Encephalartos natalensis, Their Nutrient-Cycling Microbes and Enzymes: A Story of Successful Trade-Offs.

Encephalartos spp. establish symbioses with nitrogen (N)-fixing bacteria that contribute to soil nutrition and improve plant growth. Despite the Encephalartos mutualistic symbioses with N-fixing bacteria, the identity of other bacteria and their contribution to soil fertility and ecosystem functioning is not well understood. Due to Encephalartos spp. being threatened in the wild, this limited information presents a challenge in developing comprehensive conservation and management strategies for these cycad species. Therefore, this study identified the nutrient-cycling bacteria in Encephalartos natalensis coralloid roots, rhizosphere, and non-rhizosphere soils. Additionally, the soil characteristics and soil enzyme activities of the rhizosphere and non-rhizosphere soils were assayed. The coralloid roots, rhizosphere, and non-rhizosphere soils of E. natalensis were collected from a population of >500 E. natalensis in a disturbed savanna woodland at Edendale in KwaZulu-Natal (South Africa) for nutrient analysis, bacterial identification, and enzyme activity assays. Nutrient-cycling bacteria such as Lysinibacillus xylanilyticus; Paraburkholderia sabiae, and Novosphingobium barchaimii were identified in the coralloid roots, rhizosphere, and non-rhizosphere soils of E. natalensis. Phosphorus (P) cycling (alkaline and acid phosphatase) and N cycling (ß-(D)-Glucosaminidase and nitrate reductase) enzyme activities showed a positive correlation with soil extractable P and total N concentrations in the rhizosphere and non-rhizosphere soils of E. natalensis. The positive correlation between soil enzymes and soil nutrients demonstrates that the identified nutrient-cycling bacteria in E. natalensis coralloid roots, rhizosphere, and non-rhizosphere soils and associated enzymes assayed may contribute to soil nutrient bioavailability of E. natalensis plants growing in acidic and nutrient-poor savanna woodland ecosystems.

Soil Acidification in Nutrient-Enriched Soils Reduces the Growth, Nutrient Concentrations, and Nitrogen-Use Efficiencies of Vachellia sieberiana (DC.) Kyal. & Boatwr Saplings.

Nitrogen (N) and phosphorus (P) nutrient enrichment is important for grasslands. This study aimed to determine how soils enriched with N and P influenced soil concentration correlations and affected the growth kinetics, mineral nutrition, and nitrogen-use efficiencies of Vachellia sieberiana grown in a greenhouse experiment. The soils used as the growth substrate were analysed and showed extreme acidity (low soil pH, 3.9). Nitrogen-enriched soils were more acidic than P-enriched soils. Exchangeable acidity was strongly negatively correlated with an increase in soil pH, with soil pH between 3.9 and 4.1 units showing the strongest decline. Plant saplings showed increased root biomass, shoot biomass, total biomass, and plant N and P concentrations when grown in soils with high soil P concentrations. Extreme soil acidification in N-enriched soil was one of the main factors causing P unavailability, decreasing sapling growth. Extreme soil acidification increased concentrations of toxic heavy metals, such as Al which may be alleviated by adding lime to the extremely acidic soils. Research implications suggest that soil pH is an important chemical property of the soil and plays a significant role in legume plant growth. Legume species that are unable to tolerate acidic soils may acquire different strategies for growth and functioning.

Soil microbes and associated extracellular enzymes largely impact nutrient bioavailability in acidic and nutrient poor grassland ecosystem soils.

Understanding the role of soil microbes and their associated extracellular enzymes in long-term grassland experiments presents an opportunity for testing relevant ecological questions on grassland nutrient dynamics and functioning. Veld fertilizer trials initiated in 1951 in South Africa were used to assess soil functional microbial diversity and their metabolic activities in the nutrient-poor grassland soils. Phosphorus and liming trials used for this specific study comprised of superphosphate (336 kg ha-1) and dolomitic lime (2250 kg ha-1) (P + L), superphosphate (336 kg ha-1) (+ P) and control trials. These soils were analyzed for their nutrient concentrations, pH, total cations and exchange acidity, microflora and extracellular enzyme activities. The analysed soil characteristics showed significant differences except nitrogen (N) and organic carbon (C) concentrations showing no significant differences. P-solubilizing, N-cycling and N-fixing microbial diversity varied among the different soil treatments. ß-glucosaminidase enzyme activity was high in control soils compared to P-fertilized and limed soils. Alkaline phosphatase showed increased activity in P-fertilized soils, whereas acid phosphatase showed increased activity in control soils. Therefore, the application of superphosphate and liming influences the relative abundance of bacterial communities with nutrient cycling and fixing functions which account for nutrient bioavailability in acidic and nutrient stressed grassland ecosystem soils.

Alien invasive Leucaena leucocephala successfully acquires nutrients by investing in below-ground biomass compared to native Vachellia nilotica in nutrient-amended soils in South Africa.

Soils in grasslands and savannas of southern Africa are acidic and nutrient-poor. Legume plants, such as Vachellia nilotica and alien invasive Leucaena leucocephala, are a major component of the vegetation there. Vachellia nilotica can establish in drought-prone environments, and is invasive in high rainfall areas. Leucaena leucocephala is an emerging invasive in South Africa and is ranked among the world's 100 most invasive alien species. Alien plants can invade native habitats through their adaptability to low-resource soils, and thus can out-compete and displace native vegetation. We investigated the effects of phosphorus (P) deficiency and soil acidity on legume-microbe symbiosis, nitrogen (N) nutrition and carbon (C) growth costs of these two legumes in grassland soils. We used as inoculum and growth substrate soils collected from a long-term (>65 years) nutrient and lime-addition trial, the Veld Fertilizer Trial (VFT), located at Ukulinga Research Farm near Pietermaritzburg in South Africa. We used soils from three VFT treatments: soils fertilized with superphosphate (336 kg ha-1) applied once per year (+P), soils fertilized with superphosphate (336 kg ha-1) applied once per year with dolomitic lime (2250 kg ha-1) applied once every 5 years (P+L) and soils with no superphosphate and no dolomitic lime applications (Control). Seeds of V. nilotica and L. leucocephala were germinated and grown independently in these soils in green house conditions and harvested after 125 days for measurement of growth, legume-microbe symbiosis, N nutrition and C growth costs. Results showed that the two legumes had different growth adaptations. Vachellia nilotica grown in control soils and +P soils nodulated with various Burkholderia spp., while L. leucocephala did not nodulate in all soil treatments. Both legumes utilized for growth both atmospheric- and soil-derived N across all treatments thereby decreasing C growth costs. Vachellia nilotica grown in +P soils accumulated the most biomass and N nutrition. Leucaena leucocephala maximized specific N assimilation rates by investing in below-ground biomass accumulation in control soils. This shows that L. leucocephala possesses traits that are successful in acquiring nutrients by investing in below-ground biomass and relying on utilization of N from both the soil and the atmosphere.

Nitrogen Source Preference and Growth Carbon Costs of Leucaena leucocephala (Lam.) de Wit Saplings in South African Grassland Soils.

Leucaena leucocephala (Fabaceae) is native to Central America and has invaded many climatic regions of the tropics. In South Africa, the species is categorized as an emerging or incipient weed used as fodder, timber, firewood and in erosion control on degraded habitats. The species is common along the eastern subtropical regions of KwaZulu-Natal (KZN) Province, where it invades grasslands, savannas and edges of forests. Soils of these ecosystems are characterized as nutrient deficient and acidic. Using a pot trial, we determined the effects of the nutrient addition treatments on microbial symbiosis, N nutrition and biomass accumulation of L. leucocephala under greenhouse conditions. After 180 days of growth, plants were harvested, and their utilization of N derived from the atmosphere and from the soil was quantified through determination of δ15N values. L. leucocephala maintained growth and N nutrition by relying on both atmospheric- and soil-derived N across all soil treatments. The NDFA was significantly higher in high P (N1 + P, N2 + P and N3 + P) soils. L. leucocephala was able to nodulate with intermediate and fast-growing strains from the Mesorhizobium and Rhizobium genus in N2 + P grown plants. This shows that L. leucocephala possesses traits that are successful in acquiring nutrients, especially in nutrient limited conditions, by establishing plant symbiosis with multiple bacteria and relying on extracting N from the soil and from the atmosphere through the symbiosis.

Altering Nitrogen Sources Affects Growth Carbon Costs in Vachellia nilotica Growing in Nutrient-Deficient Grassland Soils.

Vachellianilotica (L.) Willd. Ex Del. is a multipurpose leguminous tree that is common in grassland and savanna ecosystems in southern and eastern Africa. These ecosystem soils are reported to be acidic and nutrient-limited, specifically with regards to nitrogen (N) and phosphorus (P). The presence of this plant in these terrestrial ecosystems improves soil fertility benefiting the surrounding vegetation due to its ability to fix atmospheric N. This study seeks to understand the N-fixing bacteria symbiosis and physiological adaptations of V. nilotica in these acidic and nutrient-deficient KwaZulu-Natal soils. The soils used for this study were collected from the Ukulinga Grassland Nutrient Experiment located at the Ukulinga research farm of the University of KwaZulu-Natal, Pietermaritzburg, South Africa. Due to long-term soil nutrient addition treatments, these soils offered a diverse nutrient variation for better understanding the effects of acidity and nutrient variation on microbial symbiosis, plant nutrition, and biomass accumulation of V. nilotica. V. nilotica was able to maintain growth by relying on both atmospheric and soil-derived N across all treatments decreasing carbon (C) growth costs. There was an increased reliance on atmospheric-derived N of un-nodulated high N-treated plants. The plants grown in high N + P soils were able to nodulate with various species from the Mesorhizobium genus, which resulted in increased biomass compared to other plants. The results of this study show that V. nilotica can alter N sources to reduce C growth costs. In addition, both nodulating and free-living soil N2 fixing bacteria such as Caulobacter rhizosphaerae, Sphingomonas sp. and Burkholderia contaminans identified in the experimental soils may play an important role under P-deficient conditions.

Soil nutrient status of KwaZulu-Natal savanna and grassland biomes causes variation in cytokinin functional groups and their levels in above-ground and underground parts of three legumes.

Cytokinins (CKs) are involved in several developmental stages in the life-cycle of plants. The CK content in plants and their respective organs are susceptible to changes under different environmental conditions. In the current study, we profiled the CK content in the above and underground organs of three legumes (Lessertia frutescens, Mucuna pruriens and Pisum sativum) grown in soils collected from four locations (Ashburton, Bergville, Hluhluwe and Izingolweni) in KwaZulu-Natal province, South Africa. The quantified CK contents in the three legumes were categorized on the basis of their side chains (isoprenoid, aromatic and furfural) and modifications (e.g. free bases and glucosides). Legume and soil types as well as their interaction significantly influenced the concentrations of CKs. Lessertia frutescens, Mucuna pruriens and Pisum sativum had CK content that ranged from 124-653, 170-670 and 69-595 pmol/g DW, respectively. Substantial quantity (> 600 pmol/g DW) of CK were observed in plants grown in Bergville (above-ground part of Lessertia frutescens) and Izingolweni (underground part of Mucuna pruriens) soils. A total of 28 CK derivatives observed in the legumes comprised of isoprenoid (22), aromatic (5) and furfural (1) side-chain CKs. However, the 16 CK derivatives in Mucuna pruriens were isoprenoid-type based on the side-chain. Generally, a higher ratio of cis-zeatin (cZ) relative to the trans-zeatin (tZ) was evident in the above-ground part of Lessertia frutescens and Pisum sativum for the four soil treatments. In terms of functional and physiological importance of the CKs, the free bases (active form) and ribosides (translocation form) were the most abundant CK in Lessertia frutescens and Pisum sativum. However, N-glucoside, a deactivation/detoxicification product was the most dominant CK in Mucuna pruriens from Hluhluwe and Izingolweni soils. The total CKs in the underground parts of the legumes had a positive significant correlation with the total phosphorus and nitrogen content in the plant as well as the soil nitrogen. Overall, the CK profiles of the legumes were strongly influenced by the soil types. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01021-2.

Glutamate dehydrogenase is essential in the acclimation of Virgilia divaricata, a legume indigenous to the nutrient-poor Mediterranean-type ecosystems of the Cape Fynbos.

Glutamate dehydrogenase (NAD(H)- GDH, EC 1.4.1.2) is an important enzyme in nitrogen (N) metabolism. It serves as a link between C and N metabolism, in its role of assimilating ammonia into glutamine or deaminating glutamate into 2-oxoglutarate and ammonia. GDH may also have a key in the N assimilation of legumes growing in P-poor soils. Virgilia divaricata is such a legume, growing in the nutrient limited soils of the mediterranean-type Cape fynbos ecosystem. In order to understand the role of GDH in the nitrogen nutrition of V. divaricata, the aim of this study was to identify the GDH gene transcripts, their relative expressions and enzyme activity in P-stressed roots and nodules during N metabolism. During P deficiency there was a reduction in total plant biomass as well as total plant P concentration. The analysis of the GDH cDNA sequences in V. divaricata revealed the presence of GHD1 and GHD2 subunits, these corresponding to the GDH1, GDH-B and GDH3 genes of legumes and non-legume plants. The relative expression of GDH1 and GDH2 genes in the roots and nodules, indicates that two the subunits were differently regulated depending on the organ type, rather than P supply. Although both transcripts appeared to be ubiquitously expressed in the roots and nodules, the GDH2 transcript evidently predominated over those of GDH1. Furthermore, the higher expression of both GDH transcripts in the roots than nodules, suggests that roots are more reliant on on GDH in P-poor soils, than nodules. With regards to GHD activity, both aminating and deaminating GDH activities were differently affected by P deficiency in roots and nodules. This may function to assimilate N and regulate internal C and N in the roots and nodules. The variation in GDH1 and GDH2 transcript expression and GDH enzyme activities, indicate that the enzyme may be regulated by post-translational modification, instead of by gene expression during P deficiency in V. divaricata.

Differential patterns of nitrogen nutrition and growth cost of the indigenous Vachellia sieberiana and the introduced Chromolaena odorata in the savannah environment.

Vachellia sieberiana fixes atmospheric nitrogen (N) and distributes it back into ecosystems. We hypothesize that biological nitrogen fixation in this plant species is limited by competition from the invasive shrub, Chromolaena odorata. Competition would therefore result in the legume plant switching its limited nitrogen (N) sources in phosphorus-poor soils in savannah ecosystems when resources have to be shared. This study investigated the different patterns of N use and growth costs by a native and an introduced leguminous shrubby species. We propose that the two species sharing the same environment might result in competition. The competitive effect would induce in the indigenous legume to better utilize atmospheric-derived N modifying plant growth kinetics and plant mineral concentrations. Seedlings of V. sieberiana were cultivated in natural soil inoculum with low levels of phosphorus (mg L-1 ± SE) of 3.67 ± 0.88. The experiments were divided into two treatments where (i) seedlings of V. sieberiana were subjected to competition by cultivating them together with seedlings of C. odorata, and (ii) seedlings of V. sieberiana were cultivated independently. Although V. sieberiana was subjected to competition, the N2-fixing bacteria that occupied the nodules was Mesorhizobium species, similar to plants not subjected to competition. Total plant biomass was similar between treatments although V. sieberiana plants subjected to competition accumulated more below-ground biomass and showed higher carbon construction costs than plants growing individually. Total plant phosphorus and nitrogen decreased in seedlings of V. sieberiana under competition, whereas no differences were observed in percent N derived from the atmosphere (%NDFA) between treatments. The specific nitrogen utilization rate (SNUR) was higher in V. sieberiana plants subjected to competition while specific nitrogen absorption rate (SNAR) showed the opposite response. Vachellia sieberiana is highly adapted to nutrient-poor savannah ecosystems and can withstand competition from invasive shrubs by utilizing both atmospheric and soil nitrogen sources.

Variable P supply affects N metabolism in a legume tree, Virgilia divaricata, from nutrient-poor Mediterranean-type ecosystems.

Virgilia divaricata Adamson is a forest margin legume that is known to invade the N- and P-poor soils of the mature fynbos, implying that it tolerates variable soil N and P levels. It is not known how the legume uses inorganic N from soil and atmospheric sources under variable P supply. Little is known about how P deficiency affects the root nodule metabolic functioning of V. divaricata and the associated energy costs of N assimilation. This study aimed to determine whether P deficiency affects the metabolic status of roots and nodules, and the impact on the routes of N assimilation in V. divaricata.V. divaricata had reduced biomass, plant P concentration and biological nitrogen fixation during P deficiency. Based on adenylate data, P-stressed nodules maintained their P status better than P-stressed roots. V. divaricata was able to alter C and N metabolism differently in roots and nodules under P stress. This was achieved via internal P cycling by possible replacement of membrane phospholipids with sulfolipids and galactolipids, and increased reliance on the pyrophosphate (PPi)-dependent metabolism of sucrose via UDP-glucose (UDPG) and to fructose-6-phosphate (Fru-6-P). P-stressed roots mostly exported ureides as organic N and recycled amino acids via deaminating glutamate dehydrogenase. In contrast, P-stressed nodules largely exported amino acids. Compared with roots, nodules showed more P conservation during low P supply. The roots and nodules of V. divaricata metabolised N differently during P stress, meaning that these organs may contribute differently to the success of this plant in soils from forest to fynbos.