Department of Plant Sciences, The University of Tennessee, Knoxville, Tennessee, USA.
Department of Plant Pathology, University of Florida, Homestead, Florida, USA.
Microbiologyopen. 2022 Jun;11(3):e1286. doi: 10.1002/mbo3.1286.
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. A total of 3256 candidate microsatellite markers were identified (2236, 789, 137 di-, tri-, and tetranucleotide motifs, respectively), with 2011, 703, 101 di-, tri-, and tetranucleotide motifs, respectively, 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. Five novel microsatellite markers (GOBS9, GOBS10, GOBS41, GOBS43, and GOBS50) were developed based on the G. obscura genome. These species-specific microsatellite markers are available as a tool for use in molecular diagnostics and can assist future surveillance studies.
地衣菌属真菌与蛀干和树皮甲虫之间的共生关系很少导致植物宿主发病。然而，也有例外，如导致核桃千疮百孔病（TCD）的地衣菌属莫比达和导致橡树泡沫状树皮溃疡病的地衣菌属 sp.41。从田纳西州东部的黑胡桃树上和至少一个分离株中回收了地衣菌属 obscura 的分离株，在人工接种后会引起溃疡。由于假定的致病性和缺乏从自然病变中回收地衣菌属 obscura，因此使用从地衣菌属 obscura 基因组中挖掘的微卫星标记开发了一种分子诊断筛选工具。总共鉴定出 3256 个候选微卫星标记（分别为 2236、789、137 个二核苷酸、三核苷酸和四核苷酸基序），其中分别有 2011、703、101 个二核苷酸、三核苷酸和四核苷酸基序包含带有引物的标记。从中随机选择了 75 个微卫星标记进行筛选和优化，得到了 28 个多态性标记，这些标记产生了单一、一致回收的条带，用于下游分析。其中 5 个微卫星标记对地衣菌属 obscura 具有特异性，不会与其他密切相关的物种交叉扩增。虽然其余经过测试的标记可能有用，但它们在地衣菌属的不同物种内交叉扩增，因此对于地衣菌属 obscura 的检测不可靠。基于地衣菌属 obscura 基因组开发了 5 个新的微卫星标记（GOBS9、GOBS10、GOBS41、GOBS43 和 GOBS50）。这些种特异性微卫星标记可作为分子诊断的工具，并可协助未来的监测研究。
