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PubMed2003

Within-field manipulation of potato leafhopper (Homoptera: Cicadellidae) and insect predator populations using an uncut alfalfa strip.

The effects of a 3-m wide uncut alfalfa strip on the within field distribution of Empoasca fabae (Harris) (Homoptera: Cicadellidae) and several species of insect predators were examined for 3 yr (1998-2000). The objectives were to determine whether this uncut strip would serve as a trap crop for E. fabae and a refuge for insect predators at first harvest. Empoasca fabae and insect predators in the families Coccinellidae, Nabidae, Anthocoridae, Chrysopidae, and Hemerobiidae were collected weekly using sweep samples and sticky traps from the uncut strips and up to 40 m into the alfalfa regrowth. For 2 yr, both small- (0.34 ha) and large-scale (approximately 11.3 ha) field trials showed higher numbers of E. fabae in 73% of the uncut strips for 2-3 wk after harvest. Similarly, the number of insect predators found within <50% of the uncut strips was also higher during the first or second week after harvest. In 1999, however, we did not observe higher numbers of E. fabae in the uncut strips. Differences may be because of higher E. fabae population numbers in 1999 compared with 1998 and 2000. This research provides alfalfa growers a potential cultural management technique for E. fabae while conserving predatory insects.

Animals↗

PubMed2005

Strain distribution of alfalfa weevil (Coleoptera: Curculionidae) in an intergrade zone.

Strain distribution for the alfalfa weevil, Hypera postica (Gyllenhal), was determined for the five major alfalfa-growing regions of New Mexico. Mitochondrial DNA from field-collected weevils at each location was digested and sequenced to differentiate among strains. Eastern, Egyptian, and western strains were recovered, along with a population not matching any known strain. The majority of sites examined had populations of two strains intermingled within a field. This is the first confirmation of the presence of all three strains within the same geographic region. Possible implications for alfalfa weevil management are discussed.

Animals↗

PubMed1984

The digestion of total and cell wall monosaccharides of alfalfa by sheep.

The effect of dietary starch on the digestibility of total and cell wall (CW) monosaccharides of alfalfa hay, was examined in sheep equipped with rumen and duodenal cannulas. The experiment consisted of two treatments. In one of them the sheep received 803 g dry matter (DM) of alfalfa hay (A), and in the second, 242 g DM alfalfa hay + 553 g of a purified starchy concentrate mixture (A + conct ). Rumen volume was not significantly different between treatments, but the mean retention time of markers in the rumen was shorter, and their rate of passage was faster in the A treatment than in the A + conct treatment. The overall digestibility of total CWs and CW constituents was not depressed in the A + conct treatment, the values for total CW being: 58.2 and 58.9% for the A and A + conct treatments, respectively. However, the digestion of CW monosaccharides in the rumen of the A + conct sheep was reduced despite the 100% longer retention time of particles in the rumen. The complimentary digestion of the potentially digestible CW monosacharides has been completed in the hindgut. The digestibility of CW glucose was higher (72%) than that of xylose (57%) in both treatments. CW glucuronic acid was the least digestible constituent (40%). Soluble uronic acids, representing the pectic material, were highly digestible (86.5% in A), but this was reduced to 80.3 in the A + conct treatment.

Animals↗

PubMed1989

Polyadenylated H3 histone transcripts and H3 histone variants in alfalfa.

Histone H3 mRNAs were found in polyA(+) fractions of total RNA prepared from alfalfa plants, calli and somatic embryos. The sequence analysis of cDNAs revealed the presence of a polyA tail on independent alfalfa H3 mRNAs. A highly conserved sequence motif AAUGAAA identified about 20bp upstream from the 3' ends of the alfalfa H3 cDNAs was suggested to be one of the possible regulatory elements in the 3' end formation and polyadenylation. Three out of the four analysed H3 cDNAs have more than 97% homology with a genomic clone and encode the same protein. While the fourth represents a minor species with only 78.8% homology to the coding region of the genomic clone and encodes a H3 histone with four amino acid replacements. On the basis of compilation analysis we suggest a consensus sequence for plant H3 histones which differs from that of animal's by four amino acid changes.

Amino Acid Sequence↗

PubMed1989

Purification of beta-amylase from alfalfa (Medicago sativa L.) seeds.

An amylase from alfalfa (Medicago sativa L. c.v. Moapa) seeds was purified by column chromatography and gel filtration, followed by chromatofocusing on Mono P HR 5/20. The last step was effective for separation of the alfalfa amylase to a homogeneous state. The purified amylase was identified as beta-amylase from the fact that only beta-maltose was formed by the enzymatic degradation of soluble starch. The molecular weight and specific activity of the beta-amylase (E1%(280 nm) = 18.3) were determined to be 61,000 and 1,077 A.U./mg, respectively. The beta-amylase activity was inhibited by the modification of sulfhydryl groups with p-chloromercuribenzoic acid. The optimum pH and isoelectric point of alfalfa beta-amylase were 7.0 and 4.8, respectively, which were different from other plant beta-amylases.

Amylases↗

PubMed2006

Effects of alfalfa and fructooligosaccharide on molting parameters and bone qualities using dual energy X-ray absorptiometry and conventional bone assays.

The aim of this study was to evaluate effects of alfalfa and fructooligosaccharides (FOS) on molting performance and bone parameters compared with the conventional feed withdrawal molting procedure. A total of 36 Single Comb White Leghorn hens (84 wk of age) were used for this experiment. The hens were divided into 6 treatment groups with 6 birds per treatment: pre-trial control (PC), full fed (FF), feed withdrawal (FW), 100% alfalfa (A100), A100 + 0.375% FOS (A100L), and A100 + 0.75% FOS (A100H). At the end of the 9-d molt period, hens were euthanized, and tibia and femurs were collected to evaluate bone qualities using dual-energy x-ray absorptiometry (DXA), Instron (Model 1011 Instron Universal Testing Machine, Instron Corp., Canton. MA), and conventional bone assays. Egg production was recorded during the molting period to evaluate first day out of production, and ovary was also collected to measure ovary weight. Alfalfa molting diets had comparable molting parameters, such as percentage of BW loss, ovary weight, and first day out of egg production, to the conventional feed withdrawal molting procedure, and FOS supplementation did not have any detrimental effects on molting performance. Conventional bone assay and DXA results suggest that hens lose a considerable amount of bone minerals during a molting period. The tibia and femur bone strengths of the FF, FW, A100, and A100L hens were significantly lower than the PC hens, whereas hens fed A100H had similar tibia bone breaking strength to that of the PC hens. The bone parameters measured by conventional assays, bone breaking strength measured by Instron, and bone density and mineral content measured by DXA were highly correlated to each other.

Absorptiometry, Photon↗

PubMed2005

Phosphorus-free membrane lipids of Sinorhizobium meliloti are not required for the symbiosis with alfalfa but contribute to increased cell yields under phosphorus-limiting conditions of growth.

The microsymbiont of alfalfa, Sinorhizobium meliloti, possesses phosphatidylglycerol, cardiolipin, phosphatidylethanolamine, and phosphatidylcholine as major membrane phospholipids, when grown in the presence of sufficient accessible phosphorus sources. Under phosphate-limiting conditions of growth, S. meliloti replaces its phospholipids by membrane lipids that do not contain any phosphorus in their molecular structure and, in S. meliloti, these phosphorus-free membrane lipids are sulphoquinovosyl diacylglycerols (SL), ornithine-containing lipids (OL), and diacylglyceryl-N,N,N-trimethylhomoserines (DGTS). In earlier work, we demonstrated that neither SL nor OL are required for establishing a nitrogen-fixing root nodule symbiosis with alfalfa. We now report the identification of the two structural genes btaA and btaB from S. meliloti required for DGTS biosynthesis. When the sinorhizobial btaA and btaB genes are expressed in Escherichia coli, they cause the formation of DGTS in this latter organism. A btaA-deficient mutant of S. meliloti is unable to form DGTS but can form nitrogen-fixing root nodules on alfalfa, demonstrating that sinorhizobial DGTS is not required for establishing a successful symbiosis with the host plant. Even a triple mutant of S. meliloti, unable to form any of the phosphorus-free membrane lipids SL, OL, or DGTS is equally competitive for nodule occupancy as the wild type. Only under growth-limiting concentrations of phosphate in culture media did mutants that could form neither OL nor DGTS grow to lesser cell densities.

Base Sequence↗

PubMed1999

The alfalfa (Medicago sativa) TDY1 gene encodes a mitogen-activated protein kinase homolog.

Development of root nodules, specifically induction of cortical cell division for nodule initiation, requires expression of specific genes in the host and microsymbiont. A full-length cDNA clone and the corresponding genomic clone encoding a MAP (mitogen-activated protein) kinase homolog were isolated from alfalfa (Medicago sativa). The genomic clone, TDY1, encodes a 68.9-kDa protein with 47.7% identity to MMK4, a previously characterized MAP kinase homolog from alfalfa. TDY1 is unique among the known plant MAP kinases, primarily due to a 230 amino acid C-terminal domain. The putative activation motif, Thr-Asp-Tyr (TDY), also differs from the previously reported Thr-Glu-Tyr (TEY) motif in plant MAP kinases. TDY1 messages were found predominantly in root nodules, roots, and root tips. Transgenic alfalfa and Medicago truncatula containing a chimeric gene consisting of 1.8 kbp of 5' flanking sequence of the TDY1 gene fused to the beta-glucuronidase (GUS) coding sequence exhibited GUS expression primarily in the nodule parenchyma, meristem, and vascular bundles, root tips, and root vascular bundles. Stem internodes stained intensely in cortical parenchyma, cambial cells, and primary xylem. GUS activity was observed in leaf mesophyll surrounding areas of mechanical wounding and pathogen invasion. The promoter was also active in root tips and apical meristems of transgenic tobacco. Expression patterns suggest a possible role for TDY1 in initiation and development of nodules and roots, and in localized responses to wounding.

3' Untranslated Regions↗

PubMed2000

Expression profiles of 22 novel molecular markers for organogenetic pathways acting in alfalfa nodule development.

During symbiotic nodule development, a variety of molecular signals of rhizobia and plant origin are likely to be involved in the control of the expression of specific genes in the legume Medicago sativa (alfalfa). Twenty-two new, nodule-associated Expressed Sequence Tags (ESTs, MsNod clones) as well as 16 clones for previously reported alfalfa nodulins were identified by cold-plaque screening. Protein homologs were found for 10 of the 22 MsNod-encoded polypeptides, revealing putative novel functions associated with this symbiosis. Expression of these MsNod genes was investigated in spontaneous nodules (generated in the absence of bacteria), in nodules induced by a Sinorhizobium meliloti wild-type strain and Eps- and Bac- mutant derivatives, as well as in roots inoculated with a Nod- mutant strain. This analysis enabled us to correlate plant gene expression with the different stages of nodule ontogeny and invasion. The effect of phytohormones on MsNod gene expression was analyzed in cytokinin- and auxin-treated alfalfa roots. Cytokinin induced the accumulation of seven MsNod transcripts, four of them were also regulated by the synthetic auxin 2,4-D (2,4-dichlorophenoxyacetic acid). Comparison of MsNod expression profiles in wild-type and transgenic M. truncatula roots overexpressing the early nodulin Enod40 suggested that one clone, the M. sativa L3 ribosomal protein homolog (MsNod377), is a putative component of an Enod40-dependent pathway acting during nodule development. These novel molecular markers may help in the investigation of gene networks and regulatory circuits controlling nodule organogenesis.

Cytokinins↗

PubMed2000

Constitutive accumulation of a resveratrol-glucoside in transgenic alfalfa increases resistance to Phoma medicaginis.

Alfalfa (Medicago sativa) was transformed with a peanut (Arachis hypogaea) cDNA encoding resveratrol synthase (RS) transcriptionally regulated by an enhanced Cauliflower mosaic virus (CaMV) 35S promoter. Transgenic plants accumulated a new compound, not present in wild-type or vector-transformed alfalfa, that was identified as trans-resveratrol-3-O-beta-D-glucopyranoside (RGluc) by high-pressure liquid chromatography (HPLC), UV, 1H- and 13C-nuclear magnetic resonance (NMR) analyses. RGluc concentration was highest in the youngest leaves (>15 microg per g fresh weight) and oldest stem internode segments (>10 microg per g fresh weight) while roots contained only trace amounts (<0.2 microg per g fresh weight). RS transcript levels were highest in leaves and stems, with comparatively little transcript accumulation in the roots, while an inverse pattern was observed for chalcone synthase (CHS) transcript levels. CHS directly competes with RS for the metabolic precursors p-coumaroyl CoA and malonyl CoA, and may also contribute to the developmental variations in RGluc levels by limiting the availability of substrates. Agar-plate bioassays indicated that both RGluc and resveratrol greatly inhibit hyphal growth of the alfalfa fungal pathogen Phoma medicaginis. Subsequently, RGluc-containing leaves were wound inoculated and showed a significant reduction (relative to control leaves) in the size of necrotic lesions, intensity of adjacent chlorosis, and number of fungal reproductive structures (pycnidia). Decreasing sporulation of this pathogen may greatly reduce disease spread and severity throughout the field.

Acyltransferases↗

PubMed2001

Expression studies of superoxide dismutases in nodules and leaves of transgenic alfalfa reveal abundance of iron-containing isozymes, posttranslational regulation, and compensation of isozyme activities.

The composition of antioxidant enzymes, especially superoxide dismutase (SOD), was studied in one nontransgenic and three transgenic lines of nodulated alfalfa plants. Transgenic lines overproduced MnSOD in the mitochondria of nodules and leaves (line 1-10), MnSOD in the chloroplasts (line 4-6), and FeSOD in the chloroplasts (line 10-7). In nodules of line 10-7, the absence of transgene-encoded FeSOD activity was due to a lack of mRNA, whereas in nodules of line 4-6 the absence of transgene-encoded MnSOD activity was due to enzyme inactivation or degradation. Transgenic alfalfa showed a novel compensatory effect in the activities of MnSOD (mitochondrial) and FeSOD (plastidic) in the leaves, which was not caused by changes in the mRNA levels. These findings imply that SOD activity in plant tissues and organelles is regulated, at least partially, at the posttranslational level. All four lines had low CuZnSOD activities and an abundant FeSOD isozyme, especially in nodules, indicating that FeSOD performs important antioxidant functions other than the scavenging of superoxide radicals generated in photosynthesis. This was confirmed by the detection of FeSOD cDNAs and proteins in nodules of other legumes such as cowpea, pea, and soybean. The cDNA encoding alfalfa nodule FeSOD was characterized and the deduced protein found to contain a plastid transit peptide. A comparison of sequences and other properties reveals that there are two types of FeSODs in nodules.

Amino Acid Sequence↗

PubMed2002

Roles for riboflavin in the Sinorhizobium-alfalfa association.

Genes contributing to riboflavin production in Sinorhizobium meliloti were identified, and bacterial strains that overproduce this vitamin were constructed to characterize how additional riboflavin affects interactions between alfalfa (Medicago sativa) and S. meliloti. Riboflavin-synthesis genes in S. meliloti were found in three separate linkage groups and designated as ribBA, ribDribC, and ribH for their similarities to Escherichia coli genes. The ribBA and ribC loci complemented corresponding E. coli rib mutants. S. meliloti cells containing extra copies of ribBA released 10 to 20% more riboflavin than a control strain but grew at similar rates in a defined medium lacking riboflavin. Cells carrying extra copies of ribBA colonized roots to densities that were 55% higher than that of a control strain. No effect of extra rib genes was detected on alfalfa grown in the absence or presence of combined N. These results support the importance of extracellular riboflavin for alfalfa root colonization by S. meliloti and are consistent with the hypothesis that this molecule benefits bacteria indirectly through an effect on the plant.

Amino Acid Sequence↗

PubMed2004

Expression of MsLEC1 transgenes in alfalfa plants causes symbiotic abnormalities.

Legume lectins have been proposed to have important symbiotic roles during Rhizobium-legume symbioses. To test this hypothesis, the symbiotic responses of transgenic alfalfa plants that express a portion of the putative alfalfa lectin gene MsLEC1 or MsLEC2 in either the antisense or sense orientation were analyzed following inoculation with wild-type Sinorhizobium meliloti 1021. MsLEC1-antisense (LEC1AS) plants were stunted, exhibited hypernodulation, and developed not only abnormally large nodules but also numerous small nodules, both of which senesced prematurely. MsLEC2-antisense plants were intermediate in growth and nodule number compared with LEC1AS and vector control plants. The symbiotic abnormalities of MsLEC1-sense transgene plants were similar to but milder than the responses shown by the LEC1AS plants, whereas MsLEC2-sense transgene plants exhibited symbiotic responses that were identical to those of vector and nontransgenic control plants. MsLEC1 mRNA accumulation was not detected in nodule RNA by Northern blot analysis but was localized to alfalfa nodule meristems and the adjacent cells of the invasion zone by in situ hybridization; transcripts were also detected in root meristems. A similar spatial pattern of MsLEC2 expression was found by using a whole-mount in situ hybridization procedure. Moreover, mRNAs for an orthologous lectin gene (MaLEC) were detected in white sweetclover (Melilotus alba) nodules and root tips.

Gene Expression Regulation, Plant↗

PubMed2003

Effect of soil amendments on radiocesium transfer to alfalfa.

Greenhouse experiments were carried out to evaluate the effect of different soil-based countermeasures on radiocesium transfer to Medicago saliva (alfalfa) grown on artificially contaminated loamy-clayey soil. Various rates of potassium, ammonium, and Prussian Blue supplements were applied, and the uptake of radiocesium by control and treated alfalfa plants was monitored during four growth periods. Transfer factors ranging between 0.06 and 0.02 were determined for control plants. Application of potassium at rates higher than 0.1 meq per 100 g soil was found to suppress radiocesium uptake, the effect being more pronounced at increasing fertilization rates. On the contrary, soil treatment with ammonium enhanced the bio-accumulation of radiocesium, indicating that Cs+ ions, previously unavailable to plant roots, were released from soil particles. Prussian Blue supplements had practically no effect on soil-to-alfalfa transfer of the radionuclide.

Agriculture↗

PubMed1997

The Rhizobium meliloti PII protein, which controls bacterial nitrogen metabolism, affects alfalfa nodule development.

Symbiotic nitrogen fixation involves the development of specialized organs called nodules within which plant photosynthates are exchanged for combined nitrogen of bacterial origin. To determine the importance of bacterial nitrogen metabolism in symbiosis, we have characterized a key regulator of this metabolism in Rhizobium meliloti, the uridylylatable P(II) protein encoded by glnB. We have constructed both a glnB null mutant and a point mutant making nonuridylylatable P(II). In free-living conditions, P(II) is required for expression of the ntrC-dependent gene glnII and for adenylylation of glutamine synthetase I. P(II) is also required for efficient infection of alfalfa but not for expression of nitrogenase. However alfalfa plants inoculated with either glnB mutant are nitrogen-starved in the absence of added combined nitrogen. We hypothesize that P(II) controls expression or activity of a bacteroid ammonium transporter required for a functional nitrogen-fixing symbiosis. Therefore, the P(II) protein affects both Rhizobium nitrogen metabolism and alfalfa nodule development.

Bacterial Proteins↗

PubMed1993

Alfalfa (Medicago sativa L.) Root Exudates Contain Isoflavonoids in the Presence of Rhizobium meliloti.

Root exudates of alfalfa (Medicago sativa L.) inoculated with symbiotic Rhizobium meliloti bacteria contained three isoflavonoids that were not found in exudates of uninoculated plants. Data from proton nuclear magnetic resonance, mass spectrometry, and ultraviolet-visible absorbance analyses indicated that root exudates of inoculated plants contained aglycone and glycoside forms of the phytoalexin medicarpin and a formononetin-7-O-(6"-O-malonylglycoside), a conjugated form of the medicarpin precursor formononetin. The medicarpin molecules did not induce nod gene transcription in R. meliloti, but the formononetin-7-O-(6"-O-malonylglycoside) induced nod genes regulated by both NodD1 and NodD2 proteins in R. meliloti. Hydrolysis of either the malonyl or the glycosyl linkage from the formononetin conjugate eliminated nod gene-inducing activity. The nod gene-inducing activity of crude root exudates was increased 200 and 65% upon inoculation with R. meliloti or R. leguminosarum bv phaseoli, respectively. When root exudate from uninoculated alfalfa was incubated with R. meliloti, high performance liquid chromatography analyses showed no evidence that bacterial metabolism produced medicarpin. These results indicate that alfalfa responds to symbiotic R. meliloti by exuding a phytoalexin normally elicited by pathogens and that the microsymbiont can use a precursor of the phytoalexin as a signal for inducing symbiotic nod genes.

Journal Article↗

PubMed1994

The Effects of Heavy Metals and Root Immersion on Isoflavonoid Metabolism in Alfalfa (Medicago sativa L.).

Modest increases in the concentration of medicarpin, 6-fold in leaves and 4-fold in roots, were observed in alfalfa (Medicago sativa L.) seedlings treated with 1 mM metal salts for 72 h. However, medicarpin-3-O-glucoside-6"-O-malonate (MGM) and formononetin-7-O-glucoside-6"-O-malonate (FGM) levels were up to 50-fold lower in metal-treated compared to control roots. Approximately 10% of the "missing" conjugates could be accounted for in the root treatment solution, where FGM and MGM transiently accumulated prior to their hydrolysis. Time-course studies revealed that total isoflavonoid content (roots plus solution) increased slightly after CuCl2 treatment, whereas the levels of FGM and MGM increased rapidly in alfalfa roots immersed in water. This increase was reduced by aeration. The phenylalanine ammonia-lyase inhibitor L-[alpha]-aminooxy-[beta]-phenylpropionic acid was used to show that immersion of the roots reduced conjugate rates of degradation, which explains their accumulation. In contrast, conjugate rates of degradation were elevated in CuCl2-treated roots, with 50% of the increase being due to hydrolysis. Up to 90% of formononetin and medicarpin produced in response to CuCl2 treatment arose via conjugate hydrolysis. Our results demonstrate that both immersion/anaerobiosis and abiotic elicitation modify isoflavonoid metabolism in alfalfa, and that metal-stimulated accumulation of phytoalexins may arise through the release from preformed stores rather than de novo synthesis.

Journal Article↗

PubMed1995

Lipo-chitooligosaccharide Nodulation Signals from Rhizobium meliloti Induce Their Rapid Degradation by the Host Plant Alfalfa.

Extracellular enzymes from alfalfa (Medicago sativa L.) involved in the degradation of nodulation (Nod) factors could be distinguished by their different cleavage specificities and were separated by lectin affinity chromatography. A particular glycoprotein was able to release an acylated lipo-disaccharide from all tested Nod factors having an oligosaccharide chain length of four or five residues. Structural modifications of the basic lipo-chitooligosaccharide did not affect the cleavage site and had only weak influence on the cleavage efficiency of Nod factors tested. The acylated lipo-trisaccharide was resistant to degradation. When alfalfa roots were preincubated with Nod factors at nanomolar concentrations, the activity of the dimer-forming enzyme was stimulated up to 6-fold within a few hours. The inducing activity of Nod factors decreased in the order NodRm-IV(C16:2,Ac,S) > NodRm-IV(C16:2,S) and NodRm-V(C16:2,Ac,S) > NodRm-V(C16:2,S) > NodRm-IV(C16:0,S) > NodRm-IV(C16:2). Pretreatment with NodRm-III(C16:2) as well as unmodified chitooligosaccharides did not stimulate the dimer-forming enzyme. Roots preincubated with Rhizobium meliloti showed similar stimulation of the dimer-forming activity. Mutant strains unable to produce Nod factors did not enhance the hydrolytic activity. These results indicate a rapid feedback inactivation of Nod signals after their perception by the host plant alfalfa.

Journal Article↗