Stephen W. Fraedrich

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.

Biology and host associations of redbay ambrosia beetle (Coleoptera: Curculionidae: Scolytinae), exotic vector of laurel wilt killing redbay trees in the Southeastern United States

Abstract The redbay ambrosia beetle, Xyleborus glabratus Eichhoff (Coleoptera: Curculionidae: Scolytinae), and its fungal symbiont, Raffaelea sp., are new introductions to the southeastern United States responsible for the wilt of mature redbay, Persea borbonia (L.) Spreng., trees. In 2006 and 2007, we investigated the seasonal flight activity of X. glabratus, its host associations, and population levels at eight locations in South Carolina and Georgia where infestations ranged from very recent to at least several years old. Adults were active throughout the year with peak activity in early September. Brood development seems to take 50–60 d. Wood infested with beetles and infected with the Raffaelea sp. was similar in attraction to uninfested redbay wood, whereas both were more attractive than a nonhost species. Sassafras, Sassafras albidium (Nutt.) Nees, another species of Lauraceae, was not attractive to X. glabratus and very few beetle entrance holes were found in sassafras wood compared with redbay. Conversely, avocado, Persea americana Mill., was as attractive to X. glabratus as swampbay, P. palustris (Raf.) Sarg., and both were more attractive than the nonhost red maple, Acer rubrum L. However, avocado had relatively few entrance holes in the wood. In 2007, we compared X. glabratus populations in areas where all mature redbay have died to areas where infestations were very active and more recent. Trap catches of X. glabratus and numbers of entrance holes in trap bolts of redbay were correlated with the number of dead trees with leaves attached. Older infestations where mature host trees had been eliminated by the wilt had low numbers of beetles resulting in trap catches ranging from 0.04 to 0.12 beetles per trap per d compared with 4–7 beetles per trap per d in areas with numerous recently dead trees. Our results indicate beetle populations drop dramatically after suitable host material is gone and provide hope that management strategies can be developed to restore redbay trees. The lack of attraction of X. glabratus to sassafras suggests that spread of X. glabratus may slow once it is outside the range of redbay.

Isolations from the redbay ambrosia beetle, Xyleborus glabratus, confirm that the laurel wilt pathogen, Raffaelea lauricola, originated in Asia

The laurel wilt pathogen Raffaelea lauricola was hypothesized to have been introduced to the southeastern USA in the mycangium of the redbay ambrosia beetle, Xyleborus glabratus, which is native to Asia. To test this hypothesis adult X. glabratus were trapped in Taiwan and on Kyushu Island, Japan, in 2009, and dead beetles were sent to USA for isolation of fungal symbionts. Individual X. glabratus were macerated in glass tissue grinders, and the slurry was serially diluted and plated onto malt agar medium amended with cycloheximide, a medium semiselective for Ophiostoma species and their anamorphs, including members of Raffaelea. R. lauricola was isolated from 56 of 85 beetles in Taiwan and 10 of 16 beetles in Japan at up to an estimated 10 000 CFUs per beetle. The next most commonly isolated species was R. ellipticospora, which also has been recovered from X. glabratus trapped in the USA, as were two other fungi isolated from beetles in Taiwan, R. fusca and R. subfusca. Three unidentified Raffaelea spp. and three unidentified Ophiostoma spp. were isolated rarely from X. glabratus collected in Taiwan. Isolations from beetles similarly trapped in Georgia, USA, yielded R. lauricola and R. ellipticospora in numbers similar to those from beetles trapped in Taiwan and Japan. The results support the hypothesis that R. lauricola was introduced into the USA in mycangia of X. glabratus shipped to USA in solid wood packing material from Asia. However differences in the mycangial mycoflora of X. glabratus in Taiwan, Japan and USA suggest that the X. glabratus population established in USA originated in another part of Asia.

Quantification of Propagules of the Laurel Wilt Fungus and Other Mycangial Fungi from the Redbay Ambrosia Beetle, Xyleborus glabratus

The laurel wilt pathogen, Raffaelea lauricola, is a fungal symbiont of the redbay ambrosia beetle, Xyleborus glabratus, which is native to Asia and was believed to have brought R. lauricola with it to the southeastern United States. Individual X. glabratus beetles from six populations in South Carolina and Georgia were individually macerated in glass tissue grinders and serially diluted to quantify the CFU of fungal symbionts. Six species of Raffaelea were isolated, with up to four species from an individual adult beetle. The Raffaelea spp. were apparently within the protected, paired, mandibular mycangia because they were as numerous in heads as in whole beetles, and surface-sterilized heads or whole bodies yielded as many or more CFU as did nonsterilized heads or whole beetles. R. lauricola was isolated from 40 of the 41 beetles sampled, and it was isolated in the highest numbers, up to 30,000 CFU/beetle. Depending on the population sampled, R. subalba or R. ellipticospora was the next most frequently isolated species. R. arxii, R. fusca, and R. subfusca were only occasionally isolated. The laurel wilt pathogen apparently grows in a yeast phase within the mycangia in competition with other Raffaelea spp.

Three genera in the Ceratocystidaceae are the respective symbionts of three independent lineages of ambrosia beetles with large, complex mycangia

The genus Ambrosiella accommodates species of Ceratocystidaceae (Microascales) that are obligate, mutualistic symbionts of ambrosia beetles, but the genus appears to be polyphyletic and more diverse than previously recognized. In addition to Ambrosiella xylebori, Ambrosiella hartigii, Ambrosiella beaveri, and Ambrosiella roeperi, three new species of Ambrosiella are described from the ambrosia beetle tribe Xyleborini: Ambrosiella nakashimae sp. nov. from Xylosandrus amputatus, Ambrosiella batrae sp. nov. from Anisandrus sayi, and Ambrosiella grosmanniae sp. nov. from Xylosandrus germanus. The genus Meredithiella gen. nov. is created for symbionts of the tribe Corthylini, based on Meredithiella norrisii sp. nov. from Corthylus punctatissimus. The genus Phialophoropsis is resurrected to accommodate associates of the Xyloterini, including Phialophoropsis trypodendri from Trypodendron scabricollis and Phialophoropsis ferruginea comb. nov. from Trypodendron lineatum. Each of the ten named species was distinguished by ITS rDNA barcoding and morphology, and the ITS rDNA sequences of four other putative species were obtained with Ceratocystidaceae-specific primers and template DNA extracted from beetles or galleries. These results support the hypothesis that each ambrosia beetle species with large, complex mycangia carries its own fungal symbiont. Conidiophore morphology and phylogenetic analyses using 18S (SSU) rDNA and TEF1α DNA sequences suggest that these three fungal genera within the Ceratocystidaceae independently adapted to symbiosis with the three respective beetle tribes. In turn, the beetle genera with large, complex mycangia appear to have evolved from other genera in their respective tribes that have smaller, less selective mycangia and are associated with Raffaelea spp. (Ophiostomatales).

Susceptibility to Laurel Wilt and Disease Incidence in Two Rare Plant Species, Pondberry and Pondspice

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.

Ambrosiella roeperi sp. nov. is the mycangial symbiont of the granulate ambrosia beetle, Xylosandrus crassiusculus

Isolations from the granulate ambrosia beetle, Xylosandrus crassiusculus (Coleoptera: Curculionidae: Scolytinae: Xyleborini), collected in Georgia, South Carolina, Missouri and Ohio, yielded an undescribed species of Ambrosiella in thousands of colony-forming units (CFU) per individual female. Partial sequences of ITS and 28S rDNA regions distinguished this species from other Ambrosiella spp., which are asexual symbionts of ambrosia beetles and closely related to Ceratocystis spp. Ambrosiella roeperi sp. nov. produces sporodochia of branching conidiophores with disarticulating swollen cells, and the branches are terminated by thick-walled aleurioconidia, similar to the conidiophores and aleurioconidia of A. xylebori, which is the mycangial symbiont of a related ambrosia beetle, X. compactus. Microscopic examinations found homogeneous masses of arthrospore-like cells growing in the mycangium of X. crassiusculus, without evidence of other microbial growth. Using fungal-specific primers, only the ITS rDNA region of A. roeperi was amplified and sequenced from DNA extractions of mycangial contents, suggesting that it is the primary or only mycangial symbiont of this beetle in USA.

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.

First report of laurel wilt, caused by Raffaelea lauricola, on sassafras (Sassafras albidum) in Alabama.

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.

Progression and Impact of Laurel Wilt Disease within Redbay and Sassafras Populations in Southeast Georgia

Abstract Laurel wilt disease (LWD), caused by the fungus Raffaelea lauricola and transmitted by Xyleborus glabratus (Redbay Ambrosia Beetle [RAB]), has killed millions of Persea borbonia (Redbay) trees throughout the southeastern Coastal Plain. Laurel wilt also has been detected in Sassafras albidum (Sassafras) in widely dispersed locations across the southeastern US. We established long-term laurel wilt disease-progression plots in Redbay and Sassafras stands in southeastern Georgia and monitored them through 4 years to document mortality rates and investigate long-term effects of LWD on Redbay and Sassafras survival and regeneration. Laurel wilt disease killed 87.3% of Redbay and 79.5% of Sassafras trees in the plots. The time from initial LWD detection to inactivity (no new mortality) in Redbay stands ranged from 1.1 to 3.6 years, with rate of disease progression positively related to host-tree size and abundance. Larger trees died at a higher rate in both Redbay and Sassafras stands, and mortality curves were similar for both species. All diseased Redbay trees died to the ground level, but the majority produced persistent below-ground basal sprouts, rapidly providing potential replacement stems. Few below-ground basal sprouts were observed on Sassafras trees killed by LWD, but over a quarter had epicormic shoots that survived up to several years after infection, and small trees remained alive on most sites, suggesting some level of tolerance to LWD. Substantial numbers of RAB were only captured in baited traps located adjacent to plots in an advanced-active stage of disease progression with abundant infested trees, both in Redbay and Sassafras stands. However, lingering presence of small numbers of RAB in post-epidemic areas and scattered LWD mortality in small-sized Redbay regeneration sprouts and seedlings suggest that secondary disease cycles may occur as Redbay trees there reach greater numbers and size in the future. Documentation of RAB and LWD spreading in Sassafras in the absence of Redbay supports concern that LWD will continue to spread into areas with abundant, large Sassafras trees, which would increase the probability that RAB and LWD will expand into extensive populations of other laurel species present in the western US and Central and South America.