In-vitro evaluation of different bactericides and bioagents against Ralstonia solanacearum

Introduction

Tomato (Solanum lycopersicum L.) are not only a staple in global cuisines but also hold significant economic value as a widely cultivated crop. Yet, their cultivation is often threatened by numerous pathogens, particularly the notorious Ralstonia solanacearum. This bacterial agent is responsible for bacterial wilt, a severe disease that can devastate yields and inflict financial strain on tomato producers. Ralstonia solanacearum presents a major challenge due to its extensive host range and its ability to persist in the soil, complicating management efforts once it infiltrates a field. The pathogen targets the vascular system of tomato plants, causing wilting, stunted growth, and ultimately leading to plant death. The economic ramifications of Ralstonia solanacearum extend beyond mere yield losses, encompassing heightened production costs related to disease management and control strategies. As the global demand for tomato continues to rise, the urgency for effective and sustainable solutions to combat this pathogen becomes even more pronounced. Conventional methods, such as crop rotation and soil fumigation, often have limitations and may not sufficiently safeguard against this resilient threat. To address these challenges, innovative strategies for managing bacterial wilt in tomato plants are essential. One promising approach is the investigation of bactericides chemical agents capable of inhibiting or eliminating Ralstonia solanacearum. Conducting in vitro studies is critical for evaluating the effectiveness of these bactericides, as they provide controlled settings for initial screenings, determine minimum inhibitory concentrations, and assess impacts on the pathogen's virulence. This research aims to advance the fight against bacterial wilt in tomato plants by thoroughly evaluating various bactericides in vitro for their effectiveness against Ralstonia solanacearum. The outcomes of this study could empower tomato growers with practical tools for disease management, help lower production costs, and promote the stability of tomato production amid challenges posed by this resilient pathogen.

Materials and Methods

The present investigation on "In-vitro Evaluation of different bactericides and bioagents against Ralstonia solanacearum" was studied during 2023-2024 at Department of Plant Pathology, College of Agriculture, Nagpur. The details of the material and methods used are as follows.

Isolation

Tomato plants showing symptoms of bacterial wilt, such as yellowing and wilting, were collected from affected fields. Infected root samples were cut into small pieces, disinfected with sodium hypochlorite, rinsed with distilled water, and air-dried. These pieces were then placed on sterilized nutrient agar (NA) medium and incubated at 28±2 °C to promote bacterial growth. Once growth was observed, cultures were purified. Ralstonia solanacearum was identified based on its morphological characteristics using a compound microscope and confirmed through additional biochemical tests.

In -vitro evaluation of bactericides

The in vitro assessment of bactericides and bioagents evaluated the susceptibility of bacterial isolates to these treatments. Sensitivity tests were conducted using the paper disc method to determine the efficacy of each treatment.

Procedure

1. The study involved preparing the recommended doses of bactericides in sterile nucleus-free water and storing them in vials. 2. Paper discs, punched out from autoclaved filter paper, were soaked in the bactericides for 2 hours. 3. The discs were then placed on nutrient agar plates inoculated with Ralstonia solanacearum culture, which had been incubated for 72 hours. 4. The plates were further incubated and observed for the production of inhibition zones around the paper discs. 5. The results were analyzed statistically, with sterile distilled water-soaked paper discs serving as controls. after growth was spread on plates containing nutrient agar with help of spreader. 5. Fungal discs of 5mm diameters from margin of actively growing four days old culture removed and placed in the center of agar plates. 6. The plates were incubated at 28+2 °C for 4 days. 7. The observation on the zone of inhibition around the mycelial disc against Ralstonia solanacearum was recorded after the incubation period.

In -vitro

Results and Discussion

This study focused on tomato wilt caused by Ralstonia solanacearum. It involved several key steps: collecting samples, isolating the pathogen, identifying the isolated strain, and conducting pathogenicity tests to fulfill Koch's postulates. Additionally, the research evaluated the efficacy of bactericides and bioagents against Ralstonia solanacearum in vitro. The findings from these experiments are presented below.

In -vitro evaluation of bactericides

Efficacy of 6 bactericides at 300 ppm concentration was tested in-vitro by following the paper disc diffusion method against Ralstonia solanacearum. The data presented in Table . 1 revealed that Among the six bactericides tested against Ralstonia solanacearum, Streptocycline demonstrated the highest inhibitory activity, with a zone of inhibition of 20.43 mm at a concentration of 300 ppm. This was followed by Streptomycin sulphate + Copper oxychloride and Streptomycin sulphate + Streptocycline, which exhibited zones of inhibition of 17.37 and 16.73 respectively at concentrations of 300 ppm and In contrast, Validamycin showed the least efficacy, with a zone of inhibition of only 9.97 mm at a concentration of 300 ppm.

In a similar way Singh and Jagtap (2017) [15] studied In vitro the efficacy of antibacterial chemicals against. Bawari (2014) [3] . He reported that Streptocycline was highly effective with the maximum inhibition zone of 31.25 to 36.00 mm at 500 to 750 ppm. Gupta and Razdan (2013) [6] evaluated in vitro eleven antibiotics against R.solanacearum, causing brinjal wilt and reported that antibiotic plantomycin (streptomycin sulphate + tetracycline) at 100, 250 and 500ppm gave highest inhibition zone of 10.83,18.63 and 28.77 mm,

In -vitro evaluation of bio-agents against Ralstonia solanacearum

The effectiveness of various bioagents against Ralstonia solanacearum was evaluated using the paper disc diffusion method, and the results are presented in the following tables. These results demonstrate significant variations in the efficacy of different bioagents, indicating that some are more effective than others in controlling this bacterial isolate. Among the four bioagents tested against Ralstonia solanacearum (Table 2), Pseudomonas fluorescens demonstrated the highest inhibitory activity, with a zone of inhibition of 13.75 mm. Bacillus subtilis also showed significant activity, with a zone of inhibition of 10.63 mm. In contrast, the fungal bioagents tested were ineffective, failing to produce any inhibition zones and instead being inhibited by the bacterial isolate itself. These findings are in consonance with reports of several earlier workers. Singh, R. and Jagtap, G. P., (2017) [15] bioagents against Ralstonia solanacearum infecting bacte rial wilt in ginger. Khan et al., (2018) [8] evaluted the three potential microbial bioagents, viz. Trichoderma harzianum, Bacillus thuringiensis, Pseudomonas fluorescens against bacterial wilt of lettuce under hydroponic culture system.

Conclusion

The salient features of the present investigation are as under 1. In vitro evaluation of bactericides against Ralstonia solanacearum showed that Streptocycline had the highest inhibitory effect, with a 20.43 mm inhibition zone at 300 ppm. 2. In vitro evaluation of bioagents against Ralstonia solanacearum showed that Pseudomonas fluorescens had the highest inhibitory effect at 13.75 mm

Acknowledgement

I am thankful to the Head of the Plant Pathology section, College of Agriculture, Nagpur for providing all the necessary facilities for conducting the research work.

References

1. Bawari MR, Narendrappa T. In vitro evaluation of bioagents and antibiotics against Ralstonia solanacearum causing brinjal wilt. J Pharmacogn Phytochem. 2019;8(5):2038-2041.
2. Dutta P. Management of bacterial wilt of tomato through an innovative approach. J Biol Control. 2012;26(3):288-290.
3. Gupta G, Razdan. Evaluation of antagonists and antibiotics against bacterial wilt of brinjal caused by R. solanacearum. Bionfolet. 2013;10(3A):851-852.
4. Kataky M, Tamuli AZ, Teron R. Efficacy of biocontrol agent against bacterial wilt on Naga King tomato (Capsicum chinense Jacq.). Interna J App Pure Sci Agric (IJAPSA), 2016, 2(1).
5. Makhlouf AH, Hamedo HA. Suppression of bacterial wilt disease of tomato plants using bacterial strains. Life Sci J. 2013;10(3):1732-1741.
6. Mondal B, Bhattacharya I, Khatua DC. Crop and weed host of Ralstonia solanacearum in West Bengal. J Crop Weed. 2011;7(2):195-199.
7. Murthy KN, Srinivas C. In vitro screening of bioantagonistic agents and plant extracts to control bacterial wilt of tomato. J Agric Tech. 2012;8(3):9991015.
8. Owoseni AA, Sangoyami TE. Effect of solvent extract of some plants on Ralstonia solanacearum. Br Microbiol Res J. 2014;4(1):89-96.
9. Ramesh R, Ghanekar MP, Joshi AA. Potential rhizobacteria for the suppression of bacterial wilt pathogen, Ralstonia solanacearum in eggplant (Solanum melongena L.). Veg Sci. 2009;36(2):193-199.
10. Shahbaz MU, Mukhtar T, Ul-Haque MI, Begum N. Biochemical and serological characterization of Ralstonia solanacearum associated with tomato seeds from Pakistan. Int J Agric Biol. 2015;17:31-40.
11. Singh R, Jagtap GP. In vitro evaluation of antibacterial chemicals and bioagents against Ralstonia solanacearum infecting bacterial wilt in ginger. Int J Curr Microbiol App Sci. 2017;6(5):2034-2045.
12. Sultana T, Naz F, Haq MIU, Butt S, Abas MF. Characterization and relative contribution of fungal and bacterial pathogens involved in sudden death syndrome of tomatoes. Pakistan J Phytopath. 2014;26(01):53-61.