2006 Annual Progress Report

The goal of our project during the first year (Aug 2005-Aug 2006) was to develop an efficient, sensitive, and reliable diagnostic assay to detect foliar nematodes (Aphelenchoides spp.) in host plant tissues, and then in the following year (2006-2007) to utilize the assay as a research tool to study the life cycle of this pathogen in a greenhouse/nursery setting, and to evaluate the effectiveness of potential cultural, chemical, and biological methods of control. Early detection of foliar nematodes is a key component in managing this pest through exclusion of the pathogen from growing areas; however, the identification and development of new methods of control will be critical in situations where eradication is required due to prior establishment of the pathogen. Our overall goal during the second year of this research is to determine why foliar nematodes, once introduced into a growing facility, continue to persist over multiple growing seasons, and to develop or identify effective management strategies for foliar nematode control. A second year of funding will provide us with the means to utilize our diagnostic assay to optimize the timing and implementation of control measures to when they are most likely to be effective based on the biology of the nematode, as well as to identify and quantify the efficacy of experimental methods of control.

Over 100 naturally infected plant species were collected over the past 18 months from commercial greenhouses and nurseries throughout North Carolina. These host plants represent 58 different, and diverse, plant families ranging from the polypodiaceae (terrestrial ferns) to the verbenaceae (flowering herbs and woody shrubs). Fifteen of the plants are genera that had not previously been reported as hosts, and another 37 plant samples represent new host species. Interestingly, each of these plant samples was infested with A. fragariae. This is in sharp contrast to reports from Europe where the primary species reported on ornamentals is A. ritzembosi. The traditional extraction method to detect foliar nematodes in plant tissue relies on the presence of symptomatic tissue, as well as the ability of the nematode to emerge from infected tissue when submerged in water. The success of detection is, therefore, influenced by both the selection of plant tissue to be sampled, as well as the developmental stage and population of the nematodes. Identification to species is further complicated by subtle differences in morphology, requiring a trained taxonomist to differentiate species. The overall objective of this project was to develop a highly sensitive diagnostic assay, as well as a more accurate sampling method, for early detection of A. fragariae in host plant tissue. PCR primers were developed using the unique ITS1 region of A. fragariae.

Our goals during the first year of the project were to 1) verify the utility and integrity of our molecular markers to detect foliar nematodes in plant tissue, 2) test our detection assay against other pests (whiteflies, aphids, mites, mealybugs) to confirm that we are not seeing cross-reactivity, 3) perform sensitivity assays to determine minimum and maximum number of nematodes that can be detected, and 4) test our detection assay against a background of plant tissues from a range of plant species to insure that the primers do not target DNA sequences in these plant genomes, and to insure that the plant tissues do not contain inhibitors that could interfere with the assay.

 

We have developed a highly specific, sensitive, and reproducible diagnostic assay. Through collaboration with Dr. Sergei Subbotin, a professor of nematology at the University of Ghent, we were able to test our primers against DNA of A. besseyi and have determined that our assay is specific to A. fragariae. Based on the DNA sequence information kindly provided by Dr. Subbotin, we are continuing to work on developing primers specific to A. besseyi, and A. ritzembosi, however, our ability to work with these nematode species is limited since we have not detected them in any of the plant materials collected to date (all plant samples are assayed by both traditional water extraction and PCR; and any nematodes found are identified by microscopy).

 

Are assay has shown a high degree of selectivity. We have not detected any cross-reactivity with whiteflies, aphids or mites. Our assay is capable of detecting a single nematode in a background of host plant tissue. We a have not yet surpassed the maximum number of nematodes that can be detected in a single reaction., but have determined that the number is in excess of 1,000 nematodes.

The source of foliar nematode problems in ornamental crops is often traced to infested cuttings, liners, or rootstocks. Early detection of foliar nematodes can be problematic because infested plants may not initially show symptoms. As a result, infested material is often unknowingly propagated or shipped to other locations. Keeping this in mind, the next step in the development of our detection assay and sampling scheme was to optimize and test the effectiveness (and practicality) of the assay in a greenhouse/nursery production setting in collaboration with two perennial nurseries in North Carolina.

The traditional water extraction method to detect nematodes was compared to our PCR-based diagnostic assay to detect the presence of foliar nematodes in blocks of plants grown in a commercial greenhouse. A block of 44 ferns, and a second block of 73 anenome plants were sampled. Plants were emerging from dormancy, and just beginning to send up new growth. Several plants exhibiting symptoms of foliar nematode damage were present, but the grower was unaware that the plants were infested. Three leaves were removed from each plant and six 6 mm leaf disks were removed from each leaf. Symptomatic leaves were collected when present. Water extraction was used to assay one half of the leaf disks and the remaining three disks were assayed by

PCR. Three ferns tested positive for foliar nematodes by both detection methods, with an additional 24 ferns testing positive only by the PCR method. The remaining 17 ferns were negative by both methods. Three of the anemone plants were positive by both methods, while 48 of the plants tested positive only by PCR. The remaining 22 anenomes were negative by both methods. The traditional water extraction method failed to detect nematodes in 48% and 62% of the fern and anenome samples, respectively. In addition, 12 of the ferns has no visible symptoms of foliar nematode damage; however, nematodes were detected in two of these plants using water extraction and in all 12 plants using our diagnostic assay.

We sampled the fern and anenome plants again six weeks after the first sampling and obtained similar results. We are continuing to sample additional blocks of plants at a second location with a known history of foliar nematodes, and will assay these plants throughout the season comparing both the traditional water extraction method of detection and our assay. A nursery block containing naturally infested verbena plants was sampled to determine the foliar distribution of nematodes.  The majority of nematodes (65%) were found on the foliage near the base of the plant at or below the second internode. The youngest, uppermost foliage of the plants contained 7% of the total number of foliar nematodes detected. Foliar nematodes were found in both symptomatic and asymptomatic leaves. Our final goal is to apply this information in the development of a sampling scheme for screening plant populations for the presence of foliar nematodes (based on the plant tissues most likely to harbor the nematodes, as well as how much tissue must be sampled to reliably detect the nematodes if present). We expect to have a sampling scheme developed near the end of the first year. Our diagnostic assay will provide a more accurate, high throughput method for screening vegetatively propagated plant materials helping to eliminate foliar nematodes at their source. Early detection of this pest, through screening of incoming or onsite plant materials will help growers maintain nematode-free stock plants as well as nematode-free growing facilities. Our assay has immediate use for screening stock plants for the presence of foliar nematodes.

During the second year of the study, our diagnostic assay will be used as a tool to study the life cycle and epidemiology of the nematode in a nursery/greenhouse setting. Because our assay detects genomic DNA, we have the ability to detect eggs as well as the juvenile and adult stages of the nematode. This is a key point, as the traditional extraction method of detection requires that the nematode be motile in order to swim out of the plant tissue where it can be viewed microscopically (in which case, eggs or the first stage juveniles would not be detected by traditional methods). We will be investigating the effects of environmental parameters such as temperature, humidity, and leaf wetness on the nematode population. Commonly accepted knowledge is that the population of foliar nematodes peaks during the summer months when temperatures are warmest. Preliminary work in our nursery trials has suggested that foliar nematode symptoms are most pronounced

during midsummer in North Carolina, but the nematode population actually plummets during this same period which could have important implications as to when to implement control measures. We will be sampling and monitoring nematode populations throughout the year in a commercial greenhouse in an effort to determine where the nematodes are over-wintering, and what is the most probable means of survival from crop-to-crop.

During the second year we will also be evaluating several methods of foliar nematode control, using the detection assay to evaluate the efficacy of each method. In a related research project, we are developing chitosan films in collaboration with a polymer chemist at NC State University for use as a potential method of foliar nematode control (based on the ability of chitosan to form an anti-microbial barrier on the leaf surface when applied as an aqueous solution). In addition, we have received several experimental as well as commercial chemical and biological compounds to test for efficacy against foliar nematodes.

The diagnostic assay will provide an invaluable 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. The ability to perform quick and accurate nematode identifications will be beneficial to regulatory agencies such as the USDA Animal and Plant Health Inspection Service in screening and releasing shipments of domestic and foreign plant materials.

 

Successful implementation of this research (routine testing of vegetative stock plant material) will be dependent upon highlighting the importance of foliar nematodes as a serious and emerging pest within the ornamental and landscape industries, and the economic ramifications that can result when foliar nematodes are introduced into a growing facility. Each year, Dr. Warfield presents educational talks and programs at regional and national trade shows and conferences. She presented a talk on foliar nematodes at the SAF Pest Management Conference, at the Southeast Greenhouse Growers Conference, and the Ohio Short Course in 2005. Our research results will be reported to the scientific community in August 2006 at the annual American Phytopathological Society meeting in Quebec City. The diagnostic technology will be made available to regional diagnostic laboratories within the National Plant Diagnostic Network.