(Schumach. & Thonn.) Daniell juveniles induced by water and inorganic nutrient
management
Dèdéou Apocalypse Tchokponhoué , Sognigbé N'Danikou , Iago Hale ,
Allen Van Deynze , Enoch Gbènato Achigan-Dako1
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 12 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] 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] ), 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.
Introduction
The miracle plant, Synsepalum dulcificum (Schumach. & Thonn.) Daniell (Sapotaceae), is a perennial shrub originating from West Africa and is the only known natural source of miraculin, a glycoprotein with remarkable edulcoration properties. 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. The fruit of the species (miracle berry) is a rich source of vitamin C, leucine, flavonols, and anthocyanin and its modern utilizations include many applications in cosmetics, food, and pharmaceuticals. Recently, additional scientific evidences were highlighted on the ability of the species to substitute sugar, particularly in sour beverages. Despite the nutritional, economic, and medicinal promise of the species, S. dulcificum remains a neglected crop that is not widely cultivated. 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. An important step toward the systematic improvement of S. dulcificum would be to accelerate the transition to reproductive maturity, thus shortening generation times.
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).
There was only one seedling per pot and each pot had 15 l volume. The experiment was made up of twelve treatments, out of which the absolute control 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. 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.
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 to ensure that seedlings had similar size. 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. 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.
Statistical analysis
Prior to analysis we explored the datasets, and outliers were identified using the boxplot approach. 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.
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. 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.
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. 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. 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).
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. Regard ing 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.
Flowering and fruiting responses
Budding and flowering.
The proportion of budding juveniles was significantly affected by the treatment and ranged from 0–100%. The contrast analysis on the average time to budding revealed a significant effect of treatment (P = 0.02;). 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. After 10 months, NPK fertilized seedlings produced a significantly greater number of buds than unfertilized plants (six times, P = 0.05). The proportion of flowering juveniles was also highly affected by the treatment. 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 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.
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. 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. 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.
Phenophases length
The lengths of the various phenophases observed during the reproductive growth of S. dulcificum. 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.
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. 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. 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.
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 acrossAfrica. 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.
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.
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