Control of phytopathogenic fungi in horticultural crops by natural plant extracts
Author(s)
Švecová, Eva
Date Issued
March 19, 2010
Type
Doctoral Thesis
Abstract
Fungal pathogens represent a constant threat for vegetable crops under field and
protected conditions. Historically, losses of crop yield from fungal diseases have had severe
effects even on the human race causing famines and epidemics. Therefore, the proper
management of fungal diseases is of global importance. Even today many of these diseases
are difficult to control. In addition, some fungi have developed resistance against certain
fungicides. Therefore, other alternatives to manage plant pathogenic fungi are of main
interest. Recently, plant extracts represent a valid possibility for managing fungal diseases as they are a natural source of antifungal compounds.
In the present thesis, methanolic extracts of five plants such as Siparuna guianensis
(Monimiaceae), Cordia leucocephala (Boraginaceae), Boerhavia diffusa (Nyctaginaceae), Vitex agnus-castus (Verbenaceae), and Phyllanthus niruri (Euphorbiaceae) were screened for
their antifungal activity under both in vitro and in vivo conditions. All these plants come from tropical or subtropical regions, some of them with known antimicrobial properties commonly
used in indigenous medicine throughout the world. Nevertheless, no reports on their activity against fungal plant pathogens have been still reported in literature. Once identified the extract effective to a specific host-pathogen interaction, this was studied with innovative
methodologies by use of flow cytometry and gene expression analysis.
All plant extracts tested showed antifungal activity against phytopathogenic fungi
under in vitro conditions. The percentage of mycelial growth inhibition increased with the
concentration of plant extract. Only some extracts were efficient under in vivo conditions too.
S. guianensis extract reduced mycelial growth of Botrytis cinerea and Ascochyta
rabiei at the concentration of 6 ml·l-1, as confirmed also by detached tomato leaf assay. In addition, it inhibited completely spore germination at the concentration of 6 and 3 ml·l-1 for B. cinerea and A. rabiei, respectively. In greenhouse tests, S. guianensis extract was efficient to
control gray mold in cucumber leaves and young tomato plants at the concentrations of 3 ml·l-1 and 6 ml·l-1, respectively. However, the extract had no efficiency in controlling B. cinerea on strawberry and on young cucumber plants and fruits. S. guianensis extract significantly
controlled Ascochyta blight in chickpea at all concentrations tested.
C. leucocephala extract inhibited fungal growth of three Fusarium spp. at the
minimum inhibitory concentration (MIC) of 7 ml·l-1 for F. oxysporum f. sp. basilici, and 14 ml·l-1 both for F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. melonis. Extract concentration of 7 ml·l-1 inhibited of 92 and 99 % spore germination of F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. melonis, respectively. Tomato plants treated with the
extract of C. leucocephala at all concentrations showed significant reduction of disease development respect to the inoculated control. Significant inhibitory effect on Fusarium wilt in melon was observed after treatments by high concentrations of plant extract. In addition, its efficiency increased when applied immediately after inoculation. The plant extract did not
control Fusarium wilt in basil.
B. diffusa extract inhibited mycelial growth of both P. infestans and P. cactorum at the
concentrations of 2.5 ml·l-1, and 10 ml·l-1 for P. capsici. The extract had fungitoxic effect on the mycelial growth of P. infestans and P. cactorum. At the concentrations of 2.5 ml·l-1 and 5
ml·l-1, showed a moderate inhibition effect against P. infestans in tomato plants. B. diffusa extract reduced also disease symptoms of P. capsici in pepper at all concentrations but the concentration of 10 ml·l-1 showed the best efficacy in controlling the oomycete.
V. agnus-castus extract showed strong antifungal activity against Pythium ultimum.
The extract delayed the mycelial growth of the 7 day cultured fungus in comparison with the control; the minimal inhibitory concentration was 2 ml·l-1. In addition, V. agnus-castus extract showed significant antifungal activity against P. ultimum on tomato seedlings.
P. niruri extract, at the concentration of 16 ml·l-1, was fungitoxic on Monosporascus
cannonballus. Minimum inhibitory concentration was 8 ml·l-1. Although P. niruri extract had no significant inhibition effect on M. cannonballus in melon plants under greenhouse conditions, an improvement of general health status of the plants was observed when treated with P. niruri extract at the concentration of 28 ml·l-1.
As the efficiency of the extracts of B. diffusa and V. agnus-castus under greenhouse
conditions was comparable to that of synthetic fungicides, these extracts can be considered as potential valuable alternatives to chemical control of P. capsici and P. ultimum, respectively.
Additionally and after the screening of the different plant extracts on various hostpathogen interactions, the antifungal activity of selected extracts was studied by innovative techniques. The effect of C. leucocephala extract against F. oxysporum f.sp melonis was investigated by flow cytometry, an innovative method for antifungal screening. Cytometric
analysis of over time fungal development dynamics was verified also by microscope
observations. It was possible to observe an immediate effect of plant extract on spore
viability; the extract at the concentration of 14 ml·l-1 inhibited completely spore germination.
The technique showed many advantages in comparison with classical methods of antifungal screening, especially higher velocity and precision. Therefore, flow cytometry can be considered as a valuable method for screening antifungal activity of plant extracts enabling to determine the interaction of the tested fungus with the plant extract in early stages of fungal
growth.
To determine the involvement of plant extract in pathogenesis-related (PR) gene
induction, tomato seedlings were treated with Vitex agnus-castus and/or inoculated with Pythium ultimum. The expression of four PR genes (PR-1, PR-4, PR-5, PR-6) was monitored for 48 hours after treatments and artificial inoculations. The treatments with plant extract resulted in significant activation of genes encoding PR-1 and PR-4 proteins already after three
hours. The amount of PR genes expressed in the plants treated both with plant extract and fungus increased over time, while decreased in the plants treated just with plant extract.
Twenty-four hours after treatments, PR-1, PR-4, and PR-6 genes were activated very strongly in plants treated with both plant extract and fungus, and the expression increased also 48 hours after the treatments. These results indicated that the plant extract contributes to the
activation of plant defence mechanisms. Apart the direct induction of PR genes, the extract acted also as a priming agent conferring to the plant enhanced defence responses upon pathogen inoculation. The activation of various PR genes suggests that the induction of defence responses by V. agnus-castus extract in tomato can be regulated by more signalling pathways.
Additional information
Dottorato di ricerca in Ortoflorofrutticoltura
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