This study was designed to determine the effect of exogenous spermidine (Spd) (30 μM) on white clover seed germination under water stress induced by polyethylene glycol 6000. Use of seed priming with Spd improved seed germination percentage, germination vigor, germination index, root viability and length, and shortened mean germination time under different water stress conditions. Seedling fresh weight and dry weight also increased significantly in Spd-treated seeds compared with control (seeds primed with distilled water). Improved starch metabolism was considered a possible reason for this seed invigoration, since seeds primed with Spd had significantly increased α-amylase/β-amylase activities, reducing sugar, fructose and glucose content and transcript level of β-amylase gene but not transcript level of α-amylase gene. In addition, the physiological effects of exogenous Spd on improving seeds’ tolerance to water deficit during germination were reflected by lower lipid peroxidation levels, better cell membrane stability and significant higher seed vigour index in seedlings. Enhanced antioxidant enzyme activities (superoxide dismutase, peroxidase, catalase and ascorbate peroxidase), ascorbate-glutathione cycle (ASC-GSH cycle) and transcript level of genes encoding antioxidant enzymes induced by exogenous Spd may be one of the critical reasons behind acquired drought tolerance through scavenging of reactive oxygen species (ROS) in water-stressed white clover seeds. The results indicate that Spd plays an important function as a stress-protective compound or physiological activator.

To develop a silage fermentation technique to adapt to global climate changes, the microbiome and fermentation dynamics of corn silage inoculated with heat-resistant lactic acid bacteria (LAB) under high-temperature conditions were studied. Corn was ensiled in laboratory silo, with and without two selected strains, Lactobacillus salivarius LS358 and L. rhamnosus LR753, two type strains L. salivarius ATCC 11741^T and L. rhamnosus ATCC 7469^T. The ensiling temperatures were designed at 30 °C and 45 °C, and the sampling took place after 0, 3, 7, 14, and 60 days of fermentation. The higher pH and dry matter losses were observed in the silages stored at 45 °C compared to those stored at 30 °C. Silages inoculated with strains LS358 and LR753 at 30 °C had a lower ratio of lactic acid/acetic acid. The dominant bacterial genera gradually changed from Pediococcus and Lactobacillus to Lactobacillus in silages during ensiling at 30 °C, while the bacterial community became more complex and fragmented after 7 d of ensiling at 45 °C. The high temperatures significantly led to a transformation of the LAB population from homo-fermentation to hetero-fermentation. This study is the first to describe microbial population dynamics response to high temperature during corn ensiling, and the results indicate that L. rhamnosus 753 shows potential ability to improve silage fermentation in tropics and subtropics.

Increased transcriptional levels of genes encoding antioxidant enzymes play important protective roles in coping with excessive accumulation of reactive oxygen species (ROS) in plants exposed to various abiotic stresses. To fully elucidate different evolutions and functions of ROS-scavenging enzymatic genes, we isolated iron superoxide dismutase (FeSOD), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) from white clover for the first time and subsequently tested dynamic expression profiles of these genes together with previously identified other antioxidant enzyme genes including copper zinc superoxide dismutase (Cu/ZnSOD), manganese superoxide dismutase (MnSOD), glutathione reductase (GR), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) in response to cold, drought, salinity, cadmium stress and exogenous abscisic acid (ABA) or spermidine (Spd) treatment. The cloned fragments of FeSOD, DHAR and MDHAR genes were 630, 471 and 669 bp nucleotide sequences encoding 210, 157 and 223 amino acids, respectively. Phylogenetic analysis indicated that both amino acid and nucleotide sequences of these three genes are highly conservative. In addition, the analysis of genes expression showed the transcription of GR, POD, MDHAR, DHAR and Cu/ZnSOD were rapidly activated with relatively high abundance during cold stress. Differently, CAT, APX, FeSOD, Cu/ZnSOD and MnSOD exhibited more abundant transcripts compared to others under drought stress. Under salt stress, CAT was induced preferentially (3–12 h) compared to GR which was induced later (12–72 h). Cadmium stress mainly up-regulated Cu/ZnSOD, DHAR and MDHAR. Interestingly, most of genes expression induced by ABA or Spd happened prior to various abiotic stresses. The particular expression patterns and different response time of these genes indicated that white clover differentially activates genes encoding antioxidant enzymes to mitigate the damage of ROS during various environmental stresses.

A two-year field experiment was conducted to address the effects of mixture composition and legume-grass seeding ratio on the biomass yield and nutritional quality of legume–grass mixtures. Alfalfa (Medicago sativa L.), white clover (Trifolium repens L.), red clover (Trifolium pratense L.), orchardgrass (Dactylis glomerata L.), perennial ryegrass (Lolium perenne L.), and tall fescue (Festuca arundinacea Schreb.) were selected as plant materials. A total of seven legume–grass mixtures (A1: white clover, orchardgrass, and tall fescue; A2: alfalfa, orchardgrass, and tall fescue; B1: alfalfa, white clover, orchardgrass, and tall fescue; B2: red clover, white clover, orchardgrass, and tall fescue; C1: alfalfa, white clover, orchardgrass, tall fescue, and perennial ryegrass; C2: red clover, white clover, orchardgrass, tall fescue, and perennial ryegrass; and D: alfalfa, red clover, white clover, orchardgrass, tall fescue, and perennial ryegrass) were sown in two legume-grass seeding ratios (L:G) of 4:6 and 5:5. The results showed that A2 produced a higher two-year average biomass yield (14.20 t/ha) in L:G of 4:6 than that of other mixtures. The grasses biomass yield proportion decreased while legume biomass yield proportion increased with prolonged establishment time. A2 showed a higher crude protein yield (2.5 t/ha) in L:G of 4:6. C2 and A1 showed lower neutral detergent fiber (4.6 t/ha) and acid detergent fiber (2.8 t/ha) yields in L:G 5:5, while diverse mixtures showed higher water-soluble carbohydrate yields. Overall, A2 showed a relative feed value of 146.50 in L:G of 4:6, indicating that it has not only produced the higher biomass yield but also had a better nutritional quality.

Oat (Avena sativa L.) is one of the important forage crops in the world. However, oat grown in Southwest China has higher moisture content and their preservation face significant challenges. In addition, existing commercial lactic acid bacteria (LAB) have poor fermentation effects in hot and humid regions. Consequently, the current study investigated the response of oat fermentation quality and microbial community to self-selected LAB inoculation. The treatments were: CK, sterilized water; LP694, Lactobacillus plantarum 694; LR753, Lactobacillus rhamnosus 753; and LPLR, LP694 combined with LR753, followed by 1, 3, 7, 14, and 60 days (d) of fermentation. The results showed that LAB inoculation significantly raised the lactic acid content, and decreased the level of pH value, acetic acid, and ammonia-N in oat silage. The LR753 group had a significantly higher (p < 0.05) lactic acid content (60.95 g kg^–1 DM), and lower pH value (3.95) and ammonia-N content (10.1 g kg^–1 DM) followed by the LPLR group. The LR753 showed lower NDF (54.60% DM) and ADF (39.73% DM) contents than other groups. The Lactobacillus was a prevalent genus in LAB-treated groups, and its relative abundance reached maximum in LP694 (69%) on day 3, while in the LR753 group (72%) on 60 days. The Lactobacillus rhamnosus, Lactobacillus plantarum, and Lactobacillus fermentum became the dominant species in LAB-treated groups with fermentation time. The Lactobacillus genus was positively correlated with WSC (R = 0.6, p < 0.05), while negatively correlated with pH (R = −0.5, p < 0.05), and BA (R = −0.5, p < 0.01). Overall, the LR753 group had better fermentation quality and preservation of nutritional components providing theoretical support and guidance for future oat silage production in Southwest China.