Albert E. Mayfield

A fungal symbiont of the redbay ambrosia beetle causes a lethal wilt in redbay and other lauraceae in the Southeastern United States

Extensive mortality of redbay has been observed in the coastal plain counties of Georgia and southeastern South Carolina since 2003 and northeastern Florida since 2005. We show that the redbay mortality is due to a vascular wilt disease caused by an undescribed Raffaelea sp. that is a fungal symbiont of Xyleborus glabratus, an exotic ambrosia beetle. Trees affected by the disease exhibit wilt symptoms that include a black discoloration of the sapwood. Redbay trees and containerized seedlings died within 5 to 12 weeks after inoculation with the Raffaelea sp. When redbay seedlings were challenged with X. glabratus, the beetles tunneled into 96% of the plants, 70% died, and the Raffaelea sp. was recovered from 91%. X. glabratus and the Raffaelea sp. have also been associated with mortality of sassafras, and the Raffaelea sp. has been isolated from wilted pondberry and pondspice. Additional inoculation studies have shown that the Raffaelea sp. is pathogenic to sassafras, spicebush, and avocado, but not to red maple. Female adults of X. glabratus have paired mycangia near the mandibles, and the Raffaelea sp. is routinely isolated from the heads of beetles. The fungus is apparently introduced into healthy redbay during beetle attacks on stems and branches. The wilt currently affecting redbay and sassafras represents a major threat to other members of the Lauraceae indigenous to the Americas, including avocado in commercial production.

Development of a rain down technique to artificially infest hemlocks with the hemlock woolly adelgid, Adelges tsugae.

Abstract The hemlock woolly adelgid Adelges tsugae Annand (Hemiptera: Adelgidae), is a non-native invasive pest that has caused widespread decline and mortality of eastern hemlock (Tsuga canadensis (L.) Carr. (Pinales: Pinaceae)) and Carolina hemlock (T. caroliniana Engelm.) in the eastern United States. Our preliminary experiments evaluated the utility of a rain-down technique to induce artificial infestations of A. tsugae on hemlock seedlings en masse. Experiments were conducted in PVC (1 m3) cages topped with poultry wire for placement of A. tsugae-infested branches, and with 1 m2 gridded glue sheets and/or hemlock seedlings placed below to capture adelgid abundance, distribution, and infestation rate data. In the March 2011 experiment, the density of progrediens crawlers (adelgid nymphs, first instars) that rained down inside the PVC cages was significantly higher in the high ovisac treatment compared to the low ovisac treatment, with an estimated 513,000 and 289,000 crawlers per m2 falling beneath each treatment, respectively. Resulting A. tsugae infestation rates on Carolina hemlock seedlings placed inside the cages did not differ between the treatments but were at or above established damage threshold densities for the adelgid. Infestation rates on eastern hemlock seedlings that were placed in cages nine days after the experiment started were below damage threshold levels and did not differ between the treatments. In the May 2011 experiment, the density of sistens crawlers raining down was substantially lower, with 17,000 and 33,000 falling per m2 in the low and high ovisac treatments, respectively. Resulting infestation rates on Carolina hemlock seedlings were extremely low and well below damage threshold levels. Although A. tsugae crawlers were well distributed across the 1 m2 gridded glue sheets placed at the bottom of each cage, hot spots of unusually high crawler density did occur in both experiments. This rain-down technique shows potential for use in an operational tree-breeding program where screening large numbers of hemlock seedlings for resistance to A. tsugae is required.

First Report of Laurel Wilt Disease Caused by a Raffaelea sp. on Avocado in Florida

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.

North American Lauraceae: Terpenoid Emissions, Relative Attraction and Boring Preferences of Redbay Ambrosia Beetle, Xyleborus glabratus (Coleoptera: Curculionidae: Scolytinae)

The invasive redbay ambrosia beetle, Xyleborus glabratus, is the primary vector of Raffaelea lauricola, a symbiotic fungus and the etiologic agent of laurel wilt. This lethal disease has caused severe mortality of redbay (Persea borbonia) and swampbay (P. palustris) trees in the southeastern USA, threatens avocado (P. americana) production in Florida, and has potential to impact additional New World species. To date, all North American hosts of X. glabratus and suscepts of laurel wilt are members of the family Lauraceae. This comparative study combined field tests and laboratory bioassays to evaluate attraction and boring preferences of female X. glabratus using freshly-cut bolts from nine species of Lauraceae: avocado (one cultivar of each botanical race), redbay, swampbay, silkbay (Persea humilis), California bay laurel (Umbellularia californica), sassafras (Sassafras albidum), northern spicebush (Lindera benzoin), camphor tree (Cinnamomum camphora), and lancewood (Nectandra coriacea). In addition, volatile collections and gas chromatography-mass spectroscopy (GC-MS) were conducted to quantify terpenoid emissions from test bolts, and electroantennography (EAG) was performed to measure olfactory responses of X. glabratus to terpenoids identified by GC-MS. Significant differences were observed among treatments in both field and laboratory tests. Silkbay and camphor tree attracted the highest numbers of the beetle in the field, and lancewood and spicebush the lowest, whereas boring activity was greatest on silkbay, bay laurel, swampbay, and redbay, and lowest on lancewood, spicebush, and camphor tree. The Guatemalan cultivar of avocado was more attractive than those of the other races, but boring response among the three was equivalent. The results suggest that camphor tree may contain a chemical deterrent to boring, and that different cues are associated with host location and host acceptance. Emissions of α-cubebene, α-copaene, α-humulene, and calamenene were positively correlated with attraction, and EAG analyses confirmed chemoreception of terpenoids by antennal receptors of X. glabratus.

First report of laurel wilt disease caused by Raffaelea lauricola on camphor in Florida and Georgia.

Laurel wilt is a recently described (1) vascular disease of redbay (Persea borbonia (L.) Spreng) and other plants in the family Lauraceae. The wilt is caused by Raffaelea lauricola, a fungus vectored by the nonnative redbay ambrosia beetle (Xyleborus glabratus Eichhoff) (1,2). Since 2003, laurel wilt has caused widespread mortality of redbay in Georgia, South Carolina, and Florida (1) and has recently been found on avocado in Florida (4). Since June of 2007, wilted shoots and branch dieback have been observed in several camphor trees (Cinnamomum camphora (L.) Sieb.) in residential areas of McIntosh and Glynn counties in Georgia and Baker County in Florida. Symptomatic camphor trees ranged from 4.5 to 12 m high and occurred in areas where redbay mortality due to laurel wilt has been frequently observed during the last 2 to 3 years. In some camphor trees, only the smaller branches (<2 cm in diameter) were wilting or dead, whereas in other trees (e.g., Baker County, Florida), the larger branches and substantial portions of the crown were also symptomatic. Rapid wilt that affects entire trees that is usually observed in redbay, has not been observed in camphor. Some camphor trees in residential areas of Jekyll Island, Georgia (Glynn County), where extensive wilt of redbay has occurred, have exhibited only localized wilt of some shoots or branches and other camphors remain asymptomatic. Removal of bark from wilted branch sections revealed black-to-brownish staining in the sapwood, characteristic of laurel wilt. Although no evidence of ambrosia beetles was observed on these samples, more extensive surveys are needed to determine the role of this vector in laurel wilt of camphor. Wood chips from symptomatic areas of branches were surface sterilized and plated on cycloheximide-streptomycin malt agar as previously described (1,4) and R. lauricola was routinely isolated. Small subunit (18S) sequences from rDNA were amplified by PCR and sequenced using primers NS1 and NS4 (3). BLASTn searches revealed homology to R. lauricola C2203 (GenBank Accession No. EU123076, 100% similarity, e-value of 0.0, and a total score of 1,886). The small subunit rDNA sequence for this isolate has been deposited into GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/index.html ) and has been assigned Accession No. EU 853303. The presence of laurel wilt in camphor provides an opportunity to understand the pathogen distribution and possible resistance mechanisms in this host, which could have implications for efforts to remediate the impacts of the disease in redbay and other species in the Lauraceae 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) M. A. Innis et al. PCR Protocols, A Guide to Methods and Applications. Academic Press. San Diego, CA, 1990. (4) A. E. Mayfield, III et al. Plant Dis. 92:976, 2008.

First report of laurel wilt disease caused by Raffaelea lauricola on sassafras in Florida and South Carolina.

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). Kochs 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.

Effect of tree species and end seal on attractiveness and utility of cut bolts to the redbay Ambrosia beetle and granulate Ambrosia beetle (Coleoptera: Curculionidae: Scolytinae)

ABSTRACT The redbay ambrosia beetle, Xyleborus glabratus Eichhoff, is a non-native invasive pest and vector of the fungus that causes laurel wilt disease in certain trees of the family Lauraceae. This study assessed the relative attractiveness and suitability of cut bolts of several tree species to X. glabratus. In 2009, female X. glabratus were equally attracted to traps baited with swampbay (Persea palustris (Rafinesque) Sargent) and camphortree (Cinnamomum camphora (L.) J. Presl), which were more attractive than avocado (Persea americana Miller), lancewood (Ocotea coriacea (Swartz) Britton), and sweetbay (Magnolia virginiana L.). These species were more attractive than loblolly bay (Gordonia lasianthus (L.) J. Ellis). X. glabratus entrance hole density and emergence from caged bolts were highest on swampbay and camphortree. In 2010, swampbay was significantly more attractive to X. glabratus than sassafras (Sassafras albidum (Nuttall) Nees), yellow poplar (Liriodendron tulipifera L.), and eastern redbud (Cercis canadensis L.). Sassafras bolts end sealed with a liquid wax-and-water emulsion were more attractive to X. glabratus than end-sealed bolts of yellow poplar and redbud. Relative to unsealed bolts, end seal decreased X. glabratus entrance hole density on swampbay and decreased granulate ambrosia beetle (Xylosandrus crassiusculus (Motschulsky)) trap catch, entrance hole density, and adult emergence from swampbay. X. crassiusculus was not attracted to sassafras, yellow poplar, and redbud and was not more attracted to manuka oil than to unbaited traps. Sassafras was more attractive to X. glabratus than previously reported and supported reproducing populations of the insect. End sealing bolts with a wax-and-water emulsion may not be optimal for attracting and rearing ambrosia beetles in small logs.

Recovery plan for laurel wilt on redbay and other forest species caused by Raffaelea lauricola and disseminated by Xyleborus glabratus

This recovery plan is one of several disease-specific documents produced as part of the National Plant Disease Recovery System (NPDRS) called for in Homeland Security Presidential Directive Number 9 (HSPD-9). The purpose of the NPDRS is 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. Each disease-specific plan 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. These documents are not intended to be stand-alone documents that address all of the many and varied aspects of plant disease outbreak and all of the decisions that must be made and actions taken to achieve effective response and recovery. They are, however, documents that will help USDA guide further efforts directed toward plant disease recovery.

Suitability of California bay laurel and other species as hosts for the non-native redbay ambrosia beetle and granulate ambrosia beetle.

The redbay ambrosia beetle Xyleborus glabratus Eichhoff is a non‐native vector of the pathogen that causes laurel wilt, a deadly disease of trees in the family Lauraceae in the southeastern U.S.A. Concern exists that X. glabratus and its fungal symbiont could be transported to the western U.S.A. and cause damage to California bay laurel Umbellularia californica (Hook. & Arn.) Nutt. in California and Washington. The present study evaluated in‐flight attraction, attack density and emergence of X. glabratus and another invasive ambrosia beetle Xylosandrus crassiusculus (Motschulsky) on cut bolts of California bay laurel and eight related tree species in an infested forest in South Carolina. Xylosandrus crassiusculus is not a vector of the laurel wilt pathogen but is a pest of nursery and ornamental trees. Mean catch of X. glabratus on California bay laurel bolts was not significantly different from catches on bolts of known X. glabratus hosts sassafras Sassafras albidum (Nutt.) Nees and swampbay Persea palustris (Raf.) Sarg. Mean attack density and adult emergence of both beetle species from California bay laurel was equal to or greater than all other tree species tested. Both beetle species readily produced brood in California bay laurel bolts. The results obtained in the present study suggest that California bay laurel may be negatively impacted by both of these invasive ambrosia beetles if they become established in the trees native range.

Efficacy of Heat Treatment for the Thousand Cankers Disease Vector and Pathogen in Small Black Walnut Logs

ABSTRACT Thousand cankers disease, caused by the walnut twig beetle (Pityophthorus juglandis Blackman) and an associated fungal pathogen (Geosmithia morbida M. Kolarík, E. Freeland, C. Utley, and N. Tisserat), threatens the health and commercial use of eastern black walnut (Juglans nigra L.), one of the most economically valuable tree species in the United States. Effective phytosanitary measures are needed to reduce the possibility of spreading this insect and pathogen through wood movement. This study evaluated the efficacy of heat treatments and debarking to eliminate P. juglandis and G. morbida in J. nigra logs 4–18 cm in diameter and 30 cm in length. Infested logs were steam heated until various outer sapwood temperatures (60, 65, and 70°C in 2011; 36, 42, 48, 52, and 56°C in 2012) were maintained or exceeded for 30–40 min. In 2011, all heat treatments eliminated G. morbida from the bark, but logs were insufficiently colonized by P. juglandis to draw conclusions about treatment effects on the beetle. Debarking did not ensure elimination of the pathogen from the sapwood surface. In 2012, there was a negative effect of increasing temperature on P. juglandis emergence and G. morbida recovery. G. morbida did not survive in logs exposed to treatments in which minimum temperatures were 48°C or higher, and mean P. juglandis emergence decreased steadily to zero as treatment minimum temperature increased from 36 to 52°C. A minimum outer sapwood temperature of 56°C maintained for 40 min is effective for eliminating the thousand cankers disease vector and pathogen from walnut logs, and the current heat treatment schedule for the emerald ash borer (60°C core temperature for 60 min) is more than adequate for treating P. juglandis and G. morbida in walnut firewood.