Taxonomy annotation
‘Greengenes’ database was used to generate taxonomic classification. The taxonomic abundance information in all seven taxonomy levels (kingdom, phylum, class, order, family, genus, species) of all the three samples was determined. Taxonomy analysis of total OTUs from three root samples revealed 17 phyla including three major phyla namely Cyanobacteria (37%), Proteobacteria (35%) and OD1 (25.48%). Root samples of Black Njavara and Rakthasali were dominated by Cyanobacteria followed by Proteobacteria while, that of Vaishak was dominated by Proteobacteria followed by OD1 (Fig. 1). In an investigation on the endophytic bacterial community associated with black scented rice, 30 phyla were identified and Proteobacteria was the dominating phylum in all samples (Singha et al., 2021). Taxonomy analysis identified significant differences in abundance at different developmental stages in root and shoot.
Cyanobacteria, commonly known as blue-green algae, are prokaryotic microorganisms found in environments like rice fields. They engage in photosynthesis and some, such as Anabaena, play a role in biological nitrogen fixation and phosphate solubilization. Singha et al. (2021) reported on the abundance of Cyanobacteria, which decreased in the mature roots of all varieties of Black scented rice, while in shoot it remained consistent. Cyanobacteria are known for synthesizing various metabolic compounds, including phycobiliproteins, crucial in biomedical research (Gonzalez-Medina and Medina-Franco, 2019). Additionally, they produce metabolites with pharmaceutical properties, such as antibiotic, anti-inflammatory, antioxidant, and anti-Alzheimer's functions (Chaubey et al., 2019).
Proteobacteria has been reported as the most abundant phylum in rice tissues, by several researchers. In Indian rice roots, Proteobacteria, Firmicutes, Cyanobacteria, and Actinobacteria were found to be abundant (Sengupta et al., 2017). A study across different regions of India also highlighted the prevalence of Proteobacteria, followed by Bacteroidetes, Firmicutes, and Actinobacteria in rice (Kumar et al., 2021). Singha et al. (2021) reported Proteobacteria dominance, especially in black scented rice roots (> 80%) and shoots (> 60%). The relative abundance of Proteobacteria increased from young to mature plants, with higher levels in roots (48.13–84.44%) compared to shoots (48.32–70.71%).
Parcubacteria, or Candidate phylum OD1 bacteria, are part of the Patescibacteria superphylum, also known as Candidate Phyla Radiation (CPR). These microbes are uncultured, and their genetic information is available only through metagenome assembled genomes (MAGs). With highly reduced genomes (∼1 Mb), Parcubacteria predominantly feature hypothetical proteins with unknown functions, showing no homology to well-studied microbes. The genomes of Parcubacteria reveal limited metabolic capabilities, lacking complete pathways for synthesizing essential molecules such as vitamins, lipids, amino acids, and nucleotides. This deficiency suggests a likely symbiotic lifestyle, where Parcubacteria depend on obtaining nutrients, metabolites, and energy directly from their host or partner cells (Nelson and Stegen, 2015).
In taxonomy classification, 19 classes were present in Vaishak and 11 classes were present in both Black Njavara and Rakthasali. Root samples of all three varieties were dominated by Class Alphaproteobacteria.
Genus level taxonomy classification of OTUs of root samples showed significant diversity among the three rice varieties. The number of genera recorded in Black Njavara was 14, Rakthasali was 19 and Vaishak was 26. In total, Azospirillum was recorded as the most dominant genus, followed by Sphingomonas, Caulobacter and Novosphingobium (Fig. 2 &3).
The study focused on the microbial diversity in different rice varieties, emphasizing the abundance and potential roles of various genera. Azospirillum emerged as the most dominant genus, known for its diazotrophic nature and its ability to act as a plant growth-promoting rhizobacterium (PGPR). Azospirillum species, prevalent in rice and other grasses, directly stimulate plant development through phytohormone production and enhanced nitrogen nutrition via biological nitrogen fixation (Rodrigues et al., 2008). Ding et al. (2019) reported the nitrogen-fixing ability of rice endophytic Azospirillum, and Edwards et al. (2015) explored the diversity of nitrogen-fixing bacteria, highlighting higher abundance in organically cultivated rice compared to conventionally grown rice.
The genus Sphingomonas, comprising Gram-negative, rod-shaped, chemoheterotrophic bacteria, occupied second position in abundance in root samples of all three rice varieties. Wang et al. (2016) recorded the presence of Sphingomonas in sprout, stem, and root, while Raweekul et al. (2016) associated it with plant growth promotion through indole-3-acetic acid (IAA) production. Additionally, Khan et al. (2014) correlated the abundance of Sphingomonas with the production of plant growth hormones ; gibberellins and IAA.
Caulobacter, a dominant genus, was highlighted for its potential in enhancing plant biomass. Berrios and Ely (2020) reported on the ability of Caulobacter to shape plant-microbe and microbe-microbe interactions in various ecosystems, positioning it as a model organism for plant microbiome studies. Berrios (2022) compared the genomes of three plant growth-promoting Caulobacter strains, revealing higher metabolic gene abundance in plant growth-promoting strains.
Novosphingobium, belonging to the class Alphaproteobacteria, stood out as one of the dominating genera in the study. The glycosphingolipids of Novosphingobium were noted for their applications in food and pharmaceutical products (Pollock, 1993). The bacterium exhibited stress-reducing properties, decreasing transpiration and stomatal conductance in citrus under salt stress conditions (Vives-Peris et al., 2018). Rangjaroen et al. (2015) isolated diazotrophic Novosphingobium from rice roots, emphasizing its unique endophytic colonization and plant growth-promoting potentials.
Magnetospirillum, exclusively found in Rakthasali roots, belonged to the class Alphaproteobacteria and demonstrated characteristics of facultative anaerobe/ microaerophiles. The genus, known for biomineralization of magnetite crystals, has implications in fabricating carbon nanotubes (Matsunaga and Takeyama, 1998). Singh et al. (2022) reported Magnetospirillum as one of the most abundant nifH bacteria in sugarcane, indicating its diazotrophic nature. Knief et al. (2012) found that Magnetospirillum bacteria, known for converting nitrogen, were discovered in the soil around rice plants in the Philippines. This suggested that these bacteria in plant growth. These bacteria have also been studied for breaking down certain aromatic compounds. However, whether it contributes to the synthesis of pharmaceutical compounds in the medicinal rice Rakthasali, needs further investigation.
Sulfurospirillum, exclusively present in Rakthasali root tissues, included anaerobic microbes with potential roles in phytoremediation. Li et al. (2011) suggested that endophytic Sulfurospirillum might enhance phytoremediation through sulfur metabolism, removing toxic materials or organic compounds from water.
Agrobacterium, recorded only in Rakthasali, has been identified as an endophytic bacterium in various plant species (Moronta-Barrios et al., 2018). Wang et al. (2006) reported the isolation of Agrobacterium tumefaciens from root nodules of leguminous plants, while Tan et al. (2001) highlighted its role in promoting rice growth by enhancing nutrient uptake and the accumulation of indole-3-acetic acid (IAA).
Rhodoblastus, a phototrophic genus belonging to class Alphaproteobacteria, was exclusively recorded in Rakthasali roots, in the present study. Earlier, a positive correlation between its abundance and sugarcane stalk diameter and sugar content was reported (Fallah et al., 2023).
Asticcacaulis, Elstera, Mycoplana, Asteroleplasma, Clostridium and Bdellovibrio, were observed exclusively in medicinal rice varieties, Black Njavara and Rakthasali. Clostridium is an anaerobic nitrogen-fixing bacterium, reported as endophytes in many crops, challenging the traditional notion that Clostridia are not bacterial endophytes (Minamisawa et al., 2004; Saito et al., 2008). Saito et al. (2008) isolated and characterized clostridia from cultivated and wild species of rice. They concluded that endophytic clostridia are distributed in agricultural crops as well as natural grasses. The proposed cycle of endophytic-enteric-soil-endophytic microbes by Martinez-Romero (2020) suggested that Clostridia from plants may become part of animal gut microflora.
Bdellovibrio belonging to Class Deltaproteobacteria is reported to be a predatory bacterium on other Gram-negative bacteria. While its endophytic role in rice and maize has been reported (Mano and Morisaki, 2008; Adeleke and Babalola, 2022), its specific function remains unclear.
Out of the 418 OTU groups in the species level, 17 were assessed into specific species groups while 401 were unclassified. In these 76 groups were classified up to genus level, 59 to family level, 45 to order level, seven to class level, 100 to phylum level and 114 only to kingdom level. Highest species diversity was observed in Vaishak (253) followed by Rakthasali (106) and Black Njavara (86). These unclassified species are considered novel or lack sufficient characterization and have not been included in the database. Identification and characterization of these novel microbes can be accomplished through specific culturing techniques or in-silico methods.