Isolation of Native Strains of Entomopathogenic Nematodes against Fall armyworm, Spodoptera frugiperda (Smith.) in Maize

INTRODUCTION

            Entomopathogenic nematodes (EPNs) are obligate parasites that belong to the family Steinernematidae and Heterorhabditidae and thus the Genera were Steinerema and Heterorhabditis, respectively [1]. About 100 Steinernema species and 21 Heterorhabditis species have been discovered worldwide; Of these, 12 species of Steinernema and 3 species of Heterorhabditis exist in India [2]. The free-living infective juveniles (IJs), which are members of the genera Xenorhabdus and Photorhabdus, can kill the host insect by carrying symbiotic species-specific bacteria in their intestines. Nematode-bacteria complexes are used to kill insect pests most frequently within 48-72 h post-infection, and the host itself serves as the site of subsequent reproduction. EPNs are well amenable to being used as a bio-control agent due to their wide host range and large-scale in vitro and in vivo cultureeability [3]. In recent decades, insects have developed resistance due to the indiscriminate use of insecticides [4]. Therefore, the major objective of this study was to isolate native EPN strains from the maize-grown tracts of the Tuticorin district, Tamil Nadu, India and to test the pathogenicity of native EPN strains against an exotic pest, fall armyworm, S. frugiperda in maize.

Materials and Methods

Isolation of entomopathogenic nematode

Site selection: Soil samples were collected from the maize-growing regions of the Tuticorin district as well as from the fields of Agricultural College and Research Insitute, Killikulam campus during 2022-2023 in order to isolate native EPN strains.

Fig. 1. Map showing the blocks of Tuticorin, Tamil Nadu, India

Soil Sampling: By adopting methods described by [5], hand shovels were used to collect 200-250 g of samples from each sample site at a depth of 10 to 15 cm near the rhizosphere region and vegetation [6]. Samples were packaged in polythene bags, stored at the optimal room temperature to conserve moisture, and labelled with the location, kind of soil and crop ecosystem. Using 70% ethanol, hand shovels were sterilized prior to sampling another site.

Culturing of host insect, Corcyra cephalonica Staint.

EPN was isolated from soil using the Rice moth, C. cephalonica as a bait insect. Additionally, employed for mass rearing of EPN as well [7]. C. cephalonica was mass reared on half half-broken pearl millet in the biocontrol laboratory at Agricultural College and Research Institute, Killikulam in accordance with standard protocol by [8].

Soil baiting technique

Ten final instar larvae of C. cephalonica were employed as a bait along with 250 g of soil from each sampling site was placed in a plastic container under laboratory conditions and sprayed a small quantity of water to retain moisture; holes are made in the lids to allow aeration. Containers were then incubated in a dark environment at a temperature of 25±2 °C. Dead cadavers were extracted from the soil after an incubation period of 6–7 days [7]. Cadavers were transferred to White traps after being completely rinsed with sterile distilled water.

White trap method

White The white trap contains a 90 mm petri dish filled to a depth of 1 cm with 0.01 per cent formalin solution. Inverted petri dishes of 50 mm diameter were placed within 90 mm petri dish. Whatmann No. 1 filter paper was placed on top of the dish. Dead cadavers were arranged over filter paper. To ensure that the filter paper came into contact with the formalin water. Petri– plates were stored in a dark environment at a temperature of 28 °C. IJs start to emerge from the cadavers and gather in the formalin solution [9].

Harvesting and Enumeration of EPNs

Harvested EPNs were added to a 250 ml Erlenmeyer flask, which was then rinsed thrice with distilled water and allowed to settle them, supernatants were removed out. Process The process was repeated until to it attain clear EPN suspension. For counting, 1 ml of EPN suspension was spread over a nematode counting chamber (8 x 8 x 1.5 cm) and examined using a stereozoom microscope (40 x).

Mass culturing of Fall armyworm, Spodoptera frugiperda Smith.

From the corn fields, egg masses and larvae were collected and they were reared separately in plastic containers measuring 30 mm in diameter by 40 mm in height, fed with an artificial diet based on the TNAU artificial diet composition. Pupa were was placed inside a 30 x 30 x 30 cm oviposition chamber with adult feeding solution as provided by TNAU and young nerium Nerium plant branches were used as a substrate for egg laying. First and second instar larvae are reared with maize leaves and shifted to a semi-synthetic diet.

Based on preliminary bioassays conducted in the laboratory, effective strain is carried over to the pathogenicity study.

Pathogenicity of effective EPN strain against S. frugiperda

For pathogenicity assays of FAW, EPN suspensions of different doses (50, 100, 200, 250, 400, and 600 IJs/larva) against third and fifth instar larvae. To avoid cannibalism, one larvaee per petri-plate (90 mm), was fed with maize leaf. To conserve moisture, plates were sealed with parafilm tape and kept in a polythene cover under a dark room temperature at of 28 °C. Larval mortality was examined 48, 72, and 96 hours of the inoculation period.

Statistical analysis

The “Probit analysis in Excel” application was used to compute the LC₅₀ [10].

Results and Discussion

Isolation and Identification of native entomopathogenic nematodes

A total of 87 soil samples were collected from maize maize-growing areas of Tuticorin district, Tamil Nadu, India. Out of 87 samples, nine samples were EPN positive. From the soils, EPN were isolated by the soil baiting method using C. cephalonica. One of nine samples was determined to have Heterorhabditis from Kayathar, which was distinguished by the brick red colour of the Corcyra cadaver. Based on the cadaver’s creamy white and greyish colouring, the remaining 8 samples were discovered to have Steinernema.

 [11] reported that soil surveys showed an EPN recovery rate of 6.00-35.00%. With a frequency of 10.34%, retrieved 9 EPN positive samples out of 87 samples from the Tuticorin district’s maize-growing tracts. In the Northern regions of Tamil Nadu, a rate of 16.30% was observed [12]. Recovery rates in the Karur district were 0.25 per cent [13] while,, Andhra Pradesh were was 1.20 per cent [14]. The method of detection and sample intensity used in the various surveys showed differences in the recovery rate of EPN.

The prevalence of EPN in maize fields of the Tuticorin district ranged widely from 10.00 to 20.00 per cent, with Killikulam (10.00%) having the lowest prevalence and Kayathar and Srivaikuntam having the most (20%), followed by Kovilpatti (13.33%) having the moderate. The discrepancy could be a result of the EPN’s patchy distribution, which is caused by temporal and spatial fluctuation and also relies on the species [15].

Additionally, only Steinernematids are reported in soil samples taken from several locations in Tamil Nadu [16]. Steinernematids alone were also reported by [10]. Furthermore, Steinernema (2.20 %) was most prevalent than Heterorhabditis (0.30 %) [13]. Steinernema (74.00 %) was more predominant in the cotton ecosystem of Tamil Nadu [17].

Furthermore, samples that tested positive for EPN ranged from sandy loam (lower) to clayey (higher) which supports EPN’s survival and mobility [18] [19].

Preliminary study

Based on the preliminary bio-efficacy study, Steinernema sp. (Kayathar strain) was found more effective than others. So this strain was further tested against S. frugiperda at different concentrations of EPN.

Pathogenicity of Steinernema sp. (Kayathar strain) against S. frugiperda

Based on the computed LC ₅₀ values, Steinernema sp. required 33.13 IJs/larva and 37.09 IJs/larva at 96 hours after infection, and 37.80 IJs/larva and 54.33 IJs/larva at 72 hours after infection for third and fifth instar larva of S. frugiperda, respectively. According to [20] Heterorhabditis indica consumed 20.26 and 62.07 IJs per third and fifth instar larva at 72 hours. According to [21], S. riobrave 500 IJs/larva were ineffective against FAW. The present findings, are in line with those of [22], who found that the isolated strain of S. carpocapsae RW14-GR3a-2 from Rwanda was the most virulent. Additionally, S. riobrave MEX-15 was the most virulent strain.

Additionally, third instar mortality was 95.00% to 100.00% at 96 hours after infection due to 100 – 600 IJs per larva. For the fifth instar, 96-hour infections with 250, 400, and 600 IJs/larvae resulted in mortalities of 94.50% to 99.80%. The data suggest that FAW caterpillar mortality rises, as anticipated, with an increase in EPN concentration applied, but declines with increasing instar.

Conclusion

The prevalence and geographic distribution of EPN in the Tuticorin district’s maize-growing regions are highlighted in this study. Furthermore, this study shows that soils collected from maize fields are fairly rich in widespread EPN dispersion. However, the Steinerematids and Heterorhabditis discovered during the investigation may both have the ability to control insect pests in the maize ecosystem. Entomopathogenic nematodes are the most secure biocontrol agents and a potential substitutes for pesticides. Because of their wide host range, EPNs can be used on a variety of crops. The isolated Steinernema sp. (Kayathar strain), was extremely pathogenic to S. frugiperda. EPN as is one of the promising bio-control agents in the maize environment, further research is needed to determine their host range, lethal effects, pathogenicity, seasonal abundance, and biotic and abiotic variables. Implying that the EPN could be an effective biocontrol agent for thecontrol of pests that impact maize crops.

Future scope of the study

The findings of the present study will undoubtedly contribute to decrease decreasing the indiscriminate use of insecticides in the maize crop ecosystem. Additionally, EPN is more virulent than many other bio-control agents, also as an alternative to insecticides.

Acknowledgement

The Department of Agricultural Entomology, Agricultural College and Research Institute, Killikulam, Tuticorin, Tamil Nadu, India is acknowledged by the authors for providing all the necessary resources for the study.

Conflict of interest

The authors declare that they have no conflict of interest in the publication.

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