The tea agro-ecosystems face persistent problems of disease incidences such as blister blight, grey blight, brown blight and red rust, as well as attacks of diverse tea pests (Sen et al. 2020). The use of inorganic chemical-based fertilizers and broad-spectrum organosynthetic insecticides is a common approach to control these pathogens and to sustain maximum productivity (Lin et al. 2019). Rampant applications of the inorganic compounds detrimentally affect soil fertility as well as native soil-microbe activities (Meena et al. 2020). Inorganic agricultural inputs used in tea cultivations carry additional risks of deteriorating air and groundwater quality; imparting undesirable residual effect on the processed tea; emergence and re-emergence of primary pests leading to concomitant episodes of primary and secondary pest outbreaks; variations in antagonistic action, and development of resistance mechanisms in the pests (Lin et al. 2019). Pesticides such as organochlorides, organophosphates, carbamates and synthetic pyrethroids that are regularly used in conventional intensive tea cultivation system also exert toxic effects on non-target species and natural regulatory agents, and pose associated health hazards to human handlers and grazing animals (Bhattacharyya and Kanrar 2013). As an alternative, biological approaches can be implemented in organic tea farming practices for promoting plant growth and yield as well as rejuvenating soil health and productivity (Han et al. 2018). Organic farming involving bioinocula has advantages of high profitability, cost reduction, financial subsidies, climate adaptability and lesser health risks with promises of sustainable crop production (Morshedi et al. 2017). These approaches can modulate above- and below-ground microbial diversity-composition towards the formation of long-term disease suppressive soils (Lupatini et al. 2017).
Microbial metabolism attributed to majority of the COG annotations followed by ‘Cellular processing’ but the relative abundance of metabolic genes, pathways, orthologies was more in rhizospheric metagenome than in the endospheric one. An increase in the relative abundance of few agriculturally important traits (genes and metabolic pathways) in the endosphere suggested that the plant system can selectively permit the penetration and establishment of an elite group of symbionts which help the plant in overcoming various stress conditions. For instance, KEGG-based analogies showed that abundance of several previously reported genes related to vitamin B6 and B9 metabolism (ko00750, ko00790), steroid biosynthesis (ko00100), glutathione metabolism (ko00480) etc. was relatively higher in the endosphere than in the rhizosphere. These pathways and metabolisms improve plant biomass production and empower the plant defense mechanism against various biotic and abiotic stress conditions (Zhu et al. 1999; del Barrio-Duque et al. 2019). Endophytic bacteria Sphingomonas SaMR12 enhanced cadmium (Cd) accumulation and upregulated glutathione synthase gene expression in Sedum alfredii Hance (Pan et al. 2016). Another endophytic bacteria Bacillus subtilis CBR05 induced Pyridoxine (vit. B9) biosynthesis (PDX) genes in tomato plants under the pathogenic attack of Xanthomonas campestris pv. vesicatoria (Chandrasekaran et al. 2019). In bacteria, the ATP binding cassette (ABC) superfamily comprises mainly ATP-dependent pump proteins dedicated towards active mobilization of metabolites (Lewis et al. 2012). These superfamily proteins mobilize hormones, lipids, peptides, and primary as well as secondary metabolites etc. and therefore, have immense significance in plant growth and adaptations (Vasiliou et al. 2009). A few members also have intrinsic channel properties with functions related to drug resistance, heavy metal detoxification etc. (Lewis et al. 2012). In addition, these proteins also have defense-related implications as evidenced by the presence of an early defense gene in rice which is induced in presence of salicylic acid and phytopathogenic Magnaporthe grisea (Lee et al. 2005). The two-component system consists of a first component protein with transmembrane domain and a second component protein with phosphotransfer histidine kinase property (Zschiedrich et al. 2016). These systems play an important role in plant-microbe interaction by recognizing the active metabolites present in the root exudates and thereby facilitates chemotaxic movement of the microbial population towards the root surface. The GacS/GacA present in pseudomonads and enteric bacteria exemplifies such systems that are responsible for microbial root colonization (Heeb and Haas 2001).
Based on Shannon’s diversity indices, it was evident that the microbial richness and diversity was greater in the rhizosphere than the endosphere which could definitely be attributed to the rhizosphere being the primary and dynamic interface of plant-microbe interactions (Mohanram and Kumar 2019). These interacting microbes produce an array of hormones and metabolites which are known to signal and promote plant growth, antagonize pathogens and increase availability of essential micro- and macronutrients for plant uptake (Singh et al. 2019). In this study, we observed that the tea rhizosphere harbored a diverse array of the soil microbiota such as Arthrobacter, Azospirillum, Azotobacter, Bacillus, Burkholderia, Caulobacter, Chromobacterium, Enterobacter, Erwinia, Flavobacterium, Klebsiella, Micrococcus, Pseudomonas, Serratia, Xanthomonas etc.; while, the endosphere harbored functionally and strategically important subset of the rhizospheric microbiota viz., symbiotic Bradyrhizobium, Mesorhizobium, Rhizobium and non-symbiotic rhizobacteria viz., Azotobacter, Azospirillum, Azomonas, Bacillus, Klebsiella, Pseudomonas. Most of these bacteria have been reported to either promote plant growth or protect the plants from pests and diseases (Aeron et al. 2011; Fernández-González et al. 2017; Verma et al. 2019). Microorganisms such as rhizobia, mycorrhizal fungi, actinomycetes, diazotrophic are already known to be aggressive colonizers which mobilize and allocate nutrients through symbiotic and non-symbiotic associations with plant roots (Barea et al. 2005). Healthy stature of the tea bushes indicated to the rhizosphere-microbial taxa playing a key role in suppressing disease incidence, nutrient cycling and healthy status of the soil. Rhizobacteria viz., Azotobacter, Azospirillum, Bacillus amyloliquefaciens, Bacillus pumilus, Serratia marcescens and Pseudomonas have already been tested for PGP activities in tea plantations of North East India. Most of these bacteria carried promising plant growth promoting traits such as solubilization of minerals (zinc and phosphate), production of siderophore and indole acetic acid and exhibition of biocontrol activities which successfully contributed right from tea seed germination to seedling growth in nursery and subsequent field establishment (Nath et al. 2013; Bhattacharyya and Sarmah 2018).
The potentials of free-living N2-fixing bacteria such as Azotobacter (aerobic) and Azospirillum (anaerobic, microaerobic), that play an important role in nitrogen geocycle have been realized in the form of organic amendments in tea soils (Gebrewold 2018). These bacteria derive nutrients from the root exudates and in return, efficiently provide fixed nitrogen to the host plant (Tejera et al. 2005). Besides fixing atmospheric nitrogen, Azotobacter also secretes important phytohormones (IAA, gibberellic acid and cytokinins), vitamins (thiamine, riboflavin) and various antipathogenic agents which promote growth as well as suppress disease incidences (Sumbul et al. 2020). Azotobacter chroococcum can aid in seed germination and change root architecture in response to phytopathogen attacks (Romero-Perdomo et al. 2017). Azospirillum is another important bacterium which exhibited nutrient supplementation in vegetatively propagated tea seedlings and cuttings (Thomas et al. 2010). Azospirillum once inoculated into soil, multiplies and propagates rapidly in the microaerobic sites of plant roots, intercellular spaces and symbiotically assist the host plant in nutrient uptake (Fukami et al. 2018). Azospirillum is also reported to produce an array of bioactive compounds such as phytohormones (IAA and GA), siderophores, poly β-hydroxybutyrate which can influence root architecture through mineral nutrition, root hair development etc. (Egamberdieva et al. 2017). Both Azotobacter and Azospirillum are suitable candidates for biofertilizer development targeted for better tea productivity; while Azotobacter is suited for aerated, light textured soil, Azospirillum is indicated for waterlogged hard-textured soils (Aquilanti et al. 2004).
Phosphorus (P) is considered as a vital nutrient essential for plant growth and immunity status. Despite of its high abundance in soils, the availability of this non-metallic ions remain low due to insolubility issues (Dordas 2008). Phosphate solubilization ability of rhizosphere microorganisms is considered to be one of the most important traits associated with plant phosphate nutrient cycling. Such kind of microorganisms under the aegis of ‘phosphate solubilizing microbes (PSM)’ convert the insoluble forms of phosphorus into soluble form through acidification, organic acid secretion and chelation of bound cations (Alori et al. 2017). The most promising candidate phosphate solubilizing tea rhizobacterial species revealed in our study were Azotobacter chroococcum, Bacillus circulans, Bradyrhizobium japonica, Pseudomonas chlororaphis and Pseudomonas putida. In a P-deficient soil, PSMs can effectively modulate root architecture through the promotion of lateral root and root hair system (Péret et al. 2014). These bacteria can survive and thrive in soils with a neutral pH range (6.0-7.5). Among the fungi, Aspergillus sp., Penicillium sp. and Schwanniomyces occidentalis are considered to be the predominant members that convert insoluble inorganic phosphate in soil into soluble plant-usable forms (Gizaw et al. 2017; Alori et al. 2017). Earlier, culture-dependent approaches revealed that Bacillus subtilis and Aspergillus niger were most dominant in P-deficient acidic soils under tea cultivation (Bhattacharyya and Sarmah 2018). A considerable amount of soil phosphorus is also found in the form of phytate (inositol phosphate) which is a complex of the phosphorus with other minerals (Findenegg and Nelemans 1993). Most of the Bacillus species identified in this study have been reported to produce phytase, an enzyme which cleaves phytate into soluble form (Borgi et al. 2015). The abundance and efficacy of phytase producing microbes has been already assessed in tea garden soils of NE India (Pramanik et al. 2014).
Microbial pesticides intended against a range phytopathogens including tea pests are an important component of integrated pest and disease management system in tea cultivation (Idris et al. 2020). Several microbial species revealed in this metagenomics based study viz., Bacillus spp., Pseudomonas spp., Aspergillus sp., Beauveria bassiana, Gliocladium sp., Metarhizium anisopliae, Paecilomyces sp., Penicillium sp., Trichoderma spp., Lecanicillium, Streptomyces sp. have reported biopesticidal activities and as such, their introduction as biocontrol agents have the potential to elevate tea productivity (Krauss et al. 2004; Scheepmaker and Butt 2010; Sandhu et al. 2012). At microbe-microbe interaction level, the biopesticides suppress the growth of phytopathogens either through the secretion of antimicrobial agents (antibiotics, hydrogen cyanide, hydrolytic enzymes, volatile and non-volatile compounds etc.) or by outcompeting through better nutrient acquisition (mineral solubilization, iron chelation through siderophore etc.) (Cheong et al. 2017). Experimental evidences suggest that most of these activities occur in tandem and in a concerted way towards the suppression of phytopathogens and enhanced host-plant growth (Vurukonda et al. 2018). These fungi could be broadly categorized into three groups: (A) ubiquitous soil inhabitant with no- or latent pathogenicity (B) phytopathogens responsible for various plant diseases (C) ecologically-important fungi with potential implications as plant growth promoters. Species of the genus Tilletiopsis, Humicola, Chaetomium, Alternaria, Curvularia and Xylaria have been reported to be ubiquitous in various environmental metrices such as dead or living plant materials, decomposing soils etc. Most members from this group carry potentials to cause serious plant diseases as revealed by several confirmed reports surfacing recently. The second group consisted of members belonging to genus Nigrospora, Cladosporium, Fusarium, Lecanicillium, Phoma, Phomopsis, Colletotrichum and Rhizoctonia which are essentially phytopathogens responsible for plant diseases such as leaf spots, scab, rot, wilt etc.
The third group consisting of ecologically-important fungi exhibit diverse functionality such as biocontrol activities (mycoparasitic activity: Coniothyrium, Gliocladium, Trichoderma and Penicillium; entomopathogenic activity: Beauveria, Metarhizium and Lecanicillium) and plant-growth promoting activities (Aspergillus and Trichoderma). Coniothyrium minitans (a coelomycete) is a potential biocontrol candidate with mycoparasitic activity against Sclerotinia sclerotiorum (Zhao et al. 2020). Sclerotinia sclerotiorum under favorable environment can cause white mold disease or blossom blight disease. Many studies have reported that C. minitans can inhibit mycelial growth and sclerotia formation of Sclerotinia ascospores thereby suppressing leaf blight infection (Smolińska and Kowalska 2018). Gliocladium virens is another important mycoparasitic fungus which exhibits broad-spectrum biocontrol activity (van Tilburg and Thomas 1993). This common saprophytic fungus is known to show strong hyperparasitism against plant-pathogens viz., Rhizoctonia solani, Sclerotium rolfsii and Pythium aphanidermatum (Sreenivasaprasad and Manibhushanrao 1990). The presence of enzymes viz., 1-glucanase, N-acetylglucosaminidase, lipase, and proteinase makes it a successful wide-spectrum biocontrol agent (van Tilburg and Thomas 1993).
Entomopathogenic fungi such as Beauveria and Metarhizium are very much effective in controlling insect population and are considered as dominant candidates in various biopesticide programme worldwide (Kirkland et al. 2004). Members of the genus Beauveria (especially, Beauveria bassiana) have the ability to colonize multiple plant hosts without causing any apparent disease or showing secondary symptoms, yet retaining the capacity to infect insects and invoke induced systemic resistance (Wei et al. 2020). A field trial in Northeast India has reported Beauveria bassiana to be effective against Helopeltis theivora (a sucking pest of tea-leaves) and other tea pests (Borkakati and Saikia 2019; Kumhar et al. 2020). Another entomopathogenic fungus Metarhizium (especially, M. anisopliae) is also regularly used as a broad spectrum insect biopesticide (Singha et al. 2011). In Brazil, this fungal species is commercially produced in large scale for controlling spittlebugs (adults and nymphs) in sugarcane cultivation (Iwanicki et al. 2019). Metarhizium biopesticide is considered as one of the most successful biocontrol agents in the world with its application in about 2 million hectares of sugarcane cultivation (Mascarin et al. 2019). In Assam, field application of this biopesticide has been found to be effective against tea wood-eating termite infestation (Roy et al. 2020). Similar to Beauveria bassiana and Metarhizium anisopliae, another entomopathogen, Lecanicillium spp. (formerly known as Verticillium lecanii) is also regularly used as a biopesticide against insect pests and pathogenic fungi. This fungus with sheer mechanical force and hydrolytic enzymes can directly invade into the insect integument or the cell wall of the target fungi (Xie et al. 2015; Reddy and Sahotra 2020).
In terms of plant-growth promoting endofungal agents, it is generally observed that fungi belonging to Trichoderma genus interact with plants by inducing their defense system and promoting plant growth (Zhang et al. 2016). The endophytic ability of Trichoderma has been reported in many plants like maize, cucumber, cotton and tomato (Yuan et al. 2017; Contreras-Cornejo et al. 2018; Harman et al. 2019). Trichoderma can colonize the intracellular spaces, as well as the spaces between the plasma membrane and the cell wall of the root tissue (Ramírez-Valdespino et al. 2019). Once in the endosphere, the fungus can promote seed germination, development of root, shoot and biomass, increase tiller number and overall crop yield (Hajieghrari and Mohammadi 2016). Trichoderma can not only modulate levels of auxin through a crosstalk with MAP-kinases based signaling pathway but also contributes its own hormone or hormone intermediates thereby synergistically influencing the root architecture (Viterbo et al. 2005; Brotman et al. 2013). In addition, Trichoderma can exhibit antagonistic activities against its adversaries through competition for space and nutrients, antibiosis and mycoparasitism (Chen et al. 2016). Aspergillus is a group of multifaceted fungi which are known to exert additive plant growth promoting effects when applied in combination with other PGP microorganisms. The additive effects of Aspergillus strain NPF7 have been found prominent in wheat and chickpea with significant increase in germination index and, root and shoot length (Pandya et al. 2018).
In conclusion, our present culture-independent study provides a comparative insight into the microbial diversity and function associated with an organically-grown tea ecosystem of Assam. EggNOG based COG analysis of the metagenomes revealed that microbial metabolism was most prevalent in both the rhizosphere and endosphere environments. The endospheric metagenome carried a higher relative abundance of genes linked to ‘information storage and processing’ which is integral to any kind of plant-microbe interaction (mutualism or antagonism). KOBAS-mediated KEGG Orthology analysis of the metagenomes showed that the ‘ABC transporter pathway’ genes were highly abundant followed by the ‘Two-component system pathway’ related genes in both the environments. As compared to the rhizosphere, the endosphere contained higher abundances of PMI-important genes linked to bacterial chemotaxis, vitamin B6 and folate metabolism, glutathione metabolism etc. The Shannon diversity indices confirmed higher bacterial diversity in the rhizosphere than in the endosphere. The endosphere bacterial microflora was mostly dominated by Proteobacteria (relative abundance 89.17%). Bacterial species such as Serratia, Methylobacterium, Yersinia, Burkholderia etc. populated the endosphere. The endosphere also had minor representations of few agriculturally important symbiotic and non-symbiotic taxa such as Bradyrhizobium, Mesorhizobium, Rhizobium, Azotobacter, Azospirillum, Azomonas, Bacillus, Klebsiella, Pseudomonas etc. The fungal population could be roughly divided into three categories (i) ubiquitous soil inhabitant/potent pathogenicity; (ii) phytopathogens; (iii) agriculturally important fungi. The presence of few fungal species such as Chaetomium, Coniothyrium, Cladosporium etc. in the endosphere could have resulted from hyphal penetration into the plant tissues. Overall, a vast array of agronomically important microbes linked to phytohormone secretion, nitrogen fixation, phosphate and potash solubilization, biotic-abiotic stress tolerance and biocontrol (entomopathogenesis) activity were detected.
Successful organic tea cultivation must have to circumvent the dependence on inorganic inputs and adopt an eco-friendly yet cost-effective pest and nutrient management system. Microbe-based plant-growth promoting and biocontrol agents have the potentials to be the indispensible candidates in integrated pest management practices towards overcoming the issues of economic and environmental sustainability in tea cultivation. To this, native tea-soil microflora can be targeted for the isolation and screening of highly function-specific candidate microbes. One major limitation of this current study is that despite the exposition of microbial diversity, function and indicators, we could not perform an effective screening procedure to ascertain various PGP activities. Nevertheless, we expect that this study will pave a way for identifying natural enemies prevalent in tea plantation as well as towards prospecting bioinocula for tea plant growth promotion and plant-protection from various pests and diseases. Further studies on the cross-talks of a tripartite interaction encompassing tea plants, bioinoculum and natural enemies can be considered for the promotion of a sustainable and healthy tea ecosystem.