Contrasting plant height can improve the control of rain-borne diseases in wheat cultivar mixture: modelling splash dispersal in 3-D canopies.

Background and Aims: Growing cultivars differing by their disease resistance level together (cultivar mixtures) can reduce the propagation of diseases. Although architectural characteristics of cultivars are little considered in mixture design, they could have an effect on disease, in particular through spore dispersal by rain splash, which occurs over short distances. The objective of this work was to assess the impact of plant height of wheat cultivars in mixtures on splash dispersal of Zymoseptoria tritici, which causes septoria tritici leaf blotch. Methods: We used a modelling approach involving an explicit description of canopy architecture and splash dispersal processes. The dispersal model computed raindrop interception by a virtual canopy as well as the production, transport and interception of splash droplets carrying inoculum. We designed 3-D virtual canopies composed of susceptible and resistant plants, according to field measurements at the flowering stage. In numerical experiments, we tested different heights of virtual cultivars making up binary mixtures to assess the influence of this architectural trait on dispersal patterns of spore-carrying droplets. Key Results: Inoculum interception decreased exponentially with the height relative to the main inoculum source (lower diseased leaves of susceptible plants), and little inoculum was intercepted further than 40 cm above the inoculum source. Consequently, tall plants intercepted less inoculum than smaller ones. Plants with twice the standard height intercepted 33 % less inoculum than standard height plants. In cases when the height of suscpeptible plants was doubled, inoculum interception by resistant leaves was 40 % higher. This physical barrier to spore-carrying droplet trajectories reduced inoculum interception by tall susceptible plants and was modulated by plant height differences between cultivars of a binary mixture. Conclusions: These results suggest that mixture effects on spore dispersal could be modulated by an adequate choice of architectural characteristics of cultivars. In particular, even small differences in plant height could reduce spore dispersal.

A White Paper on Global Wheat Health Based on Scenario Development and Analysis.

Scenario analysis constitutes a useful approach to synthesize knowledge and derive hypotheses in the case of complex systems that are documented with mainly qualitative or very diverse information. In this article, a framework for scenario analysis is designed and then, applied to global wheat health within a timeframe from today to 2050. Scenario analysis entails the choice of settings, the definition of scenarios of change, and the analysis of outcomes of these scenarios in the chosen settings. Three idealized agrosystems, representing a large fraction of the global diversity of wheat-based agrosystems, are considered, which represent the settings of the analysis. Several components of global changes are considered in their consequences on global wheat health: climate change and climate variability, nitrogen fertilizer use, tillage, crop rotation, pesticide use, and the deployment of host plant resistances. Each idealized agrosystem is associated with a scenario of change that considers first, a production situation and its dynamics, and second, the impacts of the evolving production situation on the evolution of crop health. Crop health is represented by six functional groups of wheat pathogens: the pathogens associated with Fusarium head blight; biotrophic fungi, Septoria-like fungi, necrotrophic fungi, soilborne pathogens, and insect-transmitted viruses. The analysis of scenario outcomes is conducted along a risk-analytical pattern, which involves risk probabilities represented by categorized probability levels of disease epidemics, and risk magnitudes represented by categorized levels of crop losses resulting from these levels of epidemics within each production situation. The results from this scenario analysis suggest an overall increase of risk probabilities and magnitudes in the three idealized agrosystems. Changes in risk probability or magnitude however vary with the agrosystem and the functional groups of pathogens. We discuss the effects of global changes on the six functional groups, in terms of their epidemiology and of the crop losses they cause. Scenario analysis enables qualitative analysis of complex systems, such as plant pathosystems that are evolving in response to global changes, including climate change and technology shifts. It also provides a useful framework for quantitative simulation modeling analysis for plant disease epidemiology.

Establishment and characterization of a new cell line (SSP-9) derived from Atlantic salmon Salmo salar that expresses type I ifn.

In the present work, the establishment and biological characterization of a new cell line, SSP-9, derived from the pronephros of the Atlantic salmon Salmo salar, are reported. These cells grew well in Leibovitz's (L15) medium supplemented with 10% foetal calf serum at temperatures from 15 to 25° C, and they have been sub-cultured over 100 passages to produce a continuous cell line with an epithelial-like morphology. The SSP-9 cells attached and spread efficiently at different plating densities, retaining 80% of cell viability after storage in liquid nitrogen. When karyotyped, the cells had 40-52 chromosomes, with a modal number of 48. Viral susceptibility tests showed that SSP-9 cells were susceptible to infectious pancreatic necrosis virus and infectious haematopoietic necrosis virus, producing infectious virus and regular cytopathic effects. Moreover, these cells could be stimulated by poly I:C, showing significant up-regulation in the expression of the genes that regulate immune responses, such as ifn and mx-1. SSP-9 cells constitutively express genes characteristic of macrophages, such as major histocompatibility complex (mhc-II) and interleukin 12b (il-12b), and flow cytometry assays confirmed that SSP-9 cells can be permanently transfected with plasmids expressing a reporter gene. Accordingly, this new cell line is apparently suitable for transgenic manipulation, and to study host cell-virus interactions and immune processes.

Using virtual 3-D plant architecture to assess fungal pathogen splash dispersal in heterogeneous canopies: a case study with cultivar mixtures and a non-specialized disease causal agent.

BACKGROUND AND AIMS: Recent developments in plant disease management have led to a growing interest in alternative strategies, such as increasing host diversity and decreasing the use of pesticides. Use of cultivar mixtures is one option, allowing the spread of plant epidemics to be slowed down. As dispersal of fungal foliar pathogens over short distances by rain-splash droplets is a major contibutor to the spread of disease, this study focused on modelling the physical mechanisms involved in dispersal of a non-specialized pathogen within heterogeneous canopies of cultivar mixtures, with the aim of optimizing host diversification at the intra-field level. METHODS: Virtual 3-D wheat-like plants (Triticum aestivum) were used to consider interactions between plant architecture and disease progression in heterogeneous canopies. A combined mechanistic and stochastic model, taking into account splash droplet dispersal and host quantitative resistance within a 3-D heterogeneous canopy, was developed. It consists of four sub-models that describe the spatial patterns of two cultivars within a complex canopy, the pathway of rain-splash droplets within this canopy, the proportion of leaf surface area impacted by dispersal via the droplets and the progression of disease severity after each dispersal event. KEY RESULTS: Different spatial organization, proportions and resistance levels of the cultivars of two-component mixtures were investigated. For the eight spatial patterns tested, the protective effect against disease was found to vary by almost 2-fold, with the greatest effect being obtained with the smallest genotype unit area, i.e. the ground area occupied by an independent unit of the host population that is genetically homogeneous. Increasing both the difference between resistance levels and the proportion of the most resistant cultivar often resulted in a greater protective effect; however, this was not observed for situations in which the most resistant of the two cultivars in the mixture had a relatively low level of resistance. CONCLUSIONS: The results show agreement with previous data obtained using experimental approaches. They demonstrate that in order to maximize the potential mixture efficiency against a splash-dispersed pathogen, optimal susceptible/resistant cultivar proportions (ranging from 1/9 to 5/5) have to be established based on host resistance levels. The results also show that taking into account dispersal processes in explicit 3-D plant canopies can be a key tool for investigating disease progression in heterogeneous canopies such as cultivar mixtures.

Antiviral activity of casein and αs2 casein hydrolysates against the infectious haematopoietic necrosis virus, a rhabdovirus from salmonid fish.

Salmonid fish viruses, such as infectious haematopoietic necrosis virus (IHNV), are responsible for serious losses in the rainbow trout and salmon-farming industries, and they have been the subject of intense research in the field of aquaculture. Thus, the aim of this work is to study the antiviral effect of milk-derived proteins as bovine caseins or casein-derived peptides at different stages during the course of IHNV infection. The results indicate that the 3-h fraction of casein and α(S2) -casein hydrolysates reduced the yield of infectious IHNV in a dose-dependent manner and impaired the production of IHNV-specific antigens. Hydrolysates of total casein and α(S2) -casein target the initial and later stages of viral infection, as demonstrated by the reduction in the infective titre observed throughout multiple stages and cycles. In vivo, more than 50% protection was observed in the casein-treated fish, and the kidney sections exhibited none of the histopathological characteristics of IHNV infection. The active fractions from casein were identified, as well as one of the individual IHNV-inhibiting peptides. Further studies will be required to determine which other peptides possess this activity. These findings provide a basis for future investigations on the efficacy of these compounds in treating other viral diseases in farmed fish and to elucidate the underlying molecular mechanisms of action. However, the present results provide convincing evidence in support of a role for several milk casein fractions as suitable candidates to prevent and treat some fish viral infections.

Immune responses elicited in rainbow trout through the administration of infectious pancreatic necrosis virus-like particles.

Virus like particles (VLPs) against viral pathogens not only constitute a novel approach for the development of antiviral vaccines for an specific virus, but also for the creation of multivalent vaccines in which antigens from other pathogens may be expressed on the surface of these VLPs. Despite positive results on protection for many of these VLPs in both fish and mammals, not many studies have focused on the immune response triggered by these particles; studies that may provide hints for the identification of immune mechanisms responsible for antiviral protection, which are mostly unknown in fish. In the current work, we have studied the levels of transcription of several immune genes in the spleen of rainbow trout (Oncorhynchus mykiss) intraperitoneally injected with VLPs from infectious pancreatic necrosis virus (IPNV) focusing on the chemokine response as well as the response of genes related to interferon (IFN) production. Surprisingly, the capacity of VLPs to induce chemokines differed from that of live IPNV, suggesting a direct effect of viral replication on the chemokine response in this organ. While VLPs up-regulated the transcription of CK3, CK10 and CXCd and down-modulated CK5B, CK6 and CK9 transcription, a previous study in which the transcription of γIP, CXCd, CK1, CK3, CK5B, CK6, CK7A, CK9 and CK12 had been studied demonstrated that IPNV only significantly up-regulated CK6 and down-modulated CK3 in the spleen. On the other hand, the administration of VLPs produced a strong mobilization to the peritoneum of CD4(+), IgM(+), IgT(+) and CD83(+) leukocytes similar to that induced by the live viral infection. In both cases, this leukocyte recruitment seemed to be greatly mediated through CK3, CK5B, CK9 and CK10 chemokine production. These results together with the fact that VLPs strongly induced non-specific lymphocyte proliferation and specific anti-IPNV antibody production point to VLPs as excellent candidates for vaccine development.

An active DNA vaccine against infectious pancreatic necrosis virus (IPNV) with a different mode of action than fish rhabdovirus DNA vaccines.

Although there are some commercial vaccines available against infectious pancreatic necrosis virus (IPNV), the disease still continues to be a major problem for aquaculture development worldwide. In the current work, we constructed a DNA vaccine against IPNV (pIPNV-PP) by cloning the long open reading frame of the polyprotein encoded by the viral RNA segment A. In vitro, the vaccine is properly translated giving the functional IPNV polyprotein since preVP2, VP2 and VP3 proteins were detected because of the VP4-protease cleavage. EPC cells transfected with the vaccine plasmid expressed the viral proteins and induced the expression of type I interferon (IFN)-induced Mx genes. Furthermore, IPNV synthesized proteins seemed to assemble in virus-like particles as evidenced by electron microscopy. In vivo, rainbow trout specimens were intramuscularly injected with the vaccine and expression of immune-relevant genes, the presence of neutralizing antibodies and effect on viral load was determined. The pIPNV-PP vaccine was expressed at the injection site and up-regulated MHC Ialpha, MHC IIalpha, type-I interferon (IFN), Mx, CD4 and CD8alpha gene expression in the muscle, head kidney or spleen, although to a much lower extent than the up-regulations observed in response to an effective DNA vaccine against viral hemorrhagic septicaemia virus (VHSV). However, the IPNV vaccine was also very effective in terms of acquired immunity since it elicited neutralizing antibodies (in 6 out of 8 trout fingerlings) and decreased 665-fold the viral load after IPNV infection. The effectiveness of this new IPNV DNA vaccine and its possible mechanism of action are discussed and compared to other viral vaccines.

Immunogenic and protective effects of an oral DNA vaccine against infectious pancreatic necrosis virus in fish.

DNA vaccines and oral DNA-based immunotherapy against infectious pancreatic necrosis virus (IPNV) have scarcely been studied in salmonid fish. Here, a vector with the capsid VP2 gene inserted was encapsulated in alginate microspheres to avoid the aggressive gastrointestinal conditions experienced following oral administration. Alginate microspheres were effective to protect the pDNA encoding VP2, which was expressed early in different organs of the vaccinated trout and that persisted for at least 60 days. The vaccine induces innate immune responses, raising the expression of IFN more than 10-fold relative to the fish vaccinated with the empty plasmid, at 7 and 15 days post-vaccination. Likewise, maximal expression of the IFN-induced antiviral Mx protein was recorded 15 days post-vaccination and neutralizing antibodies were also detected after 15 days, although their titre rose further at 21 days post-vaccination. Protection was high in the immunized fish, which showed around an 80% relative survival when challenged 15 and 30 days after vaccine delivery. Very low viral load with respect to the control group was detected in the vaccinated fish that survived 45 days after challenge. Thus, this study demonstrates the potential of the encapsulation technique for IPNV-DNA vaccine delivery and the relevance of the IPNV-VP2 gene for future plasmid constructs.

Salmonid fish viruses and cell interactions at early steps of the infective cycle.

A flow cytometric virus-binding assay that directly visualizes the binding and entry of infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) and virus haemorrhagic septicaemia virus (VHSV) to several cell lines was established. The highest efficiency of binding was shown by the BF-2 cell line and this was used to study, at the attachment level, the interactions of these cells with salmonid fish viruses in coinfections, and to further determine if the earliest stage of the viral growth cycle could explain the previously described loss of infectivity of IHNV when IPNV is present. Our results demonstrated that IPNV binds to around 88% of cells either in single or dual infections, whereas IHNV attachment always decreased in the presence of any of the other viruses. VHSV binding was not affected by IPNV, but coinfection with IHNV reduced the percentage of virus-binding cells, which suggests competition for viral receptors or co-receptors. Internalization of the adsorbed IHNV was not decreased by coinfection with IPNV, so the hypothetical competence could be restricted to the binding step. Treatment of the cells with antiviral agents, such as amantadine or chloroquine, did not affect the binding of IPNV and VHSV, but reduced IHNV binding by more than 30%. Tributylamine affected viral binding of the three viruses to different degrees and inhibited IPNV or IHNV entry in a large percentage of cells treated for 30 min. Tributylamine also inhibited IHNV cytopathic effects in a dose-dependent manner, decreasing the virus yield by 4 log of the 50% endpoint titre, at 10 mm concentration. IPNV was also inhibited, but at a lower level. The results of this study support the hypothesis that IHNV, in contrast to VHSV or IPNV, is less efficient at completing its growth cycle in cells with a simultaneous infection with IPNV. It can be affected at several stages of viral infection and is more sensitive to the action of antiviral compounds.

Detection of lymphocystis disease virus (LCDV) in asymptomatic cultured gilt-head seabream (Sparus aurata, L.) using an immunoblot technique.

An immunoblot technique for the detection of lymphocystis disease virus (LCDV) in naturally infected gilt-head seabream (Sparus aurata, L.) has been developed. A specific antiserum against a 60 kDa viral protein has been proven to be an appropriate tool for LCDV diagnosis either from inoculated cell cultures or from fish tissues using the immunoblot assay. The sensitivity of this technique varied between 10(-1) and 10(2) TCID50. LCDV has also been detected in fish tissues from both, diseased and asymptomatic gilt-head seabream. For the asymptomatic fish detection, a viral amplification step in cell culture and a subsequent viral concentration using polyethylene glycol (PEG) (600 wt) are required. On the contrary, immunoblot allowed the detection of LCDV antigens directly from tissue homogenates of diseased fish. The method described in this study shows higher sensitivity than classical detection techniques based on cell culture inoculation.

Study of the reactivity and in vitro bioactivity of Sr-substituted alpha-TCP cements.

In this study the effect of strontium substitution on the hydrolysis of alpha -tricalcium phosphate (alpha-TCP) toward the formation of calcium deficient hydroxyapatite (CDHA) was investigated. For that purpose substituted alpha-TCP powders with 1, 5 and 10 mol% Sr substitution for Ca were synthesized by reacting at 1500 degrees C stoichiometric amounts of CaCO(3), SrCO(3), and Ca(2)P(2)O(7), followed by rapid quenching in air. XRD analysis of the powders revealed the presence of alpha-TCP (traces of beta-TCP) with enlarged unit cell volume at increased Sr contents, indicating the incorporation of Sr in the crystal structure. Strontium was also incorporated in the apatite phase as revealed by XRD analysis of the set cements. The hydrolysis of milled alpha-SrTCP powders and a pure alpha-TCP (control) was monitored by isothermal calorimetry and the compressive strength of set cements was tested. The results showed a decrease in the reactivity with increasing Sr content and similar final mechanical strength within the Sr series, though lower than the control. The in vitro bioactivity of the set cements after soaking in simulated body fluid for 4 weeks was also tested. The formation of a bone-like apatite layer on the surface of the set cements indicated a potential in vivo bioactivity.

Hydration characteristics of alpha-tricalcium phosphates: Comparison of preparation routes.

Alpha tricalcium phosphate ( á -TCP) cement powders were obtained by solid state reaction and milling (M1) and by precipitation from aqueous solution followed by heating (M2). The materials were hydrated to form calcium-deficient hydroxyapatite (CDHA) with a 2.5 wt% solution of Na2 HPO4 (liquid to powder ratio = 0.34 ml/g, temperature = 37.5 degrees C) and subjected to isothermal calorimetry, mechanical compression tests, X-ray powder diffraction, at various times during hydration, as well as scanning electron microscopy (SEM), laser diffraction and gas adsorption. The particle characteristics of the two powders were similar, but M2 exhibited two reaction events in the thermal power curve, while M1 showed a single event. Both reaction events were attributed to á -TCP dissolution and CDHA recipitation. The minimum in the reaction rate response of M2 was probably due to the formation of a passivating product layer. No such layer was formed on the milled M1 due to its rougher surfaces. Both preparations reached a compressive strength of 30-40 MPa after 24 hr.

Virulence of infectious hematopoietic necrosis virus and Infectious pancreatic necrosis virus coinfection in rainbow trout ( Oncorhynchus mykiss) and nucleotide sequence analysis of the IHNV glycoprotein gene.

The outcomes of a coinfection of rainbow trout ( Oncorhynchus mykiss) with Infectious hematopoietic necrosis virus (IHNV) strain S46 and Infectious pancreatic necrosis virus (IPNV) strain S46 was determined after waterborne infection. Trout infected with the IHNV/IPNV.S46 sample, (a mixed sample containing equal infectious titers of the viruses) showed 50% less mortality than fish infected with either of the reference viruses alone. Forty-five days after the coinfection, IPNV antigens were detected by flow cytometry in 49 to 63% of the leukocytes from the surviving trout; whereas, only 9-15.6% of the leukocytes expressed IHNV viral antigens. IPNV was easily detected by reverse transcription-polymerase chain reaction (RT-PCR), whereas, for IHNV, a second step of amplification of a 753 bp fragment corresponding to the internal sequences of the IHNV G gene was necessary to optimize viral detection. The sequence of the IHNV gene involved in virulence, the glycoprotein (G) gene, was determined for the IHNV.S46 and compared with other sequences available in the GenBank. Changes found were not located in the antigenic domains of the glycoprotein and were considered not significant.

Comparative evaluation of five serological methods and RT-PCR assay for the detection of IPNV in fish.

In the present study, six diagnostic methods for the detection of infectious pancreatic necrosis virus (IPNV) (indirect immunofluorescence, flow cytometry, immunoperoxidase, immunodot blot, immunostaphylococcus-protein A, and RT-PCR) have been comparatively evaluated using the seroneutralization as the reference assay, and 83 Spanish isolates and 3 reference strains. The most reliable methods were flow cytometry and RT-PCR which could detect virus at titers of 1x10(2) and 1x10(3) TCID50/ml, respectively. At a multiplicity of infection of 50, both assays allowed the earliest detection of IPNV at 4 h post-inoculation. Indirect immunofluorescence and immunoperoxidase assays required at least 6 h post-inoculation to detect viral antigens. The immunodot blot assay possesses low sensitivity and the immunostaphylococcus-protein A test cannot be applied for routine examination of IPNV. Positive reactions were obtained in 100% of the samples tested by seroneutralization and RT-PCR, 90.4% by the flow cytometry, 80.7% by the indirect immunofluorescence assay, 67.5% by the immunoperoxidase, 62.6% by the immunodot blot, and only 27.7% by immunostaphylococcus-protein A test. Therefore, RT-PCR and flow cytometry were the most appropriate and sensitive methods for the routine detection of IPNV from affected fish.

Virus susceptibility of the fish cell line SAF-1 derived from gilt-head seabream.

The recently reported SAF-1 cell line from fins of gilt-head seabream was evaluated for susceptibility to lymphocystis disease virus (LDV) and to several salmonid fish viruses, such as infectious haematopoietic necrosis virus (IHNV), viral haemorrhagic septicemia virus (VHSV) and several strains of infectious pancreatic necrosis virus (IPNV). LDV, VHSV and IHNV replicated well in the cultured fin cells as demonstrated by cell lysis and increases in viral titer. The potential use of this cell line to detect viruses from fish marine species is discussed.