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Solidago canadensis L. Essential Oil Vapor Effectively Inhibits Botrytis cinerea Growth and Preserves Postharvest Quality of Strawberry as a Food Model System
• Department of Food Science and Engineering, Ningbo University, Ningbo, China
This study investigated the anti-fungal properties of Solidago canadensis L. essential oil (SCLEO) against Botrytis cinerea in vitro, and its ability to control gray mold and maintain quality in strawberry fruits. SCLEO exhibited dose-dependent antifungal activity against B. cinerea and profoundly altered mycelial morphology, cellular ultrastructure, and membrane permeability as evaluated by scanning electron microscopy, transmission electron microscopy, and fluorescence microscopy. SCLEO vapor at 0.1 mL/L maintained higher sensory acceptance and reduced decay of fresh strawberry fruit, and also reduced gray mold in artificially inoculated fruit. SCLEO treatment did not, however, stimulate phenylalanin ammonia-lyase, polyphenol oxidase, or chitinase, enzymes related to disease resistance. This suggests that SCLEO reduces gray mold by direct inhibition of pathogen growth. SCLEO vapor may provide a new and effective strategy for controlling postharvest disease and maintaining quality in strawberries.
Introduction
Strawberries are in high demand because of their delicious flavor and nutritional value, but often become unmarketable due to mechanical injury and fungal contamination. Gray mold, caused by the fungus Botrytis cinerea, is one of the major causes for the reduced post-harvest storage life of strawberries (Lazar et al., 2010). In past decades, chemical fungicides were widely used to control postharvest fungal disease in fruit. However, the indiscriminate and excessive use of synthetic fungicides has been a prime cause for the development of resistant fungal pathogen populations, resulting in the use of even greater quantities of antifungal compounds in agriculture and the appearance of increased levels of toxic residues in food products (da Cruz Cabral et al., 2013). Alternative control methods are therefore urgently needed (Sukorini et al., 2013). Plant essential oils (EOs) and extracts have been used for thousands of years in food preservation, pharmaceuticals, alternative medicine and natural therapies (Prabuseenivasan et al., 2006). EOs, which are naturally synthesized in different plant organs as secondary metabolites, are characterized as oily fragrant liquids extracted from aromatic plant materials (El Asbahani et al., 2015). EOs have recently attracted interest as control agents for postharvest disease due to their volatility, relative safety, broad acceptance by consumers, and eco-friendly and biodegradable properties (Tzortzakis and Economakis, 2007). Numerous studies have documented antifungal effects for different EOs used to control deterioration in postharvest fruit such as citrus (Fan et al., 2014; Tao et al., 2014a; Shao et al., 2015), strawberry (Shao et al., 2013a), blueberry (Mehra et al., 2013), peach (Elshafie et al., 2015), and cherry tomato (Guerra et al., 2015).
Solidago canadensis L. is an herbaceous perennial of the family Asteraceae that is widely distributed in South America, Europe, and Asia (Skrzypczak and Budzianowski, 2001). It was intentionally introduced to eastern China as an ornamental plant in 1913 (Jin et al., 2004). Since the 1980s, S. canadensis has spread rapidly and has become one of the most destructive invasive weeds in southeastern China (Guo and Fang, 2002). It has significantly reduced the abundance and diversity of native plant communities, and poses a growing threat to important ecosystems and regional economies (Guo, 2005). Questions concerning potential control strategies and/or possible uses for the plant are now of great interest. S. canadensis has been used in European phytotherapy for 700 years for the treatment of chronic nephritis, cystitis, urolithiasis, rheumatism, and as an antiphlogistic drug (Apati et al., 2003). Its leaves contain a wide range of active ingredients that are responsible for its antioxidant, antimicrobial, anti-inflammatory and spasmolytic and diuretic properties (Wang et al., 2006; Deng et al., 2015). α-Pinene, germacrene D, and 6-epi-β-cubebene are the major components of the EO found in leaves from several Solidago species (Kalemba et al., 1990; Kasali et al., 2002; El-Sherei et al., 2014). These compounds may contribute to the antibacterial ability observed against Listeria monocytogenes, Staphylococcus aureus (Deng et al., 2015), and B. cinerea (Wang et al., 2006). Importantly, acute toxicity assays show that the S. ÿanadensis extracts have no obvious toxicity (Nie et al., 2008).
To the best of our knowledge, Solidago canadensis L. essential oil (SCLEO) has not yet been applied to strawberry fruit during storage. The aims of this study were to (1) investigate the effects and possible mechanisms of SCLEO treatment against B. cinerea in vitro, (2) examine effects of SCLEO treatment on the postharvest quality of fresh strawberry, and (3) measure the induction of disease resistance, and assess the control of gray mold, in artificially inoculated fruit treated with SCLEO vapor.
Materials and Methods
Essential Oil, Pathogen, and Fruit
Leaves of fresh Solidago canadensis L. were collected in October 2014, dried in the dark at room temperature, and then powdered with a pulverizer. EO was isolated by hydro-distillation using a distillation apparatus and a mixture of approximately 150 g fresh leaves in 1800 ml distilled water. Subsequently, EOs were dried using anhydrous sodium sulfate, filtered, and stored in amber flasks (4°C) until tested. The oil obtained from the plant species possessed a iridescent coloration and a characteristic odor.
Highly virulent B. cinerea was isolated from spoiled, greenhouse-raised strawberry fruit. Isolate identity was confirmed using morphological and molecular criteria (Shao et al., 2013a). Fungal cultures were maintained on potato dextrose agar (PDA) medium at 25°C. Spore suspensions were harvested from 10 day old cultures and adjusted to 1 × 106 spores/mL by hemocytometer.
Strawberries (Fragaria ananassa Duch. cv. Hongyan) were harvested by hand at the mature red stage from a commercial greenhouse near Ningbo University, PR China, and transferred to the laboratory within 1 h. All fruit used in experiments were uniform in size and free of defects.
Effects of SCLEO on Mycelial Growth
The toxicity of the SCLEO against B. cinerea was assessed using the method of Shao et al. (2013a). Plates were subjected to different SCLEO vapor concentrations (0.5, 1, 2, and 3 mL/L air) and then incubated at 25°C for 3 days. Treatment efficacy was evaluated by measuring and averaging two perpendicular diameters for each colony. Mycelial inhibition rate = (dc-dt)/(dc-di) × 100, where dc is the mean colony diameter of the control sets, dt is the mean colony diameter of the treatment sets, and di is the initial colony diameter of fungal PDA disks. All tests were repeated five times.
Effects of SCLEO on Fungal Morphology and Ultrastructure
One hundred and fifty milliliter potato dextrose broth (PDB) medium was inoculated with 1 mL B. cinerea spore suspension (106 spores/mL) and incubated at 25°C with shaking at 150 revolutions per minute (rpm) for 72 h. SCLEO was then added to the medium to a final concentration of 16.5 mL/L, and incubation continued for 2 h before samples were collected. Cultures without oil were used as controls. Samples were centrifuged at 4000 rpm for 10 min and washed with cold phosphate buffer solution (PBS, 0.1 M, pH = 7.4) three times collect fungal mycelia. Mycelia were fixed with 2.5% glutaraldehyde for 2 h at 4°C. Three replicates were prepared for the treated and control groups. The effects of SCLEO on hyphal morphology and cell ultrastructure of B. cinerea were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), using our previously described methods (Yu et al., 2015).
Effects of SCLEO on Fungal Membrane Integrity
Membrane integrity was assayed by fluorescent microscopy (FSM) method, following the method of Liu et al. (2010). B. cinerea was treated with 16.5 mL/L SCLEO and collected as described in Section "Effects of SCLEO on Fungal Morphology and Ultrastructure". The collected mycelium was stained with 50 mg/L propidium iodide (PI) for 30 min at 4°C in the dark. Residual dyes were removed by washing twice with phosphate buffered saline. Samples were observed with a Zeiss Axioskop 40 microscope (Carl Zeiss, Oberkoch en, Germany) equipped with a single fluorescein rhodamine filter set (Zeiss no.15: excitation BP 546/12 nm, emission LP 590 nm). fields of view from each cover slip were chosen randomly, and all experiments were repeated three times.
Effects of SCLEO on the Postharvest Quality of Fresh Strawberry Fruit
Fresh strawberries were divided into two groups. For the treated group (SCLEO), fruits were placed into 8 L polystyrene containers with snap-on lids and minitype shelves. A 20 W heater was placed in the container and 800 μL SCLEO was pipetted into a glass dish (80 mm diameter), placed on the heater, and then the fruit container was immediately sealed. The heater was powered for 30 min to promote oil volatilization before being turned off. The SCLEO concentration in the chamber was regarded as 0.1 mL/L air, which is the ratio of SCLEO volume (800 μL) to the container volume (8 L). The container remained sealed for 12 h and was maintained at 25°C. Samples without EO treatment were used as controls. After treatment, all strawberry fruits were removed from the sealed containers, and then stored at 20°C for 4 days. Each day, 15 fruits were randomly selected for sensory evaluation and to assess changes in weight, firmness, total soluble solids (TSS), and titratable acidity (TA) content. Each group was replicated three times and the entire experiment was performed twice.
Quality Measurements
Weight loss was expressed as the reduction in weight as a percentage of total weight. Fruit firmness was measured by a hand penetrometer (GY-1, Hangzhou Top Instrument Co., LTD, China) and expressed in Newtons (N). Soluble solid content (TSS) and titrated acid (TA) were measured by the method of Shao et al. (2012).
At the end of storage, the extent of decay was evaluated by decay index. Decay was evaluated visually using 10 fruit per replicate according to a 4-level scale, where 0 = no decay; 1 = slight decay, covering <25% of the fruit surface; 2 = moderate decay, covering >25% but <50% of the fruit surface; 3 = severe decay, covering >50% of the fruit surface. The decay index was calculated using the following formula: [(1 × N1 + 2 × N2 + 3 × N3 + 4 × N4) × (4 × N)], where N is the total number of fruit measured and N1, N2, N3, and N4 are the number of fruit showing the different degrees of decay (Cao et al., 2010).
Sensory Evaluation
Sensory profiles were assessed using a twenty-category scale (1-5 = dislike extremely, 6-10 = neither like nor dislike, 11-15 = like, 16-20 = like extremely) for color, aroma and decay. The approach is modeled after the method described by Gol et al. (2013). Ten panelists were trained at the beginning of the experiment to evaluate relevant fruit characteristics. Sensory tests were conducted in a sensory laboratory equipped with individual sensory compartments.
Effects of SCLEO on Gray Mold and the Induction of Disease Resistance in Artificially Inoculated Strawberries
Strawberries with no physical defects were surfaced-disinfected with 75% ethanol and air-dried for two hours. A single artificial wound (depth 2 mm) was made in each fruit using a nail 2 mm in diameter then 15 μL of B. cinerea suspension (106 spores/mL) was inoculated into each wound. Inoculated fruits were randomly divided into control and SCLEO-treated groups. SCLEO treatment was as described in section of 2.5. Following incubation, the decay index was measured.
To evaluate the induction of active defense responses by SCLEO treatment, tissue samples (1 cm distance from the edge of the wound or decay area) of ten fruits from each replicate were collected at 0, 12, 24, 36, 48, 60, and 72 h after inoculation. Samples of each time point from each replicate were mixed and frozen immediately in liquid nitrogen, and then stored at -80°C. All enzyme extraction procedures were conducted at 4°C. Phenylalanin ammonia-lyase (PAL) was extracted with 0.1 M PBS (pH 8.8) containing 5 mM β-mercaptoethanol and 2% polyvinyl polypyrrolidone (PVPP, m/v). polyphenol oxidase (POD) was extracted with 200 mM PBS (pH 6.4) and 2% PVPP. Chitinase (CHI) and β-1,3-Glucanase were extracted with 0.05 M sodium acetate buffer solution (pH 5.0) with 2% PVPP (m/v). All extracts were homogenized and centrifuged at 10,000 g at 4°C for 20 min. The supernatants was used for the assay...
http://dx.doi.org/10.3389/fmicb.2016.01179
Conclusion
Solidago canadensis L. essential oil exhibited high antifungal activity against B. cinerea in vitro, reduced decay development in fresh strawberry fruits, and successfully controlled gray mold in artificially inoculated strawberries. SCLEO vapor treatment also resulted in higher sensory acceptance of strawberries during storage, but did not induce enzymes related to disease resistance in fruit. It was propose that SCLEO-mediated disease inhibition occurs as a result of direct interactions with the fungus itself, and that SCLEO vapor treatment is a potential alternative to synthetic fungicides for the control of phytopathogenic fungi in strawberry fruits. It is also important to reveal the effects of SCLEO on the strawberry fruits stored at different temperature and evaluate the cost of SCLEO application in future.
Source: Frontiers