Diversity and structure of bacterial communities in saline and non-saline rice fields in Cilacap Regency, Indonesia

Reginawanti Hindersah; Etty Pratiwi; Yuli Siti Fatma; Tualar Simarmata

doi:10.1515/opag-2022-0225

Artikel Open Access

Diversity and structure of bacterial communities in saline and non-saline rice fields in Cilacap Regency, Indonesia

Reginawanti Hindersah , Etty Pratiwi , Yuli Siti Fatma und Tualar Simarmata

Veröffentlicht/Copyright: 28. September 2023

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Abstract

Increased concerns for sustainable agriculture have led to increased use of beneficial rhizobacteria as biofertilizers. Soil bacteria play a significant role in the nutrient cycle of soil, but their presence can be affected by abiotic stress, such as salinity. This study aimed to compare the chemical characteristics of slightly saline and non-saline rice soil and examine the bacterial community structure in both rhizosphere and bulk soil. We utilized 16SrRNA gene sequencing and performed arithmetic means clustering, a type of hierarchical clustering, on the samples collected from the rice fields of Cimrutu and Rawaapu Village in Cilacap Regency, Indonesia. Although the nutrient content was similar in both soils, there was a noticeable difference in their electrical conductivity (EC) despite the two locations being less than 4 km apart. The EC value in the Cimrutu soil suggests that it is non-saline, while the Rawaapu soil exhibits a low salinity level. The study found that Proteobacteria was the most prevalent phylum in saline rhizospheric soil. In contrast, Firmicutes was the most abundant group in saline bulk soil and non-saline rhizospheric and bulk soil. Additionally, Halothiobacillus, Thioalkalispira-Slvurivermis, and Acidothermus genera dominated the saline rhizospheric soil, suggesting that halotolerant microbes play a significant role as plant growth-promoting rhizobacteria in saline soil. The study provides valuable insights into cultured or uncultured bacterial populations and structure in saline and non-saline soil to develop future strategies related to salinity by introducing beneficial microbes.

Keywords: bacterial diversity; bulk soil; rhizosphere; soil chemical characteristics.

1 Introduction

The richness of beneficial microbes in agricultural soil plays a crucial role in determining food crop productivity. The bacterial communities in soil dictate various pathways of the nutrient cycle, including nitrogen (N), phosphorous (P), potassium (K), and even micronutrients such as iron (Fe), zinc (Zn), and manganese (Mn). Moreover, certain soil bacteria play a significant role in providing phytohormones. The rhizosphere is a dynamic area around the plant roots which is the most active area of soil as it is rich in nutrients released by roots. Moreover, the community structure of microbes in the rhizosphere is more varied resulting in a higher microbial population than in bulk soil.

The composition of bacteria in soil is influenced by biotic and abiotic factors. Bacterial composition depends mostly on soil salinity which reduces the water availability rather then temperature and pH [1]. Some lowland rice farms in tropical areas located in coastal and tidal-influenced regions are the most affected by increased salinity. Rice farming in the coastal areas of the Cilacap district of Central Java, Indonesia, often experiences floods due to climate change. Moreover, the major challenge of growing rice in the tidal area is low productivity due to salinity; during the rainy season, the salinity will reduce and vice versa in the dry season. Due to inundation, the salinity of soil in Cilacap increased from 1.47 to 7.36 dS m⁻¹ [2].

Farmers grow rice varieties that are not tolerant enough to submerge conditions. Frequent floods over a longer period cause abiotic stress which adversely affects the growth of rice plants. Rice fields in the coastal area of Cilacap Regency had low rice productivity of 3.78–4.97 t ha⁻¹ [3]. Farmers rely on inorganic fertilizers to promote plant productivity, but integrated plant nutrition by using chemical, organic, and biofertilizer is recommended for maintaining soil health and saving the environment. The biofertilizer formulation required plant growth-promoting rhizobacteria to supply nutrition, mainly nitrogen and phosphor, and stimulate plant growth. In general, the bacteria for biofertilizer formulation are isolated from plant rhizosphere.

The diversity of bacterial communities in bulk soil is usually lower than that in the rhizosphere due to the richest nutrients from root exudates [4,5]. Based on the 16sRNA gene, the abundance and diversity of Bacillus, Pseudomonas, Staphylococcus, nitrogen-fixing bacteria, and denitrifying bacteria are reported in the rice rhizosphere [6,7]. The rhizobacteria Bacillus sp., Bulkholderia sp. Pseudomonas sp., Streptococcus sp., and Staphylococcus sp. in rice produce indole acetic acid [8,9], while Pseudomonas aeruginosa and a different strain of Bacillus subtilis provide available P for plant growth [10]. Moreover, indigenous Bacillus spp. control Rice blast (Pyricularia oryzae) diseases and increase rice yield [11]. For decades, the role of rhizospheric microbes to withstand abiotic stress conditions is reported [12]. Despite the abundance of data about the bacterial community in bulk soil in the rice field, the studies of bacterial groups in the rice rhizosphere grown in tropical slightly saline soil by 16SrRNA gene sequencing are limited.

The composition and community structure of bacteria in soil largely depend on organic matter and salinity. The effect of organic matter on the abundance of bacteria in soil has been reported for heterotrophic microbes [13,14]. High level of dissolved inorganic salts in the soil can increase the osmotic pressure inside the microbial cells and induce a rapid flow of cell water out of the cell along the osmotic gradient, resulting in decreased turgor and cytoplasmic dehydration [15]. Certain soil microbes accumulate the osmolytes as a mechanism to withstand the high salinity environment; the osmo-protectant produced by halotolerant microbes includes glycine, betaine, carnitine, and proline [16,17].

According to a meta-analysis of global studies, salinity, rather than pH, temperature, or other physicochemical environmental factors, is the most important environmental determinant of microbial community composition [1]. The reduced microbial diversity and richness due to saline conditions are reported elsewhere [18,19]. The objectives of this research were to compare the chemical characteristics of saline and non-saline rice soil, to study the biodiversity in saline and non-saline rice soil in Cilacap, Central Java, and to identify the bacterial community structure in the rice rhizosphere and bulk soil of both soils.

2 Materials and methods

2.1 Study area

The soil samples were collected from a rice field in Rawaapu and Cimrutu Village in Patimuan District, Cilacap Regency, Central Java, Indonesia (Figure 1). Patimuan District is located in the tropic with an equatorial climate. According to the Indonesian Central Bureau of Statistics [20], the average annual temperature in Patimuan was 32–36°C, the average relative humidity was 55–70%, and the rainfall distribution of 21–50 mm per month. Most of this area is lowland with an average slope of 4% and is located at an average altitude of 25–100 m above sea level; the study area has slightly wavy to hilly topography. The Patimuan area has a high clay Vertisol configuration; the soil expands during the rainy season and cracks in the dry season.

Figure 1

Location map of study sites in Cimrutu (A soil) and Rawaapu (B soil) of Cilacap Regency, Central Java, Indonesia. Source: Google map.

2.2 Site description and soil sampling

Soil samples were collected from two rice fields: non-saline (A) and saline (B) areas at Cimrutu and Rawaapu Village, respectively (Table 1). Both rice fields were grown with different rice varieties. The soil of Cimrutu was non-saline while that of Rawaapu was slightly saline.

Table 1

Locations of sampling sites in Patimuan District, Cilacap Regency, Central Java Province

Location code	Geographical coordinates	Descriptions
A	−7.630251, 108.787954	Cimrutu village Rice variety: Mendawak Non-saline soil (salinity: 0.21 dS m⁻¹)
B	−7.653744, 108.805172	Rawaapu village Rice variety: Ciherang Saline soil (salinity: 2.30 dS m⁻¹)

The rhizosphere and bulk soil samples were collected by composite method from five sampling points of each location. Rhizospheric soil samples were collected from the remaining soil attached to the roots of rice plants using a clean brush. Soil samples taken from the five plant roots in each location were mixed well, put in sealed plastic bags, and stored in a cold box. Meanwhile, bulk soil samples were taken from the 20 cm-deep top soil around the roots of five rice plants in each location. Soil samples were put in a plastic bag and stored in a cold box. Double sampling was performed for either rhizosphere or bulk soil samples of both locations. Composite soil samples were transported in the cold box to the laboratory for analysis of physicochemical parameters and 5 g of each sample was stored at −20°C for metagenomic analysis.

2.3 Physico-chemical soil analysis

Already established procedures of soil analysis [21,22] were used to evaluate the physical and chemical properties of the soil. The pH of the soil was measured from the soil suspended in deionizer water of 1:2.5 soil–water ratio by using a glass electrode. The Walkley and Black wet oxidation techniques were used to detect organic carbon. The total N was calculated by using the Kjeldahl method. The total P₂O₅ and K₂O contents were measured after soil extraction with using 25% HCl. Exchangeable bases such as K⁺, Ca²⁺, Na⁺, and Mg²⁺ were measured of soil extraction using 1 M NH₄OAc, and their atomic absorption spectra were then used to measure the cationic concentration. The soil texture was determined by using the pipet approach [22]. The cation exchange capacity (CEC) and base saturation (BS) of soil were determined by calculation. The electrical conductivity (EC) of soil was measured by a conductivity meter on the soil–water suspension (1:5).

2.4 Molecular analysis of microbial community

Total soil DNA was extracted from a 0.25 g soil sample using a MoBio PowerSoil DNA Isolation Kit (Mobio Laboratory, Carlsbad, CA, USA) following the manufacturer’s instructions. DNA concentration and purity were monitored on 1% (w/v) agarose gels. According to the concentration, DNA was diluted to 1 ng/μL using sterile water. Bacterial DNA was amplified from each soil sample for Illumina sequencing analysis. The V4 region of the bacterial 16S rRNA gene was amplified using the forward primer 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and the reverse primer 806R (5′-GGACTACHVGGGTWTCTAAT-3′) [23].

Polymerase chain reactions (PCRs) were carried out using Phusion® High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA). The PCR amplification program consisted of an initial heating to 98°C for 1 min, 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and extension at 72°C for 60 s, followed by a 5 min extension at 72°C. The PCR products were mixed with the same volume of 1× loading buffer (containing SYBR green) and were detected through electrophoresis on 2% agarose gel. A Qiagen Gel Extraction Kit (Qiagen, Hilden, DE) was used to purify PCR amplification products. Sequencing libraries were generated with NEBNext^® UltraTM DNA Library Prep Kit for Illumina and quantified via Qubit and qPCR. The libraries were sequenced on an Illumina platform (Illumina, Inc., San Diego, CA, US), generating 250 bp paired-end reads, by the Novogene Bioinformatics Technology Company (Beijing, China).

2.5 Sequencing data processing

Paired-end reads were assigned to samples based on their unique barcodes and truncated by cutting off the barcode and primer sequences. The 250 bp paired-end reads were merged using Fast Length Adjustment of short reads of FLASH version 1.2.7 [24]. Quality filtering on the raw tags was performed under specific filtering conditions to obtain high-quality clean tags [25], according to the QIIME (version 1.7.0) quality-controlled process [26]. The tags were compared with the reference database (GOLD database) using the UCHIME algorithm to detect chimera sequences [27]. The chimera sequences were removed [28]. Subsequently, the effective tags were finally obtained.

Sequence analysis was performed by UPARSE software (Uparse version 7.0.1001) using all the effective tags [29]. Sequences with ≥97% similarity were assigned to the same operational taxonomic units (OTUs). The representative sequence for each OTU was screened for further annotation. For each representative sequence, Mothur software analysis was performed against the SSUrRNA database of SILVA Database available at http://www.arb-silva.de/ [30] for species annotation at each taxonomic rank (Threshold:0.8–1), including kingdom, phylum, class, order, family, genus, and species [31]. In order to obtain the phylogenetic relationship of all OTU representative sequences, MUSCLE (version 3.8.31) was used to compare multiple sequences rapidly [32]. The OTU abundance information was normalized using a standard sequence number corresponding to the sample with the least sequences. Subsequent analyses of alpha diversity and beta diversity were all performed based on these output normalized data.

The Alpha diversity (Observed-species, Chao1, Shannon, Simpson, ACE, and Good-coverage) was calculated with QIIME (version 1.7.0) and displayed with R software (version 2.15.3). Beta diversity was calculated by QIIME software (version 1.7.0). Unweighted pair-group method with arithmetic means (UPGMA) clustering was performed as a type of hierarchical clustering method to interpret the distance matrix using average linkage and was conducted by QIIME software (Version 1.7.0).

3 Results

3.1 Soil properties

The texture of location A and B soil was clay loam and clay, respectively (Table 2), which agrees with vertisol texture in general. The chemical properties of the soil samples from locations A and B were slightly different (Table 2). The pH of soil sample A was highly acidic, while the pH of soil sample B was approximately neutral. At both sites, organic-C and total-N were very high, but the C/N ratio was low, indicating an advanced decomposition. The available-P content in locations A and B was very low, only 1.44 and 2.37 mg kg⁻¹, respectively, relating to a high level of P fixation, and so P became unavailable. Soil salinity at location A was low with an EC of only 0.21 dS m⁻¹, indicating that it is non-saline soil. Meanwhile, the soil at location B has low salinity with an EC of 2.30 dS m⁻¹.

Table 2

Texture and chemical properties of soil samples taken from Cimrutu and Rawaapu Village

Properties	Location A (Cimrutu)	Criteria¹	Location B (Rawaapu)	Criteria¹
Texture
Sand (%)	3.97	Clay loam	6.57	Clay
Silt (%)	36.08		38.32
Clay (%)	59.96		55.12
pH (in H₂O)	4.99	Acid	6.65	Neutral
C-organic (%)	5.46	Very high	7.00	Very high
Total N (%)	0.99	Very high	0.99	Very high
C/N ratio	5.45	Low	7.07	Low
P (mg kg⁻¹)	1.44	Very low	2.37	Very low
K (cmol kg⁻¹)	2.34	Very high	2.03	Very high
Ca_ex (cmol kg⁻¹)	12.67	High	26.59	Very high
Mg_ex (cmol kg⁻¹)	10.18	High	10.34	High
Na_ex (cmol kg⁻¹)	2.54	High	9.11	High
CEC³ (cmol kg⁻¹)	29.44	High	41.17	Very high
Al_ex (cmol kg⁻¹)	0.87	Low	0.77	Low
H_ex (cmol kg⁻¹)	0.30	Low	0.25	Low
BS⁴ (%)	95.89	Very high	97.93	Very high
EC⁵ (dS m⁻¹)	0.21	Non-saline²	2.30	Low salinity²

¹[33]; ² [34]; ³CEC – cation exchange capacity; ⁴BS – base saturation; ⁵EC – electrical conductivity.

3.2 Bacterial diversity and composition

A total of 429,729 high-quality sequences were obtained after quality filtering. The high-quality sequences in all samples ranged between 96,488 and 118,592 sequences with an average length of 420 bp. Table 3 shows the alpha diversity of the bacterial community in the bulk soil and rhizospheric soil of rice plants. Good coverage (Table 3) and rarefaction curves (Figure 2) indicated that the DNA libraries were sufficient to estimate most bacterial diversity in the samples. The results showed that non-saline soil (AR and AB) had more diverse bacteria than low-saline soil (BR and BB) according to observed species, Shannon and Simpson indices.

Table 3

Alpha diversity of the bulk soil and rhizospheric soil of rice plants grown in flooding areas

Sample name	Observed species	Shannon	Simpson	Chao1	ACE	Good’s coverage	PD whole tree
AR	2,383 ± 102	7.81 ± 0.63	0.96 ± 0.03	2,541 ± 85	2,570 ± 77	0.996	162.46 ± 6.24
AB	1,902 ± 193	8.01 ± 0.47	0.99 ± 0.00	1,931 ± 183	1,968 ± 197	0.998	186.05 ± 1.87
BR	1,842 ± 82	6.06 ± 1.29	0.85 ± 0.13	2,025 ± 78	2,051 ± 76	0.996	133.59 ± 6.60
BB	1,441 ± 240	3.85 ± 1.12	0.64 ± 0.14	1,635 ± 199	1,682 ± 204	0.996	115.53 ± 15.76

Data were calculated at 3% genetic distance level with standard deviation based on the same number of sequences from each replicate in Mothur.

Values are the mean values of two replicates ± SE. AR – non-saline rhizospheric soil; AB – non-saline bulk soil; BR – saline rhizospheric soil; BB – saline bulk soil.

Figure 2

Rarefaction curves based on the V4 region of the 16S rRNA gene obtained from bulk soil and rhizospheric rice soil samples. Error bars represent the standard error of two replicates. AR – non-saline rhizospheric soil; AB – non-saline bulk soil; BR – saline rhizospheric soil; BB – saline bulk soil.

The Venn diagram shows that a total of 4,075 OTUs were successfully detected across all samples, in which 1,305 OTUs were common to all samples. The shared OTUs mainly belonged to Firmicutes (266 OTUs), Proteobacteria (258 OTUs), Chloroflexi (135 OTUs), and Actinobacteria (98 OTUs) at the phylum level (Table 4). The AB sample had more specific OTUs (688 OTUs) in comparison with other samples (Figure 3).

Table 4

Top ten bacterial phyla shared among the samples

Bacterial phylum	OTU number
Firmicutes	266
Proteobacteria	258
Chloroflexi	135
Actinobacteria	98
Patescibacteria	66
Bacteroidetes	58
Myxomycota	49
Acidobacteria	41
Cyanobacteria	15
Others	302

Figure 3

Venn diagram of bacterial OTUs shared among different samples. AR – non-saline rhizospheric soil; AB – non-saline bulk soil; BR – saline rhizospheric soil; BB – saline bulk soil.

The diversity of bacterial communities in different samples was revealed by Illumina-based high-throughput sequencing at different taxonomic levels. The top ten bacterial phyla with each relative abundance >1% are presented in Figure 4. Proteobacteria was the most abundant phylum in BR (54.07%), followed by Actinobacteria (14.38%) and Firmicutes (13.68%). On the other hand, Firmicutes became the most dominant phylum in BB, AR, and AB, accounting for 48.53, 32.73, and 29.39%, respectively. Proteobacteria (BB: 37.91%, AR: 27.93%, and AB: 20.43%) and Actinobacteria (BB: 4.63%, AR: 9.72%, and AB: 16.45%) were the second and third most abundant phyla in these three samples. We observed Acidobacteriota (0.9%) in BB, which were less abundant compared to other samples (BR: 2.46%, AR: 6.97%, and AB: 3.95%). In addition, Halobacterota was observed as less dominant in BB (0.0075%) than in other samples (BR: 0.12%, AR: 0.37%, and AB: 1.51%).

Figure 4

The relative abundances of bacteria at the phylum level in different samples. AR – non-saline rhizospheric soil; AB – non-saline bulk soil; BR – saline rhizospheric soil; BB – saline bulk soil.

Figure 5 shows the relative abundance of bacterial order in each sample. At the order level of taxonomy, Bacillales had the most abundance in AR (19.67%) and BB (38.69%), followed by Burkholderiales as the second most prevalent group in AR (10.92%) and BB (31.96%). Meanwhile, Burkholderiales was the most abundant group in AB (8.29%) and BR (35.74%). Bacillales was the second most dominant group in AB (7.20%). In contrast, Frankiales was observed as the second most common order in BR, accounting for 7.74%.

Figure 5

The relative abundances of bacteria at the order level in different samples. AR – non-saline rhizospheric soil; AB – non-saline bulk soil; BR – saline rhizospheric soil; BB – saline bulk soil.

The most abundant 35 bacterial genera were clustered as shown in Figure 6. Bifidobacterium (3.12%), Clostridioides (1.15%), Paenibacillus (1.03%), WPS-2 (1.86%), Candidatus_Levybacteria (1.84%), Streptomyces (0.76%), Enterobacter (0.86%), and Clostridium_sensu_stricto_1 (2.53%) were predominantly distributed in the AB. Meanwhile, AR had more species belong to Sideroxydans (2.51%), RCP2-54 (1.30%), Lysinibacillus (12.70%), Thiobacillus (0.99%), Magnetospirillaceae (2.85%), TM7a (1.11%), Streptococcus (1.13%), Subgroup_2 (2.25%), and Subgroup_13 (0.84%). Staphylococcus (9.11%), Bacillus (37.19%), and Veillonella (8.26%) were mainly distributed in the BB, while Thioalkalispira-Sulfurivermis (7.58%), Halothiobacillus (2.27%), and Acidothermus (7.03%) were the most dominant group in the BR.

Figure 6

Heatmap showing the relative abundance of the most abundant bacterial genera in different samples. AR – non-saline rhizospheric soil; AB – non-saline bulk soil; BR – saline rhizospheric soil; BB – saline bulk soil.

The results showed two distinct clusters at the phylum level in the UPGMA tree, i.e., cluster 1 (AR and AB, non-saline soil) and cluster 2 (BR and BB, slightly-saline soil), as shown in Figure 7. This finding revealed that the bacterial community in AR was more similar to those of the AB. In addition, BB and BR had more similar bacterial diversity and composition. It is observed that the clustering was based on soil salinity (non-saline and saline soil) rather than soil type (rhizospheric and bulk soil).

Figure 7

UPGMA clustering tree of the bacterial community structure based on Weighted UniFrac Distance at the phylum level. AR – non-saline rhizospheric soil; AB – non-saline bulk soil; BR – saline rhizospheric soil; BB – saline bulk soil.

4 Discussion

The impact of salinity on bacterial diversity in rice field can be complex and depends on several factors, including the severity and duration of salinity, the specific bacterial taxa present, and soil environmental conditions. This complexity was also observed in a study conducted in Rawaapu and Cimrutu Village, Cilacap Regency, where the impact of salinity on the bacterial community in rice fields was investigated.

In general, the nutrient composition of the two soils is quite similar. Both soils exhibit a significantly high level of organic carbon, total nitrogen, and potassium but a markedly low level of phosphorus content. However, there was a noticeable difference in the EC of the soils in Cimrutu and Rawaapu, despite the locations being less than 4 km apart. The EC in Cimrutu soil is inadequate, indicating that the soil is non-saline, whereas the Rawaapu soil has a low level of salinity according to the salinity classification proposed by food and agriculture organization (FAO) [34]. The cause of the difference in salinity between Cimrutu and Rawaapu is unclear. There are several possible causes for the difference in salinity between the two adjacent locations, including differences in water source, irrigation practices, and drainage system.

The non-saline soil exhibited an acidic reaction, whereas the low-salinity soil had a neutral pH. Furthermore, the sodium content in the Rawaapu soil was higher than that in the non-saline Cimrutu soil, which is consistent with the previous findings [35], which reported an increase in soil pH and exchangeable Na in saline soils. In addition, the neutral pH in the Rawaapu soil may be attributed to a higher ex-Ca concentration compared to the Cimrutu soil. This is in line with the observation that soils with a high Ca concentration tend to have a higher pH [36].

The high nitrogen content in both soils can be attributed to the frequent use of urea and NPK chemical fertilizers in rice cultivation. Such high nutrient levels can have an impact on the abundance and diversity of bacteria in the rhizosphere. Specifically, excessive nitrogen levels can inhibit the activity of N₂-fixing bacteria, while low levels of available phosphorus can stimulate the growth of phosphate-solubilizing microbes [37]. Additionally, the high level of organic carbon in the soil supports the growth of heterotrophic bacterial communities that utilize organic carbon for their oxidative metabolism [38].

The results of this study indicated that the EC values of A and B soils were 0.21 and 2.30 dS m⁻¹, respectively (Table 2), which fell within the tolerable salinity range of less than 4 dS m⁻¹ recommended by FAO [34]. Rice is sensitive to the high salt content in the soil: more than 50% loss was recorded in soil with EC up to 6 dS m⁻¹ [39]. Outside of this range, rice yield may be significantly reduced, and the plants may exhibit symptoms of salt stress, such as stunted growth, reduced tillering, and decreased photosynthetic activity.

The findings of this study indicated that the diversity of bacteria in slightly saline soils in Rawaapu (BR and BB) was lower than that in non-saline soils in Cimrutu (AR and AB), as evidenced by the Observed species, Shannon, and Simpson indices (Table 3). Furthermore, non-saline soils (AR and AB) exhibited a greater number of specific OTUs than slightly saline soils (BR and BB) depicted in Figure 3. These results are in agreement with findings that salinity is a significant factor influencing the community structure of soil microbes [1,40]. Saline conditions generally reduce microbial diversity and richness. Elevated salt levels can alter microbial physiology and activity directly by modifying microbial communities or indirectly via changes in pH and nutrient availability [18]. High levels of ambient salt can cause osmotic stress in microbial cells, thereby reducing their physiological fitness [41]. Microbes that fail to adjust their intracellular osmolarity will eventually perish or become inactive. As a result, salinization may alter the microbial community directly by favoring halophilic or halotolerant microbes [40].

The diversity of bacteria in soil is influenced by several factors, including soil characteristics, environmental conditions, and plant–microbe interactions [42]. When it comes to slightly saline soil, the presence of salt can limit bacterial diversity. As a result, the diversity of bacteria in slightly saline soil is typically less than in non-saline soil. Salt can alter the physical and chemical properties of soil, reducing water availability, which in turn limits the growth and survival of some bacterial species. Additionally, salt can change the soil’s ionic composition, impacting nutrient availability and altering microbial communities. This can create a selective pressure in favor of salt-tolerant bacteria, which can outcompete other bacterial species and reduce bacterial diversity.

Our results revealed an enriched population of salt-tolerant species, indicating that microbial communities were abundant and diverse even in high-salinity soils. Non-saline bulk soil Cimrutu (AB) is dominated by Bifidobacterium, Clostridioides, Paenibacillus, WPS-2, Candidatus_Levybacteria, Streptomyces, Enterobacteria, and Clostridium_sensu_stricto_1 (Figure 6). On the other hand, non-saline rhizospheric soil Cimrutu (AR) had more bacterial species belonging to Sideroxydans, RCP2-54, Lysinibacillus, Thiobacillus, Magnetospirillaceae, TM7a Streptococcus, Subgroup_2, and Subgroup_23 (Figure 6), which was identified as having a function as a biofertilizer. Sideroxydans are microaerobic, neutrophilic iron-oxidizing Beta-proteobacteria in rice soils [43]. Lysinibacillus are naturally colonizing rice roots and can potentially be highly resistant biofertilizers due to their spore production [44]; moreover, Thiobacillus is a nitrate-dependent neutrophilic Fe-oxidizer in rice soils. The members of the family Magnetospirillaceae, Magnetospirillum, are also found as diazotrophs in rice roots [45,46]. The diversity of bacteria that can serve as biofertilizers in high-salinity rice fields is extensive, and different bacterial species can offer various advantages to rice plants. The saline rice soil provides a valuable opportunity to explore and discover new beneficial indigenous microbes that have a main function as biofertilizers and enable to withstand high-salinity stress, offering potential benefits to rice cultivation.

In our study, rice varieties under different salinity affected the abundance and diversity of bacteria around the roots. Rice plants release exudates rich in organic acids, such as malate and citrate, which are known to chelate and solubilize toxic ions in the rhizosphere under salinity stress [47]. Some rice varieties may produce specific exudates in response to salinity stress, which can modify the rhizosphere’s physicochemical properties, such as pH and ion concentrations [48]. Salinity stress affects the composition and quantity of exudates released by rice plants, which, in turn, influences the selection of specific microbial communities in the rhizosphere [47].

The difference in bacterial community structure in the rhizosphere among the two locations might also be due to the root exudate differences between the rice cultivar Mendawak and Ciherang. Plant genotype may influence root exudation, and hence the rhizosphere microbiota. Root exudate composition depends on the expression level of specific genes [49]. The rice genotype is one of the factors affecting the microbial communities in the endosphere, rhizoplane, and rhizosphere [50]. Rice variety significantly altered the active fungal and Gram-negative bacterial groups [51]. Moreover, the rice plant with the seedling salt-tolerant gene under salt stress influence the rhizosphere bacterial community, proving that the key genes of plants play a significant role in shaping the rhizosphere microbiome [52].

5 Conclusions

The soil nutrient content was similar in both Cimrutu (location A) and Rawaapu (location B) rice fields, but the soil reaction in Cimurutu was acidic while the pH in Rawaapu was near neutral. There was a noticeable difference in their EC despite the two locations being less than 4 km apart. The EC value in the Cimrutu soil suggests that it is non-saline, while the Rawaapu soil exhibits a low level of salinity. The soil in both locations had high C-organic and total-N but a low ratio of C to N. The soils were very low in P and very high in K; the CEC and BS of soils were high. Those soil properties indicated that the soils were fertile.

The predominant phylum in saline rhizospheric soil Rawaapu (BR) was observed to be Proteobacteria, while Firmicutes was the most abundant group in non-saline rhizospheric soil Cimrutu (AR) and non-saline bulk soil Cimrutu (AB) as well as saline bulk soil Rawaapu (BB). Halothiobacillus, Thioalkalispira-Slvurivermis, and Acidothermus were the predominant genera in saline rhizospheric soil Rawaapu (BR), indicating the significant roles played by halotolerant microbes as plant growth-promoting rhizobacteria in saline soil. The current study showed that the salinity instead of chemical fertility was the main driving force for the different bacterial community diversity in rice field. The difference was possibly related to the root exudates in the rhizosphere of rice grown in saline and non-saline soil, as well as metabolic characteristics of predominant species. The study provided an important understanding of cultured or uncultured bacterial populations and structure in saline and non-saline soil to develop future strategies related to salinity by introducing beneficial microbes. Further, a more comprehensive study to determine the role and contribution of saline-resistance microbes found in this study is needed for better plant nutrient management in rice fields near the coastal area.

Acknowledgments

This research was funded by the Academic Leadership Grant of Universitas Padjadjaran year 2021.

Funding information: This study was funded by Universitas Padjadjaran, Indonesia.
Author contributions: RH and EP composed the research proposal and methods; RH and TS performed the sample collection and soil analysis; EP and YSF carried out the metagenomic analysis and its data analysis; RH, EP, and YSF wrote the manuscript. The authors have reviewed the manuscript and agreed to submit the manuscript.
Conflict of interest: The authors state no conflict of interest.
Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Received: 2023-03-30

Revised: 2023-08-01

Accepted: 2023-08-24

Published Online: 2023-09-28

This work is licensed under the Creative Commons Attribution 4.0 International License.

Artikel in diesem Heft

https://doi.org/10.1515/opag-2022-0225

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bacterial diversity; bulk soil; rhizosphere; soil chemical characteristics.

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