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Moringa (Moringa oleifera) is used for treating several diseases, feeding humans and for reducing water pollution (Bancessi et al. 2019). Its nutritional quality, digestibility, degradability and palatability make it a good animal feed (Su and Chen 2020). Its seed can be sown directly in the field or in a nursery. The latter, although it requires greater effort, allows to take the most resistant plantlets to stressful environmental conditions into the field. As substrates of a nursery, worm humus, organic matter and livestock excrement can be used (González and Crespo 2016). Moringa plantlets show good development when 25% of any of the aforementioned substrates is included (Silvestre 2019). However, livestock production systems are developed under different conditions and possibilities, so this recommendation cannot always be fulfilled. Due to this, it is necessary to develop studies that propose other substrate alternatives for nursery development of M. oleifera.
Sawdust is a material that is commonly discarded. Due to its physical characteristics, it maintains higher humidity in the soil, which guarantees seed germination and their emergence (Babatunde et al. 2019). This avoids having to water seeds daily during the early stages of development, and saves water and effort. For this substrate, it is necessary to clarify the protocols that indicate how much sawdust and time the plants should remain in the nursery, as well as the frequency and amount of irrigation they should receive. It is necessary to know the advantages of its use, beyond the growth in height and leaf production. Therefore, the objective of this study was to compare the effect of adding sawdust to the substrate on M. oleifera growth under nursery conditions.
Materials and Methods
Study area. The study was developed in the nursery of the Estación Experimental de Pastos y Forrajes de la Unidad de Ciencia y Técnica Básica La Habana, belonging to the Instituto de Investigaciones de Pastos y Forrajes. This is located at 22099'60.1'' N and 82037'87.1 '' W and has a red ferralitic soil (Hernández et al. 2015).
Plant material. Moringa oleifera seeds were obtained from the Instituto de Investigaciones de Pastos y Forrajes. They were stored at 240C for 3 months. To accelerate the germination process, seeds were soaked in water at room temperature for 12 hours (Njehoya et al. 2014). Subsequently, 300 seeds were placed on wet paper for a period of three days, and 60 mL of water were daily added. An amount of 150 germinated seeds with homogeneous root development were selected.
Sowing conditions. Two treatments were used, based on red ferralitic soil (soil) and a mixture of 1:1 soil-sawdust for their development under nursery conditions. The soil was collected at 15 cm deep and sieved with a 0.25 cm2 mesh. The sawdust was obtained from pine, with around 2 mm in size. The field capacity of both substrates was determined using Kirkham (2004) methodology. The bags (0.8 L) containing soil weighed 0.87 kg and soil-sawdust bags weighed 0.56 kg. The field capacity of substrates was 0.32 L kg-1 and 1.1 L kg-1, respectively. Two weekly irrigations were carried out, adding 0.35 L and 0.7 L to each bag of substrate so that each treatment would remain at 100% of its field capacity at the time of irrigation, since red ferralitic soil has a high drainage capacity (Hernández et al. 2015).
Five germinated seeds were sown per bag, at 2 cm deep (Cardoso et al. 2006). To guarantee destructive samplings, each treatment consisted of 15 bags, with a total of 75 plants. The nursery was located under 4 m high trees, so plantlets were protected from direct incidence of solar radiation.
Growth analysis. Non-destructive samplings were carried out at 7, 14, 21 and 28 days, for which 20 plants were taken at random from each treatment. Variables measured were: stem height and diameter, number of leaves and length and width of the last two compound leaves, as recommended by Valdés et al. (2014).
For destructive samplings, 20 plants were harvested to calculate growth rates at 10, 20 and 28 days, and four plants to estimate the concentration of photosynthetic pigments. Fresh and dry weights of leaves, stem and roots were measured, except in the first sampling, because roots were very fragile and their extraction from substrate without damaging them was compromised. Samples were placed in an oven at 800C for 72 hours until constant weight was reached to obtain the biomass value. An analytical balance (± 0.1 mg) was used for weighing.
Growth rates. With the data obtained in the destructive samplings, the following indices were calculated in the intervals 10-20 days and 20-28 days, according to Hunt (1990):
Where: W: biomass (g), t: time (d)
Determination of photosynthetic pigments. The third and fourth leaves of four plants were taken at 28 days. Leaves were macerated in a mortar with alcohol at 95%. Concentration of chlorophyll and carotenoids was estimated from the absorbances of the extracts, according to Ortega and Rodés (1986).
Statistical analysis. Statistical analysis was performed with the Statistica v.10 program (StatSoft Inc 2011). Fulfillment of normality premises was verified using Kolmogorov-Smirnov, Liliefors, and Shapiro-Wilk tests, and homoscedasticity using Levene test.
Results
The 57.3% of moringa seeds germinated after three days on wet paper. This was considered adequate for this study, considering incubation time and three months of storage.
Plants grown in soil, during the first week, produced four leaves of approximately 6 cm wide, higher values than those of plants that grew in soil:sawdust (two leaves of 3 cm). However, at 28 days, leaves of the plants in soil:sawdust were longer (figure 1). Width and number of leaves did not show significant differences after the first week (figure 1). The type of used substrate did not influence on these variables when plants reached 28 days of development. Stems of plantlets in soil were 24.0 cm high at 28 days of growth. Meanwhile, those grown in soil:sawdust reached 19.8 cm. No differences were found in stem diameter (figure 2).
Figure 1 Length, width and number of leaves of M. oleifera for 28 days under nursery conditions, in soil and soil:sawdust (1:1) substrates
Figure 2 Height and diameter of the stem of M. oleifera for 28 days under nursery conditions, in soil and soil: sawdust (1: 1) substrates
Fresh (FW) (figure 3a and c) and dry weight (DW) (figure 3 b and d) of leaves and stems at 10 days was different for moringa in the different substrates. The production of aerial biomass was higher for plants grown in soil compared to soil: sawdust. These differences were maintained at 20 and 28 days in the DW of the stem. FW and DW of roots was higher at 28 days in the plants that grew in soil:sawdust (figure 3e and f). The substrates used had no influence on the aerial biomass/root biomass ratio at 20 and 28 d, according to Mann-Witney analysis (p <0.05, n=20) (table 1), although there was an effect of time factor on this proportion.
Figure 3 Fresh weight and dry weight of leaf, stem and root of M. oleifera for 28 days under nursery conditions in soil and soil: sawdust (1: 1) substrates.
Table 1 Aerial biomass / root biomass proportion of M. oleifera under nursery conditions, at 20 and 28 days
| Days | Treatments | |
|---|---|---|
| soil | soil:sawdust | |
| 20 | 3.9 ± 0.9 ns | 3.8 ± 1.6 ns |
| 28 | 3.0 ± 1.0 ns | 1.7 ± 1.6 ns |
ns no significant differences by Mann-Witney (p <0.05)
n = 20
Absolute growth rate (AGR) and relative growth rate (RGR) were superior in the interval 20-28 days of plants in soil:sawdust (table 2). Leaf weight ratio (LWR) was higher at 20 days for plants grown in soil, while it was higher for plants grown in soil:sawdust at 28 days. Furthermore, a higher LWR was observed at 20 days than at 28 days. In addition, there were significant differences in the content of chlorophyll a, chlorophyll b and carotenoids between both treatments. In all cases, the highest values belong to plants grown in soil (figure 4).
Table 2 Leaf weight ratio, absolute growth rate and relative growth rate of M. oleifera under nursery conditions at 20 and 28 days
| Treatments | LWR20 (g g-1) | LWR28 (g g-1) | AGR20-28 (g d-1) | RGR20-28 (mg mg-1 d-1) |
|---|---|---|---|---|
| Soil | 0.38 * | 0.33 * | 0.01 | 0.02 |
| Soil:sawdust | 0.42 * | 0.28 * | 0.02 | 0.04 |
* indicates significant differences by Mann-Witney (p <0.05) n = 20
Discussion
Moringa rapidly decreases its viability over time (Ruiz et al. 2017). Most germination tests are performed from 7 to 21 days, and seeds germinate between 5 and 10 d (Padilla et al. 2017). The quality of seeds used in the experiment can be considered high considering germination was quantified three days after the imbibition started.
Decrease of stem height, leaf dimensions and number of leaves (figure 1 and 2 a) is related to the increase of rainfall (INSMET 2019) in the first week of the experiment, since moringa does not tolerate water excess for a long time in poorly drained substrates (Yang et al. 2015). The greater field capacity and water retention capacity of soil:sawdust substrate caused stress due to waterlogging, without causing death or damage due to pests or diseases. It is suggested that when stress stops, surviving plants can maintain good growth (Patricio and Palada 2017), which was verified during this experiment.
Despite the stressful conditions during the first week (10 days of growth), moringa reached higher heights with respect to those reported by Valdés et al. (2014) for the same development time in sandy, sandy-loam, clayey substrate and sugarcane filter cake mud compost. Plant height was higher at 28d compared to those reported by Toral et al. (2013) at 40 d in red ferralitic soil and bovine manure (3:1). Red ferralitic soil is characterized by having a pH between 6 and 7 (Hernández et al. 2015) and a level of organic matter of 3.3% (Rosales et al. 2020). These conditions favor M. oleifera growth (Yang et al. 2015). The soil:sawdust combination incorporates nutrients such as nitrogen, carbon, phosphorous, potassium, and calcium, which can contribute to a superior growth of moringa (Babatunde et al. 2019). Under the studied experimental conditions, results show that plants did not reach 40 cm at 28 d, necessary height for their transplantation, as proposed by Medina et al. (2007). However, the possibility of transplantation was not discarded, since plants were vigorous.
In the applied irrigation regime, soil:sawdust combination stimulated the increase of leaf length (figure 1a) since it retained higher amount of water than soil substrate, without being excessive. The short length of plant leaves in soil substrate could be related to the fact that this treatment presented a greater drainage, which could contribute to the appearance of stress due to water deficit. It is reported that plants, in response to a water deficit, decrease the development of their leaves, since certain levels of turgidity is needed for cell expansion to occur (Taiz and Zeiger 2014).
Both treatments stimulated an equal production of leaves at 28 days (figure 1a). Valdés et al. (2014) and Silvestre (2019) reported similar leaf amounts to those of the current study, for combinations of substrates that included manure, rice husk, compost, sugarcane filter cake mud compost and sandy and clay soils. It can be argued that, regardless of the type of substrate used, moringa does not have a high leaf production during its first four weeks of development in the nursery.
During the initial growth of the moringa, it is necessary not only to reach great heights, but to obtain a greater stem diameter, since this influences on the amount of reserve substances that plant stores and provides it with greater resistance to adverse environmental conditions (Solorio and Solorio 2002 and Ledea-Rodríguez et al. 2020). Stem diameter was not affected by the conditions imposed at 28 days, as reported by Medina et al. (2007) and Sarwar et al. (2017) in other substrate combinations. However, Silvestre (2019) obtained diameters superior to 7 mm, when substrate contains 50% of compost. Substrate is not the only factor that influences on growth capacity of moringa, because there is also a genetic effect due to open pollination (Selvakumari and Ponnuswami 2017 and Wu et al. 2018).
Low values of FW and DW of leaves and stem at 10 days (figure 3 a, b, c and d), may be related to the excess of rains during the first week and is consistent with the results of non-destructive samplings (figure 1 and 2). The stress due to puddled culture reduces plant growth (Abud et al. 2018), and its effect was evidenced in the low biomass accumulation, especially plants from the soil:sawdust treatment. However, once the stress was removed, a higher DW was observed in the stem of plants grown in soil, which indicated a recovery.
The highest FW and DW of the root system of plants in soil:sawdust (figure 3 e and f) may be due to the great thickening observed its roots, which is an adaptation for survival to adverse conditions (Casanova et al. 2018). Because this combination of substrates has a greater field capacity, plants can take advantage of water availability for the development of the root system (figure 3 e and f). Mora and García (2017) observed an increase in root length under nursery conditions in soil:sawdust (1:3). Sawdust has the ability to reduce the effect of temperature on the substrate, which allows a constant root growth (Conceição et al. 2005). The high development of the root system in soil:sawdust is an indication that quantity and frequency of irrigation in this study did not affect plantlets that are susceptible to water excess. Regarding the observations during the first week of the experiment, soil:sawdust combination is recommended for dry season, because rainfall could become an excess of water for plants in this stage and affect their growth.
Stressing factors in plants can modify their content of photosynthetic pigments (Abud et al. 2018, Sharma et al. 2019). Moringa is resistant to drought, which does not exclude it to suffer from stress due to lack of water. Plants grown in soil, with less field capacity, could face a certain degree of water stress, which increased their content of chlorophyll and carotenoids (figure 4). Abud et al. (2018) observed a higher chlorophyll content in moringa when it received less irrigation. The increase of carotenoid content may be related to its role in identifying and protecting from stress, as proposed by Nisar et al. (2015). Plants in soil had a higher growth in height and biomass accumulation in the stem, compared to plants in soil:sawdust (figure 2a and 3d), which may be related to the highest pigment concentration. Saini et al. (2012) reported that higher content of chlorophyll and carotenoids in moringa is associated with a more vigorous plant growth, since the concentration of pigments gives a measure of plant capacity to carry out photosynthesis and produce biomass (Taiz and Zeiger 2014).
In moringa, shoot/root weight ratio is a quality index that shows good development when it is between 1.5 and 2.5 (Sáenz et al. 2010). Only the plants in soil:sawdust reached a shoot/root ratio within the proposed limits at 28 days (table 1), a sign of a more equitable development in both systems. This allows the root to support the aerial part, and the aerial part to fix the carbon necessary for plant growth.
The highest LWR, reached by plants in soil:sawdust at 20 days, was influenced by the fact that dry biomass of its stems was lower compared to that of soil plants in soil (figure 3d). This tendency was reversed at 28 days, because DW of leaves between 20 and 28 days did not considerably vary (figure 3b) or increased biomass accumulation in stems and roots. Plants in soil had higher DW of the stem than those in soil:sawdust, and the latter had greater growth in their root system (figure 3f), which determined the performance of the LWR index. Biomass distribution of plants in each treatment had an effect on AGR and RGR, because they had twice the value for plants sown in soil:sawdust than in soil. Although these last presented higher height, they could be subjected to some stress due to water deficit. This could decrease, to a certain extent, stomatal conductance to avoid water loss causing a lower photosynthetic rate (Núñez and 2017), and lower total biomass accumulation with respect to plants in soil:sawdust (figure 3 b, d and f).
The establishment of M. oleifera in a nursery with red ferralitic soil stimulates the production of photosynthetic pigments, growth in height, and dry biomass in the stem. Soil:sawdust (1:1) substrate constitutes a good alternative for places in which rains are infrequent, as it increases the dimensions of leaves and root biomass. In addition, an equitable growth between root system and the aerial part of plants is obtained.
