Investigation of the antibacterial properties of Moringa oleifera Extracts against Xanthomonas axonopoidespv. glycines the incitant of Bacterial Pustules of Glycine max

Investigation of the antibacterial properties of Moringa oleifera Extracts against Xanthomonas axonopoidespv. glycines the incitant of Bacterial Pustules of Glycine max | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Investigation of the antibacterial properties of Moringa oleifera Extracts against Xanthomonas axonopoidespv. glycines the incitant of Bacterial Pustules of Glycine max Farhana Khan, Arwa Talat, Abhishek Joshi, Bhanupriya Kanthaliya, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7550249/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract Bacterial pustule disease triggered by Xanthomonas axonopodis pv. glycines (Xag) is one of the major constraints affecting the cultivation of soybean in India. According to the recent reports, the disease severity was prevalent in moderate to severe intensities and damaged soybean crop and its yield. The current investigation examined the in vitro and in vivo preventive efficacies of Moringa oleifera lam. different extracts on bacterial pustules disease of soybean. The number of leaves per plant, Plant, soluble protein, carbohydrate and chlorophyll content were examined. A significant control of disease was recorded with seed extract (SE4). Results showed the treatment with seed extract notably increased plant height, carbohydrate, chlorophyll, carotenoids. However, soluble protein and carbohydrate content was observed to be less in higher concentrations along with reference antibacterial agent streptomycin. According to this current research seed extracts with 250µg/500 µl could be essential towards sustainable agricultural crop management for soybean. Therefore, compounds of seed can be evaluated and commercially explored for agricultural use. Soybean bacterial pustules disease Xanthomonas M.oleifera Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Sustainable food production for a rapidly growing human population is one of the major challenges faced by the agriculture sector globally [ 1 – 3 ].Therefore, the increased uses of pesticides and synthetic agrochemicals have become essential to maximize agricultural productivity. Despite their beneficial role in agriculture, they can be hazardous to humans and other non-targeted organisms [ 4 , 5 ]. According to the Food and Agriculture Organization of the United Nations, an estimated 3.2 million tones of pesticides are used on crops each year [ 6 ]. In recent years, unrestrained use of commercial and synthetic agrochemicals in protection of crop has raised significant worry about environmental contamination and resistance enhancement in phytopathogenic microbes [ 7 ]. To address these issues, the development of bio-based/non-synthetic biocides for agriculture has become an important research direction. Soybean [ Glycine max (L.)] is significant and major pulse crop worldwide that delivers two-third of calories derived from agriculture [ 8 ] and accounts for half of the global demand for oil and vegetable protein. It is high in edible oil (18–22%) with major essential amino acids and proteins (38–42%). Its oil and food make up about 85% of the global soybean edibles including soy milk, tofu, soy sauce and tempeh [ 9 , 10 ]. Federation of Indian Chamber of Commerce and Industry (FICCI) claims that India currently produces 25% of its oil and 43% of its oilseeds from soybean, making it the fourth-largest producer of soybeans in the world, behind the United States, Brazil, Argentina and China [ 11 ]. Despite all these aspects, soybean yield in India currently stands at around 1T/ha (minimum) to 3.5t/ha (maximum), there is still an urgent need to increase production to meet the demand of ever increasing population [ 12 ]. Because it is a rainfed crop, it is highly dependent on monsoon rains. However, bacterial pustule disease caused by a gram negative bacterium Xag severely damage the soybean crops, it is among the most serious diseases that reduces the yield and quality of the crop [ 13 ]. It limits the soybean production worldwide by 20–40%. This disease runs rampant in most soybean-growing fields and may cause yield loss as high as 53% under favorable conditions [ 14 ]. Xag may enter soybean leaves via natural openings such as stomata and wounds and proliferate in intercellular spaces [ 15 ]. Typical symptoms are small, light yellow colored pustules surrounded by chlorotic hallows on the underside of soybean leaves [ 16 ]. The pustules spots range in size from small brown patches to large. Numerous management strategies, including the use of synthetic agrochemicals, commercial antibiotics and nanoformulations, have been studied [ 13 , 17 ] but have side effects on overall plant growth and development. Therefore environment-friendly strategies and plant based products for disease management is necessary. Some studies on biocontrol investigations on stem rot by the fungus Sclerotinia sclerotiorum , root rot by the fungus Phytophthora sojae and cyst by the nematode Heterodera glycines were evaluated in previous years [ 18 ], also bio based formulations were investigated [ 19 ] but plant based disease prevention and management studies of bacterial pustules disease of soybean have not been identified yet. M. oleifera Lam. (Family: Moringaceae, commonly known as horseradish, drumstick, ben oil, miracle, and Mother’s best friend), one of the 13 species of the monogeneric family Moringaceae, native to the sub-Himalayan lowlands in northwest India, shows remarkable medicinal potential [ 20 ]. Traditionally, M. oleifera has been associated with various medicinal benefits, including pain relief, controlling blood sugar levels, liver protection, anti-inflammatory effects, promotion of urination, reduction of pain sensitivity, prevention of artery hardening, blood pressure regulation, antioxidant properties and tumor growth inhibition [ 21 , 22 ]. Different phytochemicals like phenols, omega fatty acids, flavonoids, carotenoids, kaempferol, terpenoids, tocopherols, tannins, vanillin, glycosides, β-sitosterol, triterpenoids, ascorbates, quercetin and sterols have been isolated from the flowers, leaves, roots, seeds and fruits [ 23 , 24 ]. In particular, this plant family is rich in compounds containing the simple sugar rhamnose and it is rich in a fairly unique group of compounds called glucosinolates (β-thioglucoside N -hydroxysulfates) and isothiocyanates(4-α-L-rhamnopyranosyloxy)benzyl isothiocyanate [ 20 , 25 ]. About 20% of all plants have undergone biological and pharmacological testing and a significant number of new antibiotics introduced in agriculture sector are from natural or semi-synthetic resources. For this, In the present investigation, we report for the first time the in vitro and in vivo efficacy of different plant parts methanolic extract against Xanthomona saxonopoidis pv. glycine along with the antibiotic streptomycine which is widely used for controlling this disease in soybean field. Our results convincingly establish effective control of bacterial pustule disease of soybean. Materials and Method 3 .1) Chemicals: Folin–Ciolcateu’s reagent, diphenyl-1-picrylhydrazyl-(DPPH), sodium bicarbonate(Na 2 CO 3 ), HCl, TPTZ (2,4,6-tri[2-pyridyl]-s-triazine), ferric chloride (FeCl 3 ), methanol, gallic acid, acetic acid, sodium acetate, nutrient broth (modified), Letheen agar (modified), JS 27 certified seeds of soybean and Streptomycin were of analytical grade purchased from Himedia, India. 3.2) Collection of plant material and preparation of extract: The leaves, flowers, seeds, gum and bark of M. oleifera were collected from the University College of Science campus MLSU Udaipur, Rajasthan. The plant was identified on the basis of herbarium sheet by Botanical Survey of India (BSI), Jodhupur, India (Vide letter Number: BSI/AZRC/I.12012/Tech./2021-22 (PI.-Id.)/412 dated: 14-12-2021). Different plant parts (Fig. 1) except gum were washed thoroughly with sterile water, then shade dried at room temperature and ground to coarse powder by using a blender. The powdered material was kept in airtight jars until further experiment. 50g dried powder of each plant part were extracted with methanol (50 × 250ml) using a soxhlet apparatus. While gum was kept in methanol (5 × 25) for 4 days at room temperature. The extract was filtered through a membrane filter by using Whatman No. 1 filter paper. The filtrate was reduced through a rotary vacuum evaporator at 50 0 C, 80 rpm to obtain the concentrated extract (Fig. 1), which was then frozen and freeze-dried at -20 0 C until analysis. 3.3) Total phenolic content: Total phenolic content of M. oleifera different extracts was determined by the Folin-Ciocalteu method [26]. 2mg of each extract was dissolved in 1ml methanol. The freshly prepared Folin- Ciocalteu reagent (2.5 ml) was mixed with 500 μl of extract, then 400 μl of sodium bicarbonate was added and kept for 1 min in a boiling water bath. For calibration curve gallic acid (30-300 μg/ml) was used and data was recorded at 650 nm. TPC was expressed as mg gallic acid equivalent/g DW of extract. 3.4) Antioxidant activity: 3.4.1) DPPH radical scavenging capacity: This antioxidant activity was calculated by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) [27] . Different extracts of M. oleifera (2mg/100µl) each were added to the reaction mixture having 1 ml of DPPH and 1.5 ml ethanol and 500 μl acetate buffer (pH 5.5). This reaction mixture was kept in a dark place at room temperature. After half an hour, the absorbance was measured at 517 nm. Methanol served as the blank. The radical scavenging activity was expressed in percentage using the equation. Where A s = absorbance of sample solution, A c = absorbance of control 3.4.2) FRAP analysis The ferric reducing power of different extracts was determined by the method given by [28]. The FRAP reagent was incubated for 30min at 37 0 C in water bath. 100 μl extract was added to FRAP reagent (3ml) and shake vigorously. Absorbance was measured at 593 nm using spectrophotometer. Results were expressed in μM Fe 2+ /G and FeSO4.7H 2 O (100-1000 μg/ml) was used as a standard for calibration curve. 3.5) Isolation and purification of X. anopodis pv. Glycine The pathogen was isolated from the infected soybean leaves showing typical disease symptoms collected from soybean fields of RCA, MPUAT, Udaipur, Rajasthan during 2023. The yellowish brown infected portion of the leaves was subjected for the isolation of the pathogen (Fig. 3) on Leethen agar media incubated at 29 0 C ± 2 0 C. The colony developed on the periphery was streaked on fresh media [29]. Pure cultures were established by single colony isolation and maintained in refrigerator at 5 0 C by sealing 48 h old cultures on media. 3.6) Pathogenecity and host range Bacterium pathogenicity was confirmed on 30 days old soybean plants grown in (25cm) pots in a glasshouse. The plants were kept in humid chamber (30±1 0 C, 80-90% RH), 24 h prior to inoculation for stomata opening and to optimize high intercellular humidity in the surrounding tissues of natural openings. 48 h old bacterial culture was gently scrapped from slants and suspended in DDW. An inoculation (2×108 cfu/ml) was made by spray and pinprick to runoff the methods. When the disease symptoms occur (Fig. 3) the pathogen was again isolated from the inoculated plant and compared with the original culture. Suitable controls were maintained, and pathogenicity was proved by following Koch’s postulates. 3.7) Morphological and biochemical characterization of bacterium The isolated bacterium was examined for cell flagellation, Gram staining, KOH solubility and catalase. Methyl red test, urease, and growth in NaCl concentration were also tested [30]. 3.8) Antibacterial activity: Antibacterial activity of different extracts from M. oleifera was evaluated by the agar well diffusion method with slight modifications given by [31]. Active cultures were prepared by transferring a loop full of cells from the stock cultures to test tubes containing nutrient broth for bacteria that were incubated at 24 hrs at 29 0 C ± 2 0 C. To prepare the samples for antibacterial assay 50-300 μg/500 μl of each extract was dissolved in 500 μL methanol and sonicated for 30 minutes. Pure methanol was used as a negative control and standard antibiotic; streptomycin (30μg/ml) was used as a positive control. Petri plates containing 20 ml Letheen agar medium were seeded with 24hr old culture of bacterial strains. 100μl of each test solutions was poured in the respective wells and incubated for 24 hrs at 29 0 C±2 0 C. At the end of the incubation period (24 hrs.) The antimicrobials present in the extract are allowed to diffuse out into the medium and interact in a plate freshly seeded with the test organism. The resulting diameter of the zone of inhibition was measured in millimeters. 3.9) pot experiment: JS27 non-resistant variety of soybeans acquired from an authorized local vendor at Krishi Kendra, Udaipur, Rajasthan was used for plantation. The seeds were sown in pots with autoclaved sandy soil in late July and were allowed to grow under ambient atmospheric conditions. A total of eight pots were prepared for experimentation, with five allocated for assessing the impact of seed extract treatment, while the remaining three served as positive control (streptomycin), diseased plant negative control (NC) and non treated (NT) healthy plants. While bark extract also showed significant inhibition in agar well plate method but it was not considered in in-vivo experiment because it was partially dissolved in solvent both water and methanol and a thin extract layer was observed on leaves and seeds, so the penetration of extract was blocked, and it clogged the natural openings. Before sowing, the seeds were soaked in broth containing the pathogen for thirty minutes. Subsequent to sowing, the soil was inoculated with the pathogen by spraying 10 ml water-suspended bacterial solution to ensure timely disease development. The plant was allowed to grow for 10 days above the surface without further inoculation. Upon achieving a decent growth, approximately 35 days after sowing the plants were artificially inoculated with 24-hour-old culture of Xag for two consecutive days [32]. Disease manifestation was observed in ten to twelve days. Following the onset, different concentrations of M.oleifera seed extract (100-300 μg/500 μl) were sprayed on the potted plants after 48 hrs of the second foliar spray. Also biochemical parameters were conducted after second foliar spray. Plant height and leaf per plant were observed and recorded after 10 days of last spray. Three replications per concentration were maintained and for each replication 4 plants per pots were placed. 3.10) Biochemical analysis 3.10.1) Chlorophyll and carotenoid The quantitative estimation of chlorophyll and carotenoid was performed by using the method of [33], fresh samples from healthy and inoculated plant leaves were collected after 10 d of inoculation from green house. 1 g of plant leaves was chopped and homogenized to a fine pulp by using 85% acetone for 5 min and centrifuged for 10 min at 2000 rpm. The supernatant was made up to 30 ml with 80% acetone; aqueous acetone is used as blank. The absorbance was measured at 665, 663 and 450 nm by using a spectrophotometer. Chlorophyll a, b and total chlorophyll content were calculated by these formulas: a.) Total chlorophyll (mg/g) = (20.2A645+8.02 A663×V) b.) Chlorophyll a (mg/g) = (12.7A663-2.69A645×V)/AC1000×W c.) Chlorophyll b (mg/g) = (22.7 A645+4.68 A663×V)/ A×1000×W d.)Carotenoids (mg/g tissue) = (10A450×V×F)/2500×W Where, A (absorbance) nm in a 1 cm cell, V= volume of extract in ml, W= fresh weight of the sample, F= dilution factor 3.10.2) Determination of soluble protein content Soluble proteins were estimated by the method given by [34] using bovine serum albumin as a standard. 1g of fresh leaves was ground in 5 ml chilled phosphate buffer (0.1M, pH 7.5) and centrifuged at 12000 rpm for 15 minutes. Supernatant was used for analysis. 500 μl extract was added to Coomassien brilliant blue reagent and vortexed for 30 seconds after color development absorbance was noted by using spectrophotometer at 595 nm and the results were expressed in mg g-1 dry weight through the calibration curve with bovine serum albumin. 3.10.3) Determination of total carbohydrate content: Estimation of carbohydrate content was carried out by the method given [35]. 5 g of powdered sample was homogenized in 4 ml of 80% ethanol and the total volume was made up to 25 ml this solution was subjected to a centrifuge for 10 min at 2500 rpm and supernatant solution was used for analysis. Suitably diluted aliquot was mixed with 4.5 ml of freshly prepared anthrone reagent and heated for 10 min. the absorbance was noted at 620 nm and total carbohydrate content was calculated with the help of a calibration curve with glucose and expressed as mg g -1 dry weight of samples. 3.11.) Isolation and HPLC analysis of phenolic compounds M. oleifera seeds dry powder (500g) was exhausted in a reflux apparatus with methanol: water 7:3 v/v (500×1L) solvent system at 50 0 C for 5×3 hrs. The extract was filtered and concentrated under vacuum at 45 0 C. This extract was subjected to partition thrice with hexane for the removal of photosynthetic pigments and lipid substances. Remaining methanolic phase was concentrated in vacuum with silica gel 60-120 mesh size to make silica slurry and stored until use. 100gm of free flowing extract with silica slurry was eluted with gradient of dichloromethane/methanol (100:00, 95:5, 90:10, 85:15. 80:20, 75:25, 50:50) and with pure methanol (100ml each) to afford 55 fractions. After TLC analysis with DCM: Methanol (9:1) the column fractions were pooled in five groups) MOF 1-5). These pooled fractions were analyzed by sub columns and visualized on TLC plates in UV chamber. HPLC analyses were performed with Waters Alliance 2695 separations maintained at 25 0 C with PDA detector. Test samples (20 μl) were injected into a reverse phase column C18 (250 mm length, 4.6 mm and 5μm). The samples were dissolved in methanol and filtered with 0.45 μm nylon filters. 1ml/min flow rate, 254 nm λ, mobile phase A 0.05% TFA in ACN and B 0.05% TFA in water were used as chromatographic conditions. Gradient elution (T/A %) from 0/5, 30/25, 40/40, 50/63, 60/5 and 66/5 and 20μg/20 μl concentration of samples were used. The compositions of the mobile phase were rising over the period of 20 minutes. Statistical analysis Data recorded from the experiments are expressed as mean ± standard deviation of triplicate determination (n = 3).All the statistical analysis were accomplished by using graph pad prism10.4.2 software. The significant differences among treatments in pot experiment and antioxidant potential were determined by Tukey-Kramer HSD test at p = 0.05. Dunnett’s post hoc test was used for comparative analysis in zone of inhibition at p = 0.05. Results 5.1) Total Phenol Content It was reported that the antioxidant activity of plant extracts is well correlated with the content of their phenolic compounds. Total phenolic content of the extracts was calculated using the standard curve of gallic acid. Phenol molecules possess hydroxyl group (-OH) making them polar. This polarity allows them to dissolve readily in polar solvents. Total phenolic content was found in the bark extract (56.10 ± 2.38 GAE /g) followed by seeds (42.00 ± 2.65 GAE /g), leaves (33.13 ± 0.50 GAE/g) and flower (13.6 ± 1.32 GAE/g). The lowest total phenolic content (5.63 ± 0.25 GAE/g) was obtained from gum extract (Fig. 2 ). Results indicated that bark and seed extract contained a higher amount of phenols as compared to other extracts. 5.2) DPPH radical scavenging capacity: In the present study the extracts of M. oleifera plant parts were investigated for the antioxidant activity using 2,2-diphenyl − 1picrylhydrazyl (DPPH) free radical scavenging assay. The antioxidant present in plant extract neutralizes the DPPH by the transfer of an electron or hydrogen atom. The methenolic extracts of different plant parts showed varying scavenging ability. Among these five, bark extract showed highest free radical scavenging effect (FRS) (74.70 ± 1.95 followed by seed extracts (70.39 ± 0.07), leaf extract (54.63 ± 0.39 and flower extract (42.40 ± 0.30) (Fig. 2 ). Again lowest DPPH activity was observed in gum extract (10.61 ± 1.50). 5.3) FRAP analysis: FRAP analysis was performed with a similar concentration used in DPPH activity by using the standard curve. Seed extract displayed highest activity with 168.61 µM Fe + 2 /g, followed by bark extract 162.11 µM Fe + 2 /g and leaves extract 124.25 µM Fe + 2 /g (Fig. 2 ). However, flower and gum extract did not show any significant result. 5.4) Isolation, morphological identification of test bacterium The bacteria were isolated from naturally infected leaves of soybean on Letheen agar media after 72 h of inoculation. Single semitransparent gummy, circular pale yellow colonies were observed. When this purified bacterium inoculated on soybean plant leaves showed typical pustules symptoms as pale yellow spots near the interveinal area which become large brown patches on leaves. All the inoculated plants yielded the same bacteria on reisolation within 15–17 days after inoculation with circular light yellowish brown spots on leaves (Fig. 3 ). Isolated bacteria were rod shaped (0.9×1.6 µm), gram negative, capsulated with single polar flagella. These observations of morphological parameters indicated its similarity with the genus Xag . 5.5) Biochemical characterization of test organism The isolated bacterium showed growth between 12-37 0 C, pH 5–8.0, and NaCl concentration at 2.5–4.5%. It was KOH soluble and negative for urea hydrolysis. The bacteria did not utilize the carbohydrate but derived energy and produced acid from fructose, mannose, sucrose, dextrose and manitol. Based on biochemical characteristics the isolates were identified as Xanthomonas axonopodis and based on host range and specificity of the bacterium for soybean it was related to variant glycine . 5.6) in vitro Antibacterial activity by different extracts of M.oleifera The agar well plate method is widely used to evaluate the antimicrobial activity of different extracts. Contact and diffusion of the extract into the media is better in the well plate method Evaluation of the antibacterial activity of M. oleifera plant extracts against plant pathogenic bacteria Xag was illustrated in (Fig. 4 ). The results revealed that plant extracts were potentially effective in suppressing microbial growth of Xag in sequential manner, when the concentration of extract increased, the zone of inhibition also increased (Fig. 5 ). Therefore it showed bigger sized inhibition zone. Each well is of the diameter. The results were recorded after 24 hours. Seed extract was the most effective extract for retarding microbial growth of pathogenic bacteria at 250µg/500µL concentration, showing an 18.86 mm zone of inhibition, followed by bark extract at 300 µg/500µL (18.16 mm) flower extract 300µg/500µL (15.36), leaves extract at 300µg/500µL (15.26mm) and gum extract (5.24 mm) (Fig. 4 ). It was observed that methanol extract of leaves and flowers were slightly less active against test organism. However, reference plate of streptomycin is showed wider zone of inhibition as compared to seed and bark extract. 5.7) Biochemical analysis 5.7.1) Chlorophyll and carotenoids content The result of preventive effect of seed extracts on chlorophyll and carotenoid contents summarized in (Table 1 ). Data revealed that seed extract was positively correlated with chlorophyll and carotenoid content. The maximum chlorophyll content was observed in SE4 (1.98 mg/g) followed by SE5 (1.96 mg/g), SE3 (1.66 mg/g) and SE1 (1.22 mg/g) as compared to STRP (1.81 mg/g). However it was lower than healthy NT plants (2.12mg/g) but higher than NC (1.13mg/g). Similar results were observed in Chlorphyll a and b content. Highest weight was observed in SE4 (1.19 mg/g, 0.84 mg/g) followed by SE5 (1.14 mg/g, 0.82 mg/g), SE3 (1.01 mg/g, 0.76 mg/g), SE2 (0.97 mg/g, 0.72) and SE 1(0.91 mg/g, 0.67). In control groups Ch a, b content were significantly different, where NT showed highest content (1.33 mg/g, 0.97 mg/g) and NC showed lowest (0.88mg/g, 0.59 mg/g). While in carotenoids content extracts results were positively correlated with it. SE4 showed highest carotenoids (0.98 mg/g) followed by SE5 (0.97 mg/g), SE3 (0.89 mg/g), SE2 (0.81 mg/g) and SE1 (0.74 mg/g). As compared to STRP (0.94 mg/g) SE5 and SE4 were significant. 5.7.2) Protein and carbohydrate content The result of the effect of seed extract prepared from M. oleifera on biochemical parameters i.e. carbohydrate and protein content of Soybean is summarized in (Table 1 ). Data clearly indicated that seed extract increases carbohydrate and protein content of the host plant. The maximum carbohydrate content was observed in SE4 (4.022 mg/g) followed by SE5 (4.01 mg/g), SE3 (3.34 mg/g), SE 2(3.16 mg/g) and SE 1(3.06 mg/g) except NC (2.61mg/g). For carbohydrate streptomycin (3.94 mg/g) and healthy plant (5.08 mg/g) was observed, which were significant. Similarly, protein content was increased as the concentration of seed extract increased. The highest protein content was observed in SE 4 (9.81 mg/g) followed by SE5 (9.24 mg/g), SE3 (7.76mg/g), SE 2(5.48mg/g) and SE 1(4.05 mg/g). All the treatments are significant as compared to streptomycin (8.43 mg/g) and healthy plant (13.95 mg/g) for protein except NC (1.67 mg/g). 5.8) Plant height and leaf per plant Results of the effect of seed extracts on number of leaves per plant are given in (Table 1 Fig. 6 ). The data indicated that there was slight increment in the number of leaves as extract concentration increases. The maximum increase in the number of leaves/ plant was observed in treatments with SE 4 (24.33) followed by SE 5 (21.67), SE 3 (19.33), SE 2 (16.33) and SE 1(15.00). On the other hand data given in (Table 1 ) indicated that there was significant increment in the plant height due to treatment of seed extracts. The maximum increase in plant height was observed in SE4 (50.33 cm.) followed by SE5 (50.18 cm.), SE3 (49.95 cm.), SE2 (49.88 cm.) and SE1 (48.76 cm.). However seedling length and growth were found in SE4 and STRP which indicated that SE4 had a good capacity to influence seedling growth as compared to other treatments. All the treatments are significant as compared to both control groups except NC (48.33 cm.) for both parameters plant height and leaf per plant as shown in (Table 1 ). Table 1 Estimation of growth parameters (leaf per plant and plant height) and biochemical parameters (Chlorophyll a, b, carotenoids, protein and carbohydrate) in soybean by different concentrations of seed extract against bacterial pustule disease. T Leaf/plant Plant height (cm) Carbohydrate (mg/g) Chlorophyll and carotenoids (mg/g) fresh weight Total ch Cha Chb carotenoids Protein (mg/g) SE1 15.00 ± 1.00 e 48.76 ± 0.56 d 3.06 ± 0.13 d 1.22 ± 0.04 ef 0.91 ± 0.06 b 0.67 ± 0.22 c 0.74 ± 0.20 c 4.05 ± 0.44 f SE2 16.33 ± 1.15 de 49.88 ± 0.06 c 3.16 ± 0.02 d 1.41 ± 0.17 de 0.97 ± 0.13 b 0.72 ± 0.13 ab 0.81 ± 0.06 b 5.48 ± 0.44 e SE3 19.33 ± 0.58 cd 49.95 ± 0.07 bc 3.34 ± 0.03 c 1.66 ± 0.09 cd 1.01 ± 0.15 ab 0.76 ± 0.09 ab 0.89 ± 0.17 ab 7.76 ± 0.59 d SE4 24.33 ± 1.15 ab 50.33 ± 0.05 bc 4.02 ± 0.09 b 1.98 ± 0.03 ab 1.19 ± 0.19 ab 0.84 ± 0.07 b 0.98 ± 0.15 a 9.81 ± 0.22 b SE5 21.67 ± 1.15 bc 50.18 ± 0.04 bc 4.01 ± 0.09 b 1.96 ± 0.12 ab 1.14 ± 0.06 ab 0.82 ± 0.03 ab 0.97 ± 0.09 a 9.24 ± 0.50 bc STRP 24.67 ± 1.53 ab 50.49 ± 0.05 b 3.94 ± 0.04 b 1.81 ± 0.01 bc 1.11 ± 0.12 ab 0.81 ± 0.01 ab 0.94 ± 0.06 ab 8.43 ± 0.29 cd NT 26.00 ± 1.00 a 51.84 ± 0.06 a 5.08 ± 0.03 a 2.12 ± 0.03 a 1.33 ± 0.11 a 0.97 ± 0.20 a 1.01 ± 0.20 a 13.95 ± 0.36 a NC 13.33 ± 1.53 e 48.33 ± 0.51 d 2.61 ± 0.15 e 1.13 ± 0.13 f 0.88 ± 0.08 cd 0.59 ± 0.11 d 0.64 ± 0.16 d 1.67 ± 0.44 g SE (1–5) seed extract concentration µg/500µl, NT (non treated), STRP (streptomycin) and NC (negative control). The results are expressed as mean ± SD of three replicates. Different significantly different letters (P < 0.05) as determined by Tukey-Kramer HSD test 5.9) Isolation of phenolic compounds and HPLC analysis In order to obtain the antibacterial phenolic compounds from the seeds of M. oleifera repeated column chromatography and TLC were performed. For their purity HPLC analysis was used which revealed the presence of three major phenolic compounds. The fraction number 2 give quercetine (MOF 2.3.1 with solvent ratio toluene: ethyl acetate: formic acid 5:4:1). P-coumaric acid and cinnamic acid were isolated from fraction 5.4 and 5.7.Cinnamic acid was eluted with ethyl acetate: n hexane (70:30) and p coumaric acid was isolated with the solvent system of chloroform:ethyl acetate (60:40).HPLC showed single compound profile with highest peak area percentage of quercetin at 16.34 RT, p coumaric acid 13.18 RT and cinnamic acid 17.20 RT (Fig. 7 ). Among all three compounds peak area percentage of cinnamic acid was highest 99.13%. Remaining two phenolic compounds also had good percentage area in which p coumaric acid had 98.04% and quercetin had 90.67%. However in quercetin minor peaks also observed indicated that impurity in isolated quercetin at certain level. Discussion Worldwide, soybean is an essential food crop but it is susceptible to different microbial diseases. Bacterial pustule disease is one of them, which causes severe damage to its yield and overall production [ 13 ]. Disease management by different commercially available chemicals can control the severity but there are several negative impacts on soybean crop physiologically and morphologically and it also affects the quality of seeds. Entering in food chain is another concern, as it damages other populations as well as the environment. In the current examination we report for the first time the efficacy of M. oreifera plant to pursue the defense reaction against bacterial pustules disease in soybean and regulate sustainable plant growth. Several reports are available related to plant based natural and integrated approaches for bacterial disease control and management [ 36 – 38 ]. In the present study, M. oleifera seed extract was recorded in both the pot experiment and agar well plate method against the disease. Among all the treatments, seed extract was recorded with significant in vitro and in vivo control of pustule disease in soybean. The defense response of seed extract might be due to the presence of phenolic compounds which can stimulate plant immune response by enhancing its antioxidant defense enzyme system and contribute to their resilience and adaptability towards environmental challenges including microbial stress. Seeds of M. oleifera contain a wide array of beneficial phenolic compounds such as gallic acid, ferulic acid, caffeic acid, catechin, epicatechin, vanillin, quercetin, cinnamic acid, p-coumaric acid etc.[ 39 ] and function as signaling compounds that trigger the development of systemic acquired resistance (SAR) and activation of defense genes. In the present study three phenolic molecules Quercetin, cinnamic acid and p coumaric acid were successfully isolated from the seeds of M. oleifera . Many studies suggests that these compounds inhibited bacterial growth specially quercetin exhibited antibacterial potential against Xanthomonas axonopodis pv. citri [ 40 ]. In other study p coumaric acid induced phenolic metabolites alleviates the disease symptoms caused by Xanthomonas campestris pv. campestris in cabbage [ 41 ]. In peach tree bacterial shot hole disease is a global concern caused by pathogen Xanthomonas arboricolapv. pruni. this bacteria was effectively controlled and inhibited by the derivatives of cinnamic acid [ 42 ]. They also contains phenylpropenoids such as 4-hydroxyphenyl acetate, 3- hydroxyl-1-(4-hydroxy-3-methoxyphenyl)-1-propanone, 3-hydroxy-1-(4-hydroxyphenyl)-propyl-1-one, erythro-guaiacylg-lycerol and threo-guaia-cylglycerol [ 43 ]. They can be involved in the synthesis of cell wall strengthening compounds, such as lignin and suberin so it is positively correlated with fortifying plant core structure against environmental stress [ 44 ]. They also function as signaling compounds that trigger the development of systemic acquired resistance (SAR) and activation of defense genes [ 45 ]. Glucosinolates and its derivative byproduct (isothiocyanates) also present in the seeds of M. oliefera , can act as antibacterials. While it is not influenced photosynthesis directly but indirectly through their impact on plant physiology and stress response [ 46 ]. It also influences the growth promoting consequences like transcription and translation processes and prevents bacterial growth. Plant based extracts and products considerably enriched the growth and development of soybean in pot experiments as compared to streptomycin treated plants. However, streptomycin treatment also showed good results in agar well plate method but in pot experiment protein, chlorophyll and carbohydrate quantities were low as compared to non treated (NT) healthy plants and SE 4, which indicated that streptomycin negatively impacted the plant own defense system, growth, post germinative development and weakening the plant overall defense capacity. Whereas increased sugar content in treated plants especially in SE4 can be directly correlated with enhanced photosynthesis. Because Reactive oxygen species are byproducts of photosynthesis and phenolic compounds can help in regulating ROS levels, potentially influencing photosynthesis efficiency [ 47 – 49 ]. Many researchers have used various kinds of bioformulations, nanoformulations and PGPR based formulations but these approaches are very costly, time consuming. Therefore these antimicrobial rich seed extracts can act as environmental friendly by eliminating phytopathogenic microbes specially Xag. Such sustainable approach has the capacity to revolutionize agriculture and reducing reliability on chemical pesticides. Conclusion M.oleifera has been reported to produce a large number of primary and secondary metabolites. In our study higher total phenol content and antioxidant activities were obtained, which showed antibacterial activity in both in in vivo and in vitro . Based on these results, it could be concluded that seed extract of M. oleifera showed antibacterial activity as it contain different bioactive molecules including quercetin, cinnamic acid and p coumaric acid against Xag . It can be used against the management of bacterial pustules of soybean. However, it is important to isolate and identify other compounds except these one through spectroscopic methods like HPLC, NMR responsible for the antimicrobial activity as well as to establish the mechanism of action of the extract and its bioactive molecules to come to a definite conclusion. Since demand for food free from synthetic constituents has exponentially increased in the last few decades, an herbal approach is imperative to be amended to reinforce the immunity of plants to survive and for sustained plant growth. Declarations Conflict of interest: The authors declare that there is no conflict of interest. Clinical trial, ethics approval and consent to participate The manuscript does not contain any studies with human participants or animals performed by any of the authors. Consent for publication All authors read and approved the final manuscript. Data availability All data underlying the results are available as part of the article and no additional source data are required. Author’s Contributions : Conceptualization, F.K. and J.A.; methodology, F.K., A.T. and J.A.; investigation, F.K., A.T. and J.A.; resources, F.K., B.K. and J.A.; data curation, F.K., A.J. and J.A.; writing—original draft, F.K., A.T. and J.A.; writing—review F.K., A.J., B.K., and J.A.; visualization, F.K. and J.A.; supervision, J.A. All figures have been made by F.K; Revision, final update and editing, R.K., F.K. and J.A. All authors have read and agreed to the published version of the manuscript. 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Bar diagrams represent the mean±SD for three replicates of each extract followed by different significantly different letters (P\u0026lt;0.05) as determined by Tukey-Kramer HSD test\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2moallgraphbiochemicalfinal.png","url":"https://assets-eu.researchsquare.com/files/rs-7550249/v1/87fb65d544698b3a74fcc4da.png"},{"id":93944343,"identity":"c6a24285-5a7c-4cf7-a258-f231aad553f7","added_by":"auto","created_at":"2025-10-20 14:07:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4507241,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIsolation of inoculum (A) Infected leaves collected from soybean field (B)Pure \u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eXag\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e colony (red arrow) on Letheen agar media (C) Disease symptoms on leaves in pot experiment (D) \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eXanthomonas\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e colony observed again on media from inoculated plant (red arrow denotes pure colonies isolated from the infected part of soybean leaves).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.isolationofinoculum.png","url":"https://assets-eu.researchsquare.com/files/rs-7550249/v1/534e8a3f386e6a7733b76573.png"},{"id":93944356,"identity":"60df1d79-4601-4946-b227-84273ed1a25d","added_by":"auto","created_at":"2025-10-20 14:07:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3997858,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZone of inhibition for the test organism \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eXag\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e by different extracts of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM.oleifera\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.zoneofinhibition.png","url":"https://assets-eu.researchsquare.com/files/rs-7550249/v1/c9da6c53946fd918a2b72fbd.png"},{"id":93945888,"identity":"d66e1fc8-1916-4a9e-ab02-37e7dd5af614","added_by":"auto","created_at":"2025-10-20 14:23:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":197335,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBar diagrams represents the effect of different \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. oleifera\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e plant part extracts against \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eX. pv. glycine\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ein agar well plate method. Bar diagrams represent the mean±SD for three replicates of each extract followed by different asterisks showed comparative significant difference (p\u0026lt;0.05) as determined by Dunnett’s post hoc test. Streptomycin (STRP), B (bark), F (flower), L (leaves), G (gum), S (seeds)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5zoiallgraphmergfinal.png","url":"https://assets-eu.researchsquare.com/files/rs-7550249/v1/97386a91d4f00d44fca2cae9.png"},{"id":93944324,"identity":"9db7c84a-3387-4a00-b2e4-d019bbfd6327","added_by":"auto","created_at":"2025-10-20 14:07:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":9085496,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePotential efficiency of seed extract against pustules disease of soybean in pot experiment [B] and [A] control group [(healthy plant (NT) and streptomycin treated plant as positive control and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eXag\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e inoculated plant as negative control (NC)]\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7potexperiment.png","url":"https://assets-eu.researchsquare.com/files/rs-7550249/v1/05c4e6a882c17908281cd47a.png"},{"id":93944322,"identity":"bf9dc4f3-9908-429f-a3a6-15d604f423a3","added_by":"auto","created_at":"2025-10-20 14:07:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1630357,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHPLC chromatograms of isolated phenolic compounds from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM.oleifera\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e seed extract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6moringahplcjpg.png","url":"https://assets-eu.researchsquare.com/files/rs-7550249/v1/8f88c590eb65c8270548a4df.png"},{"id":93949057,"identity":"04aa8454-a299-4f90-b697-bd2eb629ece0","added_by":"auto","created_at":"2025-10-20 14:39:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":24420133,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7550249/v1/b9911278-1173-4268-ac7a-081b8a808d49.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Investigation of the antibacterial properties of Moringa oleifera Extracts against Xanthomonas axonopoidespv. glycines the incitant of Bacterial Pustules of Glycine max","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSustainable food production for a rapidly growing human population is one of the major challenges faced by the agriculture sector globally [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].Therefore, the increased uses of pesticides and synthetic agrochemicals have become essential to maximize agricultural productivity. Despite their beneficial role in agriculture, they can be hazardous to humans and other non-targeted organisms [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. According to the Food and Agriculture Organization of the United Nations, an estimated 3.2\u0026nbsp;million tones of pesticides are used on crops each year [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In recent years, unrestrained use of commercial and synthetic agrochemicals in protection of crop has raised significant worry about environmental contamination and resistance enhancement in phytopathogenic microbes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. To address these issues, the development of bio-based/non-synthetic biocides for agriculture has become an important research direction.\u003c/p\u003e\u003cp\u003eSoybean [\u003cem\u003eGlycine max\u003c/em\u003e (L.)] is significant and major pulse crop worldwide that delivers two-third of calories derived from agriculture [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and accounts for half of the global demand for oil and vegetable protein. It is high in edible oil (18\u0026ndash;22%) with major essential amino acids and proteins (38\u0026ndash;42%). Its oil and food make up about 85% of the global soybean edibles including soy milk, tofu, soy sauce and tempeh [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Federation of Indian Chamber of Commerce and Industry (FICCI) claims that India currently produces 25% of its oil and 43% of its oilseeds from soybean, making it the fourth-largest producer of soybeans in the world, behind the United States, Brazil, Argentina and China [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite all these aspects, soybean yield in India currently stands at around 1T/ha (minimum) to 3.5t/ha (maximum), there is still an urgent need to increase production to meet the demand of ever increasing population [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Because it is a rainfed crop, it is highly dependent on monsoon rains. However, bacterial pustule disease caused by a gram negative bacterium Xag severely damage the soybean crops, it is among the most serious diseases that reduces the yield and quality of the crop [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. It limits the soybean production worldwide by 20\u0026ndash;40%. This disease runs rampant in most soybean-growing fields and may cause yield loss as high as 53% under favorable conditions [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Xag may enter soybean leaves via natural openings such as stomata and wounds and proliferate in intercellular spaces [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Typical symptoms are small, light yellow colored pustules surrounded by chlorotic hallows on the underside of soybean leaves [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The pustules spots range in size from small brown patches to large. Numerous management strategies, including the use of synthetic agrochemicals, commercial antibiotics and nanoformulations, have been studied [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] but have side effects on overall plant growth and development. Therefore environment-friendly strategies and plant based products for disease management is necessary. Some studies on biocontrol investigations on stem rot by the fungus \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e, root rot by the fungus \u003cem\u003ePhytophthora sojae\u003c/em\u003e and cyst by the nematode \u003cem\u003eHeterodera glycines\u003c/em\u003e were evaluated in previous years [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], also bio based formulations were investigated [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] but plant based disease prevention and management studies of bacterial pustules disease of soybean have not been identified yet.\u003c/p\u003e\u003cp\u003e\u003cem\u003eM. oleifera\u003c/em\u003e Lam. (Family: Moringaceae, commonly known as horseradish, drumstick, ben oil, miracle, and Mother\u0026rsquo;s best friend), one of the 13 species of the monogeneric family Moringaceae, native to the sub-Himalayan lowlands in northwest India, shows remarkable medicinal potential [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Traditionally, \u003cem\u003eM. oleifera\u003c/em\u003e has been associated with various medicinal benefits, including pain relief, controlling blood sugar levels, liver protection, anti-inflammatory effects, promotion of urination, reduction of pain sensitivity, prevention of artery hardening, blood pressure regulation, antioxidant properties and tumor growth inhibition [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Different phytochemicals like phenols, omega fatty acids, flavonoids, carotenoids, kaempferol, terpenoids, tocopherols, tannins, vanillin, glycosides, β-sitosterol, triterpenoids, ascorbates, quercetin and sterols have been isolated from the flowers, leaves, roots, seeds and fruits [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In particular, this plant family is rich in compounds containing the simple sugar rhamnose and it is rich in a fairly unique group of compounds called glucosinolates (β-thioglucoside\u003cem\u003eN\u003c/em\u003e-hydroxysulfates) and isothiocyanates(4-α-L-rhamnopyranosyloxy)benzyl isothiocyanate [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAbout 20% of all plants have undergone biological and pharmacological testing and a significant number of new antibiotics introduced in agriculture sector are from natural or semi-synthetic resources. For this, In the present investigation, we report for the first time the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e efficacy of different plant parts methanolic extract against \u003cem\u003eXanthomona saxonopoidis pv. glycine\u003c/em\u003e along with the antibiotic streptomycine which is widely used for controlling this disease in soybean field. Our results convincingly establish effective control of bacterial pustule disease of soybean.\u003c/p\u003e"},{"header":"Materials and Method","content":"\u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003cstrong\u003e.1) Chemicals:\u0026nbsp;\u003c/strong\u003eFolin\u0026ndash;Ciolcateu\u0026rsquo;s reagent, diphenyl-1-picrylhydrazyl-(DPPH), sodium bicarbonate(Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e), HCl, TPTZ (2,4,6-tri[2-pyridyl]-s-triazine), ferric chloride (FeCl\u003csub\u003e3\u003c/sub\u003e), methanol, gallic acid, acetic acid, sodium acetate, nutrient broth (modified), Letheen agar (modified), JS 27 certified seeds of soybean and Streptomycin were of analytical grade purchased from Himedia, India.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2)\u003c/strong\u003e \u003cstrong\u003eCollection of plant material and preparation of extract:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe leaves, flowers, seeds, gum and bark of \u003cem\u003eM. oleifera\u0026nbsp;\u003c/em\u003ewere collected from\u0026nbsp;the University College of Science campus MLSU\u0026nbsp;Udaipur, Rajasthan. The plant was identified on the basis of herbarium sheet by Botanical Survey of India (BSI), Jodhupur, India (Vide letter Number: BSI/AZRC/I.12012/Tech./2021-22 (PI.-Id.)/412 dated: 14-12-2021).\u003c/p\u003e\n\u003cp\u003eDifferent plant parts (Fig. 1) except gum were washed thoroughly with sterile water, then shade dried at room temperature and ground to coarse powder by using a blender. The powdered material was kept in airtight jars until further experiment. 50g dried powder of each\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eplant part were extracted with methanol (50\u003cstrong\u003e\u0026times;\u003c/strong\u003e250ml) using a soxhlet apparatus. While gum was kept in methanol (5\u003cstrong\u003e\u0026times;\u003c/strong\u003e25) for 4 days at room temperature. The extract was filtered through a membrane filter by using Whatman No. 1 filter paper. The filtrate was reduced through a rotary vacuum evaporator at 50\u003csup\u003e0\u0026nbsp;\u003c/sup\u003eC, 80 rpm to obtain the concentrated extract (Fig. 1), which was then frozen and freeze-dried at -20\u003csup\u003e0\u0026nbsp;\u003c/sup\u003eC until analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3) Total phenolic content:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal phenolic content of \u003cem\u003eM. oleifera\u003c/em\u003e different extracts was determined by the Folin-Ciocalteu method [26]. 2mg of each extract was dissolved in 1ml methanol. The freshly prepared Folin- Ciocalteu reagent (2.5 ml) was mixed with 500 \u0026mu;l of extract, then 400 \u0026mu;l of sodium bicarbonate was added and kept for 1 min in a boiling water bath. For calibration curve gallic acid (30-300 \u0026mu;g/ml) was used and data was recorded at 650 nm. TPC was expressed as mg gallic acid equivalent/g DW of extract.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4)\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Antioxidant activity:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.1) DPPH radical scavenging capacity:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis antioxidant activity was calculated by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) [27]\u003cem\u003e.\u0026nbsp;\u003c/em\u003eDifferent extracts of \u003cem\u003eM. oleifera\u003c/em\u003e (2mg/100\u0026micro;l) each were added to the reaction mixture having 1 ml of DPPH and 1.5 ml ethanol and 500 \u0026mu;l acetate buffer (pH 5.5). This reaction mixture was kept in a dark place at room temperature. After half an hour, the absorbance was measured at 517 nm. Methanol served as the blank. The radical scavenging activity was expressed in percentage using the equation.\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/58895_8739fc6c57c1c19a/58895_custom_files/img1760968694.png\" width=\"583\" height=\"102\"\u003e\u003c/p\u003e\n\u003cp\u003eWhere A\u003csub\u003es\u003c/sub\u003e= absorbance of sample solution, A\u003csub\u003ec\u003c/sub\u003e= absorbance of control\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.4.2) FRAP analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ferric reducing power of different extracts was determined by the method given by [28]. The FRAP reagent was incubated for 30min at 37\u003csup\u003e0\u003c/sup\u003eC in water bath. 100 \u0026mu;l extract was added to FRAP reagent (3ml) and shake vigorously. Absorbance was measured at 593 nm using spectrophotometer. Results were expressed in \u0026mu;M Fe\u003csup\u003e2+\u003c/sup\u003e/G and FeSO4.7H\u003csub\u003e2\u003c/sub\u003eO (100-1000 \u0026mu;g/ml) was used as a standard for calibration curve.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5)\u003c/strong\u003e \u003cstrong\u003eIsolation and purification of \u003cem\u003eX.\u003c/em\u003e \u003cem\u003eanopodis\u0026nbsp;\u003c/em\u003epv. \u003cem\u003eGlycine\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe pathogen was isolated from the infected soybean leaves showing typical disease symptoms collected from soybean fields of\u003cem\u003e\u0026nbsp;\u003c/em\u003eRCA, MPUAT, Udaipur, Rajasthan during 2023. The yellowish brown infected portion of the leaves was subjected for the isolation of the pathogen (Fig. 3) on Leethen agar media incubated at 29\u003csup\u003e0\u003c/sup\u003eC\u003cspan dir=\"RTL\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e2\u003csup\u003e0\u003c/sup\u003eC. The colony developed on the periphery was streaked on fresh media\u0026nbsp;[29]. Pure cultures were established by single colony isolation and maintained in refrigerator at 5\u003csup\u003e0\u003c/sup\u003eC by sealing 48 h old cultures on media.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6) Pathogenecity and host range\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacterium pathogenicity was confirmed on 30 days old soybean plants grown in (25cm) pots in a glasshouse. The plants were kept in humid chamber (30\u0026plusmn;1\u003csup\u003e0\u003c/sup\u003eC, 80-90% RH), 24 h prior to inoculation for stomata opening and to optimize high intercellular humidity in the surrounding tissues of natural openings. 48 h old bacterial culture was gently scrapped from slants and suspended in DDW. An inoculation (2\u0026times;108 cfu/ml) was made by spray and pinprick to runoff the methods. When the disease symptoms occur (Fig. 3) the pathogen was again isolated from the inoculated plant and compared with the original culture. Suitable controls were maintained, and pathogenicity was proved by following Koch\u0026rsquo;s postulates.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7) Morphological and biochemical characterization of bacterium\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe isolated bacterium was examined for cell flagellation, Gram staining, KOH solubility and catalase. Methyl red test, urease, and growth in NaCl concentration were also tested [30].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.8)\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Antibacterial activity:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntibacterial activity of different extracts from \u003cem\u003eM. oleifera\u003c/em\u003e was evaluated by the agar well diffusion method with slight modifications given by [31].\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eActive cultures were prepared by transferring a loop full of cells from the stock cultures to test tubes containing nutrient broth for bacteria that were incubated at 24 hrs at 29\u003csup\u003e0\u003c/sup\u003eC \u0026plusmn; 2\u003csup\u003e0\u003c/sup\u003eC. To prepare the samples for antibacterial assay 50-300 \u0026mu;g/500 \u0026mu;l of each extract was dissolved in 500 \u0026mu;L methanol and sonicated for 30 minutes. Pure methanol was used as a negative control and standard antibiotic; streptomycin (30\u0026mu;g/ml) was used as a positive control. Petri plates containing 20 ml Letheen agar medium were seeded with 24hr old culture of bacterial strains. 100\u0026mu;l of each test solutions was poured in the respective wells and incubated for 24 hrs at 29\u003csup\u003e0\u003c/sup\u003eC\u0026plusmn;2\u003csup\u003e0\u003c/sup\u003eC. At the end of the incubation period (24 hrs.) The antimicrobials present in the extract are allowed to diffuse out into the medium and interact in a plate freshly seeded with the test organism. The resulting diameter of the zone of inhibition was measured in millimeters. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.9) pot experiment:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJS27 non-resistant variety of soybeans acquired from an authorized local vendor at Krishi Kendra, Udaipur, Rajasthan was used for plantation. The seeds were sown in pots with autoclaved sandy soil in late July and were allowed to grow under ambient atmospheric conditions. A total of eight pots were prepared for experimentation, with five allocated for assessing the impact of seed extract treatment, while the remaining three served as positive control (streptomycin), diseased plant negative control (NC) and non treated (NT) healthy plants. While bark extract also showed significant inhibition in agar well plate method but it was not considered in \u003cem\u003ein-vivo\u003c/em\u003e experiment because it was partially dissolved in solvent both water and methanol and a thin extract layer was observed on leaves and seeds, so the penetration of extract was blocked, and it clogged the natural openings. Before sowing, the seeds were soaked in broth containing the pathogen for thirty minutes. Subsequent to sowing, the soil was inoculated with the pathogen by spraying 10 ml water-suspended bacterial solution to ensure timely disease development. The plant was allowed to grow for 10 days above the surface without further inoculation. Upon achieving a decent growth, approximately 35 days after sowing the plants were artificially inoculated with 24-hour-old culture of Xag for two consecutive days [32]. Disease manifestation was observed in ten to twelve days. Following the onset, different concentrations of \u003cem\u003eM.oleifera\u003c/em\u003e seed extract (100-300 \u0026mu;g/500 \u0026mu;l) were sprayed on the potted plants after 48 hrs of the second foliar spray. Also biochemical parameters were conducted after second foliar spray. Plant height and leaf per plant were observed and recorded after 10 days of last spray. Three replications per concentration were maintained and for each replication 4 plants per pots were placed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.10) Biochemical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.10.1) Chlorophyll and carotenoid\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe quantitative estimation of chlorophyll and carotenoid was performed by using the method of [33],\u0026nbsp;fresh samples from healthy and inoculated plant leaves were collected after 10 d of inoculation from green house. 1 g of plant leaves was chopped and homogenized to a fine pulp by using 85% acetone for 5 min and centrifuged for 10 min at 2000 rpm. The supernatant was made up to 30 ml with 80% acetone; aqueous acetone is used as blank. The absorbance was measured at 665, 663 and 450 nm by using a spectrophotometer. Chlorophyll a, b and total chlorophyll content were calculated by these formulas:\u003c/p\u003e\n\u003cp\u003ea.) Total chlorophyll (mg/g) = (20.2A645+8.02 A663\u0026times;V)\u003c/p\u003e\n\u003cp\u003eb.) Chlorophyll a (mg/g) = (12.7A663-2.69A645\u0026times;V)/AC1000\u0026times;W\u003c/p\u003e\n\u003cp\u003ec.) Chlorophyll b (mg/g) = (22.7 A645+4.68 A663\u0026times;V)/ A\u0026times;1000\u0026times;W\u003c/p\u003e\n\u003cp\u003ed.)Carotenoids (mg/g tissue) = (10A450\u0026times;V\u0026times;F)/2500\u0026times;W\u003c/p\u003e\n\u003cp\u003eWhere, A (absorbance) nm in a 1 cm cell, V= volume of extract in ml, W= fresh weight of the sample, F= dilution factor\u003c/p\u003e\n\u003cp\u003e3.10.2) \u003cstrong\u003eDetermination of soluble protein content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSoluble proteins were estimated by the method given by [34] using bovine serum albumin as a standard.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e1g of fresh leaves was ground in 5 ml chilled phosphate buffer (0.1M, pH 7.5) and centrifuged at 12000 rpm for 15 minutes. Supernatant was used for analysis. 500\u0026nbsp;\u0026mu;l extract was added to Coomassien brilliant blue reagent and vortexed for 30 seconds after color development absorbance was noted by using spectrophotometer at 595 nm and the results were expressed in mg g-1 dry weight through the calibration curve with bovine serum albumin.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.10.3) Determination of total carbohydrate content:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEstimation of carbohydrate content was carried out by the method given [35]. 5 g of powdered sample was homogenized in 4 ml of 80% ethanol and the total volume was made up to 25 ml this solution was subjected to a centrifuge for 10 min at 2500 rpm and supernatant solution was used for analysis. Suitably diluted aliquot was mixed with 4.5 ml of freshly prepared anthrone reagent and heated for 10 min. the absorbance was noted at 620 nm and total carbohydrate content was calculated with the help of a calibration curve with glucose and expressed as mg g\u003csup\u003e-1\u003c/sup\u003edry weight of samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.11.) Isolation and HPLC analysis of phenolic compounds\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eM. oleifera\u003c/em\u003e seeds dry powder (500g) was exhausted in a reflux apparatus with methanol: water 7:3 v/v (500\u0026times;1L) solvent system at 50\u003csup\u003e0\u003c/sup\u003eC for 5\u0026times;3 hrs. The extract was filtered and concentrated under vacuum at 45\u003csup\u003e0\u003c/sup\u003eC. This extract was subjected to partition thrice with hexane for the removal of photosynthetic pigments and lipid substances. Remaining methanolic phase was concentrated in vacuum with silica gel 60-120 mesh size to make silica slurry and stored until use. 100gm of free flowing extract with silica slurry was eluted with gradient of dichloromethane/methanol (100:00, 95:5, 90:10, 85:15. 80:20, 75:25, 50:50) and with pure methanol (100ml each) to afford 55 fractions. After TLC analysis with DCM: Methanol (9:1) the column fractions were pooled in five groups) MOF 1-5). These pooled fractions were analyzed by sub columns and visualized on TLC plates in UV chamber. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHPLC analyses were performed with Waters Alliance 2695 separations maintained at 25\u003csup\u003e0\u003c/sup\u003eC with PDA detector. Test samples (20 \u0026mu;l) were injected into a reverse phase column C18 (250 mm length, 4.6 mm and 5\u0026mu;m). The samples were dissolved in methanol and filtered with 0.45 \u0026mu;m nylon filters. 1ml/min flow rate, 254 nm \u0026lambda;, mobile phase A 0.05% TFA in ACN and B 0.05% TFA in water were used as chromatographic conditions. Gradient elution (T/A %) from 0/5, 30/25, 40/40, 50/63, 60/5 and 66/5 and 20\u0026mu;g/20 \u0026mu;l concentration of samples were used. The compositions of the mobile phase were rising over the period of 20 minutes.\u003c/p\u003e\n\u003ch3\u003eStatistical analysis\u003c/h3\u003e\n\u003cp\u003eData recorded from the experiments are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of triplicate determination (n\u0026thinsp;=\u0026thinsp;3).All the statistical analysis were accomplished by using graph pad prism10.4.2 software. The significant differences among treatments in pot experiment and antioxidant potential were determined by Tukey-Kramer HSD test at p\u0026thinsp;=\u0026thinsp;0.05. Dunnett\u0026rsquo;s post hoc test was used for comparative analysis in zone of inhibition at p\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e5.1) Total Phenol Content\u003c/h2\u003e\u003cp\u003eIt was reported that the antioxidant activity of plant extracts is well correlated with the content of their phenolic compounds. Total phenolic content of the extracts was calculated using the standard curve of gallic acid. Phenol molecules possess hydroxyl group (-OH) making them polar. This polarity allows them to dissolve readily in polar solvents. Total phenolic content was found in the bark extract (56.10\u0026thinsp;\u0026plusmn;\u0026thinsp;2.38 GAE /g) followed by seeds (42.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65 GAE /g), leaves (33.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50 GAE/g) and flower (13.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32 GAE/g). The lowest total phenolic content (5.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 GAE/g) was obtained from gum extract (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Results indicated that bark and seed extract contained a higher amount of phenols as compared to other extracts.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e5.2) DPPH radical scavenging capacity:\u003c/h2\u003e\u003cp\u003eIn the present study the extracts of \u003cem\u003eM. oleifera\u003c/em\u003e plant parts were investigated for the antioxidant activity using 2,2-diphenyl \u0026minus;\u0026thinsp;1picrylhydrazyl (DPPH) free radical scavenging assay. The antioxidant present in plant extract neutralizes the DPPH by the transfer of an electron or hydrogen atom. The methenolic extracts of different plant parts showed varying scavenging ability. Among these five, bark extract showed highest free radical scavenging effect (FRS) (74.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.95 followed by seed extracts (70.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07), leaf extract (54.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39 and flower extract (42.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Again lowest DPPH activity was observed in gum extract (10.61\u0026thinsp;\u0026plusmn;\u0026thinsp;1.50).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e5.3) FRAP analysis:\u003c/h2\u003e\u003cp\u003eFRAP analysis was performed with a similar concentration used in DPPH activity by using the standard curve. Seed extract displayed highest activity with 168.61 \u0026micro;M Fe\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e/g, followed by bark extract 162.11 \u0026micro;M Fe\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e/g and leaves extract 124.25 \u0026micro;M Fe\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e/g (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, flower and gum extract did not show any significant result.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e5.4) Isolation, morphological identification of test bacterium\u003c/h2\u003e\u003cp\u003eThe bacteria were isolated from naturally infected leaves of soybean on Letheen agar media after 72 h of inoculation. Single semitransparent gummy, circular pale yellow colonies were observed.\u003c/p\u003e\u003cp\u003eWhen this purified bacterium inoculated on soybean plant leaves showed typical pustules symptoms as pale yellow spots near the interveinal area which become large brown patches on leaves. All the inoculated plants yielded the same bacteria on reisolation within 15\u0026ndash;17 days after inoculation with circular light yellowish brown spots on leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Isolated bacteria were rod shaped (0.9\u0026times;1.6 \u0026micro;m), gram negative, capsulated with single polar flagella. These observations of morphological parameters indicated its similarity with the genus \u003cem\u003eXag\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e5.5) Biochemical characterization of test organism\u003c/h2\u003e\u003cp\u003eThe isolated bacterium showed growth between 12-37\u003csup\u003e0\u003c/sup\u003eC, pH 5\u0026ndash;8.0, and NaCl concentration at 2.5\u0026ndash;4.5%. It was KOH soluble and negative for urea hydrolysis. The bacteria did not utilize the\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ecarbohydrate but derived energy and produced acid from fructose, mannose, sucrose, dextrose and manitol. Based on biochemical characteristics the isolates were identified as \u003cem\u003eXanthomonas axonopodis\u003c/em\u003e and based on host range and specificity of the bacterium for soybean it was related to variant \u003cem\u003eglycine\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e5.6) \u003cem\u003ein vitro\u003c/em\u003e Antibacterial activity by different extracts of \u003cem\u003eM.oleifera\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe agar well plate method is widely used to evaluate the antimicrobial activity of different extracts. Contact and diffusion of the extract into the media is better in the well plate method Evaluation of the antibacterial activity of \u003cem\u003eM. oleifera\u003c/em\u003e plant extracts against plant pathogenic bacteria \u003cem\u003eXag\u003c/em\u003e was illustrated in (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe results revealed that plant extracts were potentially effective in suppressing microbial growth of \u003cem\u003eXag\u003c/em\u003e in sequential manner, when the concentration of extract increased, the zone of inhibition also increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Therefore it showed bigger sized inhibition zone. Each well is of the diameter. The results were recorded after 24 hours. Seed extract was the most effective extract for retarding microbial growth of pathogenic bacteria at 250\u0026micro;g/500\u0026micro;L concentration, showing an 18.86 mm zone of inhibition, followed by bark extract at 300 \u0026micro;g/500\u0026micro;L (18.16 mm) flower extract 300\u0026micro;g/500\u0026micro;L (15.36), leaves extract at 300\u0026micro;g/500\u0026micro;L (15.26mm) and gum extract (5.24 mm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). It was observed that methanol extract of leaves and flowers were slightly less active against test organism. However, reference plate of streptomycin is showed wider zone of inhibition as compared to seed and bark extract.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e5.7) Biochemical analysis\u003c/h2\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003e5.7.1) Chlorophyll and carotenoids content\u003c/h2\u003e\u003cp\u003eThe result of preventive effect of seed extracts on chlorophyll and carotenoid contents summarized in (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Data revealed that seed extract was positively correlated with chlorophyll and carotenoid content. The maximum chlorophyll content was observed in SE4 (1.98 mg/g) followed by SE5 (1.96 mg/g), SE3 (1.66 mg/g) and SE1 (1.22 mg/g) as compared to STRP (1.81 mg/g). However it was lower than healthy NT plants (2.12mg/g) but higher than NC (1.13mg/g). Similar results were observed in Chlorphyll a and b content. Highest weight was observed in SE4 (1.19 mg/g, 0.84 mg/g) followed by SE5 (1.14 mg/g, 0.82 mg/g), SE3 (1.01 mg/g, 0.76 mg/g), SE2 (0.97 mg/g, 0.72) and SE 1(0.91 mg/g, 0.67). In control groups Ch a, b content were significantly different, where NT showed highest content (1.33 mg/g, 0.97 mg/g) and NC showed lowest (0.88mg/g, 0.59 mg/g). While in carotenoids content extracts results were positively correlated with it. SE4 showed highest carotenoids (0.98 mg/g) followed by SE5 (0.97 mg/g), SE3 (0.89 mg/g), SE2 (0.81 mg/g) and SE1 (0.74 mg/g). As compared to STRP (0.94 mg/g) SE5 and SE4 were significant.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section3\"\u003e\u003ch2\u003e5.7.2) Protein and carbohydrate content\u003c/h2\u003e\u003cp\u003eThe result of the effect of seed extract prepared from \u003cem\u003eM. oleifera\u003c/em\u003e on biochemical parameters i.e. carbohydrate and protein content of Soybean is summarized in (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Data clearly indicated that seed extract increases carbohydrate and protein content of the host plant. The maximum carbohydrate content was observed in SE4 (4.022 mg/g) followed by SE5 (4.01 mg/g), SE3 (3.34 mg/g), SE 2(3.16 mg/g) and SE 1(3.06 mg/g) except NC (2.61mg/g). For carbohydrate streptomycin (3.94 mg/g) and healthy plant (5.08 mg/g) was observed, which were significant.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSimilarly, protein content was increased as the concentration of seed extract increased. The highest protein content was observed in SE 4 (9.81 mg/g) followed by SE5 (9.24 mg/g), SE3 (7.76mg/g), SE 2(5.48mg/g) and SE 1(4.05 mg/g). All the treatments are significant as compared to streptomycin (8.43 mg/g) and healthy plant (13.95 mg/g) for protein except NC (1.67 mg/g).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e5.8) Plant height and leaf per plant\u003c/h2\u003e\u003cp\u003eResults of the effect of seed extracts on number of leaves per plant are given in (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The data indicated that there was slight increment in the number of leaves as extract concentration increases. The maximum increase in the number of leaves/ plant was observed in treatments with SE 4 (24.33) followed by SE 5 (21.67), SE 3 (19.33), SE 2 (16.33) and SE 1(15.00).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOn the other hand data given in (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) indicated that there was significant increment in the plant height due to treatment of seed extracts. The maximum increase in plant height was observed in SE4 (50.33 cm.) followed by SE5 (50.18 cm.), SE3 (49.95 cm.), SE2 (49.88 cm.) and SE1 (48.76 cm.). However seedling length and growth were found in SE4 and STRP which indicated that SE4 had a good capacity to influence seedling growth as compared to other treatments. All the treatments are significant as compared to both control groups except NC (48.33 cm.) for both parameters plant height and leaf per plant as shown in (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEstimation of growth parameters (leaf per plant and plant height) and biochemical parameters (Chlorophyll a, b, carotenoids, protein and carbohydrate) in soybean by different concentrations of seed extract against bacterial pustule disease.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLeaf/plant\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePlant height\u003c/p\u003e\u003cp\u003e(cm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCarbohydrate\u003c/p\u003e\u003cp\u003e(mg/g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c8\" namest=\"c5\"\u003e\u003cp\u003eChlorophyll and carotenoids (mg/g) fresh weight\u003c/p\u003e\u003cp\u003e\u003cem\u003eTotal ch Cha Chb\u003c/em\u003e carotenoids\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eProtein\u003c/p\u003e\u003cp\u003e(mg/g)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSE1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e48.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e4.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSE2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e49.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSE3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e19.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e49.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e7.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSE4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e9.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSE5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e21.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e9.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSTRP\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e8.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNT\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e26.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e51.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e13.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNC\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e48.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003cb\u003eSE (1\u0026ndash;5) seed extract concentration \u0026micro;g/500\u0026micro;l, NT (non treated), STRP (streptomycin) and NC (negative control). The results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three replicates. Different significantly different letters (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) as determined by Tukey-Kramer HSD test\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\u003ch2\u003e5.9) Isolation of phenolic compounds and HPLC analysis\u003c/h2\u003e\u003cp\u003eIn order to obtain the antibacterial phenolic compounds from the seeds of \u003cem\u003eM. oleifera\u003c/em\u003e repeated column chromatography and TLC were performed. For their purity HPLC analysis was used which revealed the presence of three major phenolic compounds. The fraction number 2 give quercetine (MOF 2.3.1 with solvent ratio toluene: ethyl acetate: formic acid 5:4:1). P-coumaric acid and cinnamic acid were isolated from fraction 5.4 and 5.7.Cinnamic acid was eluted with ethyl acetate: n hexane (70:30) and p coumaric acid was isolated with the solvent system of chloroform:ethyl acetate (60:40).HPLC showed single compound profile with highest peak area percentage of quercetin at 16.34 RT, p coumaric acid 13.18 RT and cinnamic acid 17.20 RT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmong all three compounds peak area percentage of cinnamic acid was highest 99.13%. Remaining two phenolic compounds also had good percentage area in which p coumaric acid\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ehad 98.04% and quercetin had 90.67%. However in quercetin minor peaks also observed indicated that impurity in isolated quercetin at certain level.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWorldwide, soybean is an essential food crop but it is susceptible to different microbial diseases. Bacterial pustule disease is one of them, which causes severe damage to its yield and overall production [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Disease management by different commercially available chemicals can control the severity but there are several negative impacts on soybean crop physiologically and morphologically and it also affects the quality of seeds. Entering in food chain is another concern, as it damages other populations as well as the environment. In the current examination we report for the first time the efficacy of \u003cem\u003eM. oreifera\u003c/em\u003e plant to pursue the defense reaction against bacterial pustules disease in soybean and regulate sustainable plant growth. Several reports are available related to plant based natural and integrated approaches for bacterial disease control and management [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn the present study, \u003cem\u003eM. oleifera\u003c/em\u003e seed extract was recorded in both the pot experiment and agar well plate method against the disease. Among all the treatments, seed extract was recorded with significant \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e control of pustule disease in soybean. The defense response of seed extract might be due to the presence of phenolic compounds which can stimulate plant immune response by enhancing its antioxidant defense enzyme system and contribute to their resilience and adaptability towards environmental challenges including microbial stress. Seeds of \u003cem\u003eM. oleifera\u003c/em\u003e contain a wide array of beneficial phenolic compounds such as gallic acid, ferulic acid, caffeic acid, catechin, epicatechin, vanillin, quercetin, cinnamic acid, p-coumaric acid etc.[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] and function as signaling compounds that trigger the development of systemic acquired resistance (SAR) and activation of defense genes. In the present study three phenolic molecules Quercetin, cinnamic acid and p coumaric acid were successfully isolated from the seeds of \u003cem\u003eM. oleifera\u003c/em\u003e. Many studies suggests that these compounds inhibited bacterial growth specially quercetin exhibited antibacterial potential against \u003cem\u003eXanthomonas axonopodis pv. citri\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In other study p coumaric acid induced phenolic metabolites alleviates the disease symptoms caused by \u003cem\u003eXanthomonas campestris pv. campestris\u003c/em\u003e in cabbage [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In peach tree bacterial shot hole disease is a global concern caused by pathogen \u003cem\u003eXanthomonas arboricolapv. pruni.\u003c/em\u003e this bacteria was effectively controlled and inhibited by the derivatives of cinnamic acid [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThey also contains phenylpropenoids such as 4-hydroxyphenyl acetate, 3- hydroxyl-1-(4-hydroxy-3-methoxyphenyl)-1-propanone, 3-hydroxy-1-(4-hydroxyphenyl)-propyl-1-one, erythro-guaiacylg-lycerol and threo-guaia-cylglycerol [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. They can be involved in the synthesis of cell wall strengthening compounds, such as lignin and suberin so it is positively correlated with fortifying plant core structure against environmental stress [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. They also function as signaling compounds that trigger the development of systemic acquired resistance (SAR) and activation of defense genes [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Glucosinolates and its derivative byproduct (isothiocyanates) also present in the seeds of \u003cem\u003eM. oliefera\u003c/em\u003e, can act as antibacterials. While it is not influenced photosynthesis directly but indirectly through their impact on plant physiology and stress response [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIt also influences the growth promoting consequences like transcription and translation processes and prevents bacterial growth. Plant based extracts and products considerably enriched the growth and development of soybean in pot experiments as compared to streptomycin treated plants. However, streptomycin treatment also showed good results in agar well plate method but in pot experiment protein, chlorophyll and carbohydrate quantities were low as compared to non treated (NT) healthy plants and SE 4, which indicated that streptomycin negatively impacted the plant own defense system, growth, post germinative development and weakening the plant overall defense capacity. Whereas increased sugar content in treated plants especially in SE4 can be directly correlated with enhanced photosynthesis. Because Reactive oxygen species are byproducts of photosynthesis and phenolic compounds can help in regulating ROS levels, potentially influencing photosynthesis efficiency [\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Many researchers have used various kinds of bioformulations, nanoformulations and PGPR based formulations but these approaches are very costly, time consuming. Therefore these antimicrobial rich seed extracts can act as environmental friendly by eliminating phytopathogenic microbes specially Xag. Such sustainable approach has the capacity to revolutionize agriculture and reducing reliability on chemical pesticides.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e\u003cem\u003eM.oleifera\u003c/em\u003e has been reported to produce a large number of primary and secondary metabolites. In our study higher total phenol content and antioxidant activities were obtained, which showed antibacterial activity in both in \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. Based on these results, it could be concluded that seed extract of \u003cem\u003eM. oleifera\u003c/em\u003e showed antibacterial activity as it contain different bioactive molecules including quercetin, cinnamic acid and p coumaric acid against \u003cem\u003eXag\u003c/em\u003e. It can be used against the management of bacterial pustules of soybean. However, it is important to isolate and identify other compounds except these one through spectroscopic methods like HPLC, NMR responsible for the antimicrobial activity as well as to establish the mechanism of action of the extract and its bioactive molecules to come to a definite conclusion. Since demand for food free from synthetic constituents has exponentially increased in the last few decades, an herbal approach is imperative to be amended to reinforce the immunity of plants to survive and for sustained plant growth.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial, ethics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe manuscript does not contain any studies with human participants or animals performed by any of the authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data underlying the results are available as part of the article and no additional source data are required.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s Contributions\u003c/strong\u003e: Conceptualization, F.K. and J.A.; methodology, F.K., A.T. and \u0026nbsp;J.A.; investigation, F.K., A.T. and J.A.; resources, F.K., B.K. and \u0026nbsp;J.A.; data curation, F.K., A.J. and J.A.; writing\u0026mdash;original draft, F.K., A.T. and J.A.; writing\u0026mdash;review F.K., A.J., B.K., and J.A.; visualization, F.K. and J.A.; supervision, J.A. All figures have been made by F.K; Revision, final update and editing, R.K., F.K. and J.A. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eThe authors are highly thankful to the Ministry of Education and SPD-RUSA Rajasthan for the Infrastructure support created under funds received from the RUSA 2.0 projects. All the authors acknowledge their host institute for infrastructure support. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e: No Funding support provided to this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eF. C. McKenzie and J. 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Rao \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Antioxidant defense system in plants: Reactive oxygen species production, signaling, and scavenging during abiotic stress-induced oxidative damage,\u0026rdquo; \u003cem\u003eHorticulturae\u003c/em\u003e, vol. 11, no. 5, p. 477, 2025. https://doi.org/10.3390/horticulturae11050477.\u003csub\u003e \u003c/sub\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-applied-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Applied Sciences](https://link.springer.com/journal/42452)","snPcode":"42452","submissionUrl":"https://submission.springernature.com/new-submission/42452/3","title":"Discover Applied Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Soybean, bacterial pustules disease, Xanthomonas, M.oleifera","lastPublishedDoi":"10.21203/rs.3.rs-7550249/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7550249/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBacterial pustule disease triggered by \u003cem\u003eXanthomonas axonopodis pv. glycines\u003c/em\u003e (Xag) is one of the major constraints affecting the cultivation of soybean in India. According to the recent reports, the disease severity was prevalent in moderate to severe intensities and damaged soybean crop and its yield. The current investigation examined the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e preventive efficacies of \u003cem\u003eMoringa oleifera\u003c/em\u003e lam. different extracts on bacterial pustules disease of soybean. The number of leaves per plant, Plant, soluble protein, carbohydrate and chlorophyll content were examined. A significant control of disease was recorded with seed extract (SE4). Results showed the treatment with seed extract notably increased plant height, carbohydrate, chlorophyll, carotenoids. However, soluble protein and carbohydrate content was observed to be less in higher concentrations along with reference antibacterial agent streptomycin. According to this current research seed extracts with 250\u0026micro;g/500 \u0026micro;l could be essential towards sustainable agricultural crop management for soybean. Therefore, compounds of seed can be evaluated and commercially explored for agricultural use.\u003c/p\u003e","manuscriptTitle":"Investigation of the antibacterial properties of Moringa oleifera Extracts against Xanthomonas axonopoidespv. glycines the incitant of Bacterial Pustules of Glycine max","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-20 14:07:03","doi":"10.21203/rs.3.rs-7550249/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-30T07:36:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-28T11:23:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-28T04:51:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-21T18:56:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"149959146326698790245897149828057263044","date":"2025-10-12T12:40:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"115106411784251438393959937417858736423","date":"2025-10-10T18:56:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36321203561637311427686023801289135922","date":"2025-10-09T12:51:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46100728168602025611903100144138522361","date":"2025-10-09T05:07:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"189686757574140273143194000005012535449","date":"2025-10-07T14:46:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"232629544536229011388118187131079376271","date":"2025-10-07T12:52:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-07T12:24:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-26T05:46:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-24T15:31:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Applied Sciences","date":"2025-09-24T15:27:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-applied-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Applied Sciences](https://link.springer.com/journal/42452)","snPcode":"42452","submissionUrl":"https://submission.springernature.com/new-submission/42452/3","title":"Discover Applied Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"86372251-41f7-4ec2-887c-8f662d89bb3d","owner":[],"postedDate":"October 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T11:28:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-20 14:07:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7550249","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7550249","identity":"rs-7550249","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}