A high-throughput method for isolation of salicylic acid metabolic mutants

Background  

Salicylic acid (SA) is a key defense signal molecule against biotrophic pathogens in plants. Quantification of SA levels in plants is critical for dissecting the SA-mediated immune response. Although HPLC and GC/MS are routinely used to determine SA concentrations, they are expensive and time-consuming. We recently described a rapid method for a bacterial biosensor Acinetobacter sp. ADPWH_lux-based SA quantification, which enables high-throughput analysis. In this study we describe an improved method for fast sample preparation, and present a high-throughput strategy for isolation of SA metabolic mutants.     

Resistance mechanisms of wild tomato germplasm to infection of Oidium neolycopersici

Tomato powdery mildew (Oidium neolycopersici) is one of the most devastating diseases of cultivated tomatoes worldwide. Although the first epidemics were recorded more than 25 years ago many aspects of this host-pathogen interaction are still not well understood. Detailed morphological and molecular studies of the anamorphs confirmed that O. neolycopersici is phylogeneticaly close to Erysiphe aquilegiae var. ranunculi. Host range is rather broad, apart from Solanaceae hosts were found in the families Apocynaceae, Campanulaceae, Crassulaceae, Cistaceae, Cucurbitaceae, Linaceae, Malvaceae, Papaveraceae, Pedialiaceae, Scrophulariaceae, Valerianaceae a Violaceae. Non-host resistance within these families is not based on inhibition of formation of primary haustorium, however, on post-haustorial hypersensitive reponse and another type of non-hypersensitive resistance. Screening of wild Solanum species (previous Lycopersicon spp.) germplasm revealed valuable sources of resistance (S. habrochaites, S. pennellii, S. cheesmaniae, S. chilense, S. peruvianum). The main resistance mechanism was found to be a hypersensitive response (HR), in some cases followed by limited development of the pathogen. However, there is a broad variation in resistance response on the histological and cytological level. Interaction between many wild Solanum spp. and O. neolycopersici is race-specific, at least three races were differentiated. In some interspecific crosses (S. lycopersicum × S. habrochaites) adult plant resistance was observed. Biochemical studies focusing on production of reactive oxygen species (ROS) and peroxidase activity during infection of O. neolycopersici showed that production of ROS and activity of corresponding enzymes is related to activation of defence responses in genotypes of wild Solanum sect. Lycopersicon. The significance of nitric oxide (NO) in O. neolycopersici pathogenesis was supported by experiments with NO donors and scavengers. In moderately resistant genotype S. chmielewskii, treatment by heat stress caused slight deceleration of pathogen development, increased production of jasmonic acid (JA) and abscisic acid (ABA) and increased peroxidase activity in infected plants. The different degree of tomato resistance/susceptibility did not markedly change the rate and extent of photosynthetic response to O. neolycopersici; only minimal impairment of photosynthesis was found in both susceptible and moderately resistant genotypes during the first 9 days after inoculation. The accumulated evidence confirm a crucial role of localised increased production of ROS and reactive nitrogen species (RNS) in response to pathogen penetration into plant tissue and its involvement in the plant resistance responses including the initiation and progression of plant cell death in host wild Solanum species. Crucial points of further research are discussed.     

Evaluation of the ability of colistin,amoxicillin (components of Potencil®), and fluoroquinolones to attenuate bacterial endotoxin‐ and Shiga exotoxin‐mediated cytotoxicity—In vitro studies

Escherichia coli is one of the major pathogens in humans and animals causing localized and systemic infections, which often lead to acute inflammation, watery diarrhea, and hemorrhagic colitis. Bacterial lipopolysaccharide (LPS) and Shiga exotoxins (Stx) are mostly responsible for such clinical signs. Therefore, highly effective treatment of E. coli infections should include both eradication of bacteria and neutralization of their toxins. Here, for the first time, we compared the in vitro ability of common antibiotics to decrease LPS‐ and Stx‐mediated cytotoxicity: colistin, amoxicillin (used separately or combined), enrofloxacin, and its metabolite ciprofloxacin. Three experimental scenarios were realized as follows: (a) the direct effect of antibiotics on endotoxin, (b) the effect of antibiotic treatment on LPS‐mediated cytotoxicity in an experiment mimicking “natural infection,” (c) the effect of antibiotics to decrease Stx2e‐mediated cytotoxicity. Two cell lines, A549 and Vero cells, were used to perform cytotoxic assays with the methyl tetrazolium (MTT) and lactate dehydrogenase leakage (LDH) methods, respectively. Colistin and amoxicillin, especially used in combination, were able to attenuate LPS toxic effect, which was reflected by increase in A549 cell viability. In comparison with other antibiotics, the combination of colistin and amoxicillin exhibited the highest boster or additive effect in protecting cells against LPS‐ and Stx2e‐induced toxicity. In summary, in comparison with fluoroquinolones, the combination of colistin and amoxicillin at concentrations similar to those achieved in plasma of treated animals exhibited the highest ability to attenuate LPS‐ and Stx2e‐mediated cytotoxicity.     

Performance of three infiltration models under surge irrigation

Three models were developed to predict infiltration under a surge-flow irrigation regime. One model (2P) utilized the Kostiakov-Lewis infiltration equation. The other two models (2PF and 3P) utilized the modified Kostiakov-Lewis infiltration equation. All three models combined continuous-flow furrow irrigation volume-balance hydraulic models presented by Reddell and Latortue (1986, 1988) with a numerical variation of the Cycle Ratio-Time Model (CRTM) for surge irrigation presented by Blair and Smerdon (1987). The volume–balance component of the models estimated the soil's continuous-flow furrow irrigation infiltration function using first surge-cycle advance data. The numerical CRTM component used the estimated infiltration function in conjunction with the surge-flow irrigation infiltration opportunity-time to predict infiltration. An assessment of overall model accuracy was based upon an infiltrated volume root mean square error (RMSE) statistic. Overall, the 2PF model returned the lowest RMSE values and the 3P model was the next most accurate. The 2P model was the least accurate. While the 2PF model was the most accurate, this model has limited applicability since it requires inflow and outflow measurements throughout an irrigation event to evaluate the soil's basic infiltration constant. Despite its good overall performance, anomalous results from the 3P model suggest that further testing is needed. Although the 2P model was the least accurate of the three models evaluated here, and although it consistently underpredicted infiltration, it appears to be the most stable and effective of the three models evaluated. Received: 22 May 2000     

Characterization of flowering time and SSR marker analysis of spring and winter type Brassica napus L. germplasm

Flowering dates and life forms of all available Brassica napus accessions conserved at the North Central Regional Plant Introduction Station (NCRPIS) were characterized, and a survey of molecular variation was conducted by using simple sequence repeats (SSR) in order to support better management of accessions with diverse life forms. To characterize flowering phenology, 598 B. napus accessions from the NCRPIS collection were planted in Iowa and Kansas field sites together with a current commercial cultivar and observed for days to flowering (first, 50% and 100% flowering) in 2003. Days from planting to 50% flowering ranged from 34 to 83 in Iowa and from 53 to 89 in Kansas. The mean accumulated growing degree days (GDD) to 50% flowering were 1,997 in Iowa, and 2,106 in Kansas. Between locations, the correlation in flowering time (r = 0.42) and the correlation in computed GDD (r = 0.40) were both significant. Differences in flowering-time rank were observed for several accessions. Accessions that failed to flower in Iowa in a single growing season comprised 28.5% of the accessions; of the flowering accessions, 100% plant flowering was not always achieved. Accessions were grouped according to flowering time. A stratified sample of 50 accessions was selected from these groups, including 10 non-flowering and 40 flowering accessions of diverse geographic origins and phenological variation. The flowering time observed in the sampled accessions when grown in the greenhouse were found to be significantly correlated to the flowering time observed in the field locations in Iowa (r = 0.79) and Kansas (r = 0.49). Thirty SSR markers, selected across 18 Brassica linkage groups from BrassicaDB, and 3 derived from Brassica expressed sequence tags (ESTs) were scored in the stratified sample. An average of three bands per SSR primer pair was observed. Associations of SSR marker fragments with the life forms were determined. Analysis of molecular variation by using cluster analysis and ordination resulted in recognizable, distinct groups of annual and biennial life-form types, which may have direct applications for planning and management of future seed regenerations. Mention of commercial brand names in this paper does not constitute an endorsement of any product by the U.S. Department of Agriculture or cooperating agencies.     

Trace Metal Fluxes in a Sphagnum Peatland – Humic Lake System as a Consequence of Drainage

The study was conducted in a small (7.4 ha) peatland system with a humic lake concentrically surrounded by Sphagnum mat (Caricetum limosae) and Sphagnum bog (Ledo-Sphagnetum) in the Mazurian Lakeland in North-Eastern Poland. The peatland was situated in a forested catchment with a total area of 16.25 ha, and was influenced by surface drainage, artificially designed for forestry purposes. The content of zinc (Zn), cadmium (Cd), lead (Pb), copper (Cu) and hydrogen (H) ions was analysed in waters from precipitation, humic lake, mat, bog and surface outflow. The yearly budget of these ions, expressed as difference between atmospheric inflow and surface outflow, for the whole catchment was presented. The drainage of the peatland system over three years was accompanied by an increase in pH of lake, mat and bog waters. The whole catchment retention of H⁺ and trace metals studied decreased yearly in absolute values under these conditions. The peatland system began to leach Pb into surface waters and lost its ability to intercept Cd. These effects could be caused by intensity of outflow of water and sulphates and lowering of water storage. However, the potential to retain Zn and Cu ions was preserved.     

The effect of biostimulators on the content of crude oil and total protein in winter oilseed rape (Brassica napus L.) seeds

The objective of the study was to determine the effect of the experimental factors on the seed quality of three winter oilseed rape cultivars. The following factors were studied: I-three winter oilseed rape morphotypes: population, hybrid restored characterised by a semi-dwarf growth pattern, hybrid restored characterised by a conventional growth pattern; II-two sowing methods: a spacing of 22.5?cm, a spacing of 45.0?cm; III-four types of biostimulants: control-no biostimulants, the biostimulant Tytanit^®, Asahi^®SL, Silvit^®. Seed samples were subjected to chemical analysis to determine: crude oil, total protein and crude fibre. Natural stimulants of plant growth and development contributed to an increase in crude oil ranging from 7.4 to 9.5?g?kg^–1 d.m., on average, compared with control. The biostimulants applied did not affect total protein content which was, however, affected by meteorological conditions in individual study years as well as the genetic factor. Biostimulants increased crude fibre content compared with control.     

Geochemistry of the Hyperalkaline Gorka Pit Lake (pH?>?13) in the Chrzanow Region, Southern Poland

The Gorka pit lake was formed in an inactive Jurassic limestone quarry after cessation of open-pit dewatering. The main problem of the water quality in this area is linked to a large volume of extremely alkaline leachate disposed in the flooded quarry. The lake is meromictic due to a large density contrast between shallow and deep water layers. Water in the lake is of the Na?CCO₃?COH type, pH is in the range from 11.5 to 13.3, and there are high concentrations of sulfate and several toxic elements (Al, As, Cr, Mo, P, and V). The chemical composition of the extremely alkaline leachate was formed as a result of the groundwater interaction with the industrial red mud wastes containing 5?C10 wt.% of sodium carbonate. There is a trend of increasing concentrations and pH values with depth, mainly due to the in-gassing of atmospheric CO₂ into the surface layer and due to density stratification in the water column. Similar stratification is observed in groundwater wells around the lake. High dissolved concentrations of oxyanionic contaminants such as As, Cr, and V are caused by their high mobility and desorption under extremely alkaline conditions. In spite of reducing conditions at the bottom of the lake, caused by high concentrations of dumped organic matter, sulfate behaves conservatively because sulfate reducing bacteria do not survive in this pH range.     

Novel biological properties of Oenothera paradoxa defatted seed extracts: effects on metallopeptidase activity

In this study, for the first time, we used the in vitro metallopeptidase model for the identification of a potential novel activity of defatted evening primrose seed extracts. Prepared extracts of different polarity (aqueous, 60% ethanolic, isopropanolic, and 30% isopropanolic) at concentrations of 1.5-100 microg/mL exhibited a significant and dose dependent inhibition of three tested enzymes. The 50% inhibition of enzymes activity showed that aminopeptidase N (APN) was the enzyme affected to the greatest extent with IC50 at the level of 2.8 microg/mL and 2.9 microg/mL for aqueous and 30% isopropanolic extracts, respectively. The activity of neutral endopeptidase (NEP) was quite strongly inhibited by the extracts as well. The HPLC-DAD analysis and bioguided fractionation led to the identification of four active compounds: (-)-epicatechin gallate, proanthocyanidin B3, oenothein B, and penta-O-galloyl-beta-D-glucose (PGG). Oenothein B has been shown previously to inhibit metallopeptidases. The three other compounds are known to inhibit angiotensin-converting enzyme (ACE), but they have not been previously reported to inhibit the NEP and APN activity. PGG and procyanidins with different degrees of polymerization, as the dominating compounds in O. paradoxa seeds, seemed to play a role in the crude extract activity.