【作者】 刘超；

【导师】 吴海燕；

【作者基本信息】 山东农业大学 ， 植物病理学， 2012， 硕士

【摘要】 小麦孢囊线虫病己在世界范围内造成严重的危害,是制约小麦和大麦生产的主要因素之一。各地区地理位置、气候条件不同,小麦孢囊线虫群体的孵化侵染特点也不同。为明确山东泰安小麦孢囊线虫(cereal cyst nematode, CCN)群体对环境的响应,2010年和2011年对自然、室内条件下小麦孢囊线虫群体的孵化特性,小麦孢囊线虫群体对种植模式的响应,不同小麦品种对小麦孢囊线虫的抗病性进行了系统的研究,并对小麦孢囊线虫滞育卵与非滞育卵全蛋白进行差异比较。研究结果如下：1.小麦孢囊线虫的孵化对人工和自然条件改变的响应小麦孢囊线虫孢囊卵和自由卵的孵化对温度处理的响应不同。孢囊卵的孵化最适温度处理为4℃低温-30d,然后转入15℃中温处理,其次是15℃恒温处理,两处理间差异不显著。卵孵化最适条件为15℃恒温,且显著大于其他处理。其次为4-C低温-30d转入15℃中温处理。4℃恒温处理的孢囊卵和自由卵均无孵化高峰出现。小麦孢囊线虫孵化与外界环境温度的变化相适应。4月土壤中孢囊绝大多数为空孢囊,未进行孵化试验,11月至次年3月,田间孢囊置于15℃下孵出的J2明显高于其他月份。4月份开始有少量白色孢囊形成,8月份土壤中新孢囊数量达全年最高值。土壤中空孢囊与总的孢囊量的全年变化趋势大体一致。6月土壤中新孢囊及空孢囊较多,出现峰值。随着秋季气温的降低,土壤中J2开始渐渐增多,3月J2数量达到全年高峰值,显著高于其他月份。总体来讲,全年孢囊内卵的数量要多于J2数。4月、5月新孢囊内无J2,夏季滞育期间,孢囊内有J2,但不活动。在小麦生长期间,10月至次年3月,孢囊内的卵呈下降趋势,J2数量变化不明显。2.小麦品种(系)对Heterodera avenae群体的抗性鉴定供试20个小麦品种中无高抗(HR)和中抗(MR)品种,所有品种表现高度感病(HS)。在接种量相同的条件下,小麦根系的生长状况与最终形成的孢囊量没有明显相关性。3.小麦孢囊线虫群体对种植模式的响应本试验是种植模式循环体系中第一年,非寄主作物播前收后各种植模式间孢囊线虫群体减少差异不显著,两季小麦播种前各处理小麦孢囊线虫群体的变化差异不显著。为明确种植模式对小麦孢囊线虫群体数量的影响,正继续试验。4.小麦孢囊线虫滞育与非滞育卵全蛋白差异比较试验根据小麦孢囊线虫的性质及实验条件,统一采用机械匀浆法破碎孢囊,选用7种全蛋白提取试剂提取小麦孢囊线虫全蛋白,进行电泳分析。通过比较电泳谱带及提取经济适用性,得出E(20%蔗糖,50mM Tris-HCl(pH7.1),0.5%Triton X-100,0.1%SDS)为提取小麦孢囊线虫全蛋白较优的提取试剂。运用提取试剂E提取线虫全蛋白,进行SDS-PAGE电泳,比较滞育、非滞育小麦孢囊线虫全蛋白差异。滞育小麦孢囊线虫蛋白总量最高,4℃低温处理CCN蛋白含量最低。从蛋白谱带数量上看,滞育孢囊蛋白电泳图谱及4℃-40d处理均得到33条蛋白谱带,经升温的滞育解除处理得到的蛋白条带数最多。与滞育线虫蛋白相比,4℃-40d处理缺少分子量为200KD蛋白谱带,多出12.69KD的小分子量蛋白谱带；4℃-40d,转入15℃-7d处理,多5条蛋白带；在解除滞育的两处理中,4℃-40d,转15℃处理比4℃-40d处理多5条蛋白带。尿素直接提取法提取小麦孢囊线虫全蛋白,2-D电泳后,经ImageMasterTM2D Platinum5.0软件分析,两组电泳图谱共检测到230个蛋白点,非滞育线虫蛋白2-D电泳图谱检蛋白点109个,滞育线虫蛋白则检测到蛋白点121个。相互匹配的蛋白点有90对,匹配率为74.38%；特异性蛋白点共31个,其中滞育孢囊有14个,非滞育有17个。更多还原

【Abstract】 The Cereal Cyst Nematode (CCN) has caused important crop damage all over the world, and has been one of the major constraints on wheat and barly production. Hatch of H. avenae varies according to region and climate. In order to make clear the responses of H. avenae from Tai’an to environment, experiments were carried out in vitro and field on hatching of second stage larvae (J2) for the Tai’an population of H. avenae, responses of H. avenae to different planting patterns, wheat cultivars’ resistance against H. avenae, and comparison of total protein on diapause and non-diapause H. avenae in2010and2011. The main results as follows:1. Response of CCN to nature and artificial condition changesResponse of eggs in CCN cysts was different from free eggs to the same temperature treatments. The optimum temperature treatment for emergence of J2from cysts of H. avenae was15℃after pre-treated at4℃for30d, then the treatment of15℃constant temperature, and there was no significant difference between the two treatments. J2hatched the most at15℃for free eggs at different temperature treatments and was significantly higher than other temperature treatments, then the treatment of15℃after pre-treated at4℃for30d. There were no hatch peaks at4℃constant temperature treatment for free eggs and eggs in cyst.Hatch of H. avenae was adapted to changes of external temperatures. Cysts in soil were almost empty in April, so it was removed from hatch experiments. Cysts collected from the field treated at15℃had high emergence rate from November to March the next year and were significantly higher than the other months of round year.A little white females produced in April, and number of new cyst reached highest value in August. Number changes of empty cysts in soil were similar to that of total cysts in round year. Numbers of empty and new cysts were high in June and peak appeared in the same month. The number of J2in soil was increased as temperature dropped, reached peak value in March and significantly higher than the leftover11months. In general, number of eggs was higher than that of J2in cysts in one year. There were no J2s in new cysts in April and May. J2s existed in cysts during CCN diapause in summer and were inactive. Number of eggs in cysts was downtrend from October to March next year, while number of J2s has no significant changes.2. Identification of wheat variety resistance to Heterodera avenaThere were no resistance varieties to Heterodera avenae among tested wheat variety (line)., all cultivars were high susceptable (HS). In the present study, there was no relative between growth character of wheat root and numbers of new cysts under the same inoculation number of J2.3. CCN response to different planting paternsThe experiment was the fist year of planting pattern cycle systems, there were no significant difference at decreased number of CCN before nonhost crops seeding and harcestion, so were at two different wheat sowing period. Experiment is being studied in order to make clear CCN response to different planting paterns.4. Comparision of total protein between diapause and non-diapause CCN eggsCCN cell crushed with grinding homogenate method accronding to character of cyst and experimental conditions.7protein extraction reagents were used to extract total protein, and then examined by SDS-PAGE. E (20%sucrose,50mM Tris-HCl (pH7.1),0.5%Triton X-100,0.1%SDS) was selected for further experiment by coparing electrophoretic band and extraction cost between7protein extraction reagents.Differences of total protein between diapause and non-diapaues CCN eggs were examined by SDS-PAGE and compared using reagent E. Total protein content was highest in diapause CCN eggs, and lowest in new CCN eggs at4℃-40d treatment.33electrophoretic bands were examined in diapause CCN eggs, so were in CCN eggs at4℃-40d treatment, while the most electrophoretic bands were examined in treatment of new CCN eggs placed at4℃-40d then transferred to15℃for7days. There was no200KD protein band and existed12.7KD protein spectrum in CCN eggs of treatment at4℃-40d; There was5more protein bands in treatment of CCN eggs placed at4℃-40d then transferred to15℃for7days compared with diapause CCN egg protein.5protein bands were exist in treatment of4℃-40d then transferred to15℃-7d, while none in treatment of4℃-40d.Total protein of CCN eggs was extracted using urea direct extraction solution, then was investigated by two-dimensional electrophoresis (2-DE) and image analysis software ImageMaster M2D Platinum5.0. A total of230protein spots were detected in the2-DE pattern of diapause and non-diapause CCN eggs, of which109belonged to non-diapause eggs and121belonged to diapause eggs. Comparison showed that90pairs were matched in the2-DE patterns of both diapause and non-diapause CCN eggs, with the match percentage as high as74.4%. There are31specific protein spots, of which14protein spots specific to diapause and17specific to non-diapause eggs.更多还原