In vitro efficacy of chemicals and antibiotics against Pectobacterium sp. causing soft rot disease in dragon fruit

Introduction

Dragon fruit [Selenicereus monacanthus (Lem.) D. R. Hunt], earlier known as Hylocereus polyrhizus (F. A. C. Weber) Britton & Rose, is a perennial, semi-epiphytic cactus belonging to the family Cactaceae (Patwary et al., 2013) [8] . It originated in South and Central America and Mexico and is now cultivated in many tropical and subtropical countries, including several parts of Asia (Barthlott and Hunt, 1993; Mizrahi and Nerd, 1999) [2,6] . The recent taxonomic classification places H. polyrhizus under S. monacanthus (Govaerts et al., 2021) [3] . Dragon fruit has gained importance due to its attractive fruit colour, nutritional value and good market demand. Among the different types, red-fleshed dragon fruit is widely preferred for commercial cultivation (Nangare et al., 2020) [5] . The crop is generally propagated through stem cuttings for maintaining the desirable characters of the selected plant (Ortiz-Hernández, 1999) [7] . With the increase in area under dragon fruit cultivation, problems related to diseases are also becoming important. Bacterial soft rot is one such problem, particularly because infected fruits undergo rapid softening and decay, resulting in loss of fruit quality and market value. Pectobacterium spp. are important bacterial pathogens associated with soft rot and cause tissue maceration through the production of pectinolytic enzymes. The disease development is favoured by high moisture, wounds and suitable temperature conditions. Management of bacterial soft rot is difficult once the infection becomes established, and therefore identification of effective chemicals and antibiotics is important.

Previous studies have reported good antibacterial activity of copper-based compounds such as copper hydroxide and copper oxychloride against Pectobacterium (Thiyagarajan, 2016; Pavitra et al., 2025) [13,9] . Streptomycin and other antibiotics have also been reported to inhibit Pectobacterium under laboratory conditions (Sang et al., 2015; Shen et al., 2025) [10,11] . However, the effectiveness of these treatments may vary with the pathogen, concentration and environmental conditions. Hence, the present study was carried out to evaluate the in-vitro efficacy of selected chemicals and antibiotics against Pectobacterium sp. causing soft rot disease of dragon fruit.

Materials and Methods

Collection, isolation and maintenance of the pathogen

Diseased dragon fruit samples showing typical soft rot symptoms were collected from affected fruits and stems from infected plants. The collected samples were brought to the laboratory in clean polythene bags and processed for isolation of the associated bacterial pathogen. Small pieces of diseased tissue were cut from the margin of healthy and infected portions using a sterilized scalpel. The tissue pieces were surface sterilized with suitable disinfectant, washed with sterile distilled water and transferred aseptically to sterile nutrient agar (NA) medium.The inoculated plates were incubated at 27±1 °C and observed periodically for bacterial growth. Bacterial colonies developing from the infected tissues were selected based on their colony characteristics and purified by repeated streaking on nutrient agar medium. Pure cultures were maintained on NA slants and used for further studies. The bacterial isolate obtained from diseased fruit and stem tissues was identified as Pectobacterium sp. based on its cultural, morphological and biochemical characteristics. A fresh 48-hour-old bacterial broth culture was prepared and used for evaluating the invitro efficacy of chemicals and antibiotics.

In-vitro evaluation of chemicals and antibiotics

The efficacy of selected chemicals and antibiotics against Pectobacterium sp. was evaluated by the paper disc method described by Thornberry (1950) [14] . Nutrient agar medium was prepared, sterilized in an autoclave and poured into sterile Petri plates. After solidification, each plate was inoculated with 1 ml of 48-hour-old bacterial broth culture and spread uniformly over the surface of the medium using a sterile spreader under aseptic conditions.

For chemical evaluation, eight chemicals, namely hydrogen peroxide, sodium hypochlorite, ammonium bicarbonate, lactic acid, citric acid, copper sulphate, copper oxychloride and copper hydroxide, were tested at 1 and 2 percent concentrations. For antibiotic evaluation, eight treatments consisting of streptocycline, erythromycin, chloramphenicol, ciprofloxacin, tetracycline, amoxycillin, streptocycline + copper oxychloride and streptocycline + copper hydroxide were tested at 250 and 500 ppm concentrations. Sterile Whatman No. 1 filter paper discs of 5 mm diameter were separately soaked in the respective treatment solutions for 5 minutes. Two discs representing the lower and higher concentrations of each treatment were placed on opposite sides of each inoculated Petri plate. Three replications were maintained for each treatment. Discs soaked in sterile distilled water were used as the untreated control. The plates were incubated at 27±1 °C for 48-72 hours. After incubation, the antibacterial activity of each treatment was assessed by measuring the diameter of the clear zone of inhibition formed around the paper discs. The zone of inhibition was measured in millimetres and the mean values of three replications were recorded.

Results

In-vitro efficacy of chemicals

Eight chemicals were evaluated at 1 and 2 percent concentrations against Pectobacterium sp. All the chemicals exhibited antibacterial activity, whereas no inhibition was observed in the untreated control. At 1 percent concentration, copper sulphate recorded the highest inhibition zone of 14.85 mm. It was followed by copper oxychloride (13.20 mm), copper hydroxide (12.40 mm) and ammonium bicarbonate (10.90 mm). Citric acid and sodium hypochlorite produced inhibition zones of 8.36 and 8.15 mm, respectively. Hydrogen peroxide and lactic acid recorded comparatively lower inhibition zones of 7.11 and 6.14 mm, respectively. At 2 percent concentration, copper hydroxide recorded the maximum inhibition zone of 17.42 mm. Copper oxychloride, ammonium bicarbonate and sodium hypochlorite recorded inhibition zones of 14.96, 14.68 and 14.62 mm, respectively. Copper sulphate and citric acid produced inhibition zones of 12.62 and 12.43 mm, respectively. Hydrogen peroxide and lactic acid showed comparatively lower activity, with inhibition zones of 10.41 and 8.36 mm, respectively. The untreated control did not show any inhibition zone at either concentration. All the chemicals tested showed inhibition against Pectobacterium sp., but their effectiveness differed with treatment and concentration. At 1 percent, copper sulphate gave the highest inhibition zone (14.85 mm), followed by copper oxychloride (13.20 mm) and copper hydroxide (12.40 mm). At 2 percent, copper hydroxide was the best treatment (17.42 mm), followed by copper oxychloride (14.96 mm), ammonium bicarbonate (14.68 mm) and sodium hypochlorite (14.62 mm). Similar results were reported by Thiyagarajan (2016) [13] , who found copper hydroxide effective against bacterial soft rot. In another study, Ashwini et al., (2022) [1] found that 3% sodium hypochlorite gave the highest inhibition zone (12.17 mm), followed by ammonium bicarbonate (11.70 mm), sodium bicarbonate (9.50 mm) and acetic acid (6.70 mm) against Pectobacterium carotovorum. Pavitra et al., (2025) [9] reported copper oxychloride as the best chemical against P. carotovorum subsp. carotovorum. Yaganza et al., (2009) [15] also reported that different salts could inhibit the growth of Pectobacterium spp.

Note: In each Petri plate, the inner paper disc represents the lower concentration (1%), whereas the outer paper disc represents the higher concentration (2%) of the respective chemical. Sodium hypochlorite showed better activity at 2 percent (14.62 mm) than at 1 percent (8.15 mm). Hong et al., (2021) [4] reported that sodium hypochlorite reduced soft rot caused by P. carotovorum. Most chemicals showed better inhibition at 2 percent, but copper sulphate gave a lower zone at 2 percent (12.62 mm) than at 1 percent (14.85 mm). Hydrogen peroxide, citric acid and lactic acid were less effective. The difference in activity may be due to the sensitivity of the bacterial isolate and the nature of the chemicals. Overall, copper hydroxide, copper oxychloride, ammonium bicarbonate and sodium hypochlorite were the better treatments under in-vitro conditions.

In-vitro efficacy of antibiotics

Eight antibiotic treatments were evaluated at 250 and 500 ppm. All the antibiotics produced inhibition zones against Pectobacterium sp., although their effectiveness varied considerably.

At 250 ppm, ciprofloxacin recorded the highest inhibition zone of 16.66 mm, followed by streptocycline ( [10] reported good activity of streptomycin against P. carotovorum. Pavitra et al. (2025) [9] also reported good activity of streptomycin sulphate against P. carotovorum subsp. carotovorum. Most antibiotics showed higher inhibition at 500 ppm than at 250 ppm. However, streptocycline + copper hydroxide showed lower inhibition at 500 ppm (9.26 mm) than at 250 ppm (13.76 mm). The difference may be due to the response of the bacterial isolate or the interaction between the antibiotic and copper compound. Shen et al. (2025) [11] reported strong activity of streptomycin against P. carotovorum and also highlighted the problem of streptomycin resistance. Therefore, the effective treatments found in the present study, particularly streptocycline + copper oxychloride, should be further tested under field conditions before practical recommendation.

Conclusion

The present study confirmed the occurrence of bacterial soft rot of dragon fruit caused by Pectobacterium sp. In the invitro chemical evaluation, all the tested chemicals showed inhibitory activity against Pectobacterium sp. Copper hydroxide recorded the highest inhibition zone of 17.42 mm at 2%, followed by copper oxychloride (14.96 mm), ammonium bicarbonate (14.68 mm) and sodium hypochlorite (14.62 mm). Among the antibiotics, streptocycline + copper oxychloride recorded the highest inhibition zone of 22.90 mm at 500 ppm, followed by streptocycline (20.36 mm) and ciprofloxacin (18.66 mm). Therefore, copper hydroxide, copper oxychloride, sodium hypochlorite, streptocycline and streptocycline + copper oxychloride showed good in vitro activity against Pectobacterium sp. These treatments may be useful for further field evaluation and management of bacterial soft rot of dragon fruit.

References

1. Ashwini E, Nandeesha SV, Sakthi Priya P. Eco-friendly post-harvest management of Pectobacterium carotovora subsp. carotovora causing bacterial soft rot of carrot in Meghalaya, India. Biological Forum-An International Journal. 2022;14(4):55-63.
2. Barthlott W, Hunt DR. Cactaceae. In: Kubitzki K, Rohwer JG, Bittrich V, editors. The Families and Genera of Vascular Plants. Vol. 2. Berlin: SpringerVerlag; 1993. p. 161-197.
3. Govaerts R, Nic Lughadha E, Black N, Turner R, Paton A. The World Checklist of Vascular Plants, a continuously updated resource for exploring global plant diversity. Scientific Data. 2021;8:215.
4. Hong SJ, Park NI, Park Y, Kim BS, Eum HL. Effect of disinfecting harvesting knives with sodium hypochlorite on soft rot infection of Kimchi cabbage. Food Science and Biotechnology. 2021;30(8):1139-1150.
5. Nangare DD, Taware PB, Singh Y, Kumar PS, Bal SK, Ali S, et al. Dragon fruit: a potential crop for abiotic stressed areas. Technical Bulletin No. 47. Baramati, Pune: ICAR-National Institute of Abiotic Stress Management; 2020.
6. Nerd A, Gutman F, Mizrahi Y. Ripening and postharvest behaviour of fruits of two Hylocereus species (Cactaceae). Postharvest Biology and Technology. 1999;17(1):39-45.
7. Ortiz-Hernández YD, Livera-Muñoz M. Manual sobre la propagación de la pitahaya (Hylocereus spp.). Oaxaca, Mexico: SIBEJ-CONACYT-FMCN-IPN; 1999.
8. Patwary MMA, Rahman MH, Barua H, Sarkar S, Alam MS. Study on the growth and development of two dragon fruit (Hylocereus undatus) genotypes. The Agriculturists. 2013;11(2):52-57.
9. Pavitra, Yenjerappa ST, Amaresh YS, Ajithkumar K, Ashok H, Mahadevaswamy. in vitro management of Pectobacterium carotovorum subsp. carotovorum, the causal organism of carrot soft rot disease, using chemicals and antibiotics. International Journal of Advanced Biochemistry Research. 2025;9(12S):983988.
10. Sang MK, Dutta S, Park K. Influence of commercial antibiotics on biocontrol of soft rot and plant growth promotion in Chinese cabbages by Bacillus vallismortis EXTN-1 and BS07M. Research in Plant Disease. 2015;21(4):255-260.
11. Shen Y, Li Y, Wang L, Wu C, Su X, Tian Y. Carvacrol and streptomycin in combination weaken streptomycin resistance in Pectobacterium carotovorum subsp. carotovorum. Plants. 2025;14(6):908.
12. Thammaiah N, Kanamadi VC, Shirol AM, Satyanarayana Reddy B, Gangadharappa PM. Management of rhizome rot or tip-over disease of banana caused by Erwinia chrysanthemi. Journal of Asian Horticulture. 2005;2(1):62-63.
13. Thiyagarajan V. Investigations on tip-over disease of banana caused by Erwinia carotovora subsp. carotovora (Jones) Holland [MSc thesis]. Raichur: University of Agricultural Sciences; 2016.
14. Thornberry HH. A paper-disc method for the quantitative evaluation of fungicides and bactericides. Phytopathology. 1950;40:419-420.
15. Yaganza ES, Tweddell RJ, Arul J. Physicochemical basis for the inhibitory effects of organic and inorganic salts on the growth of Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum. Applied and Environmental Microbiology. 2009;75(5):1465-1469.