Entomopathogenic fungi (EPFs), Isaria fumosorosea and Beauveria bassiana, are efficient biological agents in the management of multiple arthropod pests. In this study, the effects of both EPF species on various life stages of Spodoptera litura (F.) (Lepidoptera: Noctuidae) and its natural enemy Rhynocoris marginatus (Fab.) (Hemiptera: Reduviidae) were determined under laboratory conditions. I. fumosorosea significantly (P < .05) reduced the growth rate of the third and fourth instar larvae of S. litura. For relative consumption rate (RCR), the maximum impact was recorded for I. fumosorosea, which reduced the RCR of the larvae. The larvae of S. litura treated with I. fumosorosea showed significantly lower efficiency of conversion of ingested food (ECI) and the larval mortality rate (58.0%) was also higher compared with B. bassiana (33.3%). Similarly, I. fumosorosea had a significant effect on the pupal formation of S. litura; however, no significant effect was found on adult emergence percentage. To determine the effect of EPF-infected prey on the adult predator, their handling time, predatory rate, consumption rate, and the survival rate were recorded. No significant effect of EPF species on the predation rate was found. Furthermore, no significant difference was found in the survival rate of predators fed on either EPF-infected prey or healthy larvae. The interaction of these EPFs with a reduviid predator suggested that both EPF species, especially I. fumosorosea, could be used together with the predator to boost the biological control of S. litura in commercial crops.
Introduction
Armyworm, Spodoptera litura (F.) (Lepidoptera: Noctuidae), is an economically important insect pest known to attack various agricultural crops and is widely distributed in the tropical and temperate zones of Asia, Australasia, and Pacific Islands.1 It is native to India and South-East Asia2 and is well established in Pakistan.3,4 It is reported to potentially cause 35% to 55% yield losses at the blossom and vegetative stages of the crops.5 In south Punjab, Pakistan, it causes damage to the economically important crops6 like cotton, tomatoes, tobacco, groundnut, soybean, lucerne, cabbage, sunflower, castor, cauliflower, onion, brinjal, and turnip.789-10 Recently, it has also been reported to feed on citrus cultivars in Sargodha region of Pakistan.10
Due to its economic importance and widely known losses to agricultural crops, insecticide application is considered the best method to manage S. litura.11 However, repeated applications and extensive use of insecticides have resulted in ecological imbalances such as toxic effects on natural enemies and humans.12 Development of insecticide resistance has also been reported for S. litura.131415-16 Hence, it is important to explore eco-friendly (ecological and economical) insect pest management (IPM) strategies using natural enemies providing similar efficacy against S. litura. The use of microorganisms has achieved a prominent position among different options to control insect pests that cause considerable losses to agroecosystems.1718-19 Use of entomopathogens is one of the management strategies against notorious insect pests. Different entomopathogens, such as Pseudomonas fluorescens (Trevisan) Migula, Metarhizium anisopliae (Metsch.) Sorokin, and S. litura nucleopolyhedrovirus (SpltNPV), have been used against different pests.19,20 Entomopathogenic fungi (EPFs), namely, Isaria fumosorosea and Beauveria bassiana, have been reported to effectively reduce the population of lepidopterous insect pests.21,22
Furthermore, the release of natural enemies including predators and parasitoids is another strategy to manage insect pests in an eco-friendly way. Previously reported biological control predators of S. litura are Platymeris laevicollis (Distant),23 Zelus renardii Kolenati,24 Rhynocoris marginatus (Fab.),25 Rhynocoris kumarii Ambrose and Livingstone, Rhynocoris fuscipes (Fab.), Pristhesancus plagipennis Walker (Reduviidae: Hemiptera), Acanthaspis pedestris (Stål), Catamiarus brevipennis (Serville), and Ectomocoris tibialis Distant26; all belong to the family Reduviidae. A number of insect species from different orders like Lepidoptera, Coleoptera, Hemiptera, Orthoptera, and Isoptera have been controlled by hunter reduviid predators (Reduviidae).23 These are commonly found in the agricultural fields and suppress the population of many important pests including Creontiades dilutus (Stål) (Miridae: Hemiptera), S. litura, Helicoverpa armigera (Hubner), Anomis flava F. (Noctuidae: Lepidoptera), Phenacoccus solenopsis (Tinsley) (Pseudococcidae: Hemiptera), Dysdercus cingulatus (Fab.) (Pyrrhocoridae: Hemiptera), and Aphis gossypii Glover (Aphididae: Hemiptera).27,28 R. marginatus (Heteroptera: Reduviidae), a polyphagous reduviid predator, feeds on more than 20 economically important insect pests29 and has paralytic potential using salivary gland extract against its host.30 Sahayaraj and Ravi31 reported a 66.6% reduction of S. litura population after releasing R. marginatus into groundnut field.
Few control strategies focus on limiting the use of insecticides because of their hazardous and non-target effects. Sometimes, insecticides are used synergistically at low doses with microbes to suppress pest populations.32 However, the non-target effect of these microbial insecticides should be determined before recommendation. Other alternate strategies involve the use of natural enemies entirely.
Due to lack of information, the possible combination of these natural enemies in several pest management programs has not yet been explored. No study has been reported on the reduviid predation against S. litura in Pakistan. This study focused on investigating the interaction of EPFs, B. bassiana and I. fumosorosea, with reduviid predators, especially R. marginatus (Reduviidae: Hemiptera), and their combined effect on S. litura. Hence, in this study, the effectiveness of EPFs was checked against various life stages of S. litura and the performance and survival of its predator, R. marginatus, was investigated.
Materials and Methods
To determine the effect of EPFs on S. litura and their non-target effect on reduviid predator R. marginatus, experiments were conducted in the Entomology Laboratory at College of Agriculture, University of Sargodha, Pakistan.
S. litura culture
Egg batches and larvae of S. litura were collected from the lucerne field nearby the University (32°07′42.9″N 72°41′27.2″E). The culture was maintained at 27°C ± 2°C temperature and 75% ± 5% relative humidity (RH) in the laboratory. Newly hatched larvae were provided with artificial diet till pupation. Artificial diet was prepared in accordance with Sorour et al.33 The adults were shifted into clean plastic cages (120 mm × 116 mm × 95 mm) covered with muslin cloth where they were fed on 10% sugar solution. The cotton wool strips (1 cm wide, 5-10 cm long) were kept in plastic cages as a suitable oviposition substrate to collect eggs. In the experiments, the F2 generation was used.
Entomopathogenic fungi
The commercial formulations of B. bassiana NCIM 1216 ATCC 26851 and I. fumosorosea IF-171201 (Agri Life, India) were used and tested at 1 × 108 cfu. Both EPF species were purchased from the Ali Akbar Group of Companies, Lahore, Pakistan. At the time of treatment, spore viability was determined by spraying 1 mL aliquots of suspension on potato dextrose agar (PDA) and incubated at 25°C. The viability of both fungi was more than 90%.
R. marginatus culture
The egg batches of R. marginatus were collected from the tobacco fields (32°07′38.8″N 72°40′33.6″E). The eggs were placed in clean glass Petri plates lined with a filter paper. The culture was maintained at 25°C ± 2°C and 70% ± 5% RH. R. marginatus was reared for second generation prior to the experiments. Newly hatched nymphs were provided with early instar larvae of S. litura and the mature nymphs and adults were provided the later instars of S. litura. About 2 larvae of hosts were provided to each predator nymph and adult on a daily basis. S. litura larvae were collected from the reared culture in the laboratory.
Effect of EPFs on eggs and larvae of S. litura
Freshly laid egg batches were collected and placed in separate Petri plates lined with a filter paper. The eggs were sprayed with EPFs (B. bassiana and I. fumosorosea) at 1 × 108 cfu using a hand sprayer (Taizhou Longshixiang Plastic, China). Distilled water was used in control treatment. Each treatment was replicated 3 times and 1 batch of the egg was considered as 1 replication. The total number of eggs was counted per batch before application. The average number of eggs was 130 per batch in each treatment. The eggs were kept in an incubator maintained at 25°C ± 1°C and 70% ± 5% RH. The color changes in eggs were observed daily. After 3, 5, and 7 days of treatment application, egg hatchability was recorded.
The efficacy of EPFs was also tested on the third and fourth instar larvae of S. litura. Each treatment was replicated 4 times and 5 larvae of each instar were tested in each treatment. The experiment for both eggs and larvae was repeated thrice. The topical bioassay was performed to test the efficacy of EPFs. About 1-µL drop of each treatment was applied on the thorax of each larva. The treated larvae were shifted into new Petri plates containing sunflower leaves. The leaves were collected from unsprayed field and brought into laboratory. Leaves were washed with water to remove contaminants and dried at room temperature. The leaves were cut into disk size (6 cm) of Petri dish and changed daily. The weights of larvae before and after 24, 72, and 120 hours of the application were recorded. The survival rates of larvae were also recorded at 24-hour interval for a total of 10 days. To determine the consumption rate, the leaves were weighed before and after 24 hours of application. Dead larvae due to the application of EPFs were separated into clean Petri plates and sealed with parafilm. The Petri plates were kept in an incubator at 26°C ± 2°C and 70% ± 5% RH. The pupal formation and adult emergence rate of S. litura were also recorded. The digestion, consumption, and use of the third and fourth instar larvae of S. litura after infection were calculated using the formulae described by Waldbauer34
where ΔB is the change in body weight of the insect (mg), BI is the initial larval weight, T is the duration of the feeding period (days), I is the dry weight of food (mg) consumed, and B is the insect dry weight gain (mg).
Effect of EPFs on the performance of R. marginatus predator
S. litura larvae were treated with EPFs for the reduviid R. marginatus predator. Freshly molted third and fourth instar larvae of S. litura were starved for 12 hours and then treated with either B. bassiana or I. fumosorosea on an artificial diet. Larvae for control treatment were fed on diet inoculated with distilled water. Larvae that consumed the entire diet within 24 hours were separated and transferred to clean Petri dishes containing fresh uncontaminated diet and reared under controlled conditions. Larvae that did not eat the diet were discarded. The microbial infection was identified based on their sluggish behavior, food consumption, and later the growth of conidia.
To record the effect of EPFs on the performance of the predator, newly emerged R. marginatus adults from the lab culture were selected that were reared on healthy S. litura larvae. Throughout their nymphal instar, they were provided the healthy S. litura larvae. However, the third and fourth nymphal instars and adults were starved for 12 hours and then EPF-infected third and fourth instar prey were provided separately to each category of R. marginatus. For the experimental treatment, 10 nymphs/adult predators (10 replicates/treatment) were provided 5 EPF-infected prey for each category separately. In the case of control treatment, healthy larvae were provided to the predators. Handling time (paralyzing plus sucking), predator rate (number of prey/predator/day), number of preys consumed, and the survival rate were recorded daily for 5 days. A digital video camera (DSC-WX60, 16.2 MP HD, China) was set over the experimental setup for 4 hours daily to estimate the handling time (paralyzing + sucking act). Food consumption of R. marginatus adults was calculated using the formula35
where FC is the food consumption, PWB is the prey weight before providing to predators, and PWA is the prey weight after feeding of predators.
To record the effect of EPF-infected prey on the developmental biology of R. marginatus, 10 pairs of newly emerged adults from nymphs were fed on healthy S. litura larvae throughout their lives, and then they were selected for further experiment. In total, 10 newly emerged nymphs were fed on EPF-treated prey and 10 were fed on healthy S. litura larvae that served as the control treatment. Data on the number of days required for the completion of each stage were recorded.
Data analysis
To check the significance of EPFs on the various stages of S. litura, 1-way analysis of variance (ANOVA) was performed. Mortality was analyzed using Kaplan-Meier survival analysis with a log-rank test. Similarly, 1-way ANOVA was also performed to check the effect of EPFs on the handling time, predatory rate, food consumption, and developmental biology of predators. Means were separated with the least significant difference (LSD) test at a probability level of 5%. All the analyses were performed using the Minitab 17.0 software.
Results
Effect of EPFs on eggs and larvae of S. litura
There was a significant effect of EPFs on the hatchability of S. litura after 3 (F = 48.7, P < .05), 5 (F = 10.6, P < .05), and 7 (F = 14.1, P < .05) days of exposure. After 7 days, egg hatchability was found lower (55.6%) by the application of I. fumosorosea followed by B. bassiana (71.1%) compared with the control treatment (92.9%) (Figure 1). A significant effect (P < .001) of EPFs was observed on all nutritional indices in both the third and fourth instar larvae of S. litura. The relative growth rates (RGRs) of the third (0.07 mg/mg/day) and fourth (0.13 mg/mg/day) instar larvae were affected more by I. fumosorosea compared with B. bassiana (0.20 mg/mg/day for the third instar and 0.26 mg/mg/day for the fourth instar). The values of the relative consumption rate (RCR) and efficiency of conversion of ingested food (ECI) indices were also significantly (P < .001) lower in the I. fumosorosea treatment (Table 1). Both microbial treatments had a similar effect on the third and fourth larval instars of S. litura for the different nutritional indices following a general trend: I. fumosorosea < B. bassiana < control. The survival rate of S. litura larvae was significantly (Kaplan-Meier log rank: df = 2, χ2 = 32.34, P < .001) affected after the application of EPFs. However, 33.3% of larvae survived after 10-day exposure of I. fumosorosea followed by 53.3% in the B. bassiana treatment (Figure 2).
Figure 1.
Percent (mean ± SE, n = 3) egg hatchability of Spodoptera litura after the application of entomopathogenic fungi. BB indicates Beauveria bassiana; Ifr, Isaria fumosorosea.
Table 1.
Different nutritional indices: relative growth rate (RGR), efficiency of conversion of ingested food (ECI), and relative consumption rate (RCR) of 2 larval instars of Spodoptera litura sprayed with Ifr and BB after exposure to sunflower leaf disks.
Figure 2.
Effect of entomopathogenic fungi on survival rate (%) (mean ± SE, n = 20) of Spodoptera litura larvae. BB indicates Beauveria bassiana; Ifr, Isaria fumosorosea.
Entomopathogenic fungi had a significant effect (F = 7.00, P < .05) on pupal formation of S. litura. However, no significant (F = 0.44, P > .05) effect of EPFs was observed on adult emergence. I. fumosorosea affected the pupal formation and adult emergence of S. litura comparatively more than B. bassiana. In comparison with control, I. fumosorosea lowered 53.3% pupal formation and 14.4% adult emergence rate (Figure 3).
Effect of EPFs on R. marginatus
During predation, R. marginatus showed the sequential pattern of behavior such as locating, capturing, sucking, and paralyzing the prey. Food consumption, handling time, and predatory rate of adults of R. marginatus fed on EPF-infected S. litura prey are presented in Table 2. Food consumption of R. marginatus was not significantly (P > .05) affected by the application of EPFs. However, a significant (P < .001) difference in the handling time of R. marginatus on EPF-treated and untreated prey was found. The handling time was longer (172.2 minutes) on untreated prey compared with the EPF-treated one. Between the EPF treatments, the handling time of R. marginatus fed on I. fumosorosea-infected prey was longer. The R. marginatus consumed almost the same amount (P > .05) of S. litura larvae that were either untreated or EPF treated (Table 2).
Table 2.
Effect of EPF-infected Spodoptera litura larvae (third and fourth instars) on food consumption (FC), handling time (HT), and predatory rate (PR) of the Rhynocoris marginatus predator.
When B. bassiana-infected prey was fed to the third and fourth instar nymphs and adults of R. marginatus, the developmental period delayed compared with I. fumosorosea and untreated prey. Similarly, the total nymphal period was also affected significantly (F = 5.68, P < .05) fed on EPF-treated prey. The total nymphal period of R. marginatus was 61.0 days in the control treatment followed by 62.0 days in the I. fumosorosea and 64.3 days in the B. bassiana treatment. The adults lived 23.2 days fed on healthy S. litura larvae (control treatment). Total adult longevity of predator was 22.7 days fed on I. fumosorosea-treated prey followed by 26.9 days when they fed on B. bassiana-treated prey. No significant effect of EPFs was found on the survival rate of the third (F = 0.82, P > .05), fourth (F = 0.15, P > .05), and adult longevity (F = 2.21, P > .05) of R. marginatus (Table 3).
Table 3.
Effect of EPF-treated and untreated Spodoptera litura prey consumed by Rhynocoris marginatus nymphal stages and the total nymphal developmental period (days) and survival rate (%).
Discussion
As this study was based on EPFs and their effects, it was observed that EPFs significantly affected the egg hatchability of S. litura. The abnormal reduction in egg hatchability of S. litura treated with EPFs was in accordance with Leckie et al36 where lower egg hatchability rate and delayed development of Heliothis zea (Boddie) (Lepidoptera: Noctuidae) larvae fed on diet treated with B. bassiana were reported. Similarly, Malarvannan et al37observed complete arrest in fecundity of S. litura by the application of B. bassiana (2.4 × 107 spores/mL). Gindin et al38 observed an 80% to 82% reduction in the hatchability rate of EPF-treated red palm weevil adults, Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae).
In this study, among both EPFs, I. fumosorosea proved to be more effective as it reduced the consumption rate and affected the relative growth of S. litura larvae compared with B. bassiana. Among larval instars, I. fumosorosea reduced the consumption rate of S. litura by 14.8% and had more effect in reducing the growth rate of small larvae (RGR: 46.1%) compared with large larval instars. Due to the application of I. fumosorosea, the values of RGR and RCR remained lower in both larval instars and, consequently, the conversion of ingested food remained low. In this investigation, I. fumosorosea-treated larvae showed a higher reduction in ECI which ranged from 32% to 38% with respect to the third and fourth instar larvae. In a similar study by Moorthi et al,21 a 46% reduction in ECI in S. litura larvae treated with I. fumosorosea was observed. This finding is suggestive of the altered digestive activity of S. litura after treatment with I. fumosorosea which could be used as a suitable biological control agent after testing it in the field. I. fumosorosea might have a direct effect on the metabolic process of S. litura as a significant decrease in food consumption (FC) and growth rate (GR) was observed (Table 1). It has been reported that entomopathogenic fungi (EPF) degrades the insect cuticle through the enzymes and enters hemocoel where they take on host nutrients and multiply in numbers.39
I. fumosorosea reduced the feeding indices and was effective in killing the S. litura larvae. The current results were well supported by Moorthi et al,21 Tefera and Pringle,40 and Asaff et al41 who observed more decline in food consumption of S. litura after I. fumosorosea application in comparison with B. bassiana and Paecilomyces variotii. A significant effect on the pupal formation of S. litura was observed after treatment with EPFs; however, no significant effect was recorded for the adult emergence rate. EPFs reduced the larval weight of S. litura during development, due to which the formation of shriveled pupa was observed.42 Many researchers have reported the effectiveness of I. fumosorosea against wide host range, especially lepidopterous insect pests.434445-46
Before considering the application of these EPFs in field conditions either individually or in combination with other control strategies, such as the release of natural enemies (predators or parasitoids), it is essential to understand the interactions and compatibility of these EPFs with natural enemies. The current results showed that the provision of EPF-infected S. litura larvae to R. marginatus predator did not affect its food consumption. Furthermore, in the presence of EPFs, the predator took less time to handle the prey compared with untreated prey. This could be due to the sluggish behavior or slow movement of EPF-infected larvae that were easy for predators in locating, capturing, consuming, and digesting the larvae. These findings were well supported by Sahayaraj et al47 where no significant effect of EPF-infected larvae of S. litura on the performance of R. kumarii predator was reported. When the EPF-infected prey fed to different life stages of R. marginatus, the developmental period was prolonged but did not reduce the survival rate of nymphal and adult R. marginatus. Zhang et al48 have confirmed that Isaria cateniannulata fungus has no deleterious effects on the vitality and fertility of the predator Euseius nicholsi (Ehara & Lee) (Acari: Phytoselidae). Similarly, Scorsetti et al49 also reported that B. bassiana-infected Rhopalosiphum padi L. (Hemiptera: Aphididae) did not affect the development of the predator Eriopis connexa (Germar) (Coleoptera: Coccinellidae). The efficacy of these entomopathogens may vary under the field condition where the predator interacts with the pest and environment. However, Nalepa and Weir50 reported that even if the EPF invades the cuticle of coccinellid predators, there are no known deleterious impacts on the host.
The non-target effect of EPFs on the reduviid predator R. marginatus under laboratory conditions was mainly studied to assess the usefulness of the integration of natural predator and FPF. The findings demonstrated that there is a potential for combining R. marginatus with other entomopathogenic microbes, especially I. fumosorosea. Many researchers have encouraged the integration of commercial formulations of EPFs with other components for IPM.5152535455-56 Reduviid as generalist predators, abundant in many agroecosystems and distributed worldwide, have been recommended for IPM programs23 and could be used together with I. fumosorosea.
Conclusions
The EPFs, I. fumosorosea and B. bassiana, proved to be effective in reducing the egg hatchability, food consumption, and growth rate of S. litura. However, among both treatments, I. fumosorosea significantly proved to affect the growth and development parameters in prey. Moreover, the integration of I. fumosorosea with the reduviid predator, R. marginatus, also yielded beneficial results as the predator easily handled and captured the EPF-infected prey due to its altered behavior. These findings allude to the consideration of I. fumosorosea as an effective eco-friendly mycoinsecticide against S. litura. However, limited numbers of studies are available on the interaction of microbes with natural enemies under field conditions. Such studies where more combinations of EPFs and natural enemies are exploited would be helpful in developing effective pest control programs.
Acknowledgements
We are thankful to two anonymous reviewers for useful comments on an earlier version of the manuscript.
REFERENCES
Notes
[1] Financial disclosure The author(s) received no financial support for the research, authorship, and/or publication of this article.
[2] Conflicts of interest The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
[3] Contributed by SM and NA performed the experimental work and analysis in conducting this research study. MIU and MAf contributed in concept and designing of this study and also research supervision. MAr, NM, MR, SA, AA contributed equally in literature search, interpretation of results, manuscript drafting, improvement and critical reviewing of drafted manuscript. All authors approved and agreed for the submission of this article for publication.
[4] Muhammad Irfan Ullah 
https://orcid.org/0000-0002-2463-2665
Naunain Mehmood https://orcid.org/0000-0001-7852-9113
Muhammad Riaz https://orcid.org/0000-0002-5524-7735
