*3.3. Antimicrobial Activity on Stored Fruits and Vegetables: In Vivo Assays*

The results of in vivo antimicrobial tests against the *B. cinerea* by using CH on kiwifruits, are summarized in Figure 8. Until the 25th day after artificial inoculation, kiwifruits treated with chitosan hydrochloride showed a lower level of disease severity with respect to the control kiwifruits and to those treated with chemical compounds, both at external and internal levels (Figure 8).

**Figure 8.** Gray mold severity on kiwifruits artificially inoculated with *B. cinerea* after treatment with the fungicide fenhexamid or CH. Negative controls were not inoculated. For each evaluation date, columns with different letters are significantly different according to Tukey's HSD test (*p* = 0.01). Green histograms related to negative controls (only sterile distilled water, SDW) are absent because non-microbial growth was recorded in this thesis. Significant differences among the bars are marked with different letters; if there is no significant difference among the bars they get the same letter.

The kiwifruits, inoculated with this fungal pathogen, but not protected by any chemical product, started showing the typical gray mold at the petiole end, where the inoculum was deposited, starting from day 10. After that, the tissue rotting continued to expand reaching, after 25 days, an average of 3.2 in the symptom scale, which corresponds to about 70% of the entire surface of the fruit. The initial symptoms on kiwifruits treated with chitosan was slightly delayed, appearing around the 15th day but, interestingly, the rotting process was limited and was markedly less than those observed on kiwifruit controls and to those on kiwifruits submitted to chemical treatment.

The in vivo antimicrobial assays of the CH coating applied on lettuce surfaces with respect to *P. c.* subsp. *carotovotum* bacterial plant pathogen were investigated. The results, after seven days (data not shown), demonstrated an important reduction of damage (value 2–3), induced by chitosan hydrochloride-based coating. The damage, in the presence of the active coating, appeared (see Figure 9), in fact, much less severe that those recorded for the controls (only inoculated by *P. c.* subsp. *carotovotum*, 10<sup>6</sup> CFU/mL) even if the resulted in being much less effective with respect to the damage level developed on/in the samples treated with chemical compounds (value 1–2).

All the results obtained by in vitro and in vivo antimicrobial tests were statistically significant. On the basis of our evidence, the selected grade of chitosan hydrochloride, able to form a stable solution in a green solvent as water and to form a homogeneous coating on food, could be preventively applied to prolong the postharvest shelf-life of important fresh products. Considering the high concentrations used for fungal and bacterial artificial inoculations (1 × <sup>10</sup><sup>6</sup> conidia/mL and 1 × 106 CFU/mL, respectively) it is reasonable to consider the effectiveness of the chitosan hydrochloride-based coating against these plant pathogens up to five days on Romaine lettuce and up to 20 days on kiwifruits. Mechanisms by which chitosan hydrochloride coating reduced the decay of lettuce and kiwifruits with respect to *P. c.* subsp. *carotovorum* and to *B. cinerea*, not even studied here in depth, seems to be related to its bacteriostatic/fungistatic properties. Until now, antimicrobial activity of chitosan has been

widely studied against clinically-important microorganisms; this can be considered a new contribution with respect to dangerous foodborne (bacteria and fungi) plant pathogens as other active principles of natural origin recently resulted in being able to control plant pathogens in greenhouses and in open fields [38–40]. Finally, it is well known that gray mold of kiwifruits is mainly caused by latent or wound infections that are produced in the orchard [41]; thus, effective postharvest control means need to provide curative activity (the treatment should be applied after the pathogen inoculation). Here the potentiality of the chitosan hydrochloride treatment, as a preventive strategy, in contrast to *B. cinerea*, was considered and studied to reduce further treatments. Future research activities will be also addressed for curative activity.

**Figure 9.** In vivo symptoms (external and internal damages) developed after five days due to artificial inoculation by *Pectobacterium carotovorum* subsp. *carotovorum* (DSM 30184 [Leibniz Institute German Collection of Microorganisms and Cell Cultures, Germany]) bacterial plant pathogen on Romaine lettuce after preventive treatments by chitosan hypochloride and chemical compound (Sodium hypochlorite) respect to the control thesis (treated only by *P. c.* subsp. *carotovorum* and, as control negative, only by SDW). (**a**) Control positive: *P. c.* subsp. *carotovorum* bacterial plant pathogen at 1 <sup>×</sup> <sup>10</sup><sup>6</sup> CFU/mL; (**b**) Control negative, SDW; (**c**) Chitosan hydrochloride (1 g/L) solution; and (**d**) Sodium hypochlorite (1 mL/L) solution (*p* = 0.01). Chitosan hydrochloride and Sodium hypochlorite (Sigma-Aldrich®, St. Louis, MO, USA).
