Non-Chemical Approaches to Control Postharvest Gray Mold Disease in Bell Peppers

: Bell pepper ( Capsicum annuum ) is a widely grown vegetable crop that is nutritious and ﬂavorful and economically important for growers worldwide. A signiﬁcant limiting factor in the postharvest storage and long-distance transport of peppers is gray mold caused by Botrytis cinerea . The pathogen is widespread in nature, highly aggressive, and able to cause disease at cool refrigerated temperatures during transport and storage. Fungicides have been relied on in the past to reduce bell pepper rots in storage; however, concern over residues on the fruit and environmental degradation have heightened the importance of natural and generally recognized as safe (GRAS) solutions that effectively limit disease. Essential oils, plant extracts, inorganic biocontrols, activators, hot water treatments, and modiﬁed storage conditions have been tested to reduce losses from gray mold. Despite signiﬁcant amounts of research on natural methods of control of B. cinerea postharvest, research speciﬁc to gray mold in peppers is limited. The objective of this review is to summarize the research conducted with environmentally friendly alternatives to chemical fungicides to control this important pathogen of peppers postharvest. To ensure a steady supply of healthy and nutritious produce, more research is needed on the development, use, and application of non-hazardous Botrytis control methods. Until an effective solution is found, using a combined approach including environmental controls, sanitation, and GRAS products remain paramount to limit Botrytis fruit rot of peppers postharvest.


Introduction
Bell pepper is one of the most important vegetable crops grown worldwide for fresh consumption and processing. There are five domesticated species of capsicum and over 20 wild ancestors; however, Capsicum annuum is the most widely cultivated [1]. C. annuum has high horticultural value due to its pungency, flavor, and nutritional qualities, adding color, flavor, and texture to many fresh and processed products. In the Mediterranean region, the pepper crop is grown during the winter and summer months for export and local markets. High-quality fruits are uniform in shape, bright in color, large, firm, and without blemishes [2,3]. In North America, the blocky bell pepper with four lobes is preferred, as well as pungent chili peppers [1]. Bell peppers are typically produced in the open field and in greenhouses [4]. In many regions, the pepper crop is grown in shade structures and other forms of protected cultivation [5]. A major constraint to pepper production are diseases from fungal and bacterial pathogens [6]. The most important pathogens infecting pepper fruit during the growing season include Botrytis cinerea, Colletotrichum spp., Alternaria spp., Phytophthora capsici, and Xanthomonas spp. [7,8]. Protected cultivation reduces some stresses on the plants from intense sun and wind that provide entry for pathogens; however, temperature and humidity fluctuation still occurs, allowing for dew Aspects of the Postharvest Disease Cycle B. cinerea affects many specialty crops and flowers postharvest. The pathogen is able to cause disease from 0-26 • C [7,20], allowing it to cause disease in postharvest storage at cool temperatures [21]. The source of postharvest outbreaks is not always evident; fruit can be infected directly from inoculum present on neighboring fruit, from latent infections, or at wound sites [7,14]. Rots are insidious and often do not appear until the fruit arrive at retail setting; shipments with a significant percentage of infected fruit may be rejected by the broker. The disease cycle of postharvest fruit rot of pepper caused by B. cinerea has not been fully elucidated but likely shares similarities with other fruiting vegetables [7]. Postharvest fungal diseases of pepper fruit caused by Alternaria alternata and Fusarium spp. enter the fruit via natural openings. A. alternata was able to enter pepper fruit via the blossom end of the fruit and cause internal rots postharvest [22], while the calex-end of the pepper appears to be an important area of entry for Fusarium subglutinans [23] and Erwinia carotovora [24]. These may also be avenues for B. cinerea infection in addition to direct penetration of the cuticle. The pepper cuticle is very thick and lacking stomata [25]; however, microscopic cracks are apparent, which may be an entry site or provide nutrients for B. cinerea conidia [26]. Available nutrients and sugars on the host surface are important for successful infection [27,28]. B. cinerea is known to produce numerous enzymes, including cutinase, pectolytic enzymes, and cellulase, which allow for dissolution of the cuticle and infection to progress [28]. B. cinerea has also been observed on sunscalded areas of pepper fruit and anywhere where wounds are apparent [14]. Extended periods at cool temperatures were suggested as possibly enhancing gray mold postharvest. Cold temperatures weaken the cuticle and cell structure, which can reduce resistance to the pathogen [29]. Using early harvesting as a technique to limit disease as is practiced with citrus, avocado, and other fruits is not an option for the fresh market sales of bell peppers due to the defined changes in antioxidants, carotenoids, flavonoids, and sugars of the fruit and other desired characteristics that develop with fruit ripening [30].

Trends in Fungicide Use
Intensive agriculture has resulted in an increase in agrochemical use across all cropping systems [31]. Fungicides have been heavily relied upon in specialty crop production to limit losses from B. cinerea infection pre-and postharvest [10,13,32,33]. Over the last 15 years, scrutiny of agrochemicals has increased, and numerous compounds have been removed from the market [34]. Restrictions in agrochemical use due to governmental regulations, consumer concerns over residues and environmental degradation, and the development of extensive fungicide resistance has led to the search for natural compounds with fungicidal activity [31,35]. Maximum residue limits are used by governmental agencies to determine if the residues present in foodstuffs are dangerous to consumers [36]. In general, agrochemical residues on produce in developed countries are low [37]; however, the potential for toxic residues exists. Residues on fruit are a heightened concern, making control of postharvest diseases of fruiting vegetables a unique challenge to the industry. There are very few fungicides that can be applied to produce postharvest to limit losses from B. cinerea, depending on the crop and market. For freshly consumed vegetables, there are often no fungicides labeled for use, with few notable exceptions, including citrus and apples [38]. Research on biological compounds with fungicidal activity has increased significantly to meet market demands and alleviate concerns over residues [39], and these compounds are increasingly important for growers. In addition to toxic residue concerns, fungicide resistance is regularly observed in many fungi [40]. B. cinerea is a highly variable pathogen and well documented as able to overcome commonly used fungicides [41,42]. Resistance mechanisms to benzimidazole and dicarboximide classes of chemistries have been described [43,44]. Over-production of ATP-binding cassette transporters was the mechanism of resistance to the anilinopyrimidines, an important class of fungicides [32]. Reports of resistance to Fludioxanil [45] cyprodinil [46] and the recently developed succinate dehydrogenase inhibitors (SDHIs) fluopyram, fluxapyroxad, and penthiopyrad [47] have highlighted the need to find alternatives. Liu et al. [33] tested B. cinerea isolates from tomato greenhouses in China and found that resistance to the SDHI fungicide Boscalid was moderate, potentially reducing the effectiveness of fungicide management programs. The challenges of managing B. cinerea with traditional fungicide chemistries are manifold and an integrated approach that reduces reliance on chemicals is needed for continued successful production and postharvest storage of bell peppers.

Alternatives to Fungicides
Due to the high level of interest in natural and generally regarded as safe (GRAS) products to control gray mold postharvest, numerous compounds including essential oils and volatiles, plant extracts, agricultural by-products, inorganic chemicals, biocontrols, and plant defense activators have been researched [48][49][50][51][52]. In addition, UV light, environmental regulation, hot water treatment, and sanitation have been researched and found to have an effect on B. cinerea development of fruiting vegetables in storage (Table 1). Postharvest processing and storage is considered an ideal location for the use of biocontrols because it is a controlled environment, protected from UV and precipitation, and the implementation of control strategies is practical with equipment already present in many operations. There is a good opportunity to optimize formulations to take advantage of organisms that are adapted for specific conditions postharvest [53]. Table 1. Reports of physical, chemical, microbial, or environmental treatments effective against gray mold of bell pepper (Capsicum annuum L.) postharvest.

Essential Oils and Agricultural By-Products
A strong desire to identify compounds that are safe to humans and the environment has resulted in the development of numerous plant oils and extracts as biofungicides [61,62]. These compounds are bioactive compounds with high antifungal activity [38]. Essential oils have been researched intensively for activity against B. cinerea and other pathogens. Wilson, et al. [63] tested over 300 essential oils and extracts for activity against B. cinerea isolates and found 13 with high antifungal activity. The concentrations tested ranged from 0.39-100%. The researchers suggested that these compounds could be used as safe alternatives to conventional pesticides as both fungicides and fumigants. Hinokitiol, an oil extract from Japanese Cyprus, inhibited B. cinerea growth in vitro and prevented gray mold on bell peppers dipped in a solution at 750 µL/L [49]. This compound disintegrates the plasma membrane of B. cinerea and genes for pathogenicity are downregulated [64]. Vapors from sage essential oils reduced postharvest pepper rot [65], while volatiles of cinnamon oil did not prevent B. cinerea infection [56]. Peppers that were inoculated prior to cinnamon oil treatment rotted. Cinnamon oil at 500 ppm in vitro completely inhibited mycelial growth of B. cinerea, and 100 ppm reduced growth; conidial germination was reduced but not prevented at any concentration [56]. Clove and olive oil (0.125-0.5%) have also been tested and found to reduce B. cinerea in vitro and limit fungal growth on peppers when applied as a pre-harvest spray [66]. Natural agricultural by-products such as compost water extracts [67] and olive oil mill waste water [57] can control gray mold of pepper; however, formulations must be optimized to facilitate practical application. The reduction in fungal growth from olive oil mill waste water may have been due to a systemic response or direct fungicidal action, as both peroxidase and phenolperoxidase activity were increased in fruits after 2-5 days in storage and may be involved with the defense response of the fruit [57].

Chitin and Chitosan
Chitin and chitosan are natural polymers that are safe to consumers and the environment and able to prevent diseases of numerous crops caused by B. cinerea postharvest [68]. Chitin is an abundant biopolymer extracted from exoskeletons of crustaceans and is widely regarded as safe for human consumption. Chitosan, a polymer of β-1,4-linked D-glucosamine, has known activity against B. cinerea, damaging hyphal cell walls that come in contact with the compound [69] and activating the fruits defense response against the pathogen [70]. These compounds have been tested against numerous pathogens for effective and safe disease control [71]. Chitin and chitosan are available in various forms, which may provide different levels of disease control when used alone or in combination with a microbial formulation or essential oil [72,73]. The yeast Candida utilis and chitosan prevented tomato fruit rot at 0.25 and 0.5%, and chitosan at concentrations from 0.025-1.0% inhibited A. alternata and Geotrichum candidum germination in vitro [74]. Cucumber plants treated with chitosan (0.1%) showed lower gray mold disease severity levels than control plants when inoculum was applied before and after treatment [75]. Longer time intervals after treatment prior to inoculation resulted in greater levels of control. Tomatoes treated with chitosan plus Ruta graveolens essential oil coating had significantly lower incidence of gray mold growth and higher quality than untreated fruit [76]. A chitosan-oil dip for peppers improved storage quality after 35 days at 8 • C due to smoother skin surface, better sensory quality, and increases in certain antioxidant enzymes [77]. Chitosan mixed with edible coatings reduced disease severity on bell peppers and improved storage quality by increasing firmness, reducing weight loss, and reducing disease [50]; general decay was reduced below 5%, although specific disease-causing agents were not recorded. Waxes and edible coatings are known to improve certain quality characteristics of fruiting vegetables postharvest such as firmness and reduce water loss [78,79]. Adding chitosan or other biopreparations to these coatings has been shown to be beneficial in postharvest produce storage [50,80]. Wax mixtures with natural fungicidal compounds have been tested on citrus to reduce the reliance on synthetic chemistries [81] and could be adopted to pepper postharvest.

Calcium and Natural Compounds
The prevalence of calcium, low cost, and effectiveness make this an optimum postharvest treatment for gray mold control. High calcium content in plant tissues is known to reduce B. cinerea severity [19], and calcium is an important natural fungicide alternative for control of gray mold [82,83]. Increasing tissue calcium content increases cross linking of polygalacturonate polymers and reduces damage from decay causing organisms [84]. Fungal polygalacturonase is also inhibited by calcium [85]. Kamara et al. [86] found that calcium chloride sprays reduced gray mold severity and increased the storage life of Egyptian pepper cultivars. Apples were protected from Botrytis fruit rot postharvest by a calcium (2%) dip prior to storage [87]. Postharvest dips of calcium may also be beneficial for peppers during the postharvest washing and packaging process [2,55,88]. Calcium-containing salts tested in vitro against B. cinerea from pepper demonstrated that calcium hydroxide was the most effective at controlling the pathogen [89]. Other researchers have highlighted the importance of combining calcium compounds and biocontrol agents to improve disease control. For example, Botrytis fruit rot of pear was significantly reduced when CaCl 2 and the yeast Cryptococcus laurentii were combined and spread over fruit wounds [90]. The authors noted that peroxidase activity in the fruits treated with calcium continually increased up to 96 h after application (the last time point recorded), suggesting an induced response of the fruit as well as a potential physical protection against the pathogen. Hydrogen peroxide (Sanosil), a GRAS-certified disinfestant, inhibited B. cinerea conidial germination and mycelial growth at 0.5 and 1.5%, respectively [48]. Dipping red bell peppers for 60 s in a 0.5% solution improved storage quality and reduced decay. Vapors of acetic acid, a naturally derived acid, completely prevented Botrytis fruit rot of tomato and other fruits after 1 h exposure, and the treatment was effective at cool temperatures as low as 1 • C demonstrating usefulness in cool storage conditions [91]. Elad [51] sprayed different antioxidants known to scavenge free radicals onto pepper leaves and found that four provided significant reduction in B. cinerea at 0.1, 1.0, and 10 mM. Inhibition of ethylene production by the antioxidants may have played a role in the disease reduction observed due to the known effects of this compound on plant tissues and B. cinerea. Storage duration and quality of green bell peppers was greatly enhanced by delaying the ripening process with hexanal vapors [92]. Multiple fruit quality evaluations were made, including water loss and CO 2 evolution, which decreased with hexanal treatments from 0.005 to 0.02% w/w.

Microbial Biological Control
Numerous microorganisms have shown activity against B. cinerea and other frequently cited storage pathogens; however, limited research has been specifically conducted on control of postharvest fruit rots [39]. Microbial compounds are applied pre-or postharvest and may be used alone or with another compound [53]. Bacillus licheniformis reduced gray mold on tomato plants and flowers [93], and Trichoderma harzianum was able to significantly reduce B. cinerea development on beans [52]. Other microbial species such as Bacillus subtilis, Rhodosporidium paludigenum, and Pichia guilliermondii have provided control against Botrytis fruit rot of tomato [94][95][96]. The biocontrols can have direct action on the pathogen and activate the plant's defense response. Bacillus amyloliquefaciens effectively controlled gray mold of tomatoes [97]. This biocontrol has known PGPR qualities when sprayed on pepper plants [98] and improves antioxidant quality in the fruit [99]. Udalova, et al. [100] tested foliar sprays of a humic acid (0.1%) product with and without B. subtilius on two pepper cultivars and found that there was a significant increase in overall yield, which may have been a result of plant activation. The fruits from treated plants also have had higher total carbohydrates, ascorbic acid, and dry matter (%) at maturity. Microbial formulations that have a beneficial effect on plant health could be applied at optimal times to reduce gray mold. When the endophyte Beauveria bassiana was applied to the roots of tomato and chili pepper, it provided protection against foliar B. cinerea [101]. This area of plant protection needs further research to optimize formulations and application timing to improve disease control postharvest.

Systemic Acquired Resistance
The potential to spray resistance inducers near harvest to prevent postharvest rots would benefit farmers by reducing inputs and lowering the risk of surpassing MRLs. Resistance inducers benefit plant health and protect the fruit by increasing quantities of antifungal compounds, phytoalexins, or other protective factors [102]. Salicylic acid (SA) is a well-known activator of induced resistance in plants that has been researched to control bacterial and fungal plant pathogens [103]. SA, abscisic acid, and methyl jasmonate all acted as resistance inducers of peppers, reducing Botrytis gray mold severity in peppers harvested from plants treated with different concentrations of the SAR compounds [86]. Phenol content and defense related enzymes increased in fruits of all varieties tested, which may have increased resistance levels. Spraying pepper plants in the field with SA at 8 mM and citric acid at 30 mM reduced gray mold of peppers in storage [66] and mycelial growth of the pathogen was sensitive to SA in vitro. When bell peppers were sprayed at 50 g/L with harpin protein, a known resistance activator, B. cinerea growth was reduced [59]. Greenhouse grown fruits from plants regularly sprayed with harpin protein had higher soluble solids and acidity [104]. These treatments can also extend the storage life of peppers under cool (7 • C) temperatures and modified atmosphere [105]. After application, resistance inducers likely increase the levels of certain antifungal compounds in the fruit. More research is needed to determine which SAR inducers enhance these compounds in the fruit and to what magnitude and duration.

Heat Treatment and UV-C Application
Heat treatment of pepper fruit postharvest prior to storage can directly inhibit pathogens and increase the resistance of the fruit [106,107]. Treating peppers with hot water at 55 • C for 12 s reduced decay and improved storability [55]. Physical removal of spores and possible beneficial effects to the fruit's cuticle were suggested to have reduced disease (Figure 1). Heat treatments from 45 to 55 • C reduce spore germination and germ tube elongation. Using irradiation can reduce gray mold by killing pathogen spores and inhibiting latent infections. UV-C light was germicidal to B. cinerea conidia and prevented gray mold of bell pepper [108]. This treatment was also found to reduce damage from chilling injury and general decay from Alternaria spp. and B. cinerea [109]. More research is needed on this control strategy to determine practicality and economic feasibility for the pepper industry. Effects on pepper quality would also need to be tested on additional cultivars and fruit-types; high rates of UV-C applied to tomato resulted in undesirable effects on fruit color [110].

Heat Treatment and UV-C Application
Heat treatment of pepper fruit postharvest prior to storage can directly inhibit pathogens and increase the resistance of the fruit [106,107]. Treating peppers with hot water at 55 °C for 12 s reduced decay and improved storability [55]. Physical removal of spores and possible beneficial effects to the fruit's cuticle were suggested to have reduced disease (Figure 1). Heat treatments from 45 to 55 °C reduce spore germination and germ tube elongation. Using irradiation can reduce gray mold by killing pathogen spores and inhibiting latent infections. UV-C light was germicidal to B. cinerea conidia and prevented gray mold of bell pepper [108]. This treatment was also found to reduce damage from chilling injury and general decay from Alternaria spp. and B. cinerea [109]. More research is needed on this control strategy to determine practicality and economic feasibility for the pepper industry. Effects on pepper quality would also need to be tested on additional cultivars and fruit-types; high rates of UV-C applied to tomato resulted in undesirable effects on fruit color [110].

Packaging and Controlled Atmosphere
Environmental regulation and temperature have a major effect on B. cinerea and disease development [111]. This is a well-established area of research, and in controlled agricultural settings, such as greenhouses, reducing relative humidity is known to limit gray mold during production [27]. In storage, RH levels and atmospheric conditions are largely dependent on the crop being stored and the quantity. Disease severity levels are reduced when storage containers are kept at low O2 and high CO2 levels [112]. In studies on controlled storage, the severity of gray mold of eggplant was reduced for fruits stored at 13 °C at 7.5% CO and 1.5% O2 [21]. Storage at 10 °C was found to significantly reduce rots of pepper compared to 21 °C [113]. Although cold storage helps to reduce fungal diseases, it will not eliminate B. cinerea, especially where crops are held for long periods [114]. Peppers are typically washed after harvest, which can remove spores of B. cinerea but also contributes to high humidity in postharvest packaging. Typical packaging techniques for pepper may result in increased humidity during storage and transport unless perforations are used [115]. Artificially reducing the humidity in packaging for peppers greatly reduced B. cinerea and other pathogens [54]. Additionally, modifying packaging atmosphere using moisture absorbance packets greatly increased the storage of green bell peppers by reducing the respiration levels of the fruit [116]. Low temperatures in combination with modified atmosphere were important for maintaining high ascorbic acid content.

Packaging and Controlled Atmosphere
Environmental regulation and temperature have a major effect on B. cinerea and disease development [111]. This is a well-established area of research, and in controlled agricultural settings, such as greenhouses, reducing relative humidity is known to limit gray mold during production [27]. In storage, RH levels and atmospheric conditions are largely dependent on the crop being stored and the quantity. Disease severity levels are reduced when storage containers are kept at low O 2 and high CO 2 levels [112]. In studies on controlled storage, the severity of gray mold of eggplant was reduced for fruits stored at 13 • C at 7.5% CO and 1.5% O 2 [21]. Storage at 10 • C was found to significantly reduce rots of pepper compared to 21 • C [113]. Although cold storage helps to reduce fungal diseases, it will not eliminate B. cinerea, especially where crops are held for long periods [114]. Peppers are typically washed after harvest, which can remove spores of B. cinerea but also contributes to high humidity in postharvest packaging. Typical packaging techniques for pepper may result in increased humidity during storage and transport unless perforations are used [115]. Artificially reducing the humidity in packaging for peppers greatly reduced B. cinerea and other pathogens [54]. Additionally, modifying packaging atmosphere using moisture absorbance packets greatly increased the storage of green bell peppers by reducing the respiration levels of the fruit [116]. Low temperatures in combination with modified atmosphere were important for maintaining high ascorbic acid content.

Host Resistance
Genetic host resistance is an important component of integrated disease management of specialty crops and has been used successfully against many diseases. Resistance to B. cinerea has proven to be challenging due to the aggressive nature of the pathogen; however, there are examples of successful breeding in some crops [117,118]. For vegetables, limited research has been conducted other than on tomatoes [119], where breeding techniques were used to introduce qualitative resistance [120]. Complete resistance to B. cinerea was not observed on other fruit crops and is likely difficult to find in wild host sources [121]. Breeding resistance into specialty crops is difficult due to the many stages during development at which B. cinerea can infect and the different mechanisms involved in the resistance response [122]. Pepper fruit resistance has not been looked at significantly. Kamara et al. [86] studied four local Egyptian pepper cultivars and found one that exhibited lower levels of gray mold after artificial inoculations. The mechanisms of this reduced susceptibility are not known. Additional extensive screening of cultivars and breeding efforts are needed to identify resistant fruit with desired horticultural traits.

Capsacin and Phytoalexins
Although not specifically associated with resistance, biological compounds in peppers may confer resistance during infection and improve the fruits resistance. Capsacin is a well-studied compound that is produced by hot peppers to give flavor and pungency and is only found in the genus Capsicum. Numerous factors affect capsacin production in the fruit, including the nutrition of the plant, the ability to produce peroxidase, and other host factors [123,124]. Xing et al. [125] found that capsaicin was inhibitory to B. cinerea in vitro, and that pH 5 was the most optimum for growth inhibition. Substantial research on the production and metabolism of a similar metabolite capsidiol, a terpenoid phytoalexin, has demonstrated that it is important in resistance to certain pathogens of pepper and provides a source of resistance to non-pathogens [126][127][128][129]. Interestingly, B. cinerea is known to degrade capsidiol to less toxic compounds [130]. The role of this phytoalexin in susceptibility of pepper to Botrytis fruit rot is not completely clear. Other phytoalexins such as 6-methoxymellein from carrots can limit B. cinerea growth [131]. Enhancing metabolites within the fruit without negatively affecting fruit quality could improve postharvest storage duration. This is an important research area that needs more attention to ensure fruit quality remains high, even if fungicides or other biocontrols are not used.

Conclusions
B. cinerea remains a significant threat to bell peppers in postharvest storage and transport [7]. Although agrochemical use has decreased in some production settings, and research to find alternative methods of controlling pathogens has increased [34,39], fungicides are still used widely during production to control gray mold and other diseases of peppers. These practices will likely continue until satisfactory alternatives are found that are safe and provide disease control. The importance of B. cinerea as a postharvest pathogen complicates initiatives to reduce fungicides and the use of GRAS products due to the difficulties inherent in managing this important pathogen. High fruit quality and nutritious produce are essential for healthy diets, and society continues to focus on increasing produce consumption. Consumers remain concerned about fungicide residues on produce, and governmental restrictions are increasing in developed countries [36,132]. Additionally, environmental degradation due to extensive agrochemical use is an important factor in the need to find novel solutions. Natural products and resistance activators have been found to improve the postharvest quality of bell peppers and reduce gray mold severity in many studies, and continued research in this area is paramount. Despite the use and availability of natural and GRAS certified products and even the use of fungicides, B. cinerea remains a significant problem postharvest when conditions are favorable for disease. Future research needs to address this discrepancy so that bell peppers can be stored and transported without losses. Integrated control remains an important strategy and includes careful control of environmental conditions, sanitation, applying natural and GRAS products, and using fungicides judiciously to improve and maintain pepper quality postharvest.