Efficacy of heat treatment for the thousand cankers disease vector and pathogen in small black walnut logs.

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 C. 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 degrees C in 2011; 36, 42, 48, 52, and 56 degrees 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 degrees C or higher, and mean P. juglandis emergence decreased steadily to zero as treatment minimum temperature increased from 36 to 52 degrees C. A minimum outer sapwood temperature of 56 degrees 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 degrees 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.

Fusarioid community diversity associated with conifer seedlings in forest nurseries across the contiguous USA.

Introduction: Fusarioid fungi that cause damping-off and root diseases can result in significant losses to conifer crops produced in forest nurseries across the USA. These nurseries are vital to reforestation and forest restoration efforts. Understanding the diversity of Fusarioid fungi associated with damping-off and root diseases of conifer seedlings can provide an approach for targeted management techniques to limit seedling losses and pathogen spread to novel landscapes. Methods: This study identifies 26 Fusarium spp. (F. acuminatum, F. annulatum, F. avenaceum, F. brachygibbosum, F. clavus, F. commune, F. cugenangense, F. diversisporum, F. elaeagni, F. elaeidis, F. flocciferum, F. fredkrugeri, F. fujikuroi, F. grosmichelii, F. ipomoeae, F. lactis, F. languescens, F. luffae, F. odoratissimum, F. oxysporum, F. queenslandicum, F. redolens, F. torulosum, F. triseptatum, F. vanleeuwenii, & F. verticillioides), 15 potential species within Fusarium and Neocosmospora species complexes (two from F. fujikuroi species complex, nine from F. oxysporum species complex, three from F. tricinctum species complex, and one from Neocosmospora species complex), and four Neocosmospora spp. (N. falciforme, N. metavorans, N. pisi, & N. solani) and associated host information collected from conifer-producing nurseries across the contiguous USA. Results: Phylogenetic analyses identified Fusarioid fungi haplotypes that were associated with 1) host specificity, 2) localization to geographic regions, or 3) generalists found on multiple hosts across diverse geographic regions. Discussion: The haplotypes and novel species identified on conifer seedlings should be considered for further analysis to determine pathogenicity, pathogen spread, and assess management practices.

First Report of Laurel Wilt Caused by Raffaelea lauricola on Sassafras in Mississippi.

Laurel wilt is caused by the fungus Raffaelea lauricola T.C. Harrin., Aghayeva & Fraedrich and is lethal to redbay (Persea borbonia (L.) Spreng.), sassafras (Sassafras albidum (Nutt.) Nees), and other species in the Lauraceae (1). The fungus is carried by the redbay ambrosia beetle (Xyleborus glabratus Eichh.), which is native to Asia. After being discovered in Georgia in 2002 (1), X. glabratus and R. lauricola have spread rapidly, causing extensive redbay mortality in South Carolina, Georgia, Florida, and Mississippi (1,4). The disease has also been confirmed on sassafras in Florida, South Carolina (1), and Georgia. Questions remain as to whether laurel wilt will continue to spread on sassafras, which often occurs as scattered trees in the eastern United States. In June 2010, a homeowner reported that a sassafras tree north of Van Cleave, MS (30.668°N, 88.686°W) had begun wilting in late May. This landscape tree had three 10-m high stems (~20 cm in diameter at breast height). Dark staining in the xylem was observed around the entire circumference of all three stems and nearly all leaves were bronze colored and wilted. No ambrosia beetle tunnels were observed in the stems. No other symptomatic Lauraceae were encountered in the wooded area within 300 m. The nearest known location with laurel wilt on redbay was ~15 km away (4). A Lindgren funnel trap baited with manuka oil (2) was placed at the site in June and monitored biweekly until November, but no X. glabratus adults were captured. Chips from discolored xylem of the sassafras were surface sterilized, plated on cycloheximide-streptomycin malt agar, and R. lauricola was readily isolated (1). Identity of the fungus (isolate C2792 in collection of T. Harrington) was confirmed by using partial sequences of the 28S rDNA (3). The sassafras sequence was identical to that of all known sequences of R. lauricola in the United States, including GenBank No. EU123076 (the holotype isolate from redbay). To confirm pathogenicity, isolate C2792 was grown on malt extract agar and three redbay (average: 141 cm high and 12 mm in diameter at soil interface) and three sassafras (average: 170 cm high and 17 mm in diameter at soil interface) potted plants were wound inoculated with 0.2 ml of a spore suspension (4.9 × 106 conidia/ml) (1). Three control plants of each species were inoculated with sterile deionized water. After 8 weeks in a growth chamber at 26°C, all inoculated redbay and sassafras plants exhibited xylem discoloration above and below the inoculation point, two of the redbay and two of the sassafras had died, and the other plant of each species exhibited partial wilt (the main terminal or one or more branches). All control plants were asymptomatic. R. lauricola was reisolated from all inoculated symptomatic plants but not from controls. To our knowledge, this is the first report of laurel wilt on sassafras in Mississippi. Both redbay (4) and sassafras appear to be highly susceptible to the disease as it moves westward. Sassafras is less attractive than redbay to X. glabratus and it was thought that this might contribute to slowing the spread of laurel wilt once outside the range of redbay (2). Nonetheless, our observations confirm that sassafras can be infected where laurel wilt on redbay is not in the immediate vicinity. References: (1) S. W. Fraedrich et al. Plant Dis. 92:215, 2008. (2) J. L. Hanula et al. J. Econ. Entomol. 101:1276, 2008. (3) T. C. Harrington et al. Mycotaxon 111:337, 2010. (4) J. J. Riggins et al. Plant Dis. 94:634, 2010.

Shoot Blight Caused by Sirococcus tsugae on Eastern Hemlock (Tsuga canadensis) in Georgia.

Eastern hemlock (Tsuga canadensis) is an ecologically and economically important conifer from the north-central United States to the east coast of North America to the southern Appalachian Mountains. In early spring 2010, blighted shoot tips of eastern hemlock were observed at widely separated locations in the Chattahoochee National Forest in north Georgia. Damage did not appear to be directly related to hemlock woolly adelgid (Adelges tsugae) activity, which was sporadic or absent in some areas where symptoms were observed. A preliminary survey in March 2010 revealed that incidence of blighted shoots on individual trees varied, but was as high as 70%. Stems of shoots produced the previous year were frequently necrotic, had lost needles, and bore pycnidia with hyaline, two-celled conidia consistent with those of Sirococcus tsugae (2,3). Later in the spring and summer, shoots of the current year's growth became blighted, with sporulation of S. tsugae also on dead and dying needles. While S. tsugae previously has been reported on T. heterophylla, T. mertensiana, Cedrus atlantica, and C. deodara in western North America, it has only recently been reported on eastern hemlock (1), and its ability to induce shoot blight has not been proven. Pure cultures (2,3) were obtained on streptomycin-amended potato dextrose agar (PDA) and their identity was confirmed by species-specific PCR primers (4). Nuclear rDNA internal transcribed spacer sequence (554 nucleotides) also was obtained for isolate 10-05 and deposited in GenBank (Accession No. HQ256769). This sequence was found to be identical to sequences previously deposited for S. tsugae isolates. Isolate 10-05 and a second isolate (10-06) were used to inoculate potted 2-year-old eastern hemlock seedlings in a growth chamber at 20°C with a 16-h photoperiod. Conidia were collected by flooding 1-month-old colonies on PDA with sterile water. Expanding shoots on one branch of each seedling were wounded using scissors to cut the tips off needles and stems, while another branch remained nonwounded. Ten seedlings per isolate were inoculated by spraying to runoff with a suspension of 5 × 106 conidia ml-1 in sterile water, and five similarly treated control seedlings were sprayed with sterile water. Seedlings were covered with plastic bags to maintain high humidity for 4 days. Germination of conidia of each isolate incubated on water agar in this growth chamber was >80% after 24 h. Symptoms were evaluated and reisolation was attempted on streptomycin-amended PDA 2 months after inoculation. Symptoms of seedlings inoculated with either isolate included chlorotic and necrotic needle spots, browning of cut edges of needles, browning and death of needle tips and entire needles, death of stem tips with retention of dead needles, and needle loss. Symptoms of control seedlings were limited to slight browning of cut edges of needles. The fungus was reisolated from wounded shoots of 17 of 20 inoculated seedlings and nonwounded shoots of 5 of 20 inoculated seedlings and was not cultured from control seedlings. To our knowledge, this is the first report of S. tsugae in Georgia and also the first demonstration of its ability to produce symptoms that have been attributed to it on any tree species. References: (1) M. Miller-Weeks and W. Ostrofsky. USDA. Forest Service. Online publication. NA-PR-01-10, 2010. (2) A. Y. Rossman et al. For. Pathol. 38:47. (3) D. R. Smith et al. For. Pathol. 33:141, 2003. (4) D. R. Smith and G. R. Stanosz. For. Pathol. 38:156, 2008.

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.

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.

Diplodia pinea, the Cause of Diplodia Blight of Pines, Confirmed in Alabama, Louisiana, and Mississippi.

Loblolly pine (Pinus taeda) is the major commercial pine species cultivated in the Gulf Coast Region of the southern United States. Symptoms of Diplodia shoot blight (including yellow and brown needles and resin-soaked, dead, small twigs), pycnidia with conidia typical of Diplodia pinea on blighted shoots, and damaged, immature seed cones were observed during the summer of 2007 in loblolly pine seed orchards near Ward, AL, Winn Parish, LA, and Moselle, MS. Similar conidia also were obtained from pycnidia on opened seed cones of longleaf pine (P. palustris) collected on the campus of Mississipi State University, Starkville. Pure cultures obtained from specimens collected at each location were confirmed as D. pinea using species-specific PCR primers (3) that allow differentiation of D. pinea from the similar pine shoot blight pathogen D. scrobiculata. Isolates from loblolly pines in Alabama (07-58), Louisiana (07-38), and Mississippi (06-45) were used individually to inoculate potted 6- to 7-month-old loblolly pine seedlings grown from seed in a greenhouse in each of two independent trials. Elongating terminal shoots of seedlings to be inoculated were wounded by removing a needle fascicle ∼2 cm below the shoot apex. A 4-mm-diameter plug cut from an actively growing colony on water agar (WA) was placed on the wound, mycelium side toward the stem. Noncolonized WA plugs were placed in the same manner on similarly wounded control seedlings and nonwounded control seedlings also were used. Parafilm was wrapped around the shoots to hold the agar plugs in place and was removed after 1 week. Each of the five isolate-treatment combinations was applied to seven (trial 1) or eight (trial 2) seedlings (35 and 40 seedlings per trial, respectively). One week after inoculation, small, brown lesions were visible at the point of inoculation on stems of most of the inoculated seedlings. At 25 days after inoculation, all inoculated seedlings exhibited needle browning and stem cankers ranging from 0.6 cm to 9.0 cm long (mean 2.5 cm) that girdled and killed distal portions of the shoots of ∼25% of the inoculated seedlings in each trial. Wounded control and nonwounded control seedlings did not develop symptoms. Stem segments including the point of inoculation (or comparable segments of wounded and nonwounded control seedlings) were excised, surface disinfested, and incubated on tannic acid agar with sterile red pine needles. D. pinea was cultured from all inoculated seedlings and also from one wounded control seedling. Although occurrence of D. pinea on Cedrus spp. is included in an index (1), to our knowledge this is the first confirmed report of D. pinea on pines in Alabama, Louisiana, and Mississippi. The degree of risk presented by D. pinea to loblolly pine, longleaf pine, and other pine species native to the southern United States when grown in their native ranges is unknown. Reports of Diplodia shoot blight of southern U.S. pines when grown as exotics in the southern hemisphere (4) and the potential for epidemics to develop suddenly under severe weather conditions (2,4) justify additional studies to evaluate the potential for damage to these hosts in their native ranges. References: (1) Anonymous. Page 333 in: Index of Plant Diseases in the United States. Agric. Handb. 165, U.S. Dep. Agric. Washington, DC, 1960. (2) T. H. Nicholls and M. E. Ostry. Plant Dis. 74:54, 1990. (3) D. R. Smith and G. R. Stanosz. Plant Dis. 90:307, 2006. (4) W. J. Swart and M. J. Wingfield. Plant Dis. 75:761, 1991.

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). 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.

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.

California Laurel Is Susceptible to Laurel Wilt Caused by Raffaelea lauricola.

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.

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.

Morphological and Molecular Characterization of Longidorus americanum n. sp. (Nematoda: Longidoridae), a Needle Nematode Parasitizing Pine in Georgia.

We describe and illustrate a new needle nematode, Longidorus americanum n. sp., associated with patches of severely stunted and chlorotic loblolly pine, (Pinus taeda L.) seedlings in seedbeds at the Flint River Nursery (Byromville, GA). It is characterized by having females with a body length of 5.4-9.0 mm; lip region slightly swollen, anteriorly flattened, giving the anterior end a truncate appearance; long odontostyle (124-165 microm); vulva at 44%-52% of body length; and tail conoid, bluntly rounded to almost hemispherical. Males are rare but present, and in general shorter than females. The new species is morphologically similar to L. biformis, L. paravineacola, L. saginus, and L. tarjani but differs from these species either by the body, odontostyle and total stylet length, or by head and tail shape. Sequence data from the D2-D3 region of the 28S rDNA distinguishes this new species from other Longidorus species. Phylogenetic relationships of Longidorus americanum n. sp. with other longidorids based on analysis of this DNA fragment are presented. Additional information regarding the distribution of this species within the region is required.

Suitability of Southern Pines, Other Selected Crops, and Nutsedge to a Longidorus sp. Associated with Stunting of Loblolly Pine Seedlings.

An undescribed needle nematode (Longidorus sp.) has been associated with severely stunted loblolly pine seedlings at a south Georgia nursery. Containers with selected crop and weed species were infested with 100 or 200 adults and juveniles of the Longidorus individuals to evaluate host suitability. Nematode populations increased in containers with slash, loblolly, and longleaf pine seedlings. The Longidorus sp. significantly reduced the dry root weights of slash (P = 0.008) and loblolly (P = 0.047) but not longleaf (P = 0.095) pine compared with controls. Populations of Longidorus decreased on nutsedge and small grains, including wheat, rye, oat, sorghum, and millet; and, in most experiments, populations decreased to levels found in fallow containers. Populations decreased on tomato and cabbage but increased slightly on red oak. The periodic removal of fields from pine production to grow cover crops consisting of small grains or to maintain fallow fields may be an important practice for management of this Longidorus sp.