Clover Lines Research Articles

The tolerance of three transgenic subterranean clover (Trifolium subterraneum L.) lines with the bxn gene to herbicides containing bromoxynil

A bxn gene, coding for the nitilase enzyme that catalyses the detoxification of the herbicide bromoxynil, was inserted into subterranean clover (Trifolium subterraneum L. subsp. yanninicum). The agronomic characteristics of 3 transgenic lines (BXN 5, 7, 10) and their response to herbicides containing bromoxynil, were compared with that of the non-transgenic parent, cv. Gosse, in 1 glasshouse and 2 field experiments.The application of bromoxynil at 1.5 L/ha resulted in a 50% reduction in the spring herbage yield of the non-transgenic control but no reduction in BXN 5 and 7. The level of the phyto-oestrogens, genistein and biochanin A, in the leaf tissue were low in Gosse and the 2 transgenic lines, BXN 7 and 10, but the level of genistein increased significantly (P < 0.05) from 0.85% in Gosse to 1.43% dry weight in BXN 5, and biochanin A increased from 0.35% in Gosse to 0.73% in BXN 5. The application of the herbicides bromoxynil, bromoxynil+MCPA, and Jaguar (bromoxynil+diflufenican) at the cotyledon stage in a glasshouse study significantly reduced the leaf area of the non-transgenic Gosse, but did not reduce the leaf area of the 3 transgenic lines. When applied at the 4–5 leaf stage, the leaf area of Gosse was again reduced by all the herbicides, but only the bromoxynil+MCPA treatment reduced the leaf area of the transgenic lines relative to the control. The application of bromoxynil or Jaguar at the 4–5 leaf stage depressed the herbage yield of Gosse by about 62% compared with 0–24% in the 3 transgenic lines. In the field studies, BXN 5 and 10 had similar seed yields to Gosse in the absence of herbicide application, but the seed yield of BXN 7 was significantly less than the other 2 transgenic lines. The level of hard-seed in BXN 7 was also lower at 25% compared with 55–57% in the other transgenic lines and Gosse.These findings stress the need to carefully assess the agronomic qualities of transgenic lines prior to release as although tolerance to bromoxynil may be greatly increased by the bxn gene, other important agronomic characteristics, such as seed yield, hardseededness, and isoflavone content, may change as result of the gene or transformation process.

Red clover - a legume showing tolerance to clover root weevil

Clover root weevil (CRW) has now spread through the northern North Island, and south into Taranaki and Hawke's Bay r egions. Sever e infestations can decimate white clover in pasture. In 1998, 2000 and 2002, trials were estab lished on a Warkworth Northland dairy farm to test a range of legume species for resistance or tolerance to CRW damage. Trial lines were regularly assessed for relative vigour and levels of CRW damage. No plant resistance w as identified among white clover, red clover, lucerne, Lotus corniculatus, Lotus pedunculatus or Caucasian clover seed lines. A few lines were adapted to the site and showed tolerance to CRW. Red clover suf fered the least damage and tolerant selections were made from a creeping red clover and nine white clover lines. Two further trials confirmed that red clover showed the least damage. Further cycles of selection are required to develop the potential tolerance shown in field tests. Red clover or a well-adapted white clover offers potential to cope with the CRW stress. Keywords: clover root weevil, clover selections, legume tolerance, Sitona lepidus

A simple model of feedback regulation for nitrate uptake and N2 fixation in contrasting phenotypes of white clover.

A simple three equation model is proposed for the feedback regulation of nitrate uptake and N2 fixation, based on the concentration of the organic N substrate pool within the plant and two parameters denoting the N substrate concentrations at which half-maximal inhibition occurs. This model simulated three contrasting phenotypes of white clover (Trifolium repens L.) inbred lines with (1) normal rates of nitrate uptake and N2 fixation (NNU); (2) low rates of nitrate uptake (LNU); and (3) very low rates of N2 fixation (VLF). The LNU phenotype was simulated by a decrease in the value of the inhibition parameter for nitrate uptake and the VLF phenotype was simulated by a decrease in the value of the N2 fixation inhibition parameter. The model was tested against nitrate uptake data obtained from white clover plants growing in flowing nutrient culture. There was an accurate prediction of the increase in nitrate uptake caused by N2 fixation activity of the NNU and LNU inbred lines being interrupted by a switch in gas phase from air to Ar : O2. The model was also tested against data for nitrate uptake, N2 fixation and %N from fixation for the three inbred clover lines grown in flowing nutrient culture at 0, 5 or 20 mmol m(-3) N(3-). Again there was accurate prediction of nitrate uptake, although simulated values for N2 fixation were more variable. The simple model has potential use as a sub-routine in larger models of legume growth under field conditions.

Differences in symptom development in subterranean clover infected with Kabatiella caulivora Race 1 and Race 2 are related to host resistance

Clover scorch (Kabatiella caulivora) is a severe fungal disease of Trifolium spp. contributing to the collapse of pasture swards across southern Australia during warm, humid spring weather. Host plant responses associated with resistance to the disease were determined in 2 cultivars of subterranean clover (T. subterraneum) separately inoculated with K. caulivora Race 1 or Race 2. Germination of conidia of both races reached a maximum 5 days post-inoculation on cv. Woogenellup (susceptible to both races) and 4 days post-inoculation on cv. Daliak (resistant to Race 1 but susceptible to Race 2). Germ tube growth of Race 1 was inhibited on cv. Daliak and the percentage of conidia penetrating leaf surfaces was lowest on this race–cultivar combination. Susceptibility was characterised by large petiole lesions, with invasive hyphae extending through the mesophyll tissue into the pith and then through the phloem tissue of vascular bundles, eventually causing the petioles to collapse. Resistance was characterised by small, black lesions with invasive hyphae extending no further than the fourth layer of mesophyll cells. A suberin-based material was observed beneath infected mesophyll cells in the incompatible interaction, beyond which no further growth of hyphae occurred. Race 2 caused a faster rate of host tissue necrosis than Race 1 and also the breakdown of starch grains in uninvaded petiole tissues. Starch grains in plants infected with Race 1 were evenly distributed in uninvaded tissue. Sporulation was rarely observed in the incompatible interaction but was common in compatible interactions within 15 days post-inoculation. These responses to K. caulivora can now be used as a breeding tool in evaluating and selecting improved resistance to clover scorch disease among breeding lines of subterranean clover.

Proliferation and rol gene expression in hairy root lines of Egyptian clover

Hairy roots were efficiently induced from seedlings of Egyptian clover (berseem clover, Trifolium alexandrinum L.) by infection with Agrobacterium rhizogenes strain DC-AR2 (harboring mikimopine-type pRi1724) and cultured on Murashige-Skoog's (MS) medium containing 1 mg l−1 naphthaleneacetic acid (NAA). Each of the hairy root lines showed variations in growth on solid MS medium containing various concentrations of NAA and these variations were also found in culture in liquid MS medium containing 0.5 mg l−1 NAA. To investigate whether the growth capacity is related to the endogenous auxin IAA and the expression of oncogenes on Ri plasmid T-DNA, the concentration of IAA was determined by ELISA using IAA-specific antibody and the extent of transcripts of oncogenes rolB and rolC was analyzed by Northern blot hybridization. From these results, in hairy roots of Egyptian clover, a rolB expression seemed to influence the intrinsic growth capacity and to call for the proper level in order that the hairy roots grew vigorously, while the endogenous IAA seemed to directly affect the growth capacity on medium without exogenous auxin.

A rapid semi-quantitative procedure for screening hydrocyanic acid in white clover (Trifolium repens L.)

The presence of cyanogenic glucosides in white clover (Trifolium repens L.) is an anti-nutritional factor due to the potential for cyanide to pre-dispose selenium deficiency in grazing animals. Considerable genotypic variation in cyanogenesis occurs in white clover and it is important that highly cyanogenic white clover lines are identified to ensure that germpasm used in breeding programs does not lead to the release of cultivars that exceed safe levels. A procedure for rapid semi-quantitative screening of large white clover germplasm collections is described. This procedure is based on the picrate assay and utilises computer imagery and calibration relationships between spectral intensity (red, green, blue bands) of the colour reaction on picrate paper with cyanide in standard solutions to predict hydrocyanic acid concentration in white clover leaf.

Searching for clover root weevil (Sitona lepidus) resistance/tolerance - A progress report

The decimation of white clover in Waikato and Bay of Plenty pastures owing to the invasion clover root weevil (CRW) initiated an extensive screening of legume germplasm to identify plant material that could survive and remain productive under high CRW pressure. A total of 50 000 plants from 275 lines and six species, were planted on seven commercial farms and evaluated for tolerance/ resistance to CRW under conventional farming practices. Plant productivity and level of CRW damage were both highest on white clover. Significant CRW damage was also observed on red clover and caucasian clover, but not on birdsfoot trefoil, lotus major or lucerne. However, the productivity under stock grazing of the latter three species was not sufficient to consider them suitable alternatives to white clover. Within the white clover lines there was encouraging variability in the level of CRW damage, and in the ratio of damage to shoot production. While root damage is of greater importance than shoot damage, the latter reflects CRW activity and egg deposition and is a good indicator of likely larval development and root damage. Factors such as farm type (dairy farms versus dry stock farms) had some influence on shoot production, but very little influence on CRW damage. Climatic conditions appeared to have strong influences on clover shoot production and CRW damage, with dry conditions from late spring to early autumn having a particularly suppressive effect on CRW damage and CRW population growth. Top performing lines have been selected from this screening and are currently being evaluated in more detail under high CRW pressure. Keywords: clover, clover root weevil, plant screening, Sitona lepidus, tolerance, Trifolium repens

Adaptation to low fertility hill country in New Zealand of white clover lines selected for differences in response to phosphorus

White clover breeding lines developed as high and low P‐responsive, in glasshouse selection trials, were compared, over three years, with 17 other breeding lines and cultivars for growth in four soil‐fertility treatments under two defoliation treatments on a hill‐country farm. Differences between P‐response groups were transitory and biologically insignificant. Selection for differences in response to P in a controlled environment was not successful in identifying white clover germ‐plasm adapted to low P hill‐country soils. White clover breeding lines and cultivars that achieved high growth over the 3‐year duration of the trial had New Zealand parentage and were all medium to small leaf types.

Progress in recurrent selection and in crossing cultivars with white clover resistant to the clover root‐knot nematode Meloidogyne trifoliophila

New Zealand pastures are generally deficient in clover content due in part to root‐invading nematodes. A recurrent selection programme for resistance to the clover root‐knot nematode (Meloidogyne trifoliophila) assessed individual white clover (Trifolium repens) genotypes for the number of root galls and root dry weight. Low galling (resistant) and a few high galling (susceptible) genotypes were crossed to provide divergent selections and progeny screened. In the sixth generation, the resistant selections (n = 581) averaged 45% of the susceptible selections (n = 146) for number of galls per gram of root dry weight. The seventh generation equivalent figure improved to 38% (n = 112 resistant and n = 81 susceptible genotypes). In the seventh generation, survival of the resistant group of lines was poorer than other groups. Resistant genotypes were crossed with established cultivars and progeny screened and crossed. The resistant × cultivar progeny averaged 83% of the susceptible × cultivar progeny's galls per gram in the first cross (ns), 58% (P < 0.001) in the second, and 44% (P < 0.001) in the third cross. Progeny of Sustain × resistant crosses had fewer galls per gram than progeny of crosses with other cultivars. Foliage dry weights were assessed every 3 weeks for 6 months from M. trifoliophila‐infected and nematode‐free pairs of resistant and susceptible white clover lines. Yields of all four lines were reduced by the invasion of the M. trifoliophila second‐stage juveniles but the two resistant lines outyielded the susceptible ones. There was little yield from infected susceptible lines from 127 to 191 days after inoculation. More M. trifoliophila eggs were recovered from one resistant line mainly because it survived and grew better than the other lines.

Production of Bean yellow mosaic virus resistant subterranean clover (Trifolium subterraneum) plants by transformation with the virus coat protein gene

SUMMARYTransgenic lines of subterranean clover were constructed that contained three different Bean yellow mosaic virus (BYMV) coat protein (CP) gene constructs; full‐length CP, the core region of the CP, and full‐length CP plus the 3′ untranslated region of the viral genome. Transgenic plants containing the full‐length and core CP gene constructs showed high and moderate levels of BYMV resistance. Resistance was measured as a lack or amelioration of viral disease symptoms, which was correlated with a reduction in virus levels and yield loss. A range of different resistance phenotypes was observed. They included reduced infection rates, delay and reduction in local lesion development, and delay and reduction in severity of systemic symptom development. Resistance levels were not correlated with transgene mRNA levels and no transgene‐encoded protein was detected in any of the transgenic lines. This is the first example of genetically engineered virus resistance in a clover.

Enhanced expression of a cold-induced gene coding for a glycine-rich protein in regenerative somaclonal variants of red clover (Trifolium pratense L.)

Cold hardiness and in vitro regeneration are closely related traits in selected alfalfa and red clover germplasms. We investigated the cold-induction of three genes, MsaCIA, MsaCIB and MsaCIC, implicated in the cold tolerance of alfalfa, in non-regenerative and regenerative somaclonal variants of red clover by Northern blot analysis using alfalfa cDNA clones as hybridization probes. Only the MsaCIA gene exhibited increased steady-state mRNA levels in cold-acclimated red clover. We observed, however, enhanced-cold induction of steady-state levels of MsaCIA gene transcripts in all regenerative somaclonal variants compared to non-regenerative red clover lines. Moreover, these two traits, regeneration and enhanced cold-induction of MsaCIA, were concomitantly transmitted to progeny suggesting that enhanced expression of MsaCIA and the regenerative trait are either linked, or the expression of both traits are regulated by a ‘master’ gene whose expression is modified by somatic embryogenesis.

Genetic variation for the ratio of inorganic to total phosphorus in white clover leaves

The inorganic phosphorus (P.) and total P (PT) concentrations of leaf tissue were measured for 9 genotypes from each of 4 white clover lines—Grasslands Sustain, Grasslands Prestige, a low‐P site ecotype, and a high‐P site ecotype— grown at 50 and 400 ug added P g‐1 soil, in a glasshouse. The cultivars/ ecotypes varied significantly for Pi/PT ratio at the high level of P supply, but not at the lower level. The two ecotypes were not significantly different from each other for Pi/PT ratio at either P supply level. Among the 36 genotypes, P/ PT ratio varied 2‐fold at the high P level and 2.5‐fold at the low P level. Broad sense heritabilities for Pi/PT ratio were, however, low, particularly at the high level of P supply.

Genetic variation for rate of establishment in caucasian clover

Slow establishment of caucasian clover (Trifolium ambiguum L.) is hindering the use of this legume in pasture mixtures. Improved genetic material is one strategy of correcting the problem. Newly harvested seed of hexaploid caucasian clover germplasm covering a range of origins, together with white and red clover and lucerne, were sown in 1 m rows in a Wakanui soil at Lincoln in November 1995. After 21 days, the caucasian clover material as a group had similar numbers of emerged seedlings as white clover and lucerne, but was inferior to red clover. There was wide variation among caucasian clover lines (48-70% seedling emergence), with the cool-season selection from cv. Monaro ranked the highest. Recurrent selection at low temperatures could be used to select material with improved rates of seedling emergence. Red clover and lucerne seedlings produced significantly greater shoot and root dry weight than caucasian and white clover seedlings. Initially, caucasian clover seedlings partitioned 1:1 shoot to root dry weight compared with 3:1 for white clover. After 2 months, caucasian clover seedlings had similar shoot growth but 3 times the root growth of white clover. Between 2 and 5 months, caucasian clover partitioned more to root and rhizome growth, resulting in a 0.3:1 shoot:root ratio compared with 2:1 for white clover. Both clover species had similar total dry weight after 5 months. Unhindered root/ rhizome devel-opment is very important to hasten the establishment phase of caucasian clover. The caucasian clover lines KZ3 and cool-season, both selections from Monaro, developed seedlings with greater shoot and root growth than cv. Monaro. KZ3 continued to produce greater root growth after 5 months, indicating the genetic potential for improvement in seedling growth rate. Different pasture estab-lishment techniques are proposed that take account of the seedling growth characteristics of caucasian clover. Keywords: establishment, genetic variation, growth, seedling emergence, Trifolium ambiguum

Host Plant Acceptance by Redlegged Earth Mite, Halotydeus destructor (Tucker) (Acarina: Penthaleidae)

Host plant acceptance behavior of redlegged earth mites (Halotydeus destructor) on cotyledons of subterranean clover (subclover) lines was investigated in the laboratory. Sustained feeding consisted of a series of short bouts of feeding; rather than one long period of feeding. H. destructor preferred to feed at mite-damaged rather than undamaged sites on cotyledons. With time the numbers of mites feeding in aggregations increased, as mites were attracted to damaged patches. Most feeding occurred in aggregations, and mites in such groups benefited by greater weight gain. The sequence of foraging leading to feeding was similar between subclover lines, however, feeding and aggregation activities were markedly reduced on a resistant line. Host plant acceptance occurred during probing but only after some feeding had occurred.

Effects of improved, adapted white clover (Trifolium repens L.) germplasm on the productive properties of a hill pasture

White clover (Trifolium repens L.) herbage accumulation rate, nitrogen (N) fixation rate and efficiency, and plant growth characteristics were compared in pastures based on eight white clover lines on north‐ and south‐facing sites under sheep grazing in southern North Island hill country during years 4–7 following oversowing. The lines compared included three cultivars ('Grasslands Tahora’, ‘Grasslands Prestige’, and Prop) bred for hill country and/or sheep grazing systems with (notionally, at least) a moderate‐high degree of adaptation to the environment, and two cultivars ('Grasslands Huia’ and ‘Grasslands Kopu') known to have moderate—low adaptation. The control treatment comprised pastures based on the resident clover ecotype of the site, seed of which was sown at the same rate as the other lines. Two of the adapted lines (Tahora, Prestige) yielded significantly more white clover herbage than the resident ecotype over 3 full years of measurement. Prestige displayed consistently greater cool‐season growth in late winter‐early spring than all other lines. All three adapted cultivars fixed significantly more N than the resident ecotype over 12 months (mean for resident = estimated 64 kg N/ha per year compared to mean for three cultivars = 100 kg N/ha per year). There was a close linear relationship between white clover herbage accumulation rate and clover stolon density in most years and on both aspects. The adapted lines developed greater stolon densities through superior branching ability (higher frequency of occurrence of branches at stolon nodes). There was evidence that the adapted clover lines were able to compete more effectively with grasses for growth resources at some times of the year, e.g., for mineralised N in late winter‐early spring, resulting in lower N fixation efficiencies for Tahora and Prestige (mean 43 kg N fixed/t clover DM grown, versus 122 kg N/t clover DM for the resident ecotype). There were no statistically significant effects of clover line on total annual pasture herbage accumulation, but a difference between Prestige and all other lines in total pasture growth in winter gradually strengthened over 4 years of measurement so that, in the final year measured (year 7 after sowing), a statistically significant effect of line was recorded. In this winter, Prestige‐based pastures grew 1130 kg DM/ ha over 65 days from early June to late August, whereas resident pastures grew 570 kg DM/ha. The results are discussed within the context of pasture improvement strategies in hill country. Several important effects of adapted germplasm on the productive properties of hill pastures are identified.

Mass rearing Halotydeus destructor (Tucker) (Acari : Penthaleidae) for use in summer screening of Trifolium subterraneum (L.) for mite resistance

Summary. Controlled environment experiments were conducted to establish some of the requirements for successful mass rearing of Halotydeus destructor (redlegged earth mite). Numbers of mites reared on Vicia sativa (common vetch) cv. Blanchefleur grown alone or on a mixture of vetch with Trifolium subterraneum (subterranean clover) cv. Goulburn, were significantly higher than those on subterranean clover or Arctotheca calendula (capeweed) alone. Populations reared on vetch grown in a sandy soil were significantly higher than those reared on vetch grown in a loamy soil, pure sand or pure loam. Covering the soil surface with a natural pasture mulch increased mite numbers compared with leaving the soil bare or placing plant pots inside ventilated cages. Subsequent changes in rearing methodology produced enough mites to enable summer screening of subterranean clover lines for resistance to H. destructorfor the first time. Over 20 000 mites can be produced from vetch at one time for screening tests throughout the year.

Response of Native Eastern North American Trifolium spp. to Root‐Knot Nematodes

The true clovers (Trifolium spp.) are important pasture plants, but most of the cultivated species have low resistance to root‐knot nematodes (RKN) (Meloidogyne spp.). Plant introductions of seven Trifolium spp. native to eastern North America (T. bejariense Moric., T. calcaricum Collins &amp; Weiboidt; T. carolinianum Michx., T. polymorphum Poir. ex Lam. &amp; Poir.; T. reflexum L., T. stoloniferum Muhlenberg, and T. virginicum Small ex Small &amp; Vail.) were evaluated in two greenhouse experiments, conducted in spring 1992 and 1993, for response to four RKN species, Meloidogyne arenaria (Neal) Chitwood race 1, M. hapla Chitwood, M. incognita (Kofoid and White) Chitwood race 3, and M. javanica (Treub) Chitwood. Additionally, T. riograndense Burkhart and one accession each of T. ambiguum M.B. and T. repens L. were evaluated. Responses determined were numbers of galls or egg masses 8‐wk after inoculation rated on a 0 to 5 scale where 0 is no galling or egg masses per plant and 5 is greater than 100. Among the annual species, T. carolinianum and T. bejariense were the most resistant annual species. Among the perennials, T. calcaricum and T. stoloniferum were highly resistant (ratings &lt; 1.0) except that T. stoloniferum had an intermediate response to M. hapla (ratings of 2.5–3.5). Ttifolium polymorphum was variable in response among RKN. The kura and white clover lines and T. riograndense were highly susceptible to RKN (ratings &gt; 4.5). The federally endangereds pecies, T. stoloniferum, rated less than 1.0 in response to M. arenaria, M. incognita, and M. javanica. The recently described rare species, T. calcaricum, had the most resistant response of any species evaluated. These native eastern North American clovers may contain valuable genes for RKN resistance, but due to sexual incompatibility with the cultivated clover species, exploitation of such genes will require molecular methods of gene transfer.

Variation in white clover for root‐knot nematode resistance

Abstract Root‐knot nematode (Meloidogyne sp.) reduces growth and nutrition of white clover (Trifolium repens L.). Breeding resistant cultivars is the preferred control method. A range of overseas and New Zealand white clover lines was screened for resistance, measured as a reduction in the number of galls per root size. Highly significant differences between white clover lines were found, although no plants were immune. There was variation in both gall numbers and root size, and root size was a major factor determining the galls/ root ratio. The most resistant genotypes generally were from larger‐leaved lines, whereas the most suceptible genotypes generally were from smaller‐leaved lines. Resistant genotypes (and some susceptible genotypes) were re‐screened, some twice, using five clonal propagules per genotype. While there were significant correlations between the re‐screens for galls, roots, and galls/root, the values showed that repeatability was low. Comparisons with other nematode and clover species ...

Third generation progress in breeding white clover for resistance to root-knot nematode

Root-knot nematode (Meloidogyne sp.) reduces growth and nutrition of white clover (Trifolium repens L.) in New Zealand, and breeding resistant cultivars (with low galls per gram of root) is the preferred control method. Resistant and susceptible selections were bred from a wide range of white clover lines for three generations. In the third generation there were significant differences between seed lines from the selections for number of galls, root dry weight, visual growth score and galls/gram of root dry weight. Resistant selections had 43% of the susceptible selections' galls per gram, and 50% of the number of galls. Germplasm showing resistance to Meloidogyne spp. in the USA showed partial resistance to the local Meloidogyne sp. Two resistant and two susceptible genotypes were also compared for nematode egg production; resistant genotypes had a mean of 3,460 eggs/plant, compared to 25,030 for susceptible genotypes. Keywords: breeding, Meloidogyne sp., resistance, rootknot nematode, screening, selection, Trifolium repens, white clover

Resistance of the white clover variety G49 and its parent lines to stem nematode (Ditylenchus dipsaci)

Abstract Stem nematode (Ditylenchus dipsaci) infection of white clover variety G49 was compared to that of other white clover lines in two experiments. The G49 parent lines were also screened for infection in a third experiment. Symptoms of stem nematode infection were assessed at 8, 14, and 21 days after inoculation. The lower infection rate of G49 by D. dipsaci was not sufficient, as a stand‐alone trait, to distinguish G49 from other existing lines. However, G49 had a significantly lower infection rate than Pitau so these can be distinguished for the purposes of a Plant Variety Right application. The variation among the parent lines of G49 indicated scope for further selection to improve resistance. Three techniques for applying inoculum to seedlings were compared; the best one uses 1 ml of nematode suspension placed over a 3‐day‐old seedling lying on the surface of peat‐based potting mix which is then lightly covered.