ABSTRACT
One of the most challenging aspects of long-term research based on microorganisms is the maintenance of isolates under ex situ conditions, particularly the conservation of phytopathological characteristics. Our research group has worked for more than 10 years with Gaumannomyces graminis var. tritici (Ggt), the main biotic factor affecting wheat. In this sense we preserved the microorganisms in oil overlaid. However, several strains preserved for a long time lost their pathogenicity. These strains show white and non-infective mycelia. In this sense, we hypothesized that this is attributable to low melanin content. Melanin is a natural pigment mainly involved in UV protection, desiccation, salinity, oxidation, and fungal pathogenicity. Therefore, understanding the melanin role on Ggt pathogenicity is fundamental to developing melanin activation strategies under laboratory studies. In this study, we induce melanin activation by UV-A light chamber, 320 to 400 nm (T1) and temperature changes of 30 °C, 15 °C, and 20 °C (T2). Fungal pathogenicity was evaluated by determination of blackening roots and Ggt was quantified by real-time PCR in inoculated wheat plants. Results revealed that Ggt grown under UV-A (T1) conditions showed around 40% higher melanin level with a concomitant effect on root infection (98% of blackened roots) and 4-fold more Ggt genome copy number compared with the control (non-infective mycelia) being T1, a more inductor factor compared with T2. These findings would support the role of melanin in pathogenicity in darkly pigmented fungi such as Ggt and could serve as a basis for activating pathogenicity under laboratory conditions.
ABSTRACT
This study assesses the effect of bovine sperm (obtained from three bulls) separation using density gradients (Percoll and BoviPure) and Swim-up on sperm function and gene expression. Sperm evaluations included the plasma membrane integrity (SYBR14/PI), acrosomal integrity (PNA-FITC/PI), oxidative stress (ROS; CH2FDDA), DNA fragmentation (TUNEL assay) and mitochondrial membrane potential (ΔYm; TMRM) using flow cytometry. Sperm motility was evaluated by computer-assisted sperm analysis (CASA) and gene expression using RT-qPCR. The results showed that separation by Percoll achieves a higher proportion of sperm with intact plasma and acrosomal membranes (89.8 and 87.5%, respectively) than the unseparated control (70.3 and 62.4%, respectively), as well as by Swim-up (74.9 and 63.3%, respectively) and BoviPure (83.3 and 80.4%, respectively). No differences were observed in the proportion of spermatozoa with high ΔΨm between Percoll and BoviPure (84.3% and 83.5%, respectively), which were higher than Swim-up and the unseparated control (72.8% and 43.8%, respectively). The ROS levels were higher in the spermatozoa separated by Percoll and no differences were observed in the sperm DNA integrity between all groups. The motility analysis showed that the separation methods improve (p<0.05) total and progressive motility compared to the control, with Percoll proving the most efficient in this regard. Finally, the gene expression analysis of leptin (LEP), aromatase cytochrome P450 (CYP19) and protamine I (PRM1), after validation of 6 reference genes, showed no differences between groups. In conclusion, bovine sperm separation using density gradient improves the parameters of motility and sperm function without affecting the gene expression.
