文献类型：期刊文献

中文题名：哈密瓜镰刀果腐菌的鉴定、生物学特性和室内防治药剂的筛选

英文题名：Identification,biological characteristics and indoor fungicide selection of Fusarium incarnatum in Hami melon

作者：杜莉芳[1];曾晴[1];徐晶[1];张思怡[1];俞国相[1];郭天荣[1];莫亿伟[1]

机构：[1]绍兴文理学院生命科学学院,浙江绍兴312000

年份：2022

卷号：39

期号：5

起止页码：855

中文期刊名：果树学报

外文期刊名：Journal of Fruit Science

收录：CSTPCD、、CSCD2021_2022、Scopus、北大核心、CSCD、北大核心2020

基金：浙江省基础公益研究计划项目(LGN20C150018,LGN22C150002)。

语种：中文

中文关键词：哈密瓜(Cucumis melo L.);果腐镰刀菌;生物学特性;纳他霉素;硼酸

外文关键词：Hami melon(Cucumis melo L.);Fusarium incarnatum;Biological characteristics;Natamycin;Boric acid

中文摘要：【目的】鉴定引起哈密瓜(Cucumis melo L.)果蒂处的果肉腐烂病原菌种类,以及对不同宿主的致病性检测,探讨光照、温度、pH值、碳源、氮源及不同药剂处理对该菌生长的影响,并筛选出哈密瓜镰刀果腐病的有效防治方法。【方法】采用组织分离法,从发病的哈密瓜果实中分离病原菌,结合形态学与核糖体内转录间隔区(rDNA-ITS)、转录延伸因子EF-1α和组蛋白Histone3序列的分子鉴定确定病原菌种类。测定不同的培养温度、pH、碳源、氮源、光照及需氧条件对其生长的影响。用NaHCO;、硼酸、施保功、苯甲·嘧菌酯和纳他霉素处理,观察各种药剂的抑菌效果。再用罗丹明123、健那绿B、线粒体红色荧光探针(Mito Tracker;Red CMXRos)及溴化丙啶(PI)染色,了解不同药剂处理对病原菌细胞膜透性及线粒体功能的影响,用安全性较高的纳他霉素对哈密瓜进行室内防效实验,了解纳他霉素对病原菌的防治效果。【结果】病原菌为哈密瓜果腐变红镰刀菌(Fusarium incarnatum,编号ZJHM-01),孢子形态呈镰刀状,长度为25.4~35.5μm,宽度为3.3~5.4μm;病原菌生物学特性测定结果表明,宿主具广谱性,光照能促进生长,最适生长温度为30℃,最适p H为7~9,最适碳源为淀粉、山梨醇和葡萄糖,最适氮源为蛋白胨和牛肉膏。1.0%NaHCO;、1.2%硼酸、0.025%施保功、0.016%苯甲·嘧菌酯和40 mg·L^(-1)纳他霉素均能完全抑制病原菌生长。罗丹明123染色结果发现,1.0%NaHCO;、1.2%硼酸和30 mg·L^(-1)纳他霉素处理使菌丝的细胞膜透性增加;健那绿B和Mito Tracker;Red CMXRos染色发现,药剂处理降低了菌丝的线粒体活性;PI染色结果表明,病原菌细胞膜完整性遭到破坏,线粒体活性和线粒体膜电位受损。纳他霉素室内防效表明,纳他霉素处理的果实发生病害时间明显延后且症状减轻,说明纳他霉素具有较好的防病效果。【结论】果实采收期,生产上用施保功和苯甲·嘧菌酯防治哈密瓜镰刀菌果腐病。果实采收后,用1.0%NaHCO;、1.2%硼酸和40 mg·L^(-1)纳他霉素浸泡果实处理以降低哈密瓜镰刀菌果腐病的发生率。从食品安全性考虑,纳他霉素应该作为哈密瓜采后镰刀菌果腐病防治的首选药剂。研究结果为哈密瓜镰刀菌果腐病防治提供了新的方法。

外文摘要：【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-1αand 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 50μL volume containing a 2μL DNA template as well as 2μL of each primer(10μmol),25μL 2×PCR Taq MasterMix(Tsingke Biotech,China),and 21μL ddH;O.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 O;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 NaHCO;,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 25.4-35.5μm and the width between 3.3-5.4μm.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%NaHCO;,1.2%boric acid and 40 mg·L-1natamycin 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%NaHCO;,1.2%boric acid and 30 mg·L-1natamycin 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%NaHCO;,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.