Mitigation of bacterial spot disease induced biotic stress in Capsicum annuum L. cultivars via antioxidant enzymes and isoforms

Bacterial spot, caused by a group of Xanthomonads (Xanthomonas spp.), is a devastating disease. It can adversely affect the Capsicum annum productivity. Scientists are working on the role of antioxidants to meet this challenge. However, research is lacking on the role of antioxidant enzymes and their isoforms in the non-compatible pathogen and host plant interaction and resistance mechanisms in capsicum varieties. The present study was conducted to ascertain the defensive role of antioxidant enzymes and their isoforms in chilli varieties Hybrid, Desi, Serrano, Padron, and Shehzadi against bacterial spot disease-induced Xanthomonas sp. The seedlings were inoculated with bacterial pathogen @ 107 CFU/mL, and samples were harvested after regular intervals of 24 h for 4 days followed by inoculation. Total plant proteins were extracted in phosphate buffer and quantified through Bradford assay. The crude protein extracts were analyzed through quantitative enzymatic assays in order to document activity levels of various antioxidant enzymes, including peroxidase (POD), Catalase (CAT), Ascorbate peroxidase (APX), and Superoxide dismutase (SOD). Moreover, the profiles appearance of these enzymes and their isoforms were determined using native polyacrylamide gel electrophoresis (PAGE) analysis. These enzymes exhibited maximum activity in Hybrid (HiR) cultivar followed by Desi (R), Serrano (S), Padron, and Shehzadi (HS). Both the number of isoforms and expression levels were higher in highly resistant cultivars compared to susceptible and highly susceptible cultivars. The induction of POD, CAT, and SOD occurs at the early stages of growth in resistant Capsicum cultivars. At the same time, APX seems to make the second line of antioxidant defense mechanisms. We found that modulating antioxidant enzymes and isoforms activity at the seedling stage was an important mechanism for mitigating plant growth inhibition in the resistant ones.

) 28 . Kuznaik and Sklodowska 29 analyzed peroxisomal antioxidant enzymes viz. SOD, CAT, Glutathione peroxidase (GPX), and ascorbate-glutathione (AA-GSH) cycle activities in tomato infected with B. cinerea. It was early noticed increases in SOD, CAT and GPX indicating that initial infection induced their activities and consequently the activation of antioxidant plant defense, which was followed by a progressive inhibition concomitant with disease symptoms development. Depending upon the severity of pathogenic activity and its interaction with host, scavenging potential of antioxidants i.e., SOD, CAT and peroxidase (POX) produced by host also become variable 30 . Furthermore, the activities of AA-GSH cycle enzymes APX, monodehydroascorbate (MDHAR), dehydroascorbate (DHAR) and glutathione reductase (GR) as well as ascorbate and glutathione concentrations and redox reactions were significantly decreased with the differential rate and timing of activity. However, research is lacking on the role of antioxidant enzymes and their isoforms in the non-compatible pathogen and host plant interaction and resistance mechanisms in capsicum varieties.
This study's primary goal was to differentiate capsicum varieties based on their ability to cope with the stress induced by the bacterial spot pathogen. The study was further extended to get an insight into the mechanistic details of the defense mechanisms. Thus, in the current study, we investigated the role of different antioxidant enzymes (SOD, POD, CAT, and APX) in the defense mechanism of capsicum cultivars, enabling them to mitigate the infection damage on them. It is hypothesized that antioxidant enzymes (SOD, POD, CAT, and APX) could be evolved as a defense of capsicum mechanism against the bacterial spot pathogen during their coevolution process in the past time.

Results
Screening of five chilli cultivars for bacterial spot disease was performed. Disease score different levels were observed ranging 0-40%. The chilli pepper cultivar Hybrid (HiR) was completely free from bacterial spot disease. Desi pepper was found to be resistant, and Serrano pepper was considered susceptible. Two cultivars (Padron and Shehzadi) were susceptible to bacterial spot disease (Table 1) and MDA contents in chilli seedlings after 72 h of inoculation than non-treated plants ( Table 2). In contrast, MDA and H 2 O 2 contents were increased in Shehzadi followed by Padron, whereas Hybrid exhibited the least H 2 O 2 and MDA contents. Table 1. Bacterial spot severity on five chilli cultivars challenged by a Xanthomonas spp., bacterial spot disease agent. Screening results of five Capsicum varieties under bacterial spot disease stress in greenhouse conditions. Chilli plants were grown in plastic pots, and after 21 days of growth, seedlings were inoculated with Xanthomonas campestris. Bacterial spot disease incidence was observed up to 60 days from the day of bacterial inoculation. HIR highly resistant, R resistant, S susceptible, and HS highly susceptible.

S No
Varieties Bacterial spot incidence (%) Categorization  1A) CAT (Fig. 1B), APX (Fig. 1C), and SOD ( Fig. 1D) activities were observed in control and inoculated seedlings of HS and HiR varieties. A steady enhancement in POD activity was observed in all varieties. In resistant seedlings, a drastic enhancement in POD activity was observed initially after inoculation of the pathogen, which reached the peak at 48 h.p.i (54.2 min −1 g −1 FW) whereas, in highly susceptible seedling, POD activity was found to be 50.2 min −1 g −1 FW at 48 h.p.i. after inoculation ( Fig. 2A).  Native PAGE analysis of POD. The POD isoforms detection where analyzed with the total protein samples of HS and HiR, cultivars. A total of four isoforms appeared, and their strength varied between control and inoculated seedlings. Infected plants have an expression of more POD isoforms as compared to their respective controls. Infected HS and HiR cultivar had an equal number of isoforms (four), whereas the non -inoculated plant has three isoforms. POD isoform's protein bands were low in intensity in HS over HiR seedlings (Figs. 3,4).
CAT activity temporal changes. The  Temporal changes in SOD activity. Figures 1D and 2D shows variable patterns of SOD activity in control and inoculated seedlings of HS and HiR varieties. In HiR, after pathogen inoculation, a noticeable enhancement in SOD activity was observed that reached the peak at 48 h (78 min −1 g −1 FW) whereas, in HS (Padron) seedlings, SOD activity was found to be 70 min −1 g −1 FW at 48 h after pathogen inoculation.
Native PAGE analysis of SOD. SOD expression was not detected in highly resistant (Hybrid) variety in case of both inoculated and un-inoculated. Three isoforms of SOD were detected in control seedlings of resistant variety, while only one isoform was expressed in seedlings of treated resistant variety. Susceptible variety showed www.nature.com/scientificreports/ only one isoform expression, but the expression level was low in control seedlings compared to treated seedlings. Control chilli pepper (Padron) has shown only one isoform in gel detection assay (Figs. 3c, 4c).
Temporal changes in APX activity. A continuous increase in APX activity was noticed for all cultivars.
In HiR seedlings, after inoculation of pathogen, a significant increase in APX activity was noticed and reached at its peak (3.   The early and rapid increase of various antioxidants is an important feature of plant-resistance to pathogenic agents 32 . In the present study, peroxidase activity in chilli significantly increased after pathogen inoculation. POD activity was maximum in cv. Hybrid (HiR) at 48 h after pathogen inoculation compared to control and least in Shehzadi pepper at 24 h. Such observations are consistent with Xie et al. 33 ; their results revealed that POD activity increased in Patchouli against Ralstonia solanacearum the causal agent of bacterial wilt disease on solanaceous crops. Peroxidase native PAGE results showed that treated plants had increased POD isoforms compared to untreated plants. Padron (S) seedling isoforms had a low expression when compared to the cv. Hybrid (HiR) seedlings. In chilli infected by Colletotrichum and brinjal (eggplant-USA or aubergine-UK) by Ralstonia solanacearum exhibited dense and dark isoform in resistant compared to susceptible plants 34,35 .
After bacterial inoculation, CAT showed significant activity. In highly resistant cv. Hybrid at 48 h.p.i, CAT temporal changes were highest whereas least CAT activity was observed at 48 h.p.i. in treated cv. Padron pepper. However, Debona et al. 36 revealed that the leaves of a resistant P. oryzae-infected wheat cultivar produced more CAT than susceptible cultivar. CAT detection on Native PAGE further confirmed the results of the spectrophotometric estimation. Hybrid (HiR) and Desi (R) Capsicum cultivars express three CAT isoforms upon bacterial inoculation, whereas infected cv. Padron (HS) had an expression of just one isoform. www.nature.com/scientificreports/ Spectrophotometric SOD assay showed maximum activity in cv. Hybrid (HiR) after 48 h of post-inoculation of pathogen compared to susceptible cultivars. POD and SOD enzymatic activities increased significantly in patchouli inoculated with R. solanacearum 34 , and the findings of them and those of the present study were consistent with Chai et al. 37 studies of antioxidant isozymes to some extent. Lobna et al. 38 reported that POX, CAT, PPO, and SOD enzyme activity after pathogen infection increased in resistant cultivar compared to a susceptible one. The detection of SOD isoforms by PAGE showed the expression of two isoforms in control and one in infected S (Serrano) and R (Desi) variety, according to the results of Xie et al. 33 .
Rojas-Beltran et al. 39 revealed that the chloroplast of most angiosperms has an expression of two SOD isoforms. A similar number of isoforms were detected in resistant and susceptible Capsicum cultivars as reported by Garcıá-Limones et al. 40 . APX enzyme activity was maximum in resistant (cv. Desi) and least in susceptible cultivars. Vuleta et al. 41 have shown that the APX in the Iris pumila leaves was the main H 2 O 2 scavenger when exposed to high light intensities. In the present investigation, just one type of APX isoform was observed in all cultivars. Chandrashekar and Umesha 17 , found similar results. During the bacterial spot pathogen attack, all four antioxidant enzymes (POD, CAT, SOD, and APX) participated in ROS-scavenging metabolism in chilli pepper varieties, in agreement with the results of 27 .

Materials and methods
The experiments were done in a greenhouse at the Department of Botany, The Islamia University of Bahawalpur (IUB), Pakistan (29.37 latitude and 71.75 longitude) in consecutive three trials. River soil, peat moss, and sandy loam 39 were mixed (50:20:30%) and utilized at 30-35 °C and 61% (humidity) as growing medium. For 15-20 days soil was exposed to sun and placed in plastic pots (9 cm diameter). 42 . Seeds were collected from the regional vegetable research agriculture institute of Bahawalpur, and surface-sterilized with sodium hypochlorite solution (0.1 %). After that 3 washing were given with sterilized water 43 . Seeds were sown in the growing medium in pots. Twenty-one days old seedlings of chilli pepper cultivars Hybrid, Desi, Serrano, Padron, and Shehzadi were thinned for maintaining 3 healthy seedlings in each pot. Pots were arranged in a completely randomized design. Un-inoculated pots (n = 5) were used as control. The bacterial inoculum level used was at approximately 10 -7 /mL. The inoculation was carried out after 2 days of seedling thinning. The bacterial spot infected chili leaves were collected from greenhouse of Department of Botany, IUB. The seedlings were harvested at the time of inoculation (0 h) and 24 h intervals up to 72 h, initial inoculation. For bacterial source and growth in the laboratory, an ooze test was performed by cutting 1 cm of the infected portion of the plant, sterilized gently squeezed to get a suspension in a sterile test tube containing 3 mL sterilized saline. After dilution, it is plated on YDC Agar medium. After 24 h, incubation light yellow color colonies were developed resembling Xanthomonas sp. biochemical assay was carried out to confirm X. campestris species. Recovered colonies were harvested and suspended in flasks containing sterilized distilled water and incubated for 2-4 h at 32 ± 1 °C. Inoculum suspension was adjusted with a spectrophotometer. Plants were observed daily for bacterial spot symptoms. Depending on disease severity, the cultivars were divided into highly resistant (0%), resistant (0.1% to 10%), slight marginal spot, and 1-20% of leaves become brown, susceptible (10.1% to 20%) of plants showing sectorial spots and 20-40% of leaves become brown and highly susceptible (> 25%) of the plants showing leaf collapse and more than 40% of leaves become brown 17 .
Determination of oxidative stress. H 2 O 2 and Malondialdehyde (MDA) contents of chloroplast were examined by a previously described method of Diao et al. 44 .
Temporal study of antioxidant enzymes. The antioxidant enzyme activity studied were POD, CAT, SOD, and APX, which were analyzed using the highly resistant (cv. Hybrid) and highly susceptible (cv. Padron), which were selected based on their performance on the pathogenicity tests carried out previously. This study aimed to find out the early response of antioxidant enzymes and their isoforms in resistance mechanism; for this purpose the chilli pepper seedlings were grown as previously enlightened, and seedlings were harvested at 0, 24, 48, and 72 h after Xanthomonas pathogen inoculation. Distilled water inoculated samples were used as a negative control. One gram of each chilli seedling was homogenized in 1 mL of 50 mM buffer (potassium-phosphate) of pH 7. The homogenate was centrifuged at 12,000 rpm for 15 min at 4 °C. Catalase assay. CAT activity was analyzed by following the method described by Chance and Maehly 45 .
The reaction mixture (3 mL) containing 50 mM phosphate buffer (pH 0.0), 5.9 mM H 2 O 2, and 0.1 mL enzyme extract was assayed. The CAT activity was measured by a change in absorbance due to H 2 O 2 intake at 240 nm after every 20 s. Superoxide dismutase assay. The SOD activity was evaluated by observing its ability to inhibit the photochemical reduction of nitro blue tetrazolium (NBT) according to Giannopolitis

Native-PAGE analysis of POD, CAT, SOD and APX. Expression of enzyme isoforms of Hybrid (HiR)
and Padron (HS) pepper varieties were detected by discontinues native PAGE using a Mini-Protean II electrophoresis system 48   Ethics approval and consent to participate. We all declare that manuscripts reporting studies do not involve any human participants, human data, or human tissue. So, it is not applicable.

Complies with international, national and/or institutional guidelines. Experimental research and
field studies on plants (either cultivated or wild), comply with relevant institutional, national, and international guidelines and legislation.

Conclusions
Present results also suggest that increased levels of built-up antioxidant enzymes also decrease the scavenging potential of ROS produced under bacterial stress. Increased activity of peroxidase, catalase, and superoxide dismutase in chilli cultivars are associated with the plant resistance to bacterial spot disease and also are involved in the early response to is causal agent, whereas ascorbate peroxidase is less associated with stress in the early growth process. Furthermore, Isoforms synthesis confirmed that antioxidant enzymes are produced in many cell organelles due to the biotic stress and lead to resistance expression. Present investigations cover the expression of antioxidants as isoforms. In the future, the transcription expression of these enzymes must be further investigated. There is a need to explore the mechanistic details and factors that regulate antioxidant enzymes' expression.