Development of a TaqMan allelic discrimination assay for the distinction of two major subtypes of the grapevine yellows phytoplasma Bois noir

Bois noir is a grapevine yellows disease that is gaining importance in many regions of Europe. The Bois noir phytoplasma (“Candidatus Phytoplasma solani”) was shown to be transmitted by the planthopper Hyalesthes obsoletus, which normally feeds on herbaceous weeds, and occasionally also on grapevines. Three subtypes of the Bois noir phytoplasma have been described and were shown to be associated with distinctive host plants. In this study, we developed a novel and rapid real-time PCR allelic discrimination assay for the distinction of the two major Bois noir phytoplasma subtypes, VK type I and II. Two TaqMan probes carrying different fluorescent dyes were designed to specifically bind to a polymorphism characteristic for the two Bois noir phytoplasma subtypes, thereby allowing discriminative amplification in a single-tube and single-step assay. A total of 259 bois noir-positive grapevine samples collected over 5 years were analysed using the conventional PCR-RFLP method and our newly developed TaqMan allelic discrimination assay. 257 out of 259 samples could be typed with the TaqMan method, compared to 200 out of 259 samples when using the conventional method. The overall concordance of the two methods was 100%. Our newly developed TaqMan assay represents a useful tool for fast and reliable determination of Bois noir phytoplasma subtypes in infected grapevine, insect vector, and host plant samples. The test is suitable for high-throughput analysis and will thereby facilitate further characterisation of Bois noir epidemiology.     

Chemical composition and antioxidant and antimicrobial activity of essential oil of Artemisia annua L. from Bosnia

Hydrodistilled volatile oil obtained from the aerial parts of Artemisia annua L., cultivated near Sarajevo, Bosnia, was analyzed by GC-MS. More than one hundred compounds were identi?ed, representing 95.5% of the total oil. The major constituents of essential oil were oxygenated monoterpenes, artemisia ketone (30.7%) and camphor (15.8%). Isolated essential oil was tested for radical-scavenging ability using the stable DPPH radical, the ABTS radical, for reducing power ability with a test based on the reduction of ferric cations, for reducing ability of hydroxy radical in ORAC assay, and for metal chelating ability using the ferrozine assay. In all tests oil did not show a prominent antioxidant activity, but still comparable with thymol, an already known antioxidant. The screening of antimicrobial activity of oil was individually evaluated against representatives of Gram-positive, Gram-negative bacteria and fungi, using the agar diffusion method. All tested microorganisms were inhibited by essential oil. To the best of our knowledge, this is the first report of antimicrobial activity of essential oil of A. annua against Haemophilus influenzae, Enterococcus faecalis, Streptococcus pneumoniae, Micrococcus luteus and Candida krusei microbial strains. The antioxidant, antibacterial and antifungal activity of essential oil of A. annua from Bosnia is presented here for the first time and extends our knowledge in the range of valuable biological activities and possible roles in therapy associated with this medicinal herb.     

Effect of recombination in the maize breeding population with exotic germplasm on the yield stability

A little knowledge exists about the probability that recombination in the parental maize populations will enhance the chances to select more stable genotypes. The synthetic parent maize population ((1601/5 × ZPL913)F₂ = R₀) with 25% of exotic germplasm was used to assess: (i) genotype × environment interaction and estimate stability of genotypes using nonparametric statistics; (ii) the effect of three (R₃) and five (R₅) gene recombination cycles on yield stability of genotypes; (iii) relationship among different nonparametric stability measures. The increase of mean grain yield was significant (P < 0.01) in the R₃ and R₅ in comparison to the R₀, while it was not significant between R₃ and R₅. Analysis of variance showed significant (P < 0.01) effects of environments, families per set, environment × set interaction, family × environment interaction per set on grain yield. The non-significant noncrossover and significant crossover (P < 0.01) G × (E) interactions were found according to Bredenkamp procedures and van der Laan-de Kroon test, respectively. The significant (P < 0.01) differences in stability were observed between R₀-set 3 and R₅-set 3 determined by S_i⁽³⁾ S_{i}^{(3)} , R₃-set 1 and R₅-set 1 determined by S_i⁽³⁾ S_{i}^{(3)} (P < 0.05), and R₀-set 3 and R₅-set 3 determined by S_i⁽⁶⁾ S_{i}^{(6)} (P < 0.05). The significant parameters were those which take into account yield and stability so the differences could be due to differences in yield rather than stability. Findings can help breeders to assume the most optimum number of supplementary gene recombination to achieve satisfactory yield mean and yield stability of maize genotypes originating from breeding populations.     

Biological properties of a naturally attenuated infectious bursal disease virus isolated from a backyard chicken flock

The biological properties of an infectious bursal disease (IBD) virus isolated from bursas collected during an outbreak in a village chicken flock in Macedonia are described. The mortality rate was 50%. Two viruses coexisted in the bursas of infected chickens (IBDVwt and IBDVtc). The virus termed IBDVtc grows on chicken embryo fibroblast (CEF) cells from the first passage. Specific pathogen free chickens inoculated with IBDVtc at passage level 4 did not develop any clinical signs of disease. Some discrete bleeding on the leg muscles was seen and the bursa of Fabricius revealed pathological lesions similar to those caused by classical strains. However, the bursa recovered quickly (bursa lesion score 2) by 14 days post infection (PI). We also found evidence of bursal repopulation by means of perinuclear antigen staining. Strong CD3 influx was evident at 4 days PI, and at 33 days PI the CD3+ cell finding was comparable to the control. The mean antibody titre was 9.2 log 2 at 14 days PI. The amino acid composition of VP2 in IBDVwt (222 Ala, 242 Ile, 253 Gln, 256 Ile, 279 Asp, 284 Ala, 294 Ile and 299 Ser) is described. The same sequence was found in IBDVtc, except for two point mutations, at Gln253→His and Ala284→Thr. Such amino acid substitution is responsible for partial attenuation and the ability of the strain to replicate in cell culture. None of the commercial vaccine viruses has a similar arrangement of amino acids in the variable domain of IBDV. This strongly suggests that IBDVtc originates from a very virulent strain. To the best of our knowledge, this is the first report of a concomitant infection of chickens with highly pathogenic IBDV and its mutant counterpart.     

Distribution of porcine circovirus 2 cap antigen in the lymphoid tissue of pigs affected by postweaning multisystemic wasting syndrome

The lymphatic organs of 50 pigs from a total of eight farms located at different sites in the epizootiological region of North Ba?ka County were studied to obtain data on the prevalence of circoviral infections in Serbia. All of the pigs examined had clinical signs suggestive of postweaning multisystemic wasting syndrome (PMWS). All pigs underwent necropsy and tissue samples were taken for histopathological, immunohistochemical (IHC) and PCR analysis. The presence of porcine circovirus 2 (PCV2) was established by PCR analysis in the organs of the pigs tested. The most frequent histopathological lesions of lymphoid tissue linked with the presence of positive immunostaining for PCV2 Cap antigen confirmed the existence of PMWS in all farms tested in North Ba?ka County. Using PCR, histopathological and IHC techniques, the presence of PMWS was proved in the Republic of Serbia. During necropsy, generalised enlargement of the lymph nodes was evident. The most common histopathological finding was lymphocyte depletion in the follicular and perifollicular areas of lymph nodes. Infiltration by macrophages was also recorded. By IHC analysis, the cytoplasm of macrophages was shown to contain a large amount of the ORF2-coded Cap antigen of PCV2. Lymphocyte depletion and large numbers of macrophages were recorded in the tonsils, spleen, intestinal lymphatic tissue, Peyer's patches and ileocaecal valve. The presence of typical granulomatous lesions with multinuclear giant cells (MGCs) was also recorded in the lymphatic tissue. Cap antigen was shown to be present in macrophages and less often in lymphocytes.     

Does the Intensity of Anisakis Infection Affect the Quality of Hake Muscle?

The distribution of Anisakis larvae in fresh caught hake was studied, and the influence of three infection levels of the viscera (low, medium, high) on quality changes of deep frozen muscle was followed over 220 days. Differences in quality were attributed more to the size of the individuals than to the infection degree of the three groups. The removal of belly flaps diminished the risk of consumers’ infection and sensitization as prevalence and intensity was reduced to 75.6 and 5.9%, respectively. Allergens were detected after 220 days frozen storage, which represents a risk for consumers already allergic to Anisakis.     

Dietary Elements and Quality Parameters of 34 Old and Eight Commercial Apple Cultivars Grown at the same Site in South Tyrol,Italy

Apple is one of the most widely produced and consumed fruits worldwide and hence, complete data of apple composition are important for human diet. Currently, a limited number of cultivars dominate the market, while many others, with a potentially higher nutritional value, are neglected by consumers. The present work reports the content of the dietary elements potassium (K), phosphorus (P), calcium (Ca) and magnesium (Mg) as well as the content of the macroelement nitrogen (N) of 34 old cultivars grown at the same site under identical conditions in South Tyrol, Italy. Their elemental composition was assessed along with quality parameters such as fruit weight, firmness, and soluble solid content and total acidity at harvest and post storage. For selected cultivars the measurements were performed over two or even three different harvest years. Comparison with eight commercial cultivars chosen to represent the fruit currently dominating the market was performed.Besides offering a valuable insight in the variation of dietary elements among old and commercial apple cultivars in up to three harvest years, this study, that complements current nutritional databases, recommends several old cultivars with high content of dietary elements for further study and eventual re-introduction in niche markets.