Access to this full-text is provided by EDP Sciences.
Content available from BIO Web of Conferences
This content is subject to copyright. Terms and conditions apply.
Effectiveness of Mycorrhiza to the Growth and
Essential Oil Content of Eucalyptus citriodora
seedling
O Trisilawati11,IDarwati2,HKuntyastuti3, R. Vitri Garvita4,EFidiyawati5
1National Research and Innovation Agency, Jl. Raya Cibinong km43 Indonesia
Abstract. Eucalyptus citriodora is one of the forest plant produced
essential oil which contains various beneficial chemical compounds,
including 1,8-cineol. Arbuscular Mycorrhizal Fungi (AMF) is a
biofertilizer widely used to increase biomass and bioactive content in
cultivated plants. The objective of this study was to determine the impact
of an AMF consortium on the growth, biomass yield, and 1,8-cineol
content of E. citriodora seedlings. Research was conducted six months, in
the greenhouse of Indonesian Spice and Medicinal Crops Research Institut
(ISMCRI), Bogor, by using randomized complete block design, with six
types of inoculum treatments and four replications. The AMF consortium
inoculum were explored from various acidity land under several stand of
plants, namely consortium inoculum of M1, M2, M3, M4 and M5. The
result showed that AMF inoculum treatments had positive effect to the
growth (plant height, number of leaves and branch) and weight of leaf,
stem and root of E. citriodora. The oil, citronellal and 1.8 cineole content
of Eucalyptus sp leaves were increased by the application of AMF. The
highest oil content, citronellal and 1.8 cineole content of E. citriodora
leaf were resulted from M4 consortium application, were 2.38%, 76.07%,
0.52% respectivelly. M4 consortium application showed the highest
effectivenesss to the E. citriodora.
Keywords: Eucalyptus citriodora, mycorrhiza, growth, oil and 1,8-cineole
content
1 Introduction
Eucalyptus citriodora is one of the ten most significant Eucalypts for the annual increase in
world wood production, and it has a significant role in the production of wood in Asian
plantations, where it ranks ninth overall.[1]; [2]. Eucalyptus produces essential oils from its
leaves in addition to wood. About 1.36% of the essential oil found in E. citriodora's leaves
is citronellal (57%), followed by citronellol (15.89%), citronellyl acetate (15.33%), and
other substances.[3]; [4]. The oil contains several volatile monoterpenes, including cineole,
citronellol, citronellal, limonene, linalool, and -terpinene [5]. The perfumery and
1Corresponding author: trisilawati03@gmail.com
BIO Web of Conferences 69, 01001 (2023) https://doi.org/10.1051/bioconf/20236901001
2nd ICAFE 2023
© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution
License 4.0 (https://creativecommons.org/licenses/by/4.0/).
pharmaceutical industries use Eucalyptus essential oil, which has significant commercial
value and ranks highly in both quality and quantity. In addition, it exhibited a broad
spectrum of antimicrobial [6]; [7], antifungal [8], anticandidal [9], antibacterial [10]; [11],
expectorant, and cough stimulant activity [12] properties.
E. citriodora is one of the cultivated Eucalyptus species which developed by using plant
material derived from vegetative propagation such as cuttings. The plant is widely
cultivated in areas, which have low soil fertility statusTo successfully grow and develop in
the field, it needs suitable seed conditions. AMF make up a significant portion of the soil
microbial biomass; they are widespread throughout various ecosystems and have the
potential to infect the roots of more than 80% of terrestrial plants, resulting in a mutualistic
symbiosis.. AMF can broaden plant roots to increase the uptake of various macro and
micronutrients, which affects plant development, growth, productivity, and quality.[13]. The
AMF acquires up to 20% of photosynthates from plants in exchange for mineral nutrients
and water [14]; [15];. Some evidence suggests that AMF is a biofertilizer that is widely
used on cultivated plants to enhance nutrient availability, plant yield, and bioactive content
[16]; [17]. AMF may significantly contribute to accelerating seedling growth in nurseries
and helping plants survive in hostile environments.
The research aimed to identify the effectivity of several AMF consortiums toward the
growth, biomass yield, and the content of oil, 1.8-cineole and citronellal of E. citriodora at 6
months after planting.
2 Materials and methods
Research was conducted for seven months, in the greenhouse and laboratory of Indonesian
Spice and Medicinal Crops Research Institute, Bogor, West Java, since February to October
2021.
One month of E. citriodora seedlings (Manoko accession) were planted in polybags (3
kg volume) containing media soil (sterilized by using Basamid D soil fumigant) : cow dung
(3:1) for six months. The AMF consortium inoculums were explored from various acidity
land under several stand of plants, namely consortium inoculum of M1, M2, M3, M4 and
M5.
The research was conducted using a random block design that was repeated four times.
The factor was mycorrhiza application, which consisted of five consortium types.:
M0 = no AMF applied
M1 = Mycorrhiza isolated from vanilla rhizosphere in West Java
M2 = Mycorrhiza isolated from clove rhizosphere in North Sumatera
M3 = Mycorrhiza isolated from Ultisol soil
M4 = Mycorrhiza isolated from Cinnamomum rhizosphere in West Java
M5 = Mycorrhiza isolated from pine rhizosphere in forest soil
The AMF inoculum was prepared using colonized Zea mays root fragments in
quantities of 50 g per polybag of rhizosphere soil containing spores in order to obtain high
levels of root colonization.. There were 120 plants in the population, and five plants were
used and observed for each treatment and repetition.
Six months after planting (MAP), a number of quantitative traits of the seedlings were
evaluated, including plant height, the number of leaves and branches, the weight of the
leaves, stems, and roots, the oil content of the leaves, the concentration of 1.8-cineol and
citronella, the content of macronutrients nitrogen, phosphorus, potassium, calcium, and
magnesium (N, P, K, Ca, Mg), and the infectivity of the mycorrhizal roots (AMF
colonization). Oil and citral content were measured by distillation, and Gas
2
BIO Web of Conferences 69, 01001 (2023) https://doi.org/10.1051/bioconf/20236901001
2nd ICAFE 2023
Chromatography method [18]; [19]. Nutrient uptake of leave was calculated from nutrient
content and leaves weight. The infectivity of mycorrhizal roots was assessed using the [20]
approach in [21]. The presence of hyphae, vesicles, arbuscules, or any combination of the
three, is a sign of root colonization. Using the Kjeldahl, spectrophotometer, and AAS
techniques, the N, P, K, Ca, and Mg content of leaves was evaluated [22]. To ascertain
whether there were significant differences, the statistical studies used analysis of variance
(Anova) and Duncan's Multiple Range Test (DMRT) at the 5% level.
3 Results and discussion
The plant height, number of leaves, and number of branches of Eucalyptus species at 6
MAP were not substantially impacted by the inoculation of AMF consortium treatments,
and the control treatment had the slowest growth among the five AMF consortium
treatments.(Table 1). This can be caused by the young age of the plant, however there were
still positive effect of AMF application on the growth of Eucalyptus sp. by increasing plant
height (3.2% to 21.8%), number of leaves (27.3% to 41.6%) and branches (50% to
107.7%). [23] also found that the mycorrhizae application did not significantly affect the
growth of Eucalyptus sp. (height, diameter and root shoot ratio) at 4 months after planting.
AMF application resulted in the higher fresh weight of leaves, stems and roots of E.
citriodora compared to non mycorrhiza treatments (Figure 1). The increasing fresh weight,
stems and roots of E. citriodora leaves were 6.2% to 25.2%, 74% to 151% and 78% to
160% respectively, compared to non mycorrhiza treatments. [24] found an increase of 45%
of E. globulus shoot dry weight, inoculated with Glomus deserticola in greenhouse
condition.
Table 1. E. citriodora plant height, leaves, and branches on mycorrhiza treatments at 6 MAP
Mycorrhiza
treatments
Plant height
cm
Number of leaves
Number
of
branches
M
0
71.25 ± 9.03
38.70 ± 7.59
1.30 ± 0.58
M
1
76.23 ± 11.74
49.25 ± 5.04
2.30 ± 0.48
M
2
74.89 ± 9.71
54.80 ± 8.09
2.05 ± 0.77
M
3
86.81 ± 6.06
49.35 ± 4.87
1.95 ± 0.57
M
4
80.36 ± 11.69
53.60 ± 11.03
2.55 ± 1.31
M
5
73.51 ± 17.05
51.65 ± 14.13
2.70 ± 1.59
Notes
M0
=
no AMF applied
M3
=
AMF isolated from Ultisol soil
M1
M2
=
=
AMF isolated from vanilla rhizosphere
in West Java
AMF isolated from clove rhizosphere
in North Sumatera
M4
M5
=
=
AMF isolated from cinnamomum
rhizosphere in West Java
AMF isolated from pine
rhizosphere in forest soil
3
BIO Web of Conferences 69, 01001 (2023) https://doi.org/10.1051/bioconf/20236901001
2nd ICAFE 2023
Notes
M0
=
no AMF applied
M3
=
AMF isolated from Ultisol soil
M1
M2
=
=
AMF isolated from vanilla rhizosphere
in West Java
AMF isolated from clove rhizosphere
in North Sumatera
M4
M5
=
=
AMF isolated from cinnamomum
rhizosphere in West Java
AMF isolated from pine
rhizosphere in forest soil
Fig. 1. The weight of leaves, stem and root of E. citriodora on mycorrhiza treatments at 6 MAP
The oil, cineole and citronella content of six months plant leaves treated with
mychorrhiza consortium were varied (Table 2). The oil content ranged from 0.85 to 2.38%,
the cineole content range from 0.01 to 0.52%, and citronella content ranged from 48.28 to
76.07%. M4 consortium application resulted in the highest content of oil, cineole and
citronella, were 2.38%, 0.52% and 76.07%. Control treatment showed the lowest content of
oil, cineole and citronella, were 0.85%, 0.01% and 48.28%. The oil yield of three different
Eucalyptus species (E. citriodora, E. urophylla, and E. viminalis) from diverse areas in
Indonesia varies from 1.6 percent to 3.3 percent (w/w, dry weight) depending upon the type,
plant location, and leaf age. 1,8-cineole (18.2%–45.5%) and citronellal (69.27–82.81%)
have been named as the two main ingredients of eucalyptus oil [25]. Mycorrhizal fungus
may be used as a bioinoculant to increase the essential oil concentration of various herbal
medicine since it likely triggered or encouraged the isopentenyl diphosphate production
pathway.[26]; [27].
Table 2. The oil, cineole and citronella content of E. citriodora on mycorrhiza treatments
Mycorrhiza
treatment
Oil content Cineole content Citronella content
%
M0 0.85 0.01 48.28
M1 1.14 - 66.28
M2 1.33 0.01 68.26
M3
1.56 0.26 68.32
M4
2.38 0.52 76.07
M5
1.55 0.19 64.85
Notes
4
BIO Web of Conferences 69, 01001 (2023) https://doi.org/10.1051/bioconf/20236901001
2nd ICAFE 2023
M0
=
no AMF applied
M3
=
AMF isolated from Ultisol soil
M1
M2
=
=
AMF isolated from vanilla rhizosphere
in West Java
AMF isolated from clove rhizosphere
in North Sumatera
M4
M5
=
=
AMF isolated from cinnamomum
rhizosphere in West Java
AMF isolated from pine
rhizosphere in forest soil
Table 3. The macro nutrients content of E. citriodora leaves
Mycorrhiza
treatment
N
P
K
Ca
Mg
%
M0
2.12
0.19
2.01
0.17
0.2
M1
1.60
0.15
1.88
0.16
0.19
M2
2.03 0.16 2.08 0.21 0.18
M3
2.13
0.17
1.83
0.09
0.17
M4
1.95
0.16
2.01
0.15
0.2
M5
2.02 0.16 2.09 0.16 0.19
Notes
M0
=
no AMF applied
M3
=
AMF isolated from Ultisol soil
M1
M2
=
=
AMF isolated from vanilla rhizosphere
in West Java
AMF isolated from clove rhizosphere
in North Sumatera
M4
M5
=
=
AMF isolated from cinnamomum
rhizosphere in West Java
AMF isolated from pine
rhizosphere in forest soil
The macronutrients N, P, K, Ca and Mg content and uptake of E. citriodora leaves
showed that N and K were the highest nutrient content and uptake, followed by P, Ca and
Mg nutrients (Table 3 and 4). The M4 consortium treatment showed higher N, P, K, Ca and
Mg uptake of E citriodora leaves. Most NO3—N that is transported to leaves is quickly
transformed to amino N, which can either be used in the leaf to create protein or transferred
directly to other plant parts for protein synthesis. [32]. One of the key macronutrients for
plant growth and development is potassium, which also activates over 60 plant enzymes and
participates in the osmotic regulation of plant cells, particularly the opening and shutting of
stomata [28, 33]. In the symbiotic connection with plants, AM hyphae are critical because
they extend from the root and make it easier for water and vital inorganic nutrients like
phosphate, nitrogen, and micronutrients to be transported from the soil to the plant. AM
hyphae play a crucial role in the symbiotic relationship with plants because they extend
from the root and facilitate the transportation of water and essential inorganic nutrients
including phosphate, nitrogen, and micronutrients from the soil to the plant. They actively
take up carbon from the host plants to maintain their own existence, as detailed in reference
[29]. AM hyphae are important in the symbiotic relationship with plants because they
extend from the root and make it easier for water and vital inorganic nutrients like
phosphate, nitrogen, and micronutrients to be transported from the soil to the plant. For
their own survival, they actively take up carbon from the host plants, as mentioned in
reference [29]. Better seedling growth of Eucalyptus pellita f. Muell was obtained with
double inoculation of Glomus sp. and P. arrhizus, which might also improve nutrient uptake
of N and P by 64% and 155%, respectively, in comparison to non-inoculated control
seedlings [30].
Table 4. Macro nutrients uptake of E citriodora leaves
5
BIO Web of Conferences 69, 01001 (2023) https://doi.org/10.1051/bioconf/20236901001
2nd ICAFE 2023
Mycorrhiza
treatment
N P K Ca Mg
mg/plant
M0
128.48 11.51 121.81 10.30 12.12
M1
115.10
10.79
135.24
11.51
13.67
M2
129.08 10.17 132.26 13.35 11.45
M3
149.24 11.91 128.22 6.31 11.91
M4
140.05 11.49 144.36 10.77 14.36
M5
125.35
9.93
129.69
9.93
11.79
Notes
M0
=
no AMF applied
M3
=
AMF isolated from Ultisol soil
M1
M2
=
=
AMF isolated from vanilla rhizosphere
in West Java
AMF isolated from clove rhizosphere
in North Sumatera
M4
M5
=
=
AMF isolated from cinnamomum
rhizosphere in West Java
AMF isolated from pine
rhizosphere in forest soil
Fig. 2. Mycorrhiza colonization on E. citriodora root
The average mycorrhiza infectivity on root of E. citriodora inoculated with M4, M5 and
M2 consortiums were 72.5%, 67.5%, and 46.8% whereas the control was 15%. M4
consortium showed significantly the highest AMF root colonization compared to others
AMF consortium. A positive response to inoculation was expressed by an increase in AM
root colonization levels and in yields as compared to non-inoculated controls [31].
4 Conclusion
Treatments with AMF inoculum led to greater growth in terms of plant height, the number
of leaves and branches, and the weight of the leaves, stem, and root of E citriodora. AMF
application boosted the E. citriodora leaf's oil, 1.8 cineole, and citronellal contents. The M4
consortium application produced E citriodora leaf oil with the greatest concentrations of
citronellal and 1.8 cineole, which were 2.38 percent, 76.07 percent, and 0.52 percent,
respectively. The M4 consortium application demonstrated the greatest efficacy with E
citriodora.
6
BIO Web of Conferences 69, 01001 (2023) https://doi.org/10.1051/bioconf/20236901001
2nd ICAFE 2023
We grateful to thank Mr. Redy Aditya Permadi and Mr. Hikmat Mulyana from Indonesian Spice and
Medicinal Crops Research Institute.
References
[1] J. Davidson, "Ecological aspects of Eucalyptus plantations," in Proceedings of the
regional expert consultation on Eucalyptus, pp. 35-72, 1993.
[2] S. Midgley and K. Pinyopusarerk, "The role of eucalypts in local development in the
emerging economies of China, Viet Nam and Thailand," 1996.
[3] Y. Tian, X. Liu, Y. Zhou, and Z. Guo, "Extraction and determination of volatile
constituents in leaves of Eucalyptus citriodora," Chinese J. Chromatogr., vol. 23, no. 6,
pp. 651-654, 2005.
[4] J. C. Chalchat, A. Muhayimana, J. B. Habimana, and J. L. Chabard, "Aromatic plants of
Rwanda. II. Chemical composition of essential oils of ten Eucalyptus species growing
in Ruhande arboretum, Butare, Rwanda," J. Essent. Oil Res., vol. 9, no. 2, pp. 159-165,
1997, doi: 10.1080/10412905.1997.9699453.
[5] M. I. Brooker and D. Kleinig, "Field Guide to Eucalypts 3. Northern Australia,"
Blooming Books, Victoria, Australia, 2004.
[6] F. Hajji, S. Fkih-Tetouani, and A. Tantaoui-Elaraki, "Antimicrobial activity of
twenty-one Eucalyptus essential oils," Fitoterapia, vol. 64, no. 1, pp. 71-77, 1993.
[7] N. Chanegriha, F. Cherif, A. Baailouamer, and B. Meklati, "Antimicrobial activity of
Algerian cyprus and eucalyptus essential oils," Riv. Ital. EPPOS, pp. 11-16, 1998.
[8] H. Ramezani, "Fungicidal activity of volatile oil from Eucalyptus Citriodora Hook.
against Alternaria triticina," Commun. Agric. Appl. Biol. Sci., vol. 71, no. 3B, pp.
909-914, 2006.
[9] B. K. Dutta, S. Karmakar, A. Naglot, J. C. Aich, and M. Begam, "Anticandidial activity
of some essential oils of a mega biodiversity hotspot in India," Mycoses, vol. 50, no. 2,
pp. 121-124, 2007, doi: 10.1111/j.1439-0507.
[10] K. Cimanga, "Correlation between chemical composition and antibacterial activity of
essential oils of some aromatic medicinal plants growing in the Democratic Republic
of Congo," J. Ethnopharmacol., vol. 79, no. 2, pp. 213-220, 2002, doi:
10.1016/S0378-8741(01)00384-1.
[11] D. Low, B. D. Rawal, and W. J. Griffin, "Antibacterial action of the essential oils of
some Australian Myrtaceae with special references to the activity of chromatographic
fractions of oil of Eucalyptus citriodora," Planta Med., vol. 26, no. 6, pp. 184-189,
1974, doi: 10.1055/s-0028-1097987.
[12] A. O. Oyedeji, O. Ekundayo, O. N. Olawore, B. A. Adeniyi, and W. A. Koenig,
"Antimicrobial activity of the essential oils of five Eucalyptus species growing in
Nigeria," Fitoterapia, vol. 70, no. 5, pp. 526-528, 1999, doi:
10.1016/S0367-326X(99)00083-0.
[13] E. Sieverding, "Function of Mycorrhiza," in Vesicular Arbuscular Mycorrhiza
Management in Tropical Agrosystems, Eschborn, Germany, 1961, pp. 57-70.
[14] B. Bago, P. E. Pfeffer, and Y. Shachar-Hill, "Carbon metabolism and transport in
arbuscular mycorrhizas," Plant Physiol., vol. 124, no. 3, pp. 949-958, 2000, doi:
10.1104/pp.124.3.949.
7
BIO Web of Conferences 69, 01001 (2023) https://doi.org/10.1051/bioconf/20236901001
2nd ICAFE 2023
[15] S. Smith and D. Read, "Mycorrhizal Symbiosis, 3rd Edn.," San Diego, CA: Academic
Press, 2008.
[16] S. Sharma, A. K. Sharma, R. Prasad, and A. Varma, "Arbuscular mycorrhiza: a tool for
enhancing crop production," in Mycorrhiza-nutrient uptake, biocontrol, ecorestoration,
2017, pp. 235-250.
[17] P. Bhandari and N. Garg, "Dynamics of arbuscular mycorrhizal symbiosis and its role
in nutrient acquisition: an overview," in Mycorrhiza-nutrient uptake, biocontrol,
ecorestoration, 2017, pp. 21-43, doi: 10.1007/978-3-319-68867-1_2.
[18] E. Guenther, "The Essential Oils," D.Van Nostrand Co., New York, 1961, pp. 1-236.
[19] Balittro, "Metode Pengujian dan Penelitian Analisis Minyak Atsiri Tanaman Rempah
dan Obat," Bogor, 2006.
[20] J. Phillip and D. Hayman, "Improved procedures for cleaning and staining parasitic
and vesicular arbuscular mycorrhizal fungi," Trans. Br. Mycol. Soc., vol. 55, pp.
158-161, 1970.
[21] M. Brundrett, L. Melvill, and R. L. Peterson, "Practical methods in mycorrhizal
research. (Mycologue Publications: Waterloo, Ontario, Canada)," 1994, p. 161.
[22] I. Indonesian Soil Research Institute, "Analisis Kimia Tanah, Tanaman, Air dan Pupuk.
Petunjuk Teknis. 3rd edn," Indonesian Center of Agricultural Land Resources Research
and Development, Indonesian Agency for Agricultural Research and Development,
2012.
[23] J. Pasaribu, "Aplikasi kompos kulit kayu eukaliptus dan Mikoriza terhadap
pertumbuhan Stek pucuk eukaliptus ( Eucalyptus sp)," Fakultas pertanian Universitas
Sumatera Utara, 2009.
[24] C. Arriagada, P. Pacheco, G. Pereira, A. Machuca, M. Alvear, and J. A. Ocampo,
"Effect of arbuscular mycorrhizal fungal inoculation on Eucalyptus globulus seedlings
and some soil enzyme activities under application of sewage sludge amendment," Rev.
la Cienc. del suelo y Nutr. Veg., vol. 9, no. 2, pp. 89-101, 2009, doi:
10.4067/S0718-27912009000200001.
[25] A. Achmad, H. N. Rana, I. Fadilla, A. Fajar, R. Manurung, and M. Y. Abduh,
"Determination of yield, productivity and chemical composition of eucalyptus oil from
different species and locations in Indonesia," Biol. Nat. Resour. Eng. J., vol. 1, no. 1,
pp. 36-49, 2018.
[26] A. Copetta, G. Lingua, and G. Berta, "Effects of three AM fungi on growth,
distribution of glandular hairs, and essential oil production in Ocimum basilicum L.
var. Genovese," Mycorrhiza, vol. 16, no. 7, pp. 485-494, 2006, doi:
10.1007/s00572-006-0065-6.
[27] D. Strack, T. Fester, B. Hause, W. Schliemann, and M. H. Walter, "Arbuscular
mycorrhiza: biological, chemical, and molecular aspects," J. Chem. Ecol., vol. 29, no.
9, pp. 1955-1979, 2003.
[28] Y. Yang, H. W. Xiang, Y. Y. Xu, L. Bai, and Y. W. Li, "Effect of potassium promoter
on precipitated iron-manganese catalyst for Fischer–Tropsch synthesis," Appl. Catal. A
Gen., vol. 266, no. 2, pp. 181-194, 2004, doi: 10.1016/j.apcata.2004.02.018.
[29] M. Bucher, "Functional biology of plant phosphate uptake at root and mycorrhiza
interfaces," Tansley Rev., pp. 11-26, 2007.
[30] R. S. Irianto, "Inokulasi Ganda Glomus sp. dan Pisolithus arrhizus Meningkatkan
Pertumbuhan Bibit Eucalyptus pellita F. Muell," J. Penelit. Hutan dan Konserv. Alam,
vol. 6, no. 2, pp. 159-167, 2009, doi: 10.20886/jphka.2009.6.2.159-167.
8
BIO Web of Conferences 69, 01001 (2023) https://doi.org/10.1051/bioconf/20236901001
2nd ICAFE 2023
[31] R. Rivera, F. Fernandez, K. Fernandez, L. Ruiz, C. Sanchez, and M. Riera, "Advances
in the management of effective arbuscular mycorrhizal symbiosis in tropical
ecosystems," Mycorrhizae Crop Prod., pp. 151-196, 2007.
[32] L. E. Schrader, "Function and transformations of nitrogen in higher plants," in
Nitrogen in Crop Production, 1984, pp. 55-65.
[33] M. A. A. H. A. Bukhsh, R. Ahmad, A. U. Malik, S. Hussain, and M. Ishaque,
"Profitability of three maize hybrids as influenced by varying plant density and
potassium application," J. Anim. Plant Sci., vol. 21, no. 1, pp. 42-47, 2011.
9
BIO Web of Conferences 69, 01001 (2023) https://doi.org/10.1051/bioconf/20236901001
2nd ICAFE 2023
Available via license: CC BY 4.0
Content may be subject to copyright.
