Quantitative PCR and histopathological assessment of cereal infection by fusarium pseudograminearum

The assessment of crown rot (F. pseudograminearum) infections in winter cereals was explored using a quantitative polymerase chain reaction (PCR) approach. A range of cereal genotypes of varying resistance to crown rot were monitored during seedling and adult growth stages. A histopathological investigation of F. pseudograminearum (Fp) growth during pathogenesis in seedling and adult growth stages was also performed across a range of cereal genotypes. This utilised a novel staining method developed during this project. Visual assessment of crown rot symptoms in wheat seedlings is commonly conducted in order to rapidly identify resistant genotypes. Ratings rely heavily on discolouration of seedling tissues, predominantly the leaf sheaths. This study is the first to explore the relationship between seedling tissue discolouration and Fp DNA content in wheat genotypes of differing resistance. The partially resistant wheat 2-49 exhibited lower visual and qPCR values than the susceptible wheat Puseas. The rate of disease development and Fp growth in seedling leaf sheaths was slower in 2-49 than Puseas. The rates of symptom development in intermediate genotypes EGA Wylie and EGA Gregory were not significantly different from that recorded in 2-49. A comparison of visual ratings and qPCR values of Fp DNA indicated a strong correlation (r = 0.89, p < 0.01) between these characters at 14 days after inoculation across all genotypes. The correlation weakened over time. Furthermore, qPCR revealed differences between partially resistant and susceptible genotypes to a much greater extent than possible using visual discolouration. Crown rot infections of adult cereal stems are typically rated at maturity by recording the amount of discolouration on individual internodes. A comparison was performed between visual ratings and Fp DNA content of four cereal genotypes (two bread wheats, one durum wheat and one barley) at anthesis (16 weeks after planting) and maturity (22 weeks after planting). At anthesis a strong correlation (r = 0.86, p < 0.01) was present between visual and qPCR values. At maturity this relationship was modest (r = 0.58, p < 0.01). Furthermore, differences between partially resistant and susceptible genotypes were greatest at anthesis. The strong correlations between visual discolouration and fungal DNA content indicate that visual discolouration is a useful measure of fungal load in seedling and adult cereal tissues. However, the degree to which these two parameters correlate varies with the time elapsed since tissue infection. Fluorescence microscopy revealed no major differences between fungal growth patterns or structural characteristics in the host genotypes assessed. Leaf sheaths were most frequently penetrated via stomata, indicated by initial lesions forming at the guard cells. Leaf sheath tissues became extensively colonised in most cell types, except for the vascular bundles and abaxial silica cells. Colonisation of leaf sheaths resulted in the re-emergence of hyphae and occasionally conidiophores from stomata. Colonisation of culm tissues frequently originated in the parenchymatous hypoderm, which became greatly discoloured, resulting in the visual discolouration used for disease rating. Early infection of pith parenchyma cells was also frequent. Infections typically spread from the culm base upwards through the tissues, typically only to internode three, with a much slower lateral spread of hyphae. Colonisation of sclerified cells occurred later in the infection process. Vascular tissues were frequently colonised by anthesis. This was more rapid in susceptible genotypes. Occlusion of large xylem vessels was rare during moderate infections. The ability of quantitative PCR to accurately describe the extent of crown rot infection suggests that it could be utilised as a powerful technique for detecting new resistance sources or for identifying quantitative trait loci for resistance. This method, along with further microscopic and biochemical assessments of partially resistant and susceptible genotypes, may provide new information on the pathogenesis of crown rot and the nature of host resistance responses.