Effects of Combined Application of Salicylic Acid and Proline on the Defense Response of Potato Tubers to Newly Emerging Soft Rot Bacteria ( Lelliottia amnigena ) Infection

: Potato soft rot, caused by the pathogenic bacterium Lelliottia amnigena ( Enterobacter amni-genus ), is a serious and widespread disease affecting global potato production. Both salicylic acid (SA) and proline (Pro) play important roles in enhancing potato tuber resistance to soft rot. However, the combined effects of SA and Pro on defense responses of potato tubers to L. amnigena infection remain unknown. Hence, the combined effects of SA and Pro in controlling newly emerging potato soft rot bacteria were investigated. Sterilized healthy potato tubers were pretreated with 1.5 mM SA and 2.0 mM Pro 24 h before an inoculation of 0.3 mL of L. amnigena suspension (3.69 × 10 7 CFU mL − 1 ). Rotting was noticed on the surfaces of the hole where the L. amnigena suspension was inoculated. Application of SA and Pro with L. amnigena lowered the activity of pectinase, protease, pectin lyase, and cellulase by 64.3, 77.8, 66.4 and 84.1%, and decreased malondialdehyde and hydrogen peroxide contents by 77.2% and 83.8%, respectively, compared to the control. The activities of NADPH oxidase, superoxide dismutase, peroxide, catalase, polyphenol oxidase, phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, 4-coumaryl-CoA ligase and cinnamate-4-hydroxylase were increased in the potato tubers with combined treatments by 91.4, 92.4, 91.8, 93.5, 94.9, 91.3, 96.2, 94.7 and 97.7%, respectively, compared to untreated stressed tubers. Six defense-related genes, pathogenesis-related protein, tyrosine-protein kinase, Chitinase-like protein, phenylalanine ammonia-lyase, pathogenesis-related homeodomain protein, and serine protease inhibitor, were induced in SA + Pro treatment when compared with individual application of SA or Pro. This study indicates that the combined treatment of 1.5 mM SA and 2.0 mM Pro had a synergistic effect in controlling potato soft rot caused by a newly emerging bacterium.


Introduction
According to Lim et al. [1], potato is the fourth most important food crop and staple food in the world. Potato tubers are in most cases stored for 3-6 months before being processed and/or consumed by humans. The average annual loss due to potato soft rot is estimated to be between 6 and 25%, with up to 60% of tubers in some cases being damaged [2,3]. One of the most common potato diseases that reduce tuber quality during storage is soft rot caused by Pectobacterium carotovorum subsp. Carotovorum [4]. Postharvest diseases in fruits and vegetables are caused by bacteria during storage. Throughout the growing season and during storage, members of the families Enterobacteriaceae and Pectobacteriaceae are the primary cause of potato soft rot [4]. These pathogens cause infection either during the preharvest stage in the field or after harvesting during storage and transportation. Soft rot bacteria degrade pectate molecules, which bind plant cells together, eventually causing the The L. amnigena culture was inoculated into 250 mL of bacterial liquid (1.5 g peptone, 2.0 g glucose, 0.75 g beef extract, and 250 mL water) with and without SA and Pro, and kept for 2 d at 37 • C in an Honour Instrument Shaker Machine (HNYC-202T, Guangdong, China). One mL for each test of cultured bacterial liquid was taken and centrifuged at 10,000× g for 10 min at 4 • C. Supernatants were collected to perform the production levels of protease, pectinase, pectin lyase, and cellulase, following protocol assay kits (Solarbio Science and Technology Co., Ltd., Beijing, China). Pectinase, protease, pectin lyase, and cellulose were measured at 540, 450, 235 and 540 nm, respectively, using a spectrophotometer (EPOCH2 Plate Reader, BioTek, Santa Clara, CA, USA). The activities were expressed as U mL −1 . Each activity was carried out three times.

Effect of SA and Pro on Potato Soft Rot
The combined effect of SA and Pro on the severity of potato soft rot was investigated in this experiment. The experiment was carried out in a laboratory. Uniform healthy potato tubers were sterilized for 1 min with 75% ethanol before being washed three times in distilled water. The tubers were dried at room temperature. Using a sterile cork borer (BML505-15 mm, Wuhan Servicebio Technology Co., Ltd., Wuhan, China), a hole (about 5 mm in diameter and 10 mm in depth) was made in the center of each sterilized tuber. The tubers were pretreated with 1.5 mM SA and 2.0 mM Pro by pipetting them into the holes of the tubers using a 1000 µL Filtered Pipette Tip (ISO9001:2015, Wuhan Servicebio Technology Co., Ltd., Wuhan, China) 1 d before inoculation with the L. amnigena inoculum. A total of 0.3 mL of the L. amnigena inoculum was pipetted into the holes of the pretreated and untreated tubers with a 1000 µL Filtered Pipette Tip, and water as a control (CK). The inoculated tubers were placed in sterilized sealed plastic containers packed with sterilized moist cotton (moderate) and kept for 7 d at room temperature. The experiment Sustainability 2022, 14, 8870 4 of 20 was repeated with three replicates and for each experiment; 15 tubers were used for all the treatments.

Disease Assessment
The disease index (DI) of the tubers was measured up to 7 d after inoculation with PC3 inoculum. The disease index was determined using a method described by Scherf et al. [41], with a 5 degrees scale (0-4), where 0 = no disease, 1 = trace to 25% of the tubers were rotted, 2 = 26%-50% of the tubers were rotted, 3 = 51%-75% of the tubers were rotted, and 4 = 76%-100% of the tubers were rotted. The DI was determined using the formula DI (%) = [∑ (number of diseased tubers × disease index)/(total number of tubers investigated × highest disease index)] × 100.

Sampling
Tissues were collected from inoculated tubers according to Zhang et al. [42] after 7 d of treatment with SA and Pro using a sterilized sharp blade. Tissues of untreated tubers were collected. Liquid nitrogen (Henan Boss Liquid Nitrogen Container Co., Ltd., Dongtai, China, 78.0% by volume, 75.5% by weight) was used to freeze the collected tissues, ground with a pestle and mortar into a powdery form, and then stored at −80 • C until use.

Malondialdehyde and Hydrogen Peroxide Content in Potato Tuber
The malondialdehyde (MDA) content was determined following the assay kit provided (BC0025, Solarbio Science and Technology Co., Ltd., Beijing, China). In brief, 0.1 g of frozen potato tuber was ground in liquid nitrogen (Henan Boss Liquid Nitrogen Container Co., Ltd., Dongtai, China, 78.03% by volume, 75.5% by weight). One mL of extract solution was added and centrifuged at 8000× g for 10 min at 4 • C, and the supernatant was collected. Regents were added according to the manufacturer's instructions. MDA was measured at 600, 532 and 450 nm, and expressed as µmol kg −1 FW. This was repeated three times. The hydrogen peroxide (H 2 O 2 ) content was estimated following the protocol of the assay kit provided (BC3595, Solarbio Science and Technology Co., Ltd., Beijing, China). In brief, 0.1 g of frozen potato tuber was crushed in liquid nitrogen and placed on an ice bath in 1 mL of acetone. One mL of the extract solution was added and centrifuged at 8000× g for 10 min at 4 • C, and the supernatant was collected. Other regents were added according to the manufacturer's instructions. The H 2 O 2 content was measured at 415 nm and expressed as µmol kg −1 FW. All the results are expressed on a fresh weight (FW).

Assay of Some Enzymatic Activities
NADPH oxidase (NOX; EC 1.6.3.1) activity was measured following the protocol of the assay kit provided (BC0630, Solarbio Science and Technology Co., Ltd., Beijing, China). 0.1 g of the frozen potato tuber was ground in liquid nitrogen. 1 mL of extract solution was added to 0.1 g of frozen powder of potato tuber and centrifuged at 600× g for 5 min at 4 • C. The supernatant was transferred to another centrifuge tube and centrifuged at 11,000× g for 10 min at 4 • C. The various reagents were added as instructed by the manufacturer. The absorbance (OD) value was determined at 600 nm, then NOX activity was expressed as U g kg −1 FW. Peroxidase (POD; EC 1.11.1.7) activity was measured using the instructions of the assay kit provided (BC0090, Solarbio Science and Technology Co., Ltd., Beijing, China). 0.1 g of the frozen potato tuber was ground in liquid nitrogen. One mL of the extract solution was added to 0.1 g of frozen powder of potato tuber and centrifuged at 8000× g for 10 min at 4 • C, and the supernatant was collected. Reagents were added as instructed. The OD value was measured at 470 nm using a spectrophotometer and POD activity was expressed as U g kg −1 FW.
Catalase (CAT; EC 1.11.1.6) activity was analyzed following the instructions of the assay kit provided (BC0200, Solarbio Science and Technology Co., Ltd., Beijing, China). 0.1 g of the frozen potato tuber was ground in liquid nitrogen. One mL of the extract solution was added to 0.1 g of frozen powder of potato tuber and centrifuged at 8000× g Sustainability 2022, 14, 8870 5 of 20 for 10 min at 4 • C, and the supernatant was collected. Reagents were added as instructed. The OD value was performed spectrophotometrically at 240 nm and CAT activity was expressed as U g kg −1 FW. Superoxide dismutase (SOD; EC 1.15.1.1) activity was analyzed using the instructions of the assay kit provided (BC0170, Solarbio Science and Technology Co., Ltd., Beijing, China). 0.1 g of the frozen potato tuber was ground in liquid nitrogen and 1 mL of the extract solution was added, then centrifuged at 8000× g for 10 min at 4 • C, and the supernatant was collected. Reagents were added as instructed by the manufacturer. The OD value of SOD was measured at 560 nm and SOD activity was expressed as U g kg −1 FW. Polyphenol oxide (PPO; EC 1.14.81.1) activity was analyzed according to the protocol of the assay kit provided (BC0195, Solarbio Science and Technology Co., Ltd., Beijing, China). 0.1 g of the frozen potato tuber was ground in liquid nitrogen. One mL of the extract solution was added and centrifuged at 8000× g for 10 min at 4 • C, and the supernatant was collected. Reagents were added as instructed by the manufacturer. The OD value of PPO was measured at 420 nm and PPO activity was expressed as U g kg −1 FW.
Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) activity was determined using the protocol of the assay kit provided (BC0210, Solarbio Science and Technology Co., Ltd., Beijing, China). 0.1 g of the frozen potato tuber was ground in liquid nitrogen, 1 mL of the extract solution was added and centrifuged at 8000× g for 10 min at 4 • C, and the supernatant was collected. The OD of PAL was measured spectrophotometrically at 290 nm, then expressed as U g kg −1 FW. Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) activity was determined according to the instructions of the assay kit provided (BC4170, Solarbio Science and Technology Co., Ltd., Beijing, China). One mL of extract solution was added to 0.1 g of frozen powder of potato tuber, then centrifuged at 10,000× g for 10 min at 4 • C, and the supernatant was collected. Other reagents were added as instructed by the manufacturer. The OD was determined at 340 nm and CAD activity was expressed as U g kg −1 FW. 4-coumaryl-CoA ligase (4CL; EC 6.2.1.12) activity was assayed following the instructions of the assay kit provided (BC4220, Solarbio Science and Technology Co., Ltd., Beijing, China). One mL of the extract solution was added to 0.1 g of frozen powder of potato tuber, then centrifuged at 8000× g for 10 min at 4 • C, and the supernatant was collected. Other reagents were added as instructed by the manufacturer. The OD was measured at 333 nm and 4CL activity was expressed as U g kg −1 FW. Cinnamate-4hydroxylase (C4H; EC 1.14.13.11) activity was determined using a kit provided (BC4080, Solarbio Science and Technology Co., Ltd., Beijing, China). One mL of the extract solution was added to 0.1 g of frozen powder of potato tuber, then centrifuged at 12,000× g for 15 min at 4 • C, and the supernatant was collected. Regents were added as instructed The OD was measured at 340 nm and expressed as U g kg −1 FW.

Statistical Analysis
The data were subject to one-way ANOVA using the SPSS package (SPSS V16.0; SPSS, Inc., Chicago, IL, USA). Treatment effects were determined using Duncan's multiple range test and significant results were expressed at p < 0.05.

Disease Assessment
The results show that SA or Pro and their combined treatments reduced the disease index of potato soft rot. However, L. amnigena-treated tubers experienced a high incidence of soft rot compared to potato tubers treated with SA and Pro ( Figure 2). The results show that applied SA reduced the disease index by 67.9% after 6 d of treatment. In addition, applied Pro reduced the disease index by 64.6% after 6 d of treatment. However, the combined application of SA and Pro reduced the disease index by 72.5% after 6 d of treatment.

Disease Assessment
The results show that SA or Pro and their combined treatments reduced the disease index of potato soft rot. However, L. amnigena-treated tubers experienced a high incidence of soft rot compared to potato tubers treated with SA and Pro ( Figure 2). The results show that applied SA reduced the disease index by 67.9% after 6 d of treatment. In addition, applied Pro reduced the disease index by 64.6% after 6 d of treatment. However, the combined application of SA and Pro reduced the disease index by 72.5% after 6 d of treatment. The combination of SA and Pro treatment provided better disease control than either SA or Pro application alone ( Figure 2). The combination of SA and Pro treatment provided better disease control than either SA or Pro application alone ( Figure 2).

Effects of SA and Pro on MDA and H2O2 Content in Potato Tubers Inoculated with L. amnigena
Our results in Figure 3 show that L. amnigena stress significantly increased MDA and H2O2 contents in potato tubers by 50.0 and 40.7%, respectively, compared to the control. In Figure 3, the beneficial impact of SA or proline or SA + Pro on decreasing oxidative

Effects of SA and Pro on MDA and H 2 O 2 Content in Potato Tubers Inoculated with L. amnigena
Our results in Figure 3 show that L. amnigena stress significantly increased MDA and H 2 O 2 contents in potato tubers by 50.0 and 40.7%, respectively, compared to the control. In Figure 3, the beneficial impact of SA or proline or SA + Pro on decreasing oxidative stress and MDA and H 2 O 2 is observed. These treatments resulted in a significant decrease in MDA and H 2 O 2 , and the best treatment was SA + Pro (77.2 and 83.8%), followed by SA then Pro (Figure 3).

Figure 2.
Effect of salicylic acid (SA) and proline (Pro) on the disease index of tubers inoculated with L. amnigena (PC3). Data are presented as mean ± standard error (SE), based on two independent experiments with three replicates. Means with the same lowercase letters are not significantly different at p < 0.05 according to Duncan's multiple range test.

Effects of SA and Pro on MDA and H2O2 Content in Potato Tubers Inoculated with L. amnigena
Our results in Figure 3 show that L. amnigena stress significantly increased MDA and H2O2 contents in potato tubers by 50.0 and 40.7%, respectively, compared to the control. In Figure 3, the beneficial impact of SA or proline or SA + Pro on decreasing oxidative stress and MDA and H2O2 is observed. These treatments resulted in a significant decrease in MDA and H2O2, and the best treatment was SA + Pro (77.2 and 83.8%), followed by SA then Pro (Figure 3).  The activities of NOX, SOD, POD, CAT, and PPO as stress indicators were higher in potato tubers exposed to L. amnigena than in controls. The data presented in Figure 4A show that antioxidant enzymes NOX, SOD, POD, PPO, and CAT activity significantly increased by 69.3, 80.9, 78.0, 86.0, and 83.0%, respectively, in potato tubers under L. amnigena stress treated with SA, respectively, compared with untreated stressed tubers. In addition, Pro-treated tubers increased NOX, SOD, POD, CAT, and PPO activity by 62.1, 69.8, 70.4, 66.9, and 73.9%, respectively, compared with untreated stressed tubers. However, the best results of NOX, SOD, POD, CAT, and PPO activity were recorded with SA + Pro treatment compared with untreated stressed tubers ( Figure 4A-E).

SA and Pro Treatment Increased PAL, CAD, 4CL, and C4H Activity in Potaato Tubers Inoculated with L. amanigena
In the current study, the results show that PAL, CAD, 4CL, and C4H activities increased significantly by exposure to L. amnigena stress ( Figure 5). However, the application of SA led to an increase in PAL, CAD, 4CL, and C4H activities by 77.7, 73.6, 79.1, and 76.0%, respectively, compared with untreated stressed tubers. Additionally, application  In the current study, the results show that PAL, CAD, 4CL, and C4H activities increased significantly by exposure to L. amnigena stress ( Figure 5). However, the application of SA led to an increase in PAL, CAD, 4CL, and C4H activities by 77.7, 73.6, 79.1, and 76.0%, respectively, compared with untreated stressed tubers. Additionally, application of Pro increased PAL, CAD, 4CL, and C4H activities by 70.4, 60.4, 72.1, and 64.9%, respectively, as compared to the control. Similarly, the activities of PAL, CAD, 4CL, and C4H were higher in the SA + Pro treatments than in the two single treatments ( Figure 5). were higher in the SA + Pro treatments than in the two single treatments ( Figure 5).

Discussion
Extracellular enzymes can be used as a primary mechanism by pathogens, including bacteria, to develop plant diseases. The activity of the extracellular enzymes permits the pathogen to penetrate host tissues by breaking down the host cells' defensive outer layers. Extracellular enzymes secreted by microorganisms play an important role in disease progression. These degrade plant materials into smaller particles that pathogens can easily absorb and use for growth and development. In this current study, pectinase, protease, pectin lyase, and cellulase were produced by L. amnigena, aiding it to cause potato soft rot. Application of SA or Pro alone to L. amnigena reduced the synthesis of pectinase, protease, pectin lyase, and cellulase. However, combined SA and Pro further reduced the production of these enzymes. This means that, while using SA and Pro alone can limit extracellular enzyme production by L. amnigena, combining the two is the most effective. Similarly, Bandara et al. [44] discovered that applied SA had an impact on the secretion of protease and elastase by Pseudomonas aeruginosa, which is involved in the development of microbial keratitis. Plant pathogens have evolved complex penetration, invasion, and colonization tactics to disable susceptible hosts' plant defense mechanisms and cause disease [45]. The pathogen normally infects the plant by producing components that are utilized to manipulate plant tissue to get physical access to the tissues and to draw nutrients [46]. These factors allow the plant both to induce disease and to advance the infection into the tissue's core, resulting in a worsening of the disease situation. Pathogens attack plants in different ways. One mode necessitates that the plants and living plant tissues cooperate. Within the invaded plant tissues, the pathogen produces penetration structures and a network of flagella [47]. This mass of cell wall structures generated in the plant's intercellular gaps extracts nutrients from the plant.
The reduction in disease severity caused by SA and Pro could be the result of the antibacterial consequence of SA and Pro, which reduces L. amnigena production of extracellular enzymes. The results show that the treatment of SA and Pro significantly reduced potato soft rot induced by L. amnigena; we suggested that combined SA and Pro treatment in the present study has synergistic effects on the control of potato soft rot caused by L. amnigena. Our results could also be attributed to either a direct toxicity effect of SA and Pro on L. amnigena growth or an indirect plant defense-related effect by inducing resistance in infected tissues. Although the molecular mechanism of SA and Pro-induced L. amnigena resistance is unknown, it appears to include inhibition of pathogen-secreted extracellular enzymes. SA was found to be effective in generating localized acquired tolerance to Pectobacterium carotovora subsp. carotovora infection [48]. Lastochkina et al. [49] previously discovered that SA can boost potato resistance to F. oxysporum and Phytophthora infestans postharvest disease. Eshgpour et al. [50] also reported that applied SA reduced infection of Pectobacterium carotovorum causing potato soft rot in vitro studies, which are similar to the current study's findings. Our findings are comparable with those of Bawa et al. [51], who found that applying SA to soybean seedlings decreased disease severity and induced resistance to Fusarium solani. According to Li and Zou [52], foliar spraying tomato plants with SA at a dosage of 2.0 mM resulted in a substantial reduction in disease severity. According to Yao and Tian [22], the roles of SA in reducing brown rot may be due to the direct toxicity of SA on fungal mycelia and/or an indirect plant defense-related effect by activation of some defense enzymes that play an important role, such as: (i) breaking down the fungus cell wall (such as chitinase and -1,3-glucanase); (ii) saving the plant cell wall; or (iii) increasing antioxidants (such as PAL or POD). According to Cecchini et al. [53], Pro is a defense compound contributing to hypersensitive response and tolerance to diseases. Pro treatments have thus been shown in several investigations to reduce the negative impacts of environmental pressures such as pathogen infections [54]. Ben et al. [55] reported that Pro application induces plant resistance to various pathogens through mechanisms such as oxidative scavenger and gene expression regulation. Qian et al. [56] also found that applied Pro mitigated superficial scald incidence and index in pear fruit.
A well-known side impact of stress is a buildup of oxidatives (reactive oxygen species, ROS) [57]. To minimize ROS damage effectively, plants have evolved scavenging systems like antioxidants. SOD, POD, PAL, and CAT, which are essential enzyme systems for ROS scavenging mechanisms, are essential metrics used for measuring plant resistance to stress. ROS scavenging mechanisms, are mediated by antioxidant enzymes and are the first edge of defense against stress. To reduce ROS generation and interference, as well as to alleviate the negative impacts of stress on plant growth and development, effective antioxidant capacity is required [57]. Protein denaturation occurs in plant cells due to oxidative stress, and significant amounts of MDA and H 2 O 2 accumulate, which may act as primary stress mediators and triggers of plant defense systems [58]. While reactive oxygen species (ROS) can help improve plant tissue resistance to pathogen infections, high accumulations of ROS also lead to lipid peroxidation and loss of plant organ membrane virtue. In this study, potato tubers responded to L. amnigena treatment by producing more oxidants such as MDA and H 2 O 2 . SA reduced oxidant content in tubers inoculated with L. amnigena by acting as an antibiotic against bacteria pathogens, which is consistent with the study of Mishra and Baek [59]. Bawa et al. [60] found a decrease in H 2 O 2 and MDA levels in soybean plants treated with SA, compared to untreated soybean plants under biotic stress. Studies by Estaji [61] and Sayyari et al. [62] discovered that applying SA reduced oxidants such as MDA and H 2 O 2 content in pepper and purslane, respectively, under drought stress. According to Naeem et al. [63], SA at 0.5 mM may be administered to tomato plants under saline conditions up to 90 mM, significantly alleviating the harmful effect of salt stress. Iqbal et al. [64] demonstrated that SA supplementation reduced the negative effects of salt stress on wheat cultivar development. The results show that the content of MDA and H 2 O 2 in Pro-treated tubers compared to untreated inoculated tubers indicates that proline has efficacy in alleviation of L. amnigena-induced oxidative damage in potato tubers. This finding agrees with the findings of Hayat et al. [65], who discovered that applied Pro decreased MDA and H 2 O 2 contents in pigeon peas subjected to cadmium stress. Pro application enhances plant resistance to biotic stress [66]. However, the combined therapy of SA and Pro with L. amnigena-infected tubers significantly reduced MDA and H 2 O 2 content. De Carvalho et al. [67] discovered that applied Pro reduced the negative impacts of ROS as a result of increased SOD, POD, and CAT. The results of the current study collaborate with those of Abdelaal et al. [32], who found that combined SA and Pro lowered MDA and H 2 O 2 levels in barley plants under drought stress. Sanchez-Rodriguez et al. [68] demonstrated that tomato plants treated with Pro reduced MDA and H 2 O 2 levels, while increasing the activity of the antioxidant enzymes.
In a variety of plants, the role of antioxidant enzymes in plant defense against pathogen stress has been studied [69]. The current study found that either SA or Pro alone increased the activity of NOX, SOD, POD, CAT, and PPO in tubers subjected to L. amnigena stress. However, combined treatment of SA and Pro with L. amnigena-infected tubers further increased the activity of NOX, SOD, POD, CAT, and PPO, compared to the controls. The role of SA in the antioxidative system is widely assumed to be that of a signal molecule. This result suggests that SA treatment of L. amnigena-inoculated tubers may induce resistance because it activates the plant defense system by increasing some enzymes (NOX, SOD, POD, CAT, and PPO). Previous research has shown that, under stress, SA can maintain the activity of antioxidant enzymes to some extent, and it may also help to limit the impact of oxidative processes associated with disease development and spread, implying that SA may play an important antioxidant role in oxidative processes associated with plant defense responses [70,71]. Furthermore, SA-induced protein synthesis could be used to activate antioxidants [72]. The findings support the findings of Ma et al. [73], who found that during salt stress, SA raised the antioxidant enzyme activity in Dianthus superbus. Similar results were obtained in a previous work on Portulaca oleracea L., in which SA increased antioxidant enzyme activity, lowering ROS concentrations under diverse environmental conditions [74]. By adhering to hydrogen bonds, Pro can increase protein stability and safeguard membrane integrity [75]. Pro may also protect cells by enhancing water absorption capacity and promoting enzyme activity [76]. Pro, in addition to being an osmolyte, is a powerful antioxidative defense molecule, a metal chelator, a protein stabilizer, a ROS scavenger, and an inhibitor of programmed cell death [77,78]. Exogenous Pro has been shown in several studies to improve plant stress tolerance [7,79]. This type of treatment may activate stress avoidance systems by increasing stress tolerance and enhancing reactivity to stress triggers during a later stressful condition [54]. Pro contributes to the plant's stress response by enhancing antioxidant enzymes [65]. Pro was found to increase the enzyme's activity associated with the ascorbate-glutathione revolution [80], implying a role in improving cell antioxidant capacity. Abdelhamid et al. [81] discovered that applied Pro raised SOD, CAT, and POD in Phaseolus vulgaris L. plants subjected to salt stress. Furthermore, Tabssum et al. [82] reported that applied Pro at 50 mM increased SOD and CAT activity in rice under salt conditions. Ghaffari et al. [83] reported that Pro-mediated changes in antioxidant enzymatic activities and the physiology of sugar beet under drought stress. Pro regulates the activity of SOD, CAT, and POX enzymes in plant cells, as well as their involvement in metabolic response development in response to environmental factors, according to Abdallah and El-Bassiouny [84]. The results of the present study are also in agreement with those of Abdelaal et al. [32], who discovered that combining SA and Pro increased drought resistance in barley plants through modulation of antioxidant activities. Based on the findings, it is proposed that the reduction of oxidative stress by activated antioxidant enzymatic systems may contribute to the synergistic effects of SA and Pro in the control of L. amnigena-caused potato soft rot.
Phenylpropanoid metabolism aids in the reduction of potato soft rot by providing substrates for the synthesis of phenolic and monolignin with antibacterial properties [85]. PAL is an important enzyme that initiates this metabolism by deaminating L-phenylalanine to trans-cinnamic acid [86]. In the current study, SA and Pro treatments significantly increased the production of phenylpropanoids such as PAL, CAD, 4CL, and C4H in potato tubers inoculated with L. amnigena. This could be due to an increase in the expression levels of phenylpropanoid-related genes following treatment with SA or Pro, as previous research has shown that different types of elicitors increase the expression levels of phenylpropanoid-related genes and increase phenolic compound accumulation. Thus, SA and Pro treatment may promote the phenylpropanoid pathway by increasing enzyme activity and the synthesis of phenol compounds and lignin at the tuber's rotting site. SA may affect the phenylpropanoid pathway by inducing key enzymes such as PAL, CAD, 4CL, and C4H, resulting in phenolic compound accumulation. PAL, CAD, 4CL, and C4H also act as plant defense mechanisms, and the activities of these phenylpropanoids help the potato tubers to reduce the excess reactive oxygen species which is the source of oxidative stress during pathogen infection [87]. Plants can successfully induce PAL, CAD, 4CL, and C4H activity when infected with microorganisms such as bacteria pathogens [20,88]. Our results confirm this; PAL, CAD, 4CL, and C4H activities increased in L. amnigena-infected tubers treated with SA and Pro.
The important role of SA and Pro in protecting plants from stress may be due to their capability to increase the gene expression of PR proteins [89]. In this current study, potato tubers infected with L. amnigena increased relative expression of NOX, PAL, CAD, 4CL, and C4H compared with the control. However, combined treatment of SA and Pro with L. amnigena-infected tubers significantly up-regulated NOX, PAL, CAD, 4CL, and C4H genes. Lavrova et al. [90] reported that applied SA promotes gene expression in tomato plants infected with pathogens. These findings also support those of El-Esawi et al. [91], who found that bZIP62, DREB2, ERF3, and OLPb were increased in rosemary plants treated with SA, implying that applied SA-modulated genes enhanced the rosemary plants' resistance to the salinity condition. SA reduces oxidative damage caused by L. amnigena stress by up-regulating antioxidant defense mechanisms, making potato tubers more resistant to this type of biotic stress [92]. The Pro is a radical scavenger, in addition to being a suitable osmolyte [93]. As a result, Pro served as both an osmolyte molecule and an antioxidant [94]. Pro also serves as an antioxidative defense molecule and a signaling molecule during stress, in addition to being an effective osmolyte [93].
Plant defenses defined by pathogen gene-for-gene recognition by plants containing resistance genes have also been connected to the stimulation of SA-dependent R-genes. Higher expression of PR proteins has been linked to activated resistance attained with SA in earlier research [95]. In addition, SA-primed stressed-infected plants had 1.7, 2.9, 2.1, 2.5, and 2-fold IAA27, MPK1, GPX, chitinase, and 1,3-glucanase, respectively, than non-primed susceptibility inoculation controls. Since these genes are critical for developing resistance during the host-pathogen interaction, the high level of IAA27, MPK1, GPX, chitinase, and 1,3-glucanase correlates with disease protection studies. The increased stimulation of defense gene processes in potato tubers treated with SA or Pro alone suggests the molecular mechanisms governing both SA and Pro to alleviate L. amnigena infection in tubers. Combined treatment of SA and Pro with L. amnigena-infected tubers induced the up-regulation of PR1, PR2, CTL1, PAL, PRH3, and SPI1, compared to the controls, which led to the most important effector genes for systemic acquired resistance (SAR) mediated by SA and Pro [96,97]. Chen et al. [98] discovered that applied Pro at 0.5 mM activated PR gene expression. The discovery that Pro raises the OxyR gene implies that Pro metabolism raises ROS scavengers. OxyR reacts with H 2 O 2 to form a disulfide bond between Cys199 and Cys208, resulting in OxyR regulon transcriptional activation [99].

Conclusions
The use of SA and Pro could help to reduce potato soft rot caused by L. amnigena. It could be attributed to the stimulation of antioxidant enzymes and the alleviation of oxidative damage caused by pathogenic infection, which stabilized intracellular redox homeostasis. SA and Pro treatment also increased the activities of PR1, PR2, CTL1, PAL, PRH3, and SPI1 in the tuber, which may slow the growth of pathogenic bacteria (L. amnigena). These findings suggest that antioxidants and plant defense-related genes, particularly in potato tubers, are important in the defense response to pathogen infection.