First Report of Laurel Wilt Caused by Harringtonia lauricola (previously Raffaelea lauricola) on Northern Spicebush in Kentucky and Tennessee.

Laurel wilt (LW) is a vascular disease caused by the fungus Harringtonia lauricola (previously Raffaelea lauricola) and transmitted by its primary vector, the redbay ambrosia beetle (Xyleborus glabratus, RAB), both of which were first detected in the United States (US) in 2002, likely introduced from their native range in Asia (Fraedrich et al. 2008; Harrington et al. 2008). LW has since spread across the southeastern US causing the death of millions of native redbay, sassafras, silk bay, swamp bay and other native Lauraceae species (Hughes et al. 2017). Detection of LW on the deciduous understory shrub northern spicebush (Lindera benzoin) was previously reported in South Carolina (Fraedrich et al. 2016) and Louisiana (Olatinwo et al. 2021) and is hereby confirmed in Kentucky and Tennessee. Spicebush plants displaying typical LW symptoms were observed in September 2020 on a property spanning the KY/TN border (Christian Co., KY and Montgomery Co., TN). Several dense stands of spicebush exhibited leaf wilt, early fall leaf coloration, dead leaves on branches, and black streaks of discolored xylem. LW was already confirmed on sassafras in the area (Loyd et al. 2020), and there were abundant dead sassafras nearby. Ambrosia beetle boring dust was observed and callow female RABs emerged from containerized bolts of spicebush collected from the site, indicating that the vector used spicebush as a brood host. Samples of spicebush sapwood tissue collected from two symptomatic plants were plated onto CSMA (cycloheximide-streptomycin malt extract agar) medium. The cultures were grown at room temperature in ambient light, and a fungus was recovered and further transferred onto MEA (malt extract agar) and PDA (potato dextrose agar) media. The morphology of the two fungal isolates, referred to as LW415 and LW416, matched the typical white mucoid growth and ovoid conidia of H. lauricola (Harrington et al. 2008). DNA was extracted from conidia harvested from two-week-old MEA cultures using a modified method of Dreaden et al. (2014). The identity of the fungus was confirmed by performing PCR with the H. lauricola-specific microsatellite primer sets IFW and CHK (Dreaden et al. 2014, Parra et al. 2020). A positive amplification product was obtained for LW415 and LW416 for both primer sets, validating their identification as H. lauricola. To confirm pathogenicity, four spicebush seedlings (mean height 22.5 cm; mean ground line diameter 3.3 mm) were inoculated: two with H. lauricola isolate LW415 grown on PDA for two weeks at room temperature in the dark, and two were mock-inoculated with sterile PDA as a control. A scalpel was used to nick the spicebush stem at a bud about 5 cm above groundline, and a 3 mm2 agar plug was placed in the wound and wrapped with parafilm. The spicebush seedlings were maintained in a growth chamber with an average temperature of 24°C and a 15 h photoperiod. Wilt symptoms were evident on inoculated seedlings after two weeks, while the control plants remained healthy. Four weeks post-inoculation, black staining of the vascular tissue was present in the symptomatic seedlings, and a fungus matching the morphology of H. lauricola was consistently recovered, while no fungus was isolated from the control plants. These results provide additional evidence that northern spicebush populations may be threatened by LW and could serve as a reservoir for the pathogen and vector (Gramling 2010). The spread of LW and RAB on spicebush may gain importance as preferred hosts (e.g., sassafras) are killed.

Use of Telomere Fingerprinting to Identify Clonal Lineages of Colletotrichum fioriniae in Kentucky Mixed-Fruit Orchards.

Multiple species in the fungal genus Colletotrichum cause anthracnose fruit rot diseases that are responsible for major yield losses of as much as 100%. Individual species of Colletotrichum typically have broad host ranges and can infect multiple fruit species. Colletotrichum fioriniae causes anthracnose fruit rots of apples, blueberries, and strawberries in Kentucky orchards where these fruits grow in close proximity. This raises the possibility of cross-infection, which may have significant management implications. The potential occurrence of cross-infection was investigated by using telomere fingerprinting to identify C. fioriniae clones in several mixed-fruit orchards. Telomere fingerprints were highly polymorphic among a test group of C. fioriniae strains and effectively defined clonal lineages. Fingerprints were compared among apple, blueberry, and strawberry isolates of C. fioriniae from three different orchards and similarity matrices were calculated to build phylograms for each orchard group. Multiple clonal lineages of C. fioriniae were identified within each orchard on the same fruit host. Related lineages were found among isolates from different hosts, but the results did not provide direct evidence for cross-infection of different fruit species by the same clones. Recovery of the same clonal lineages within orchards across multiple years suggested that local dispersal was important in pathogen population structure and that C. fioriniae strains persisted within orchards over time. Isolates from blueberry were less diverse than isolates from apple, perhaps related to more intensive anthracnose management protocols on apple versus blueberry. Telomere fingerprinting is a valuable tool for understanding population dynamics of Colletotrichum fruit rot fungi.

Diversity and Cross-Infection Potential of Colletotrichum Causing Fruit Rots in Mixed-Fruit Orchards in Kentucky.

Fungi in the genus Colletotrichum cause apple, blueberry, and strawberry fruit rots, which can result in significant losses. Accurate identification is important because species differ in aggressiveness, fungicide sensitivity, and other factors affecting management. Multiple Colletotrichum species can cause similar symptoms on the same host, and more than one fruit type can be infected by a single Colletotrichum species. Mixed-fruit orchards may facilitate cross-infection, with significant management implications. Colletotrichum isolates from small fruits in Kentucky orchards were characterized and compared with apple isolates via a combination of morphotyping, sequencing of voucher loci and whole genomes, and cross-inoculation assays. Seven morphotypes representing two species complexes (C. acutatum and C. gloeosporioides) were identified. Morphotypes corresponded with phylogenetic species C. fioriniae, C. fructicola, C. nymphaeae, and C. siamense, identified by TUB2 or GAPDH barcodes. Phylogenetic trees built from nine single-gene sequences matched barcoding results with one exception, later determined to belong to an undescribed species. Comparison of single-gene trees with representative whole genome sequences revealed that CHS and ApMat were the most informative for diagnosis of fruit rot species and individual morphotypes within the C. acutatum or C. gloeosporioides complexes, respectively. All blueberry isolates belonged to C. fioriniae, and most strawberry isolates were C. nymphaeae, with a few C. siamense and C. fioriniae also recovered. All three species cause fruit rot on apples in Kentucky. Cross-inoculation assays on detached apple, blueberry, and strawberry fruits showed that all species were pathogenic on all three hosts but with species-specific differences in aggressiveness.