Identification, biological characteristics and indoor fungicide selection of Fusarium incarnatum in Hami melon

Abstract: 【Objective】Hami melon(Cucumis melo L.) is native to Xinjiang, with attractive smell, delicious taste and rich nutrition and is also famous in China. Hami melon is a kind of typical respiratory climacteric fruit with high sugar and water. Thus, it is very easy to decay caused by the fruit temperature increase and to be soft due to the acceleration of physiological metabolism in the process of sales and transportation. Meanwhile, the soft fruit is also easy to be infected by pathogenic fungus. The Hami melon storage quality and safety control system is not perfect, which leads to low quality of Hami melon in the market, and brings with serious influence on the reputation of Hami melon. Storage rots,caused by Penicillium, P. digitatum, Botritis cinerea, Aspergillus, Richoderma viridescens and Monilinia fruictigena are the most common postharvest diseases, which erode the fruit, cause it soft and result in great loss for farmers. Meanwhile, several synthetic fungicides with different modes of action are available to control fruit rots including benzoxystrobin, iprodione, prohydantoin and dysenine. However, to our best knowledge, few researches have been focused on the identification of the major postharvest diseases at the species level by morphological characterization and molecular analysis based on multiple DNA sequences. Meanwhile, chemical treatment efficacy frequently decreases by development of postharvest pathogen resistance. There is also increasing concern about the environmental issues and human health problems caused by synthetic fungicides due to possible toxicological risks. Therefore,the need is urgent for alternative and effective strategy to control postharvest disease of Hami melon fruits. The objective of the present experiment was to identify the pathogenic fungi species in the pedicle of Hami melon fruit by morphological characterization and molecular analysis based on multiple DNA sequences, to detect the hosts of pathogenic fungi, and to optimize growth environments including illumination, temperature, pH value, carbon and nitrogen sources and so on. Meanwhile, the effective prevention measures of Hami melon rot causing by the fungus were needed to be screened out.【Methods】Firstly, the pathogenic fungus was isolated from the diseased Hami melon fruits when it decayed with typical symptoms. Then the pathogen was sterilized with 70% alcohol for 2 min, soaked with 0.1% NaClO for 5 min, rinsed with sterile water for 3 times, and purified and cultured for several times after being transferred to potato-dextrose agar medium. The pathogen species was determined by using morphological observation and molecular identification methods, including DNA sequence analysis of ribosomal transcription spacer(rDNA its), transcription elongation factor EF-1α and histone 3. Fungal isolates were incubated on PDA at 25 ℃ for 4 d and the fresh plugs from colony margins were plated in the center each plate. The colors of each colony were recorded every day. The micro-morphological results were measured under a microscope. Mycelia were collected from the surface of colonies incubated for seven days. Samples of total DNA were then extracted from the purified isolate using the method described. PCR was performed in a 50 µL volume containing a 2 µL DNA template as well as 2 µL of each primer(10 μmol), 25 µL 2×PCR Taq MasterMix(Tsingke Biotech, China), and 21 µL ddH2O. After repeated purification of the pathogen, different fruits including cucumber, apple, peach, pear, apricot, plum, jujube, watermelon, tomato, and so on were inoculated by pathogen using stabbing method to detect the pathogenicity to other fruits until the obvious plaque on the fruit surface was observed. Furthermore, the pathogenic fungus cultured in the incubator at different growth temperatures, pH values,carbon sources, nitrogen sources, light intensities, and O2 concentrations was to determine the optimal cultural environment for the pathogen growth. Finally, in order to understand their antifungal effects,the pathogenic fungus was treated with NaHCO3, boric acid, prochloraz, difenoconazole azoxystrobin and natamycin. Further staining by Rhodamine 123, Janus Green B, Mito TrackerTMRed CMXRos, and propidium bromide(PI) was used to understand the effect of the treatments on the cell membrane and mitochondrial structure of pathogenic fungus. Indoor prevention experiment was conducted to understand the antifungal effect of natamycin in practical applications.【Results】The results showed that the pathogen infecting Hami melon was F. incarnatum (No. ZJHM-01). The spores were sickle-shaped with the length between 25.4-35.5 μm and the width between 3.3-5.4 μm. The hosts were broad and it could cause disease for 10 kinds of fruits such as tomatoes, apricots and Chinese dates. Light promoted the colony growth. The optimum growth temperature, pH, carbon and nitrogen sources for the fungus were30 ℃, 7-9, starch, sobitol, glucose, and peptone, beef extract, respectively. 1.0% NaHCO3, 1.2% boric acid and 40 mg·L^-1 natamycin could significantly inhibit colony growth. Meanwhile, 0.025% prochloraz and 0.016% Difenoconazole azoxystrobine could completely inhibit pathogens growth, which indicated that F. incarnatum was very sensitive to Prochloraz and Difenoconazole azoxystrobine. The staining results by Rhodamine showed that 1.0% NaHCO3, 1.2% boric acid and 30 mg·L^-1 natamycin treatments could completely increase the cell membrane permeability of F. incarnatum cell, and staining results by Janus Green B and Mito TrackerTMRed CMXRos showed the mitochondrial activity was destroyed significantly, while the staining result by PI indicated that the integrity of the cell membrane,mitochondrial activity and mitochondrial membrane potential of the pathogenic fungi were destroyed.The delayed disease occurrence and the mitigated symptoms were observed in 40 mg·L^-1 natamycintreated fruit, indicating that natamycin had an excellent antifungal effect in practical applications.【Conclusion】Prochloraz and Difenoconazole azoxystrobine are recommended to be used before harvest to prevent Hami melon fruit from F. incarnatum for sake of its toxicity. After fruit harvest, 1.0% NaHCO3,1.2% boric acid and 40 mg·L-1 natamycin could be used to soak the fruit to reduce the occurrence of F. incarnatum rot. In consideration of food safety, natamycin was recommended as the optimal fungicide for the control of F. incarnatum rot in Hami melon. Thus, the results of this study provided a new method for preventing F. incarnatum rot before and after harvesting of Hami melon.