Foliar nematode population dynamics: Changes in the population of A. fragariae in the foliage of naturally-infected, container-grown plants of L. camara ‘Miss Huff’ (a woody perennial hardy in USDA zones 8-11) was followed at a commercial ornamental nursery in Pender County, North Carolina, over three growing seasons. In each growing season, a new set of 30 naturally-infected lantana plants were sampled from May through October. Sampled plants had been naturally infected with A. fragariae during the previous growing season and overwintered in a Quonset-type polyhouse. In April of each year, plants from the polyhouse were transplanted into larger 13.2 liter containers of pine bark and placed 60-cm apart on a nursery pad. The plants were sprinkler irrigated during the growing season for 5-10 min three times each day. Plants were pruned to half their size once per month after sampling to shape the plants and mimic typical nursery practices. Air temperature, relative humidity, and rainfall data were recorded at the nursery (GroWeather, Davis Instruments, Hayward, California) so that correlations between environmental variables and nematode populations could be made. Environmental variables were measured every 2 hr for a total of twelve readings per day. When occasional data skips occurred, missing data was supplemented with weather data recorded at the North Carolina State University, Horticultural Crops Research Station located 50 km away in Castle Hayne, NC. Daily high temperatures ranged from 26.9°C to 32.6°C in 2006, 24.5°C to 36.5°C in 2007, and 20.4°C to 32.4°C in 2008. Daily low temperatures ranged from 17.2°C to 23.1°C in 2006, 13.4°C to 26.7°C in 2007, and 14.6°C to 23.3°C in 2008. Relative humidity levels ranged from 71-81% in 2007 to 69-84% in 2008.
Plant sampling: Only symptomatic leaves were collected during the 2006 growing season, from 6 June to 25 September. Each of the 30 plants was sampled every 14 d by removing a total of five symptomatic leaves from the bottom and middle tier of each plant. During the 2007 and 2008 growing seasons, from 16 May to 17 October, and 28 May to 27 August, respectively, symptomatic, asymptomatic, and abscised leaves were collected from each of the 30 plants. Three symptomatic and three adjacent asymptomatic leaves were removed from both the middle and bottom tiers of each plant canopy. If symptomatic leaves were not present, asymptomatic leaves were arbitrarily collected from at least two branches of each plant in accordance with the sampling pattern. Two to four abscised leaves with apparent symptoms were collected at each sampling date from the surface of the container substrate. The substrate surface of each container was cleared of fallen leaf debris after sampling so that all abscised leaf samples collected at the next sample date would have a maximum time of 14 d on the substrate surface. A total of 150 symptomatic leaves were collected at each sampling date in 2006. In 2007 and 2008, 180 symptomatic, 180 asymptomatic and 60-120 abscised leaves were collected at each sampling date. Leaf samples from each plant were placed by sample type into individual 50-ml centrifuge tubes and capped prior to transport inside an insulated cooler to the lab where samples were refrigerated at 4°C prior to assay within 24 to 48 hr.
Beginning in 2007, each plant was visually assessed twice per month for characteristic symptoms of foliar nematode damage. An overall disease severity rating, based on the percentage of symptomatic leaves, was assigned to each plant at each assessment date using the Horsfall-Barratt Scale (Horsfall and Barratt, 1945
). Symptomatic leaf samples collected in 2007 and 2008 were photographed, and the percent leaf area of diseased tissue was calculated with ASSESS Image Analysis Software (APS Press, St. Paul, Minnesota). For those leaf samples in which the presence of foliar nematodes was confirmed, the percent area of diseased tissue was averaged across all infected leaves to obtain an average percentage of symptomatic leaf area at each sampling date. Correlation analysis of the percentage of symptomatic leaf area with the nematode population at each sampling date was performed using the Spearman rank correlation test (SAS, SAS Institute, Cary, NC).
All collected leaves of one sample type (symptomatic, asymptomatic or abscised) from each plant were pooled, weighed, cut into small pieces and incubated in 10-15 ml of deionized water in a 50-ml centrifuge tube at room temperature to stimulate foliar nematode emergence (Esser and Riherd, 1981
). After 48 hr, the leaf pieces and extraction water were passed through a set of nested sieves with a large mesh sieve to first remove leaf debris, followed by a 500-mesh (25-μm openings) sieve to collect any nematodes that had emerged. Nematodes were washed from the 500-mesh sieve in 5-10 ml of deionized water. The nematode samples then were refrigerated at 4°C until counted and quantified in a 3- × 7.5-cm counting dish under an inverted microscope at x40. The total number of A. fragariae
in the dish was corrected for the total fresh weight of leaf tissue in each respective sample from each plant to obtain a nematode count per gram fresh weight per plant at each sampling date. Since leaf samples were always less than 1 g, counts were extrapolated. For comparison purposes, one gram of chopped, fresh, leaf tissue was equivalent to approximately 10 fully expanded, intact leaves. While both saprophytic and parasitic nematodes were recovered in leaf samples, the only pathogenic nematode species found during each growing season and overwintering period was A. fragariae
based on morphological features and morphometrics (Sanwal, 1961
; Siddiqi, 1975
; and Hunt 1993
Correlation analysis: The average nematode count per gram fresh weight at each sampling date, for each leaf sample type, was tested for correlation with the environmental data using the Spearman rank correlation test. This correlation test was used because it is a robust, non-parametric statistical method of analysis that makes no prior assumptions about the data. Environmental data including daily high air temperature, daily low air temperature, daily average air temperature, relative humidity, and rainfall collected 3, 7, 14 and 20 d prior to each sampling date was used for analysis.
Overwintering study: Each growing season, a companion group of 30 newly-rooted lantana ‘Miss Huff’ plants were grown in trade gallon (2.8 liter) containers of pine bark. These plants were placed on the nursery pad around the perimeter of the block of larger, infected plants in 13.2 liter containers in the current season's population trial. The smaller plants became naturally infected with A. fragariae by splash dispersal from the larger plants. In October of 2006 and 2007 the smaller plants were moved into the polyhouse at the Pender County nursery site for overwintering. Plants were tightly spaced, allowing adjacent plant canopies to touch per standard industry practice. The polyhouse was equipped with supplemental heat to maintain minimum temperatures just above freezing to prevent plant damage but daily fluctuations on sunny days could be quite large, requiring venting. Plants were sprinkler irrigated as needed. The top of each plant canopy was pruned once during winter 2006-07. Plants were not pruned in 2007-08 to prevent removal of potentially nematode-infected plant tissue. During 2007-08, leaf debris was removed from the surface of the container substrate after each sampling.
A maximum of six attached, symptomatic leaves (three from the lower canopy and three from the middle plant canopy) on each plant and a maximum of four abscised leaves from the substrate surface of each container were sampled at 6-wk intervals during overwintering. When plants were first moved to the polyhouse, infected leaves became senescent and defoliated, so six symptomatic leaves were not always available per plant at the first sampling dates in the polyhouse. Later as plant growth was stimulated during warm winter periods more leaves became symptomatic. Attached, asymptomatic leaves directly adjacent to symptomatic leaves also were collected during winter 2007-08. Foliar nematodes were detected by water extraction of collected leaf tissue and counts extrapolated to nematodes per gram as previously described. Environmental variables including daily minimum, maximum and average air temperature, and relative humidity collected 3, 7, 14 and 20 d prior to each sampling date were used in Spearman correlation tests with nematode counts for each leaf sample type. Daily high temperatures in the polyhouse ranged from 5.5°C in mid February 2006 to 29.6°C in late March 2007 and 26.2°C in mid December to 2.1°C in early January in 2007-08. Daily low temperatures ranged from -4.7°C in mid February to 17.6°C in late March during the 2006-07 period and 16.9°C in late December to -9.7°C in early January in 2007-08. The average daily temperatures ranged from 0.9°C to 23.0°C in 2006-07 and –1.9°C to 20.7°C in 2007-08. Relative humidity ranged from 42% to 98% in 2006-07 and from 43% to 98% in 2007-08.
The pine bark substrate in each container was sampled for foliar nematodes in winter 2006-07. A 2-cm diameter soil sampling tube was used to remove a core of substrate from each container. The resulting hole was re-filled with fresh pine bark. Substrate samples were placed in Baermann funnels in a modified Seinhorst mist apparatus (Barker et al., 1986
) for 24 hr. The resulting water solution from each sample was drained into a test tube then examined microscopically in a counting dish for the presence of Aphelenchoides
. Foliar nematodes were not detected in any substrate sample in 2006-07; therefore, container substrate was not sampled in 2007-08.
Disease gradient study: Plant-to-plant dispersal of foliar nematodes was evaluated in a simulated nursery setting at the Horticultural Field Lab located at North Carolina State University in Raleigh, North Carolina from 3 August to 12 October 2007, and 20 July to 7 October 2008. Healthy, asymptomatic lantana ‘Miss Huff’ plants grown in trade gallon containers of pine bark were placed on a nursery pad into 15 blocks, each composed of four lantana plants spaced equidistantly around a Salvia farinaeae (2007) or a lantana (2008) plant naturally infected with A. fragariae. For each of the blocks, one of three container spacings was used to separate the canopy of the nematode-infected plant from the canopies of the four, healthy lantana plants: 0-cm (touching), 30-cm, or 100-cm apart. Spacing distance was replicated five times, for a total of 20 lantana plants placed at each spacing distance in each year. Blocks were separated by a minimum of 100 cm to avoid inter-plot interference. The average canopy size of the lantana plants at the start of each trial was 30 × 38 cm. As the plants grew, they were re-spaced to maintain the original distance between plant canopies. Plants were watered twice daily (2.8-cm water/day) by sprinkler irrigation except on days when at least 1.2 cm of rain occurred. Natural rainfall during the trial periods totaled 13.1 and 31.4 cm in 2007 and 2008, respectively. The ambient air temperature during the trial period in each year ranged from a minimum of 12°C to a maximum of 40°C.
The lantana plants were evaluated visually once per week for characteristic symptoms of foliar nematode infection. Symptomatic leaves, when observed, were sampled and processed by water extraction to confirm the presence of foliar nematodes. If foliar nematodes were detected in a leaf sample, the corresponding plant was recorded as positive. The data are presented as a series of plot maps representing the weekly sampling periods throughout the duration of each year's trial. At the end of each trial, a disease gradient was estimated by regression of the percentage of infected plants at each spacing distance against the spacing distance to determine the best fit equation.