Inoculation with Lentilactobacillus buchneri alone or in combination with Lentilactobacillus hilgardii modifies gene expression, fermentation profile, and starch digestibility in high-moisture corn.

Inoculants combining Lentilactobacillus buchneri and Lentilactobacillus hilgardii have been shown to improve the aerobic stability of high-moisture corn (HMC) and whole-plant corn silage, but the mode of action of this co-inoculation remains to be elucidated. This study used metatranscriptomics to evaluate the effects of inoculation with L. buchneri alone or combined with L. hilgardii on the bacterial community, gene expression, fermentation profile, and starch digestibility in HMC. High-moisture corn not inoculated (Control) or inoculated with L. buchneri NCIMB 40788 (LB) or L. buchneri NCIMB 40788 combined with L. hilgardii CNCM-I-4785 (Combo) was ensiled in mini silo bags for 30, 60, 120, and 180 days. The fermentation profile was evaluated at all time points. Metatranscriptomics was performed on samples collected on day 120. Combo had a greater alpha diversity richness index of contigs than LB and Control, and inoculation with Combo and LB modified the beta-diversity of contigs compared to Control. Out of 69 genes of interest, 20 were differentially expressed in LB compared to Control and 25 in Combo compared to Control. Of those differently expressed genes, 16 (10 of which were associated with carbohydrate metabolism and six with amino acid metabolism) were differently expressed in both LB and Combo compared to Control, and all those genes were upregulated in the inoculated silages. When we compared Combo and LB, we found seven genes expressed differently, four associated with carbohydrate metabolism and downregulated in Combo, and three associated with amino acid metabolism and upregulated in Combo. At day 120, the inoculated silages had more culturable lactic acid bacteria, higher Lactobacillus relative abundance, and lower Leuconostoc relative abundance than Control. The concentration of acetic acid remained low throughout ensiling in Control, but in LB and Combo, it increased up to day 60 and remained stable from day 60 to 180. The 1,2-propanediol was only detected in LB and Combo. Inoculation did not affect the concentration of starch, but starch digestibility was greater in Combo than in Control. Inoculation of HMC with Combo modified the gene expression and fermentation profile compared to Control and LB, improving starch digestibility compared to uninoculated HMC.

Farm management practices and season dependent factors affect the microbial community and chemical profile of corn and grass-legume silages of farms in Ontario, Québec, and Northern New York.

The effects of farm management practices and seasonal variation on the microbial community and chemical composition of corn and grass-legume silage are largely understudied due to the advantages of controlled mini-silo experiments. This study aims to investigate the effects that some key farm factors (use of an inoculant, farm region, and bunker or tower silo) and seasonal variations have on corn and grass-legume silage from farms across Ontario, Quebec, and New York. The silage was either treated with a commercial inoculant (Lallemand Biotal Buchneri 500® or Chr Hansen SiloSolve FC®) or left untreated. The bacterial communities of silage were compared to those of raw bulk tank milk from the same farm to determine if they were similarly affected by management practices or seasonal variations. Family level analysis of the 16S rRNA V3-V4 gene amplicon bacterial community, the ITS1 amplicon fungal community, NMR water soluble metabolome, and mycotoxin LC-MS were performed on silage over a two-year period. Chemical compounds associated with the use of inoculants in corn and grass-legume silage were higher in inoculated corn (acetate, propane-1,2-diol, γ-aminobutyrate; p < 0.001) and grass-legume (propionate; p = 0.011). However, there was no significant difference in the relative abundance (RA) of Lactobacillaceae in either silage type. Leuconostocaceae was higher in non-inoculated corn (p < 0.001) and grass-legume (p < 0.001) silage than in inoculated silage. Tower silos had higher RA of Leuconostocaceae (p < 0.001) and higher pH (p < 0.001) in corn and grass-legume silage. The one farm that used liquid manure with no other fertilizer type had higher RA of Clostridiaceae (p = 0.045) and other rumen/fecal (p < 0.006) bacteria in grass-legume silage than all other farms. Seasonal variation affected most of the key silage microbial families, however the trends were rarely visible across both years. Few trends in microbial variation could be observed in both silage and bulk tank milk: two farms had higher Moraxellaceae (p < 0.001) in milk and either corn or grass-legume silage. In farms using an inoculant, lower Staphylococcaceae was observed in the raw bulk tank milk.

Effects of a mixture of Lentilactobacillus hilgardii, Lentilactobacillus buchneri, Pediococcus pentosaceus and fibrolytic enzymes on silage fermentation, aerobic stability, and performance of growing beef cattle.

This study evaluated the effects of novel silage inoculants containing lactic acid bacteria (LAB) and fibrolytic enzymes on ensiling, aerobic stability (AS), and the performance of growing beef cattle. Whole-plant corn forage was either uninoculated (CON) or inoculated with a mixture of LAB containing (cfu g-1 fresh forage) 1.5 × 105 L. hilgardii (CNCM I-4785), 1.5 × 105 L. buchneri (NCIMB 40788) and 1.0 × 105 P. pentosaceus (NCIMB 12455) for a total of 4.0 × 105 cfu g-1 fresh forage LAB (IB), or a combination of IB plus fibrolytic enzymes (xylanase + ß-glucanase) (IC). All treatments were ensiled in mini-silos, whereas CON and IC were also ensiled in silo bags for the growth performance study. Total bacteria (TB) counts were lower (P = 0.02) for IC than CON after 14 d of ensiling, whereas TB counts of IC and IB were greater (P ≤ 0.01) than CON after 60 d of ensiling in mini-silos. The LAB in IC and IB ensiled in mini-silos were greater than CON on d 60 (P ≤ 0.01) and 90 (P ≤ 0.001) of ensiling and after 3 d (P ≤ 0.01) of aerobic exposure (AE). Silage pH of IC ensiled in silo bags was lower than CON on d 3 (P < 0.01), 7 (P < 0.001), and 14 (P = 0.02) of AE. Yeast counts were lower for IC than CON in terminal silage (P < 0.001), and after 3 (P < 0.001) and 7 d (P < 0.01) of AE. Acetate (AC) concentrations were higher (P ≤ 0.02) for IC than CON throughout AE, whereas lactate (LA) concentrations of IC were greater than CON on d 3 (P < 0.001), 7 (P < 0.01), and 14 (P < 0.001) of AE. Greater AC concentration and lower yeast counts resulted in greater (P < 0.001) stability for IC ensiled in silo bags than CON after 14 d of AE. Growth performance of steers was similar (P > 0.05) as the nutrient composition of silage was similar across diets. Improved AS of IC could potentially have a greater impact on DMI, production efficiency, and growth performance in large-scale commercial feedlot operations where silage at the silo face may be exposed to air for longer periods of time.

Changes to the microbiome of alfalfa during the growing season and after ensiling with Lentilactobacillus buchneri and Lentilactobacillus hilgardii inoculant.

AIMS: This study evaluated changes in epiphytic microbial population of alfalfa (Medicago sativa) during the growing season. First cut forage was harvested to study the effects of an inoculant combining two obligate heterofermentative lactic acid bacteria strains on the bacterial and fungal communities and the fermentation of alfalfa silage. METHODS AND RESULTS: The epiphytic microbiome of alfalfa was evaluated 10-times during the growing season. Alfalfa wilted to 395.0 g/kg was treated with water (Control) or with a combination of L. buchneri NCIMB 40788 and L. hilgardii CNCM-I-4785 (LBLH). Mini-silos were opened after 1, 4, 8, 16, 32, and 64 days of ensiling. The relative abundance (RA) of the epiphytic bacterial and fungal families varied during the growing season. After 1 day, Weissella was the most abundant genus and present at similar RA in the two treatments (average 80.4%). Compared with Control, LBLH had a higher RA of Lactobacillus at day 1, 16, 32, and 64, and a lower RA of Weissella from day 8 to 64. Control contained more bacteria belonging to the Enterobacteriales than LBLH up to day 16. Inoculated silage had more acetate than Control at day 32 and 64. The fungal population were similar between treatments. The enhanced development and dominance of Lactobacillus in inoculated silage led to greater accumulation of acetate and propionate, which reduced the numbers of culturable yeasts but did not markedly affect the fungal community structure. CONCLUSIONS: The bacterial community composition of alfalfa stands in the filed changed over time and was affected by cutting. For the ensiling trial, inoculation modified the composition of the bacterial community of alfalfa, increasing the RA of Lactobacillus while reducing the RA of Weissella and of Enterobacteriaceae. SIGNIFICANCE AND IMPACT OF STUDY: Inoculation increased the RA of Lactobacillus, hampering the dominance of Weissella in the early stages of ensiling, improving antifungal compounds production and reducing the numbers of culturable yeasts.

A Novel Microbial Zearalenone Transformation through Phosphorylation.

Zearalenone (ZEA) is a mycotoxin widely occurring in many agricultural commodities. In this study, a purified bacterial isolate, Bacillus sp. S62-W, obtained from one of 104 corn silage samples from various silos located in the United States, exhibited activity to transform the mycotoxin ZEA. A novel microbial transformation product, ZEA-14-phosphate, was detected, purified, and identified by HPLC, LC-MS, and NMR analyses. The isolate has been identified as belonging to the genus Bacillus according to phylogenetic analysis of the 16S rRNA gene and whole genome alignments. The isolate showed high efficacy in transforming ZEA to ZEA-14-phosphate (100% transformation within 24 h) and possessed advantages of acid tolerance (work at pH = 4.0), working under a broad range of temperatures (22-42 °C), and a capability of transforming ZEA at high concentrations (up to 200 µg/mL). In addition, 23 Bacillus strains of various species were tested for their ZEA phosphorylation activity. Thirteen of the Bacillus strains showed phosphorylation functionality at an efficacy of between 20.3% and 99.4% after 24 h incubation, suggesting the metabolism pathway is widely conserved in Bacillus spp. This study established a new transformation system for potential application of controlling ZEA although the metabolism and toxicity of ZEA-14-phosphate requires further investigation.

Microbiota succession during aerobic stability of maize silage inoculated with Lentilactobacillus buchneri NCIMB 40788 and Lentilactobacillus hilgardii CNCM-I-4785.

Aerobic deterioration of silage following feeding out is responsible for the deterioration of its quality. Inoculation of silage with lactic acid bacteria is one strategy to limit these effects. A trial was performed using whole-plant corn ensiled in bag silo, and forage was inoculated with Lentilactobacillus buchneri NCIMB 40788 (Lactobacillus buchneri) and Lentilactobacillus hilgardii CNCM-I-4785 (Lactobacillus hilgardii) or not (Control silage). After 159 days of fermentation, the silos were opened and the silage was sampled at 24-h intervals during a 10-day aerobic stability assay to measure pH, the fermentation profile, mycotoxins, and microbial and fungal populations. In inoculated silage, lactic acid concentrations and pH remained stable during the aerobic phase and higher microorganism alpha-diversity was observed. Treated silage was characterized by a high abundance of Saccharomycetes and maintenance of Lactobacillus throughout the aerobic stability assay. The high aerobic stability of the inoculated silage contrasted with the decrease in lactic acid contents and the increase in pH observed in the Control silage, concomitantly with an increase in lactate-assimilating yeast (Pichia and Issatchenkia), and in Acetobacter and Paenibacillus OTUs. Remarkably, Penicillium and roquefortine C were detected in this silage by day 8 following exposure to air. Our study highlighted the fact that the use of L. buchneri with L. hilgardii modified the consequences of exposure to air by maintaining higher microbial diversity, avoiding the dominance of a few bacteria, and preventing fungi from having a detrimental effect on silage quality.

Testing selectivity of bacterial and fungal culture media compared to original silage samples using next generation sequencing.

Plate counts using selective culture media is still the most frequently used method for the enumeration of the different microbial groups that colonize silage, including lactic acid bacteria, yeasts and molds. Since different culture media have specific composition, they may allow the growth of specific populations. To date, no study has used next generation sequencing technology to compare the selective capacity of these different culture media although this approach could provide comprehensive insight into the relevance of using one culture medium over another. Sequencing of the 16S rDNA and ITS amplicon were performed to compare the selectivity of different culture media used in silage microbiology. Corn silage, grass-alfalfa silage and total mixed ration extracts were plated on five selective media for lactic acid bacteria, incubated under aerobic and anaerobic conditions, and on eight selective media for yeast and molds to compare their selectivity. Ensiling provided a pre-selection environment for specific microorganisms over forage and reduced the number of observed OTUs: only 12 OTUs of bacteria were observed in corn silage sampled in the center of a bunker silo, while the mean number of OTUs identified in samples taken closer to the side of the silo, influenced by higher oxygen and humidity level, increased to 79. Still, MRS and Rogosa plates had less than 12 different OTUs in the center and 24 at the side, mainly Lactobacillaceae, Acetobacteraceae, and Leuconostocaceae. Incubating the plates under anaerobic conditions was selective against Acetobacteraceae. MRS supplemented with acetic acid increased selectivity of lactic acid bacteria. When plated on culture media specific for yeast and molds, from 17 to 68 different OTUs were observed in corn silage. Mixed grass-alfalfa silage and total mixed ration samples usually had more observed OTUs and the diversity profile of the corresponding culture media was similar to that of the original samples. For yeasts and molds, Dichloran Rose Bengal Chloramphenicol Agar revealed a diversity profile close to the that of the corn silage.

Effects of inoculation of corn silage with Lactobacillus hilgardii and Lactobacillus buchneri on silage quality, aerobic stability, nutrient digestibility, and growth performance of growing beef cattle.

This study evaluated the effects of inoculation of whole crop corn silage with a mixture of heterofermentative lactic acid bacteria (LAB) composed of Lactobacillus hilgardii and Lactobacillus buchneri on ensiling, aerobic stability, ruminal fermentation, total tract nutrient digestibility, and growth performance of beef cattle. Uninoculated control corn silage (CON) and silage inoculated with 3.0 × 105 cfu g-1 of LAB containing 1.5 × 105 cfu g-1 of L. hilgardii CNCM I-4785 and 1.5 × 105 cfu g-1 of L. buchneri NCIMB 40788 (INOC) were ensiled in silo bags. The pH did not differ (P > 0.05) between the two silages during ensiling but was greater (P < 0.001) for CON than INOC after 14 d of aerobic exposure (AE). Neutral detergent insoluble crude protein (NDICP) content (% of DM and % of CP basis) of terminal INOC silage was greater (P ≤ 0.05) than that of CON. In terminal silage, concentrations of total VFA and acetate were greater (P < 0.001), while water-soluble carbohydrates were lower (P < 0.001) for INOC than CON. Yeast and mold counts were lower for INOC than CON (P ≤ 0.001) in both terminal and aerobically exposed silages. The stability of INOC was greater (P < 0.001) than that of CON after 14 d of AE. Ruminal fermentation parameters and DMI did not differ (P > 0.05) between heifers fed the two silages, while there was a tendency (P ≤ 0.07) for lower CP and starch digestibility for heifers fed INOC than CON. Total nitrogen (N) intake and N retention were lower (P ≤ 0.04) for heifers fed INOC than CON. Dry matter intake as a percentage of BW was lower (P < 0.04) and there was a tendency for improved feed efficieny (G:F; P = 0.07) in steers fed INOC vs. CON silage. The NEm and NEg contents were greater for INOC than CON diets. Results indicate that inoculation with a mixture of L. hilgardii and L. buchneri improved the aerobic stability of corn silage. Improvements in G:F of growing steers fed INOC silage even though the total tract digestibility of CP and starch tended to be lower for heifers fed INOC are likely because the difference in BW and growth requirements of these animals impacted the growth performance and nutrient utilization and a greater proportion of NDICP in INOC than CON.

Dynamic Succession of Microbiota during Ensiling of Whole Plant Corn Following Inoculation with Lactobacillus buchneri and Lactobacillus hilgardii Alone or in Combination.

Lactic acid bacteria (LAB) used as silage additives have been shown to improve several fermentation parameters, including aerobic stability. Inoculation with a combination of Lactobacillus buchneri NCIMB40788 and Lactobacillus hilgardii CNCM-I-4785, contributes to an increase in aerobic stability, compared to each strain inoculated independently. To understand the mode of action of the combination on the LAB community, a fermentation-kinetic study was performed on corn. Four treatments, Control, Lb. buchneri, Lb. hilgardii, and a combination of the two strains, were fermented 1, 2, 4, 8, 16, 32, and 64 days. Corn silage inoculated by both strains had a lactate:acetate ratio of 0.59 after 64 days and a higher concentration of lactate than Lb. buchneri. Analysis of the microbiota by 16S and ITS amplicon metasequencing demonstrated that inoculation led to lower bacterial diversity after 1 day, from 129.4 down to 40.7 observed operational taxonomic units (OTUs). Leuconostocaceae represented the dominant population by day 1, with 48.1%. Lactobacillaceae dominated the succession by day 4, with 21.9%. After 32 days, inoculation by both strains had the lowest bacterial alpha diversity level, with 29.0 observed OTUs, compared to 61.3 for the Control. These results confirm the increased fermentation efficiency when the two Lactobacillus strains are co-inoculated, which also led to a specific yeast OTUs diversity profile, with Hannaella as the main OTU.

Effects on microbial diversity of fermentation temperature (10°C and 20°C), long-term storage at 5°C, and subsequent warming of corn silage

Objective: To evaluate the effects on microbial diversity and biochemical parameters of gradually increasing temperatures, from 5 to 25 °C on corn silage which was previously fermented at ambient or low temperature. Methods: Whole-plant corn silage was fermented in vacuum bag mini-silos at either 10 or 20 °C for two months and stored at 5 °C for two months. The mini-silos were then subjected to additional incubation from 5 to 25 °C in 5 °C increments. Bacterial and fungal diversity was assessed by PCR-DGGE profiling and biochemical analysis from mini-silos collected at each temperature. Results: A temperature of 10 °C during fermentation restricted silage fermentation compared to fermentation temperature of 20 °C. As storage temperature increased from 5 to 25 °C, little changes occurred in silages fermented at 20 °C, in terms of most biochemical parameters as well as bacterial and fungal populations. However, a fairly high number of enterobacteria and yeasts (4-5 log10 CFU g FM-1) were detected at 15 °C and above. PCR-DGGE profile showed that Candida humilis predominated the fungi flora. For silage fermented at 10 °C, no significant changes were observed in most silage characteristics when temperature was increased from 5 to 20 °C. However, above 20 °C, silage fermentation resumed as observed from the significantly increased number of LAB colonies, acetic acid content, and the rapid decline in pH and WSC concentration. DGGE results showed that Lactobacillus buchneri started to dominate the bacterial flora as temperature increased from 20 to 25 °C. Conclusion: Temperature during fermentation as well as temperature during storage modulates microorganism population development and fermentation patterns. Silage fermented at 20 °C indicated that these silages should have lower aerobic stability at opening because of better survival of yeasts and enterobacteria.

Rhizobial strains exert a major effect on the amino acid composition of alfalfa nodules under NaCl stress.

Specific amino acids have protective functions in plants under stress conditions. This study assessed the effects of rhizobial strains on the amino acid composition in alfalfa under salt stress. Two alfalfa cultivars (Medicago sativa L. cv Apica and salt-tolerant cv Halo) in association with two Sinorhizobium meliloti strains with contrasting growth under salt stress (strain A2 and salt-tolerant strain Rm1521) were exposed to different levels of NaCl (0, 20, 40, 80 or 160 mM NaCl) under controlled conditions. We compared root and shoot biomasses, as well as root:shoot ratio for each association under these conditions as indicators of the salt tolerance of the symbiosis. Amino acid concentrations were analyzed in nodules, leaves and roots. The total concentration of free amino acids in nodules was mostly rhizobial-strain dependent while in leaves and roots it was mostly responsive to salt stress. For both cultivars, total and individual concentrations of amino acids including asparagine, proline, glutamine, aspartate, glutamate, γ-aminobutyric acid (GABA), histidine and ornithine were higher in Rm1521 nodules than in A2 nodules. Conversely, lysine and methionine were more abundant in A2 nodules than in Rm1521 nodules. Proline, glutamine, arginine, GABA and histidine substantially accumulated in salt-stressed nodules, suggesting an enhanced production of amino acids associated with osmoregulation, N storage or energy metabolism to counteract salt stress. Combining the salt-tolerant strain Rm1521 and the salt-tolerant cultivar Halo enhanced the root:shoot ratios and amino acid concentrations involved in plant protection which could be in part responsible for the enhancement of salt tolerance in alfalfa.

Plant maturity and nitrogen fertilization affected fructan metabolism in harvestable tissues of timothy (Phleum pratense L.).

Timothy (Phleum pratense L.) is an important grass forage used for pasture, hay, and silage in regions with cool and humid growth seasons. One of the factors affecting the nutritive value of this grass is the concentration of non-structural carbohydrates (NSC), mainly represented by fructans. NSC concentration depends on multiple factors, making it hardly predictable. To provide a better understanding of NSC metabolism in timothy, the effects of maturity stage and nitrogen (N) fertilization level on biomass, NSC and N-compound concentrations were investigated in the tissues used for forage (leaf blades and stems surrounded by leaf sheaths) of hydroponically grown plants. Moreover, activities and relative expression level of enzymes involved in fructan metabolism were measured in the same tissues. Forage biomass was not altered by the fertilization level but was strongly modified by the stage of development. It increased from vegetative to heading stages while leaf-to-stem biomass ratio decreased. Total NSC concentration, which was not altered by N fertilization level, increased between heading and anthesis due to an accumulation of fructans in leaf blades. Fructan metabolizing enzyme activities (fructosyltransferase-FT and fructan exohydrolase-FEH) were not or only slightly altered by both maturity stage and N fertilization level. Conversely, the relative transcript levels of genes coding for enzymes involved in fructan metabolism were modified by N supply (PpFT1 and Pp6-FEH1) or maturity stage (PpFT2). The relative transcript level of PpFT1 was the highest in low N plants while that of Pp6-FEH1 was the highest in high N plants. Morevoer, transcript level of PpFT1 was negatively correlated with nitrate concentration while that of PpFT2 was positively correlated with sucrose concentration. This distinct regulation of the two genes coding for 6-sucrose:fructan fructosyltransferase (6-SFT) may allow a fine adequation of C allocation towards fructan synthesis in response to carbon and N availability. Contrary to fructans, starch content increased in low N plants, suggesting different regulatory mechanisms and/or sensitivity of starch and fructan metabolism in relation to the N status.

Species composition of saproxylic fungal communities on decaying logs in the boreal forest.

Coarse woody debris supports large numbers of saproxylic fungal species. However, most of the current knowledge comes from Scandinavia and studies relating the effect of stand or log characteristics on the diversity and composition of decomposer fungi have not been conducted in Northeastern Canada. Logs from five tree species were sampled along a decomposition gradient in nine stands representing three successional stages of the boreal mixed forest of Northwestern Quebec, Canada. Using a molecular fingerprinting technique, we assessed fungal community Shannon-Weaver diversity index, richness, and composition. We used linear mixed models and multivariate analyses to link changes in fungal communities to log and stand characteristics. We found a total of 33 operational taxonomic units (OTUs) including an indicator species for balsam fir (similar to Athelia sp.) and one found only in aspen stands (similar to Calocera cornea). Spruce logs supported the highest fungal Shannon-Weaver diversity index and OTU number. Our results support the hypothesis that log species influences fungal richness and diversity. However, log decay class does not. Stand composition, volume of coarse woody debris, and log chemical composition were all involved in structuring fungal communities. Maintaining the diversity of wood-decomposing communities therefore requires the presence of dead wood from diverse log species.

Tolerance to agricultural pesticides of strains belonging to four genera of Rhizobiaceae.

In order to determine their tolerance to pesticides, 122 strains of rhizobia isolated from different geographical regions, and belonging to the genera Rhizobium, Mesorhizobium, Sinorhizobium and Bradyrhizobium were tested against eight herbicides, four fungicides and five insecticides. Sensitivity to the pesticides was measured by using the filter paper disk method at four concentrations, 0.45, 4.5, 45 and 450 µg per disk. When the pesticides were used at 0.45 µg per disk, a concentration similar to that found when pesticides are applied under field conditions, no inhibition was observed. Strains growth was affected at concentrations of 45 and 450 µg pesticide per disk. These higher concentrations can be encountered when seeds are treated with pesticides. Pesticides tolerance level was correlated to pesticide function, i.e rhizobial strains were more tolerant to insecticides, followed by herbicides and then fungicides. Two fungicides, captan and mancozeb, inhibited the highest number of strains. Only one insecticide, carbaryl, affected the growth of some rhizobial strains. Strains isolated from the arctic (Mesorhizobium spp. and R. leguminosarum bv. viciae), a putative pesticides-free environment, were either less or equally affected by pesticides compared to strains isolated from agricultural regions.

Sources of clostridia in raw milk on farms.

A PCR-denaturing gradient gel electrophoresis (DGGE) method was used to examine on-farm sources of Clostridium cluster I strains in four dairy farms over 2 years. Conventional microbiological analysis was used in parallel to monitor size of clostridial populations present in various components of the milk production chain (soil, forage, grass silage, maize silage, dry hay, and raw milk). PCR amplification with Clostridium cluster I-specific 16S rRNA gene primers followed by DGGE separation yielded a total of 47 operational taxonomic units (OTUs), which varied greatly with respect to frequency of occurrence. Some OTUs were found only in forage, and forage profiles differed according to farm location (southern or northern Québec). More clostridial contamination was found in maize silage than in grass silage. Milk represented a potential environment for certain OTUs. No OTU was milk specific, indicating that OTUs originated from other environments. Most (83%) of the OTUs detected in raw milk were also found in grass or maize silage. Milk DGGE profiles differed according to farm and sampling year and fit into two distinct categories. One milk profile category was characterized by the presence of a few dominant OTUs, the presence of which appeared to be more related to farm management than to feed contamination. OTUs were more varied in the second profile category. The identities of certain OTUs frequently found in milk were resolved by cloning and sequencing. Clostridium disporicum was identified as an important member of clostridial populations transmitted to milk. Clostridium tyrobutyricum was consistently found in milk and was widespread in the other farm environments examined.

Correlation of long-range membrane order with temperature-dependent growth characteristics of parent and a cold-sensitive, branched-chain-fatty-acid-deficient mutant of Listeria monocytogenes.

Listeria monocytogenes is a food-borne, pathogenic, psychrotolerant bacterium that grows at refrigeration temperatures. Long-range membrane order of the parent (10403S) and of a cold-sensitive mutant ( cld-1) deficient in odd-numbered, branched-chain fatty acids was measured using the width of the central line of spectra of an electron paramagnetic resonance probe, 4,4-dimethyl-2-heptyl-2-hexyloxazolidine- N-oxyl (7N14), that locates deep in the hydrocarbon region of the membranes. The line width decreased from 0.9 to 0.5 milliTesla (mT) over the temperature range of 0-10 degrees for strain 10403S and -5 to 32 degrees C for strain cld-1 independent of protein state (heat denatured or intact). This provided new evidence for phase transitions in the membranes. When strain cld-1 was grown in medium supplemented with 2-methylbutyric acid, which restores anteiso fatty acids and the ability to grow at low temperature, the change in central line width as a function of temperature resembled that of strain 10403S. The temperatures at which the central line width became 0.8 mT corresponded to those at which growth became very slow in both strains (3-5 degrees C for 10403S, 15 degrees C for cld-1) as determined by Arrhenius plots. These data underscore the critical role of odd-numbered anteiso fatty acids in influencing the lower temperature limits of growth through their effects on long-range membrane fluidity.

Classification of rhizobia based on nodC and nifH gene analysis reveals a close phylogenetic relationship among Phaseolus vulgaris symbionts.

The nodC and nifH genes were characterized in a collection of 83 rhizobial strains which represented 23 recognized species distributed in the genera Rhizobium, Sinorhizobium, Mesorhizobium and Bradyrhizobium, as well as unclassified rhizobia from various host legumes. Conserved primers were designed from available nucleotide sequences and were able to amplify nodC and nifH fragments of about 930 bp and 780 bp, respectively, from most of the strains investigated. RFLP analysis of the PCR products resulted in a classification of these rhizobia which was in general well-correlated with their known host range and independent of their taxonomic status. The nodC and nifH fragments were sequenced for representative strains belonging to different genera and species, most of which originated from Phaselous vulgaris nodules. Phylogenetic trees were constructed and revealed close relationships among symbiotic genes of the Phaseolus symbionts, irrespective of their 16S-rDNA-based classification. The nodC and nifH phylogenies were generally similar, but cases of incongruence were detected, suggesting that genetic rearrangements have occurred in the course of evolution. The results support the view that lateral genetic transfer across rhizobial species and, in some instances, across Rhizobium and Sinorhizobium genera plays a role in diversification and in structuring the natural populations of rhizobia.