**3. Disease control**

Obtaining healthy pomegranates is the early step to ensure an extended postharvest life, so fruit sorting during the production chain is relevant. Cold-storage temperature significantly influences the incidence of postharvest diseases and related storage life. Although optimal cold-storage temperature slightly changes among cultivars, in general, 6.5 ± 1°C is fine for most of them in presence of 90–95% relative humidity (RH). Weight loss and decay incidence are increased by higher temperatures, instead chilling injuries are enhanced by lower temperatures; similarly, higher RH favors fungal growth and lower one causes weight loss [75]. In addition, arranging pomegranates in microperforated plastic bags can reduce fruit dehydration allowing transpiration and minimizing condensation, indeed it is possible to further extend the storage life of fruit. These bags create a modified atmosphere [76].

Latent and wound pathogens display different modes of infection, so it is important to take action otherwise; particularly, to control postharvest latent pathogens is needed preharvest application of fungicides during the blooming stage; instead to reduce infections caused by wound pathogens during postharvest processing is useful to act on harvested pomegranates. However, good agronomical practices based on pruning, mummy and debris removal, adequate irrigation, and fertilization are key steps in fungal disease prevention. Nevertheless, in general, fruit are just postharvest treated [77], also because often are no chemical fungicides labeled for preharvest application, like happen in Florida and Italy [6, 77] except for temporary emergency registration. In agreement with the United Nations Priorities, disease prevention is basic to reduce food waste from 30 to 15% and discarded fruit by 20%; finally, UNO requests the reduction of postharvest pesticides by 20% by 2030. This "One Health" approach is useful to reduce fruit-waste and economic losses, defend human health

by reducing exposure to chemical fungicides, and decrease fungal resistance to chemical molecules. Regardless of cultivar susceptibility and conventional/alternative nature of the treatment, fungicide application timing is significant to nip-in-the-bud infections.

Tested chemical fungicides aim to stroke main pomegranate postharvest fungal pathogens, such as *B. cinerea*, *A. alternata*, *C. granati,* and *C. gloeosporioides*. In addition, being a minor crop, no fungicides are fully registered in most of the producer countries, such as Italy, where regulations change year by year. As an example, in this country in 2019 and in 2020, to reduce gray mold incidence, fludioxonil postharvest application was allowed, and in 2021 preharvest employment of the beneficial microorganism *Bacillus amyloliquefaciens* sbs. *plantarum,* strain D747, was temporarily approved in addition to sulfur and copper. Particularly, since 2021 employment of copper, well-known for its broad-spectrum anti-bacterial properties, has been banned due to toxicity to the whole ecosystem (humans, animals, plants, and environment). Therefore, the already allowed microorganism, together with boscalid, fludioxonil, commercial formulates based on *Trichoderma asperellum* or *Trichoderma atroviridae* strain T11, and essential oils (geraniol, thymol, and eugenol) have been temporarily approved for 2022. Finally, for treatments on leaves, *Coniothyrium minitans* is temporarily allowed as well as dazomet, metam-potassium, and metam-sodium for treatment in soil.

Fludioxonil belongs to phenylpyrroles that originated from pyrrolnitrin antibiotic, which is produced by different species within the *Pseudomonas* genus [78]. It is a nonsystemic fungicide, exploitable both pre- and postharvest, and broad-spectrum; it is effective against *B. cinerea* and other fungal pathogens, such as *Alternaria* spp. and *Aspergillus* spp. [75, 79]. Although its mode of action is not fully understood, fludioxonil inhibits spore germination, germ tube elongation, and mycelium growth of *B. cinerea*. Its mode of action involves the hyperactivation of the high osmolarity glycerol (HOG) signaling pathway through group III hybrid histidine kinases (HK). Being part of the multistep phosphorelay systems (MSP), HKs of group III are needed for the variability of cellular signal transduction in eukaryotic organisms. These signaling systems allow interaction and response between microorganisms and environments through cellular homeostasis regulation, but HOG is responsible for fungicide action also. In the HisKA domain, the histidine H736 is the putative signaling switch [80]. As argued by Xavier and colleagues [77] treating fungicide effectiveness, almost two treatments during the blooming stage are enough to significantly reduce gray mold incidence; in addition, he suggests to alternatively use different active molecules to avoid fungal resistance.

Concerning boscalid, it strikes different stages of fungal development from germ tube elongation and spore germination till appressoria formation or mycelial growth; in addition, during absorption through leaf surface, it is translaminarily and acropetally transported and distributed. Boscalid is a carboxamide fungicide that blocks fungal respiration inhibiting succinate dehydrogenase (SDH) activity. It acts by binding itself to the ubiquinone reduction site within complex II of the mitochondrial electron transport chain. Its site-specific mode of action makes boscalid a broadspectrum fungicide, but this implies high risk to develop resistant fungal strains also [81]. Generally, these resistant strains are related to point mutations that reach to the substitution of histidine to arginine (H272R) or tyrosine (H272Y) within SDH.

The US Environment Protection Agency classified fludioxonil as reduced-risk compound, but some researchers described its potentially hazardous effects due to mitochondrial oxidative damages that are reflected in organ-specific responses and diseases acting as comorbid factors. Indeed, European Union (EU) set a maximum residue limit (MRL) of 3 ppm within 7 days after pomegranate treatment [82, 83]. However, fludioxonil residues half from the 7th till the 30th day after application [75] and, as observed by Usanmaz et al., fludioxonil concentration decreases below 0.25 ppm after 7 days from the application [79]. Similar considerations concern boscalid that is considered a new-generation fungicide; as other members belonging to SDH inhibitors, it is considered safe. Although, due to its brief lifetime (it is tuned in 2003), EU set 2 mg/kg as MRL value for pomegranate [84].

Hence, in this scenario, the balance of advantages and disadvantages should be considered when choosing the chemical fungicides employment. Therefore, alternative control means may be the safe solution to control postharvest losses caused by fungal pathogens, reducing putative mycotoxin contamination, and avoiding health hazardous compounds like chemical fungicide residues. As stated above, among approved and commercially available microorganisms, there is *B. amyloliquefaciens*. Its efficacy is related to competition for space and nutrients, induction of resistance, and antibiosis. Lipopeptides, such as iturines, fengycin, and surfactine, are produced by *B. amyloliquefaciens* to control gray mold [85]. During the blooming stage, in the field application of *B. amyloliquefaciens* significantly allows controlling postharvest losses caused by *Botrytis* spp.; treated pomegranates enhance the activity of enzymes involved in defense mechanisms, like polyphenol oxidase, peroxidase, phenylalanine ammonia lyase, superoxide dismutase, and catalase (86). By these defense mechanisms, substances (i.e., quinones, reactive oxygen species) and lignin, which are active against fungi, are produced [86]. Furthermore, being elicitors of the induced systemic resistance, chitinase and β-1,3 glucanase are enhanced and boosted by iturin A and surfactine [87]. Repeated treatments during the blooming stage should improve *B. amyloliquefaciens* efficacy due to gradual physiological reduction of defense mechanisms during fruit ripening [88, 89].

Among EU-approved basic substances there is chitosan hydrochloride. Basic substances are "active substances, not predominantly used as plant protection products but which may be of value for plant protection and for which the economic interest of applying for approval may be limited" [https://www.efsa.europa.eu/en/supporting/pub/en-1900]. Chitosan is a d-glucosamine linear polymer available in several commercial formulates differing in composition. It acts in three different ways: it elicits host defense mechanisms, it discloses antimicrobial activity, and it has coating properties [90]. Its effectiveness changes based on the origin (it can be obtained by exoskeleton of crustaceans or insects or fungal mycelium), deacetylation degree (from 60 to 100%), molecular weight (between 3800 and 20,000 Da), production process (chemical or biological synthesis), environmental features (like pH and temperature), and not least sensitivity of fungal species [90–92]. Its water solubility, bio-adhesive properties, and related user-friendliness depend on just mentioned parameters and hydrochloride. Antifungal properties are also highly influenced by its composition [92], although its effectiveness in controlling postharvest rots of pomegranates is proven. Therefore, chitosan hydrochloride display both direct and undirect antifungal effects as described by Munhuweyi et al. [78]: halving of mycelial growth of *C. granati*, *Botrytis* sp., and *Penicillium* sp. is validated by *in vitro* trial; *in vivo* assay displays a reduction by 18–66% of rot incidence caused by the same fungi. Main postharvest fungal pathogens of pomegranates, such as *Botrytis* spp., *Penicillium* spp., and *C. granati*, are controlled by using chitosan concentration between 0.5 and 2.2 g/L, with an efficacy comparable to fludioxonil chemical control [78]. Chitosan could be applied as fruit coating also. It changes respiration and transpiration rate, so delaying ripening, reducing decay incidence, and maintaining qualitative parameters

*Pomegranate: Postharvest Fungal Diseases and Control DOI: http://dx.doi.org/10.5772/intechopen.109665*

till 6 months [93]. However, its employment is more popular regarding fresh arils. Often chitosan effectiveness is improved by combination with essential oils for both whole fruit and arils [78, 94]. On the other hand, geraniol, thymol, and eugenol have recently been approved, indeed no reliable data are available, although preliminary *in vitro* trials regarding their efficacy is in progress [95].

Chitosan is also usable combined with sulfur nanoparticles, this element is historically known for its antifungal properties: Greek and Romans used it as drug and disinfectant [96, 97]. Its broad-spectrum efficacy contributed to sulfur success, which over time has been included in other fungicide formulations, such as nanoparticles and dots, often displaying stronger antimicrobial activity and greater environmental friendliness [96, 98, 99]. Sulfur success is related to wide efficacy, high sensitivity, and selectivity to fungi, in addition, fungal resistance due to its employment is limited. Based on low toxicity to humans and animals and rapid dissipation, elemental sulfur is safe enough also, but hydrogen sulfide and sulfur dioxide are hazardous to humans and animals [97]. Common sulfur mode of action is not clearly understood; the most popular theory suggests involvement of sulfur permeability of fungal cell walls due to ergosterol content. Hence, sulfur passes into the cell cytoplasm and damages both cytochrome *b* and *c* and the proteic and non-proteic sulfhydryl groups implicated in the mitochondrial electron transport chain [97].

#### **4. Conclusions**

Although in recent years pomegranate is worldwide spreading due to high market value and nutraceutical properties, it is still considered a minor crop, so conventional and alternative fungicides registered for this crop are scarce. This entails important postharvest yield and economic losses mainly related to fungal diseases. Albeit losses are evident in the post-harvest phase, most of the infections start in the field during the blooming stage*,* so preharvest treatments are needed to control them. Remaining infections are caused by wound pathogens that attack fruit through injuries; these infections being reparteed along the whole processing chain imply a particular care of fruit from harvest to the retail. The effects of good agronomical practices, preharvest treatments during the blooming stage, fruit sorting along the production chain, optimal storage conditions, and good hygiene conditions decrease the incidence of postharvest pomegranate rots and extend commercial pomegranate availability. Alternative control means deeply supporting the reduction of postharvest pomegranate disease incidence defending human and animal health by both fungicide residues and fungal mycotoxins and ensuring a One Health approach to saving food waste production.

#### **Acknowledgements**

This work was conducted within PRIMA StopMedWaste "Innovative Sustainable technologies to extend the shelf-life of Perishable Mediterranean fresh fruit, vegetables, and aromatic plants and to reduce WASTE," which is funded by the Partnership for Research and Innovation in the Mediterranean Area (PRIMA), Project ID: 1556, a program supported by the European Union, and Euphresco Basics "Basic substances as an environmentally friendly alternative to synthetic pesticides for plant protection" projects. Authors aknowledge "Masseria Fruttirossi Srl" (Castellaneta, Taranto, Italy), for their collaboration in the research activities.
