Project Title Apple scab diagnostic test development

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					                 Project Title: Apple scab diagnostic test development
                               Project Termination Report
                  Project leaders: Andrew M. Jarosz and Jan Byrne

Objective: We are developing a fast, reliable method of screening for resistance to
strobilurin fungicides in the pathogen causing apple scab, Venturia inaequalis. The
assay will be offered as a service through Michigan State University’s Plant Diagnostic
Services. It will provide real time information on the resistance status of V. inaequalis in
a grower’s orchards, allowing them to quickly adjust disease management procedures if
strobilurin resistance is found.

Results: Activities in 2005 were designed to determine three factors crucial to the final
implementation of the assay. The first factor was the time course of spore germination
over the growing season. Second, we needed to determine the best method of shipping
samples to Plant Diagnostic Services. Lastly, we wanted to develop an inexpensive but
efficient technique for harvesting pathogen spores from infected lesions. The first two
factors were designed to validate data collected in 2004.

Spore germination over the growing season: Spore germination was variable across
the growing season (Figure 1). Germination percentages for most samples were
between 30 and 60%, except for samples obtained on 20 and 27 June and 25 July
where germination percentages were 20% or less. We do not know why germination
was so low on these days. While all three samples were collected during periods with
little rain, other samples with significantly higher germination were collected deeper into
hot, dry periods experienced in 2005. Regardless, some spores remained viable at all
collection periods, and we adjusted the assay to score 200 spores rather than 100.

Figure 1. Germination percentage of conidia collected from lesions at different times
over the 2005 growing season.
Doubling the number of spores scored means that the assay can detect strobilurin
resistance even when germination percentages are low. These results are similar to
data obtained in 2004. The combined data gives us confidence that infected leaves can
be collected at any time during the growing season and sent to Plant Diagnostic
Services for analysis.

Transport of samples: Samples collected from an orchard were divided into thirds; one-
third of the sample was kept on ice and transported directly back to the lab, the second
third was mailed to Plant Diagnostic Services, while the remaining third was shipped by
an overnight delivery service. Average germination for samples transported by car was
35%. Mailing samples seemed to have no affect on spore viability since germination
percentages for mailed samples averaged 41%. The percentage germination for
samples shipped via overnight delivery was 29%. While this level of germination does
not differ statistically from samples transported by car, it was significantly lower than
germination percentages obtained for samples mailed to Plant Diagnostic Services.
These data are similar to what we found in 2004. Therefore, we will recommend that
samples be mailed to Plant Diagnostic Services since it is less expensive than overnight
delivery ($1.50 vs. $14.75) and germination rates are superior.

Methods of harvesting spores: Last year we found that reliable results could be obtained
when lesions were wiped directly onto agar Petri dishes amended with the appropriate
concentration of strobilurin. This method is not ideal because the number of lesions that
can be wiped onto a plate is limited to approximately 10, and each Petri dish is wiped
with a different set of lesions. The method is also relatively expensive because of the
cost of agar and plates. In 2004, we tried to dislodge spores by excising lesions and
shaking them in a water bath for 20 minutes. This second method is less expensive
because spores can be scored in droplets on cover slips. It also has a sampling
advantage because spores from hundreds of lesions can be mixed together so each
assay treatment is scored using a single population sample. Unfortunately, this water
bath method proved unreliable. A survey of the orchards suggested that failures were
associated with fungicide applications during the week prior to sample collection.

In 2005, we attempted to circumvent this problem by placing small drops of water
directly on lesions and gently rubbing with a dissecting needle to dislodge the spores.
We hoped this method would collect spores for use in droplets, but avoid contact with
leaf material where fungicide residues may be found. However, this new method
proved unreliable; 11 out of 31 samples processed using this method failed. In contrast,
only 2 out of 57 samples processed by rubbing lesions directly onto agar failed.
Therefore, we will use the agar method when implementing the assay. While more
expensive, it is the only method that has proven to be reliable.

Reference cultures: During the extension period of the grant, single spore cultures
from five orchards were prepared for long term storage. These cultures will serve as a
reference collection for future work. They may be useful if resistance to strobilurins is
found in Michigan, since the cultures can serve as a baseline data to determine genetic
Activities: We are in the process of doing a cost analysis of the procedure. The assay
will be offered in 2006 on a fee basis by Plant Diagnostic Services at Michigan State
University. Instructions for collecting and submitting samples will be posted on the Plant
Diagnostic Services Web page, and availability of the service will be advertised through
the MI Fruit network.

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