Mark W. Sutherland

The generation of oxygen radicals during host plant responses to infection

Recent evidence points to significant oxygen radical production by some plant tissues in response to pathogenic challenge. These findings have proved quite controversial, in part because of an inadequate appreciation of the behaviour of oxygen radicals in biological systems. This review critically discusses the evidence to date and outlines several potential roles for oxygen species in host-pathogen interactions. The production of oxygen radicals during plant defence responses is compared to the respiratory burst of mammalian phagocytic cells.

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.

A QTL located on chromosome 4A associated with dormancy in white- and red-grained wheats of diverse origin

Improved resistance to preharvest sprouting in modern bread wheat (Triticum aestivum. L.) can be achieved via the introgression of grain dormancy and would reduce both the incidence and severity of damage due to unfavourable weather at harvest. The dormancy phenotype is strongly influenced by environmental factors making selection difficult and time consuming and this trait an obvious candidate for marker assisted selection. A highly significant Quantitative Trait Locus (QTL) associated with grain dormancy and located on chromosome 4A was identified in three bread wheat genotypes, two white- and one red-grained, of diverse origin. Flanking SSR markers on either side of the putative dormancy gene were identified and validated in an additional population involving one of the dormant genotypes. Genotypes containing the 4A QTL varied in dormancy phenotype from dormant to intermediate dormant. Based on a comparison between dormant red- and white-grained genotypes, together with a white-grained mutant derived from the red-grained genotype, it is concluded that the 4A QTL is a critical component of dormancy; associated with at least an intermediate dormancy on its own and a dormant phenotype when combined with the R gene in the red-grained genotype and as yet unidentified gene(s) in the white-grained genotypes. These additional genes appeared to be different in AUS1408 and SW95-50213.

Construction of three linkage maps in bread wheat, Triticum aestivum

Genetic maps were compiled from the analysis of 160-180 doubled haploid lines derived from 3 crosses: Cranbrook × Halberd, CD87 × Katepwa, and Sunco × Tasman. The parental wheat lines covered a wide range of the germplasm used in Australian wheat breeding. The linkage maps were constructed with RFLP, AFLP, microsatellite markers, known genes, and proteins. The numbers of markers placed on each map were 902 for Cranbrook × Halberd, 505 for CD87 × Katepwa, and 355 for Sunco × Tasman. Most of the expected linkage groups could be determined, but 10-20% of markers could not be assigned to a specific linkage group. Homologous chromosomes could be aligned between the populations described here and linkage groups reported in the literature, based around the RFLP, protein, and microsatellite markers. For most chromosomes, colinearity of markers was found for the maps reported here and those recorded on published physical maps of wheat. AFLP markers proved to be effective in filling gaps in the maps. In addition, it was found that many AFLP markers defined specific genetic loci in wheat across all 3 populations. The quality of the maps and the density of markers differs for each population. Some chromosomes, particularly D genome chromosomes, are poorly covered. There was also evidence of segregation distortion in some regions, and the distribution of recombination events was uneven, with substantial numbers of doubled haploid lines in each population displaying one or more parental chromosomes. These features will affect the reliability of the maps in localising loci controlling some traits, particularly complex quantitative traits and traits of low heritability. The parents used to develop the mapping populations were selected based on their quality characteristics and the maps provide a basis for the analysis of the genetic control of components of processing quality. However, the parents also differ in resistance to several important diseases, in a range of physiological traits, and in tolerance to some abiotic stresses.

Curation of wheat maps to improve map accuracy and QTL detection

Three Australian doubled haploid populations were used to illustrate the importance of map curation in order to improve the quality of linkage maps and quantative trait locus (QTL) detection. The maps were refined and improved by re-examining the order of markers, inspection of the genetic maps in relation to a consensus map, editing the marker data for double crossovers, and determining estimated recombination fractions for all pairs of markers. The re-ordering of markers and replacing genotypes at double crossovers with missing values resulted in an overall decrease in the length of the maps. Fewer apparent genotyping errors, associated with the presence of double recombinants, were identified with restriction fragment length polymorphisms (RFLPs) than with other types of markers used in this study. The complications that translocations may cause in the ordering of markers and subsequent QTL analysis were investigated. QTL analysis using both the original and revised maps indicated that QTL peaks were more sharply located or had improved log-likelihood (LOD) scores in the revised maps. An accurate indication of the QTL peak and a significant LOD score are both essential for the identification of markers suitable for marker-assisted selection. Recommendations are provided for the improvement of the quality of linkage maps.

Production of reactive oxygen species during non-specific elicitation, non-host resistance and field resistance expression in cultured tobacco cells

We examined production of reactive oxygen species (ROS) and induction of cell death in tissue-cultured tobacco cells undergoing different disease resistance responses. A superoxide-dependent hypersensitive response occurs during both the race-specific resistance response of tobacco cells challenged with incompatible zoospores of Phytophthora nicotianae and during non-specific elicitation of tobacco cells challenged with Phytophthora glucan elicitors extracted from the fungal cell wall. Inhibition studies are consistent with dependence upon endogenous Ca2+ levels, and with involvement of NAD(P)H oxidase and peroxidases in production of ROS during both specific and non-specific elicitation. The patterns of resistance expression during non-host resistance or field resistance responses appear to be similar to race-specific resistance expression with regard to the timing and order of events. However, the intensity of the response is very much reduced. In contrast, during non-specific elicitation, these temporal patterns are significantly altered. The differences in timing, intensity and extent of responses during different modes of disease resistance expression indicate that stimulation of cultured plant cells with non-specific soluble fractions in order to model in planta events during plant / Oomycete and, by implication, plant / fungal interactions, has significant limitations.

An investigation of genetic variation among Australian isolates of Bipolaris sorokiniana from different cereal tissues and comparison of their abilities to cause spot blotch on barley

Bipolaris sorokiniana (teleomorph: Cochliobolus sativus), the causal agent of common rootrot (CRR) and foliar spot blotch (SB) diseases in barley and wheat, is an economically important fungal pathogen worldwide. However, the relationship between these two diseases is poorly understood. Differences within Australian B. sorokiniana populations were revealed by cluster analysis of amplified fragment length polymorphisms in genomic DNA of 48 B. sorokiniana isolates collected from the northern grain-growing region of Australia. Isolates collected from SB infections clustered apart from isolates collected from CRR infections. A subset of 31 B. sorokiniana isolates was assessed for their abilities to cause SB infections on barley leaves using a differential set of 15 barley genotypes and three other cereal species. The pathogen samples included 14 isolates from CRR infections of either wheat or barley and 14 isolates from SB infections of barley. Phenotypic experiments revealed that isolates of B. sorokiniana collected from barley SB infections showed a high level of pathogenic variability across the differential set. In contrast, isolates from CRR infections produced significantly less SB disease on inoculated barley leaves. Cluster analysis of the phenotypic infection response scores grouped isolates into three pathogenicity clusters demonstrating low, intermediate or high pathogenicity. The results of this study suggest divergence within Australian populations of B. sorokiniana in relation to host tissue specificity.

Confirmation of QTL mapping and marker validation for partial seedling resistance to crown rot in wheat line '2-49'

We have tested the efficacy of putative microsatellite single sequence repeat (SSR) markers, previously identified in a 2-49 (Gluyas Early/Gala) × Janz doubled haploid wheat (Triticum aestivum) population, as being linked to partial seedling resistance to crown rot disease caused by Fusarium pseudograminearum. The quantitative trait loci (QTLs) delineated by these markers have been tested for linkage to resistance in an independent Gluyas Early × Janz doubled haploid population. The presence of a major QTL on chromosome 1DL (QCr.usq-1D1) and a minor QTL on chromosome 2BS (QCr.usq-2B1) was confirmed. However, a putative minor QTL on chromosome 2A was not confirmed. The QTL on 1D was inherited from Gluyas Early, a direct parent of 2-49, whereas the 2B QTL was inherited from Janz. Three other putative QTLs identified in 2-49 × Janz (on 1AL, 4BL, and 7BS) were inherited by 2-49 from Gala and were not able to be confirmed in this study. The screening of SSR markers on a small sample of elite wheat genotypes indicated that not all of the most tightly linked SSR markers flanking the major QTLs on 1D and 1A were polymorphic in all backgrounds, indicating the need for additional flanking markers when backcrossing into some elite pedigrees. Comparison of SSR haplotypes with those of other genotypes exhibiting partial crown rot resistance suggests that additional, novel sources of crown rot resistance are available.

Superoxide release is necessary for phytoalexin accumulation in Nicotiana tabacum cells during the expression of cultivar-race and non-host resistance towards Phytophthora spp.

Abstract The relationship between superoxide radical generation, the accumulation of the phytoalexin capsidiol and hypersensitive cell death has been examined in Nicotiana tabacum following challenges by compatible and incompatible races of Phytophthora nicotianae , and the non-host species Phytophthora palmivora . Challenging suspension cell cultures of N. tabacum with zoospores of incompatible isolates of P. nicotianae elicits a biphasic burst of superoxide release. The maximum rate of capsidiol accumulation between 9 and 12 h after challenge coincides with the second oxidative burst and the onset of hypersensitive cell death. Addition of superoxide dismutase or Mn (III) desferal, which scavenge superoxide anions and quench the superoxide burst, suppresses both phytoalexin accumulation and hypersensitive cell death. Mevastatin, an inhibitor of the sesquiterpenoid biosynthesis enzyme HMG-CoA reductase, has no effect on the oxidative burst or hypersensitive cell death, but abolishes capsidiol accumulation. Zoospores of the non-host pathogen P. palmivora also elicit superoxide release, but in a single, broad burst between 3 and 12 h after challenge. Capsidiol accumulates to levels similar to those seen in incompatible host reactions, although the onset of capsidiol accumulation is more rapid in the non-host interaction. As in the incompatible interaction, phytoalexin accumulation and hypersensitive cell death are both inhibited by superoxide scavengers, although scavenging does not render host cells susceptible to infection by non-host zoospores. Our findings indicate that phytoalexin accumulation and hypersensitive cell death in both incompatible and non-host interactions are regulated by pathways that diverge downstream of superoxide release. While hypersensitive cell death and phytoalexin accumulation appear to be necessary for gene-for-gene resistance, other factors cause non-host infection to fail.

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.