Molecular Microbiology Research 2024, Vol.14, No.6, 259-270 http://microbescipublisher.com/index.php/mmr 265 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, andAlt a 1genes. 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 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: 1200 m; geographic position: 108.9 E, 29.3 N).
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