Characterization and phylogenetic analysis of the chitinase gene from the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus

A putative chitinase gene was identified within the fragment EcoRI-K of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HearNPV, also called HaSNPIV) genome. The open reading frame (ORF) contains 1713 nucleotides (nt) and encodes a protein of 570 amino acids (aa) with a predicted molecular weight of 63.6 kDa. Transcription started at about 18 h post infection (p.i.) and the protein was first detected at 20 h p.i. The times of transcription and expression are characteristic of a late baculovirus gene. 5' and 3' RACE indicated that transcription was initiated from the adenine residue located at -246 nt upstream from the ATG start site and the poly (A) tail was added at 267 nt downstream from the stop codon. This is the first report on the molecular characterization of a chitinase from a single nucleocapsid NPV. The phylogeny of baculoviral chitinase genes were extensively examined in comparison with chitinases derived from bacteria, fungi, nematode, actinomycetes, viruses, insects and mammals. Neighbor-joining and most parsimony analyses showed that the baculoviral chitinases were clustered exclusively within gamma-proteobacteria. Our results strongly suggest that baculoviruses acquired their chitinase genes from bacteria. (C) 2004 Elsevier B.V. All rights reserved.

Some nematodes of fishes from central China, with the redescription of Procamallanus (Spirocamallanus)fulvidraconis (Camallanidae)

The present paper comprises a systematic survey of nematodes based on helminthological examinations of 176 specimens of freshwater fishes, belonging to 22 species, from central China (mostly lakes in Hubei Province) collected during the autumn of 2001. The following six species were recorded: Procamallanus (Spirocamallanus) fulvidraconis Li, 1935, Camallanus cotti Fujita, 1927, Dentiphilometra monopteri Moravec et Wang, 2002, Pingus sinensis Hsu, 1933, Proleptinae gen. sp. larv., and Eustrongylides sp. larv. Data on their morphology, morphological variability, host range, prevalence, intensity and distribution are provided. SEM studies of P. fulvidraconis and larval Physalopterinae, used for the first time in these species, revealed some additional morphological details and made it possible to redescribe the former. In contrast to the existing description of P. fulvidraconis, this species was found to possess two spicules and a V-shaped gubernaculum with unequal arms (originally mistaken for the left spicule), as well as deirids, whose location can be considered an important taxonomic feature. Larvae of the Physalopterinae have not previously been reported from fishes in China. The finding of larval Eustrongylides in Paramisgurnus dabryanus represents a new host record. All but one nematode species from this zoogeographically interesting region are briefly described and illustrated.

Advances in phylogenetic studies of Nematoda

Nematoda is a metazoan group with extremely high diversity only next to Insecta. Caenorhabditis elegans is now a favorable experimental model animal in modern developmental biology, genetics and genomics; studies. However, the phylogeny of Nematoda and the phylogenetic position of the phylum within animal kingdom have long been in debate. Recent molecular phylogenetic studies gave great challenges to the traditional nematode classification. The new phylogenies not only placed the Nematoda in the Ecdysozoan and divided the phylum into five clades, but also provided new insights into animal molecular identification and phylogenetic biodiversity studies. The present paper reviews major progress and remaining problems in the current molecular phylogenetic studies of Nematoda, and prospects the developmental tendencies of this field.

Two new species of Actinolaimidae thorne, 1939 (Nemata : Dorylaimida) from China

Two new species of Actinolaimidae are described from China. Trachactinolaimus brevicaudatus n. sp. is 3.4-4.4 mm long; a = 40-60, b = 3.5-5.2, c = 20-21 in female and 34-39 in male; odontostyle length is 29-33 mu m; spicules are 67-77 mu m long; and the stoma has four onchia with numerous mural denticles. The female has a longitudinal vulva, and the male has 20 to 23 contiguous ventromedian supplements. Egtitus sinensis n. sp. is 1.7-2.2 mm long; a = 24-33, b = 3.1-3.9, c = 16-19 in female and 0.7-0.9 in male; odontostyle length is 25-29 mu m; spicules are 55-56 mu m long; and the prerectum 53-77 pm long. The cardia is short and blunt conoid, 13-19 mu m long. The male has 12 to 13 ventromedian supplements at intervals of 2-3 mu m.

松辽平原农田土壤线虫多样性研究

本研究立足于松辽平原农田生态系统，研究土壤线虫物种多样性，营养类群多样性，生活史多样性和功能多样性的分布规律，为进一步揭示线虫主要功能类群与土壤C、N等土壤环境因子之间的相互关系提供基础性数据。 在松辽平原沿不同纬度梯度，选择北至黑龙江海伦，南至辽宁大石桥共7个地点的玉米地，按照0–20 cm、20–40 cm、40–60 cm、60–80 cm和80–100 cm五个层次分层采集土壤样品。研究发现，土壤线虫多样性的纬度分布格局在0–40 cm土层表现得比较明显。在不同农田生态系统中，线虫属的丰富度在0–100 cm的土壤剖面内，随着土层深度的加深而降低；在0–40 cm土层，线虫属的丰富度随纬度梯度的降低而表现出增加的趋势，最高值（18属）出现在大石桥的0–20 cm土层，最低值出现在海伦采样点的20–40 cm土层，仅观察到10个线虫属。通过对松辽平原农田土壤线虫的生活史策略组成进行研究发现，在不同采样地点，不同生活史的土壤线虫表现出不同的分布特征：在0–20 cm土层，cp-2类群线虫的相对多度随纬度的降低表现出降低的趋势，而cp-3-5的线虫则表现出增加的趋势。在不同土层研究发现， 线虫的营养多样性指数没有表现出明显的纬度分布格局，而在0–20 cm土层，食细菌线虫和捕食/杂食线虫的相对多度随纬度的降低表现出降低的趋势，而植物寄生线虫则随纬度的降低而呈增加的趋势。土壤线虫区系分析结果表明，海伦样点土壤食物网受到的扰动较小，而处于结构化状态；而哈尔滨，公主岭和沈阳采样点的土壤食物网处于退化状态，受到外界环境扰动较大。通过对松辽平原农田土壤线虫群落与理化因子的典型对应分析，可以看出相关的土壤理化性质如阳离子交换量、有机碳、全氮和粘粒含量等能够在一定程度上解释松辽平原农田土壤线虫群落的分布特征。

辽河油田采油井场污染土壤线虫群落特征研究

通过对辽河油田新老两个年限井场的污染状况开展调查研究，对比不同开采年限井场土壤石油总烃、重金属污染状况及线虫群落分布特征，为油田污染土壤评价和治理提供科学依据。研究结果表明，油田井场污染土壤的石油烃和重金属均高于对照，老井场的污染程度重于新井场。井场污染土壤石油总烃含量（TPHs）均随土层深度增加而降低，而重金属含量总体随土层深度增加而增加。与夏季相比，土壤有效态重金属含量有降低的趋势。 对照的苇田土壤线虫优势类群为植物寄生线虫，而井场污染土壤则为食细菌线虫，且其相对多度随污染程度加重而增加。井场污染土壤线虫属的数目显著减少，且随污染程度加重而减少。苇田土壤优势属为Helicotylenchus，井场污染土壤为Cuticularia。在重污染老井场土壤中发现Seinura线虫，且数量明显多于新井场，可能说明Seinura受长期污染而驯化。 井场土壤污染一定程度上促进了cp1线虫数量及相对多度增加，而对cp2、cp3-5线虫有明显抑制作用（尤其cp3-5线虫）。 井场和对照土壤线虫数量总体上均表现为随土层深度加深而减少的趋势，表层土壤夏季高于秋季，下层则表现出秋季高于夏季的趋势。 井场土壤污染导致线虫群落在属水平上的生物多样性以及丰富度和均匀度降低，且随污染程度加重而降低；优势度指数则表现为随污染程度加重而升高的趋势。两井场污染土壤线虫群落均受到高度干扰，趋于富集化，线虫种类趋于单一，稳定性下降。

沿城乡梯度土壤重金属和线虫群落时空分布研究——以沈阳南部为例

城市化及其生态效应是当前国际上主要研究热点之一。本文通过在沈阳南部沿城乡梯度采集0~10 cm、10~20 cm和20~30 cm三个层次土壤样品，研究城市化效应对土壤重金属和土壤线虫群落时空分布的影响，探讨了土壤线虫对重金属污染的指示作用。 研究发现，沿城乡梯度土壤重金属Cu、Zn、Pb和Cd全量及有效态含量呈现明显的梯度效应，说明城区比农村重金属污染严重。土壤线虫总数在不同采样时期、不同采样点和不同土层表现出显著差异，沿城乡梯度显著增加，并且随土壤深度的增加而显著减少。土壤线虫属的丰富度、香农-威纳多样性、优势度和均匀度指数也表现出显著的时空差异性。不同取样季节线虫各营养类群表现出极其显著的时间差异。城市土壤处理植物寄生线虫相对多度明显低于郊区和农村土壤处理，而食细菌线虫的相对多度显著高于郊区和农村处理。通过线虫区系分析可以看出，土壤食物网和环境状态沿城乡梯度呈现明显不同的分布格局。 通过对土壤线虫群落分布与有效态重金属分布的相关性研究可知，城市化所造成的土壤重金属污染主要对线虫总数、植物寄生线虫数量以及线虫群落多样性指数产生了显著影响，而对土壤线虫食物网相关指数的影响不显著。以上结果也表明，土壤线虫对城市化带来的重金属污染具有一定的指示作用。

重金属对矮小拟丽突线虫的毒性效应研究

为了探讨重金属对矮小拟丽突线虫（Acrobeloides nanus）的毒性作用，本文采用室内培养的方法，研究了不同浓度Cu、Zn、Pb、Cd对矮小拟丽突线虫死亡率、生长发育和繁殖的影响，并对线虫世代间的毒性效应进行了比较。结果表明：1）Cu、Zn、Pb、Cd对矮小拟丽突线虫24 h的半致死浓度LC50值分别为 0.80、71.20、12.94 和 1.42 mg • L-1，48 h 的半致死浓度LC50值分别为 0.71、35.08、2.65 和 0.32 mg • L-1。不同重金属离子对矮小拟丽突线虫产生的毒性具有一定的差异，24 h四种重金属离子对矮小拟丽突线虫的毒性大小依次为Cu > Cd > Pb > Zn，48 h毒性大小依次为Cd > Cu > Pb > Zn；2）与对照组相比，各暴露组当代（P0）和后代（F1）线虫平均体长均显著降低（P<0.05, P<0.01）；随着暴露浓度的增加，当代（P0）和后代（F1）线虫平均体长呈逐渐降低的趋势。生物量比体长更能敏感地反应重金属对矮小拟丽突线虫生长发育的影响，可作为重金属污染的敏感检测指标；3）供试线虫的产卵数随着Cu、Zn、Pb、Cd暴露浓度的增加而降低；其中Cu对线虫产卵数的72h-EC50、EC20和EC10值分别为1.35、0.49 和 0.2mg • L-1，Zn的72h-EC20和EC10值分别为330.29 和 163.9 mg • L-1，Pb的72h-EC20、EC10分别为17.41、4.36 mg•L-1，而Cd对线虫产卵数的72h-EC50、EC20和EC10分别为4.47、0.91 和 0.53 mg • L-1。研究结果表明，重金属Cu、Zn、Pb、Cd暴露可显著抑制矮小拟丽突线虫的生长发育和繁殖；线虫体长、产卵数表现出明显的重金属浓度依赖性。

CO2浓度升高和氮肥管理对土壤线虫群落的影响

大气CO2浓度升高能够对农田生态系统产生一系列的影响。土壤线虫在农田生态系统腐屑食物网中占有重要的地位，能够对外界环境变化作出较迅速的响应。本文利用江苏省江都市小记镇的稻-麦轮作FACE系统研究平台，在2007-2008年小麦生长季，研究了大气CO2浓度升高和不同氮肥处理（高N和低N）对农田土壤线虫群落的影响。 研究结果表明：高氮肥施用情况下, CO2浓度升高显著降低了麦田土壤铵态氮和硝态氮含量。不同氮肥处理中CO2浓度升高条件下土壤可溶性碳的含量显著低于对照，而土壤总有机碳和微生物量碳含量高于对照。 大气CO2浓度升高条件下，麦田土壤线虫群落组成和多样性与对照相比表现出显著差异。CO2浓度升高显著增加了麦田土壤线虫总数、食细菌线虫、食真菌线虫和植物寄生线虫数量。在小麦拔节期和成熟期，低N和高N施用条件下，FACE处理中土壤线虫多样性指数（H’）、成熟度指数（MI和PPI）均低于对照处理，而结构指数（SI）高于对照处理。线虫生态指数的结果表明，大气CO2浓度升高条件下，土壤线虫群落多样性降低，土壤环境受到一定的干扰，食物网趋于结构化。

大气CO_2浓度升高对农田土壤线虫的影响

大气CO_2浓度升高能够对生态系统产生一系列的影响。土壤线虫在农田生态系统腐屑食物网中占有重要的地位，能够对环境变化作出较迅速的反应。本文利用江苏省无锡市稻一麦轮作FACE系统研究平台，研究了大气CO_2浓度升高对农田土壤线虫群落的影响。在麦田生态系统中共观察到土壤线虫29科40属。研究发现大气CO_2浓度升高对土壤线虫的影响存在季节性的波动，不同营养类群的土壤线虫在不同生长关键期受大气CO2浓度升高影响的程度不同。在大气CO_2浓度升高（FACE）条件下，土壤线虫总数，食细菌线虫和食真菌线虫数量显著增加。由于土壤温湿条件的季节变化，只有在适宜的条件下，大气CO_2浓度升高对土壤线虫的影响才比较显著。在稻田生态系统中共观察到土壤线虫27科40属。研究发现，大气CO_2浓度升高能使土壤线虫总数和植物寄生线虫数量增加。在O-5cm土层，FACE系统中食真菌线虫数量显著低于对照。在5一10 cm土层，FACE系统中植物寄生线虫的潜根属（Hirschmanniella）和散香属（Boleodorus）线虫数量显著高于对照，对CO2浓度升高的反应较敏感。本研究试图为在全球变化条件下进一步认识土壤动物对农田生态 系统生态过程产生的影响提供参考。

沈阳城市森林生态系统健康与管理研究

本文系统研究了沈阳城市森林的布局与结构、城市森林功能、城市森林病虫害发生与树木健康状况和城市自然资源与社会经济状况等指标对沈阳城市森林生态系统健康与管理的影响。同时一，采用2种生态系统健康评价方法对沈阳城市森林生态系统健康状况进行了评价，并提出了沈阳城市森林生态系统健康管理的对策。研究结果如下：1、截至2004年末，沈阳城市森林植被覆盖率已经达到35％，城市森林林地分布基本合理，但需要进一步加强道路林地、居住区林地和城郊大面积生态林建设。2、沈阳城市森林以乔木为主，乔灌株数比为1.7：1，乔灌的覆盖度比约为7:1。3、沈阳城市森林不同类型林地中植物组成不同。公园林地中有74个属，137个种（变种）；庭院林地中有53个属，104个种（变种）；居住区林地中有45个属，81个种（变种）；道路林地中有43个属，94个种（变种）；运河风景林地中有75个属，142个种（变种）；棋盘山风景林地中有48个属，118个种（变种）。4、公园林地、庭院林地、居住区林地、道路林地和运河风景林地的Shannon一Wiener多样性指数分别为2.78、3.05、3.15、3.18和3.18，均匀度指数分别为0.56、0.66、0.72、0.70和0.64。除了棋盘山风景林地外，沈阳城市森林中栽植总量超过乔木总量5％的乔木树种有7个属，分别为李、柳树、杨树、桧柏、榆树、槐树和银杏，7种树木总量达到了全部乔木总量的82.09％；栽植总量超过灌木总量5％的灌木树种也有7个属，分别为水腊、丁香、李属，小聚、玫瑰、忍冬和连翘，7个属灌木总量达到了全部灌木总量的87.92％。5、公园林地、庭院林地、道路林地和防护林地中OBH＜20cm、20cm＜DBH＜60cm和DBH＞60cm树木的比例分别为：57.9％、40.0％、2.1％，49.2％、47.8％、3.0％，65.3％、33.1％、1.6％和64.6％、34.9％、0.5％，表明沈阳城市森林树木的规格总体上偏小。6、经样方调查和CITYgreen模型计算，沈阳城市森林的生态效益约2.0亿USD／yr.。公园林地、庭院林地和风景林地的景观指标相对较高；道路林地和居住区林地的景观效果一般；防护林地的景观效果较差。7、目前已经发现的沈阳城市森林病害约600余种，虫害约700余种，其中杨树主要病虫害39种，柳树的主要病虫害有33种，榆树和槐树的主要病虫害均为，1种。杨柳树腐烂病、光肩星天牛、天幕毛虫、桃红颈天牛和美国白蛾等是近10年来沈阳城市森林中普遍发生和造成严重危害的主要病虫害。沈阳城市森林主要树木的平均健康指数为2.68，处于一般健康状态。8、沈阳城市森林的土壤和水资源状况均不利于树木的健康生长，沈阳的社会经济发展也有待于进一步提高。9、经过生物指示物法（光肩星天牛为生物指示物）、专家权重法、公众问卷调查和对比研究，沈阳城市森林生态系统总体上处于亚健康状态。10、通过对沈阳城市森林资源、管理状况的调查研究和健康状况的评价，本文提出了沈阳城市森林生态系统健康管理的对策，包括合理规划沈阳城市森林林地布局，增加道路林地、居住区林地和城郊林地的面积和植被覆盖率；调整树木种类组成，避免单一或少数树种的大量栽植，提高生物多样性水平；保护大树和古树；增加城市森林管理资金的投入；应用先进技术，采取科学的病虫害防治和植物养护方法，促进树木的健康生长等。This project systematically studied the urban forest ecosystem health and management in Shenyang. The study explored factors, such as urban forest structure, distribution, pests, aesthetic value, ecological benefit, natural resources and socieo-economic status, that affecting the urban forest ecosystem health and management. Two methods were used to evaluate the ecosystem health. This project also proposed Shenyang's urban forest ecosystem health management strategies. The research results can be summarized as follows: 1. As of the end of 2004, urban forest coverage in Shenyang is about 35%, and is in relatively even patch distribution pattern. However, the street trees and roadside forest patches, residential block forest patches should be enhanced. 2. Trees are the major component of the Shenyang s urban forest, followed by shrubs. The quantity ratio of tree to shrub is about 1.7:1, and the coverage ratio of trees to shrub is about 7:1. 3. Species composition varies by location. There are 74 genera, 137 species (including varieties) in the public parks; 53 genera, 104 species (and var.) in the green spaces of the institution (including school), factory, and company; 45 genera, 81 species (var.) in residential blocks; 43 genera, 94 species (var.) in streets and roadside forest patches; 75 genera, 142 species (var.) in the Canal landscape forest patches; 48 genera, 118 species (var.) in the Qipan Mountain recreation forest. 4. The Shannon-Woener indices varies in parks, in institution, factory, and company yards, in streets and roadside forest patches, in residential blocks.there are 2.78, 3.05, 3.18, 3.15, 3.18, respectively; and the evenness indices are 0.56, 0.66, 0.70, 0.72, 0.64, respectively. Besides the Qipan Mountain forest patches, trees of 7 genera, Prunus spp., Salix spp., Populus spp., Sabina spp., Ulmus spp., Robinia spp. and Ginkgo biloba are of more than 5% the total urban trees, respectively. In fact, trees from these 7 genera are about 82% of all trees in Shenyang's urban forests. In terms of shrubs, species of 7 genera, Ligustrum spp., Syringa spp., Prunus spp., Berberis spp., Rosa spp., Lonicera spp., and Forsythia spp. are more than 5% the total urban shrubs, respectively. 88% of all the shrubs in Shenyang s urban forest are from these 7 genera. 5. The diameter class of DBH<20cm, 20cm60cm of trees in parks, in institution, factory, and company yards, in in streets and roadside forests, and in protective forests are 57.9%, 40.0%, 2.1%; 49.2%, 47.8%, 3.0%; 65.3%, 33.1%, 1.6%; and 64.6%, 34.9%, 0.5%, respectively. The result indicated that the DBH is relatively small in Shenyang s urban forests. 6. Using random plots sampling and CITYgreen model calculation, the ecological benefit of Shenyang's urban is about 0.2 billion US dollars per year. The aesthetic value of parks, institution and company yards, and scenary forest patches is relative high; however, the aesthetic value of protective forest patches is low. 7. There are more than 600 types of diseases and more than 700 kinds of insects in Shenyang's urban forest. Among them, Populus spp., Salix spp., Ulmus spp. and Robinia spp., are attached mainly by 39, 33, 11, and 11, difference types of pests respectively. The rot disease of the Populus spp. and Salix spp., and the insects such as, Anoplophora glabripennis, Malacosoma neustria, Aromia bungii, and Hyphantria cunea are major pests in Shenyang s urban forest for the past 10 years. The trees health condition is relatively poor by having the average health index of 2.68 for major species. 8. The urban soil and water resource are not favorable to tree's growth in Shenyang, and so is the socieo-economic status. 9. Through using bio-indicator, professional and public evaluation, and comparision with other urban forests, it can be concluded that the status of Shenyang s urban forest is not very healthy in general. 10. To improve the urban forest ecosystem health in Shenyang, a better urban forest design need to be implemented to enhance the area and vegetation coverage of the street and roadside forests, the residential block forests, and the surburb forest Species biodiversity need to be enhanced by adjust the species composition of urban forest trees to reduce the single or several tree species populations and to plant more varieties. A better fundings for urban forest health management need to be budgeted. Advanced technologies in tree care and scientific measures to control pests need to be adopted to prove better care for plants and to keep trees grow healthfully.

不同利用方式下潮棕壤线虫群落时空分布特征

中国东北潮棕壤不同利用方式下线虫群落随季节和土壤层次而产生的分布格局的变化的研究显示：不同土地利用方式下线虫总数、植物寄生线虫、食细菌线虫、食真菌线虫、捕食一杂食线虫数量绝大多数分布在0-20cm土壤层次。水田主要以食细菌线虫为优势营养类群，玉米地、撂荒地、林地的优势营养类群为植物寄生线虫。潮棕壤不同土地利用方式下共发现线虫104个属。水田土壤线虫群落表现为低密度、低物种多样性的基本特征。不同土地利用方式下线虫总数在不同取样时期垂直分布的变化趋势有一定的相似性。植物寄生线虫数量在0-5cm或5-10cm土层达到了峰值；捕食一杂食线虫数量在撂荒地和林地0-5cm和5～10cm土层显著高于水田和玉米地。不同利用方式下，线虫总数、营养类群、属的组成在季节和垂直分布格局上表现出了不同的变化特征。主成分分析、聚类分析、线虫区系分析结果表明：土壤线虫总数、营养类群、属对不同土地利用方式产生明显的响应。时间和空间格局是不同土地利用方式土壤线虫群落的重要属性，由于土地利用方式改变了土壤环境条件，并通过不同种群生态位的变化来改变线虫群落多样性。线虫区系分析表明，撂荒地和林地食物网是结构化食物网，水田、玉米地食物网是受到胁迫食物网。

氮肥对黄瓜根际土壤线虫群落组成、结构及其多样性的影响

土壤线虫是土壤动物的主要功能类群之一，在土壤养分分解和循环中起到重要的作用。本研究通过施用两种形态氮肥，硝态氮(NO-3–N)和铵态氮(NH+4–N)，对黄瓜整个生长期内根际土壤线虫的群落组成、结构及其多样性等的影响进行了比较研究。为增进土壤健康，提高土壤质量以及合理施用氮肥提供科学的理论依据。研究结果如下： 1. 氮肥处理后，不同浓度NO-3–N处理提高了根际土壤线虫数量，而NH+4–N处理（202.5 kg N/ hm2）抑制了线虫数量的增加。线虫群落结构相对较稳定，营养类群变化不大。且少量优势科/属会对土壤线虫群落特征起着至关重要的作用。 2. 在整个生长季节内，非寄生线虫的群体动态变化与寄生线虫的群体的动态变化具有相反的变化趋势。其中，NO-3–N处理和NH+4–N处理后植物寄生线虫出现频率的变化趋势相近，都是由高到低；非植物寄生线虫出现频率的变化趋势则都是由低到高。这说明非植物寄生线虫数量增长和空间占位对植物寄生线虫群体有一定的抑制作用。另外，也反映了适量氮肥在一定程度上能够减轻植物寄生线虫对黄瓜的危害。 3. 由多样性指数变化可知，NO-3–N和NH+4–N在低肥（67.5 kg N/hm2）和高肥（202.5 kg N/hm2）处理较中肥（135.0 kg N/hm2）处理，更不利于提高土壤线虫多样性地提高和线虫群落的稳定性。中肥不同形态氮肥处理与对照相比，H´指数在初花期和结果期显著增加了土壤线虫的H´指数，说明施入两种形态的氮肥能够提高线虫生物多样性程度。NH+4–N处理在初花期和结果期显著降低了土壤线虫种类的丰富性。土壤线虫生物多样性变化中，H´和SR指数在一定程度上能够反映施用无机氮肥对土壤线虫的多样性的影响，而J指数和λ指数效果不明显。NO-3–N和NH+4–N处理相比较，NO-3–N处理对黄瓜土壤线虫的多样性指数影响更大，促进了土壤线虫群体的多样化和种类的丰富度，更有利于提高土壤线虫的多样性，增加其稳定性。这些结果表明适量无机氮肥特别是NO-3–N的施用对黄瓜土壤线虫的生物多样性有一定的维护和提高作用。 4. 线虫数量与土壤质量指标的相关分析表明：线虫数量与有关土壤理化生指标，如土壤NO-3–N、NH+4–N、有机质含量等的正相关程度高，与总酚含量等显著负相关；与根际土壤微生物，细菌、真菌、放线菌数量等呈显著正相关。另外，线虫数量与土壤含水量未表现出显著相关关系。 Nematodes play a major role in decomposition and nutrient cycling in soil. Nematode community analyses are useful in assessing soil ecosystem status and function. The effects of two forms of mineral nitrogenous fertilizers (NO-3–N and NH+4–N) on nematode community composition, structure and diversity in rhizosphere of cucumber were investigated during different growing seasons of cucumber. Systematically research of effects of nitrogenous fertilizers could help to obtain better undstanding of a healthy soil and using nitrogenous fertilizers in reason. The main results are as follows: 1. The total numbers of nematode were more abundant in NH+4–N treatments than other teatments. However, NH+4–N teatment（202.5 kg N/hm2）dramatically inhibited it. All the tropic groups in the soil nematode communities were stable, and the dominant family or genus had an important function in the nematode community structure. 2. There was similar trend of the frequency of plant parasitic nematodes between NO-3–N and NH+4–N treatment, the similar trend of the frequency of non-plant parasitic nematodes was also found. But the frequency of plant parasitic nematodes exhibited a contrary trend to that of plant parasitic nematodes after different nitrogenous fertilizer treatments. The results showed that the increasing trend of the frequency and the niche of non-plant parasitic nematodes inhibited the plant parasitic nematodes, and indicated that right chemical fertilizers dosage could abate plant parasitic nematodes harm to cucumber. 3. The changes of the biodiversity index showed that the nitrogen treatment（135.0 kg N/hm2）promoted the stabilization of soil nematode diversity than other nitrogen treatments(67.5 kg N/hm2 and 202.5 kg N/hm2). In the treatment（135.0 kg N/hm2）,The changes in nematode diversity between the control plots and treated plots were compared by the biodiversity index (H´, J, SR, λ). Among these tested index, H´ and SR were effective in reflecting the effects of different nitrogenous fertilizers on the diversity of soil nematodes. In comparison with the NH+4–N treatment, the NO-3–N treatment promoted the stabilization of soil nematode diversity. 4. Correlation coefficients between nematode abundance and soil quality indices indicated that the total numbers of nematode were affected positively by NO-3–N, NH+4–N and the organic matter, and negatively by total phenolic acids; the total num- bers of nematode had positive correlation with bacteria, fungi and actinomycetes nu- mbers. Soil water contents had only a weak negative influence on it.

来自易变山羊草抗禾谷孢囊线虫基因的克隆与序列分析

禾谷孢囊线虫（Heterodera avenae）是严重危害禾谷类作物的病原线虫之一,它广泛分布于澳大利亚、欧洲、北美、印度和中国等世界主要小麦产区,使作物严重减产,造成巨大的经济损失。目前最有效的防治措施之一是将外源抗性基因导入栽培小麦(Triticum aestivum L.)，培育抗禾谷孢囊线虫的新品种。但迄今为止抗禾谷孢囊线虫基因克隆研究的相关报道却很少。 本实验根据此前从抗禾谷孢囊线虫材料E-10扩增得到的与来自节节麦（Aegilops tauschii）的抗禾谷孢囊线虫基因Cre3高度同源的序列Rccn4,设计出三条嵌套引物,采用SON-PCR(single oligonucleotide nested PCR)方法,从E-10基因组DNA中得到一个长为1264 bp的扩增产物(命名为Rccn-L),测序比对结果显示,这一序列将Rccn4的3’端延伸了1209 bp,与抗禾谷孢囊线虫Cre3基因核苷酸同源性为86﹪，核苷酸编码区长1026 bp，含一个不完整的开放阅读框,一个终止密码子，没有起始密码子和内含子结构,编码一个342个氨基酸残基的蛋白质。该蛋白质等电点为5.19，分子量为38112.6Da。从序列的第113位开始到第332位是NBS-LRR类抗病性基因LRR区,呈现XXLXXLXXL重复。LRR编码区内亮氨酸残基的含量达17﹪，与抗禾谷孢囊线虫Cre3基因LRR编码区的核苷酸和氨基酸同源性分别为89﹪和78﹪。本实验首次将SON-PCR成功地运用于植物基因克隆，为植物基因克隆提供了又一有效方法。 此外,还根据Cre3基因及其他的NBS-LRR类植物抗性基因的NBS和LRR区保守序列设计了两对特异性引物，从禾谷孢囊线虫抗性材料易变山羊草基因组DNA中扩增到两个相应的目标条带。测序分析结果表明，它们的长度分别为532bp和1175bp，构成了一个有32bp的共同序列的NBS-LRR编码区。其序列总长为1675bp（命名为RCCN），含有一个不完整的开放阅读框，没有起始密码子、终止密码子和内含子结构。其中编码序列为1673bp，可编码一个557个氨基酸的蛋白质，等电点（pI）为5.39，分子量为63537.5Da。与Cre3的核苷酸和氨基酸同源性分别为87.8﹪和77﹪。RCCN氨基酸序列中含有已知抗病基因NBS区域的几个保守模体：kinase2区的ILDD、kinase3的（ⅰ）ESKILVTTRSK，（ⅱ）KGSPLAARTVGG，（ⅲ）RRCFAYCS及EGF。RCCN NBS区与Cre3 NBS区的核苷酸和氨基酸的同源性分别为96.4﹪和94﹪。从氨基酸序列的274位到548位为LRR保守区，呈现不规则的aXXLXXLXXL（其中a代表I，V，L，F或M）重复，其中亮氨酸的含量为15.6﹪。该区域与Cre3的LRR区的核苷酸和氨基酸同源性分别为80.8﹪和74﹪。推测该序列可能为一个抗禾谷孢囊线虫的新基因。 本文对抗禾谷孢囊线虫基因的克隆研究，为进一步克隆基因全序列，探索其结构与功能，和研究该基因表达与调控提供了关键信息。同时也为通过基因工程途径将抗性基因向优良小麦品种高效、定向转移，最终培育出小麦抗禾谷孢囊线虫新品种奠定了基础。 Cereal cyst nematode (CCN) is a damaging pathogen of broad acre cereal crops in Australia, Europe, North America, India and China. It affects wheat, barley, oat and triticale and causes yield loss of up to 80%. At present, Transferring resistance genes against CCN into wheat cultivars and breeding varieties are considered one of the most effective methods for controlling the CCN. However, there are very limited reports concerning the cloning studies of resistance genes against the cereal cyst nematode. According to the sequence of Rccn4 which had high similarity to the nucleotide binding site (NBS) coding region of cereal cyst nematode resistance gene, Cre3， We designed three 3’ nested primers. Using single oligonucleotide nested PCR (SON-PCR) we successfully amplified one band, Rccn-L, of 1264bp from E-10 which is the wheat-Ae.variabilis translocation line containing the cereal cyst nematode resistance gene of Ae.variabilis. We found that this band of interesting is the 3’ flanking sequence of 1209bp in size of Rccn4. The coding region was 1026bp, which contained an incomplete open reading frame and a terminator codon, without initiation codon and intron, encoding a peptide of 342 amino acid residues, and shared 86﹪nucleotide sequence identity with Cre3. This peptide had a conserved LRR domain, containing the imperfect repeats,XXLXXLXXL, which contains 17﹪ leucine residues and shares, respectively, 89﹪ nucleotide sequence and 78﹪ amino acid sequence identity with the LRR sequence of Cre3 locus. This research firstly used SON-PCR in the research of plant genome successfully, which indicated that SON-PCR is another method of cloning plant gene. At the same time, According to the conversed motif of NBS and LRR region of cereal cyst nematode resistance gene Cre3 from wild wheat (Triticum tauschlii L.) and the known NBS-LRR group resistance genes, we designed two pairs of specific primers for NBS and LRR region respectively. One band of approximately 530bp was amplified using the specific primers for conversed NBS region and one band of approximately 1200bp was amplified with the specific primers for conversed LRR region. After sequencing, we found that these two sequences included 32bp common nucleotide sequence and have 1675 bp in total, which was registered as RCCN in the Genbank. RCCN contained a NBS-LRR domain and an incomplete open reading frame without initiation codon, terminator codon and inxon. Its exon encodes a peptide of 557 amino acid residues. The molecular weight of the protein from the amino acid was 63.537 KDa. The amino acid sequence of RCCN contained conserved motif: ILDD, ESKILVTTRSK, KGSPLAARTVGG, RRCFAYCS, EGF，LRR. RCCN shares 87.8﹪ nucleotide sequence and 77﹪ amino acid sequence identity with cereal cyst nematode gene Cre3. It might be a novel cereal cyst nematode resistance gene. These research results of cloning the resistance genes against cereal cyst nematode bring a great promise for transferring resistance genes into wheat cultivars and breeding new wheat varieties against cereal cyst nematode by gene engineering. And these results also lay the hard foundation for the expressing researches of these genes.

小麦抗禾谷孢囊线虫（ Heterodera avenae）相关基因的研究

禾谷孢囊线虫严重影响禾谷类作物的产量，在小麦中由禾谷孢囊线虫引起的产量损失可达30-100%。尤其在澳大利亚、欧洲、印度和中东危害严重，目前禾谷孢囊线虫已成为危害我国作物的主要病源。控制禾谷孢囊线虫的方法主要有：作物轮作、杀线虫剂、寄主抗性等等，其中基因工程方法培育抗线虫小麦品种被认为是最经济有效的方法。分离抗禾谷类孢囊线虫基因对揭示抗性基因结构与功能及其表达调控具有重要意义。 尽管小麦是重要的粮食作物，在小麦中已发现的抗禾谷孢囊线虫的基因很少，而比其近缘属如节节麦、易变山羊草、偏凸山羊草中含有丰富的抗源。目前已鉴定出禾谷孢囊线虫抗性位点Cre，并发现了9个禾谷孢囊线虫抗性基因(Cre1,2, 3, 4, 5, 6, 7, 8, and R) ，其中只有Cre1和Cre8直接从普通小麦中获得。从节节麦中获得的Cre3基因能最有效的控制线虫数量，其次是Cre1和Cre8。这些基因的克隆对于了解禾谷孢囊线虫抗性机制及进一步的育种应用都是非常关键的。然而，目前为止仅有Cre3基因通过图位克隆的方法从节节麦中被分离得到。该基因已被克隆得到的多数线虫抗性基因一样均属于核苷酸结合位点区（NBS）－亮氨酸重复序列区（LRR）基因家族。目前，已有很多抗性基因被分离，这些已知的NBS-LRR类抗性基因的保守序列为应用PCR的方法克隆新的抗性基因提供了可能。 因此本课题的目的是采用保守区同源克隆、3′RACE 和5′RACE 等方法从抗禾谷孢囊线虫小麦-易变山羊草小片段易位系E10 中克隆小麦抗禾谷孢囊线虫基因全序列，进而通过半定量PCR 和荧光定量PCR 研究该基因的表达模式。同时通过mRNA 差别显示技术和任意引物PCR（RAP-PCR）技术分离克隆植物禾谷孢囊线虫抗性基因及其相关基因，为阐明植物抗病性分子机制以及改良作物抗病性和作物育种提供基础，为通过分子标记辅助育种和基因工程方法实现高效、定向转移抗病基因到优良小麦品种奠定了重要的理论和物质基础。主要研究结果： 1． 本实验根据此前从抗禾谷孢囊线虫材料E-10 扩增得到的与来自节节麦的抗禾谷孢囊线虫Cre3 基因及其他的NBS-LRR 类抗性基因的NBS 和LRR 保守区序列设计了两对特异性引物，从E10 中扩增到532bp 和1175bp 的两个目标条带，它们有一个32bp 的共同序列，连接构成总长为1675bp 的NBS-LRR 编码区（命名为RCCN）。根据RCCN设计引物，利用NBS-LRR区序列设计引物，通过5′RACE 和3′RACE 技术采用3′-Full RACE Core Set(TaKaRa)和5'-Full RACE Kit (TaKaRa)试剂盒，反转录后通过嵌套引物GSP1 和GSP2 分别进行两轮基因特异性扩增，分别将NBS_LRR 区向5′端和3′端延伸了1173bp 和449bp，并包含了起始密码子和终止密码子。根据拼接的得到的序列重新设计引物扩增进行全基因扩增的结果与上面获得的一致。拼接后得到全长2775 bp 的基因序列（记作CreZ, GenBank 号：EU327996）。CreZ 基因包括完整的开放阅读框，全长2775 bp，编码924个氨基酸。序列分析表明它与已知的禾谷孢囊线虫抗性基因Cre3的一致性很高，并且它与已经报到的NBS-LRR 类疾病抗性基因有着相同的保守结构域。推测CreZ基因可能是一个新的NBS-LRR 类禾谷孢囊线虫抗性基因，该基因的获得为通过基因工程途径培育抗禾谷孢囊线虫小麦新品种奠定了基础，并为抗禾谷孢囊线虫基因的调控表达研究提供了参考。 2． 通过半定量PCR和SYBR Green荧光定量PCR技术对CreZ基因的相对表达模式进行了研究。以α-tubulin 2作为参照，采用半定量PCR 分析CreZ 基因在不同接种时期1d, 5d, 10, 15d 的E-10的根和叶的的表达情况。在内参扩增一致的条件下，CreZ 在E-10的根部随着侵染时间的增加表达量有明显的增加，在没有侵染的E-10的根部其表达量没有明显变化，而在叶中没有检测表达，说明该基因只在抗性材料的根部表达。SYBR Green定量PCR分析接种前后E10根部基因CreZ基因的表达水平为检测CreZ基因的表达建立了一套灵敏、可靠的SYBRGreen I 荧光定量PCR 检测方法。接种禾谷孢囊线虫后E10根内CreZ基因的相对表达水平显著高于接种前。随接种时间的延长持续增加，最终CreZ基因的相对表达量达到未接种的对照植株的10.95倍。小麦禾谷孢囊线虫抗性基因CreZ的表达量与胁迫呈正相关，表明其与小麦的的禾谷孢囊线虫抗性密切相关，推测CreZ基因可能是一个新的禾谷孢囊线虫候选抗性基因。 3． 针对小麦基因组庞大、重复序列较多，禾谷孢囊线虫抗性基因及其相关基因的片断难以有效克隆的问题，通过mRNA 差别显示技术及RAP-PCR 技术分离克隆植物禾谷孢囊线虫抗性及其相关基因。试验最终得到154 条差异表达条带，将回收得到的差异条带的二次PCR 扩增产物经纯化后点到带正电的尼龙膜上，进行反向Northern 杂交筛选，最终筛选得到102 个阳性差异点。将其中81 个进行测序，并将序列提交到Genbank 中的dbEST 数据库，分别获得登录号(FE192210 -FE192265，FE193048- FE193074 )。序列比对分析发现，其中26 个序列与已知功能的基因序列同源；有28 条EST 序列在已有核酸数据库中未找到同源已知基因和EST，属新的ESTs 序列；另外27 个EST 序列与已知核酸数据库中的ESTs 具有一定相似性，但功能未知。其所得ESTs 序列补充了Genbank ESTs 数据库，为今后进一步开展抗禾谷类孢囊线虫基因研究工作打下了基础。结合本试验功能基因的相关信息，对小麦接种禾谷孢囊线虫后产生的抗性机制进行了探讨。接种禾谷孢囊线虫后植物在mRNA 水平上的应答是相当复杂的，同时植物的抗病机制是一个复杂的过程，涉及到多个代谢途径的相互作用。 The cereal cyst nematode (CCN), Heterodera avenae Woll, causes severe yieldreductions in cereal crops. The losses caused by CCN can be up to 30-100% in somewheat fields. At present, cereal cyst nematode has become the major disease sourcein China and it also damaged heavily in Australia, Europe, India and Middle East.The damage caused by CCN can be mitigated through several methods, includingcrop rotation, nematicide application, cultural practice, host resistance, and others.Of these methods, incorporating resistance genes into wheat cultivars and breedingresistant lines is considered to be the most cost-effective control measure forreducing nematode populations. Although wheat is an economically important crop around the world, far fewergenes resistant to CCN were found in wheat than were detected in its relatives, suchas Aegilops taucchi, Aegilops variabilis and Aegilops ventricosa. Cloning these genesis essential for understanding the mechanism of this resistance and for furtherapplication in breeding. Because of the huge genome and high repeat sequencescontent, the efficient methods to clone genes from cereal crops, are still lacking. A resistance locus, Cre, has been identified and 9 genes resistant to CCN (designatedCre1, 2, 3, 4, 5, 6, 7, 8, and R) have been described, in which Cre1 and Cre8 werederived directly from common wheat. The Cre3 locus, which was derived from Ae.tauschii, has the greatest impact on reducing the number of female cysts, followed byCre1 and Cre8. Cloning these genes is essential for understanding the mechanism ofthis resistance and for further application in breeding. However, to this point, only Cre3, a NBS-LRR disease resistance gene, has been obtained through mappingcloning in Ae. tauschii. The majority of nematode resistance genes cloned so far belong to a super familywhich contains highly conserved nucleotide-binding sites (NBS) and leucine-richrepeat (LRR) domains. To date, many NBS-LRR resistance genes have been isolated.The conserved sequences of these recognized NBS-LRR resistance genes provide thepossibility to isolate novel resistance genes using a PCR-based strategy. The aim of the present study was to clone the resistance gene of CCN fromWheat/Aegilops variabilis small fragment chromosome translocation line E10 whichis resistant to CCN and investigate the espression profiles of this gene withsemi-quantitative PCR and real-time PCR. Another purpose of this study is cloningthe relational resistance gene for CCN by mRNA differential display PCR andRAP-PCR. These works will offer a foundation for disease defence of crop andbreeding and directional transferring resistance gene into wheat with geneengineering. Primary results as following: 1．According to the conversed motif of NBS and LRR region of cereal cystnematode resistance gene Cre3 from wild wheat (Triticum tauschlii) and the knownNBS-LRR group resistance genes, we designed two pairs of specific primers for NBSand LRR region respectively. One band of approximately 530bp was amplified usingthe specific primers for conversed NBS region and one band of approximately 1175bpwas amplified with the specific primers for conversed LRR region. After sequencing,we found that these two sequences included 32bp common nucleotide having 1675bpin total, which was registered as RCCN in the Genbank. Based on the conservedregions of known resistance genes, a NBS-LRR type CCN resistance gene analog wasisolated from the CCN resistant line E-10 of the wheat near isogenic lines (NILs), by5′RACE and 3′ RACE.designated as CreZ (GenBank accession number: EU327996) .It contained a comlete ORF of 2775 bp and encoded 924 amino acids. Sequencecomparison indicated that it shared 92% nucleotide and 87% amino acid identitieswith those of the known CCN-resistance gene Cre3 and it had the same characteristic of the conserved motifs as other established NBS-LRR disease resistance genes. 2． Usingα-tubulin 2 as exoteric reference, semi-quantitative PCR and real-timePCR analysis were conducted. The expression profiling of CreZ indicated that it wasspecifically expressed in the roots of resistant plants and its relative expression levelincreased sharply when the plants were inoculated with cereal cyst nematodes. therelative expression level of the 15days-infected E10 is the 10.95 times as that ofuninfected E10,ultimately. It was inferred that the CreZ gene be a novel potentialresistance gene to CCN. 3．We cloned the relational resistance gene for CCN by mRNA differentialdisplay PCR and arbitrarily primed PCR fingerprinting of RNA from wheat whichpossess huge and high repeat sequence content genomes. Total 154 differentialexpression bands were separated and second amplified by PCR. The products werenylon membrane. The 102 positive clones were filtrated by reverse northern dot blotand 81 of those were sent to sequence. The EST sequences were submitted toGenbank (Genbank accession: FE192210 - FE192265, FE193048 - FE193074). Thesequences alignment analysis indicated 26 of them were identical with known genes;28 were not found identical sequence in nucleic acid database; another 27 ests wereidentical with some known ests, but their functions were not clear. These ESTsenriched Genbank ESTs database and offered foundation for further research ofresistance gene of CCN.