Transfer of clover cyst nematode resistance from Trifolium nigrescens Viv. to T. repens L. by interspecific hybridisation

 Resistance to clover cyst nematode (Heterodera trifolii) has been successfully transferred from Trifolium nigrescens to T. repens by interspecific hybridisation. A sterile triploid hybrid (H-6909-5, 2n=3x=24) was initially produced with the aid of embryo culture. The hybrid was chromosome-doubled from axillary meristems by an in vitro colchicine method. Three chromosome-doubled plants were obtained, and these showed a marked increase in pollen stainability from 10% in 3x H-6909-5 to an average of 89% (range 88–91%) in 6x H-6909-5. T. nigrescens was a source of clover cyst nematode resistance. A mean of 23 (range 0–150) cysts per plant was recorded for T. nigrescens in comparison to a mean of 150 (range 50–240) cysts per plant for T. repens. The 3x and 6x interspecific hybrids were shown to be as resistant as the most resistant. T. nigrescens genotype and were significantly lower in cyst number per gram of root dry weight than the susceptible T. nigrescens and T. repens genotypes. Received: 11 July 1997 / Accepted: 15 July 1997     

Genetic analysis of shikimate dehydrogenase allozymes in Trifolium repens L.

 A genetic analysis was carried out on progeny families from pair crosses among plants polymorphic for shikimate dehydrogenase (SDH) isozymes in white clover (Trifolium repens L.). SDH was controlled by two independently assorting disomic loci. This result is consistent with the presence of a single gene (Sdh) represented in this putative allotetraploid by one locus in each of the genomes. One of the Sdh loci is linked (6.0±2.0 cM) to the linamarase (Li) locus. There was no evidence for differentiation of the duplicate Sdh loci (both carry common alleles). White clover behaves genetically as a diploidised allotetraploid but the possibility of a low frequency of multivalent formation and homoeologous pairing has not been ruled out. The SDH locus is likely to be useful for the marker-assisted selection of agronomic traits. Received: 5 June 1997 / Accepted: 19 November 1997     

Development and characterisation of simple sequence repeat (SSR) markers for white clover (Trifolium repens L.)

Highly informative molecular markers, such as simple sequence repeats (SSRs), can greatly accelerate breeding programs. The aim of this study was to develop and characterise a comprehensive set of SSR markers for white clover (Trifolium repens L.), which can be used to tag genes and quantitative trait loci controlling traits of agronomic interest. Sequence analysis of 1123 clones from genomic libraries enriched for (CA) _n repeats yielded 793 clones containing SSR loci. The majority of SSRs consisted of perfect dinucleotide repeats, only 7% being trinucleotide repeats. After exclusion of redundant sequences and SSR loci with less than 25 bp of flanking sequence, 397 potentially useful SSRs remained. Primer pairs were designed for 117 SSR loci and PCR products in the expected size range were amplified from 101 loci. These markers were highly polymorphic, 88% detecting polymorphism across seven white clover genotypes with an average allele number of 4.8. Four primer pairs were tested in an F₂ population revealing Mendelian segregation. Successful cross-species amplification was achieved in at least one out of eight legume species for 46 of 54 primer pairs. The rate of successful amplification was significantly higher for Trifolium species when compared to species of other genera. The markers developed in this study not only provide valuable tools for molecular breeding of white clover but may also have applications in related taxa. Received: 3 April 2000 / Accepted: 12 May 2000     

Effect of pollen competition and stigmatic receptivity on seed set in white clover (Trifolium repens L.)

The effect of competition between incompatible and compatible pollen grains on the seed production of white clover was studied. Stigmatic receptivity was also studied. A selection line of white clover that has red leaves was used as a pollen donor and as a genetic marker to allow determination of the pollen donor responsible for ovule fertilisation. Results show that incompatible pollen did not inhibit compatible pollen grains from fertilising ovules and producing seeds, although it did slightly impair seed yield. At temperatures of 20/10°C (day/night) stigmas of white clover remained receptive to additional pollination up to 32 h after an initial compatible pollination and 40 h after an incompatible pollination. The results imply that factors other than inadequate pollination and the self-incompatibility system are responsible for failure of white clover to attain its potential seed yield. Received: 31 August 1999 / Revision accepted: 28 February 2000     

Neighbourhood effect of genotypes of Rhizobium leguminosarum biovar trifolii, Trifolium repens and Lolium perenne

 Ryegrass, white clover and Rhizobium isolated from the corresponding clover nodules, were harvested from a natural pasture in the Massif Central mountains (France). The specificity between Lolium, Trifolium and Rhizobium, and the genetic diversity of Rhizobium were examined. This study showed that: 1) Natural neighbouring combinations of white clover and ryegrass, re-planted together in pots, accumulated a higher biomass than non-neighbouring ones. This increase of mass is higher in the presence of the native strain of Rhizobium. 2) When white clover was inoculated with a mixture of Rhizobium strains, nodules were more often formed by its native strain. 3) The genetical diversity of the Rhizobium leguminosarum biovar trifolii was very high, as revealed by electrophoresis of esterases on seven substrates. These results support the hypothesis that there is a co-adaptation between white clover, ryegrass and Rhizobium Received: 25 March 1996 / Accepted: 13 September 1996     

Genetic polymorphism for cyanogenesis and linkage at the linamarase locus in Trifolium nigrescens Viv. subsp. nigrescens

Polymorphisms at two genetic loci conditioning the cyanogenic glucoside linamarin (Ac) and the glucosidase linamarase (Li) are reported for the first time in Trifolium nigrescens Viv. subspecies nigrescens (2n=2x=16). T. nigrescens is one of several possible ancestral species that may have donated a genome to the allotetraploid species white clover (T. repens L., 2n=4x=32). T. nigrescens is a strong candidate because it is the only very close relative that, like white clover, is cyanogenic. Genetic analysis showed that in T. nigrescens, cyanogenesis was inherited as a two-locus genetic system in a similar way to that in white clover. Furthermore, Li, which is linked to the locus Sdh (shikimate dehydrogenase, SDH) at a distance of 6 cM in one genome of white clover, also showed linkage (12 cM) in T. nigrescens. It is concluded that one of the subspecies of T. nigrescens is a likely donor of a genome to white clover. Received: 27 December 2000 / Accepted: 12 April 2001     

气候因子和非气候因子对白三叶草叶片生长的影响

本文采用裂区试验设计,研究了刈割频率（强度）和品种对永久性混播草地组分白三叶草叶片生长的影响,并通过回归分析探讨了气候因子与白三叶草叶片生长的相关性。刈割对叶片出生率和每个葡萄茎现存叶片数的处理效应具季节依赖性特性,在夏季生长期,高频率刈割 处理。三叶草品种对叶片出生率和每葡萄茎观存叶片数均无显著的处理效应,但刈割与品种对秋初生长期叶片的出生率有显著的互作效应,对秋季生长期每葡萄茎现存叶片数有微弱     

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。     

Spectrophotometer-aided evaluation of cyanogenic potential in white clover (Trifolium repens L.)

In comparison with ordinary methods of colorimetric evaluation of cyanogenic potential based on visual evaluation of the alkaline picrate reaction, a spectrophotometer-aided method could be more accurate (since it determines the exact amount of hydrogen cyanide released by the plant material), and less time-consuming as it can be performed on bulk material rather than on a number of individual plants. Ten white clover populations were evaluated by a spectrophotometer-aided method and by two visual evaluation criteria. All methods indicated the presence of large variation between populations. Visual methods gave almost identical results and allowed only for the distinction between cyanogenic and substantially acyanogenic populations. The results were only moderately consistent with those obtained by the spectrophotometer-aided method, which could detect the presence of variation also between cyanogenic populations. The effects of various incubation times (from 4 to 48 h) and of the addition of β-glucosidase on hydrogen cyanide release were also investigated. Comparable results for ranking of populations could be obtained over a range of incubation times, but at least 24 h were needed for a reliable estimation of the hydrogen cyanide produced by plants. The addition of enzyme did not increase the released cyanide. The effect of season and/or conditions of evaluation was marked on mean cyanogenic potential but limited on ranking of populations. Copyright © 2000 John Wiley & Sons, Ltd.     

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。     

铜胁迫下链格孢菌对白车轴草生理生态的影响

通过盆栽实验研究了Cu胁迫下接种病原菌链格孢菌(Alternaria tenuis Nees)对白车轴草(Trifolium repens L.)生理生态指标的影响.结果表明:在未接种的对照组中,随着Cu浓度的增大(0~3000mg.kg-1),植株叶片失绿,生物量下降,叶绿素a、b、a b和类胡萝卜素及叶片可溶性蛋白质含量下降;植物体内丙二醛(MDA)高度积累,细胞膜结构遭到损坏,电导率增大;植株活性氧清除系统受损,不能保持原有平衡,超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性显著降低,过氧化物酶(POD)活性升高.接种链格孢菌后,植株叶片色素、膜系统及保护酶系统的损伤均加重,与不接种对照组相比,相同浓度Cu处理的叶绿素a、b、a b和类胡萝卜素、可溶性蛋白质含量、SOD和CAT活性均呈不同程度的下降,而电导率、MDA含量和POD活性则有所上升.     

Population differences in<Emphasis Type="Italic"> Trifolium repens</Emphasis> L. response to ultraviolet-B radiation: foliar chemistry and consequences for two lepidopteran herbivores

White clover growing in New Zealand is experiencing increasing levels of ultraviolet-B (UV-B) radiation as a result of ozone depletion. We evaluated the effects of UV-B radiation on the foliar chemistry of two populations of white clover (Trifolium repens L.), ’Huia’ and ’Tienshan,’ and the consequences for the performance of armyworms (Spodoptera litura) and cutworms (Graphania mutans). Plants were grown in controlled environment rooms with and without supplemental UV-B radiation at a dose of 13.3 kJ m^–2 day^–1, corresponding to a 25% mid-summer ozone depletion above Palmerston North, New Zealand. In both white clover populations, UV-B radiation elicited changes in foliar chemistry, including slight increases in nitrogen concentrations and decreases in carbohydrate concentrations. In addition, the ’Huia’ population showed decreases in fiber concentrations and marked increases in cyanogenic activity. No change in UV-absorbing compounds was detected in either population. Long- and short-term feeding trials were conducted to assess dietary effects on insect growth, consumption, and food utilization. Changes in the performance of both insect species were generally small. The most pronounced effect was a 36% reduction in weight of S. litura after 2 weeks of feeding on Huia grown at high UV, but larval development times were only slightly prolonged and pupal weights were unaffected. S. litura short-term performance was affected by differences in white clover population. The long-term performance of G. mutans was not affected and its short-term performance (stadium duration and consumption rate) was only marginally affected by the high-UV treatment. We conclude that the effects of elevated UV-B radiation on white clover plant chemistry can be specific to certain plant populations. The differences in sensitivity of the two generalist insect species suggest that effects may also be specific to certain plant-herbivore associations. These results indicate that future UV-B herbivory studies should examine genotypic effects in both plants and animals. Received: 18 May 1999 / Accepted: 25 July 1999     

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     

Effects of VA mycorrhizal colonization on photosynthesis and biomass production of Trifolium repens L.

The influence of vesicular–arbuscular mycorrhizal (M) colonization on biomass production and photosynthesis of Trifolium repens L. was investigated in two experiments in which the foliar nitrogen and phosphorus contents of non-mycorrhizal (NM) plants were manipulated to be no lower than that of M plants. Throughout both experiments there was a stimulation in the rate of CO₂ assimilation of the youngest, fully expanded leaf of M compared with NM plants. In addition, M plants exhibited a higher specific leaf area compared with NM plants, a response that maximized the area available for CO₂ assimilation per unit of carbon (C) invested. Despite the increased rate of photosynthesis in M plants there was no evidence that the additional C gained was converted to biomass production of M plants. It is suggested that this additional C gained by colonized plants was allocated to the mycorrhizal fungus and that it is the fungus, by acting as a sink for assimilates, that facilitated the stimulation in the rate of photosynthesis of the plant partner.     

Mycorrhizal sink strength influences whole plant carbon balance of Trifolium repens L.

A comparative analysis of daily carbon (C) budgets and aspects of the C physiology of clover ( Trifolium repens L.) colonized by vesicular-arbuscular (VA) mycorrhizal fungi was carried out over a 70 d growth period under conditions designed to ensure that shoots of mycorrhizal (M) and non-mycorrhizal (NM) plants were of similar nutrient status. C budgets did not differ on day 24 but by day 42 M plants had a significantly higher rate of photosynthesis than their NM counterparts when expressed on a whole shoot basis or unit dry weight basis. As both sets of plants were of the same size it was concluded that this greater C gain was the result of increased sink strength provided by the mycorrhizal fungus. By day 53 M plants had become larger than their uncolonized counterparts and a sink-induced stimulation in the rate of photosynthesis was no longer apparent. M plants had higher root sucrose, glucose and fructose pools from day 24. Analyses suggested that these sugars were utilized for trehalose and lipid synthesis, for the production of the large extramatrical mycelium and for the support of the respiratory demands of the M root system. Increased C allocation to roots of M plants was associated with a stimulation of the activities of cell wall and cytoplasmic invertases and of sucrose synthase in roots colonized by VA fungi. Such increases in enzyme activity may provide the mechanism enabling increased partitioning of carbohydrate both to the M root system and the fungal symbiont.     

光照强度和氮水平对白三叶幼苗生长与光合生理特性的影响

采用植物生长箱溶液培养方式,对白三叶幼苗进行了不同光强(2个水平)和氮浓度(5个水平)处理,探讨其生长、生物量和光合生理特征对生境变化的响应.结果表明:两种光强下白三叶幼苗茎和叶生物量随氮素浓度呈先升高后降低,而根系生物量和根冠比则随氮素浓度增高而降低.光照强度降低使白三叶幼苗根、茎、叶和整株生物量分别降低67.8%、29.9%、42.5%和45.2%;低光处理使幼苗的根冠比显著下降,而比叶面积(SLA)明显提高.幼苗根系体积随氮素浓度增高而降低,高生长光强根系体积显著高于低生长光强下的白三叶.幼苗根系表面积、根系长度和根系直径随氮素浓度增加呈先增加后降低趋势,两种不同生长光强下幼苗根系长度和根系直径差异显著,而根系表面积差异不明显.白三叶叶片光合速率(Pn)随氮素浓度增加呈先增加后降低趋势,高生长光强白三叶Pn显著高于低生长光强下的白三叶.两种生长光强间叶片气孔导度(Gs),胞间CO2浓度(Ci)和蒸腾速率(Tr)无显著差异,但氮素浓度对叶片Gs、Ci和Tr均有显著影响.光、氮及其交互作用对白三叶幼苗生长发育产生了显著影响,光照不足和氮缺乏都将导致白三叶幼苗生长减弱,但幼苗对这些不利环境具有较强的调节和适应能力.     

刈割频次对白三叶能量分配及构型的影响

4～ 10月 ,在亚热带高山人工草地 ,通过生长季割 2、4和 7次的刈割试验后发现 ,不同刈割频次对翌年白三叶能量分配、构件密度、分枝数量及分枝角度影响显著 .随刈割频次的增加 ,白三叶匍匐茎的能量分配递增 ,分别为 42 .8%、44 .9%、47.7% ,而单位长度茎所含能量递减 ,分别为 13 .0、12 .2和 11.1kJ·m-1;叶密度、茎密度、分枝数量及节间长度出现低→高→低的变化趋势 .提高刈割频次后 ,白三叶的分枝强度由 15分枝·m-1递增到 2 3 .7分枝·m-1;分枝角度由 49.5°递增到 60 .2° ,从而提高了白三叶种群在刈割干扰下对土壤微生境资源的利用率     

Studies on the feeding of Sitona lineatus L. (Coleoptera: Curculionidae) on white clover (Trifolium repens L.) seedlings

Measurements of feeding damage by sitona weevil (Sitona lineatus L.) adults on differing numbers of seedlings of white clover (Trifolium repens L.) at the first and fourth trifoliate leaf stage were made in the glasshouse at 20°C. S. lineatus consumed more of the trifoliate component of the seedling. Sitona adults caused significant yield reduction at all levels of plant population. Total clover consumption increased with increasing size of sitona population, but consumption per adult weevil was reduced.     

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。     

A developmentally based categorization of branching in Trifolium repens L.: influence of nodal roots

This study describes the successive stages of development of branches from axillary buds in fully rooted plants of Trifolium repens grown in near optimal conditions, and the way in which this developmental pathway differs when nodal root formation is prevented as plants grow out from a rooted base. Cuttings of a single genotype were established in a glasshouse with nodal root systems on the two basal phytomers and grown on so that nodal rooting was either permitted (+R) or prevented (-R). In +R plants, axillary tissues could be assigned to one of four developmental categories: unemerged buds, emerged buds, unbranched lateral branches or secondarily branched lateral branches. In -R plants, branch development was retarded, with the retardation becoming increasingly pronounced as the number of -R phytomers on the primary stolon increased. Retarded elongation of the internodes of lateral shoots on -R plants resulted in the formation of a distinct fifth developmental category: short shoots (defined as branches with two or more leaves but with mean internode length equal to, or less than, 10% of that of the immediately proximal internode on the parent stolon) which had reduced phytomer appearance rates but retained the potential to develop into lateral branches. Transfer of +R plants to -R conditions, and vice versa, after 66 d demonstrated that subsequent branch development was wholly under the control of the youngest nodal root present, regardless of the age and number of root systems proximal to it.