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.

New Raffaelea species (Ophiostomatales) from the USA and Taiwan associated with ambrosia beetles and plant hosts.

Raffaelea (Ophiostomatales) is a genus of more than 20 ophiostomatoid fungi commonly occurring in symbioses with wood-boring ambrosia beetles. We examined ambrosia beetles and plant hosts in the USA and Taiwan for the presence of these mycosymbionts and found 22 isolates representing known and undescribed lineages in Raffaelea. From 28S rDNA and ß-tubulin sequences, we generated a molecular phylogeny of Ophiostomatales and observed morphological features of seven cultures representing undescribed lineages in Raffaelea s. lat. From these analyses, we describe five new species in Raffaelea s. lat.: R. aguacate, R. campbellii, R. crossotarsa, R. cyclorhipidia, and R. xyleborina spp. nov. Our analyses also identified two plant-pathogenic species of Raffaelea associated with previously undocumented beetle hosts: (1) R. quercivora, the causative agent of Japanese oak wilt, from Cyclorhipidion ohnoi and Crossotarsus emancipatus in Taiwan, and (2) R. lauricola, the pathogen responsible for laurel wilt, from Ambrosiodmus lecontei in Florida. The results of this study show that Raffaelea and associated ophiostomatoid fungi have been poorly sampled and that future investigations on ambrosia beetle mycosymbionts should reveal a substantially increased diversity.

Geographic variation in mycangial communities of Xyleborus glabratus.

Factors that influence fungal communities in ambrosia beetle mycangia are poorly understood. The beetle that is responsible for spreading laurel wilt in SE USA, Xyleborus glabratrus, was examined at three sites along a 500 km N-S transect in Florida, each populated by host trees in the Lauraceae. Fungal phenotypes were quantified in mycangia of individual females that were collected from a site in Miami-Dade County (MDC), 25.8N, with swamp bay (Persea palustris), one in Highlands County (HC), 27.9N, with silkbay (P. humulis) and swamp bay and another in Alachua County (AC), 29.8N, with redbay (P. borbonia). Based on combined LSU, SSU and beta-tubulin datasets the most prominent phenotypes were Raffaelea lauricola (cause of laurel wilt), R. subalba, R. subfusca, R. fusca, R. arxii and an undescribed Raffaelea sp. Mean numbers of colony forming units (CFUs) of R. lauricola varied by location (P < 0.003), and a multivariate analysis, which accounted for the presence and relative abundance of fungal species, indicated that there were significant variations in mycangial communities among the sites; thus climate and vegetation might have affected fungal diversity and the relative abundance of these fungi in the mycangia of X. glabratus Statistically it was unlikely that any of the species influenced the presence and prevalence of another species.

The U.S. Culture Collection Network Lays the Foundation for Progress in Preservation of Valuable Microbial Resources.

The U.S. Culture Collection Network was formed in 2012 by a group of culture collection scientists and stakeholders in order to continue the progress established previously through efforts of an ad hoc group. The network is supported by a Research Coordination Network grant from the U.S. National Science Foundation (NSF) and has the goals of promoting interaction among collections, encouraging the adoption of best practices, and protecting endangered or orphaned collections. After prior meetings to discuss best practices, shared data, and synergy with genome programs, the network held a meeting at the U.S. Department of Agriculture (USDA)-Agricultural Research Service (ARS) National Center for Genetic Resources Preservation (NCGRP) in Fort Collins, Colorado in October 2015 specifically to discuss collections that are vulnerable because of changes in funding programs, or are at risk of loss because of retirement or lack of funding. The meeting allowed collection curators who had already backed up their resources at the USDA NCGRP to visit the site, and brought collection owners, managers, and stakeholders together. Eight formal collections have established off-site backups with the USDA-ARS, ensuring that key material will be preserved for future research. All of the collections with backup at the NCGRP are public distributing collections including U.S. NSF-supported genetic stock centers, USDA-ARS collections, and university-supported collections. Facing the retirement of several pioneering researchers, the community discussed the value of preserving personal research collections and agreed that a mechanism to preserve these valuable collections was essential to any future national culture collection system. Additional input from curators of plant and animal collections emphasized that collections of every kind face similar challenges in developing long-range plans for sustainability.

Phylogeny of ambrosia beetle symbionts in the genus Raffaelea.

The genus Raffaelea was established in 1965 when the type species, Raffaelea ambrosia, a symbiont of Platypus ambrosia beetles was described. Since then, many additional ambrosia beetle symbionts have been added to the genus, including the important tree pathogens Raffaelea quercivora, Raffaelea quercus-mongolicae, and Raffaelea lauricola, causal agents of Japanese and Korean oak wilt and laurel wilt, respectively. The discovery of new and the dispersal of described species of Raffaelea to new areas, where they can become invasive, presents challenges for diagnosticians as well as plant protection and quarantine efforts. In this paper, we present the first comprehensive multigene phylogenetic analysis of Raffaelea. As it is currently defined, the genus was found to not be monophyletic. On the basis of this work, Raffaelea sensu stricto is defined and the affinities of undescribed isolates are considered.

Development of Multilocus PCR Assays for Raffaelea lauricola, Causal Agent of Laurel Wilt Disease.

Laurel wilt, caused by the fungus Raffaelea lauricola, is an exotic disease that affects members of the Lauraceae plant family in the southeastern United States. The disease is spreading rapidly in native forests and is now found in commercial avocado groves in south Florida, where an accurate diagnostic method would improve disease management. A polymerase chain reaction (PCR) method based on amplifying the ribosomal small-subunit DNA, with a detection limit of 0.0001 ng, was found to be suitable for some quantitative PCR applications; however, it was not taxon specific. Genomic sequencing of R. lauricola was used to identify and develop primers to amplify two taxon-specific simple-sequence repeat (SSR) loci, which did not amplify from related taxa or host DNA. The new SSR loci PCR assay has a detection limit of 0.1 ng of R. lauricola DNA, is compatible with traditional and real-time PCR, was tested in four labs to confirm consistency, and reduces diagnostic time from 1 week to 1 day. Our work illustrates pitfalls to designing taxon-specific assays for new pathogens and that undescribed fungi can limit specificity.