Sex ratio determination in bovine semen: a new approach by quantitative real time PCR

Sex preselection of livestock offspring in cattle represents, nowadays, a big potential for genetic improvement and market demand satisfaction. Sperm sorting by flow cytometer provides a powerful tool for artificial insemination and production of predefined sexed embryos but, an accurate verification of the yield of sperm separation remains essential for a field application of this technique or for improvement and validation of other related semen sexing technologies. In this work a new method for the determination of the proportion of X- and Y-bearing spermatozoa in bovine semen sample was developed by real time PCR. Two sets of primers and internal TaqMan probes were designed on specific X- and Y-chromosome genes. To allow a direct quantification, a standard reference was established using two plasmid cDNA clones (ratio 1:1) for the specific gene targets. The method was validated by a series of accuracy, repeatability and reproducibility assays and by testing two sets of sorted and unsorted semen samples. A high degree of accuracy (98.9%), repeatability (CV=2.58%) and reproducibility (CV=2.57%) was shown. The results of X- and Y-sorted semen samples analysed by real time PCR and by flow cytometric reanalysis showed no significant difference (P>0.05). The evaluation of X-chromosome bearing sperms content in unsorted samples showed an average of 51.11+/-0.56% for ejaculates and 50.17+/-0.58% for the commercial semen. This new method for quantification of the sexual chromosome content in spermatozoa demonstrated to be rapid and reliable, providing a valid support to the sperm sexing technologies.     

Emerging new crown symptoms on Castanea sativa (Mill.): Attempting to model interactions among pests and fungal pathogens

In the 2015–2016 growing seasons, two novel symptoms were assessed on the crown of trees in orchards and coppices of chestnut groves in Central Italy. The first symptom was flagging of annual shoots with green leaves undergoing sudden wilt and turning brown later in the season. The second symptom consisted of leaves on annual shoots turning yellow before wilting in absence of flagging represented the second symptom. Samples were collected along transects in early summer, late summer and winter, and processed in the laboratory. The flagging symptom was associated in early summer with the presence of C. parasitica in cryptic dried buds on stems from the previous year's growth. The pathogen was also found in dormant buds in winter, suggesting that the infection could take place in summer during the Chinese gall wasp oviposition period. Cryphonectria parasitica was also isolated from abandoned galls in winter supporting the hypothesis that galls are a potential source of inoculum for crown infections. Aetiology of yellowing was not clarified and no fungal taxa were specifically associated with this symptom. Gnomoniopsis castanea, C. parasitica and, in early summer, Colletotrichum acutatum were the most abundant fungal taxa isolated from chestnut shoots and buds.     

The fungal community associated with the ambrosia beetle Xylosandrus compactus invading the mediterranean maquis in central Italy reveals high biodiversity and suggests environmental acquisitions

In summer 2016 a severe infestation of the alien ambrosia beetle Xylosandrus compactus was recorded from the Mediterranean maquis in the Circeo National Park in Central Italy. Trees and shrubs were infested and displayed wilting and necrosis of terminal branches caused by the combined impact of the insect and associated pathogenic fungi. A preliminary screening carried out on captured adults resulted in the isolation of a discrete number of fungal taxa with different life strategies, ranging from true mutualist (e.g. Ambrosiella xylebori) to plant pathogens (Fusarium spp.). In the present study, high-throughput sequencing was applied to determine the total diversity and functionality of the fungal community associated with X. compactus adults collected in the galleries of three Mediterranean woody hosts, Quercus ilex, Laurus nobilis, and Ceratonia siliqua. The effect of season and host in determining the composition of the associated fungal community was investigated. A total of 206 OTUs composed the fungal community associated with X. compactus. Eighteen OTUs were shared among the three hosts, including A. xylebori and members of the Fusarium solani complex. All but two were previously associated with beetles.Sixty-nine out of 206 OTUs were resolved to species level, identifying 60 different fungal species, 22 of which already reported in the literature as associated with beetles or other insects. Functional guild assigned most of the fungal species to saprotrophs and plant pathogens. Effects of seasonality and host on fungal community assemblage were highlighted suggesting the acquisition by the insect of new fungal taxa during the invasion process. The consequences of enriched fungal community on the risk of the insurgence of novel threatful insect–fungus association are discussed considering direct and indirect effects on the invaded habitat.     

Plant Growth‐Promoting Bacteria from Solarized Soil with the Ability to Protect Melon Against Root Rot and Vine Decline Caused by Monosporascus cannonballus

This study was undertaken to isolate indigenous plant growth‐promoting (PGP) bacteria from solarized soil effective in the biocontrol of Monosporascus cannonballus, the cause of root rot and vine decline of melon, which is one of the most destructive soilborne diseases of this crop worldwide. The screening strategy resulted in the selection of two interesting PGP bacteria as biocontrol candidates against M. cannonballus belonging to the same microbial community. The two bacterial species, identified according to phenotypic, physiological tests and analysis of the 16S rDNA sequence as Bacillus subtilis/amyloliquefaciens (BsCR) and Pseudomonas putida (PpF4), showed PGP traits and in vitro antagonistic activity towards M. cannonballus. Antagonism by BsCR was characterized by a consistent inhibition of the pathogen in vitro growth; PpF4 strongly inhibited the development of perithecia of the pathogen. Under greenhouse conditions, the selected bacteria were tested for their biocontrol activity in the pathosystem melon‐M. cannonballus. BsCR alone and in combination with PpF4 determined a consistent decrease in the disease symptoms. BsCR and the combination of the bacterial strains significantly increased root biomass in both inoculated and un‐inoculated plant. Upon seed treatment with BsCR, the accumulation and isoenzyme induction of peroxidase in roots as biochemical marker for induction of resistance were found, thus indicating that BsCR may reduce the disease severity also by the activation of the plant defence responses. The study highlights the synergistic biocontrol potential of B. subtilis BsCR and P. putida PpF4 in the integrated management of root rot and vine decline of melon caused by M. cannonballus.     

Plasma estrone sulfate,clinical biochemistry,and milk yield of dairy cows carrying a fetus from a bull or its clone

The aim of this article was to compare plasma estrone sulfate (E₁SO₄), clinical biochemistry, and milk yield of dairy cows carrying a female fetus from a bull (BULL) or from its clone (CLONE), evaluating also the relationship between the former variables and the birth weight of the newborn. Sixteen recipient dairy Friesian heifers (10 BULL and 7 CLONE) received a female embryo, obtained by in vitro embryo production and sexing by polymerase chain reaction with the semen of the BULL or the CLONE. Blood samples on all cows were obtained before feed distribution in the morning from jugular vein from 4 weeks before to 4 weeks after calving, to be analyzed for metabolic profile. The samples from late gestation were also analyzed for E₁SO₄ concentration. To separately assess the effect of calf birth weight (CBW), data were categorized as follows: low (<39 kg; BWT-A), mid (39–46 kg; BWT-B), and high (>46 kg; BWT-C). The plasma concentrations of β-hydroxybutyric acid (BHB, P = 0.019), Na (P = 0.002), Cl (P = 0.026), strong cation–anion balance (P = 0.020), total bilirubin (P = 0.054), and α₁-globulin (P = 0.044) were higher in prepartum BULL recipients than those in CLONE, whereas BHB (P = 0.021) and Mg (P = 0.090) were higher in postpartum BULL recipients, while no differences were recorded in the remaining postpartum parameters. The CBW class had significant interaction with week of gestation on antepartum plasma estrone sulfate (P = 0.021), whereas CBW per se affected antepartum plasma BHB (P = 0.021), and nonesterified fatty acids (NEFA; P = 0.011) being higher in BWT-C which also had the lower NEFA concentration during postpartum. Milk yield was unaffected by the sire used, both for quantitative and qualitative aspects. Cows carrying heavier fetus (BWT-C) had a different lactation affected by month compared with the other 2 CBW groups. From these results, there were no differences between BULL and CLONE recipients. Estrone sulfate, BHB, and NEFA may be used to predict CBW and provide different nutritional management during gestation.     

Body growth, hematological profile, and clinical biochemistry of heifer calves sired by a bull or its clone

The aim of this paper was to compare body growth, hematological profile development, and clinical biochemistry in the female progeny of a sire with the female progeny of its clone. Sixteen Friesian female calves, 9 daughters from a tested bull (BULL) and 7 from its somatic cell nuclear transfer clone (CLONE) were monitored from birth to 60 wk of life. Body weight (BW), wither height (WH), hip height (HH), body length (BL), and hearth girth (HG) were measured at birth and 4, 8, 12, 16, 20, 24, 36, and 50 wk. Blood samples were taken from jugular vein at 12 to 48 h from birth and 1, 2, 3, 4, 8, 12, 16, 20, 24, and 36 wks of age, to be analyzed for hematological, serum protein, and metabolic profiles. At the same time, rectal temperature (RT) was recorded. Age at puberty was assessed on surviving heifers by measuring weekly plasma progesterone levels. Data were evaluated using a mixed model, taking into account the repeated measures in time on the calf. For each variable, different covariance structures were tested, choosing the best according to the Akaike's Information Criteria. Significant was set at P < 0.05, and a trend was considered for P < 0.10. At 24 wk of age, WH was lower in CLONE daughters than BULL daughters. Around 20 wk of age, there was a trend for lower BW in CLONE daughters than BULL daughters, confirmed from differences in HG. There was no difference in RT due to sire effect. Blood glucose concentration decreased in both groups during the first 4 wk of life; at birth, only a trend for higher blood glucose in CLONE daughters was recorded, whereas an opposite trend was observed for plasma creatinine. Total leukocyte count did not differ between progenies. Circulating lymphocytes tended to be lower in CLONE than BULL daughters. The neutrophil: lymphocyte ratio tended to be higher in CLONE than BULL calves. No difference was demonstrated for erythrocyte features, whereas mean platelet volume tended to be lower in CLONE than BULL progeny. From these results, there were no differences between progenies from BULL and its clone that suggest welfare problems in the first 6 mo of life.     

Assessment of suitability and suppressiveness of on-farm green compost as a substitute of peat in the production of lavender plants

Suppression of root rot diseases of ornamental plants is a potential benefit of formulating soilless container media with compost. A green compost (green nursery compost, GNC), obtained by a circular-economy approach from residues of pruning of woody plants and grass clippings during the nursery activities was analysed for its suppressiveness of root rot diseases using lavender. To this end, a bioassay was develop by formulating potting mixes containing GNC with two rates of peat substitution (25% and 50%) and infested with the root rot pathogens Sclerotinia sclerotiorum, Rhizoctonia solani and Phytophthora nicotianae. Contrasting results were obtained by using both substrates with a significant reduction of root rot by S. sclerotiorum, no effect on the containment of that by P. nicotianae, and an increase of symptoms caused by R. solani. The specific suppressiveness observed may be attributed to the colonisation of compost by specific groups of antagonistic microorganisms. This hypothesis was investigated by the analysis of culturable fungal community, which resulted in the isolation of Trichoderma harzianum and T. atroviride as preponderant fungal species. Trichoderma representative isolates exerted in vitro antagonistic activities against the target pathogens with varying efficiencies indicating the employment of multiple complementary mechanisms, which may have contributed to the observed specific suppressiveness. Both substrates containing GNC resulted suitable for nursery cultivation of lavender, showing a growth performance similar to that obtained with peat-based substrate. Present results indicate that on-farm compost is a suitable component of mixed-peat substrates capable to support plant growth and provide specific disease suppression.     

Induced Resistance by β‐Aminobutyric Acid in Artichoke against White Mould Caused by Sclerotinia sclerotiorum

β‐aminobutyric acid (BABA) was assessed for the ability to protect two artichoke cultivars, C3 and Exploter, against white mould caused by Sclerotinia sclerotiorum, which represents a major problem in the cultivation of this crop in many growing areas of Central Italy. Changes in the activity and isoenzymatic profiles of the pathogenesis‐related (PR) proteins β‐1,3‐glucanase, chitinase and peroxidase in plantlets upon BABA treatment and following inoculation of the pathogen in plantlets and leaves detached from adult plants were also investigated as molecular markers of induced resistance and priming. BABA treatments by soil drenching induced a high level of resistance against S. sclerotiorum in artichoke plantlets of both cultivars C3 and Exploter with a similar level of protection and determined a consistent increase in peroxidase activity paralleled with the differential induction of alkaline isoenzyme with a pI 8.6. A consistent change was found in Exploter in the peroxidase activity following BABA treatments and pathogen inoculation and was paralleled with the expression of an anionic band in plantlets and both anionic and cationic bands in leaves. Our results showed a correlation between BABA‐induced resistance (BABA‐IR) and a augmented capacity to express basal defence responses, more pronounced in cultivar C3 and associated β‐1,3‐glucanase accumulation in both plantlets and leaves inoculated with the pathogen, whereas chitinase resulted affected only at plantlet stage. The present results represent the first one showing the effect of BABA in inducing resistance in artichoke and associated accumulation of selected PRs. If confirmed in field tests, the use of BABA at early plant stages may represent a promising approach to the control soilborne pathogens, such as the early infection of S. sclerotiorum.     

Lower HIV Provirus Levels Are Associated with More APOBEC3G Protein in Blood Resting Memory CD4+ T Lymphocytes of Controllers In Vivo

Immunodeficiency does not progress for prolonged periods in some HLA B57- and/or B27-positive subjects with human immunodeficiency virus type 1 (HIV) infection, even in the absence of antiretroviral therapy (ART). These “controllers” have fewer HIV provirus-containing peripheral blood mononuclear cells than “non-controller” subjects, but lymphocytes that harbor latent proviruses were not specifically examined in studies to date. Provirus levels in resting memory cells that can serve as latent reservoirs of HIV in blood were compared here between controllers and ART-suppressed non-controllers. APOBEC3G (A3G), a cellular factor that blocks provirus formation at multiple steps if not antagonized by HIV virion infectivity factor (Vif), was also studied. HLA-linked HIV control was associated with less provirus and more A3G protein in resting CD4+ T central memory (Tcm) and effector memory (Tem) lymphocytes (provirus: p = 0.01 for Tcm and p = 0.02 for Tem; A3G: p = 0.02 for Tcm and p = 0.02 for Tem). Resting memory T cells with the highest A3G protein levels (>0.5 RLU per unit of actin) had the lowest levels of provirus (<1,000 copies of DNA per million cells) in vivo (p = 0.03, Fisher''s exact test). Using two different experimental approaches, Vif-positive viruses with more A3G were found to have decreased virion infectivity ex vivo. These results raise the hypothesis that HIV control is associated with increased cellular A3G that may be packaged into Vif-positive virions to add that mode of inhibition of provirus formation to previously described adaptive immune mechanisms for HIV control.