1. Isolation, Survival and Growth of Rhodococcus fascians from Plants with Shoot Proliferation and/or Leafy Galls Eva Sandberg Marilyn Miller, Melodie Putnam Department of Botany and Plant Pathology Oregon State University

2. Relevance In Oregon, the nursery industry is the largest component of agriculture in terms of sales. Disease is a huge threat to wholesale production nurseries distributing ornamental plants. Abnormal growth or disease? Throwing out millions of dollars in plants vs. spreading disease. http://www.awatergarden.com/plant.htm

3. Symptoms Oenethera plant submitted to Plant Clinic with leafy gall (natural infection).

4. Rhodococcus fascians Gram positive, aerobic bacterium. The only phytopathogen within its genus. Pleiomorphic Strains used in this experiment were extremely virulent: they produce dramatic symptoms on a wide range of hosts.

5. Hypothesis R. fascians is the causal agent of these symptoms in submissions to the plant clinic. -How can it be established that the Rhodococcus isolates are responsible for the symptoms seen on the original host?

6. Testing the hypothesis according to Koch’s postulates Physician Robert Koch developed the following postulates as a criteria for proving the relationship between a microorganism and a specific disease: 1. The microorganism must be present in every case of the disease but absent from healthy organisms. 2. The suspected microorganism must be isolated and grown in a pure culture. 3. The same disease must result when the isolated microorganism is inoculated into a healthy host. 4. The same microorganism must be isolated again from the diseased host.

7. Testing the hypothesis A44a & A25f strains isolated from host plants using selective and non-selective media. Pea seedlings inoculated with isolates. Presumed virulent isolates inoculated into original hosts and observed for symptom development. Plants with symptoms of LG and/or SP used for re- isolations. Purification and identification of bacterium performed with Biolog software.

8. Materials and methods

9. Bud proliferation on Tiarella. Shoot proliferation on Iberis.Infected Iberis and negative control about 4 mo. post-inoculation. Leafy gall on Oenethera.

10. Re-isolations from inoculated plants using D2C medium LG = Leafy gallSP = Shoot proliferation* = Many symptoms Rhodococcus strainHostSymptoms A44aErysimum ‘Bowle’s mauve’LG A44aNemesia ‘Vanilla’LG, SP* A44aIberis ‘Gibraltarica’LG A44aTiarellaSP on underside A25fOenethera ‘Siskiyou’LG A3bErysimum ‘Variegata’LG, SP 02-815CampanulaSP on underside A78OenetheraLG 05-340-1OenetheraLG, SP*

11. A25f Oenethera ‘Siskiyou’ after re-isolation. 02815 Campanula after re-isolation.

12. Identification of bacterium Biolog: A company that created a database for microbial identification based on substrate utilization by bacteria. GP2 plates require a bacterial suspension of 19% transmittance. Plates are incubated at 27°C for 16-24 hours. Plates are read at 590 nm wavelength.

13. Conclusions The symptoms are produced by a pathogen and are not due to abiotic factors. Rhodococcus produces symptoms identical to those that were originally submitted to the lab.

14. Future work Determine the pH at which best growth occurs to enhance recovery from symptomatic plants. - Bacterium is primarily on surface of plants - Isolations are in broth at a single pH – are we killing the bacteria? - Establish growth curve at different pH’s to see which is the best for isolations.

15. Acknowledgements Howard Hughes Medical Institute Dr. Kevin Ahern Dr. Chris Mathews Marilyn Miller Melodie Putnam Andre Spycher Kelly Collins Peoria Gardens, Inc.