Development of expression vectors based on pepino mosaic virus

Background  

Plant viruses are useful expression vectors because they can mount systemic infections allowing large amounts of recombinant protein to be produced rapidly in differentiated plant tissues. Pepino mosaic virus (PepMV) (genus Potexvirus, family Flexiviridae), a widespread plant virus, is a promising candidate expression vector for plants because of its high level of accumulation in its hosts and the absence of severe infection symptoms. We report here the construction of a stable and efficient expression vector for plants based on PepMV.     

In planta and soil quantification of Fusarium oxysporum f. sp. ciceris and evaluation of Fusarium wilt resistance in chickpea with a newly developed quantitative polymerase chain reaction assay

Fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris can be managed by risk assessment and use of resistant cultivars. A reliable method for the detection and quantification of F. oxysporum f. sp. ciceris in soil and chickpea tissues would contribute much to implementation of those disease management strategies. In this study, we developed a real-time quantitative polymerase chain reaction (q-PCR) protocol that allows quantifying F. oxysporum f. sp. ciceris DNA down to 1 pg in soil, as well as in the plant root and stem. Use of the q-PCR protocol allowed quantifying as low as 45 colony forming units of F. oxysporum f. sp. ciceris per gram of dry soil from a field plot infested with several races of the pathogen. Moreover, the q-PCR protocol clearly differentiated susceptible from resistant chickpea reactions to the pathogen at 15 days after sowing in artificially infested soil, as well as the degree of virulence between two F. oxysporum f. sp. ciceris races. Also, the protocol detected early asymptomatic root infections and distinguished significant differences in the level of resistance of 12 chickpea cultivars that grew in that same field plot infested with several races of the pathogen. Use of this protocol for fast, reliable, and cost-effective quantification of F. oxysporum f. sp. ciceris in asymptomatic chickpea tissues at early stages of the infection process can be of great value for chickpea breeders and for epidemiological studies in growth chambers, greenhouses and field-scale plots.     

Performance of Sheep and Goats Fed Arctostaphylos canescens With and Without Polyethylene Glycol Supplementation

Arctostaphylos canescens Eastw. is considered an important element in the chaparral fire matrix and an invasive plant in coniferous forest plantations in California. Previous studies reported that dry matter intake of Arctostaphylos was low, presumably because of its low nutritional quality and high condensed tannin (CT) content. We hypothesized that intake and digestibility of Arctostaphylos could be increased by the provision of a tannin-complexing agent polyethylene glycol (PEG). This study determined the effects of PEG (MW 4000) supplementation on intake (I) and digestibility (D) of Arctostaphylos in goats and sheep. Polyethylene glycol was added to drinking water at four levels (0.3%, 0.15%, 0.05%, and 0%) of body weight (BW). Alfalfa pellets were used as diet supplement at 1.5% of BW. Nutritional quality of Arctostaphylos was low as compared with alfalfa pellets. Arctostaphylos crude protein (CP) levels were low (4.5% vs. 17.9%) and CT concentration was high (23.1% vs. 0%), whereas estimates of in vitro organic matter digestibility (OMD, 36.6%) and metabolizable energy (5.1 MJ · kg^?1 dry matter [DM]) in Arctostaphylos were almost half of those found for alfalfa pellets (70.3% and 9.5 MJ · kg^?1 DM). A curvilinear increase (P < 0.05) in nutrient intake (per g · d^?1 and per kg BW^0.75) was observed in goats and sheep as PEG levels increased, although a linear increase (P < 0.001) was observed in CP intake (g · d^?1) of Arctostaphylos by goats. Addition of PEG curvilinearly increased (P < 0.05) digestibility of DM, CP, and neutral and acid detergent fiber, but quadratically increased (P < 0.05) that of OM in goats and sheep. Incorporation of PEG in drinking water at the level of 0.15% BW in sheep and goats was effective to maximize inactivation of CT in Arctostaphylos. However, the success in adopting this practice as a useful tool in vegetation management programs will depend on the cost–benefit ratio.     

Induction and scale-up of Billbergia zebrina nodule cluster cultures: Implications for mass propagation,improvement and conservation

The Tropical Atlantic Rain Forest is a biome of high diversity and endemism of plant genetic resources. High diversity of bromeliad species occurs in this biome, but part of them is now endangered. Tissue culture based techniques comprise valuable tools for the mass propagation and conservation of endangered bromeliads. In the present work the in vitro regenerative system based on the induction and development of NC from nodal segments of Billbergia zebrina showed different morphogenetic features from those observed in organogenesis and somatic embryogenesis. Different levels of TDZ, NAA, and 2i-P promoted the induction of NC with different morphologies and regenerative potential. NC induced in response to 0.01 μM of TDZ and subcultured on BM free of PGR resulted in high regenerative frequency. NC originated from BM supplemented with TDZ (0.1 μM) when subcultured on BM with NAA (2 μM) and 2-iP (4 μM) resulted in higher mean number of shoots. Elongated shoots were successfully acclimatized in ex vitro conditions and derived plantlets had normal phenotype. Histological analyses revealed the morphogenesis of the NC related to organization of the meristematic zone in the subepidermic region composed by organized layers with small and isodyametric cells followed by the induction of shoot buds. SEM analysis showed that the induction of NC occurred from the basal region of the explants.     

Infectious bronchitis virus in testicles and venereal transmission

Even though males represent only 8%-12% of the birds of a breeder flock, their role in infectious bronchitis virus (IBV) dissemination is largely unknown. We first assessed the effect of IBV replication in the chicken testes. Ten-week-old males were inoculated with Arkansas (Ark) or Massachusetts (Mass) IBV virulent strains. Seven days postinoculation (DPI) IBV RNA was detected in testicles of 100% of M41- and in 96% of Ark-infected males. Marginal nonsynonymous variation was detected in spike (S) gene of the predominant population of IBV replicating in the testes compared to the S gene of the predominant population of viruses prior to inoculation. IBV M41 and Ark were detected in spermatogonia and Sertoli cells of testicles of infected roosters by immunofluorescence, without evident histopathological changes. We next assessed venereal transmission of IBV by artificially inseminating 54-wk-old hens either with semen from IBV-infected roosters or with IBV suspended in na?ve semen. IBV RNA was detected in the trachea of all hens inseminated with IBV-spiked semen and in 50% of hens inseminated with semen from IBV-infected males. The egg internal and external quality was negatively affected in hens inseminated with semen containing IBV. These results provide experimental evidence for IBV venereal transmission.     

Avian influenza mucosal vaccination in chickens with replication-defective recombinant adenovirus vaccine

We evaluated protection conferred by mucosal vaccination with replication-competent adenovirus-free recombinant adenovirus expressing a codon-optimized avian influenza (AI) H5 gene from A/turkey/WI/68 (AdTW68.H5ck). Commercial, layer-type chicken groups were either singly vaccinated ocularly at 5 days of age, singly vaccinated via spray at 5 days of age, or ocularly primed at 5 days and ocularly boosted at 15 days of age. Only chickens primed and boosted via the ocular route developed AI systemic antibodies with maximum hemagglutination inhibition mean titers of 3.9 log2 at 32 days of age. In contrast, single vaccination via the ocular or spray routes maintained an antibody status similar to unvaccinated controls. All chickens (16/16) subjected to ocular priming and boosting with AdTW68.H5ck survived challenge with highly pathogenic AI virus A/chicken/Queretaro/14588-19/95 (H5N2). Single ocular vaccination resulted in 63% (10/16) of birds surviving the challenge followed by a 44% (7/16) survival of single-sprayed vaccinated birds. Birds vaccinated twice via the ocular route also showed significantly lower (P < 0.05) AI virus RNA concentrations in oropharyngeal swabs compared to unvaccinated-challenged controls.     

Regulatory factors and structure of a component population of the spionid Polydora bioccipitalis infesting the surf clam Mesodesma donacium

Spionidae, particularly polydorids, are common polychaete parasites of edible mollusks around the world. However, our understanding of the regulatory factors and population structure of these parasites is scant. In this study involving Polydora bioccipitalis and the surf clam Mesodesma donacium we evaluated (1) the environmental correlates of the prevalence and mean intensity of the infestation, (2) the relationship between the number of egg capsules and juvenile and adult parasites and the time elapsed since infestation, and (3) the spatial patterns of juveniles and adults within the host. Environmental factors showed no significant correlations with prevalence and mean intensity, suggesting that these factors do not act directly as regulators. Rather, storm surges seemingly induced clam stranding, which in turn affected both the prevalence and intensity of the infestation. The numbers of juveniles and egg capsules in blisters were significantly related to the time since infestation, suggesting mechanisms of use and expansion of the space within the host. Juvenile worms showed an aggregated distribution that was probably related to the episodic nature of infestation events, whereas adults exhibited uniform distributions that probably reflect territorial behavior and reproductive strategies.     

In vitro invasion efficiency and intracellular proliferation rate comprise virulence-related phenotypic traits of Neospora caninum

ABSTRACT: In this study, we examined the in vitro invasion and proliferation capacities of the Nc-Liv and ten Spanish Neospora caninum isolates (Nc-Spain 1 H - Nc-Spain 10). The invasion rate was determined as the number of tachyzoites that completed their internalisation into MARC-145 cells at 2, 4, and 6 h post-inoculation (pi). The proliferation rate was evaluated by determining the doubling time during the exponential proliferation period. Significant differences in the invasion rates of these isolates were detected at 2 and 4 h pi (P < 0.0001, Kruskal-Wallis test). At 4 h pi, the Nc-Spain 4 H and Nc-Liv isolates displayed the highest, while the Nc-Spain 3 H and Nc-Spain 1 H isolates had the lowest invasion rates (by Dunn's test). Variations in the proliferation kinetics of these isolates were also observed. Between different isolates, the lag phase, which occurs before the exponential growth phase, ranged from 8 to 44 h, and the doubling time ranged from 9.8 to 14.1 h (P = 0.0016, ANOVA test). Tachyzoite yield, which combines invasion and proliferation data, was also assessed and confirmed marked differences between the highly and less prolific isolates. Interestingly, a direct correlation between the invasion rates and tachyzoite yields, and the severity of the disease that was exhibited by infected pregnant mice in previous works could be established for the isolates in this study (Spearman's coefficient > 0.62, P < 0.05). The results of this study may help us to explain the differences in the pathogenicity that are displayed by different isolates.     

Strawberry recovers from iron chlorosis after foliar application of a grass‐clipping extract

Bare‐root transplants of strawberry (Fragaria × ananassa Duch. cv. Selva) were transferred to nutrient solutions with or without iron. After 35 d of growth, plants in the solution without iron became chlorotic and had morphological changes in roots typical of iron‐deficiency chlorosis (IDC). Acidification of the nutrient solution was also observed. We tested a grass‐clipping extract to correct IDC in strawberry plants by foliar application to some chlorotic plants. We also assessed the effects of this product on plant growth, Fe allocation, as well as morphological and physiological parameters related with IDC. After the second spray, leaf chlorophyll increased in the youngest expanded leaves. The total content of iron in plants increased from 1.93 mg to 2.37 mg per plant after three sprays, accounting for 80% of the total iron supplied by the extract. Newly formed roots from sprayed plants had a normal morphology (no subapical swollen zone) but a higher ferric chelate–reductase (FC‐R; EC 1.16.1.17) activity per root apex compared with roots from plants grown with iron or untreated chlorotic plants. Acidification of the nutrient solution continued in sprayed recovered plants. The results suggest an uncoupling of the regulation of morphological and physiological mechanisms related to IDC: FC‐R activity seems to be controlled by roots on their own or together with shoots, while morphological changes in roots are apparently regulated only by the level of iron in shoots.     

Mobilisation of AS and trace metals in saline,acidic Sopolic Technosols: the role of the rhizosphere and flooding conditions

Purpose  

The aim of this work was to study the risk of As and metal pollution in saline wetlands affected by slightly acidic mine wastes, in the presence or absence of a plant rhizosphere, under different flooding regimes. Some guidelines for management will be proposed in order to minimise the risk of metal leaching in metal-polluted salt marshes.