Presence and Prevalence of Raffaelea lauricola, Cause of Laurel Wilt, in Different Species of Ambrosia Beetle in Florida, USA.

We summarize the information available on ambrosia beetle species that have been associated in Florida with Raffaelea lauricola T.C. Harr., Fraedrich & Aghayeva, the primary symbiont of Xyleborus glabratus Eichhoff and cause of laurel wilt. In total, 14 species in Ambrosiodmus, Euwallacea, Premnobius, Xyleborus, Xyleborinus, and Xylosandrus were either reared from laurel wilt-affected host trees or trapped in laurel wilt-affected stands of the same, and assayed for R. lauricola. In six collections from native species in the southeastern United States [Persea borbonia (L.), Persea palustris (Raf.) Sarg., and Persea humilis Nash] and four from avocado (Persea americana Mill.), extracted mycangia or heads (taxa with mandibular mycangia) or intact bodies (taxa with mycangia in other locations) were surface-disinfested before assays on a semi-selective medium for the isolation of Raffaelea (CSMA+). Raffaelea lauricola was identified based on its characteristic phenotype on CSMA+, and the identity of a random subset of isolates was confirmed with taxon-specific microsatellite markers. The pathogen was recovered from 34% (246 of 726) of the individuals that were associated with the native Persea spp., but only 6% (58 of 931) of those that were associated with avocado. Over all studies, R. lauricola was recovered from 10 of the ambrosia beetle species, but it was most prevalent in Xyleborus congeners. This is the first record of R. lauricola in Ambrosiodmus lecontei Hopkins, Xyleborinus andrewesi (Blandford), and Xyleborus bispinatus Eichhoff. The potential effects of R. lauricola's promiscuity are discussed.

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.

Discordant phylogenies suggest repeated host shifts in the Fusarium-Euwallacea ambrosia beetle mutualism.

The mutualism between xyleborine beetles in the genus Euwallacea (Coleoptera: Curculionidae: Scolytinae) and members of the Ambrosia Fusarium Clade (AFC) represents one of 11 known evolutionary origins of fungiculture by ambrosia beetles. Female Euwallacea beetles transport fusarial symbionts in paired mandibular mycangia from their natal gallery to woody hosts where they are cultivated in galleries as a source of food. Native to Asia, several exotic Euwallacea species were introduced into the United States and Israel within the past two decades and they now threaten urban landscapes, forests and avocado production. To assess species limits and to date the evolutionary diversification of the mutualists, we reconstructed the evolutionary histories of key representatives of the Fusarium and Euwallacea clades using maximum parsimony and maximum likelihood methods. Twelve species-level lineages, termed AF 1-12, were identified within the monophyletic AFC and seven among the Fusarium-farming Euwallacea. Bayesian diversification-time estimates placed the origin of the Euwallacea-Fusarium mutualism near the Oligocene-Miocene boundary ∼19-24 Mya. Most Euwallacea spp. appear to be associated with one species of Fusarium, but two species farmed two closely related fusaria. Euwallacea sp. #2 in Miami-Dade County, Florida cultivated Fusarium spp. AF-6 and AF-8 on avocado, and Euwallacea sp. #4 farmed Fusarium ambrosium AF-1 and Fusarium sp. AF-11 on Chinese tea in Sri Lanka. Cophylogenetic analyses indicated that the Euwallacea and Fusarium phylogenies were largely incongruent, apparently due to the beetles switching fusarial symbionts (i.e., host shifts) at least five times during the evolution of this mutualism. Three cospeciation events between Euwallacea and their AFC symbionts were detected, but randomization tests failed to reject the null hypothesis that the putative parallel cladogenesis is a stochastic pattern. Lastly, two collections of Euwallacea sp. #2 from Miami-Dade County, Florida shared an identical cytochrome oxidase subunit 1 (CO1) allele with Euwallacea validus, suggesting introgressive hybridization between these species and/or pseudogenous nature of this marker. Results of the present study highlight the importance of understanding the potential for and frequency of host-switching between Euwallacea and members of the AFC, and that these shifts may bring together more aggressive and virulent combinations of these invasive mutualists.