Salinity of irrigation water selects distinct bacterial communities associated with date palm (Phoenix dactylifera L.) root

Saline water irrigation has been used in date palm (Phoenix dactylifera L.) agriculture as an alternative to non-saline water due to water scarcity in hyper-arid environments. However, the knowledge pertaining to saline water irrigation impact on the root-associated bacterial communities of arid agroecosystems is scarce. In this study, we investigated the effect of irrigation sources (non-saline freshwater vs saline groundwater) on date palm root-associated bacterial communities using 16S rDNA metabarcoding. The bacterial richness, Shannon diversity and evenness didn’t differ significantly between the irrigation sources. Soil electrical conductivity (EC) and irrigation water pH were negatively related to Shannon diversity and evenness respectively, while soil organic matter displayed a positive correlation with Shannon diversity. 40.5% of total Operational Taxonomic Units were unique to non-saline freshwater irrigation, while 26% were unique to saline groundwater irrigation. The multivariate analyses displayed strong structuring of bacterial communities according to irrigation sources, and both soil EC and irrigation water pH were the major factors affecting bacterial communities. The genera Bacillus, Micromonospora and Mycobacterium were dominated while saline water irrigation whereas contrasting pattern was observed for Rhizobium, Streptomyces and Acidibacter. Taken together, we suggest that date-palm roots select specific bacterial taxa under saline groundwater irrigation, which possibly help in alleviating salinity stress and promote growth of the host plant.

www.nature.com/scientificreports/ to promote plant growth 15 through iron mobilization (i.e. siderophores), extracellular polysaccharides (EPS) production, phosphate solubilization and hormone production (i.e. indole acetic acid). In a recent study, it has been shown that irrigation water salinity as a major determinant of bulk soil bacterial communities from arid agroecosystem 16 . Apart from salinity, additional factors such as soil pH and total nitrogen are also known to structure cotton field soil bacterial communities between irrigation sources (non-saline vs saline water) 6 . We also expect similar structuring of bacterial communities in date palm roots under non-saline freshwater and saline groundwater irrigation. At compositional level, saline groundwater enrich Gemmatimonadetes and Actinobacteria phyla in cotton field soil 6,17 , while barley field soil enrich specific genera namely Rhodanobacter, Candidatus Koribacter and Burkholderia 18 . Previous studies in date palm show compartmentspecific selection of taxa 15,19 , wherein Gammaproteobacteria was dominant 19 , while another study show enrichment of Pseudomonas and Rahnella genera 20 in the roots. Apart from the compartment effect, geographical factor (i.e. location) also affect the root-associated bacterial communities of date palm through consistant selection of Gammaproteobacteria and Alphaproteobacteria 19 in high abundance. Furthermore, climatic factors (i.e. temperature and precipitation) are also important for structuring date palm root-associated bacterial communities 15,20 . Considering the enrichment and selection of several bacterial taxa specifically under high saline conditions of below-ground compartments of agroecosystems, we predict to have a similar response from date palm rootassociated bacterial communities as well under saline groundwater irrigation.
In this study, we investigated the impact of irrigation sources (non-saline freshwater and saline groundwater) on date palm root-associated bacterial diversity and community structure. We also explored how soil and water chemistry is related with bacterial community patterns. We hypothesize that bacterial community composition while saline groundwater irrigation would be different from non-saline freshwater irrigation due to salinity stress and concomitant change in the soil chemistry. To test our hypothesis, we collected date palm root samples from different sites that receive non-saline freshwater or saline groundwater as an irrigation source and assessed the bacterial communities using 16S rDNA metabarcoding. Overall, the understanding of the date palm rootassociated bacterial communities under saline conditions will help in assessing the ability to use these organisms as beneficial bio-inoculants in date palm agriculture.

Materials and methods
Site description, experimental setup and sampling. The study was conducted in the Al-Ain region located approximately 160 km east of Abu Dhabi (UAE; Supplementary Table S1). The region is classified under the hyper-arid category and has a mean annual rainfall of 9.03 mm and average temperature ranges between 23 °C and 35 °C in the sampling year 2020 (https:// www. world weath eronl ine. com/ al-ain-weath er-avera ges/ abudhabi/ ae). In our study, we selected the most commonly grown variety of date palm i.e. Khalas and sampling was performed during March 2020. The date palm trees across sites were irrigated with 50-60 L of non-saline freshwater or saline groundwater. In total, we collected 42 root samples (7 sites × 2 treatments × 3 replicates). To perform soil OM analysis, we have also collected bulk soil samples from each hole left after root excavation (approx. 10-15 cm deep). In addition, we have also collected water samples (non-saline freshwater and saline groundwater) from across sites for chemical analyses. All the samples were transported from field to lab in cooled condition, stored in − 20 °C and root samples were processed for DNA extraction within 48 h. We confirm that the use of plants in the present study complies with international, national and institutional guidelines.
Soil and water chemistry analyses. Bulk soil samples were homogenized and passed through a 2 mm sieve. One gram of soil sample was dissolved in 9 mL of Milli-Q water and mixed for 1 h at 200 rpm. The soilwater mixture was then filtered through Whatman grade 42 filter paper and the filtrate was used for measuring soil EC and pH 21 using Hanna pH and EC bench top meter, USA. Similarly, the irrigation water was also analyzed for EC and pH. The soil OM was analyzed using mass loss on ignition (LOI) method 22 . Briefly, 5 g of air-dried desiccated soil was incubated at 360 °C for 4 h. The difference between the mass of the soil before and after heating was used for determining the soil OM. DNA extraction and Illumina sequencing. The fine roots were ground with liquid nitrogen using a sterile mortar and pestle. DNA extraction was performed using one gram of the ground root tissue by DNeasy Plant Mini Kit (Qiagen, Germany) following manufacturer's protocol. We amplified V3-V4 region of the 16S rRNA gene using 10 base pair (bp) barcoded primer combination of 341F (CCT ACG GGNGGC WGC AG) and 805R (GAC TAC HVGGG TAT CTA ATC C) 23 . We set-up a 50 μL PCR reaction, consisting of forward and reverse primers (1 μM each), 250 µM dTNPs (0.5 µM of each), 0.02 U Phusion High-Fidelity DNA Polymerase (Finnzymes OY, Espoo, Finland), 0.3 mg/mL BSA (Bovine Serum Albumin) and 5× Phusion HF buffer containing 1.5 mM MgCl 2 . The applied PCR conditions were as follows: initial denaturation at 95 °C for 5 min, 25 cycles of denaturation (95 °C for 40 s), annealing (55 °C for 30 s) and extension (72 °C for 1 min), a final extension step (72 °C for 7 min). The purification and normalization of the PCR products were done using DNA Normalization Kit (Charm Biotech). The amplicon libraries were sequenced using Illumina MiSeq system (2 × 300 bp) at IMR lab, Halifax, Canada (https:// www. imr. bio. com) following standard Illumina protocol. The demultiplexed raw sequence files used in this study are deposited in the Zenodo repository (https:// doi. org/ 10. 5281/ zenodo. 60782 92).

Bioinformatic analyses.
A total of 5,50,566 raw sequence reads were analyzed using Divisive Amplicon Denoising Algorithm 2 (DADA2_v1.12) R package (Benjamin Callahan 2016). We removed primer sequences using rbind function, performed quality filtering and sequence trimming (> 275 bp for forward, > 225 bp for reverse reads) (maxN = 0, truncQ = 2, maxEE = 2) using function filterAndTrim. The trimmed sequences were www.nature.com/scientificreports/ denoised using error models (learnErrors), and amplicon sequence variants (ASVs) were inferred for both forward and reverse reads, then contigs were generated by merging (mergePairs). We further removed chimera using removeBimeraDenovo function 24  Statistical analyses. All the statistical analyses were performed in R v4.0.3 (R Core Development Team, 2020) using respective packages unless stated otherwise. To perform community structural analyses, OTU abundance data was arcsine-transformed to increase the homogeneity of variances. Water and soil chemistry variables including pH, EC and soil OM were standardized using Z transformation and expressed on a 0-1 scale, which helps in the comparison of values from disparate distributions. To examine the effects of irrigation source (nonsaline freshwater vs saline groundwater) on soil (pH, EC and OM) and water chemistry (pH and EC) variables, we used analyses of variance (ANOVA) test, followed by Tukey's HSD post-hoc test using R package agricolae.
The between site variation of environmental metadata was performed using analyses of variance (ANOVA) test, followed by Tukey's HSD pair-wise test using R package agricolae. The impact of irrigation sources (non-saline freshwater vs saline groundwater) on bacterial richness, Shannon diversity index and Pielou's evenness index was also analyzed using similar statistical test. Linear regression analyses were performed using R package vegan, to investigate the relationship of soil and water chemistry variables with bacterial richness, Shannon diversity index and Pielou's evenness index (P < 0.05) 29 .
To perform multivariate analyses, we calculated dissimilarities in OTUs matrices using Bray-Curtis distances. The relationship of irrigation sources (non-saline freshwater vs saline groundwater), soil (pH, EC and OM) and water (pH and EC) chemistry variables in explaining bacterial community structuring was assessed by permutational analysis of variance (PERMANOVA) using Adonis function vegan. We calculated pseudo-F statistics following 9999 times permutation of the OTUs matrices. A forward selection procedure was exercised to optimize the final model for PERMANOVA analyses 30 . At first, we tested single factor models, and thereafter significant factors were added in the order of their R 2 values to obtained final significant model. To investigate bacterial community structural patterns, we also performed Nonmetric Multi-Dimensional Scaling (NMDS) ordination analysis using metaMDS function of vegan. NMDS was performed with following settings, dimensions (k) = 2, maximum iterations = 1000, initial configurations = 100, minimum stress improvement in each iteration cycle = 10 -5 and P value and R 2 values were calculated. Bacterial community structural relationship with irrigation water sources, soil and water chemistry variables were inferred using envfit function of vegan and vector variables were fitted on NMDS ordination space.
Saline groundwater irrigation effect on soil chemistry and bacterial diversity. We found that soil OM and irrigation water pH as well as EC differed significantly between non-saline freshwater and saline groundwater irrigation (Fig. S2). Irrigation water pH and soil OM were significantly higher in the samples where non-saline freshwater was the irrigation source, and pattern contrasted for irrigation water EC.
The saline groundwater irrigation did not affect bacterial diversity including richness, Shannon diversity index and Pielou's evenness index (Fig. 1a-c). However, the OTU accumulation curve displayed presence of relatively higher number of total OTUs in the samples where non-saline freshwater was used as a source of irrigation (Fig. 2a). 40.5% of total OTUs were exclusively detected in roots of date palm under non-saline freshwater irrigation and 26% were unique to saline groundwater irrigation (Fig. 2b). Only 33.6% were shared between both irrigation sources. We also found that all the unique bacterial OTUs detected under saline groundwater irrigation belonged to rare taxa (< 0.1% occurrences) (Supplementary Table S3). The linear regression analyses showed that the bacterial richness and the Shannon diversity index were negatively correlated soil EC (Fig. 3a,b). Similarly, the Pielou's evenness index also showed negative correlation to soil pH (Fig. 3c). Shannon diversity index also showed a significant positive correlation with the soil OM concentrations (Fig. 3d). The between site variations of water and soil chemistry is given in Supplementary Tables S3 and S4. Non-saline water irrigation showed highest soil pH in site 5, meanwhile highest soil EC was observed in site 2. Saline water irrigation showed highest soil pH and soil OM in site 4, while site 5 recorded highest soil EC. www.nature.com/scientificreports/ Saline groundwater irrigation affects bacterial community structural patterns. The multivariate methods, including PERMANOVA and NMDS ordination analyses, indicated the presence of distinct root-associated bacterial communities between different irrigation sources i.e. non-saline freshwater and saline groundwater (R 2 = 0.06, P = 0.002) (Fig. 4, Table 1).Two distinct clusters belonging to non-saline freshwater and saline groundwater irrigation was observed in the NMDS ordination space indicating presence of distinct bacterial communities when irrigation source differ. Further, the PERMANOVA analyses indicated that soil EC (R 2 = 0.07, P = 0.001) and water chemistry [pH (R 2 = 0.05, P = 0.006) and EC (R 2 = 0.05, P = 0.015)] were the significant factors affecting bacterial community structural patterns.
Saline groundwater irrigation effect on bacterial community composition. Phylum Chloroflexi was relatively more abundant in root samples where non-saline freshwater was the irrigation source and abundance patterns were contrasted for the phylum Firmicutes, being more common while saline groundwater irrigation (Fig. 5a, Supplementary Table S2). At order level, we found that relative abundance of Rhizobiales, Streptomycetales, Actinomarinales, and Burkholderiales was higher while non-saline freshwater irrigation compared to saline groundwater and opposite patters were observed for orders Bacillales, Micromonosporales, Corynebacteriales, and Steroidobacterales (Fig. 5b). The hierarchical clustering analysis using proportional abundances of bacterial genera revealed that Bacillus, Rhizobium, Acidibacter and Streptomyces were more abundant in the sample having non-saline freshwater irrigation (P < 0.05) (Fig. 6, Table 2). Similarly, bacterial genera such as Mycobacterium, Micromonospora, Steroidobacter and Pseudomonas were abundant while saline groundwater irrigation (P < 0.05) (Fig. 6, Table 2). The most common OTUs with taxonomic affinity to Actinocorallia, Bacteroides and SWB02 were more abundant in the samples when non-saline freshwater was the irrigation source   Table S4).

Discussion
We find that date palm root-associated bacterial diversity does not change but number of unique bacterial OTUs associated with date palm roots vary under different irrigation sources. Saline groundwater irrigation strongly alters the bacterial community structure and soil EC as well as irrigation water pH are the major factors affecting their pattern. Abundance of genera Rhizobium, Streptomyces and Acidibacter is higher under non-saline freshwater irrigation, whereas Bacillus, Micromonospora, Pseudomonas and Mycobacterium are more common in the samples with saline groundwater irrigation. We didn't assess the role of geo-climatic factors (i.e. MAT and MAP) since focus of our study is to understand saline groundwater irrigation impact on root-associated bacterial communities, but they may also act as possible factor(s) of soil community structure across sites in this study.
Saline groundwater irrigation altered soil OM. The irrigation water source (non-saline freshwater and saline groundwater) did not significantly affect soil EC and pH (Fig. S2a,b) across sites in our study, which may attribute to rapid percolation of water through soil layers since soil types (i.e. Entisol and Aridsol) prevalent in arid agroecosystems of UAE 31 are known to have lower water holding capacity. On contrary, the irrigation water source altered soil OM between treatments (Fig. S2c), the decrease in soil OM concentrations under saline groundwater irrigation is possibly primed by root exudates which enhance bacterial degradation of soil OM through rhizosphere priming effect 32 . In addition, saline groundwater can also potentially affect the quantities of root-derived carbon 33 , which is evident from decreased soil OM. www.nature.com/scientificreports/ Saline groundwater irrigation changes community structure patterns but not diversity. We found that saline groundwater irrigation affected the date palm root-associated bacterial community structural patterns but not the diversity including richness, Shannon diversity index and Pielou's evenness index. Although diversity didn't change but we observed altered presence of unique OTUs while irrigating date-palm with different water sources indicating selection of specific bacterial species. Moreover, the entire set of unique OTUs detected under saline groundwater irrigation of this study belonged to rare taxa. Therefore, irrigation water source driven changes in unique OTUs and their proportions possibly structured the bacterial communities. Our results are in agreement with the previous studies, reporting irrigation source specific distinct bacterial communities in plants (i.e. cotton and barley) grown in arid environment 6,17,18 . The observed community structural pattern can be explained by a deterministic salinity filtering process, wherein the selection of bacterial assemblages depend on the salinity tolerance of date palm roots. The plant roots under salinity stress can act as "gatekeepers" by selecting soil bacteria from the rhizosphere and salinity trait may play a critical environmental filter in selectively enriching bacterial species in root niches. The salinity filtering is perhaps governed by high salt concentration, which could affect the survival and replication of bacteria in root through plasmolysis 34 . Soil EC and irrigation water pH were the most important factors affecting root-associated bacterial community structural patterns. Firstly, salinity (which is an indirect measure of soil EC) is one of the key determinants  Table 1. PERMANOVA analysis showing effects of different irrigation sources (non-saline vs saline water) on date palm root-associated bacterial community compositional structure. Effects of irrigation source, irrigation water pH, irrigation water EC (dS/m); soil (pH, EC, and organic matter (OM) chemistry were tested on overall bacterial communities. P-values were obtained using 9999 permutations, and boldface indicates statistical significance (P < 0.05). www.nature.com/scientificreports/  www.nature.com/scientificreports/ for soil microbial communities in a desert ecosystem 35 and it is known to affect desert plant root-associated bacterial communities 18,35 . The soil EC mediated alteration in bacterial taxa can potentially influence nutrient availability since salinity and nitrogen concentrations are reportedly inter-connected in the rhizosphere 36 . Increase in soil EC negatively impacted bacterial richness and Shannon diversity index, indicating bactericidal effect of salinity 34 as reported by a previous study on desert plants irrigated with high-saline water 17 . Secondly, the irrigation water pH is known to regulate bacterial communities either directly and/or indirectly by affecting the availability of cations or anions in the plant rhizosphere 37 . The relative increase in irrigation water pH under non-saline freshwater irrigation could be contributed by lime dissolution associated increase in calcium levels 38 , which in turn possibly structured bacterial communities by selecting bacteria depending on their pH preference. The alterations in irrigation water pH also affects the soil pH due to water flow and percolation in soil layers. Moreover, pH of soil was reported as one of the major predictors of bacterial diversity 39,40 and its increase negatively affected evenness of root-associated bacteria in this study is in line with a previous study 38 .

Source of variation
Enrichment of specific bacterial taxa by date palm roots. The pattern of distribution of the major phyla Proteobacteria, Actinobacteria and Acidobacteriota was similar in roots under both types of irrigation water sources. The higher abundance of these phyla seems to be common for plants (i.e. cotton and barley) while growing in desert environments under different irrigation water salinities 6,17 , which play a vital role in ROS homeostasis and maintain ionic balance in the roots of date palm, therefore indirectly supports host plant under drought conditions 41 . In addition, these phyla also play an important role in nitrogen fixation (Actinobacteria and Proteobacteria), decomposition of soil OM (Actinobacteria) and biological control (Actinobacteria and Acidobacteriota) [42][43][44] . We found higher dominance of Chloroflexi in date palm roots under non-saline freshwater irrigation, which are important in breaking down complex carbohydrates and polymeric organic compounds into low molecular weight substances within the rhizosphere 45 . On contrary, Firmicutes was relatively higher in saline groundwater irrigation, whose members are known to be involved in salinity stress tolerance 46 . We found lower dominance of the following orders, Rhizobiales, Streptomycetales, Actinomarinales and Burkholderiales and their representative genera in roots under non-saline freshwater irrigation indicate their aversion to higher salinity. Rhizobiales was represented by Rhizobium in roots under non-saline freshwater irrigation, a fast growing rhizobacteria 47 observed generally in the nodules of leguminous plants 48 . Previous studies show that nonleguminous date palms also contain Rhizobium in the rhizosphere 15,19 , which is possibly involved in the promotion of plant growth 49 . The reason for the higher abundance of Rhizobium in roots of non-saline freshwater irrigation may be due to lack of salinity stress and higher soil OM, which possibly enabled Rhizobium to multiply faster in roots. The abundance of Streptomyces was also higher in roots under non-saline freshwater irrigation, www.nature.com/scientificreports/ which is known to carry out anti-phytopathogenic and biocontrol roles in the plant rhizosphere 50 . Acidibacter detected in the roots of non-saline freshwater irrigation, is an acidophilic iron-reducing bacterium 51 possibly involved in contributing iron to roots. In contrary, bacterial species found in this study and belonging to Bacillales, Micromonosporales, Corynebacteriales and Steroidobacterales were more common under saline groundwater irrigation, which is in line with previous reports on plants growing under saline conditions [52][53][54][55] . Bacillales consisted of Bacillus genus, which produces spores for survival under saline stress conditions 56,57 , is also capable of producing rhizosheath made up of EPS around plant roots and confer salinity resistance by excluding Na + ions from plant roots 55 . Another saline groundwater irrigation specific order detected was Micromonosporales represented by Micromonospora genus, which is known to perform complex polysaccharide degradation 58 , mobilize nutrients for the plant through metallophores 59 and perform plant growth promotion through aminocyclopropane-1-carboxylic acid (ACC) deaminase activity 60 . The Micromonospora is also reported to contain genes related to osmotic stress tolerance (betC and proU) and plant growth promotion (indole-3-glycerol phosphate synthase) 61 . The other dominant genera Pseudomonas and Mycobacterium detected in this study were also earlier reported in the plant rhizosphere 19,62 . The Mycobacterium genus is known to perform plant growth promoting activity under high temperature and hyper-saline conditions 62 , thus providing an advantage to the survival of date palms under salinity stress. The Pseudomonas genus was one of the major genera in the date palm rhizosphere 19 in this study, is known to provide biocontrol properties and increase plant fitness by triggering salicylic acid-mediated systemic acquired resistance (SAR) in plants 63 . The other dominant OTUs Chryseobacterium and Blastocatella were detected only in date palm roots under saline groundwater irrigation, are known to perform plant growth promotion (ACC deaminase, siderophore, ammonia and hydrogen cyanide production) under saline stress conditions 64,65 . These results suggest that date palm roots select a set of bacterial species at higher abundance depending on irrigation water sources (non-saline freshwater vs saline groundwater). These selected taxa potentially help host plants to allevite salinity-induced stress and increase plant growth and performance.

Conclusion
Our findings show that saline groundwater irrigation does not affect date palm root-associated bacterial diversity, but alter compositional patterns, which is mainly affected by soil EC and irrigation water pH. The enrichment of taxa (Bacillus, Micromonospora, Mycobacterium and Pseudomonas) under saline groundwater irrigation revealed how date palm roots adapt to salinity by selectively enriching specific bacteria. The increased abundance of these salinity resistant bacteria under saline groundwater irrigation is very important for the survival of the date palms under drought conditions due to their potential role in plant growth promotion and nutrient mobilization. Overall, this study revealed that saline groundwater induce perturbations in enrichment or reduction of certain bacterial species, which may potentially help the host plant to alleviate salinity stress.