Biological control potential of local entomopathogenic nematodes against the different stage larvae of cotton leafworm Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae)

Background: The cotton leafworm, Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), is an economic pest on various crops worldwide. Farmers generally used to apply chemical pesticides to control the pest. The bio-control potential of the entomopathogenic nematodes (EPNs) as alternatives to harmful synthetic pesticides was examined in this study. The study aimed to determine the efficacy of EPNs isolates against the different stages of larvae of the cotton leafworm S. littoralis and the effect of time on mortality rate under laboratory conditions. Results: EPNs isolates were tested at 4 different concentrations (0, 250, 500 and 1000 IJs/ml) in 150 ml plastic containers on last instar larvae (sixth instar) of S. litorallis . Experiments for third, fourth and fifth instar larvae of S. litorallis were carried out in petri dishes at different concentrations (0, 50, 100 and 200 IJs/ml) at 25 °C temperature. Mortality rates of larvae were calculated at 3 different times (48, 72, 96 h) after inoculation. The high mortality rate of last instar larvae (98.81%) of S. littoralis was caused by Steinernema feltiae (Tokat-Emir), followed by S. carpocapsae (Tokat-Bakışlı05) (95.24%) and then H. bacteriophora (11 KG) with (90.47%) at the highest concentration (1000 IJ/ml). The highest mortality rate of fifth instar larvae was caused by S. feltiae (Tokat-Bakışlı05) and S. carpocapsae (Tokat-Emir) with (100%) and (92.12%). In addition, the highest mortality rate of the 4th instar larvae was determined S. feltiae (Tokat-Bakışlı05) and S. carpocapsae (Tokat-Emir) isolates (98.87%) and (97.74%), respectively. Additionally, the highest larval mortality rate in the third stage by S. feltiae (Tokat-Bakışlı05) and S. carpocapsae (Tokat-Emir) was (100%) and (97.74%) at the highest concentration. Mortality rates of larvae were calculated at 3 different times after inoculation. The highest mortality rate counted in all isolates was determined 96 and 72 h after inoculation of EPNs. Conclusions: All indigenous EPN isolates were found to be effective at different rates against S. littoralis . The results showed that these nematode species could be used against S. littoralis biological control programs.


Background
The cotton leafworm, Spodoptera littoralis (Boisduval, 1833) (Lepidoptera: Noctuidae), is one of the most damaging lepidopterous pests in numerous commercial crops, inflicting significant economic losses on a wide range of ornamental, industrial, and crops in both Open Access Egyptian Journal of Biological Pest Control *Correspondence: myagci0645@gmail.com greenhouses and open fields (Mohamed et al. 2019). S. littoralis is difficult to be controlled due to its resistance to synthetic pesticides (Ghulam et al. 2017). Biological agents such as parasitoids, predators, and entomopathogens (bacteria, fungi, nematodes, and viruses) have been used to control several pests (Atia et al. 2016). EPNs have high adaptability to different conditions, a wide variety of insect hosts, simple mass rearing, and the ability to resist some chemical pesticides. EPNs belong mainly to the 2 families: Heterorhabditidae and Steinernematidae, which are the most economically used against several pests (Koppenhöfer 2007). Around 100 valid species of Steinernema and 21 species of Heterorhabditis have been described from different countries of the World (Bhat et al. 2020). Many factors efficacious the distribution of EPNs to several regions (Laznik and Trdan 2012).

Mass rearing of Spodoptera littoralis
Spodoptera littoralis larvae were collected from cotton fields in the vicinity of Adana, Turkey, transferred to the laboratory, and reared at 25 ± 2 °C and 65-75 RH%. The larvae were fed on an artificial (Chen et al. 2000). The ingredients were mixed in a blender and stored at 4˚C for not more than one week. Diet-fed larvae were reared in plastic boxes (10 cm height × 20 cm width × 30 cm length) containing the artificial diet. Adults were placed in the plastic cages (10 cm diameter by 20 cm high) closed on the upper extremity with tissue and were fed on 10% sugar solution (Santos et al. 2005). The culture was kept at 25 ± 2 °C, 65 ± 5% RH, and 16: 8 h (L: D) photoperiod.

Galleria mellonella larval growth
Last stage larvae of Galleria mellonella (L.) (Lepidoptera: Pyralidae) were mass-reared on a diet containing 445 g milk powder, 890 g flour, 222 g dry baker's yeast, 500 g honey, 500 g glycerin, 125 g beeswax. Diet was prepared according to Mohammed and Coppel, (1983). Glycerin, beeswax, and honey melted after adding other materials. Eggs were placed onto the food in one-liter jars and kept in an incubator at 23-24 °C.

Rearing of entomopathogenic nematodes
Entomopathogenic nematode juveniles of Steinernema feltiae (Tokat-Emir), S. carpocapsae (Tokat-Bakışlı05), Heterorhabditis bacteriophora , H. bacteriophora (11 KG) obtained from the Plant Protection Department of Gaziosmanpaşa University were used in this study. Infective juveniles of the EPNs isolates were reared on last instar larvae of greater wax moth G. mellonella according to Kaya and Stock (1997). Sixth (last) instar larvae of Galleria larvae were used for mass rearing of the EPNs juveniles at all experiments. Four different EPNs isolates (S. carpocapsae (Tokat-Bakışlı05), H. bacteriophora (11 KG), H. bacteriophora (TOK-20), S. feltiae (Tokat-Emir) were tested. Ten larvae were placed in Petri dishes (6 cm diameter) with Whatman No. 1 paper soaked with distilled water. Infective juveniles of nematode species were applied on G. mellonella larvae. The lid of the Petri dishes was wrapped by a parafilm and placed in an incubator at 23-25 °C. Larval mortality was counted and recorded daily. EPNs were collected from infected wax moth larvae using the "White trap" method (White 1927). The traps were placed for 3-7 days under the same conditions. EPN (IJs) were placed in flasks and then stored in a refrigerator at 10 °C. This process was repeated every 2 months to prevent the nematodes from losing their activity.

Bioassays
Studies were carried out in 150 ml plastic containers on last instar larvae (sixth instar) of S. litorallis, which were consisted of soil mixture (80% sand, 15% soil and 5% clay) sterilized at 121 °C (Chen et al. 1995). Last instar larvae were placed into soil mixture in plastic containers. Four different nematode concentrations (0, 250, 500 and 1000 IJs/ml) were tested. The soil moisture level was adjusted to 10% (w/w) by adding distilled water. Control plastic cups were added only distilled water. EPNs were inoculated onto the soil using a pipette in each cup. The treated plastic cups were kept at 25 °C, concentration was located onto soil and cups were covered by tulle. Laboratory trials were conducted with 10 individuals for each concentration of EPN isolate (n = 10 per EPN isolate per cont.) In each container, only one S. littoralis last stage larva was added. Trials were repeated 3 times under the same conditions on different dates. Other experiments were conducted for other stage larvae (third, fourth and fifth) S. litorallis in plastic Petri dishes (9 cm) that included artificial diet at the same conditions. Two laboratory-reared larvae of S. litorallis, taken from the stock culture, were placed in a new Petri dish. EPN isolates were prepared, using distilled water at 0, 50, 100, and 200 IJs/ml for third, fourth and fifth instar larvae and applied directly into the Petri dish and then covered with a parafilm. All experiment was repeated 3 times at different dates. Experiments were conducted under the laboratory conditions (25 °C and 65% RH). Mortality rates of larvae were calculated at 3 different times (48, 72, 96 h) after inoculation. Dead S. littoralis larvae were placed using the White trap method. After one week, insect cadavers were examined in distilled water under a stereomicroscope. EPNs juveniles were obtained from each infected S. littoralis larvae.

Data analysis
Data obtained in the trials were converted to measurements' percentage, transformed using arc-sin transformation. Comparisons between concentrations were done using Duncan's range comparison test. And then analyzed with analysis of variance (One-way ANOVA). Comparisons between concentrations were done using Duncan's range comparison test. All statistical analyses were made SPSS 23.0 package program (IBM Corp 2013).

Results
Results were evaluated, 48, 72, 96 h after EPNs inoculation. The interaction among the parameters (isolates and doses)(times and isolates) investigated was statistically significant in the study. The results showed differences among the nematode concentrations and isolates.
According to the results, EPNs concentrations against larvae of S. littoralis were found more effective than controls (soils treated with distilled water). In addition, it was observed that the mortality rate increased proportionally as the exposure time increased. When the effect of time on larval death was examined, it was determined that the highest mortality rate was within 96 h post-infection.

Discussion
Obtained results revealed that EPNs virulence differed significantly among EPN species. According to results, the highest mortality rates were found in S. carpocapsae (Bakışlı05) and S. feltiae (Tokat-Emir) isolates, followed by H. bacteriophora (11 KG),, respectively. In addition, the highest mortality rate was observed on S. littoralis larvae 72 and 96 h after inoculation and mortality started from the first count in the trials. This study may provide the basis for