Infectivity and reproductive potential of the Oswego strain of Heterorhabditis bacteriophora associated with life stages of the clover root curculio,Sitona hispidulus

The infectivity and reproductive potential of the entomopathogenic nematode Heterorhabditis bacteriophora (Oswego strain), at different concentrations, was studied. Seventy to 80.0% mortality to late instar larvae of the clover root curculio, Sitona hispidulus, and 40.0-76.0% mortality to pupae, was observed at concentrations of 15-100 infective juveniles. There were no significant differences in mortality among nematode concentrations. LC(50) levels of 4.0 and 21.4 nematodes were determined for clover root curculio larvae and pupae, respectively. Nematodes did not cause significant mortality to adult or first instar clover root curculio. H. bacteriophora was able to complete its development and reproduce in 74.0-95.0% of clover root curculio late instar larvae and pupae. Reproductive potential in curculio larvae and pupae ranged from 0 to 7040 infective juveniles per host. Larvae exposed to 100 nematodes had a reproductive potential significantly higher than in those larvae exposed to 15 and 50 nematodes. Reproductive potential in pupae decreased with an increased nematode dose, indicating potential crowding effects. Host larval and pupal mass were positively correlated with nematode progeny production.     

Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.)

Isoflavonoids play important roles in plant defense and exhibit a range of mammalian health-promoting activities. Isoflavone reductase (IFR) specifically recognizes isoflavones and catalyzes a stereospecific NADPH-dependent reduction to (3R)-isoflavanone. The crystal structure of Medicago sativa IFR with deletion of residues 39-47 has been determined at 1.6A resolution. Structural analysis, molecular modeling and docking, and comparison with the structures of other NADPH-dependent enzymes, defined the putative binding sites for co-factor and substrate and potential key residues for enzyme activity and substrate specificity. Further mutagenesis has confirmed the role of Lys144 as a catalytic residue. This study provides a structural basis for understanding the enzymatic mechanism and substrate specificity of IFRs as well as the functions of IFR-like proteins.     

Profiling phenolic metabolites in transgenic alfalfa modified in lignin biosynthesis

Soluble phenolics, wall-bound phenolics and soluble and core lignin were analyzed in transgenic alfalfa with genetically down-regulated O-methyltransferase genes involved in lignin biosynthesis. High performance liquid chromatography and principal component analysis were used to distinguish metabolic phenotypes of different transgenic alfalfa genotypes growing under standard greenhouse conditions. Principal component analysis of HPLC chromatograms did not resolve differences in leaf metabolite profiles between wild-type and transgenic plants of the same genetic background, although stem phenolic profiles were clearly different between wild-type and transgenic plants. However, the analytical methods clearly differentiated two non-transgenic alfalfa cultivars based on either leaf or stem profiles. Metabolic profiling provides a useful approach to monitoring the broader biochemical phenotypes of transgenic plants with altered expression of lignin pathway enzymes.     

Metabolic adaptations to mercury-induced oxidative stress in roots of Medicago sativa L

Alfalfa (Medicago sativa) roots were treated with mercuric ions in a concentration- and time-dependent manner, and lipid peroxidation was studied biochemically as well as histochemically along with other physiological responses. Histochemical staining with Schiff's reagent and Evans blue revealed that the peroxidation of membrane lipids and loss of plasma membrane integrity in Hg-treated roots occurred in the meristem and the elongation zone. The histochemical observations were supported by the quantitative determinations of thiobarbituric acid reactive substances (TBARS). However, under the mercuric ions stress, the alfalfa plants showed no significant alteration of hydrogen peroxide in roots. Analysis of lipoxygenase activity by non-denaturing polyacrylamide gel electrophoresis (PAGE) showed that there were two isoforms in the root of alfalfa plants, but they showed quite different patterns under the Hg exposure. Also, using non-denaturing PAGE, activities of superoxide dismutase (SOD) and peroxidase (POD) were determined in roots after treatment with Hg ions. The total activities of SOD and POD increased in roots after Hg treatment of roots. Activity of ascorbate peroxides (APX) was stimulated at relatively high concentration of Hg (40microM), and after prolonged Hg exposure (20microM, 24h). In contrast, glutathione reductase activity was depressed at higher concentrations of Hg (10-20microM). Treatments of seedlings with 10-40microM Hg decreased the ascorbate and glutathione amounts but increased total non-protein thiols. The above results indicated that Hg exerted its toxic effect on the root growth of alfalfa by induction of oxidative stress.     

Identification of Fructose-1,6-bisphosphate aldolase cytosolic class I as an NMH7 MADS domain associated protein

We are interested in identifying proteins that interact with the MADS domain protein NMH7 of Medicago sativa. We use an affinity column with a synthetic peptide derived from the MADS domain of NMH7 which has been reported to mediate protein-protein interaction with non-MADS domain interacting proteins. We identified ∼40 and ∼80 kDa specifically bound proteins as the monomeric and dimeric forms of Fructose-1,6-bisphosphate aldolase cytosolic class I. NiNTA pull down assays revealed that K- and C-terminus regions of NMH7 are not required for the interaction with aldolase. Aldolase enzymatic activity is not required for the interaction with NMH7. NMH7 and aldolase were coimmunoprecipitated from non-inoculated seed and seedlings extracts. Colocalization studies using confocal microscopy showed that aldolase and NMH7 are localized in the cytoplasm and the nucleus of the cortical cells. These data together show that M. sativa aldolase is a novel MADS domain binding protein, and suggest a broader functional repertory for this enzyme, as has been proposed for other glycolytic enzymes.     

Effect of Meloidogyne incognita on Reproduction of Pratylenchus penetrans in Red Clover and Alfalfa

Roots of seedlings of red clover and alfalfa growing on 10⁻¹ Hoagland and Arnon solution agar were inoculated with various combinations of Meloidogyne incognita and Pratylenchus penetrans. Egg-laying by P. penetrans decreased as the number of nematodes, the ratio of entrant M. incognita to entrant P. penetrans, and the priority of invasion of roots by M. incognita increased. Embryogeny and hatching of eggs of P. penetrans, and development of larvae of M. incognita, were not affected. In red clover, the greatest red uction occurred when there were 65 entrant nematodes, the ratio of M. incognita:P. penetrans was 4:1 and M. incognita was inoculated four days prior to P. penetrans. In alfalfa, the less-favorable host for both nematodes, the greatest reduction occurred when there were 45 entrant nematodes, the ratio of M. incognita:P. penetrans was 2:1, and M. incognita was inoculated 4 days prior to P. penetrans.     

O-Methylation of benzaldehyde derivatives by "lignin specific" caffeic acid 3-O-methyltransferase

Although S-adenosyl-l-methionine (SAM) dependent caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) is one of the key enzymes in lignin biosynthesis, the present work demonstrates that alfalfa COMT methylates benzaldehyde derivatives more efficiently than lignin pathway intermediates. 3,4-Dihydroxy, 5-methoxybenzaldehyde and protocatechuic aldehyde were the best in vitro substrates for OMT activity in extracts from developing alfalfa stems, and these compounds were preferred over lignin pathway intermediates for 3-O-methylation by recombinant alfalfa COMT expressed in Escherichia coli. OMT activity with benzaldehydes was strongly reduced in extracts from stems of transgenic alfalfa down-regulated in COMT. However, although COMT down-regulation drastically affects lignin composition, it does not appear to significantly impact metabolism of benzaldehyde derivatives in alfalfa. Structurally designed site-directed mutants of COMT showed altered relative substrate preferences for lignin precursors and benzaldehyde derivatives. Taken together, these results indicate that COMT may have more than one role in phenylpropanoid metabolism (but probably not in alfalfa), and that engineered COMT enzymes could be useful for metabolic engineering of both lignin and benzaldehyde-derived flavors and fragrances.     

Assessment of agroeconomic indices in polycultures of lettuce, rocket and carrot through uni- and multivariate approaches in semi-arid Brazil

The polyculture among vegetables is an activity that to have good results, needs a proper planning. Although it often requires more labor, has several advantages over monoculture, among them is that polycultures are generally are more productive, provide with productivity of various plant constituents and a more balanced human diet, contribute to economic return, economic and yield stability, social benefits and farmer's direct participation in decision-making. The objective of this study was to evaluate agroeconomic indices of polycultures derived from the combination of two cultivars of lettuce with two cultivars of rocket in two cultures strip-intercropped with carrot cultivar ‘Brasilia’ through uni-multivariate approaches in semi-arid Brazil. The experimental design used was of randomized complete blocks with five replications and the treatments arranged in a factorial scheme of 2 × 2. The treatments consisted of the combination of two lettuce cultivars (Babá de Verão and Tainá) with two rocket cultivars (Cultivada and Folha Larga) in two cultures associated with carrot cv. Brasília. In each block were grown plots with two lettuce cultivars and two rocket cultivars, and carrot in sole crop. In each system was determined the lettuce leaf yield, rocket green mass yield and carrot commercial yield. Agrieconomic indices such as operational cost, gross and net income, monetary advantage, rate of return, profit margin, land equivalent ratio and yield efficiency for DEA were used to measure the efficiency of intercropping systems. In the bicropping of lettuce and rocket associated with carrot cv. ‘Brasilia’, suggests the use of lettuce cultivar ‘Tainá’ combined with rocket cultivars ‘Cultivada’ or ‘Folha Larga’. It was observed significant effect of lettuce cultivars in the evaluation of polycultures of lettuce, carrot and rocket, with strong expression for the lettuce cultivar ‘Tainá’. Both uni- and multivariate approaches were effective in the discrimination of the best polycultures.     

Influence of Concomitant Pratylenchus brachyurus and Meloidogyne spp. on Root Penetration and Population Dynamics

Populations of Pratylenchus brachyurus on cotton were increased significantly in the presence of either Meloidogyne incognita or M. arenaria.This occurred with either simultaneous inoculation or prior invasion by M. incognita. P. brachyurus penetrated cotton roots previously invaded by, or simultaneously inoculated with, M. incognita, as well as, or better than, in the absence of M. incognita. Prior invasion by M. incognita, however, suppressed P. brachyurus populations on tomato, while it had no effect on alfalfa and tobacco. Populations of M. incognita on cotton were generally inhibited by the presence of P. brachyurus. Simultaneous inoculation with, or previous invasion by, P. brachyurus also inhibited root penetration by M. incognita. These findings emphasize the importance of host susceptibility in the study of concomitant nematode populations.     

Down-regulation of hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase in transgenic alfalfa affects lignification, development and forage quality

The recently discovered enzyme hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) catalyzes the reactions both immediately preceding and following the insertion of the 3-hydroxyl group into monolignol precursors. A number of independent transgenic lines of alfalfa (Medicago sativa L.) were generated in which the levels of HCT were reduced through antisense HCT expression under control of the bean PAL2 promoter which is preferentially expressed in vascular tissue. Reduction of enzyme activity in these lines was from at least 15-50%. The most severely down-regulated lines exhibited significant stunting, reduction of biomass and delayed flowering. HCT down-regulation resulted in strongly reduced lignin content and striking changes in lignin monomer composition, with predominant deposition of 4-hydroxyphenyl units in the lignin. Vascular structure was impaired in the most strongly down-regulated lines. Analysis of forage quality parameters showed strong reductions of neutral- and acid-detergent fiber in the down-regulated lines, in parallel with large increases (up to 20%) in dry matter forage digestibility. Although manipulation of lignin biosynthesis can greatly improve forage digestibility, accompanying effects on plant development need to be better understood.     

Mass spectral characterization of fatty acid amides from alfalfa trichomes and their deterrence against the potato leafhopper

A homologous series of N-(3-methylbutyl)amides of normal saturated C14, C15, C16, C17 and C18 fatty acids were identified as major components of glandular trichome extracts from Medicago sativa G98A, an alfalfa genotype resistant to the potato leafhopper, Empoasca fabae. A second homologous series of N-(2-methylpropyl)amides of C14 through C18 normal fatty acids were minor components. Saturated free fatty acids C12, C13, C14, C15, C16, C17 and C18 were present in trace amounts, as was the N-(3-methylbutyl)amide of linoleic acid (C18:2). N-(3-methylbutyl)amides and N-(2-methylpropyl)amides of C14 through C18 fatty acids, along with the N-(3-methylbutyl)amide of linoleic acid, were synthesized and bioassayed for leafhopper deterrence by applying the compounds to the surface of a sachet containing an artificial diet. Leafhoppers were then offered a two-way choice between diet surfaces treated with the synthetic amides or an untreated control. N-(3-methylbutyl)amides and N-(2-methylpropyl)amides of C14 through C18 fatty acids did not deter leafhopper settling in a dose-dependent fashion. In contrast, when tested singly, N-(3-methylbutyl)amide of linoleic acid exhibited dose-dependent deterrence against leafhopper settling. Fatty acid amides localized in alfalfa glandular trichomes likely contribute to leafhopper resistance.     

Effect of Ditylenchus dipsaci on Alfalfa Mortality,Winterkill, and Yield

Ditylenchus dipsaci-infected and noninfected alfalfa plants in a naturally infested field were studied from July 1980 to September 1982. Forty-one percent of the plants died during the study. Ninety-seven percent of the plants that died were infected with D. dipsaci. Sixty-nine percent of the observed mortality occurred during winter. Forage yield of infected plants was significantly lower than yield of noninfected plants at each harvest. Stored carbohydrates in infected plants were significantly lower than in noninfected plants. In a controlled environment test, significantly greater mortality occurred in frozen severely infected plants than in frozen noninfected plants, while no mortality occurred in severely infected or noninfected plants that were not frozen. Both forage yield and stored carbohydrates were significantly lower in severely infected than noninfected, non-frozen plants. Mortality in greenhouse-grown plants that were transplanted to field plots was significantly greater in D. dipsaci-infected plants than in noninfected plants after one winter.     

Phytotoxic activity and conformational analysis of thymol analogs from Hofmeisteria schaffneri

Bioassay-guided fractionation of two phytotoxic extracts (a CH(2)Cl(2)-MeOH (1:1) and an aqueous) prepared from the aerial parts of Hofmeisteria schaffneri led to isolation of thymol analogs 3-5, along with seven known compounds, 1, 2 and 6-10. Compounds 3-5 were identified by spectroscopic methods as 1,4-bis(2'-hydroxy-4'-methylphenyl)butane-1,4-dione (3), 2-isopropyl-5-methylphenyl (2Z)-2-methylbut-2-enoate (4) and 2-hydroxy-2-(2-hydroxy-4-methylphenyl)propane-1,3-diyl (2Z,2'Z)bis(2-methylbut-2-enoate) (5) and designated trivial names of hofmeisterins II-IV, respectively. Their conformational behavior was also studied by molecular modeling using density functional theory calculations at the B3LYP/DGDZVP level. Compounds 1-4 and 6-10 significantly inhibited radicle growth of seedlings of Amaranthus hypochondriacus and Echinochloa crus-galli in the Petri dish bioassay with IC(50)'s10(-4)M. Furthermore, the northymol analog 3 provoked significant bleaching of seedlings of A. hypochondriacus. However, none of the isolates affected either seedling growth or germination of Medicago sativa.     

Mass Culturing of Ditylenchus dipsaci to Yield Large Quantities of Inoculum

Methods are described for rearing large quantities of Ditylenchus dipsaci on alfalfa tissues. Nematodes and alfalfa seed were disinfected and nematodes were reared in quantities sufficient to provide a continuous supply of inoculum for our alfalfa-breeding program. Nematodes reproduced best in darkness at 20-25 C. Cultures reached maximum numbers in 3-6 wk.     

(n-7) and (n-9) cis-Monounsaturated fatty acid contents of 12 Brassica species

cis-Vaccenic acid or cis-11-octadecenoic acid, a C18:1 (n-7) isomer of oleic acid (C18:1 (n-9)) has been found in several oilseeds. It is synthesized from palmitic acid (C16:0) via production of C16:1 (n-7) by a Delta9 desaturase and elongation by an elongase giving C18:1 (n-7). In this study, the fatty acid composition of 12 Brassica species was analyzed by GC-FID and confirmed by GC-MS. All species contained C18:1 (n-7), C20:1 (n-7) and C22:1 (n-7) fatty acid isomers, suggesting that C18:1 (n-7) was elongated. The levels of these fatty acids varied according to the species. C18:1(n-7)) represented from 0.4% to 3.3% of the total relative fatty acid contents of the seeds. The contents of C20:1(n-7) and C22:1(n-7) levels were lower than C18:1(n-7) contents; the relative fatty acid composition varied from 0.02% to 1.3% and from below the limit of detection to 1.3% for C20:1 (n-7) and C22:1 (n-7), respectively. The ratios of (n-7)/(n-9) ranged from 2.8% to 16.7%, 0.6% to 29.5% and 0% to 2.6% for C18:1, C20:1 and C22:2, respectively. Using statistical similarities or differences of the C18:1 (n-7)/(n-9) ratios for chemotaxonomy, the surveyed species could be arranged into three groups. The first group would include Brassica napus, B. rapa, and B. tournefortii with Eruca sativa branching only related to B. napus. The second group would include B. tournefortii, Raphanus sativus and Sinapis alba. The last group would include B. juncea, B. carinata and B. nigra with no similarity/relationship between them and between the other species. Results suggested that the level of C20:1 (n-7) influenced the levels of all monounsaturated fatty acids with chain length higher than 20 carbons. On the other hand, palmitoleic acid (C16:1) levels, C16:1 being the parent of all (n-7) fatty acids, had no statistically significant correlation with the content of any of the fatty acids of the (n-7) or (n-9) family.     

Monolignol biosynthesis in microsomal preparations from lignifying stems of alfalfa (Medicago sativa L.)

Microsomal preparations from lignifying stems of alfalfa (Medicago sativa L.) contained coniferaldehyde 5-hydroxylase activity and immunodetectable caffeic acid 3-O-methyltransferase (COMT), and catalyzed the S-adenosyl L-methionine (SAM) dependent methylation of caffeic acid, caffeyl aldehyde and caffeyl alcohol. When supplied with NADPH and SAM, the microsomes converted caffeyl aldehyde to coniferaldehyde, 5-hydroxyconiferaldehyde, and traces of sinapaldehyde. Coniferaldehyde was a better precursor of sinapaldehyde than was 5-hydroxyconiferaldehyde. The alfalfa microsomes could not metabolize 4-coumaric acid, 4-coumaraldehyde, 4-coumaroyl CoA, or ferulic acid. No metabolism of monolignol precursors was observed in microsomal preparations from transgenic alfalfa down-regulated in COMT expression. In most microsomal preparations, the level of the metabolic conversions was independent of added recombinant COMT. Taken together, the data provide only limited support for the concept of metabolic channeling in the biosynthesis of S monolignols via coniferaldehyde.