2.4.3. Antifungal Activity on Apples

Results from the trial carried out on apple fruits inoculated with *P. expansum* CECT 2278 evidenced that, three days post inoculation (Figure 7), "Nitric-extract" as such (ID02), at 75% (ID04) and at 50% significantly reduced rot severity in comparison with any other

treatment and controls. Five days post inoculation, only "Nitric-extract" as such (ID02) still significantly reduced rot severity (Figure 8).

**Figure 4.** Rot severity caused by *Penicillium digitatum* strain P1PP0 in orange (*Citrus* × *sinensis*) fruits cv. Valencia treated with water (ID01) or nitric-extract as such (ID02), 75% Nitric-extract (ID04), 50% Nitric-extract (ID06), 25% Nitric-extract (ID08) and respective controls (NaNO3 0.17 g/mL—ID03; NaNO3 0.17 g/mL diluted in sterile distilled water (sdw) at 75%—ID05; NaNO3 0.17 g/mL diluted in sdw at 50%—ID07; NaNO3 0.17 g/mL diluted in sdw at 25%—ID09) 5 days after inoculation. Values sharing the same letters are not statistically different according to Tukey's HSD test (*p* ≤ 0.05). Bars represent SD.

**Figure 5.** Rot severity caused by *Penicillium digitatum* strain P1PP0 in lemon (*Citrus* × *limon*) fruits cv. Femminello Siracusano treated with water (ID01) or Nitric-extract as such (ID02), 75% Nitricextract (ID04), 50% Nitric-extract (ID06), 25% Nitric-extract (ID08) and respective controls (NaNO3 0.17 g/mL—ID03; NaNO3 0.17 g/mL diluted in sterile distilled water (sdw) at 75%—ID05; NaNO3 0.17 g/mL diluted in sdw at 50%—ID07; NaNO3 0.17 g/mL diluted in sdw at 25%—ID09) 3 days after inoculation. Values sharing the same letters are not statistically different according to Tukey's HSD test (*p* ≤ 0.05). Bars represent SD.

**Figure 6.** Rot severity caused by *Penicillium digitatum* strain P1PP0 in lemon (*Citrus* × *limon*) fruits cv. Femminello Siracusano fruits treated with water (ID01) or Nitric-extract as such (ID02), 75% Nitric-extract (ID04), 50% Nitric-extract (ID06), 25% Nitric-extract (ID08) and respective controls (NaNO3 0.17 g/mL—ID03; NaNO3 0.17 g/mL diluted in sterile distilled water (sdw) at 75%—ID05; NaNO3 0.17 g/mL diluted in sdw at 50%—ID07; NaNO3 0.17 g/mL diluted in sdw at 25%—ID09) 5 days after inoculation. Values sharing the same letters are not statistically different according to Tukey's HSD test (*p* ≤ 0.05). Bars represent SD.

**Figure 7.** Rot severity caused by *Penicillium expansum* strain CECT 2278 in apple (*Malus domestica*) fruits cv. Braeburn treated with water (ID01) or Nitric-extract as such (ID02), 75% Nitric-extract (ID04), 50% Nitric-extract (ID06), 25% Nitric-extract (ID08) and respective controls (NaNO3 0.17 g/mL—ID03; NaNO3 0.17 g/mL diluted in sterile distilled water (sdw) at 75%—ID05; NaNO3 0.17 g/mL diluted in sdw at 50%—ID07; NaNO3 0.17 g/mL diluted in sdw at 25%—ID09) 3 days after inoculation. Values sharing the same letters are not statistically different according to Tukey's HSD test (*p* ≤ 0.05). Bars represent SD.

**Figure 8.** Rot severity caused by *Penicillium expansum* strain CECT 2278 in apple (*Malus domestica*) fruits cv. Braeburn treated with water (ID01) or Nitric-extract as such (ID02), 75% Nitric-extract (ID04), 50% Nitric-extract (ID06), 25% Nitric-extract (ID08) and respective controls (NaNO3 0.17 g/mL—ID03; NaNO3 0.17 g/mL diluted in sterile distilled water (sdw) at 75%—ID05; NaNO3 0.17 g/mL diluted in sdw at 50%—ID07; NaNO3 0.17 g/mL diluted in sdw at 25%—ID09) 5 days after inoculation. Values sharing the same letters are not statistically different according to Tukey's HSD test (*p* ≤ 0.05). Bars represent SD.

### **3. Discussion**

This study evaluated, for the first time, the potentialities of minimally processed shrimp wastes in the *in vitro* inhibitory activity on fungal and oomycete plant pathogens, and their effectiveness in controlling post-harvest rots caused by *Penicillium* spp. in citrus and apple fruits. To this aim, wastes from the shrimp species *Parapenaeus longirostris* were dried and grounded to result in a "dry-powder", which was further processed leading to four different extracts "Water-extract", "EtOAc-extract", "MetOH-extract" and "Nitricextract". Acid hydrolysis is mandatory for the mineralization of calcium-containing shrimp waste, and hydrolysis is commonly performed by hydrochloric, acetic, phosphoric, sulfuric, nitric and lactic acids. Nitric acid was selected as, among the above-mentioned acids, it has the slowest reaction kinetics [28], which allows for better digestion control. All these substances, "Water extract", "EtOAc-extract", "MetOH-extract" and "Nitric-extract, were analyzed to determine their composition in metabolites and phenolic compounds. Then, the "dry-powder", "EtOAc-extract", "MetOH-extract" and "Nitric-extract" were also preliminarily tested *in vitro*, in order to select the substance with the highest mycelial growth inhibitory activity. "Nitric-extract" was the most effective substance and was further investigated to determine its antifungal properties (in terms of MIC and MFC) and *in vivo* antifungal activity.

Results from the chemical analysis showed that all substances extracted from the shrimp waste were miscellaneous mixtures of a conspicuous number of metabolites and phenolic compounds. Interestingly, a high relative abundance of the 2-Hydroxyisocaproic, 3-(4-Hydroxyphenyl) propionic and 4-Aminobenzoic acids in "MetOH-extract", and of docosahexaenoic acid in "EtOAc-extract" were reported. Various studies reported fungicidal activity for these molecules when tested as pure substances; 2-hydroxyisocaproic acid was effective against *Candida* and *Aspergillus* species [29]; 3-(4-Hydroxyphenyl) propionic acid contains the hydroxyl group, which has been reported as one of the substance responsible for the antifungal activity of *Lactobacillus paracasei* [30]. Moreover, the para-aminobenzoic acid showed antibiotic activity toward *Staphylococcus aureus* [31]; a *Pseudomonas aeruginosa*bioconverted oil extract of docosahexaenoic acid was effective against the mycelial growth of several plant pathogens, including *Botrytis cinerea*, *Colletotrichum capsici*, *Fusarium oxys-* *porum*, *F. solani*, *Phytophthora capsici*, *Rhizoctonia solani* and *Sclerotinia sclerotiorum* [32]. However, in the present study, two extracts, "MetOH-extract" and "EtOAc-extract", containing a higher amount of the above-mentioned acids, showed no inhibitory activity on mycelial growth.

An additional interesting metabolite present in all substances was phenylalanine, which was also detected in high amount in "Water-extract" and "MetOH-extract". A recent study [33] reported that post-harvest treatments of mango, avocado and citrus fruits with phenylalanine induced resistance against infections caused by *Colletotrichum gloeosporioides*, *Lasiodiplodia theobromae* and *P. digitatum*, respectively, although *in vitro* tests carried out in the same study evidenced no inhibitory effects toward the same pathogens. Therefore, although lacking of fungicidal action, the "Water-extract" and "MetOH-extract", which showed a high amount of phenylalanine, could provide strong resistance induction properties to control post-harvest disease. It goes without saying that, since phenylalanine was also detected in "EtOAc-extract" and "Nitric-extract", these samples could also have resistance induction properties, as demonstrated for other extracts of natural origin [34]. This possibility assumes a particular significance of the extract "Nitric-extract", which was the only substance tested that demonstrated clear and strong *in vitro* antifungal activity as well as significant *in vivo* control of infective processes. Additional studies are, therefore, ongoing, to verify possible resistance induction properties of all the minimally processed shrimp wastes produced in this study. Quite interestingly, although the exoskeleton of shellfish is the main raw material for the extraction of chitosan, whose inhibitory activity on post-harvest fruit rots is well documented [35], this biopolymer was not present in the extracts examined in this study. As a consequence, it can be inferred that other substances are responsible for the antimycotic activity showed by the "Nitric-extract".

With reference to composition in phenolic compounds, analyses evidenced the presence, in all tested substances, of molecules whose antimicrobial activity is supported by a wide range of literature [2,36–44]. Some of these compounds have been also applied as eco-friendly alternatives to synthetic fungicides [1,45]. Among the phenolic compounds, the molecules that recurred in all analyzed substances were the benzoic, caffeic and pcoumaric acids and the vanillin. Benzoic and caffeic acids have important preservative properties that determine the inhibition of fungal growth [43,46]. Vanillin (4-hydroxy-3-methoxybenzaldehyde) is considered one of the most important additives used in the food industry; it is characterized by effective inhibitory activity toward a wide range of microorganisms, thus causing a delay in the growth of yeasts and fungi [36,40]. The p-coumaric acid (4-hydroxycinnamic acid), which, in "Nitric-extract", had the highest concentration, is the main phenolic acid contained in the peel of sweet oranges [44], and is well known for its efficacy in negatively affecting the growth of post-harvest pathogens, such as *Monilinia fructicola, Botrytis cinerea* and *Alternaria alternata* [2]. Interestingly, "Nitricextract" also reported the highest concentration of catechol (1-2-dihydroxybenzene) and the exclusive presence of dihydroferulic (3-(4-hydroxy-3-methoxyphenyl) propionic acid) and ellagic acids. Catechol shows significant activity in the control of *Fusarium oxysporum* and *Penicillium italicum* [38]. Dihydroferulic acid significantly inhibits the *in vitro* growth of *Saccharomyces cerevisiae, Aspergillus fumigatus* and *A. flavus* [39]. Moreover, ellagic acid, which possesses well-documented antibacterial activity [37], shows extraordinary antifungal effects toward *Botrytis cinerea* [41], as well as a significant growth inhibition of several fungal species belonging to the genera *Trichophyton* and *Candida* [42]. Finally, phenolic compounds are hypothesized to be, at least in part, responsible for the strong broad-spectrum antifungal activity shown by a pomegranate peel extract [34].

Overall, unlike the "Water-extract", "EtOAc-extract" and "MetOH-extract", "Nitricextract" results were characterized by p-coumaric acid and catechol, both present at high concentrations, and by the exclusive presence of the acids dihydroferulic and ellagic; these molecules could be, therefore, responsible for the antifungal activity of this extract. Synergetic action of some of the molecules detected in "Nitric-extract" also cannot be excluded. This effect has already been observed for the active components of extracts

from different natural matrices. This is the case, for example, of pomegranate, whose high biological value is recognized as being the result of the synergistic chemical action of the total phytoconstituents of the fruit rather than of single extracted components [47–49].

The quantity and quality of the molecules that were active (individually or in synergy) in determining the *in vitro* antifungal activity of the tested substances could also be related to the extraction process. By comparing the compositions of the three extracts, namely, "EtOAc-extract", "MetOH-extract" and "Nitric-extract", the three applied extraction processes had different efficiencies. The choice of the best solvent for the extraction of precise bioactive components from a specific matrix is a crucial aspect for reaching the expected qualitative and quantitative yield of the desired molecules in the final extract [34]. Examples of this aspect are provided by studies carried out on pomegranate extracts; Al-Zoreky [50] observed that the 80% methanolic extract was richer in polyphenols compared to hot water and diethyl ether extracts and, therefore, led to higher antimicrobial activity against pathogenetic bacteria. Tayel et al. [51] found that, regardless the concentration of specific bioactive components, a methanolic pomegranate peel extract was more effective than ethanol and water extracts in controlling *Penicillium digitatum*. In view of these aspects, it is quite surprising that, among the extracts, only "Nitric-extract" provided *in vitro* antifungal efficacy and, at the same time, neither "EtOAc-extract" nor "MetOH-extract" resulted in an inhibitory effect on mycelial growth.

Results from the *in vitro* preliminary test together with those from MIC and MFC tests overall demonstrated that the pathogens mostly affected by "Nitric-extract" were *Pl. tracheiphilus* Pt 2, *C. gloeosporioides* C2 and *Ph. Nicotianae*—both tested isolates. *Plenodomus tracheiphilus* is the causal agent of 'mal secco, one of the most destructive diseases affecting lemon trees [52]. Because of the vascular propagation of the pathogen in all aerial parts of the infected plant, the management of the disease is complicated [53]. It is commonly carried out by the pruning of diseased twigs, withered shoots and suckers, followed by the spraying of the canopy with copper-based fungicides, which can reduce the occurrence of new *Pl. tracheiphilus*-infections. However, many copper-based treatments are not cost effective in commercial lemon groves, and also represent a significant source of environmental pollution [53]. Another copper-susceptible pathogen is *C. gloeosporioides*, the causative agent of anthracnoses in several fruits and vegetables [54] as well as of twig and shoot dieback in citruses [55]. *Phytophthora nicotianae* is very likely the most widespread and destructive *Phytophthora* species worldwide, affecting a very wide host range of more than 255 plant species [8,56,57]. Control strategies may be different depending on the specific situation, although the pathogen is markedly sensitive to Metalaxyl and Fosetyl Al, fungicides which are commonly used for controlling plant diseases affecting roots, collars and stems [56]. Results from this study pose "Nitric-extract" as a promising alternative to the use of conventional fungicides in controlling not only *Pl. tracheiphilus*, *C. gloeosporioides* and *Ph. nicotianae*, but all pathogens tested in the present study. To this aim, further investigations are needed to evaluate the phytotoxicity, if any, of the extract, its attitude to systemic translocation, which is of particular relevance in the case of tracheomycoses, such as 'mal secco' caused by *Pl. tracheiphilus,* as well as the most effective method of application, e.g., by drenching, spraying or incorporation into fruit coatings, which also depends on the type of disease.

As a preliminary step towards the application of "Nitric-extract" to control plant diseases, its effectiveness was tested *in vivo* against molds caused by *Penicillium* species in orange, lemon and apple fruits, which are the most economically important post-harvest diseases affecting these fruits [58,59]. Post-harvest molds of citrus and apple fruits are traditionally controlled by the application of highly effective chemicals, such as imidazole and bendimidazole (thiabendazole) fungicides [60,61]. More recently, as a consequence of the selection of imidazole- and bendimidazole-resistant strains of *Penicillium*, several other synthetic fungicides, including azoxystrobin, fludioxonil, cyprodinil and pyrimethanil, have been proposed as alternatives for the chemical control of these post-harvest fruit

diseases [7,60–63]. Like imidazoles and benzimidazoles, all these fungicides are effective at relatively low doses but are characterized by a high acute toxicity [64–69].

There is boundless literature evaluating the efficacy of alternative strategies to the use of conventional synthetic fungicides for the control of postharvest molds of *Penicillium* species [70–78]. A novelty in the present study is the *in vivo* control of *Penicillium* spp. using a natural substance that is derived from minimum waste treatment.

Overall, treatments with "Nitric-extract" at the highest concentrations were the most effective in positively affecting the reduction of rot severity in all tested fruits. Additionally, an interesting weak positive effect was also observed in all control treatments, including NaNO3 in water solution (ID03, ID05, ID07 and ID09), although, *in vitro,* they were not effective in inhibiting the mycelial growth of all pathogens included in this study. As already observed for other inorganic salts [74], it cannot be excluded that the *in vivo* effectiveness of NaNO3 was not the consequence of direct antifungal activity, but the possible result of the triggering of defense mechanisms in fruits. Further tests are ongoing to verify this hypothesis.

The results from the treatments with "Nitric-extract" demonstrated that three days post-treatment, "Nitric-extract" as such determined a significant reduction of rot severity over any other treatment in all fruits (oranges, lemons and apples). Additionally, "Nitricextract" at 75% significantly reduced rot severity in lemon and apples over controls; "Nitricextract" at 50% had a significant effect over controls only on apples; the concentration of 25% was as effective as the controls in all fruits. Five days post-treatment, "Nitricextract" as such still maintained significant effects in reduction of rots only in lemons and apples; "Nitric-extract" at 75% demonstrated significant reduction of rot severity only in lemons; finally, "Nitric-extract" at 50% and at 25% were as effective as the controls in all tested fruits. Overall, the results showed an interesting performance of "Nitricextract" in controlling postharvest mold caused by *Penicillium* spp., although the effective dose was much higher than that of traditional synthetic fungicides [7], and, as with other eco-friendly alternatives to synthetic fungicides [7,74], its use may not provide complete protection. A successful strategy for improving its efficacy or reducing fungicide residues from post-harvest fruit treatments could include the use of "Nitric-extract" in a mixture with conventional fungicides applied at a concentration lower than the standard dose, or by incorporating it in a fruit coating.

This study is part of a research program aimed at exploring the antifungal activity of extracts obtained from minimally processed shrimp wastes and their possible application in agriculture. The antifungal activity shown *in vitro* against a wide range of fungal and oomycete pathogens by the nitric extract appears promising and could be exploited in the context of new strategies for the management of plant diseases caused by these pathogens. *In vivo* preliminary results suggest a possible use of nitric extract for postharvest treatments against citrus and apple molds caused by *Penicillium* species. To this aim, and to optimize the efficacy of treatments, next steps will be to define the methods and times of application. In this study, nitric extracts were applied to fruits 24 h after inoculation with the pathogen, indicating curative efficacy. However, an additional aspect that would merit further investigation is whether nitric extract, like other natural substances, is able to elicit plant defense mechanisms against infections by pathogens. In this case, the treatment of fruits with this extract might also have preventive efficacy against infections by molds. Regarding this, it cannot be ruled out that the other shrimp waste extracts, which, in preliminary *in vitro* tests did not show inhibitory activity on the mycelium growth, may also be effective *in vivo* acting as resistance elicitors. Last but not least, a prerequisite for the use of nitric extracts of shrimp waste to prevent post-harvest molds is to evaluate if the treatment leaves unpleasant odors on the fruits. A sensory analysis using an electron nose is planned to clarify this aspect. Although the effective dose of nitric shrimp waste extract is far higher than the label dose of synthetic fungicides used to control post-harvest fruit diseases, this extract, as a natural substance, could be an interesting alternative to

traditional post-harvest chemical treatments, as it is more eco-friendly and far less toxic than synthetic fungicides.
