Bio-stimulants derived from seaweed enhance true shallot seed (TSS) growth

The obstacles that farmers face by using the True Shallot Seed (TSS) due to the germination ability. In a laboratory level, the application of bio-stimulants was tested to enhance the TSS germination. Two types of bio-stimulants namely Bio A and Bio B with 16 treatment were tested with the TSS. Hypocotyl length, speed of seed germination, germination percentage, seedling growth rate, number of dead seeds and abnormal seeds, number of fungi-infected and bacteria-infected seeds were all measured. The results showed that soaking in Bio A for 1-3 hours reduced 38%-46% of seeds attacked by seed-borne bacteria compared to the control. The Bio B treatment had a better effect on the speed of seed germination and germination percentage parameters, compared to Bio A.


Introduction
In addition to chili, garlic, and potatoes, shallot (Allium cepa L. var aggregatum) is also an important vegetable commodity in Indonesia.Apart from being the main seasoning in cooking, shallot can also be fried as a sprinkling of a dish [1].Shallot farming provides benefits (feasible) because it has an R/C ratio of more than 2 both conventionally and organically [2] and has a comparative advantage [3] because farmers prefer to plant shallots compared to other crops.Therefore, the planting area of shallots in Indonesia has increased.From Central Bureau of Statistics data, Indonesian shallot production increased about 15% from 2019 to 2020 [4].As the planting area increases, the need for seeds also increases.
True Shallot Seed (TSS) has been known by Indonesian farmers as an alternative material for propagation of shallots other than bulbs [5].The advantages of TSS over tuber seeds include the need for less seed; lower seed storage and transport costs, healthier cropping [6] larger tuber size [7], and higher yields and profits [8,9,10].This is in accordance with the results of studies in shallot farming which states that efficient use of seeds can increase shallot production [11].The TSS technology, which is still new, causes some farmers to be not so proficient in its cultivation, especially at the time of seeding.Some farmers apply seed treatment before sowing to improve plant performance.Seed treatment that is usually done by TSS farmers includes the use of fungicides, growth hormones, or with microbes.Materials other than fertilizers or microorganisms that are given to plants that can increase plant growth if applied in small amounts are called bio-stimulants.The use of microbes or other substances to induce plant growth is said to be a bio-stimulant [12].Utilization of bio-stimulant is able to stimulate and modify plant physiological processes such as respiration, photosynthesis, nucleic acid synthesis and ion absorption [13].According to [14] there are several sources of bio-stimulants that have been developed in agriculture field, namely microbial inoculants (bacteria, fungi), humic acid [15] fulvic acid, amino acids, extracts seaweed, and plant extracts.These bio-stimulants can be used separately or together with more than one type of biostimulant.
Bio-stimulant from seaweed extract contains elements that stimulate plant metabolism so that it can increase plant growth and yield [16].The nutrients contained in seaweed are able to act as fertilizers that play a role in germination, the formation of new plants, and better development [17].Many studies related to the use of one type of seaweed bio-stimulant have been carried out, including on chickpeas [18,19], dates [20], tomato seedlings [21], leeks [22], chili [23,24], and few reported the use of seaweed [25] on shallot plants.The use of beneficial bacteria as bio-stimulants in various plants has also been carried out, including in lettuce [26], rice and corn [27], strawberries [28], rice [29], maize [30,31], potatoes [32], soybean [33], kailan [34], chili [35,36] etc.Several studies are known to use beneficial bacteria to increase the growth of shallots [37,38,39,40].
The use of beneficial bacteria or endophytic bacteria is utilizing natural hormones (growth regulators) produced by these bacteria.The use of synthetic growth regulators in shallots has been widely practiced [41,42,43] but the results were still varied, and further research was needed.The use of a consortium of endophytic bacteria producing growth regulators enriched by seaweed can be an alternative to synthetic growth regulators, especially to support organic agriculture [44,45].Previous studies have been conducted using seaweed enriched with endophytic microbes in chili peppers, onions, and shallots in the field [46,47,48].The results of the study showed that the use of endophytic microbes enriched with seaweed can increase the yield of chili fruit [46], and bio-stimulants can reduce the use of chemical fertilizer on shallot plants [48].However, no studies have reported testing using endophytic microbial-enriched seaweed at the seed level with the aim of seed invigoration.Therefore, an experiment was conducted to determine the effect of using bio-stimulants derived from seaweed and a consortium of bacteria on enhancing TSS germination at the laboratory level.

Materials and methods
This research was conducted at the Microbiology Laboratory of the Indonesian Center for Agricultural Biotechnology and Genetic Resource Research and Development (ICABIOGRD), Bogor from February to April 2021 and the Indonesian Vegetable Research Institute (IVEGRI), Lembang from August to September 2021.Bio-stimulant A and B were formulated in the Microbiology laboratory of ICABIOGRD and applied on the TSS germination in the seed laboratory of IVEGRI.

Bio-stimulants formulation
Production of bio-stimulants began with preparing Ulva seaweed and the selected endophytic bacteria from shallot plants that have been identified molecularly in previous research [46].This bio-stimulants formula, which are a consortium of endophytic bacteria with the addition of seaweed extract, was made into 2 formulas, namely formula A (Bio-stimulant A) with seaweed preparation using oven technique (pre-dried treatment at 60 0 C for 72 hours), and formula B (Bio-stimulant B) with seaweed preparation without oven technique.The endophytic bacteria consortium used were three types of endophytic bacteria isolated from the shallot plant (DBS2, TK2Na, and T2), which are ICABIOGARD Culture Collection (ICABIOGARD CC).
The bio-stimulant was formulated by mixing bacterial suspension and carrier made from Ulva seaweed extracts.Some organic and mineral ingredients were added into the bio-stimulant formula.The seaweed extract was prepared to follow the extraction method developed by [49] with some modifications.The seaweed i.e., Ulva sp.collected from the Bayah seashore of Banten were put into a heat -resistant plastic containers and then dried in oven at 60 0 C temperature for 72 hours (for biostimulant A with pre-dried treatment using oven technique).The dried seaweed was powdered by using blender.Finally, a total of 200 g of seaweed powder was diluted in 400 mL distilled water (1:2 w/v) and centrifuged for 15 minutes at a speed of 10,000 rpm to obtain seaweed extract.
Furthermore, the complete phytohormone levels were measured using HPLC.Measurement of the IAA and GA phytohormone extracts followed the method of [50].The HPLC column used was C-18 with the mobile phase methanol: water: acetic acid (30:70:1).Measurements will be carried out at the SEAMEO BIOTROP service laboratory.

The evaluation of bio-stimulants in TSS
The materials and the equipment used were Bima variety TSS seeds, plastic box, styrofoam, newsprint paper, nylon net, tweezers, petri-dish, gauze, scales, oven.The design used was a completely randomized design with 16 levels (Table 1), and it consists of four replications with Treatment 16 as the control (not soaked).Bio A -Bio B Not soaked T16 Note: Bio A = Bio-stimulants A; Bio B = Bio-stimulants B TSS seeds that have been weighed as much as 1.5 g for each treatment are placed in a petri-dish that has been lined with gauze or water according to the treatment.The seeds were then arranged in the TSS germination box.The germination box is a modification of the Top of Paper (TP), in the form of a plastic box filled with distilled water, styrofoam pieces on four sides, then covered with nylon netting and 10 sheets of wet newsprint.Each box filled with 100 TSS seeds was tightly closed and placed in the incubation room (temperature ± 25 °C and RH ± 70%).
The observed parameters included hypocotyl length, speed of seed germination, germination percentage, seedling growth rate, percentage of abnormal seedling and percentage of dead seeds.
1. Hypocotyl length (HL) Ten normal sprouts from the test results for seedling growth rate on 6 th day, were measured for hypocotyl length using a ruler.The percentage of abnormal seedling, dead seeds, seeds infected with fungi, and seeds infected with bacteria were obtained by counting the number of abnormal sprouts, dead seeds, fungusinfected seeds, and bacteria-infected seeds at the end of the observation.Calculated by the formula: Note: C = percentage of abnormal seedling/ dead seeds/ fungus-infested seeds/ bacterial-infected seeds A = number of abnormal seedling/ dead seeds/ fungus-infected seeds/ bacteria-infected seeds B = total number of seeds 5. Seedling growth rate (SGR) Seeds from each treatment were germinated using the TOP method.Observations were made on the 6 th day of normal seedling.Normal seedlings were dried in an oven at 80 °C for 24 hours and then weighed.Seedling growth rate (mg/seedling) is the dry weight of normal seedling divided by the number of normal seedling [51].
All the data obtained were then analyzed for variance and further tested with Tukey's HSD test with a level of 5%.

Result and discussion
Because this bio-stimulant is made with seaweed extract, which is superior in its phytohormone content, phytohormone measurements were carried out.Based on the results of the phytohormone analysis (Table 2), it can be seen that the two bio-stimulant formulas contain phytohormones of the types IAA, GA, zeatin, and kinetin.Formula bio-stimulant B has a relatively higher content of phytohormones than formula bio-stimulant A.
The results of the analysis of variance are shown in Table 3.There was an interaction between the types of bio-stimulants and the treatments given to abnormal seedlings (AS), dead seeds (DS), fungusinfected seeds 6 th day (FIS6), bacteria-infected seeds 6 th day (BIS6), fungus-infected seeds 12 th day (FIS12), bacteria-infected seeds 12 th day (BIS12) parameters.While SSG and GP parameters are influenced by a single factor of bio-stimulant and treatment separately.Moreover, HL is only affected by the treatment.Note: HL = hypocotyl length; SSG = speed of seed germination; GP = germination percentage; DS = dead seeds; AS = abnormal seedling; FIS6 = fungus-infected seeds at 6 th days after sowing; BIS6 = bacteria-infected seeds at 6 th days after sowing; FIS12 = fungus-infected seeds at 12 th days after sowing; BIS12 = bacteria-infected seeds at 12 th days after sowing; SGR = seedling growth rate; * = significant effect on P<0.05, ** = significant effect on P<0.01, ns = no significant effect The AS, DS, FIS6, BIS6, FIS12 and BIS12 parameters were influenced by the interaction between the type of bio-stimulant and the combined treatment dose and soaking time (Table 4).The lowest percentage of abnormal seeds (AS) was shown by immersing TSS seeds in 10% bio-stimulant A for 1 hour (Bio A-T13) of 3.75% and 5% of Bio B for 1 hour (Bio B-T7) of 4%, while the highest was by immersing TSS seeds in 5% bio-stimulant B for 3 hours by 15.00% (Bio B-T9).The lowest percentage of dead seeds (DS) was shown by the control (Bio A-T16) of 47.25% while the highest was the treatment of soaking TSS seeds in 10% bio-stimulant A for 1 hour by 68% (Bio A-T13).
The lowest percentage of seeds infected with fungi at 6 days after sowing (FIS6) was the lowest shown by the treatment of TSS seed immersion in 10% bio-stimulant B for 1 hour (Bio B-T13) of 1.75% while the highest was the treatment of TSS seed immersion in 10% bio-stimulant A for 1 hour by 18% (Bio A-T13).The lowest percentage of seeds infected with bacteria at 6 days after sowing (BIS6) was shown by several treatments including soaking TSS seeds in 5% bio-stimulant A for 2 hours (Bio A-T8); 7.5% Bio A for 2 hours (Bio A-T11); 10% Bio A for 2 hours (Bio A-T14) and for 3 hours (Bio A-T15); and soaking for 1 hour in the water (Bio B-T1) while the highest was in the treatment of soaking TSS seeds in water for 2 hours by 19.75% (Bio A-T2) and soaking in 7.5% Bio B for 3 hours (Bio B-T12) of 20.50%.The lowest percentage of seeds infected with fungi at 12 days after sowing (FIS12) was the lowest shown by the treatment of TSS seed immersion in 7.5% biostimulant A for 2 hours (Bio A-T11) of 9.5% while the highest was the treatment of TSS seed immersion in 5% bio-stimulant B for 1 hour by 18% (Bio B-T7).The lowest percentage of seeds infected with bacteria at 12 days after sowing (BIS12) was shown by several treatments including the treatment of soaking TSS seeds in 5% bio-stimulant A for 1 hour (Bio A-T7); 5% bio-stimulant A for 2 hours (Bio A-T8); 10% bio-stimulant A for 2 hours (Bio A-T14); 10% bio-stimulant A for 3 hours (Bio A-T15); and the control (Bio A-T16) while the highest was in the treatment of TSS seed immersion in 10% bio-stimulant B for 3 hours of 20.75% (Bio B-T15).Note: T = Treatment; AS = abnormal seedling; DS = dead seeds; FIS6 = fungus-infected seeds at 6 th days after sowing; BIS6 = bacteria-infected seeds at 6 th days after sowing; FIS12 = fungus-infected seeds at 12 th days after sowing; BIS12 = bacteria-infected seeds at 12 th days after sowing; yellow bold = the lowest number; blue bold = the highest number; the numbers followed by the same letter in the same column were not significantly different, at 5% Tukey test In the parameter of bacteria-infected seeds at 6 th (BIS6) and 12 th days after sowing (BIS12), most of the lowest number, which is preferred condition that only a few seeds were attacked by the seedsborne bacteria, were experienced by the Bio A treatment ranged from 5% to 10% dosages (T7, T8, T11, T14, T15) with soaking range between 1 to 3 hours (T7, T8, T11, T14, T15).In the parameter of BIS6 in the Bio A, the lowest number was range between 1.75% -2% which was lower than the control (T16) at 3.25%.It showed that Bio A can reduce around 38%-46% the bacteria-infected seeds compared to the control.In this study, Bio A was the bio-stimulant with the pre-dried seaweed extract treatment.The seaweed extraction method affects the alginate yield [53] and the difference in drying method affects the total phenol, alginate and proximate content of seaweed [54] temperature treatment and extraction time of the extraction liquid Sargassum sp.seaweed, had a significant effect on the amount of nutrients, viscosity, total plate number (ALT), phosphate, Organic C, and EC [55].Alginate derived from seaweed has been reported to have anti-bacterial, anti-biofilm [56], and anti-fungal properties [57].The proportion of alginate contained in Bio A, could stimulate this bio-stimulant to prevent and reduce bacterial and fungal infections at the seedling stages.
The speed of seed germination and germination percentage parameters were influenced by the biostimulants (Table 5), it is showed that Bio B had a better performance than Bio A in those two parameters.The Bio B experienced 4.93 Etmal for speed of seed germination, which is 11% higher than Bio A, and 32.56% of germination percentage parameter, which is 8% higher than Bio A. The content of Gibberellins and IAA hormones in Bio B, which was higher than Bio A, enhanced the ability to promote seedling growth.Gibberellins are known to be one of the main hormones that support plant growth.It is reported that Gibberellins (GA3) which used as a seed priming in some TSS cultivars, could increase seed germination and hypocotyl vigor index [58].However, another study presented different results [58], GA3 combined with NAA and BAP were applied as plant growth regulators on TSS, did not affect the TSS germination, plant growth, the number of living plants, and yields in the field.The HL, SSG, GP, DS, FIS6, BIS6, FIS12 and BIS12 parameters were influenced by the soaking time treatment (Table 6).For the parameters of hypocotyl length, speed of seed germination, germination percentage and the percentage of dead seeds, the control (T16) showed the best result with the highest score for each parameter.This result is similar to a study on soybeans seeds treatment that was given a rhizobium as bio-stimulant, which reported that it has not able to increase soybean production and reduce seedling populations, compared to control without treatment [60].However, the parameters of fungus-infected seeds and bacteria-infected seeds in the 6 th and 12 th days after sowing showed the best result, which is the lowest number, in the various treatments.The lowest number for FIS6 parameter, was from soaking in 5% bio-stimulants for 3 hours (T9) at 2.63%, then, the same dosage of bio-stimulants soaking for 2 hours showed the best result for BIS6 parameter by 3.50% (T8) and soaking for 1 hour resulted the lowest number for BIS12 parameter at 5.63% (T7), which was the same condition for the control (T16).For the FIS12 parameter, the best result was shown by soaking 10% of bio-stimulant for 3 hours (T15) at 10.63% of seeds that were infected by fungi.
In this study, a consortium of endophytic bacteria which were extracted from shallot plants (DBS2, TK2Na, and T2) with the addition of seaweed extract used as bio-stimulants.Our results found that bio-stimulant with pre-dried treatment (Bio A) has a better effect on promoting antibacterial properties in the seedling stage than Bio B, while Bio B treatment presented a better performance in speed of seed germination and germination percentage parameters than Bio A. This result is similar to [48] where the application of bio-stimulants on shallot plants can reduce the chemical fertilizer by 25%, while the result is same as 100% of chemical fertilizer application.Nevertheless, the seedling growth parameters in both bio-stimulants in all concentrations and soaking times, were not better than control.Those responses were same as TSS germination treated by dark septate endophyte (DSE) [61], which can reduce the number of seed-borne fungi and bacteria, while some seedling growth parameters tend to be lower than controls.Further research was needed to test whether there is a better effect on growth seedling characteristics with the addition of bio-stimulants derived from a consortium of endophytic bacteria and seaweed extract at more various concentrations and soaking time.Note: T = Treatment; HL = hypocotyl length; SSG = speed of seed germination; GP = germination percentage; DS = dead seeds; FIS6 = fungus-infected seeds at 6 th days after sowing; BIS6 = bacteria-infected seeds at 6 th days after sowing; FIS12 = fungus-infected seeds at 12 th days after sowing; BIS12 = bacteria-infected seeds at 12 th days after sowing; yellow bold = the best result; the numbers followed by the same letter in the same column were not significantly different, at 5% Tukey test

Conclusions
Soaking the seeds of TSS Bima variety in Bio A for 1-3 hours reduced 38%-46% of seeds attacked by seed-borne bacteria compared to the control.The Bio B treatment had a better effect on the speed of

Table 1 .
The treatment and bio-stimulant dosage levels used in this research.
[51]d of seed germination (SSG)The test for speed of seed germination was measured per Etmal (per 24 hours).Seeds from each treatment were germinated using the TOP method.Observations were made on 6 th day (First Day Count/ FDC) to 12 th day (Last Day Count/ LDC) on number of normal seedlings, abnormal seedlings, and dead seeds.The speed of seed germination was calculated by the method of the Association of Official Seed Analysts[51].

Table 2 .
Hormone content in bio-stimulants A and B.

Table 3 .
Analysis of various (P value) treatment combinations of doses and soaking time of TSS seeds on two types of bio-stimulants.

Table 4 .
Interaction between bio-stimulants dosages and soaking times on abnormal seeds, dead seeds, percentage of seeds attacked by fungi and bacteria at 6 and 12 days after sowing.

Table 5 .
The effect of bio-stimulants in the SSG and GP parameters
1230 (2023) 012024 IOP Publishing doi:10.1088/1755-1315/1230/1/0120249 seed germination and germination percentage parameters, compared to Bio A. Further study is needed to check the effect of bio-stimulants with more various concentration and soaking time.