from a 5-day-old fungal culture. The flasks were incubated at 28°C in the dark for 20 days, with shaking occurring at least once daily. Once colonized, the grains were transferred into paper pockets, dried and ground. The inoculums were subsequently mixed with sterilized soil at a rate of 10 g per kg of soil (Etebarian et al., 2000).
 
Greenhouse experiment
 
Twelve rhizobacterial isolates were assessed in greenhouse examinations (Ministry of Agriculture, Department of Plant Protection, Iraq) for their ability to manage prevalent soil-borne pathogenic fungi, specifically R. solani (AG4), M. phaseolina and P. aphanidermatum, while also evaluating their potential for promoting plant growth.
       
The seeds of tomato (cv. Super Marmande) underwent superficial disinfection by being soaked in a 2% sodium hypochlorite solution for one minute. Following this, they were rinsed three times with sterile distilled water and allowed to dry overnight on sterile filter paper within a sterile laminar flow hood. Subsequently, the seeds were immersed in a formulated bacterial solution in 1% CMC, shaken at 150 rpm at room temperature for three hours and then air dried on sterilized filter paper under the laminar hood overnight. The starting population of bacterial cells on the seed was approximately 10-9 to 10-11 colonies per seed (Qaisy et al., 2016; Alwan, 2018; Hussin, 2018).
       
Plastic pots measuring 17 cm in diameter and 15 cm in height were filled with a mixture of sterile field soil and peat moss in a 2:1 volume ratio. This mixture underwent sterilization via autoclaving on two consecutive days at 121°C for 60 minutes. For the treatments involving pathogenic fungi, an inoculum of 10 g per kg of pathogens was incorporated into the pots seven days prior to the planting of seeds. Each pot contained five coated seeds, with the exception of the negative control treatment, which was treated solely with a 1% CMC solution and did not include any bacterial inoculum. The pots received watering every two days using tap water and were maintained in a greenhouse with day/night temperatures of 32/25°C, 40% humidity and a light cycle of 16 hours brightness followed by 8 hours of darkness, without the application of any fertilizer during the experiment. The experiment was carried out with four replicates.
       
The treatments examined included negative control plants subjected to mock inoculation. Positive control includes R. solani (Rs), M. phaseolina (Mp) and P. aphanidermatum (Pa) individually. Isolates of rhizobacteria free from pathogens, along with treatment of a combination of 12 distinct rhizobacterial isolates (COR12). Isolates of pathogenic fungi R. solani, M. phaseolina and P. aphanidermatum were tested individually against each rhizobacterial isolate individually, as well as against the combination isolates (COR12).
       
After 15 days of planting, the percentage of seed germination was calculated according to the method of Hussein (2019) as follows:
 

  

       
The plants were harvested 60 days after seed planting. 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; where: 0 - Plants with healthy stems and roots, 1 - Plants with minimal stem/ root rot (less than 10%), 2 - Plants with slight stem/ root rot (25%), 3 - Plants with medium stem/ root rot (50 %), 4 - Plants with severe stem /root rot (75%) and 5 - Dead Plants (100%) (Castro et al., 2017) and the disease severity index (%) was calculated (Hussein and Al Zubidy, 2019) as follows:
 

  

       
Control disease value (CDV) was calculated according to the method of Song et al., (2004) as follows:
 

  

               
Further, the growth parameters comprising of length, fresh and dry weight of shoot and root systems were measured and recorded, plants were dried in an oven at 70°C until they reached a consistent weight for dry weight (Bhamra et al., 2022; Lavanya et al., 2023; Bekele et al., 2024). 

Greenhouse experiment
 
Seed germination
 
The findings illustrated in (Fig 1) reveal the impact of various rhizobacterial treatments on the germination rates of tomato seeds in the presence of pathogenic fungi that include R. solani, M. phaseolina and P. aphanidermatum. Even with the existence of these pathogens, the findings indicate that rhizobacterial isolates can significantly enhance germination rates. The results indicate that when pathogenic fungi are present, the germination rate of tomato seeds was notably enhanced by all rhizobacterial isolates, with the exception of SDV1 and BZD3. The two outliers demonstrated germination rates of 63.3%, aligning with the positive control treatment (pathogenic fungi alone), which also showed a seed germination rate of 63.3% (Fig 1). Interestingly, the COR12 combination treatment achieved a germination rate equivalent to that of the negative control treatment (seeds free of pathogens) in the presence of pathogens, resulting in a 100% germination rate. This finding suggests that the COR12 treatment effectively mitigates the detrimental effects of pathogenic fungi, likely due to the advantageous interactions of the bacterial strains that enhance the health and resilience of seeds.


 
Biological control of root rots disease
 
The findings shown in Table 2 offer compelling evidence regarding the efficacy of various rhizobacterial isolates in mitigating both the incidence and severity of diseases in tomato plants attributed to the pathogenic fungi R. solani, M. phaseolina and P. aphanidermatum. These findings hold significant value for the development of effective and sustainable biocontrol strategies aimed at managing agricultural diseases. The results indicate a notable reduction in disease incidence caused by pathogenic fungi, attributed to six unique rhizobacterial isolates: DMC8, BZD2, SKE2, NAS1, TRS4 and TNK1. The positive control treatment exhibited a disease incidence of 81.7%, whereas the disease incidence in these treatments ranged from 48.3% to 58.3% (Table 2). The most significant reduction in disease incidence was observed with the combination treatment COR12, which lowered it to 5.0% (Fig 2).




       
Relative to the positive control treatment, which demonstrated a disease severity of 53.3% (Fig 2), all tested rhizobacterial isolates showed a significant decrease in the disease severity index, with reductions varying from 30.7% to 48.7% (Table 2). This indicates that the intensity of the disease’s symptoms can still be mitigated by employing isolates that are less effective in decreasing incidence. The COR12 treatment demonstrated superiority, with the degree of disease lowered to 1.0%, statistically comparable to the negative control (0%).
       
The research demonstrates considerable heterogeneity in disease control efficacy across the twelve rhizobacterial strains examined. The control disease values in these treatments ranged from 8.7% to 42.4% (Table 2), reflecting the differential efficacy of the isolates in combating pathogenic fungus in tomato plants. The treatment with COR12 demonstrates an outstanding disease control effectiveness of 98.1%. The value far exceeds the results of each treatment, underscoring the possibility of combining several bacterial strains to have a synergistic effect. The remarkable efficacy of disease suppression in COR12 is ascribed to the synergistic metabolic activity, antibiotic production and nutritional competition among the consortium of strains (Glick, 2012).
       
These results align with previous research on the topic. Research by Kloepper et al., (2004) has shown that rhizobacteria may successfully protect plants from phytopathogens and enhance their growth and general health, this is accomplished through many mechanisms, including induced systemic resistance (ISR), nutritional competition. Additionally, synthesize phytohormones, which may enhance plant growth and stress resilience while indirectly mitigating disease impacts. Abdeljalil et al., (2016) investigated 25 rhizobacterial isolates sourced from the rhizosphere of healthy tomato plants obtained from various tomato cultivation sites in Tunisia. They assessed the antifungal efficacy of bacterial isolates from Bacillus spp., E. cloacae, Chryseobacterium jejuense and Klebsiella pneumoniae against R. solani, the pathogen responsible for Rhizoctonia root rot in tomato plants. The tomato-associated rhizobacteria demonstrated a substantial reduction in disease severity, ranging from 47% to 100%, compared to the control group infected with the pathogen. Diaz-Diaz et al.  (2023) discovered that biocontrol agents from Streptomyces sp. strains CBQ-EA2 and CBQ-B-8 significantly influenced seed germination and mitigated disease in bean (Phaseolus vulgaris) plants afflicted by root rot complex disease induced by M. phaseolina and R. solani.
 
Efficacy of rhizobacterial isolates on plant growth metrics

Effect on plant height
 
The research indicated that all rhizobacterial isolates significantly enhanced the shoot length of tomato plants under pathogen-free conditions. The combined treatment COR12 exhibited the most significant development, with a shoot length of 33.5 cm (Table 3). COR12 surpassed all opponents, achieving a remarkable shot length of 32.2 cm (Table 3).


       
The rhizobacterial isolates significantly augmented the root length of tomato plants, with the treatments of COR12, TNK1, BZD2 and NAS1 exhibiting the most substantial increases in root length (Table 3). Upon exposure to detrimental fungi, six particular strains exhibited enhanced root lengths, illustrating COR12’s robustness and ability to promote root growth.
 
Effect on plant fresh weight
 
The findings indicated that rhizobacterial isolates, especially COR12, notably enhanced the shoot fresh weight of tomato plants in pathogen-free environments, achieving a peak shoot fresh weight of 8.296 g (Table 3). In the context of disease, the isolates led to an increase in shoot weight, varying from 2.363 g to 3.010 g, in contrast to the positive control’s weight of 2.144 g (Table 3). COR12 showed significant effectiveness in promoting plant growth under stress conditions, highlighted by its impressive shoot fresh weight of 8.153 g.
       
The root system’s weight displayed a similar trend, as all isolates showed a significant rise in root fresh weight when grown in pathogen-free conditions. COR12 consistently exhibited the greatest root fresh weight, measuring 0.791 g (Table 3). Upon exposure to pathogens, eight isolates showed a significant enhancement in root fresh weight, suggesting that COR12 facilitates root growth, thereby improving the uptake of nutrients and water, essential for plant development and stress resilience.
 
Effect on plant dry weight
 
 All rhizobacterial isolates notably enhanced the dry weight of the plant’s shoot system in treatments free of pathogenic fungi. The COR12 combination treatment achieved the highest dry weight of 1.659 g (Table 3), demonstrating the effectiveness of the bacterial mixture in enhancing shoot development more effectively than the individual isolates.

The effectiveness of the rhizobacterial isolates varied when pathogenic fungi were present. Eight isolates demonstrated a significant increase in the dry weight of the shoot system, with values ranging from 0.473 g to 0.602 g, in comparison to the positive control treatment. The COR12 combination treatment demonstrated significant protective and growth-promoting effects, even under conditions of pathogen-induced stress.
       
Isolates from rhizobacteria notably increased the dry weight of the root systems of tomato plants when pathogenic fungi were not present, with the COR12 combination treatment achieving the highest dry weight of 0.158 g (Table 3). The COR12 treatment exhibited remarkable results, achieving an average root dry weight of 0.153 g, which is significantly higher than that of both the negative and positive controls.
       
Prior investigations have demonstrated the positive effects of rhizobacteria on the growth of plants. Abdeljalil et al., (2016) demonstrated that rhizobacterial isolates of B. thuringiensis B2, B. subtilis B10 and E. cloacae B16 notably enhanced the growth of tomato plants affected by the pathogenic fungus R. solani, leading to a 62-76% increase in plant height, a 53-86% increase in the fresh weight of roots and a 34-67% increase in the fresh weight of aerial parts. Abdel-Monaim​ et al. (2012) demonstrated that employing PGPR strains of Azotobacter sp., B. cereus and B. megaterium enhanced several growth parameters of tomato plants. Kang et al., (2021) observed notable differences in plant development traits when cucumber seeds were infected with isolates of L. adecarboxylata MO1. Zhang et al., (2015) demonstrated that six distinct bacterial strains significantly enhanced the growth of tomato plants in greenhouse pot experiments. Suprapta et al., (2014) demonstrated that five isolates of E. cloacae derived from cogongrass plants positively influenced plant growth and enhanced rice seedling yield. Patel et al., (2023) noted remarkable characteristics that promote plant growth when fenugreek seeds were treated with the B. subtilis ER-08 strain in greenhouse conditions.

The greenhouse trials showed that rhizobacterial isolates can enhance tomato seed germination, reduce disease incidence and severity and promote plant development under pathogenic stress conditions. The COR12 combination treatment, consisting of twelve bacterial strains, consistently outperformed individual isolates, achieving a 100% germination rate and significantly reducing disease incidence and severity. The collaborative function of these bacteria highlights the potential for employing bacterial groups in comprehensive pest management strategies. The COR12 treatment also significantly enhanced plant development metrics, including shoot and root length and the weight of fresh and dried plant material. The effectiveness of disease control varied among the rhizobacterial isolates, with COR12 achieving the highest efficacy at 98.1%. Specific strains like B. subtilis NAS1, B. halotolerans DMC8, S. lentus BZD2 and B. megaterium TNK1 exhibit significant potential as biocontrol agents, positioning them as viable options for sustainable agriculture.

The authors express gratitude to Mustansiriyah University, Iraq; and Tarbiat Modares University, Faculty of Agriculture, Department of Plant Pathology, Iran for their support and contributions to scientific and technological advancement, as well as to the Ministry of Agriculture, Department of Plant Protection, Iraq for supplying the requisite facilities.
 
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.

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.