种间竞争梯度对白车轴草与其病原菌白车轴草单孢锈菌相互作用的影响

白车轴草（Trifolium repens）在与其病原菌白车轴草单孢锈菌(Uromyces trif olii-repentis) 的长期相互作用中分化形成了抗病型(Resistance)无性系和易感型(Sus ceptibility)无性系。该研究工作旨在了解： 1）在种间竞争不断增强的环境梯度中,抗病型无性系和易感型无性系的生长表现有何区别？2）在同样的实验条件下,分别对抗病型无性系和易感型无性系进行接种感染后,两者的生长表现又有何区别？在一严重感病的白车轴草自然种群中,分别标定17个抗病型无性系和14个易感型无性系,从各无性系的匍匐茎上分别剪取长5 cm,具两个叶两个腋芽的小段作实验材料。接种孢子采自同一植物种群植株的病叶。实验在一个处在次生演替阶段的草本植物群落上进行,在该群落的种间竞争梯度上选定 3个分别具有弱、中等和强种间竞争的生境（Habitat）建立实验站,每站设置等量的实验组和对照组。结果显示：1）随着环境中种间竞争强度的增加,无论接种与否,抗病型和易感型白车轴草的叶生长量均递减;2）在弱或强种间竞争环境条件下,无论接种与否,易感型无性系的生长均好于抗病型无性系;但在中等种间竞争强度的环境条件下,对照实验中易感型无性系的生长显著好于抗病型无性系,而接种实验中的结果相反。这一研究结果表明,种间竞争强度可能是影响给定生境中寄主植物抗病型和易感型遗传型比率的重要因子之一,这一影响还将随着病原菌存在与否而发生改变。     

种间竞争梯度对白车轴草与其病原菌白车轴草单孢锈菌相互作用的影响（英文） 刘登义 Lars Ericson

 白车轴草(Trifolium repens)在与其病原菌白车轴草单孢锈菌(Uromyces trifolii-repentis)的长期相互作用中分化形成了抗病型(Resistance)无性系和易感型(Susceptibility)无性系。该研究工作旨在了解：1)在种间竞争不断增强的环境梯度中,抗病型无性系和易感型无性系的生长表现有何区别?2)在同样的实验条件下,分别对抗病型无性系和易感型无性系进行接种感染后,两者的生长表现又有何区别?在一严重感病的白车轴草自然种群中,分别标定17个抗病型无性系和14个易感     

白车轴草（Trifolium repens）植株抗病性和生长与植物病史的关系

从白车轴草（Trifolium repens）自然种各中采集无白车轴草单孢锈菌病史的无性系（clones）17个,有白车轴草单孢锈菌病史的无性系14个,分别作为抗病型和感受型植物实验材料;采集白车轴草单孢锈菌（Uromyces trifolii-repentis）菌系（strains）10个,作为病菌实验材料,分别设置并进行了两个温室实验、一个田间盆栽实验和一个原生长地移栽实验,实验处理上分对照、单菌系接种和10个菌系接种等3种。实验结果表明,无论是用单菌系接种还是10个菌系接种,植株发病的概率和程度均与其抗病性有关,抗病型植株（无病史）发病的概率和程度显著低于感受型（有病史）植株,在相同处理的实验中（无论是田间实验还是温室实验）,无病史植株和有病史植株的生长无显著差异;不同处理田间实验植株的生长有显著差异,病情愈重,生长愈差,无病史植株的抗病性明显强于有病史植株,但是,原生长地的移栽实验结果表明,在无病原菌存在的情况下,有病史植株的（叶）生长显著好于无病史植株,可以认为,研究生物个体对环境因子反应性差异的实验应当在自然条件下和自然梯度范围内进行。     

白车轴草(Trifolium repens)植株抗病性和生长与植物病史的关系

从白车轴草(Trifolium repens)自然种群中采集无白车轴草单孢锈菌病史的无性系(clones)17个,有白车轴草单孢锈菌病史的无性系14个,分别作为抗病型和感受型植物实验材料;采集白车轴草单孢锈菌(Uromyces trifolii-repentis)菌系(strains)10个,作为病菌实验材料.分别设置并进行了两个温室实验、一个田间盆栽实验和一个原生长地移栽实验,实验处理上分对照、单菌系接种和10个菌系接种等3种.实验结果表明,无论是用单菌系接种还是10个菌系接种,植株发病的概率和程度均与其抗病性有关,抗病型植株(无病史)发病的概率和程度显著低于感受型(有病史)植株.在相同处理的实验中(无论是田间实验还是温室实验),无病史植株和有病史植株的生长无显著差异;不同处理田间实验植株的生长有显著差异,病情愈重,生长愈差.无病史植株的抗病性明显强于有病史植株.但是,原生长地的移栽实验结果表明,在无病原菌存在的情况下,有病史植株的(叶)生长显著好于无病史植株.可以认为,研究生物个体对环境因子反应性差异的实验应当在自然条件下和自然梯度范围内进行.     

白车轴草病原菌链格孢菌及其症状研究

通过对不同白车轴草无性系病叶样本的病原菌分离、接种、鉴定及致病力测定,并结合野外观察,研究了安徽铜陵铜尾矿区白车轴草叶斑病病原菌链格孢菌(Alternaria tenuisNees)的致病、发病状况和寄主植物的抗病性、研究表明,不同白车轴草无性系抗病性有明显差异,根据寄主植物发病情况,以及病斑大小、形状、颜色等,将白车轴草无性系分为抗病型(R)和感病型(S)。     

铜胁迫下链格孢菌对白车轴草生理生化特性的影响

通过盆栽实验研究了Cu胁迫下接种链格孢菌(AlternariatenuisNees)对源自Cu污染区(记作种群Ⅰ)和非污染区(记作种群Ⅱ)白车轴草(TrifoliumrepensL)生理生化特性的影响。结果表明,两种群白车轴草在Cu处理后接种链格孢菌,0~1500mg·kg-1时叶片电导率、MDA含量均高于未接种组,且随浓度增加而升高,2000mg·kg-1时电导率低于未接种组,MDA含量降低但仍高于未接种组。两种群接种后叶绿素、蛋白质含量较未接种组下降,且随Cu浓度增加而降低,其中叶绿素含量与浓度呈显著或极显著负相关,种群Ⅰ和种群Ⅱ相关系数分别为-0.954*和-0.961**。链格孢菌使白车轴草SOD活性低浓度(0和500mg·kg-1)高于未接种组,高浓度则降低。接种后POD活性随Cu浓度增加先增后减,500mg·kg-1浓度下接种组POD高于未接种组,而在其他浓度下,显著低于未接种组。0~1000mg·kg-1时接种组CAT活性高于未接种组,随土壤Cu浓度增加先增后减。3种保护酶对胁迫的敏感性为POD>SOD>CAT。高浓度Cu(2000mg·kg-1)胁迫下,白车轴草保护酶系统的受损打破了体内活性氧产生与清除之间的正常平衡状态,积累了过量的活性氧,膜脂过氧化程度加重,对白车轴草产生毒害,且这种毒害在接种链格孢菌后表现更为严重。两种群白车轴草相比,种群Ⅰ植物在土壤Cu<2000mg·kg-1时可生存,感染链格孢菌后生长土壤Cu浓度应控制在1500mg·kg-1以内;种群Ⅱ在Cu胁迫浓度不超过1500mg·kg-1,链格孢菌和Cu双重胁迫时不超过1000mg·kg-1可生长。种群Ⅰ在Cu处理、链格孢菌单一或双重胁迫下表现的抗逆性较Ⅱ强。     

温度胁迫对白车轴草水浸提液化感作用的影响

采用蚕豆根尖细胞微核试验和染色体畸变试验,研究了温度胁迫下白车轴草水浸提液对蚕豆根尖细胞的化感作用。结果表明:不同温度(5℃、25℃和35℃)处理后的白车轴草的水浸提液对蚕豆根尖细胞有丝分裂指数的影响为低浓度促进,高浓度抑制。在白车轴草水浸提液的作用下,蚕豆根尖细胞内出现了多种染色体畸变,如微核、染色体断片、染色体桥、落后染色体等,对微核率和染色体畸变率的诱导效应表现为随水浸提液浓度的增大而增大。温度胁迫后的化感作用明显大于胁迫前,其化感效应表现为低温(5℃)下白车轴草水浸提液高温(35℃)常温(25℃)白车轴草水浸提液。推测原因可能是温度胁迫改变和增加了化感物质的释放。     

光照、磷和AMF对白车轴草生长的影响

外来植物成功入侵受非生物因素与生物因素的共同调控。本研究以典型入侵植物白车轴草（Trifolium repens L.）为研究对象,通过三因素两水平正交实验,探究光照（L）、磷（P）和丛枝菌根真菌（Arbuscular mycorrhizal fungi,AMF）及其交互作用对其生长的影响。结果显示：（1）高光照、高磷和接种AMF均显著提高白车轴草的生物量及生长速率,且接种AMF对生物量的促进作用随着光照的增加而增强,低磷条件下AMF对总生物量和相对生长速率的促进效应更明显;（2）高光照降低比叶面积;高磷和接种AMF显著增加叶片数和总叶面积,在高光强下尤为明显;（3）高光照可显著提高根表面积、根直径和根质量分数,但降低细根占比和比根长。高磷显著降低根质量分数。不接种AMF时,高磷增加根表面积与根直径,降低细根长占比;但接种AMF后,高磷则降低根表面积与根直径,增加细根长占比。AMF显著降低根质量分数和比根长。研究结果表明,三种因素对白车轴草的生长具有显著影响,且磷和AMF对其生长及地上性状的影响与光照强度相关,AMF对其地下性状的影响与磷浓度相关。     

豆链格孢菌对白车轴草生理代谢及活性氧清除系统酶的影响

通过盆栽实验,研究了接种豆链格孢菌对白车轴草叶组织细胞膜透性、色素含量、丙二醛(MDA)含量以及超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性的影响.结果表明,豆链格孢菌使白车轴草叶组织细胞膜透性上升,电导率显著升高,膜脂过氧化加强,MDA水平上升,色素含量下降.电导率和MDA含量均与感病时间呈显著正相关,而色素含量与感病时间呈显著负相关.豆链格孢菌刺激其寄主白车轴草叶片膜脂过氧化作用发生,积累大量的活性氧,使其超出防御酶的清除能力,致使活性氧清除系统遭到破坏,保护酶系统失衡,其中SOD和CAT活性显著下降,而POD活性明显上升.接种12d后,SOD和CAT分别降低了55.2%和37.8%,而POD比对照升高约1.6倍.     

白车轴草和紫花苜蓿根瘤的显微及超微结构

通过对白车轴草(Trifolium repens Linn.)和紫花苜蓿(Medicago sativa Linn.)根瘤的显微及超微结构进行观察,发现其根瘤显微结构都由4部分组成,由外向内依次为：保护层、皮层、鞘细胞层和中心组织(侵染组织)。在中心组织的侵染细胞中,分布有大量的线粒体、高尔基体、核糖体及内质网,白车轴草根瘤侵染细胞中的细菌圆形或椭圆形,有明显的周膜、细菌细胞壁和质膜,在细菌发育过程中,周膜活动旺盛,有时相邻细菌的周膜发生融合,在周膜附近常分布有大量的内质网、高尔基体以及高尔基体小泡,似与周膜融合有关。紫花苜蓿细菌椭圆形、长棒形,甚至有的细菌呈分枝状。二者细菌细胞质中分布着大量的核糖体和纤维状的核物质。在白车轴草中还有染色很深的多聚磷酸盐颗粒。     

Direct amplification of nodD from community DNA reveals the genetic diversity of Rhizobium leguminosarum in soil

Sequences of nodD , a gene found only in rhizobia, were amplified from total community DNA isolated from a pasture soil. The polymerase chain reaction (PCR) primers used, Y5 and Y6, match nodD from Rhizobium leguminosarum biovar trifolii , R. leguminosarum biovar viciae and Sinorhizobium meliloti . The PCR product was cloned and yielded 68 clones that were identified by restriction pattern as derived from biovar trifolii [11 restriction fragment length polymorphism (RFLP) types] and 15 clones identified as viciae (seven RFLP types). These identifications were confirmed by sequencing. There were no clones related to S. meliloti nodD . For comparison, 122 strains were isolated from nodules of white clover ( Trifolium repens ) growing at the field site, and 134 from nodules on trap plants of T. repens inoculated with the soil. The nodule isolates were of four nodD RFLP types, with 77% being of a single type. All four of these patterns were also found among the clones from soil DNA, and the same type was the most abundant, although it made up only 34% of the trifolii -like clones. We conclude that clover selects specific genotypes from the available soil population, and that R. leguminosarum biovar trifolii was approximately five times more abundant than biovar viciae in this pasture soil, whereas S. meliloti was rare.     

Involvement of reactive oxygen species in the response of resistant (hypersensitive) or susceptible cowpeas to the cowpea rust fungus

A previous study had indicated that scavengers of reactive oxygen species (ROS) delayed cell death (the hypersensitive response (HR)) triggered in epidermal cells of intact, resistant, cowpea ( Vigna unguiculata (L.) Walp) leaves by the monokaryotic stage of the cowpea rust fungus ( Uromyces vignae Barclay race 1). This HR had been monitored by cell autofluorescence, which occurs after protoplast collapse. In the present study, when cytoplasmic disorganization was used to monitor cell death more directly, ROS-scavengers, superoxide dismutase, catalase, horseradish peroxidase, and desferal-Mn(IV) had no effect on HR development. Cytological staining for superoxide or hydrogen peroxide generation also did not reveal the presence of ROS before or during the early stages of the HR, but did, as in the previous study, suggest a role in the autofluorescence and browning of invaded cells that occur following protoplast collapse. Staining of plant mitochondria with nitroblue tetrazolium, possibly attributable to increased dehydrogenase activity but not superoxide generation, occurred transiently around invasion hyphae (monokaryotic stage of the fungus) or haustoria (dikaryotic stage) of the fungus as they entered a cell in the susceptible or resistant cultivar. Around invasion hyphae in epidermal cells in resistant plants, this staining diminished as cytoplasmic streaming stopped, and gradually disappeared as cell death progressed. These data are consistent with other evidence that rust fungi initially negate non-specific defensive responses in both resistant and susceptible cells as part of the establishment of biotrophy. They also suggest that the HR in the cowpea–cowpea rust fungus pathosystem is not triggered by an oxidative burst.     

PR-1 protein inhibits the differentiation of rust infection hyphae in leaves of acquired resistant broad bean

Treatment of broad bean leaves with salicylic acid (SA) or 2, 6-dichloro-isonicotinic acid (DCINA) induces resistance against the rust fungus Uromyces fabae resulting in reduced rust pustule density. Light-microscopy studies showed that in induced resistant plants the rust fungus is inhibited immediately after penetration through the stomatal pore. The differentiation of infection structures growing within the intercellular space of the leaf, i.e. infection hyphae and haustorial mother cells, is inhibited. Furthermore, low-temperature scanning electron microscopy studies of freeze fractures revealed protrusions at the tips of infection hyphae growing in induced resistant broad bean leaves. Treatment of in vitro-differentiating rust infection structures with intercellular fluids (IFs) from induced resistant plants confirmed that the fungus is sensitive towards an apoplastic anti-fungal activity only after having formed appressoria. Other legume rusts such as U. vignae and U. appendiculatus were likewise inhibited in the presence of IF from SA-treated broad bean leaves. Heterologous antibodies were used to study changes in the extracellular pathogenesis-related (PR) protein pattern after resistance induction. Western blots indicated that chitinases and beta-1,3-glucanases were present in both induced and control plants. In contrast, PR-1 proteins were newly synthesized in response to SA or DCINA application. The major induced PR-1 protein was purified and exhibited strong differentiation-inhibiting activity towards U. fabae infection structures. We conclude that the inhibition of rust infection hyphae in acquired resistant broad bean plants is mainly due to the anti-fungal activity of this induced basic PR-1 protein.     

小豆VaDREB1A基因克隆及其应答锈菌侵染的表达分析

为明确小豆VaDREB1A在抗锈菌侵染过程中的作用,本研究分别从小豆基因组DNA及cDNA中克隆了该基因,测序结果表明VaDREB1A无内含子,全长660 bp编码219个氨基酸,氨基酸序列包含1个AP2结构域,序列特征及系统发育分析表明VaDREB1A属于DREB/CBF亚家族A1亚类。启动子顺式元件分析发现,VaDREB1A启动子包含乙烯、水杨酸及生物和非生物胁迫等响应元件。对该基因响应豇豆单胞锈菌(Uromyces vignae)侵染的表达分析发现,与不接种对照相比,VaDREB1A在抗病品种中于接种后24 h和120 h显著下调,而在感病品种中于接种后120 h和192 h显著上调。在1-氨基环丙烷-1-羧酸(ACC)诱导小豆抗锈病过程中,VaDREB1A的表达与其在抗病品种中的表达相似,分别于ACC处理并接种后24 h和192 h显著下调。上述结果说明VaDREB1A可能参与小豆对锈病的抗性。     

Effect of Host Plant Resistance on Haustorium Formation in Cereal Rust Fungi

Haustoria of Puccinia triticina (wheat leaf rust fungus) and P. hordei (barley leaf rust fungus) were isolated from susceptible and partially resistant wheat lines, and susceptible, hypersensitive and partially resistant barley lines. Haustoria were counted and measured. The size of haustoria was similar in the partially resistant and susceptible genotypes but haustoria were smaller in the hypersensitive barley line L94+Pa7. The number of haustoria was reduced in both partially and hypersensitive lines when compared with susceptible ones. Therefore it seems that the reduction in the number of haustoria is a consequence of the resistance that can be attributable either to early abortion of infection units or reduced colony growth. The reduction of the number of haustoria was more pronounced in the adult plant stage.     

Rhizobial polysaccharide-degrading enzymes from roots of legumes

The surface structure and chemistry of symbiotic bacteria from the genus Rhizobium are probably important for the outcome of the infection of legume hosts. Exopolysaccharide, capsular polysaccharide, lipopolysaccharide and a low-molecular-weight polysaccharide were isolated from R. trifolii UTC 110-1 and R. leguminosarum UTC 114-5 and partially characterized. No or only minor differences in sugar composition could be found for the corresponding fractions from the two organisms. A general method to measure low activities of polymer-degrading enzymes was developed, and used to determine enzyme activities in root extracts of Trifolium repens L. cv. Lena and Pisum xativiini L. cv. Little Marvel against the isolated rhizobial polysaccharides. An enzyme preparation from T. repens partially degraded all polysaccharides isolated from its symbiont R. trifolii while polysaccharides from R. leguminosarum , symbiont of P. sativum , were degraded to a much lesser extent. Correspondingly, an enzyme preparation from P. sativum degraded all polysaccharides isolated from both its symbiont R. leguminosarum and its non-symbiont R. trifolii. The amount of symbiont polysaccharides degraded was larger than the amount of polysaccharides degraded from the non-symbiont R. trifolii.     

Transfer of the Rhizobium leguminosarum biovar trifolii symbiotic plasmid pRtr5a to a strain of Rhizobium sp. that nodulates on Hedysarum coronarium

The Rhizobium leguminosarum biovar trifolii symbiotic plasmid pRtr5a was transferred to the Rhizobium sp. (Hedysarum coronarium) strain RB16. Transconjugants carrying pRtr5a ineffectively nodulated Trifolium repens, T. pratense and T. alexandrinum and were unable to nodulate H. coronarium plants. Agarose gel electrophoresis of transconjugants showed that all had lost an indigenous plasmid (230 Md). These results suggest that this plasmid harbours the symbiotic determinants for nodulation on H. coronarium.