Introduction

Gray mold, caused by Botrytis cinerea, is a serious postharvest fungal disease of rose flowers. Previous studies showed that many rose cultivars are susceptible to infection with B. cinerea and the storage period of cut rose flowers is often strongly dependent on their susceptibility to this necrotrophic pathogen (Friedman et al. 2010;Hazendonk et al. 1995). Once rose petals are infected with B. cinerea, the symptom develops on the infected petals as very small lesions. Under favourable conditions, these lesions become necrotic and spread to the whole petals and the receptacle and pedicel of flowers, resulting in failure of the flower opening and petal abscission (Elad 1988;Williamson et al. 1995;Williamson et al. 2007). Thus, infection by B. cinerea significantly reduces the postharvest quality and ornamental values of cut rose flowers. The petals of cut roses sometimes do not show symptoms during harvest, but high incidence of gray mold is often observed during storage and transport to flower markets (Williamson et al. 1995).

Many chemical fungicides have been applied to reduce B. cinerea infection on cut rose flowers (Elad and Shtienberg 1995). However, recently, chemical fungicides treatments for controlling gray mold disease in cut flowers are becoming less acceptant due to other undesired residues, the environmental pollution, and the development of fungal resistance (William et al. 2007). Many alternative methods including gamma irradiation, the treatment with carbon dioxide and calcium sulfate, and photochemical treatments have been applied to control B. cinerea infection on cut rose flowers after harvest (Baka et al. 1999;Capdeville et al. 2005;Chu et al. 2015). However, these alternative methods did not efficiently reduce the damages of this necrotrophic pathogen in cut flowers. Thus, it is necessary to develop an effective solution to control gray mold disease as well as improve postharvest quality of cut rose flowers during storage and transportation.

Nano silver (NS) and sodium hypochlorite (NaOCl) are non-residual chemicals and widely used in water purification, medical industry, and vegetable disinfection (Fukuzaki 2006;Kim et al. 2012). NS and NaOCl effectively reduced fungal diseases including Fusarium sp. and B. cinerea on vegetables and fruits (Kim et al. 2012;van Doorn et al. 1990). Recently, NS and NaOCl have been also used as vase solutions for extending the vase life of cut flowers (Ha et al. 2019;Naing et al. 2017). Salicylic a cid (SA) i s one k ind of r esistance inducer, and can induce resistance of plants against fungal pathogens (Hua et al. 2018). SA was used to enhance the resistance of fruits against fungal disease (Chan et al. 2008). Compared to fungicides, NS, NaOCl, and SA are used with relative safe evaluation of various plant pathogens (Kim et al. 2012;Koo et al. 2020;Macnish et al. 2010). However, there has not been a lot of research into the effects of treatment of NS, NaOCl, and SA on B. cinerea infection on cut rose flowers.

In the present study, we investigated the effects of NS, NaOCl, and SA treatments on B. cinerea infection on cut flowers to develop an effective preservative solution for reducing the infection of B. cinerea on rose flowers and improving postharvest quality of cut roses during export processes.

Materials and Methods

Plant materials and treatments

Cut roses (Rosa hybrida L. 'Revival') were harvested from a commercial greenhouse in Daegu, Korea in January 2020. After harvest, cut roses were placed in the buckets containing tap water and transferred to the laboratory in Andong National University for 2 h. For NS, NaOCl, and SA treatments, cut flowers were placed and sprayed with 20 mg·L^-1 NS, 200 μL·L^-1 NaOCl, and 2 mM SA solutions. Non-treated (NT) and control (CON) flowers were held in distilled water without treatment solutions. After treatment with the preservative solutions, cut flowers, with the exception of NT flowers, were sprayed with Botrytis cinerea solution (3.4x10⁵ conidia mL^-1). Subsequently, cut flowers were held at 10 ± 2°C and 80 - 90% RH in the dark condition for 4 d for export simulation. B. cinerea was obtained from Agricultural Genetic Resource Information Center, Korea. For a pure culture, isolated conidia were transferred to potato dextrose agar media. Prior to experiment, all cultures were grown on the potato dextrose agar media at 25°C for 5 d. For harvest the B. cinerea conidia, 22 mL of sterile distilled water was added to the culture dishes and the conidia were gently collected by filtration through four layers of gauze. The concentration of B. cinerea conidia was determinated using a hemacytometer and the final concentration was adjusted to 3.4x10⁵ conidia mL^-1 for the experiment. After the transport simulations, rose flowers were trimmed to 50 cm of length with three upper leaves. Each cut rose was placed in a glass vase containing 400 mL distilled water. For vase life evaluation, the flowers were maintained in a controlled environment room at 25 ± 1°C, 50 - 60% RH, and a photoperiod of 12 h of fluorescent tubes at 20 μmol·m^-2·s^-1.

Assessment of B. cinerea infection and flower quality

Visual symptoms of B. cinerea on petals, number of rose stems with leaf and flower damage, changes in flower diameter, fresh weight and water uptake of cut flowers were determined daily. The extent of development of visual symptoms of B. cinerea was assessed on a relative scale of 0-3 according to the extent of the infected rose petal area, as follows: 0, n o infection; 1 , 1 - 2 5%; 2 , 26 - 5 0%; a nd 3, > 50%. The vase life of cut roses was considered terminated when cut flowers showed scale 2 or 3 of B. cinerea growth in petals. Other senescence symptoms, such as bluing, bent neck, wilting of leaf and flower were also recorded when flowers reached the senescent stage. For evaluation of B. cinerea growth in rose petals, three petals were detached from each cut flower and placed into a falcon tuble containing 20 mL sterile distilled water, subsequently vortex vigorously for 1 min. After vortexing, 1 mL of each sample was spread onto an aerobic count plate (Petrifilm 6440; 3M Heath Care, St. Paul, MN, USA). After 4 d of incubation at 25 °C, the B. cinerea growth was evaluated.

Experimental design and data analysis

Vase life experiment was performed with nine replicates for all treatments and one flower per replication. All data were shown as means ± standard errors. One-way analyses of variance were conducted using SPSS 20.0 (IBM, Somers, NY, USA). When significant effects were detected, post hoc pairwise comparisons of group means were executed with Duncan's multiple range test, with a significant difference at p = 0.05.

Results

The visual symptom of Botrytis cinerea infection in cut roses was observed on day 5 of vase life in CON and SA flowers and on day 6 in NT and NaOCl flowers (data not shown). CON, NaOCl, and SA treatments showed the highest incidence rate of B. cinerea in rose plants on day 7 of vase life. Whereas, treatment with NS significantly reduced the incidence rate of gray mold on rose plants during vase periods (Fig. 1A).

After cut rose flowers were infected with B. cinerea, the lesions in rose petals were also observed and recorded daily to evaluate the effectiveness of preservative solutions on inhibition of B. cinerea infection. The incidence rate of B. cinerea on rose petals was significantly higher in CON, NT, NaOCl, and SA flowers on day 7 of the vase life (Fig. 2). Whereas, the rate of B. cinerea infection on petals of NS flowers was reduced during vase life (Fig. 2). This indicated that NS treatment can inhibit the development of B. cinerea completely on cut rose petals, whereas NaOCl and SA treatments have no inhibitive effects on the lesion development.

Changes in vase life, flower diameter, fresh weight, and water balance of cut flowers were also determined daily to assess the effect of preservative solution on postharvest quality of cut rose flowers. In the present study, the rapid termination of vase life of cut roses was due to B. cinerea infection in NT, CON, NaOCl, and SA treatments ( Figs. 1 B and 2). The shortest vase life was recorded in CON, NaOCl, and SA f lowers (Fig. 1 B). Compared t o NT f lowers, the vase life of NS flowers was significantly extended by 2.3 d (Fig. 1 B). Interestingly, t here was n o significant difference in flower opening of cut rose flowers after treatments (Fig. 3A). Whereas, the initial fresh weight and positive water balance of cut flowers were maintained effectively during vase periods after treatment with NS (Figs. 3B and C). These results showed that treatment with NS not only reduced the B. cinerea infection on cut roses but also effectively improved the postharvest quality of cut flowers during vase life.

Discussion

Gray mold affects different tissues in rose plants, including flowers, stamens and ovary, sepals, stems, and leaves. The disease infection in the petals often causes a heavy economic damage in cut rose flowers (Muñoz et al. 2019). Therefore, management of gray mold disease on rose plants is economically important. There are numerous researches of the antibacterial activity of silver ions, but antifungal activity of silver ions has not been examined fully. Results from this study showed that NS was the most effective solution that reduced the B. cinerea infection in cut rose flowers during vase life. It has been known that silver ions attack a wide range of biological processes in microorganism including cell membrane structure and functions (Dakal et al. 2016). Silver ions also inhibit the expression of protein associated with ATP production in microbial cells. In addition, silver ions are well known to effectively delay fungal spore development by releasing reactive oxygen species via the reaction with oxygen and triggering damage to nucleic acids, lipids, and proteins of cells (Dakal et al. 2016). Therefore, in the present study, NS treatment effectively inhibited the development of B. cinerea in rose petals and prolonged the vase life of cut flowers, compared with other treatments. Previous study indicated that cut flowers susceptible to fungal infection accelerate ethylene production by flowers. Also, ethylene can make the host plant tissues more susceptible to B. cinerea (Elad 2008). Recently, NS has been used as a vase solution and effectively decreases ethylene production in the cut flowers and microbial growth at the cut stem ends (Liu et al. 2009;Naing et al. 2017). Thus, in this study, NS treatment could suppress ethylene synthesis, reduce B. cinerea susceptibility in petals, and delay senescence of cut rose flowers. In addition, placing cut roses into NS solution effectively decreased bacterial growth at the cut rose stem ends, enhanced water uptake and maintained fresh weight and positive water balance of cut flowers.

Previous studies showed the effectiveness of NaOCl and SA on suppressing fungal disease and increasing the disease resistance in many plants (Hua et al. 2018;Macnish et al. 2010). However, NaOCl and SA treatments did not reduce the incidences of B. cinerea infection in cut rose flowers in this study. The vase life of the cut rose flowers treated with these solutions was considerably decreased, compared with NT flowers. Probably the concentration of NaOCl used in this experiment was low and it is important to test different higher concentrations to assess its ability to reduce gray mold in cut rose flowers. Although SA has resistance effects on different microbial pathogen, the role of SA in plant defense against necrotrophic pathogens is not fully understood yet (Koo et al. 2020). In addition, the effectiveness of exogenous SA treatment to enhance disease resistance is dependent on different plants (Koo et al. 2020). In some cases, treatment with exogenous SA ehanced B.cinerea susceptibility in tomatoes and broad beans by increasing the lesion size and disease serverity (El Oirdi et al. 2011;Khanam et al. 2005). Therefore, SA treatment could induce the necrotrophic pathogen infection in cut rose flowers in this study.

Overall, treatment with NS solution significantly reduced the damage by B. cinerea, decreased the incidence of senescence of cut flowers, improved postharvest quality, and extended the vase life of cut rose flowers. Our results demonstrate that NS is an effective preservative solution to control gray mold disease and improve postharvest quality and vase life of cut ‘Revival’ roses that are exported overseas.