Progress 09/25/03 to 08/30/05
Outputs
4d Progress report. This report serves to document research conducted under a grant agreement between ARS and the Washington State University. Additional details of research can be found in the report for the parent project 5358-22000-030- 00D Biology and Control of Foliarand Fruit Diseases of Horticultural Crops. Objective 1: Determine the relationship between a novel carlavirus and black death. In spite of a reputation for being susceptible to relatively few diseases, a number of viruses have been reported in Helleborus spp. including Chrysanthemum B carlavirus, Helenium S carlavirus, Helleborus mosaic carlavirus, Broad bean wilt 1 fabavirus, and Cucumber mosaic cucumovirus (Murant & Roberts, 1977; Koenig, 1985; Kleinhempel, 1991; Waterworth, personal communication), and additional unidentified carlaviruses have been found in hellebores with black death symptoms (Koenig, 1985; Mansour et al., 1998). This has complicated the search for the identity of the
etiological agent(s) of black death. Samples of Helleborus x hybridus that expressed symptoms of black death were tested by serological methods (enzyme-linked immunosorbent assays, or ELISAs) for a number of viruses known to infect ornamental plants. In addition, a group specific molecular assay for the Carlavirus group was applied. The latter was the only test to consistently yield a positive reaction in association with black death symptoms. From the products of the group-specific test, a pair of specific primers was designed and a robust assay based on RT-PCR was developed. Specimens of Helleborus spp. were obtained from the States of Washington, Oregon, Pennsylvania and West Virginia. Over seventy plants with black death symptoms have been tested and all symptomatic plants react positively with the diagnostic RT-PCR assay indicating the reliability of this procedure for detecting the causal agent of black death disease. The accumulated data demonstrate that a distinct
Carlavirus is associated with black death. We have completed characterization of the entire genome of this virus and demonstrated that it is different from any previously reported virus. We propose the name Helleborus net necrosis virus (HeNNV) for the agent associated with black death of hellebores. Objective 2: Develop diagnostic capabilities to test Helleborus for black death. We have developed a prototype serological test for HeNNV. We have not been able to isolate this virus from symptomatic hellebores, nor has the virus been transmitted to other plant hosts that are more amenable to laboratory studies (e.g., Nicotiana spp. or Chenopodium spp.). Therefore, we cloned and sequenced the genome of HeNNV, and identified the coat protein gene. The putative virus coat protein was synthesized in bacteria by subcloning the gene into a bacterial protein-expression system. Subsequently, polyclonal antibodies were produced in response to this bacterially-synthesized protein.
Successful development of the prototype ELISA has verified that this concept is valid for detection of HeNNV. The serological test has detected the virus from all sources examined to date, and the test has been used at the request of several nurseries to screen new plants and existing plants in their breeding programs. Objectives 3 and 4: Determine the major populations of aphid occurring on Helleborus and the role of Macrosiphum hellebori and Aulacorthum circumflexum aphids in the transmission of black death. The vast majority of aphids collected from hellebores in the U.S.A. is the Helleborus aphid. These aphids appear to flourish on hellebores, and thus constitute potential vectors for the movement of Carlaviruses and the spread of black death disease. The association between a virus and its insect vector is often very specific; not all aphid species are able to transmit all viruses. Aphid transmission studies were carried out to investigate the role of aphids in transmission
of HeNNV and development of black death. Exposure of the Helleborus aphid to virus-infected and virus-free plants for one month resulted in transmission of HeNNV to the healthy plants. A prolonged assessment period of 3 to 18 months was necessary because of the long latent period reported (Rice & Strangman, 1993). We also identified and established colonies on hellebores of the crescent-marked lily aphid, Aulacorthum circumflexum (Buckton) and the violet aphid, Myzus ornatus (Laing). Preliminary trials did not acheive transmission of HeNNV with either of the latter two aphid species. Objective 5: Develop effective management strategies for aphid populations on Helleborus. Most Carlaviruses have a restricted natural host range, and are transmitted by aphids non-persistently. As a consequence, the viruses can be transmitted by investigative probing by the aphid into mesophyll cells; feeding in the phloem is not required. A strategy to minimize aphid movement and transmission of
viruses is a key element of an integrated disease control program. The use of neonicotinyl insecticides is promising for the control of black death because, in other agroecosystems, these chemicals have exhibited anti-feeding behavior with respect to aphids. Inhibition of feeding and probing activity of aphids would effectively prevent the transmission of carlaviruses. Tests in greenhouse studies have indicated that the systemic insecticide imidacloprid (Merit) is effective in controlling aphids on infested hellebores. Merit is registered for use on landscape and nursery plants in some States and this option appears to be available for nursery production of hellebores. Since both HeNNV and the predominant aphid vector have narrow host ranges, there is little risk of influx of this disease from outside sources if appropriate management recommendations are implemented. Thus, control of aphid populations combined with readily available testing methods should put the control of black
death within practical reach. Objective 6: Extension activities to provide growers with necessary tools to control black death. In addition to research reports, results of this study are being made available to growers and nurseries that have participated in the study as well as their respective networks. Several nurseries that specialize in Hellebores and regional groups have utilized newsletters to disseminate new information to the nursery community. We have already reached a wide audience through these informal channels. Many nurseries are submitting samples fro virus testing to help minimize the spread of this disease. We reported the results of our studies to diagnosticians, extension agents and States department of agriculture personnel attending the 2004 Western Turf & Ornamental Disease Conference in Portland, OR. This meeting was attended by representatives from most western states and has resulted in contact with several Hellebore nurseries. Nurseries from Oregon,
Washington and West Virginia have submitted samples for virus testing to insure that new acquisitions are virus free, and to assist in eliminating infected plants from their breeding stocks. A book chapter will appear soon describing this disease and its control.
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Progress 10/01/03 to 09/30/04
Outputs
4. What were the most significant accomplishments this past year? This report serves to document research conducted under a grant agreement between ARS and Oregon State University. Additional details of research can be found in the report for the parent project 5358-22000-030-00D Biology and Control of Foliar and Fruit Diseases of Horticultural Crops. K. Eastwell and colleagues at Washington State University are examining 'black death', the common name for a disease that quickly degrades the marketability and appearance of several Helleborus spp. RESEARCH PROGRESS TOWARDS GOALS: Objective 1: Determine the relationship between a novel carlavirus and 'black death'. Leaf and flower specimens from healthy and 'black death' symptomatic tissue were obtained from several independent sources in the USA, including nurseries, breeders and growers. Samples were analyzed by molecular and serological techniques developed in this project; the results revealed a strong
correlation between the presence of this unique carlavirus and the occurrence of disease symptoms. From these same samples, potential bacterial and fungal pathogens were also isolated and identified. Pathogenicity tests carried out in the greenhouse were unable to establish a link between these microbial isolates and the presence of 'black death' symptoms. This provided further support to the hypothesis that the carlavirus is the cause of 'black death'. Another virus, Helleborus mosaic carlavirus (HeMV) has been described in H. niger (Koenig R. 1985. Acta Horticulturae 164:21-31). We obtained authenticated cultures of HeMV from Germany, and the antiserum that had been prepared against this virus. Our subsequent research has demonstrated that although the carlavirus isolated from 'black death'- diseased Hellebores does react with HeMV antiserum, these two viruses are distinct viruses. The coat protein gene of the 'black death' associated virus shares less than 50% homology with
the coat protein gene of HeMV. Members of the genus Carlavirus typically share very similar amino acid sequences in the C-terminus region of the coat protein, so it is not uncommon for different carlaviruses to react with antiserum produced against other carlaviruses. Objective 2: Develop diagnostic capabilities to test Helleborus for 'black death'. The coat protein gene from the 'black death'-associated carlavirus has been successfully inserted into a bacterial expression vector. This allowed us to purify significant amounts of protein encoded by this gene; the purified protein was used to induce antibody production in rats. The resulting antisera have been used successfully at a 1:2000 dilution to detect the virus in extracts of diseased plants using an enzyme-linked immunosorbent assay (ELISA). This result provides strong evidence that the gene that we have isolated does encode the coat protein of the virus. More significantly, this provides the first practical diagnostic
test for the pathogen associated with 'black death' disease. The molecular assay (RT-PCR) that we reported in our previous progress report is a valuable research tool, but it is not realistic or economical for routine diagnostics of large numbers of nursery samples. Therefore, this ELISA is the first practical system available for detecting the disease agent in nursery and genetic stocks of Helleborus spp. Rats provided a small amount of antibody appropriate for test development and evaluation of this strategy. The bacterially-expressed coat protein has now been injected in sheep for the production of larger amounts of antibodies. A larger amount of antiserum will enable the technology to be accessed by a broader spectrum of growers and nurseries. Objective 3: Determine the role of Macrosiphum hellebori and Aulacorthum circumflexum aphids in the transmission of 'black death'. The association between a virus and its insect vector is often very specific. Although many carlaviruses
are aphid-transmitted, this association must be established for the newly identified carlavirus linked to 'black death'. Moreover, not all aphid species are able to transmit all viruses. Established colonies of the Helleborus aphid (Macrosiphum hellebori) and the crescent-marked lily aphid (Aulacorthum circumflexum) were used to attempt transmission of the novel carlavirus. Of the approximately 100 plants exposed to aphids from diseased plants, none have developed 'black death' symptoms. This may indicate that these aphid species are not vectors of the virus, or that the virus has not yet reached detectable levels as suggested by the long latent period previously reported for 'black death'. Discussions with breeders have indicated that white flies are periodic pests on Hellebores. Since some carlaviruses are transmitted by white flies, we have established a white fly colony on H. x hybridus. In an experiment that parallels the aphid transmission studies, diseased and non-diseased
plants are caged in the presence of white flies, with a mesh barrier to prevent direct plant-to-plant contact. We have allowed one month for white fly-mediated transmission and the plants will be maintained and assayed in the future to determine whether transmission of the virus has occurred. Objective 4: Determine the major populations of aphid occurring on Helleborus. Although Macrosiphum hellebori (Theobald & Walton) and Aulacorthum circumflexum (Buckton) have been discovered on Helleborus x hybridus and shown to colonize these plants; other aphid species may be involved in the transmission of 'black death'. We have collected aphids from Helleborus plants throughout the growing season in three states. Macrosiphum hellebore has been isolated with greatest frequency from Hellebores. This season, Myzus ornatus (Laing), the violet aphid, was also identified on Hellebores; a colony derived from this population has been established. Efforts to transmit the 'black death'-associated
virus will continue. With the availability of the ELISA, we can identify tissue types with the greatest virus titer that may be more appropriate sources of virus for acquisition by the aphid or white fly species. Objective 5: Develop effective management strategies for aphid populations on Helleborus. Aphid-attacking parasitic wasps have been used successfully to reduce aphids to below plant damaging levels in other plant systems. These successes are generally linked to parasitoids that co-evolved with the target pest. That is, in most cases of successful biological control, the parasitoid was introduced from the place of origin of the pest aphid. Both the Helleborus aphid and the crescent-marked lily aphid are European or Eurasian in origin, and should be at least partially controlled by Old World parasitoids. New systemic neonicotinyl insecticide chemistries have the potential to control 'black death' because, in other agroecosystems, these chemicals have exhibited
anti-feeding behavior. Inhibition of feeding and probing activity of aphids would effectively prevent the transmission of carlaviruses. The evaluation of either biocontrol or chemical control strategies will be initiated pending confirmation of the insect vector of the 'black death' virus. Objective 6: Extension activities to provide growers with necessary tools to control 'black death'. In addition to research reports, results of this study are being made available to growers and nurseries that have participated in the study as well as their respective networks. Several nurseries that specialize in Hellebores and regional groups have utilized newsletters to disseminate new information to the nursery community. We have already reached a wide audience through these informal channels. We reported the results of our studies to diagnosticians, extension agents and State Department of Agriculture personnel attending the 2004 Western Turf & Ornamental Disease Conference in Portland,
OR. This meeting was attended by representatives from most western states and has resulted in contact with several Hellebore nurseries. Nurseries from Oregon, Washington and West Virginia have submitted samples for virus testing to insure that new acquisitions are virus free, and to assist in eliminating infected plants from their breeding stocks.
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