The development of LENTICULES™ as reference materials for noroviruses.

AIMS: To investigate the potential for LENTICULES™ to act as reference materials (RMs) for noroviruses (NoV) [genogroups I (GI) and II (GII)] by determining their homogeneity and stability characteristics. METHODS AND RESULTS: NoV used in this study originated from human faecal material, screened for the absence of other faecally transmitted pathogens. The norovirus strains present in the faecal material were characterized by sequencing, and samples containing GI and GII strains representative of genotypes commonly circulating in the community were selected. RMs were produced utilizing modified lenticulating technology. A batch comprising 500 LENTICULES™ containing both norovirus genogroups was produced according to ISO Guide 34. The batch was tested and quantified using an ISO 17025 accredited quantitative real-time RT-PCR assay. Sufficient homogeneity was established using procedures described by Fearn and Thompson (2010), while stability at less than -15°C and ambient temperature (17-22°C) was assessed over 52 weeks and 7 days, respectively. CONCLUSIONS: Lenticulation was shown to be an effective means of preservation of detectable NoV. LENTICULES™ were sufficiently homogeneous and stable throughout medium-term frozen and short-term storage at room temperature to serve as RMs. Virus LENTICULES™ have the advantages of being easy to manipulate, provide assigned values and do not require the manipulation of high titre clinical material. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study show that norovirus LENTICULES™ can be used as stable RMs for quantitative real-time RT-PCR assays. They can be utilized as in-run positive extraction controls and potentially for method calibration and to enable more easy comparison of data generated by the variety of differing norovirus determination methods that have emerged in recent years. LENTICULES™ have the potential to provide essential elements of laboratory quality assurance systems for laboratories implementing these new methods for virus testing in foodstuffs and for those running routine analyses.

Quantitative trait loci in the Ogle/TAM O-301 oat mapping population controlling resistance to Puccinia coronata in the field.

Crown rust is the most damaging disease of cultivated oat (Avena sativa) and genetic resistance is the primary means of controlling the disease. Quantitative trait loci (QTL) with major and minor effects have been identified in Ogle1040 and TAM O-301 (most notably, Pc58 and PcNQMG/LGCG from TAM O-301 and OT-27 from Ogle1040) through single-isolate greenhouse and field tests. To map loci and determine the effectiveness of previously identified QTL against naturally occurring pathogen populations in highly disease-conducive environments, the Ogle/TAM O-301 (OT) recombinant inbred line (RIL) population was grown in Texas and Louisiana over 2 years and in Manitoba, Canada. The genetic region characterized by the Pc58 resistance gene complex, particularly Pc58a, accounted for most of the diseased leaf area (DLA) and infection type (IT) variance in all five experiments. Additionally, the genetic region characterized by PcNQMG/LGCG accounted for a portion of the IT variance in three experiments. Although no QTL was detected on OT-27 in this study, all the markers on this linkage group were associated (P < 0.0001) with reducing both IT and DLA using single-marker analysis. Screening with 25 Puccinia coronata isolates from six different states indicated that Pc58abc and Pc58a were highly effective, while characterization using F(2) populations derived from OT RILs containing the two main genetic regions responsible for crown rust resistance in TAM O-301 (Pc58 and PcNQMG/LGCG) and a minor QTL in Ogle (OT-27) indicated that Pc58a, in combination with a locus in Ogle1040, provided high levels of resistance to natural races in Texas. This study provides new information and key loci in OT mapping population and may be useful for effective control of crown rust in North America.

Detached-Leaf Method for Propagating Puccinia coronata and Assessing Crown Rust Resistance in Oat.

The crown rust pathogen Puccinia coronata is an obligate biotroph with wind-disseminated propagules and numerous races. These characteristics make propagation of single-race cultures difficult. Genetic studies using single races in field and greenhouse environments are also problematic because pure cultures can easily become contaminated. In this study, we developed an isolated propagation system for P. coronata and tested its ability to assess host resistance. Oat (Avena sativa) leaf sections (10 cm each) were harvested, disinfested, and suspended in sterile plastic boxes by enclosing 3.5-cm linear sections of each leaf end between 4% agar blocks amended with various chemical constituents. The exposed sections (approximately 3 cm) were inoculated with P. coronata urediniospores suspended in water. Boxes were sealed and incubated in a lighted growth cabinet until the pathogen sporulated. Viable spores were produced on leaves in all treatments, whereas 6-benzylaminopurine (BAP) and kinetin treatments sustained the leaves longer and yielded the most viable spores. Based on these data, the BAP treatment was adopted and used for additional studies. Detached leaves of differential oat cultivars produced the same reactions as whole plants screened under standard conditions in a growth chamber. The proposed detached-leaf system should be useful for the propagation of numerous single-race cultures of P. coronata as well as evaluation of host resistance under highly controlled conditions.

Characterization and mapping of oat crown rust resistance genes using three assessment methods.

ABSTRACT Resistance is the primary means of control for crown rust of oat (Avena sativa L.), caused by Puccinia coronata f. sp. avenae, and better knowledge of the genetics of resistance will enhance resistance breeding. Disease data were generated in the field and greenhouse for parents and recombinant inbred lines of the Ogle/TAM O-301 (OT) oat mapping population using (i) a new quantitative assay that employs quantitative real-time polymerase chain reaction (q-PCR) to estimate fungal growth in the host, (ii) digital image analysis, and (iii) visual ratings. The objectives of this study were to evaluate each assessment method's ability to map a major gene from cv. Ogle and potential quantitative trait loci (QTL) contributed by Ogle and TAM O-301. All three assessment methods identified the major gene in Ogle, which was mapped to linkage group OT6. The resolution produced by q-PCR, however, enabled more precise mapping of the major gene. Quantitative analysis indicated that 64% of the phenotypic variation was accounted for using q-PCR, whereas 41 and 52% were accounted for using visual and digital assessments, respectively. Data generated by q-PCR permitted identification of QTL on linkage groups OT32, accounting for 6% of the phenotypic variation, and OT2, accounting for 4% of the variation. QTL on both OT32 and OT2 were conferred by TAM O-301, one of which (OT2) was indiscernible using data from the visual and digital assessments. The new method of precisely phenotyping crown rust resistance provided a more accurate and thorough means of dissecting resistance in the OT mapping population. Similar methods could be developed and applied to other important cereal rust diseases.

A Quantitative Assay of Puccinia coronata f. sp. avenae DNA in Avena sativa.

Crown rust, caused by Puccinia coronata f. sp. avenae, is the most damaging disease of oat. Quantification of the disease can be done by visual or digital assessments of diseased leaf area, lesion number, lesion size, and latent period. Laborious measurements of sporulation can also be made. As an alternative to these methods, a new quantitative assay was developed. The method employs simple inoculum application, quantitative sampling from inoculated areas, a closed tube DNA extraction method restricting loss of tissue, and real-time polymerase chain reaction (PCR) using a pathogen-specific TaqMan primers/probe set. Image analyses of genotypes with varying levels of crown rust resistance were compared to fungal DNA (FDNA) estimations by the new assay. The moderately resistant genotype IA98822-2 was distinguished from susceptible genotypes at the seedling stage, and the moderately resistant genotype O × T 107 was distinguished from the resistant cultivar TAM-O-301 at seedling and adult plant stages using FDNA. These separations were not evident with digital image analysis. The new assay also detected fungal development earlier and more rapidly in genotypes with lower levels of resistance. The assay can consistently estimate disease and should be useful for studying many aspects of the crown rust host-pathogen interaction where precise assessment of pathogen development is needed.