Fe2+ protects postharvest pitaya (Hylocereus undulatus britt) from Aspergillus. flavus infection by directly binding its genomic DNA.

Aspergillus flavus (A. flavus) is a postharvest fungus, causing pitaya fruit decay and limiting pitaya value and shelf life. However, safer and more efficient methods for preventing A. flavus contamination for pitaya fruit remain to be investigated. In this study, we successfully proved exogenous Fe2+ could inhibit A. flavus colonization in pitaya fruit and extend pitaya's shelf life after harvest. Moreover, gel electrophoresis, CD analysis and Raman spectrum tests revealed Fe2+ could more effectively and thoroughly promote conidial death by directly binding to A. flavus DNA. Increased expression of DNA damage repair-related genes after Fe2+ treatment was observed by transcription analysis, which might eventually lead to SOS response in A. flavus. These results indicated Fe2+ could prevent A. flavus infestation on pitaya in a novel, quickly responsive mechanism. Our results shed light on the potential application of Fe2+ in the food industry and provided a more universal antifungal agent against food pathogens.

Introduction

Pitaya (Hylocereus undulatus britt) has become one of the most popular trends in the fruit consumption market, owing to its desirable taste and rich sources of health-promoting antioxidant compounds (Ong, Wen, Mohamad, Sieo, & Tey, 2014). Nevertheless, the quality of pitaya is often challenged by postharvest fungal disease. It has been reported that more than 40 % of postharvest fruit and vegetables are lost, which is largely due to decay caused by fungal attack. Among these, Aspergillus flavus (A. flavus) is believed to be the main contributor to pitaya fungal contamination, which results in pitaya deterioration and rapid senescence and inevitably leads to physiological responses, including shrinkage, softening, weight loss, decay of the fruit and changes in flavor and antifungal-related matter. (Zahid et al., 2013, Toivonen and Brummell David, 2008). Apart from the ill effect of A. flavus on fruit quality, the secondary metabolite aflatoxin B1 (AFB1) it produced has been put into Group 1 carcinogen by IARC (the International Agency for Research on Cancer), and the toxin can be transferred to humans and animals through contaminated foods, provoking life-threatening diseases to humans, such as acute poisonings, immune-system dysfunctions, and stunted growth in children. High-dose intake of AFB1 even leads to death (Fiore et al., 2018, Umesha et al., 2017). With global warming, the infection of pitaya with fungal diseases, especially A. flavus, has increased, thus limiting its development and marketing potential.

For the past few decades, the control of fungi in pitaya has mainly been achieved by application of synthetic fungicides (Jiang et al., 2020, Pace and Cefola, 2021). However, the successive and extensive use of these fungicides not only causes drug resistance but also raises concerns about the toxicity of chemical fungicides to both humans and the environment. Therefore, safer and more environmentally friendly strategies to protect pitaya from fungal infection are urgently needed. Fe2+, as an essential chemical element, is a bio efficient and inexpensive iron nutrient fortifier and is often used as an iron supplement for children and pregnant women (Harvey, Zkik, Auges, & Clavel, 2016). It is one of the most important metallic elements in life, participates in many biological processes and is considered as a better antimicrobial agent since it can be degraded by human body and does not cause toxicity. (Jomova & Valko, 2011). Newly studies have suggested that Fe2+ facilitated ferroptosis in Magnaporthe oryzae, eventually affected the growth of hyphae and reduced infection ability of fungi (Shen, Liang, Yang, Deng, & Naqvi, 2020). Our previous study proved that exogenous 0.5 mM Fe2+ induced ferroptosis in A. flavus spores and suppressed growth of A. flavus (Yao, Ban, Peng, Xu, Li, & Mo, 2021). A. flavus is one of the most important fungi causing pitaya decay after harvest; however, few studies have focused on the antifungal effects of Fe2+ on food preservation. To test the effect of Fe2+ on preservation and sterilization of pitaya, FeSO4 was used to treat postharvest pitaya fruit. The conidial mass, weight loss ratio, fruit decay rate, total soluble solids, titratable acidity, soluble sugars, total betalain content, phenols, flavonoids, malondialdehydehyde content and antioxidant-related enzyme activity (CAT, SOD, POD) were measured, and the possible physiological mechanism was explored in our study. The results will provide a theoretical basis for scientific storage and raise the potential application of Fe2+ as a safer, less expensive and more eco-friendly antifungal agent for food storage.

Materials and methods

Chemicals and strains

A. flavus NRRL3357 was obtained from Sun Yat-sen University (Guangdong, China) and cultivated on potato dextrose agar (PDA) medium (Becton, Dickinson and Company, USA) at 30 °C. The spore suspension was harvested with 0.05 % Triton X-100, and the spore number was counted using a hemocytometer under a microscope. DNA was extracted using a Biospin fungus genomic DNA extraction kit from cultured A. flavus hyphae, and a Nanodrop (Nanodrop ND-1000 UV, Thermo Scientific) was used to measure the purity of DNA using an absorption ratio of 260 to A280, the DNA concentration was measured by Nanodrop and adjusted to a final concentration of 2 × 103 ng/μL. Ferrous sulfate heptahydrate (FeSO4·7H2O) was purchased from Sigma. Chemical kits used for testing activity of CAT (BC0200-50 T/48S), POD (BC0090-50 T/48S) and SOD (BC0170-50 T/24S) were purchased from Solarbio (Beijing Solarbio Science & Technology Co., ltd.). All other chemical agents were analytical grade.

Spore viability measurement

Spore viability assay was performed to evaluate the inhibitory effect of Fe2+ on spore according to our previous methods (Yao et al., 2021). Briefly, in spot dilution assay, 5 μL of serial-diluted A. flavus spore suspension (107, 106, 105, 104, 103 CFU/mL) were dropped onto each of Sabouraud solid plate with preadded Fe2+ to the final concentration of 0, 0.25, 0.5, 1, 2, 3 mM, respectively, and these plates were subjected to a stationary culture for 3 days at 30 °C.

Plant materials and treatments

White pitaya fruit with similar weight, size, color, maturity and no obvious external mechanical damage were purchased at local market and picked for preservation and sterilization experiments. A total of 105 CFU/mL A. flavus spores were used for the infection test, and 3 mM Fe2+ was prepared. A total of 48 fruit were selected, after disinfecting the surface of the fruit, one group (at least 12) fruit were soaked in the solution of Fe2+ for 10 min, dried naturally, repeated the treatment three times, one group fruit with only disinfecting the surface as control; in the sterilization test, fruit were soaked in A flavus spore suspension for 10 min, then soaked again after natural air drying, repeated three times, then divided into two groups, one as control, the other was soaked in 10 mL Fe2+ solution for 10 min, dried naturally, repeated three times. All fruit were sampled, observed and measured after 7 d cultured in a 25℃ incubator. The pulp on the equatorial line was cut and quickly frozen in a −80℃ refrigerator with liquid nitrogen for index measurement. Each index was measured three times, and the average value was taken.

Conidial mass assay

The spores on the pitaya surface were removed and soaked with phosphate-buffered saline (PBS, pH 7.4) and then stained with 1 μg/mL SYTO™ 9 Green Fluorescent (Beyotime Institute of Biotechnology, China) at 4 °C for 30 min in the dark, followed by observation under a flow cytometer (Beckman CytoFLEX FCM).

Weight loss ratio (WLR)

The pitaya fruit before and after the test was weighed, and the weight loss ratio was calculated according to the following formula:

Fruit decay rate (FRI) and firmness

The decay severity for each fruit was divided into five grades according to the percentage of decay area in the whole fruit: 1 = 0–1 %, 2 = 2 %-25 %, 3 = 25 %-50 %, 4 = 50 %-75 %, and 5 = 75 %-100 %. This grade was then converted into the fruit decay rate (FRI) as follows:

The firmness of pitaya fruit was measured at three equatorial points of the peeled fruit using a handheld fruit hardness tester (GY-3, Shanghai Grows Precision Instrument Co., ltd., Shanghai, China) with two probes (diameters: 8 and 11 mm) and expressed as N.

Total soluble solid (TSS), titratable acidity (TA) and soluble sugar content (SSC)

The total soluble solids were measured by a portable refractometer. Briefly, pitaya juices were dropped on the clean prism surface of the refractometer, and the refractometer was faced to the light. The scale shown in the refractometer is the total soluble solid of the fruit. The titratable acidity (TA) was determined using extracted pitaya juices by volumetric titration with NaOH, TA in the juice was a percentage of malic acid as the dominant organic acid in the fruit. The total soluble sugar content was measured through the anthrone-sulfuric acid colorimetric method using a spectrophotometer (HIRP V1700G, Hirp International Trade Co., ltd., Shanghai, China) at 620 nm and calculated with a glucose standard curve and is expressed as g/L.

Malondialdehyde (MDA) content and total betalain, phenol and flavonoid contents

The MDA content was determined by thiobarbituric acid method. One gram of fresh pitaya fruit was ground into a homogenate in 5.0 mL of 10 % TCA (trichloroacetic acid) and centrifuged at 10,000 × g for 20 min at 4 °C. Then, 2.0 mL of supernatant was taken, 2.0 mL of 0.67 % TBA (thiobarbituric acid) was added, and the supernatant was boiled for 20 min. After cooling, the supernatant was centrifuged again, and the absorbance values were measured at 450 nm, 532 nm and 600 nm.

Under high temperature and acidic conditions, MDA can react with TBA to form a reddish-brown trimethyl complex (3,5,5-trimethyloxazole-2,4-dione), which has a maximum absorbance value at 532 nm. However, soluble carbohydrates in fruit tissues can interact with MDA-TBA products and have absorbance values at 532 and 450 nm. To eliminate this interference, the following empirical formula was used to eliminate the soluble carbohydrate interference:

Based on the calculated MDA concentration in the supernatant, the MDA in the whole fresh pitaya was attained as follows:

The flesh of pitaya (2 g) was soaked with 20 mL of methanol (80 %, v/v) and sonicated for 10 min in a bath (KQ5200DE, Prevnext). The methanolic extract was stirred for 20 min at room temperature and centrifuged at 5,000 × g for 10 min (Thermo Scientific, Legend Micro17, Waltham, MA, USA). One milliliter of supernatant was collected and fourfold diluted, and the concentrations of betalain were determined by spectrophotometry at 538 nm wavelength. The total content of betalain was expressed as g per kg of fresh pitaya weight:

where B is the content of betalain, A538 is the absorbance at 538 nm, W is the molecular weight (550 g·mol−1), V is the volume of the extracted solution (mL), Df is the dilution factor, ε is the molar extinction coefficient (65,000 L·mol−1··cm−1), m is the mass of the sample (2 g), and l is the length of the cell (1 cm).

According to Folin-Ciocalteu’s procedure, the content of total phenols was determined from the reaction of 1 mL of the methanolic extracts and 2.5 mL of 1:1 (v/v) diluted Folin-Ciocalteu’s reagent. Then, 2 mL of 7.5 % (w/v) Na2CO3 solution was added and incubated for 5 min at 50 °C, and the absorbance was measured at 760 nm. A standard curve of gallic acid was made to estimate phenol content, and the results were expressed as g/kg.

Flavonoid content was assessed as followed: First, 3.7 mL of the methanolic extracts, 0.15 mL of 10 % AlCl3·6H2O and 0.15 mL of 5 % NaNO2 were mixed. After 5 min, 1 mL of 1 M NaOH was added prior to the measurement at 510 nm. The concentration of flavonoids was estimated from the calibration curve using rutin as the standard and expressed as (g/kg).

Assessment of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities

The activities of SOD, POD and CAT were measured using a biochemical kit (Beijing Solarbio Science & Technology Co., ltd.) following the manufacturer’s guidelines. Briefly, SOD is widely present in animals, plants, microorganisms and cultured cells, and catalyzes the disorganization of superoxide anions (O2), O2 can reduce nitro-blue tetrazolium to generate formazan, which is absorbed at 560 nm; SOD activity can be reflected by absorption at 560 nm through inhibiting the formation of formazan; POD has the dual effect of eliminating H2O2 and toxicity of phenolic and amine, POD catalyzes specific substrates with characteristic absorption at 470 nm; CAT is the most important H2O2 scavenging enzyme and plays an important role in reactive oxygen species scavenging systems, H2O2 has a characteristic absorption peak at 240 nm, which decreases when CAT decomposed H2O2, so that CAT activity can be calculated according to the change of absorbance. The activity of these enzymes was expressed as units (U) kg−1.

Genomic DNA break detection

The A. flavus suspension (105 CFU/mL) exposed to 2 mM Fe2+ for 20 min and 1 h were stained with TUNEL (10 μg/mL) for 30 min at 4 °C. The broken genomic DNA was observed with a flow cytometer (FCM, Beckman CytoFLEX).

RNA-seq analysis of A. Flavus spores after Fe2+ treatment

A 106 CFU/mL spore suspension exposed to 2 mM Fe2+ for 20 min and 1 h was sampled and ground in liquid nitrogen. Total RNA was extracted using a total RNA extractor (TRIzol) kit (Yili, Shanghai, China). The integrity and quality of RNA were evaluated using a 1 % agarose gel and an Agilent Technologies 2100 Bioanalyzer. High-quality RNA samples were prepared for cDNA library construction and sequencing. FastQC software was used for data quality evaluation, and sequenced clean reads were obtained by filtering out low-quality reads and adaptor contamination, then mapped against predicted transcripts of the A. flavus NRRL 3357 genome. The differentially expressed genes (DEGs) were filtered by p value < 0.05. DEGs related to DNA repair between the control and treatments were selected at significant levels calculated by Fisher, and DEGs were visualized by a heatmap. All data are the average of three replications.

Gel electrophoresis

A gel electrophoresis system was used to test Fe2+ damage on purified genomic DNA, amplified PCR products and process of PCR. A 10 µL interaction system was set containing 2 µL of Fe2+ solution, which was adjusted to final concentrations of 1 mM, 2 mM, 3 mM and 8 µL of DNA or PCR products. Then, 1 µL of 10 × DNA loading buffer was added to the system after 30 min treatment, and 1 % gel was prepared for electrophoresis. The system was under electrophoresis for 30 min at 120 V and 400 mA, and then the image was observed under a UV lamp. To test the effects of Fe2+ on the process of PCR, Fe2+ solution was added to the PCR system at final concentrations of 1 mM, 2 mM, and 3 mM. A 20 µL PCR system used was shown in Table S1, the procedure was as shown in Table S2, and the product was measured using gel electrophoresis as mentioned above.

Circular dichroism (CD) experiments

CD spectral analysis were determined on Chirascan Plus equipment (V-100, Applied Photophysics, UK). Briefly, DNA samples were prepared and mixed with Fe2+ saline solution for 30 min, and the solution was adjusted to pH 7.3. The temperature was controlled by water circulation. The CD spectra were collected between 200 and 500 nm with the bandwidth at a 1 nm interval using a JASCO-720CD spectropolarimeter. All spectral data were the average of three technical duplicates and three biological duplicates.

Confocal Raman microspectrometer

Well-prepared A. flavus DNA solution was used in our test and Fe2+ was added to DNA solutions to achieve a final concentration of 2 mM, then the liquids were placed on specific liquid-used glass plates. A Renishaw (Gloucestershire, UK) inVia confocal Raman spectrometer coupled to a DMLM Leica microscope was used for the measurement. A 785 nm laser source, 600 notched grating, 50X long focal length was adopted, and the laser power was 30 mW. Raman spectra in the range of 800–2000 cm−1 were collected using a 50 (NA = 0.5) focal length lens and 10.5 mm working distance, the spectra were acquired at 30 min afterward. The integration time was 8 s, and the integration was performed in triplicate. All solutions were maintained at room temperature during the experiment.

Statistical analysis

IBM SPSS Statistics v20 Software was used for statistical analysis and quantitative detection data in each result is presented as the mean ± standard deviation (SD) of at least three replicated measurements. A t test was used to analyze the significant differences between two groups, multiple groups’ comparison was achieved by one way ANOVA analysis and Duncan. Statistically significant difference was considered at p value < 0.05. All charts were accomplished using OriginPro 2021.

Results and discussion

Exogenous 3 mM Fe2+ completely inhibited A. favus spore germination

In order to evaluate the influence of exogenous Fe2+ on A. flavus growth and germination, different concentrations of Fe2+ were applied to A. flavus spore suspension. As shown by spot dilution assay, the antifungal activity of Fe2+ was observed in a dose dependent manner, exogenous 3 mM Fe2+ could completely inhibited A. flavus germination (Fig. 1), which was consistent with our previous study (Yao et al., 2021). Therefore, 3 mM Fe2+ was selected in our following test to explore the effect of Fe2+ on pitaya preservation and sterilization experiments.

Fig. 1

Inhibitory effects of Fe2+ on the spore growth of A. flavus NRRL3357.

Exogenous Fe2+ extended pitaya shelf life and inhibited fungal infestation

To test the potential effects of Fe2+ on extending shelf life and preventing pathogenic colonization of pitaya fruit, 3 mM Fe2+ was applied for preservation and sterilization tests (Fig. 2). As shown in Fig. 2, Fe2+ obviously alleviated the decay phenotypes after long time storage and reduced contamination caused by A. flavus.

Fig. 2

Phenotypes of Fe Pitaya without any treatment as a control (A); pitaya inoculated with 105 CFU/mL A. flavus spore suspension (B); pitaya inoculated with Fe2+ for the preservation test (C); pitaya inoculated with 105 CFU/mL A. flavus spore suspension and soaked with Fe2+ for the sterilization test (D). All fruit were stored at 25 ℃ for 7 d.

Water and organic matter in the fruit can be consumed by respiratory metabolism and pathogen colonies, resulting in weight loss, reduced firmness and decay on the surface of the fruit. As shown in Fig. 3A, the application of Fe2+ alleviated the total conidial mass after A. flavus contamination, and weight loss rate (WLR) and fruit decay rate (FRI) were suppressed by 34.07 % and 49.98 %, respectively, in Fe2+-treated pitaya fruit compared to untreated fruit. Fe2+ also retained fruit weight and constrained fruit decay after A. flavus infection, which suppressed weight loss and fruit rot by 7.87 % and 40 %, respectively (Fig. 3B-C).

Fig. 3

Conidial mass (A), weight loss rate (WLR) (B) and fruit decay rate (FRI) (C) of pitaya fruit after Fe2+ treatment in preservation and sterilization tests.

Fe2+ could retain pitaya firmness after A. flavus treatment and maintain flavor in pitaya under A. flavus treatment, where Fe2+ markedly retained TA content. Decreased TA in fresh pitaya fruit could be attributed to accelerated respiratory intensity, which caused consumption of carbohydrates and organic acids. In our study, Fe2+ suppressed the decline in TA during storage, indicating that Fe2+ probably acted as an inhibitor, affecting the respiratory mode in pitaya fruit. However, no statistically significant difference was observed in total soluble solids (TSS) and soluble sugar content (SSC) after Fe2+ treatment in either the preservation or sterilization test. It indicated that exogenous Fe2+ treatment could not affect the ripening degree of pitaya after harvest, for total soluble solids (TSS) and soluble sugar content (SSC) were believed to be an indicator of fruit ripening. (Fig. 4A-D).

Fig. 4

Firmness (A), total soluble solid (TSS) (B), titratable acidity (TA) (C), soluble sugar content (SSC) (D), malondialdehyde (MDA) content (E), total betalain content (F), total phenol (G), flavonoid (H), antioxidant-related enzyme (DPPH (I), CAT (J), SOD (K) and POD (L)) activities in pitaya fruit after Fe2+ treatment in preservation and sterilization tests. NS represents no significant difference between the compared groups; * represents p < 0.05, ** represents p < 0.01.

Effects of Fe2+ on MDA, total betalain, phenol and flavonoid contents

Malondialdehyde (MDA), as an indicator of cell membrane oxidation, is greatly induced during pitaya storage due to fruit senescence and pathogen attack. MDA content was measured based on Fe2+ treatment during preservation and A. flavus contamination. The result showed that MDA content remained unchanged after Fe2+ treatment during preservation. Considerable decreased MDA was detected after Fe2+ exposure under A. flavus treatment (Fig. 4E). These results suggested that increased MDA content might prior be attributed to pathogen invasion, and Fe2+ could protect cell membranes from oxidative damage, thus reducing MDA content. The result indicated that Fe2+ could not only extend pitaya shelf life but also shield cell membrane from oxidation after A. flavus. Betalain, as a natural water-soluble polyphenolic pigment, is responsible for its high antioxidant capacity, which is beneficial for human health. Fig. 4F showed that Fe2+ suppressed the reduction in betalain concentrations during preservation and defense against A. flavus.

Effects of Fe total phenol, flavonoid, DPPH, SOD, POD, and CAT expression

As the main active antioxidant response, the phenol content was higher after Fe2+ treatment than in the control during preservation and A. flavus treatment, while the total flavonoid content was unchanged (Fig. 4G-H). The expression of DPPH, SOD, POD and CAT showed a similar gradually elevated trend, and improved activity of these enzymes was detected after A. flavus contamination; moreover, Fe2+ exposure could significantly induce their expression, except for DPPH, which was decreased during the preservation process (Fig. 4I-L). These results revealed that Fe2+ could activate antioxidant reaction of pitaya fruit by promoting expression of antioxidant-related enzymes, especially in response to A. flavus infection.

Above all, in terms of postharvest preservation and resistance to A flavus infestation, Fe2+ showed potential capability to extend the shelf life of pitaya fruit and ability to combat colonization of A. flavus.

Fe2+ induced DNA damage of A. Flavus

To explore whether Fe2+ induced A. flavus DNA damage, TUNEL staining of A. flavus spores after different concentrations of Fe2+ treatment was conducted. The phenomenon of DNA breakage after disposal to Fe2+ was observed, and a right shift under a flow cytometer was detected after 1 mM Fe2+ treatment, indicating that damage occurred in part of the DNA under Fe2+ treatment (Fig. 5A).

Fig. 5

Exogenous Fe DNA damage was detected by TUNEL staining and flow cytometry (A); transcriptome analysis of DNA repair-related genes after 20 min (B) of Fe2+ treatment and 1 h of treatment (C); Fe2+ damage to gDNA of A. flavus (D); different concentrations of Fe2+ damage to gDNA of A. flavus (E); Fe2+ damage to the PCR product of A. flavus (F). Two microliters of Fe2+ solution was added to DNA at final concentrations of 1 mM, 2 mM, and 3 mM, and all DNA solutions were adjusted to the same concentration.

Fe2+ activated DNA damage repair genes and triggered the SOS response

DNA damage further interferes with cellular replication and transcription, even results in genetic mutations and chromosomal lesions, and severe DNA damage usually eventually leads to the SOS response (Loeb, James, Waltersdorph, & Klebanoff, 1988). To further investigate the possible DNA damage-related genes under Fe2+ pressure, RNA sequencing analysis in A. flavus spores after Fe2+ treatment was performed. The data indicated that a total of 49 genes enriched in the DNA damage repair pathway were detected, with only minor variations in the transcription levels between the three replicates, but Fe2+ treatment differed significantly from control (Fig. S1), where Fe2+ increasingly upregulated DNA damage repair-related genes compared to control, including RAD gene, a nucleotide excision repair homologous gene, which was reported to be involved in repair of aflatoxin-induced DNA damage in yeast by removing bulky DNA lesions formed by UV light, environmental mutagens or chemicals (Fig. 5B-C) (Guo, Breeden, Zarbl, Preston, & Eaton, 2005). These results suggested that Fe2+ may trigger DNA damage repair system under exogenous Fe2+ accumulation, which was corresponded to previous studies showing that exogenous stress could upregulate DNA damage-inducible genes (Christmann & Kaina, 2013). We assumed that this might be related to the SOS response, because DNA repair genes are usually activated by the SOS response, and this was a survival strategy for microorganisms to survive challenging pressure (Keseler et al., 2013). However, how Fe2+ triggers this reaction requires further investigation.

Fe2+ chelated gDNA and affected the PCR process

DNA, as the material basis of life, is of great importance to all living organisms, and its molecular integrity is also necessary for life activities of living organisms. Any lesion or injury to DNA will affect not only its integrity but also the transcription it participates in. Gel electrophoresis is known as a straightforward and convenient method to detect the integrity of DNA and PCR procedures. Therefore, gel electrophoresis analysis under different concentrations of Fe2+ was performed in our study to investigate the effects of Fe2+ on DNA and PCR processes. Stripe retention was observed in the Fe2+-treated lane both in the gDNA and in the process of PCR, and the levels of retention increased along with the concentrations of Fe2+ (Fig. 5D-F). These results indicated that Fe2+ could cause damage to gDNA and influence PCR procedure. We suspected that Fe2+ could directly interact with DNA and might affect DNA conformation and chelate DNA fragments to form a macromolecular substance that finally stuck in the gel hole (Fig. 5D-F). It has been proven in similar reports that exposure to Co and Ni could specifically affect DNA polymerization and the accuracy of replication, repair and transcription processes (Kumar, Mishra, Kaur, & Dutta, 2017).

Effects of Fe2+ on the conformation of DNA

CD spectra are good indicator to investigate DNA conformation, to investigate the effects of Fe2+ on DNA conformation, a CD experiment was performed in our study. Fig. 6 showed a strong positive peak in the region at approximately 280 nm of A. flavus DNA, but the peak decreased in the Fe2+-pretreated DNA trait. It seemed that Fe2+ slightly reduced the compactness of DNA structure. A large negative peak was observed in the region of 200–250 nm, Fe2+-pretreated DNA showed opposite spectra compared to DNA and Fe2+ solely in this region, which indicated that DNA conformation might change in the presence of Fe2+. In addition, there was a peak at 400 nm, but no difference was observed in the CD spectra among DNA, Fe2+ and their Fe2+-pretreated DNA trait. In conclusion, there was a strong interaction between Fe2+ and DNA. Fe2+ influenced DNA conformation at 200–250 nm and reduced the density of the CD spectrum of DNA. The changes in DNA structure might further affect the function of DNA binding proteins.

Fig. 6

CD spectroscopy and Raman spectra analysis of DNA upon binding to Fe ion solution.

Raman spectra of Fe-DNA complexes

In addition, Raman spectra were utilized to experimentally interrogate the binding and possible intercalation of Fe2+ to DNA. Fig. 6B indicated a typical spectrum of Fe2+ alone at approximately 1400 cm−1 and a spectrum of DNA alone at approximately 1370–1390 cm−1, which had been reported to derive from the ring stretching mode of thymine, guanine and adenine bases (Punihaole et al., 2018). When Fe2+ premixed with DNA for 30 min, a decrease in the viscosity of solution was observed, and an apparent drop in the spectrum at approximately 1400 cm−1 occurred. This result suggested that there was an interaction between Fe2+ and DNA. To better highlight the changes, we subtracted the spectrum in Fig. 6C. DNA and Fe2+ interaction lowered the significant peak of Fe2+ and reduced the peak of DNA, but both Raman shifts of Fe2+ and DNA did not change under their interaction. These results might be attributed to the nomadic Fe cation could coordinate with the backbone phosphate group of DNA and change the cleavage ability of DNA, finally resulting in the disappearance of the Raman spectra of Fe2+ and DNA. In conclusion, exogenous even low concentration of Fe2+ could change the DNA conformation.

Discussion

Postharvest decay is the main limiting factor for commercial value and shelf life of pitaya fruit (Xu et al., 2021), which causes 40–50 % loss of postharvest fruit due to pathogen contamination and improper postharvest handling (Bordoh, Ali, Dickinson, Siddiqui, & Romanazzi, 2019). Several approaches, such as cold storage, edible-coating, X-ray, plant hormones, antioxidation additives and antimicrobial agents, have been reported to resist postharvest diseases and hold fruit quality (Ba et al., 2022, Xu et al., 2021). However, these treatments are basic means of delaying fruit senescence and retaining flavor characteristics to various degrees after pathogenic invasion. Despite the additional coating of antibacterial substances, it is still impossible to eliminate pathogens, particularly A. flavus. Our study proved that exogenous Fe2+ (3 mM) could efficiently suppress weight loss, constrain fruit decay, reduce titratable acid content and retain total betalian content both during the preservation and infection of A. flavus (Fig. 1, Fig. 2, Fig. 3). Moreover, Fe2+ showed strong potential to suppress malondialdehyde (MDA) content in pitaya fruit after A. flavus infection, indicating that Fe2+ could not only extend pitaya shelf life but also shield cell membrane from oxidation after A. flavus infection (Fig. 4E).

In addition, Fe2+ treatment increased activity of antioxidant-related enzymes, such as CAT, SOD, and POD, in pitaya, especially after A. flavus attack (Fig. 4I-L). It has been reported in some studies that ROS dyshomeostasis during pathogen attack is strongly related to fruit senescence and decay processes (Lin et al., 2017, Xu et al., 2021). ROS dyshomeostasis is the result of an imbalance between ROS production and antioxidant activity. CAT, SOD and POD are the most studied antioxidant enzymes that act to resist the production of ROS and disintegrate H2O2 into H2O and O2 (Tan et al., 2020). Enhanced activity of these enzymes is believed to delay senescence and quality deterioration in different kinds of fruit (Mirshekari et al., 2020, Jiang et al., 2021). Xu (Xu et al., 2021) proved that increased SOD, CAT, POD and their associated genes in pitaya during storage could efficiently lower the level of ROS and mitigate oxidative damage, development of decay and senescence. On the other hand, fungi can suppress antioxidant-induced pathways, such as hormone signal transduction pathways and enzymatic systems, which accelerate the impairment of host cells (Choudhary, Kumar, & Kaur, 2019). However, it has also been suggested that low concentrations of ROS play a central role in host defense by transmitting stimulatory signals to neighboring cells and activating host defense responses under both biotic and abiotic conditions (Mor et al., 2014). This might explain why MDA content of pitaya pulp in our study was suppressed after A. flavus contaminated pitaya cuticle, despite its cuticle senescence and rotting symptoms had been noticed, we speculated that in our study, exogenous Fe2+ could produce low dose of ROS and transmit stress signal on the fruit cuticle to inner cells.

It is commonly accepted that Fe2+ induced an ROS burst, accelerated cell membrane lipid peroxidation and accumulated MDA by ferroptosis, but our study proved that exogenous Fe2+ protected pitaya fruit from infection by postharvest pathogen A. flavus in a novel but potentially universal and easily applied way, which was different from classical ferroptosis, by directly inducing pathogen DNA breakdown. We found that Fe2+ might induce DNA breakage and upregulate DNA damage repair-related genes by directly binding to DNA and changing the DNA conformation, thus affecting its nature and blocking SOS repair pathways in A. flavus (Fig. 5A-C). It has been proven in some studies that metal ions are inextricably involved in nucleic acids due to their polyanionic nature and are responsible for large conformational changes. Additionally, the dynamic nature of nucleic acids is reflected in the characteristics of interactions with metal ions (Pechlaner & Sigel, 2012). In particular, the importance of Fe2+ binding to DNA has been considered in relation to DNA damage and mutations. (Loeb et al., 1988). It is assumed that there might exist some interactions between Fe2+ and DNA, and although some studies using calf thymine DNA as materials have shown that Fe2+ might bind to the backbone phosphate group and affect cleavage in DNA (Ouameur, Arakawa, Ahmad, Naoui, & Tajmir-Riahi, 2005), few studies have focused on the direct affinity of Fe2+ and DNA under physiological conditions. In this study, we performed agar electrophoresis analysis, the results showed that the levels of DNA fade with increasing concentrations of Fe2+, and similar results were observed in the PCR products and in the process of PCR (Fig. 5D-F). Circular dichroism (CD) (Fig. 6A) and Raman (Fig. 6B-C) experiments were even more intuitive in that there was an interaction between Fe2+ and DNA, exogenous even low concentration of Fe2+ could change DNA conformation. As a result, the SOS response in A. flavus might be initiated because the SOS response is believed to be the mode of the survival strategy and allows microorganisms to evolve from an unfavorable environment (Keseler et al., 2013).

Recently, an increasing number of studies have reported that several nontoxic inorganic compounds, such as Ca2+, Zn2+, Cu2+ and Ni2+, have been effectively utilized as additives in postharvest food to maintain flesh and extend shelf life of many fruit (Milosevic et al., 2017, Ahankari et al., 2021, Meena et al., 2020). For example, Ca2+ treatment could alleviate fruit postharvest chilling damage by protecting membrane structure and reducing cell wall breakdown, thus reducing the chance of pathogen infection (Manganaris et al., 2010, Gupta et al., 2011). It has been reported in some studies that pretreatment with Ca2+ reduced the occurrence of anthracnose and brown rot severity of pitaya fruit (Ghani, Awang, & Sijam, 2011). In addition to protecting the membrane, it has also been suggested that Ca2+, as an intracellular second messenger, can transmit stress signals and activate various physiological responses in cells (Hepler, 2005). Sardella proved that Zn2+ had antifungal efficiency against several fungal contaminants (Sardella, Gatt, & Valdramidis, 2017). ZnO micro- and nanoparticle-based edible coatings have been successfully regarded as antibacterial agents and exhibit remarkable efficiency in extending shelf life of fruit, which is attributed to their ability to promote the production of reactive radicals. (La et al., 2021, Khla et al., 2021). Cu2+ combined chitosan nanoparticles show the ability to extend shelf life of stored tomato (Meena et al., 2020), and Tsvetkov et al. showed that Cu2+-dependent death occurred by means of direct binding to lipoylated components of the tricarboxylic acid (TCA) cycle (Tsvetkov et al., 2022).

These studies raise the potential application of metal iron in postharvest food, and they are superior in their durability, low toxicity and ability to withstand severe conditions, such as high temperature and high pressure. The antifungal effect triggered by Fe2+ found in this study would be a more efficient strategy to prevent postharvest decay because it can kill pathogens at nucleic acid level, and Fe2+ might be a universal antifungal agent against other postharvest pathogens. Combining Fe2+ and other postharvest protection methods will be a higher priority to prevent fruit postharvest fungal contamination to extend fruit shelf life.

Conclusion

In this study, we demonstrated that exogenous low dose within safe limit (3 mM) of Fe2+ could defend A. flavus infection and extend shelf life of postharvest pitaya fruit, by suppressing weight loss, constraining fruit decay, retaining fruit firmness, reducing titratable acid content and retaining total betalain content both during preservation and during infection with A. flavus. Antioxidant-related substances, including total phenols, CAT, SOD and POD, were enhanced after Fe2+ treatment. Moreover, these beneficial effects of Fe2+ might be achieved by two aspects. One was activating the antipathogen-related pathway inside the fruit by transmitting the stress response signal from the pericarp after A. flavus infection. On the other hand, Fe2+ could control colonization of A. flavus by means of directly binding to A. flavus DNA and affecting the conformation of DNA, eventually leading to DNA breakage. Furthermore, transcription analysis revealed that Fe2+ could elicit DNA damage repair-related genes.

This is the first report about Fe2+ directly binding to fungal DNA, resulting in DNA breakage to eliminate fungi, and the study raises the possibility of Fe2+ application as a safer, less expensive and more eco-friendly antifungal agent.

CRediT authorship contribution statement

Lishan Yao: Project administration, Conceptualization, Writing – original draft, Methodology. Tao Zhang: Project administration, Methodology. Shurui Peng: Writing – review & editing. Dan Xu: Writing – review & editing. Zhenbin Liu: Writing – review & editing. Hongbo Li: Writing – review & editing. Liangbin Hu: Funding acquisition, Supervision, Writing – review & editing. Haizhen Mo: Writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.