Characterization and microsatellite marker development for Geosmithia obscura, a common bark and ambrosia beetle associate

Background. Symbioses between Geosmithia fungi and wood-boring and bark
beetles seldom result in disease induction within the plant host. Yet
exceptions exist such as Geosmithia morbida, the causal agent of Thousand
Cankers Disease (TCD) of walnuts and wingnuts and Geosmithia sp. 41, the
causal agent of Foamy Bark Canker disease of oaks. Isolates of G. obscura
were recovered from black walnut trees in eastern Tennessee and at least
one isolate induced cankers following artificial inoculation. Due to the
putative pathogenicity and lack of recovery of G. obscura from natural
lesions, a molecular diagnostic screening tool was developed using
microsatellite markers mined from the G. obscura genome. Results. A total
of 3,256 candidate microsatellite markers were identified (2236, 789, 137
di-, tri-, and tetra- motifs were identified, respectively), with 2011,
703, 101 di-, tri-, and tetra- motifs containing markers with primers.
From these, 75 microsatellite markers were randomly selected, screened,
and optimized, resulting in 28 polymorphic markers that yielded single,
consistently recovered bands which were used in downstream analyses. Five
of these microsatellite markers were found to be specific to G. obscura
and did not cross-amplify into other, closely related species. Although
the remaining tested markers could be useful, they cross-amplified within
different Geosmithia species, making them not reliable for G. obscura
detection. Conclusion. Five novel microsatellite markers (GOBS9, GOBS10,
GOBS41, GOBS43, GOBS50) were developed based on G. obscura genome. These
species-specific microsatellite markers are available as a tool for use in
molecular diagnostics and can assist future surveillance studies.