Virulence of new South Carolinian heterorhabditid isolates (Rhabditida: Heterorhabditidae) to the beet armyworm, Spodoptera exigua

The virulence of new nine heterorhabditid isolates from South Carolina (Heterorhabditis megidis LEX, H. zealandica EDS and CHR, and H. bacteriophora WPS, SMP, PD, CFG, MF and CFM strains) against the beet armyworm was compared with two known heterorhabditid nematodes (H. bacteriophora Hb and HP88 strains) under laboratory conditions. The Petri-plate bioassay procedure was used to evaluate the susceptibility of the Spodoptera exigua larvae to the heterorhabditids at concentrations of 10, 25, 50, and 100 infective juveniles (IJs) per larva. Mortalities were counted for 4 days. At the final count, mortalities were 53.6-100, 72-100, 79.8-100, and 92.9-100% for all nematode species/strains at the concentrations of 10, 25, 50, and 100 IJs per larva, respectively. H. megidis LEX strain was superior and differed than others by having 100% mortality in all of the concentrations. It was second to cause early mortality. It had the highest mortality rate at 10 nematodes per larva and H. bacteriophora WPS, H. zealandica CHR, H. bacteriophora HP88 and H. zealandica EDS strains followed it with 92.9, 89.3, 85.7 and 82.1% mortality, respectively. LC50 for most of the nematodes was relatively low (10 IJs per larva). Virulence of H. bacteriophora WPS, HP88 and SMP and H. zealandica CHR strains were similar. The least virulent heterorhabditid was H. bacteriophora CFM strain with LC50 value of 14.8 IJs per larva. The LT50 value of H. bacteriophora WPS strain was the smallest and it was followed by H. megidis LEX, H. zealandica CHR and EDS, H. bacteriophora SMP, HP88, MF, PD, Hb, CFG and CFM strains, respectively.

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) are obligate parasites of insects (Poinar, 1990;Adams and Nguyen, 2002).They are mutualistically associated with bacteria (Xenorhabdus spp.and Photorhabdus spp.for steinernematids and heterorhabditids, respectively).Infective juveniles (IJs), the only free-living stage, enter hosts through natural openings (mouth, anus, and spiracles), or in some cases, through the cuticle.After entering the host's hemocoel, nematodes release their symbiotic bacteria, which are primarily responsible for killing the host, defending against secondary invaders, and providing the nematodes with nutrition (Dowds and Peters, 2002).The nematodes molt and complete up to three generations within the host after which IJs exit the cadaver to search out new hosts (Kaya and Gaugler, 1993).
These nematodes are effective biocontrol agents of a variety of economically important insect pests (Klein, 1990;Shapiro-Ilan et al., 2002;Grewal et al., 2005) and they have been used in controlling insect pest for about 25 years, extending their usage from high value markets to large area crops, including forestry (Peters, 2010).A number of studies indicate that applications of EPN can result in high levels of control for a variety of noctuid pests including S. exigua (Feaster and Steinkraus, 1996;Medeiros et al., 2000;González-Ramírez et al., 2000;Kim et al., 2006;Kepenekçi and Evlice, 2009).
Despite the progress that has been made in the use of EPN (Laznik et al., 2010a), knowledge about their natural host range and their efficacy on insect populations as biological control agents is still limited (Ansari et al., 2007).Our overall goal was to determine the potential use of several heterordabditid nematodes for S. exigua suppression.
A crucial element to be successful in any biological control program with EPN is the pairing the most suitable nematode with the defined host, and relative pathogenicity among various nematodes is one of the important factors to consider in determining suitability (Georgis and Gaugler, 1991;Shapiro-Ilan et al., 2002;Shapiro et al., 2006).Therefore, this research aimed to compare the virulence of new nine heterorhabditid isolates from South Carolina against the beet armyworm with two known heterorhabditid nematodes (Heterorhabditis bacteriophora Hb and HP88 strains) under laboratory conditions.

MATERIALS AND METHODS
The larvae of S. exigua were reared on artificial pinto bean diet (Burton, 1969).Heterorhabditis bacteriophora Hb strain was obtained from Dr. David I. Shapiro-İlan, Integrated BioControl Systems, Inc. (Aurora, Indiana) and H. bacteriophora HP88 strain was provided by Dr. Khoung B. Ngyuen and Dr. Byron J. Adams of the University of Florida.The other nine heterorhabditids; H. megidis LEX, H. zealandica EDS and CHR, and H. bacteriophora WPS, SMP, PD, CFG, MF and CFM strains were obtained from soil on a survey in South Carolina, USA (Canhilal and Carner, 2006a).
EPN were produced on last-instar of the greater wax moth, Galleria mellonella (L.) (Lepidoptera: Pyralidae) following the standard rearing method described by Woodring and Kaya (1988).A modified White Trap (Canhilal and Carner, 2006b), consisting of a folded 11-cm filter paper (3 mm in depth after folding) in a Petri dish (100 x 15mm) with 15-20 ml of distilled water, was used to collect the infective juveniles (IJs).These IJs were stored at 7-8C in tissue culture flasks for 15-20 days before being used for experiments (Kung et al., 1990).Before the assays, viability was confirmed by observing nematode activity (rapid wiggling) under a binocular microscope (Laznik et al., 2010b).
The Petri-plate bioassay procedure was used to evaluate the susceptibility of the beet armyworm larvae to heterorhabditids at concentrations of 10, 25, 50, and 100 IJs per larva in 1 ml of sterile distilled water, following the procedures described by Woodring & Kaya (1988).Petri dishes (100 x 15 mm) were lined with two Whatman No.1 filter paper pieces (9 cm diameter).1 h before the beginning of the experiment, the IJs were applied and distributed evenly on the filter paper.For each treatment concentration, four groups of seven 4 th instars of S. exigua were placed per dish containing IJs.The Petri dishes were placed in a double plastic bag and put in a dark incubator at 25 ± 1°C (Glazer et al., 1991).Controls consisted of 1 ml of sterile distilled water without nematodes.The bioassay was repeated two times.
S. exigua mortality was recorded every 24 h for 4 days (Epsky and Capinera, 1994).Dead insects were incubated on modified White Traps at room temperature (25 ± 1°C) and examined to confirm the presence of nematodes.
The mortalities were converted to percentages and adjusted for control mortality, using Abbott's correction formula.The data were analyzed as a completely randomized factorial design and Least Significant Difference (LSD) mean separation procedure was used to detect differences among treatments.Lethal concentration (LC50) values and median lethal time (LT50) values at 10, 25, 50, and 100 nematode concentrations for each nematode strain were estimated by probit analysis (SPSS, 2003).
Low mortality occurred during the first day after treatment except 100 nematode concentrations of some nematodes and 50 nematode concentration of H. bacteriophora WPS strain.H. bacteriophora WPS and H. zealandica EDS strains were the best performer at the first day count (Figure 1).In general, mortality rates increased from day 2 to day 4 (Figures 1 and 2).
On the second day; mortalities at 50 and 100 nematode rates reached usually over 80%.All rates of H. megidis LEX strain, 50 and 100 nematode rates of H. zealandica CHR strain, 25, 50 and 100 nematode rates of H. bacteriophora WPS strain, and 100 nematode rate of H. zealandica EDS strain killed all larvae in the treatments (Figure 1 75.6 and 92.3% mortality, respectively.They were followed by H. bacteriophora MF and HP88 strains with 73.2 and 65.5% mortality, respectively (Figure 1).There was similar trend at 10 nematode rate with 25.The lowest rate of H. megidis LEX strain which gave 100% mortality was not significantly different than H. bacteriophora WPS with 85.7% mortality.They were followed by H. zealandica CHR, H. bacteriophora HP88 and SMP, H. zealandica EDS strains with 75, 71.4,67.9 and 50% mortalities, respectively.Others produced less than 50% mortality (Figure 1).
On the 3rd day count; only same strains killed all larvae in the second day caused 100% mortality  2).
At 50 nematode concentrations, while H. megidis LEX, H. zealandica CHR, and H. bacteriophora WPS strains were producing 100% mortality, the others caused mortality mostly over 90%.All treatments were at the same group statistically.H. megidis LEX and H. bacteriophora WPS strains again were superior with 100% mortality at 25 IJs per larva and it was followed by H. bacteriophora SMP, H. zealandica CHR and EDS, H. bacteriophora HP88 an MF strains (Figure 2).
LC 50 for most of the nematodes was relatively low (˂10 IJs per larva).The LC 50 and LC 90 data are summarized in Table 1.The lowest LC 50 value was obtained by H. megidis LEX strain (3.5 IJs per larva).Virulence of H. bacteriophora WPS, HP88 and SMP and H. zealandica CHR strains were similar (LC 50 s ranging from 5.5 IJs per larva in H. bacteriophora WPS strain to 7.2 IJs per larva in H. zealandica CHR strain).The least virulent heterorhabditid was H. bacteriophora CFM strain with LC 50 value of 14.8 IJs per beet armyworm larva.The LT 50 and LT 90 data are given in Table 2.The LT 50 values ranged from 1.5 to 3.4 days for H. megidis LEX and H. bacteriophora CFM strains at 10 IJS per larva, from 1.1 to 2.6 days for H. bacteriophora WPS and H. bacteriophora CFG and CFM strains at 25 IJs per larva, from 0.8 to 2.1 days for H. bacteriophora WPS and CFG strains at 50 IJs per larva, and from 0.6 to 1.7 days for H. bacteriophora WPS and CFM strains at 100 IJs per larva, respectively.The LT 50 value of H. bacteriophora WPS strain was the smallest and it was followed by H. megidis LEX, H. zealandica CHR and EDS, H. bacteriophora SMP, HP88, MF, PD, Hb, CFG and CFM strains, respectively when the average of LT 50 values at four concentrations were evaluated.

DISCUSSION
In determining an entomopathogenic nematode as a biological control agent, it is important to look at several attributes of the agent such as attraction, penetration, movement, host defense mechanisms, and biotic and abiotic environmental factors.Although many factors are responsible for the level of infectivity, some basic data may be gathered through lab studies (Mannion and Jansson, 1992;Shapiro-Ilan et al., 2002;Laznik et al., 2011).The virulence of nematodes to noctuids and other lepidopters varies significantly (Mbata and Shapiro-Ilan, 2005;Mederios et al., 2000).Mederios et al. (2000) reported that H. bacteriophora Az33 strain caused 23% mortality to the sixth instar of another noctuid, Pseudaletia unipuncta, the armyworm at 200 IJs per larva, whereas, the mortality of H. bacteriophora strains in our research was much higher even at lower rates.This may be because of different host and difference of strain which was collected from different locality.Some heterorhabditid nematodes may possess additional positive attributes compared with others as demonstrated with ranging percent mortalities on large scales (53.6-100%) in the current study.Although the beet army worm larva was susceptible to each nematode species and strain tested, there were differences among these nematodes in their ability to kill the insect.H. megidis LEX, H. bacteriophora WPS, HP88 and SMP, H. zealandica CHR and EDS strains were more efficacious than others against S. exigua larva as it was reflected in the LC 50 , LT 50 and percent mortality data.Mortalities were higher, LC 50 values were lower and LT 50 values were shorter for these nematodes.Kim et al. (2006) reported that LC 50 value of H. bacteriophora Hamyang strain was 5.5 IJs per 4 th instar of the beet armyworm.This value is the same as LC 50 s of H. bacteriophora WPS and HP88 strains (5.5 IJs per larva) and close to H. bacteriophora SMP strain (6.1 IJs per larva) in our study.On the other hand, it was quite different than LC 50 s of other H. bacteriophora strains which ranged from 12.3 to 14.8 IJs per larva.These differences may be due to difference of the origins of the strains (Mannion and Jansson, 1992).
No statistical difference was obtained among nematode strains at 50 and 100 nematode concentrations.Therefore 10 and 25 nematode per larva were distinctive rates to differentiate the nematodes' biological efficacy on the beet armyworm.H. megidis LEX strain was superior and differed than others by having 100% mortality in all of concentrations and it was second to cause early mortality.
All the heterorhabditid strains tested showed high virulence to 4th instars of the beet armyworm, producing a significantly higher mortality (53.6-100%) at all concentrations than the untreated control at the final count.Apparently, they are effective bio-control agents of S. exigua and our results corroborate the finding of the study of Kim et al. (2006).However, environmental factors such as soil structure, temperature, humidity and host density under greenhouse and field conditions have huge impact on the efficacy of EPN (Koppenhöfer, 2000;Georgis et al, 2006).Therefore future studies need be directed to the greenhouse and field conditions with these heterorhabditid isolates.
Our results suggest that H. megidis LEX strain, H. bacteriophora WPS, SMP and HP88 strains, and H. zealandica EDS and CHR strains should be consider first to be studied further as potential biocontrol agents of the beet armyworm and other similar lepidopters.The others, H. bacteriophora PD, CFG, MF, Hb and CFM strains may also be valuable material to be studied.

Figure 1 .
Figure 1.Mean percent mortality of 4th instars of the beet armyworm after 1 and 2 d by heterorhabditid species/strain in a Petri-plate bioassay at 10, 25, 50 and 100 IJs per larva.

Figure 2 .
Figure 2. Mean percent mortality of 4th instars of the beet armyworm after 3 and 4 d by heterorhabditid species/strain in a Petri-plate bioassay at 10, 25, 50 and 100 IJs per larva.