Much of soils in African continent are inherently low in fertility. Part of the land is degraded due to overgrazing; wind and water erosion (Henao and Baanante, 1999). This low in soil fertility reduces productivity and crop yield are often low leading to low organic matter inputs. In most cases, N is the key mineral nutrient limiting plant growth(Vance, 2001). An application of synthetic N fertilizers is the one way to enhance soil fertility and crop yield(Crews and Peoples, 2004). However, they are very expensive particularly to smallholder farmers. Additionally, they can contribute to environmental pollution(Crews and Peoples, 2004). Kraal manure can be used to improve soil fertility, but they are also found in smaller quantities to fertilize the whole cropping field.
Leguminous plants are extensively cultivated under agricultural systems in Africa as a source of protein. Legumes play a major role in nitrogen supply to the nearby plant and the supply of nitrogen to the soil. Through their symbiotic relationship with rhizombium, they have ability to fix atmospheric nitrogen into ammonia the form in which can be utilized by plants. They also help with increasing soil organic matter. In these circumstances fast-growing leguminous trees are suggested to be alternative forimproving soil fertility(Briske and Richards, 1995). Because of their ability to nodulate and fix N2 in association with members of rhizobia, they can accumulate abundant N in their biomass. The transportation of N from the roots to the shoot differs with type of legume plant. Some plant transport N as amides (aspargine and glutamine) whereas tropical legumes export ureides (allantoine)(Unkovich and Baldock, 2008, Herridge et al., 1988).
Pigeon pea (Jacanuscajan) is a perennial shrub legume belonging to the family Leguminosae. Pigeon pea originated from India, it is the third most important legume species in terms of grain after cowpea and groundnut (Fabunmi et al., 2010, PEOPLES et al., 1989). It has ability to nodulateandfixes atmospheric N2in association with members of rhizobium bacteria (Egbe and Bar-Anyam, 2011). It has ability to survive in harsh environment such as poor soil fertility and drought stress (Egbe and Bar-Anyam, 2011, Fabunmi et al., 2010). There are different methods that have been used to estimate N2 fixation in plants.Fixed N can also be estimated calorimetrically using the ureide technique(Unkovich et al., 2008), however earlier studies have indicated that xylem sap collection for determination of ureide concentration in pigeon pea must be done between 10h00 and 16h00 because of the diurnal variation in ureide concentration in xylem sap(Herridge, 1982, PEOPLES et al., 1989). In hedgenous (also known as alley) cropping system, pigeon pea trees are often subjected to periodical pruning to enhance light availability to associate crops or generate pruning to be used as soil amendment(Briske and Richards, 1995, Fabunmi et al., 2010). Some studies have demonstrated that pruning have detrimentaleffect on nodulation and the subsequent N2 fixation process(Briske and Richards, 1995, Fabunmi et al., 2010).To date, there are no studies that show how pruning could affect diurnal variation in xylem sap of leguminous trees.
Research question for this study are: Is there a difference in diurnal fluctuation in ureide concentration of clipped and unclipped pigeon pea plants? Does clipping affect gas exchange parameters on pigeon pea plants? The aim of this study is to determine the effect of clipping ondiurnal variation of ureide concentration in xylem sap of pigeon pea. To measure diurnal fluctuation in ureide concentration in the xylem sap of pigeon pea plants at different times of the day. To optimize the method for sampling ureide concentration of pigeon pea plants. It was hypothesized that the mean ureide concentration in xylem sap of pigeon pea is the same in all treatments, and that clipped and unclipped pigeon pea plants do not show similar pattern in diurnal variation of ureides concentration in the xylem sap.It was predicted that unclipped pigeon pea will have a high ureide concentration in the xylem sap due to undisturbed N2 fixation,
2. Methods and material
2.1 Study Site
The field sampling experiment were carried out at fountain hill Estate, 29o 27’ 02.84†S30o 32’ 31.68†E Wartburg, North East of the city of Pietermaritzburg, KwaZulu Natal South Africa. The study site is a nature reserve supporting wildlife and agricultural practice. The area has a warm and temperate climate with an average rainfall of 905mm per year.
Data collection
Seeds of pigeon pea plants were obtained from agro-forestry research area at Empangeni KwaZulu Natal. The plants were planted 1 m apart in a plot area of 70m2(7m x10m), randomized complete block design during November 2016. The plants were at mature stage. Three plots with 98 plants in total were selected. Out of 98 plants 72 were selected randomly for sampling. In each pot 50% of the trees were clipped at 75 cm height and 50% of the plants were left unclipped. Branches were removed from the clipped plants and only two branches were left for sampling in all the clipped plants.
Sampling for ureide
After 10 days the xylem sap were collected from stem segments in all plants clipped and unclipped. Sampling was done for 24 hours at 2 hour interval from 4am to 2am the next day. The stems were detached from the branches and were connected into a vacutainer that was connectedto a vacuum pump, 3 – 4cm of stem segments were cut from the top of the shoot to the bottom; this was done to allow the air to displace the xylem content into the vacutainer. A pressure of 20-25 kpa was applied to pump the sap from the stems. It took about 10 minutes to extract the sap in one plant. Xylem saps were extracted from 4 plants per block at each time interval. The xylem sap was stored in the ice and was placed in the freezer for later analyses. Total ureideallantion were estimated calorimetrically as described by Unkovich (2008). Chlorophyll was measured from 12 clipped and 12 unclipped pigeon pea plants. At each tree chlorophyll was measured from 4 trifoliate leaves using Dualex Dx_10_151 Chlorophyll and polyphenol meter made in France.
Analyses of xylem sap
Ureideallantoin was measured using a (Agilent Technologies Cary 60 UV- Vis) spectrophotometer as described by Herridge (1982) and Unkovich et al. (2008). A total of 72 sap samples with three water blanks and three internal standard of (0.02mM) concentration were analyzed. A sap sample of 0.05 ml and 1.2 ml of distilled water was added into each test tube. 0.5 ml of 0.5 NaOH was added to each test tube and the test tube was placed in boiling water for 8 minutes. The test tube was removed from boiling water and was placed into cool water for 10 minutes. 1.0 ml of 0.65N HCL/ Penylhdrazine mixture was added to each test tube and was placed into boiling water for 2 minutes. The test tube were removed immediately from boiling water and placed into an ice bath for 15 minutes. 2.5 lm of 10N HCL/ 1.67 KFeCN was added to each test tube, the test tubes were placed into ice bath for 20- 25 minutes for the development of reddish color. The absorbance was measured at 520nm on a spectrophotometer the absorption of the final colour from the spectrophotometer determines the presence of ureide concentration in the sap.Sample concentration were calculated using the formula from Unkovich et al. (2008), sample concentration = standard concentration *(O.D. sample/ O.D.standard)* dilution (25).(faka design yakho)
Statistical analyses
IBM statistical analysis (SPSS) software and Microsoft excel were used to plot the graphs and Two Way analysis of variance (ANOVA) were used to compare the means and to test the significance of treatments on ureide concentration of pigeon pea plants.