The production of a halo zone around the bacterial colonies in a plate containing ZnO could be due to proton extrusion, siderophore, or organic acid production by bacteria [31-32]. Quantitative Zn dissolution in flasks containing ZnO indicated the ability of isolates to release Zn, although the dissolution amount varied between the two studied isolates. The decrease in pH of the inoculated medium containing insoluble forms of Zn and phosphorus could confirm the isolates’ mechanism to dissolve these mineral forms of elements.
The decrease in pH of liquid media and Zn dissolution by bacteria through different organic acid production methods have been reported previously [16]. Organic acid production by bacteria through insoluble phosphorus dissolution can be the cause of pH reduction [33]. Mumtaz et al. [34] screened 13 Zn solubilising isolates from a maise rhizosphere with maximum Zn dissolutions of 27.15 and 27.66 µg mL-1 for Bacillus subtilis and Bacillus aryabhattai, respectively. The potential of Bacillus sp., Bacillusaryabhattai (ZM31), and Bacillus aryabhattai (S10) to decrease the pH of a medium, as well as the dissolution of phosphorus from the source of Ca3(PO4)2, has been recorded by Mumtaz et al. [34].
Impact of Zn solubilising bacteria on Zn in soil
The results indicated a positive effect of bacteria on the increase of available Zn (DTPA-extractable Zn) in soil. Increased soil DTPA-extractable Zn could be attributed to a decrease in soil pH (Table 4). This conclusion is supported by the negative correlation between DTPA-extractable Zn values and soil pH (R2=-0.80**). Soil microbial inoculation diminished the pH of soil [10, 35] probably by organic acids production. The results of soil pH and DOC measurements confirmed that bacteria could reduce soil pH and increase DOC by producing organic acids and proton secretion. This proton secretion could ultimately increase the amount of available Zn in the soil, as previously reported by Vaid et al. [13]. A negative and significant correlation between DOC and pH (R2 = -0.95**) indicated the impact of DOC on reducing soil pH (Table 4). The enhancement of soluble organic carbon content due to the organic matter degradation in the soil and organic acid production by bacteria has been reported [36]. Zn solubility reduction has also been documented by sorbing Zn on functional groups of solid organic matter [37], whereas DOC has been found to increase Zn solubility [38]. Iratkar et al. [39] reported more availability of soil Zn with greater organic matter content. Our results indicate that there is a positive correlation between DTPA-extractable Zn values and DOC (Table 4). Based on the findings of this study, applied Zn-solubilizing bacteria increased EXCH-Zn and ORG-Zn and decreased CAR-Zn, FeMnOX-Zn, and RES-Zn. However, these changes varied depending on the bacteria. The increase in the concentration of EXCH-Zn in the treatment of plant growth-stimulating bacteria might be due to the transition of Zn from its less dynamic and unavailable forms to more dynamic and available forms.
In the present study, the reduction of CAR-Zn in the treatment of growth-stimulating bacteria was accompanied by an increase in EXCH-Zn and ORG-Zn when compared with the control. The increase in the solubility of carbonate-bound Zn and the conversion of this form to other forms reduced the concentration of carbonate-bound Zn in plant growth-stimulating bacteria treatment when compared with the control. It is noteworthy that the decrease in the carbonates bound form was accompanied not only by an increase in the exchangeable component but also by an increase in the organically bound form. Therefore, it seems that in addition to pH, the secretion of bacteria caused the release of Zn bounded to calcium carbonate and transformed it into exchangeable and organically bound forms, therefore Zn availability increased [40, 41].This result was confirmed by the positive correlation between DOC and ORG-Zn (R2= 0.89**). The increased EXCH-Zn due to the influence of pH and DOC in the soil might also be related to the negative and positive relationships between EXCH-Zn values and pH (R2= -0.80**) and DOC (R2= 0.87**).
The role of DOC in the enhancement of Zn-exchangeable fraction in calcareous soil has been reported previously [42, 43]. Reductions in the FeMnOX and RES fractions of Zn due to the bacterial inoculation have been accompanied by increases in OM and EXCH fractions of Zn. Huang et al. [41] reported that the inoculation of soil with Rhizobium fredii reduced the amount of Zn bound to iron and manganese oxides. Our results agree with the findings of Bharti et al. [44], who indicated that the co-inoculation of AMF (Glomus mosseae), Burkholderia cepacia, and Azospirillum brasilense increased OM and EXCH fractions of Zn and reduced FeMnOX-Zn. The application of Zn fertiliser increased the RES, FeMnOX and CAR fractions of Zn, whereas bacterial inoculation reduced these fractions. These results demonstrate that Zn fertilisers, even soluble salts of Zn (Zn sulfate), dissolve slowly in soil and release Zn, which is then converted into carbonates, oxides, and residual forms [45]. Our data (Table 2) suggest that bacterial inoculation increased the EXCH and OM fractions of Zn under deficient conditions and even Zn fertiliser application. The replenishment of EXCH-Zn by OM-Zn fraction for plant uptake can be concluded by a highly positive correlation between EXCH-Zn and OM-Zn fractions (Table 4).
The relative contents of Zn fractions in the soil for all treatments were in the following order: RES > FeMnOX > CAR > OM>EXCH. For all treatments, up to 37-41% of the Zn was associated with the RES fraction, and 27.5-29.5% and 24-27% resided in the FeMnOX and CAR-Zn fractions, respectively (Figure 1). The residual form of Zn is the most stable fraction in soil, and according to others, the highest and lowest proportions of Zn fractions at all treatments were the RES and EXCH fractions, respectively [44, 46, 47].
Our results also indicate that the application of Zn fertiliser and bacteria inoculation cause a shift in Zn distribution from unavailable forms to the EXCH-Zn fraction (0.84, 1.28, 1.68, 2.41, and 2.5% for Zn0B0, Zn1B0, Zn2B0, Zn1B3, and Zn2B3, respectively) (Figure 1). An increase in EXCH-Zn content was observed when Zn fertiliser treatments [46] and bacterial inoculation treatments [44] were applied.
Zn uptake, protein content, and molar ratio of PA/Zn in wheat grains
The bacterial inoculation increased Zn uptake at all ZnSO4 application levels. The increase in Zn uptake might be related to the different mechanisms such as IAA production [48], root growth improvement, mineralization and transformation of Zn by isolates [49]. A close relationship was observed between EXCH and OM-Zn fractions and Zn uptake in grains, thus confirming that the exchangeable and organically bound fractions were the most significant contributors towards Zn uptake in grain (R2= 0.94** and 0.95**, respectively), followed by RES-Zn (R2= 0.33*). These results agree with the findings of Bharti et al. [44]. The EXCH and OM fractions of Zn mainly influenced the available Zn concentration and its uptake by the plant [50, 47]
Soil inoculation of Bacillus megaterium and Enterobactercloacae in combination with Zn fertilizer application was more effective than the application of each treatment lonely on Zn uptake in wheat grains. Rehman et al [10] recorded more Zn uptake by wheat using endophytic bacterial Pseudomonas sp. MN12 in combination with Zn application.
In our study, soil application of Zn caused to enhance grain yield, possibly due to plant growth improvement, seed establishment, nutrients uptake and Zn involvement in carbohydrate metabolism, IAA production and RNA polymerase expression [10].
Moreover, inoculation of bacteria further improved grain yield of wheat due to better root growth [10, 51, 52], photosynthesis [52, 53] and Zn uptake [51]. More improvement in grain yield, protein content and grain quality was observed by soil inoculation of Bacillus megaterium and Enterobactercloacae in combination with Zn fertilizer application. The same results have been reported by Co-application of Zn and endophyte Enterobacter sp. MN17 on improved nodulation, leghemoglobin, grain yield, bioavailable Zn and grain quality of chickpea [12].
The grain yield was increased by enhancing Zn uptake, which is indicated by the positive and significant correlation between Zn uptake and grain yield (R2= 0.99**, Table 4B). Solubility and availability of Zn present in soil enhanced by inoculation of Bacillus megaterium and Enterobactercloacae [54].
The increases in grain protein content in treatments of bacterial inoculation and ZnSO4 application are likely due to Zn’s role in the expression of RNA polymerase enzymes and synthesis protein [55, 56].
Increasing Zn uptake in grains reduced the molar ratio of PA/Zn, which could be due to the negative and significant correlation between Zn uptake and PA/Zn ratio (R2= -0.90**).
According to the World Health Organization [57], foodstuffs are categorised into three groups in terms of Zn availability: low availability (molar ratio of PA/Zn > 15), moderate availability (molar ratio of PA/Zn: 5 to 15), and high availability (molar ratio of PA/Zn < 5). Hence, increasing Zn concentration in wheat is not enough to increase its nutritional value since phytate (the phosphorus storage in plants) is a combination that tends to complex iron, Zn, calcium, and magnesium and prevents their absorption in the human body [58].
In the present study, the co-inoculation of Bacillus megaterium and Entrobacter cloacae, together with ZnSO4 application, decreased the molar ratio of PA/Zn in wheat grains from 17.59 (control) to 12.07 (Zn2B3) and 12.77 (Zn1B3). Based on the classification of food by the World Health Organization [57], treatments with a PA/Zn molar ratio of less than 15 are considered to have a satisfying Zn adsorption. The increase in Zn uptake and the reduction of PA/Zn ratio in wheat grains, have been associated with an increase in the amount of applied ZnSO4 fertiliser [59] and inoculation of soil with Zn solubilising bacteria, as well [10, 11, 60].
Among Zn application levels and bacterial treatments, the highest soil available Zn (extracted using DTPA), Zn uptake and the lowest phytate/Zn molar ratio was recorded due to application of 10.1mgkg-1 zinc sulfate (Zn2) and a mixed inoculation of both bacteria (B3) which confirmed the efficiency of biological methods to trigger Zn uptake and improve nutritive status of wheat grain by changing morphology of roots and solubilizing Zn in soil [61].