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 20o
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.