2. REVIEW OF LITERATURE The available literature on entomopathogenic nematodes (EPNs) in relation to the investigations reported in the thesis has been reviewed under the following heads. 2.1 Nematodes associated with insects and their distribution 2.2 Symbiotic relationship of entomopathogenic nematodes with bacteria 2.3 Mass production of entomopathogenic nematodes 2.4 Storage and shelf life of entomopathogenic nematodes 2.5 Efficacy of entomopathogenic nematodes against insect-pests. 2.1 Nematodes associated with insects and their distribution 2.1.1. Distribution of entomopathogenic nematodes in world excluding India Entomopathogenic nematodes are known since the 17 th century and perhaps the earlier than this (Nguyen and Smart, 2004), however, extensive studies on entomopathogenic nematodes were carried out in the 19 th and 20 th centuries. These nematodes have a ubiquitous world-wide distribution. Different species/ genera/ families of these parasites occur in different habitats/ ecosystems depending on their insect hosts (Mracek 2008). Heterorhabditids and steinernematids are found in many areas of all continents, excluding Antarctica. It has been reported that the steinernematids are found in cooler, temperate regions, whereas heterorhabditids are in warmer, tropical conditions (Hominick et al . 1996). Hominick et al. (1995) demonstrated that at least some steinernematids show a distinct habitat preference that may reflect the distribution of suitable hosts which are adapted for the habitat. Even though the entopmopathogenic nematodes are ubiquitous, their recovery from the field is influenced by a number of biotic factors, including host range that is dependent on the suitability for penetration of different insect hosts by nematodes, possibility of finding a suitable host in the habitats and by the natural population density of the nematodes creating epizootics in outbreak sites (Peters 1996). Mracek and Beevas (2000) emphasized an essential impact of host aggregations on the incidence of entomopathogenic nematodes.
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5
5
2. REVIEW OF LITERATURE
The available literature on entomopathogenic nematodes (EPNs) in
relation to the investigations reported in the thesis has been reviewed under the following
heads.
2.1 Nematodes associated with insects and their distribution
2.2 Symbiotic relationship of entomopathogenic nematodes with bacteria
2.3 Mass production of entomopathogenic nematodes
2.4 Storage and shelf life of entomopathogenic nematodes
2.5 Efficacy of entomopathogenic nematodes against insect-pests.
2.1 Nematodes associated with insects and their distribution
2.1.1. Distribution of entomopathogenic nematodes in world excluding India
Entomopathogenic nematodes are known since the 17th
century and perhaps the
earlier than this (Nguyen and Smart, 2004), however, extensive studies on
entomopathogenic nematodes were carried out in the 19th
and 20th centuries. These
nematodes have a ubiquitous world-wide distribution. Different species/ genera/ families
of these parasites occur in different habitats/ ecosystems depending on their insect hosts
(Mracek 2008). Heterorhabditids and steinernematids are found in many areas of all
continents, excluding Antarctica. It has been reported that the steinernematids are found
in cooler, temperate regions, whereas heterorhabditids are in warmer, tropical conditions
(Hominick et al. 1996). Hominick et al. (1995) demonstrated that at least some
steinernematids show a distinct habitat preference that may reflect the distribution of
suitable hosts which are adapted for the habitat.
Even though the entopmopathogenic nematodes are ubiquitous, their recovery
from the field is influenced by a number of biotic factors, including host range that is
dependent on the suitability for penetration of different insect hosts by nematodes,
possibility of finding a suitable host in the habitats and by the natural population density
of the nematodes creating epizootics in outbreak sites (Peters 1996). Mracek and Beevas
(2000) emphasized an essential impact of host aggregations on the incidence of
entomopathogenic nematodes.
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6
The first entomopathogenic nematode was described by Steiner as Aplectana
kraussei (now Steinernema kraussei) in 1923 followed by Neoplectana glaseri (Steiner,
1929) from material isolated by Glaser and Fox (1930). It was not until Weiser (1955)
described a European population of Neoplectana carpocapsae from codling moth larvae
and Dutky and Hough (1955) isolated the DD-136 strain of an undescribed
steinernematid from codling moth larvae in eastern North America there after the serious
studies on entomopathogenic nematodes began.
The distribution of entomopathogenic nematodes on a global scale is probably
strongly influenced by climate and chance dispersal events, including those associated
with human activities. The juveniles of heterorhabditids disperse vertically and
horizontally, both actively and passively (Parkman et al. 1994; Timper et al. 1988).
Passively, they may be dispersed by rain, wind, soil, humans or insects (Smart and
Nguyen 1994). Soil texture, vegetation and availability of suitable hosts are amongst the
factors that have been implicated in affecting local distribution patterns. There is growing
evidence of preferences of nematode species for certain habitats. S. affine is found largely
in arable lands and grasslands, and virtually absent in forests (Hominick 2002). More
striking is the association of some species with soil of particular texture, in particular
sand. Heterorhabditis megidis and H. indica are almost exclusively found in sandy soils,
resulting in a mainly coastal distribution (Hara et al. 1991; Amarsinghe et al. 1994;
Griffin et al. 1994, 2000). In Germany, the rate of prevalence of steinernematids was
highest in woodland (50.3%) where S. affinis, S. feltiae, S. intermedium and Steinernema
sp. were the predominant species (Sturhan 1999). Similarly, heterorhabditids were
equally abundant in turf and weedy habitats, but never found in closed-canopy forest
(Stuart and Gaugler 1994).
Of the nematodes associated with insects those belonging to the orders
Mermethida, Aphelenchida, Tylenchida and Rhabditida have been most intensively
studied. However, at present only the Rhabditid genera Heterorhabditis and Steinernema
are widely used for insect control due to their high and rapid infectivity and pathogenicity
and easy manipulation (Mracek 2008).
The heterorhabditids are soil inhabiting and have free living, parasitic and
saprophytic stages. The infective third stage juveniles survive outside the insect and
search for hosts and wait for host to pass by (Mracek 2008). The two genera
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7
Sterinernema and Heterorhabditis contain the most important species of
entomopathgogernic nematodes. Currently, there are 25 species in Steinernema and 11
species in Heterorhabditis. All members of the order Rhabditida are bacteriophagous and
many of them have phoretic associations with insects ((Burnell and Stock 2000)).
Therefore, genera Steinernema and Heterorhabditis are considered here and their
distribution on a global scale is reviewed here under. The available information on
various species of these genera with respect to their distribution, hosts, etc. is summarised
in Table 2.1.
Table 2.1 Distribution of entomopathogenic nematodes in different parts of the
world
Species Habitat Host Country Reference
S. feltiae,
H. bacteriophora
Coastal,
wooded and herbaceous
habitats
- Lebanon Noujeim et al. 2011
H. bacteriophora S. glaseri
S. scarabaei
S. feltiae
- Galleria mellonella
Czech Republic
Hyrsl 2011
H. indica, S. abbasi,
S. cholashanense,
S. feltiae
S. siamkayai
Forest, Agriculture
land
River bank
(weeds) Paddy
Walnut
G. mellonella Nepal Chhetri et al. 2010
S. khoisanae,
Steinernema sp, H. bacteriophora
Fruit orchards Whitegrub and
black vine weevil
(Otiiorhynchus
sulcatus)
South
Africa
Hatting et al. 2009
S. pakistanense, S. asiaticum,
S. abbasi,
S. simkayai, S. feltiae,
H. bacteriophora,
H. indica
- - Pakistan Fayyaz and Javed 2009
S. feltiae, S. affine,
Steinernema sp.,
H. bacteriophora
Cultivated field (vine and
vegetables),
coastal regions, oak forest
- France Emelianoff et al. 2008
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8
Species Habitat Host Country Reference
S. carpocapsae - - Slovenia Laznik et al. 2008 S. anatoliense,
S. carpocapsae,
S. feltiae,
H. bacteriophora
Coniferous,
oak forests,
grasses and
palm
- Stock et al. 2008
Heterorhabditis sp.
Steinernemaspp.
Forest, Pasture,
Coffee,
Vegetables
G. mellonella Kenya Nyasani et al. 2008
S. abassi H. bacteriophora
Agricultural fields
- Pakistan Shahina et al. 2007
S. feltiae,
H. bacteriophora,
H. downesi, H. megidis
Oak and
deciduous
forests, new plantations,
fruit orchards
Melolontha
melolontha
Hungary Toth 2006
S. oregonense
S. riobrave
Oak forest - Arizona Stock and Gress 2006
S. yirgalemense H. bacteriophora
- - Ethiopia Makete et al. 2005
S. carpocapsae
S. arevarium
S. weiseri S. silvaticum
H. bacteriophora
Deciduous,
coniferous
forests, fruit orchards and
crop fields
- Czech
Republic
Mracek et al. 2005
Steinernema n. sp. H. indica
- - Costa Rica Lorio et al. 2005
S. aciari
- - China Qiu et al. 2005
Heterorhabditis sp. Greenhouse Bradysia
agrestis
Korea Kim et al. 2004
S. monticolum Heterorhabditis sp.
Turfgrass Agrotis ipsilon A. segetum
Korea Kang et al. 2004
Steinernema sp.
H. bacteriophora
H. indica
- - Egypt Atwa et al. 2004
S. feltiae
- - Netherland Jgdale et al. 2004
Heterorhabditis sp.
- - Korea Kim et al. 2004
S. hermaphroditum
n sp.
- - Indonesia Stock et al. 2004
S. feltiae
Potato Tecia
solanivora
Colombia
Venezuela
Saenz 2003
S. carpocapsae
S. glaseri
S. longicaudum
Greenhouse Bradysia
agrestis
Korea Kim et al. 2003
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9
Species Habitat Host Country Reference
H. bacteriophora
S. carpocapsae Palm Rhynchophorus
ferrugineus
Japan Iiboshi and Izono 2003
S. carpocapsae H. bacteriophora
Korea Jeon et al. 2003
S. feltiae
S. affine
Steinernema n. sp. H. bacteriophora
- - Turkey Hazir et al. 2003
H. indica
H. baujardi
- - Vietnam Phan et al. 2003
S. carpocapsae
H. bacteriophora
Greenhouse Frankiniella
occidentalis
Korea Kim et al. 2003
S. rarum
S. glaseri
S. carpocapsae
H. bacteriophora
- - USA Shapiro-Ilan et al. 2003
S. arenarium
S. glaseri
S. carpocapsae
Heterorhabditis sp.
Sugarcane Mahanarva
Wmbriolata
Brazil Leite et al. 2003
S. carpocapsae
H. bacteriophora
Cassava and
other crops
Cyrtomenus
bergi
Panama Aguilar 2003
H. megidis
S. feltiae
Grains
Maize
Spodoptera
frugiperda Helicoverpa
zea
Mexico Molina–Ochoa et al.
2003a,b,c
S. carpocapsae
- - Japan Iiboshi and Izono, 2003
S. carpocapsae
Heterorhabditis sp.
Coffee Hypothenemus
hampei
- Molina-Acevedo and
Lopez-Nunez 2002, 2003
H. bacteriophora Maize White grubs
Mexico Ruiz-Vega and Aquino-
Bolaños 2002
S. carpocapsae,
S. glaseri
H. bacteriophora
Turfgrass Exomala
orientalis
Korea Choo et al. 2002
S. glaseri H. bacteriophora
Turfgrass Adoretus tenuimaculatus
Korea Lee et al. 2002
S. carpocapsae S. monticolum
H. bacteriophora
Chestnut Parapediasia teterrella
Korea Choo et al. 2001
H. bacteriophora,
H. m
- - USA Mannion et al. 2001
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10
Species Habitat Host Country Reference
S. carpocapsae S. glaseri
S. longicaudam
H. bacteriophora
Vegetables Palpita indica Korea Kim et al. 2001
Heterorhabditis sp.
- - Netherland Wardlow et al. 2001
S. abbasi
- - Taiwan Liao et al. 2001
S. feltiae
Potato Tecia
solanivora
Colombia
Venezuela
Fan et al. 2000
S. carpocapsae Apple Carposina
niponensis
China Yang et al. 2000
Steinernema sp. - -
UK Piggott et al. 2000
S. feltiae
S. carpocapsae
- - Russia Ivanova et al. 2000
Heterorhabditis sp.
Steinernema sp.
- - Azores Rosa et al. 2000
S. krausei S. feltiae
S. affine
S. intermedium S. bicornutum
S. glaseri
- - Czech Republic
Mracek et al. 1999
S. ragum,
S. feltiae, H. bacteriophora
- - Argentina Doucet et al. 1999
S. feltiae
Potato Tecia
solanivora
Colombia
Venezuela
Alvarado et al. 1998
S. glaseri
S. carpocapsae
Turfgrass S. depravata
Parapediasia teterrella
Japan Kinoshita and
Yamanaka 1998
Heterorhbditis sp. - - UK Bennison et al. 1998
H. indica
H. bacteriophora Steinernema sp.
- - Guadeloupe
Islands
Constant et al. 1998
S. feltiae
H. bacteriophora
- - Hungary Lucskai and Mracek
1998
S. bibionis
S. carpocapsae H. bacteriophora
H. indicus
Heterorhabditis spp.Strains HV1,
HV2, HV3, HV4,
HV5, HV6
- - Venezuela Rosales and Saurez
1998
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Species Habitat Host Country Reference
H. indicus Steinernema spp.
Crop fields, lawn, turf
grass, sea shore
- Pakistan Shahina et al. 1998
S. kushidai
H. indicus H. megidis
- - Japan Yoshida et al. 1998
S. carpocapsae
S. glaseri
H. bacteriophora S. longicaudum
Turfgrass A. ipsilon
A. segetum
Korea Lee et al. 1997
S. monticolum sp.
nov.
- - Korea Stock et al. 1997
S. carpocapsae Sweet potato Cylas
formicarius Euscepes
postfasciatus
Japan Yamaguchi and
Kawazoe 1997
S. carpocapsae
Banana Cosmopolites
sordidus
Venezuela Rosales and Suarez
1997
Steinernema sp. Potato Premnotrypes
vorax
Chile Garzon et al. 1996
S. carpocapsae
S. glaseri H. bacteriophora
Outhouse Calliphora
lata Muscina
stabulans
Korea Choo et al. 1996
S. carpocapsae,
S. feltiae, S. riobrave,
H. bacteriophora
- - USA Schroeder et al. 1996
S. carpocapsae
- - China Han et al. 1996
S. affinis
S. feltiae S. kraussei
Steinernema spp.
- - Scotland Gwynn and Richardson
1996
H. marelatus n. sp.
- - Oregon Jie and Berry 1996
Steinernema spp. Heterorhabditis
spp.
- - Korea Lee et al. 1996
S. feltiae
S. affinis
- - Belgium Miduturi et al. 1996
S. feltiae Heterorhabditis sp.
- - Spain Pino and Palomo 1996
S. carpocapsae Banana,
Vegetables
Odoiporus
longicollis
China Peng and Han 1996
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Species Habitat Host Country Reference
Phyllotreta
striolata
S. carpocapsae Litchi Aristobia
testudo
China Han et al. 1996
H. bacteriophora
- - Cuba Rodriguez et al. 1996
S. carpocapsae Fig Psacothea
hilaris
Japan Tsutsumi and Yamada
1995
S. carpocapsae Litchi Aristobia testudo
China Xu et al. 1995
S. carpocapsae
S. monticolum H. bacteriophora
Forest
Chestnut
Glyphodes
perspectalis
Dichocrocis punctiferalis
Curculio
sikkimensis
Korea Choo et al. 1995
S. carpocapsae
- - China Xu et al. 1995
S. feltiae
S. affinis
H. megidis
- - U.K. Hominick et al. 1995
S. feltiae S. carpocapsae
S. scapterisci
H. bacteriophora
- - Argentina Stock. 1995
S. glaseri
Turfgrass Anomala sp. Japan Yamanaka et al. 1995
S. feltiae Pig sty Musca
domestica
China Xu et al. 1994
S. carpocapsae Palm Sagalassa
valida
Colombia Ortiz-Sarmiento 1994
Heterorhabditis spp.
Steinernema spp.
- - Sri Lanka Amarsinghe et al. 1994
S. affinis
S. feltiae S. intermedia
S. kraussei
S. RFLP type E1 Heterorhabditis sp.
- - Swiss Alps Steiner 1994
S. carpocapsae Apple Carposina
niponensis
China Wang 1993
S. carpocapsae Avenue trees Holcocerus
insularis
China Yang et al. 1993
H. bacteriophora
H. megidis
- - Israel Glazer et al. 1993
Steinernema spp. - - Norway Haukeland 1993
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13
Species Habitat Host Country Reference
S. feltiae
Steinernema spp. H. megidis
Western
Canada
Mracek and Webster
1993
S. carpocapsae
Banana Cosmopolites
sordidus
Brazil Schmitt et al. 1992
S. carpocapsae S. glaseri
H. bacteriophora
Rice
Forest
Chilo suppressalis
Agelastica
coerulea
Korea Choo et al. 1991
Heterorhabditis sp.
- - Peurto Rica Figueroa et al. 1991
Steinernema sp. Heterorhabditis sp.
- - Hawaiian Islands
Hara et al.1991
N. bibionis - - U.K. Hominick and Briscoe
1990
Heterorhabditis sp. S. bibionis
- - U.K. Hominick and Briscoe 1990
S. carpocapsae Strawberry Spodoptera
litura
Japan Gupta et al. 1987
2.1.2. Distribution of entomopathogenic nematodes in India
India is a tropical country having diverse agroclimatic conditions ranging from
the humid, high rainfall north eastern zone to north western semi-arid and arid zones
(Rahaman et al. 2000). The climate is conducive for entomopathogenic nematodes having
no environmental limitation for their commercial exploitation.
In India, the work on entomopathogenic nematodes was first initiated by Rao and
Manjunath (1966) who demonstrated the use of DD-136 strain of S. carpocapsae for the
control of insect-pests of rice, sugarcane and apple. Now there are several reports of
entomopathogenic nematodes parasiting insect pests of rice, maize, groundnut, potato
with a wide host range. The initial work with entomopathogenic nematodes in India was
conducted primarily with exotic species/strains of S. carpocapse, S. glaseri, S. feltiae and
H. bacteriophora. In many cases, these nematodes were found less effective, probably
due to their poor adaptability to the local agro-climatic conditions. India, as in the case
with many other parts of the world, has a rich biodiversity resource because of its varied
geographic, climatic and weather conditions. In India, the search for indigenous strains
have resulted in a number of Indian isolates from different parts of India (Ganguly 2003).
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Among the indigenous nematode isolates, two have been described as new
species, H. indica (Poinar et al. 1992) from Tamil Nadu and S. thermophilum Ganguly
and Singh (Ganguly and Singh 2000, 2003) from New Delhi. Other species identified as
indigenous isolates include S. carpocapsae (Hussaini et al. 2001), S. bicornutum
(Hussaini et al. 2001), S. riobrave (Ganguly et al. 2002), S. feltiae and H. bacteriophora
(Sivakumar et al. 1989). Hussaini et al. (2001) also identified some of the native
populations of Steinernema by restriction fragment length polymorphism (RFLP)
analysis and analysis of the PCR amplified ITS-rDNA region using 17 restriction
enzymes. In addition, surveys have revealed natural occurrence of several species/strains
of Steinernema and Heterorhabditis in Andaman and Nicobar islands (Prasad et al.
2001), Gujarat (Vyas 2003), Kerala (Banu et al. 1998), New Delhi (Ganguly and Singh
2000) and Tamil Nadu (Bhaskaran et al. 1994). The detailed list showing occurrence and
distribution of entomopathogenic nematodes in India is given in Table 2.2.
Table 2.2 Distribution of entomopathogenic nematodes in India
Species Habitat Host State Reference
H. indica (Meerut
strain)
- - Uttar Pradesh Prasad et al.
2012
H. indica (Meerut
strain)
- - Uttar Pradesh Pal and Prasad
2012
S. meghalayensis sp. - - Meghalaya Ganguly et al.
2011
S. carpocapsae,
H. indica
- - Meghalaya Gitanjalidevi
2011
S. thermophilum, S. riobrave,
S. harryi,
S. meghalayensis
- G. mellonella New Delhi, Gujarat,
Tamil Nadu,
Meghalaya
Kumar and Ganguly 2011
H. indica, S. thermophilum,
S. glaseri
- - Meghalaya Yadav and Lalramliana
2011
Steinernema sp.,
Heterorhabditis sp., Neosteinernema
- - Uttar Pradesh Khan and Haque
2011
H. indica,
S. thermophilum,
S. glaseri
Dry land, wet
land, jhum
land and forests
- Meghalaya Lalramliana and
Yadav 2010
15
15
Species Habitat Host State Reference
S. carpocapsae - - Gujarat Shinde et al.
2010
S. siamkayai Coastal
ecosystem
- Puducherry Adiroubane et al.
2010
H. indica,
S. carpocapsae
- - Kerala,
Karnataka
Shakeela and
Hussaini 2009
S. abbasi, H. indica
- - Haryana Sunanda 2009
H. indica (Meerut
strain)
Deserted
honey comb
G. mellonella Andhra
Pradesh
Sankar et al.
2009
H. bacteriophora Apple orchards and
forest
- Himachal Pradesh
Chandel et al. 2009
S. carpocapsae - - Jammu and
Kashmir
Gupta et al. 2008
S. masoodi - - Uttar Pradesh
Khan et al. 2007
H. bacteriophora,
S. feltiae,
Steinernema sp.
Apple
orchards and
forest
- Himachal
Pradesh
Singh and Gupta
2006
H. indicus Grape garden
Scelodonta
strigicollis
Karnataka Prabhuraj et al.
2006
S. carpocapsae,
H. indica
Agriculture
soil
- Karnataka Hussaini et al.
2004
S. abbasi,
S. tami,
S. carpocapsae,
H. indica, H. bacteriophora
Agriculture
soil
- Karnataka
Hussaini et al.
2004
S. bicornutum
H. indica
Potato
Maize
Crucifers
Tobacco
Brinjal (egg
plant)
Banana
Phthorimaea
operculellaChilo
zonellus Swinhoe
Plutella
xylostella S. litura
Lasioderma
serricorne Odoiporus
longicollis
Karnataka Hussaini 2003
Heterorhabditis sp.,
Steinernema sp.
- - Gujarat Vyas 2003
Heterorhabditis sp., Steinernema sp.
- - Gujarat Vyas et al. 2002
Steinernema sp. Mango - Tamil Nadu Ambika and
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Species Habitat Host State Reference
orchard, millet
field
Sivakumar 2002
S. carpocapsae Tobacco
nursery
S. litura Karnataka Sitaramaiah et al.
2003
Heterorhabditis sp., - - Rajasthan Rajkumar et al.
2001
Heterorhabditis sp. Cultivated area, forest,
scrub land and
coastal sandy region
- South Andamans
Prasad et al. 2001
S. thermophilum - - New Delhi Ganguly and
Singh 2000
H. indica,
S. riobrave, S. feltiae,
S. carpocapsae
S. Bicornutum
- - Karnataka Hussaini et al.
2000
Steinernema spp. and Heterorhabditis sp.
- - - Kaushal et al. 2000
S. bicornutum,
S. carpocapsae,
H. indica
- - Karnataka Hussaini et al.
2000
H. indica - - Kerala Banu et al. 1998
H. indicus, Steinernema sp.
- - Tamil Nadu Josephrajkumar and Sivakumar
1997
S. glaseri,
S. feltiae, Steinernema sp.,
H. bacteriophora,
Heterorhabditis sp.
- - Rajasthan Bareth et al.
1997
S. feltiae - P. brassicae, Alphitobius
diaperinus,
Oryzaephilus mercator
- Mathur et al. 1994
S. carpocapsae,
H. bacteriophora,
Heterorhabditis sp.
Groundnut Amsacta
albistriga
- Bhaskaran et al.
1994
S. feltiae. Potato A. ipsilon A. segetum
- Singh 1993
H. bacteriophora - - Tamil Nadu Poinar et al.
1992
17
17
Species Habitat Host State Reference
S. feltiae - - Andhra
Pradesh
Singh et al.1992
N. bibionis,
Neoplectana sp. and H.
bacteriophora
Apple orchard
and forest soil
- Himachal
Pradesh
Singh 1990
H. bacteriophora - - Tamil Nadu Sivakumar et al.1989
S. feltiae Tobacco S. litura Karnataka Narayanan and
Gopalakrishnan
1987
S. carpocapsae Vegetables Anomala sp. - Rajeswari et al. 1984
S. carpocapsae Maize Rice
C. zonellus Tryporyza
incertulas
Karnataka Rao et al. 1971
S. carpocapsae Maize C. zonellus - Mathur et al.
1966
2.2 Symbiotic relationship of entomopathogenic nematodes with bacteria
Knowledge of the nematode-bacterial symbiosis is essential to understand the
pathogenicity of the complex for target insects and is important for successful mass
production (Grewal et al. 2004). The nematode-bacterial interaction is symbiotic and
each partner can be cultured separately, but when combined they present a high degree of
specificity (Grewal et al. 2004). Sudhaus and Schulte (1998) has suggested that
Heterorhabditis and Steinernema most probably evolved from necromenic nematodes
which developeded a symbiotic association with an entomopathogenic bacterium. Such a
symbiosis specialized for parasiting animals has not been described so far for any other
group of nematodes (Burnell and Stock, 2000). Thomas and Poinar (1979) reported that
symbionts associated with Steinernema are placed in the genus Xenorhabdus, whereas the
bioluminescent symbionts associated with Heterorhabditis are placed in the genus
Photorhabus (Boemare et al. 1993).
According to Bird and Akhurst (1983), the Xenorhabdus occurs in a special
intestinal vesicle of Steinernema infective juveniles. The Photorahabus are mainly
located in the anterior part of the intestine in Heterorhabditis (Boemare et al. 1996).
Symbiont bacteria of both genera are motile, gram negative and belong to the
Enterobacteriaceae (Burnell and Stock 2000). Both genera are negative for nitrate
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reductase and Xenorhabdus are negative for catalase (Grewal et al. 2004). Hazir et al.
(2003) also reported that Photorhabdus spp. are luminescent and catalase positive,
whereas Xenorhabdus spp. have no luminescence and are catalase negative. The colonies
of Xenorhabdus are smooth and somewhat granular in appearance, whereas
Photorhabdus colonies are smooth and mucoid in appearance with irregular margins.
Initially they are pale yellow but change to deep yellow and usually red with age
(Thomas and Poinar 1979). The infective stage of nematode carry bacteria within the
intestinal lumen (Poinar and Leuteregger 1968).
The relationship of steinernematids and heterorhabditids with Xenorhabdus spp. is
one of the classical mutualism. The nematodes provide entry to the bacterium and destroy
its inducible antibacterial system and the bacteria optimize nematode reproduction by
providing nutrients and inhibiting many contaminating microorganisms (Poinar and
Thomas 1967). Hu and Webster (2000) reported that an antibiotic 3, 5-dihydroxy-4-
isopropylstibene is produced by P. luminescens C9 which helps in minimizing the
competition from other microorganisms and prevents the putrification of the nematode
infected insect cadaver.
Both Xenorhabdus and Photorhabdus occur in two phenotypic forms. Phase I
cells are larger than phase II cells and produce significantly greater amounts of
exoenzymes, toxins, antibiotics than phase II forms. The phase I cells are normally the
cells carried by the infectives (Burnell and Stock 2000). When symbiont bacteria are
released by the nematode into the insect haemolymph, the bacterial cells begin to grow
and the death of insect ensues, either from toxaemia or septicemia, depending on the
sensitivity of the insect and the symbiont strain (Frost et al. 1997). Some strains of
Xenorhabdus and Photorhabdus are highly virulent and an injection of less than 10 cells
of the bacterium into the haemocoel may be sufficient to kill a susceptible insect such as
G. mellonella or Manduca sexta (Poinar and Thomas 1967).
Nagesh et al. (2002) isolated Xenorhabdus and Photorhabdus species from both
surface sterilized infective juveniles of the indigenous isolates of Steinernema and
Heterorhabditis spp., and the haemolymph of G. mellonella larval cadavers infected with
the nematodes. Further, Raman and Bhatnagar (2002) purified the insecticidal toxin
complex produced by the bacterium P. luminescens sub sp. akhurstiiwhich was active
against the larvae of Spodoptera litura and G. mellonella. Similarly, Mahar et al. (2000)
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also recorded high mortality in the larvae of G. mellonella due to P. luminescens isolated
from H. bacteriophora. Lengyel et al. (2005) described four new species viz. X.
budapestensis from S. biocornutum, X. ehlersii from S. serratum, X. innexi from S.
scapterisci and X. szentirmaii from S. rarum.
There are no reports of the isolation of Xenorhabdus and Photorhabdus from soil
and it has been generally assumed that these bacteria cannot exist in soil environment in
the absence of their nematode associates (Burnell and Stock 2000).
There exists a close relationship between the taxonomy of the symbiont species
and their nematode host. Each nematode species is specifically associated with one
symbiont species, although a symbiont species may be associated with more than one
nematode species (Akhurst and Boemare 1990). Grewal et al. (2004) reported that
Xenorhabdus bovienii is associated with four species of Steinernema and X. poinarii with
two species of Steinernema. However, P. luminiscens and P. temperate are both
associated with H. bacteriophora. Kaya and Gaugler (1993) reported that, the nematode
relies upon the bacterium for killing the insect host, creating a suitable environment for
its development by producing antibiotics that suppress competing secondary
microorganisms, breakdown the host tissues into usable nutrients, and serve as food
source. The bacterium requires the nematode for protection from external environment,
penetration into hosts haemocoel, and possibly inhibition of the hosts antibacterial
proteins.
Mohan et al. (2003) tested P. luminiscens isolated from entomopathogenic
nematode, H. indica against Pieris brassicae. They sprayed bacterial formulation
containing 1.8 x 106 CFU/ ml uniformly on the foliage of ornamental nasturtium heavily
infested with larvae of P. brassicae and recorded 100 per cent mortality within 24 hours.
There are reports of successful management of mango mealy bug, Drosicha mangiferae
by using P. luminescens isolated from H. indica (Mohan et al. 2004). Mahar et al. (2004)
reported lethal effects of cell and cell free filtrates of X. nematophila taken from S.
carpocapsae against diamondback moth. It was observed that cells can penetrate into
insects even in the absence of the nematode vector. Cell-free solution containing
metabolites was found equi-effective as bacterial cell suspension.
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Chongachitmate (2005) reported association of symbiotic bacteria Xenorhabdus
sp. with S. siamkayai isolated from haemolymph of Helicoverpa armigera. The bacteria
yielded perfect colonies on NBTA media after 18 hours of infection. Direct application of
either cell solutions or cell-free filterates from X. nematophila has provided good control
of S. exigua, Plutella xylostella, Otiorhynchus sulcatus and nymphs of Schistocera
gregaria. The toxicity of cell suspensions and cell free filtrates persisted for upto 5
months in soil (Mahar et al. 2008). Gerritsen et al. (2005) listed oral toxicity of excretion
products of 52 Photorhabdus and Xenorhabdus strains on Frankliniella occidentalis and
Thrips tabaci and only 6 P. temperate isolates from North America were toxic against
these thrips. There was 90 per cent mortality of thrips after 7 days of feeding from P.
temperate supernatant. Thrips were also killed after sucking from leaves covered with the
toxins.
In France, molecular characterization of isolated entomopathogenic nematodes
depicted three different species of Steinernema, one species of Heterorhabditis, and H.
bacteriophora. The Steinernema species were identified as S. feltiae and S. affine and an
undescribed species. Xenorhabdus symbionts were identified as X. bovienii for both S.
feltiae and S. affine. The Xenorhabdus symbionts from Steinernema species was
identified as X. kozodoii. The bacterial symbionts of H. bacteriophora were identified as
P. luminiscens ssp. kayaii and P. luminescens ssp. laumondii (Emelanoff et al. 2008).
Tsai et al. (2008) isolated a symbiotic bacterium of entomopathogenic nematode S.
abbasi from Taiwan which was determined to be a species of Xenorhabdus. This species
was found similar to X. indica of S. abbasi Oman isolate as based on sequence analysis of
16S r DNA.
2.3 Mass production of entomopathogenic nematodes
For a biocontrol agent to be successful it should be amenable for production on
large scale the ready availability of the organism in required quantity and at competitive
cost makes them acceptable among entrepreneurs and farmers (Rabindra and Hussaini
2003). Mass production of entomopathogenic nematodes has evolved from the first large
scale in vitro solid media production by Glaser et al. (1940), to the in vivo production by
Dutky et al. (1964), to the three dimensional solid media in vitro process (Bedding 1981;
1984) and to the in vitro liquid fermentation production method (Friedman 1990).
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Currently, commercial nematodes are produced monoxenically using the solid media
process developed by Bedding (1981; 1984) or the liquid-fermentation method. The
solid-media process has successfully produced pathogenic steinernematids and
heterorhabditids.
During the past few years, a distinct cottage industry has emerged that produces
entomopathogenic nematodes mostly in vivo for the home lawn and garden markets. The
in vivo process requires minimal expertise and capital investments and may be an
important future sector in nematode commercialization for specific niche markets
(Koppenhofer and Kaya 2001).
Dutky et al. (1964) used larvae of the greater wax moth, G. mellonella to multiply
the DD-136 strain of Neoaplectana carpocapsae in vivo and obtained up to 2,00,000
infective juveniles per larva. House et al. (1965) devised a dog food based medium to
produce the DD- 136 strain of N. carpocapsae on a commercial scale. This method was
later refined by Hara et al. (1981) who stressed monoxenicity, and produced 125 million
nematodes/week from 100 dog food agar Petri dishes at a cost of $ 0.28 per million.
Milstead and Poinar (1978) mass multiplied H. bacteriophora on larvae of G. mellonella
and produced higher yield of up to 3,50,000 infective juveniles per host.