CHAPTER can be found on the top layer

CHAPTER 2LITERATURE REVIEW2.1 Soil MicrobiologySoil is a multi-layered surface that consist of minerals and organic components present in solid, liquid, and gaseous states. The minerals that are present in the soil depends on the action of weathering and erosion on rock.

As example, a broad soil type (sand, slit, and clay) is identified by the size of the particles. The particle surfaces, pore spaces, and plant roots are usually a habitat for microorganism (Baumgardner, 2012). The soil in Malaysia can be divided into two groups.

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The first group is the sedentary soil that forms the interior on various soil types and the second group is the soils of the coastal alluvial plains (Olaniyi et al., 2013). Soil biological activities happen by aerobic unicellular and just a small part of the activities have been done by the anaerobic microorganism which usually located in deep depths and layers. Aerobic microorganisms can be found on the top layer of soil which consisted of humus and organic matter. There are many oxygens presents in the underground level which maximized the microorganism activity which shows the higher number of organism that can established in the underground. So, when the oxygen level were reduced from the upper layers, the activity and the value of microorganism cannot survive in the underground level (Miri et al.

, 2014). Some of the biological activities in soil are consequences from fungi.The Ultisols and Oxisols that contains kaolinite, gibbsite, and hematite in a clay fraction is usually found in Malaysia (Shamshuddin and Kapok, 2010).

These soils are existing under tropical environment with the high temperature and rainfall throughout the year and give the plant nutrients and accumulation of sesquioxides which makes them highly weathered. The silicate minerals in the soil become unstable because of the changes in the chemistry of their environment due to the high temperature in Malaysia. Other than that, soils in Peninsular Malaysia have a pH value in the range of 4 to 5.

The soil pH remain stable in these range because the soil have a greater stability when the potential in them is zero (Shamshuddin and Daud, 2011).Faculty of Applied Sciences Universiti Teknologi MARA Shah Alam is located i Selangor which is 28 kilometres west of the country’s capital Kuala Lumpur. The physical properties of Shah Alam soil was identified based on the composition characteristic, grain size distribution, specific gravity (Gs), and water content. The natural water contents are generally higher for the phyllite soil in the range of 26 – 35%. For the quarzite soil, the phyllite soil is related to the significantly higher clay contents of the soil, therefore the high absorption or retention of soil moisture (Tan and Miasin @ Awang, 2005). The very soft soil found in Shah Alam Expressway is also broadly subdivided into two tin mine tailing (slimes) and marine clays (Raju, 2004). The mine tailings were generally clayey silts with fine sand content of about 15% and this soil can be found in the Kuala Lumpur and the marine clays can be found in the port city of Klang. The specific gravity can show the relative density of the soil solids, and the Gs value of soil solid are higher for the quartzite soil that ranging from 2.

40 to 2.69. the phyllite soils have lower Gs value that ranging in between 2.40 to 2.

52, reflecting differences in soil mineralogy. In the grain size distribution, the residual of soil derived from phyllite and those from the quartzite were compared and the different source of rock is obvious. The residual soil develops from the fine grained phyllite that contain unstable minerals that readily weather into clay minerals and clay size particles therefore they more clay in nature. Fine grained soils dominated by clays in general will yield low compacted densities (Tan and Miasin @ Awang, 2005).2.2 Fungus in SoilMost of microorganisms in the soil play a critical control on ecosystem processes such as nutrient cycling, decomposition, and soil organic matter formation. The habitat scale pattern in the functioning and composition of microorganisms are associated with the biotic, and climatic, and the other factor that can influenced the soil such as temperature and precipitation, plant community composition, and soil carbon, pH, and nitrogen (Waldrop et al.

, 2017). The growth of these microorganisms are based on the soil criteria and may also involve complex life cycles including animal hosts and amoebae. There are various types of soil-related fungal pathogens that can cause major diseases to human and their ability to grow in diverse conditions such as harsh microenvironment can promote pathogenesis which gives infection by direct inoculation or ingestion, through inhalation, and ingestion of contaminated food. Other than that, systemic fungi are usually obtain via inhalation from contaminated soil and can cause death for those with compromised immune system (Baumgardner, 2012).

 2.3 Filamentous FungiFilamentous fungi are a wide-ranging group of the eukaryotic organism with a common characteristic in their nutrition. Fungi are heterotrophic (chemo-organo-heterotrophs) in the environment that are not capable of doing photosynthesis processes and needed organic matter for energy formation and growth. Other than that, fungi can live as saprophytes on dead animals or plants or parasites assimilating tissue of living plants and animals, or their own waste.

Their natural growth in nature is extremely variable and reflected by many methods that have been developed for their isolation and cultivation. A common fungal life cycle involved the formation of threadlike vegetative hyphae which capable of effective adaptation of nutrient and antagonistic growth. Hyphae arise from germinating spore that is conidia that might be uninucleate or multinucleate, haploid or diploid structure. Fungi can be cultured in organic matter, liquids, or soil on a Petri dish that contain a rich medium such as potato dextrose agar (PDA) and malt extract agar that can support the growth of many types of fungi (Nevalainen et al.

, 2014). Aspergillus and Penicillium species are predominant genera of filamentous fungi that are most abundant in the soil and they are prevalent because the genera have a high number of species and are able to survive in a dry environment (Coutinho et al., 2010).2.4 Introduction to Aspergillus speciesAspergillus species is a divergent genus with high social impact and a high economic. These species can be found enormously in diverse habitat and they are also known to spoil food, produce mycotoxins, and usually reported as animal and human pathogens (Samson et al., 2014).2.

4.1 History of Aspergillus speciesAspergillus species are ubiquitous fungal saprophytes that can be found in various ecological niches all around the world (Garcia-Rubio et al., 2017). In 1729, Micheli introduced the name Aspergillus. He named, described, and illustrated the fungus enchanted in his known works.

He also claims that the pattern of the conidial head of the Aspergillus, which the spore heads extend from their central structure and resemble as aspergillum, thus he named the genus as Aspergillus (Prakash and Jha, 2014). A few years later, Haller validating the genus in 1768, and Fries has been authorized the generic name for Aspergillus species (Samson et al., 2014). Aspergillosis was first described in an animal by Mayer in 1815 when he observed the infection in the lungs and air sacs of a jackdaw.

In 1842, the first human case associated with the aspergillosis was reported by Bennett in Edinburgh (Prakash and Jha, 2014) and 184 anamorphic Aspergillus names and 70 associated with teleomorph names (Samson et al., 2014). In the list are mostly based on the morphological of the species concept at that time. However, old names were commonly reintroduced as accepted, distinct species, therefore, the updating list of the species that have been accepted were crucial for the taxonomic and nomenclatural stability of Aspergillus (Samson et al., 2014). Aspergillus species live as septate molds and usually cause of opportunistic mycoses in the immunocompromised host. The genus Aspergillus has been identified over 200 species and around 20 species of them has reported act as causative agents of opportunistic infections which can give a harm to human (Prakash and Jha, 2014) which can cause a variety infections in the skin, eyes, ears, lungs, and many other organs (Shah and Hazen, 2013). 2.

4.2 Morphological of Aspergillus species The morphological form is a crucial part of species concept of Aspergillus. Colony character was used for characterising species include texture, the degree of sporulation, colony growth rates, production of sclerotia or cleistothecia, colours of mycelia, sporulation, soluble pigments, exudates, colony reverses, sclerotia, Hulle-cells and cleistothcia. Aspergillus species can reproduce by using both sexual and asexual reproduction and the microscopic component of these structure also a major thing. Ascospore sizes and morphology are essential to identify the species.

Other than that, there is a certain point that can affect the morphological characters such as the media that were used, inoculation technique and incubation conditions (Zulkifli and Zakaria, 2017). Figure 2.0 shows the structure of Aspergillus species and Figure 2.

1 to 2.8 show various Aspergillus species that has been grown in different media and the structure and characteristic of them under a microscope. Table 2.0 shows the size and observation of different Aspergillus species after 7 days of incubation periods. Figure 2.

1: Aspergillus flavus colonies on MEA (a and c); CYA (b and d); and (e) biseriate conidia heads (Nyongesa et al., 2015).Figure 2.2: Aspergillus parasiticus A colonies on PDA (a and b); CYA (c and d); and (e) conidiophores and uniseriate conidia heads (Nyongesa et al., 2015).Figure 2.

3: Aspergillus parasiticus B colonies on PDA (a and b); MEA (c and d); and (e) uniseriate conidia heads and stipe (Nyongesa et al., 2015). Figure 2.4: Aspergillus tamarii on CYA (a and b); MEA (c and d); and (e and f) biseriate, conidia heads, vesicle, and conidiophores (Nyongesa et al., 2015).Figure 2.5: Aspergillus fumigates colonies on PDA (a and b); MEA (c and d); CYA (e and f); and (g) stipe and short conidia head (Nyongesa et al.

, 2015).Figure 2.6: Aspergillus heteromorphus colonies on MEA (a and b); CYA (c and d); (e) biseriate, conidia head, and vesicle (Nyongesa et al., 2015).Figure 2.7: Aspergillus niger colonies on PDA (a and b); MEA (c and d); CYA (e and f); and (g) biseriate, globose conidia heads and vesicle (Nyongesa et al., 2015).

Figure 2.8: Aspergillus candidius colonies on PDA (a and b); MEA (c and d); CYA (e and f); and (g) conidiophores, conidia heads and conidia (Nyongesa et al., 2015). Species Size (mm) on MEA Stripe Texture Seriation Vesicle shape Vesicle Diameter (?m) Conidia Size (?m) Colour, Texture/Shape Conidia Head/ShapeA.

Parasiticus (A) 15 – 20 Smooth u/b P/G 24 – 30 4 – 5.8 yg/r/G RadiateA. Parasiticus (B) 30 – 60 Rough u/b P/G 19 – 35 3 – 7 g/r/G RadiateA.

tamarii 45 – 55 Rough b G 26 – 43 3 – 5 g/G RadiateA. flavus 50 -55 Rough u/b R 18 – 36 3.5 – 5 yg/r/G RadiateA. fumigatus 24 – 40 Smooth u Spathulate to Clavate 19 – 31 2 – 3 g/r/G S/CA. carbonarius 40 – 60 Smooth B Umbrella Shape 41 – 60 6 – 10 b/r/G S/G to GA. niger 45 – 55 Smooth B S/G 37 – 52 4 – 6 b/r/G GA. japonicus 45 – 50 Smooth U G/ Elliptical 29 – 45 3 – 5 b/s/G RadiateA. rhizopodus 54 – 60 Rough U Clavate 32 – 43 1.

6 – 3.2 g/s/G S/CA. clavatus 35 – 40 Smooth U R/Clavate 32 – 40 3 – 5 g/s/E RadiateA. nidulans 45 – 55 Smooth B Spathulate/P 9 – 16 3 – 4 g/s/S L/CA. candidus 32 – 40 Smooth u SG/G 17 – 30 2 – 3 w/s/G RadiateA. heteromorphus 35 – 45 Rough b S/G 12 – 16 3.

5 –  4 b/r/G S/G to GA. ochaceaus 25 – 30 Rough b/u G 26 – 55 2.5 – 4 o/s/G RadiateA.

ostians 33 – 45 Rough B G 28 – 35 4 – 5 o/s/G GTable 2.0: The size of the colonies after 7 days of incubation; Seriation; u = Uniseriate, b = Biseriate; Vesicle Shape; P = Pyriform, G = Globose, R = Radiate, Conidia Colour, Texture Shape; yg = Yellow Green, g = Green b = Brown, Orange, w = White, Rough, s = Smooth, G = Globose, E = Elliptical, S = Spherical, S/C = Short Columnar, L/C = Long Columnar, S/G = Subglobose (Nyongesa et al., 2015).   2.5 Introduction of Penicillium speciesPenicillium species are common and abundant species in the environment which have good and bad influence on human activities.

Some of these are very important for the industrial field, while some others cause illnesses (Biyik et al., 2016).2.5.1 History of Penicillium speciesPenicillium species are famous as one of the most frequent fungi that can be found in a variety places, from soil to vegetation to air, indoor and outdoor environment and in the diverse food products.

It has a global dispensation and give huge economic impact on our daily life (Visagie et al., 2014). Penicillium is a genus within ascomycetes fungi which extraordinary importance in the natural environment, as well as in the drugs production and food (Li et al., 2015). Other than that, Penicillium species also considered among the frequent genera that can be found indoors and often related with unique food items.

The colonies can also produce millions of conidia at certain times. The abundant sporulation may account for the ease which some species are isolated. Since Link introduced the generic name Penicillium in 1809 that is more than 200 years ago, it is more than 1000 names were introduced in the genus. The generic name Penicillium means ‘brush’ and described the three species that is P. candidum, P.

glaucum, and the generic type P. expansum. Most of the names cannot be identified because the observation is undone by modern characteristic and published invalidly also considered have a synonym name with the other species (Visagie et al., 2014).

All the species that reported until 1930 and accepted more than 300 species. After that, in 1949, the scientist have accepted 137 species, in 1979, 150 species was accepted, and in 1982, another 252 species was accepted (Gupta and Rodriguez-Couto, 2017). Penicillium classification and identification was identified based on the morphological species concept at that time and continued with the DNA sequencing to identify the species during 1990’s. 2.5.2 Morphological of Penicillium speciesFigure 2.9 shows the structure of Penicillium species which consist of conidia, phialides, metulae, rami, and conidiophore. Figure 2.

10 to Figure 2.14 show the structures of Penicillium species macromorphologically and micromorphologically. Table 2.1 describes the characteristics that are used in phenotypic based identification of Penicillium species.Figure 2.10: Penicillium paradoxum sexual reproduction. A–C.

Cleistothecia. D, E. Asci. F. Ascospores. Scale bars: A, B = 500 ?m; C–F = 10 ?m (Visagie et al., 2014).

 .Figure 2.11: Penicillium crystallinum.

A.Colonies: top row left to right, observe CYA, YES, DG18, and MEA; bottom row left to right, reverse CYA, reverse YES, reverse DG18 and CREA. B.

Colony texture on MEA. C-G. Conidiophores. H. Conidia.

Scale bars: C-H = 10 ?m (Visagie et al., 2014).Figure 2.12: Penicillium paradoxum. A. Colonies: top row left to right, observe CYA, YES, DG18, and MEA; bottom tow left to right, reverse CYA, reverse YES, reverse DG18 and CREA. B. Young sclerotia.

C. Phototrophic conidiophores after two weeks growth. D-H. Conidiophores. I. Conidia.

Scale bars: C-H= 10 ?m (Visagie et al., 2014). Figure 2.13: Penicillium malodoratum. A.

Colonies: top row left to right, obverse CYA, YES, DG18 and MEA; bottom row left to right, reverse CYA, reverse YES, reverse DG18 and CREA. B. Colony texture on MEA. C–G. Conidiophores. H. Conidia.

Scale bars: C–H = 10 ?m (Visagie et al., 2014).Figure 2.14: P. chrysogenum. 7-day old colonies at CYA, MEA, YES. B, P. canescens, C, P.

griseofulvum, D, P. polonicum, E, P. glabrum, F, P. expansum, G, P. italicum, H, P.

ratstrickii, I, P. meanoconidium, J, P. paltans (Abastabar et al.

, 2016) Table 2.1: The criteria used in Phenotypic Based Identification (Abastabar et al., 2016).Species identification by Phenotypic criteria Conidia Phialide Conidiophore Branching Pattern MetulaeP. polonicum Smooth, globose, 3?m Cylindrical, 10?m Terverticillate, Biverticillate Cylindrical, 10?mP. glabrum Smooth, ellipsoidal, 3?m Cylindrical, 10?m Monoverticillate Not presentP. expansum Smooth, ellipsoidal, 3?m Cylindrical, 8?m Terverticillate Cylindrical, 11?mP.

raistrickii Smooth, globose, 2.5?m Cylindrical, 8?m Biverticillate Cylindrical, 10-12?mP. italicum Smooth, ellipsoidal, 3.5?m Cylindrical, 7-9?m Terverticillate Cylindrical, 14?mP. melanoconidium Smooth, globose, 3?m Cylindrical, 8-10?m Terverticillate Cylindrical, 12?mP.

grisefulvum Smooth, ellipsoidal, 2?m Cylindrical, 4-5?m Terverticillate Cylindrical, 9?mP. chrysogenum Smooth, globose, 3?m Cylindrical, 7?m Bi, ter and quarterverticillate Cylindrical, 10?mP. canescens Finely roughened, globose, 2?m Cylindrical, 8?m Biverticillate, monoverticillate Cylindrical, 10-16?mP. palitans Smooth globose, 3-4?m Cylindrical, 10?m Terverticillate Cylindrical, 10-14?m Definitions: Monoverticillate, conidiophore without branching; Biverticillate, conidiophore one-stage branching; Terverticillate, conidiophore two-stage branch. 2.6 Molecular IdentificationInternal Transcribed Sequences (ITS) is used for molecular identification. The official DNA barcode for fungi is the nuc rDNA internal transcribed spacer rDNA region (ITS1-5.8S-ITS2).

This kind of PCR will be used because it is the most common sequence marker in fungi and the primers that work universally (Samson et al., 2014).  More than 90,000 fungal internal transcribed spacer (ITS) region sequences as in Figure 2.15 shows one of the most widely used barcoding regions of fungi that have been deposited in public databases.

This shows that the ITS sequences of most of, much of fungi remain to be revealed. The universality of PCR primers can limit the efficiency of cataloguing and identify the specimen, the development and selection of high coverage barcoding primers are regarded as crucial steps in the DNA barcoding and many attempts have been made to design high-coverage primers for amplification of the fungal ITS region. Some of the primers can mismatch with target fungal ITS sequences at negligible frequencies, potential precluding the DNA barcoding of some subgroup of fungi or hampering the accurate description of fungal community structure.

A high coverage PCR primers are targeting the ITS1 and ITS2 regions that were designed at conserved nucleotide positions of the aligned consensus sequences (Toju et al., 2012). Table 2.2 shows the primer that was used in ITS-PCR and the annealing temperature used for amplification and sequencing.

  Table 2.2: Primers and annealing temperatures used for amplification and sequencing (Samson et al., 2014).

Locus Amplification Annealing temp (?) Cycles Primer name Direction Primer sequence(5′-3′)Internal Transcribed Spacer (ITS) standard 55 (alt. 52) 35 ITS1 Forward TCC GTA GGT GAA CCT GCG G ITS4 Reverse TCC TCC GCT TAT TGA TAT GC  2.7 Antimicrobial properties of Aspergillus and Penicillium speciesAspergillus species are known to act in three different clinical settings in humans either in allergic states, opportunistic infections, and toxicoses. Immunosuppression is the main factor that leads to the development of opportunistic infections. This infection can be found in a huge spectrum, starting from the local involvement to dissemination and is called aspergillosis. Aspergillus is the most frequently isolated in invasive infections among all filamentous fungi.

Table 2.3 and the Figure 2.16, Figure 2.17, Figure 2.18 and Figure 2.

19 shows the inhibitory effects of fungi isolated against pathogenic bacteria after 24 hours of incubation (Prakash and Jha, 2014). Aspergillus species strains YSN038, YSN052, and YSN064 which shows inhibitory zones is shown in Table 2.4. Figure 2.20 shows the diameter of growth inhibition zones of Aspergillus fumigatus and Penicillium chrysogenum after treatment using various antifungal agents in mean ± standard deviation (N = 40) and Table 2.

5 shows the antagonistic of bacteria on the fungal pathogens.Table 2.3: Inhibitory effect of fungal isolates against pathogenic bacteria after 24 hours of incubations period (Miri et al.

, 2014).Bacterial isolate Inhibition zone diameter (mm)* Aspergillus niger Penicillium sp. AlternariaP.

aeruginosa 1 15 10 -P. aeruginosa 2 10 7 -S. aureus 1 22 18 -S.

aureus 2 25 19 -S. epidermidis 1 30 20 -S. epidermidis 2 25 15 -Bacillus sp 1 32 18 -Bacillus sp 2 29 15 -Figure 2.16: Antibacterial activity of fungal isolates against Staphylococcus aureus grown on BHI agar incubated at 37?C for 24 hours (Miri et al., 2014).

 1: Filtrate of Aspergillus niger, 2: Filtrate of Penicillium sp.,  3: Filtrate of Alternaria sp, 4: Control Figure 2.17: Antibacterial activity of fungal isolates filtrate against Staphylococcus epidermidis isolated from inflamed eye grown on BHI agar incubated at 37?C for 24 hours (Miri et al., 2014).

1: Filtrate of Aspergillus niger. 2: Filtrate of Penicillium sp. 3: Filtrate of Alternaria sp. 4: Control.

Figure 2.18: Antibacterial activity of the fungal isolates against Bacillus sp isolated from inflamed eye grown on nutrient agar incubated at 37?C for 24hours (Miri et al., 2014).1: Filtrate of Aspergillus niger, 2: Filtrate of Penicillium sp.3: Filtrate of Alternaria sp.

, 4: Control.Figure 2.19:  Antibacterial activity of fungal isolates on P. aeruginosa isolated from in flamed eye grown on nutrient agar incubated 37?C for 24hours (Miri et al., 2014).1: Filtrate of Aspergillus niger, 2: Filtrate of Penicillium sp.3: Filtrate of Alternaria sp., 4: Control.

Table 2.4: Inhibitory zone of Aspergillus species strains YSN038, YSN052, and YSN064 crude extracts (80?g/disk) (Wang et al., 2017).Bacteria for antibacterial test Inhibitory zone (mm) YSN038 YSN052 YSN064B. pumilus 13.2±0.

8 NA 13.1±0.3B. subtilis 13±0.4 NA 15.

3±0.4S. aureus 11.2±0.2 NA 11.9±0.7K.

rhizophila 10.2±0.2 NA 13.2±0.

2E. coli NA NA NAR. solanacearum NA NA NAFigure 2.

20: Diameter of growth inhibition zones of Aspergillus fumigatus and Penicillium chrysogenum after treatment using various antifungal agents. Mean ± SD (N = 40) (Rogawansamy et al., 2015).Table 2.5: Antagonistic potential of bacteria on the fungal pathogens (Mushtaq et al., 2010).Bacterial strains Fungal growth (cm) Aspergillus niger Aspergillus flavus Penicillium simplicissmum Penicillium italicumControl 9.0±0.00 8.3±0.33 2.5 0.00 3.5±0.00Escherichia coli 1.6±0.08 1.2±0.08 0.1±0.02 0.3±0.06Bacillus fortis 6.5±0.86 0.6±057 0.6±0.08 2.7±2.6Bacillus foragiris 2.0±0.09 1.1±0.09 1.0±0.12 0.8±0.02Pseudomonas fluorescence 3.5±0.28 4.4±0.26 1.3±0.05 0.2±0.02Pseudomonas maliphilia 3.2±0.14 0.2±0.08 0.3±0.08 0.1±0.02