© 2013 Plant Management Network.
Geosmithia morbida, Thousand Cankers Disease of Black Walnut Pathogen, Was Found for the First Time in Southwestern Ohio
John R. Fisher, Ohio Department of Agriculture, Plant Health Diagnostic Laboratory, Plant Health Division, Reynoldsburg, OH 43068; and David P. McCann and Nancy J. Taylor, C. Wayne Ellett Plant and Pest Diagnostic Clinic, The Ohio State University, Reynoldsburg, OH 43068
Corresponding author: John R. Fisher. JFisher@agri.ohio.gov
Fisher, J. R., McCann, D. P., and Taylor, N. J. 2013. Geosmithia morbida, thousand cankers disease of black walnut pathogen, was found for the first time in southwestern Ohio. Online. Plant Health Progress doi:10.1094/PHP-2013-1201-01-BR.
Thousand cankers disease (TCD) of walnut is a recently described disease caused by a newly described phytopathogenic fungus, Geosmithia morbida (5), which has been isolated from walnut species (Juglans californica, J. hindsii, J. major, J. nigra) in Arizona, California, Colorado, Idaho, New Mexico, Oregon, Utah, and Washington (5,6). Walnut mortality in those states has been attributed to the fungus and its association with the walnut twig beetle (WTB), Pityophthorus juglandis (6,7). The decline and death of walnut trees is the result of repeated attacks by the beetle and concurrent inoculation of phloem and cambium tissues by the fungus. Numerous coalescing cankers girdle the tree, eventually causing death (7). In 2010, TCD was detected for the first time east of the Mississippi River near Knoxville, TN (2). It was found in Virginia (4) and Pennsylvania in 2011 (media release, 12 August 2011, Pennsylvania Department of Agriculture), and North Carolina in 2012 (3).
Eight adult WTB were caught over a three month period in a trap set by the Ohio Department of Natural Resources at a walnut veneer mill in southwestern Ohio in the fall of 2012. The identity of the beetles was confirmed by the U.S. Forest Service (USDA Forest Service Pacific Southwest Research Station, Davis, CA). The Ohio Department of Agriculture conducted a more intensive trapping survey of Butler Co. in the vicinity of the veneer plant in 2013. This time the beetle catches were in the hundreds to thousands. In early June a residence was identified approximately three miles from the veneer mill with dead and dying black walnut (J. nigra) trees. The trees were exhibiting end stage TCD symptoms including foliage yellowing, canopy thinning, and branch dieback (7), but branch and/or trunk cankers were not readily observed in the field and beetle emergence holes were rarely found (Fig. 1). Ohio Plant Diagnostic Network scientists collected 3-5-cm diameter branch samples having clear transition from living to dead tissue to attempt fungal isolations.
Branches with thinner bark were peeled with a sterile scalpel and chips (~5-10 mm × 3-5 mm containing discolored phloem tissue) not clearly associated with beetle holes or galleries (Fig. 1) were plated without surface sterilization directly onto ¼-strength potato dextrose agar amended with streptomycin and chloramphenicol (¼ PDA++) (7). The plates were sealed with Parafilm, incubated at room temperature (22°C), and monitored for fungal growth. Five days after plating, three plates (10%) had fungal growth with condiophore and spore morphology (Fig. 2) consistent with Geosmithia sp. (5,7). The isolates were transferred to new ¼ PDA++ plates where colonies developed that had irregular margins and profuse white sporulation becoming tan with age as described for G. morbida (Fig. 2) (5,7). Purification of fungal DNA from mycelial scrapings of agar plates yielded poor results so 100 mL liquid cultures of sterilized malt extract broth (10 g malt extract, 4 g yeast extract, 4 g dextrose, final vol. 1 L) amended with 100 µL of 250 mg/mL ampicillin were inoculated with ~3 × 3-mm agar plugs and grown for five days at 22°C with gentle shaking (Fisher orbital shaker, 150 rpm).
DNA was extracted using the DNAeasy plant mini kit (Qiagen, Venlo, The Netherlands). Mycelia from liquid cultures were pelleted by centrifugation, frozen in liquid N2, ground with a mortar and pestle, and then 100 mg and 250 mg of resulting powder extracted according to the manufacturers protocol. Alternatively, mycelia growing on agar plugs used to inoculate the liquid cultures were recovered and ground directly in 400 µL of lysis solution and extracted as above. PCR amplifications of the ITS1, 5.8S, and ITS2 regions of the rDNA were performed using ITS1-ITS4, and ITS4-ITS6 primer pairs (1,7,8) in 25 µL reactions [1× GoTaq buffer, 2.5 mm MgCl2, 0.2 mm dNTP mix, 0.4 µM primer pair, 0.625 units GoTaq polymerase (Promega Inc., Madison, WI)] with the cycling conditions: 95°C (2 min), 35 cycles of 95°C (30 sec), 55°C (30 sec), 50°C (30 sec), 72°C (60 sec), and a final 72°C extension (10 min). Both primer pairs amplified strong, distinct products of approximately 600 bp for each of the DNA extraction preparations. The DNA was excised from the agarose gels, purified (QiaQuick gel extraction kit, Qiagen), and sequenced directly with ITS1, ITS4, and ITS6 primers (The Ohio State University Plant Microbe Genomics Facility, Columbus, OH). DNA sequences were trimmed (Chromas v. 2.4, Technelysium Pty. Ltd., South Brisbane, Australia), contigs were assembled, and pairwise and multiple sequence alignments were performed (Vector NTI Advance, Invitrogen Inc., Carlsbad, CA).
Six ITS-1 and ITS-4, and eight ITS-4 and ITS-6 amplicon sequences were used to assemble 564 nucleotide (nt) and 572 nt contigs, respectively. Two consensus sequences were deposited in GenBank under accession numbers KF656716 and KF656717. A BLASTn search of the NCBI database using default settings with the ITS1-4 sequence (100% query coverage) resulted in four G. morbida matches that were 100% identical to the Ohio isolate. A BLASTn search using the ITS4-6 sequence (100% query coverage) resulted in two G. morbida matches that were 100% identical and several others that were 100% identical with 98% query coverage. A summary is shown in Table 1.
Table 1. Geosmithia morbida isolates with the greatest percent nucleotide (nt) sequence identities to the Ohio isolate ITS1-4 and ITS4-6 PCR product nt sequences.
w G. morbida type isolate.
x American type culture collection strain MYA-4903.
y ITS 1-4% nt sequence identity.
z ITS 4-6% nt sequence identity.
Geosmithia morbida is established in the Ohio landscape and has likely been present longer than initially suspected since the fungus was isolated from mature declining black walnut trees in a wooded residential environment nearly three miles from the initial WTB catches. The veneer mill where the WTB was first discovered has been importing walnut material from California for over a decade. The fungal conidiophore and spore morphology, and the colony morphology on ¼ PDA++ agar are consistent with those reported for G. morbida (5,7). Furthermore, the ITS1-4 and ITS 4-6 DNA sequence data is 100% identical to isolates from California, Colorado, and Oregon, including the G. morbida type isolate (accession number FN434082.1) (3,7). It is important to emphasize that we isolated the fungus from discolored phloem tissue resembling small cankers that were not associated with WTB galleries or exit holes, and were much smaller than TCD cankers reported in the literature (7). Our recovery rate isolating G. morbida from small cankers was only 10% (3/30 plated bark chips) but demonstrates that in the absence of large cankers associated with beetle galleries it is worthwhile isolating from the smaller cankers. These results expand the known geographic range of Geosmithia morbida and thousand cankers disease to include southwestern Ohio. Black walnut is native to Ohio and is an important forest canopy species. The state ranks third in standing walnut inventory behind Indiana and Missouri. This detection is concerning because the Butler county site is approximately 16 miles from Indianas eastern border and 16 miles from Kentuckys northern border, so WTB and TCD may very well be in those states already. Its also possible the insect and disease may be present in Ohio at locations distant from the original site, so WTB and TCD surveillance will continue.
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