Ph.D. THESES
GHAZALA M. FURGANI
2006.
GNOTOBIOLOGICAL ANALYSIS OF
ENTOMOPATHOGENIC NEMATODE /
BACTERIUM SYMBIOTIC COMPLEXES
Ph.D. THESES
GHAZALA M. FURGANI
SUPERVISOR: DR. ANDRÁS FODOR
Accredited PhD program: Molecular, classical and evolutionary genetics
(Head: Prof. Gábor Vida, Full Member of the Hungarian Academy of
Sciences)
DEPARTMENT OF GENETICS, FACULTY OF NATURAL SCIENCES
EÖTVÖS LORÁND UNIVERSITY
BUDAPEST, HUNGARY
BUDAPEST, 2006.
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CONTENT
INDEX OF FIGURES ................................................................................................................................ 6
INDEX OF TABLES ................................................................................................................................. 7
1. INTRODUCTION ..................................................................................................................................... 8
1.1. REVIEW OF THE LITERATURE ON ENTOMOPATHOGESIS IN NEMATODES (EPN) AND
BACTERIS (EPB)...................................................................................................................................... 8
1.1.1. Taxonomic position of the EPN species ...................................................................................... 8
1.1.1.1. Taxonomic position of the EPN species within Phylum Nematoda ..................................................... 9
1.1.2. The Steinernema genus .............................................................................................................. 10
1.1.3. The Heterorhabditis genus ........................................................................................................ 12
1.1.4. The biological background of insect pathogenesis caused by EPN/EPB symbiotic complexes 14
1.1.5. Entomopathogenic symbiotic bacteria ....................................................................................... 15
1.1.6. Mutualism between entomopathogenic nematodes and bacteria ............................................... 18
1.2. OBJECTIVES AND RATIONALE .................................................................................................. 21
2. MATERIALS AND METHODS............................................................................................................ 23
2.1. ENTOMOPATHOGENIC NEMATODE AND BACTERIA USED IN THIS STUDY .................. 23
2.1.1. EPN STRAINS .......................................................................................................................... 23
3.1.2. EPB STRAINS .......................................................................................................................... 24
2.2. METHODS AND TECHNIQUES .................................................................................................... 25
2.2.1. MICROBIOLOGICAL METHODS.......................................................................................... 25
2.2.1.1. SOLUTIONS AND MEDIA .............................................................................................................. 25
M9 a buffered physiological salt solution for nematodes ........................................................................... 25
Liquid (or solid) LB media (Luria Bertani medium) (without or with agar): ............................................. 25
Liquid (or solid) TSY media (without or with agar): ................................................................................. 25
Liquid (or solid) NA media (without or with agar): ................................................................................... 26
NBTA an indicator medium ....................................................................................................................... 26
MacConkey Indicator Agar ........................................................................................................................ 26
Wouts” agar media ..................................................................................................................................... 26
2.2.1.2. METHODS ......................................................................................................................................... 27
ISOLATION OF THE SYMBIOTIC BACTERIA FROM DAUER JUVENILES .................................... 27
FREEZING OF BACTERIA FOR LONG-TERM STORAGE ................................................................. 27
SURFACE STERILIZATION OF INFECTIVE DAUER JUVENILES ................................................... 28
2.2.2. METHODS FOR GNOTOBIOLOGY ...................................................................................... 28
3. RESULT AND DISCUSSION ................................................................................................................ 30
3.1.
ANTIBIOTICS PRODUCTION OF THE EPB STRAINS ....................................................... 30
3.1.1. ISOLATION OF EPB SYMBIONTS DIRECTLY FROM DAUER LARVAE OF
STEINERNEMA SPECIES .................................................................................................................. 30
3
3.1.2. ANTIBIOTICS PRODUCTION OF DIFFERENT XENORHABDUS AND PHOTORHABDUS
STRAINS TESTED ON BACILLUS CEREUS ................................................................................... 31
3.1.3. THE EFFECT OF ANTIBIOTIC PRODUCTION ON OTHER ENTOMOPATHOGENIC
BACTERIA ......................................................................................................................................... 34
3.1.4. EFFECTS OF THE XENORHABDUS ANTIBIOTICS ON ERWINIA AMYLOVORA ............. 39
3.1.4.1. EFFORTS TO CONTROL THE FIRE BLIGHT DISEASE IN APPLE ORCHARDS ..................... 39
3.1.4.2. LABORATORY TESTS ON ERWINIA AMYLOVORA ..................................................................... 40
3.2. FERMENTATIVE PRODUCTION, CHEMICAL IDENTIFICATION AND APPLICATION OF
EMA ANTIBIOTICS ............................................................................................................................... 43
3.2.1. FERMENTATION OF THE EMA STRAIN AND TESTING THE ANTIMICROBIAL
ACTIVITY OF THE FERMENTATION SOUP ................................................................................ 43
3.2.2. ISOLATION OF COMPOUNDS OF ANTIMICROBIAL ACTIVITY FROM FERMENTOR
LIQUID CULTURE OF XENORHABDUS SP. EMA STRAIN ......................................................... 44
3.3. THE EMC STRAIN .......................................................................................................................... 50
3.3.1. THE PHENOTYPIC CHARACTERIZATION OF THE EMC STRAIN ................................. 50
3.3.1.1. COLONY MORPHOLOGY AND SWARMING BEHAVIOR ......................................................... 50
3.3.2. EXO-CRYSTAL PRODUCTION ............................................................................................. 51
3.3.3. ANTIBIOTICS PRODUCTION OF EMC ................................................................................ 57
3.3.4. CONCLUSIONS ....................................................................................................................... 58
3.4.
GNOTOBIOLOGY .................................................................................................................... 59
3.4.1. RESULTS IN GNOTOBIOLOGY ............................................................................................ 59
3.4.2. CONCLUSIONS ....................................................................................................................... 63
3.5. FIRST ATTEMPTS ON LABORATORY FERMENTATION OF A NEW STEINERNEMA
ISOLATE ................................................................................................................................................. 64
SUMMARY ........................................................................................................................................ 64
3.5.1. INTRODUCTION ..................................................................................................................... 64
5.5.2. MATERIALS AND METHODS .............................................................................................. 64
3.5.3. ISOLATION AND CHARACTERIZATION OF EPB SYMBIONTS FROM SOME NEW
STEINERNEMA STRAINS OF LONG DAUER PHENOTYPE ........................................................ 65
3.5.1.1. PHENOTYPIC CHARACTERIZATION OF THE FRESHLY ISOLATED BACTERIAL
SYMBIONTS .................................................................................................................................................. 65
3.5.1.2. PHOSPHOLIPASE (LECITINASE TEST ON YOLK AGAR) ......................................................... 65
3.5.1.3. PROTEOLYSIS ON GELATIN AGAR (FRAZIERS’S METHOD) ................................................. 66
3.5.1.4. ANTIBIOTICS PRODUCTION ......................................................................................................... 66
3.5.1.6. AMPICILLIN SENSITIVITY ............................................................................................................ 66
3.5.1.7. GNOTOBIOLOGICAL TESTS ......................................................................................................... 66
3.5.2. RESULTS AND DISCUSSION ................................................................................................ 67
3.5.3. LABORATORY FERMENTATION OF THE MOROCCO STRAIN ..................................... 69
3.5.4. CONCLUSIONS ....................................................................................................................... 70
3.6. MOLECULAR, GENETIC AND GNOTOBIOLOGICAL IDENTIFICATION OF NEW
STEINERNEMA ISOLATES .................................................................................................................... 72
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SUMMARY ........................................................................................................................................ 72
3.6.1. INTRODUCTION ..................................................................................................................... 72
3.6.2. MATERIALS AND METHODS .............................................................................................. 73
3.6.3. RESULTS AND DISCUSSION ................................................................................................ 74
4. SUMMARY ............................................................................................................................................. 79
5. REFERENCES ........................................................................................................................................ 80
6. ACKNOWLEDGEMENTS .................................................................................................................... 84
5
Index of Figures
Fig. 1: Life cycle of Steinernema species
Fig 2: The life cycle of the Heterorhabditis species
Fig. 3: The biological role of the EPB symbiont
Fig. 4: Isolation of EPB symbionts from EPN infective dauer juveniles
Fig. 5. Antibiotics production of EMC (left) and X. sp. (bibionis) (right) determined by
using B. cereus or B. subtilis as indicator strains.
Fig 6. A qualitative and quantitative comparison of the antibiotics produced by different
Xenorhabdus strains
Fig. 7. Demonstration of Bibionis inhibiting the growth of Photorhabdus temperata ssp.
temperata strain HL81.
Fig. 8: Outlines of laboratory tests on Erwinia amylovora
Fig. 9: Tests on EMA antibiotics on E. amylovora on Petri plate, liquid culture and
phytotrone.
Fig. 10A: Fractionation of the biologically active compounds adsorbed to charcoal.
Fig. 10B: Fractionation of the biologically active compounds adsorbed to DOWEX 50
Fig.11: Swarming phenotype of EMC. The color of the colonies is also unusual.
Fig. 12: The metallic-like crystals appear not only in the media but also on the surface of
the older colonies in agar plates
Fig. 13 The exo-crystal production of two isolates, EMC and EMD.
Fig 14 A Crystal production of EMC in liquid minimum media
Fig 14 B Crystal production of EMC in liquid minimum media
Fig. 15A. Light microscopic picture of the isolated crystal. 125X magnification.
Fig. 15B. Light microscopic picture of the isolated crystal. 250X magnification
Fig. 15C. Light microscopic picture of the isolated crystal. 250X magnification
Fig. 16. EM picture and image analysis of the exo-crystal
Fig. 17 The proof of the cytotoxic activity of the antimicrobial compound of EMC and
EMA.
Fig. 18. Effect of EMC antibiotics on the colony formation of Phytophtora sp.
Fig. 19: The new Xenorhabdus isolates (Morocco, SP2, Italy, Slovak) and control (X.
nematophila (AN6/1), X. poinarii (DSM 4766) strains on Luria Broth (LB) agar plates
Fig. 20: Lecitinase activities of my new isolates. Control: DSM 4766 (X. poinarii).
Fig. 21: Lipase activities of my new Xenorhabdus isolates
Fig. 22: In the article: (Fig. 6). Antibiotics activities of my new Xenorhabdus isolates
Fig 23. Ampicillin resistance of the new Xenorhabdus isolates
Fig. 24: The laboratory fermentation unit.
Fig 25: GenePhore PCR-RFLP Analysis 1.
Fig. 26: GenePhore PCR-RFLP Analysis 2
Fig. 27: GenePhore Analysis 3
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32
35
36
41
45
47
49
51
52
53
54
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59
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70
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71
72
79
80
81
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Index of Tables
Table 1. The Steinernema strains in our stock collection and their natural symbionts
21
Table 2. The Xenorhabdus strains used in this study
22
Table 5 Antibiotics production of the EPB strains
Table 6: Antagonism of Xenorhabdus vs. Photorhabdus. I
Table 7. Antagonism of Xenorhabdus vs. Photorhabdus. II
Table 8. Antagonism of Xenorhabdus vs. Photorhabdus. III.
Table 9. Antagonism of Xenorhabdus vs. Photorhabdus. IV
Table 10: The effectivity of EPB antibiotics on 10 E. amylovora isolates (by comb
test)
Table 9: The stability of the water solution of the EMA antibiotics at + 4 Co after half
a year storage
Table 11: Comparison of the biological activities of the compounds with
antimicrobial activity on B. subtilis
Table 12. Summary of the gnotobiological tests of Heterorhabditis / Photorhabdus
complexes (data of E. Böszörményi and Katalin Lengyel)
Table 13: Growth of S. carpocapsae and S. feltiae strains on symbionts of S. feltiae
and closely related species
Table 14: Growth of S. carpocapsae and S. feltiae strains on Xenorhabdus symbionts
of S. carpocapsae and related species
Table 15: Growth of S. carpocapsae and S. feltiae strains on Xenorhabdus symbionts
of other Steinernema species of short dauer phenotype
Table 16: Growth of S. carpocapsae and S. feltiae strains on Xenorhabdus symbionts
of other Steinernema species of long dauer phenotype
Table 17: Growth of S. carpocapsae and S. feltiae strains on Xenorhabdus symbionts
of taxonomically undetermined Steinernema species
Table 18: (In the paper: Table 1). Growth and propagation of Steinernema strains on
each others’ symbionts
Table 19: (In the article: Table 2) The results of cross breeding tests
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39
40
42
46
50
60
61
62
63
64
64
77
78
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1. INTRODUCTION
1.1. REVIEW OF THE LITERATURE
Many types of associations exist between nematodes and insects ranging from
phoresis to parasitism and pathogenesis. The families Steinernematidae,
Neosteinernematidae and Heterorhabditidae are unique because they are the only
nematodes which have developed the ability to carry and introduce symbiotic bacteria
into the body of insects, they are the only insect pathogens with a host range which
includes the majority of insect orders and families and they can be cultured in large scale
on or in artificial solid or liquid media (Poinar, 1990).
The nematodes that serve as vectors of pathogenic bacteria show considerable
potential in biological control of insect pests. They are called entomopathogenic
nematode (EPN) and used as biological control agents. They are insect parasitoids, since
the immature forms of EPN species develop at the expense of one host individual. They
are also ecologically similar to both parasitoids and arthropod predators because the host
individual is eventually killed (Kaya et. at 1985).
EPN species belonging to the different families possess many qualities that make
them excellent biological control agents. They have a broad host range, can easily be
mass produced, possess the ability to seek out their host, kill their host rapidly and are
environmentally safe. These attributes are combined with others like the easy application
by using standard spray equipment, compatibility with many chemical pesticides and no
evidence of mammalian pathogenicity (Ehlers and Peters, 1995, Ehlers and Hokanean,
1996, Boemare et al, 1996). The soil environment is one that offers an excellent site for
insect nematode interactions, because more than 90% of insect pests spend part of their
life cycle in the soil, and the soil is the natural reservoir of steinernematid and
heterorhabditid nematodes.
1.1.1. Taxonomic position of the EPN species
When considering the number of species of Animal Kingdom, Phylum nematoda
is the second following Arthropoda. According to estimates, several hundred thousands
species belong to this taxonomic group. They inhabit mainly soil, but also freshwater and
sea. The majority of them are neutral concerning their relation with mankind, mostly
8
feeding on soil bacteria or decomposing organic material. A group of them is harmful as
plant pathogens, while others cause tropical diseases, the EPN species are beneficial and
commercially useful nematodes.
1.1.1.1. Taxonomic position of the EPN species within Phylum Nematoda
The classification Phylum Nematoda (after Andrássy, 1976)
Class Adenopohora
Order Mermithida
Suborder Mermithina
Superfamily Mermithoidea
Family Mermithidae
Family Tetradonematidae
Class Secernentea
Order Rhabditida
Suborder Rhabditina
Superfamily Diplogasteroidea
Family Diplogasteridae
Superfamily Rhabditoidea
Family Steinernematidae
Family Neosteinernematidae
Family Heterorhabditidae
Family Steinernematidae
Genus: Steinernema Travassos, 1927
Type strain: Steinernema carpocapsae
Species that was discovered first: Steinernema kraussei (Steiner, 1923, Travassos,
1927).
Most important species: S. carpocapsae (for general use); S. feltiae (controlling
mushroom flies), S. glaseri, S. scarabeaiae (both controlling scarabs); S. scapterisci (to
control mole cricket and cockroaches); S. abassi, S. Riobravia (used in hot conditions).
Other species involved in this study: S. rarum, S. bicornutum, S. serratum, S. cubanum,
Family Heterorhabditidae
Genus: Heterorhabditis Poinar, 1976
Type strain: Heterorhabditis bacteriophora Poinar, 1976.
Other species involved in this study: H. megidis (see review of Adams, 2002); H.
downesii (Griffin et al., 1997); H. marelata (Liu et al., 1994), H. indica (see review of
Adams, 2002)
The EPN species are phylogenetically close to Caenorhabditis elegans (Brenner,
1974; The C. Elegans Consortium, 1998), which provides the possibility to adopt the
information gathered by the C. elegans researchers to EPN.
The EPN species belong to three families, namely Steinernematidae,
Neosteinernematidae and Heterorhabditidae. Each EPN family is comprised by a single
genus. In the family Steinernematidae the species have been more or less unambiguously
9
identified by their morphological differences, supplemented by crossbreeding tests.
Despite some problems with misidentification and nomenclature, there has not been too
much difficulty in species recognition. In contrast, when working with Heterorhabditis, it
is complicated to obtain crossbreeding data and there have been some difficulties in
defining
taxonomically
useful
morphological
characters
(Ilona
Dix,
personal
communication). The taxonomy of Heterorhabditis spp. will likely be based on
morphological analysis and construction of phylogenetic and derived classification
system (Curran 1990, Stock et al, 1996 Adams et al, 1998). The major purpose of
molecular techniques in EPN taxonomy is the identification of sibling species,
subspecies, and other intraspecific groupings. In addition, it contributes to understanding
the biology and evolution of EPNs. This role may become of critical importance in
identifying species and isolates for registration, quarantine and proprietary protection
purposes. This study mainly focuses on Steinernema species and their symbionts.
1.1.2. The Steinernema genus
The first Steinernema species was named by G. Steinernema, and the species was
Steinernema kraussei. He named the nematode, isolated in Germany, Aplectana kraussei
(Poinar, 1990). In 1927, Travassos determined a new genus, Steinernema for the species.
However, apparently two different species are included in the original description of S.
kraussei. Thus that species is presently considered as a species inquirenta, and the name
“kraussei” as nomen dubium. In 1929, Steiner erected the genus Steinernema. In 1934
Filipjev noted the resemblance between the two genera and placed them in subfamily
Steinernematidae. This subfamily was erected to family level by Chitwood and Chitwood
in 1937. Both genera, Neoaplectana and Steinernema, were considered valid, but all
recognized species were placed in the former genus because it was more completely
defined and the type species (glaseri) was intensively studied in the 1930 and 1940s.
Species belonging to Steinernema are obligate entomopathogenic nematodes that
are capable of infecting a wide variety of insects. As shown on Figure 1., they have a life
cycle including a third stage alternative, the so called infective dauer juvenile (IJ). This is
the only developmental variant, which can be found outside the insect, and this stage is
the vector for the entomopathogenic bacterial symbiont (belonging to the Xenorhabdus
genus) that eventually kills the insect after brought to its body cavity and hemolymph.
Each Steinernema species, and even each strain of the same species, carries different
10
Xenorhabdus strains. The bacterium is an obligate symbiont of the steinernematid
nematode. The symbiont has a primary and a secondary variant form. The primary (10) –
secondary (20) transition is spontaneous and is usually a “one-way street.” Only the
primary form is capable to establish an intimate symbiosis with the nematode, probably
based on a signal transduction mechanism between some gut cells of the alimentary tracts
of the IJ and the 10 of the EPB symbiont. Therefore the EPN / EPB symbiosis is
developmentally controlled from both sides. The infective dauer juvenile is capable of
surviving in the soil, entering the body cavity of a host and then developing into a female
or male. After two or three generations in the host the nematodes the infective juvenile
stage appears again, leaves the host and begins searching for a new host. The infective
stage juvenile is an alternative third stage juvenile, often still inside the second stage
cuticle. The mouth and anus are closed, the pharynx and intestine is collapsed. In every
(but one) Steinernema species the IJs develop in to an amphimictic female or a male but
never into hermaphrodites (see Fig. 1.). The EPN species have developmental larvae
stages (J1 – J4). An alternative developmental pathway leads to IJ, which leaves the
insect host and searches for another one. The choice between the two pathways is made at
the end of the first stage is based on chemical stimulus. When the nematodes are
crowding, the concentration of a pheromone (DRIF) overpasses the threshold
concentration, which, just like in case of C. elegans, induces dauer (IJ) formation (Fodor
et al., 1994). The number of the IJ is correlated with the number of nematodes present in
the insect cadaver. At this stage there is usually food storage and the “food signal”
competes with the pheromon (Golden and Riddle, 1982, 1984, Riddle 1988). The
reduction of food does not only induce IJ formation, but also blocks the recovery from the
IJ stage. A pheromone is a chemical stimulus to the late J1 stage, which responds by
developing into the alternative (J2D – dauer) pathway.
The intimate relation of the nematode and bacterium happens in the gut of the IJ
through an active retention of the bacteria by the cells of the vesiculum or bursa
intestinalis (in steinernematids). (No specialized cells were found for retaining the EPN
symbiont in Heterorhabditis. Maybe the whole intestine is responsible for the prokaryotic
/ eukaryotic cell – cell communication). Almost all phase 1 (1o) bacterial cells are
consumed prior to the dauer larva formation. Consequently, the primary stage bacteria
and the dauer larvae form monoxenic symbiotic combinations. When the IJ enters the
body of the insect, it releases the bacteria from its alimentary tract into the haemocoel.
11
After the death of the larvae the whole insect becomes monoxenic in which the nematode
and its symbiont are present.
Fig. 1: Life cycle of Steinernema species
LEGEND to Fig. 1. All but one Steinernema species are amphimictic. There sex is determined by XX / X0
mechanisms. There are males and females. In the insect cadaver the fertilized eggs hatch as first larval
juvenile (J1). These animals molt into J2, J3, J4 and adults, respectively. When the nematode population is
overcrowded (usually in the third generation in the nature), a chemical signal (called dauer inducing
pheromone, DRIF) passes a threshold concentration which induce in the available J1 larvae an alternative
developmental pathway. These worms grow to predauer (J2d) and then infective dauer juveniles (IJ). The IJ
worms represent a non-feeding, non-ageing developmental variant. They leave the cadaver and search for
another insect hosts. They then molt to J4, release a few cells of the primary form of their natural
Xenorhabdus symbiont. The bacteria kill the insect by their toxins and then colonize the insect cadaver. The
bacteria convert the insect tissues consumable for the nematodes, serve as food as well and even protect the
monoxenic culture by producing antimicrobial compounds.
1.1.3. The Heterorhabditis genus
The Heterorhabditidae family, Heterorhabditis genus was described in 1975 with
H. bacteriophora as the type species (Poinar, 1975). These are obligatory parasitic
rhabditids with biology similar to the Steinernematids. The Heterorhabditis species have
also a similar, but not identical life cycle including a third alternative - infective dauer
juvenile (IJ).
An important difference between the two genera is the mechanism of sex
determination. Heterorhabditids have alternative life cycles with hermaphrodites and
amphimictics.
As for the history of Heterorhabditidae, the nematode H. heliothidis was originally
described as a member of the family Steinernematidae, but later on it was synonymized
with Heterorhabditis bacteriophora (Poinar, 1990). Each IJ develops into a
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hermaphrodite (anatomically, just like C. elegans, the female is producing both sperm and
ova). However, the second generation consists of automictic hermaphrodites and
amphimictic (female and male) adults (Dix et al., 1992). This heterogamy, and the
occurrence of amphimictic and automictic adults make the life cycle and sex
determination of heterorhabditid nematodes very complex.
Fig 2: The life cycle of the Heterorhabditis species
LEGEND to Fig 2.Each Heterorhabditis species has an automictic and an amphimictic life cycle. The
mechanism of sex is determination is unknown. From the IJs 100 % self-fertilizing hermaphrodites develop.
Their progenies are of 50% males and females. In the insect cadaver the fertilized eggs hatch as first larval
juvenile (J1). These animals molt to J2, J3, J4 and amphimictic adults, respectively. When the nematode
population is overcrowded (usually in the third generation in the nature) a chemical signal (called dauer
inducing pheromone, DRIF) passes a threshold concentration, which induce an alternative developmental
pathway in the available J1 larvae. These worms grow to predauer (J2d) and then infective dauer juveniles
(IJ). The IJ worms represent a non-feeding, non-ageing developmental variant. They leave the cadaver and
search for another insect host. They then molt to hermaphroditic J4, release some cells of the primary form
natural Photorhabdus symbiont. The bacteria kill the insect by their toxins and then colonize the insect
cadaver. The bacteria convert the insect tissues consumable for the nematodes, serve as food as well as, and
protect the monoxenic culture by producing antimicrobial compounds.
In the last 10 years new species have been determined by morphology (Poinar et
al, 1994, Luanda Berry 1995, Stock et al, 1996, Adams et al, 1998 Blaxter et al, 1998).
The Heterorhabditis species are all obligate EPNs, capable of infecting a wide
variety of insects. Alternative third-stage infective juveniles (IJ), are often enclosed in a
second stage cuticle. Cells of a symbiotic bacterium (Photorhabdus spp.) occur in the
infective juvenile’s alimentary tract. Infective juveniles are capable of surviving adverse
13
environmental conditions that would be impossible for all other developmental variants
(see Fig. 2).
The IJs enter the body cavity of host or penetrate directly throughout the insect
cuticle. After infecting and releasing the symbiotic bacteria into the haemocoel, they
develop into J4 and then the adult stage, a self - fertilizing hermaphroditic female. Thus in
infection, a single hermaphroditic Heterorhabditis IJ is sufficient to initiate nematode
development and proliferation within the haemocoel (Zioni et al, 1992). These first
generation Heterorhabditis hermaphrodites give rise to a second generation, which
consists of amphimictic females and a few hermaphrodites (Fig. 1.). There are large
differences in the percentage of male progeny produced by the first generation
hermaphrodites in different species and strains of Heterorhabditis (Dix et al, 1992).
Hermaphroditic and amphimictic populations are found only inside infected cadavers in
nature (Poinar, 1992).
Heterorhabditis spp. have been recognized by molecular identification as H.
bacteriophora, H. indica, H. marelata, H. megidis, H. zealandica, and H. argentinensis
(Adams et al., 1998). It was reported that Heterorhabditis strains belonging to the North
Western European (NWE) group were considered as H. megidis when compared to the
original OH1 H. megidis strain (Poinar et al., 1987). Heterorhabditis strains isolated in
Ireland and classified as Irish type are represented by strains such as K122 or M145 (Dix
et al., 1992, Griffin et al., 1994). The new name of the species is Heterorhabditis
downesii (Griffin et al., 1994).
1.1.4. The biological background of insect pathogenesis caused by
EPN/EPB symbiotic complexes
Infection by both Steinernema and Heterorhabditis is initiated by a third stage
juvenile, which is morphologically and physiologically adopted to remain in the
environment for a prolonged period (without taking nourishment). The IJ stage is the only
survival stage in the life cycle of these nematodes. Morphological adaptations for this
survival period include a collapse of certain body tissues. The alimentary tract essentially
nonfunctional, because the walls of the intestine and pharynx have closed together,
thereby greatly reducing the lumen of the digestive tract. The mouth and anus are closed.
Symbiotic bacteria (Xenorhabdus or Photorhabdus), which play an important role for
nutrition inside the host, are found in the alimentary tract of the infective stage. In
Steinernema, the great majority of the bacteria are found in the modified ventricular
14
portion of the intestine. In Heterorhabditis .Photorhabdus is found in this location as
well, but can also occur throughout the intestine and in the pharyngeal lumen.
1.1.5. Entomopathogenic symbiotic bacteria
The entomopathogenic bacteria that are symbiotic with the nematodes were first
placed in the genus Xenorhabdus. On the basis of physiological and genomic analysis
data, it was later proposed that the bacteria, which emit light (X. luminescens), should be
given genus status and this taxon become designated Photorhabdus luminescens. The
symbiotic nematode partner is Heterorhabditis spp. The Xenorhabdus species are
obtained from nematodes belonging to the genus Steinernema (Szállás et. al., 1997).
Xenorhabdus and Photorhabdus spp. are Gram negative gamma Protobacteria that form
entomopathogenic symbiosis with soil nematodes. Their life cycle involves a symbiotic
stage in which they are carried in the gut of the nematode and a pathogenic stage in which
the insect prey is killed by the combined action of the nematode and the bacteria (Forst et.
al., 1996). As mentioned above, the bacteria are carried in the intestine of the infective
(dauer) juvenile stage of nematode. The nematode enters the digestive tract of the larvae
of diverse insects and penetrates into the haemocoel. Nematodes also enter through the
respiratory spiracles or by penetrating directly throughout the insect cuticle (Forst et. al.,
1997). Upon entrance into the haemocoel the nematodes release the bacteria into the
hemolymph. Within the haemocoel of larva, the bacteria grow to stationary phase while
the nematodes develop and sexually reproduce. In the stationary phase, bacteria break
down the host by secreting several extracellular products including lipase(s),
phospholipase(s), protease(s), and several different broad spectrum antibiotics (Forst et.
al., 1997). These products are believed to be secreted into the insect hemolymph. When
bacteria enter stationary phase conditions, the degrading enzymes break down the
macromolecules of the insect cadaver to provide the nematode with the nutrient supply,
while the antibiotics prevent the contamination of the cadaver with other microorganisms. Cytoplasm inclusion bodies - composed of highly expressed crystalline
proteins - are also produced by both bacteria during the stationary phase (Couche et. al.,
1987).
Nematode reproduction is optimal when the natural symbiont dominates the
microbial flora, suggesting that the bacteria can serve as food source and provide
essential nutrients that are required for efficient nematode proliferation (Akhurst et. al.,
15
1986). This stage of the bacteria which provides several compounds for the nematodes, is
named Phase 1 or primary phase (1o). The bacteria may also enter into Phase 2 or the
secondary phase (2o). Both phases can serve as food supply, but only the Phase 1 cells
produce antibiotics. A possible explanation of the primary - secondary phase shift may
influence the survival of the nematode (Poinar, 1978), especially in laboratory or
fermentation conditions. It should be emphasized, that although both phase variants are
taken up by the nematodes and used as food, only the Phase 1 cells are retained by
infective dauer juveniles and exists as symbionts for a longer period of time. In case of
steinernematids several other bacteria including (E. coli) could be used as nutrients.
Freshly hatched first stage (J1) juveniles can develop up to adult age on E. coli, but the
adults are infertile (Vanfleteren and Fodor, unpublished) if the Phase 1 cells of the
symbiont are not present. When Steinernema spp. dauer larvae are transferred into a
culture of either a non - symbiotic or Phase 2 cells of their associated bacteria, they may
recover and grow to low fertility or infertile adults. When growing steinernematids in
axenic media or fertilized chicken eggs, they may grow, but poorly produce infective
dauer juveniles. Heterorhabditids are different. They grow and reproduce exclusively on
the Phase 1 cells of their symbionts (Fodor et al., unpublished).
During the final stage of development, the nematode and bacteria re-associate and
the nematode subsequently develops into its infective dauer juvenile (IJ) stage. The IJ
carries the bacteria in its intestinal tract then emerges from the insect carcass and searches
a new insect host.
All the Xenorhabdus isolates studied so far have been obtained from nematodes
harvested from soil samples. Free-living forms of the bacteria have not been isolated from
soil or water (Forst et. al., 1997).
The above findings suggest that the symbiotic association may be essential for the
survival of the bacteria in the soil environment. It is a little bit contradictory to the fact,
that there is no problem of growing Xenorhabdus spp. or Photorhabdus spp. on
conventional bacterial media in the laboratory. The only known thing about these
symbiotic bacteria is that they are proline auxotrophs (Nealson et al., 1990). Except for a
very few known cases, the bacteria in turn are essential for effective killing of the insect
host and they are required for the nematode to complete its life cycle. There is only one
known Steinernema species (isolated from Azores), which does not have any bacterial
symbiont. This Steinernema glaseri strain is in facultative symbiosis with X. poinarii. In
any population of this nematode strain one can always find nematodes without bacteria.
16
On the other hand, some insects are resistant to S. glaseri,
(when the bacteria are not
present), but not to the symbiotic complex (Ray Akhurst, Ralf - Udo Ehlers, personal
communication).
Both bacterial genera belong to the Enterobacteriaceae but are nitrate reductase
negative. Photorhabdus are catalase positive and are the only known terrestrial bacteria
able to emit light similar to that of the marine bioluminescent bacteria. The taxonomy of
Xenorhabdus and Photorhabdus has been discussed elsewhere (Boemare et al., 1993,
Akhurst and Boemare, 1994, Boemare et al.,1993) used a method of total genomic DNA
hybridization to re-evaluate the designation of Xenorhabdus species which had been
determined by classical methods of bacterial taxonomy. Their work confirmed that X.
beddingii, X. nematophila, X. bovienii and X. poinarii are valid species whereas the fifth
species, X. luminescens was transferred to a new genus, as Photorhabdus luminescens.
Photorhabdus and Xenorhabdus spp. are facultative anaerobic Gram negative
rods, non sporulating oxidase negative chemoorganotrophic heterotrophs with respiratory
and fermentative metabolisms. They belong to the family Enterobacteriaceae in groups 5
and 1 (Akhurst, 1980, Boemare et al, 1993, Hoot et al, 1994). The general features of the
life cycles of these bacteria are quite similar. Xenorhabdus and Photorhabdus spp. are
carried as symbionts in the intestine of infective juvenile stage of nematodes belonging to
the families Steinernematidae and Heterorhabditidae, respectively (Akhurst, 1993, Forst
and Nealson, 1996, Poinar, 1990).
The bacterial strains belonging to the Photorhabdus genus are usually symbionts
of entomopathogenic nematode (EPN) strains. The only exceptions are those isolated
from clinical material (human wounds, Colepicolo et al, 1989) and have recently been
recorded as the novel species P. asymbiotica (La Saux et al, 1999). Photorhabdus species
other than those belonging to P. asymbiotica are carried in the gut of the infective dauer
juveniles (IJ).
While Xenorhabdus spp. and Photorhabdus spp. are similar in numerous
characteristics, they differ in several salient features. Xenorhabdus spp. is specifically
found associated with EPNs in the group Steinernematidae, while Photorhabdus spp.
only associated with the nematode group Heterorhabditidae. A primary property
distinguishing Photorhabdus from Xenorhabdus spp. is the ability of the former to emit
light under stationary phase culture conditions and in the infected host insect (Poinar et
17
al., 1980). On the other hand, Xenorhabdus is catalase negative which is an unusual
property for bacteria in the Enterobacteriaceae family (Boemare, 1990 Farmer 1984).
1.1.6. Mutualism between entomopathogenic nematodes and bacteria
As discussed above, the bacteria need the nematode as a vector to bring them from
one insect hemolymph to the next. The bacteria do not survive well in soil (Poinar et al.,
1980), and are generally not pathogenic for insect when ingested. The nematode needs the
bacteria to kill the insect. Heterorhabditids are not able to kill an insect without their
symbiont (Han et al., 1990) and the steinernematids are less pathogenic without their
symbiont (Akhurst, 1986, Dunphy et al., 1985, Ehlers et al., 1990). The nematodes help
the bacteria by suppressing the antibacterial immune system of the insect (Götz et al.,
1981). The nematodes also need the symbiont to get access to right nutrient. Again,
Heterorhabditids are more specific in this process than Steinernematids. Steinernematids
can be cultured in vitro on a complex medium without bacteria while Heterorhabditids
can be grow only in the presence of their own symbiont or a very closely related
symbiont strain (Luan et al., 1993). The bacteria produce antibiotics to inhibit growth of
other microorganisms in the insect cadaver (Akhurst, 1982). Although adult and
developing nematodes can digest bacterial cells, the bacteria are not digested inside the
infective juveniles, since they need the bacteria to kill a new host. The close relationship
between nematodes and bacteria is also illustrated by the fact that most nematode species
are associated with a single bacterium species (Akhurst and Boemare 1988, Smith and
Ehlers, 1991). Steinernema carpocapsae is associated with Xenorhabdus nematophila, S.
glaseri with X. poinarii, S. feltiae and S. affinis with X. bovienii, etc. Currently all
symbionts of Heterorhabditis spp. are classified as Photorhabdus but different species.
The biological role of the EPB symbiont is summarized in Fig. 3. The symbiont
demolishes the immune system of the insect and kills it by toxins. The EPB cells released
into the haemocoel convert the insect tissue consumable for the nematode by their exoenzymes. Finally, they protect the monoxenic system against soil microorganisms by
producing antimicrobial compounds of large spectrum.
18
Fig. 3: The biological role of the EPB symbiont
LEGEND to Fig. 3.. The symbiont demolishes the immune system of the insect and kills it by toxins. The
EPB cells released into the haemocoel convert the insect tissue consumable for the nematode by their exoenzymes. Finally, they protect the monoxenic system against soil microorganisms by producing
antimicrobial compounds of large spectrum.
Entomopathogenic bacteria belonging to the genera of Xenorhabdus and
Photorhabdus are known to produce several compounds with antimicrobial activity,
effective against large spectrum of Gram (+) and Gram (-) bacteria and against several
fungi. The antibiotics production profile of the different strains is different.
Entomopathogenic nematodes can be used to control several agricultural
insect pests. All known species can be used successfully against most Lepidoptera
especially if some developmental stage is in the soil. S. feltiae is used against mushroom
(Sciaridae) flies in the US and several EU countries. They are also effective against
Othyorhynchus species. In Hungary, S. carpocapsae Mexicana and All strains proved to
be effective against Colorado potato beetle (Leptinotarsa decemlineata) larvae in heavily
irrigated conditions (Fodor and Sáringer, 1986). S. glaseri and several Heterorhabditis
strains are widely used against scarabid grubs with successes all over the world. We have
recently tested the effect of Heterorhabditis sp. HU86 in our laboratory with promising
results. The agricultural potential of the toxins and antibiotics produced by
entomopathogenic bacteria is a new and growing challenge.
19
The natural product of biologically active materials produced by the EPB strains
(as well as of their biological role) is summarized in Fig. 3. Both the primary and the
secondary forms of each EPB strains produce toxins of protein nature (see the review of
french-Constant, 2003). They cause septicemia after being released to the blood of the
infected insects while others exert oral toxic activity as well. The phase 1 variant bacteria
also produce proteases and other exo-enzymes aiming it inactivate the immune system of
the insect hosts. (Some exo-enzymes are also produced by the phase 2 forms as well).
Again, phase 1 variant bacteria produce antibiotics effective against almost all Gram
positive as well as a large number of Gram negative bacteria. It is not known whether the
antifungal activity of the fermentation soup of the phase 1 EPB strains are due to the
same molecules as the antibacterial activity. What is known, that (i) only the primary
variant produces compounds of antimicrobial activity, and (ii) the spectrum and the
amount of these compounds differs from one species to another.
The primary forms of each EPB strain also produces intracellular crystals of
protein nature. These proteins are not toxic, and their physiological role is unknown. The
genes, cipA and cipB coding for the crystal proteins in Photorhabdus are known. The
Xenorhabdus intracellular crystals are smaller and the genes coding for them are not
related to those of Photorhabdus (see the review of Forst, 2002). Mutants or phase
variants not producing intracellular crystals are neither producing antibiotics nor building
symbiosis with entomopathogenic nematodes.
As it will be discussed in Chapter 3 (Results) we have discovered an exo-cellular
crystal produced by only one Xenorhabdus strain (EMC) isolated in our laboratory. This
crystal is not protein, but a polysaccharide metal complex. Although we have crystalminus mutants, and have isolated the compound, we do not know anything about its
biological role.
20
1.2. OBJECTIVES AND RATIONALE
This study is aimed at revealing some details of the nature and evolution of the
developmentally controlled,
intimate,
and
highly taxon-specific
symbiosis
of
entomopathogenic nematodes (EPN) and bacterium (EPB) species. The ultimate goal is
benefiting from this information for improved biological pest control in the agriculture. I
intended to provide examples of using selected EPN strain against target insect pests and
EPB strains against target microbial pests and focus on the potential of producing new
EPN/EPB symbiotic complexes.
Biological control is characterized by the utilization of living microorganisms to
control pests. Gnotobiology comprises a study of germ-free plants and animals to which
specific microorganisms are coupled by experimental methods. When one or more known
species of microorganisms are added experimentally to a germ-free animal, the host is no
longer germ – free. Both the host and the introduced species are gnotobiological.
Gnobiological research seeks to explore the effects of microorganisms in natural diseases,
to identify the specific causative agents in infectious diseases. Gnotobiology is a part of
the microbial ecology, which studies the relationships between animals and their
associated microbial populations. An axenic animal is an animal living and reproducing
free from any microorganism.
Entomopathogenic nematodes are a welcome addition to the natural enemy pool
of insects and can be integrated with various control measures for management of those
target pests where individual tactics alone are inadequate. Entomopathogenic nematodes
play a role underground reminiscent of that played by insect parasitoids. Like parasites or
predators they have chemoreceptors and are motile. Like pathogens they are highly
virulent, killing their hosts quickly and can be cultured easily in vivo or in vitro.
Entomopathogenic nematodes are among the best known of an otherwise poorly
studied group of natural soil insect enemies. Interest in these beneficial organisms has
increased rapidly in recent years and research are being conducted in many laboratories
world-wide (Gaugler et al, 1997). New species are described every year and many more
isolates are waiting for identification and study (Koppenhofer and Kaya, 1999).
21
The aims of the study are:
1.
Screening the EPB bank of our laboratory to determine the antibiotic
production of different Xenorhabdus and Photorhabdus strains, to compare
the effects of the antibiotics on closely related strains and on taxonomically
unrelated bacteria, such as E. coli, Erwinia amylovora B. subtilis, and B.
cereus.
2.
To determine the symbiotic partner range of a large number of
Steinernema and Xenorhabdus strains (gnotobiological analysis).
3.
To identify new Steinernema isolates by using a molecular tool (GeneGel
Excel kit), cross fertilization and gnotobiological studies.
4.
To establish a liquid fermentation technique of a new Steinernema isolate.
22
2. MATERIALS AND METHODS
2.1. ENTOMOPATHOGENIC NEMATODE (EPN) AND
BACTERIA (EPB) USED IN THIS STUDY
2.1.1. EPN STRAINS
Table 1. Summarizes the Steinernema strains from our laboratory collection and
their natural Xenorhabdus symbionts.
Table 1. Steinernema strains and their natural symbionts.
Steinernema species
Strains
Origin
Xenorhabdus symbiont
S. feltiae
Filipjev
Umea
Russia
Sweden
X. bovienii
X. bovienii
SF22
Finland
X. bovienii
Vija Norway
Norway
X. bovienii
Scp
Poland
X .bovienii
1048
Galyatetõ
X. bovienii
1003
Hortobágy
X. bovienii
Nyíregyháza
Nyíregyháza
X. bovienii
IS6
Israel
X. bovienii
Sulcatus
USA
X. bovienii
Mexican
Mexico
X. nematophila
T1
Azores
X. nematophila
T2
Azores
X. nematophila
SK98
Unknown
X. nematophila
NCI
N. Caroline
X. poinarii
NCI LU
N. Caroline
X. poinarii
NC 513
N. Caroline
X. poinarii
KMD 15
Ohio, USA (A. Lucskai)
X. spp.
S. anomali
S. intermedia
Anomali
W. Europe
X. spp.
BIOSYS
?
X. spp.
S. rarum
Rarum
S. America
X. spp.
S. bicornutum
Bicornutum
Europe (B. Tallósi)
X. spp.
S. affinis
Affinis
?
X. spp.
S. cubana
Cubana
Cuba
X. spp.
S. scapterisci
Scapterisci
S. America
X. spp.
S. riobrave
Riobrave
Brasilia
X. spp.
S. spp.
SK 27
?
X. spp.
S. kraussei
Kraussei
Alps
X. spp.
S. bibionis
Bibionis
USA
X. spp.
S. spp.
336
?
X. spp.
S. spp.
Mongolian
Mongolia (H. Pamjav)
X. spp.
S. carpocapsae
S. glaseri
S. ohioensis
23
3.1.2. EPB STRAINS
Table 2. Summarizes the Xenorhabdus strains in the gnotobiological study.
Table 2. The Xenorhabdus strains. A. Known, B. unknown (newly identified) strains.
XENORHABDUS
X. bovienii DSMZ 4766
X. poinarii DSMZ 4768
X. beddingii DSMZ 4764
X. nematophila DSMZ 3370
X. nematophila ATTC 19061 2.
X. nematophila ATTC 19061 1.
X. nematophila ANT 1
X. nematophila AN6 2.
X. nematophila AN6 1.
X. nematophila 703
X. nematophila N2-4 SS (2.)
X. nematophila N2-4 SA (2.)
X. nematophila N2-4 P (1.)
X. nematophila T1
Xenorhabdus spp. SK 98
X. scapterisci 1
X. scapterisci 2
X. bovienii SF22
X. bovienii Norwegian
“Mongol”
X. bovienii Sulcatus
X. bovienii Filipjev
X. bovienii Umea
X. bovienii HU1003
X. bovienii IS6
X. bovienii Nyíregyháza
X. spp. kraussei
X. spp. „affinis”
X. spp. „rarum”
X. spp. „kushidai”
X. bibionis
X. spp. „serratum”
X. spp. cubanum
S47A
X. riobrave 1.
X. riobrave 2.
KMD15
X. bicornutum
X. intermedium
X. intermedia BIOSYS
X. anomali Lucskai
X. anomali Azores
A. SYMBIOTIC PARTNERS
STEINERNEMA
S. feltiae
S. glaseri
S. intermedium
S. carpocapsae
S. scapterisci
S. feltiae
S. kraussei
S. affinis
S .rarum
S. kushidai
S. bibionis
S. serratum
S. cubanum
Steinernema sp.
S. riobrave
S. glaseri (strain Ohioensis)
S. bicornutum
S. intermedium
S. anomali
24
Table 2. (continued)
XENORHABDUS
B. SYMBIOTIC PARTNERS
STEINERNEMA
Z 06
Z3 1/1
H5
FA 019 (Randy Gaugler)
FA 013
49. HW 7
50. HK 2001
51. S 47A
52. KMD 44
Strain 3905
Unknown Steinernema from Transylvania
Unknown Steinernema from Ohio (M. Klein)
Unknown Steinernema, Washington State
2.2. METHODS AND TECHNIQUES
2.2.1. MICROBIOLOGICAL METHODS
2.2.1.1. SOLUTIONS AND MEDIA
M9 a buffered physiological salt solution for nematodes (Brenner, 1974)
KH2PO4---------------3 g
Na2HPO4------------- 6 g
NaCl-------------------5 g
Fill it up with distilled H2O up to1000 ml
After sterilization add:
MgSO4 1 M------------1ml
Liquid (or solid) LB media (Luria Bertani medium, Miller, 1972) (without or
with agar):
Bacto- trypton---------------- 10 g
Bacto-yeast extract----------- 5 g
NaCl-----------------------------10g
Distilled H2O up to1000 ml
(Agar: 15 g, if LB agar media is needed).
Fill it up to 1 liter. Shake until the solutes have dissolved. Adjust the pH to 7.0 with 5N
NaOH (0.2ml). Adjust the volume of the solution to 1 liter with deionized H2O. Sterilize
by autoclaving for 20 minutes at 15 lb/sq. in. liquid cycle.
Liquid (or solid) TSY media (without or with agar):
Yeast extract----------------------5 g
Soy peptone----------------------30g
25
Cholesterol------------------------3 ml/l
Distilled H2O up to1000 ml
(Agar: 15 g, if TSY agar media is needed).
Liquid (or solid) NA media (without or with agar):
Beef extract------------------------3 g
Peptone-----------------------------5 g
Sodium chloride-------------------5 g
(Agar: 15 g, if NA agar media is needed).
Fill it up with distilled H2O up to1000 ml
NBTA an indicator medium
Meat extract-----------3 g
Pepton------------------5 g
Agar--------------------15 g
Fill it up with dH2O up to-1000 ml
After sterilization and moderate cooling:
BTK 25mg /ml---------1 ml /L (Bromothymol blue)
TTC 40 mg /ml---------1 ml /L (Triphenyl tetrazoliumchloride)
(An indicator media used to distinguish between primary and secondary forms of
Xenorhabdus and Photorhabdus strains.)
MacConkey Indicator Agar
MacConkey Agar 47 g
Bidistilled water 1 liter
Expected results distinguishing between primary and secondary forms
Media
Primary
Secondary
MacConkey
NBTA
Tween
Blood agar
Brown
Greenish
Granules
Hemolysis
The colony is clear
Red to Brown
No granules
No hemolysis
Wouts” agar media
Per liter
Agar------------------------------12 g
Bacto Peptone------------------10 g
Beef extract----------------------6 g
Vegetable or fish oil------------5 g
Bidistilled water to 1000ml
26
2.2.1.2. METHODS
ISOLATION OF THE SYMBIOTIC BACTERIA FROM DAUER JUVENILES
The bacterium strains were isolated from cadavers of Galleria mellonella infected with
Heterorhabditis spp or with Steinernema spp strains as described by Akhurst (1980). The
protocol was modified as follows (A. Lucskai, unpublished):
1.
The covering part of a sterile (Falcon) Petri plate was taken in a laminal box under
a binocular microscope.
2.
Add 200µl of 5% sodium-hypochlorite (NaOCl, chlorox).
3.
Add 4X200 µl of sterile M9 buffer (Brenner, 1974) on the plate.
4.
After washing the dauer with sterile tap water (1x) and M9 (3X) by centrifugation,
transfer them into the drop of Sodium hypochlorite with the platinum wire and
keep them there for five to seven minutes.
5.
Transfer the dauers into the 1st drop of sterilized M9 buffer. Then transfer to the
2nd, and so on.
6.
Transfer the nematodes into the last drop of sterilized M9 buffer by a platinum
wire.
7.
Cut the dauer into two or more parts.
8.
Transfer about 2 µl of the suspension into LB or directly on LBTA indicator plates
with a sterile automatic pipette.
9.
The bacteria were grow on the plate at 30 °C. On indicator plates the primary form
of the bacteria can be distinguished from the secondary forms and the
contamination. (On the NBTA plate the color of the primary form colony is blue,
all the others (contamination or secondary variants) are red. Bacterial strains were
selected based on colony morphology on nutrient agar (Difco), ability to absorb
dye when grown on (NBTA) medium and MacConkey Agar (DIFCO). Production
of antibiotic substance was detected as described by Akhurst (1982). The
secondary form lacks inclusion bodies as well as antibiotic activity as described by
Boemare and Akhurst (1988).
FREEZING OF BACTERIA FOR LONG-TERM STORAGE
1. One colony of any strain of bacteria was inoculated into sterilized liquid LB
medium (5ml).
2. 1 ml suspension was placed into a sterile Eppendorf tube.
27
3. 0.5 ml glycerol (8.7%) was added.
4. Vortex for 30”.
5. Eppendorf tube was put into - 80 ˚C.
6. When taken out of the freezer 100 µl of the melt suspension was placed on LB
agar plate.
SURFACE STERILIZATION OF INFECTIVE DAUER JUVENILES
1. The dauer juveniles were washed 3 times with autoclaved tap water and
centrifuged at low speed (~1,000 rpm) for 1 min.
2. The nematodes were surface sterilized in 0.05 % hyamine solution with vortexing
every minute for 10 sec.
3. The IJs were centrifuged at 2,000 rpm for 1 min. and washed quickly with sterile
M9 (hyamine kills the dauer stage when held too long).
4. The dauer juveniles were washed 3 times with sterile tap water.
5. Galleria mellonella (wax month) larvae were infected with the sterile nematodes.
2.2.2. METHODS FOR GNOTOBIOLOGY
Some EPN species and strains were usually grown only on their own symbionts. Some of
them can grow on each other symbionts in agar in Petri plates:
Media
TSY
Woots
NGM
Bacterium
X. bovienii Norwegian
P. temperata ssp. temperata HSH1
X. bovienii Norwegian
P. temperata ssp. temperata HSH1
E. coli OP50
Nematode
Steinernema feltiae
Heterorhabditis megidis NEW HSH1
All strains of S. feltiae
All strains of H. megidis NWE
C .elegans
For all gnotobiological analysis the following procedure was used:
1. Liquid cultures of the primary form of Xenorhabdus or Photorhabdus were grown
overnight aseptically in 5 ml of nutrient broth at 30 °C in a test tube (one tube per
flask to be inoculated). The bacteria then were transferred to TSY or lipid agar
plates and allowed to grow for 2-3 days at 25 °C.
2. Axenic J1 juveniles of Steinernema or Heterorhabditis strains were transferred to
bacterial lawn on TSY or lipid agar plates. Next generation infective dauer
juveniles were collected.
28
3. Infective dauer juveniles (IJ) of several Steinernema or Heterorhabditis strains
were surface sterilized and put on the bacterial lawn of the bacteria under
gnotobiological investigation.
The plates were scored based on:
a. EPN grew (+) or did not grow (-) in into fertile adults.
b. In the progeny population IJ appeared (+) or did not (-) appear.
c. The IJ proved (+) or did not prove to be pathogenic for insect hosts (4th
instar larvae of Galleria mellonella).
d. The new generation of IJs left (+) or did not leave (-) the insect cadaver on
water traps.
e. The bacteria isolated from the IJs could be identified as the “new” (+) or
the original (-) symbiont.
29
3. RESULT AND DISCUSSION
3.1. ANTIBIOTICS PRODUCTION OF THE EPB STRAINS
3.1.1. ISOLATION EPB SYMBIONTS DIRECTLY FROM DAUER
LARVAE OF STEINERNEMA SPECIES
The technique of direct isolation EPB symbiont from a single dauer larva was
originally elaborated by A. Lucskai. The method is considerably safer than isolating the
symbionts from an infected insect, since the contamination inside the carcass can be
eliminated. The method was further modified in our laboratory (Fig. 4.).
Fig. 4. Isolation of EPB symbionts from EPN infective dauer juveniles
LEGEND to Fig. 4. Isolation of the symbiotic EPB bacterium from surface sterilized IJ nematode. The
dauer larvae obtained from water trap are washed with M9, bleach, and with M9 again. The nematodes
were cut into pieces in sterile M9. Drops of the M9 solution containing the bacteria coming out from the gut
of the IJ are transferred to indicator (NBTA) plates. Blue colonies are considered to be the primary form of
the bacterial symbiont.
30
3.1.2. ANTIBIOTICS PRODUCTION OF DIFFERENT
XENORHABDUS AND PHOTORHABDUS STRAINS TESTED ON
BACILLUS CEREUS
The antibiotics production of 103 strains was tested using Bacillus cereus as
indicator bacteria based on Akhurst’s method (Akhurst, 1980). Figure 5. demonstrates
how the effect of the antibiotics produced by EPN strains on B. cereus was determined.
An EPB colony was grown (from a 10µl of overnight suspension) in the center of the LB
agar plates. After 5 days the bacteria are killed in a chloroform fume and overlaid by the
indicator bacteria (B. cereus spores) suspended in soft agar. The size of the inactivation
ring is proportional to the antibiotics activity of the EPB strain studied. The antibiotic
productions of different Xenorhabdus strains are variable. The results are presented in
Table 3.
Fig. 5. Antibiotics production of EMC (left) and X. bibionis (right) determined by
using B. cereus as indicator strains.
Inhibition zone
LEGEND to Fig. 5. EPB bacteria were grown for 5 days in the center of the Petri dish and then the EPB
was overlaid by the indicator strain resuspended in soft agar. The inhibition zone is shown as a ring around
the EPB colonies. The strain EMC (left) produced excessive amount of antibiotics inhibiting the growth of
the indicator strain almost completely. X. bibionis produced less antibiotics against B. subtilis, the inhibition
zone is clearly visible on the plate.
As it can be seen in Table 3. the best antibiotics producing strain was Ema with
inhibition zones between 80-90 mm. The Ema strain (Ema new) was reisolated from the
original nematode symbiont and the bactera were tested again for producing antibiotics.
31
The inhibition zone of the new Ema isolate was 87mm, and so there is a slight decrease
compared to the original bacterial stock stored in the freezer. It is possible, that storing
the nematodes in stock and allowing the reproduction “artificially” in G. mellonella does
not help the bacterial strain to keep the antibiotics production (or other traits) at the same
level and by time the laboratory conditions decrease or diminish the antibiotics
production. The KMD15 and the DSM3370 X. nematophila strains also produced larger
amount of antibiotics, the inhibition zones for B. cereus were 56 and 49 mm, respectively.
The antibitotics production was tested with the colony on the plates and with the
removed EPB colonies as well (Table 3.). In all cases the inhibition zones were larger (the
antibiotics production was higher) when the colonies were present on the plates. The
inhibitions zones were approximately one third smaller without the colonies. It is possible
that the bacterial colony is releasing another – and not very stable - compound that makes
the antibitotics more effective. Alternatively, if some of the produced antibiotics are
membrane bound (the membranes of the bacteria are destroyed with the chloroform
treatment) some of the activity will be lost as well. This way removing the colony means
less “active” antibiotics in the media.
When the fermentation soup was used, the inhibition zones were even smaller in
each case. If the produced antibiotics and their activity are depending on a factor that is
membrane bound, with fermentation of the bacteria, the inhibition zone would be smaller.
Alternatively, liquid fermentation conditions are less preferable for the antibiotics
production than the solid surface aerobic growth. As control for the EPB antibiotics
production Ampcillin (100ppm) was used.
32
Table 3. Antibiotics production of the EPB strains against B. cereus.
Petri dishes overlaid by B. cereus spore suspension in soft agar
EPB strains
1.
2.
3.
4.
5.
6.
7.
8
9.
10.
11.
12.
13.
5-days old EPB colony
Diameter of the inhibition zone (mm)
1.
2.
Average
X.
Ema1 old
90
90
90
>80
X.
Ema1 new
87
87
87
X.
Kmd15
56
56
X. n.
N2-4/I
25
X. n.
N2-4/Ii
200 μl of fermentation soup
Diameter of the inhibition
zone (mm)
Average
1.
2.
Average
>80
>80.0
30
30
30
77
77
77.0
30
30
30
56
46
46
46.0
17
17
17
23
24
12
9
10.5
20
20
20
14
12
13
3
2
2.5
13
13
13
DSM
3370
55
43
49
53
43
43.0
15
15
15
P.
Jun old
29
28
28.5
20
19
19.50
12
12
12
P.
Jun new
27
26
26.5
18
17
17.5
12
12
12
P.
Eg2
24
24
24
15
14
14.5
14
14
14
P.
Eg2 new
23
24
23.5
12
13
12.5
15
15
15
P.
Arg
41
34
37.5
32
25
28.5
13
13
13
P.
IS5
28
29
28.5
19
20
19.5
14
14
14
P.
OH-I
13
14
13.5
3
4
3.5
16
16
16
28
23
26.5
Colony
removed
Ampicillin 100 ppm
LEGENDS to Table 3. Comparison of the antibiotic production of several Xenorhabdus (X.) and
Photorhabdus (P) isolates. There was no significant difference between old and new isolates of the same
strain. Abbreviations: Ema: Xenorhabdus symbiont of S. bicornutum strain Tallósi; old: refrozen stock;
new: freshly isolated from the nematode just for this experiment; P. Jun: Photorhabdus stackebrandtii;
33
isolated from Heterorhabditis megidis (NWE) strain H. Jun by Emília Szállás; and re-isolated by Attila
Lucskai. X. Kmd15: Xenorhabdus sp. isolated from S. glaseri strain Kmd 15 (Ohio); discovered by Attila
Lucskai, and the symbiont was also isolated and re-isolated by him; X.n.: X. nematophila (natural symbiont
of S. carpocapsae). I: phase I (primary form); phase II (secondary form); Ii: an intermediate form. P.Eg2:
P. luminescens ssp. akhurstii; isolated from S. indica strain EG2 (from Egypt) by Emília Szállás and reisolated by Attila Lucskai. P. arg: P. luminescens ssp. laumondii from H. bacteriophora strain
Argentinensis Emília Szállás and re-isolated by Attila Lucskai. P. IS5: P. luminescens ssp. akhurstii;
isolated from S. indica strain IS5 (originated from the Negev desert, Israel, got from Prof. Itamar Glazer) by
Emília Szállás and re-isolated by Attila Lucskai. P. OHI: P. temperata (originated from the Mohican
National Park, Ohio, USA, got from Prof. Michael G. Klein) by Emília Szállás and re-isolated by Attila
Lucskai. The reference strains (DSM) are originated from the DSMZ collection, Braunschweig, Germany,
provided by Prof. Erko Stackebrandt.
3.1.3. THE EFFECT OF ANTIBIOTIC PRODUCTION ON OTHER
ENTOMOPATHOGENIC BACTERIA
The Xenorhabdus strains producED antibiotics against Photorhabdus and
Photorhabdus strains are also producing antibiotics against Xenorhabdus. A comparison
of the different EPB strains is demonstrated in Fig 6. The antibiotics produced by one
EPB strain might be also effective against other EPB strains. It was supposed that
antimicrobial compounds including antibiotics and colicins have an important role in
competition between EPB (and related EPN) strains in nature. The sensitivities of the
different Photorhabdus strain to antibiotics produced by a Xenorhabdus strain differed
from each other. Figure 6 and Figure 7 give examples that the antibiotics of the symbiont
of S. kraussei (X. bibionis) and S. affinis could be effective against Photorhabdus strains.
As it is shown on Figure 6 the K122 strain showed to be the least sensitive against the
antibiotics produced by the Xenorhabdus symbionts of S. kraussei and S. affinis, the most
antibiotics were produced by X. bibionis and against HL81 (Fig 6. and Fig. 7).
As shown in Table 4, the antibiotics produced by the symbionts of S. scaptersi, S.
bicornutum, S. rara and S. intermedia were the most effective against the symbionts of H.
bacteriophora. The symbionts of S. affinis and S. kraussei produced effective antibiotics
against most of the P. bacteriophora strains. The symbionts of S. feltiae and S. serratum
did not produce antibiotics effective against the P. bacteriophora strains tested. It is also
shown that the A1 and Koh strains were the most insensitive to the antibiotics produced
by Xenorhabdus. Table 5 shows the antibiotics produced by the symbionts of S. scaptersi,
S. bicornutum, S. rara and S. intermedia were the most effective against the symbionts of
H. megidis, H. marelatus and the Wisconsin isolates. The symbionts of S. affinis
produced effective, but less, antibiotics against most of the Photorhabdus symbionts.
Again, the symbionts of S. feltiae and S. serratum did not produce antibiotics effective
34
against most of the Photorhabdus strains tested. It is also shown that the A1 and Koh
strains were the most insensitive to the antibiotics produced by Xenorhabdus.
From the data it might be concluded that in some cases the coexistance between
the symbionts of Xenorhabdus and Photorhabdus is possible if there are no other
products of the nematodes or the bacteria that would inhibit the growth. For instance, H.
bacteriophora A1 strain could infect the same insects that S. affinis already infected.
Based on our data, theoretically symbiotic bacteria of different nematode species could
coexist in the same invironment allowing horizontal gene flow. Explain Table 4., table 5.,
table 6.
Data also show that different Photorhabdus strains of H. bacteriophora symbionts
belonging to the presented subspecies of the P. luminescens species show a similar degree
of sensitivity to the different Xenorhabdus antibiotics. Interestingly, the toxin-producing
P. luminescens ssp. akhurstii strains (symbionts of H. indica) are very sensitive to the
Xenorhabdus antibiotics, as demonstrated in Table 5.
Fig. 6. A qualitative and quantitative comparison of the antibiotics produced
by different Xenorhabdus strains.
LEGEND to Fig. 6. A comparison of affectivity of the antibiotics of different Xenorhabdus strains against
different Photorhabdus strains. The symbiont of S. kraussei (X. bibionis) is in the middle and the indicator
strains are A. K122, B. HP88, C. HL81. The symbiont of S. affinis is in the middle and the indicator strains
are D. K122, E. Hb1, F. OH10. The most effective antibiotics were produced by X. bibionis against
HL81strain among the strains tested in this experiment.
35
Fig. 7. X. bibionis strain inhibiting the growth of Photorhabdus temperata ssp.
temperata strain HL81.
Error!
Inhibition zone
LEGEND to Fig. 7. X. bibionis generally produced antibiotics with low effectivity against indicator
bacteria (see Table 5. and Fig. 6), but exerts a stronger inhibiting activity against Photorhabdus strain
HL81.
36
Table 4. Antagonism between Xenorhabdus and Photorhabdus. I as shown in
inhibition zones (mm)
Symbiont(s)
of
Steinernema
scapterisci
bicornutum
rara
intermedia
affinis
kraussei
(X.bibionis)
anomali
Symbionts of H. bacteriophora strains
P. luminescens subspecies
luminescens
laumondii
boemarii
Ind
IS5
EG2
Hm1
Hb1 HP88
A1 Koh
MOL
38
22
38
43
41
44
37
38
37
24
28
13
12
11
25
19
20
15
14
27
30
18
11
10
26
25
13
8
8
25
30
15
14
10
29
15
7
10
7
30
34
16
0
0
32
37
17
11
0
21
30
17
0
8
0
0
0
0
14
0
0
0
0
0
13
8
23
14
13
0
12
0
0
0
0
0
0
0
0
5
0
0
6
0
0
0
14
14
0
0
0
0
0
0
4
0
0
0
0
0
7
0
5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
carpocapsae (X. nematophilus)
DSM 3370
N2-4
0
15
glaseri (X. poinarii)
DSM 4768
Kmd 15
Cubana
8
12
0
feltiae (X. bovianii)
DSM 4766
Nyíregyháza
0
0
serratum (X.beddingi)
DSM 4764
0
LEGENDS to Table 4. Antagonism between Xenorhabdus and Photorhabdus. The symbionts of
Steinernema species (S. scapterisci, S. bicornatum, S. rara, S. intermedia, S. affinis, S. kraussei, S. anomali,
S. carpocapsae, S. glaseri, S. feltiae and S. serratum) were grown for two days on TSY agar plates and the
sensitivity of P. luminescens strains (the symbionts of H. bacteriophora) for the produced antibiotics was
measured through the size of the inhibition zone. The data are representative, the experiments were repeated
three times.
37
Table 5. Antagonism of Xenorhabdus vs. Photorhabdus. II.
Photorhabdus symbionts of
Symbiont
of Steinernema
scapterisci
bicornutum
rara
intermedia
affinis
kraussei
(X. bibionis)
carpocapsae
(X. nematophilus)
DSM 3370
N2-4
glaseri
(X. poinarii)
N2-4
glaseri
(X. poinarii)
DSM 4768
Kmd 15
Cubana
feltiae
(X. bovianii)
DSM 4766
Nyíregyháza
serratum
(X.beddingi)
DSM 4764
H. megidis
NWE
(ssp.temperata)
US
H. marelatus
Wisconsin
isolates
HSH2
31
HL81
49
H4
35
Meg1
47
OR10
41
Hepialius
42
Wx6
45
Wx8
50
14
35
21
10
0
35
49
28
0
27
20
28
14
7
0
38
36
0
12
0
32
17
20
10
5
35
28
19
13
0
38
42
18
8
8
29
41
19
12
0
0
0
0
27
18
17
0
0
0
0
0
0
5
10
0
0
0
0
8
0
0
0
0
0
0
37
7
0
0
0
0
0
0
11
0
14
20
0
4
0
0
0
0
0
5
0
0
0
0
0
8
0
0
0
0
0
0
0
0
14
0
0
0
0
0
0
0
0
0
0
0
0
0
0
7
0
0
0
0
0
LEGENDS to Table 5. Antagonism between Xenorhabdus and Photorhabdus III. The symbionts of Steinernema
species (S. scapterisci, S. bicornatum, S. rara, S. intermedia, S. affinis, S. kraussei, S. anomali, S. carpocapsae,
S. glaseri, S. feltiae and S. serratum) were grown for two days on TSY agar plates and the sensitivity of
Photorhabdus strains (the symbionts of H. megidis, H. marelatus and the Wisconsin isolates) for the produced
antibiotics was measured through the size of the inhibition zone. The data are representative, the experiments
were repeated three times.
38
Table 6. Antagonism of Xenorhabdus vs. Photorhabdus. IV
Photorhabdus symbionts of
H. downesii
Xenohabdus
strain, symbiont
of Steinernema
scapterisci
bicornutum
rara
intermedia
affinis
kraussei
anomali
carpocapsae
DSM 3370
N2-4
glaseri
DSM 4768
Kmd 15
Cubana
feltiae
X. bovianii
DSM 4766
X. bovianii
Nyíregyháza
serratum
Irish
ssp. temperata
Hungarian
H. megidis
P. stackebrandtii
H. sapiens
Wisconsin
isolate
P.asymbiotica
P.nealsonii
WX13
46
K122
45
HIT
42
Jun
41
ATTC
43951
13
33
30
17
10
0
0
36
15
17
10
0
0
25
11
15
7
4
0
0
15
14
9
12
0
37
39
12
9
12
24
0
0
0
0
0
0
0
16
16
0
0
0
0
0
0
0
7
0
0
0
10
10
9
15
12
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
LEGENDS to Table 6. Antagonism of Xenorhabdus vs. Photorhabdus IV. The data are representative, the
experiments were repeated three times. Data concerning the effects of Xenorhabdus antibiotics on Photorhabdus
strains belonging to the species Xenorhabdus antibiotics to Photorhabdus strains belonging to P. stackebrandtii, P.
asymbiotica and P. nealsonii species. K122 strain belongs to P. temperata ssp. temperata and a symbiont of a strain
(K122) of H. downesii. HIT strain, on the other hand, belongs to P. stackebrandtii and is a symbiont of another
strain (HIT) of H. downesii. There is not too much difference between the sensitivity to Xenorhabdus antibiotics of
the two, taxonomically different Photorhabdus strains. The most interesting data are those of P. nealsonii (WX13)
and the human pathogen P. asymbiotica strain. The latter is the less sensitive the earlier is the most sensitive
Photorhabdus strain we examined.
3.1.4. EFFECTS OF THE XENORHABDUS ANTIBIOTICS ON
ERWINIA AMYLOVORA
3.1.4.1. EFFORTS TO CONTROL THE FIRE BLIGHT DISEASE IN
APPLE ORCHARDS
Fire blight is a destructive bacterial disease of apples and pears that kills
blossoms, shoots, limbs, and, sometimes, entire trees. The disease is generally common
throughout the mid-Atlantic region of the US and in Europe. The outbreaks are typically
very erratic, causing severe losses in some orchards in some years, and little or no
39
significant damage in others. This erratic occurrence is attributed to differences in the
availability of overwintering inoculum, the specific requirements governing infection,
variations in specific local weather conditions, and the stage of development of the
cultivars available. The destructive potential and sporadic nature of fire blight, along with
the fact that epidemics often develop in several different phases, make this disease
difficult and costly to control.
Fig. 8. Fire blight disease
in apple orchads.
Overwintering cankers harboring the fire blight pathogen are often clearly visible
on trunks and large limbs as slightly to deeply depressed areas of discolored bark, which
are sometimes cracked about the margins. The largest number of cankers, however, are
much smaller and not so easily distinguished. These occur on small limbs where blossom
or shoot infections occurred the previous year and often around cuts made to remove
blighted limbs (P.W. Steiner, University of Maryland, T. van der Zwet, USDA and A. R.
Biggs, West Virginia University).
Strains of the pathogen that are resistant to streptomycin are present in some
orchards in the eastern USA and in Europe., and are widespread in most apple and pear
regions of the western U.S. Biological control agents, although not widely used, have
provided partial control of blossom infections. More effective biological agents are
required if their use is to become widespread.
3.1.4.2. LABORATORY TESTS ON ERWINIA AMYLOVORA
The majority of the tests were carried out in the Quarantine Laboratory of the
Szent István University of Horticultural Sciences in Budapest, Hungary within a
framework of co-operation. Some of the experiments were performed by M. Hevesi and
S. Pekár and their data with their permission, are also incorporated in Table 7. Different
Erwinia isolates were used as “indicator” bacteria to test different Xenorhabdus and
40
Photorhabdus strains for antibiotics production. The results were very reproducible. The
results of the Erwinia laboratory tests are summarized in Table 7.
Table 7. The effect of EPB antibiotics on ten E. amylovora isolates.
Erwinia
isolates:
EPB strain
Ea2
Ea60
Ea67
Ea9
Ea26
Ea56
Ea78 Ea47 Ea17 Ea28 E. coli
K12
X. Ema1
X. Kmd15
X. N2-4-P
X. N2-4Ii
26.6
6.0
28.5
23.0
25.5
6.0
28.5
23.0
23.0
5.5
27.0
24.0
28.0
6.0
28.0
23.5
26.5
3.0
23.0
22.5
22.5
2.0
25.5
24.5
21.5
4.0
25.0
24.5
24.0
2.0
25.5
24.5
27.5
3.0
24.5
22.5
24.5
1.0
22.0
21.0
25.5
3.8
22.7
23.3
X. N2-4/II
X. DSM3370
P. Hit
P. Jun
P. K122
P. Az29
P. Eg2
P. OH-10
P. Arg
P. IS5
P. Brecon
P. HE86
P. indica
P. Wx6
P. Az36
P. HP88
E. coli K12
26.5
28.0
18.0
19.0
26.5.
21.0
19.0
6.5
18.5
28.0
21.5
10.0
20.0
14.0
26.5
25.5
0
26.5
26.0
21.0
20.0
28.5
28.5
21.0
8.0
18.0
30.0
19.0
12.0
18.5
15.0
35.0
24.0
0
26.0
30.0
25.0
18.0
26.0
25.5
22.5
9.0
20.5
25.0
22.0
15.0
26.5
16.5
30.0
22.0
0
22.0
25.0
24.0
19.0
27.0
20.5
23.0
8.5
17.0
27.5
21.5
12.0
24.0
15.0
26.5
22.0
0
22.5
20.0
22.0
15.5
12.5
16.0
20.0
6.0
10.0
25.0
0
10.0
15.0
11.0
17.0
23.0
0
20.0
19.5
25.0
10.5
13.5
16.0
20.0
8.0
10.0
25.0
24.5
8.0
14.5
9.0
15.0
18.0
0
20.0
19.0
23.5
13.0
12.0
18.0
19.5
8.0
10.0
23.5
25.0
9.0
13.0
8.5
22.0
25.0
0
20.0
22.0
28.0
14.2
12.5
19.0
23.5
7.0
14.0
26.0
22.5
6.0
18.0
11.0
23.5
19.0
0
20.0
22.0
28.0
13.5
14.0
19.0
19.0
6.0
15.0
22.5
19.0
5.0
18.5
13.0
21.0
30.0
0
17.0
20.0
23.0
11.5
12.5
14.5
17.5
5.0
14.0
29.0
18.0
6.0
13.0
11.0
19.0
30.0
0
22.0
23.1
23.7
15.4
16.1
19.8
20.5
7.2
14.7
24.1
21.4
9.3
18.1
12.4
23.5
23.8
0
LEGENDS to Table.7. The symbionts of Steinernema and Heterorhabditis species were grown for two
days on TSY agar plates and the sensitivity of the Erwinia isolates (Ea2, Ea60, Ea67, Ea9, Ea26, Ea56,
Ea78, Ea47, Ea17 and Ea28) for the produced antibiotics was measured by the size of the inhibition zone.
The data are representative, all experiments were repeated three times. The inhibition zones with the size
above or equal to 30 are labeled with blue, and with green if the inhibition zone was above or equal to 24.
E. coli strain K12 was used as control. The best antibiotics producing strains were among Photorhabdus
strains (IS5, Az36 and HP88, labeled by blue), but the effectiveness was specific to certain Erwinia isolates.
Xenorhabdus strains Ema1 and N2-4 produced antibiotics widely usable for most of the tested Erwinia
isolates (green). Generally, all EPB strains produced antibiotics against the Erwinia isolates.
The best results (antibiotic activity equivalent to 100-200 ppm streptomycin
sulfate) were produced by the Xenorhabdus strains EMA and N2-4, and the
Photorhabdus strain IS5 and Az36. Other EPB strains proved to be also very effective
against the Erwinia isolates. In other tests (data are not shown) antibiotics produced by 20
EPB strains were also effective against Agrobacterium, Clavibacter, Pseudomonas, and
Xanthomonas species (M. Hevesi, personal communication). The Erwinia isolates were
collected from different plants and geographical locations by M. Hevesi.
In larger scale EPB antibiotics were not tested so far against any plant diseases
directly. Our results clearly indicate that the application of the EPB antibiotics might be a
41
very important tool for fighting the fire blight disease and presents a novel tool for
fighting other bacterial parasites as well. In our laboratory the first attempt was made to
investigate the antibiotics production of several strains of Xenorhabdus and
Photorhabdus against Erwinia, and the data are promising for the development os
commercially useful strains, or even mixture of strains, that kills Erwinia.
42
3.2. FERMENTATIVE PRODUCTION, CHEMICAL
IDENTIFICATION AND APPLICATION OF EMA
ANTIBIOTICS
3.2.1. FERMENTATION OF THE EMA STRAIN AND TESTING THE
ANTIMICROBIAL ACTIVITY OF THE FERMENTATION SOUP
We have optimized the conditions for growing several Xenorhabdus strains in liquid
culture. We used both shake cultures, a small fermentor (G. Furgani, F. Főglein and A.
Fodor) and a bioreactor (A. Szentirmai). In the bioreactor we grew the bacteria using 10
liters of volume in a 35-liter fermentor in Debrecen (Hungary). The products of
fermentation (fermentation cell free supernatant) were directly tested for antibiotics
production in the laboratory and phytotrone. The fractions of the fermentation soup were
used to isolate and identify the biologically active compounds. The results of the tests are
summarized in Figure 9. The figure shows that the antimicrobial activity of EMA (X.
bicornutum) is extremely effective, the Erwinia test bactera were almost completely
killed. In the phytotrone test the EMA strain produced antibiotics were as effective as
kasumycin or streptomycin when the infection and the treatment were performed at the
same time. Antibiotics were more effective when the plants were treated 24-48 h before
the artificial infection. Even in diluted forms the fermentation soup proved to be very
effective against Erwinia, although with dilution the efficiency decreased, the examined
length of the infected area increased. The project was completed in cooperation with M.
Hevesi.
The results of the agar assays and the phytotrone tests are promising for fighting the
fire blight in the apple and pear plantations and raise questions about the chemistry and
biology of the active compounds.
43
Fig. 9. EMA antibiotics tests on E. amylovora on Petri dishes, liquid culture and
phytotrone.
Inhibition
zone
LEGENDS TO Fig.9. The antimicrobial activity of the fermentation soup of EMA (isolate 262). A:
Antimicrobial activity of Xenorhabdus sp. EMA strain on E. amylovora agar plate. B: Phytotrone test of
EMA fermentation soup. The biological activity of the EMA antibiotics can be compared to those of
streptomycin and kasumycin. With the treatment of kasumycin the length of the infected area is greatly
decreased. The fermentation soup (FS) of EMA is effective against Erwinia and the length of the infected
area is decreased in the case of application of the FS in diluted (1:1 and 1:10) and undiluted form. With
dilution of FS the infection increases. The experiment was comleted in duplicates twice in July (18. and
31.).
3.2.2. ISOLATION OF COMPOUNDS OF ANTIMICROBIAL
ACTIVITY FROM FERMENTOR LIQUID CULTURE OF
XENORHABDUS SP. EMA STRAIN
Recently elaborated techniques have been identified for isolating active
compounds from the fermentation soup of Xenorhabdus cultures. The experiments were
carried out in co-operation, within the framework of OTKA grant T 035010 (B.
Sztaricskai, Batta, M. Szentirmai, A. Fodor, G. Furgani, 2003). The strains were chosen
for the study based on their ablility to produce antibiotics on Patri dishes using B. subtilis
as indicator bacterium. The active compounds could be adsorbed to charcoal from the
supernatant of the liquid culture or from the suspension of broken cells and eluted by a
mixture of HCl and methanol (Fig. 10A.). The diluted compounds could be fractionated
by paper chromatography. The biological activity was tested on B. subtilis. Both the
supernatant and the cell fraction contained biologically active fractions. Comparing the
activities to each other, with 11 mg of the charcoal separated EMA (isolate 262)
supernatant fraction the inhibition zone was 12 mm, while with 200 mg of the active cell
fraction the inhibition zone was 16 mm. With linear regression for 200 mg supernatant
fraction the inhibition zone would be 218 mm, so it might be concluded that the
supernatant fraction is biologically more active.
44
Two different isolates of the EMA strain (262 and 282) were tested for their
biological activity against B. subtilis after the charcoal separation process. Table 8. shows
that with fermenting of the isolate 262 the antibiotic activity was better, since with 1100
g/0.1 ml active supernatant compound the inhibition zone average was 18.5 mm, while
the isolate 282 at 2000 g/0.1 ml active supernatant compound concentration the
inhibition zone average was 20.5 mm. With linear regression the isolate 282 would have
produced an 11 mm inhibition zone if the concentration of the active compounds was
1100 g/0.1 ml. It is also possible that the isolate 262 active compounds separated with
charcoal process are more stable than that of the isolate 282.
The stability of the water solution of the charcoal isolated EMA antibiotics was
also tested for two different isolates of the EMA strain (262 and 282).
Table 8. The stability of the water solution of the charcoal isolated EMA antibiotics
at 4 Co after 6 months storage.
Sample
EMA
Cc
Isolate 262
1100 g/0.1 ml
Isolate 282
2000 g/0.1 ml
Streptomycin
50 g/0.1 ml
B. subtilis
Inactivation zone (mm)
Right after isolation
6 month later
19
17
18
16
21
16
20
16
23
19
23
21
LEGENDS to Table 8. Comparison of the antimicrobial compounds of the same samples before and after
storage in refrigerator. The antibiotics activity of EMA compounds is stabile, with 6 months of refrigation
the inactivation zone caused fraction 1. (fermentation soup water soluble compounds) only decreased
slightly. The experiments were performed in duplicates.
1. The material obtained from cell free supernatant. Several ninhidrin-positive
fractions could be separated by chromatography, but only one fraction (Compound 1,
comprising about 3.2%) proved to be biologically active against B. subtilis.
2. The biologically active compound could be also obtained from the cell mass,
separated by chromatography to several ninhidrin-positive fractions, and one of them
(Compound 2, comprising about a 2.6 %) proved to be biologically active, but its activity
was significantly lower than that of the one originated from the cell-free supernatant. The
two biologically active compounds were chemically very different. No synergism could
be detected. The biological activity of Compound 1 was severely pH-dependent.
Compound I was further purified by Kieselgel 60 [CHCl3-MeOH-NH4OH (8:2:0.25)]
45
column chromatography. We finally found a ninhidrin-positive, UV-active compound of
low biological activity. By using 1H-, and13CNMR-, as well as mass spectrum (EI)
analysis it was identified as Triptamin.
3. The rest of the biologically active compounds of unknown chemical structure
were isolated by using an alcohol: hydrochloride elution. Both the pellet (Compound 2
about a 0.40 g) and the freeze-dried pellet (Compound 3, about 1.34 g) are active. The
data on biological activity of the compounds isolated by the charcoal and Dovex50
method on B. subtilis are presented in Fig. 10 A and 10 B, respectively. The purified
material obtained by the Dowex technique was hydrolyzed with 6 n HCl (105oC, 24 hrs).
The identification of the 9 peptide fragments is in progress.
Considering the presence of Triptamin amongst the active compounds we
supposed it was nematophin (Webster et al, Patent number: 5,569,668. USA Patent, 27
October, 1996). Therefore we have synthesized nematophin in the following way: racem
Triptamin was vacillated by 2-oxo-3-metil-penthane-carbonyl-acid. Briefly, the structure
of the isolated Triptamin was proved by 1H and 13C NMR: the presence of four aromatic
proton; three quaternary C; one vinyl-proton and two – CH2-- group. This compound was
treated with 2-oxo-3-metil-penthane-carbonyl-acid in the presence of sulphonylic acid
(SOCl2). In the presence of absolute pyridine 1 molecule of HCl was lost, and the product
of the synthesis was the nematophin (D, L 3-indolethyl-3’-methyl-2’-oxo-pentanamid).
The identity of the molecule was proven by E+MS, the molecular peak was 160 m/z.
46
Fig. 10A: Fractionation of the biologically active compounds adsorbed to charcoal.
LEGENDS to Fig 10A. Concentration of the biological active compounds by using charcoal adsorption
followed by fractionation. The supernatant of the fermentation soup was separated from the cells by
centrifugation. The supernatant was incubated with Cellit to remove the lipids from the supertanatnt and
filtrated. The filtrate was incubated with charcoal to bind the active compounds and the bound fraction was
separated from the inactive suparnatant with centrifugation. The charcoal-active compound was washed
with methanol and then the active compound was eulted from the charcoal with methanol/1N HCl (9:1).
After neutralization with ammonium hydroxide the active compound was freeze-dryed.With processing 500
ml cell-free supernatant 0.53 g active crude product could be gained. After centrifuging 500 ml
fermentation product, 63 g of cells could be processed. The incubation with 96 % ethanol/2N HCl (9:1) and
centrifugation removes almost all the cell debris and leaves the active compound in the liquid. With freezedrying the weight of the active compound was 0.49 g. The biological activity of the two fractions was
compared using B. subtilis as indicator bacteria and was compared to that of the Streptomycin.
47
Fig. 10B. Fractionation of the biologically active compounds adsorbed to DOWEX
50.
LEGENDS to Fig 10B. Concentration of the biological active compounds by using charcoal adsorption
followed by fractionation. 500 ml fermentation soup supernatant was incubated with pretreated resin and
after filtration the inactive filtrate was removed. The compound was bound to Dowex50 and gradually
eluted with 2-50 % methanol. The fractions were tested for biological activity. The active fractions were
incubated with 80 % methanol and freeze-dried. The pellet was further purified with the incubation of 80 %
methanol/1N HCl (99:1), neutralization and filtration and the filtrate was also biologically active. After
freeze-drying the same product kept the biological activity. Biologically the most active fraction was the
second with 24 mm inhibition zones compared to that of the first (12 mm) and third (18 mm) fractions.
48
Table 9. Comparison of the biological activities of the compounds with
antimicrobial activity on B. subtilis.
Compounds of
antimicrobial activity
Charcoal method
Fermentation soup
Compound 1
Compound 2
DOWEX50 method
Compound 1
(Triptamin)
Compound 2
Compound 3
Streptomycin
Amount
Origin
Isolated by
B. subtilis
Inactivation zone 
(mm)
0.1 ml
11
200
Supernatant
Supernatant
Cells
Charcoal
96% ethanol:
2n HCl (9:1)
15
12
16
0.5
Supernatant
80% methanol
12
0.5
0.5
50
Supernatant
Supernatant
-
80% methanol:
1n HCl (9:1)
-
24
18
20
Table 9. summarizes the biological acticities of the fractions from the
fermentation soup. The greatest biological activity belonged to Compound 2 isolated with
the DOWEX50 method. The DOWEX50 method is more efficient, than the charcoal
method. The final product (crude product) active ingredients give 12 mm inhibition zone
for the indicator bacteria when using 11 mg of the product, while with DOWEX50
method Compound 3 gives 18 mm inhibition zone, when using 0.5 mg of the product.
Since storing the freeze-dried product is the best way of keeping the activity for longer
periods, the final active (crude) products should be compared in the freeze-dried form.
The active compounds isolated with the DOWEX50 method is 36 times more efficient
against the indicator bacteria than the ones isolated with the charcoal method.
49
3.3. THE EMC STRAIN
The EMC strain was isolated from an S. rarum strain by E. Szállás (Eötvös University,
Dept. of Genetics, Budapest, Hungary). Unusual phenotypic characteristics make this
strain different from other Xenorhabdus strains: swarming behavior, color of the colony
and the unique crystal production.
3.3.1. THE PHENOTYPIC CHARACTERIZATION OF THE EMC
STRAIN
3.3.1.1. COLONY MORPHOLOGY AND SWARMING BEHAVIOR
On indicator plates (NBTA and MacConkey agar) EMC exhibits typical Xenorhabdus
phenotypes. On conventional media such as LB or TSY the EMC colony is brown, and
the cells are pigmented within the colony. This phenotypic character is unusual for
Xenorhabdus, in the EPB families only the Photorhabdus strains produce pigments. EMC
exhibits an extreme swarming phenotype as well. Swarming behaviour is different from
swimming, since swarming motility is very specific to solid agar surfaces and supposedly
the flagella that are produced and responsible for swarming are different from those of
swimming. The characteristic color of an EMC colony, and the swarming activity of the
cells are shown in Fig. 11.
Fig.11. Extreme swarming phenotype and color of EMC.
50
3.3.2. EXO-CRYSTAL PRODUCTION
This is the first study to describe the spectacular and unique exo-crystals that
appear in the medium and on the surface of the EMC colonies. The exo-crystals
especially appear on rich solid media (Fig. 12. and 13). In older agar media, the colored
exo-crystals appear on the surface of the colony as well (Fig. 12). Surprisingly, a different
Xenorhabdus (EMD) isolate from another S. rarum strain does not produce exo-crystals
and the color of the colony - similar to other Xenorhabdus strains - is blue on NBTA agar
media (Fig. 13). Re-isolating the crystal-producing EMC strain from the original S. rarum
was successful.
Fig. 12. The metallic-like crystals appear not only in the media but also on the
surface of older colonies in agar plates.
LEGEND TO Fig. 12 The Xenorhabdus sp. EMC strain produces exotic crystals of unknown function and
chemical character. In older cultures (especially in sugar-rich media) the crystals could be detected not only
in the agar but also on the surface of the colonies.
51
Fig. 13. The exo-crystal producing EMC and no exo-crystal producing EMD.
EMC
EMD
LEGEND TO Fig. 13. Exo-crystals produced by Xenorhabdus sp. EMC (left): not characteristic to other
isolates (EMD, right) isolated from S. rarum. On NBTA the colonies of EMC and EMD are blue, the EMC
crystals exhibit red color.
The exo-crystals can be found in older liquid cultures and even in minimum
media, if the carbon sources are adonitol or maltose (A. Máthé, G. Furgani, A. Fodor,
2003, unpublished). In the API tests the minimum media is supplemented with one single
carbon source. With API 50 tests EMC was shown to be able to metabolize trehalose and
N-acetyl-glucose-amine and under these conditions the EMC strain produced only small
amount of crystal. EMC metabolizes D-glucose, D-fructose, D-ribose, sorbitol, inositol,
glycerol, gluconate and 5-aceto-gluconate, but with the compounds as an exclusive
carbon sources it does not produce exo-crystals. EMC does not metabolize lactose, Lxylose and methyl-β-xylopyranoside. The crystals seen in the API 50 “pockets” are
demonstrated on Fig 14.
52
Fig. 14 A. Exo-crystal production of EMC in liquid minimum media.
growth
L-xylose
crystals
Adonitol
No growth
Methyl-βxylopyranoside
LEGENDS to Fig. 14 A. If the carbon source is L-xylose, EMC can grow, but does not produce exocrystals. If adonitol is used, both growth and exo-crystal production is detectable. In methyl-βxylopyranoside supplemented minimal medium, EMC can not grow, the carbon source can not be
metabolized.
Since the exo-crystals are completely insoluble in water or ethanol, it was difficult
to understand how the bacteria could secrete them into the medium. One plausible
explanation might be that a water-soluble monomer is released, which forms a polymer in
the media. The polymer is colored, making the colonies brown.
53
Fig. 14 B. Crystal production of EMC in liquid minimum media.
D-cellobiose
Maltose
Lactose
LEGENDS to Fig 14B. If the carbon source is D-cellobiose, EMC can grow, and produces some exocrystals. If maltose is used, both growth and exo-crystal production is detectable. In lactose supplemented
minimal medium, EMC can not grow, the carbon source can not be metabolized.
The exo-crystals isolated from the media could be observed by light microscopy
(Fig. 15 A, B and C) and scanning electron microscopy (Fig. 16). The crystals can be
identified by their specific shape and brownish red color, the later might make the
colonies appear brown. The exo-crystals can be dissolved by chloroform or DMSO. The
shape of the crystals depends on the C-source. The data of image (ThermoNoran) analysis
is also presented (Fig. 16). The exo-crystal does not contain nitrogen, but contains carbon,
54
oxygen and boran. We propose that the chemical nature of the exo-crystal is a large
molecular weight carbohydrate – boran complex.
Fig. 15A. Light microscopic picture of the isolated exo-crystal (125X magnification).
LEGEND to Fig 15 A. Magnification: 125X; Jenaval Light Microscope.
Fig. 15B. Light microscopic picture of the isolated crystal (250X magnification).
LEGEND to Fig 15 B. Magnification: 250X; Jenaval Light Microscope.
55
Fig. 15C. Light microscopic picture of the isolated crystal (1000X magnification).
LEGEND TO Fig. 15 C. The exo-crystal in 1,000X magnification (Jenaval light microscope).
Fig. 16. EM picture and image analysis of the exo-crystal.
LEGEND TO Fig 16. EM picture (right) and its (ThermoNoran) image analysis (left). The exo-crystal does
not contain N, but contains C, O and B. Supposedly, the chemical nature of the exo-crystal is a large
molecular weight carbohydrate – boran complex.
56
3.3.3. ANTIBIOTICS PRODUCTION OF EMC
EMC is one of the best antibiotics producing strains tested in our laboratory. The
compounds of the fermentation soup exert cytotoxic activity reproducibly on both E. coli
and E. amylovora (Fig. 17). The effect of the antibiotics is cytotoxic and not cytostatic.
The antimicrobial compounds of Xenorhabdus from fermentative productions effectively
reduced the number of Erwinia cells in liquid media, and within 60 minutes no living
Erwinia cell could be counted.
Fig. 17. The cytotoxic activity of EMC and EMA.
LEGEND to Fig. 17. Comparison of the cytotoxic (bactericide) activities of the 1:1 dilutions of the
fermentation soup of EMC (blue) and EMA (pink) against E. amylovora. The EMA strain is an isoltate
living in symbiosis with S. rarum as well. The antimicrobial compounds of both strains effectively reduced
the number of Erwinia cells in liquid media and within 60 minutes no living Erwinia cell could be counted.
Abscissa: the time in minutes; ordinate: the number of E. amylovora cells. (The exponentials of 100, 102,
104, 106, 108, 1010 are labelled as 1,00+00, 1,00+02, 1,00+04, 1,00+06, 1,00+08 and 1,00+10, respectively).
EMC antibiotics also inhibit the growth of Phytophtora and Trichoderma species
(Fig. 18). The tests were carried out in cooperation with Prof. Tibor Érsek (Institute of
Plant Protection, Hungarian Academy of Sciences). The results indicate that the isolated
antibiotics from EMC fermentation soup (DOWEX50 method) inhibit the colony forming
of Phytophtora sp. With the application of 12.5 ppm antibiotics some growth still can be
detected, but the concentration of 25 ppm inhibits the growth of Phytophtora completely.
57
Fig. 18. Effect of EMC antibiotics on the colony formation of Phytophtora sp.
LEGEND to Fig 18. The antibiotics isolated from EMC fermentation soup (DOWEX50 method) inhibit the
colony forming of Phytophtora sp. The concentrations are given in ppm.
3.3.4. CONCLUSIONS
EMC is one of the most interesting strains of our stock collection. The swarming
behavior as well as the color of the colony is unusual for Xenorhabdus. The crystal
production is unique. The biological role of this carbohydrate – polymer metal complex
(Furgani et al., in preparation) is unknown. The data from API tests confirm that the
crystals are synthesized from sugars, which might be metabolized by EMC when the
carbon sources are depleted. The genes that are responsible for the production of exocrystals are unknown.
Screening 7,000 Tn10 transposon-induced EMC colonies we have generated 10
mutants, which do not produce exocrystals. None of the (10) mutants lacked the antibiotic
production. Thus the genes that regulates the production of exo-crystals and the
antimicrobial compounds may be independently regulated. The genomic regions where
the transposon inserted are yet to be cloned and sequenced.
58
3.4. GNOTOBIOLOGY
3.4.1. RESULTS IN GNOTOBIOLOGY
The Steinernema strains are capable of reproducing and infecting/killing
caterpillars while carrying EPB symbionts of other Steinernema strains. In our laboratory
Katalin Lengyel and Erzsébet Böszörményi (PhD students) performed gnotobiological
analysis of Heterorhabditis /Photorhabdus complexes as well. The complete
gnotobiological analysis of S. carpocapsae and S. feltiae isolates - with their permission is presented in Table 13-16.
Table 10. Summary of the gnotobiological tests of Heterorhabditis / Photorhabdus
complexes (with kind permission of E. Böszörményi and K. Lengyel).
Bacteria
EPB
Hepialiu
s
H4
OHI
Jun
HSH2
K122
HIT
Brecon
HP88
A1
Az29
IS5
Wx4
Wx6
Wx11
Nematodes
EPN
H. marelata
natural host
H. mareliata
Hepialius
+++
H4
+
H. megidis
OHI
+
Jun
+
K122
-
H. downesi
HIT
+++
H. bacteriophora
A1
+++
H. megidis
unknown
unknown
unknown
H.downesi
unknown
H.
bacteriophora
unknown
unknown
unknown
H.indica
unknown
unknown
unknown
+*
+++
+++
+++
+++
+++
+++
+++
+
+++
+
+
+++
+
+
+++
+
+
+++
+
+++
+++
+++
+++
nt
+++
nt
+++
+
+
+++
+++
+++
+
++
++
++
++
++
++
+++
+++
+++
+++
+++
+++
+++
+
+++
+++
+++
+++
+
+
+
nt
nt
+++
+
nt
nt
nt
nt
nt
+++
nt
+
+
+
+++
+++
+++
+++
+++
+++
++
+++
+++
LEGEND to Table 10. -: not growing at all; +: J1 develops to fertile adult; ++: the new EPB symbiont is
retained and was reisolated after several generations; nt: not tested; +*: J1 could not be recovered, but the IJs
developed into fertile adults.
Table 10. summarizes the gnotobiological analysis of the Heterorhabditis /
Photorhabdus complexes. The tests show that H. marelata and H. bacteriophora can
develop into fertile adults and IJs on almost all Photorhabus strains tested, but the
nematode strains belonging H. megidis and H. downesi are not as successful, although
three and two strains were tested, respectively.
59
Table 11. shows how the strains of S. carpocapsae (Mexicana and T1) and S.
feltiae (SF22 and IS6) can develop on several strains of X bovienii (S. feltiae symbionts)
and on the closely related X. affine, X. bibionis and X. kraussei. Most of the nematodes
from both species developed into fertile adults, but the non-natural symbion could not be
reisolated after several generations. The most successful nematode was the strain IS6,
which could develop into fertile adults on almost all non-natural symbiont and the
bacteria could be reisolated after several generations of growth.
Table 11. Growth of S. carpocapsae and S. feltiae strains on the symbionts of S.
feltiae and closely related species.
Steinernema carpocapsae
Mexicana
T1
S. feltiae symbionts
X. bovienii Nyíregyháza
X. bovienii DSMZ4766
X. bovienii 1003
X. bovienii 1S6
X. bovienii Umea
X. bovieni Finnish
X. bovieni Norwegian
X. bovieni Mongolian
Closely related species
X. affine
X. bibionis
X. kraussei
Steinernema feltiae
SF22
IS6
+
+
+
+
+
+
+
+
+
+++
++
+
++
++
+
++
+
+
+++
++
+
+
+
++
+++
+++
+++
+++
+++
+++
-
+
+
+
+
+
+
+
++
+++
+
+++
LEGENDS to Table 11. Practically all S. feltiae strains (but not the S. carpocapsae strains) can
grow, propagate and produce infective dauer juveniles on the other Steinernema symbiont. Thy can
also grow on the symbiont of S. kraussei and S. affine. -: not growing at all; +: J1 develops to
fertile adult; ++: the new EPB symbiont is retained and was reisolated after several generations;
+++: even more nematodes were produced, than at ++.
The data in Table 12. records the same nematode strains as Table 11., but with
the isolates of X. nematophila symbionts. The symbiotic relationship of the same
nematodes to X. nemtophila looks less efficient, less strains support the growth of S.
feltiae and even the strains of S. carpocapsea were not retaining the bacterial strains
form different isolates of X. nematophila. Interestingly, although ATCC19061 primary
strain supported the growth of S. feltiea Mexicana strain, N2-4 secondary and X.
riobrave secondary strains supported the growth while their primary pair did not.
Table 13. shows how the nematode strains of S. carpocapsea and S. feltiae can
develop and reproduce on the symbionts of Steinernema species with short dauer
phenotypes. The results clearly indicate that the nematodes only sporadically develop
60
into fertile adults retaining the bacteria. S. feltiae Mexicana strain is not able to retain
these bacteria at all after several generation. Table 14. contains the data of the nematodes
developing on symbiotic bacteria with unknown origin. The bacteria seem to support the
nematode development better than the previously described species (Table 13.), and
most of them provide good conditions for the S. feltiae Mexicana strain.
Table 12. Growth of S. carpocapsae and S. feltiae strains on the symbionts of S.
carpocapsae and related species.
Steinernema carpocapsae
Mexicana
T1
S. carpocapsae symbionts
X.nematophila DSMZ3370
X.nematophila T1
X.nematophila ATTC19061/2
X.nematophila ATTC19061/1
X. nematophila ANT 1
X.nematophila AN6 2
X.nematophila AN6 1
X.nematophila 703
X.nematophila N2-4 SS (2)
X. nematophila N2-4 SA (2)
X. nematophila N2-4P (1)
S. riobrave symbionts
X. riobravis/1
X. riobrave/2
S. kushidai symbionts
X.kushidai
Steinernema feltiae
SF 22
Mexicana
+
+
+
+
+
+
+
+
++
+++
+
+
++
+
++
+
+
+
+++
+
+
+
-
+++
+
-
+
+
+++
++
+
+
-
+++
-
-
LEGENDS to table 12. Almost all S. carpocapsae strains (but not the
propagate and produce infective dauer juveniles on other symbionts.
Xenorhabdus symbiont of S. riobrave but not that of and S. kushidai.
develops to fertile adult; ++: the new EPB symbiont is retained and
generations; +++: even more nematodes were produced, than at ++.
S. feltiae strains) can grow,
Thy can also grow on the
-: not growing at all; +: J1
was reisolated after several
61
Table 13. Growth of S. carpocapsae and S. feltiae strains on Xenorhabdus
symbionts of other Steinernema species of short dauer phenotypes.
Steinernema
carpocapsae
Mexicana
T1
Steinernema feltiae
SF 22
Mexicana
Symbionts of Steinernema species with short dauer phenotypes
S. rarum symbionts
X. rara
S. scapterisci symbionts
X. scapterisci/1
X. scapterisci/2
S. bicornutum symbionts
X. bicornutom
S. serratum symbionts
X. serratum
S. anomali symbionts
X. anomaly Azores
X. anomali Lucskai
S. intermedium symbionts
X. intermedia
X. intermedia Biosys
X. bedingii DSMZ 4764
S. glaseri symbionts
X. poinarii DSMZ4768
KMD15
S. cubanum symbionts
X. cubana
+
+
+
-
-
+
-
+
-
-
-
+
+
+
+
++
+
-
-
++
-
++
++
+
-
+
+
++
+/++
-
+/+/-
-
+
+
+
+/+/-
+
+
+
++
+/-
-
LEGENDS to Table 13. -: not growing at all; +/-: not all J1 develop into fertile adult, +: J1 develops
to fertile adult; ++: the new EPB symbiont is retained and was reisolated after several generations.
Table 14. Growth of S. carpocapsae and S. feltiae strains on Xenorhabdus
symbionts of taxonomically undetermined Steinernema species.
Steinernema
carpocapsae
Mexicana
T1
Steinernema feltiae
SF 22
Mexicana
Symbionts of unknown Steinernema Isolates
Z06
Z31/1
3905
S47A
H5
FA019 (from R. Gaugler)
FA013
HW7
HK2001
HK98
KMD44
-
+/-
++
++
+
+
+
+
+
-
++
+/++
+
++
+/-
++
+
+
+
+
++
+++
++
+
+++
+
+
+/+
+++
+
++
++
-
+++
+++
-
LEGENDS to table 15. -: not growing at all; +/-: not all J1 develop into fertile adult, +: all J1
develop into fertile adult; ++: the new EPB symbiont is retained and was reisolated after several
generations; +++: even more nematodes were produced, than at ++.
62
3.4.2. CONCLUSIONS
These data may help to clarify some questions concerning the conception of cospeciation, like what bacterial symbiont might be a better symbiotic partner for a
nematode strain, and what might happen in nature when two different symbiotic bacteria
mix within the caterpillar. It is known that the symbiotic partners of Steinernema and
Heterohabditis inhibit each others growth, and so the nematodes can not develop if mixed
in the carcass. The gnotobiological analysis of the Steinernema and Heterorhabditis strain
shows that the Steinernema species are more strain specific for their symbionts than the
Hetrorhabditis species. The tests prove that the growth of almost all tested
Heterorhabditis strains is supported by Photorhabdus strains from different species. The
results showed less readiness of the S. carpocapsea and S. feltiae species to be in
symbiotic relationship with the others bacterial symbiont, and the symbionts of the
Mongolian X. bibionis strains cannot be utilized at all by any S. feltiae strains. It can be
concluded that neither S. feltiae nor S. carpocapsae could grow and propagate on the
symbiont of other Steinernema species of short dauer phenotype.
The gnotobiological analysis might be able to help in the future to understand the
molecular bases of the specificity of the symbiotic relations between the EPB and EPN
strains. Also it is possible for specific pest controls, better symbiotic relations could be
generated as the research moves forward.
63
3.5. FIRST ATTEMPTS TO LABORATORY FERMENTATION
OF A NEW STEINERNEMA ISOLATE
This chapter is based on the published version for COST 850 (edited by
Prof. R.-U. Ehlers).
SUMMARY
The bacterium symbionts of the Morocco, SP2, Italian and Slovak Steinernema strains of
long dauer phenotype were isolated and phenotypically characterized. Each proved to be
Xenorhabdus according to cell and colony morphology, dye uptake, catalase negativity,
as well as on the basis of their lecitinase, lipase and proteolytic activities. In
gnotobiological tests the nematodes from which they had been isolated grew on it and
produced fertile progeny in agar plates as well as in liquid culture in our laboratory
fermentor. Each bacterium strains produced antibiotics of medium level and proved to be
ampicillin resistant. The Morocco strain, however, expressed a low degree of ampicillin
resistance. The sequence analysis and taxonomic identification will be accomplished
soon.
3.5.1. INTRODUCTION
Steinernema species of long dauer phenotypes are a potential tool for controlling scarab
grubs. In Hungary the Maybeetle (Melolontha melolontha) is a harmful agricultural pest
and entomopathogenic nematode species to control it is needed. We are to produce the
effective nematode in liquid culture. As a first step, the bacterial symbionts should be
isolated, identified and characterized. Herein we are to publish the first data on the
bacterial symbionts of Steinernema sp. Morocco, Steinernema sp. SP2, Steinernema sp.
Italy, and Steinernema sp. Slovak strains.
5.5.2. MATERIALS AND METHODS
Nematode strains: All strains were kindly provided by the participants of the COST 850
Meeting and Workshop in Debrecen, Hungary. 2002. Namely, the Italian Steinernema
strain (“Italy”) was kindly provided by Prof. Trigiani Oreste, (Bari, Italy); the Spanish
64
(SP2) one by Fernando Garcia del Pinto (Barcelona, Spain), the Morocco strain was
kindly provided by Ralf-Udo Ehlers (Raisdorf, Germany).
3.5.3. ISOLATION, CHARACTERIZATION OF EPB SYMBIONTS
OF SOME NEW STEINERNEMA STRAINS OF LONG DAUER
PHENOTYPE
Infective dauer juveniles (IJ) freshly harvested from Galleria white traps were
collected and washed by centrifugation with sterilized tap water three times. Some were
then put in to a drop of physiological saline (M9) solution in a sterile Petri plates and then
transferred to 5% chlorox. After 2 minutes the animals were transferred to a series of
sterilized M9 drops and finally fragmented by using a sterile platinum wire (Fig 4).
Different steps of isolation of entomopathogenic bacterial (EPB) symbionts from
infective dauer juveniles (IJ) of entomopathogenic nematodes (EPN). The washing,
surface sterilizing, and the removal of the chlorox were made in more steps by direct
transfer of the worms from one drop to another. The indicator plates were NBTA, on
which the bacterium wanted form black colonies and the agar plate around the colonies
was clear.
The drop of M9 was diluted and plated on NBTA indicator plate. Blue colonies
were picked and retested again. From the positive colony a stock culture was established
and frozen. Samples from the stock cultures were then tested for Xenorhabdus primary
phenotypes.
3.5.1.1. PHENOTYPIC CHARACTERIZATION OF THE FRESHLY
ISOLATED BACTERIAL SYMBIONTS
Cell morphologies were examined under light microscopy. Colony morphology
and dye uptake: LB, NBTA, LBTA and MacConkey agar plates were used as described
by Boemare et al (1996).
3.5.1.2. PHOSPHOLIPASE (LECITINASE TEST ON YOLK AGAR)
An egg was surface disinfected with 70% ethanol. Under the laminar flow the yolk
was collected aseptically and poured into an equal volume of (0. % NaCl) sterile saline
solution. After homogenization, 10% vol/vol of this yolk solution was added to nutrient
65
agar at 45 oC. An opaque halo around the inoculation line meant a positive lecitinase
reaction.
3.5.1.3. PROTEOLYSIS ON GELATIN AGAR (FRAZIERS’S METHOD)
12 g/liter of gelatin were added to LB agar powder and the plates were streaked. After the
prerequisite incubation, plates were flooded with a solution of 12 g HgCl2; 16 ml of 12N
HCl and 80 ml distilled water. A clear halo due to the proteolysis was evidenced.
3.5.1.4. ANTIBIOTICS PRODUCTION
5 µl of an overnight culture of the Xenorhabdus examined were pipetted into the center of
an LB agar plate. After a 2-5 days incubation at 30 oC, the plate was overlaid with 5ml
soft agar (0.6 g agar/100 ml LB solution) including 1 ml suspension of B. subtilis spores
(containing about 108 spores). The plates were incubated overnight at 37 oC. The diameter
of the clear (inactivation) zone, as well as that of the bacterium colony were determined
3.5.1.6. AMPICILLIN SENSITIVITY
In general, LB plates containing 0, 25, 50, 75, 100, 125, and (150 mg/ml) ampicillin
sodium (Sigma) were made. In case of the Morocco strain the ampicillin doses in the LB
plates were as follows: 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.563 and 0 ug/ml. An
overnight culture (the number of cells per ml was about of about 10-8order of magnitude)
was serially diluted to 1:10, 1:102, 1:103, 1:104, 1:105, 1:106, a 0, :107 ratio. 0.1 ml of each
dilution was plated into each ampicillin plates. The colonies were then counted and
presented as a function of the ampicillin dose. The MIC values were also determined as
follows: 5 ml LB liquid cultures containing 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.563 and
0 ug ampicillin sodium /ml respectively were inoculated with 0.1 ml of overnight
bacterium culture and incubated overnight at 30 oC. The OD values were determined at
600 nm and presented as a function of the ampicillin dose. The latter was deduced from
the previous one.
3.5.1.7. GNOTOBIOLOGICAL TESTS
One colony of the stock culture was transferred into 5 ml of LB media. The overnight
culture was seeded on one half of a large (of 9 cm diameter) TSY agar plate (in glass
66
Petri plate). Surface sterilized IJ larvae were transferred to the bacterium-free part of the
agar plate. The animals were moving to the bacteria. The gnotobiological test was
considered positive if the majority of the dauers molted into L4 and grew to fertile adults
producing viable progeny.
3.5.2. RESULTS AND DISCUSSION
The colonies of the new isolates can be seen and compared on LB, NBTA, egg yolk and
tween 20 agar plates, on( Fig 19-22), respectively.
Fig. 19: The new Xenorhabdus isolates (Morocco, SP2, Italy, Slovak) and control (X.
nematophila (AN6/1), X. poinarii (DSM 4766) strains on Luria Broth (LB) agar
plates
LEGEND to Fig 19. Phenotypes of the isolates on LB agar plates. Upper row, left to right: Xenorhabdus
symbiont of the Spanish SP2, Morocco and Italian strains. Lower row: Slovak, DSM 4466 (reference strain)
and X. nematophila AN6/1 reference strain.
67
Fig. 20. Lecitinase activities of my new isolates. Control: DSM
4766 (X. poinarii).
LEGEND to Fig.20. Comparison of the lecitinase activities of the new isolates
on NBTA agar plates. The new Xenorhabdus isolates: Morocco, SP2, Italy,
Slovak and control strain, X. poinarii (DSM 4766) can be detected as cleaning
up the agar around the colonies.
Fig. 21. Lipase activities of my new Xenorhabdus isolates.
LEGEND TO Fig 21. Comparison of the exo-lipase activities of the new isolates on
tween 20 agar plates. The new Xenorhabdus isolates: Morocco, SP2, Italy, and
Slovak. Control strains: X. nematophila (AN6/1) and X. poinarii (DSM 4766). The
lipase decomposes the triglycerides into glycerol and fatty acids, and the Ca-salt of
the free fatty acid is precipitated around the bacterium colonies.
68
On the basis of their cell (not shown) and colony morphology (Fig. 19.), catalase
negativity, dye uptake (not shown), exo lecitinase (Fig. 20), and exo-lipase (Fig. 21)
activities all the four bacterium isolates belong to the Xenorhabdus genus, but definitely
different from both the X. nematophilus AN6/1, and X. poinarii reference (control)
strains. All of the produce compounds of antimicrobial activity (Fig. 22).
Fig. 22. Antibiotic activities of the new Xenorhabdus isolates.
Ampicillin resistance: All strains but the Morocco strain proved to be resistant to
ampicillin. A low fraction of the cells seems to keep their ampicillin resistance, and so the
Morocco strain can grow at 200 µg/ml ampicillin can survive and grow.
Gnotobiology: The infective dauer juveniles recover, grow and propagate on TSY agar
plates, where they were released on their own symbionts. The nematodes, as expected,
can only grow poorly on each other’s bacterial symbionts.
3.5.3. LABORATORY FERMENTATION OF THE MOROCCO
STRAIN
A new 3.7-liter laboratory fermentor was developed in our laboratory (Fig. 23.). All the
Morocco strains can propagate well on their symbiont in the laboratory liquid fermentor.
The fermenting conditions were tested with the new Steinernema strain isolated with its
own symbiont as well. The results indicate that the Morocco strain has the best
fermentation qualities (yield and surival properties of the nematodes) of the strains
studied so far. The Morocco strain grown in the fermenter did not prove to be very toxic
towards either Manduca sexta orally (J. Marokházi, personal communication) or
69
Melolontha grubs when injected. When injected, the Morocco strain killed Galleria
mellonella efficiently (S. Pekár and J. Marokházi, personal communication).
Fig. 23: The laboratory fermentation unit.
LEGEND to Fig. 24. Laboratory fermentor constructed by Imre Kurucz and Csaba Sisak. 2 X TSY (liquid).
medium was used in the fermentor The oxygen was provided by (moderate) stirring and simultaneous aeration.
The inoculum for the monoxenic bacteria population was grown on TSY in agar.
3.5.4. CONCLUSIONS
The best source of the natural EPB symbionts of an EPN strain is the infective dauer
juvenile. Xenorhabdus strains can easily be identified by using the conventional indicator
plates. The antibiotics production is an advantage but not a precondition of successful
liquid cultures of EPNs. The ampicillin resistance of the Xenorhabdus and Photorhabdus
strains is a general feature, but there is significant variation between strains. The unusual
behavior of the Morocco strain needs further analysis. The fermentation qualities of a
given strain influenced by several factors, but the most important are to have the proper
EPB symbionts. The Morocco strain was the easiest to propagate in a 3.7 liter laboratory
fermenter.
Acknowledgements: This research was supported by a Hungarian National Fundamental
research Program (OTKA) T035010 Grant. We would like to express our thanks for the
nematode strains to the participants of the COST 850 Meeting and Workshop in
Debrecen, Hungary. 2002. For Italian Steinernema strain (“Italy”) to Prof. Trigiani
Oreste, (Bari, Italy); for the Spanish (SP2) one to Dr. Fernando Garcia del Pinto
70
(Barcelona, Spain); and Morocco strain to Dr. Ralf-Udo Ehlers (Raisdorf, Germany). We
acknowledge the professional help of Prof. A. Szentirmai in the MIC determination, to
Dr. Csaba Sisak for his help in operating the fermentor, Mrs. Simon Sára for taking care
of the Steinernema strains. The research was also supported by the Research and
Extension Centre for Fruit Growing, headed by Dr. Ferenc Inántsy. Thanks to Judit
Marokházi and Szilvia Pekár for making the unpublished data available for us.
71
3.6. MOLECULAR, GENETIC AND GNOTOBIOLOGICAL
IDENTIFICATION OF NEW STEINERNEMA ISOLATES
In this chapter is based on the published version for COST 850 (edited by
Prof. R.-U. Ehlers).
SUMMARY
Thirteen Steinernema strains of long dauer phenotypes were compared on the
basis of their PCR – RFLP profiles based on the GeneGel Excel kit analysis. They were
also grown on some Xenorhabdus symbionts isolated from S. glaseri, S. arenarium, as
well as from unidentified Steinernema isolates, such as SP2, Slovak and “Italy”. They
were also intercrossed to study their species differences. We found that one group of
strains belong to the S. glaseri group, characterized by very similar RFLP profiles and
unlimited cross fertility. They grew on each other is symbionts. Another group
comprised the S. arenarium group of rather similar RFLP patterns and gnotobiological
similarity, although their cross fertility has shown limitations. Two strains (Morocco,
Palestine) definitely belong to two, different species. The taxonomic positions of the SP2
and the Italy strains are still ambiguous.
3.6.1. INTRODUCTION
Thirteen Steinernema strains of long dauer phenotypes, potential tools for grub
control were studied for their identification within the framework of a COST 850
Workshop. We have studied their growth and development on each others’ bacterial
symbionts (gnotobiological analysis), cross fertility and PCR - RFLP profiles obtained
by GeneGel excel kit (Blaxter, 1998).
PhastSystem PCR RFLP is an excellent method for identifying entomopathogenic
nematodes (Filipjev, 1935). On the other hand, the classic species definition is that those
individuals which are capable of producing fertile progeny do belong to the same
species. We intended to determine which steinernematids of long dauer phenotype
belong to the same species and intended to compare their PCR-RFLP patterns by using
the GeneGel Excel kit.
72
Considering the species specific symbiosis of Steinernema and Xenorhabdus
(bacterium) strains, and as a consequence of it. entomopathogenic nematodes can grow
and propagate only on their own symbionts, a precondition for the successful crosses is
to find bacteria on which both parental strain could somehow grow and propagate. We
have isolated the Xenorhabdus symbionts of several Steinernema strains and tested the
others for growth and reproduction. Finally, we found bacterium for each intended
crosses.
3.6.2. MATERIALS AND METHODS
Bacterium strains: Xenorhabdus bacterium have been isolated straight from the
surface sterilized infective dauer juveniles as has been described by Lucskai. Bacteria
were tested on indicator plates, for their catalase negativity and for other taxonomic
characters. We have isolated Xenorhabdus from the nematode strains KMD15, S.
anomali, SP2, Slovak, Morocco and Italy and used also the DSM reference strain of X.
poinarii (natural symbionts of S. glaseri).
Nematode strains: In our stock collection we have two Steinernema arenarium
(anomali) strains: one from N. Simoes (Portugal Azores), one from Ramon Georgis
(Biosys, USA). S. glaseri strains NC1 (from Michael G. Klein, USDA-Ohio, USA),
NC513, NCLU (from Pierre Abad’s collection via Attila Lucskai) and AZ26 (from N.
Simoes, Portugal Azores). The Italian Steinernema strain (“Italy”) was kindly provided
by Prof. Trigiani Oreste, (Bari, Italy); the Spanish (SP2) one from Fernando Garcia del
Pinto (Barcelona, Spain), the “Polish” strain from Marek Tomalak, Poznan, Poland), the
“Slovak” strain from Zdenek Mracek, Ceske Budejovice, Check Republic, The
“Russian” strain from Sergei Spiridonov Moscow, Russia, the “Gel” (probably the
original Russian isolate) from Z. Mracek; the Morocco strain was kindly provided by
Ralf-Udo Ehlers while the “Palestine” one come from Dr. Nair Iraki).
For in vitro culturing nematodes, large glass Petri dishes of (9 cms) diameter were
filled with TSY agar medium2 and one half was seeded with the proper bacterium. A
sterile Sartorius filter of 0.2 µm pore size was put on the bacterium free part of the TSY
agar. Surface sterilized IJs were then transferred to filter. Couple of hours later, when the
nematodes left the filter and moved toward the bacterial lawn the filter was removed. If
the nematodes are in conform to the bacteria, they molt to L4 and grow to fertile adults
and propagate. Otherwise they may or may not recover and even if they recover they die
73
or become unfertile. For intercross experiments, small plastic Petri dishes of 3.5 cm
diameter were used with TSY agar. A drop of an overnight bacterium culture was
dropped in the center of the plates, one virgin (L4) female from the maternal strain, and
1-9 young males from the paternal strain were transferred to the plate by a platinum wire.
The Petri plates were closed by using parafilm and put in a “humidity chamber” (e.g.
glass Petri plates with water). We observed that the males started to court immediately
whoever the females were and usually mated successfully. In the control groups, where
the males and the females originated from the same strain, the females became gravid
carrying embryos, the majority of which developed to larvae inside their mother (entokia
matricida) (Akhurst, 1987, Al-Samarrai et al., 2000). In case of absence of cross fertility
the oocytes remained unfertile and no progeny developed.
The DNA isolation from single nematodes as well as the PCR amplification of the
ITS1 – 5.8S rDNA – ITS2 region of the 18S- 5.8S – 28S rDNA operon was carried out
by using the protocol of Triga et al (2000). The molecular analysis was conducted
according to the manufacturer’s original protocol, without any modification. Based on
our previous studies3, we used four enzymes (AluI, MseI, TaqI and RsaI) to cut the DNA
and compare the restriction patterns.
3.6.3. RESULTS AND DISCUSSION
Gnotobiology: Morocco strain could grow on almost all Xenorhabdus strains, but the
growth was the most efficient on its own symbiont. For crosses only the KMD15 and the
Anomali bacterium plates could be used. The data for gnotobiology are summarized in
Table 18.
Crossbreeding: On the basis of the classic species definition, EPN strains NC1, Kmd15,
NC513, NCLU, and AZ 26 do belong to the same (S. glaseri) species, while strains
Anomali, Gel, and Russian comprise another species (S. arenarium). On the basis of
cross fertility, neither SP2, nor Italy, nor Polish, nor Slovak could be sorted into either of
these two species, in spite of some the similarities between the molecular markers (see
later). Morocco does definitely belong to another (unknown) species.
74
Table 16. Growth and propagation of Steinernema strains on each others’ symbionts.
Xenorhabdus: strains:
Steinernema isolates:
S. glaseri NC1
NCLU
NC513
Kmd15
AZ26
S. anomali
Polish
Slovak
Gel
Russian
SP2
Italy
Morocco
X. poinarii
Palestine
X. sp.
X. sp.
X. sp.
X. sp.
X. sp
KMD11
Anomali
SP2
Italy
Morocco
+++
+++
+++
+++
+++
-
+++
+++
+++
+++
+++
+
+
+
+
+
+
+
++
+++
+++
++
++
+++
++
++
++
++
++
++
++
++
+++
++
++
+
+
+
+
+
+
+++
++
+++
-
-
-
-
Not
tested
-
-
LEGENDS to Table 16. Data show that hardly any growth of the majority of the strains could be observed on any
artificial symbionts. Of the S. glaseri symbionts only a selected line of Kmd15 bacteria could serve as food for
some other strains, while the symbionts of S. anomali (Azores) could moderately be used to grow one or two
generations of other steinernematids. Morocco strain could not be grown on any other bacteria, therefore we could
not test them in intercross studies. The molecular identification of the bacterial symbionts is in progress.
Table 17. The results of cross breeding tests.
Maternal
strains:
NC1
NCLU
NC513
AZ26
Kmd15
Ano-mali
Gel
Russian
S. g. NC1
+
+
+
+
+
-
-
-
NCLU
+
+
+
+
+
-
-
NC513
+
+
+
+
+
-
AZ26
+
+
+
+
+
Kmd15
+
+
+
+
S. anomali
-
-
-
Polish
-
-
Gel
-
Russian
Paternal strains:
Sp2
Italy
Polish
Morocco
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
+
+
+
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-
-
+
+
+
-
-
-
-
-
-
-
-
-
+
+
+
-
-
-
-
SP2
-
-
-
-
-
-
-
-
+
-
-
-
Italy
-
-
-
-
-
-
-
-
-
+
-
-
Morocco
-
-
-
-
-
-
-
-
-
-
-
+
-
LEGENDS to Table 17. The data confirm that EPN strains can be grown and propagate properly on their own natural
symbiont or symbionts of other strains of the same species. NCLU, NC513, AZ26 as well as NC I all belong to the same
species, S. glaseri, therefore they can utilize each other’s symbiont. In the EPN strains belonging to the S. arenarium
species all but the Polish strain can utilize each other’s symbionts, but those of S. glaserii. SP2, Italy, and especially
Morocco strain definitely belong to a different species.
75
Molecular analysis: Results of the PCR-RFLP analysis are demonstrated on
figures 25, 26 and 27, respectively.
Fig 25. GenePhore PCR-RFLP Analysis 1.
GenePhore PCR RFLP
Analysis
M
M 7 8 9 10 11 12
M
• M = Molecular marker
(Azores) / Rsa I
• 7 S. anomali
(Fragment sizes 120, 130, 190)
• 8 Kmd15
/ Rsa I
(Fragment sizes 130, 210)
•9
S. anomali
(Azores) / /Mse I
( Fragment sizes 120, 250)
• 10
/
Kmd15 / Mse I
(Fragment sizes
120, 300)
• 11 S. anomaly
( Fragment sizes
• 12 Kmd15
(Azores) /Taq I
100, 110,125,210, 260)
/ Taq I
(Fragment sizes
100, 110, 125, 150, 230)
LEGEND to Fig 25. RsaI, MseI and TaqI PCR-RFLP restriction profiles of the ITS1 – 5.8S – ITS2 regions of the
18S – 5.8.S – 28S operons of entomopathogenic Steinernema strains of long dauer phenotypes belonging to
Steinernema arenarium (S. anomali from Nelson Simoes’s collection in Portugal Azores) and Steinernema
glaseri (KMD 15). The patterns are individual and reproducible.
The RsaI pattern of S. anomali (7) consists of a 120, 130 and 190 Dalton
fragments and is different from that of KMD15 (8), which has a profile of 130 and 210
Dalton fragments. Similarly, the two strains also differ in their MseI and TaqI profiles,
respectively.
Before we started the intercross experiments, based on the RFLP analysis Kmd15
was believed to be belonging to a new species, S. ohioensis (Lucskai and Klein,
unpublished) and S. anomali was believed to exist as a separate species. Kmd15,
however, can be crossed with all the other four S. glaseri strains (NCI, NCLU, NC 513,
AZ26), and - as we could see on Fig. 26. and 27 - their GenPhore PCR-RFLP restriction
profiles are also similar. As for our S. anomali strains they could cross freely with each
other as well as with the Gel and “Russian” strains. The intercross experiments show that
76
they belong to the same species, (S. arenarium). But the restriction profiles of these
strains are not identical. The Polish, Italian and SP2 strains are not identical either. Since
the intercross experiments gave also negative results, we suppose that even if all these
strains belong to a large S. arenarium group, they are not only geographically, but also
sexually isolated.
Fig. 26. GenePhore PCR-RFLP Analysis 2.
LEGEND TO Fig. 26. Comparison of the TaqI and AluI PCR-RFLP restriction profiles of the ITS1 – 5.8S –
ITS2 regions of the 18S – 5.8.S – 28S operons of strains belonging to the S. glaseri (S.g.), S. arenarium (S.
a.) groups as well as those of taxonomically unidentified Steinernema strains of long dauer phenotypes
from Poland (Polish), Italy, (Italy), Spain (SP2) and Morocco. The AluI profiles of KMD15, NC513, and
NCI seem to be identical but AZ26 differs in some smaller fragments significantly. The characteristic
“glaseri” pattern, however, cannot be mixed up with any other. The AluI patterns of the Anomali (Biosys)
and Gel strains are identical, and that of the Polish strain is very similar to them, almost identical, but the
Italian strain, however, seems completely different. The Morocco pattern is completely unique. The TaqI
profiles of KMD15, NC513 and AZ26 are unambiguously identical and NCI slightly differ from them.
However, all the other RFLP patterns significantly differ from the “glaseri profile”. Those of S. anomali
(Biosys) and Gel are similar but not identical and the Polish profile differs from both. The Italy just like the
Morocco shows unique pattern.
77
Fig. 27. GenePhore Analysis 3
GenePhore PCR-RFLP Analysis
1. Molecular Marker
2. S. a. (anomali) (Biosys) / MseI
3. S. g. (Kmd15) / MseI
4. S. g. (NC 513) / MseI
5. Steinernema sp. Polish
/ MseI
6. S. g. NCI /Mse I
7. Steinernema sp. (Morocco)/ MseI
8. S. a. (Gel) / MseI
9. S. g. (Az26) / MseI
I
/ Italy /
10. Steinernema sp.
Mse
11. Steinernema sp. (Slovak) / MseI
12. Steinernema sp. (SP2) / MseI
19. Gel / Rsa I
13. S. a. (anomali) (Biosys) / Rsa I
20. S. g. sp.(Az26) / RsaI
14. S. g. Kmd / RsaI
/ Italy / Rsa I
21 Steinernema sp.
15. S. g. Nc513 / Rsa I
I
22. Steinernema .sp (Slovak) /
I
16. Steinernema sp. Polish /
23. Steinernema sp.(SP2)/ RsaI
17. S. g. NCI / RsaI
Rsa
Rsa
18. Steinernema sp. (Morocco) / RsaI 24. Molecular Marker
LEGEND to Fig 27. Comparison of the MseI and RsaI PCR-RFLP restriction profiles of the ITS1 – 5.8S –
ITS2 regions of the 18S – 5.8.S – 28S operons of strains belonging to the S. glaseri (S.g.), S. arenarium (S.
a.) groups as well as those of taxonomically unidentified Steinernema strains of long dauer phenotypes
from Poland (Polish), Italy, (Italy), Spain (SP2), Slovak and Morocco.
The MseI profiles of KMD15 and NC513 seem identical, and very similar to that
of AZ26. This enzyme (as we had found before) 3 could not cut the DNA of S. glaseri
NCI. The characteristic “glaseri” pattern, however, cannot be mixed up with any other
one. The MseI patterns neither of the anomali (Biosys), nor of Gel are strains different,
since the Gel DNA was not very well digested by this enzyme. The Polish strain was
almost identical to that of anomali. The Italian strain showed completely different pattern
of lots of fragments, but was very similar to that of SP2. The pattern of the Slovak strain
was something between the Polish and the Italian, but was definitely different from both
of them. The Morocco pattern is completely unique.
78
4. SUMMARY
The entomopathogenic nematode (EPN) / bacterium (EPB) symbiotic complexes
have great agricultural potential with the key of success in bacterium. The bacterium
produces toxins, killing the insect and produce antibiotics to protect the monoxenic EPN
/ EPB complex against other microorganisms. In my dissertation I aimed to answer some
questions related to the taxon specificity of symbiosis and the role of the antibiotics.
Several strains available in our stock were tested for antibiotic production. The
data demonstrate that different compounds may play a role in the antibacterial action of a
EPB and the competition with another EPB strain. Two Xenorhabdus strains, EMA and
EMC produce antibiotics extremely effective against Erwinia amylovora (the cause of
fire blight disease in apple orchard) both in laboratory and phytotrone tests. These
compounds are also effective against Phytophtora and Trichoderma. We are to
concentrate and chemically characterize the antibiotics for liquid fermented culture of
EMA.
The EMC strain produces unusual exo-crystals of carbohydrate nature. The role
and exact chemical nature of the crystals are yet to be answered.
In different gnotobiological studies it was confirmed that the Steinernema species
can grow and propagate only their own symbionts or on symbionts of other nematode
species. In this study we have compared large number of strains of Photorhabdus and
Xenorhabdus species and we can conclude that the Photorhabdus strains are better
source for more Hererorhabditis strains in vivo and in vitro than the Xenorhabdus strains
for the Steinernema species. The Steinernema strains are more “picky” in terms of
finding proper symbionts.
We have isolated the EPB symbiont of the Morocco strain and elaborated a 3.7liter laboratory fermentation technique for growing them. The fermentation technique
can be used for other entomopathogenic nematode strains as well.
We are recommending a new molecular technique to identify new Steinernema
isolates. The GenePhore PCR-RFLP System might be useful for further molecular
identification of unknow and known bacterial strains.
79
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6. ACKNOWLEDGEMENTS
I would like to start out by thanking God, and all the people who helped me in
accomplishing my PhD. I am grateful to Professor Gábor Vida, the full member of the
Hungarian Academy of Science for all his help and for allowing my work in the
Department of Genetics at Eötvös Lóránt University, which is headed by him.
I want to express my thanks and appreciation to my supervisor Dr. habil András
Fodor, Associate Professor of the Department of Genetics of the Eötvös Lóránt
University, who proposed the project to me and helped me in accomplishing it with his
guides. My best regards to Professor Sándor Koch, who helped me so much with all his
advises and who was like a father to me.
To Dr. Károly Márialigeti, head of the Microbiology Department, who also helped
in so many ways both professionally and privately. For teaching me how to study and
how to prepare for an exam.
I would also like to say thanks to Professor Michel Klein for helping with the
correction and proofreading of my work.
In the microscopic studies and making the pictures, thanks to Dr. Géza Zboray and
Attila Kovács Associate Professors at the Zoology Department, who were my Mentors
Many thanks for all your help.
I have to thank my fellow students in the laboratory for the everyday help they gave
me. Andrea Máthé, Sára Simon and Piroska Magyar, also Andrea and Sára as
well as Emilia Szállás for previously keeping the nematode and bacterium strains in
order, and provided me with the samples whenever I needed. My deepest gratitude to
Antonia Völgyi, Katalin Lengyel, Triga Dimitra, Horolma Pamjav, Csaba
Ortutay, Takács-Sántha, Tibor Vellai, Bayar Baatar, Antal Vancsó and Kiss
Zsuzsanna Halima, who helped me a lot in typing and many things.
Last but not least I would like to thank my mother and father and brothers
Mohamed Abdorahman and Dr. Yosif and my sisters Fawzia and Turkia and my
daughter Mawada for being there for me. Special thanks to my country, Libya, for
generously supporting my studies. Thank you all for your time and help!
84