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.     

Seroprevalence and risk factors for Neospora caninum in sheep in the state Minas Gerais, southeastern Brazil

The aim of this study was to determine the prevalence and risk factors associated with infection due to Neospora caninum in serum samples from 488 sheep originating from 63 farms in 63 municipalities distributed across eight of the twelve mesoregions of the state Minas Gerais, southeastern Brazil. For detection of N. caninum the sheep serum samples were subjected to the indirect fluorescence antibody test (IFAT ≥ 50). To identify the risk factors associated with infection due to N. caninum a questionnaire was filled out for each herd by interviewing, the individual responsible for the herd, demanding information on the general characteristics of the property. Sixty-four sheep sera (13.1%; 95% CI=10.3-16.4) presented IgG-specific anti-N. caninum antibodies with the following titers: 50 (49; 76.6%), 100 (7; 10.9%), 200 (4; 6.2%), 400 (3; 4.7%) and 800 (1; 1.6%). The prevalence of infected sheep per mesoregion ranged from 0 to 28.1%. Out of the 63 farms sampled, 31 (49.2%; 95% CI=36.4-62.1) presented at least one seropositive sheep. No significant association was found between the presence of anti-N. caninum antibodies and the risk factors evaluated on the farms, except for the mesoregion variable (p=0.004; OR=0.429; CI95%=0.182-1.008). These results indicate that there is a need for additional research to define the epidemiological importance of this parasite as a cause of reproductive problems in sheep herds in Minas Gerais.     

Effects of spent mushroom substrates and inorganic fertilizer on the characteristics of a calcareous clayey‐loam soil and lettuce production

We evaluated the effects of the addition of two types of spent mushroom substrate (SMS), SMS from an Agaricus bisporus crop (SMS1) and a mixture of SMSs from an A. bisporus crop and a Pleurotus crop (50% v/v each) (SMS2), on the characteristics of a calcareous clayey‐loam soil and the yield and nutritional status of lettuce (Lactuca sativa L.), relative to crops receiving mineral fertilizer (M) and a control (C) without amendment. The application of SMS, especially SMS1, improved soil fertility compared with C and M soils. Moreover, the use of these organic substrates as soil amendments did not harm the plants and gave yields similar to that obtained with mineral fertilizer. The nutritional contents of the lettuce plants were greater than or similar to those of the plants from treatment C or M, except for the plant tissue concentrations of K, Fe and Zn, which were significantly reduced by SMS application. However, this latter fact did not reduce the lettuce yield in the amended soils. Therefore, the use of SMSs as organic amendments contributes to residue utilization, in an environmentally friendly way, and to improved soil fertility and crop yield.     

Effect of Caffeine Added to the Activating Solution on Sperm Motility of Fresh and Thawed Semen of Pacu,Piaractus mesopotamicus,and Curimba,Prochilodus lineatus

The aim was to evaluate the effect of different concentrations of caffeine added in activating solution over sperm motility in fresh and thawed semen of pacu, Piaractus mesopotamicus, and curimba, Prochilodus lineatus. The activating solutions were prepared with sodium bicarbonate solution of 0.76% (NaHCO₃) and caffeine was added at concentrations of 2.5, 5.0, 10.0, and 20.0 mM. As control, a solution of NaHCO₃ 0.76 without caffeine was used. Eight males of pacu and 20 males of curimba were used. Aliquots of 200 μL of semen were diluted in 800 μL extender solution (DMSO 10% and BTS 5%), placed in 0.5 mL straws and cryopreserved for 7 d in a liquid nitrogen tank. There was a linear increase in sperm motility for fresh semen of pacu, and for curimba fresh and thawed semen (P < 0.05), due to the increase in the concentration of caffeine. There was a quadratic response for duration of motility for thawed semen of pacu and for fresh semen of curimba (P < 0.05), respectively. These results indicate that addition of caffeine in the activator solution can improve sperm motility parameters, however, is dependent on the species and concentration used.     

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.     

Effects of vaccination against brucellosis and clostridia on the intake,performance, feeding behavior,blood parameters,and immune responses of dairy heifers calves

The aim of this study was to identify possible effects of different vaccination strategies (concomitantly or not) against brucellosis and clostridia on intake, performance, feeding behavior, blood parameters, and immune responses of dairy heifers calves. Fifty heifers calves were enrolled [38 Gyr (Zebu, Bos taurus indicus) and 12 5/8 Holstein Gyr]. At 120 d of age, animals were randomly distributed among 3 groups: B (n = 18), vaccinated against brucellosis; C (n = 14), vaccinated against clostridia and CB (n = 18), vaccinated concomitantly for both. Rectal and thermographic temperatures were evaluated on days 1, 0, 1, 2, 3, 5, 7,10, 14, and 28 relatives to the vaccination day. Feed and water intake, body weight (BW), and feeding behavior were monitored daily by an electronic feeding system. Blood was sampled on days 0, 3, 7, 14, and 28, relative to the vaccination day for determination of glucose and -hydroxybutyrate (BHBA) concentrations. Blood sampled on day 0 (prevaccination) and on days 28 and 42 were used to evaluate the immune response against Brucella abortus and clostridia. There was an increase in rectal temperature between the first and the third day postvaccination in the 3 groups. The thermography revealed an increase of local temperature for 7 d on groups B and CB. Group C had increased local temperature for a longer period, lasting for up to 14 d. Dry mater intake was reduced for groups B and CB, but no alteration was observed for group C. No alterations regarding initial BW, final BW, average daily weight gain, and feed efficiency were observed. No differences were observed for the 3 vaccination groups for blood parameters throughout the evaluation period. The concomitant vaccination against brucellosis and clostridia led to lower neutralizing antibody titers against epsilon toxin of Clostridium perfringens and botulinum toxin type C of C. botulinum (C > CB > B). When cellular proliferation assay and serological tests to B. abortus were evaluated, no differences were observed between groups B and CB. The present results indicate that the concomitant vaccination against brucellosis and clostridia has no relevant impact on the intake, performance, and feeding behavior of dairy calves. However, the concomitant vaccination of vaccines against these 2 pathogens impacts animal immunity against clostridial infections.