Aspergillus terreus is a common fungus, which has an important role in desert soils (Amin et al., 2010). Arid soil is complex ecosystem with topographically distinct myco floras. Its microbial communities are subjected to surface heating, cycles of rainfall and extreme desiccation, changing water table, CO₂ enrichment and UV light supplementation and attenuation; it is directly exposed to sun light due to the absence or abundance of vegetation variation (Lipson et al., 1999).

A. terreus is a widely dispersed species among the soilmycoflora; it belongs to the genus Aspergillus, and of the sub-genus Terrei Nidulantes (Raper and Fennell, 1965; Varga et al., 2005). Many isolates of this fungus show morphological variation in growth criteria (Rath et al., 1999). Most of the A. terreus isolates produce a variety of secondary metabolites that are of pharmaceutically significance; they are used for producing lovastatin drug (Casas L´opez et al., 2003). Morphological characters include making weak classification at the species and genus levels, while molecular methods are powerful tools in species identification (Hong et al., 2008).

In advanced fungal molecular studies, ribosomal marker genes have been used, such as universal genes found in all fungi. Fungal communities are commonly genotyped by the multiple genes of rDNA (ITS, IGS, 18S and 25SrRNA, 25S and their introns) (Imran and Al-Asadi, 2014). These genes allow for phylogenetic analysis, and assist in taxonomic classification (Chase and Fay, 2009).

Various genotypic methods have been used to successfully do fingerprinting in several fungi such as A. fumigatus, Fusarium solani and Candida spp. (Crowhurst et al., 1991; Loudon et al., 1993; Nariasimhan and Asokam 2010, Imran and Al-Shukry 2014). Ribotyping analysis of the ITS region revealed detailed information about molecular analysis and showed reproducible polymorphism in several studies (Chase and Fay, 2009). Random Amplification of Polymorphic DNA (RAPD) assay revealed polymorphic DNA patterns of Aspergillus spp. (Anderson et al., 1995). Furthermore, RAPD-PCR assay is adapted for doing fingerprinting of fungi, as it reveals an accurate and simple method for differentiation among fungal isolates (Aubin et al., 1991; Symones et al., 2000; Nariasimhan and Asokan, 2010).

It is not clear whether the variations in the characteristics of the colony of A. terreus isolates are due to environmental response or genetic variation. As a result, there is the need for a reliable molecular ecolo-gical typing of A. terreus isolates in arid soils. Furthermore, the important roles played by these fungi in the arid regions include biodegradation, drug biosynthesis, recycling of materials and microbial activities and are potential source of novel pharmaceuticals (Amin et al., 2010). Few comparable data in the literature are available for A. terreus in the world (Lass-Florl et al., 2007).

The role of genetic variants on phenotypic traits often depends upon environmental and physiological conditions, but such gene-environment interactions are poorly understood. The beneficial role of soil mycoflora is the focus of this study.

Several studies have been attempted to evaluate the ability of A. terreus isolates to produce lovastatin drug (Alberts et al., 1980; Juzlova et al., 1996; Casas L´opez et al., 2003; Miyake et al., 2006). The wild and mutant strain of A. terreus appears to be the most commonly producer of lovastatin, although it uses various ways to produce biological and significant product (Novak et al., 1997; Hajjaj et al., 2001).

Unfortunately, there is no information on the molecular ecology of fungal communities in the desert of Iraq and also there are no previous studies on the role of genetic variants on phenotypic traits of A. terreus based on environmental changes. The aim of our study was to evaluate the phenotypic and genotypic variations based on ITS typing region, using RAPD-PCR of the environmental isolates of A. terreus; it aimed to perform a phylogenic analysis, explains their variations by molecular ecological diagnosis and how this understanding enables fungal diagnosis and screening of lovastatin produced by the wild type A. terreus cultures.

Site descriptions and samples collection

The study area is located around the geographical coordinates of 33° 20' N. 44° 23' E. in Iraq. Western and southern Iraq cover a vast desert area of about 168.000 square km: south-west (Najaf and Kerbela provinces) and north-west (Ramadi and South Mosul provinces) (Figure 1). The common vegetation observed in the area includes: Alhaji, Tamarix and Salsols. Soil pH ranges from 6.8 to 8.8 (Guest and Al-Rawi, 1966).

A total of 45 soil samples (150-200 g each) were collected from October to April 2011-2012, in different localities in the desert regions. Soil temperatures were approximately around 5°C in January, 56°C in August and 37°C in October at the time the collections were made. Soil samples were taken from a depth of 5 cm and stored in polythene bags at 4°C.

Culturing and isolating of A. terreus

The pure cultures of A. terreus were isolated by the serial dilution technique using Potato Dextrose Agar (PDA) medium. Suspected yellow isolates of A. terreus were sub cultured on the PDA medium in separate triplicate plates for each isolate and incubated at 28°C for 7 days (Suhail et al., 2007). Microscopic examination was performed using mounted hyphal inoculums from the colony margins. This was done by using adhesive transparent tape placed on a slide with a drop of lacto phenol cotton blue stain. A. terreus isolates were identified phenotypically using the taxonomic key created by Raper and Fennel (1965). They were maintained on PDA slants at 28°C for four days and were kept in refrigerator at 4°C until use; they were sub cultured every two weeks. The frequency of a fungus is denoted by the number of samplings in which it is recorded against the total: Frequency (%) = No. of observation in which colony appears / total number of observation recorded x 100 (Adhikari et al., 2004).

Genomic DNA extraction

The culture media for each of the 19 A. terreus isolates were frozen for 1 h and tiny portions of the mycelia mat were harvested into 1.5 ml tube. The harvested mats were suspended in 400 µl of lysis extraction buffer (400 mM  Tris-HCl, 20 mM EDTA,150 mM  NaCl , and 0.5% SDS adjusted 8.5 pH) then vortexed for 5 min and added to 10 µL protinase K. Tubes were incubated in 65°C water bath overnight. A mixture of phenol: chloroform: Isoamyl alcohol (25:24:1) was added to the tubes. Tubes were centrifuged at 5000 rpm for 10 min. The aqueous supernatant was transferred to a new tube. An equal volume of cool isopropanol was added and agitated many times; it was centrifuged at 1000 rpm for 10 min. The supernatant was poured out. The pellet containing DNA was rinsed with 70% ethanol. It was air dried; pellets were re-suspended with 100 µL TE and placed in 70°C water bath. 6 µL of RNase A was added, and incubated at 37°C for 30 min. The tubes were centrifuged at 5000 rpm for 2 min. The supernatant was transferred to a new tube and frozen at -20°C until use (Saghai-Maroof et al., 1984; Edwards et al., 1991).

Genotyping of A. terreus

The 19 A. terreus isolates were confirmed by amplification, using specific pair primer: ATE1:CTA TTG TAC CTT GTT GCT GGCG; ATE2 :AGT TGC AAA TAA ATG CGT CGG CGG (Logotheti et al., 2009). Ribotyping of the targeted rDNA (ITS1 -5.8S-ITS2 region) of the 19 isolates was done with primer pairs of ITS1/ITS4 and ITS1/ITS2 that amplified ITS1-5.8S-ITS2 and ITS1 regions. The reaction was performed in a thermal cycler (LABENAT, USA) with 1.2 µL of each primer (20 pmole) and 0.8 µL of genomic DNA; the water was adjusted to a final volume of 25 µL. PCR protocol consisted of the initial denaturation at 95°C for 5 min; followed by 35 cycles at 94°C for 1 min, 59°C for 1 min, and 72°C for 1.5 min; and a final elongation of 72°C for 5 min. Finally, 10 µL of PCR products was loaded onto 1.2% agarose gel that was premixed with ethidium bromide stain (0.5 µg/ml) and TBE running buffer for 45 min at 100 V at room temperature. The products were visualized under a UV transilluminator and then photographed.

RAPD-PCR ecological typing

The primer R108 (5´-GTATTGCCCT-3´), described by Aufauvre-Brown et al. (1992), was used for RAPD-PCR typing of the 19 environmental isolates of A. terreus. Amplification reactions were done in a final volume of 20 µL containing 0.5 µL of genomic DNA, 1 µL Primer (50 pmole) , and 12.5 µL master mix PCR buffer. Water was adjusted to the final volume reaction. PCR was performed in a thermal cycler (LABENAT, USA) with the following temperature profile: 1 cycle of 5 min at 94°C, followed by 35 cycles of 45 s at 94°C, 45 s at 36°C and 1 min  at 72°C and a final extension step at 72°C for 10 min. 10 µL of amplification products were loaded onto 2% agarose gel, which was premixed with ethidium bromide stain  (0.5 µg/ml) and TBE running buffer for 1.30 h at 80 V at room temperature. The products were photographed using a UV transilluminator. (Lass-Florl et al., 2007).

Phylogenetic analysis

A phylogenetic tree dendrogram (UPGMA) of the 19 environmental isolates of A. terreus was constructed by using UVI band software, and the similarity coefficient factor was evaluated according to Ute et al. (1994).

Cultivation of the A. terreus isolates for production lovastatin

Three A. terreus isolates were selected to produce lovastatin. They were selected based on their color variation on agar plate. Production was performed in two complex media: The  first culture was prepared in 250-ml Erlenmeyer flask containing 40 ml of medium A (10 g of glucose, 5 g of corn steep liquor, 40 g of tomato paste, 10 g of oatmeal, and 10 ml of trace elements, 1 g of FeSO₄·7H₂O, 1 g of MnSO₄ · 4H₂O, 200 mg of ZnSO₄·7H₂O, 100 mg of CaCl₂·2H₂O, 25 mg of CuCl₂·2H₂O, 56 mg of H₃BO₃, and 19 mg of (NH₄)₆Mo₇O₂₄·4H₂O per liter of solution (Alberts et al., 1980); it was inoculated with 1·10⁷ conidiospores. The flasks were shaken at 200 rpm for a day at 28°C. The second culture was prepared by inoculating 200 ml of medium B (containing [per liter] 45 g of glucose, 24 g of peptone, 2.5 g of yeast extract)  (Alberts, 1990) with 6 ml of the previous culture in a 1-liter Erlenmeyer flask. The flasks were shaken at 200 rpm and incubated at 28°C for 12 days. For cultivations in fermentors, a 1-litre Erlenmeyer flask containing 200 ml of medium A was inoculated with 4 ml (10⁷) of conidiospore suspension. The flask was shaken at 200 rpm for 1 day and then transferred to the fermentor (Alberts, 1990).

Cultural and microscopic characteristics of A. terreus

A. terreus grew fast on PDA medium. Macroscopic characters of the colonies included difference in their colors, from pale-yellow to dark yellow. There was reverse pigmentation, from yellow to dark gray color. The texture of the colonies showed raised surfaces which are velvety, yet tough, yellowish and powdery (Figure 2a). Microscopic characters did not show more variations among the isolates; conidiophores were typically long, hyaline and smooth giving rise to sub-spherical vesicles that were biseriate. Conidia had smooth wall, and were slightly elliptical) Figure 2b).

Isolation and Identification

A total of 143 desert A. terreus isolated from South-west Najef Province (n=28 isolates), Ramadi Province (n=40), South Mosul Province (n=46 isolates), and Western Kerbela province (n=25 isolates) (Table 1) were studied.

Molecular ecological typing

Molecular diagnosis of A. terreus isolates

The specific pair of primers for A. terreus ATE1 and ATE2 was successfully annealed, and the targeted regions of the 19 environmental isolates of A. terreus were amplified. The PCR product showed monomorphic bands of 450 bp in length (primer included) (Figure 3a).These results confirmed the diagnosis that all the isolates used in this study belonged to A. terreus.

Ribotyping the ITS region for environmental isolates of A. terreus

The targeted rDNA (ITS1 -5.8S-ITS2 region) of the 19 isolates was amplified with primer pairs: ITS1/ITS4 that produced an amplicon length of approximately 550 -600 bp, which was obtained for all the tested isolates of A. terreus (Figure 3b); and ITS1/ITS2 primer that amplified the ITS1 region, which produced an amplicon length of approximately 280-380 bp (Figure 3c).

RAPD-PCR ecological typing

Several bands (1 to 5 bands) in various sizes ranging from about 100 to 1000 base pairs were obtained by using primer R108. This primer generated by RAPD-PCR patterns can discriminate between very closely related environmental isolates, but incidental similarities among the typing of distantly related isolates may also occur. We highlighted that similar typing patterns of bands correspond to the same ecological genotype having the same locus or loci and generating similar or different patterns with identical band sizes in the different environmental isolates of A. terreus (Figure 4).

Phylogenetic tree of the A. terreus isolates based on RAPD-PCR

The results show that there was a wide range of degrees of similarity among the 19 isolates of A. terreus: J and L isolates showed 100% similarity coefficient value (0% distance coefficient), C and D showed 80% similarity, both S, T and N, O showed 68% similarity; H,I isolates showed 50% similarity coefficient value, while the R isolate showed a distinct ecological genotype with coefficient value of 0% similarity. Figure 5 shows all the similarity coefficient values.

Screening of fungal cultures for lovastatin production

The fungal cultures were grown under submerged fermentation conditions to assess their potential to produce lovastatin. From the results (data not shown), it is clear that all the three A. terreus cultures were able to produce lovastatin. Lovastatin production was confirmed by using thin layer chromatography. It was observed that, both commercial lovastatin and the sample spots had approximately same R_f value = 0.32, with light brown in both treatments and standard drug. 

Our study enhances understanding of the impact of extreme environmental factors and shows high variation in the characteristics of the colonies of the A. terreus isolate in each site of study. We also concluded that variations in the colonies in all the soil samples were governed by dominant environmental characteristics of the desert soil such as soil texture (sandy – gravel texture), dry areas, and sites containing varied densities and low diversity of vegetation as well as scarcity of rain  (Guest and Al-Rawi, 1966). The results of this study described the taxonomic criteria that allow rapid typing of extreme environmental factors and shows high variation in the characteristics of the colonies of the A. terreus isolate in each site of study. We also concluded that variations in the colonies in all the soil samples were governed by dominant environmental characteristics of the desert soil such as soil texture (sandy – gravel texture), dry areas, and sites containing varied densities and low diversity of vegetation as well as scarcity of rain  (Guest and Al-Rawi, 1966). The results of this study described the taxonomic criteria that allow rapid typing of high salt concentrations, low pH, and high radiation. Some physical factors also influence fungal growth and metabolite production (Gautam et al., 2009). The biotechno-logical potential of microorganisms to produce is based on their special adaptations to their environment (Gautam et al., 2010). Sunlight, salinity and soil ecology by natural selection, genetic drift and gene flow and gene mutation are representatives of the domain sources of genetic variations (Kurtzman, 1985; Carlile et al., 2001; Fe´ral, 2002; Terry et al., 2004; Lass-Florl et al., 2007; Smith and Kruglyak, 2008).

Molecular typing based on RAPD-PCR patterns used for the 19 isolates of A. terreus showed distinctive patterns. This result agrees with that of Lass-Florl et al. (2007) who classified clinical isolates of A. terreus, using RAPD-PCR patterns. These variations in the pattern may help explain the sources of variation; provide solution to several phenotypic variations in A. terreus colony and explain the difference in colors of reverse pigmentations. A phylogeny tree based on RAPD-PCR profile was sufficient in genotyping A. terreus isolates collected from the arid regions of Iraq; it showed variable degrees of similarity among the 19 isolates of A. terreus and divided them into many genotypes. Only two isolates showed 100% similarity coefficient values. Other isolates showed 0-80% similarity coefficient values (Figure 5). These results agree with those of Lasker (2002) whose genotyped A. fumigatus isolates, and also the results of Raclasky et al. (2006) and Nariasimhan and Asokan (2010).

The RAPD-PCR patterns used for A. terreus isolates were more effective than monomorphic ribotyping patterns used for ecological genotyping (Loudon et al., 1993; Symones et al., 2000). Finally, ecological genotyping find minor differences among isolates at the species to genus level (Birch et al., 1995).

Our results concur with that of Lewington et al. (2007), in which the wavelength of statin produced by fungal isolates ranges between 200-400 nm. On the other hand, this result conflicts with some earlier studies in which pH, medium and choice of wild type or mutant govern lovastatin production. We found that no specific pH or media induce lovastatin production. This result is in line with that of Kumar et al. (2000) who reported that lovastatin is generally produced by batch fermentation in complex media. A. terreus fermentations are typically carried out at 28?C and pHs of 5.8–6.3.

This study may encourage future research of ecological genotyping of closely related environmental isolates. It showed highly discriminatory profiles of RAPD –PCR. RAPD–PCR could identify genetic diversity among closely related isolates in the same species. The molecular genotyping of A. terreus based on ITS region was  reliable,  but  not as discriminating as RAPD – PCR.These methods are useful tools in taxonomical studies, give precise, rapid results with low cost and no time consuming. This study confirmed genotyping as an important method to find solution to fungal ecological diversity problems.

Both authors hereby declare that all actions have been examined and approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.

The authors did not declare any conflict of interest.