Molecular characterization of potential zinc solubilizing bacterial isolates from onion rhizosphere and validation of solubilization ability of PantoeaeucrinaZSC9 using FE-SEM and EDS

doi:10.21203/rs.3.rs-3980990/v1

Zinc (Zn) is essential for optimal growth and nutrition of plant and zinc solubilizing bacteria (ZSB) enhance its accessibility for plants by converting insoluble forms into usable ones. The primary objective of current research was to isolate and identify Zn solubilizing strains from onion rhizosphere and evaluate their ability to solubilize different insoluble Zn compounds, including ZnO, ZnCO3 and Zn3(PO4)2. Out of the nineteen bacterial isolates retrieved, fifteen were found to be proficient in solubilizing inorganic Zn minerals based on plate assay techniques. The Zn solubilizing bacterial strains chosen through qualitative assessment were subjected to quantitative testingin the broth culture using AAS and FE-SEM-EDS. Seven most potential bacterial isolates with the ability to solubilize Zn were identified using 16S rRNA gene amplification and sequence analysis. The isolates were found to be affiliated with Pantoeaeucrina, Pantoeadispersa, Pseudomonas aeruginosa, Bacillus velezensis and Pseudomonas fluorescens. To the best of our knowledge, this appears to be the first finding demonstrating Pantoeaeucrina as a potential ZSB. The maximum Zn solubilization index (8.85) and the highest soluble Zn content (624 mg/l) among the three insoluble Zn salts was exhibited by the strain Pantoea eucrina ZSC9 on the 10th day of incubation in ZnO enriched basal medium. Among the three insoluble Zn compounds, all of the bacterial isolates were more effective at solubilizing ZnO compared to ZnCO3 and Zn3(PO4)2. The solubilization of Zn led to a significant drop in pH of the broth and Pantoeaeucrina ZSC9 exhibited the maximum reduction in pH (3.82) in ZnO supplemented medium. A negative correlation was observed between the pH of broth and Zn solubilization by all the isolates. Based on our results, it is suggested that the identification of promising ZSB isolates and their application as biofertilizers has the potential to enhance plant growth and development.

Zn solubilizing bacteria

Onion rhizosphere

AAS

FE-SEM-EDS

pH reduction

Fifteen bacterial isolates were found to be proficient in solubilizing various inorganic Zn minerals
Quantitative testing in the broth culture using AAS and FE-SEM-EDS
Measurement of pH reduction in culture medium and correlation of pH with zinc solubilization
16s rRNA sequencing revealed the affiliation of selected bacterial isolates to the genera Pantoea, Pseudomonas and Bacillus
Pantoeaeucrinawas found to be the most potent zinc solubilizing bacterium with solubilizing ZnO more effectively compared to ZnCO₃ and Zn₃(PO₄)₂

Zinc (Zn) plays a vital role as a micronutrient aiding the growth, development, reproduction and metabolic processes of agricultural crops (Sindhuet al., 2019).Znserves as a fundamental element inphysiological (structural composition of numerous proteins, structural and functional integrity of cellular membranes, chloroplast development, cofactor for a variety of enzymes etc.), biochemical and metabolic processeslike photosynthesis, carbohydrate and auxin metabolism, nodulation, protein synthesis, lipid and nucleic acid metabolism (Kamalakannanet al., 2019; Bhatt and Maheshwari, 2020; Karnwal, 2021a). Italso plays a pivotal role in enzymes like tryptophan synthetase, which is responsible for the synthesis of tryptophan in the biosynthesis of indole-3-acetic acid (Hashemnejadet al., 2021).Despite the fact that Zn is needed in trace amounts, its deficiency has been documented as the most prevalent micro-nutritional deficiency in food crops. This deficiency affects various crops such as cereals, pulses, vegetables, and oilseeds globallycausingvarious adverse effects like stunted shoot growth, decreased leaf size, chlorosis, reduction in flowering and fruit development, heightened vulnerability to light, heat and infections (Rani et al., 2023). Furthermore, it has negative impact on grain yield, pollen formation, root development, water uptake, and the overall transport of nutrients within the plant (Tavallaliet al., 2010).

Plants uptake Zn in soluble state as divalent cation (Zn²⁺) only, whereas a significant portion of Zn in the soil exists in insoluble forms [ZnO, ZnCO₃ and Zn₃(PO₄)₂]. Therefore, the global occurrence of Zn deficiency in crops primarily results from the limited solubility of Zn in the soil rather than a lack of Zn availability (Karnwal, 2021b).It is estimated that major area under cultivation in India are deficient in Zn. Zinc deficiency is associated with various factors that are related to soil conditions.For instance, the soils with higher pH valuesleads to decreased solubility of Zn from insoluble Zn compounds available naturally. Additionally, high contents of organic matter, calcite, bicarbonate ions, magnesium-to-calcium ratio, and elevated levels of phosphorus and iron availability in the soil also contribute to Zn deficiency (Gontia-Mishra et al., 2017; Kumar et al., 2019). When Zn is applied to agricultural fields in the form of ZnSO₄, it gets converted into different insoluble forms under specific soil conditions. For instance, in high pH soils it can transform into Zn(OH)₂, in calcium-rich alkali soils it may become ZnCO₃, and in near-neutral to alkali soils with significant P fertilizer application, it can turn into Zn₃(PO₄)₂and ZnS under reduced condition.These findings underscore that application ofZn fertilizers in field results in theirconversion to insoluble forms that plants struggle to absorb, causing Zn deficiency in crops (Ayyaret al.,2020).Although Zn deficiency in crops can be overcome by using Zn-based chemical fertilizers in agricultural fieldsbut widespread use of these often has a detrimental impact on the environment.However, in addition to fertilization, various strategies have been employed to address the problem of Zn deficiency, and these include the practices of conventional breeding (Veluet al., 2019), genetic modification(Ibrahim et al., 2022) and transgenic procedures (Harrington et al. 2023). Nevertheless, each of these methods proves to be more expensive, time-consuming, and demanding in terms of manpower (Upadhayayet al., 2022c). Therefore, to encounter this challenge, it is necessary to implement environmentally safe and economically efficient approaches.Hence, researchers have shifted their focus towards exploring alternative approaches,like external application of zinc solubilizing bacteria (ZSB) such as Bacillus, Pseudomonas, Rhizobium etc. (Kushwahaet al.,2021; Ali et al., 2023; Chaudharyet al., 2023).Thisstrategy seems promising and eco-friendly for enhancing the accessibility of native Zn for absorption by roots and thus enhancing overallplant growth.

Numerous plant growth-promoting rhizobacterial (PGPR) strains have been identified as proficient agents for enhancing the solubility of Zn. These bacteria inhabit the rhizosphere and transform complex insoluble Zn compounds into more easily accessible forms contributing increased plant growth and development (Kushwahaet al., 2020). Solubilization of these complex insoluble Zn minerals is done by adverse range of mechanisms, encompassing the production of organic acids (Vidyashreeet al., 2018), generation of chelated ligands (Singh and Prasanna, 2020), proton release, and the involvement of oxidoreductive systems on the cell membrane (Upadhayayet al., 2022b).ZSB release diverse organic acids, leadinglower pH of surrounding soil that results in the sequestration of Zn cations(Alexander, 1997; Kamran et al., 2017). Furthermore, it has been observed that anions can also form chelates with Zn, thereby enhancing the solubility of Zn (Kumar et al., 2019). Additional mechanisms involve the release of iron-chelating compounds known as "siderophores," which are thought to have a significant role in the solubilization of micronutrients like iron and Zn (Ali et al., 2023).

Numerous ZSB have been characterized from diverse soil conditions, thereby facilitating the provision of soluble Zn to plants and promoting their growth. Findings from in vitro research indicated the efficacy of various species belonging to the genera Gluconacetobacter, Arthrobacter, Agrobacterium, Rhizobium, Bacillus, Burkholderia, Pseudomonas, Enterobacter,Pantoea, Serratia, Acinetobacter, and Klebsiella in solubilizing insoluble Zn compounds, indicating their potential as Zn biofertilizers (Saravananet al., 2007; Singh et al., 2017; Khanghahiet al., 2018; Bhakat et al., 2021; Hashemnejad et al., 2021; Kushwahaet al., 2021; Upadhayayet al., 2022a, 2022c; Ali et al., 2023).Considering the importance of eco-friendly sources of micronutrients to promote plant growth and crop yield, as well as the desire to minimize environmental pollution resulting from chemical fertilizers, this study was conducted with the aim of isolating, characterizing, and identifying ZSB that are associated with the onion rhizosphere. Additionally, the research sought to evaluate their ability to solubilize various insoluble Znminerals both qualitatively and quantitatively under controlled laboratory conditions.

2.1 Collection of rhizospheric soil samples-

Soil samples were collected from rhizosphere of onion grown at CCS HAU field, DDUCE-OF, Hisar (29.1492°N, 75.7217°E) and G.V.M. High School, Fatehabad (29.5132°N, 75.4510°E), Haryana, India. To gather rhizospheric soil samples, the plants were carefully removed from the ground, and the soil adhering to their roots was collected under sterile conditions. The collected soil samples were then promptly placed in sterile polythene bags and transported to the laboratory and were stored in the refrigerator at 4℃ for isolation of ZSB.

2.2 Isolation of zinc solubilizing rhizobacteria-

The dilution plate technique was employed to isolate ZSB from soil samples taken from the rhizosphere. Once serial dilutions were made from the soil samples, 0.1 ml from 10^− 4, 10^− 5 and 10^− 6 dilutions were plated evenly onto a basal medium (dextrose − 10.0 g; (NH₄)₂SO₄ − 1.0 g; KCl − 0.2 g; K₂HPO₄ − 0.1 g; MgSO₄ − 0.2 g in 1000 ml distilled water at pH 7.0) supplemented with 0.1% ZnO (w/v) (Saravananet al., 2004). The petri-plates were incubated at 28–30℃ for up to 7 days. After incubation, the bacterial cultures showing clear halo zones around their colonies were chosen as Zn solubilizers, transferred on NA plates, sub-cultured for purification and stored at 4℃ for further use.

2.3 Qualitative assessment of zinc solubilization-

All of the bacterial isolates that showed solubilization zone were further screened for their ability to solubilize 3 insoluble Zn compounds (zinc oxide, zinc carbonate and zinc phosphate).For this purpose,48 hours grown colonies were spot inoculated aseptically on to basal medium having various Zn sources separately.The petri plates were incubated at 30℃ for 10 days. Bacterial isolates with higher zone of clearance were selected and the diameter of bacterial colony and clear halozone around the colony were determined at 3rd, 5th, 7th and 10th day. The halozone formation around the bacterial colony is a sign of Zn solubilization. Zinc solubilization index (SI)was calculated using the solubilizing indexformula (Gandhi and Muralidharan, 2016),

2.4 Quantitative assay for zinc solubilization in broth media-

Quantitative Zn solubilizing potential of the bacterial isolates was determined in the liquid basal medium following the method of Saravananet al.(2007). The bacterial isolates (1 ml overnight grown culture at 30℃, 120rpm) were inoculated into 100 ml basal medium broth containing 0.1% of insoluble Zn compounds [ZnO, ZnCO₃ and Zn₃(PO₄)₂] added separately.Every treatment was subjected to three replications and incubated at 30°C in an incubator shaker at 120rpm for a duration of 10 days.A flask containing an un-inoculated medium with insoluble Zn sources was kept as the control during the experiment. The samples were processed at intervals of the 3rd, 5th, 7th, and 10th days. Subsequently, these were aseptically transferred to centrifuge tubes and subjected to centrifugation at 8000 rpm for 10 min. Following the infiltration of the supernatants through a 0.45-µm filter, a 1-ml filtrate was diluted with sterile distilled water to a total volume of 50 ml and subsequently utilized for estimating the soluble Zn content using Atomic Absorption Spectrophotometry (AAS).

2.5 pHmeasurement during the process of zinc solubilization-

In addition to assessment ofZn solubilizationquantitatively, the pH variations in both the culture broth and un-inoculated medium were estimated on the 3rd, 5th, 7th, and 10th days of incubation.In order to achieve this, the pH of the basal medium was adjusted to 7.0 by adding 0.1 M HCl and NaOH as needed. Aliquots of the samples were subjected to centrifugation, and subsequently the pH of the resulting supernatant was measured using a pH meter (Bhakatet al., 2021).Pearson product-moment correlation analysis program was utilized to observe the correlationwithin the Zn solubilization index, quantity of Zn solubilized by the bacterial isolates and the corresponding pH reduction of the culture medium.

2.6 Field Emission-Scanning Electron Microscopy and Energy Dispersive X-ray Spectroscopy-

To investigate the practical Zn solubilizing ability of the most potential bacterial isolate(ZSC9), Field Emission-Scanning Electron Microscopic (FE-SEM) and Energy Dispersive X-ray Spectroscopic (EDS) analysis was conducted following the established standard procedure. The bacterial isolateZSC9 was inoculated into the liquid basal medium containing 0.1% ZnO and incubated at 30°C, 120 rpm for 10 days. A flask with un-inoculated medium supplemented with ZnOwas used as the control in the experiment. On the 10th day after incubation, the dried samples were prepared using the procedure outlined by Upadhayayet al. (2022c). The dried samples underwent platinum coating and analysis of elemental components was then carried out utilizing a JEOL IT800FE-SEM along with EDS.

2.7 Biochemical and molecular characterization of zinc solubilizing bacteria-

The most potential bacterial isolates capable of solubilizing Zn were selected for further analysis through biochemical and molecular characterization. The colony's morphological characteristics and other fundamental traits, such as gram staining, motility, Methyl Red (MR) test, Voges-Proskauer (VP) test, spore formation, catalase, oxidase, citrate etc., were examined using the procedure outlined by Tindalet al. (2007).The 16S rRNA gene sequencing was conducted to identify seven bacterial isolates with the ability to solubilize Zn. The genomic DNA of bacterial isolates was extracted using the GenElute™ Bacterial Genomic DNA kit (Sigma Aldrich, St. Louise, Missouri, USA). PCR amplification of the 16S ribosomal DNA fragment was performed using 16s forward (5′GGATGAGCCCGCGGCCTA-3′) and reverse (5′CGGTGTGTACAAGGCCCGG-3′) primers specifically designed to target the ribosomal DNA, spanning approximately 1500 base pairs. ABI3130 genetic analyzer was used to purify and sequence the amplicons in both directions using BDT v3.1 chemistry cycle sequencing kit. The collected sequence information from bacterial isolates' 16S rRNA gene was matched against sequences in the NCBI database via the BLAST tool. Sequence alignment was achieved using ClustalW, and a phylogenetic tree based on the maximum likelihood method was constructed using Mega 11 software.

2.8 Statistical Analysis-

All statistical analyses were carried out using R software (version 4.3.1). The data from three parameters (solubilization index, soluble Zn and pH) was collected and each treatment was replicated three times to estimate mean values and standard deviations.The data obtained in this study was analyzed using one-way ANOVA and Tukey's Honest Significant Difference (HSD) test was used to compare the significant difference between the mean values of different parameters at p < 0.05. Distinct letters are used to denote values that are significantly different from each other.Pearson product-moment correlation analysis program was used to assess the correlation between the parameters.

3.1 Isolation and screening of zinc solubilizing bacteria-

There was a total of 19 ZSB isolated from the rhizospheric soil of onion plants and labeled as ZSC1-ZSC19. These bacterial strains were examined to assess their ability for Zn solubilization using three types of insoluble Zn compounds, namely ZnO, ZnCO₃ and Zn₃(PO₄)₂.Out of these 19 isolates, 4 (ZSC1, ZSC3, ZSC11, ZSC12) have shown least solubilization for the above said Zn compounds, so they were not utilized for further studies. The solubilization index of 15 bacterial isolates was calculated on3rd, 5th, 7th and 10th day and the results revealed that these isolates were more efficient in solubilizing ZnO compared to ZnCO₃ and Zn₃(PO₄)₂.The bacterial isolate ZSC9 demonstrated the highest solubilization index (8.85 and 5.64), with ZSC10 (6.74 and 5.04) and ZSC5 (6.23 and 4.73) showing the next highest index after 10 days of incubation, when grown on basal medium containing ZnOand ZnCO₃ separately (Tables 1 and 2).However, the bacterial isolate ZSC18 exhibited the maximum solubilization index (3.18) forZn₃(PO₄)₂ followed by ZSC9 (3.04) and ZSC14 (2.80) (Table 3).

Table 1

Evaluation of solubilization index of zinc solubilizing bacterial isolates in solid medium containing ZnO at different time intervals
Bacterial Isolates	Solubilization Index
Bacterial Isolates	3 days	5 days	7 days	10 days
ZSC2	1.54 ± 0.13^c	1.60 ± 0.15^cd	2.25 ± 0.18^de	2.62 ± 0.11^jk
ZSC4	1.47 ± 0.06^c	1.50 ± 0.06^d	2.05 ± 0.07^de	2.60 ± 0.06^jk
ZSC5	3.51 ± 0.16^ab	3.83 ± 0.12^b	4.67 ± 0.11^b	6.23 ± 0.15^b
ZSC6	1.42 ± 0.10^c	2.20 ± 0.15^c	2.55 ± 0.12^d	4.34 ± 0.09^de
ZSC7	1.44 ± 0.07^c	1.65 ± 0.17^cd	2.30 ± 0.09^de	3.62 ± 0.06^fgh
ZSC8	1.42 ± 0.17^c	1.64 ± 0.10^cd	2.32 ± 0.12^de	3.35 ± 0.10^ghi
ZSC9	3.97 ± 0.13^a	4.81 ± 0.11^a	6.59 ± 0.13^a	8.85 ± 0.10^a
ZSC10	3.60 ± 0.19^ab	3.91 ± 0.19^b	4.93 ± 0.14^b	6.74 ± 0.14^b
ZSC13	1.47 ± 0.08^c	1.70 ± 0.18^cd	2.31 ± 0.10^de	3.65 ± 0.08^fg
ZSC14	3.03 ± 0.14^b	3.57 ± 0.15^b	4.42 ± 0.15^b	5.59 ± 0.12^c
ZSC15	1.32 ± 0.10^c	1.42 ± 0.08^d	1.82 ± 0.12^e	2.35 ± 0.08^k
ZSC16	1.61 ± 0.13^c	1.78 ± 0.13^cd	2.38 ± 0.17^de	2.81 ± 0.11^ijk
ZSC17	1.55 ± 0.10^c	1.63 ± 0.10^cd	3.20 ± 0.16^c	4.73 ± 0.16^d
ZSC18	1.38 ± 0.09^c	1.58 ± 0.09^cd	2.49 ± 0.12^d	4.01 ± 0.13^ef
ZSC19	1.70 ± 0.16^c	2.06 ± 0.14^cd	2.31 ± 0.09^de	3.05 ± 0.14^hij

Data are presented as means ± standard error, n = 3. The means with similar letter(s) in each column did not differ significantly from each other.

Table 2

Evaluation of solubilization index of zinc solubilizing bacterial isolates in solid medium containing ZnCO₃ at different time intervals
Bacterial Isolates	Solubilization Index
Bacterial Isolates	3 days	5 days	7 days	10 days
ZSC2	1.10 ± 0.05^e	1.11 ± 0.06^f	1.18 ± 0.10^fg	1.23 ± 0.21^gh
ZSC4	1.09 ± 0.06^e	1.12 ± 0.08^f	1.15 ± 0.29^g	1.18 ± 0.18^h
ZSC5	2.03 ± 0.14^b	2.33 ± 0.14^b	3.31 ± 0.13^a	4.73 ± 0.25^b
ZSC6	1.37 ± 0.09^cde	1.50 ± 0.12^cde	1.76 ± 0.28^cde	2.14 ± 0.14^e
ZSC7	1.27 ± 0.12^cde	1.34 ± 0.13^def	1.52 ± 0.11^defg	1.76 ± 0.28^ef
ZSC8	1.26 ± 0.10^cde	1.40 ± 0.10^def	1.55 ± 0.14^def	1.93 ± 0.19^ef
ZSC9	2.50 ± 0.15^a	2.66 ± 0.15^a	3.45 ± 0.16^a	5.64 ± 0.33^a
ZSC10	2.45 ± 0.19^a	2.61 ± 0.14^ab	3.43 ± 0.19^a	5.04 ± 0.28^b
ZSC13	1.18 ± 0.08^de	1.28 ± 0.09^def	1.56 ± 0.21^def	1.80 ± 0.10^ef
ZSC14	1.54 ± 0.14^c	1.76 ± 0.12^c	2.35 ± 0.15^b	3.74 ± 0.34^c
ZSC15	1.14 ± 0.07^de	1.24 ± 0.07^ef	1.51 ± 0.11^defg	1.61 ± 0.14^fgh
ZSC16	1.17 ± 0.14^de	1.22 ± 0.10^ef	1.37 ± 0.20^efg	1.64 ± 0.16^fg
ZSC17	1.42 ± 0.12^cd	1.58 ± 0.12^cd	1.99 ± 0.19^bc	3.02 ± 0.26^d
ZSC18	1.33 ± 0.17^cde	1.48 ± 0.10^cde	1.86 ± 0.14^cd	2.86 ± 0.27^d
ZSC19	1.17 ± 0.08^de	1.24 ± 0.09^ef	1.50 ± 0.12^def	1.74 ± 0.24^ef

Data are presented as means ± standard error, n = 3. The means with similar letter(s) in each column did not differ significantly from each other.

Table 3

Evaluation of solubilization index of zinc solubilizing bacterial isolates in solid medium containing Zn₃(PO₄)₂ at different time intervals
Bacterial Isolates	Solubilization Index
Bacterial Isolates	3 days	5 days	7 days	10 days
ZSC2	1.09 ± 0.09^f	1.13 ± 0.06^g	1.18 ± 0.07^f	1.19 ± 0.18^g
ZSC4	1.13 ± 0.05^ef	1.15 ± 0.05^fg	1.18 ± 0.09^f	1.20 ± 0.09^g
ZSC5	1.76 ± 0.22^bc	1.65 ± 0.12^cd	2.45 ± 0.25^b	2.58 ± 0.26^c
ZSC6	1.39 ± 0.19^de	1.48 ± 0.17^de	1.66 ± 0.12^cd	1.71 ± 0.11^e
ZSC7	1.36 ± 0.21^def	1.41 ± 0.19^def	1.58 ± 0.19^de	1.64 ± 0.14^ef
ZSC8	1.07 ± 0.17^f	1.08 ± 0.16^g	1.14 ± 0.07^f	1.17 ± 0.17^g
ZSC9	2.31 ± 0.11^a	2.49 ± 0.21^a	2.79 ± 0.21^a	3.04 ± 0.23^ab
ZSC10	1.76 ± 0.15^bc	1.82 ± 0.29^bc	2.43 ± 0.13^b	2.75 ± 0.13^bc
ZSC13	1.17 ± 0.08^ef	1.26 ± 0.27^efg	1.33 ± 0.15^ef	1.37 ± 0.18^fg
ZSC14	1.83 ± 0.07^b	2.06 ± 0.16^b	2.64 ± 0.18^ab	2.80 ± 0.29^bc
ZSC15	1.13 ± 0.14^ef	1.13 ± 0.08^g	1.18 ± 0.09^f	1.23 ± 0.27^g
ZSC16	1.23 ± 0.09^def	1.33 ± 0.19^efg	1.42 ± 0.17^def	1.45 ± 0.21^efg
ZSC17	1.52 ± 0.22^cd	1.61 ± 0.15^cd	1.96 ± 0.14^c	2.15 ± 0.25^d
ZSC18	2.39 ± 0.10^a	2.64 ± 0.11^a	2.91 ± 0.22^a	3.18 ± 0.11^a
ZSC19	1.15 ± 0.08^ef	1.21 ± 0.18^efg	1.28 ± 0.11^ef	1.33 ± 0.12^fg

Data are presented as means ± standard error, n = 3. The means with similar letter(s) in each column did not differ significantly from each other.

3.2 Quantitative assay for zinc solubilization in broth media-

The potential of 15 bacterial isolates to solubilize Zn was additionally assessed through quantitative analysis in liquid basal medium at 3rd, 5th, 7th and 10th day.Each of the bacterial isolates demonstrated the ability to solubilize the insoluble Zn compounds introduced into the culture medium and Zn solubilization was observed to increase with prolonged incubation time (Fig. 1). The highest concentration of Zn released was observed by the isolate ZSC9 (624 mg/l and 503 mg/l) followed by ZSC10 (564 mg/l and 487 mg/l) and ZSC5 (557 mg/l and 462 mg/l) in the medium amended with ZnO and ZnCO₃ individually on the 10th day after incubation. Whereas, ZSC18 exhibited notably greater efficiency in solubilizing zinc phosphate (375 mg/l) as compared to ZSC9 (361 mg/l).

3.3 pH measurement during the process of zinc solubilization-

During the solubilization of various insoluble Zn compounds in broth culture, a noticeable decrease in pH was commonly observed, indicating a connection between Zn solubilization and pH alteration in the culture medium.Out of the various Zn salts, the medium enriched with zinc oxide exhibited the most significant decrease in pH in comparison to the medium containing zinc carbonate and zinc phosphate. The assessment of pH revealed a reduction in pH levels reaching a minimum of 3.82 from the initial pH of 7.0 over the course of 7 days during the incubation period and then a slight rise in pH till the 10th day (Fig. 2). Among the bacterial isolates, cell-free culture supernatant of ZSC9 displayed the most acidic pH value of 3.82, while ZSC10 and ZSC5 showed a slightly higher pH value of 4.0 and 4.07 respectivelyin ZnO amended medium. There was a major shift in pH from 7.0 to 4.35 in ZnCO₃ supplemented mediumand from 7.0 to 4.89 in basal medium enriched with Zn₃(PO₄)₂ by the isolates ZSC9 and ZSC18 respectively on the 7th day of incubation.Subsequently, there was a gradual pH increase in both mediums until the 10th day.

Pearson product moment correlation within the Zn solubilization index, amount of soluble Zn content and pH reduction was analyzed using 3 insoluble Zn compounds viz. ZnO, ZnCO₃ and Zn₃(PO₄)₂. During the process of solubilizing ZnO, a significant negative correlation coefficient of -0.60 was noticed between the solubilization index and pH with the p value of < 0.001. Conversely, the correlation between soluble Zn and pH exhibited a less pronounced negative association, with a correlation coefficient of-0.50. However, a positive correlation was noted between solubilization index and soluble Zn with r = 0.89.A significant inverse relationship was identified between solubilization index and pH, denoted by a strong negative correlation with r=-0.69 during ZnCO₃ solubilization, whereas the correlation between soluble Zn and pH was less negative, with a value of r= -0.56 and the correlation was found to be positive between solubilization index and soluble Zn (r = 0.89). A comparable pattern was noticed with Zn₃(PO₄)₂ solubilization, where a stronger negative correlation was found between solubilization index and pH (r=-0.86), while the soluble Zn and pH was relatively less correlated (r=-0.46) and solubilization index vs soluble Zn was positively correlated with r = 0.75. This analysis suggests that pH reduction of the culture medium was inversely related to the Zn solubilization by the bacterial isolates (Fig. 3).

3.4 FE-SEM and EDS analysis-

On the 10th day of incubation, the SEM images revealed enhanced Zn solubilization in the liquid basal medium supplemented with zinc oxide for the bacterial isolate ZSC9, in comparison to the control without any inoculation (Fig. 4). A large amount of dense and amorphous residues of insoluble substances have been found in untreated control whereas treated bacterial samples were found with lesser mineral residues. The analysis conducted using EDS indicated a reduction in the proportion of Zn and a rise in the carbon concentration in ZSC9 treated sample (C-77.5%, Zn-0.2%) when contrasted with the untreated control (C-51.4%, Zn-24.6%).

Figure 5 Scanning Electron Microscopy (SEM) images depict increased Zn solubilization in the liquid basal medium containing ZnO by the isolate ZSC9 (A) on the 10th day as compared to the uninoculated control (B) and EDS analysis reveals a reduction in the Zn percentage and an increase in the carbon content in the residues of ZSC9 inoculated samples (C) than the control (D).

3.5 Biochemical characterization and identification of potential ZSB isolates-

The identification of selected bacterial isolates was determined through analysis ofmorphological and biochemical characteristics (Table 4). In terms of their morphology, all of the isolates (except ZSC17) exhibited characteristics of being Gram-negative and motile, and all were having a rod-shaped structure. The 16S rDNA-based analysis revealed the most potent ZSB isolate ZSC9 as Pantoeaeucrina (Accession no. OQ547246.1).After BLAST investigation of the 16S rRNA gene sequences, the isolates ZSC5 and ZSC10 were found to be similar with Pantoeadispersa (Accession no. OQ569483.1 and OQ547235.1), while ZSC6 and ZSC14 showed maximum similarity with Pseudomonas aeruginosa (Accession no. OQ547241.1 and OQ402584.1).The remaining 2 isolates ZSC17 and ZSC18 were found to be closely related with Bacillus velezensis(Accession no. OQ410447.1) and Pseudomonas fluorescens(Accession no. OQ392599.1) respectively (Fig. 5).

Table 4

Morphological and biochemical properties of selected zinc solubilizing bacterial isolates
Serial no.	Morphological and biochemical properties	Bacterial isolates
Serial no.	Morphological and biochemical properties	ZSC5	ZSC6	ZSC9	ZSC10	ZSC14	ZSC17	ZSC18
1.	Gram’s reaction	-ve	-ve	-ve	-ve	-ve	+ve	-ve
2.	Cell shape	Small rod	Rod	Small rod	Small rod	Rod	Rod	Rod
3.	Spore formation	-	-	-	-	-	+	-
4.	Motility	+	+	+	+	+	+	+
5.	Indole	-	-	-	-	-	-	-
6.	Methyl red	+	-	+	+	-	-	+
7.	Voges-proskauer	+	-	+	+	-	-	-
8.	Citrate utilization	+	+	+	+	+	+	+
9.	Oxidase	+	-	+	+	-	+	+
10.	Urease	-	+	+	-	+	+	+
11.	Catalase	+	+	+	+	+	+	+
12.	Gelatin liquefication	+	+	-	+	+	+	+
13.	Nitrate reduction	+	+	+	+	+	-	-
( + = Positive result, - = Negative result)

"Microbial-facilitated biofortification" represents an innovative idea within the realm of agricultural microbiology. It involves enhancing the nutritional value of crop yields by utilizing microorganisms to introduce vital micronutrients like zinc, iron, and selenium.Zn holds a pivotal role in plant constituents, playing a vital role in their growth and maturation. The lack of Zn stands as the prevalent shortfall of micronutrients in crops globally, leading to significant reductions in agricultural productivity and subsequently results in malnutrition in human populations. Numerous strategies exist to combat Zn deficiency, employing various approaches to enhance the intake of dietary Zn. An example of such a strategy involves harnessing soil microorganisms, which have the ability to make inaccessible Zn ineasily available form. This, in turn, enhances Zn absorption, facilitates plant development, and ultimately improves crop productivity (Ramesh et al., 2014; Kamran et al., 2017; Upadhayayet al., 2022b). Thepresent research illustrates the isolation, identification and characterization of the strains with the ability to solubilize insoluble Zn compounds. A total of nineteen bacterial isolates were obtained from the rhizospheric soil samples of onion plants. Among these, seven bacterial isolates exhibited the highest proficiency in effectively solubilizing different forms of Zn salts. These strains showed evolutionary relationships with Pantoeaeucrina, Pantoeadispersa, Pseudomonas aeruginosa, Bacillus velezensis and Pseudomonas fluorescens. The earlier findings illustrate that how Pseudomonas aeruginosa and Pseudomonas fluorescens can effectively solubilize Zn from different types of insoluble Zn salts (Pawaret al., 2015; Gontia-Mishraet al., 2017; Hashemnejadet al., 2021; Macwanet al., 2022; Nasirzadehet al., 2023).Numerous species of Bacillus, including B. aryabhattai, B. megaterium, B. subtilis,andB. cereus, have been thoroughly investigated concerning their role in Zn solubilization (Ramesh et al., 2014; Khandeet al., 2017; Mumtazet al., 2017; Bhatt and Maheshwari, 2020). There have been a few reports indicating the Zn solubilizing potential of B. velezensis (Ramavathet al., 2019; Mahdi et al., 2022; Naseemet al., 2022), however, our research yielded contrasting findings compared to the outcomes reported by de Meloet al.(2021) and Kadiriet al.(2023), where B. velezensis was unable to solubilize Zn compounds.Kamran et al. (2017) reported two species of Pantoea (P. dispersa and P. agglomerans) having Zn solubilizingability, however, our finding appears to be the first report demonstrating Pantoeaeucrina as a potential Zn solubilizing bacterium.

All of the bacterial isolates were screened for their Zn solubilizing ability using plate assay and Zn solubilization index was calculated.PantoeaeucrinaZSC9 demonstrated the highest solubilization index (8.85) when grown on the ZnO amended basal medium on the 10th day after incubation, which is significantly greater than the previously documented Zn solubilization results (Mumtazet al., 2017; Naseemet al., 2021).Ali et al. (2023) demonstrated that out of their 69 examined strains, which were evaluated for their Zn solubilizing ability against two insoluble Zn compounds (ZnO and ZnCO₃) using plate assay, Raoultella sp. exhibited the maximum solubilization index (4.3) using ZnO. In another investigation, Pseudomonas putida displayed the highest Zn solubilization index (3.31) in the culture medium containing ZnO (Hashemnejadet al., 2021). The bacteria used in current study exhibited different levels of solubilization, as evidenced by the variability in the solubilization index. The results revealed that among the three insoluble Zn compounds [ZnO, ZnCO₃ and Zn₃(PO₄)₂], all of the bacterial isolates were more effective at solubilizing ZnO compared to ZnCO₃ and Zn₃(PO₄)₂.Our results align with the conclusions of previous studies, which also indicated better Zn solubilization in ZnO amended medium followed by ZnCO₃ and Zn₃(PO₄)₂(Sunithakumariet al., 2016; Dinesh et al., 2018; Bhakatet al., 2021). However, our findings contradict the outcomes reported by Sharma et al. (2012) and Vidyashreeet al. (2016). The solubilizing potential of various strains might be associated with distinctions in the solubility product constant (Ksp). The values of ksp are3.86×10^− 10, 1.4×10^− 11 and 9.0×10^− 33forZnO, ZnCO₃ andZn₃(PO₄)₂, respectively. A greater Ksp corresponds to increased solubility of the compound. Among the investigated salts, ZnO displays the most elevated Ksp, closely pursued by ZnCO₃, both significantly surpassing the Ksp of Zn₃(PO₄)₂ (Bhattacharya and Elzinga 2018).

The reliability of the qualitative assessment for the Zn solubilization potential of bacterial strains is questionable.To further clarify the doubts, all of the bacterial isolates were subsequently subjected to quantitative analysis in a liquid medium enriched with insoluble Zn sources separately [ZnO, ZnCO₃ and Zn₃(PO₄)₂] (Bashan et al., 2013) and a distinct variation in the pattern of Zn solubilization was found. The results revealed that all of the bacterial isolates were able to solubilize all the three Zn compounds and solubilization was observed to be greater in a medium enriched with ZnO than ZnCO₃ and Zn₃(PO₄)₂which ranged from 245 to 624 soluble Zn (mg/l). The maximum Zn solubilization was exhibited by PantoeaeucrinaZSC9 (624 mg/l) on the 10th day after incubation, indicating that the bacteria solubilized 62.4% of the initially added Zn followed by Pantoeadispersa ZSC10 which solubilized 56.4% of the introduced Zn. Our results are consistent with earlier studies where bacterial strains demonstrated the most effective ability to solubilize ZnO in comparison to ZnCO₃ and Zn₃(PO₄)₂ (Gontia-Mishra et al., 2017;Bhakatet al., 2021). Additional research involving P. fluorescens strain Ur22 indicated the highest rate of Zn solubilization (35.6 mg/l) when grown in medium supplemented with ZnO (Hashemnejadet al., 2021). However, our findings contradict the conclusions of Ramesh et al. (2014), who reported that strains MDSR7 and MDSR14 were more efficient in solubilizing Zn₃(PO₄)₂ (148.09 and 153.47 µg/ml) as compared to ZnO and ZnCO₃.

The ability of ZSB to make Zn available from insoluble sources could be attributed to the medium acidification by secretion of organic acids (Costerousseet al., 2018). Previous research has indicated that among various organic acids produced by bacterial strains, gluconic acid is the most prevalent acid for Zn solubilization (Saravananet al., 2007; Dinesh et al., 2018; Kushwahaet al., 2021).However, secretion of other organic acids like malonic, citric, succinic (Vidyashreeet al., 2018) lactic, acetic acid (Rani et al., 2023), have also been noticed by ZSB. Martino et al. (2003)have also observed the release of some organic acids (malic and citric acids) in the culture medium containing insoluble Zn metals to facilitate solubilization by ericoid mycorrhizal fungi derived from heavy metal polluted sites. In current investigation, we observed a noteworthy decline in the pH level of the medium, indicating that all bacterial isolates secreted organic acids that led to pH reduction (from 7 to less than 4). This acidic environment subsequently facilitated the solubilization of the initially introduced insoluble Zn salts. For all the bacterial isolates, a significant negative correlation between the pH reduction and Zn solubilization (both qualitative and quantitative) was observed and the results are in accordance with the study of Gontia-Mishra et al. (2017) and Bhakat et al. (2021). The isolate Pantoeaeucrina ZSC9 exhibited the maximum reduction in pH (3.82) in ZnOsupplemented medium on the 7th day of incubation. Similar findings were documented by Hinaet al. (2018), wherein the bacterial isolate H-103 exhibited the highest pH reduction (4.05). This decline in pH was directly linked to the increased Zn solubilization. In current study, a gradual increase in pH of culture medium was observed after 7th day of incubation and the medium likely became more alkaline due to the hydrolysis of amino acids, leading to the release of ammonium ions (Costerousseet al., 2018).

In present investigation, FE-SEM was employed to validate the effectiveness of most potent ZSB isolate Pantoeaeucrina ZSC9 to enhance the Zn solubilization in the medium amended with zinc oxide. The results indicated that the bacterial isolate ZSC9 demonstrated a higher efficiency in the solubilization of zinc oxide in comparison to the uninoculatedcontrol on the 10th day after incubation.The treated samples had shown minimum mineral residues in comparison to untreated control that is found with dense, amorphous and opaque insoluble residues. Similarly, Gandhi and Muralidharan (2016) also found that the SEM images of Acinetobacter (AGM3) revealed superior ZnO solubility compared to ZnCO₃ on the 12th day of incubation when grown in tris-mineral salt medium. In our study, EDS analysis suggested that the percentage of Zn was decreased and carbon content was increased in the treated sample in comparison to the non-inoculated control due to the solubilization of Zn and aggregation of organic compounds secreted by bacterial isolate with mineral residues.The results, however, are in accordance with the study of Upadhayayet al. (2022c), where the SEM images of Burkholderiacepacia and Pantoearodasiishowed superior mobilization of ZnO compared to the untreated control on the 10th day of incubation. The EDS analysis further confirmed a reduction in the Zn percentage and an increase in the carbon content in the residues of the inoculated samples than the control.

Out of the 19 bacterial isolates obtained from the onion rhizosphere, seven bacterial strains were found to have significant ability to dissolve insoluble Zn compounds. The identification of the selected ZSB isolates through 16s rRNA gene sequencing revealed the affiliation of all strains to the generaPantoea, Pseudomonas and Bacillus.All of the strains were more effective at solubilizing zinc oxide followed by zinc carbonate and zinc phosphate. The maximum Zn solubilization index (8.85) and the highest concentration of available Zn (624 mg/l) was exhibited by the strain Pantoeaeucrina ZSC9 on the 10th day of incubation when grown in zinc oxide enriched basal medium. The analysis conducted through FE-SEM-EDS showed a significant dissolution of Zn, indicating the mobilization of Zn from the ZnO residues by Pantoeaeucrina ZSC9.The primary reason for medium acidification and the subsequent solubilization of Zn appears to be the production of gluconic acid by the bacteria.The potential of these isolates to solubilize Zn will be further evaluated for their beneficial effects on plant growth promotion, making them a viable option for exploration as biofertilizers for economic crops.This approach holds promise as a sustainable strategy for reducing the reliance on chemical fertilizers and mitigating the associated environmental pollution.

Acknowledgement: The authors are sincerely thankful to Directorate of Research and Department of Microbiology, COBS&H, CCS-HAU, Hisar for providing all the facilities to carry out the research work and support. The CSIR-UGC is greatly acknowledged for providing Junior Research Fellowship and Senior Research Fellowship to Shivi Choudhary (UGC Grant no. 528/(CSIR-UGC NET DEC. 2018).

Author contributions: BSS have contributed to the conceptualization and design & Execution of experiment; SC have contributed in, sample collection, lab work and reviewing; SK sample collection: AG has contributed in statistical analysis; BSS and RG has monitored the overall progress and approval of the work.

Ethical Approval

Not Applicable

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Availability of data and materials

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

Competing Interest

There are no Competing Interests

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No competing interests reported.

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Graphical abstract

Molecular characterization of potential zinc solubilizing bacterial isolates from onion rhizosphere and validation of solubilization ability of PantoeaeucrinaZSC9 using FE-SEM and EDS

Status:

Version 1

Abstract

Figures

Highlights

1. Introduction

2. Materials and methods

2.1 Collection of rhizospheric soil samples-

2.2 Isolation of zinc solubilizing rhizobacteria-

2.3 Qualitative assessment of zinc solubilization-

2.4 Quantitative assay for zinc solubilization in broth media-

2.5 pHmeasurement during the process of zinc solubilization-

2.6 Field Emission-Scanning Electron Microscopy and Energy Dispersive X-ray Spectroscopy-

2.7 Biochemical and molecular characterization of zinc solubilizing bacteria-

2.8 Statistical Analysis-

3. Results

3.1 Isolation and screening of zinc solubilizing bacteria-

3.2 Quantitative assay for zinc solubilization in broth media-

3.3 pH measurement during the process of zinc solubilization-

3.4 FE-SEM and EDS analysis-

3.5 Biochemical characterization and identification of potential ZSB isolates-

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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