Practical on Blumeria graminis f.sp hordei

(Formerly known as Erysiphe graminis f.sp. hordei.)

You have segments of seedling leaves from two sets of barley isogenic lines inoculated with B. graminis f.sp. hordei race CC1 and incubated for 48h at 20^o C before fixation. Fixation was by immersion in 3:1 ethanol: glacial acetic acid (v:v) for 24h by which time all chlorophyll had been removed. Segments were then washed in water for 5 min. and then cleared in lactoglycerol (lactic acid: glycerol: water; 1:1:1, v:v). The fungus was stained for 2h in 0.1% Trypanblue in lactoglycerol. Tissues then stored in lactoglycerol and mounted in same.

One set of barley lines was produced from a cultivar called “Algerian” (abbreviation = Alg). The Alg lines (Alg-R and Alg-S) have different alleles at the Mla locus. Alg-R has the dominant allele Mla-1 which conditions a rapid hypersensitive reaction to attack by avirulent races of B. graminis f.sp. hordei (including CC1), while Alg-S has the equivalent but recessive mla-1 allele for susceptibility.

The Risø lines were selected from a mutagenesis programme at Risø National laboratory in Denmark. Risø-R and Risø-S have different alleles at the Mlo locus. Risø-R has the recessive mlo allele which conditions a papilla-based resistance to penetration to all known isolates of B. graminis, while Risø-S has the equivalent dominant Mlo allele for susceptibility.

Have a general look around the slides to get an impression of what is going on. You should be able to see conidia, primary germ tubes and appressorial germ tubes, and, if infection has succeeded, haustoria and secondary hyphae. If the host cell has died (hypersensitivity), it should have taken up the Trypanblue stain. Papillae should be visible as bright, refractive bodies which take up some stain, under appressoria.

Score ten spores on each slide and describe their stage of development and the host response. Score germlings according to whether they have only formed an appressorium (call them “A”s), or whether they have formed a haustorium and secondary mycelium (“H”s). Score host cells according to whether they are living (“L”s) or dead (“D”s). So for each of the ten spore/host cell interactions you score, you should produce two letters e.g. AL or AD, or HL or HD

Workshop Requirements.

I would like to see you write up your data as a little mini- paper. Ask me if you are unsure how to do this. You do not have to produce an “introduction” or a “material and methods”. However, you must present and describe your results in a coherent manner (i.e Table / Graphs as relevant). What kind of statistical analysis would you think would be appropriate? Most of your marks will be obtained from a full discussion of your data.

Also in a real paper you must consider what your data actually means when set against your knowledge of Blumeria pathogenesis and host responses. Thus in your discussion you MUST consider the following points –

What is gene-for-gene resistance?

Are you observing this in ALL of your experiments? If not how would you test if this were so?

What is the difference in the resistance mediated by dominant and recessive resistance genes?

Would you consider gene-for-gene resistance mechanisms to be a source of long-term filed resistance? Explain you answer.