Fall armyworm collection and rearing
Fall armyworm larvae were collected from maize fields in Thailand's Phitsanulok (16°49′29.32" N, 100°15′30.89" E, elevation: 50 msl.), Sukhothai (17° 19′1.6608" N, 9°33′42.12" E, elevation: 93.54 msl.) and Uttaradit provinces (17°54′1.4537" N, 100°30′48.89" E, elevation: 108 msl.). The larvae were identified and confirmed according to the identification procedures provided by Visser (2017). Larvae were placed in a 20-ml plastic container and fed on fresh maize leaves grown without the use of chemical pesticides. The pupae were collected and placed in a plastic container inside a rearing cage (30 × 30 × 30 cm) once they had developed. Adults were fed on a 10% sugar solution in a rearing cage when they emerged, and then transferred for the experiments. For egg-laying, a young plant was dipped in a glass of water and placed inside the chamber. The larvae were transferred for the experiments when they had reached the relevant stages.
Entomopathogenic nematodes collection and multiplication
The study used 2 EPN isolates, Heterorhabditis indica isolate AUT 13.2 and Steinernema siamkayai isolate APL 12.3. These EPNs were collected from agricultural areas which H. indica isolate AUT13.2 was collected from a mango orchard (17°26′13.4" N, 100°05′40.4" E, elevation 57 msl.), while S. siamkayai isolate APL12.3 was collected from a vegetable garden (17°02′12.8" N, 100°10′01.0" E, elevation 48 msl.). Final instar larvae of the Greater wax moth, Galleria mellonella L., were used to multiply the EPNs. The White trap technique (White 1927; Kaya and Stock 1997) was utilized to obtain infective juveniles (or IJs) of the EPNs from dead larvae to be used in the experiments.
Testing of the efficacy of EPNs in the laboratory
Experimental design and application of the EPNs
The experiment was designed in a completely randomized design (CRD), with 6 treatments consisting of 6 different numbers of IJ nematode suspension, namely: 50, 100, 150, 200, 250 and 300 IJs ml−1, and a control consisting of the same volume of sterilized distilled water. The 2nd and 5th larval instars were tested separately on each EPN isolate and nematode suspension. The tests were repeated 4 times (4 replications), with 10 larvae/replication. Individually, the larvae were placed in a 5.5-cm-diameter Petri dish with a detached maize leaf as food. One milliliter of nematode suspension containing a different density of IJs was applied topically to the larvae and maize leaf in each treatment, with a similar application in the control treatment. The food was changed daily and the larvae were kept at 25 °C under a 14:10 (light: dark) photoperiod with 60 ± 10% relative humidity in the insect rearing room.
Assessment of mortality
Mortality of the larvae was assessed 48 h after inoculation, and the observations were recorded for 10 days. When larvae failed to respond to the forceps' touch, they were marked as dead. The dead larvae were kept separately to observe emergence of nematodes from the cadaver using the White trap technique. Only those larvae that showed evidence of nematode emergence were recorded as nematode killed. The following formula was used to compute the 10-day accumulated mortality percentage of the tested samples.
$$Observed\; mortality = \frac{Total \;number \;of\; dead\; larvae}{{Total \;exposed \;larvae}} \times 100$$
The tests were rejected if the control treatment mortality was more than 20%. When control mortality was less than 20%, Abbott's (1925) formula was used to correct observed mortality, as shown below.
$$Corrected\;Mortality = \frac{\% test\; mortality - \% control\; mortality}{{100 - \% control\; mortality}} \times 100$$
Testing of the efficacy of EPNs in the greenhouse
Planting maize in greenhouse and release of the FAW
Super-sweetcorn maize variety was grown in the earthen pot (50 cm in diameter), and planting soil was added in a pot at 2/3 in each pot’s capacity. Initially, 10 maize seeds were grown in each pot and watered daily and the seedlings were reduced to 5 per pot after germination. The seedlings were ready to utilize in testing when they reached the 4 leaf fully emerged stage, which took around 2 weeks following emergence. Prior the larvae reach their 2nd instar, 10 fully grown 1st instar larvae were manually placed into the maize plant in each container with some detached maize leaves. The pots were caged and covered vertically and from the top with insect mesh when they were infested.
Experimental design and application of the EPNs
The greenhouse experiments were carried out using a randomized complete block design (RCBD). There were 3 treatments, each with 2 different densities of EPNs, 20,000 IJs ml−1, 50,000 IJs ml−1, and a sterile water as a control. Each treatment was carried out 8 times. Because the FAW was in its dispersal stage, only 2nd instar larvae were chosen to be studied. The nematode suspension was applied 24 h after the FAW larvae were released. Using a hand sprayer, each pot was sprayed by 100 ml of the described densities of EPNs suspension directly on the entire leaves. EPNs were applied 3 times, with two-day interval between applications. In the greenhouse experiment, the assessment of larval mortality followed a similar procedure to that used in the laboratory experiment.
Data analysis
Mortality percentages the FAW caused by the EPNs both from the laboratory and the greenhouse conditions were normalized using square root transformation. The number of dead larvae in different treatments (density of EPNs) was subjected to statistical analysis of variance (ANOVA). The mean comparison of the laboratory condition was made using Duncan multiple range tests (DMRT) to find a significant difference between treatments (p ≤ 0.05). The Tukey test was carried out to determine a significant difference of the means between treatments (p ≤ 0.05) of the greenhouse experiment.
