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Bulletin UASVM Agriculture 69(1)/2012
Print ISSN 1843-5246; Electronic ISSN 1843-5386
Characterization of Botrytis cinerea Pers. Isolates and their Biological Control with
Hydroalcoholic Plant Extracts
Raluca Vasilica MICLEA, Carmen Emilia PUIA
University of Agricultural Sciences and Veterinary Medicine, Cluj Napoca, Faculty of Agriculture,
Manastur Street, No. 3-5, 400372, Cluj-Napoca, Romania,
E-mail: [email protected]
Abstract. Gray mould fungus, Botrytis cinerea is a plant pathogenic fungus with an extensive host
range that causes damage to a wide range of crops including vegetables, soft fruit, ornamental plants
and grapevines. Control of fungal diseases is a key problem in agriculture worldwide. To develop
effective controls for this gray mould, various characteristics of Botrytis cinerea Pers., such as its life
cycle and mode of infection, must be determined. Epidemiological studies are difficult because of the
genetic and phenotypic variability within this species. Under this consideration the purpose of our
study was the observation of the fungus cultural and morphological characteristics but also the testing
of the antifungal effect of some plant hydroalcoholic extract. In our experiments we’ve identified and
isolated the pathogen Botrytis cinerea Pers., inoculated it on three culture media and we observed the
growth rate of the colonies, the sporulation and sclerotia appearance, the shapes and the dimensions of
conidia, the number and distribution of the sclerotia. The different extracts in three different
concentrations were tested in 80 mm diameter Petri plates incubated at 25ºC and assessed after three,
six and nine days of incubation. Plates were examined for the presence or the absence of the colony
growth and the diameters of the colonies were measured establishing the colony area and the
inhibition percent of the tested plant extracts.
Keywords: mould, rot, sclerotia, conidia, tincture, plant extracts
Introduction. Gray mould fungus, Botrytis cinerea Pers. is a plant pathogenic
fungus with an extensive host range that causes damage to a wide range of crops including
vegetables, soft fruit, ornamental plants and grapevines. The pathogen overwinters as
mycelium or sclerotia (Elmer and Michailides, 2007). Control of fungal diseases is a key
problem in agriculture worldwide. To develop effective controls for this gray mould, various
characteristics of Botrytis cinerea Pers., such as its life cycle and mode of infection must be
determined (Eksteen et al., 2001). Plant extracts are a very important source of antifungal
products and these plant extracts have the potential to develop into effective disease
management tools, they have the ability to suppress multiple pathogens (Schilder et al. 2002),
are compatible with other products, including synthetic fungicides (Jeandet et al. 1996,
Jeandet et al. 2000) and biofungicides and pathogen suppression can range from moderate
(Schilder et al. 2002) to high levels, achieved by conventional fungicides (Schmitt et al.
2002).
Aims and objectives. Epidemiological studies are difficult because of the genetic
and phenotypic variability within Botrytis cinerea Pers specie. Under this consideration the
purpose of our study was the observation of cultural and morphological characteristics of the
fungus but also the testing of the antifungal effect of some plant hydroalcoholic extract on
these isolates of the pathogen.
Materials and methods. In our experiments we’ve identified and isolated the
pathogen Botrytis cinerea Pers., we’ve inoculated it on three culture media and we observed
the growth rate of the colonies, the sporulation and sclerotia appearance, the shapes and the
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dimensions of conidia, the number and distribution of the sclerotia. We’ve tested different
plant extracts (Aloe arborescens, Satureja hortensis, Helleborus purpurascens, Aristolochia
clematitis) in three different concentrations (4%, 6%, 8%) which were added to the medium
by poison food technique. The centre of each test plate was inoculated with a 1 mm size plug
and incubated for 9 days on PDA at 25 ± 2°C. Radial growth was determined by calculating
the mean of four perpendicular colony diameters on each replicate and the establishment of
the colony area and the inhibition percent of the tested plant extracts compared with control.
Analysis of variance (ANOVA) was performed on the data, using the Polifact statistical
program to identify differences between treatments.
Results and discussions. Our study shows that there were substantial differences in
the morphology, the growth rate and the density of sclerotia but only minor differences in the
size of the macroconidia.
The conidia were ovate, ellipsoidal or sometimes globose to subglobose, smooth,
usually unicellular. Conidial dimension had the biggest values on the MA medium in the case
of pepper isolate (12.04 – 10.18 µm), on Czapek-agar medium in the case of lettuce isolate
(12.78-11.02 µm) and on MA medium for the tomato isolate (12.73-10.09 µm). The sclerotia
were black, shinny black or white at first, becoming black with the age. They were variable in
shape and size, abundant, rare or absent in others. The number and the distribution of the
resistant bodies varied with the type of the media on with the isolate were grown. Thus, the
highest number of sclerotia, at 30 days after the inoculation, was obtained in the lettuce
isolate’s case, on PDA medium (33 sclerotia) and on MA medium in the tomato isolate (31
sclerotia) and in the case of the pepper isolate (11 sclerotia). The distribution pattern of the
sclerotia formation, on the surface of the three culture media was all over the surface of the
medium and in the edges of the plates.
Regarding the effect of the extracts against Botrytis cinerea Pers. the highest
inhibition percentage was obtained in the lettuce isolate’s case with the Helleborus
purpurascens and Aristolochia clematitis extracts (100%). The Satureja hortensis extract had
also a 100% inhibition on the tomato isolate at all the tested concentrations.
Conclusion. Our study shows that there were substantial differences in the
morphology, the growth rate and the density of sclerotia but only minor differences in the size
of the macroconidia.
The fungistatic effects of the extracts indicate the importance of some plant species
as a possible natural source of fungicidal material. Antifungal activity was confirmed in all
the plants tested, although the results showed that different plant extracts varied in their
effectiveness in inhibiting the mycelia growth of the tested pathogen.
The development of the Botrytis cinerea Pers. isolates was affected by all the
extracts. In all cases the percentage inhibition of the mycelia growth of Botrytis cinerea Pers.
was higher in comparison to the untreated control, approximately above 85%.
Among the four plant species tested, the Helleborus purpurascens and Aristolochia
clematitis extracts were the most effective against the three isolates compared to control.
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