Skip to main content

Storage and efficacy of entomopathogenic nematode species as a biocontrol agent against the armyworm, Spodoptera litura (FABRICIUS) (Lepidoptera: Noctuidae)



The armyworm, Spodoptera litura (FABRICIUS) (Lepidoptera: Noctuidae) is a widely distributed polyphagous pest that is seriously affecting more than 100 different hosts. Globally, biopesticides are becoming the mainstream as chemicals and biocontrol agents gain more of the market share every year, because some of these biocontrol measures prevent the economic disruption of food crops.


For storage, 3 substrates were tested for entomopathogenic nematodes (EPNs), viz., polyether polyurethane sponge, distilled water and soil. Best storage medium was found to be polyether polyurethane sponge for 2–12 months of storage at 15–18 °C. For biocontrol evaluation four species of Steinernematidae; Steinernema pakistanense, S. siamkayai, S. ceratophorum and S. bifurcatum, and one species of Heterorhabditidae; Heterorhabditis indica, were used against the armyworm. At 350 IJs/ml S. pakistanense; S. siamkayai, S. ceratophorum, S. bifurcatum and H.indica, showed 95, 78, 74, 90 and 87% mortality, respectively.


Nematode species, long-term storage and shelf life are important considerations for the success of EPNs against target insect pests which in turn are a prerequisite for successful commercialization and this can be achieved by maximum survival of IJs in a formulation for a long period before their utilization in the field.


The armyworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), has a wide geographical distribution and it is a serious pest of many ornamentals, vegetables and crops (Ahmad and Mehmood 2015). Under favorable conditions, it has a great potential to cause outbreaks and inflict a massive yield loss to the growers. Globally, it has been reported as a major pest for 120 cultivated and uncultivated plant species distributed in 44 families (Gao et al. 2014). Heavy resistance to all conventional and some new chemistry insecticides have also been observed which need a proper rotation and alternatives for long-term benefits in pest management (Saleem et al. 2008). The exploit of alternative methods in a pest management has a number of profits and has proved their efficiency in integrated pest management in some rural crops. These benefits are: drop off in usage of chemicals; lesser manufacturing expenses; avoidance of extreme deposits levels and minimum hazard of pests mounting resistance, reduce the opportunity of insects recurrence (Shahina et al. 2017).

The nematodes of the family Steinernematidae Travassos, 1927, and Heterorhabditidae Poinar, 1975 along with symbiotic bacteria Xenorhabdus (Thomas and Poinar 1979) and Photorhabdus Boemare et al. 1993 have come to be regarding as optimistic biological control agent of a broad range of farming crop pests and the key constituent in IPM (Tabassum and Salma 2020). Their virulence mechanism and the infection process are based on the free-living infective juveniles (IJs), which are highly mobile and responsible for the dispersal of the nematodes (Ciche et al. 2008). Globally many researchers are now investigating the possible exploit of EPNs as biological controls to convene the innovative values, require approximately residue free fruits and vegetables (Shahina et al. 2017). Successful commercialization can be achieved by a maximum survival of IJs in a formulation for a long period before their utilization in the field. Nematode mortality may vary from 70 to 100%, if storage life of the formulated product has expired (Grewal 2002). The approval of beneficial nematode grounded products influenced on their permanency during shipment, healthy shelf life and their ease of practice and reliable enactment in field conditions (Piggot and Wardlow 2002).

Therefore, the present study was designed to evaluate different substrates for storage of EPNs and their efficacy against S.litura under laboratory conditions.


Storage of entomopathogenic nematodes on different substrates

Entomopathogenic nematodes species, viz., S. pakistanense; S. siamkayai, S. ceratophorum, S. bifurcatum and H. indica, were stored in incubating chamber on polyether polyurethane foam, distilled water and soil medium for 2, 4, 8, 10 and 12 months duration at 15–18 °C and tested out their survival rates. The average number of live IJs was calculated under stereo microscope (40 x) on counting chamber. Experiment was completely randomized block design, with three replicates of each EPNs species and months.

Polyether polyurethane sponge

Polyurethane sponge was cut into small pieces (1.25 × 1.25 cm) cleanse with 75% ethanol and sterilized at 60 ºC for 10 min. Freshly emerged from infected Galleria mellonella larvae, 1000 infective juveniles of each species were evenly poured on 240 gm pieces of sponge separately. Sponge-coated pieces were stored in a plastic bag sealed with tips and tied with rubber band. To check viability of nematodes, sponge was soaked separately (each species and treatment) in 20 ml water filled in a 100-ml sterilized beaker for half an hour and squeezed to release the nematodes.

Distilled water

Freshly harvested IJs of the five species of EPNs were stored in 80 ml distilled water in 250-ml conical flask separately at the concentration of 200 IJs/ml.

Soil medium

A 100-ml sterilized beakers was filled up to 80 ml with sterilized sand. Newly propagated 1000 IJs of mentioned nematodes species were pipetted in a beaker with 5 ml water suspension separately. For incubation beakers were covered with aluminum foil tied with elastic band. After completion of time survival of IJs was calculated by Cobb’s sieving and decanting method using 150 mesh sieves over a 500 mesh sieve. Nematodes were retained on the bottom sieve and then collected to estimate the survival percentage.

Efficacy of EPNs against Spodoptera litura

Batches of 25 S. litura last stage larvae were placed in each 6-cm plastic Petri dish bottomed with moist filter paper and treated with nematode concentrations @ 150, 250 & 350 IJs in 1 ml distilled water with few drops of 2% Tween 80 as emulsifier. Plates were sealed with Parafilm and incubated at 28–30 °C. Mortality was determined after 48 h and untreated as a control, with 1 ml of distilled water without nematodes. Each treatment had four replicates and the experiments were repeated 3 times on different dates. The cause of mortality of insect was confirmed by dissecting the insect and observing the presence of nematodes.

Statistical analysis

Statistical data were analyzed by multifactor analysis of variance (ANOVA, followed by Duncan’s multiple range test (P < 0.05) for separation of means (Duncan 1955).


Polyether polyurethane sponge

In sponge after 2 months of storage, the average survival rates of S. ceratophorum, S. pakistanense were 72% and S. siamkayai 71%, S. bifurcatum 77% while H. indica showed 75% survival rate. As the time duration increased, the survival rates of H. indica declined up to 50%, while S. ceratophorum, S. pakistanense showed 45%, S. bifurcatum 42% and S. siamkayai 35% (Fig. 1). S. pakistanense showed a high survival during 2–6 months in sponge medium (ANOVA F = 3.6; df = 3, 15; P < 0.05), whereas S. bifurcatum showed a maximum survival in sponge during 2–6 months (ANOVA F = 3.2; df = 3, 15; P < 0.05), while H. indica showed a minimum survival in sponge during 2–6 months (ANOVA F = 0.2; df = 3, 15; P < 0.05), whereas S. ceratophorum, S. siamkayai showed less significant survival rates in sponge during 2–6 months (ANOVA F = 0.135; df = 3, 15; P < 0.05).

Fig. 1
figure 1

Survival rate of entomopathogenic nematodes on different substrates

Distilled water

After 2 months of storage in distilled water, the average survival rates of S. ceratophorum were 65%, S. pakistanense 72%, S. bifurcatum 68% and S. siamkayai and H. indica showed 64% of viability. After half year of storage the survival percentage of S. ceratophorum and S. pakistanense was 56 and 60%, respectively. S. siamkayai and H. indica showed 52% and S. bifurcatum 42%. After completion of one year of storage all species survival rates in distilled water were found to be 32–36% (Fig. 1). In water, S. pakistanense showed a significant high survival during 2–6 months (ANOVA F = 8.4; df = 4, 15; P < 0.05), while S. ceratophorum, S. bifurcatum, S. siamkayai and H. indica showed a moderate significance during 2–6 months (ANOVA F = 4.0; df = 3,15; P < 0.05).

Soil medium

In soil medium, viability of nematodes is shown in (Fig. 1) from 2 to 12 months of storage. In soil medium after 2 months of storage less number of nematodes survived of all tested species as compared to other substrate. The survival rates ranged from 35 to 60%at 6 months of storage, declined up to 22–30%. In soil, all type of nematodes showed nonsignificant survival rates during 2–6 months (ANOVA F = 0.1; df = 4, 15; P < 0.05).

Efficacy of EPNs against Spodoptera litura

Isolates of S. bifurcatum, S. ceratophorum, S. pakistanense, S. siamkayai and H. indica were evaluated to find, infectivity and mortality S.litura last stage larvae at different concentrations at 28–30 °C by filter assay in laboratory experiments (Fig. 2). The ANOVA showed significant differences among effectiveness of nematode species on army worm (ANOVA F = 24; df = 4; P = 0.0005). Concentration of nematodes also diverged significantly (RCBD-one-way ANOVA F = 23; df = 4; P < 0.05) and impact of the three concentrations with five nematode species also had the remarkable results on army worm (ANOVA F = 30; df = 4; P < 0.05). Outcome verified that the EPNs could restrain the frequency of S.litura. S. pakistanense, S. bifurcatum and H. indica showed higher mortality rates (87–95%) at high application concentrations than S. ceratophorum and S. Siamkayai (74–78%). Maximum mortality rates were attained at the concentrations of 350 IJs/ml (Fig. 2) after 48 h. At the lowest concentration (150 IJs/ml) 60% of army worms were killed by S. bifurcatum, after 48 h of exposure time showing significant differences with 50% against H. indica and S. ceratophorum. The mortality rates of S. litura increased depends upon the concentrations. At 350 IJs/ml, S. pakistanense showed the highest mortality rates (95%), while the lowest one was (74%) by S. ceratophorum. Nematode juveniles reproduced in the larvae of S. litura, which were clearly seen when dead larvae were transferred to vacant cavity block.

Fig. 2
figure 2

Efficacy of EPNs against armyworm, Spodoptera litura


For field application of EPNs as biocontrol agents against agricultural pests, key necessities are the suitable packing of IJs at optimum temperature according to species requirement, so that infective juvenile scan persists with maximum shelf life (Lalramliana and Yadav 2009). In the present study, five species of EPNs, viz., S. bifurcatum, S. ceratophorum, S. pakistanense, S. siamkayai and H. indica, were stored in three different substrates sponge, water and soil medium to check the longevity for 2–12 months. The best storage medium was sponge where the maximum number of infective juveniles remained alive, followed by water and then soil medium. Lewis and Shapiro-Ilan (2002) used sand, starch substrate and soil (high clay content) to store S. carpocapsae IJs. It was observed that, depending on the substrate used for storage, the IJs moved and obtained oxygen more or less easily and were able to increase or decrease their energy expenditures. The starch substrate, in which the space between particles is small, was not suitable to extend IJs survival. The soil used in the experiment was considered poorer for storage than sand; this can be explained by the fact that nematode movement is more difficult in that substrate, increasing energy expenditure and decreasing oxygen diffusion. Andalo et al. (2011) evaluated substrates to extend the survival of entomopathogenic nematodes, suspensions of 3000 IJ ml−1 of Heterorhabditis sp. JPM4 and Steinernema carpocapsae. All were added to dirt, fine sand, coarse sand, foam, expanded clay, phenolic foam, agar, corn starch, Plantmax®, and water. S. carpocapsae IJs were still alive after 180 days in the foam treatment as compared to other treatments, while expanded clay, Plantmax® and phenolic foam were not effective in maintaining the survival rate. Foam, coarse sand and fine sand provided greater Heterorhabditis species IJs survival at 180 days. Agar, phenolic foam and Plantmax® had lower survival indices than the control. The polythene sponges used as carriers for infective juvenile storage have been successful in many studies (Ley and Mundo-Campos 2004). The physical carriers can reduce the stress as compared to water suspension by simulating a natural environment because it provided a large surface area for oxygenation through perforation (San-Blas 2013). The use of storage substrates can reduce stress by creating an environment more similar to natural conditions than storage in water alone, as those substrates provide better oxygenation and conditions for IJ movement (Andalo et al. 2010).

In the present investigation, five species of EPNs, viz., were also evaluated against the last stage larvae of S. litura. As nematode concentration increases the mortality response increases. Radhakrishan and Shanmugam (2017) also reported that the percentage mortality increased with increase in the concentration of H. indica and S. glaseri. H. indica proved to be more effective. Rashad et al. (2018) found that the highest mortality rates of the second instar larvae were observed by using the mixtures of S. carpocapsae or H. indica. Similar result was reported by Acharya et al. (2020) with H. bacteriophora, H. indica, S. carpocapsae and S. longicaudum against fall armyworm. Yan et al. (2020) screened 15 EPN isolates against S. litura larvae and stated that EPN are effective against S. litura and can reduce the overuse of chemical insecticides. Other researchers were also reported that H. indica and S. carpocapsae are highly effective against S. litura and other lepidopteran pests, viz., (Holajjer et al. 2014). Park et al. (2001) studied that the last instar larvae of S. litura were more sensitive than younger one of S. litura upon infection of EPN because the size of the last larva is larger to provide more chance for IJ penetration.


It was concluded that S. bifurcatum, S. pakistanense and H. indica have higher virulence, penetration and reproduction rates than S. ceratophorum against S. litura. For sustainable agriculture farming, EPNs as a biocontrol agent is vital implement in pest management approach. One of the significant desires in employing beneficial nematodes is the appropriate storing of infective juveniles of EPNs, in a given inhabitants and also at optimal temperature, so that maximum IJs can persist for extensive times for application in field conditions. The current study showed that the best storage medium was sponge where the maximum number of infective juveniles remains alive followed by water and soil medium. Long-term storage and persistence is the need of the hour for successful applications.

Availability of data and materials

Not applicable.



Analysis of variance


Randomized completely block design


Entomopathogenic nematodes


Infective juveniles


  • Acharya R, Hwang H-S, Mostafiz MM, Yu YS, Lee KY (2020) Susceptibility of various developmental stages of the Fall Armyworm, Spodoptera frugiperda, to entomopathogenic Nematodes. Insects 11:1–13

    Article  Google Scholar 

  • Ahmad M, Mehmood R (2015) Monitoring of resistance to new chemistry insecticides in Spodoptera litura (Lepidoptera: Noctuidae) in Pakistan. J Econ Entomol 108:1279–1288

    Article  CAS  Google Scholar 

  • Andalo V, Cavalcanti RS, Molina JP, Moino A Jr (2010) Substrates for storing entomopathogenic nematodes (Rhabditida: Steinernematidae, Heterorhabditidae). Sci Agricol 67:342–347

    Article  Google Scholar 

  • Andalo V, Moino A Jr, Maximiniamo C, Campos VP, Mendonca LA (2011) Influence of temperature and duration of storage on the lipid reserves of entomopathogenic nematodes. Rev Colomb Entomol 37:203–209

    CAS  Google Scholar 

  • Boemare NE, Akhurst RJ, Mourant RG (1993) Deoxyribonucleic acid relatedness between Xenorhabdus spp. (Enterobacteriaceae), symbiotic bacteria of entomopathogenic nematodes, with a proposal to transfer Xenorhabdus luminescens to a new genus, Photorhabdus gen.nov. Int J Syst Bacteriolo 43:249–255

    Article  CAS  Google Scholar 

  • Ciche TA, Kim KS, Kaufmann-Daszczuk B, Nguyen KCQ, Hall DH (2008) Cell invasion and matricideduring Photorhabdus luminescens transmissionby Heterorhabditis bacteriophora nematodes. App Environ Microbiol 74:2275–2287

    Article  CAS  Google Scholar 

  • Duncan DB (1955) Multiple range and multiple F-test. Biometrics 11:1–41

    Article  Google Scholar 

  • Gao M, Mu W, WangW ZC, Li X (2014) Resistance mechanisms and risk assessment regarding indoxacarb in the beet armyworm, Spodopteraexigua. Phytoparasitica 42:585–594

    Article  CAS  Google Scholar 

  • Grewal PS (2002) Formulation and application technology. In: Gaugler R (ed) Entomopathogenic nematology. CAB International, Wallingford, pp 265–287

    Chapter  Google Scholar 

  • Holajjer P, Patil JB, Harish G, Nataraja MV (2014) Evaluation of entomopathogenic nematodes, Steinernema carpocapsae and Heterorhabditis indica for their virulence against Spodoptera litura. Ann Plant Prot Sci 22:163–165

    Google Scholar 

  • Lalramliana Yadav AK (2009) Effect of relative humidity on theemergence and reproduction of three entomopathogenic nematode species (Rhabditida: Steinernematidae and Heterorhabditidae) from Meghalaya, India. Sci vis 9:92–96

    Google Scholar 

  • Lewis E, Shapiro-Ilan S (2002) Host cadavers protect entomopathogenic nematodes during freezing. J Invert Pathol 81:25–32

    Article  Google Scholar 

  • Ley P, Mundo-Campos M (2004) Cultivation of nematodes. In: Chen ZX, Chen SY, Dickson DW (eds) Nematology advances and perspectives. CABI Publishing, Wallingford, pp 541–619

    Google Scholar 

  • Park SH, YuY S, Park JS, Choo HY, BaeS D, NamM H (2001) Biological control of tobacco cutworm, Spodoptera litura Fabricius with entomopathogenic nematodes. Biotechnol Bioproc Eng 6:139–143

    Article  CAS  Google Scholar 

  • Piggot S, Wardlow L (2002) A fresh solution for the control of western flower thrips: dramatic results in trials. Commer Greenh Grower 5:20–23

    Google Scholar 

  • Radhakrishnan S, Shanmugam S (2017) Bioefficacy of entomopathogenic nematodes against Spodoptera litura (Lepidoptera: Noctuidae) in Bhendi. Int J Curr Microbiol Appl Sci 6:2314–2319

    Article  CAS  Google Scholar 

  • Rashad RK, Rameesha A, Ali AA, Muhammad A, Shahid M, Sohail A, Sajid AK, Muhammad A (2018) Compatibility of entomopathogenic nematodes (Nematoda: Rhabditida) and thebiocide, spinosad for mitigation of the armyworm, Spodoptera litura (F) (Lepidoptera: Noctuidae). Egypt J Biol Pest Control 28:58

    Article  Google Scholar 

  • Saleem MA, Ahmad M, Ahmad M, Aslam M, Sayyed AH (2008) Resistance to selected organochlorine, organophosphates, carbamates and pyrethroids in Spodoptera litura (Lepidoptera: Noctuidae) from Pakistan. J Econ Entomol 101:1667–1675

    Article  CAS  Google Scholar 

  • San-Blas E (2013) Progress on entomopathogenic nematology research: a bibliometric study of the last three decades: 1980–2010. Biol Control 66:102–124

    Article  Google Scholar 

  • Shahina F, Salma J, Christos I R, Christos GA (2017) Control of stored grain pests by entomopathogenic nematodes. In: Biocontrol agents, entomopathogenic and slug parasitic nematodes. Elagawad, Tariq and James, p 629

  • Tabassum AK, Salma J (2020) Virulence of four Steinernema species as abiological control agent in controlling the termite, Coptotermes heimi (Wasmann) (Isoptera: Rhinotermitidae). Egypt J Biol Pest Control 30:26

    Article  Google Scholar 

  • Yan X, Shahid Arain M, Lin Y, Gu X, Zhang L, Li J, Han R (2020) Efficacy of Entomopathogenic Nematodes Against the Tobacco Cutworm, Spodoptera litura (Lepidoptera: Noctuidae). J Econo Entomolo 113:64–72

    Google Scholar 

Download references


Not applicable


Not applicable.

Author information

Authors and Affiliations



SM performed storage; TAK analyzed the data. AA carried out the biocontrol experiment. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Salma Javed.

Ethics declarations

Ethics approval and contents to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

We have no potential conflicts of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Javed, S., Khanum, T.A. & Ali, A. Storage and efficacy of entomopathogenic nematode species as a biocontrol agent against the armyworm, Spodoptera litura (FABRICIUS) (Lepidoptera: Noctuidae). Egypt J Biol Pest Control 32, 6 (2022).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: