Does the source of nitrogen affect the response of subterranean clover to prolonged root hypoxia?

Nitrogen (N) is taken up by most plant species in the form of nitrate (NO ) or ammonium (NH ). The plant response to continuous ammonium nutrition is species‐dependent. In this study, the effects of the source of N nutrition (NO , NH , or the mixture of NO and NH ) on the response of clover (Trifolium subterraneum L. cv. 45C) plants to prolonged root hypoxia was studied. Under aerobic conditions, plant growth was strongly depressed by NH , compared to NO or mixed N nutrition, as indicated by the significant decrease in root and shoot‐dry‐matter production (DW), root and shoot water contents (WC), leaf chlorophyll concentration, and chlorophyll fluorescence parameters (F₀, F_v/F_m). However, the N source had no effect on chlorophyll a–to–chlorophyll b ratio. Under hypoxic conditions, the negative effects of root hypoxia on plant‐growth parameters (DW and WC), leaf chlorophyll concentration, and chlorophyll fluorescence parameters were alleviated by NH rather than NO supply. Concomitantly, shoot DW–to–root DW ratio, and root and leaf NH concentrations were significantly decreased, whereas root and leaf carbohydrate concentrations, glutamine synthetase activities, and protein concentrations were remarkably increased. The present data reveal that the N source (NO or NH ) is a major factor affecting clover responses to hypoxic stress, with plants being more tolerant when NH is the N form used. The different sensitivity is discussed in terms of a competition for energy between nitrogen assimilation and plant growth.     

Vitamin C and elemental composition of some bushfruits

Vitamin C and elemental analysis of a range of indigenous bushfruit from the Northern Territory, Darwin, Australia, was carried out. The results suggest that bushfruit may be a valuable food resource. In the majority of cases, vitamin C and elemental composition of bushfruits were comparable to those of western fruits in the same fruit category. Billy goat plums (Terminalia fertinandiana) have the highest vitamin C content, while K concentration is generally high as compared to other elements. Morinda citrifolia gave strong test for the presence of terpenes.     

Agricultural intensification exacerbates female-biased primary brood sex-ratio in tree swallows

Impacts of agriculture practices are documented at every ecosystem level from landscape structure to biodiversity. Birds are especially affected by agricultural modifications as shown by the decline of farmland species in Europe and North America. Few studies have assessed the effects of such modifications on individual characteristics directly influencing population dynamics. Several bird studies showed that sex-ratio may be adaptive and that mother condition affects the production of sons and daughters. However, little is known about the connections between environmental and individual characteristics on sex allocation. Here we quantified the variation in primary sex-ratio in tree swallows (Tachycineta bicolor) nesting in contrasted environments associated with agricultural intensification in southern Québec, Canada. We found that intensive agricultural practices affected female sex-ratio allocation in this area, resulting in more biased sex-ratio towards daughters throughout most of the hatching period. Yet, this bias towards daughters was reduced as the season progressed in the most intensively cultivated areas, suggesting that tree swallows have problems foreseeing the difficult growth and postfledging conditions that their nestlings will experience in such environments. Our results thereby support the hypothesis that intensive agricultural areas act as an ecological trap in our study system. We also found that effects of agricultural intensification on sex allocation differed among years and affected the relationships between sex-ratio allocation and hatching date. Our results suggest that agricultural intensification modifies female sex allocation in tree swallows, but the importance of the effects might vary among years and depend on timing of breeding.     

An optimized duplex real-time PCR tool for sensitive detection of the quarantine oomycete Plasmopara halstedii in sunflower seeds

Plasmopara halstedii, the causal agent of downy mildew of sunflower, is an oomycete listed as a quarantine pathogen. This obligate parasite resides in a quiescent state in seeds of sunflower and can be spread from seed production areas to areas of crop production by international seed trade. To prevent the spread or the introduction of potentially new genotypes or fungicide-tolerant strains, an efficient method to detect P. halstedii in sunflower seed is required. This work reports the optimization of a real-time detection tool that targets the pathogen within sunflower seeds, and provides statistically validated data for that tool. The tool proved to be specific and inclusive, based on computer simulation and in vitro assessments, and could detect as few as 45 copies of target DNA. A fully optimized DNA extraction protocol was also developed starting from a sample of 1,000 sunflower seeds, and enabled the detection of <1 infected seed/1,000 seeds. To ensure reliability of the results, a set of controls was used systematically during the assays, including a plant-specific probe used in a duplex quantitative polymerase chain reaction that enabled the assessment of the quality of each DNA extract.     

Broad-range PCR-TTGE for the first-line detection of bacterial pathogen DNA in ticks

Ticks are known or suspected vectors for a wide range of bacterial pathogens. One of the first steps for tick-borne risk assessment is the detection of these pathogens in their vectors. In the present study, a broad-range PCR amplification of the eubacterial gene encoding the 16S rRNA gene combined with Temporal Temperature Gradient gel Electrophoresis (TTGE) was evaluated as a method allowing the one-step detection of bacterial pathogen DNA in ticks. Firstly, DNA extracts from bacteria known to be tick-borne pathogens, i.e., Borrelia burgdorferi lato sensu, Anaplasma phagocytophilum, Spotted Fever Group (SFG) Rickettsia spp., were used to establish a TTGE pathogen DNA reference marker. Secondly, we used broad-range PCR-TTGE to detect the presence of DNA from these three pathogens in 55 DNA extracts from pools of 10 nymphal Ixodes ricinus ticks, which have been previously shown to carry DNA from at least one of those bacteria by specific PCR. Among the 20 B. burgdorferi specific-PCR samples, 15 (75%) were also found to be positive using PCR-TTGE. Sixteen of the seventeen (94%) Rickettsia spp. PCR-specific samples were positive using PCR-TTGE detection and all PCR-specific positive extracts (11/11, 100%) for A. phagocytophilum were also positive using PCR-TTGE. Moreover, we identified unexpected bacterial sequences that were not related to any of the three pathogens such as a sequence related to Spiroplasma sp. Thus, broad-range PCR-TTGE allowed the single step detection of DNA from up to 3 pathogens in the same co-infected samples as well as detection of DNA from unexpected bacteria.     

Rapid in planta detection of Chalara fraxinea by a real-time PCR assay using a dual-labelled probe

Chalara fraxinea is a fungus currently threatening ash trees (Fraxinus excelsior) in several European countries. This emerging pathogen was assigned to the EPPO’s alert list and therefore accurate detection and identification tools are needed. Because of its slow growth rate on agar media and the frequent presence of fast-growing saprotrophic fungi within the host tissue, classical isolation techniques are time-consuming and sometimes inefficient. In this study, we used species-specific polymorphisms observed within the internal transcribed spacer region to design a primer pair and a dual-labelled probe to be used in a real-time PCR assay for the detection of C. fraxinea. The test proved to be specific, based on in silico and in vitro assessments, and could detect as little as 20 fg of C. fraxinea DNA. A protocol was developed in order to detect the pathogen directly in plant tissue and proved to be more efficient and rapid than isolation on agar plates. This new tool should be useful both for monitoring and to conduct epidemiology research on this emerging pathogen.     

SCAR–based PCR primers to detect the hybrid pathogen <Emphasis Type="Italic">Phytophthora alni</Emphasis> and its subspecies causing alder disease in Europe

Since the 1990s, a new Phytophthora species hybrid has been jeopardizing the natural population of alders throughout Europe. This new Phytophthora, P. alni, has been suggested as a natural hybrid between two closely related species of Phytophthora. Little is known about the epidemiology of this pathogen, because its direct isolation is not always satisfactory. In this study we developed three pairs of Polymerase Chain Reaction (PCR) primers derived from Sequence Characterized Amplified Regions (SCAR) that allow discrimination among the three subspecies of P. alni: P. alni subsp. alni, P. alni subsp. uniformis and P. alni subsp. multiformis. These molecular tools were successfully used to detect P. alni directly in different substrates such as infested river water and soil, and necrotic alder bark, without the need for any prior baiting or isolation stages. An Internal Amplification Control (IAC) was included to help discriminate against false negative samples due to the potential presence of inhibitory compounds in DNA extracts. These molecular tools should be useful for epidemiological studies on this emerging disease.     

Effects of dietary fat and energy on body weight and composition after gonadectomy in cats

OBJECTIVE: To evaluate the effect of dietary fat and energy density on body weight gain, body composition, and total energy expenditure (TEE) in neutered and sexually intact cats. ANIMALS: 12 male and 12 female cats PROCEDURE: Male cats were castrated (castrated male [CM]) or underwent no surgical procedure (sexually intact male [IM]). Female cats underwent ovariectomy (spayed female [SF]) or laparotomy and ligation of both uterine tubes without ovary removal (sexually intact female [IF]). Cats were fed either the low-fat (LF) or high-fat (HF) diet for 26 weeks, with the final allocation consisting of 8 groups: IF-LF IF-HE SF-LF, SF-HF IM-LF, IM-HF, CM-LF, and CM-HF. Mean food intake for each group was recorded daily, and body weight was monitored weekly throughout the study. Body composition and TEE were measured before surgery in week 0 and at the end of the study (week 26) by isotope dilution (double-labelled water). RESULTS: N eutered cats gained significantly more body fat and body weight (53.80+/-5.79%) than sexually intact cats (27.11+/-5.79%) during the study. Body weight gain of neutered cats fed the HF diet was greater than those fed the LF diet. Following correction for body composition, TEE was similar in all groups and no pattern towards increased food intake was evident. CONCLUSIONS AND CLINICAL RELEVANCE: Weight gain in neutered cats was decreased by feeding an LF, low energy-dense diet. To prevent weight gain in cats after neutering, a suitable LF diet should be fed in carefully controlled meals rather than ad libitum.     

Determination of an efficient and reliable method for DNA extraction from ticks

Molecular detection of pathogenic microorganisms in ticks is based on DNA amplification of the target pathogen; therefore, extraction of DNA from the tick is a major step. In this study, we compared three different tick DNA extraction protocols based on an enzymatic digestion by proteinase K followed by DNA extraction by a commercial kit (method 1), or on mortar crushing, proteinase K digestion and phenol/chloroform DNA extraction (method 2) and fine crushing with a beads beater, proteinase K digestion and DNA extraction using a commercial kit (method 3). The absence of PCR inhibitors and the DNA quality were evaluated by PCR amplification of the tick mitochondrial 16S rRNA gene using tick-specific primers. With method 1, 23/30 (77%) of the samples were extracted; with method 2, 30/31 (97%) of the samples were extracted and with method 3, 30/30 (100%) of the samples were extracted. DNA extraction efficiency using method 3 is significantly higher than DNA extraction efficiency using method 1 (100% versus 77%, P < 0.05). There was no significant difference between methods 2 and 3. Method 3 was however more adapted to cohort studies than method 2. This technique was validated for cohort tick DNA extraction and applicable to the treatment of small samples such as nymphs and soft ticks with 100% efficiency.