Red Clover Plants Research Articles

Heterologous rhizobial lipochitin oligosaccharides and chitin oligomers induce cortical cell divisions in red clover roots, transformed with the pea lectin gene.

Division of cortical cells in roots of leguminous plants is triggered by lipochitin oligosaccharides (LCOs) secreted by the rhizobial microsymbiont. Previously, we have shown that presence of pea lectin in transgenic white clover hairy roots renders these roots susceptible to induction of root nodule formation by pea-specific rhizobia (C. L. Díaz, L. S. Melchers, P. J. J. Hooykaas, B. J. J. Lugtenberg, and J. W. Kijne, Nature 338:579-581, 1989). Here, we report that pea lectin-transformed red clover hairy roots form nodule primordium-like structures after inoculation with pea-, alfalfa-, and Lotus-specific rhizobia, which normally do not nodulate red clover. External application of a broad range of purified LCOs showed all of them to be active in induction of cortical cell divisions and cell expansion in a radial direction, resulting in formation of structures that resemble nodule primordia induced by clover-specific rhizobia. This activity was obvious in about 50% of the red clover plants carrying hairy roots transformed with the pea lectin gene. Also, chitopentaose, chitotetraose, chitotriose, and chitobiose were able to induce cortical cell divisions and cell expansion in a radial direction in transgenic roots, but not in control roots. Sugar-binding activity of pea lectin was essential for its effect. These results show that transformation of red clover roots with pea lectin results in a broadened response of legume root cortical cells to externally applied potentially mitogenic oligochitin signals.

Interaction Effects of Nitrogen and Phosphorus on Nodulation in Red Clover (Trifolium pratense L.)

Red clover (Trifolium pratense L.) is one of the most important plants in forage production, especially in northern areas. Fertilisation practices are focused on high yield and forage quality but effects of nutrients on nodulation and N2 fixation are poorly understood. The aim of this work was to study how nitrogen (N) and phosphorus (P) separately as well as in combination affected nodulation. Red clover plants were grown in pots with gravel in a greenhouse for 11 weeks. To resemble field conditions the root temperature was kept lower than the shoot temperature. Plants were given five different combinations of N and P concentrations during growth. The result showed that at high N concentrations P had a counteracting effect on the N inhibition. The N2-fixation parameters, nodule number, nodule dry matter and specific nitrogenase activity, were six times higher in plants grown with high N and high P than in plants with high N and low P. When the N2-fixation parameters and the dry matter of roots and shoots were related to total plant dry matter, there was a stronger effect of P on nodulation parameters than on roots and shoots. This indicates that P has a direct effect on the N2-fixation parameters, rather than an indirect effect via increased plant growth. These results demonstrate the importance to studying the effects of more than one nutrient at a time.

Selection for resistance to Fusarium wilt in red clover

In Wisconsin, Fusarium oxysporum, Schlect., a pathogen causing vascular wilt, is the most prevalent fungal pathogen recovered from diseased red clover (Trifolium pratense L.) plants. This study was conducted to determine the mode of inheritance for red clover resistance to this pathogen and to develop resistant germplasm. Virulent isolates of this pathogen, collected from red clover plants at the Ashland Research Station, Ashland, Wisconsin, were used to screen three populations, the red clover cultivars Arlington and Marathon and the C11 germplasm, for resistant plants. Plants were inoculated with the pathogen and evaluated for reaction, using a disease-severity index (DSI) score from 1 to 5 (1 = no reaction, 5 = plant dead). Selected plants from each cycle were intercrossed to produce subsequent generations. After two and three cycles of selection, the developed populations were simultaneously evaluated for gain from selection. The gain from selection for resistance in these populations (cycle 0 minus cycle 2) ranged from 0.31 to 0.48, 0.12 to 0.75, and 0.13 to 0.83 DSI units, respectively, for Arlington, Marathon, and C11. Estimated narrow-sense heritabilities, based on cycle-1 and cycle-2 progeny, were, respectively, 0.20 and 0.37 for Arlington, 0.15 and 0.13 for Marathon, and 0.06 and 0.17 for C11. These results indicate that resistance is a quantitative trait controlled by many loci, each contributing some portion to overall resistance in the host. Key words: Red clover, Trifolium pratense L., Fusarium oxysporum, vascular wilt, resistance

Nitrogen mineralization and crop uptake of N from decomposing 15N labelled red clover and yellow sweetclover plant fractions of different age

Nitrogen mineralization from 15N labelled red clover (Trifolium pratense L.) and yellow sweetclover (Melilotus officinalis Lam.) plant fractions of three different ages (8-, 14- and 20-week old) was studied in an out-door pot experiment during 8.5 months. Individual plant fractions (leaves/stems/roots/flowers), 23 g dry matter pot-1 (corresponding to 7300 kg ha-1), were incorporated into a sandy soil. The net mineralization of N was measured as 15N recovery in spring wheat (Triticum aestivum L.), perennial ryegrass (Lolium perenne L.) following the wheat and in the soil mineral N pool.

Harvest Management Effects on Red Clover Forage Yield, Quality, and Persistence

Forage cutting management recommendations emphasize early and frequent harvests to maximize forage nutrient yields. This study was conducted to determine forage yield, quality, and persistence of five modern red clover (Trifolium pratense L.) cultivars harvested at different frequencies and stages of maturity throughout the growing season. Red clover plots were established at Arlington, Marshfield, Spooner, and Ashland, WI, in 1989 and at Marshfield, WI, in 1990. During the establishment year, forage was removed once or twice when red clover plants reached 20% bloom. Five cultivars were harvested for 2 yr after the seeding year under five harvest management systems (HMS): A = 3 summer cuts at bud stage + late fall cut; B = 3 summer cuts at bud stage; C = 2 summer cuts at 20% bloom + late fall cut; D = 2 summer cuts at 40% bloom + late fall cut; E = 2 summer cuts at 40% bloom. Cutting red clover twice at 20% bloom and once in late fall (HMS C) produced maximum seasonal forage yield during both harvest years at all locations. In the northern Wisconsin locations, HMS D and E yielded the same as HMS C, while southern locations favored the earlier, more frequent cutting schedules (HMS A, B, and C). Fall harvest of red clover before the second winter did not significantly reduce forage yield in the third year. When comparing forages cut at bud stage, 20% bloom, or 40% bloom, maximum red clover yield is attained by 20% bloom. Forage quality of spring growth forage was lower than subsequent summer harvests as measured by crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF). Forages cut at bud stage had the highest concentration of CP and lowest concentration of NDF and ADF, and forage quality declined with increasing maturity. Persistence of red clover was not greatly affected by HMS, but where differences occurred, plant stands and fourth year yields were highest for management systems C, D, and E where plots were cut less frequently or at later growth stages. Current red clover cultivars should be harvested three times (2 summer cuts + 1 late fall cut) during the year when plants are between bud stage and 20% bloom stage for optimum forage yield, quality, and persistence.

Growth, mineral nutrition and mycorrhizal colonization of red clover and cucumber plants grown in a soil amended with composted urban wastes

The utility of an urban solid waste, either freshly composted or vermicomposted, for improvement of plant growth in a soil B horizon was investigated. Growth, mineral nutrition and arbuscular mycorrhizal (AM) colonization of cucumber and red clover plants were studied in an experiment carried out under controlled growing conditions, using different mixtures of soil and composts as plant substrates. Soil inoculation with the AM fungus Acaulospora sp. did not benefit growth of plants when soil was used as the only substrate, possibly due to its poor fertility. Results showed that neither mycorrhizal plant species grew when soil was mixed with composted urban waste or when compost was used as the only substrate. However, amendment of soil with 10 or 50% vermicompost significantly increased dry matter yields of red clover and cucumber plants, compared to treatments where soil was the only substrate. Addition of vermicompost also increased Olsen-P and other mineral elements in soil and shoot P, Ca, Mg, Cu, Mn and Zn concentrations, but caused a significant reduction on root length colonized by AM fungi in red clover plants. It is concluded that application of high amounts of vermicompost from composted urban wastes to soils might cause a significant reduction of activity of AM fungi, which must be taken into account when using these organic amendments in agricultural systems.

A Cluster-Based Approach to Means Separation

A plant breeder is frequently faced with analyzing a particular variable such as mean yields for a group of cultivars. Usually this follows an F test from a standard ANOVA which indicates that there are significant differences among cultivars. Many breeders then employ one of the large variety of available range tests to aid in digesting the experimental results. Most of the tests construct groups which may have substantial overlaps. An example of one such analysis was cited by Steel and Torrie (1980), beginning on page 140 with the data and continuing on page 176 with Duncan's multiple comparison procedure. The original experiment measured nitrogen content of red clover plants inoculated with six different rhizobium treatments. Duncan s procedure produced three groups. Group 1 contained four of the treatments and Group 2 contained three of the treatments. The disconcerting part of this analysis was that two of the treatments in Group 1 were also two of the treatments in Group 2. In the same way, the third treatment in Group 2 was also one of the two treatments in Group 3. This situation presents a problem since it gives rise to ambiguous interpretations. The proposed procedure eliminates that problem and simulation results indicate that the procedure is quite good when compared with several others. In the statistics literature, there are many types of multiple range or multiple comparison techniques as well as other approaches that are neither multiple range tests nor multiple comparisons. Examples include Fisher's Least Significant Difference (Fisher 1935), Tukey's Honestly Significant Difference (Tukey 1953), Student-Newman-Keuls (Student 1927; Newman 1939; Keuls 1952), Waller and Duncan (Waller and Duncan 1969), Duncan's New Multiple Range Test (Duncan 1955), Scheff6s method (Scheff6 1959),

Biodiversity of endophytic bacteria which colonize red clover nodules, roots, stems and foliage and their influence on host growth

The aim of this study was to identifiy the endophytic bacteria recovered from the foliage, tap roots and nodules of red clover plants (Trifolium pratense L.); and to assess the effects of the nodule bacteria, alone and in combination with Rhizobium spp., on the growth and development of red clover seedlings. Thirty-one bacteria species from 14 different genera were recovered from within the foliage, roots and nodules of red clover plants cv. AC Charlie. Genera diversity and species number were greatest in foliage tissues. Pantoea agglomerans (59.6%) was the most frequent species recovered in foliage tissues, Agrobacterium rhizogenes A in the tap root (49.2%) and Rhizobium leguminosarum BV phaseoli and R. loti B in the nodules (27.2% each). Recovery of Rhizobium species was not restricted to the nodules, and species of this genus were systemic throughout the plant. Clover root nodules were host to 12 bacteria species other than rhizobia, of which 8 were specific to this tissue. Using non-selective media, R. leguminosarum BV trifolii constituted only 8.8% of all the root nodule bacteria recovered. In root bacterization experiments, species of nodule bacteria promoted growth of red clover more often when applied in combination with R. leguminosarum BV trifolii than when applied singly. However, Bacillus megaterium, Bordetella avium and Curtobacterium luteum consistently promoted growth either individually or in combination with R. leguminosarum BV trifolii. Nodulation was promoted when R. leguminosarum BV trifolii was coinoculated with Bacillus insolitus, B. brevis or A. rhizogenes A. Single isolate applications of Rhizobium species to roots always led to the depression of clover growth, but mixtures of R. leguminosarum BV trifolii and R. leguminosarum BV phaseoli resulted in growth promotion. The latter is considered further evidence of the beneficial allelopathic side effect of strain competition for the same ecological niche.

Combining Ability Analysis of Resistance of Southern Root‐knot Nematode in Red Clover

Production of red clover, Trifolium pratense L., is reduced in areas infested with root‐knot nematodes (RKN) in the southeastern USA. Although progress has been made in improving RKN resistance in red clover through recurrent selection, little information is available on the mode of inheritance of red clover resistance to any of the RKN species. This study was conducted from 1993 to 1995 in Florida to determine the mode of inheritance and relative importance of general combining ability (GCA) and specific combining ability (SCA) effects in controlling resistance to Meloidogyne incognita (Kofoid & White) Chitwood Race 3 in red clover. By means of a breeding population selected for low root galling and egg mass production when infested with M. incognita, four red clover plants were identified as resistant, three as intermediate, and two additional susceptible individuals were selected from ‘Kenstar’. These individuals were cloned and used as parents in a diallel genetic study. Forty‐eight progeny from each of 36 F1 crosses, without reciprocals, were evaluated under greenhouse conditions during 1995 for response to inoculation with 1500 M. incognita eggs per plant. Eight weeks after inoculation, the number of galls and egg masses on roots of each plant were counted under low power magnification (10–20 ✕). Resistant genotype 119 produced progeny with the least M. incognita reproduction and galling. Crosses involving the two susceptible genotypes, N1 and K4, resulted in the highest numbers of galls and egg masses. The GCA effects were significant for both traits, whereas SCA effects were not significant. These results indicate that for this set of parents, breeding procedures that utilize GCA effects to maximize exploitation of additive gene action should be most effective in improving resistance to M. incognita galling and reproduction.

Production of Red Clover Transgenic for Neomycin Phosphotransferase II Using Agrobacterium

Genetic variability for resistance to several of the more important diseases of red clover (Trifolium pratense L.) appears to be limited. Incorporation of resistant genes from other species via genetic transformation offers potential. This research was conducted to evaluate methodology for production of transgenic red clover plants via Agrobacterium tumefaciens mediated transformation. The effects of red clover genotypes and Agrobacterium strains on transformation success were evaluated in two experiments. Plants determined to be transgenic for the β‐glucuronidase (GUS) and neomycin phosphotransferase II (NPTII) genes were crossed with normal plants and the inheritance of GUS was determined. Putative transgenic callus and plants were produced on B5 medium containing 50 and 300 mg L−1 kanamycin and carbenicillin, respectively. Both transformation experiments showed significant red clover genotype, Agrobacterium strain, and genotype × strain interactions (P< 0.05) for percentage of petiole pieces regenerating plants. Southern blots of DNA isolated from various GUS+ plants showed evidence of single and multiple copies of the NPTII gene. Petiole pieces from various putative transgenic plants produced near normal amounts of callus when plated on B5 callus induction medium containing 50 mg L−1 kanamycin. Progeny from crossing transgenic with normal plants segregated 1:1 as expected. The protocoloutlined in this paper should be useful for incorporation of genes of agronomic importance into red clover.

A hierarchical analysis of population genetic structure in Rhizobium leguminosarum bv. trifolii

AbstractLittle is known about the population processes that shape the genetic diversity in natural populations of rhizobia. A sample of 912 Rhizobium leguminosarum biovar trifolii isolates were collected from naturalized red clover populations (Trifolium pratense) and analysed for 15 allozyme loci to determine the levels and distribution of genetic diversity. Hierarchical analyses compared different sampling levels, geographical separation, and temporal separation. Total genetic diversity across all isolates was H= 0.426, with 57.6% of the total diversity found among isolates obtained from individual red clover plants. Relatively low genetic differentiation among populations and high differentiation among plants within populations was observed; this suggests that gene flow and founder effect act differently at geographical and local scales. Significant differences were observed in (i) allele frequencies among populations and among plants within populations, and (ii) the frequency distribution of the most widespread and the most abundant strains. When multilocus linkage disequilibrium was calculated, significant levels of disequilibrium were observed in the total sample and in three of the eight populations.

A hierarchical analysis of population genetic structure in Rhizobium leguminosarum bv. trifolii

Little is known about the population processes that shape the genetic diversity in natural populations of rhizobia. A sample of 912 Rhizobium leguminosarum biovar trifolii isolates were collected from naturalized red clover populations (Trifolium pratense) and analysed for 15 allozyme loci to determine the levels and distribution of genetic diversity. Hierarchical analyses compared different sampling levels, geographical separation, and temporal separation. Total genetic diversity across all isolates was H = 0.426, with 57.6% of the total diversity found among isolates obtained from individual red clover plants. Relatively low genetic differentiation among populations and high differentiation among plants within populations was observed; this suggests that gene flow and founder effect act differently at geographical and local scales. Significant differences were observed in (i) allele frequencies among populations and among plants within populations, and (ii) the frequency distribution of the most widespread and the most abundant strains. When multilocus linkage disequilibrium was calculated, significant levels of disequilibrium were observed in the total sample and in three of the eight populations.

Evaluation of ‘Grasslands Puna’ chicory (Cichorium intybusL.) in various grass mixtures under sheep grazing

Abstract Growth of chicory (Cichorium intybus) in mixtures with different companion grass species and white clover (Trifolium repens) was examined over 4 years in a trial rotationally grazed by sheep. A mixture containing red clover (T. pratense) instead of chicory, and ryegrass (Lolium perenne), was also included. Over summer and autumn, pastures were either grazed frequently (every 3–4 weeks) or infrequently (4–6 weeks), and at other times of the year every 4 weeks. Chicory establishment was high (82% of viable seed sown), with 46 plants/m2, declining to 15/m2 by Year 4, whereas red clover plant numbers declined from 101/m2 to 13/m2. Dry matter (DM) yields of chicory and red clover increased from Year 1 to Year 3, then declined in Year 4. Over the 4 years, chicory contributed 34, 80, 85, and 57%, respectively, to green DM yield; red clover contributed 9, 24, 77, and 13%. Yields of sown grass were inversely related to chicory or red clover yields (P < 0.001, r= 0.82), particularly for chicory‐based pastu...

Virulence, Legume Host Specificity, and Genetic Relatedness of Isolates ofFusarium oxysporumfrom Red Clover

Fusarium oxysporum is the most prevalent fungal pathogen recovered from symptomatic red clover (Trifolium pratense) plants in Wisconsin and contributes to stand loss and reduced productivity. Three field isolated and 44 single-conidium isolates of F. oxysporum were recovered from red clover plants with vascular wilt symptoms and assessed for virulence, host specificity, and source of variation in host reaction. In addition, genetic relatedness of selected isolated was determined by vegetative compatibility groups (VCG). Twenty-one populations of red clover, two populations of alfalfa (Medicago sativa), and one population each of alsike clover (Trifolium hybridum), ladino clover (Trifolium repens), and birdsfoot trefoil (Lotus corniculatus) were tested for their reaction to isolates or single-conidium isolates. Isolates differed significantly in their ability to elicit reactions both among and within host species. VCGs were not useful in predicting host reaction because isolates from distinct groupings elicited similar host reactions. The reaction of red clover to F. oxysporum is attributable to genetic diversity in isolate virulence as well as to variation within the host population for resistance

Effect of manganese on vesicular-arbuscular mycorrhizal development in red clover plants and on soil Mn-oxidizing bacteria

The effect of manganese on the development of vesicular-arbuscular (VA) mycorrhizae and on the population of Mn-oxidizing soil bacteria was studied using red clover as host plant and Glomus mosseae or G. aggregatum as VA-colonizing fungi. The addition of Mn to the substrate in which plants grew had a detrimental effect on root colonization, G. aggregatum being more susceptible than G. mosseae. Mn uptake was lower in mycorrhizal than in non-mycorrhizal plants, especially when colonized by G. mosseae. The development of mycorrhizae seems to favour the Mn-oxidizing bacteria population.

Regenerative trait and cold hardiness in highly productive cultivars of alfalfa and red clover

In earlier work on improvement of persistance in forage legumes, we selected genotypes from highly productive cultivars of alfalfa, Algonquin and Apica (Euphytica 45: 105–112, 1990) and cv. Florex red clover (Plant Cell Reports 8: 395–398, 1989) capable of in vitro regeneration from callus and cell culture. The alfalfa germplasm and its F1 progeny as well as an F2 red clover population were tested for cold stress tolerance. Plantlets were hardened in culture tubes at 2 or 5°C, 8h photoperiod, for at least four weeks and then subjected to freezing temperatures, −16 or −10°C for alfalfa and red clover, respectively. Survival of regenerative genotypes was significantly higher than of the non-regenerative ones in both species. A strong oositive correlation (r=0.78) between the regenerative trait and plant survival was found in alfalfa. The experiments indicate that in vitro selection for regenerative trait may improve cold stress tolerance of alfalfa and red clover.

Selection for Resistance to Aphanomyces euteiches in Red Clover

Red clover (Trifolium pratense L.) is known to be a moderate host for Aphanomyces euteiches Drechs. Recently, an isolate of A. euteiches (Ae‐572) was observed to be highly virulent on red clover. This research was conducted to determine inheritance of, and select for, resistance to A. euteiches in red clover using an inoculated seedling assay. Resistant and susceptible red clover plants were intercrossed to study the inheritance of resistance. Frequency distributions of resistant and susceptible F1 plants indicated that resistance to A. euteiches is inherited as a quantitative trait. Three cycles of selection for resistance to A. euteiches in red clover resulted in significant improvement in resistance from the original selected population (Cycle 0 = 3.64 mean disease severity index (DSI); Cycle 3 = 2.43 mean DSI). Narrow‐sense heritability estimates were high enough ((h2n = 51% for Cycle 3) to assure that a recurrent selection program utilizing family selection could be used to improve red clover populations for resistance to A. euteiches.

Effect of several root pathogenic fungi on growth of red clover under field conditions

Abstract Effects of Cylindrocladium scoparium Morgan, Trichocladium basicola (Berk. & Br.) Carmichael), Fusariumoxysporum Schlecht., and F. solani (Mart.) Sacc., on survival and growth of red clover (Trifolium pratense L.) plants were investigated in two field experiments using plots of fumigated soil. In the first experiment, the survival of plants inoculated at 5 weeks by dipping roots in conidial suspensions of the test fungi before planting, was significantly reduced by C. scoparium and T. basicola, but not by F. oxysporum or F. solani. In the second, C. scoparium, T. basicola, and to a lesser extent F. solani, reduced survival of plants grown from seed in clay tile microplots for 76 weeks. C. scoparium, T. basicola, F. solani, and F. oxysporum reduced cumulative shoot dry matter (DM) yields by 25-50%, compared with the control microplots. All test fungi caused symptoms of disease on roots, and reduced root DM production in this experiment. Both experiments thus demonstrated that C. scoparium, T. basi...

Relationship between spring vigor and the presence of adventitious roots in established stands of red clover (Trifolium pratense L.)

Two experiments were undertaken to investigate the relationship between the presence of adventitious roots growing from the crown of red clover plants and the spring vigor of those plants. Experiment 1 was conducted in the springs of 1988 and 1989 in commercial stands of red clover-timothy mixtures entering their third year. Two hundred and thirty-seven plants were visually rated for spring vigor and degree of adventitious rooting using an ordinal scale. Experiment 2 was carried out on a research plot entering its fourth year and consisting of mixtures of red clover and timothy. Visual scores, taproot and adventitious root volumes and foliage weight were recorded. In all experiments significant differences (P &lt; 0.01) in average spring vigor score were found among the five root classes. Correlation coefficients between spring vigor and adventitious root scores were 0.49, 0.65 and 0.58 for exp. 1 (springs of 1988 and 1989) and exp. 2, respectively. Adventitious root volume made up 86% of total root volume in exp. 2. Significant positive linear relationships were found between foliage weight and adventitious root and taproot volumes. It was concluded that profuse production of adventitious roots imparts better spring vigor to red clover. Key words: Taproot, adventitious root, red clover

Fungal invasion of red clover roots in a soil naturally infested with a complex of pathogens: Effects of soil temperature and moisture content

A quantitative method employing tissue maceration and plating was used to determine the internal fungal flora of roots of red clover ( Trifolium pratense) plants, 4, 8 and 12 weeks after sowing in field plots, and in pots of field soil kept at different constant temperatures (10. 15. 20 or 25°C) and moisture contents (40, 60 or 80% of soil water holding capacity) in the glasshouse. In field plots, the total number of colonies isolated per root increased at each successive harvest whereas the extent of fungal invasion (number of fungal colonies g 1− root tissue) decreased. A similar decrease in the invasion of roots of plants in pots reflected a slow rate of fungal infection and an accelerating production of lateral roots. Plant growth in pots was greatest at 25 C and 60% WHC but slowed in most treatments between 8 and 12 weeks as plants became potbound. The fungi which dominated at 4 weeks ( Verticillium dahliae and Fusarium solani) were isolated most commonly from plants grown at 10°C. while at 12 weeks most colonies of the dominant fungi ( Cylindrocladiun scoprium, F. oxysporum, F. solari, and Trichocladium basicola) were obtained from plants grown at 20 and 25 C. Roots from soil at high moisture content (80% WHC) yielded more fungal colonies in total than those from less moist soil at 4 and 12 weeks, however, during the first 8 weeks, F. solani was most often recovered from dry soil (40% WHC). Since the root mycoflora of field-grown and pot-grown plants was similar, choice of appropriate soil temperature and moisture conditions for glasshouse experiments would allow simulation of events in the field.