Effects of Nematicides and Plant Resistance on White Clover Performance and Seasonal Populations of Nematodes Parasitizing White Clover in Grazed Pasture

Root-infecting nematodes are a major cause of white clover, Trifolium repens, not reaching its potential in New Zealand pastures. Resistance and/or tolerance are the preferred control options. Greenhouse-based, recurrent selection programs have developed resistance to Meloidogyne trifoliophila and Heterodera trifolii, and a field-based program has developed tolerance. Lines from these programs were compared with commercial cultivars as controls in a series of field trials at four sites over 4 years. Resistant lines from the CCN program performed better than susceptible lines and as well as most cultivars, reflecting the high level of resistance developed in this greenhouse-based program. In stained root from Cambridge, numbers of CCN were lower in resistant lines than in cultivars; numbers in susceptible lines were intermediate. CCN resistance was also reflected to a lesser extent in the number of cysts counted in soil under resistant lines in Palmerston North. The root-knot nematode-resistant material performed better than the susceptible and as well as most cultivars. In one trial of CRKN-resistant lines, resistant and susceptible lines had similar numbers of CRKN which were both lower than the numbers in the cultivars; in the second trial, there were fewer CRKN in resistant than in susceptible lines or cultivars. The tolerant selections, developed under field conditions, performed as well as or better than the cultivars. The selections from the breeding programmes have exhibited strong agronomic potential across locations and years, and the best material has been crossed; progeny are being assessed in current field trials.     

Variations in Host Preference among and within Populations of Heterodera trifolii and Related Species

Seven populations of Heterodera trifolii from Arkansas, Kentucky, Pennsylvania, and Australia plus 3 or 4 single-cyst isolates (SCI) from each population were tested for reproduction on seven species of plants to compare the host preferences among and within populations. Common lespedeza, Kummerowia striata cv. Kobe, was a good host for all populations and isolates. Therefore, a plant was considered to be a host if the number of females produced on it was 10% or more of the number on Kobe. All seven populations reproduced on Trifolium repens and T. pratense. None reproduced on Beta vulgaris or Glycine max. One single-cyst isolate from the Australian population produced a few females on T. pratense. The Australian population maintained on carnation, Dianthus caryophyllus, produced females on carnation but not on curly dock, Rumex crispus. However, its subpopulation maintained on T. repens produced females on R. crispus but not on carnation. Four of the other six populations produced females on R. crispus, and four produced females on carnation. Differences in host range were observed among seven of the mother populations and their SCI, and among isolates within each population. Five host range patterns were found in populations and SCI of H. trifolii. Significant quantitative differences occurred among populations in the numbers of females on most hosts, between isolates and their original populations, and among isolates from the same population. SCI selected from white clover produced fewer females on a series of test hosts and had host ranges the same as or narrower than those of the original populations. However, SCI selected from Kobe lespedeza had more females on some hosts and had host ranges the same as or wider than those of the original populations. The host ranges of all populations and SCI of H. trifolii were different from those of populations and SCI of race 3 of H. glycines and H. lespedezae.     

Ditylenchus dipsaci Infestation of Trifolium repens. II. Dynamics of Infestation Development

Trifolium repens (white clover) stolons were inoculated with Ditylenchus dipsaci (stem nematode), and the development of resulting infestations was monitored. Nematodes initially remained confined to superficial locations, concentrating in petiole axils near inoculation points. They were able to migrate slowly from the inidal inoculation points and infest adjacent axils, especially in regions near the stolon tip. As time progressed, in some axils, nematodes migrated through the stolon epidermis and colonized slowly expanding subepidermal pockets of host tissue (ca. 0.2-mm length of stolon/day). In these loci nematodes established exponentially increasing populations, but the rates of locus expansion remained constant, indicating that locus expansion was limited by unidentified host-dependent factors. As a result of increasing population pressure within subepidermal loci, J4 entered a "diapause" state and the rate of egg production by adults declined, thereby reducing rate of population growth to more sustainable levels. Typically, these populations peaked at ca. 10,000 individuals in ca. 160 days occupying 3-cm lengths of stolon. Thereafter, heavily infested regions of stolons started to die, leading to the formation of longitudinal splits in their epidermis. In other axils, nematodes did not migrate into the stolons but remained confined to axils. Some of these populations increased a hundred-fold in 95 days, with population growth ending when petioles started to die. Host plant stolon morphology was affected only when subepidermal stolon populations developed high population levels (>100 nematodes) within close proximity (<2 cm) to active terminal meristems. This occurred either when axillary buds became active on previously infested nodes or when nematodes established endoparasitic populations at locations near the stolon tip during winter and spring, when the rate of stolon extension was limited by low light intensity. Affected stolon tips could "escape" from the influence of such infestations when light intensity and temperature increased. Nematode activity was limited by low temperature rather than light intensity. Global warming is likely to lead to greater damage to infested plants during the winter and early spring because the predicted milder winter temperatures will enhance nematode activity but not necessarily promote stolon growth.     

Comparative Electrophoretic Analyses of Soluble Proteins from Heterodera glycines Races 1-4 and Three Other Heterodera Species

Modified polyacrylamide gel and SDS-polyacrylamide gel electrophoretic systems using a low molarity tris-HCl buffer and equal pH of homogenizing buffer and stacking gel provided improved stacking for separation of soluble proteins from Heterodera schachtii, H. trifolii, H. lespedezae, and H. glycines races 1, 2, 3, and 4, compared with previous studies with cyst nematodes, The four Heterodera species were easily distinguished using the polyacrylamide gel system, but H. trifolii and H. lespedezae had similar protein patterns. H. glycines races were not separable by that system. The SDS-polyacrylamide gel system produced different protein patterns for all four Heterodera species although H. trifolii and H. lespedezae differed by only a single band, suggesting that these two may be subspecifically related. A protein band unique to H. glycines races 3 and 4 was not detected in SDS-polyacrylamide gel profiles from races 1 and 2. Molecular weight determinations were 55,000 for distinctive proteins in profiles of H. trifolii and 75,000 for H. glycines races 3 and 4.     

Variation in Resistance of Soybean Lines to Races of Heterodera glycines

The objective of this study was to determine the interrelationships of Heterodera glycines races based on their resistance to soybean (Glycine max) cultivars and lines against which they were tested. Greenhouse tests determined the numbers of females of each of eight races of H. glycines that developed on 277 to 522 soybean cultivars and lines. A Female Index (number of females on a test cultivar as a percentage of the number on ''Lee 74'') was calculated and used in frequency distributions, correlations, and duster analyses of the resistance reactions to the different races in an attempt to determine relationships among cultivars. Frequency distribution patterns of all cultivars and lines tested against each race were skewed in favor of resistance, and in some cases bimodality was observed. The majority of correlations between pairs of races were highly significant. Cluster analyses based on the correlations divided eight races into four clusters that explained 73% of the variation in resistance. Cluster 1 was comprised of races 2, 4, and 14; Cluster 2 was comprised of races 6 and 9; Cluster 3 was comprised of races 1 and 3; and Cluster 4 was comprised of race 5. The information obtained in this study could increase the efficiency of testing resistant soybean breeding lines for resistance to H. glycines.     

Genetic Diversity for Resistance to Heterodera glycines Race 5 in Soybean

Heterodera glycines is a serious pest of soybean in the United States. Plant introductions 90763 and 424595 are reported to be resistant to H. glycines race 5; however their genetic relationship for resistance is unknown. Crosses between these two lines and the susceptible cultivar Essex were studied in the F₁, F₂, and F₃ generations to determine the number of genes involved in inheritance of resistance. The plants were screened using conventional techniques based on the index of parasitism. The data were subjected to analyses using chi-square test to determine goodness of fit between observed and expected genetic ratios. The cross PI 424595 x Essex segregated 1 resistant:63 susceptible in the F₂ generation, which indicated the presence of three recessive genes controlling resistance to race 5. In the cross PI 90763 x Essex, resistance was conditioned by one dominant and two recessive genes. The cross between PI 424595 and PI 90763 segregated into 13 resistant:3 susceptible. The data fit a four-gene model with two recessive and two dominant genes with epistasis. PI 90763 has a dominant gene, whereas PI 424595 has a recessive gene; both share two additional recessive genes for resistance to race 5. This information is important to geneticists and soybean breeders for the development of cultivars resistant to H. glycines.     

Screening Subterranean Clover (Trifolium spp.) Germplasm for Resistance to Meloidogyne species

This study was conducted to identify lines of subterranean clover (Trifolium spp.) with resistance to Meloidogyne arenaria (Neal, 1989) Chitwood, 1949, race 1; M. incognita (Kofoid and White, 1919) Chitwood, 1949, race 3; and M. javanica (Treub, 1885) Chitwood, 1949. A collection of 134 subterranean clover lines was evaluated and all had intermediate to high susceptibility. Root galling was negatively correlated with both seed and dry matter yields. Soil fumigation significantly reduced the nematode population in the field. Results indicate there is limited genetic resistance to root-knot nematodes among subterranean clover lines. Alternative sources of variation for this trait should be investigated.     

利用P5CS基因转化白三叶的研究

为了培育抗旱白三叶,构建了植物表达载体pBPC-P5CS,利用基因枪法转化白三叶的愈伤组织,PCR检测和Southern blot鉴定证实白三叶中已导入P5CS基因。对转P5CS基因白三叶植株的不同抗旱指标进行了分析。结果表明,与对照相比,转P5CS基因株系的抗旱能力得到了较大的提高。干旱胁迫下,与对照相比,转P5CS基因植株的脯氨酸含量和相对含水量分别比对照高20.0%-21.2%和5.6%-8.5%。     

Effects of Zinc Fertilization of Corn on Hatching of Heterodera glycines in Soil

Experiments were conducted to determine the effects of zinc fertilizers on hatching and soil population densities of Heterodera glycines. In vitro egg hatching in solutions of reagent-grade zinc sulfate and zinc chloride and fertilizer-grade zinc sulfate was significantly greater than hatching in deionized water, whereas zinc chelate fertilizer significantly inhibited egg hatching relative to deionized water. In greenhouse experiments, no differences in cumulative percentage egg hatch were detected in soil naturally infested with H. glycines amended with fertilizer-grade zinc sulfate and zinc chelate at rates equivalent to 0, 1.12, 11.2, and 112 kg Zn/ha and subsequently planted with corn (Zea mays L.). In a field experiment, no significant differences in H. glycines egg population densities and corn yields were detected among plots fertilized with 0, 11.2, and 22.4 kg Zn/ha rates of zinc chelate. Yields of H. glycines-susceptible soybean planted in plots 1 year after zinc fertilization of corn plots also were not significantly affected. Zinc compounds significandy affected H. glycines egg hatching in vitro, but had no effect on hatching in natural soils.     

Effect of Soil Temperature and pH on Resistance of Soybean to Heterodera glycines

Soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is a major pest of soybean, Glycine max L. Merr. Soybean cultivars resistant to SCN are commonly grown in nematode-infested fields. The objective of this study was to examine the stability of SCN resistance in soybean genotypes at different soil temperatures and pH levels. Reactions of five SCN-resistant genotypes, Peking, Plant Introduction (PI) 88788, Custer, Bedford, and Forrest, to SCN races 3, 5, and 14 were studied at 20, 26, and 32 C, and at soil pH''s 5.5, 6.5, and 7.5. Soybean cultivar Essex was included as a susceptible check. Temperature, SCN race, soybean genotype, and their interactions significantly affected SCN reproduction. The effect of temperature on reproduction was quadratic with the three races producing significantly greater numbers of cysts at 26 C; however, reproduction on resistant genotypes remained at a low level. Higher numbers of females matured at the soil pH levels of 6.5 and 7.5 than at pH 5.5. Across the ranges of temperature and soil pH studied, resistance to SCN in the soybean genotypes remained stable.     

Structural Changes Associated with Resistance of Soybean to Heterodera glycines

Subcellular responses to infection by Race 3 of Heterodera glycines in susceptible (''Lee'') and resistant (''Forrest'' and ''Bedford'') soybean cultivars were compared. Syncytial formation, initiated in susceptible as well as resistant soybean cultivars, was characterized by wall perforations, dense cytoplasm, and increased endoplasmic reticulum, In susceptible plants, syncytia developed continuously until nematode maturity. This included hypertrophy of nuclei, increase of rough endoplasmic reticulum in early stages of infection, and formation of wall ingrowths at a late stage of infection. In the resistant reaction in Forrest, a necrotic layer surrounded syncytium component cells demarcating them from surrounding normal cells and leading to syncytial necrosis. Wall appositions were prominently formed near the necrotic layer, and the cytoplasm of the syncytium component cells was extremely condensed. The whole syncytium became necrotic at a late stage of infection. Bedford had nuclear degeneration prior to cytoplasmic degradation. Chromatin was often scattered throughout the syncytial cytoplasm. Finally the whole syncytium became degenerated with plasmalemma completely detached from the syncytial cell walls. The differences in resistant responses reflect a difference in genetic composition of the soybean cultivars tested.     

Variation in growth and ion accumulation between two selected populations of Trifolium repens L. differing in salt tolerance

Two divergent populations of T. repens cv. Haifa developed from two generations of recurrent selection for shoot chloride concentration, were grown in the greenhouse at 0 and 40 mol m^–3 NaCl. Over two harvest cycles at 40 mol m^–3 NaCl, the population selected for a low concentration of chloride in the shoot maintained a significantly lower chloride and sodium concentration compared with those plants selected for a high shoot chloride concentration. The distribution of chloride in the shoots was further examined in a subsample of plants from both populations. In all plants, concentrations of chloride were lower in the expanding and fully expanded leaves than in the older leaf tissue or petioles.While there were no significant differences in the photosynthetic rates between lines, shoot yields and relative leaf expansion rates were higher in the low chloride population. Plant death was greater in plants selected for high shoot chloride. These results suggest that selections based on measurements of low shoot chloride concentrations may be successful in developing a cultivar of T. repens with improved salt tolerance.     

Identification of Cyst Nematodes of Agronomic and Regulatory Concern with PCR-RFLP of ITS1

The first internally transcribed spacer region (ITS1) from cyst nematode species (Heteroderidae) was compared by nucleotide sequencing and PCR-RFLP. European, Asian, and North American isolates of five heterodefid species were examined to assess intraspecific variation. PCR-RFLP patterns of amplified ITS1 DNA from pea cyst nematode, Heterodera goettingiana, from Northern Ireland were identical with patterns from Washington State. Sequencing demonstrated that ITS1 heterogeneity existed within individuals and between isolates, but did not result in different restriction patterns. Three Indian and two U.S. isolates of the corn cyst nematode, Heterodera zeae, were compared. Sequencing detected variation among ITS1 clones from the same individual, between individuals, and between isolates. PCR-RFLP detected several restriction site differences between Indian and U.S. isolates. The basis for the restriction site differences between isolates from India and the U.S. appeared to be the result of additional, variant ITS1 regions amplified from the U.S. isolates, which were not found in the three India isolates. PCR-RFLP from individuals of the U.S. isolates created a composite pattern derived from several ITS1 types. A second primer set was specifically designed to permit discrimination between soybean (H. glycines) and sugar beet (H. schachtii) cyst nematodes. Fok I digestion of amplified product from soybean cyst nematode isolates displayed a uniform pattern, readily discernible from the pattern of sugar beet and clover cyst nematode (H. trifolii).     

Eco-geographically divergent diploids, Caucasian clover (Trifolium ambiguum) and western clover (T. occidentale), retain most requirements for hybridization

Background and Aims

DNA sequence similarities and hybridization patterns in Trifolium (clovers) section Trifoliastrum suggest that rapid radiation from a common ancestral source led to this complex of diverse species distributed across Europe, western Asia and North Africa. Two of the most geographically and ecologically divergent of these species are the rhizomatous T. ambiguum from high altitudes in eastern Europe and western Asia and the stoloniferous T. occidentale from sea level in western Europe. Attempts were made to hybridize these species to ascertain whether, despite this separation, gene flow could be achieved, indicating the retention of the genetic factors necessary for hybridization.

Methods

Three F₁ hybrids formed after embryo rescue were described, characterized by conventional and molecular cytogenetics, subjected to fertility tests and progeny generations were developed.

Results and Conclusions

Partially fertile hybrids between Trifolium ambiguum and T. occidentale were obtained for the first time. The F₁ hybrids produced seeds after open-pollination, and also produced triploid progeny in backcrosses to T. occidentale from the functioning of unreduced gametes in the hybrids. These plants were fertile and produced progeny with T. occidentale and with T. repens. Meiotic chromosome pairing in the F₁ showed six to eight bivalents per pollen mother cell, indicating pairing between the parental genomes. A chromosome-doubled form of one hybrid, produced using colchicine, showed some multivalents, indicative of interspecific chromosome pairing. The hybrid plants were robust and combined phenotypic characteristics of both species, having stolons, thick roots and a few rhizomes. Results show that despite separation by the entire breadth of Europe, the speciation process is incomplete, and these taxa have partially retained most of the genetic compatibilities needed for hybridization (possibly except for endosperm development, which was not tested). The fertile progeny populations could lead to new clover breeding strategies based on new hybrid forms.     

A Technique for Evaluating Heterodera glycines Development in Susceptible and Resistant Soybean

A technique was developed to evaluate Heterodera glycines development in susceptible and resistant soybean. Roots of 3-day-old soybean were exposed to infective juveniles of H. glyci.nes in sand for 8 hours followed by washing and transfer to hydroponic culture. The cotyledons and apical meristem were removed and plants were maintained under constant light, which resulted in a dwarfed plant system. After 15 or 20 days at 27 C, nematodes were rated for development. Emerged males were sieved from the culture water and females were counted directly from the roots. Nematodes remaining in the roots were rated for development after staining and clearing the tissues. The proportion of nematodes at each stage of development and the frequency of completed molts for each stage were calculated from these data. This technique showed that resistance to H. glycines was stage related and did not affect males and females equally in all resistant hosts. The resistance of plant introduction PI 209332 primarily affected development of third and fourth-stage juveniles; ''Pickett'' mainly affected second and third-stage juveniles, whereas PI 89772 affected all stages. Male development was markedly affected in PI 89772 and ''Pickett'' but not in PI 209332.     

Temperature Effects on Race Determination in Heterodera glycines

Currently there are 16 possible races for Heterodera glycines, and these are differentiated based on ability of a nematode population to develop on a set of four differential soybean genotypes. Because results are based on numbers of nematode females that develop to a specific stage rather than on the reproductive capability of these females, race determinations based on female indices may not represent results obtained after several reproductive cycles of H. glycines. Counting numbers of eggs and juveniles, and then developing corresponding indices, would allow reproduction to be considered in making race determinations. Our objectives were to compare the present race identification scheme for H. glycines based on female indices with those using egg and juvenile indices and to examine the effect of temperature on race designations using female, egg, and juvenile indices. Race designations for H. glycines populations from two locations in Illinois were determined at 20, 27, and 30 °C in a water bath. The numbers of females, eggs, and juveniles (at 19 days) were recorded, and an index based on each life stage was calculated. Race determinations based on female, egg, or juvenile indices were inconsistent when conducted at 20 °C, which demonstrates that this temperature is not suitable for identifying races of H. glycines. However race designations at 27 and 30 °C were consistent for all three indices. This indicates that counting females, eggs, or juveniles should be equally reliable when race determinations are conducted at these two temperatures, and choice of method would depend on investigator preference or research objective.     

Relationship Between Heterodera schachtii,Meloidogyne hapla,and Nacobbus aberrans on Sugarbeet

Heterodera schachtii, Meloidogyne hapla, and Nacobbus aberrans either alone, or in various combinations with each other, can, when inoculated at a concentration of 12 second-stage juveniles/ cm³ of soil, cause a significant (P = 0.01) suppression of growth of sugarbeet (cv. Tasco AH14) seedlings. M. hapla and H. schachtii decreased growth of sugarbeet more than N. aberrans over a 60-day period. The adverse effect of N. aberrans on the final population/initial population (Pf/Pi) ratio for either M. hapla or H. schachtii was dependent on time, and was more accentuated on that of M. hapla than on that of H. schachtii. Neither M. hapla nor H. schachtii had an adverse effect on the Pf/ Pi ratio of N. aberrans. N. aberrans is considered to be less aggressive on sugarbeet than either H. schachtii or M. hapla. Sections of sugarbeet roots infected simultaneously with H. schachtii and N. aberrans showed scattered vascular elements between the N. aberrans syncytium located in the central part of the root and that of H. schachtii in the peripheral position.     

FMRFamide-like Immunoactivity in Heterodera glycines (Nemata: Tylenchida)

Material antigenically related to the neuromodulatory peptide FMRFamide was detected and examined in preparations of the soybean cyst nematode, Heterodera glycines, and in the free-living nematodes Caenorhabditis elegans and Panagrellus redivivus. FMRFamide-related peptides were quantified by an enzyme-linked immunosorbent assay. Specific activities were remarkably similar among all of the vermiform members of the three species. FMRFamide-related peptide immunoactivity was present in both sexes and all stages of H. glycines examined. The highest specific activity was present in second-stage juveniles and in males, and the lowest in white and yellow females. Total FMRFamide-related peptide level per individual was highest in brown females, with 90% of the activity associated with the eggs. Peptide levels in these eggs and in second-stage juveniles were comparable and increased in adults, especially in females. Chromatographic analysis of FMRFamide-related peptide preparations from H. glycines juveniles, C. elegans, and P. redivivus revealed distinct qualitative differences between the infective plant parasite and the free-living nematodes.