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Diversity of ascomycetes in peat soil from three selected areas in Selangor.

INTRODUCTION

For a long time, soil is known to be the storehouse for a wide variety of filamentous fungi and other microorganisms. The interest in soil microbial diversity has emerged due to its critical function in maintaining and controlling the quality of the soil. Peat soil ecosystems are found to be the best habitats for a large number of organisms from all domains of life including archaea, bacteria, fungi, protists, plants and animals[1]. Peat is defined as an organic soil having mineral content that is not exceeding 35% where it basically comprises of plant materials that are partially decomposed in addition of mineral fractions such as silt, clay and sand. Area of peat soil with depth of 0.5 m and more has been classified into shallow peat soil; (with organic matter depth less than 0.45 m) and deep peat soil (with organic matter depth more than 0.5 m)[2]. Since peat soil is important as major carbon storage but little is known of their ecology and microbial diversity, the interest in studying microorganisms isolated from this type of soil has increased.

Among the microorganisms in many peatland ecosystems, fungi are found to be the main decomposer where they have an extensive hyphal network with fast growth rate which give them the ability to translocate nutrients to vast area in the ecocystem[3]. Diverse group of fungi have been recovered from peatland soils worldwide such as Ascomycota, Basidiomycota and Zygomycota[4]. Ascomycota is the largest phylum of Fungi with over 64,000 species and the members of this phylum are known as Ascomycetes or the sac fungi[5]. This fungal group has a defining feature called "ascus", a microscopic sexual structure in which ascospores, a nonmotile spores are formed. However, some species of Ascomycetes are asexual, where they do not have sexual cycle thus do not form ascospores. These asexual Ascomycetes are identified and classified based on their morphological and physiological similarities to the ascus-bearing taxa by phylogenetic analyses of DNA sequences[6,7].

The Ascomycetes are represented in all land ecosystems worlwide[8]. The hyphal fragments and spores are dispersed through the atmosphere and freshwater environments, as well as beaches and tidal zone. Malaysia is known as a country that is rich in tropical regions. Previous studies showed that fungi isolated from ordinary surroundings like soils and plants have been the reservoir of a wide range of bioactive natural products. Tropical regions have also proven to offer a much larger wealth of biodiversity but yet, very little work has been carried out on tropical microbes. This is one of the reasons why soil microorganisms from Malaysian soil can be a rich source of the new leading compounds in term of drug discovery.

2. Objective:

The aim of this study is to investigate the diversity of Ascomycetes that can be isolated from three selected peat soil areas in Selangor.

MATERIALS AND METHODS

3.1. Soil Sampling:

The sites of sampling involved in this study were Felcra Sijangkang, Pulau Kempas and Pulau Carey. Three points of sampling sites were determined by GPS at peat area in Selangor. Soils were sampled at three different depth, top soil (30cm), midway between top soil and water table (60cm) and water table (90cm). Collected soil samples were kept in sterile plastic bags and labeled. All soil samples were air-dried at room temperature for 48 to 72 hours. The dried samples were sieved through a 0.5 mm sieve to remove stones and plant residues. Sieved soils were stored in sterile container until further analysis. Plastic bags were not used to store soil samples as these bags do not allow the soil to dry completely and may encourage bacterial growth[9].

3.2. pH Measurements:

A 30 g of each soil samples were weighed in 100 mL beaker and added with 75 mL distilled water. The samples were mixed well before being incubated at room temperature for 24 hours. The reading of pH were taken and recorded after 24 hours of incubation. Average pH was calculated to ensure the accuracy of the results.

3.3. Soil Moisture Content:

A 30 g of soil samples were put in petri dishes and weighed. The samples were then incubated at 105[degrees]C for 48 to 72 hours. The samples were then cooled down at room temperature and weighed again after 72 hours of incubation.

3.4. Growing Culturable Fungi:

Soil samples were serially diluted up to [10.sup.-4]. Potato dextrose agar was used for the fungal isolation. All cultures were incubated at 30[degrees]C for 5 to 7 days. Cycloheximide was added to suppress the unwanted bacterial growth. Putative fungal isolates from growth medium were transferred into new plates of potato dextrose agar (PDA) to maintain their purity. New plates were incubated at room temperature for 5 to 7 days to produce abundant pure fungal cultures.

3.5. Mycological Slide Preparation:

Characteristics of pure cultures of filamentous fungi were identified by observing the colonies produced in plates after 7 days of incubation. Mycological slide were prepared using adhesive tape preparation method. Small amount of spores and conidia were taken by adhesive tape and placed onto a clean slide. A drop of lactophenol blue was added as the counterstain. The images of filamentous fungi were observed under light microscope.

RESULTS AND DISCUSSION

A total of 106 isolates were recovered from all soil samples. Among the soil sampling sites, Pulau Kempas has the highest number of Ascomycetes isolated in which 43 isolates were obtained, followed by Pulau Carey with 35 isolates, and Felcra Sijangkang with 28 isolates, respectively. Table 1 showed the recorded pH reading and moisture contents for samples collected at three selected areas in Selangor. The pH of peat soil from Felcra Sijangkang ranged from 3.59 to 3.98 while the moisture content ranged from 63.72 to 84.51%. For peat soil sampled at Pulau Kempas, the pH readings were ranging from 3.33 to 3.59 while the soil moisture ranged from 62.40 to 68.54%. As the samples collected from Pulau Carey, the pH went from 3.09 to 3.23 with the soil moisture ranged from 15.18 to 42.16%.

The different depths of the soil samples collected affect the moisture content of each soil samples but not the pH reading and the fungal distribution. All of the soil samples collected at the highest depth of 90 cm from all three sampling sites showed the highest percentage of moisture content but the pH and the number of isolates varies. The highest number of isolates in Felcra Sijangkang and Pulau Carey was obtained at 60 cm depth with pH of 3.72 and 3.09, with 82.63% and 41.80% of moisture content, respectively. While in Pulau Kempas, deepest area of peat soil which is 90 cm with highest moisture content of 68.54% at pH 3.59 showed the most number of isolates. Although the optimal pH and moisture content varies greatly, majority of fungi grow best at pH 3.59 with 10 isolates from Felcra Sijangkang and 18 isolates from Pulau Kempas, respectively in this study.

The moisture content of tropical peat soil ranged from 338% to 398% [10] meanwhile in this study, the moisture content range were found to be much lower. This large difference of moisture content may be due to the different type of peat soil that is maintained in the ground water, the water that is found underground in the cracks and spaces in soil. This reduced water content may also be caused by lower soil organic content and water evaporation[11]. The higher moisture content in deeper peat soil may due to the removal of vegetation and lesser soil compaction in soils located at the water table[12].

Pulau Carey, located to the south of Port Klang is not considered as a real island due to its proximity to the mainland and the river that separates it is practically a stream. The heat of sunlight and wind are among the factors that might influenced the lower moisture content of soil samples collected from Pulau Carey as compared to Pulau Kempas and Felcra Sijangkang.

The pH readings obtained from all three sampling sites varies where soil samples collected from Pulau Carey showed lower pH reading as compared to Pulau Kempas and Felcra Sijangkang. However, the results did not differ much from previous studies. Tropical peat soils are commonly acidic with pH ranging from 3 to 4.3. It was found to be the favorable conditions for most Ascomycetes[13] and Ascomycetes were found to be the most isolated fungi in this study.

From the result obtained, it can be seen that the content of organic matter would affect the fungal distribution in peat soil. The result showed that Ascomycetes prefer a location which is high in organic content such as plant roots as their habitat. Soil samples from Pulau Kempas and Pulau Carey were collected in the area of undisturbed peat soil from human and animal activities while soil samples from Felcra Sijangkang were collected from disturbed peat soil in the palm oil plantation site. The use of chemical fertilizers could be the cause of lesser amount of microfungi isolated from Felcra Sijangkang as they may disturb the organic matter content in the soil. The content of organic matter in soils is a potential source of nitrogen, phosphorus and sulphur for fungal growth. Organic matter decomposition by microorganisms is essential to release the bound nutrients in organic residues that then utilised by the microfungi [14].

Table 2 showed the most abundant fungal isolated according to sampling sites. Based on the morphological characteristics, nine genera of filamentous fungi were identified namely, Aspergillus sp., Penicillium sp., Trichoderma sp.. Trichophyton sp., Chrysosporium sp., Cladosporium sp., Paecilciyces sp., Hypocrea sp. and Chaetomium sp. From the results showed in Table 2, Aspergillus sp., Penicillium sp., and Trichoderma sp. represented in all three sampling sites and the most abundant Ascomycetes isolated were found to be Aspergillus sp., with 34.91% of total isolates, followed by Penicillium sp. with 21.69% of total isolates, respectively.

Aspergillus sp., a well-known Ascomycetes can be easily found throughout the world as it fits well in the soil niche. Aspergillus adapt well in a wide range of environment especially in air and soil. It is natural for fungi to obtain moisture and nutrients without having the rays from the sun[15]. Penicillium sp., another well-known fungi genera often found in soils rather than any other sources[16]. Trichoderma sp. is considered as the most versatile fungi due to its high adaptability to its environment where it may produce both sexually and asexually, depending on the environmental conditions. Other soil fungi like Paecilomyces sp. and Trichophyton sp. have also been isolated widely from agricultural soil where these fungi species favors the abundance of plant roots [17]. This explained why Ascomycetes were able to be isolated in this study.

Figure 1 showed the microscopic observation of some Ascomycetes isolated from this study after 7 days of incubation. Most species of Ascomycetes grow as filamentous with microscopic structure called hyphae. The interconnected hyphae form mycelium, which when visible to the naked eye is commonly called mold or thallus. The Ascomycetes cell walls often contain chitin and [beta]-glucans. The internal boundaries of the individual cells called septa, which is the divisions within the hyphae. The cell wall and septa gives these Ascomycetes the stability and rigidity that may prevent the loss of cytoplasm in case of local damage to the cell membrane and cell wall.

During sexual reproduction, Ascomycetes will typically produce a large number of asci where it often contained in multicellular fruiting structure, ascocarp. Ascocarp comes in multiple colors, red, orange, yellow, brown, black and rarely green or blue. Some ascomyceous fungi grow as single-celled yeasts, which do not form fruiting bodies as it developed into an ascus during sexual reproduction. Several species of Ascomycetes are biological model organisms in laboratory research. Aspergillus is used in many genetics and cell biology studies and many Penicillium sp. are those fungal producing antibiotics that are important for treating bacterial infectious diseases.

Ascomycetes fulfill a central role in most land-based ecosystems. They are important decomposers as they have evolved the capacity of breaking down almost every organic substance to obtain their nutrients. They are able to use their own enzymes to digest plant biopolymers like cellulose or lignin thus have crucial roles in nutrient cycling such as carbon cycle. The fruiting bodies of the Ascomycetes provide food for many animals ranging from insects and slugs to larger mammals such as deer and wild boars. Ascomycetes make many contributions to the good of humanity, but also have many ill effects as they also play as the agent of many plant diseases.

This group of fungi is also form important relationship with plants; as mycorrhizal fungi and as endophytes. Mycorrhiza, a symbiotic association between the root systems and the fungi is crucial for growth and persistence of the plants. The fungi are able to increase the uptake of mineral salts that occur at the low levels in soil with the help of their fine mycelial network. The plants will provide metabolic energy from the process of photosynthesis to the fungus in return. In the other hand, the endophytic fungi live inside the plants. They form a mutual relationship with their host and do not harm the host. The exact nature of this relationship depends on the species involved. In some cases, the fungal colonization of plants give a higher resistance against insects and bacteria where the endophytes produces poisonous alkaloids that are harmful to the plant-eating mammals[18]. Conclusion:

All peat soils samples collected from three sampling sites namely Felcra Sijangkang, Pulau Kempas and Pulau Carey demonstrated that they can be a valuable natural sources of Ascomycetes. Out of 106 isolates from all three sampling sites, the most abundant fungi isolated was found to be Aspergillus sp. with 34.91% of total isolates. Other common fungi found in this study were Penicillium sp., Cladosporium sp., Trichoderma sp., Trichophyton sp., Paecilomyces sp. and Hypocrea sp. Since there is very little information on this, further research in the field of diversity and occurrence of fungi from tropical peat soil need to be performed.

ARTICLE INFO

Article history:

Received 25 July 2015

Accepted 1 September 2015

Available online 19 September 2015

ACKNOWLEDGEMENT

The authors would like to thank the Faculty of Applied Sciences, Universiti Teknologi MARA, Shah Alam, Selangor for supporting this study.

Authors' Contribution:

Dr. Zainon Mohd Noor and Dr Adzmi Yaacob developed the idea and had an important role in the result and material section. Norzatushima perform the analysis, discussion and abstract submission.

Financial Disclosure:

There is no conflict of interest.

Funding/Support:

The project was funded by Universiti Teknologi MARA, Shah Alam, Selangor, Malaysia.

REFERENCES

[1] Nuyim, T., 2000. Proceedings of the International Symposium on Tropical Peatlands, Bogor.

[2] Morison, J., E. Vanguelova, S. Broadmedow, M. Perks, S. Yamulki, T. Randle, 2010. The Research Agency of the Forestry Commission Scotland.

[3] Williams, R.T. and R.L. Crawford, 1983. Microbial diversity of Minnesota peatlands. Microbial Ecology 9: 201-214.

[4] Cavalier-Smith, T., 1998. A revised six-kingdom of Life.

[5] Kirk, P.M., P.F. Cannon, D.W. Minter, J.A. Stalpers, 2008. Dictionary of the Fungi. 10th Ed. Wallingford, CABI. ISBN 0-85199-826-7.

[6] Lutzoni, F., et al., 2004. Assembling the fungal tree of life; progress, classification, and evolution of subcellular traits. American Journal of Botany, 91(10).

[7] James, T.Y., et al., 2006. Reconstructing the early evolution of Fungi using a six-gene phylogeny. Nature 443(7113).

[8] Laybourn-Parry, J., 2009. Microbiology. No place too cold. Science, 324(5934).

[9] Leslie, J.F., B.A. Summerell, 2006. The Fusarium Laboratory Manual, 1st ed. UK, Blackwell Publishing.

[10] Firdaus, M.S., S. Gandaseca, O.H. Ahmed, 2011. Int j. Physi Sci., 6(23): 5462-5466.

[11] Firdaus, M.S., S. Gandaseca, O.H. Ahmed, N.M.A. Majid, 2010. Am. j. Environ. Sci., 6: 402-405.

[12] Moduying, V., Y. Yangkat, C.H. Laugesen, T. Greer, 2000. Environmental Impact Assessment (EIA) Guildlines Oil Palm Plantation Development. State Environmental Conservation Department (ECD) Sabah, Malaysia.

[13] Smit, E., P. Leeflang, B. Glandorf, J.D. van Elsas, K. Warnels, 1999. Appl. Environ. Microbiol., 65(6): 2614-2620.

[14] Subba Rao, N.S., 2001. Soil Microbiology. 4th Ed. of soil microorganisms and plant growth. New Hampshire; Science Publisher Inc.

[15] O'Gormann, C.M., 2011. Fungal Biology Reviews, 25(3): 151-157.

[16] Dayalan, S.A.J., P. Darwin, S. Prakash, 2011. Asian Pacific Journal of Tropical Biomedicine, pp: 315-319.

[17] Harman, G.E., T. Bjorkman, K. Ondik, M. Soresh, 2008. Outlooks in Pesticide Management, 9: 24-29.

[18] Schulz, B., C. Boyle, 2005. The endophytic continuum. Mycological Research, 109(6).

(1) Norzatushima, M.J., (2) Zainon, M.N., (3) Adzmi, Y.

(1,2) School of Biology, Faculty of Applied Sciences, Universiti Teknologi MARA (UiTM), Shah Alam, 40450 Selangor Darul Ehsan, Malaysia

(3) Faculty of Plantation and Agrotechnology, Universiti Teknologi MARA (UiTM), Shah Alam, 40450 Selangor Darul Ehsan, Malaysia

Corresponding Author: Norzatushima M.J., School of Biology, Faculty of Applied Sciences, Universiti Teknologi MARA (UiTM), Shah Alam, 40450 Selangor Darul Ehsan.

E-mail: dzainonmn@salam.uitm.edu.my

Table 1: pH measurements, moisture contents and number of isolates
from three selected sites of soil sampling.

Sampling site       Depth    pH    Moisture (%)   Number of
                    (cm)                          isolates

Felcra Sijangkang    30     3.98      63.72           8
                     60     3.72      82.63          11
                     90     3.59      84.51          10

Pulau Kempas         30     3.33      62.40           9
                     60     3.48      65.61          16
                     90     3.59      68.54          18

Pulau Carey          30     3.23      15.18          12
                     60     3.09      41.80          15
                     90     3.17      42.16           8

Table 2: Most abundant fungal isolates according to sampling site.

Sampling site       Total no. of      Fungi genus      Percentage of
                      isolates                           genus (%)

Felcra Sijangkang        28         Aspergillus sp.        42.86
                                    Penicillium sp.        14.29
                                    Trichoderma sp.        10.71
                                   Paecilomyces sp.        10.71
                                   Cladosporium sp.        7.14
                                    Chaetomium sp.         7.14
                                   Chrysosporium sp.       7.14

Pulau Kempas             43         Aspergillus sp.        34.88
                                    Penicillium sp.        16.28
                                    Trichoderma sp.        6.98
                                   Cladosporium sp.        4.65
                                   Trichophyton sp.        4.65
                                     Hypocrea sp.          4.65
                                      Fungal sp.           20.83

Pulau Carey              35         Aspergillus sp.        28.57
                                    Penicillium sp.        34.29
                                    Trichoderma sp.        8.57
                                   Chrysosporium sp.       8.57
                                      Fungal sp.           14.58
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Author:Norzatushima, M.J.; Zainon, M.N.; Adzmi, Y.
Publication:Advances in Environmental Biology
Article Type:Report
Date:Sep 1, 2015
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