Efficacy of Thai indigenous entomopathogenic nematodes for controlling fall armyworm (

Background: Under laboratory and greenhouse conditions, the virulence of 2 isolates of Thai indigenous entomopathogenic nematodes (EPNs) in controlling the fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith) (Lepidoptera; Noctuidae), was demonstrated. Six EPNs dosages were tested against 2 larval instars of FAW under the laboratory conditions, while 2 different concentrations were tested under the greenhouse conditions. Results: The results of a laboratory experiment revealed that 2 Thai indigenous EPNs isolates (Heterorhabditis indica isolate AUT 13.2 and Steinernema siamkayai isolate APL 12.3) were efficient against the FAW, 2nd and 5th larval instars. Six different nematode concentrations (50,100, 150, 200, 250 and 300 infectious juveniles (IJs) ml−1) were evaluated, and all were proven to be effective, with the mortality rate associated with concentration. Inoculated larvae in the 2nd instar was more vulnerable than that in the 5th instar. H. indica isolate AUT 13.2 was more destructive than S. siamkayai isolate APL 12.3. The greatest mortality rate of 2nd instar larvae was 83% when H. indica AUT 13.2 was applied at the concentration of 250 IJs ml−1, and 68% when the nematode S. siamkayai APL 12.3 was used at the concentration of 300 IJs ml−1. At 250 IJsml−1, the highest mortality rate of the 5th instar larvae was 45% for H. indica AUT 13.2 and 33% for S. siamkayai APL 12.3, respectively. To customize the concentration and volume of nematodes suspension evaluated in the greenhouse settings, the most sensitive stage of FAW and the optimum concentration that caused the highest mortality were used. The concentrations of both indigenous nematodes’ isolates were 20,000 and 50,000 IJsml−1 per pot, respectively, and the results showed that the mortality rates were lower than that in the laboratory. FAW mortality rate was the highest (58%) in case of the nematode H. indica isolate AUT 13.2, against (45%) in case of S. siamkayai isolate APL 12.3, at the 50,000 IJs ml−1 concentrations. Conclusions: The study revealed the 2 Thai indigenous EPNs isolates (H. indica isolate AUT 13.2 and S. siamkayai isolate APL 12.3) were capable of controlling the FAW in both laboratory and greenhouse environments. The 2 Thai EPNs showed the potential to be considered as a biological control agent.


Background
In Thailand, maize forms an essential part of food and feed system and contributes significantly to income generation for rural households (Ekasingh et al. 2004). Fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is indigenous to tropical and subtropical regions of the Americas (Day et al. 2017). This pest has not been reported outside America until 2016, when it was reported for the first time in Africa (Goergen et al. 2016). Since its invasion, the pest is causing considerable economic losses in Africa (De Groote et al. 2020). This pest was first detected in Thailand in December 2018, in a few sub-districts of Kanchanaburi and Tak provinces in the west of the country, near the Myanmar border (IPPC 2018). This insect is a polyphagous pest that has been documented to impact over 353 plant species from 76 plant families (Montezano et al. 2018). They are said to be a significant economic pest in maize and other Poaceae crops (Silva et al. 2017). Younger larvae feed on the tissue of the leaves, while older ones cause severe defoliation. It also burrows into the growing point (bud, whorl, etc.), leading "dead heart," wilting and death of the unfurled leaves (Day et al. 2017).
Pesticides are currently being utilized to control and minimize the spread of FAW in maize crops. Chemical pesticides may reduce insect pest attacks in the short term, but they may not be sustainable in the long run. Many studies have showed insecticide-resistant populations in FAW (Yu et al. 2003). Furthermore, synthetic chemical pesticides have the potential to harm both humans and the environment (Carvalho 2017). As a result, a new method of controlling FAW is required to decrease the damage caused by this destructive insect pest. Entomopathogens, such as bacteria, nematodes, fungi and virus, are an essential option for the management of diverse arthropod species and are appropriate approaches for a long-term sustainability of the ecosystem (Charnley and Collins 2007). Entomopathogenic nematodes (EPNs) are roundworms that live as parasites in insects (Lacey and Georgis 2012). Out of 23 nematode families, Steinernematidae and Heterorhabditidae are the two most prevalent nematode families studied as biological agents (Lacey and Georgis 2012). The two most important genera within the family are Steinernema and Heterorhabditis. All Steinernema species are symbiotic with Xenorhabdus bacteria, whereas all Heterorhabditis nematode species are symbiotic with Photorhabdus bacteria (Boemare et al. 1993). These symbiotic bacteria are vital in the death of insect hosts (Hominick et al 1996). These EPNs have a wide host range, making them a viable option for biological insect pest control (Arthurs et al. 2004). The EPNs can be used solely as a biological control agent or combined with other biocontrol agents, such as entomopathogenic bacteria and fungi in order to improve their efficacy in controlling insect pests (Laznik et al. 2012).
In the Americas and freshly invaded areas like Africa, FAW larvae have been found to be vulnerable to EPNs species (Lacey and Georgis 2012). Noosidum et al. (2010) documented the existence of EPNs in Thailand. Other researchers have recently discovered several species of those indigenous entomopathogenic nematodes in Thailand (Thanwisai et al. 2021).
The goal of this study was to test the efficacy of several Thai indigenous EPNs isolates against the FAW in both laboratory and greenhouse environments.

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.

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 Page 3 of 7 Wattanachaiyingcharoen et al. Egyptian Journal of Biological Pest Control (2021) 31:149 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.
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.

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 Observed mortality = Total number of dead larvae Total exposed larvae × 100 Corrected Mortality = %test mortality − %control mortality 100 − %control mortality × 100 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. The efficacy of H. indica isolate AUT 13.2 and S. siamkayai isolate APL12.3 against the 5th instar larvae of FAW gave a high mortality percentage when they were treated with a high density of the EPNs ( Table 2). The mortality percentage was statistically significant between treatments as determined by one-way ANOVA (F (6, 21) = 13.75, p = 0.05 for H. indica isolate AUT 13.2 and F = 39.25; df = 6, 21; p = 0.05 for S. siamkayai APL 12.3, respectively). The highest mortality occurred in the FAW larvae (45.0%) when they were infected by the EPN H. indica isolate AUT 13.2 at the density of 250 IJs ml −1 , which was non-significantly different from the application at 300 IJs ml −1 . The EPN, S. siamkayai isolate APL12.3 caused the highest mortality percentage when they were infected with 250 IJs ml −1 (at 32.5%), but was not different from those at the density of 300 IJs ml −1 (42.5%). However, when compared to the mortality of 2nd instar larvae, this larval stage had a low mortality rate.

Efficacy of EPNs in the greenhouse
By comparing mean percentage mortality of FAW larvae among the treatments at the end of 10 days, the efficacy of H. indica isolate AUT13.2 and S. siamkayai isolate APL12.3 on mortality rate of FAW larvae at the 2nd instar larvae under greenhouse conditions was determined. After the application for 10 days, the results showed nonsignificant difference between those two EPN isolates (Table 3) at the EPN density at 20,000 IJs ml-1 . The EPN H. indica isolate AUT 13.2 caused a high mortality rate (37.92%), while S. siamkayai isolate APL12.3 killed only (28.75%) (F = 28.63; df = 2, 21; p = 0.000). Mortality percentages of the FAW raised when the EPN density was increased to 50,000 IJs ml −1 , and significant difference between the two isolates was observed. H. indica isolate AUT 13.2 had a high mortality rate (57.78%), compared to (44.72%) S. siamkayai isolate APL 12.3 (F = 64.09; df = 2, 21; p = 0.05).

Discussion
Entomopathogenic nematodes (EPNs) have already been found to be effective against a wide range of insect pests. They have been widely utilized to manage pests both below and above ground (Bhairavi et al. 2021). Many researchers previously reported the FAW's susceptibility to EPNs (Caccia et al. 2014). However, the efficacy of EPNs is governed by their virulence and their capability to find out their hosts (Cutler and Webster 2003). In Thailand, several isolates of indigenous EPNs have been documented (Thanwisai et al. 2021). This is the first study evaluating the efficacy of Thai indigenous EPNs; H. indica isolate AUT13.2 and S. siamkayai isolate APL12.3 against the FAW. The result of the study demonstrated that H. indica isolate AUT 13.2 and S. siamkayai isolate APL12.3 were capable of infecting and killing both the 2nd and 5th larval instars of FAW under laboratory conditions. Both isolates showed varied efficacy depending on the different concentrations used. The isolate H. indica AUT 13.2 caused more mortality larvae than S. siamkayai APL 12.3. With an increase in the density of infective juveniles per milliliter of pre-sterilized distilled water, there was a proportional increase in FAW larval mortality; however, increasing concentration above 250 IJs ml −1 had non-significant difference. The 2nd instar larvae were particularly more sensitive to both EPN isolates examined.
Environment, stages of the host and innate characteristics of the nematodes, like the capability to find the host and the presence of symbiotic bacteria, all influence the potentials of EPNs (Batalla-Carrera et al. 2010). The discrepancies in efficiency reported in the laboratory where most settings are controlled and uniform may be attributable to the potential of the nematode isolates to infect the host and the efficacy of the symbiotic bacteria that is responsible for killing the host. Presence of symbiotic bacteria Photorabdus and Xenorhabdus in the nematode H. indica isolates AUT 13.2 over S. siamkayai isolate APL 12.3 in this study may explain the increased mortality reported by H. indica isolates AUT 13.2 over S. siamkayai isolate APL 12.3. As a result, it appears that the ability of H. indica isolates AUT 13.2 to kill FAW larvae over S. siamkayai isolate APL 12.3 was the most likely owing to the symbiotic bacterium Photorabdus contained within these EPNs. The mortality of FAW larvae has been demonstrated to vary in previous studies using different strains and isolates of Heterorhabditis spp. and Steinernema spp. Acharya et al. (2020) investigated the effectiveness of 7 EPN species and found that H. indica, S. carpocapsae, S. arenarium and S. longicaudum were highly virulent against various stages of the FAW larvae.
In the present study, the 2nd instar FAW larvae were more vulnerable to both isolates of EPNs than the 5th instar larvae. Some authors have reported differences in susceptibility of different stages of the FAW. For example, Acharya et al. (2020) reported that younger larvae (e.g., first-, second-and third-instar larvae) of the FAW were more susceptible to H. indica and S. carpocapsae, while elder larvae (e.g., 4th, 5th and 6th larval instars) were susceptible to S. arenarium and S. longicaudum.
The greenhouse experiment was applied to test how effective EPNs are on 2nd instar larvae that feed on maize plants. The results demonstrated that the EPN isolates were successful in killing the FAW larvae; however, the mortality rate was lower than in the laboratory. It is possible that the lowest mortality of FAW larvae in the greenhouse experiment was attributable to the difference in environmental conditions. UV light, warmer temperatures, desiccation and the features of exposed foliage can promote lesser EPN activation in greenhouse circumstances (Vashisth et al. 2013). These features make it more difficult for infective juvenile nematodes to locate the host larvae, because EPNs' adequate host interaction is crucial in infecting and killing larvae (Kaya and Gaugler 1993). Furthermore, because FAW larvae were placed on maize plants and had the freedom to move from plant to plant, EPNs may have had a difficult time making good host contact and attacking them. The mortality of FAW larvae was a high when the EPNs' concentrations increased to 50,000 IJs ml −1 , and the EPNs application targeted the feeding larvae site. However, in field conditions, some environmental factors, especially soil moisture and temperature, may affect the efficiency of those EPNs as they need some moisture for survival. According to obtained results, under the greenhouse conditions, the rate and application frequency of EPNs increased, according to providing adequate moisture, to achieve better outcomes. There are many ways to optimize the efficacy of EPNs for greenhouse and field application like preparing EPNs formulation. EPNs can be combined with other control agents which can be some appropriate chemical insecticides and other biological control agents like fungus and bacteria (Koppenhöfer et al. 2020). The effectiveness of EPNs can also be improved by genetic improvement through selection and transgenic methods (Abd-Elgawad 2019). The efficacy of the 2 isolates used in the present study can be improved by developing the formulations or combining EPNs with other control agents.

Conclusions
Both isolates of Thai indigenous EPNs, H. indica isola-teAUT13.2 and S. siamkayai isolate APL12.3, evaluated against FAW larvae under both laboratory and greenhouse situations. The EPNs performed much effective in the laboratory than in greenhouse. Virulence