【作者】 于佰双；

【导师】 段玉玺；

【作者基本信息】 沈阳农业大学 ， 植物病理学， 2009， 博士

【摘要】 大豆起源于中国,我国种植大豆已有5000年历史。我国大豆栽培区域概括为北方春大豆区,黄淮海春夏大豆区和南方多熟制大豆区。大豆胞囊线虫（Heterodera glycinesIchinohe,Soybean Cyst Nematode,简称SCN）病害是世界大豆生产上的重要病害。1899年俄国科学家雅切夫斯基在我国东北首次发现了大豆根线虫（即大豆胞囊线虫）。直到1915年日本首次公开报道了该病害,而后朝鲜（1936年）、美国（1954年）、埃及（1968年）、原苏联（1978年）、哥伦比亚（1983年）、印度尼西亚（1984年）、加拿大、巴西、阿根廷等国家相继报道该线虫的发生和危害。在我国,SCN主要分布于东北和黄淮海2个大豆主产区;东北大豆主产区的黑龙江省、吉林省、辽宁省、内蒙古自治区及黄淮海大豆主产区的山东、河北、山西、安徽、河南、北京等省市。尤其东北地区多年连作大豆的干旱、盐碱地发生严重,黑龙江省是我国北方春大豆重要产区,大豆胞囊线虫病是大豆生产上的主要病害之一。由于大豆连作面积扩大和时间的延长,大豆胞囊线虫病在黑龙江省有蔓延趋势。目前黑龙江省SCN发生面积达66.7万hm²以上,是仅次于大豆花叶病毒病的第二大病害。大豆受其危害后,轻者减产20%-30%,严重的减产达到70%-80%,并且每年都有大面积地块绝产。目前选育抗病品种是最广泛应用的防治措施。本研究立足于提高黑龙江省大豆产量、降低大豆胞囊线虫对黑龙江省大豆生产带来的危害,在黑龙江省大豆胞囊线虫生理小种分布;抗病基因对大豆胞囊线虫3号生理小种的选择作用;黑龙江省主要大豆抗源对胞囊线虫抗性遗传;大豆抗胞囊线虫的分子标记辅助证明;抗感SCN3号生理小种的大豆品种及后代受SCN侵染后根尖超微结构变化,大豆胞囊线虫防治技术体系6个方面进行研究。取得以下研究进展:1.大豆胞囊线虫3号生理小种仍然是黑龙江省大豆胞囊线虫的优势小种,大庆市和安达市由原来SCN3号生理小种分别变为4号和14号生理小种,表明黑龙江省SCN生理小种稳中有变;绘制了黑龙江省大豆胞囊线虫分布图,明确了黑龙江省SCN生理小种的类型和分布,为抗线育种及抗线品种合理布局提供依据。2.在盆栽条件下,强迫SCN3号生理小种的群体在抗病品种上繁殖10代,应用Golden等使用的一套鉴别寄主进行鉴定。原来为3号生理小种的线虫群体,经在抗病品种抗线1号、抗线2号、抗线3号、抗线4号、抗线5号上连续选择10代之后,变为6号小种;经在灰皮支黑豆上选择之后变为10号小种;经在Peking上选择之后变为14号小种,经在哈尔滨小黑豆上选择变为15号小种。上述鉴定结果说明,原大豆胞囊线虫生理小种群体,经过在抗病品种上连续强迫繁殖后,形成新的生理小种,并使原抗病品种变为感染品种,认为在生产上采用轮作方式是保持抗线品种抗性稳定性的有效途径。3.合丰25×抗线4号正反交组合的F₂群体抗感分离遗传分析表明,抗线4号对胞囊线虫3号生理小种的抗性受三对隐性等位基因控制;大豆对大豆胞囊线虫3号生理小种抗性的遗传不存在细胞质效应;在抗线育种中选育兼抗品种难度大,但通过分子标记辅助选择来进行基因聚合选育兼抗品种是可行的途径。该结果为抗SCN育种提供了遗传基础,有利地指导了抗SCN育种工作。4.抗病品系哈98-4598在Satt082位点上具有与抗病亲本抗线4号不同的等位变异,这可能是由于两者具有不同的抗病基因,在Satt082位点上,感病亲本合丰25与感病后代材料3（合丰25×抗线4号F₂）、材料12（合丰25×抗线4号F₃）均有相同的第三个等位变异位点,母体效应在这两种材料中表现更为显著;在Satt309位点上,存在两个等位变异,供试材料1、3、5、9、10、11和12存在第一等位变异位点,供试材料2、3、4、7、8、11存在第二个等位变异位点,而3、11号材料同时存在两个等位变异位点;根据两个位点上的5个等位变异的聚类结果表明,在合丰25×抗线4号正反应组合中,抗线4号与7、8号抗病后代聚为一类,感病亲本合丰25与感病材料3被聚为一类,则将抗感材料分开。其它后代材料则聚为一类,即聚类结果没有将抗感材料分开,这可能是由于本研究采用的引物数量较少,未能充分揭示供试材料间的遗传变异,也可能是这些杂交后代仍存在对SCN3生理小种的抗性分离。5.在黑龙江省内首次报道了大豆抗感SCN亲本（抗:抗线4号和哈98-4598;感:合丰25）受SCN3号生理小种侵染后根尖细胞超微结构变化存在明显差异,这些抗感亲本被大豆胞囊线虫3号生理小种侵入后的细胞和组织病理学反应,揭示了大豆抗感胞囊线虫的机制;大豆杂交后代受SCN3号生理小种侵染后根尖细胞超微结构,不同组合及同一组合个体间存在明显差异,合丰25×抗线4组合F₁代的表现,说明大豆对大豆胞囊线虫3号生理小种的抗性遗传可能主要受细胞核控制与细胞质无关,这与SCN抗性遗传合丰25×抗线4号正反交F₁代结果表现没有细胞质效应相一致;但是,合丰25×哈98-4598正反交组合F₁代表现趋向母本反应与细胞质效应有关;这一结果尚未见报道,受SCN侵染后根尖,在感病品种合丰25形成明显增长的合胞体,而在抗病品种抗线4号合胞体细胞较小,退化中空,细胞质降解,细胞质膜与细胞壁发生分离,细胞器稀少,抗病品种不能为侵入的线虫提供养分,使其发育不良或死亡,这是大豆对大豆胞囊线虫的抗病机制之一。6.综合前人和本研究结果,在黑龙江省首先提出了大豆胞囊线虫防治技术体系（1）将大豆、胞囊线虫及自然环境视为一体,进行生态调控,改善大豆生长发育的生态环境,生长季节按需灌溉,改良土壤,培肥地力,进行土壤耕暄,深松,为大豆生长发育创造一个良好的耕层,建立轮作体系,其中,采用拮抗植物,例如万寿菊做为轮作植物,能有效抑制大豆胞囊线虫的发生,减轻危害。避免大豆重迎茬种植,使大豆生产进入良性生态循环。（2）掌握大豆胞囊线虫的发生规律和危害特点,采取播种时将杀线剂与肥料一同施入土壤。为了减少对环境的污染,应选择低毒杀线剂或种衣剂,并适当减少使用剂量,在大豆初花期采取叶面施肥（惠满丰活性液肥等）缓解胞囊线虫危害,以保证大豆植株健康生长。（3）充分利用天敌微生物,组装联合调控技术,实现以菌治虫的生物调控,人为增加豆田有益菌的数量,这些菌可寄生到胞囊上,被寄生的胞囊不能正常孵化,目前应用的主要有豆丰1号（厚垣轮枝菌）5kg/667m²和大豆根保菌剂,防治效果60%左右,降低大豆产量损失15%以上。（4）选用抗线品种,抗病品种的应用是防治大豆胞囊线虫病的最经济有效措施之一。建议在黑龙江省安达地区主要推广抗线2号、抗线3号、抗线4号,在嫩江地区推广嫩丰15号。在抗线育种中应选育水平抗性和多抗品种,以防止抗病品种抗性丧失。更多还原

【Abstract】 Soybean originated in China which has 5,000 years’ history of soybean cultivation. Generally speaking, soybean cultivation area in China could be normally distinguished the Northen spring soybean area, Huang-Huai-Hai spring - summer soybean area and southern multiple cropping soybean area in China. Soybean Cyst Nematode （ SCN） , Heterodera glycines Ichinohe, is the major diseases of soybean production worldwide. In 1899, Russian scientist Jaczevski found soybean root nematode （Heterodera glycines ）in the northeast of China. In 1915 , the disease was firstly reported in Janpan, followed by North Korea （1936）, the United States （1954）, Egypt （1968）, the former Soviet Union （1978）, Colombia （1983）, Indonesia （1984）, Canada, Brazil, Argentina and other countries have reported the occurrence of the nematodes and hazards. In our country, SCN is mainly distributed in the Northeast and Huanghuaihai soybean-producing areas; Heilongjiang, Jilin, Liaoning, Inner Mongolia Autonomous Region in the Northeast soybean-producing area and Shandong, Hebei, Shanxi, Anhui , Henan, Beijing and other provinces and cities in the Huang-Huai-Hai soybean-producing area,especially for the northeastern region. To Heilongjiang Province-China’s major northern soybean producing areas, Soybean Cyst Nematode is one of the major diseases in Soybean production. Due to the expansion and extension of time for continuous cropping of soybean, the trend of Soybean Cyst Nematode has spreaded in Heilongjiang Province . At present, 667000 ha of soybean production area suffered with SCN, which is just next to soybean mosaic virus disease as the second-largest. SCN could cause 20% -30% yield reduction normally, 70% -80% yield loss some circumstance and enven worse, production lost on large-scale could be found every year.At present, breeding for disease-resistant varieties is the most widely used prevention and control strategy. This study will be based on the following 6 aspects: enhancing and reducing the damage on the production of soybean in Heilongjiang Province, which brought by soybean cyst nematode and soybean cyst nematode races distribution in Heilongjiang Province; the selecting function for resistance gene to Soybean Cyst Nematode race 3 ; the inheritance of resistance of the main soybean resistance resource to cyst nematode in Heilongjiang Province ; the molecular marker-assisted proof for the resistance of soybean to cyst nematode; ultrastructural changes in the root tip of resistant and susceptable soybean varieties and their progenies after the SCN race 3 infection as well as soybean cyst nematode control system. The study obtained the following process:1. The dominante race of SCN is still race 3 in Heilongjiang Province. But the SCN race 3 changed into race 4 and 14 in Daqing City and Anda City, indicating some changes happened in Heilongjiang. A distribution map of soybean cyst nematode in Heilongjiang was drawn , which made clear the style and distribution of SCN race in Heilongjiang, and provide the basis for soybean breeding for SCN resistance species and the rational distribution of resistant cultivars.2. Soybean cyst nematode （SCN）, Heterodera glycines Ichinohe, populations were selected on eight soybean [Glycine max （L.） Merr.] cultivars in SCN race 3 infested soil. This was done to isolate any populations within SCN race 3 field populations which could reproduce successfully on soybean cultivars with SCN race 3 resistance. The populations were selected by growing the soybean cultivars in infested soil for 20 days, then plant roots were cleaned with water and moved to the soil without SCN .After 10 generations of selection, each soil sample was tested for SCN race type. The race designation of the SCN population changed from Race 3 to Race 6 when resistant Kangxian 1, 2, 3, 4, 5 were grown, and from Race 3 to 10, 14 ,15 when Huipizhiheidou ,Peking and Harbinxiaoheidou were grown respectively. The results indicated that the original soybean cyst nematode race groups form the new race after continuous forced selecting on disease-resistant varieties,and changed the original disease-resistant varieties into susceptible varieties. Adopting rotation mode in production is saw as the effective way for maintaining the stability of resistant varieties.3. Genetic analysis of SCN resistance segregation in F₂ of Hefeng 25×Kangxian 4 reciprocal cross shown that the resistance for Kangxian 4 to SCN 3 is controlled by three pairs of recessive alleles. The cytoplasmic effect did not exist in the genetic resistance for soybean to SCN 3. Soybean breeding for multiresistant to SCN is very difficult, but molecular marker-assisted selection is a feasible way which could help to pyramid multigenes from different resistant sources. The results provided genetic base for SCN resistance breeding, and the guidance of SCN resistance breeding.4. Resistant soybean line Ha98-4598 has different allele variation on Satt082 from resistance Kangxian 4,this may be due to two different resistance genes. On the Satt082, Susceptible parent Hefeng 25 and its susceptible progeny material 3（Hefeng 25×Kangxian 4 ）F2、material 12（Hefeng 25×Kangxian 4） F3 have the third allele variation locus, maternal effect shows more significant in these two materials. There are two allelic variations existed on the Satt309, the test materials 1,3,5,9,10,11 and 12 have the first allelic variation locus,the test material 2,3,4,7,8 ,11 have the second allelic variation locus, while the test materials 3,11 exist two allelic variation points at the same time. According to the clustering analysis results of 5 allele variation on the two loci, in the Hefeng 25×Kangxian 4 reciprocal cross, Kangxian 4 and resistant offspring 7,8 were in the same class, susceptible parent Hefeng 25 and susceptible material 3 were in another class, so the materials could be separated into resistance and susceptable. Other materials in the progeny are clustered in one class, that is the result of clustering did not separate the susceptible and resistant materials, which may be caused by the small number of primers and failed to fully reveal the genetic variation of test material, and may also for the reason of these progeny materials still existing on SCN3 resistance segregation.5. It is firstly reported that the significant differences of root tip cells ultrastructure changes existed after resistante and susceptible soybean parents were infected by SCN in Heilongjiang Province （resistance: Kangxian 4 and Ha 98-4598;susceptible: Hefeng 25 ）.The materials infected by SCN3,which invasive cells and histopathological response to soybean cyst nematode race 3,shown soybean cyst nematode resistance mechanism , Soybean hybrids infected by SCN 3,root tip cells ultrastructure, and the same cross and different crosses individuals existed significant differences . （Hefeng 25×Kangxian 4） F₁ shown that the genetic resistance of soybean to soybean cyst nematode race 3 may mainly be controlled by nucleus and has nothing to do with the cytoplasm. This result is confirmed with the result of SCN inheritance of resistance of （Hefeng25xKangxian4）reciprocal hybrid F₁ which shown no cytoplasmic effect. However, F₁ of （Hefeng 25×Ha 98-4598） shown the trend to parent reaction,which is relative to cytoplasmic effect. The root tip infected by SCN,and formed obvious growing syncytial in infected varieties Hefeng 25,while smaller cells,degradation hollow cytoplasmic degradation,cell membrane and cell wall separation,less cell path;Resistant varieties Kangxian 4 can not provide nutrients to invasive nematode and make them dysplasia or death,this is one of resistance mechanism of soybean to soybean cyst nematode.6. Intergrated and the results of this study and former research conducted by other scientists, the soybean cyst nematode control system is firstly proposed in Heilongjiang Province:（1） Insist on making the soybean cyst nematode and the natural environment as an integral whole part, adopting ecological control, improving soybean growth and ecological environment, enhance the system of irrigating and draining, improving the soil fertility, soil tillage and deep tillage in order to create a good tilth for soybean growth and development, establish crop rotation system to avoid soybean continuous cropping cultivation, so that soybean production into the benign ecological cycle.（2）According to the occurrence rules and characteristics of soybean cyst nematode, due to the soybean high planting density and large area, Pharmacy irrigation is not feasible. Therefore,put the nemacide with fertilizers into soil together when planting so that kill the larvae hatch. In order to reduce the pollution of the environment , a low toxic nemacide or seed coating should be selected, and reduce the dose appropriately. Applying the leave fertilizer （huimanfeng liquid fertilizer et.al）during the early flowering period to ensure healthy growth of soybean plants.（3） Make full use of natural microorganisms, assemble joint control technology, realize the biological control of making bacteria to control pest, artificially increase the number of soybean fields. These bacteria can be parasitic on the cyst, which can not hatch normally. The current applications mainly contain Doufeng No. 1 （thick fungi Verticillium Tanzania） 5kg/667m² and soybean root-holding-bactoria agents. All these can get about 60% of control effect, and reducing soybean yield loss of more than 15%.（4）The selection of SCN-resistant soybean varieties. The application of disease-resistant varieties is one the most of cost-effective measures for controlling the Soybean Cyst Nematode. Our suggestion to Heilongjiang Province: planting kangxian 2, 3, 4 in Anda area in and Nenfeng 15 in Nenjiang area. In resistant breeding for SCN, level resistance and multi-resistant varieties should be used to protect soybean resistance for the future .更多还原