The effect of lichen extracts obtained from Cladonia substellata and its main constituent, on the germination and growth of onion (Allium cepa L.) seedlings was investigated. No significant inhibitory effect of the lichen extracts and usnic acid on germination was found, except of usnic acid at the concentration of 726.7 mM. Growth inhibition of the radicle and of the hypocotyl of the seedlings was found for the total extract and for usnic acid in concentrations of 290.6 mM and 726.7 mM

Filho, Lauro Xavier

Vicente, Carlos

Yano-Melo, Adriana Mayumi

English

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11Tropical Bryology 16: 11-15, 1999Influence of Cladonia substellata Vainio extracts and usnic acidon germination and growth of Allium cepa L. seedlingsAdriana Mayumi Yano-Melo¹, Carlos Vicente² & Lauro Xavier-Filho³¹ Departamento de Micologia, Centro de Ciências Biológicas, Universidade Federal dePernambuco, Av. Moraes Rego, s/n, Cidade Universitária, 50670-420, Recife – Pernam-buco, Brasil² Laboratory of Plant Physiology, Faculty of Biology, Complutense University of Madrid,Madrid, Spain³ Laboratório de Tecnologia Farmacêutica, Universidade Federal da Paraíba, CaixaPostal 5009, 58051-970, João Pessoa – Paraíba, Brasil.Abstract: The effect of lichen extracts obtained from Cladonia substellata and its main constituent,usnic acid, on the germination and growth of onion (Allium cepa L.) seedlings was investigated. Nosignificant inhibitory effect of the lichen extracts and usnic acid on germination was found, except ofusnic acid at the concentration of 726.7 mM. Growth inhibition of the radicle and of the hypocotyl ofthe seedlings was found for the total extract and for usnic acid in concentrations of 290.6 mM and726.7 mM.IntroductionA lichen from north-east Brazil, Clado-nia substellata (Ahti et al., 1993), is known tohave the highest concentration of usnic acidamong lichens, c. 98% of its chemical constituti-on, representing c. 7.3% of the thallus dry weight(Huovinen & Ahti, 1986). Usnic acid is a corti-cal substance broadly distributed among lichens(Rundel, 1978) and due to this fact, considerableresearch has been undertaken to show its efficien-cy in antineoplasic, antimicrobial and antimitoticactivities (Henningsson & Lundstrom, 1970;Lima et al., 1990; Pereira et al., 1991).Dalvi et al. (1972), Vicente et al. (1983)and Vavasseur et al. (1991) have shown that us-nic acid is an inhibitor of oxygen catchment tosome organisms, causing growth reduction and,in some cases, impeding the germination of seeds.However, decrease in germination and growthby usnic acid was not observed by Lawrey (1977)and Nishitoba et al. (1987) studying its effect ongermination of moss spores and on growth oflettuce seedlings, respectively.The present work evaluates the effect ofpure usnic acid and extracts from Cladonia sub-12stellata on germination and growth of Alliumcepa seedlings.Materials and MethodsOnion seeds (cv. IPA-6) were supplied bythe Agricultural Research Agency of Pernambu-co (IPA), Recife-PE, Brazil.The lichen material was collected in theregion of Tabuleiro of Santa Rita, Paraíba State(Brazil) (7° 52' 45'’ S and 35° 30' W), during thedry season (October/November) of 1991. Sam-ples of 30g of thallus were dried and stored atroom temperature (28° C), and pulverized for thepreparation of extracts.From this material aqueous, phosphate-buffered and total extracts, and pure usnic acid-solutions were prepared. The aqueous andphosphate buffered extracts were prepared using,respectively, distilled water and phosphate buf-fer 50 mM in pH 7.0. For the total extract, threesolvents were used: ethylic ether, acetone, andethanol/water (8:2 v/v). The solvents remainedin a shaker with the thallial material for one hour.The solution of total extract for use in the germi-nation test was prepared according to the metho-dology of Dalvi et al. (1972). The concentrati-ons of the extracts were 166 mg/l, comparable tothe second level (290.6 mM) of the pure usnicacid solutions.To determine the lichen acid compositi-on of the extracts, HPLC analyses were perfor-med in a VARIAN 5000 apparatus under the fol-lowing conditions: RP - C8 column of 40 x 0.4cm of i.d.; mobile phase acetonitrile:acetic acid(80:20 v/v):water (98:2 v/v); flux 0.3 cm/min.;temperature 26° C; pressure 84 atmos. and UVdetector at 280 nm (Table 1).For the preparation of pure usnic acid so-lutions, the substance was isolated by the metho-dology of Asahina and Shibata (1954), utilizingthe „Soxlet“ apparatus with the addition of 500ml of ethylic ether as solvent, and extracting for72 hours. The preparation of the usnic acid solu-tion followed the same procedures as the totalextract solution. For the tests three concentrati-ons were utilized: 72.6 mM, 290.6 mM and 726.7mM.In the germination tests, onion seeds wereplaced in „gerbox“ plates containing filter paper(100 seeds in each „gerbox“ plate for each repli-cation). Measurements were made on the sixthand tenth days when the following parameterswere evaluated: germination rate, radicle andhypocotyl lengths. The experimental design wascompletely randomized, with four replications.Results and DiscussionThe Cladonia substellata extracts contai-ned usnic, stictic and constictic acids in variableconcentrations (Table 1). In the total extract ofC. substellata usnic acid appeared to have thehighest concentration (Table 1), which is in ac-cordance with the observation of Huovinen andAhti (1986). However, in the aqueous extract ithad the lowest concentration while the highestconcentration was of stictic acid. This is probab-ly due to the low solubility of usnic acid in water(Vicente, 1975; Rundel, 1978; Hale, 1983), whi-le stictic acid is highly soluble, as shown by As-caso and Galvan (1976).The C. substellata extracts did not inhibitthe germination of A. cepa (cv. IPA-6) seeds,which exhibited a high germination rate (over90%) (Table 2). Among the treatments, only290.6 mM and 726.7 mM of usnic acid seemed tolower germination rates, germination decreasingwith increasing concentrations of usnic acid.However, only the treatment at 726.7 mM con-centration showed a germination rate below themean which was statistically different from thecontrol (Table 2).Dalvi et al. (1972) had observed inhibiti-on of germination of seeds of Phaseolus mungoand Triticum aestivum by usnic acid in concen-trations of 290.6 mM and 726.7 mM. The resultsof the present work indicate that the inhibitioneffect depends also on the genotype of the seedthat is being used.The growth of A. cepa seedlings wasstrongly affected by treatments with usnic acidin concentrations of 290.6 mM and 726.7 mM andwith the total extract of C. substellata. Affectedwere both the radicle and hypocotyl lengths (Ta-bles 3 and 4). In the concentration of 72.6 mMusnic acid did not show an inhibitory effect onthe growth of A. cepa. Likewise there was noeffect from both the aqueous and phosphate buf-fered extracts.The inhibiting influence of the total ex-tract, contrary to the aqueous and phosphate-buf-13Table 1 – Lichen substance composition of the aqueous-, phosphate buffer- and totalextracts of Cladonia substellata, as shown by HPLC.EXTRACT            SUBSTANCES   RETENTION TIME AREA                         (min.)       (%)Aqueous stictic acid                 1.12     60.45constictic acid     1.34     15.37usnic acid            3.53 - 7.98     22.16Phosphate buffered stictic acid                 1.00     35.53constictic acid      1.40     31.02usnic acid            2.47 - 4.56     32.24Total stictic acid                 0.83     25.67constictic acid      1.47     13.98usnic acid            2.10 - 6.0     59.58Table 2 - Germination rates of Allium cepa (cv. IPA 6) seeds on usnic acid and C.substellata extracts.   TREATMENT  GERMINATION RATE  (%)      Aqueous extract                   95.3  a *      Total extract                   93.5  a b      Control                   93.5  a b      Phosphate buffered extract                  92.0  a b      Usnic acid - 72.6 mM                   91.6  a b      Usnic acid - 290.6 mM                   87.1     b c      Usnic acid - 726.7 mM                   81.2        c* - values with the same letter do not differ statistically by the Tukey Test (0.05).Table 3 - Mean length of Allium cepa radicle growth on usnic acid and C. substellataextracts.                          TREATMENT                      RADICLE LENGTH                            (cm)Usnic acid - 72.6 mM                       2.138  a *Control                        2.075  aPhosphate buffered extract        1.761  aAqueous extract                        1.718  aUsnic acid - 290.6 mM                    0.901     bTotal extract                        0.698     bUsnic acid - 726.7 mM                    0.508     b* - values with the same letter do not differ statistically by the Tukey Test (0.05).14fered extracts, suggests that usnic acid is the ac-tive agent: it constitutes around 60% of the totalextract, while the other extracts contain much less(Table 1). This assumption is supported by thefact that the usnic acid-solutions have a similareffect in high concentrations, and none in lowconcentration.These results are not in agreement withthose of Nishitoba et al. (1987), but support theresults of Goldner et al. (1986) and Lascève &Gaugain (1990). The decline in development ofthe root system may be explained by the obser-vations of Vicente et al. (1983) and Vavasseur etal. (1991), that usnic acid plays a role in the re-spiratory process and thus affects growth.AcknowledgementsThe authors thank Dr Vânia Canuto(EMBRAPA/IPA, Brazil) for making available thefacilities of the Seed Laboratory of the AgriculturalResearch Agency of Pernambuco for the germinationtests, Dr Marçal de Queiroz Paulo (Dept. of Chemistry-UFPB, Brazil) for his criticism and suggestions, andDr Eduardo Assis Menezes (Embrapa Semi-Árido,Brazil) and Dra. Leonora Costa Maia (UFPE, Brazil)for reviewing the manuscript.BibliographyAsahina, Y. & Shibata, S. 1954. Chemistry of LichenSubstances. Tokyo: Japan Society for thePromotion of Science, 240 pp.Ascaso, C. & Galvan, J. 1976. Studies on thepedogenetic action of lichen acids. Pedobiologia16: 321-331.Ahti, T., Stenroos, S. & Xavier-Filho, L. 1993. Thelichen family Cladoniaceae in Paraiba,Pernambuco and Sergipe, northeast Brazil.Tropical Bryology 7: 55-70.Dalvi, R.R., Singh, B. & Salunkhe, O.K. 1972.Physiological and biochemical investigation onthe phytotoxicity of usnic acid. Phyton (BuenosAires) 29(1/2): 63-72.Goldner, W R., Hoffman, F.M. & Mede, R.J. 1986 .Allelopathic effects of Cladonia cristatella onectomyco-rrhizal fungi common to bituminousstrip-mine spoils. Canadian Journal of Botany64: 1586-1590.Hale Jr, M.E. 1983. The Biology of Lichens. 3rd ed.London, Edward Arnold. 190 pp.Henningsson, B. & Lundstrom, H. 1970. Theinfluence of lichens, lichen extracts and usnicacid on wood destroying fungi. Material undTable 4 - Mean length of Allium cepa hypocotyl developed on usnic acid and C.substellata extracts.TREATMENT         HYPOCOTYL LENGTH(cm)Control                       5.007  a *Phosphate buffered extract                   4.760  a bUsnic acid - 72.6 mM                       4.341  a bAqueous extract                        4.157    bUsnic acid - 290.6 mM                        3.171       cTotal extract                        2.786       cUsnic acid - 726.7 mM                        2.590       c* - values with the same letter do not differ statistically by the Tukey Test (0.05).15Organismen 5: 19-31.Huovinen, K. & Ahti, T. 1986. The composition andcontents of aromatic lichen substances in thegenus Cladonia section Unciales. AnnalesBotanici Fennici 23: 173-188.Lascève, E. & Gaugain, F. 1990. Effects of usnicacid on sunflower and maize plantlets. Journalof Plant Physiology 136: 723-727.Lawrey, J.D. 1977. Adaptative significance ofmethylated lichen depsides and depsidones.Lichenologist 9: 137-142.Lima, R.C., Nascimento, S.C., Pereira, E.C.G. &Campos-Takaki, G.M. 1990. Atividadecitotóxica e antitumoral de extratos liquênicos.Boletim da Sociedade Broteriana 63 (2): 339-348.Nishitoba, Y., Nishimura, H., Nishiyama, T. &Mizutani, J. 1987. Lichen acids, plant growthinhibitors from Usnea longissima.Phytochemistry 26: 3181-3186.Pereira, E.C.G., Campos-Takaki, G.M., Silva, N.H.,Vicente, C., Legaz, M.E. & Xavier-Filho, L.1991. Fractionation of Cladonia substellatacrude extracts and detection of antimicrobialactivity. Boletim da Sociedade Broteriana 64 (2):173-186.Rundel, P.W. 1978. The ecological role of secondarylichen substances. Biochemical Systematics andEcology 6: 155-170.Vavasseur, R., Gautier, H., Thiband, M.C. &Lascève, G. 1991. Effects of usnic acid on theoxygen exchange properties of mesophyll cellprotoplasts from Commelina communis L..Journal of Plant Physiology 139: 9094.Vicente, C. 1975. Fisiologia de las SubstanciasLiquenicas. Madrid: Alhambra. 16 pp.Vicente, C., Legaz, M.E. & Xavier-Filho, L. 1983.Estudio biologico y farmacologico de losantibioticos de origem liquenico. In: SimpósioNacional de Farmacologia e Química deProdutos Naturais 2, Anais do Simpronat, JoãoPessoa, PB: UFPB, LTF, p. 319-343.16

Allelopathic effects of Cladonia cristatella on ectomyco-rrhizal fungi common to bituminous strip-mine spoils.

Chemistry of Lichen Substances.

Physiological and biochemical investigation on the phytotoxicity of usnic acid.

Studies on the pedogenetic action of lichen acids.

The influence of lichens, lichen extracts and usnic acid on wood destroying fungi.

The lichen family Cladoniaceae in Paraiba, Pernambuco and Sergipe, northeast Brazil.

Influence of Cladonia substellata Vainio extracts and usnic acid on germination and growth of Allium cepa L. seedlings

Influence of Cladonia substellata Vainio extracts and usnic acid on germination and growth of Allium cepa L. seedlings

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