CHAPTER global carbon cycle (Chavez et al., 2015).

 

 

CHAPTER 5

 

 

DISCUSSION

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Soil
is a complex biological system that give a major function for the living
things.  Fungi in the soil can be used as
bioindicator for biodiversity conservation (Barbosa
et al., 2016) and fungi are crucial as a component
of soil microbiota depending on the soil depth and nutrient condition as it can
constitute more in the soil rather than the bacteria. Fungi help in
decomposition of plant structural polymers such as cellulose, hemicellulose and
lignin that can lead to maintenance of the global carbon cycle (Chavez
et al., 2015). Fungi such as Aspergillus and Penicillium species
were the most abundant filamentous fungi, thus, most of the fungi isolated from
the soil at the Block A and behind DK Delta in the Faculty of Applied Sciences,
UiTM Shah Alam are these genera.

 

In
this research, four fungal cultures were isolated from block A and another
seven fungal cultures was isolated from behind DK Delta by serial dilution
technique. This technique was done to reduce the cell concentration of the
fungi systematically so that the number of colonies of the fungi not too
crowded in one plate (Sanders, 2012).  The fungi were then sub-cultured and purified
to reduce any contamination that can cause a major problem in the
identification methods. The isolated fungi were identified by classical way
which is by using morphological identification and molecular identification.
Even though, molecular identification was able to identify rapidly and more
complete, the morphological identification remains as one of the methods that
mostly used for identification of fungi. This method also can helps the researchers
to better understand fungal growth and their diversity (Gautam and Bhadauria, 2012).

 

In
the macromorphological identification, the isolated fungi were grown in different
types of media which are Czapek Dox Agar (CDA), Malt Extract Agar (MEA), and
Potato Dextrose agar (PDA) so as to differentiate the colony morphology, form
and elevation, and the reverse and surface colour of the fungi (Kadhim and Al-Hussaini, 2015). Other than that,
these different media used for macromorphological can influence the
pigmentation and sporulation, vegetative growth and colony morphology depending
to the composition of the media (Stanly
Pradeep et al., 2013). Physical and
chemical factors can also differentiate effects in characterisation of the fungi
by using these several types of media the sporulation and mycelial growth on
artificial media are crucial in biological characteristics (Chaudhuri
et al., 2017). Most of isolated fungi in this
study grown on CDA, MEA and PDA showed the various colours colony such as cream,
dark green, yellow, and white in the colony morphology and reverse and surface
colour. Most of the isolated fungi showed irregular flat and circular flat forms.

 

Micromorphology
characteristics within a species are more stable and informative o be compared
with the colony appearance. The ornamentation of ascospores and their size are
the major informative phenotypic characteristic for species recognition (Chen
et al., 2017). In the micromorphological
characteristic, the suspected fungi were observed under 1000x magnification
with immersion oil after grown them in MEA in about seven days. MEA was used in
this micromorphological study due to the stripes on MEA are usually more
consistent and distinctly rough than other media (Samson and Pitt, 2000). In this
magnification, the structure of the Aspergillus
and Penicillium species were
observed based on their structure of the phialide, vesicle, conidia,
conidiophore, hyphae, metulae, and rami. Aspergillus
species is characterised by having a spore bearing structure known as
conidial head, basal foot or hyphae and aseptate. They also have a conidiophore
that terminated into the vesicle. The vesicles of Aspergillus species have one or two layer of same cells and conidial
heads that is asexually formed spore produced by the phialides (Nyongesa
et al., 2015). Seven samples of suspected Penicillium species were identified
based on the basic micromorphological characteristics such as conidia,
conidiophore, phialides, rami, metuale, and hyphae that are typical element for
Penicillium species. The comparison
in micromorphology of Penicillium species  was based on their branching of conidiophore,
and the shape of the ornamentation of the conidia (Houbraken
et al., 2010).

 

The
260nm/280nm of QuickDrop Spectrophotometer reading of the eleven samples detected
and the positive controls, most of the samples were less than 1.8 which indicating
that were presence of protein phenol or others. Other than that, only one
sample showed the results in the range of 1.8 to 2.0 which indicated the sample
has pure DNA (Tan and Yiap, 2009). In addition,
there are no sample that showed the reading that is more than two which
indicated presence of RNA. The reading was repeated three times to obtain the
best result. However, even though most of the samples was not in the range of
the pure DNA, the samples were used for further analysis since the value of the
purities is close to the range of pure DNA.

 

Even
though identification of Aspergillus and
Penicillium species was done by using
morphology (phenotypic characters) which also known as classical methods, this
method was commonly being misleading due to the hybridization, cryptic
speciation, and convergent evolution. This method also did not provide an
precise grouping within the evolutionary framework typically at species level (Raja
et al., 2017). DNA barcoding is a molecular
identification also known as modern procedure by using ITS region was used to
alignment the gene sequence and to identify the suspected Aspergillus and Penicillium species.
Internal transcribed space (ITS) region in identification of various types of
fungi has high success rate which give the clearest define barcode gap between
inter and intra-specification variation. DNA barcoding used standardise range between
500 to 800 bp sequences to identify species of fungi by using the primer that
can be used to a wide range of taxonomic groups (Schoch
et al., 2012). Based our study, the PCR product of
all fungi samples in 1.4% agarose gel, showed that all bands were in the range
of 500 to 700 bp which is in the same range of the Aspergillus and Penicillium species
and matched with the range size of the primers used (Henry
et al., 2000; Demirel
et al., 2013).

 

The
PCR purification was done after the size of PCR product of the sample is the
same as the expected size for Aspergillus
and Penicillium species. The purified
PCR also in the same size range with these species. Two samples of purified PCR
products (F3 and F11) then was send to the third party for the sequencing
because they have the highest concentration of DNA and highest DNA purity. The
sequencing result for both samples is shown in figure 4.5, 4.6, 4.7, 4.8, 4.9,
4.10, 4.11, and 4.12. Based on the sequencing result after being cleaned, the
size of contig for F3 and F11 sample are 662 bp and 577 bp. However, both
samples are not in Aspergillus or Penicillium species but Scolecobasidium sp. and Microsphaeropsis arundinis. Figure 4.7 and 4.8 showed the BLAST results for F3.
The results showed the description of the alignment of the contig sequences.
The identity of both accession number for F3 is KC790476.1 and KJ942584.1 were
84% indicate that the query’s length was 84% identical to the first hit in the
nucleotide-to-nucleotide alignment. The identity is not totally similar because
the DNA sequences that was sent by the third-party showed there are multiple
peaks throughout the sequences due to the sample quality. GenBank Graphics of
the accession number KC790476.1 was with 36 gaps. The species that was
identified in the F3 sample is Scolecobasidium
sp. The identity of accession number
for F11 is KJ774054.1 was 100% identical to the first hit in the
nucleotide-to-nucleotide alignment. The identity is similar because the DNA
sequences that was sent by the third-party showed cleans peaks with low noise
peaks throughout the sequences. This sequence showed that the sample quality
for that purified PCR is good. The species that was identified in the F11
sample is Microsphaeropsis arundinis.

 

Even though both species are not Aspergillus and Penicillium species,
they shared some of the characteristics of these two fungi. Scolecobasidium sp. is commonly found in soil and water and is a thermophilic
dematiaceous fungus (Pundhir
et al., 2017)
and an anamorphic ascomycetes (Renker
et al., 2005).
Microsphaeropsis arundinis is a
dematiaceous mould that inhabits terrestrial plant host and in ubiquitous in
soil and fresh water (Pendle
et al., 2004). Scolecobasidium sp. and Microsphaeropsis arundinis are
deuteromycetes also known as imperfect fungi because they are artificial group
of fungi. Deuteromycetes are an artificial assemble that join ascomycetes and basidiomycetes
asexual stages because there is no sexual reproductive structure that have been
discovered yet (Gams and Seifert, 2001; Lockey and Ledford, 2014).  Aspergillus and Penicillium species belong to the phyla Zygomycota, Ascomycota and
Deuteromycetes. These three phyla have different ways to reproduce. Ascomycota
have a mycelium barrier that can reproduce by producing a spore bags, while
zygomycota have the ability to multiply vegetative and produce a spore whereas deuteromycetes
only reproduce vegetatively by conidia (?ukiewicz-Sobczak,
2013).
The molecular approach has confirmed deuteromycetes as a lineage to Ascomycetes
or Basidiomycetes (Pringle, 2013), so it is not possible if Scolecobasidium sp. and Microsphaeropsis arundinis has some similarity
with the Aspergillus and Penicillium species.

 

In the antimicrobial activity of the isolated fungi
towards pathogenic bacteria only one sample showed the inhibition zone and it
was towards the pathogenic bacteria S.
epidermidis. It is Gram-positive bacteria. F4 fungi sample was suspected to
be Penicillium species. This species
can produce penicillin that can slow down the peptidoglycan of Gram-positive
bacteria (Lobanovska and Pilla, 2017). In general, about 70% fungal strains can shows the
antimicrobial properties against pathogenic bacteria and fungi that slow down
the growth bacteria was higher than showing the antibacterial properties. Other
than that, most of antimicrobial properties was frequently against
Gram-positive bacteria than Gram-negative bacteria. This is due to the
complexity of the cell wall of the Gram-negative bacteria than the
Gram-positive bacteria and Gram-negative bacteria can provide additional degree
of protection against antimicrobial properties that aim the cell wall (Zainuddin
et al., 2010).
The antimicrobial properties of these fungi can also influence by their habitat
and the lack of significant difference in the size of inhibition can exist due to
the same biochemical pathways used by the bacteria (Waithaka
et al., 2017).