Detection and Partial Characterization of Pelargonium Viruses with Emphasis on Pelargonium Flower Break Virus 1993 Proposal
VIRUSES WITH EMPHASIS ON PELARGONIUM FLOWER BREAK
VIRUS
A. Project Summary:
The objectives of the proposed research will be: 1) Initiate a detailed
study on pelargonium
flower break virus (PFBV) in an effort to better characterize this
virus; 2) To develop and
refine state-of-the art methods of geranium virus detection, identification
and
characterization and to apply these methods to a detailed evaluation
of viruses or virus-like
agents that infect geranium.
There continues to be a great deal of confusion and lack of understanding
associated with the
viruses that infect geranium. Because of this, many viruses which have
in the past been
associated with diseases of geranium today may no longer be of major
importance. In some
cases these viruses may no longer even exist. Furthermore viruses which
were not previously
described may now play an important role in geranium production.
Most viruses which infect geranium possess a similar if not an identical
morphology, a quality
which has added to the confusion surrounding identification and diagnosis.
Newer, more
sophisticated methods of virus diagnosis, identification and characterization
are now available.
These methods, some of which need to be adapted to the geranium system,
would allow for
more rapid and accurate identification, diagnosis, and characterization
of the viruses or virus-
like agents that infect geranium. This project will focus on diagnosis,
detection and
characterization of geranium viruses along with a detailed study of
pelargonium flower break
virus.
The geranium production industry would benefit from the proposed research
by gaining a
greater understanding of geranium viruses, thus allowing for better
control measures and the
production of healthier plants.
B. Detailed Proposal:
Introduction and Review of the Significant Literature:
Zonal geranium (Perlargonium x hortorum, Bailey) is one of the most
economically important
floral crops in the United States. In 1987, zonal geranium sales (as
measured by wholesale
value) were third behind poinsettias and chrysanthemums among potted
flowers and first in
number of actual plants sold (12). Although in recent years increasing
numbers of geraniums
have been grown from seed, many are still propagated vegetatively from
cuttings (12).
Vegetative propagation makes the transmission of plant viruses from
stock plants to cuttings
a serious problem because infected plants are often symptomless (11).
While estimation of
the importance of viruses in geraniums is difficult (12), a commercial
grower can suffer
devastating losses when cuttings taken from virus-infected, asymptomatic
stock plants are
later exposed to environmental conditions that induce symptom expression
in young plants.
Reduced rooting and flowering as well as cosmetic problems have been
attributed to virus
infection (12).
Pelargonium Flower-Break Virus (PFBV) is the predominant virus associated
with zonal
geraniums. Symptoms in susceptible cultivars include light colored
streaks in the petals
(flower color breaking), smaller reduced flower size, rugose petals,
and overall stunting. Until
recently PFBV has been reported to cause disease in rare instances
and therefore thought to
be of little importance to commercial geranium production (11). However,
approximately 50
percent of the geraniums assayed for viruses (using serology and double-stranded
RNA
analysis) in the Plant and Pest Diagnostic Clinic at The Ohio State
University have tested
positive for PFBV (1). The results of virus-indexing via indicator
plants and/or serological
methods have proven to be unreliable in the detection of PFBV in geranium
stock plants
(personal communication, Michael Klopmeyer). Because of this, the geraniums
that are sold
as cultured, virus-indexed (CVI) cannot always be guaranteed as virus-free.
This is partly
due to the fact that PFBV is present in the geranium plant at a very
low titer (concentration).
It has been proposed that PFBV is in the Carnation Mottle Virus (CarMV)
group based on
certain physical characteristics. No detailed analysis has been conducted
on PFBV since it
was first described (10). Determination of the sequence of this small
genome (approximately
4000 nucleotides single-stranded RNA) will provide complete characterization
of PFBV. In
addition, sequence comparison and molecular hybridization will enable
us to determine if
PFBV belongs to the proposed CarMV group as suggested by Hull (4),
and Morris and
Carrington (5). But more importantly the molecular reagents used in
these characterizations
will provide highly sensitive and specific methods of virus detection.
In 1959, Kivilaan and Scheffer (5) observed that “Many brief reports
in the literature describe
various virus diseases of Pelarponium, but little work has gone beyond
description” because
of difficulties in working with this host-pathogen system. Remarkably
little has changed in
the ensuing thirty years. The stagnation in the field of geranium viruses
is probably due in
large part to the lack of new technology applied to this area. Much
of the geranium virus
research was conducted using electron microscopy and indicator hosts
prior to the advent of
new techniques in molecular biology in the mid 1970’s. With the notable
exception of TMV,
the majority of the commonly encountered geranium viruses are isometric
particles of about
30 nm diameter (10). This fact limits the usefulness of electron microscopy
in the
identification of these viruses. Coupled with the similarity of symptoms
on indicator hosts,
this more than likely has led to misidentification of some of the geranium
viruses.
Within the last ten years, several researchers have applied basic serological
techniques to the
study of geranium viruses, most often using it to examine for relatedness
between various
isometric viruses (7, 9, 10). Serology is an extremely powerful technique
but its results are
only meaningful if all of the viruses being used are correctly identified
and characterized.
Some of the new technologies developed in molecular biology may allow
for faster and more
accurate identification and characterization of geranium viruses thereby
leading to a greater
understanding of the geranium virus situation. Recent work conducted
by this researcher
indicates a much higher incidence of PFBV than previously reported
(1). Also pelargonium
line pattern virus which has been reported as a “true” virus in previous
literature (8) may not
be a “true” geranium virus at all (1). As of yet, this researcher has
found no virus particles
associated with this disease.
It is obvious there is a great deal of confusion surrounding geranium
viruses and the diseases
they induce. This confusion needs to be addressed if the geranium industry
within the
United States is to continue to grow and flourish. The research outlined
in this proposal
will give greater understanding to the area of geranium viruses and
the diseases they induce.
This in turn will allow for better, more efficient virus monitoring
and control strategies.
Objectives and Rational of Proposed Research:
1. Initiate a detailed study on pelargonium flower break virus
(PFBV) in an effort tobetter characterize this virus.
PFBV is at the present time a tentative member of the Carnation Mottle
Virus(CarMV) Group, a newly proposed virus group. The nucleotide sequence
of two othermembers of the CarMV group - Carnation Mottle Virus (CarMV) and Turnip
CrinkleVirus (TCV) have been determined (2, 3). We intend to determine the
relatedness ofPFBV to these two viruses by direct sequence comparisons. In addition
to theinvestigation of the fundamental gene structure of this virus, the
development ofhighly sensitive assays for PFBV in geranium tissues will allow the
detection of thevirus at low titers. This will provide geranium growers with a reliable
method todetect PFBV earlier in the propagation cycle, thus decreasing the frequency
andimportance of this virus in zonal geraniums.
2. To continue developing state-of-the-art methods of virus detection,
identification andcharacterization and to apply those methods to a detailed evaluation
of viruses orvirus-like agents that infect geranium.
Current methods of geranium virus detection and identification fall
short of thedegree of sensitivity and specificity needed to properly identify and
diagnose virusinduced diseases of geranium. Research needs to be conducted on the
developmentand application of state-of-the-art methods of virus identification.
Currenttechnologies employing nucleic acid probes, monoclonal antibodies,
andpolyacrylamide gel electrophoresis are all methods of virus detection
which could beapplied to geranium.
Materials and Methods (Brief Description)
Objective 1: Detailed study of pelargonium flower break virus (PFBV)
Pelargonium flower break virus will be purified from leaf tissue taken
from geranium or
Chenopodium quinoa plants showing symptoms of infection with PFBV.
Once successful,
virus preparations will be used for further characterization of this
virus. Virus morphology,
RNA content, RNA size, coat protein size and various other characteristics
of PFBV will be
defined.
Methods will be employed to determine the true nature of this virus
and its relationship to
other viruses in the Carnation Mottle Virus Group. These methods include:
cloning and
sequencing viral RNA and using this cloned RNA to compare PFBV to other
members of the
CarMV group. Once cloned the RNA will be used as a sensitive and highly
specific
diagnostic probe.
Objective 2: State-of-the-art of diagnostic methods
Work will continue on the use of viral-associated dsRNA analysis as
a method of virus
diagnosis (1). The dsRNA analysis technique will continue to be refined
in an effort to
produce a greater degree of sensitivity. New methods for virus extraction
from plant tissue,
nucleic acid purification, and polyacrylamide electrophoresis will
be employed. Serological-
based and nucleic acid hybridization-based methods of virus diagnosis
will also be explored.
Facilities and Equipment
The Department of Plant Pathology at The Ohio State University and
the Ohio Agricultural
Research and Development Center, Columbus is well equipped to conduct
the research
outlined in this proposal. New greenhouse facilities, state-of-the-art
equipment and adequate
laboratory space place no restriction on the types and amount of research
carried out.
All the equipment needed for plant virus identification and characterization
exists and no
new, major equipment purchases are needed.
C. Project Leader Qualifications
Dr. Nameth is an associate professor in the Department of Plant Pathology,
the Ohio
Agricultural Research and Development Center, The Ohio State University,
Columbus, OH.
Dr. Nameth’s primary research interest lies in the area of virus disease
diagnosis, virus
identification, and virus characterization. Dr. Nameth is also Director
of the Plant and Pest
Diagnostic Clinic. This facility serves commercial floral crop growers
and producers both
within Ohio as well as the rest of the United States. Dr. Nameth has
published papers on
virus identification and diagnosis in geraniums as well as other hosts.
Dr. Nameth has written
a chapter on viruses in “Geraniums IV - A manual on the Culture of
Geraniums.” Within the
last year Dr. Nameth has had two graduate students working on projects
involving viruses that
infect geranium. In 1990 Dr. Nameth received a grant for $6,500.00
from the Fred C.
Gloeckner Foundation for the study of a virus-like agent associated
with line pattern and ring
pattern in geranium. Dr. Nameth has also received $2,000.00 from Ball
Floral Plant for
studies on tomato ringspot virus on geranium.
LITERATURE CITED
1. Adkins. S. T. and S. T. Nameth. 1989. Detection of viruses in florist
geraniums using a
simplified method of dsRNA analysis. Phytopathology 79:1197 (Abstr.)
2. Carrington, J. C., Heaton, L. A., Zuidema, D., Hillman, B. I., and
Morris, T. J. 1989. The
genome structure of turnip crinkle virus. Virology 170:219-226.
3. Guilley, H., Carrington, J. C., Balazs, E., Jonard, G., Richards,
K., and Morris, T. J. 1985.
Nucleotide sequence and genome organization of carnation mottle virus
RNA. Nucleic Acids
Research 13 (18):6663-6677.
4. Hull, R. 1977. The grouping of small spherical plant viruses with
single RNA components.
Journal of General Virology. 36:289-295.
5. Kivilaan, A. and R. P. Scheffer. 1959. Detection, Prevalence and
Significance of Latent
Viruses in Pelargonium. Phytopathology 49:282-286.
6. Morris, T. J. and Carrington, J. C. 1988. Carnation mottle virus
and viruses with similar
properties. The Plant Viruses. Volume 3. Polyhedral Virions with Monopartite
RNA
Genomes. Plenum Press, New York and London.
7. Plese, N. and Z. Stefanac. 1980. Some properties of a distinctive
isometric virus from
pelargonium. Acta Hort. 110:183-190.
8. Ryden, K. 1972. Pelargonium ringspot - a virus disease caused by
tomato ringspot virus in
Sweden. Phytopath. Z. 73:178-182.
9. Stefanac, Z., Plese, N. and M. Wrischer. 1982. Intracellular changes
provoked by
pelargonium line pattern virus. Phytopath. Z. 105:288-292.
10. Stone, O. M. 1980. Nine viruses isolated from Pelargonium in the
United Kingdom. Acta
Hort. 110-177-182.
11. Strider, D. L. 1985. “Geranium.” pp. 166-180 in Diseases of Floral
Crom. David L. Strider,
ed. New York: Praeger.
12. Welvaert, W. and G. Samyn. 1985. Relative importance of pelargonium
viruses in cutting
nurseries. Acta Hort. 164:341-346.
Budget
Year 1:
- Partial cost of technical assistant or graduate student $2,500.00
- Supplies and Expendibles (i.e … chemicals, reagents,
plasticware) $2,000.00
- Photographic and publication printing costs $500.00
Total (year 1) $5,000.00
Year 2:
- Partial cost of technical assistant or graduate student $2,500.00
- Supplies and Expendibles (i.e … chemicals, reagents,
plasticware) $2,000.00
- Photographic and publication printing costs $500.00
Total (year 2) $5,000.00
Year 3:
- Partial cost of technical assistant or graduate student $2,500.00
- Supplies and Expendibles (i.e … chemicals, reagents,
plasticware) $2,000.00
- Photographic and publication printing cost $500.00
Total (year 3) $5,000.00
Total for Entire Project $15,000.00
