Ecofriendly Natural Extracts as Biobactericides Against Potato Pathogenic Bacteria

. Results: In

harvest storage of potatoes are substantially jeopardized by the presence of bacterial soft rot disease (Hadizadeh et al., 2019).The significant economic losses incurred during the transportation and storage of crops can be attributed mostly to the pathogenic effects of Pectobacterium carotovorum (Charkowski, 2018).
The utilization of several chemical bactericides for the management of soft-rot bacteria has been hindered due to their potential hazardous risks to both human health and the environment.The utilization of biocontrol strategies has been investigated as a means to address several plant infections (Abd-El-Khair et al., 2021).In recent times, there has been a growing need to decrease the utilization of chemical pesticides in the field of agriculture as a result of apprehensions over their ecological consequences and potential health hazards.As a result, there has been a growing recognition of the need to develop safer antimicrobial treatments, including extracts derived from plants, for the purpose of managing potato bacterial soft rot (Körpe et al., 2013;Yuliar et al., 2015).According to Ribera and Zuñiga (2012), plant antimicrobial metabolites, which can be either natural bioactive molecules known as phytoanticipins or those produced in response to pathogen infections called phytoalexins, have the ability to suppress pathogens.
Several studies have demonstrated that some plants possess secondary metabolites that exhibit antimicrobial capabilities.These compounds may be utilized to manage plant diseases either by inducing systemic resistance or by directly inhibiting bacterial growth (Wagura et al., 2011;Malkhan et al., 2012;Rahman et al., 2012;Körpe et al., 2013).Although water is commonly used as a universal solvent for extracting antimicrobial chemicals, organic solvents such as methanol and acetone are generally favored due to their capacity to provide more reliable antibacterial activity.These solvents are preferred due to their elevated polyphenol concentration in comparison to aqueous extracts, as well as their enhanced capacity to permeate cellular membranes, hence aiding in the extraction of intracellular constituents from plant matter.The extraction of antimicrobial components from plants is commonly carried out using acetone or methanol as solvents due to the prevalence of aromatic or saturated organic compounds in these plant sources (Prashanth et al., 2011).
The objective of this research is to examine the capacity of plant extracts that are readily accessible, economically feasible, and ecologically sustainable to effectively control potato bacterial soft rot through their antimicrobial capabilities.This study investigates the utilization of different organic solvents, such as methanol and acetone, in the extraction process of antimicrobial compounds from certain plant sources.The objective is to develop an environmentally friendly and sustainable method for managing phytopathogens and safeguarding potato crops.

Isolation of the Pathogens:
The process of isolation was conducted on potato tubers that exhibited varied degrees of natural infection with symptoms associated with potato infection.During the 2020-2021 seasons, potato tubers were gathered from many areas throughout Alexandria governorate, Egypt.The tubers were subjected to surface sterilization by submerging them in a sodium hypochlorite solution with a concentration of 1% (v/v) for 2 minutes.Subsequently, the specimens underwent two rounds of rinsing with sterile water.Subsequently, a minute fraction of the afflicted tissues was subjected to maceration using 5 ml of nutrient broth media.Following a 24-hour duration, a single loopful of the resultant suspension was applied in a streaking manner over a nutritional agar medium (NA), as outlined by Abo-El-Dahab and El-Goorani (1969).The plates were thereafter placed in an incubator set at a temperature of 28°C for a period of 48 hours.Subsequently, an analysis was conducted to assess the occurrence of bacterial proliferation on the plates.The researchers utilized the single colony method in order to get a culture that is free from contamination.This involved preserving colonies that grew on slanted media at a temperature of 4 °C, which allowed for further tests to be conducted.

Molecular Identification Through The 16S rRNA Gene:
In accordance with the bacterial DNA isolation protocol outlined by Ashmawy et al. (2020), utilizing the CTAB approach, the bacterial samples that were obtained underwent amplification of the whole 16S rRNA gene, which encompasses a length of 1550 base pairs.The amplification process was accomplished by employing the P0 and P6 primers.The PCR amplification was performed using a total volume of 20 µL, comprising 10 µL of master mix, 0.5 µL of each P0 or P6 primer, 2 µL of bacterial genomic DNA.The remaining volume was supplemented with molecular-grade water to attain a final volume of 20 µL.The PCR protocol was executed as follows: an initial denaturation step comprising a single cycle at a temperature of 95°C for a duration of 5 minutes, followed by 35 cycles of denaturation at 95°C for 45 seconds, annealing at 50°C for 60 seconds, and elongation at 72°C for 120 seconds.The duration of the last extension stage was 7 minutes, during which the temperature was maintained at 72°C.Extraction and Disk Assessment Microbiology Procedure: Plant Material Preparation: Juniperus phoenicea and Cicer arietinum plant materials were collected and subjected to a shade-drying process at room temperature conditions for four weeks.Subsequently, the materials were further dried in an oven at 40 ºC for an additional two days to ensure thorough drying.Solvent Selection: Based on prior research by Malkhan et al. (2012) and Rahman et al. (2012), acetone and methanol were selected as solvents for extracting polar metabolites.Extraction Process: Ten grams of dried and powdered plant material were macerated in a mixture of 40 milliliters of methanol and acetone (at a 1:4 ratio).The mixture was then placed on a rotary shaker and left undisturbed for three days.Subsequently, the mixture was filtered using Whatman filter paper and placed in a petri dish.The plates were then incubated at 28 •C for two days.The resulting extracts were diluted to four different concentrations (3000 ppm, 5000 ppm, 7000 ppm, and 10000 ppm) using a 1% dimethyl sulfoxide (DMSO) solution, following the method described by Mwitari et al. (2013).The extracts were stored at 4 °C until further use.Antimicrobial Testing: Two separate plates were prepared for each plant extract, Juniperus phoenicea and Cicer arietinum.Each plate was coated with a nutrient agar medium (NA) inoculated with bacteria.Five paper discs were placed on the agar, consisting of one disc containing an antibiotic and four discs containing different concentrations of the plant extracts (each disc containing 20 µl).The fifth disc was soaked in distilled water, serving as the negative control.Amoxicillin at a concentration of 25 mg was employed as the positive control.The plates were then incubated for two days, and the inhibitory zone (mm) was measured after a 48-hour incubation period.

Statistical Analysis:
The results of the measured parameters underwent computerized statistical analysis using the COSTAT package for analysis of variance (ANOVA).Subsequently, treatment means were compared using the LSD (Least Significant Difference) test at a significance level of 0.05.

Effect of Juniperus phoenicea Methanol Extract:
Table 1 and Figure 1 illustrate the results of disc diffusion assays, demonstrating the antimicrobial activity of the Juniperus phoenicea 25% methanol extract.The extract displayed significant bioactivity against Pectobacterium carotovorum, P. atrosepticum, Ralstonia solanacearum, and Streptomyces scabies at concentrations of 10,000, 7,000, 5,000, and 3,000 ppm, as well as in both negative and positive control groups.
Specifically, the 10,000-ppm concentration exhibited notable bioactivity with inhibition zone (IZ) values of 6.33, 11.00, 10.00, and 8.00 mm against the respective pathogens.At 7,000 ppm, the IZ values ranged from 9.67 to 8.67 mm, and at 5,000 ppm, they varied from 9.76 to 7.00 mm.It's worth noting that the positive control (antibiotics) displayed wider inhibition zones, especially against P. atrosepticum, with an impressive 22.33 mm IZ.The highest IZ value for the J. phoenicea extract was recorded against P. atrosepticum, measuring 11.00 mm.

Effect of Cicer arietinum Acetone Extract:
The results, as presented in Table 2 and Figure 2, from the disc diffusion assays, reveal a significant level of antimicrobial activity associated with the Cicer arietinum 75% acetone extract.The extract exhibited notable bioactivity against P. carotovorum, P. atrosepticum, R. solanacearum, and S. scabies at varying concentrations of 10,000, 7,000, 5,000, and 3,000 ppm, as well as in both negative and positive control groups.
Specifically, the 5,000-ppm concentration demonstrated robust bioactivity, resulting in inhibition zone (IZ) values of 7.00, 8.00, 9.00, and 10.00 mm against the respective pathogens.At 7,000 ppm, the IZ values ranged from 7.67 to 8.67 mm.It's important to note that the positive control (antibiotics) exhibited wider inhibition zones, particularly against P. atrosepticum, with an impressive 20.33 mm IZ.
The highest IZ values for the Cicer arietinum extracts, specifically at a concentration of 7,000 ppm, were observed against P. atrosepticum, measuring 10.00 mm, and S. scabies at a concentration of 5,000 ppm.Hence, numerous studies have delved into the use of natural extracts to combat potato diseases.For instance, Salem (2013) uncovered that bark extracts from Delonix regia and Erythrina humeana exhibited moderate antibacterial activity against various strains of potato soft rot bacteria, including D. dianthicola, P. wasabiae, P. carotovorum, P. atrosepticum, and D. chrysanthemi.Furthermore, extracts derived from Tecoma stans leaves and branches displayed notable activity in comparison to those from Callistemon viminalis against the same bacterial strains.
In a related study, Salem et al. (2014) demonstrated that wood and bark extracts from Picea abies and Larix decidua exhibited moderate activity against the growth of P. atrosepticum, P. carotovorum, and D. solani.Notably, Stenotrophomonas maltophilia, isolated from the rhizosphere of eggplant cultivated in Egypt's Nile Delta region, emerged as a potential biocontrol agent against R. solanacearum (Messiha et al., 2007).These findings align with previous reports indicating that certain plant species harbor bioactive compounds with antimicrobial properties (Yuliar et al., 2015;Sharma et al., 2016).
Moreover, an in vitro experiment highlighted the potency of methanol leaf extracts from castor beans, surpassing ethanol and water extracts in their activity against both grampositive and gram-negative bacteria (Naz and Bano, 2012).
In conclusion, the utilization of natural extracts to combat potato diseases presents a promising avenue for eco-friendly and sustainable agricultural practices.The diverse range of plant-based bioactive compounds underscores the potential of harnessing nature's arsenal in our ongoing efforts to protect vital crops like potatoes from detrimental pathogens.Further research and exploration in this field hold the key to developing effective, environmentally conscious strategies for disease management in agriculture.

Conclusions
In summary, our research highlights the potential of natural extracts as eco-friendly biobactericides against potato pathogens.The 25% methanol extract from Juniperus phoenicea showed significant antibacterial activity, particularly against Pectobacterium carotovorum.Additionally, the Cicer arietinum 75% acetone leaf extract demonstrated efficacy against P. atrosepticum and P. carotovorum.These findings suggest that natural compounds could play a role in protecting potatoes from disease-causing agents in a sustainable and environmentally friendly manner.

Table 1 .
Effect of Juniperus phoenicea methanol extract against pathogenic potato bacterial isolates

Table 2 .
Effect of Cicer arietinum acetone extract against pathogenic potato bacterial isolates