Boron (B) is a vital micronutrient that is indispensable for proper crop growth (Bhupenchandra et al., 2021a). Boron is a necessary micro-element for plant cell wall structural integrity and is involved in various plant processes like cell division, calcium utilization, pollen production, and anther development during the reproductive phase (Nadeem et al., 2019). Currently, the B deficits in soils are widespread globally causing B micronutrient malformations that impinge on agricultural production (Shorrocks, 1997; Liu, 2000; Choudhary et al., 2014; Behera et al., 2022). Due to excess deficiency symptoms manifested as an implication of B deficit, the assessment of key functions of B in plants has long been the main concern from a nutritional point of view. For plants, managing B is difficult because the optimum B range is narrow which can fluctuate from soil to soil (Gupta, 1993; Marschner, 1995). Normally, B averaged nearly 30 mg kg^–1 soil depending on the main rock wherein its content varies extensively. Satisfactory B content for flora in soils is more or less 25 mg kg^–1 (GreenFacts, 2002). Soil microbial biomass holds a vital role in nutrient-cycling, plant-pathogen suppression, the disintegration of debris, and decay of pollutants establishing the vibrant living entity of soil, and thus, attributing to ecological sustainability owing to their diverse existence, enormous effective genetic pools, catabolic adaptability, and stress tolerance ability in a holistic manner (Deluca et al., 2019). The dimension and activity of the microbial biomass determine the nutrient availability and production potential of the agro-ecosystems (Friedel et al., 1996; Singh et al., 2020, 2021). Therefore, it becomes obligatory to determine microbial biomass nitrogen (MBN). Since it becomes vital for the quantification of N-dynamics in agro-ecosystems as it controls the soil inorganic-N accessibility and loss and its contribution to the primary N-sources of potentially mineralizable-N (PMN) in the soil (Bonde et al., 1988). Microbial biomass carbon (MBC), MBN, and microbial respiration, have further garnered added interest owing to their sensitivity to crop management practices than the bulk soil organic matter (Awale et al., 2017). Soil microbes, the existing fraction of soil organic matter (SOM) acts as a transitory nutrient-sink and are accountable for unleashing nutrients from SOM for exploitation by plants (Bollag et al., 2017). Basal respiration (BR) and C-mineralization are ample indicators of microbial activity, depending on the substrate accessibility and the soil edaphic environment (Balota et al., 2003). On the whole, the CO₂ respired during a year in terrestrial ecosystems is the consequence of C-mineralization of the minute active fraction pools, which are mainly accountable for unleashing nutrients in the soil (Brdar-Jokanović, 2020).

It is implicit to address that B acts a crucial function in the biological activities of living organisms as proven earlier by establishing the necessity of B for diatoms and cyanobacteria (Bonilla et al., 1997). Soil microorganisms are by far the most important producers of soil enzymes that perform many ecological processes such as bio-geochemical cycling and decomposing pollutants and debris from flora and fauna and the microbes (Goswami et al., 2017; Furtak and Gajda, 2018). Soil microbes are accountable for the transformation of SOM and soil nutrients (Mooshammer et al., 2014; Singh et al., 2020, 2021). The microbes and their enzymatic outputs are indispensable to plants, while plant roots generate organic substances that are vital to the populace expansion of microbes (Jjemba and Alexander, 1999). Soil microbial indices is touted as a substitute for organic carbon cycling and its related nutrients viz., N, P, and S, signifying that elevated microbial action implied increased soil productivity and vice-versa (Pavan et al., 2005). Soil enzymes are touted as an index for examining the activities of microbes, soil productivity and soil quality owing to the symbiosis of microbes and flora (Dick, 1994; Bandick and Dick, 1999). Evaluation of diverse soil extracellular enzymes established it as a potent means for assessment of the soil functions for nutrient-cycling and microbial nutrient requirements (Sinsabaugh et al., 2012). We also hypothesized that in lieu of specific enzyme activity, an index merging diverse enzymes would be a more efficient and appropriate indicator of soil quality, since it could specify an inclusive diversity of soil functions. Relationships between crop yields, nutrient availability, and these enzyme activities are also obscure, as information on the enzymes’ ability to predict soil quality attributes is scarce. With the induction of intensive crop management practices like fertilizer application exhibiting complicated and harmful implications on plants and microbial associations, the studies on impact assessment of B-fertilization on soil microbes and enzymes for recuperating agricultural output in B-deficient soils become highly imperative (Dick, 1994; Tabatabai, 1994). The necessity of B-fertilization for the augmentation and maturity of plants has already been established (Shelp, 1993; Marschner, 1995). Boron acts as a vital function in the translocation and assimilation of complex carbohydrates in the plant, production of plant hormones and nucleic acids, germinating pollen, flower induction and fruiting, and water utilization. The main significant roles of B in plants are its structural role in cell wall growth and stimulating or inhibiting of precise metabolic pathways (Ahmad et al., 2009). In addition, B plays a crucial role in N assimilation, N fixation, and the growth of legume root nodules (Bolaños et al., 2004a; Bellaloui et al., 2014).

Boron accessibility depends on many criteria that exist in the soil–plant system such as SOM, soil texture, cultivation, soil moisture, temperature, soil pH and liming, soil fertility, and microbial activity (Shorrocks, 1997; Kumar et al., 2016; Shireen et al., 2018). Microbes assimilate SOM, which in turn, helps in releasing the B from organic complexes. Boron present in the soil is considered as a vital fraction related to SOM and is unleashed via microbial activities (Berger and Pratt, 1963). Despite the prime role of B on floral growth and functioning, no credibility has been established to explain that B is an enzyme component and possesses a direct role in enzyme actions. It is obscure to claim that these processes are precursor of the direct functioning of B or the indirect role of B. The biological effects of B are better understood in plants, where it has been proven that it can influence physiology and biological activities (Grattan et al., 2015). Though the impact of B on the soil microbial community is little known, there is a paucity of information on the element’s consolidative impacts on several characteristics of the soil microbial community, including activity, biomass, and diversity. Most of the soil fertility experiments stressed the aspects of the changes in soil chemical pools without giving much attention to biological attributes in soil rendering a lacuna on comprehensive fertility evaluation in soil fertility research. To date, the study on the role of B on soil biological attributes is obscure and very limited. Consequently, advanced research is necessitated to have a clear insight into the functioning of B in plant development and soil biology. The study hypothesized B-fertilization would improve the soil physio-chemical and biological properties and productivity of cauliflower-cowpea-okra cropping system. The objective of the study is to assess the impact of graded levels of B-fertilization on the soil physio-chemical and biological properties and their relationships in a cauliflower-cowpea-okra cropping system in North East India.

Boron is one of the indispensable nutrients for the ideal growth, development, produce, and quality of crops (Shireen et al., 2018). In general, B being a vital nutrient plays a role in plant growth, phenols, lignification, tissue expansion, membrane-related reactions, ribose nucleic acid (RNA) metabolism, hydrocarbon metabolism, pollen germination and seed development which are directly implicated in increasing crop yield (Goldbach and Monika, 2007). The enhancement in crop yield as a result of B-fertilization could be ascribed to the improved availability and accessibility of nutrients to plants (Kumar et al., 2016, 2017), hence producing and mobilizing surplus carbohydrates and proteins along with its role in enhancing their translocation from the site of synthesis to the storage organs (Takkar and Randhawa, 1978; Verma et al., 2012). Moreover, B acts as a key role in many metabolic processes such as cell wall differentiation, cell development, N-metabolism, fertilization, fat metabolism, hormone metabolism, active salt absorption, and photosynthesis (Nason and McElory, 1963), which in turn contributed to higher fresh and dry matter yield of cauliflower. Similar findings in okra were also reported earlier (Saha et al., 2010; Rahman et al., 2017). Kumar and Sen (2004) reported that the application of B and Zn improved the yield and quality of okra seed. The beneficial impacts of B on curd quality and yield of cauliflower were acknowledged by Gupta (1993).

The decrease in BD under CCOCS might be due to an improvement in soil structure and porosity due to the addition of FYM. A slight increment in soil pH could be possibly due to the ligand exchange between OH^–groups of soil Al and Fe(OH)₂ and organic compounds, and the disintegration and binding of organic complexes of the applied FYM (Xu et al., 2006). Similarly, SOC in soil remained significantly (p ≤ 0.05) (Table 1) unaffected after two years of B application, however, an increment in SOC was observed possibly due to the SOC build-up through regular addition of FYM and desirable changes in biochemical and physical properties of soil (Ghosh et al., 2012; Bhupenchandra et al., 2022; Harish et al., 2022; Kumar et al., 2022). Also, another reason for the improvement in SOC could be due to the formation of a strong diol complex of B with organic matter in soils and the capacity of organic matter to improve CEC of soils (Bhupenchandra et al., 2021b). Increase in available N could be due the release of mineralized N by the addition of organic matter along with the concurrent release of N via symbiotic biological N fixation by cowpea roots, since B plays a vital role in biological N fixation and upsurges the number of effective nodules (Bolaños et al., 2001). The improvement in the status of available P could be explicated by the existence of positive interaction between P and B in the soil as both are in anionic forms and might have been involved in anion exchange (Bhupenchandra et al., 2021b).

Microbial biomass carbon is the measure of the C present within the living constituent of soil organic matter. Soil respiration (SR) is the CO₂ released by the biological activities of soil organisms, involving plant roots, microbes, and soil animals are usually calculated as a flux of CO₂ from the soil surface. Escalating B appliance quickly altered soil MBC content and soil respiration (Bilen et al., 2011). Improvement in MBC and CO₂-C production in soil with the application of graded B could also be due to the continuous application of FYM in all the crops for two years in the sequence, which in turn, might have created a conducive atmosphere for intensified microbial activities in soil (Singh et al., 2020, 2021). Soil MBC, being an active and labile component of SOC dependent on the SOM (Chen et al., 2005; Kumar et al., 2022). There is no direct evidence of the effect of B application on microbial properties of soil. But, the enhancement in these biological properties with the incremental doses of B indicated a possible relationship between the B-fertilization and microbial activity of the soils. Subsequently, microbes are implicated in the assimilation of SOM, which further led to the release of B from organic complexes in soil (Kumar et al., 2016, 2017). Also, total B present in the soil fraction is closely related to SOM and was unleashed via microorganism action (Berger and Pratt, 1963). Upon intensifying the B appliance, the soil microbial biomass load in the soil quickly transformed and escalated vibrantly. Moreover, the accessibility of readily mineralized C and N, and improvement in soil physico-chemical properties might have enhanced the microbial population in soil (Bhardwaj and Datt, 1995; Kumar et al., 2022). Application of FYM improves the SOC pool by supplying organic matter in greater mineralizable form, thereby, delivering substrate for microbial utilization and this could be the cause of higher MBC and SR in the current study (Goldberg, 1997; Liu, 2000; Rajpoot et al., 2021).

The PMC, also known as biodegradable C, is the entirety of organic matter which can be decomposed through microbial action (Guo et al., 2019). Potentially mineralizable-N (PMN) is a measure of the active fraction of soil organic-N, predominantly accountable for the discharge of mineral-N via microbial accomplishment (Campbell and Curtin, 2007). The PMN is availed to plants and microorganisms in the form of NO₃^– by aerobic mineralization. It is a fraction of N linked to the microbial biomass which is positively related to MBC. With crop growth, PMN content in soil is augmented as it is the quantum of N that mineralize with time at the most favorable temperature and moisture. It comprises a diverse group of organic complexes which encompass microbial biomass, crop residues and humus. The increment of PMC content might be attributed to the soil application of well-decomposed FYM which acts as a substrate for microbial entities. The enhanced N-mineralization was observed during the symbiotic biological N-fixation by cowpea roots since B acts a vital role in the biological fixation of N and augments the number of effectual nodules (Bonilla et al., 1997; Bolaños et al., 2001), and hence, might have created signaling compound through the rhizobia infection on roots of legume crop (Dénarié and Cullimore, 1993; Spaink, 2000).

It is established that B is vital for symbiont/plant signaling, namely nod-gene activation by root plant exudates and nodule invasion (Redondo-Nieto et al., 2001). Moreover, B is essential for infectivity thread advancement and nodule initiation (Bolaños et al., 1996) due to the function of B as a modulator of the interactions amidst plants derived infection thread matrix glycoproteins and the bacteria cell surface. Boron stabilizes membranes, which aids the relationship between bacterial cell surfaces and the peri-bacteroid membranes, helping them in regulating symbiotic setup (Bolaños et al., 1996). Specifically, B is indispensable for the target of nodule-specific plant-derived glycoproteins that are critical for signaling bacteroid differentiation into a N₂-fixing form (Bolaños et al., 2004b). Thus, all these factors created a conducive atmosphere for augmenting the microbial population in the soil. Several B tolerant bacterial strains belonging to the genus Bacillus, Chimaereicella, Pseudomonas, Microbacterium, Shewanella, Mycobacterium, and Rhodococcus have been reported with the ability to accumulate B from soil (Ahmed et al., 2007; Raja and Omine, 2013). There are reports on increased rhizosphere microbial populations by B in soybean (Sun et al., 2013). There were reports of B improving the population of diverse bacterial orders (Burkholderiales, Nitrosospherales, and Rhodospirales) (Vera et al., 2019). Boron aids in the enhancement of endomycorrhizae in roots owing to the action of indole 3-acetic acid (IAA) oxidase activity that activates IAA intensities eventually augmenting the translocation of carbohydrates to roots thereby improving fungi–mycorrhizal interaction and its fungal population (Lambert et al., 1980; Kumar et al., 2017). Related findings were reported by Bilen et al. (2011), where the highest population of bacteria and fungi production were observed with 2 kg B ha^–1 in altered growth periods of the plant and diverse soil depths.

Even though the direct role of the effect of B on soil enzymes could not be established, however, the improvement in the status of enzyme activities (AS, DH, FDA, and PMA) was observed during the two years of experimentation. Possibly it was speculated that the enhanced enzyme activities could be attributed to improved soil condition due to the continuous addition of organic matter in the form of FYM in all the crops for two years which enhanced greater microbial activities. Since mineralised C and N from FYM enhanced the soil physico-chemical properties and the quantum of applied-B, the microbial populace and soil enzyme activities increased (Bhardwaj and Datt, 1995; Kumar et al., 2022). The improvement in the soil enzymatic activities might be attributed to the readily degradable organic matter added to the soil, which increases soil microorganisms and soil enzyme activities (Perucci, 1992). Also, DH enzyme activities in soil improved under graded B-fertilization. A similar finding was reported by Bilen et al. (2011) who observed a significant (p < 0.01) positive correlation with B and DH enzyme activity. DHA is a key indicator of microbial activity and organic matter stability since it is directly implicated in microbial respiration (Nikaeen et al., 2015). Improvement in the rhizosphere soil enzyme activity of soybean on the appliance of B was also reported (Goldberg, 1997; Liu, 2000). It was reported that surface residues encouraged the conservation of mineralizable C via residues that bettered the activities of soil enzymes ensuing in higher soil microbial biomass carbon and enhanced soil quality (Mohammadi et al., 2012). The increment in the soil microbial populations enhanced the rhizosphere metabolisms and bettered the soil enzyme activities on the appliance of B (Sun et al., 2013). Urease (urea amidohydrolase, EC 3.5.1.5) is N-related extracellular enzyme, the enzyme implicated in the degradation of urea cleaving urea to NH₃ and CO₂ (Kappaun et al., 2018). Evidently, UE enzyme activity de-escalation in all the crops at different growth stages was observed in B applied to soil as compared to control. The most likely reason may be due to the fact that B containing acids acts as a UE inhibitor in soil (Vera et al., 2019). It was reported that UE activity was inhibited and minimized by the application of higher B levels. Furthermore, this profound effect of B on the UE enzyme activity might be attributed to its action on water-soluble N, apart from the structural impairment to the enzyme and improved availability of N, as amino-boranes, which might have inhibited the urease activity via feedback mechanism (Kappaun et al., 2018). It was reported that the enzyme UE was immobilized on a membrane of microbes as inhibited B (Zaborska, 1995).

Soil functional diversity is governed by substrate amount, quality, and microbial accessibility. (Bending et al., 2002). Consequently, the plots which received higher C sources could alter microbial load of organic matter and the functional diversity of the microbial community in soils (Sall et al., 2006). Higher H values observed under the plots receiving escalated 2 kg B ha^–1 might be due to the higher receipt of B dose which in turn improves SOC contents in the soil as there existed a positive correlation between B and SOC had been earlier documented (Bilen et al., 2011). Similarly, in the current investigation also, higher SYI values hold true in those plots where the highest B levels were applied and this is attributed to the greatest C availability by catabolic diversity (Lagomarsino et al., 2011) ensuing in better soil functional diversity.

The forming of clusters (Figure 4) with MBC, MBN, and SR in the soil in CCOCS may be possibly due to the fact that MBC is on the whole readily decomposable pool of organic material owing to the simplest structure and high quality of C and nutrients where soil bacteria used to nourish (Singh et al., 1992). The primary activation of microbial activity possibly ensued from swift catabolism of simple soluble C compounds, thereby, augmenting microbial biomass load in soil (Singh and Singh, 1991). Due to the possibility of similarity in their function, the soil enzymes were found to accumulate in similar clusters in the soil.

Normally, the first component explains most of the variability contained in the data set (Johnson and Wichern, 2002). The loading plot (Figure 5) exhibited that BP, MBN, PMC and MBC, PMN, actinomycetes, soil enzymes barring urease and subsequently followed by FP, and are highly correlated to each other in CCOCS. Because these parameters are highly correlated to each other as the angle within the variables of 0 or 180° reveals a correlation of 1 or −1, respectively (Kohler and Luniak, 2005). However, UE activity was negatively correlated with PC1 which is ascribed to the inverse link between levels (Vera et al., 2019). Results of the path analysis (Figure 6) also showed that barring urease enzymes, all twelve biological parameters indirectly contributed to MYCS by largely linking to the BP and their indirect path coefficients through BP. The contributions of the biological entities to mean yield of the cropping sequence followed the trend of BP > MBN > PMC > AP > PMC > PMN > AP > BP > FP > AS > DH > FDA > PMA > UE, respectively. The execution of SMLR regression models is considered best fitting (R² > 0.9), as, R² is in the range of 0.90 and 1 (Ogwueleka and Ogwueleka, 2010). In general, an R² value ranging between 0.8 and 0.9 implies a good fitting and values lesser than 0.8 signify a poor model.

The results of the foregoing study revealed a tangible and significant impact of the graded levels of B-fertilization on soil biological entities under cauliflower-cowpea-okra cropping system in an acid Inceptisol. The key biological properties like BP, MBC, MBP, PMC, microbial population, and soil respiration were enhanced significantly with the incremental dose of B-fertilization. The order of the B treatments in respect of MBC, MBN and soil respiration at different crop growth stages was 2.0 kg B ha^–1> 1.5 B kg ha^–1> 1.0 B kg ha^–1> 0.5 B kg ha^–1> 0 kg B ha^–1, respectively. Higher recoveries of PMC and PMN were noticed under 2 kg B ha^–1 in cauliflower, cowpea and okra, respectively, at all the crop growth stages over control. Barring urease, the activities of all other important soil enzymes (AS, DH, FDA, and PMA) were increased significantly up to the application of 2 kg B ha^–1. The positive impact of B-fertilization on these biological properties was observed at different growth stages of all three crops in the sequence which ultimately led to higher and sustainable crop production. A significant and positive relationship between these properties and crop yield greatly supported this observation. Multivariate analysis also confirmed the role of B-fertilization in the augmentation of the soil’s biological properties and yield enhancement. Overall, it was concluded that different soil physico-chemical and biological properties under the cauliflower-cowpea-okra cropping sequence can be invariably improved by the application of graded levels of B up to 2 kg B ha^–1 in an acid Inceptisol. Future research entails more advanced research between B with soil microbial pools, microbial populations and soil enzymes to explore the precise mechanism of their interaction in soil. Comprehension of the mechanisms underlying established functions of B may explicate the significance of B and, in the end, lead to an advanced perception of its biological function, which has vital pragmatic implications in agriculture. The continuity of the residual impact as well as the beneficial effect of B-fertilization in such cropping sequences is therefore an important subject for future empirical research to elucidate its sustainability.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

IB: conceptualization, methodology formulation and implementation, data analysis, project administration, resource, and review. AB and DS: analysis and graphical works, editing, and results validation. AKC: data curation, reviewing, and editing. AdK and SC: editing of original and revised versions of the manuscript. AS, ED, GG, and LO: data collection and processing and original draft preparation. SB and MDS: review of literature and results compilation. MS and BAG: review of literature and basic analysis. AmK and SHD: results compilation and draft preparation. BG and MH: graphs and maps preparation. YD, KS, and ST: review of literature, results compilation, and English language correction. MR: review and editing of the final draft of manuscript, validation of statistical analysis, and formatting the manuscript. All authors contributed to the article and approved the submitted version.