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 Phaseolus 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.

Ensiled forage, particularly corn silage, is an important component of dairy cow diets worldwide. Forages can be contaminated with several mycotoxins in the field pre-harvest, during storage, or after ensiling during feed-out. Exposure to dietary mycotoxins adversely affects the performance and health of livestock and can compromise human health. Several studies and surveys indicate that ruminants are often exposed to mycotoxins such as aflatoxins, trichothecenes, ochratoxin A, fumonisins, zearalenone, and many other fungal secondary metabolites, via the silage they ingest. Problems associated with mycotoxins in silage can be minimized by preventing fungal growth before and after ensiling. Proper silage management is essential to reduce mycotoxin contamination of dairy cow feeds, and certain mold-inhibiting chemical additives or microbial inoculants can also reduce the contamination levels. Several sequestering agents also can be added to diets to reduce mycotoxin levels, but their efficacy varies with the type and level of mycotoxin contamination. This article gives an overview of the types, prevalence, and levels of mycotoxin contamination in ensiled forages in different countries, and describes their adverse effects on health of ruminants, and effective prevention and mitigation strategies for dairy cow diets. Future research priorities discussed include research efforts to develop silage additives or rumen microbial innocula that degrade mycotoxins.

Silage making can be conveniently divided into field, ensiling, storage, and feed-out phases. In all of these stages, controllable and uncontrollable components can affect silage quality. For instance, silages produced in hot or cold regions are strongly influenced by uncontrollable climate-related factors. In hot regions, crops for silage are influenced by (1) high temperatures negatively affecting corn yield (whole-crop and grain) and nutritive value, (2) butyric and alcoholic fermentations in warm-season grasses (Panicum, Brachiaria, and Pennisetum genera) and sugarcane, respectively, and (3) accelerated aerobic deterioration of silages. Ensiling expertise and economic factors that limit mechanization also impair silage production and utilization in hot environments. In cold regions, a short and cool growing season often limits the use of crops sensitive to cool temperature, such as corn. The fermentation triggered by epiphytic and inoculated microorganisms can also be functionally impaired at lower temperature. Although the use of silage inoculants has increased in Northern Europe, acid-based additives are still a good option in difficult weather conditions to ensure good fermentation quality, nutritive value, and high intake potential of silages. Acid-based additives have enhanced the quality of round bale silage, which has become a common method of forage preservation in Northern Europe. Although all abiotic factors can affect silage quality, the ambient temperature is a factor that influences all stages of silage making from production in the field to utilization at the feed bunk. This review identifies challenges and obstacles to producing silages under hot and cold conditions and discusses strategies for addressing these challenges.

This study demonstrated the impact of temperature gradient on the diversity and some important population shift of lactic acid bacteria communities during fermentation of corn silage.

Ensiling of forages was recognized as a microbial-driven process as early as the late 1800s, when it was associated with the production of "sweet" or "sour" silage. Classical microbiological plating techniques defined the epiphytic microbial populations associated with fresh forage, the pivotal role of lactic acid-producing bacteria in the ensiling process, and the contribution of clostridia, bacilli, yeast, and molds to the spoilage of silage. Many of these classical studies focused on the enumeration and characterization of a limited number of microbial species that could be readily isolated on selective media. Evidence suggested that many of the members of these microbial populations were viable but unculturable, resulting in classical studies underestimating the true microbial diversity associated with ensiling. Polymerase chain reaction-based techniques, including length heterogeneity PCR, terminal RFLP, denaturing gradient gel electrophoresis, and automated ribosomal intergenic spacer analysis, were the first molecular methods used to study silage microbial communities. Further advancements in whole comparative genomic, metagenomic, and metatranscriptomic sequencing have or are in the process of superseding these methods, enabling microbial communities during ensiling to be defined with a degree of detail that is impossible using classical microbiology. These methods have identified new microbial species in silage, as well as characterized shifts in microbial communities with forage type and composition, ensiling method, and in response to aerobic exposure. Strain- and species-specific primers have been used to track the persistence and contribution of silage inoculants to the ensiling process and the role of specific species of yeast and fungi in silage spoilage. Sampling and the methods used to isolate genetic materials for further molecular analysis can have a profound effect on results. Primer selection for PCR amplification and the presence of inhibitors can also lead to biases in the interpretation of sequence data. Bioinformatic analyses are reliant on the integrity and presence of sequence data within established databases and can be subject to low taxonomic resolution. Despite these limitations, advancements in molecular biology are poised to revolutionize our current understanding of the microbial ecology of silage.

No abstract

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.

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.