Isolation and Characterization of Steinernema seemae Entomopathogens Against Helicoverpa armigera (Hubner) from North-Eastern Uttar Pradesh, India

Received 20 Jun, 2022

Accepted 05 Sep, 2022

Published 31 Mar, 2023

Background and Objective: In India, the application of natural Entomopathogenic Nematodes (EPNs) are considered potential biological control agents against soil-borne insect pests last 4-5 decades. Local isolates of EPNs have strong potential and are usually more effective for controlling indigenous insect pests as they are adapted to the local environmental conditions and the insect pest species. The present study aims to isolate indigenous EPN from the eastern part of Uttar Pradesh, India, to test the occurrence and their use as a biological control agent in IPM. Materials and Methods: The distribution, occurrence and diversity of Steinernema seemae entomopathogenic nematode community were investigated and isolation of S. seemae was carried out by using the galleria wax baiting technique and morphological identification methods which reveal that it belongs to the genus Steinernema. Further study was done to examine S. Seemae virulence’s impact on soil temperature and soil moisture. Results: The effect of temperature, 12, 15, 20, 25 30 and 37°C and relative humidity, 35, 55, 85 and 100% on S. seemae was observed and they were listed against the  Helicoverpa armigera  larvae. The results reveal that, at 25°C temperature and 85% relative humidity, S. seemae is a more effective bio-control agent in terms of penetration and multiplication against host  H. armigera larvae under controlled conditions. Conclusion: The EPNs isolated from the soil samples increase the information of S. seemae species showing bio-control activities, which was available for developing suitable bio-control strategies as well as enabling us the recognition of bio-control EPN. EPN species would serve as an alternative to chemical pesticides and fit well in integrated pest management.

INTRODUCTION

India is an agro-based country more than two-thirds of the Indian population depends on agriculture for their livelihood^1,2. It has always been the country’s most important economic sector and constitutes one of the important Gross Domestic Product (GDP) contributors³. Food has always scared a problem for human beings in context with its fast-growing human population². To meet this end, the production of food has to be boosted through various multiple ways, such as, by enhancing food grain production, increasing crop intensity, agricultural land reforms, and protecting crop damage against pests in the fields as well as during storage in the godowns etc². High yield production can be achieved by using hybrid high vigour seeds implementation via biotechnological tools^4,5 (green revolution phase II). Land reforms and crop intensity increases can be yielded through government policies and agricultural practices, respectively.

Pest control leads to major destruction of the crop at both stages i.e., during agricultural practices as well as at storage time^2,6 and will cause huge damages to crops which create an overburden to the economy (GDP). Entomopathogenic Nematodes were second to Bacillus thuringiensis used as bio-insecticides around the world⁷.

Pest control can be achieved in many ways i.e., chemical control includes, the use of chemicals known as herbicides, insecticides, pesticides etc⁸. Initially, it was thought to be a great success and the people sighted a relief, but soon that they create more problems than were supposed to solve, or in other words, the insecticidal crisis proved to be a multifaceted one affecting society and the environment in diverse ways⁹. Secondly, biological control includes the utilization of natural organisms against harmful pathogens¹⁰. They seem to be environmentally friendly and don’t seem to disturb the environment in any way i.e., environmental pollution such as soil, water, and air pollution is negligible¹¹. Recent reports revealed that biological control of the pest also enhances crop yield via helping them through various means such as making plant nutrients available to plants^12,13, secretion of plant hormones, chelation of essential micro and macro elements etc¹⁴. Thus the effort shifted from chemical control towards biological control, which refers to direct or indirect manipulation by man, of living natural control agents to increase their attack on pest species¹⁵. Further, indiscriminate and injudicious use of chemical pesticides made conditions more severe as well as awareness of public concern for safe food and a healthy environment has catalyzed the search for more environmentally friendly procedures.

With due emphasis on the safety of human beings and the environment including benefits, the search for suitable potential biological control agents in the control of H. armigera pest against the chickpea crop is an utmost urge of researchers. The objective of the present study is to isolate indigenous entomopathogenic nematodes from the eastern part of Uttar Pradesh, India, a biodiversity hotspot region, to record the occurrence and their further use as a biological control agent.

MATERIALS AND METHODS

Study area: The study was carried out at the Department of Zoology, D.D.U. Gorakhpur, U.P. Gorakhpur, from 2015 to 2017.

Geographical location: Soil samples were collected from geographical locations in triplicates from the different North Eastern divisions of Uttar Pradesh, India. The sampling location and sample type were tabulated. The study site includes the Gorakhpur division with four districts (Gorakhpur, Kushinagar, Deoria and Maharajganj). The four districts had coordinates 26°75'88"N 83°36'97"E (Gorakhpur), 26°74'1"N 83°88'8"E (Kushinagar), 26°30'36"N 83°46'48"E (Deoria) and 27°07'48"N 83°34'12"E (Maharajganj), respectively.

Soil characterization: Soil texture estimation was carried out by wet sieving methodology while, soil moisture was determined through the direct gravimetric method. Soil pH values of the suspension were determined with the help of a laboratory pH meter (Cyber scan pH 1500, Eutech Instruments, Singapore).

Organic content was determined according to the Walkley-Black method¹⁶. Percentage C and organic matter were calculated by using the following method:

Easily oxidizable organic C (%):

where:

B	=	mL of Fe2+ solution used to titrate blank
S	=	mL of Fe2+ solution used to titrate sample
12/4000	=	Milliequivalent weight of C in g

To convert easily oxidizable organic C into total C, divide it by 0.77 (or multiply by 1.30). To convert total organic C to organic matter use the following equation:

Organic matter (%):

All analytical grade chemicals were procured from Merk India, Bangalore, Karnataka, India.

EPN isolation and culture: The isolation of indigenous entomopathogenic soil nematode will be carried out via the Helicoverpa pupae/larvae bait method¹⁷ in the laboratory. The surface-sterilized Helicoverpa pupae/larvae of different age groups will be placed below the surface of collected soil samples in a sterilized beaker (250 mL) and will be incubated for 12-18 days (25±2°C, 75±5% RH). Almost 3-4 pupae/larvae of different age groups will be taken for each soil sample. After 12-18 days of the incubation period, the treated pupae will be taken out for isolation and identification.

Evaluation of the factors affecting infectivity of S. seemae: Evaluation of the effects of abiotic stress such as temperature and relative humidity on the infectivity of the S. seemae will be tested at six different constant temperatures (12, 15, 20, 25 (control), 30, 35 and 37°C) and four relative humidity (35, 55, 85 and 100%) in the laboratory.

RESULTS AND DISCUSSION

The occurrence and distribution of entomopathogenic nematodes were assessed throughout an extensive soil survey in the Gorakhpur, Deoria, Kushinagar and Maharajganj regions of the Gorakhpur division. A total of 11826.4 km² sampling area covered i.e., 2873.5 km² (Gorakhpur), 2,873.5 km² (Kushinagar), 2,535 km² (Deoria), 2,934.1 km² (Maharajganj) regions. Gorakhpur division comes under a humid subtropical climate. Annual temperatures of all districts were reported as 26°C. Soil characterization was also examined (Table 1) and results revealed that soil texture varies from sandy loam to clay loam whileminimum to maximum pH, organic matter and soil humidity (w/w) were recorded to be 7.0-7.8, 0.13-0.36 and 20-24, respectively. Entomopathogenic nematode S. seemae isolation results revealed that the distribution of S. seemae varies as they were procured from only cultivated soils from Gorakhpur, Deoria, Kushinagar and Maharajganj districts respectively. For long decades, many researchers reported entomopathogenic nematodes from all over the world^18,19. The distribution of entomopathogenic nematodes in our results showed a positive correlation with soil physiological properties such as soil temperature, soil porosity as well as relative humidity. Other researchers also reported the effect of soil physiological parameters such as soil texture, soil temperature, pH and relative humidity limits the distribution of EPN in soils^19,20.

Fig. 1: Evaluation of penetration rate and multiplication rate of S. seemae on H.armigera larvae at different temperatures

Table 1:

Sampling location and characterization of soil samples isolated from Gorakhpur division of North-Eastern Uttar Pradesh, India

Sampling site (Districts with Coordinates)	Sampling types	Soil texture	pH	Organic matter (%)	Soil humidity (w/w)
Gorakhpur	Cultivated	Sandy loam, sandy clay loam, clay loam	7.1 -7.6	0.29	23
(26°75'88"N83°36'97"E)	Uncultivated	Sandy loam		0.13	22
	Garden/Forest	Sandy loam, sandy clay loam, clay loam		0.34	24
Deoria	Cultivated	Sandy loam, sandy clay loam, clay loam	7.0-7.6	0.26	23
(26°30'36"N 83°46'48"E)	Uncultivated	Sandy, sandy clay loam, clay loam		0.12	22
	Garden/Forest	Sandy loam, sandy clay loam, clay loam		0.28	24
Kushinagar	Cultivated	Loam	7.1-7.8	0.32	22
(26°74'1"N 83°88'8"E)	Uncultivated	Sandy loam		0.36	21
	Garden/Forest	Sandy, sandy clay loam, clay loam		0.35	24
Maharajganj	Cultivated	Loam	7.0-7.5	0.3	20
(27°07'48"N 83°34'12"E)	Uncultivated	Loam		0.16	20
	Garden/Forest	Sandy loam		0.33	22

For control of particular insect pest selection of entomopathogenic nematodes based on several factors like nematode’s host range, host finding, tolerance of environmental factors and their effects on survival and efficacy (temperature, moisture, soil type, exposure to ultraviolet light, salinity and organic content of the soil, means of application, agrochemicals etc). Continued prospection for new EPN species or races that are infective for H. armigera is highly warranted^20-22. The foremost critical factors include moisture, temperature, pathogenicity for the targeted insect etc¹⁰.

Experimental evaluations of several species for biological control including H. bacteriophora, H. megidis, Heterorhabditis spp., Steinernema kushidai and Steinernema spp. were recently carried out^23,24, However, virulence habits of S. seemae were on H. armigera have not been reported at all. In this effort, we tried to evaluate the relative infectivity of S. seemae on H. armigera pest and our results showed that maximum penetration and multiplication rate of S. seemae in H. armigera at 25°C and 85% relative humidity respectively. Temperature effect on penetration and multiplication rate increasing with the increase of temperature up to 25°C followed by decreases upto 37°C (Fig. 1). Similarly, relative humidity effect on penetration and multiplication rate of S. seemae was also observed and results revealed that the penetration and multiplication rate increases relative humidity from 35% up to 85%. However, at 100% relative humidity, the penetration and multiplication rate decrease drastically (Fig. 2). Thus our experimental results revealed that at 25°C and 85% (w/w) temperature and relative humidity S. seemae showed effective biocontrol agents against H. armigera larvae pest, respectively. The temperature range for survival and infectivity will depend on the species of EPN and its native habitat and centre of origin^25-27. Our result was also confirmed by other researchers, they recently evaluated two commercially produced species, S. riobrave and H. indica as effective biocontrol agents at temperatures (27±2°C) in coarse sandy soils²⁸.

Fig. 2: Evaluation of penetration rate and multiplication rate of S. seemae in H. armigera larvae at different relative humidity (w/w) (%)

Therefore, in this investigation, we find that the isolation of S. seemae entomopathogenic nematode in North-Eastern Uttar Pradesh, India of Gorakhpur division has contributed to our knowledge of the distribution and population diversity of S. seemae which further helps us to understand its ecological distribution as well as physiological variation in a particular niche. Further biocontrol study results revealed that S. seemae showed best biocontrol activity against H. armigera at temperature and humidity of 25°C and 85% (w/w) at controlled conditions respectively.

CONCLUSION

It was concluded that the EPNs are of great importance to use in biological control. It is possible to rely on the results of this research in the control of H. armigera for use in the field application of pests. EPNs species would serve as an alternative to chemical pesticides and fit well in integrated pest management programs against larvae as well as pupae and adults of many economic insect pests which inhabit the soil.

SIGNIFICANCE STATEMENT

This study discovers the biocontrol agent in the form of EPN that have great importance to use in biological control. This study will help the researcher to increase the information to the researcher that entomopathogenic nematodes show biocontrol activities, which was available for developing suitable biocontrol strategies as well as enable us the recognition of biocontrol EPN from the North-Eastern region of India in place of chemical control.

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