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.     

The response of phenolic compounds in grapes of the variety ‘Chardonnay’ (Vitis vinifera L.) to the infection by phytoplasma Bois noir

The study was carried out on phytoplasma susceptible grapevine variety ‘Chardonnay’ (Vitis vinifera L.). The changes in total and individual phenolics, with a focus on hydroxycinnamic acids, flavanols and flavonol contents, were studied in phytoplasma-symptomatic and non-symptomatic berries of Bois noir (BN) infected and uninfected vines. Evident responses to BN infection at veraison have been monitored in a decreased accumulation of caftaric and coutaric acids, p-coumaroyl hexose, procyanidin B1, procyanidin trimer as well as of quercetin-3-O-glucoside, quercetin-3-O-glucuronide and quercetin-3-O-xyloside. At berry softening BN infection statistically increased the content of total phenolics, hydroxycinnamic acids and flavanols, but decreased the flavonol contents, especially at phytoplasma-symptomatic berry skins. Later, at harvest, the BN infection caused an additional significant decrease of coutaric acid and p-coumaroyl pentose contents, moreover of procyanidin B1 and procyanidin dimmers (1, 2, and 3), trimer, kaempferol-3-O-glucoside and of most identified quercetins, except of quercetin-3-O-xyloside. At harvest, non-symptomatic berries from infected plants showed similar dynamics in the total phenolic content compared to berry skins from uninfected plants, but in total flavanols and flavonols content similarity to those symptomatic was observed. The latter decreases grape quality and its antioxidant potential. The Bois noir disease showed specific, local and growth-phase-induced responses regarding the content of phenolics in berry skins, where in particular the differences between phytoplasma-symptomatic and non-symptomatic grapes have to be underlined.     

Haptoglobin and ceruloplasmin as determinants of inflammation in dogs.

Assay procedures for determining serum haptoglobin concentration and ceruloplasmin oxidase activity in dogs were validated, and reference values were established. Serum haptoglobin concentration is reported as milligrams per deciliter of cyanmethemoglobin binding capacity, whereas serum ceruloplasmin oxidase activity was determined by use of p-phenylenediamine as substrate. Both assays were used to analyze serum samples from 288 dogs. In each dog's case record, clinical history and final diagnosis were evaluated to determine whether the dog had an inflammatory condition. Complete blood cell counts were performed in 265 dogs, using simultaneously collected blood samples. Plasma fibrinogen concentration was determined for 161 dogs. A positive correlation (P less than 0.01) for serum haptoglobin concentration and for ceruloplasmin oxidase activity, compared with WBC counts, segmented neutrophil and band neutrophil counts, and plasma fibrinogen concentration. Ceruloplasmin oxidase activity and haptoglobin concentration were up to 6 times more sensitive than fibrinogen concentration or leukocyte counts in detecting inflammation. Specificity of ceruloplasmin oxidase activity was comparable to fibrinogen concentration and leukocyte counts, whereas haptoglobin concentration was found to be slightly less specific. Specificity of haptoglobin concentration improved slightly (from 0.82 to 0.88) when dogs with a history of glucocorticoid administration were excluded from analysis. Predictive value of a negative test result (haptoglobin concentration less than 125 mg/dl; ceruloplasmin oxidase activity less than 20 IU/L) and predictive value of a positive test result for haptoglobin concentration and ceruloplasmin activity were comparable to or better than fibrinogen concentration or various oxidase leukocyte counts in detection of inflammation in a variety of disease conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of the acylurea insect growth regulator teflubenzuron on the adults of two endolarval parasitoids of Plutella xylostella,Cotesia plutellae and Diadegma semiclausum

The acylurea chitin synthesis inhibitor, teflubenzuron (‘Nomolt’® SC) showed no apparent activity against adult males of Diadegma semiclausum Hellén or Cotesia plutellae Kurdj. when applied to foliage at a rate ( = 50 g a.i. ha^?1) approximating to 2 × field usage. In contrast, the pyrethroid cypermethrin (‘Cymbush’® EC) showed appreciable foliar activity (at rates = 10–50 ga.i. ha^?1) to D. semiclausum but showed low activity against C. plutellae. When single adult female D. semiclausum were pre-treated with teflubenzuron (= 50 g a.i. ha^?1) and then exposed to 5. 10. 25, 50 or 100 second-instar host larvae, the number of purasitoid cocoons formed was significantly reduced compared with controls except at the lowest host density (P 0-05). However, there was no evidence that teflubenzuron had a host density-dependent effect on parasitism by D. semiclausum. Teflubenzuron had no significant effect on host parasitism in an equivalent experiment with C. plutellae (P > 0-05) except at the median host density. When batches of ten host larvae were introduced at hourly intervals to female D. semiclausum pre-treated with teflubenzuron (= 50 g a.i. ha^?1), the number of cocoons formed in the first two batches was not significantly different from untreated controls (P > 0-05), whereas teflubenzuron significantly reduced the number of cocoons in the next three batches of host larvae (P < 0-05). This suggests that teflubenzuron may affect chitin synthesis in developing eggs within female D. semiclausum but has little or no effect on pre-formed eggs which are more likely to be oviposited first. Teflubenzuron had no significant (P > 0-05) effect on parasitism by C. plutellae when presented with batches of host larvae. When female D. semiclausum and C. plutellae were placed in the same host arena, the former showed a 3-fold greater rate of parasitism. Pre-treatment with teflubenzuron significantly reduced parasitism by D. semiclausum (P > 0-05) but had no significant effect with C. plutellae (P > 0-05) although the latter species failed to exploit the reduction in parasitism by D. semiclausum. The differential response of D. semiclausum and C. plutellae to insecticides is discussed in relation to the relative rates of establishment of these species in Malaysia.     

Recovery of floristic diversity,composition and structure of regrowth forests on fallow lands: implications for conservation and restoration of degraded forest lands in Laos

New Forests - In Laos, there are extensive shifting cultivation areas and regrowth forests spontaneously established on fallow lands. The aims of the study were to conduct a comparative study of...     

Triacylglycerol and fatty acid compositions of French virgin olive oils. Characterization by chemometrics

There is no data concerning the fatty acid and triacylglycerol composition of French virgin olive oil. Thus, these compositions were determined using 564 samples coming from four olive harvests (1998-1999 to 2000-2001). Among these 564 samples, 372 came from the four main French cultivars (Aglandau, Cailletier, Picholine, and Salonenque) and from both of the oldest French protected designations of origin: "Nyons" (cv. Tanche) and "Vallée des Baux". The fatty acid compositions took the different isomeric monounsaturated fatty acids (C16:1 and C18:1) into account. The eicosenoic acid is gondoic acid (20:1n-9) and was determined by dimethyl disulfide adduct using GC/MS. The use of propionitrile as a mobile phase for the HPLC analysis of the triacylglycerols led to better resolutions between triacylglycerols than those resolutions obtained with the mix of solvents recommended by the normalized method (acetone/acetonitrile). Of the samples, 88 had a 9-heptadecenoic acid level (17:1n-8) higher than 0.3% and 33 had a linolenic acid level higher than 0.9%, which are maximal values accepted by the International Olive Oil Council and the European Union. A linear discriminant analysis was carried out on 372 samples with the SAS system and particularly with STEPISC and CANDISC procedures. Variables (n = 37) representing the different fatty acids, the sum of saturated, monounsaturated, and polyunsaturated fatty acids, squalene, and triacylglycerols were used, thus allowing us to classify samples into six groups defined with 100% of well classified samples. These results constitute an original data bank that can be used to identify the origin of virgin olive oils.