Cancer bush-microbe symbiosis and their nutrient cycling efficiency in Limpopo Province.

Abstract

Cancer bush (Sutherlandia frutescens (L.) R. Br) is one of most important medicinal plant native to Southern Africa. It is currently facing extinction threats due to overharvesting from the wild. In the wild, cancer bush grows mostly in arid terrains characterized by nutrient poor soils. As a results, the plant establishes symbiotic relationship with beneficial microorganisms such as the root-nodulating bacteria which through N-fixation and other nutrient cycling abilities is able to enhance growth and development of the plant. However, the cancer bush symbionts remain unknown, together with their specific roles. The current study intended to investigate the diversity of microbes in the root nodules of cancer bush across two sites (Tubatse and Makgupheng) in Limpopo Province over two seasons, winter and summer (Objective 1) and examine the rhizosphere microbial enzyme activities on N-fixation, C-cycling, and P-solubilisation, together with potential of soil physicochemical properties in influencing the above (Objective 2). To achieve Objective 1, the bacteria were extracted from cancer bush root nodules and their morphological and molecular characteristics were determined. Morphological characteristics were described based on colony color, shape, elevation, surface and margins. Molecular analysis was based on the isolation and sequencing of the bacterial 16S RNA ribosomal gene. The bacteria were further grown on Simmons citrate and Pikovskaya’s media to test their ability to fix nitrogen and solubilize phosphorus, respectively. To achieve Objective 2, the calorimetric analysis method was conducted to determine rhizosphere soil bacteria extracellular enzyme activities on carbon (using the glucosidase and ꞵ-glucosaminidase tests), nitrogen (using the nitrate reductase test) and phosphorus (using phosphatase alkaline and phosphatase acid test). Soil chemical tests were further conducted to determine the total extractable micro-and macro nutrients in cancer bush rhizosphere soil, organic carbon, organic matter as well as soil pH. Based on morphological analysis, the study isolated a total of 30 bacteria species in winter and 70 species in summer which were further classified into 16 and 13 different morphological taxa, respectively. Of the 100 isolates combined, a total of 19 symbiotic bacteria were successfully characterized based on molecular characteristics and were affiliated with 12 different genera: Cellulosimicrobium, Sphingobacterium, Rhizobium, Bacillus, Micrococcus, Lysinibacillus, Alcaligenes, Stenotrophomonas, Enterobacter, Leucobacter, Serratia and Kosakonia. In Makgupheng a total of 3 genera: Serratia, Leucobacter and Sphingobacterium were detected, while in Tubatse 4 genera: Micrococcus, Alcaligenes, Rhizobium and Bacillus were detected. A total of 5 genera: Lysinibacillus, Stenotrophomonas, Cellulosimicrobium, Kosakonia and Enterobacter occurred at both study sites. The Stenotrophomonas and Leucobacter spp. emerged as the dominant genera for Makgupheng, while Stenotrophomonas spp. was dominant in Tubatse for the winter season. During the summer season, Bacillus emerged as the dominant group in Tubatse, while Serratia was dominant in Makgupheng. The comparisons of the neighbour-joining (NJ) tree indicated a strong homology between the generated DNA sequences with those from NCBI database with 80 to 100% homology of the species identified. A maximum homology 99.66% was found with Serratia sp. (CP055161.1) and minimum homology 78.33% with Lysinibacillus sp. (FJ528593.1). The Shannon-Wiener diversity index (H′) showed that both localities had diversity indices greater than 1. The H′ value observed in Makgupheng was 1.61 in summer and 2.40 in Winter. The H′ value of Tubatse was 2.00 in summer and 2.04 in winter. The Sampson indices (Ds) in Tubatse were 0.94 and 0.93 for winter and summer, respectively. The Ds values observed in Makgupheng were 0.91 and 0.85 for winter and summer, respectively. Both populations were evenly distributed with a Pielou’s evenness (J) value closer to 1. In addition, Tubatse, with 94% average had highest number of bacteria that tested positive (+) for nitrogen cycling test compared with Makgupheng, 90% average. The enzyme activities of bacteria in the soil were not significantly different. Moreover, no correlation was observed between soil edaphic factors, particularly primary nutrients and enzyme activities, except for P alkaline with soil pH and K. No correlation was observed between season and edaphic factors, except pH. A strong correlation was observed between location and edaphic factors (soil nutrient availability). The soil analysis report showed that Makgupheng had low pH (5.43) (overly acidic) in summer and pH neutral (7.23) in winter. In Tubatse there were no significant differences in pH between seasons, the pH range was between 6.82-7.14. Soils from Tubatse had a higher Ca (> 10 Cmolc/kg) and ECEC (15 - 25 Cmolc/kg), slightly higher Mg (> 4 Cmolc/kg), higher K (0.2 - 0.6 Cmolc/kg) and clay (> 40%) content compared with Makgupheng which had low Ca (< 4 Cmolc/kg), ECEC (< 15 Cmolc/kg), Mg (0.5 - 4 Cmolc/kg), K (< 2 Cmolc/kg) and clay (< 15%). The P levels (0 - 5 mg. Kg-1), organic carbon and organic matter percentage were relatively low (< 4%) for both sites in both seasons. Both site had low C: N, Makgupheng (12.57 :1) and Tubatse (11.09: 1), lower than the general C: N of legume plant soils (20: 1 or < 25: 1). Moreover, plants growing in Makgupheng derived most of their N from the atmosphere than plants in Tubatse as indicated by a higher percentage nitrogen derived from the atmosphere (%NDFA) in the leaves. In conclusion, diverse group of PGPRs were isolated from the root nodules of cancer bush plants in both sites. Bacteria occurrence, abundance and diversity were more site (nutrient status and pH) specific rather than due to seasonal shift. Season indirectly influenced the abundance and diversity by altering soil edaphic factors (particularly pH). Soil pH was the major driving factor of bacterial diversity followed by total soil nutrient (carbon, nitrogen and with phosphorus to less extent). Neutral pH seems to support optimal growth and functioning of most organisms and also influenced enzyme activity. High ECEC increase nutrient availability in the soil, increase soil fertility thus supporting/providing good conditions for plant growth and microbial functioning. Lastly, the study reports on the potential role of these microbes in improving plant growth through nutrient cycling and acquisition mechanisms confirmed by the high % NDFA levels, specifically in plants from Makgupheng.