Assays with commercial fungicides against sclerotia formation in flowers
infected by Ciborinia camelliae
Salinero M.C.*, Couselo J.L., Vela P., Neves A., González M., Mansilla P.
Estación Fitopatolóxica do Areeiro, Deputación de Pontevedra, Subida a la Robleda s/n,
36153 Pontevedra, Spain. E-mail: [email protected]
Abstract. Camellia flower blight is caused by sclerotia-forming fungus Ciborinia camelliae (Kohn).
Current control strategies are focused on the interruption of the fungal cycle to reduce the incidence of
the disease. This work summarises the effects of five commercial fungicides against sclerotia formation
in camellia flowers infected by C. camelliae in a field trial. Neither of these fungicides was capable of
preventing the formation of a large number of sclerotia or reducing their size. In contrast to previous in
vitro assays with these fungicides, our results indicate that petal tissues provide protection to the
mycelium of the fungus that develops inside of the petals. This allows the formation of sclerotia without
interrupting the cycle of the fungus at this point. Moreover, differences in size and number of sclerotia
formed in flowers of different cultivars have been registered for the first time. There is a clear
correlation between the number of petals of the flower and the number of sclerotia produced. Since the
number of flower petals is a feature inherent of each cultivar, it is clear that not all cultivars contribute
equally to the spread and incidence of the disease.
Keywords: petal blight, fungus, control.
Introduction
Ciborinia camelliae Kohn is a fungal pathogen highly specialized that attacks the
flowers of many species of the genus Camellia. This fungus causes the disease known
as Camellia Flower Blight which is the main plant sanitary problem affecting Camellia
cultivars of ornamental value. Most cultivars are susceptible to the disease, although in
the last decade several cultivars resistant to the disease have been described
(Vingnanasingam et al. 2001; Taylor, 2004). The fungus affects flower petals, which
turn brown and causes premature flower drop, although sometimes the whole flower
turns brown but still remains in the plant. Once flower petals are infected, fungus
develops inside the flower and subsequently a grey mycelium appears between the
calyx and the corolla. Then sclerotia (Figure 1), a hardened mass of mycelium that
forms at the petal base, which is resistant to adverse environmental conditions, once
the flower has dropped, overwinters in the soil until the next camellia flowering period
when they become active. Sclerotia can remain dormant in the soil for several years
before activating for the first time, or they can activate repeatedly, producing
apothecia during successive years. The sclerotia produce beige to brown mushroom
alike structures named apothecia. When apothecia are mature they produce
ascospores that are disseminated by the wind and when reaching camellia petal
surface, they infect the flower that blights, and the life cycle of the fungus starts again.
Several attempts have been made to control of the disease. All of them have
been focused on the interruption of C. camelliae life cycle to reduce the incidence of
the disease. These include direct methods such as preventive cultural measures, the
2014 International Camellia Society Congress
Pontevedra (Spain) 11- 15 March
more effective being the immediate removal of fallen flowers to reduce sclerotia
formation. Other preventive measures are the elimination of weeds around the plants
and pruning of low branches to allow plant base ventilation and to create unfavourable
conditions for sclerotia development.
Figure 1. Ciborinia camelliae life cycle (Northern hemisphere)
However, these preventive measures have not been enough to control the
disease, thus in the last years research studies have been developed to find an
effective method to control C. camelliae. The effects of different fungicides and
biological control agents (BCAs) on sclerotia (van Toor, 2002; McLean et al, 2004;
Montenegro et al. 2010) have been assessed. Although some of them have been
effective in reducing the viability of the fungus in vitro (inhibiting mycelia growth or
sclerotia viability), none of them has been able to significantly reduce the viability of
natural sclerotia in field trials (van Toor, 2002; van Toor et al. 2005b) and only the
application of calcium cyanamide has proved to be effective preventing apothecial
development (van Toor et al. 2004). Different fungicides and BCAs have also been
tested on flowers (van Toor et al, 2002, 2005a) but only frequent application of azoletype fungicides has been shown to protect the flowers against ascospore infection
(van Toor et al, 2003). Despite these progresses, so far there is no known fungicide or
BCA that prevents the formation of new sclerotia on infected flowers so that the
disease cycle continues. This paper summarises the results of experiments that
evaluated the effects of commercial fungicides against sclerotia formation in flowers
infected by C. cameliae.
2014 International Camellia Society Congress
Pontevedra (Spain) 11- 15 March
Material and methods
In a field trial, five fungicide treatments were applied to infected flowers of
camellia (Table 1). Two treatments were performed with biological fungicides and
three were carried out using chemical fungicides. At the same time an untreated
control was conducted. Flowers from ten different cultivars were used for each
treatment (Figure 2).
Table 1. Fungicides used against sclerotia formation in flowers infected by Ciborinia cameliae.
Active ingredient / Dose
Coniothyrium minitans
(strain CON/M/91-08)
4kg/Ha
Tebuconazole
1g/L
Boscalid
1g/L
Brand
Mode of action
Contans®
Coniothyrium minitans is a parasite that attacks
the resting stage (sclerotia). This breaks the "cycle
of disease" by reducing or eliminating the
disease-causing fungus from treated soil.
Folicur®
Systemic fungicide of the azole group. Like many
other azoles, affects the fungal organism's sterol
biosynthesis inhibiting mycelia growth.
Cantus®
Contact and systemic fungicide through the
inhibition of complex II in the respiratory chain, it
inhibits spore germination, germ tube elongation,
mycelia growth, and sporulation.
Azoxystrobin
1mL/L
Ortiva®
Trichoderma atroviride
(strain MUCL 45632)
4Kg/Ha
Condor®
Contact and systemic fungicide that is relatively
non-toxic to humans and the environment.
Azoxystrobin act at the Quinol outer binding site
of the Complex III of the mitochondrial electron
transport chain. They inhibit electron transfer in
mitochondria, disrupting metabolism and
preventing spore germination and the early
stages of fungal development.
The fungus Trichoderma atroviride is a fungus
with direct antagonist action (predation,
metabolites production and competition) against
many pathogenic fungi (Fusarium spp.,
Rhizoctonia spp., Verticillium spp., Armillaria spp.,
Phyrochaeta spp., Phytophtora spp., Botrytis spp.
etc.).
Four to five replicates for each treatment were conducted and ten flowers per
replicate were used. A total of 2,520 flowers were used throughout the trial. The
flowers were placed on peat in wooden boxes. The trial was conducted in a shaded
area away from camellia bushes and boxes covered with a reticulated mesh to protect
the trial. Overall the trial covered an area of 46 m2 (Figure 3A). The flowers were
collected from the camellia plants of the Diputacion of Pontevedra living collection
located in the Estacion Fitopatoloxica do Areeiro. The flowers affected by C. camelliae
but where had not yet been formed sclerotia were selected (Figure 3B, C, D). The
2014 International Camellia Society Congress
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application of fungicides was performed according to manufacturer's instructions, by
spraying and using the maximum dose recommended for ornamentals (Table 1). The
flowers remained on the field for 8 weeks. Then the flowers were collected and the
number and weight of sclerotia that were formed in each one were recorded.
Figure 2. Flowers of the ten cultivars used in the assays with fungicides against C. camelliae. CV, number
of the cultivar in field trial. In brackets, average number of petals of each cultivar.
2014 International Camellia Society Congress
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Statistical analysis of the effect of fungicides on the number and weight of
sclerotia was performed by two-way ANOVA (significance p <0.05) using the SPSS 10.0
statistical software.
Figure 3. A, spraying of fungicides over camellia flowers placed on peat in wooden boxes. The boxes
were divided into two parts. Each part was an independent replicate (10 flowers). Each treatment was
applied to 420 flowers arranged in groups of 21 boxes. B, C, D, Flowers used in the trial. Observe for
symptoms of infection, including mycelial ring. Note that flowers where sclerotia had been formed were
rejected for the trial.
Results and discussion
Effect of fungicides on the number and size of sclerotia
The aim of the treatments was to stop the growth of C. camelliae mycelium
within the tissues of the petals of the camellia flower so that sclerotia were not
formed. However, a large number of sclerotia were formed in all treatments (in the
whole trial 15,238 sclerotia were recorded) and none was able to reducethe size of
sclerotia formed in the flowers (Tables 2, 3).
2014 International Camellia Society Congress
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Table 2. Number of sclerotia formed in each flower (mean ± SE) according to the cultivar to which it
belongs (CV) and the fungicide applied. Cultivars correspondence: CV1 Orandakô; CV2 Lavinia Maggi;
CV3 Dona Herzília de Freitas Magalhães; CV4 Joshua E. Youtz; CV5 Vilar d'Allen; CV6 Rubescens Major;
CV7 Tomorrow; CV8 Triumphans; CV9 Mary Phoebe Taylor; CV10 Mikuni-no-homare. The averages
among cultivars followed by the same letter (columns) are not significantly different at p <0.05.
CV1,a
CV2,b
CV3,c
CV4,c
CV5,d
CV6,e
CV7,f
CV8,g
CV9,c
CV10,c
Contans®
(Coniothyrium
minitans)
11.09±
0.78
17.38
±1.09
2.52
±0.29
2.30
±0.34
4.33
±0.41
10.55
±0.88
7.10
±0.52
4.77
±0.45
2.33
±0.27
2.90
±0.30
Folicur®
(Tebuconazole)
11.57±
0.81
16.98
±1.48
2.13
± 0.29
2.20
± 0.28
4.03
±0.41
11.13
±1.12
6.31
±0.59
5.06
±0.59
2.89
±0.32
3.23
±0.23
Cantus®
(Boscalid)
12.17±
0.72
18,30
±1.10
2.63
±0.32
3.06
±0.41
4.18
±0.39
10.13
±0.95
6.57
±0.62
5.27
±0.47
2.80
±0.20
3.03
±0.29
Ortiva®
(Azoystrobin)
11.18±
0.67
17.73
±0.95
1.96
±0.41
2.65
±0.33
4.15
±0.46
10.63
±0.91
6.83
±0.56
4.93
±0.46
2.23
±0.29
2.83
±0.38
Condor®
(Trichoderma
atroviride)
10.93±
0.73
18.75
±1.18
2.00
±0.34
1.97
±0.31
4.75
±0.37
10.97
±0.89
6.5
±0.60
5.50
±0.40
2.4
±0.34
2.96
±0.42
Control
11.05±
0.69
18.09
±1.05
2.83
±0.36
2.16
±0.38
4.03
±0.50
10.33
±1.03
7. 16
±0.65
5.16
±0.48
2.13
±0.38
3.14
±0.35
In previous in vitro assays, these fungicides were capable of preventing the
growth of the mycelium of C. camelliae. However, in view of our results it is clear that
the petal tissues protect the mycelium of the fungus that grows inside from the
adverse effect of these fungicides. Even direct exposure of mycelial ring to fungicides
(Figure 3B) cannot prevent the formation and development of sclerotia.
In addition, as the size of the sclerotia is an indicator of the viability of sclerotia
(van Toor, 2002), viability is not adversely affected by the treatments because the size
of the sclerotia formed in the treated flowers did not differ from that reached in
untreated control flowers (Table 3).
Effect of the cultivar on the formation of sclerotia
Both the number of sclerotia formed inside a flower as well as its size clearly
depends on the cultivar involved (Tables 2 & 3).Thus, while in the cultivar Lavinia
Maggi (CV2) an average of 18 sclerotia per flower were formed, in the cultivar Dona de
Freitas Herzíla Magalhães (CV3) only 2 were formed (Table 2).
We did not find relation between the number and size of sclerotia formed in a
flower. For example, while in the cultivar Mary Phoebe Taylor (CV9) 3 sclerotia per
flower were formed with an average of 0.044 g, in the cultivar Rubescens Major (CV6)
11 sclerotia were formed with a very similar average weight (Table 3). The cultivars
with the largest number of sclerotia are those with a greater number of petals, namely
Lavinia Maggi (50), Orandakô (50), and Rubescens Major (45) (Figure 2).
2014 International Camellia Society Congress
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Table 3. Average weight (g) of sclerotia formed in the flowers (mean ± SE) according to the cultivar to
which they belong (CV) and the fungicide applied. Cultivars correspondence: CV1 Orandakô; CV2 Lavinia
Maggi; CV3 Dona Herzília de Freitas Magalhães; CV4 Joshua E. Youtz; CV5 Vilar d'Allen; CV6 Rubescens
Major; CV7 Tomorrow; CV8 Triumphans; CV9 Mary Phoebe Taylor; CV10 Mikuni-no-homare. The
averages among cultivars followed by the same letter (columns) are not significantly different at p <0.05.
Contans®
(Coniothyrium
minitans)
Folicur®
(Tebuconazole)
Cantus®
(Boscalid)
Ortiva®
(Azoystrobin)
Condor®
(Trichoderma
atroviride)
Control
CV1,a
CV2,b
CV3,c
CV4,d
CV5,e
CV6,c
CV7,c
CV8,e
CV9,e
CV10,c
0.0135
±0.001
0.0179
±0.001
0.0490
±0.010
0.0410
±0.021
0.0482
±0.008
0.0480
±0.005
0.0641
±0.012
0.0338
±0.003
0.0497
±0.007
0.0628
±0.019
0.0116
±0.001
0.0166
±0.001
0.0548
±0.012
0.0401
±0.010
0.0438
±0.010
0.0469
±0.004
0.0580
±0.023
0.0476
±0.006
0.0468
±0.007
0.0613
±0.010
0.0136
±0.001
0.0169
±0.002
0.0519
±0.021
0.0383
±0.010
0.0501
±0.013
0.0511
±0.006
0.0625
±0.008
0.0341
±0.005
0.0466
±0.015
0.0613
±0.013
0.0132
±0.001
0.0172
±0.004
0.0552
±0.016
0.0450
±0.018
0.0431
±0.004
0.0479
±0.011
0.0657
±0.005
0.0414
±0.007
0.0447
±0.012
0.0598
±0.017
0.0142
±0.002
0.0174
±0.001
0.0508
±0.009
0.0429
±0.010
0.0466
±0.007
0.0494
±0.003
0.0589
±0.006
0.0448
±0.004
0.0415
±0.012
0.0557
±0.015
0.0123
±0.004
0.0181
±0.005
0.0522
±0.007
0.0391
±0.012
0.0485
±0.009
0.0502
±0.006
0.0584
±0.014
0.0418
±0.002
0.0436
±0.008
0.0575
±0.010
Conclusion
The results obtained in this work highlight the limited value of the in vitro assays
against the mycelium of C. camelliae. In nature, this mycelium develops just inside the
petals and these effectively protect the mycelium against the negative effects of the
fungicides. The protective effect is probably due to the fact fungicides are not able to
contact with the mycelium of the fungus. Future work should be addressed to ensure
that the fungicide can penetrate/or be distributed inside the petal to stop the infection
and stop the life cycle of C. camelliae to prevent the formation of new sclerotia.
The number and size of sclerotia formed in a flower depends largely on
morphological features, such as the as number of petals, which in turn are associated
with each cultivar. It follows that, at least partially, the ability of the disease to spread
and its impact on the next flowering season depends on the cultivars present in each
region.
Acknowledgements
We thank Alicia del Carmen Marcelino for her skillful technical assistance.
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2014 International Camellia Society Congress
Pontevedra (Spain) 11- 15 March
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2014 International Camellia Society Congress
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