BIOLOGICAL ACTIVITES AND FUNDAMENTAL VARIATIONS BETWEEN FUNGAL ISOLATES BELONG TO ASCOMYCETES

Sexuality in fungi has long been a matter of concern and debates that always necessitated extensive analysis of the relationship between organisms assumed to represent different developmental forms of the same organism. Random amplification of polymorphic DNA (RAPD), amino acids, fatty acids and secondary metabolites profiles were performed for four isolates belong to ascomycetes fungi; Aspergillus nidulans and Aspergillus chevalieri and their teleomorph Emercilla nidulans and Eurotium chevalieri. Comparison of their activities as antibacterial and antifungal potency against many of Gram positive, Gram negative bacterial and several fungal species including yeast were determinate. Changes in fatty acid, secondary metabolite and RAPD profiles of sexual and their corresponding asexual isolates were observed. Oleic acid was of lower concentrations in A.nidulans and A. chevalieri than in E. nidulans and E. chevalieri while the opposite was observed for linoleic and linolenic acids. RAPD bands of molecular weights of 559 and 790 bp were the only different ones between A. chevalieri and E. chevalieri using primer 5 while those of molecular weights of 1239 and 1757 bp using primer 3 as well as that of 1209 bp using primer 5 represented the only different bands between A. nidulans and E. nidulans. Some intraand extracellular secondary metabolites were undetected in the imperfect isolates while were detected in the corresponding perfect ones as contract with amino acids percentage detection where exhibited in imperfect isolates much more than perfect one. Deep relationship among cleistothecia formation, amino acids, fatty acid and secondary metabolite biosynthesis has been shown. Study of biological potency of four ascomycetes fungi exhibited great activities of Emercilla nidulans and Eurotium chevalieri (cleistothecia producing isolates) against Gram negative bacterial growth and yeast like fungi on contrast Aspergillus nidulans and Aspergillus chevalieri (two non-cleistothecia producing ones) show high inhibition of growth of Gram positive bacterial and filamentous fungi isolates.


INTRODUCTION
Fungi are ubiquitous eukaryotes that are estimated to comprise a quarter of the entire biomass on earth and consist of nearly 1.5 million species.They are enrichment with intra and extra cellular chemical compounds including protein and enzymes where they are the primary degraders of cellulose and lignin and devastating pathogens of plants and animals (Pitt and Hocking, 1997).The genus Aspergillus was first described almost 300 years ago and is an important genus in foods, both from the point of view of spoilage, and because many species produce mycotoxins which can cause liver carcinogen and effect on the both central nervous system and kidney function.Although a few species have been used in production of fermented food and the early discovery of their ability to produce organic acids were made at the turn of the century.It is therefore not surprising that the aspergilli used or encountered in biotechnology play a significant role.They are extremely common in stored commodities such as grains, nuts and spices, and occur more frequently in tropical and subtropical than in temperate climates (Calvo et al., 2001).
A unique property of many fungi is their ability to propagate by both sexual and asexual spores.The integration of the teleomorph (meiotic sexual morph) and anamorph (mitotic asexual morph) stages, Aspergillus nidulans (teleomorph: Emericella nidulans) and Aspergillus chevalieri (teleomorph: Eurotium chevalieri) are a homothallic ascomycete with a defined asexual and sexual cycle that has long served as model system for understanding the genetic regulation of asexual development and secondary metabolism in filamentous fungi.The asexual cycle is characterized by the production of haploid conidiophores that bear single-cell asexual spores called conidia.Sexual development commences with the formation of multinucleate globular cells, called Hülle cells that surround the cleistothecium, the ascocarp that contains sexual spores called ascospores (Calvo et al., 2008).
The program of asexual and sexual sporulation is characterized by many developmental stages including temporal and spatial regulation of gene expression, cell specialization and intercellular communication. A. nidulans asexual reproductive cycle can be divided into at least three different stages: (i) a growth phase required for cells to acquire the ability to respond to induction signals (competence phenomenon), (ii) initiation of the developmental pathway and (iii) execution of the developmentally regulated events leading to sporulation.A development-specific array of transcription factors is activated that control the expression of multiple sets of genes required for conidiophore morphogenesis.Sexual fruiting body formation is influenced by several environmental and genetic determinants; however, the molecular pathways for this developmental stage are not well dissected (Braus et al., 2002).
Many studies are focusing on the characterization of mutants defective in sexual reproduction.Normal sexual and asexual development in A. nidulans and A. chevalieri requires the function of the velvet (veA) gene.VeA is known to have a role in activating sexual development and/or inhibiting asexual development, since asexual sporulation in the veA1 mutant is promoted and increased, while sexual development is significantly delayed and reduced (Sambrook et al. 1989).Furthermore, veA1 mutants do not exhibit light-dependent development of conidia and ascospores in contrast to the wild type where light induces asexual and delays and reduces sexual spore production.The veA1 mutant gene differs from that of the wild type by one nucleotide in the initiation codon resulting in a putative truncated protein by 37 amino acids.veA null (ΔveA) mutants do not form cleistothecia; by contrast, overexpression of veA gene leads to formation of cleistothecia even in liquid culture and to formation of fewer conidial heads than in a wild type on solid media (Yager, 1992).

Calvo et al. (2001)
identified two mutants (acoB202, acoC193) in A. nidulans that fail to become competent and are blocked in both sexual and asexual sporulation.These mutants overproduce hormone-like fatty acid derived oxylipins collectively termed psi factor (precocious sexual inducer).Psi factor serves as signals that modulate sexual and asexual sporulation by affecting the timing and balance of asexual and sexual spore development.
Diseases caused by bacteria and fungi are common worldwide and major causes of death, disability, and social and economic hindrance for millions of people.According to World Health Organization, over 9.5 million people die each year due to infectious diseases and up to 19% patients are infected from hospital visits throughout the world.Hospital-acquired infections cause a wide range of severe infections including pneumonia, infections of the bloodstream, urinary tract among other organs of the body.Most of the nosocomial pathogens are difficult to treat because they are resistant to many antibiotics (Talbot et al., 2006).
The treatment of infections caused by pathogenic fungi also faces enormous challenges.In the past 30 years, the incidence of fungal infections has significantly increased around the world, and current antifungal drugs such as polyene macrolides (amphotericin B), azoles fluconazole, miconazole, itraconazole, and voriconazole), flucytosine, and the candins (caspofungin acetate and micafungin), are not ideal in terms of efficacy, antifungal spectrum, or safety.Furthermore, recent reports support that invasive candidiasis and aspergillosis has increased dramatically and spread rapidly (Gullo, 2009).Amphotericin B is efficacious against both candidiasis and aspergillosis.However, it exhibits severe side effects such as renal toxicity (Maschmeyer and Ruhnke, 2004).Hence, the urgent need of new agents to combat bacterial and fungal infections is immense.Fungi offer a treasure trove for the discovery of structurally unique natural products with potential biomedical applications.Recently, we isolated and identified eight secondary metabolites from the fungus aspergillus (Gao et al., 2012).In this study, we report the antibacterial and antifungal activities of these compounds.
It has been noted since the earliest days of fungal manipulation that many species of filamentous fungi readily synthesize complex compounds named secondary metabolites that are putatively helpful but not necessary for survival and whose production is presumably costly to maintain.Natural products are often produced late in fungal development, and their biosynthesis is complex.This complexity is due to a number of factors that affect secondary metabolite production.These include (i) the influence of a number of external and internal factors on natural product biosynthesis, (ii) the involvement of many sequential enzymatic reactions required for converting primary building blocks into natural products, (iii) tight regulation of natural product enzymatic gene expression by one or more transcriptional activators, (iv) close association of natural product biosynthesis with primary metabolism, and (v) close association of natural products with later stages of fungal development, particularly sporulation.Furthermore, the genes required for biosynthesis of some natural products are clustered, perhaps as a consequence of these factors (Calvo et al., 2008).
Lipids have been shown to regulate virulence and development, including both spore and secondary metabolite production in fungi.Shared intracellular signaling pathways for sporulation and secondary metabolite production suggest a common trigger(s) for both these processes.Lipid signals also affect secondary metabolites and sporulation.Among lipid signals, of particular note are the previously mentioned oxylipins.Additionally, the processes of sporulation has been demonstrated to share common regulatory elements, for instance, in Colletotrichum lagenarium, deletion of a mitogen-activated kinase (MAPK) gene lowers both production of conidia and expression of melanin genes (Brodhagen and Keller, 2006).
The genus Aspergillus is a relatively large taxon among the Hyphomycetes, and still many aspects of its biology have not been fully understood.With the increasing awareness of the beneficial and deleterious impacts of aspergilli, not only screening for new members in the genus is required, but also multi-disciplinary investigation of the currently identifiable members ranks high in the field of mycology.Techniques from molecular biology have provided a series of new tools for the analysis of sexuality in fungi, thus in the current study, fatty acid and secondary metabolite profiles as well as RAPD patterns and biological activities as antifungal, antibacterial activities of two Aspergillus cultures uncapable of producing sexual spores (A.nidulans and A. chevalieri) and two other perfect ones (E.nidulans and E. chevalieri) were investigated in an attempt to figure out reasons for cleistothecia unproductivity by the imperfect cultures taking into account these three interconnected parameters.

MATERIALS AND METHODS
A: Fungal isolates used: Aspergillus isolates (Aspergillus chevalieri, Eurotium chevalieri, Aspergillus nidulans and Emercilla nidulans) and all bacterial and fungal isolates used in the present assay for biological activity were obtained from the culture collection of The Regional Center for Mycology and Biotechnology.

B: DNA-Based Techniques:
1-Fungal DNA Extraction Using Qiagen kit: The mycelial growth from 5-7 day old cultures on Malt Extract Agar (MEA) slopes were scraped by using 2 ml of sterile distilled water.The two ml of spore suspension were used to inoculate a 100 ml of Yeast Extract Sucrose (YES) medium in a universal 250ml flask and incubated with gentle shaking (180 rpm at 22<C for 48hrs).The mycelia from the flasks were harvested by filtration under aseptic conditions using a microcloth, washed with sterile distilled water and stored at -20 overnight in a sterile Petri dishes.The mycelia were lyophilized in a Heto lyophilizer system model Maxi Dry Plus.The freeze-dried mycelia were ground in a mortar using a sterile pestle, and the powdery samples were placed in eppendorf tubes (1.5 ml).DNA extraction was conducted using DNeasy kit (Qiagen-Germany) (Sambrook et al., 1989 andScazzocchio, 2006).

2-RAPD-PCR:
Ready to go PCR beads kit (purchased from Amershambioscience) was used to amplify DNA genomic fragments using a thermal cycler machine (gradient Robocycler 96 Stratagene, USA) by combining the lyophilized bead, 25 pmole of each primers, 100 ng DNA as a template in 25ml of total reaction volume.The mixture was then placed to the thermal cycler machine directly to start the appropriate PCR program including a universal denaturation cycle (5 min at 94°C), 45 cycles of annealing/extension reactions (1 min at 94°C, 1 min at an optimum annealing temperature 36°C for each used universal primer and 2 min at 72°C) and cycle of final extension step (5 min at 72°C) was followed by soaking at 4°C.The primers used in this study were supplied with the Ready to go kit and are of the following sequences: Primer3: 5'-d {GTAGACCCGT}; Primer5: 5'-{AACGCGCAAC}; Primer 6: 5 '-d{CCCGTCAGCA} (Sambrook et al., 1989 andScazzocchio, 2006).

D-Secondary Metabolite Analyses:
Seven day old fungal isolates were grown on 100 ml of YES medium for the determination of extracellular secondary metabolites while CYA medium was used for the determination of intracellular secondary metabolites using agar plug technique, incubate for 21 days at 28 o C. Extraction was carried whereas the flasks were aseptically filtered and the filtrate was concentrated using speed vacuum device (Maxi Dry Plus).Methanol/chloroform (1:2, v/v) was used for extraction of fungal secondary metabolites.Concentrated YES broth obtained from fungal cultures was mixed individually with chloroform/methanol.The mixture was shaken vigorously in a separating funnel and left to settle down forming a dense lower aqueous layer containing the secondary metabolites.Each extract was concentrated by evaporation of solvent under reduced pressure using an evaporator at a temperature not exceeding than 50 (±2) °C.Analysis and identification of intracellular and extracellular metabolites were carried out according Paterson and Bridge (1994), using the automatic HPTLC system (CAMAG, model scanner 3-Switzerland).

C-Amino acids Analysis:
Preparation of cell-free extract for amino acids was analyzed according to Barrett and Elmore (1998).Fifty ml sterile MEA medium were employed in each flask and inoculated with fungal spore suspension using 7 days old cultures for each fungus.The flasks were incubated for 7 days at 28ºC.Mycelia were harvested by filtration using a Buchner funnel.The mycelia were then washed thoroughly with distilled water.The harvested mycelia were then ground with clean glass using 70% ethyl alcohol.The obtained slurry was then centrifuged at 6000 rpm for 20 minutes.After centrifugation, the supernatant was decanted to be used for the analysis.Amino acid composition was carried out by using an amino acid analyzer (LC 3000 Eppendorf Biotronik).

E-Fatty Acid Analysis:
Intracellular fatty acids were extracted according Peter and Michael (1996).Gas chromatography analysis was achieved using Dani GL\C-FID 1000 at the Central Laboratory of Ain Shams University.The fatty acid standard was manufactured by Supelco tm containing mixture of 37 fatty acid methyl esters (C4 -C24).

F-Biological activities assay:
Antimicrobial Screening: Disc diffusion method was used for the antimicrobial susceptibility testing.Antifungal potentialities were expressed as the diameter of inhibition zones.Intra and extra of fungal metabolites were examined as antimicrobial agent against twelve bacterial isolates (six Gram positive and other Gram negative) and sixteen fungal isolates including six isolates of yeast.Inoculum suspensions of all bacteria and fungi isolates were spread on the surface media [Nutrient agar "NA" medium for bacterial growth and Malt Glucose Agar "MGA" medium for fungal growth].Six equidistant (1 cm diameter) holes were made in the agar using sterile cork borer in media plates (10x 10 cm), which had previously been seeded with bacteria and/or fungi tested, were filled by 100 µL with each fungal extracts (after evaporation of solvent).Control holes were failed with organic solvents, which were used in the extraction methods.Plates were left in a cooled incubator at 4 (±2)°C for one hour and then incubated at 37 (±2) °C for 24 hour for bacterial growth and 28ºC for 48 hours for fungal growth.Inhibition zones developed due to active fungal extract ingredients were measured after 24-48 hours of incubation (Fagbemi et al., 2009).

RESULTS:
The current work studies biochemical and genomic differences between an asexual culture of A. nidulans and another capable of reproducing sexually (E.nidulans) as well as between A. chevalieri (asexual culture) and E. chevalieri (sexual culture) in an attempt to investigate genomic and metabolic differences between the sexual and the interestingly asexual culture through studying the three interconnected chosen parameters; fatty acids, intracellular and extracellular secondary metabolites representing the biochemical investigations, as well as RAPD analysis using three universal primers (primer 3, 5 and 6) representing the molecular investigations.
Teleomorphs are sexual, or perfect, states of fungi.Aspergillus anamorphs (imperfect states) are found in at least eight teleomorphic Ascomycete genera; however only three of these, Eurotium, Neosartorya and Emericella, occur in foods.All form cleistothecia.The cleistothecia are surrounded by Hülle cells, which are thick, walled, highly refractile, roughly spherical cells resembling chlamydoconidia.Eurotium species are the most common and significant of the foodborne genera with Aspergillus anamorphs.They produce bright yellow cleistothecia and pale yellow ascospores.Heads producing conidia are formed from phialides only (figure 1).RAPD-PCR: Three primers (primers 3, 5 and 6) were used to evaluate the genomic profile of each of A. chevalieri; E. chevalieri; A. nidulans as well as E. nidulans.Exactly the same RAPD pattern was obtained for both A. chevalieri and E. chevalieri using primer 3 (Figure 2 and Table 1).For primer 5, only two bands (559 bp and 790 bp) were different; the band of molecular weight of 559 bp was present in A. chevalieri while absent in E. chevalieri, on contrast the band of molecular weight of 790 bp present in E. chevalieri while absent in A. chevalieri, (Figure 3 and Table 2).Primer 6, like primer 3, resulted in exactly the same RAPD pattern (Figure 4 and Table 3).
In case of A. nidulans and E. nidulans, primer 3 resulted in differences in two bands of molecular weights of 1239 bp (present in A. nidulans while absent in E. nidulans) and 1757 bp (present in E. nidulans while absent in A. nidulans) (Figure 5 and Table 4).Amplification of their DNA using primer 5 resulted in differences in only one band of molecular weight of 1209 bp which was present in E. nidulans while absent in A. nidulans (Figure 6 and Table 5).Exactly the same RAPD pattern was developed using primer six (Figure 7 and Table 6).
Intracellular Secondary Metabolite Profiles: Eight intracellular secondary metabolites were detected.E. chevalieri being the richest in secondary metabolites for detecting seven of the eight secondary metabolites in its cell free extract whereas all secondary metabolites were found except Viridicatum toxin.While, A. nidulans represented the poorest isolate for possessing only three metabolites (ochratoxin A, carlosic acid and 2carboxy -3, 5-dihydroxy phenyl acetyl carbinol).E. nidulans exceeded its anamorph by Viridicatum toxin.As well as, E. chevalieri exceeded its anamorph by schizopaltic, genestic and psoromic acids (Table 7).
Extracellular Secondary Metabolite Profiles: Six extracellular secondary metabolites were detected in the culture filtrate of the investigated aspergilla (Table 8).E. chevalieri was the richest for possessing five of the six detected metabolites followed by E. nidulans.Both Asexual cultures produced poor extracellular secondary metabolites whereas 2-pyruvoylaminobenzamide and rosepurpurine for A. chevalieri while alectronic and α-collatolic acids for A. nidulans.Amino acids analysis: thirty one amino acids were detected.A. nidulans being the richest in amino acids for detecting twenty eight of amino acids in its cell free extract except Glycine; isoleucine and phenyl alanine followed by A. chevalieri were twenty four amino acids were exhibits except taurine; theronine; cysteine; arginine; γamino butric acid; tyrosine and 3-methyl histidine.In addition, E. chevalieri and E. nidulans represented the lowest isolate for possessing only twenty and nineteen amino acids respectively.Serine; alanine; 1-methyl-histidine were absent in E. chevalieri and E. nidulans while present in theirs anamorph that may explant might be used in clestithecia formation (sexual reproduction) (Table 9).
Fatty Acid Profiles: Sixteen fatty acids (eleven saturated and five unsaturated) were detected in the cell free extract of A. chevalieri, A. nidulans and their corresponding isolates capable of cleistothecia production; E. chevalieri and E. nidulans, respectively (Table 10).A. chevalieri represented the isolate with the maximum number of fatty acids (thirteen out of the sixteen detected); only tridecanoic, stearic and oleic acids were undetected, followed by E. chevalieri (twelve detected fatty acids) and then E. nidulans and A. nidulans (each possessing ten fatty acids) (Figure 8).The detected unsaturated fatty acids were palmitoleic, oleic, elaidic, linoleic and linolenic.Palmitoleic was only detected in the cultures producing asexual conidia (A.chevalieri, 3.7 μg/ml and A. nidulans, 6.2 μg/ml) while oleic acid was only detected in the cultures producing sexual cleistothecia (E.chevalieri, 0.9 μg/ml and E. nidulans, 3.73 μg/ml).Linoleic acid was detected in the four investigated isolates with lower concentrations in the sexual cultures (E.chevalieri, 0.1 μg/ml and E. nidulans, 0.2 μg/ml) than in the asexual ones (A.chevalieri, 1.9 μg/ml and A. nidulans, 2.3 μg/ml).Elaidic acid was also present in the four investigated cultures with the highest concentration being detected in E. chevalieri (22.9 μg/ml) while with much lower concentrations in the rest of the investigated isolates (A.chevalieri, 1μg/ml; A. nidulans, 3 μg/ml; E. nidulans, 0.5 μg/ml).

Antimicrobial Assay: antimicrobial potency of intra and extra A. chevalieri; E. chevalieri; A. nidulans and E. nidulans metabolites were recorded in table (11).
Susceptibilities of pathogenic bacterial and fungal isolates to different fungal extracts were investigated by measuring their inhibitory effect on isolates growth, compared to the solvent used.In the current data show that extra-secondary metabolites extracts of asexual fungi "A.chevalieri and A. nidulans" had strong antibacterial and antifungal activities on Gram positive and filamentous fungal growth, while Gram negative bacterial isolates and yeast like fungi exhibited high sensitivity to extra-secondary metabolites extracts of sexual fungi "E.chevalieri and E. nidulans".On the other hands, intracellular metabolites of all four ascomycetes fungi tested had moderate effects against the growth of all bacterial and fungal isolates used.

DISCUSSION:
The genus Aspergillus includes fungi of importance in the food and biotechnology industries, as well as pathogens.Aspergillus is a large genus containing more than 100 ecognized species.Teleomorphs are sexual, or perfect, states of fungi.Aspergillus anamorphs (imperfect states) are found in at least eight teleomorphic Ascomycete genera; however only three of these, Eurotium, Neosartorya and Emericella, occur in foods.All form cleistothecia.It would therefore be of major economic and medical advantage to be able to study their biochemistry as well as the inheritance of genes of interest and to bring together desirable genetic traits in the aspergilli.Unfortunately, such genetic efforts have been impeded because most Aspergillus species are only known to reproduce asexually, thus the sexual cycle cannot be used for strain improvement and inheritance studies (Calvo et al., 2001).In the current study, RAPD patterns of the four investigated fungal isolates, it could be observed that the maximum number of different bands was only two bands using primer 5 with A. chevalieri and E. chevalieri and primer 3 with A. nidulans and E. nidulans.Primer 5 resulted in only one band difference with A. nidulans and E. nidulans.The rest of the investigated primer-isolate combinations resulted in no band differences.This might suggest that the asexual isolates unable of cleistothecia production suffered sexual, fatty acid or secondary metabolite gene mutations as these three criteria are connected.
Regarding the oleic acid was undetected in the imperfect cultures (A.nidulans and A. chevalieri) while was of concentrations of 0.9 ppm in E. chevalieri and 3.7 ppm in E. nidulans suggesting its increased requirement in conidia formation (asexual reproduction).While, in case of linoleic acid, it was of lower concentrations in perfect cultures (0.1 ppm for E. chevalieri and 0.2 ppm for E. nidulans) than in imperfect ones (1.9 ppm for A. chevalieri and 2.3 ppm for A. nidulans) suggesting its increased demand in sexual spore production.The latter was also observed for linolenic acid which was undetected in the perfect cultures while was of concentrations of 3.05 and 1.2 ppm in A. chevalieri and A. nidulans respectively.Also, increase in stearic and decrease in palmitic acids agree with the current results were in cases with increased oleic acid (0.9 and 3.37 ppm for E. chevalieri and E. nidulans respectively) production there were also increased stearic acid (3 and 9.69 ppm for E. chevalieri and E. nidulans respectively) but decreased palmitic acid production (0.5 and 0.2 ppm for E. chevalieri and E. nidulans respectively).It could also be observed that the fatty acid profile of the asexual culture of A. nidulans and A. chevalieri was different than that of their corresponding sexual ones (E.nidulans and E. chevalieri, respectively).This agrees with the results of Tsitsigiannis et al. (2004) who reported that in A. nidulans, mutations in ppoA (encoding the dioxygenase PpoA contributing to the generation of 8-hydroxy linoleic acid, psiBα or 8-HODE) enhanced the ratio of asexual to sexual spore production, reflecting the role of linoleic acid in sexual spore 3.73 -0.9 -Oleic acid (18:1∆ 9 cis) 0.5 3 22.9 1.0 Elaidic acid(18: 1 ∆ 9 trans) 0.2 2.3 0.1 1.9 Linoleic acid (18:2) -1.2 -3.05 Linolenic acid (18:3) production.However, ∆ppoC (encoding the dioxygenase PpoC necessary for optimal production of 8-hydroxy oleic acid, psiBβ or 8-HOE) mutant exhibited an increase in sexual spore production reflecting the role of oleic acid in asexual reproduction.These effects on sporulation are reflected in expression levels of the sporulation-specific transcriptional regulatory genes, brlA and nsdD.
Recently, extra-metabolites of E. chevalieri and E. nidulans exhibited great potency against Gram negative bacterial isolates and yeast like fungi while intra  2012) who we examined in vitro antibacterial and antifungall activities of secondary metabolites isolated from the many fungus belong to Eurotium sp. the results showed mild to moderate antibacterial or antifungal or both activities except E. chevalieri was the best of fungi tested.The ability or inability of the investigated fungal cultures to produce cleistothecia (which also resulted in different fatty acid profiles and concentrations) resulted in different intracellular and extracellular secondary metabolite profiles, where certain metabolites were detected in case of cleistothecia production which were ceased when the culture lost its ability to produce cleistothecia.It has been reported that reproduction in fungi is accompanied by developmental changes among which are changes in secondary metabolite profiles (Brodhagen and Keller, 2006).Fungal secondary metabolism and sporulation are associated both temporally and functionally (Calvo et al., 2001).
The accompanying changes in secondary metabolite and fatty acid profiles between the sexual and asexual cultures with RAPD profile differences agrees with the results of Tsitsigiannis and Keller (2006) who reported that deletion of ppo genes (affecting sexuality) affected the production of at least three different secondary metabolites in A. nidulans, including sterigmatocystin, the antibiotic penicillin and an octaketide, shamixanthone, where for sterigmatocystin and penicillin, these effects were reflected and supported by levels of biosynthetic gene transcription.Comparing the RAPD results with fatty acid results, it could be concluded that variations in the DNA are very low when compared to fatty acid variations, this reflects that it might be the expression of the genes that govern fungal sexuality which might be repressed or blocked, or the parts of the gene regulators are mutated so these isolates need DNA repair (differences in some of the bands with this great DNA similarity might reflect the possibility of some mutations).
Hence mutations in fatty acid synthesis, secondary metabolite synthesis and/or sexuality genes as well as differences in the promoter sequence of either genes (or gene clusters) might be responsible for the ceasing of fungal sexual reproduction especially that it has been recently reported that sexual genes exist in the formerly thought asexual forms (Dyer, 2007).

Table 4 :
Molecular weights of fragments generated using primer 3 for A. nidulans and E.nidulans +, band present; -, band absent.

Table 7 :
Intracellular secondary metabolites of sexual and asexual states of the investigated aspergilla.(+) detected ; (-) not detected

Table 8 :
Extracellular secondary metabolites of sexual and asexual states of the investigated aspergilla.(+) detected ; (-) not detected

Table 9 :
Percentage of amino acids (%) detected in the cell free extract of sexual and asexual states of the investigated aspergilla.(-) not detected

Table 10 :
Concentration of fatty acids (μg/ml) detected in the cell free extract of sexual and asexual states of the investigated aspergilla.(-) not detected

Table 11 :
In vitro antimicrobial activity of fungal intra and extra-metabolites used (measurement of inhibition zones by cm, Ext.= Extracellular metabolites; Int.= intracellular metabolites)Trichosporon cutameum metabolites showed mild effects against the growth of all bacterial and fungal isolates used.That in agree with Char