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Boletín de la Sociedad Chilena de Química

versão impressa ISSN 0366-1644

Bol. Soc. Chil. Quím. v.46 n.4 Concepción dez. 2001 



**Departamento de Ciencias Biológicas, #Departamento de Química de los Materiales,
Facultad de Química y Biología, Universidad de Santiago de Chile
Casilla 40-Correo 33, Santiago-Chile
(Received: March 21, 2001 - Accepted: July 31, 2001)


The activity of the extracts obtained from the resinous exudates of the plants Pseudognaphalium cheiranthifolium, P. heterotrichium, P. robustum and P. vira vira on mycelial growth of the phytopathogenic fungus Botrytis cinerea was analyzed. Ten flavones, two flavanones and three diterpenoids isolated from these extracts were also tested for antifungal activity against B. cinerea. The extracts reduced mycelial growth and the inhibitory activity of the pure compounds was higher. Flavones with two hydroxyl groups on ring- A showed higher antifungal activity. Flavanones were inactive. The diterpenoid, 3b -hydroxy-kaurenoic acid was the most active compound of this set against mycelial growth of B. cinerea. This compound also retarded the germination of conidia of the fungus.

Keywords: Botrytis cinerea; Pseudognaphalium spp.; diterpenoids; flavonoids; antifungal activity


Se analizó la actividad de extractos resinosos obtenidos de las plantas Pseudognaphalium cheiranthifolium, P. heterotrichium, P. robustum y P. vira vira sobre el crecimiento micelial del hongo fitopatógeno Botrytis cinerea. Adicionalmente, se determinó la actividad antifúngica contra B. cinerea de diez flavonas, dos flavanonas y tres diterpenos aislados de estos extractos. Se encontró que los extractos disminuyeron el crecimiento del hongo y que la actividad inhibitoria de los compuestos puros fue mayor. Las flavonas con dos grupos hidroxilos en el anillo A fueron las más activas contra el hongo. Las flavanonas fueron inactivas. El diterpenoide, ácido 3b -hidroxi-kaurenoico fue el compuesto más activo de este conjunto sobre el crecimiento micelial de B. cinerea. Este compuesto también retardó la germinación de los conidios del hongo.

Palabras claves: Botrytis cinerea; Pseudognaphalium spp.; diterpenos; flavonoides; actividad antifúngica


Bunch rot, an important disease in Chile, is caused in grapes by the phytopathogenic fungus Botrytis cinerea1). Climatic conditions such as high relative humidity and low temperatures lead to a high incidence of this disease. This fungus affects grapes in the field and during storage1). In Chile, B. cinerea has traditionally been controlled by commercial fungicides (dicarboximides and benzimidazoles). The use of these fungicides has caused serious problems such as the appearance of highly resistant strains and the contamination of soil and water2, 3).

Some naturally occurring substances found in higher plants, terpenoids, flavonoids, alkaloids and stilbenes are an important group of molecules with antifungal properties4-9).

The antifungal activity of some of these compounds against B. cinerea has been previously studied. Germination of conidia of B. cinerea was inhibited by sakuranetin, a flavonoid isolated from the surface of Ribes nigrum6). Three chalcones from the wood of Bauhinia manca, showed activity against the myceliar growth of B. cinerea6). Resveratrol, a stilbene produced by grapes, inhibited the spread of B. cinerea infection10). Strains of B. cinerea that degraded resveratrol were more pathogenic to in vitro cultures of grapevine than those unable to degrade this phytoalexin11). Besides, a -tomatine, a steroidal glycoalkaloid produced by tomato affected the growth of this fungus12,13). Finally, cucurbitacin a tetracyclic triterpenoid protected cucumber tissue against infection by B. cinerea14).

The aim of the present study was to examine the effect on mycelial growth and germination of B. cinerea of some flavonoids and diterpenoids isolated from resinous exudates of Pseudognaphalium spp.


Fungal isolate and culture conditions

The strain U29 of B. cinerea isolated from infected grapes was used in this study. The fungus was grown and maintained in Petri dishes on malt-yeast extract agar (1.5% agar, 2% malt extract and 0.2% yeast extract). Cultures were incubated at 22ºC.

Isolation of secondary metabolites from Pseudognaphalium spp.

The surface extracts of P. cheiranthifolium (Lam.) Hilliard & Burtt, P. heterotrichium (Phil.) A. Anderb., P. robustum (Phil.) A. Anderb and P. vira vira (Mol.) A. Anderb. were obtained as described by Urzúa15). The flavonoids and diterpenoids used in this study were isolated from the resinous exudates of Pseudognaphalium spp. as has been described15,16). Quercetine was obtained commercially (Aldrich Chemical Co., Milwaukee, WI, USA).

Fungitoxicity assay

1.- Effect on mycelial growth of B. cinerea

Fungitoxicity of extracts, pure natural products and the commercial fungicide iprodione was assesed using the radial growth test on malt-yeast extract agar (2% malt extract, 0.2% yeast extract and 1.5% agar)17). Total extracts, pure plant compounds or iprodione were dissolved in methanol to different final concentrations. One hundred mL of this solution were added to 5 mL of medium (0.6% agar, 2% malt extract and 0.2% yeast extract). The final methanol concentration was identical in controls and treatment assays. The medium with or without test substances was poured into 9 cm diameter Petri dishes containing malt-yeast extract agar. Dishes were left open in a laminar-flow hood for 30 min to remove methanol. After evaporation of the solvent, the Petri dishes were inoculated with 0.5 cm agar discs with thin mycelium of B. cinerea. Cultures were incubated at 22ºC for seven days. Mycelial growth diameters were measured daily. The mean value of at least three different experiments was used for calculation. Each experiment was done in triplicate.

2-. Effect on germination of B. cinerea conidia

Germination assays were carried out on microscope slides coated with malt-yeast extract agar (2% malt extract, 0.2% yeast extract and 0.6 % agar) with methanol or methanol and the pure compounds to give a final concentration of 40 mg/mL. Methanol was allowed to evaporate prior to inoculation. The slides were inoculated with dry conidia, placed in a humid chamber and incubated at 22º C for ten hours. Conidial germination was determined directly on the slides at hourly intervals. Conidia were judged to have germinated when germ tube length was equal to or greater than conidial diameter. The mean value of at least three different experiments was used for calculation. Each experiment was done in triplicate.


The activity of the surface extracts obtained from Pseudognaphalium spp. on mycelial growth of B. cinerea was analyzed. Figure 1 shows the percentage of growth inhibition of B. cinerea in the presence of the extracts. At 40 m g/mL, all extracts reduced the growth of this fungus. The percentage of inhibition varied between 13 and 21%. The chemical composition of these extracts had been previously determined15,16). Extracts from P. heterotrichium are mainly constituted by the flavonoid 7 and the diterpenoids 13, 14 and 15 (Figure 2). Diterpenoids 13, 14 and 15 are the main components of the extract from P. cheiranthifolium and the flavonoids 1, 2, 5 and 7 are minor components of this extract. P. vira vira extracts mainly contain diterpenoids 13 and 14. Flavonoids 4, 6, 7, 8, 9 and 11 are components of the resinous exudates of P. robustum15, 16). Flavonoid 8 has been sometimes called by the non systematic name of "gnaphalin". This name is derived from its botanical source Gnaphalium robustum18). However, the term "gnaphalin" was used before to name the diterpenoid ent-4b ,18:15,16-diepoxy-19-hydroxy-6-oxo-13(16),14-clerodadien-20,12-olid which was isolated from Teucrium gnaphaloide19). We have therefore decided to avoid the use of "gnaphalin" refering to this compounds by its IUPAC name.

Figure 1: Inhibitory effect of extracts obtained from resinous exudates of Pseudognaphalium spp. on the growth of B. cinerea.
Total extracts dissolved in methanol were added to a final concentration of 40 m g/mL Controls contained methanol at the same concentration as treatment assays. Percentages of inhibition of growth relative to controls with methanol were calculated after four days of incubation. Extracts from P. heterotrichium (P. h) , P. cheiranthifolium (P. ch), P. vira vira (P. vv) and P. robustum (P. r).

Besides the compounds previously described in these extracts other components have been identified (Urzúa, unpublished results). This author analyzed the four resinous exudates by GLC/EI-MS. In the resinous exudates of P. cheiranthifolium and P. vira vira monoterpenes, sesquiterpenes and n-alkanes were also identified. The resinous exudate of P. heterotrichium also contained monoterpenes, sesquiterpenes, n-alkanes, organic acids and esters. In the resinous exudate of P. robustum, monoterpenes, sesquiterpenes, n-alkanes, alcohols and esters were also identified.

To determine if flavonoids and diterpenoids are the active compounds in the Pseudognaphalium spp. extracts, the effect of each of these compounds on B. cinerea mycelial growth was assessed by calculating the percentage of growth reduction, relative to the control, after four days of incubation. Tested compounds are shown in Figure 2.

Figure 2: Structures of flavonoids and diterpenoids used in this study

The effect of flavonoids on the mycelial growth of B. cinerea is shown in Table I. Flavone 8 was the most active inhibitor of hyphal growth. At 40 mg/mL, it inhibited the growth by 32.1%. At the same concentration, flavone 2 reduced hyphal growth by 14.9%. Flavones 1, 3 and 7 reduced growth by about 9%. Flavones 4, 5, 6 and 10 did not affect the growth of B. cinerea significantly. Our results suggest that the presence of two hydroxyl groups on ring A is important for the antifungal activity of oxygenated flavones against B. cinerea. The only exception was compound 3 which has only one hydroxyl group on ring A. Quercetin (10), a highly hydroxylated flavone, had no inhibitory effect on B. cinerea. In general, it has been demonstrated that high polarity due to several hydroxyl groups seems to reduce antifungal activity of flavones5,20,21). Previous studies have shown that the unsubstituted flavone and flavanone were more active against Aspergillus spp., Cladosporium herbarum, Fusarium oxysporum, Trichoderma harzianum and Verticillium albo-atrum than hydroxylated flavonoids5, 20, 21). Consequently, the antifungal activity was ascribed to the absence of polar groups on the molecules5, 21).

aThe compounds were added as described in Materials and Methods at a concentration of 40 m g/mL. The percentage of inhibition was determined after four days of incubation.
Values are means ± S. E.

On the other hand, flavanones pinocembrin (11) and naringenin (12) tested in this study showed no antifungal activity against B. cinerea (Table I). Naringenin, however, has been showed to inhibit the growth of V. albo-atrum21). Therefore, the effect of flavonoids on the different fungi may be specific to each fungal species.

The antifungal activity of a mixture of 15-(2-methylbutenyloxy) kaurenoic acid and kaurenoic acid against Sclerotinium sclerotium and V. dahliae had been previously reported22), but there is no information on the structure-activity relationship of diterpenoids in this regard.

Among the diterpenoids tested in this study, 3b -hydroxy-kaurenoic acid (14) was the most active compound against B. cinerea. At 40 m g/mL, it inhibited hyphal growth by 38.3%. At the same concentration, kaurenoic acid (13) reduced hyphal growth by 10.3%. It seems reasonable to suggest that the hydroxyl group at C-3 increases the polarity of the rather lipophilic kaurenoic acid and that this characteristic might be important for the fungitoxic effect. Another explanation could be that the carboxyl group in 14 might be protected by formation of an intramolecular hydrogen-bond with the b -hydroxyl group at C-323), with the result that the carboxyl group would be less available for detoxification reactions by the fungus. Further work is necessary to confirm this hypothesis.

Therefore, the antifungal activity against B. cinerea of the extracts obtained from resinous exudates of Pseudognaphalium spp. could be mainly related to the presence of flavonoids and diterpenoids in these extracts.

Finally, the fungitoxic effect of the flavonoids and diterpenoids on the mycelial growth of B. cinerea was compared with the effect of the commercial fungicide iprodione (Table I). This product is used to control B. cinerea in the field. At 40 mg/mL, the fungicide inhibited mycelial growth by 57.3 %, higher than the most active flavonoids and diterpenoids analyzed in this study.

Concentration-response curves for some of the more potent flavones (1, 2 and 7), 3b -hydroxy-kaurenoic acid (14) and 13-epi-sclareol (15) were determined (Figure 3). The inhibitory effect of flavones 1 and 2 on mycelial growth was dependent on concentration until 120 mg/mL. If concentration-response curves from the flavones 1 and 7 are compared, a similar inhibitory effect was observed at low concentration (40 mg/mL); instead, at higher concentrations, 1 showed a higher inhibitory effect. At 40 mg/mL, 3b -hydroxy-kaurenoic acid (14) showed a stronger effect than flavones 1 and 2, but at 120 mg/mL flavone 2 showed a similar inhibitory effect to b -hydroxykaurenoic acid. The inhibitory effect of compound 15 decreased when the compound concentration was increased.

Figure 3: Effect of the concentration of flavones and diterpenoids isolated from Pseudognaphalium spp. on antifungal activity.
Percentage of inhibition was calculated after four days of incubation, relative to the controls with methanol (A) Flavones 1 (¨), 2 (!) and 7 (+). (B) diterpenoids 14 (# ) and 15 ()).

Furthermore, the effect of the diterpenoids 13 and 14 on conidia germination of B. cinerea was analyzed (Figure 4). Kaurenoic acid (13) did not affect the germination of the conidia, but 3b -hydroxy-kaurenoic acid (14) retarded germination. The control without diterpenoids attained 100% germination after 8 h of incubation. In the presence of 40 m g/mL of 3b -hydroxy-kaurenoic acid (14) 100% germination was reached after 10 h of incubation.

Figure 4: Effect of the diterpenoids 13 and 14 on conidia germination of B. cinerea.
The diterpenoids were dissolved in methanol and added to a final concentration of 40 m g/mL Control contained methanol at the same concentration as treatment assays. ('…) Control, (!) 13 and ()) 14.


We thank Alejandro Urzúa for his thoughtful comments. This research was supported by the Departamento de Investigaciones Científicas y Tecnológicas (Dicyt) of the Universidad de Santiago de Chile and by the International Foundation for Science (Grant No C/2807-1).


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