Introduction

Gray mold caused by Botrytis cinerea (B. cinerea) is a destructive fungal disease affecting more than 500 plant species, including roses, chrysanthemums, and gerberas (Elad 1988b;Williamson et al. 2007). This necrotrophic pathogen is able to be present in various plant organs such as stem, leaf, flower, fruit, and seed and causes a huge economical loss in various important crops (Dean et al. 2012). This pathogen can trigger conspicuous disease symptoms in the preharvest stage or remain latent until postharvest stage (Fillinger 2016). Gray mold rot has been considered as one of the major postharvest diseases in flowers, vegetables, and fresh fruits.

Among various pathogens such as bacteria, fungi, and viruses, gray mold disease is the most economically impactful pathogens in roses. The gray mold can develop very rapidly in all parts of rose plants and cause damage and death of cells and tissues (Elad 1988b). The control of B. cinerea in plants is difficult due to its broad host range, various attack modes, and both sexual and asexual periods to survive in favourable and unfavorable conditions (Fillinger and Elad 2016). To date, the main strategies to control B. cinerea in rose flowers remain as the application of synthetic fungicides (Elad and Shtienberg 1995). However, the effectiveness of the synthetic fungicides on B. cinerea is not satisfactory because its genome is prone to form drug resistance genes. Furthermore, synthetic fungicides are not safe for the environment and humans (Droby et al. 2009). Thus, it is important to deeply understand the pathogenic mechanisms of B. cinerea and develop new effective strategies to control gray mold disease in roses.

In this review, we mainly discussed the latest information about pathogenesis of B. cinerea to understand the pathogenic mechanism, as well as the current strategies against gray mold disease in rose flowers.

Botrytis cinerea Pathosystem in Rose Flowers

B. cinerea mainly enters the host via natural openings or wounds in plants (Holz et al. 2007). Infection of unripe or non-senescent plant organs often lead to limited damage and latent infections. Three various types of B. cinerea latent have been reported in plants such as delaying of conidia germination or growth arrest after germination, endophytic symptomless growth in the apoplast, and colonization of abscising flower organs such as petals followed by growth into receptacles or ovaries where growth captures (Bristow et al. 1986;Jarvis 1994;Sowley et al. 2010). Mechanisms of the B. cinerea infection have been explored in model organisms (Staats and van Kan 2012;Van Kan et al. 2017). Briefly, in the initial phase, B. cinerea releases sRNAs and virulence factors to inhibit premature host cell death and immune responses, that allows the fungus to growth inside the host and then increase biomass before the necrotrophic stages (Veloso and van Kan 2018). Secreted virulence factors are deployed from B. cinerea which can lead to host cell death including enzymes and toxins involved in reactive oxygen species (ROS) production, and effector proteins (Schumacher 2016). In addition, B. cinerea also releases oxalic acid which can decrease pH value of the host tissues and enhance the activity and production of fungal enzymes such as proteases, laccases, and pectinases. The accumulation of oxalic acid can lead to calcium chelation, which deteriorates the pectin structure of plant cell walls and suppresses the accumulation of callose (Chakraborty et al. 2013;Manteau et al. 2003;Sharon et al. 2007). B. cinerea also can lysis plant cells and loosen walls to simplify tissue penetration by secreting cell wall degrading enzymes (Blanco-Ulate et al. 2016). B. cinerea is also known to produce hormones or its analogues to interrupt immune responses and the metabolism of the host cells. However, the relevance of this mechanism for the capacity of B. cinerea to infect rose flowers is still poorly understood.

Roses are vulnerable to infection by B. cinerea and their storage capacity is highly dependent on their susceptibility to this pathogen. Susceptibility to B. cinerea infection has been studied for various fresh cut rose cultivars (Friedman et al. 2010). The infection process in rose flowers begins with deposition of conidia on petals during flower development (Kerssies et al. 1995). Disease symptoms are usually first visualized on petals after harvest as small quiescent lesions or pocks (Pie and De Leeuw 1991). When exposed to favorable conditions, such as high humidity and low temperature during storage and transport, lesions become necrotic and can spread to infect entire petals as shown in Fig. 1 (Williamson et al. 1995). Infection by B. cinerea reduces the postharvest quality of cut roses leading to substantial economic loss by growers, wholesalers, and retailers. The necrotrophic pathogen can influence different development stages and various rose plant organs, including flowers, stems, and leaves (Fig. 2). However, the most severe economic damage in roses occurs when the pathogen infects the flower petals (Elad 1988b).

Control Strategies of B. cinerea in Rose Flowers

Due to the huge economic losses in rose flowers brought by B. cinerea, many considerable control strategies have been conducted to reduce gray mold disease, including chemical control, radiation control, biological control, resistance inducer, and using ethylene antagonists.

Chemical Control

Pre- or postharvest treatment with synthetic fungicides for controlling gray mold disease is the main method in cut flowers (Elad and Shtienberg 1995). The benzimidazoles and the dicarboximides are two groups of synthetic fungicides, which were effective against gray mold disease and have been used for many years (Elad and Shtienberg 1995). Mechanisms of the synthetic fungicides to B. cinerea are acting on the respiration, sterol biosynthesis, osmoregulation, microtubule assembly, and those whose influences can be inverted by methionine (Leroux 2007). Previous study indicated that the combination of biocontrol agent Trichoderma harzianum with the fungicides fenpiclonil, polyoxine B, and tebuconazole reduced 50 - 70% B. cinerea infection in rose flowers by spraying once a week in the greenhouses (Elad et al. 1993). However, there are two important problems existing in the application of synthetic fungicides in rose flowers as well as in other crops. On one hand, B. cinerea is often resistant to different synthetic fungicides due to a multinucleate conidia. This causes a constant change during generations of B. cinerea and leads to form different resistance strains (Bollen and Scholten 1971;Katan 1982). On the other hand, application of synthetic fungicides are harmful to the human beings and environment due to their toxicological residues. Additionally, synthetic fungicides are expensive and the control of gray mold disease in plants usually needs a higher concentration of treatment than other fungal diseases. Therefore, some alternative chemicals for controlling gray mold disease in roses have been applied including treatment with CaSO₄, NaOCl, ClO₂, SO₂, CO₂, or dipping cut roses with hot water (Baka et al. 1999;Capdeville et al. 2005;Macnish et al. 2010;Kwon et al. 2013;Lee et al. 2016;Lee et al. 2016;Lee and Kim 2019). However, none of these applications consistently provided complete control of this necrotrophic pathogen in rose flowers. For examples, postharvet treatment with 200 μL·L^-1 NaOCl by dipping for 10 s at 20°C decreased B. cinerea infection in ‘Gold Strike’ and ‘Akito’ rose flowers (Macnish et al. 2010). However, another previous studies indicated that application of the high concentration of 1% NaOCl or 200 μL·L^-1 NaOCl did not reduce gray mold disease in rose petals (Fig. 3) (Elad 1988; Ha et al. 2020).

Radiation Control

Radiation is an effective non-chemical application for control of postharvest fungal diseases in many crops (Hallman and Loaharanu 2016). Previous study showed that ionizing irradiation treatment used for sterilizing or killing pests without damaging cut flowers. Furthermore, application of irradiation can improve longevity and postharvest quality of cut flowers (Cia et al. 2007;Ha et al. 2020a). The effectiveness of radiation in cut flowers is often dependent on the development of fungi, moisture condition, treatment dose, and storage conditions. Previous study showed that the combined application of 0.2 kGy gamma irradiation and 70 ppm sodium dichloroisocyanurate, an eco-friendly chemical, significantly reduced B. cinerea damage in cut rose flowers by directly influencing the structures of the pathogen (Chu et al. 2015). Electron beams suppressed spore germination and mycelial development of B. cinerea in cut roses with increasing irradiation doses from 0.1 to 20 kGy (Kwon et al. 2014). Treatment with 1080 J·m^-2 UV-C every 24 h for five days in a humid chamber significantly decreased the B. cinerea germination and the area of the lesions by the gray mold disease in rose petals (Vega et al. 2020). However, in some cases, the application of radiation with a high dose exhibits a negative effect such as bent necks of the cut flowers or abscission of leaves. The control of B. cinerea in cut rose flowers often requires high doses of gamma irradiation, which causes changes in physico-chemical properties and thereby affects the postharvest quality of rose flowers (Chu et al. 2015). Thus, the combined application of irradiation with the preservative solutions often requires to reduce this negative effect in cut rose flowers.

Biological Control

In recent years, use of biological microbe agents for control of gray mold disease in vegetables, fruits, and flowers is a potential technology instead of synthetic fungicides (Pertot et al. 2017;Sharma et al. 2009). Among biological microbe agents, antagonistic yeasts and Bacillus subtilis are highly recommended since they are non-toxic secondary metabolites and safer than bacteria (Pertot et al. 2017). Isolation of microbial agents from living petals, leaf and petal residues of rose flowers, and from laboratory collections were applied for controlling B. cinerea infection in rose flowers (Elad et al. 1993). Isolations of Trichoderma inhamatum and Gliocladium roseum effectively reduced 90% of sporulation of the necrotrophic pathogen in rose leaf residues (Elad et al. 1993). Combined treatment with yeasts and Bacillus subtilis completely reduced lesion development by gray mold in detached rose petals. Isolates of Bacillus subtilis, C. cladosporioides, and C. oxysporum effectively worked against B. cinerea in rose flower buds by reducing 42 - 65% number of lesions (Elad et al. 1993;Tatagiba et al. 1998). Biological control of B. cinerea in plants via microbes can work with different modes of action, including limitation of space, competition for nutrients, and host resistance. However, the effectiveness of biological control is not that remarkable as synthetic fungicides and usually call for the combination of fungicides or exogenous substances to gain a satisfying result (Droby et al. 2009;Elad et al. 1993). Additionally, use of biological products in commercial cut rose productions is limited due to their insufficient applicability in the supply chain or in the field.

Resistance Inducer

Jasmonic acid (JA) and salicylic acid (SA) are plant hormones that can induce resistance of many plants against necrotrophic pathogens (Yang et al. 2019). JA and SA can activate plant inducible genes, thereby leading to synthesizing secondary products which function as antimicrobial compounds of central importance to the plant defense response (Meir et al. 1998;Yang et al. 2019). Methyl jasmonate (MeJA), a derivative of JA, is also a natural growth regulator that induces plant defense responses for fungal pathogens (Meir et al. 1998). Recently, MeJA was examined for control of postharvest gray mold disease in various cut rose cultivars (Meir et al. 1998). MeJA (200 μM) effectively decreased lesion size and appearance of B. cinerea, as assessed by a detached petal bioassay (Meir et al. 1998). Treatment with higher concentrations of MeJA (300 - 400 μM) also significantly inhibited spore germination and germ-tube elongation of B. cinerea in both in vivo and in vitro. Interestingly, at high concentrations, MeJA did not impair postharvest quality and vase life of cut rose flowers (Meir et al. 1998). Application of exogenous JA (50 μM) significantly reduced the diameter of the gray mold disease lesions in rose petals (Cao et al. 2019). Additionally, JA was suggested to play an important role in rose flower defense against B. cinerea (Cao et al. 2019).

However, different from JA, the role of SA in defense against B. cinerea is various in plants (Koo et al. 2020). In some cases, application of SA enhanced B. cinerea susceptibility in plants by increasing the lesion size and disease severity (Koo et al. 2020). Multiple previous studies showed that SA treatment had no inhibitive effects on B. cinerea infection in cut rose flowers (Cao et al. 2019;Ha et al. 2020b). Our previous study indicated that treatment with exogenous SA did not reduce the incidence of B. cinerea infection in cut ‘Revival’ rose flowers (Fig. 3). The vase life of the cut rose flowers treated with SA was rather significantly decreased, compared with non-treated flowers (Ha et al. 2020b).

Control B. cinerea in Rose Flowers by Ethylene Inhibitors

Ethylene production by B. cinerea infection can accelerate senescence processes that can favour the disease development, since rose flower becomes more susceptible to gray mold with ageing (Elad 1997). Ethylene-accelerated senescence in many flower species, including roses, is related to increased membrane permeability. The deteriorating membrane can release oxidation compounds which enhance the susceptibility of the host to disease development (Elad 1997). In addition, changes in cell wall chemistry associated with senescence might make the plant cell walls more susceptible to the hydrolases secreted by the pathogen (Elad 1997). Previously, rose and carnation flowers produced high amounts of ethylene during development of necrosis in petals which was caused by B. cinerea infection (Elad 1988a;Seglie et al. 2012). However, the ethylene production was decreased since the host became completely macerated at late stage of B. cinerea infection (Elad 1988a;Seglie et al. 2012). Cut roses were sprayed with methionine, a precursor of ethylene production, or incubated with exogenous ethylene increased gray mold disease incidence considerably in flowers as shown in Fig. 4 (unpublished data).

Treatment with ethylene inhibitors such as aminoethoxyvinylglycine (AVG), aminooxyacetic acid (AOA), and silver thiosulphate (STS) by spraying significantly reduced gray mold disease severity in rose petals and leaves which inoculated with conidia or mycelial plugs (Elad 1988a). Cut rose flowers treated with AOA by spraying or with STS by dipping also effectively decreased B. cinerea infection under simulated transport conditions (Elad 1988a). Another ethylene action inhibitor, 1-methylcyclopropene (1-MCP), is also effective in suppressing the development of gray mold disease in cut flowers (Seglie et al. 2012). Compared to single treatments, the combination treatment with AVG and 1-MCP was the most effective in suppressing B. cinerea infection in cut ‘Pink Beauty’ roses under simulated transport conditions (Fig. 4). Ethylene inhibitors can retain the membrane integrity of plant tissues by delaying senescence, thus reducing Botrytis blight in rose flowers (Elad 1997;Elad and Shtienberg 1995). Recently, nano silver (NS) was applied to prolong vase life of cut flowers by suppressing ethylene synthesis and control B. cinerea infection (Ha et al. 2020b;Ha et al. 2021;Park et al. 2017). Our previous study showed that NS was the most effective in suppressing B. cinerea growth in cut ‘Revival’ roses during vase life (Fig. 3) (Ha et al. 2020b). The silver ion, which is released from nano silver, can replace the cofactor Cu²⁺ for the ethylene receptor and thereby effectively suppresses ethylene binding (Rodríguez et al. 1999). The suppression of ethylene action by NS prevented the degradation of ethylene receptors and the downstream protein kinase, resulting in the inhibition of the ethylene responses and the necrosis development in rose petals (Fig. 5) (Ha et al. 2021). Silver ions also delay the growth of fungal spores by releasing ROS via the reaction with oxygen and causing damage to nucleic acids, lipids, and proteins of the cells of B. cinerea (Dakal et al. 2016). AVG, 1-MCP, and NS can be promising agents for control gray mold disease in cut roses, since they are known as safe and non-toxic chemicals for human and environment and highly efficient in inhibiting ethylene damage in many crops. However, the relationship between ethylene, B. cinerea infection, and ethylene inhibitors should be further studied in rose flowers.

Conclusion

B. cinerea is the necrotrophic fungal pathogen inducing severe necrosis on roses, thereby resulted in huge economical loss in the flower industry. It has been established that extracellular proteins and ROS production are associated with the regulation of B. cinerea growth, development, and virulence. However, many details about B. cinerea in roses still remain unknown. Further studies are necessary to characterize the gene expression, biochemical components, and genetic pathways which relate to the interactions between ethylene biology and B. cinerea infection in rose flowers. Molecular analyses of the B. cinerea infection process in rose flowers may provide the important information to develop effective methods against the gray mold disease. Herein, we have discussed the advantages and disadvantages of current control strategies of gray mold disease in rose flowers. Among above mentioned strategies, use of the ethylene antagonist such as AVG, 1-MCP, and nano silver may be a promising strategy for control and prevention of B. cinerea in cut rose flowers.