Belonolaimus longicaudatus is considered native to the sandy regions of the coastal southern states (Perry and Rhoades, 1982). This region is most likely the point of origin of both B. longicaudatus and its Pasteuria parasites. Candidatus Pasteuria usgae (CPu) is a parasite of B. longicaudatus (Giblin-Davis et al., 2003) that has been shown to naturally suppress B. longicaudatus on turfgrasses (Giblin-Davis, 2000). Suppression of B. longicaudatus was transferable through inoculation with CPu infested soil (Giblin-Davis et al., 2000). However, because CPu is an obligate parasite its use as a commercial biopesticide was not considered practical until recently.

Pasteuria Bioscience LLC has developed techniques for in vitro production of Pasteuria sp. Among the Pasteuria spp. being produced in this manner is a Pasteuria sp. isolate found infecting B. longicaudatus. This isolate is morphologically similar to the description of CPu, (Luc et al., 2010 a; Giblin-Davis et al., 2001) but it has not been confirmed as CPu with molecular sequencing data. Greenhouse trials with the in vitro produced Pasteuria sp. from B. longicaudatus showed that 280,000 endospores/cm³ of soil was capable of suppressing B. longicaudatus in pots (Luc et al., 2010 a, 2010 b, 2011). A comparison of formulations found that a liquid formulation was more effective at suppressing B. longicaudatus in pots than was a granular formulation created from the liquid formulation (Luc et al., 2010 b).

In 2010, Pasteuria Bioscience launched Econem^™, the first commercial bionematicide with in vitro produced Pasteuria sp. endospores as the active ingredient. Econem contains Pasteuria sp. from B. longicaudatus (0.002% a.i.) on a clay carrier. It is designed to be a topical treatment for the inundative management of B. longicaudatus on turfgrass. After application, and subsequent irrigation, the endospores are released from the clay and move into the soil profile. The product is labeled for 98 to 488 kg/ha per application. According to the manufacturer, 98 kg/ha and 293 kg/ha of Econem adds the equivalent of 100,000 and 300,000 endospores/cm³ of soil to the top 5-cm of soil profile, respectively (T. E. Hewlett, Pasteuria Bioscience, pers. comm.). Therefore, a single application of 293 kg/ha or three applications of 98 kg/ha of Econem should add enough endospores to be in excess of the amount shown to induce suppression of B. longicaudatus in greenhouse trials (Luc et al., 2010 a, 2010 b, 2011). This assumes that all the endospores applied stay in the top 5-cm of the soil profile.

The current industry standard for management of B. longicaudatus on golf and sports turf in the coastal southeastern states is the nematicide 1,3-dichloropropene (1,3-D; Curfew Soil Fumigant, 97.5% a.i., Dow Agrosciences, Indianapolis, IN). While 1,3-D is very effective against B. longicaudatus in turf (Crow et al., 2003, 2005), rapid resurgence in population density of B. longicaiudatus following putting green applications has been observed in some locations (Crow et al., 2005). A possible explanation for this resurgence is lack of natural enemies like Pasteuria spp. in these locations. It is unknown if application with Econem following a 1,3-D application could slow down or prevent this effect by inoculating the green with the Pasteuria parasite of B. longicaudatus.

The objectives of our research were: i) to determine if three applications of 98 kg/ha or a single application of 293 kg/ha of Econem is sufficient to suppress B. longicaudatus and improve turf health in the field, ii) to compare Econem to 1,3-D for management of B. longicaudatus on putting greens, iii) to determine if Econem is effective is reducing resurgence of B. longicaudatus population densities following a 1,3-D application, and iv) to determine if much higher rates or reapplication strategies with Econem suppressed B. longicaudatus or led to recycling of Pasteuria sp. in greenhouse pot studies on turfgrass hosts.

All field experiments were conducted on sites that had population densities of B. longicaudatus in excess of the “high risk” threshold (25/100 cm³ of soil) for bermudagrass used by the Florida Cooperative Extension Service (Crow, 2011). Econem applications were made with a walk-behind drop spreader (Gandy, Owatonna, MN). After each application all plots, including the controls, were irrigated with 0.64-cm of water. Nematode samples consisted of nine 1.9-cm-diam. × 10-cm-deep cores from each plot. The top layer of the cores consisting of leaves, stolons, and rhizomes, along with associated organic thatch layer was discarded and the remaining soil fraction was thoroughly mixed. Nematodes were extracted from a 100 cm³ subsample by centrifugal-flotation (Jenkins, 1964). The plant-parasitic nematodes extracted were then identified and counted. Turf percent green cover is a measurement 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. Root lengths were measured from two 3.8-cm-diam. × 15.25-cm-deep (174 cm³ volume) cores taken from each plot and combined. Roots were extracted and root lengths determined using WinRHIZO (Regent Instruments, Quebec, Canada) software according to the method of Pang et al., (2011a).

Fairway Experiment: Two trials were conducted on golf course fairways in 2010 to evaluate Econem as a bionematicide for use on turf for management of B. longicaudatus. One site was located at the Babe Zaharius Golf Club in Tampa, FL, the other was at the SummerGlen Golf Club in Ocala, FL. Both sites were planted to ‘Tifway’ bermudagrass (Cynodon dactylon × C. transvaalensis) that was overseeded during the winter with perennial ryegrass (Lolium perenne). Winter overseeding is used in the region to provide turf cover during the winter when bermudagrass is dormant. During the late spring perennial ryegrass dies under Florida conditions, roughly concurrent with bermudagrass breaking dormancy. In addition to B. longicaudatus the Babe Zaharius site was infested with below-threshold population densities of Helicotylenchus sp., Hemicriconemoides annulatus, Hemicycliophora sp., Meloidogyne sp., Mesocriconema ornatum, Peltamigratus christiei, Trichodorus obtusus, and Tylenchorhynchus sp., and the SummerGlen site with Helicotylenchus sp., H. annulatus, Meloidogyne sp., M. ornatum, and Nanidorus minor.

Plots were established using a randomized block design, with blocking based on the initial population density of B. longicaudatus. There were five replications of each treatment. Plots were 21 m² with 1.5 m untreated borders between plots. A 2.3 m² subplot in the center of each plot was used for all data collection. Treatments were: i) untreated control, ii) Econem applied at 98 kg/ha at each of three applications at four wk intervals, and iii) Econem applied once at 293 kg/ha. Initial treatment applications were made on 15 April at Babe Zaharius and 5 May at SummerGlen.

Nematode and root samples were collected 1 wk prior to the initial treatment applications, 4 wk after each treatment application, and 8 wk after the final treatment application. Turf percent green cover was measured approximately every 2 wk.

To evaluate treatment effects on B. longicaudatus, nematode data were subjected to analysis of variance and treatment means separated according to Fisher's protected least significant difference (LSD) test (P ≤ 0.1) using SAS software (Cary, NC). For turf percent green cover and root length data, orthogonal contrasts between the Econem treatments and the untreated controls at each measurement date (P ≤ 0.1) were used to determine if either Econem treatment was different from the untreated.

Tee Box Experiment: To evaluate the efficacy of Econem for suppression of B. longicaudatus on turf, two field trials were conducted on golf course tee boxes in 2011. One tee box was located at Miromar Lakes Golf and Beach Club in Estero, FL, the other was at the University of Florida Plant Science Research Unit in Citra, FL. The Miromar Lakes site was planted to Tifway bermudagrass, the Plant Science site was planted to ‘Celebration’ bermudagrass. In addition to B. longicaudatus, the Miromar Lakes site was infested with below-threshold population densities of Helicotylenchus sp., H. annulatus, Hoplolaimus galeatus, Meloidogyne sp., M. ornatum, and N. minor, and the Plant Science site with Helicotylenchus sp., H. annulatus, Hemicycliophora sp., H. galeatus, P. christiei, Pratylenchus hippeastri, Meloidogyne sp., M. ornatum, and N. minor. The experimental design was completely randomized block with 5 replications. Blocks were based on initial population densities of B. longicaudatus. Plots were 1.5 m² with 0.6 m untreated borders between plots.

Treatments were untreated control and a single application of 293 kg/ha of Econem. Treatments were made on 8 March at the Miromar Lakes site and 15 March at the Plant Science site. Nematode samples were collected 2 wk before the Econem applications, and 2 and 10 wk after the Econem applications. Turf percent green cover was evaluated every 2 wk. Initial root length samples were collected the day of treatment and 4, 8, and 18 wk after treatment.

To evaluate treatment effects on B. longicaudatus, nematode data were subjected to analysis of variance and treatment means separated according to Fisher's protected LSD test (P ≤ 0.1). For turf percent green cover and root length data, analysis of covariance was conducted with the initial measurement used as the covariate. The Econem treatment was compared to the untreated control and the P-value generated (P ≤ 0.1, 0.05, 0.01) for the comparison was used to determine differences.

Putting Green Experiment: In 2010, two trials were conducted to compare the effects of Econem to the nematicide, 1,3-D on golf course putting greens. One trial was conducted at Venetian Bay Golf Club in New Smyrna Beach, FL, the other at the Avila Club in Tampa, FL. Both sites were putting greens planted with ‘Tifdwarf’ bermudagrass. In addition to B. longicaudatus the Venetian Bay site also was infested with below-threshold population densities of Dolichodorus sp., Hemicycliophora sp., Meloidogyne sp., M. ornatum, and N. minor, and the Avila Club site with Helicotylenchus sp., H. annulatus, Meloidogyne sp., M. ornatum, and N. minor. The experimental design was completely randomized block with four replications. Blocks were based on spatial location on the green and not nematode population densities. Plots were 26 m² with 1 m untreated borders between plots. In the center of each plot a 3.34 m² subplot was established for collection of all data.

Treatments were: an untreated control, Econem applied at 98 kg/ha at each of three applications at 4 wk intervals, and 1,3-D applied at 53 kg a.i./ha once by slit injection as described by Crow et al. (2005). 1, 3-D was applied by a commercial applicator (Southern Soils Turf Management, Lake Mary, FL). The initial Econem applications were made on 5 April at Venetian Bay and 16 March at Avila Club. The 1,3-D applications were made on the same day as the second Econem applications.

Nematode samples were collected 2 wk before the initial Econem applications, 2 wk after the 1,3-D/second Econem applications, and 4 wk after the final Econem applications. Turf percent green cover was measured every two wk. Root samples were collected 2 wk after the final Econem application. To evaluate treatment effects on B. longicaudatus, nematode data were subjected to analysis of variance and treatment means separated according to Fisher's LSD test (P ≤ 0.1). For turf percent green cover and root length data, orthogonal contrasts between each experimental treatment and the untreated controls at each measurement date (P ≤ 0.1) were used to determine if experimental treatment was different from the untreated.

Population Resurgence Experiment: Two field trials were conducted in 2010 to determine if applications of Econem could slow down resurgence in B. longicaudatus population density following treatment with 1,3-D. One trial was conducted at the MetroWest Golf Club in Orlando, FL, the other was at the Continental Country Club in Wildwood, FL. In addition to B. longicaudatus the MetroWest site was infested with below-threshold population densities of Helicotylenchus sp., H. galeatus, Meloidogyne sp., and M. ornatum, and the Continental site with Helicotylenchus sp., Meloidogyne sp., M. ornatum, and T. obtusus. Both sites had a history of resurgence of B. longicaudatus within a couple of months following application of 1,3-D.

Both golf courses had commercial applications of 1,3-D at 53 kg a.i./ha made to all their putting greens for management of B. longicaudatus 2 wk prior to the initiation of the trials; therefore, the entire experimental area was treated with 1, 3-D. The experimental design was randomized complete block with five replications. Blocks were based on spatial location on the green and not nematode population densities. Plots were 13.4 m² with 1 m untreated borders between plots. In the center of each pot a 3.34 m² subplot was established for collection of all data. The two treatments were untreated control and Econem applied at 98 kg/ha at each of three applications at 4 wk intervals.

Nematode population density, turf percent green cover, and root length data were obtained as described for the previous experiments. Nematode samples were collected the day of the initial Econem applications, and 4 and 8 wk after the final Econem application. Turf percent green cover was evaluated every 2 wk. Root samples were collected 4 wk after the final Econem application.

Nematode and turf percent green cover and data was subjected to analysis of covariance with the initial measurement used as the covariate. The Econem treatment was compared to the untreated control and the P-value generated (P ≤ 0.1, 0.05, 0.01) for the comparison was used to determine differences. For root length data, orthogonal contrasts between the Econem treatment and the untreated controls (P ≤ 0.1) were used to determine differences.

Greenhouse Pot Experiments: Two separate greenhouse experiments were conducted in 2010-2011 to determine if different rates or reapplication strategies with Econem suppressed B. longicaudatus or led to recycling of Pasteuria sp. in model bentgrass or St. Augustinegrass hosts. Both experiments were conducted at greenhouses at the UF-IFAS Fort Lauderdale Research and Education Center (FLREC). Soil used in these experiments was a Margate fine sand with 3.8% OM.

Experiment 1 was conducted to determine if Econem in a single or multiple applications at 488, 4,883, or 48,830 kg/ha (or 5 × 10⁵, 5 × 10⁶, and 5 × 10⁷ endospores/cm³ of soil to the top 5-cm of soil profile, respectively) suppressed B. longicaudatus in pots of cultured Pasteuria-free nematodes on a St. Augustinegrass (Stenotaphrum secundatum) host. Single aerial cuttings of ‘FX-313’ St. Augustinegrass was sprigged with into bulb pots (17 cm effective diam. × 8 cm depth) filled to within 2 cm of the top with autoclaved soil on 1 June 2010. Biweekly fertilization was started on 4 June 2010 with each pot receiving 10 ml of solution containing 18-18-21 + micros fertilizer (Miracle-Gro, Scotts, Marysville, OH) at 4.9 cm³/liter. All pots receiving B. longicaudatus were inoculated with ca 488 B. longicaudatus/pot in 130 ml of soil on 11 August 2010. The soil was from B. longicaudatus stock cultures on FX-313 St. Augustinegrass (Giblin-Davis et al., 1992) to avoid handling damage to the nematodes during inoculation. Soil for the noninoculated control was treated with boiling water to kill the nematodes and allowed to cool before inoculation. Pots were completely randomized on a bench in a greenhouse receiving overhead irrigation every other day. The B. longicaudatus populations were allowed 48 days to establish before they were sampled to estimate the pre-treatment population density by pulling two random cores with a #8 cork borer (1.5 cm diam. × 11.5 cm depth) to produce a measured volume of 25 ml which was extracted using sugar flotation and counted as described above. There were eight treatments with four replicates in a completely randomized design. The treatments were applied to the Pasteuria-free B. longicaudatus pots on 8 October 2010 and included; 1) a control with no Econem, 2) a single application of Econem at 488 kg/ha, 3) a single application of Econem at 4,883 kg/ha, 4) a single application at 48,830 kg/ha, 5) a single application at 488 kg/ha followed by four additional monthly applications at same rate, 6) a single application at 4,883 kg/ha followed by four additional monthly applications at same rate, 7) a single application at 48,830 kg/ha followed by two additional monthly applications at same rate, and 8) a “no nematode” and “no Econem” control to be used only in turf quality assessments. Originally, treatment 7 was going to receive four additional applications as for treatments 5 and 6 instead of just two. However, the pots were too full to add additional Econem after the third application.

Nematode population dynamics (nematode soil counts) were determined with subsampling as done for pretreatment counts 6 wk, 12 wk, and 26 wk post-treatment using sugar flotation extraction. Turf quality in each pot was scored separately for color and coverage using a 0-10 scale to evaluate overall plant performance prior to taking nematode samples. Roots from the final nematode harvest at 26 wk were collected, cleaned of adhering sand and dried for 48 hr before being weighed. Foliar plant performance was examined weekly for phytotoxicity. Nematode count data (square root transformed), averaged color and coverage scores, and root dry weights were analyzed using PROC ANOVA followed with a Waller-Duncan k-ratio test for means separation (P ≤ 0.05). In addition, a subsample of at least 10 B. longicaudatus harvested from each pot at each harvest date were stained with 1% crystal violet and examined under a compound microscope to look for endospore encumbrance and/or spore-filled cadavers, indicative of attachment of and/or recycling of Pasteuria sp. in the pots.

Tee Box Experiment: At the Miromar Lakes site there were no differences in population density of B. longicaudatus between treatments at any sampling date (Table 2). However, plots treated with Econem exhibited less (P ≤ 0.1) root length than the untreated plots on the third root sampling date, root lengths were 352 cm and 216 cm for the untreated and Econem treated, respectively. At the Plant Science site, Econem treated plots had a lower (P ≤ 0.1) population density of B. longicaudatus than the untreated control plots on the third sampling date only (Table 2). The Econem treated had greater (P ≤ 0.1) root length than the untreated control on the second sampling date only, root lengths were 394 cm and 710 cm for the untreated and Econem treated, respectively. There were no differences in turf percent green cover between treatments on any observation date in either trial (Data not shown).

Putting Green Experiment: At the Venetian Bay site, population densities of B. longicaudatus in all treatments dropped from ≥ 55/100 cm³ of soil two wk before treatment to ≤ 5/100 cm³ of soil 2 wk after treatment, and remained low for the rest of the study. This is below the “moderate risk” threshold of 10/100 cm³ of soil for B. longicaudatus on bermudagrass used by the Florida Cooperative Extension Service (Crow, 2011). There were no differences (P > 0.1) in population density of B. longicaudatus (Table 3) or in root lengths (Figure 1) among treatments at the Venetian Bay site, although there were increases (P ≤ 0.1) in turf percent green cover from 1,3-D compared to the untreated on two of the observation dates (Figure 2). At the Avila Club site population densities of B. longicaudatus were reduced (P ≤ 0.1) by 1,3-D compared to the untreated control two wk after application (Table 3), but the population densities rebounded by the final sample date. Similarly, 1,3-D increased (P ≤ 0.1) turf root lengths, and improved (P ≤ 0.1) turf percent green cover on one observation date compared to the untreated control (Figures 1, ​,2).2). Econem was not different from the untreated control for any parameter evaluated in either trial (P > 0.1).

Population Resurgence Experiment: Population densities of B. longicaudatus increased in both trials over the course of the experiment (Table 4). However, population density of B. longicaudatus in Econem treated plots was not different from the untreated at any sampling date in either trial. Similarly, there were no differences in root length or percent cover between treatments in either trial (data not shown).

Greenhouse Pot Experiments: In experiment 1, there were no differences (P > 0.05) in B. longicaudatus/100 cm³ of soil among treatments. No typical looking attached CPu endospores as described by Giblin-Davis et al. (2001), or any spore-filled cadavers were observed. Econem at the highest rate (48,830 kg/ha with single or multiple applications) consistently produced turf coverage and color comparable with the noninoculated/nontreated control pots. These treatments were significantly improved over the other Econem treatments and the inoculated control without Econem at 12, and 26 weeks post-treatment. At 6 weeks post-treatment, Econem at the highest rate (48,830 kg/ha with single or multiple applications) produced better turf coverage and color performance than all other treatments (P > 0.05). Root dry weights for the noninoculated/nontreated control were significantly greater than any of the other treatments with or without Econem, which were extremely variable but statistically similar. No phytotoxicity was observed during the experiment.

During earlier experiments, in vitro produced Pasteuria sp. applied at 280,000 endospores/cm³ of soil suppressed population densities of B. longicaudatus on creeping bentgrass (Agrostis palustris) in pots (Luc et al., 2010 a, 2010 b, 2011), but not in the current studies using the Econem formulation. Even though the Pasteuria sp. in Econem is derived from the same isolate as the one used by Luc et al. (2010 a, 2010 b, 2011), the in vitro process used to produce the endospores and the formulation were different between these studies. While the process used for in vitro production of Pasteuria sp. is proprietary, the endospores used by Luc et al. (2010 a, 2010 b, 2011) were produced using laboratory-scale fermenters and the formulation was simply endospores in liquid growth media, whereas the endospores contained in Econem are produced in large-scale commercial fermenters that are then formulated into a commercial granular product. It is possible that during the scale-up in production or during the formulation process the endospores lost virulence. The granular formulation evaluated by Luc et al. (2010 b) was made by pipetting the liquid growth media containing endospores onto clay granules and letting it dry, versus the concentrated, formulated endospores on clay granules contained in Econem. The lab formulated product was not as effective as the straight liquid formulation but it did have some efficacy (Luc et al., 2010b), whereas the commercially formulated Econem product used in the current studies was not effective.

From the greenhouse experiment 2, examination of B. longicaudatus harvested from each pot at each harvest date with a compound microscope was conducted. While endospores observed attaching to the cuticle and occasionally filling cadavers with the earlier liquid formulation of in vitro-produced Pasteuria sp. (Luc et al., 2010 a, 2010 b; 2011) appeared similar to those reported in natural populations of CPu, , nothing that could unambiguously be assigned to CPu was observed attaching to or filling B. longicaudatus in the pot studies conducted with Econem herein. Although visual improvement in turf was noted in greenhouse experiment 1, no effects on B. longicaudatus or roots were observed. This suggests that the very high rates of clay added in these treatments may have improved plant growth without affecting B. longicaudatus numbers.

Environmental factors also might have played a part in the lack of efficacy of Econem. While bermudagrass has the genetic potential to establish roots as deep as 2 m or more (Lunt, 1968), under Florida golf course conditions the root depth in putting greens seldom exceeds 10-cm-deep (W. T. Crow, pers. observation). Therefore, the treatment zone for turfgrass nematicides in Florida is considered 5 to 10-cm-deep. Luc et al. (2010 a, 2010 b) found that 280,000 endospores/cm³ of soil of in vitro produced Pasteuria sp. endospores were required to suppress population densities of B. longicaudatus. While 293 kg/ha of Econem adds enough endospores to reach this target rate, this assumes all the endospores come off the clay and that most do not leach beyond 5-cm of the soil profile. Currently, the release rate of Pasteuria sp. endospores from Econem with irrigation is unknown. Also, Luc et al. (2011) found that Pasteuria sp. endospores are leachable in sand, with many of the endospores moving beyond the target 5-cm of soil profile with increasing amounts of irrigation. Therefore, it is possible that the amount of Pasteuria sp. endospores in the top 5-cm of soil profile at any given time is insufficient to adequately suppress B. longicaudatus.

As mentioned previously, both B. longicaudatus and its Pasteuria parasites may be native to Florida and therefore commonly occur concomitantly in most turf sites in the region. In an attempt to locate turf field sites for experiments having B. longicaudatus but no detectible level of preexisting Pasteuria attachment, 43 golf course and athletic field sites across the state of Florida were sampled. However, Pasteuria sp. attachment to B. longicaudatus was observed in all 43 sites (W. T. Crow, unpubl. data). In Florida, it can be assumed that the vast majority of bermudagrass locations infested with B. longicaudatus already have some level of biological suppression of the nematodes from Pasteuria spp. occurring. While biological suppression of B. longicaudatus is likely occurring in most locations and preventing turf damage from being as severe as it would otherwise, in many cases this natural suppression is insufficient to prevent root damage to a degree acceptable for most turf managers. This lack of acceptable suppression could be due to low CPu endospore production in B. longicaudatus and environmental conditions in the turf root zone. Belonolaimus longicaudatus causes severe root reductions and moreover, the carrying capacity for B. longicaudatus on most bermudagrass cultivars is < 300/150 cm³ of soil (Pang et al., 2011 b). Individual B. longicaudatus cadavers were found to produce 1,483-4,700 CPu endospores/cadaver (Giblin-Davis, 2000) depending on nematode life-stage and sex. Therefore, CPu endospore production is much lower than would be expected for P. penetrans on Meloidogyne spp. Due to low endospore production and leaching, it is likely a rare occurrence that naturally occurring CPu endospore densities in soil reach the 280,000/cm³ of soil required to prevent B. longicaudatus population increases as reported by Luc et al. (2010 a).