Development of Stem-base Pathogens on Different Cultivars of Winter Wheat Determined by Quantitative PCR

The progress of development of stem-base pathogens in crops of second winter wheat was plotted in nine experiments in three years. The amount of each pathogen present was determined by quantitative PCR. Where Tapesia yallundae was present in quantifiable amounts, it usually developed earlier than the other eyespot pathogen, T. acuformis. Both species were usually present in greater amounts on cultivars which are more susceptible to eyespot. The sharp eyespot pathogen, Rhizoctonia cerealis, developed more erratically than either of the Tapesia spp. and there were no consistent effects on different cultivars. Fusarium spp., the cause of brown foot rot, were rarely present in quantifiable amounts, but Microdochium nivale was usually present as one or both of the varieties nivale and majus. Late-season (after anthesis) decreases in M. nivale suggest that any brown foot rot symptoms attributable to this fungus would have fully developed earlier. Cultivar differences in amounts of M. nivale were most clear in stems during internode extension and when relatively large amounts of DNA were present. Such differences approximately reflected eyespot susceptibility, cv. Soissons containing most and cv. Lynx containing least DNA. The results emphasise the difficulty in relating diagnoses, by quantitative PCR or other means, at early growth stages when decisions to apply fungicides against stem-base disease are made, to later disease severity.     

Repression of deoxynivalenol accumulation and expression of Tri genes in Fusarium culmorum by fungicides in vitro

A defined medium was developed in which to monitor deoxynivalenol (DON) accumulation, fungal growth and expression of genes involved in trichothecene biosynthesis (designated Tri genes). In liquid culture, DON accumulated shortly after maximum expression of Tri6 and coincident with expression of Tri5. This was generally 96 h after inoculation. The effects of sublethal concentrations of the fungicides azoxystrobin, trifloxystrobin, kresoxim-methyl and tebuconazole on biosynthesis of the trichothecene DON by Fusarium culmorum were studied using this medium. The strobilurin fungicides trifloxystrobin and azoxystrobin significantly reduced the accumulation of DON in culture medium at a range of concentrations. Kresoxim-methyl, also a strobilurin, and tebuconazole, a triazole, did not significantly reduce the accumulation of DON, although levels were lower than those in nonamended cultures. Trifloxystrobin significantly reduced the accumulation of DON when added to cultures before initiation of trichothecene biosynthesis. RT-PCR assays of the expression of Tri6 and Tri5 genes indicated that trifloxystrobin acted by inhibiting the initiation of trichothecene biosynthesis.     

Accumulation of sorghum phytoalexins induced by Colletotrichum graminicola at the infection site

Microspectrophotometry was performed on intact, pigmented vesicle-like inclusions within living sorghum cells that were accumulating phytoalexins as a response to attempted fungal infection. The results indicate that the deoxyanthocyanidin phytoalexins are present in inclusions. Moreover, the phytoalexin concentration within a single inclusion, based on luteolinidin, was calculated to be 0·15 m. The amounts of luteolinidin and apigeninidin in cells involved in the phytoalexin response at individual infection sites were also determined. The data showed that luteolinidin accumulated to levels of 0·48–1·20 ng/cell whereas apigeninidin accumulated to levels of 0·24–0·91 ng/cell. The results of both analyses confirmed that at the infection site the deoxyanthocyanidins accumulate to levels in substantial excess of those required for inhibition of the fungus Colletotrichum graminicola.     

Antifungal Activity Toward Fusarium culmorum in Soluble Wheat Extracts

ABSTRACT This study investigated antifungal activity in soluble extracts from seed of a range of wheat cultivars differing in susceptibility to Fusarium head blight. Antifungal activity was assessed in terms of beta-D-glucuronidase (GUS) activity of a Fusarium culmorum GUS transformant using a sensitive laboratory assay. Significant antifungal activity was detected in seed extracts from WEK0609, CM 820036, and Arina. Initial characterization of the Arina seed extract indicated that it contained antifungal proteinaceous compounds. The Arina extract yielded two (60 and 80%) ammonium sulfate fractions containing inhibitory compounds. Gel filtration chromatography and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of antifungal fractions showed that the antifungal activities detected in the Arina 60 and 80% ammonium sulfate fractions were associated with putative proteinaceous compounds with apparent molecular masses of approximately 60 and 28 kDa, respectively.     

Mutation and virulence assessment of chromosomal genes of Rhodococcus equi 103

Rhodococcus equi can cause severe or fatal pneumonia in foals as well as in immunocompromised animals and humans. Its ability to persist in macrophages is fundamental to how it causes disease, but the basis of this is poorly understood. To examine further the general application of a recently developed system of targeted gene mutation and to assess the importance of different genes in resistance to innate immune defenses, we disrupted the genes encoding high-temperature requirement A (htrA), nitrate reductase (narG), peptidase D (pepD), phosphoribosylaminoimidazole-succinocarboxamide synthase (purC), and superoxide dismutase (sodC) in strain 103 of R. equi using a double-crossover homologous recombination approach. Virulence testing by clearance after intravenous injection in mice showed that the htrA and narG mutants were fully attenuated, the purC and sodC mutants were unchanged, and the pepD mutant was slightly attenuated. Complementation with the pREM shuttle plasmid restored the virulence of the htrA and pepD mutants but not that of the narG mutant. A single-crossover mutation approach was simpler and faster than the double-crossover homologous recombination technique and was used to obtain mutations in 6 other genes potentially involved in virulence (clpB, fadD8, fbpB, glnA1, regX3, and sigF). These mutants were not attenuated in the mouse clearance assay. We were not able to obtain mutants for genesfurA, galE, and sigE using the single-crossover mutation approach. In summary, the targeted-mutation system had general applicability but was not always completely successful, perhaps because some genes are essential under the growth conditions used or because the success of mutation depends on the target genes.     

A rapid method for estimating longitudinal growth stresses in logs

Summary A method of rapidly determining the longitudinal growth stress present on the surface of logs and standing trees is described. Application of the technique will enable detailed examination of stress present about the circumference and along the length of trees and logs. In addition it will enable comparisons to be made between trees, as well as monitoring changes occurring in a particular log as the result of treatment to reduce stress. Finally, its use will enable the selection of low stressed trees for genetic studies and propagation trials.The author would like to thank members of the staff of the Division, and in particular Mr. J. Barnacle, for valuable discussion relating to this work. In addition, thanks are due to Mr. K. Murray for participation in the design and construction of the equipment described.     

Multiple‐Aetiology Enteric Infections Involving Non‐O157 Shiga Toxin‐Producing Escherichia coli – FoodNet, 2001–2010

We describe multiple‐aetiology infections involving non‐O157 Shiga toxin‐producing Escherichia coli (STEC) identified through laboratory‐based surveillance in nine FoodNet sites from 2001 to 2010. A multiple‐aetiology infection (MEI) was defined as isolation of non‐O157 STEC and laboratory evidence of any of the other nine pathogens under surveillance or isolation of >1 non‐O157 STEC serogroup from the same person within a 7‐day period. We compared exposures of patients with MEI during 2001–2010 with those of patients with single‐aetiology non‐O157 STEC infections (SEI) during 2008–2009 and with those of the FoodNet population from a survey conducted during 2006–2007. In total, 1870 non‐O157 STEC infections were reported; 68 (3.6%) were MEI; 60 included pathogens other than non‐O157 STEC; and eight involved >1 serogroup of non‐O157 STEC. Of the 68 MEI, 21 (31%) were part of six outbreaks. STEC O111 was isolated in 44% of all MEI. Of patients with MEI, 50% had contact with farm animals compared with 29% (P < 0.01) of persons with SEI; this difference was driven by infections involving STEC O111. More patients with non‐outbreak‐associated MEI reported drinking well water (62%) than respondents in a population survey (19%) (P < 0.01). Drinking well water and having contact with animals may be important exposures for MEI, especially those involving STEC O111.