Agro-Insects- M. ABID HUSSAIN

Role of entomopathogenic nematodes in the management of insect pests

M. ABID HUSSAIN

Department of Entomology, M.S. Swaminathan School of Agriculture, Centurion University of Technology and Management, Village Alluri Nagar, R. Sitapur, Uppalada, Paralakhemundi, Gajapati – 761221, Odisha, India

Email ID: abidentonema@gmail.com

One of the major constraints in agricultural production in India is losses sustained due to attack by pests and diseases. Chemical pesticides are widely adopted for pest control despite of well-known hazards like development of resistance, persistent residues, contamination of food and water resources, etc. Sole reliance on pesticides is not sustainable because of the associated problems of environmental degradation, secondary outbreak of insect pests due to elimination of natural enemies. Other effective and environment-friendly methods have to be adopted, which has gained momentum in recent years. Biological control is an ideal alternative, is economical and has long-term control, without risk to human beings, cattle and other non-target organisms. When used as a part of IPM programmes, microbial pesticides (bacteria, fungi, nematodes, protozoans and viruses, etc.) are viable, safe and eco-friendly alternatives. Entomopathogenic nematodes represent an important part of the spectrum of biocontrol agents.

Nematodes associated with insects that parasitize, cause disease and kill the insects are called Entomopathogenic nematodes (EPN). Entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae are obligate parasites of insect species and are associated with symbiotic bacteria Xenorhabdus and Photorhabdus spp., respectively which also show potent insecticidal properties (Stock & Goodrich-Blair, 2012). Their potential as insecticidal agents has been investigated against a wide range of insect species such as coleopteran, lepidopteran, and dipteran pests by many researchers around the world (Begley, 1990; Grewal et al., 2005; Hussain & Ahmad, 2011; Lacey & Georgis, 2012; Prasad et al., 2012). Entomopathogenic nematodes persist in the soil as non-feeding, third stage infective juveniles (IJs), detecting their potential hosts by physical and chemical cues. Infective juveniles penetrate the host via natural body openings, such as mouth, anus, spiracles and/or also directly through cuticle. The symbiotic bacteria are regurgitated in the host, which then multiply rapidly and cause host death by septicaemia, often within 48 h of infection. The bacterial attack causes break down of the host tissue, thereby providing nourishment to the nematodes; the juvenile nematodes develop into adults and reproduce, raising its next generation (Grewal et al., 2005). The dauver juveniles recover in response to food signals present in the insect’s haemolymph, feed on proliferating bacteria and decomposing tissues, develop into adults, mate and produce two or more generations. When the food resources get exhausted, IJs exit the cadaver and search for new hosts in the soil.

World scenario

EPN-based products are available worldwide for control of insect pests largely due to the progress made in mass production, formulations and field efficacy. The exemption of nematodes from registration, their ability to infect and reproduce on a broad spectrum of insects and safety to non-target organisms have made them an alternative proposition commercially.

Entomopathogenic nematodes are available commercially for large-scale application in many crops world over and now accepted as commercially viable biopesticides. End users became more interested in their use and can vary depending on the crop, market or country. The most obvious reason may be the non-availability of alternative conventional chemical insecticides. Most products are sold through local and established distribution companies who have long standing relationship with their customers (Table 1).

Table 1. Nematode producers in Europe and USA*

Company                             EPN species**                 Formulation        Commodity

Andermatt Biocontrol, Switzerland  Sc, Sf, Hme     Clay Greenhouse, home gardening

Asa Jung Lab, California   Sc, Sf        Clay                Varied

BioLogic Willow Hill, Pennsylvania Sc, Sf               Bulk, WDG, sponge     Varied

Bionema, Sweden               Sc, Sf        Polymer         Home gardening

Becker Underwood Ames, Iowa        Sc, Sf, Sg, Sk,  Sprayable, WDG         Greenhouse, mushrooms,

                                              Ss, Hme                           mole crickets, turfgrass

Certis Columbia, Maryland               Sc, Sf, Sr          Bulk, WDG         Varied

e-nema, Germany               Sc, Sf, Hb Clay, polymer         Greenhouse, mushroom,

                                                                                      nurseries, turfgrass

Hydrogardens, Colorado  Sc, H. sp. Sponge            Greenhouse

Integrated Biocontrol Greendale, Indiana                Hb, Hi, Hma      Paste, sponge                                              Citrus, turf grass

Koppert, The Netherlands Sf, Hb      Clay                Greenhouse, turfgrass

M&R Durango, Colorado  Sc, Sf, Hb Sponge            Varied

Owiplant, Poland               Sf              Clay                Greenhouse, mushrooms

* Reproduced from Kaya et al. (2006).

**Sc = Steinernema carpocapsae, Sf = S. feltiae, Sg = S. glaseri, Sk = S. kraussei, Ss = S. scapterisci,

  Sr = S. riobrave, Hb = Heterorhabditis bacteriophora, Hi = H. indica, Hma = H. marelatus, Hme = H. megidis

In Japan, EPN are used against billbugs on golf courses. In China, they are used to fight peach borers. In Europe, blackvine weevils and mushrooms flies are combated with nematodes. In Canada, EPN are used to control caterpillars and root weevils. In USA, various species of EPN are used against white grubs, mole crickets, fungus gnats, citrus weevils and artichoke plume moth. Bill bugs, sod webworms, fleas and mint borers are best controlled by Steinernema carpocapsae. Blackvine weevil, strawberry root weevil and cranberry girdler are major pests in strawberry and cranberry industries and are best controlled by S. carpocapsae and Heterorhabditis. Two other nematodes, S. scapterisci and S. riobrave are nematode industries success stories. S. scapterisci was detected in South America and introduced into Florida, which kept the population of mole cricket under check. S. riobrave was highly effective against citrus weevil on Florida, which was tried against the weevil pests.

Indian scenario

Most of the work in India was focused on the biological control potential of insect parasitic nematodes, especially Steinernema carpocapsae. For example, Rao & Manjunath (1966) suggested that DD-136 strain of S. carpocapsae could be used in the control of insect pests of rice, sugarcane and apple. Different workers have studied the use of EPN against cutworms, ragi pink borer, rice leaf folder, stem borers, paddy gall midge, sugarcane borers, white grubs, red hairy caterpillars, etc. in laboratory and field. Table 2 includes some of the insect pests that could be controlled with EPN. Although efficacy research has been conducted in India with S. carpocapsae since mid-1960s, research programmes dealing with other aspects of EPN are still not finalized and up to marketable stage, which can be delivered to end users.

In 1980s, one company (Ecomax Pvt. Ltd.) brought out two internationally marketed products of Biosys Company (USA) for marketing in India, namely, S. carpocapsae and H. bacteriophora under the trade name soil commando and green commando. These products were tested through multilocational trials in the country during 1995-1996. However, these nematodes were not efficacious against insects under field conditions probably because of their poor adaptability to environmental conditions in India or production/formulation problems. Finally, these nematodes were withdrawn from the market and presently no company is marketing EPN in India.

Several national-level research laboratories such as National Institute of Plant Health Management (Hyderabad), Indian Agricultural Research Institute (New Delhi), National Bureau of Agricultural Insect Resources (Bengaluru) and a few commercial companies such as Multiplex Biotech Pvt. Ltd., and Pest Control (India) Pvt. Ltd. are conducting research to bring their formulated products with enhanced shelf-life. Efforts were made to improve the formulations with respect to storage, shelf-life, application technology, infectivity, etc. The attempts were made to formulate different species of entomopathogenic nematodes (e.g., H. indica, S. abbasi, S. bicornutum, S. carpocapsae, S. riobrave) in different carrier materials (e.g., talc, alginate capsule, wheat bran pellets, sodium alginate pearl, vermiculite, spray-adjuvants, or hydrogel) (Hussaini et al., 2001, 2003; Gupta, 2003; Vyas et al., 2001). However till date, it is under extensive research phase only and other aspects of nematodes are still not finalized and up to marketable stage.

Table 2. Target pests of EPN in India

Scientific name          Common name     Commodity        EPN species**

Agrotis ipsilon/A. segetum                         Black cutworm   Potato        Sf

Anomala sp.                Potato chafer grub                             Vegetable  Sc

Amsacta albistriga        Red hairy caterpillar                        Groundnut          Sc, Hb

Chilo sacchariphagus indicus                      Sugarcane internode borer   Sugarcane  Sc, Sg, Sf, Hi

Chilo partellus              Maize tissue borer                             Maize         Sc, Sb, Sf, Sg, Hi

Earias vittella               Spotted bollworm Okra                    Sf, Sr, H. sp.*

Helicoverpa armigera    Pod borer              Pulses, cotton, vegetable       Sc, Sf, Sg, St, Hb, Hi

Holotrichia sp.             White grub            Sugarcane           H. sp.*

Leucinodes orbonalis    Early shoot & fruit borer                  Egg plant   Sc, Sb, Hi, H. sp.*

Odoiporus longicollis   Banana pseudostem weevil              Banana      Sb, Hi

Phthorimaea operculella                               Potato tuber moth        Potato        Sb, Hi

Plutella xylostella         Diamondback moth                          Crucifers    St, Sb, Hi

Scirpophaga incertulas Yellow stem borer                             Rice  Sc, Os

Spodoptera litura          Tobacco cutworm Tobacco, vegetable,     Sc, Sa, Sb, Sg, Sf, Sr,

                                                                   groundnut           Hb, Hi, H. sp.*

* Recently work has been carried out at Biocontrol Laboratory, SBVPUAT, Meerut (Prasad et al., 2012).

**Sa = Steinernema abbasi, Sb = S. bicornutum, Sc = S. carpocapsae, Sf = S. feltiae, Sg = S. glaseri, Sr = S. riobrave, St = S. thermophilum, Os = Oscheius sp., Hb = Heterorhabditis bacteriophora, Hi = H. indica, H. sp. = Heterorhabditis sp.

Nematode diversity

The initial research with EPN in India was conducted primarily with exotic species/strains of S. carpocapsae, S. glaseri, S. feltiae and H. bacteriophora imported by researchers. In many cases, these nematodes yielded inconsistent results in field trials probably due to their poor adaptability to the local agro-climatic conditions.

India has a rich biodiversity resources because of its varied geographical, climatic and weather conditions. Therefore, a search for indigenous species/strains resulted in a number of nematode isolates from different parts of India.

Among the indigenous nematode isolates, four have been described as new species, namely, Heterorhabditis indica from Tamil Nadu, Steinernema thermophilum from New Delhi, S. masoodi and S. seemae from Uttar Pradesh. Other species identified as indigenous isolates include S. abbasi, S. carpocapsae, S. bicornutum, S. glaseri, S. riobrave, S. feltiae, S. siamkayai, S. tami and H. bacteriophora. Apart from these, there are several unidentified Steinernema and Heterorhabditis species reported from different parts of India.

Recently, one isolate of Heterorhabditis sp. and one Steinernema sp. were isolated from the soil samples brought from mango orchard and guava respectively of Meerut District indicating a possible significant role in regulating the population of soil insect pests or even to those insects which undergo pupation in the soil. Encouraging results were obtained in the laboratory bioassays of Heterorhabditis sp. against Galleria mellonella (greater wax moth), Corcyra cephalonica (rice meal moth), Leucinodes orbonalis (brinjal shoot & fruit borer), Earias vittella (spotted bollworm), Spodoptera litura (tobacco cutworm) and Holotrichia sp. (white grub) (Prasad et al., 2012). Further studies will establish their possible role in formulating insect pest management strategies.

Mass production

In the past, EPN have been cultured on a variety of substrates such as potato mash, ground veal pulp, peptone-glucose agar and pork kidney, homogenized animal tissues, dog food, chicken offal homogenate, etc.

Entomopathogenic nematodes infect and reproduce on a broad spectrum of insects. They may be reared in vivo in laboratory, Galleria mellonella is the most used host all over the world because it can be reared easily, are very susceptible and an excellent host for nematode reproduction. A population of 465,000 IJs of S. abbasi and up to 535,000 H. indica have been harvested from a single larva of G. mellonella (Shakeela & Hussaini, 2006). However, other locally available host insects like S. litura, Corcyra cephalonica, Chilo sacchariphagus indicus, Scirpophaga exceptalis and Bombyx mori may also be utilized for this purpose.

Production on a commercial scale had been accomplished by using inexpensive protein and sterol sources. Bedding (1984) pioneered the development of an inexpensive monoxenic, 3D culture matrix using animal byproducts. EPN have also been produced in large-scale, liquid fermentors using a combination of plant and animal proteins. Thus, mass production of EPN has been evolved from the first large-scale in vitro solid media production by Glaser et al. (1940), to in vitro production by Dutky et al. (1964) to 3D solid media in vitro process (Bedding, 1984) and to the in vitro liquid fermentation production method (Ehlers, 2001). Currently, commercial nematodes are produced monoxenically using the solid media process developed by Bedding or liquid fermentation method. The solid media process has successfully produced EPN but high labour costs limit economics of scale. This technology is most adaptable for countries where labour costs are minimal. The liquid fermentation process is highly efficient for several steinernematids but not for heterorhabditids and is economical for industrialized countries.

At present S. carpocapsae, S. feltiae, S. glaseri, S. riobrave and H. indica can be consistently and efficiently produced in 7,500 – 80,000 litre fermentor with an yield capacity as high as 150,000 IJs/ml. In India where labour costs are minimal, the solid media technology is adaptable and can be developed into a cottage industry utilizing locally available ingredients as well as nematodes.

Formulation and application

An effective formulation provides a suitable shelf-life, stability of product from transport to application, and ease of handling. Shelf-life in most formulation is obtained by reducing nematode metabolism and immobilization, which may be accomplished through refrigeration and partial desiccation. The potential for using desiccation for long-term storage is limited because EPN apparently cannot reach a true cryptobiotic state (fully arrested metabolism) upon desiccation.

Entomopathogenic nematodes have been formulated for commercial application in various carriers such as activated charcoal, sponge, vermiculite, peat, alginate gels, infected cadavers, etc. (Georgis, 1990). A breakthrough in formulation technology was cited in the introduction of water dispersible granules (WDG) in which nematode enter a partially anhydrobiotic state allowing them to survive up to 6 months at 4–25 ^oC. Infective juveniles are encased in 10–20 mm dia. granules consisting of mixtures of various types of silica, clays, cellulose, lignin and starches (Grewal, 2000).

A new nematode formulation based on polymeric material has played a major part in the acceptance of foliar application and the use of weekly, low volume, low concentration sprays has proved more robust than one innundative release (Shapiro-Ilan et al., 2005).

In India, efforts have been made to formulate and use EPN for insect control. Commercial formulations green/soil commandos of yesteryears are not available and its production has been discontinued. Different laboratories of Indian Council of Agricultural Research and State Agricultural Universities have tried out various EPN formulations with varying degree of success. Efforts are on to improve the formulations with respect to storage, shelf-life, application technology, infectivity, etc. A list of EPN formulations developed or reported in the experimental research are given in Table 3.

Table 3. EPN formulations

EPN species                           Production/formulation       Reference

Heterorhabditis indica              Talc                               Hussaini et al. (2003)

Steinernema abbasi                   Talc                               Hussaini et al. (2003)

S. bicornutum                          Bait as alginate capsule        Hussaini et al. (2001)

S. carpocapsae                          Talc                               Hussaini et al. (2003)

                                                Alginate capsule          Hussaini et al. (2001)

                                                Wheat bran pellets      Hussaini et al. (2001)

                                                Pearl (sod. alginate)    Gupta (2003)

                                                Vermiculite                  Hussaini et al. (2003)

S. riobrave                                Spray-adjuvants           Vyas et al. (2001)

The comparison of three (talc-, alginate-, and water-based) formulations of entomopathogenic nematodes revealed better survival of IJs in water formulations than the talc and alginate-based formulations of Steinernema sp. (Hussaini, 2003). In comparison, H. indica survived better in talc-based formulation than in other formulations. A NemaGel-based hydrogel formulation of S. thermophilum (at the concentration of 100,000 infective juveniles per gram of suspension) was claimed to survive up to 9 months at 15 °C (Ganguly et al., 2008). The latest formulation, developed by Multiplex Biotech Pvt. Ltd., comprised of hydrogel-based semi-solid cream containing 1,000,000 infective nematodes suspension in a polythene pouch that can be readily mixed in a water spray tank without blocking the nozzles (Divya & Sankar, 2009). They achieved shelf-life of 3 months by keeping the formulation at 27-28 ^oC with 80% nematode survival by increasing the nematodes' metabolic rate when mixed with silica powder (0.1%).

During the last two decades, testing on EPN against several insect pests of agricultural and horticultural crops was continued, tested in field and variable results were obtained. Some of the promising cases are cited in Table 4.

Table 4. Field efficacy of EPN against various insect pests in India

EPN species                           Insect pests                   Reference

Heterorhabditis bacteriophora   Amsacta albistriga          Bhaskaran et al. (1994)

H. indica                                  Helicoverpa armigera      Hussaini (2003)

                                                Leucinodes orbonalis       Hussaini (2003)

Steinernema carpocapsae          Spodoptera litura            Sezian et al. (1996),

                                                                                      Hussaini (2003),

                                                                                      Sitaramaiah et al. (2003)

                                                H. armigera, L. orbonalis          Hussaini (2003)

                                                A. albistriga                    Bhaskaran et al. (1994)

Hi, Hb, Sc, Sa                          Holotrichia longipennis  Hussaini et al. (2005)  

S. glaseri                                  S. litura, H. consanguinea       Vyas & Yadav (1993)

S. riobrave                                Agrotis ipsilon                Mathasoliya et al. (2004)

Steinernema sp.                       Papilio sp.                      Singh (1993)

Heterorhabditis sp.                   H. armigera                    Vyas et al. (2002)

Future thrusts

Even though EPN has been proved to be potential biocontrol agents against a number of insect pests world over, India lag behind and still is in developing stage. There is need for intensive surveys for isolation of heat and desiccation tolerant isolates of EPN from different agro-climatic zones of the country and setting up of EPN identification services based on morphometrics and molecular techniques.

There are several issues, which require immediate attention for their commercialization such as mass production, formulation and utilization in field as a component of IPM. Successful control of insect pests can only be achieved when the nematode materials reaches the end user in good condition. Improvement in storage condition, formulation shelf-life and transport facilities must provide optimum conditions to guarantee maximum survival and infectivity.

To make EPN successful, realistic strategies are needed through IPM programmes, improved delivery systems to overcome their inherent cost, formulation instability and limited field efficacy towards certain insect pests. Application methods used to deliver EPN require critical assessment or standard pesticides application methods which would ensure better efficacy under field condition.

Conclusion

With EPN’s impressive attributes, which make them an excellent alternative tool for control of insect pests, EPN has sparked heightened interests amongst the biocontrol workers and lead to immense opportunities to test them against a wide variety of insect pests. EPN are particularly virulent against lepidopterous and coleopterous pests and is being used to control insect pests in high-value crops. They can be used on a large-scale in integrated pest management, organic farming and sustainable agricultural systems to control insect pests.

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