Development of real-time PCR systems based on SYBR? Green I and TaqMan? technologies for specific quantitative detection of Phoma tracheiphila in infected Citrus

Real-time PCR assays based on SYBR? Green I and TaqMan? technologies were developed for in planta detection and quantification of Phoma tracheiphila, the mitosporic fungus causing ‘mal secco’ disease on citrus. Primers and a hybridization probe were designed on the basis of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The real-time PCR assays were compared with a classic isolation method in two separate experiments carried out on 6 and 24 month-old sour orange seedlings, artificially inoculated with a conidial suspension of the pathogen. Both technologies made it possible to follow the progression of infection by P. tracheiphila, enabling detection and quantification of the target fungus prior to the development of symptoms. The detection limit was 10 copies of the cloned target sequence and 15 pg of genomic DNA extracted from fungal spores. The values of the cycle threshold (Ct) were linearly correlated with the concentration of the target DNA, indicating that the method is suitable as a qualitative and quantitative assay. The presence of non-target fungal DNA had no effect on the specificity of the assay, but resulted in a 10-fold reduction of sensitivity. Total inhibition of the reaction occurred when conidia of the target pathogen were mixed with an organic soil substrate before extracting DNA using the standard protocol, while an alternative purification kit resulted in a significant decrease in sensitivity. Compared to classic methods, real-time PCR proved faster and easier to perform and showed a higher sensitivity. These results suggest that real-time PCR, based on both chemistries, has a great potential for early diagnosis of ‘mal secco’ disease and for quantitative estimation of fungal growth within host tissue.     

First results of embryonic development,spawning and larval rearing of the Mediterranean spider crab Maja squinado (Herbst ) under laboratory conditions,a candidate species for a restocking program

The Mediterranean spider crab, Maja squinado, is depleted due to overfishing. The crab has virtually disappeared from areas where it was abundant, such as the Balearic Islands and the Catalan coast. Maja squinado, is economically and ecologically very valuable, and it is essential to obtain information on its biology and rearing conditions to attempt to repopulate the damaged stocks of the species in the Mediterranean basin. Herein, we describe the first successful rearing of M. squinado under laboratory conditions. Our results show that M. squinado is an excellent candidate for restocking using cultured juveniles. Two consecutive broods with a 1–4 day interbrood period were observed in the laboratory in wild‐caught females, the maximum observed duration of embryonic development of the egg mass being 32 days at 18.4 ± 0.9°C, and went through four different stages. The complete larval and first juvenile development was studied in laboratory cultures fed enriched Artemia metanauplius. At 19.6 ± 0.6°C, development from hatching to first crab moult took 17 days, and it comprised two zoeae stages and one megalopa stage. The survival rate at the different stages was monitored, and 7.13 ± 2.3% was achieved at the first crab instar.     

Proteomic profiling of sea bass muscle by two-dimensional gel electrophoresis and tandem mass spectrometry

In this study, the proteome profile of European sea bass (Dicentrarchus labrax) muscle was analyzed using two-dimensional electrophoresis (2-DE) and tandem mass spectrometry with the aim of providing a more detailed characterization of its specific protein expression profile. A highly populated and well-resolved 2-DE map of the sea bass muscle tissue was generated, and the corresponding protein identity was provided for a total of 49 abundant protein spots. Upon Ingenuity Pathway Analysis, the proteins mapped in the sea bass muscle profile were mostly related to glycolysis and to the muscle myofibril structure, together with other biological activities crucial to fish muscle metabolism and contraction, and therefore to fish locomotor performance. The data presented in this work provide important and novel information on the sea bass muscle tissue-specific protein expression, which can be useful for future studies aimed to improve seafood traceability, food safety/risk management and authentication analysis. This work is also important for understanding the proteome map of the sea bass toward establishing the animal as a potential model for muscular studies.     

Molecular and biological characterization of an isolate of <Emphasis Type="Italic">Tomato mottle mosaic virus</Emphasis> (ToMMV) infecting tomato and other experimental hosts in eastern Spain

Fusarium Head Blight is a major disease of wheat and an important contributor to the reduced cultivation of wheat in South Africa, where the crop often is grown under irrigation. We collected Fusarium isolates from 860 Fusarium Head Blight-infected wheat heads in seven irrigated wheat-growing areas of South Africa. Six Fusarium species, i.e., F. chlamydosporum, F. crookwellense, F. culmorum, F. equiseti, F. graminearum and F. semitectum were recovered, three of which, i.e., F. chlamydosporum, F. equiseti and F. semitectum, were not previously associated with Fusarium Head Blight in South Africa. Fusarium graminearum occurred at high frequencies at all seven locations. Based on polymerase chain reaction (PCR) assays of diagnostic sequences, more isolates were predicted to produce deoxynivalenol than nivalenol. Fusarium graminearum (sensu lato) appears to be the primary causal agent of Fusarium Head Blight in irrigated wheat in South Africa, which may not be the case for wheat cultivated under rain-fed conditions. Rotations of irrigated wheat with other graminaceous crops and maize could increase fungal inoculum and disease pressure. The establishment of Fusarium Head Blight in the irrigated wheat region of the country means that resistant lines and alternative agronomic practices are needed to limit disease severity, yield losses and mycotoxin contamination.     

Fine‐scale analysis of heavily invaded Italian freshwater fish assemblages

Inland waters are highly vulnerable to the introduction and spread of non‐native species, due to heavy human use of aquatic ecosystems and the natural linkages among streams and lakes. This is particularly noticeable in freshwater fish communities. To better evaluate how these communities are affected by non‐native species introductions, we conducted a fine‐scale analysis of the changes in Italian freshwater fish assemblages after species introduction. For this analysis, we collected information on fish species present in 44 basins. The present Italian freshwater fish fauna is composed of 48 native and 41 established introduced species, while a further 15 introduced species have been reported but are not yet considered naturalized. The changes in the fish assemblages mostly took place in the past 2 centuries and have increased recently, with nearly 60% of the species introduced in the past 3 decades. The number of species introduced per basin ranged from 0 to 35 (mean 10.85 ± 7.77 species/basin), and in 10 basins the number of species introduced is now equal to or even higher than the number of native species. In the past, introduced species mainly originated from America, but over the past three decades, an increase of introductions from other parts of Europe and Asia has been recorded. Our results show that basins already rich in native species tend to become even richer as a consequence of the establishment of introduced species. This confirms the trend toward a biotic homogenization of ecosystems even at a local scale, due to an increase in the human‐mediated spread of generalist species.     

Sweet Pepper (Capsicum annuum L.) Organic Seedling Production: The Role of Compost,Cultivar, and Protein Hydrolyzate

In the past decade, the increasing environmental concerns regarding the use of non-renewable resources, such as peat, for growing media production has led to a search for alternative materials as substrate components. Until now, compost represents the most investigated one. Despite the critical aspects related to compost use (i.e., its high pH, salinity, and potential content of toxic elements), partial peat substitution with this material represents an achievable objective. In this study, green compost was evaluated as a component of peat substrates for organic sweet pepper (Capsicum annuum L.) seedling production. Three compost rates (CR) were compared: 0 (peat without compost), 30, and 60% of compost v/v. Two pepper cultivars, one derived from a local (medium-Adriatic) germplasm (L) and a commercial hybrid (H), were tested. Furthermore, organic fertilizers based on hydrolyzed proteins, of animal (A) and vegetal (V) origin, were compared. Substrates were analyzed for characterization (pH, EC, nutrient contents, heavy metals). Seedling performances were evaluated in terms of size, biomass, nutrient uptake, and toxic element contents. Indices able to assess seedling resistance to transplant stress were also calculated. Results showed that CR60 treatment was able to produce seedlings with the same biomass of control. CR30, instead, assured the same chance to resist the transplanting stress of CR0. The performances of fertilizer A were significantly higher than those of fertilizer V. Local germplasm (L) showed the highest resistance to transplant stress, while H was more performing in nitrogen uptake. The risk of seedling contamination by toxic elements resulted negligible.     

Landscape,Soil, and Plant Analysis of Japanese Stiltgrass (Microstegium vimineum) Invasion in the Piedmont Region,SC

Japanese stiltgrass (Microstegium vimineum), an exotic invasive plant, is native to Southeast Asia. This study was conducted to determine the chemical composition of Japanese stiltgrass as well as soil and landscape characteristics that correlate with invasion of Japanese stiltgrass around Lake Issaqueena in the upper Piedmont of South Carolina. Geographic Information Systems (GISs) and Light Detection and Ranging (LiDAR) were used to determine the spatial pattern of invasion with respect to the aspect, slope, canopy cover, soils, and distance to roads and trails. Japanese stiltgrass was distributed on both sides of Lake Issaqueena in Pacolet and Madison soil map units (Fine, kaolinitic, thermic Typic Kanhapludults) on the average slopes of 21%, but it was particularly common on the eastern shore of the lake in low-lying wet and shaded areas (mean canopy cover 51%). In addition, invasion by Japanese stiltgrass was correlated with the proximity to roads and trails. Plant tissue analysis revealed many differences in the distribution of macronutrients, macrominerals, and micronutrients in the leaves, stems, and roots of Japanese stiltgrass, although those differences were not always statistically significant. Concentrations of nitrogen (N), phosphorus (P), and calcium (Ca) were the highest in leaves while zinc (Zn) concentrations were the highest in stems and concentrations of magnesium (Mg), copper (Cu), manganese (Mn), iron (Fe), and sodium (Na) tended to be higher in roots. Carbon (C), sulfur (S), and potassium (K) concentrations were generally higher in above-ground tissues versus roots. Soil chemical analysis revealed no statistical differences between control and invaded plots. Our findings suggest that watershed areas surrounding lakes may be particularly susceptible to the invasion of Japanese stiltgrass due to their microclimates, low-lying wet pathways for seed distribution and recreational uses.     

Factors Affecting the Growth of Microalgae on Blackwater from Biosolid Dewatering

This paper discusses the possibility of including the culturing of microalgae within a conventional wastewater treatment sequence by growing them on the blackwater (BW) from biosolid dewatering to produce biomass to feed the anaerobic digester. Two photobioreactors were used: a 12 L plexiglas column for indoor, lab-scale tests and a 85 L plexiglas column for outdoor culturing. Microalgae (Chlorella sp. and Scenedesmus sp.) could easily grow on the tested blackwater. The average specific growth rate in indoor and outdoor batch tests was satisfactory, ranging between 0.14 and 0.16 day^?1. During a continuous test performed under outdoor conditions from May to November, in which the off-gas from the combined heat and power unit was used as the CO₂ source, an average biomass production of 50 mgTSS L^?1 day^?1 was obtained. However, statistical analyses confirmed that microalgal growth was affected by environmental conditions (temperature and season) and that it was negatively correlated with the occurrence of nitrification. Finally, the biochemical methane potential of the algal biomass was slightly higher than that from waste sludge (208 mLCH₄ gVS^?1 vs. 190 mLCH₄ gVS^?1).     

Effect of spineless cactus intake (<Emphasis Type="Italic">Opuntia ficus-indica</Emphasis>) on blood glucose levels in lactating sows and its impact on feed intake,body weight loss,and weaning-estrus interval

The effect of spineless cactus intake (Opuntia ficus-indica) on blood glucose (BG) levels in lactating sows and its impact on daily and total feed intake (dFI^?1 and TFI, respectively), body weight loss (BWL), and weaning-estrus interval length (WEI) were evaluated. Thirty-four hybrid (Yorkshire × Landrace × Pietrain) sows in lactation phase were used. Sows were divided into two groups: G1 (n = 17) where they received commercial feed and G2 (n = 17) provided with commercial feed plus an average of 2.0 ± 0.5 kg spineless cactus, based on a sow’s body weight. The variables evaluated were BG, dFI^?1, TFI, BWL, and WEI. Statistical analysis was performed by using a fixed and mixed model methodology, under a repeated measurements experiment. Group effects were found on all analyzed variables (P < 0.05). The BG was lower in G2 (55.2 and 64.5 mg/dL pre- and post-prandial, respectively), compared to that in G1 (70.9 and 80.1 mg/dL pre- and post-prandial, respectively) (P < 0.05). G2 showed better performance than G1 for dFI^?1, BWL, and WEI (P < 0.05) whose averages were 5.5 ± 1.8 kg, 7.4 ± 4.5%, and 5.3 ± 1.2 days, respectively. Averages for these variables in G1 were 4.7 ± 1.5 kg, 16.8 ± 4.6%, and 6.1 ± 1.6 days, respectively. Intake of spineless cactus reduced BG levels in lactating sows, generating greater dFI^?1, lower BWL at the end of lactation, and a lower WEI.