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Research Article

Early fruiting in Synsepalum dulcificum (Schumach. & Thonn.) Daniell juveniles induced by water and inorganic nutrient management

[version 1; peer review: 2 approved]
PUBLISHED 30 Mar 2017
Author details Author details
OPEN PEER REVIEW
REVIEWER STATUS

This article is included in the Agriculture, Food and Nutrition gateway.

Abstract

Background. The miracle plant, Synsepalum dulcificum (Schumach. & Thonn.) Daniell is a native African orphan crop species that has recently received increased attention due to its promise as a sweetener and source of antioxidants in both the food and pharmaceutical industries. However, a major obstacle to the species’ widespread utilization is its relatively slow growth rate and prolonged juvenile period. Method. In this study, we tested twelve treatments made up of various watering regimes and exogenous nutrient application (nitrogen, phosphorus and potassium, at varying dosages) on the relative survival, growth, and reproductive development of 15-months-old S. dulcificum juveniles. Results. While the plants survived under most tested growing conditions, nitrogen application at doses higher than 1.5 g [seedling]-1 was found to be highly detrimental, reducing survival to 0%. The treatment was found to affect all growth traits, and juveniles that received a combination of nitrogen, phosphorus, and potassium (each at a rate of 1.5 g [seedling]-1), in addition to daily watering, exhibited the most vegetative growth. The simple daily provision of adequate water was found to greatly accelerate the transition to reproductive maturity in the species (from >36 months to an average of 23 months), whereas nutrient application affected the length of the reproductive phase within a season, as well as the fruiting intensity. Conclusions. This study highlights the beneficial effect of water supply and fertilization on both vegetative and reproductive growth in S. dulcificum. Water supply appeared to be the most important factor unlocking flowering in the species, while the combination of nitrogen, phosphorus and potassium at the dose of 1.5 g (for all) consistently exhibited the highest performance for all growth and yield traits. These findings will help intensify S. dulcificum’s breeding and horticultural development.

Keywords

Mineral fertilization, juvenility phase, precocity, environmental induction, growth, flowering, miracle berry

Introduction

The miracle plant, Synsepalum dulcificum (Schumach. & Thonn.) Daniell (Sapotaceae), is a perennial shrub originating from West Africa (Inglett & May, 1968) and is the only known natural source of miraculin, a glycoprotein with remarkable edulcoration properties (Lim, 2013). In West Africa, the sweetening activity of the fruit is valued in drink-making, whereas the leaves, roots, and bark of the species are used in traditional treatments of diabetes, enuresis, kidney, hyperthermia, coughing, and stomach afflictions (Burkill, 2000; Oumorou et al., 2010). The fruit of the species (miracle berry) is a rich source of vitamin C, leucine, flavonols, and anthocyanin (Du et al., 2014; Njoku et al., 2015); and its modern utilizations include many applications in cosmetics, food, and pharmaceuticals (Achigan-Dako et al., 2015). With its many unique properties, some writers have suggested that miracle berry would currently have a much larger market in the USA, and therefore globally, if it had not been misclassified in the 1970's as a food additive instead of a sweetener (http://www.gayot.com/Lifestyle/Health/Benefits/Miracle-Fruit; http://www.theweek.co.uk/politics/27131/sweet-and-sour-tale-miracle-berry). Recently, additional scientific evidences were highlighted on the ability of the species to substitute sugar, particularly in sour beverages (Rodrigues et al., 2016).

Despite the nutritional, economic, and medicinal promise of the species, S. dulcificum remains a neglected crop that is not widely cultivated. In addition, according to Adomou (2005), the species is in depletion and is also suspected to exhibit recalcitrant seed storage behavior (Chen et al., 2012). One of the major constraints to economic cultivation of miracle berry is the very slow growth rate and the prolonged juvenile phase of the plant. According to Joyner (2006), the species seedling size at four years old is a maximum of 60 cm and fructification occurs only after three to four years; however, information regarding the growing conditions of the seedlings in that study was lacking. In Benin, where the plants are also reported to exhibit a relatively slow growth rate and to be late maturing, the species is almost wholly neglected. When encountered in its natural habitat (open field), the species exhibits relatively poor fitness in the face of weed competition, as well as anthropogenic and animal disturbances (Houeto, 2015).

An important step toward the systematic improvement of S. dulcificum would be to accelerate the transition to reproductive maturity, thus shortening generation times. According to Wilkie et al. (2008), there are three possible ways to induce flowering in horticultural trees, thereby reducing the length of the juvenile phase, or increasing precocity: environmental induction, autonomous induction, and the use of growth regulators. A plant’s ability to favorably respond to any of these flowering induction techniques greatly depends on its origin. While tropical and subtropical species tend to respond better to environment stimuli (e.g. mango, Mangifera indica L.; lychee, Litchi chineensis Sonn.), those from temperate regions exhibit autonomous floral induction (e.g. apple, Malus domestica Borkh.; sweet cherry, Prunus avium L.) (Wilkie et al., 2008). Given that S. dulcificum is a tropical species, we hypothesize that an accelerated transition to reproductive maturity can be triggered through proper environmental manipulation. Additionally, in woody angiosperms, cold treatment, nutrient supply, photoperiod, and water stress were found to be the main environmental stimulations that could induce flowering (Meilan, 1997).

One important factor limiting plant growth is nitrogen and phosphorus deficiency (King et al., 2008; Poothong & Reed, 2014). Nutrient status has been reported to affect gene activity and protein synthesis in plant species (e.g. Japanese red pine, Pinus densiflora Sieb. & Zucc.) (Nakaji et al., 2001). For instance, a high C/N ratio was reported to favor flowering in fruit trees (Hanke et al., 2007). Fertilization management thus appears to be a promising means of promoting plant growth and early flowering in horticultural species; and yet, different plant species tend to react to nutrient supply in unpredictable ways. For example, while phosphorus fertilization was found to be beneficial for the lobolly pine (Pinus taeda L.) growth, nitrogen fertilization on the same species was rather detrimental (Faustino et al., 2013). In another study, phosphorus fertigation was shown to be harmful to the fan flower (Scaevola aemula R. Br.) when applied at a rate exceeding 43.5 g.ml-1 (Zhang et al., 2004). In many other species, such as marula, Sclerocarya birrea (Hochst.) and wild loquat, Uapaca kirkinia (Muell.Arg.), the benefit of fertilizer application remains elusive (Akinnifesi et al., 2008). Similarly, water availability is considered to be one of the three most important factors controlling a plant's transition to flowering (Bernier et al., 1993), in addition to affecting the phenological rhythm of tropical species; and yet plant response to water stress (excess/deficiency) also tends to be species-specific. While water deficiency was found to promote flowering in Citrus spp. (Davenport, 2003), it reduced vegetative growth in Mangifera indica L. (Pavel & De villiers, 2004).

To the best of our knowledge, the response of S. dulcificum to fertilization and regular water supply has never been documented. Furthermore, detailed phenological data, especially in juveniles, are not available despite their importance to pioneering breeding programs. Understanding how nutrient and water supply affect fruiting in S. dulcificum juveniles is critical to the development of this promising species.

In this study, we analyzed the growth, flowering, and fruiting response of S. dulcificum to water and mineral fertilizer treatments with the objective of reducing the species natural (in reference to stands evolving in natural habitat) production cycle, while significantly enhancing overall growth and fruit yield.

Methods

Experimental site

The experiment was carried out from December 2013 to April 2016 in the municipality of Abomey-Calavi (southern Benin), at the experimental site of the Faculty of Agronomic Sciences, University of Abomey-Calavi (06°25’00.8”N, 002°20’24.5”E), and in a neighboring open field (06°27’00.”N, 002°21’00”E) to simulate natural rain fed conditions (no irrigation or exogenous nutrient application). Abomey-Calavi is located in the Guinean phytogeographical region of Benin largely characterized by a ferralitic soil type (Röhrig, 2008). During the experimental timeframe, the mean annual rainfall was 1,329 mm and the mean monthly temperature was 24°C.

Experimental system

In December 2013, mature, ripe and fresh fruits of S. dulcificum were collected from a single tree located in the district of Toffo (6°92’N; 2°27’E), where the soil is ferralitic, the mean annual rainfall is around 1,000 mm, and the mean annual temperature varies from 27°C to 30°C. Fruits collected were processed and sown at ambient temperature (25–27°C) in black polystyrene nursery bags (0.75 l) filled with sand to produce seedlings that were monitored in the nursery until they reached 13 months old. At that time, seedlings of a similar size were transplanted either in pots on the site of University of Abomey-Calavi or directly at soil in the open field and monitored for two months before being used in the watering and fertilization experiment. There was only one seedling per pot and each pot had 15 l volume.

The experiment was made up of twelve treatments (Table 1), out of which the absolute control (Cont: rain fed seedlings with no nutrient supply) was established at soil in the open site and the other 11 treatments were established in pots (to control the amount of water supply and its efficiency) filled with soil collected at 0–10 cm depth on the site of University of Abomey-Calavi. Each seedling in pots received two liters of water daily. Nutrients were brought to each pot (seedlings) separately; the nitrogen was applied as urea (46% N), the phosphorus as simple superphosphate (46% P2O5) and the potassium as potassium sulfate (48% K2O). Fertilizers were applied using the sub-surface method at 8 cm beneath the soil and at a frequency of one application every two months. The first application occurred in March 2015. Physico-chemical characteristics of the experimental medium in pots were as follows: pH (KCl) = 5.48, pH (H2O) = 5.88, silt = 25.75%, clay = 12.27%, sand = 61.98%, organic carbon = 1.03%, N = 0.06%, Mg = 2.37 (meq/100g), Ca = 0.63 (meq/100g), P = (2.08 meq/100g), and assimilable P = 23.06 ppm. The experiment design was of completely randomized design and each treatment was made up of a cohort of 10 seedlings of the same age (15 months). We used this sample size because S. dulcificum is a recalcitrant perennial, and obtaining progeny individuals of similar age and size was challenging.

Table 1. Treatments, amount of water supplied and nutrient doses applied at each fertilization event.

TreatmentsDaily watering
(l.seedling-1)
N (g. seedling-1)P (g. seedling-1)K (g. seedling-1)
Control----
W2---
N1.521.5--
N323.0--
N4.524.5--
P1.52-1.5-
P32-3.0-
P4.52-4.5-
K1.52--1.5
K32--3.0
K4.52--4.5
NPK21.51.51.5

Data collection

Measuring growth parameters. Before treatment application, initial stem collar diameter, plant height, number of branches, and number of leaves were measured for all seedlings (Table 2) to ensure that seedlings had similar size. At the end of the experiment (April 2016), the same traits were also measured to evaluate the increments.

Table 2. Initial growth parameters in juveniles of Synsepalum dulcificum at experiment onset.

Values are means ± SE (n = 10 seedlings).

TreatmentsStem collar
diameter (mm)
Height (cm)Number of
leaves
Branching
Cont4.28 ± 0.29a16.33 ± 1.35a40.2 ± 4.36a5.30 ± 0.21a
W3.97 ± 0.41a14.08 ± 1.16a36.30 ± 8.31a4.50 ± 0.5a
N1.53.91 ± 0.32a16.54 ± 0.92a39.20 ± 6.32a4.90 ± 0.43a
N33.96 ± 0.37a15.84 ± 1.33a41.80 ± 7.24a5.00 ± 0.59a
N4.54.30 ± 0.33a16.58 ± 1.29a46.10 ± 8.11a5.00 ± 0.74a
P1.54.12 ± 0.27a16.84 ± 1.62a42.80 ± 6.95a5.30 ± 0.53a
P34.41 ± 0.33a15.72 ± 1.45a44.20 ± 7.42a5.20 ± 0.48a
P4.53.98 ± 0.33a17.03 ± 1.57a39.10 ± 5.64a5.50 ± 0.71a
K1.54.23 ± 0.23a16.36 ± 1.13a37.60 ± 5.68a4.60 ± 0.5a
K34.00 ± 0.27a14.40 ± 1.51a35.30 ± 7.22a4.40 ± 0.8a
K4.54.38 ± 0.28a17.90 ± 1.42a45.10 ± 6.51a5.30 ± 0.21a
NPK4.31 ± 0.25a18.38 ± 0.92a47.4 ± 7.22a4.80 ± 0.35a
P-value 0.97ns 0.57ns 0.97ns 0.94ns

ns= Not significant at 5%.

Leaf area was measured following the method by Cornelissen et al. (2003). The most mature and fully sun exposed leaf was harvested from each seedling. Harvested leaves were photocopied on paper, which were cut-out and weighed according to the shape of the leaves. The weight of the cut-out paper was multiplied by the known area/weight ratio of the paper to get the leaf area. Growth was assessed based on the increment recorded for each vegetative growth parameter between the onset and the end of the experiment.

TreatmentsD0H0L0B0
Cont2.610.3264
Cont4.1815275
Cont3.69.5345
Cont5.318.9426
Cont3.713245
Cont4.3220.4456
Cont4.616.9525
Cont4.518.8325
Cont418636
Cont622.5576
W3.3812235
W2.9711153
W5.6521688
W3.9614.2273
W2.5810.733
W3.615.5375
W6.1117.4735
W5.5517.6753
W311.4155
W2.9610275
N1.55.2620.3714
N1.52.6213.8193
N1.54.8620.1606
N1.54.818.2507
N1.53.212.895
N1.54.2518465
N1.53.6914.8357
N1.54.7518.5514
N1.53.4316.4344
N1.52.2712.5174
N34.5518337
N32.6911.3265
N34.4720437
N33.3914.3324
N33.213233
N36.34221048
N33.6412.8334
N35.0621.8432
N33.9514405
N32.3711.2415
N4.54.1817.5543
N4.53.0115.1253
N4.55.2318.5405
N4.55.0121.2596
N4.53.2310304
N4.56.082010110
N4.55.3222697
N4.54.0917446
N4.53.5212.4192
N4.53.3312.1204
P1.54.6318.5555
P1.53.5617348
P1.54.9716.1464
P1.54.0714.6277
P1.53.4715314
P1.55.0326.5875
P1.54.318.5554
P1.55.322588
P1.53.2612.7254
P1.52.627.5104
P35.3518454
P33.4414.5173
P34.5216506
P3414.7364
P34.1815.2294
P3518777
P36.121.5827
P35.3121.8606
P33.7111347
P32.496.5124
P4.54.2524.5538
P4.53.6814.8193
P4.5417.8465
P4.54.5317.5496
P4.53.1613.8222
P4.54.1515386
P4.55.2125577
P4.55.7819.9679
P4.52.2910216
P4.52.7612193
K1.53.5514.5324
K1.54.3316.5336
K1.54.918.7574
K1.55.5323.5598
K1.53.3313.7132
K1.54.517625
K1.5418.7354
K1.54.7217476
K1.54.313.5234
K1.53.2210.5153
K33.6813.5324
K33.6812252
K35.2420664
K34.4516.5416
K32.558.361
K34.8518.5518
K35.1320.2769
K33.6111.5193
K33.5817214
K33.286.5163
K4.52.7310.3264
K4.54.1818165
K4.53.6412.5345
K4.55.3520586
K4.53.612.9245
K4.54.3219.2566
K4.54.7922525
K4.54.518.8405
K4.55.0121.5636
K4.55.7223.8826
NPK5.5225.4596
NPK3.2617.5505
NPK5.3820.1685
NPK417396
NPK3.5815.5375
NPK5.2619.5905
NPK4.2518125
NPK3.6815182
NPK417.5505
Dataset 1.Initial growth parameters at the fertilization experiment onset.

Tracking flowering phases. From the first day of treatment application to the end of experiment, we monitored each seedling development daily. Within the so-called generative phase, starting with budding and ending with fruit ripening, we distinguished seven main events (budding, flowering, flower bloom, fructification onset, fruit physiological maturity, ripening onset, and full ripening) demarcating six distinct phases (S1: budding to flowering, S2: flowering to flower bloom, S3: flower bloom to fructification onset, S4: fructification onset to physiological maturity, S5: physiological maturity to fruit ripening onset, and S6: fruit ripening onset to full ripening; see Figure 1). The occurrence date of each event was recorded and the total number of buds, flowers, and fruits per seedling were counted. The number of buds and the number of flowers were monitored until the tenth month (to avoid flower drop) of the experiment (December 2015) and only the fruiting was monitored to the end of the experiment (April 2016).

2bfa1224-40be-4af9-9cbd-211a20692a2a_figure1.gif

Figure 1. Main generative phases observed in Synsepalum dulcificum juveniles.

(A) Budding; (B) Flowering; (C) Flower bloom; (D) Fructification onset; (E) Physiological maturing; (F) Fruit ripening onset; (G) and (H) Fruit full ripening. S1 A→B; S2 B→C; S3 C→D; S4 D→E; S5 E→F; S6 F→G, H.

Statistical analysis

Prior to analysis we explored the datasets, and outliers were identified using the boxplot approach (Crawley, 2007). These outliers contained in Datasets 3 and 4 ((Tchokponhoué et al., 2017c; Tchokponhoué et al., 2017d) were removed from further vegetative growth analysis. Following this approach, outliers are considered as more than 1.5 times the interquartile range above the third quartile and below the first quartile. To test the effects of treatments on seedling survival, we performed a survival analysis. To analyze stem collar diameter, height, and leaf area variation in response to treatments, we performed analyses of variance followed by Tukey post hoc test for means separation. We employed orthogonal contrasts to dissect the effect of daily watering, as well as to analyze trends in growth response to progressive doses of nutrients when significant effects were observed. To analyze how the treatments affected the proportion of plants bearing buds, flowers, and fruits, we used prop.test. The number of branches, the number of leaves, the length of each generative phase, the number of buds, the number of flowers and the number of fruits were analyzed using a generalized linear model (glm) with poisson error structure (or quasi error structure to account for over-dispersion) where necessary. Apart from survival analysis, other statistical analyses were only performed for treatments that had at least two surviving seedlings at the end of the experiment. Also, since all seedlings considered in vegetative growth have not reached reproductive stages (e.g. budding, flowering), there is a discrepancy in the number of seedling between vegetative and reproductive growth datasets. Analyses were performed using “agricolae”, “car”, “gvlma”, ‘‘multcomp’’ and ‘‘survival’’ packages in R version 2.15.3 (R Developement Core Team, 2013) and results are presented as means ± standard errors (SE).

Results

Effect of treatments on the survival of seedlings

At the end of the experiment, the survival rate in the juveniles was highly affected by the treatment (P < 0.001), with the lowest survival rates observed in nitrogen-based treatments (Table 3). For this specific nutrient type (N), the higher the dose, the lower the survival and the more abrupt the survival decline. For instance, while the average time to death in juveniles that received 1.5 g nitrogen each was 12.00 ± 0.5 weeks, times to death in juveniles that received 3.0 g and 4.5 g nitrogen were 4.22 ± 0.3 weeks and 3.50 ± 0.3 weeks, respectively (Figure 2).

Table 3. Proportion and number of surviving seedling at the end of the experiment (n = 10 seedlings).

TreatmentsSurviving
seedlings (%)
Number of
surviving
seedlings
Cont100a10
W90a9
N1.580a8
N310b1
N4.500c0
P1.590a9
P390a9
P4.5100a10
K1.5100a10
K390a9
K4.5100b10
NPK90a9
P-value<0.001***-

Means with different letters within a column denote significant differences. ***= Significant at 1‰

2bfa1224-40be-4af9-9cbd-211a20692a2a_figure2.gif

Figure 2. Survival trends for Synsepalum dulcificum juveniles under various treatments (n =10 seedlings).

Cont = rain fed, no exogenous nutrients; W = Daily watering, no exogenous nutrients; N1.5 = Daily watering + 1.5 g N [seedling]-1; N3 = Daily watering +3 g N [seedling]-1; N4.5 = Daily watering + 4.5 g N [seedling]-1; P1.5 = Daily watering + 1.5 g P [seedling]-1; P3 = Daily watering + 3 g P [seedling]-1; P4.5 = Daily watering + 4.5 g P [seedling]-1; K1.5 = Daily watering +1.5 g K [seedling]-1; K3 = Daily watering + 3 g K [seedling]-1; K4.5 = Daily watering + 4.5 g K [seedling]-1; NPK = Daily watering + 1.5 g N + 1.5 g P + 1.5 g K [seedling]-1.

Treatment;Fertilizer;Dose;Time;Status
Cont;C;0;60;0
Cont;C;0;60;0
Cont;C;0;60;0
Cont;C;0;60;0
Cont;C;0;60;0
Cont;C;0;60;0
Cont;C;0;60;0
Cont;C;0;60;0
Cont;C;0;60;0
Cont;C;0;60;0
W;C;0;60;0
W;C;0;60;0
W;C;0;60;0
W;C;0;60;0
W;C;0;1;1
W;C;0;60;0
W;C;0;60;0
W;C;0;60;0
W;C;0;60;0
W;C;0;60;0
N1.5;N;1.5;60;0
N1.5;N;1.5;60;0
N1.5;N;1.5;60;0
N1.5;N;1.5;60;0
N1.5;N;1.5;16;1
N1.5;N;1.5;60;0
N1.5;N;1.5;60;0
N1.5;N;1.5;60;0
N1.5;N;1.5;60;0
N1.5;N;1.5;8;1
N3;N;3;1;1
N3;N;3;1;1
N3;N;3;1;1
N3;N;3;1;1
N3;N;3;2;1
N3;N;3;8;1
N3;N;3;8;1
N3;N;3;8;1
N3;N;3;8;1
N3;N;3;60;0
N4.5;N;4.5;2;1
N4.5;N;4.5;1;1
N4.5;N;4.5;8;1
N4.5;N;4.5;8;1
N4.5;N;4.5;2;1
N4.5;N;4.5;1;1
N4.5;N;4.5;8;1
N4.5;N;4.5;2;1
N4.5;N;4.5;2;1
N4.5;N;4.5;1;1
P1.5;P;1.5;60;0
P1.5;P;1.5;60;0
P1.5;P;1.5;60;0
P1.5;P;1.5;60;0
P1.5;P;1.5;60;0
P1.5;P;1.5;60;0
P1.5;P;1.5;60;0
P1.5;P;1.5;60;0
P1.5;P;1.5;60;0
P1.5;P;1.5;16;1
P3;P;3;60;0
P3;P;3;60;0
P3;P;3;60;0
P3;P;3;60;0
P3;P;3;60;0
P3;P;3;60;0
P3;P;3;60;0
P3;P;3;60;0
P3;P;3;60;0
P3;P;3;24;1
P4.5;P;4.5;60;0
P4.5;P;4.5;60;0
P4.5;P;4.5;60;0
P4.5;P;4.5;60;0
P4.5;P;4.5;60;0
P4.5;P;4.5;60;0
P4.5;P;4.5;60;0
P4.5;P;4.5;60;0
P4.5;P;4.5;60;0
P4.5;P;4.5;60;0
K1.5;K;1.5;60;0
K1.5;K;1.5;60;0
K1.5;K;1.5;60;0
K1.5;K;1.5;60;0
K1.5;K;1.5;60;0
K1.5;K;1.5;60;0
K1.5;K;1.5;60;0
K1.5;K;1.5;60;0
K1.5;K;1.5;60;0
K1.5;K;1.5;60;0
K3;K;3;60;0
K3;K;3;60;0
K3;K;3;60;0
K3;K;3;60;0
K3;K;3;60;1
K3;K;3;60;0
K3;K;3;60;0
K3;K;3;60;0
K3;K;3;60;0
K3;K;3;60;0
K4.5;K;4.5;60;0
K4.5;K;4.5;60;0
K4.5;K;4.5;60;0
K4.5;K;4.5;60;0
K4.5;K;4.5;60;0
K4.5;K;4.5;60;0
K4.5;K;4.5;60;0
K4.5;K;4.5;60;0
K4.5;K;4.5;60;0
K4.5;K;4.5;60;0
NPK;NPK;4.5;60;0
NPK;NPK;4.5;60;0
NPK;NPK;4.5;60;0
NPK;NPK;4.5;12;1
NPK;NPK;4.5;60;0
NPK;NPK;4.5;60;0
NPK;NPK;4.5;60;0
NPK;NPK;4.5;60;0
NPK;NPK;4.5;60;0
Dataset 2.Survival data.

Vegetative growth response to treatments

The survival data indicated a survival rate less than 20% in treatments N3 and N4.5; consequently they were discarded from subsequent analyses.

Stem collar diameter, plant height, and branching. The increment in the seedlings stem collar diameter was highly affected by treatment (Figure 3A). The daily watered juveniles performed better than the rain fed ones (P < 0.001). The extent of the stem collar diameter growth also greatly differed among nutrient types. For instance, the average increment in juveniles fertilized with NPK (10.36 ± 0.96 mm) was nearly twofold higher than that in juveniles fertilized with nitrogen only (4.73 ± 1.31 mm). The stem collar diameter growth with phosphorus was as good as potassium (P = 0.52), but higher than N (P = 0.007), and lower than with NPK (P = 0.04). We observed a highly significant effect of treatment on plant height (Figure 3B). Contrast analysis indicated that combined N, P and K application increased plant height better than single nutrient application (P = 0.01). Plants also better responded to phosphorus or potassium supply than to nitrogen (P = 0.002). Meanwhile, rain fed seedlings grew taller than daily watered plants receiving a single nutrient (P < 0.01).

2bfa1224-40be-4af9-9cbd-211a20692a2a_figure3.gif

Figure 3. Vegetative growth response of Synsepalum dulcificum juveniles under various treatments.

(A) Stem collar diameter; (B) Height; (C) Branching; (D) Leaf production and (E) Leaf area. Values are means ± SE (n = 8 – 10 seedlings). Means with different letters denote significant differences at P < 0.05, ANOVA, Tukey Test. Cont = rain fed, no exogenous nutrients; W = Daily watering, no exogenous nutrients; N1.5 = Daily watering + 1.5 g N [seedling]-1; N3 = Daily watering +3 g N [seedling]-1; N4.5 = Daily watering + 4.5 g N [seedling]-1; P1.5 = Daily watering + 1.5 g P [seedling]-1; P3 = Daily watering + 3 g P [seedling]-1; P4.5 = Daily watering + 4.5 g P [seedling]-1; K1.5 = Daily watering +1.5 g K [seedling]-1; K3 = Daily watering + 3 g K [seedling]-1; K4.5 = Daily watering + 4.5 g K [seedling]-1; NPK = Daily watering + 1.5 g N + 1.5 g P + 1.5 g K [seedling]-1.

The branching intensity also greatly varied following treatments (Figure 3C). The average branches gain in rain fed seedlings was 3.75 ± 0.53, whereas the set of daily watered juveniles gained on average nearly double (7.33 ± 1.35; P < 0.001). The effect of nutrient supply was also significant (P < 0.001) on the seedling branching, with plants fertilized with NPK gaining on average 12.33 ± 1.8 branches against 6.74 ± 1.25 for plants fertilized with a single nutrient.

Increase in leaf number and size. The variation in leaf production based on treatment is presented in Figure 3D. The differences in the increment of the number of leaves due to water supply and to exogenous nutrient application were all highly significant (P < 0.001). Grouped together, daily watered juveniles produced on average fourfold more leaves than rain fed juveniles. Regarding the fertilizer type, daily watered juveniles fertilized with NPK gained on average 925 ± 154 leaves, representing for instance 2.51 times the average leaf gain in simply watered juveniles without exogenous nutrient (W). Furthermore, NPK particularly improved leaf production comparatively to single nutrient application (P < 0.001). Likewise, the treatment significantly affected the leaf size with daily watered juveniles presenting a larger leaf area (1539.06 ± 55.46 mm2) than rain fed juveniles (695.37 ± 86.87 mm2), and leaf area in juveniles fertilized with NPK was greater than those of juveniles fertilized with a single nutrient (Figure 3E). However, the juveniles responded better when P or K was supplied than when N was supplied.

Treatments;Fertilizer;Dose;Diam_inc;Height_inc;Branch_inc;Leaves_inc
Cont;C;0;6;17.1;4;125
Cont;C;0;4.3;15.1;5;116
Cont;C;0;5.8;15;3;98
Cont;C;0;5;17;4;100
Cont;C;0;3.06;14.5;2;77
Cont;C;0;4.5;16.1;6;110
Cont;C;0;4;10;3;73
Cont;C;0;3.9;8.9;3;91
Cont;C;0;0.1;30;15;98
Cont;C;0;0.07;14;13;22
W;C;0;4.8;7.6;3;325
W;C;0;3.16;3.8;6;138
W;C;0;4.62;1.5;5;277
W;C;0;7.77;12.1;12;548
W;C;0;10.59;10.2;9;548
W;C;0;6.23;6.7;7;395
W;C;0;17.1;20.9;18;1299
W;C;0;6.4;6.9;8;408
W;C;0;6.1;7.2;6;330
N1.5;N;1.5;6.82;2.2;11;538
N1.5;N;1.5;0.97;0.7;0;4
N1.5;N;1.5;7.83;9.9;6;374
N1.5;N;1.5;5.4;5.8;4;394
N1.5;N;1.5;0.6;1.3;4;0
N1.5;N;1.5;9.31;11.58;7;697
N1.5;N;1.5;2.23;0;4;34
N1.5;N;1.5;19.3;25.5;17;1554
P1.5;P;1.5;8.25;12;2;241
P1.5;P;1.5;9.22;12.5;6;348
P1.5;P;1.5;8.23;9.5;3;412
P1.5;P;1.5;1.55;2.9;2;82
P1.5;P;1.5;8.93;9;4;820
P1.5;P;1.5;10.1;15;13;475
P1.5;P;1.5;5.52;3.2;1;128
P1.5;P;1.5;1.28;1.3;4;1
P1.5;P;1.5;6.65;7.9;12;1229
P3;P;3;8.54;11;8;397
P3;P;3;4.31;5.5;5;121
P3;P;3;0.21;11.58;5;405
P3;P;3;9.33;14.2;7;478
P3;P;3;6.11;6.8;7;215
P3;P;3;6;9.3;5;278
P3;P;3;11.55;20;10;714
P3;P;3;6.64;6;4;500
P3;P;3;10.3;13.7;9;663
P4.5;P;4.5;12.68;15.8;7;1263
P4.5;P;4.5;8.78;14.8;7;830
P4.5;P;4.5;6.49;9.6;7;439
P4.5;P;4.5;6.32;9.8;5;206
P4.5;P;4.5;7.24;17;10;627
P4.5;P;4.5;1.07;0.7;7;263
P4.5;P;4.5;1.73;13.9;13;2000
P4.5;P;4.5;8.42;17;5;394
P4.5;P;4.5;10.33;11.6;18;497
P4.5;P;4.5;9.17;13.7;4;399
K1.5;K;1.5;16.9;13.1;8;575
K1.5;K;1.5;3.34;5;3;101
K1.5;K;1.5;7.8;18.5;7;416
K1.5;K;1.5;11.59;13.5;14;975
K1.5;K;1.5;8.74;13.5;10;636
K1.5;K;1.5;6.5;13.8;8;211
K1.5;K;1.5;7.74;8.3;8;269
K1.5;K;1.5;4.32;6;4;125
K1.5;K;1.5;7.3;8.4;10;231
K1.5;K;1.5;8.1;12.6;19;1488
K3;K;3;8.12;16;9;371
K3;K;3;8.23;8.5;10;198
K3;K;3;13.41;15;9;819
K3;K;3;8.47;9.5;7;381
K3;K;3;5.3;12.4;5;157
K3;K;3;7.21;9;4;312
K3;K;3;0.98;9.5;4;20
K3;K;3;5.59;5.9;7;101
K3;K;3;7.2;6;7;296
K4.5;K;4.5;11.89;18.1;8;496
K4.5;K;4.5;12.16;19.9;20;1057
K4.5;K;4.5;5.65;6.5;2;148
K4.5;K;4.5;9.09;9.5;7;512
K4.5;K;4.5;3.68;6;2;83
K4.5;K;4.5;10.51;9.8;4;630
K4.5;K;4.5;12.33;16.5;9;917
K4.5;K;4.5;6.87;8.2;8;523
K4.5;K;4.5;8.5;5.5;8;473
K4.5;K;4.5;17.74;23.2;19;1601
NPK;NPK;4.5;8.98;13.3;7;644
NPK;NPK;4.5;9.3;16.5;14;895
NPK;NPK;4.5;9.49;14.2;12;858
NPK;NPK;4.5;11.8;14.5;14;744
NPK;NPK;4.5;12.72;14.4;14;1393
NPK;NPK;4.5;10.73;24;18;1285
NPK;NPK;4.5;9.1;16.3;11;1023
NPK;NPK;4.5;11.63;18;14;1051
Dataset 3.Growth parameters (increment) at the end of the experiment for vegetative growth.
Treatments;Fertilizer;Dose;Leaf area
Cont;C;0;107
Cont;C;0;599
Cont;C;0;704
Cont;C;0;984
Cont;C;0;618
Cont;C;0;714
Cont;C;0;498
Cont;C;0;762
Cont;C;0;118
Cont;C;0;684
W;C;0;1250
W;C;0;750
W;C;0;1125
W;C;0;1625
W;C;0;1425
W;C;0;2330
W;C;0;20
W;C;0;1355
W;C;0;1570
N1.5;N;1.5;1875
N1.5;N;1.5;1625
N1.5;N;1.5;1000
N1.5;N;1.5;2956
N1.5;N;1.5;1625
N1.5;N;1.5;500
N1.5;N;1.5;125
N1.5;N;1.5;1134
P1.5;P;1.5;3210
P1.5;P;1.5;1875
P1.5;P;1.5;1250
P1.5;P;1.5;1750
P1.5;P;1.5;875
P1.5;P;1.5;79
P1.5;P;1.5;1875
P1.5;P;1.5;1625
P1.5;P;1.5;1625
P3;P;3;1625
P3;P;3;1000
P3;P;3;2125
P3;P;3;1125
P3;P;3;1000
P3;P;3;2125
P3;P;3;1375
P3;P;3;2000
P3;P;3;3635
P4.5;P;4.5;2000
P4.5;P;4.5;1375
P4.5;P;4.5;1000
P4.5;P;4.5;1500
P4.5;P;4.5;1750
P4.5;P;4.5;2775
P4.5;P;4.5;1750
P4.5;P;4.5;1625
P4.5;P;4.5;2800
P4.5;P;4.5;1625
K1.5;K;1.5;220
K1.5;K;1.5;1375
K1.5;K;1.5;1625
K1.5;K;1.5;1500
K1.5;K;1.5;1250
K1.5;K;1.5;180
K1.5;K;1.5;1375
K1.5;K;1.5;1250
K1.5;K;1.5;1875
K1.5;K;1.5;1465
K3;K;3;1500
K3;K;3;1625
K3;K;3;1625
K3;K;3;2125
K3;K;3;1250
K3;K;3;1625
K3;K;3;750
K3;K;3;1750
K3;K;3;100
K4.5;K;4.5;2000
K4.5;K;4.5;2000
K4.5;K;4.5;1625
K4.5;K;4.5;1625
K4.5;K;4.5;1350
K4.5;K;4.5;1750
K4.5;K;4.5;1750
K4.5;K;4.5;1500
K4.5;K;4.5;1275
K4.5;K;4.5;2125
NPK;NPK;4.5;1625
NPK;NPK;4.5;1500
NPK;NPK;4.5;2500
NPK;NPK;4.5;4255
NPK;NPK;4.5;2375
NPK;NPK;4.5;1500
NPK;NPK;4.5;3925
NPK;NPK;4.5;2000
Dataset 4.Growth parameters at the end of the experiment for leaf area.

Flowering and fruiting responses

Budding and flowering. The proportion of budding juveniles was significantly affected by the treatment and ranged from 0–100% (Table 4). The contrast analysis on the average time to budding revealed a significant effect of treatment (P = 0.02; Figure 4A). Though the shortest times to budding, 190 ± 5.92 days and 201 ± 24.51 days were recorded in daily watered unfertilized juveniles and in daily watered and NPK fertilized juveniles, respectively, the highest number of buds was observed in juveniles fertilized with NPK (Table 5). After 10 months, NPK-fertilized seedlings produced a significantly greater number of buds than unfertilized plants (six times, P = 0.05).

Table 4. Proportion and number of budding, flowering and fruiting juveniles of Synsepalum dulcificum based on treatments (n = 8 – 10 seedlings).

TreatmentsBudding
seedlings (%)
Flowering
seedlings (%)
Fruiting
seedlings (%)
Budding
seedlings (n)
Flowering
seedlings (n)
Fruiting
seedlings (n)
Cont0.00d 0.00c 0.00e 000
W33.33c 33.33b 22.22d 332
N1.562.50b 50.00b 50.00c 544
P1.566.66b 55.55b 55.55c 655
P388.88a 88.88a 88.88b 888
P4.570.00b 70.00b 60.00c 776
K1.560.00b 60.00b 60.00c 666
K388.88a 88.88a 88.88b 888
K4.5100.00a 100.00a 100.00a 101010
NPK100.00a 100.00a 100.00a 999
P-Value < 0.001*** < 0.001*** < 0.001*** - - -

Means with different letters within a column denote significant differences. ***= Significant at 1‰.

Table 5. Average numbers of buds and fruits produced by juveniles of Synsepalum dulcificum under various treatments.

Values are means ± SE (n = 3 – 10 seedlings).

TreatmentsNumber of buds$ Number of fruit
W30.66 ± 15.05b17.00 ± 6.24b
N1.543.60 ± 10.47b25.50 ± 8.43b
P1.567.83 ± 62.47b17.80 ± 12.61b
P338.62 ± 19.79b19.87 ± 5.93b
P4.524.71 ± 13.79b24.20 ± 5.69b
K1.511.5 ± 6.73b13.00 ± 3.34b
K322.62 ± 20.18b29.50 ± 18.5b
K4.574.10 ± 31.42b21.88 ± 6.95b
NPK187.55 ± 84.95a52.50 ± 15.64a
P-value 0.05* 0.01*

$: assessed at the tenth month of the experiment, : assessed at the end of the experiment (thirteenth month of the experiment).

Means with different letters within a column denote significant differences. *= Significant at 5%.

The proportion of flowering juveniles was also highly affected by the treatment (Table 4). The highest flowering percentages were observed in NPK-fertilized juveniles and those fertilized with potassium at 4.5 g [juvenile]-1 (100%). The time to flowering (Figure 4B) was shorter for NPK-fertilized juveniles (P = 0.004), which flowered after 242.0 ± 21.97 days compared to 299.65 ± 7.41 days for the set of single-nutrient fertilized juveniles. Within the potassium-based treatments, the effect of application dose was significant (P = 0.01) and the time to flowering decreased as the potassium dose increased with a quadratic relationship between the two variables (P = 0.02). The regression equation reads: Time to flowering = 300.52 + 49.32 Potassium dose -19.51 (Potassium dose)2.

2bfa1224-40be-4af9-9cbd-211a20692a2a_figure4.gif

Figure 4. Reproductive performance in juveniles of Synsepalum dulcificum under various treatments.

(A) Time to budding; (B) Time to flowering; (C) Time to fruiting and (D) Total fruit production. Values are means ± SE (n = 5 – 10 seedlings). Means/Values with different letters denote significant differences. Generalized linear model, Tukey Test. W = Daily watering, no exogenous nutrients; N1.5 = Daily watering + 1.5 g N [seedling]-1; N3 = Daily watering +3 g N [seedling]-1; N4.5 = Daily watering + 4.5 g N [seedling]-1; P1.5 = Daily watering + 1.5 g P [seedling]-1; P3 = Daily watering + 3 g P [seedling]-1; P4.5 = Daily watering + 4.5 g P [seedling]-1; K1.5 = Daily watering +1.5 g K [seedling]-1; K3 = Daily watering + 3 g K [seedling]-1; K4.5 = Daily watering + 4.5 g K [seedling]-1; NPK = Daily watering + 1.5 g N + 1.5 g P + 1.5 g K [seedling]-1.

Fructification. The proportion of fruiting juveniles ranged from 0% in rain fed juveniles to 100% in NPK-fertilized plants and was highly affected by the treatment (Table 4). Likewise, the time to fruiting in S. dulcificum juveniles significantly differed among treatments (P = 0.004) and varied from 286 ± 9.33 days to 377 ± 5.43 days (Figure 4C). The earliest fruiting individuals included NPK-fertilized plants. Here also, the time to fruiting was affected by the potassium dose (P = 0.02). We also observed a significant quadratic relationship between the time to fruiting and the potassium application dose (P = 0.03). The equation reads: Time to fruiting = 355.48 + 39.18 Potassium dose -16.99 (Potassium dose)2.

Furthermore, the highest cumulative fruit number per treatment (Figure 4D) and average fruit number yielded by each plant (Table 5) were observed in NPK-fertilized juveniles. For instance, NPK-fertilized juveniles produced twofold more fruits than those that received a single nutrient (N or P or K) and threefold more fruits than juveniles that received no nutrients (Table 5). The fruit mass significantly differed among treatments (P = 0.01) and ranged from 1.08 ± 0.17 g (in juveniles fertilized with 1.5 g phosphorus) to 1.47 ± 0.04 g (in juveniles fertilized with 3 g phosphorus).

Treat;Fertilizer;Dose;BUDTIME
W;Control;0;202
W;Control;0;182
W;Control;0;188
N1.5;N;1.5;216
N1.5;N;1.5;192
N1.5;N;1.5;307
N1.5;N;1.5;188
N1.5;N;1.5;226
P1.5;P;1.5;293
P1.5;P;1.5;230
P1.5;P;1.5;307
P1.5;P;1.5;112
P1.5;P;1.5;256
P1.5;P;1.5;241
P3;P;3;207
P3;P;3;315
P3;P;3;232
P3;P;3;308
P3;P;3;261
P3;P;3;179
P3;P;3;220
P3;P;3;227
P4.5;P;4.5;220
P4.5;P;4.5;300
P4.5;P;4.5;305
P4.5;P;4.5;207
P4.5;P;4.5;315
P4.5;P;4.5;116
P4.5;P;4.5;233
K1.5;K;1.5;265
K1.5;K;1.5;240
K1.5;K;1.5;228
K1.5;K;1.5;269
K1.5;K;1.5;324
K1.5;K;1.5;315
K3;K;3;295
K3;K;3;299
K3;K;3;193
K3;K;3;272
K3;K;3;302
K3;K;3;254
K3;K;3;304
K3;K;3;298
K4.5;K;4.5;295
K4.5;K;4.5;203
K4.5;K;4.5;297
K4.5;K;4.5;217
K4.5;K;4.5;302
K4.5;K;4.5;254
K4.5;K;4.5;179
K4.5;K;4.5;295
K4.5;K;4.5;172
K4.5;K;4.5;177
NPK;NPK;4.5;179
NPK;NPK;4.5;224
NPK;NPK;4.5;114
NPK;NPK;4.5;202
NPK;NPK;4.5;194
NPK;NPK;4.5;298
NPK;NPK;4.5;201
NPK;NPK;4.5;218
Dataset 5.Dataset 5. Reproductive performance (time to budding).
http://dx.doi.org/10.5256/f1000research.11091.d155627 This dataset was used to prepare Figure 4A and to perform related analysis.
Treat;Fertilizer;Dose;FLOWERTIME
W;Control;0;269
W;Control;0;269
W;Control;0;261
N1.5;N;1.5;263
N1.5;N;1.5;245
N1.5;N;1.5;368
N1.5;N;1.5;266
P1.5;P;1.5;323
P1.5;P;1.5;335
P1.5;P;1.5;135
P1.5;P;1.5;287
P1.5;P;1.5;285
P3;P;3;268
P3;P;3;368
P3;P;3;304
P3;P;3;369
P3;P;3;335
P3;P;3;261
P3;P;3;280
P3;P;3;278
P4.5;P;4.5;278
P4.5;P;4.5;350
P4.5;P;4.5;351
P4.5;P;4.5;252
P4.5;P;4.5;375
P4.5;P;4.5;255
P4.5;P;4.5;300
K1.5;K;1.5;315
K1.5;K;1.5;297
K1.5;K;1.5;289
K1.5;K;1.5;341
K1.5;K;1.5;382
K1.5;K;1.5;358
K3;K;3;335
K3;K;3;335
K3;K;3;254
K3;K;3;304
K3;K;3;349
K3;K;3;295
K3;K;3;350
K3;K;3;347
K4.5;K;4.5;332
K4.5;K;4.5;262
K4.5;K;4.5;325
K4.5;K;4.5;271
K4.5;K;4.5;339
K4.5;K;4.5;286
K4.5;K;4.5;198
K4.5;K;4.5;325
K4.5;K;4.5;193
K4.5;K;4.5;198
NPK;NPK;4.5;219
NPK;NPK;4.5;269
NPK;NPK;4.5;173
NPK;NPK;4.5;235
NPK;NPK;4.5;224
NPK;NPK;4.5;332
NPK;NPK;4.5;244
NPK;NPK;4.5;218
Dataset 6.Reproductive performance (time to flowering).
Treatments;Fertilizer;Dose;FRUITINGTIME
W;Control;0;328
W;Control;0;312
N1.5;N;1.5;312
N1.5;N;1.5;307
N1.5;N;1.5;423
N1.5;N;1.5;334
P1.5;P;1.5;401
P1.5;P;1.5;399
P1.5;P;1.5;182
P1.5;P;1.5;365
P1.5;P;1.5;356
P3;P;3;320
P3;P;3;415
P3;P;3;354
P3;P;3;418
P3;P;3;379
P3;P;3;307
P3;P;3;325
P3;P;3;319
P4.5;P;4.5;326
P4.5;P;4.5;401
P4.5;P;4.5;402
P4.5;P;4.5;300
P4.5;P;4.5;414
P4.5;P;4.5;280
K1.5;K;1.5;358
K1.5;K;1.5;348
K1.5;K;1.5;344
K1.5;K;1.5;388
K1.5;K;1.5;424
K1.5;K;1.5;400
K3;K;3;392
K3;K;3;384
K3;K;3;290
K3;K;3;345
K3;K;3;395
K3;K;3;337
K3;K;3;392
K3;K;3;392
K4.5;K;4.5;382
K4.5;K;4.5;318
K4.5;K;4.5;372
K4.5;K;4.5;324
K4.5;K;4.5;387
K4.5;K;4.5;330
K4.5;K;4.5;242
K4.5;K;4.5;372
K4.5;K;4.5;236
K4.5;K;4.5;238
NPK;NPK;4.5;256
NPK;NPK;4.5;318
NPK;NPK;4.5;213
NPK;NPK;4.5;277
NPK;NPK;4.5;278
NPK;NPK;4.5;377
NPK;NPK;4.5;293
NPK;NPK;4.5;273
Dataset 7.Reproductive performance (time to fruiting).
Treatement;Total_ number_fruit
W;51
N1.5;56
P1.5;79
P3;159
P4.5;121
K1.5;78
K3;59
K4.5;197
Dataset 8.Cumulative fruiting.
Treatments;Fertilizer;Dose;Number_bud
W;Control;0;2
W;Control;0;53
W;Control;0;37
N1.5;N;1.5;63
N1.5;N;1.5;27
N1.5;N;1.5;41
N1.5;N;1.5;48
N1.5;N;1.5;39
P1.5;P;1.5;2
P1.5;P;1.5;255
P1.5;P;1.5;13
P1.5;P;1.5;1
P1.5;P;1.5;71
P1.5;P;1.5;65
P3;P;3;70
P3;P;3;9
P3;P;3;2
P3;P;3;124
P3;P;3;13
P3;P;3;14
P3;P;3;43
P3;P;3;34
P4.5;P;4.5;65
P4.5;P;4.5;14
P4.5;P;4.5;16
P4.5;P;4.5;3
P4.5;P;4.5;30
P4.5;P;4.5;21
P4.5;P;4.5;24
K1.5;K;1.5;1
K1.5;K;1.5;13
K1.5;K;1.5;30
K1.5;K;1.5;2
K1.5;K;1.5;8
K1.5;K;1.5;15
K3;K;3;63
K3;K;3;1
K3;K;3;4
K3;K;3;22
K3;K;3;35
K3;K;3;18
K3;K;3;30
K3;K;3;8
K4.5;K;4.5;66
K4.5;K;4.5;36
K4.5;K;4.5;3
K4.5;K;4.5;152
K4.5;K;4.5;4
K4.5;K;4.5;184
K4.5;K;4.5;75
K4.5;K;4.5;58
K4.5;K;4.5;84
K4.5;K;4.5;79
NPK;NPK;4.5;188
NPK;NPK;4.5;13
NPK;NPK;4.5;488
NPK;NPK;4.5;36
NPK;NPK;4.5;213
NPK;NPK;4.5;200
NPK;NPK;4.5;92
NPK;NPK;4.5;245
Dataset 9.Budding intensity.
Treatments;Fertilizer;Dose;Diameter;Height_F;Branches_F;Leaves;Leaf_area;Fruiting
W;C;0;13.88;29.5;17;621;1625;20
W;C;0;16.14;27.8;14;623;1750;14
N1.5;N;1.5;12.08;22.5;15;609;1875;23
N1.5;N;1.5;12.69;30;12;434;1625;28
N1.5;N;1.5;10.2;24;11;444;1000;5
N1.5;N;1.5;14.06;30.08;14;748;1625;46
P1.5;P;1.5;12.88;30.5;7;296;1875;3
P1.5;P;1.5;12.78;29.5;10;382;1250;5
P1.5;P;1.5;13.2;25.6;10;458;1750;3
P1.5;P;1.5;13.96;35.5;12;907;1875;68
P1.5;P;1.5;14.4;33.5;17;530;1625;10
P3;P;3;13.89;29;12;442;1625;23
P3;P;3;7.75;20;8;138;1000;8
P3;P;3;13.85;30.2;12;528;2125;14
P3;P;3;10.11;21.5;11;251;1125;5
P3;P;3;10.18;24.5;9;307;1000;5
P3;P;3;16.55;38;17;791;2125;50
P3;P;3;12.74;27.5;11;582;1375;40
P3;P;3;15.61;35.5;15;723;2000;14
P4.5;P;4.5;16.93;40.3;15;1316;2000;38
P4.5;P;4.5;11.77;34.5;16;676;1500;18
P4.5;P;4.5;12.57;32;11;432;1750;14
P4.5;P;4.5;15.54;36.6;20;554;1750;13
P4.5;P;4.5;14.95;33.6;13;466;1625;38
P4.5;P;4.5;14.95;32;15;688;1725;25
K1.5;K;1.5;12.13;35;13;449;1375;4
K1.5;K;1.5;16.49;32.2;18;1032;2125;15
K1.5;K;1.5;14.27;37;18;695;1500;25
K1.5;K;1.5;11.74;27;12;304;1250;18
K1.5;K;1.5;9.04;23;10;172;1625;12
K1.5;K;1.5;11.6;21.9;14;254;1250;4
K3;K;3;18.65;36.5;13;885;1625;48
K3;K;3;12.34;31;13;388;1625;11
K3;K;3;14.72;40.3;13;1111;1958;35
K3;K;3;14.5;34;15;704;2014;48
K3;K;3;15;29;17;786;2291;24
K3;K;3;12.00;17.98;18;620;1801;23
K3;K;3;115;34.5;18;665;1348;18
K3;K;3;15.9;34.1;18;983;1480;29
K4.5;K;4.5;14.62;28.4;12;522;2000;3
K4.5;K;4.5;16.34;37.9;25;1073;2000;55
K4.5;K;4.5;9.29;19;7;182;1625;5
K4.5;K;4.5;14.44;29.5;13;570;1625;20
K4.5;K;4.5;14.83;29;10;686;1750;4
K4.5;K;4.5;17.12;38.5;14;969;1750;46
K4.5;K;4.5;11.37;27;13;563;1500;9
K4.5;K;4.5;13.51;27;14;536;1625;10
K4.5;K;4.5;23.46;47;25;1683;2125;45
K4.5;K;4.5;14.99;29.8;15;754;1778;22
NPK;NPK;4.5;15.06;32;19;794;2375;25
NPK;NPK;4.5;18.1;34.5;19;1461;2500;83
NPK;NPK;4.5;15.99;43.5;23;1375;2000;20
NPK;NPK;4.5;15.88;36;19;1063;1500;55
NPK;NPK;4.5;9.98;24.8;9;453;1500;20
NPK;NPK;4.5;14.5;38.7;13;703;1625;112
NPK;NPK;4.5;16.34;37.9;25;1073;2000;54
NPK;NPK;4.5;17.1;38;18;1360;2100;78
Dataset 10.Fruiting intensity and correlation between growth parameters and fruiting.

Table 6. Correlation matrix of vegetative growth and development parameters in Synsepalum dulcificum's juveniles.

Stem collar
diameter
HeightNumber of
branches
Number of
leaves
Leaf areaNumber of
fruits
Stem collar
diameter
Height0.81***
Number of
branches
0.69***0.66***
Number of
leaves
0.84***0.74***0.75***
Leaf area0.68***0.53***0.47***0.61***
Number of
fruits
0.57***0.59***0.54***0.7***0.34*

* Significant at 5%, ** = Significant at 1%, ***= Significant at 1‰

Phenophases length

The lengths of the various phenophases observed during the reproductive growth of S. dulcificum are presented in Figure 5. The effect of treatments on the times from budding to flowering (S1), from flower bloom to fructification onset (S3), and from fructification onset to physiological maturity (S4) were very significant (P < 0.01), highly significant (P < 0.001) and significant (P < 0.05), respectively. The shortest length for S1 was observed in juveniles fertilized with 1.5 g phosphorus (32.33 ± 6.97 days), whereas the longest time for S1 was recorded in daily watered unfertilized juveniles (87.00 ± 12.52 days). NPK-fertilized juveniles rapidly started fruiting (within 16.66 ± 3.32 days), once their flowers bloomed. The longest time from fructification onset to physiological maturity (S4) was recorded in daily watered unfertilized juveniles (W) (28.66 ± 3.52 days).

2bfa1224-40be-4af9-9cbd-211a20692a2a_figure5.gif

Figure 5. Phenophases duration in juveniles of Synsepalum dulcificum under various treatments (n = 5 – 10 seedlings).

(S1 ) Time from budding to flowering; (S2) Time from flowering to flower bloom; (S3) Time from flower bloom to fructification onset; (S4) Time from fructification onset to physiological maturity; (S5) Time from physiological maturity to fruit ripening onset; (S6) Time from fruit ripening onset to full ripening. ns = not significant, * Significant at 5%, ** = Significant at 1%, ***= Significant at 1‰. W = Daily watering, no exogenous nutrients; N1.5 = Daily watering + 1.5 g N [seedling]-1; N3 = Daily watering +3 g N [seedling]-1; N4.5 = Daily watering + 4.5 g N [seedling]-1; P1.5 = Daily watering + 1.5 g P [seedling]-1; P3 = Daily watering + 3 g P [seedling]-1; P4.5 = Daily watering + 4.5 g P [seedling]-1; K1.5 = Daily watering +1.5 g K [seedling]-1; K3 = Daily watering + 3 g K [seedling]-1; K4.5 = Daily watering + 4.5 g K [seedling]-1; NPK = Daily watering + 1.5 g N + 1.5 g P + 1.5 g K [seedling]-1.

Treatment;Phases;Phases_Length
W;S1;99
W;S2;6
W;S3;25
W;S4;32
W;S5;7
W;S6;6
W;S1;88
W;S2;6
W;S3;17
W;S4;29
W;S5;7
W;S6;6
W;S1;74
W;S2;5
W;S3;24
W;S4;25
W;S5;6
W;S6;5
N1.5;S1;48
N1.5;S2;5
N1.5;S3;26
N1.5;S4;21
N1.5;S5;7
N1.5;S6;6
N1.5;S1;54
N1.5;S2;7
N1.5;S3;31
N1.5;S4;27
N1.5;S5;7
N1.5;S6;4
N1.5;S1;62
N1.5;S2;7
N1.5;S3;28
N1.5;S4;23
N1.5;S5;4
N1.5;S6;5
P1.5;S1;31
P1.5;S2;8
P1.5;S3;50
P1.5;S4;23
P1.5;S5;5
P1.5;S6;4
P1.5;S1;33
P1.5;S2;8
P1.5;S3;46
P1.5;S4;18
P1.5;S5;7
P1.5;S6;5
P1.5;S1;29
P1.5;S2;8
P1.5;S3;37
P1.5;S4;22
P1.5;S5;5
P1.5;S6;5
P1.5;S1;24
P1.5;S2;5
P1.5;S3;27
P1.5;S4;18
P1.5;S5;3
P1.5;S6;3
P1.5;S1;32
P1.5;S2;7
P1.5;S3;49
P1.5;S4;25
P1.5;S5;6
P1.5;S6;6
P1.5;S1;45
P1.5;S2;8
P1.5;S3;47
P1.5;S4;19
P1.5;S5;4
P1.5;S6;6
P3;S1;62
P3;S2;7
P3;S3;14
P3;S4;34
P3;S5;5
P3;S6;3
P3;S1;54
P3;S2;9
P3;S3;21
P3;S4;20
P3;S5;4
P3;S6;4
P3;S1;73
P3;S2;6
P3;S3;19
P3;S4;28
P3;S5;7
P3;S6;5
P3;S1;62
P3;S2;7
P3;S3;23
P3;S4;22
P3;S5;5
P3;S6;3
P3;S1;75
P3;S2;8
P3;S3;16
P3;S4;23
P3;S5;6
P3;S6;4
P3;S1;83
P3;S2;8
P3;S3;17
P3;S4;24
P3;S5;5
P3;S6;4
P3;S1;61
P3;S2;9
P3;S3;18
P3;S4;24
P3;S5;4
P3;S6;4
P3;S1;62
P3;S2;7
P3;S3;15
P3;S4;22
P3;S5;5
P3;S6;5
P4.5;S1;59
P4.5;S2;6
P4.5;S3;18
P4.5;S4;27
P4.5;S5;6
P4.5;S6;4
P4.5;S1;51
P4.5;S2;6
P4.5;S3;23
P4.5;S4;25
P4.5;S5;2
P4.5;S6;6
P4.5;S1;47
P4.5;S2;4
P4.5;S3;32
P4.5;S4;18
P4.5;S5;6
P4.5;S6;3
P4.5;S1;46
P4.5;S2;4
P4.5;S3;24
P4.5;S4;23
P4.5;S5;5
P4.5;S6;4
P4.5;S1;61
P4.5;S2;5
P4.5;S3;17
P4.5;S4;20
P4.5;S5;4
P4.5;S6;4
P4.5;S1;140
P4.5;S2;4
P4.5;S3;12
P4.5;S4;12
P4.5;S5;5
P4.5;S6;9
P4.5;S1;68
P4.5;S2;4
P4.5;S3;26
P4.5;S4;18
P4.5;S5;3
P4.5;S6;3
K1.5;S1;51
K1.5;S2;6
K1.5;S3;21
K1.5;S4;19
K1.5;S5;7
K1.5;S6;4
K1.5;S1;58
K1.5;S2;7
K1.5;S3;26
K1.5;S4;21
K1.5;S5;4
K1.5;S6;5
K1.5;S1;62
K1.5;S2;6
K1.5;S3;27
K1.5;S4;25
K1.5;S5;5
K1.5;S6;6
K1.5;S1;73
K1.5;S2;5
K1.5;S3;22
K1.5;S4;23
K1.5;S5;4
K1.5;S6;5
K1.5;S1;59
K1.5;S2;5
K1.5;S3;17
K1.5;S4;22
K1.5;S5;4
K1.5;S6;5
K1.5;S1;40
K1.5;S2;7
K1.5;S3;23
K1.5;S4;17
K1.5;S5;4
K1.5;S6;4
K3;S1;41
This is a portion of the data; to view all the data, please download the file.
Dataset 11.Phenophase length.

Relationship between growth traits and fruit production

The correlation matrix overall indicated positive and highly significant correlation between growth traits; a higher correlation was observed between the stem collar diameter and the number of leaves (Table 6). Similarly, correlations between fruit production and growth traits are all positive but higher with leaves production than other growth traits. The regression equation for fruit production in juveniles reads: ln (Number of fruit) = -4.51 + 1.15 ln (Number of leaves).

Discussion

Growth and reproductive responses of S. dulcificum seedling to watering and fertilization treatments

In S. dulcificum’s juveniles the use of appropriate fertilizer at a relevant dose is critical to avoid detrimental effects. The present study showed that while seedlings with phosphorus and potassium supply maintained survival at a high rate, nitrogen fertilization decreased survival rate with an increasing prevalence of dead seedlings as the dose increased. Similar negative effects of a larger nitrogen supply on survival was also reported in Trifolium medium L. (Chmelíková & Hejcman, 2014) and in Eucalyptus pauciflora Sieber ex Sprengel (Atwell et al., 2009). Likewise, in Betula pubescens Ehrh., Larix sibirica Ledeb., and Picea sitchensis (Bong.) Carr seedlings fertilized with nitrogen at the rate of 3.7 g [seedling]-1 had lower survival than those fertilized with 1.2 g [seedling]-1 (Oskarsson et al., 2006). Therefore, for 15 month-old juveniles of S. dulcificum we should limit the nitrogen dose to 1.5 g [seedling]-1 to encourage further growth and development.

Juvenility represents a crucial stage in survival, functional and productive traits of plant species (Trubat et al., 2010), and improving the performance of plant species at this stage through fertilization is desirable. Though the beneficial effect of fertilization on juveniles of tree species is questionable (Akinnifesi et al., 2008; Ebert et al., 2002), our results revealed that in the case of S. dulcificum, all vegetative growth traits positively responded to water supply and fertilization. We observed two main morphotypes in juveniles of S. dulcificum in response to treatments. The first morphotype was ‘thin’ and exclusively observed in the field where juveniles were rain fed, and where the plant mainly grew in height as an adaptation strategy to cope with weed competition for the light and gained a limited number of branches and leaves. In contrast, when water and/or nutrients were supplied, this induced a ‘well-branched’ morphotype. The characteristics of this morphotype included a high stem collar diameter, a high number of leaves and branches and a dense crown. NPK application to 15 months old seedlings improved vegetative growth. For instance, at the end of the experiment, initial stem collar diameter and leaf number increased by 1.6 fold and 18 folds, respectively, in 15-month old juveniles watered and supplied with NPK; whereas in control juveniles (without watering and fertilization), initial stem collar diameter, height, and number of leaves increased by 1.36 fold and 6.41 folds, respectively. This performance of NPK-fertilized seedlings highlighted the additive effect of those three nutrients (N, P and K) (Chang, 2003).

At 28 months old, juveniles were 47 cm tall after 13 months of fertilization with a 23.2 cm gain. Existing literature reported that the species height at four years old was 50–60 cm (Joyner, 2006). Even under a fertilization regime, S. dulcificum height growth did not dramatically improve, particularly compared to other tropical fruit species, such as Vitex doniana Sweet in which seedlings in nursery reached 75 cm before one year old (N’Danikou et al., 2015). However, the effect of NPK on the vegetative growth was reflected in increased branch and leaf numbers, which represents an interesting prerequisite to further investigation of the species’ response to increased dose of the N, P, and K combination.

More importantly, our findings provided evidence (for the first time) of the beneficial effect of water supply and fertilization on S. dulcificum flowering and fructification. Only juveniles that were daily watered entered in the generative phase. No bud and flower were observed in juveniles evolving in natural conditions, i.e. rain-fed juveniles. This suggested water supply as the key determinant for S. dulcificum juveniles’ entrance into reproductive phase. This finding is in line with Bernier et al. (1993) who indicated that any environmental factors that have the ability to change regularly (e.g. photoperiod, temperature, water availability) can control plant development towards flowering. While perennial species were reported to exhibit, in general, a long juvenile phase (Hanke et al., 2007) that could reach up to five years (e.g. Olea europea L., Malus domestica Borkh.) (Santos-Antunes et al., 2005; Zimmerman, 1972), this juvenile phase (ending with budding) can be shortened in S. dulcificum from > 36 months to 21 months with simple daily water provision. Our results also revealed that when suitable fertilization scheme was combined to daily watering, first flowering occurred in S. dulcificum at an average age of 23 months (less than two years old) and at 16 months old for early flowering individuals. This highlighted the importance of nutrient balance to the development of fruit tree species. First fruiting occurred at the average age of 24 months (20 months for extra early individuals). This achievement represented a major progress in the improvement of the species reproduction, as previous reports indicated that S. dulcificum bears fruit after 3 to 4 years (Joyner, 2006). Although water supply was crucial for S. dulcificum to initiate generative phase, our findings also suggested that nutrient supply is of paramount importance for the species productivity. This is illustrated by the fruit production that is fivefold higher in juveniles receiving NPK in addition to daily watering than in juveniles that benefited just of daily watering.

Our findings also expand the current knowledge on the phenology and reproductive biology of S. dulcificum. In juveniles of S. dulcificum, budding is continuous once it started, provided water is available. Flowering occurred one to three months after budding. In the first production round, flower production started from within the crown outward. This same “centrifugal” flowering pattern was also reported in Acer platanoides L. (Tal, 2011). Flower bloom occurred five to seven days after flowering and was always observed at the hot hours of the day (from 11 a.m. to 4 p.m.). In this study, we observed that flowers fully exposed to sun bloomed quicker than those hidden in the plant crown. This was well observed in NPK-fertilized seedlings and we suspected the flower bloom time in S. dulcificum to be light-dependent. This suspicion could even be expanded to the whole reproductive stage length of the species, since Xingway & Abdullah (2016) reported that four year old juveniles kept under shelter took 200 days from budding to fruiting stage, whereas in this study, sun exposed juveniles fruited within 100 – 160 days after budding. The growth stage also played a key role in the length of S. dulcificum phenophases. In adult trees, the timeframe from flowering to fruiting was estimated at seven days (Oumorou et al., 2010), while in juveniles, we observed that flowering to fruiting lasted 46 to 57 days.

Implications for crop improvement and increased production

S. dulcificum as a sweetener and source of secondary metabolites has a lot of potential as a future crop that can be used to reduce the prevalence of diabetes, high blood pressure, and other diseases due to inadequate nutrition. The species has suffered from lack of interest and is rarely included in breeding programs. Moreover, strategies to develop cultivars are still obscure. Also, agronomic practices to improve production and seed management require increased mobilization of resources. Our study is the first of its kind, and reports on the effect of water and nutrient management on flowering and fruiting in S. dulcificum. When the suitable nutrient was combined to regular water supply, fructification time in S. dulcificum can be reduced to half of its natural duration.

Inorganic fertilization significantly improved S. dulcificum growth; however, the most efficient fertilizer formulation is yet to be determined. Moreover, the use and the effects of organic fertilization on the species growth and fruit production should be explored. A major reason of the renewed interest in S. dulcificum is its high content in secondary metabolites. In our study, the effect of fertilization on metabolite content was not assessed and future studies should shed light on that effect, as well as on the metabolite production across ecological gradients.

To date only limited knowledge is available on the genetic variation in S. dulcificum and the distribution of genotypes across Africa. S. dulcificum is reported to be native to West Africa and thrives in Ghana, Benin, Togo, and Nigeria. Assessment of the genetic diversity and the definition of heterotic groups, as well as a region-wide collection of germplasms, are necessary to gather ecotypes and cultivars to increase the range of diversity and enable the development of breeding populations.

S. dulcificum is a shrub that naturally matures after three to four years. Although regular watering and nutrient supply can accelerate fruit production, it will be useful to identify secondary traits related to yield so as to increase predictive accuracy and efficient breeding plan (e.g. efficient time management, selection of high-yielding population). In this regard, leaf production represents an interesting secondary trait to consider in correlative selection of high yielding genotypes. In our study, high leaf production was positively correlated with higher fruit production. To increase the accuracy of the selection programme, the use of quantitative traits loci might be an option. So far there are no data on genes involved in leaf and fruit production. The sequencing of the species’ genome could then enable rapid identification of such genes and other useful ones so as to strengthen the development of cultivar and the economic return of the species.

Heat and drought stresses are yet to be assessed in S. dulcificum. Empirical observation from the first and last authors revealed that shaded seedlings were more vigorous than sun-exposed ones. Understanding how various genotypes of S. dulcificum respond to environmental stresses will shed light onto which cultivar would be appropriate to which locations and help adapt to climate changes. In addition, juveniles submitted to rainfall survived as well as those regularly watered. Such a response opens room for the investigation of the adaptation potential of the species to drier environments and the side-effects of such adaptation on cultivar selection.

Phenology data presented in this study remains incomplete since it did not cover the whole year. A follow up experiment will be necessary to provide a wider view on the phenological timeframe, including analysis of the fructification frequency, the period of flowering and fructification peak, and their variation across dry and rainy reasons.

Conclusions

This study has highlighted the beneficial effect of water supply and fertilization on both vegetative and reproductive growth in S. dulcificum. Water supply appeared as the most important factor unlocking flowering in the species, while nutrient supply was crucial in accelerating entrance into reproductive phase and enhancing fruit production. Throughout the experiment, the combination of nitrogen, phosphorus and potassium at the dose of 1.5 g (for all) consistently exhibited the highest performance for all growth and yield traits. These findings represent a crucial progress towards the species breeding and production scaling up.

Data availability

Dataset 1. Initial growth parameters at the fertilization experiment onset. D0 = Initial diameter, H0 = Initial height, L0 = Initial number of leaves, and B0 = Initial branching. This dataset was used to prepare Table 2. doi, 10.5256/f1000research.11091.d155614 (Tchokponhoué et al., 2017a)

Dataset 2. Survival data. This dataset was used to prepare Figure 2 and Table 3 and to perform related analysis. “Status” refers to whether the seed died (1) or was still alive at the end of the experiment (0) and “Time” refers to the number of weeks after each the seedling died (for dead seedlings) or the last time we saw surviving seedling (for seedlings still alive at the end of the experiment). doi, 10.5256/f1000research.11091.d155615 (Tchokponhoué et al., 2017b)

Dataset 3. Growth parameters (increment) at the end of the experiment for vegetative growth. This dataset was used to prepare Figures 3A–D and to perform related analysis. doi, 10.5256/f1000research.11091.d155616 (Tchokponhoué et al., 2017c)

Dataset 4. Growth parameters at the end of the experiment for leaf area. This dataset was used to prepare Figure 3E and to perform related analysis. doi, 10.5256/f1000research.11091.d155626 (Tchokponhoué et al., 2017d)

Dataset 5. Reproductive performance (time to budding). This dataset was used to prepare Figure 4A and to perform related analysis. doi, 10.5256/f1000research.11091.d155627 (Tchokponhoué et al., 2017e)

Dataset 6. Reproductive performance (time to flowering). This dataset was used to prepare Figure 4B and to perform related analysis. doi, 10.5256/f1000research.11091.d155628 (Tchokponhoué et al., 2017f)

Dataset 7. Reproductive performance (time to fruiting). This dataset was used to prepare Figure 4C and to perform related analysis. doi, 10.5256/f1000research.11091.d155629 (Tchokponhoué et al., 2017g)

Dataset 8. Cumulative fruiting. This dataset was used to prepare Figure 4D and to perform related analysis. doi, 10.5256/f1000research.11091.d155630 (Tchokponhoué et al., 2017h)

Dataset 9. Budding intensity. This dataset was used to prepare Table 5 and to perform related analysis. doi, 10.5256/f1000research.11091.d155631 (Tchokponhoué et al., 2017i)

Dataset 10. Fruiting intensity and correlation between growth parameters and fruiting. This dataset was used to prepare Table 5 and to generate Table 6 (correlation matrix), and to perform related analysis. doi, 10.5256/f1000research.11091.d155632 (Tchokponhoué et al., 2017j)

Dataset 11. Phenophase length. This dataset was used to generate Figure 5 and to perform related analysis. doi, 10.5256/f1000research.11091.d155633 (Tchokponhoué et al., 2017k)

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Tchokponhoué DA, N'Danikou S, Hale I et al. Early fruiting in Synsepalum dulcificum (Schumach. & Thonn.) Daniell juveniles induced by water and inorganic nutrient management [version 1; peer review: 2 approved]. F1000Research 2017, 6:399 (https://doi.org/10.12688/f1000research.11091.1)
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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
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Reviewer Report 05 Jun 2017
Nur Ashikin Psyquay Abdullah, Department of Crop Science, Faculty of Agriculture and Food Science, Universiti Putra Malaysia, Sarawak, Malaysia 
Approved
VIEWS 12
When the title presented was as “induced by water and inorganic nutrient”, I was expecting different watering different regimes. It comes to no surprise that the control will have least significant effects on the plant growth since it received no ... Continue reading
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Abdullah NAP. Reviewer Report For: Early fruiting in Synsepalum dulcificum (Schumach. & Thonn.) Daniell juveniles induced by water and inorganic nutrient management [version 1; peer review: 2 approved]. F1000Research 2017, 6:399 (https://doi.org/10.5256/f1000research.11963.r23248)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Reviewer Report 26 Apr 2017
Emil Luca, Department of Horticulture and Landscaping, University of Agriculture and Veterinary Medicine, Cluj-Napoca, Romania 
Laura Cristina Luca, University of Agriculture and Veterinary Medicine, Cluj-Napoca, Romania 
Approved
VIEWS 16
It was a great and pleasant surprise for me to read such a well-documented paper. The results obtained by the authors are revealing the intensive research conducted in the almost 3 years of experiments, and those results are properly highlighted ... Continue reading
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Luca E and Luca LC. Reviewer Report For: Early fruiting in Synsepalum dulcificum (Schumach. & Thonn.) Daniell juveniles induced by water and inorganic nutrient management [version 1; peer review: 2 approved]. F1000Research 2017, 6:399 (https://doi.org/10.5256/f1000research.11963.r21888)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.

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Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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