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El-Meleigy M.A., Hoseiny E.N., Ahmed S.A. and Al-Hoseiny A.M., 2010. Isolation, Identification, Morphogenesis and ISSN 0126-2807 Ultrastructure of Obligate Halophilic Fungi. Volume 5, Number 2: 201-212, April-July, 2010 © T2010 Department of Environmental Engineering Sepuluh Nopember Institute of Technology, Surabaya & Indonesian Society of Sanitary and Environmental Engineers, Jakarta Open Access http://www.trisanita.org

This work is licensed under the Creative Commons Attribution 3.0 Unported License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Research Paper

ISOLATION, IDENTIFICATION, MORPHOGENESIS AND ULTRASTRUCTURE OF OBLIGATE HALOPHILIC FUNGI

EL-MELEIGY M.A.*, HOSEINY E.N., AHMED S.A. and AL-HOSEINY A.M.

Botany and Microbiology Department, Faculty of Science (For Girls), Al-Azhar University - Cairo Egypt.

*Corresponding Author: Phone: (002)0101230466; Email: [email protected]; [email protected] Received: 20th April 2010; Revised: 27th May 2010; Accepted: 31st May 2010

Abstract: Two halophilic fungal isolates named Trichoderma piluliferum fs. Halophila AZ and Aspergillus restrictus were isolated from Sharm El-Sheikh and from Raas Sader regions at Sinai­Egypt. They tolerates till 30 and25 %( w/v) NaCl (respectively). The former required at least 5% (w/v) NaCl, while the second grew poorly at salt free medium. Morphogenesis and ultrastructure of the two fungal isolates revealed that, 10 ­ 20 % NaCl concentration was the best for normal growth and cell ultrastructure and .It is worth to mention that this is the first record for true obligate halophilic fungal isolate (Trichoderma piluliferum fs. halophila AZ). The two fungal isolates could be used in many biotechnological applications.

Keywords: Halophiles, salt stress, fungi, morphogenesis, ultrastructure

INTRODUCTION Microbes must sense environmental stresses, transduce these signals and mount protective responses to survive in hostile environments [1]. A variety of microbes inhabit extreme environments .Extreme is a relative term, which is viewed compared to what is normal for human beings. Extreme environments include high temperature, pH, pressure, salt concentration, and low temperature, pH, nutrient concentration and water availability, and also conditions having high levels of radiation, harmful heavy metals and toxic compounds (organic solvents [2]. According to Gunde-Cimerman et al.[3,4] the recent definition of halophilic fungi are those species which are isolated regularly with high frequency on selective saline media from environments at salinities above 10% and are able to grow in vitro on media with at least 17% NaCl. Candida halophila CBS 4019 (syn. C. versatilis), is an extremely salttolerant yeast, studied by Silva-Graca et al.[5], it is a glycerol accumulator under high salt concentrations, while H. werneckii is constitutively melanized under various salinity conditions. Ultrastructural study 201

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showed localization of melanin in the outer parts of the cell wall as a distinct layer at optimal salinity (0.86 M NaCl), whereas cell-wall melanization diminished at higher salinities. The role of melanized cell wall in the effective retention of glycerol is already known, and was also demonstrated in H. werneckii by lower retention of glycerol in cells with blocked melanization compared to melanized cells. Halophilic microorganisms form a tremendously diverse group. Within the phylogenetic tree of life, halophilic and halotolerant microorganisms are found within each of the three domains; Archaea, Bacteria and Eukarya [6]. In their natural environments microorganisms have to cope with changing conditions of various kinds. This study was intended to isolate and identify halophilic fungi and determine the morphogenesis and ultrastructure under salt stress. MATERIALS AND METHODS Soil Samples The soil samples were collected from Sharm El-Shaikh and Raas Saddr at Sinai, Egypt. These soil samples were used for isolation of halophilic fungi. Media and Methods used for isolation of halophilic fungi Dox`s peptone -agar medium supplemented with different NaCl concentrations (0-30 % g (w/v)) was used for isolation and maintenance of fungal isolates. Two methods were used for isolation of halophilic fungi, direct inoculation method by spreading 0.5 g of soil sample on the surface of the isolation medium Dox ,s peptone agar medium supplemented with different concentrations of NaCl ( 0.0 to 30.0% g (w/v)) and serial dilution method using 1ml of dilutions of soil samples from(10-1 - 10-6 ). Identification of fungal isolates: Identification of fungal isolates was carried out using [7-10] for Trichoderma piluliferum and [11-13] for Aspergillus restrictus. Microscopic examination The two fungal isolates Trichoderma piluliferum [7] and Aspergillus restrictus [11] were inoculated at Capek's Dox peptone agar medium amended with different concentrations of NaCl (5%, 15%, and 25% (w/v)) for T. piluliferum and (0.0%, 10%, and 20% (w/v)) for A. restrictus. Scanning Electron Microscopy (SEM): Coated specimens of each fungal isolate were examined using high vacuum mode of JEOL JSM-5500LV Scanning Electron Microscope at Regional Center for Mycology and Biotechnology (RCMB) at Al-Azhar University. Transmission Electron Microscopy (TEM): Specimens of each fungal isolate were examined using a JEOL 1010 Transmission Electron Microscope at 80 KV at Regional Center for Mycology and Biotechnology (RCMB) at Al-Azhar University. Determination of mycelial dry weight The mycelial dry weights were determined after filtration through dry pre-weighed filter papers and washing carefully with distilled water three times for medium removing, and then dried at 60-70 oC to a constant weight. Statistical analysis :Statistical analysis was carried out using one way Analysis of Variance (ANOVA) (n=3) with post test if P < 0.05 and using software GraphPad InStat 3.06 Guide. RESULTS AND DISCUSSION Isolation of halophilic fungi Two isolates of halophilic fungi were isolated on peptone dox,s agar medium supplemented with different concentrations of NaCl( 0 ­ 30 % w/v), the first one(isolate A) was isolated at 30% 202

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(w/v)NaCl, the second isolate (isolate G) was isolated at 25% (w/v) NaCl. The former required at least 5 % ( w/ v ) NaCl for growth. Colonies of isolate A are white relatively slow growing on peptone Dox ,s agar medium ,malt extract agar medium, Saburad agar medium and oat meal agar medium, (supplemented with at least 5 % (w/v) NaCl) forming some discrete white pustules (Fig. 1).

Fig. 1: Trichoderma piluliferum fs. halophila AZ grown at different concentrations of NaCl.

Fig. 2: Trichoderma piluliferum fs. halophila AZ cells grown at 5 %( w/v). Conidiophores are branching more or less symmetrically at 90o, progressively longer with distance from the tip and branches often at right angles. Conidiophores are flask shape often constricted sharply below the tip (Fig. 2). Conidia are white, hyaline, globose, smooth and sometimes rough. Hyphae are rouphened using oil immersion. Characteristics features of this 203

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isolate resemble the features of Trichoderma piluliferum [7] While T. piluliferum grow on media devoid of NaCl ,isolate A couldn't grow on media devoid of NaCl, so this isolate may be new strain of T. piluliferum. Isolate A may has the name T. piluliferum fs. halophila AZ. It is worth to mention that , this is the first obligate halophilic fungal isolate isolated all over the world.The colony diameter of this isolate was increased by increasing NaCl concentration till 15-20% (w/v) and then the colony diameter began to decrease. No growth was obtained above 30% (w/v) NaCl. Isolate G is characterized by very small colonies on CZA peptone medium (Fig. 3). On NaCl free medium has dirty green color while by increasing NaCl have olive green color .The colony diameter increased by increasing NaCl concentration till 15% (w/v) , then the diameter decreased sharply and the colony color became more darker till 25 % (w/v) NaCl. Poor growth at 30 % (w/v) NaCl. Conidiophores were long , hyaline , and smooth walled with a flask­shaped or hemispherical vesicle, lacking metula ,conidial heads columnar ,conidia narrowly ellipsoidal and echinulate (Fig.4) .Upon morphological and colonial morphology this isolate resembling Aspergillus restrictus [8,11-12].

Fig. 3: Aspergills restrictus grown at different concentrations of NaCl.

Fig. 4: Aspergills restrictus grown at NaCl free medium.

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Until recently, no true halophilic representatives were thought to exist within the kingdom of Fungi. Halophiles characterized based on their requirement of salt for growth in hypersaline conditions. In contrast, halotolerant bacteria do not require NaCl for growth, although they grow in high salinity and in environments devoid of high concentration of salt [13]. Reports have, however, emerged arguing that the orders Dothideales, Eurotiales (Ascomycota) and Wallemiales (Basidiomycota) generally include genera and species adapted to growth under hypersaline conditions that represent part of the mycota of solar salterns [14-18]. Growth of fungal isolates on different concentrations of NaCl Growth of Tichoderma piluliferum fs. halophila AZ was increased by increasing NaCl concentration ,reached its maximum at 20% (w/v) NaCl ,then the growth was sharply decreased by increasing the concentration to 25% (w/v) NaCl. The increase in dry weight was extremely significant compared by 5% (w/v) NaCl till 20%(w/v) NaCl concentration ,while there was extremely significance decrease in dry weight at 25% and 30%(w/v) NaCl compared by 15%(w/v) NaCl .No growth was obtained in medium devoid of NaCl, 5% NaCl was required at least for the beginning of growth of Tichoderma piluliferum fs.halophila AZ (Fig. 5).

Trichoderma

1600

Aspergillus

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1200

1000

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Fig. 5: Influence of sodium chloride concentration on growth of Tichoderma piluliferum fs. halophila AZ and Aspergillus restrictus. The growth of Aspergillus restrictus was initiated at NaCl free medium, the growth was increased by increasing NaCl concentration till 15 % (w/v) NaCl. Then the growth was begun to decrease. No growth was obtained at 30 %(w/v) NaCl concentration. There was a significant increase in dry weight of A. restrictus at 10% and 15% (w/v) NaCl comparing with control (NaCl free medium). 205

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The fungal growth was extremely significant increased by increasing NaCl concentration till 15 and 20 (w/ v) NaCl (respectively). Andrews and Pitt [19] recorded that halophilic and not just halotolerant character was display by Basipetospora halophila, Exophiala werneckii and Polypaecilum pisce since their growth rate in saline medium exceeded that recorded in media supplemented with glucose / fructose or glycerol, also Blomberg and Adler [20] were added the species examined for their growth response in NaCl medium , nearly all adhere to a generalized growth response curve with a distinct growth rate optimum in relation to the NaCl concentration. Morphological and Ultrastructure of the isolated fungi Electron micrograph of Trichoderma piluliferum fs. halophila AZ grown at 5% sodium chloride(Fig.6a) showing rough hyphae ,flask shape conidiophores with smooth and rough conidia, by increasing salt concentration to 15% (w/v) sodium chloride showing conidiophores branching more or less symmetrically near the tip and with a short stipes , branches arising at 90o progressively longer with distance from the tip, branches often re-branching at right angles and often constricted sharply below the tip.This concentration (15­20 %) sodium chloride (w/v) (Fig.6b) appeared as the normal condition of this fungus. The phialides of T. piluliferum fs. halophila AZ differs from the typical pachybasium morphology in being more divaricate .The phialides typically were slightly narrower than the cell from which they arose. Electron micrograph of Trichoderma piluliferum fs. halophila AZ grown at 25% sodium chloride showing rough and spiny mycelia and conidiophores. While hyphae aggregate together and winding with no or little conidiophores, with very small rounded conidia. Chlamydospores are terminal and intercalary in chains.Conidiophores have very small globose and rough conidia, sometimes with papilla (Fig 6c).

(a)

(b) (c) Fig. 6: SEM micrographs of Tichoderma piluliferum fs. halophila AZ at (a) 5% NaCl; (b)at 15% NaCl and (c)at 25% NaCl.

(a =x2.700: and Bar=5µm; b =x2.300: and Bar=10µm; C =x2.700: and Bar=5µm).

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(a)

(b) (c) Fig. 7: SEM micrographs of Aspergillus restrictus at (a) 0.0%NaCl; (b)at 10% NaCl and (c)at 20% NaCl.

(a =x3.700: and Bar=5µm; b =x4000: and Bar=5µm; c =x2.500: and Bar=10µm).

Electron micrograph of Aspergillus restrictus grown on sodium chloride free medium showing that some A. restrictus were unable to produce phialides or conidia and appeared as (naked) sterile heads, these heads become sapthulate to globose in shape (Fig. 7a). Metamorphosed heads of A. restrictus had triangle, or sapathulate head with sterile phialides, sometimes had one row of conidia. Electron micrograph of Aspergillus restrictus grown at 10% sodium chloride showing conidiophores, long, hyaline and smooth -walled with hemispherical or flask shape vesicle lacking metulae (Fig. 7b). Conidial heads columnar, conidia narrowly ellipsoidal ,echinulate , while at 20% sodium chloride showing flask shape phialides with chain of globose rough conidia (Fig. 7c). Vesicles have cylindrical shape and completely covered with one row of short flask shaped phialides with one or no conidia. Conidia are globose with rough surface. Sometimes vesicles are sterile (naked). The same result again this concentration of sodium chloride (15 ­ 20 %) appeared as normal conditions of this fungus too. Metamorphoses in morphological structures under stress conditions were observed under salt and osmotic stress[21],under anaerobic stress [22], and under heavy metal stress [23,24]. Helmy [21] reported that vesicles of A.flavus subjected to 10 % NaCl was no longer able to produce neither phialides nor conidia. Phialides appeared cylindrical to oval shape losing their normal flask shape and covered with spines. Upon subjection of A.flavus to anaerobic conditions Ashoor [22] noticed that the fungal phialides were shorten swollen and pitted losing their normal flask shape became 207

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barrel shape and covered with spines. Vesicles became oblong to sapathulate and very small. ElMeleigy [23] and Osman [24] discussed the effect of heavy metals on different Aspergillus sp, they recorded changes in vesicles , phialides and marked decrease in conidiations.

(a)

(b) (c) Fig. 8: TEM Microphotograph of Trichoderma piluliferum fs. halophila AZ cells grown at: (a&b) 5 %( w/v) NaCl: L.S.in hyphae: b,: conidia. (c) cells at 15 %( w/v) NaCl: (d) T.S.in hyphae at 25% NaCl: CW, cell wall; CM, cell membrane; M, mitochondria; N, nucleus; V, vacuole; Vm, vacuole membrane; cm, conidial membrane; S, septum and ER, endoplasmic reticulum.

(a =x30000: and Bar=500nm; b =x40000: and Bar=500nm; C =x40000: and Bar=500nm d =x60000: and Bar=500nm).

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Thin sections of hyphae of T. piluliferum fs.halophila AZ cultivated on 5, 15,and 25% (w/v) sodium chloride (Fig. 8a-c) appeared normal and their ultrastructures were characterized. The hyphal wall, plasma membrane, nucleus, endoplasmic reticulum and mitochondria appeared electron dense and well developed. A characteristic feature in this fungus was the presence of lipid globules and some granules. Hyphal septa are well formed(Fig. 8a), there are some Woronin bodies near the septal pores.

(a)

(b) (c) Fig. 9: TEM Microphotograph of Aspergills restrictus cells grown at (a)NaCl free medium: (b) cells grown at 10 %( w/v) NaCl, (c&d):cells grown at 20 %( w/v) NaCl. CW, cell wall; CM, cell membrane; C, cytoplasm; M, mitochondria; V, vacuole; Vm, vacuole membrane; S, septum ; P, pigment ; L, lysosome ;W, Woronin body; M, mitochondria; Inv, invagination.

(a =x40000: and Bar=500nm; b =x10000: and Bar=500nm ; C =x50000: and Bar=500nm; d =x40000: and Bar=500nm).

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Examination of ultrathin sections of Aspergillus restrictus cultivated on sodium free medium, 10, and 20% (w/v) sodium chloride appeared healthy and well characterized structure hyphae. Hyphal membrane, hyphal walls, nucleus, vacuoles, mitochondria and dense cytoplasm (Fig. 9ac). Also lipid globules and melanin granules appeared along in front of the cell membrane (Fig. 9a). Many lysosomes appeared in the hyphal cytoplasm. Well developed septa and smaller vacuoles appeared in some hyphae. At 10% (w/v) sodium chloride the ultrathin section (Fig. 9b) of the fungal hyphae was characterized by dense granular cytoplasm, large mitochondria with different shapes, well developed cell wall, while the cytoplasmic membrane appeared undulate along the hyphal wall. The characteristic feature in this concentration ( 15 % (w/v) NaCl is the disappearance of the different shapes of biggish mitochondria, only normal and small mitochondria appeared Also melanin granules appeared in the cytoplasm in front of the cytoplasmic membrane (Fig. 9c). After treatment the unicellular green alga Micrasterias denticulata with 200 mM NaCl for 3 h, mitochondria showed normal shape but an electron dense matrix . In contrast to KCl treatment, dictyosomes did not reveal any specific morphological changes. After 24 h, ER compartments were swollen and dilated and their number was increased. In these cells dictyosomes were disintegrated into numerous small vesicles. Autophagy by enclosure of organilles such as microbodies by double membranes could be observed [31]. CONCLUSION Our studies showed that T.piluliferum fs. halophila AZ and Aspergillus restrictus are promising sources of salt tolerant transgenic for agriculture. Adaptation of T.piluliferum fs. halophila AZ and Aspergillus restrictus for salt stress will be our future work.

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