INTRODUCTION:

Agrobacterium tumefaciens is a gram-negative, non-sporing, motile, soil bacterial pathogen known to infect dicot plants (Eg: Apricot, Pear, Apple, Cherry, Grapevine, Almond, Walnut) by causing crown gall disease.^1-3 It is one of the deadly soil-borne bacterial plant diseases around the world. It is known to be saprophytic surviving on decaying organic matter. The isolated strains from the various infected dicot plants such as Tectona grandis, Artocarpus heterophyllus, Anthocephalus codomba, Terminalia arjuna, Rosa chinensis and Solanum lycopersicum in the Rajshahi University campus, Rajshahi, Bangladesh were investigated by carrying out the pathogenicity, antibiotic resistance and biochemical tests to identify the causative agent in crown gall.

It was reported that the characteristics of the isolate matched with the characteristics of the Agrobacterium tumefaciens (ATCC23308T).⁴ The isolated bacterial strains from the galls of tobacco were also identified as Agrobacterium tumefaciens by Naruto et al.⁵

The disease prevalence is found to be high particularly in dicot plants due to the selective absorption of Acetosyringone, a phenolic metabolite from the soil which is not absorbed or least absorbed by monocot plants. The causative agent, Agrobacterium tumefaciens possesses the VirA gene in its Ti (Tumor inducing) plasmid which can metabolize the Acetosyringone, a phenolic compound. The crown gall disease affects severely the growth of the trees since the size of the gall increases proportionally with the growth of the trees. Agrobacterium tumefaciens can able to transfer its T-DNA to various plant parts through transient and stable transformation.⁶ Gowthami and his research team⁷ isolated the stem gall-induced bacterium from the agricultural farm to conclude them as Agrobacterium tumefaciens by carrying out the biochemical analysis.

Bobbrala et al.⁸ demonstrated the antimicrobial potential of fifty different methanolic plant extracts against Agrobacterium tumefaciens except for the following six plants such as A. officinalis, A. ilcifolius, P. rubra, C. sativum, T. pumila and R. communis. Agrobacterium tumefaciens, which develop resistance against the Agrocin antibiotics⁹. Therefore, the other soil microbes also develop resistance against the antibiotic. Further, the grown plants in such soils yield fruits and vegetables with inbuilt antibiotic resistance. Plants with several galls are unable to move water and nutrients through the trunk which affects the growth of the plant and fertility. Finally, there is a very low crop yield due to this infection. As far as agriculture is concerned, commercial antibiotics are expensive, and hence natural remedies are proven to work effectively against such tumor-forming bacteria without side effects in a cost-effective manner for farmers.

This disease causes severe vulnerability in Pongamia pinnata. The leaves of this plant have been studied and shown to have antimicrobial and immune-enhancing properties.^10,11Allium sativum extract, similarly, possesses significant compounds¹² that may have antibacterial, anti-diabetic, antioxidant,¹³ and antiplatelet effects.^14-16 The plant's extract is considered to use against plant pathogens.^17-19Therefore, the present study intends to isolate and identify the Agrobacterium tumefaciens which causes the crown gall in Pongamia pinnata (syn. Millettia pinnata) leaf. Its pathogenicity was also confirmed by detecting tumor formation. Finally, the antimicrobial activity of crude and ethanolic extract of Allium sativum (Garlic) was examined against Agrobacterium tumefaciens to control crown gall in dicot plants.

MATERIALS AND METHODS:

Isolation of crown galls:

Crown galls were collected from the leaf of Pongamia pinnata in the Hindustan College of Arts and Science campus, Padur, Chennai, India. The collected samples were kept in a sterile container and transferred immediately to the laboratory for surface sterilization to prevent contamination. The processed sample was further authenticated by botanical experts in the National Institute of Siddha, Tambaram, Chennai, India.

Preparation of crown gall extract from the leaf of Pongamia pinnata:

The crown galls (Fig. 1a) collected from the leaf of Pongamia pinnata were washed and sterilized with 95% ethyl alcohol to remove the soil impurities (Fig. 1b). The outer layer was removed with a sterile scalpel and the Gall tissues were crushed into small particles to make a mother inoculum. The gall tissue was again crushed using sterile mortar and pestle using ethanol.⁴ Then it was made as a paste to develop the starter culture for the isolation of Agrobacterium tumefaciens.

Fig: 1 a) Leaf of Pongamia pinnata with crown gall and b) Crushed crown galls (surface sterilized)

Scanning Electron Microscopic Analysis of Gall tissues:

The gall tissues were properly fixed and dehydrated to perform SEM analysis. The gall tissues were kept in phosphate buffer containing 3.5 % glutaraldehyde for 1.5 – 2 hrs at 0^oC prior to pre-fixation. It was rinsed with sodium phosphate buffer (0.1 M) at pH 7.2 for 15 min. Then the post-fixation process was carried out with equal parts of sodium phosphate buffer (0.2M) and 2 % osmium tetroxide for 1.5–2 hrs in an ice bath. The tissue was washed again with sodium phosphate buffer (in 0.1 M) at pH 7.2 for 15 min. It was dehydrated in different concentrations of diluted ethyl alcohol solutions (25%, 40%, 60% and 80%). The dehydration process was done with 100 % ethyl alcohol. Then it was dried and sputter-coated with Au-palladium.²⁰

Isolation and Identification of Agrobacterium tumefaciens:

In brief, the gall tissue sample was crushed and the homogenized tissues were streaked in Congo Red-Yeast Extract Mannitol Agar (CRYEMA) medium (pH- 6.8) and Hofer’s Alkaline Medium (pH-11) (selective medium). Then the plates were kept for incubation at 25±2˚C for the development of the microbial colonies.^21,
22. The developed colonies were morphologically identified and biochemically confirmed.²³ The biochemical tests are given as follows: a. Gram’s staining, b. IMViC (Indole, Methyl Red, Voges-Praskaur, Citrate), c. TSI (Triple Sugar Iron agar), d. Urease, e. Catalase, f. Oxidase.

Pathogenicity analysis:

The pathogenicity of the isolates can be confirmed by tumorigenesis assay using potato disc. Water agar medium (distilled water with pure 2% of agar agar) and surface sterilized sliced potato (Solanum tuberosum L.) disc were used for this study. In brief, the sliced potato disc was kept in the middle of the water agar and each disc was inoculated with 100 μl of grown inoculum obtained from crown gall tissues. Then, it was incubated at 30^oC. The discs were monitored to confirm any development of the tumors²⁴after 3^rd week of incubation.

Antibacterial activity of garlic extract:

The antibacterial activity of garlic extract against the isolate was carried out by a well diffusion method.²⁵ The assay used a sterilized Muller Hinton Agar (MHA) medium. In brief, the isolate was spread on MHA plates using a sterile swab. Agar wells were made with sterile gel puncture. The crude and ethanolic garlic extracts (Allium sativum) with a concentration of 10 μg/ml were separately loaded in the wells. Ampicillin disc (10μg concentration) was used as control.

RESULTS AND DISCUSSION:

Isolation of crown gall-inducing microbes from tissues of Pongamia pinnata:

The obtained crown gall tissues from the leaf of Pongamia pinnata were inoculated in four different nutrient media such as MacConkey agar, Congo Red-Yeast Extract, Mannitol Agar (CRYEMA) and Hofer’s Alkaline Medium to identify the crown gall diseases causing microbes. In the Nutrient agar medium, the isolate produced cream-colored, confluent, slimy, crowded, glistening colonies (Fig. 2a). In MacConkey agar medium, the isolate developed pink-colored, mucoid, confluent, pin-pointed, circular, elevated, slimy glistening colonies with pungent odor production (Fig. 2b). MacConkey agar medium restricted the growth of gram-positive bacteria as it contained bile salt which inhibited the gram-positive bacterial growth. Hence, the sustained organism in this medium may be a gram-negative organism. which was observed by the change in color from dark red to pink.²⁶

The appearance of the colony formation in CRYEMA medium was pink-centered, colorless, glistening, mucoid, moderately sized, opalescent, slightly elevated and circular colonies with a pungent odor (Fig. 2c). Two different organisms such as Agrobacterium tumefaciens and Rhizobium sp can able to sustain in this medium. The obtained isolate produces pink-centered colonies as it absorbed by the Congo Red dye (pH 6.8). The Agrobacterium species usually absorb congo red dye present in the CRYEMA medium, whereas Rhizobium sp cannot absorb Congo red.⁹ Hence, the isolate may be Agrobacterium tumefaciens. In Hofer’s alkaline medium, the appearance of the colonies seemed to be Yellow-colored, mucoid, confluent, pinpointed, circular, moderately sized, opalescent, elevated and slimy glistening colonies (Fig. 2d). The fermentation of mannitol agar occurred in the presence of bromothymol blue dye (pH 11) which led to the formation of yellow colonies. Therefore, the obtained isolate is concluded as Agrobacterium tumefaciens. In this medium, Rhizobium sp cannot absorb the dye that led to the formation of blue colonies. The growth characteristics of the crown gall in all four media are in good agreement with the characteristics of Agrobacterium species.

Fig. 2: Growth of crown gall isolate on a) nutrient agar, b) Mac Conkey agar, c) CRYEMA and d) Hofer’s alkaline

Isolation and Identification of Agrobacterium tumefaciens:

Microscopic analysis:

The selected isolates from the CRYEMA medium were subjected to gram staining to conclude their morphology. The cell color and shape of the isolates were concluded by this analysis. The isolates were observed to be slender, long, rod-shaped and pink in color under 10X magnification (Fig. 3a). It confirms that the isolate belongs to gram-negative. The SEM image of crown gall also revealed the long, slender and rod-shaped cells (Fig. 3b). This confirms the presence of Agrobacterium tumefaciens in the crown gall tissue.²⁷

Fig. 3: a) Optical microscope image of Crown gall and b) SEM image of Crown gall

Biochemical analysis:

The biochemical analysis was carried out as per Bergey’s manual (Figs. 4 and 5). The obtained results of this analysis are listed in Table 1. Indole test was performed to monitor the production of tryptophanase enzymes by the isolate to breakdown the tryptophan amino acid. However, the selected isolate was negative for this analysis. The study of Spiers²⁸ reported that the Agrobacterium spices could not able to split tryptophan. MR and VP analyses were carried out to evaluate the microbial potential towards the fermentation process. The isolate was negative for these tests since the absence of fermentation during this analysis. The citrate test was employed to monitor the production of the Citrase synthase enzyme by the isolate towards the degradation of the sodium citrate and inorganic ammonium dihydrogen phosphate. This was confirmed by the change in color from green to blue. Citrate synthase mutants of Agrobacterium species showed reduced virulence potential.^29,30 The selected isolate was positive according to this analysis.

The fermentative ability of the microbes was evaluated by the Trible Sugar Iron test. The obtained isolate produced alkaline slant/acid butt with H₂S production. This proved the sugar fermentation ability of the isolate. The test isolate was subjected to Urease analysis to assess its Urease enzyme production. It was understood that the test organism produced urease enzyme with the formation of pink color. The Agrobacterium tumefaciens produced a catalase enzyme to detoxify the H₂O₂while inducing tumors in plants.³⁰ Catalase enzymes act as a virulence factor.³¹ In this study, the crown gall isolates produced catalase enzyme which was evidenced by the formation of bubbles while adding H₂O₂to it. The Oxidase test was performed to conclude the production of cytochrome c oxidase enzyme by the microbes. The test isolate showed a positive response for the oxidase test.

Fig. 4: Biochemical analysis of Test and Control for a) Indole, b) MR, c) VP, d) Citrate, e) TSI and f) Urease test

Fig. 5 Biochemical analysis of a) catalase and b) oxidase tests

Table 1: Biochemical analysis of crown gall isolate

S. No	Bio-chemical Test	Result
1	Indole	-ve
2	Methyl Red (MR)	-ve
3	Voges-Proskaur (VP)	-ve
4	Citrate	+ve
5	Triple Sugar Iron (TSI)	+ve
6	Urease	+ve
7	Catalase	+ve
8	Oxidase	+ve

Tumerogenesis Assay:

Agrobacterium tumefaciens induces tumor formation while infecting dicotyledon plants. This tumor-inducing potential can be determined under in-vitro condition by placing potato or carrot disc in a water agar medium without supplementing any nutrients to identify the isolate. The inoculated microbes utilized the nutrients from the plant disc and produced a tumor-like appearance. In this study, the isolated crown gall tissue was examined for tumor development on the potato (Solanum tuberosum) disc. After 1^st week of incubation, the potato disc started to produce tumor-like spots (Fig. 6a). It gradually became thick in 2^nd week of incubation (Fig. 6b). Then it showed intensified appearance of the tumor during 3^rd week of incubation (Fig. 6c). In this assay, the potato (Solanum tuberosum) acts as a host cell.³² The morphology of the tumors was found to be a thick, white-colored, dense mat-like structure on the potato disc. Moreover, the formation of tumors on the potato disc was only due to the Agrobacterium tumefaciens. It is easily differentiated from the fungal contamination,³³ since the fungal spores possess loosely bound aerial structures that enable them to attach weekly to the media. The tumor-causing potential of the isolate was authentically confirmed by the Potato disc assay. Hence, the isolate was concluded as Agrobacterium tumefaciens.^34,35.

Fig. 6: Tumor formation on potato disc (Tumerogenesis assay) a) 1^stweek of incubation, b) 2^nd week of incubation and c) 3^rdweek of incubation)

Anti-bacterial sensitivity test using garlic extracts:

The antibiotic sensitivity test helps to determine the resistance pattern of the microbes against antibiotics and plant extracts. In this study, the agar well diffusion method was used to evaluate the antibacterial efficacy of the garlic extract against the isolate. After 24 hrs of incubation, the ethanolic extract of garlic at the concentration of 10μg/ml produced an inhibitory zone of 15mm (Table 2). The ampicillin and crude extract of garlic (10 μg/ml) showed an inhibitory zone of 10mm and 5mm (Fig. 7) respectively, against the crown gall isolates. The study of Mounyr et al.¹⁵ on antimicrobial activity of garlic showed that the ethanolic extract exhibited significant inhibition potential against crown gall isolate than the crude garlic extract. Similarly, the ethanolic extract of garlic showed a high inhibitory potential against crown gall in the present work.Hence, it may be used to control the spread of Agrobacterium tumefaciens infections in dicotyledons at the field level.

Fig.7: Antibiotic sensitivity assay against crown gall isolate using garlic (Allium sativum) extract

Table 2: Antibacterial analysis of ethanolic garlic extract against Crown gall

S. No	Samples used	Concentration (μg)	Zone Diameter (mm)
1	Ethanolic extract of Allium sativum (garlic)	10	15
2	Crude extract of Allium sativum (garlic)	10	5
3	Ampicillin	10	10

CONCLUSION:

The isolated bacterial strains from the crown gall tissue of the infected leaf of Pongamia pinnata were identified as Agrobacterium tumefaciens. The appearance of the isolated colonies in four different nutrient media also confirmed the existence of Agrobacterium tumefaciens in crown gall tissue. The morphology of the Agrobacterium tumefaciens was observed as pink, slender and rod-shaped by Grams’ staining and SEM analyses. The potato disc assay also proved the characteristic tumor formation potential of Agrobacterium tumefaciens. The ethanolic extract of garlic significantly inhibited the growth of Agrobacterium tumefaciens with the inhibitory zone of 15 mm. Therefore, the crown gall disease in Pongamia pinnata can be effectively controlled in a cost-effective manner using ethanolic extract of garlic to increase the crop yield.

CONFLICT OF INTEREST:

There is no conflict of interest among the authors.

REFERENCES:

1. Kerpen L, Niccolini L, Licausi F, van Dongen J.T., Weits, D.A. Hypoxic Conditions in Crown Galls Induce Plant Anaerobic Responses That Support Tumor Proliferation. Frontiers in Plant Science. 2019 10:56. https://doi.org/10.3389/fpls.2019.00056

2. Gohlke, J., Deeken, R. Plant responses to Agrobacterium tumefaciens and crown gall development. Frontiers in Plant Science. 2014; 5: 155. https://doi.org/ 10.3389/fpls.2014.00155

3. Daniel P, Hans, T.-C.,Monica, L.P., Doru, P., Constantin, B., Catherine, B. Agrobacterium tumefaciens: From Crown Gall tumors to genetic transformation. J. Physilogic. Moleculecular Plant Pathology. 2011; 76(2): 76-81. https://doi.org/ 10.1016/J.Pmpp.2011.06.004.

4. Soriful, I., Munsina, A., Atikur, R, Mostafizur, R., Mauluda, A., Firoz, A. Isolation of Agrobacterium tumefaciens strains from Crown Gall Samples of Dicot plants in Bangladesh. Current Research in Bacteriology. 2010; 3(1): 27-36. DOI: 10.3923/crb. 2010.27.36.

5. Naruto F., Fumika, S., Yutaka, N., Kishiko, N., Minoru, T., Nobuaki, M., Kayo, M., Youichi, T. Crown Gall of tobacco caused by Agrobacterium tumefaciens biovar 1 in tobacco fields. Journal of General Plant Pathology. 2004; 70(1):39-44, https://doi.org/10.1007/s10327-003-0088-1.

6. Shih, P.Y., Chou, S.J., Müller, C., Halkier, B.A., Deeken, R., Lai, E.M. Differential roles of glucosinolates and camalexin at different stages of Agrobacterium-mediated transformation. Molecular Plant Pathology. 2018; 19(8): 1956–70. doi: 10.1111/mpp.12672

7. Gowthami, Y., Chandrasehar, G., Sathya, T.N. Isolation of Agrobacterium tumefaciens strains from stem Gall disease on Manilkara zapota tree in Padappai, Tamil Nadu, India. Biotechnological Research. 2004; 1(3):156-159.

8. Bobbrala, V., Rao, G.S., Maduri, D.B., Naidu, K. C. Biocide Potentialities of Different Plant Methanolic Extracts Against Crown Gall Bacteria viz Agrobacterium tumefaciens. Biomedical and Pharmacology Journal. 2009; 2(1): 79-84.

9. Kado, C.I. 2002. Crown Gall, The Plant Health Instructor, https://doi.org/10.1094/PHI-I-2002-1118-01.

10. Sumathi, R., Pavni, S., Sivakumar, T. Antimicrobial Evaluation of Lipid Extract of Pongamia pinnata Leaves. Research J. Pharm. and Tech. 2009; 2(4): 714-718.

11. Manikannan, M., Arumugam, S., Rangasamy, B., Selvaraj J. Immune Stimulation effects of Pongamia pinnata extracts, an In vitro Analysis. Research J. Pharm. and Tech. 2020; 13(1): 308-312. doi: 10.5958/0974-360X.2020.00062.1

12. Padmini, R., Sitrarasi, R., Razia, M. Molecular Docking Studies of Bioactive Compounds from Allium sativum Against EML4-ALK Receptor. Research J. Pharm. and Tech. 2017; 10(11): 3741-3747. doi: 10.5958/0974-360X.2017.00679.5

13. Hussein, J., Hussein, Imad Hadi, H., Mohammed Y.H. A Review: Anti-microbial, Anti-inflammatory effect and Cardiovascular effects of Garlic: Allium sativum. Research J. Pharm. and Tech. 2017; 10(11): 4069-4078. doi: 10.5958/0974-360X.2017.00738.7

14. Lolok, N., Muthmainnah, H., Annah, M.I., Saleh, A., Yuliastri, W.O., Isrul, M. Antidiabetic Effect of the Combination of Garlic Peel Extract (Allium sativum) and Onion Peel (Allium cepa) in Rats with Oral-Glucose Tolerance Method. Research J. Pharm. and Tech. 2019; 12(5): 2153-2156. doi: 10.5958/0974-360X.2019.00357.3

15. Fatimah, A.J., Hameed, S.A.H. Chemical Datasets, Antioxidant, Free Radicals Scavenger activities estimate in Aqueous Garlic (Allium sativum) extract. Research Journal of Pharmacy and Technology. 2021; 14(10): 5157-2. doi: 10.52711/0974-360X.2021.00897

16. Lia, A., Emilia, G., Ninis, Y., Giftania, W., Sudjarwo. In vitro Antiplatelet Activities of Aqueous Extract of Garlic (Allium sativum) and black Garlic in Human Blood. Research Journal of Pharmacy and Technology. 2022; 15(4): 1579-2. doi: 10.52711/0974-360X.2022.00263

17. Keshav, S., Deepak Kumar, B. Effect of liquid biofertilizer with Herbal Insecticide Garlic (Allium sativum L.) on productivity of Gram (Cicer arietinum) and Infestation of Helicoverpa armigera (Hübner). Research J. Pharm. and Tech. 2020; 13(6): 2567-2572. doi: 10.5958/0974-360X.2020.00457.6

18. Manjula, R., Syed Abdul, H., Aravind Yaswanth, C., Gowtham, V., Padma, T. Formulation and Characterization of Cedrus deodara Oil Emulsion and studies on its activity against representative food and plant pathogens. Research J. Pharm. and Tech. 2019; 12(3): 1333-1337. doi: 10.5958/0974-360X.2019.00223.3

19. Parvathi, P. Garlic- A Golden Wonder. Research J. Pharm. and Tech. 2018; 11(1): 393-396. doi: 10.5958/0974-360X.2018.00072.0

20. Anand, P.P., Ramani, N. Dynamics of limited neoplastic growth on Pongamia pinnata (L.) (Fabaceae) leaf, induced by Aceria pongamiae (Acari: Eriophyidae). BMC Plant Biology. 2021; 21: 1. https://doi.org/10.1186/s12870-020-02777-7

21. Hamed, K. Characterization and Biocontrol of Crown Gall Disease in Jordan. Jordan Journal of Agricultural Sciences. 2006; 2(3): 265-272.

22. Wise, A. A., Liu, Z., Binns, A.N. Culture and Maintenance of Agrobacterium Strains. Agrobacterium Protocols. 2006; 3–14. https://doi.org/10.1385/1-59745-130-4:3

23. Raffaele, P., Aida, R., Fabio, M., Astolfo, Z. Physiological, biochemical and molecular analyses of an Italian collection of Agrobacterium tumefaciens strains. Europe. Journal of Plant Pathology. 2003; 109: 291–300. https://doi.org/ 10.1023/A:1023556108085

24. Hussain, A., Zia, M., Mirza, B. Cytotoxic and antitumor potential of Fagonia cretica L. Turkish Journal of Biology. 2007 31: 19-24.

25. Mounyr, B., Moulay, S., Saad, K. I. Methods for in vitro evaluating antimicrobial activity: A review. Journal of Pharmaceutical Analysis. 2016; 6(2): 71-79. https://doi.org/10.1016/j.jpha.2015.11.005

26. Erican, A. Inhibition of Crown Gall Tumorigenesis with Plant Extracts. Journal of Pharmaceutical Biology. 2009; 47(5): 463-466, https://doi.org/10.1080/1388020090824873

27. Abdul, Q.S., Palash, C. M., Soriful, I., Mohammad, F.A. Identification of Virulent Agrobacterium tumefaciens Strains from some Dicotyledonous Plants in Bangladesh. Agriculturae Conspectus Scientificus. 2011; 76 (2): 147-152.

28. Spiers, A.G. Isolation and characterisation of Agrobacterium species. New Zealand Journal of Agricultural Research. 1979; 22(4): 631-639, https://doi.org/10.1080/00288233.1979.10417834

29. Suksomtip, M., Liu, P., Wood, D., Nester, E.W. Citrate synthase mutants of Agrobacterium are attenuated in virulence and display reduced vir gene induction. J. Bacteriology. 2005; 187: 4844–4852.

30. Nester, E.W. Agrobacterium: nature’s genetic engineer. Frontiers in Plant Sciences. 2015; 5: 730. https://doi.org/ 10.3389/fpls.2014.00730

31. Bosmans, L., Moerkens, R., Wittemans, L. De Mot, R., Rediers, H., Lievens, B. Rhizogenic agrobacteria in hydroponic crops: epidemics, diagnostics and control Plant Pathology. 2017; 66:1043. https://doi.org/10.1111/ppa.12687

32. Thompson, M.A., Onyeziri, M.C., Fuqua, C. Function and Regulation of Agrobacterium tumefaciens Cell Surface Structures that Promote Attachment. Curr. Topic. Microbiology and Immunology. 2018; 418: 143–184. https://doi.org/10.1007/82_2018_96

33. Aysan, et al. First report of Crown Gall of apricot (Preunus armenica) caused by Agrobacterium tumefaciens in Turkey. Journal of Plant Pathologys. 2003; 52(6): 793. https://doi.org/ 10.1111/j.1365-3059.2003.00902.x.

34. Rahman, S., Kafeel, A., Niaz A., Muhammad, I., Waqar, A. Isolation and Characterization of Agrobacterium tumefaciens Strains from Malakander Farm, University of Agriculture, Peshawar. Pakistan Journal of Zoology. 2018; 52(5): 2019-2021. https://dx.doi.org/10.17582/journal.pjz/20181021061044

35. Ganjeh, A., Heshmatollah, R., Esmaeil, B. Agrobacterium tumefaciens causing crown and stem gall disease of citrus propagation nursery in Iran. Journal of Plant Pathology. 2021; 103:355. https://doi.org/10.1007/s42161-020-00679-z

Received on 11.11.2022 Modified on 30.04.2023

Research J. Pharm. and Tech 2023; 16(12):5597-5602.

DOI: 10.52711/0974-360X.2023.00905