Fungicidal Activity of Some Metallic Ions, Fungicides and Essential Oils for Preventing Biodeterioration of Old Manuscripts

Aims: The main goal of this work to investigate the fungicidal activity of some metallic ions and essential oils which were to be applied as alternative protective of synthetic fungicides for old manuscripts and documents. Place and Duration of Study: Sample: Studies were conducted at three floors (as indoor sampling sites) of the National Library and Archives of Dar El-Kottob, Egypt during one year, between November 2012 to October 2013. Methodology: By using the Food Poisoned Technique 11 metallic ion, 5 fungicides and 5 essential oils were used to investigate their effective against the two selected fungal isolates Fusarium oxysporum and Trichoderma viride to evaluate these protective agents by measuring their effect on fungal mycelial (linear) growth and cellulolytic activity. Results: The results revealed that of 11 metallic ions tested only metallic ions of CoCl2, FeSO4, Original Research Article Ahmed et al.; JAMB, 3(2): 1-14, 2017; Article no.JAMB.33853 2 NiCl2, CuSo4 and ZnSO4 at 100 mM were completely inhibited the linear growth of F. oxysporum, while the same concentration of CoCl2, FeSO4, FeCl3 and NiCl2 also completely inhibit the growth of T. viride. For cellulases activity, all metallic ions at different concentrations inhibit the activity, except CoCl2, which increased the avicelase enzyme activity of F. oxysporum. Results for T. viride were little different than F. oxysporum. In vitro effect of fungicides, formaldehyde was found to be toxic to F. oxysporum and T. viride causing complete inhibition at all concentrations with percentage inhibition of 100%. Present study also indicated that, all tested essential oils were found to highly effective and gave 100% reduction in the growth of the two tested fungi at the higher concentration of 0.4%. The Anise essential oil was most effective against F. oxysporum and T. viride responsible for 91.80 and 100 mean % inhibition respectively followed by Rocket essential oil responsible for 82.90 and 89.97 mean% inhibition respectively. Conclusion: Metallic ions (CH3COOH)2 Pb completely inhibited the linear growth of F. oxysporum and T. viride at low concentration (10 mM), as well as formaldehyde which gave percentage inhibition of 100% for both organisms. Anise essential followed by Rocket gave 100% reduction in the growth of the two tested fungi at the higher concentration of 0.4%.


INTRODUCTION
Although several heavy metal ions are trace elements necessary for the growth of fungi, however at high concentrations they are toxic. The toxic effect of metals upon the growth and activity of microorganisms may result from the fact that metals can bind to various biomolecules by covalent and coordinate bonds. Metals may also un-specifically affect many cell structures and influence metabolic processes through a blockage of enzyme activity [1,2]. The ions of Fe, Mn at concentrations of 10 and 20 ppm did not cause decreased in the growth of Trichoderma viride [3]. Several researches reported that, 100% growth of T. viride up to the concentration of 3g /L of Cu (II) only 55% and 14% growth were observed at 4 and 5g/L of Cu (II) respectively. Also, the responses of Trichoderma isolates to zinc ions were connected with concentrations of this metal [4]. Zinc ions at 1000 and 3000 ppm inhibited the mycelial growth and spore germination of Trichoderma, while Zn at 16 ppm did not decrease the growth [3]. In addition, zinc apparently inhibited mycelial growth, whereas manganese ions stimulated spore germination of T. viride [5].
Additional work declared that, calcium chloride at concentrations of 1% and 2% gradually reduced mycelial growth of F. solani and F. oxysporum by 22.2% and 33.3%, respectively, whereas no further reductions were observed at a higher concentration (4%) [6]. On the other hand, some researchers found that Fe ++ , Ca ++  Endoglucanase activity of Trichoderma viride was stimulated slightly by Mg +2 , Co +2 , Fe +3 , Ca +2 and Zn +2 , and strongly by Co+2 and reached the highest level when activated by 75 mM of Mg+2, [8,9]. Cellulase activity of T. harzianum was greatly inhibited by Cu +2 , Ag + and Hg +2 [10], while Fpase (Filter paperase) and CMCase (Carboxy Methyl Cellulase) enzymes were completely inhibited in the presence of 10 μM Hg +2 . The inhibitory effect of Hg +2 on purified cellulases of T. viride were shown whereas; the enzymes were activated by Co +2 and Mn +2 at a concentration of 1 mM [11].
A fungicide is an agent or a chemical that kills the fungi. Therefore, it was necessary to use a less toxic biocide or scheme but with an equally efficient effect on microbiota [12]. Specialists in the laboratories of museums, libraries, and archives most often applied biocides used in other fields: medical disinfectants and chemical protection of plants [13].
Fumigation by formaldehyde at 1.5% has been used in the treatment of 8.1 million books in Russia, but it was restricted due to its toxicity and irritation effect [14]. He also reported that, thymol vapor was used extensively by many conservators using a chamber named "thymol cabinets." This compound is no longer used because of its health hazard and deleterious effects on the objects. Rizolex and Topsin have been used successfully against F. oxysporum [15] and Trichoderma spp. [2,16]; they gave complete inhibition in-vitro at 100 ppm for blocking mycelium growth and spore germination. Mancozeb (Dithane M-45) was the most effective chemical against F. oxysporum and F. solani, only at higher concentrations of 50 and 100 ppm [17]. On the other hand, mancozeb could not inhibit the growth T. viride [18]. Copper oxychloride showed weak inhibition against Trichoderma spp, as T. viride and T. harzianum [19,20].
Great importance is given to essential oils by the industry and scientific research for their antifungal activity and safety which make them useful as natural preservatives [21,22,23,24]. The antifungal activity of essential oils of thyme, rosemary, lemongrass, armoise, clove, boldo, eucalyptus, ravensare, lavender, tea tree, thuya, wormseed and their main components were tested in the vapour phase [25,26,27].
Thus, the present study explore the in-vitro efficacy of some metallic ions, fungicides and essential oils for their antifungal and cellulases activities against F. oxysporum and T. viride isolated from deteriorated old documents.

Fungal Culture
Through studies on fungal isolation involved on biodeterioration of ancient manuscripts, two isolates of fungi namely Fusarium oxysporum and Trichoderma viride were isolated and identified [28]. The two fungal isolates were maintained on Czapek´s agar medium supplemented with 5% avicel and were further confirmed by the Department of plant pathology, NRC of Cairo, Egypt.

Antifungal Activity
Antifungal activity of microelements, fungicides, and essential oils were studied on the selected fungal isolates by mixing them with the culture using a contact assay method.

Effect of microelements on fungal mycelial (linear) growth
Sterilized PDA solid medium amended with serial concentrations of some metallic ions, as MgSo 4 , MnSO 4 , CuSO 4 , NiCl 2 , ZnSO 4 , CoCl 2 , CaCl 2 , FeSO 4 , FeCl 3 , NaCl and (CH3COOH) 2 Pb at concentrations of 10, 50, and 100 mM was tested. Microelements were added to the medium before autoclaving and the experiment was performed in triplicates. The linear growth was estimated until any plate was completely covered by fungal growth. The efficacy of the microelements was expressed as % of inhibition of mycelial growth over control, using formula by [29].
Where: IP = Inhibitory percentage (%). C = Average colony diameter in Check (control). T = Average colony diameter in treatment.

Effect of fungicides on fungal mycelial (linear) growth
The efficacy of each of five fungicides (Rizolex-T50, Topsin-M70, mancozeb-64%, Cuprosan 35% WP, and Formaldehyde) against selected fungal isolates was conducted with the method recommended for laboratory tests with fungicides [30]. Chemical formulas of these tested fungicides are given in Table 1.
Potato dextrose agar (PDA) molten medium was mixed with different fungicides at concentrations 0, 200, 400, 600, 800 and 1000 ppm of each, and a set of three replicates were used for each concentration. The radius of the mycelia growth in each concentration and the inhibition percentage was calculated as mentioned before.

Effect of essential oils on fungal mycelial (linear) growth
The efficacy of each of five essential oils (Anise, Fennel, Rosemary, Rocket and Tea tree oil) against the selected fungal isolates were evaluated using formula of [29]. Potato dextrose agar (PDA) molten medium was mixed with different essential oils at concentrations of 0, 0.05, 0.1, 0.2 and 0.4% (v/v) of each, and a set of three replicates were used for each concentration. The radius of the mycelia growth and the percentage of inhibition were calculated as mentioned before.

Preparation of essential oil
Seeds (250 g) of Anise (Pimpinella anisum), Fennel (Foeniculum vulgare) and Rocket (Eruca sativa), and the leaves of Rosemary  [31]. Two layers were formed, upper organic layer of oil and lower aqueous layer of water. Lower aqueous layer was discarded and upper layer of oil was collected. The resulted essential oil of each treatment was separately dehydrated with anhydrous sodium sulfate [32] and preserved in a sealed vial at 4°C until further analysis of alkanes.

Statistical Analysis
The collected data were statistically computed using the software Mstate-c for Windows. Results were expressed with the standard error of the treatment means for 95% confidence limits.

RESULTS AND DISCUSSION
The two isolates of Fusarium oxysporum and Trichoderma viride isolated from deteriorative old documents in the storage area of National Library and Archives of Dar El-Kottob, Egypt were used for the experiment. The choice of Czapek's medium, pH and temperature were made from our earlier work as the most potent growth medium and environmental factors for the test in terms of radial growth rate.

Influence of metallic ions on growth
The effects of thirteen aforementioned metal ions at different concentrations on the growth and cellulolytic activity of F. oxysporum and T. viride were evaluated. As seen there was a general trend to reduce the linear growth of the two fungi as the dose of metallic ions applied to medium increased, except MgSO 4 , NaCl and CaCl 2 which showed the same linear growth of T. viride as control Table 2. Metallic ion of (CH 3 COOH) 2 Pb at low concentration (10 mM) proved in this concentration to be the most effective metallic ion as it completely stopped the linear growth of both fungi. Data also showed that, the growth rate of F. oxysporum significantly decreased at the higher level (100 mM) of CoCl 2 , FeSO 4 , NiCl 2 , CuSO 4 , FeCl 3 and ZnSO 4 metallic ions, while the growth of T. viride at the same concentration of CoCl 2 , FeSO 4 , FeCl 3 and NiCl 2 was also completely inhibited. The other metallic ions were less effective on the growth of both fungi at different concentrations. The results were in agreement with those recorded by many authors [33,34]. Similar results were also observed by [17,35] who emphasized that, Zn, Mn, and Cu as sulphate reduced the linear growth and sporulation of F. oxysporum and T. viride. Also, present results are greatly supported by [36,37] who stated that, although micronutrients are essential for microbes, however elevation of concentrations above certain threshold rendered them toxic to microorganisms.

Influence of metallic ions on cellulolytic activity
It was shown from Table 2 that avicelase activity of F. oxysporum was increased by adding NaCl at (10 mM), CoCl 2 at (100 mM), and MgSO 4 at (100 mM) more than the control. On the other hand, the addition of other used metal ions to the medium affected adversely the activity of avicelase produced by F. oxysporum. Concerning T. viride the same result was obtained for the same metal ions of CoCl 2 at (50 & 100 mM), CaCl 2 at (10 and 50 mM), NiCl 2 at (10 mM), and MnSO 4 at (10, 50 and 100 mM). It is clear also that all metallic ions added to the culture media of F. oxysporum and T. viride affected drastically the CMCase activity in the culture filtrate, except, metallic ion of CoCl 2 at (100 mM) which increased the CMCase activity of T. viride more than the control. While, the effect of these metallic ions on Fpase was little different, as the Fpase activity of F. oxysporum was slow increased by adding CoCl 2 at all concentrations (10, 50 and 100 mM), MgSO 4 at (10 and 50 mM), CaCl 2 at (10 mM). On the other hand, data showed that, the addition of other used metal ions to the medium affected adversely the activity of Fpase. Concerning T. viride results showed that, there was an increase in the activity of Fpase in the filtrate more than the control by adding CaCl 2 and NaCl at (10 mM), MgSO 4 and MnSO 4 at (50 and 100 mM), while the other tested metals affected badly the activity of Fpase.
These results are in agreement with other scientific researchers, who stated that; the salts had been noticed directly related to metabolism, stimulation or inhibiting enzyme production in microorganism [38]. Previous studies detected an increase in the activity of cellulases of T. viride by adding cobalt up to 10 ppm and then declined as cobalt was further increased to 100 ppm [3].
The production of endoglucanase (CMCase) by F. oxysporum was enhanced in the presence of MgCl 2 , CoCl 2 , MnCl 2 and CaCl 2 metal cations at 10 mM, while inhibition was occur to some extent by FeCl 2 and to great extent by HgCl 2 [39].
The effect of balance between different metal ion concentrations could be more important than their individual effects [40].

Effect of Fungicides on the mycelial Growth
The effect of systemic and non-systemic fungicides (Topsin M70, Rizolex, Mancozeb and Cuprosan) and formaldehyde were evaluated for their efficacy on mycelial growth of F. oxysporum and T. viride by food poisoning technique. Based on the experiment it was found that the effect of fungicides upon growth depends on both the concentration and the fungal isolate (Tables 3 and 4).

Effect on F. oxysporum mycelial (linear) Growth
The analysis of the obtained results revealed that formaldehyde caused complete inhibition to F. oxysporum at all concentrations with percentage inhibition of 100%, so it was considered the most effective fungicides ( Table 3). The present results agreed with Mishchenko et al. [41] who stated that, formaldehyde was the most effective in suppressing growth of several fungi.
Generally, growth of tested fungi was reduced gradually as the fungicide concentration increased. Mancozeb and Topsin gave complete inhibition to F. oxysporum at concentrations of 200 ppm and 1000 ppm, respectively with percentage inhibition of 100%. While, Rizolex and Cuprosan applied to the medium significantly decreased the linear growth from 90 mm in control to 12.5 and 16.3 mm at 1000 ppm with percentage inhibition of 86.1and 81.1%, respectively. The results of this investigation partially confirm those of previous authors [42,43,44].
The inhibitory effect of Cuprosan on F. oxysporum was also observed by many investigators [45,46]. As reported by Dłużniewska [2] fungicides containing copper ions block the enzyme activity in the energy processes.

Effect on T. viride mycelial (linear) growth
The analysis of obtained results revealed that Topsin M 70% was considered the best fungicide causing complete reduction of mycelial growth at low concentration and Mancozeb at 800 ppm with percentage inhibition of 100% (Table 4). Rizolex and Cuprosan at higher concentration of 1000 ppm caused percentage inhibition of 72.2%, and 65% respectively. Almost, similar results were obtained by several investigators [2,47,19,48]. The differential response of T. viride to various fungicides in the present study might be due to their inherent resistance to the fungicides, and their ability to degrade these chemicals [48].

Antifungal Activity of Essential Oils
Essential oils were also evaluated in the laboratory for their inhibition of mold growth of the two tested fungi. The analysis of the obtained results revealed that there was a wide variation in the linear growth of F. oxysporum which ranged from 7.33 to 58 mm (Table 4 and Plate1) and between 0.0 to 75mm in T. viride (Table 4 and Plate 2) according to the essential oil and its concentration.

viride
Anise and Rocket showed the highest effect on F. oxysporum, as the linear growth were 34.33, 16.00 and 7.33 mm at 0.05, 0.1 and 0.2% respectively for Anise and were mm respectively for Rocket. The same trend was also observed on the effect , although the higher concentration (0.2%) of the two essential oils caused complete inhibition to linear growth of hese results are conformity with the Anethole was found in anise as the main compound, and this compound has more fungicidal effect, the same results were also reported before Besides, Shukla and Tripathi [61] trans-anethole from anise essential oil was found to be responsible for its antifungal activity. Also, Sabry [ oxysporum viride found in anise as the main compound, and this compound has more fungicidal effect, the same results were also reported before [52][53][54][55][56][57][58][59][60]. [61] reported that anethole from anise essential oil was found to be responsible for its antifungal activity. Also, and Khoobchandani et al. [62] reported that, erucin followed by Carvacrol and Thymol were the main components of rocket as erucin accounted for approximately 78.69% of the rocket extracts which play an important role as an antifungal agent. Previous studies have highlighted rocket as a rich source of glucosinolate compounds which have a great role in the antimicrobial activity [63].
In contrast to Saleem et al. [64] who reported that, essential oil of anise show moderate and weak inhibition effect against T. viride.
It is well known that, essential oils have antimicrobial properties [25,65]. Antimicrobial activity of these oils can be attributed to the presence of an aromatic nucleus and a phenolic OH group that are known to be reactive and can form hydrogen bonds with -SH groups in the active sites of target enzymes, resulting in deactivation of enzymes in fungi [66,67]. They thought it may be the result of phenolic compounds of essential oils that cause an altering of microbial cell permeability by interaction with membrane proteins. This would cause a deformation in cell membrane and functionality, and permit the loss of macromolecules from their interior [68,69].
The Tea tree essential oil comes at the third level against the growth of the two fungi as the linear growth of F. oxysporum and T. viride were 17.33 and 13.33mm at the higher concentration (0.2%) respectively. The antifungal activity of tea tree oil had been reported by many authors [28,70]. Contrary with Yang et al. [25] who reported that, Tea tree vapors did not inhibit the growth of mold fungi (Aspergillus niger, Trichoderma viride, and Penicillium chrysogenum).
Present work showed that Rosemary and Fennel have moderate effect on the growth of F. oxysporum and T. viride, as the linear growth of F. oxysporum were 38.0 and 26.0 mm at concentration of 0.2% respectively and were 24.0 and 20 mm for T. viride at the same concentration respectively. Similarly, the antifungal effects of Rosmarinus officinalis oil can be attributed to the monoterpens combination and in particular α-pinene whose antifungal effects of this combination has been proved by Okamura et al. [71,72]. Rosemary vapor was found by other workers to be inhibiting in-vitro against T. viride and P. chrysogenum for 12 weeks. These findings suggest that ketone volatilization may play a role in preventing spore germination for rosemary oil [25].

CONCLUSION
The main goal of this work to investigate the fungicidal activity of different microelements (metallic ions), commercial fungicides and essential oils. Present study revealed (CH3COOH) 2 Pb, completely inhibited the linear growth of F. oxysporum and T. viride at low concentration (10 mM). The present study also detected that Formaldehyde followed by Mancozeb were the most effective fungicides inhibiting the growth of the two fungi at low concentration (200 ppm). Regarding to essential oils the study noted that, Anise and Rocket oils showed the highest effect on the linear growth of F. oxysporum and T. viride. composition, antimicrobial and antioxidant activities of the essential oil and the ethanol extract of