Evaluation of Entomopathogenic Nematode Isolates against Subterranean Termites under Laboratory and Field Conditions

Ethics approval

This experiment was conducted within an appropriate ethical framework of Ethiopian Institute of Agricultural Research as confirmed by institutional letter with Ref. No.: 8.8/154/2023 written on 07 August 2023.

Description of the study area

The laboratory experiment was conducted in the laboratory of the Entomology program at Ambo Agricultural Research Center, and the field evaluation was done at Bila district, East Wollega zone which is an insect pest-prone area. Termites, Macrotermes spp., were collected and established following Addisu et al. (2014) methods from Bako areas of the insect-prone maize field. Termite mounds were dug up using a spade, and soil containing termites was put on plastic sheets. The insects were collected from the plastic sheets using a camel-hair brush and placed in plastic boxes. Wooden plants (termite-infested materials) were added to the plastic boxes as feed for the termites. The top parts of the plastic boxes were covered with a mesh cloth that allowed air ventilation. A moistened wad of cotton was placed in the plastic boxes to maintain the moisture level for the survival of termites. The boxes carrying the termites were transported to the Ambo agricultural research center’s Entomology laboratory. Periodically, dry wooden materials were provided to the termite population, and the plastic boxes were inspected for maintenance of the required moisture level.

Evaluation of EPN against termites under laboratory and field conditions

37 newly collected and preserved entomopathogenic nematode isolates were used for this experiment (Table 1). Culturing and preparation of EPN were performed using standard methods described by Kaya and Stock (1997). Harvested nematodes were kept at 14°C in 250 ml flasks and used within a week of culture preparation. Each isolate was tested at 800 infective juveniles per ml concentrations and negative control (treated with sterile water) for comparison. 10 soldier termites were placed in a petri dish on filter paper and sprayed with the infective juvenile suspension. This study was conducted under laboratory conditions at 25 ± 2°C and 60% RH in dark places. The experiment was replicated three times and laid out in a completely randomized design.

For the field experiment, Jibat variety of the crop used, and studied under natural infestation of termite. Two EPN (AEH and S#50) isolates were applied at a rate of 800 IJ/ml as a basal application at the seedling and tasseling growth stages of maize. Likewise, the recommended rate of diazinon at 60% EC (2.5 lit/ha) was applied. Untreated check plots were neither treated with the insecticide nor with isolates and recommended agronomic practices were applied. The plot size was 4 m long and 3 m wide, with intra-row spacing of 75 cm and inter-row spacing of 25 cm, with 2 m and 3 m spacing between plots and blocks, respectively. Two seeds per hill were sown, and then after emergence, the seedlings were thinned to adjust the population of the crop to one plant per hill. This is based on the maize production manuals of Ethiopia, which recommend 25 kg of maize seed per hectare (Nigussie et al., 2009). Data collection started two weeks after the first treatment application and continued every two weeks until physiological maturity of the crop. 10 plants were randomly sampled per plot to assess termite damage.

Design, treatment arrangements, and data collection

The screening of EPN isolates was laid out in a completely randomized design, and the field evaluation used a completely randomized block design. Treatments were replicated three times in the experiments. AEH and S#50 isolates that recorded complete mortality of termites were considered promising isolates from laboratory results and taken to field conditions along with positive and negative checks. The following data were collected.

Damaged root: all plants in the plot were inspected for the termite damage on maize root at harvesting time, and plant roots that showed damage from termites were counted and expressed in percentages. Damaged stem: all plants in the plot were inspected for the termite damage on the maize stem, and plants that showed damage from termites were counted. Damaged cob: at physiological maturity, all cobs of maize in the plot were checked for damage by termites and counted. Damage severity: five maize plants were randomly taken from the plot, and a 0 – 9 scale were used to assess their damage. 0 indicates the plant was not attacked by termites, and 9 indicates that the plant was eaten and lodged to ground. Lodged plants: at physiological maturity, all maize plants in the plot were checked for lodging, and the numbers of plants lodged were counted. Foraging termites: all foraging termites observed in the plot were counted. Yield: at physiological maturity, maize cobs were harvested from each plot. The threshed grains moisture content was adjusted at 11 %, maize grains were weighed to determine the amount of yield, and the plot yields were converted to kg ha^-1. The insect and crop data collected were analyzed using statistical analysis software version 9.4 (SAS, 2023). The least significant difference (LCD) at the 5% level was used to compare treatment means (Gomez and Gomez, 1984).

Screening of entomopathogenic nematodes in laboratory condition

All entomopathogenic nematode isolates screened caused mortality in termites under laboratory conditions (Abate, 2023). These isolates showed significant differences among each other and with the negative control across the exposure time (Table 1). 18% of tested isolates caused greater than 50% mortalities on the insect pest after the 3^rd day of the treatments’ application (Table 1). On this date, S#50 and AEH isolates recorded higher mortalities at 70% and 63.33%, respectively, while the lower mortalities were from the APPRC-PL0056 isolate (20%) and negative control (10%). On the other hand, half of the tested isolates recorded greater than 50% termite mortalities after the 6^th day of the treatments’ application (Table 2). But S#50 and AEH isolates achieved 90% and 80% mortality on the insect pest on the same date, respectively. 10.53% of isolates recorded greater than 75% of mortalities on insect pests after the 9^th day of their applications. Similarly, 92.11% of isolates caused greater than 50% of mortalities in the insect pest on this date. After the 12^th day of treatments applications, tested isolates showed significant differences with the negative control (p < 0.001) (Table 1). On this date, AEH and S#50 isolates achieved complete mortality on the tested insect pest and showed significant differences with other. Similarly, 31.58 % and 94.74% of tested isolates recorded greater than 75% and 50% of mortalities on the insect pest, respectively. However, S#8 isolate recorded the minimum mortality (43.33%) among the entomopathogenic nematode isolates evaluated.

This result revealed that the tested entomopathogenic nematode isolates were pathogenic to the termites and caused mortality. The effectiveness of all isolates showed an increasing trend across the exposure durations. Some isolates achieved complete mortality of the insect pest within 12 days of exposure. The finding indicated that AEH and S#50 were the more pathogenic and virulent isolates on termites under laboratory conditions. Based on this result, AEH and S#50 isolates were considered as promising under field conditions. Similarly, microbials bioagent (Metarhizium anisoplie and Beauveria bassiana) had showed promising results under laboratory conditions for Macrotemes spp management (Addisu et al., 2014). Entomopathogenic nematodes (EPNs) are helpful nematodes that have a high potential for controlling insects in soil environments and are commercially employed to treat a wide range of pests (Verma et al., 2018). In a filter paper and sand test, Shahina and Tabassum (2010) found that EPN produced increased mortalities in the subterranean termite. Yu et al. (2010) investigated the pathogenicity of three new strains of Steinernema riobrave (3-8b, 7-12, and TP) against workers of Heterotermes aureus, Reticulitermes flavipes, and Coptotermes formosanus. Heterotermes aureus was shown the most sensitive to all S. riobrave strains, and termites in all nematode treatments died after four days. The TP strain caused 75% and 91% mortality in R. flavipes and C. formosanus, respectively. More research was suggested to determine S. riobrave (TP)’s potential to control the targeted termite species in the field condition (Yu et al., 2010).

Evaluation of promising entomopathogenic nematode isolates under field condition

The effectiveness of AEH and S#50 entomopathogenic nematode isolates showed a non-significant difference in the number of foraging termites and maize root damage in maize fields (Table 3). However, treatments indicated a significant difference in stem damage, lodged plant, cob damage, the severity of insect damage, and yield (p < 0.001) (Table 3). The lower stem damage was recorded by the S#50 isolate and positive control, while the higher stem damage was recorded by the AEH isolate. Similarly, the AEH isolate and negative control (untreated plot) showed higher cob damage and lodged plants and were more severely attacked by the insect pest, whereas S#50 and positive control showed significant differences, and lower lodged plants and cobs were damaged. Similarly, negative control and AEH isolates gave a lower maize yield (2111 kg/ha), while the highest yield was obtained from positive control (Table 3).

The experiment indicated that the S#50 entomopathogenic nematode isolate showed a non-significant difference with the positive control on all considered parameters except yield data. This revealed that the isolate is more pathogenic and reduced the infestation and severity of the insect pest on the maize crop under field conditions. Additionally, the S#50 isolate increased the maize yield by 13.63% over the negative control. This result showed that the entomopathogenic nematode isolates have the potential to manage termites in the maize field. Subterranean termites’ dwell and forage in damp, cool, and shaded environments such as soil or wood materials. These characteristics are favorable for the survival and mobility of nematodes which enhances their role in termite management. Wang et al. (2002) and Yu et al.’s (2006) studies showed that entomopathogenic nematodes have the potential to be used as an environmentally safe alternative termite control method. Steinernema spp. and Heterohabditis spp. provided some limited control of subterranean termites in field studies but were largely ineffective for long-term suppression (Mauldin and Beal, 1989). However, entomopathogenic nematodes have been used in classical, conservation, and augmentative biological control strategies. In addition to other nuisance insects, commercially generated EPNs are being used to manage scarab larvae in lawns and turf, fungus gnats in mushroom production, invasive mole crickets in lawns and turf, black vine weevils in nursery plants, and Diaprepes root weevils in citrus (Lacey and Georgis, 2012). Similarly, Aslam et al.’s (2023) study indicated that Steinernema carpocapsae, Heterorhabditis bacteriophora and Heterorhabditis indica species recorded higher mortality of termites under field conditions. As a result, they suggested that indigenous EPNs can provide more effective termite control, presumably due to their direct interaction with pest species in the soil and the likelihood of secondary infection via infected cadavers.