Application technique and concentration of biofertilizer from rhizosphere of cocoa (Theobroma cacao L.) given biosurfactant dietanolamide oil palm on the growth of cocoa seedlings

This research aimed to know the influence of application technique and biofertilizer that given concentration of biosurfactant diethanolamide (DEA) for the growth of cocoa seedlings and also to know the interaction between application technique and concentration level of biofertilizer given DEA growth of cacao seedling (Theobroma cacao L.). This research was conducted in UPTD SITANDU, Banten and the Soil Agroclimate Laboratory, Faculty of Agriculture, University of Sultan Ageng Tirtayasa from December 2020 until March 2021, by using randomize complete block design with two factors. The first factor is technique application T1 at soil, T2 at leaf, and T3 with combination soil and leaf fertilization), and the second factor is concentration level of biofertilizer given DEA P1 10 ml and 5%, P2 10 ml and 10%, P3 20 ml and 5%, P4 20 ml and 10%. As a result, there were twelve possible treatment combinations, and each was applied three times. Parameters total chlorophyll (unit), root length (cm), stem diameter (mm), seedlings dry weight (g) and root shoot rasio (g). The results there was no interaction effect between the two treatments, namely application technique and concentration level of biofertilizer with DEA biosurfactant, on all observed parameters of cocoa seedlings.


Introduction
In the last five years, cocoa bean production has continued to increase.The decline is in line with the decline in cocoa plantations.In 2018, cocoa bean production reached around 767,280 tons.Then it continues to decline until 2022; cocoa bean production only reached around 650,612 tons [1].The Ministry of Agriculture has set a main target to increase exports threefold, one of which is cocoa, namely to increase production by 7% until 2024.Therefore, it is necessary to increase cocoa production in Indonesia.
Appropriate cultivation technology, one of which is the technique of applying the right fertilizer, can increase the effectiveness of plant absorption of nutrients.The amount or percentage of fertilizer nutrients taken by plants determines how effective fertilization is.When a plant absorbs the majority of the nutrients from the fertilizer, the fertilization is considered effective.According to [2], providing biological fertilizer can help increase plant growth because it contains live Plant Growth Promoting Rhizobacteria (PGPR), which functions to help provide nutrients for plants and can reduce disease or damage by insects.Biological fertilizers also produce phytohormones that can increase plant growth.1302 (2024) 012019 IOP Publishing doi:10.1088/1755-1315/1302/1/012019 2 [3] argue that aspects of fertilization management are also important to study so that the implementation of fertilization is in accordance with the standard operational standards implemented by a company and that the use of fertilizer is effective and efficient.
Then, to facilitate the entry of oxygen and nitrogen into the soil during its application, palm oil DEA biosurfactant can be added.The addition of surfactants can significantly increase the bioavailability of the soil.Surfactants are able to expand the surface contact of oil with water through the formation of micro-emulsions and biological availability for the needs of microorganisms.Diethanolamide (DEA) is a non-ionic surfactant, i.e., a surfactant that has no charge but has a high affinity for water [4].Palm kernel oil, distillate palm fatty acids, and coconut oil are vegetable ingredients that have great potential in the manufacture of diethanolamide surfactants [5].
Based on the description above, it is important to carry out research on the effect of application techniques and concentration levels of biofertilizers from the rhizosphere of cocoa plants (Theobroma cacao L.) and palm olein diethanolamide biosurfactant on the growth of cocoa plant seeds (Theobroma cacao L.).

Time and Place
This research was conducted in November 2020-March 2021 and took place in the Integrated Agriculture System Area, Unit Horticultural Food Crops and Plantation Protection Seeds (UHFCPPS), Curug District, Serang Regency, Banten, and the Soil and Agro-climate Laboratory, Faculty of Agriculture, Sultan Ageng Tirtayasa University.

Methods
The research design used was a factorial randomized block design.The first factor was the application technique of biofertilizers (T) with three levels, first Aplication at the leaf (T1), second Aplication at the soil (T2) and Third is Combination aplication on leaf and soil (T3).The second factor was the level of concentration of biofertilizers treated with biosurfactant diethanolamide palm olein (P) with four level, First Biofertilizer 10 ml and biosurfactant 5% (P1), second Biofertilizer 10 ml and biosurfactant 10% (P2), Third Biofertilizer 20 ml and biosurfactant 5% (P3) and Biofertilizer 20 ml and biosurfactant 10% (P4).Where each treatment was repeated three times, thus obtaining 36 experimental units.If the results of the variance test show a significant to very significant difference, then further tests will be carried out.In this research, the Duncan Multiple Range Test (DMRT) follow-up test was carried out at the 5% level.The Biofertilizer used microbial from rhizosfer cacao with identification is Bacillus cereus and Bacillus Amiloliquefaciens [6].

Observed Paramaters
The parameters observed in this research are: 1.Total chlorophyll (units) Total chlorophyll was measured using a Soil Plant Analysis Development (SPAD) meter.This measurement is carried out every two weeks and starts from day 0 to 12 week after planting (WAP).These measurements were carried out on three green parts of the leaf, namely, the tip, middle, and base of the leaf.2. Root Length (cm) Root length is measured from the base of the stem to the tip of the longest root.
Using a ruler, do this once at the age of the plant, at 12 WAP.3. Plant Dry Weight (g) Plant dry weight was measured using an analytical balance.Plants are cut into three parts: roots, stems, and leaves.Then it dried in the oven for 2 x 24 hours at 70 °C.Done when the plant is 12 WAP.4. Root-Stem Ratio (g) The root-to-shoot ratio is obtained by dividing the dry weight of the roots by the dry weight of the shoot.The dry weight of the roots and shoots was determined after the roots and crowns of the plants were oven-treated at 70 °C for 2 x 24 hours.Carried out when the plant is

Total Chlorophyll
The results of variance in the total chlorophyll observation parameters of cocoa plants aged 0-12 WAP showed that application techniques and doses of biological fertilizers given biosurfactant DEA palm olein had no significant effect on the total chlorophyll of cocoa plant seedlings, both from application technique factors and biological fertilizer doses and interactions.between application technique and dosage of biological fertilizer.The effect of application techniques and concentrations of biological fertilizers added with biosurfactant diethanolamide palm olein on the total chlorophyll of cocoa plants can be seen in Table 1.
Table 1.Average total chlorophyll (units) of cocoa plants under the influence of application technique and concentration of biofertilizer added with DEA palm olein biosurfactant.
A 10 ml dose of biological fertilizer with a concentration of 5% biosurfactant DEA palm olein gave the highest total chlorophyll value with an average of 39.25 units.This is presumably because the successful use of beneficial microorganisms in plants is not only influenced by the quantity of cells in

Time application
Application technique the inoculant.[7] stated that when plants are treated with biological fertilizers, they can fix N2, which causes the chlorophyll content in plants to be high so that the rate of photosynthesis increases.The increased rate of photosynthesis also causes carbohydrate synthesis to increase.The formation of carbohydrates caused by the rate of photosynthesis will increase the vegetative growth of plants, including the growth of leaf formation.While the lowest average total chlorophyll value was at a dose of 10 ml of biofertilizer with a 10% concentration of biosurfactant DEA palm olein, namely 34.83 units.This is presumably because the leaves are given biological fertilizers with too high a concentration, causing the leaves to burn, and the leaf area index is reduced, so the leaves do not carry out photosynthesis optimally, which causes total chlorophyll to not be optimal.This is in accordance with the statement from [8], which states that the chlorophyll content of plants is related to the leaf area that receives light for photosynthesis and nutrient content.photosynthesis and the amount of leaf chlorophyll Then, because all the fertilizer is given through the leaves, it results in a reduction in nutrients in the soil, which causes a deficiency in the leaves.In line with the statement from [8], which states that when the amount of nutrients in the soil is insufficient, especially the element N, it will reduce the total leaf area of the plant because N is a constituent element of the plant body, especially leaves, Nitrogen and potassium (K) are active nutrients; when the supply from the soil is insufficient, the N and K that are in the old tissue will be translocated to the young tissue, so that the old tissue will show symptoms of deficiency.A deficiency in leaves can result in leaf fall.

Root Length (cm)
Based on the results of various treatments, application techniques, and doses of biofertilizer added with DEA palm olein biosurfactant on the parameters of observing the root length of cocoa plants aged 12 WAP, the results show that there is no interaction and no significant effect independently on the root length of cocoa seedlings, both on the fertilization technique factor and the dosage of biological fertilizer.Root length is the root's response to the availability of water and nutrients.The treatment of application techniques and concentrations of biological fertilizer added with DEA palm olein biosurfactant on the root length of cocoa plants can be seen in Table 2. Based on the results of the analysis of variance in the treatment of application techniques through soil with a dose of 20 ml of biological fertilizer, which was given 5% DEA biosurfactant, the best average root length was 16.22 cm.It can be seen that by providing biofertilizer with the appropriate dosage and application technique, it is possible to increase the length of the roots of cocoa seedlings.According to [7], apart from the nutrient content, the function of several bacteria in biological fertilizers can increase plant root development.Then [9] stated that beneficial microorganisms actively influence soil microorganisms to increase soil fertility.Azospirillum sp. are bacteria that live in the root area of plants.This bacteria breeds mainly in the root extension area and at the base of the root hairs.Furthermore, [10] stated that this IAA hormone stimulates the division of root tip cells and lateral roots so as to provide an optimal environment for roots.

Plant dry weight (g)
Based on the results of variance on the parameters of the dry weight observation of cocoa plants aged 12 WAP, it showed that the application technique and concentration of biofertilizers treated with DEA biosurfactant did not occur between treatments and had no significant independent effect on plant dry weight either from fertilization technique factors or biofertilizer dosage.The effect of the application technique and dosage of biofertilizers with the addition of biosurfactant DEA on the dry weight of cocoa seedlings can be seen in Table 3.Based on the data in Table 3, the dry weight results for the soil application technique treatment with an average value of shoot dry weight was 6.77 g, root dry weight was 1.90 g, and plant dry weight was 8.68 g.Then for the dose of biofertilizer that gave the highest average value, namely at a dose of 10 ml with a concentration of 5% DEA and an average dry weight of the plant of 6.76 g, this is because this dose and the application technique through the soil are appropriate application techniques for the growth of cocoa plants.According to [11], plant dry weight is the result of the accumulation of available carbohydrates for plant growth during its lifetime.So, if the physiological processes that occur in plants run well and are supported by the efficient application of fertilizer, they can increase the dry weight of the plant.
Application of fertilizer through leaves with a biosurfactant concentration (10% DEA) that is too high causes less than optimal plant growth, especially in the number of leaves, resulting in a low dry weight of the plant.[12] stated that biogeochemical processes occur in the rhizosphere involving enzymes and secondary metabolites to increase nutrient uptake from the soil through roots.[13] state that applying fertilizer through the leaves absorbs nutrients in low concentrations.The mechanism for nutrient uptake through leaves begins with the entry of nutrients through the stomata, exodesmata, and cuticle in the epidermis into the cytoplasm.Apart from being a place for exchanging gases such as CO2 and O2 from the atmosphere, stomata also act as a place for the entry of NH2 and NO2 and other nutrients in the form of gases and solutions.Exodesmata are canals or cavities for the movement of nutrient solution through the cuticle layer.The cuticle is traversed by a number of hydrophilic pathways that are permeable to water and small solute molecules.
The low nutrient uptake through leaves is because leaves have an adaptive response to the environment (drought stress and extreme temperatures).Therefore, when environmental conditions do not support nutrient absorption, it cannot occur because the stomata will close.While the application of biological fertilizers through the soil gives a more optimal effect because absorption is more efficient, according to [13], apart from agronomic matters related to soil fertilization, specifically the uptake of nutrients from fertilizer by plants through the roots, this can also be increased through optimizing rhizosphere conditions.The rhizosphere is a root zone with a high intensity of active nutrient uptake, symbiosis of microorganisms, and release of root exudates.

Root Shoot Ratio
Root shoot ratio is the ratio of the dry weight of plant shoots and roots.This parameter can be used as an indicator of water shortage events in plants.Lack of water inhibits crown growth more than root growth, which is a ratio value [14].The effect of application technique and concentration of biofertilizer treated with palm oil DEA biosurfactant on the root shoot ratio can be seen in Table 4 Based on the analysis of variance results on the observation parameters of the root shoot ratio of cocoa seedlings aged 0-12 WAP, the application technique treatment did not have a real influence on the root shoot ratio, and there was no interaction between the application technique and the dose of biofertilizer treated with DEA biosurfactant on the root shoot ratio of cocoa plant seedlings.Based on these data, it shows that the biofertilizer concentration treatment has a real influence on the root-stem ratio of cocoa plants, where the highest ratio value is in the treatment dose of 20 ml with a concentration of 20% DEA palm olein biosurfactant with an average value of 5.41.Meanwhile, the lowest yield was at a dose of 10 ml of biofertilizer with a 5% concentration of DEA palm olein biosurfactant.This shows that the P1 treatment of 10 ml of biofertilizer, which was given 5% DEA palm olein biosurfactant, had more optimal growth because the dose was appropriate and the microorganisms in it worked well.The functional of microbial in biofertilizer used in this study can be seen on [14].There fore can be seen from the observation parameters, where the growth and development in the P1 treatment showed The highest yield was in the number of leaves, plant height, and plant dry weight.This is because a low root shoot ratio value indicates better root development.According to [16], if the root shoot ratio value is low, the proportion of roots will be greater than the proportion of the crown.Roots with good development will influence canopy growth which can then influence plant growth.So that nutrient absorption is more optimal and plant production is good because the nutrients are fulfilled.In line with the statement from [17] the lower the root shoot ratio, the better.The greater the number of leaves, stem diameter and root length, the smaller the root shoot ratio.

Conclusion
Cocoa (Theobroma cacao L) seedlings observed parameters did not show any interaction between the two treatments, namely the biological fertilizer's application method and concentration level treated with DEA palm olein biosurfactant.Therefore, further observations need to be made at all treatment levels.

Table 2 .
Average root length (cm) of cocoa (Theobroma cacao L.) plants under the influence of application techniques and concentrations of biological fertilizers added with biosurfactant DEA palm olein

Table 3 .
Average dry weight (g) of cocoa plants (Theobroma cacao L.) on the influence of application techniques and concentrations of biological fertilizers added with biosurfactant DEA palm olein

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Tabel 4. Average root shoot ratio (g) of cocoa plants (Theobroma cacao L.) on the influence of application techniques and concentrations of biological fertilizers added with biosurfactant DEA palm olein Note: Numbers followed by the same letter in the same row show no significant difference in the DMRT test at the 5% level.