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PubMed2001

Purification and characterization of molybdenum species from Medicago sativa (alfalfa) grown on mine tailings.

Extraction, purification, and identification procedures were developed for the chemical investigation of molybdenum (Mo) in freeze-dried aerial portions of alfalfa (Medicago sativa L.) collected from a reclaimed tailings pond at the Highland Valley Copper mine in British Columbia. The purification procedures were guided by ICP and colorimetric analyses. The methods included development of an efficient aqueous extraction protocol, sample cleanup by partitioning against n-butanol, and filtration through diatomaceous earth. Further purification was achieved by anion-exchange chromatography, elution with aqueous NaCl, and desalting by gel filtration. Final purification of the Mo-containing fraction was carried out using preparative anion-exchange HPLC. Molybdenum was found to be present in its purified form as the molybdate anion (MoO(4)(2-)) primarily on the basis of multinuclear ((1)H, (13)C, and (95)Mo) NMR studies.

Chromatography, Gel↗
PubMed2006

Selenium species in aqueous extracts of alfalfa sprouts by two-dimensional liquid chromatography coupled to inductively coupled plasma mass spectrometry and electrospray mass spectrometry detection.

The complementary use of two different liquid chromatographic mechanisms coupled to inductively coupled plasma mass spectrometry (ICP-MS) for selenium (Se) specific detection has permitted the screening of the most abundant Se-containing fractions in selenized alfalfa sprouts (Medicago sativa). Aqueous extracts of the sprouts were fractionated first by size exclusion chromatography (SEC) using a Superdex Peptide column and a mobile phase containing an ammonium acetate buffer (pH 7). Further purification of the individual SEC Se-containing fractions was carried out using two different chromatographic systems: a Shodex Ashaipack column, with a mixed mechanism of size exclusion and ion exchange, and a conventional reversed phase C8 using ion-pairing reagents. In both cases, the columns were coupled to an inductively coupled plasma mass spectrometer equipped with an octapole reaction system for Se specific detection. This system allowed the on-line monitoring of the most abundant Se isotopes (78Se, 80Se) by reducing the possible polytomic interferences affecting these ions by adding hydrogen (2 mL min(-1)) to the octapole reaction cell. The results obtained by both separation mechanisms were highly comparable, revealing the presence of Se-methionine and Se-methyl selenocysteine. Both compounds were then confirmed by analyzing the corresponding fractions by electrospray quadrupole-time-of-flight (ESI-Q-TOF) mass spectrometry. Finally, an additional Se-containing species showing Se isotope distribution was detected at a molecular ion m/z 239 in the ESI-Q-TOF. The collision-induced dissociation of the m/z 239 and 237 ions (corresponding to 80Se and 78Se isotopes, respectively) revealed the possible presence as well of a derivative of the Se-2-propenyl selenocysteine.

Chromatography, High Pressure Liquid↗
PubMed1999

Saponins in alfalfa (Medicago sativa L.) root and their structural elucidation.

Twenty-four saponins have been identified in alfalfa roots, including 13 medicagenic acids, 2 zanhic acids, 4 hederagenins, 1 soyasapogenol A, 2 soyasapogenol B's, 1 soyasapogenol E, and 1 bayogenin glycoside. Ten of the identified compounds, including 3-O-[beta-D-glucopyranosyl(1-->3)-beta-D-glucopyranosyl]-28-O-beta-D- glucopyranoside medicagenate, 3-O-[alpha-L-rhamnopyranosyl(1-->2)-beta-D-glucopyranosyl(1-->2)-beta -D-glucopyranoside] medicagenic acid, 3-O-[beta-D-glucopyranosyl(1-->2)-beta-D-glucopyranosyl(1-->2)-beta-D -glucopyranosyl]-28-beta- D-glucopyranoside medicagenate, 3-O-[beta-D-glucuronopyranosyl methyl ester]-28-O-[beta-D-xylopyranosyl(1-->4)-alpha-L-rhamnopyranosyl(1--> 2)-alpha-L-arabinopyranoside] medicagenate, 3-O-[alpha-L-rhamnopyranosyl(1-->2)-beta-D-galactopyranosyl(1-->2)-be ta-D-glucuronopyranosyl]-21-O-alpha-L-rhamnopyranoside soyasapogenol A, 3-O-[beta-D-glucopyranosyl(1-->2)-beta-D-glucopyranosyl(1-->2)glucopy ranosyl]-28-O-[beta-D-xylopyranosyl(1-->4)-alpha-L-rhamnopyranosyl (1- ->2)-alpha-L-arabinopyranoside] medicagenate, 3-O-[beta-D-glucopyranosyl(1-->2)-beta-D-glucopyranosyl(1-->2)glucopy ranosyl]-28-O-¿beta-D-xylopyranosyl(1-->4)-)-[beta-D-apiofurano syl-(1 -->3)]- alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopyranoside¿ medicagenate, 3-O-[beta-D-glucopyranosyl(1-->2)-beta-D-glucopyranosyl(1-->2)-beta-D -glucopyranosyl]-28-O-[beta-D-xylopyranosyl(1-->4)-alpha-L-rhamnopyra nosyl(1-->2)-alpha-L-arabinopyranoside] zanhic acid, 3-O-[beta-D-glucopyranosyl(1-->2)-beta-D-glucopyranosyl(1-->2)-beta-D -glucopyranosyl]-28-O-¿beta-D-xylopyranosyl(1-->4)-[beta-D-apiofurano side-(1-->3)]- alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopyranoside¿zanhic acid, and 3-O-[beta-D-galactopyranosyl(1-->2)-beta-D-glucuronopyranosyl]-28- O-b eta-D-glucopyranoside bayogenin, were not reported before, and their structures were established by spectral (FAB-MS and NMR) techniques. In addition, 3-O-[alpha-L-rhamnopyranosyl(1-->2)-beta-D-galactopyranosyl(1-->2)-be ta-D-glucuronopyranoside] soyasapogenol E was identified in the roots for the first time.

Carbohydrate Conformation↗
PubMed1997

Differential accumulation of two glycine-rich proteins during cold-acclimation alfalfa.

Two mRNAs, MsaCiA and MsaCiB, encoding for proteins harboring glycine-rich motifs, accumulate in alfalfa during cold acclimation. Fusion polypeptides containing the amino acid sequences deduced from these mRNAs were produced in Escherichia coli and used to raise antibodies. Each antibody cross-reacted specifically with soluble polypeptides, MSACIA-32 and MSACIB, respectively. These polypeptides were detectable only in crowns of cold-acclimated plants, even though MsaCiA mRNA accumulated in both crows and leaves during cold acclimation. The analysis of parietal proteins showed that several MSACIA-related proteins, with a molecular mass of 32, 41 and 68 kDa, did accumulate in leaf cell walls and one of 59 kDa crown cell walls. This diversity is most probably due to a tissue-specific maturation of MSACIA. A discrepancy was found between the time-course of accumulation of MSACIB and the one of the corresponding transcript. These results indicate that timing and localization of MSACIA and MSACIB expression are different, and suggest that this differential expression involves both transcriptional and post-transcriptional events. Comparisons made among six cultivars of contrasting freezing tolerance suggest that low tolerance could be explained by failure to accumulate proteins like MSACIA and MSACIB at a sufficient level.

Acclimatization↗
PubMed1997

A promoter directing epidermal expression in transgenic alfalfa.

We describe the isolation, cloning and expression of a 2.8 kb promoter fragment of the Blec4 gene from pea (Pisum sativum cv. Alaska) and demonstrate that it is capable of directing the expression of the beta-glucuronidase coding region to the developing epidermal tissue of vegetative and floral shoot apices of transgenic alfalfa (Medicago sativa cv. RegenSY). The Blec4 promoter represents a useful tool with which to target the expression of foreign genes to the epidermal layer of actively growing shoots. The activity of the Blec4 promoter in the epidermis of the shoot apex makes it particularly suitable for genetically engineering defense against insects and diseases that attack the growing shoot apex.

Fabaceae↗
PubMed1999

Down-regulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility.

To improve the digestibility of the forage crop alfalfa (Medicago sativa L.), cinnamyl alcohol dehydrogenase (CAD), which catalyses the last step in the biosynthesis of the lignin monomers, was down-regulated by using an antisense approach. A subset of six transgenic lines with reduced CAD activity and control lines were analysed when grown in the greenhouse and in the field. The down-regulation of the CAD enzyme was associated with a red coloration of the stem. The lignin quantity remained unchanged, but the lignin composition, as determined by thioacidolysis, was altered. The highest reduction of CAD activity was associated with a lower syringyl/guaiacyl (S/G) ratio and a lower S+G yield, mainly because of a decreased amount of S units. An increase in in situ disappearance of dry matter and of cell wall residue was detected in one of the transgenic lines grown in the greenhouse, and for two of the lines grown in the field the rate of disappearance of dry matter slightly improved. Furthermore, these two lines had a higher solubility in alkali as shown by the lower yield of saponified residue. This study opens perspectives for improving forage crop digestibility by the modulation of enzymes involved in lignin biosynthesis.

Alcohol Oxidoreductases↗
PubMed1998

A core promoter hairpin is essential for subgenomic RNA synthesis in alfalfa mosaic alfamovirus and is conserved in other Bromoviridae.

The nucleotide sequence immediately in front of the initiation site for subgenomic RNA 4 synthesis on RNA 3 minus strand, which has been proved to function as a core promoter, was inspected for secondary structure in 26 species of the plant virus family Bromoviridae. In 23 cases a stable hairpin could be predicted at a distance of 3 to 8 nucleotides from the initiation site of RNA 4. This hairpin contained several conserved nucleotides that are essential for core promoter activity in brome mosaic virus (R.W. Siegel, S. Adkins and C.C. Kao, Proc. Natl. Acad. Sci. USA 94, 11238-11243, 1997). Phylogenetic evidence and evidence from the effect of artificial mutations reported in the literature (E.A.G. van der Vossen, T. Notenboom and J.F. Bol, Virology 212, 663-672, 1995) indicate that the stem-loop structure is essential for promoter activity in alfalfa mosaic virus and probably in other Bromoviridae. Stability of the hairpin is most pronounced in the genera Alfamovirus and Ilarvirus which display genome activation by coat protein. The hypothesis is put forward that with these viruses the coat protein is needed for the viral RNA polymerase to interact with the core promoter hairpin leading to access for the enzyme to the initiation site of RNA 4.

Alfamovirus↗
PubMed2002

Effects of water stress on antioxidant enzymes of leaves and nodules of transgenic alfalfa overexpressing superoxide dismutases.

The antioxidant composition and relative water stress tolerance of nodulated alfalfa plants (Medicago sativa L. x Sinorhizobium meliloti 102F78) of the elite genotype N4 and three derived transgenic lines have been studied in detail. These transgenic lines overproduced, respectively, Mn-containing superoxide dismutase (SOD) in the mitochondria of leaves and nodules, MnSOD in the chloroplasts, and FeSOD in the chloroplasts. In general for all lines, water stress caused moderate decreases in MnSOD and FeSOD activities in both leaves and nodules, but had distinct tissue-dependent effects on the activities of the peroxide-scavenging enzymes. During water stress, with a few exceptions, ascorbate peroxidase and catalase activities increased moderately in leaves but decreased in nodules. At mild water stress, transgenic lines showed, on average, 20% higher photosynthetic activity than the parental line, which suggests a superior tolerance of transgenic plants under these conditions. However, the untransformed and the transgenic plants performed similarly during moderate and severe water stress and recovery with respect to important markers of metabolic activity and of oxidative stress in leaves and nodules. We conclude that the base genotype used for transformation and the background SOD isozymic composition may have a profound effect on the relative tolerance of the transgenic lines to abiotic stress.

Journal Article↗
PubMed2003

Overexpression of alfalfa cytosolic glutamine synthetase in nodules and flowers of transgenic Lotus japonicus plants.

Legumes can obtain nitrogen from symbiotic nitrogen fixation in root nodules. The glutamine synthetase/glutamate synthase cycle is responsible for the initial nitrogen assimilation. This work reports the analysis of transgenic Lotus japonicus plants with the chimeric gene containing the alfalfa cytosolic glutamine synthetase (GS1) (EC 6.3.1.2) gene controlled by the Sesbania rostrata leghemoglobin gene promoter (Srglb3p). Surprisingly, all of the transgenic primary transformants analysed were sterile. Two transformants designated GS39 and GS44 were further analysed. GS in nodules of GS39 and GS44 plants was upregulated, at the level of transcript and protein. The transgenic plants had 2-fold higher nodule GS activity and similar root GS activity compared to control plants. The GS39 and GS44 sterile plants showed morphological alterations in pollen grains and in ovules. An increase in GS transcript abundance and enzyme activity was measured during early and late stages of flower development of GS plants. Flowers of GS plants showed higher glutamine content, resulting in an increased glutamine/glutamate ratio. The GS transcript and protein were detected in ovules. These data indicate that overexpression of GS1 in reproductive organs critically affects their development and might be a reason for sterility of L. japonicus plants.

Journal Article↗
PubMed2006

Sinorhizobium meliloti nifA mutant induces different gene expression profile from wild type in Alfalfa nodules.

Several studies have demonstrated that the Rhizobium nifA gene is an activator of nitrogen fixation acting in nodule bacteria. To understand the effects of the Sinorhizobium meliloti nifA gene on Alfalfa, the cDNA-AFLP technique was employed to study the changes in gene expression in nifA mutant nodules. Among the approximately 3,000 transcript-derived fragments, 37 had differential expression levels. These expression levels were subsequently confirmed by reverse Northern blot and RT-polymerase chain reaction. Sequence analyses revealed that 21 cDNA fragments corresponded to genes involved in signal communication, protein degradation, nutrient metabolism, cell growth and development.

Bacterial Proteins↗
PubMed2001

Comparison of an immunochromatographic method and the TaqMan E. coli O157:H7 assay for detection of Escherichia coli O157:H7 in alfalfa sprout spent irrigation water and in sprouts after blanching.

An immunochromatographic-based assay (Quixtrade mark E. coli O157 Sprout Assay) and a polymerase chain reaction (PCR)-based assay (TaqMan E. coli O157:H7 Kit) were used to detect Escherichia coli O157:H7 strain 380-94 in spent irrigation water from alfalfa sprouts grown from artificially contaminated seeds. Ten, 25, 60, or 100 seeds contaminated by immersion for 15 min in a suspension of E. coli O157:H7 at concentrations of 10(6) or 10(8) cfu/ml were mixed with 20 g of non-inoculated seeds in plastic trays for sprouting. The seeds were sprayed with tap water for 15 s every hour and spent irrigation water was collected at intervals and tested. E. coli O157:H7 was detected in non-enriched water by both the TaqMan PCR (30 of 30 samples) and the immunoassay (9 of 24 samples) in water collected 30 h from the start of the sprouting process. However, enrichment of the spent irrigation water in brain heart infusion (BHI) broth at 37 degrees C for 20 h permitted detection of E. coli O157:H7 in water collected 8 h from the start of sprouting using both methods, even in trays containing as few as 10 inoculated seeds. The TaqMan PCR assay was more sensitive (more positive samples were observed earlier in the sprouting process) than the immunoassay; however, the immunoassay was easier to perform and was more rapid. At 72 h after the start of the sprouting process, the sprouts were heated at 100 degrees C for 30 s to determine the effectiveness of blanching for inactivation of E. coli O157:H7. All of the 32 samples tested with the TaqMan assay and 16 of 32 samples tested with the Quixtrade mark assay gave positive results for E. coli O157:H7 after enrichment of the blanched sprouts at 37 degrees C for 24 h. In addition, the organism was detected on Rainbow Agar O157 in 9 of 32 samples after 24 h of enrichment of the blanched sprouts. In conclusion, E. coli O157:H7 was detected in spent irrigation water collected from sprouts grown from artificially contaminated seeds by both the TaqMan and Quixtrade mark assays. The data also revealed that blanching may not be effective to completely inactivate all the E. coli O157:H7 that may be present in sprouts.

Chromatography↗
PubMed1980

Purification and properties of alpha-D-mannosidase from the germinated seeds of Medicago sativa (alfalfa).

alpha-Mannosidase of Medicago sativa (alfalfa) was purified 1340-fold. The purification method included dialysis of the crude extract against a citrate/phosphate buffer, pH 3.9, (NH4)SO4 precipitation, hydroxyapatite chromatography, chromatography on Sephadex G-200 and finally a preparatory electrophoresis on polyacrylamide-gel gradient by Doly & Petek's [(1977) J. Chromatogr. 137. 69--81] method. Each step of purification was checked by polyacrylamide-gel disc electrophoresis. The purified enzyme showed a single band, corresponding to alpha-mannosidase activity. alpha-Mannosidase has a mol.wt. 230 000 as estimated by Hedrick & Smith's [(1968) Arch. Biochem. Biophys. 126, 155--164] method and also by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate by Weber & Osborn [(1969) J. Biol. Chem. 244, 4406--4412]. The enzyme comprises four subunits of different molecular weight. Optimum pH and Km values were determined with p-nitrophenyl alpha-D-mannoside as substrate. When incubated at a temperature between 20 and 62 degrees C before assay, alpha-mannosidase initially shows an increase in activity. alpha-Mannosidase is stable when the pH is about neutrality. It can be inactivated by several metal ions, including Zn2+. At a pH below 5 the enzyme undergoes irreversible inactivation. The presence of EDTA at acid pH considerably enhances the inactivation of the enzyme. This inactivation due to EDTA can be specifically reversed by incubation with Zn2+.

Cations↗
PubMed1988

Adenylate cyclase activity in a higher plant, alfalfa (Medicago sativa).

An adenylate cyclase activity in Medicago sativa L. (alfalfa) roots was partially characterized. The enzyme activity remains in the supernatant fluid after centrifugation at 105,000 g and shows in crude extracts an apparent Mr of about 84,000. The enzyme is active with Mg2+ and Ca2+ as bivalent cations, and is inhibited by EGTA and by chlorpromazine. Calmodulin from bovine brain or spinach leaves activates this adenylate cyclase.

Adenylyl Cyclase Inhibitors↗
PubMed2001

Genetic diversity of indigenous Rhizobium leguminosarum bv. viciae isolates nodulating two different host plants during soil restoration with alfalfa.

A total of 360 Rhizobium leguminosarum bv. viciae strains was isolated from three brown-coal mining restoration fields of different age and plant cover (without and in the first and second year of alfalfa, Medicago sativa, cultivation) using two host species (Vicia hirsuta and Pisum sativum) as capture plants. The strains were genetically typed by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-generated 16S-23S ribosomal DNA intergenic spacer regions (IGS-RFLP) and characterized by plasmid profiles and RFLP analysis of amplified nodABC genes. The R. leguminosarum bv. viciae population was dominated by the same group of strains (irrespective of the trap plant used). According to type richness, the genetic diversity of indigenous R. leguminosarum in the second year of restoration was lower than in the first year and it resembled that of the fallow field, except for plasmid types, in which it was higher than that of the fallow field. Some of the less frequent nodABC genotypes were associated with distinct chromosomal IGS genotypes and symbiotic plasmids (pSyms) of different sizes, indicating that horizontal transfer and rearrangements of pSym can occur in natural environments. However, the dominant pSym and chromosomal genotypes were strictly correlated suggesting a genetically stable persistence of the prevailing R. leguminosarum bv. viciae genotypes in the absence of its host plant.

DNA, Bacterial↗
PubMed1998

Alfalfa malate dehydrogenase (MDH): molecular cloning and characterization of five different forms reveals a unique nodule-enhanced MDH.

Malate dehydrogenase (MDH) catalyzes the readily reversible reaction of oxaloacetate reversible malate using either NADH or NADPH as a reductant. In plants, the enzyme is important in providing malate for C4 metabolism, pH balance, stomatal and pulvinal movement, respiration, beta-oxidation of fatty acids, and legume root nodule functioning. Due to its diverse roles the enzyme occurs as numerous isozymes in various organelles. While antibodies have been produced and cDNAs characterized for plant mitochondrial, glyoxysomal, and chloroplast forms of MDH, little is known of other forms. Here we report the cloning and characterization of cDNAs encoding five different forms of alfalfa MDH, including a plant cytosolic MDH (cMDH) and a unique novel nodule-enhanced MDH (neMDH). Phylogenetic analyses show that neMDH is related to mitochondrial and glyoxysomal MDHs, but diverge from these forms early in land plant evolution. Four of the five forms could effectively complement an E. coli Mdh- mutant. RNA and protein blots show that neMDH is most highly expressed in effective root nodules. Immunoprecipitation experiments show that antibodies produced to cMDH and neMDH are immunologically distinct and that the neMDH form comprises the major form of total MDH activity and protein in root nodules. Kinetic analysis showed that neMDH has a turnover rate and specificity constant that can account for the extraordinarily high synthesis of malate in nodules.

Amino Acid Sequence↗
PubMed2000

Inhibition of serine/threonine-specific protein phosphatases causes premature activation of cdc2MsF kinase at G2/M transition and early mitotic microtubule organisation in alfalfa.

Reversible phosphorylation of serine/threonine residues of cell cycle-regulatory proteins is one of the key molecular mechanisms controlling eukaryotic cell division. In plants, the protein kinase partners (i.e. p34cdc2/CDC28-related kinases) have been extensively studied, while the role of counter-acting protein phosphatases is less well understood. We used endothall (ET) as a cell-permeable inhibitor of serine/threonine-specific protein phosphatases to alter cytological and biochemical characteristics of cell division in cultured alfalfa cells. A high concentration of ET (10 and 50 microM) inhibited both protein phosphatases 1 and 2 (PP1 and PP2A), while a low concentration (1 microM) of ET-treatment primarily reduced the PP2A activity. High concentrations of the inhibitor increased the frequency of hypercondensed early and late prophase chromosomes that could not enter metaphase. In contrast, a low concentration of ET did not interfere with chromosomal events but caused significant alterations in the organisation of microtubules. Exposure of cells to 1 microM ET resulted in disturbance of preprophase band formation, increase in the number of nuclei with prophase microtubule assembly, premature polarisation of the spindle, and abnormal phragmoplast maturation. Under the same conditions, the ET-treated cells exhibited an early increase in cdc2MsF kinase activity. These results suggest that PP2A contributes to the control of mitotic kinase activities and microtubule organisation. Normal chromosome condensation and mitotic progression are dependent on both PP1 and PP2A activities. The presented data support the functional role of protein phosphatases in the co-ordination of chromosomal and microtubule events in dividing plant cells.

Chromosomes↗
PubMed2003

Expression of a stress-induced hemoglobin affects NO levels produced by alfalfa root cultures under hypoxic stress.

Transgenic alfalfa root cultures expressing sense and antisense barley hemoglobin transcripts were examined under varying levels of atmospheric oxygen. Root cultures overexpressing the hemoglobin gene (Hb+) maintained root growth when placed under 3% oxygen, whereas control cultures or cultures underexpressing hemoglobin (Hb-) experienced 30-70% declines in growth under the same conditions. ATP levels and ATP/ADP ratios for Hb+ lines did not significantly differ in 40 and 3% oxygen, whereas the ATP levels and ATP/ADP ratios in control and Hb- lines were significantly lower under 3% oxygen. Large increases in the production of nitric oxide (NO) were measured in root cultures grown under hypoxic conditions compared to aerobic conditions. The amount of NO accumulated in an Hb- line was 2.5-fold higher than that in the Hb+ line. Treatment of transgenic root lines under 40% oxygen with NO resulted in significant declines in the ATP levels and ATP/ADP ratio of an Hb- line and the control line, with no significant change in an Hb+ line. The root cell structure of an Hb- line showed evidence of cell breakdown under hypoxic growth, whereas an Hb+ line had no evidence of cell breakdown under similar growth conditions. These results lead us to hypothesize that NO is involved in the response of plants to hypoxia and that hemoglobin modulates the levels of NO in the hypoxic cell.

Adenosine Diphosphate↗
PubMed2001

Growth of Escherichia coli O157:H7 during sprouting of alfalfa seeds.

AIMS: Escherichia coli O157:H7 was monitored daily during sprouting of alfalfa seeds inoculated at high (3.92 log10 cfu g(-1)) and low (1.86 log10 cfu g(-1)) levels to assess the extent of pathogen growth during production. METHODS AND RESULTS: Sprouts and rinse water were tested by direct and membrane filter plating on modified sorbitol MacConkey agar and BCM O157:H7(+) agar; the antibody-direct epifluorescent filter technique; and rapid immunoassays. The pathogen reached maximum populations after one and two days of sprouting seeds inoculated at high and low levels, respectively; in either case, populations of 5-6 log10 cfu g(-1) were reached. Detection limits of two rapid immunoassays, Reveal and VIP, without enrichment were determined to be 5-7 log10 cfu ml(-1). CONCLUSION: These results show the ability of E. coli O157:H7 to grow to high levels during sprouting; however, because these levels may be below detection limits, it is necessary to include enrichment when monitoring sprout production for E. coli O157:H7 by the rapid test kits. SIGNIFICANCE AND IMPACT OF THE STUDY: The data indicate that sprouts may harbor high levels of pathogens. The appropriate use of rapid test methods for pathogen monitoring during sprouting is indicated.

Colony Count, Microbial↗