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Rhizobium meliloti Rm1021 requires a Calcofluor-binding exopolysaccharide, termed succinoglycan or EPS I, to invade alfalfa nodules. We have determined that a strain carrying a mutation in the exoZ locus produces succinoglycan that lacks the acetyl substituent. The exoZ mutant nodules alfalfa normally.
The soil bacterium Sinorhizobium meliloti is capable of entering into a nitrogen-fixing symbiosis with Medicago sativa (alfalfa). Particular low-molecular-weight forms of certain polysaccharides produced by S. meliloti are crucial for establishing this symbiosis. Alfalfa nodule invasion by S. meliloti can be mediated by any one of three symbiotically important polysaccharides: succinoglycan, EPS II, or K antigen (also referred to as KPS). Using green fluorescent protein-labeled S. meliloti cells, we have shown that there are significant differences in the details and efficiencies of nodule invasion mediated by these polysaccharides. Succinoglycan is highly efficient in mediating both infection thread initiation and extension. However, EPS II is significantly less efficient than succinoglycan at mediating both invasion steps, and K antigen is significantly less efficient than succinoglycan at mediating infection thread extension. In the case of EPS II-mediated symbioses, the reduction in invasion efficiency results in stunted host plant growth relative to plants inoculated with succinoglycan or K-antigen-producing strains. Additionally, EPS II- and K-antigen-mediated infection threads are 8 to 10 times more likely to have aberrant morphologies than those mediated by succinoglycan. These data have important implications for understanding how S. meliloti polysaccharides are functioning in the plant-bacterium interaction, and models are discussed.
During September 1999, a multistate outbreak of Salmonella serovar Muenchen infection associated with eating raw alfalfa sprouts was identified in Wisconsin. Despite use of a calcium hypochlorite sanitizing procedure to pretreat seeds before sprouting, at least 157 outbreak-related illnesses were identified in seven states having sprouters who received alfalfa seed from a specific lot. The continued occurrence of sprout-related outbreaks despite presprouting disinfection supports the concern that no available treatment will eliminate pathogens from seeds before sprouting and reinforces the need for additional safeguards to protect the public. A lack of consumer knowledge regarding exposure to sprouts documented in this investigation suggests that more-targeted outreach to high-risk individuals may be needed to reduce their risk.
The production of eggs with the sporophytic chromosome number (2n eggs) in diploid alfalfa (Medicago spp.) is mainly associated with the absence of cytokinesis after restitutional meiosis. The formation of 2n eggs through diplosporic apomeiosis has also been documented in a diploid mutant of M. sativa subsp. falcata (L.) Arcang. (2n = 2x = 16), named PG-F9. Molecular tagging of 2n-egg formation appears to be an essential step towards marker-assisted breeding and map-based cloning strategies aimed at investigating and manipulating reproductive mutants of the M. sativa complex. We made controlled crosses between PG-F9 and three wild type plants of M. sativa subsp. coerulea (Less.) Schm. (2n = 2x = 16) and then hand-pollinated the F1 progenies with tetraploid plants of M. sativa subsp. sativa L. (2n = 4x = 32). As a triploid embryo block prevents the formation of 3x progenies in alfalfa because of endosperm imbalance, and owing to the negligible selfing rate, seed set in 2x-4x crosses was used to discriminate the genetic capacity for 2n-egg production. F1 plants that exhibited null or very low seed sets were classified as normal egg producers and plants with high seed sets as 2n-egg producers. A bulked segregant analysis (BSA) with RAPD (random amplified polymorphic DNA), ISSR (inter-simple sequence repeat), and AFLP (amplified fragment length polymorphism) markers was employed to identify a genetic linkage group related to the 2n-egg trait using one of the three F1 progenies. This approach enabled us to detect a paternal ISSR marker of 610 bp, generated by primer (CA)8-GC, located 9.8 cM from a putative gene (termed Tne1, two-n-eggs) that in its recessive form determines 2n eggs and a 30% recombination genomic window surrounding the target locus. Eight additional RAPD and AFLP markers, seven of maternal, and one of paternal origin, significantly co-segregated with the trait under investigation. The minimum number of quantitative trait loci (QTLs) controlling seed set in 2x-4x crosses was estimated by ANOVA and regression analysis. Four maternal and three paternal independent molecular markers significantly affected the trait. A paternal RAPD marker allele, mapped in the same linkage group of Tne1, explained 43% of the variation for seed set in 2x-4x crosses indicating the presence of a major QTL. A map of the PG-F9 chromosome regions carrying the minor genes that determine the expression level of 2n eggs was constructed using selected RAPD and AFLP markers. Two of these genes were linked to previously mapped RFLP loci belonging to groups 1 and 8. Molecular and genetic evidence support the involvement of at least five genes.
Improving commercial utilization of perennial Medicago collections requires developing approaches that can rapidly and accurately characterize genetic diversity among large numbers of populations. This study evaluated the potential of using amplified fragment length polymorphism (AFLP) DNA markers, in combination with DNA bulking over multiple genotypes, as a strategy for high-throughput characterization of genetic distances (D) among alfalfa (Medicago sativa L.) accessions. Bulked DNA templates from 30 genotypes within each of nine well-recognized germplasms (African, Chilean, Flemish, Indian, Ladak, Medicago sativa subsp. falcata, Medicago sativa subsp. varia, Peruvian, and Turkistan) were evaluated using 34 primer combinations. A total of 3754 fragments were identified, of which 1541 were polymorphic. The number of polymorphic fragments detected per primer combination ranged from 20 to 85. Pairwise D estimates among the nine germplasms ranged from 0.52 to 1.46 with M. sativa subsp. falcata being the most genetically dissimilar. Unweighted pair-group method arithmetic average (UPGMA) analysis of the marker data produced two main clusters, (i) M. sativa subsp. sativa and M. sativa subsp. varia, and (ii) M. sativa subsp. falcata. Cluster-analysis results and D estimates among the Chilean, Peruvian, Flemish, and M. sativa subsp. varia germplasms supported the hypothesis that Peruvian was more similar to original Spanish introductions into Central and South America than Chilean. Hierarchical arrangement of the nine germplasms was supported by their respective geographic, subspecific, and intersubspecific hybrid origins. Subsets of as few as seven highly informative primer pairs were identified that produced comparable D estimates and similar heirarchical arrangements compared with the complete dataset. The results indicate that use of primer-pair subsets for AFLP analysis of bulk DNA templates could serve as a high-throughput system for accurately characterizing genetic diversity among large numbers of alfalfa populations.
Polymerase chain reaction was used, with single 10-mer primers of arbitrary sequence, to amplify random regions of genomic DNA from a diploid cultivated alfalfa backcross population. Segregation of the random amplified polymorphic DNA (RAPD) fragments was analysed to determine if RAPD markers are suitable for use as genetic markers. Of the 19 primers tested, 13 amplified a total of 37 polymorphic fragments, of which 28 (76%) segregated as dominant Mendelian traits. RAPD markers appear useful for the rapid development of genetic information in species like alfalfa where little information currently exists or is difficult to obtain.
A DNA fragment of approximately 750 bp was cloned from EcoRI-digested nuclear DNA of alfalfa (Medicago sativa). Southern blot and sequence analysis showed that the cloned DNA fragment represents a tetramer of a highly tandemly repeated DNA sequence of 185-188 bp (E180). The consensus sequence deduced from the four repeating units is 189 bp in length with an AT content of 67%. The copy number of the satellite DNA was estimated to be approximately 1.8 x 10(5) per genome and constitutes about 1% of the alfalfa genome. Sequence comparison revealed no identity to any repetitive DNA sequences that have been published to date. Digestion with HpaII and MspI indicated a high degree of methylation at the internal C of the restriction site CCGG within E180.
A genome map of cultivated alfalfa was constructed using segregating restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNAs (RAPDs) in a diploid backcross population generated from noninbred parents. Among the 153 loci scored in 87 progeny, four segregation ratios were observed for codominant and dominant markers: 1:1, 1:2:1, 1:1:1:1, and 3:1. Deviations from expected Mendelian ratios (p < 0.05) were observed for 34% of the loci studied. A genome map was assembled from two separate linkage maps, each constructed from a subset of the segregation data. One linkage map was constructed from 46 RFLP and 40 RAPD markers segregating 1:1 from the F1 parent of the backcross and the other linkage map was constructed from 33 RFLP and 28 RAPD markers segregating 1:1 from the recurrent parent. Sixteen loci with alleles segregating 1:1 from both parents were used as locus bridges to align individual linkage groups between the two maps. The combined use of RFLPs and RAPDs was an effective method for developing an alfalfa genome map.
A velocity sedimentation procedure was described to fractionate bacteroids of alfalfa nodules into four subpopulations. Bacteroids in these subpopulations were different in size and nucleic acid content as determined by microscopy and flow-microfluorometry (FMF). The slowest-sedimenting bacteroids (fraction I) were small and resembled free-living Rhizobium meliloti both in size and nucleic acid content. The fastest-sedimenting bacteroids (fraction IV) were 2 to 3 times longer and contained 3 to 4 times more nucleic acid than the small bacteroids in fraction I and free-living R. meliloti. A positive correlation was established between bacteroid size and relative nucleic acid content of bacteroids in alfalfa nodules.
Light and electron microscopy was used to establish the structural organisation of the developing nodule of alfalfa. In these nodules three distinct regions were noted: (1) the base region, site of original infection where the nodule is attached to the root and now composed of degenerating nodule tissue, (2) the central region, or active region composed of nodule cells containing tightly packed bacteroids surrounding a central vacuole, and (3) the meristematic region, a site of new growth, behind which newly formed cells are continually invaded. The ongoing infection process accompanying continued nodule development provided the opportunity to study the release of Rhizobium cells from the infection threads. In the nodules of alfalfa it would appear that the Rhizobium cells are released from infection thread into the nodule tissue in two different ways: (i) release with infection thread membrane and (ii) release in thin-walled vesicular structures. Thus it is concluded that Rhizobium cells are surrounded by the infection thread membrane when they are released from the infection thread into nodule tissue.
Small isometric virus particles containing double-stranded RNA have been independently reported in Europe and Japan from apparently healthy alfalfa (Medicago sativa), beet (Beta vulgaris), and white clover (Trifolium repens). They have been called cryptic viruses in Europe and temperate viruses in Japan. Serological comparison using immunoelectron microscopy, and polyacrylamide gel electrophoresis of the RNAs indicate that alfalfa cryptic and temperate viruses are the same, beet cryptic virus is probably a mixture of two different viruses, one of which is similar to or the same as beet temperate virus, and white clover temperate virus is a mixture of at least three different viruses, two of them indistinguishable from white clover cryptic viruses 1 and 2, respectively, and the third very likely the same as white clover cryptic virus 3.
We determine the effects of alfalfa top saponins on cholesterol and bile acid balance in eight cynomolgus macaques (Macaca fascicularis). The monkeys ate semipurified food containing cholesterol with or without added saponins. The saponins decreased cholesterolemia without changing the levels of high density lipoprotein-cholesterol; hence, they reduced the total cholesterol/high density lipoprotein-cholesterol ratio. Furthermore, they decreased intestinal absorption of cholesterol, increased fecal excretion of endogenous and exogenous neutral steroids and bile acids, and decreased the percent distribution of fecal deoxycholic and lithocholic acids. The fecal excretion of fat was also slightly increased, but steatorrhea did not occur. We saw no signs of toxicity in the monkeys after 6 or 8 wk of saponin ingestion. The data suggest that alfalfa top saponins may be of use in the treatment of patients with hypercholesterolemia, but long-term studies on possible toxicity are needed before this therapy can be recommended for humans.
The alfalfa leafcutting bee, Megachile rotundata (F.), is a solitary, cavity-nesting bee that has been managed in large numbers to pollinate alfalfa, Medicago spp., seed crops since the 1960s. Propagation of these bees from 1 yr to the next has been seriously hampered by chalkbrood, a larval disease caused by the fungus Ascosphaera aggregata Skou. In the United States, attempts to control the disease have been fairly unsuccessful, but include removing nests from the nesting boards and then disinfecting the boards with heat treatments or a fumigant. The problem is that many boards are made of polystyrene (so heat cannot be used), and very few fumigants are registered for this use. In this study, ozone was tested as a fumigant and compared with heat treatments and methyl bromide fumigation. Ozone was found to be inadequate for killing A. aggregata spores and for reducing chalkbrood levels in the field. Methyl bromide and heat treatments did greatly reduce spore viability in the boards, but did not reduce chalkbrood levels in the field. Surprisingly, larvae in new nesting boards (boards free of contamination) and chalkbrood infection levels were similar to those from nests in contaminated, used boards. Disinfecting nesting boards may be necessary for controlling chalkbrood, but the results reported here indicate that it is not sufficient in and of itself. Some other source of spores was present in the field that was greater than the effect of contamination from the boards, but the source still needs to be determined.
Laboratory experiments were conducted to characterize the trichome-based defense of glandular-haired alfalfa, Medicago sativa L., against the potato leafhopper, Empoasca fabae (Harris). Within-plant variability in leafhopper resistance was examined by caging adult leafhoppers to either basal or apical stem internodes of the leafhopper-resistant, glandular-haired M. sativa genotype G98A or the susceptible, nonglandular-haired M. sativa 'Ranger'. Young, actively secreting glandular trichomes are located on apical internodes of G98A, whereas senesced gland heads are found on older, basal internodes of G98A. After 96 h, the highest cumulative leafhopper mortality and lowest number of excretory droplets were associated with apical internodes of G98A. No difference was detected in mortality and feeding levels among insects caged to basal internodes of G98A and basal and apical internodes of Ranger. The influence of abiotic factors on leafhopper resistance was evaluated by caging adult leafhoppers to either G98A or Ranger under four combinations of low and high light (250 and 1,000 micromol s(-1) m(-2)) and temperature regimes (17 and 30 degrees C). After 96 h, the highest cumulative mortality was associated with leafhoppers confined to G98A under high light and high temperature conditions. Temperature level and plant type also had an effect on the production of excretory droplets, resulting in the highest number of excretory droplets being associated with Ranger under the high temperature regime. These results indicate that certain regions of M. sativa G98A are better protected against the potato leafhopper than others and that temperature influences resistance levels of glandular-haired alfalfa.
Seed yield of alfalfa (Medicago sativa L.) is important in determining the effective distribution of new cultivars to farmers. Many genetic and environmental factors affect seed yield. This study was conducted to explain seed yield variation induced by either environmental conditions or cultivars. We analyzed seed yield, aboveground phytomass, harvest index, and seed yield components for a set of 12 cultivars at four locations across France in each of three years. Each location x year combination was considered an environment. Seed weight, number of pods per inflorescence, number of seeds per pod, and mean seed weight were measured. Mean seed yield was 801 kg ha(-1). Large variation in seed yield was found among cultivars and environments. The cultivar x environment interaction was significant. Among environments, seed yield was highly correlated with aboveground phytomass at harvest (r = 0.94) as the lowest seed yields were obtained in the seeding year. The cultivars most adapted to grazing showed the lowest seed yields. Seed yield was genetically correlated with lodging resistance (r = -0.89) and harvest index (r = 0.99). The mean harvest index was 12.7%. The seed weight per inflorescence showed a high broad-sense heritability (0.58) and a high genetic correlation with seed yield (r = 0.91) and with harvest index (r = 0.96). Variation in seed weight per inflorescence was associated with variation in the number of seeds per pod and number of pods per inflorescence. Seed weight per inflorescence appears to have a strong genetic association with seed yield in alfalfa. Environments with high aboveground phytomass potential also have high seed yield potential.
Seventy-two castrated male four-way composite (Chester White x Landrace x Large White x Yorkshire) weanling pigs (17 kg initial BW) were used to determine the compensatory response of specific organs and whole body following a 3-wk period of restricted energy intake imposed by ad libitum-feeding an 80% alfalfa meal diet or pair-feeding an adequate corn-soybean meal diet to attain equal weight loss. Six pigs were killed at d 0 to provide baseline data. Six pigs from each diet group (control, C; alfalfa, A, restricted, R) were killed at d 21, after which all remaining pigs were fed C ad libitum to 35 d, 105 d or 126 d. Six pigs per group were killed at 35 and 105 d and four per group were killed at 126 d. Organ weight and carcass data were recorded for all pigs at all time intervals. Regression of organ and trimmed lean tissue weights on time was tested for linearity (d 1 to 126 for C and d 21 to 126 for A and R except d 35 to 126 for gastrointestinal tract traits for A and R); differences in organ weights between C, A and R at d 21 and d 35 were tested by a two-way analysis of variance. There was no evidence of a compensatory growth response to dietary energy restriction for 21 d except for rate of change in weights of liver and kidneys and backfat depth from 21 to 126 d and of gastrointestinal tract components from 21 to 35 d. Specific organs may exhibit compensatory growth over different time spans in young pigs in the absence of observable weight compensation at the whole-animal level.
Twelve artificially reared, male Holstein calves, ruminally cannulated at 53 d of age, were used in a split-plot design to study the effects of no milk replacer reduction (CON), or reduction by 30% (30R) or 60% (60R) of this value on alfalfa hay intake and digestibility, ruminal fermentation and digestive kinetics. Milk replacer reduction began at 53 d of age and continued until 135 d of age, after which no milk replacer was fed. All calves had ad libitum access to long-stemmed alfalfa hay from birth. Five collection periods were conducted at average calf ages of 72, 87, 108, 129 and 151 d. Reducing the amount of milk replacer fed resulted in a linear increase (P less than .05) in forage OM intake; however, total OM intake (forage + milk) was not different (P greater than .10) among milk reduction groups. Size of particles in feces exhibited quadratic effects in response to milk replacer reduction (P less than .05) but only in the small (less than 150 microns) size groupings. Ruminal pH and ammonia and individual VFA concentrations (except isobutyrate) were not altered by milk reduction (P greater than .10) but increased (P less than .01) with calf age. Milk replacer reduction had a quadratic effect (P less than .05) on fluid outflow rate from the rumen, increasing as milk replacer was reduced. Other fluid and particulate kinetic data, as well as NDF digestion rate and DM digestion showed no effects (P greater than .10) from milk replacer reduction but changed with calf age. Milk replacer reduction increased forage intake but had minimal effects on digestive variables evaluated, suggesting that intake of milk replacer by calves can be reduced by up to 60% without disturbing forage fermentation and passage.
Six steers (BW = 436 +/- 15 kg) were assigned randomly to alfalfa, bromegrass, or prairie hay diets in a replicated 3 x 3 Latin square design to evaluate the efficacy of different internal markers for estimating OM digestibility (OMD). Internal markers used to estimate OMD included ADL, alkaline peroxide lignin (APL), AIA, and indigestible ADF (IADF), which consisted of a 144-h in vitro incubation (IADFWOUT), indigestible ADF with acid/pepsin pretreatment of feed and orts samples (IADFFEED), or acid/pepsin pretreatment of feed, orts, and fecal samples (IADFALL). Marker-derived estimates of OMD were compared with OMD measured by total fecal collection (TFC). For the alfalfa diet, all marker estimates differed (P less than .05) from the TFC measurement; however, lignin-based procedures (ADL and APL) and IADF with acid/pepsin pretreatment (IADFFEED, IADFALL) were numerically closest to TFC values. Estimates of bromegrass and prairie hay OMD by ADL, APL, and AIA ratio were not different (P greater than .05) from TFC measurement, although AIA seemed to provide the most accurate estimate for prairie hay. All indigestible ADF procedures yielded estimates of forage OMD that differed (P less than .05) from TFC for all forages; acid/pepsin pretreatment of samples (IADFFEED and IADFALL) improved accuracy of the OMD estimates in all forages. Likewise, recovery of indigestible ADF was consistently least among the markers evaluated. These results indicate that APL ratio performed similarly to ADL ratio in estimating forage OMD, that AIA estimated grass OMD very accurately, and that estimation of OMD by indigestible ADF ratio was improved when the procedure was accompanied by an acid/pepsin pretreatment of samples.