Transcript Isolation, Survival and Growth of Rhodococcus facians from plants

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
Relevance
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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
Symptoms
Oenethera plant submitted to Plant Clinic with
leafy gall (natural infection).
Rhodococcus fascians
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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.
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?
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.
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 reisolations.
Purification and identification of bacterium
performed with Biolog software.
Materials and methods
Infected Iberis and negative control
about 4 mo. post-inoculation.
Leafy gall on Oenethera.
Shoot proliferation on Iberis.
Bud proliferation on Tiarella.
Re-isolations from inoculated plants
using D2C medium
Rhodococcus strain
Host
Symptoms
A44a
Erysimum ‘Bowle’s mauve’
LG
A44a
Nemesia ‘Vanilla’
LG, SP*
A44a
Iberis ‘Gibraltarica’
LG
A44a
Tiarella
SP on underside
A25f
Oenethera ‘Siskiyou’
LG
A3b
Erysimum ‘Variegata’
LG, SP
02-815
Campanula
SP on underside
A78
Oenethera
LG
05-340-1
Oenethera
LG, SP*
LG = Leafy gall
SP = Shoot proliferation
* = Many symptoms
02815 Campanula
after re-isolation.
A25f Oenethera ‘Siskiyou’
after re-isolation.
Identification of bacterium
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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.
Conclusions
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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.
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.
Acknowledgements
Howard Hughes
Medical Institute
Dr. Kevin Ahern
Dr. Chris Mathews
Marilyn Miller
Melodie Putnam
Andre Spycher
Kelly Collins
Peoria Gardens, Inc.