Endophytes are ubiquitous in nature. Colonization of particular endophyte by plants depends on various edaphic factors. Factors such as geographic locations, soil source, host genotype and cultivation practice influence microbial communities in soil which in turn play a critical role in establishing the endospheric microbes (Edwards et al., 2015). Moreover, the composition of root exudates changes with developmental stages of plant which also affects the microbiome assembly (Imchen et al., 2019). Endophytic bacteria are generally considered as a subset of rhizospheric bacteria (Afzal et al., 2019). Therefore any changes in the rhizosphere can also alter the endophytic component. Soils rich in nutrients like organic carbon and nitrogen make the soil fertile (Sahoo et al., 2019) as well as productive and promote higher plant growth which in turn is an indication of rich rhizospheric as well as endophytic diversity in plants. Reports indicate that plants enriched with nutrients tend to have increased microbial diversity (Afzal et al., 2019). In our present study we tried to unravel the endophytic diversity in roots of rice cultivated across the different agro-ecological zones in West Bengal to understand the community change and explore the variation in bacterial community composition across the zones.
Understanding the variation in environmental parameters:
The six agro-ecological zones of West Bengal differ from each other in almost all the environmental parameters tested which are expected since the zones vary in their soil combination, landform and climate characteristics (Banerjee et al., 2019). The differences could also be attributed to different cultivation practices across the zones, as primarily it is in the farmer’s hand to maintain crop yield and production (Kunda et al., 2020). In our result, the southernmost part of West Bengal represented by CSZ reported the highest EC, which is quite obvious given the fact that saline zones are characterised based on high EC (Sen & Maji, 1994). PCA results also corroborated that separation of CSZ from the rest of the zones was mainly due to its high values of EC. The northern part of West Bengal represented by the zones – NHZ and TTAZ was seen to be rich in TOC which is in line with reports that indicated these regions have experienced low biological activity and lower decomposition of biomass. Soil organic carbon is one of the most important soil quality indicators which indicates fertility and productivity of soil (Sahoo et al., 2019). The farmers from these regions mostly cultivate high-yielding variety of rice (Kumar Bag, 2011) which justifies the fact that there is no requirement of additional fertilizers and the soil also remains untouched thus experiencing high organic carbon levels. These two zones also reported the highest available N, one of the most important nutrients for plant growth. Reports are there which indicate that soil of these regions is generally high in nitrogen content (Devi & Sherpa, 2019) making it nutrient rich. As per PCA, VAZ experienced highest pH in soil among all the samples. The pH of VAZ was nearly neutral but in rest of the zones pH of soil is acidic, low pH could be accounted for greater rainfall in these regions that washes the basic cations rendering the soil acidic.
Microbial richness across the zones:
Alpha diversity indicates that there was significant difference in species richness among the zones. Reports are there that higher soil fertility is related to greater species diversity (Furtak et al., 2019). The high microbial species richness of rice plants in GAZ is reflected by the fact that this soil is most fertile (Banerjee et al., 2019), whereas low species richness of CSZ could be due to its high salinity, since excess salinity decreases species diversity and alters the community composition in plants (K. Zhang et al., 2019). The difference between GAZ and NHZ is also due to the fact that NHZ is not as fertile as GAZ and it faces difficulty in cultivation (Banerjee et al., 2019). Hence NHZ is low in endophytic microbial diversity because although the soil is nutrient dense but it is not productive, resulting in lower plant growth.
Microbial community variation in the different zones:
The members of Gammaproteobacteria, found universally in rice (Kunda et al., 2018) and abundantly in rice endorhizosphere (Moronta-Barrios et al., 2018), was seen to be dominant in case of fertilized soils (Fierer et al., 2011), intensively cultivated soil (Hamamoto et al., 2018), agricultural soil (Kuramae et al., 2012) or soil rich in N (Q. Wang et al., 2018) which is in line with our results that indicates its high abundance in GAZ, NHZ and TTAZ. These zones are characterised by soil rich in nutrients like organic carbon and nitrogen. As endophytes are a part of rhizopsheric soil therefore, we can say that enrichment of Gammaproteobacteria as root endophyte is seen in nutrient rich environment. Another class, Bacilli is also present abundantly in permanent grasslands and arable land (Mendes et al., 2013), which justifies their highest occurrence in GAZ. Bacteroidetes being abundant in nutrient rich soil (He et al., 2017) explains the high proportion of class Bacteroidia as rice root endophyte in GAZ. Abundance of Firmicutes have been seen in zones like CSZ, RLZ and TTAZ which are categorised as nutrient low groups. As reported by Mukhtar et al., 2019, this phylum is found in abundance in the rhizosphere of plants grown in extreme environments (Mukhtar et al., 2019). Firmicutes was also reported under high saline conditions (Zhang et al., 2020) and hence its representative class Clostridia is dominant in CSZ as 95% abundance of CSZ is due to Clostridia. This class has also been reported as an abundant phyla in rice seed (Raj et al., 2019) as well as is found in rice soil (Hayat et al., 2010). Association of Alphaproteobacteria have been found to be enriched in soils having higher N supply (Fierer et al., 2011) as well as organic carbon (Kim et al., 2014). Alphaproteobacteria was mostly dominant in NHZ which is characterised based on both high TOC and N. Abundance of this class in NHZ can also be related to its plant growth promoting properties. As per Hardoim et al., 2011, this class occur in rice roots endophytes largely irrespective of plant genotype because their universal adaption in rice is believed to be associated with their beneficial functions which might be the driving force for their selection (Hardoim et al., 2011). It has been reported that Planctomycetes have more stable and resistant life-strategy (De León-Lorenzana et al., 2018) and hence are found in zones like RLZ, NHZ and TTAZ where soil is not perfectly suitable for cultivation. Planctomycetes is also reported to be abundant in drought condition (Dai et al., 2019) which is a characteristic of RLZ.
The most common genera found as rice root endophytes among all the sampled zones are Clostridium sensu stricto (33.7%), Sulfurospirillum (8.3%), Uliginosibacterium (7.7%), Aeromonas (5.2%), Veillonellaceae Incertae sedis (2.8%,) Acidaminococcaceae Incertae sedis (2.4%), Lachnospiraceae Incertae sedis (1.6%), Bacillus (1.1%), Burkholderiaceae Incertae sedis (1.1%),Shewanella (1%), Massilia (1%), etc. These genera are already reported as rice endophytes (Hardoim et al., 2011; Kunda et al., 2018; Walitang et al., 2017) and their presence in all the sampling sites may be because they represent the core endophytic microbiomes of rice in West Bengal. Most of these genera are known diazotrophs and are reported to have plant growth promoting properties. They being present inside plants may help their host by promoting growth or tolerate stressful conditions or produce allelopathic substances to compete with other species. Genus like Massilia is reported to reduce nitrate have an important role in nitrogen cycle and thus act as a plant growth promoting bacteria (Wemheuer et al., 2017). This genus also induces production of napthoquinones like alkannin and shikowin in root cultures of a medicinal plant and thus possesses anti-microbial properties (Rat et al., 2021). Another genus, Shewanella is reported to alleviate salt stress (Paul & Lade, 2014). Moreover, as revealed in our analysis, two zones have some specific genera that are unique to them. The unique genera for GAZ are – Dickeya, Lactococcus and Prevotellaceae Incertae sedis while for TTAZ they are - Azonexus, Pectobacterium, and Diplorickettsia.Dickeya and Lactococcus are known rice endophytes (Kunda et al., 2018; Marag & Suman, 2018) and Prevotellaceae has no known functions in plants although it has been reported as an endophyte of fruit Pitaya (Ren et al., 2018). Among the unique genera of TTAZ, Azonexus is reported as rice endophytes (Kunda et al., 2018) but the other two genera are not reported as rice endophytes so far, Diplorickettsia is reported as an insect endosymbiont (Mathew et al., 2012) and Pectobacterium as plant pathogen (Davidsson et al., 2013). Pectobacterium possess a large number of plant cell-wall degrading enzymes (Davidsson et al., 2013) and thus may have colonised rice roots. Maybe these endophytes are signatorial bacterial genera of the particular zones whose functions are yet to be discovered.
According to dot plot, out of the 17 differentially significant OTUs many are reported as known plant growth promoting bacteria (PGPB). Genera such as Clostridium, Bacillus, Comamonas, Aeromonas, Aquitalea, Burkholderia and Enterobacter have plant growth promoting abilities by fixing nitrogen, solubilising phosphorus, potassium, zinc, producing phytohormones like IAA as well as can protect plant from pathogen attack by producing HCN and siderophore (Ishizawa et al., 2017; Mendes et al., 2013; Nath Yadav et al., 2017; Radhakrishnan et al., 2017; Saxena et al., 2020; J. Wang et al., 2020). Clodtridium apart from being a plant growth promoter (Doni et al., 2014; Emami et al., 2019) is also reported to tolerate and mitigate soil salinity (Rahman et al., 2017). Interaction of these bacteria with rice roots can contribute to the growth of the plants and can also help plants to grow under normal as well as stressful conditions.
It is worth mentioning that due to some logistical problems we were unable to complete sampling at once. We have done sampling in a span of two years in two different seasons. This could also contribute to any differences in microbial community composition. Since all the samples were not collected in the same season any direct relation of seasonal variation with endophytic composition could not be drawn.