Using gas chromatography mass spectrometry and the PacBio single molecule with real-time sequencing technology (SMRT), we analyzed the detailed metabolomic profiles and microbial community dynamics involved in ensiledMedicago sativa(alfalfa) inoculated without or with the homofermenterLactobacillus plantarumor heterofermenterLactobacillus buchneri. Our results revealed that 280 substances and 102 different metabolites were present in ensiled alfalfa. Inoculation ofL. buchneriled to remarkable up-accumulation in concentrations of 4-aminobutyric acid, some free amino acids, and polyols in ensiled alfalfa, whereas considerable down-accumulation in cadaverine and succinic acid were observed inL. plantarum-inoculated silages. Completely different microbial flora and their successions during ensiling were observed in the control and two types of inoculant-treated silages. Inoculation of theL. plantarumorL. buchnerialters the microbial composition dynamics of the ensiled forage in very different manners. Our study demonstrates that metabolomic profiling analysis provides a deep insight in metabolites in silage. Moreover, the PacBio SMRT method revealed the microbial composition and its succession during the ensiling process at the species level. This provides information regarding the microbial processes underlying silage formation and may contribute to target-based regulation methods to achieve high-quality silage production.

The traditional concept of the genusHumicolaincludes species that produce pigmented, thick-walled and single-celled spores laterally or terminally on hyphae or minimally differentiated conidiophores. More than 50 species have been described in the genus. Species commonly occur in soil, indoor environments, and compost habitats. The taxonomy ofHumicolaand morphologically similar genera is poorly understood in modern terms. Based on a four-locus phylogeny, the morphological concept ofHumicolaproved to be polyphyletic. The type ofHumicola,H. fuscoatra, belongs to theChaetomiaceae. In theChaetomiaceae, species producing humicola-like thick-walled spores are distributed among four lineages:Humicola sensu stricto,Mycothermus,Staphylotrichum,andTrichocladium. In our revised concept ofHumicola, asexual and sexually reproducing species both occur. The re-definedHumicolacontains 24 species (seven new and thirteen new combinations), which are described and illustrated in this study. The species in this genus produce conidia that are lateral, intercalary or terminal on/in hyphae, and conidiophores are not formed or are minimally developed (micronematous). The ascospores of sexualHumicolaspecies are limoniform to quadrangular in face view and bilaterally flattened with one apical germ pore. Seven species are accepted inStaphylotrichum(four new species, one new combination). Thick-walled conidia ofStaphylotrichumspecies usually arise either from hyphae (micronematous) or from apically branched, seta-like conidiophores (macronematous). The sexual morph represented byStaphylotrichum longicolleum(=Chaetomium longicolleum) produces ascomata with long necks composed of a fused basal part of the terminal hairs, and ascospores that are broad limoniform to nearly globose, bilaterally flattened, with an apical germ pore. TheTrichocladiumlineage has a high morphological diversity in both asexual and sexual structures. Phylogenetic analysis revealed four subclades in this lineage. However, these subclades are genetically closely related, and no distinctive phenotypic characters are linked to any of them. Fourteen species are accepted inTrichocladium,including one new species, twelve new combinations. The type species ofGilmaniella,G. humicola, belongs to the polyphyletic familyLasiosphaeriaceae(Sordariales), butG. macrosporaphylogenetically belongs toTrichocladium. The thermophilic genusMycothermusand the type speciesMy. thermophilumare validated, and one newMycothermusspecies is described. Phylogenetic analyses show thatRemersonia, another thermophilic genus, is sister toMycothermusand two species are known, including one new species.Thermomyces verrucosusproduces humicola-like conidia and is transferred toBotryotrichumbased on phylogenetic affinities. This study is a first attempt to establish an inclusive modern classification ofHumicolaand humicola-like genera of theChaetomiaceae. More research is needed to determine the phylogenetic relationships of “humicola”-like species outside theChaetomiaceae.

In tropical regions, as in temperate regions where seasonality of forage production occurs, well-preserved forage is necessary for animal production during periods of forage shortage. However, the unique climate conditions (hot and humid) and forage characteristics (high moisture content and low soluble carbohydrate) in the tropics make forage preservation more difficult. The current study used natural ensiling of tropical forage as a model to evaluate silage characteristics under different temperatures (28°C and 40°C). Four tropical forages (king grass, paspalum, white popinac, and stylo) were ensiled under different temperatures (28°C and 40°C). After ensiling for 30 and 60 days, samples were collected to examine the fermentation quality, chemical composition and microbial community. High concentrations of acetic acid (ranging from 7.8 to 38.5 g/kg dry matter [DM]) were detected in silages of king grass, paspalum and stylo with relatively low DM (ranging from 23.9% to 30.8% fresh material [FM]) content, acetic acid production was promoted with increased temperature and prolonged ensiling. Small concentrations of organic acid (ranging from 0.3 to 3.1 g/kg DM) were detected in silage of white popinac with high DM content (50.8% FM). The microbial diversity analysis indicated that Cyanobacteria originally dominated the bacterial community for these four tropical forages and was replaced by Lactobacillus and Enterobacter after ensiling. The results suggested that forage silages under tropical climate conditions showed enhanced acetate fermentation, while high DM materials showed limited fermentation. Lactobacillus and Enterobacter were the most probable genera responsible for tropical silage fermentation.

The objective of this study was to select lactic acid bacteria (LAB) isolated from the rumen fluid and feces of dairy cows, and evaluate their effects on silage quality of alfalfa after 30 or 60 days of ensiling. One hundred and four LAB strains were isolated from rumen fluid and feces of six dairy cows, of which four strains (Lactobacillus plantarum F1, L. plantarum F50, Lactobacillus salivarius L100, and Lactobacillus fermentum L120) and one commercial inoculant (GFG) isolated from forage were employed for further study. The silages treated with F1 had the lowest (P < 0.05) pH value and the highest (P < 0.05) lactic acid (LA) content in all treatments. Besides, higher (P < 0.05) in vitro digestibility was also observed in F1-treated silage after 60 days of ensiling. The microbial analysis showed that the Lactobacillus abundance in the F1-treated silages increased to 60.32%, higher than other treatments (5.12–47.64%). Our research indicated that strain F1 could be an alternative silage inoculant, and dairy cows could be a source for obtaining excellent LAB for ensiling.

Silage quality remains an important issue in farming, as do limitations in the range of products suitable for animal fodder. We therefore explored the microorganisms that are critical for the fermentation quality of paper mulberry silage. Low (unwilted) and high (wilted) dry matter (DM) paper mulberry were harvested at two cutting times. These were ensiled for 0, 3, 7, 14, and 56 days, respectively. Compared with unwilted silages, wilting significantly decreased (p < 0.05) silage pH value, ammonia‐N concentration, and yeast counts but increased (p < 0.05) lactic acid content. In addition, higher (p < 0.05) crude protein (CP) contents were also observed in wilted silages. Next‐generation sequencing (NGS) analysis revealed that wilting reduced the abundance of Enterobacter, while increasing that of Lactobacillus. Single‐molecule real‐time sequencing (SMRT) revealed that the silage was enriched in the lactic acid bacteria (LAB), Lactobacillus rhamnosus after wilting, which showed a positive correlation with CP and lactic acid content. We conclude that wilting may help preserve paper mulberry silage, facilitating its use as a new fodder resource. Moreover, L. rhamnosus has the potential to be developed as a new inoculant for the modulation in wilted silages, particularly paper mulberry silage.