Agronomía Colombiana

Study site

This study was conducted in a glass greenhouse of the Biotechnology Department, Forestry Research Institute of Nigeria (FRIN), Ibadan, Oyo - State, Nigeria, located at longitudes 07°23'18"N to 07°23'43"N and latitudes 03°51'20"E to 03°23'43"E. The greenhouse was situated at 215 m a.s.l. The weather reports of the location were obtained from FRIN Meteorological station and indicated average maximum and minimum temperatures of 32.4°C and 23.6°C, respectively. The mean relative air humidity was 79.2%, while mean monthly rainfall was 114 mm during the period of the study from March to August, 2023.

Treatments and experimental design

This experiment consisted of 7x4 factorial treatment combinations. The first factor was the type of nutrient solution (NS): complete nutrient solution (CNS) with macro and micronutrients, CNS minus (-) each of nitrogen (N), phosphorus (P), potassium (K), magnesium (Mg), calcium (Ca), and sulphur (S). The second factor was four volumes of nutrient solution: 0 (control), 100, 200, and 300 ml. These volumes per plant were applied twice a week. The control did not receive a nutrient solution, and 200 ml of distilled water per plant was applied in the control treatment. Application volumes varied in order to expose the plants to adequate nutrient concentrations, as there were no reports establishing a particular volume of Hoagland solution for the optimum growth of the noni seedlings. The resulting 28 treatments were laid out in a randomized complete block design with four replicates, where each replicate corresponded to one plant per pot.

Growth medium preparation

Sand culture and "minus one element technique" (MOET) were used for this experiment (Burns, 1992). The river sand used was collected from the Asanmagbe riverbank in FRIN. The sand was sieved to remove particles larger than 4.5 mm, washed several times under flowing tap water until the water became clear, and then washed with distilled water. The sand was then leached with 2 N HCl solution containing 1% oxalic acid for 18 h and later washed with deionised water (Taxiarchou et al., 1997). It was sterilized at 100°C for 1 h in a hot air oven and was filled into polythene pots of 2 kg size, perforated at the base.

Preparation of the nutrient solutions

Stock solutions of the essential macronutrients were prepared, and the treatment solutions were made by adding the correct proportions of the required stock solutions according to Hoagland and Arnon (1938), as shown in Table 1.

Initial growth and transplanting of seedlings

Mature noni (Morinda citrifolia var. citrifolia) fruits were collected from the FRIN arboretum. The seeds were extracted and sundried. Seeds of similar size were sorted, clipped at the tip and propagated in leached, sterilized river-sand substrate at 100 seeds per basket. Seedlings began to emerge three weeks after sowing. Seedlings of uniform height at the four-leaf stage were carefully removed, their roots rinsed and transplanted into the growth media (sand) at one seedling per pot. The seedlings were watered with distilled water for the first week, followed by the application of 50% complete Hoagland solution during the second week to stabilize the plants. Subsequently, nutrient solutions according to each treatment were applied by the slop method, twice a week (McCall & Nakagawa, 1970). A plastic plate was placed under each pot to prevent outside root extension, while leachate was not reused.

Data collection

The biometric response of the plants in terms of stem height (cm), stem diameter (mm), number of leaves, and leaf area (cm²) was assessed at four-week intervals, starting from the 4^th week after transplanting (WAT). Total dry matter (shoot + roots) (g/plant) and leaf chlorophyll content were determined at 20 WAT. The seedlings were carefully uprooted, the sand rinsed off the roots and then separated into leaves, stems and roots. The plant parts were then put in separate envelopes, labelled according to treatments and oven dried to constant weight at 70°C in an electric oven. The oven-dried weights of each of the plant parts were added together to obtain the total dry biomass. Nutrient deficiency symptoms in the plants were visually assessed along with the collection of biometric data. Stem height was determined with a meter rule. Stem diameter at the base was evaluated using a digital vernier calliper. Leaf area was measured with a portable leaf area meter (YMJ-B®, Hinotek), while the number of leaves was assessed by counting. Relative contents of chlorophyll (SPAD units) were measured in a third or fourth pair leaf from the top with a hand-held chlorophyll meter (TYS-B®, Hinotek).

Statistical analysis

The data collected were subjected to analysis of variance using GenStat (version 4), while the significantly different means were separated using the Duncan Multiple Range Test at 5% probability.

TABLE 1.: Composition of the "minus one element culture" solution.

+ indicates presence and - indicates absence of a particular mineral element. Ferric-sodium salt of EDTA (18.4 g L^-1) was used to replace Fe tartrate in the Hoagland solution. Trace element solution consisted of H₃BO₃ (2.86 g), MnCl₂·4H₂O (1.81 g), ZnSO₄·7H₂O (0.22 g), CuSO₄·5H₂O (0.08 g), and H₂MoO₄·H₂O (0.02 g) in 1 L of distilled water.

Plant height

The growth of noni seedlings, as affected by different nutrient solutions, indicated significant differences (P≤0.05) between the treatment means for plant height at 8 to 20 weeks after transplanting (WAT) (Fig. 1A). Moreover, results at 12, 16, and 20 WAT followed a similar pattern, except for the plants treated with CNS-Ca. Seedlings treated with complete nutrient solution CNS had the highest mean heights of 20.4, 34.7, and 43.0 cm at 12, 16, and 20 WAT, respectively, which were significantly taller than the seedlings in each of the other treatments at the respective growth periods. These were followed by seedlings treated with CNS-S, which were taller than seedlings of other treatments, except the CNS treatment, at the same periods. The plant height in the CNS-Ca treatment decreased gradually towards 20 WAT. The smallest heights of 7.4, 8.7, and 9.7 cm at 12, 16, and 20 WAT, respectively, were observed in seedlings treated with CNS-N (Fig. 1A).

Stem diameter

The analysis of variance revealed significant differences (P≤0.05) between the nutrient solutions for stem diameter at 8, 12, 16, and 20 WAT. Seedlings treated with CNS had significantly higher stem diameter compared to those from other treatments throughout the period of study (Fig. 1B). At 8 WAT, the stem diameter of seedlings treated with CNS-Mg, CNS-S, and CNS-Ca were comparable, with each significantly higher than the stem diameter of seedlings treated with CNS-K and CNS-N. The smallest stem diameter was observed in seedlings treated with CNS-P at 8 WAT. At 12 and 16 WAT, the seedlings treated with CNS-S had thicker stems than seedlings treated with CNS-Ca. Similarly, the stem diameter of seedlings treated with CNS-K and CNS-P were similar and significantly higher than that of seedlings treated with CNS-N, with the smallest values at 12 and 16 WAT. At 20 WAT, the stem diameter of seedlings subjected to CNS-S was significantly higher than in the other treatments, while the seedlings treated with CNS-N had the smallest stem diameter (Fig. 1B).

Number of leaves

The number of leaves of noni seedlings treated with various NS significantly differed (P≤0.05) among the treatments from 8 to 20 WAT (Fig. 1C). The seedlings treated with CNS-Ca produced the highest number of leaves at 8 WAT, while those supplied with CNS-N had the least number of leaves. At 12 WAT, the seedlings treated with CNS had the highest number of leaves, whereas seedlings treated with CNS-Mg had the least number of leaves. Moreover, the number of leaves produced by seedlings treated with CNS-Mg decreased gradually from 8 to 20 WAT, while the number of leaves in the CNS-Ca treatment decreased from 12 WAT onward, reaching the lowest values at 16 and 20 WAT. At 16 and 20 WAT, the seedlings treated with CNS produced the highest number of leaves, closely related to those treated with CNS-K, and significantly higher than the number of leaves for other treatments at 20 WAT (Fig. 1C).

Leaf area

Significant variations (P≤0.05) in leaf area were found between the treatment means at successive weeks of growth, except at 4 WAT. At 8 WAT seedlings treated with CNS-Ca had the highest leaf area (135.9 cm²), which was not significantly different from values observed for seedlings treated with CNS (133.1 cm²), CNS-Mg (123 cm²) or CNS-S (117.7 cm²) (Fig. 1D). The smallest leaf area (52.9 cm²) during this period was observed in seedlings treated with CNS-P, which was closely related to the value obtained for seedlings treated with CNS-N. At 12 WAT, the seedlings treated with CNS had significantly higher leaf area compared to other treatments. Seedlings treated with CNS-N had the smallest leaf area at 12 WAT. Results at 16 and 20 WAT followed a similar trend. Seedlings treated with CNS had the highest leaf area at 16 and 20 WAT (1097.7 and 1363.5 cm², respectively), significantly exceeding the leaf area of other treatments at both periods. The seedlings treated with CNS-N had the smallest leaf area at both periods. The leaf area of the seedlings treated with CNS-Ca declined gradually from 12 WAT to 20 WAT (Fig. 1D).

FIGURE 1.: Effects of the nutrient solutions on (A) plant height, (B) stem diameter, (C) number of leaves, and (D) leaf area of noni seedlings during 20 weeks after transplanting into sand culture

Total dry matter accumulation

The analysis of variance indicated that the effects of NS, application rates, and their interactions were highly significant (P≤0.01) on the total dry matter yields of noni seedlings at 20 WAT. Seedlings treated with CNS had the highest total dry matter (15.45 g/plant), which was significantly higher than values for each of the other treatments (Fig. 2A). This was followed by seedlings treated with CNS-S with 8.08 g/plant. The dry matter of seedlings treated with CNS-K (5.42 g/plant) was similar to that of seedlings treated with CNS-P (4.40 g/plant), which was also not different from the dry matter of seedlings treated with CNS-Mg (3.36 g/plant). Moreover, the dry weight of seedlings treated with CNS-Mg was closely related to those of the CNS-Ca treatment (2.65 g/plant). The least biomass (1.52 g/plant) was observed in seedlings treated with CNS-N at the same period (Fig. 2A). Between the NS volumes, the seedlings under the least application volume (100 ml NS) accumulated the highest dry matter (8.91 g/plant), which was significantly higher than the seedling weights obtained for each of the other volumes (200 or 300 ml of NS) and control which had the least seedling weight at 20 WAT (Fig. 2B).

Furthermore, results of the effects of nutrient solution volumes on plant height, stem diameter and leaf area showed significant differences (P≤0.05) for means of the variables at successive weeks of growth (Fig. 3). Seedlings treated with 100 ml NS had the highest plant height, stem diameter and leaf area, while the control seedlings had the least values for these variables at 8 to 20 WAT. Conversely, the control seedlings had the highest number of leaves, while seedlings treated with 100 ml, 200 ml, or 300 ml of NS decreased in number of leaves from 12 to 20 WAT (Fig. 3C). The results of the interactive effects of NS and application volumes on the seedlings also revealed significant differences for means of all variables at successive weeks of growth.

FIGURE 2.: Effects of (A) nutrient solutions and (B) application volumes on total dry matter of noni seedlings at 20 weeks after transplanting into sand culture. Means with different letters indicate significant differences according to the Duncan test (P≤0.05)

FIGURE 3.: Effects of nutrient solution application volumes on (A) plant height, (B) stem diameter, (C) number of leaves, and (D) leaf area of noni seedlings during 20 weeks after transplanting into sand culture

Leaf deficiency symptoms

Observations on leaf deficiency symptoms of noni seedlings indicated that plant growth was affected by the different NS treatments (Fig. 4). The growth of the seedlings was uniform with no visible deficiency symptoms at 4 WAT. However, at 8 WAT, seedlings treated with nutrient-deficient solutions began to show deficiency symptoms, which persisted and became severe at 16 and 20 WAT (Fig. 4). Seedlings treated with CNS showed no deficiency symptoms throughout the periods of observation (Fig. 4A). In contrast, stunted growth and chlorosis, which started in the older leaves and progressed to the younger leaves, were observed in seedlings treated with CNS-N (Fig. 4B). Similarly, small leaves and retarded growth were identified in seedlings treated with CNS-P (Fig. 4C), whereas brown patches in the third pair of leaves and crinkled stems were observed in seedlings treated with CNS-K (Fig. 4D). Seedlings treated with CNS-Ca produced interveinal chlorosis in young fully expanded leaves, burned leaf margins, and shoot tips die-back (Fig. 4E). Similarly, necrosis and premature leaf fall were noticed in seedlings treated with CNS-Mg (Fig. 4F), while seedlings treated with CNS-S had interveinal chlorosis on young and recently matured leaves (Fig. 4G) compared with seedlings treated with CNS at the same growth period.

FIGURE 4.: Macronutrient deficiency symptoms in noni seedlings at 16 weeks after transplanting into sand culture as affected by nutrient solutions (200 ml applied twice a week). The seedlings were treated with: A) Complete Nutrient Solution (CNS); B) CNS - N; C) CNS - P; D) CNS - K; E) CNS - Ca; F) CNS - Mg; and G) CNS - S.

Chlorophyll content

The chlorophyll (CHL) content in the leaves of noni seedlings varied significantly with respect to different nutrient solutions applied (Fig. 5). The seedlings treated with CNS had the highest CHL content (71.7 SPAD value), which was significantly higher than values for each of the other treatment solutions at 20 WAT (Fig. 5A). The lowest CHL content was recorded for seedlings treated with CNS-Ca at 20 WAT. The CHL content in ascending order was CNS-Ca < CNS-N < CNS-Mg < CNS-S < CNS-K < CNS-P < CNS (Fig. 5). The CHL content was also affected by the levels of NS applied (Fig. 5B). The seedlings treated with only distilled water had CHL contents similar to those treated with 100 ml NS. The CHL values for these treatments were significantly higher than those recorded for seedlings treated with 200 ml or 300 ml NS at 20 WAT.

FIGURE 5.: Effects of (A) nutrient solutions and (B) application levels on leaf chlorophyll content of noni seedlings at 20 weeks after transplanting into sand culture. Means with different letters indicate significant differences according to the Duncan test (P≤0.05)