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Effect of Plant Growth-Promoting Rhizobacteria and Biochar on Ricinus communis Growth, Physiology, Nutrient Uptake and Soil Enzyme Activities

Vadivel Anbuganesan, Ramasamy Vishnupradeep, Viswanathan Subhadra Varshini, Ashok Suma Archana, Sundaramoorthy Soundarya, L. Benedict Bruno, Mani Rajkumar*

Department of Environmental Sciences, Bharathiar University, Coimbatore - 641046, India

Abstract: The objective of the present study was to assess the combined effect of plant growth promoting rhizobacteria (PGPR) and biochar on Ricinus communis growth, physiological response, nutrient uptake and soil enzyme activity. A total of 16 bacterial strains isolated initially, two strains namely ST1NI01 and ST1NI15, demonstrating maximum in vitro plant growth promoting (PGP) potential were selected and identified as Enterobacter hormaechei and Bacillus thuringiensis, respectively. These isolates showed various PGP traits including production of indole-3-acetic acid, siderophores, ammonia and solubilization of phosphate. Similarly, the biochars were prepared from coconut husk by slow pyrolysis at 550°C. The characteristics of prepared biochar were pH 10.95, conductivity 3.52 mS cm⁻¹, cation exchange capacity 74.6 Cmol Kg⁻¹, moisture content 2.1 % and carbon 78 %. In pot experiments, inoculation of R. communis with PGPR or amendment of soil with biochar significantly increased the plant growth, protein content, nutrient uptake and soil enzyme activities, whereas decreased stress related metabolites (proline and malondialdehyde contents) and altered the activities of antioxidant enzymes (guaiacol peroxidase, catalase and ascorbate peroxidase) in plant leaves. However, the combined PGPR and biochar treatment exhibited greater effect on plant growth, physiological response, nutrient uptake and soil enzymatic activities as compared with sole treatment of PGPR or biochar. The findings suggest that both PGPR and biochar work synergistically on soil functions and plant growth, which may be utilized as an alternative to chemical fertilizers for improving soil quality and crop productivity.

Keywords: PGPR, Biochar, plant growth, soil enzyme activity, malondialdehyde

Received September 08, 2022; Revised October 12, 2022; Accepted October 24, 2022

Citation: Vadivel Anbuganesan, Ramasamy Vishnupradeep, Viswanathan Subhadra Varshini, Ashok Suma Archana, Sundaramoorthy Soundarya, L. Benedict Bruno, Mani Rajkumar, Effect of Plant Growth-Promoting Rhizobacteria and Biochar on Ricinus communis Growth, Physiology, Nutrient Uptake and Soil Enzyme Activities. Applied Ecology and Environmental Sciences, 10 (10). 640-651. January 2022. doi:10.12691/aees-10-10-5

1. Introduction

As soil is one of the essential non-renewable resources with vital role in plant growth and development, maintaining soil fertility is of immense significance for improving agriculture productivity. Thus, to improve the soil fertility and crop productivity, various organic (animal manure, compost, straw, wood chips, husk, etc.,) and inorganic (lime, alkaline fly ash, vermiculite, iron oxide, gypsum, etc.,) nutrients have been extensively used as soil amendments [1]. Although such nutrients improve the soil health and crop productivity, the excessive use of nutrients poses serious threats to soil ecosystems and human health. For instance, long-term application of chemical fertilizers to agriculture soils results soil salinity, organic matter degradation, acidification, heavy metal accumulation, water eutrophication etc., [2,3]. Similarly, amendment of organic manures also reported to cause various environmental problems as they often release various pollutants to soils including persistent organic pollutants, heavy metals, emerging contaminants, potential human pathogens, etc., [4,5]. Considering these negative effects, extensive research efforts have been focused to develop efficient alternative strategies to improve the plant growth and soil fertility.

In last few decades, a wide range of plant growth promoting rhizobacteria (PGPR) have been utilized as a promising alternative to organic and inorganic nutrients for improving soil fertility, plant growth and productivity [6,7]. PGPR improve the soil fertility through decomposition of organic matter, mobilization and immobilization mineral elements etc., [8,9]. In addition, they can increase the plant growth by improving plant nutrient assimilation (P, Fe and N), abiotic stress tolerance and disease resistance through producing various plant beneficial metabolites including siderophores, 1-aminocyclopropane-1-carboxylate deaminase (ACCD), indole-3-acetic acid (IAA), and phosphate (P) solubilization [10,11,12,13,14]. An issue with PGPR, however, is that their inconsistence nature in field levels since the prevailing unfavourable environmental conditions in soil including nutrient deficiency, low organic matter, salinity, drought, extreme temperature, pH, indigenous microbial populations etc., reduce the beneficial effects of PGPR on plant and soils through interfering their growth, survival and activity [15].

In this context, the use of carrier materials including peat, agro-waste biomass, vermiculite, and other minerals is becoming more popular for improving the growth, survival and functioning of inoculated plant beneficial microbes. Particularly, the utilization of biochar with the aim of providing favourable habitat could improve the growth, multiplication and survival of indigenous and/or inoculated microbes and thus their beneficial effects on plants [16]. Biochar is a carbonaceous material produced through thermochemical conversion of biomass at high temperatures. The highly porous nature of biochar provides a suitable micro-habitat for PGPR and also increases their activity, basal respiration and colonization with plant root [17]. Besides, biochar improves soil fertility by increasing soil nutrient retention (C and N), nutrient recycling, cation exchange capacity (CEC), water holding capacity and altering soil pH and bulk density [18,19,20].

Although, many findings have shown the individual effects of PGPR or biochar improves plant growth [21,22,23] and soil fertility [17,24], only a few studies [25,26] have reported the combined effects of PGPR inoculation and biochar amendment on plant growth, physiological response, nutrient uptake and soil enzyme activities. Thus, the aim of present study was i) to isolate, characterize and identify efficient PGPR, ii) to prepare and characterize biochar from agricultural waste and iii) to assess the individual and combined effects of PGPR inoculation and biochar amendment on plant growth, physico-chemical parameters, nutrient uptake and soil enzyme activity. To assess these parameters we have utilized the oil crop Ricinus communis (castor bean) for the pot experiment and agricultural waste (coconut husk) for biochar preparation.

2. Materials and Methods

2.1. Isolation and Characterization of PGPR

Bacterial strains were initially isolated from rhizosphere soil of Catharanthus roseus grown in magnesite mine soil, Vellakalpatti, Salem, India following the protocol of Ma et al. [27]. In order to select efficient PGPR, the bacterial strains were screened for their plant growth promoting (PGP) activity on Eleusine coracana using phytagar assay [27]. Briefly, the surface sterilized seeds of E. coracana were inoculated by soaking in bacterial culture containing 10⁸ CFU/ml for 2 h. For control, the sterilized seeds were soaked in sterile distilled water. The inoculated and non-inoculated seeds were placed in 0.5 % phytagar and incubated at 30°C under 8/16 h (light and dark) illumination. After 25 days, the growth parameters such as shoot and root length were determined. The PGPR strains selected based on PGP potential were characterized for various PGP traits including production of IAA [28], siderophores [29,30,31], ammonia [32] and solubilization of P [33] using standard protocol. The selected bacterial strains were further identified by 16S rRNA gene sequence analysis. Briefly the genomic DNA was isolated, purified, amplified and sequenced. The amplified sequences were analysed and compared using NCBI algorithm with NCBI sequences [34]. Phylogenetic tree was constructed using MEGA software Version 11 to identify the similarities with other PGPR strains.

2.2. Preparation and Characterization of Biochar

The biochars were prepared from coconut husk by slow pyrolysis at 550 °C with a residence time of 60 min following the method of Sahoo et al. [35]. The prepared biochar was ground, sieved (2 mm sieve) and analysed for proximate and ultimate parameters. Electrical conductivity (EC) and pH of the biochar were analyzed in an aqueous solution of the biochar in distilled water (1:10, w/v). Moisture content of biochar was measured by gravimetrical method and organic content was quantified based on the method described by Nelson and Sommers [36]. Cation exchange capacity was estimated by sodium acetate method described by Laird and Fleming [37]. Elemental (C and NO₂+NO₃) analyses were performed using an auto analyzer (SKALAR, Netherlands). Surface area, total pore volume, and pore radius were analyzed using Brunauer-Emmett-Teller (BET) method by Quantachrome Touch Win v1.22.

2.3. Pot Experiment

A pot experiment was conducted using agriculture soil collected from the field laboratory of Department of Environmental Sciences, Bharathiar University, Coimbatore. The biochar were mixed with dried and sieved soil samples to produce the final concentrations of 5 % (W/W). The surface sterilized seeds of R. communis were allowed to grow in soil at 28°C and a 16/8 day/night regime. In order to prepare the bacterial inoculum, cells of log phase cultures of PGPR were harvested, washed twice with sterile distilled water and suspended in sterile water. Two week-old of uniform size seedlings were selected and soaked in PGPR suspension containing 10⁸ CFU/ml for 2 h. The seedlings soaked in sterile water for 2 h were used as control. The inoculated and non-inoculated seedlings were transplanted in plastic pot containing 750 g of biochar amended or non-amended soils. The pots were kept in plant growth chamber at ambient temperature with a light cycle of 8/16 (day/night regime) and watered once a day to maintain the soil moisture at 70 % field capacity. Each treatment was performed in triplicate with four plants pot⁻¹. After 60 days, plants were harvested for further analysis.

2.4. Plant Analysis

2.4.1. Estimation of Biometric, Pigment, Biochemical and Enzymatic Activity

The uprooted plants were washed with deionized water, air dried and analysed for biometric parameters including root length, shoot length, fresh and dry weight. The photosynthetic pigments such as chlorophyll (Chl) and carotenoid contents in leaves were extracted and estimated by the method of Smith and Benitez [38]. Further, the contents of soluble protein [39], proline [40], malondialdehyde (MDA) [41] in leaf tissues were estimated. Besides, the antioxidant enzyme activities in plant leaves including superoxide dismutase (SOD) [42], guaiacol peroxidase (POD) [43], catalase (CAT) [44] and ascorbate peroxidase (APX) [45] were also analyzed as described previously.

2.4.2. Estimation of Nutrient Accumulation

Nutrient contents in leaf, shoot and root tissues of the plants were quantified after digestion of 200 mg of dried plant tissues with HNO₃ and HClO₄ (2:1 v/v) at 150°C for 3 h [27]. The total nitrogen (N) and total P content was estimated by Kjeldahl method and Vanadomolybdate method, respectively [46,47]. Potassium (K) content was quantified using flame photometry method (Accumax AIO-671, India).

2.5. Soil Analysis

2.5.1. Estimation of Soil Enzymatic Activity

The effect of PGPR inoculation and biochar amendment on soil enzymatic activity including dehydrogenase [48], acid phosphatase [49], alkaline phosphatase [49], β-glucosidase [50] and urease [51] were assayed after harvesting of R. communis. Briefly, dehydrogenase activity was analysed using 2, 3, 5-triphenyltetrazolium chloride (TTC) and expressed as µmol TPF g⁻¹ h⁻¹. Acid and alkaline phosphatases activity was assayed using ρ-nitrophenyl phosphate (р-NPP) as substrate and expressed as µg pNP g⁻¹ h⁻¹. β-glucosidase activity was determined using ρ-nitrophenyl- β-D-glucoside (PNG) and expressed as µg pNP g⁻¹ h⁻¹ at 37°C. Similarly, soil urease activity was assayed using 10 % urea solution as the substrate and expressed as mg NH₄⁺-N g⁻¹ h⁻¹.

2.6. Statistical Analysis

The experiment of this work were performed in triplicates and values were represented by calculating mean and standard deviation using SPSS (Version 20.0). Mean values of treatments were compared using Tukey’s HSD test and significant difference (P < 0.05) among the treatments were calculated using one way ANOVA. To construct the graphical representation among the treatments, the Graph-Pad was used.

3. Result and Discussion

3.1. Isolation and Characterization of PGPR

During the initial isolation process, 16 bacterial strains were isolated from the rhizosphere soil of C. roseus. Among 16 isolates, two strains namely, ST1NI01 and ST1NI15 were selected for further studies based on their potential in promoting the growth of E. coracana in phytagar assay. Among the strains tested, ST1NI01 and ST1NI15 showed maximum plant growth promoting potential, which increased in shoot length by 20 and 36 %, and root length by 137 and 117 %, respectively over the uninoculated plants (Table 1). The increase in shoot length and root length caused by PGPR inoculation indicates that ST1NI01 and ST1NI15 may possess multiple PGP traits such as siderophores, IAA, ACCD, P solubilization, etc., [52]. It is known fact that such PGP traits improve the plant growth by increasing plant nutrient assimilation, abiotic stress tolerance and root growth. Several recent studies have also confirmed a strong relationship between improved plant growth and various PGP traits of PGPR [53,54,55]. Hence, both the strains ST1NI01 and ST1NI15 were further screened their ability of production of various PGP metabolites (Table 2). Strain ST1NI01 showed the P solubilization potential by forming clear zone in Pikovskaya’s agar medium and solubilised 243 µg P/ml. It well known that the PGPR can solubilise the P and thereby improve its bioavailability for plant uptake through releasing protons and organic acids [56]. In addition to P solubilization, IAA production by PGPR is thought to play vital role in production of longer roots which improve plant water and nutrient uptake through stimulating cell division and elongation [57]. In our study both ST1NI01 and ST1NI15 produced substantial amount of IAA in L-tryptophan amended medium indicating that these strains utilized tryptophan dependent pathway for the synthesis of IAA. Previously, several PGPR producing IAA through tryptophan dependent pathway have been demonstrated to increase the plant growth and development even under environmental stress conditions (eg, drought, salinity etc.,) [58,59]. Further characterization of PGP traits showed that both the strains were capable of producing catechol and hydroxamate siderophores, under iron limiting conditions. However, strain ST1NI01 produced the highest levels of catechol (4.15 µg/mL) and hydroxamate (159.3 µg/mL) type siderphores. This result suggests that the siderophore production trait of these strains may help plants to uptake more iron by forming a complex with Fe and thereby improving its bioavailability [60]. Besides, several authors reported that the ammonia producing PGPR supply N to the host plant and thereby promote its growth. In the present study, both the strains were found to produce ammonia. However, the maximum production was recorded in ST1NI15 compared with ST1NI01. The results reflect that the in vitro plant growth promotion by selected PGPR strains might be associated with their multiple PGP traits.

Table 1. In vitro plant growth promotion potential of bacterial strains

Table 2. PGP traits of selected PGPR

The subsequent analysis of partial 16S rRNA by online BLAST program and further construction of phylogenetic tree (Figure 1) showed that the selected PGPR ST1NI01 and ST1NI15 were members of genus Enterobacter and Bacillus, respectively. The sequence of the strain ST1NI01 (1325bp) exhibited 100 % similarity with Enterobacter hormaechei and was deposited to GenBank under accession number MT509849, Similarly sequence of the strain ST1NI15 (1277bp) showed 100 % homology with Bacillus thuringiensis and was deposited under accession number MN515293.

Figure 1. Phylogenetic tree showing the relationship of partial 16S rRNA gene sequences of PGPR strains ST1NI01 and ST1NI15 with other related sequences obtained from NCBI database. The tree was clustered with the neighbor-joining method using MEGA 11 package

3.2. Characterization of Biochar

The physico-chemical characteristics of the biochar derived from coconut husk are shown in Table 3. The pH of biochar was alkaline with a value of 10.95. The alkaline nature of biochar may be due to loss of volatile organic compounds and/or an increase in the basic cations as the results pyrolysis at high temperature [61]. The conductivity and CEC of the biochar were 3.52 mS cm-1 and 74.6 Cmol Kg⁻¹, respectively. In general the high value of CEC of biochar is a common phenomenon of thermally produced biochar and vary according to production method and raw material used [62]. These properties help to exchange ions, nutrient retention and increase the nutrient solubilization by altering soil pH [63,64].

Table 3. Characteristics of coconut husk biochar

The BET results revealed that the biochar has high pore volume with an average particle size and pore radius of 0.008 nm and 21.72 nm, respectively, suggesting that the COC biochar has the capacity to retain water, nutrients and also improve the survival and viability of the soil microbes [65] and by which biochar improve soil physical, chemical and biological characteristics [66]. The elemental analysis revealed that the prepared biochar has high carbon content and low nitrogen content suggest that the biochar is highly carbonaceous with low surface polar functional groups [67].

3.3. Effect of PGPR and Biochar on R. communis

3.3.1. Plant Growth and Biochemical Parameters

The effect of PGPR inoculation and biochar amendment on R. communis growth were assessed by measuring the plant shoot length, root length, fresh weight and dry weight (Table 4). Inoculation of PGPR ST1NI01 and ST1NI15 significantly increased the plant growth parameters. However, the maximum PGP effects was observed with inoculation of ST1NI15, where the plant shoot length, root length, fresh weight and dry weight were increased by 6, 22, 44 and 40 %, respectively, over the uninoculated control plants. Similarly sole application of biochar increased the plant shoot length (10 %), root length (3 %), fresh weight (39 %) and dry weight (17 %) compared with control (untreated; uninoculated). Whereas, the combined treatment of PGPR inoculation and biochar amendment (PGPR + biochar) resulted in a further increase in plant growth. For instance, biochar amendment along with ST1NI15 inoculation remarkably increased the fresh weight (80 %) and root length (44 %) over the non-inoculated control plants. These results were in agreement with previous works [68,69,70] and suggested that the enhancement of plant growth parameters is due to beneficial properties of biochar (water retention, nutrient retention, reduction in soil bulk density, improved microbial survival and improved nutrient solubilization) and PGPR (production of PGP metabolites including IAA, siderophores and P solubilization) [71,72].

Table 4. Effect of PGPR and biochar on the growth of R. communis

The beneficial effect of PGPR and biochar on R. communis was further analyzed by measuring plant pigment and protein contents. Similar to plant growth parameters, PGPR inoculation and biochar amendment either alone or in combination significantly increased the chlorophyll (total chl, chl a and chl b) and total soluble protein contents in the leaves of R. communis (Figure 2). However, the maximum increase was recorded in plants grown with PGPR + biochar treatment. For instance, PGPR ST1NI01 + biochar treatment increased total chlorophyll and protein contents by 96 and 49 %, respectively. Whereas, a slight decrease in carotenoid content was observed in PGPR + biochar treatment compared with sole PGPR treatment. The enhancement of chlorophyll and protein contents could be explained by increased uptake of nutrients including N. This might be due to the enhancement of soil physical, chemical and biological properties as the consequence of biochar amendment which improves soil retention capacity by preventing the nutrient leaching and improving their availability [73]. Another possible mechanism behind enhanced chlorophyll content in plants could be linked to increased PGP activity of inoculated microbes as the consequence of biochar amendment. Several recent studies found that the combined application of PGPR and biochar increased plant nutrient uptake and growth as compared with sole biochar treatment [15,74] and suggested that the biochar might be improved the survival and activity of PGPR resulting in higher plant nutrient uptake and growth. The PGPR which produce PGP metabolites including ammonia, IAA [75,76] may increase the plant root length and N uptake for enhancing photosynthesis and ultimately plant growth. In our study the production of ammonia and IAA shown by STINI01 and ST1NI15 was positively related with root length of R. communis seeming that these PGP metabolites can be influencing nutrient uptake and thus chlorophyll and protein contents in plants. Our results reflect that biochars improve the plant growth not only by improving soil physico-chemical characteristics but also through providing favorable micro-environment to inoculated PGPR which can improve the plant nutrient uptake and growth by producing various PGP metabolites.

Figure 2. Effect of PGPR and biochar on a) chlorophyll a, b) chlorophyll b, c) total chlorophyll, d) carotenoid, e) protein, f) proline and g) MDA contents of R. communis. Bars represent the mean and standard deviation of three replicates. Bars index with same letters are not significant between treatments using HSD tukey’s test (p < 0.05). fw – fresh weight

The analysis of the stress related compounds in plants revealed that PGPR inoculation and biochar amendment either alone or in combination had decreased the proline and MDA contents compared with untreated control (Figure 2). However, the maximum effect was recorded when the plants were treated with PGPR + biochar although there were no significant difference between sole application of biochar and combined treatment on MDA content. For instance, the treatment of PGPR STIN115 + biochar decreased proline and MDA content by 55 and 32 %, respectively compared with untreated control plants. Our results were in agreement with findings of Khan et al. [77], Zhu et al. [78] and Tirry et al. [79] who found that the treatment of PGPR + biochar decreased proline and MDA levels in Medicago sativa, Gossypium hirsutum L and Cicer arietinum by reducing the ROS generation through improving soil properties, moisture level, triggering plant antioxidants level, improving nutrient assimilation, as well as by protecting cell organelles from degeneration.

3.3.2. Effect of PGPR and Biochar On Antioxidants of R. communis

PGPR inoculation and biochar amendment either alone or in combination significantly altered the leaf enzymatic antioxidant activities including APX, POD and CAT except SOD (Figure 3). The sole PGPR and combined treatment had slightly increased the SOD activity; however, sole biochar application had no effect on SOD activity. The results indicated that PGPR have the ability to stimulate the synthesis of SOD by modulating SOD genes [52]. Similarly, sole application of PGPR increased of APX, POD and CAT activities compared with uninoculated control. The maximum effects of APX (73 %) and POD (26 %) were observed in ST1NI15 inoculated plants, whereas maximum CAT (156 %) activity was observed with inoculation of ST1NI01. On the other hand sole biochar or PGPR + biochar treatment decreased the activities of POD, APX and CAT. Previously, Bruno et al. [52] and Sharma et al. [80] similar to our study demonstrated that the PGPR inoculation increased the antioxidant activities including SOD, CAT and APX under stressed and non-stressed condition and reported that this effect may be as a result of microbial mediated alteration of the expression of responsible genes. Whereas, a declined or no effect on antioxidant activity in non-stressed plants due to biochar amendment has also been reported previously [81] supporting that the application of biochar had no direct effect on antioxidant synthesis.

Figure 3. Effect of PGPR and biochar on a) SOD, b) POD, c) APX and d) CAT activity in R. communis. Bars represent the mean and standard deviation of three replicates. Bars index with same letters are not significant between treatments using HSD tukey’s test (p < 0.05). fw – fresh weight

3.3.3. Effect of PGPR and Biochar on Nutrient Uptake by R. communis

The significant impact on the uptake of N, P and K by R. communis was observed with PGPR inoculation and biochar amendment. For instance, inoculation of ST1NI15 increased N content by 256, 67 and 89 %, P content by 82, 39 and 97 % and K content by 13, 22 and 45 % in leaf, shoot and root tissues, respectively over the uninoculated control plant (Table 5). The increase of nutrient uptake (eg, N, P) in plants due to PGPR inoculation was observed by several authors [82,83,84] which might be due to the stimulation of root growth, solubilization of P, production of ammonia and siderophores etc. In fact, both strains in this study demonstrated the ability of ammonia production along with other PGP traits indicating these isolates might have helped plants to uptake more nutrients. Our results corroborate those of Stajkovic et al. [85] and Radhapriya et al. [86] demonstrating that the ammonia producing PGPR facilitate N bioavailability through nitrogen fixation thus improving its uptake by plants. Likewise, it was found that sole biochar amendment also increased the N content by 233, 67 and 300 %, P content by 78, 156 and 14 %, and K content by 29, 32, and 289 %, in leaf, shoot and root tissues, respectively over the uninoculated control plant. This phenomenon is due to the high alkaline nature of the biochars, which improves the solubilization of the minerals by increasing the soil pH and thereby making them available for plants [87,88]. However, the P content in plant tissues is comparatively lesser than N and K in biochar treated plants, which may be due to the increase of soil pH as a result of biochar amendment. It is well known that the alkaline nature of soils reduces P uptake in plants by decreasing P bioavailability through precipitation [89]. Moreover, a maximum increase in uptake of N, P and K was observed when plants were treated with PGPR + biochar, which reflects that the cumulative effects of PGPR and biochar on nutrient solubilization in soils and its uptake by plants. For instance, PGPR ST1NI15 + biochar treatment increased the N content in shoot, root and leaf tissues by 150, 511 and 522 %, P content by 200, 998, 139 % and K content by 92, 465, 62 %, respectively over the uninoculated control plants. Recently, Jabborova et al. [90] also reported the inoculation of Bradyrhizobium japonicum USDA 110 with biochar showed a significant increase in N (30 %), P (54 %) and K (160 %) contents in plants. This effect was probably attributed to improved PGPR activity as the result of biochar amendment. It is known fact that in addition to providing nutrients to plants, biochars afford conducive habitat for the growth, colonization and activity of inoculated PGPR which enhance nutrient bioavailability and thus its uptake by plants via producing various PGP metabolites.

Table 5. Effect of PGPR and biochar on nutrient content in shoot, root and leaf tissues of R. communis

3.4. Effect of PGPR and Biochar on the Soil Enzyme Activities

Soil enzyme activities play vital role in various biogeochemical processes such as transformation of organic matter, absorption of nutrients in soils, nutrient release, humus synthesis, various redox reactions etc., [91,92,93,94]. In the present study we assessed whether the combined PGPR + biochar treatment influence the activities of soil enzymes (Figure 4). The PGPR inoculation and biochar amendment either alone or in combination increased the soil enzymatic activities compared to the uninoculated control soil. For instance, PGPR ST1NI15 increased dehydrogenase, alkaline phosphatase, acid phosphatase, urease and β-glucosidase activity by 144, 95, 197, 53 and 42 %, respectively over the uninoculated control soil. Previously, Ju et al. [91] also observed that the inoculation of PGPR Paenibacillus mucilaginosus increased the acid phosphatase (21 %), β-glucosidase (28 %) activities. It has been hypothesized that PGPR can increase soil enzyme activities by accelerating the decomposition of soil organic compounds and providing substrates for enzymatic reactions [68]. Similarly, sole biochar treatment also exhibited an increase in dehydrogenase, alkaline phosphatase, acid phosphatases, β-glucosidase and urease activity by 1.7, 1.1, 0.6, 1.2 and 0.8 %, respectively. Our results were in agreement with others who demonstrated a significant increase in the activities of dehydrogenase [92], alkaline phosphatase [51], acid phosphatase [20], urease [93], and β-glucosidase [94] due to biochar amendment. It has been demonstrated that the increase in the activities of soil enzymes as the results of biochar treatment could be due to the biochar mediated improvement of soil characteristics including the increase of organic matter, microbial activity and its biomass C, alteration of pH, decrease of bulk density, increase of aeration etc., [95]. Further, the combined PGPR + biochar treatment led to a maximum increase in the activities of dehydrogenase, alkaline phosphatase, acid phosphatase, urease and β-glucosidase, which confirms that PGPR and biochar play a synergistic role on the activities of soil enzymes. The PGPR ST1NI15 + biochar treatment increased dehydrogenase, alkaline phosphatase, acid phosphatase and urease by 4, 3, 7.2 and 1.38 %, respectively, compared to the respective control. Similar to our results, Ashry and Hassan [92] found that when soils were treated with Paenibacillus polymyxa, Ochrobactrum intermedium and rice straw biochar exhibited greater increase in the activities of dehydrogenase, phosphatase and nitrogenase activities and concluded that increased enzyme activity should be attributed to improved microbial growth and activity as the consequence of biochar amendment because the porosity, surface area and nutrient contents of biochar may provide favorable environmental conditions for the proliferation of soil microbes. In general, the soil enzymes improve the soil health and thus act as an indicator of soil fertility. For instance, dehydrogenase involves in decomposition of organic matter improves the soil nutrient content, whereas phosphatase, urease and glucosidase involve in P, N and carbon cycle and mineralization [95,96,97]. Therefore from the results, it can be correlated that the increased activities of soil enzymes as the results of cumulative effects of PGPR + biochar might be improved nutrient bioavailability and thus their uptake by plants through decomposition of SOM and mineralization of nutrients, that can be evident from the results obtained in plant biometric parameter, pigment, protein content and nutrient uptake.

Figure 4. Effect of PGPR and biochar on a) dehydrogenase, b) alkaline phosphatase, c) acid phosphatase, d) urease and e) β-glucosidase activity in soil. Bars represent the mean and standard deviation of three replicates. Bars index with same letters are not significant between treatments using HSD tukey’s test (p < 0.05). ρNP - ρ Nitrophenol; TPF - Triphenyl formazan, NH₄⁺-N - ammonium nitrogen

4. Conclusion

The application of PGPR and biochar is an eco- friendly alternative to chemical fertilizers that can improve soil structure, plant growth and productivity. Although the sole treatment of PGPR inoculation or biochar amendment improved R. communis growth, physiology, nutrient uptake and soil enzymatic activities, the combined treatment of PGPR and biochar was found to be more effective in terms of improving plant and soil parameters. Notably, the combined application increased the soil enzymatic activities suggests that biochars provide suitable environment which may improve the growth and activity of PGPR and thus their beneficial effects on plant. However, further attempts should be made to study how the biochar characteristics influence the diversity, activity and colonization of indigenous and/or inoculated microbes and their potential on plant growth in marginal lands. This will provide not only an improved knowledge on plant microbe and biochar interaction, but also useful to develop suitable bioformulation for improving the soil fertility and crop productivity.

Acknowledgements

V.A acknowledges the Bharathiar University for providing University Research Fellowship (File No. C2/28047-2/2019). A.S.A thanks to the Department of Science and Technology (DST), Government of India for awarding Inspire Fellowship (File No. DST/INSPIRE/03/2019/000159). S.S thankfully acknowledges the financial support from DST PURSE - II, Bharathiar University (File No. BU/DST PURSE (II) /APPOINTMENT/571).

References

[1]  
Indoria, A.K., Sharma, K.L., Reddy, K.S., Srinivasarao, C.H., Srinivas, K., Balloli, S.S., Osman, M., Pratibha, G. and Raju, N.S, “Alternative sources of soil organic amendments for sustaining soil health and crop productivity in India – impacts, potential availability, constraints and future strategies,” Current science, 115(11). 2052- 2062. Dec.2018.

[2]   | Mannan, M.A., Halder, E., Karim, M.A. and Ahmed, J.U, “Alleviation of adverse effect of drought stress on soybean (Glycine max. L.) by using poultry litter biochar,” Bangladesh agronomy journal, 19(2). 61-69. 2016.
[3]   | Lin, W., Lin, M., Zhou, H., Wu, H., Li, Z. and Lin, W, “The effects of chemical and organic fertilizer usage on rhizosphere soil in tea orchards,” PLoS One, 14(5). e0217018. May.2019.
[4]   | Atidegla, S.C., Huat, J., Agbossou, E.K., Saint-Macary, H. and Kakai, R.G, “Vegetable contamination by the fecal bacteria of poultry manure: Case study of gardening sites in Southern Benin,” International journal of food science, 1-8. Mar.2016.
[5]   | Urra, J., Alkorta, I. and Garbisu, C, “Potential benefits and risks for soil health derived from the use of organic amendments in agriculture,” Agronomy, 9(9). 542. Sep.2019.
[6]   | Gupta, G., Parihar, S.S., Ahirwar, N.K, Snehi, S.K. and Singh, V, “Plant growth promoting rhizobacteria (PGPR): Current and future prospects for development of sustainable agriculture,” Journal of microbial and biochemical technology, 7. 96-102. Mar.2015.
[7]   | Dos Santos, R.M., Diaz, P.A.E., Lobo, L.L.B. and Rigobelo, E.C, “Use of plant growth-promoting rhizobacteria in maize and sugarcane: Characteristics and applications,” Frontiers in sustainable food systems, 4. 136. Sep.2020.
[8]   | Khan, N., Bano, A. and Cura, J.A, “Role of beneficial microorganisms and salicylic acid in improving rainfed agriculture and future food safety,” Microorganisms, 8(7). 1018. Jul.2020.
[9]   | Gouda, S., Kerry, R.G., Das, G., Paramithiotis, S., Shin, H. and Patra, J.K, “Revitalization of plant growth promoting rhizobacteria for sustainable development in agriculture,” Microbiological research, 206. 131-140. Jan.2018.
[10]   | Glick, B.R, “Using soil bacteria to facilitate phytoremediation,” Biotechnology advances, 28. 367-374. May-Jun.2010.
[11]   | Ma, Y., Rajkumar, M., Moreno, A., Zhang, C. and Freitas, H, “Serpentine endophytic bacterium Pseudomonas azotoformans ASS1 accelerates phytoremediation of soil metals under drought stress,” Chemosphere, 185. 75-85. Oct.2017.
[12]   | De los Santos-Villalobos, S. and Parra-Cota, F.I, “Current trends in plant growth-promoting microorganisms research for sustainable food security,” Current research in microbial sciences, 2. 100016. Dec.2021.
[13]   | Idris, R., Trifonova, R., Puschenreiter, M., Wenzel, W.W. and Sessitsch, A, “Bacterial communities associated with flowering plants of the Ni hyperaccumulator Thlaspi goesingense,” Applied and environmental microbiology, 70. 2667-2677. May.2004.
[14]   | Kumar, K.V., Singh, N., Behl, H.M. and Srivastava, S, “Influence of plant growth promoting bacteria and its mutant on heavy metal toxicity in Brassica juncea grown in fly ash amended soil,” Chemosphere, 72. 678-683. Jun.2008.
[15]   | Wang, Y., Li, W., Du, B. and Li, H, “Effect of biochar applied with plant growth-promoting rhizobacteria (PGPR) on soil microbial community composition and nitrogen utilization in tomato,” Pedosphere, 31(6). 872-881. Dec.2021.

[16]  
Egamberdieva, D., Shurigin, V., Alaylar, B., Ma, H., Muller, M.E.H., Wirth, S., Reckling, M. and Bellingrath-Kimura, S.D, “The effect of biochars and endophytic bacteria on growth and root rot disease incidence of fusarium infested narrow-leafed lupin (Lupinus angustifolius L.),” Microorganisms, 8(4). 496. Mar.2020.

[17]  
Malik, L., Sanaullah, M., Mahmood, F., Hussain, S., Siddique, MH., Anwar, F. and Shahzad, T, “Unlocking the potential of co-applied biochar and plant growth-promoting rhizobacteria (PGPR) for sustainable agriculture under stress conditions,” Chemical and biological technologies in agriculture, 9(1). 58. Aug.2022.

[18]   | Amoah-Antwi, C., Kwiatkowska-Malina, J., Thornton, S.F., Fenton, O., Malina, G. and Szara, E, “Restoration of soil quality using biochar and brown coal waste: A review,” Science of the total environment, 722. 137852. Jun.2020.

[19]  
Igalavithana, A.D., Choi, S.W., Shang, J., Hanif, A., Dissanayake, P.D., Tsang, D.C.W., Kwon, J., Lee, K.B. and Ok, Y.S, “Carbon dioxide capture in biochar produced from pine sawdust and paper mill sludge: Effect of porous structure and surface chemistry,” Science of the total environment, 739. 139845. Oct.2020.

[20]   | Jabborova, D., Ma, H., Bellingrath-Kimura, SD. and Wirth, S, “Impacts of biochar on basil (Ocimum basilicum) growth, root morphological traits, plant biochemical and physiological properties and soil enzymatic activities,” Scientia Horticulturae, 290. 110518. Dec.2021.
[21]   | Vaccari, F.P., Baronti, S., Lugato, E., Genesio, L., Castaldi, S., Fornasier, F. and Migliettaa, F, “Biochar as a strategy to sequester carbon and increase yield in durum wheat,” European journal of agronomy, 34. 231-238. May.2011.
[22]   | De Souza, R., Beneduzi, A., Ambrosini, A., da Costa, P.B., Meyer, J., Vargas, L.K., Schoenfeld, R. and Passaglia, L.M.P, “The effect of plant growth-promoting rhizobacteria on the growth of rice (Oryza sativa L.) cropped in southern Brazilian fields,” Plant and soil, 366. 585-603. 2013.
[23]   | Tripti., Kumar, A., Kumar, V., Anshumali., Bruno, L.B. and Rajkumar, M, “Synergism of industrial and agricultural waste as a suitable carrier material for developing potential biofertilizer for sustainable agricultural production of eggplant,” Horticulturae, 8. 444. May.2022.
[24]   | Kalu, S., Simojoki, A., Karhu, K. and Tammeorg, P, “Long-term effects of softwood biochar on soil physical properties, greenhouse gas emissions and crop nutrient uptake in two contrasting boreal soils,” Agriculture, ecosystems and environment, 316. 107454. Aug.2021.
[25]   | Vanek, S.J., Thies, J., Wang, B., Hanley, K. and Lehmann, J, “Pore-Size and water activity effects on survival of Rhizobium tropici in biochar inoculant carriers,” Journal of microbial and biochemical technology, 8. 296-306. Jan.2016.
[26]   | Tripti., Kumar, A., Usmani, Z., Kumar, V. and Anshumali, “Biochar and flyash inoculated with plant growth promoting rhizobacteria act as potential biofertilizer for luxuriant growth and yield of tomato plant,” Journal of environmental management, 190. 20-27. Apr.2017.
[27]   | Ma, Y., Rajkumar, M., Luo, Y. and Freitas, H, “Inoculation of endophytic bacteria on host and non-host plants-Effects on plant growth and Ni uptake,” Journal of hazardous materials, 195. 230-237. Nov.2011.
[28]   | Bric, J.M., Bostock, R.M. and Silverstone, S.E, “Rapid in situ assay for indole acetic acid production by bacteria immobilized on a nitrocellulose membrane,” Applied and environmental microbiology, 57(2). 535-538. Feb.1991.
[29]   | Schwyn, B. and Neilands, J.B, “Universal chemical assay for the detection and determination of siderophores,” Analytical biochemistry, 160(1). 47-56. Jan.1987.
[30]   | Arnow, L.E, “Colorimetric determination of the components of 3,4-dihydroxyphenylalaninetyrosine mixtures,” Journal of biological chemistry, 118. 531. Apr.1937.
[31]   | Atkin, C.L., Neilands, J.B. and Phaff, H.J, “Rhodotorulic acid from species of Leucosporidium, Rhodosporidium, Rhodotorula, Sporidiobolus, and Sporobolomyces, and a new alanine-containing ferrichrome from Cryptococcus melibiosum,” Journal of bacteriology, 103. 722-733. Sep.1970.
[32]   | Cappuccino, J.G. and Sherman, N, Microbiology, A Laboratory Manual, Second edition. The Benjamin Cummings Publishing Co. Inc, San Francisco, USA, 1996.
[33]   | Suleman, M., Yasmin, S., Rasul, M., Yahya, M., Atta, B.M. and Mirza, M.S, “Phosphate solubilizing bacteria with glucose dehydrogenase gene for phosphorus uptake and beneficial effects on wheat,” PLoS one, 13(9). e0204408. Sep.2018.

[34]  
Bruno, L.B., Anbuganesan, V., Karthik, C., Tripti., Kumar, A., Banu, J.R. and Rajkumar, M, “Enhanced phytoextraction of multi-metal contaminated soils under increased atmospheric temperature by bioaugmentation with plant growth promoting Bacillus cereus,” Journal of environmental management, 289. 112553. Jul.2021.

[35]   | Sahoo, S.S., Vijay, V.K., Chandra, R. and Kumar, H, “Production and characterization of biochar produced from slow pyrolysis of pigeon pea stalk and bamboo,” Cleaner engineering and technology, 3. 100101. Jul.2021.
[36]   | Nelson, D.W. and Sommers, L.E. Total carbon, organic carbon, and organic matter, In: Sparks, D.L., Page, A.L., Helmke, P.A., Loeppert, R.H., Soltanpour, P.N., Tabatabai, M.A., Johnston, C.T. and Sumner, M.E (eds.), Methods of Soil Analysis, Part 3, SSSA book Series, Madison, 961-1010. 1996.
[37]   | Laird, D. and Fleming, P. Analysis of layer charge, cation and anion exchange capacities and synthesis of reduced charge clays, In: Ulery, A.L. and Drees, R.L (eds.), Methods of soil analysis: Mineralogical methods, American society of agronomy, 499-501. 2008.
[38]   | Smith, J.H.C. and Benitez, A. Chlorophylls analysis in plant materials. In: Peach, K. and Tracey, M.V (eds.), Modern methods of plant analysis, Springer, Verlag, Berlin, 4. 142-196. 1955.
[39]   | Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J, “Protein measurement with the folin phenol reagent,” Journal of biological chemistry, 193(1). 265-275. Nov.1951.
[40]   | Bates, L.S., Waldren, R.P. and Teare, I.D, “Rapid determination of free proline for water-stress studies,” Plant and soil, 39. 205-207. Aug.1973.
[41]   | Heath, R.L. and Packer, L, “Photoperoxidation in isolated chloroplasts.I. Kinetics and stoichiometry of fatty acid peroxidation,” Archives of biochemistry and biophysics, 125(1). 189-98. Apr.1968.
[42]   | Kono, Y, “Generation of superoxide radical during auto oxidation of hydroxylamine and an assay for superoxide dismutase,” Archives of biochemistry and biophysics, 186(1). 189-195. Feb.1978.
[43]   | Putter, J. Peroxidase. In: Bergmeyer H.U (ed), Methods of enzymatic analysis, NewYork, Academic Press, 685-690. 1974.
[44]   | Lata, C., Jha, S., Dixit, V., Sreenivasulu, N. and Prasad, M, “Differential antioxidative responses to dehydration-induced oxidative stress in core set of foxtail millet cultivars [Setaria italica (L.)],” Protoplasma, 248(4). 817-828. Jan.2011.
[45]   | Chen, G. and Asada, K, “Ascorbate peroxidase in tea leaves: Occurrence of two isozymes and the differences in their enzymatic and molecular properties,” Plant and cell physiology, 30(7). 987-998. Oct.1989.
[46]   | Poshtdar, A., Mashhadie, A.R.A., Moradi, F., Siadat, S.A. and Bakhshandeh, A, “Effect of source and rate of nitrogen fertilizer on yield and water and nitrogen use efficiency of peppermint (Mentha piperita L.),” Iranian journal of crop sciences, 18. 13-31. 2016.
[47]   | Zhu, Q., Kong, L., Shan, Y., Yao, X., Zang, H., Xie, F. and Ao, X, “Effect of biochar on grain yield and leaf photosynthetic physiology of soybean cultivars with different phosphorus efficiencies,” Journal of integrative agriculture, 18(10). 2242-2254. Oct.2019.
[48]   | ISO 23753-1:2005 Soil quality-Determination of dehydrogenase activity in soils-Part 1: method using triphenyltetrazolium chloride (TTC). 2005.
[49]   | Eivazi, F. and Tabatabai, M.A, “Phosphatases in soils,” Soil biology and biochemistry, 9. 167-172. 1977.
[50]   | Eivazi, F. and Tabatabai, M.A, “Glucosidases and galactosidases in soils,” Soil biology and biochemistry, 20. 601-606. 1988.
[51]   | Li, Y., Feng, H., Chen, J., Lu, J., Wu, W., Liu, X., Li, C., Dong, Q. and Siddique, K.H.M, “Biochar incorporation increases winter wheat (Triticum aestivum L.) production with significantly improving soil enzyme activities at jointing stage,” Catena, 211. 105979. Apr.2022.
[52]   | Bruno, L.B., Karthik, C., Ma, Y., Kadirvelu, K., Freitas, H. and Rajkumar, M, “Amelioration of chromium and heat stresses in Sorghum bicolor by Cr6+ reducing-thermotolerant plant growth promoting bacteria,” Chemosphere, 244. 125521. Apr.2020.
[53]   | Sheng, X., Xia, J., Jiang, C., He, L. and Qian, M, “Characterization of heavy metal-resistant endophytic bacteria from rape (Brassica napus) roots and their potential in promoting the growth and lead accumulation of rape,” Environmental pollution, 156(3). 1164-1170. Dec.2008.
[54]   | Xess, N. and Sao, S, “Effect of carbon and nitrogen source on phosphate solubilization and impact of phosphate solubilizing bacteria, rock phosphate and organic waste treatments on maize plants,” Applied ecology and environmental sciences, 8(6) 499-504. Oct.2020.

[55]  
Roman-Ponce, B., Reza-Vazquea, D.M., Gutierrez-Paredes, S., De haro-Cruz, M.D.J., Maldonado-Hernandez, J., Bahena-Osorio, Y., Estrada-de lossantos, P., Wang, E.T. and Vasquez-Murrieta, M.S, “Plant growth-promoting traits in rhizobacteria of heavy metal-resistant plants and their effects on Brassica nigra seed germination,” Pedosphere, 27(3). 511-526. Jun.2017.

[56]   | Rodriguez, H. and Fraga, R, “Phosphate solubilizing bacteria and their role in plant growth promotion,” Biotechnology advances, 17(4). 319-339. Oct.1999.
[57]   | Olanrewaju, O.S., Glick, B.R. and Babalola, O.O, “Mechanisms of action of plant growth promoting bacteria,” World journal of microbiology and biotechnology, 33. 197. Oct.2017.
[58]   | Trung, N.T., Hieu, H.V. and Thuan, N.H, “Screening of strong 1-aminocyclopropane-1-carboxylate deaminase producing bacteria for improving the salinity tolerance of cowpea,” Applied microbiology and biotechnology, 2(1). 1-6. Mar.2016.
[59]   | Jochum, M.D., McWilliams, K.L., Borrego, E.J., Kolomiets, M.V., Niu, G., Pierson, E.A. and Jo, Y, “Bioprospecting plant growth-promoting rhizobacteria that mitigate drought stress in grasses,” Frontiers in microbiology, 10. 2106. Sep.2019.
[60]   | Rajkumar, M., Ae, N., Prasad, M.N.V. and Freitas, H, “Potential of siderophore-producing bacteria for improving heavy metal phytoextraction,” Trends biotechnology, 28(3). 142-149. Mar.2010.
[61]   | Tomczyk, A., Sokołowska, Z. and Boguta, P, “Biochar physicochemical properties: pyrolysis temperature and feedstock kind effects,” Reviews in environmental science and biotechnology, 19. 191-215. Feb.2020.

[62]  
Mehmood, S., Rizwan, M., Bashir, S., Ditta, A., Aziz, O., Yong, L.Z., Dai, Z., Akmel, M., Ashed, W., Adial, M., Imtiaz, M. and Tu, S, “Comparative effects of biochar, slag and ferrous-mn ore on lead and cadmium immobilization in soil,” Bulletin of environmental contamination and toxicology, 100. 286-292. 2018.

[63]   | Mendez, A., Gomez, A., Paz-Ferreiro, J. and Gasco, G, “Effects of sew-age sludge biochar on plant metal availability after application to a mediterranean soil,” Chemosphere, 89. 1354-1359. Nov.2012.
[64]   | Hossain, M.Z., Bahar, M.M., Sarkar, B., Donne, S.W., Ok, Y.S., Palansooriya, K.N., Kirkham, M.B., Chowdhury, S. and Bolan, N, “Biochar and its importance on nutrient dynamics in soil and plant,” Biochar, 2(4). 379-420. Sep.2020.
[65]   | Yaashikaa, P.R., Kumar, P.S., Varjani, S. and Saravanan, A, “A critical review on the biochar production techniques, characterization, stability and applications for circular bioeconomy,” Biotechnology reports, 28. e00570. Dec.2020.
[66]   | Baiamonte, G., De Pasquale, C., Marsala, V., Cimo, G., Alonzo, G., Crescimanno, G. and Conte, P, “Structure alteration of a sandy-clay soil by biochar amendments,” Journal of soils and sediments. 15. 816–824. 2015.
[67]   | Cantrell, K.B., Hunt, P.G., Uchimiya, M., Novak, J.M. and Ro, K.S, “Impact of pyrolysis temperature and manure source on physicochemical characteristics of biochar,” Bioresource technology, 107. 419-428. Mar.2012.
[68]   | Li, H., Qiu, Y., Yao, T., Ma, Y., Zhang, H. and Yang, X, “Effects of PGPR microbial inoculants on the growth and soil properties of Avena sativa, Medicago sativa, and Cucumis sativus seedlings,” Soil and tillage research, 199. 104577. May.2020.
[69]   | Azeem, M., Hassan, T.U., Tahir, M.I., Ali, A., Jeyasundar, P.G.S.A., Hussain, Q., Bashir, S., Mehmood, S. and Zhang, Z, “Tea leaves biochar as a carrier of Bacillus cereus improves the soil function and crop productivity,” Applied soil ecology, 157. 103732. Jan.2021.
[70]   | Murtaza, G., Ahmed, Z., Usman, M., Tariq, W., Ullah, Z., Shareef, M., Iqbal, H., Waqas, M., Tariq, A., Wu, Y., Zhang, Z. and Ditta, A, “Biochar induced modifications in soil properties and its impacts on crop growth and production,” Journal of plant nutrition, 14(11). 1677-1691. Jan.2021.
[71]   | Dey, R., Pal, K.K., Bhatt, D.M. and Chauhan, S.M, “Growth promotion and yield enhancement of peanut (Arachishypogaea L.) by application of plant growth-promoting rhizobacteria,” Microbiological research, 159. 371-394. Dec.2004.

[72]  
Vishnupradeep, R., Bruno, L.B., Taj, Z., Karthik, C., Challabathula, D., Tripti., Kumar, A., Freitas, H. and Rajkumar, M, “Plant growth promoting bacteria improve growth and phytostabilization potential of Zea mays under chromium and drought stress by altering photosynthetic and antioxidant responses,” Environmental technology and innovation, 25. 102154. Feb.2022.

[73]   | Rombel, A., Krasucka, P. and Oleszczuk, P, “Sustainable biochar-based soil fertilizers and amendments as a new trend in biochar research,” Science of the total environment, 816. 151588. Apr.2022.
[74]   | Singh, A., Singh, A.P., Singh, S.K., Rai, S. and Kumar, D, “Impact of addition of biochar along with PGPR on rice yield. Availability of nutrients and their uptake in alluvial Soil,” Journal of pure and applied microbiology, 10(3). 2181-2188. Sep.2016.
[75]   | Marques, A.P.G.C., Pires, C., Moreira, H., Rangel, A.O.S.S. and Castro, P.M.L, “Assessment of the plant growth promotion abilities of six bacterial isolates using Zea mays as indicator plant,” Soil biology and biochemistry, 42. 1229-1235. Aug.2010.
[76]   | Bhattacharyya, C., Banerjee, S., Acharya, U., Mitra, A., Mallick, I., Haldar, A., Haldar, S., Ghosh, A. and Ghosh, A, “Evaluation of plant growth promotion properties and induction of antioxidative defense mechanism by tea rhizobacteria of Darjeeling, India,” Scientific reports, 10. 1-19. Sep.2020.
[77]   | Khan, N., Bano, A.M.D. and Babar, A, “Impacts of plant growth promoters and plant growth regulators on rainfed agriculture,” PLoS one, 15(4). e0231426. Apr.2020.
[78]   | Zhu, Y., Wang, H., Lv, X., Zhang, Y. and Wang, W, “Effects of biochar and biofertilizer on cadmium-contaminated cotton growth and the antioxidative defense system,” Scientific reports, 10. 20112. Nov.2020.
[79]   | Tirry, N., Kouchou, A., Laghmari, G., Lemjereb, M., Hnadi, H., Amrani, K., Bahafid, W. and Ghachtouli, N.E, “Improved salinity tolerance of Medicago sativa and soil enzyme activities by PGPR,” Biocatalysis and agricultural biotechnology, 31. 101914. Jan.2021.

[80]  
Sharma, P., Chouhan, R., Bakshi, P., Gandhi, S.G., Kaur, R., Sharma, A. and Bhardwaj, R, “Amelioration of chromium-induced oxidative stress by combined treatment of selected plant-growth-promoting rhizobacteria and earthworms via modulating the expression of genes related to reactive oxygen species metabolism in Brassica juncea,” Frontiers in microbiology, 13. 802512. Apr.2022.

[81]   | Moradi, R., Pourghasemian, N. and Naghizadeh, M, “Effect of beeswax waste biochar on growth, physiology and cadmium uptake in saffron,” Journal of cleaner production, 229. 1251-1261. Aug.2019.
[82]   | Esitken, A., Yildiz, H.E., Ercisli, S., Donmez, M.F., Turan, M. and Gunes, A, “Effects of plant growth promoting bacteria (PGPB) on yield, growth and nutrient contents of organically grown strawberry,” Scientia horticulturae, 124. 62-66. Feb.2010.
[83]   | Zafar, M., Abbasi, M.K., Khan, M.A., Khaliq, A., Sultan, T. and Aslam, M, “Effect of plant growth-promoting rhizobacteria on growth, nodulation and nutrient accumulation of lentil under controlled conditions,” Pedosphere, 22(6). 848–859. Dec.2012.
[84]   | Pereira, S.I.A., Abreu, D., Moreira, H., Vega, A. and Castro, P.M.L, “Plant growth-promoting rhizobacteria (PGPR) improve the growth and nutrient use efficiency in maize (Zea mays L.) under water deficit conditions,” Heliyon, 6. 05106. Oct.2020.
[85]   | Stajkovic, O., Delic, D., Josic, D., Kuzmanovic, D., Rasulic, N. and Knezevic-Vukcevic, J, “Improvement of common bean growth by co-inoculation with rhizobium and plant growth-promoting bacteria,” Romanian Biotechnological Letters, 16(1). 2011.
[86]   | Radhapriya, P., Ramachandran, A., Anandham, R. and Mahalingam, S, “Pseudomonas aeruginosa RRALC3 enhances the biomass, nutrient and carbon contents of Pongamia pinnata seedlings in degraded forest soil,” PLoS one, 10(10). 0139881. Oct.2015.
[87]   | Tanure, M.M.C., Costa, L.M., Huiz, H.A., Fernandes, R.B.A., Cecon, P.R., Junior, J.D.P. and Luz, J.M.R, “Soil water retention, physiological characteristics, and growth of maize plants in response to biochar application to soil,” Soil and tillage research, 192. 164-173. Sep.2019.
[88]   | Levesque, V., Jeanne, T., Dorais, M., Ziadi, N., Hogue, R. and Antoun, H, “Biochars improve tomato and sweet pepper performance and shift bacterial composition in a peat-based growing medium,” Applied soil ecology, 153. 103579. Sep.2020.
[89]   | Liu, M., Che, Y., Wang, L., Zhao, Z., Zhang, Y., Wei, L. and Xiao, Y, “Rice straw biochar and phosphorus inputs have more positive effects on the yield and nutrient uptake of Lolium multiflorum than arbuscular mycorrhizal fungi in acidic Cd-contaminated soils,” Chemosphere, 235. 32-39. Nov.2019.

[90]  
Jabborova, D., Wirth, S., Kannepalli, A., Narimanov, A., Desouky, S., Davranov, K.,Sayyed, R.Z., Enshasy, H.E., Malek, R.A., Syed, A. and Bahkali, A.H, “Co-inoculation of rhizobacteria and biochar application improves growth and nutrients in soybean and enriches soil nutrients and enzymes,” Agronomy, 10. 1142. 2020.

[91]   | Ju, W., Jin, X., Liu, L., Shen, G., Zhao, W., Duan, C. and Fang, L, “Rhizobacteria inoculation benefits nutrient availability for phytostabilization in copper contaminated soil: Drivers from bacterial community structures in rhizosphere,” Applied soil ecology, 150. 103450. Jun.2020.
[92]   | Ashry, N.M. and Hassan, M.A.A, “Integration between biochar and plant growth promoting bacteria affecting growth of pepper (Capsicum annum L.) plant,” International journal of microbiology research, 10(2), 53-61. 2019.
[93]   | El-Bassi, L., Azzaz, A.A., Jellali, S., Akrout, H., Marks, E.A.N, Ghimbeu, C.M. and Jeguirim, M, “Application of olive mill waste-based biochars in agriculture: Impact on soil properties, enzymatic activities and tomato growth,” Science of the total environment, 755. 142531. Feb.2021.
[94]   | Lopes, E.M.G., Reis, M.M., Frazao, L.A., Terra, L.E.M., Lopes, E.F., Martins dos Santos, M. and Fernandes, L.A, “Biochar increases enzyme activity and total microbial quality of soil grown with sugarcane,” Environmental technology and innovation, 21. 101270. Feb.2021.
[95]   | Oladele, S.O, “Effect of biochar amendment on soil enzymatic activities, carboxylate secretions and upland rice performance in a sandy clay loam alfisol of southwest Nigeria,” Scientific african, 4. e00107. Jul.2019.
[96]   | Karaca, A., Cetin, S.C., Turgay, O.C. and Kizilkaya, R. Soil enzymes as indication of soil quality, In: Shukkla G, Varma A (eds.), Soil enzymology, Springer, Heidelberg, Dordrecht, London, New York, 119-148. 2003.
[97]   | Kaur, R., Kaur, A., Bhatti, R.C., Nirmala, C. and Singh, A, “Differential responses of β-glucosidase, SOC and MBC of degraded soil under various treatments in a North-Western arid region, India,” Applied ecology and environmental sciences, 6(4), 99-108. Oct.2018.