The GRAS Salts of Na2SiO3 and EDTA-Na2 Control Citrus Postharvest Pathogens by Disrupting the Cell Membrane

Sodium silicate (Na2SiO3) and ethylenediaminetetraacetic acid disodium salt (EDTA-Na2) are inorganic salts classified as ‘Generally Recognized as Safe’ (GRAS) compounds with great advantages in controlling various pathogens of postharvest fruits and vegetables. Here, we determined the median effective concentration (EC50) of Na2SiO3 (0.06%, 0.05%, 0.07% and 0.08%) and EDTA-Na2 (0.11%, 0.08%, 0.5%, and 0.07%) against common pathogens affecting postharvest citrus fruit, including Penicillium digitatum, Penicillium italicum, Geotrichum citri-aurantii, and Colletotrichum gloeosporioides. Na2SiO3 and EDTA-Na2 treatments at the EC50 decreased the spore germination rate, visibly disrupted the spore cell membrane integrity, and significantly increased the lipid droplets (LDs) of the four postharvest pathogens. Moreover, both treatments at EC50 significantly reduced the disease incidence of P. italicum (by 60% and 93.335, respectively) and G. citri-aurantii (by 50% and 76.67%, respectively) relative to the control. Furthermore, Na2SiO3 and EDTA-Na2 treatment resulted in dramatically lower disease severity of the four pathogens, while also demonstrating no significant change in citrus fruit quality compared with the control. Therefore, Na2SiO3 and EDTA-Na2 present a promising approach to control the postharvest diseases of citrus fruit.


Introduction
Citrus fruits such as orange, lemon, lime, mandarin orange, and pomelo, which are cultivated in more than 130 countries, are in high demand worldwide [1]. Citrus fruits are a rich source of vitamin C, dietary fiber, phenolic acids, and other bioactive compounds [2,3]. According to FAO, global citrus production exceeded 147 million tons in 2022, playing an essential role in the development of the global agricultural economy (http://www.fao.org/faostat/en/?#data/QC, accessed on 12 December 2020). However, various citrus diseases cause huge economic losses and seriously affect the development of the citrus industry [4]. Penicillium digitatum, Penicillium italicum, and Geotrichum citriaurantii are three pathogens responsible for causing the greatest post-harvest loss of citrus fruit. They can infect the fruit through wound during transport, packing, and post-harvest storage [5]. Anthracnose is another serious citrus disease caused by Colletotrichum spp., and can infect citrus fruit not only in the field but also during post-harvest storage, which has serious negative impacts on the yield and quality of citrus fruit [6,7]. Overall, these post-harvest losses were estimated to reach 20-50% in developing countries and 10-20% in developed countries in the fresh and juice citrus industries [8].
Chemical fungicides, including thiabendazole, imazalil, fludioxonil, pyrimethanil, and sodium o-phenylphenate, have been used for continuous control of citrus post-harvest

High-Throughput Screening of GRAS Salts for Citrus Postharvest Diseases
There are hundreds of GRAS salts that can be used for controlling citrus post-har diseases. Here, we first collected the data of all GRAS salts according to the United St Food and Drug Administration (US FDA), and then screened the most appropriate GR salts of high solubility in water, steady chemical property, no unpleasant smell, no sti lation of the skin, no harm, no toxicity, and wide availability at relatively low costs.

High-Throughput Screening of GRAS Salts for Citrus Postharvest Diseases
There are hundreds of GRAS salts that can be used for controlling citrus post-harvest diseases. Here, we first collected the data of all GRAS salts according to the United States Food and Drug Administration (US FDA), and then screened the most appropriate GRAS salts of high solubility in water, steady chemical property, no unpleasant smell, no stimulation of the skin, no harm, no toxicity, and wide availability at relatively low costs.

Evaluation of Antifungal Activity In Vitro
The antifungal activity of GRAS salts against P. digitatum, P. italicum, G. citri-aurantii, and C. gloeosporioides was evaluated using the Oxford cup technique with some modifications [31]. Briefly, 100 µL spore suspension (1.0 × 10 6 CFU mL −1 ) of these pathogens were added into PDA medium at 40 • C to avoid scalding to death. Then, sterilized Oxford cups were inserted gently in the center of the PDA medium, and 250 µL GRAS salt solution (1%) was dropped. The diameter of the inhibition zone (IZ) was measured after 5 d of incubation at 25 • C.
After selection of GRAS salts with obvious IZ, the inhibition rate of every GRAS salt at 1% was determined and calculated. Then, the EC 50 of GRAS salts with a broad spectrum of antifungal function to the four citrus post-harvest pathogens was detected, and the inhibitory effect of these salts on the growth of hyphae was determined with the agar dilution method as described in a previous study [32].
The PDA medium contained different concentrations (0, 0.02%, 0.04%, 0.06%, 0.08%, and 0.16%) of sodium silicate (Na 2 SiO 3 , SS), and ethylenediaminetetraacetic acid disodium salt (EDTA-Na 2 , EA) was prepared into final concentrations of 0, 0.03%, 0.06%, 0.12%, 0.24%, and 0.48%. Then, 2.5 µL of fresh spore suspension (10 6 CFU mL −1 ) was deposited in the center of the culture dish and incubated at 25 • C in a constant-temperature incubator (Bo er Si, BES600SH). Finally, each measurement consisted of three 90-mm Petri dishes. The logarithm of the concentration of 10 was used as the abscissa and the probability value corresponding to the inhibition rate was used as the ordinate to obtain the regression equation and the correlation coefficient R 2 for each test concentration. The EC 50 value and its 95% confidence interval were calculated based on the probability value corresponding to 50% inhibition [33].

Effect of Na 2 SiO 3 and EDTA-Na 2 on Spore and Hyphal Morphology
First, 50 µL of 10 6 CFU mL −1 fresh spore suspension (P. Digitatum, P. italicum, G. citri-aurantii, and C. gloeosporioides) was prepared and added into 50 mL PDB. After shaking for 12 h at 25 • C and 150 r min −1 , Na 2 SiO 3 and EDTA-Na 2 were added into the PDB medium to the final EC 50 concentration, respectively. The PDB without the addition of any pathogen was used as the negative control. All 12 samples continued to be incubated for 12 h. Finally, the morphology of spores and hyphae was observed via light microscopy (Nikon Eclipse E100, Nikon corporation, Tokyo, Japan).

Effect of Na 2 SiO 3 and EDTA-Na 2 on Spore Germination
The spore suspension of every sample was prepared using the same method as in the previous section. The germination of spores was recorded via light microscopy. The rate of spore germination (RSG) = (the number of germination spore/the number of total spores) × 100%.

Cell Wall Integrity, Cell Membrane Integrity, and Lipid Droplet Accumulation Assays
The spore suspension and the PDB medium with Na 2 SiO 3 and EDTA-Na 2 were prepared as described in Section 2.4 above. Then, 100 µL spore suspensions (1 × 10 6 CFU mL −1 ) of four citrus post-harvest pathogens were added in the prepared PDB medium, and these suspensions were shaken for 8 h at 25 • C, centrifuged at 8000 r min −1 for 10 min [34], and the supernatant was removed.
Cell wall integrity assay: Calcofluor white (CFW, Coolaber, Beijing, China) was used to show the cell wall because of the special etch reactive. The cell wall integrity of spores and hyphae was determined with a modified protocol [34]. Spores were dyed with 10 µL CFW containing 10% KOH [32], and then observed using a fluorescence microscope (Nikon Eclipse 90i).
Cell membrane integrity assay: The cell membrane integrity of spores and hyphae was determined with a modified method [32]. Spores and hyphae were collected and dyed with 10 µg mL −1 propidium iodide (PI, (Coolaber Technology Co., Ltd., Beijing, China)). They were stained at 37 • C for 30 min [35] and the floating dye was washed off with PBS for three times. All samples were observed using a fluorescence microscope (Nikon Eclipse 90i).
Lipid droplet (LD) accumulation assay: LDs of all samples were observed according to the modified protocol developed in a previous study [32]. Fresh spores and hyphae were dyed with the Nile Red solution (Coolaber, Beijing, China) for 3-6 min, then washed twice with 0.1 × PBS buffer. LDs were observed using a fluorescence microscope.

Release of Cell Components
Detection of the cell components for these citrus post-harvest pathogens after EC 50 Na 2 SiO 3 and EDTA-Na 2 treatment was performed with a modified method [34]. The spore suspensions were prepared and treated as described in Section 2.4 above; however, all 12 samples only continued to be incubated for 2 h after adding Na 2 SiO 3 and EDTA-Na 2 at the EC 50 . Finally, the fungal suspensions were centrifuged at 8000 r min −1 for 10 min, and then detected at 260 nm using a UV-spectrophotometer (UV-1500, AOE INSTRUMENTS (Shanghai, China) Co., Ltd.).

Fruit Decay Test
Healthy unwounded citrus fruit were soaked in 2% (v/v) sodium hypochlorite for 2 min and air-dried after washing twice with distilled water. Then, three wounds (4 mm deep and 4 mm wide) were created at equal spaces in the equator with an inoculating needle. The optimal concentrations for in vivo experiments were determined according to the in vitro results. Each wound of the fruit was inoculated with 20 µL spore suspension (10 7 CFU mL −1 ) of the four citrus post-harvest pathogens, respectively [35]. After air-drying of the wounds, the fruits were dipped in 0 (control) and EC 95 Na 2 SiO 3 and EDTA-Na 2 solution for 2 min and then stored in a plastic crisper with wet tissue at room temperature. The disease incidence and lesion size were determined after 7 d. The disease incidence (DI) = (decaying wounds number/total wounds number) × 100%. Every experimental group included five fruits, and was repeated three times.

Fruit Quality Evaluation In Vivo
To verify the practical value of Na 2 SiO 3 and EDTA-Na 2 in the citrus industry, longterm storage experiments were performed in a storehouse in Zhijiang city, Hubei province in China. The fruits were soaked in EC 95 Na 2 SiO 3 and EDTA-Na 2 solution for 2 min, with tap-water as the control. Treated fruits were stored for three months under natural conditions, during which the fruit quality (fruit weight-loss rate, soluble solid, titration acid contents, and VC) was constantly monitored. Each treatment contained 300 fruit in three replicates.

Statistical Analysis
All experiments were conducted with a completely randomized design and repeated three times. SPSS 26.0 statistical software was used to analyze the data. The results were reported as the average value of the three replicates. Moreover, the standard error and significant differences were calculated with one-way ANOVA followed by Duncan's Multiple Range test (p < 0.05).

Digital Photography of Na 2 SiO 3 and EDTA-Na 2 against Four Postharvest Pathogens
As shown in Figure 2, Na 2 SiO 3 and EDTA-Na 2 inhibited the hyphal growth of four pathogens in a concentration-dependent manner ( Figure 2). Table 2 shows that the EC 50 of Na 2 SiO 3 was 0.06%, 0.05%, 0.07%, and 0.08%, and that of EDTA-Na 2 was 0.11%, 0.08%, 0.5%, and 0.07% for G. citri-aurantii, P. digitatum, P. italicum, and C. gloeosporioides, respectively.    As shown in Figure 3, the number of spores decreased after Na 2 SiO 3 and EDTA-Na 2 treatment (red arrow); however, non-treated hyphae of the four pathogens showed a normal morphology with clear boundaries and loose arrangement. The hyphae were tightly aggregated and adhered to each other under Na 2 SiO 3 and EDTA-Na 2 treatment. Moreover, Na 2 SiO 3 and EDTA-Na 2 treatment caused abnormal bulges and swelling on the fungal hyphae (blue arrow and blue numbers of 1, 2, 3, 4, 5, 6, 7 and 8). In general, Na 2 SiO 3 and EDTA-Na 2 treatment obviously disrupted the fungal spores and hyphae of P. digitatum, P. italicum, G. citri-aurantii, and C. gloeosporioides.

Effect of Na 2 SiO 3 and EDTA-Na 2 on Cell Wall Integrity and Lipid Droplet Accumulation
As shown in Figure 5A, the blue fluorescence brightness of spore cell walls of the four pathogens generally remained unchanged after Na 2 SiO 3 and EDTA-Na 2 treatment compared with that of the control in CFW-staining assay, suggesting that Na 2 SiO 3 and EDTA-Na 2 treatments caused no obvious damage to the cell wall integrity.

Effect of Na2SiO3 and EDTA-Na2 on Cell Membrane Integrity
As shown in Figure 6A, Na2SiO3 and EDTA-Na2 treatments led to clear red fluorescence compared with the control, indicating that the two treatments disrupt the hyphal cell membrane of pathogens. The accumulation of LD in spores and hyphae was observed using LD staining with Nile red solution ( Figure 5B). The fluorescence intensity of the experiment group was obviously higher than that of the control, because of a significant increase in LD biogenesis under Na 2 SiO 3 and EDTA-Na 2 treatments.
3.6. Effect of Na 2 SiO 3 and EDTA-Na 2 on Cell Membrane Integrity As shown in Figure 6A, Na 2 SiO 3 and EDTA-Na 2 treatments led to clear red fluorescence compared with the control, indicating that the two treatments disrupt the hyphal cell membrane of pathogens.

Effect of Na2SiO3 and EDTA-Na2 on the Nucleic Acid Leakage
The nucleic acid concentration was determined to further confirm the disruption of cell membrane integrity. The nucleic acid concentration increased under Na2SiO3 and EDTA-Na2 treatments. As shown in Figure 6B, the OD260 values of P. digitatum, P. italicum, G. citri-aurantii, and C. gloeosporioides were 0.037, 0.067, 0.28, and 0.01 after Na2SiO3 treatment, and 0.022, 0.034, 0.019, and 0.006 after EDTA-Na2 treatment, respectively, which were all apparently higher than those of the control (0.017, 0.023, 0.008, and 0.003, respec-

Effect of Na 2 SiO 3 and EDTA-Na 2 on the Nucleic Acid Leakage
The nucleic acid concentration was determined to further confirm the disruption of cell membrane integrity. The nucleic acid concentration increased under Na 2 SiO 3 and EDTA-Na 2 treatments. As shown in Figure 6B, the OD 260 values of P. digitatum, P. italicum, G. citriaurantii, and C. gloeosporioides were 0.037, 0.067, 0.28, and 0.01 after Na 2 SiO 3 treatment, and 0.022, 0.034, 0.019, and 0.006 after EDTA-Na 2 treatment, respectively, which were all apparently higher than those of the control (0.017, 0.023, 0.008, and 0.003, respectively). These results indicated that Na 2 SiO 3 and EDTA-Na 2 disrupted the cell membrane, resulting in a massive leakage of nucleic acid.

Fruit Quality
As shown in Table 3, EC50 Na2SiO3 and EDTA-Na2 treatments resulted in lower weight-loss rates of fruit than the control treatment, and significantly better fruit quality in terms of soluble solid, titratable acid, and VC, suggesting that EC50 Na2SiO3 and EDTA-Na2 treatments had no negative impact on the fruit quality and could be used as preservatives to replace traditional chemicals.

Fruit Quality
As shown in Table 3, EC 50 Na 2 SiO 3 and EDTA-Na 2 treatments resulted in lower weight-loss rates of fruit than the control treatment, and significantly better fruit quality in terms of soluble solid, titratable acid, and VC, suggesting that EC 50 Na 2 SiO 3 and EDTA-Na 2 treatments had no negative impact on the fruit quality and could be used as preservatives to replace traditional chemicals.

Discussion
In previous studies, some GRAS salts were found to be capable of effectively controlling various post-harvest diseases of fruits and vegetables [36]. For example, 2% PS, 2% SB, and 2% PSi could control anthracnose severity [7]; sodium dehydroacetate [37] and polyhexamethylene biguanide (PHMB) [38] could effectively reduce citrus sour rot; and cinnamic acid (CA) could decrease the incidence of blue mold caused by Penicillium italicum in "Orah" mandarin during storage [39]. However, there have been no reports on GRAS salts with broad-spectrum inhibitory effects and their inhibition mechanism on the growth of citrus post-harvest pathogens. The Na 2 SiO 3 and EDTA-Na 2 reported in this study may provide more options for the prevention and control of citrus post-harvest diseases.
In fact, Na 2 SiO 3 , an inexpensive GRAS salt, has been widely used to control the postharvest diseases of muskmelon and grape [40]. However, there has been no study and application of Na 2 SiO 3 in controlling citrus post-harvest diseases. The in vitro experiments in this study showed that the hyphal growth and spore germination of four pathogens could be almost completely inhibited by Na 2 SiO 3 at a concentration of 1%, which is lower than that of PS, SB, and Psi (2%) used for controlling C. gloeosporioides [7]. Na 2 SiO 3 treatment significantly reduced the disease incidence of blue mold and sour rot in citrus fruit compared with the control, as well as decreased the disease severity of P. italicum, P. digitatum, G. citri-aurantii, and C. gloeosporioides. It has been reported that Na 2 SiO 3 increases the resistance of muskmelon and grape fruit against post-harvest diseases by activating reactive oxygen species metabolism and phenylpropanoid pathway, thereby maintaining the post-harvest quality of fruit [40]. This study demonstrated that Na 2 SiO 3 decreased the incidence and disease severity of citrus postharvest diseases by inhibiting the hyphal growth of pathogens, providing a theoretical foundation for the application of Na 2 SiO 3 to control citrus postharvest diseases as an important alternative.
EDTA-Na 2 is a stable food additive used in the food industry, but there are no reports about its application in the control of post-harvest diseases in fruits and vegetables. In this study, we demonstrated for the first time the antifungal ability of EDTA-Na 2 to control citrus post-harvest pathogens in vitro. Notably, the combination of EDTA-Na 2 and Na 2 SiO 3 decreased the sour rot incidence to 0 in vivo, as well as significantly reduced the lesion diameter of green mold, blue mold, and anthracnose, indicating that the combination has a better inhibitory effect on the pathogen than EDTA-Na 2 and Na 2 SiO 3 alone.
The cell wall plays an important role in maintaining the morphology and integrity of the cells [35,36,41]. In this study, the CFW and PI staining results revealed that some spores had no blue fluorescence ( Figure 5A) and all hyphae had more visible red fluorescence ( Figure 6A) after EDTA-Na 2 and Na 2 SiO 3 treatments compared with the control, suggesting that the treatments have nearly no effect on the cell wall, but disrupt the cell membrane integrity of the post-harvest pathogens. In addition, EC 50 Na 2 SiO 3 and EDTA-Na 2 treatments increased the OD 600nm , suggesting that the treatment might severely disrupt the cell membrane integrity and increase the membrane permeability, leading to nucleic acid leakage, which is consistent with the results obtained from PI staining. Moreover, Na 2 SiO 3 treatment resulted in much higher OD 600nm values of P. digitatum, P. italicum, G. citri-aurantii, and C. gloeosporioides than EDTA-Na 2 treatment, indicating that Na 2 SiO 3 causes further damage to the cell membrane. Notably, Na 2 SiO 3 caused the most significant damage to the cell membrane of G. citri-aurantii ( Figure 6B).
LDs are involved in regulating the balance of lipid metabolism by changing the LD size and number [42,43]. Furthermore, in a previous study, a significant increase in LD biogenesis was observed under rapamycin treatment in Magnaporthe grisea, Botrytis cinerea, Fusarium oxysporum, Fusarium annularis, Alternaria alternaria, and Fusarium graminearum [32]. In this study, clearly visible green fluorescence was observed under Na 2 SiO 3 and EDTA-Na 2 treatments, indicating that Na 2 SiO 3 and EDTA-Na 2 induce the accumulation of LDs of P. digitatum, P. italicum, G. citri-aurantii, and C. gloeosporioides. These results indicate a potential future research direction to study the inhibition mechanism of GRAS salts against citrus post-harvest pathogens.
During citrus post-harvest storage, fruit quality traits, including cumulative weightloss rate, soluble solid, and titratable acid, are usually determined before and after treatment to measure the applicability of antifungal agents [44]. So far, studies have confirmed that no GRAS salt would impair the fruit quality when used to control post-harvest decay in fruit [35,39]. In our previous study, KCl (K + ) was found to be capable of controlling sour rot with decreasing weight-loss rate during a 90-day storage period [30]. Moreover, US FDA and the European Food Safety Authority (EFSA) have exempted the residue detection of GRAS salts in all agricultural commodities [5,24]. Therefore, considering our in vitro and in vivo results, EDTA-Na 2 + Na 2 SiO 3 treatment might present an effective approach to control the post-harvest diseases of citrus fruit.

Conclusions
The two GRAS salts, Na 2 SiO 3 and EDTA-Na 2 , can alter the microstructure of spores and hyphal cell membrane and increase the cell membrane permeability, resulting in the leakage of nucleic acid and synergistic inhibition on the hyphal growth and spore germination of P. digitatum, P. italicum, G. citri-aurantii, and C. gloeosporioides. Moreover, the EC 50 Na 2 SiO 3 and EDTA-Na 2 treatment conspicuously induced LD accumulation and reduced both disease incidence and disease severity without posing a negative impact on the fruit quality. These findings indicate Na 2 SiO 3 + EDTA-Na 2 treatment as a promising approach in controlling the post-harvest diseases of citrus fruit.