First Report of Armillaria root disease of Celtis laevigata caused by A. gallica in South Carolina, USA.

Celtis laevigata (sugarberry, southern hackberry) is an important, shade-tolerant, deciduous hardwood tree species that occurs naturally in flood plains, along streams and rivers, and in urban landscapes of the southeastern USA (Kennedy 1990). In recent years, dieback and mortality of C. laevigata have been commonly observed in some areas of South Carolina (SC) and Georgia (GA) (Poole et al. 2021). In April/May of 2018, the crown conditions and root systems were examined for three C. laevigata trees in North Augusta, SC. The crown of each tree was visually assessed using the method of Poole et al. (2021). Root samples were obtained by excavating two main roots ca. 2 meters away from the stem of each tree. Tree SB474 (N33 29.472, W81 59.082, elev. 55.8 m) exhibited > 66% crown loss and decaying roots with white mycelial fans and dark rhizomorphs characteristic of Armillaria. Tree SB913 (N33 29.830, W81 59.349, elev. 58.8 m) exhibited ca. 34-66% crown loss, while tree SB914 (N33 29.837, W81 59.338, elev. 57 m) appeared healthy with no apparent crown loss. Roots of trees SB913 and SB914 appeared healthy, although rhizomorphs were attached to the root surfaces. Roots and/or attached rhizomorphs were surface disinfested and plated n a basidiomycete-selective medium (Hendrix and Kuhlman 1962). Three Armillaria isolates, one from each corresponding tree, were paired with each other, and two genets were identified (SB474 and SB913 = SB914). The two genets (SB474 and SB913) were used in somatic pairing tests against three known tester isolates for each of the following species: A. solidipes, A. mellea, A. gallica, A. mexicana, and Desarmillaria caespitosa (=A. tabescens). Pairing of isolates SB474 and SB913 showed the highest compatibility with A. gallica (isolates ST22, ST23, and M70) with 100% and 89%, respectively. These isolates were definitively confirmed as A. gallica by translation elongation factor 1α gene sequences (tef1; Klopfenstein et al. 2017) (GenBank accession nos. OM993577 and OM993578 for SB474 and SB913, respectively). GenBank nucleotide BLAST showed tef1 similarity for both SB474 and SB913 isolates was highest for A. gallica (≥98.7%; GenBank accession nos. MT761696, MT761697, and KF156772). This is the first report of A. gallica associated with Armillaria root disease of C. laevigata. Rhizomorphs on the surface of apparently healthy tree roots and root colonization in severely declining trees are a common sign of A. gallica (Baumgartner and Rizzo 2001). Pathogen colonization of root surfaces may provide an opportunity for infection of highly damaged trees, resulting in root disease (Gregory 1985). Primary agents of C. laevigata dieback and mortality in SC and GA remain undefined, but continued study is needed to confirm the role of A. gallica in C. laevigata dieback and mortality. Although pathogenicity tests are impractical for Armillaria, these A. gallica occurrences in SC further adds to our knowledge of this pathogen's distribution in the southeastern USA, where it has also been confirmed in Tennessee in hardwood forests (Bruhn et al. 1997), SC on Hemerocallis sp. (Schnabel et al. 2005), and GA on a Rhododendron/span> sp. and Quercus rubra (Hanna et al. 2020). The distribution and host range of A. gallica is likely more widespread in the southeastern USA than existing records indicate. Documenting Armillaria distribution, including A. gallica, is essential for predicting climate-change impacts on Armillaria root diseases (Kim et al. 2022). Baumgartner, K., and Rizzo, D. M. 2001. Plant Dis. 85:947-951. Bruhn, J. N., et al. 1997. In 11th Central Hardwood Forest Conference, USDA, FS, NC-GTR-188, 49-57. Gregory, S. C. 1985. Plant Path. 34:41-48. Hanna, J. W., et al. 2020. Plant Dis. 105: 1226. Hendrix Jr, F. F., and Kuhlman, E. G. 1962. PI. Dis. Rep. 46:674-676. Kennedy, Jr., H. E. 1990. Silvics of North America: 2. Hardwoods. USDA-FS. Agriculture Handbook 654. Kim, M.-S., et al. 2022. Front. For. Glob. Change 4:740994. Klopfenstein, N. B., et al. 2017. Mycologia 109:75-91. Poole, E. M., et al. 2021. J. For. 119:266-274. Schnabel, G., et al. 2005. Plant Dis. 89:683.

Rapid Detection of Raffaelea lauricola Directly from Host Plant and Beetle Vector Tissues Using Loop-Mediated Isothermal Amplification.

Since its introduction in 2002, laurel wilt disease has devastated indigenous lauraceous species in the southeastern United States. The causal agent is a fungal pathogen, Raffaelea lauricola, which, after being introduced into the xylem of trees by its vector beetle, Xyleborus glabratus, results in a fatal vascular wilt. Rapid detection and accurate diagnosis of infections is paramount to the successful implementation of disease management strategies. Current management operations to prevent the spread of laurel wilt disease are largely delayed by time-consuming laboratory procedures to confirm the diagnosis. In order to greatly speed up the operations, we developed a loop-mediated isothermal amplification (LAMP) species-specific assay that targets the ß-tubulin gene region of R. lauricola, and allows for the rapid detection of the pathogen directly from host plant and beetle tissues. The assay is capable of amplifying as little as 0.5 pg of fungal DNA and as few as 50 conidia. The assay is also capable of detecting R. lauricola directly from wood tissue of artificially inoculated redbay saplings as early as 10 and 12 days postinoculation, when testing high-quality and crude DNA extracts, respectively. Finally, crude DNA extracts of individual adult female X. glabratus beetles were assayed and the pathogen was detected from all specimens. This assay greatly reduces the time required to confirm a laurel wilt diagnosis and, because LAMP technology is well suited to provide point-of-care testing, it has the potential to expedite and facilitate implementation of management operations in response to disease outbreaks.

First Report of Armillaria Root Disease Pathogen, Armillaria gallica, on Rhododendron and Quercus rubra in Georgia, USA.

Armillaria root and butt diseases, which are a global issue, can be influenced by changing environmental conditions. Armillaria gallica is a well-known pathogen of diverse trees worldwide (Brazee and Wick 2009). Besides A. gallica causing root rot of Hemerocallis sp. and Cornus sp. in South Carolina (Schnabel et al. 2005), little is reported on the distribution and host range of A. gallica in the southeastern USA. In July 2017, three Armillaria isolates were obtained from two naturally occurring hosts in Georgia, USA and cultured on malt extract medium (3% malt extract, 3% dextrose, 1% peptone, and 1.5% agar). One isolate (GA3) was obtained in Unicoi State Park near Helen, Georgia (Lat. 34.712275, Long. -83.727765, elev. 498 m) from the basal portion of Rhododendron sp. with extensive root/butt decay, but no crown symptoms were evident (Supplementary Figure 1). GA4 and GA5 (Lat. 33.902433, Long. -83.382453, elev. 215 m) were isolated from wind-felled Quercus rubra (red oak) with root disease at the State Botanical Gardens in Athens, Georgia. GA4 was associated with a large root ball (ca. 4-m diameter) (Supplementary Figure 2), and GA5 was obtained from a mature tree with infected roots, with characteristic spongy rot of Armillaria root disease. Crown symptoms could not be evaluated because the crowns had been removed before the collections. Several other oaks with Armillaria root disease were noted throughout the State Botanical Gardens. Pairing tests reduced these three isolates (whiteish mycelia with a dark, brownish crust and rhizomorphs), to two genets with GA4 = GA5. Both genets (GA3 and GA4) were identified as A. gallica using translation elongation factor 1α (tef1) sequences (Genbank Nos. MT761697 and MT761698, respectively) that showed ≥ 97% identity (≥ 98% coverage) with A. gallica sequences (KF156772, KF156775). Also, nine replications of somatic pairing tests showed 33 - 67% compatibility with A. gallica (occurs in southeastern USA), compared with 0 - 22% for A. mexicana, A. mellea (occurs in southeastern USA), A. solidipes, and Desarmillaria tabescens (occurs in southeastern USA). To our knowledge, this note represents the first report of A. gallica on Rhododendron and Q. rubra in Georgia, USA, which has experienced severe drought in recent decades (e.g., Park Williams et al. 2017) that could predispose trees to Armillaria infection (e.g., Wargo 1996). Quercus rubra was previously reported as a host of A. gallica in Arkansas (Kelley et al. 2009) and Massachusetts (Brazee and Wick 2009), USA. In Missouri, USA, A. gallica has been reported as a weak pathogen with potential biological control against A. mellea (Bruhn et al. 2000). Other reports from several regions on various hosts suggest pathogenicity of A. gallica is associated with changing climate (Nelson et al. 2013, Kim et al. 2017, Kubiak et al. 2017). Wide genetic variation and/or cryptic speciation within A. gallica may account for differences in ecological behavior (Klopfenstein et al. 2017), but this is difficult to evaluate because Armillaria pathogenicity tests cannot be used on most forest tree seedlings. This study suggests that A. gallica is more widespread than previously known and its adverse impacts on woody plants may intensify over time, depending on the environmental conditions. Further studies are needed to determine environmental influences on A. gallica, the full distribution of A. gallica, and its effects in forests of the southeastern USA.

Genetic Variation in Native Populations of the Laurel Wilt Pathogen, Raffaelea lauricola, in Taiwan and Japan and the Introduced Population in the United States.

Laurel wilt is a vascular wilt disease caused by Raffaelea lauricola, a mycangial symbiont of an ambrosia beetle, Xyleborus glabratus. The fungus and vector are native to Asia but were apparently introduced to the Savannah, GA, area 15 or more years ago. Laurel wilt has caused widespread mortality on redbay (Persea borbonia) and other members of the Lauraceae in the southeastern United States, and the pathogen and vector have spread as far as Texas. Although believed to be a single introduction, there has been no extensive study on genetic variation of R. lauricola populations that would suggest a genetic bottleneck in the United States. Ten isolates of R. lauricola from Japan, 55 from Taiwan, and 125 from the United States were analyzed with microsatellite and 28S rDNA markers, and with primers developed for two mating-type genes. The new primers identified isolates as either MAT1 or MAT2 mating types in roughly equal proportions in Taiwan and Japan, where there was also high genetic diversity within populations based on all the markers, suggesting that these populations may have cryptic sex. Aside from a local population near Savannah and a single isolate in Alabama that had unique microsatellite alleles, the U.S. population was genetically uniform and included only the MAT2 mating type, supporting the single introduction hypothesis. This study suggests the importance of preventing a second introduction of R. lauricola to the United States, which could introduce the opposite mating type and allow for genetic recombination.

Molecular and Morphological Characterization of Xiphinema chambersi Population from Live Oak in Jekyll Island, Georgia, with Comments on Morphometric Variations.

A population of Xiphinema chambersi from the root zone around live oak (Quercus virginiana Mill.) trees on Jekyll Island, GA, is described using both morphological and molecular tools and compared with descriptions of type specimens. Initially, because of a few morphological differences, this nematode was thought to represent an undescribed species. However, on further examination, the morphometrics of the nematodes from live oak tend to agree with most of the morphometrics in the original description and redescription of X. chambersi except for few minor differences in V% relative to body length, slightly shorter stylet length, different c value, and the number of caudal pores. We consider these differences to be part of the normal variation within this species and accordingly image this new population of X. chambersi and redescribe the species. The new population is characterized by having females with a body length of 2.1 to 2.5 mm; lip region slightly rounded and set off from head; total stylet length 170 to 193 µm; vulva at 20.4% to 21.8% of body length; a monodelphic, posterior reproductive system; elongate, conoid tail with a blunt terminus and four pairs of caudal pores, of which two pairs are subdorsal and two subventral. Sequence data from the D2-D3 region of the 28S rRNA molecule subjected to GenBank sequence comparison using BLAST showed that the sequence had 96% and 99% similarity with X. chambersi from Alabama and Florida, respectively. Phylogenetic relationships of X. chambersi with other xiphinematids based on analysis of this DNA fragment are presented. This finding represents a new location of X. chambersi in Georgia on live oak for this species.

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.

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.

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

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.

Fumigant distribution in forest nursery soils under water seal and plastic film after application of dazomet, metam-sodium and chloropicrin.

Adequate concentration, exposure time and distribution uniformity of activated fumigant gases are prerequisites for successful soil fumigation. Field experiments were conducted to evaluate gas phase distributions of methyl isothiocyanate (MITC) and chloropicrin (CP) in two forest-tree nurseries. Concentrations of MITC and CP in soil air were measured from replicated microplots that received dazomet, metam-sodium and CP. Half of the plots were covered with high-density polyethylene tarp immediately after fumigation; the other half were not covered but received daily sprinkler irrigation for 1 week to create and maintain a water seal. The magnitude of MITC concentrations was similar between nurseries for metam-sodium in both tarp and water seal treatments and for dazomet in the tarp treatment. Consistently greater MITC and CP concentrations were found in the upper 30 cm of soil in the tarped plots compared with the water-sealed plots. Despite potential environmental and economic benefits with the water seal method, tarp covers were more reliable for achieving and maintaining higher MITC and CP concentrations and less prone to variations due to irrigation/rain, soil bulk density and other environmental conditions.

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).

The Association of a Longidorus Species with Stunting and Root Damage of Loblolly Pine (Pinus taeda L.) Seedlings.

A Longidorus species was consistently associated with patches of stunted and chlorotic loblolly pine seedlings at a forest-tree nursery in Georgia. Seedlings from affected areas had poorly developed root systems that lacked lateral and feeder roots. Longidorus population densities in composite soil samples from the margins of patches ranged from 9 to 67 nematodes per 100 cm3 of soil. In a growth chamber experiment, seedling root dry weight decreased with respect to the initial Longidorus dose as well as the final Longidorus populations in containers. The dry root weight of seedlings were 0.117, 0.090, 0.066, and 0.065 g in containers initially infested with 0, 50, 100, and 200 Longidorus, respectively. Lateral and fine roots were lacking on seedlings at the highest doses. Populations of Longidorus increased in all containers during the experiment. Damage to loblolly pine seedlings caused by Longidorus is a previously undescribed problem in southern pine nurseries. Proper diagnosis of the problem by nematode testing laboratories may require the use of extraction techniques specific for larger nematodes such as Longidorus.