Sarah L. Boggess

Development and characterization of sixteen microsatellite loci for Geosmithia morbida, the causal agent of thousand canker disease in black walnut (Juglans nigra)

Sixteen polymorphic microsatellite loci were identified from the fungal pathogen Geosmithia morbida. Loci were characterized for 13 different isolates collected in 2010 from symptomatic black walnut trees in Tennessee. A total of 77 loci were tested and 16 of those were optimized, screened and selected for diversity studies of G. morbida. Number of alleles per locus ranged from 3 to 8. These microsatellite loci will be useful for rapid disease diagnostic, population genetic analyses on a global scale as well as further epidemiological studies of G. morbida.

Confirmation of independent introductions of an exotic plant pathogen of Cornus species, Discula destructiva, on the east and west coasts of North America.

Cornus florida (flowering dogwood) and C. nuttallii (Pacific dogwood) are North American native tree species that belong to the big-bracted group of dogwoods. Cornus species are highly valued for their ornamental characteristics, and have fruits that contain high fat content for animals. Also, they are an important understory tree in natural forests. Dogwood anthracnose, caused by Discula destructiva, was observed in the late 1970s on the east and west coasts of the United States and by 1991 had quickly spread throughout most of the native ranges of C. florida and C. nuttalli. We investigated the genetic diversity and population structure of 93 D. destructiva isolates using 47 microsatellite loci developed from the sequenced genome of the type strain of D. destructiva. Clone-corrected data indicated low genetic diversity and the presence of four genetic clusters that corresponded to two major geographic areas, the eastern United States and the Pacific Northwest, and to the two collection time periods when the isolates were collected (pre- and post-1993). Linkage disequilibrium was present in five out of six subpopulations, suggesting that the fungus only reproduced asexually. Evidence of population bottlenecks was indicated across four identified genetic clusters, and was probably the result of the limited number of founding individuals on both coasts. These results support the hypothesis that D. destructiva is an exotic pathogen with independent introductions on the east and west coasts of North America. We also tested the cross-amplification of these microsatellite primers to other Discula species. Genomic DNA from 17 isolates of four other Discula species and two isolates of Juglanconis species (formerly Melanconis species) were amplified by 17 of 47 primer pairs. These primers may be useful for investigating the genetic diversity and population structure of these Discula species.

Controlling Herbicide-Resistant Annual Bluegrass (Poa annua) Phenotypes with Methiozolin

Methiozolin is an isoxazoline herbicide being investigated for selective POST annual bluegrass control in managed turfgrass. Research was conducted to evaluate methiozolin efficacy for controlling two annual bluegrass phenotypes with target-site resistance to photosystem II (PSII) or enolpyruvylshikimate-3-phosphate synthase (EPSPS)-inhibiting herbicides (i.e., glyphosate), as well as phenotypes with multiple resistance to microtubule and EPSPS or PSII and acetolactate synthase (ALS)-inhibiting herbicides. All resistant phenotypes were established in glasshouse culture along with a known herbicide-susceptible control and treated with methiozolin at 0, 125, 250, 500, 1000, 2000, 4000, or 8000 g ai ha-1. Methiozolin effectively controlled annual bluegrass with target-site resistance to inhibitors of EPSPS, PSII, as well as multiple resistance to EPSPS and microtubule inhibitors. Methiozolin rates required to reduce aboveground biomass of these resistant phenotypes 50% (GR50 values) were not significantly different from the susceptible control, ranging from 159 to 421 g ha-1. A phenotype with target-site resistance to PSII and ALS inhibitors was less sensitive to methiozolin (GR50 = 862 g ha-1) than a susceptible phenotype (GR50 = 423 g ha-1). Our findings indicate that methiozolin is an effective option for controlling select annual bluegrass phenotypes with target-site resistance to several herbicides. Nomenclature: Methiozolin, annual bluegrass, Poa annua L.

First Report of Powdery Mildew Caused by Golovinomyces spadiceus on Green and Gold (Chrysogonum virginianum) in the United States

Green-and-gold or Goldenstar (Chrysogonum virginianum L.: Asteraceae: Heliantheae) is a low-growing, shade-tolerant, perennial flowering groundcover that occurs naturally in moist, woodland environments in the eastern United States. Several variants of the plant are available commercially, and are generally considered easy to grow and relatively disease-free, except for some anecdotal reports of “mildew.” The fungus causing powdery mildew on this plant has never been identified and Koch’s postulates have not been completed. Plants growing in ornamental settings from Knoxville, TN, in 2015 to 2017 exhibited signs and symptoms of powdery mildew that included superficial, scanty and patchy, white mycelia, and hyaline conidiophores and conidia on both surfaces of the laminae, which were slightly cupped and upturned from the midvein to the margins compared with uninfected leaves. By June, mycelia were tightly appressed to the leaf surface, gray and crusty, and sporulation was greatly reduced. Infected leaf pieces were mounted in water or stained with Trypan blue. Conidia (n = 30), which lacked fibrosin bodies and were borne in chains, were ellipsoid or doliiform, rarely cylindrical, had a L:W ratio between 1.7 and 2.1, and measured 36.6 µm (32.9 to 42.0 µm) by 18.6 µm (15.6 to 20.5 µm). Conidia germinated subterminally and produced nipple-shaped appressoria. The morphology of the fungus agreed with the description for Golovinomyces spadiceus that occurs on species in the Heliantheae (Braun and Cook 2012). Chasmothecia were not detected during any of the three growing seasons. Total genomic DNA was prepared from mycelium, conidiophores, and conidia from leaves and amplified with ITS1 and ITS4 primers (White et al. 1990). Amplicons were purified before sequencing (Trigiano et al. 2016) and the sequences deposited in GenBank (MF460422). The sequence was 100% identical to G. ambrosiae reported on Helianthus annuus (KM657962) and H. verticillatus (KM657962). The sequence for C. virginianum was also entered in GenBank (MF460421). However, ITS sequencing is not sufficient to delineate G. ambrosiae from G. spadiceus (Takamatsu et al. 2013). Morphologically, G. spadiceus conidia are ≤20 µm wide, whereas G. ambrosiae conidia can be wider (Braun and Cook 2012). Five C. virginianum powdery mildew-free plants were inoculated by rubbing the adaxial surface of leaves with leaves having signs of powdery mildew; two plants were rubbed with healthy leaves as controls. Plants from all treatments were covered with plastic bags for 2 days and then removed. After 10 days, four plants inoculated with powdery mildew leaves from C. virginianum exhibited the same signs and symptoms as described originally from infected C. virginianum plants. All control plants remained disease-free. Our conclusion is that G. spadiceus is the cause of powdery mildew on C. virginianum. The disease on C. virginianum appears to be more cosmetic than detrimental as flowering and growth of the plants are not noticeably affected. This is the first report that provides completed Koch’s postulates for this disease in the United States.

First Report of Cercospora apii s. lat. Causing a Leaf Spot on Cardinal Flower (Lobelia cardinalis) in the United States

Cardinal flower (Lobelia cardinalis L.: Campanulaceae) is a wildflower common throughout much of the eastern United States and grows in a variety of habitats. It is used as an ornamental plant because its bright red flowers appear in the fall and attract hummingbirds. The foliage is generally free of disease when the plant is not flowering, although a few leaf and stem lesions may be present during the growing season. In August/September 2016 and 2017, plants established in eastern Tennessee developed numerous spots on stems and leaves. The round- to lenticular-shaped lesions had dry, white, papery necrotic centers surrounded by reddish-purple host tissue. There were no signs of a pathogen. Leaves were washed in running water for 5 min, surface disinfested with 1.05% NaClO for 15 min, and rinsed three times in sterile water. Lesions and some green tissue from leaves were transferred to half-strength potato dextrose agar (PDA) augmented with 10 mg rifampicin/liter, and incubated in the dark. Mycelium was i...

Haplotyping of Cornus florida and C. kousa chloroplasts: Insights into species-level differences and patterns of plastic DNA variation in cultivars

Chloroplast DNA is a part of plant non-nuclear genome, and is of particular interest for lineage studies. Moreover, the non-coding regions of cpDNA display higher mutation rates than the conserved coding cpDNA, which has been employed for phylogenetic and population research. We analyzed the cpDNA of 332 gDNA samples from collections of Cornus florida and C. kousa (commercial cultivars, breeding selections, and wild kousa accessions from Asia), using the chlorotyping system developed on North America-native, wild accessions of C. florida. Our results indicated significant differences in chlorotype frequencies between the two species. Cornus florida samples were represented by all major chlorotypes previously described, whereas all C. kousa samples analyzed had only one of the chlorotype patterns shown by C. florida. The chlorotyping analytic panel was then expanded by sequencing the targeted three non-coding cpDNA regions. Results indicated a major difference in the maternally-inherited cpDNA between the two closely related Big-Bracted Cornus species. Chlorotype diversity and differences in the proportion of informative sites in the cpDNA regions of focus emphasized the importance of proper loci choice for cpDNA-based comparative studies between the closely related dogwood species. Phylogenetic analyses of the retrieved sequences for the other species of Cornus provided information on the relative utility of the cpDNA regions studied and helped delineate the groups (Big-Bracted, Cornelian Cherries, Blue/White-Fruited) within the genus. Genealogical relationships based on the cpDNA sequences and the inferred chlorotype networks indicated the need for continued analyses across further non-coding cpDNA regions to improve the phylogenetic resolution of dogwoods.

First Report of Powdery Mildew on Rescuegrass (Bromus catharticus) Caused by Blumeria graminis in Tennessee

Bromus catharticus Vahl, or rescuegrass, is a South American member of the brome grass group that has been spread around the world, including introduction into the United States where it has become naturalized in many areas including agricultural fields. The plant grows either as a short-lived perennial or annual depending on location and produces copious seeds. Rescuegrass was listed in an index as infected by Blumeria graminis (DC.) Speer (powdery mildew) in Virginia (Roane 2004), but no reports exist in which the pathogen, disease symptoms and signs were described, and in which Koch’s postulates were completed. Powdery mildew-like symptoms were seen on rescuegrass growing along roadsides in Knoxville, TN in late May and June, 2016 and 2017. White, powdery masses in lens-shaped lesions were abundant on the adaxial surfaces of leaves. By mid-July, the lesions had expanded to two- to- threefold the initial size, and had become crusty and rust-colored. The mostly ellipsoid-shaped conidia (n = 30) borne in ...