Nano-zinc and plant growth-promoting bacteria is a sustainable alternative for improving productivity and agronomic biofortification of common bean

Nano-zinc (Zn) fertilizer is an easily adaptable and environmentally safe alternative option that can effectively improve growth, yield and biofortification of common bean. Plant growth-promoting bacteria (PGPBs) could promote plant growth and nutrients availability in sustainable manner. Therefore, this study aimed to investigate the influence of foliar nano-Zn application in association with seed co-inoculations of PGPBs on growth, yield, biofortification and Zn use efficiencies in common bean cultivation. Two field experiments were performed with seven co-inoculations of PGPBs and three foliar nano-Zn doses applied 50% at R5 and 50% at R8 stages of common bean to determine plant height, shoot dry matter, grain yield, Zn concentration and uptake in shoot and grains, Zn partitioning index, daily Zn intake and Zn use efficiencies for agronomic biofortification. The combined foliar nano-Zn application and co-inoculation of R. tropici + B. subtilis enhance grain yield, leaf chlorophyll index, total protein content, grain Zn concentration and uptake, daily Zn intake, Zn use efficiency, applied Zn recovery and Zn utilization efficiency in common beans in 2019 and 2020 cropping seasons. Foliar nano-Zn application at a dose of 1.5 kg ha−1 increased plant height, shoot dry matter, shoot Zn uptake, Zn partitioning and agro-physiological efficiency under co-inoculation with R. tropici + B. subtilis in both cropping years. The treatments with foliar nano-Zn application at a dose of 1.5 ha−1 and co-inoculation with R. tropici + B. subtilis improved performance, chlorophyll index, protein content, grain yield, and Zn efficiencies that can lead to better biofortification of common bean in tropical savannah. Therefore, it is recommended that applying nano-Zn via foliar along with co-inoculation of PGPBs could be the better option for productivity and biofortification of common bean. Foliar nano-zinc (Zn) fertilization can improve agronomic biofortifcation and producitvity of common beans. Plant growth-promoting bacteria (PGPBs) can sustainably increase nutrient use efficiency and zinc content in edible tissues. The combined application of nano-Zn and PGPBs can potentially alleviate food and nutritional security crises. The sustianbale mechanisms of co-application of nano-Zn and PGPBs need further investigation. Foliar nano-zinc (Zn) fertilization can improve agronomic biofortifcation and producitvity of common beans. Plant growth-promoting bacteria (PGPBs) can sustainably increase nutrient use efficiency and zinc content in edible tissues. The combined application of nano-Zn and PGPBs can potentially alleviate food and nutritional security crises. The sustianbale mechanisms of co-application of nano-Zn and PGPBs need further investigation.


Introduction
Common bean (Phaseolus vulgaris L.) is one of the most important legume crop and being a part of daily diet for human consumption around the world.Brazil is one of the largest global producer of common bean, ranked third in terms of grain production (3.2 million metric tons) and second in terms of cultivated area (2.9 million hectares) during three major sowing seasons [1].It is one of the most consumed grain food in Brazilian nutrition for being a source of important protein, vitamins, minerals and amino acids [2].Agricultural production systems and practices are responsible to ensure food and nutrition security to increasing population without environmental risks, which linking soil, plants and animals to human health for maintaining sustainable agriculture [3].Climatic extremes and variabilities are disturbing sustainable agriculture production, availability, utilization, and stability of food in both developed and developing countries, leading to global hunger [4].In addition, crop cultivation is accompanied by intensive use of synthetic fertilizers to meet food demand of increasing global population which contribute to the deficiency of most common nutrients including iron (Fe), zinc (Zn), selenium (Se) and iodine (I) in human blood plasma [5].Zinc (Zn) malnutrition is a global dietary concern and most vulnerable to agriculture soil, crop production and nutritional quality of staple field crops.Zinc is one of the most persistent deficiency in tropical regions that lead to serious health issues especially in low income countries [6].Plant-based Zn deficiency could impair a series of human metabolic reactions and affect more than 2 billion people, being ranked as the second most common deficiency and health concern [6,7].The inadequate Zn supply has affected around 17% of the global population and was ranked as 5th health risk factor with a prevalence deficiency in least developed or developing countries [8,9].To feed the raising population of the world with both sufficient and nutritious food is itself one of sustainable development goal of United Nation (https:// sdgs.un.org/ goals/ goal3).Thus, it is consider that agronomic biofortification is one the best and prompt alternative to enrich grain crops with targeted deficient nutrient in the soil and fulfill nutritional requirments [10,11].
Biofortification with nano-fertilizer could enhance nitrogen metabolism, productivity and nutrition into the edible tissues of targeted plant crops to address global malnutrition [12].Biofortification could better be define by the time of foliar spray, choice and growth stage of crop as well as source, formulation and particle size of applied fertilizers [13,14].Foliar spray of nano-Zn fertilizer has gained more attention and interest in agriculture sector for increasing crop nutrition and productivity in ecofriendly manner [11,15].Nano-zinc oxide (ZnO) is widely use Zn fertilizer that could quickly absorb via stomata and cuticles, mobilize and translocated to chloroplast thus contributing to plant nutrition and production [16,17].The efficacy of nano-ZnO could be recognized by their physical characteristics, composition and size of particle that potentially increase nutrient use efficiency and redudce dependency on synthetic fertilizers, promoting Zn concentration in stem and edible tissues, and other growth characteristics of different crops [18,19].However, excessive use of synthetically produced nanoparticles may retarder plant growth and productivity, depending on the surface and size of particulate [20].Therefore, more greener and sustainable alternative could be adapted to to improve crop production and nutritional status as well as minimizing devestating environmental impacts of current agriculture techniques [21].
Plant growth-promoting bacteria (PGPBs) application is one of the sustainable and greener approach that are enhancing food quantity without compromising quality and productivity [22].These PGPBs are able to improve nutrient use efficiency and tolerance against biotic and abiotic stresses by contributing to root architecture, improving soil fertility, and enhancing solubilization of macro-and micronutrient that all together can lead to higher plant productivity [23].Several genus of PGPBs including Bacillus sp., Pseudomonas sp., Enterobacter sp., Rhizobium sp., Acinetobacter sp. and Klebsiella sp. are being reported and studied for improving nutrient use efficiency and productivity, reducing the use of syhthetic fertilizers in different crops [24][25][26][27].Plant growth-promoting bacteria support plant health and performance by production and regulation of phyto-hormones [28,29], solubilization of nutrients [30,31], biosynthesis of siderophores and antibiotics [32,33] to resist biotic and abiotic stresses.The interaction of PGPBs with soil and plant tissues enhance fitness of the plant to drastic environemntal conditions by regulating plants metabolic and physiological triats [34].
Sustainable nutrient management in field crops is challenging issue in tropical savannah.Despite this, Brazil is being spotted in the list of Zn deficient countries (http:// www.harve stplus.org).Several strategies have being adapted to increase nutrient use efficiency and productivity; however, there still exist research gap on the use of nano-Zn foliar spray in association with PGPBs for improving nutrition, productivity and biofortification of common bean.In this context, the hypothesis of the current study was that PGPBs and nano-Zn foliar may improve nutrition, productivity, Zn use efficiencies and biofortification of common bean edible tissues.Hence, the study aimed to determine the best performing PGPBs seed co-inoculation with nano-Zn foliar application on growth, yield, concentration of Zn in shoot and grains, Zn use efficiencies and estimated Zn intake in common beans under tropical savannah of Brazil.

Description of experimental site
Two field experiments with a test crop common bean were conducted during May-August of 2019 and 2020, respectively, at the Research and Extension Farm of School of Engineering, São Paulo State University (UNESP) in Selvíria, state of Mato Grosso do Sul, Brazil.The geographical coordinates of the site are 20° 22′ S latitude, 51° 22′ W longitude, and an altitude of 335 m (Fig. 1).The experimental soil is classified as Rhodic Haplustox [35] and Red Dystrophic according Brazilian soil classification system [36], being cultivated with cereallegume cropping system for last 30 years, and no-tillage for last 13 years.
The climate of the region is characterized as Aw-Köppen with rainy summer, dry winter and humid tropical with relative humidity 70-80% [37].The climatic data during current experiment in both cropping seasons is summarized in Fig. 2.

Soil analysis
Twenty random soil samples were collected with the help of cup-auger from a soil layer of 0.00-0.25 m before initiation of common bean experiments during both cropping seasons.The samples were mixed to make a composite sample and determined for chemical characterizations [38].The physio-chemical characterizations of soil are summarized in Table 1.
Seeds of common bean were manually inoculated by mixing each inoculant of PGPBs and seeds in a plastic bag an hour before to sowing.Common bean seeds were inoculated with R. tropici, using commercial peat inoculant, strain SEMIA 4080 with 2 × 10 9 colony forming units (CFU) g −1 at a dose of 200 g per 100 kg seeds.A 10% sugar solution was used to facilitate inoculant adhesion with seeds.R. tropici is a commercially registered inoculant with Ministry of Agriculture and Livestock, Brazil for common bean cultivation.A. brasilense strains Ab-V5 (CNPSo 2083) and Ab-V6 (CNPSo 2084) seed inoculation was carried out at a dose of 200 mL liquid inoculant per 24 kg seeds with guarantee of 2 × 10 8 CFU mL −1 .Inoculations with B. subtilis strain (CCTB04), guarantee of 1 × 10 8 CFU mL −1 and P. fluorescens strain (CCTB03), guarantee of 2 × 10 8 CFU mL −1 at dose of 150 mL ha −1 per 24 kg seeds were manually performed by following the procedures of inoculants providing company (Total Biotechnology ® ), Curitiba, Brazil.It has been reported that A. brasilense strains Ab-V5 and Ab-V6 are carrying fix and nif genes to enhance nutrients availability and recycling, also has a role in biological nitrogen fixation, auxin production and induce plant tolerance against biotic and abiotic stresses [41][42][43].B. subtilis is gram positive bacterium carrying nonribosomal peptide synthetases and a beta-glucanase as phytopathogens resistant and zntR as Zn transportator possessing plant growth-promotion while resisting phyto-pathogens and heavy metals absorption [44][45][46].P. fluorescens is considered the most efficient biocontrol agent due to the synthesis of antibiotics, volatile organic compounds to resist soil pathogens, produce gluconic acid, solubilize nutrients and fixing N [47,48].Nano-Zn folair spray was carried out from a liquid Zn source (Nano-R1 zinco) of ALLPLANT ™ fertilizers industry, São Paulo-Brazil, being registered with Ministry of Agriculture, Brazil.This nano-Zn is characterized as fluid suspension with 50% p/p Zn, 1000 g L −1 solubility and 2.0 density and successfully studied for improving plant growth and productivity [11,49].All the doses of foliar nano-Zn were applied in two split applications (50% at R5 and 50% at R8 stage of common bean according to International Center for Tropical Agriculture-CIAT) [40].The application was performed through manual sprayer pump with water capicity of 6.0 L (300 L ha −1 of volume spray).The field was thoroughly visited soon after foliar nano-Zn spray and no leaf damage was observed.

Field management
The field site was sprayed with herbicides 2,4-D + glyphosate (670 + 1800 g ha −1 of a.i.) for controlling preexperiment emerged weeds.Common bean cultivar (IPR-Campos Gerais belonging to commercial Carioca group), erect and inderterminate type-II with and an avreage life cycle of 88 days [50].Two field experiments of common bean were conducted in the first half of May, 2019 and 2020 under a no-tillage system.Each plot was consisted of 5 m long 6 lines with plot size of 2.7 m × 5 m, totalizing 13.5 m 2 .The rows were spaced by 0.45 m and regulated 12 seeds m −1 .The treatments in both experiments were performed at the same phenological stages.
All the treatments were applied with a basal dose of NPK according to initial soil analysis (Table 1).A total amount of nitrogen (N: 40 kg ha −1 ) from a source of urea, phosphorus (P 2 O 5 : 80 kg ha −1 ) from triple superphosphate and potassium (K 2 O: 40 kg ha −1 ) from ammonium sulphate applied at plantation.Despite this, a recommneded dose of 90 kg ha −1 of N was applied in top dressing after 5 weeks of plantation.The region of the experiment was interpreted as boron deficient according Campinas Agronomic Institute-IAC [38].Therefore, the entire field was sprayed with 1 kg boron ha −1 from the source of boric acid through a tractor spryer at folwering stage of common bean.The crop was irrigated with a central-pivot sprinkler irrigation system (14 mm water volume on a shift of 72 h) and all cultural practices were performed, when necessary.The common bean were manully harvested on 24th and 28th August, 2019 and 2020, respectively.

Assessments and evaluations
The assessments and evalautions were carried out in all four replicates.Plant height was determined at physiological maturity by measuring plant length from ground surface to upper apex of plants.Plants from four central rows were harvested, labeled, dried and weighed with analytical balance for shoot dry matter.Each sample was mechanically threshed and transferred into kg ha −1 at 13% humidity to quantify grain yield of common bean.
Leaf chlorophyll index was measured using a nondestructive, hand-held chlorophyll Falker meter (Cloro-fiLOG ® -model CFL-1030 Falker, Porto Alegre, Brazil).Total protein content was derived from the nitrogen content (Kjeldahl method) of the current experiment multiplied with Jones's factor, which was specified as 6.25 for common beans [51].
Zinc concentrations in shoot and grains were determined with nitroperchloric digestion and quantified by atomic absorption spectrophotometry (FAAS-Model Varian SpectrAA-55B, Varian, California, USA), following the procedure of Malavolta et al. [52].In addition, uptake of Zn in shoot and grains were estimated from the ratio of Zn concentration in shoot and grains, and shoot dry matter and grain yield, respectively.Zinc partitioning index (ZPI) was derived from grains and shoot Zn concentration [53], while estimated Zn intake with common bean grains in Brazil was calculated from the biofortified common bean grains of the present study [54]: where Zn intake (g person −1 day −1 ) is daily Zn intake estimation person −1 , [Zn] (g kg −1 ) is Zn concentration in biofortified grains in the present study and C (kg person −1 day −1 ) is the mean consumption of common bean grains per person in Brazil, which is ~ 142.2 g person −1 day −1 [55].Zinc use efficiency (ZnU), agro-physiological efficiency (APE), applied Zn recovery (AZnR) and utilization efficiency were derived in the fractions aquired from Fageria et al. [56] via the following equations: where GYF = grain yield with nano-Zn foliar spray, GYC = grain yield in without nano-Zn foliar, ZnF = with nano-Zn foliar spray, ZnC = without nano-Zn foliar spray, ZnUF = grain + shoot Zn uptake in nano-Zn sprayed treatments, ZnUC = grain + shoot Zn uptake in without nano-Zn foliar treatments and PE = physiological efficiency.

Statistical analysis
The entire data were tested for normality with Shapiro-Wilk test and Levene's homoscedasticity test (p < 0.05) which showed that data were to be normally distributed (W ≥ 0.90).Then, data were subjected to analysis of variance (F test).Foliar nano-Zn doses, PGPBs inoculations and their interactions were considered fixed and replication was random effect in the model.When a main effect or interaction was observed significant by F test (p ≤ 0.05), then Tukey test (p ≤ 0.05) was used for means comparison of nano-Zn spray and Scott Knott test (p ≤ 0.05) for PGPBs inoculations using ExpDes package of R software [57].
The Pearson correlation analysis (p ≤ 0.05) was calculated and heatmap was created using corrplot package of "color" and "cor.mtest"functions to calculate coefficients (1) and evaluate relationship among growth, yield, nutritional, biochemical and metabolic attributes of maize using R software [57].

Growth and grain yield of common bean
Plant height of common bean was positively influenced by nano-Zn foliar spray and co-inoculation with plant growth-promoting bacteria (PGPBs) in 2019, while their effect in 2020 was not significant (Table 2).Nano-Zn foliar application at a dose of 1.5 kg ha −1 increased plant height by 6.4% as compared to control in first cropping season.In addtion, co-inoculation with R. tropici + B. subtilis was observed with increasing plant height by 9.7% as compared to without inoculation treatments in 2019 crop season.The interaction of nano-Zn foliar spray and inoculations with PGPBs were not significant in both cropping seasons (Table 2).
Shoot dry matter of common bean was significantly influenced by nano-Zn foliar spray and inoculation with PGPBs, whereas their interaction was not significant in both crop seasons (Table 2).Shoot dry matter was increased by 2.8% with application of 1.5 kg ha −1 of foliar nano-Zn in 2019, while an increase of 2.7% was observed with 3 kg ha −1 of foliar nano-Zn application, which was statistically at per with 1.5 kg ha −1 of foliar nano-Zn in 2020 as compared without nano-Zn foliar application treatments.In addtion, co-inoculation with R. tropici + B. subtilis increased shoot dry matter of common bean by 11.8% and 9.2% in first and second crop seasons, respectively, in comparison of without inoculation treatments (Table 2).
The effect of of nano-Zn foliar doses and inoculations with PGPBs and their interaction were significant for grain yield of common bean in both 2019 and 2020 cropping seasons (Table 2).The treatments with co-inoculation of R. tropici + B. subtilis at a dose of 1.5 kg ha −1 of nano-Zn foliar application were observed with higher grain yield as compared to no-inoculation and other inoculations treatments in 2019 and 2020 common bean cropping seasons (Fig. 3A, B).The treatments with nano-Zn foliar application and inoculations with PGPBs were observed with greater grain yield as compared to without inoculation and Zn application.The treatments with 1.5 kg ha −1 of nano-Zn foliar application was observed with greater grain yield under all inoculations treatments as compared to other Zn doses (Fig. 3A, B).The treatments without inoculations of PGPBs and nano-Zn foliar application was observed with the lowest grain yield in 2019 and 2020 in relation to other treatments (Fig. 3A,  B).

Chlorophyll index and protein content
Leaf chlorophyll index (LCI) was significantly influenced by co-inoculation and nano-Zn foliar application, while their interactive effect was not significant in both cropping seasons (Table 3).All the treatments with inoculation and co-inoculations increased LCI in both cropping seasons, when compared with control.Co-inoculation with R. tropici + B. subtilis increased LCI by 16.9% and 15.2% as compared to no-inoculation in first and second cropping season.Foliar nano-Zn application at a dose of 1.5 kg ha −1 increased LCI by 24.0% and 14.9% as compared to without nano-Zn application in 2019 and 2020 cropping season, respectively.
Inoculation and co-inoculation with PGPBs increased total protein in 2019 and 2020 cropping seasons.The foliar nano-Zn application increased protein content only in the second cropping season, while the interactive effect of inoculations and foliar nano-Zn application in both seasons was not significant (Table 3).Total protein was increased by 16.32% with co-inoculation of R. tropici + A. brasilense in 2019, which was statistically not different from the rest of co-inoculation treatment except R. tropici + A. brasilense + B. subtilis as compared to control.
In addition, co-inoculation with R. tropici + B. subtilis increased crude protein by 16.27% in 2020 cropping season, which was statistically not different from the treatments with co-inoculation as compared to control.Foliar nano-Zn application at a dose of 1.5 kg ha −1 increased total protein by 3.1% and 6.2% as compared to without foliar nano-Zn application in 2019 and 2020 cropping season, respectively.

Shoot and grain Zn nutrition, and Zn daily intake
Shoot Zn concentration of common bean was increased with nano-Zn foliar doses and co-inoculations with PGPBs, while the interactions were not significant in both 2019 and 2020 (Table 4).Nano-foliar Zn application at the dose of 1.5 kg ha −1 increased shoot Zn conentration of common bean by 31.3% and 14.4% in 2019 and 2020 cropping season in relation to the treatments without nano-Zn foliar application.Shoot Zn concentration was increased by 38.2% and 23.6% with co-inoculation of R. tropici + B. subtilis in 2019 and 2020 common bean crop seasons, respectively, as compared to un-inoculated treatments.
The interaction of nano-foliar Zn doses and inoculation with PGPBs was significant for grain Zn concentration in both 2019 and 2020 common bean crop seasons (Table 4).The treatments applied with 1.5 kg ha −1 foliar nano-Zn in combination with co-inoculation of R. tropici + B. subtilis were observed with higher grain Zn concentration in 2019 and 2020 cropping seasons as compared with all other treatments (Fig. 4A, B).The treatments with 1.5 kg ha −1 foliar nano-Zn was observed with higher grain concentration regardless of the inoculations.The treatments with no foliar nano-Zn application and without inoculation was observed with lowest grain Zn concentration in 2019, while 3 kg ha −1 of foliar nano-Zn and without inoculation treatments were noted with lowest grain Zn concentration in 2020 as compared to other treatments (Fig. 4A, B).Nano-Zn foliar doses and inoculations with PGPBs had positively improved shoot Zn uptake in 2019 and 2020 cropping seasons.The interaction of nano-Zn foliar doses and inoculations with PGPBs for shoot Zn uptake was significant in first season while non-significant in the second season (Table 4).Shoot Zn uptake was improved by 35.8% and 19.6% with foliar nano-Zn at a dose of 1.5 kg ha −1 in first and second cropping seasons, respectively, in relation to without nano-Zn fertilization.The treatments with co-inoculation of R. tropici + B. subtilis improved shoot Zn uptake by 55.0% and 34.6% in 2019 and 2020 resepctively as compared to without inoculation treatments.In addition, the intreactive effect of 1.5 kg ha −1 nano-Zn foliar application and co-inoculation of R. tropici + B. subtilis was observed with higher shoot Zn uptake in first common bean growing season as compared with other treatments (Fig. 4C).In general, the treatments with foliar nano-Zn application and inoculations of PGPBs were observed with higher shoot Zn uptake as compared to control.The treatments without foliar application of nano-Zn and without inoculation were observed with low shoot Zn uptake as compared to other treatments (Fig. 4C).
The single and interaction effects of nano-Zn foliar doses and inoculations with PGPBs were positive for grain Zn uptake in both studied years (Additional file 1: Table S1).Grains Zn uptake was improved with foliar nano-Zn at a dose of 1.5 kg ha −1 in association with co-inoculation of R. tropici + B. subtilis in both common bean copping seasons (Fig. 4D, E).Interesting, the treatments with foliar nano-Zn application and inoculations with PGPBs were observed with higher grain Zn uptake as compared to control.In addition, treatments without foliar nano-Zn application and without inoculation were observed with low grain Zn uptake in 2019 (Fig. 4D), while foliar nano-Zn at a dose of 3 kg ha −1

W i t h o u t R . t r o p i c i R . t r o p i c i + A . b r a s i l e n s e R . t r o p i c i + B . s u b t i l i s R . t r o p i c i + P . f l u o r e s c e n s R . t r o p i c i + A . b r a s i l e n s e + B . s u b t i l i s R . t r o p i c i + A . b r a s i l e n s e + P . f l u o r e s c e n s
Grain yield (kg ha -1

W i t h o u t R . t r o p i c i R . t r o p i c i + A . b r a s i l e n s e R . t r o p i c i + B . s u b t i l i s R . t r o p i c i + P . f l u o r e s c e n s R . t r o p i c i + A . b r a s i l e n s e + B . s u b t i l i s R . t r o p i c i + A . b r a s i l e n s e + P . f l u o r e s c e n s
Grain yield (kg ha -1   and without inoculation was noted with low Zn uptake in common bean grains in 2020 as compared to other treatments (Fig. 4E).In general, the treatments without inoculations of PGPBs were observed with low grain Zn uptake regardless of the foliar nano-Zn applications in both cropping seasons (Fig. 4D, E).

r o p i c i R . t r o p i c i + A . b r a s i l e n s e R . t r o p i c i + B . s u b t i l i s R . t r o p i c i + P . f l u o r e s c e n s R . t r o p i c i + A . b r a s i l e n s e + B . s u b t i l i s R . t r o p i c i + A . b r a s i l e n s e + P . f l u o r e s c e n s
Grain Zn uptake (g ha -1

W i t h o u t R . t r o p i c i R . t r o p i c i + A . b r a s i l e n s e R . t r o p i c i + B . s u b t i l i s R . t r o p i c i + P . f l u o r e s c e n s R . t r o p i c i + A . b r a s i l e n s e + B . s u b t i l i s R . t r o p i c i + A . b r a s i l e n s e + P . f l u o r e s c e n s
Grain Zn uptake (g ha Foliar nano-Zn doses and inoculation with PGPBs had positively increased partitioing of Zn to common bean grains, while their interaction was not significant in 2019 and 2020 cropping seasons (Additional file 1: Table S1).Foliar nano-Zn application at a dose of 1.5 kg ha −1 increased Zn partitioning index by 7.0% and 11.3% in 2019 and 2020, respectively, as comapred to control.The treatments with co-inoculation of R. tropici + B. subtilis were observed with 13.9% and 11.4% higher Zn partitioning to common bean grains in 2019 and 2020, respectively, as compared to without inoculations treatments.
The interactions and single effect of foliar nano-Zn doses and inoculation with PGPBs were significant for estimated daily Zn intake in Brazil with consumption of common bean grains in 2019 and 2020 (Additional file 1: Table S1).The treatments with foliar nano-Zn application at a dose of 1.5 kg ha −1 in association with co-inculation of R. tropici + B. subtilis were observed with greater daily Zn intake in first and second cropping seasons, respectively, in relation to control (Fig. 5A, B).The treatments with foliar nano-Zn application and inoculation with PGPBs were observed with greater daily Zn intake as compared to control treatments.The treatments in the absence of foliar nano-Zn application were noted with low daily Zn intake regaless of the inoculation in both studied years.However, least Zn intake with daily consumption of common bean grains was observed without foliar nano-Zn fertilization and inoculation in 2019 cropping season (Fig. 5A).In addition, foliar nano-Zn at a dose of 3 kg ha −1 and without inoculation was observed with lowest daily Zn intake in 2020 crooping season, which was statistically similar with co-application of 0.0 kg ha −1 foliar nano-Zn and without inoculation, inoculation with R. tropici, co-inoculation with R. tropici + A. brasilense + B. subtilis and R. tropici + A. brasilense + P. fluorescens (Fig. 5B).

Zinc use efficiencies
Foliar nano-Zn and inoculation with PGPBs, and their interactions were significant for Zn use efficiency in both 2019 and 2020 cropping seasons (Additional file 1: Table S2).The co-application of foliar nano-Zn at a dose of 1.5 kg ha −1 along with inoculation of R. tropici + B. subtilis increased Zn use efficiency as compared, when compared with 3 kg ha −1 foliar nano-Zn and inoculation

r o p i c i + A . b r a s i l e n s e R . t r o p i c i + B . s u b t i l i s R . t r o p i c i + P . fl u o r e s c e n s R . t r o p i c i + A . b r a s i l e n s e + B . s u b t i l i s R . t r o p i c i + A . b r a s i l e n s e + P . fl u o r e s c e n s
Zn intake (

W i t h o u t R . t r o p i c i R . t r o p i c i + A . b r a s i l e n s e R . t r o p i c i + B . s u b t i l i s R . t r o p i c i + P . fl u o r e s c e n s R . t r o p i c i + A . b r a s i l e n s e + B . s u b t i l i s R . t r o p i c i + A . b r a s i l e n s e + P . fl u o r e s c e n s
Zn intake (g person other PGPBs in both cropping seasons (Fig. 6A, B).The lowest Zn use efficiency in both common bean cropping seasons was observed at 3 kg ha −1 foliar nano-Zn application, under no-inoculation treatments (Fig. 6A, B).Agro-physiological efficency (APE) of common bean was positively influenced by foliar nano-Zn doses and inoculation with PGPBs only in 2019, while the treatments effect and interaction were not significant in 2020 cropping season (Additional file 1: Table S2).The treatments with foliar nano-Zn at a dose of 3 kg ha −1 increased APE by 19% in 2019 cropping season.The treatments with inoculation of R. tropici were observed with higher APE, which was statistically similar with triple inoculation of R. tropici + A. brasilense + B. subtilis and R. tropici + A. brasilense + P. fluorescens, co-inoculation of R. tropici + A. brasilense, and without inoculation treatments in relation to other inoculations (Additional file 1: Table S2).
The interactions and treatment effect of foliar nano-Zn and inoculation with PGPBs were significant for applied Zn recovery (AZnR) in 2019 and 2020 (Additional file 1: Table S2).The treatments with foliar nano-Zn at a dose of 1.5 kg ha −1 along with co-inoculation of R. tropici + B. subtilis in both cropping seasons as compared to other treatments (Fig. 6C, D).Interestingly, AZnR was increased with 1.5 kg ha −1 foliar nano-Zn application under inoculation, co-inoculation and without inoculation treatments as compared with 3 kg ha −1 foliar nano-Zn application.The lowest AZnR in the first cropping season was observed without inoculation and foliar nano-Zn application at a dose of 3 kg ha −1 as compared to other treatments (Fig. 6C, D).
Foliar nano-Zn doses, inoculation with PGPBs and their interaction had positively improved Zn utilisation efficiency (UE) of common bean in both studied years (Additional file 1: Table S2).The highest Zn utilization efficiency was observed with foliar nano-Zn at a dose of 1.5 kg ha −1 in combination with co-inoculation of R. tropici + B. subtilis in both 2019 and 2020 cropping seasons as compared to other treatments (Fig. 6E, F).In general, the treatments with 1.5 kg ha −1 foliar nano-Zn application performed better within all inoculation, coinoculation and without inoculation treatments as compared with 3 kg ha −1 foliar nano-Zn application.The lowest UE was noted with 3 kg ha −1 foliar nano-Zn application under without inoculation treatments as compared to other treatments (Fig. 6E, F).

Pearson's correlation
There were positive and significant correlations between zinc use efficiency and plant height, shoot dry matter, shoot-grain Zn concentration and accumulation, Zn partitioning index, Zn intake in Brazil under common beans cultivation in 2019 regardless the treatments applied (Fig. 7A).A positive correlation was observed between Zn partitioning index and shoot and grain Zn accumulation, applied Zn recovery, shoot dry matter, and grain yield of common beans in 2020 cropping season (Fig. 7B).

Discussion
Several sustainable strategies have been adapted to maintain growth, productivity and quality of crop plants under harsh tropical conditions [43].In this context, nano-Zn fertilizers have attracted the attention by providing nutrients to plants in more technical manner to minimize leaching and adsorption as well as improving fertilizer efficiency and grain tissue assimilation [11,58].Plant growth-promoting bacteria (PGPBs) are being used as sustainable and eco-friendly approach to enhance Zn availability to plants through several direct and indirect mechanisms [29,30,33], leading to better grains biofortification [59,60].Thus, the positive interaction between foliar nano-Zn and inoculation/co-inoculation with PGPBs (Fig. 7) endorsed the hypothesis of the present study.
The present results indicated that foliar nano-Zn and co-inoculation with R. tropici + B. subtilis were observed with taller plants and greater shoot dry matter (Table 2), and higher grain yield of common bean (Fig. 3).The increase might be driven by the direct mechanisms (nutrients solubilization and availaibility, and phytohormones production) of PGPBs that could enhance nutrients availability by either fixation, solubilization or alteration of hormonal activities and Zn involvement in maintaining cell division, elongation and photosynthesis and regulatory co-factor in protein synthesis [25,61].Previous studies have reported that inoculation of PGPBs either alone or with Zn could alter different enzymatic activities of soil and plant to enhance plant growth, yield and biofortification of edible tissues [27,62].Plant growth-promoting bacteria could increase vegetative growth of crop plants that contribute to higher productivity at later reproductive stage [63].In addition, Zn has critical role in regulation of cell multiplication and elongation as well as several biochemical function of plant that could ultimately increase shoot dry matter and productivity of different crops [64][65][66].The co-application of Zn and PGPBs could increase pod formation through pollen development that could contribute to better Zn use efficiency and consequently greater growth and yield of different crops [67][68][69].
Zinc is considered as one of the key element of photosynthesis that can help in the synthesis of chlorophyll and protein, which might increase biomass accumulation in common beans [70,71].The integrated use of PGPBs and zinc fertilizer has also been reported for the improvement of biochemical and yield attributes of crops [43,67].Hence, the current results exhibited that combined use of PGPBs and nano-Zn fertilizer increased leaf chlorophyll index and total protein of common bean (Table 3).Zinc is involved in the improvement of different biochemical processes of plant, such as improving chlorophyll content and protein synthesis that may results in greater growth and development of crops [64,67].Several studies reported that different bacterial strains could stimulate the optimization of stomatal conductance and nutrient transportation, which may increase chlorophyll and protein content in plants [72,73].
Zinc deficiency is an alarming issue in agricultural soils that caused stagnation in agronomic biofortification and productivity [65,66].However, Zn foliar spray could be an efficient alternative strategy to cope Zn edaphic deficiency by ameliorating its bioavailability in edible tissue, leading to biofortification [11].In this context, the present study indicated that treatments with foliar Zn and inoculation of PGPB were observed with higher Zn concentration and uptake in shoot and grains of common bean (Table 4).Foliar nano-Zn and co-inoculation with R. tropici + B. subtilis were noted with higher shoot and grain Zn concentration (Table 4, Fig. 4A, B) and also shoot and grain Zn uptake (Fig. 4C, D, E).This might be due to the influence of applied inoculants in the roots rhizosphere that may help the host plant to articulate root architecture and stimulate nutrients availability for better uptake [74].In addition, PGPB have the ability to produce organic acids, chelating agents and siderophores that could not only promote plant growth and productivity but also increasing Zn assimilation to the edible tissue to sustain biofortification [75,76].The co-application of PGPB with foliar/soil Zn could reduce phytic acid concentration in the edible tissue, thus contributing to higher Zn concentration in embryo, aleurone, endosperm and whole grains of cereal [77].
Zinc partitioning index and daily Zn estimated intake in common bean grains were improved with foliar nano-Zn fertilization along with co-inoculation of PGPBs (Additional file 1: Table S1 and Fig. 5A, B).It might be due to the positive interaction of plants and microbes that could biosynthesis several compounds including phenolic acid, siderophores, organic acids and phyto-hormones to contribute different biochemical and metabolic functions of plants [77,78].In addition, foliar Zn has also been reported a rapid strategy that could improve translocation and remobilization of applied Zn into newly generated grains to deal with human malnutrition [64,79].It has also reported in previous studied that Zn and PGPB contributed to the biosynthesis of nodules and leghemoglobin, where root nodulation could improve transportation of Zn in sucrose from leaves to nodules and leg-hemoglobin could optimize plant growth [26,80].
The combined application of Zn with PGPBs has the ability to increase Zn efficiency by dissolving carbonates and oxides of Zn as compared to individual Zn fertilization [81].Zinc efficiencies are being defined by the availability of Zn in edible tissues in Zn deficient soils [56], where foliar Zn fertilization is considered a better option for biofortification and higher yield [11].Therefore, the present results indicated that combined application of foliar nano-Zn with co-inoculations of PGPBs improved Zn use efficiency (Fig. 6A, B), agro-physiological efficiency (Additional file 1: Table S2), applied Zn recovery (Fig. 6C, D), and utilization efficiency (Fig. 6E, F) in tropical savannah of Brazil.The possible reason of higher Zn efficiencies might be due to higher Zn concentration and uptake in shoot and grains (Table 4 and Additional file 1: Table S1) and greater biomass and grain yield (Table 2).Plant growth-promoting bacteria adapt several mechanism including solubilization of nutrients, nitrogen fixation, organic acids production and enzymatic activities that could possibly increase nutrient absorption and relocalization to stem and grains tissues [31,72].It has also previously reported that Zn in combination with diazotrophic bacteria could improve bioavailability and translocation of Zn to shoot and grains, leading to higher Zn use efficiencies [26,27].Hence, current results are forefront step to understand influence of foliar nano-Zn fertilization and inoculation with PGPBs on growth, yield and biofortification of common bean, emphasizing on the integrated use of foliar ZnO and PGPBs to ameliorate Zn accumulation, yield and Zn use efficiencies in tropical savannah.

Conclusions
Foliar nano-zinc (Zn) fertilization is an efficient and rapid alternative option of nutrients delivery that enhance plant performance and this nutrient use efficiency in a sustainable manner.The multifaceted functions of plant growthpromoting bacteria (PGPBs) to increase productivity and nutrition of crop plants while maintaining sustainability of agriculture is not deniable.Hence, it has derived from the current results that foliar nano-Zn application along with co-inoculation of PGPBs improved plant growth, shoot dry matter, grain yield, leaf chlorophyll index, total protein and grain nutrition of common bean.Zinc partitioning index and estimated daily Zn intake as well as Zn efficiencies including use efficiency, agro-physiological efficiency, applied Zn recovery and utilization efficiency were also increased with foliar nano-Zn and co-inoculation of PGPBs in common bean cultivation.It has concluded that foliar nano-Zn application at a dose of 1.5 ha −1 along with co-inoculation of R. tropici + B. subtilis was observed the most effective for improving performance and grain yield, Zn nutrition and its efficiencies, leading to biofortification of common bean grains in tropical savannah.Therefore, it is recommended that applying nano-Zn via foliar along with co-inoculation of PGPBs could improve productivity and nutrition of common bean in more sustainable manner.
The present study showed integrated use of synthetic fertilizers with PGPBs; however, we believe that it is not remain an ambitious to highlight that PGPBs/bio-stimulants have the potential to replace synthetic fertilizers in near future.The molecular and physiological behavior of these PGPBs would attract more attention of agriculturists, which will bring the technology out of developing stage and can highly improve sustainability and agriculture under harsh environmental conditions.

Fig. 2
Fig. 2 Rainfall, and maximum and minimum temperatures during common bean cultivation, acquired from the weather station of Research and Extension Farm of UNESP from May to August 2019 and 2020

Fig. 3
Fig. 3 Grain yield of common bean in 2019 (A) and 2020 (B) cropping seasons as a function of foliar nano-Zn application and co-inoculations of plant growth-promoting bacteria.The upper case letters are used for the interactions of PGPBs inoculations within each dose of foliar nano-Zn application, whereas lower case letters are used for the interaction of foliar nano-Zn doses within each inoculation treatment.The identical alphabetic letters are statistically similar with each other as analyzed by Tukey test for foliar Zn doses (p < 0.05) and Scott-Knott test for PGPBs inoculations (p < 0.05) in 2019 and 2020, respectively.Error bars indicate standard error of the means (n = 4 replicates)

Fig. 4
Fig. 4 Grain zinc (Zn) concentration in 2019 (A) and 2020 (B), shoot Zn uptake in 2019 (C), grain Zn uptake in 2019 (D) and 2020 (E) common bean cropping seasons as a function of foliar nano-Zn application and co-inoculations of plant growth-promoting bacteria.The upper case letters are used for the interactions of PGPBs inoculations within each dose of foliar nano-Zn application, whereas lower case letters are used for the interaction of foliar nano-Zn doses within each inoculation treatment.The identical alphabetic letters are statistically similar with each other as analyzed by Tukey test for foliar Zn doses (p < 0.05) and Scott-Knott test for PGPBs inoculations (p < 0.05) in 2019 and 2020, respectively.Error bars indicate standard error of the means (n = 4 replicates)

Fig. 5
Fig.5 Daily zinc (Zn) intake with common bean grains in 2019 (A) and 2020 (B) cropping seasons as a function of foliar nano-Zn application and co-inoculations of plant growth-promoting bacteria.The upper case letters are used for the interactions of PGPBs inoculations within each dose of foliar nano-Zn application, whereas lower case letters are used for the interaction of foliar nano-Zn doses within each inoculation treatment.The identical alphabetic letters are statistically similar with each other as analyzed by Tukey test for foliar Zn doses (p < 0.05) and Scott-Knott test for PGPBs inoculations (p < 0.05) in 2019 and 2020, respectively.Error bars indicate standard error of the means (n = 4 replicates)

Fig. 6
Fig. 6 Zinc (Zn) use efficiency (A, B), applied Zn recovery (C, D), and utilisation efficiency (E, F) in 2019 and 2020 common bean cropping seasons, respectively, as a function of foliar nano-Zn application and co-inoculations of plant growth-promoting bacteria.The upper case letters are used for the interactions of PGPBs inoculations within each dose of foliar nano-Zn application, whereas lower case letters are used for the interaction of foliar nano-Zn doses within each inoculation treatment.The identical alphabetic letters are statistically similar with each other as analyzed by Tukey test for foliar Zn doses (p < 0.05) and Scott-Knott test for PGPBs inoculations (p < 0.05) in 2019 and 2020, respectively.Error bars indicate standard error of the means (n = 4 replicates)

Fig. 7
Fig. 7 Heat-map color scale indicating Pearson's correlation among evaluated attributes of common beans in response to plant growth-promoting bacteria and foliar nano-ZnO applications in 2019 (A) and 2020 (B) cropping seasons.* × = indicates a non-significant relationship (p ≤ 0.05).PH plant height, DM shoot dry matter, NLC number of row per cob, NGC number of grains per cob, HGs 100-grains weight, GY grain yield, SZnA shoot Zn accumulation, GZnA grain Zn accumulation, ZnUE Zn use efficiency, AZnR applied Zn recovery, APE agro-physiological efficiency, UE utilization efficiency

Table 1
Soil physio-chemical characteristics of field site before experiment initiation during 2019 and 2020 common bean growing seasons [39] cation exchange capacity; V: base saturation † Determined according to methodology of Teixeira et al.[39]

Table 2
Plant height, shoot dry matter and grain yield of common bean as a function of plant growth-promoting bacteria and nano-Zn foliar doses in 2019 and 2020 cropping seasons Means in the column followed by similar letters are statistically not different by Tukey test (p ≤ 0.05) for foliar nano-Zn dose and Scott Knott test for PGPBs (p ≤ 0.05).** and *-significant at p < 0.01 and p < 0.05, respectively, while ns -non-significant by F test.CV = Coefficient of variance.(n = 4 replicates)

Table 3
Leaf chlorophyll index and total protein of common bean as a function of plant growth-promoting bacteria and nano-Zn foliar doses in 2019 and 2020 cropping seasons Means in the column followed by similar letters are statistically not different by Tukey test (p ≤ 0.05) for foliar nano-Zn dose and Scott Knott test for PGPBs (p ≤ 0.05).** and *-significant at p < 0.01 and p < 0.05, respectively, while ns -non-significant by F test.CV = Coefficient of variance.(n = 4 replicates)

Table 4
Zinc concentration and accumulation in shoot and grains tissues of common beans as a function of plant growth-promoting bacteria and nano-Zn foliar doses in 2019 and 2020 cropping seasons Means in the column followed by similar letters are statistically not different by Tukey test (p ≤ 0.05) for foliar nano-Zn dose and Scott Knott test for PGPBs (p ≤ 0.05).**and*-significant at p < 0.01 and p < 0.05, respectively, while ns -non-significant by F test.CV = Coefficient of variance.(n=4 replicates)TreatmentsShoot Zn concentration (mg kg −1 ) Grain Zn concentration (g ha −1 ) Shoot Zn uptake (g ha −1 )