Antimicrobial potential of aqueous extracts of selected plant species against crown gall disease

 Corresponding author: Nagalakshmi Devamma M Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Antimicrobial potential of aqueous extracts of selected plant species against crown gall disease Anusha Rani Yavvari, N Haseeda, J Reddy Kumari, M Mounika, Ankanna S, Venugopal A, Vijaya T and Nagalakshmi Devamma M * Department of Botany, Sri Venkateswara University, Tirupati 517502. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 129-136 Publication history: Received on 18 March 2025; revised on 29 April 2025; accepted on 01 May 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.22.3.0415 Abstract This study investigates the antimicrobial potential of aqueous extracts from selected plant species (Lantana camara, Mentha spicata, Chromolaena odorata, Hyptis suaveolens, and Anisomeles malabarica) against Agrobacterium tumefaciens, the causal agent of crown gall disease. The plant extracts were prepared using the Soxhlet and decoction methods, and their phytochemical composition was qualitatively analyzed. The antimicrobial activity of the extracts was evaluated through in vitro and in vivo methods. Results indicated that Lantana camara, Mentha spicata, and Chromolaena odorata extracts effectively suppressed the growth of A. tumefaciens, while Hyptis suaveolens and Anisomeles malabarica did not exhibit significant antimicrobial activity. This study suggests the potential of Lantana camara, Mentha spicata, and Chromolaena odorata as natural alternatives for controlling crown gall disease. Keywords: Crown gall disease; Agrobacterium tumefaciens; Antimicrobial activity; plant extracts; Lantana camara; Mentha spicata; Chromolaena odorata 1. Introduction Crown gall is a widespread disease affecting numerous woody and herbaceous plants, caused by the bacterium Agrobacterium tumefaciens[1]. The disease leads to the formation of tumors or galls on various plant parts, causing significant economic losses in agriculture[2]. Agrobacterium tumefaciens modifies the genetic material of host cells, transferring part of its Ti-plasmid DNA into the host cell DNA, leading to uncontrolled cell proliferation and tumor formation[3]. The control of crown gall disease is challenging, and there is a need for effective and eco-friendly control strategies[4]. Plant-derived compounds have emerged as promising alternatives for disease management due to their antimicrobial properties[5]. The present study aims to evaluate the antimicrobial potential of aqueous extracts from five selected plant species (Lantana camara, Mentha spicata, Chromolaena odorata, Hyptis suaveolens, and Anisomeles malabarica) against Agrobacterium tumefaciens 2. Material and methods 2.1. Plant Material Collection and Preparation The leaves of the selected plant species, namely Hyptis suaveolens, Lantana camara, Chromolaena odorata, Mentha spicata, and Anisomeles malabarica, were collected from Tirupati, Andhra Pradesh, India. The leaves were then dried, pulverized and subjected to extraction. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 129-136 130 Figure 1 Drying, Grinding and Weighing 2.2. Extraction Methods Two extraction methods were used to obtain plant extracts: Soxhlet extraction, primarily used for extracting nonthermolabile compounds, and the decoction method, traditionally used for water-soluble and thermostable compounds. • Soxhlet Extraction: 15 grams of the powdered plant material was placed in a filter paper thimble and then placed in a Soxhlet apparatus. Ethanol, a common solvent for extracting plant compounds, was placed in a round-bottom flask and heated. The ethanol vapours rose and condensed in a condenser. The condensed ethanol dripped into the thimble containing the plant material, extracting soluble compounds. As the ethanol level in the thimble rose, it siphoned back into the flask, carrying the extracted compounds. This process was repeated to ensure efficient extraction. After extraction, the ethanol was removed by distillation, leaving the crude extract. • Decoction Method: In this method, the crude plant material was boiled in water within an open-type extractor. The ratio of crude drug sample to water was 1:4 or 1:16. The mixture was boiled, reducing the volume to onefourth of the original, and the concentrated extract was then strained or filtered. Figure 2 Soxhlet Extraction Method Figure 3 Decoction Method 2.3. Qualitative Phytochemical Analysis The plant extracts were subjected to preliminary phytochemical screening to detect the presence of various secondary metabolites. Standard methods were employed to identify the following compounds: 2.3.1. Alkaloids • Wagner's Test: Extracts were treated with Wagner's reagent (iodine in potassium iodide). A reddish-brown precipitate indicates the presence of alkaloids. 2.3.2. Flavonoids • Lead acetate test: Extracts were treated with few drops of lead acetate solution. Formation of yellow colour precipitate indicates that the presence of flavonoids. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 129-136 131 2.3.3. Steroids • Acetic Anhydride Test: Extracts were treated with acetic anhydride and sulfuric acid. A color change from violet to blue or green indicates steroids. 2.3.4. Terpenoids • Salkowski Test: Extracts were treated with two ml of chloroform and concentrated sulfuric acid. Brown colour in the lower layer indicates the presence of terpenoids. 2.3.5. Anthraquinones • Borntrager’s Test: About five mg of the extract was boiled with 10% HCl for few minutes in a water bath. It was filtered and allowed to cool. Equal volume of CHCl3 was added to the filtrate. Few drops of 10% NH3 were added to the mixture and heated. Formation of pink colour indicates the presence of anthraquinones. 2.3.6. Phenols Ferric Chloride Test: Extracts were treated with ferric chloride solution. Formation of bluish black colour indicates the presence of phenols. 2.3.7. Saponins Foam Test: Extracts were shaken vigorously with water. Persistent foam indicates the presence of saponins. 2.3.8. Tannins • Ferric Chloride Test: Extracts were treated with ferric chloride solution. A dark green or bluish black colour indicates the presence of tannins. 2.3.9. Carbohydrates • Benedict's Test: Extracts were treated with Benedict's reagent and heated. A color change and precipitate indicate reducing sugars. 2.4. Protein and Aminoacids • Biuret Test: Extracts were treated with NaOH and copper sulfate. A violet color indicates proteins. 2.4.1. Oils and Resins Extracts were applied to filter paper. A transparent appearance indicates oils and resins. 2.5. Antimicrobial Activity Assays The antimicrobial activity of the plant extracts were evaluated through in vitro and in vivo methods. 2.5.1. In Vitro Method The antimicrobial activity of the plant extracts was evaluated through an in vitro assay using the agar diffusion method. An Agrobacterium tumefaciens culture was uniformly spread on MacConkey agar. Wells were impregnated with 50 µL of the plant extracts at a concentration of 100 mg/mL and placed on the agar surface. The plates were incubated at 28°C for 24-48 hours. The zone of inhibition was measured in millimeters to assess the effect of the plant extracts on the growth of Agrobacterium tumefaciens. 2.5.2. In Vivo Method: Agrobacterium tumefaciens was inoculated into carrots by introducing 100 µL of the bacterial inoculum at a concentration of 10^8 CFU/ml into a wound created in each carrot. Gall development in the carrots was observed after two weeks, with measurements taken of gall size and number. Plant extracts were applied to the inoculated carrots to assess their ability to control gall formation, and the concentration and application method of the extracts were recorded. • Control: Carrots inoculated with A. tumefaciens but without plant extract treatment were included as a control. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 129-136 132 2.6. Gram Staining Gram staining was performed to confirm the identity of A. tumefaciens. A sample was heat-fixed on a slide, stained with crystal violet for one minute, treated with Gram's iodine for one minute as a mordant, decolorized with acetone or alcohol for about three seconds, counterstained with safranin for one minute, and observed under a microscope. 3. Results and discussion 3.1. Phytochemical screening Table 1 Phytochemical screening S. No Hyptis suaveolens Lantana camara Mentha spicata Anisomeles malabarica Chromolaena odarata Alkaloids ++ - + ++ + Flavonoids + + ++ ++ + Steroids - + - - - Terpenoids + - + + + Anthraquinones + - - - - Phenols + ++ + + + Saponins + + + + + Tannins - + + + + Carbohydrates - + - - - Proteins and Aminoacids - - + - - Oils and Resins - - + + + 3.2. In Vitro Antimicrobial Activity The disk diffusion method revealed varying zones of inhibition against A. tumefaciens: Table 2 Zone of Inhibition of selected Plants S.No Name of the Plant Zone of Inhibition 1 Lantana camara 1.5 2 Mentha spicata 1.0 3 Chromolaena odarata 0.5 4 Hyptis suaveolens 0 5 Anisomeles malabarica 0 The study revealed that extracts from Lantana camara, Mentha spicata, and Chromolaena odorata exhibited antimicrobial activity against A. tumefaciens, while extracts from Hyptis suaveolens and Anisomeles malabarica did not show significant inhibition. This variation can be attributed to the differences in the phytochemical composition of each plant species. The larger inhibition zones observed for Lantana camara, Mentha spicata, and Chromolaena odorata extracts suggest a higher concentration of bioactive compounds effective against A. tumefaciens. These compounds may include various secondary metabolites known for their antimicrobial properties. Conversely, the lack of significant inhibition by Hyptis suaveolens and Anisomeles malabarica extracts indicates a lower concentration or absence of effective antimicrobial compounds. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 129-136 133 Figure 4 Control Figure 5 Media + A.tumefaciens Figure 6 Media+selected plant species extract World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 129-136 134 Figure 7 Inoculation of media containing A.tumefaciens into the carrot Figure 8 Carrots showing growth of A.tumefacians The variations in antimicrobial activity are likely due to the presence or absence of secondary metabolites. Plant secondary metabolites are a diverse group of organic compounds that are not directly involved in the normal growth, development, or reproduction of plants. These metabolites often play a crucial role in plant defense against herbivory and microbial pathogens. Many secondary metabolites possess antimicrobial properties, and their presence and concentration in plant extracts can determine the extract's effectiveness against bacteria like A. tumefaciens. In summary, the differences in antimicrobial activity observed among the plant extracts can be attributed to variations in their secondary metabolite composition. Extracts with higher concentrations of antimicrobial secondary metabolites, such as those from Lantana camara, Mentha spicata, and Chromolaena odorata, demonstrated greater inhibitory effects on A. tumefaciens. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 129-136 135 Table 3 Growth of A.tumefaciens S.No Carrot with extracts Growth of A.tumefaciens 1 Carrot with A.tumefaciens Present 2 Carrot with Lantana camara Absent 3 Carrot with Mentha spicata Absent 4 Carrot with Chromolaena odarata Absent 5 Carrot with Anisomeles malabarica Present 6 Carrot with Hyptis suaveolens Present 3.3. In Vivo Antimicrobial Activity The in vivo experiments using carrot models confirmed the results obtained from the in vitro tests: • Carrots inoculated with A. tumefaciens alone showed gall formation after two weeks. • Carrots treated with Lantana camara, Mentha spicata, or Chromolaena odorata extracts after inoculation with A. tumefaciens showed no gall formation. • Carrots treated with Hyptis suaveolens or Anisomeles malabarica extracts after inoculation with A. tumefaciens still developed galls, indicating the ineffectiveness of these extracts against the pathogen. 3.4. Gram Staining The gram-negative A. tumefaciens appeared as red, rod-shaped microorganisms. Figure 9 Microscopic view of Agrobacterium tumeficiens 4. Conclusion In conclusion the findings of this study demonstrate the suppressive growth of crown gall on bacterium A.tumefaciens by Lantana camara, Mentha spicata, Chromolaena odorata. In carrot we also observe suppressive growth of crown gall disease causing A.tumefaciens.The plant extract of selected plants species, extraction method done by water to reduce the chemical use in fields and reduce the economically less to farmers and ecofriendly. Compliance with ethical standards Disclosure of conflict of interest No conflict of interest to be disclosed. References [1] Agrios GN. Chapter twelve-plant diseases caused by prokaryotes: bacteria and mollicutes. Plant pathology. 2005:615-703. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 129-136 136 [2] Pulawska J. Crown gall of stone fruits and nuts, economic significance and diversity of its causal agents: tumorigenic Agrobacterium spp. Journal of Plant Pathology. 2010 Sep 1:S87-98. [3] Gohlke J, Deeken R. Plant responses to Agrobacterium tumefaciens and crown gall development. Frontiers in Plant Science. 2014 Apr 23;5:155. [4] Clare BG. Agrobacterium: Biological plant disease control. InAdvanced Engineered Pesticides 2024 Nov 1 (pp. 129-146). CRC Press. [5] Savoia D. Plant-derived antimicrobial compounds: alternatives to antibiotics. Future microbiology. 2012 Aug 1;7(8):979-90. [6] Alconero R. Crown gall of peaches from Maryland, South Carolina, and Tennessee and problems with biological control 1980, 835-838. [7] Jones WP, Kinghorn AD. Extraction of plant secondary metabolites. Natural products isolation. 2012:341-66. [8] Li Q, Guo R, Li Y, Hartman WH, Li S, Zhang Z, Tringe SG, Wang H. Insight into the bacterial endophytic communities of peach cultivars related to crown gall disease resistance. Applied and environmental microbiology. 2019 May 1;85(9):e02931-18. [9] Subramanian P, Anandharamakrishnan C. Extraction of bioactive compounds. InIndustrial Application of Functional Foods, Ingredients and Nutraceuticals 2023 Jan 1 (pp. 45-87). Academic Press. [10] Ghanney N, Rhouma A. Schinus terebinthifolius Raddi (Anacardiaceae) leaf extracts: Antibacterial activity against two Agrobacterium tumefaciens strains. Journal of Crop Protection. 2015 Mar 10;4(1):85-96. [11] Shaikh JR, Patil M. Qualitative tests for preliminary phytochemical screening: An overview. International journal of chemical studies. 2020 Mar 1;8(2):603-8. [12] Njagi A, Nyamwange MM, Njeru EM, Birgen JK. Antibacterial effect of Artemisia and ginger extracts in controlling Agrobacterium tumefaciens in roses. Journal of Floriculture and Landscaping. 2021;7:1-5. [13] Kahla Y, Zouari-Bouassida K, Rezgui F, Trigui M, Tounsi S. Efficacy of Eucalyptus cinerea as a source of bioactive compounds for curative biocontrol of crown gall caused by Agrobacterium tumefaciens strain B6. BioMed Research International. 2017;2017(1):9308063. [14] Darji B, Ratani J, Doshi M, Kothari V. In vitro antimicrobial activity in certain plant products/seed extracts against selected phytopathogens. [15] Barton IS, Fuqua C, Platt TG. Ecological and evolutionary dynamics of a model facultative pathogen: Agrobacterium and crown gall disease of plants. Environmental Microbiology. 2018 Jan;20(1):16-29. [16] Coico R. Gram staining. Current protocols in microbiology. 2006 Feb(1):A-3C.