Progress 07/01/01 to 06/30/06
Outputs
The overall objective of this project is to identify sources of primary inoculum for Phytophthora diseases that affect ornamental crops in nurseries. The project was initiated in July 2001 and terminated in 2006. This project has been very successful and productive. It has included three undergraduate honor students, three MS graduate students, one PhD graduate student, and two postdoctoral research fellows. Over the duration of this project, we have developed and optimized methods for recovering Phytophthora species from water by filtration and baiting and from soil by baiting. Filtration has been very useful for recovering Phytophthora spp. from nursery waterways (e.g., runoff, irrigation, ponds, puddles, etc.), waterways in natural ecosystems (e.g., streams, creeks, lakes, etc.), and waterways in urban areas (e.g., streams, creeks, etc.). Baiting with wounded or non-wounded leaves also was very effective; however, using filtration, we recovered a greater diversity
of species more consistently. Waterways appear to be tremendous reservoirs of Phytophthora spp. Many known species were recovered, but a number of unknown "species" also were found--based on morphological and molecular genetic characters. Interestingly, the species complex in natural waterways appears to be different from the one in suburban waterways. Phytophthora pseudosyringae was recovered and identified in five streams in North Carolina; this was the first report of this species outside of California and Oregon in the USA. Identification of other "unknown" species is in progress. These detection methods were used to survey and monitor for P. ramorum, which causes sudden oak death on trees in the forests of coastal California and southwest Oregon and ramorum blight on many understory trees and shrubs in the forest as well as numerous nursery crops. P. ramorum was detected successfully in soilless container mix around symptomless container-grown plants, in soil at a residential
landscape and in a nursery, in streams in California, and in water at a nursery. We also demonstrated species of Phytophthora produce a number of fatty acids (FA) that can used to identify isolates: 15 FA were common to the species evaluated, but 5 FA typically comprised 75% of the total profile. Consequently, fatty acid methyl ester (FAME) analysis may be useful to identify species of Phytophthora; if so, it may provide another method for rapid identification of species of Phytophthora for routine diagnoses. The importance of P. nicotianae and P. palmivora as pathogens on herbaceous ornamental crops was studied. Diagnostic methods to separate these two morphologically similar pathogens were determined. P. nicotianae has a much broader host range than P. palmivora, attacking plants in numerous different families, and 13% of the isolates of P. nicotianae were resistant to the fungicide mefenoxam. New hosts of P. nicotianae were determined, and plants tolerant to this pathogen were
identified. Also, we found that isolates of this pathogen have developed host specialization on ornamental crops.
Impacts
Our goal is to identify the major sources of primary inoculum for Phytophthora diseases in ornamental crop nurseries. Based on this information, effective management strategies can be developed to prevent or limit primary infection, which should reduce the amount fungicides needed to manage these diseases and increase nursery profitability. Over the 5-year duration of this project, we evaluated methods for detecting Phytophthora spp. in nursery, natural, and suburban waterways. Filtering water sub-samples through membrane filters was more effective and efficient than baiting with wounded and non-wounded rhododendron leaves. Both methods were shown to be effective for detecting Phytophthora ramorum, which causes sudden oak death and ramorum blight. Our results have documented the wide-spread occurrence and diversity of Phytophthora spp. in waterways in natural, urban, and nursery ecosystems and have demonstrated the utility of filtration and baiting for detection of
these notorious plant pathogens--including P. ramorum. Our methods have been adopted for use by colleagues around the country and in national survey programs. In addition, we have expanded our knowledge on the importance of Phytophthora nicotianae as a pathogen of floriculture crops. The number of plant species attacked by this pathogen continues to increase. We also found out that P. nicotianae has developed resistance to one of the primary fungicides (i.e., mefenoxam) used to protect ornamental crops.
Publications
- Hong, C. X., Richardson, P. A., Kong, P., Jeffers, S. N., and Oak, S. W. 2006. Phytophthora tropicalis isolated from diseased leaves of Pieris japonica and Rhododendron catawbiense and found in irrigation water and soil in Virginia. Plant Disease 90:525.
- Jeffers, S. N. 2006. In search of Phytophthora ramorum: A perspective from the southeastern USA. (Abstr.) Phytopathology 96:S146.
- Hwang, J., Oak, S. W., and Jeffers, S. N. 2006. Monitoring occurrence and distribution of Phytophthora species in forest streams in western North Carolina. (Abstr.) Phytopathology 96:S52.
- Rebollar-Alviter, A., Madden, L. V., Jeffers, S. N., and Ellis, M. A. 2006. Sensitivity of Phytophthora cactorum isolates from strawberry to pyraclostrobin and azoxystrobin. (Abstr.) Phytopathology 96:S96.
- Wamishe, Y. A., Jeffers, S. N., and Hwang, J. 2006. Phytophthora ramorum and other species of Phytophthora detected in field soil and water at retail nurseries in the southeastern USA. (Abstr.) Phytopathology 96:S1120.
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Progress 01/01/05 to 12/31/05
Outputs
The overall objective of this project is to identify sources of primary inoculum for Phytophthora diseases that affect ornamental crops in nurseries. The project was initiated in July 2001 and terminates in 2006. This past year, we focused primarily on one project: detection and occurrence of Phytophthora spp. in waterways in natural forest ecosystems. This research effort was conducted primarily by Dr. Jaesoon Hwang, a Postdoctoral Researcher, and was done in collaboration with the Mr. Steve Oak and the USDA Forest Service-Forest Health Protection. We compared methods for detecting Phytophthora spp. in five forest streams monthly from April through December 2005. The streams came from three watersheds located in the Pisgah National Forest in western North Carolina. Three detection methods were compared: filtration of 1-liter water samples using three membrane filters with different pore sizes (5-um Durapore filters and 3- and 1-um Nuclepore filters), baiting with
wounded rhododendron leaves that were floated in streams for 3 days, and baiting with non-wounded rhododendron leaves that were floated in streams for 1-2 weeks. All leaves came from native Rhododendron maximum plants. Isolates of Phytophthora spp. were recovered on PARPH-V8 selective medium and were identified based on standard morphological characteristics as well as DNA fingerprints using the PhytID database (http://www.phytid.org). In the first seven months, April through October, 379 isolates were collected, including five known species and 10 genetically distinct groups or species. Phytophthora spp. were recovered from all five streams in each of the seven months, but species recovery varied by month, site, and detection method. Overall, filtration was most effective at detecting the greatest diversity of species and most efficient at recovery in general. Filtration detected 13 species whereas wounded and non-wounded leaves each detected eight species; however, no method
detected all 15 species. All three filters were effective, but more isolates and species were recovered on 3-um Nuclepore filters than on the other two types. More species (11) were recovered in July than in any other month. Data collected in November and December still are being analyzed. Filtration and wounded rhododendron leaves were compared for efficacy at detecting P. ramorum (causal agent of sudden oak death and ramorum blight) in three naturally-infested streams in Santa Cruz Co. California in May 2005. Both methods successfully recovered this pathogen in all three streams.
Impacts
Our goal is to identify the major sources of primary inoculum for Phytophthora diseases in ornamental crop nurseries. Based on this information, effective management strategies can be developed to prevent or limit primary infection, which should reduce the amount fungicides needed to manage these diseases and increase nursery profitability. In 2005, we evaluated methods for detecting Phytophthora spp. in forest streams. Filtering water sub-samples through membrane filters was more effective and efficient than baiting with wounded and non-wounded rhododendron leaves. Both methods were shown to be effective for detecting Phytophthora ramorum, which causes sudden oak death and ramorum blight. These results document the wide-spread occurrence and diversity of Phytophthora spp. in waterways in natural ecosystems and demonstrate the utility of filtration and baiting for detection of these notorious plant pathogens--including P. ramorum.
Publications
- Zwart, D. C., Jeffers, S. N., Waldrop, T. A., and Bridges, W. C. 2005. Effects of fuel reduction treatments on detection of Phytophthora spp. in soil in an Appalachian Mountain forest. (Abstr.) Phytopathology 95:S119.
- Jeffers, S. N. 2005. Phytophthora foliage blight (and root rot). Pages 5-6 in: Hosta Diseases and Pests. Sustainable Urban Landscapes 14. D. Mueller et al. Iowa State University Extension, Ames, IA.
- Hwang, J., and Jeffers, S. N. 2005. Detection and identification of Phytophthora species in streams in the Southern Appalachian Mountains. (Abstr.) Phytopathology 95:S46.
- Jeffers, S. N. 2005. Recovery of Phytophthora ramorum from soilless mixes around container-grown ornamental plants. (Abstr.) Phytopathology 95:S48.
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Progress 01/01/04 to 12/31/04
Outputs
The overall objective of this project is to identify sources of primary inoculum for Phytophthora diseases that affect ornamental crops in nurseries. The project was initiated in July 2001. This past year, we have focused on completing a study on the occurrence and distribution of Phytophthora spp. in a natural ecosystem in the southern Appalachian Mountains and the potential pathogenicity of these isolates. This was a portion of the M.S. thesis project of Drew C. Zwart, who graduated in December 2004. The study site for this project was the Green River Game Land in Polk Co., North Carolina, which is located in the southwestern region of the state on the eastern slope of the Blue Ridge escarpment. The study site was established as part of the national Fire and Fire Surrogate Study. Soil samples were collected to a depth of 20 cm with a 2-cm-diameter soil sampling tool, and 20 individual soil cores were combined to produce a representative composite sample from a given
area. A baiting bioassay was used to detect Phytophthora spp. in composite soil samples. Both fresh and air-dried subsamples were baited with camellia leaf pieces and hemlock needles. Two species of Phytophthora were recovered in this study: P. cinnamomi and P. heveae. Isolates of P. cinnamomi had typical morphological and physiological characteristics of this species, and all isolates were A2 mating type. Isolates of P. heveae were homothallic and produced abundant oospores on isolation plates. Identification of P. heveae isolates was confirmed using ribosomal RNA fingerprinting of the ITS1 and ITS2 sequences. P. cinnamomi was recovered in 33% of all soil samples and occurred uniformly throughout the study site. P. heveae, on the other hand, was recovered in only 2.5% of all soil samples so occurred infrequently in the study site. However, both species appear to be well-established in this Appalachian Mountain location. P. cinnamomi was recovered best in fresh soil samples using
camellia leaf-piece baits that were removed at either 24 or 72 hours. However, P. heveae was recovered best in air-dried soil when camellia leaf-piece baits were removed after 72 hours. Representative isolates of each species were tested for potential pathogenicity. Agar plugs from isolates of P. heveae were placed on wounded and non-wounded leaves of native rhododendron, hybrid rhododendron, and mountain laurel. Only wounded leaves consistently became infected; therefore, isolates of P. heveae from mountain soils were only weakly virulent. Isolates of P. cinnamomi were used to infest soilless container mix into which healthy plants were transplanted. These isolates caused root rot and mortality on mountain laurel and white pine plants, which confirmed that isolates of P. cinnamomi from mountain soils are virulent and capable of causing disease.
Impacts
Our goal is to identify the major sources of primary inoculum for Phytophthora diseases in ornamental crop nurseries. Based on this information, effective management strategies can be developed to prevent or limit primary infection, which should reduce the amount fungicides needed to manage these diseases and increase nursery profitability. During the past year, isolates of P. cinnamomi and P. heveae were found to be well-established in a forest ecosystem located in the southern Appalachian Mountains of North Carolina. Representative isolates were tested for potential pathogenicity. Isolates of P. heveae were only weakly virulent on leaves of three test plants, but isolates of P. cinnamomi were virulent and caused mortality on both white pine and mountain laurel plants. This demonstrates that natural populations of P. cinnamomi present in the southern Appalachian mountain forests are potential pathogens of ornamental crops.
Publications
- Harrison, L. A., Riley, M. B., and Jeffers, S. N. 2004. Fatty acid methyl ester (FAME) profile analysis of Phytophthora cactorum. (Abstr.) Phytopathology 94:S39.
- Huang, H. H., Jeffers, S. N., Layne, D. R., and Schnabel, G. 2004. AFLP analysis of Phytophthora cactorum isolates from strawberry and other hosts: Implications for identifying the primary source of inoculum. Plant Disease 88:714-720.
- Jeffers, S. N., Schnabel, G., Smith, J. P. 2004. First report of resistance to mefenoxam in Phytophthora cactorum in the United States and elsewhere. Plant Disease 88:576.
- Zwart D. C., Jeffers, S. N., and Waldrop, T. A. 2004. Incidence of Phytophthora spp. in a hardwood forest site in the southern Appalachian Mountains. (Abstr.) Phytopathology 94:S118.
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Progress 01/01/03 to 12/31/03
Outputs
The overall objective of this project is to identify sources of primary inoculum for Phytophthora diseases that affect ornamental crops in nurseries. The project was initiated in July 2001. This past year, we have focused on completing a study on identification, pathogenicity, and virulence of isolates of Phytophthora nicotianae from ornamental plants in South Carolina. This was the M.S. thesis project of Jessica A. Eisenmann, who graduated in December 2003. Both P. nicotianae and P. palmivora are known to attack numerous herbaceous and certain woody ornamental crops; however, these two species are morphologically similar and can be difficult to differentiate. Therefore, the morphological, physiological, and molecular characters of 145 isolates of these two species that have been recovered from diseased ornamental plants were evaluated and compared. Random fragment length polymorphism (RFLP) patterns of the internal transcribed spacer (ITS) regions I and II separated
the isolates into two groups: 114 isolates were P. nicotianae and 31 isolates were P. palmivora. Isolates of P. nicotianae were associated with a broad range of plants (e.g., Catharanthus, Buxus, Verbena, Vinca, Daphne, Hemerocallis, Gardenia, Petunia)and were both A1 and A2 mating types (35% and 65%, respectively); they produced distinct colony patterns on agar media, papillate non-caducous sporangia (which often were distorted or had multiple papillae) on loose sporangiophores, and mycelia that grew at 35C but not at 5C. In comparison, isolates of P. palmivora were associated primarily with two crops--Hedera spp. and Fatsia japonica--and were predominantly the A1 mating type (87%); they produced papillate caducous sporangia on tight sporangiophores, and mycelia that did not grow at 5 and 35C. The pathogenicity and virulence of P. nicotianae to 15 herbaceous ornamental plants not previously documented as hosts were investigated. Each plant was planted into soilless container mix
infested with each of four different isolates--one from the test plant (host isolate) and three from other plants (non-host isolates)--and grown for 80 days in a greenhouse with periodic flooding. Root and shoot weights were measured, symptom severity over time was monitored, and attempts were made to isolate the pathogen from roots. After analyses were complete, plants were rated as susceptible, resistant, or tolerant. Eight plants were susceptible: Aruncus, Ceratostigma, Kniphofia, Penstemon, Phlox, Rodgersia, Vinca, and Viola; four plants were resistant: Delosperma, Gaura, Hemerocallis 'Anzac Gold', and Sedum; and three plants were tolerant: Hemerocallis 'Stella d'Oro', Heuchera, and Lamium. Tolerant isolates pose a significant risk to the industry because they harbor the pathogen but show no symptoms. Isolates of P. nicotianae varied in virulence with the host isolate usually more virulent than the non-host isolates. Therefore, isolates of P. nicotianae have developed a certain
degree of host specialization, particularly those from Petunia, but were not host specific.
Impacts
Our goal is to identify the major sources of primary inoculum for Phytophthora diseases in ornamental crop nurseries. Based on this information, effective management strategies can be developed to prevent or limit primary infection, which should reduce the amount fungicides needed to manage these diseases and increase nursery profitability. During the past year, new hosts for Phytophthora nicotianae and P. palmivora were identified, and diagnostic profiles were prepared so these two pathogens can be easily differentiated. In addition, plant species tolerant to P. nicotianae also were identified; this is particularly noteworthy because these plants allow the pathogen to survive and produce inoculum but do not appear diseased.
Publications
- Eisenmann, J. A. 2003. Identification, pathogenicity, and virulence of isolates of Phytophthora nicotianae from ornamental plants. M.S. Thesis. Clemson University, Clemson, SC. 131 pp.
- Eisenmann, J. A., Schnabel, G., and Jeffers, S. N. 2003. Characterization of two papillate species of Phytophthora from ornamental plants. (Abstr.) Phytopathology 93:S23.
- Eisenmann, J. A., and Jeffers, S. N. 2003. Phytophthora nicotianae: A pathogen of herbaceous ornamental plants. Proceedings of the Southern Nursery Association Research Conference 48:238-242.
- Kong, P., Hong, C., Jeffers, S. N., and Richardson, P. A. 2003. A species-specific polymerase chain reaction assay for rapid detection of Phytophthora nicotianae in irrigation water. Phytopathology 93:822-831.
- Kuhajek, J. M., Jeffers, S., N., Slattery, M., and Wedge, D. E. 2003. A rapid microbioassay for discovery of novel fungicides for Phytophthora spp. Phytopathology 93:46-53.
- Zwart, D. C., and Jeffers, S. N. 2003. Incidence of Phytophthora spp. in a hardwood forest site in the southern Appalachian Mountains. Proceedings of the Southwide Forest Disease Workshop. 10-12 June 2003, Asheville, NC.
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Progress 01/01/02 to 12/31/02
Outputs
The objective of this project is to identify sources of primary inoculum for Phytophthora diseases that affect ornamental crops in nurseries. The project was initiated in July 2001. This past year, an M.S. student who worked on this project graduated and a Ph.D. student, whose research supported this project, also graduated. In addition, we validated the filtration procedure that was developed in the M.S. research project; this assay was designed to recover propagules of Phytophthora spp. from water samples. Validation was necessary before we began rigorous sampling of water sources in nurseries, landscapes, and natural ecosystems to determine the occurrence and distribution of species of Phytophthora. Briefly in this assay, replicate subsamples of water are passed through 47-mm-diameter filters under vacuum to collect fungus propagules, the filter is inverted on Phytophthora-selective agar medium in petri dishes, dishes are incubated at 20C for several days, and
developing colonies are counted and subcultured. We have routinely found Phytophthora spp. in waterways in nurseries, landscapes, natural ecosystems-which has provided ample sources of naturally infested water for our experiments. We tested several pore sizes for Nuclepore polycarbonate membrane filters for efficacy in recovering propagules of Phytophthora spp. Filters with pores that were 1 and 3 micrometers were more effective than those with pores that were 5 and 8 micrometers . The larger pores, because of pore overlap, apparently allowed zoospores to pass through the filter, often damaging to the zoospores. We then demonstrated in multiple experiments that Nuclepore filters with 3-micrometers pores, Durapore filters with 5-micrometers pores, and Millipore filters with 5-micrometers pores were equally effective at collecting propagules from naturally-infested water. However, fewer propagules were recovered on Nuclepore filters with 1-micrometer pores. The length of time filters
were left on agar medium affected number of colonies that developed; more colonies developed if filters were left for 48 and 72 hr than if they were left only 24 hr. Therefore, filters should remain in contact with the medium for at least 48 hr before being removed. The number of propagules in a water sample decreased as storage time at 15C increased. Consequently, water samples should be assayed as soon after collection as possible to optimize recovery of propagules. There was no significant effect of vacuum duration (0 vs. 1 min) or filter funnel type (clamp vs. magnetic) on propagule recovery. We have begun to identify the species of Phytophthora that have been recovered from waterways in South Carolina and North Carolina. These include: P. nicotianae, P. cinnamomi, P. citricola, P. cryptogea-like species, and some unusual isolates that do not readily match known described species. Representative isolates have been stored in our permanent culture collection. We worked with a
colleague in the department of Computer Science to develop a Microsoft Access database for our Phytophthora spp. Culture Collection. We are in the process of entering isolate information into the database and testing its utility.
Impacts
Our goal is to identify the major sources of primary inoculum for Phytophthora diseases in ornamental crop nurseries. Based on this information effective management strategies can be developed to prevent or limit primary infection, which should reduce the amount fungicides needed to manage these diseases and increase nursery profitability. If an effective FAME database can be developed, diagnostic clinics and labs around the country will have access to a rapid procedure for identification of Phytophthora spp. This procedure will utilize existing MIDI hardware and software.
Publications
- Duan, C.-h., M. B. Riley, and Jeffers, S. N. 2002. Fatty acid production by species of Phytophthora: Potential for identification. (Abstr.) Phytopathology 92:S21.
- McCracken, J. L. 2002. Recovery and pathogenic potential of Phytophthora spp. from recirculated irrigation water in South Carolina nurseries. M.S. Thesis. Clemson University, Clemson, SC. 80 pp.
- Duan, Chung-hang. 2002. Variations in fatty acid methyl ester profiles and amplified fragment length polymorphisms among and within species of Phytophthora from ornamental plants. Ph.D. Thesis. Clemson University, Clemson, SC. 153 pp.
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Progress 07/01/01 to 12/31/01
Outputs
The overall objective of this project is to identify sources of primary inoculum for Phytophthora diseases that affect ornamental crops in nurseries. The project was initiated in July 2001 and is just getting underway. We have begun to sample water sources in nurseries, landscapes, and natural ecosystems for species of Phytophthora. We are using a filtration procedure that was developed in our laboratory to assay water samples. In initial samples, Phytophthora spp. have been recovered from irrigation water at four ornamental crop nurseries, from streams in two landscape settings, and from six natural waterways removed from commercial agriculture. Usually, multiple species were recovered from a specific water source. Overall, a variety of species of Phytophthora have been recovered and tentatively identified. Currently, we are in the process of confirming identifications of these isolates using traditional morphological characteristics. Isolates from water have been
added to our permanent culture collection as have isolates from colleagues around the country and isolates from the Clemson University Plant Problem Clinic. We are in the process of developing a multi-faceted database for storing and retrieving information on isolates in the collection. We have established a standard culture procedure for growing isolates of Phytophthora spp. for fatty acid methyl ester (FAME) analysis as a rapid means of identification. Using five isolates of each of five species, isolates from the same species clustered when a dendrogram was constructed using MIDI software. Therefore, it appears that FAME analysis has good potential as a means for identifying isolates of Phytophthora spp.
Impacts
Our goal is to identify the major sources of primary inoculum for Phytophthora diseases in ornamental crop nurseries. Based on this information effective management strategies can be developed to prevent or limit primary infection, which should reduce the amount fungicides needed to manage these diseases and increase nursery profitability. If an effective FAME database can be developed, diagnostic clinics and labs around the country will have access to a rapid procedure for identification of Phytophthora spp. This procedure will utilize existing MIDI hardware and software.
Publications
- No publications reported this period
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