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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 β-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.

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., (© 2016 by The Mycological Society of America.)

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., (Copyright © 2014. Published by Elsevier Ltd.)

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.

In September 2010, live oak (Quercus virginiana Mill.) trees in an Alachua County, FL, shopping center parking lot were observed with shoot dieback and cankers on small branches. Isolations were made from canker margins by surface sterilizing tissue in 2.5% sodium hypochlorite and plating on potato dextrose agar (PDA) and incubating at 23°C. Fungi morphologically similar to Diplodia quercivora Linaldeddu & A.J.L. Phillips (mycelium initially velvety and white and later turning pale to dark olivaceous and grayish in reverse) were consistently isolated from symptomatic tissue (2). The two loci used by Linaldeddu et al. (2) in the description of D. quercivora were sequenced to identify a representative isolate (PL1345) as D. quercivora. The internal transcribed spacer (ITS) (GenBank Accession No. KF386635) and translation factor 1-alpha (EF-1α) (KF386636) regions were amplified and sequenced using primers ITS1F/ITS4 (3) and EF1-728F/EF1-986R (1). BLASTn searches of the two sequences resulted in 99% (467 of 469 and 257 of 259, respectively) homology with D. quercivora CBS 133852, confirming the fungal isolates' identity as D. quercivora. In October 2011, Koch's postulates were verified by inoculating, repeated twice, three Q. virginiana saplings (stem diameters, 12 to 14 mm; at inoculation sites approximately 50 mm above soil line) with isolate PL1345. Agar plugs (3 × 3 mm) taken from the margin of a 12-day-old culture on PDA were inserted into flaps in the stems made by a sterile blade with the mycelia facing the cambial tissue. One negative control tree was mock inoculated with a sterile PDA plug. All inoculation sites were sealed with Parafilm and maintained in a greenhouse (19 to 29°C). Trees were assessed for symptoms 90 days after inoculation. External bleeding was noted on all but one tree, and all flaps became necrotic. Pycnidia were observed on the outer surface of the flap on one inoculated tree. Negative controls showed no bleeding and their tissue flaps remained alive. Vertical length of phloem necrosis and percent of stem girdling were measured after removing the bark. Mean necrotic length and percent girdling for inoculated saplings were 48 mm (standard error [SE] = 10.6) and 26.6% (SE = 5.7) for the first inoculation and 46 mm (SE = 17) and 25% (SE = 5) for the second, respectively. Controls showed no internal necrosis and all produced healthy callus tissue at inoculation sites. Two of the pathogen-inoculated trees per inoculation were sampled and the pathogen was re-isolated from each. Recovered fungal isolates were confirmed as D. quercivora based on morphology and 100% ITS sequence homology to PL1345. D. quercivora was first described as causing shoot dieback and cankers on Q. canariensis in Tunisia and was found to be pathogenic to three additional Mediterranean oak species, Q. ilex, Q. pubescens, and Q. suber (2). To our knowledge, this is the first report of D. quercivora causing cankers on Q. virginiana and the first report of the fungus outside of Tunisia. Given the damage this pathogen has caused there, efforts to monitor the spread of this disease would seem warranted. More research is needed to assess the risk this pathogen poses to North American oaks, however. References: (1) I. Carbone et al. Mycologia 91:553, 1999. (2) B. T. Linaldeddu et al. Mycologia 105:1266, 2013. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Laurel wilt, caused by Raffaelea lauricola, threatens native and nonnative species in the Lauraceae in the southeastern United States, including the important commercial crop, avocado, Persea americana (2,4). Although the pathogen's vector, Xyleborus glabratus, was detected in Miami-Dade County, FL in January 2010, laurel wilt had not been reported (4). In February 2011, symptoms of the disease were observed on native swampbay, P. palustris, in Miami-Dade County (25°72'N, 80°48'W). Externally, foliage was brown, necrotic, and did not abscise; internally, sapwood was streaked with dark gray-to-bluish discoloration; and, in dead trees, holes of natal galleries of the vector from which columns of frass were attached were evident. On a semiselective medium for R. lauricola, a fungus with the pathogen's phenotype was isolated from symptomatic sapwood. Colonies were slow growing, light cream in color, with dendritic, closely appressed mycelium and often a slimy surface. A representative strain of the fungus was further identified with PCR primers for diagnostic small subunit (SSU) rDNA (1) and its SSU sequence (100% match, GenBank Accession No. JN578863). In each of two experiments, plants of 'Simmonds' avocado, the most important cultivar in Florida, were inoculated with three strains of the fungus, as described previously (3). Symptoms of laurel wilt developed in all inoculated plants and the fungus was recovered from each. After aerial and further ground surveys, additional symptomatic swampbay trees, some of which had defoliated, were detected in the vicinity of the original site. Since swampbay defoliates only a year or more after symptoms develop (4), the 2010 detection of X. glabratus may have coincided with an undetected presence of the disease. As of July 2011, a 6-km-diameter disease focus was evident in the area, the southernmost edge of which is 5 km from the nearest commercial avocado orchard. In August 2011, a dooryard avocado tree immediately north of the above focus was affected by laurel wilt, and an SSU sequence confirmed the involvement of R. lauricola (GenBank Accession No. JN613280). The outbreak of laurel wilt in Miami-Dade County represents a 150 km southerly jump in the distribution of this disease in the United States ( http://www.fs.fed.us/r8/foresthealth/laurelwilt/dist&#95;map.shtml ) and is the first time this disease has been found in close proximity to Florida's primary commercial avocado production area. Approximately 98% of the state's commercial avocados, worth nearly $54 million per year, are produced in Miami-Dade County. Since effective fungicidal and insecticidal measures have not been developed for large, fruit-bearing trees, mitigation efforts will focus on the rapid identification and destruction of infected trees (3,4). References: (1) T. J. Dreaden et al. Phytopathology 98:S48, 2008. (2) S. W. Fraedrich et al. Plant Dis. 92:215, 2008. (3) R. C. Ploetz et al. Plant Dis. 95:977, 2011. (4) R. C. Ploetz et al. Recovery Plan for Laurel Wilt of Avocado. National Plant Disease Recovery System, USDA, ARS, 2011.

Numerous cankers on small branches showing dieback were observed on live oak (Quercus virginiana) trees in September 2010 in Marion County, FL. Approximately 24 12-year-old landscape trees planted on a farm displayed symptoms. Samples were collected from six of the symptomatic trees and returned to the laboratory for processing. Isolations were made from canker margins after surface sterilization of samples in 2.5% sodium hypochlorite and by plating on potato dextrose agar (PDA). A suspect Botryosphaeriaceae sp. (based on colony morphology) was consistently isolated from the symptomatic branches from all six trees sampled. Fungal colonies consisted of plentiful, white, aerial mycelium that turned dark olive after 5 to 7 days at 23°C with the underside of the cultures turning black (1). Total genomic DNA from three representative Botryosphaeriaceae sp. isolates was extracted and the internal transcribed spacer (ITS1-5.8s-ITS2) region of the rDNA (GenBank Accessions Nos. JF798638, JF798639, and JF798640) using the primers ITS1 and ITS4 (3) and a portion of the β-tubulin gene (Bt), (GenBank Accession Nos. JF798641, JF798642, and JF798641) using the primers Bt2a and Bt2b (2) were amplified, sequenced, and deposited in GenBank. BLASTn searches of the ITS-rDNA sequences resulted in 100% homology (467 of 467, 467 of 467, and 540 of 540, respectively) with Diplodia corticola isolate CBS 112074 (GenBank Accession No. AY268421). BLASTn searches of the Bt sequences resulted in 99, 98, and 99% (391 of 393, 396 of 400, and 392 of 394, respectively) matches with D. corticola strain UCD2397TX, GenBank Accession No. GU294724. To complete Koch's postulates, nine seedlings of Q. virginiana, 0.6 to 0.9 cm in diameter at ground line maintained in a greenhouse, were inoculated with isolate PL949 (GenBank Accession Nos. JF798638 and JF798641) by making a 1.5-cm incision with a single-edge razor blade into the xylem 10 cm above ground line. Inoculations were done by placing mycelial plugs (1 × 0.25 cm) from cultures on PDA in the incision with the mycelium facing the center of the stem. Wounds were sealed by wrapping them with Parafilm. Three negative controls were mock inoculated as previously described except sterile PDA plugs were used. Eight weeks postinoculation, the lengths of the necrotic lesions were measured. Mean lesion length of the inoculated seedlings was 41.2 cm ± SE 4.5 and ranged between 27 and 63 cm. The negative control inoculations showed no necrotic lesions. Three of the inoculated seedlings were plated on PDA in an effort to reisolate the inoculated fungus. D. corticola was reisolated from each and all had the same ITS sequence as D. corticola strain CBS 112074. To our knowledge, this is the first report of D. corticola causing cankers on Q. virginiana and the first report of the disease occurring in Florida. D. corticola has been reported to cause cankers and dieback in several Quercus spp. in Greece, Hungary, Italy, Morocco, Portugal, and Spain and has recently been reported to cause cankers on Q. chrysolepis and Q. agrifolia in California. References: (1) A. Alves et al. Mycologia. 96:598, 2004. (2) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Laurel wilt disease, caused by Raffaelea lauricola (T.C. Harr., Fraedrich & Aghayeva sp. nov.), which is a fungal symbiont of the nonnative redbay ambrosia beetle (Xyleborus glabratus Eichhoff), has caused widespread mortality of native redbay (Persea borbonia (L.) Spreng) in Georgia, South Carolina, and Florida since 2002. The disease has been noted on other species in the Lauraceae including sassafras in Georgia (1), and more recently, on avocado and camphor in Florida (4). Since 2005, wilted shoots, branch dieback, and tree death have been observed in sassafras trees (Sassafras albidum (L.)) in Liberty, McIntosh, Chatham, Effingham, Bulloch, Evans, and Screven counties in Georgia; Bamberg, Beaufort, Charleston, Colleton, Hampton, and Orangeburg counties in South Carolina; and Putnam County in Florida. Symptomatic sassafras trees ranged from 1 to 12 m high and 2.5 to 25 cm in diameter at breast height. In contrast to red bay trees that retain wilted foliage, symptomatic sassafras defoliate rapidly as trees wilt and die. Multiple symptomatic ramets originating from a common root system have been observed. Removal of bark from stem and root sections from wilted trees revealed black-to-brownish staining in the sapwood, characteristic of laurel wilt. Wood chips from symptomatic areas of branches and roots were surface sterilized and plated on cycloheximide-streptomycin malt agar as previously described (1) and R. lauricola was routinely isolated. Small subunit (18S) sequences from rDNA were amplified by PCR and sequenced using primers NS1 and NS4 (3) for isolates from sassafras from Florida and South Carolina. BLASTn searches revealed homology to Raffaelea sp. C2203 (GenBank Accession No. EU123076, 100% similarity) described by Fraedrich et al. (1) from redbay and later named R. lauricola (2). The small subunit rDNA sequences for these isolates have been deposited into GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/index.html ) and assigned Accession Nos. EU980448 (Florida) and GQ329704 (South Carolina). Koch's postulates have been completed with R. lauricola on this host previously (1). Laurel wilt on sassafras often was geographically isolated from other symptomatic hosts in Georgia and South Carolina and appears to occur on this host independently of proximity to redbay. Further studies to determine the epidemiology of laurel wilt on sassafras, potential resistance, and impact on sassafras life history and distribution are needed. Given the clonal nature of sassafras, the disease would appear to have the potential to move through roots of trees once established in a stand. References: (1) S. W Fraedrich et al. Plant Dis. 92:215, 2008. (2) T. C. Harrington et al. Mycotaxon 104:399, 2008. (3) M. A. Innis et al. PCR Protocols, A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990. (4) J. A. Smith et al. Plant Dis. 93:198, 2009.

Laurel wilt is a vascular disease of redbay (Persea borbonia (L.) Spreng.) and other plants in the family Lauraceae in the southeastern United States. It is caused by a fungus (Raffaelea sp.) that is vectored by a non-native insect of Asian origin, the redbay ambrosia beetle (Xyleborus glabratus Eichhoff) (1). Since the initial detection of the redbay ambrosia beetle near Savannah, GA in 2002, laurel wilt has caused widespread mortality of redbay in Georgia, South Carolina, and Florida (1). In September 2007, an avocado (Persea americana Mill.) tree planted approximately 10 years earlier in a residential neighborhood in Jacksonville, FL was discovered to be infected with laurel wilt. The crown was in various stages of decline, including upper branches that were dead and leafless, those with wilted and drooping foliage, and those with healthy foliage. Removal of bark from wilted branch sections revealed black-to-brown streaks of discoloration in the sapwood and a few ambrosia beetle holes from which the discoloration extended into the adjacent wood. A Raffaelea sp. was isolated from discolored wood samples by surface sterilizing wood chips by submersion in a 5% sodium hypochlorite solution for 30 s and plating them on cycloheximide streptomycin malt agar (2). Small subunit (18S) sequences from the rDNA were amplified by PCR and sequenced with primers NS1 and NS4 (3). BLASTn searches revealed homology to Raffaelea sp. C2203 (GenBank Accession No. EU123076, 100% similarity, e-value of 0.0, and a total score of 1,886), which is known to be the causal agent of laurel wilt (1). The small-subunit rDNA sequence for this isolate has been deposited into GenBank and has been assigned accession No. EU257806. Pathogenicity of the laurel wilt pathogen on Persea spp. in growth chamber trials has been previously demonstrated (1). Laurel wilt is of concern to the commercial avocado industry and is a potential threat to the Lauraceae elsewhere in the Americas. References: (1) S. W. Fraedrich et al. Plant Dis. 92:215, 2008. (2) T. C. Harrington. Mycologia 73:1123, 1981. (3) T. J. White et al. Page 315 in: PCR Protocols, a Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.