Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 13  |  Issue : 1  |  Page : 27-32

Biologically active fungi recorded for the first time from new reclaimed soil, Egypt


1 Department of Botany, Faculty of Science, Assiut University, Assiut, Egypt
2 Department of Chemistry of Microbial Natural Products, National Research Center, Giza, Egypt

Date of Submission08-Dec-2013
Date of Acceptance17-Feb-2014
Date of Web Publication30-Jun-2014

Correspondence Address:
Waill A Elkhateeb
Department of Chemistry of Microbial Natural Products, National Research Center, Tahrir Street, 12311, Dokki, Giza
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1687-4315.135595

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  Abstract 

Objective
This work was designed to record fungal isolates of known biological activities isolated for the first time from new reclaimed soil in El-Wady El-Assiuty and Assiut Petroleum farm in Assiut, Egypt.
Materials and methods
Soil, phyllosphere, and phylloplane samples were collected from El-Wady El-Assiuty and Petroleum farm, which represent the largest distinctive regions of newly reclaimed soil at Assiut Governorate. Identification of the isolated fungi during our investigation was carried out on the basis of many reported morphological and microscopic features, which was confirmed by Assiut University Mycology Center.
Results and conclusion
Three fungal species, in addition to one unidentified ascomycete, were isolated for the first time from new reclaimed soil in El-Wady El-Assiuty and Assiut Petroleum farm in Assiut, Egypt. They were identified and recorded by the Mycological Laboratories at Assiut University. They were identified as Arthrinium sacchari (Speg.) M.B. Ellis, which was isolated from the phylloplane of Guava, Beltrania querna Harkn., which was isolated from the soil from Petroleum farm, and Papulaspora immersa Hotson, which was isolated from the phyllosphere of Wheat plants; the newly unidentified ascomyceteous fungal species was obtained from the phyllosphere of pomegranate. The endemic mycobiota of these areas is of great significance, as there are no adequate mycological studies that have been performed to describe the fungal flora of these areas.

Keywords: Identification, isolation, phylloplane fungi, phyllosphere fungi, reclaimed soil, soil fungi


How to cite this article:
Zohri ANA, Elkhateeb WA, Mazen MB, Hashem M, Daba GM. Biologically active fungi recorded for the first time from new reclaimed soil, Egypt. Egypt Pharmaceut J 2014;13:27-32

How to cite this URL:
Zohri ANA, Elkhateeb WA, Mazen MB, Hashem M, Daba GM. Biologically active fungi recorded for the first time from new reclaimed soil, Egypt. Egypt Pharmaceut J [serial online] 2014 [cited 2017 Oct 18];13:27-32. Available from: http://www.epj.eg.net/text.asp?2014/13/1/27/135595


  Introduction Top


Studying the occurrence, diversity, and distribution of mycoflora provides a clear image of the phytosanitarian state of the surrounding environment. Many papers have reported the presence and occurrence of fungi [1],[2],[3],[4]. Investigating the mycobiota inhabiting the newly reclaimed areas at Assiut Governorate was very important for many reasons, including achieving a better understanding of the fungal flora existing in these areas; in addition, it presents an opportunity to isolate and identify new fungal species. Most investigations on fungal flora have been carried out on agricultural or forest soils; however, in this study, we investigated soil, phyllosphere, and phylloplane fungi from newly reclaimed localities of El-Wady El-Assiuty and Assiut petroleum Farm. They represent two of the largest newly reclaimed areas at Assiut Governorate, cultivated with different important crops. Many investigations have been carried out worldwide on the occurrence of soil fungal flora [5],[6],[7],[8],[9],[10],[11],[12]. In Egypt, numerous investigations have been carried out on soil fungi in the upper Egypt delta area and Sinai Peninsula, as well as in some valleys in the eastern desert of Egypt [13],[14],[15],[16],[17],[18],[19].

The term phyllosphere fungi was coined by Abdel-Hafez et al. [20] and Last [21] to describe organisms found on leaf surfaces. Phylloplane, as described by Ruinen [22], is the outer skin of the leaf surface and is thus an excellent site for growth of saprophytic and parasitic fungi. Several studies have described the microorganisms on insects or on the leaf surface of plants growing or cultivated worldwide as parasites [23],[24],[25],[26],[27],[28].


  Materials and methods Top


Selected areas

Two areas representing the largest and most distinctive regions of newly reclaimed soil in Assiut Governorate were selected. They are El-Wady El-Assiuty, which lies 10 km east of Assiut, and Petroleum farm, which lies 10 km northwest of Assiut.

Collection of samples

Soil samples

Soil samples were collected from the two selected reclaimed regions in Assiut Governorate. Soil samples were collected from patches free from roots according to the method described by Johnson et al. [29], which could be summarized as follows:

  1. A sample tube was used, which was washed thoroughly before starting the sampling procedure. Samples were taken from a depth of 5 inches, and the soil was transferred directly into clean and sterilized plastic bags (at least five samples were taken at random for each replication).
  2. Five or more samples from each replication were combined to form a composite sample, which was mixed thoroughly.
  3. Finally, 10 g (on an oven-dry basis) of the mixed composite sample was used to determine fungal counts by the dilution plate method, as recommended by Johnson and colleagues [10],[29].


Phyllosphere and phylloplane samples

Leaves of plants were collected from the above-investigated localities, packed directly into polyethylene bags, and kept in a refrigerator until use.

Isolation and identification of fungi

Soil fungi

The dilution plate method was used to identify soil fungi [10],[29], as follows:

  1. Soil to be diluted was sifted through a nine-mesh sieve. Three aliquots each (5-10 g) of soil were placed in a previously weighed metal container and dried overnight in an oven at 105°C. The aliquots were then reweighed, and the moisture content of the soil sample was calculated.
  2. Ten grams of soil particles (oven-dry weighed soil) was taken in a graduated cylinder; sterilized distilled water was added to the soil to obtain a total volume of 100 ml. The suspension was stirred and poured into a 1000 ml Erlenmeyer flask. The flask containing the suspension was shaken on mechanical shaker for 30 min.
  3. Of the suspension, 10 ml was immediately drawn (while in motion) using a sterile Menzies dipper [30] and mixed with a known volume of sterile distilled water blank until the desired final dilution was reached. Each suspension was shaken by hand for a few minutes and was in motion while being drawn using the dipper.
  4. Of the desired dilution, 1 ml was transferred directly into each of the sterilized  Petri dish More Detailses; thereafter, 12-15 ml of glucose-Czapek's agar medium, cooled to just above solidifying temperature, was added to each dish. The dishes were rotated by hand in a broad swirling motion so that the diluted soil was dispersed in the agar.
  5. Five plates were used for each sample and incubated at 28 ± 1°C for 7 days, during which the developing colonies were identified and counted [expressed as colony forming units (cfu) per gram dry soil]. The average number of colonies per plate was multiplied by the dilution factor to obtain the number per gram in the original soil samples.
  6. Glucose-Czapek's agar [10],[31] was used throughout the present investigation for isolation and identification of fungi. This media was supplemented with rose bengal (66 mg/ml) and chloramphenicol (30 mg/ml) as bacteriostatic agents [32]. The composition of this medium is as follows: agar, 15.0 g; NaNO 3 , 3.0 g; KH 2 PO 4 , 1.0 g; MgSO 4 × 7H 2 O, 0.5 g; KCl, 0.5 g; FeSO 4 × 7H 2 O, 0.01 g; glucose, 10.0 g; 1000 ml distilled water.
  7. For further investigation, growing fungal species were obtained on slants of glucose-Czapek's agar medium and kept in a refrigerator at 4°C.


Phyllosphere fungi

Ten grams of plant leaf segments was taken in a sterile conical flask containing 100 ml sterile distilled water. The flask was shaken by hand in a rotating motion for 10 min; 10 ml of the suspension was transferred into another flask (250 ml) containing 90 ml sterile distilled water; thereafter, the flask was shaken for 5 min. Final dilutions were made in the same way to obtain a series of dilutions. Of the final dilution, 1 ml was transferred to a sterile Petri-dish and agar medium was poured into the dish. Five replicates were prepared for each sample and they were incubated at 28 ± 1°C for 7 days. Developing colonies were identified and counted (expressed as cfu/g fresh leaves).

Phylloplane fungi

Plant leaves were subjected to a series of washing with sterile distilled water. They were thoroughly dried between sterile filter paper and cut into equal segments (about 1 cm each). One hundred segments were selected per sample. Five segments were placed on the surface of the agar medium in each plate; thereafter, the plates were incubated at 28 ± 1°C for 7-10 days, and the developing fungi were counted, identified, and calculated as cfu/100 segments of fresh leaves.

Identification of fungal genera and species

Identification of the isolated fungi during our investigation was carried out on the basis of morphological and microscopic features, according to the following references:

  1. Ainsworth [33] as a dictionary of the fungi.
  2. Domsch et al. [34] for fungi in general.
  3. Ellis [35],[36] for dematiaceous hyphomycetes.
  4. Kendrick [37] for the genera of imperfect fungi.
  5. Moubasher [38] for fungi in general.


In addition, the isolated fungi was reviewed and compared with the same species deposited at Assiut University Mycological Center (AUMC).


  Results and discussion Top


The screening survey, which was developed at El-Wady El-Assiuty, resulted in the isolation and identification of 37 species and one species variety belonging to 21 genera from the soil, 71 species and four species varieties belonging to 33 genera from the phyllosphere on glucose-Czapek's agar at 28 ± 1°C, and 56 species apart from the isolation and identification of Papulaspora immersa from the phyllosphere of wheat plants (Triticum aestivum L.) and an unidentified ascomyceteous fungal species from the phyllosphere of pomegranate (Punica granatum L.). Finally, three species varieties belonging to 26 genera from the phylloplane on glucose-Czapek's agar at 28 ± 1°C besides the isolation and identification of Arthrinium sacchari from the phylloplane of Guava (Psidium guajava L.) were identified. From the Assiut petroleum farm 56 species and three species varieties belonging to 29 genera were isolated and identified from the soil, besides the isolation and identification of Beltrania querna, 69 species and three species varieties belonging to 30 genera from the phyllosphere on glucose-Czapek's agar at 28 ± 1°C, and 43 species and one species variety belonging to 21 genera from the phylloplane on glucose-Czapek's agar at 28 ± 1°C.

The four species that were isolated and identified for the first time are as follow:

Arthrinium sacchari (Speg.) M.B. Ellis

Colonies grow well on potato dextrose agar, attaining a diameter of 9 cm after incubation at 28 ± 1°C for 7 days; limited growth (about 1 cm) was observed on glucose-Czapek's agar after 7 days. Colonies are compact or widely diffuse, black or dark blackish brown [Figure 1] and [Figure 2]. Conidiophores are 1-1.5 μm thick and septa are numerous and brown. Conidia are one-celled, smooth, almost round in face view, lenticular, solitary, lateral or terminal, frequently flattened, and with a hyaline rim, which is either brown or dark brown, measuring 6-8 μm in face view and 3-4 μm in thickness. Arthrinium sacchari was isolated from the phylloplane of Guava (Psidium guajava L.) cultivated in El-Wady El-Assiuty. According to the literature review, several compounds with antitumor activity were isolated from the culture extract of Arthrinium sacchari, including cytochalasin E [39], decarboxyhydroxycitrinone, libertellenone F, libertellenone E, myrocin D, libertellenone C, and myrocin A [40].
Figure 1:

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Figure 2:

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Beltrania querna Harkn

Colonies are effuse, brown to black, and attain a diameter of 9 cm after 7 days of incubation at 28 ± 1°C on potato dextrose agar; stroma usually present setae that are simple, dark, smooth or verrucose, and thick-walled, arising from flat, radially lobed basal cells that are up to 400 μm long [Figure 3] and [Figure 4]. Conidiophores are straight or flexuous, pale olive to brown, smooth, septate, up to 200 × 2-7 μm conidiogenous cells terminal, polyblastic sympodial, clavate, denticulate, separating cells when present 8-12 × 4-7 μm. Conidia are solitary, biconic, appendiculate, the free end usually being spicate, without septa, smooth, and pale olive to dark reddish brown, with a hyaline band. Biconic conidia are mostly asymmetrical, with the proximal end being U-shaped, measuring 15-30 × 7-14 μm, with a 2-5 mm appendage. Beltrania querna was isolated from soil collected from Assiut petroleum farm. The following isocyclic compounds were isolated from Belternaria spp., with known antifungal activity [41]: (−)-β-eudesmol; β-eudesmol (−)-chrysanthemol rhombidiol; eudesm-4(14)-ene-9β,11-diol (−)-pterocarpol (−)-5β-hydroxy-β-eudesmol rhombitriol; 5α, 8α-dihydroxy-β-eudesmol (−)-longilobol.
Figure 3:

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Figure 4:

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Papulaspora immersa hotson

Colonies reach 6.5 cm diameter after 7 days of incubation at 28 ± 1°C on PDA and 4 cm on glucose-Czapek's agar. Papulaspores originate from intercalary cells, are pale brownish yellow, irregular in outline, 88-150 (−260) μm in diameter, often submerged in the agar central cells, and are comparatively large, angular, and darker than the peripheral cells. Reverse brownish yellow [Figure 5] and [Figure 6]. Papulaspora immersa was isolated from the phyllosphere of wheat (Triticum aestivum L.) plants cultivated in El-Wady El-Assiuty. Twelve substances of medical importance different from anticancer, antimicrobial, or antituberculosis agents were reported to be produced by Papulaspora spp. [42],[43],[44],[45],[46],[47].
Figure 5:

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Figure 6:

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Unidentified ascomyceteous fungal species

Colonies on PDA and glucose-Czapek's agar attained diameters of 2.5 and 2 cm, respectively, after 7 days of incubation at 28 ± 1°C. Mycelium at first appeared light brown, becoming dark brown with age, then brownish black, and finally reverse black; tiny drops of exudates appeared after 3 weeks of incubation; cultures produced ascomata after 3-4 weeks at 28 ± 1°C. Ascomata were dark, solitary, firmly adhering to the substratum, globose, and measured 360 mm in diameter. Asci were numerous, globose to subglobose with delicate, thin, transparent walls, and eight-spored, measuring 14-16 mm. Ascospores were ellipsoid to fusiform each, with one transverse septum, appearing light to dark brown under the microscope, measuring 10.5-12 mm in length×5.5-6.5 mm in width; no anamorph was observed [Figure 7] and [Figure 8]. This culture was isolated from the phyllosphere of pomegranate (Punica granatum L.) cultivated in El-Wady El-Assiuty.
Figure 7:

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Figure 8:

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  Acknowledgements Top


Conflicts of interest

There are no conflicts of interest.

 
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