First Report of Geosmithia morbida on English Walnut and its Paradox Rootstock in California.

Geosmithia morbida, the causal agent of thousand cankers disease (TCD), is vectored by the walnut twig beetle (WTB), Pityophthorus juglandis, causing decline in eastern black walnut, Juglans nigra (4), and canker development on many Juglans species (5). In the summer of 2012, a survey for TCD incidence in English walnut, J. regia, in orchards in California identified many trees with WTB activity and characteristic TCD symptoms. Both the J. regia scion and its Paradox hybrid rootstock (J. hindsii× J. regia) were affected. In some cases, trees exhibited bleeding on the bark surface from WTB entrance holes. Removal of the outer bark revealed cankers in the phloem around the WTB galleries. Two samples were taken from scions and three samples were collected from rootstocks of trees in orchards in northern California. Pieces (~3 to 4 mm²) of symptomatic tissue were placed in acidified potato dextrose agar (APDA), and the plates were incubated for 4 to 5 days at 30°C. Samples exhibiting fungal growth similar in morphology to G. morbida were transferred to PDA plates to obtain pure cultures and then processed to obtain single-spore cultures. Culture morphology for five single-spore isolates (Gm103, Gm104, Gm105, Gm107, and Gm108) was similar to that described by Kolařík et al. (4) for G. morbida. Conidiophores were penicillate and verrucose. Conidia were narrowly cylindrical, 5.2 ± 0.06 × 2.2 ± 0.04 μm (n = 50). Single-spore isolates were then grown in 1% yeast extract glucose liquid culture for 7 to 10 days. DNA was extracted and the ITS region was amplified, including the 5.8S region by using primers ITS1F/ITS4. Sequences were assembled and deposited in GenBank under accessions KJ664793 to KJ664797. Sequences were compared to those in GenBank; all sequences matched (99 to 100% identity) the ITS sequences of G. morbida strain CBS 124663. Pathogenicity tests were performed on 28-cm-long detached branches of J. regia. Four branches per isolate were inoculated with a 5-mm-diameter mycelial plug from a 2-week-old culture. Branches were incubated at room temperature (23 ± 2°C) in a humidified container for 3 weeks, and then canker lengths were measured. Pieces of the cankered area were placed in APDA and incubated as described above with G. morbida re-isolated from the cankers for all of the isolates, completing Koch's postulates. Average canker lengths ranged from 48.6 ± 4.3 to 72.1 ± 7.1 mm. Re-isolated G. morbida exhibited the same growth and reproductive structure morphology in culture on PDA as the original cultures. TCD in association with WTB has been observed in California English walnut orchards since 2008 (1,2,3). However, this is the first report for completion of Koch's postulates and morphological and molecular confirmation of G. morbida in J. regia and the Paradox rootstock, the predominant rootstock used in commercial orchards. TCD is a concern to the walnut industry in California with over 245,000 bearing acres reported in 2012. References: (1) M. Flint et al. CAPCA Adviser 8:36, 2010. (2) A. D. Graves et al. Walnut Twig Beetle and Thousand Cankers Disease: Field Identification Guide. UC-IPM website publication, http://www.ipm.ucdavis.edu/PDF/MISC/thousand_cankers_field_guide.pdf , 2009. (3) J. Hasey et al. (Abstr.) Phytopathology 100:S48, 2010. (4) M. Kolařík et al. Mycologia 103:325, 2011. (5) C. Utley et al. Plant Dis. 97:601, 2013.