Abstract
The aim of the study is to determine the effect of clipping on diurnal variation in ureide concentration of xylem sap of pigeon pea plants. Legumes help the poor farmers rise out of poverty as it is the most important source of protein. Field can be fertilized with nitrogen by rotating planting every year by planting legumes. Nitrogen can also be returned to the soil by rotating this crop through the process called nitrogen fixation. Most tropical legume species manufacture ureide (allantoin and allantoic acid) in the root nodules and use these compounds for transport and storage of nitrogen, but there are legume species who don’t produce ureide it usually presents in low amount those legumes produces organic nitrogen from their root as amide (asparagine and glutamine). There are different methods that have been used to estimate N2 fixation in plants. In this study the xylem ureide technique was used in a field experiment, ureide concentration were assessed using ureide N solute method. Three separate plots of pigeon pea plantation were used, 96 pigeon pea plants were randomly selected from the three plots. 50% of the plants were clipped at 75 cm height and the other 50% were unclipped. It was hypothesized that the mean ureide concentration in xylem sap of pigeon pea plants is the same in all treatments. It was also hypothesized that clipped and unclipped pigeon pea plants do not show similar pattern in diurnal variation of ureides concentration in the xylem sap.
Xylem sap was collected using a vacuum extraction pump. The analyses were done using spectrophotometer. Results from the experiment showed statistically significant effect of treatments F (11, 120) = 3.720, p = 0.000 (P < 0.05), the ureide concentration in the xylem sap of pigeon pea plants is not the same in all treatments.
1. Introduction
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 for improving 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) (Herridge et al., 1988; Unkovich and Baldock, 2008).
Pigeon pea (Jacanus cajan) 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 cow pea and groundnut (Fabunmi et al., 2010; PEOPLES et al., 1989). It has ability to nodulate and fixes atmospheric N2 in 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 detrimental effect 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 on diurnal 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,
Methods and material
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 intervals from 4am to 2am the next day. The stems were detached from the branches and were connected into a vacutainer that was connected to 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 ureide allantion 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
Ureide allantoin 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 520 nm 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).
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.
Results
A total of 72 pigeon pea plants were samples for ureide. A total of 32 plants were clipped at 75cm height and a total of 32 were not clipped. All plants were at matured stage. The experiment was a randomized block design with 3 blocks, each containing 2 replicate per treatment. Two Way analysis of variance was conducted to examine the effect of treatments (clipping and time in hours) and the interaction effect between the two on ureide concentration of pigeon pea plants. All effect were significant at p = 0.05 significant level. The main effect of time was F (11,120) = 9, p < 0.001 indicating a significant different between time intervals. The main effect for treatment was also significant clipped (Mean= 0.5139, SD= 0.16778) and unclipped (Mean = 0.3554, SD= 0.13953). Interaction between treatments and time on ureide concentration was statistically significant F= (11,120) = 3.720 p < 0.001. Clipping had an effect on ureide concentration of pigeon pea plants but the effect differed across time.
Figure 1: mean ureide concentration of clipped and unclipped pigeon pea plants. The graph shows significant difference in mean concentration of clipped and unclipped plants (0.514 mM and 0.355 mM) respectively p < 0.05. The total mean ureide concentration of clipped plants showed a higher ureide concentration than the unclipped plants.
Figure 2: Mean ureide concentration of clipped and unclipped pigeon pea plants at different times of the day.
The graph is showing the mean ureide concentration of clipped and unclipped pigeon pea plants at different times of the day. Clipped plants showed higher concentration of ureide throughout the sampling time compared to unclipped plants. At 2pm the mean ureide concentration of clipped plants was the highest (Mean= 0.8117). At 8am the mean ureide concentration between clipped and unclipped plants was significant (Mean= 0.5590 and 0.5530) respectively therefore p < 0.05.
Figure 3: Pattern in diurnal variation of ureide concentration of clipped and unclipped pigeon pea plants. The diurnal pattern in ureide concentration of clipped and unclipped plants showed different pattern throughout the sampling time p< 0.05. The mean ureide concentration of clipped plants showed the highest pick between 12pm and 2pm whereas in unclipped plants the ureide concentration was at maximum and stable between 8am and 10am and also showed a slightly stable, but decreasing concentration between 10pm and 2am.
Results and discussion
Most legumes plant species are being planted and used in agro-forestry system and agricultural practices as timber, fuel wood, plantation shades, used as green manure and improving soil fertility through mulching in cropping system, and can be used to feed livestock (people’s et al., 1997). These plants are usually subjected to periodically pruning to enhance their yield. Pigeon pea in one plant that has been used in tropical environment, because of its ability to re-grow rapidly after pruning and it produces high protein foliage that is mostly used for livestock (Peoples. 1996).
Effect of clipping in ureide concentration
Clipping has shown no effect but a positive impact on ureide concentration of pigeon pea plants (figure 1). Therefore, clipping for 10 days did not affect nitrogen fixation of the plants, since the higher ureide concentration suggested a higher nitrogen fixation in the plants.
Table 1: Analysis of variance on the effect of treatments on ureide concentration of pigeon pea plants
Source Type I Sum of Squares df Mean Square F Sig.
Corrected Model
Intercept
Time
Treatments
Time * Treatments
Error
Total
Corrected Total 2.371a
27.206
.814
.905
.653
1.914
31.491
4.285 23
1
11
1
11
120
144
143 .103
27.206
.074
.905
.059
.016 6.464
1705.589
4.640
56.709
3.720 .000
.000
.000
.000
.000
In general clipping remove most of the leaf area that is important to support n2 fixation therefore the removal of leaf area reduce N2 fixation of the plants therefore reduce ureide concentration of the plant. A study done by (Nygren, 1995) on nodulated woody legume reported the important effect of pruning on N economy of legumes. They hypothesized that pruning may disturb N circulation and C economie of the plant may become unbalanced (Nygren, 1995)
Diurnal fluctuation
In diurnal pattern fluctuation of ureide concentration in the xylem sap (figure 2, 3) Diurnal fluctuation was very high between 4am -12pm there was no fluctuation around 12pm-4pm the values reached maximum at 2pm and decreased again. At night there was no or little fluctuation in ureide concentration. In unclipped plants fluctuation was between 4am-8am then it reached maximum at 8am and it had a little fluctuation between 14-18 afternoons. A diurnal study done by Herridge at al. (1988) on soyabean at flowering and mid pod filling stage they found very little fluctuation in relative abundance of ureide it was constant between 10am- 16pm and it reached maximum at 22pm -2am. There was a significant difference in diurnal pattern in ureide concentration of clipped and unclipped plants p < 0.05 (figure 3)
At night the transpiration is low therefore nitrogen fixation is stable and ureide concentration is high. The diurnal fluctuation in ureide concentration of pigeon pea plants was measure at different times of the day staring from 4am to 2am. We found that ureide concentration of clipped plants was low in early morning and it was very high at 2pm and very low at 1400 then increased again at 16h00. The ureide concentration in the xylem sap of clipped pigeon pea fluctuated throughout 24 hours; there was no stable period. Other studies have reported a high concentration of ureide in early mornings and in the afternoons around 9h00- 12h00 and at night (Herridge et al., 1988; PEOPLES et al., 1989) whereas in this study ureide concentration was higher during the day and lower at night figure 2 and 3 is showing the trend. The higher ureide concentration during the day may be due to physiological factors of the plant such as photosynthesis and transpiration that takes place during the day. Plant growth stage and other environmental factors such as weather conditions, soil type may also influence the concentration of ureide in the xylem sap of pigeon pea plants. It was expected that clipping will reduce N2 fixation of the plants, since the clipping method remove 75% of the leaf area which is very important to support N2 fixation. Therefore, ureide concentration was predicted to be low on clipped plants.
A study by Herrige (1996) on the diurnal fluctuation in relative ureide abundance of four legume species showed higher values in the early morning and late afternoon, and they found a significant fluctuation p < 0.05. The diurnal pattern for this study was not similar to that found by (Herridge et al., 1996). Another studies by (Herridge et al., 1988; Unkovich and Baldock, 2008) on other legume species suggested that sampling of xylem sap must be done between 10am and 2pm to avoid diurnal fluctuation. In this study ureide concentration was only stable between only 8am and 10am in unclipped plants and it was low at night in both treatments. A study by (Herridge et al., 1996) suggested that sampling of ureide must be done around noon to avoid fluctuation, in this study fluctuation was very high at noon (figure 2 and 3) low fluctuation was found at night therefore this suggestion was not supported by this study.
Looking at factors that could be affected by clipping, we measured chlorophyll content in trifoliate leaves in clipped and unclipped plants which is related to photosynthesis that is the primary source of nitrogen fixation. The results showed no significant effect of clipping on the leaf chlorophyll therefore no effect on photosynthesis of the plants p > 0.05.
Figure 4: at 95% CI Mean chlorophyll content measure in clipped and unclipped pigeon pea plants showed no significant difference between the two treatments p > 0.05
Chlorophyll is the green pigment that is needed by a plant to convert CO2 and H2O using sunlight into O2 and glucose. During photosynthesis chlorophyll capture the sun rays and produce sugar and carbohydrate which gives the pant energy to grow. The amount of chlorophyll in leaves suggest a photosynthetic ability of the plant. The chlorophyll content of the leaves were examined by analysis of variance to determine the effect of clipping on photosynthesis of the plants, the results indicated that there was not significant effect of the treatment on chlorophyll content of the plants. Chlorophyll content of the clipped plants did not differ significantly from the chlorophyll content of the control (unclipped) F (1, 22) = 3.014, p = 0.097, (p > 0.05). Clipping did not affect photosynthetic ability of the plants.
A study by (Luthra et al., 1983) reported that nitrogen fixation decrease as a results of decrease in photosynthesis of legumes. When photosynthesis of a plant increases nitrogen fixation also increases therefore ureide concentration in the xylem sap of the plant increases. Our results suggested that cutting increase nitrogen fixation of the plants and did not affect photosynthesis of the plants. One reason for the increase in ureide concentration of clipped plants, may be that plant were fixing more nitrogen for the plant to survive after the reduction in leaf area that could reduce photosynthesis of the therefore clipping encourages the plant to produce new stems or to re-grow new branches therefore nitrogen fixation increases for that process.
The change in ureide concentration between the treatments was significantly different in relation to time. It was expected that the control will have higher ureide concentration than the clipped due to the undisturbed N2 fixation and photosynthetic pathways. In general reducing the total leaf area of the plant causes the reduction in carbon fixing capacity of the plants. Other studies have reported that when a plant is exposed to moderated defoliation or pruning it photosynthetic rate of the remaining foliage increases (Pinkard et al., 1998). The results from this study have showed that ureide concentration of pigeon pea plants increase in response to clipping (Pinkard et al., 1998).
The importance of developing or optimizing simple to use sampling procedure for ureide concentration in the xylem sap of pigeon pea plants, it the presence of diurnal fluctuations in the ureide content of the sap. Knowing that there is a presence of fluctuation at a particular time therefore sampling must be restricted to a particular time of the day when there is no fluctuation or where the concentration is stable thus that will limit the number of treatment comparison that could be made and minimize errors that could be caused by fluctuation.
The limitation of this study was that the study was only conducted only on field grown plants and the plants were not given enough time to respond fully on the treatment. There were no nodules in the roots of the plants to examine the effect of clipping on the total carbohydrate on the plants. I would recommend future studies to look at all the factors that could cause the fluctuation in ureide concentration of pigeon pea plants such as physiological factors of the plants and the environmental factors and the plants must be given enough time to respond to the treatments.
Appendix
Table 1. Properties of soil along the profile wall before the initiation alley experiments at Fountainhill Estate, Wartburg.
Soil properties Unit Depth (cm)
0 – 20 20 – 40 40 – 60 60 – 80
Clay % 21,75 21,75 27,25 30,5
pH KCl 4,24 4,305 4,4825 4,4925
Organic matter % 0,8 0,5 0,5 0,5
N % 0,0725 0,05 0,05 0,05
P mg/L 15,75 9,5 4 4
K mg/L 82,75 44,75 43,5 42,75
Ca mg/L 419,25 371,5 495,25 498
Mg mg/L 101,5 106,5 150,5 185,75
Total cations cmol/L 3,5125 3,29 3,965 4,2725
Exchangeable acidity cmol/L 0,375 0,45 0,155 0,1475
Zn mg/L 3,85 2,625 0,825 0,625
Mn mg/L 46,75 50 26,75 21,25
Cu mg/L 2,95 3 2,525 2,375
Table 2: Chlorophyll content measure from 12 clipped and 12 unclipped pigeon pea plants.
Source Type III Sum of Squares df Mean Square F Sig.
Corrected Model
Intercept
Treatments
Error
Total
Corrected Total 38.939a
40608.472
38.939
284.180
40931.591
323.119 1
1
1
22
24
23 38.939
40608.472
38.939
12.917 3.014
3143.730
3.014 .097
.000
.097
Table 3: Mean chlorophyll content of clipped and unclipped pigeon pea plants
Treatments Mean Std. Deviation N
Clipped
Unclipped
Total 42.4079
39.8604
41.1342 4.34108
2.64378
3.74815 12
12
24
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