PGPR’s mix treatment to Moringa improved plant growth and iron content in foliage as substantiated by biochemical and molecular methods

ABSTRACT In this study, Bacillus pumilus SE34 and B. pumilus T4 were combined with Bacillus subtilis GBO3, B. pumilus INR7 and Pseudomonas fluorescens UOM14 to form COM1, COM2 and COM3, respectively. All combinations were used to find their synergistic effect on Moringa oleifera for growth promotion and Fe accumulation efficiency in foliage. The results indicate a significant increase in Fe content in foliage using COM3 (405.70%) followed by COM2 (105.83%) in comparison to the control with a simultaneous decrease in the soil Fe content. Increased expression of iron-transport-related genes like iron-phytosiderophore oligopeptide transporter and natural-resistance-associated macrophage protein, in foliage of Moringa using real-time PCR correlates with the enriched iron content in foliage of treated plants. Increase in citric acid was in direct relation to the Fe accumulation in foliage. Growth promotion and Fe enhancement using plant-growth-promoting rhizobacteria’s combination was significantly higher, proving its synergistic effect as a great source for sustainable development in agriculture and nutrition.


Introduction
Human body requires a vast variety of nutrients to function properly and Fe plays a major role. Fe is a vital structural part of hemoglobin and necessary for the normal cell functioning and its development (Erdman et al. 2012). Since, Fe perform imperative roles in the human biology, its deficiency can lead to various diseases. Fe deficiency is the leading cause for anemia worldwide (Lopez et al. 2016), mainly because of its very low quantity in diet consumed by humans, insufficient intake, mal absorption and ineffective utilization (Roohani et al. 2013).
The easiest method to provide the system with commensurate measures of the nutrients is to take a diet rich in Fe. Bioavailability of Fe through plant sources is comparatively less than of animal sources due to the presence of phytate as in case of lentils and beans, whereas the presence of organic acids such as malic and citric acids made a plant source capable of Fe availability to the human body (Gillooly et al. 1982). Other than Fe bioavailability problem, the plants are themselves deficient in this element as they themselves do not receive Fe in sufficient amounts. One of the methods to increase the dietary intake of Fe from plant source is to make sure that the plants receive more than the sufficient amount of Fe from the soil and make them bioavailable to the human body on consumption (Saini, Nile et al. 2016;Pataco et al. 2017).
An approach to overcome poor Fe bioavailability would be to employ genetic engineering strategies by increasing the levels of siderophores, chelating agents, reducing agents, enzymes and transporter proteins in roots that help in increasing Fe uptake. Another method to increase the Fe bioavailability is to decrease the phytate-to-mineral ratio by combining cross-breeding techniques for developing low phytic acid plants with selection of highly nutrient-dense seeds (Zimmermann and Hurrell 2002). However, the introduction of genetically modified crops into the field has various ethical issues; therefore the use of plant-growthpromoting rhizobacteria (PGPR) is a simpler approach to this issue.
Growth promotional activities of PGPR such as superior germination, high vigor, enhanced yield along with endeavoring disease resistance against a wide variety of pathogens by triggering induced systemic resistance in plants are well studied (Zhang et al. 2002;Vessey 2003;Kloepper et al. 2004;Freitas et al. 2015;Rahmoune et al. 2017). PGPR enhances the levels of micronutrients in plants using various mechanisms (Loper and Henkels 1999). In many studies, PGPRs were found to enhance the Fe availability to the plants, making it easier for uptake. Pseudomonas species are known to produce siderophores that have high specificity to Fe III, and solubilize it, making it available for plant uptake (Wandersman and Delepelaire 2004). Bacillus pumilus SE34, B. pumilus T4, B. pumilus INR7, Bacillus subtilis GB03 are commercially available PGPRs that can be directly used to infect plants via seed coating, seed infection and soil drenching methods. Unlike other PGPR's these are also known for their growth-promotion-producing plant hormones and inducing systemic resistance promoting plant protection. B. pumilus SE34, B. pumilus T4, B. pumilus INR7 and B. subtilis GB03 have also been extensively studied individually or in combination for their plant growth promoting effects (Murphy et al. 2003;Kloepper et al. 2004;Kumar et al. 2011). They have also been widely employed on different plant systems such as cucumber, tomato, tobacco, pepper, millets and Arabidopsis against various fungal and viral infections. In recent years, B. subtilis GB03 has been extensively studied for its Fe accumulation ability in different plants such as Arabidopsis and cassava. B. subtilis GB03 was found to release an array of volatiles that were found to indulge in plant growth promotion, plant defense along with iron homeostasis. B.-subtilis-GB03-mediated enhancement of plant iron was found to occur via activation of the plant's own iron acquisition machinery (Zhang et al. 2009;Freitas et al. 2015). In the presence of PGPRs, plants are reported to exudate flavonoids and organic acids which perform specific functions such as biofilm formation and chemotaxis of organisms towards plant roots (Tyler and Strom 1995;Zhang et al. 2009;Ahmed et al. 2014). The role of various flavonoids and organic acids in solubilization of iron, phosphorus and other divalent such as Zn was reported from soil (Bais et al. 2006). Furthermore, these acids form complexes with Fe and allow easier uptake, wherein the role of divalent cation transporter (IRT1) gene for the transport of Fe from soil to the plant was elucidated (Vert et al. 2002). To boost the uptake of Fe from the soil, the Fe must be present in soluble form and in this regard two mechanisms were demonstrated. In strategy 1 pH of soil is dropped, leading to increase in solubility and uptake of iron via Protons' extrusion from roots of plants in the rhizosphere. In strategy 2 low molecular weight compounds; phytosiderophores (PS) are released in response to iron deficiency, and PS-Fe +3 complexes is then transported from soil to root using IRT1 and other parts of the plants through iron-phytosiderophore oligopeptide transporter (OPT) (Buckhout et al. 2009;Yang et al. 2010;Kobayashi and Nishizawa 2012). OPT is generally involved in intracellular and long distance Fe transport in plants. OPT was also found to transport other divalent metals such as Zn and Mn. OPT3 (a plasma membrane transporter) was also found to load Fe into phloem, indicating its role in Fe distribution from shoot to root and from mature organs to the developing one (Zhai et al. 2014). Advocating foliar spray and soil loading with PGPRs leads to enhanced production of bioactive metabolites in plants along with nutritional enrichment of plants (Giridhar and Parimalan 2010;Gururaj et al. 2012;Matam and Giridhar 2017).
Moringa, popularly known as drumstick tree, is a much sought-after food plant for addressing the malnutrition problems of the undernourished population of the developing world in view of its optimal levels of major nutrients' profile and also vital health-promoting oils (Anwar et al. 2003). Its foliage was found to be rich in carotenoids, tocopherols, iron, folate, fatty acids, etc. (Saini, Shetty, et al. 2014a;Saini, Shetty, et al. 2014b;Gupta et al. 2017). Use of an abiotic elicitor on plants enhanced accumulation of carotenoids and tocopherols in leaves (Saini, Prashanth, et al. 2014). The leaves were also reported for their superior bioavailability of Fe and folate in rat models (Saini, Manoj, et al. 2014;Saini, Manoj et al. 2016). Being an effective nutritional security provider, commercial cultivation of this high-value energy crop and its further improvement through biotechnological and other traditional methods to overcome cultivar and agroclimatic variation and enhancement of its nutrient profile would facilitate farmers to prefer this for better economical prospective. In view of this, Moringa oleifera was chosen as model plant for our study.
Hence in this study we have examined the effects of PGPR application on plant foliage Fe content and other growth promotional activities such as shoot length, primary root length, shoot weight, root weight and lateral root numbers. Levels of organic acids in root exudates were also analyzed as it helps in increasing the bioavailability of Fe. All the studies were restricted to foliage as it is important from food point of view.

Bacterial growth and infection to seeds and soil
An array of potential PGPR which have been widely studied were selected for the study; these include B. subtilis GB03, B. pumilus INR7, B. pumilus SE34, B. pumilus T 4 and Pseudomonas fluorescens UOM14 obtained from University of Mysore. Three combinations of these PGPRs were made, based on initial characteristic studies and their competence with each other. The three combinations are COM1 (B. pumilusSE34 Two different media were used for the growth of these microorganisms. Tryptic Soya Broth (HiMedia, Mumbai) was used for B. subtilis GB03, B. pumilus INR7, B. pumilus SE34 and B. pumilus T 4 and P. fluorescens UOM14 was grown on King's B medium (Himedia, Mumbai). Upon inoculation of the microorganisms in their respective medium, the media were kept in a shaking incubator (Orbitek, India) at 120 rpm, with growth conditions of 12 h, 27°C. M. oleifera, PKM-1 variety seeds were used for the study. The seeds were washed using Tween 20 and kept under running water for 45 min, following which the seeds were transferred to aseptic conditions and sterilized using sodium hypochlorite (NaOCl) 5% (w/v) for 5 min; these were then rinsed thrice using sterile distilled water to free the seeds of residual NaOCl and infection step was carried (Saini et al. 2012). The broth culture of bacteria obtained by growing them for 12 h was centrifuged and the pellet obtained was washed using 1/8th Hoagland solution to remove residual growth media. The pellet obtained was mixed with (1/4th) strength of Hoagland solution to achieve a culture concentration of 1 × 10 8 cfu mL −1 . To make different combinations, cultures were mixed in equal amount and were used for further study. Surface sterilized M. oleifera seeds were transferred to the respective combinations and were kept for 6 h for infection. Subsequently the infected seeds and control seeds were sowed in pots to allow germination. The pots contained soil mix consisting of 2 kg sterile soil present in the ratio of 2:1:1 of soil:vermi compost:coir dust. The soil was also drenched with respective combinations.
Experiment was planned by a randomized block design. All the experiments to study the effect of PGPR on the levels of iron in M. oleifera foliage and soil were performed in polyhouse (12 h photoperiod, 25 ± 2°C and 70% relative humidity). Sampling was done at the sixth week after seed germination.

Fe analysis by atomic absorption spectrophotometry
Acid washed glasswares were used for the study. Ten grams leaf samples from the plants were collected and desiccated in a hot air oven for 3 h at 105°C and charred on an electrical stove until it ceased to smoke. One gram of this sample was transferred into muffle furnace and heated at 550°C to acquire white or grayish ash. Aquaregia (1 mL) was added to the ash and diluted using 1 N HNO 3 prepared in Millipore water. Total Fe content in sample was estimated using direct aspiration into the Atomic Absorption Flame Emission Spectrophotometer (AA-6710F, Shimadzu). Wavelength was set to 248.3 nm with different Fe concentration ranging from 0.5 to 2 ppm. A calibration curve using four concentration of minimum range in analytical range was prepared.
Wet soil from rhizosphere (2.0 g) on the day of leaf sampling for Fe analysis was collected and extracted in low molecular weight organic acid solution (20 mL) containing acetic, lactic, citric, malic and formic acids in 0.01 M in the ratio of 4:2:1:1:1 so as to evaluate the extractability of Fe in soil treated by PGPR's combinations compared to control. Sample was extracted for 16 h, centrifuged and clear solution was taken for atomic absorption spectrophotometry after diluting to 1:1 with 1 N HNO 3 (Feng et al. 2005).

Root exudates composition
For the analysis of organic acids, 2 g rhizospheric soil was collected and extraction was carried out in 0.1% acidified methanol followed by centrifugation at 10,000 rpm for 15 min at room temperature. The supernatant collected was concentrated in the fume hood and suspended in 1 mL methanol, which was centrifuged again at 12,000 rpm for 10 min allowing the residual particles to settle. The consequential supernatant was used to carry out HPLC. C 18 reversed phase SunFire column (250 mm × 4.6 mm × 5 µM) was used for the HPLC analysis of both the compounds. Isocratic elution was performed for the detection of organic acid using 5 mM H 2 SO 4 with photodiode array (PDA) detector (Shimadzu SPD-M20A 230 V, Japan) (λ max at 210 nm) with a flow rate of 1 mL per min.

Gene expression profile
2.4.1. RNA extraction and cDNA synthesis Fresh leaves from the treatment and control plants were collected in the sixth week and frozen in liquid nitrogen. Spectrum Plant Total RNA Kit (Sigma, Bangalore) was used for the total RNA extraction from the samples, using the protocol provided by the manufacturer. The quality, quantity and integrity of extracted RNA were assessed by Nanodrop (Thermo Scientific, USA) and agarose gel electrophoresis. The RNA was treated with DNase I (Thermo Scientific) and reverse transcription was performed using Revert Aid First Strand cDNA Synthesis Kit (Thermo Fischer Scientific, USA). Gene expression analysis was then carried out using the resulting cDNA obtained from the previous step using RT-qPCR.

Quantitative real-time PCR (RT-qPCR)
Real-time studies were performed to analyze the expression pattern and to correlate it with biochemical analysis. Real-time primers were designed (Table 1) and analyzed to validate the accuracy and specificity using conventional PCR (Biorad). RT-qPCR was performed using the Applied Biosystems QuantStudio 5 Real-Time PCR System with the Maxima SYBR Green/ROX Master Mix (Thermo Fischer Scientific). To normalize sample variance, the GAPDH gene was used as reference. The data obtained were analyzed using the mean of the cycle threshold (CT) values of three biological replicates that were normalized to the mean CT values of the endogenous gene. The expression ratios were calculated using the 2 −ΔΔCt method (Livak and Schmittgen 2001). Relative transcription levels are presented graphically.

Plant growth measurements
Immediately after collecting foliage for non-destructive measurements, plants were harvested for shoot height, shoot dry weight, root dry weight; primary root length and lateral root numbers were measured manually.

Statistical analysis
The data were expressed as mean ± SD of triplicates and the experiments were repeated twice. Data were subjected to ANOVA (Sigma plot 12.5) analysis with post hoc using Tukey's test (SPSS 17) at the significance level of P < .05.

Results
All the PGPRs that were used for mix formulation were tested individually for their Fe enhancement ability in Moringa foliage. B. pumilus SE34, B. subtilis GBO3 and B. pumilus T4 were found to enhance Fe accumlation by 80-85%. B. pumilus INR7 and P. fluorescens UOM14 showed only 30-40% Fe accumulation in Moringa foliage. B. pumilus SE34 and B. pumilus T4 were kept constant in all the combinations to study their synergistic effect on Moringa foliage with PGPR related to same or other species or other genus. In COM1 B. pumilus SE34, B. pumilus T4 were combined with B. subtilis GBO3. In COM2 B. pumilus SE34, B. pumilus T4 were combined with B. pumilus INR7. In COM2 B. pumilus SE34, B. pumilus T4 were combined with P. fluorescens UOM14.

Fe analysis by atomic absorption spectrophotometry
In the present study, PGPRs' mix showed a profound influence on Fe content of M. oleifera foliage and also positively influenced shoot and root growth. Various studies involving the effect of PGPR on Fe have been conducted in economically important plants. The production of siderophores by the PGPR helped to enhance the Fe uptake in cucumber (Cucumis sativus) (Wang et al. 1993), maize (Zea mays L.) and sunflower (Helianthus annuus L.) (Masalha et al. 2000). A study conducted on Raspberry grown in soil supplied with PGPR strains of Bacillus (strains OSU-142 and M3) showed significant effects on Fe availability in the soil due to decreased pH and increased mineralization (Orhan et al. 2006). B. subtilis GBO3, B. pumilus INR7, B. pumilus SE34 and B. pumilus T4 and P. fluorescens UOM14 have been widely used for its disease resistance and growth promotional activities individually or in different combinations (Kokalis et al. 2003;Raj et al. 2003). But their effect in different combinations on Fe accumulation and growth promotional activities directly on food plant such as Moringa is scarce. The result obtained in the present study for various growth parameters and Fe accumulation in Moringa was further supported by similar studies in another plants. B. subtilis GBO3 was found to increase Fe accumulation in cassava leaves by 300% (Freitas et al. 2015). Increased uptake of Fe was attributed to various activities of plants such as volatile, organic acid and flavonoid production, which results in a decrease in soil pH (Zhang et al. 2009).

Organic acid composition of root exudates
Organic acids such as oxalic acid, citric acid, ascorbic acid and malic acid are main components in root exudates (Ling et al. 2011). Similarly, in the present study, oxalic acid and citric acid were detected in all the PGPR's treatments, including control plant root exudates. Ascorbic acid and citric acid were detected in many of the root exudates from various plants. Citric acid was reported to stimulate colonization of PGPR's, such as P. fluorescens and B. subtilis (Andersen et al. 2007) and also reduce the pH of soil, leading to the formation of citrate. Lower pH leads to increased availability of Fe. Hence, the release of citric acid in root exudates helps plants in Fe accumulation in roots and foliage (Dinkelaker et al. 1989). It is also known to induce biofilm formation within a very short period after incubation (Rudrappa et al. 2008). Reduction of oxalic acid and increase in citric acid shows effective colonization of applied PGPR combination. Malic acid was not detected in any of the samples in our study because its concentration is very low in rhizosphere and its action to biofilm formation and as biocontrol agent may not be significant, but its concentration around roots may be high, which is difficult to estimate (Rudrappa et al. 2008).

Gene expression profile
Plants treated with B. subtilis GBO3 were also found to mimic Fe deficiency condition, leading to overexpression of genes involved in Fe transport like OPT and NRAMP to facilitate Fe uptake (Vert et al. 2002;Stacey et al. 2008). In our study, significant upregulation of OPT and NRAMP was seen demonstrating plants response to cope up Fe deficiency symptoms through enhanced Fe accumulation under influence of respective PGPR's. Expression of OPT also proves long-distance transport of Fe from soil and accumulation in foliage (Zhai et al. 2014). NRAMP was found to upregulate in the present study, which can be considered as a way for the plant to mimic Fe deficiency response. Thomine et al. (2003) showed NRAMP-enhanced expression under Fedeficient condition. AtNRAMP was also found to be upregulated in Arabidopsis thaliana in the presence of biotic stress (Zimmermann et al. 2004).

Plant growth measurements
Increase of Fe content was found to associate with poor growth and low yield in most of the approaches. Use of PGPR was found to enhance Fe accumulation and growth of plant simultaneously. In fact, many endophytes (including arbuscular mycorrhizal fungi) and PGPRs are known for their growth promoting nature in plants, including enrichment of desired metabolite production (Matam and Giridhar 2017). But with reference to enrichment of Fe content in treated plant foliage, this study is the first of its kind in general and on Moringa in particular. All the PGPR combination that were used in the present study have shown positive effects on morphological characters such as germination; shoot length, primary root length, lateral root numbers, and shoot biomass, which can mainly be correlated with characters of organisms such as auxin production, phosphate  solubilization, phytohormone production and rhizosphere engineering (Kloepper et al. 2004;Kumar et al. 2011).

Conclusion
A combination of PGPRs comprising B. pumilus SE34 + B. pumilus T4 + P. fluorescens UOM14 (COM3) was found to exhibit a significant influence on enhancement of Fe accumulation in Moringa foliage. It was also found to be a good Fe extractor from soil, which can be correlated to the citric acid content in the root exudates. They also increased the expression of Fe transporters significantly. PGPR mix also modified root morphology so as to enhance the uptake of Fe. This study concludes that PGPR's performs synergistically and significantly in combinations. It can effectively be used as an alternate for breeding, transgenic, chemical fertilizer, etc. PGPR mix can successfully enhance the Fe content in edible parts of various crops. Enhancing Fe in edible parts of plants can help to restrain the problem of hidden hunger to a greater level. But more research needs to be focused on different formulations using different PGPR and their effect in open field.