1a. Objectives (from AD-416)
Conduct a thorough and systematic survey of the NCGR collection at Winters for the presence of the pathogen, with a focus on J. major and J. microcarpa. Conduct Geosmithia inoculation studies of Juglans species and accessions, with a focus on J. major and J. microcarpa. Documents SCA with UC Davis.
1b. Approach (from AD-416)
The collection at the NCGR at Winters consists of 13 different Juglans species, the majority of which is comprised of 3 species (J. californica, J. hindsii, and J. major) native to the western U.S., as well as a large collection of J. regia trees introduced from 19 different countries. A preliminary survey of the collection for symptomatic trees in Sept. 2009 will allow us to target individual accessions for isolation and identification of the pathogen. Symptomatic J. major (9.8% of the 133 individuals) and J. microcarpa (21.2% of the 33 individuals) will be given the most scrutiny, but similar exposure of all species in the collection to the high population of pathogen and vector in the vicinity makes this a unique opportunity to evaluate the impact of the disease on a range of hosts. The NCGR facility allows for the evaluation of the degree of threat to the various species in the collection under on-site conditions. In addition, the collection affords a unique opportunity to compare susceptibility of mature trees of the various species all at one location. Field trees will be evaluated as follows: Healthy, intact branches (approximately 2 cm diameter) will be inoculated with an isolate of Geosmithia obtained from the NCGR site. Small puncture wounds will be made in the bark with a needle approximately the same diameter as the entry wounds created by the beetle (~0.5 mm) and inoculated with a conidial suspension of the pathogen. The wounds will then be covered with parafilm. Controls spaced along the same branch will include a non-inoculated wound and wounds inoculated with conidia of a nonpathogenic fungus (e.g., Aspergillus nidulans). Based on the initial survey and assessment, some of the other species in the collection will serve as highly susceptible (e.g., J. hindsii & J.californica) and highly resistant (e.g., J. ailantifolia and J. mandshurica) reference species for comparison. Artificial inoculations (March/April) will be made to try to approximate when the overwintering beetles emerge to initiate new infections. After an appropriate incubation period (several weeks), inoculated branches will be excised, transported to the laboratory and evaluated for canker development. Dimensions of each canker will be recorded and analyzed. It is anticipated that 3-5 branches per tree will be routinely inoculate, with single tree reps and 3-5 trees per species of diverse accessions. It is believed that this field assessment will pose any additional risk to the collection because TCD is already present, and an isolate of the pathogen will be used that has been obtained from a diseased tree at the site, and all inoculated branches will be excised,removed from the site, and destroyed by heat inactivation after evaluation. Limb removal will ensure that the fungus does not move from the sites of inoculation. There are additional host materials, some using parent material from the NCGR, which have been developed as part of the walnut rootstock improvement program. Of particular interest are rootstock hybrids that are also under evaluation for resistance to other important walnut diseases (e.g., crown gall, Phytophthora root and crown rot).
3. Progress Report
Five Juglans species were evaluated: J. ailantifola, J. californica, J. major, J. microcarpa, and J. regia. Results indicate that although all of the five Juglans spp. examined are susceptible to G. morbida, there are differences in relative susceptibility among them. During the 2010 season, trees were artifically inoculated with two California isolates of Geosmithia morbida. Within this group J. californica and J. microcarpa are most susceptible, J. regia is of intermediate susceptibility, and J. ailantifolia and J. major are least susceptible. Results with mature trees inoculated in the field in California share similarities with results with young trees in greenhouse inoculations in Colorado, but also show some differences. Five isolates of G. morbida obtained from natural infections occurring in three different Juglans species and from different areas of northern California were compared for their virulence in laboratory assays using detached J. regia branches (cv. Chandler). All were similar in virulence. There are differences among Juglans species in susceptibility to G. morbida. Among the five species evaluated, J. californica appears to be most susceptible, and this was also apparent from a survey of naturally infected trees in the germplasm collection, where incidence of TCD was by far the greatest in the J. californica trees. Although there are differences among these species, none appear to be resistant as all produced cankers following artificial inoculation. The apparent high incidence of natural infections in J. californica, but not in the other species in the collection, results from other unknown factors. Perhaps differences in the attraction of different Juglans species to the Walnut Twig Borer may explain the differences observed in the incidence of natural infections; however, it is unresolved if this is due to inherent genetic differences or differences in site factors peculiar to the J. californica collection and will require further research. The project was monitored by maintaining a complete file of the agreement, reviewing the annual reports, and conducting meetings with the cooperator. The progress of research, so far, is satisfactory and useful for effective management of wild Juglans germplasm at the repository.