Agrobacterium tumefaciens-mediated transformation (ATMT) has long been used to transfer genes to a wide variety of plants and has also served as an efficient tool for insertional mutagenesis. In this paper, we report the construction of four novel binary vectors for fungal transformation and the optimization of an ATMT protocol for insertional mutagenesis, which permits an efficient genetic manipulation of Fusarium oxysporum and other phytopathogenic fungi to be achieved. Employing the binary vectors, carrying the bacterial hygromycin B phosphotrans-ferase gene (hph) under the control of the Aspergillus nidulans trpC promoter as a selectable marker, led to the production of 300 to 500 hygromycin B resistant transformants per 1 x 10(6) conidia of F. oxysporum, which is at least an order of magnitude higher than that previously accomplished. Transformation efficiency correlated strongly with the duration of cocultivation of fungal spores with Agrobacterium tumefaciens cells and significantly with the number of Agrobacteruium tumefaciens cells present during the cocultivation period (r = 0.996; n = 3; P < 0.01). All transformants tested remained mitotically stable, maintaining their hygromycin B resistance. Growing Agrobacterium tumefaciens cells in the presence of acetosyringone (AS) prior to cocultivation shortened the time required for the formation of transformants but decreased to 53% the percentage of transformants containing a single T-DNA insert per genome. This increased to over 80% when Agrobacterium tumefaciens cells grown in the absence of AS were used. There was no correlation between the average copy number of T-DNA per genome and the colony diameter of the transformants, the period of cocultivation or the quantity of Agrobacterium tumefaciens cells present during cocultivation. To isolate the host sequences flanking the inserted T-DNA, we employed a modified thermal asymmetric interlaced PCR (TAIL-PCR) technique. Utilizing just one arbitrary primer resulted in the successful amplification of desired products in 90% of those transformants analyzed. The insertion event appeared to be a random process with truncation of the inserted T-DNA, ranging from 1 to 14 bp in size, occurring on both the right and left border sequences. Considering the size and design of the vectors described here, coupled with the efficiency and flexibility of this ATMT protocol, it is suggested that ATMT should be regarded as a highly efficient alternative to other DNA transfer procedures in characterizing those genes important for the pathogenicity of F. oxysporum and potentially those of other fungal pathogens.

The Loess Plateau of China is one of the most fragile ecosystems worldwide; thus, human production activities need to be conducted very cautiously. In this study, MiSeq high-throughput sequencing was applied to assess the relationship between bacterial and fungal community structures and changes in vegetation and soil physical and chemical properties induced by grazing, in four grasslands with different levels of grazing intensity (0, 2.67, 5.33, and 8.67 sheep/ha) in the semiarid region of the Loess Plateau. The relative abundances of the bacterial community in the grasslands with 2.67 and 5.33 sheep/ha were significantly higher than those in grasslands with 0 and 8.67 sheep/ha, and the fungal diversity was significantly lower for grasslands with 2.67 sheep/ha than for the other grasslands. Redundancy analysis (RDA) showed that plant biomass, nitrate, and total nitrogen have significant effects on bacterial community structure, whereas nitrate and total nitrogen also significantly affect fungal community structure. Variation partitioning showed that soil and plant characteristics influence the bacterial and fungal community structures; these characteristics explained 51.9 and 52.9% of the variation, respectively. Thus, bacterial and fungal community structures are very sensitive to grazing activity and change to different extents with different grazing intensities. Based on our findings, a grazing intensity of about 2.67 sheep/ha is considered the most appropriate in semiarid grassland of the Loess Plateau.

This experiment was designed to test the effect of inclusion rate of 3-nitrooxypropanol (3-NOP), a methane inhibitor, on enteric methane emissions in dairy cows. The study was conducted with 49 multiparous Holstein cows in a randomized complete block design in 2 phases; phase 1 was with 28 cows, and phase 2 with 21 cows. Cows were fed a basal total mixed ration ad libitum and were blocked based on days in milk, milk yield, and enteric methane emissions during a 14-d covariate period. Treatments were control (no 3-NOP) and 40, 60, 80, 100, 150, and 200 mg of 3-NOP/kg of feed dry matter. Following a 14-d adaptation period, enteric gaseous emissions (methane, carbon dioxide, and hydrogen) were measured using the GreenFeed system (C-Lock Inc., Rapid City, SD) over a 3-d period. Compared with the control, inclusion rate of 3-NOP quadratically decreased daily enteric methane emissions from 22 to 40%. Maximum mitigation effect was achieved with the 3 highest 3-NOP doses (with no statistical difference among 100, 150, and 200 mg/ kg). The decrease in methane emission yield and emission intensity ranged from 16 to 36% and from 25 to 45%, respectively. Emissions of hydrogen quadratically increased 6-to 10-fold, compared with the control; the maximum increase was with 150 mg/kg 3-NOP. Treatment did not affect daily emissions of carbon dioxide, but a linear increase in carbon dioxide emission yield was observed with increasing 3-NOP doses. Dry matter intake and milk yield of the cows was not affected by 3-NOP. Milk fat concentration and yield were increased by 3-NOP due to increased concentration of de novo synthetized short-chain fatty acids in milk. Inclusion of 3-NOP also tended to increase milk urea nitrogen but had no other effects on milk components. In this shortterm experiment, 3-NOP decreased enteric methane emissions without affecting dry matter intake or milk yield and increased milk fat in dairy cows. Maximum mitigation effect was achieved at 100 to 200 mg/kg of feed dry matter.

From August to September 2015 (trial 1 [T1]) and September to November 2015 (trial 2 [T2]), the effects of replacing concentrate feed (C) with alfalfa hay (AH) on the daily gain (DG), dietary energy utilization status and the economic advantage of AH feeding for growing beef cattle were studied in crossbred male Simmental calves (n = 18) in Gansu Province, China. The target DG was set as 1 kg for both trials. Animals in each trial were allocated to a conventional feeding group (CTRL), a low‐level AH feeding group (LA), and a high‐level AH feeding group (HA). In a one‐way‐layout design, they were fed iso‐energetic experimental diets comprising harvested corn stover (CS) and C (T1‐CTRL, T2‐CTRL), diets replacing 22% (T1‐LA) or 44% (T1‐HA) of the quantity of C for T1‐CTRL with AH, and diets replacing 13% (T2‐LA) or 25% (T2‐HA) of the quantity of C for T2‐CTRL with AH. Measurements of feed intake and DG, respiration and metabolism trials were performed for 49 and 41 days in T1 and T2, respectively. Average DG did not reach the target value for HA in T1 and CTRL in T2. Energy metabolizability was slightly greater for CTRL than for LA and HA in T1 and significantly greater for CTRL than for the other groups in T2. There was no marked difference in energy metabolizability between LA and HA in both trials. Dietary substituting AH for C did not impair the feed intake of the animals, but it did not improve feed efficiency. In terms of economic feasibility, low‐level AH inclusion in the diets of growing beef cattle appeared more profitable at the 1‐kg DG level as compared with CTRL and high‐level AH inclusion, and should be practiced in the drylands of Gansu Province, China.