Progress 10/01/02 to 09/30/08
Outputs
OUTPUTS: C. Warfield is no longer employed with the University. No work has been done on this project. This project is final/terminated. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: n/a PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
C. Warfield is no longer employed with the University. No work has been done on this project. This project is final/terminated.
Publications
- C. Warfield 2007 is no longer employed with the University. No work has been done on this project. This project is final/terminated.
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Progress 10/01/05 to 09/30/06
Outputs
A sensitive diagnostic assay to detect foliar nematodes was developed using a specific primer set based on the unique ITS1 region of the Aphelenchoides fragariae genome. Sequence comparison of the ITS1 region from a subset of foliar nematodes collected throughout the North Carolina nursery industry demonstrated 100% homology with each other and with the only A. fragariae ITS1 sequence published in Genbank from a research group in Japan. Our primer set amplifies a region of ribosomal DNA that is specific only to A. fragariae, and does not amplify DNA of A. besseyi. Based on sequence data, there is no significant homology between the ITS1 region of A. fragariae and A. ritzembosi; therefore, amplification of A. ritzembosi would not be expected. Of the 95 naturally infested ornamental plant samples collected in North Carolina, 98.7% were infested with A. fragariae based on morphology and were further confirmed to species by our PCR-based assay. Our primer set did not
amplify DNA extracted from a number of common greenhouse pests and pathogens, including mealybugs, whiteflies, aphids, mites, bacteria, and fungi. In addition, no cross-reactivity or inhibition of the assay was observed when the primer set was tested against DNA extracted from healthy plant samples. A sampling protocol is being developed to screen nursery blocks of host plants for the presence of foliar nematodes to determine the minimum number of plants that must be sampled in order to detect the nematodes if present. Four morphologically diverse, perennial plant species were sampled in a commercial nursery over a period of two months, using both methods of detection. The PCR-based assay detected significantly more foliar nematodes in asymptomatic plants compared to the traditional water extraction method of detection. For leaves with symptoms, both detection methods were statistically equivalent in detecting nematodes. Using Taylor's Power Law, it was determined that the foliar
nematodes are distributed in an aggregate pattern within a nursery block, which is consistent with the biology of this microorganism. Depending on the host plant, it was determined that sampling sizes must range from 23-41 plants per 8.5 square feet in order to detect the nematodes at a precision of 0.25. Naturally infected Lantana camara plants were sampled throughout the growing season, beginning in June, at a commercial nursery. Environmental data was collected during this period to determine any relationships between temperature, humidity, leaf wetness, and wind speed and the population of nematodes present throughout the season. The data is currently being analyzed. A second set of containerized Lantana plants, naturally infected with foliar nematodes, is being monitored to determine where the foliar nematodes are over-wintering in a nursery setting. Dormant buds, leaf debris, and soil samples are being collected at periodic intervals throughout the winter months.
Impacts
Foliar nematodes are a serious, and widespread problem on herbaceous perennials and woody ornamentals grown in commercial nurseries. Once introduced into a growing facility, foliar nematodes not only thrive but also continue to persist due to the difficulty in detection and the lack of effective options for control. Exclusion of infested plant material is currently the best method of control, however, plant symptoms are generally not visible until high populations of the nematode are present. A highly sensitive diagnostic assay and nursery sampling protocol will allow for earlier detection of foliar nematodes in host plant tissues, and will provide a more accurate, high throughput method for screening vegetatively propagated nursery stock. The diagnostic assay will be used as a research tool in developing management strategies to control foliar nematodes through a better understanding of nematode biology and epidemiology. The development of more effective control
options will promote higher standards and expectations for plant quality within the ornamentals industry. Chitosan has anti-microbial activity against a range of plant pathogens and induces the expression of a variety of genes involved in plant defense responses. Chitosan films applied as aqueous solutions to leaf surfaces have the ability to form a physical barrier, thereby inhibiting foliar pathogens from colonizing and invading plant tissue. The use of chitosan as a bio-pesticide would provide an environmentally safe means of controlling plant diseases.
Publications
- McCuiston, J.L., Davis, E.L., and C.Y. Warfield. 2006. A comparison of detection methods for foliar nematodes in herbaceous ornamental crops. Phytopathology (Supplement) 96:S75.
- Warfield, C.Y. 2006. Evaluation of registered and non-registered fungicides for control of Myrothecium leaf spot on Syngonium, 2005. Fungicide and Nematicide Tests (online). Report 61:OT001. DOI: 10.1094/FN61. The American Phytopathological Society, St. Paul, MN.
- Warfield, C.Y. 2006. Evaluation of registered and nonregistered fungicides for control of Myrothecium leaf spot on pansy, 2005. Fungicide and Nematicide Tests (online). Report 61:OT002. DOI: 10.1094/FN61. The American Phytopathological Society, St. Paul, MN.
- Warfield, C.Y. 2006. Evaluation of registered and nonregistered fungicides for control of black root rot on pansy, 2005. Fungicide and Nematicide Tests (online). Report 61:OT003. DOI: 10.1094/FN61. The American Phytopathological Society, St. Paul, MN.
- Warfield, C.Y. 2005. The threat of foliar nematodes, is it real? Proceedings of the 21st Annual Conference on Pest Management and Ornamentals, Society of American Florists, Orlando, FL, pp. 14-16.
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Progress 10/01/04 to 09/30/05
Outputs
A practical and sensitive diagnostic assay to detect foliar nematodes is being developed using a specific primer set that we have developed based on the unique ITS1 region of the Aphelenchoides fragariae genome. Our primer set appears to amplify a region of ribosomal DNA that is specific only to A. fragariae. Progress to date has focused on the optimization of the assay for specificity, sensitivity, reproducibility, and the capacity to detect foliar nematodes within a range of plant species. Ninety-five plants naturally infested with foliar nematodes were collected from North Carolina nurseries. These host plants represented 47 genera from 23 different plant families. Seventy-two plants have been assayed using both conventional water extraction and our diagnostic assay. The conventional method detected A. fragariae in 56 plant samples compared to 51 samples using the PCR assay, suggesting further optimization is needed. We are developing a sampling scheme for
nurseries. The first step was to determine what plant tissues are most likely to be infected. A fully expanded (older) leaf and a new leaf were removed from each stem node of verbena plants in a nursery block, working from the base of the plants upward. Leaf disks were removed from each leaf. Half of the leaf disks were assayed for nematodes using water extraction, and the other half by the PCR assay. The sensitivity between water extraction versus our molecular diagnostic assay was compared. Of the 244 leaf samples collected, 100 samples were negative for foliar nematodes by water extraction, but 34 of these samples were found positive using the molecular assay. However, the other 100 samples were positive by water extraction but only 40 of these samples were positive by molecular detection. It was determined that the nematodes can be found throughout the entire plant; with nematodes consistently found in the lower two thirds of the plant. The population of nematodes in the expanded
(older) leaves was higher than the population in the newer leaves but this was only true for the lower half of the plant. In the top half of the plant, this trend reversed and more nematodes were found in the newer versus the older leaves. Foliar nematodes were detected in leaf tissue with and without visible symptoms of foliar nematode damage. Nematodes were also found on the internode tissue between symptomatic leaves. A high degree of anti-microbial activity against bacteria was obtained when a high molecular weight chitosan derivative (deacetylated to 98%) was tested in vitro. The bacterial pathogen Xanthomonas campestris was quickly and effectively killed by contact with aqueous chitosan. Aqueous chitosan does not have any direct anti-microbial effect on foliar nematodes. But when mobilized into films, the nematodes are unable to penetrate the films. Using zinnia plants as a model system, we have applied the chitosan to the leaf surfaces using a compressed air sprayer. There was
no observed phytotoxicity with the chitosan. The plants were then challenged with Xanthomonas. Although control was initially poor, we are experimenting with improved mobilization of the films on the leaf surface.
Impacts
Foliar nematodes are a serious and widespread problem on herbaceous and woody ornamentals grown in nurseries, due, in part, to the difficulty in detection and the lack of effective options for control. A highly sensitive diagnostic assay will allow for earlier detection and will provide a more accurate, high throughput method for screening vegetatively propagated materials. The diagnostic assay will also serve as a research tool in developing management strategies to control foliar nematodes through a better understanding of nematode biology and epidemiology. In turn, more effective control options for managing foliar nematodes will promote higher standards and expectations for plant quality within the ornamentals industry. Bacterial diseases are difficult to control due to the small number of bactericides available for use on plants as well as their relative ineffectiveness. Resistance to copper and streptomycin is widespread in commercial nurseries. Chitosan has
been shown to have anti-microbial activity against bacteria, viruses and fungi, and has been reported to induce the expression of a variety of genes involved in plant defense responses. Chitosan films applied as aqueous solutions to plant and leaf surfaces have the ability to form a physical barrier, thereby inhibiting foliar pathogens from colonizing and invading the plant tissue. The use of chitosan as a bio-pesticide would provide an environmentally safe means of controlling plant diseases, while utilizing an abundant, natural waste product.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
One hundred samples of vacuum harvested Canadian sphagnum peat were assayed for the presence of Thielaviopsis basicola to determine if peat is a source of disease inoculum in greenhouse production. Peat moss samples were collected from 17 bogs in 4 provinces. From each sample bag, five 500 ml subsamples were removed and assayed. There was no evidence of T. basicola in any of the subsamples. This does not rule out that T. basicola is not present in the peat, but the incidence of the fungus and/or the population density were below the assay detection limits. The lack of evidence of this fungal pathogen in the peat moss suggests that the incidence and population of this pathogen must be very low if actually present at all. Canadian sphagnum peat is, therefore, not a likely source of disease inoculum. A study was undertaken in 2003 and repeated in 2004 to assess how quickly foliar nematodes can spread from infested plants to healthy plants in a nursery setting under two
different irrigation regimes. Nematode-free Lantana plants were intermixed with an equal number of foliar nematode-infested plants of the same cultivar in a completely randomized design. Overhead sprinklers irrigated one block, and the second block was irrigated with low volume spray stakes. Spread of foliar nematodes from infested to healthy plants was rapid. By day 32 in 2003, foliar nematodes spread to 45% of the originally healthy plants in the low volume block, and to 55% of the healthy plants in the overhead-irrigated block. By day 62 in each year, foliar nematodes spread to 100% of the originally healthy plants in both the overhead and low volume irrigated blocks. There was no significant difference, at day 62, in the nematode population between the initially healthy plants compared to the originally infested plants. While the rate of spread was slightly faster for plants irrigated by overhead sprinklers, the foliar nematodes were equally capable of spreading from infested to
healthy plants under a low volume irrigation regime that promoted drier foliage. The efficacy of Pylon applied to naturally infected lantana plants grown in containers was evaluated in 2004. Plants were grown under overhead sprinkler irrigation and low volume irrigation. Pylon failed to eradicate foliar nematodes under either irrigation regime. We have designed Aphelenchoides spp.-specific primers that amplify unique regions of foliar nematode DNA for use in a PCR-based diagnostic assay. The design of these primers is based on a unique sequence that lies within an amplified region of the Aphelenchoides genome. GenBank accession number AF119049 (A. fragariae ribosomal RNA gene) was used to design primers specific to the ITS1 ribosomal regions of A. fragariae. Primers were designed using Primer3 software, and analyzed and rated for success using Sense Primer Report. We have confirmed specificity of our primers to the genus (Aphelenchoides) level. We will be testing the sensitivity of
our primer set using a variety of nematode-infested plant materials. Preliminary sensitivity assays have demonstrated that our assay is capable of detecting a single nematode within a background of host plant tissue.
Impacts
Foliar nematodes are becoming increasingly common and injurious to a wide range of woody ornamental plants grown in nurseries. Current management options are extremely limited. The nursery industry needs an effective management program that can be used to reduce damage to susceptible crops in the nursery and to minimize the risk of shipping foliar nematode infested plants to other nurseries or for final use in the landscape. The results of this project will help to identify effective control options, and will develop a diagnostic tool to help study the biology of these pests leading to strategies that will enhance control. Identifying sources of initial disease inoculum is important for preventative disease management. The lack of evidence of peat moss as a source of disease inoculum rules this out as a likely source. Other avenues of pathgeon introduction will need to be explored.
Publications
- Warfield, C.Y., Parra, G.R. and P.A. Hight. 2004c. Evaluation of miticides and a surface disinfestant for control of foliar nematodes on abelia, 2003. Fungicide and Nematicide Tests 59:OT001.
- Hudson, L.C., X. Wang, E.L. Davis, and C.Y. Warfield. 2004. Development of a PCR-based detection assay for foliar nematodes (Aphelenchoides spp.) Phytopathology (Supplement) 94:S43.
- Warfield, C.Y., Dudley, J.B., and P. A. Hight. 2004a. Evaluation of Chemical and Cultural Methods for Managing Foliar Nematodes on Woody Ornamental Crops in Nurseries. Proceedings of the Southern Nursery Association (SNA) Research Conference 49-2004, pp. 290-293.
- Warfield, C.Y. Parra, G.R and J.D. Dudley. 2004b. Efficacy of miticides and a disinfestant for foliar nematode control on lantana and buddleia. F & N Tests 59: OT003.
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Progress 10/01/02 to 09/30/03
Outputs
It was confirmed that re-used greenhouse plug trays are a source of Thielaviopis basicola inoculum in greenhouse production. Spores of T. basicola are not only capable of surviving on re-used plug trays, but the population of viable spores was sufficient to cause black root rot disease when pansies were directly seeded into infested, re-used trays. Commercial greenhouse disinfestants can reduce the viability of T. basicola spores when applied at label rates to naturally infested greenhouse plug trays, but they may not eradicate the pathogen when it is present in high numbers or if organic matter is present on the trays. Bleach (1:9 dilution) and ZeroTol (1:50 dilution) were both capable of eradicating the pathogen from infested trays. Greenshield reduced the number of viable spores, but did not eliminate the pathogen from the trays. The efficacy of two registered insecticides (Pylon and Floramite) and a surface disinfestant (ZeroTol) were evaluated for control of
foliar nematodes (Aphelenchoides spp)on nursery grown woody ornamentals in an on-site nursery trial in 2002, and in two on-site nursery trials in 2003. An experimental bio-rational product was also evaluated in 2003. Treatments in 2003 were applied to plants growing under either low volume irrigation (spray stakes that direct water onto the soil away from the foliage) or by overhead sprinklers. None of the treatments significantly reduced the nematode population in the 2002 nursery trial. Pylon and Floramite reduced the population of foliar nematodes in one nursery, at the second sampling date (14 days after initial application) when applied to naturally infested lantana plants grown under low volume irrigation, but had no effect on the population at the later sampling dates, or on plants irrigated with overhead sprinklers. Pylon reduced the foliar nematode population on buddleia at 28 and 42 days after initial treatment. ZeroTol was ineffective in reducing the nematode population on
any of the hosts (lantana, abelia, buddleia) in either year. The development of genus-specific or species-specific assay to detect foliar nematodes in plant host tissue appears to be a viable option. The polymerase chain reaction (PCR) was used to amplify DNA extracted from monoxenic cultures of Aphelenchoides using either rRNA (ITS) or small subunit RNA "universal" primer sets. The fragment produced with the ITS primer set displayed a high degree of homology with the other two Aphelenchoides ITS sequences in the public database. Sequences of rDNA specific to the genus Aphelenchoides appear to be available to design genus and species-specific primer sets. Experiments are now focusing upon designing species-specific Aphelenchoides primer sets and to assess the limits of nematode detection within infected plant tissues.
Impacts
Foliar nematodes are becoming increasingly common and injurious to a wide range of woody ornamental plants grown in nurseries. Current management options are extremely limited. The nursery industry needs an effective management program that can be used to reduce damage to susceptible crops in the nursery and to minimize the risk of shipping foliar nematode infested plants to other nurseries or for final use in the landscape. The results of this project will help to identify effective control options, and will develop a diagnostic tool to help study the biology of these pests leading to strategies that will enhance control. Identifying sources of initial disease inoculum is important for preventative disease management. The confirmation of greenhouse plug trays as a source of disease inoculum identifies a potential area where growers can concentrate their sanitation efforts to reduce the incidence of disease and more effectively manage this disease.
Publications
- Warfield, C.Y. and Konczal, K.M. 2003. Re-used greenhouse plug trays as a source of Thielaviopsis basicola inoculum in pansy production and efficacy of greenhouse disinfectants. Phytopathology 93:S89.
- Warfield, C.Y. and Konczal, K.M. 2003. Survival of Thielaviopsis spores on re-used plug trays and efficacy of disinfectants on spore viability. Southern Nursery Association (SNA) Research Conference Proceedings, Atlanta, GA (in press)
- Warfield, C. Y. 2003. Evaluation of pesticides for control of foliar nematodes on lantana, 2002. F & N Tests 58: OT021.
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