Effect of plant extracts on book deteriorated fungal species

Abstract The aim of the study was to evaluate the effect of leaf extracts of four plants against some isolated fungal species from deteriorated books. Aqueous, methanol and chloroform extracts of selected plant species were screened in vitro for their antifungal activity against some book deteriorating fungal species. Fifteen species belonging to 09 genera were isolated and identified from infested books in library. Aqueous and solvent extracts of leaves of Azadiracta indica, Callistemon citrinus, Eucalyptus lanceolatus and Pongamia pinnata were tested against some dominant fungal species viz. Chaetomium spiralis, Alternaria alternata, Aspergillus flavus, Aspergillus niger, Aspergillus fumigatus and Rhizopus stolonifer. Solvent extracts exhibited potent inhibitory activity than aqueous extracts. However, these plant extracts exhibited moderate activity against A. flavus, C. spiralis, R. stolonifer and A. alternata.


Introduction
Fungi are one of the major causes of biodeterioration and economical losses caused by diseases to animal, plants and other valuable cultural heritage like books and papers in libraries, as these books provide nutrient source for the growth of fungi. Due to the biodeterioration, books may be damaged, deteriorated or discoloured in pictures and prints (Hughes 1968;Ohtsuki 1982). The paper attacking fungi are not only cellulolytic, but also includes other fungi as the papers in the production phase or during handling use and acquire various amounts of dirt and debris by the use of this the nutritional requisite of other fungi are often rapidly fulfilled (Kowalik 1980a;Kowalik 1980b;Kowalik 1984).
In addition to cellulose, paper also contains lignin, hemicelluloses, pectin, waxes, tannins, proteins and minerals that are beneficial for the growth of microbes (Dhawan & nigam 2005). Fungal spores present in environment not only contaminate the books, but also cause hazardous effects on the health of visitors who come in contact with the indoor environment of library. To overcome the pre-deterioration and post-deterioration problems, excessive usage of pesticides and fungicides are available in market and were utilised in the conservation of books and documents. Spreading of such chemicals may minimise the population of harmful microflora, but this may affect the health of students, readers, visitors and working staffs in the library. by taking this fact into consideration, use of herbal products as antimicrobial agents may provide the best alternative to the wide and injudicious use of synthetic antibiotics. Therefore, researchers are progressively turning their attention to natural products, looking for new leads to develop better drugs against microbial infections and screening several medicinal plants for their potential antimicrobial activities (Mathur et al. 2011;Gul et al. 2012;Hanif et al. 2013;Gupta et al. 2014;Kakad et al. 2015;Thakur & Shama 2015).

Fungi isolated from infested books
During this period of research, 15 species belonging to 09 genera were isolated from infested books in library which were mostly observed in rainy season. The fungi were isolated from highly damaged, old and unreadable books. Some photographs of infested/deteriorated books from which fungi were isolated are shown in Figure S1, and their colours on books listed in Table 1.
Out of the above-isolated fungi from the infested books, Chaetomium spiralis, Alternaria alternata, Aspergillus flavus, Aspergillus niger, Aspergillus fumigatus and Rhizopus stolonifer were found to be highly responsible for the deterioration of books in library. by taking this fact into consideration, we have tried to find out the effect of aqueous, methanol and chloroform solvent extracts of leaves of Azadiracta indica, Callistemon citrinus, Eucalyptus lanceolatus and Pongamia pinnata on the above-selected fungi isolated from books by antifungal activity. Chaetomium globosum dark green 7.

Extraction yield
As shown in Table 2, the extraction yields of the methanol and chloroform plant extracts varied from 19.8 to 49.4% and 5 to 14.15%, respectively. A wide range of the yields among extracts was observed depending on the extraction solvent and plant material used. The maximum extraction yield obtained was 49.4% from leaves of E. lanceolatus with methanol extract. P. pinnata with chloroform extract exhibited the lowest extraction yield 5%.

Antifungal activity of aqueous extracts
Aqueous extracts of each of the four samples were tested against C. spiralis, A. alternata, A. flavus, A. niger, A. fumigatus and R. stolonifer. All the aqueous plant extracts were found to be more effective against C. spiralis and A. alternata as compared to the other fungal species. Treatment with 20% concentration of aqueous extract of A. indica resulted in 7.69% inhibitory activity against fungal radial growth of C. spiralis, C. citrinus shows 24.72% inhibitory activity, E. lanceolatus shows 25.27% inhibitory activity and P. pinnata reported 30.21% inhibitory activity against C. spiralis ( Figure  S2(a)). A. alternata inhibited 21.46% by A. indica, 24.39% by C. citrinus, 31.70% by E. lanceolatus and 12.68% by P. pinnata ( Figure S2(b)). A. indica shows 20% inhibitory activity against A. niger, C. citrinus shows 23.80% inhibitory activity, E. lanceolatus shows 19.04% inhibitory activity ( Figure S2(c)), but Pongamia pinnata does not show any inhibitory activity against A. niger.
The percentage inhibitions (%) of all the aqueous plant extracts on the tested fungi are as shown in Table 3.

Antifungal activity of solvent extracts
Among the two solvents tested, methanol extracts recorded significant activity as compared to chloroform extracts.
The chemical fungicide, Clotrimazol, shows 100% inhibitory activity against all the tested fungal species. Clotrimazol was used as standard antifungal agent tested at recommended dosage of 2 grams/litre.
The percentage inhibitions (%) of all the solvent plant extracts on the tested fungi are as shown in Table 4.

Isolation of fungal species from deteriorated books
The culture media employed for the isolation of fungi from damaged book materials were: • Potato Dextrose Agar (PDA) • Czapek Dox Agar (CzA)  For the isolation of fungi from damaged book materials, the following two methods were employed: (i) Direct Observation Method (ii) Cotton Swab Method

(i) Direct observation method
In this method, the cellotape pressed against the infested materials. The tape was peeled off and affixed to a clean glass slide (Samson, 1985). before examination, some drops of lactophenol cotton blue stain were incorporated between the tape and the object glass. In other way, the moulds present on books or papers/pages were taken off on slides with sterilised needles and were stained with lactophenol cotton blue stain and the fungi materials were observed under the microscope.

(ii) Cotton swab method
The sterilised cotton swabs were gently rubbed over the affected areas of the papers/pages of books and then pressed on sterilised Petri plates containing PDA and CzA media with dissolved streptomycin to exclude bacterial colonies. The plates were sealed with paraffin tapes and kept for 4-5 days in the biological incubator at 28 ± 1° C to allow fungal growth and were regularly examined. After the occurrence of number of colonies on mother plates, each colony was inoculated on the separate Petri plates. The plates were sealed with paraffin tapes and kept for 4-5 days in the biological incubator at 28 ± 1 °C to allow fungal growth (Leben & Keitt 1950;Dhawan & Agrawal 1986;Agrawal et al. 1988;Garg 1995). Then the fungal isolates were transferred to slants. The fungal species were identified with the help of an identification manual and identification handbook (barnett 1969; nagamani et al. 2006).
The leaves of A. indica, C. citrinus, E. lanceolatus and P. pinnata were used in this study to treat the fungi isolated from infested books in libraries. These leaves were washed thoroughly 2-3 times with running tap water and once with sterile distilled water, shade dried at room temperature. After complete drying, leaves were ground to fine powder with the help of mixer grinder machine, percolate through double layered muslin cloth and were used for preparation of solvent extracts.

Aqueous extraction
For aqueous extraction, about 100 grams of powered materials were mixed with 100 ml of sterile distilled water and kept on a rotary shaker for 12 h. Thereafter, it was filtered through a double-layered muslin cloth and then centrifuged at 4000 g for 30 min. The supernatant was filtered through Whatman no.1 filter paper and sterilised at 120 °C for 10 min, which served as 100% aqueous mother extract. The extracts were preserved aseptically in refrigerator at 5 °C for further use (Verma & Dohroo 2003).

Solvent extraction
Soxhlet extraction method was used for solvent extraction. For solvent extracts, 20 grams of plant powder was filled in the thimble and extracted successively with chloroform and methanol by adjusting each solvent extraction as per their boiling point. This process was employed by soxhlet extractor till the solvent in the siphon tube of an extractor becomes colourless. After extraction, extracts were filtered through Whatman no.1 filter paper then all extracts were evaporated to dryness under reduced pressure at 40 °C in oven and preserved at 5 °C in airtight brown bottles for further use.

Determination of extraction yield (% yield) of solvent extracts
The yield (%, w/w) from all the dried extracts has been calculated as (Díaz Dellavalle et al. 2011). where W1 is the weight of the extract after evaporation of solvent, W2 is the weight of the plant powder.

Preparation of fungal culture
The fungal species isolated from books were maintained on potato dextrose agar (PDA) medium at 27 ± 1 °C.

Aqueous extract
PDA medium with 20% concentration of 500 μl aqueous extracts of four different plants' leaves were poured into sterile Petri plates containing 15 ml of PDA media, and allowed to cool and solidify. Five millimetre mycelial discs of previously maintained seven-day-old cultures of test fungi were cut with sterilised cork borer from margins of actively growing culture of test fungi and transferred aseptically in the centre of the Petri plates containing media and incubated at 25 ± 2 °C for seven days. The PDA medium without aqueous extract but with the same concentration of sterile distilled water was treated as a control.

Solvent extract
One gram of chloroform and methanol solvent residues of four different plants' leaves was dissolved in 10 ml of chloroform and methanol, respectively. About 20% concentration of 500 μl of each solvent extracts was amended with 15 ml of PDA media per plate before solidification of the media. Pure chloroform and methanol (500 μl) amended with the medium served as control. After solidification, 5-mm mycelial discs of previously maintained seven-day-old cultures of test fungi were cut with sterilised cork borer from margins of actively growing culture of test fungi and transferred aseptically in the centre of the Petri plates containing media and incubated at 25 ± 2 °C for seven days.

Determination of antifungal activities
The diameter of the colony was measured in cm. These antifungal activities were done in triplicates. The percentage inhibition of the mycelial growth was calculated by the following formula (naz et al. 2006).

Conclusion
Among the aqueous plant extracts tested, E. lanceolatus and C. citrinus showed the most observable antifungal activity than the other two aqueous plant extracts. extracts of C. citrinus produced nearly similar inhibitory effects against C. spiralis and A. alternata. Out of the total six fungi tested A. flavus, A. fumigatus and R. stolonifer failed to show any considerable antifungal activity to all the four aqueous plant extracts. However, C. spiralis and A. alternata exhibited moderate activity to all the four aqueous plant extracts, but A. niger is the only one who exhibited antifungal activity to the three aqueous plant extracts except P. pinnata. Methanol extract of E. lanceolatus was the only plant extract which could inhibit the growth of C. spiralis. The strong inhibitory activity of chloroform extract of this plant against R. stolonifer was also noticeable. Chloroform extract of A. indica and methanol extract of C. citrinus produced nearly similar inhibitory effects against C. spiralis and was strongly inhibited by methanol extract of E. lanceolatus. Similarly, methanol extract of A. indica and E. lanceolatus showed similar inhibitory activity against R. stolonifer but was strongly inhibited by chloroform extract of E. lanceolatus. From the results obtained, it is concluded that methanol extracts of all the four plant materials exhibited maximum efficiency than the chloroform and aqueous extracts for preventing the growth of most of the tested fungi. Methanol was found to be the best solvent for the extraction because polyphenolic compounds such as flavonoids and most other reported bioactive compounds are generally soluble in polar solvents such as methanol.