Isolation, characterization of phytopathogenic bacterial types in pomegranate crops of Chitradurga district Biochemical and molecular insights

Geetha M. B¹ , Ravikumar Patil H. S¹ , Kiran Kumar H. B²

¹Department of Studies in Food Technology, Shivagangotri, Davangere University, Davangere, India

²Nrupathunga University, Nrupathunga Road, Bangalore, 560001, India

Corresponding Author Email: patil_varuni@rediffmail.com

DOI : https://doi.org/10.51470/eSL.2026.7.1.88

Abstract

Download this article as:

1.0.Introduction

Phytopathogenic bacteria infect a large spectrum of food-producing plants worldwide (1). Despite its high economic and nutritional value, pomegranate farming is hampered by a variety of phytopathogenic bacteria that reduce fruit quality and output. They induce symptoms such as spots, blights, cankers, and tissue rots when they colonise the plant’s surface or tissues, resulting in plant overgrowth, stunning, root branching, and leaf epinasty (2). Thus, they have a deleterious impact on both the qualitative and quantitative characteristics of crops and constitute a significant threat to the global food supply chain. Pomegranate (Punica granatum L.) is a member of the Lythraceae family and has long been a popular fruit in tropical and subtropical climates around the world.  The fruit’s peel, seeds, and pulp have great medicinal and pharmacological value and are a good source of carbohydrates and minerals like calcium, iron, and potassium. Additionally, they are currently marketed as a nutraceutical and functional food source with health-promoting advantages (3). Numerous bioactive substances, as well as significant concentrations of water, sugars, and organic acids like malic, citric, and ascorbic acid, are found in pomegranate fruit. According to 4, it is also abundant in polyphenols, including fatty acids (punicic acid), minerals (potassium and phosphorus), vitamins (C and K), flavonoids (anthocyanins), and tannins (ellagitannins and proanthocyanidins). The bioactive substances support antibacterial, anti-inflammatory, and antioxidant properties, among other health advantages. In addition, help prevent and treat chronic conditions such as diabetes mellitus, obesity, renal disease, cardiovascular disease, and neurodegenerative illnesses (5). Numerous bacterial and fungal diseases cause disease in the crop, affecting both its quality and quantity. Approximately 37 distinct pomegranate diseases have been linked to over 55 pathogens worldwide (6). The main pathogens that cause blasts, wood canker, branch dieback, rots, and wilts are Xanthomonas, B. cinerea, Penicillium spp., Al. alternata, and C. granati. Fruit rind and aril deterioration during storage is facilitated by pathogens, which also cause fruit to perish (7). According to 8, bacterial infections are prevalent in areas where pomegranates are grown and cause significant fruit losses during the growing season. The problem of bacterial blight is dangerous and has the potential to spread like wildfire. Pomegranate bacterial blight has been documented worldwide in a number of nations, including South Africa (9), Iran, Iraq, Pakistan (10), and Turkey (11). All plant parts, with the exception of roots and flowers, exhibit symptoms of bacterial blight; fruits are particularly susceptible. The symptoms include cankers on stems, branches, and trunks, as well as spots on fruits and foliage (12). In India, the oldest recorded blight are (13). Since then, several researchers from various regions of the nation, including Tamil Nadu, Himachal Pradesh, Haryana, Karnataka, Maharashtra, and Rajasthan, have reported blight and the resulting losses (14,15,16,17,18,19,20).

Bacterial infection in crops starts with adhering to the plant’s surface through biofilms, and enters through wounds, stomata, or hydathodes and uses enzymes to break down plant tissues (2). The majority of plant infections introduce effector proteins into plant cells via a type-III secretion system (T3SS), which interferes with host cellular functions and permits bacteria to proliferate and spread illness. To evade plant defence proteins, they also synthesise effectors (ETS) (21). The bacterial blight spreads quickly in altered environmental conditions, leading to significant defoliation and decreased photosynthesis (22). Environmental stress, extensive pesticide usage, and other control methods cause mutation or recombination, which leads to new forms of plant infectious illnesses (23). Additionally, the temporal and spatial dissemination of infectious pathogens is accentuated by human mobility. A high level of genetic variability among the strains of X. axonopodis pv. punicae has been reported, which is independent of host cultivar and geographical origins (24). Further, in the last decades, outbreaks are attributed to horizontal transmission of the pathogen aided by genetically homogenous clones of apparently healthy, but latently infected, planting material of elite pomegranate cultivars (25). In order to decipher plant defence mechanisms and create efficient management strategies, it is critical to comprehend disease resistance reactions in pomegranate genotypes against bacterial blight. Epidemiology and laboratory methods enable assessment of pathogen emergence, strain variability and outbreaks of disease symptomology (26). With this background, the present study was carried out to isolate and identify phytopathogenic bacterial types present in pomegranate crops of Chitradurga district using morphological, biochemical, and molecular methods.

2.0. Material and methods

Field Survey and Sample Collection

A single individual collected the sample, adhering to GLP (Good Laboratory Practices). A systematic field survey was conducted across five taluks in Chitradurga district in the month of May, 2025. From Hiriyur, Challakere, Molakalmuru, Holalkere, and Chitradurga taluk (longitude 76.210329°, latitude 14.361161°) respectively. Pomegranate plants with visible symptoms such as leaf spots, fruit rot, stem blight, and bacterial ooze were selected. A total of 45 symptomatic leaves (n=20), fruits (n=15), and stems (n=10) were collected in individual sterile bags and labelled and transported to the laboratory.

Isolation of Bacterial Strains

Asepsis and microbiology standards were adhered to during isolation of the bacteria. High-grade chemical and growth media were used.

Infected issue samples were surface-sterilised using 0.1% mercuric chloride followed by rinsing in sterile distilled water (27).  The tissues were macerated in phosphate-buffered saline (PBS), and aliquots were streaked onto Nutrient Agar (NA) and King’s B agar medium plates and incubated at 28°C for 48–72 hours. After incubation, the colonies with characteristic bacterial morphology were sub-cultured to obtain pure isolates (28).

Characterization of Isolates

Morphological Characteristics

Colony morphology such as shape, colour, elevation, margin, and opacity were documented using standard methods (29)

Biochemical Tests

Gram staining, Catalase and oxidase, Gelatin and starch hydrolysis were carried out as described in (30)

Molecular Identification

DNA extraction using CTAB method was completed for all isolates (31). Quality and quantity were estimated by a Spectrophotometer and 1% agarose gel. PCR amplification targeting 16S rRNA gene was carried out using primer (32). 33

PCR primer seqs- 16S rRNA primers (F-5’-GAGTTTGATCCTGGCTCA-3’; R-5’ -AGAAAGGAGGTGATCCAG-3’)

The PCR products were sequenced and the sequence was searched with the Basic Local Alignment Search Tool (BLAST) using the ncbi-blast server to find regions of similarity between sequences in the database.

In-vitro Pathogenicity Evaluation-Host Plant Inoculation (Detached Leaf Assay)

Bacterial cultures were grown in nutrient broth for 24 hours. Suspension adjusted to 10⁸ CFU/mL using spectrophotometric OD values at 600 nm. Healthy, surface-sterilized middle aged pomegranate leaves were inoculated with bacterial suspensions using the prick and drop method using a sterilised needle at several sites on the leaf. Control leaves were treated with sterile distilled water. Incubation was done in moist chambers at 28°C and leaves observed over 7 days for symptoms (32).

Scoring was based on the following criteria:

• Lesion diameter
  • Color change
  • Water-soaked appearance
  • Presence of bacterial ooze

Re-Isolation and confirmation of pathogenicity by Koch’s Postulates

Pathogens were re-isolated from infected tissues using standard methods and compared with original isolates using staining and biochemical methods to confirm the causality.

3.0. Results

A total of 17 distinct bacterial isolates were isolated and preserved on slants for further studies. The Gram staining results indicated majority of the bacteria were Gram-negative rods. Bacteria were positive for the catalase and oxidase tests. The Gelatin hydrolysis and starch hydrolysis indicated variable results, aiding in differentiation. Most isolates exhibited yellow to cream-colored mucoid colonies suggestive of Xanthomonas species. The biochemical tests of the Xanthomonas are summarised in the table-1.

Successful isolation of DNA was carried out from all bacteria and the quality and quantity. The 16S rRNA gene sequence was used as a primer for amplification of the DNA from all isolates. Figure-3 depicts the PCR products of the blight strains amplified using 16sRNA primer, electrophoresed on 2% agarose gel.

Studies on cultural, morphological, physiological, biochemical and genetic features of the pathogen are of immense use in understanding the nature of the pathogen. BlastP results of the strain Xa-1 indicated 100% alignment to 140 blast sequences in databases. Major hits (128 were to Xanthomonas (g-proteobacteria). Major alignments were to Xanthomonas axonopodis (23), Xanthomonas perforans(12), Xanthomonas citri pv. Fuscans(15), Xanthomonas euvesicatoria(24) causative agents of blight in fruits and vegetables. The Xc-4 blast indicated 100% alignment to 148 blast sequences in databases. Major hits (122) were to Xanthomonas(g-proteobacteria) and one uncultured bacterium. Major alignment was to Xanthomonas axonopodis (21), Xanthomonas perforans(10), Xanthomonas euvesicatoria (74), Xanthomonas citri pv. Fuscans (9). The data is summarised as a phylogenetic tree in figure-4A and B and respectively.

The In-vitro Pathogenicity Evaluation-Host Plant Inoculation (Detached Leaf Assay)

Symptomology was scored visually and recorded

Re-Isolation and confirmation of pathogenicity by Koch’s Postulates.

The results were Gram staining positive and similar biochemical tests as seen in the isolated bacteria confirming the pathogen its pathogenicity.

4.0. Discussion

India is the world’s largest pomegranate grower with a cultivable area of 2.3 lakh ha and an annual production of 28 lakh tonnes. Maharashtra covers the largest area (1.4 lakh ha) and is the India’s largest pomegranate producer, followed by Gujarat, Karnataka and Andhra Pradesh (34).Bacterial blight caused by Xanthomonas axonopodis pv. punicae (Xap) is one of the most devastating diseases of pomegranate, resulting in yield loss of 70-90 per cent (35 and 36). Figure-5 is a representative image of the infection. Fruit bacterial infection variability is a complicated, multifaceted phenomenon that is impacted by maturation stage, environmental factors, particular fruit species and postharvest handling (37,38,39). In recent times, Xap has spread to different geographical regions, indicating the wide adaptability of the pathogen. This variation impacts not only the kind of bacterial populations that are present (composition), but also their density and pathogenicity, which can range from minor tissue damage to serious contamination (40). Bacterial variability is determined by a number of variables, including the fact that isolates of bacteria exhibit notable variations in the course of the disease, with certain strains such as the jackfruit-bronzing disease strain JEN-14, being significantly more aggressive than others (41). b. The main pathogen in crops, such as kiwifruit, can change depending on the season. For instance, Pectobacterium carotovorum subsp. Actinidiae causes greater harm in the summer and Pseudomonas syringae pv. actinidiae in the winter and spring. c. Same species variation as demonstrated by the different, unique genotypic groups of Pseudomonas syringae pv. Syringae discovered on stone fruit (42). 43,44 and other researchers used morphological and DNA-based techniques to report similar diversity in Xanthomonas axonopodis pv punicae.

The pathogen exhibits considerable variability when cultured on different artificial media. Growth on Phyto-Xynocamp Agar Base Media enabled the characterisation of Xanthomonas. Its utility has been successfully demonstrated by its adoption by the 45. Earlier efforts to characterise the pathogen include growth and colony morphology on different media like SX, SM, BSCAA, MXP, MD-5 and Tween (46).

Biochemical test methods are employed to identify and differentiate bacteria based on their specific metabolic activities, enzyme production, and chemical utilisation. Xanthomonas spp., Pseudomonas spp., and Erwinia spp. were the main species identified in the present study based on colony, culture and biochemical tests. These tests detect phenotypic differences, such as carbohydrate fermentation or protein metabolism, by analysing changes in pH, color, or gas production, crucial for species identification. The Biochemical test of the four isolates confirmed they are Gram-positive, aerobic and have other features unique to Xanthomonas. Previous studies by researchers on bacteria on pomogranates include Pant and Shashidhar report Acinetobacter, Micrococcus, Pantoea, Microbacterium, Strenotrophomonas, Bacillus, Staphylococcus and Exiguobacterium,Gluconobacter oxydans (47), Xanthomonas (48).

Host Plant Inoculation data suggested 11 of the isolated bacteria demonstrated pathogenicity with visible symptoms on leaf tissue. Three isolates of the Xanthomonas showed strong virulence, causing necrotic lesions within 48 hours in the Host Plant inoculation assay. The data complement the observation from in vitro studies that Xanthomonas are dominant and highly virulent. A correlation was observed between colony morphology and disease severity.  It is reported pathogen causes water-soaked, angular, and dark brown/black necrotic lesions on leaves, stems, and fruits including necrotic spots, within 2–3 days (48-72 hours) of inoculation (49). It is reported that high humidity (>60%), warm temperatures (25–33°C), and rain splash accelerate the development of these necrotizing lesions and these symptoms are indicative of the Virulence (50,51).

Xanthomonas use effectors (ETS) and Extracellular polysaccharides (EPS) to overcome plant defense proteins (52). Extracellular polysaccharides (EPS), clog the xylem vessels and cause wilting. The type III secretion systems (T3SS) supply virulence factors, which decrease host immune responses and promote the spread of disease (22,53). Through the type III secretion system (T3SS), Xap secretes the effector proteins XopN and XopL to promote pathogenesis and inhibit host immune responses. The TTSS-effector inhibits plant immune responses, such as programmed cell death (PCD), and supports the proliferation and pathogenicity of XopL for a significant amount of time throughout the development of blight disease (54). Particularly in poor weather conditions, the blight can spread quickly, resulting in severe defoliation and decreased photosynthesis. Studies of the Indian strain of Xanthomonas axonopodis implicate six non-TAL or Xop effectors (55) also, highlight XopL and XopN are major contributors to virulence and (54).56 report using null mutants virulence, in planta bacterial growth, and reactive oxygen species (ROS) production studies and suggest multiple non-TAL (Xop) T3SS effectors have roles in suppression of plant immune responses. (33) report Virulence diversity in Xap for Xanthomonas axonopodis pv. Punicae isolates on Pomegranate Variety cv. Bhagwa. The results implicate infection of different isolates is directly correlated with the EPS production.

The Phylogentic distance tree grouped the Xa-1 strain along with other Xanthomonas strains with X. oryzae as the outgroup. The Xc-4 strain had two nodes with Xanthomonas campestris pv. campestris strain sequence as the outgroup. The blast sequence was not grouped in the first cluster along with other Xanthomonas strains suggesting its unique sequence. Xa-1 strain has higher similarity with Xanthomonas axonopodis, where Xc-4 strain Xanthomonas euvesicatoria suggesting the different evolutionary trajectories. Numerous clusters and subgroups of the pathogen have been found by molecular investigations employing molecular markers, suggesting a high level of genetic diversity in the Indian isolates.  56 report an multilocus sequence analysis MLSA study of 24 isolates collected from major Indian states during disease epidemics documenting phenotypic traits, pathogenicity and cultural characteristics. Sequence-based characterization at nine genetic loci, revealed that all isolates showed more than 99% similarity with X. axonopodis pv. punicae isolates for the 16S and the IGS rDNA gene sequence confirming their identity as X. axonopodis pv. punicae.  The study proposes clonal evolution and lateral transmission of bacterial blight of pomegranate as a consequence of pathogen migration with the expansion of pomegranate cultivation.

Research insights into the pathogenicity indicate that Xanthomonas axonopodis pv. punicae is highly specific to pomegranate, and there are no alternate hosts to this pathogen. Resistance to the pathogen in tolerant lines of pomegranates results from the expression of high transcript levels of defence response genes (57). Few epidemiology studies demonstrate that this variation is location-dependent (58).Also, few authors opine that outcrossing behaviour of pomegranate crop and the acquired antibiotic resistance due to overexposure to antimicrobial agents employed for the control of the disease have contributed to pathogen variability (59,60). Evidence of population shift and local adaptation is reported from Taiwan (61). Horizontal Gene Transfer (HGT), genome rearrangements, emergence of new virulent strains with modified or lost effectors are the mechanisms adopted by the pathogen as part of its virulence arsenal (62,63,64). These research findings implicate the pathogen evolves using various mechanisms to overcome resistance and differentiate at the morphological and genetic level to aid spread or overcome environmental and preventive practices.

Among the different districts surveyed by 65, maximum fruit infection of 38.29 per cent was recorded in Chitradurga district Koppal (32.40%) and Bellary (32.21%) districts. Correspondingly, average severity of the disease on fruits was observed as maximum in Bijapur district (28.95 PDI) and minimum severity of 17.86 PDI was recorded in Bellary district. 65 reported Xa₁₃ strain isolated from Hiriyur district. Further, the dendrogram analysis showed two major clusters A and B with cluster-A composed of all other isolates belonging to different isolates of Bagalkot, Bellary, Bijapur, Chitradurga Koppal of Karnataka. In India blight severity increases during June and July and reaches a maximum in September and October, attributed to the seasonal south-west monsoon and then gradually declines (12). Altitude, climate significantly influences the epiphytic bacterial community. Warm weather and high humidity are reported to increase the susceptibility of fruits to bacterial diseases. It could be hypothesised that in the present study, these could be the triggers for the strain variability. The study has few limitations such as only single season sampling, few biochemical assays and genotyping of a single locus to test strain variability.

Conclusion The current study identified a consortium of epiphytic bacteria from the pomogranante. Specifically 4 strains of Xanthomonas were identified with high Virulence. Whole genome sequencing of the strain will provide further insights into the genetic correlates of the pathogen’s virulence. The results will help better management of blight in pomegranate by designing better anti-microbials and thus reduce fruit loss. Thus the study of plant-pathogen and their interaction, molecular pathways and evolution of strain variability in response to various biotic and abiotic factors assumes significance.

References

1. Martins, P. M., Merfa, M. V., Takita, M. A., & De Souza, A. A. (2018). Persistence in phytopathogenic bacteria: do we know enough? Frontiers in microbiology, 9, 1099.
2. Agrios G.N. Plant Pathology 5Ed 2020.Elsevier Exclusive.ISBN: 9788131206393
3. Sreekumar, S., Sithul, H., Muraleedharan, P., Azeez, J. M., & Sreeharshan, S. (2014). Pomegranate fruit as a rich source of biologically active compounds. BioMed research international, 2014(1), 686921.
4. Siddiqui, S. A., Singh, S., & Nayik, G. A. (2024). Bioactive compounds from pomegranate peels-Biological properties, structure–function relationships, health benefits and food applications–A comprehensive review. Journal of Functional Foods, 116, 106132.
5. Cheng, J., Li, J., Xiong, R. G., Wu, S. X., Huang, S. Y., Zhou, D. D., A Saimaiti, A Shang, Y Feng, RY Gan & Li, H. B. (2023). Bioactive compounds and health benefits of pomegranate: An updated narrative review. Food Bioscience, 53, 102629.
6. Jain, K., & Desai, N. (2018). Pomegranate the cash crop of India: A comprehensive review on agricultural practices and diseases. Int J Health Sci Res, 8(5), 315-336.
7. Mincuzzi, A., Picciotti, U., Sanzani, S. M., Garganese, F., Palou, L., Addante, R., Ippolito, A & Ippolito, A. (2023). Postharvest diseases of pomegranate: Alternative control means and a spiderweb effect. Journal of Fungi, 9(8), 808.
8. Munhuweyi, K., Lennox, C. L., Meitz-Hopkins, J. C., Caleb, O. J., & Opara, U. L. (2016). Major diseases of pomegranate (Punica granatum L.), their causes and management – A review. Scientia Horticulturae, 211, 126–139. https://doi.org/10.1016/j.scienta.2016.08.0 16.
9. Petersen, Y., Mansvelt, E. L., Venter, E., & Langenhoven, W. E. (2010). Detection of Xanthomonas axonopodis pv. punicae causing bacterial blight on pomegranate in South Africa. Australasian Plant Pathology, 39(6), 544-546.   
10. Osdaghi, E. (2022). Xanthomonas citri pv. punicae (bacterial blight of pomegranate). In CABI Compendium
11. Icoz, S. M., Polat, I., Sulu, G., Yilmaz, M., Unlu, A., Soylu, S., Bozkurt I A, Baysal, Ö. (2014). First report of bacterial blight of pomegranate caused by Xanthomonas axonopodis pv. punicae in Turkey. Plant disease, 98(10), 1427-1427.
12. Gowda, B. H., Ramesh, P. R., & Prashanth, J. M. (2020). Weather Based Approach for Effective Management of Bacterial Blight of Pomegranate caused by Xanthomonas axonopodis pv. punicae. Int. J. Curr. Microbiol. App. Sci, 9(9), 1424-1428.
13. Hingorani, M. K., & Mehta, P. P. (1953). Bacterial leaf spot of pomegranate. Indian Phytopathology, 5, 55–56.  
14. Kanwar, Z. S. (1976). A note on bacterial disease of pomegranate (Punica granatum L.) in Haryana. Haryana J. Hort. Sci, 5, 177-180.
15. Eddy, N., Karale, M., Singh, R., & Gilotra, P. (2018). Horticulture statistics at a glance 2018. Government of India, Ministry of Agriculture & Farmers’ Welfare, Department of Agriculture, Cooperation & Farmers’ Welfare. http://agricoop.nic.in/sites/default/files/Horti culture Statistics at a Glance-2018.pdf.
16. Manjula, C. P., & Khan, A. N. A. (2002, December). Incidence of bacterial blight of pomegranate (Punica granatum L.) in Karnataka. In Annu. Meet. Symp. Plant disease scenario in southern India, held at Bangalore.
17. Pawar, B. T. (2007). Studies on the bacterial diseases of fruit plants from Aurangabad district (Doctoral dissertation). Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra, India.

18. Meena, S. C., Meena, M. K., Chattopadhyay, A., & Mali, B. L. (2017). Distribution and prevalence of bacterial blight of pomegranate in Southern Rajasthan.

19. Chowdappa, A., Kamalakannan, A., Kousalya, S., Gopalakrishnan, C., Venkatesan, K., & Raju, G. S. (2018). A survey on the incidence of bacterial blight Xanthomonas axonopodis pv. punicae of Pomegranate in Tamil Nadu. Journal of Pharmacognosy and Phytochemistry, 7(3), 3740-3742.

20. V.I Benagi, M.R. Ravi Kumar, S.B. Gowdar, Basvaraj B. Pawar. (2009).

II International Symposium on Pomegranate and Minor – including Mediterranean – Fruits: 890_71, 509 – 511. ISPMMF2009

Yenjerappa (2014). PhD thesis Dharwad agriculture university.  Dharwad.

21. Prescott, John. (2013). Bacterial Pathogenesis: Molecular and Cellular Mechanisms, C. Locht, M. Simonet (Eds.). Academic Press, Caister (2012), 369 pp., Price: US$360. Veterinary Microbiology. 162. 1000. 10.1016/j.vetmic.2012.09.022.

22. Huang, Y. C. (2024). Bacterial blight: The mechanisms behind its aggressive spread. Journal of Plant Physiology and Pathology.

23. Islam, T., Danishuddin, Tamanna, N. T., Matin, M. N., Barai, H. R., & Haque, M. A. (2024). Resistance mechanisms of plant pathogenic fungi to fungicide, environmental impacts of fungicides, and sustainable solutions. Plants, 13(19), 2737.

24. Chavan, N. P., Pandey, R., Nawani, N., Tandon, G. D., & Khetmalas, M. B. (2017). Genetic variability within Xanthomonas axonopodis pv. punicae, causative agent of oily spot disease of pomegranate. J Plant Pathol Microbiol, 8(394), 2.

25. Patil, P. G., Sharma, J., Nanjundappa, M., Singh, N. V., Bohra, A., Gunnaiah, R., Jamma S.M., Vinayaka J.,Sangnure V.R., Marathe, R. A. (2022). Identification and validation of SSR markers for Xanthomonas axonopodis pv. punicae an incitant of bacterial blight of pomegranate. 3 Biotech, 12(7), 153.

26. Jeger MJ. (2020). The Epidemiology of Plant Virus Disease: Towards a New Synthesis. Plants (Basel). 9(12):1768. doi: 10.3390/plants9121768. 

27. Chowdappa, A., Kamalakannan, A., Kousalya, S., Gopalakrishnan, C., Venkatesan, K., & Raju, G. S. (2018). Isolation and characterization of Xanthomonas axonopodis pv. punicae from bacterial blight of pomegranate. J. Pharmacogn. Phytochem, 7(4), 3485-3489.

28. Adioumani, A. E., Kakou-Ngazoa, S., Trebissou, J. N. D., Martin, T., Addablah, A. Koudou, A., … & Dosso, M. (2022). Isolation and identification of phytopathogenic bacteria in vegetable crops in West Africa (Côte d’Ivoire). African Journal of Microbiology Research, 16(4), 436D2E669026.

29. K.R. Aneja. (2022). Experiments in Microbiology, Plant Pathology, Tissue Culture and Microbial Biotechnology. Paperback – 1. New age publications.

30. Khan, M. I., Ur Rehman, M., Khan, I., Shah, T. A., Aziz, T., Alharbi, M., Alshammari, A & Alasmari, A. F. (2024). Isolation, identification and characterization of xanthomonas axonopodis pv. citri from selected species. Applied Ecology & Environmental Research, 22(1).

31. Liu, B., Wu, H., Cao, Y., Ma, G., Zheng, X., Zhu, H., X Song & Sui, S. (2025). Reducing costs and shortening the cetyltrimethylammonium bromide (CTAB) method to improve DNA extraction efficiency from wintersweet and some other plants. Scientific Reports, 15(1), 13441.

32. Dattatraya Hegde Radhika, Raghavendra Gunnaiah, Dadapeer Peerjade, Manjunath G, Sathish D, RS Jawadagi and A M Nadaf (2022). confirmation of the pomegranate bacterial blight causing pathogen xanthomonas citri pv. punicae(Xcp) to assess disease severity in pomegranate genotype. The Pharma Innovation Journal, 11(12):4098-4104.

33. Rathna, V., & Prasannakumar, M. K. (2022). Virulence Diversity in Xanthomonas axonopodis pv. Punicae isolates on Pomegranate Variety cv. Bhagwa. Mysore Journal of Agricultural Sciences, 56(2).

34. Reddy, N., Karale, M., Singh, R. and Gilotra, P. (2018), Horticulture statistics at a glance, New Delhi, India: Government of India, Ministry of Agriculture and Farmers’ Welfare, Department of Agriculture.

35. Gargade, V. A., & Kadam, D. G. (2014). Antibacterial activity of some herbal extracts and traditional biocontrol agents against Xanthomonas axonopodis punicae. Journal of International Academic Research for Multidisciplinary, 2(3), 16-25.

36. Sonyal Shreeshail., Ravichandra, N. G., & Reddy, B. (2015). Efficacy of bioagents on Ceratocystis fimbriata and Meloidogyne incognita wilt complex in pomegranate. ecialise Sp, 49(2), 350-354.

37. Tenea, G. N., & Reyes, P. (2024). Bacterial community changes in strawberry fruits (Fragaria× ananassa variety “Monterey”) from farm field to retail market stands, an indicator of postharvest contamination. Frontiers in Microbiology, 15, 1348316.

38. Ramírez-Gil, J. G., Henao-Rojas, J. C., & Morales-Osorio, J. G. (2021). Postharvest diseases and disorders in avocado cv. Hass and their relationship to preharvest management practices. Heliyon, 7(1).

39. Morales, G., Moragrega, C., Montesinos, E., & Llorente, I. (2018). Environmental and inoculum effects on epidemiology of bacterial spot disease of stone fruits and development of a disease forecasting system. European journal of plant pathology, 152(3), 635-651.

40. Basamma (2013). Molecular phylogenetic analysis of xanthomonas axonopodis pv. punicae isolates and bio-prospecting of selected botanicals and bio-agents against bacterial blight of pomegranate Thesis submitted to the University of Agricultural Sciences, Dharwad

41. Abidin, N., Ismail, S. I., Vadamalai, G., Yusof, M. T., Hakiman, M., Karam, D. S., & Zulperi, D. (2021). Pathogenic variability of the jackfruit-bronzing bacterium Pantoea stewartii subspecies stewartii infection to jackfruit varieties and its pivotal plant hosts in Malaysia. Agronomy, 11(11), 2113.

42. Popović, T., Menković, J., Prokić, A., Zlatković, N., & Obradović, A. (2021). Isolation and characterization of Pseudomonas syringae isolates affecting stone fruits and almond in Montenegro. Journal of Plant Diseases and Protection, 128(2), 391-405.

43. Peerajade, D. A., Moger, N., & Patil, H. B. (2017). Studies on variability, identification of Xanthomonas axonopodis pv. punicae isolates and screening of gamma (γ) irradiated seed derived progenies and germplasms of pomegranate. Int. J. Curr. Microbiol. App. Sci, 6(8), 2987-3002.

44. Sahane, S. P., Ambadkar, C. V., & Navgire, K. D. (2017). Genetic Variability Amongst Xanthomonas axonopodis pv. punicae Studied by RAPD Banding Pattern Analysis. Int. J. Curr. Microbiol. App. Sci, 6(11), 1973-1982.

45. ISHI-veg. (2019). Detection of Xanthomonas campestris pv. campestris pv. Raphanin, on Brassica sps seeds.  International seed federation.

46. Schaad, N. W. (1992). Xanthomonas. In: Laboratory Guide for Identification of Plant Pathogenic Bacteria, II Ed. International Book Distributing Co. Lucknow, p. 165. 

47. Armanious, H. A. (2021). Pomegranate (Punica granata L.) inner decay caused by Gluconobacter oxydans bacterium. Asian Journal of Agricultural and Horticultural Research, 7(4), 14-29.

48. Ayana Chathalingath, Anbarasi Gunasekar, Sreelakshmy Venu, (2023), Phenotypic and molecular characterisation of Xanthomonas axonopodis pv. punicae from pomegranate leaves,Physiological and Molecular Plant Pathology,Volume 128,102160, ISSN 0885-5765.

49. Dia, N. C., Morinière, L., Cottyn, B., Bernal, E., Jacobs, J. M., Koebnik, R.,  Osdaghi E  & Pothier, J. F. (2022). Xanthomonas hortorum–beyond gardens: Current taxonomy, genomics, and virulence repertoires. Molecular plant pathology, 23(5), 597-621.

50. Mincuzzi, A., Sanzani, S. M., Palou, L., Ragni, M., & Ippolito, A. (2022). Postharvest rot of pomegranate fruit in southern Italy: Characterization of the main pathogens. Journal of Fungi, 8(5), 475.

51. Patel, Jay & Parmar, Fenil & Upasani, Vivek. (2021). Isolation and Characterization of Pathogens Causing Disease in Pomegranate (Punica granatum L.), India. International Journal of Innovative Research in Science Engineering and Technology. 10. 4403. 10.15680/IJIRSET.2021.1005041.

52. Waheed, A., Haxim, Y., Islam, W., Kahar, G., Liu, X., & Zhang, D. (2022). Role of pathogen’s effectors in understanding host-pathogen interaction. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1869(12), 119347.  

53. Shafee Ur Rehman. (2024). Recent developments in sugarcane transcriptome. Int. J. Biosci., 25(5), 252–270. DOI: http://dx.doi.org/10.12692/ijb/25.5.252-270

54. Soni, M., & Mondal, K. K. (2018). Xanthomonas axonopodis pv. punicae uses XopL effector to suppress pomegranate immunity. Journal of integrative plant biology, 60(4), 341-357.

55. Kumar, R., & Mondal, K. K. (2013). XopN-T3SS effector modulates in planta growth of Xanthomonas axonopodis pv. punicae and cell-wall-associated immune response to induce bacterial blight in pomegranate. Physiological and molecular plant pathology, 84, 36-43.

56. Mondal, K. K., Soni, M., Verma, G., Kulshreshtha, A., Mrutyunjaya, S., & Kumar, R. (2020). Xanthomonas axonopodis pv. punicae depends on multiple non-TAL (Xop) T3SS effectors for its coveted growth inside the pomegranate plant through repressing the immune responses during bacterial blight development. Microbiological research, 240, 126560.

57. Kumar, P., Dashyal, M. S., Doddaraju, P., Meti, B. S., & Girigowda, M. (2021). Differential gene responses in different varieties of pomegranate during the pathogenesis of Xanthomonas axonopodis pv. punicae. 3 Biotech, 11(4), 180.

58. Giri, M. S., Prashanthi, S. K., Srikanth Kulkarni, S. K., Benagi, V. I., & Hegde, Y. R. (2011). Biochemical and molecular variability among Xanthomonas axonopodis pv. punicae strains, the pathogen of pomegranate bacterial blight.

59. Chavan, N. P., Pandey, R., Nawani, N., Tandon, G. D., & Khetmalas, M. B. (2017). Genetic variability within Xanthomonas axonopodis pv. punicae, causative agent of oily spot disease of pomegranate. J Plant Pathol Microbiol, 8(394), 2.

60. Sharma, J., Manjunatha, N., Pokhare, S. S., Patil, P. G., Agarrwal, R., Chakranarayan, M. G., Anita Aralimar, Priya Devagire &Rajiv A. Marathe Marathe, R. A. (2022). Genetic diversity and streptomycin sensitivity in Xanthomonas axonopodis pv. punicae causing oily spot disease in pomegranates. Horticulturae, 8(5), 441.

61. Chen J-R, Aguirre-Carvajal K, Xue D-Y, Chang H-C, Arone-Maxwell L, Lin Y-P, Armijos-Jaramillo V and Oliva R (2024). Exploring the genetic makeup of Xanthomonas species causing bacterial spot in Taiwan: evidence of population shift and local adaptation. Front. Microbiol. 15:1408885. doi: 10.3389/fmicb.2024.14088857471.1000394    

62. ZHANG, G. Q., Jun, Z. H. U., ZHANG, X. W., Bin, L. I., & Bo, Z. H. U. (2012). Evolution of Xanthomonas gene content: gene gain/loss history and species divergence. Journal of Integrative Agriculture, 11(6), 954-961.

63. Rafael Marini Ferreira, Amanda Carolina P. de Oliveira, Leandro M. Moreira, José Belasque Jr., Edith Gourbeyre, Patricia Siguier, Maria Inês T. Ferro, Jesus A. Ferro, Michael Chandler, Alessandro M. Varani (2015). A TALE of transposition: Tn 3-like transposons play a major role in the spread of pathogenicity determinants of Xanthomonas citri and other xanthomonads. MBio, 6(1), 10-1128.

64. Subedi, A., Iruegas-Bocardo, F., Luo, L., Minsavage, G. V., Roberts, P. D., Jones, J. B., & Goss, E. M. (2024). Amylase-associated genetic pattern in Xanthomonas euvesicatoria on pepper. Applied and Environmental Microbiology, 90(10), e01313-24.

65. Yenjerappa (2014). PhD thesis Dharwad agriculture university.  Dharwad.