Assays with commercial fungicides against sclerotia formation in flowers infected by Ciborinia camelliae

Salinero C¹., 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, España
¹ Author for correspondence: carmen.salinero@depo.es

Introduction

Ciborinia camelliae Kohn is a highly specialized fungal pathogen 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 have been reported as being resistant to the disease (Vingnana et al. 2001, Taylor 2004). The fungus affects the flower petals which turn brown, and causes premature flower drop, although sometimes the whole flower turns brown but still remains on the plant. Once flower petals are infected, fungus develops inside the flower and subsequently a grey mycelium appears between the calyx and the corolla. Sclerotia (Figure 1) are a hardened mass of mycelium that form at the petal base and which are resistant to adverse environmental conditions. Once the flower has dropped, the sclerotia overwinter in the soil until the next camellia flowering period when they becomes active. Sclerotia can remain dormant in the soil for several years before becoming active for the first time, or they can become active repeatedly, producing apothecia during successive years. The sclerotia produce beige to brown mushroom-like structures named apothecia. When apothecia are mature they produce ascospores that are disseminated by the wind and, when they reach the camellia petal surface, they infect the flower that blights, and the life cycle of the fungus starts again (Figure 1).

Figure 1. Ciborinia camelliae life cycle (Northern hemisphere).

Several attempts have been made to control of the disease. All of them have 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 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. 

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 et al, 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, 2005b) and only the application of calcium cyanamide has proved to be effective in 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 the frequent application of azole-type 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 flowersinfectedby Ciborinia camelliae.   

Material and methods

In a field trial, five fungicide treatments were applied to infected camellia flowers (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).

Four to five replicates (repetitions) 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 camellia plants of the Diputacion of Pontevedra living collection located in the Estacion Fitopatoloxica do Areeiro. The flowers selected were affected by C. camelliae but had not yet formed sclerotia (Figure 3B, C, D). The 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 in the field trial for 8 weeks. Then the flowers were collected and the number and weight of sclerotia that were formed in each one was recorded.

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.

Table 1. Fungicides used against sclerotia formation in flowersinfectedby Ciborinia camelliae.

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.

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 15238 sclerotia were recorded) and none was able to reduce the size of sclerotia formed in the flowers (Table 2, 3).

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 cultivar on the formation of sclerotia

Both the number of sclerotia formed inside a flower and their size, clearly depends on the cultivar involved (Table 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 any relationship 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).

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 that fungicides are not able to come into contact with the mycelium of the fungus. Future work should be addressed to ensure that the fungicide can penetrate and / or be distributed inside the petal to stop the infection and interrupt 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 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.

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.

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.

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