Plant-microbe interactions play an important role in plant growth, health and productivity. Despite the importance of plant growth-promoting microbes in plants, particularly in the rhizosphere, microbial species associated with resurrection plants have not been explored (Tebele et al. 2021). Our study provides one of the first high-resolution characterisations of the root microbiome of a resurrection plant. We characterised the bacterial and fungal microbiomes associated with the resurrection plant M. flabellifolia under air-dry stage (≤ 10% RWC) and investigated how the microbial community composition varied across compartments from bulk soil to rhizosphere, to endosphere. Based on our findings here, we speculate on the role of these communities and their contribution to the unique resilience of M. flabellifolia. Our analyses revealed substantial differences in the microbiome of the bulk, rhizosphere and endosphere compartment niches. A substantial species richness and microbial dynamism were observed in the bulk soil and, to a higher degree, in the rhizosphere compartment. In contrast, the endosphere microbiome was notably reduced in such properties, with only a few select species being present, suggesting that strong filtering occurs at the soil-to-root interface. These findings suggest that regardless of extreme drought conditions, M. flabellifolia is exposed to a diverse microbial community and may actively recruit beneficial microbial assemblages to different compartment niches.
The most dominant bacterial phyla across all compartments were Acidobacteriota, Actinobacteria, Chloroflexi, Planctomycetota, Proteobacteria and WPS-2, and fungal phyla were Ascomycota, Basidiomycota, Mortierellomycota and Mucoromycota. These results are parallel to previous studies that also reported the presence of Actinobacteria, Chloroflexi and Planctomycetes in Ramonda species as well as in chickpea, sorghum and other plant hosts (Ahkami et al. 2017; Đokić et al. 2010; Xu et al. 2018; Yadav et al. 2021). However, there are notable differences between the findings on Ramonda spp and the current study. Most importantly, the presence of Flavobacterium, Mucilaginibacter and Nocardioides in M. flabellifolia but absent in the Ramonda species’ rhizosphere, suggests that these microbes could be environmentally or geographically determined (M. flabellifolia occurs in southern Africa whereas Ramonda spp. occurs in Eurasia). In desiccation sensitive species, such as sorghum, Xu et al. (2018) have reported a low abundance of Acidobacteriota in roots under drought stress. On contrary, our study shows a high enrichment of Acidobacteriota in the endosphere of M. flabellifolia suggesting a possible role for these taxa in coping with extreme environmental stresses. The abundance of these distinctive microbial strains in M. flabellifolia demonstrates that extreme environmental stress restructures the plant microbiome.
Drought stress undoubtedly introduces physiological, metabolic, and genetic responses while also impacting nutrient availability in both microbiome and host plants. Bulk soil samples were clustered together more strongly compared to the rhizosphere compartment (Fig. 5). There was variability in the rhizosphere samples, this suggests that the rhizosphere of each plant hosts a slightly different microbial composition and this could be due to the landscape topography of the natural environment. We identified differences in soil physicochemical factors that significantly drive microbiome composition across soil compartments. The bioavailability of micronutrients such as Ca, Cu, Mg, Mn, Na, and Zn in the rhizosphere soil of M. flabellifolia under drought stress seems to impact microbiome abundance and hints at an intriguing plant-microbe-soil interaction. It has been reported that arbuscular mycorrhiza enhances the bioavailability of micronutrients through extraradical hyphae (Srinivasagam et al. 2013).
The enrichment of particular bacteria and fungi under extreme drought could indicate a role for those microbes in supporting drought tolerance through close linkage with the host plant. We identified numerous taxa that were uniquely enriched in different compartment niches of M. flabellifolia. The putative function of these enriched taxa could point towards a role in drought tolerance. The M. flabellifolia microbiome is extremely complex consisting of over 900 unique bacterial and fungal taxa in each compartment. To summarize this complexity, we highlight selected taxa from each compartment niche and discuss their possible role in mitigating drought stress.
This study found a significant enrichment of monoderm (Actinobacteria, Chloroflexi, Firmicutes) and diderm (Acidobacteria, Bacteriodetes and Proteobacteria) lineages in rhizosphere and endosphere compared to the bulk soil, except for Chloroflexi, which showed high enrichment in bulk soil. The bulk soil compartment of the M. flabellifolia microbiome hosted an abundance of bacterial taxa in Coleofascicus, Flavisolibacter and Leptolyngbya. These bacterial genera are known to be resistant to drought stress and can improve the physical and biological conditions of rhizosheath soil (Liu et al. 2021; Moreira et al. 2021). The finding of cyanobacteria in bulk soil is significant, as these species are normally restricted to the upper surface of soil crusts (due to their photosynthetic ability). Such species have been shown to play a significant role in soil stabilization and the addition of organic carbon (Gao et al. 2020b) and their role in the bulk soil could be significant. The significantly enriched fungal taxa in bulk soil were Epicoccum dendrobii, Metarhizium lepidiotae and Mycenastrum. These enriched fungal genera also have multiple functions related to resilience. For instance, Epicoccum dendrobii secrete penicillin to inhibit pathogens (Bian et al. 2021) and Metarhizium spp. is an endophyte that proliferates propagule levels in the rhizosphere (Steinwender et al. 2015). Remarkably, Metarhizium spp was found in the bulk soil of M. flabellifolia, instead of the endosphere compared to previous studies. Interestingly, our study revealed that the bulk soil compartment hosts unique, beneficial bacterial and fungal taxa, and this might be due to the ecological niche of M. flabellifolia. The bulk compartment serves as the major source for microbial recruitment for the rhizosphere.
A plethora of bacterial and fungal taxa was significantly enriched in the rhizosphere compared to other compartments. The bacterial genera include Flavobacterium, Nocardioides and Streptomyces. These bacterial genera have the plant growth-promoting and drought tolerance traits ranging from the secretion of phytohormones and production of primary metabolites to nitrogen fixation, phosphate solubilisation and resistance to numerous heavy metals (Cardoso et al. 2018; Khan et al. 2014; Kielak et al. 2016; Yandigeri et al. 2012; Yang et al. 2019; Zhang et al. 2020). This suggests that these species play a vital role during drought stress in M. flabellifolia by secreting, inter alia, phytohormones such as indole-3-acetic acid (IAA), which have been proposed to improve drought tolerance by enhancing root growth (Zhang et al. 2020). Moreover, high amounts of iron in the rhizosphere soil might be associated with the abundance of Acidicapsa species (Fig. S3A), which produces IAA (Kielak et al. 2016). The enriched fungal taxa included Actinomucor, Aspergillus and Penicillium spp. Actinomucor sp. has been shown to produce abundant quantities of proteases, lipases and amylases that hydrolyse the components of sorghum seeds (Gao et al. 2020a). Therefore, the presence of hydrolytic-enzyme producing species under drought conditions in the rhizosphere suggests that they might be involved in the rearrangement of metabolism through the selective degradation of short-lived proteins (Vaseva et al. 2012). The presence of trehalose in M. flabellifolia's leaves has been related to fungal species, and trehalose is a desiccation-induced osmoticum in resurrection plants (Farrant 2000; Moore et al. 2011). The selection of rhizosphere-microbiome from the bulk soil is likely influenced by the roots of M. flabellifolia. Beneficial microbes are further filtered into the endosphere compartment.
The endosphere of M. flabellifolia was significantly less diverse in microbial presence than the other two compartments, suggesting that stringent filtering occurs at the soil-root interface. Bacterial genera significantly enriched in the endosphere were Mucilaginibacter, TMa7 and unclassified Pirellulaceae and fungal taxa were Cladosporium and Penicillium. The Mucilaginibacter and Cladosporium genera are known to produce exopolysaccharides which form biofilms and adhere to root surfaces (Fan et al. 2018; Kielak et al. 2016; Mahapatra and Banerjee 2013). Exopolysaccharides also act as defence mechanisms against drought by sustaining a hydrated microenvironment and reducing water loss (Morcillo and Manzanera 2021). As the rhizosphere soil moisture was significantly greater than bulk soil, it is possible that the presence of exopolysaccharide-producing microbes may slow water loss and alleviate drought stress. These results provide further support for the hypothesis that root-associated microorganisms confer drought tolerance to their host plant. In addition, bacterial (Mucilaginibacter) and fungal genera (Penicillium and Aspergillus) are known to be resistant to heavy metals and remove or absorb Zn2+, Cd2+ and Cu2+ (Anahid et al. 2011; Fan et al. 2018). This suggests that these species do not only tolerate drought stress, but also may play a role in minimising heavy metal toxicity in M. flabellifolia.