Indian Farming 66(2): 34–38; May 2016

Effective Mass Production Technique

for entomopathogenic nematodes
Sunanda Soregaon1 and P. Jeyakumar2
National Institute of Plant Health Management, Rajendranagar, Hyderabad, Telangana 500 030

A GRICULTURE in India is
progressing towards second
green revolution. Though first green
dependence on chemical fertilizers
and pesticides simultaneously.
The heavy pest incidences force
the cost of cultivation and
dependence on chemical pesticides.
The emerging problems due to
revolution had helped in increasing the growers to resort to indiscrimate indiscriminate use of chemical
the food production to feed the use of chemical pesticides to get pesticides are pesticide resistance,
increasing population during late 60s quick respite and increased yield. The resurgence and secondary pest
and 70s, it was not sufficient due to annual pesticide consumption during outbreak, beside pesticide residues
many fold increase in population 2011-12 stood at 50.58 thousand leading to health hazards. On the
during last 4-5 decades. However, the tonnes. Although per capita other hand public concern is arising
successive revolutions such as white consumption of pesticides in India is about the potential adverse effects of
revolution, yellow revolution, blue 0.6kg/ha below the world average of chemical pesticides on human health,
revolution and brown revolution in 3kg/ha and lowest among the environment and biodiversity.
the country have also attempted to developed and developing countries Further many export commodities
fulfill the demands of milk, edible (Japan 11kg/ha, USA 7kg/ha, China such as basmati rice, mangoes, spices
oils, fish products and meat products 13kg/ha, France 4.6kg/ha, Pakistan and tea were rejected due to high
respectively. The country has achieved 1.3kg/ha and Netherlands 9.4kg/ha). level of pesticide residues and
first position in production of many USA is the most pesticide conscious detection of banned pesticides, which
agricultural commodities in the country; though it uses more adversely hit the trade and economy.
world. But over the decades climate pesticide than India and the These negative impacts of pesticides
has changed and is showing its productivity is also more due to cannot be eliminated altogether,but,
adverse impact on agriculture, judicious and scientific use of their intensity can be minimized
especially the droughts, floods and pesticides. Thus, India has to through development, dissemination
biological threats such as insect pests seriously look towards non chemical and promotion of alternative
and diseases have increased. Further ways of pest management to reduce technologies such as bio-pesticides
their changed behavior is hindering and bio-agents as well as good
Crop Losses due to Insect Pest in Major
many agricultural and horticultural agronomic practices rather than
Crops
crops in addition to forest flora and relying solely on chemical pesticides.
fauna. In order to overcome these Crop % losses
problems and maintain sufficient Rice 25
Non chemical pesticide approaches for
supply of food, feed, fodder, fuel and Wheat 5-10 insect pest management
fiber steady move toward second Maize 25 The traditional intellectual
green revolution has become Other cereals 30 knowledge of Indian farming
inevitable. The struggle is not only Pulses 30 community has the treasure of many
for food security but also to ensure Oilseeds 35 ways of non-chemical pest
food safety, i.e. to keep increasing Sugarcane 25 management. Over the centuries they
Cotton (lint) 50
food production by reducing were learnt, inherited, tried, tested

Crop protection is equally important as that of other crop management aspects. The herbivorous
insects cause loss of 25 to 28% of the potential food production. Annual crop losses due to insect
pests and diseases in India are estimated to be 18% of the agricultural output. Insects alone cause
losses worth of ` 9.5 lakh billions. Losses caused by key pests may be higher, i.e.Helicoverpa spp. in
cotton causes losses up to 50%, i.e. estimated to be more than ` 2,000 crores despite the use of
pesticides worth of ` 500 crores. The yield losses due to these insect pests reach as high as 70-80%
in severe conditions both in pre-harvest and post-harvest stages.

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Indian Farming
May 2016
and authenticated for practice among Though nematodes are commonly release of symbiotic bacteria. They
the community. However, they considered as plant pathogenic, but, are highly active in top 20 to 30 cm
started disappearing under the like other microbes, beneficial of the soil, where most of the soil
shadow of industrialization and nematodes known as borne insects thrive, perpetuate and
infusion of chemical pesticides. Entomopathogenic Nematodes cause damage to crops. They have a
However, many such vanished non (EPNs) also exist which act against broad host range comprising of more
chemical pest management many insect pests of economic than 250 insect species belonging to
approaches are reinvented under the importance. Their use and ten orders.
aegis of organic farming. The most exploitation in Indian farming is very EPNs are totally safe to
important nonchemical approach limited due to the fact that they were environment, humans and animals.
being explored currently is the use of widely studied and known for their They are already used in many
bio-control agents, bio-pesticides and beneficial characters, but little is done developed countries against different
bio-fertilizers in Indian agriculture to to bring them to main stream of insect pests. In USA, they are
reduce the use of chemical pesticides. successful field application. exempted from registration by
The rich biodiversityin Indian Environmental Protection Agency
subcontinent also encourages the Entomopathogenic nematodes for (EPA). They can be recovered from
explorers to hunt for new bio-control biological pest management soil and mass multiplied on artificial
agents and bio-pesticides. Microbes Organic farming or integrated pest diet or living hosts and can be stored
such as bacteria, viruses and fungi of management aims to reduce cost of for a long period in soil or cadaver.
different species are widely cultivation and dependence on They are compatible with many
considered as best alternative sources chemical pesticides. Like other pesticides, can be formulated as dust,
of bio-pesticides. Some of the microbial bio-pesticides, entomo- sprays, capsules, granules, etc. and
biopesticides such as Metarhizium, pathogenic nematodes (EPNs) can be applied through
Beauveria, Pseudomonas and NPV are promise both objectives at no risk routineprocedures such as spraying
considered as potential candidates of and low cost. EPNs are basically soil suspension or through irrigation
integrated pest management (IPM) dwelling nematodes, there is no system. With this wide range of
practices. requirement of special conditions for advantages EPNs certainly hold
The past one decade has witnessed their action and survival. They prefer promising to be adopted as one of
boom of many such inventions and conditions suitable for their hosts and the most ideal and suitable bio-
products in the market. However, they keep infecting and multiplying pesticide against economically
their field application is not upto the as long as hosts are available. They important insect pests.
expectations and has not yielded have ability to search the target EPNs and their mode of action: Like
satisfactory results. Most of them are insects in soil, plant surface, partly plant parasitic nematodes,
applied against foliar insects, but little embedded insects in plant tissues and Entomopathogenic nematodes
is done against soil dwelling insects. quick kill of the target insect through (EPNs) are soft bodied, non-
segmented group of nematodes that
Success story are capable of causing death of
National Institute of Plant Health Management (NIPHM) has started popularizing the insects. They live parasitically inside
use of EPN with special focus on management of root grub in sugarcane. Root grub is a the infected insect host as
major pest which feeds on sugarcane roots. It was damaging thousands of sugarcane endoparasites. They infect different
fields in Maharashtra. The NIPHM took an initiative to popularize EPN as an alternative
types of soil dwelling insects. EPNs
strategy for root grub management in sugarcane of Maharashtra. It opened a new chapter
in management of root grub in this area. Initially EPN culture was supplied to sugarcane are capable of living in a wide range
belt of Sangli and Kolhapur district. Best practices of EPN application were demonstrated. of ecologically diverse habitats. The
Farmers were also trained in EPN mass rearing technology for root grub management. most commonly used and studied
With the assistance of district agriculture officersthe technology was successfully EPNs are members of
demonstrated in farmers’ fields. Based on the success in management of root grub with
Steinernematidae and Heterorhabditidae
EPN, farmer’s co-operatives in Sangli, Kolhapur and neighboring districts started
approaching the institute for training on use of EPNs against root grub. They established families. Both have similar life
a laboratory for in vivo production with technical support of NIPHM. The outstanding histories. The non-feeding,
results convinced the farmers that EPNs are most ideal bio agents for root grub developmentally arrested infective
management in sugarcane. The technology spread to more area during 2013-14 due to juvenile seeks out insect hosts and
its promising results. Total 2500 extension officers, farmers from farmers’ associations,
initiates infections. When they sense
groups of famers and contract farmers of sugar factories were trained successfully and
they are mass producing and using these nematodes against root grub control in the temperature and carbon dioxide
sugarcane field. Within short time, the newly introduced bio-control agent reduced the emissions of soil-dwelling insects,
pest population very drastically. The smiles returned on drowsy cane growers was proof they move towards their prey by
of ability of this technology developed at NIPHM. The entire sugar bowl of Maharashtra is means of two strategies i.e.
now looking to adopt this technology, many district agriculture officials are collecting
ambushing and cruising. Ambusher
EPNs culture from NIPHM and successfully demonstrating in farmers’ fields to show the
way forward in mitigating the menace caused by root grub in sugarcane.NIPHM is EPNs most effectively control insect
committed to build the capasity of different stake holders to popularize means production pests that are highly mobile on the
of EPN and their utilization as an alternative to excessive reliance on chemical pesticides, soil surface, such as cutworms,
the usage of which is determental to soil health. armyworms, mole crickets, etc.

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May 2016
 red if the insects are killed by USA, Israel, Germany, UK and other
Heterorhabditids and brown or parts of Europe, mass production,
tan if killed by Steinernematids. commercialization and application of
The color of the host body is EPN has been carried out.
indicative of the pigments
produced by the monoculture of Mass production of EPN
mutualistic bacteria growing in In India, though vast research
the hosts. The death of the insects works were carried out on use of
is mainly due to the toxins EPNs for long time, but the results
released by the bacteria and are yet to see the light in
nematodes acts as vector for their commercialization and field
transportation inside the insect application. Efforts done so far were
Isolation of EPNs by using mud pots body. When purified bacterium is limited to academic interest with
applied to insects, their mortality mainly of laboratory/net house
Cruiser EPNs are most effective is observed. A trilateral interaction experiments only. Few private
against sedentary and slow-moving exists between bacteria, nematodes entrepreneurs tried to bring them on
insect pests at various soil depths, and insects. Specific toxins released main stream of pesticides market
such as white grubs, root weevils, etc. by each bacteria and production of chain, but failed due to various
Generally, several Infective urease are the main factors reasons. The mass production of
Juvenile (IJ) stages of EPNs may responsible for insecticidal properties EPN is normally being done
infect a single insect host. IJs usingWhite Trap method, which
penetrate the insect’s body cavity Different insects managed by EPN involves technical skills and
either through natural body openings within India and outside India sophisticated laboratory facilities, this
(mouth, anus, genital pore, or Biological pest management is method has been modified by using
spiracles) or by penetrating the outer widely practiced in developed low cost materials to enable the
cuticle of the insect; countries. EPNs are equally progressive farmers to produce the
Heterorhabditids do this using a considered as potential bio-control quality EPN with locally available
dorsal “tooth” or hook. After agents in addition to bacteria, fungi materials is described here.
penetration inside the host, the and viruses. Large-scale application Isolation and Mass production of
IJsrelease bacteria that live and demonstration of the efficacy of EPN by “White Trap” method:
symbiotically within the EPN’s gut, EPN was carried out in the western Entomopathogenic nematodes are
but do not harm the nematode. The countries covering thousands of currently mass-produced by either in
nematode-bacterium relationship is hectares of land with different crops vivo or in vitro (solid and liquid
highly specific: only Xenorhabdus spp. and pests. So far, more than 30 culture). In vivo production is the
bacteria co-exist with species of EPNs have been described, most appropriate technology for
Steinernematids, and only but only nine species of Steinernema developing countries where labor is
Photorhabdusspp. bacteria co-exist and three species of Heterorhabditis less expensive. It involves simple
with Heterorhabditids. Once released have been commercially exploited process of culturing specific EPNs in
into the host, the bacteria multiply using in vivo and in vitro techniques live insect hosts at less capital and
quickly and under optimal conditions worldwide. In USA, only seven minimal technical expertise. In vivo
cause the host to die within 24 to 48 species are being commercially production system is based on the
hours. The insect cadaver becomes available. Only in few countries viz., white trap, which took advantage of
the IJ’s natural migration away from
Commercial use of EPNs against different insect pests host cadaver upon emergence. The
EPN species Target insect hosts most common insect host used is the
Steinernema glaseri White grubs
last instar larvae of the greater wax
Steinernema kraussei Black vine weevil
moth (Galleria mellonella), because of
Steinernema carpocapsae Cutworms, armyworms, sod webworms, chinch
its high susceptibility to most
bugs, codling moth, cranberry girdler, dogwood entomopathogenic nematodes, ease
borer and other clearwing borer species, black in rearing, wide availability and
vine weevil, peachtree borer, shore flies ability to produce high yields.
Steinernema feltiae Fungus gnats, shore flies, western flower Initially soil samples are collected
thrips
from specific locations. Around 250-
Steinernema scapterisci Mole crickets
300gm of soil with 60-70% field
Steinernema riobrave Citrus root weevils
Heterorhabditis bacteriophora White grubs (scarabs), cutworms, black vine
moisture is placed in mud pots. The
weevil, flea beetles, corn root worm Galleria larvae are placed inside the
Heterorhabditis megidis Weevils soil for baiting. After three to four
Heterorhabditis indica Fungus gnats, root mealy bug, grubs days, discolored dead insects are
Heterorhabditis marelatus White grubs (scarabs), cutworms, black vine collected, rinsed in water and placed
weevil for White trap method.

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Inoculation of EPN maintained between the cadavers incubator required for incubation.
Take a small petri plate (9 mm). and the top cover. Instead of Petri plates and watch
 • Place the white trap in the glasses, earthen plates of their
Place a filter paper along the inner incubator maintained at 25±10 corresponding shape and different
wall of the petriplate and observe daily for nematode sizes are used. In this simplified
 activity inside the body of the method, instead of plastic box the
Inoculate nematodes. larvae. Nematodes (especially moist soil samples are directly placed
 infective dauers) after exhausting in similar size moistened earthen pots
Release four grown up last instar the host will start emerging out of and kept closed their lid with earthen
wax moth larvae into petriplate and the cadavers and move to the plate. Once the insects are infested,
close it. bottom of the Petri dish they are placed on White trap like
 containing water. arrangement of earthen plates. A
(Write the date, host and species of • Decant water along with smaller earthen plate with convex
nematodes) nematodes daily into a beaker and bottom upward is placed in a larger
 replace with fresh distilled water. sized plate. Water is placed like in
After two days, carefully check the • Clean the harvested nematodes White trap till the edges of smaller
condition of the wax moth larvae. repeatedly by washing with plate immerse. The infested cadavers
Look for the nematode infected distilled water and stored with 0.1 are placed on top of the inverted
cadavers per cent formalin solution in a plate and upon emergence, the EPNs
conical flask. Such harvested migrate towards water. The
Extraction of entomopathogenic nematodes should be further procedure is repeated using same
nematodes infected to fresh culture of wax earthen plates after washing. On
• Take a watch glass (52 mm) and moth to confirm the authenticity every 15 to 18 days interval one cycle
place with its convex side up in a of EPN (Koch’s postulate) of production can be completed. The
large petri dish (110 x 25 mm). infection and to obtain pure EPNs are harvested and formulated
Place a filter paper (Whatman#1, strain. using different carrier materials such
dia. 90 mm) on the watch glass in as sponge sheet or in cocopeat clay
such a way that, the lower edge of Materials required for white trap soil pith and coir powder.
the filter paper should touch the method The entire process maintains the
bottom of the Petri dish. • Glass wares (petriplates, watch similar conditions as that of white
• Pour the distilled water over the glass etc.) trap and can be done in a clean room.
filter paper and allow spreading on • Sophisticated laboratory The capillary movement of water in
the petri dish. The level of the • Biological Oxygen Demand earthen plates takes care of
water should be maintained such (BOD) maintaining the moisture required for
that, it should always be in contact • Technical skilled personal nematodes migration.
with the edges of the filter paper • Filter paper (Whatman)
only. Modified White Trap Method (New Materials required for modified white
• Place wax moth cadavers on the method): The simplified low cost high trap method
top of the watch glass. Cover the production method has principles of • Mud plates with 12 cm diameter
watch glass with top cover of the White trap method but replaces the and 15 cm height
petri dish and care should be taken glassware, filter paper and even • Convex mud plates with 12 cm
that; sufficient gap should be laboratory conditions including diameter 10 cm height

Ray diagram of new method of EPN extraction New method of EPN extraction

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May 2016
Advantages of modified white trap
method
• The process is very easy, simple,
economical and has high output
(production) compared to white
trap.
• No specific skill or training is
required.
• The glass plates and watch glasses
can be replaced by earthen plates Infested wax moth larvae
and much of the investment can
be reduced. to fields such as lawns and turf grass
• The quality of harvested mats etc. Cocopeat formulation
nematodes is also very high
qualitatively and quantitatively. Field application methodologies plant, for lawns and large scale field
• This method holds very promising Infective juveniles of EPNs can be applications, infected cadavers can be
in popularizing EPNs by its mass applied using various spray directly broadcasted mixed with FYM
production in rural areas at farm equipment and standard irrigation or cocopeat. After landing in to soil,
level at very low-cost. systems. They require a film of water the IJs start emerging gradually. The
• The method can be explored as a around soil particles to move through advantage of EPNs over other insect
cottage industry and encourage the soil profile in search of a host. bio-control agents is that EPNs can
self-employment for many rural Therefore, ensure adequate agitation be applied in combination with
youth. Most suitable for during application. Enhanced efficacy insecticides and other bio-control
cooperatives and group of farmers in EPN applications can be facilitated agents and its controls for long term.
engaged. through improved delivery
SUMMARY
mechanisms or optimization of spray
Formulation and storage of EPN equipment. Application can be made Sugarcane is a commercial and
Entomopathogenic nematodes can by using water-can, irrigation system, industrial cash crop grown across
be stored and formulated in different and knapsack or hand sprayer. While many states in the country. The crop
ways. Use of polyurethane sponge, using sprayer, use maximum pressure is source of employment for millions
cocopeat, and in aqueous suspension to avoid blockage. The sprayer nozzle and earns huge foreign exchange
of nematodes and baits are much in opening should be at least 1/2 mm. annually. Every year sugarcane suffers
practice. Bait formulations and insect Spray the solution evenly on the area seriously from many pests, of which
host cadavers can enhance EPN to be treated. Ensure continuous white grub is economically
persistence and reduce the quantity of mixing to prevent the nematodes important. This pest causes heavy
nematodes required per unit area. from sinking to the bottom. After yield losses annually, in ratoon crops
Formulated EPNs can be stored for 2 application keep the soil moist during under severe cases the losses will be
to 7 months depending on the the first two weeks for the nematodes up to 100%. It has threatened the
nematode species and storage media to get establish. Irrigate the area sugar bowl of many states and a
and conditions. Unlike other bio- before application if it is dry and major concern for many growers and
control agents (fungi, bacteria and apply whenever symptomatic damage scientists. There is a need for
virus), EPNs do not have a fully from insects is detected. Apply EPNs popularizing the use of EPN for
dormant resting stage. They use their only when pests are noticed in soil insect pest management both soil
limited energy during storage. In otherwise repeat the application to borne and foliar insect pests. EPN is
order to keep them viable and get the desired results. Application the best solution for the management
virulent for longer period moistened rate of EPN is 1 billion nematodes of root grub manace in sugarcane. By
cocopeatis found very ideal media for per acre or 2-4 infected cadavers per the modified method farmers can
their formulation. Due to its high mass produce EPN and use
moisture holding capacity, it will effectively. EPN are not availeble
retain the nematodes viable for commercially, no private companies
longer period. It can be mixed with are producing EPN for large scale.
vermicompost or compost before use Utilization of EPN by the farmers
to increase the volume enough for can be increased only when
broadcasting or soil application. The production of EPN is feasible at farm
cocopeat can also be used for level, besides reduction in cost of
maintenance of EPNs culture as well production for commercial operators.
as infected cadavers. Often the
1 and 2
infected cadavers can also be used as Scientists, PHM Division, NIPHM,
Hyderabad
formulation for direct broadcasting in Infested cadavers

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