Exploring infection of wheat and carbohydrate metabolism in Mycosphaerella graminicola transformants with differentially regulated green fluorescent protein expression

A Mycosphaerella graminicola strain transformed with the green fluorescent protein (GFP) downstream of either a carbon source-repressed promoter or a constitutive promoter was used to investigate in situ carbohydrate uptake during penetration of the fungus in wheat leaves. The promoter region of the acu-3 gene from Neurospora crassa encoding isocitrate lyase was used as a carbon source-repressed promoter. The promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase was used as a constitutive promoter. Fluorometric measurement of GFP gene expression in liquid cultures of acu-3-regulated transformants indicated that the N. crassa acu-3 promoter functions in M. graminicola as it does in N. crassa, i.e., acetate induced and carbon source repressed. Glucose, fructose, and saccharose triggered the repression, whereas mannitol, xylose, and cell wall polysaccharides did not. Monitoring the GFP level during fungal infection of wheat leaves revealed that acu-3 promoter repression occurred after penetration until sporulation, when newly differentiated pycnidiospores fluoresced. The use of GFP transformants also allowed clear visualization of M. graminicola pathogenesis. No appressoria were formed, but penetration at cell junctions was observed. These results give new insight into the biotrophic status of M. graminicola.     

Rapid detection and diagnosis of Septoria tritici epidemics in wheat using a polymerase chain reaction/PicoGreen assay

In order to detect and quantify Septoria tritici infection levels in wheat leaves, a polymerase chain reaction (PCR) assay was developed using the β-tubulin gene as target. Specific PCR primers were designed by aligning and comparing β-tubulin sequences from other fungi. The final primer set was selected after being tested against several fungi, and against S. tritici -infected and uninfected wheat leaves from different localities. A single DNA fragment (496 bp) was amplified from S. tritici, whereas no products were generated from DNA of the host plant or other micro-organisms associated with wheat leaves. Using agarose gel analysis, approximately 2 pg S. tritici genomic DNA could be detected in each assay. However, for rapid quantification of PCR-amplified products, a fluorometric microtitre plate-formatted PicoGreen assay was used; this could detect as little as 10 pg S. tritici DNA in the presence of 200 ng wheat leaf DNA. The PCR/PicoGreen assay was applied successfully to study the colonization, infection and subsequent disease development of S. tritici on wheat, both under controlled conditions in the glasshouse and in the field.