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Laurel wilt (LW), caused by Raffaelea lauricola (RL), is a vascular fungal disease affecting species in the Lauraceae that has rapidly spread across the United States. This disease has caused significant tree losses in natural forests and Florida’s commercial avocado orchards. RL spreads through ambrosia beetle vectors and root grafts. Early detection and eradication are recommended to contain outbreaks. Therefore, rapid diagnosis is key for the timely implementation of mitigation strategies. Current LW diagnosis can take up to 10 days and involves pathogen isolation and the amplification of two microsatellite regions. To reduce diagnosis time, we optimized the standard PCR-based detection technique and assessed its potential use in the testing of woody samples. We further screened the microsatellite primers IFW and CHK on a higher number of fungal taxa as well as 11 host genotypes. Sensitivity was evaluated using RL-DNA at different concentrations in pure and mixed solutions. There was no cross-amplification in non-RL species. Both primers amplified all tested RL strains (100); however, the IFW primers were more sensitive than the CHK primers. Using the IFW primers, we detected RL in 89% of sapwood samples (76/85). This protocol provides a rapid and effective molecular-based approach that reduces the diagnostic time from 10 days to 24 h. This method can be an important tool for diagnostic laboratories. Altogether, our data and fungal collection, represent a robust foundation for future transferability of this protocol to more sensitive detection technologies (qPCR, LAMP) and for its application to samples from diverse origin (beetles, roots).

Laurel wilt is a destructive disease of redbay (Persea borbonia) and other species in the laurel family (Lauraceae). It is caused by Raffaelea lauricola, a fungal symbiont of the redbay ambrosia beetle, Xyleborus glabratus (Coleoptera: Curculionidae), cointroduced into the United States around 2002. During assessments of fungi associated with bark beetles from loblolly pine, an unknown fungus was isolated that appeared to have broad-spectrum antifungal activities. In this study, we identified the unknown fungus and determined the inhibitory effect of its secondary metabolites on R. lauricola. DNA analysis identified the fungus as Acaromyces ingoldii (GenBank accession no. EU770231). Secondary metabolites produced by the A. ingoldii completely inhibited R. lauricola mycelial growth on potato dextrose agar (PDA) plates preinoculated with A. ingoldii and reduced R. lauricola growth significantly on malt extract agar plates preinoculated with A. ingoldii. R. lauricola isolates inoculated on PDA plates 7 days after A. ingoldii were completely inhibited with no growth or spore germination. Direct evaluation of A. ingoldii crude extract on R. lauricola spores in a multi-well culture plate assay showed inhibition of spore germination at 10% and higher concentrations. Secondary metabolites from A. ingoldii could be potentially useful in managing the future spread of laurel wilt.

Invasive ambrosia beetles (Coleoptera: Curculionidae: Scolytinae: Xyleborini) have recently become established in several regions of the US where they vector fungal pathogens responsible for vascular wilt diseases in avocado (Persea americana), woody ornamentals, and native forest trees. These include the redbay ambrosia beetle (Xyleborus glabratus) that vectors the fungus responsible for laurel wilt, and a group of cryptic species morphologically similar to tea shot-hole borer (Euwallacea fornicatus) that vectors the pathogen of Fusarium dieback. Ecologically, these wood-boring beetles function as primary colonizers, capable of attacking healthy unstressed trees. Thus, they are attracted to the volatile terpenoids emitted from host wood (i.e., kairomones used by females for host location). Since essential oils consist of concentrated plant terpenoids, they have provided an ideal substrate for development of lures for these pests. This chapter (a) summarizes the succession of essential oil lures used for redbay ambrosia beetle over the last decade, (b) outlines development of the current lure which is highly enriched in (−)-α-copaene, (c) presents chemical analysis of the α-copaene lure, and (d) describes its recent applications for detection of Euwallacea pests in Florida avocado groves.

Survival of ambrosia beetles relies on obligate nutritional relationships with fungal symbionts that are cultivated in tunnels excavated in the sapwood of their host trees. The dynamics of fungal associates, along with the developmental biology, and gallery construction of the ambrosia beetle Xyleborus bispinatus were elaborated. One generation of this ambrosia beetle was reared in an artificial medium containing avocado sawdust. The developmental time from egg to adult ranged from 22 to 24 d. The mean total gallery length (14.4 cm and 13 tunnels) positively correlated with the number of adults. The most prevalent fungal associates were Raffaelea arxii in the foundress mycangia and new galleries, and Raffaelea subfusca in the mycangia of the F1 adults and the final stages of the galleries. Raffaelea sp. PL1001, Raffaelea subalba and seven yeast species were also recovered. These results indicate flexibility in the association between X. bispinatus and its symbiont species, and that this beetle may use more than one Raffaelea species as a food source. These results are important, as X. bispinatus has been associated with the transmission of Raffaelea lauricola, the causal agent of laurel wilt, a lethal disease affecting avocado trees.

Redbay ambrosia beetle, Xyleborus glabratus, is native to Southeast Asia, but subsequent to introduction in Georgia in 2002, it has become a serious invasive pest in the USA, now established in nine southeastern states. Females vector Raffaelea lauricola, the fungus that causes laurel wilt, a lethal vascular disease of trees in the family Lauraceae. Laurel wilt has caused extensive mortality in native Persea species, including redbay (P. borbonia), swampbay (P. palustris), and silkbay (P. humilis). Avocado (P. americana) is now impacted in Florida, and with continued spread, laurel wilt has potential to affect avocado and native Lauraceae in California, Mexico, and throughout the American tropics. Effective lures for detection and control of X. glabratus are critical to slow the spread of laurel wilt. No pheromones are known for this species; primary attractants are volatile terpenoids emitted from host Lauraceae. This report provides a concise summary of the chemical ecology of X. glabratus, highlighting research to identify kairomones used by females for host location. It summarizes development of essential oil lures for pest detection, including discussions of the initial use of phoebe and manuka oil lures, the current cubeb oil lure, and a newly-developed distilled oil lure enriched in (-)-α-copaene.

Native Lauraceae (e.g. sassafras, redbay) in the southeastern USA are being severely impacted by laurel wilt disease, which is caused by the pathogen Raffaelea lauricola T. C. Harr., Fraedrich and Aghayeva, and its symbiotic vector, the redbay ambrosia beetle (Xyleborus glabratus Eichhoff). Cold temperatures are currently the only viable limitation to the establishment of X. glabratus in northern populations of sassafras. The observed lower lethal temperature of X. glabratus (− 10.0 °C) is warmer than its supercooling point (− 22.0 °C), indicating the beetle is a freeze intolerant and chill susceptible species. Empirically derived X. glabratus lower lethal temperature thresholds were combined with host distribution and microhabitat-corrected climate data to produce species distribution models for X. glabratus in the eastern USA. Macroclimate data (30-year mean annual minimum temperature) were corrected (− 1.2 °C) to account for thermal buffering afforded to X. glabratus while living inside sassafras trees. Only 0.1% of the current US sassafras spatial extent experiences sufficiently harsh winters (locales where mean annual minimum winter temperatures ≤ − 6.2 °C for ≥ 12 h) to exclude X. glabratus establishment in our species distribution model. Minimum winter temperatures will likely cause some X. glabratus mortality in ~ 52% of the current spatial extent of sassafras, although current data do not allow a quantification of X. glabratus mortality in this zone. Conversely, ~ 48% of the current spatial extent of sassafras is unlikely to experience sufficiently cold winter temperatures to cause any significant impediment to X. glabratus spread or establishment. A modest climate change scenario (RCP4.5) of + 1.4 °C would result in 91% of the current spatial extent of sassafras in the eastern USA occurring where winter minimum temperatures are unlikely to cause any mortality to X. glabratus.

Like other ambrosia beetles, Xyleborus volvulus Fabricius (Coleoptera: Curculionidae) lives in a mutualistic symbiotic relationship with fungi that serve as food source. Until recently, X. volvulus was not considered a pest, and none of its symbionts were considered plant pathogens. However, recent reports of an association between X. volvulus and Raffaelea lauricola T.C. Harr., Fraedrich & Aghayeva (Ophiostomatales: Ophiostomataceae), the cause of the laurel wilt disease of avocado (Persea americana Mill. [Laurales: Lauraceae]), and its potential role as vector of the pathogen merit further investigation. The objective of this study was to evaluate three artificial media containing sawdust obtained from avocado or silkbay (Persea humilis Nash) for laboratory rearing of X. volvulus. The effect of R. lauricola in the media on the beetle's reproduction was also evaluated. Of the three media, the one with the lowest content of sawdust and intermediate water content provided the best conditions for rearing X. volvulus. Reproduction on this medium was not affected by the sawdust species or the presence of R. lauricola. On the other two media, there was a significant interaction between sawdust species and R. lauricola. The presence of R. lauricola generally had a negative effect on brood production. There was limited colonization of the mycangia of X. volvulus by R. lauricola on media inoculated with the pathogen. From galleries formed within the best medium, there was 50% recovery of R. lauricola, but recovery was much less from the other two media. Here, we report the best artificial substrate currently known for X. volvulus.

Ambrosia beetles subsist on fungal symbionts that they carry to, and cultivate in, their natal galleries. These symbionts are usually saprobes, but some are phytopathogens. Very few ambrosial symbioses have been studied closely, and little is known about roles that phytopathogenic symbionts play in the life cycles of these beetles. One of the latter symbionts, Raffaelea lauricola, causes laurel wilt of avocado, Persea americana, but its original ambrosia beetle partner, Xyleborus glabratus, plays an uncertain role in this pathosystem. We examined the response of a putative, alternative vector of R. lauricola, Xyleborus bispinatus, to artificial diets of R. lauricola and other ambrosia fungi. Newly eclosed, unfertilized females of X. bispinatus were reared in no-choice assays on one of five different symbionts or no symbiont. Xyleborus bispinatus developed successfully on R. lauricola, R. arxii, R. subalba and R. subfusca, all of which had been previously recovered from field-collected females of X. bispinatus. However, no development was observed in the absence of a symbiont or on another symbiont, Ambrosiella roeperi, recovered from another ambrosia beetle, Xylosandrus crassiusculus. In the no-choice assays, mycangia of foundress females of X. bispinatus harbored significant colony-forming units of, and natal galleries that they produced were colonized with, the respective Raffaelea symbionts; with each of these fungi, reproduction, fecundity and survival of the beetle were positively impacted. However, no fungus was recovered from, and reproduction did not occur on, the A. roeperi and no symbiont diets. These results highlight the flexible nature of the ambrosial symbiosis, which for X. bispinatus includes a fungus with which it has no evolutionary history. Although the “primary” symbiont of the neotropical X. bispinatus is unclear, it is not the Asian R. lauricola.

Avocado trees in Florida and California are under attack, respectively, from laurel wilt and Fusarium dieback, two diseases caused by fungi that live in symbiosis with ambrosia beetle species. As a first step to identify possible bacteria that can be utilized as biocontrol agents against these important diseases of avocados, we isolated microorganisms from avocado trees in Florida to determine their potential for use as biocontrol antagonists against the pathogens responsible for laurel wilt and Fusarium dieback. In addition, we assessed the potential of other strains not isolated from avocado to determine their effectiveness as biocontrol antagonists. A total of 50 strains were identified as strong antagonists against the laurel wilt pathogen, Raffaelea lauricola. Fifteen of the most active strains were, in turn, evaluated for antagonism against Fusarium euwallaceae, the causal agent of Fusarium dieback on avocados. Four strains representing three Paenibacillus species and one Bacillus species were found to be active against both R. lauricola and F. euwallaceae. This study is the first to report antifungal or antagonist activity of bacterial strains effective against avocado pathogens vectored by ambrosia beetles, and it is also the first to report the effectiveness of Paenibacillus thiaminolyticus or Paenibacillus apiarius against a phytopathogen.

We compared the flight activity of Xyleborus glabratus Eichhoff, vector and symbiont of the causal agent of laurel wilt disease (Raffaelea lauricola), with a native species Monarthrum mali (Fitch) using flight mills. Flight mills were operated either for 24 h or for three 3-h time intervals. During the 3-h interval experiment, the shortest time to flight initiation for X. glabratus occurred at 1600-1900 hours. The average flight time and total flying distance during 1600-2100 hours were also higher than those quantified during the other two recording times investigated. However, total flight duration and proportion of fliers was highest at 1000-1300 hours. We compared several flight parameters. About 64.0% of tested X. glabratus flew

Laurel wilt is an extraordinarily destructive exotic tree disease in the southeastern United States that involves new-encounter hosts in the Lauraceae, an introduced vector (Xyleborus glabratus) and pathogen symbiont (Raffaelea lauricola). USDA Forest Service Forest Inventory and Analysis data were used to estimate that over 300 million trees of redbay (Persea borbonia sensu lato) have succumbed to the disease since the early 2000s (ca 1/3 of the pre-invasion population). In addition, numerous native shrub and tree species in the family are susceptible and threatened in the Western Hemisphere. Genetic markers were used to test the hypothesis that the vector and pathogen entered North America as a single introduction. With a portion of the cytochrome oxidase I gene, a single X. glabratus haplotype was detected in the USA. Similarly, Amplified Fragment Length Polymorphisms indicated that 95% (54 of 57) of the isolates of R. lauricola that were examined were of a single clonal genotype; only minor variation was detected in three polymorphic isolates. Similar levels of disease developed after swamp bay (P. palustris) was inoculated with each of the four genotypes of R. lauricola. It is proposed that a single founding event is responsible for the laurel wilt epidemic in the United States.

Volatile organic compounds (VOCs) in the headspace of the fungus Raffaelea lauricola have been monitored and identified over a twenty-eight day growth period. R. lauricola is an invasive and phytopathogenic fungus that was first identified in the United States in the mid-2000s. It is believed to be spread by a host beetle, Xyleborus glabratus, and is detrimental both to wild members of the Lauraceae family and to commercial avocado groves particularly in the Southeastern region of the country. The fungus causes the fatal laurel wilt disease, a result of the host tree shutting down its vascular system in order to halt the spread of the fungus. The current study identified the VOCs present in the headspace of R. lauricola over the initial growth stage using headspace solid phase microextracion-gas chromatography-mass spectrometry (HS-SPME-GC–MS). Results revealed the VOC dynamics of the fungus in culture, indicating that the initial growth period of the fungus may coincide with potential responses from the host trees that may recognize and respond to the pathogen when the fungal VOCs are produced as a result of primary metabolic processes. As fungal growth progresses past initial growth phases, the predominant compounds seen in the odor profile are hydrocarbons and terpenes, produced from secondary metabolic processes. The odor profile pattern for the twenty-eight day growth period did change with the stages of growth. Based on the information learned from this pilot study, a discussion is presented of possible host tree reactions to R. lauricola and implications for future experiments.

Raffaelea lauricola, a fungal symbiont of the ambrosia beetle Xyleborus glabratus, causes laurel wilt in members of the Lauraceae plant family. North American species in the family, such as avocado (Persea americana) and swamp bay (P. palustris), are particularly susceptible to laurel wilt, whereas the Asian camphortree (Cinnamomum camphora) is relatively tolerant. To determine whether susceptibility is related to pathogen colonization, a green fluorescent protein-labeled strain of R. lauricola was generated and used to inoculate avocado, swamp bay, and camphortree. Trees were harvested 3, 10, and 30 days after inoculation (DAI), and disease severity was rated on a 1-to-10 scale. By 30 DAI, avocado and swamp bay developed significantly more severe disease than camphortree (mean severities of 6.8 and 5.5 versus 1.6, P < 0.003). The extent of xylem colonization was recorded as the percentage of lumena that were colonized by the pathogen. More xylem was colonized in avocado than camphortree (0.9% versus 0.1%, P < 0.03) but colonization in swamp bay (0.4%) did not differ significantly from either host. Although there were significant correlations between xylem colonization and laurel wilt severity in avocado (r = 0.74), swamp bay (r = 0.82), and camphortree (r = 0.87), even severely affected trees of all species were scarcely colonized by the pathogen.

Laurel wilt kills American members of the Lauraceae plant family, including avocado (Persea americana). The disease threatens commercial production in the United States and other countries, and currently impacts the avocado industry in Florida. As laurel wilt spreads, the National Germplasm Repository for avocado in Miami (USDA-ARS) and commercial and residential production in other states (e.g., California and Hawaii), U.S. protectorates (Puerto Rico), and other countries are at risk. In the United States, value-added production of avocado of more than $1.3 billion/year is threatened. This recovery plan was produced as part of the National Plant Disease Recovery System (NPDRS), called for in Homeland Security Presidential Directive Number 9 (HSPD-9) to insure that the tools, infrastructure, communication networks, and capacity required to mitigate the impact of high-consequence plant disease outbreaks are such that a reasonable level of crop production is maintained. It is intended to provide a brief primer on the disease, assess the status of critical recovery components, and identify disease management research, extension, and education needs.

Extensive mortality of redbay (Persea borbonia (L.) Spreng.) has been observed in the southeastern United States since 2003. The mortality is due to laurel wilt caused by Raffaelea lauricola T. C. Harr., Fraedrich & Aghayeva, a fungal symbiont of the recently introduced redbay ambrosia beetle (RAB), Xyleborus glabratus Eichhoff (1,2). The wilt is known to affect other members of the Lauraceae including sassafras (Sassafras albidum (Nuttall) Nees) and avocado (Persea americana Mill.) (1,3). Two inoculation experiments were conducted to evaluate the susceptibility of California laurel (Umbellularia californica (Hook. & Arn.) Nutt.) to R. lauricola. Seedlings, averaging 73 cm high and 13 mm in diameter, were wounded with a drill bit (2.8 mm) to a depth of one-half the diameter of the stems. In each experiment, 10 seedlings were inoculated with one of two isolates of R. lauricola (five seedlings per isolate) obtained as previously described (1) from wilted redbays on Hilton Head Island, South Carolina and Fort George Island, Florida. In the first experiment, seedlings were inoculated with spore suspensions (0.1 ml) ranging from 1.9 to 2.3 × 106 spores/ml and produced as previously described (1). In the second experiment, seedlings were inoculated with mycelial plugs obtained from the edge of 10-day-old cultures growing on malt extract agar (MEA). Five seedlings in each experiment served as controls and were inoculated with sterile deionized water or plugs of sterile MEA. Inoculation points were wrapped with Parafilm M (Pechiney Plastic Packaging, Menasha, WI). Seedlings were grown in growth chambers (daytime temperature 26°C, nighttime 24°C, and a 15-h photoperiod) for 13 to 15 weeks. At the end of the first experiment, 7 of 10 seedlings inoculated with R. lauricola exhibited wilt that appeared as a dieback of a few to the majority of branches. Nine of the ten seedlings exhibited sapwood discoloration and the fungus was isolated from eight of these seedlings. At the end of the second experiment, 8 of 10 seedlings exhibited wilt that again appeared as a dieback of a few branches to most branches. All seedlings with wilt exhibited sapwood discoloration and the fungus was recovered from these seedlings. Two seedlings inoculated with R. lauricola exhibited no symptoms of disease and the fungus was not recovered. Control seedlings remained healthy in both experiments with no evidence of wilt or sapwood discoloration and R. lauricola was not isolated. These results indicate that California laurel is susceptible to laurel wilt caused by R. lauricola. Furthermore, the disease on California laurel may appear as a branch dieback affecting individual branches one at a time rather than a rapid wilt of the entire crown as is often observed in redbay (1). Currently, the RAB is not known to occur on the West Coast and it is also not known if this beetle is capable of attacking and producing brood on California laurel. Nonetheless, if the RAB and R. lauricola become established on the West Coast, laurel wilt could pose a serious threat to natural ecosystems as well as the avocado industry in California. References: (1) S. W. Fraedrich et al. Plant Dis. 92:215, 2008. (2) T. C. Harrington et al. Mycotaxon 104:399, 2008. (3) A. E. Mayfield, III et al. Plant Dis. 92:976, 2008.

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.

Laurel wilt is a lethal, nonnative vascular wilt disease of redbay (Persea borbonia), sassafras (Sassafras albidum), and other trees in the Lauraceae (1,4). It is caused by a fungus (Raffaelea lauricola) and transmitted by the redbay ambrosia beetle (Xyleborus glabratus), a nonnative insect first detected in Georgia in 2002 (1,2). Since introduction of the pathogen and vector (presumably from Asia), laurel wilt has caused extensive mortality to redbays in Georgia, Florida, and South Carolina (1). In June 2009, a landowner in Gautier, MS reported dead redbay trees. Signs and symptoms were identical to those reported for laurel wilt along the Atlantic Coast (wilted, bronze red foliage, and dark gray-to-black vascular discoloration) (1). Infected trees have subsequently been confirmed in and near the Pascagoula River Basin. Size of infected redbays ranged from 5 to 20 cm (diameter at breast height). No heavily decomposed or fallen redbays were noted. Many individual specimens exhibited extensive drying of stem wood and dry, wilted, light brown foliage. This indicates that introduction to the area may have occurred within the last 3 years. X. glabratus adults were collected (30°26′44.45″N, 88°39′41.83″W) in a Lindgren funnel trap baited with phoebe and manuka oil lures. Beetle identification was confirmed by USDA-APHIS, and voucher specimens were submitted to the Smithsonian National Museum of Natural History and the Mississippi Entomological Museum. Symptomatic redbay wood chips from the same location were surface sterilized and plated on cycloheximide-streptomycin malt agar and R. lauricola was isolated. A 1,026-bp portion of 18S rDNA (GenBank No. GQ996063) was amplified by PCR and sequenced using primers NS1 and NS4. BLASTn searches revealed perfect homology to R. lauricola isolate PL 697 (GQ329704). Two isolates of R. lauricola were recovered and prepared into separate spore suspensions (1 × 108 CFU/ml). Each isolate was inoculated into two healthy redbays. The inoculated redbays were placed in a growth chamber with two water-only controls. All inoculated plants, and none of the controls, exhibited wilt symptoms and died within 20 days. R. lauricola was recovered from the discolored sapwood of the inoculated plants, completing Koch's postulates. A model prediction for the natural dispersion of X. glabratus and R. lauricola estimated that these organisms may not reach Mississippi for 10 to 15 years (3). The current detection of laurel wilt in Mississippi is substantially ahead of this estimate. Currently, no records of laurel wilt have been reported from western Georgia, all of Alabama, or the panhandle of Florida. Confirmed locations in Mississippi are in Jackson County, along the Interstate 10 corridor and the Pascagoula River drainage. Due to the relatively large extent of the infestation (~64 km2, including hundreds of infected trees) eradication is not being attempted. Surveys, remote sensing, and phylogeographic analysis are underway to delineate the extent of infestation and discover the mode of introduction. The current outbreak of laurel wilt in Mississippi is likely the result of human transport of infested wood, either from Asia as a separate, new introduction or from previously infested areas in the southeastern United States. References: (1) S. W Fraedrich et al. Plant Dis. 92:215, 2008. (2) T. C. Harrington et al. Mycotaxon 104:399, 2008. (3) F. Koch and W. Smith. Environ. Entomol. 37:442, 2008. (4) J. A. Smith et al. Plant Dis. 93:198, 2009.

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 fungal vascular disease of redbay (Persea borbonia (L.) Spreng) and other plants in the family Lauraceae in the southeastern United States (1). The disease is caused by Raffaelea lauricola T. C. Harr., Fraedrich & Aghayeva, which is vectored by the exotic redbay ambrosia beetle (Xyleborus glabratus Eichhoff) (2). Pondspice (Litsea aestivalis (L.) Fern.) is an obligate wetland shrub listed as endangered in Florida and Maryland and threatened in Georgia (4). On 29 August 2008, 369 of 430 (85%) pondspice trees observed at St. Marks Pond in St. John's County, Florida were dead and/or dying (4). Stem samples were collected from plants with wilted and reddened foliage, entrance holes with boring dust characteristic of ambrosia beetle attack, and dark discoloration in the outer sapwood. Discolored stem sections were surface disinfested for 30 s in a 5% sodium hypochlorite solution and then plated onto cycloheximide streptomycin malt extract agar (1). Smooth, cream-buff, submerge hyphae with uneven margins resembling R. lauricola (2) was observed growing from all sapwood pieces. DNA was extracted from a single isolate (PL 392) and the 18s small subunit rDNA was PCR amplified and sequenced with primers NS1 and NS4 (3), resulting in a 1,026-bp amplicon. A BLASTn search showed identical homology to R. lauricola strain PL 159 (GenBank Accession No. EU257806). The 18s small subunit rDNA sequence was deposited into GenBank (FJ514097). In May 2011, a spore suspension was made by flooding a single-spore culture plate of isolate PL 392 with 2 ml of sterile water, collecting the spores by pipette, and quantification by hemacyometer to 1.5 × 106 spores/ml. Pathogenicity tests were conducted on 1 to 1.5 m tall pondspice plants. Six saplings were wounded by a 3/32-inch drill bit, with four receiving 50 μl of the spore suspension and two serving as water-inoculated controls. All plants were kept in a greenhouse under ambient temperature. Within 21 days, all fungal-inoculated saplings displayed complete canopy wilt, typical of laurel wilt. R. lauricola was later recovered from all four infected plants, completing Koch's postulates. To determine if the vector can reproduce in pondspice, infected stem sections were placed in a plastic rearing box indoors at room temperature, and both callow and mature adult female X. glabratus emerged in October and November 2008. Although laurel wilt has been previously observed on pondspice in South Carolina and Georgia (1), this is the first confirmation of the disease on pondspice in Florida and the first confirmation of the vector from stem material of this host. 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. Surdick and A. M. Jenkins. Pondspice (Litsea aestivalis) Population Status and Response to Laurel Wilt Disease in Northeast Florida. Florida Natural Areas Inventory, Tallahassee, FL, 2009.

Laurel wilt, caused by Raffaelea lauricola, has been responsible for extensive losses of redbay (Persea borbonia) in South Carolina and Georgia since 2003. Symptoms of the disease have been noted in other species of the Lauraceae such as the federally endangered pondberry (Lindera melissifolia) and the threatened pondspice (Litsea aestivalis). Pondberry and pondspice seedlings were inoculated with R. lauricola from redbay, and both species proved highly susceptible to laurel wilt. Field assessments found substantial mortality of pondberry and pondspice, but in many cases the losses were not attributable to laurel wilt. R. lauricola was isolated from only 4 of 29 symptomatic pondberry plants at one site, but the fungus was not recovered from three plants at another site. R. lauricola was isolated from one of two symptomatic pondspice plants at one site, and from five of 11 plants at another site, but not from any plant at a third site. Insect bore holes, similar to those produced by Xyleborus glabratus (the vector of laurel wilt), were found in some pondberry and pondspice stems, but adults were not found. Damage caused by Xylosandrus compactus was found in pondberry stems, but this ambrosia beetle does not appear to be a vector of R. lauricola. Xyleborinus saxeseni adults were found in a dying pondspice with laurel wilt, and R. lauricola was recovered from two of three adults. Isolates of R. lauricola from pondberry, pondspice, and X. saxeseni had rDNA sequences that were identical to previously characterized isolates, and inoculation tests confirmed that they were pathogenic to redbay. Because pondberry and pondspice tend to be shrubby plants with small stem diameters, these species may not be frequently attacked by X. glabratus unless in close proximity to larger diameter redbay.

Laurel wilt, caused by the fungus Raffaelea lauricola, affects the growth, development, and productivity of avocado, Persea americana. This study evaluated the potential of visible-near infrared spectroscopy for non-destructive sensing of this disease. The symptoms of laurel wilt are visually similar to those caused by freeze damage (leaf necrosis). In this work, we performed classification studies with visible-near infrared spectra of asymptomatic and symptomatic leaves from infected plants, as well as leaves from freeze-damaged and healthy plants, both of which were non-infected. The principal component scores computed from principal component analysis were used as input features in four classifiers: linear discriminant analysis, quadratic discriminant analysis (QDA), Naïve-Bayes classifier, and bagged decision trees (BDT). Among the classifiers, QDA and BDT resulted in classification accuracies of higher than 94% when classifying asymptomatic leaves from infected plants. All of the classifiers were able to discriminate symptomatic-infected leaves from freeze-damaged leaves. However, the false negatives mainly resulted from asymptomatic-infected leaves being classified as healthy. Analyses of average vegetation indices of freeze-damaged, healthy (non-infected), asymptomatic-infected, and symptomatic-infected leaves indicated that the normalized difference vegetation index and the simple ratio index were statistically different.

Silk bay (Persea humilis Nash) is a member of the Lauraceae precinctive to the scrub forests of central and southern Florida and a sister species to the primary laurel wilt host, redbay (P. borbonia (L.) Spreng), which is generally not found in these ecosystems. In November 2011, observations of silk bay mortality near Lake Placid in Highlands County, FL, were reported to Florida Forest Service agents. A subsequent visit to roadside and homeowners' properties in the area revealed many dead and dying silkbays with characteristic laurel wilt symptoms, including wilted crowns with brown persistent foliage, frass accumulated at tree bases, sapwood with dark streaking, and ambrosia beetle entrance holes (1). Twig samples were taken and later confirmed as silk bay by the Florida Division of Plant Industry in Gainesville. Trunk samples were taken from four trees for fungal isolation. Stem sections with vascular discoloration were surface disinfested for 30 s in a 5% sodium hypochlorite solution and then plated onto cycloheximide streptomycin malt extract agar (CSMA) (1). All sapwood pieces from the four samples resulted in the same cream-buff submerged fungal growth characteristic of the laurel wilt pathogen Raffaelea lauricola T. C. Harr., Fraedrich & Aghayeva (2). DNA was extracted from a single-spore derived isolate, PL1389, and an 18S small subunit ribosomal RNA gene sequence was generated with primers NS1 and NS4, resulting in a 1,031-bp amplicon (3). A BLASTn search showed identical homology to R. lauricola strains PL159 and PL382 (GenBank Accessions No. EU257806 and JF797171, respectively, 100% similarity, e-value 0.0, and a total score of 1,982). The sequence was deposited into GenBank and assigned the accession No. JQ247569. In December 2011, a spore suspension was made by flooding a PL1389 culture plate with 2 ml of sterile water, collecting by pipette, and quantification and adjusting to 3.25 × 106 spores/ml by hemacytometer. Pathogenicity was tested on potted plants in a growth chamber experiment. Five silk bays and three redbays were drill-wounded with a 3/32” drill bit and inoculated with 20 μl of the spore suspension. Three silk bays and two redbays served as water-inoculated controls. Within 5 weeks, all inoculated plants displayed the wilt and vascular discoloration characteristic of laurel wilt disease, while all water-inoculated controls remained healthy. Sapwood samples from all plants were plated onto the same CSMA media. R. lauricola was later recovered only from the wilted plants inoculated with PL1389, while no fungal growth was recovered from the asymptomatic water-inoculated controls. Silk bay, which plays a significant role in the limited scrub ecosystems of Florida, has now become another host in the laurel wilt epidemic, with its implications upon the scrub forests yet to be seen. 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.

Laurel wilt, caused by Raffaelea lauricola, a fungal symbiont of the redbay ambrosia beetle, Xyleborus glabratus, is responsible for extensive mortality of native redbays (Persea borbonia and P. palustris) in the coastal plains of the southeastern United States (1). The wilt also affects the more widespread sassafras, Sassafras albidum, particularly in areas where diseased redbays are common and populations of X. glabratus are high. Because sassafras stems were thought to lack chemicals that are attractive to the beetle, and sassafras tends to be widely scattered in forests, it was believed that the advance of the laurel wilt epidemic front might slow once it reached the edge of the natural range of redbay, which is restricted to the coastal plains of the Gulf and Atlantic Coasts (2). In July and August of 2011, wilt-like symptoms (i.e., wilted and dead leaves, and streaks of black discoloration in the xylem) were observed on 1 to 10 sassafras trees (15 to 23 cm diameter; 6 to 9 m height) at each of three locations, which were approximately 6 km from one another in Marengo Co., Alabama. Samples of the discolored wood from five trees were plated on malt agar amended with cycloheximide and streptomycin (CSMA), and a fungus morphologically identical to R. lauricola was isolated from each tree (1). For confirmation, a portion of the large subunit (28S) of the rDNA region of three of the isolates was sequenced (3); in each case, the sequence matched exactly that of other isolates of R. lauricola (EU123077) from the United States. Symptomatic trees were found at all three sites when revisited in April 2012, and approximately 20 sassafras trees in various stages of wilt were observed at one location, where only one diseased tree had been noted in 2011. Bolts were cut from the main stem of a symptomatic tree, and eggs, larvae, and adults of X. glabratus were commonly found in tunnels, and R. lauricola was isolated from the discolored xylem. Three container-grown sassafras saplings (mean height 193 cm, mean diameter 2.1 cm at groundline) were inoculated as previously described (1) with conidia (~600,000) from an isolate of R. lauricola. Three additional sassafras saplings were inoculated with sterile, deionized water, and all plants were placed in a growth chamber at 25°C with a 15-h photoperiod. Inoculated plants began to exhibit wilt symptoms within 14 days, and at 30 days all inoculated plants were dead and xylem discoloration was observed. Control plants appeared healthy and did not exhibit xylem discoloration. Pieces of sapwood from 15 cm above the inoculation points were plated on CSMA, and R. lauricola was recovered from all wilted plants but not from control plants. This is the first record of laurel wilt in Alabama and is significant because the disease appears to be spreading on sassafras in an area where redbays have not been recorded (see http://www.floraofalabama.org ). The nearest previously documented case of laurel wilt is on redbay and sassafras in Jackson Co., Mississippi (4), approximately 160 km to the south. The exact source of the introduction of X. glabratus and R. lauricola into Marengo Co. is not known. The vector may have been transported into the area with storms, moved with infested firewood, or shipped with infested timber by companies that supply mills in the area. References: (1) S. Fraedrich et al. Plant Dis. 92:215, 2008. (2) J. Hanula et al. Econ. Ent. 101:1276, 2008. (3) T. Harrington et al. Mycotaxon 111:337, 2010. (4) J. Riggins et al. Plant Dis. 95:1479, 2011.

Gulf licaria, Licaria trianda (Sw.) Kosterm., is a federally endangered member of the Lauraceae plant family in Miami-Dade County, Florida. It was never common in the area, and urban development has extirpated it from most of its former range; as of 2001, fewer than 10 trees remained in a single, remnant habitat in the continental United States, Simpson Park (25°45′31″N, 80°11′46″W) (2). Laurel wilt, caused by the fungus Raffaelea lauricola T. C. Harr., Fraedrich & Aghayeva, has recently devastated members of the Lauraceae in the southeastern United States, most notably redbay, Persea borbonia (1). As R. lauricola and its vector, the redbay ambrosia beetle Xyleborus glabratus, have spread in the region, an increasing number of taxa in this plant family have been affected by this disease (1). In 2012, seedlings of gulf licaria and redbay were obtained from local nurseries; they were grown in 30 liter pots, 1.3 m tall, had stems 3 cm in diameter 20 cm above the soil line, and were maintained with standard watering and fertilization practices. In two pathogenicity experiments on July 6 and September 25, 2012, three plants each of gulf licaria and redbay were inoculated with an isolate of R. lauricola, RL4, as described in previous experiments (3), and two plants each were mock inoculated (water control). RL4 is deposited as CBS 127349 at the Centraalbureau voor Schimmelcultures (CBS Fungal Biodiversity Centre, Utrecht, The Netherlands), and a SSU rDNA sequence for it is deposited in GenBank under Accession No. HM446155. Beginning 2 weeks after inoculation, plants were rated on a weekly basis for the development of external symptoms, on a subjective 1 (no symptoms) to 10 (dead) scale (3). After 5 weeks, inoculated plants of redbay in each experiment (positive control) had died after first developing symptoms of wilt and necrotic foliage that are typical for this disease (1). In contrast, inoculated plants of gulf licaria developed severe symptoms by the time experiments were terminated 6 and 11 weeks after inoculation; chlorosis developed on some of the leaves of all plants and these eventually abscised (mean external severities of 7.3 and 6.5, respectively), but plants did not die. Brown to greyish discoloration of sapwood developed in all inoculated plants, and the pathogen was recovered from symptomatic sapwood on CSMA (3). No symptoms developed on mock inoculated plants and the pathogen was not recovered from them. It is concluded that gulf licaria is susceptible to laurel wilt, but that it is apparently less susceptible than redbay. Whether X. glabratus is attracted to, or will bore into, gulf licaria is not known, but will play a significant role in the extent to which this rare tree is affected by laurel wilt. References: (1) S. W. Fraedrich et al. Plant Dis. 92:215, 2008. (2) G. D. Gann et al. Rare Plants of South Florida: Their History, Conservation, and Restoration. Institute for Regional Conservation, Miami, 2002. (3) R. C. Ploetz et al. Plant Pathol. 61:801, 2012.

Bay laurel (Laurus nobilis L.) is an economically important evergreen tree of the family Lauraceae. It is native to Asia Minor and the Balkans and was introduced into the United States for its ornamental and culinary uses (4). In September 2013, a 6-m-tall bay laurel in Gainesville, FL, attracted our attention because it had wilted leaves, discolored sapwood, and ambrosia beetle entrance holes, all symptoms of laurel wilt. In addition, the tree was growing close to an avocado that succumbed to the disease months earlier. In an effort to determine whether the laurel wilt pathogen (Raffaelea lauricola T.C. Harr., Fraedrich & Agaveya) was, indeed, involved in the decline of the tree of current interest, discolored sapwood was sectioned into 5-mm2 pieces, surface disinfested for 30 s in a 4% sodium hypochlorite solution, and plated onto CSMA media (1,2). Within 7 to 14 days, cream-colored, adpressed fungal growth typical of R. lauricola grew from the sapwood pieces (2). DNA was extracted from an isolate of a single conidium (PL1634) and a portion of the 18S rRNA gene was PCR-amplified with primers NS1/NS4, resulting in a 1,021-bp amplicon (GenBank Accession No. KF913344.1), with a BLASTn search revealing 100% homology to several R. lauricola isolates (3). To confirm pathogenicity, six bay laurel seedlings (0.5 m) and a silk bay (0.65 m) (Persea humilis, susceptible control) were wounded twice with a 0.5-mm-diameter drill bit. Then, 30 μl of a spore suspension of PL1634 (1.38 × 105 condia/plant) were introduced into the xylem by pipette and the wounds were wrapped in Parafilm (1). Negative controls consisted of a mock-inoculated (water) and non-inoculated bay laurel plus a mock-inoculated silk bay. Plants were placed in a growth chamber set to a 16/8 h (25/22°C) diurnal light/temperature cycle. After 60 days, all fungal-inoculated plants were completely wilted with dead leaves and subsequent necrosis of stems, while mock- and non-inoculated controls remained asymptomatic. Sapwood dissection revealed xylem discoloration similar to the original infected tree, and fungi morphologically similar to PL1634 were recovered from all inoculated plants upon isolation on CSMA media. Mock- and non-inoculated controls lacked vascular discoloration and fungal growth on media. In order to determine if the redbay ambrosia beetle, Xyleborus glabratus Eichoff (laurel wilt vector) could successfully reproduce in this host, symptomatic branches (7 cm in diameter) of L. nobilis with external evidence of ambrosia beetle attack (frass “toothpicks”) were placed in a plastic rearing box within a growth chamber (25°C). Within 4 weeks of incubation, dozens of immature and mature X. glabratus beetles emerged. This is the first record of Koch's postulates being completed for R. lauricola on L. nobilis and the ability of X. glabratus to infest and breed in its stems. This information may be of importance in the event of an introduction of X. glabratus and its fungal associate to Mediterranean areas where bay laurel is either growing wild or being cultivated as valuable commercial crop. 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., eds. PCR Protocols: A Guide to Methods and Applications. Academic Press. San Diego, CA, 1990. (4) A. O. Sari et al. New Forest 31:403, 2006.

Redbay ambrosia beetle, Xyleborus glabratus Eichhoff, is a wood-boring pest that has now invaded nine states in the southeastern United States. The beetle's dominant fungal symbiont (Raffaelea lauricola) is phytopathogenic, inducing laurel wilt in trees within the family Lauraceae. Members of the genus Persea are particularly susceptible to the lethal disease, including native redbay (P. borbonia) and swampbay (P. palustris), as well as commercial avocado (P. americana). Cubeb oil lures are the current standard for detection of X. glabratus, but recently eucalyptol and a 50% α-copaene oil have been identified as additional attractants. This study used a combination of binary-choice bioassays, field cage release-and-recapture assays, and a 12-wk field trial to compare efficacy of eucalyptol and copaene lures relative to commercial cubeb lures. In addition, GC-MS was used to quantify emissions from lures field-aged for 12 wk. In laboratory bioassays, copaene lures were more attractive than eucalyptol lures. In field cage assays, copaene lures recaptured a higher percentage of released beetles than cubeb lures. In the field test, cubeb lures captured fewer beetles than copaene lures, and lowest captures were obtained with eucalyptol lures. Combining eucalyptol with either copaene or cubeb lures did not increase captures over those lures deployed alone. Both copaene and cubeb lures were effective in attracting X. glabratus for 12 wk, but field life of eucalyptol lures was only 4 wk, consistent with the quantification of lure emissions. Results suggest that the 50% α-copaene lure provides the best pest detection currently available for X. glabratus.

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.

The exotic redbay ambrosia beetle, Xyleborus glabratus Eichhoff, has become a serious invasive pest in the USA, now established in eight southeastern states. Females are the primary vectors of a fungal pathogen, Raffaelea lauricola, that causes laurel wilt, a lethal disease of trees in the family Lauraceae. Laurel wilt has caused extensive mortality in native Persea species, including redbay (P. borbonia), swampbay (P. palustris), and silkbay (P. humilis), and currently it threatens avocado (P. americana) in Florida. With continued spread, laurel wilt may impact additional Lauraceae throughout the Americas. Currently, the most effective lures for X. glabratus contain cubeb oil, an essential oil composed of a complex mixture of terpenoids. To elucidate the primary attractants of X. glabratus, fractional distillation was used to separate whole cubeb oil into 17 fractions. All fractions were used initially as substrates for electroantennographic (EAG) analyses to assess olfactory response; then eight representative fractions were chosen for binary-choice bioassays to assess behavioral response. Although fractions containing monoterpenes elicited the strongest EAG responses, significant attraction of X. glabratus was observed only with fractions that contained high percentages of the sesquiterpenes α-copaene and α-cubebene. This information was used to prepare two prototype lures, one of which contained copaiba oil (an essential oil consisting of 10 % α-copaene), and the other a proprietary product enriched to 50 % (−)-α-copaene content. In field trials, the copaiba and commercial cubeb lures captured equal numbers of X. glabratus, but the 50 % copaene lure captured significantly more beetles and had field longevity of 3 months.

The redbay ambrosia beetle, Xyleborus glabratus Eichhoff (Coleoptera: Curculionidae: Scolytinae), due to its association with Raffaelea lauricola T.C. Harr., Fraedrich & Aghayeva (Ophiostomatales: Ophiostomataceae), a pathogen that causes laurel wilt, is considered one of the most damaging pests of Persea species (Laurales: Lauraceae) including avocado. Currently, there is no satisfactory method to control this pest. Biological control is being examined as an additional tool to be used to lower the pest population and slow its spread. The objective of this study was to determine the natural enemy community associated with X. glabratus in Florida and Taiwan by using 3 methods: 1) field-collected wood naturally infested with X. glabratus (Florida), 2) bolts of avocado artificially infested with X. glabratus (Florida) and 3) direct collection of natural enemies from a trap area baited with infested wood and known X. glabratus lures (Florida and Taiwan). Among the predacious insects, there were 8 species of Laemophloeidae, an unidentified species of Staphylinidae, Microsicus spp. (Zopheridae), and Europs sp. (Monotomidae) (all Coleoptera). Among the parasitoids, hymenopterans of the families Braconidae, Eulophidae, Pteromalidae, Encyrtidae, Eupelmidae, and Bethylidae emerged from wood containing various species of Scolytinae. However, the only specimens that emerged from logs in which X. glabratus was present were Bethylidae, Braconidae, Encyrtidae (perhaps Closterocerus sp.), and Scelionidae. Four hymenopteran species were collected using attractants in Florida and Taiwan. However, more studies are needed to clarify their role as natural enemies of X. glabratus.

Redbay ambrosia beetle, Xyleborus glabratus Eichhoff (Coleoptera: Curculionidae: Scolytinae), is a nonnative pest that transmits the pathogenic fungus Raffaelea lauricola T.C. Harr., Fraedrich & Aghayeva (Ophiostomatales: Ophiostomataceae), which causes laurel wilt disease in trees of the family Lauraceae. Laurel wilt is present in the commercial avocado (Persea americana Mill.; Laurales: Lauraceae) growing areas of Florida and poses a potential threat to the avocado industries of California and Mexico. The life cycle of X. glabratus was studied in avocado logs at 16, 20, 24, 28, and 32 °C. Xyleborus glabratus successfully completed its life cycle at 24, 28, and 32 °C, with the greatest oviposition and development rate at 28 °C. Development of the egg and pupal stages was studied at 12, 16, 18, 20, 24, 28, 32, and 36 °C. One linear and 7 nonlinear developmental models were used to estimate the temperature-dependent development of both stages. The linear model estimated the lower threshold temperatures for egg and pupal development to be 13.8 °C and 11.1 °C, respectively, and the degree-days (DD) for egg and pupal development to be 55.1 DD and 68.2 DD, respectively. The Brier-2, Ratkowsky, Logan, and polynomial models gave the best estimates for the temperature-dependent development of the egg stages, whereas the Brier-1, Logan, and polynomial models gave the best estimates of temperature-dependent development of the pupal stages. Our results suggested that the optimal temperature for development of X. glabratus was around 28 °C, and that temperature will play an important role in the spread and successful establishment of X. glabratus.

graphical a bstract abstract The redbay ambrosia beetle (RAB), Xyleborus glabratus, is a wood-boring insect that vectors the fungal pathogen, Raffaelea lauricola, which causes laurel wilt, a lethal disease of avocado. The objective of this study was to determine the susceptibility of RAB to infection and subsequent death by exposure to three commercial strains of entomopathogenic fungi (two strains of Isaria fumosorosea (Ifr 3581 and PFR), and strain GHA of Beauveria bassiana). RAB females were dipped in fungal spore solutions and their median survivorship times (MST) determined. Contact with any of the biopesticides resulted in death of all RAB females. MSTs of RAB females ranged from 3 days (B. bassiana) to 5 days (I. fumosorosea PFR). B. bas- siana killed RAB females faster, followed by Ifr 3581 and PFR. RAB females dipped in B. bassiana suspen- sions had the highest number of viable spores attached to their bodies, followed by Ifr 3581. Beetles dipped in PFR suspension had significantly less viable spores attached to their bodies. No significant dif- ferences were observed in the mortality of beetles exposed to entomopathogenic fungi by dipping in a fungal suspension or walking on treated avocado bolts. Beetles bored into the logs and constructed gal- leries, but they were found dead inside the galleries a few days after exposure to the entomopathogens. Entomopathogenic fungal infection in dead beetles was confirmed through molecular techniques. This is the first study to demonstrate that entomopathogenic fungi are potential biological control agents against RAB.

During a survey of nematode associates of ambrosia beetles from dead and dying red bay and avocado trees affected by the laurel wilt epidemic in southern Florida, a Ruehmaphelenchus species was isolated from the non-native ambrosia beetle, Xylosandrus crassiusculus. The new species is characterised by its possession of an oral disc at the stoma opening, three lines in the lateral field, male spicule with clear dorsal and ventral limbs connected by elongated triangular cuticle, thin membrane-like tissue and cuticular bridge-like structure, conical tail with pointed tip of males and conical tail with digitate mucro of females. The new species is very similar to four previously described species: R. asiaticus, R. digitulus, R. thailandae and R. sirisus, and can be distinguished only by some minor morphological differences in male tail characters, i.e., spicule morphology, position of genital papillae and tail tip shape, and morphometric values. However, the new species is phylogenetically unique, i.e., it is the basal taxon of the Ruehmaphelenchus clade and close to Bursaphelenchus spp. Ruehmaphelenchus juliae n. sp. is therefore proposed based on its morphological diagnostic characters and molecular sequences of near-full-length of SSU, internal transcribed spacer region, D1, D2 and D3 expansion segments of LSU ribosomal RNA and partial mitochondrial COI genes.

Bark and ambrosia beetles are highly specialized weevils (Curculionidae) that have established diverse symbioses with fungi, most often from the order Ophiostomatales (Ascomycota, Sordariomycetes). The two types of beetles are distinguished by their feeding habits and intimacy of interactions with their symbiotic fungi. The tree tissue diet of bark beetles is facilitated by fungi, while ambrosia beetles feed solely on fungi that they farm. The farming life history strategy requires domestication of a fungus, which the beetles consume as their sole food source. Ambrosia beetles in the subfamily Platypodinae originated in the mid-Cretaceous (119-88Mya) and are the oldest known group of farming insects. However, attempts to resolve phylogenetic relationships and the timing of domestication events for fungal cultivars have been largely inconclusive. We sequenced the genomes of 12 ambrosia beetle fungal cultivars and bark beetle associates, including the devastating laurel wilt pathogen, Raffaelea lauricola, to estimate a robust phylogeny of the Ophiostomatales. We find evidence for contemporaneous diversification of the beetles and their associated fungi, followed by three independent domestication events of the ambrosia fungi genus Raffaelea. We estimate the first domestication of an Ophiostomatales fungus occurred ~86Mya, 25 million years earlier than prior estimates and in close agreement with the estimated age of farming in the Platypodinae (96Mya). Comparisons of the timing of fungal domestication events with the timing of beetle radiations support the hypothesis that the first large beetle radiations may have spread domesticated ‘ambrosia’ fungi to other fungi-associated beetle groups, perhaps facilitating the evolution of new farming lineages. This article is protected by copyright. All rights reserved.

Summary Laurel wilt, caused by the fungus Raffaelea lauricola and transmitted by the exotic ambrosia beetle Xyleborus glabratus, has killed members of the Lauraceae plant family throughout the southeast United States. A series of experiments were conducted to examine the effects of inoculum concentration on the development of laurel wilt in swamp bay, Persea palustris, and avocado, Persea americana. In each experiment, host plants were inoculated with aqueous suspensions of 102, 103, 104 or 105 conidia of R. lauricola, and plants were rated periodically for external symptom development (wilting and foliar dieback). At the end of experiments, plants were rated for internal symptoms of the disease (discoloration of sapwood) and assayed for R. lauricola on a semi-selective medium. Symptom severity in swamp bay was significantly lower for the 102 treatment than at higher (103–105) concentrations, whereas 102 and 103 conidia caused less disease than 104 and 105 conidia in avocado. At the lowest inoculum concentration, 67% of the swamp bay plants and 20% of the avocados died by the time the respective experiments were terminated. The pathogen was recovered from a high proportion of the symptomatic sapwood of swamp bay (100%) and avocado (94%), and sapwood discoloration and recovery of R. lauricola from inoculated stems of swamp bay were highly correlated with recovery of the pathogen and symptom development in roots. Clearly, swamp bay and avocado are very sensitive to R. lauricola. The ability of only 100 conidia of this pathogen to kill these hosts suggests that few individuals of X. glabratus or other ambrosia beetles that carry low levels of the pathogen would be sufficient to transmit conidia that infect and lead to disease development. The results are also relevant to the development of disease-tolerant host selections, as they indicate levels of the pathogen appropriate for use in screening plants for disease resistance.

Summary Laurel wilt, caused by Raffaelea lauricola, is responsible for extensive mortality of redbay and other American members of the Lauraceae in the southeastern United States. Raffaelea lauricola is a mycangial symbiont of the redbay ambrosia beetle (Xyleborus glabratus), and the beetle and fungus were accidentally introduced from Asia. Branch dieback of camphortree (Cinnamomum camphora), an Asian member of the Lauraceae, has been occasionally observed in areas where laurel wilt has decimated redbay populations, and R. lauricola was isolated from such camphortrees. However, the role of X. glabratus and R. lauricola in this branch dieback remains unclear. Examination of camphortrees on Jekyll Island, Georgia showed that healthy-appearing trees and those with branch dieback had been attacked by X. glabratus, but the trees with branch dieback had four times as many beetle attacks. Raffaelea lauricola was routinely isolated from discoloured xylem near beetle tunnels in healthy trees and those with dieback. Single-point inoculations with R. lauricola on stems of mature, healthy camphortree trees failed to induce wilt-like symptoms or branch dieback, although areas of discoloration were scattered throughout the xylem, and R. lauricola was reisolated irregularly at various heights in some inoculated trees. In growth chamber experiments, single-point inoculations with R. lauricola resulted in systemic colonization but no wilt symptoms or branch dieback in camphortree saplings. In contrast, inoculations at multiple points along the stem (simulating multiple attacks by the vector) caused branch dieback and wilt-like symptoms, including a brownish, diffuse discoloration of the xylem. Camphortree appears to be more resistant than American species of Lauraceae to the vascular wilt caused by R. lauricola. The fungus does colonize camphortrees systemically, however, and can apparently cause branch dieback. This suggests that the fungus may provide brood material for X. glabratus in Asia as it does in the southeastern United States.

Summary The addition of a trap baited with manuka oil or longer lasting cubeb oil lures (Hanula et al. 2013) along with those baited with ethanol for surveys targeting detection of non-native, invasive Curculionidae could improve these surveys by also targeting the destructive redbay ambrosia beetle.

In the southeastern US, laurel wilt disease (LWD) is causing widespread mortality of species in the Lauraceae. The principal target, Persea borbonia, is the primary larval host of Papilio palamedes, which is known to feed on other Lauraceae species. Among these potential hosts, the exotic Cinnamomum camphora is the only species that has shown resistance to LWD. We hypothesized that oviposition preference for C. camphora and P. borbonia would correspond to larval performances on these species and that the relative host suitability of C. camphora would indicate an opportunity for host-switching. We used laboratory experiments and field observations to compare performance and preference of P. palamedes between C. camphora and P. borbonia foliage. Our results indicate moderate survivorship on C. camphora compared to P. borbonia and no differences in first and fourth instar growth rates between treatments. Fourth instars consumed relatively less of C. camphora foliage compared to that of P. borbonia, but metabolic efficiency did not differ between treatments. Rearing on the foliage of P. borbonia stump sprouts from LWD-infected trees resulted in significantly higher growth rates and metabolic efficiency as first and fourth instars, respectively. In the field and laboratory, we found no oviposition preference for C. camphora. While females laid eggs on C. camphora during laboratory trials, the same number of eggs was also laid on inanimate objects. We conclude that C. camphora is suitable for larval development but host-switching to this species by P. palamedes will be primarily constrained by the ecological factors that govern oviposition behaviors.

The redbay ambrosia beetle, Xyleborus glabratus, is an invasive wood-boring beetle that has become established in the southeastern United States. The beetle transmits the causal pathogen of lethal laurel wilt to susceptible host trees, which include redbay, an important forest community species, and avocado, a valuable food crop. By examining odors of redbay wood, we developed an artificial lure that captured X. glabratus in redbay forests. Eucalyptol was a critical component of the blend for beetle attraction, and eucalyptol alone in large quantities attracted X. glabratus. Furthermore, eucalyptol stimulated boring by X. glabratus into paper arenas. The results suggest that eucalyptol contributes to host selection behavior of X. glabratus and may be useful for management of this pathogen vector.

Dreaden, T. J., Davis, J. M., Harmon, C. L., Ploetz, R. C., Palmateer, A. J., Soltis, P. S., and Smith, J. A. 2014. Development of multilocus PCR assays for Raffaelea lauricola, causal agent of laurel wilt disease. Plant Dis. 98:379-383. 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.

The redbay ambrosia beetle, Xyleborus glabratus Eichhoff, vectors laurel wilt, Raffaelea lauricola T.C. Harr., Fraedrich & Aghayeva, that quickly kills all large diam (> 2.5cm) redbay trees [Persea borbonia (L.) Sprengel] in an area but smaller diam trees ( 2.5 cm diam near ground level were still living at the beginning of the study in 2007. Despite having no large trees available, populations of the beetle persisted at 2 of the ...

Vegetative propagation experiments were conducted on redbay ( Persea borbonia (L.) Spreng. [Lauraceae]) to evaluate disease resistance and to conserve germplasm. We developed a primary framework for redbay vegetative propagation to address limita tions of long-term seed storage and the need to preserve and screen putatively laurel wilt-resistant redbays. Healthy/asymptomatic redbay individuals were chosen from 6 field sites with high levels of disease pressure and mortality. These individuals were exposed to various IBA forms, bottom heat, and rooting media mixtures. The use of IBA was required for rooting redbay; however, we found no significant differences among IBA type or bottom heat on rooting success. Parent tree provenance significantly affected root length. A 3:1 (v:v) perlite:vermiculite mixture yielded significantly more cuttings with roots and more roots per cutting. We observed an average root ing percentage of 20 to 37%, and ramets that were still alive after 7 mo averaged 46 to 72% rooting across treatments. Therefore, we recommend treating with a 0.3% IBA gel and striking them in 3:1 (v:v) perlite:vermiculite without bottom heat under intermittent mist.

Redbay ambrosia beetle, Xyleborus glabratus, is an exotic wood-borer that vectors the fungal agent that induces laurel wilt. Since its introduction into Georgia in 2002, X. glabratus has spread throughout the southeastern U.S., and laurel wilt has decimated native Persea trees, particularly redbay (P. borbonia) and swampbay (P. palustris). The lethal disease now threatens avocado (P. americana) in Florida. To control the spread of laurel wilt, effective attractants are needed for early detection of the vector. Phoebe oil lures are the best known attractant, but they are no longer available. Current detection relies on manuka oil lures, but our previous research indicated they have a field life of only 2–3 weeks in Florida. Therefore, we evaluated seven essential oils as attractants for X. glabratus, and ethanol as a potential synergist. Field tests and electroantennography (EAG) were conducted to compare attraction and olfactory response to angelica seed, cubeb, ginger root, manuka, phoebe, tea tree, and orange oils. The highest captures were obtained with cubeb, manuka, and phoebe oils; ginger and angelica oils were intermediate, and tea tree and orange oils were not attractive. Addition of ethanol to oil lures did not increase captures. In subsequent tests with commercial formulations, cubeb lures captured more X. glabratus than manuka lures, were better for early detection at low population levels, and had longevity of at least 8 weeks. The highest EAG responses were elicited with phoebe and cubeb lures. Our results indicate that cubeb lures are the best attractant currently available for detection of X. glabratus.

Xyleborus glabratus Eichhoff (Coleoptera: Curculionidae: Scolytinae) is an invasive ambrosia beetle that vectors laurel wilt, a new disease that threatens avocado and other species in the Lauraceae Family. The lethal concentrations (LC50 & 90) of nine commercial insecticides to X. glabratus were determined by using a bolt-dip bioassay. Different formulations of bifenthrin, permethrin, fenpropathrin, z-cypermethrin + bifenthrin, l-cyhalothrin + thiamethoxam, malathion, chlorpyrifos, carbaryl, and methomyl were tested. Four concentrations of each insecticide were tested (0.5, 0.1, 0.03, and 0.01 of the label rate) and with water as a control. Beetles were exposed to treated bolts and mortality registered 48 h later. After 2 wk, bolts were destructively sampled to determine the number of beetles that constructed galleries and were alive inside the wood. Probit analysis was used to determine the LC50 & 90. Six pesticides were applied directly to the trunk and limbs of avocado trees in a commercial gr...

The redbay ambrosia beetle, Xyleborus glabratus Eichhoff (Coleoptera: Curculionidae: Scolytinae), is an exotic wood-boring pest that vectors Raffaelea lauricola T.C. Harr., Fraedrich & Aghayeva (Ophiostomatales: Ophiostomataceae), the etiologic agent of laurel wilt. To date, all confirmed U.S. hosts of X. glabratus and suscepts of laurel wilt are members of the family Lauraceae. However, in previous research, an unknown variety of lychee, Litchi chinensis Sonn. (Sapindales: Sapindaceae), was found to be highly attractive to X. glabratus and elicited boring behaviors. Therefore, a study was undertaken to evaluate two commercial cultivars of lychee, ‘Brewster’ and ‘Mauritius’, for susceptibility to attack by X. glabratus, for transmission of R. lauricola, and for development of laurel wilt disease. In no-choice laboratory bioassays, 35 and 44% of females bored into cut bolts of ‘Mauritius’ and ‘Brewster’, respectively. Similar boring was observed on the trunks of two live ‘Brewster’ trees; but afte...

The redbay ambrosia beetle, Xyleborus glabratus Eichoff, (Coleoptera: Curculionidae: Scolytinae) is a severe pest of North American trees and shrubs in the family Lauraceae. Supercooling point (SCP) is an important physiological baseline for cold tolerance studies and could provide useful insights into the invasive potential of X. glabratus in northern latitudes of North America. The supercooling point (SCP) of X. glabratus was experimentally determined on field-collected and artificially cold hardened specimens. Field-collected beetles were captured in Jackson County, Mississippi using Lindgren funnel traps baited with manuka oil lures. Testing was conducted from June through August 2011. The mean SCP for field-collected X. glabratus was -21.7 ± 0.5 °C (± SE). A significant negative trend in the SCPs of field-collected beetles occurred over the summer testing period. Xyleborus glabratus specimens were reared from redbay (Persea borbonia (L.) Sprengel bolts in June 2012 and artificially cold hardened in a low temperature incubator at a thermo-photoperiod of 7 °C:2 °C (10:14 h L:D) for 31 days. Artificially cold hardened X. glabratus supercooled to a mean temperature of -23.9 ± 0.4 °C (± SE), which was significantly lower than that of field-collected beetles. Biometric indices of beetles (size, weight, and size x weight interaction) had no effect on the mean supercooling SCPs of either field-collected or artificially cold hardened beetles. Results from environmentally conditioned beetles suggest that X. glabratus has a high degree of thermal plasticity. Based on the artificially cold hardened mean SCP, X. glabratus and laurel wilt disease have the possibility to impact sassafras and northern spicebush throughout eastern North America. The data, although preliminary, suggests that a previous spatio-temporal model based on climate match data may have substantially underestimated the geographical area that may be affected by X. glabratus. This study will help form the basis of building and validating models to better predict the North American invasion potential of X. glabratus.

Redbay ambrosia beetle Xyleborus glabratus is an invasive wood boring beetle that has become established in the southeastern U.S.A. and transmits a fungus Raffaelea lauricola that causes lethal laurel wilt. Among susceptible Lauraceae hosts are redbay Persea borbonia and avocado Persea americana. There is a crucial need for detection of this pest as it moves into new areas. Consequently, our goal was to create a better lure for the monitoring and control of redbay ambrosia beetle. We analyzed volatile emissions of R. lauricola, created a synthetic odour blend based on this analysis and tested this odour blend as a potential attractant in a redbay forest infested with X. glabratus. The synthetic Raffaelea odour blend was not attractive to the beetles by itself. However, it synergistically increased attraction to host-mimic volatiles. We tested four commercial release devices for dispensing Raffaelea odour at various release rates. Two prototypes with the highest release rate, when paired with commercial manuka oil lures, captured more beetles than manuka oil lures alone. These results indicate that a synthetic blend of volatiles based on the odour of the symbiotic fungus of X. glabratus may be useful for the development of more sensitive monitoring lures for this invasive pathogen vector.

Significant mortality of redbay trees (Persea borbonia (L.) Spreng.) in the southeastern United States has been caused by Raffaelea lauricola, T.C. Harr., Fraedrich, & Aghayeva (Harrington et al. 2008), a fungal symbiont of the exotic redbay ambrosia beetle, Xyleborus glabratus, Eichhoff (Fraedrich et al. 2008). This pathogen causes laurel wilt, which is an irreversible disease that can kill mature trees within a few weeks in summer. R. lauricola has been shown to be lethal to most native species of Lauraceae and cultivated avocado (Persea americana Mill.) in the southeastern United States. In this study, we examined the survival of X. glabratus and R. lauricola in wood chips made from infested trees by using a standard tree chipper over a 10-wk period. After 2 wk, 14 X. glabratus were recovered from wood chips, whereas 339 X. glabratus emerged from nonchipped bolts. R. lauricola was not found 2 d postchipping from wood chips, indicating that the pathogen is not likely to survive for long inside wood chips. In contrast, R. lauricola persisted in dead, standing redbay trees for 14 mo. With large volumes of wood, the potential for infested logs to be moved between states or across U.S. borders is significant. Results demonstrated that chipping wood from laurel wilt-killed trees can significantly reduce the number of X. glabratus and limit the persistence of R. lauricola, which is important for sanitation strategies aimed at limiting the spread of this disease.

The redbay ambrosia beetle, Xyleborus glabratus Eichhoff (Coleoptera: Curculionidae: Scolytinae), is a wood-boring pest that transmits the fungal pathogen Raffaelea lauricola, the causal agent of laurel wilt disease in American Lauraceae. This study documents the gallery formation patterns of X. glabratus as well as its life cycle and development at 25 ± 2 °C in logs of 3 natural hosts: avocado (Persea americana), redbay (P. borbonia) and swampbay (P. palustris). Females were observed to excavate galleries perpendicular to the tree trunk; galleries were characterized by a main entrance tunnel, from which branched secondary tunnels that, in turn, gave rise to tertiary tunnels. By dissecting infested logs daily, the length of time was determined for each developmental stage, and found to be comparable in all 3 hosts. Eggs were first encountered in avocado, redbay, and swampbay at 7, 11, and 10 days after gallery initiation (agi), respectively; larvae at 14, 20, and 14 days agi; pupae at 24, 26, and 26 days agi; and teneral adults at 31, 30, and 27 days agi. Despite comparable rates of development in all hosts, there were fewer progeny per female produced in avocado. Oviposition by the founding female extended over a broad time-span, and all stages were observed in the gallery at 1 month agi. Three larval instars were present, with mean head capsule widths of 0.21, 0.26, and 0.37 mm, respectively. Long term rearing of X. glabratus was achieved on swampbay logs soaked in water prior to infestation. Emergence of new females from logs was first observed at 60 d agi, indicating that teneral adults remain in hosts for ~1 month prior to dispersal. Emergence continued for up to 240 days, with maximum emergence observed between 120-150 days agi.