Cultural Characters of Soil Fungi and their Antimicrobial Activity

Candidate: Ma Han Wai Zin (Roll No. 2 Mahar Yakha-4)

Specialization: Microbiology
Supervisor: Dr Khin Ohn Myint, Associate Professor, Dept. of Botany, Dagon
University

ABSTRACT

In this study, the two soil samples were collected from the two different depths (20 cm and 40
cm) near Bago River in (94) Ward, Dagon Seikkan Township, Yangon Region. The twelve
soil fungi were isolated from the collected samples on three different media by the serial
dilution method of Booth, et al. 2006. Three different media were Nutrient Agar Medium
(NA), Sucrose Yeast Extract Agar Medium (SY), Lactose Yeast Extract Agar Medium (LY)
according to Atlas, 1993. Five soil fungal strains were isolated from 20 cm depth and seven
soil fungal strains were isolated from 40 cm depth. The morphological and microscopical
characters of all fungal strains will be carried out at Microbiological Laboratory, Department
of Botany, Dagon University according to Dubey and Maheswari, 2014; Barnett, 1992 and
other available literatures. The colony appearance, margins, elevation, surface color and
reverse color of all fungal strains will be investigated. Antimicrobial activity of all fungal
strains will be evaluated by paper disc diffusion method with pathogenic test organisms such
as Agrobacterium tumefaciens, Bacillus subtilis, Candida albicans, Escherichia coli,
Malassezia furfur, etc by the method of Davis and Stout, 1971; Strobel and Sullivan, 1999. It
is hoped that some of isolated strains will be exhibited antimicrobial activity on some test
organisms.
INTRODUCTION
Soil microorganisms are classified into bacteria, actinomycetes, fungi, algae and
protozoa. Each of these groups has characterstic functions in soils (Mendes et al, 2013). Soils
contain about about 8 to 15 tons of bacteria, fungi, protozoa, nematodes, earthworms, and
arthropods (Brady, Weil, 2012). Ingham (2009, pg. 18) states that “Bacteria are tiny one-
celled organisms generally 4/100,000 of an inch wide (1 µm). A teaspoon of productive soil
generally contains between 100 million and 1 billion bacteria. That is as much mass as two
cows per acre. A ton of microscopic bacteria may be active in each acre.” While bacteria may
be small, they make up both the largest number and biomass (weight) of any soil
microorganism. Bacteria are similar in size to clay soil particles (<.2 µm) and silt soil
particles (2-50 µm). Bacteria are so simple in structure that they have often been called a bag
of enzymes and/or soluble bags of fertilizer (Dick, R., 2009).
Soil fungi are microscopic plant-like cells that grow in long threadlike structures or
hyphae that make a mass called mycelium. The mycelium absorbs nutrients from the roots it
has colonised, surface organic matter or the soil. It produces special hyphae that create the
reproductive spores. Some fungi are single celled (eg yeast). Fungi have many different
structures but they can act in similar ways and thus are not as plant specific in their needs as
some soil bacteria such as Rhizobia. Fungi perform important functions within the soil in
relation to nutrient cycling, disease suppression and water dynamics, all of which help plants
become healthier and more vigorous.
Fungi are found wherever there is hard, carbon-rich woody organic matter. This could
be dead rotting trees in a forest, leaf litter on the surface of orchard soils, or plant roots. They
can survive in the soil for long periods even through periods of water deficit by living in dead
plant roots and/or as spores or fragments of hyphae.
Fungi are an important part of the microbial ecology. The majority of fungi decompose
the lignin and the hard-to-digest soil organic matter, but some fungi consume simple sugars.
Fungi dominate in low pH or slightly acidic soils where soils tend to be undisturbed (Lavelle
& Spain, 2005). Fungi break down the organic residues so that many different types of
microbes can start to decompose and process the residues into usable products.
Fungi prefer slightly acidic conditions, low disturbance soils, perennial plants, internal
nutrient sources directly from the plant, and highly stable forms of organic residues with high
carbon to nitrogen (C:N) values and slower recycling time. Bacteria dominate in highly
disturbed ecosystems with fast nutrient recycling, low C:N values, prefer annual plants, and
external nutrient additions outside the plant. (Lavelle & Spain, 2005). fungi are multi-celled
organisms that grow rapidly and in great lengths in the soil (feet or meters). This allows fungi
to bridge gaps in the soil so as to transport nutrients relatively far distances back to the plants
(Lowenfels &Lewis, 2006).
There are at least 70,000 different species of fungi identified but it is estimated that there
may be 1.5 million species worldwide (Hawksworth, 1991, 2001). Genetically, fungi evolved
a billion years ago and are closely related to plants and animals. Membrane bound organelles
present in each cell are similar to those found in insects, plants, and animals. Fungi have
80percent or more of the same genes as humans (Dick, R., 2009).
There are four major groups of soil fungus: Zygomycota, Ascomycota, Basidiomycota,
and Deuteromycota. Zygomycota are less than 1,000 species and are mostly common bread
molds. Ascomycetes have about 30,000 species and are mostly yeasts used in baking.
Basidiomycetes include most mushrooms, toadstools, and puffballs while Deuteromycota
include the lichens and the mycorrhizal fungus (Biological Diversity: Fungus, n. d.; Lavelle
& Spain, 2005). Fungi are classified as heterotrophs so the carbon source originates from the
decomposition of organic compounds or residues (Sylvia et al., 2005).
Fungi benefit most plants by suppressing plant root diseases and fungi promote
healthier plants by attacking plant pathogens with fungal enzymes. Fungi also use antagonism
to reduce competition by producing antibodies, which suppress other microorganisms from
growing. They produce many vitamins which promote plant growth. By competing with other
fungus for nutrients, beneficial fungi prevent pathogenic and disease causing organisms from
getting established. Beneficial fungi along with some bacteria may also form protective webs
and nets around roots and even leaves to protect the host plant (Lowenfels & Lewis, 2006;
Sylvia et al., 2005). The fungus Trichoderma protects plant roots from attack by harmful
microorganisms. Fungi also protect plants by supplying a protective sheath to supply both
water and phosphorus to the plant roots during droughts (Magdoff & Van Es, 2009).

MATERIALS AND METHODS

Collection of Soil Samples
The two soil samples were collected from the two different depths near Bago River in
Dagon Seikkan Township, Ward (94), Yangon Region (Figure 1). The soil samples (10g for
each sample) were taken between twenty centimeter depth and fourty centimeter depth and
put them into the clean plastic bags. The pH of each sample was measured in Figure (2). Soil
sample were record by using the Global Positioning System (GPS) Table (1).

Table (1) Location of the collected soil sample

Collection area Location Depth

Isolation of Soil Fungi

Serial dilutions of soil, plating and streaking techniques described by Salle, 1948 and
Collins, 1965 were used for the isolation of microbes.
An appropriate amount (1g) of soil was introduced into a conical flask containing 99ml of
distilled water to make a soil-water dilution ratio of 1:100. The flask was then shaken for
about 30 minutes in order to make the soil particles free from each other. This solution was
then serially diluted into 10 to 10 dilution in separate test tubes. 1ml each of the above
dilutions was separately transferred into sterile petridishes under aseptic condition. A sterile
pipette was used for each transfer. 0.5ml of 10 diluted tube was added on each agar medium,
then the plate was gently shaken to get the uniform solution on the medium. These plates
were incubated at room temperature for 2 weeks.

Composition of culture media (Atlase, 1993)

Medium 1
Nutrient Agar Medium (NA)
Nutrient Agar 3.5 g
Distilled Water 100 ml
Agar 1.0 g

Medium 2
Sucrose/Yeast Extract Medium (SY)
Sucrose 1.0 g
Yeast 0.3 g
Distilled Water 100ml
Agar 2.5 g

Medium 3
Lactose/Yeast Extract Medium (LY)
Lactose
Yeast
Distilled Water 100 ml
Agar 2.5 g

Morphological Characters of Isolated Fungi

The morphological and colonial characters such as colony appearance, margin, surface and
reverse colours, and elevation of all isolated strains were recorded as revealed in the
reference of Dubey and Maheswari, 2014.

Microscopical Characters of Isolated Fungi

The microscopical characters of all isolated strains (HAN-1 to HAN-12) were carried out
under light microscope with high magnification at Department of Botany, Dagon University.
The main characters of hyphae, mycelium, sporangiophores, spore, color formation on upper
as well as lower surface were comparatively studied. These are compared to those of fungi
with available literatures such as Barnett, 1992

Antimicrobial Activities of Isolated Fungal Strains

Fermentation of isolated strains
The isolated 12 fungi were inoculated on the three selective culture media at room
temperature for 5 days. All isolated fungi were inoculated in the conical flasks containing
seed medium (SY medium), and incubated at room temperature for 3 days. The twelve
conical flasks (50ml) containing SY fermentation medium (20ml in each flask) were
autoclaved at 121°C for 15 minutes. After 3 days, 1% of seed culture was transferred into
each fermentation flask, and incubated at room temperature under shaken condition. The
fermentation was carried out for 17 days. The fermented culture was then tested for
antimicrobial activity against five test organisms by paper disc diffusion method (Strobel and
Sullivan, 1999)

Sucrose/Yeast Extract Medium (SY)

Sucrose 1.0g
Yeast extract 0.3g
NaCl 0.3g
CaCO3 0.1g
Agar 2.5g
Distilled Water 100ml
PH 6.8

Paper disc diffusion method

After solidification, the paper discs impregnated with fermented broth samples were applied
on the test plates from 4 days to 17 fermentation days. These plates were incubated at 30°C
for 24 hrs. The fermented broth (10 µl) was applied for each disc.
After 24 hrs, clear zones (inhibitory zones) surrounding the test discs were measured. These
zones indicate the presence of the bioactive compounds which inhibit the growth of test
organisms. The plate size was 9.0 cm. The paper disc size was 6.0 mm (Davis and Stout,
1971).

Test organisms
All soil isolate were screened for antimicrobial activity. The five kinds of test organisms were
Agrobacterium tumefaciens, Bacillus subtilis, Candida albicans, Escherichia coli, and
Malassezia furfur in Table 2.