Synergistic impacts of plant growth promoting Pseudomonas geniculata TIU16A3 and green-synthesized iron-oxide nanoparticles from Eichhornia crassipes for the amelioration of heavy metal stress in Vigna radiata L.

Synergistic impacts of plant growth promoting Pseudomonas geniculata TIU16A3 and green-synthesized iron-oxide nanoparticles from Eichhornia crassipes for the amelioration of heavy metal stress in Vigna radiata L. | 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 Synergistic impacts of plant growth promoting Pseudomonas geniculata TIU16A3 and green-synthesized iron-oxide nanoparticles from Eichhornia crassipes for the amelioration of heavy metal stress in Vigna radiata L. Barkha Madhogaria, Sangeeta Banerjee, Sohini Chakraborty, Prasanta Dhak, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4863542/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Plants are often affected by deleterious effects of heavy metals (HM). This HM stress leads to growth and production capacity issues. The most hazardous trace metals in agricultural areas are lead (Pb), cadmium (Cd), and chromium (Cr). It is not only carcinogenic to humans, but also negatively affects plants' phenotypic, biochemical, and genetic properties. Bioremediation can be used to economically remove contamination of heavy metals. The study aims to test the bioremediation potential of biogenic iron oxide nanoparticles (Fe-O-NP) along with Pseudomonas geniculata strain TIU16A3 (accession number OR507186). Plants of Eichhornia crassipes were used to create Fe-O-NP. Individually iron oxide nanoparticles exhibited enhancement of phenotypic traits in Vigna Radiata L. under heavy metal stress. We used four concentrations (10, 20, 40, and 80 mg/L) of Cr, Cd, and Pb for stress conditions. Biogenic nanoparticles and TIU16A3 in combination act synergistically with the increase in the amount of chlorophyll content and growth in distinct phenotypic traits. In plants, under HM stress, levels of antioxidant enzymes were observed to be high including superoxide dismutase, peroxidase, and catalase and H 2 O 2 content and electrolyte leakage, when they were treated with biogenic NPs and TIU16A3 together the antioxidants decreased significantly. TIU16A3 and Fe-O-NP synergistically in the presence of Cd and Cr enhanced the expression of the Aux/IAA gene more than the expression observed in the presence of Fe-O-NP only. Due to the enhancement of intrinsic properties, and ability to remove Cr, and Cd by TIU16A3 when used in combination with Fe-O-NP for bioremediation exhibits promising results. Bioremediation Vigna radiata L. Eichhornia crassipes nanoparticle Fe-O-NP TIU16A3 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Highlights Nanoparticles are reported to aid in plant growth promotion and alleviate heavy metals and the sustainable approach of synthesizing potential iron oxide nanoparticles (Fe-O-NP) has been studied. Water hyacinth serves as a reducing and capping agent for the production of desired Fe-O-NP. Efficiency of TIU16A3 (accession number OR507186) and biogenic Fe-O-NP to bio-remediate soil contaminated with heavy metals have been first reported in the present study. Combinatorial strategy effect on the alleviation of heavy metal and growth of economically important legume Vigna radiata L. is a breakthrough research so far. Economical and sustainable eradication of water hyacinths by using them to produce non-toxic nanoparticles. 1. Introduction The tanning process used in the leather industry uses chromium (Cr) and other hazardous chemicals. It is said that 80–90% of leather is tanned with basic chromium sulfate (BCS) and only 50–70% of Cr is fixed in chromium tanning; the remaining Cr is released into wastewater(Saravanabhavan et al. 2017 ). According to(Gupta et al. 2007 ), the leather industry is the largest producer of heavy metals in a nearby water facility. The sludge is also used for irrigation, which causes heavy metal (HM) contamination in agricultural land. When sludge is used for irrigation, heavy metals such as Cd, Zn, Cr, Ni, Pb, and Mn can accumulate in surface soils and enter groundwater or soil solutions that can be taken up by plants(Mondal et al. 2017 ). Before the independence of West Bengal, three agglomerations of Kolkata - Tangra, Topsia, and Tilala - saw the activity of the leather industry. However, the leather industry moved about 20 kilometres from Kolkata to Bantala, where they are now located at the Calcutta Leather Complex (CLC). More than 300 tanneries moved to CLC due to the Supreme Court decision. CLC is supplied with a Common Effluent Treatment Plant (CETP), which uses several methods to treat a significant amount of wastewater from nearby tanneries before it is released back into the environment. Liquid and solid waste from tanneries and other factories entering water bodies adversely affects the natural ecosystem of the Ramasar area and can damage major waterways due to the 12,500-hectare East Kolkata Wetland adjacent to Bantala. It is generally known that heavy metal pollution negatively affects plant germination, photosynthesis, yield, and mineral nutrition (Kundu et al. 2019 ; Banerjee et al. 2023 ). When animals and humans eat plants contaminated with heavy metals, the metals are absorbed through the food chain into their cells and tissues can damage the intestinal microbiota and cause several chronic diseases in humans (Madhogaria et al. 2022 ). Heavy metals negatively affect dry matter, root and shoot growth (Naz et al. 2015 ). Many studies have shown that certain bacterial species can effectively promote bioremediation of metal-contaminated soil. Pseudomonas sp., Enterobacter cloacae , Bacillus sp., and other metal-resistant PGPBs are already been studied for the purpose (Zulfiqar et al. 2022 ). Biosorbents are one of the many technologies available to remove heavy metals from the environment(Banerjee et al. 2022 ; Madhogaria et al. 2024b ). In recent decades, nanotechnology has become a central area of ​​research. Researchers are paying more attention to a green approach to produce nanoparticles (NPs) without harming the environment by using an aqueous solution of plant extract. The effect of metal oxide nanoparticles on plants has been studied previously(Kiwumulo et al. 2022 ). Plant extracts contain sugars, terpenoids, polyphenols, etc. which can stabilize and reduce metal nanoparticles. Functional groups and phenolic compounds have been shown to contribute to the formation of metal nanoparticles. The International Union for Conservation of Nature (IUCN) has listed water hyacinth ( Eichhornia crassipes ) as one of the 100 most popular invasive and aggressive species that reproduce quickly. Under the right conditions, biomass can double in just a week, causing some problems for waterways. The green synthesis of Fe-O-NPs by E. crassipes has not yet been investigated, and the plant growth properties of these NPs have hardly been documented in the literature. Iron is oxidizable, so instead of reducing Fe 2+ to FeO, the phytochemicals in the extract react with iron ions to form iron oxide nanoparticles. Iron oxide nanoparticles, or Fe-O-NPs, were chosen over other iron oxide phases due to their infrared, magnetic, and optical properties and stability. TEM, FTIR, UV-Vis spectroscopy, and X-ray diffraction confirm the morphology and presence of nanoparticles. Fe affects important physiological functions in plants, such as respiration, redox reactions, and chlorophyll production. In addition, the large surface area of ​​Fe-O-NPs acts as a platform to reduce nutrient losses and ensure the timely delivery of nutrients to plants. Fe-O-NPs have recently been used directly to improve plant growth and alleviate drought stress and heavy metal pollution(Rizwan et al. 2019 ; Yang et al. 2020 ). Treatment of plants with Fe-O-NPs has also been studied in connection with the solubilization of heavy metals. Agricultural applications of Fe-O-NPs are an exciting area of ​​current research. Compared to other nanoparticles, Fe-O-NPs are toxic to soil bacterial communities. In addition, they support seed germination and maintain soil pH and nutrient levels(Rastogi et al. 2019 ). Previous studies have investigated how Fe-O-NPs can increase crop growth and yield by accelerating photosynthesis and altering several other metabolic processes. Although PGPR and Fe-O-NP have established distinct roles, little is known about their functions. Their small size and large surface area help ​​them to cause strong adsorption of various molecules, which increases their effectiveness(Nayana et al. 2020 ). There are no studies on the synergistic and comparative use of Fe-O-NP and PGPR to reduce heavy metal stress in Vigna radiata L. There is a theory suggesting that PGPR and Fe-O-NP are important in stressed plants. This work aimed to investigate the relative and synergistic effects of heavy metal-resistant PGPR and Fe-O-NP isolated from tannery wastewater-affected soil on germination and growth of V. radiata L. under heavy metal stress, especially Cr and Cd. 2. Materials and Methods 2.1. Chemicals The raw materials utilized in the production of Fe-O-NPs from Eichhornia crassipes , namely Fe 2 SO 4 .7H 2 O (98.5%), ethanol (99%), and NaOH (97%), were procured from LOBA CHEMIE PVT. LTD. Eichhornia crassipes , utilized herein, were procured from the pond site situated near Techno India University in West Bengal. Luria Bertini broth, Mercuric chloride (95%), Mueller Hinton agar, Czapek dextrose agar, phosphate buffer solution (PBS), H 2 O 2 (95%), NaCl (95%), and EDTA (95%), and Triton-X100 were purchased from Hi-media. 2.2. Preparation of Water hyacinth-mediated biogenic production of iron oxide nanoparticles With some additional adaptations, we followed the protocol of (Periakaruppan et al. 2021 ) for the green synthesis of Fe-O-NP. Five grams of water hyacinth leaves were weighed. Thorough cleaning and washing were done under running water and again with double distilled water. Leaves were ground and 100 ml distilled water was added and boiled for 20–30 mins with constant stirring. After boiling the extract was filtered through Whatman No. 1 filter paper. The filter was then stored at 4°C in a sterile beaker for later use. After continuous stirring, 20 mL of water hyacinth leaf extract was added dropwise to 80 mL of aqueous ferrous sulfate heptahydrate (Fe 2 SO 4 .7H 2 O) (0.1 M). NaOH was added to the mixture to obtain and then maintained a pH of 12. After centrifuging the precipitated liquid at 10,000 rpm for 30 minutes, impurities were removed by washing two or three times with Millipore water. To air dry the assortment, it was placed in a hot air oven at 120°C for 60 minutes. For further analysis and evaluation, the pellet was crushed and kept in a sterile container. (Fig. 1 ). 2.3. Material characterization X-ray diffraction was used to study the crystallinity, phase purity, and crystallite size of Fe-O-NP. The hydrodynamic size of Fe-O-NP and their distribution pattern were measured using dynamic light scattering, with some modifications as described (Dhak et al. 2011 ). The UV-visible spectrum of Fe-O-NP synthesized by green methods was done using a Hitachi 330 spectrophotometer. Elemental composition of Fe-O-NP was done using energy-dispersive X-ray spectroscopy (EDS), while scanning electron microscopy (SEM) was used to study and record the surface morphology and approximate shape of the particles. 2.4. Antibacterial capacity of Fe-O-NPs against PGPR TIU16A3 The antibacterial activity of iron oxide nanoparticles prepared from E. crassipes extracts against TIU16A3 was evaluated by the agar well diffusion method. Zones of inhibition were evaluated to determine the degree of inhibition of bacterial growth. 2.5. Antifungal analysis of Fe-O-NPs on growth of Fusarium sp. The antifungal activity of Fe-O-NP was analyzed with Fusarium sp., obtained from the Department of Plant Biology, Bose Institute, and Kolkata. Using Fe-O-NP, the fungal strain was inoculated onto Cazpek dextrose agar (CDA). Growth was examined on days three, six, and nine. Analysis was done based on colony diameter measurements using the formula for PIRG (percentage inhibition in radial growth) % = (R1‒ R2 ÷ R1) × 100 (Eq. 2), where R1 is the radial growth of the control and R2 is the radial growth of each treatment was used to determine the percentage of inhibition of fungal growth(Yazid et al. 2023 ). 2.6. Hemolytic analysis The hemolysis assay typically uses fresh human blood samples from healthy adult volunteers stabilized with ethylenediaminetetraacetic acid (EDTA). With some modifications, the test was performed as previously mentioned (Modi et al. 2023). From serum, Red blood cells (RBC) were first separated by centrifugation at 2000 rpm for 10 min. After that, 5 ml of blood was added to 10 ml of 50 mM PBS. RBCs were then washed three more times using 50 mM PBS. A hematocrit of 5% was then used to dilute the purified erythrocytes in PBS. 100 µl of erythrocyte suspension was added to an eppendorf tube containing 100 µl of Fe-O-NPs at different concentrations (50, 100, 150, and 200 µg/mL). After one hour incubation at 37°C, centrifugation at 1500 rpm for ten minutes was done. The 80 µl supernatant was then added to PBS. Absorbance at 414 nm OD was measured using a plate reader. A positive control of 100% hemolysis was achieved using 0.1% Triton X-100 (v/v), while a negative control using PBS. The percentage of hemolysis was calculated using formula % hemolysis = (OD of sample-OD of PBS) / (OD of positive control-OD of PBS) × 100 (Eq. 3) 2.7. Priming seeds of Vigna radiata L., germination, and transfer of seedlings in hydroponics culture For seed priming, the seed of Vigna radiata L. (mung bean) procured from West Bengal State Seed Corporation Limited, Kolkata, West Bengal was used. Surface sterilization, priming with Fe-O-NP alone and synergistic with Pseudomonas geniculata TIU16A3, germination, and seedlings were transferred to a hydroponics solution for further experiments were done following the method(Kundu and Ganesan 2020 , 2023a ; Madhogaria et al. 2024a ). 2.8. Standardization of Fe-O-NP for plant growth promotion Treatments included control and different concentrations of Fe-O-NPs (25, 50, and 100 µg/mL) in three replicates were chosen for this analysis. Fe-O-NP treatment with maximum germination of V. radiata L. seedlings and maximum increment in different phenotypic plant traits (root length, shoot length, fresh weight. dry weight, secondary root number, secondary root length, leaves breadth, and leaves length)was selected for further experiment for analysis of heavy metal alleviation and plant growth promotion of V. radaiata L. in the presence of Cd, Cr, and Pb stress following the methods with some modifications by(Kundu and Ganesan 2023b ). 2.9. Phenotypic differences between untreated, individually treated, and co-treated Vigna radiata L. plants and total biomass studies in the presence of heavy metal toxicity The difference in plant growth was observed after 14 days of reading with the following treatments: untreated (UT), Fe-O-NP 50 µg/ml treated (T), TIU16A3 P. geniculata treated (T), and Fe-O-NP (50 µg/ml) + TIU16A3 treated plants in the presence and absence of heavy metals (chromium, cadmium, and lead) was done as described by(Madhogaria et al. 2024a ). The traits analyzed for plant growth variation are primary root length, shoot length, dry weight, wet weight, leaf width, leaf length, total chlorophyll content, number of secondary roots, and secondary root length(Kundu and Ganesan 2023b ). 2.10. Antioxidant SOD, POD, CAT, H 2 O 2 content, and Electrolyte leakage (EL) analysis Determination SOD, POD, CAT, H 2 O 2, and electrolyte leakage estimation was done as described by (Madhogaria et al. 2024a ) with some modifications. 2.11. Determination of HMs (Cr, Cd, and Pb) content A 3:1 mixture of sulfuric and nitric acid was used to digest 100 mg of dry plant material. Further analysis was done as described by ( Madhogaria et al. 2024a ) with some modifications. 2.12. Analysis of gene expression by RT-qPCR The transcript levels of gene Aux/IAA of shoot tissue of Vigna radiata L. plant in the presence of 10, and 20 µg/mL of HMs Cr, and Cd stress with Fe-O-NP alone and P. geniculata + 50µg/mL Fe-O-NPs were determined by real-time PCR following the method with some medication by (Madhogaria et al. 2024a ). 2.13. Statistical analysis All the tests were performed in triplicates to avoid any errors and to increase the accuracy. All graphical analyses were performed using Graph pad prism 8.0.2 and Origin Pro 8.5.0. Statistical significance differences among different treatments were done using Duncan’s multiple range test (DMRT) using software IBM SPSS version 16. Statistical probability P < 0.05 was followed. 3. Results and Discussions 3.1. Material characterization 3.1.1. UV-visible spectroscopy UV–Vis absorption spectrum showed a wide absorption peak at 595 nm. The firm transmission peaks were observed at 568nm with 84.54% transmission, 588nm with 89.8% transmission, 595nm with 91.38% transmission, and 629nm with 91.51% transmission. Figure 2 depicts the transmission spectra of E.crassipes mediated biogenic synthesis of Fe-O-NPs falls between 400–700 nm. According to (Basavegowda et al. 2014 ) using A. annua and P. frutescens green synthesized iron oxide NP had a similar range by which we can confirm that metal-oxide nanoparticles are being formed. 3.1.2. X-ray diffraction analysis The X-ray diffraction (XRD) patterns were analyzed with the JCPDS card file 19–0629 89–0596, confirming the formation of amorphous magnetite iron oxide nanoparticles. The scanning speed is 10 0 /min. The amorphous nature and composition of Fe-O-NP were evaluated over a 2θ range from 30 o to 70 o (Fig. 3 ). The diffraction peaks were present at 35 ◦ (104), 42 ◦ (113), 52 ◦ (018), and 64 ◦ (024). The average crystallite size of Fe-O-NP was 15.5 nm, calculated using the Scherrer equation. The intensity of diffraction peaks signifies good crystallinity of the synthesized nanoparticles. Moreover, similar results have been reported by (Yew et al. 2017 ; Yadav et al. 2020 ). 3.1.3. Dynamic light scattering analysis The intensity-average diameter of Fe-O-NP was calculated at 1026 nm by using dynamic light scattering (DLS) analysis. The hydrodynamic size distribution of Fe-O-NP is shown in (Fig. 4 ). It is the core size and the presence of capping elements around it. NPs were at moderate stability and exhibited excellent dispersion in an aqueous medium. 3.1.4. Scanning electron microscopy wit henergy-dispersive X-ray mapping Structural analysis of biogenic Fe-O-NP can be done precisely by scanning electron microscopy (SEM). Fe-O-NPs surface morphology and elemental composition are illustrated in (Fig. 5 ). As obtained by using SEM analysis, respectively. Energy dispersive X-ray (EDX) analysis reported the presence of 32.62% iron and 34.97% oxygen along with 31.88% carbon due to the availability of phyto-phenolic compounds in Fe-O-NP synthesized from E. crassipes (Fig. 6 ). Similar results were reported by (Periakaruppan et al. 2021 ). 3.2. Antibacterial evaluation Minimum inhibitory concentration MIC is evaluated for our synthesized Fe-O-NP against TIU16A3 (accession no. OR507186). We observed that Green synthesized Fe-O-NP has no antibacterial effect against TIU16A3 as no zone of inhibition was formed (Fig. 7 ). We can imply from this result that the green synthesized Fe-O-NPs from E. crassipes andTIU16A3can be used in combination on the seed of V. radiata L.to study synergistic effect on heavy metal mitigation and plant growth promotion. 3.3. Antifungal evaluation Biogenic Fe-O-NP synthesized in this study revealed remarkable antifungal potency against Fusarium sp. when compared to the control Fig. 8. Radial growth size of fungus in the presence of 25, 50, and 100µg/mL was measured to be 25mm, 16mm, and 17mm respectively. Percent inhibition of radial growth (PIRG) was calculated with the help of Eq. 2 and PIRG% for 25 µg/mL was 44.4%, similarly for 50 µg/mL, the PIRG% was 64.4%, and for 100 µg/mL 62.2% was the PIRG%. This result suggested that as the concentration of Fe-O-NP is increasing, inhibition of the fungus growth is also increasing. The gradual increment of the inhibition potential rate of green-synthesized Fe-O-NPs may be due to their high effect on the growth of fungal mycelium. The biogenic Fe-O-NPs synthesized from the leaf extract of Euphorbia herita showed excellent antifungal activity against Aspergillusniger , Arthogrophis cuboida , and Aspergillus fumigatus in the range of 10–30 mg/mL (Win et al. 2021 ). Another study confirmed the potential antifungal activity of Fe-O-NPs synthesized from Chlorella -K01 extract against Fusarium oxysporum , Fusarium tricinctum , and Fusarium maniliforme (Cruz-Luna et al. 2023 ). Thus, the present findings and previous reports signify the efficiency of biogenic Fe-O-NPs usage as potent natural fungicides in comparison with chemical-toxic fungicides. In addition, the application of biologically synthesized Fe-O-NPs affirms fungal resilience resulting from the extensive use of chemical fungicides. The interaction of NPs with fungal cells might lead to the elimination of cytoplasmic membranes (Ahmed et al. 2021 ). The disruption of the fungal cell membrane is attributed to the secretion of micro-ions such as potassium and phosphate radicals, along with large-sized molecules such as RNA, DNA, and other intracellular substances (Ojoma et al. 2021 ). 3.4. Hemolytic activity The hemolytic effect of biogenic Iron oxide NP was carried out by Human RBC hemolytic assay in the presence of different Fe-O-NP concentrations (Fig. 9 ). At concentrations of 50, 100, 150, and 200 µg/mL of biogenic Fe-O-NP, the hemolysis level was below 15% even at the highest concentration (200 µg/mL) in comparison of the value of 0.1% Triton X100 (positive control) mediated hemolysis. The reason behind the non-toxic behavior against human RBC of our green synthesized is that it has been previously studied that biogenic Fe-O-NPs are biocompatible, biodegradable, and potentially nontoxic to humans (Wahajuddin and Arora 2012). 3.5. Standardization of different concentrations of biogenic Fe-O-NP for the promotion of plant growth Fe-O-NP effect on seedling growth and different growth parameters, in V. radiata L. plants, to understand its mode of action and to establish an optimal concentration for plant growth promotion. We used 25, 50, and 100 µg/mL for the test. Seed priming was done, Non-primed seeds were kept as control. We found out that seed germination was highest at 50 µg/mL of Fe-O-NP concentration at which root length was 65.2 mm in length when compared to control 23.5 mm (Fig. 10 ) Analysis of different phenotypic plant traits was done and we found out that at 50 µg/mL of Fe-O-NP concentration, shoot length increased up to 38% in comparison to 25 and 100 µg/mL. The secondary root number and its length showed more growth in 50µg/mL of Fe-O-NP concentration (Fig. 11 ). Similar results were seen in the case of the length and breadth of leaves. If we cumulatively analyze the growth in all the plant traits in different concentrations we can say that growth was 54.4% higher in 50 µg/mL than in 25, and 100 µg/mL of Fe-O-NP treated plants. Fe-O-NPs release iron. Iron affects plant metabolism, photosynthesis, and respiration. Electron carriers like cytochromes, ferredoxins, etc. are being affected by iron (Hochmuth 2011 ). The vigorous growth of V. radiata L. plants under the effect of Fe-O-NPs is because of enhancement in auxin, metabolism enhancement, cell expansions, and enhanced biochemical activities which ultimately lead to growth in various morpho-physiological traits of the plant. Similar results were reported by (Iqbal et al. 2019 ) 3.6. Effect of green synthesized Fe-O-NPs 50µg/mL on V. radiata L. growth and photosynthetic content under heavy metal stress The seedlings exposed to Fe-O-NP (50 µg/mL) exhibits increased nutrient uptake, followed by stimulation of plant growth, and improvement of morphological and physiological characteristics of V. radiata L. seedlings (Fig. 12 ). Compared with control and NP-treated seedlings, heavy metal treatment negatively affected the growth of V. radiata L. seedlings. Compared to HM treated seedlings, Fe-O-NP treated seedlings showed better root length (54.2%) and shoot length (62.8%) difference (P ≤ 0.05) (Fig. 13 a), (Fig. 13 b). V. radiata L. seedlings treated with Fe-O-NPs showed a significant (P ≤ 0.05) increase in both fresh and dry weight compared to the control group (Fig. 13 c) (Fig. 13 d).The root length increased up to 73%, and 67%and shoot length 75%, and 71% for Cr and Cd HM stress in presence of Fe-O-NP. A negligible growth range from 21.4–31% increment was observed in lead stress for Fe-O-NP 50 µg/mL treatment. Fresh weight and dry weight of the Fe-O-NP treated test crop were increased 79% and 85.2% respectively in the presence of 10 µg/mL of Cr to control followed by increase in fresh and dry weight of Fe-O-NP treated V. radiata L. in the presence of Cd 10 µg/mL which was 65.4% and 69.7% respectively. At 20 µg/mL of Cr and Cd fresh and dry weight were observed in the range of (61.4–63.7%) fresh weigh and dry weight respectively. In presence of lead in all concentrations Fe-O-NP treated V. radiata L. exhibited minimal growth in fresh and dry weight ranging from 24–31%. Which is less than the Fe-O-NP treated V. radiata L. growth observed in Cr and Cd stress in 10 and 20 µg/mL concentrations. A similar pattern was observed in leaf length, breadth, and chlorophyll content in Fe-O-NP-treated V. radiata L. in the presence of different concentrations of Cr, Cd, and Pb. At 10 and 20 µg/mL of Cr and Cd increase in leaf breadth was prominent with 24.3–24.1% was no prominent difference in leaf breadth and length was observed in other concentrations (Fig. 13 e) (Fig. 13 f) (Fig. 13 g).Thus we can say that the best variations in phenotypic traits root length, shoot length, fresh and dry weight, leaf length, leaves breadth, and photosynthetic pigment were observed in Fe-O-NP treated V. radiata L. in Cr and Cd at 10 µg/mL and 20 µg/mL of stress. Roots appear to transport green synthesized Fe-O-NPs into plant tissues when they are treated with the synthesized nanoparticles and grown in Hoagland's solution. The increased uptake of potassium, phosphorus, and nitrogen in the presence of iron is thought to be the main reason for the increase in growth and total biomass of plants treated with Fe-O-NP. Fe-O-NP also works against the negative effects of sodium and chloride ions (Tawfik et al. 2021 ). According to reports, cadmium accumulation in crops decreased when the concentration of Fe-O-NPs increased (Sebastian et al. 2017 ; Rizwan et al. 2019 ). Similarly, Fe and Fe 3 O 4 NPs were found to be more effective than other methods in reducing arsenic (As) in rice plants (Lux et al. 2011 ; Huang et al. 2020 ), have reported that cadmium and iron have the same transport pathways during their uptake in plants, and under the situation of iron deficiency, iron transporters are activated, reducing the cadmium uptake and accumulation in crop plants. (Bashir et al. 2018 ) have suggested that due to competitive adsorption, the accumulation of heavy metals like cadmium is reduced in the presence of higher concentrations of iron. 3.7. Effect of Fe-O-NPs 50µg/mL and TIU16A3 on V. radiata L. growth and photosynthetic content under heavy metal stress In that study, different concentrations of Cr, Cd, and Pb (i.e. 0 µg/mL, 10 µg/mL, 20 µg/mL, 40 µg/mL, and 80 µg/mL) were used to grow V. radiata L. seedlings. In addition, TIU16A3, seeds were treated with 50 µg/mL green synthesized Fe-O-NP. Figures 12 and 13 show information on shoot length, root length, fresh weight, dry weight, secondary root length, and number. Figure 14 shows the data on the length and width of the leaves and the total chlorophyll content. The findings showed that, in V. radiata L. seedlings in the presence of rising levels of Cr, and Cd considerably reduced biomass when compared to plants grown with the application of TIU16A3 and Fe-O-NP, the application of these two substances to the seeds of V. radiata L resulted in a significant increase in shoot length, root length, fresh weight, dry weight, secondary root length, leaves length, breadth, and content of total chlorophyll of Cr, Cd and Pb stress. The Fe-O-NP with PGPB ( P. geniculata ) had the best effect on Cr and Cd (10 µg/mL, 20 µg/mL), followed by 40 µg/mL and 80 µg/mL, according to our results. Pb exhibited growth but was least effective which is why our further studies were focused on the effective HM concentrations. There is a direct correlation between plant growth and biomass, which increased up to 90.89% more after the augmentation of TIU16A3 and 50 µg/ml Fe-O-NP in combination under heavy metal toxicity. Total chlorophyll contents were 69% and 63% respectively in the presence of Fe-O-NP alone, but the increment was 83% in combination as depicted in Fig. 15 .Fe-O-NP 50µg/mL + TIU16A3 exhibited a synergistic effect on root growth under heavy metal Cr, and Cd stress and the effectiveness of combined treatment was maximum in Cr 84.6%, followed by Cd 92% as illustrated in Fig. 12 . This is maximum growth compared to TIU16A3 treatment and Fe-O-NP 50µg/mL. Thus, we can say that the combinatorial effect is best for V. radiata L. growth in the presence of Cr and Cd strain is most effective at lower concentrations of Cd and Cr. This exponential increase in the phenotypic traits of V. radiata L. in the presence of TIU16A3 and Fe-O-NP 50 µg/mL together than individually could be due to the acceleration in the accumulation of mineral nutrients from NPs. Compared to the use of PGPR or NP alone, the combined use of both can improve crop productivity, plant height, dry-fresh biomass, and seed germination frequency (Medina-Velo et al. 2017 ). This combination can work in many different ways. Improved plant performance and productivity result from direct mechanisms including PGPR, regular production of plant hormones (e.g., indoleacetic acid, siderophores, etc.), and increased soil mineral availability through phosphate solubilization and N 2 fixation. The presence of NPs can help PGPR tolerate higher density while providing a nutrient-rich substrate that improves PGPR efficiency and increases plant production (Rani et al. 2008 ; Mushtaq et al. 2020 ). In the presence of heavy metal contamination by exploiting the ability of PGPR to release exopolysaccharides under dry conditions, plants can increase their stress tolerance (Bishnoi Saran 2015 ; Vurukonda et al. 2016 ). 3.8. Effect of 50 µg/mL Fe-O-NPs and TIU16A3 on superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), H 2 O 2 content, and electrolyte leakage (EL) in Vigna radiata L. under heavy metal stress Cd and Cr stress significantly increased H 2 O 2 content and EL value; however, when treatment of 50 µg/mL Fe-O-NP and TIU16A3 were given on Vigna radiata L. the leakage percentages of H 2 O 2 and EL of the respective controls were reduced by 37.5% and 43% and 21.5% and 26%, respectively (Fig. 15 d), and (Fig. 15 e). Activities of invertase and catalase in soil were significantly higher when P. geniculata was used with NPs than when NPs were used alone (Khanna et al. 2021 ). In summary, the results show that PGPR with NPs increases the enzymatic activity of sucrose, urease, protease, and invertase in addition to soil phosphomonoesterase under both acidic and basic conditions. There is a direct correlation between soil nutrients and the enhanced enzyme activity of antioxidant-responsive genes caused by PGPR and NPs (Mushtaq et al. 2020 ). 3.9. Effect of 50µg/mL Fe-O-NPs and TIU16A3 on metal uptake by plant Vigna radiata L. By Flame Atomic Absorption Spectroscopy (FAAS) analysis of the total metal contents and the accumulation was done. The results show that the plant tissues accumulate more Cr and Cd (10 µg/mL, and 20 µg/mL) in non-inoculated plants in comparison to 50 µg/ml Fe-O-NP and TIU16A3inoculated. FAAS data of Cr and Cd metals in the presence of synergistic treatment of 50 µg/mL Fe-O-NP and TIU16A3 showed that there was a reduction in metal content by30.6% in comparison to individual treatment as depicted in (Fig. 16 a), and (Fig. 16 b). Fe-O-NP and TIU16A3 combined increased soil enzyme (dehydrogenase) more than NPs, and P. geniculata alone which must have resulted in better biological remediation. Previous studies have shown that the combined use of PGPR and NP decreased Cd levels more effectively than the use of NP and PGPR alone. TIU16A3 and NPs together reduced significantly the harmful effects of chromium and promoted plant growth. This may be because PGPRs and NPs limit the availability of metals for uptake and translocation in soil (Mitra et al. 2018 ). 3.10. Effect of 50µg/mL Fe-O-NPs andTIU16A3 synergistically on gene expression of Aux/IAA of shoot tissue in V. radiata L. under heavy metal stress In the present study, gene expression profiling of auxin-responsive genes in seedlings under Cr and Cd metal stress has been studied through qRT-PCR which exhibits a high expression of the gene of interest in the presence of 50 µg/mL Fe-O-NPs and TIU16A3 together as shown in Fig. 17 . 4. Conclusion This study aimed to determine whether Eichhornia crassepis plant extract and green synthesized iron oxide nanoparticles (Fe-O-NP) could protect Moong bean plants against heavy metal stress. The deleterious effects of metal stress on plants were confirmed by significantly increased electrolyte leakage and hydrogen peroxide (H 2 O 2 ) levels. Increases in biomass, photosynthetic pigments, nutrients, and indole acetic acid in plants treated with Fe-O-NP and PGPR and exposed to all concentrations of heavy metal stressors, all of which improved plant growth. Reduction of heavy metals by treatment is associated with improved plant growth based on Flame Atomic Absorption Spectrophotometry. TIU16A3 and Fe-O-NPs work together to reduce antioxidant enzymes, mainly catalase, peroxidase, and superoxide dismutase, which signifies that the synergistic treatment reduced the stressed condition in the plant. In addition, significant reductions in electrolyte leakage and H 2 O 2 indicated that this treatment alleviated heavy metal stress. The results of this study indicated that plants can be protected against single and combined heavy metal stress caused by Cr, and Cd through the complementary effects of PGPR and Fe-O-NPs. Therefore, Fe-O-NPs are suitable for agricultural applications in living organisms instead of chemically synthesized Fe-O-NPs due to their negligible side effects. Eco-friendly production and development of super-stabilized nanoparticles is promising because it uses few harmful chemicals and high energy consumption. In addition to enhancing plant tolerance, the interaction of PGPR and Fe-O-NPs opens the door to long-term increases in yield. By combining the strengths of these two factors, we can open new opportunities to improve plant health and productivity, an important step forward in the search for sustainable agricultural methods. Declarations Acknowledgment The authors thank Techno India University, West Bengal for their encouragement and support during this study. Author’s contribution P.D. and A.K. designed and conceptualized the experiment. B.M. performed the experiments, synthesis and material characterization, data analysis, and paper writing. S.B. performed the synthesis and material characterization of the sample, data analysis, and paper writing. S.C. helped in editing. All authors have checked the current form of this manuscript. Data availability This article incorporates all the data examined during this research. Compliance with ethical standards Ethics statement This study did not involve any human or animal participants to conduct the research work. Informed consent No patient data were reported. Competing interests The authors have no competing interests to declare that are relevant to the content of this article. 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PLoS One 17:e0277101. https://doi.org/10.1371/journal.pone.0277101 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4863542","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":348960748,"identity":"19f25fc9-9642-4121-a2ea-b25dced90ed4","order_by":0,"name":"Barkha Madhogaria","email":"","orcid":"","institution":"Techno India University","correspondingAuthor":false,"prefix":"","firstName":"Barkha","middleName":"","lastName":"Madhogaria","suffix":""},{"id":348960751,"identity":"b2d9070e-b6c3-4906-b308-16162e9c8349","order_by":1,"name":"Sangeeta Banerjee","email":"","orcid":"","institution":"Techno India 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8","display":"","copyAsset":false,"role":"figure","size":96994,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFusarium sp.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e on Czapex dox agar Fig a, b, and c in absence of biogenic Fe-O-NP fig c, d, and e in the presence of biogenic Fe-O-NP.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4863542/v1/c72316c879bddb9735284053.png"},{"id":64055868,"identity":"060e1707-0df2-47d0-ab44-481041311520","added_by":"auto","created_at":"2024-09-05 18:51:03","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":9570,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHemolytic efficacy of biogenic Fe-O-NP at different concentrations. Similar alphabets in the plotting area depict no significance at P\u0026lt;0.05, and different alphabets in the plotting area depict significant differences in different treatment groups. Each value indicates the mean ± SE (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e =3)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4863542/v1/21cfc4e62cc2ac9f9f7e0c81.png"},{"id":64055878,"identity":"3d9081da-c309-4feb-9e3e-7389ee303227","added_by":"auto","created_at":"2024-09-05 18:51:04","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":157495,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of 25 µg/mL, 50 µg/mL, and 100 µg/mL Fe-O-NP on growth in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eV. radiata \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eL.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-4863542/v1/5cc0191bdc52c28d03707fef.png"},{"id":64055879,"identity":"15969d22-ea08-4866-9145-2bf027fdc153","added_by":"auto","created_at":"2024-09-05 18:51:04","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":678693,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of 25 µg/mL, 50 µg/mL, and 100 µg/mL Fe-O-NP on morpho-physiological parameters, shoot length, root length, fresh weight, dry weight, number of secondary roots, length of secondary root, leaves length, and leaves breadth in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eV. radiata \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eL. . Similar alphabets in plotting area depict no significance at P\u0026lt;0.05, and different alphabets in plotting area depict significant difference in different treatment groups. Each value indicates the mean ± SE (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e =15)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-4863542/v1/9589e1be5007c987be9764f1.png"},{"id":64055876,"identity":"29e8b3da-761d-42a7-a853-3787988720b8","added_by":"auto","created_at":"2024-09-05 18:51:04","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":493201,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlant body exhibiting detrimental effect of Cd, Cr and Pb stress on phenotypic variation in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVigna radiata\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e L. Recovery of phenotypic traits of plants treated with Fe-O-NP (50 µg/mL).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-4863542/v1/ea6ab89214c84e334f71761f.png"},{"id":64055871,"identity":"060cf889-6edd-4756-b0cd-ff307435c5c6","added_by":"auto","created_at":"2024-09-05 18:51:04","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":127076,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of standardized Fe-O-NP on morpho-physiological parameters in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eV. radiata \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eL. under different concentrations of Cd, Cr, and Pb stress. (a)-(g) HM induced deterioration in different morphological parameters and Fe-O-NP induced recovery in root length, shoot length, fresh weight, dry weight, leaves length, leaves breadth, and chlorophyll content. Similar alphabets in plotting area depict no significance at P\u0026lt;0.05, and different alphabets in plotting area depict significant difference in different treatment groups. Each value indicates the mean ± SE (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e =15).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-4863542/v1/406b0013416d73f71343d9bb.png"},{"id":64056063,"identity":"ec5c2190-b04e-4c47-9275-057867deba04","added_by":"auto","created_at":"2024-09-05 18:59:04","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":767239,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of standardized Fe-O-NP + TIU16A3 on morpho-physiological parameters in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eV. radiata \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eL. under different concentrations of Cd, and Cr stress. (a)-(i) HM induced deterioration in different morphological parameters and Fe-O-NP + TIU16A3 induced recovery in root length, shoot length, fresh weight, dry weight, secondary root number, secondary root length, leaves length, leaves breadth, and total chlorophyll content. Similar alphabets in the plotting area depict no significance at P\u0026lt;0.05, and different alphabets in the plotting area depict significant differences in different treatment groups. Each value indicates the mean ± SE (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e =15)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-4863542/v1/b25419a299062838953cc8d1.png"},{"id":64056061,"identity":"d116ca7a-1d71-45e9-978f-6f90e36621a2","added_by":"auto","created_at":"2024-09-05 18:59:04","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":38041,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of standardized Fe-O-NP + TIU16A3 on morpho-physiological parameters in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eV. radiata \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eL. under different concentrations of Cd, and Cr stress. (a)-(e) HM induced deterioration in different morphological parameters and Fe-O-NP + TIU16A3 induced recovery in POD, CAT, SOD, H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2,\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e and EL % content. Similar alphabets in the plotting area depict no significance at P\u0026lt;0.05, and different alphabets in the plotting area depict significant differences in different treatment groups. Each value indicates the mean ± SE (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;=15)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"15.png","url":"https://assets-eu.researchsquare.com/files/rs-4863542/v1/bb5d1a2582e9ef62ef385cfe.png"},{"id":64056275,"identity":"f960d68a-f61a-42b1-8410-a02f1f91c168","added_by":"auto","created_at":"2024-09-05 19:07:04","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":23956,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlame Atomic absorption spectrophotometer analysis of heavy metal content in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eV. radiata\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e L. Fig. a cadmium (Cd) content in TIU16A3 + 50 µg/mL Fe-O-NP treated plant. Fig. b chromium (Cr) content in TIU16A3 + 50 µg/mL Fe-O-NP treated plant. Similar alphabets in the plotting area depict no significance at P\u0026lt;0.05, and different alphabets in the plotting area depict significant differences in different treatment groups. Each value indicates the mean ± SE (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e =15)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"16.png","url":"https://assets-eu.researchsquare.com/files/rs-4863542/v1/2a4cc491b2c4c6c28307397f.png"},{"id":64055881,"identity":"24d5cb58-0d55-4204-b0a3-fc204ae8b004","added_by":"auto","created_at":"2024-09-05 18:51:04","extension":"png","order_by":17,"title":"Figure 17","display":"","copyAsset":false,"role":"figure","size":251325,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene expression analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAux/IAA \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egene of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVigna radiata \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eL. by RT-qPCR under the stress of heavy metals chromium (Cr), and cadmium (Cd) treated with Fe-O-NP (50 µg/mL) alone and Fe-O-NP (50 µg/mL) + TIU16A3. Similar alphabets in the plotting area depict no significance at P\u0026lt;0.05, and different alphabets in the plotting area depict significant differences in different treatment groups. Each value indicates the mean ± SE (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e =15)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"17.png","url":"https://assets-eu.researchsquare.com/files/rs-4863542/v1/fb2c03310791a66773a22c7a.png"},{"id":64059764,"identity":"03292696-a889-414f-a5b9-207a32a576bd","added_by":"auto","created_at":"2024-09-05 22:48:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5509506,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4863542/v1/8ad78b4d-ad0d-4736-b14b-29c1d4e3366b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synergistic impacts of plant growth promoting Pseudomonas geniculata TIU16A3 and green-synthesized iron-oxide nanoparticles from Eichhornia crassipes for the amelioration of heavy metal stress in Vigna radiata L.","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eNanoparticles are reported to aid in plant growth promotion and alleviate heavy metals and the sustainable approach of synthesizing potential iron oxide nanoparticles (Fe-O-NP) has been studied.\u003c/li\u003e\n \u003cli\u003eWater hyacinth serves as a reducing and capping agent for the production of desired Fe-O-NP.\u003c/li\u003e\n \u003cli\u003eEfficiency of TIU16A3 (accession number OR507186)\u0026nbsp;and biogenic Fe-O-NP to bio-remediate soil contaminated with heavy metals have been first reported in the present study.\u003c/li\u003e\n \u003cli\u003eCombinatorial strategy effect on the alleviation of heavy metal and growth of economically important legume \u003cem\u003eVigna radiata\u003c/em\u003e L. is a breakthrough research so far.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eEconomical and sustainable eradication of water hyacinths by using them to produce non-toxic nanoparticles.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eThe tanning process used in the leather industry uses chromium (Cr) and other hazardous chemicals. It is said that 80\u0026ndash;90% of leather is tanned with basic chromium sulfate (BCS) and only 50\u0026ndash;70% of Cr is fixed in chromium tanning; the remaining Cr is released into wastewater(Saravanabhavan et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). According to(Gupta et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), the leather industry is the largest producer of heavy metals in a nearby water facility. The sludge is also used for irrigation, which causes heavy metal (HM) contamination in agricultural land. When sludge is used for irrigation, heavy metals such as Cd, Zn, Cr, Ni, Pb, and Mn can accumulate in surface soils and enter groundwater or soil solutions that can be taken up by plants(Mondal et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Before the independence of West Bengal, three agglomerations of Kolkata - Tangra, Topsia, and Tilala - saw the activity of the leather industry. However, the leather industry moved about 20 kilometres from Kolkata to Bantala, where they are now located at the Calcutta Leather Complex (CLC). More than 300 tanneries moved to CLC due to the Supreme Court decision. CLC is supplied with a Common Effluent Treatment Plant (CETP), which uses several methods to treat a significant amount of wastewater from nearby tanneries before it is released back into the environment. Liquid and solid waste from tanneries and other factories entering water bodies adversely affects the natural ecosystem of the Ramasar area and can damage major waterways due to the 12,500-hectare East Kolkata Wetland adjacent to Bantala. It is generally known that heavy metal pollution negatively affects plant germination, photosynthesis, yield, and mineral nutrition (Kundu et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Banerjee et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). When animals and humans eat plants contaminated with heavy metals, the metals are absorbed through the food chain into their cells and tissues can damage the intestinal microbiota and cause several chronic diseases in humans (Madhogaria et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Heavy metals negatively affect dry matter, root and shoot growth (Naz et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Many studies have shown that certain bacterial species can effectively promote bioremediation of metal-contaminated soil. \u003cem\u003ePseudomonas\u003c/em\u003e sp., \u003cem\u003eEnterobacter cloacae\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e sp., and other metal-resistant PGPBs are already been studied for the purpose (Zulfiqar et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Biosorbents are one of the many technologies available to remove heavy metals from the environment(Banerjee et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Madhogaria et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). In recent decades, nanotechnology has become a central area of ​​research. Researchers are paying more attention to a green approach to produce nanoparticles (NPs) without harming the environment by using an aqueous solution of plant extract. The effect of metal oxide nanoparticles on plants has been studied previously(Kiwumulo et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Plant extracts contain sugars, terpenoids, polyphenols, etc. which can stabilize and reduce metal nanoparticles. Functional groups and phenolic compounds have been shown to contribute to the formation of metal nanoparticles. The International Union for Conservation of Nature (IUCN) has listed water hyacinth (\u003cem\u003eEichhornia crassipes\u003c/em\u003e) as one of the 100 most popular invasive and aggressive species that reproduce quickly. Under the right conditions, biomass can double in just a week, causing some problems for waterways. The green synthesis of Fe-O-NPs by \u003cem\u003eE. crassipes\u003c/em\u003e has not yet been investigated, and the plant growth properties of these NPs have hardly been documented in the literature. Iron is oxidizable, so instead of reducing Fe\u003csup\u003e2+\u003c/sup\u003e to FeO, the phytochemicals in the extract react with iron ions to form iron oxide nanoparticles. Iron oxide nanoparticles, or Fe-O-NPs, were chosen over other iron oxide phases due to their infrared, magnetic, and optical properties and stability. TEM, FTIR, UV-Vis spectroscopy, and X-ray diffraction confirm the morphology and presence of nanoparticles. Fe affects important physiological functions in plants, such as respiration, redox reactions, and chlorophyll production. In addition, the large surface area of ​​Fe-O-NPs acts as a platform to reduce nutrient losses and ensure the timely delivery of nutrients to plants. Fe-O-NPs have recently been used directly to improve plant growth and alleviate drought stress and heavy metal pollution(Rizwan et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Treatment of plants with Fe-O-NPs has also been studied in connection with the solubilization of heavy metals.\u003c/p\u003e \u003cp\u003eAgricultural applications of Fe-O-NPs are an exciting area of ​​current research. Compared to other nanoparticles, Fe-O-NPs are toxic to soil bacterial communities. In addition, they support seed germination and maintain soil pH and nutrient levels(Rastogi et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Previous studies have investigated how Fe-O-NPs can increase crop growth and yield by accelerating photosynthesis and altering several other metabolic processes. Although PGPR and Fe-O-NP have established distinct roles, little is known about their functions. Their small size and large surface area help ​​them to cause strong adsorption of various molecules, which increases their effectiveness(Nayana et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). There are no studies on the synergistic and comparative use of Fe-O-NP and PGPR to reduce heavy metal stress in \u003cem\u003eVigna radiata\u003c/em\u003e L. There is a theory suggesting that PGPR and Fe-O-NP are important in stressed plants. This work aimed to investigate the relative and synergistic effects of heavy metal-resistant PGPR and Fe-O-NP isolated from tannery wastewater-affected soil on germination and growth of \u003cem\u003eV. radiata\u003c/em\u003e L. under heavy metal stress, especially Cr and Cd.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals\u003c/h2\u003e \u003cp\u003eThe raw materials utilized in the production of Fe-O-NPs from \u003cem\u003eEichhornia crassipes\u003c/em\u003e, namely Fe\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO (98.5%), ethanol (99%), and NaOH (97%), were procured from LOBA CHEMIE PVT. LTD. \u003cem\u003eEichhornia crassipes\u003c/em\u003e, utilized herein, were procured from the pond site situated near Techno India University in West Bengal. Luria Bertini broth, Mercuric chloride (95%), Mueller Hinton agar, Czapek dextrose agar, phosphate buffer solution (PBS), H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (95%), NaCl (95%), and EDTA (95%), and Triton-X100 were purchased from Hi-media.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of Water hyacinth-mediated biogenic production of iron oxide nanoparticles\u003c/h2\u003e \u003cp\u003eWith some additional adaptations, we followed the protocol of (Periakaruppan et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) for the green synthesis of Fe-O-NP. Five grams of water hyacinth leaves were weighed. Thorough cleaning and washing were done under running water and again with double distilled water. Leaves were ground and 100 ml distilled water was added and boiled for 20\u0026ndash;30 mins with constant stirring. After boiling the extract was filtered through Whatman No. 1 filter paper. The filter was then stored at 4\u0026deg;C in a sterile beaker for later use. After continuous stirring, 20 mL of water hyacinth leaf extract was added dropwise to 80 mL of aqueous ferrous sulfate heptahydrate (Fe\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO) (0.1 M). NaOH was added to the mixture to obtain and then maintained a pH of 12. After centrifuging the precipitated liquid at 10,000 rpm for 30 minutes, impurities were removed by washing two or three times with Millipore water. To air dry the assortment, it was placed in a hot air oven at 120\u0026deg;C for 60 minutes. For further analysis and evaluation, the pellet was crushed and kept in a sterile container. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Material characterization\u003c/h2\u003e \u003cp\u003eX-ray diffraction was used to study the crystallinity, phase purity, and crystallite size of Fe-O-NP. The hydrodynamic size of Fe-O-NP and their distribution pattern were measured using dynamic light scattering, with some modifications as described (Dhak et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The UV-visible spectrum of Fe-O-NP synthesized by green methods was done using a Hitachi 330 spectrophotometer. Elemental composition of Fe-O-NP was done using energy-dispersive X-ray spectroscopy (EDS), while scanning electron microscopy (SEM) was used to study and record the surface morphology and approximate shape of the particles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Antibacterial capacity of Fe-O-NPs against PGPR TIU16A3\u003c/h2\u003e \u003cp\u003eThe antibacterial activity of iron oxide nanoparticles prepared from \u003cem\u003eE. crassipes\u003c/em\u003e extracts against TIU16A3 was evaluated by the agar well diffusion method. Zones of inhibition were evaluated to determine the degree of inhibition of bacterial growth.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Antifungal analysis of Fe-O-NPs on growth of \u003cem\u003eFusarium\u003c/em\u003e sp.\u003c/h2\u003e \u003cp\u003eThe antifungal activity of Fe-O-NP was analyzed with \u003cem\u003eFusarium\u003c/em\u003e sp., obtained from the Department of Plant Biology, Bose Institute, and Kolkata. Using Fe-O-NP, the fungal strain was inoculated onto Cazpek dextrose agar (CDA). Growth was examined on days three, six, and nine. Analysis was done based on colony diameter measurements using the formula for PIRG (percentage inhibition in radial growth) % = (R1‒ R2\u0026thinsp;\u0026divide;\u0026thinsp;R1) \u0026times; 100 (Eq.\u0026nbsp;2), where R1 is the radial growth of the control and R2 is the radial growth of each treatment was used to determine the percentage of inhibition of fungal growth(Yazid et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Hemolytic analysis\u003c/h2\u003e \u003cp\u003eThe hemolysis assay typically uses fresh human blood samples from healthy adult volunteers stabilized with ethylenediaminetetraacetic acid (EDTA). With some modifications, the test was performed as previously mentioned (Modi et al. 2023). From serum, Red blood cells (RBC) were first separated by centrifugation at 2000 rpm for 10 min. After that, 5 ml of blood was added to 10 ml of 50 mM PBS. RBCs were then washed three more times using 50 mM PBS. A hematocrit of 5% was then used to dilute the purified erythrocytes in PBS. 100 \u0026micro;l of erythrocyte suspension was added to an eppendorf tube containing 100 \u0026micro;l of Fe-O-NPs at different concentrations (50, 100, 150, and 200 \u0026micro;g/mL). After one hour incubation at 37\u0026deg;C, centrifugation at 1500 rpm for ten minutes was done. The 80 \u0026micro;l supernatant was then added to PBS. Absorbance at 414 nm OD was measured using a plate reader. A positive control of 100% hemolysis was achieved using 0.1% Triton X-100 (v/v), while a negative control using PBS. The percentage of hemolysis was calculated using formula\u003c/p\u003e \u003cp\u003e% hemolysis = (OD of sample-OD of PBS) / (OD of positive control-OD of PBS) \u0026times; 100 (Eq.\u0026nbsp;3)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Priming seeds of \u003cem\u003eVigna radiata\u003c/em\u003e L., germination, and transfer of seedlings in hydroponics culture\u003c/h2\u003e \u003cp\u003eFor seed priming, the seed of \u003cem\u003eVigna radiata\u003c/em\u003e L. (mung bean) procured from West Bengal State Seed Corporation Limited, Kolkata, West Bengal was used. Surface sterilization, priming with Fe-O-NP alone and synergistic with \u003cem\u003ePseudomonas geniculata\u003c/em\u003e TIU16A3, germination, and seedlings were transferred to a hydroponics solution for further experiments were done following the method(Kundu and Ganesan \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e; Madhogaria et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Standardization of Fe-O-NP for plant growth promotion\u003c/h2\u003e \u003cp\u003eTreatments included control and different concentrations of Fe-O-NPs (25, 50, and 100 \u0026micro;g/mL) in three replicates were chosen for this analysis. Fe-O-NP treatment with maximum germination of \u003cem\u003eV. radiata\u003c/em\u003e L. seedlings and maximum increment in different phenotypic plant traits (root length, shoot length, fresh weight. dry weight, secondary root number, secondary root length, leaves breadth, and leaves length)was selected for further experiment for analysis of heavy metal alleviation and plant growth promotion of \u003cem\u003eV. radaiata\u003c/em\u003e L. in the presence of Cd, Cr, and Pb stress following the methods with some modifications by(Kundu and Ganesan \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.9. Phenotypic differences between untreated, individually treated, and co-treated\u003c/b\u003e \u003cb\u003eVigna radiata\u003c/b\u003e \u003cb\u003eL. plants and total biomass studies in the presence of heavy metal toxicity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe difference in plant growth was observed after 14 days of reading with the following treatments: untreated (UT), Fe-O-NP 50 \u0026micro;g/ml treated (T), TIU16A3 \u003cem\u003eP. geniculata\u003c/em\u003e treated (T), and Fe-O-NP (50 \u0026micro;g/ml)\u0026thinsp;+\u0026thinsp;TIU16A3 treated plants in the presence and absence of heavy metals (chromium, cadmium, and lead) was done as described by(Madhogaria et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e). The traits analyzed for plant growth variation are primary root length, shoot length, dry weight, wet weight, leaf width, leaf length, total chlorophyll content, number of secondary roots, and secondary root length(Kundu and Ganesan \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Antioxidant SOD, POD, CAT, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content, and Electrolyte leakage (EL) analysis\u003c/h2\u003e \u003cp\u003eDetermination SOD, POD, CAT, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2,\u003c/sub\u003e and electrolyte leakage estimation was done as described by (Madhogaria et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e) with some modifications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Determination of HMs (Cr, Cd, and Pb) content\u003c/h2\u003e \u003cp\u003eA 3:1 mixture of sulfuric and nitric acid was used to digest 100 mg of dry plant material. Further analysis was done as described by \u003cb\u003e(\u003c/b\u003eMadhogaria et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e) with some modifications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Analysis of gene expression by RT-qPCR\u003c/h2\u003e \u003cp\u003eThe transcript levels of gene \u003cem\u003eAux/IAA\u003c/em\u003e of shoot tissue of \u003cem\u003eVigna radiata\u003c/em\u003e L. plant in the presence of 10, and 20 \u0026micro;g/mL of HMs Cr, and Cd stress with Fe-O-NP alone and \u003cem\u003eP. geniculata\u003c/em\u003e\u0026thinsp;+\u0026thinsp;50\u0026micro;g/mL Fe-O-NPs were determined by real-time PCR following the method with some medication by (Madhogaria et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll the tests were performed in triplicates to avoid any errors and to increase the accuracy. All graphical analyses were performed using Graph pad prism 8.0.2 and Origin Pro 8.5.0. Statistical significance differences among different treatments were done using Duncan\u0026rsquo;s multiple range test (DMRT) using software IBM SPSS version 16. Statistical probability P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was followed.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussions","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Material characterization\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1. UV-visible spectroscopy\u003c/h2\u003e \u003cp\u003eUV\u0026ndash;Vis absorption spectrum showed a wide absorption peak at 595 nm. The firm transmission peaks were observed at 568nm with 84.54% transmission, 588nm with 89.8% transmission, 595nm with 91.38% transmission, and 629nm with 91.51% transmission. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e depicts the transmission spectra of \u003cem\u003eE.crassipes\u003c/em\u003e mediated biogenic synthesis of Fe-O-NPs falls between 400\u0026ndash;700 nm. According to (Basavegowda et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) using \u003cem\u003eA. annua\u003c/em\u003e and \u003cem\u003eP. frutescens\u003c/em\u003e green synthesized iron oxide NP had a similar range by which we can confirm that metal-oxide nanoparticles are being formed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2. X-ray diffraction analysis\u003c/h2\u003e \u003cp\u003eThe X-ray diffraction (XRD) patterns were analyzed with the JCPDS card file 19\u0026ndash;0629 89\u0026ndash;0596, confirming the formation of amorphous magnetite iron oxide nanoparticles. The scanning speed is 10\u003csup\u003e0\u003c/sup\u003e/min. The amorphous nature and composition of Fe-O-NP were evaluated over a 2θ range from 30\u003csup\u003eo\u003c/sup\u003e to 70\u003csup\u003eo\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The diffraction peaks were present at 35\u003csup\u003e◦\u003c/sup\u003e (104), 42\u003csup\u003e◦\u003c/sup\u003e (113), 52\u003csup\u003e◦\u003c/sup\u003e (018), and 64\u003csup\u003e◦\u003c/sup\u003e (024). The average crystallite size of Fe-O-NP was 15.5 nm, calculated using the Scherrer equation. The intensity of diffraction peaks signifies good crystallinity of the synthesized nanoparticles. Moreover, similar results have been reported by (Yew et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yadav et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.1.3. Dynamic light scattering analysis\u003c/h2\u003e \u003cp\u003eThe intensity-average diameter of Fe-O-NP was calculated at 1026 nm by using dynamic light scattering (DLS) analysis. The hydrodynamic size distribution of Fe-O-NP is shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). It is the core size and the presence of capping elements around it. NPs were at moderate stability and exhibited excellent dispersion in an aqueous medium.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.1.4. Scanning electron microscopy wit henergy-dispersive X-ray mapping\u003c/h2\u003e \u003cp\u003eStructural analysis of biogenic Fe-O-NP can be done precisely by scanning electron microscopy (SEM). Fe-O-NPs surface morphology and elemental composition are illustrated in (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). As obtained by using SEM analysis, respectively. Energy dispersive X-ray (EDX) analysis reported the presence of 32.62% iron and 34.97% oxygen along with 31.88% carbon due to the availability of phyto-phenolic compounds in Fe-O-NP synthesized from \u003cem\u003eE. crassipes\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Similar results were reported by (Periakaruppan et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Antibacterial evaluation\u003c/h2\u003e \u003cp\u003eMinimum inhibitory concentration MIC is evaluated for our synthesized Fe-O-NP against TIU16A3 (accession no. OR507186). We observed that Green synthesized Fe-O-NP has no antibacterial effect against TIU16A3 as no zone of inhibition was formed (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e). We can imply from this result that the green synthesized Fe-O-NPs from \u003cem\u003eE. crassipes\u003c/em\u003e andTIU16A3can be used in combination on the seed of \u003cem\u003eV. radiata\u003c/em\u003e L.to study synergistic effect on heavy metal mitigation and plant growth promotion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Antifungal evaluation\u003c/h2\u003e \u003cp\u003eBiogenic Fe-O-NP synthesized in this study revealed remarkable antifungal potency against \u003cem\u003eFusarium\u003c/em\u003e sp. when compared to the control Fig.\u0026nbsp;8. Radial growth size of fungus in the presence of 25, 50, and 100\u0026micro;g/mL was measured to be 25mm, 16mm, and 17mm respectively. Percent inhibition of radial growth (PIRG) was calculated with the help of Eq.\u0026nbsp;2 and PIRG% for 25 \u0026micro;g/mL was 44.4%, similarly for 50 \u0026micro;g/mL, the PIRG% was 64.4%, and for 100 \u0026micro;g/mL 62.2% was the PIRG%. This result suggested that as the concentration of Fe-O-NP is increasing, inhibition of the fungus growth is also increasing. The gradual increment of the inhibition potential rate of green-synthesized Fe-O-NPs may be due to their high effect on the growth of fungal mycelium. The biogenic Fe-O-NPs synthesized from the leaf extract of \u003cem\u003eEuphorbia herita\u003c/em\u003e showed excellent antifungal activity against \u003cem\u003eAspergillusniger\u003c/em\u003e, \u003cem\u003eArthogrophis cuboida\u003c/em\u003e, and \u003cem\u003eAspergillus fumigatus\u003c/em\u003e in the range of 10\u0026ndash;30 mg/mL (Win et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Another study confirmed the potential antifungal activity of Fe-O-NPs synthesized from \u003cem\u003eChlorella\u003c/em\u003e-K01 extract against \u003cem\u003eFusarium oxysporum\u003c/em\u003e, \u003cem\u003eFusarium tricinctum\u003c/em\u003e, and \u003cem\u003eFusarium maniliforme\u003c/em\u003e (Cruz-Luna et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Thus, the present findings and previous reports signify the efficiency of biogenic Fe-O-NPs usage as potent natural fungicides in comparison with chemical-toxic fungicides. In addition, the application of biologically synthesized Fe-O-NPs affirms fungal resilience resulting from the extensive use of chemical fungicides. The interaction of NPs with fungal cells might lead to the elimination of cytoplasmic membranes (Ahmed et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The disruption of the fungal cell membrane is attributed to the secretion of micro-ions such as potassium and phosphate radicals, along with large-sized molecules such as RNA, DNA, and other intracellular substances (Ojoma et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Hemolytic activity\u003c/h2\u003e \u003cp\u003eThe hemolytic effect of biogenic Iron oxide NP was carried out by Human RBC hemolytic assay in the presence of different Fe-O-NP concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). At concentrations of 50, 100, 150, and 200 \u0026micro;g/mL of biogenic Fe-O-NP, the hemolysis level was below 15% even at the highest concentration (200 \u0026micro;g/mL) in comparison of the value of 0.1% Triton X100 (positive control) mediated hemolysis. The reason behind the non-toxic behavior against human RBC of our green synthesized is that it has been previously studied that biogenic Fe-O-NPs are biocompatible, biodegradable, and potentially nontoxic to humans (Wahajuddin and Arora 2012).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Standardization of different concentrations of biogenic Fe-O-NP for the promotion of plant growth\u003c/h2\u003e \u003cp\u003eFe-O-NP effect on seedling growth and different growth parameters, in \u003cem\u003eV. radiata\u003c/em\u003e L. plants, to understand its mode of action and to establish an optimal concentration for plant growth promotion. We used 25, 50, and 100 \u0026micro;g/mL for the test. Seed priming was done, Non-primed seeds were kept as control. We found out that seed germination was highest at 50 \u0026micro;g/mL of Fe-O-NP concentration at which root length was 65.2 mm in length when compared to control 23.5 mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e) Analysis of different phenotypic plant traits was done and we found out that at 50 \u0026micro;g/mL of Fe-O-NP concentration, shoot length increased up to 38% in comparison to 25 and 100 \u0026micro;g/mL. The secondary root number and its length showed more growth in 50\u0026micro;g/mL of Fe-O-NP concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). Similar results were seen in the case of the length and breadth of leaves. If we cumulatively analyze the growth in all the plant traits in different concentrations we can say that growth was 54.4% higher in 50 \u0026micro;g/mL than in 25, and 100 \u0026micro;g/mL of Fe-O-NP treated plants. Fe-O-NPs release iron. Iron affects plant metabolism, photosynthesis, and respiration. Electron carriers like cytochromes, ferredoxins, etc. are being affected by iron (Hochmuth \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The vigorous growth of \u003cem\u003eV. radiata\u003c/em\u003e L. plants under the effect of Fe-O-NPs is because of enhancement in auxin, metabolism enhancement, cell expansions, and enhanced biochemical activities which ultimately lead to growth in various morpho-physiological traits of the plant. Similar results were reported by (Iqbal et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.6. Effect of green synthesized Fe-O-NPs 50\u0026micro;g/mL on\u003c/b\u003e \u003cb\u003eV. radiata\u003c/b\u003e \u003cb\u003eL. growth and photosynthetic content under heavy metal stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe seedlings exposed to Fe-O-NP (50 \u0026micro;g/mL) exhibits increased nutrient uptake, followed by stimulation of plant growth, and improvement of morphological and physiological characteristics of \u003cem\u003eV. radiata\u003c/em\u003e L. seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e). Compared with control and NP-treated seedlings, heavy metal treatment negatively affected the growth of \u003cem\u003eV. radiata\u003c/em\u003e L. seedlings. Compared to HM treated seedlings, Fe-O-NP treated seedlings showed better root length (54.2%) and shoot length (62.8%) difference (P\u0026thinsp;\u0026le;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003ea), (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eb). \u003cem\u003eV. radiata\u003c/em\u003e L. seedlings treated with Fe-O-NPs showed a significant (P\u0026thinsp;\u0026le;\u0026thinsp;0.05) increase in both fresh and dry weight compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003ec) (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003ed).The root length increased up to 73%, and 67%and shoot length 75%, and 71% for Cr and Cd HM stress in presence of Fe-O-NP. A negligible growth range from 21.4\u0026ndash;31% increment was observed in lead stress for Fe-O-NP 50 \u0026micro;g/mL treatment. Fresh weight and dry weight of the Fe-O-NP treated test crop were increased 79% and 85.2% respectively in the presence of 10 \u0026micro;g/mL of Cr to control followed by increase in fresh and dry weight of Fe-O-NP treated \u003cem\u003eV. radiata\u003c/em\u003e L. in the presence of Cd 10 \u0026micro;g/mL which was 65.4% and 69.7% respectively. At 20 \u0026micro;g/mL of Cr and Cd fresh and dry weight were observed in the range of (61.4\u0026ndash;63.7%) fresh weigh and dry weight respectively. In presence of lead in all concentrations Fe-O-NP treated \u003cem\u003eV. radiata\u003c/em\u003e L. exhibited minimal growth in fresh and dry weight ranging from 24\u0026ndash;31%. Which is less than the Fe-O-NP treated \u003cem\u003eV. radiata\u003c/em\u003e L. growth observed in Cr and Cd stress in 10 and 20 \u0026micro;g/mL concentrations. A similar pattern was observed in leaf length, breadth, and chlorophyll content in Fe-O-NP-treated \u003cem\u003eV. radiata\u003c/em\u003e L. in the presence of different concentrations of Cr, Cd, and Pb. At 10 and 20 \u0026micro;g/mL of Cr and Cd increase in leaf breadth was prominent with 24.3\u0026ndash;24.1% was no prominent difference in leaf breadth and length was observed in other concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003ee) (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003ef) (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eg).Thus we can say that the best variations in phenotypic traits root length, shoot length, fresh and dry weight, leaf length, leaves breadth, and photosynthetic pigment were observed in Fe-O-NP treated \u003cem\u003eV. radiata\u003c/em\u003e L. in Cr and Cd at 10 \u0026micro;g/mL and 20 \u0026micro;g/mL of stress.\u003c/p\u003e \u003cp\u003eRoots appear to transport green synthesized Fe-O-NPs into plant tissues when they are treated with the synthesized nanoparticles and grown in Hoagland's solution. The increased uptake of potassium, phosphorus, and nitrogen in the presence of iron is thought to be the main reason for the increase in growth and total biomass of plants treated with Fe-O-NP. Fe-O-NP also works against the negative effects of sodium and chloride ions (Tawfik et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). According to reports, cadmium accumulation in crops decreased when the concentration of Fe-O-NPs increased (Sebastian et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Rizwan et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Similarly, Fe and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs were found to be more effective than other methods in reducing arsenic (As) in rice plants (Lux et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), have reported that cadmium and iron have the same transport pathways during their uptake in plants, and under the situation of iron deficiency, iron transporters are activated, reducing the cadmium uptake and accumulation in crop plants. (Bashir et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) have suggested that due to competitive adsorption, the accumulation of heavy metals like cadmium is reduced in the presence of higher concentrations of iron.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.7. Effect of Fe-O-NPs 50\u0026micro;g/mL and TIU16A3 on\u003c/b\u003e \u003cb\u003eV. radiata\u003c/b\u003e \u003cb\u003eL. growth and photosynthetic content under heavy metal stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn that study, different concentrations of Cr, Cd, and Pb (i.e. 0 \u0026micro;g/mL, 10 \u0026micro;g/mL, 20 \u0026micro;g/mL, 40 \u0026micro;g/mL, and 80 \u0026micro;g/mL) were used to grow \u003cem\u003eV. radiata\u003c/em\u003e L. seedlings. In addition, TIU16A3, seeds were treated with 50 \u0026micro;g/mL green synthesized Fe-O-NP. Figures\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e and \u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e show information on shoot length, root length, fresh weight, dry weight, secondary root length, and number. Figure\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003e shows the data on the length and width of the leaves and the total chlorophyll content. The findings showed that, in \u003cem\u003eV. radiata\u003c/em\u003e L. seedlings in the presence of rising levels of Cr, and Cd considerably reduced biomass when compared to plants grown with the application of TIU16A3 and Fe-O-NP, the application of these two substances to the seeds of \u003cem\u003eV. radiata\u003c/em\u003e L resulted in a significant increase in shoot length, root length, fresh weight, dry weight, secondary root length, leaves length, breadth, and content of total chlorophyll of Cr, Cd and Pb stress. The Fe-O-NP with PGPB (\u003cem\u003eP. geniculata\u003c/em\u003e) had the best effect on Cr and Cd (10 \u0026micro;g/mL, 20 \u0026micro;g/mL), followed by 40 \u0026micro;g/mL and 80 \u0026micro;g/mL, according to our results. Pb exhibited growth but was least effective which is why our further studies were focused on the effective HM concentrations.\u003c/p\u003e \u003cp\u003eThere is a direct correlation between plant growth and biomass, which increased up to 90.89% more after the augmentation of TIU16A3 and 50 \u0026micro;g/ml Fe-O-NP in combination under heavy metal toxicity. Total chlorophyll contents were 69% and 63% respectively in the presence of Fe-O-NP alone, but the increment was 83% in combination as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003e.Fe-O-NP 50\u0026micro;g/mL\u0026thinsp;+\u0026thinsp;TIU16A3 exhibited a synergistic effect on root growth under heavy metal Cr, and Cd stress and the effectiveness of combined treatment was maximum in Cr 84.6%, followed by Cd 92% as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e. This is maximum growth compared to TIU16A3 treatment and Fe-O-NP 50\u0026micro;g/mL. Thus, we can say that the combinatorial effect is best for \u003cem\u003eV. radiata\u003c/em\u003e L. growth in the presence of Cr and Cd strain is most effective at lower concentrations of Cd and Cr. This exponential increase in the phenotypic traits of \u003cem\u003eV. radiata\u003c/em\u003e L. in the presence of TIU16A3 and Fe-O-NP 50 \u0026micro;g/mL together than individually could be due to the acceleration in the accumulation of mineral nutrients from NPs.\u003c/p\u003e \u003cp\u003eCompared to the use of PGPR or NP alone, the combined use of both can improve crop productivity, plant height, dry-fresh biomass, and seed germination frequency (Medina-Velo et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This combination can work in many different ways. Improved plant performance and productivity result from direct mechanisms including PGPR, regular production of plant hormones (e.g., indoleacetic acid, siderophores, etc.), and increased soil mineral availability through phosphate solubilization and N\u003csub\u003e2\u003c/sub\u003e fixation. The presence of NPs can help PGPR tolerate higher density while providing a nutrient-rich substrate that improves PGPR efficiency and increases plant production (Rani et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Mushtaq et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the presence of heavy metal contamination by exploiting the ability of PGPR to release exopolysaccharides under dry conditions, plants can increase their stress tolerance (Bishnoi Saran \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Vurukonda et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.8. Effect of 50 \u0026micro;g/mL Fe-O-NPs and TIU16A3 on superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), H\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eO\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003econtent, and electrolyte leakage (EL) in\u003c/b\u003e \u003cb\u003eVigna radiata\u003c/b\u003e \u003cb\u003eL. under heavy metal stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCd and Cr stress significantly increased H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content and EL value; however, when treatment of 50 \u0026micro;g/mL Fe-O-NP and TIU16A3 were given on \u003cem\u003eVigna radiata\u003c/em\u003e L. the leakage percentages of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and EL of the respective controls were reduced by 37.5% and 43% and 21.5% and 26%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003ed), and (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003ee). Activities of invertase and catalase in soil were significantly higher when \u003cem\u003eP. geniculata\u003c/em\u003e was used with NPs than when NPs were used alone (Khanna et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In summary, the results show that PGPR with NPs increases the enzymatic activity of sucrose, urease, protease, and invertase in addition to soil phosphomonoesterase under both acidic and basic conditions. There is a direct correlation between soil nutrients and the enhanced enzyme activity of antioxidant-responsive genes caused by PGPR and NPs (Mushtaq et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.9. Effect of 50\u0026micro;g/mL Fe-O-NPs and TIU16A3 on metal uptake by plant \u003cem\u003eVigna radiata\u003c/em\u003e L.\u003c/h2\u003e \u003cp\u003eBy Flame Atomic Absorption Spectroscopy (FAAS) analysis of the total metal contents and the accumulation was done. The results show that the plant tissues accumulate more Cr and Cd (10 \u0026micro;g/mL, and 20 \u0026micro;g/mL) in non-inoculated plants in comparison to 50 \u0026micro;g/ml Fe-O-NP and TIU16A3inoculated. FAAS data of Cr and Cd metals in the presence of synergistic treatment of 50 \u0026micro;g/mL Fe-O-NP and TIU16A3 showed that there was a reduction in metal content by30.6% in comparison to individual treatment as depicted in (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e16\u003c/span\u003ea), and (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e16\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eFe-O-NP and TIU16A3 combined increased soil enzyme (dehydrogenase) more than NPs, and \u003cem\u003eP. geniculata\u003c/em\u003e alone which must have resulted in better biological remediation. Previous studies have shown that the combined use of PGPR and NP decreased Cd levels more effectively than the use of NP and PGPR alone. TIU16A3 and NPs together reduced significantly the harmful effects of chromium and promoted plant growth. This may be because PGPRs and NPs limit the availability of metals for uptake and translocation in soil (Mitra et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.10. Effect of 50\u0026micro;g/mL Fe-O-NPs andTIU16A3 synergistically on gene expression of\u003c/b\u003e \u003cb\u003eAux/IAA\u003c/b\u003e \u003cb\u003eof shoot tissue in\u003c/b\u003e \u003cb\u003eV. radiata\u003c/b\u003e \u003cb\u003eL. under heavy metal stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn the present study, gene expression profiling of auxin-responsive genes in seedlings under Cr and Cd metal stress has been studied through qRT-PCR which exhibits a high expression of the gene of interest in the presence of 50 \u0026micro;g/mL Fe-O-NPs and TIU16A3 together as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e17\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study aimed to determine whether \u003cem\u003eEichhornia crassepis\u003c/em\u003e plant extract and green synthesized iron oxide nanoparticles (Fe-O-NP) could protect Moong bean plants against heavy metal stress. The deleterious effects of metal stress on plants were confirmed by significantly increased electrolyte leakage and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) levels. Increases in biomass, photosynthetic pigments, nutrients, and indole acetic acid in plants treated with Fe-O-NP and PGPR and exposed to all concentrations of heavy metal stressors, all of which improved plant growth. Reduction of heavy metals by treatment is associated with improved plant growth based on Flame Atomic Absorption Spectrophotometry. TIU16A3 and Fe-O-NPs work together to reduce antioxidant enzymes, mainly catalase, peroxidase, and superoxide dismutase, which signifies that the synergistic treatment reduced the stressed condition in the plant. In addition, significant reductions in electrolyte leakage and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e indicated that this treatment alleviated heavy metal stress. The results of this study indicated that plants can be protected against single and combined heavy metal stress caused by Cr, and Cd through the complementary effects of PGPR and Fe-O-NPs. Therefore, Fe-O-NPs are suitable for agricultural applications in living organisms instead of chemically synthesized Fe-O-NPs due to their negligible side effects. Eco-friendly production and development of super-stabilized nanoparticles is promising because it uses few harmful chemicals and high energy consumption. In addition to enhancing plant tolerance, the interaction of PGPR and Fe-O-NPs opens the door to long-term increases in yield. By combining the strengths of these two factors, we can open new opportunities to improve plant health and productivity, an important step forward in the search for sustainable agricultural methods.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Techno India University, West Bengal for their encouragement and support during this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP.D. and A.K. designed and conceptualized the experiment. B.M. performed the experiments, synthesis and material characterization, data analysis, and paper writing. S.B. performed the synthesis and material characterization of the sample, data analysis, and paper writing. S.C. helped in editing. All authors have checked the current form of this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article incorporates all the data examined during this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not involve any human or animal participants to conduct the research work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo patient data were reported.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors did not receive support from any organization for the submitted work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhmed B, Rizvi A, Syed A, et al (2021) Differential responses of maize (\u003cem\u003eZea mays\u003c/em\u003e) at the physiological, biomolecular, and nutrient levels when cultivated in the presence of nano or bulk ZnO or CuO or Zn2+ or Cu2+ ions. 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Mycotoxin Res 39:177\u0026ndash;192. https://doi.org/10.1007/s12550-023-00484-4\u003c/li\u003e\n\u003cli\u003eYew YP, Kamyar S, Miyake M, et al (2017) An Eco-Friendly Means of Biosynthesis of Superparamagnetic Magnetite Nanoparticles via Marine Polymer. IEEE Transactions on Nanotechnology PP:1\u0026ndash;1. https://doi.org/10.1109/TNANO.2017.2747088\u003c/li\u003e\n\u003cli\u003eZulfiqar U, Yasmin A, Fariq A (2022) Metabolites produced by inoculated Vigna radiata during bacterial assisted phytoremediation of Pb, Ni and Cr polluted soil. PLoS One 17:e0277101. https://doi.org/10.1371/journal.pone.0277101\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bioremediation, Vigna radiata L., Eichhornia crassipes, nanoparticle, Fe-O-NP, TIU16A3","lastPublishedDoi":"10.21203/rs.3.rs-4863542/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4863542/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlants are often affected by deleterious effects of heavy metals (HM). This HM stress leads to growth and production capacity issues. The most hazardous trace metals in agricultural areas are lead (Pb), cadmium (Cd), and chromium (Cr). It is not only carcinogenic to humans, but also negatively affects plants' phenotypic, biochemical, and genetic properties. Bioremediation can be used to economically remove contamination of heavy metals. The study aims to test the bioremediation potential of biogenic iron oxide nanoparticles (Fe-O-NP) along with \u003cem\u003ePseudomonas geniculata\u003c/em\u003e strain TIU16A3 (accession number OR507186). Plants of \u003cem\u003eEichhornia crassipes\u003c/em\u003e were used to create Fe-O-NP. Individually iron oxide nanoparticles exhibited enhancement of phenotypic traits in \u003cem\u003eVigna Radiata \u003c/em\u003eL. under heavy metal stress. We used four concentrations (10, 20, 40, and 80 mg/L) of Cr, Cd, and Pb for stress conditions. Biogenic nanoparticles and TIU16A3 in combination act synergistically with the increase in the amount of chlorophyll content and growth in distinct phenotypic traits. In plants, under HM stress, levels of antioxidant enzymes were observed to be high including superoxide dismutase, peroxidase, and catalase and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content\u003csub\u003e \u003c/sub\u003eand electrolyte leakage, when they were treated with biogenic NPs and TIU16A3 together the antioxidants decreased significantly. TIU16A3 and Fe-O-NP synergistically in the presence of Cd and Cr enhanced the expression of the \u003cem\u003eAux/IAA\u003c/em\u003e gene more than the expression observed in the presence of Fe-O-NP only. Due to the enhancement of intrinsic properties, and ability to remove Cr, and Cd by TIU16A3 when used in combination with Fe-O-NP for bioremediation exhibits promising results.\u003c/p\u003e","manuscriptTitle":"Synergistic impacts of plant growth promoting Pseudomonas geniculata TIU16A3 and green-synthesized iron-oxide nanoparticles from Eichhornia crassipes for the amelioration of heavy metal stress in Vigna radiata L.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-05 18:50:58","doi":"10.21203/rs.3.rs-4863542/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a1bb8a1f-bc46-468b-93d4-b56f0a2d4b2b","owner":[],"postedDate":"September 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-05T22:40:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-05 18:50:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4863542","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4863542","identity":"rs-4863542","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}