EFFECTIVENESS ENDOPHITE BACTERIA OF AUXIN HORMONE

PRODUCING AS A BIOFERTILIZER IN TEA (Camellia sinensis L)

PLANTS

RESEARCH PROPOSAL

By :

NAME : SIGIT INDRA ZIT

NPM : 1913010186
STUDY PROGRAM : AGROTECHNOLOGY

FACULTY OF SCIENCE AND TECHNOLOGY

AGROTECHNOLOGY STUDY PROGRAM
UNIVERSITY PEMBANGUNAN PANCA BUDI
MEDAN
2023
 TABLE OF CONTENTS

TABLE OF CONTENTS ................................................................................. i

PRELIMINARY .............................................................................................. 1
Background ............................................................................................ 1
Research purposes .................................................................................. 2
Research Hypothesis ............................................................................... 2
Research Usability .................................................................................. 3
MATERIALS AND METHODS ..................................................................... 4
Place and time of research .......................................................................4
Materials and tools .................................................................................. 4
Data Analysis Methodology ....................................................................5
Data Analysis Methods ...........................................................................6
RESEARCH IMPLEMENTATION ................................................................. 8
Preparation of Research Sites .................................................................. 8
Endophytic Bacteria Isolation ................................................................. 8
Characteristics of Endophytic Bacteria .................................................... 8
Bacterial Activity Test in Producing IAA Hormones ............................... 9
Potential Test of IAA Producing Bacteria ...........................................9
DNA 16 S rRNA analysis and sequencing ............................................ 10
Isolation and Extraction of DNA ...................................................... 10
DNA sequencing process 16s rRNA ................................................. 11
16s rRNA Gene amplification by PCR ............................................. 11
Seed selection ....................................................................................... 12
Preparation of Planting Media ............................................................... 12
Planting Media Sterilization .................................................................. 12
Seed Soaking ........................................................................................ 12
Planting ................................................................................................ 12
Plant Maintenance ................................................................................ 13
Making Microcapsules of Endophytic Bacteria as Biofertilizers ............ 13
Preparation of 0.1 M CaCl2 Solution .................................................... 13
Preparation of Sodium Alginate Solution .............................................. 13
Manufacturing of Microcapsules ........................................................... 13
Microcapsule analysis ........................................................................... 14
Microcapsule application ...................................................................... 14
Observation Parameters ........................................................................ 14
Seed Germination Rate ..................................................................... 14
Plant Height (cm) ............................................................................. 14
Number of Leaves (strands) .............................................................. 14
Stem Diameter(mm) ......................................................................... 15
Leaf Area (cm2) ............................................................................... 15
Plant Wet Weight (g)........................................................................ 15

i
BIBLIOGRAPHY .......................................................................................... 16
ATTACHMENT ............................................................................................ 18
Appendix 1. Experiment Chart. ............................................................. 18
Appendix 2. Spacing in the field ........................................................... 19
Appendix 2. Description of Plants ......................................................... 20

ii
 PRELIMINARY

Background

Tea (Camellia sinensis) is an agricultural commodity that is in great

demand by the wider community. Tea plant nursery is a step that needs to be done

before rejuvenation and planting. In general, the propagation technique is carried

out by vegetative propagation because it can meet the needs of planting materialin

large quantities (Hindersah et al., 2016). The thing that must be considered in tea

plant nurseries is the fertilization process for soil fertility and plant health. So far,

the process of fertilizing tea nurseries still uses inorganic fertilizers such as urea,

SP 36, and Kcl.

The use of inorganic fertilizers continuously and without the application of

the right dosage can degrade soil fertility, even changing the physical, chemical

and biological properties of the soil (Maghfoer 2018). Soil pollution due to

inorganic fertilizers and pesticides can also cause the balance of soil elements to

change (Puspawati & Haryono 2018).

Another alternative to fulfilling some nutrients is through the use of

organic fertilizers (Rachmiati et al, 2014). One of the newest organic fertilizer

innovations is by utilizing endophytic bacteria. Organic fertilizers that contain lots

of microbes are called biofertilizers. Biofertilizer is obtained from a consortium of

several types of microbes, both microbes obtained from the environment and

microbes associated in plant tissues (endophytic microbes) (Setiawati et al., 2014).

Several studies have used endophytic bacteria which have proven effective

as a source of nitrogen nutrients. The results of research conducted by Pranoto and

Mieke (2014) show that some microbes are known to be effective as a source of

1
nitrogen nutrients. Endophytic bacteria can also produce growth hormones such as

auxins, ethylene and cytokinins, so that they can help plants meet their hormone

needs (Herlina et al. 2016). The availability of the auxin hormone can stimulate

root growth which allows the roots to reach water and nutrients widely (Syaputra

& Arista 2017).

Currently research on endophytic bacteria from tea plants as a biofertilizer

is still very minimal. In an effort to explore endophytic bacterial isolates that have

the potential as biofertilizers, further research is needed. The bacterial isolates

obtained are expected to be able to add references in the context of utilizing

microbes as biofertilizers in tea plants.

Research purposes

To determine the characteristics and diversity of endophytic bacteria

present in the tea plant (Camellia sinensis L).

To determine the effect of soaking time of tea plant seeds with a

suspension of endophytic bacteria on the growth of tea plants (Camellia sinensis

L).

To determine the response of encapsulation from endophytic bacteria to

the growth of tea plants (Camellia sinensis L).

Research Hypothesis

There are several types of endophytic bacteria that are symbiotic with the

tea plant (Camellia sinensis L).

2
 There is an effect of soaking time of tea plant seeds with suspension of

endophytic bacteria on the growth of tea plants (Camellia sinensis L).

There is the effectiveness of endophytic bacteria administration to

stimulate the generative growth of tea plants (Camellia sinensis L).

The presence of auxin hormone levels from endophytic bacteria

originating from the tea plant (Camellia sinensis L).

Research Usability

As reference material and information for readers, especially

students and scholars who want to do tea plant nurseries (Camellia sinensis)

3
 MATERIALS AND METHODS

Place and time of research

This research was carried out in November 2022 until completion. The

stages of isolation, characterization and microencapsulation of endophytic bacteria

were carried out at the Microbiology Laboratory, Faculty of Science and

Technology, Pembangunan Panca Budi University, Medan. The observation stage

of bacterial microcapsules using an electron microscope was carried out at the

Integrated Laboratory, University of North Sumatra. For the analysis of the

content of the planting media was carried out at the Laboratory of the Center for

Seeding and Plantation Plant Protection, Medan. Endophytic bacterial DNA

extraction was carried out at the Genetics and Molecular Laboratory, Department

of Biology, University of North Sumatra. Analysis and sequencing of the bacterial

16s rRNA gene was carried out at the Macrogen Europe Laboratory, Amsterdam,

The Netherlands. The biofertilizer application stage was carried out in the screen

house of the Faculty of Science and Technology, University Pembangunan Panca

Budi.

Tools and materials

The tools used in this study were petri dishes, test tubes, test tube racks,

measuring cups, beaker glass, Erlenmeyer, autoclave, oven, spatula, needle loops,

incubator, hot plate, stir bar, analytical balance, sprayer, laminar water flow,

shaker, glass bottle, aluminum foil, cotton, electron microscope, cutter knife, poly

bag.

While the materials used in this study were tea roots and stems, Media

Nutrient Agar (NA), distilled water, 70% alcohol, chlorine solution, CaCl2,

4
Sodium Alginate from seaweed extract, inulin, poultry manure, top soil, rice husk

charcoal. , Physiological NaCl, Crystal violet, Safranin, Acetone, alcohol, iodine.

Data Analysis Methodology

This study used a factorial completely randomized design (CRD)

consisting of 2 treatment factors, 16 treatment combinations, 2 replications and 32

research plots, namely:

A. Treatment factor I is the soaking time of the seeds. This treatment factor is

given the symbol S.

S0 = Control (plain water)

S1 = 24 jam

S2 = 36 jam

S3 = 48 jam

B. Treatment Factor II is a capsule containing a consortium of endophytic

bacteria from roots and stems. This treatment factor is given the symbol I.

I0 = 0 grams/plot (control)

I1 = 5 g capsules

I2 = 10 g capsules

I3 = 15 g capsules

C. The treatment combination consisted of 16 combinations (t):

S0I0 S1I0 S2I0 S3I0

S0I1 S1I1 S2I1 S3I1

S0I2 S1I2 S2I2 S3I2

S0I3 S1I3 S2I3 S3I3

5
 Number of repetitions (n):

(t-1) (n-1) ≥15

(16-1) (n-1) ≥15

15(n-1 ) ≥15

15n-15 ≥15

15n ≥15+15

15n ≥30

n ≥30/15

n ≥2 (2 connected)

Data Analysis Methods

The linear model for this study is as follows:

γijk = μ + αj + βκ + (αβ) jκ + εϳκ

Where :

Γijk :The results of observations on the experimental unit i which obtained a

combination of treatments of variations in seed soaking time and

endophytic bacterial capsule fertilizer.

Μ : Middle value

αj : Effect of the jth level of the seed soaking time factor

βκ : Effect of the κ level of the factor of giving various capsule fertilizers

from endophytic bacteria.

(αβ)ϳκ : The effect of the interaction between the jth level of the seed soaking

time factor and the κth level of the capsule fertilizer administration factor

from endophytic bacteria

6
eϳk : Error rate at the jth level of the variation factor of soaking time and at

the κth level of the factor of capsule fertilizer application from

endophytic bacteria.

The data obtained statistically based on analysis of variance on each

observation that was measured was followed by using Duncant's Multiple Range

Test (Harsojuwono et al, 2011).

7
 RESEARCH IMPLEMENTATION

Preparation of Research Sites

The place of research used is the screen house. Before use the screen house

area is cleaned of all weeds, then level the floor of the screen house, then cover

the floor of the screen house with black plastic, and cover the walls with white

plastic so that sunlight can enter the screen house, the following steps the

sterilization of the inside of the screen house is sprayed with 0.4% formalin

evenly.

Endophytic Bacteria Isolation

Endophytic bacteria was isolated from tea roots and stems. As for the

types of types. Isolation of endophytic bacteria using the method of Singh et al.

(2022) modified. Prior to isolation, the surface of the roots and stems of tea was

sterilized.

Fresh tea root and stem samples were cleaned under running water and

then cut into 1-3 cm long pieces and separated according to the part of the plant.

Sample pieces were immersed in 70% ethanol for 1 minute, 5.25% sodium

hypochlorite solution for 5 minutes, and washed with 70% ethanol three times.

The sample pieces were sliced sterile and then planted in sterile nutrient agar

(NA) media in a petri dish. The media containing the sample was incubated in an

incubator at 370 C in the dark and observed every day until there was growth of

tea colonies.

Characterization of Endophytic Bacteria

The growing endophytic bacteria were purified one by one and cultivated

in a petri dish containing Nutrient Agar medium. Pure endophytic bacterial

8
isolates were identified morphologically based on colony color, colony shape,

colony margins, and colony elevation (Lay, 1994). Characterization of cell

morphology observed included cell shape, cell arrangement and gram staining

(Pelczar et al., 1988). The gram staining procedure was carried out by cleaning the

object glass with 96% alcohol then fixing it on a spirit lamp, then taking the active

isolate aseptically and placing it on the object glass and then flattening it. Then fix

it again on the spirit lamp. After cold, drip Gram A paint (Crystal violet) 2-3 drops

for 1 minute, then wash with Gram B (Lugol Iodine) for 1 minute, wash with

running water and dry in air or air dry. Then dripped with Gram C (96% alcohol)

for 30 seconds, then washed with running water and dried in air. The last step is

dripping Gram D (Safranin) for 45 seconds, then washing it with running water

and removing excess water with absorbent paper. This observation was made by

looking at the shape and color of the cells under a microscope with 1000x

magnification. Bacterial isolates that had been purified and characterized were

then cultivated in oblique agar media in test tubes.

Bacterial Activity Test in Producing IAA Hormones

Potential Test of IAA Producing Bacteria

IAA Production Potential Test Endophytic bacterial isolates were

inoculated on flat Nutrient Agar media supplemented with tryptophan at a

concentration of 100 ppm using the streak plate method. Then incubated at room

temperature for 48 hours. The Salkowski reagent is dripped onto the endophytic

bacterial isolates that have grown in the Nutrient Agar medium until it is evenly

distributed. Furthermore, the isolate that has been dripped with Salkowski reagent

is stored in a dark room for 30 minutes. A positive result is indicated by a change

9
in the color of the isolate colony to red. To determine the ability of endophytic

bacteria to produce IAA in vitro, the first pure endophytic bacterial isolate was

rejuvenated onto NA medium (Nutrient Agar) and incubated for 48 hours. Then

the young isolate was made into a suspension of 10 ml with the Mac Farland

standard in order to obtain a bacterial suspension with a cell density of 108

CFU/ml (Bresson & Borges, 2003).

3 ml of bacterial culture suspension was taken and put into 30 ml of liquid

LB medium (Luria Bertani) + tryptophan (Bric et al., 1991). Each treatment was

repeated 3 times and incubated at 280 C for 5 days in a shaker at 150 rpm

(Bhutani et al., 2018). Then centrifuged at 5000 rpm for 25 minutes to obtain

supernatant and pellets. Analysis of IAA levels using the Colorimetric method.

The supernatant was taken as much as 2 ml plus 1 ml salkowsky reagent (Gordon

& Weber, 1951) or with a ratio of 2: 1 (Zahir et al., 1997). Allowed to stand for 60

minutes, the absorbance was measured with a 530 nm spectrophotometer. The

regression equation is substituted with the absorbance value of the sample.

DNA 16 S rRNA analysis and sequencing

Isolation and Extraction of DNA

Genomic DNA isolation from bacterial culture was carried out with a

modified Genomic DNA Mini Kit (Geneaid). This process was carried out at the

Genetics and Molecular Laboratory, Department of Biology, University of North

Sumatra. DNA extraction using the Genomic DNA Mini Kit (Geneaid). The lysis

process (damaging or destroying the membrane and cell wall) is carried out using

GP1 buffer. The heating temperature is 60ºC for 15 minutes. The GP2 buffer

10
functions as a neutralizing buffer, and the GP3 buffer functions to bind DNA to

the buffer and the spin column. The washing process was carried out successively

using buffer W1 (to remove the remnants of protein attached to the DNA), Wash

Buffer (to wash the previous buffer salts), and lastly the DNA was dissolved in the

elution buffer (to release the DNA from the membrane).

The extracted DNA results were then amplified by PCR (Polymerase

Chain Reaction) using primers for the 16S rRNA genes BKXF (forward) and

BKXR (reverse) which can amplify the 16S rRNA gene up to 1,200 bp

(Kolondam, in press). In this amplification process, DNA multiplication occurs in

certain areas limited by primers. The PCR product in the form of amplified DNA

was then electrophoresed as a qualitative test to measure the concentration of

DNA obtained from the PCR process. (Rahayu and Nugroho, 2015).

DNA sequencing process 16s rRNA

The 16rs rRNA DNA sequencing process was carried out at the Macrogen

Europe Laboratory, Amsterdam, The Netherlands. Sequencing was performed to

determine the nucleotide sequence of the detected DNA fragments from the

visualization of amplified DNA in the PCR process using an autosequencing

machine.

16s rRNA Gene amplification by PCR

Amplification was performed using a combi block PCR machine. The

primers used for the PCR process were BKXF (forward) and BKXR (reverse)

primer pairs. The template used for 16S rRNA gene amplification is the isolated

bacterial genomic DNA. Amplification by PCR technique was carried out with

various reagent compositions and modified PCR reaction conditions.

11
 Seed Selection

Selection of tea seeds is done manually and visually by selecting seeds that

are old and have a blackish brown color and normal seed shape.

Preparation of Planting Media

The planting media used in this study were top soil that had been cleaned

of weeds, broiler manure and rice husk charcoal. With a ratio (1: ½ : ½ ), top soil:

50%, broiler manure: 25% and rice husk charcoal: 25%.

All the media is mixed and stirred evenly then, Filling the media into

polybags measuring 2 kg and isolating the top of the polybag tightly and without

gaps using insulating tape, is done after the isolation process and battery

characteristics are complete.

Planting Media Sterilization

Sterilization is done to avoid contamination with other organisms that can

affect the growth of the tea plant. Sterilization was carried out at the oyster

mushroom cultivation site using a drum where the sterilization was carried out for

5 hours 2 times at 1000C.

Soaking Seeds With Endophytic Bacteria

Collection of endophytic bacteria solution was carried out by adding 10 ml

of NaCl solution in 1 petri, and stirring using a triangular stir bar. Then the tea

seeds were soaked at a ratio of 24 hours, 36 hours and 48 hours with a container

covered with aluminum foil to keep it sterile.

Planting

Planting was carried out when the seeds were soaked with each treatment.

Then planted into a sterile poly bag that has been prepared.

12
 Plant maintenance

Plant maintenance includes watering, weeding and pest and disease

control. Watering is done 1 time a day in the morning and evening using hype and

a volume of 0.5 liter/polybag sterile water. The weeding process is carried out at

the age of 1 and 2 months of seedlings manually, by removing weeds that are in

the plant media in polybags.

Making Microcapsules of Endophytic Bacteria as Biofertilizers.

Preparation of 0.1 M CaCl2 Solution

A total of 14.7 g of CaCl2 was weighed and then dissolved with 1000 ml

of distilled water in a volumetric flask, stirred until homogeneous. The solution

was sterilized by autoclaving at 121°C for 15 minutes.

Preparation of Sodium Alginate Solution

Preparation of Sodium Alginate Solution Containing Endophytic Bacteria

Suspension Preparation of sodium alginate solution was first made of alginate

solution with a concentration of 2% (w/v) with distilled water (v/v). 2% inulin

was added to it while stirring until homogeneous and made up to 50 ml, then

sterilized by autoclaving at 121°C for 15 minutes. Then 50 ml of endophytic

bacterial suspension was added.

Manufacturing of Microcapsules

A sterile alginate solution containing a suspension of endophytic bacteria

was put into a spit needle and then dropped into a 0.1M CaCl2 solution, then

allowed to stand for one hour until a dense microencapsulation was formed, then

the formed microcapsules were transferred into sterile distilled water and stirred

13
slowly using a shaker for one hour. to remove CaCl2 residue, then filtered and

rinsed using distilled water (Junaidi, 2018).

Microcapsule analysis

As much as 1 g of endophytic bacterial microcapsules were analyzed for

morphology and bead diameter using an electron microscope which was sent to

the Integrated Laboratory, University of North Sumatra.

Application of Microcapsules

The application of endophytic bacterial micropaslas was carried out 4

weeks after planting with a predetermined dose treatment level.

Observation Parameters

Seed Germination Rate

Plant germination was observed from the moment the plant seeds were

planted into the media. And the observation of germination is done every day.

Plant Height (cm)

Observation of plant height began in the first week at the time of shoot

emergence, and was measured at intervals of 1 week until 4 weeks after the

growth of shoots was measured using a ruler.

Number of Leaves (strands)

Observation of the number of leaves begins in the first week at the time of

shoot emergence, and is measured at intervals of 1 week to 4 weeks after the

growth of shoots, and for leaves whose data can be collected are leaves that have

opened completely.

14
Stem Diameter(mm)

Calculation of plant diameter is done by measuring using a digital stem

diameter gauge.

Leaf Area (cm2)

Calculation of leaf area using the constant method with the formula P x L

x K, by measuring the length of the leaf and the width of the leaf

Plant Wet Weight (g)

Determination of the wet weight of rubber plants is done by weighing the

plants that have been cleaned.

15
 BIBLIOGRAPHY

Gordon, S.A., Weber, R.P. (1951). Colorimetric Estimation of Indole acetic Acid.
Plant Physiol. 26:192–195.

Harsojuwono, B. A., Arnata, I. W., & Puspawati, G. A. K. D. (2011). Theoretical

Experiment Design, SPSS and Excel Applications. Cross Publishing:
Malang.

Herlina, L., Pukan, K. K., & Mustikaningtyas, D. (2016). Study of IAA (Indole
Acetic Acid) Producing Endophytic Bacteria for Plant Growth. Journal of
Science and Technology (Sainteknol), 14(1), 51–58.

Hindersah R, Bagu A, Pujawati S (2016) Population of Bacteria and Fungus and

Growth of Tea Plant (Camellia Sinensis L.) in Two Types of Planting
Media. Agrology. 5 (1): 1 – 9

Junaidi, M. 2018 Viability Test of Loctobactillus Acidophilus Microcapsulation

Using Chitosan Sodium Alginate Polymer on Simulation of Gastric Acid
Liquid

Lay, B. W. 1994. Microbial Analysis in the Laboratory. PT Raja Grafindo

Persada. Jakarta.

Maghfoer MD. 2018. Environmentally Friendly Eggplant Fertilization

Techniques. Malang (ID): Universitas Brawijaya Press.

Pelczar, M. J. and Chan, E. C. S., 1988, Fundamentals of Microbiology, translated

by Hadioetomo, R. S., Publisher, University of Indonesia, Jakarta.

Puspawati C, Haryono P. 2018. Environmental Health Teaching Materials for Soil

Health. Jakarta (ID): Center for Health Human Resources Education 2018
Edition, Ministry of Health of the Republic of Indonesia.

Pranoto, E. and M. Setiawati. 2014. Comparison of several exogenous phosphate

solubilizing bacteria in andisol soil as the dominant tea growing area in
Indonesia Journal of Research on Tea and Quinine. 18(2):159-164.

Rachmiati, Y., Karyudi, B. Sriyadi, S.L. Dalimoenthe, P. Rahardjo, and E.

Pranoto. 2014. Fertilization Technology and Technical Culture Adaptive to
Climate Anomalies in Tea Plants. Conference Paper "Efforts to increase
productivity in plantations with technology.

16
Rahayu, D. A. and Nugroho, E. D. 2015. Molecular Biology in a Conservation
Perspective. Plataxia, Yogyakarta.

Setiawati, M.R., Wulansari, R. and Pranoto, E. 2014. Comparison of the

Effectiveness of Consortium Biological Fertilizers and Endophytic
Biological Fertilizers on the Productivity and Health of Tea Plants to
Produce GMB Clones 7. Journal of Tea and Quinine Research, 17 (2): 71-
82.

Singh, R.; Pandey, K.D.; Singh, M.; Singh, S.K.; Hashem, A.; Al-Arjani, A.-B.F.;
Abd_Allah, E.F.; Singh, P.K.; Kumar, A. Isolation and Characterization of
Endophytes Bacterial Strains of Momordica charantia L. and Their
Possible Approach in Stress Management. Microorganisms 2022, 10, 290.
https://doi.org/10.3390/ microorganisms10020290

Syaputra, R., & Arista, A.M. (2017). Isolation and Characterization of Root
Endophytic Bacteria of Sugar Cane (Saccharum officinarum L.) Producing
Indole Acetic Acid (IAA) Hormone. National Seminar, (April), 143–152.

17
 ATTACHMENT

Appendix 1. Experiment Chart

S0I0 S1I1 S2I2 S3I3

S1I0 S2I1 S3I2 S0I3

S2I0 S3I1 S0I2 S1I3

S3I0 S0I1 S1I2 S2I3

II

S0I0 S1I1 S2I2 S3I3

S1I0 S2I1 S3I2 S0I3

S2I0 S3I1 S0I2 S1I3

S3I0 S0I1 S1I2 S2I3

Information : U

Number of polybags per repetition : 16 polybags

The total number of repetitions of polybags : 4 lines

The total number of polybags : 32 polybags

18
Appendix 2. Spacing in the field

x X

25 cm

50 cm X
100 cm

x
X

Information :

x : Not a Sample Plant

X : Sample Plants

19
Appendix 3. Description of Plants

The description of the GMB 7 clone is as follows:

Origin : Cross Mal 2 x PS 1

Group : Assamese variety

Stem shape : Cylinder

Stem surface : Short furrowed slightly crusted white

Branching system : Fine, 47-600

Bud segment : 1,3-5,2 cm

Stem color : Chocolate

Wake up leaves : Eliptic oblong (2.0:1)

Leaf size : 40.17 cm square

Leaf stalk : 0,2-0,6 cm

Leaf position : 29-490

Leaf base : Pointy

Bone leaves : 18-24 pieces (9-12 pairs)

Leaf edge : Serrated small irregular

Leaf tip : Taper

Leaf face : Wavy rather shiny

Leaf color : Light green

Seed Color : Black

Rooting : Very well

Pest resistance : Resistant to mites

Illness : Resistant to smallpox disease tea leaves

20
Development area : Suitable in low, medium to high areas, Has wide

adaptability, well planted in lowland, medium to high

areas,

21