An Improved Detached-Leaf Assay for Phenotyping Net Blotch of Barley Caused by Pyrenophora teres.

Net blotch, caused by Pyrenophora teres, is a major barley (Hordeum vulgare) leaf disease worldwide. P. teres occurs as two forms-P. teres f. teres, and P. teres f. maculata-inducing net and spot-like symptoms, respectively. An intact-seedling assay, where entire seedlings are inoculated by spraying with a conidial suspension, is frequently used for phenotyping net blotch. However, this presents a biosecurity risk in the glasshouse when nonlocal isolates are being screened. Alternatively, a detached-leaf assay (DLA-droplet method) can be used in which leaf segments laid out in a covered tray are inoculated with droplets of a conidial suspension, confining the inoculum. However, using this method, net and spot form symptoms cannot be distinguished from each other. We have developed an improved DLA (DLA-spray method) in which detached whole leaves are sprayed with the inoculum to produce distinct lesions. We compare the results for the three phenotyping methods above using four isolates from both net and spot forms of the disease to inoculate a standard set of eight barley genotypes. Results indicate that the DLA-spray method is a functional, informative and rapid test that readily differentiates the two forms of the pathogen in a biosecure environment.

Post-head-emergence frost in wheat and barley: defining the problem, assessing the damage, and identifying resistance.

Radiant frost is a significant production constraint to wheat (Triticum aestivum) and barley (Hordeum vulgare), particularly in regions where spring-habit cereals are grown through winter, maturing in spring. However, damage to winter-habit cereals in reproductive stages is also reported. Crops are particularly susceptible to frost once awns or spikes emerge from the protection of the flag leaf sheath. Post-head-emergence frost (PHEF) is a problem distinct from other cold-mediated production constraints. To date, useful increased PHEF resistance in cereals has not been identified. Given the renewed interest in reproductive frost damage in cereals, it is timely to review the problem. Here we update the extent and impacts of PHEF and document current management options to combat this challenge. We clarify terminology useful for discussing PHEF in relation to chilling and other freezing stresses. We discuss problems characterizing radiant frost, the environmental conditions leading to PHEF damage, and the effects of frost at different growth stages. PHEF resistant cultivars would be highly desirable, to both reduce the incidence of direct frost damage and to allow the timing of crop maturity to be managed to maximize yield potential. A framework of potential adaptation mechanisms is outlined. Clarification of these critical issues will sharpen research focus, improving opportunities to identify genetic sources for improved PHEF resistance.

Markers for seedling and adult plant crown rot resistance in four partially resistant bread wheat sources.

KEY MESSAGE: QTL identified for seedling and adult plant crown rot resistance in four partially resistant hexaploid wheat sources. PCR-based markers identified for use in marker-assisted selection. Crown rot, caused by Fusarium pseudograminearum, is an important disease of wheat in many wheat-growing regions globally. Complete resistance to infection by F. pseudograminearum has not been observed in a wheat host, but germplasm with partial resistance to this pathogen has been identified. The partially resistant wheat hexaploid germplasm sources 2-49, Sunco, IRN497 and CPI133817 were investigated in both seedling and adult plant field trials to identify markers associated with the resistance which could be used in marker-assisted selection programs. Thirteen different quantitative trait loci (QTL) conditioning crown rot resistance were identified in the four different sources. Some QTL were only observed in seedling trials whereas others appeared to be adult plant specific. For example while the QTL on chromosomes 1AS, 1BS, and 4BS contributed by 2-49 and on 2BS contributed by Sunco were detected in both seedling and field trials, the QTL on 1DL present in 2-49 and the QTL on 3BL in IRN497 were only detected in seedling trials. Genetic correlations between field trials of the same population were strong, as were correlations between seedling trials of the same population. Low to moderate correlations were observed between seedling and field trials. Flanking markers, most of which are less than 10 cM apart, have now been identified for each of the regions associated with crown rot resistance.

Current and emerging screening methods to identify post-head-emergence frost adaptation in wheat and barley.

Cereal crops can suffer substantial damage if frosts occur at heading. Identification of post-head-emergence frost (PHEF) resistance in cereals poses a number of unique and difficult challenges. Many decades of research have failed to identify genotypes with PHEF resistance that could offer economically significant benefit to growers. Research and breeding gains have been limited by the available screening systems. Using traditional frost screening systems, genotypes that escape frost injury in trials due to spatial temperature differences and/or small differences in phenology can be misidentified as resistant. We believe that by improving techniques to minimize frost escapes, such 'false-positive' results can be confidently identified and eliminated. Artificial freezing chambers or manipulated natural frost treatments offer many potential advantages but are not yet at the stage where they can be reliably used for frost screening in breeding programmes. Here we describe the development of a novel photoperiod gradient method (PGM) that facilitates screening of genotypes of different phenology under natural field frosts at matched developmental stages. By identifying frost escapes and increasing the efficiency of field screening, the PGM ensures that research effort can be focused on finding genotypes with improved PHEF resistance. To maximize the likelihood of identifying PHEF resistance, we propose that the PGM form part of an integrated strategy to (i) source germplasm;(ii) facilitate high throughput screening; and (iii) permit detailed validation. PGM may also be useful in other studies where either a range of developmental stages and/or synchronized development are desired.

Retention of D genome chromosomes in pentaploid wheat crosses.

The transfer of genes between Triticum aestivum (hexaploid bread wheat) and T. turgidum (tetraploid durum wheat) holds considerable potential for genetic improvement of both these closely related species. Five different T. aestivum/T. turgidum ssp. durum crosses were investigated using Diversity Arrays Technology (DArT) markers to determine the inheritance of parental A, B and D genome material in subsequent generations derived from these crosses. The proportions of A, B and D chromosomal segments inherited from the hexaploid parent were found to vary significantly among individual crosses. F(2) populations retained widely varying quantities of D genome material, ranging from 99% to none. The relative inheritance of bread wheat and durum alleles in the A and B genomes of derived lines also varied among the crosses. Within any one cross, progeny without D chromosomes in general had significantly more A and B genome durum alleles than lines retaining D chromosomes. The ability to select for and manipulate this non-random segregation in bread wheat/durum crosses will assist in efficient backcrossing of selected characters into the recurrent durum or hexaploid genotype of choice. This study illustrates the utility of DArT markers in the study of inter-specific crosses to commercial crop species.

Pyramiding QTL increases seedling resistance to crown rot (Fusarium pseudograminearum) of wheat (Triticum aestivum).

Crown rot of wheat (Triticum aestivum), predominantly caused by the fungus Fusarium pseudograminearum, has become an increasingly important disease constraint in many winter cereal production regions in Australia. Our group has previously identified a range of quantitative trait loci (QTL) for partial resistance to crown rot in various bread wheat sources. Here, we report on work that has assessed the effectiveness of pyramiding QTL to improve resistance to crown rot. Two doubled haploid populations were analysed--one from a cross between two previously characterised sources of partial seedling resistance (2-49 and W21MMT70; n = 208) and one from a cross between 2-49 and the commercial variety Sunco, a source of adult field resistance (n = 134). Both populations were phenotyped for seedling resistance to crown rot. Microsatellite and DArT markers were used to construct whole genome linkage maps for use in composite interval mapping (CIM) to identify QTL. Three QTL were detected in both trials conducted on the 2-49/W21MMT70 population. These were located on chromosomes 1D (QCr.usq-1D.1), 3B (QCr.usq-3B.1) and 7A. QCr.usq-1D.1 and the previously undetected 7A QTL were inherited from 2-49. QCr.usq-3B.1, inherited from W21MMT70, was the most significant of the QTL, explaining up to 40.5% of the phenotypic variance. Three QTL were identified in multiple trials of the Sunco/2-49 population. These were located on chromosomes 1D (QCr.usq-1D.1), 2B (QCr.usq-2B.2) and 4B (QCr.usq-4B.1). Only QCr.usq-2B.2 was inherited from Sunco. QCr.usq-4B.1 was the most significant of these QTL, explaining up to 19.1% of the phenotypic variance. In the 2-49/W21MMT70 population, several DH lines performed significantly better than either parent, with the best recording an average disease severity rating of only 3.8% of that scored by the susceptible check cultivar Puseas. These lines represent a new level of seedling crown rot resistance in wheat.

Allelopathy, DIMBOA production and genetic variability in accessions of Triticum speltoides.

A screening was conducted to study the allelopathic potential of Australian-held accessions of Triticum speltoides. Of 26 accessions, four were found to inhibit root growth in the indicator species, lettuce (Lactuca sativa). The methanol leaf extracts of these accessions significantly reduced the root length of wild oat (Avena spp.). In all but one case, alellopathic accessions contained higher amounts of DIMBOA than did nonallelopathic accessions. Since some variation in allelopathic response was detected within lines, random amplified polymorphic DNA (RAPD) markers were used to estimate genetic diversity between and within the allelopathic accessions of Triticum speltoides L. The average genetic similarity between all possible pairs of selected accessions was found to be 55% and ranged from 44% to 88%. Comparison of DNA extracted from different single seedlings within the same accession revealed significant intraaccession genetic diversity (4-24%), although this was much less than that observed between accessions tested. This intraaccession diversity has significant implications for the selection of T. speltoides accessions in breeding or screening programs.

Hydrogen peroxide yields during the incompatible interaction of tobacco suspension cells inoculated with Phytophthora nicotianae.

Rates of H(2)O(2) production by tobacco suspension cells inoculated with zoospores from compatible or incompatible races of the pathogen Phytophthora nicotianae were followed by direct measurement of oxygen evolution from culture supernatants following catalase addition. Rates of HO(2)(*)/O(2)(-) production were compared by following the formation of the formazan of sodium, 3'-[1-[phenylamino-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate. In the incompatible interaction only, both reactive oxygen species (ROS) were produced by the cultured host cells in a minor burst between 0 and 2 h and then in a major burst between 8 and 12 h after inoculation. Absolute levels of H(2)O(2) could not be accurately measured due to its metabolism by host cells, but results are consistent with the majority of H(2)O(2) being formed via dismutation of HO(2)(*)/O(2)(-). The effects of inhibitors of endogenous Cu/Zn superoxide dismutase (diethyldithiocarbamate) and catalase (3-amino-1,2,4-triazole and salicylic acid) were also examined. Yields of ROS in the presence of the inhibitors diphenylene iodonium, allopurinol, and salicylhydroxamic acid suggest that ROS were generated in incompatible host responses by more than one mechanism.

The tetrazolium dyes MTS and XTT provide new quantitative assays for superoxide and superoxide dismutase.

The tetrazolium dyes MTS and XTT were reduced to their soluble formazans by superoxide radical anions (O2-) produced by the oxidation of xanthine by xanthine oxidase under standard conditions. These reactions were compared to the well-known reductions of NBT and cytochrome c by the xanthine/xanthine oxidase system. Reduction of the dyes was completely inhibited by superoxide dismutase (SOD). Rate constants for the reaction of MTS and XTT with O2- were estimated at 1.3 +/- .1 x 10(5) M-1S-1 and 8.6 x 10(4) M-1S-1 respectively. The stable MTS and XTT formazans have high extinction coefficients in the visible range which enable sensitive detection and quantification of superoxide radicals, avoiding some of the problems inherent in assays based on production of the insoluble NBT formazan. MTS and XTT have considerable potential both for the quantitative assay of radical production in living tissues and for the assay of superoxide dismutase activity in tissue extracts. Implications for the interpretation of cell culture growth assays which employ these dyes are discussed.

The prevention of violent spinal cord injuries.

Interpersonal violence has been defined by the USPHS as a national critical health care problem. Of the 10,000 to 12,000 new spinal cord injuries in the United States each year, about a 1,000 will be caused by gunshot wounds. Violence is identified as part of our heritage, inherent in our entertainment and, sadly, part of our everyday life. Community response to these intentional injuries has indicated they will no longer be ignored. Conventional prevention efforts are often not relevant. Violence prevention efforts require the focus to be upon the critical precipitating factors. The development of school, community and youth-based violence-specific injury prevention programs are essential.

Effects of t-butyl hydroperoxide on NADPH, glutathione, and the respiratory burst of rat alveolar macrophages.

The effects of t-butyl hydroperoxide on glutathione and NADPH and the respiratory burst (an NADPH-dependent function) in rat alveolar macrophages was investigated. Alveolar macrophages were exposed for 15 min to t-butyl hydroperoxide in the presence or absence of added glucose. Cells were then assayed for concanavalin A-stimulated O2 production or for NADPH, NADP, reduced glutathione, glutathione disulfide, glutathione released into the medium and glutathione mixed disulfides. Exposure of rat alveolar macrophages to 1 X 10(-5) M t-butyl hydroperoxide causes a loss of concanavalin A-stimulated superoxide production (the respiratory burst) that can be prevented or reversed by added glucose. Cells incubated without glucose had a higher oxidation state of the NADPH/NADP couple than cells incubated with glucose. With t-butyl hydroperoxide, NADP rose to almost 100% of the NADP + NADPH pool; however, addition of glucose prevented this alteration of the NADPH oxidation state. Cells exposed to 1 X 10(-5) M t-butyl hydroperoxide in the absence of glucose showed a significant increase in the percentage GSSG in the GSH + GSSG pool and increased glutathione mixed disulfides. These changes in glutathione distribution could also be prevented or reversed by glucose. With 1 X 10(-4) M t-butyl hydroperoxide, changes in glutathione oxidation were not prevented by glucose and cells were irreversibly damaged. We conclude that drastic alteration of the NADPH/NADP ratio does not itself reflect toxicity and that significant alteration of glutathione distribution can also be tolerated; however, when oxidative stress exceeds the ability of glucose to prevent alterations in oxidation state, irreversible damage to cell function and structure may occur.

Selenium deficiency potentiates paraquat-induced lipid peroxidation in isolated perfused rat lung.

Glutathione peroxidase (GSHPx), a seleno-enzyme, reduces lipid hydroperoxides while producing oxidized glutathione (GSSG), which can efflux from cells. To study the role of GSHPx in antioxidant defense, isolated lungs from selenium-deficient rats were perfused for 2 h with or without 1 mM paraquat. Perfusate GSSG was measured as an index of GSHPx activity, and malondialdehyde (MDA) as an index of lipid peroxidation. Selenium deficiency decreased lung GSHPx activity 75-80%. During perfusion control lungs showed GSSG efflux of 8.5 +/- 4.5 nmol/h and with paraquat 49.1 +/- 12.1 nmol/h. Selenium-deficient lungs with or without paraquat showed GSSG efflux of 16.4 +/- 5.3 and 13.7 +/- 8.9 nmol/h, respectively. MDA efflux occurred only in paraquat-perfused selenium-deficient lungs (7.8 +/- 2.7 nmol/h). Lung homogenates from this group had lower GSH + GSSG than the other three groups. These results indicate an inverse correlation between GSSG efflux and MDA accumulation from paraquat-perfused lungs and suggest that increased turnover of the GSHPx reaction protects paraquat-perfused lungs from lipid peroxidation.

Oxygen toxicity: loss of lung macrophage function without metabolite depletion.

Hyperoxia inhibited concanavalin A stimulated O2- release (respiratory burst) of alveolar macrophages obtained by bronchoalveolar lavage from rats. After 36 h of normobaric 100% O2, a partial reversal (48%) of the inhibition was produced by addition of glucose. Since oxidant-induced, reversible NADPH depletion correlates with reversible inhibition of the respiratory burst, intracellular NADPH was assayed to determine whether irreversible inhibition of the respiratory burst was related to persistent changes in this metabolite. The cellular concentrations of ATP, glutathione, and ascorbate were also measured. After 36 h of hyperoxia, NADPH concentration in alveolar macrophages rose slightly while ATP and glutathione content remained at control levels. Ascorbate levels fell significantly but were not responsible for respiratory burst inhibition. Thus, irreversible loss of cellular function in hyperoxia is not due to persistent alterations in these metabolites. Significant amounts of both glutathione and ascorbate were found in extracellular fractions of lung washings, indicating high concentrations in the aqueous subphase in the lung fluid lining. There was no change in total content of these extracellular antioxidants following O2 exposure.

Studies of the reactivity of HO2/O2- with unsaturated hydroperoxides in ethanolic solutions.

A study of the reactivity of HO2/O2- with unsaturated hydroperoxides/peroxides was carried out in a stopped-flow spectrophotometer equipped with an O2--generating plasma lamp. The results show that, in 80% aqueous ethanol solution containing either 0.05 M H2SO4 (for HO2 studies) or 0.005 M KOH (for O2- studies), these oxy-radicals do not react with oleic acid hydroperoxide, linoleic acid hydroperoxide, 1-hydroperoxy-2-cyclooctene, and tert-butyl allyl peroxide. These findings are discussed in the light of conflicting evidence concerning the reaction of HO2/O2- with organic hydroperoxides/peroxides.

A study of the reactivity of HO2/O2- with unsaturated fatty acids.

The reaction of perhydroxyl radical (HO2) with linoleic, linolenic, and arachidonic acids has been studied in aqueous ethanolic solutions by the stopped flow technique. The corresponding rate constants are 1.2 x 10(3), 1.7 x 10(3), and 3.0 x 10(3) M-1 S-1, respectively. While kinetic results suggest that the HO2 radical reacts with the double allylic H atom of the polyunsaturated fatty acids, thermodynamic approximations indicate that the reaction is exothermic by approximately 10 kcal/mol. The relevance of this reaction to membrane damage observed in biological systems that have been exposed to HO2/O2- radicals is discussed.

Purification of oleic acid and linoleic acid.

To permit kinetic studies of the reactivity of unsaturated fatty acids towards oxygen radicals, it is essential to remove traces of hydroperoxides and other conjugated lipid impurities commonly present in commercial samples. Removal of these impurities has been satisfactorily achieved for oleic and linoleic acids by anaerobic low temperature recrystallization from acetonitrile. The UV spectra of commercial and purified samples are compared.