Anti-phytopathogenic activities of macro-algae extracts

Abstract

Aqueous and ethanolic extracts obtained from nine Chilean marine macro-algae collected at different seasons were examined in vitro and in vivo for properties that reduce the growth of plant pathogens or decrease the injury severity of plant foliar tissues following pathogen infection. Particular crude aqueous or organic extracts showed effects on the growth of pathogenic bacteria whereas others displayed important effects against pathogenic fungi or viruses, either by inhibiting fungal mycelia growth or by reducing the disease symptoms in leaves caused by pathogen challenge. Organic extracts obtained from the brown-alga Lessonia trabeculata inhibited bacterial growth and reduced both the number and size of the necrotic lesion in tomato leaves following infection with Botrytis cinerea. Aqueous and ethanolic extracts from the red-alga Gracillaria chilensis prevent the growth of Phytophthora cinnamomi, showing a response which depends on doses and collecting-time. Similarly, aqueous and ethanolic extracts from the brown-alga Durvillaea antarctica were able to diminish the damage caused by tobacco mosaic virus (TMV) in tobacco leaves, and the aqueous procedure is, in addition, more effective and seasonally independent. These results suggest that macro-algae contain compounds with different chemical properties which could be considered for controlling specific plant pathogens.

Keywords: Botrytis cinerea; Phytophthora cinnamomi; macro-algae; plant pathogen; tobacco mosaic virus (TMV).

Figure 1.

Effect of extracts obtained from Chilean seaweeds on growth of Erwinia carotovora and Pseudomonas syringae. Crude aqueous (A and C) and 50% ethanolic extracts (B and D) isolated from Lessonia trabeculata (LT), Macrocystis pyrifera (MP), Gracilaria chilensis (GC), Gigartina skottsbergii (GS), Porphyra columbina (PC), Ulva costata (UC), Lessonia nigrescens (LN), Durvillaea antarctica (DA) and Macrocystis integrifolia (MI) collected in season 1 (black boxes), season 2 (white boxes), season 3 (light gray boxes) and season 4 (dark gray boxes) at a concentration of 10,000 ppm were incubated with E. carotovora (A and B, respectively) or with P. syringae (C and D) as described in Experimental Section. Negative control [C(–)] represents bacteria growing in media without algae extracts whereas positive control [C(+)] corresponds to bacteria growing in media containing 5 μM streptomycin. The activities of the extracts were evaluated in microculture assays by growing bacteria as described in the Experimental Section. All values represent mean of triplicate determinations ± standard deviation. Significant differences (P < 0.05) from control cell cultures are marked with an asterisk.

Figure 1.

Effect of extracts obtained from Chilean seaweeds on growth of Erwinia carotovora and Pseudomonas syringae. Crude aqueous (A and C) and 50% ethanolic extracts (B and D) isolated from Lessonia trabeculata (LT), Macrocystis pyrifera (MP), Gracilaria chilensis (GC), Gigartina skottsbergii (GS), Porphyra columbina (PC), Ulva costata (UC), Lessonia nigrescens (LN), Durvillaea antarctica (DA) and Macrocystis integrifolia (MI) collected in season 1 (black boxes), season 2 (white boxes), season 3 (light gray boxes) and season 4 (dark gray boxes) at a concentration of 10,000 ppm were incubated with E. carotovora (A and B, respectively) or with P. syringae (C and D) as described in Experimental Section. Negative control [C(–)] represents bacteria growing in media without algae extracts whereas positive control [C(+)] corresponds to bacteria growing in media containing 5 μM streptomycin. The activities of the extracts were evaluated in microculture assays by growing bacteria as described in the Experimental Section. All values represent mean of triplicate determinations ± standard deviation. Significant differences (P < 0.05) from control cell cultures are marked with an asterisk.

Figure 2.

Effect of algae extracts on growth of Phytophthora cinnamomi. Fungal mycelium was grown on medium containing 10,000 ppm either of (A) aqueous or (B) ethanolic extracts obtained from red alga Gracillaria chilensis in four different seasons (S1: summer; S2: autumn; S3:spring; S4: spring–summer) as described in Experimental Section. Different concentrations (0.1 = 100 ppm; 1 = 1000 ppm and 10 = 10,000 ppm) of the (C) active aqueous extracts obtained from samples collected in season S4. Ethanolic extracts obtained in S1 were also tested (D). Negative control [C(–)] represents mycelium growing in media without algae extracts whereas positive control [C(+)] corresponds to fungal growing in media containing 400 ppm Metalaxil. Activities of the extracts were evaluated as described in Experimental Section. All values represent the mean of triplicate determinations ± standard deviation. Significant differences (P < 0.05) from control fungal cultures are marked with an asterisk.

Figure 3.

Effect of algae extracts on tomato leaves following infection with Botrytis cinerea. Tomato leaves were used for evaluating the protecting properties of Chilean algae extracts. Control leaves without previous treatment were infected with fungal conidial suspension. Another set of tomato leaves were treated with different concentrations of either aqueous or ethanolic extracts before pathogen challenge as described in Experimental Section. The picture represents an average example of (A) control leaves and (B) leaves treated with commercial fungicide Captan before to B. cinerea infection.

Figure 4.

Protecting effect of ethanolic extracts from Lessonia trabeculata on tomato leaves challenged with Botrytis cinerea. Tomato leaves were treated before pathogen challenge with solutions containing 1000 (black boxes), 5000 (white boxes) or 10,000 (light gray boxes) ppm of ethanolic extracts obtained from different seasons (summer, autumn, spring, and spring–summer). Negative control [C(–)] represents B. cinerea mycelium growing on leaves without previous treatment with extracts whereas positive control [C(+)] corresponds to fungal growing on leaves which were pre-treated with solutions containing 600 ppm of Captan. Activities of the extracts were evaluated as described in Experimental Section. All values represent the mean of triplicate determinations ± standard deviation. Significant differences (P < 0.05) from negative control leaves are marked with an asterisk.

Figure 5.

Effect of algae extracts on the infection symptoms in tobacco leaves caused by tobacco mosaic virus (TMV). In vivo assays using tobacco leaves were performed to determine the influence of macroalgae extracts on tobacco leaves challenged with TMV. Therefore, (A) non-treated tobacco leaves or (B) treated ones with solutions containing different amounts of D. antarctica extracts were subsequently infected with TMV. Damage symptoms were evaluated as described in Experimental Section.

Figure 6.

Protective effect of D. antarctica extract on tobacco leaves following TMV infection. Leaves of tobacco plants were treated with algae extracts from samples collected at different times (S1–S4), with Ribavirin [C(+), dotted boxes] or non-treated [C(–), narrow horizontal boxes] before infection with TMV. (A) Aqueous and (B) ethanolic extracts were applied by spraying at different concentrations (black boxes: 1000 ppm; white boxes: 5000 ppm; gray boxes: 10,000 ppm) and the activities of the extracts were evaluated as described in Experimental Section. All values represent the mean of triplicate determinations ± standard deviation. Significant differences (P < 0.05) from negative control leaves are marked with an asterisk.

Figure 7.

Protective effect of algal aqueous extracts on tobacco. Leaves of tobacco plants were treated with different concentrations ((0.01–10) × 10³) of aqueous extracts obtained from samples collected in season 1 before infection with TMV (A). Negative control [C(–), narrow horizontal box] represents non-treated tobacco leaves whereas positive control [C(+), dotted box] corresponds to those pre-treated with Ribavirin. Activities of the extracts were evaluated as described in Experimental Section. All values represent the mean of triplicate determinations ± standard deviation. Significant differences (P < 0.05) from control leaves are marked with an asterisk.