Antifungal activity in vitro of propolis solutions from Argentina against two plant pathogenic fungi: Didymella bryoniae and Rhizotocnia solani

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Journal of Apicultural Research
53(4): 438-440 (2014) © IBRA 2014
DOI 10.3896/IBRA.1.53.4.08
NOTES AND COMMENTS
Antifungal activity
in vitro
of propolis solutions from
Argentina against two plant pathogenic fungi:
Didymella
bryoniae
and
Rhizotocnia solani
Liliana Gallez1*, Mirta Kiehr2, Leticia Fernández1, Rolf Delhey2 and Débora Stikar1.
1Laboratorio de Estudios Apícolas - Departamento de Agronomía - Universidad Nacional del Sur, San Andrés s/n Altos del
Palihue, Bahía Blanca 8000, Buenos Aires, Argentina.
2Departamento de Agronomía - Universidad Nacional del Sur, San Andrés s/n Altos del Palihue, Bahía Blanca 8000, Buenos
Aires, Argentina.
Received 11 September 2013, accepted subject to revision 2 June 2014, accepted for publication 9 June 2014.
*Corresponding author: Email:
labea@uns.edu.ar
Keywords: Argentine propolis, antifungal activity, phytopathogenic fungi,
Didymella bryoniae, Rhizoctonia solani
Didymella bryoniae
(Auersw.) Rehm and
Rhizoctonia solani
Kühn
cause some of the most destructive fungal plant diseases in the
southern Pampas Region of Argentina. The gummy stem blight
caused by
D. bryoniae
affects melon crops, damaging leaves and
fruits (Fiori
et al.,
2000).
R. solani
is a well-known species complex
which causes seedling death in several crops (Nicoletti
et al.,
1999).
Chemical control of fungi has increased the productivity and
quality of various crops, but abuse of chemical products has caused
soil pollution and harmful effects on human beings (Fiori
et al
., 2000).
The replacement of synthetic fungicides by natural products,
particularly those which are of plant origin such as propolis, could
present advantages such as efficacy and low toxicity (Agüero
et al.,
2010).
Propolis is an extremely complex resinous material gathered by
honey bees from various plant sources. The antibacterial and
antifungal activities are the most popular and the most extensively
investigated biological actions of propolis. However, the use of
propolis for control of phytopathogens has been poorly assayed
(Quiroga
et al
., 2006). In this context, we studied the possible use of
propolis as a biological alternative for control of phytopathogenic
fungi. The objective of this work was to evaluate the effect of an
alcoholic propolis solution against
D. bryoniae
and
R. solani
colony
growth
in vitro
by the mycelial growth test.
The propolis (poplar-type) was collected from the southern
Pampas region in Argentina. Sensory, chemical and physical
characteristics of the sample were determined by Luis Maldonado in
the Experimental station of INTA EEA Famaillá (Tucumán, Argentina).
The propolis sample was ground into small pieces and sieved through
a 2 mm mesh before the extraction. The alcoholic propolis solution
was prepared by dissolving 20 g of propolis per 200 ml of 96 %
ethanol, agitated at 40±2°C for 18 h and filtered with Whatman Nº 40
paper. In order to concentrate this propolis solution, it was agitated
for 72 h under the same conditions, and then stored at 4ºC in the
dark until used. The final concentration of propolis soluble compounds
was determined (Bedascarrasbure
et al
., 2006). Phytopathogenic
fungi were obtained from Plant Phytopathology (Universidad Nacional
del Sur, Argentina).
D. bryoniae
was isolated from fruits of
Cucurbita
moschata
(squash) while
R. solani
was from
Allium
(onion) roots.
They were grown at 22ºC on potato dextrose agar (PDA) (potato 200
g, agar 13 g, dextrose 20 g and water to 1l) and stored at 4ºC.
The antifungal activity of the alcoholic propolis solution was
assayed
in vitro
by the mycelial growth test. A propolis treatment
(called P) and two different control treatments (C1 and C2) were
performed. Propolis alcoholic solution (P) and alcohol 96º (C1) were
both dropped in separated plastic Petri dishes (1.5 ml/dish) and left in
a laminar flow chamber until total evaporation. Then, 15 ml of PDA
were carefully poured into the dishes. Another treatment called C2,
which consisted of only PDA, was added. Once the medium solidified,
circles of mycelium of 10 mm diameter were obtained from colonies
with 72 h of incubation and were placed in the centre of the plates.
Plates were incubated in the dark for 30 days at 22ºC. The colony
growth of
Didymella bryoniae
and
Rhizoctonia solani
was measured
daily. The experimental design was completely randomized with four
replicates per treatment. Multiple comparisons were performed with a
one way analysis of variance. Least significant difference (Fischer
LSD) was used for comparison of means.
The propolis was composed of granulated fragments of soft
consistency. The aroma of the sample mainly corresponded to “soft
resinous”. The flavour was sweet and the predominant colour was
yellow / yellowish brown. The propolis composition showed an
average value of 63.38 % of total resins, 24.37 % of total phenolic
compounds, 7.65 % of total flavonoids and 3.27 % of impurities

439 Gallez
et al.
(beeswax and other debris). UV spectrograms showed that the
propolis extract analysed displayed a maximum absorbance range
between 270 and 315 nm, with a main absorption peak at 294 nm
which is indicative of an important biological activity (Sosa López
et
al.,
2003). It is interesting to notice that physicochemical and sensory
characteristics previously described were consistent with data
recorded from other propolis samples from the Pampas Region of
Argentina (Bedascarrasbure
et al.,
2006). The propolis sample
characteristics and the abundance of poplar trees within the foraging
area indicate that the main resin origin was
Populus
sp.
Table 1 shows the fungal growth in mm measured by day. The
final propolis soluble compounds concentration in the alcoholic
solution was 127.83 mg/ml (12.27 %). Both plant pathogenic fungi
were sensitive to this tested concentration. The mean diameters of
D. bryoniae
and
R. solani
colonies in the propolis treatment were
significantly smaller (
p
≤0.01) than in their respective controls until the
end of the experiment. The colonies of
R. solani
in both control
treatments covered the whole surface of the dish at day 10, and those
of
D. bryoniae
at day 17, whereas none of the colonies reached the
edge of the Petri dishes in the propolis treatment at day 30
(Figs 1a,b). Myceliar growth stopped developing in the propolis
treatment on day 27 for
Didymella
and on day 28 for
Rhizoctonia
and
could not reach the Petri dish diameter (85 mm diameter) as was the
Fig. 1b.
Inhibitory effect of propolis solution on
D. brioneae
growth at
day 12. Three treatments were compared: propolis (P) (12% in
ethanol 96% plus PDA); Control 1 (C1) (ethanol 96% plus PDA); and
Control 2 (C2) (PDA). Small portions of mycelium of 10 mm diameter
were streaked onto the agar. Incubation was carried out in the
darkness at 22ºC.
Table 1.
Effect of propolis solution on myceliar growth of
D. bryoniae
and
R. solani
over 30 days. Portions of mycelium of 10 mm diameter
were streaked onto the agar. Incubation was carried out in darkness for 30 days at 22ºC. P: propolis (12.78 % of propolis soluble compounds
in ethanol 96 % plus potato dextrose agar); C1: Control 1 (ethanol 96 % plus potato dextrose agar) and C2: Control 2 (potato dextrose agar).
*Myceliar diameters in mm 1are the mean of 4 determinations ± SD. Means with different letters differ significantly at
p
≤ 0.01 according to
the Fisher LSD.
Fungus Treatment
Days
7 10 17 30
D. bryoniae
P 15.6*±2.41a 32±3.7b 67.9±3.1b 81.4±0.5a
C1 50.9± 0.5b 72.5±0.4c 85±0c 85±0b
C2 51.6±2.1b 71.9±2.0c 85±0c 85±0b
R. solani
P 14.9±2.7a 27±3.2a 56.9±4.1a 80.6±2.4a
C1 66.4±0.8c 85±0d 85±0d 85±0b
C2 67.7±1.3c 85±0d 85±0d 85±0b
Fig. 1a.
Inhibitory effect of propolis solution on
R. solani
growth at
day 9. Three treatments were compared: propolis (P) (12 % in
ethanol 96 % plus PDA); Control 1 (C1) (ethanol 96 % plus PDA); and
Control 2 (C2) (PDA). Small portions of mycelium of 10 mm diameter
were streaked onto the agar. Incubation was carried out in darkness
at 22ºC.

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case for both controls. The mycelial growth test revealed that growth
of both fungi was inhibited within the first week in the propolis
treatment compared to the controls. This is particularly important
because this period is crucial in crop development (Agrios, 1996).
Many other researchers also found promising results with propolis
for control of fungal pathogens using the same
in vitro
technique.
Pineda
et al
. (2010) evaluated antifungal property of
Apis mellifera
propolis on isolates of
Colletotrichum gloeosporioides
that affect fruits
of avocado (
Persea americana
), papaya (
Carica papaya
) and passion
fruit (
Passiflora edulis
) from Venezuela. Quiroga
et al.
(2006)
determined the antifungal and cytotoxic activities of partially purified
propolis extract on plant pathogenic fungi such
Aspergillus niger
,
Fusarium
sp. and
Macrophomina
sp. Chaillou y Nazareno (2009)
evaluated antimycotic activity of different propolis samples from
Argentina against
Fusarium
sp.,
Macropohomina
sp.,
Phomopsis
sp.,
Aspergillus niger
and
Trichoderma
sp.
In conclusion, we assessed the antifungal activity of an alcoholic
propolis solution against two plant pathogenic fungi:
Didymella
bryoniae
and
Rhizotocnia solani
by the mycelial growth test. Propolis
showed a fungistatic effect on the growth of both plant pathogenic
fungi. Further investigations of
in vivo
studies are necessary to make
sure propolis has the potential to effectively control fungal diseases
under field conditions.
Acknowledgements
The authors are very thankful to Luis Maldonado for his valuable help
with the analysis of the propolis sample and to Jorge Espié for his
technical assistance. We would like to thank Soledad Villamil for her
careful review of the manuscript.
References
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Antifungal activity of Argentinian propolis. 440