Susceptibility of immature Telenomus remus, an egg parasitoid of Spodoptera frugiperda (J.E. Smith), to entomopathogenic fungi from South Sumatra, Indonesia

Abstract

Background

The fall armyworm (FAW) Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) is a newly introduced pest that damages maize production in Indonesia. To control this pest in maize fields, better solution is to use the egg parasitoid, such as Telenomus remus Nixon (Hymenoptera: Scelionidae), as another better option to apply topically entomopathogenic fungi (EPF). Therefore, it is necessary to study the effect of the EPF on the egg parasitoid of T. remus. The objective of this research was to evaluate susceptibility of immature T. remus to the EPFs, Beauveria bassiana, Chaetomium sp., Curvularia lunata, Penicillium citrinum, and Metarhizium anisopliae. The EPFs (1 × 10⁶ conidia mL⁻¹) were sprayed topically on one-day-old mummies (immature T. remus) in post-parasitism periods.

Results

The results showed that the cumulative percentage of T. remus adult emergence from the mummies treated with EPF on 11 days after treatment ranged 54–100% and was non-significantly different than those of control (untreated with EPF) (90.48%). Therefore, the immature stage of T. remus was not susceptible to the EPF topical application. The EPFs were harmless to the immature stage of T. remus. Percentage of aborted mummies (embryonic death) of T. remus after treated with the EPF was also non-significantly different than those of control. However, the EPFs could significantly affect developmental times of immatures stages of T. remus. The EPF also could shorten the adult longevity of the egg parasitoid.

Conclusions

The immature T. remus is less sensitive to the EPFs; B. bassiana, Chaetomium sp., C. lunata, P. citrinum, and M. anisopliae. It can be considered integrating the EPF with T. remus inundation in maize field. However, it is necessary to limit the topical application of the EPF to avoid negative effects on the adult longevity of the egg parasitoid. Thus, it needed to be further investigated that the application of the endophytic EPFs by inoculating the fungi within the plant tissue could be harmless to the egg parasitoids.

Background

The fall armyworm (FAW), Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae), is a new pest introduced to Indonesia, West Sumatra in March 2019 (Sartiami et al. 2020). The pest originates from South America. However, it has now spread to several provinces in Indonesia, including South Sumatra (Hutasoit et al. 2020), Bengkulu (Ginting et al. 2020), West Java (Russianzi et al. 2021), and Bali (Supartha et al. 2021). The pest is polyphagous and it has more than 353 species from 76 host plant families in the world (Montezano et al. 2018). In Indonesia, damage can reach 100% in maize attacked at the beginning of vegetative stage. Two strains of this pest have been found in Indonesia, namely the rice and corn strains (Herlinda et al. 2022b). S. frugiperda is destructive in the larval stage and can destroy the leaves, shoots, and growing points of maize or corn plant (Zea mays L) (Herlinda et al. 2022b). In order to reduce the FAW population, it must be controlled from the egg stage by using egg parasitoids, and if there are still some escaping into larvae, control is continued in the larval stage by using entomopathogenic fungi (EPFs).

Each of the life stage of FAW has natural enemies, such as parasitoids (Agboyi et al. 2020), entomopathogens (Herlinda et al. 2022a), and predatory arthropods (Anandhi and Saminathan 2021). Previous study in South Sumatra, Indonesia showed that S. frugiperda eggs could be parasitized by egg parasitoids, such as Telenomus remus Nixon (Hymenoptera: Scelionidae), Trichogramma spp. (Hymenoptera: Trichogrammatidae), and larval parasitoids, such as Chelonus formosanus Sonan, C. oculator F., C. annulipes Wesm. and C. cautus (Cresson) (Hymenoptera: Braconidae),. T. remus is most dominantly found attacking S. frugiperda eggs in Indonesia (Herlinda et al. 2023) and other countries (Kenis et al. 2019). Other larval parasitoid species have been found attacking the larvae of FAW was Cotesia ruficrus (Haliday) (Hymenoptera: Braconidae) (Gupta et al. 2019). Use of the EPFs to control S. frugiperda in maize fields can damage the parasitoids, particularly the egg parasitoids exposed on the leaf surface. Previous research showed that the EPF did not harm pupae and adults of egg parasitiods (Battisti et al. 2022), but synthetic insecticides apparently harmed the parasitoids (Amaro et al. 2018). Other study reported that azadirachtin-based insecticides also did not harm an egg parasitoid, Trichogramma minutum Riley (Lyons et al. 2003). However, information on the adverse effects of the EPF on immature stages of egg parasitoids is limited.

Abundance of parasitoids and the effectiveness of the EPF in controlling S. frugiperda larvae in maize fields need to be integrated to support the implementation of Integrated Pest Management (IPM). Therefore, it is necessary to investigate whether the spraying of the EPF can have a negative impact or even synergy with the EPF. For this reason, it is necessary to find isolates that kill S. frugiperda larvae but do not kill egg parasitoids. The results of the previous research showed that there were 10 isolates spread across several EPF species, namely Beauveria bassiana (Balsamo) Vuillemin, Chaetomium sp., Curvularia lunata (Wakker), Penicillium citrinum Thom., Metarhizium anisopliae (Metschn.) Sorokin causing the highest mortality against larvae of S. frugiperda (Herlinda et al. 2021a). The fungal isolates have never been studied whether they selectively kill only pest larvae or can even kill parasitoid eggs. Therefore, it is necessary to investigate the sensitivity of egg parasitoids to the EPF. The novelty of this research is that looking to the EPF isolates that do not have a negative impact on the immature stage of the egg parasitoids, but are still effective in killing S. frugiperda, so that in the future they can be integrated into an integrated pest management (IPM) approach in maize fields. A pest control approach that successfully combines the release of egg parasitoids and spraying of the EPF is an integrated management approach that is safe and sustainable (Dean et al. 2012). The present research aimed to evaluate susceptibility of immature T. remus to the EPF, B. bassiana, Chaetomium sp., C. lunata, P. citrinum, and M. anisopliae.

Methods

Preparation and reculture of entomopathogenic fungi

This research was carried out at the Entomology Laboratory, Department of Plant Protection, Faculty of Agriculture, Universitas Sriwijaya, Indonesia from March to September 2023. The fungal species isolates consisted of ten isolates were JgSPK isolate of B. bassiana (acc. No. MZ356494), JaGiP isolate of B. bassiana (acc. No. MZ356495), JaSpkPGA(2) isolate of B. bassiana (acc. No. MZ356496), JgCrJr isolate of B. bassiana (acc. No. MZ356497), and JaTpOi (1) isolate of B. bassiana (acc. no. MZ356498), PiCrPga isolate of Chaetomium sp. (acc. no. MZ359735), JaMsBys isolate of C. lunata (acc. no. MZ359819), JaSpkPga(3) isolate of C. lunata (acc. no. MZ359818), JaTpOi(2) isolate of P. citrinum (acc. no. MZ359812), and CaTpPga isolate of M. anisopliae (acc. no. MZ242073) (Table 1). Our previous research has isolated the ten isolates of the EPF from plants in South Sumatra, and the isolates had been identified molecularly (Herlinda et al. 2021a). The fungi were confirmed as endophytic EPF because they were able to colonize plants as endophytes and to kill host insect as entomopathogens (Herlinda et al. 2021a). The EPF were first cultured by modifying the method of Sumikarsih et al. (2019) by adding Tenebrio molitor L. flour in to a Petri dish (Ø 9 cm) containing agar medium (SDA, Sabouraud Dextrose Agar), then incubated for 14 days. Finally, the fungi were re-grown in SDA medium. In a laminar flow cabinet, fungal solutions were prepared using agar medium (65 g of SDA in 1 L of sterile distilled water). The medium was boiled to dissolve it completely and then sterilized by autoclaving at 121 °C for 20 min. The fungus with a diameter of 10 mm developed in Petri dishes were added and incubated for 14 days in room temperature.

Table 1 Isolates and species of entomopathogenic fungi from South Sumatra, Indonesia used in this research

Mass-rearing of egg parasitoid and Spodoptera frugiperda

The initial culture of egg parasitoid, T. remus was collected from maize planting centres in Ogan Ilir Regency (3°1′12″S, 104°28′48″E) of South Sumatra. T. remus was collected from S. frugiperda eggs on maize plants, then the egg parasitoid species was identified morphologically by an insect taxonomist from Universitas Sriwijaya, Dr. Chandra Irsan. Based on observations in the field, T. remus was the most dominant species found. Therefore, it was the most abundant species that was used in this research. The parasitoid was maintained and mass-reared in the laboratory until it reached the third generation, after that it was used in the experiments. Mass-rearing of the T. remus was carried out on the eggs of the factitious host, Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae) following the method of Chen et al. (2021). To prepare the colony of C. cephalonica, its larvae was fed on chicken feed and maize meal.

Mass-rearing of S. frugiperda modified the method of Faddilah et al. (2022) and was carried out in the laboratory at room temperature (27 ± 2 °C) and 82 ± 5% relative humidity (RH) Light was controlled at a photoperiod of 12: 12 (light: dark) hours. Larval colonies were taken from laboratory collections that had been cultured for many generations and had been molecularly identified by Herlinda et al. (2022b). Larvae were reared individually in plastic containers with covers (Ø 6 cm high 4 cm) because the third until last instars were cannibalistic. The 1st instar larvae were fed leaves of pigweed (Amaranthus hybridus L.) and the 2nd instars were fed fresh corn leaves, then the 3rd to 6th instars were reared on an artificial diet. Artificial diet was made following the method of Sreelakshmi and Mathew (2017). More than 100 prepupae were placed in a Petri dish (Ø 12 cm) containing sterile soil (10 mm thick) for pupae habitat, the Petri dish was transferred to a transparent wire mesh cage (50 × 50 × 50 cm³) containing a 7-day-old maize plant for adults (emerging from the pupae) to lay eggs (Sari et al. 2022).

Observation of the sensitivity of egg parasitoids to entomopathogenic fungi.

To measure the level of sensitivity of egg parasitoids to the EPF, variable development of the immature parasitoid has been observed, modifying the method Potrich et al. (2017). Cards (1.5 × 11 cm) attached to a egg mass (containing 50 eggs) of S. frugiperda were placed in a test tube (Ø 3.0 cm, 20 cm long) and ten mated females of T. remus (less than 24-h-old) were released into the tube and allowed to lay eggs for 24 h. The experiment was conducted at room temperature (27 ± 2 °C) and 82 ± 5 RH %. The card containing parasitized S. frugiperda eggs (1-day-old mummies) was topically dripped with 1 mL of fungal suspension (concentration 1 × 10⁶ conidia.mL⁻¹) (post-parasitism), dried in air and then placed in a sterile test tube (Ø 3.0 cm, 20 cm long). The fungal concentration (1 × 10⁶ conidia.mL⁻¹) was chosen because it could kill T. remus host (S. frugiperda). For the control containing parasitized S. frugiperda eggs (1-day-old mummies), 1 mL of sterile distilled water was dripped. Changes in the color of S. frugiperda eggs were observed daily until 6 days after application, and the emergence of parasitoid adults was recorded daily for 11 days (after all adults had emerged). This experiment used a completely randomised design with 10 fungal treatments (isolates) and control (11 treatments in total) and was repeated four times. The variables observed were changed in color of S. frugiperda parasitized eggs, developmental times of immatures stages of T. remus, percentage of aborted mummies of T. remus, the percentage of T. remus adult emergence, adult longevity, and sex ratio. To confirm that dead mummies were infected with fungus, the mummies were placed in a Petri dish containing agar-water medium and incubated for 7 days, and fungus-infected mummies were recorded.

Data analysis

Differences in data on developmental times of immature stages of T. remus, percentage of aborted mummies of T. remus, the percentage of T. remus adult emergence, percentage of parasitized eggs, adult longevity, and sex ratio among treatments (10 treatment isolates and control) were analyzed using analysis of variance (ANOVA). If a difference was found, continue with the Tukey’s test (P < 0.05), but if there is no difference, Tukey’s test was not performed.

Results

Developmental times of immatures stages of Telenomus remus

The morphology of healthy, unparasitized S. frugiperda eggs is greenish white, whereas parasitized eggs are grey to black (Fig. 1). S. frugiperda eggs containing immature stages of the parasitoid, T. remus began to show a color change to grey when the mummies were two days old, and all eggs could be black when the mummies were 5-day old (Table 2). The color of the mummies in both the control (which was dripped with sterile distilled water) and the fungal treatment showed non-color difference. No mycelia or conidia were found in the mummies treated with the EPF (B. bassiana, Chaetomium sp., C. lunata, P. citrinum, M. anisopliae).

Fig. 1

Morphology of Spodoptera frugiperda eggs: healthy (A), unhealthy (parasitized) (B)

Table 2 Changes in color of Spodoptera frugiperda eggs after treated with EPF (1 × 10⁶ conidia.mL⁻¹)

Percentage of parasitized eggs after treatment with the EPFs (1 × 10⁶ conidia.mL⁻¹) were not significantly different from those of the control (untreated with EPF) (P > 0.05). However, developmental times of immature stages of T. remus treated with the EPF was significantly different from those of the control (untreated EPF) (P < 0.0001) (Table 3). Developmental times of immature stages of T. remus were calculated from the time the adult females lay their eggs until the new adults emerge from their mummies. The developmental times of the immature stages of T. remus ranged from 164.98 h (6.87 days) to 221.58 h (9.23 days). The longest developmental times of the immature stages occurred on T. remus treated with B. bassiana JaTpOi(1) isolate and was significantly different from those of other treatments (P < 0.0001). The shortest developmental times of the immature stages occurred on T. remus treated with B. bassiana JgSPK isolate and was non-significantly different from those of B. bassiana JaSpkPGA (2) and JgCrJr isolates, M. anisopliae CaTpPga isolates, and control (untreated fungus).

Table 3 Parasitized eggs of Spodoptera frugiperda and developmental times of immatures stages of Telenomus remus after treated with EPF (1 × 10⁶ conidia.mL⁻¹)

Aborted mummies and emergence of Telenomus remus adults

The percentage of T. remus adult emergence from eggs sprayed with EPF (1 × 10⁶ conidia.mL⁻¹) was non-significantly different from those of the control (untreated EPF) (P > 0.05) (Table 4 and 5). On the last day of observation, the percentage of T. remus adult emergence varied from 54.00 to 100% and was non-significantly influenced by the application of the EPF. The percentage of aborted mummies of T. remus after treatment with the EPF (1 × 10⁶ conidia.mL⁻¹) was non- significantly different from that of the control (untreated with EPF) (P > 0.05) (Table 6). The high percentage of aborted mummies in both fungal treatment and control indicated that aborted mummies were not caused by the influence of the EPF application. All aborted mummies (fungal treated and untreated) were grown in water-agar medium to detect the fungal infection, however no indication of the mummies infected by the EPF was found. The longest adult longevity of T. remus occurred on the immature stage untreated with the fungi (control) was significantly different from those of treated with EPF (P < 0.0001) except those of C. lunata JaSpkPGA (3) isolate. The sex ratio of the control (untreated EPF) was non-significantly different from those of treated EPF (P > 0.05).

Table 4 Percentage of Telenomus remus adult emergence after its mummies treated with EPF (1 × 10⁶ conidia.mL⁻¹) on one up to six days after fungal aplication

Table 5 Percentage emergence of Telenomus remus adults after its mummies treated with EPF (1 × 10⁶ conidia.mL⁻¹) on seven up to 11 days after fungal aplication

Table 6 Percentage of aborted mummies, adult longevity, and sex ratio of Telenomus remus after treated with EPF (1 × 10⁶ conidia.mL⁻¹)

Discussion

The percentage of parasitized eggs was non-significantly different, and it indicated that fungal treatments did not affect preference of adult T. remus in parasitising eggs of S. frugiperda. In line with previous study, if no-choice parasitism experiment was carried out, it could force the parasitoid to parasitise the host egg provided (Potrich et al. 2017). The color of parasitized eggs began to be grey or black when the mummies were two days old. In present study, the parasitized eggs of S. frugiperda turned to be black when the mummies were 5-day old. The previous study showed that if the parasitized host eggs became dark or black color or melanised eggs, it indicated that parasitoid embryos or immature progenies were developing within the host egg (Lyons et al. 2003). The results of the present study showed that until the 12th day of observation (11 days after application), neither conidia nor hyphae/mycelia of the EPF were found on eggshells of S. frugiperda and aborted mummies (fungal treated and untreated) grown in water-agar medium. The high percentage of aborted mummies in untreated (control) and the fungal treated mummies was not caused by the EPF infection, but it could be due to genetic or internal parasitoid factors, future research is needed to confirm it. Previous research also showed that no indication of fungal presence was found within the tissues of an egg parasitoid (Trichogramma pretiosum Riley) treated with the fungus (M. anisopliae) (Potrich et al. 2017).

The EPF could affect the developmental times of the immature stages of T. remus. B.bassiana JgSPK isolate and B. bassiana JaSpkPGA (2) and JgCrJr isolates, and M. anisopliae CaTpPga isolates could reduce the developmental times of the immature stages of T. remus. However, B.bassiana JaTpOi(1) isolate could prolong the developmental times of the immature stages of T. remus. Previous experiment showed that no difference in the developmental times of the immature stages of T. pretiosum from eggs sprayed with M. anisopliae (Unioeste 22 strain) (Potrich et al. 2009). The prepupal stage (72 h post-parasitism) of T. pretiosum did not influence the developmental times of the immature stages (Potrich et al. 2017). However, in this present research, the EPF sprayed to the 24 h post-parasitism eggs indicated that the 24 h post-parasitism eggs were more sensitive to the EPF. So that, it could alter the duration of the T. remus immature stages.

In this present study, adult emergence of T. remus from the parasitized eggs sprayed with EPF was non-significantly different from those of the control. It indicated that the EPF of the study were harmless to the immature of T. remus. Amaro et al. (2015) found that B. bassiana had non- significant effect on progeny viability or mortality of an egg parasitoid, T. pretiosum. However, when the dose was increased to 1.0 × 10⁹ conidia.mL⁻¹, it could jeopardise the immature of egg parasitoids, such as M. anisopliae decreased T. pretiosum adult emergence and caused mortality (Potrich et al. 2009). B. bassiana with a dose of 1.0 × 10⁷ conidia.mL⁻¹ could also reduce significantly larval parasitism of Plutella xylostella L. by Oomyzus sokolowskii (Kurdjumov) (dos Santos et al. 2006). The present research used a dose of 1.0 × 10⁶ conidia.mL⁻¹ did not harm immature stage of T. remus. However, a previous research found that Metarhizium spp. with 1.0 × 10⁶ conidia.mL⁻¹ applied topically can kill S. frugiperda larvae up to 78.67% (Herlinda et al. 2020). Thus, when the EPF will be applied to maize to control S. frugiperda larvae, it cannot harm the mummies of S. frugiperda eggs. The results of this study could provide a new solution to integrate the release of egg parasitoids and spraying of the EPF simultaneously (Integrated Pest Management, IPM). Therefore, the EPF could be used in pest management studies in order to control the pests in the fields.

The percentage of T. remus adult emergence from the parasitized host eggs sprayed with the EPF did not differ from untreated eggs because the immature parasitoid was protected within the host egg (the endoparasitoid egg). The immature stage of endoparasitoid egg is not more susceptible compared to the free-living adult stage (Amaro et al. 2018) or ectoparasitoid (Wei et al. 2023). In addition, the EPF cannot cause disease in T. remus adult by contact when walking on a treated surface (Amaro et al. 2018). However, in the present experiment, almost all isolates of the EPF, except C. lunata JaSpkPGA (3) isolate could decrease the adult longevity of T. remus that emerged from host eggs. The present result is in line with the findings of Potrich et al. (2017) that the longevity of T. pretiosum adults emerged from host eggs sprayed with M. anisopliae could reduce significantly compared to controls. The sex ratio of T. remus adults emerging from S. frugiperda eggs was not influenced by spraying the EPF. Likewise, the sex ratio of T. pretiosum emerging from Anagasta kuehniella (Zeller) (Lepidoptera: Pyrallidae) eggs was not affected by M. anisopliae (Potrich et al. 2009).

This study found that the EPF applied topically (contact) to the parasitized S. frugiperda eggs in post-parasitism periods (24 h) could affect the developmental time of the egg parasitoid immature stage and shorten the adult longevity of the parasitoid. However, parasitoid preference for host eggs sprayed with the EPF showed non-significant effect when host eggs were limited and no choice experiment. The preference of parasitoids for host eggs sprayed with the EPF with many other egg choices in the field needed to be further investigated. Thus, it was necessary to limit the topical application of the EPF to avoid negative effects on the egg parasitoid, T. remus. However, when this data research was referred to our previous research, the EPF used in this present research were the endophytic and EPF because our previous research confirmed that the fungi were able to colonise plants as endophytes and to kill host insects as entomopathogens (Herlinda et al. 2021a). Our other previous experiment found that the EPF used in this present research could be applied by seed treatment and could penetrate and colonize within maize leaf tissue (Sari et al. 2023a). On the other hand, the endophytic B. bassiana and M. anisopliae had negative effect on growth (Sari et al. 2023b) and development of S. frugiperda (Lestari et al. 2022). Therefore, it is needed to be further investigated that the use of the EPF by inoculating the fungi within the plant tissue (endophytic) could be less harmful to egg parasitoids but negatively affects on growth and development of S. frugiperda.

Conclusions

The immature stage of Telenomus remus is not susceptible or less sensitive to the the EPFs, Beauveria bassiana, Chaetomium sp., Curvularia lunata, Penicillium citrinum, and Metarhizium anisopliae. Hyphae and conidia of the fungi were not detected in morphological observations, next study could observe them histologically. Application of the fungi could be considered compatible to T. remus inundation, therefore, future research is needed to confirm it. Nevertheless, it was necessary to limit the topical application of the fungi to avoid negative effects on the adult longevity of the egg parasitoid. Therefore, the use of the endophytic EPF by inoculating the fungi within the plant tissue could be developed to be harmless to the egg parasitoids.