Antifungal Activity of Squid Pen Chitosan Nanoparticles against Three Fungal Pathogens in Various Citrus Fruits In Vitro and In Vivo

Fungal infections have been considered a primary cause of the postharvest losses of citrus fruits. Therefore, it is necessary to find low-cost and high antifungal activity materials for preventing the decay of citrus fruits after harvest. In this study, squid chitosan nanoparticles (SCNs) were prepared from squid pen chitosan and used as a biofungicide against three citrus fungal pathogens in both in vitro and in vivo evaluations. The prepared SCNs had a mean size of ca. 56 nm and a high zeta potential of +98.7 eV with a narrow size distribution. At a range of 50–250 ppm, the SCN concentration of 200 ppm exhibited the highest activity in totally inhibiting the growth of Lasiodiplodia pseudotheobromae, Alternaria alternate, and Penicillium digitatum in in vitro tests where these fungi were isolated from symptomatic fruits and identified. Furthermore, after 12 days of incubation at 30 ± 0.2 °C and high relative humidity in in vivo studies, the infection area of the sample treated at 250 ppm completely suppressed fruit disease symptoms. The results demonstrate that prepared SCNs efficiently control postharvest citrus fruit diseases. These findings recommend applying SCNs as a potential candidate for citrus fruit storage.


Introduction
Citrus is an important fruit with a world production estimated at over 100 million tons per year [1]. In Vietnam, citrus is widely grown as a high economic fruit in the Northern and Southern provinces. However, citrus fruits have a low pH value, high moisture content, and many nutrients making them very susceptible to attack by pathogenic fungi [2]. So, the postharvest losses usually are high during the storage stage, and the loss rate is about 15-35% of the total output, which causes severe economic losses after harvest. It was reported that Lasiodiplodia pseudotheobromae, Alternaria alternate, and Penicillium digitatum were the main disease problems with blue mold, green mold, and watery rot and unexpected flavor during postharvest storage [3,4]. So far, an effective fungicide has mainly managed these fungi [5], but its intensive use may cause resistant isolates to develop and pose several risks for human and environmental health. Therefore, it is necessary to create safe alternatives for controlling postharvest rots [6].
Chitosan is a biodegradable, nontoxic, and abundant polymer [7,8]. Furthermore, it is well-known to have a broad spectrum of antimicrobial activities [9]. Chitosan has been reported to have high fungal activity against many phytopathogenic fungi [10,11], which cause several fungal plant diseases [12][13][14]. Recently, chitosan-based nanoparticles have been widely studied for various applications because of their small size, good biocompatibility and degradability, high permeability, high antimicrobial property, low cost, and facile preparation [15][16][17][18][19]. Practically, chitosan nanoparticles have affected and prevented the growth of fungi on citrus fruits, such as P. steckii and A. oryzae [17], C. albicans [20,21], A. fumigatus [22], and Xanthomonas oryzae pv. Oryzae [23]. It has been indicated that the antifungal activity of chitosan nanoparticles depended on its concentration, the molecular weight of base initial chitosan or functional groups added to the chitosan, and the target pathogen. However, these published works have mainly focused on the antifungal activities of chitosan nanoparticles prepared from shrimp chitosan [24][25][26]. So far, no investigations on the antifungal activity of squid chitosan nanoparticles against the fungal pathogens in citrus fruits have been reported.
Based on these above considerations, the present study aims to determine the antifungal activity of squid chitosan nanoparticles against Lasiodiplodia pseudotheobromae, Alternaria alternate, and Penicillium digitatum caused by the decay of postharvest citrus fruits; the fungi were isolated from symptomatic fruits and identified. The antifungal tests were firstly performed under in vitro conditions. In addition, the antifungal activity of SCNs was evaluated on the development of three fungi in postharvest citrus fruits under in vivo conditions to expand the potential application of SCNs.

Preparation of SCNs
Sodium tripolyphosphate (TPP, >99%, Aldrich) and acetic acid (CH 3 COOH, 99.6%, Merck) were used as received. Chitosan (M w of ca. 20 kDa, deacetylated degree of ca. 92%) was prepared in the laboratory [27]. SCNs were prepared from squid chitosan by the ionic gelation method based on our previous report [28]. Typically, 20 mL of TPP solution (0.05 wt.%) was added dropwise to 100 mL of chitosan solution (0.05 wt.% in 1.0% (v/v) acetic acid) under stirring at 1500 rpm and room temperature. After stirring for 10 min, CSNs were received by centrifugal, followed by washing with deionized water three times to remove free chitosan, TTP, and acetic acid. The CSN solutions were prepared by dispersing CSNs in DI water for characterization and antifungal tests.
The particle size and zeta potential were determined by a Nano Particle Analyzer SZ-100 (Horiba Scientific, Kyoto, Japan) at 25 • C and transmission electron microscopy (TEM, Philips, CM-120, Eindhoven, The Netherlands) at an acceleration voltage of 120 kV. The prepared chitosan particles had a mean size of about 56 nm and high zeta potential of +98.7 eV with a narrow size distribution, as shown in Figure 1.

Fruits Samples
The three kinds of citrus fruits considered in this study include Citrus maxima, Citrus reticulata sinensis, and Citrus reticulata Blanco. The nondiseased and diseased citrus fruits were collected at Hon Quan and Binh Long districts, and Dong Xoai city in Binh Phuoc province, Vietnam. The fruits were packed in a cool box and then transferred to the laboratory.

Pathogen Isolation and Identification
Small pieces were cut from infected fruits by a sterile scalpel blade for isolating and identifying the pathogen of blue mold, green mold, and watery rot diseases. These pieces were dipped in a 10 wt.% NaClO solution for 3 min and then washed with distilled water twice. The obtained samples were put in between two sterilized filter papers and dried. After that, the dried samples were placed on potato dextrose agar (PDA) containing a streptomycin sulfate (STS) at a ratio of STS/PDA 1:20 (mg/mL). The strain was grown at 30 ± 0.2 • C for 12 days [29]. Afterward, obtained spores were suspended in PDA medium at the same conditions. Finally, the identification of three isolated fungi was based on morphological characteristics as well as physiological and biochemical properties by the Sanger method by ABI 3130/3500 equipment in the company Nam Khoa. For further investigations, the identified colonies were maintained at 5 • C on a PDA medium.

Antifungal Test
In in vitro tests, various SCN solutions (50, 100, 150, 200, and 250 ppm) were mixed with the PDA. The obtained mixtures were sterilized at 121 • C for 20 min and poured into the Petri dishes. Growing fungal mycelial plugs (3 mm × 3 mm) were inoculated in the center of the dishes and incubated at 28 ± 0.2 • C for 23 days [29]. The colony diameters of the fungi were measured after culturing for 1, 2, and 7 days. PDA without SCNs was tested as the negative control [30].
In in vivo tests, healthy citrus fruits (Citrus maxima; Citrus reticulata × sinensis, and Citrus reticulata Blanco) of uniform maturity and size were selected, washed with tap water, and dipped in a 10% NaClO solution, rinsed with sterile water, and finally air dried under aseptic conditions. Then, each cleaned fruit was wounded at 3 mm deep and 5 mm wide by a sterile needle. Different concentrations of SCN solutions (0, 50, 100, 150, 200, and 250 ppm) were prepared and sprayed wholly through the cleaned fruit surface before adding 25 µL of isolated fungal suspension (10 6 CFU spores ml −1 ) into the wound using a micropipette [12]. Treated fruits were placed under aseptic conditions for 3 h. The treated fruits (5 fruit per treatment) were placed and sealed in plastic boxes containing a cup of water in order to maintain a high humidity. All treatments were incubated at 25 ± 0.2 • C [31,32]. The fruits were visually observed for fungal symptoms, and the percent of decay was calculated through lesion diameters (mm) that were measured by a digital micrometer daily until the fruits were destroyed entirely [12]. All the fruit treatments were performed twice.

Statistical Analysis
All experiments were repeated three times. Minitab determined significant differences with a statistical variation among the samples at p < 0.05.

Isolation and Identification of the Fungi from Citrus Fruits
Three fungi were isolated from diseased citrus fruits collected at four different locations in the Binh Phuoc province, Vietnam. Figures 2-4 show that the plate and fruit were injected with a spore fungal suspension and incubated at 30 • C, and the 16s rRNA gene sequences of these fungi were recorded identified. After 12 days of incubation, the colonies of Lasiodiplodia pseudotheobromae on the PDA medium were turquoise in color (Figure 2a), while the color of the Alternaria alternate and Penicillium digitatum colonies was dark brown (Figures 3a and 4a). All fungi developed very quickly after being injected with a spore fungal suspension, and the fruits were almost completely destroyed after 12 days, as shown in Figures 2b, 3b and 4b. In addition, the obtained fungal isolates were then identified by the Sanger sequencing method. These sequencing results were deposited on BLAST search and were indicated as being 100%, 99.88%, and 99.78% homologous with Lasiodiplodia pseudotheobromae (Figure 2c), Alternaria alternate (Figure 3c), and Penicillium digitatum, respectively (Figure 4c). The morphological and culture characteristics of the obtained fungi are similar to those of previous reports [4][5][6]11,31,32]. These identified fungi were kept at 5 • C on a PDA medium and used for further in vitro and in vivo tests.

Pathogenicity Test
In in vitro studies, the antifungal activity of SCNs against Lasiodiplodia pseudotheobromae ( Figure 5), Alternaria alternate (Figure 6), and Penicillium digitatum (Figure 7) was examined on a PDA medium using SCN concentrations of 50, 100, 150, 200, and 250 ppm and incubated at 28 ± 0.2 • C. PDA without SCNs was tested as the negative control. Three 3 days after the inoculation, the colony of three fungi in the control samples was observed in white mold (Figures 5b and 6b), and green mold (Figure 7b). In contrast, the SCNs significantly inhibited the growth and biomass of mycelium. The antifungal activity of SCNs may be explained by the fact that nanoparticles have a high surface area to volume ratio and unique chemical and physical properties, which increase their contact with microbes and the ability to permeate cells [33]. The potent inhibitory effect of chitosan on fungal growth was according to the damage of the plasma membrane of the mycelia and spores [32]. In addition, the higher the concentration used, the higher the antifungal activity observed in our tests. The concentration-dependent phenomenon was also reported in previous research [17,33,34], which may be due to the strong interactions between chitosan's positive charges and the cell's negatively charged membranes [35]. As can be seen from the figures, SCNs wholly suppressed the mycelium linear growth, and biomass tested at a concentration of 200 ppm. No significant growth of the fungi was observed at 250 ppm of SCNs.
In in vivo tests, three citrus fruits, including Citrus maxima; Citrus reticulata×sinensis, and Citrus reticulata were first injected with fungal suspensions of P. digitatum, L. pseudotheobromae, and A. alternate and then treated with SCNs at different concentrations. The samples were incubated at 25 ± 0.2 • C for a week under high relative humidity. Concentrationdependent inhibition of the mycelial growth of three fungi by SCNs was observed, in agreement with the behavior in in vitro tests. Table 1 and Figure 8 show that the disease reduction was enhanced by increasing the concentration up to 250 ppm. The SCNs treated at 200 ppm significantly reduced the growth of rot symptoms of citrus maxima caused by P. digitatum, L. pseudotheobromae, and A. alternate. However, the SCNs displayed a higher antifungal activity against L. pseudotheobromae than those of P. digitatum and A. alternate. Practically, the SCNs highly reduced the mold infection and the lesion diameter to 15.6% and 2.1 cm (P. digitatum), 1.7 cm (L. pseudotheobromae), and 2.3 cm (A. alternate) after a week of incubation, respectively compared with the control of 100% and 15.0 cm. Moreover, as can be seen in Figure 8, the control fruits appeared blighted, and there were water-soaked dark brown areas up to the seventh day of incubation. Conversely, no blight and rot symptoms were observed on the treated fruits at 250 ppm in all cases. These results are somewhat compatible with those found on antifungal tests in vitro.    The antifungal activity of SCNs against P. digitatum, L. pseudotheobromae, and A. alternate caused the decay on Citrus reticulata×sinensis fruits as presented in Table 2 and Figure 9. Compared to that of the citrus maxima tests, the SCNs showed weaker antifungal activity against three fungi. It may be explained that citrus maxima fruits and their peels contain large numbers of bioactive compounds, such as vitamins, minerals, dietary fibers, pectins, and active phytochemicals, including phytophenolics (e.g., flavanones, flavones, flavonols, and phenolic acids) [36]. The infection and the lesion diameter were 25.6% and 3.1 cm (P. digitatum), 4.7 cm (L. pseudotheobromae), and 2.3 cm (A. alternate) after being treated with the SCN concentration at 200 ppm for a week, while those of the control sample was 100% and 9.5 cm, respectively. It was not complete but the suppressed decay was still observed at the SCN concentration of 250 ppm for all fungi. The high antifungal activities of SCNs could be explained by the higher surface charge density (higher zeta potential), which provided them greater binding affinity for negatively charged fungal membrane [37].  9. The antifungal activity of SCNs against three fungi on Citrus reticulata sinensis after being treated for a week. Table 3 and Figure 10 show a high antifungal activity of the SCN against P. digitatum, L. pseudotheobromae, and A. alternate on Citrus reticulata Blanco. Similarly, the antifungal activity enhanced by increasing the SCN concentrations. By treating with an SCN concentration of 200 ppm for a week, the mold infection and the lesion diameter were 15.5% and 1.1 cm (P. digitatum), 2.7 cm (L. pseudotheobromae), and 1.2 cm (A. alternate), respectively compared with those of the control samples of 100% and 6.5 cm. When the SCN concentration was 250 ppm, the rot symptoms were not observed for all fungi, as shown in Figure 10. However, slight rot symptoms were observed in the sample incubated with the A. alternate suspension and treated with SCNs at 250 ppm. These findings are in agreement with those found in antifungal tests in vitro.

Conclusions
This study demonstrated the antifungal effectiveness of squid chitosan nanoparticles against Lasiodiplodia pseudotheobromae, Alternaria alternate, and Penicillium digitatum, which cause decay in the postharvest conservation of citrus fruits. SCNs exhibited a preservative capable of effectively controlling these common diseases in in vitro and in vivo tests as a biological fungicide. Practically, the samples treated with the SCN concentrations at 200 ppm (in vitro) and 250 ppm (in vivo) showed the best control of mold growth and biomass that causes diseases in citrus fruits. The findings suggest that the SCN suspension is a potential candidate to reduce the spoilage rate of decomposition by various diseases, resulting in expanding the storage time and shelf life of citrus fruits to meet export requirements. However, the antifungal mechanism of the SCNs against Lasiodiplodia pseudotheobromae, Alternaria alternate, and Penicillium digitatum as well as the toxicity test of the treated fruits need further study.