EVALUATION OF THE ANTIFUNGAL ACTIVITY BY PLANT EXTRACTS AGAINST Colletotrichum gloeosporioides PENZ

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.


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 o C.After seven days at 22 o C, 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 4.0x10 4 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 22 o C, 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 freezedried 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.
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.

Table 1
Percentage of Colletotrichum gloeosporioides germinated spores after treatment with plant extracts at a constant concentration.To be continued...

Table 1
Continued... Means of six replicates with the same letter do not differ significantly (P 0.05).CV values varied between 3.5 and 14.3 %. *A mixture of leaves and inflorescences was used to prepare the extract.

Table 2
Percentage of Colletotrichum gloeosporioides germinated spores after treatment with plant extracts at different concentrations.

Table 3
Percentage of Colletotrichum gloeosporioides germinated spores after treatment with plant extracts before and after freeze-drying.