Seed priming using moringa leaf extract and application of Tithonia compost on shallot growth

Early establishment of shallot growth is important in order to ensure optimum yield. This study aimed to evaluate the effect of seed priming with moringa leaf extract (MLE) and the application of Tithonia compost on the growth of shallots. The research was conducted at the experimental farm belonging to the Faculty of Agriculture, Hasanuddin University, Makassar, from September to December 2022. The study was arranged in a factorial design with three replications. The first factor was priming, consisting of five levels: unprimed, hydropriming, 12.5% MLE, 25% MLE, and 37.5% MLE. The second factor was the level of Tithonia compost, consisting of four levels: 0, 5, 10, and 15 tons ha -1 . The results revealed that shallots treated with a combination of priming 37.5% MLE followed by application of 15 tons ha -1 of Tithonia compost in the field had the highest chlorophyll index (25.97). Priming with 37.5% solely resulted in the highest average shallots height (38.23 cm), the number of plant leaves (10.55), leaf weight (15.67 g), and bulb weight (29.92 g). Application of Tithonia compost solely of 15 tons ha -1 produced shallot with the highest average plant height (37.72 cm), the number of plant leaves (9.74), leaf weight (13.68 g), and bulb weight (27.01 g). Seed priming using MLE and application of Tithonia compost are prospective to enhance shallots growth, however, it is important to further evaluate the effect of priming on germination traits and economic evaluation on using Tithonia compost for practical applications in the field.


INTRODUCTION
Shallot (Allium ascalonicum L.) is an important commodity in Indonesia for domestic consumption as well as an export product. In 2022, the export volume of fresh and processed shallots reached 47,955 tons (BPS, 2022a). Domestic consumption of shallots in Indonesia reaches 56 g per capita per week (BPS, 2022b). However, domestic shallot production fluctuates resulting in the scarcity of shallot bulbs in particular periods. Such scarcity causes price spikes in the market that stimulates price inflation. As a result, Kustiari (2017) noted that shallot is classified as a sensitive agricultural product because it directly affects national inflation. Therefore, stabilizing shallots production is important in Indonesia.
Recently, true shallot seeds (TSS) are getting popular as planting material in Indonesia. There are various advantages of shallot cultivation using TSS, including the need for less planting material, free from soil-borne diseases, and higher productivity results in increasing farmers' profit (Basuki, 2009;Sembiring et al., 2014Palupi et al., 2017. However, propagation using TSS required a longer time than that of bulb, therefore, early establishment of shallots growth such as through seed priming and compost application is important in order to ensure optimum yield using TSS. Seed priming has many benefits, such as accelerating the germination process, better seed uniformity, and helping plants to adapt to environmental stress (Rhaman et al., 2020). The effect of seed priming is not only on seedling establishment, but the effect continues into the crop production in the field as concluded by many studies on soybean, long bean, rice, and onion (Agawane & Pahre, 2015;Thejeshwini et al., 2019;Karim et al., 2020;El-Sanatawy et al., 2021). One of priming agent that can be utilized is moringa leaf extract. Moringa leaves contain active compounds of cytokinin, antioxidant compounds, amino acids, flavonoids, carotenoids, and vitamins that are able to stimulate plant growth and production (Sheikha et al., 2022).
In the cultivation of shallots, farmers usually apply a large number of inorganic fertilizers. In the long run, applying excess inorganic fertilizers causes negative effects on soil fertility and environmental pollution (Putri et al., 2012). Therefore, the application of organic fertilizers such as compost to the soil can be a solution to enhance soil fertility with a low negative impact on the environment. Although many sources of compost are already known, selected compost ideally should consider the availability and easiness of materials (Nugroho et al., 2019;Ayilara et al., 2020).
Tithonia or Mexican sunflower (Tithonia diversifolia (Hemsl.) A. Gray) is invasive weed species (Ajao & Moteetee, 2017) and abundant in Makassar, South Sulawesi. Biomass has been widely used to improve soil fertility because of its ability to decompose quickly and release nitrogen, phosphorus, and potassium into the soil (Ajao & Moteetee, 2017). Turmundi et al. (2019) reported that applying Tithonia compost on peanuts at a level of 20 t ha -1 increased plant height, plant dry weight, and production by 71%. Furthermore, research conducted by Hafifah et al. (2016) also found a positive effect on applying Tithonia compost on cauliflower because the compost contains organic C, total N, C/N ratio, total P, and total K of 31.76%, 4.46%, 7.12, 0.61%, and 3.75%, respectively; the nutrient content is higher than cow manure. The objective of the research was to evaluate the effect of seed priming with MLE and supplementing shallots growing with Tithonia compost on their growth.

Location and experimental design
The research was conducted in the experimental field belonging to the Faculty of Agriculture, Hasanuddin University, Makassar. The research was conducted from September to December 2022. The average daily temperature was 29.51 ± 0.55 °C.
The study was arranged in a factorial design of two factors with three replications. The first factor was priming, consisting of five levels: unprimed, hydropriming, 12.5% MLE, 25% MLE, and 37.5% MLE. The second factor was the level of Tithonia compost consisting of four levels: without compost, 5 tons ha -1 , 10 tons ha -1 , and 15 tons ha -1 . True shallot seeds of Sanren F1 variety were used. The seed was obtained from a commercial market.

Preparing Tithonia compost
Tithonia plant biomass (Tithonia diversifolia) was obtained from the Experimental Farm, Faculty of Agriculture, Hasanuddin University. Harvesting the plants was done by cutting the leaves and the young stems, then chopping them into smaller sizes (Figure 1a). The biomass was then dried under sunshine for three days.
Fungi of Gliocladium virens Miller were added to the dry biomass. The fungi suspension was dissolved with water as much as 100 g in 5 L, and then evenly sprinkled on the plant biomass for about 50 kg. To make 1,000 kg of Tithonia compost it was required at least 2,500 kg of fresh biomass. After being sprinkled and mixed, the biomass was incubated in a plastic bag for 30 days (Figure 1b).

Priming materials
Moringa leaf extract was used as priming material. The procedure to develop the extract followed Yaseen and Hájos (2020). In brief, moringa leaves were obtained from the local market in Makassar City. Selected moringa leaves were separated from the petioles and primary veins, and it was washed thoroughly with running water. The leaves were crushed using an electric blender by adding distilled water with a ratio of 1:1 (v/w). The solution was filtered using filter paper and poured into a sterile bottle.
Shallots seeds were soaked in moringa leaf extract (MLE), with a ratio of seed to MLE solution was 1:5 (W/V). The weight of seeds for each treatment was about 20 g. The soaking was carried out for 20 hours at room temperature, and the supplement aeration was added using a bubbling pump ( Figure 2). After the priming process, the seeds were dried at room temperature.

Seedling preparation
Seeds after priming were sown in seedling beds of fine soil. Maintenance during seedling was watering. Here, seeds from different priming treatments were planted separately. After 40 days, the seedlings were collected and transferred to cultivation beds. Only healthy seedling was collected in this stage.

Cultivation process
The planting bed was sized 100 cm × 150 cm, with a height of 30 cm and a distance between beds of 50 cm. The compost was spread on the bed at seven days before transplanting. The beds were situated in the open field.
After transplanting, watering was done twice a day. Fertilizers were applied before and after transplanting. Seven days before transplanting, P2O5 was applied at a level of 62.5 kg ha -1 . After transplanting, K2O and N at rates of 60 kg ha -1 and 90 kg ha -1 , respectively were applied. The potassium and nitrogen fertilizers were split into three times of application at 15, 30, and 45 days after transplanting (DAT). Each application time used one-third of total fertilizer levels. Weeding was done manually. Pest control was carried out by manual and biological agents of Beauveria bassiana.
Harvesting is done at 70 days after transplanting. The shallots bulb was cleaned and separated from the leaves. Fresh and dry masses were measured after harvest. A dry mass was obtained after drying under sunshine for seven days.

Measurement and data analysis
Observed traits included plant height (cm), number of leaves, leaf weight (g), bulb weight (g), leaf and bulb ratio, stomatal opening area, and leaf chlorophyll index. Plant height and the number of leaves were observed at 20, 40, and 60 days after transplanting. The weight of the leaves and bulbs was measured after harvest. The stomatal opening area was calculated using a formula based on Song et al. (2020) as follows: Stomatal Opening Area = × × ; a = ½ length of stomata and b = ½ wide of stomata. The chlorophyll index was measured using CCM-200 Plus at 50 DAT.
The data obtained were then analyzed for variance (ANOVA). For any significant effect, a further Tukey test was performed with α = 0.05. Data processing used RStudio software version 4.2.1.

Plant height and number of leaves
Variance analysis showed no interaction effect between priming and Tithonia compost application on plant height at 20, 40, and 60 DAT (data not shown). However, priming with MLE and applying Tithonia compost solely affected plant height (Table 1). In general, increasing the concentration of MLE increased plant height in all observation periods. Priming with 37.5% MLE recorded the highest average plant height as compared to other treatments. The plant height significantly increased in the 37.5% MLE treatment by 20.47%, 12.01%, and 10.07% at 20, 40, and 60 DAT, respectively, as compared to unprimed ones. It is known that MLE contains hormones such as gibberellic acid, zeatin, and indole acetic acid (Ali et al., 2018;Alkuwayti et al., 2020;Yap et al., 2021) that can regulate plant growth and development. According to Castro-Camba et al. (2022), gibberellin promotes plant elongation.
Tithonia compost treatment increased plant height (Table 1). Increasing the level of Tithonia compost increased plant height. Application of 15 tons ha -1 significantly increased the average plant height by 9.04%, 6.62%, and 3.52% at 20, 40, and 60 DAT, respectively as compared to control (0 tons ha -1 ). Analysis of variance showed no interaction effect between priming and application of Tithonia compost on the average number of leaves at the age of 20, 40, and 60 DAT (data not shown). However, priming and Tithonia compost treatment individually affected the average number of leaves (Table 2). At 60 DAT, shallots from the treatment of priming 12.5% MLE and 37.5% MLE had an about single leaf in different, indicating the seed priming had a significant effect. It is probable that the cytokinin content of MLE (Alkuwayti et al., 2020) might affect leaf number. Sakakibara (2021) states that cytokinin regulates shoot growth. Application of Tithonia compost at a level of 15 tons ha -1 significantly increased the number of leaves (Table 2), irrespective of plant age, as compared to control no compost application. The laboratory analysis showed that Tithonia compost contained 0.95% N, 0.14% P, and 0.32% K. Therefore, a higher level of Tithonia application likely increases the availability of NPK nutrients. According to Fathi (2022), sufficient nitrogen in plants ensures high performance in the photosynthetic process. Nevertheless, providing 15 tons ha -1 compost required 37.5 tons of fresh Tithonia biomass. As shown in Table 2 application of 5 tons ha -1 Tithonia compost had no significant difference in leaf number at 60 DAT to 15 tons ha -1 ; thus 5 tons ha -1 could be considered.

Leaf and bulb weight
Interaction between seed priming and Tithonia compost did not show any significant effect (data not shown). The application of priming treatment significantly affected leaf and bulb weight (Table 3). Priming treatment at a concentration of 37.5% significantly increased leaf and bulb weight by 61.88% and 53.34%, compared to unprimed. Likewise, treating the Tithonia compost application affected both parameters (Table 3). Application at a dose of 15 tons ha -1 increased leaf and bulb weight by 27.01% and 22.49%, compared to 0 tons ha -1 . It was evident that shallot leaf and bulb weight was heavier after priming than that without priming ones (Table 3). Such phenomenon might correlate with increasing plant height and leaf number (Table 1 and 2), which produced more assimilates. Kirschbaum (2011) stated that some portion of assimilate from photosynthesis is allocated to support plant growth and development, while Paat (2011) stated that assimilate is used to form new cells. In the present study, the application of Tithonia compost increased the weight of the leaf and shallots bulb. The finding is in line with Pelu et al. (2020) where pak choi vegetable grows more vigorously and produces more biomass after the application of Tithonia compost.
Applications of seed priming and Tithonia compost had no effect on the leaf bulb ratio ( Table 3). The interaction of the two treatment factors did not significant (data not shown). The average leaf-to-bulb ratio ranged from 0.039 to 0.043. This means that assimilates allocation to leaf and bulb is constant, irrespective of priming and compost applications. According to Irinato et al. (2017), a high ratio of leaf to bulb indicates more assimilates are allocated to vegetative organs especially leaves.

Stomatal opening area
Seed priming treatment and Tithonia compost application did not significantly affect the stomatal opening area (Table 3). There is no interaction between the two factors (data not shown). The area of stomatal openings in both treatments ranged from 126.59 μm 2 to 200.83 μm 2 . The stomatal opening area tended to be higher in the 37.5% MLE treatment than in the unprimed treatment. Tithonia compost application at a dose of 15 tons ha -1 tended to have a larger stomatal opening area than without Tithonia compost application.
The insignificant effect of priming and compost applications on the stomatal opening area is likely due to the stomatal opening being strongly affected by environmental factors. According to Driesen et al. (2020), guard cells that regulate stomata opening are controlled by environmental conditions including light, carbon dioxide concentration, temperature, and air relative humidity.

Clorophyll index
Observation of shallot leaf chlorophyll index was influenced by the interaction between priming treatment with MLE and the application of Tithonia compost (data not shown). The interaction between seed priming with 37.5% MLE and the application of 15 tons ha -1 Tithonia compost recorded the highest chlorophyll index value (Table 4). The increase in the chlorophyll index value was 158.92%, compared to the lowest in the interaction between unprimed and without Tithonia compost application and the interaction between hydropriming and without Tithonia compost application. The present study envisages the prospect of MLE and Tithonia compost to enhance shallot growth. Research on maize conducted by Bakhtavar et al. (2015) revealed that seed priming using MLE increases the leaf chlorophyll. It is likely that the high nitrogen content of Tithonia compost might contribute to chlorophyll formation in shallot leaves. According to Anas et al. (2020), nitrogen is an essential macronutrient for plants, and one of its functions is as a constituent of green leaf substance or chlorophyll. Nevertheless, it needs further study the availability of nutrients after the application of the Tithonia compost because nutrient analysis after the application of the compost was absent in the present experiment.

CONCLUSIONS
Combination of TSS priming with MLE at a concentration of 37.5% and the application of Tithonia compost of 15 tons ha -1 increased the chlorophyll index by 158.92% as compared to the combination of no priming and no application of compost. Priming shallot seeds using MLE at a concentration of 37.5% increased plant height (24.71, 32.72, and 38.23 cm) and the number of leaves (4. 83, 8.38, and 10.55) at the age of 20, 40, and 60 DAT, respectively as compared to unprimed treatment. Likewise, the leaf weight (15.67 g) and bulb weight (29.92 g) increased significantly compared to the control. In addition, applying Tithonia compost of 15 tons ha -1 increased the average plant height (23.76, 32.21, and 37.72 cm) and the number of plant leaves (4.46, 7.72, and 9.74) at the age of 20, 40, and 60 DAT, respectively, as compared to no application of compost. Leaf weight (13.68 g) and bulb weight (27.01 g) were recorded as the heaviest in applying Tithonia compost at a level of 15 tons ha -1 .