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Assessment of the potential for Listeria monocytogenes survival and growth during alfalfa sprout production and use of ionizing radiation as a potential intervention treatment.

Alfalfa seeds (Australian, nondormant, nonscarified) were treated with 20,000 ppm active chlorine, sprouted in canning jars for 5 days, and packaged and stored at 5 degrees C for up to 9 days. Seeds or sprouts were inoculated with a three-strain cocktail of Listeria monocytogenes at one of three points during the process-day 0 (before 24-h aqueous seed soak), day 1 (after 24-h aqueous seed soak), or day 5 (after sprouting, before prepackaging 10 ppm chlorine rinse)--or control (no inoculum), and the ability of the inoculum to survive and grow was evaluated. Total bacterial numbers on uninoculated seeds increased dramatically during the first 24-h the seeds were soaked, from 3.5 to ca. 8.0 log CFU/g, and remained at this level during refrigerated storage. When the seeds were inoculated with a cocktail of L. monocytogenes (log 5 CFU/10 ml) on day 0 or 1, the population of the pathogen increased dramatically, to within 1 to 2 logs of the total, and remained high during refrigerated storage. When sprouted seeds were inoculated with L. monocytogenes later in the process (day 5), the inoculum survived but did not grow more than ca. 1 log CFU/g, regardless of whether the inoculation level in each jar was low (10(3)) or high (10(5)). Irradiation of sprouts with beta radiation at 3.3 or 5.3 kGy, but not 1.5 kGy, was effective at eliminating L. monocytogenes from inoculated sprouts (6 log CFU/g) without causing noticeable changes in appearance or odor. In summary, L. monocytogenes can grow on sprouts during production, can survive on refrigerated sprouts, and may be eliminated on sprouts with beta radiation.

Beta Particles↗

Alfalfa sprouts and Salmonella Kottbus infection: a multistate outbreak following inadequate seed disinfection with heat and chlorine.

Raw sprouts have been implicated in a number of foodborne disease outbreaks. Because contaminated seeds are usually responsible, many sprout producers attempt to disinfect seeds before germination and detect sprout contamination during production. In March 2001, we detected an increased number of Salmonella serotype Kottbus isolates in California. Overall, we identified 31 cases from three western states. To identify the cause, we conducted a case-control study with the first 10 identified case-patients matched to 20 controls by age, sex, and residential area. Our case-control study found illness to be statistically associated with alfalfa sprout consumption. The traceback investigation implicated a single sprouter, where environmental studies yielded Salmonella Kottbus from ungerminated seeds and floor drains within the production facility. Pulsed-field gel electrophoresis patterns of all patient, seed, and floor drain Salmonella Kottbus isolates were indistinguishable. Most implicated sprouts were from seeds that underwent heat treatment and soaking with a 2,000-ppm sodium hypochlorite solution rather than the Food and Drug Administration (FDA)-recommended 20,000-ppm calcium hypochlorite soak. Other implicated seeds had been soaked in a calcium hypochlorite solution that, when tested, measured only 11,000 ppm. The outbreak might have been averted when screening tests of sprout irrigation water detected Salmonella in January; however, confirmatory testing of these samples was negative (but testing improperly utilized refrigerated irrigation water). Producers should use the enrichment broth of positive screening samples, not refrigerated irrigation water, for confirmatory testing. Until other effective disinfection technologies are developed, producers should adhere to FDA recommendations for sprout seed disinfection.

Adult↗

Changes in growth and antioxidant status of alfalfa sprouts during sprouting as affected by gamma irradiation of seeds.

Viking 3000 alfalfa seeds irradiated with gamma rays to doses of 0, 1, 2, 3, or 4 kGy were sprouted and allowed to grow for up to 8 days at 23 degrees C. Germination, growth (yield and length), antioxidant capacity, and ascorbic acid (AA) were measured during sprouting. Results showed percent germination of the seeds and the rates of growth of the sprouts were inversely related to the radiation dose absorbed by the seeds. Both antioxidant capacity and AA content expressed on a fresh weight basis decreased during growth of the sprouts. Sprouts grown from irradiated seeds had greater antioxidant capacity and AA content on a fresh weight basis than those grown from nonirradiated seeds. However, when the nutritive values were expressed on a per gram of seed basis, irradiation had no effect on the nutritive values of sprouts.

Antioxidants↗

Inhibition of Salmonella enterica by plant-associated pseudomonads in vitro and on sprouting alfalfa seed.

Foodborne illness due to the consumption of contaminated raw or lightly cooked sprouts is a continuing food safety concern. In this study, we tested several plant-associated pseudomonads for their ability to inhibit the growth of Salmonella enterica both in vitro and in situ. An agar spot bioassay method was used with three different media. Only Pseudomonas fluorescens 2-79 produced clear zones of inhibition when tested against five serovars of S. enterica, and activity was dependent on media type and serovar. The antibiosis by derivative strains of P. fluorescens 2-79 defective in the production of phenazine-1-carboxylic acid and fluorescent siderophore was not reduced, indicating that these known antimicrobial metabolites were not responsible for the inhibition observed in our studies. However, mutants defective in the regulatory gene gacS (global antibiotic and cyanide control) were severely reduced in inhibitory activity. In tryptic soy broth, the control cultures of a cocktail of S. enterica strains reached approximately 10 log CFU/ml by 24 h but, when coinoculated with P. fluorescens 2-79, reached only approximately 5 log CFU/ml. The addition of P. fluorescens 2-79 to the seed soak water prior to the germination of alfalfa seed previously inoculated with a cocktail of S. enterica strains led to an average reduction of 5 log CFU/g at 6 days of sprouting without an adverse effect on sprout yield or appearance. Time course studies indicated that S. enterica outgrowth was controlled on days 1 through 6 of sprouting. Competitive exclusion as a potential food safety intervention for seed sprouts merits further study.

Animals↗

Inactivation of escherichia coli 0157:H7 and Salmonella on mung beans, alfalfa, and other seed types destined for sprout production by using an oxychloro-based sanitizer.

The efficacy of a stabilized oxychloro-based food grade sanitizer to decontaminate seeds destined for sprout production has been evaluated. By using mung bean seeds as a model system, it was demonstrated that the sanitizer could be used to inactivate a five-strain cocktail of Escherichia coli O157:H7 or Salmonella introduced onto beans at 10(3) to 10(4) CFU/g. Salmonella was more tolerant to stabilized oxychloro than was E. coli O157:H7, with sanitizer levels of >150 and >50 ppm, respectively, being required to ensure pathogen-free sprouts. The decontamination efficacy was also found to be dependent on treatment time (>8 h optimal) and the seed-to-sanitizer ratio (>1:4 optimal). Stabilized oxychloro treatment did not exhibit phytotoxic effects, as germination and sprout yields were not significantly (P > 0.05) different as compared with untreated controls. Although human pathogens could be effectively eliminated from mung beans, the aerobic plate count of native microflora on sprouts grown from treated seed was not significantly (P > 0.05) different from the controls. The diversity of microbial populations (determined through 16S rRNA denaturing gradient gel electrophoresis analysis) associated with bean sprouts was not significantly affected by the sanitizer treatment. However, it was noted that Klebsiella and Herbasprillum (both common plant endophytes) were absent in sprouts derived from decontaminated seed but were present in control sprouts. When a further range of seed types was evaluated, it was found that alfalfa, cress, flax, and soybean could be decontaminated with the stabilized oxychloro sanitizer. However, the decontamination efficacy with other seed types was less consistent. It appears that the rate of seed germination and putative activity of sanitizer sequestering system(s), in addition to other factors, may limit the efficacy of the decontamination method.

Chlorine Compounds↗

Decreased NADH glutamate synthase activity in nodules and flowers of alfalfa (Medicago sativa L.) transformed with an antisense glutamate synthase transgene.

Legumes obtain a substantial portion of their nitrogen (N) from symbiotic N2 fixation in root nodules. The glutamine synthetase (GS, EC 6.3.1.2)/glutamate synthase (GOGAT) cycle is responsible for the initial N assimilation. This report describes the analysis of a transgenic alfalfa (Medicago sativa L.) line containing an antisense NADH-GOGAT (EC 1.4.1.14) under the control of the nodule-enhanced aspartate amino-transferase (AAT-2) promoter. In one transgenic line, NADH-GOGAT enzyme activity was reduced to approximately 50%, with a corresponding reduction in protein and mRNA. The transcript abundance for cytosolic GS, ferredoxin-dependent GOGAT (EC 1.4.7.1), AAT-2 (EC 2.6.1.1), asparagine synthase (EC 6.3.5.4), and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) were unaffected, as were enzyme activities for AAT, PEPC and GS. Antisense NADH-GOGAT plants grown under symbiotic conditions were moderately chlorotic and reduced in growth and N content, even though symbiotic N2 fixation was not significantly reduced. The addition of nitrate relieved the chlorosis and restored growth and N content. Surprisingly, the antisense NADH-GOGAT plants were male sterile resulting from inviable pollen. A reduction in NADH-GOGAT enzyme activity and transcript abundance in the antisense plants was measured during the early stages of flower development. Inheritance of the transgene was stable and resulted in progeny with a range of NADH-GOGAT activity. These data indicate that NADH-GOGAT plays a critical role in the assimilation of symbiotically fixed N and during pollen development.

Antisense Elements (Genetics)↗

[Analysis of various types of competition in Tn5-mutants of alfalfa rhizobium bacteria (Sinorhizobium meliloti)].

Nodulation, rhizospheral, and saprophytic types of competitiveness (NC, RC, and SC, respectively) were studied in the highly active strains CXM1-105 and CXM1-188 of the alfalfa rhizobium Sinorhizobium meliloti. The competitiveness was estimated with the use of markers of antibiotic resistance. It was found that the mutant strain T37, which was characterized by a drastically decreased NC, had higher SC and RC than the parental strain. The mutant T107 (with a moderately decreased NC) did not differ from the parental strain with respect to RC but had a higher SC. The mutant T27 (with the lowest NC) did not differ from the parental strain with respect to SC or RC. In the mutant Tb1, the NC and RC were decreased and the SC was the same as in the parental strain. In Tb7, the SC was decreased and RC was increased. In the mutant T795, all of the three types of competitiveness were decreased. The difference between the mutants studied and the parental strain with respect to NC and RC was confirmed using an indirect method (the ability to form effective symbiosis after mixed inoculation together with the an ineffective tester strain CXM1-48) and the X-Gluc staining method (using the S. meliloti RmM4gus tester strain carrying the gene of beta-glucuronidase). However, the decreased SC that the mutants exhibited when they were cultivated together with parental strains in a plant-growth substrate (vermiculite) was not observed in the case of their cocultivation in liquid media. The independent variation of different types of competitiveness indicate that rhizobia have several separate gene systems determining their survival in in planta and ex planta ecological niches.

DNA Transposable Elements↗

Outbreak of Salmonella serotype Kottbus infections associated with eating alfalfa sprouts--Arizona, California, Colorado, and New Mexico, February-April 2001.

On March 12, 2001, the California Department of Health Services (CDHS) identified a cluster of Salmonella Kottbus isolates with indistinguishable pulsed-field gel electrophoresis (PFGE) patterns. During February 1-May 1, CDHS identified 23 patients with S. Kottbus infections in several California counties and an additional patient from Arizona. This report summarizes the results of the investigation of this outbreak, which identified cases in four states and implicated alfalfa sprouts produced at a single facility.

Adolescent↗

[Isolation and characterization of holocellulose from Alfalfa].

Holocellulose isolated from the aerial parts of alfalfa (Medicago sativa) contains a polysaccharide complex of cellulose and hemicelluloses, the major structural components of cell walls. Holocellulose is highly hydrophilic and has a dense biopolymer packing. The carboxylic groups of hemicelluloses and cellulose determines the ability of holocellulose to adsorb polyvalent metal cations.

Cations↗

Controlled test of anthelmintic activity of levamisole administered to calves via drinking water, subcutaneous injection, or alfalfa pellet premix.

A controlled test of the activity of 3 formulations of levamisole, at the dose level of approximately 8 mg/kg, against naturally occurring infections of gastrointestinal parasites and lungworms was made in 24 calves allotted to 4 groups of 6 calves each. Levamisole was administered to group I calves in the drinking water, to group II calves by subcutaneous injection, and to Group III calves by feeding alfalfa pellets mixed in corn silage; group IV calves were nontreated controls. Group I calves consumed the medicated water between 4 hours and 20 minutes and 9 hours and 40 minutes; group III calves consumed the medicated feed within 2 hours and 15 minutes. For calves of group I, II, and III, removals of 4th-stage Ostertagia sp were 64, 23, and 0%; of mature Ostertagia ostertagi, 90, 93, and 83%; and of mature Trichostrongylus axei, 92, 99, and 92%, respectively. For all 3 treated groups of calves, removal was 100% for 4th-stage Cooperia sp and for mature Trichostrongylus colubriformis, Cooperia oncophora, Cooperia punctata, and Oesophagostomum radiatum. Removals of Dictyocaulus vivipara were 90, 90, and 94% for calves of groups I, II, and III, respectively. There was no evidence of toxicosis. At necropsy, 2 calves in group II had small areas of edema at the sites of injection of levamisole.

Administration, Oral↗

The effect of grassmeal and alfalfa meal on daily gain, thickness of backfat and on the esophagogastric region in bacon pigs.

In pigs dietary factors often are involved as causes of lesions in pars esophagea of the stomach. In experiments the primary purpose of which was to clarify the effect of grassmeal or alfalfa meal on growth, feed efficiency and carcass quality in bacon pigs, an influence of these feedstuffs on the growth rate and degree of esophagogastric lesions was detected. Pigs with these lesions had as an average a lower daily gain and less backfat than pigs without lesions.

Animal Feed↗

Nitrogen control of bacterial signal production in Rhizobium meliloti-alfalfa symbiosis.

Under nitrogen-depleted conditions nitrogen-fixing soil bacteria of the family Rhizobiaceae are able to induce symbiotic nodules on the roots of leguminous plants where bacteroids convert atmospheric nitrogen to ammonia. The presence of exogenous nitrogen source inhibits the development and the functioning of bacterium-plant symbiosis. Earlier experiments demonstrated that nitrate inhibited all stages of symbiotic interaction, affecting primarily the host functions. The investigation of the possible involvement of the microsymbiont in nitrogen regulation showed that two signalling steps were controlled by ammonium. The synthesis of the first bacterial signal, the Nod factor was repressed by ammonium. The nitrogen signal is conveyed to nodulation (nod) genes by the general nitrogen regulatory (ntr) system and by the nodD3-syrM self-amplifying system. The fine control also involves a negative regulatory factor, ntrR. When ntrR is mutated, more efficient nodule formation and nitrogen fixation is observed in symbiosis with alfalfa even in the presence of ammonium. The biosynthesis of the second bacterial signal succinoglycan is also controlled by ammonium. SyrM, a common regulatory factor for nod and exo gene expression, may contribute to the adjustment of the amount of succinoglycan and the ratio of its biologically active form.

Genes, Bacterial↗

Host and virus specific RNA polymerases in alfalfa mosaic virus infected tobacco.

The particulate fraction of a homogenate of alfalfa mosaic virus infected tobacco was found to contain the viral replicase, and one or more host-specific RNA-polymerases. Using an endogeneous template, the membrane-bound replicase incorporates 3H-CTP in virion-type RNA. The synthesized RNA is part of a replicative intermediate; after RN-ase-treatment the product comigrates with viral double-stranded RNA in polyacrylamide gels. A preliminary characterization of this double-stranded RNA was made by RNA-RNA hybridization. Treatment of the particulate fraction with lubrol releases a host-specific RNA-polymerase. The activity of this enzyme is completely dependent on exogeneous template-RNA. Probably only a very small region of the template is transcribed. After washing with lubrol, the particulate fraction still contains the viral replicase. When this fraction is resuspended in a Mg++-deficient buffer, about 60% of the enzyme activity is released into the supernatant. No such activity is found in a comparable extract of healthy leaves.

Cytosine Nucleotides↗

[Use of three-hybrid system to detect RNA-binding activity of alfalfa mosaic virus coat protein].

We used yeast three-hybrid system, for studying interaction of alfalfa mosaic virus coat protein AMVCP (AMVCP) with RNA4, which codes this protein. We have shown that AMVCP with high affinity is bound to plus-chain of RNA4 in vivo. The mutational analysis has shown, that the N-terminal part of AMVCP (aa 1 to 85) contains RNA-binding domain. C-terminal part of this protein (aa 86 to 221) does not participate in direct interaction with RNA4. However activity of the reporter-gene LacZ, which codes beta-galactosidase, in case of interaction only N-terminal part of AMVCP is five times lower, in comparison with full-length hybrid protein, that confirms that the tertiary structure of full-length AMVCP is more favourable for interaction with RNA4.

Blotting, Western↗

Heritable paternal cytoplasmic organelles in alfalfa sperm cells: ultrastructural reconstruction and quantitative cytology.

Sperm cells within pollen grains and pollen tubes of alfalfa (Medicago sativa L.) were observed at the ultrastructural level, and their plastid DNA was detected by DAPI (4,6-diamidino-2-phenylindole) staining. One sperm pair within the pollen grain and three sperm pairs within pollen tubes were reconstructed in three-dimensions from serial ultrathin sections. The two sperm cells are linked by cytoplasmic bridges in both pollen grains and tubes, and the vegetative nucleus is closely associated with the sperm cells within the pollen tube. The number of plastids and plastid nucleoids (DNA aggregates) in the sperm cell pair, collectively, is not significantly different from that in the generative cell; however, over 60% of the sperm cell plastids contain no DNA detectable with DAPI. The mean number of mitochondria in sperm cells is reduced from that in the generative cell (from 54 to 17), which suggests that paternal mitochondrial inheritance probably does not occur in the genotype investigated. Sperm cells of a pair may vary in their shape within the pollen grain and tube, but the number of plastids and mitochondria is not significantly different between the sperm cells. Therefore, heterospermy is not a factor determining cytoplasmic inheritance patterns in this species.

Cytoplasm↗

Correlations and heritability of the characters determining the seed yield of the long-raceme alfalfa (Medicago sativa L.).

Phenotypic correlation coefficients and heritability of the characters controlling seed yield of long-raceme forms of alfalfa was determined. It was found that seed yield per plant, which was positively correlated with 10 out of 12 analysed characters, depended upon the number of pods per raceme and the number of seeds per pod. Variability of these characters determined about 60% of the variability of seed yield. Multiple linear regression and phenotypic correlations show that simultaneous selection for increased pod number per raceme and increased seed number per pod and raceme length resulted in enhanced seed yield potential. The share of the additive genetic effects in the phenotypic variance for number of pods per raceme was low and about 21-23%, while for number of seeds per pod and per raceme amounted to about 50%. The expected genetic progress in recombination breeding for number of seeds per pod and number of seeds per raceme will be of medium magnitude, while one cannot expect any rapid and considerable progress in the number of pods per raceme. Considering the high positive correlation between raceme length and number of pods and seeds per raceme, one should conclude that raceme length can be an important criterion in selection of plants showing a high seed productivity.

Hybridization, Genetic↗

Endogenous protein kinase-C activity and phosphorylated proteins in messenger ribonucleoprotein complexes of developing embryos of alfalfa.

Developing somatic and zygotic embryos of alfalfa (Medicago sativa L.) exhibited endogenous protein kinase activity and protein acceptors of phosphate groups using both cell-free translational extracts and oligo(dT)-cellulose-column-purified mRNPs. The cell-free-translation extracts from pre-cotyledonary-stage somatic embryos had approximately 50- and 100-fold more protein kinase activity than cotyledonary-stage somatic and zygotic embryos. Several polypeptides were phosphorylated; some of them were unique to the early stage and some to the late-stage developing embryos. A 65 kDa protein was phosphorylated heavily in pre-cotyledonary-stage somatic embryos. This phosphorylated protein was comprised of three main components, two of which were phosphorylated heavily. Heat-shock treated-embryos lost their exitant kinase activity and at the same time another form of protein kinase activity was activated which phosphorylated a novel 28 kDa protein. Endogenous protein kinase activity was also observed within the mRNPs of polysomal and non-polysomal fractions of developing embryos, and this phosphorylated only 65, 43 and 30 kDa proteins within these fractions. A 30 kDa protein from the pre-cotyledonary-stage somatic embryos showed a higher affinity for accepting phosphate groups than the proteins from cotyledonary-stage somatic or zygotic embryos. The activity of protein kinase was largely c-AMP-independent, but was dependent on Ca2+, phospholipid and phorbol ester. The enzyme belongs to the protein kinase-C family; the 65 kDa protein cross-reacts with antibodies made against protein kinase-C (alpha- and beta-isoforms) and it may be an autophosphorylated protein.

Cotyledon↗

Quantitation of pyrimidine dimers in DNA from UVB-irradiated alfalfa (Medicago sativa L.) seedlings.

Depletion of stratospheric ozone will increase the solar ultraviolet radiation in the range from 290-320 nm (UVB) that reaches the surface of the earth, placing an increased UV burden on exposed organisms. One consequence of increased UVB may be decreased productivity of crop plants. A principal lesion caused by UV in DNA is the cyclobutyl pyrimidine dimer. We have adapted a method for measuring these dimers in nanogram quantities of non-radioactive DNA for use in UV-irradiated plants. We find that biologically relevant doses of broad band UVB radiation induce easily detectable frequencies of pyrimidine dimers in the DNA of irradiated alfalfa sprout leaves and that the dose response for dimer formation is linear up to doses of at least 690 J m-2. We also find easily measurable frequencies of dimers in the leaves of seedlings grown in glass filtered sunlight but not exposed to additional UVB, suggesting that significant numbers of dimers are formed in plants exposed to normal sunlight.

DNA↗