ANALYSIS OF 16S RRNA FOR IDENTIFICATION ANALYSIS OF 16S RRNA FOR IDENTIFICATION OF MICROBIAL COMMUNITIES IN THE RHIZOSPHERE OF LJ. ANALYSIS OF 16S RRNA FOR THE IDENTIFICATION OF MICROBIAL ASSOCIATIONS IN THE RHIOSPHERE OF GROWN DATE PALMS UNDER SALT STRESS.
Introduction
- Overview
- Plant and Micro-Organisms Interaction
- Salinity and Effect of Saline Water Irrigation
- Problem Statement and Missing Knowledge
It is estimated that approximately 20% of the world's total irrigated land is adversely affected by salinity (Egamberdieva et al., 2019). Therefore, salinization is one of the most consequential land degradation problems facing the agricultural sector (Yan et al., 2015).
Literature Review
- Farming in Arid Land of United Arab Emirates
- Plant Abiotic Stress
- Salinity Stress
- What is Holobiont?
- Impact of Microbial Communities on Plant Salinity Stress
- Metabarcoding
Abiotic stress is the harmful effect of non-living variables on living organic beings in a certain environment (He et al., 2018). Due to the low osmotic potential, it is more difficult for roots and microbes to remove water from the soil (Yan et al., 2015). However, some microorganisms can accumulate osmolytes to adapt or tolerate salinity (Sagot et al., 2010; Yan et al., 2015).
Phoenix dactylifera expressing heterologous ACCD can facilitate the colonization of a variety of beneficial endophytes (Hazzouri et al., 2020; Sessitsch et al., 2012). Metabarcoding is one of the most widely used molecular approaches for the detection of biodiversity in environmental samples such as soil, water, plants and feces (Alberdi et al., 2018). It is a molecular tool that aims to determine the species composition of the sample (Ruppert et al., 2019).
About 5~10 copies of 16S rRNA are present in each bacterium, making the detection sensitivity of the 16sRNA very efficient (Johnson et al., 2019). Metabarcoding using the 16S rRNA marker is widely used in the study of various microbial communities (Hamady & Knight, 2009; Wang et al., 2005). Specifically, using different regions of 16S rRNA regions leads to an estimation of different taxa (Table: 1) and a real composition of the sample can be obtained (Bukin et al., 2019).
Materials and Methods
- Site Description, Experimental Setup, and Sampling
- Materials and Equipment's
- Soil and Water Chemistry Analyses
- EC and pH
- Soil Organic Matter
- DNA Extraction and Gel Electrophoresis
- PCR process using 16s rRNA
- Bioinformatic Analyses
- Statistical Analyses
Soil collected from date palm roots was analyzed to obtain various metadata related to bacterial community composition. For analyzing soil organic matter (SOM), the method used is the loss of mass on ignition (LOI). Then, total DNA extraction of root tissues was performed using one gram of ground tissue and the DNeasy Plant Mini kit (Qiagen, Germany).
To avoid any cross-contamination of the samples, the process was performed with sterile equipment. The quantity and quality of extracted DNA were assessed using a Nanodrop spectrophotometer (Thermo Fisher Scientific). The quantity and quality of extracted DNA were analyzed by spectrophotometry using an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and by agarose gel electrophoresis.
The first step was to perform quality filtering and sequence trimming, followed by dereplication, generation of error models and denoising, further assembly into contigs, generation of an amplicon sequence variant (ASV) table, and then finally removal of chimeras. Taxonomic assignment of OTUs was performed using vsearch in the Silva database v 138.1 (downloaded from https://zenodo.org/record/4587955) and the taxonomic table containing kingdom, phylum, class, order, family, genus is was created, and species-level resolution was also added if the database was available. The envfit function of the vegan package was used to investigate the significance of the aforementioned factorial and vector variables with community structural patterns, where R2 and p-values were calculated by regressing individual variables with the NMDS1 and NMDS2 axes.
Results
- Data Characteristics
- Saline Water Irrigation Effect on Soil Chemistry and
- Saline Water Irrigation Affects Bacterial Community
- Saline Water Irrigation Effect On Bacterial Community
c) soil organic matter (OM); (d) pH of the irrigation water; and (e) EC of irrigation water, measured in this study under different conditions of irrigation water sources (non-saline versus saline). However, the accumulation curve of OTU showed that relatively more OTU was detected at sites with non-saline water as an irrigation source compared to saline water as an irrigation source (Figure: 10a). Of the total number of OTUs, nearly 40.5% were found exclusively at sites with non-salt water irrigation, while only 26% were unique to sites with salt water as the source of irrigation.
Most of the saline samples were placed close to the ordination space and compared with non-saline samples, and 95% ellipses for different locations also do not overlap, indicating different groups of communities in both environments. The NMDS analyzes also confirmed that vector variables soil EC and irrigation water pH are an important determinant of community structure, as their ordering space arrows target saltwater irrigation samples. Based on relative abundance by phylum, Proteobacteria is the most common phylum among the bacterial communities found, and alone comprised 82 bacterial families.; it had approximately similar relative abundance in both saline and non-saline samples; similarly, the relative abundance of Actinobacteria and Acidobacteriota also did not differ in either condition.
Comparison of the relative abundance at the order level shows that there is an increased abundance of Rhizobiales, Streptomycetales, Actinomarinales and Burkholderiales among the non-saline root samples; however, order such as Bacillales, Micromonosporales, Corynebacteriales and Steroidobacterales were relatively more abundant in the saline root samples than non-saline (Figure: 13 (b); Table: S1). The hierarchical clustering for proportional abundances of different bacterial genera and their affinity towards the saline and non-saline samples shows that genera such as Bacillus, Rhizobium, Acidibacter, Streptomyces have a high abundance in non-saline conditions compared to saline, where they are either absent or their presence is negligible. Total reads (%) and occurrences among samples were calculated for the overall database, non-saline sample and saline samples subset.
Discussion
Saline Water Irrigation Does Not Change The Diversity
Similarly, the Shannon diversity index showed a significant positive correlation with increasing soil organic matter content, and this correlation is seen because SOM reflects soil organic carbon content, which is an essential source of energy required by bacterial community to thrive. and multiply (Ngatia et al., 2020). Also, soil microbial communities break down organic materials present in the soil, helping to form soil organic matter (SOM). Increased SOM enhances the growth of specific species and is responsible for increasing the diversity of beneficial microbial species (Ngatia et al., 2020).
The growth of plants produces more root secretions, which serve as the carbon source for the survival and proliferation of microbes. While some bacteria are able to alter soil pH by acidifying their environment to outcompete others, most bacteria thrive at a neutral pH. The present results were similar to a study conducted by Hollister et al. 2010) who found that the shifts in microbial communities were largely linked to soil chemistry (pH, EC and SOM).
However, opposite results were observed in another study in oil palm, which showed no significant changes in overall microbial diversity in relation to changes in soil characteristics, especially pH, EC and SOM (Berkelmann et al., 2020). We found that overall bacterial diversity parameters including bacterial richness, Shannon diversity index and Pielou's evenness index were not significantly different between the two types of irrigation. This was also consistent with a previous study (Sharaf et al., 2019) that concluded that similar indicators of richness and diversity were found even when irrigated with different water sources.
Saline Water Irrigation Changes Community Compositional
Another study on sugarcane found that the most abundant classes of root and soil bacteria were Proteobacteria, similar to the current date palm study (Supriadi et al., 2020). In another study on Sorghum bicolor, similar bacterial phyla were identified Proteobacteria (Alpha and Beta), Actinobacteria and Firmicutes (Guazzaroni et al., 2018). In a recent study on maize plants, Firmicutes were more abundant in saline than non-saline soils, similar to the results found in the present study (Hou et al., 2021).
Studies have shown that Bacillus-like organisms play a key role in the biogeochemical cycles in saline soils and marine waters (Mehnaz et al., 2017; . Thanapun Taprig, 2013). The rhizosphere community and diversity within soybean plantations was found to vary significantly in different soil types, with a decrease in saline soils as in the present study (Han et al., 2020). This result is correlated with the study conducted by Ferjani et al. 2015), as it was indicated that the date palms in the desert ecosystem of Tunisia had a high dominance of Pseudomonas, Pantoea and Mycobacterium genera.
Furthermore, it was indicated that the halophytic plant Salicornia bigelovii soil was occupied by large communities of Micromonospora, as found in the current study (El-Tarabily et al., 2019). The identified microbial communities Micromonospora, Pseudomonas and Mycobacterium in the saline soil of date palms in the current study indicated the potential effect of PGPR of these microbial communities, which has been demonstrated in different studies (Ahemad & Kibret, 2014; El-Tarabily et al., 2019). Different studies showed that Pseudomonas played a role in the salinity tolerance of cotton (Egamberdieva et al., 2015).
Conclusion
The present study extends the knowledge of the microbial communities present in the rhizosphere of date palms grown in saline and non-saline irrigated farms. It indicates the possible roles of the identified microbial communities for the adaptations of the date palm to salinity stress. The present study will be further developed with a focus on a complete description of the metagenome of the date palm saline environments that will report metabolic pathways of the identified microbial communities with an understanding of how these microbial communities remain active and recover under the salinity stress.
The taxonomic and functional profiling of microbial communities will continue current research to elucidate functional features that enable the proliferation of the identified microbial communities under salt stress. The study will take a closer look at analyzing the potential of PGPR or ACC gene activity of the phyla Micromonospora, Pseudomonas and Mycobacterium found in the saline soils of date palms. Another future perspective will be the investigation of the integration of the identified microbial community in the rhizosphere as a factor in determining desirable traits in date palms, such as yield or salt tolerance.
The research perspectives of date palm rhizosphere show the achievement of great potential of rhizosphere microbial communities for remediation of salinity-stressed agricultural ecosystems, and the application of this green biotechnology will have more positive effects on expected climate change. Soil microbiome of post-mining sites in polar ecosystems around Nadym, Western Siberia, Russia. Soil salinity: a serious environmental problem and plant-promoting bacteria as one of its mitigation tools.
Diversity of bacteria in the roots of sugarcane used to produce Wasanbon in Kagawa, Japan. Changes in the bacterial populations of the highly alkaline saline soil of the former Lake Texcoco (Mexico) after flooding.
