Unraveling the Effects of Pruning Frequency on Biomass Productivity, Nonstructural Carbohydrates and Nitrogen Fixation Rates of Sesbania sesban

Abstract

Tree pruning is a management tool in agroforestry systems for reducing shade, enhancing nutrient cycling or providing fodder. However, little information is available on the effect of pruning management on plant growth, nonstructural carbohydrate (NSC) levels in the roots and N₂ fixation of Sesbania sesban. A glasshouse experiment was conducted to assess the effect of pruning frequency on biomass production, NSC levels and N₂ fixation of Sesbania sesban. Pruning treatments consisted of a control (PF0), one pruning at 3 months after transplanting (MAT) (PF1), two successive prunings at 3 and 6 MAT (PF2), and three successive prunings at 3, 6 and 9 MAT (PF3), with each pruning removing shoot biomass above 50% of the initial height. The experiment was laid out in a completely randomized design (CRD) with four replications. Results showed that increasing pruning frequency resulted in decreased nodulation and nonstructural carbohydrate levels in the roots. Above and below ground biomass, root length, percentage N derived from the atmosphere and amount of N₂ fixed were decreased in a similar manner whether plants were successively pruned twice or thrice. It can be concluded that two or three successive prunings in nine months significantly reduce nonstructural carbohydrates, DM productivity and N₂ fixation of S. sesban, and might result in supply of insufficient biomass required for improving soil N fertility and livestock production.

1. Introduction

Sesbania sesban (L.) Merr is an important multipurpose woody legume capable of providing fuelwood, fodder and/or nutrient-rich biomass for nutrient cycling. It has been widely used in agroforestry in Eastern and Southern African regions for replenishing soil fertility in degraded agricultural landscapes [1,2], and for providing forage in “cut and carry” systems. It has a deep root system, which allows it to capture and recycle nutrients efficiently. Rhizobia that nodulate on the roots of Sesbania spp. are fast-growing strains and are found in a wide range of African soils [3]. S. sesban trees, through their symbiotic association with soil rhizobia, have been shown to contribute 84 kg N ha⁻¹ in Zimbabwe [1] and between 280 and 360 kg of N ha⁻¹ in Kenya [2].

In many agroforestry systems, smallholder African farmers manage woody trees by pruning branches, cutting soft twigs or allowing their livestock to browse on trees [4]. Management factors such as the frequency and intensity of pruning are often not considered by some farmers when harvesting tree prunings. For instance, smallholder farmers in Kenya have been reported to prune trees at a height of 0.60 m or less whilst others pruned between heights of 0.75–1.2 m [5]. In assessing fodder harvesting techniques, it was observed that Xymalos monospora and Rinorea convallarioides were often severely pruned by farmers in three montane forests in Kenya [6].

Leguminous woody trees are capable of providing high-quality prunings for various purposes, including nutrient cycling and livestock fodder. Consequently, farmers often shorten intervals between pruning events or increase the intensity of pruning. Earlier studies have shown that pruning more frequently decreased survival and biomass production of Sesbania grandiflora (L.) Pers. [7]. Shortening the harvest frequency from 8–12 weeks to 4 weeks has been shown to result in decreased leaf yields and increased mortality rates of woody legume trees by 50% [8]. In cases where a greater proportion of the leaf area is removed following pruning, the energy requirements for regrowth of the new shoot will be obtained from C assimilation in the residual photosynthetic tissue and mobilization of nonstructural carbohydrates (NSCs) stored in branches, stems and roots [9,10]. The pool of carbohydrate reserves is replenished once sufficient leaf area is re-established [9,11]. This means that the amount of carbohydrate reserves stored in the plant tissues and their subsequent translocation largely determine the success of new leaf and shoot regrowth [11,12].

Since tree regrowth largely depends on the reserve carbohydrates [12], pruning at short time intervals (more frequently) may result in depletion of these energy resources [10], reduced regenerative capacity or even death of trees [13]. Differences in post-cutting biomass production due to various cutting frequencies have been reported in Leucaena leucocephala and Gliricidia sepium that were linearly correlated with carbohydrate reserves [14].

S. sesban can derive between 5 and 90% of its N requirements from atmospheric N₂ [1,2]. Because leaves are the main source of carbon assimilates required for nodule functioning, tree pruning may negatively affect photosynthate supply to nodules [15]. Earlier studies show that, after shoots are removed, nodule biomass and nodule functioning are significantly reduced in some leguminous species [15,16,17], thus leading to suppression of symbiotic N₂ fixation. The reduction in N₂ fixation of agroforestry species following pruning will not only have implications on provision of N-rich fodder but also on soil N fertility improvement in degraded agricultural landscapes.

Despite intense research on the effects of pruning on leguminous species, limited information is currently available on the effects of increased pruning frequency on biomass productivity, nonstructural carbohydrate levels and N₂ fixation of S. sesban. Moreover, because growth and N₂-fixing capacity of woody legumes may vary depending on the species, they are most likely to react differently to frequent pruning. The objectives of this study were to (i) evaluate the effects of pruning frequency on biomass productivity and nonstructural carbohydrate reserves, (ii) assess if there is a relationship between biomass productivity and nonstructural carbohydrate root reserves, and (iii) assess the effects of pruning frequency on nodulation and N₂ fixation of S. sesban. It was hypothesized that increased pruning frequency will reduce biomass productivity, nonstructural carbohydrate levels, nodulation and percentage of N derived from the atmospheric N₂ (%Ndfa).

2. Materials and Method

2.1. Experimental Site and Plant Culture

The experiment was carried out under controlled greenhouse conditions at the University of KwaZulu-Natal, South Africa. The species being investigated was Sesbania sesban (L.) Merr (var. nubica), but the experiment required the use of Senna siamea as a reference plant for the estimations of N₂ fixation using the ¹⁵N natural abundance technique because this requires a non-N₂-fixing leguminous species with similar characteristics to the species being investigated. The N uptake pattern and rooting phenology of S. siamea are very similar to that of S. sesban [18]. Seeds of S. sesban (var. nubica) and S. siamea used in this study were obtained from natural populations near Empangeni (28°39′ S, 31°57′ E), KwaZulu-Natal Province, South Africa. The seeds of S. sesban and S. siamea were surface scarified by immersing in boiled water for 10–15 min and cooled with five rinses of cool tap water. The seeds were planted into seedling trays containing a mixture of ‘Umgeni River’ sand and seedling growth mix (Farmyard Organics). The sand was purchased from commercial suppliers in Pietermaritzburg. The seedling trays were placed in a glasshouse (temperature 21–32 °C and relative humidity of 60%–90%) and watered with fresh tap water once or twice a week, depending on the water requirements.

At 60 days after planting, sixteen healthy seedlings of S. sesban were transplanted into 5 L free-draining pots containing a 7:1 kg mixture of local unsterilized Umgeni River sand and sterile vermiculite. One seedling was transplanted per pot. Sixteen seedlings of S. siamea were also transplanted into separate 5 L pots containing a mixture of sand and vermiculite.

During the course of the experiment, plants were fed with 1 L of modified Hoagland N-free nutrient solution once a week, and in some cases, particularly on hot days, 1 L of water was used to supplement irrigation. Whenever aphids or red spidermites were observed on plants, a solution of Aphicide Plus (Chloro-nicotinyl) and Red Spidermicide (Tetradifon) was sprayed on plants at a rate of 62.5 mL per pot.

2.2. Nodulation

To ensure the presence of compatible rhizobia essential for symbiotic N₂ fixation, rhizosphere soils were also collected from S. sesban populations during seed collection. The soils were collected using shovels to prepare soil inoculum. About 700–800 g of soil from rhizospheres of S. sesban was collected in the top 0.5–20 cm of the soil profile and stored in a freezer (10 °C) prior to preparation of the soil inoculum. The inoculum was prepared by adding 1200 mL of sterile distilled water to 350 g of soil in a 2000 mL container. The contents were stirred for 20 to 30 min and left to settle before applying the soil suspension to seedlings. To guarantee effective nodulation, the seedlings were inoculated immediately after transplanting and 14 days after transplanting at a rate of 15 mL per pot per event.

2.3. Pruning Treatments

At approximately 100 days after transplanting when mean plant height (measured from root collar to the terminal bud) reached 91.4 cm and the mean number of leaves per plant was 87, the pruning treatments were applied to the pots. Pruning treatments consisted of a control (PF0), one pruning at 3 months after transplanting (MAT) (PF1), two successive prunings at 3 and 6 MAT (PF2), and three successive prunings at 3, 6 and 9 MAT (PF3), subsequently referred to as PF0, PF1, PF2 and PF3, respectively. At three MAT, all plants (except control) were pruned by removing shoot biomass above 50% of the initial height. At six MAT, only plants from the PF2 and PF3 treatments were pruned, and at nine MAT, only plants from the PF3 treatment were pruned. Each treatment consisted of four plants which were regarded as replicates. The experiment was laid out in a completely randomized design (CRD). Reference plants were not pruned throughout the duration of the experiment.

2.4. Plant Sampling and Processing

At 4 weeks after the final pruning, all plants were destructively harvested by decapitating at 2 cm above the soil line, and the aboveground biomass was partitioned into leaves, twigs, branches and main stem. The roots were carefully recovered from pots, washed free of soil over a sieve and root length was measured using a ruler. For determination of nonstructural carbohydrates, a 5 cm sample was taken from the uppermost portion of the tap root and put into an envelope. The envelopes were kept in a cooler box containing crushed ice and later oven-dried. The nodules were detached from roots and counted.

Non-N₂-fixing Senna plants were also harvested, and the biomass was partitioned into leaves, twigs, branches and main stem. All collected samples were oven-dried separately at 60 °C for 72 h and weighed for determination of dry matter yield. All the leaves and petioles of S. sesban and S. siamea were ground into fine powder for the analysis of total N (%N) and ¹⁵N natural abundance (δ¹⁵N). Root samples for the determination of starch and soluble sugars were ground into fine powder using a hammer mill and later homogenized (Precellys Evolution, Bertin Technologies, Thiron-Gardais, France).

2.5. Carbohydrate Analyses

Starch and soluble sugar concentrations were analyzed using a method described in [19]. However, in this study, hydrochloric acid rather than amyloglucosidase was used to hydrolyze the starch and sugars to glucose [20]. The root sugar and starch concentrations were used to calculate total nonstructural carbohydrates and the ratio of sugar-to-starch concentrations.

2.6. Analysis of Isotopic Composition

To determine the ¹⁵N/¹⁴N ratio of S. sesban and S. siamea, aliquots of 1.1 to 1.2 mg subsample of finely ground plant material were weighed into aluminum tin capsules that have been precleaned in toluene. The isotopic analysis was performed on a Flash EA 1112 Series coupled to a Delta V Plus stable light isotope ratio mass spectrometer via a ConFlo IV system (all equipment supplied by Thermo Fisher, Bremen, Germany), housed at the Stable Isotope Laboratory, Mammal Research Institute, University of Pretoria. During analysis, two laboratory running standards, Merck Gel (δ¹⁵N = 7.89‰; N% = 15.29) and DL-Valine (δ¹⁵N = –6.15‰; N% = 11.86), and a blank sample were run after every 11 unknown samples.

The ¹⁵N abundance, expressed as δ¹⁵N, i.e., the per million (‰) ¹⁵N excess over atmospheric N₂, was determined as [21]:

$${\delta }^{15}\mathrm{N}=\frac{{{(}^{15}\mathrm{N}{/}^{14}\mathrm{N})}_{\mathrm{sample}}-{{(}^{15}\mathrm{N}{/}^{14}\mathrm{N})}_{\mathrm{standard}}}{{{(}^{15}\mathrm{N}{/}^{14}\mathrm{N})}_{\mathrm{standard}}}\times 1000$$

(1)

where the ¹⁵N/¹⁴N sample and ¹⁵N/¹⁴N standard are, respectively, ratios of the sample and the standard (atmospheric N₂). The international standard for atmospheric N₂ is = 0.0036765 [22].

The proportion of N derived from the atmosphere (%Ndfa) was obtained by comparing the ¹⁵N natural abundance of N₂-fixing S. sesban with that of non-N₂-fixing S. siamea, which, in this case, is assumed to represent a measure of the isotopic signature of plant available soil mineral N for S. sesban [23]:

$$\%\mathrm{Ndfa}=\frac{{({\mathsf{\delta }}^{15}\mathrm{N})}_{\mathrm{non-fixing}\mathrm{leg}}-{({\mathsf{\delta }}^{15}\mathrm{N})}_{\mathrm{fixing}\mathrm{leg}}}{{({\mathsf{\delta }}^{15}\mathrm{N})}_{\mathrm{non-fixing}\mathrm{leg}}-\mathrm{B}\mathrm{value}}$$

(2)

where the B value is the ¹⁵N natural abundance of the nodulated test legume when grown with N₂ fixation as the sole source of N for its N nutrition. The B value replaces the value of atmospheric N as it incorporates the isotopic fractionation associated with N₂ fixation. The B value for S. sesban used in this study was −1.76 and was obtained from Reference [24]. The mean δ¹⁵N of S. siamea used for the estimation of %Ndfa was +1.28.

The amount of N₂ fixed in dry matter was determined from the %Ndfa and the amount of N accumulated in the dry biomass [25]:

$${\mathrm{N}}_2\mathrm{fixed}\left({\mathrm{g}\mathrm{plant}}^{-1}\right)=\frac{\%\mathrm{Ndfa}}{100}\times \mathrm{N}\mathrm{content}$$

(3)

where N content is the product of %N and dry matter yield:

$$\mathrm{N}\mathrm{content}\left({\mathrm{g}\mathrm{plant}}^{-1}\right)=\frac{\%\mathrm{N}}{100}\times \mathrm{DM}\mathrm{yield}$$

(4)

2.7. Statistical Analysis

Analysis of variance was carried out to compare treatment means and where significant differences were found, the Duncan multiple range test (DMRT) was used to separate treatment means at p ≤ 0.05 and significance level at * p < 0.05, ** p < 0.01 and *** p < 0.001 is indicated; NS = not significant. Correlation analysis was performed using Pearson’s simple correlation coefficients to test the relationships between DM yield and nonstructural carbohydrate reserves.

3. Results

3.1. Above- and Belowground Biomass Productivity

Aboveground dry matter (DM) yield of S. sesban plants was significantly reduced by the frequency of pruning (

Table 1. Aboveground dry matter (DM) productivity of S. sesban as affected by pruning frequency.

Treatment 1	Aboveground DM Yield (g plant−1)
	Stem	Branch	Twig	Leaf	Total
PF0	52.82 ± 2.26a	33.40 ± 2.91b	1.82 ± 0.11a	11.70 ± 0.49a	99.73 ± 4.48a
PF1	16.56 ± 1.37b	49.50 ± 4.77a	0.70 ± 0.11c	9.33 ± 0.31b	76.10 ± 6.18b
PF2	13.20 ± 1.19bc	15.17 ± 1.25c	1.83 ± 0.22a	6.50 ± 0.54c	36.70 ± 2.25c
PF3	17.88 ± 0.78c	8.00 ± 0.54c	1.67 ± 0.16b	5.83 ± 0.66c	33.38 ± 1.32c
Probability	≤0.001	≤0.001	≤0.01	≤0.001	≤0.001

¹ PF0 = unpruned; PF1 = pruned once; PF2 = pruned twice; PF3 = pruned 3 times. Values in columns (mean ± SE) with dissimilar letters are significantly different at p ≤ 0.01 and p ≤ 0.001.

). Except for twigs, the PF2 and PF3 plants achieved significantly lower branch, leaf and therefore total aboveground DM yield. Thus, the unpruned plants had significantly greater DM yield as compared with their unpruned counterparts.

As with total aboveground DM yield, root DM yield and root length were significantly reduced by the frequency of pruning (

Table 2. Root DM yield and root length of S. sesban as affected by pruning frequency.

Treatment 1	DM Yield	Length
	g plant−1	cm
PF0	77.35 ± 3.74a	71.50 ± 3.49a
PF1	65.45 ± 3.72b	58.75 ± 4.57ab
PF2	47.02 ± 2.97c	47.85 ± 4.24bc
PF3	42.78 ± 3.81c	44.20 ± 3.17c
Probability	≤0.001	≤0.001

¹ PF0 = unpruned; 1 = pruned once; PF2 = pruned twice; PF3 = pruned 3 times. Lowercase letters denote significant differences between treatments at p ≤ 0.05.

). In comparison with PF0 plants, pruning frequency reduced root DM yield by 15, 39 and 45% in PF1, PF2 and PF3 plants, respectively. The root length of PF1-, PF2- and PF3-treated plants were significantly reduced by 18, 33 and 38%, respectively, when compared with that of PF0 plants (Table 2).

3.2. Reserve Carbohydrate Concentrations

There was a significant effect of pruning frequency on root starch, sugar and total nonstructural carbohydrate (TNC) concentrations in roots of S. sesban (

Table 3. Concentration of starch, sugar and total nonstructural carbohydrates (TNC) in roots of S. sesban plants subjected to different pruning frequencies.

Treatment 1	Nonstructural Carbohydrates (mg g−1)
	Starch	Sugar	TNC
PF0	173.52 ± 3.74a	66.02 ± 5.49a	239.54 ± 8.88a
PF1	161.27 ± 5.84ab	58.82 ± 1.87ab	220.09 ± 7.17a
PF2	144.09 ± 3.08bc	48.91 ± 1.41b	193.01 ± 4.31b
PF3	140.34 ± 4.71c	43.89 ± 2.87c	184.23 ± 6.19b
Probability	≤0.001	≤0.01	≤0.001

¹ PF0 = unpruned; PF1 = pruned once; PF2 = pruned twice; PF3 = pruned 3 times. Lowercase letters denote significant differences between treatments at p ≤ 0.05.

). Plants that were pruned twice or thrice in nine months had lower starch concentrations than the less frequently pruned ones (PF1) and unpruned controls. Relative to PF0 plants, the starch concentration of PF1, PF2 and PF3 plants was decreased by 7, 17 and 19%, respectively. Similarly, the concentration of sugar in roots of PF1-, PF2- and PF3-treated plants decreased by 11, 26 and 33%, respectively, as compared with PF0 plants. Relative to PF0 plants, the TNC of PF1, PF2 and PF3 plants was decreased by 8, 19 and 23%, respectively (Table 3).

3.3. Relationship between Aboveground DM Yield and Carbohydrate Concentrations

Starch concentration correlated positively with aboveground DM yield (R² = 0.75, p < 0.001), just as sugar concentration correlated positively with aboveground DM yield (R² = 0.77, p < 0.001) (Figure 1a,b).

3.4. Nodulation and Symbiotic Performance

Nodulation of S. sesban, measured as nodule dry weight (DW) and nodule number per plant, was significantly affected by pruning frequency. Nodule DW decreased with increasing pruning frequency (

Table 4. Nodule dry weight (DW) and number of S. sesban as affected by pruning frequency.

Treatment 1	Nodule DW	Nodule Number
	g Plant−1	No. Plant−1
PF0	3.20 ± 0.10a	185 ± 10a
PF1	2.82 ± 0.09ab	163 ± 15ab
PF2	2.71 ± 0.10b	140 ± 9b
PF3	1.97 ± 0.12c	110 ± 9c
Probability	≤0.001	≤0.01

¹ PF0 = unpruned; PF1 = pruned once; PF2 = pruned twice; PF3 = pruned 3 times. Lowercase letters denote significant differences between treatments at p ≤ 0.05.

). When compared with nodule DM of the PF0 plants, nodule DM of PF1, PF2 and PF3 plants declined by 12, 15, and 38%, respectively. The number of nodules per plant also decreased with increasing pruning frequency. Nodule numbers of PF1, PF2 and PF3 plants were reduced by 12, 24 and 40%, respectively, as compared with their unpruned counterparts.

Isotopic analysis revealed that pruning frequency significantly affected N content, ¹⁵N natural abundance (δ¹⁵N), percent N derived from the atmosphere (%Ndfa) and amount of N₂ fixed by S. sesban but not total N (%N) (

Table 5. Symbiotic performance (measured as %N, N content, δ¹⁵N, %Ndfa and N₂ fixed) of S. sesban as affected by pruning frequency.

Treatment 1	N	N Content	δ15N	Ndfa	N2 Fixed
	%	g Plant−1	‰	%	g Plant−1
PF0	3.36 ± 0.29a	3.35 ± 0.12a	−1.45 ± 0.04b	90.72 ± 1.19a	3.04 ± 0.11a
PF1	3.39 ± 0.27a	2.59 ± 0.34b	−1.24 ± 0.04ab	84.80 ± 1.23a	2.17 ± 0.26ab
PF2	3.48 ± 0.31a	1.29 ± 0.18c	−1.16 ± 0.10a	81.99 ± 3.06b	1.05 ± 0.11b
PF3	4.00 ± 0.21a	1.33 ± 0.08c	−1.03 ± 0.08a	78.28 ± 2.46b	1.04 ± 0.05b
Probability	≤0.25	≤0.001	≤0.01	≤0.01	≤0.001

¹ PF0 = unpruned; PF1 = pruned once; PF2 = pruned twice; PF3 = pruned 3 times. Lowercase letters denote significant differences between treatments at p ≤ 0.05.

). As expected, unpruned plants and the less frequently pruned plants (PF1) recorded the highest N content in biomass as compared with the most frequently pruned plants (PF3). The δ¹⁵N signatures of plants significantly increased with increasing frequency of pruning. As a result, %Ndfa estimates of PF1, PF2 and PF3 plants decreased by 6, 10 and 14%, respectively, as compared with PF0 plants. The %Ndfa estimates varied from 88%–93%, 82%–87%, 75%–90% and 73%–85% for PF0, PF1, PF2 and PF3 plants, respectively. The amount of N₂ fixed was significantly decreased by pruning frequency (Table 5). As compared with PF0 plants, the amounts of N₂ fixed were decreased by 29, 65 and 66% for PF1, PF2 and PF3, respectively.

4. Discussion

4.1. Above- and Belowground Biomass Productivity

Pruning frequently significantly decreased total biomass productivity of S. sesban trees, and the pattern of dry matter production for PF2 and PF3 plants was almost identical (Table 1). The findings of this study agree with earlier results, which have demonstrated that increased pruning frequency decreases subsequent biomass production in leguminous woody species [7,11,13,26]. For example, it was shown that pruning frequency of 3 months significantly reduced biomass production of Sesbania grandiflora (L.) [7]. In the same study, monthly pruning frequency resulted in 100% mortality of S. grandiflora plants within six months of repeated pruning. In assessing the effects of pruning frequency (4, 6 and 8 weeks) on the productivity of S. sesban var. nubica, it was found that the total dry matter yield increased with increased pruning frequency over 16 weeks. More recently, it was also found that increased pruning frequency significantly reduced biomass productivity of Tithonia diversifolia [13]. Previous studies have shown that tree pruning limits C assimilation by reducing the total leaf area, and when it is done more frequently, it is known to decrease biomass productivity [13] and carbohydrate reserves [10]. The poor recovery of shoot growth of PF2 and PF3 plants could be associated with reduced photosynthetic capacity of plants and low levels of carbohydrate reserves in the roots, as has also been found for G. sepium and L. leucocephala [14] and Terminalia sericea [10]. This means that the frequent reduction in photosynthetic tissue of PF2 and PF3 plants resulted in decreased photosynthetic C supply that is required to maintain rapid regrowth of the developing shoots.

In the present study, pruning twice or three times in nine months was also found to decrease root DM and root length in S. sesban (Table 2), a finding consistent with data from many studies conducted on other important N₂-fixing agroforestry legumes [16,27,28]. For example, shoot removal reduced root biomass of Albizia lebbeck and L. leucocephala by 40 and 20%, respectively [27]. The reduction in root DM and length of PF2 and PF3 plants observed in this study could be interpreted to mean that S. seban sacrificed energy for development of belowground biomass in favor of shoot biomass. Reduced belowground DM production could also be due to cessation of root growth and root decomposition. Indeed, pruning of trees seems to impact root turnover, as suggested by significant reduction in live root abundance observed in the subsoil of pruned trees as compared to unpruned ones [28]. Similar responses were also observed in other agroforestry systems for Erythrina poeppigiana [12].

4.2. Reserve Carbohydrate Concentrations

The results showed that increased pruning frequency resulted in a progressive decline in the concentration of nonstructural carbohydrates in roots of the test species, suggesting that pruned trees relied on NSC reserves to overcome the reduction in C assimilates induced by shoot pruning. In fact, the PF3 plants, which were most affected by the pruning treatment, had significantly lower levels of starch, sugar and total nonstructural carbohydrates. The PF1 trees had significantly higher starch, sugar and TNC concentration compared to PF2 and PF3 trees because there was a regrowth period of seven months, during which the trees had the opportunity to replenish mobilized carbohydrates following pruning. The results of this study strongly support the assertion that nonstructural carbohydrates (comprised mainly of sugar and starch) stored in plant parts play an important role in resprouting trees during periods of negative carbon balance induced by shoot pruning, seasonal loss of leaves, fire disturbance or defoliation [9,10,14,29]. An earlier study also showed that frequent pruning progressively decreased starch and total reserve carbohydrates in Leucaena leucocephala and Gliricidia sepium [14]. A progressive decline in total NSC pool was also reported in Salix nigra following shoot removal at different intensities [30]. As opposed to pruning every 12 months, pruning every 2 and 6 months for harvesting forage decreased whole-tree carbohydrate levels in G. sepium [9]. The total NSC levels of Terminalia sericea that was cut seven times in two years were one-third that of uncut trees, while levels in trees cut once were half the levels in uncut trees [10]. The findings of this current study might mean that, when the supply of C assimilates was limited by losses of photosynthetic tissue, the demand for C required to rebuild photosynthetic tissue was met from mobilization of nonstructural carbohydrates, thus leading to reduction of these reserves in the roots. It is therefore reasonable to assume that S. sesban, as with other woody legume species, mobilize NSC reserves as a physiological mechanism to compensate for the reduction in photosynthetic C supply following shoot pruning.

4.3. Relationship between Aboveground DM Yield and Carbohydrate Concentrations

The relationship between dry matter productivity and nonstructural carbohydrates was determined to assess whether the effects of frequent pruning on biomass productivity are related to nonstructural carbohydrates in roots of S. sesban. The results showed a linear relationship between root starch levels and aboveground DM productivity (Figure 1a) and between root sugar levels and aboveground DM productivity (Figure 1b), indicating a greater depletion of these energy sources for re-establishment of leaf area following shoot pruning. It is very difficult to establish the actual role and regulation of reserve carbohydrates during regrowth of woody legume species following tree pruning. However, significant positive correlations between dry matter production and reserve carbohydrate levels have been shown before in frequently pruned L. leucocephala and G. sepium [14].

4.4. Nodulation and Symbiotic Performance

In the present study, nodule DM and nodule numbers of S. sesban tended to decrease progressively with increased pruning frequency (Table 4). The finding is consistent with data of [27] which showed that pruning frequency of 3 times in 16 months decreased nodule mass of A. lebbeck, G. sepium and L. leucocephala by 34.8, 26.8 and 11.6%, respectively, as compared with unpruned controls. In an experiment with E. poeppigiana, a 71% decline in nodule mass of completely pruned plants was found as compared with partially pruned plants [16]. The lower nodule biomass and nodule number recorded in PF3 plants in this study could be caused by intermittent reductions in C supply to nodules, thus leading to decomposition of more nodules. Removal of aerial parts of nodulated plants has been shown to limit the supply of photosynthates to the nodules thus inducing nodule decay and sloughing off of individual nodules [16].

There were no significant differences observed between the pruning frequencies in terms of %N in plant biomass. A similar finding was also reported for G. sepium subjected to varying pruning regimes [31]. Although the δ¹⁵N signatures of plants tended to increase with increasing pruning frequency (Table 5), they remained relatively close to the ¹⁵N natural abundance value of −1.76‰ for S. sesban grown with N₂ fixation as the sole source of N [24]. The lower δ¹⁵N signatures of S. sesban are indicative of higher reliance on N₂ fixation, hence greater %Ndfa values (Table 5).

Relative to PF0, pruning frequency decreased %Ndfa estimates by 6, 10 and 14% in PF1, PF2 and PF3, respectively (Table 5). The decline in %Ndfa estimates of S. sesban observed in this study is consistent with the results of a previous study which showed that 15, 25 and 40% weekly defoliation of Alnus tenuifolia seedlings reduced %Ndfa by 23, 33 and 67%, respectively, as compared with untreated control [17]. In another greenhouse study of G. sepium, a tropical N₂-fixing woody tree, it was found that the nitrogenase activity of partial and complete defoliated plants was decreased by 10 and 60%, respectively, as compared with that of undefoliated plants [32]. The findings of this study suggest that pruning twice or thrice in nine months led to recurrent fluctuations in leaf area, and under these conditions, photosynthesis from the residual leaf area was in some cases insufficient to supply C assimilates required for nodule functioning and maintenance of high N₂ fixation levels. Therefore, the decline in %Ndfa values of PF2 and PF3 plants could be due to limited supply of C assimilates to nodules, leading to reduced nodule functioning [15,32], death and their decomposition [16,27]. Nodule sucrose, malate and α-ketoglutarate contents were demonstrated to decrease 7 days following shoot removal, concluding that shoot removal caused a decline in C availability, thus leading to reduced nodule functioning in Medicago sativa (alfalfa) [15].

The effects of pruning frequency on amount of N₂ fixed in biomass followed trends similar to those of DM productivity. Since N content and amount of N₂ fixed depends mainly on the legume DM yield, the lower N content and amount of N₂ fixed in PF2 and PF3 plants was due to lower DM yields [25]. The N₂ fixed values obtained in this study show similar trends to those reported for G. sepium but slightly lower than those reported for A. lebbeck and L. leucocephala subjected to different pruning frequencies [27].

There were several limitations associated with repeating this experiment under field conditions. Firstly, it was very difficult to manage unpruned plants in this experiment because they regularly experienced transpiration-induced leaf shedding due to greater aerial biomass. This could be because the irrigation supply from 5 L pots was not sufficient to satisfy the water requirements of plants. Secondly, under field conditions, it is practically impossible to recover the entire root mass of S. sesban for determination of belowground biomass productivity and for measuring root lengths. Additionally, nodules of most woody species (including S. sesban) are highly delicate, and a majority of them can be lost from the root system during digging, with only a minor percentage being recovered. The study used Senna species as a reference plant for the estimations of N₂ fixation under controlled conditions. Although it is considered as a suitable reference plant, establishment of Senna species in the field experimental site was prohibited, as it is considered an alien plant in South Africa [33].

5. Conclusions

The results of this study showed that greater biomass was achieved when S. sesban trees were pruned once in ten months (less frequent pruning). However, two or three successive prunings significantly reduced DM production of S. sesban. The effects on nonstructural carbohydrate levels and nodulation appeared to be decreasing consistently with increasing frequency of pruning, which is in line with the hypothesis that increased pruning frequency reduces nonstructural carbohydrate levels and nodulation. The majority of other measured variables, including above- and belowground biomass, root length, percentage N derived from the atmosphere and amount of N₂ fixed, were decreased in a similar manner whether plants were successively pruned twice or thrice in ten months. It can be concluded that two or three successive prunings in ten months significantly reduce nonstructural carbohydrates, DM productivity and N₂ fixation of S. sesban trees. The reduction in DM productivity and N₂ fixation following frequent pruning might result in supply of insufficient biomass required for improving soil N fertility and livestock production in agroforestry systems. The findings of this study have implications for designing management of biological nitrogen fixation in agroforestry systems.