Phytopathology

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INTRODUCTION
Citrus is an imperative fruit crop in Pakistan, contributing 34.9% to the overall fruit production (FAO, 2017).The province of Punjab, known for its favorable climate, contributes 94.8% of the total citrus production (1.9074 million tons) in the country (FAO, 2017).Citrus fruits are known for their nutritional value, being rich in nutrients such as amino acids, fiber, vitamin C, carbohydrates, and various phytochemicals like terpenes, carotenoids, flavonoids, limonoids, and alkaloids (Alexander, 2019).Pharmacological studies have provided evidence of the diverse health benefits associated with citrus fruits, including anti-inflammatory, anticancer, anthelmintic, antimicrobial, antioxidant, anti-diabetic, immunomodulatory, gastroprotective, analgesic, insect repellent, and other pharmacological effects (Al-Snafi, 2016).However, citrus is susceptible to several diseases, including sooty mold, phytophthora root rot, tristeza complex, gummosis, citrus greening, slow decline, citrus wither tip, and citrus canker (Anjum and Javaid, 2005).Among them, citrus canker, caused by Xanthomonas citri subsp.citri (Xcc), is most prevalent (Tehmina and Arshad, 2005), resulting in 5% to 30% yield losses in Pakistan (Ashraf et al., 2014;Hameed et al., 2022;Sahi et al., 2007).Xanthomonas citri subsp.citri is a gram-negative, rodshaped, aerobic bacterium with a single polar flagellum measuring 0.5-0.75µm in length, primarily thriving at temperatures of 35-39°C (Sahi et al., 2007).Xanthomonas citri subsp.citri spreads through a combination of wind and rain within a certain distance from tree to tree (Sahi et al., 2007).Asian leaf miners have also been identified as facilitation factor of infection by rupturing the cuticle, providing entry points for the pathogen into the mesophyll of leaves, twigs, fruits and stems (Graham and Leite, 2004).Asian leaf miners may also act as vectors for Xcc dispersal (Nirvan, 1961;Sohi and Sandhu, 1968;Sinha et al., 1972;Cook, 1988).The pathogen can easily suspend in water and disseminate with the help of its mucilaginous coat.Rainwater typically carries the bacterium as it drips from drips down from surfaces of lesions to non-infected parts of plants or other plants (Stall et al., 1980).
Various strategies have been employed to manage plant diseases, such as the application of fungicides, utilization of plant defense activators, and implementation of antagonistic organisms.However, in cases where resistant varieties are unavailable and the disease reaches epidemic proportions, farmers often resort to the use of synthetic chemicals due to their rapid action and easy availability.During severe disease prevalence use of chemicals is the only option to overcome the high yield losses.Recognizing importance of chemicals, the current study was aimed to manage citrus canker by employing nine antibiotics (Enrofloxacin, Enco-

MATERIALS AND METHODS Isolation, Purification, and identification
The diseased samples (Xanthomonas citri subsp.citri) of citrus were collected from the botanical garden, University of Agriculture, Faisalabad (UAF) and brought to Molecular Phyto pathological Laboratory, Department of Plant Pathology, UAF.The disease samples were rinsed through tap water, cut into small pieces, surface sterilized by dipping in 1% Sodium hypochlorite (NaOCl) for 30 seconds and then washed by using distilled water for two minutes and placed on sterile Nutrient Agar (NA) medium already poured in Petri plates and incubated at 28°C for 48 hours.The bacterial ooze on NA media were transferred to new Petri plates containing NA media through streaking method by using sterile inoculating loop and incubated at 28°C.The isolated pathogenic bacteria were purified by following the dilution plate technique on NA medium.The purified bacterium was identified on the bases of literature and following assays (Nethravathi and Yadahalli, 2016).

Pathogenicity test
One year old disease-free citrus plants were collected from Nursery of Institute of Horticultural Sciences, UAF.The seedlings were transposed in pots containing soil and farmyard manure (2:1).Under natural conditions, the seedlings were inoculated after 7-10 days of transplanting through pin pricking technique.The seedlings were observed after a one-week interval for the appearance of the visual canker symptoms.The bacteria were reisolated from leaves of inoculated citrus seedlings and cultured on NA media.After purification, further microscopic and biochemical tests were performed to confirm the pathogen (Nethravathi and Yadahalli, 2016).Copper oxychloride and Copper acetate with three concentrations (0.5, 0.75, 1.00%) were evaluated against citrus canker under field conditions by using foliar spray.
The disease incidence was recorded after 5, 10, and 15 days of treatments' application.

Evaluation of copper-based chemicals and antibiotics against citrus canker under field condition
Most effective antibiotic in laboratory experiments and copper-based chemical in field trials were examined against citrus alone and in combination.Treatments were applied through foliar spray method after one week of inoculation except control.Disease incidence was recorded after 5, 10 and 15 days of treatments' application.

Pathogenicity test
The pathogen was confirmed by following Koch's postulates.Symptoms observed on leaves, morphology on NA medium, and microscopic identification confirmed the disease was citrus canker caused by Xanthomonas citri subsp.citri.

Biochemical analysis
A clear zone was formed around the bacterial growth in Hankins's media indicating the pectin degradation due to secretion of pectate lyase by the bacterium thus confirmed the isolated bacterium as Xcc in Pectinolytic activity test.Acid was produced by trehalose, maltose, sucrose and lactose while sorbitol was not consumed on King's media B and the change in color and production of gas indicated positive results.The rotten egg smell in MacConkey broth confirmed the release of H2S that also indicated positive results for Xcc.Similarly, red color appeared in indole production test after addition of Kovac's reagents also confirmed the positive result.The production of canker and visible change in color of Simon's citrate agar media from green to blue Simon's citrate agar plates indicated the results were positive.αmethyle D glucoside was fermented by culture of bacterium which was recognize by pink result that indicated the positive result (Table 1).  2 and 3).

DISCUSSION
Citrus canker, caused by one of the most damaging subfamilies of the bacterial phytopathogen Xanthomonas citri subsp.citri (Xcc), is a serious threat to the world's most valuable citrus fruit crop.Symptoms of a severe infection include leaf loss, early fruit drop, dieback, severe fruit blemishes or discoloration and a decline in fruit quality (Ali et al., 2023).Present experiments were designed to examine the efficacy of different antibiotics and copper-based chemicals.On the basis of average data Enrofloxacin proved to be the best one which inhibited the growth of Xcc in laboratory by 35.68mm (Inhibition zone) followed by Enco-Mix, Penivet-5, Kanamycin sulphate, Sinobiotic, Streptomycin sulphate, Gentam-20% but Benzyl penicillin sodium and Tylofurcin gave no result.Earlier studies have reported the efficacy of a number of antibiotics against Xcc but the efficacy of Enrofloxacin has never been reported by any researcher.Outcomes of present study was supported by the findings of Jaskani who studied the effect of different antibiotics for controlling citrus canker and concluded that kanamycin sulphate expressed the higher inhibition zone followed by Streptomycin Sulphate and Lincomycin against Xanthomonas citri subsp.citri (Jaskani et al., 2021).Already, nine antibiotics against citrus canker caused by Xanthomonas citri subsp.citri revealed Levofloxacin was most significant at 700 ppm concentration (Hameed et al., 2022).Another study supported our finding under in vitro circumstances and concluded streptomycin sulphate and bromopol (each @ 100, 250, and 500 ppm) were the most efficient antibiotics against Xanthomonas citri subsp.citri (Kharat et al., 2020).Enrofloxacin involves in inhibiting the bacterial DNA gyrase and topoisomerase IV enzymes, which are essential for the replication, transcription, and repair of bacterial DNA (Bush et al., 2020).Enrofloxacin interferes with the activity of DNA gyrase, an enzyme found in bacteria that is responsible for introducing negative supercoils into the bacterial DNA molecule.By inhibiting DNA gyrase, enrofloxacin prevents the relaxation of supercoiled DNA during replication and transcription, leading to the inhibition of bacterial DNA synthesis (Bush et al., 2020).Among copper-based chemicals, Copper Nitrate proved to be the best one which controls the disease 16.07%(disease incidence), followed by copper hydroxide, Amistar top, Copper oxychloride and Copper acetate.The consequences of the current study were presented by Malik who came to the conclusion that the bordeaux mixture was best for eradicating the canker on leaves and fruit.Another therapy that successfully reduced the occurrence of disease on leaves and fruit was copper hydroxide (Malik et al., 2020).Canker incidence was significantly lower in citrus plants that were treated by copper oxychloride (Rehman et al., 2020).Mean data of combined treatments (antibiotic + copper-based chemical) field experiments exhibited minimum disease incidence (18.22%) in case of Enrofloxacin+ Cu (NO2)3, followed by Enrofloxacin (21.28%) and Cu (NO2)3 (22.28%).Earlier studies have supported our experiments regarding the efficacy of copper-based chemicals and antibiotics alone and combined but Enrofloxacin with Cu (NO2)3 combined has never been reported by any researcher.Present findings depict by Rehman who ecaluated the combination of antibiotics and chemicals.The treatments were streptomycin+ copper oxychloride followed by kasugamycin + copper oxychloride and copper oxychloride + validamycin.The minimum disease severity was found by the combination of copper nitrate + streptomycin (Sharif et al., 2021).
Copper ions have the ability to disrupt the integrity and function of bacterial cell membranes (Shams et al., 2020).They can interact with the phospholipids in the cell membrane, causing structural damage and increased permeability.This disruption compromises the bacterial cell's ability to maintain proper osmotic balance and essential cellular processes, eventually leading to cell death.Moreover, it can directly interact with bacterial DNA, leading to DNA strand breaks and damage (Li et al., 2019).This interference with the DNA structure and stability disrupts bacterial replication and transcription processes, preventing the bacteria from proliferating and surviving.

CONCLUSION
Enrofloxacin and copper nitrate exhibited remarkable efficacy among nine antibiotics and six copper-based compounds, respectively against Xanthomonas citri subsp.citri under in vitro conditions.The combination of Cu (NO2)3 and Enrofloxacin was revealed as the most effective antibacterial agent among all evaluated treatments against citrus canker under field conditions.Cu (NO2)3 + Enrofloxacin would be a successful amendment to manage citrus canker and other bacterial pathogens.

ACKNOWLEDGMENT
This paper is a part of the PhD thesis of Mr. Hamza Shahbaz.I pay my vote of thanks to the researchers of Citrus Pathology Lab and Higher Education Commission for providing research facilities.

CONFLICT OF INTEREST
The authors declare that they have no conflicts of interest.

CONTRIBUTION OF AUTHORS
Hamza Shahbaz conducted research and wrote original manuscript; Muhammad Atiq conceived the idea and edited manuscript; Nasir Ahmad Rajput member supervisory committee; Ahsan khan member supervisory committee.

Figure 1 .
Figure 1.Mean inhibition zone (mm) exhibited by different antibiotics at different concentrations and time periods in the growth of Xanthomonas citri subsp.Citri.

Figure 3 .
Figure 3. Mean inhibition zone (mm) exhibited by different antibiotics at different concentrations in the growth of Xanthomonas citri subsp.Citri.
p e r o x y c h l o r i d e C o p p e r N i t r a t e C o p p e r H y d r o o x i d e C o p p e r a c e t a

Figure 7 .
Figure 7. Mean disease incidence (percent) of citrus canker disease at different intervals (days) after application of different antibiotics (alone and in combination) under field conditions.

activity test of Xcc Xcc
was cultivated on Hankins's media and incubated at 32°C.Pectinolytic activity of Xcc was confirmed on the base of a clear zone appearance around bacterial.

Trehalose, maltose, sorbitol, sucrose and lactose test:
Xcc culture grown on Kings Medium B were relocated to α-methyle D glucoside medium and incubated for 48 hours at 32°C.Xcc was recognized by the appearance of pink color of the Xcc colony.
Utilization of Citrate by Xcc Xcc culture were inoculated on Simmon's Citrate Agar plates and placed in the incubator for 48 hours at 32°C.Citrate positive results were confirmed on the bases of color changes from green to blue.α-methyle D glucoside test Xcc.The data were recorded after 24, 48 and 72 hours.In vivo evaluation of copper-based chemicals against citrus canker: One-year old citrus plants were collected from Nursery of Institute of Horticultural Sciences, UAF and placed in pots at Department of Plant Pathology, UAF.Five chemicals including Copper nitrate, Copper hydroxide, Amistar top,

Table 2 .
Mean inhibition zone (mm) exhibited by different antibiotics at different concentrations and time periods in the growth of Xanthomonas citri subsp.Citri.

Table 3 .
Mean inhibition zone (mm) exhibited by different antibiotics at different concentrations in the growth of Xanthomonas citri subsp.citri

Table 4 .
Effect of different copper-based chemicals (at different concentration and number of sprays) on the mean disease incidence (percent) of citrus canker under field conditions.

Table 5 .
Effect of different copper-based chemicals at different concentrations on the mean disease incidence (percent) of citrus canker under field conditions.

Table 6 :
Effect of different copper-based chemicals on the mean disease incidence (percent) of citrus canker, recorded after different number of sprays under field conditions.

Table 7 .
Mean disease incidence (percent) of citrus canker disease after application of different antibiotics (alone and in combination) under field conditions.

Table 8 .
Mean disease incidence (percent) of citrus canker disease at different intervals (days) after application of different antibiotics (alone and in combination) under field conditions