Seasonal Effect on Bacterial Communities Associated with the Rhizospheres of Polhillia, Wiborgia and Wiborgiella Species in the Cape Fynbos, South Africa

The Cape fynbos biome in South Africa is home to highly diverse and endemic shrub legumes, which include species of Aspalathus, Polhillia, Wiborgia and Wiborgiella. These species play a significant role in improving soil fertility due to their ability to fix N2. However, information regarding their microbiome is still unknown. Using the 16S rRNA Miseq illumina sequencing, this study assessed the bacterial community structure associated with the rhizospheres of Polhillia pallens, Polhillia brevicalyx, Wiborgia obcordata, Wiborgia sericea and Wiborgiella sessilifolia growing at different locations during the wet and dry seasons in the Cape fynbos. The results showed that the most dominant bacterial phylum was Actinobacteria during both the dry (56.2–37.2%) and wet (46.3–33.3%) seasons. Unclassified bacterial genera (19.9–27.7%) were the largest inhabitants in the rhizospheres of all five species during the two seasons. The other dominant phyla included Bacteroidetes, Acidobacteria, Proteobacteria and Firmicutes. Mycobacterium and Conexibacter genera were the biggest populations found in the rhizosphere soil of all five test species during both seasons, except for W. obcordata soil sampled during the dry season, which had Dehalogenimonas as the major inhabitant (6.08%). In this study plant species and growth season were the major drivers of microbial community structure, with W. obcordata having the greatest influence on its microbiome than the other test species. The wet season promoted greater microbial diversity than the dry season.


Introduction
South Africa is a biologically diverse country with nine terrestrial biomes. The fynbos biome is located at the southwestern tip of Africa, and recognized as one of the world's biological hotspots due to its high plant diversity and endemicity, including shrub legumes [1][2][3][4]. The high diversity of nodulated legume species in the Cape fynbos region may suggest that this region is a hotspot for bacterial diversity [5][6][7]. Shrub legumes such as Polhillia, Wiborgia and Wiborgiella grow naturally in the fynbos [8,9], and they are characterized by bright yellow and/ or white flowers, a major attraction for tourists. These plants can also be used as cut-flowers in the ornamentals industry. Furthermore, they have the potential for reforestation and for control of soil erosion, which may be caused by heavy rains and wind [10].
Similar to other shrub legumes in the Cape fynbos [5,11,12]; Polhillia, Wiborgia and Wiborgiella species are able to nodulate with a diversity of soil bacteria, including Mesorhizobium and Rhizobium species [13], indicating their potential in fynbos biodiversity improvement. Furthermore, they are reported to derive over 61% of their N nutrition from biological nitrogen fixation [8], which means they depend on soil microbes for survival. Moreover, these species are also known to exhibit high acid and alkaline phosphatase

Site Description and Soil Sampling
Sampling of rhizosphere soils from Polhillia, Wiborgia and Wiborgiella species was performed during the dry (February-March, 2017) and wet (August-September, 2017) seasons at five different locations in the Cape fynbos, as indicated in Table 1. Three replicate soil samples were collected from 20 cm depth at each location, pooled, and thoroughly mixed to obtain a single representative soil sample per location. For each species, there were five soil samples per season. As shown in Table 2, 100 g soil was weighed from each sample and determine water content. Soil water content was determined using the gravimetric water content (θ m ) [29]. Soil analysis was performed at the Institute for Plant Production, Elsenburg, in the Western Cape, for only the wet season (Table 3). Total bacterial genomic DNA was extracted from each rhizosphere soil sample, as described by Jaiswal et al. [22]. Bacterial DNA was extracted from 0.5 g of each rhizosphere soil using PowerSoil™ DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA), according to the manufacturer's instructions. After determining the DNA concentrations using the Qubit™ 4 Fluorometer (Invitrogen, cat. #Q33226).

Library Preparation
Random fragments of the DNA samples were used to prepare the library, followed by 5 and 3 adapter ligation. PCR amplification was performed using adapter-ligated primer pairs 5 TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACGGGNGGC WGC AG3 and 5 GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTACHVGGG TAT CTA ATC C3 targeting variable regions V3-V4 of the 16S rRNA gene [30] (Klindworth et al. 2013) in 25 µL reaction volume containing 12.5 µL KAPA HiFi hotstart ready mix (2×), 5 µL each of forward and reverse primer (1 µM) and 2.5 µL sterilized double distilled water with the standard temperature profiles (30 The PCR-amplified products were purified using AMPure XP beads. The library was loaded onto a flow cell for cluster generation.

Sequencing and Data Assembling
Illumina SBS technology was employed for paired-end sequencing. During data analysis and alignment, the newly identified sequence reads were aligned using FLASH to the reference genome. The taxonomical abundance (%) of microbial communities in each rhizosphere soil was calculated using read files as queries against removed and dereplicated sets of sequences from the small subunit (SSU) UCLUST. After normalization of sequences of each sample, rarefaction analysis was performed at the species level for sampling adequacy using alpha_rarefaction.py of QIIME. The data were submitted to NCBI Sequence Read Archive SUB11923586 under Bioproject ID PRJNA880697.

Site Elevation and Physical Properties of Rhizosphere Soils
Uitvlucht and Ghwarriekop farms, where rhizosphere soils of Polhillia brevicalyx and Polhillia pallens were respectively collected, had an elevation of 310 and 152 m in that order. The soil texture was clayey at both locations ( Figure 1). The water content from the rhizosphere of P. pallens and P. brevicalyx was, respectively, 9.26 and 8.21 % for the dry season and 26.55% and 25.47% for the wet season ( Table 2). Soils from the rhizosphere of Wiborgia sericea and Wiborgia obcordata collected at Traveller's Rest farm and Bushmans Kloof areas were sandy and at 120 and 441 m elevations, respectively. The gravimetric water contents were 6.37% and 20.0% for W. sericea in the dry and wet seasons, respectively, and 3.93% and 11.90% for W. obcordata in the dry and wet seasons, respectively ( Table 2). The Bredasdorp area had loamy soil and was at 311 m elevation (Table 1). Wiborgiella sessilifolia rhizosphere soil recorded a water content of 5.42% (Table 2). Uitvlucht and Ghwarriekop farms, where rhizosphere soils of Polhillia brevicalyx and Polhillia pallens were respectively collected, had an elevation of 310 and 152 m in that order. The soil texture was clayey at both locations ( Figure 1). The water content from the rhizosphere of P. pallens and P. brevicalyx was, respectively, 9.26 and 8.21 % for the dry season and 26.55% and 25.47% for the wet season (Table 2). Soils from the rhizosphere of Wiborgia sericea and Wiborgia obcordata collected at Traveller's Rest farm and Bushmans Kloof areas were sandy and at 120 and 441 m elevations, respectively. The gravimetric water contents were 6.37% and 20.0% for W. sericea in the dry and wet seasons, respectively, and 3.93% and 11.90% for W. obcordata in the dry and wet seasons, respectively ( Table 2). The Bredasdorp area had loamy soil and was at 311 m elevation (Table 1). Wiborgiella sessilifolia rhizosphere soil recorded a water content of 5.42% (Table 2).

Chemical Properties of Rhizosphere Soils
Except for Wiborgiella sessilifolia rhizosphere soil from Bredasdorp, which had a pH of 8.2, the pH of all the other rhizosphere soils was acidic and ranged from pH 4.3 for W. obcordata to pH 5.5 for P. brevicalyx. The concentrations of macronutrients (namely, Mg, K, Na, P and C) were consistently higher in the rhizosphere soil of P. pallens, followed by P. brevicalyx, and much lower for Wiborgia obcordata. The concentration of soil micronutrients (Cu, Zn and B) mirrored those of the macronutrients, with high levels recorded for the rhizosphere soil of P. pallens and P. brevicalyx and lowest for W. sericea and W. obcordata. Fe concentration was generally high at all sites, with P. pallens showing the highest level (174.6 mg/kg), followed by Wiborgia sericea (129.2 mg/kg), with Wiborgiella sessilifolia the lowest (46.9 mg/kg). Wiborgiella sessilifolia recorded the highest NH4 + concentration (0.67 mg/kg), and Wiborgia sericea the lowest (0.03 mg/kg) ( Table 2).

Quantity and Quality of Raw Sequences Generated
The quality and quantity of total sequences generated from the analysis were generally adequate. The total sequence bases and read counts were relatively higher for the rhizosphere soils collected during the wet season than the dry season. The least sequence bases recorded after quality control of all samples were from rhizosphere soils of P. brevicalyx (4,444,959 bp), followed by rhizosphere soils of Wiborgiella sesilifollia (4,521,971 bp); while the highest sequence bases were observed in the rhizosphere soils of P. pallens (5,537,882 bp) followed by Wiborgia sericea (5,180,741 bp). Furthermore, the reads outputs varied across samples, with ˃10,000 reads being obtained for each sample and the trend mirroring that of total sequence bases (Table S1). For example, the lowest read count was recorded for P. brevicalyx rhizosphere soil (11,820 bp), followed by Wiborgiella sessilifolia

Chemical Properties of Rhizosphere Soils
Except for Wiborgiella sessilifolia rhizosphere soil from Bredasdorp, which had a pH of 8.2, the pH of all the other rhizosphere soils was acidic and ranged from pH 4.3 for W. obcordata to pH 5.5 for P. brevicalyx. The concentrations of macronutrients (namely, Mg, K, Na, P and C) were consistently higher in the rhizosphere soil of P. pallens, followed by P. brevicalyx, and much lower for Wiborgia obcordata. The concentration of soil micronutrients (Cu, Zn and B) mirrored those of the macronutrients, with high levels recorded for the rhizosphere soil of P. pallens and P. brevicalyx and lowest for W. sericea and W. obcordata. Fe concentration was generally high at all sites, with P. pallens showing the highest level (174.6 mg/kg), followed by Wiborgia sericea (129.2 mg/kg), with Wiborgiella sessilifolia the lowest (46.9 mg/kg). Wiborgiella sessilifolia recorded the highest NH 4 + concentration (0.67 mg/kg), and Wiborgia sericea the lowest (0.03 mg/kg) ( Table 2).

Quantity and Quality of Raw Sequences Generated
The quality and quantity of total sequences generated from the analysis were generally adequate. The total sequence bases and read counts were relatively higher for the rhizosphere soils collected during the wet season than the dry season. The least sequence bases recorded after quality control of all samples were from rhizosphere soils of P. brevicalyx (4,444,959 bp), followed by rhizosphere soils of Wiborgiella sesilifollia (4,521,971 bp); while the highest sequence bases were observed in the rhizosphere soils of P. pallens (5,537,882 bp) followed by Wiborgia sericea (5,180,741 bp). Furthermore, the reads outputs varied across samples, with >10,000 reads being obtained for each sample and the trend mirroring that of total sequence bases (Table S1). For example, the lowest read count was recorded for P. brevicalyx rhizosphere soil (11,820 bp), followed by Wiborgiella sessilifolia (12,006 bp), while the highest reads recorded were from P. pallens rhizosphere soils (14,904 bp). Moreover, Over 90 and 80% of the raw sequence bases passed the quality score of over 20 and 30%, respectively (Table S1) 3.4. Rhizosphere Bacterial Communities 3.4.1. Polhillia Brevicalyx Actinobacteria formed the largest population in the rhizosphere of P. brevicalyx and ranged from 45.8% to 46.3% in soil collected during the dry and wet seasons, respectively, followed by unclassified bacteria, which were 15.2% and 12.2% in the dry and wet seasons, respectively. During the dry season, Firmicutes (9.5%), Proteobacteria (7.0%) and Bacteroidetes (5.2%) showed relatively larger populations, in contrast to Bacteroidetes (9.1%), Firmicutes (6.9%) and Acidobacteria (6.0%) which recorded much larger populations during the wet season. Although they differed in population sizes, most of the bacteria found in the wet season were also available in the dry season ( Figure 2). Actinobacteria formed the largest population in the rhizosphere of P. brevicalyx and ranged from 45.8% to 46.3% in soil collected during the dry and wet seasons, respectively, followed by unclassified bacteria, which were 15.2% and 12.2% in the dry and wet seasons, respectively. During the dry season, Firmicutes (9.5%), Proteobacteria (7.0%) and Bacteroidetes (5.2%) showed relatively larger populations, in contrast to Bacteroidetes (9.1%), Firmicutes (6.9%) and Acidobacteria (6.0%) which recorded much larger populations during the wet season. Although they differed in population sizes, most of the bacteria found in the wet season were also available in the dry season ( Figure 2). At the genera level, unclassified bacteria were dominant and formed about 19.7 and 17.5 %, respectively, in soils collected during the dry and wet seasons. Mycobacterium, Conexibacter and Nocardioides, respectively, recorded 8.6%, 7.0% and 6.8% in the dry season, and 10.8%, 11.0% and 4.4% in the wet seasons. Interestingly, Bacillus, Bradyrhizobium, Anaerotruncus and Pseudonocardia were only present in soils sampled during the dry season, while Frankia, Chitinophaga, Pedobacter and Dehalogenimonas were present in only soil collected during the wet season. Of the 13 genera identified, nine were found in both dry and wet seasons (Figure 3). From the rarefaction analysis, there was high species diversity in both seasons, as 2000 species could be counted using 140,000 sequence reads. At the genera level, unclassified bacteria were dominant and formed about 19.7 and 17.5 %, respectively, in soils collected during the dry and wet seasons. Mycobacterium, Conexibacter and Nocardioides, respectively, recorded 8.6%, 7.0% and 6.8% in the dry season, and 10.8%, 11.0% and 4.4% in the wet seasons. Interestingly, Bacillus, Bradyrhizobium, Anaerotruncus and Pseudonocardia were only present in soils sampled during the dry season, while Frankia, Chitinophaga, Pedobacter and Dehalogenimonas were present in only soil collected during the wet season. Of the 13 genera identified, nine were found in both dry and wet seasons (Figure 3). From the rarefaction analysis, there was high species diversity in both seasons, as 2000 species could be counted using 140,000 sequence reads. Microorganisms 2022, 10, x FOR PEER REVIEW 6 of 12
At the genus level, unclassified bacteria were 21.8% in the dry season and 20.2% in the wet season. Conexibacter, Mycobacterium, Nocardioides and Chthoniobacter, however, showed a high number of sequences in both seasons. Together, they accounted for 19.1% and 24.3% of the bacterial communities in the dry and wet seasons, respectively. Candidatus Solibacter (3.7%), Clostridium (3.4%) and Thermoleophilum (1.9%) were only present in soil from the dry season, while Cicer (2.9%), Pseudonorcadia (1.7%) and Kribbella (1.7%) were only found in soil from the wet season ( Figure 3). The rarefaction analysis showed that about 2500 species were present in P. pallens rhizosphere during both wet and dry seasons based on 160,000 sequence reads obtained ( Figure S1).

Wiborgia Sericea
As found with the two Polhillia species, Actinobacteria dominated in the rhizosphere of W. sericea and, respectively, formed 44.6 and 37.7% of the sequences in the wet and dry seasons. Unclassified bacteria were the second largest group in both seasons (18.7 and 19.0%). Although their population sizes differed significantly between seasons, the five major phyla found in W. sericea rhizosphere soil were Chloroflexi (8.1 and 5.4%), Bacteroidetes (6.9 and 10.0%), Firmicutes (4.7 and 7.4%) and Proteobacteria (4.6 and 8.6%) during the dry and wet seasons, respectively. However, all the phyla were present in soils collected from the two seasons, except for Synergistetes (0.22%) and Cyanobacteria (0.85%), which were only detected in soil from the dry and wet seasons, respectively ( Figure 2). About 26.6% of the bacteria belonged to unclassified genera. Of the classified group, Mycobacterium was dominant in both dry (10.6%) and wet (9.4%) seasons, followed by Dehalogenimonas and Conexibacter in both dry (6.6% and 4.0%) and wet (3.6% and 3.7%) seasons, respectively. Isosphaera (5.4%) was present in only soil from the dry season, and Bacillus (3.7%) in only the wet season. Of the 13 classified bacterial genera listed, six were found in both wet and dry seasons (Figure 3). Species analysis using the rarefaction tool revealed 2000 species out of 160,000 sequences studied ( Figure S1).

Wiborgia Obcordata
During the two seasons, Actinobacteria (33.34-39.34%) was most dominant, followed by an unclassified phylum (18.5-19.1%) and Chloroflexi (9.8-10.8%). In the dry season, 9.01% and 6.1% of the bacteria belonged to Bacteroidetes and Proteobacteria, respectively, while in the wet season, 7.9% and 6.4% were Proteobacteria and Firmicutes phyla, respectively. Cyanobacteria were present in only soil collected during the dry season and Basidiomycota in only the wet season ( Figure 2).
As found with the other species, W. obcordata rhizosphere soil recorded a much higher number of unclassified bacterial genera in both seasons (27.7% and 24.2%). The genera Dehalogenimonas (6.0%), Ktedonobacter (5.6%) and Isosphaera (5.0%) were dominant in the dry season, while Conexibacter (6.7%), Dehalogenimonas (6.6%) and Mycobacterium (5.8%) were higher in the wet season ( Figure 3). The rarefaction studies showed that, of a total number of 160,000 sequences, only 1600 bacterial species were found in the dry season and 2000 species in the wet season ( Figure S1).

Wiborgiella Sessilifolia
With W. sessilifolia, only the dry season soil sequence results were included due to the poor concentration of DNA from soils collected in the wet season. Over half (56.2%) of the bacteria from dry season soil of W. sessilifolia belonged to Actinobacteria. Unclassified bacteria formed 12.08% of the total sequences, followed by Firmicutes (6.8%), Bacteroidetes (6.6%) and Proteobacteria (4.4%) (Figure 2). At the genus level, Mycobacterium (15.3%), Pseudonocardia (5.4%) and Conexibacter (4.7%) were dominant in the dry season after the unclassified genera (16.9%) (Figure 3). The number of species detected using 120,000 sequences was 1800 for soils sampled in the dry season ( Figure S1).

Discussion
The microbial community structure in the fynbos is rather complex and includes bacteria, fungi and archaea. These microbes play a vital role in supporting plant growth in the acidic, nutrient-poor soils of the fynbos [28,31]. This study was conducted to ascertain any changes in microbial community structure during wet and dry seasons, and the results showed that the phylum Actinobacteria was very dominant (≥30%) in the rhizosphere of Polhillia, Wiborgia and Wiborgiella species during both wet and dry seasons. More specifically, up to 56.2% of the bacteria belonged to this Actinobacteria phylum in the rhizosphere soil of Wiborgiella sessilifolia. The dominance of Actinobacteria in the rhizospheres of the five shrub legumes could suggest the presence of these bacteria in the entire fynbos ecosystem. Actinobacteria have been reported to occur in abundance in rhizosphere soils from other biomes [32,33]. In fact, the phylum Actinobacteria has been reported to account for a large proportion of the microbial population in the rhizosphere of many plant species [34] and is also known to colonize root nodules of legumes, including lupin [35] An earlier study found that the phylum Actinobacteria was more abundant in undisturbed ecosystems than farmlands [36], a finding consistent with results from this study, which was conducted on undisturbed sites in the fynbos. The Actinobacteria phylum plays a major role in plant growth promotion through processes such as biological N 2 fixation, production of IAA, antibiotics and siderophores, as well as the solubilization of P, K and Zn [37][38][39]. In fact, antibiotic resistance, the production of IAA and the solubilization of P are common functional attributes of bacterial symbionts associated with Polhillia, Wiborgia and Wiborgiella species [13].
Although their dominance differed per species and season, Bacteroidetes, Acidobacteria, Proteobacteria and Firmicutes were the major bacterial phyla with a higher number of sequences in the rhizosphere soils of the five legumes tested, a finding consistent with the results of earlier studies in the Cape fynbos [23,40]. This suggests that the rhizosphere microbial communities of the Polhillia, Wiborgia and Wiborgiella species are very diverse and high in numbers. Similar high diversity of bacterial community has been found in the rhizosphere of crops growing in undisturbed forest soil when compared to cultivated fields [41].
This study also discovered Acidobacteria in the rhizospheres of Polhillia brevicalyx, Polhillia pallens, Wiborgia sericea and Wiborgia obcordata, and this was not surprising given the acidic nature of the test rhizospheres (pH 4.3 to 5.5). Other studies similarly found Acidobacteria in the acidic and low-nutrient soils of the Cape fynbos [25,42]. It was, however, intriguing that the rhizosphere soil of Wiborgiella sessilifolia, with an alkaline pH of 8.2, could harbor up to 2.0% of Acidobacteria. Other studies have, however, also found Acidobacteria in alkaline habitats, including water [43].
In this study, a high number of unclassified bacterial genera were detected in the rhizosphere soils studied, a finding constant with earlier reports that found an abundance of uncultured microbes in soils from various study sites [22]. Of the classified bacteria, Mycobacterium and Conexibacter genera were more abundant in the rhizospheres of all test legumes during both wet and dry seasons, except for rhizosphere soil of Wiborgia obcordata collected during the dry season. Mycobacterium, and more specifically Mycobacterium flavum, has been reported to improve the growth of many crop species, including maize, wheat, sunflower, chickpea, cotton, soybean and rice [44][45][46][47][48]. Conexibacter was also dominant in the rhizosphere soil of sunflowers in South Africa [49]. Dehalogenimonas from the phylum Chloroflexi dominated the rhizosphere of W. obcordata during both wet (6.6%) and dry (6.0%) seasons and could play a significant role in plant defense against pathogenic microbes in the soil [50,51]. The high populations of plant growth-promoting bacteria found in the rhizosphere of legumes endemic to the Cape fynbos could explain the ability of these legumes to thrive under the nutrient-poor conditions of the fynbos [8].
Furthermore, the presence of N 2 -fixing Burkholderia and Dehalogenimonas bacteria in the acidic rhizospheres of the two Wiborgia species (Table 2; Figure 2) but not in the acidic rhizospheres of the two Polhillia species (Table 2; Figure 1) during the dry season, and the absence of Streptomyces and Candidatus koribacter during the wet season, could suggest the effect of plant species in shaping the structure of rhizosphere microbial communities in the fynbos [52]. The Wiborgia species probably released antimicrobial compounds that select for a specific type of microbiome in the rhizosphere [22]. The presence of Burkholderia in the fynbos has been associated with acidic soils, and the bacterium is known to fix N 2 when in symbiosis with compatible legume hosts [5]. The fact that Bacillus and Bradyrhizobium were found in the rhizosphere of only P. brevicalyx, but not the other test legume species, and Burkholderia detected in the rhizospheres of only W. sericea and W. obcordata, attest to the effect of host plant in selecting its symbiont partner [53]. Furthermore, the effect of host plant is further supported by the nutrient content availability in the rhizosphere soil (Table 3). For instance, the two Wiborgia species (W. obcordata and W. sericea) contained relatively lower contents Mg, K, Na and P compared to Polhillia and Wiborgiella species.
W. obcordata was found to influence its rhizosphere more than the other test species during the two seasons. For example, although Nocardioides was absent in the rhizosphere of W. obcordata during both wet and dry seasons, it was present in the rhizosphere of the other four legumes. Furthermore, in the dry season, only the rhizosphere soil of W. obcordata contained populations of the plant growth-promoting Bacillus and Bradyrhizobium genera. However, in the wet season, the same soil harbored four unique bacterial genera (namely, Ktedonobacter, Bradyrhizobium, Candidatus Solibacter and Acidobacterium). These results probably suggest that Wiborgia obcordata released phytoalexins that were selected for a specific type of microbiome in the rhizosphere, a further indication that the microbial community in the rhizosphere soils is plant-regulated in the Cape fynbos [23,54,55].
Using partial chromosomal and symbiotic gene sequences, Mpai et al. [13] found a marked diversity of Bradyrhizobium bacteria present in the rhizosphere soil of Wiborgia obcordata at Bushmans Kloof. In fact, Bradyrhizobium was present in soil collected during the wet season (2.6%) but absent in soil from the dry season. The fact that this genus was absent in the microbial community during the dry season could suggest that this group of bacteria is highly endophytic and reside mainly inside root nodules, and available in the rhizosphere soils in very small numbers (<0.5%), especially during the unfavorable dry seasons. Similarly, Mesorhizobium, Rhizobium, Agrobacterium and Pseudomonas bacteria, which were isolated from root nodules of these five shrub legumes [8,13], were also absent in this study, which is further evidence of the endophytic nature of these root-nodule bacteria.
Furthermore, the fact that Bradyrhizobium species were present in the rhizosphere soil of W. obcordata only in the wet but not the dry season could explain the increased nodulation, greater symbiotic functioning and high phosphatase secretion observed in this legume during the wet season in the Cape fynbos [56][57][58]. In fact, season and soil moisture have been found to be the main drivers of microbial community structure in both agricultural and natural ecosystems [59][60][61].

Conclusions
To our knowledge, this is the first report on total soil bacterial community structure associated with Polhillia pallens, Polhillia brevicalyx, Wiborgia sericea, Wiborgia obcordata and Wiborgiella sessilifolia, which are endemic to the Cape fynbos. In this study, plant species and season were the main drivers of bacterial community structure in the Cape fynbos. The wet season was more dominant in shaping microbial diversity relative to the dry season. Furthermore, our results also showed that Wiborgia obcordata had a greater influence on microbial community structure than the other four shrub legumes. Moreover, Actinobacteria was found to be the most common and dominant microbial phylum in the rhizosphere of all five test legumes regardless of season, followed by Bacteroidetes, Acidobacteria, Proteobacteria and Firmicutes. Dehalogenimonas was the major inhabitant of Wiborgia obcordata rhizosphere during the wet season, while Mycobacterium and Conexibacter were the major inhabitants in the rhizosphere soils of P. pallens, P. brevicalyx and W. sericea during both wet and dry seasons, as well as W. sessilifolia in the dry season. This study also found that a high number of bacteria in the Cape fynbos are still unclassified. Bacterial classification studies are therefore recommended to resolve the microbial community structure of legume rhizospheres in the Cape fynbos.