1 Introduction

Gardenia jasminoides Ellis is a shrub in the Rubiaceae family. Its desiccative fruit, often called Zhizi in China, is used not only as a supereminent natural colorant but also as excellent traditional medicine for various ailments. This includes promoting blood circulation, eliminating stasis, clearing heat, and removing toxins. It is also used to treat diabetes and has anti-inflammatory, diuretic, antidepressant, and anticancer properties (Jin et al., 2023).

G. jasminoides is native to China, Japan, India, and Thailand, and it is now widely grown in 16 provinces in China, especially along the Yangzi River (Chen et al., 2020). Among these provinces, Enshi in Hubei Province is the main production region; However, the warm and humid climate from May to July promotes the development of various diseases, resulting in large outbreaks of leaf spot disease, which seriously affect the yield of G. jasminoides.

Leaf spot disease can be caused by various fungi, such as Alternaria (Liu et al., 2021a; Yang et al., 2022), Cercospora (Tan et al., 2023), Phyllosticta (Yang, 2023), Pestalotiopsis (Liu et al., 2021b), Fusarium oxysporum (Xue et al., 2023), Pseudocercospora (Crous et al., 2021), Cylindrocladium (Polizzi et al., 2006), Myrothecium (Mmbaga et al., 2010), and others. Among them, Alternaria spp. are the most common cause of fungal foliar diseases in vegetables, cereals, fruit trees, and medicinal plants during production and storage, resulting in field and post-production losses. Alternaria is a large ubiquitous dictyosporic genus, and the variability of the isolates within this genus is high even within the same species (Lawrence et al., 2013). Consequently, various approaches based on morphology, physiology, pathogenicity, and genetics have been proposed to establish identification and classification systems, but the resulting taxonomy remains unclear (Pryor et al., 2002; Andrew et al., 2009; Woudenberg et al., 2015). Molecular sequence analysis of nuclear genes such as intron spacer regions (ITSs) and the mitochondrial ribosomal large subunit (mtLSU), Alternaria major allergen (Alt a 1), glyceraldehyde-3-phosphate dehydrogenase (gapdh), and the RNA polymerase II second largest subunit (rpb2) is useful for evaluating phylogenetic relationships among species (Zhu et al., 2015; Ma et al., 2021).

Chemical fungicides are an effective strategy for managing diseases caused by Alternaria spp. in many plants. Hou et al. (2024) reported that flutolanil, phenamacril, pyraclostrobin, and boscalid can effectively prevent and control black spot disease in tree peony, which is caused by Alternaria suffruticosae. Pyraclostrobin can especially suppress the conidial germination, mycelial growth, germ tube elongation, and sporulation quantity of A. suffruticosae. Moreover, prochloraz was shown to have a stronger effect on leaf spot disease in blue honeysuckle, which was caused by A. tenuissima (Liu et al., 2024a). However, over-reliance on fungicides results in the pathogens becoming resistant to these fungicides, posing dangerous health and environmental safety concerns. Therefore, Alternaria spp. could be controlled in an environmentally friendly way by using beneficial fungi or bacteria and harmless chemicals.

Melatonin (nacetyl-5-methoxytryptamine) was first identified in 1985 and given this name because of its role in the darkening effects of melanocyte-stimulating hormones (Lerner et al., 1958). It is a naturally occurring small-molecule indole neurohormone, which contains no obvious toxicity to vertebrates, including humans, and is safe and harmless to plants and the environment, so it has good potential for its application in plants. Melatonin also plays an important role in plant defense against biotic stresses. Exogenous melatonin treatment of apple fruits that were inoculated with Botrytis cinerea effectively reduced the incidence of gray mold and the area of spots, resulting in significant resistance to gray mold (Zhu et al., 2021). Root application of 0.1 mmol/L melatonin significantly enhanced the resistance of apples to brown spot disease (caused by Diplocarpon mali) (Yin et al., 2013). Melatonin inhibited the expansion of Litchi chinensis fruit spots infected with Peronophythora litchii and induced the resistance of litchi to the pathogenic fungus (Zhang et al., 2021). The treatment of fresh pistachios (Pistacia vera) inoculated with Aspergillus flavus and treated with exogenous melatonin inhibited Aspergillus flavus spore germination and shoot tube elongation; this effectively slowed down the fruit rot caused by the fungus and reduced the accumulation of aflatoxins (Aghdam et al., 2020). In addition, exogenous melatonin treatment reduced the incidence of leaf blight in rice (Chen et al., 2020). Sun et al. (2020) sprayed strawberry leaves and stolons inoculated with Alternaria alternata with 0.5 mmol/L of melatonin to effectively inhibit the growth of the mycelium of the pathogen. However, although these studies have shown that melatonin can alleviate plant diseases by increasing the level of plant autoimmunity and inhibiting the pathogenicity of plant pathogenic microorganisms, the specific mechanism of melatonin in the plant immune response is still unclear, and the pathogenic mechanisms of fungi, bacteria, viruses, and other pathogenic bacteria are different.

Considering the significant influence of leaf spot disease in the G. jasminoides industry and the limited understanding of its causal agent, it has become imperative to identify and control the phytopathogenic agent. The key objective of this research was to isolate and identify Alternaria spp. which causes leaf spot disease in G. jasminoides. Furthermore, alternative approaches using melatonin as an environmentally friendly chemical to control leaf spot disease were also investigated.

2 Results

2.1 Sample collection

In June 2023, high temperature, high humidity, and poor ventilation accelerated the incidence rate of leaf spot disease in G. jasminoides Ellis in Enshi, Hubei Province, China. According to the field observations, 40%~60% exhibited leaf spot symptoms, as the infected leaves of G. jasminoides Ellis had dark brown or black lesions with a surrounding concentric ring. The diameter of the lesions ranged from 3 mm to 2 cm, and larger lesions had a chlorotic halo (Figure 1).

Figure 1 Symptoms of leaf spot disease on G. jasminoides Ellis. Bar, 1 cm

2.2 Fungal isolates and morphological characterization

A total of six Alternaria isolates were obtained from infected leaf samples and named ES1-1, ES2-1, ES2-2, ES2-3, ES4-2, and ES4-3. The six isolates were morphologically consistent with Alternaria alternata (Fonseca-Guerra et al., 2023). The colonies of these isolates on PDA varied. ES1-1 (Figure 2a) and ES4-2 (Figure 2f) displayed an olive-green color, and colony margins were white with a feathery texture. Moreover, the growth rates of these two isolates were slower than those of the other four species. The whole mycelium of the other four isolates had a dense, cottony texture, and the color was dark olive. Furthermore, the surface of all six colonies exhibited concentric rings. The conidia were ovoid to obclavate in shape and ranged from 14.6 μm to 34.9 μm×7.0 μm to 16.4 μm (length×width) in size (n=15). All types of conidia were muriformly septate with 1 to 6 transverse septa and 0 to 5 longitudinal septa. The beak size varied from 1.3 μm to 9.8 μm (n=15). Therefore, based on these characteristics, the isolates were identified as A. alternata. There were no significant differences in the length (P=0.532) and width (P = 0.166) of the conidial body or length (P = 0.665) of the beak among the isolates (Figure S1).

Figure 2 Colony appearance and conidia morphology of six isolates Image caption: a~f: Colonies of six isolates, ES1-1, ES2-1, ES2-2, ES2-3, ES4-2, and ES4-3, respectively, cultured on PDA plates for 7 d at 25 ℃ in the dark; g~l: Conidia of six isolates, ES1-1, ES2-1, ES2-2, ES2-3, ES4-2, and ES4-3, respectively

2.3 Phylogenetic analysis of PCR-generated DNA sequences

The Sanger sequencing results showed that the fragments of ITS, gapdh, rpb2, and Alt a 1 were 514 bp to 515 bp, 572 bp to 579 bp, 752 bp, and 444 bp to 453 bp, respectively. When the 24 sequences of genes of the Alternaria isolates were searched using the blast tool in GenBank of the National Center for Biotechnology Information (NCBI), the isolates were 99% to 100% similar to other A. alternata sequences reported in the GenBank. Further phylogenetic trees were constructed based on maximum likelihood analysis with these sequences, and all these isolates were identified as A. alternata (Figure 3), which was consistent with previous morphological characterization. Furthermore, ES2-2, ES2-3, and ES2-1 were grouped into the same subclade; ES4-2, ES4-3, and ES1-1 formed another subclade. Sequences of Alternaria alternantherae (CBS 124392; HSAUP2798) were used as outgroups (Woudenberg et al., 2015) (Figure 3).

Figure 3 Phylogenetic tree based on maximum likelihood analysis. Bar: the estimated nucleotide substitutions per site are 0.02

2.4 Pathogenicity assays

The pathogenicity of these six isolates was identified by wound inoculation. Three days after inoculation, tiny, round, and necrotic spots appeared on the leaves. These spots gradually expanded by 7 dpi (days post inoculation) and were similar to those found under field conditions (Figure 4). Furthermore, all fungal isolates were re-isolated from these diseased leaves while no Alternaria spp. were isolated from control leaves. All the leaves that were inoculated with the isolates of A. alternata were infected, and the average incidence was 100%. ANOVA results indicated no significant differences (P=0.238) in pathogenicity among the different isolates (Figure S2).

Figure 4 Symptoms of leaf spot disease 7 d after inoculation of G. jasminoides leaves. Bars, 1 cm

2.5 Effects of melatonin treatment on the inhibition of the growth of A. alternata

Melatonin is a natural molecule that plays an important role in plant defense against fungal pathogens. We tested the direct antifungal properties of melatonin on A. alternata. The direct effect of melatonin on mycelial growth was evaluated. We applied different concentrations of melatonin to the PDA medium. As shown in Figure 5, the colony diameter gradually decreased with increasing melatonin concentration. Seven days after treatment, DMSO also demonstrated a minor inhibitory effect on mycelial growth. However, the inhibition property was limited, and there was no significant difference in the effect of DMSO and double-distilled water (ddH₂O) 14 d after treatment. Furthermore, treating A. alternata with 5 mM to 10 mM of melatonin significantly affected the growth of ES4-2 after 14 d. Therefore, melatonin can effectively restrict the growth of A. alternata.

Figure 5 Melatonin treatment applied to A. alternata on PDA plates Image caption: a: The colony morphology of A. alternata treated with different concentrations of melatonin after 7 d and 14 d. b, c: The colony diameter 7 d and 14 d, respectively, after treatment. Bars, 2 cm. Significance was determined according to Duncan’s multiple range test at P < 0.05. The data (mean ± SD) were calculated using three replicates

2.6 Effects of melatonin treatment on G. jasminoides leaves infected with A. alternata

We also investigated whether melatonin affected the disease resistance of G. jasminoides leaves against A. alternata. We treated detached leaves with different melatonin concentrations before and after inoculation with A. alternata. The lesion areas of both treatments (before and after inoculation) were significantly reduced on melatonin-treated leaves compared to ddH₂0-treated leaves, and treatment before A. alternata inoculation showed better efficacy than treatment after A. alternata inoculation. Similar to DMSO inhibiting the mycelial growth of ES4-2, DMSO also affected the infection of A. alternata in G. jasminoides leaves, but the inhibitory effect was significantly lower than that of the high-concentration melatonin treatment. The concentration of 1 mM melatonin after inoculation with A. alternata 12 h was the most effective in reducing the infection of ES4-2 (Figure 6a). However, treatment with 5 μM, 10 μM, and 100 μM melatonin did not result in significant differences in the lesion area (Figure 6b). For the melatonin treatment before inoculation, 1 mM of melatonin significantly enhanced the resistance to A. alternata infection (Figure 6a). The lesion area of leaves treated with 1 mM melatonin was 4.1% of those treated with H₂O and 14.7% with DMSO (Figure 6b). Taken together, these results suggest melatonin can control the infection of A. alternata on G. jasminoides leaves.

Figure 6 Effects of melatonin treatments on the resistance of G. jasminoides leaves against A. alternata Image caption: (a) Leaf lesions 14 d after melatonin treatment before and after inoculation. (b) Lesion area 14 d after melatonin treatment before and after inoculation. Bars, 1 cm. Significance was determined according to Duncan’s multiple range test at P < 0.01. The data (mean ± SD) were calculated using three replicates

3 Discussion

Leaf spot disease has already plagued the G. jasminoides industry with a high incidence rate in Hubei Province, China. In this study, six isolates of Alternaria spp., identified as causal agents of Gardenia jasminoides Ellis leaf spot, were determined to belong to A. alternata based on the phylogenetic analysis and morphological characteristics. The pathogenicity test showed that all these isolates can cause leaf spots on Gardenia jasminoides Ellis, indicating that these isolates were pathogenic to gardenia leaves. Furthermore, we studied the effect of melatonin on the prevention and treatment of A. alternata.

Alternaria fungi have always been identified with a combination of morphological and molecular methods. We also used these two approaches to characterize Alternaria isolates collected from symptomatic leaves. The Alternaria spp. isolated in this study can be categorized into two groups. There was no significant difference in their pathogenicity, spore size, and colony growth rate. All six isolates on PDA resulted in typical A. alternata morphological features reported previously (Xu et al., 2022). Subsequent molecular analysis also identified them as A. alternata.

A. alternata is a common causal agent that can cause foliar disease on many plants, such as Panax notoginseng (Liang et al., 2015), Pinus bungeana (Zhang et al., 2023), Elaeocarpus decipiens (Liu et al., 2024b), Punica granatum (Yang et al., 2022), Prunus avium (Pan et al., 2023), etc. Therefore, A. alternata has a wide host range and can infect a variety of plants. In Gardenia jasminoides Ellis, Li (2022) reported that Diaporthe gardeniae can cause branch blight in Zhejiang Province, China. The phytoplasma 16SrI-B was associated with yellowed leaves, stunted growth, and small flowers in G. jasminoides (Sun and Zhao, 2012). Recently, the leaf blight disease of G. jasminoides has been reported, and the pathogen was also identified as A. alternata. However, only one isolate was reported and identified by rDNA–ITS (Zhao et al., 2021). In our study, we identified six isolates and conducted a phylogenic analysis using 24 sequences of ITS, gapdh, rpb2, and Alt a 1 genes.

Melatonin is an environmentally friendly chemical that plays an important role in various processes of plant growth, such as seed germination (Shaheen et al., 2024), leaf senescence (Bai et al., 2020), flowering (Xu et al., 2024), fruit ripening (Brüning et al., 2018), abiotic stress (Arnao et al., 2020), and biotic stress resistance (Ahmad et al., 2023). Previous studies have shown that melatonin affects plant–pathogen interactions by directly inhibiting fungal pathogens and enhancing plant immunity to pathogens. Arnao (2015) demonstrated that 4 mM of melatonin inhibited the growth of mycelium, such as that of Alternaria spp., Botrytis spp., and Fusarium spp. Likewise, 100 μM to 5 mM of melatonin also suppressed the growth of P. infestans (Zhang et al., 2017), Colletotrichum gloeosporioides, and Colletotrichum acutatum (Ali et al., 2020). Our results also show that 5 mM to 10 mM melatonin significantly reduced 70% of mycelial growth in A. alternata. Moreover, melatonin contributes to improving plant immunity to pathogens. When roots of apple plants (Malus prunifolia) were treated with 50 μM~500 μM melatonin, their resistance to Marssonina apple blotch (Diplocarpon mali) improved through reactive oxygen species (ROS) scavenging and the activation of defense genes (Yin et al., 2013). The melatonin treatment (1 000 ppm) of cucumber (Cucumis sativus) leaves reduced both the disease index and severity of Phytophthora capsici by increasing the activity of antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase, and peroxidase (SOD, CAT, APX, and POD, respectively), along with the activation of antioxidant genes (Mandal et al., 2018). Cowpea seedlings underwent with 400 μM melatonin will improve cowpea resistance to Fusarium oxysporum (Gan et al., 2024). Additionally, the innate immunity of Arabidopsis to Pseudomonas syringae pv. tomato (Pst) DC3000 was improved by regulating nitric oxide (NO)-mediating defense signaling when 20 μM of melatonin was added to the culture media (Shi et al., 2015). In this study, we estimated the effect of exogenous melatonin on A. alternata infection with G. jasminoides leaves. The melatonin was treated both before and after A. alternata. As shown in Figure 6, applying melatonin at 5 μM~1 mM could effectively reduce the lesion area compared with the control. Moreover, melatonin treatment before inoculation was more effective.Therefore, in agricultural production, we recommend using melatonin spray to prevent plant diseases. For the prevention of G. jasminoides leaf spot disease, a melatonin concentration of more than 5 μM can be applied to effectively prevent leaf spot disease. It is worth noting that, in our study, DMSO, as a solvent for melatonin, had certain effects on both the mycelial growth of A. alternata and its infection of G. jasminoides leaves. However, this is not the first time that DMSO has been reported to inhibit fungal growth. DMSO is thought to be able to alter the permeability of fungal cell membranes, thereby affecting the growth and survival of fungi (Hazen, 2013; Huang et al., 2020). 14 days after treatment, the effect of DMSO was only consistent with that of 0.5 μM melatonin. In addition, the amount of DMSO added was consistent with the amount of DMSO solvent required in 1 mM of melatonin, while the actual amount required in 5 μM of melatonin was less. Thus, it had less effect on the growth and infection of mycelia. Therefore, exogenous melatonin treatment may be a promising strategy and can be used in the field to prevent gardenia leaf spots in future. However, the mechanism through which melatonin mediates pathogen resistance and its effect on field application should be studied further.

4 Materials and Methods

4.1 Fungal Isolation and identification

In June 2023, G. jasminoides leaves with typical leaf spots were collected in Enshi city, Hubei Province, China (elevation: 1 200 m; geographic position: 108.9 E, 29.3 N).

After the diseased leaves were cleaned with sterile water, the leaves at the junction of necrotic spots and healthy tissues were cut into 5 mm×5 mm pieces and disinfected in 75% alcohol for 30 s and in a 2% commercial sodium hypochlorite solution (4% active chlorine) for 2 min. After each disinfection, the samples were washed several times with distilled water. Then, surface moisture was sucked up, and the samples were placed on potato dextrose agar (PDA) plates, with 3 pieces per plate. Mycelia around the disinfected tissue were selected for purification, and the pure cultured isolates were obtained through continuous transfer and stored at 4 ℃.

4.2 Pathogenicity tests

After the healthy G. jasminoides leaves were wiped clean with 75% alcohol, a wound was made on each side near the middle main vein using a 1 mL sterilized syringe. Then, agar plugs (5 mm in diameter) of the isolates were inoculated into these wounds. The leaves were then moisturized and placed in an incubator at 25 ℃. Five leaves were treated per isolate. The agar plugs (5 mm in diameter) of the PDA plate were used as the negative control. After 72 h of moisturizing culture, the agar plugs were removed to continue observing and recording the disease symptoms that were captured with a digital camera (Nikon, D750, Japan). Subsequently, the tissues with obvious symptoms were re-isolated from the inoculated leaves to determine whether the inoculated isolates were the causal agents of the G. jasminoides leaf spots.

4.3 Identification based on cultural and morphological characteristics

The pathogenic strains were inoculated on a PDA plate and cultured at 25 ℃ in the dark. For each isolate, three replicates were used. After 7 days, all cultures were assessed for colony color, margin, and texture. The colony morphology was photographed.

To observe the spore morphological characterization, isolates from PDA plates were transferred to potato–carrot agar (PCA) and incubated for 14 days. For each isolate, conidia were harvested from the same plates and suspended in distilled water. The conidial suspension was observed under a Nikon Eclipse microscope (Japan), and more than 10 pictures were taken for each isolate. These pictures were analyzed using ImageJ 1.47 software (National Institutes of Health, Bethesda, MD) to measure the length and width of each conidia per isolate.

4.4 Identification based on molecular characteristics

The DNA of pathogenic fungi was extracted from the mycelia that had been growing for 7 days, using the plant Genomic DNA kit (Tiangen, China), according to the instructions. Polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS, ITS1/ITS4), the Alternaria major allergen sequence (Alt a 1), the second largest subunit of RNA polymerase II gene (rpb2), and the glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene was conducted using specific primers: ITS1/ITS4 (White, 1990), Alt1-F/Alt1-R (Chruszcz et al., 2012), RPB2-5f2/RPB2-7cr (Liu et al., 1999), and gpd1/gpd2 (Berbee et al., 1999). The extracted pathogenic DNA served as a template for this process. The PCR was carried out using 2×Phanta® Flash Master Mix Dye Plus (Vazyme, Nanjing, China). The volume of each reaction system was 30 μL, including 15 μL of Taq enzyme, 1 μL of each primer, 1 μL of DNA template, and 12 μL of sterile water; the PCR system without a DNA template served as a negative control. The PCR amplification conditions were set as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 98 ℃ for 10 s; annealing at 54 ℃ (ITS) for 30 s, 59 ℃ (rpb2) and 57 ℃ (Alt a 1) for 45 s, and 61 ℃ (gapdh) for 40 s. This process depended on the fragment size, with an extension time of 72 ℃ for 10 s, followed by 30 cycles, and concluded with a final extension of 72 ℃ for 5 min. Finally, 5 μL of PCR products were taken and detected by 1% (v/v) agarose gel electrophoresis, and the products with correct bands obtained were sequenced by Sangon Biotech Co., Ltd. All sequences in this study were uploaded to GenBank of the National Center for Biotechnology Information (NCBI), and the accession numbers are listed in Table 1. The multi-locus sequences were aligned with the previously deposited sequences in the GenBank database using BLAST, and a phylogenetic tree was constructed using the maximum likelihood method in MEGA 7.0 (https://www.megasoftware.net) software to clarify the pathogenic fungi.

Table 1 GenBank accession numbers of isolates obtained from G. jasminoides with leaf spot disease

4.5 Melatonin control of G. jasminoides leaf spot disease

To investigate the inhibitory effect of melatonin on the mycelial growth of A. alternata, we added various concentrations of melatonin to the PDA. In brief, 2.32 g of melatonin (SIGMA; Chemical Abstracts Service (CAS) Number: 73-31-4; total chromatographic limit (TCL)≧98%) was dissolved in dimethyl sulfoxide (DMSO), and then water was added to reach a volume of 10 mL (DMSO: H₂O=5:3/v:v) to create a mother liquor for storage. The PDA medium (potato, 200 g/L; glucose, 20 g/L; agar, 15 g/L) was configured, and different concentrations of the melatonin solution (based on the mother liquor, diluted to concentrations of 0.05 mM, 0.1 mM, 0.5 mM, 1 mM, 5 mM, and 10 mM) were added before sterilization, and DMSO and sterile water were added to the PDA medium as a control. We used ES4-2 (one of the six Alternaria isolates) for the melatonin control experiment; the isolate was cultured on PDA medium for approximately 7 d. Then, we took agar plugs measuring approximately 5 mm to inoculate each treatment, using 4~5 dishes for each treatment. The colony diameters were measured at 7 d and 14 d, the inhibition rate was calculated, and the experiment was repeated three times.

The effects of melatonin on leaf spot disease were tested by applying melatonin both before and after inoculation with ES4-2. We collected G. jasminoides leaves and rinsed the leaf surface using deionized water; then, we wiped the leaves clean with skimmed cotton dipped in alcohol. These leaves were placed in a square Petri dish with two pieces of filter paper. Before treatment, we wrapped the injured part of the petiole with skimmed cotton and added 5 mL of sterile water to the Petri dish. Melatonin was configured in the same way as before and diluted to concentrations of 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 100 μM, and 1 mM, with DMSO and sterile water as controls. To inoculate the G. jasminoides leaves, two small holes were made using a syringe in the middle of both sides of the large leaf veins; ES4-2 was then inoculated into the small holes. The agar plug was removed three days after inoculation, and the leaves were sprayed with different concentrations of melatonin (0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 100 μM, and 1 mM), with DMSO and sterile water sprayed as controls. Spraying was repeated on the 7th d after the initial application, and the incidence of disease was assessed 14 d after melatonin spraying. The experiment was repeated three times. For melatonin treatment before inoculation, two small holes were created on both sides of the large leaf veins in the middle of leaves and then sprayed with different concentrations of melatonin (0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 100 μM, and 1 mM), with DMSO and sterile water sprayed as controls. All these leaves were incubated under dark conditions for 12 h, ES4-2 was inoculated into the positions of the small holes, and the agar plugs were carefully removed after 3 d. Then, the leaves were incubated in an incubator and were sprayed again on the 7th d. The disease incidence was investigated 14 d after melatonin spraying. This experiment was repeated three times.

4.6 Statistical analysis

All data in this study are presented as averages±standard deviations (SDs). One-way analysis of variance (ANOVA) and Duncan's multiple tests were used for the statistical analysis by SPSS 18.0 (IBM, Armonk, USA), and significant differences at P<0.05 or P<0.01 were marked using distinct letters.

Acknowledgments

This work was supported financially by Zhejiang Province's Pairing Assistance Project-Construction of Gardenia jasminoides scalable planting demonstration base and industrialization of gardenia flower hydrolat extraction technology (Counterpart Project 2021C04026).

Conflict of Interest Disclosure

The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

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