Ken C. Goulter

Marker-assisted selection for two rust resistance genes in sunflower

In this study we report on the identification of molecular markers, OX20600 and OO04950, linked to the geneRAdv in the proprietary inbred line P2. This gene confers resistance to most of the pathotypes of Puccinia helianthi identified in Australia. Analysis indicates these RAPD markers are linked to the resistance locus at 0.0 cM and 11 cM respectively. SCAR markers SCX20600 and SCO04950 derived from these two RAPD markers, and SCT06950 derived from a previously reported RAPD marker linked at 4.5 cM from the R1 rust resistance gene were developed. SCX20600 and SCO04950 were linked at similar distances from their resistance locus as the RAPD markers. SCTO6950 co-segregated completely with rust resistance. The robustness of the R1 SCAR marker was demonstrated through the amplification of the marker in a diverse range of sunflower germplasm considered to possess the R1 gene. The SCAR markers forRAdv were not amplified in the sunflower rust differential set thereby supporting the contention that this is a novel resistance gene. They did amplify in a number of proprietary lines closely related to the line P2. This locus is under further investigation as it will be useful in our attempts to use molecular-assisted breeding to produce durable resistance in sunflower to P. helianthi.

Induction of Cell Death in Transgenic Plants Expressing a Fungal Glucose Oxidase

Hydrogen peroxide (H2O2) has been implicated in the induction of plant defense genes and programmed cell death. Expression of a chimeric fungal glucose oxidase (GO) gene driven by a pathogen- and wound-inducible promoter was evaluated in transgenic tobacco and canola as a possible tool for engineering plant cell death and defense gene induction. Expression of this gene under the control of a peroxidase gene promoter resulted in the accumulation of relatively low levels of H2O2 in the young leaves of transgenic tobacco plants and this was not sufficient to cause any visible cell death and defense gene induction as measured by PR-1a mRNA induction. Older leaves of transgenic tobacco plants, however, exhibited visible necrotic lesions and constitutively expressed PR-1a mRNA when grown under high light conditions. Inoculation of cotyledons of control and transgenic canola with Leptosphaeria maculans resulted in rapid cotyledon senescence in the transgenic plants. Strong activators of the peroxidase promoter, ...

Enhanced quantitative resistance to Leptosphaeria maculans conferred by expression of a novel antimicrobial peptide in canola (Brassica napus L.)

The novel antimicrobial peptide MiAMP1, originally isolated from the seeds of Macadamia integrifolia, was constitutively expressed in transgenic tobacco and canola plants to test its effect on disease resistance. Analysis of plants transformed with 35S-MiAMP1 construct by northern and western blot analyses demonstrated the presence of MiAMP1 mRNA and the mature peptide in the transgenic plants. The MiAMP1 purified from the leaves of transgenic plants was biologically active with the same in vitro antifungal activity as native MiAMP1 purified from the seeds of macadamia. The effect of MiAMP1 expression on the economically important canola pathogen Leptosphaeria maculans (causal agent of blackleg disease) was evaluated in comparison with an untransformed control line and an azygous segregant derived from one of the transgenic lines. Lesion development on the cotyledons of the inoculated canola seedlings was significantly reduced in the T2 progeny of seven independently transformed transgenic lines. These results suggested that, transgenic canola expressing MiAMP1 may be useful for the management of blackleg disease.

Expression of a pathogenesis-related peroxidase of Stylosanthes humilis in transgenic tobacco and canola and its effect on disease development

Abstract In this study, we have investigated the effect of the expression of a cDNA (Shpx6a) encoding a pathogenesis-related peroxidase in the tropical forage legume Stylosanthes humilis on fungal disease resistance in transgenic tobacco and canola plants. Constitutive expression of Shpx6a cDNA was obtained using the 35S CaMV promoter in transgenic tobacco and canola plants and resulted in two to three fold increases in total peroxidase activity using guaiacol as a substrate. The peroxidase encoded by Shpx6a was shown to be located in the apoplast. Inoculation experiments using fungal pathogens Phytophthora parasitica var nicotianae (black shank disease of tobacco) and Leptosphaeria maculans (blackleg disease of canola) showed that expression of Shpx6a peroxidase resulted a small (20–30%) but statistically significant reduction in lesion development in the progeny of transgenic tobacco and canola lines. The enhanced level of tolerance to fungal diseases in both species appears to correlate with the level of peroxidase activity across several lines of transgenic plants. These results provide evidence for a role for the Shpx6a peroxidase in plant defence and suggest that it may be employed as a component of strategies aimed at the engineering of disease resistance.

Carpogenic germination of Sclerotinia minor and potential distribution in Australia

This study confirms that Australian isolates of Sclerotinia minor can produce fertile apothecia and further demonstrates that ascospores collected from these apothecia are pathogenic to sunflower (Helianthus annuus). Sunflower is a known host of the related fungus Sclerotinia sclerotiorum and is grown in some regions where S. minor is known to occur. Head rot symptoms were produced following inoculation with S. minor ascospores. Predictive modeling using CLIMEX software suggested that conditions suitable for carpogenic germination of S. minor probably occur in Australia particularly in southern regions. Carpogenic germination is probably a rare event in northern regions and, if it does occur, probably does not coincide with anthesis in sunflower crops, therefore allowing disease escape.

Identification of Sclerotinia species

A variety of morphological and molecular characters were compared for their ability to separate the three plant pathogenic species that comprise the genus Sclerotinia: Sclerotinia sclerotiorum, Sclerotinia minor and Sclerotinia trifoliorum. Restriction fragment length polymorphism (RFLP) probes generated from cloned genomic DNA fragments of S. sclerotiorum were used for accurate species designation and to compare against other markers, before further use in population genetics and breeding studies. Other characters used for comparison included host species, sclerotial diameters, ascospore morphism and breeding type. Several RFLP probes, either singly or in combination, enabled clear separation of the Sclerotinia species. Sclerotial diameters remain a good criterion for separating S. minor from S. sclerotiorum and S. trifoliorum, but the host species criterion was inadequate for accurately differentiating the 3 species of Sclerotinia.

Diversity in the sunflower:Puccinia helianthi pathosystem in Australia

Sunflower rust caused by Puccinia helianthi is the most important disease of sunflower in Australia with the potential to cause significant yield losses in susceptible hybrids. Rapid and frequent virulence changes in the rust fungus population limit the effective lifespan of commercial cultivars and impose constant pressure on breeding programs to identify and deploy new sources of resistance. This paper contains a synopsis of virulence data accumulated over 25 years, and more recent studies of genotypic diversity and sexual recombination. We have used this synopsis, generated from both published and unpublished data, to propose the origin, evolution and distribution of new pathotypes of P. helianthi. Virulence surveys revealed that diverse pathotypes of P. helianthi evolve in wild sunflower populations, most likely because sexual recombination and subsequent selection of recombinant pathotypes occurs there. Wild sunflower populations provide a continuum of genetically heterogeneous hosts on which P. helianthi can potentially complete its sexual cycle under suitable environmental conditions. Population genetics analysis of a worldwide collection of P. helianthi indicated that Australian isolates of the pathogen are more diverse than non-Australian isolates. Additionally, the presence of the same pathotype in different genotypic backgrounds supported evidence from virulence data that sexual recombination has occurred in the Australian population of P. helianthi at some time. A primary aim of the work described was to apply our knowledge of pathotype evolution to improve resistance in sunflower to sunflower rust. Molecular markers were identified for a number of previously uncharacterised sunflower rust R-genes. These markers have been used to detect resistance genes in breeding lines and wild sunflower germplasm. A number of virulence loci that do not recombine were identified in P. helianthi. The resistance gene combinations corresponding to these virulence loci are currently being introgressed with breeding lines to generate hybrids with durable resistance to sunflower rust.

Aggressiveness among isolates of Sclerotinia sclerotiorum from sunflower

Isolates of Sclerotinia sclerotiorum differ significantly in aggressiveness on sunflower. In total, 120 isolates were collected from head and basal stem rots of sunflower in two locations in south-east Queensland, Australia. The inoculation of sunflower stems with mycelial plugs and the measurement of lesion development were used to compare aggressiveness between isolates. Rank ordering of isolates indicated differences, with a few isolates displaying high and low aggressive abilities. Differences in aggressiveness were more indicative of a continuous variation rather than discrete groups. Isolate aggressiveness did not correspond to the location of collection nor to the mode of pathogen reproduction or infection from which they were derived. Statistically significant differences between the isolates differed according to the statistical test employed, as different multiple comparison procedures had a greater influence in interpretation of aggressiveness than the isolates themselves. Results indicate individual isolates do not maintain discrete aggressive abilities across the multiple comparison procedures, with the exception of weakly aggressive isolates.

Population structure of Sclerotinia sclerotiorum on sunflower in Australia

Sunflower (Helianthus annuus) is host to infections by Sclerotinia sclerotiorum originating from either homothallic sexually-derived ascospores (stem and head rots) or asexually-derived sclerotia (root rot). While sunflower can be infected by either ascospores or sclerotia this study found no association between the genotypes found in lesions and the type of infection (stem, head or root rot). Multicopy Restriction Fragment Length Polymorphisms (RFLPs) showed individual sclerotia comprised of only one genotype, and that all eight ascospores within an ascus also had only one genotype. Mycelial Compatibility Groups (MCGs), Random Amplified Polymorphic DNAs (RAPDs), single and multicopy RFLP analyses all showed the majority of sunflower plants were infected by only one genotype. A sample of 250 isolates collected hierarchically from sunflowers in Queensland and New South Wales were shown to belong to one large genetic population of S. sclerotiorum. Temporal studies revealed genetic uniformity was maintained across years, further confirming one genetic population. A range of molecular markers were used to genotype 120 isolates, resulting in differing levels of resolution of a genotype. Between 13 and 24 genotypes were identified with similarities and differences in the assemblages of isolates within each genotype depending on the marker used.

Altered fungal sensitivity to a plant antimicrobial peptide through over-expression of yeast cDNAs

A yeast cDNA expression library was screened to identify genes and cellular processes that influence fungal sensitivity to a plant antimicrobial peptide. A plasmid-based, GAL1 promoter-driven yeast cDNA expression library was introduced into a yeast genotype susceptible to the antimicrobial peptide MiAMP1 purified from Macadamia integrifolia. Following a screen of 20,000 cDNAs, three yeast cDNAs were identified that reproducibly provided transformants with galactose-dependent resistance to MiAMP1. These cDNAs encoded a protein of unknown function, a component (VMA11) of the vacuolar H+-ATPase and a component (cytochrome c oxidase subunit VIa) of the mitochondrial electron transport chain, respectively. To identify genes that increased sensitivity to MiAMP1, the yeast cDNA expression library was introduced into a yeast mutant with increased resistance to MiAMP1. From 11,000 cDNAs screened, two cDNA clones corresponding to a ser/thr kinase and a ser/thr phosphatase reproducibly increased MiAMP1 susceptibility in the mutant in a galactose-dependent manner. Deletion mutants were available for three of the five genes identified but showed no change in their sensitivity to MiAMP1, indicating that these genes could not be detected by screening of yeast deletion mutant libraries. Yeast cDNA expression library screening therefore provides an alternative approach to gene deletion libraries to identify genes that can influence the sensitivity of fungi to plant antimicrobial peptides.