Introduction

Fertilization practices in agro-ecosystems influence soil quality, but the effect depends on the nature of the applied fertilizers. The application of chemical fertilizers has adverse impacts on the environment in terms of different categories, such as nutrient leaching, salinity and acidification of agricultural soils, emission of greenhouse gases, and accumulation of chemical residues (Noorhosseini and Damalas 2018). The maintenance of adequate soil organic carbon (SOC) levels in cultivated fields is an important issue due to reduction in the use of organic fertilizers in soil (Chesworth 2008; Nie et al. 2018). SOC plays a crucial role in the improvement of soil physicochemical and biological properties through improving water retention, reducing soil erosion and nutrient leaching, sequestering carbon (C), exchanging nutrients, energy, and C with the soil environment, which are important for crop productivity (Jeffery et al. 2015; Lehmann and Kleber 2015; Li et al. 2018; Rasmussen et al. 2018; Wood and Bradford 2018; Oldfield et al. 2019).

Application of organic amendments derived from animals, plants, and minerals can significantly improve SOC (Wiszniewska et al. 2016; Peltre et al. 2017; Li et al. 2018). Among soil amendments, rock phosphate enriched compost (CM) and biochar (BC) are well-known organic sources that can be used to reduce fertilizer requirements, promote soil biological properties, microbial enzyme activities, and water-holding capacity, thus mitigating drought (Smebye et al. 2016; Ali et al. 2017; Batista et al. 2018; Moharana et al. 2018). These organic amendments can favor microbial community growth and diversity and correlate with soil biological fertility and SOC (Chakraborty et al. 2011; Juan et al. 2015; Montiel-Rozas et al. 2018). SOC released from organic amendments is chemically recalcitrant, does not release greenhouse gases (GHGs), and suppresses soil-borne pathogens, thus favoring the growth of fungal mycelia and ultimately enhances crop productivity (Scotti et al. 2015; Koivunen et al. 2018; Montiel-Rozas et al. 2018).

Soil microorganisms are major players in nutrient recycling, such as SOC mineralization and nitrogen (N) fixation, thus they enhance the sustainability of nutrient sources (Bertrand et al. 2015; Kallenbach et al. 2016; Mus et al. 2016). These microbes secrete extracellular enzymes to obtain C and/or nutrients and help in the decomposition of soil organic matter and complex SOC in to smaller molecules that depend on microbial diversity and abundance (Sistla and Schimel 2012; Jian et al. 2016). Plant growth-promoting rhizobacteria (PGPR) can enhance plant growth by protecting plants from environmental stress and soil-borne pathogens, while improving plant growth through producing phytohormones and extracellular enzymes, promoting ACC deaminase activity, antifungal activity, nutrient availability, N fixation, and solubilization of phosphorus (P), potassium (K), and zinc (Zn) (Hussain et al. 2015, 2019; Pii et al. 2015; Mumtaz et al. 2017; Lin et al. 2018; Gouda et al. 2018; Ahmad et al. 2019; Kumar et al. 2019). Bacterial strains of many species support plant growth and this study focuses on Alcaligenes sp. (Sabry et al. 1997; Munoz-Rojas and Caballero-Mellado 2003; Aloni et al. 2006; Govindarajan et al. 2007; Sangeeth et al. 2012; Ahmad et al. 2013; El-Akhal et al. 2013; Wani et al. 2013; Hussain et al. 2015, 2019; Mumtaz et al. 2017, 2018). Alcaligenes sp. strains are gram-negative bacteria found in soil and aquatic environments and have the ability to promote plant growth (Bal et al. 2013; Hussain et al. 2019). Previous research showed that Alcaligenes sp. can solubilize P, produce siderophores, indole acetic acid (IAA), and promote ACC deaminase activity and resistance to heavy metals stress (Belimov et al. 2001). Such strains can promote plant ability to resist biotic and abiotic stresses through reducing the ethylene level and promoting plant growth by solubilizing nutrients, such as P and iron (Fe), as well as producing IAA. Moreover, the application of Alcaligenes sp. suppressed the incidence of rice blast and sheath blight diseases in the greenhouse, while significantly improved plant growth, enriched mineral nutrient content in seedlings, and promoted the expression of major defence-related rice genes (Kakar et al. 2018).

The individual role of soil amendments (e.g., BC) in the presence/absence of PGPR has been studied by Hale et al. (2014, 2015), but there is little information about the interactive effect of PGPR and soil amendments such as BC, CM, and HA. Nadeem et al. (2017) used BC and CM with Pseudomonas fluorescens and reported an increase in plant physiology, growth, and microbial colony-forming unit (CFU) in the soil. This strategy could be effective in increasing plant growth even under stressed conditions. Taking into account the above information, the current research work was framed to evaluate the integrated application of BC, CM, and HA with PGPR on soil microbial dynamics and plant growth and yield of maize (Zea mays L). The environmental impact associated with current fertilization practices applied in conventional crop production (Noorhosseini and Damalas 2018) and the necessity to improve soil health and plant nutrition in a way that could support more sustainable agricultural systems further motivated this research. In this context, the current study tested the hypothesis that the application of Alcaligenes sp. AZ9 improves the efficiency of organic amendments in soil and enhances plant growth. To the best of our knowledge, the integrated effect of Alcaligenes sp. with organic amendments has not been studied so far.

Materials and Methods

Collection and Analysis of Biochar (BC), Rock Phosphate Enriched Compost (CM), and Humic Acid (HA)

For the preparation of BC, corn straw was oven-dried at 67 °C to remove moisture and then crushed into small pieces. Nearly, 200 g of ground maize straw were pyrolyzed at 300 °C in 2 L U-shaped Pyrex flasks and an outlet placed in a muffle furnace to remove gases. Pyrolysing heat rate was 8–10 °C min−1 with 20-min residence time (Naeem et al. 2016). The CM was prepared through mixing crushed rock phosphate with fruit and vegetable compost (Ditta et al. 2015) and was obtained from the Environmental Microbiology Laboratory, ISES, UAF, Pakistan. The 50% HA (plus raw material) in black powder form (Warble Pvt. Ltd. Pakistan) was purchased from the local market of Bahawalpur, Punjab, Pakistan.

Chemical analysis of BC and CM were performed in the Soil Microbiology and Biotechnology Laboratory, Department of Soil Science, UCA & ES, IUB, Islamia University of Bahawalpur, Pakistan following standard methods (Ryan et al. 2001). Substrate acidity pH and electrical conductivity (EC) of 1:20 m/v ratio of both BC and CM in deionized water were measured using a pH meter and an EC meter, respectively. Carbon content (SOC) was estimated by adopting the method of Yeomans and Bremner (1988). Total N contents were determined with Kjeldahl digestion distillation apparatus (Jackson 1973). For the determination of P and K, BC and CM samples were digested through hydrogen peroxide (H2O2) and sulfuric acid (H2SO4) (McGill and Figueiredo 1993). Phosphorus contents in the digested filtrate were determined with a spectrophotometer (Model G6860A, Cary 60 UV–Vis Agilent Technologies, Australia). Potassium contents were determined using the digested filtrate on a flame photometer (Model BWB-XP, BWP Technologies, UK). The observed chemical properties of BC and CM are given in Table 1, while for the HA, similar measurements were not performed because this was a ready-to-use commercial material.

Table 1 Physicochemical properties of organic amendments and soil used in incubation and pot trials
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Collection and Determination of Bacterial Characteristics

The bacterial strain Alcaligenes sp. AZ9 was isolated from the rhizosphere of maize plants (Hussain et al. 2015) from the Environmental Microbiology Laboratory, ISES, UAF, Pakistan. The strain was screened for multiple plant growth-promoting attributes, viz. P solubilization, Zn solubilization, ACC deaminase activity, formation of IAA and siderophores. All these characteristic assays were repeated twice with three replicates. The P solubilization ability of the bacterial strain was determined qualitatively using modified Pikovskaya’s (PVK) agar media (Pikovskaya 1948), while P solubilization index (PSI) and P solubilization efficiency (PSE) were determined by measuring the P halo zone and colony growth zone (Vazquez et al. 2000). The P solubilization was quantified by inoculating strain Alcaligenes sp. AZ9 in Pikovskaya broth medium for 2 weeks at 28 ± 1 °C and 100 rpm. After incubation, the final pH of the culture was detected using a pH meter and the concentration of solubilized P was estimated by adopting the colorimetric method described by Schoenau and Karamanos (1993).

For qualitative Zn solubilization, the freshly grown strain was centrally inoculated on Bunt and Rovira’s media containing 0.1% insoluble Zn and then incubated at 30 ± 1 °C for 72 h. The Eqs. 1 and 2, as given below, were used to calculate Zn solubilization efficiency (ZSE) and Zn solubilization index (ZSI), respectively, through observing the zinc halo zone and the bacterial colony diameter. The Zn solubilization ability of the strain was also quantified by inoculating strain in Bunt and Rovira broth amended with 0.1% Zn and incubated at 28 ± 1 °C in a shaking incubator for 2 weeks (Saravanan et al. 2007). The final pH of culture was estimated through a pH meter and solubilized Zn concentration was determined by direct injection of culture filtrate in an atomic absorption spectrophotometer (Model 240FS AA, Agilent Technologies Australia) as reported by Hussain et al. (2015) and Mumtaz et al. (2017).

$${\text{Solubilization index (SI) }} = \, \frac{{{\text{Colony diameter }} + {\text{ Halo zone diameter}}}}{{\text{Colony diameter}}}$$
(1)
$${\text{Solubilization efficiency (SE) }} = \, \frac{{\text{Solubilization diameter}}}{{\text{Growth diameter}}} \times 100$$
(2)

The ability of strain Alcaligenes sp. AZ9 for IAA production both in the presence and absence of l-tryptophan was measured by the modified method of Mumtaz et al. (2017). l-tryptophan is the precursor of IAA (a well-known phytohormone of the auxin class) that is naturally present in root exudates of plants. For the IAA assay, 2-day-old broth culture was inoculated in DF minimal broth treated with 100 μg mL−1 of l-tryptophan and incubated at 28 ± 1 °C in a shaking incubator for 48 h. The auxins production was estimated by adding Salkowski coloring reagent, while the intensity of sample color was read at 530 nm using a spectrophotometer (Bric et al. 1991). Similarly, the above procedure was repeated for auxin production without l-tryptophan, apart from the addition of l-tryptophan. To detect siderophore production for Fe availability, strain Alcaligenes sp. AZ9 was centrally inoculated on chrome azurol S (CAS) agar with hexadecyltrimethylammonium bromide (HDTMA) as indicators (Schwyn and Neilands 1987). The detailed step-by-step procedure was performed using blue dye (prepared form CAS, FeCl3·6H2O and HDTMA) and mixture solution [minimal media 9 (MM9), casmino acid, glucose (20%) and NaOH stock]. CAS agar was prepared using MM9 media (100 mL) piperazine (3.24 g), and bacto agar (15 g) in ddH2O (750 mL) and autoclaved. Further, sterile casmino acid (30 mL) and glucose stock were added in MM9/piperazine mixture. Slowly, blue dye (100 mL) was added along glass wall with enough agitation to mix carefully, while plates were poured aseptically. The strain was centrally inoculated and incubated at 30 ºC for 48 h to observe the halo zone around the bacterial growth (Louden et al. 2011). The method of Honma and Smmomura (1978) and Penrose and Glick (2003) were used to access the ACC deaminase activity of strain Alcaligenes sp. AZ9 through measuring the amount of α-ketobutyric acid generated from the cleavage of ACC. The strain was grown in DF minimal broth containing 500 µmol mL−1 ACC as a sole source of N and incubated at 30 ± 1 ºC for 48 h. Further, the culture was centrifuged and the supernatant was read at 540 nm and compared with standard cure of α-ketobutyric acid.

Identification Through 16S rRNA Gene Sequencing

The strain Alcaligenes sp. AZ9 was identified through 16S rRNA partial gene sequence. Initially, extraction of DNA was done using the proteinase K treatment from cell culture (Chèneby et al. 2004). The PCR reaction was performed according to Hussain et al. (2015), further purified and sequenced by Macrogen, Korea. The 16S rDNA sequence was amplified in a thermocycler (Eppendorf, USA) using the universal primers, for forward the 27F 5′ (AGA GTT TGA TCM TGG CTC AG) 3′ and for reverse the 1492R 5′ (TAC GGY TAC CTT GTT ACG ACT T) 3′ reactions. The reactions were carried out using 2.5 μL crude DNA, 2 μL 27F (10 μM), 2 μL 1492R (10 μM) and 5 μL of H2O per reaction. One μL of control/template DNA was added just before the analysis on the instrument. Thermal cycling conditions were: 1 cycle at 95 °C for 1 min and then underwent 30 amplification cycles of 94 °C for 1 min, and 55 °C for 20 s. Fluorescence was read after each 55 °C step. The size of the amplified 16S rRNA was confirmed by separating on 1% agarose gel along with GeneRuler 1 kb DNA (Fermentas). The PCR product was sequenced for 16S rRNA using commercial service of MACROGEN Seoul, Korea (https://macrogen.com/eng/). The resultant sequences were compared with already existing nucleotide sequences using the NCBI genome database. The phylogenetic tree was constructed using the method of Thompson et al. (1997), i.e., a windows interface displaying the sequence alignment in a window on the screen, while a versatile sequence-coloring scheme allows the user to highlight conserved features in the alignment. The data processing was done using NJ Plot for neighbor-joining method (Perrière and Gouy 1996). The partial sequence was submitted in the GenBank database to obtain accession number for Alcaligenes sp. AZ9. The accession number for Alcaligenes sp. AZ9 is KU494828. The phylogenetic tree of the bacterial strain Alcaligenes sp. AZ9 is given in Fig. 1.

Fig. 1
figure 1

Neighbor-joining phylogenetic analysis resulting from the multiple alignment of 16S rRNA gene sequence of Alcaligenes sp. AZ9 (accession #KU494828) with those of other bacterial strains found in the NCBI Gene Bank database

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Soil Analysis

The sandy loam surface soil (0–15 cm) was obtained from experimental fields of the Department of Soil Science, UCA & ES, IUB, Bahawalpur, Pakistan, located at 29.40 N, 71.68E, and 116 m above the sea level. The soil was air-dried, sieved through a 2-mm mesh size, and used for incubation and pot experiments. Before performing the experiments, a soil sample (1 kg) was taken and analyzed for pH and EC through a pH meter and an EC meter in a soil-saturated paste (made in deionized water with 1:5 m/v ratio). The colorimetric method described by Schulte and Hoskins (1995) was adopted for the determination of soil organic matter. According to this method, 1.0 g of soil was taken in an Erlenmeyer flask and 10 mL of digestion solution (dichromate-sulfuric acid) was pipetted including a blank without soil. The flasks were covered with glass marbles to minimize loss of chromic acid and heated at 90 ºC for 90 min by placing in a digestion oven. After that, samples were removed from the oven to cool for 10 min and 25 mL of water were added through pipettes. The solution was allowed to stand for 3 h and then transferred in a 10-mL colorimeter tube. The blue color intensity of the supernatant was read in a spectrophotometer at 645 nm along with the reagent blank set to give 100% transmittance. The cation exchange capacity (CEC) was estimated by the ammonium acetate method (Jaremko and Kalembasa 2014).

For the determination of N, P, and K concentrations, the method of Schoenau and Karamanos (1993) was adopted in which soil was extracted by taking 2.5 g of air-dried ground soil in an Erlenmeyer flask and 50 mL of 0.5 M sodium bicarbonate (NaHCO3) along with 0.4 mL of charcoal suspension. The charcoal suspension was prepared by mixing 300 g of charcoal with 900 g of deionized water. The extract was filtered twice through filter paper (Whatman No. 40). The standard Kjeldahl method was used to determine total N concentration in soil extract (Jackson 1973), whereas the available P and the extractable K concentrations in the soil extract were measured through Olsen’s method and a flame photometer, respectively (Jackson 1973; Schoenau and Karamanos 1993). The soil particle sizes were accessed by the hydrometer method (Bouyoucos 2006) and the obtained values were drawn against the textural triangle to classify the soil textural class. SOC was estimated by adopting the method of Yeomans and Bremner (1988). Water content of saturated soil was calculated by the following equation (Eq. 3) reported by Sarfraz et al. (2017). For the preparation of the saturated pastes, 350 g of air-dried soil were used and the soil pastes were left for 24 h to reach equilibrium. All soil characterization analyses were completed in triplicate. Physicochemical characteristics of soil used for incubation and pot experiments are given in Table 1.

$${\text{Water saturation percentage (\%) }} = \, \frac{{{\text{Weight of soil paste (g) }} - {\text{ Weight of oven dried paste (g)}}}}{{\text{Weight of oven dried paste (g)}}} \times 100$$
(3)

Incubation Experiment

The incubation study was conducted in the wirehouse of the Department of Soil Science, UCA & ES, IUB, Bahawalpur, Pakistan, under natural conditions of ambient light and temperature. This study was performed to evaluate the effect of organic amendments with strain Alcaligenes sp. AZ9 on microbial dynamics in soil. For this experiment, pots having a capacity of 12 kg were filled with 10 kg soil. One mL of PGPR strain Alcaligenes sp. AZ9 having 0.5 OD at 600 nm was grown overnight in 1 L nutrient broth and mixed with CM. Mixtures of bacterial strain and CM along with BC and HA were added in the soil of each pot according to treatment plan which included: T1: control, T2: bacterial strain AZ9, T3: rock phosphate enriched compost (CM) 0.5 tonnes ha−1, T4: humic acid (HA) 24.7 kg ha−1, T5: biochar (BC) 0.5 tonnes ha−1, T6: CM (0.5 tonnes ha−1) + AZ9, T7: HA (24.7 kg ha−1) + AZ9, T8: BC (0.5 tonnes ha−1) + AZ9, T9: CM (0.5 tonnes ha−1) + HA (24.7 kg ha−1) + BC (0.5 tonnes ha−1), and T10: CM (0.5 tonnes ha−1) + HA (24.7 kg ha−1) + BC (0.5 tonnes ha−1) + AZ9. Tap water was flooded to saturate soil three times with a 25-day interval. The pots were incubated without plants for 80 days and changes in water-holding capacity in terms of water content of saturated soil and soil microbial dynamics in terms of SOC, bacterial CFU, and microbial biomass carbon were recorded.

Crop Growth Experiment

The pot experiment was conducted in the wirehouse of Department of Soil Science, UCA & ES, IUB, Bahawalpur, Pakistan to evaluate the synergistic effects of BC, CM, HA, and strain Alcaligenes sp. AZ9 for improving maize growth and yield as well as nutrient concentration in maize grain. The air-dried, ground, and sieved 21 kg soil was mixed thoroughly and filled in pots having a capacity of 25 kg. The experiment was conducted twice in a completely randomized design (CRD) with three replications. The fresh inoculum of bacterial strain Alcaligenes sp. AZ9 was prepared by using 1 mL (0.5 OD at 600 nm) culture in 1 L nutrient broth and grown overnight in nutrient broth. The organic amendments alone, with or without strain Alcaligenes sp. AZ9, as well as their mixture, as stated in the incubation study were hand mixed in soil before sowing. The control was maintained by applying the recommended fertilizer without application of bacterial strain and organic amendments. The treatment composed of bacterial strain without organic amendments were applied by dipping maize seeds in bacterial inoculum, while treatments composed of bacterial strains with organic amendments were applied by mixing inoculum with organic amendments. The recommended doses of N, P, and K (175, 160, and 125 kg ha−1) was applied as urea, diammonium phosphate, and potassium sulfate, respectively, uniformly in all treatments including control (according to recommendations of the Government of Punjab, Pakistan). The recommended doses of P and K were applied at the time of sowing, while N was applied twice, i.e., at the time of sowing and surface applied after 35 days of maize sowing. At sowing time, soil and organic amendments in each pot were mixed to homogenize the applied fertilizers and organic amendments. Maize variety Pioneer 31R88 (Pioneer Pvt. Ltd. Pakistan) was used as a test crop. Five seeds of maize were sown in each pot, but only one healthy plant was maintained in each pot by thinning after 15 days of germination. Irrigation was applied through flooding at regular intervals to keep the pots at optimum moisture level for plant growth. Each pot was provided a bottom hole to drain excess water to maintain field capacity, but no significant leaching of water was observed in the bottom of the pots.

Plant Growth and Yield Attributes

Data regarding growth attributes were recorded at physiological maturity 55 days after sowing. Plant height and root length were recorded with a measuring tape. Fresh and dry biomass of roots and shoots were recorded after harvesting maize. Grain and stover yield pot−1 and weight of 100-grains were recorded after crop harvesting.

Nutrients Concentration in Grain

The oven-dried maize grains from each pot were ground and 0.1 g of each grain sample was taken in a digestion tube. The samples were digested using sulfuric acid (H2SO4) followed by hydrogen peroxide (H2O2) by adopting the procedure reported by McGill and Figueiredo (1993). The concentration of nutrients in maize grain was determined by repeating analysis twice with three replications for each treatment. Nitrogen in plant extracts was determined by the standard Kjeldahl method (Jackson 1973). The vanadomolybdo-phosphoric acid yellow color method was used to estimate P concentration (Jackson 1973). Potassium in grain samples was determined by a flame photometer (Chapman and Pratt 1961).

Microbial Dynamics in Soil

Soil samples for analyzing biological characteristics were collected and preserved in polythene bag at 4 ºC after maize harvesting (Ryan et al. 2001). Standard serial dilutions were made to estimate the colony forming units (CFU) of bacteria following the method described by Alexander (1982). Microbial biomass carbon was estimated by the direct chloroform fumigation-extraction method (Jenkinson 1981), while the method of Yeomans and Bremner (1988) was used for SOC estimation. These soil attributes were determined from each treatment in triplicate and the analysis was repeated twice to confirm the results.

Data Analysis

Data regarding growth, yield, nutrient concentration, and soil biological parameters were analyzed statistically using Statistix 8.1®. Treatments included: four soil amendments (BC, CM, HA, and MX) at two inoculation levels with Alcaligenes sp. A29 (without and with inoculation), plus an inoculated control without soil amendments, plus a non-amended control. Analysis of variance (ANOVA) was employed using a CRD design. Means were compared with the least significant difference (LSD) test (Steel 2007).

Results

Plant Growth-Promoting Characteristics and Identification of Strain AZ9

Alcaligenes sp. AZ9 showed multiple plant growth-promoting characteristics upon inoculation (Table 2). Qualitative P solubilization revealed the appearance of a halo zone in the agar medium. It also showed solubilization of P quantitatively by lowering the pH of medium from 7.0 to 5.25 and solubilized P. The tested strain produced a halo zone in ZnO amended Bunt and Rovira agar and showed a high Z solubilizing index and Z solubilizing efficiency. Upon inoculation, Alcaligenes sp. AZ9 reduced the pH from 7.0 to 5.04 of Zn amended Bunt and Rovira broth and solubilized Zn. Alcaligenes sp. AZ9 showed ACC deaminase activity in terms of α-ketobutyrate concentration. Production of IAA both in absence and presence of l-tryptophan by strain Alcaligenes sp. AZ9 was also observed. This strain was positive for siderophores production by showing a change in color from blue to orange. The tested strain was identified as Alcaligenes sp. AZ9 through 16S rRNA gene-sequencing (V5 region). The sequences were submitted in NCBI gene bank and accession number KU494828 was obtained.

Table 2 Growth-promoting potential of Alcaligenes sp. AZ9
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Soil Microbial Dynamics

Inoculation with Alcaligenes sp. AZ9 significantly increased the effect of each amendment alone and in combination on soil bacterial CFU and SOC (Table 3) as well as MBC and SP (Table 4) both at 40th and 80th days of incubation periods. In general, Alcaligenes sp. AZ9 showed better ability to improve the efficiency of soil amendments (i.e., soil decomposition ability and increased water holding capacity) at 80th days of incubation compared with 40th days. This strain showed the highest ability to improve the efficiency of BC followed by HA in most cases. Alcaligenes sp. AZ9 showed the highest increase in bacterial CFU, SOC, SP, and MBC at 40th and 80th days of incubation over control in the case of integrated use of soil amendments. The maximum bacterial CFU (0.4 cfu × 104) in the pot experiment conducted with plants was observed by integrated use of BC, CM, HA, and AZ9) which was 27% higher than that of control (data not shown).

Table 3 Effect of biochar (BC), rock phosphate enriched compost (CM), humic acid (HA), mixture (MX) and AZ9 on bacterial CFU of soil and soil organic carbon at different incubation times
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Table 4 Effect of biochar (BC), rock phosphate enriched compost (CM), humic acid (HA), mixture (MX) and AZ9 on microbial biomass carbon of soil and water content of saturated soil at different incubation times
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Growth Attributes of Maize

The sole application of BC, CM, and HA promoted plant height of maize compared with control, while the increase in these attributes was higher when these soil amendments were applied with Alcaligenes sp. AZ9 (Fig. 2). Root length of maize was promoted only when these soil amendments were applied with Alcaligenes sp. AZ9. The highest values of plant height and root length were observed with the mixed soil amendment (mixture) treatment applied with Alcaligenes sp. AZ9. A similar trend was also observed in fresh and dry weight of maize roots (Fig. 3) as well as in fresh and dry weight of maize shoots (Fig. 4). The maximum increase in root and shoot biomass was observed in the case of integrated use of Alcaligenes sp. AZ9 with the mixture of BC, CM, and HA compared with control.

Fig. 2
figure 2

Effect of biochar (BC), rock phosphate enriched compost (CM), humic acid (HA), mixture (MX), and Alcaligenes sp. AZ9 on maize plant height and root length (different letters indicate significant differences according to least significance difference test at P < 0.05). Values are means of three replicates ± standard errors of means

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Fig. 3
figure 3

Effect of biochar (BC), rock phosphate enriched compost (CM), humic acid (HA), mixture (MX), and Alcaligenes sp. AZ9 on maize root weight (different letters indicate significant differences according to least significance difference test at P < 0.05). Values are means of three replicates ± standard errors of means

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Fig. 4
figure 4

Effect of biochar (BC), rock phosphate enriched compost (CM), humic acid (HA), mixture (MX), and Alcaligenes sp. AZ9 on maize shoot weight (different letters indicate significant differences according to least significance difference test at P < 0.05). Values are means of three replicates ± standard errors of means

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Yield Attributes of Maize

Maize yield attributes, namely grain yield, stover yield, and 100-grains weight, were enhanced with the integrated use of BC, CM, HA, and the increase was more evident in the treatment of integrated soil amendments with Alcaligenes sp. AZ9 (Fig. 5). This integrated treatment significantly promoted grain yield by 19%, stover yield by 28%, and 100-grains weight up to 26% compared with control; however, these values were not statistically different from the results of BC inoculated with Alcaligenes sp. AZ9. Sole application of soil amendments with Alcaligenes sp. AZ9 also showed an increase in those attributes. Sole application of BC exhibited better increase in grain yield by 7%, stover yield by 12%, and 100-grain weight by 15% over control, but the increase in these attributes was higher when BC was applied with Alcaligenes sp. AZ9.

Fig. 5
figure 5

Effect of biochar (BC), rock phosphate enriched compost (CM), humic acid (HA), mixture (MX), and Alcaligenes sp. AZ9 on maize yield (different letters indicate significant differences according to least significance difference test at P < 0.05). Values are means of three replicates ± standard errors of means

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NPK Concentrations in Grains

Seed inoculation with Alcaligenes sp. AZ9 in the presence of soil amendments had a significant influence (P < 0.05) on NPK concentrations in maize grains (Fig. 6). The integrated use of Alcaligenes sp. AZ9 with BC, CM, and HA promoted N concentration up to 12%, P concentration up to 11%, and K concentration up to 11% in maize grains compared with control. In addition to integrated effect observed, sole application of BC also improved the concentration of N up to 5%, P up to 6%, and K up to 5% over control.

Fig. 6
figure 6

Effect of biochar (BC), rock phosphate enriched compost (CM), humic acid (HA), mixture (MX), and Alcaligenes sp. AZ9 on nutrient concentration in maize grain (different letters indicate significant differences according to least significance difference test at P < 0.05). Values are means of three replicates ± standard errors of means

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Discussion

The replenishment of soil nutrients reserve after crop harvest is necessary to improve productivity of farming systems. The nutrients can be renewed through combined application of PGPR and organic amendments. Soil microbial CFU, MBC, and SOC along with maize growth and yield were increased in the present experiment due to use of organic amendments along with PGPR in the soil. In the present study, the pre-isolated rhizobacterial strain Alcaligenes sp. AZ9 (Hussain et al. 2015) was identified as Alcaligenes sp. AZ9 through 16S rRNA and evaluated for plant growth-promoting characteristics. Alcaligenes sp. AZ9 demonstrated its ability to solubilize P and Zn qualitatively as well as quantitatively, as evaluated during in vitro tests. The presence of halo zone on media inoculated with insoluble P and Zn due to Alcaligenes sp. AZ9 was similar to that previously reported by Hussain et al. (2015) and Mumtaz et al. (2017). Bal et al. (2013) reported solubilization of insoluble P by Alcaligenes sp. SB1-ACC2, but the current study reports for the first time about solubilization of insoluble Zn by Alcaligenes sp. AZ9. This solubilization of minerals by the bacterial strain could be due to production of organic acids, such as citric acid, lactic acid, gluconic acid, and 5-keto gluconic acid (Fasim et al. 2002; Tayyab et al. 2018) that decrease pH of the medium and cause conversion of the insoluble form to the soluble form. In the current study, a decrease in pH of medium containing insoluble P and Zn from 7.0 to 5.25 and 5.04, respectively, solubilized P up to 30.85 µg mL−1 and Zn up to 25.83 µg mL−1 (Table 2). A similar range of pH decrease and solubilized P and Zn contents were documented by Hussain et al. (2015) and Mumtaz et al. (2017) due to inoculation with strains of Bacillus spp. These solubilized forms of P and Zn will be available for plant uptake and improve plant growth and grain yield.

Soil microorganisms produce ACC deaminase, which sequesters and cleaves ACC produced by plants and stimulates plant growth by lowering ethylene level in the plant. Reduced ethylene levels due to bacterial ACC deaminase ameliorate a wide variety of environmental stresses in plants. In the current study, Alcaligenes sp. AZ9 was found positive for ACC deaminase activity and production of IAA and siderophores (Table 2). The ACC deaminase activity of Alcaligenes sp. AZ9 reduces stress ethylene level and can help plants to withstand biotic and abiotic stresses. Previously, Bal et al. (2013) reported the ability of Alcaligenes sp. SB1-ACC2 for ACC deaminase activity (2664.08 nmol α-ketobutyrate mg−1 protein h−1) that had a direct impact on root elongation and development of a better root system. In the current study, Alcaligenes sp. AZ9 produced IAA in presence of L-tryptophan (16.29 µg mL−1) as well as in absence of L-tryptophan (11.75 µg mL−1) (Table 2). Both ACC deaminase and IAA promoted root growth in a coordinated fashion (Glick et al. 2007). Siderophore-producing Alcaligenes sp. AZ9 can promote plant biomass directly through supplying Fe to the plant and indirectly by controlling the supply of Fe to disease-causing pathogenic fungus (Ahmad et al. 2008). The use of Alcaligenes sp. AZ9 with various plant growth-promoting (PGP) traits is expected to achieve better and sustainable crop productivity as well as reducing environmental stress (Rubin et al. 2017).

Application of soil amendments served as a soil conditioner and improved soil fertility and subsequently crop yield. In the current incubation and pot experiments, integrated application of soil amendments, viz. BC, CM, and HA resulted in the highest improvement of microbial dynamics (bacterial CFU, MBC, and SOC in soil) and of most parameters of maize growth, while single treatments did not improve yield over non-treated control. In addition, inoculation with Alcaligenes sp. AZ9 boosted the efficiency of integrated soil amendments in terms of microbial dynamics in soil, owing to bacterial ability of mineral solubilization, ACC deaminase production, production of phytohormones, and production of siderophores. Soil amendments have favorable characteristics, viz. porous structure, high surface area, availability of organic matter, nutrients, and change in soil pH and porosity which may provide suitable habitat for Alcaligenes sp. AZ9 to colonize and reproduce. The high porosity of organic amendments increases water-holding capacity and soil saturation percentage, which are also favorable for microbes to colonize (Han et al. 2017; Sarfraz et al. 2019). In the present study, higher metabolic efficiency of the bacterial community improved MBC and SOC that could lead to a reduction in CO2, stabilization of organic matter (OM), and improvement in microbial community biomass, structure, catabolic activity, quality and quantity of SOM and soil fertility (Tayyab et al. 2019). The microbial community would have higher metabolic efficiency with organic amendments due to labile OM that is the main precursor of stable SOM and is efficiently consumed by microbes. Decomposition of OM and increase in microbial community could determine the stabilization of SOM (Cotrufo et al. 2013, 2015). Our experiment also showed that organic amendments enhanced the abundance of microbial community and decomposition of SOM.

Application of Alcaligenes sp. AZ9 alone and/or in combination with soil amendments, viz. BC, CM, and HA increased the nutrient availability for maize uptake through plant growth-promoting characteristics of Alcaligenes sp. AZ9 and had the ability to promote microbial dynamics in the soil, which helped in improving maize growth, yield, and nutrient concentration in grains. In the present study, application of Alcaligenes sp. AZ9 showed its ability to improve the efficiency of applied soil amendments in terms of yield and nutrients concentration in maize grains. Alcaligenes sp. AZ9 may mobilize nutrients through acidification and chelation that promote grain nutrient uptake and thus yield. In addition to increased nutrient availability, application of Alcaligenes sp. AZ9 along with organic amendments promotes C sequestration and increases organic acids to balance soil pH. A similar work on the application of PGPR with soil amendment was previously performed by Zafar-ul-Hye et al. (2013), who reported increase in lentil yield through the integrated incorporation of organic fertilizers and biogas slurry.

Nutritional analysis of grains in the current study revealed increase in nutrients uptake in maize grain due to the integrated application of Alcaligenes sp. AZ9 and soil amendments. Organic amendments contain N, P, and K, while PGPR have the potential to produce ammonia, which is utilized by plants as a source of N (Tarin et al. 2018; Zafar-ul-Hye et al. 2013). Alcaligenes sp. AZ9 had the capacity to solubilize the fixed P, K, and Zn in soil through secreting organic acids. The synergistic use of Alcaligenes sp. AZ9 with soil amendments could benefit farmers to gain optimum yield from various crops as directed by numerous organic factors, including animal and plant wastes. On the contrary, the optimum yield, which is the main target of farmers, may not be achieved with the use of a single soil amendment without Alcaligenes sp. AZ9, based on the findings of this study.

Conclusions

Inoculation with Alcaligenes sp. AZ9-enhanced soil microbial dynamics, plant growth, yield, and nutrient uptake in maize in the presence of BC, CM, and HA under the conditions of our experiments. From these findings, it can be concluded that the application of BC, CM, and HA with Alcaligenes sp. AZ9 could be very effective for improving PGP bacterial CFU and organic matter in soil, which ultimately promote plant growth, nutrient uptake, and yield. Field experiments would be useful for assessing the magnitude of benefits by the presence of Alcaligenes sp. AZ9 in different soils and under different growth conditions. Given the environmental impact associated with current fertilization practices applied in conventional crop production, a necessary research priority would be to improve soil health and plant nutrition in a way that could support more sustainable agricultural systems.