【作者】 刘霞；

【导师】 郑晓冬；

【作者基本信息】 浙江大学 ， 食品科学与工程， 2009， 博士

【摘要】 酸腐病是柑橘贮运中最常见、最难防治的病害之一,由此带来的损失也是巨大的。但目前,国内外对酸腐病害的侵染机制及生物防治研究甚少,因而开展柑橘G citri-aurantii侵染规律及安全的防治技术的研究对于降低损失,增加农民收入具有重要意义。本文对柑橘G. citri-aurantii的侵染途径及侵染时期进行了初步的研究,并对从柑橘果实表面及根际土壤中分离获得的采后致病G. citri-aurantii不同菌株间的形态学特征、致病力及生物学特性等方面进行了鉴定,研究了不同拮抗菌、天然植物提取物及低剂量杀菌剂等对柑橘采后病害（主要是酸腐病害）的防治效果,并初步探讨了其可能产生的抑菌机理。主要研究结果如下：1对柑橘不同侵染途径的研究结果表明,在柑橘的苞片、花器、根、茎及叶上均没有分离到柑橘G. citri-aurantii;在柑橘果皮分离到多株G citri-aurantii;在根际土壤中分离到一株G. citri-aurantii。2对柑橘果实发育期带菌的分离结果表明,在果实发育过程中,从幼果期、转色期和成熟期的柑橘果实中没有分离到G citri-aurantii。这说明,G.citri-aurantii可能是柑橘采后在运输和贮藏过程中通过伤口进行侵染从而导致果实发病。3应用rDNA-ITS分子标记分析和传统真菌形态学鉴定相结合的方法,对从不同产地、不同品种的柑橘上分离获得的能引起柑橘贮藏期间腐烂的7株酸腐菌（G₁、G₂、G₄、G₁0、G₂7、G₅0、T₁0）进行了鉴定。结果表明：这7株菌株均属于酸腐菌（G. citri-aurantii）。4对分离到的7株G. citri-aurantii的致病性进行了研究,发现G50对柑橘果实致病能力最强,条件适宜的情况下,从果实发病到全果腐烂只要7d-10d时间；其次为G₂、G₄、G₁0、G₂7、T₁0;而G1的致病力最弱,接种后的柑橘贮藏在26℃下,在第四天的发病率仅为20%,在随后的贮藏时间内,发病率不变,病斑直径略有增加。成熟度、伤口和带菌量是柑橘采后致病的关键因子。5对不同菌株的生物学特性的研究结果表明,在同一培养基上培养,平均日生长速率、菌落颜色、以及菌落厚度不同菌株之间没有显著不同。G.citri-aurantii生长温度范围为5-35℃,菌丝体生长和孢子形成适宜温度为15-30℃。孢子致死温度试验表明：G. citri-aurantii致死温度在80-85℃。6用百里香精油、肉豆蔻精油、桉树精油、鼠尾草精油和温郁金精油对柑橘主要采后病原菌在体外熏蒸和直接接触抑菌效果进行了研究。结果发现采用熏蒸法进行抑菌试验时,百里香精油对柑橘三种主要致病菌表现了较好的抑菌效果,桉树精油的抑菌效果次之,肉豆蔻精油、鼠尾草精油和温郁金精油只对柑橘G.citri-aurantii有抑制效果。当采用直接接触法进行抑菌试验时,百里香精油显示了较强的抑菌活性,温郁金精油次之,鼠尾草精油、桉树精油和肉豆蔻精油对柑橘G citri-aurantii没有抑菌活性。7用百里香精油以直接接触的方法处理柑橘果实时,对柑橘三种采后病害均显示较好的抑制效果。在体外试验中发现,碳酸钠能够加强百里香精油的抑菌效果。在体内试验中,百里香精油与适量浓度的碳酸钠结合时,能显著的提高植物精油的抑菌效果,而且能够完全的抑制柑橘酸腐病害的发生。8在26℃培养温度下,1×lO⁶CFU/mL的三种拮抗酵母悬浮液均能够显著的抑制病菌孢子浓度为1×10⁶个/mL时柑橘果实酸腐病（G. citri-aurantii）的发生。三种拮抗酵母菌对柑橘青霉病和绿霉病也有一定的抑制效果。而且拮抗酵母与低浓度的杀菌剂混合使用,可增强拮抗酵母对柑橘酸腐病害的抑制效果。9拮抗菌在室温下能迅速在柑橘果实伤口定植,接种酵母菌28h后,数量可增加20倍以上。拮抗菌和病菌孢子的接种时间与生物防治效果有关,先接种拮抗菌的抑菌效果显著的好于同时或后于病菌接种的效果。10柑橘果实采后酸腐病害防治中试结果表明,R. paludigenum与50μl/L维鲜或400μl/L百里香精油与1%的Na₂CO₃结合使用能显著提高对柑橘果实采后酸腐病害的防治效果。更多还原

【Abstract】 In this paper, we studied infection path and period of Geotrichum citri-aurantii and morphological characters, pathogenicity, part of biological characteristics of different isolates of Geotrichum spp which were isolated from citrus fruit surface and in rhizosphere soil. Worthwhile, we researched the inhibition effects and mechanisms of different antagonists, plant extracts and low dose fungicides against citrus postharvest diseases, especially sour rot. Main conclusions were drawn as follows:1 The results about infection path showed that Geotrichum spp could not be found on foliage, bract, blossom, and stalk of citrus. We isolated several Geotrichum spps on the surface of citrus which was the most major and important carrier. A G. citri-aurantii was isolated from the rhizosphere soil.2 We isolated no Geotrichum spps from citrus surface at different growth phase. The isolation results showed that Geotrichum spps may infect citrus fruits and make them rot by wounds in transport and storge.3 Seven typical pathogenic strains includingG₁, G₂, G₄, G₁0, G₂7, G₅0, T₁0, which were isolated from the surface of citrus fruits in different areas, were identified as Geotrichum spp by morphological characters and internal transcribed spacer （ITS） analysis.4 The incidence and development of development of disease were determined by pathogen, host, environment and interaction of each other. Those factors （including pathogen capacity, the way and period of infection, temperature and humidity of environment and so on） influenced disease development. It showed that sour rot incidence and severity both increased when temperature increased during citrus storage. Pathogenicity of G. citri-aurantii （G50） was the best, the incidence disease could be whole only for 5-7d while in suitable conditions. Sequentially was G. citri-aurantii （G2、G₄、G₁0、G₂7、T₁0）. G. citri-aurantii （Gi） was the worst. The sour rot of inoculated citrus fruits was only 20%after 4 days at 26℃, then the number of sour rot would not change while the diameter increased a bit. Wound, degree of maturity, and inoculation quantity of spores would increase succession of infection, and it is the key factors for the incidence and development of disease.5 The results indicate that there were not significant difference in growth characteristics （including growth rate, color of colony and thickness of colony） in PDA media. The growth temperature range from 10℃to 70℃the hyphal could survived, and the suitable temperature for mycelia growth and spores germination was 15℃to 30℃. In the deadly temperature test, the result showed that the strains were died from 80℃to 85℃.6 Thyme oil had strong fumigant toxicity followed by eucalyptus oil to all three postharvest pathogens in citrus fruits. Nutmeg oil, sage oil and curcuma wenyujin presented weak fumigant toxicity to Penicillium digitatum and Penicillium italicum while they exhibited a moderate action to G. citri-aurantii （G50）. Thyme oil exhibited strong contact antifungal activities against the tested fungi. Curcuma wenyujin exhibited a moderate contact action while nutmeg oil, sage oil and eucalyptus oil present no contact antifungal activity to G. citri-aurantii （G50）.7 Thyme oil showed a significant inhibitory effect against all three pathogens in citrus fruits. It reduced the percentage of decayed citrus caused by fungal pathogens. The antifungal effect of thyme oil against G. citri-aurantii （G50） was enhanced significantly by combining with Na₂CO₃ in vitro and in vivo. In vivo, sour rot could be completely inhibited by 1600μl/L thyme oil with 0.5%Na₂CO₃.8 Antagonists Cryptococcus laurentii, Rhodosporidium paludigenum, and Rhodotorula glutinis at 1x1O⁶ CFU/mL could effectively control sour rot disease in citrus fruits at 26℃. They also could control green and blue mold diseases.In addition, the antifungal effect of R. paludigenum against G. citri-aurantii （G50） was enhanced significantly by combining with low doses fungicide.9 Rapid colonizations of all three yeast antagonists in wounds were observed at 26℃. The population increased more than 20 folds after 28 h inoculation compared to the start point. Efficacy of yeasts against sour rot disease was maintained when applied simultaneously with or before G. citri-aurantii, but when applied after spores, biocontrol efficacy was significantly reduced.10 On pilot-scale experiment tests, R. paludigenum and thyme oil had great biocontrol activity against sour rot of citrus fruits,1×10⁸ CFU/mL R. paludigenum in combination with low doses fungicide （50μl/L） or 400μl/L thyme oil in combination with 1%Na₂CO₃ showed great biocontrol activity to sour rot in citrus fruits, which showed potential of biocontrol agents to control the postharvest decay of citrus fruits.更多还原