【作者】 刘毅；

【导师】 许玲；

【作者基本信息】 华东师范大学 ， 植物学， 2009， 硕士

【摘要】 西兰花(Brassica oleacea L.var.italica)营养价值丰富而且具有公认的抗癌功效。但是在采后阶段,它会受到各种病原菌的侵染并且迅速衰老黄化,失去原有的商品性。对引起西兰花采后病害的病原进行分离鉴定,进而深入研究其致病机理可为今后制定控制措施提供理论依据。本研究对引起西兰花采后黑斑病的主要病原真菌进行了形态学和分子生物学的鉴定,并对该菌促进西兰花衰老黄化的机理进行了探讨。研究结果表明:1.对上市销售的西兰花的调查发现,黑斑病是西兰花销售过程中最常见的采后病害之一,西兰花的采后黑斑病往往与其花体的衰老相伴,感染黑斑病的花球会快速衰老黄化,失去商品性。2.根据柯赫氏法则,对分离自西兰花黑斑病的病原进行致病性鉴定的结果表明,导致西兰花采后病害的主要病原为链格孢属真菌,其中菌株HLYB-4为引起花球黑斑病的主要病原菌,该菌的菌落呈青褐色,分生孢子青褐色、手雷状呈链状排列,大小约为24～51.5×7.5～23μm,2～6个横隔,0～3个纵隔,喙部不发达,为单细胞假喙,形态学初步鉴定为芸苔生链格孢菌(Alternaria brassicicola)应用rDNA-ITS序列比对法和十字花科链格孢属菌的特异标记引物Abra1和Abra2扩增鉴定进一步将引起西兰花采后黑斑病病原定名为A.brassicicola.3.对西兰花黑斑病病原A.brassicicola的适宜生长温度和pH的研究表明,该菌生长的最适温度为25℃,最适pH5～8。5℃低温不能完全阻止其菌丝生长和孢子形成,而35℃可有效地抑制该菌在体外和植物体上的生长和产孢;A.brassicicola的分生孢子在pH2～12均可萌发,在pH5～9三小时内萌发率接近100%。此外,50ppm的外源乙烯能够促进其孢子萌发。4.对接种A.brassicicola西兰花的生理代谢指标测定结果表明,接种第72小时的花球开始黄化,叶绿素迅速降解,乙烯释放量和呼吸速率显著升高,接菌组乙烯释放高峰比对照组提早24小时形成。然而,西兰花采后Vc的迅速下降与病原菌侵染没有显著的对应关系。5.对西兰花衰老调节基因转录水平的分析表明,接种A.brassicicola后西兰花的ACC氧化酶基因BoACO2比对照组提前24小时上调表达;ACC合成酶基因BoACS2也在接种后的72小时内明显上调;叶绿素分解酶基因BoCLH1在前72小时的转录活性也明显高于对照组,生理生化分析与分子证据证明A.brassicicola是诱导西兰花采后的黄化衰老的重要因素之一。上述研究结果显示,提高西兰花采后贮藏的品质,延长货架期必须严格控制西兰花的采后黑斑病菌A.brassicicola的发生。更多还原

【Abstract】 Broccoli(Brassica oleacea L.var.italica) is nutritional and regarded as one anti-cancer vegetable.However,it is vulnerable to various pathogens and senescences rapidly after harvest,which remarkably shortens its market life.So it’s meaningful to separate and identify these broccoli post-harvest pathogens so as to make effective control protocol.Our research identified the black spot disease pathogen of post-harvest broccoli with the help of morphological and molecular methods and explored its contribution to the senescence of the plants.The results are as follows,a.According to our market research,Black spot disease seems to be one of the most common diseases on post-harvest broccoli.This pathogen is also related with the aging of the plants.The infected plants senescenced rapidly and lost its market value.b.According to Koch’s Postulates,pathogens on post-harvest broccoli are identificated as Alternaria spp.And strain HLYB-4 was proved to be the cause of the black spot,which produced a green-brown colony and whose spores are 24 to 51.5×7.5 to 23μm grenade-like with two to six transverse septa,zero to three mediastinal septa and fake rostrum.It was identified as Alternaria brassicicola by morphologically analysis and further proved by the rDNA-ITS blast and particular primers Abral andAbra2.c.The optimal temperature and pH of A.brassicicola are 25℃and pH 5-8 respectfully.Low storage temperature(5℃) can’t block its hypha growth and sporation totally.However,high temperature(35℃) seems to effectively restrain its growth and sporation both in vitro and in vivo.In addition,its spores could germinate from pH 2-12 and the germination rate reached 100%within 3 hours at pH 5-9.Moreover,50ppm exogenous ethylene seems to contribute to its germination.d.After pathogen inoculating on the healthy head of broccoli,obvious yellowing could be seen within 72 hours with the degradation of its chlorophyll.High ethylene output and respiration rate could be measured while the inoculated group could be tested 24 hours earlier than the control.However,the rapid descendant of Vitamin C seems not to be related with the infection.e.The RT-PCR result indicates that the transcription of Bo-ACS2 enhanced in the first 72h in the inoculated group and Bo-ACO2 have up-regulated in the first 24 hours in the inoculated group as well,which is one day earlier than the control group.And the transcription of Bo-CLH1 of infected group in the first 72 hours is obviously higher than the control group.So both physiological and molecular results indicated that A.brassicicola induced the senescence of post-harvest broccoli.Above all,in order to improve the preservation of broccoli and extend its market life,it is critical to prevent the occurrence of black spot caused by A.brassicicola.更多还原