In order to evaluate the potential of the different amino acid materials a series of experiments were conducted. In these experiments several amino acids from commercial sources and two experimental formulations were included. The following amino acids were obtained from commercial sources: DL-methionine (Evonik Degussa, Theodore, AL), L-threonine (Evonik Degussa, Theodore, AL), lysine (Biolys®, Evonik Degussa, Theodore, AL), and methionine hydroxyl analog (Alimet®; Novus International, St. Charles, MO). Two experimental methionine formulations also were included: sodium methionate and potassium methionate. Among these amino acid materials, DL-methionine, L-threonine, and lysine were dry powder materials, whereas methionine hydroxyl analog, sodium methionate and potassium methionate were liquids. The percentage amino acid content (w/w) of each treatment was: DL-methionine (99%), L-threonine (98.5%), lysine (50.7%), sodium methionate (46%), potassium methionate (35%), and methionine hydroxyl analog (88%). Application rates of each treatment were based on the weight of amino acid and not the weight of the formulation.
The first set of experiments involved bench screening of the materials to determine the relative nematicidal activity of the alternative amino acids compared with DL-methionine. After this, the top performers were selected to move forward to growth room experiments that further evaluated efficacy and also to study effects on plants. From these experiments the amino acid treatments that had the best performance was selected to move ahead to the final stage, field trials.
: Laboratory bench trials were conducted twice using a randomized complete block design with eight replications at the Entomology and Nematology Department at the University of Florida, Gainesville, FL. All of the amino acids were evaluated at two rates (224 and 448 kg amino acid/ha), for their ability to suppress activity of B. longicaudatus
(juveniles and adults) or M. incognita
(J2) in soil. Soil infested with B. longicaudatus
was collected from greenhouse pot cultures or an infested field site, for trials 1 and 2, respectively. The greenhouse pot culture of B. longicaudatus,
originally isolated from a golf course in Sun City, FL, was maintained on ‘FX 313’ St. Augustinegrass (Stenotaphrum secundatum
) in United States Golf Association (USGA) putting green specification sand (Anonymous, 1993
). The B. longicaudatus
infested field soil was collected from an athletic field in Spring Hill, FL planted to ‘Tifway’ bermudagrass (Cynodon dactylon × C. transvaalensis
). Both trials in which M. incognita
suppression was evaluated used infested field soil from an alyce clover (Alysicarpus ovalifolius
) field located in Sumter County, FL. For field soils, 100 cm3
of dry soil was used to determine soil texture by a hydrometer method (Bouyoucos, 1936
). Another 2 g of dry soil was used to determine percentage organic matter using a loss-on-ignition method (Ben-Dor and Banin, 1989
). After soil analysis, all the soil samples from the field were found to have >90% sand content. Soil from the field in Spring Hill contained 5% organic matter, whereas soil from Sumter County had <2% organic matter.
Soil containing B. longicaudatus
was hand mixed to ensure that nematodes were evenly distributed in the soil. Next, 200 cm3
aliquots were placed into 600 cm3
plastic pots. Powder treatments were applied topically followed with 50 ml of water to carry the materials into the soil. Liquid materials were mixed into water and then 50 ml of solution was poured onto the soil. Untreated controls had 50 ml of water poured onto the soil. Three days after treatments were applied, the soil was removed from the pots and placed onto modified Baermann funnels (McSorley and Frederick, 1991
) and collected after 24 hr. Room temperature for amino acid exposure and nematode extraction was maintained at 26 °C. Nematodes were counted using an inverted light microscope at ×40 magnification. Data were subjected to analysis of variance as a 7×3 factorial experiment with seven treatments and three rates using SAS software (SAS Institute, Cary, NC). Treatment means were separated according to Duncan's multiple-range test (P
Growth room trials: Two trials were conducted in a growth room on the campus of the University of Florida, Gainesville, FL. The trials were arranged in a randomized-block design and were identical except for the number of replications, eight replications for trial 1 and five replications for trial 2. These trials compared the effects of methionine formulations on B. longicaudatus in inoculated pots of creeping bentgrass (Agrostis palustris). Parameters evaluated included number of B. longicaudatus in soil, plant phytotoxicity, and soil pH.
Treatments were: 1) untreated control, 2) DL-methionine, 3) sodium methionate, 4) potassium methionate, and 5) methionine hydroxyl analog. Each amino acid was evaluated at three rates; 112, 224, and 448 kg amino acid/ha.
In each trial, 10-cm-diam. pots were filled with 400 cm3
of USGA specification greens sand, and seeded with ‘Penncross’ creeping bentgrass at 0.08 g per pot. After seed germination, B. longicaudatus
was collected from greenhouse cultures using a decant and sieve technique (Cobb, 1918
). Each pot was inoculated with 100 mixed life stages of B. longicaudatus
which were then allowed to reproduce for 4 wk. Following turf and nematode establishment, treatments were applied. The liquid materials were mixed with water and 50 ml of solution was applied topically to the pots as a drench. Powder DL-methionine was applied topically and then 50 ml water added to the surface. The untreated control treatment received 50 ml of water. Turfgrass was monitored visually for phytotoxic response three, eight, and fourteen days after treatment. A visual damage rating scale was used to evaluate phytotoxicity, where 0 = no phytotoxicity, -1 = slight phytotoxicity, -2 = moderate phytotoxicity, and -3 = severe phytotoxicity.
Fourteen days following treatment application the soil was analyzed for treatment effects on B. longicaudatus
and soil pH. Soil was removed from each pot, mixed, and a 100 cm3
subsample was removed for quantification of nematodes. Nematodes were extracted using the sugar floatation centrifugation method (Jenkins, 1964
) and quantified as described above. An additional 20 cm3
subsample was used for measuring soil pH (Mylavarapu, 2009
) with a pH meter (AR10 pH/mV/degC Meter, Standardize accumet®
Research, Fisher Scientific, USA). Data were subjected to analysis of variance as a 5×4 factorial experiment with 5 treatments and 4 rates using SAS software (SAS Institute, Cary, NC). Treatment means were separated according to Duncan's multiple-range test (P
≤ 0.05). The results of both trails were analyzed for heterogeneity and the results did not differ among the trials except for the phytotoxicity measurements. Therefore, data other than the phytotoxicity data were combined from both trials prior to analysis.
: In 2010, two field trials were conducted to compare the effects of DL-methionine and potassium methionate on nematode suppression and turf health. Two sites were used for this experiment; both were located at the University of Florida Plant Science Research Unit (PSRU), Citra, FL. One site was planted with ‘Tifdwarf’ bermudagrass, and the other site was planted with ‘Celebration’ bermudagrass. Both sites were infested with population densities of B. longicaudatus
in excess of the high risk thresholds for bermudagrass (25/100 cm3
of soil) used by the Florida Cooperative Extension Service (Crow, 2011
). The Tifdwarf bermudagrass site was managed under putting green conditions, whereas the Celebration bermudagrass site was managed as a tee box.
The experimental design was a randomized block with five replications. Thirty plots were used at each site. Plots were 1.5 m2 with a 0.6 m untreated border between plots. The treatments included: DL-methionine and potassium methionate applied at rates of 112 and 224 kg amino acid/ha, fenamiphos (Nemacur® 10G, Bayer CropScience, Research Triangle Park, NC) applied at 11.2 kg a.i./ha, and an untreated control. DL-methionine and fenamiphos treatments were applied topically using a drop spreader (Gandy, Owatonna, MN). Potassium methionate was mixed with water and sprayed topically on the plots using a CO2 powered backpack sprayer (Weed Systems, Hawthorne, FL). Fenamiphos was applied only once. Plots received DL-methionine or potassium methionate twice, on a 4 week interval. After treatment, each plot (including the untreated controls) was irrigated with 15 liter of water using a sprinkler can. An additional 0.6 cm of water was applied to the entire field after all the treatments were applied on the first application date only. The turf was maintained by the PSRU staff using standard maintenance practices. Turf was maintained at a 0.45 cm mowing height on the ‘Tifdwarf’ bermudagrass site, and 1.4 cm on the ‘Celebration’ bermudagrass site.
Nematode samples consisting of nine cores (10-cm-deep × 1.9-cm-diam.) were collected from each plot before treatment and 2 wk after each treatment application date. Leaves, stolons, rhizomes, and organic thatch layer were discarded and the soil was mixed by hand. Nematodes were extracted from a 100-cm3 subsample of soil using the sugar floatation centrifugation method. Plant-parasitic nematodes were identified and counted using an inverted light microscope at ×40 magnification. Another 100-cm3 of dry soil was used for soil texture and percent organic matter analysis as described previously. After soil analysis, soil at both sites was found to have >90% sand and <2% organic matter.
To evaluate turf health percentage green cover was used. Turf percentage green cover is a measurement of the percentage of the plot surface covered by green turf. A digital photo was taken of the center m2
of each plot. The percentage of the pixels in each photo that were “green” was determined using a macro developed by faculty at the University of Arkansas (Karcher and Richardson, 2005
) for use with SigmaScan Pro5 software (SPSS Inc., Chicago, IL). Percentage of the total pixels in the image that were green was the measure of turf percent green cover.
Treatments were applied on 16 June and 15 July at the Tifdwarf bermudagrass site and 23 June and 20 July at the Celebration bermudagrass site. Nematode samples were collected two weeks before the first application date and two weeks after each application date. Turf percentage green cover was measured approximately every two weeks.
To determine if individual treatments were effective, data were subjected to analysis of covariance with the initial nematode population density as the covariant using SAS software (Cary, NC). Each individual treatment was compared to the untreated control and the P-value for the comparison used to determine differences.