Characterization of the dynamics of initial bacterial colonization of nonconserved forage in the bovine rumen

Microbial colonization is central to ruminal degradation of dietary material yet little is known about the dynamics of this process. The aim of this study was to characterize the initial stages of bacterial colonization of forage, and to assess the impact that different postsample processing and analysis methods had on the results obtained. Bacterial 16S rRNA gene-based analysis of damaged, nonconserved perennial ryegrass, incubated in sacco in the bovine rumen, required the development and validation of new quantitative PCR and denaturing gradient gel electrophoresis (DGGE) primers. Analysis with previously available primer sets was compromised due to dominant amplification of forage-derived chloroplast 16S rRNA genes. DGGE analysis of incubated samples demonstrated that a diverse and consistent population of ruminal bacteria colonized rapidly. Postsampling methodologies did not affect overall population profiles whereas the washing method appeared to influence bacterial numbers. However, regardless of processing methodology, bacterial numbers increased rapidly within 5 min, stabilizing after 15 min of incubation. These findings reveal for the first time the dynamics of bacterial colonization of forage within the rumen.     

Effect of a blend of essential oil compounds on the colonization of starch-rich substrates by bacteria in the rumen

AIMS: To investigate the mode of action of a blend of essential oil compounds on the colonization of starch-rich substrates by rumen bacteria. METHODS AND RESULTS: Starch-rich substrates were incubated, in nylon bags, in the rumen of sheep organized in a 4 x 4 latin square design and receiving a 60:40 silage : concentrate diet. The concentrate was either high or low in crude protein, and the diet was supplemented or not with a commercial blend of essential oil compounds (110 mg per day). The total genomic DNA was extracted from the residues in the bags. The total eubacterial DNA was quantified by real-time PCR and the proportion of Ruminobacter amylophilus, Streptococcus bovis and Prevotella bryantii was determined. Neither the supplementation with essential oil compounds nor the amount of crude protein affected the colonization of the substrates by the bacteria quantified. However, colonization was significantly affected by the substrate colonized. CONCLUSIONS: The effect of essential oils on the colonization of starch-rich substrates is not mediated through the selective inhibition of R. amylophilus. SIGNIFICANCE AND IMPACT OF THE STUDY: This study enhances our understanding of the colonization of starch-rich substrates, as well as of the mode of action of the essential oils as rumen manipulating agents.     

Biohydrogenation of linoleic acid by rumen fungi compared with rumen bacteria

AIMS: To investigate biohydrogenation of linoleic acid by rumen fungi compared with rumen bacteria, and to identify the fungus with the fastest biohydrogenation rate. METHODS AND RESULTS: Biohydrogenation of linoleic acid by mixed rumen fungi and mixed rumen bacteria were compared in vitro. With mixed rumen bacteria, all biohydrogenation reactions were finished within 100 min of incubation and the end product of biohydrogenation was stearic acid. With mixed rumen fungi, biohydrogenation proceeded more slowly over a 24-h period. Conjugated linoleic acid (CLA; cis-9, trans-11 C18 : 2) was an intermediate product, and vaccenic acid (VA; trans-11 C18 : 1) was the end product of biohydrogenation. Fourteen pure fungal isolates were tested for biohydrogenation rate. DNA sequencing showed that the isolate with the fastest rate belonged to the Orpinomyces genus. CONCLUSIONS: It is concluded that rumen fungi have the ability to biohydrogenate linoleic acid, but biohydrogenation is slower in rumen fungi than in rumen bacteria. The end product of fungal biohydrogenation is VA, as for group A rumen bacteria. Orpinomyces is the most active biohydrogenating fungus. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate that rumen fungi can biohydrogenate fatty acids. Fungi could influence CLA content of ruminant products.     

Effect of rumen fill on intake of fresh perennial ryegrass in young and mature dairy cows grazing or zero-grazing fresh perennial ryegrass

Rumen fill may be a strong intake constraint for dairy cows fed on pasture, even though pasture is highly digestible in the grasslands of temperate climates. This constraint may also depend on the cows' maturity. Moreover, indoor feeding of fresh herbage may not always be a good model for the study of intake regulation at grazing. To test these hypotheses, four mature (6.3 ± 0.72 year old) and four young (3.8 ± 0.20 year old) dairy cows were offered fresh perennial ryegrass indoors or at grazing. The impact of rumen fill on intake was evaluated by addition of rumen inert bulk (RIB; coconut fiber, 15 l) compared to a control. The experimental design was a double 4 × 4 Latin square with four 14-day periods and a 2 × 2 factorial arrangement of two feeding methods (indoor feeding v. grazing), combined with the addition, or not, of RIB (RIB v. control), repeated for four mature and four young cows. Digestibility of offered herbage was 0.81. The average ytterbium measured dry matter intake (Yb DMI) was 19.0 and 15.5 kg/day for mature and young cows respectively (P = 0.019). The effect of RIB on predicted Yb DMI interacted with feeding method and cow age (P = 0.043). The presence of RIB decreased Yb DMI by 4.4 kg/day in mature cows at grazing and by 3.4 kg/day in young cows indoors, whereas it did not affect the Yb DMI of mature cows indoors or grazing young cows. Both grazing and young age constituted a clear constraint on the feeding behavior of the cows. Grazing cows had fewer ingestion and rumination sequences, which were longer and less evenly distributed throughout the day and night. Young cows had lower intake rates that were less adaptable to the feeding method and the presence of RIB. Mature cows clearly decreased their daily intake rate at grazing compared to indoor feeding, and with RIB compared to control, whereas the intake rate of young cows did not vary. These results indicate that rumen fill can represent a constraint on intake in grazing cows, even when highly digestible perennial ryegrass is offered. The study also shows that the impact of RIB on intake is highly dependent upon other constraints applied to the chewing behavior, which in this experiment were methods of offering herbage and cow age.     

Volatiles in perennial ryegrass infected with strains of endophytic fungus: impact on African black beetle host selection

Perennial ryegrass (Lolium perenne) is often infected with the fungal‐endophyte Neotyphodium lolii. In addition to the ‘wild‐type’ strain (E^WT), several ‘selected’ strains of N. lolii are now being marketed as AR1 (E^AR1) and AR37 (E^AR37). Each of these strains impact positively on L. perenne's resistance against many insects, including the African black beetles (Heteronychus arator). The impact of volatile oils produced specifically by each strain in the endophyte–grass association in enhancing the grass's resistance to insects is still largely unknown. Keeping these in view, we determined the volatile oil profiles produced by L. perenne infected with either E^WT or E^AR1 or E^AR37 and determined the impacts of these volatiles on the host‐selection behaviour of H. arator adults. In the absence of endophyte infection (E^–), L. perenne produced 18 different volatile oils. In L. perenne E^WT, quantities of 2‐ethyl‐1‐hexanol acetate (Rt = 14.5 min), (Z)‐2‐octen‐1‐ol (Rt = 22.2 min), and butylated hydroxyl toluene (Rt = 23.2 min) were 24, 16 and 26%, respectively, greater than L. perenne E^–. The strains E^AR1 and E^AR37 affected differently the quantities of the volatile compounds but not their identity. In the four‐choice bioassay, males and females of H. arator were equally attracted to each strain. In Y‐tube olfactometer, compared against E^–, H. arator adults were less attracted to L. perenne E^WT and E^AR1. The attractiveness of E^AR37 was similar in effect to E^– to H. arator. The results indicate that each strain of N. lolii alters the profile of volatile oils in L. perenne differently and that alteration can influence H. arator adult‐host selection.     

Production of indolic compounds by rumen bacteria isolated from grazing ruminants

AIM: To screen rumen bacterial cultures and fresh ruminal isolates for indole and skatole production. METHODS AND RESULTS: Culture collection strains and fresh bacterial isolates from rumen contents of sheep and dairy cows were screened for the production of indolic compounds. Clostridium aminophilum FT, Peptostreptococcus ssp. S1, Fusobacterium necrophorum D4 produced indole and Clostridium sticklandii SR produced indoleacetic acid. Fresh isolates from sheep (TrE9262 and TrE7262) and dairy cows (152R-1a, 152R-1b, 152R-3 and 152R-4) produced indole, indolepropionic acid, tryptophol and skatole from the fermentation of tryptophan and indoleacetic acid. Glucose altered the indolic compounds produced in some, but not all, isolates. TrE7262 and 152R-4 were identified as Clostridium sporogenes and 152R-1b as a new Cl. aminophilum strain. Isolates TrE9262, 152R-1a and 152R-3 were not closely related to any described species but belong to Megasphaera, Prevotella and Actinomyces genera, respectively. CONCLUSIONS: Rumen bacteria that produced a range of indolic compounds were identified. Some isolates are distinct from the previously described bacteria and may represent novel species. SIGNIFICANCE AND IMPACT OF THE STUDY: These observations will contribute to understanding skatole and indole formation in the rumen and will lead to methods that control the formation of indolic compounds in pasture-grazed ruminants.     

Dynamics of initial colonization of nonconserved perennial ryegrass by anaerobic fungi in the bovine rumen

Anaerobic fungi (Neocallimastigales) are active degraders of fibrous plant material in the rumen. However, only limited information is available relating to how quickly they colonize ingested feed particles. The aim of this study was to determine the dynamics of initial colonization of forage by anaerobic fungi in the rumen and the impact of different postsampling wash procedures used to remove loosely associated microorganisms. Neocallimastigales-specific molecular techniques were optimized to ensure maximal coverage before application to assess the population size (quantitative PCR) and composition (automated ribosomal intergenic spacer analysis) of the colonizing anaerobic fungi. Colonization of perennial ryegrass (PRG) was evident within 5 min, with no consistent effect of time or wash procedure on fungal population composition. Wash procedure had no effect on population size unlike time, which had a significant effect. Colonizing fungal population size continued to increase over the incubation period after an initial lag of c. 4 min. This dynamic differs from that reported previously for rumen bacteria, where substantial colonization of PRG occurred within 5 min. The observed delay in colonization of plant material by anaerobic fungi is suggested to be primarily mediated by the time taken for fungal zoospores to locate, attach and encyst on plant material.     

Magnesium requirement of some of the principal rumen cellulolytic bacteria

Information available on the role of Mg for growth and cellulose degradation by rumen bacteria is both limited and inconsistent. In this study, the Mg requirements for two strains each of the cellulolytic rumen species Fibrobacter succinogenes (A3c and S85), Ruminococcus albus (7 and 8) and Ruminococcus flavefaciens (B34b and C94) were investigated. Maximum growth, rate of growth and lag time were all measured using a complete factorial design, 2(3)×6; factors were: strains (2), within species (3) and Mg concentrations (6). R. flavefaciens was the only species that did not grow when Mg was singly deleted from the media, and both strains exhibited a linear growth response to increasing Mg concentrations (P<0.001). The requirement for R. flavefaciens B34b was estimated as 0.54 mM; whereas the requirement for R. flavefaciens C94 was >0.82 as there was no plateau in growth. Although not an absolute requirement for growth, strains of the two other species of cellulolytic bacteria all responded to increasing Mg concentrations. For F. succinogenes S85, R. albus 7 and R. albus 8, their requirement estimated from maximum growth was 0.56, 0.52 and 0.51, respectively. A requirement for F. succinogenes A3c could not be calculated because there was no solution for contrasts. Whether R. flavefaciens had a Mg requirement for cellulose degradation was determined in NH₃-free cellulose media, using a 2×4 factorial design, 2 strains and 4 treatments. Both strains of R. flavefaciens were found to have an absolute Mg requirement for cellulose degradation. Based on reported concentrations of Mg in the rumen, 1.0 to 10.1 mM, it seems unlikely that an in vivo deficiency of this element would occur.     

Leaf functional trait variation associated with salt tolerance in perennial ryegrass

Salinity is one of major environmental stresses that dramatically threaten plant growth, and variations in genetic structure and functional traits have important effects on the salt tolerance of perennial ryegrass (Lolium perenne L.). The objectives of this study were to: (i) assess the inter‐clonal variation of functional traits of accessions among geographic groups or between wild and commercial groups in response to salt stress; (ii) develop a mathematical model to effectively assess salt tolerance of perennial ryegrass accessions originating from different geographic populations; and (iii) determine the relation between spatial genetic structure and salt tolerance in perennial ryegrass. Wide variations were found among the accessions for seven functional traits. One regression model (F = 0.49 × F₁ + 0.303 × F₂ + 0.207 × F₃) was established to ascertain salt tolerance of each accession. The highest variation of the traits and salt tolerance were obtained for accessions from the European group. Wild accessions exhibited more variation in functional traits and salt tolerance than commercial cultivars. Both molecular marker techniques and functional traits were used to conduct phylogenetic analysis, and the majority of accessions from the same or adjacent regions were clustered into the same group or subgroup. The perennial ryegrass accessions with similar salt tolerance had a close phylogenetic background. The patterns in functional trait variations associated with salt tolerance might allow acceleration of the process for improving salt stress resistance in perennial ryegrass.     

Degradation of terpenes and terpenoids from Mediterranean rangelands by mixed rumen bacteria in vitro

This in vitro study aimed at estimating the disappearance rates of 14 terpenes and terpenoids after 24-h incubation with mixed bacteria from caprine rumens. These compounds comprised nine monoterpene hydrocarbons (δ-3-carene, p-cymene, β-myrcene, (E)- and (Z)-β-ocimene, α-phellandrene, α-terpinene, γ-terpinene and α-terpinolene), four oxygenated monoterpenes ((E)- and (Z)-linalool oxide, 4-terpinenol, α + γ terpineol) and one sesquiterpene hydrocarbon (β-cedrene). They were individually exposed to goat rumen microflora for 24 h in 70 ml culture tubes at an input level of 0.5 ml/l. Terpenoids were the least degraded, 100% of (E)-linalool oxide, 95% of (Z)-linalool oxide, 91% of 4-terpinenol and 75% of terpineol remained intact after 24-h incubation. In contrast, α-terpinolene concentration in fermentation broth extracts was below quantification limit, thus indicating an extensive, if not complete, degradation by rumen bacteria. Only 2% of the initial amounts of α-phellandrene were recovered. The other monoterpenes and β-cedrene were partly degraded, with losses ranging from 67% for δ-3-carene to 90% for (E)-β-ocimene. The corresponding rates of disappearance were between 2.67 and 4.08 μmol/ml inoculum per day.     

植物内生细菌的分离、分类、定殖与应用

植物内生细菌已成为国内外的研究热点,目前已从植物中分离得到许多不同种属的植物内生细菌,其中有的具有促生与防治病害等对宿主植物有利的作用,而一些则具有潜在的致病性。研究人员利用多种方法对植物内生细菌的内生定殖行为进行研究,发现了有意义的定殖规律,例如,某种植物内生细菌对其宿主的侵染能力明显强于另一种内生细菌。目前,尽管植物内生细菌还有不少问题有待解决,但它在农业上的巨大应用潜力业已彰显。     

Overexpression of ubiquitin‐like LpHUB1 gene confers drought tolerance in perennial ryegrass

HUB1, also known as Ubl5, is a member of the subfamily of ubiquitin‐like post‐translational modifiers. HUB1 exerts its role by conjugating with protein targets. The function of this protein has not been studied in plants. A HUB1 gene, LpHUB1, was identified from serial analysis of gene expression data and cloned from perennial ryegrass. The expression of this gene was reported previously to be elevated in pastures during the summer and by drought stress in climate‐controlled growth chambers. Here, pasture‐type and turf‐type transgenic perennial ryegrass plants overexpressing LpHUB1 showed improved drought tolerance, as evidenced by improved turf quality, maintenance of turgor and increased growth. Additional analyses revealed that the transgenic plants generally displayed higher relative water content, leaf water potential, and chlorophyll content and increased photosynthetic rate when subjected to drought stress. These results suggest HUB1 may play an important role in the tolerance of perennial ryegrass to abiotic stresses.     

Divergent evolutionary processes associated with colonization of offshore islands

Oceanic islands have been a test ground for evolutionary theory, but here, we focus on the possibilities for evolutionary study created by offshore islands. These can be colonized through various means and by a wide range of species, including those with low dispersal capabilities. We use morphology, modern and ancient sequences of cytochrome b (cytb) and microsatellite genotypes to examine colonization history and evolutionary change associated with occupation of the Orkney archipelago by the common vole (Microtus arvalis), a species found in continental Europe but not in Britain. Among possible colonization scenarios, our results are most consistent with human introduction at least 5100 bp (confirmed by radiocarbon dating). We used approximate Bayesian computation of population history to infer the coast of Belgium as the possible source and estimated the evolutionary timescale using a Bayesian coalescent approach. We showed substantial morphological divergence of the island populations, including a size increase presumably driven by selection and reduced microsatellite variation likely reflecting founder events and genetic drift. More surprisingly, our results suggest that a recent and widespread cytb replacement event in the continental source area purged cytb variation there, whereas the ancestral diversity is largely retained in the colonized islands as a genetic ‘ark’. The replacement event in the continental M. arvalis was probably triggered by anthropogenic causes (land‐use change). Our studies illustrate that small offshore islands can act as field laboratories for studying various evolutionary processes over relatively short timescales, informing about the mainland source area as well as the island.