SCREENING OF RHIZOSPHERIC BACTERIA THAT PLAYS

ROLE IN INCREASING PLANT GROWTH

Name : Pratiwi Kusuma Kurniawati

SID : B1B017007
Entourage : III
Group :3
Assistant : Azma Nurizqi Isnasari

BACTERIOLOGY LABORATORY REPORT

MINISTRY OF RESEARCH, TECHNOLOGY AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGY
PURWOKERTO
2019
 I. INTRODUCTION

Rhizosphere is a soil around a plant root which is inhabited by a diverse

population of microbes, comprising bacteria, fungi, actinomycetes and algae affected
by the chemicals which are released from the roots of plant. The organic materials
from roots provide the driving force for the development of active microbial biomass
around the root than in the bulk soil. The different compounds secreted by plant roots
into the rhizosphere perform multiple functions. The common genera of bacteria are
Azotobacter, Micrococcus, Pseudomonas, Arthrobacter, Flavobacter,
Mycobacterium, Agrobacterium, Alcaligenes, Cellulomonas and others have been
found to be either abundant or less populated in the rhizosphere (Geetanjali & Jain,
2016). Microbes in the rhizosphere are known to affect plant health by assisting with
nutrient acquisition, play a role in nutrient cycling, protecting against biotic and
abiotic stress, influencing the activity of microorganisms, as well as biological
control of root pathogens and also altering plant growth and physiology (Haney et
al., 2015). Rhizosphere supports complex plant-associated microbial communities
that play a vital role in maintaining plant health (Kalam et al., 2017).
Many microorganisms are attracted by nutrients exuded from plant roots and
this effect is known as “rhizosphere effect”. These microorganisms are found in
association with the roots. The region of soil surrounding the roots which is highly
active for the metabolism and is influenced by associated microorganisms is called
rhizosphere. Rhizosphere is the rich source of microbes and microbial activity and
thus better known as store house of microbes. The rhizoshpere consists of large
number of microorganisms mainly bacteria. These bacteria can be symbiotic or non-
symbiotic on the basis that not only the plants get benefitted by their presence but
bacteria also derive the nutrients for their survival. The major role of rhizosphere
bacteria are to supply nutrients to crops, to stimulate plant growth, e.g., through the
production of phytohormones, to control or inhibit the activity of plantpathogens, to
improve soil structure, and bioaccumulation or microbial leaching of inorganic
compounds (Kundan et al., 2015).
Plant root area is a good breeding ground for microbial growth, especially for
rhizobacteria. The relationship between bacteria and plant roots will increase the
availability of nutritional needs for both. The root surface of a plant can be called
rhizoplane (Maier et al., 2009). Rhizoplane is the root surface zone where
microorganisms attach themselves using surface structures such as flagella, fimbriae
or cell surface polysaccharides. The boundary between rhizoplane and rhizosphere is
very thin and therefore this habitat is largely considered as a continuum. The
rhizosphere is a thin layer of soil immediately surrounding plant roots. This is an
extremely important and active area for root activity and metabolism (Mwajita et
al.,2013). Rhizoplane microbiome is an important factor in the development of plants
but for their part, microorganisms, being particularly sensitive to changes in the
environment, in most cases respond negatively to NPs. Other effects may occur in the
soil, where physicochemical properties, texture, and organic matter, may change the
properties of NPs. Such an interaction may result in increased or decreased
bioavailability and toxicity of NPs for plants and microorganisms (Gorczyca et al.,
2018).
Plant growth promoting rhizobacteria (PGPR) are the soil bacteria inhabiting
around or on the root surface and are directly or indirectly involved in promoting plant
growth and development via production and secretion of various regulatory chemicals
in the vicinity of rhizosphere(Ahemad & Kibret, 2014). The function of PGPR for
plants is to stimulate root growth and physiology and to reduce disease or damage by
insects. Another function is to add to compost and speed up the composting process.
Pesticide reduction and crop rotation can spur population growth from beneficial
bacteria such as PGPR, examples of non-symbiotic nitrogen fixation bacteria
(Azotobacter sp. And Azospirillum sp.) And phosphate solvent bacteria (Bacillus
megaterium and Bacillus subtilis) (Permatasari & Nurhidayati, 2014).
There are array of mechanisms by which PGPR stimulate the growth of plants.
They are broadly classified as direct and indirect mechanisms. The direct mechanism
of PGPR is the major step involved to support plant growth in a forward and direct
manner. Direct mechanism includes nitrogen fixation, phytohormones production,
phosphate solubilization and increasing iron availability. These mechanisms influence
the plant growth activity directly but the ways by which it influences will vary from
species to species as well as strain to strain. In the presence of PGPR direct
enhancement of mineral uptake has been reported due to increases in specific ion
fluxes at the root surface. Organic substances that stimulate plant growth are known
as plant growth regulators. They stimulate plant growth by influencing the
physiological and morphological processes at very low concentrations. Indirect
mechanism involves the ability of PGPR to reduce the deleterious effects of plant
pathogens on the growth. This involves synthesizing the lytic enzymes including
chitinases, cellulases, 1,3-glucanases, proteases, and lipases that can lyse a portion of
the cell walls of many pathogenic fungi. There is another mechanism called induced
systemic resistance (ISR). This is the mechanism of increased resistance at particular
sites of plants at which induction had occurred. The defense mechanism of ISR is
activated only when there is an attack of pathogenic agent. ISR isnot specific against
particular pathogen but helps the plant to control diseases. Another mechanism is the
siderophore production which prevents plants from some pathogens to acquire
adequate amount of iron and suppresses their ability to grow. Other mechanism is HCN
production, cyanide being toxic is produced by most microorganisms including
bacteria, algae, fungi and plants as a means of survival by competing with the
counterparts (Kundan et al., 2015).
One of the factors that influences the rhizosphere bacterial community is
rhizodepocytes which are plant rhizosphere materials or around the soil that are
transformed and utilized by the rhizosphere biota and combined with soil organic
materials. As for these materials coming out of plant roots include exudates in the
form of dissolved, secreted insoluble material, lysate, CO2 and ethylene. The
statement can be termed as an organic component released by living plant roots into
the environment. Biotic and abiotic factors such as soil type, season, stage of plant
development, root shape, type of plant, and root depth affect the structure of the
bacterial community at the root. Plant roots have the ability to influence surrounding
microbiological activity, rhizosphere microbiomas, through the formation of special
chemical niches in the soil mediated by the release of phytochemicals such as root
exudates which depend on a number of factors, such as plant genotypes, soil
properties, plant nutritional conditions, and climatic conditions. Analysis of the
bacterial population reported that in two plant species with a state of Fe deficiency
and Fe adequacy could support differences in rhizosphere microbiomas (Amelia &
Aditiawati, 2016).
Soil temperature is one of the factors of soil physics that greatly determines the
presence and density of soil organisms, thus soil temperature will determine the level
of decomposition of soil organic matter. Measurement of soil pH is also very necessary
in conducting research on soil fauna. There is soil fauna that live on acidic pH and
some are happy to live on soil that has an alkaline pH. The regional climatic conditions
and various plants that grow on the soil and the abundance of microorganisms that
inhabit an area greatly affect the relative diversity of microorganism populations (Suin,
1997). Other factors that have an influence on the relative diversity of microorganism
populations are reactions that take place in the soil, moisture content and harmonious
conditions (Sutedjo et al., 1991).
The objective of the practice is to know how to screen the isolates of rhizosphere
bacteria that capable of fixing free nitrogen from the air, dissolving organic phosphate,
producing HCN and IAA hormone which plays a role in increasing plant growth.
 II. MATERIALS AND METHODS

A. Material

The tools that we used in this laboratory activity are petri dish, reaction tube,
ose needle, bunsen burner, tray isolate, tube tray, and pinset.
The materials that we used in this laboratory activity are isolate of
Sclerotium sp., and Fusarium sp., isolate A, B, C, Potato Dextrose Aagar medium,
Nutrient Agar+Glycine medium, Picric acid, Sodium carbonate 2%, Pikovskaya
medium, medium of TSA+Tryptophan 5%, salkowsky reagent, Nitrogen free
Bromothymol blue (NfB) medium, whatmann paper, sterile stick, label, and
alcohol 70%.

B. Method

The method used in this laboratory activity are :

1. Re-Culture
Isolate A, B and C was taken out using ose needle, then streaked continue
into NA medium. Each of isolates was incubate 2 x 24 hours in room
temperature.
2. Antagonism Test Against Fungi and Pathogenic Bacteria
Isolate of Fusarium sp. and Sclerotium sp. are prepared. Two of PDA
medium are prepered, than isolate A, B, C are inoculated on medium by
continous streak technique that has been divided into three parts. After that,
put 1 plug of the isolate Fusarium sp. on each side of the Petri dish. Do the
same step of the isolate Sclerotium sp. on the other PDA medium. Incubation
were for 3×24 hours at room temperature. Observe, the positive interpretation
is the growth of fungi are inhibited.
3. HCN Production Test
Isolate A, B, C are streaked on a NA+Glycine medium. The whatmann
paper is dipped in picric acid 0,5% and sodium carbonate 2% solution and
then put down into the petri dish. Incubation were for 3×24 hours at room
temperature. Observe the colour changes of watmann paper. Positive
interpretation can be suspected if there is colour changes from yellow into
dark brown.
4. Phosphate Solving Test
Isolate A, B, C are inoculated by continous streak technique on a
Pikovskaya medium. Incubation were for 3×24 hours at room temperature.
Phosphate solving bacteria can be suspected by observing the presence of
clear zone around colonies on Pikovskaya medium.
5. IAA Production Test
Isolate A, B, C are inoculated by continous streak technique at
TSA+Tryptophan 5% medium that has been divided into three parts.
Incubation were for 3×24 hours at room temperature. After that, drop
salkowsky reagent into bacterial culture and allowed to stand for 30 minutes
in the dark room. Observe, positive interpretation if the medium around the
colonies turned into pinkish red.
6. Nitrogen Fixing Test
Isolate A, B, C are inoculated by stab technique of NfB semisolid medium.
Incubation were for 3×24 hours at room temperature. Then, observe the
presence of turbid ring on the subsurface part or NfB medium and the medium
changes to be blue. If its present, nitrogen fixing bacteria can be suspected.
 III. RESULT AND DISCUSSION

Table 3.1 Screening Result of Isolates A, B, and C

Assay Isolates
A B C
Antagonism of Fusarium sp - - -
Antagonism of Sclerotium sp. - - -
HCN Production + + +
Phosphate Solubilizing - + -
IAA Production - - -
Nitrogen Fixing - + -

a
A

B C

b
C

B A

Figure 3.1. The Result of Antagonism Test Against Fungi and Pathogenic
Bacteria A, B, C Isolates to (a) Sclerotium sp. and (b) Fusarium sp.
on PDA Medium
Based on the results of the antagonism test against fungi and pathogenic
bacteria, in antagonism test on PDA media that has been inoculated by Fusarium sp.
and Sclerotium sp. isolate A, B, and C of our group are showed no clear zone and failed
to inhibit the growth of both fungi (Fusarium sp. and Sclerotium sp.). These results are
in line with the statement from Devi et al. (2011), that antagonistic plant-associated
bacteria (APB) are an important functional group of beneficial bacteria responsible for
the control of soil-borne fungal pathogen Occurrence of plant growth promoting (PGP)
attributes in APB may reinforce their effectiveness as microbial inoculants The
absences of inhibition zone indicated that three isolates we used were not as effective
in inhibition of the reference fungal pathogens. Added by Sari (2015), the growth of
inhibitor (competition) assay can identify how much the antimicrobial compounds or
nutrient competition that produce by inhibitor strain. Also this assay could be detect
the positives interaction. Actually, where the precense of the inhibitor strain could be
improve the growth of competitor strain.

A C

B
Figure 3.2. The Result of HCN Production Test to Isolates A, B, C on
NA+Glycine Medium
Based on the results of the HCN Production Test, all of isolate A, B, and C in
our group are successfully to produce HCN in NA+ glycine medium. This positive
result showed by changes the color of whatman's paper from yellow to dark brown.
The result is in accordance to the statement of Babu et al. (2017), that the color changes
from yellow to light brown, moderate or strong reddish brown was taken as indication
of HCN production. The HCN production by PGPR has been looked at both beneficial
as well as harmful trait with respect to their effect on plant growth. The beneficial
effects have mostly been discussed in their antifungal role and hence, suppression of
plant pathogens. Production of HCN in excess may play a critical role in suppression
of fungal disease

A
C

Figure 3.3 The Result of Phosphate Solving Test to Isolates A, B, C on

Pikovskaya Medium
Based on the result of phosphate solvent bacteria test, bacteria’s isolate A, B,
and C showed negative interpretations in isolates A and C with no clear zone formed,
and positive in isolate B with clear zone forming which proved that the bacterial
isolates produced phosphate. This is in accordance with the statement of Maryanti
(2006), signs that a bacterium can dissolve phosphate, namely by the presence of a
clear zone in the surrounding bacterial colonies and the addition of bacterial colony
size on Pikovskaya media, this is because these bacteria can dissolve phosphate
(Ca(PO4))2) found in the Pikovskaya medium formulation.

B
C
A

Figure 3.4 IAA Production Test to Isolates A, B, C on TSA+Tryptophan 5%

Medium
Based on the results of the IAA Production Test, obtained that all of the isolate
showed negative results or the medium around the colonies did not turned into pinkish
red. Actually, is supposed to be change to pink after we add a reagent Salkowski. One
of wrong mistake is the incubation time is less and not optimal. Then there is must be
a factor the isolates itself even the medium that we must supposed to know. Based on
the references, effect of incubation time on IAA production can influenced the
optimization of IAA production. The effect of the incubation time on IAA production
was performed in triplicate. Important role in enhancing for IAA production are
phytohormones production, siderophores production, and the production of pathogen
cell wall degrading enzymes to prevent plant pathogens (Nutaratat et al., 2017).

A B C

Figure 3.5 The Result of Nitrogen Fixing Test to Isolates A, B, C on NfB

Medium
 Based on the results of observation nitrogen fixing test, obtained that only the
isolate B showed positive results or the presence of turbid ring on the subsurface part
or NfB medium and the medium changes to be blue which indicate bacteria have the
ability to bind free nitrogen. According to Kuan et al. (2016), stated that the color
change phenomenon from green (pH 7.0) to blue (above pH 7.6) is due to the
bromothymol blue content which changes color with increase in pH above neutral.
This increase in pH is due to the formation of fixed-ammonia from atmospheric N2
through natural N fixation phenomenon. This ability to fix nitrogen is due to the
possession of the nitrogenase enzyme which catalyzes the nitrogen-fixing reaction.
Found also there, white pellicle at the bottom of NfB media. Added from Okon et al.,
(1977), the formation of a pellicle in the test could not be due to the medium being
solid so that the pellicle which should be microaerophilic could not be formed.
 IV. CONCLUSION AND SUGGESTION

A. Conclusion

Based on the objective, to screen the isolates of rhizosphere bacteria, there

are five ways to do that namely antagonism test against fungi and pathogenic
bacteria, HCN production test, phosphate solving test, IAA production test, and
nitrogen fixing test. The result of the group 3 on antagonism tests are negative on
Fusarium sp. and Scleretium sp., HCN test is positive, IAA test is negative,
phosphate solving test is positive on isolate B, and nitrogen fixing test is positive
on isolate B.

B. Suggestion

Suggestion in this laboratory activity is it could be better to be more careful of

doing the laboratory wokrs thus everyone would be safe and the data would be
valid as well.
 REFERENCES

Ahemad, M. & Kibret, M., 2014. Mechanisms and applications of plant growth
promoting rhizobacteria: Current perspective. Journal of King Saud University
Science, 26(1), pp. 1–20.

Amelia, R. & Aditiawati, P., 2016. Keanekaragaman bakteri rizosfer pemacu

pertumbuhan tanaman (Plant Growth Promoting Rhizobacteria/PGPR) selama
pertumbuhan ubi jalar cilembu (Ipomoea batatas L var. Rancing). Jurnal
SNIPS, 21(22), pp. 899 – 901.

Babu, S.V., Triveni, S., Reddy, R. S. & Sathyanarayana, J., 2017. Screening of Maize
Rhizosperic Phosphate Solubilizing Isolates for Plant Growth Promoting
Characteristics. International Journal of Current Microbiology and Applied
Sciences, 6(10), pp. 2090-2101.

Devi, S. I., Talukdar, N. C., Sharma, K. C., Jeyaram, K. & Rohinikumar, M., 2011.
Screening of Rhizobacteria for Their Plant Growth Promotion Ability and
Antagonism against Damping off and Root Rot Diseases of Broad Bean (Vicia
faba L). Indian J. Microbiol, 51(1), pp.14-21.

Geetanjali & Jain, P., 2016. Antibiotic Production By Rhizospheric Soil Microflora.
Internasional Journal Of Pharmaceutical Sciences And Research, 7(11), pp.
4304-4314.

Gorczyca, A., Przemieniecki, S. W., Kurowski, T. & Oćwieja, M., 2018. Early plant
growth and bacterial community in rhizoplane of wheat and flax exposed to
silver and titanium dioxide nanoparticles. Environmental Science and
Pollution Research, 25(1), pp. 33820-33826.

Kalam, S., Das, S. N., Basu, A. & Podile, A. R., 2017. Population densities of
indigenous Acidobacteria change in the presence of plant growth promoting
rhizobacteria (PGPR) in rhizosphere. Journal of Basic Microbiology, 57(5), pp.
376–385.

Kuan, K. B., Othman, R., Abdul Rahim, K. & Shamsuddin, Z. H., 2016. Plant Growth-
Promoting Rhizobacteria Inoculation to Enhance Vegetative Growth, Nitrogen
Fixation and Nitrogen Remobilisation of Maize under Greenhouse Conditions.
PLOS ONE, 11(3), pp. 1-19.

Kundan, R., Pant, G., Jadon, N. & Agrawal, P. K., 2015. Plant Growth Promoting
Rhizobacteria: Mechanism and Current Prospective. Journal of Fertilizers &
Pesticides, 6(2), pp. 1-9.

Maeier, R. M., Pepper, I. L. & Gerba, C. P., 2009. Environmental Microbiology. New
York: Academic Press.

Maryanti, D. 2006. Isolasi dan Uji Kemampuan Bakteri Pelarut Fosfat dari Rhizosfir
 Tanaman Pangan dan Semak. Skripsi. Fakultas Pertanian Universitas Andalas

Mwajita, M., Murage, H., Tani, A. & Kahangi, E. M., 2013. Evaluation of rhizosphere,
rhizoplane and phyllosphere bacteria and fungi isolated from rice in Kenya for
plant growth promoters. SpringerPlus, 2(1), pp. 1-9.

Nutaratat, P., Monprasit, A. & Srisuk, N., 2017. High-yield production of indole-3-
acetic acid by Enterobacter sp. DMKU-RP206, a rice phyllosphere bacterium
that possesses plant growth-promoting traits. Biotechnology, 7(5), pp. 1-15.
Okon, Y., Albrecht, S.L. & Burris, R.H., 1977. Methods for growing Spirillum
lipoferum and for counting it in pure culture and in association with plants.
Appl Environ Microbiol, 33(1), pp. 85-88.
Permatasari, A. & Nurhidayati, T. 2014. Pengaruh Inokulan Bakteri Penambat
Nitrogen, Bakteri Pelarut Fosfat dan Mikoriza Asal Desa Condro, Lumajang,
Jawa Timur terhadap Pertumbuhan Tanaman Cabai Rawit. Jurnal Sains dan
Seni Pomits, 3(2), pp. 2337-3520.

Sari, D. R., 2015. Isolasi Dan Identifikasi Bakteri Tanah Yang Terdapat Di Sekitar
Perakaran Tanaman. Bio Site, 1(1), pp. 21-27.
Suin, N. M., 1997. Ekologi Hewan Tanah. Jakarta: Penerbit Bumi Aksara.

Sutedjo, M. M., Kartasapoetra. & Sastroadmodjo., 1991. Mikrobiologi Tanah. Jakarta:

PT Rineka Cipta.