Virulent and fungicide-tolerant races of loose smut (Ustilago nuda and U. tritici) in Ireland

Following the severe outbreaks of loose smut in the early 1980s, a survey was carried out over the period l984-86of the Ustilago nuda and U. tritici populations in the Republic of Ireland. U. tritici was very rare, only one infected wheat plant being reported in the 3 years of the survey. This fungus was represented by a virulent race distinct from those identified in Britain in the 1950s. U. nuda was widespread, and two races were identified in 1984. A virulent race, capable of overcoming the recessive resistance gene present in differential variety Missouri Early Beardless, was isolated from a single crop grown from imported seed. Several crops grown from seed treated with Vitavax (active ingredient carboxin), which had been imported from France, contained a carboxin-tolerant race of U. nuda. Subsequent surveys in 1985 and 1986 failed to recover these U. nuda races. The role of imported infected seed in the introduction of new races of the loose smut fungi is discussed.     

Control of loose smut (Ustilago nuda and U. tritici) infections in barley and wheat plants by foliar application of triadimefon

Foliar application of the broad-spectrum systemic fungicide triadimefon to loose smut-infected plants of wheat and barley produced a marked curative effect. Triadimefon was particularly effective when applied at the beginning of and during stem extension. In addition to reducing percentage plant infection, foliar application of this fungicide (particularly prior to flag leaf emergence) resulted in an increased proportion of infected ears which were only partially smutted. The curative effect of these early fungicide applications resulted in significant yield increases, as a consequence of increased grain number per m². Application of triadimefon to crops at ear emergence had no significant effect on percentage plant infection but resulted in lower levels of re-infection following artificial or natural inoculation of healthy ears, apparently as a result of a protectant effect of fungicide residues on or in the ovaries.     

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of loose smut of barley (Ustilago nuda)

Polyclonal antibodies were raised against mycelium from the logarithmic growth phase of a shake culture of Ustilago nuda, and a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) with biotinylated detection antibodies was developed. The detection limit of the assay was 15 ng total protein ml^–1 for the homologous antigen and 50 ng ml^–1 for a spore extract, respectively. Other species of Ustilago reacted with the antibodies. Cross-reactivity was highest with U. tritici. No signal was obtained with the tested isolates of Tilletia, Rhizoctonia, Pythium and Fusarium. With naturally infected barley seeds, the results of the ELISAs were always in good agreement with those obtained with the routinely used seed embryo test. However, when seeds grown from artificially inoculated florets were used, the ELISA indicated significantly higher infestation levels than the embryo test. Results of assays with halved seeds from the same lot showed that high amounts of mycelium were present in the non-embryo half. This and especially the relatively long duration of the assay suggested that the ELISA (as conducted here) may not be suitable as a routine method for analysing seed infection with U. nuda. With samples from barley seedlings grown from infected seeds the results of the immunoassay again corresponded very well with the infection level determined by staining of the seed embryo, irrespective of the mode of floret inoculation (natural or artificial). Potential fields of application of the ELISA include the early prediction of the efficacy of protection agents, e.g. in screenings for seed treatments, the elucidation of the biology of the fungus and characterisation of resistance mechanisms.     

Assessment of the loose smut fungi (Ustilago nuda and U. tritici) in tissues of barley and wheat by fluorescence microscopy and real-time PCR

Loose smut fungi of barley and wheat (Ustilago nuda and U. tritici, respectively) colonize the plant without causing obvious disease symptoms before heading. The availability of diagnostic methods to detect and follow the growth of these pathogens in the plant would therefore be highly advantageous for both resistance breeding and the development of effective seed treatments. Using seed lots of barley and wheat highly infected with loose smut, we studied the early establishment of the loose smut pathogens in the plant by fluorescence microscopy. In hand-cut sections stained with the fluorochrome Blankophor?, fungal hyphae were observed to invade the shoot apical meristem and leaf primordia during the first days after the onset of germination. At the first node stage the ear and leaf primordia were generally extensively colonized. Hyphae of U. nuda were also regularly observed in high density in the nodes. A protocol was developed for the specific amplification of U. nuda and U. tritici DNA extracted from infected plant tissue. PCR screening of U nuda in seedlings from infected and healthy seed lots was compared to ELISA, microscopy and ultimately head infection of mature plants derived from tillers of the tested seedlings. The results indicated that a prediction of loose smut infection by real-time PCR is possible at the second leaf stage, and that the assay is equally suited for use with spring and winter varieties of barley and wheat.     

Detection of virulence to septoria tritici blotch (STB) resistance conferred by the winter wheat cultivar Cougar in the Irish Zymoseptoria tritici population and potential implications for STB control

Septoria tritici blotch (STB) caused by the fungal pathogen Zymoseptoria tritici continues to be the most economically destructive disease of winter wheat throughout Ireland. Due to the widespread development of fungicide resistance in the Irish Z. tritici population, integrated strategies to control STB are increasingly necessary. A key component of such strategies will be the deployment of winter wheat cultivars with improved levels of STB resistance. Unfortunately, due to the nature of Z. tritici, such resistances are at risk of being overcome by the pathogen. In late summer 2020, foci of STB were observed across a range of winter wheat cultivars under evaluation for recommendation in Ireland. Common amongst these was the cultivar Cougar in each of their pedigree. To determine if the foci observed in 2020 resulted from strains virulent to Cougar, isolate collections were established and virulence screens conducted on Cougar and a range of the cultivars currently under evaluation. These confirmed the presence of Cougar-virulent strains in the Irish Z. tritici population, and that this virulence affects not just Cougar, but also cultivars derived from it. Although the foci observed in 2020 were in both fungicide-untreated and -treated plots, there was no evidence that these strains are more sensitive or resistant to fungicides compared to the wider Irish Z. tritici population, with moderate resistance to the SDHIs and azoles dominating. Combined, the present study confirms the need to ensure a diversity of control measures for STB, including ensuring a range of STB resistances are used.     

Systemic resistance to bacterial leaf speck pathogen in Arabidopsis thaliana induced by the culture filtrate of a plant growth-promoting fungus (PGPF) Phoma sp. GS8-1

The culture filtrate (CF) from the plant growth-promoting fungus Phoma sp. GS8-1 was found to induce systemic resistance in Arabidopsis thaliana against the bacterial leaf speck pathogen Pseudomonas syringae pv. tomato DC3000 (Pst), and the underlying mechanism was studied. Roots of A. thaliana were treated with CF from GS8-1, and plants expressed a clear resistance to subsequent Pst infection; disease severity was reduced, and proliferation of pathogen was suppressed. Various mutants of A. thaliana were used to test whether the CF induced resistance through one of the known signaling pathways: salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). The CF was fully protective against Pst in Arabidopsis mutants jar1 and ein2 similar to wild-type plants. However, its efficacy was reduced in plants containing transgene NahG. Examination of systemic gene expression revealed that CF modulates the expression of SA-inducible PR-1, PR-2 and PR-5 genes, the JA/ET-inducible ChitB gene, and the ET-inducible Hel gene. Moreover, the expression of these genes was further enhanced upon subsequent stimulation after attack by Pst. Our data suggest that in addition to a partial requirement for SA, the signals JA and ET may also play a role in defense signaling in Arabidopsis.     

Explaining variation in the effects of take-all (Gaeumannomyces graminis var. tritici) on nitrogen and water uptake by winter wheat

Two field experiments, in 1999 and 2000, were used to test whether reductions in root growth and function explained the effects of take-all on crop water and nitrogen uptake. The fungicide silthiofam was used to manipulate take-all independently of other factors. Soil water content was manipulated from heading to determine effects on disease progress and resource capture. Epidemic progress was significantly delayed in the presence of silthiofam, leading to reductions in disease in both experiments. Effects of silthiofam were reduced by increasing soil water late in the season, although only in 2000 did increased soil water content have a direct effect, leading to a higher rate of disease increase. Higher levels of disease in the absence of silthiofam did not affect root growth as measured by total root length density (TRLD), but did lead to significantly reduced healthy root length density (HRLD, a measure of functional roots) in both experiments. Only in 2000 were there any significant effects of increased take-all on water and nitrogen uptake. This was attributed to the higher TRLD in the (1999) crop, which allowed HRLDs to be maintained above a critical threshold (where water and nitrogen uptake start to be severely affected) despite loss of functional root to disease. The effects of take-all on nitrogen uptake were more likely to affect crop canopy size and duration than the relatively small effects observed on water uptake. Increasing soil water content allowed the crop to take up more water in absolute terms despite, in 2000, increasing levels of disease and reducing HRLD.