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Article

Organic Farming Favors phoD-Harboring Rhizospheric Bacterial Community and Alkaline Phosphatase Activity in Tropical Agroecosystem

by
Neha
1,
Yashpal Bhardwaj
1,2,
Bhaskar Reddy
1,3 and
Suresh Kumar Dubey
1,*
1
Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi 221005, India
2
Survey of Medicinal Plant Unit, Regional Ayurveda Research Institute, Itanagar 791111, India
3
Virus Research and Diagnostic Laboratory, Government Institute of Medical Sciences, Greater Noida 201310, India
*
Author to whom correspondence should be addressed.
Plants 2023, 12(5), 1068; https://doi.org/10.3390/plants12051068
Submission received: 4 January 2023 / Revised: 17 February 2023 / Accepted: 21 February 2023 / Published: 27 February 2023
(This article belongs to the Special Issue Plant-Microbes Interactions in the Context of Abiotic Stress)
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Abstract

:
The bacteria harboring phoD encodes alkaline phosphatase (ALP), a secretory enzyme that hydrolyzes organic phosphorous (P) to a usable form in the soil. The impact of farming practices and crop types on phoD bacterial abundance and diversity in tropical agroecosystems is largely unknown. In this research, the aim was to study the effect of farming practices (organic vs. conventional) and crop types on the phoD-harboring bacterial community. A high-throughput amplicon (phoD gene) sequencing method was employed for the assessment of bacterial diversity and qPCR for phoD gene abundance. Outcomes revealed that soils treated for organic farming have high observed OTUs, ALP activity, and phoD population than soils managed under conventional farming with the trend of maize > chickpea > mustard > soybean vegetated soils. The relative abundance of Rhizobiales exhibited dominance. Ensifer, Bradyrhizobium, Streptomyces, and Pseudomonas were observed as dominant genera in both farming practices. Overall, the study demonstrated that organic farming practice favors the ALP activity, phoD abundance, and OTU richness which varied across crop types with maize crops showing the highest OTUs followed by chickpea, mustard, and least in soybean cropping.

1. Introduction

Phosphorous is one of the most important macronutrients essential for the growth and development of plants. Regardless of its abundance in soils, the availability of phosphorous is very limited in the terrestrial environment. Generally, two forms of P are present in the soil, organic and inorganic forms; however, only inorganic orthophosphate (PO43−) ions in the soil solution are easily accessible for plant use [1]. A huge amount of P fertilizers is utilized in agriculture as mineral P fertilizers and organic fertilizers (compost/manure) to maintain crop productivity. Soon after its application in the soil, a portion of inorganic P is readily used by the plants and microbes, whereas the residual P is immobilized as an insoluble form in the soil. Microbes play a vital role in recycling phosphorous from a recalcitrant unavailable form of P by solubilization and mineralization of inorganic and organic forms of P, respectively [2]. Inorganic P in soil mainly comprises minerals, such as apatite, oxyapatite, and hydroxyapatite, which are poorly soluble and assimilable. The inorganic P is solubilized by soil microbes by excreting organic acids [3]. The organic P mineralization is through the enzymatic process by the P-hydrolyzing extracellular enzyme phosphatases [1,2] which comprise phosphomonoesterases, phosphodiesterases, phosphotriesterases, etc. The phosphomonoesterases are composed of alkaline and acid phosphatases, nucleotidases, and phytases. Activities of both alkaline and acid phosphatases in soil have been analyzed to assess the organic phosphorous mineralization to inorganic phosphorous form [4]. Alkaline phosphatases (ALP) primarily originate from soil microorganisms, especially bacteria, which are involved in the hydrolysis of organic phosphorous [4]. To date, three homologous ALP encoding gene families have been identified, namely, phoA, phoD, and phoX, as a component of Pho regulon [5]. According to metagenomics datasets, based on the sequence similarity, phoD is the most frequently available gene than phoA and phoX. phoD-harboring bacteria have been widely identified and ubiquitously distributed among terrestrial and aquatic ecosystems [6]. Hence, phoD is considered a good biomarker to bestow an understanding of P transformation in an agroecosystem. Some studies showed a negative correlation between the activity of the ALP enzyme and available phosphorous [7,8]. There are reports that bacteria enhance the production of ALP during scarcity of P by upregulating the expression of functional gene encoding phosphatase enzyme [4]. Moreover, some studies reported a positive correlation between alkaline phosphatase activity and phoD copy number [9,10,11]. However, more evaluation of the origin/source of phoD is required to amplify our knowledge to understand the relationship between the potential activity of alkaline phosphatase and available P in the soil [12].
Researchers have shown the shift in phoD-gene-containing bacterial population under different environmental sites and experimental conditions, including fertilizer management practices in agriculture [6,8,11], soil pH [12], amendment of organic matter [11,13,14], and extreme environments [15]. Tan et al. studied that in pasture soil phosphorous fertilizer incorporation enhanced the phoD community [5]. Xie et al. carried out a field experiment in the temperate monsoon climate of China to study the effect of fertilization treatment, crop rotation, and wheat varieties on functional communities of the rhizosphere associated with P cycling [16]. They found that crop rotation changed the community composition of bacteria having phoD genes in the wheat rhizosphere, whereas fertilization management had no effect. A negative correlation was seen between the phoD abundance and available P, P uptake, and wheat biomass. In another study, Fraser et al. reported that phoD copy number correlated positively with ALP activity in manure- and mineral-phosphorous-treated soils [9]. Results from the Illumina high-throughput approach revealed that the response of phoD bacteria to the P status of the soil is asynchronous, and nitrogen, carbon, and phosphorus soil stoichiometric ratios were the most dominant regulatory parameters for the phoD bacterial population in soils of Inner Mongolia [17]. Most of the studies regarding ALP activity and phoD-containing bacterial diversity to date have been carried out in the temperate and subtropical zones. In a recent study, Hegyi et al. reported that Actinobacteria, Acidobacteria, Cholroflexi, Firmicutes, and Proteobacteria are the superior phyla analyzed through next-generation sequencing of the 16S rRNA gene in the agricultural soil of Vietnam [18]. The study showed a positive correlation between soil phosphatase activities and soil organic C and also between acid phosphatase and total P. Similarly, a significant positive relationship was found between the abundance of the phoD gene and the diversity of the bacterial community of soil. There are still very limited studies with reference to the influence of various factors on phoD soil bacterial abundance and community and ALP activities, especially in an agricultural system with different fertilizer management practices.
However, as per the reviewed literature, information regarding the phoD-containing bacterial distribution, diversity, and community composition is still lacking, particularly in the Indian tropical agroecosystem. To procure a better understanding of the process involved through which bacteria participate in P turnover, there is a need for more studies focusing on the phoD bacterial population in the soil. In this study, we employed a high-throughput targeted amplicon sequencing to investigate the phoD-containing bacterial population in response to different farming practices vegetated with varied crops. In the present study, we hypothesized that the impact of farming practices (organic vs. conventional) and crop types would alter the abundance and composition of phoD-gene-harboring bacterial communities and soil alkaline phosphatase activity. The pivotal goal of this study was to (1) evaluate the influence of farming practices (organic vs. conventional) on ALP enzyme activity, phoD bacterial abundance, and diversity in soil under the influence of different crops and (2) assess the relationship between the ALP activity and phoD abundance and community.

2. Results

2.1. Soil Variables and Crop Biomass

The value of pH of the soil was significantly increased in soils treated for organic farming than the conventional (Figure 1a). Mineral-N content was highest in conventional farming (Figure 1b). A significant increase in available P was detected in conventional farming compared to organic treatments, and the effect of crops on the available P was also significant (Figure 1c; p < 0.001). The microbial biomass P (MBP) showed a significantly higher value in organic farming, and the effect of the crop on microbial biomass P was also significant (Figure 1d; p < 0.001). The root and shoot biomass were observed highest in organic farming practice, and the effect of crops and farming practice on the root and shoot biomass was also significant (Figure 1e,f; p < 0.001).

2.2. Soil Alkaline Phosphatase (ALP) and Abundance of phoD Gene

Variations in soil ALP activity and phoD gene copy number in rhizosphere soils of crops vegetated in both farming fields are depicted in Figure 2a,b. The ALP activity ranged from 2.45–3.59 µmol g−1soil h−1 in organic to 1.46–2.96 µmol g−1soil h−1 in conventional farming soils. Within the crops, maize showed the highest ALP activity and soybean the least (Figure 2a). MANOVA showed a significant (p < 0.001) effect of farming practices and crops on ALP enzyme activity. Tukey’s HSD test indicated significant variation in ALP activity between different crops in the same farming practice. qPCR analysis showed that, compared to conventional farming (4.5 × 106–1.1 × 107 copies g−1 dws), the phoD population was substantially higher among organic farming, which ranged from 6 × 106 to 1.3 × 107 copies g−1 dws (Figure 2b). The abundance of phoD gene copy was highest in soil samples of maize crops, followed by chickpea, mustard, and soybean soils. The MANOVA result showed a significant (p < 0.001) effect of farming practices and types of crops on the abundance of phoD gene copy. Tukey’s HSD test suggested phoD abundance to differ significantly between different crops.

2.3. phoD-Gene-Containing Bacterial Community and Relationship with Soil Parameters

Initially, a total of 674,076 raw reads were generated from phoD amplicon 2 × 300 pair-end sequencing, with an average of 84,260 reads per sample. The stringent poor-quality filtration resulted in a total of 254,895 high-quality reads subjected for downstream analyses. To compare phoD-gene-harboring bacterial community among different samples, rarefaction of sequences/reads was performed to obtain the OTUs assigned by an equal number of sequences. We rarified the sequences at minimum library size to 2629 reads for each sample. The phoD OTUs ranged between 346 and 672 with ~457 average OTUs per sample (Table 1). The observed OTUs composition of phoD gene bacteria was greater in soils treated for organic farming than the conventional one (Table 1). The highest OTUs were reported in soils of maize crop under organic farming, followed by chickpea-, mustard, and soybean-planted soils.
We estimated the Pearson correlation coefficient between the diversity indices, soil parameters, and phoD copy number (Table 2). The Pearson correlation showed a significant (p < 0.01) positive correlation between OTUs richness, Shannon and Simpson index with that of phoD abundance, and ALP activity of soil (Table 2).
The results of CCA (canonical correspondence analysis) showed that the structure of phoD bacterial community significantly correlated with ALP activity, MBP, and available P and to a lesser extent with pH and mineral-N (Figure 3). The phoD community structure in different farming practices in all four crops changed along the first axis. The soil pH and mineral-N showed a strong correlation with the CCA2 axis that governs 24.69% of the overall variance in the phoD community.

2.4. Relative Abundance of phoD Bacterial Community

All the phoD reads were affiliated with the phyla Proteobacteria and Actinobacteria. Proteobacteria was the dominant phylum accounting for 65.24–94.57% of all the sequences. These OTUs were classified into eight bacterial orders that include Rhizobiales, Streptomycetales, Pseudomonadales, Rhodobacteriales, Rhodospirallales, Burkholderiales, Pseudonocardiales, and Micromonosporales. The Rhizobiales were dominant among all samples and accounted for 37.28–78.27% (Figure 4a). The phoD-containing bacterial order varied among farming practice and crops, but no particular trend was observed. A total of 29 genera were identified, among which the dominant genera present were Bradyrhizobium (4.41–42.73%), Ensifer (1.04–49.65%), Streptomyces (3.29–37.08%), Mesorhizobium (3.36–29.12%), Sinorhizobium (2.28–23.55%), Pseudomonas (0.15–15.62%), and Skermanella (3.75–15.43%) (Figure 4b). The relative abundance of Bradyrhizobium was most predominant in organic farming practice under chickpea cropping. Moreover, Pseudomonas also showed higher relative abundance in organic treatment, whereas Streptomyces is enriched in soybean cropping with conventional farming soil.

3. Discussion

In the current study, organic farming practice stimulates TN, SOC, and TP [19]. Microbial biomass P (MBP) increases as a result of organic farming practice that stimulates the biological activity of soil in comparison to conventional farming. The organically amended soil is well established to increase organic matter in the fields [20]. Similar studies have shown an increase in organic matter content in organic farming and organic matter-rich soil to have long-term potential to sustain nutrient release [21,22]. The pH is increased in organic farming soil in comparison to the conventional counterpart. The lowering of pH in conventional farming soil maybe because of nitrification of NH4+ and thus H+ ion is produced which results in enhanced soil acidity. Chakraborty et al. showed a decrease in pH with increasing chemical fertilizer application [23]. In this study, the available P is significantly (p < 0.001) increased under the conventional farming practice. Similar results are also reported by Liu et al. [24]. Fraser et al. reported the same trend that indicated low available P in organic management [10]. This may be because fertilizer P administers orthophosphate usually amplifying labile inorganic P [25]. However, Sakurai et al. reported the opposite trend [13]. This discrepancy in available P maybe because of confounding features, including the quantity of phosphorous in fertilizers, crop P demand, and diverse agricultural practices.
Consistent with our hypothesis, the farming practices and crop types influenced the abundance and phoD gene bacterial communities. In the present investigation, organic farming stimulated the alkaline phosphatase activity of soil. Higher ALP activity was determined in organic farming than in the conventional one with significant differences (p < 0.001) (Figure 2a). It has been reported that organic matter application enhances the ALP activity in the soil [9,10,11,13]. The possible reason for the increased ALP activity in organic farming may be increased SOC that allows bacteria to proliferate due to an additional carbon source [6]. In addition, the amendment of cattle manure in soil results in a significant increase in alkaline phosphatase activity [26]. phoD gene copy number was significantly higher (p < 0.001) in soils of organic farming fields (Figure 2b). The earlier studies also reported similar results in the long-term manure-fertilized soil [8,9,10]. It is suggested that phoD abundance was highest in organic treatment with the highest alkaline phosphatase activity and SOC that increases organic matter suggesting higher nutrient content which accordingly increases bacterial abundance [27]. In organic farming practice, the organic fertilizer, low in available P content, and plenty of C-rich substrate possibly influenced the proliferation of various phoD-gene-containing bacterial population, hence increasing the phoD abundance and alkaline phosphatase activity [28]. In addition, we found a significant (p < 0.01) and strong positive correlation between OTUs, Shannon index, and Simpson index and alkaline phosphatase activity and phoD copy number (Table 2). The previous report indicated a significant positive correlation between phoD gene copy number and the bacterial community such as Shannon and Chao 1 diversity [18]. Zhu et al. showed a positive correlation between Chao 1 index and alkaline phosphatase activity, suggesting that phoD-gene-harboring bacteria may be highly activated while secreting ALP enzyme [17]. Similarly, a significant (p < 0.001) variation was observed among crop species for the activity of alkaline phosphatase and phoD abundance. The elevated ALP activity and the abundance of phoD bacteria exhibited a trend of maize > chickpea > mustard > soybean fields in soil samples (Figure 2a,b). This may be due to the influence of varied plant species on soil microbes that exhibit variability among plant physiological attributes including exudates of root [29] that possibly differ with crops and physiological attributes [30].
Recently, many studies have been reported demonstrating the shifts in phoD-containing bacterial population in soils with organic and inorganic fertilization [5,6,8,13,28]. As existing literature in the context of Indian agroecosystem suggests scarcity of knowledge about phoD encoding bacterial assemblages; however, agroecosystems situated in other countries have been analyzed for the same. Wei et al. studied that the P fertilization influences phosphorous mineralizing microbes in paddy soil, and all the sequences are classified into five classes: Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Cyanobacteria, and Actinobacteria [31]. The dominant genera of this study were Methylobacterium, Methylomonas, and Bradyrhizobium. Here, in this study, phoD taxonomic classification revealed that Proteobacteria and Actinobacteria were the two phyla among which Proteobacteria showed dominance in both farming practices. The investigations on phoD bacterial population identified similar genera [14,28]. However, Proteobacteria was the most dominant in organic farming samples. The previous study also detected Proteobacteria as the dominant phylum [6,8,28]. The dominance of phylum Proteobacteria may be because of the supplement of compost which contributed to an increased level of soil nutrients (e.g., SOC, TN, and TP) and stimulated the copiotrophic bacterial growth and resulted in a shift of phoD-harboring bacterial community. Tian et al. reported a higher abundance of Proteobacteria in swine manure added to soil with increased soil nutrients and promoted the proliferation of copiotrophic phyla, i.e., Proteobacteria [32]. We have also observed an increased level of Actinobacteria in conventional farming as compared to the organic amendment. Actinobacteria are more apparent to be an index for inorganic fertilization as compared to organic fertilization [33]. The genera Ensifer belonging to the phylum Proteobacteria was the most predominant genera and its relative abundance was highest in organic farming practice with soybean cropping. The previous studies on total and P-mineralizing bacterial communities in maize cropping detected similar genera Ensifer in soil fertilized with compost than chemical and nonfertilized soil [34].
The higher abundance of Ensifer sp. in soils managed with organic farming compared to the conventional suggests that organic fertilization perhaps enriched this genus in soil and accordingly strengthen their abundance in the rhizosphere [34]. The relative abundance of Bradyrhizobium, a free-living and nitrogen fixer that is present in all the samples, indicates that this genus perhaps plays a major role in linking nitrogen (N) and phosphorous (P) cycle [28]. Therefore, further study is imperative to acknowledge the role of Bradyrhizobium sp. in establishing the possible link between nitrogen cycling and phosphorous turnover under diverse conditions, which may contribute to accelerating ALP activity as well as phosphorous transport rates and also alter soil nitrogen pools [28]. In another study, Gitonga et al. [35] observed the effect of farm management (conventional and organic farming) system on Bradyrhizobia species abundance and diversity and reported proliferation of Bradyrhizobium in organic farming practice and hence enhanced Bradyrhizobial diversity. This may be because organic farming has been revealed to increase soil organic carbon which provides the required energy for microbes and thus increases their abundance and diversity as compared to conventional farming practice [36]. Here, in this study, we observed that organic farming stimulated the proliferation of Pseudomonas (relative abundance: 15.62%) in soils planted with chickpeas. Hu et al. also reported an increased relative abundance of Pseudomonas in the organic amendment [6]. It is reported that several species of Pseudomonas grow rapidly under straw as the only carbon source, on the basis of their plant lignin and hemicellulose degradation capacity [37]. Moreover, Pseudomonas sp. inhabits several P-mineralizing bacterial populations [38,39]. Another dominant genus identified in this study was Streptomyces. Hegyi et al. also reported Streptomyces as abundant, potential phosphate-solubilizing bacteria in soil and Streptomyces liacinus as a phoD gene encoding isolate [18]. It is established that apart from plant growth-promoting capabilities, Streptomyces species are generally associated with a phosphorous transformation, including phosphorous solubilization and mineralization [11,18]. Moreover, several Streptomyces species reported secreting alkaline phosphatase enzyme, namely, S. griseus, S. hiroshimensis, and S. hygroscopicus [40,41]. The organic farming practice showed the greater OTUs richness and Shannon diversity index compared to conventional farming (Table 1).
The observed OTUs were positively correlated with ALP activity and phoD abundance. These findings are in accordance with Sakurai et al. [13] showing that in organic matter amended soils phoD encoding bacterial assemblages shifted differently from those in conventional farming supplied with chemical fertilizers. Several earlier studies have also reported a change in phoD encoding bacterial assemblages in response to different fertilization management [11,12,42]. Our findings, as well as the previous reports, suggested that the change in phoD bacterial community by organic fertilizer might be caused by community composition turnover, i.e., existing OTUs replacement with the new OTUs. Moreover, organic farming showed higher alkaline phosphatase activity than conventional farming, and the variation in OTU composition of phoD may have enhanced enzymatic activity [42]. According to Watts et al. [43], the bacterial diversity in soil could be enhanced by the bacteria supplemented through the organic fertilizer.
Therefore, in the present study, we found that organic fertilizer incorporation strengthens the phoD-containing bacterial richness in comparison to conventional farming, suggesting a substantial effect of the introduced bacterial population from organic fertilizer on the alpha diversity of microbes. Our results revealed the effect of crops on the phoD bacterial population, but no particular trend was observed in test crops at the genera level. The OTU richness of phoD was highest in soils vegetated with maize compared to chickpea, mustard, and soybean field soils. Neal et al. have also reported that the effect of crop type on the phosphohydrolase genes was significant [44]. It is well-established fact that plants excrete a composite mixture of chemical compounds from the roots in the soil, which could possibly favor discrete microbial communities towards the rhizospheric zone [45,46,47]. Through enzymatic hydrolysis (phosphatase activity) of organic P and solubilization of mineral phosphate, these microbes can induce a supply of orthophosphate in soil [48].

4. Materials and Methods

4.1. Study Site and Experimental Design

The sampling sites were located at the agriculture field of Dagmagpur (Mirzapur district of Uttar Pradesh) (25°9′ N, 83°34′, 80 m above MSL), India. This zone has a seasonal tropical monsoonal climate with an average rainfall of 849.9 mm annually and the mean temperature (minimum to maximum) generally ranges between 8 °C and 10 °C in January and 38 °C and 42 °C in June. The soil of the study site was Alfisol with a silty sandy texture (32:64:4, sand:silt:clay). The agricultural farm was governed by the farmers, and the study site has a long (approximately 30 years) agricultural history. In this area, besides selected test crops for the present study, rice and wheat are the major crops. The soil total P was 152.17 µg g−1 in organic and 127.08 µg g−1 in conventional farming practice. The main soil physicochemical properties were given in [19].
Two agricultural fields having two different farming practices were selected: one field received compost (organic farming) and another field mineral fertilizer (conventional farming). The experimental plots were designed in randomized complete block design including three blocks (5 m × 4 m with 1 m gap) per site and a treatment combination of 4 crops × 2 farming practices. To avoid edge effects, the organic and conventional farming experimental plots were separated by a 100 m distance. In the organic farming plot, compost was added as an organic supplement. The compost applied is composed of crop residues and cow dung prepared by NADEP (Narayan Deorao Pandharipande) technique [49]. After maturation, the compost was dispersed manually at the rate of 15 tons ha−1 and plowed up to 15 cm depth to mix and homogenize before the cropping season of Rabi and Kharif. No other nutritional supplement was added apart from cow urine (1:50; urine:water dilution ratio), which is added twice, during the vegetative and flowering stages of the crops as a nitrogen source at the interval of 40 days. The cow urine used in the present study comprises 15 g N L−1. In conventional farming, as per the standard practice, NPK (chemical fertilizer) was applied at the rate of 120, 40, and 60 kg ha−1 in Rabi crops, and for the Kharif crops, the rate of NPK application was 20, 40, and 60 kg ha−1. This was executed twice annually, i.e., one-time application in both seasons as a basal dose. There were four crops selected in this study. Two each from Rabi (chickpea: Cicer arietinum L. var Pusa-256, and mustard: Brassica campestris var. T-151) and Kharif (maize: Zea mays var. Ganga II and soybean: Glycine max var. PS-1225). The experiments were conducted for two cropping seasons (2017–2019). No pesticides or fungicides were added, and weeds were removed manually.

4.2. Soil Sampling and Soil Physicochemical Analyses

Rhizosphere soil samples in triplicate (0–15 cm) from both (organic and conventional) farming fields were collected randomly at the mid-flowering stage of a crop growth cycle. Soil adhered to the rhizosphere zone was collected after tapping the root gently of each test crop in a plastic bag. Soil samples (in triplicate) were mixed and homogenized and sieved (2 mm mesh) to remove plant debris. The homogenized soil samples are divided into two parts: one part stored at −20 °C for downstream analyses of phoD-harboring bacterial community and qPCR experiments and the other kept (at 4 °C) for the analyses of ALP activity and soil properties. Soil physicochemical properties, alkaline phosphatase activity, and phoD gene abundance were measured for two consecutive years (2017–2019) in soil samples, and data were pooled as average data for two years and the phoD diversity analysis was performed only for second-year soil samples. All the basic physical and chemical properties of soil were analyzed following the standard protocols [50]. Total P was analyzed as per Allen et al. [51]. To measure soil available P, the method of Olsen et al. was followed [52]. Microbial biomass phosphorus (MBP) was measured following the standard protocol of Brookes et al. [53], and crop biomass was measured as per Neha et al. [19].

4.3. Assay of Soil Alkaline Phosphatase (ALP) Activity

Using p-nitrophenol phosphate (p-NPP) as substrate, ALP activity was measured [54]. The analysis was carried out by taking rhizosphere soil (1 g) with 1 mL modified universal buffer (pH 11), p-NPP, and incubating at 37 °C for 1 h. After 1 h, 0.5 M NaOH was mixed to terminate the reaction. The reaction mixture was filtered, and the p-nitrophenol (p-NP) was measured spectrophotometrically (at 420 nm). The activity was recorded as µmol of p-NP g−1 soil h−1.

4.4. Soil DNA Extraction and phoD Gene Quantification

Total genomic soil DNA was extracted from 0.5 g frozen soil by FastDNA Spin Kit. NanoDrop 2000 spectrophotometer was used to measure DNA concentration and quality. The phoD gene copy number (abundance) was quantified by qPCR (iCycler iQ5 thermocycler; Bio-Rad, Hercules, CA, USA). The extracted DNA was amplified for phoD using ALPS F-730 and ALPS R-1101 primers as described by Sakurai et al. [13]. The PCR reaction mixture (20 µL) contains 10 µL of PowerUpTM SYBR Green Master Mix, 0.5 µL of each primer concentration (10 µM), DNA template (2 µL), and water (nuclease-free) to make up the final volume to 20 µL. The PCR conditions for amplification of phoD gene were as follows: 3 min at 94 °C (initial denaturation), 40 cycles at 94 °C for 1 min (denaturation), at 61 °C for 45 s (annealing), and final extension at 72 °C for 45 s. Data were tested for PCR amplification efficiencies which were 114.5% and R2 = 0.963.

4.5. Illumina MiSeq High-Throughput Sequencing for phoD Gene Amplicons and Data Analysis

The Illumina Miseq 300 bp paired-end sequencing platform was used to assess the phoD-gene-containing bacterial population. The target gene (pho D) was amplified in rhizosphere soil DNA using ALPS-F730 and ALPS-R1101 primers [13]. The indexed paired-end library was prepared by adding Illumina Nextera XT compatible adapters to the forward and reverse primer sequences. The reaction mixture comprises template DNA (50 ng), KAPA Hifi HotStart Ready Mix (KAPA Biosystems, Wilmington, NC, USA), and modified primers (100 nM) ALPS-F730 and ALPS-R1101. PCR conditions were set as initial denaturation (at 94 °C for 3 min), followed by denaturation (30 cycles at 94 °C, 1 min), annealing (61 °C, 45 s), extension (72 °C, 30 s), and terminal extension (72 °C for 7 min). The amplicons were cleaned up and subjected to quantitate libraries using a quantitation assay of Qubit DNA high sensitivity (ThermoScientific, Grand Island, NE, USA). The quantity of the library was corroborated with the help of the D7500 DNA kit and Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Illumina MiSeq 2 × 300 bp platform (Illumina, San Diego, CA, USA) was used for sequencing as per the manufacturer’s protocol. The generated raw targeted phoD gene amplicon sequences have been deposited to the NCBI sequence read archive (SRA) database under the BioProject accession number PRJNA797670.

4.6. Sequence Analysis

The sequences were analyzed as per Bhardwaj et al. [55] with slight modifications for the phoD gene. The FastQC was used to overview raw forward (R1) and reverse (R2) reads for basic quality control [56]. Raw sequences were quality filtered and trimmed using Trimmomatic V0.35 with criteria such as (i) adaptor sequences removal and (ii) eliminating unclear reads (reads having undefined nucleotides “N” > 5%) and low-quality sequences. The quality-passed (forward and reverse) joining of paired reads was executed by PEAR (Paired-End reAd mergeR), and the remaining single reads were discarded [57]. The joined reads with a quality score of < 30 and sequence size with less than 250 bp and more than 380 bp sequences were filtered out to obtain high-quality sequences (HQS). An appropriate pipeline Quantitative Insights Into Microbial Ecology (QIIME), version 1.9.0, was used for the analysis of high-quality reads [58] for bacterial diversity estimation. Initially, the quality-passed reads were screened against the funGene database regarding phoD sequences using the HMMER model with default settings. This allowed us to obtain only the phoD-gene-specific amplicon reads and filtered the unwanted reads such as 16S rRNA contamination and chimeric reads. Then, the phoD-gene-featured sequences were subjected to operational taxonomic units (OTUs) determination at 75% similarity clustering employing pick_otus.py script [59]. The OTUs were rarefied to the least library size (2629 reads) to find out the taxonomic diversity alpha indices, such as observed OTUs, Good’s coverage, Chao1, Simpson, Shannon, after discarding OTUs with <10 reads count. Furthermore, the taxonomic classification of reads was obtained using a Kaiju metagenome classifier with the k-mer setting of 31, integrated into MGX software.

4.7. Statistical Analyses

To study the effect of farming practices and crops on the physicochemical and microbiological properties of soil, MANOVA (multivariate analysis of variance) was conducted with Tukey’s post hoc test (p < 0.05). Pearson correlation test was used to study the relationship between diversity indices and soil parameters (IBM SPSS Statistics 20). Canonical correlation analysis (CCA) was employed to evaluate the effect of farming practices and crops to explore the correlation between soil variables and phoD-gene-harboring bacterial community using PAST v 3.20.

5. Conclusions

The farming practices and crop types change the composition of phoD-containing bacterial communities. The present study suggests that alkaline phosphatase activity, phoD abundance, and OTUs richness were increased in response to organic farming practice. Rhizobiales showed dominance at the order level. Bradyrhizobium, Ensifer, Streptomyces, and Pseudomonas were detected as the dominant members. This study will provide a better understanding of the significance of the alkaline phosphatase enzyme and its role in P mineralization to improve P management strategy to maintain sustainable agriculture. Future research is needed to address the influence of various types and quantities of fertilizers under different cropping systems on phoD communities for stimulating P availability in agricultural soils.

Author Contributions

N. conducted wet labs, data analysis, and wrote the manuscript; Y.B. performed statistical and sequence analysis; B.R. carried out NGS data analysis; S.K.D. did conceptualization and reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

The University Grant Commission, New Delhi-Centre for Advanced Study (UGC-CAS), Department of Botany, Banaras Hindu University, Varanasi, India, has provided fund (File No-R/Dev/IX-Sch.(SRF-JRF-CAS-Botany)/51186 in the form of JRF and SRF to Neha for this study.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The phoD gene amplicon sequences have been deposited to the NCBI sequence read archive (SRA) database under the BioProject accession number PRJNA797670.

Acknowledgments

We are thankful to the coordinator of the Centre of Advanced Study, Department of Botany, Banaras Hindu University, Varanasi, for desired facilities.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Plants 12 01068 g001 550
Figure 1. pH value (a), content of mineral-N (b), available P (c), MBP (d), shoot biomass (e), and root biomass (f) in organic and conventional farming. Different lowercases indicate significant differences (Tukey’s post hoc test) between crops in the same farming practice (p < 0.05). The crops (C) and farming practice (FP) specify the results of MANOVA indicating p-value at different significance levels, *** p < 0.001, NS: not significant). The values are two-year average mean ± SD. MBP, microbial biomass P; ALP, alkaline phosphatase.
Figure 1. pH value (a), content of mineral-N (b), available P (c), MBP (d), shoot biomass (e), and root biomass (f) in organic and conventional farming. Different lowercases indicate significant differences (Tukey’s post hoc test) between crops in the same farming practice (p < 0.05). The crops (C) and farming practice (FP) specify the results of MANOVA indicating p-value at different significance levels, *** p < 0.001, NS: not significant). The values are two-year average mean ± SD. MBP, microbial biomass P; ALP, alkaline phosphatase.
Plants 12 01068 g001
Plants 12 01068 g002 550
Figure 2. ALP activity (a) and phoD gene copy number (b) in organic and conventional farming. Different lower cases indicate significant differences (Tukey’s post hoc test) between crops in the same farming practice (p < 0.05). The crops (C) and farming practice (FP) specify the results of MANOVA indicating p-value at different significance levels (* p < 0.05, *** p < 0.001). The values are two-year average mean ± SD. MBP, microbial biomass P; ALP, alkaline phosphatase.
Figure 2. ALP activity (a) and phoD gene copy number (b) in organic and conventional farming. Different lower cases indicate significant differences (Tukey’s post hoc test) between crops in the same farming practice (p < 0.05). The crops (C) and farming practice (FP) specify the results of MANOVA indicating p-value at different significance levels (* p < 0.05, *** p < 0.001). The values are two-year average mean ± SD. MBP, microbial biomass P; ALP, alkaline phosphatase.
Plants 12 01068 g002
Plants 12 01068 g003 550
Figure 3. Ordination plot of CCA (Canonical Correspondence Analysis) to exhibit the correlation between soil variables and phoD encoding community structure. CO, chickpea organic farming; CI, chickpea conventional farming; MO, mustard organic farming; MI, mustard conventional farming; SO, soybean organic farming; SI, soybean conventional farming; ZO, maize organic farming; ZI, maize conventional farming; MBP, microbial biomass P.
Figure 3. Ordination plot of CCA (Canonical Correspondence Analysis) to exhibit the correlation between soil variables and phoD encoding community structure. CO, chickpea organic farming; CI, chickpea conventional farming; MO, mustard organic farming; MI, mustard conventional farming; SO, soybean organic farming; SI, soybean conventional farming; ZO, maize organic farming; ZI, maize conventional farming; MBP, microbial biomass P.
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Plants 12 01068 g004 550
Figure 4. (a) Relative abundance of phoD-harboring bacterial communities under organic and conventional farming practices and different crops at the order level. (b) Relative abundance of phoD-harboring bacterial communities under organic and conventional farming practices and different crops at the genera level. CO, chickpea organic farming; CI, chickpea conventional farming; MO, mustard organic farming; MI, mustard conventional farming; SO, soybean organic farming; SI, soybean conventional farming; ZO, maize organic farming; ZI, maize conventional farming.
Figure 4. (a) Relative abundance of phoD-harboring bacterial communities under organic and conventional farming practices and different crops at the order level. (b) Relative abundance of phoD-harboring bacterial communities under organic and conventional farming practices and different crops at the genera level. CO, chickpea organic farming; CI, chickpea conventional farming; MO, mustard organic farming; MI, mustard conventional farming; SO, soybean organic farming; SI, soybean conventional farming; ZO, maize organic farming; ZI, maize conventional farming.
Plants 12 01068 g004
Table
Table 1. Diversity indices (rarefied at minimum library size, i.e., 2629 reads).
Table 1. Diversity indices (rarefied at minimum library size, i.e., 2629 reads).
Crop TypeFarming PracticeObserved OTUsChao 1ShannonSimpsonGood’s Coverage
ChickpeaOrganic4927687.71.00.9
Conventional4778057.01.00.9
MustardOrganic3966126.41.00.9
Conventional3838256.10.90.9
SoybeanOrganic3765986.61.00.9
Conventional3465406.10.90.9
MaizeOrganic67211218.11.00.9
Conventional5157367.61.00.9
Table
Table 2. Pearson correlation coefficient between diversity indices and soil parameters in different crops and farming practices (* p < 0.05, ** p < 0.01). ALP, alkaline phosphatase; MBP, microbial biomass P.
Table 2. Pearson correlation coefficient between diversity indices and soil parameters in different crops and farming practices (* p < 0.05, ** p < 0.01). ALP, alkaline phosphatase; MBP, microbial biomass P.
pHAvailable PALP
Activity		MBPMineral NphoD Gene Copy NumberRoot
Biomass		Shoot
Biomass
Observed OTUs0.23−0.460.840 **0.790 *0.220.885 **0.90 **0.88 **
Chao10.08−0.290.590.640.230.680.861 **0.69
Shannon Index0.23−0.530.906 **0.815 *0.260.935 **0.78 *0.81 *
Simpson Index0.27−0.580.867 **0.795 *0.210.879 **0.670.63
Good’s coverage−0.150.34−0.64−0.67−0.18−0.725 *− 0.894 **−0.747 *
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Neha; Bhardwaj, Y.; Reddy, B.; Dubey, S.K. Organic Farming Favors phoD-Harboring Rhizospheric Bacterial Community and Alkaline Phosphatase Activity in Tropical Agroecosystem. Plants 2023, 12, 1068. https://doi.org/10.3390/plants12051068

AMA Style

Neha, Bhardwaj Y, Reddy B, Dubey SK. Organic Farming Favors phoD-Harboring Rhizospheric Bacterial Community and Alkaline Phosphatase Activity in Tropical Agroecosystem. Plants. 2023; 12(5):1068. https://doi.org/10.3390/plants12051068

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Neha, Yashpal Bhardwaj, Bhaskar Reddy, and Suresh Kumar Dubey. 2023. "Organic Farming Favors phoD-Harboring Rhizospheric Bacterial Community and Alkaline Phosphatase Activity in Tropical Agroecosystem" Plants 12, no. 5: 1068. https://doi.org/10.3390/plants12051068

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