Assessing The Impact Of Floral Crop Species And Control Strategies On TSWV/INSV Population In Commercial Production Progress Reports - June 2000
Proposal - June 2000
Accessing the Impact of Floral Crop Species and Control
Stategies on TSWV/INSV in Commercial Production
James Moyer
North Carolina State University
EXECUTIVE SUMMARY
The goal of this research is to improve control strategies for tomato spotted
wilt virus and impatiens necrotic spot virus. These two viruses belong to the
same taxonomic group of plant viruses (Tospoviruses) and although they are
distinct viral species, they share many biological properties, are spread by the
same insect vectors and have an overlapping host range. These two viruses have
been among the top priorities for research on floral crops since they were first
recognized as the cause of widespread devastating losses. Although the magnitude
of losses in greenhouses have diminished, they still pose a significant threat
to many floral crops as evidenced by their continuous detection by clinics and
commercial testing services. The NC Disease and Insect Clinic continues to
detect these two viruses in at least a dozen species of floral crops annually.
Bedding plants and chrysanthemum have been the predominant crops affected by
these viruses, but other plants such as African violet, kalanchoe rannunculus
and scheffiera are also impacted by these viruses. Thus, any improvement in
controls would impact a wide segment of the industry.
Practical control optiOns continue to rely on using virus tested propagation
stock and stringent thrips monitoring and control programs. The development of
cultivars with resistance to these two viruses may have impact on a limited
number of species and recent research has shown both natural and engineered
resistance to be short lived due to the viruses ability to adapt to new hosts.
Thus, improvement of control strategies will be dependent upon refinement of
r?±an?£gement tools. In this study we will build on recent research monitoring
thrips and on our own fundamental research on the evolution of TSWV to determine
the impact of various floral crops on the spread of the virus and on changes in
the virus population. This information will be directly applicable for growers
in high risk areas in choosing crops and cropping patterns.
INTRODUCTION and LITERATURE REVIEW
TSWV and INSV remain significant threats to the floral crop industry and are
a high priority. Current controls are based on removal of the inoculurn sources
and in controlling the vectors Daughtery et al, 1997). In instances where there
are external inoculum sources or all sources have yet to be identified, growers
continue to employ intensive, preventative thrips (the virus’s vector) control
programs. We have experimentally identified risk factors for field crops in
North Carolina and are beginning to test those modifications. In that research
alternate hosts (weeds) were identified and evaluated as TSWV reservoirs. \Veed
control and planting date provide two management opportunities for control in
the field and have been widely recommended for peanuts in Texas and other
states. Management of TSWV/INSV in floral crops provides new opportunities for
identifying factors that will reduce the risk of loss. In this research our goal
is to test changes in floral crops that will lead to more efficient risk
management strategies for TSWV/NSV in floral crops. Substantial effort has been
given to the thrips element of the disease equation. In this research, we will
build on that information by determining the affect of various floral crops on
natural populations of TSWV/INSV.
TSWV has been a recognized pathogen of floral and other crops for many years,
however 1NSV was only recently discovered (Law & Moyer, 1990; Law et al
1991) It has long been known that TSWV and now INSV, exist as complex,
heterogeneous mixtures of isolates or populations (Best and Gallus, 1953; Moyer
unpublished). This is in sharp contrast to most other viruses that exhibit far
less heterogeneity and tend to occur as single strains. We have recently shown
that TSWV genome segment mixtures can reassort into different combinations,
resulting in an isolate with a different set of characteristics or phenotypes (Qiu
et al, 1998). In one instance, a reassortant isolate with a specific combination
of genome segments was formed that was capable of breaking resistance¬ówhile
both viral parental isolates were suppressed by the resistance. We have further
shown that host genotypes can exert a specific selection pressure on TSWV virus
populations which changes the attributes of the isolate or population (Qiu et
al, 1999). In that study we demonstrated that when a TSWV population is passed
several times through a ‘neutral’ host, the population remains a random
mixture, while passages on a resistant host rapidly select for a narrowly
defined segment of the population.
In other research, also with TSWV, serial passage through specific hosts have
been shown to affect viral populations. The observation that resistant hosts
select for resistance breaking strains of TSWV has been widely reported (eg.
Finlay, 1952; Latham and Jones, 1998), although the mechanisms of
adaptation were only recently reported (Qiu et al 1998). Other host effects have
also been reported. For example, passage through certain Nicotiuna spp. generate
defective virus that toads to attenuate virus symptoms while serial passage
through other hosts has little or no detectable affect on the viral population (Resende
et al 1991).
INSV was first recognized as being distinct from TSWV only about 10 years ago
(Law and Moyer 1990; Law et al., 1991). TNSV has a host range that
includes many floral crops, especially bedding plants that is similar to TSWV.
However, it is distinct from TSWV in that many of the commonly used diagnostic
hosts in the solanaceae are only local lesion hosts for INS V. Until recently
1NSV was only rarely observed in field crops and even in those instances
propagation material could be traced back to greenhouses used for floral crop
production (Daughtery et al 1997). During 1999 there were reports of INSV
infections in field crops for the first time that could not be traced back to
floral crops. The virus was detected in peanuts in Texas (personal
communication, M. Black) and in tobacco in Kentucky (personal communication, W.
Nesmith). Symptoms were particularly striking in tobacco resulting in severe
veinal necrosis. INSV has received relatively little attention in the past five
years beyond reports of occurrence in additional floral crops and in additional
geographic areas. INSV is now widespread in the US, Europe, around the
Mediterranean area and in Japan.
However, there is some evidence to suggest that INSV is also sensitive to
selection pressure. Changes in the viral population related to high and low
temperature have been reported. High temperature (33 C) was shown to prevent
systemic infection of pepper by INSV (Deangelis et al 1994). In addition passage
at high temperatures has resulted in changes in the virus population that may be
attributed to mutation or selection (Lawson et al 1993). The influence of high
temperature was thought to have given rise to a new high temperature isolate. 1
However, it now appears that this may have been a new Tospovirus.
Variation in the cytopathology caused by defective isolates has also been
reported (Lawson eta! 1996). Despite the continued occurrence of these viruses
there have been no reports of new controls since the initial studies were
completed nearly ten years ago (Jones and Moyer, 1991; reviewed in Daughtry et
al 1997). This is surprising as the available research clearly suggests that the
virus is sensitive to changes in environment and hosts, thus providing
opportunities to interrupt the disease cycle.
In summary, the implications of this research are two fold: First the
literature provides numerous examples of how environment and host can alter the
virus. Most of the research on TSWV has been aimed at describing TSWV’s
propensity to rapidly adapt to resistant hosts and our own research which exams
genetic mechanisms of adaptation. Second, the sensitivity of TSWV/INSV
populations to selection pressure strongly suggests that additional opportunity
existz exploiting this trait to identify management practices that would reduce
risk. This opportunity is further supported by the initial success that risk
assessment has met with in managing TSWV in peanuts in Georgia (www.ces.uga.edu/pubcd/bl
165-w.html). Risk assessment is a tool that could be implemented in floral crop
production to reduce costs attributable to TSWV/INSV.
OBJECTIVES and ANTICIPATED BENEFITS
Characterize the molecular and biological diversity that exists in TSWV and
INSV populations in naturally infected floral crops. \Ve have considerable
experience with TSWV and INSV as well as having the tools to conduct the
molecular analysis (see references below). At the present time there is no
active The scope of the proposed research has two subobjectives:
1. Conduct fundamental research being conducted on INSV which may partially
explain why INSV remains the predominant tospovirus in floral crops and a
threat to the industry. \Ve will collect samples from field and greenhouse
sites currently used in investigations being conducted by Mike Parella and
Diane Uliman as well as selected other cites. From combined molecular and
biological analyses, we will identify characteristics of each population. In
addition we will also attempt to identify individual isolates as pure cultures
that may be further characterized to serve as controls in future experiments.2. Determine how specific floral crops and vector species influence
changes in viral populations. As it is now widely recognized that TSWV and
INSV are sensitive to changes in environmental and host, our overall goal is
to identify hosts that will reduce virus pressure. The measures of viral
diversity identified in objective 1 will be used to monitor changes in natural
populations as a result of passages through various floral crop species and
changes in vector species. Our ultimate goal would be to use this information
to supplement existing control procedures and then monitor changes in viral
populations following various control procedures, hbw?´ver that maybe beyond
the scope of this project.
Benefits: Due to the broad spectrum of crops that are susceptible to
TSWV/INSV and the worldwide distribution of the viruses in the floral crop
industry the research has the potential to benefit a large segment of the
industry. Information will be dissemeninated through scientific and trade
publications as in the past.
MATERIALS and METHODS
Objective 1. Characterize the molecular and biological diversity that exists
in TSWV and INSV populations in naturally infected floral crops. This
information is necessary to provide the baseline from which changes in virus
populations can be measured. Both molecular and biological criteria will be
applied so that whereever possible molecular markers can be used to efficiently
monitor changes in the biological characteristics of virus populations.
Virus sources: Plants naturally infected with TSWV and INSV will be collected
from commercial production facilities. Attempts will be made to collect TSWV and
INSV from field as well as greenhouse operations and from geographically diverse
locations (eg., California and North Carolina). Natural field ‘populations’
of virus will be maintained in the original plants and in Emelia sonchifolia (TSWV)
and impaticns (INSV).
Biological characterization of natural populations: To assess the biological
diversity of the virus isolates, single lesion transfers will be made from the
natural population. A minimum of 20 isolates will be obtained from at least four
natural populations of each virus. Controls will include the natural population
and isolates we obtained from floral crops during the last ten years. Each
isolate will be inoculated onto commonly used hosts for Tospoviruses which
include: Cucurnis sativuin, Datura strarnonium, Emelia sonchifolia, Gomphrena
globosa, various Nicotiana spp., and Petunia hybrida. In
addition, each isolate will be inoculated onto a range of floral crop species
which would include begonia, chrysanthemum, cineraria, cyclamen, New Guinea
Impatiens, double Impatiens, gloxinia, and kalanchoe.
Symptom type (local or systemic), kind of symptom, general estimate of virus
titer and extent of virus invasion will be determined, Estimate of virus
invasion (proportion and location) is important not only to assess impact but
also as one component of the plants suitability to serve as a source or
reservoir of virus for thrips transmission.
Molecular characterization of the virus populations will be done with the
ultimate objective being to find markers which can be used to characterize and
to monitor changes in the virus population. We have previously developed markers
for genetic studies of the TSWV (Qiu et a!., 1998). We will extend this to
include INSV. In those previous studies we have identified hypervariable regions
of the genome ofTSWV. We will use this information in focusing our search for
useful markers for INSV. The strategy will be to amplify and sequence the
hypervariable regions of the genomes of the biologicaIly variant isolates
discovered above. Using this approach we found useful restriction enzyme sites
that differentially digest different isolates and thus provide a molecular tool
to monitor shifts in virus populations.
Objective 2 Determine how specific floral crops and vector species influence
changes in viral populations.
Virus sources: Natural populations of TSWV and INSV will be used in these
experiments. Host Plants: A series of floral crops will be selected that exhibit
different svmptoni types and therefore would be predicted to exert a range of
selection pressures on natural virus populations. Initially, we will use
begonia, chrysantemum, gloxinia, and a tolerant and a sensitive New Guinea
Impatiens cultivar. This list may be modified pending the outcome of Objective
1. Using a protocol we have used to study virus adaptation to resistant hosts (Qiu
et al 1999), we will assess the impact of selected hosts oii virus populations.
We will also conduct measurements on the suitability of the host to serve as
a vector source for the virus. Vector studies will he conducted with the aid of
advice from Drs. George Kennedy, Dept of Entomology, NCSU and Diane Uliman, Dept
of Entomology, UC-Davis. In these experiments, groups of thrips larvae will be
placed on infected plants for virus acquisition. The plants containing thrips
will be placed in cages with ten healthy plants. Transmission efficiency
will be calculated using maximum likelihood estimator to provide and estimate
of individual efficiency of transmission. Emelia sonchifolia will be used as the
virus source when measuring inoculation efficiencies and effects of a given host
on the virus population. Emelia sonchifolia will be used as the recipient when
evaluating floral crops as sources. The effects of vector species on viral
populations will use Emelia sonchifolia as the source and recipient. All
experiments will be statistically analysed with the aid of the Statistical
Consulting service provided by the NCSU Statistics Department.
The information derived form these experiments will provide fundamental
knowledge concerning the evolutionary biology of TSWV and INSV as impacted by
host and vector, it will provide information relevant to crop selection for
growers in high risk areas and it will provide an excellent training vehicle for
a graduate student. The next phase of this research would be to develop a model
for risk assessment that would be valid for the floral crop industry.
