Detection of the Defoliating Pathotype of Verticillium dahliae in Infected Olive Plants by Nested PCR

Spread of Verticillium wilt into newly established olive orchards in Andalucía, southern Spain, has caused concern in the olive industry in the region. This spread may result from use of Verticillium dahliae-infected planting material, which can extend distribution of the highly virulent, defoliating (D) pathotype of V. dahliae to new areas. In this study, a molecular diagnostic method for the early in planta detection of D V. dahliae was developed, aimed especially at nursery-produced olive plants. For this purpose, new primers for nested PCR were designed by sequencing a 992-bp RAPD marker of the D pathotype. The use of the specific primers and different nested-PCR protocols allowed the detection of V. dahliae pathotype D DNA in infected root and stem tissues of young olive plants. Detection of the pathogen was effective from the very earliest moments following inoculation of olive plants with a V. dahliae pathotype D conidia suspension as well as in inoculated, though symptomless, plants.     

Induction of an antioxidant enzyme system and other oxidative stress markers associated with compatible and incompatible interactions between chickpea (Cicer arietinum L.) and Fusarium oxysporum f. sp.ciceris

To ascertain if active oxygen species play a role in fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris, the degree of lipid peroxidation (malondialdehyde formation) and the activity levels of diamine oxidase (DAO), an apoplastic H₂O₂-forming oxidase, and several antioxidant enzymes, namely ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), guaiacol-dependent peroxidase (GPX) and superoxide dismutase (SOD), were determined spectrophotometrically in roots and stems of ‘WR315’ (resistant) and ‘JG62’ (susceptible) chickpea cultivars inoculated with the highly virulent race 5 of the pathogen. Moreover, APX, CAT, GPX and SOD were also analysed in roots and stems by gel electrophoresis and activity staining; and the protein levels of APX and SOD in roots were determined by Western blotting. In roots, infection by the pathogen increased lipid peroxidation and CAT and SOD activities, although such responses occurred earlier in the incompatible compared with the compatible interactions. APX, GPX and GR activities were also increased in infected roots, but only in the compatible interaction. In stems, infection by the pathogen increased lipid peroxidation and APX, CAT, SOD and GPX activities only in the compatible interaction, and DAO activity only in the incompatible one. In general, electrophoregrams agreed with the activity levels determined spectrophotometrically and did not reveal any differences in isoenzyme patterns between cultivars or between infected and non-infected plants. Further, Western blots revealed an increase in the root protein levels of APX in the compatible interaction and in those of SOD in both compatible and incompatible interactions. In conclusion, whereas enhanced DAO activity in stems, and earlier increases in lipid peroxidation and CAT and SOD activities in roots, can be associated with resistance to fusarium wilt in chickpea, the induction of the latter three parameters in roots and stems along with that of APX, GR (only in roots) and GPX (only in stems) activities are rather more associated with the establishment of the compatible interaction.     

Distribution of alpha‐neoendorphin,ACTH (18–39) and beta‐endorphin (1–27) in the alpaca brainstem

Using an immunocytochemical technique, we have studied in the alpaca brainstem the distribution of immunoreactive structures containing prodynorphin (alpha‐neoendorphin)‐ and pro‐opiomelanocortin (adrenocorticotrophin hormone (18–39) (ACTH), beta‐endorphin (1–27))‐derived peptides. No peptidergic‐immunoreactive cell body was observed. Immunoreactive fibres were widely distributed, although in most of the brainstem nuclei the density of the peptidergic fibres was low or very low. In general, the distribution of the immunoreactive fibres containing the peptides studied was very similar. A close anatomical relationship occurred among the fibres containing alpha‐neoendorphin, ACTH or beta‐endorphin (1–27), suggesting a functional interaction among the three peptides in many of the brainstem nuclei. The number of fibres belonging to the prodynorphin system was higher than that of the pro‐opiomelanocortin system. A moderate/low density of immunoreactive fibres was observed in 65.11% (for alpha‐neoendorphin (1–27)), 18.18% (for ACTH) and 13.95% (for beta‐endorphin) of the brainstem nuclei/tracts. In the alpaca brainstem, a high density of immunoreactive fibres was not observed. The neuroanatomical distribution of the immunoreactive fibres suggests that the peptides studied are involved in auditory, motor, gastric, feeding, vigilance, stress, respiratory and cardiovascular mechanisms, taste response, sleep‐waking cycle and the control of pain transmission.     

Herbage intake of dairy cows in mixed sequential grazing with breeding ewes as followers

This study aimed to evaluate the hypothesis that mixed sequential grazing of dairy cows and breeding ewes is beneficial. During the seasons of spring–summer 2013 and autumn–winter 2013–2014, 12 (spring–summer) and 16 (autumn–winter) Holstein Friesian cows and 24 gestating (spring–summer) and lactating (autumn–winter) Pelibuey ewes grazed on six (spring–summer) and nine (autumn–winter) paddocks of alfalfa and orchard grass mixed pastures. The treatments “single species cow grazing” (CowG) and “mixed sequential grazing with ewes as followers of cows” (MixG) were evaluated, under a completely randomized design with two replicates per paddock. Herbage mass on offer (HO) and residual herbage mass (RH) were estimated by cutting samples. The estimate of herbage intake (HI) of cows was based on the use of internal and external markers; the apparent HI of ewes was calculated as the difference between HO (RH of cows) and RH. Even though HO was higher in CowG, the HI of cows was higher in MixG during spring–summer and similar in both treatments during autumn–winter, implying that in MixG the effects on the cows HI of higher alfalfa proportion and herbage accumulation rate evolving from lower residual herbage mass in the previous cycle counteracted that of a higher HO in CowG. The HI of ewes was sufficient to enable satisfactory performance as breeding ewes. Thus, the benefits of mixed sequential grazing arose from higher herbage accumulation, positive changes in botanical composition, and the achievement of sheep production without negative effects on the herbage intake of cows.     

How does water and salt stress affect the germination and initial growth of Brazilian soya bean cultivars?

The effects of water and salt stress on rate of germination and seedling growth were investigated under laboratory conditions in 46 soya bean genotypes from Central-West region of Brazil to verify how these stresses may limit crop establishment during the initial growth stage and also to identify the most tolerant genotypes to drought and salinity. Mild water and salt stresses were imposed by seed exposure to –0.20 MPa iso-osmotic solutions with polyethylene glycol—PEG 6000 (119.57 g/L) or NaCl (2.357 g/L) for 12 days at 25°C. The germination percentage, seedling length and seedling dry matter were measured, and then, salt or drought tolerance indexes were calculated. The “NS 5909 RG,” “NS 7000 IPRO,” “NS 7338IPRO,” “FPS Solimões RR,” “NS 5151 IPRO,” “SYN 13610 IPRO,” “LG 60177 IPRO,” “NS 6909 IPRO” and “BMX Desafio RR” were identified as the most drought-tolerant genotypes, whereas under salinity conditions, the genotypes “5D 615 RR,” “BMX Desafio RR,” “5D 6215 IPRO” and “BMX Ponta IPRO” were identified as tolerant. The “BMX Desafio RR” is the genotype most adapted to both stress conditions and, therefore, should be used under conditions of water shortage and excess salt in the soil at sowing time.     

Swine viscera preservation in hypersaturated salt solution after alcohol fixation as a preparation method for educational purposes

The use of live animals for educational purposes is an old practice that is still employed in teaching and research institutions. However, there are several objections to this practice, whether for ethical or humanitarian reasons. Surgical techniques teaching using anatomical pieces and/or preserved cadavers promotes greater learning efficiency, provides exercise repetition and increases the confidence and satisfaction of the students when compared to the use of live animals. The current work aimed to analyse the feasibility of using fresh swine urinary bladder and small intestines (jejunum), obtained from slaughterhouses, fixed in 99.8% ethyl alcohol (EA) and preserved in sodium chloride hypersaturated solution (SCHS) at 30%, for 7, 14 and 21 days, as an alternative method for surgical skills training (SST). Swine viscera, fixed in EA and preserved in SCHS, presented a realistic appearance, absence of odour and maintained the viable morphological characteristics during the performance of the operative techniques. Preservation solutions had low cost, were easy to acquire and did not offers risks to human health. Therefore, urinary bladders and small intestines fixed in 99.8% EA for 30 days and maintained in 30% SCHS at different periods were demonstrated as a good viable option as a preservation method for surgical skills training.     

Differentially expressed genes identified through RNA‐seq with extreme values of principal components for beef fatty acid in Nelore cattle

The aim of this study was to identify differentially expressed genes (DEG) in the Longissimus thoracis muscle of Nelore cattle related to fatty acid (FA) profile through RNA sequencing and principal component analysis (PCA). Two groups of 10 animals each were selected containing PC1 and PC2 extreme DEG values (HIGH × LOW) for each FA group. The intramuscular fat (IMF) was compared between cluster groups by ANOVA, and only the sum of monounsaturated FA (MUFA) and ω3 showed significant differences (p < .05). Interestingly, the highest percentage (95%) of phenotypic variation explained by the sum of the first two PC was observed for ω3, which also displayed the lowest number of DEG (n = 1). The lowest percentage (59%) was observed for MUFA, which also revealed the largest number of DEG (n = 66). Since only MUFA and ω3 exhibited significant differences between cluster groups, we can conclude that the differences observed for the remaining groups are not due to the percentage of IMF. Several genes that have been previously associated with meat quality and FA traits were identified as DEG in this study. The functional analysis revealed one KEGG pathway and eight GO terms as significant (p < .05), in which we highlighted the purine metabolism, glycolytic process, adenosine triphosphate binding and bone development. These results strongly contribute to the knowledge of the biological mechanisms involved in meat FA profile of Nelore cattle.     

Effects of thermal energy on extrusion characteristics,digestibility and palatability of a dry pet food for cats

The influence of specific thermal energy (STE) applications on extruder preconditioner was evaluated in a dry food for cats. In the first study, six STE applications were tested with mass temperatures of 45°C, 55°C, 65°C, 75°C, 85°C and 95°C. The extrusion parameters, starch gelatinization and kibble formation were evaluated. Diets were given to cats to evaluate digestibility, faecal characteristics and palatability. In the second experiment, three treatments were compared: low STE—a preconditioner temperature of 45°C (_LSTE); high STE—a preconditioner temperature of 95°C (_HSTE); high STE (preconditioner temperature of 95°C) combined with an increase in the mass flow rate to obtain a motor amperage similar to that of the _LSTE (_HSTE_flow). Data were analysed by polynomial contrasts (Experiment 1) or Tukey's test (Experiment 2; p < 0.05). An increase in STE reduced motor amperage, mass pressure and specific mechanical energy (SME) implementation (p < 0.001) and increased total specific energy (TSE) and mass temperature (p < 0.01). The increase in STE induced greater kibble expansion and starch gelatinization (p < 0.001). No changes in apparent nutrient digestibility or faeces characteristics were observed (p > 0.05). Lower STE and starch gelatinization induced higher butyrate and total volatile fatty acid (VFA) contents in faeces (p < 0.01). Cats showed greatest preference for the formulation with the highest STE (p < 0.01). In the second experiment, when the motor amperage was increased in the _HSTE_flow treatment to a value similar to that of the _LSTE, the mass flow rate increased 40%, and the electric energy consumption remained unchanged (p < 0.001), with gains observed for efficiency and cost. In conclusion, STE application is important for sufficient TSE implementation, enhancing kibble expansion, starch gelatinization, cat preferences for food, extruder productivity and reducing SME application. Foods with lower starch gelatinization lead to increased VFA in faeces, with possible implications for gut health.