International Biodeterioration & Biodegradation

Use And Effect of Essential Oils in the Maintenance and Preservation of

Historical Monuments
--Manuscript Draft--

Manuscript Number:

Article Type: Full Length Article

Keywords: Biodeterioration; Black microfungi; Essential oils; Fungal deterioration; Monuments

Corresponding Author: Hacer Sert

Akdeniz University
TURKEY

First Author: Hatice Yıldız Acar

Order of Authors: Hatice Yıldız Acar

Hacer Sert

Abstract: This study has been carried out to determine the antifungal effects of essential oils,
obtained from plants belonging to the Lamiaceae family, on the historical structures
that were corroded by black microcolonial fungi in the Antique City of Side. Black
microfungi taken from historical structures in the ancient city between the years of
2016-2018 and Origanum majorana L., Origanum minutiflorum O. Schwarz et. P. H.
Davis and Mentha longifolia L. subsp. typhoides (Brig.) Harley constitute the subject
of the study. Essential oils obtained from plants, whose antifungal effects would be
examined, were applied on microfungi, which were grown in In vitro conditions, at three
different concentrations (%0,1, %1 and %3) and then the cell counts of microfungi were
made before and after the application. At the end of the study, when the essential oils,
obtained from O. majorana , O. minutiflorum and M. longifolia , were applied on
species of Coniosporium , Sarcinomyces and Phaeococcomyces , which caused
corrosion in the historical structures within the study field, it was determined that their
fungal effects increased as their concentration increased and this suppressed the
growth of these black microfungal species.

Suggested Reviewers: Katja Sterflinger

[email protected]

Anna Gorbushina
[email protected]

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Cover Letter

COVER LETTER

We would like to affirm,

1) that this paper has not been published before in any form;
2) that it is not under consideration by another journal at the same time as IBB;
3) and that all authors approve of its submission to IBB.

Yours sincerely,
14.09.2022
Hacer Sert
Hatice Yıldız Acar
Highlights

Highlights

* Historical artifacts are exposed to the destructive effects of organisms such as algae, lichens,
bacteria, microfungi, as well as physical and chemical factors.

* Especially black microfungi play an important role in the degradation of historical artifacts.

* Black microfungi cause color changes, swellings, fractures and crater-shaped pits in historical
artifacts.

* The antimicrobial properties of essential oils are also effective in preserving historical artifacts.

* Antimicrobial effects of essential oils have been tested on black fungi that cause deterioration in
historical artifacts, and positive results have been obtained.
Manuscript File Click here to view linked References

1 Use And Effect of Essential Oils in the

2 Maintenance and Preservation of Historical
3 Monuments
4
5 Hatice Yıldız ACAR1, Hacer Bakır SERT*2
6
1
7 Akdeniz University, Institute of Science Antalya, Turkey
8 ORCID: 0000-0003-3867-2593
9 *Corresponding author: [email protected]
2
10 Akdeniz University, Manavgat Tourism Faculty, Manavgat, Antalya, Turkey
11 ORCID: 0000-0002-3081-2072
12
13 ARTICLE INFO
14
15 Keywords:
16 Biodeterioration
17 Black microfungi
18 Essential oils
19 Fungal deterioration
20 Monuments
21
22
23 ABSTRACT
24
25 This study has been carried out to determine the antifungal effects of essential oils, obtained from plants belonging
26 to the Lamiaceae family, on the historical structures that were corroded by black microcolonial fungi in the Antique
27 City of Side. Black microfungi taken from historical structures in the ancient city between the years of 2016-2018
28 and Origanum majorana L., Origanum minutiflorum O. Schwarz et. P. H. Davis and Mentha longifolia L. subsp.
29 typhoides (Brig.) Harley constitute the subject of the study. Essential oils obtained from plants, whose antifungal
30 effects would be examined, were applied on microfungi, which were grown in In vitro conditions, at three different
31 concentrations (%0,1, %1 and %3) and then the cell counts of microfungi were made before and after the
32 application. At the end of the study, when the essential oils, obtained from O. majorana, O. minutiflorum and M.
33 longifolia, were applied on species of Coniosporium, Sarcinomyces and Phaeococcomyces, which caused
34 corrosion in the historical structures within the study field, it was determined that their fungal effects increased as
35 their concentration increased and this suppressed the growth of these black microfungal species.
36
37 1. Introduction
38
39 Historical buildings and artifacts are very important for the transfer of the cultures of the societies that lived in the
40 past years. However, these structures are subject to deterioration due to climatic factors such as air pollution,
41 temperature, humidity, wind, heavy rain, and microorganisms such as fungus and lichen. Due to these negative
42 factors, cracks, tears, exfoliation, color changes and abrasions occur on the surfaces of historical buildings and
43 monuments that spoil the physical appearance. Since these factors, which deteriorate the aesthetic appearance and
44 physical structure of historical artifacts, must be determined and controlled, some methods of struggle are being
45 developed. Processes using chemical drugs can often be harmful to structures consisting of marble, sandstone and
46 limestone. For this reason, priority is given to biological control studies. Among these studies, the use of essential
47 oils has come to the fore in recent years (Antonelli et al., 2020; Vito et al., 2015; Fidanza and Caneva, 2019;
48 Neween et al., 2018; Jeong et al., 2018; Palla et al., 2022).
49 Plants and their products are used in many fields (Bal, 2021). The most important areas of use are medicine,
50 perfume, cosmetics and toothpaste, soap, confectionery industry. In addition, they are consumed as oil, tea and
51 spice (Tanker, 1990; Uçan, 2008; Uyanık, 2013). The aromatic and medicinal Lamiaceae family, which includes
52 plants of great importance, is also widely distributed in the Mediterranean basin (Baytop,1999; Biçer et al., 2003).
53 The active ingredients it contains determine the importance of medicinal and aromatic plants. These active
54 substances found in plants, especially essential oils; it may vary depending on the genetic structure of the plant,
55 the climate in which it grows, environmental factors, cultural practices, different parts of the plant, growth periods
56 and temperature changes during the day (Biçer et al., 2003).
 57 Essential oils have antibiotic and antiseptic properties against bacteria, molds and yeasts. Possible mechanism of
58 antifungal activity; it is thought that cell-bound lipophilic compounds are caused by the breakdown of the
59 membrane and the decrease in lipophilicity with the addition of the hydroxyl group (Antonelli et al., 2020; Davis
60 et al., 1988; Di Vito et al., 2015; Fidanza and Caneva, 2019; Tanker, 1990; Uçan, 2008). Structure and function
61 changes are seen in the cytoplasm and membrane, which has the antifungal effect of essential oils. Antifungal
62 agents obtained from plants can stop the enzymatic reactions of microbial metabolism, reduce the uptake of
63 nutrients in the environment. By inhibiting enzyme synthesis in the nucleus, it can change the structure of the
64 membrane and inhibit it at the ribosomal level (Brayner et al., 2006; Di Martino, 2022; Evren and Tekgüler, 2009;
65 Geweely et al., 2018; Gravesen et al., 1994; Pursel, 2020; Radaelli et al., 2016).
66 Historical artifacts are exposed to the mechanical and chemical effects of microfungi. By secreting acid,
67 microfungi cause mechanical perforation on the surface of the stone, and subsequently deterioration of the structure
68 of the stones by growing and crumbling (De Leo et al. 2022; Eckhardt., 1985; Gaylarde et al., 2022; Gorbushina,
69 2022; Gravesen et al., 1994; Isola et al., 2022; Liu Xiaobo, 2022; Sand, 1997; Sterflinger et al. 2018; Urzi and
70 Krumbein, 1994; Urzi, 2018). When fungi come into contact with the stone, they first adhere firmly to the surface.
71 Then they penetrate into the crystal structure of the stone with the help of very thin hyphae. The hyphae form new
72 colonies in the cracks and voids found in the stone. These colonies create high internal pressure as they grow inside
73 the stone; causes the stone to crack and the piece to break and fall. Contamination, adhesion and loss of parts is a
74 recurring process. The hyphae continually move deeper into the stone, forming new colonies in new spaces (Pinar
75 and Sterflinger, 2021; Sert et al., 2007a; Sert et al., 2007b; Sert et al., 2007c; Sterflinger and Krumbein, 1997;
76 Sterflinger et al., 1999; Sterflinger et al. 2018).
77
78 In this study is the antifungal effects of essential oils obtained from plants belonging to Lamiaceae family on the
79 development of members of the genera Coniosporium, Sarcinomyces, Phaeococcomyces, black microcolonial
80 fungi that play an important role in the degradation of historical artifacts. In addition, by preventing excessive and
81 irregular use of synthetic chemicals, it is aimed to minimize the negative effects of chemicals used to protect
82 historical artifacts from microfungi on the structure, human and environmental health, non-target beneficial
83 organisms, soil microflora and soil fertility.
84
85 2. Materials and methods
86
87 2.1. Preparation of the specimens
88
89 The aim of this study is to determine the antifungal effects of essential oils on historical artifacts damaged by black
90 microcolonial fungi, especially Coniosporium, Sarcinomyces, Phaeococcomyces species in the Ancient City of
91 Side (Manavgat, Antalya, Turkey). Origanum majorana, O. minutiflorum and Mentha longifolia subsp. typhoides
92 were extracted with clevenger apparatus, and antifungal activity was analyzed in vitro using direct contact on agar
93 against isolates of Coniosporium, Sarcinomyces, Phaeococcomyces species. Three different concentrations (%0,1,
94 %1 and %3) of essential oils were used and then the cell numbers of microfungi were counted before and after the
95 application. The data obtained in the study were evaluated with Duncan Multiple Range Test. The identification
96 of black microfungi was carried out according to Ellis., 2001a, Ellis, 2001b; Sert et al., 2007. The following
97 literature was used for the identification of plant species performed by morphological methods: Turkey's Flora
98 Davis (Davis, 1985) and Davis et al. (Davis, 1998).
99
100 2.2. Obtaining essential oil
101
102 Antifungal effects of plants we studied were collected and dried in different seasons according to developmental
103 periods (Fig. 1). Drying plants ready to clevenger device to get into smaller parts by passing through the robot is
104 provided (Fig. 2). Oil was obtained by using clevenger device. The oils obtained were collected in sterile glass
105 tubes with the type and plant date of the plants and stored in the refrigerator for use at the appropriate time (Fig.
106 3).
107
 a b
108

c
109
110
111 Fig. 1 a. Drying of O. majorana before extraction. b. M. longifolia (dried). c. O. minutiflorum (dried)
112

a b
113
114 Fig. 2. a. b Powdering of O. majorana
115
116

a b
117
118
119 Fig. 3 a. Clevenger Device (double) b. Essential oil from O. majorana

3
120 2.3. Isolation and Cultivation of Microfungi
121
122 Specimens taken from corroded historical monuments (Fig. 4) in the ancient city of Side were brought to the
123 laboratory. The corroded stone particles were removed with the help of hammer and scalpel and put into the
124 envelopes with the date and locality inscribed on them. For the isolation of black microfungi, stone particles were
125 washed with the %70 alcohol and examined under stereo microscope. Colonies were carefully taken with sterile
126 cannulated needles and planted on dichloron rose bengal (DRBC, MERCK, Darmstadt, Germany) medium. Where
127 the developing black microfungi were then transferred to the malt extract agar (MeA, MERCK, Darmstadt,
128 Germany) medium. Microfungi which were developed and not contaminated were finally cultured in MeA and
129 CzA (Czapek agar, MERCK, Darmstadt, Germany) media (Sert et al., 2007b)(Fig. 4 a ,b, c, d).
130

a
131

b c
132
133
134 Fig. 4. Monuments heavily infected by black microfungi in Side Antique City a. Lion statute b. Column title
135 c. Colonies of microcolonial fungi on marble floor of column title.
136
137
138 2.4 TTC (Triphenyl tetrasolium chloride) Application
139
140 After the samples taken from the study area were arranged by numbering, 0.1%, 1% and 3% essential oil solutions
141 prepared with pure water were applied directly to the stone particles by spraying method. After the oil application,

4
142 stone particles infected by micro fungi were subjected to TTC test. For the control application, culturing and TTC
143 processes were carried out in a certain area of the same sample without applying essential oil. Infected stone
144 particles were incubated for 24-48 hours at 28°C in tubes containing 0.2% TTC solution and covered with
145 aluminum foil outside (to prevent light transmission). The areas colored in red at the end of the incubation period
146 show that there is still vitality in that part. After the application, the stone particles were examined under a
147 microscope in order to observe the development of the hyphae in detail (Fig. 5).
148

a b
149
150
151 Fig. 5. TTC application a. Stone sample in TTC solution b. Observation at stereo microscope after application.
152
153 2.5. Collection and counting of spores
154
155 The colonies were scraped from about 3mm. area with sterile scalpel and transferred to sterile tubes containing
156 %1 Tween 80 solution. Spores were collected from the suspension which was thoroughly homogenized by vortex
157 treatment. The spores collected were counted on thoma slides and suspension was prepared as 6 spores/ml. over
158 10 in 1 ml3. Then 1 ml. of this spore solution and transferred to the prepared petri dishes containing MeA sterile
159 drigalski spatula to spread the spores completely on the surface of the medium is provided. 50’l µl was pipetted
160 onto the medium from different doses of solutions obtained from essential oils. After pipetting was completed, it
161 was allowed to incubate at 28 ° C for 48 hours. For control application, one solution of each suspension containing
162 spore suspension prepared with Tween 80 solution was separated from the sample without application of essential
163 oil and seeded into petri dish. Microfungi, whose growth was completed in cultures after incubation, were
164 evaluated by cell counting on thoma slide according to conventional colony counting methods in microbiology.
165
166 2.6. Statistical Analysis
167
168 The results obtained were evaluated with Duncan Multiple Range Test. In the evaluation, descriptive statistics are
169 presented with frequency and percentage values. General linear models were used to analyze the difference
170 between the numerical data of more than two groups. Analyzes were made with the SAS 9.4 program and P <0.05
171 was considered statistically significant.
172
173 3. Results and Discussion
174
175 In the study conducted in the Ancient City of Side, antifungal effect of essential oils obtained from plants was
176 observed on the development of black microfungi isolated from historical monuments. Coniosporium,
177 Sarcinomyces, Phaeococcomyces species isolated from corroded monuments (Table 1) were grown in the
178 laboratory on MeA and CzA media, and essential oils obtained from O. majorana, O. minutiflorum and M.
179 longifolia plants were applied to microfungi at different concentrations and the results were evaluated. As a result
180 1cm%1 and %3 solutions of essential oils and applying them on stone samples, some
of the preparation of % 0.1,
1cm
181 samples showed vitality while others did not. Parts showing signs of vitality were colored red after the TTC
182 (Triphenyl Tetrasolium Chloride) test. The reason for the red color is; TTC, which is dissolved and a vital dye, is
183 reduced and insoluble when taken by microfungi and turns into a red colored substance (acid formazana). At the
184 same time, microfungi whose growth was completed in cultures after incubation and applied essential oil were
185 evaluated by cell count on thoma slide according to conventional colony count methods in microbiology. Macro
186 and microscopic appearance of DRBC, MeA and CzA media of black microcolonial fungi grown in the laboratory
187 by isolating them from historical monuments are given below (Figs. 6-9) and the spore numbers of the species

5
188 belonging to Coniosporium, Sarcinomyces, Phaeococcomyces are given in the tables (Table 1-9). The results
189 obtained in this study were evaluated with Duncan Multiple Range Test. According to statistical evaluation, a
190 significant difference was found because of the application of essential oils obtained from O. majorana, O.
191 minutiflorum and M. longifolia plants applied on species belonging to the genus Coniosporium, Sarcinomyces,
192 Phaeococcomyces. It was also found that the higher the concentration, the higher the antifungal effects of the
193 essential oils, hence suppressing the growth of these black microfungi species. The results of the Duncan Multiple
194 Range Test and variance analysis according to genera are given in the tables below. When the effects of essential
195 oil applications were compared according to the genera, it was determined that the antifungal effect on
196 Phaeococcomyces species was observed more and the development of these species decreased considerably.
197 However, it is observed that the effect rate of essential oil is decreased in Coniosporium, Sarcinomyces species.
198 In addition, if the statistical evaluation is made in terms of oils, the highest reduction in fungal cell count was
199 observed with the application of Origanum minutiflorum (3%) and the application of Mentha longifolia (3% rate).
200
201 Table 1. The presence of fungi and localities in the research area
Specimen
Locality Coniosporium sp. Sarcinomyces sp. Phaeococcomycs sp.
number
Ancient theater entrance right
HA 1001 +
wall
Column heading (Ancient
HA 1002 +
theater)
Ceiling
HA 1003 +
cassette(Embellishment)
HA 1004 Sarcophagus (Ancient theater) +

HA 1005 Stage part (Ancient theater) +

Architectural relief
HA 1006 +
(Ancient theater)
HA 1007 Theater staircase +
Marble block (Apollon
HA 1008 +
temple)
HA 1009 Vault wall (Ancient theater) +

HA 1010 Column base (Ancient theater) +

HA 1011 Latrina +

HA 1012 Entrance of Agora +

HA 1013 Theater gallery +

HA 1014 Lettered table reconstruction

HA 1015 Three-pool fountain +

HA 1016 Sarcophagus (Side Museum) +

HA 1017 Lion statue (Side Museum) +

Remains of a children’s grave
HA 1018 +
(Museum)
HA 1019 Entrance of Agora +

HA 1020 Well mouth (Side museum)

HA 1021 Door lintel (Museum) +

HA 1022 Vespasian monument

Plinth remnant (Ancient
HA 1023 +
theater)
HA 1024 Nympheum ( Nine fountains) +

HA 1025 Mascot wall ( Theater) +

6
202
203 3.1. Essential oil application on the species of Phaeococcomyces
204
205 This slow growing, rock inhabiting Phaeococcomyces species forms dark brown to black colony (Fig. 6). It has
206 fine hairs on culture. Number of spores before the applications, with application of Tween 80 (%1) solution and
207 essential oil applications with concentrations of % 0,1, %1, %3 are given in table 2 (Table 2 a, b, c). The results
208 of the Duncan Multiple Range Test and variance analysis according to genera are given in the table 3.
209

1cm
1cm
a b c 10µm.

210
211
212 Fig. 6. Phaeococcomyces sp. (1 month development) a. on MeA, b. on CzA c. Conidiophore
213
214
215 Table 2. Spore numbers of Phaeococcomyces species before and after the applications a. HA 1016, b. HA 1023,
216 c. HA 1024
217
Tween 80
Before the application Concentrations Origanum Origanum Mentha
applications (Control of oils majorana minutiflorum longifolia
group)
0,1% 553x103 592x103 520x103

674x103 652x103 1% 400x103 426x103 350x103

a 3% 322x103 300x103 295x103

218
219

Tween 80
Before the Concentrations Origanum Origanum
application Mentha longifolia
applications of oils majorana minutiflorum
(Control group

0,1% 616x103 640x103 670x103

674x103
668x103 1% 450x103 316x103 534x103

3% 224x103 212x103 280x103

b
220
221
Tween 80
Concentra
application Origanum Origanum Mentha
Before the applications tions of
(Control majorana minutiflorum Longifolia
oils
group
0,1% 636x103 666x103 516x103
688x103
696x103
1% 500x103 440x103 465x103

7
 c 3% 305x103 310x103 264x103
222 Table 3. Variance analysis results of Phaeococcomyces species (The same letter space icons are not significantly different)
223
Source of change
DF Tip III SS Average square F Value Pr > F

Essence 9 18400,557 2044,5063 6,21 <.0001

Source Sd KT KO F P
Model 9 18400,557 2044,5063 6,21 <.0001

Tolerance 90 29639,411 329,32679

Adjusted total 99 48039,968

224
225 Duncan Grouping Meaning N Essence
A 48.133 10 9
A
A 46.839 10 6
A
B A 39.900 10 3
B A
B A 38.565 10 8
B A
B A C 34.976 10 5
B C
B D C 28.215 10 2
D C
E D C 19.999 10 4
E D C
E D C 19.681 10 7
E D
E D 15.427 10 1
E
E 5.294 10 10
226
227
228 3.2. Essential oil application on the species of Coniosporium
229
230 Thallus on the rock black, meristematic, moriform, located in pits. Colonies on MEA black, dry, hard, cerebriform
231 with age, margin sharp, slightly lobed. Colonies on czapek agar black, dry, hard, flat, margin irregular. Mycelium
232 consisting of pale brown, olivaceous to dark brown hyphae with cylindrical to spherical cells, thin to thick-walled.
233 The growth of Coniosporium sp. on MeA, CzA and on DRBC media and observation under light microscope is
234 shown in the pictures below (Fig. 7-8).

8
 4 cm. 10 cm. 4 cm.
a b c
235
236
237 Fig. 7. a. Coniosporium sp.1 month development (MeA) b. 2 month development (CzA); c. 1 month development
238 (DRBC)
239

a b
240
241
242 Fig. 8. a.Coniosporium sp. a. Conidiophores (40x), b.Conidiospores (100x)
243
244 Number of spores before the applications, with application of Tween 80 (1%) solution and essential oil applications
245 with concentrations of %0.1, % 1, % 3 are given in tables (Table 4).
246
247 Table 4. Number of spores of Coniosporium sp. before and after the applications a. HA 1001, b. HA 1002, c.
248 HA1003, d. HA1004 e. HA 1005, f. HA 1013, g. HA 1015, h. HA 1018, i. HA 1019, j. HA 1025
249
Tween 80
Concentrations of Origanum Origanum Mentha
Before the applications application
oils majorana minutiflorum longifolia
(Control group)

0,1% 722x103 616x103 650x103

748x103 (1 cm2)
730x103 1% 476x103 504x103 668x103

a.
3% 414x103 410x103 600x103

250
Tween 80 Concentrations of
Before the application oils Origanum Origanum
Mentha longifolia
applications (Control majorana minutiflorum
group)
0,1%
510x103 408x103 526x103
672x103 1%
666x103 410x103 366x103 448x103

b. 3%
304x103 330x103 370x103

9
 Tween 80
Before the application Concentrations of Origanum Origanum
Mentha longifolia
applications (Control oils majorana minutiflorum
group)

0,1% 656x103 648x103 638x103

640x103 652x103 1% 630x103 632x103 620x103

c. 3% 554x103 500x103 608x103

Tween 80
Before the Concentrations of Origanum Origanum Mentha
application
applications oils majorana minutiflorum longifolia
(Control group)

0,1% 738x103 994x103 868x103

998x103 996x103 1% 606x103 390x103 746x103

d. 3% 578x103 376x103 410x103

Tween 80
Before the Origanum Origanum Mentha
application Concentrations of oils
applications majorana minutiflorum longifolia
(Control group)

0,1% 746x103 518x103 960x103

972x103
950x103
1% 700x103 500x103 288x103
e. 3% 712x103 340x103 218x103
Tween 80
Before the Concentrations of Origanum Origanum Mentha
application
applications oils majorana minutiflorum longifolia
(Control group)

0,1% 690x103 582x103 562x103

990x103
886x103 1% 576x103 388x103 470x103

f.
3% 404x103 356x103 362x103

Tween 80
Before the Concentrations of Origanum Origanum Mentha
application
applications oils majorana minutiflorum longifolia
(Control group)
0,1% 728x103 720x103 520x103
880x103
765x103 1% 560x103 640x103 170x103
g.
3% 280x103 600x103 164x103
Tween 80
Before the Concentrations of Origanum Origanum Mentha
application
applications oils majorana minutiflorum longifolia
(Control group)
0,1% 665x103 680x103 582x103

720x103 600x103 1% 540x103 655x103 467x103

= 460x103 540x103 354x103

h.

10
 Tween 80
Before the Concentrations of Origanum Origanum Mentha
application
applications oils majorana minutiflorum longifolia
(Control group)
0,1% 712x103 666x103 602x103

920x103 852x103 1% 690x103 542x103 360x103

i. 3% 586x103 366x103 350x103

Tween 80
Before the Concentrations of Origanum Origanum Mentha
application
applications oils majorana minutiflorum longifolia
(Control group)
0,1% 760x103 740x103 702x103

776x103 765x103 1% 700x103 600x103 670x103

3% 640x103 450x103 640x103

j.
251
252 The results of the Duncan Multiple Range Test and variance analysis according to genera are given in the tables
253 below (Table 5-6).
254
255 Table 5. Variance analysis results of Conisporium species

Source of change DF Tip III SS Average square F Value Pr > F

Essence 9 18400,56 2044,50631 6,21 <.0001

Source Sd KT KO F P

Model 9 18400,557 2044,50631 6,21 <.0001

Tolerance 90 29639,411 329,32679

Adjusted total 99 48039,968

256

257 Table 6. Duncan's multiple range test results of Conisporium species

Duncan Grouping Meaning N Essence
A 48.133 10 9
A
A 46.839 10 6
A
B A 39.900 10 3
B A
B A 38.565 10 8
B A
B A C 34.976 10 5
B C

11
 B D C 28.215 10 2
D C
E D C 19.999 10 4
E D C
E D C 19.681 10 7
E D
E D 15.427 10 1
E
E 5.294 10 10

258 The same letter space icons are not significantly different.

259 3.3. Essential oil application on the species of Sarcinomyces

260 Colonies on malt extract agar dark grey to black, dry, initially soft, later hard; mature colonies consisting of pale
261 grey aerial mycelium, cerebriform with age; margin sharp, slightly lobed. Colonies on czapek agar; soft, initially
262 black, later becoming white to pale grey aerial mycelium; flat; margin fimbriate and irregular. The growth of
263 Sarcinomyces sp. on MeA, CzA and on DRBC media and observation under light microscope is shown in the
264 pictures below (Fig. 9).
265

20 µm.
a 3 cm. b 100 µm. c .
266

d 3 cm. e 5 cm. f 2 cm.

267
268
269 Fig. 9. Sarcinomyces sp. (1 month development) a. MeA, b. Conidiophore c. Conidiospore
270 d. MeA e. DRBC f. CzA
271
272 Number of spores before the applications, with application of Tween 80 (1%) solution and essential oil applications
273 with concentrations of % 0,1, % 1, %3 are given in tables (Table 7).
274
275 Table 7. Number of spores of Sarcinomyces species before and after the applications a. HA 1006, b. HA 1007,
276 c. HA 1008, d. HA 1009, e. HA 1010, f. HA 1011, g. HA 1012, h. HA 1017, i. HA 1021

12
 Tween 80
Before the application Concentrations of Origanum Origanum Mentha
applications (Control oils majorana minutiflorum longifolia
group)

0,1% 490x103 530x103 602x103

600x103 605x103 1% 480x103 526x103 458x103

a 3% 192x103 370x103 368x103

Tween 80
Before the application Concentrations of Origanum Origanum Mentha
applications (Control oils majorana minutiflorum longifolia
group)

0,1% 518x103 574x103 414x103

600x103 580x103 1% 406x103 534x103 396x103

3% 370x103 494x103 344x103

b
Tween 80
Before the application Concentrations of Origanum Origanum Mentha
applications (Control oils majorana minutiflorum longifolia
group)

0,1% 688x103 572x103 642x103

708x103 700x103 1% 638x103 586x103 528x103

c 3% 624x103 554x103 478x103

Tween 80
Before the application Concentrations of Origanum Origanum Mentha
applications (Control oils majorana minutiflorum longifolia
group)

0,1% 764x103 862x103 784x103

1000x103 930x103 1% 704x103 708x103 652x103

d 3% 680x103 664x103 526x103

Tween 80
Before the Concentrations of Origanum Origanum Mentha
application
applications oils majorana minutiflorum longifolia
(Control group)

0,1% 526x103 470x103 456x103

870x103 660x103 1% 516x103 410x103 416x103

e 3% 408x103 322x103 370x103

13
 Tween 80
Before the Concentrations of Origanum Origanum Mentha
application
applications oils majorana minutiflorum longifolia
(Control group)

0,1% 592x103 550x103 582x103

690x103 670x103 1% 504x103 494x103 484x103

3% 456x103 446x103 424x103

f
Tween 80
Before the Concentrations of Origanum Origanum Mentha
application
applications oils majorana minutiflorum longifolia
(Control group)

0,1% 634x103 654x103 646x103

710x103 674x103 1% 536x103 606x103 518x103

e 3% 478x103 554x103 472x103

Tween 80
Origanum Mentha
Before the application Concentrations of Origanum
minutiflorum longifolia
applications (Control group) oils majorana

0,1% 520x103 594x103 554x103

916x103 860x103 1% 506x103 570x103 430x103

f
3% 476x103 536x103 376x103

Tween 80
Before the Origanum Origanum Mentha
application Concentrations of oils
applications majorana minutiflorum longifolia
(Control group)

0,1% 370x103 320x103 980x103

952x103 870x103 1% 320x103 296x103 976x103

g 3% 230x103 270x103 550x103

277
278
279 The results of the Duncan Multiple Range Test and variance analysis according to genera are given in the tables
280 below (Table 8-9).
281

14
282 Table 8. Variance analysis results table of Sarcinomyces species
283
284
285
286
Average 287
Source of change DF Tip III SS square F Value Pr >288
F
289
Essence 9 18400.55677 2044.50631 6.21 290
<.0001
291
Source Sd KT KO F P292
293
Model 9 18400.55677 2044.50631 6.21 <.0001
294
295
Tolerance 90 29639.41116 329.32679 296
297
298
Adjusted total 99 48039.96793
299
300
301
302 Table 9. Duncan's multiple range test results of Sarcinomyces species
Duncan Grouping Meaning N Essence
A 48.133 10 9
A
A 46.839 10 6
A
B A 39.900 10 3
B A
B A 38.565 10 8
B A
B A C 34.976 10 5
B C
B D C 28.215 10 2
D C
E D C 19.999 10 4
E D C
E D C 19.681 10 7
E D
E D 15.427 10 1
E
E 5.294 10 10

303 The same letter space icons are not significantly different.
304
305
306
307 Valuable elements of cultural heritage are over time attacked by biodeteriogens such as algae, mosses
308 and lichens, followed by fungi and other organisms (up to higher plants and even humans). Various methods must
309 be taken to control (prevention, stop to development or at least slow down) the deterioration of historical
310 monuments. As an alternative to chemical biocides, which are widely used but known to be harmful to the
311 environment and human health, natural products, especially essential oils, are increasingly taking their place as
312 effective solutions for the protection of cultural heritage.

15
313 Natural products derived from plants, such as essential oils, contain a wide variety of secondary metabolites that
314 can act against biological systems and can be considered environmentally acceptable pesticides. Based on these
315 properties, scientists use certain essential oils to create green procedures for the preservation of cultural heritage.
316 Effects of essential oils at different concentrations on biodeteriogenic microorganisms and its antimicrobial effect
317 has been tried by many researchers and positive results have been obtained, as in our study (De La Paz et al., 2006;
318 Ghalem and Mohamed, 2008; Guiamet et al., 2008; Karakaya et al. 2011; Palla et al., 2020; Rotolo et. Al., 2016).
319
320 In this study on the antifungal effect of some essential oils on biodeteriogens, the results were evaluated with the
321 with the Duncan Multiple Range Test. As a result of the statistical evaluation, a significant difference was found
322 because of the application of essential oils obtained from O. majorana, O. minutiflorum and M. longifolia plants
323 applied on species belonging to the genera Coniosporium, Phaeococcomyces and Sarcinomyes. It has been
324 determined that as the concentration of essential oils increases, the antifungal effects of essential oils increase, thus
325 suppressing the growth of these black microfungi species. In addition to these, the effects of essential oil
326 applications are compared according to the genus, it was determined that the antifungal effect of the species
327 belonging to the genus Phaeococcomyces was observed more and the development of these species was
328 significantly reduced. This can be explained by the thinner and permeable cell walls of the species of the genus
329 Phaeococcomyces, thus facilitating the penetration of essential oils into the cells.
330
331 Furthermore, the aging of essential oils is accompanied by an increase in the peroxide content with acid formation
332 and finally with resinification, which is accompanied by a color change from light yellow to dark yellow and a
333 change in consistency from liquid to viscous (Avoxa, 2004; Grassmann, 2000; Leunng and Foster, 1996). For this
334 reason, it is recommended to use essential oils in low concentrations determined under control in accordance with
335 the essential oil and stone structure used in order not to cause harm instead of benefit on historical artifacts.
336
337 It is thought that essential oils used in optimum doses will be beneficial especially in the protection and restoration
338 of historical monuments, as they are both environmentally friendly and have high antifungal effects.
339
340
341 5. Acknowledgements
342
343 We thank to Akdeniz University, Scientific Research Project Unit for financial support (FYL-2018-3758) and
344 Ministry of Culture and Tourism for allowing collection of samples. We would also like to thank Asst. Prof. Dr.
345 İlker Çinbilgel for help in identifying plant species and Prof. Dr. Hüseyin Çetin for help in extracting essential
346 oils.

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477
478
479

18
480
481
482
483 Received:
484 Accepted:
485
486 CORRESPONDING AUTHOR:
487
488 Hacer Sert
489
490 E-mail: [email protected]
491

19
Conflict of Interest

Declaration of interests

☐The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☒The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:

Hacer Sert reports equipment, drugs, or supplies was provided by Akdeniz University.