The rhizosphere is characterised by high microbial activity and an array of complex and dynamic physical, chemical and biological interactions termed the ‘rhizosphere effect’ (Helliwell et al. 2019; Hiltner 1904). These interactions play a crucial role in plant nutrition (Raaijmakers et al. 2009; Sørensen 1997) and ecosystem functioning in terrestrial ecosystems (Singh et al. 2004). Microbes associated with the rhizosphere (i.e. the rhizosphere microbiome or rhizobiome) represent a vast reservoir of microbial diversity that includes fungi, bacteria, nematodes, protozoa, algae, viruses and arthropods (Berg & Smalla 2009; Bonkowski et al. 2000; Bonkowski, Villenave & Griffiths 2009; Bulgarelli et al. 2013). Plant species actively determine the composition of their rhizobiome by secreting root exudates that attract or repel specific bacteria from the surrounding bulk soil (Berg & Smalla 2009; Doornbos, Van Loon & Bakker 2012). The composition of the rhizobiome is thus determined by numerous edaphic variables of the immediately surrounding bulk soil such as the soil mineralogy, soil moisture content and pH (Bardgett et al. 2005; Fierer & Jackson 2006; Myers et al. 2001). However, comparative studies on the effect of specific edaphic variables on the composition of the rhizobiome are rare because of a lack of suitable study sites where the influence of different soil types on the same plant species in close proximity can be compared effectively.

The Kruger National Park (KNP), located in the north-eastern part of South Africa, is one of the largest natural reserves in Africa, with vast areas that are mostly pristine (Carruthers 2017). This area is mostly subtropical, with deciduous savanna landscapes inhabited by diverse herbivore and carnivore populations, leading to the integrity of most ecosystems within the park (Rughöft et al. 2016). To study these pristine areas, four ‘research supersites’ were chosen (Figure 1), each representing unique ecological, geological and climatic features within the park (Smit et al. 2013), making these sites ideal to study microbial diversity.

The Stevenson–Hamilton supersite (S-Hs) in the KNP is a granite catena (a hydrologically linked hill slope with dynamic solute, particle and colloid transport systems along the slope) (Rughöft et al. 2016) located 7 km south of Skukuza (Smit et al. 2013). The granite catena of the S-Hs is characterised by pronounced differences in soil differentiation along the slope of the catena with sandy soils in the upper region, clay accumulation along the slope and clayey, duplex soils with sporadic sodic patches on the foot slope (Levick et al. 2010) (Figure 1). The site was shown to have a unique bacterial community when compared to other soil ecosystems (Rughöft et al. 2016), and therefore it is ideal in terms of conducting a comparative study of the diversity of the rhizobiome of the same plant species growing in differing soil types. In most soil ecosystems, Acidobacteria and (Alpha-)Proteobacteria are the two dominant phyla, usually accompanied by Actinobacteria as another abundant group (Janssen 2006; Lauber et al. 2009; Nacke et al. 2011; Prober et al. 2015; Rughöft et al. 2016). However, the most abundant phyla in the granite soils from the S-Hs were Actinobacteria, Chloroflexi and Firmicutes (Rughöft et al. 2016). Acidobacteria, Planctomycetes, Alphaproteobacteria and Deltaproteobacteria were the phyla that most frequently occurred at a lower relative abundance (Rughöft et al. 2016).

The first objective of the present study was to assess the structural and functional diversity of bacteria in the respective rhizobiomes of the three most prevalent herbaceous plant species growing in the S-Hs in sandy soil at the top (Clovelly) and clay soil at the bottom (Sterkspruit) of the slope. A second objective was to determine whether the structural diversity of bacteria in each plant species was influenced by the surrounding bulk soil – such as the sandy soil at the top and clay soil at the bottom of the slope. A third objective was to determine the most dominant bacterial phyla in the two respective sites and to compare this with the findings of Rughöft et al. (2016). This is the first study to investigate the diversity of the bacterial rhizobiome of Kyphocarpa angustifolia (Amaranthaceae, Caryophyllales), Melhania acuminata (Malvaceae, Malvales) and Sida cordifolia (Malvaceae, Malvales) as well as to ascertain how different soil types affect microbial diversity.

The rhizobiome is shaped by complex interactions between the soil type, plant species and root zone location (Marschner et al. 2001). Although there were no statistically significant differences between the rhizobiome of the Clovelly (top) and Sterkspruit (bottom) soil types or between the three different plant species, it was evident that the soil type and plant species did have a role in shaping the rhizobiome, as unique OTUs were observed in all five different rhizosphere samples. This has previously been observed, with soil type (Bachman & Kinzel 1992) and plant species (Wieland, Neumann & Backhaus 2001) both being reported as the driving factor for microbial soil and rhizosphere diversity. Although the sampling area spanned only 200 m, differences in plant diversity were observed in previous studies of the plant diversity of the S-Hs granite catena (Theron, Van Aardt & Du Preez 2020). This was also reflected in the bulk soil microbiome diversity of the S-Hs granite catena (Rughöft et al. 2016) along the slope and is thus expected for the rhizobiome of the resident plants. There was, however, a significant amount of shared OTUs (47.72% of the total number of sequences) regardless of the soil type and specific plant rhizosphere they occurred in.

The invasive plant, S. cordifolia, is known to have antimicrobial properties (Pallela et al. 2018; Thangavel, Raveendran & Kathirvel 2006); however, there is no information available on its rhizobiome. Similarly, now information is available for the rhizobiome of the two native plants, K. angustifolia and M. acuminata. The soil surrounding the rhizosphere serves as the main reservoir for the microorganisms that form part of the rhizobiome (Berg & Smalla 2009). Actinobacteria and Proteobacteria were amongst the most dominant phyla in all rhizosphere samples, whilst Verrucomicrobia and Planctomycetes were the dominant phyla in the rhizosphere samples from K. angustifolia at the Sterkspruit site (bottom), Verrucomicrobia in K. angustifolia, M. acuminata and S. cordifolia at the Clovelly site (top) and Firmicutes and Chloroflexi in the S. cordifolia rhizosphere samples from the Sterkspruit site (bottom) (Figure 3). Previous studies on the soil in the KNP S-Hs showed that Actinobacteria, Chloroflexi and Firmicutes were dominant, whilst Planctomycetes, Proteobacteria and Verrucomicrobia most frequently had the lowest relative abundance (Rughöft et al. 2016). Therefore, although the abundance of specific bacterial phyla may differ between the rhizobiome and bulk soil (Rughöft et al. 2016), our results are consistent with those of Berg and Smalla (2009), who suggested that root exudates in the rhizosphere select specific microbes from the surrounding bulk soil. To our knowledge, there are no studies comparing the bacterial rhizobiome of native and invasive plants within the study site. Gryzenhout et al. (2020), however, studied the fungal rhizobiome of the same plant species in S-Hs and found that S. cordifolia only attracted half the number of OTUs compared to the native plants, M. acuminata and K. angustifolia, which could be attributed to the antimicrobial properties of S. cordifolia. However, this was not observed for the bacterial rhizobiome.

Actinobacteria, Chloroflexi, Firmicutes and Verrucomicrobia are well adapted to survival in the nutrient- and water-poor soils of the KNP S-Hs (Rughöft et al. 2016). The increase in abundance of Proteobacteria in the rhizosphere compared to the surrounding soil could be because they have been shown to benefit from the higher nutrient levels, especially carbon availability (Leff et al. 2015; Wang, Ji & Gao 2016), that will be expected because of root exudates in the rhizosphere (Haichar et al. 2014). Planctomycetes and Verrucomicrobia, although not dominant in all samples, were present (>1%) in all samples and have been reported in other studies on rhizobiomes (Gkarmiri et al. 2017; Zeng et al. 2018; Zhou et al. 2019). Similar to Proteobacteria, Planctomycetes and Verrucomicrobia could benefit from the increased availability of nutrients in the rhizosphere. Planctomycetes are known to play a role in carbohydrate metabolism (Fuerst 1995), and a lineage in the Planctomycetes has also been shown to play a role in anaerobic oxidation of ammonium (Strous et al. 1999). Planctomycetes present in the rhizosphere and bulk soil samples from the S-Hs granite catena could therefore play a role in the oxidation of ammonium reported in this soil (Rughöft et al. 2016).

According to the bacterial functional group analyses, soil type (i.e. Clovelly vs. Sterkspruit) (Figure 6) played a role in the composition of the bacterial functional groups. These two soil types present different environments for the rhizospheres. Clovelly soils have low fertility and water-holding capacity, when compared with those of other soils of the same clay content, which would affect the rhizosphere diversity and thus functionality when compared to the Sterkspruit soil type. The Sterkspruit soil type is characterised by a higher clay content, leading to a higher CEC. The most important factors pertaining to these soils that affect the rhizosphere biome are the elevated sodium concentration in the Sterkspruit soil type (3802 mg/kg vs. 1062 mg/kg) and more acidic pH in the Clovelly soil type (5.85 vs. 6.4) (Sandoval-Denis et al. 2018; Theron et al. 2020). Higher sodium could lead to lower osmotic potential, making it more difficult for microbes and plant roots to extract water from soils (Yan et al. 2015). Differences in pH would also mediate nutrient availability in soils (Zhalnina et al. 2015), affecting the rhizobiome.

Chemoheterotrophy and aerobic chemoheterotrophy were the most abundant identified functions in all rhizobiomes (Figure 5). However, this is expected for bacterial communities that use organic carbon as the main food source (heterotrophic bacteria) (Zhang et al. 2019). In the absence of oxygen, a wide range of compounds can serve as terminal electron donors (anaerobic respiration). The availability of oxygen in the rhizosphere is one of the most changing factors and anaerobic/anoxic conditions may develop (Philippot et al. 2013), leading to bacteria adapting to using various anaerobic respiration strategies. This was observed with iron, sulphur, sulphite and thiosulphate respiration, present at the Sterkspruit site (bottom) in both K. angustifolia and S. cordifolia, and arsenate respiration in K. angustifolia (Figure 5). It is possible that the higher clay content of the Sterkspruit soil could also play a role, as anaerobic respiration was only observed in the rhizobiomes associated with this particular soil type.

Soil type (Clovelly vs. Sterkspruit) and differences in pH and salinity (Figure 1) in the two soil types played a role in the rhizobiome bacterial diversity, as unique OTUs could be identified in all respective rhizosphere soil samples. There was, however, also a large overlap in bacterial diversity in the different soil types and in the rhizobiome of the three plant species. Soil type also played a significant role in the bacterial functional groups, with anaerobic respiration only being reported in the Sterkspruit soil.