Antifungal and antibiofilm activities of the essential oil of leaves from Lippia gracilis Schauer against phytopathogenic fungi.

AIMS: This study aimed to evaluate the antifungal and antibiofilm effects of essential oil (EO) from leaves of Lippia gracilis and its major constituents, thymol and carvacrol, against phytopathogenic fungi. METHODS AND RESULTS: The leaves of L. gracilis were hydrodistilled to obtain the EO and the chemical composition was determined by GC/MS analysis. The antifungal activity of EO of L. gracilis was evaluated on the vegetative and mycelial growth of Colletotrichum gloeosporioides, Colletotrichum lindemuthianum, Fusarium oxysporum and Fusarium solani. In addition, the ability of the oil to inhibit fungal biofilm formation was assessed by total biomass quantification using crystal violet staining, analysis of metabolic activity, and scanning electron microscopy (SEM). Moreover the antifungal and antibiofilm activities of the monoterpenes, thymol and carvacrol, present in EO of L. gracilis were evaluated against F. oxysporum. The analysis of the chemical composition of EO extracted from L. gracilis, revealed the presence of monoterpenes (94·13%), which included carvacrol (48·57%) and thymol (7·78%), and 4 sesquiterpenes (3·74%). In general, EO showed significant antifungal activity and inhibited the formation of fungal biofilms. Furthermore, thymol and carvacrol showed significant antifungal and antibiofilm activities against F. oxysporum. SEM images showed structural changes in fungal morphology upon treatment with EO of L. gracilis. CONCLUSION: The results presented in this study showed promising antifungal and antibiofilm effects of EO of L. gracilis and its major components, carvacrol and thymol. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate that the EO extracted from L. gracilis, and the monoterpenes, carvacrol and thymol have a great potential as antifungal and antibiofilm agents. Furthermore, this is the first report of the antibiofilm activity of the EO of L. gracilis and its major components against phytopathogenic fungi.

Fungal Planet description sheets: 625-715.

Novel species of fungi described in this study include those from various countries as follows: Antarctica: Cadophora antarctica from soil. Australia: Alfaria dandenongensis on Cyperaceae, Amphosoma persooniae on Persoonia sp., Anungitea nullicana on Eucalyptus sp., Bagadiella eucalypti on Eucalyptus globulus, Castanediella eucalyptigena on Eucalyptus sp., Cercospora dianellicola on Dianella sp., Cladoriella kinglakensis on Eucalyptus regnans, Cladoriella xanthorrhoeae (incl. Cladoriellaceae fam. nov. and Cladoriellales ord. nov.) on Xanthorrhoea sp., Cochlearomyces eucalypti (incl. Cochlearomyces gen. nov. and Cochlearomycetaceae fam. nov.) on Eucalyptus obliqua, Codinaea lambertiae on Lambertia formosa, Diaporthe obtusifoliae on Acacia obtusifolia, Didymella acaciae on Acacia melanoxylon, Dothidea eucalypti on Eucalyptus dalrympleana, Fitzroyomyces cyperi (incl. Fitzroyomyces gen. nov.) on Cyperaceae, Murramarangomyces corymbiae (incl. Murramarangomyces gen. nov., Murramarangomycetaceae fam. nov. and Murramarangomycetales ord. nov.) on Corymbia maculata, Neoanungitea eucalypti (incl. Neoanungitea gen. nov.) on Eucalyptus obliqua, Neoconiothyrium persooniae (incl. Neoconiothyrium gen. nov.) on Persoonia laurina subsp. laurina, Neocrinula lambertiae (incl. Neocrinulaceae fam. nov.) on Lambertia sp., Ochroconis podocarpi on Podocarpus grayae, Paraphysalospora eucalypti (incl. Paraphysalospora gen. nov.) on Eucalyptus sieberi, Pararamichloridium livistonae (incl. Pararamichloridium gen. nov., Pararamichloridiaceae fam. nov. and Pararamichloridiales ord. nov.) on Livistona sp., Pestalotiopsis dianellae on Dianella sp., Phaeosphaeria gahniae on Gahnia aspera, Phlogicylindrium tereticornis on Eucalyptus tereticornis, Pleopassalora acaciae on Acacia obliquinervia, Pseudodactylaria xanthorrhoeae (incl. Pseudodactylaria gen. nov., Pseudodactylariaceae fam. nov. and Pseudodactylariales ord. nov.) on Xanthorrhoea sp., Pseudosporidesmium lambertiae (incl. Pseudosporidesmiaceae fam. nov.) on Lambertia formosa, Saccharata acaciae on Acacia sp., Saccharata epacridis on Epacris sp., Saccharata hakeigena on Hakea sericea, Seiridium persooniae on Persoonia sp., Semifissispora tooloomensis on Eucalyptus dunnii, Stagonospora lomandrae on Lomandra longifolia, Stagonospora victoriana on Poaceae, Subramaniomyces podocarpi on Podocarpus elatus, Sympoventuria melaleucae on Melaleuca sp., Sympoventuria regnans on Eucalyptus regnans, Trichomerium eucalypti on Eucalyptus tereticornis, Vermiculariopsiella eucalypticola on Eucalyptus dalrympleana, Verrucoconiothyrium acaciae on Acacia falciformis, Xenopassalora petrophiles (incl. Xenopassalora gen. nov.) on Petrophile sp., Zasmidium dasypogonis on Dasypogon sp., Zasmidium gahniicola on Gahnia sieberiana.Brazil: Achaetomium lippiae on Lippia gracilis, Cyathus isometricus on decaying wood, Geastrum caririense on soil, Lycoperdon demoulinii (incl. Lycoperdon subg. Arenicola) on soil, Megatomentella cristata (incl. Megatomentella gen. nov.) on unidentified plant, Mutinus verrucosus on soil, Paraopeba schefflerae (incl. Paraopeba gen. nov.) on Schefflera morototoni, Phyllosticta catimbauensis on Mandevilla catimbauensis, Pseudocercospora angularis on Prunus persica, Pseudophialophora sorghi on Sorghum bicolor, Spumula piptadeniae on Piptadenia paniculata.Bulgaria: Yarrowia parophonii from gut of Parophonus hirsutulus. Croatia: Pyrenopeziza velebitica on Lonicera borbasiana.Cyprus: Peziza halophila on coastal dunes. Czech Republic: Aspergillus contaminans from human fingernail. Ecuador: Cuphophyllus yacurensis on forest soil, Ganoderma podocarpense on fallen tree trunk. England: Pilidium anglicum (incl. Chaetomellales ord. nov.) on Eucalyptus sp. France: Planamyces parisiensis (incl. Planamyces gen. nov.) on wood inside a house. French Guiana: Lactifluus ceraceus on soil. Germany: Talaromyces musae on Musa sp. India: Hyalocladosporiella cannae on Canna indica, Nothophoma raii from soil. Italy: Setophaeosphaeria citri on Citrus reticulata, Yuccamyces citri on Citrus limon.Japan: Glutinomyces brunneus (incl. Glutinomyces gen. nov.) from roots of Quercus sp. Netherlands (all from soil): Collariella hilkhuijsenii, Fusarium petersiae, Gamsia kooimaniorum, Paracremonium binnewijzendii, Phaeoisaria annesophieae, Plectosphaerella niemeijerarum, Striaticonidium deklijnearum, Talaromyces annesophieae, Umbelopsis wiegerinckiae, Vandijckella johannae (incl. Vandijckella gen. nov. and Vandijckellaceae fam. nov.), Verhulstia trisororum (incl. Verhulstia gen. nov.). New Zealand: Lasiosphaeria similisorbina on decorticated wood. Papua New Guinea: Pseudosubramaniomyces gen. nov. (based on Pseudosubramaniomyces fusisaprophyticus comb. nov.). Slovakia: Hemileucoglossum pusillum on soil. South Africa: Tygervalleyomyces podocarpi (incl. Tygervalleyomyces gen. nov.) on Podocarpus falcatus.Spain: Coniella heterospora from herbivorous dung, Hymenochaete macrochloae on Macrochloa tenacissima, Ramaria cistophila on shrubland of Cistus ladanifer.Thailand: Polycephalomyces phaothaiensis on Coleoptera larvae, buried in soil. Uruguay: Penicillium uruguayense from soil. Vietnam: Entoloma nigrovelutinum on forest soil, Volvariella morozovae on wood of unknown tree. Morphological and culture characteristics along with DNA barcodes are provided.

Effects of the essential oil of Lippia gracilis Schauer on caulinary shoots of heliconia cultivated in vitro / Efeito do óleo essencial de Lippia gracilis Schauer sobre ápices caulinares de heliconia cultivadas in vitro

The effects of thymol and carvacrol and the essential oil of Lippia gracilis on caulinary shoots of heliconia were evaluated. After disinfection, the shoots were inoculated into MS medium and subjected to the treatments with 420 µL L-1 of essential oil (EO) of L. gracilis; 420 µL L-1 of thymol; 420 µL L-1 of carvacrol; 210 µL L-1 of thymol and 210 µL L-1 of carvacrol. The control treatment consisted of the MS medium without any phytoregulators. The main components of EO from L. gracilis are carvacrol, ρ-cimene, and thymol. Seven days after the initiation of the experiments, 36.3% of the control treatment shoots were necrotized, but 90% of the caulinary shoots exposed to EO, thymol, or carvacrol appeared necrotized. Transmission electron microscopy of the shoots revealed that the treatment with EO, thymol, or carvacrol caused the destruction of the plasma cell membranes, and the cell organelles and the nucleus were hardly evident. The EO and its main constituent were toxic to caulinary shoots of heliconia.

O efeito do timol, carvacrol e óleo essencial de Lippia gracilis foi observado sobre ápices caulinares de heliconia. Após a desinfestação os ápices foram inoculados em meio MS com os tratamentos de 420 µL L-1 do óleo essencial (OE) de L. gracilis; 420 µL L-1 de timol; 420 µL L-1 de carvacrol; 210 µg L-1 de timol e 210 µL L-1 de carvacrol. O tratamento controle consistiu de meio MS sem fitorreguladores. Os principais componentes do OE foram carvacrol, ρ-cimeno e timol. Sete dias após o início do experimento 36,3% dos ápices submetidos ao tratamento controle e 90% dos ápices caulinares expostos ao EO, timol ou carvacrol necrosaram. A microscopia eletrônica de transmissão dos ápices caulinares revelou que os tratamentos com OE, timol e carvacrol provocaram desestruturação da membrana plasmática das células. As organelas e o núcleo não estavam evidentes. O OE e seus principais constituintes foram tóxico para os ápices caulinares de helicônias.

Estabelecimento de protocolo para micropropagação de Lippia gracilis Schauer / Establishment of protocol for Lippia gracilis Schauer micropropagation

A espécie Lippia gracilis SCHAUER (Verbenaceae) é nativa do Nordeste brasileiro e se destaca pela capacidade de acumular nos tricomas glandulares óleos essenciais com atividade antimicrobiana. Tendo em vista que não constam trabalhos na literatura sobre o estabelecimento in vitro dessa espécie, este trabalho teve como objetivo estabelecer protocolo para micropropagação de L. gracilis. Para tanto, ramos contendo folhas foram coletados de plantas matrizes no habitat natural para a confecção de estacas. Em laboratório, os explantes provenientes do processo de estaquia foram assepticamente tratados e inoculados em meio MS, acrescido de fitorreguladores, com o intuito de se estabelecer a melhor dose para o desenvolvimento dos explantes. Em decorrência de altos níveis de contaminação, avaliou-se o efeito da cefalexina. No entanto, apesar do antibiótico ter apresentado diminuição na contaminação bacteriana, a porcentagem de oxidação foi elevada. Portanto, testou-se o carvão ativado, ácido ascórbico, ácido cítrico e metade dos sais de MS quanto a eficiência no controle da oxidação. Concluiu-se que, o antibiótico na concentração utilizada provocou a oxidação dos explantes e os fitorreguladores, bem como os métodos antioxidantes testados, não apresentaram resultados consistentes para o melhor desenvolvimento dos explantes e controle da oxidação, respectivamente.

The species Lippia gracilis Schauer (Verbenaceae) is native to Northeastern Brazil and has been important for its ability to accumulate essential oils with antimicrobial activity in the glandular trichomes. Since there are no reports in the literature on the micropropagation of this species, the present work aimed to establish a protocol for L. gracilis micropropagation. Thus, branches containing leaves were collected from plant matrices in their natural habitat to prepare cuttings. In the laboratory, explants from cutting were aseptically treated and inoculated onto MS medium plus plant growth regulators in order to establish the best dose for the development of explants. Due to high levels of contamination, the effect of cephalexin was evaluated. Although the antibiotic decreased the bacterial contamination, the percentage of oxidation was high. Then, activated charcoal, ascorbic acid, citric acid and half the salts of MS were tested for their effectiveness to control oxidation. In conclusion, the used antibiotic concentration resulted in oxidation of explants. Furthermore, plant growth regulators and antioxidant methods did not show consistent results for a better development of explants and control of oxidation, respectively.

Tympanometry in neonates with normal otoacoustic emissions: measurements and interpretation.

UNLABELLED: Tympanometry is used in evaluating middle ear functional conditions. Before six months of age its results may be misleading. High frequency studies aim to provide more valid procedures. AIM: To describe and discuss tympanometric measurements and the interpretation in normal hearing neonates at 226, 678 and 1000Hz. METHOD: 110 neonates that were analyzed had normal otoacoustic emissions and no risk for hearing impairment. The age range was 6 to 30 days. Curves were obtained using the GSI-33-II, at the Divisão de Educação e Reabilitação dos Distúrbios da Comunicação, São Paulo, in 2004. STUDY DESIGN: Clinical prospective. RESULTS: There was a balance between single and double peak curves at 226Hz. Most of the curves were asymmetric at 678Hz, and single-peaked at 1000Hz. quantitative measurements showed a significant gender difference in the Equivalent Ear Canal Volume at 226Hz and on the Peak Compensated Static Acoustic Admittance at 1000Hz. The English protocol showed that almost 100% of ears were normal at 678 and 1000Hz. CONCLUSION: 1000Hz yielded superior results for characterizing normality. The English protocol was efficient to reduce the variability of tympanometric measurements. Data from this study may be used as a guide for diagnosis using tympanometry in neonates.

Achados timpanométricos em neonatos com emissões otoacústicas presentes: medidas e interpretações / Tympanometry in neonates with normal otoacoustic emissions: measurements and interpretation

Timpanometria avalia condições funcionais da orelha média. Quando realizada em neonatos abaixo de quatro meses de idade pode gerar dúvidas. Sondas de alta freqüência têm sido investigadas na busca por resultados mais confiáveis. OBJETIVO: Descrever e analisar interpretações de características e medidas obtidas na timpanometria em neonatos com freqüências de 226, 678 e 1000Hz. MATERIAL E MÉTODO: Os 110 neonatos avaliados tinham idade entre 6 e 30 dias, não apresentaram fatores de risco e tinham emissões otoacústicas presentes. A timpanometria foi realizada com o analisador GSI-33-II, na Divisão de Educação e Reabilitação dos Distúrbios da Comunicação, São Paulo, em 2004. FORMA DE ESTUDO: Clínico prospectivo. RESULTADOS: Observou-se equilíbrio nas curvas pico único e pico duplo em 226Hz, maior ocorrência de assimétrica em 678Hz e de pico único em 1000 Hz. Houve significância de gênero na medida volume equivalente do meato acústico externo, em 226Hz, e na medida admitância acústica estática de pico compensado, em 1000Hz. O protocolo inglês apresentou quase 100 por cento de normalidade em 678 e 1000Hz. CONCLUSÃO: A sonda de 1000Hz apresentou melhores resultados na caracterização da normalidade. O protocolo inglês mostrou-se eficiente em diminuir variabilidades das medidas. Os resultados podem ser usados como guia no diagnóstico da timpanometria.

Tympanometry is used in evaluating middle ear functional conditions. Before six months of age its results may be misleading. High frequency studies aim to provide more valid procedures. AIM: To describe and discuss tympanometric measurements and the interpretation in normal hearing neonates at 226, 678 and 1000Hz. METHOD: 110 neonates that were analyzed had normal otoacoustic emissions and no risk for hearing impairment. The age range was 6 to 30 days. Curves were obtained using the GSI-33-II, at the Divisão de Educação e Reabilitação dos Distúrbios da Comunicação, São Paulo, in 2004. STUDY DESIGN: Clinical prospective. RESULTS: There was a balance between single and double peak curves at 226Hz. Most of the curves were asymmetric at 678Hz, and single-peaked at 1000Hz. quantitative measurements showed a significant gender difference in the Equivalent Ear Canal Volume at 226Hz and on the Peak Compensated Static Acoustic Admittance at 1000Hz. The English protocol showed that almost 100 percent of ears were normal at 678 and 1000Hz. CONCLUSION: 1000Hz yielded superior results for characterizing normality. The English protocol was efficient to reduce the variability of tympanometric measurements. Data from this study may be used as a guide for diagnosis using tympanometry in neonates.