Antifungal Activity of Actinomycete Isolates Against Plant Pathogens

This study aims to determine the antifungal activity of actinomycete isolates from soil samples against plant pathogens. The experiment was performed using experimental design, and the results will contribute to the development of novel antimicrobial compounds for combating antibiotic resistance. The study was conducted at the University of Gondar, and the materials and methods used in the research are described.

i
UNIVERSITYOF GONDAR
INSTITUTE OF BIOTECHNOLOGY
DEPARTEMENT OF BIOTECHNOLOGY
ANTI-FUNGAL ACTIVITY OF ACTINOMYCETE ISOLATES AGAINST KNOWN PLANT
PATHOGENS
Name of student ID
1. Melkamu Debash …………………………………… 02246/12
2. Netsanet Mulat………………………………………..01071/12
3. Simon Ferede ………………………………………….02250/12
4 .Tesfaye Mengistu …………………………………....00590/12
6. Zeritu Elias………………………………………….…... 01629/12
Principal advisor: Yalemwork Jemberu (MSc)
A senior project submitted to institute of Biotechnology Department of
Biotechnology, University of Gondar for partial fulfillment of the requirements for
Bachelor of Science degree (B.Sc.) in Biotechnology
August 2023
Gondar, Ethiopia

ii
Acknowledgments
First of all we are extremely greateful to thank the almighty God for helping us in every single
thing. Then we would like to thank University of Gondar for providing us the opportunity to
perform this research.
Also we would like to express our deepest gratitude to our advisor miss Yalemwork Jemberu for
her helpful advice, comments and guidance throughout this research.
Finally, we would like to thank our Microbiology laboratory assistants for their valuable support
and encouragements during conduction of the research.

iii
Declaration

iv
Approval sheet

v
Abbreviation and acronyms
⁰C........................................ Degree Celsius
E ……………………………………….East
g........................................ Gram
G/L…………………………………….Gram per liter
H₂0₂ …………………………………..Hydrogen peroxide
KKM.................................... Ken knight media
Long …………………………………..Longitude
Lat....................................... Latitude
Ml……………………………………….Milliliter
N …………………………………………North

vi
Contents
Acknowledgments ii
Abbreviation and acronyms iii
ABSTRACT - 1 -
Introduction - 2 -
1.1 Statement of problem - 3 -
1.2 Significance of the study - 4 -
2 Objective of the study - 5 -
2.1 General objective - 5 -
2.2 Specific objectives - 5 -
3 Materials and methods - 6 -
3.1 study area - 6 -
3.2 Study period - 6 -
3.3 Study design - 6 -
3.4 Materials - 6 -
3.5 Methods Soil Samples collection - 6 -
3.6 Laboratory methods - 6 -
3.6.1 Isolation of Pure Culture of Actinomycetes............................................................................- 6 -
3.6.2 Color Grouping of the Isolates................................................................................................- 7 -
3.6.3 Gram staining.........................................................................................................................- 7 -
3.6.4 Catalase test...........................................................................................................................- 7 -
3.6.5 L-asparaginase production.....................................................................................................- 7 -
3.6.6 Amylase Production Test........................................................................................................- 8 -
3.6.7 Test Organisms.......................................................................................................................- 8 -
6.3.8 Characterization of Inhibitory activity of actinomycetes......................................................- 8 -
3.7 Data analysis - 8 -
Dissemination and utilization of the result - 8 -
4. Work plan - 9 -
References - 10 -

vii
List of tables
Table 1; Composition of KKM (ken knight media) (g/L)............................................7

1
ABSTRACT
Introduction: The development of resistance to multiple drugs is a major problem in the
treatment of infections by pathogenic microorganisms. Drug resistance is a rising concern in the
treatment of infectious diseases and necessitates the need for discovery of novel, potent
antimicrobial compounds to combat antibiotic resistance. Since natural environment remains a
potential source of novel antimicrobial products, Actinomycetes have been and remain the most
fruitful source of microorganisms for all types of bioactive metabolites including agro activate
type.
Objective: This preliminary study was performed to determine antagonistic activity of
actinomycetes isolates from different soil samples from Gondar, Ethiopia against plant
pathogens.
Method: The experiment study was performed using experimental design. Soil samples was
collected from three distinct areas around the campus from a depth of between 3cm - 5cm from
the surface of the soil profile. The actinomycetes was isolated from the mixed culture by using
serial dilution and streak plate method and the final result was seen by using cross-streak assay
method.
Result:
Conclusion:
.
Key words: Actinomycetes, Antibiotics, Incubation, Inoculation, Inhibition zone, Spread, Streak

2
Introduction
Actinomycetes are soil microorganisms, which have common characteristics to bacteria and
fungi. Unlike slimy distinct colonies of true bacteria, which grow quickly, actinomycetes colonies
grow slowly show powdery consistency and sticky firmly on the agar surface(1). Actinomycetes
differ from fungi in their composition of cell wall. They don’t have chitin and cellulose. The
number of actinomycetes increases in the presence of decomposing organic matter. The
commonest genera of actinomycete found in soil are Streptomyces, Nocordia and
Micromonospora(2).
Actinomycetes are gram-positive bacteria with high guanine+cytosine content of over 55% in
their DNA, which have been recognized as sources of several secondary metabolites,
antibiotics, and bioactive compounds that affect microbial growth Actinomycetes have
filamentous nature, branching pattern, and conidia formation, which are similar to those of
fungi. For this reason, they are also known as ray fungi (3).Actinomycetes occur in great
abundance in the soil and have a great role in the management of microbial stability with the
production of antibiotic substances (4).
Actinomycetes are the most widely distributed group of microorganisms in nature which
primarily inhabit the soil. They have been noted to serve as rich reservoirs of medicinal
antibiotics and are therefore extremely relevant to scientists, pharmaceutical industries and
agricultural industries. They have provided many important bioactive compounds of high
commercial value and continue to be routinely screened for new bioactive compounds(5).
Approximately two thirds of naturally occurring antibiotics have been isolated from
actinomycetes(6).
The richness and diversity of actinomycetes present in any specific soil, is greatly influenced by
the soil type, geographical location, cultivation and organic matter among other factors.
Numerous studies have been done by scientists to isolate actinomycetes, as sources of
antibiotics. However, because actinomycetes occur widely in nature, only a small percentage
of the globe and a small proportion of actinomycetes species have been screened(7).

3
Statement of problem
Plant pathogenic fungi causes significant economic loses and are a major threat for the food
security. The use of chemical fungicides to control plant pathogenic fungi is often costly and
environmentally damaging. The development of resistance to chemical antifungal compounds is
the major concern in agriculture. Repeated use of the same chemical antifungal compounds can
lead to the selection of fungal strains that are resistant to the compounds. This can reduce the
effectiveness of compound and lead to the treatment failure. Moreover, the development of
resistance can also cross over to other related compounds, thereby reducing the number of
available antifungal agents for disease control.
Environmental contamination is another impact of using chemical antifungal compounds. These
compounds can persist in the soil and water and can potentially harm non target organisms
such as beneficial insects, birds and mammals. Long-term exposure to these compounds can
lead to the accumulation of toxic residues in the environment, which can have far-reaching
ecological consequences.
These chemicals can also poses risks to human health. Exposure to these compounds can cause
acute and chronic toxicity, skin irritation and respiratory problems. Moreover, farmers and
farmworkers who handle these compounds are at a higher risk of exposure, which can have
long-term health consequences.

4
1.2 Significance of the study
In worldwide, different studies were conducted on potential importance of actinomycete
isolates against different plant pathogens a showed mixed result. Some isolates were specific
and others had antimicrobial effect on different plant pathogens. Actinomycetes are known to
produce biologically active compounds including antibiotics and antifungal agents. Using
actinomycetes for the purpose of antimicrobial activity can replace chemical compounds that
can be used for plant pathogens in order to reduce environmental contamination, destruction
of important insects,birds and mammals and to maintain ecosystem health. So this study will
determine the antimicrobial effect of actonomyectes from soil sample collected from Gondar
city and university of Gondar. Also it will be a bench mark study for future researchers and can
be taken as input for educational purpose.

5
2 Objective of the study
2.1 General objective
To determine antifungal activity of actinomycetes isolates against selected fungal
pathogens
2.2 Specific objectives
To isolate actinomycetes from different soil Samples.
To characterize isolated actinomycetes morphologically and biochemically
To examine the antifungal activity of characterized actinomycetes to different fungal
plant pathogens.
To identify the most effective actinomycetes isolate.

6
3 Materials and methods
3.1 study area
The study was conducted at University of Gondar, Central Gondar of Amhara region which is
located at about 749 km away from Addis Ababa, the capital city of Ethiopia. It is particularly
located at latitude12°36'N and longitude 37°28E with elevation of 2133 m above sea level.
3.2 Study period
This was conducted from June, 2023 to July, 2023 in Microbiology Laboratory, Institute of
Biotechnology, Gondar University, and Gondar city
3.3 Study design
The experiment was performed using experimental design, such as extraction of antibiotic from
actinomycete and antibacterial testing.
Materials Chemicals
Sensitive balance Agar-agar
Measuring cylinder Glucose
Flask KH2PO4
Aluminum foil MgSO4
Autoclave NaCl
Laminar Air Flow hood 70% alcohol
Inoculating needle Hydrogen peroxide
Petridishes Safranin
Bunsen burner Gram’s iodine
Spreader (L-rod) Distilled water
Incubator Crystal violate
Soil sample
Bacterial strains
Microscope
Ruler
Cotton
Spatula
Test tube
Hot plate
Glove
Micropipete
Labler

7
3.5 Methods Soil Samples collection
Three different soil Samples was collected from various depth of the earth surface, ranging
from layers just beneath the upper surface to between 3cm- 5cm depth. They was collected in
the sterile small plastic tubes and properly labeled with the date of collection and places of
collection like.
3.6 Laboratory methods
3.6.1 Isolation of Pure Culture of Actinomycetes
From each soil sample, 5gram of soil was suspended in 45mL of distilled water blanks, and
successive serial dilutions was made by transferring 1mL of aliquots to 1st test tube containing 9
ml of distilled water and in this way dilutions up to 10‾³ was prepared. Each time the contents
were shaken with hand to form uniform suspension. An aliquot of 0.1 mL of each dilution was
taken and spread evenly over the surface of ken knight medium (KKM) on 16 cm petri dishes.
Plates will be incubated at 37°C and monitored for 5 day
Table 1; Composition of KKM (kenknight media) (g/L)
Number Component g/L
1 Glucose 0.5
2 KH2Po4 0.05
3 NaCl 0.05
4 MgSo4 0.05
5 KNO3 0.5
6 Agar-Agar 10
3.6.2 Color Grouping of the Isolates
The color of the aerial mycelia and pigment production by the isolates was determined on KKM
plates after 5 days of incubation at 37°C. The color of the substrate mycelia and those of the
soluble pigment was determined and ten different actinomycetes will be isolated based on their
color.

8
3.6.3 Gram staining
Smear of the given all culture was prepared by inoculating needle. Dried the smear and then
fixed the smear by passing flames 2-3 times on lower slides. The smear was stained with crystal
violet for one minute. After one minute, the smear was washed with tap water to remove
excess stain. Then the smear was flooded with alcohol which is a decolonizing agent for one
minute and again the smear was washed with tap water. Finally the smear was counter stained
with safrainin for one minute and washed with tap water. Each smear was blotted, dried and
focused under 40× objective.
3.6.4 Catalase test
Catalase is an enzyme that decomposes H₂O₂ in to water and oxygen. Excluding streptococci
most aerobic and facultative bacteria possess catalase activity. Three different bacterial
samples were taken from previously prepared bacterial sub culture. The samples were taken
and smears were formed in each slide by using inoculating needle. Then two drops of H2O2
added to each slide. After few minutes bubles were not formed.
3.6.5 L-asparaginase production
A mineral base agar containing glucose as carbon source and L-asparagine as nitrogen source
with phenol red as indicator system will be used. The liberated ammonia after L-asparagine
break down leads to change in color from yellow to pink around the colony. Pink coloration
around the colony will be noted (13)
3.6.6 Amylase Production Test
Starch Agar Medium containing soluble starch as carbon source will be prepared as per 2012
Starch Agar Protocol(14).
Test Procedure
Pick a few colonies of the test organism using a sterile swab or loop.
Streak a starch plate in the form of a line across the width of the plate. Several cultures
can be tested on a single agar plate, each represented by a line or the plate may be
divided into four quadrants for this purpose.
Incubate plate at 37 °C for 48 hours.
Add 2-3 drops of 10% iodine solution directly onto the edge of colonies. Wait 10-15
minutes and record the results. Read plates immediately after the addition of iodine, as
the blue color fades
3.6.7 Test Organisms
The test organisms will be used to test the antibiotic activity of the isolates one from The
Laboratory and one from infected cabbage around the campus. Those test Organisms are
xantomaycete and cabbage black root which are bacterial pathogens and Aspergiles niger and
Fusariem oxyphora which are fungal pathogens.

9
6.3.8 Characterization of Inhibitory activity of actinomycetes
Secondary screening of the actinomycetes will be carried out to understand their inhibitory
profile against selected bacterial and fungal plant pathogens following the modified cross-
streak assay of Lemos et al. (1985). Four plant pathogens xantomycete , fusarium oxyphora ,
cabbage black root and aspergilus niger will be used in the assay. For streaking, the inoculation
loop will be straightened out and bent into L-shape so as to get a streak of 8-10 mm width.
After the incubation, young culture of each of the selected plant test pathogens will be streaked
perpendicular to the central streak of the actinomycetes culture, leaving a gap of 2 mm from
the central streak of the actinomycete culture. After incubation at 37⁰C for 24h, the inhibitory
activity of actinomycete isolates will be indicated by the clear zone near the central streak, due
to growth inhibition of test pathogens and this clear zone will be measured in millimeters (mm)
and recorded. The kenknignt agar plates with only the test pathogens served as control.
3.7 Data analysis
Data will be entered in to Microsoft excel spread sheet and analyzed using SPSS version 21
software. The zone of inhibition will be expressed as mean ± SD. Statistical analysis will be also
undertaken by analysis of variance (one way ANOVA) Duncan's multiple range Result will be
considered statistically significant at P<0.05. Results will be described in terms of percentages,
numbers, means, medians and will be displayed on tables, bar graphs and pie charts.
Dissemination and utilization of the result
The study result will be presented to department of biotechnology. Then it will be disseminated
to UOG libraries, different biotechnology institutes and interested governmental and
nongovernmental organization. Publication to national and international scientific journals will
be carried out.

10
6 References
1. Chaudhary HS, Yadav J, Shrivastava AR, Singh S, Singh AK, Gopalan N. Antibacterial
activity of actinomycetes isolated from different soil samples of Sheopur (A city of central
India). Journal of advanced pharmaceutical technology & research. 2013;4(2):118-23.
2. C. S. Cummins1 HH. Studies on the Cell-Wall Composition and Taxonomy of
Actinomycetales and Related Groups.
3. Ghai R, Mehrshad M, Mizuno CM, Rodriguez-Valera F. Metagenomic recovery of phage
genomes of uncultured freshwater actinobacteria. The ISME Journal. 2017;11(1):304-8.
4. Raaijmakers JM, Vlami M, de Souza JT. Antibiotic production by bacterial biocontrol
agents. Antonie van Leeuwenhoek. 2002;81(1):537-47.
5. Bhatti AA, Haq S, Bhat RA. Actinomycetes benefaction role in soil and plant health.
Microbial pathogenesis. 2017;111:458-67.

11
6. Kumar N, Singh R, Mishra S, Singh A, Pachouri UC. Isolation and screening of soil
Actinomycetes as source of antibiotics active against bacteria. International Journal of
Microbiology Research. 2010;2:12-6.
7. Sapkota A, Thapa A, Budhathoki A, Sainju M, Shrestha P, Aryal S. Isolation,
Characterization, and Screening of Antimicrobial-Producing Actinomycetes from Soil Samples.
International Journal of Microbiology. 2020;2020:2716584.
8. Mahajan GB, Balachandran L. Antibacterial agents from actinomycetes - a review.
Frontiers in bioscience (Elite edition). 2012;4(1):240-53.
9. Catalano A, Iacopetta D, Ceramella J, Scumaci D, Giuzio F, Saturnino C, et al. Multidrug
Resistance (MDR): A Widespread Phenomenon in Pharmacological Therapies. Molecules (Basel,
Switzerland). 2022;27(3).
10. Mancuso G, Midiri A, Gerace E, Biondo C. Bacterial Antibiotic Resistance: The Most
Critical Pathogens. Pathogens (Basel, Switzerland). 2021;10(10).
11. Selim MSM, Abdelhamid SA, Mohamed SS. Secondary metabolites and biodiversity of
actinomycetes. Journal, genetic engineering & biotechnology. 2021;19(1):72.
12. El Karkouri A, Assou SA, El Hassouni M. Isolation and screening of actinomycetes
producing antimicrobial substances from an extreme Moroccan biotope. The Pan African
medical journal. 2019;33:329.
13. Doriya K, Kumar DS. Isolation and screening of l-asparaginase free of glutaminase and
urease from fungal sp. 3 Biotech. 2016;6(2):239.
14. Pepper I, Gerba C, Gentry T. Preface. In: Pepper IL, Gerba CP, Gentry TJ, editors.
Environmental Microbiology (Third Edition). San Diego: Academic Press; 2015. p. xvii.