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.     

Physiological responses of Quercus oleoides (Schltdl & Cham) to soils contaminated by diesel

ABSTRACT

Oil pollution is a worldwide threat to the environment that affects the development of plants. The effect of soil contaminated by diesel on the physiological responses of seedlings of Quercus oleoides was investigated in two independent experiments. We proposed that physiological performance will decrease when seedlings are exposed to higher concentration of contamination. At the first experiment, levels of pollution with diesel were of 0%, 5%, 10% and 15%, and 0%, 2% and 3.5% in the second one. In the first experiment, photosynthetic rate, stomatal conductance, transpiration and total chlorophyll of Q. oleoides were higher in the control seedling, and lower in treatments of pollution with 5%, 10% and 15% of diesel during 3 days of treatment. Only, seedlings in soil polluted with 5% of diesel survived up to 16 days; they showed a photosynthetic rate of 5 μmol m^?2 s^?1, which was lower than control seedlings (9 μmol m^?2 s^?1). This pattern was observed in stomatal conductance, transpiration and relative water content. Surprisingly, in the second experiment, seedlings showed a higher photosynthetic rate and growth at 2% of diesel-contaminated soil than control seedlings, a phenomenon known as hormesis. In both experiments, soil respiration was proportional to soils contaminated. We concluded that Q. oleoides is highly vulnerable in soils contaminated with above 5% of diesel, but it maintains its physiological activities in soils contaminated below 2%, suggesting that seedlings can grow under low concentration of diesel contaminant, and may be used in phytoremediation of soils with low concentrations of diesel contamination.     

Design and validation of a RT‐qPCR procedure for diagnosis and quantification of most types of infectious pancreatic necrosis virus using a single pair of degenerated primers

Infectious pancreatic necrosis virus (IPNV) is an important virus which affects the salmonid aquaculture industry worldwide; therefore, it is important to develop rapid and reliable methods of diagnosis to detect the disease at early stages. Nowadays, RT‐qPCR is replacing other methods because it provides additional information on the viral load, which is important to have a better understanding of the virus replication level and of the stage of the infection and its risk level. The main problem stems from the high diversity of this virus, which can compromise the reliability of the diagnosis. In this study, we have designed an RT‐qPCR procedure for diagnosis and quantification of IPNV based on a single pair of primers targeted to segment B. The procedure has been validated, in vitro and in vivo, testing two different types of standards against seven reference strains and 23 field isolates from different types. The procedure is reliable for the detection of any type, with a detection limit of 31 TCID₅₀ mL^?1, 50 pfu mL^?1 or 66 RNA copies mL^?1, depending on the standard. All the standard curves showed high reliability (R² > 0.95). The results support the high reliability of this new procedure for the diagnosis and quantification of IPNV.     

Soybean molasses as an organic carbon source in the farming of Litopenaeus vannamei (Boone, 1931) in a biofloc system

Soybean molasses was evaluated as a partial replacement for sugarcane molasses as a carbon source for biofloc development in the superintensive culture of Pacific white shrimp (Litopenaeus vannamei). A 50‐day study was conducted with juvenile (3.2 g) shrimp stocked in 16 800 L tanks at a stocking density of 250 shrimp m^?3. Control of total ammonia concentration was performed by the addition of combined mixtures of soybean and sugarcane molasses to the culture water. Three different molasses treatments were evaluated using different soybean‐to‐sugarcane molasses ratios: 15–85%, 38–62% and 60–40% respectively. The control group was treated only with sugarcane molasses. Water quality, chlorophyll a concentration, heterotrophic bacterial load, Vibrio spp. concentration and zootechnical indexes were all evaluated. Total ammonia concentration was controlled by heterotrophic and chemotrophic pathways. Biofloc formation, as quantified by measuring the total suspended solids, was not altered. The Vibrio spp. concentration showed a significant reduction in treatments with soybean‐to‐sugarcane molasses ratios of 38–62% and 60–40%. All combined mixtures of soybean and sugarcane molasses could maintain water quality and productivity in the superintensive culture of L. vannamei using the biofloc system. Thus, the potential use of a residue from agroindustry as a carbon source in a biofloc culture is demonstrated.     

Effects of Broomrape Parasitism on Sunflower Plants: Growth,Development, and Mineral Nutrition

Sunflower broomrape (Orobanche cumana Wallr.) is a parasitic plant that infects sunflower (Helianthus annuus L.) plants. In this work, sunflower plants were grown under greenhouse conditions in pots with the substrate infested or non-infested with broomrape seeds. At different numbers of days after sowing, plant height, internode lengths, number of leaves, head diameter, mineral composition of leaves, and potassium (K) concentration in stem were measured. The negative effects of broomrape parasitism were assessed from 57 d after sowing, when broomrape started to emerge. Parasitized plants exhibited lower shoot dry weight, height, and head diameter than control plants. The reduction in internode lengths was associated with a decrease in the gradient of K concentration from basal to apical stem. The mineral composition of leaves was also affected in parasitized plants. The concentrations of calcium (Ca), magnesium (Mg), manganese (Mn), and zinc (Zn) in leaves of parasitized plants were lower than those of the control plants, while there were few differences for K, phosphorus (P), iron (Fe), and copper (Cu). The effects of parasitism are discussed in relation to their competition for resources and to perturbations of the host physiology such as hormonal and water balance.     

Nutritional response of seedling tomato plants under different lighting treatments

In order to describe the effect of complementary artificial light supply on the nutrient status of tomato seedlings, fluorescent lamps were compared to each other. Nitrogen (N), phosphorus (P), and potassium (K) concentrations were measured (mg g^?1 dry matter). Blue, red and far-red relationships were established. The N, P and K higher concentration and uptake have been found under high efficiency fluorescent lamps. Phosphorus uptake increasing was observed at high levels of irradiation. Potassium concentration is significantly higher under compact fluorescent lamps. The relationship between ratios and N, P and K extractions were used as indicators of limitation for plant. The ratio N/P and N/K present a low relation with N extraction. The ratio N/P presents a significant correlation with P extraction, also the ratios N/K and P/K with K extraction. Significant differences in constant of lineal equation have been found in P/K ratio and K extraction.     

Weed abundance and soil seedbank responses to tillage systems in continuous maize crops

Weed abundance in crops undergoes frequent changes, often due to changes in tillage practices. Annual species, with quick germination, a short vegetative stage, profuse seed production and long-lived seeds become problematic under zero-tillage systems. Portulaca oleracea L. and Amaranthus blitoides L. are widespread weeds in the Mediterranean area, prominent in irrigated crops. We studied the total weed abundance in the field, and specifically these two species (Portulaca oleracea and Amaranthus blitoides) with high frequency of occurrence in monoculture maize, from 2012 to 2014, in the field and soil seedbank. Results showed significant differences between zero-tillage (ZT) and conventional tillage (CT) systems on total weed abundance and relative abundance of Portulaca oleracea. Total weed abundance decreased in ZT plots (from 136 plants m^?2 to 25 and 46 plants m^?2, in 2013 and 2014 respectively). The same trend was observed in Portulaca oleracea recorded in ZT plots, but the abundance of Amaranthus blitoides did not vary in this system. Weed seedling germination and weed seed numbers both of total weed seedbank and Portulaca oleracea, were greater in ZT plots compared to CT, regarding Amaranthus blitoides seedling germination and seed count, the values did not increase with ZT, in continuous maize crops.     

Fate of phosphorus in soil during a long‐term fertilization experiment in Finland

Accumulation and depletion of soil phosphorus (P) was studied in a long‐term (37 y) field experiment in Southern Finland. The loam soil had a high pH (7.5–7.7) due to an earlier liming. Spring barley, spring wheat, oat, and ryegrass, grown in rotation, were annually fertilized with 0, 32, or 67 kg P ha^?1 y^?1 (P₀, P₁, and P₂K) and sufficient N. The average dry matter grain yield 2,600 kg ha^?1 of the P₀ plots increased by about 500 kg ha^?1 at P₁ treatment and another 600 kg ha^?1 by P₂K. Soil samples were collected in 1978 (beginning), 1995, 2005, and 2015. According to the Chang and Jackson sequential extraction, the P₂K and P₁ treatments increased the inorganic soil P by 732 and 32 kg P ha ^?1 in 37 years, respectively, while the P₀ plots were depleted by –459 kg P ha ^?1. The P₂K treatment increased all four P fractions, extracted with NH₄Cl (easily soluble), NH₄F (Al‐P), NaOH (Fe‐P), and H₂SO₄ (Ca‐P). Continuous depletion (P₀) decreased the NH₄Cl‐P and NH₄F‐P pools, NaOH‐P and H₂SO₄‐P pools remaining stable. None of the P pools changed significantly at P₁. The remarkable gap between the measured change and the balance for the P₂K and P₁ treatments cannot be explained solely by lateral soil movement, meaning that a significant proportion of the applied P was lost either in surface runoff or transported below the investigated depth of 40 cm. Despite large P applications, the degree of P saturation reached only 20% in the P₂K topsoil, assuming a 50% reactivity of Fe and Al oxides. As derived from sorption isotherms, a high EPC₀ (i.e., equilibrium P concentration at zero net P sorption or desorption) of 1.30 mg L^?1 had been built up in the P₂K treatment, while in the P₁ treatment EPC₀ (0.33 mg L^?1) had remained unchanged and P depletion (P₀) had caused a decrease to 0.12 mg L^?1. These results demonstrate that P sorption and desorption properties respond strongly to both P fertilization and null fertilization treatments and that in a long‐term field experiment only a low proportion of the residual fertilizer P can be recovered from soil.     

Nutrient limitations to bacterial and fungal growth during cellulose decomposition in tropical forest soils

Nutrients constrain the soil carbon cycle in tropical forests, but we lack knowledge on how these constraints vary within the soil microbial community. Here, we used in situ fertilization in a montane tropical forest and in two lowland tropical forests on contrasting soil types to test the principal hypothesis that there are different nutrient constraints to different groups of microorganisms during the decomposition of cellulose. We also tested the hypotheses that decomposers shift from nitrogen to phosphorus constraints from montane to lowland forests, respectively, and are further constrained by potassium and sodium deficiency in the western Amazon. Cellulose and nutrients (nitrogen, phosphorus, potassium, sodium, and combined) were added to soils in situ, and microbial growth on cellulose (phospholipid fatty acids and ergosterol) and respiration were measured. Microbial growth on cellulose after single nutrient additions was highest following nitrogen addition for fungi, suggesting nitrogen as the primary limiting nutrient for cellulose decomposition. This was observed at all sites, with no clear shift in nutrient constraints to decomposition between lowland and montane sites. We also observed positive respiration and fungal growth responses to sodium and potassium addition at one of the lowland sites. However, when phosphorus was added, and especially when added in combination with other nutrients, bacterial growth was highest, suggesting that bacteria out-compete fungi for nitrogen where phosphorus is abundant. In summary, nitrogen constrains fungal growth and cellulose decomposition in both lowland and montane tropical forest soils, but additional nutrients may also be of critical importance in determining the balance between fungal and bacterial decomposition of cellulose.     

Effect of nutrient solution salinity and ionic concentration on parsley (Petroselinum crispum Mill.) essential oil yield and content

The growth and essential oil (EO) production of parsley were evaluated in response to salinity and nutrient solution concentrations in a soilless culture. Parsley plants that were 60 days old were potted in a coconut fiber and peat moss medium and were treated with four different nutrient solutions, including T1, T2, T3 and T4. The T1 nutrient solution was the standard, the T2 and T3 solutions contained incremental macronutrient concentrations with an electrical conductivity (EC) of up to 2.2 and 3.2 dS m^?1, respectively, and the T4 solution was the same as T2 but with sodium chloride (NaCl) and an incremental macronutrient concentration with an EC of 3.2 dS m^?1. Next, these plants were grown for 90 days in a greenhouse with natural daylight in Nador, Morocco. Shoot and root growth significant decreased with increasing EC. However, the salinity that resulted from the addition of NaCl did not affect plant growth in the nutrient solutions. The optimum obtained growth and EO production were 1.2 and 2.2 dS m^?1, respectively. Consequently, the optimum EC value (based on the EO production) of parsley in the soilless culture was 1.2–2.2 dS m^?1.