Identification of Heterotrophic Zinc Mobilization Processes among Bacterial Strains Isolated from Wheat Rhizosphere (Triticum aestivum L.).

Soil and plant inoculation with heterotrophic zinc-solubilizing bacteria (ZSB) is considered a promising approach for increasing zinc (Zn) phytoavailability and enhancing crop growth and nutritional quality. Nevertheless, it is necessary to understand the underlying bacterial solubilization processes to predict their repeatability in inoculation strategies. Acidification via gluconic acid production remains the most reported process. In this study, wheat rhizosphere soil serial dilutions were plated on several solid microbiological media supplemented with scarcely soluble Zn oxide (ZnO), and 115 putative Zn-solubilizing isolates were directly detected based on the formation of solubilization halos around the colonies. Eight strains were selected based on their Zn solubilization efficiency and siderophore production capacity. These included one strain of Curtobacterium, two of Plantibacter, three strains of Pseudomonas, one of Stenotrophomonas, and one strain of Streptomyces In ZnO liquid solubilization assays, the presence of glucose clearly stimulated organic acid production, leading to medium acidification and ZnO solubilization. While solubilization by Streptomyces and Curtobacterium was attributed to the accumulated production of six and seven different organic acids, respectively, the other strains solubilized Zn via gluconic, malonic, and oxalic acids exclusively. In contrast, in the absence of glucose, ZnO dissolution resulted from proton extrusion (e.g., via ammonia consumption by Plantibacter strains) and complexation processes (i.e., complexation with glutamic acid in cultures of Curtobacterium). Therefore, while gluconic acid production was described as a major Zn solubilization mechanism in the literature, this study goes beyond and shows that solubilization mechanisms vary among ZSB and are strongly affected by growth conditions.IMPORTANCE Barriers toward a better understanding of the mechanisms underlying zinc (Zn) solubilization by bacteria include the lack of methodological tools for isolation, discrimination, and identification of such organisms. Our study proposes a direct bacterial isolation procedure, which prevents the need to screen numerous bacterial candidates (for which the ability to solubilize Zn is unknown) for recovering Zn-solubilizing bacteria (ZSB). Moreover, we confirm the potential of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as a quick and accurate tool for the identification and discrimination of environmental bacterial isolates. This work also describes various Zn solubilization processes used by wheat rhizosphere bacteria, including proton extrusion and the production of different organic acids among bacterial strains. These processes were also clearly affected by growth conditions (i.e., solid versus liquid cultures and the presence and absence of glucose). Although highlighted mechanisms may have significant effects at the soil-plant interface, these should only be transposed cautiously to real ecological situations.

Costerousse B ，Schönholzer-Mauclaire L ，Frossard E ，Thonar C ... -  《-》

Characterization of zinc solubilization potential of arsenic tolerant Burkholderia spp. isolated from rice rhizospheric soil.

Bhakat K ，Chakraborty A ，Islam E 《-》

Growth improvement of wheat (Triticum aestivum) and zinc biofortification using potent zinc-solubilizing bacteria.

Zinc (Zn) is an indispensable element for proper plant growth. A sizeable proportion of the inorganic Zn that is added to soil undergoes a transformation into an insoluble form. Zinc-solubilizing bacteria (ZSB) have the potential to transform the insoluble Zn into plant-accessible forms and are thus promising alternatives for Zn supplementation. The current research was aimed at investigating the Zn solubilization potential of indigenous bacterial strains and to evaluate their impact on wheat growth and Zn biofortification. A number of experiments were conducted at the National Agriculture Research Center (NARC), Islamabad, during 2020-21. A total of 69 strains were assessed for their Zn-solubilizing ability against two insoluble Zn sources (ZnO and ZnCO3) using plate assay techniques. During the qualitative assay, the solubilization index and solubilization efficiency were measured. The qualitatively selected Zn-solubilizing bacterial strains were further tested quantitatively using broth culture for Zn and phosphorus (P) solubility. Tricalcium phosphate was used as insoluble source of P. The results showed that broth culture pH was negatively correlated with Zn solubilization, i.e., ZnO (r2 = 0.88) and ZnCO3 (r2 = 0.96). Ten novel promising strains, i.e., Pantoea sp. NCCP-525, Klebsiella sp. NCCP-607, Brevibacterium sp. NCCP-622, Klebsiella sp. NCCP-623, Acinetobacter sp. NCCP-644, Alcaligenes sp. NCCP-650, Citrobacter sp. NCCP-668, Exiguobacterium sp. NCCP-673, Raoultella sp. NCCP-675, and Acinetobacter sp. NCCP-680, were selected from the ecology of Pakistan for further experimentation on wheat crop based on plant growth-promoting rhizobacteria (PGPR) traits, i.e., solubilization of Zn and P in addition to being positive for nifH and acdS genes. Before evaluating the bacterial strains for plant growth potential, a control experiment was also conducted to determine the highest critical Zn level from ZnO to wheat growth using different Zn levels (0.1, 0.05, 0.01, 0.005, and 0.001% Zn) against two wheat varieties (Wadaan-17 and Zincol-16) in sand culture under glasshouse conditions. Zinc-free Hoagland nutrients solution was used to irrigate the wheat plants. As a result, 50 mg kg-1 of Zn from ZnO was identified as the highest critical level for wheat growth. Using the critical level (50 mg kg-1 of Zn), the selected ZSB strains were inoculated alone and in consortium to the seed of wheat, with and without the use of ZnO, in sterilized sand culture. The ZSB inoculation in consortium without ZnO resulted in improved shoot length (14%), shoot fresh weight (34%), and shoot dry weight (37%); with ZnO root length (116%), it saw root fresh weight (435%), root dry weight (435%), and Zn content in the shoot (1177%) as compared to the control. Wadaan-17 performed better on growth attributes, while Zincol-16 had 5% more shoot Zn concentration. The present study concluded that the selected bacterial strains show the potential to act as ZSB and are highly efficient bio-inoculants to combat Zn deficiency, and the inoculation of these strains in consortium performed better in terms of growth and Zn solubility for wheat as compared to individual inoculation. The study further concluded that 50 mg kg-1 Zn from ZnO had no negative impact on wheat growth; however, higher concentrations hampered wheat growth.

Ali M ，Ahmed I ，Tariq H ，Abbas S ，Zia MH ，Mumtaz A ，Sharif M ... -  《Frontiers in Plant Science》

Solubilization of insoluble zinc compounds by zinc solubilizing bacteria (ZSB) and optimization of their growth conditions.

Khanghahi MY ，Ricciuti P ，Allegretta I ，Terzano R ，Crecchio C ... -  《-》

Zinc-solubilizing Bacillus spp. in conjunction with chemical fertilizers enhance growth, yield, nutrient content, and zinc biofortification in wheat crop.

Micronutrient deficiency is a serious health issue in resource-poor human populations worldwide, which is responsible for the death of millions of women and underage children in most developing countries. Zinc (Zn) malnutrition in middle- and lower-class families is rampant when daily calorie intake of staple cereals contains extremely low concentrations of micronutrients, especially Zn and Fe. Looking at the importance of the problem, the present investigation aimed to enhance the growth, yield, nutrient status, and biofortification of wheat crop by inoculation of native zinc-solubilizing Bacillus spp. in conjunction with soil-applied fertilizers (NPK) and zinc phosphate in saline soil. In this study, 175 bacterial isolates were recovered from the rhizosphere of wheat grown in the eastern parts of the Indo-Gangetic Plain of India. These isolates were further screened for Zn solubilization potential using sparingly insoluble zinc carbonate (ZnCO3), zinc oxide (ZnO), and zinc phosphate {Zn3(PO4)2} as a source of Zn under in vitro conditions. Of 175 bacterial isolates, 42 were found to solubilize either one or two or all the three insoluble Zn compounds, and subsequently, these isolates were identified based on 16S rRNA gene sequences. Based on zone halo diameter, solubilization efficiency, and amount of solubilized zinc, six potential bacterial strains, i.e., Bacillus altitudinis AJW-3, B. subtilis ABW-30, B. megaterium CHW-22, B. licheniformis MJW-38, Brevibacillus borstelensis CHW-2, and B. xiamenensis BLW-7, were further shortlisted for pot- and field-level evaluation in wheat crop. The results of the present investigation clearly indicated that these inoculants not only increase plant growth but also enhance the yield and yield attributes. Furthermore, bacterial inoculation also enhanced available nutrients and microbial activity in the wheat rhizosphere under pot experiments. It was observed that the application of B. megaterium CHW-22 significantly increased the Zn content in wheat straw and grains along with other nutrients (N, P, K, Fe, Cu, and Mn) followed by B. licheniformis MJW-38 as compared to other inoculants. By and large, similar observations were recorded under field conditions. Interestingly, when comparing the nutrient use efficiency (NUE) of wheat, bacterial inoculants showed their potential in enhancing the NUE in a greater way, which was further confirmed by correlation and principal component analyses. This study apparently provides evidence of Zn biofortification in wheat upon bacterial inoculation in conjunction with chemical fertilizers and zinc phosphate in degraded soil under both nethouse and field conditions.

Yadav RC ，Sharma SK ，Varma A ，Singh UB ，Kumar A ，Bhupenchandra I ，Rai JP ，Sharma PK ，Singh HV ... -  《Frontiers in Microbiology》