Sequence comparison of 16S ribosomal RNA partial sequences revealed the presence of multiple bacterial species in V. unguiculata plants growing in rhizosphere and surrounding soils. The culturable strains isolated from the nodules of V. unguiculata growing in rhizosphere and surrounding soils belonged to the Paenibacillus, Bacillus, Lysinibacillus, Brevibacillus, Alkalihalobacillus, and Peribacillus genera (Table 1).
Plant biomass and nutrition
Plants grown in rhizosphere soils had higher plant biomass than plants grown in surrounding soils (Table 2). The belowground biomass of plants grown in rhizosphere soils was significantly higher than plants grown in surrounding soils (Table 2). However, the root-to-shoot ratio was significantly higher for plants grown in surrounding soils (Table 2). Although the difference was insignificant, V. unguiculata plants grown in rhizosphere soils had a higher RGR (Table 2). Similarly, the total P concentration of plants in rhizosphere soils was higher than that of plants in the surrounding soil, but the difference was insignificant (Table 2). Plants in rhizosphere soils had a significantly higher total N concentration than plants in surrounding soils (Table 2). The SNAR, SNUR, and SPUR were significantly higher in plants grown in rhizosphere soils.
Table 2
Growth kinetics of 45 days old Vigna unguiculata grown in Encephalartos villosus rhizosphere and surrounding soils, Oceanview, Eastern Cape. Values are means ± SE, n = 60). In each row, different letters denote statistical differences (p ≤ 0.05) after an independent samples t-test.
Parameter | Treatments |
| Rhizosphere | Surrounding soils |
Biomass Total plant (g) | 0.967 ± 0.270a | 0.807 ± 0.128a |
Leaves (g) | 0.284 ± 0.112a | 0.250 ± 0.066a |
Shoots (g) | 0.269 ± 0.080a | 0.257 ± 0.053a |
Roots (g) | 0.457 ± 0.105a | 0.329 ± 0.096b |
Root: Shoot ratio | 1.269 ± 0.116c | 1.460 ± 0.160d |
Growth kinetics | | |
Relative growth rate (g day− 1) | 0.020 ± 0.007a | 0.016 ± 0.004a |
SPUR (g plant DW mg− 1 P day− 1) | 0.008 ± 0.002p | 0.006 ± 0.0004q |
SPAR (g root DW mg− 1 P day− 1) SNAR (mg N g− 1 root DW day− 1) SNUR (mg N g− 1 plant DW day− 1) Carbon costs (mmol C g− 1 ) | 103.85 ± 5.42a 21.21 ± 3.98g 4.19 ± 1.21j 0.004 ± 0.001a | 117.65 ± 11.57a 16.07 ± 1.11h 2.02 ± 0.95k 0.005 ± 0.002a |
Plant Nutrition | | |
Phosphorus (µmol g− 1) Nitrogen (mmol g− 1) | 20.17 ± 2.05a 4.51 ± 1.05a | 19.84 ± 2.30a 3.19 ± 0.53b |
Vigna unguiculata plants grown in E. villosus rhizosphere and surrounding soils were nodulated by bacterial species belonging to the Paenibacillus, Bacillus, Brevibacillus, Lysinibacillus, Alkalihalobacillus, and Bacillus genera. Some of these bacterial species are similar to those associated with E. villosus plants growing in Oceanview soils. In the current study, bacterial species belonging to the Bradyrhizobium genera were not isolated even though Bradyrhizobium has been reported to be associated with V. unguiculata (Sithole et al., 2019; Makaure et al., 2022). In a study on the competitive nature of Bradyrhizobium in pigeon peas, Chalasani et al. (2021) reported that Bradyrhizobium is a poor competitor against non-nodulating bacteria. The Bacillus, Brevibacillus, and Paenibacillus associated with V. unguiculata are non-nodulating bacteria (Rajendran et al., 2008) that may have had a competitive advantage over Bradyrhizobium. Vigna unguiculata plants growing in the rhizosphere and surrounding soils were predominantly nodulated by bacterial species belonging to the Paenibacillus genera. The Paenibacillus genera promote plant growth through P solubilisation, IAA production, siderophore secretion, and N-fixation (Grady et al., 2016; Weselowski et al., 2016). Bacterial strains such as Lysinibacillus fusiformis were identified in the coralloid roots of E. villosus growing in Oceanview. The same bacterial strain was isolated in V. unguiculata plants grown in rhizosphere soils, indicating the potential similarities in symbiotic associations involving cycads and legumes.
Encephalartos villosus is a forest understorey plant known to grow in acidic soil environments. The sampled E. villosus rhizosphere and surrounding soils showed a pH range between 5 and 5.4 and low P levels (3.86–6.1 mg/kg). Under these conditions, P is unavailable for plant uptake due to the formation of insoluble complexes with cations such as aluminum and iron (Karyotis et al., 2005). Thus, the association of V. unguiculata plants growing in E. villosus rhizosphere and surrounding soils with phosphobacteria such as Bacillus could have enhanced P uptake. White et al. (2008) reported high root biomass in plants growing in P-deficient soils, which may explain the higher root biomass than shoot biomass in V. unguiculata plants in rhizosphere and surrounding soils. According to Iqbal et al. (2020), elevated root biomass is attributed to high P utilisation rather than P availability. Therefore, the higher root biomass of V. unguiculata plants grown in rhizosphere soils may be attributed to a higher SPUR.
According to Marschner et al. (1991), nodulation is an energetically costly process affected by soil acidity. In this study, soil acidity did not affect nodulation in V. unguiculata roots, as evidenced by nodules in all plants grown in the rhizosphere and surrounding soils. The Paenibacillus sabinae and Bacillus altitudinis were isolated from the nodules of V. unguiculata roots, and these species are reported to be N -fixing (Lu et al., 2014; Zhang et al., 2021). Though this study did not assess the N preference of V. unguiculata plants grown in the rhizosphere and surrounding soils. The presence of nodules infected with culturable N-fixing bacteria and the ability of the plants to grow without being supplemented with N nutrients support the hypothesis that the plants may have partaken in BNF.
Vigna unguiculata plants grown in E. villosus rhizosphere and surrounding soils established symbioses with multiple bacterial species, some of which have been isolated from E. villosus coralloid roots, indicating similarities between cycads and legume symbiotic associations. The contributions of these microbes enabled V. unguiculata plants to grow in acidic and nutrient-deficient soils by enhancing nutrient bioavailability and uptake. Future studies can compare the symbionts and growth of V. unguiculata in contrasting ecosystem soils, e.g., heavily used agricultural soils and cycad rhizosphere soils from different localities. Also, the microbial community composition in the nodules can be assessed using techniques such as Illumina sequencing.