Entomopathogenic Nematodes: Potential Bio-pesticide in Insect Pest Management
What are Entomopathogenic Nematodes (EPNs)?
As the Greek word, entomopathogenic suggests where, entomon mean insect and pathogenic means causing disease so, EPNs are nematodes that cause disease in insects. More specifically, EPNs are soil in-habiting, soft bodied, non- segmented roundworms, obligate or sometimes facultative parasites of insects. They are also called endoparasitic as they remain parasitically inside the infected insect body. We can find them over diverse ecological habitat, feeding upon soil inhabiting insects like eggs, larvae of butterfly (caterpillars), immature and adults of beetle, grasshopper, crickets etc. Survival of EPNs in soil is affected by biotic and abiotic factors such as temperature, soil texture, moisture, radiation, other pathogens etc. Temperature extremes (below 0°C or above 40°C) are lethal for most EPN species. In general, sandy soil with large pores is the most favorable soil type for nematode movement. The virulence of EPNs usually decreases in low moisture conditions and infectivity is highest at moderate soil moistures (−10 to −100 kPa). EPNs are sensitive to UV radiations and short wavelengths (254 nm).
Mainly two families of nematode, Steinernematidae and Heterorhabditidae are pathogenic to insects. Family Steinernematidae has two genera Steinernema and Neosteinernema longicurvicauda. Family Heterorhabditidae has only one genus, Heterorhabditis . Worldwide, nearly 100 species of Steinernema and 17 species of Heterorhabditis (Hunt and Subbotin, 2016) have been discovered and studied. Commonly studied and widespread species of EPNs are S. carpocapsae, S. feltiae, S. glaseri, H. bacteriophora, and H. megidis (Peters, 1996). EPNs are mutually associated with bacteria of the family Enterobacteriaceae. Nematodes of family Steinernematidae usually carry bacteria of genus Xenorhabdus and family Heterorhabditidae usually carry species of Photorhabdus. Till the date 20 Xenorhabdus and 4 Photorhabdus species have been studied. The nematode and bacteria possess truly mutualistic relation, where bacteria get shelter, protection and ability to dispersion and on other hand bacteria supply nutrient, protect host cadaver from scavengers and provide suitable environment for reproduction of nematode.
Why EPNs are useful in Insect Pest Management ?
Synthetic chemical pesticides, which were highlighted as ultimate plant protection measures during 1900s have lost their charm now, because the world have faced many negative consequences of synthetic chemical pesticides. Chemical pesticides have resulted environment pollution, insect resistance to pesticide, pest resurgence, secondary pest outbreak, loss of natural enemies of insects, loss of biodiversity, bioaccumulation and biomagnifications of persistent pesticides in food chain, chronic health hazard on human and animal. World is looking for eco-friendly, economical and sustainable ways of pest control. In this situation use of entomo-pathogenic nematodes as biological control agents would be a smart step towards sustainable agriculture. In Nepal, pesticide use rate is low as compared to other countries (396 gm a.i./ha) but there is haphazard use of poisonous pesticides in certain vegetable production pockets due to poor knowledge of pesticide application, personal safety and pesticide poisoning among farmers. Realizing harmful effects of chemical pesticides, Government of Nepal has banned 24 synthetic chemical pesticides and many policies have been formulated encouraging organic agriculture, use of bio-pesticides and botanicals in plant protection. Beside these, EPNs are effective alternatives for insect pest control due to following characteristics.
- They are biological agents so there is no risk of persistence in food products and nature.
- They non-toxic to non-target organisms; plants and vertebrates
- They are precise to their target pests.
- They are compatible with other methods of integrated pest management.
- They have broad host range e.g. Coleoptera, Dictyoptera, Orthoptera, Lepidoptera, Hemiptera, Diptera.
- They do not pose harm to beneficial organisms hence do not disturb natural ecosystem.
- They can be applied efficiently with pesticide equipments like sprayer.
- They have high virulence; kill their host with 24-72 hours.
How do they control insects?
Entomopathogenic nematodes live in soil and locate their host in response to carbon dioxide, vibration and other chemical stimuli or physical structure of insect’s integument. EPNs have six stages in it’s life cycle egg, juvenile 1, 2,3,4 (called J1, J2 J3, J4) and adult. Third instar juvenile also called ‘dauer’ is the only free-living stage that occurs outside the host in soil, all other stages complete within host body. EPNs generally complete their life cycle (from penetration of infective juvenile into host body to emergence of new infective juvenile from host) in 12-15 days in optimum temperature i.e. 20-30o Celsius. Infective juvenile enter into host body through natural openings like mouth, anus,spiracles. Then IJ actively penetrate through the midgut wall or tracheae into the insect hemocoel containing haemolymph. Infective juvenile releases symbiotic bacteria from its intestine. And bacteria start multiplying in the nutrient-rich haemolymph. Infective juveniles recover from their arrested state (dauer stage) and start feeding on multiplying bacteria and disintegrated host tissues. Continuously developing nematodes and multiplying bacteria produce toxins in the body cavity and kill the insect host usually within 48 hours. In the presence of favorable environment nematode starts to multiply within host body. After two to three reproductive cycles, when host nutrients are depleted, large number of free living, infective juveniles are produced and released to continue their life. They leave the host body and immediately infect a new host, or remain in soil searching new host. In this way, mutual partnership between nematode and bacteria kill host insect.
Are EPNs effective in insect pest control?
Many EPNs have been used under biological control method to manage agricultural pests. Pests like white grubs, cutworms, black vine weevil, turf grass pests, fungus gnats, mole crickets, and weevils have been controlled by applying infective juveniles of Steinernema glaseri, Steinernema riobrave, H. bacteriophora, Heterorhabditis megidis, H. indica, and Heterorhabditis marelatus. EPNs have been used to control the larval stages of several wood-boring Lepidoptera that attack ornamental shade and fruit trees. Survival, search behaviour and infectivity vary among different nematode species and strains which make them more or less suitable for particular insect pest control. For successful use of EPNs as insect control, selection of correct nematode species for specific pest has great importance. Some steinernematids are adapted to specific groups of insects. For example, S. scapterisci is adapted to orthopterans, especially mole crickets while S. kushidai and S. scarabei are effective against scarabaeidae larvae. Some EPNs commercially used for insect pest control are given in the table below.
Table 1: Entomopathogenic nematodes (EPNs) used to control insect pests
|S.N.||Crops||Pest (Common Name)||Pest (Scientific Name)||EPNs|
|1||Artichokes||Artichoke plume moth||Platyptilia carduidactyla||Steinernema carpocapsae|
|2||Vegetables||Armyworm||Steinernema carpocapsae, S.feltae, S.riobrave|
|3||Bananas||Banana root borer||Cosmopolites sordidus||S. carpocapsae, S. feltiae, S.glaseri|
|4||Citrus, ornamentals||Citrus root weevil||Pachnaeus
|S. riobrave, Heterorhabdis bacteriophora|
|5||Vegetables||Corn earworm||Helicoverpa zea||S. carpocapsae, S. feltiae,
|6||Turf, ornamentals||Scarab grub||Phyllophaga rugosa||H. bacteriophora, H.zealandica , S. carpocapsae, S. glaseri, S.scarabaei|
|7||Vegetables, ornamentals||Leaf miner||Liriomyza spp.||S.carpocapsae, S. feltiae|
|8||Sweet potato||Sweet potato weevil||Cylas formicarius||H.bacteriophora, S.carpocapsae, S. feltiae|
|9||Grapes||Grape root borer||Vitacea polistiformis||H.zealandica, H. bacteriophora|
|10||Tomato||Tomato leaf miner||Tuta absoluta||S. feltiae|
How can we produce and formulate EPNs in mass?
EPNs can be produced commercially in vivo (inside their hosts) as well as in vitro (on artificial solid or liquid medium).
In vivo Production
In this method small quantity of EPNs are produced for nearby markets using locally available hosts. The hosts must be susceptible, having high multiplication potential, not prone to become crop pests and easy to rear using inexpensive materials. Galleria mellonella is widely used host for in vivo multiplication of EPNs. In vivo culture relies on production in trays and shelves. A White trap with moist paper is used to harvest the infective juvenile emerged from insect cadaver. This is labor intensive job but produces good quality nematodes. It is suitable for small farmers and farmer cooperatives in specific niche market.
In vitro Solid Production
In this method, nematodes are cultured on a crumbed polyether polyurethanee sponge impregnated with emulsified beef fat and pig’s kidney along with the appropriate symbiotic bacteria.
In Vitro Liquid Production
For large scale, in vitro liquid culture production is most cost-efficient and is potential to dominate the quantity of EPN production worldwide. At present, the in vitro liquid culture method is a commercially viable method wherever expertise, initial capital, and large bioreactors are available.
What are the commercial formulations of EPNs?
Formulation means to improve activity, absorption, delivery, storage, stability and ease of application of an active ingredient. Commercial products of EPNs come in various formulations like water dispersible granules, capsules, desiccated cadavers, baits etc. among them water dispersible granular formulation have been found the most appropriate with longer shelf life, minimum space for storage, and ease of application. ‘Living insect bomb’ are the latest formulations to increase efficacy of EPNs. Bio safe, Bio-safe N, Biovector, Exhibit, Fightagrub, Otinem, Pianbiot, Safer, Sanoplant, Terrix are some commercial products of EPNs. Additives such as surfactants, adjutants increase survival and invasion of EPNs. Alginate, clay, activated charcoal are some commonly used formulates. IJs encapsulated in calcium alginate gels edible to insects, protect infective juveniles from dessication and UV radiations. Encapsulated Steinernema species showed over 90% survival for 6 weeks at 25°C in the granules made from diatomaceous earth (DE), hydroxyethyl cellulose, amorphous silica, lignosulfonates, starch. Temperature greater than 20-30 degree Celsius cause rapid metabolic activity, reducing viability and pathogenicity of nematode. Steirnernema at 5-100 C and Heterorhabdus at 10-150 C can be stored for 5 months at room temperature and for 12 months in refrigerator. Further improvements in formulations can help to increase survival of EPNs on plant leaves.
How do we apply EPNs?
Along with the selection of suitable strain of EPN for target pest and suitable formulation appropriate application practice also have important role in success of EPNs against insect pests. EPNs can be applied through pressurized sprayers, hand sprayers, mist blowers, electrostatic sprayers, through aerial spraying equipment and through irrigation system. Before spraying spray volume, agitation, nozzle type, pressure and recycling time, system environmental conditions, and spray distribution pattern etc. has to be considered carefully. EPNs are sensitive to UV rays so, IJs has to be applied in the evening or early in the morning or on a cloudy day when the radiation and desiccation are minimal to get better result. EPNs can be integrated with other control methods, with chemical insecticides such as chlorpyrifos, dimethoate, and imidacloprid. Earthworms can be used to increase field population and soil movement of EPNs. But it should not be used along with chemical nematicides. Release of live insect host pre-infected with EPNs which are called living insect bombs is suitable in case of tall trees and tree trunks.
What is the situation, future perspectives and challenges of EPNs in Nepal?
In global market nematode-based products has become competitive with chemical insecticides in medium and high-value crops with advancement in genetic improvement, strain stabilization, mass production, formulation and application strategies. In Nepal, researches are being carried out to discover indigenous strains of EPNs from various parts of the country and to test their efficacy against insect pests in laboratory conditions. A survey of entomopathogenic nematodes was conducted for the first time in Nepal during June–December 2007 (Khatri-Chhetri et. al., 2010) and strains of Steinernema siamkayai and Steinernema abbasi were identified. In a bioassay experiment to test efficacy of two indigenous strains S. abbasi and S. siamkayai against white grub larvae (Chiloloba acuta), S. abbasi was found more efficient.
Discovery of wild strains of EPNs, scientific researches on efficiency evaluation of available strains, scientific studies on biology, ecology of EPNs and target hosts, , host finding strategy, host suitability, nematode-host relations, cost effective and large scale production of EPNs with appropriate formulations, storage facility and wide availability of EPNs in markets are future perspectives and room for improvements in use of entomopathogenic nematodes. Other difficulties like high cost of production, labor intensive work, technical knowledge and equipments for production, storage and transport, difficulty in formulation and quality control and deterioration by biotic and abiotic factors like temperature, humidity, UV radiations, natural enemies etc are challenges in this field.
In Nepalese context, farmer level in vivo production through cooperatives and IPM farmer groups can be a feasible approach to produce and distribute EPNs in local markets. Technical knowledge about production and application should be provided to farmers through research and extension. Before that sufficient desk studies, laboratory and field research should be done by concerned bodies and plant protectionists. Involvement of private sector in commercial production of tested EPNs can accelerate large scale production and marketing.