Evaluation of the antifungal activity by plant extracts against Colletotrichum gloeosporioides Penz

Avaliação da atividade anti-fúngica de extratos vegetais contra Colletotrichum gloeosporioides Penz

CIÊNCIAS AGRÁRIAS

Evaluation of the antifungal activity by plant extracts against Colletotrichum gloeosporioides Penz

Avaliação da atividade anti-fúngica de extratos vegetais contra Colletotrichum gloeosporioides Penz

Polyanna Alves Silva^I; Denilson Ferreira Oliveira^II; Ney Robson Taironi do Prado^III; Douglas Antônio de Carvalho^IV; Gilvane Aparecida de Carvalho^V

^IQuímica, Doutoranda – Departamento de Química/DQI – Universidade Federal de Lavras/UFLA – Cx. P. 3037 – 37200-000 – Lavras, MG – [e-mail]

^IIQuímico, Doutor, Professor Associado – Departamento de Química/DQI – Universidade Federal de Lavras/UFLA Cx. – P. 3037 – 37200-000 – Lavras, MG – [e-mail]

^IIIAcadêmico do curso de Química – Departamento de Química/DQI – Universidade Federal de Lavras/UFLA – Cx. P. 3037 – 37200-000 – Lavras, MG – [e-mail]

^IVEngenheiro agrônomo, Doutor, Professor Titular – Departamento de Biologia/DBI – Universidade Federal de Lavras/UFLA – Cx. P. 3037 – 37200-000 – Lavras, MG – [e-mail]

^VEngenheira agrônoma, Mestre – Departamento de Fitopatologia/DFP – Universidade Federal de Lavras/UFLA – Cx. P. 3037 – 37200-000 – Lavras, MG – [e-mail]

ABSTRACT

Aiming to develop more efficient and environmental friendly methods than those available to control Colletotrichum gloeosporioides Penz, which causes blister spot in coffee trees, a search for plants able to produce substances active against such pathogen was carried out. Thus, extracts of 48 plant species, collected at Alto Rio Grande region, in Minas Gerais, were prepared and submitted to in vitro assays with that fungus. The best results were obtained with the extracts prepared from Digitalis lanata Ehrh, Origanum manjorona L., Plantago lanceolata Hook. and Stevia rebaudiana (Bertoni) Bertoni, which inhibited C. gloeosporioides spores germination. After dilution of some active extracts with aqueous 1 % Tween 80 solution in a 1:2 or 1:3 ratio (extract:aqueous solution), their antifungal activity vanished. Some of the active extracts were also submitted to freeze drying and none of them presented any alteration in their antifungal activity. Concluding, several plants presented potential to be used in the search for new bioactive substances to control C. gloeosporioides, especially O. manjorona L., which inhibited 96 % of the fungus spores germination.

Index terms: Disease control, biocide, Coffee's blister spot.

RESUMO

Com vistas a contribuir para o desenvolvimento de métodos mais eficientes e menos agressivos ao ambiente que aqueles disponíveis para o controle de Colletotrichum gloeosporioides Penz, causador da mancha manteigosa em cafeeiros, buscou-se identificar espécies vegetais produtoras de substâncias ativas contra o referido patógeno. Para tanto, prepararam-se extratos de 48 espécies vegetais, coletadas na região do Alto Rio Grande, em Minas Gerais, para serem submetidos a testes in vitro com o mencionado fungo. Constatou-se que os melhores resultados foram obtidos com os extratos oriundos de Digitalis lanata Ehrh, Origanum manjorona L., Plantago lanceolata Hook. e Stevia rebaudiana (Bertoni) Bertoni que inibiram a germinação dos esporos de C. gloeosporioides. Alguns dos extratos ativos também foram submetidos a diluições em solução aquosa de Tween 80 a 1%, o que permitiu observar que, na proporção de 1:2 ou 1:3 (extrato: solução aquosa), os extratos perdiam a atividade antifúngica. Verificou-se também que alguns dos extratos ativos não alteraram suas atividades quando submetidos ao processo de liofilização. Desse modo, identificaram-se várias plantas promissoras na pesquisa por novas substâncias bioativas para o controle de C. gloeosporioides, com destaque para O. manjorona L., que inibiu 96% da germinação dos esporos do referido fungo.

Termos para indexação: Controle de doenças, biocida, mancha manteigosa do cafeeiro.

INTRODUCTION

Coffee is one of the most important export crops for Brazil, which is the leading producer and exporter of this commodity. With an area of 2.2 million ha devoted to coffee plants, such country produced 28.82 million sacks of coffee during the year of 2004 and exported the equivalent to US$ 1.51 billions in coffee products at the same year (BRASIL, 2005).

Among the phytopathological problems that occur during the Coffea arabica L. cultivation are those caused by fungi like Colletotrichum spp., whose presence in several coffee producing Brazilian regions has been constantly reported (CARVALHO, 2004). The symptoms of the fungal parasitism vary in accordance with the species, resulting in different names for the corresponding diseases. One of the most important is the blister spot in coffee plants, which is caused by the fungus Colletotrichum gloeosporioides Penz (MIRANDA, 2003).

Traditionally, plant fungal diseases are controlled by synthetic fungicides, which increase agricultural costs and contaminate the environment with very toxic substances (Carvalho, 2004). A possible alternative to solve such problem is the use of plants able to produce antifungal substances (MIRANDA, 2003). Among the several examples available in the literature are Zanthoxylum americanum Mill. and Piper regnellii var. pallescens (C. DC.) Yunck, whose antifungal activities are well known (BAFI-YEBOA et al., 2005; PESSINI et al., 2005).

Thus, this work aimed to contribute for the developement of new methods to control fungi in coffee plants by the identification of plant extracts active against C. gloeosporioides.

MATERIAL AND METHODS

Fresh leaves (3.0 g) of several plants (Table 1) collected in the Alto Rio Grande Region, State of Minas Gerais, Brazil, were cut in small pieces and soaked in methanol during 48 h. The resulting mixtures were filtered and the insoluble parts were soaked in more methanol during the same period of time. The new mixtures were filtered and the liquids obtained during both filtrations were combined and concentrated to dryness in a rotary evaporator. Immediately before carrying out the tests, the dry residues were dissolved in an aqueous 1 % (g/mL) Tween 80 solution (30 mL), resulting in the extracts to be studied.

C. gloeosporioides, isolated by Miranda (2003) and kept on deposit at Departamento de Fitopatologia - Universidade Federal de Lavras, was transferred to Petri dishes containing the culture medium MEA (20 g Agar, 20 g malt extract and 1 L distilled water), which was previously autoclaved at 120 ºC. After seven days at 22 ºC, under 12 h photoperiods, sterilized distilled water was poured into the dishes and, with a sterilized small paintbrush, the spores were removed. They were counted in the resulting suspension by the use of a Neubauer chamber and water was added to reach 4.0x10⁴ spores/mL.

As described by Carvalho (2004), the spore suspension (40 µL) was poured into each well of three well glass slides, which were kept inside Petri dishes. The plant extracts dissolved in aqueous Tween 80 (40 µL) were also poured into the wells and, after seven days at 22ºC, which was the best condition for maximum spore germination, 20 mL of a lactoglycerol solution (20 g lactic acid, 40 g glycerol, 20 mL distilled water and 0.05 g trypan blue) was poured into all wells to stop the germination. The wells were divided into five identical parts, in which 20 spores were counted. The percentage of germinated spores in each well was obtained after summing the values of germinated spores in each part.

All tests were carried out with three repetitions, arranged in a randomized design, employing aqueous 1 % Tween 80 solution as control. Statistical calculations were done using SISVAR software (FERREIRA, 2000) and values of germinated spores (%) underwent variance analysis using ANOVA; means were analyzed according to the Scott and Knott test (P < 0.05). According to Banzatto & Kronka (1989), the observed heterogeneity of variances (9:1) was above the recommended (7:1) for a joint statistical analysis. As a consequence, the experiments underwent statistical calculation separated from each other.

Some of the extracts affording low values of germinated spores were tested again at different concentrations: 1:0, 1:1 and 1:3 (plant extract dissolved in 1% Tween 80: 1% Tween 80). Statistical analysis were done as described above. However, as the homogeneity of variances was observed, a joint statistical calculation was performed.

The most promising extracts were once more prepared from 1.0 g of fresh plant material. Nevertheless, after the solvent removal, half of each residue were freeze-dried before dissolution in aqueous Tween 80 solution. The resulting extracts were submitted to the test with C. gloeosporioides as described above and values of germinated spores (%) underwent a non-parametric statistical analysis, employing the Wilcoxon test for matched pairs (SIEGEL, 1977). A P-value <0.05 was accepted as statistically significant.

RESULTS AND DISCUSSION

No influence on C. gloeosporioides was observed when the corresponding spores were treated with the extracts of C. jobi, C. officinalis, D. pectinata, H. rosa,M. pulegium, N. catarica, N. tabacum, Origanum sp., P. sonchifolia and T. officinale (Table 1). This seemed a reasonable result, since no report in the literature was found about the antifungal activity of such plants.

Although the activity against fungi has already been described for the extracts of A. arborescens, H. perforatum, J. curcas, P. granatum, P. graveolens, S. officinale, S. officinalis and S. nigra (DAMME et al., 2002; GIAMPERI et al., 2002; HOLETZ et al., 2002; KAWAI et al., 1998; LU et al., 2002; SENER, 1994; SHIN, 2003; THANGAVELU et al., 2004), none of them influenced the germination of C. gloeosporioides spores (Table 1).

An unexpected result was obtained for the extracts of F. vulgare, G. hederaceae, M. glabra, M. spicata and M. oleifera, whose antifungal activity has already been described in the literature (ABOU-JAWDAH et al., 2002; ADAM et al., 1998; CACERES et al., 1993; CORTEZ et al., 1998; LIMA et al., 1992; PATRA et al., 2002). Somehow, they increased the germination of C. gloeosporioides spores (Table 1).

The extracts of A. lappa, A. millefolium, Citrus sp., C. limon, C. longa, C. zedoarea, M. officinalis, O. biennis, O. gratissimum, P. alliacea, P. hysterophorus, P. major, R. graveolens and T. vulgaris were all active against C. gloeosporioides (Table 1), what is in accordance with the activity previously described against fungi from other genus (BENEVIDES et al., 2001; FICKER et al., 2003; FIORI et al., 2000; GIAMPERI et al., 2002; HOLETZ et al., 2002; KISHORE et al., 1996; MISHRA et al., 2002; NINO et al., 2003; POPOVIC et al., 2002; ROTH et al., 1998; SHRIVASTAVA et al., 1984; SHUKLA et al., 1999; SOUZA et al., 2005; TIWARI et al., 2003). Specifically for R. graveolens, the activity of the corresponding extract against C. gloesporioides was already known (OLIVA et al., 2003).

The extract of C. arabica cv. Acaiá-Cerrado MG1474 was also studied, since this coffee plant was less susceptible to C. gloeosporioides (FERREIRA, 2004) than other cultivars. Nevertheless, only a weak inhibition of spore germination was observed (Table 1).

A. eupatoria and Petiveria sp. extracts presented moderate antifungal action, while those from D. lanata, P. lanceolata and S. rebaudiana afforded low values of germinated spores. One of the best results was observed for the extract of O. manjorona, which inhibited about 96 % of the spores. These results were a surprise, since no report about the antifungal activity of such plants was found.

In order to assess the effect of extracts concentration on C. gloeosporioides spores, six of them were randomly selected among extracts presenting antifungal activity. None of them influenced spore germination in a 1:3 ratio (extract dissolved in 1 % Tween 80: 1% Tween 80)(Table 2).

The amount of germinated spores at 1:0 ratio (extract dissolved in 1 % Tween 80: 1% Tween 80) was approximately twice the value presented in Table 1 for those extracts prepared from de R. graveolens, S. rebaudiana and T. vulgaris. Regarding O. manjorona, the difference between values shown in Table 1 and Table 2 was much smaller, but for Citrus sp. the percentage of germinated spores was about four times less in the former Table. Probably, it ocurred due to differences in the plant collecting period, which can influence metabolite production (SAITO et al., 2004).

To simplify the purification process to be employed in the future to isolate the active substances and to preserve them, it is desirable to eliminate humidity. One of the softest processes to do so is freeze-drying (PASTORINI et al., 2002), whose influence on plant extract activity was evaluated in this work. With a probability of 0.109 and a level of significance fixed in 5%, it became clear after the Wilcoxon test that the freeze-drying process did not affect the antifungal activity (Table 3).

CONCLUSION

Among the several extracts studied, those from A. eupatoria, Petiveria sp. and mainly from D. lanata, P. lanceolata and S. rebaudiana, afforded very promissing results to be used for the control of C. gloeosporioides. The most active extract was that from O. manjorona, which inhibited 96 % of Colletotrichum gloeosporioides spore germination.

ACKNOWLEDGEMENTS

To Prof. Mário Sobral de Abreu (DFP-UFLA), Who kindly permitted the use of his Laboratory to carry out the experiments with the fungus.

(Received in august 4, 2006 and approved in march 15, 2007)

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Publication Dates

• Publication in this collection
  15 May 2008
• Date of issue
  Apr 2008

History

• Received
  04 Aug 2006
• Accepted
  15 Mar 2007