OF SPP. AND SPP., - Library and Archives...
Transcript of OF SPP. AND SPP., - Library and Archives...
NEMATICIDAL PROPERTIES OF XENOREMBDUS SPP. AND
PHOTOiüX4BDUS SPP., BACTERIAL SYMBIONTS OF
ENTOMOPATHOCEMC NEMATODES
B.Sc., Northwestem College of Fonstry, Yangling, China, 1985 M.Sc., The Chinese Acaderny of Fonstry, Beijing, China, 1988
THESIS SUBMïlTED IN PARTIAL, FULFLMENT OF THE REQUlREMENTS FOR THE DEGREE OF
DOCTOR OF P W S O P H Y
in the Department of
Biological Sciences
OKAIJI HU 1999
SIMON FRASER UNIVERSïïY
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ABSTRACT
Nematicidal properties of some secondary metabolites of Xenorhnbdus spp. and
Photorhabdus spp. (Enterobacteriaceae), bacterial symbiunts of the entomopathogenic
nematodes, Steinernema spp. and Heterorhabdiris spp., respectively, were identified and
evaluated.
Cell-free (CF) culture filtrates of X. bovienii, X. nematophilus and P. luminescens
isolates were shown to be nematicidal against Meloidogyne incognita and
Bursaphelenchus xylophilus. The degree of activity varied with the bacterial
isolate/species and the culture conditions, such as media composition, a p , pH and
aeration of the cultures.
Three metabolites, namel y ammonia, 3,s-dihydroxy4isopmpylstil bene (ST) and
indole, were identified from CF filtrates as having nematicidal properties. Ammonia was
common to al1 bacterial cultures tested. but ST was produced (10 - 30 pg/ml) by only P.
luminescens and indole was produced (10 - 50 pg/ml) by some straindspecies of both
Xenorhabdus and Photorhabdus.
ST and indole affected viability, mobility, egg hatch and dispersal khaviour of
nematodes in vitro. ST was active against Aphelenchoides rhytium, Bursaphelenchus spp.
and Caenorhabditis eleguns, but was not lethal to infective juveniles (Us) of H. megidis
90. or second stage juveniles (J2s) of M. incognita at 200 pg/ml. Indole was active in
immersion tests against J2s (LOO - 400 pg/ml) of M. incognito, but failed to pievent
infection of tomato seedhgs by M. inmgnita foîlowing a soi1 (a0 pdml) or foüar
application (<1,000 pglml). Indole repelled Us of some species of both Steinememu and
Heterorhabditis whereas ST repelled only some species of Steinemema.
ST, but no< indole, was detected in variable quantities (-665.2 to 4,182 pg/g wet
insect) in larval Galleria mellonella infected with Heterorhabditis spp. ST was produced
after 24 h of infection (2S°C) of the larvae, increased rapidly in quantity by 48 h to 5 d,
and nmained at a relatively hi@ and constant level even after the nematode symbiont had
completed its reproduction. Bacterial symbionts built up high populations (-10' cellslg
insect) within 24 h of entenng G. meilonella lame, and increased the cadaver pH to 7.4-
7.7.
The early production and relatively large amount of ST in nematode-infected
insect hosts, and the antibiotic, nematicidal and nematode-repelling properties of ST
suggest that it play a significant role in the symbiotic nematode-bacterium association.
The potential commercial application of these nematicidal metabolites may be limited by
their relatively narrow spectnim and low activity.
1 would like to take this opportunity to express my heartfelt thanks for ail of the
people who kindly offered their thoughts and help during my research. 1 am deeply
grateful to Dr. J. M. Webster, rny senior supervisor, for his encouragement, guidance and
support throughout the course of this snidy. I would also like to thank Drs. J. R.
Sutherland and A. Plant for their helpful suggestions and comrnents during my research
and during the revision of the thesis. My thanks are also given to the following people
who offered their thoughts, encouragement and help during my research: Dr. J. Li for his
work on chemicai characterization and for his help and invaluable suggestions: Dr. G.
Chen and Mr. K. Ng for their discussion and help; Dm. V. L. Bourne and G. Gries, Mrs.
R. Gries and Mrs. M. SieWUnen as well as Mr. B. Leighton and Mr. M. Yang for their
technical support; Mr. Ian Bercovitz for statistical consulting; those mentioned in the text
for their kindness and generosity in providing some of the test materials (nematodes,
bacteria and plant seeds); finaily, my colleagues and friends for their discussion and help.
I acknowledge the financial support of five Graduate Fellowships, a President's
Ph.D. Research Stipend and a Prototype Developrnent Fund for Student Entrepreneurs
from Simon Fraser University and of support ihrough research gants to my senior
supervisor, Dr. J. M. Webster, from the National Science and Engineering Resemh
Council of Canada.
Finally, I would like to express my heartfelt gratitude to my wife and son for their
love, patience and support throughout the course of this study.
TABLE OF CONTENTS
2.4. Biossssys for nematiciàai actidty..... .............................................................. ...33
2.4.1. Activity of celi-free culture filtrates .............................. ............................ 33
2.4.2. Activity of metabolic compounds ........................ ..................................... ..33
.......... .......*.....**...*.......*..*.***.........*.... 2.4.3. Mortality of the test nematodes .... .34
.....*........... ........... 2.4.4. Antibacterial activity .. .. ............................................................................... .... 2.5. Staüsücal andysis.. ......... ..35
CHAITER 3. NEMATICIDAL PROPERTIES OF IN VZTRO
CULTURES OF THE BA~RIAeoooiooem~omooaoooooooomoomooooo~emmoomm~oooooooooeoo o o o e o o o 0 0 3 6
3.1. htrod~~tl~~ooooooeooo~oooo~oooeooee~mooooeommmoommoeooomomoe~om~omooommooeoaooooooooooe 0 sommomoooeommooom 0 l 0 0 oo.36
3.2. Materials and ~ ~ t h o d s b m b m m b b b ~ ~ 0 b b ~ m b ~ b b 0 b b ~ b ~ b b b b b b b ~ b b ~ b ~ ~ ~ ~ ~ b m ~ b ~ ~ b ~ 0 ~ ~ b 0 b b ~ b ~ b ~ 0 ~ b ~ ~ b ~ b b b m m ~ 0 ~ 0 0 l *, b*bm m37
3.2.1. Bacteria and nematodes .... . .............................. . . . ................ . . . .. ... .... 37
3.2.2. Preparation of cell-free filtrates.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37
3.2.3. Nematicidal activity of bacterial strains and species .............. ................ .. . . . . . . . .38
3.2.4. Nematicidal activity of the bacterial cultures against different nematode
species ............... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . .. . . . . ... 39
3.2.5. Influence of bacterial culture conditions on nematicidal activity of the
culture filtrates ...... . . . . . . . . .. . . . . . . . . . . .,. . . . . . . . .. . . . . .. . . . . . . . . . . .. . . . . . . . . . . . . . . .,.. . . ..39
3.2.6. Nematicidal activity of organic and aqueous fractions of the culture
filtrates ... ...... ........... ....... ....... ........ . . . . ......... ............ . . .. ...... . .... . . . . . . . . .4 1
3.2.7. Nematicidal activity of some known antibiotics produced by
Xenorhabdus spp ..... ... .. . . . .. .... . . .. .. ..... . . . .. . ... ...... ... . . . . . ...... . . . . . . . ..... ..... . . . . . . . . .42
3.30 ~ e ~ ~ ~ ~ ~ o ~ ~ ~ ~ o ~ . o ~ ~ e ~ o o ~ ~ o b n m b o m ~ b b ~ o o ~ ~ m b ~ . b b ~ o o ~ b o m ~ b m m ~ m m b m b m m b m m m m m m b m b m b m m m m m o m m m b m m m m m m b m o m m l l memoa*mm l l l l l l l l e 4 3
3.3.1. Nematicidai activity of bacteriai strains and species. ... . . .. . . . ........................ .43
3.3.2. Nematicidal activity of different cultures against different nematodes
species ............... ..... .................................... ............ ..... . ..... ... ..... . ..... .. ........ .44
3.3.3. Influence of bacterial culture conditions on filtrate nematicidal activity ..... . . . . .44
3.3.4. Nematicidal activity of the organic and aqueous fractions of the
bacterial cultures ................. ... ..... + .............. ..... ....... ...+.... .... . ..... .... .... ......... . . . ...$ 1
3.3 S. Nematicidal activity of some known antibiotics produced by
Xenorhabdw spp ........................ .................................................. . . . . . . . . S4
3m4m D k ~ ~ ~ m m o m m m m o m ~ ~ ~ ~ ~ o m m o e ~ o m o m m m m m ~ m m o o m m m o ~ w ~ m o m m ~ ~ m m m m m o m o m m o o m w m m m m m m m l l l l l l moS4
CHAPTER 4. ISOLATION, IDENTIFICATION AND IN WTRO
vii
PRODUCTION OF NEMATICIDAL METABOLITES FRûM
.......................................................................... BACTERIAL CULTURES ..SS
................................................................................... 4.2. Materiah md metbods.... .Sa
4.2.1. Bacteria and their broth cultures ............ ...,.......... ......................................... 58
4.2.2. Generai procedures for isolation and identification of nematicidai
....................................................................................... metabolites .......... ., S9
4.2.3. Isolation and identification of nematicidal metabolites €rom cultures of
........................................ Photorhabdus luminescens and Xenorhabdus spp 62
4.2.4. In vitro production of the nematicidal substances identified from the
....................... ........... bacterial cultures ,.. 65
.............................. luminescens and Xenorhabdw spp ....................................... 71
4.3.2. In vitro production of the nematicidal metabolites produced by
Xenorhabdus spp . and Photorhobdus spp ............... ........... . .. ........................ 73 .......................................................................................................... 4.4. DISCUsslon. d g
CELIPTER 5 . NEMATICIDAL PROPERTIES OF 33-DIEiYDROXY-4-
..................................... ISOPROPYLSTILBENE (ST) AND INDOLE... ..94
........................................................................................................... 5.1 C O 94
............. 5.2. Mahtfll)S md methalSom ....m...........~......o.......o.........o.o...m.......~..~.oo.m...o..... 94
5.2.1. Test nematodes .....................~.......................................................................... 94
5.2.2. Nematicidal activiy of ST and indole against different nematode species ..... -95
viii
5.2.3. In vivo effect of indole on Meloidogyne incognita ............... .. .. ...*..... . . . . . . ... 100
5.2.4. Nematicidal activity of some indole derivatives ....................... .. ... . . ..... ... 103
5.2.5. Chemosensory effect of ST and indole on different mmatode species .... . . . . . . .104
53. R ~ ~ l f S m m o m m m m m o o m m m m * m m m m m m o m m o o m m m o m m o ~ m ~ m m m m m ~ o a m m o o m m o ~ m o o m m m o o m m m m ~ a m o m m m m m o m m m m m m l l 0 l *0 l a. 105
5.3.1. Nematicidai activity of ST and indole ............. .. .............................. . . . .. . . .. 105 5.3.2. Effect of ST and indole on egg hatch of the nematodes ............................... 1 15
5.3.3. In vivo activity of indole on Meloidogyne incognita. .. ... .. . .. . .. . . .. . . . . .. . . . . . .. 1 15
5.3.4. Nematicidal activity of some indole derivatives .............................. . . . . . . . . .. 12 1
5.3 .S. Chemosensory effect of ST and indole on nematodes .............. .... .... .. . . . . 1 2 1
5.40 D i ~ ~ ~ ~ ~ i ~ n m m m o o m o m m o m ~ m o m m m m m m o m m m m m m o m m m m m m ~ m m m ~ m m m o o m a o ~ m ~ m ~ m m m m m m m m m . m m m m m m m m m m ~ m ~ m m m m m ~ m m m m m m m m m m m m o o m l l l l l l l l l l 126
CHAPTER 6 m IN VIVO OCCURRENCE OF NEMATICIDAL
METABOLITES IN RELATION TO BACTERIAL GROWTH AND
NEMATODE D E V E L O P M E N T ~ m m m m m m m m m m m ~ m m m m m m m m m m m e m m m m m m m m m m mmmm.mmmammmmm.mmmmm l l l l l l l m a m l 3 0
60 1. I ~ t ~ d ~ ~ t i ~ ~ o m m m m . m m o m o ~ o m m m o m m m m m o m ~ e m m m m m m m m ~ m m m m o m m m m m m m m m m m m m m o m m o m m m m m m m m m o m m m m m m m m m m m m m m l 0 l l 0 l l l 0 130
6.2. Materials and ~ ~ t h o d s ~ ~ ~ m m ~ m m ~ m m m m m m m m m m m m o o m m m ~ ~ ~ ~ m ~ m o o m m o m m m m m m m m m l l l mm l l l 130
6.2.1. G. mellonella larvae and entomopathogenic nematodes .............. ... ..... . . . . .. 130
4.2.2. Detection and identification of indole from nematode-infected larval
cadavers of G. mell~nella*.~* .... +. ...... ....*.*.*eC*.**.*....* .... .,. .... *... 131
6.2.3. Detection of indole over time in larvai cadavers of G. mellonella infected
wifh P. luminescens MD.. .... .......... .*-.....~-.C~~-..~...................... . . . .. . . . . .* . .. . . ... 132
6.2.4, IsoIation and identification of ST from infected lacval cadavers of
G. mellonella ..... ........... -.........*.........e..*...*....****.L... . . . .. . .... 133
6.2.5. Quantitative analysis of ST from nematode-infected lamal cadavers of
G. mellonella ....... .,. ...................................................................................... 134
6.2.6. Occurrence of ST and indole in relation to the development of
Heterorhabditis and growth of Photorhabdus in larval G. mellonella
cadavers ........................................................................................ ,136
6.2.7. Occurrence of ST and indole in larvd G. mellonella cadavers infected
.................... by different Photorhabdur spp.-Heterorhabditis spp. complexes 139
6.3. Res~lts..,...................~..~.........~....~.....................~....~..............~......... ....... ...140
6.3.1. Detection of indole from larvai cadavers of G. mellonella infected by
H. megidis W... ......................................................................... 140
6.3.2. Detection of indole from larval cadavers of G. mellonella injected with P.
luminescens MD alone ................................................................................... 140
6.3.3. Isolation and identification of ST from larval cadavers of G. mellonella
infected by H. megidis 90 ............................ .. ........................................ .14 1
6.3.4. Quantitative analysis of ST from infected l a r d cadavers of
G. mell~nella.~ ...... .. ..................................... ........................... .............. 141
6.3.5. Occurrence of ST in relation to the development of Heterorhabditis
and growth of Photorhabdus in larval G. mellonella cadavers .......................... 145
6.3.6. In vivo production of ST by different Photorhobdus spp ................... ... .... 157
LIST OF TABLES
Table 1.
Table 2.
Table 3.
Table 4.
Table 5.
Table 6.
Species of Xenorhabdus and Photorhabdus and their respective nematode
.................................... symbionts, Steinernema and Heterorhabditis species 4
Major characteristics distinguisbing Xenorhabdus spp. from Photorhabdus
spp ............................................................................................................. ..7
Bioactive agents associated with or derived fmm the bacterial symbionts,
Xenorhabdus spp. and Photorhabdus spp., of entomopathogenic
nematodes.. ................. ,... .. ....................................................................... ..23
................... Species and sources of nematodes and bacteria used in this study 27
Percentage mortality of second stage juveniles of Meloidogyne incognita
(MI) and fourth stage juveniles and aûults of Bursaphelenchus
xylophilus BC (BX) exposed to the diluted, cell-free culture filtrates
(4 d-old filtrated culture and diluted to 1M or 114 strength) of
............................. Xcnorhabdus spp. and Photorhabdus luminescens.. ..47
Percentage mortality of second stage juveniles of Meloidogyne incognita
(MI) exposed to the diluted, cell-fne culture filtrates (4 d-old filtrated
culture and diluted to 1/2 or 114 stmgth) of Xenorhabdus nemutophilus
BC 1 grown in tryptic soy broth (TSB), Luria broth (LB) and nutrient
...... ....... broth (NB), respectively ......-..*.-......................*..*.-..*.. ..48
Table 7. Nematicidai activity of the 4 d-old cell-fiee fütrates of Xenorhabdus
bovienii A2 1 (Ml) and X. nematophius BC 1 @Cl) against second
stage juveniies of Meloidogyne incognita 0 when the filtrates were
adjusted, using 6N NaOH or HCI, to pH values ranging from 5.0 to
9.0 ................................................................................................................. 52
Table 8. Nematicidal activity of dried organic and aqueous fractions of
Xenorhubdus spp. and Photorhubdus luminescens against second stage
juveniles of Meloidogyne incognita (MI) and fourth stage juveniles and
adults of Burmphelenchus xylophilus BC (BX). ................................... 53
Table 9. Nematicidal activity of some known antibiotics produced by
Xenorhabdus spp. when tested against fourth stage juveniles and
...............*.... ...................... adults of Bursaphelenchus xylophilus .... -55
Table 10. Summary of nematicidal metabolites identified from cultures of
different isolates of Xenorhabdus spp. and Photorhabdus
luminescens.. .......................................................................... .72
Table 1 1. Concentration of ammonia and its salt(s) (NHi Wml) in 4 d-old
culture broths of Xenorhabdur spp. and Photorhabdus luminescens
gmwn in TSB ......................................................................................... 79
Table 12. pH and concentration of ammonia and its salt(s) of tryptic soy broth
(TSB), Luna broth (LB) and nutrient broth (NB) in which Xenorhabdus
.................. ............*....... nemutophilus BC 1 had k e n grown for 4 d ... .84
Table 13. Occurrence of indole in broth cultures of Xenorhubdus spp. and
Photorhabdus luminescens grown in tryptic soy broth (TSB), nutrient
broth (NB) or Luria broth (LB) for 1.2 or 4 d .................. ........... .... ..86
Table 14. Inhibitory effect of 3,s-dihydroxy4isopropylstiIbene (ST) and indole
on the percentage of egg hatch of Meloidogyne Nicognita over 5 d
xii
followed by immersion in distilled water for another 5 d ............................ 1 16
Table 15. Effect of 3,5-dihydroxy-4-isopropylsti1bene (ST) on percentage of
egg hatch of Bursaphelenchus xylophilus BC.. .................................. .1 17
Table 16. Effect of indole on infection of tomato seedlings by second stage
juveniles of Meloidogyne incognita in sand application tests ....................... 1 18
Table 17. Effect of indole on infection of tomato seedlings by second stage
juveniles of Meloidogyne incognito in foliage spray tests .......................... 120
Table 18. Nematicidal activity of some indole derivatives against
................................. Bursaphelenchus xylophilus BC in immersion tests -122
Table 19. Chemosensory effect of 3,5-dihydroxy-4-isopropylsti1ùene (ST) and
indole on different nematode species w hen tested at O. 1, 1, 10 and
100 pddisc in 1.5% agar plates ................................................................... 125
Table 20. Extraction of 3,s-dihydroxy4isopropylstilbene (ST), using different
solvents, from cadavers of Galleria mellonella infected by
. . Heterorhabdatis megidis 90 ........................ ... ........... ..............O....b...... 144
Table 2 1. Recovery of 3,s-dihydroxy4isopropylsti~bene (ST) with acetone
h m healihy Galleria rnellonella larvae injected with known arnounts
.......... .. of ST. ..o...........C.................................................................. 146
Table 22. Characteristics of Vp (primary form) and Vsm (small-colony variant)
................. of Photorhabdus luminescens MD .,,,....*.................................... .155
Table 23. Concentration of 3,5-dihydroxy4isopropylstilbene (ST) produced
by Hetetorhabditis spp. - Photorhbdus spp. complexes in
larval cadavers of Gallertu m e l l d l a at 7 d postinfection.. .................... 158
LIST OF FIGURES
Fig. 1. Generalized life cycle of entomopathogenic nematodes, Steinemema spp.
and Heterorhabditis spp ..................... ....... .............+........................... .17
Fig. 2. Percentage mortality and paralysis of second stage juveniles of Meloidogyne
incognita exposed to diluted, cell-free culture filtrates (4 d-old filtrated
cultures diluted to !A sangth) of Xenorhabdus spp. and Photorhabdus
luminescens.. ............................................................................. ..45
Fig. 3. (a) Growth. (b) pH and (c) nematicidal activity against Meloidogyne
incognita (second stage juveniles) of Xenorhabdus bovienii A2 1 (A2 1).
.......... X. nematophilus BC 1 (BC 1 ) and Photorhobdus luminescens MD (MD) .49
Fig. 4. Fiow-chart showing the general isolation process of the organic extracts
....................... from broth cultures of Xenorhabdw spp. or Photovhubdus spp ..60
Fig. 5. Structures of (a) 3.5-dihydroxy4isopropylstilbene (ST) (R=CH3) and
........................................................................... ........................... (b) indole .. 74
Fig. 6. UV spectra of (a) 3.5-dihydroxy4isopropylstilbene (ST) and (b) indole ........ .76
Fig. 7. Concentration of arnmonia and its salt(s) (NE&+ pg/mi) in culture broths
of Xenorhabdus bovienii A2 1 (A2 1 ), X nemtophilus BC 1 (BC 1) and
...... Photorhabdus Iminescens C9 (Cg) grown in tryptic soy broth over 5 days 80
Fig. 8. (a) Bacterid growth, (b) pH and (c) concentraiion of arnmonia and
its salt(s) of Xenorhabdus bovienii A21 in tryptic soy broth as infiuenced
by aeration of the culture ..................... ............................................ ....... ..82
Jtiv
Fig. 9. Production over time of 3.5-dihydroxy4isopmpylstilbene (ST) and
indole (HD) in culture broths of Photorhabdus luminescens C9 (Cg)
and Photorhabdus luminescens MD (MD) grown in tryptic soy broth.. ........ ..87
Fig. 10. Sand colurnn used in the migration tests of the second stage juveniles of
...... Meloidogyne incognita. ............................. ... . 9
Fig. 1 1. Arrangement of filter paper discs on the surface of an agar Petri dish
(100 x 15 mm) in relation to the point of introduction (0) of nematodes for
chemosensory tests .......................................................................................... -106
Fig. 12. Nematicidal activity of (a) 3.5-dihydroxy+isopropylsti1bene (ST) and
(b) indole against nematodes of different species in test solutions in
..................... small Peh dishes .. ...................................................................... 108
Fig. 1 3. Percentage mortality and paral ysis of (a) Bursaphelenchus qlophilus BC
(juveniles and adults), (b) Meloidogyne incognita (second stage juven iles)
and (c) Heterorhabditis sp. HMD (infective juveniles) following
............................. immersion in indole solutions at different concentrations 1 t 1
Fig. 14. Inhibitory effect of indole on mobility of second stage juveniles (J2s) of
............................ Meloidogyne incognita in a sand column afier 24 h treatment 1 13
Fig. 15. A diagrammatic representation showing the influence of
3.5-dihydroxy4-isopropylstilbene (ST) and indole on dispersal
behaviour of different nematde species on Petri dishes .................................. 123
Fig. 16. Cornparison of HPLC chromatograms of a typical test sample extracted
from Galleria mellonella larvae infected with Heterorhabditis megiàis 90,
as detected at two different wavelengths (254 nm and 3 15 nm) ........................ 142
Fig. 17. (a) Occurrence of 3.5-dihydroxy4isopropyIstilbene (ST), (b) population
dynarnics of Photorhobdus luminescens C9 and (c) pH of larval
cadavers of Galleria mellonella infected by Heterorhabditis megidis 90
over time in two repeat experiments (Exp-1 and Exp-2) ................................. .147
Fig. 18. TU: chromatogram of 3.5-dihydroxy4isopropylstilbene (ST) and of
two test samples extracted from Galleria mellonella larvae infected
............ with Heterorhabdiris megidis 90,2 and 5 d (2d and 5d) after infection 149
Fig. 19. (a) Occurrence of 3.5-dihydroxy-44sopropylstilbene (ST), (b) population
dynamics of the pnmary form (Vp) and a smallsolony variant (Vsm) of
Photorhabdus luminescens MD and (c ) pH of larval cadavers of Galleria
mellonrlla infected by Heterorhabditis sp. HMD over time in two
repeat experiments (Exp- 1 and Exp-Z).. ..................................................... .153
AW: average weight;
CF: cell-free;
CFU: colony-forming unit;
DMSO: dimethyl sulfoxide;
ECm : concentration causing paralysis and mortality in 50% of test nematodes;
HD: indole;
HPLC : high performance liquid c hromatography ;
U(s): infective juvenile(s) of Steinemema spp. and Heterorhabditis spp.
12s: second stage juveniles of Meloidogyne incognita;
J4s: fourth stage juveniles of the nematodes;
LB: Luna broth (base, Miller) (Sigma@);
LCm: concentration causing mortality in 50% of test nematodes;
Mg: a buffer solution formulated specially for the nematode Caenorhabditis elegans;
MS: mass spectmm;
NB: nutrient broth;
PBS : phosphate-buffered saline;
PDA: potato dextrose agar;
PEG: polyethylene glycol;
SD (water): sterilized distilled (water);
SMS: streptornycin sulfate;
ST: 3,Sdi hydroxy4isopropylsti1 bene;
TLC: thin-l ayer chromatopph y;
TSA: agar medium of TSB;
TSAD: TSA plus dye (25 mg bromothyrnol blue/L TSA);
TSB: tryptic soy broth wlo dextrose @ifco@);
UV: ultraviolet;
VM: volatiIe and nematicidal materials;
Vp: primary form of Phofurhubdus luminescens MD;
xvii
Vsm: small-colony variant of the primary forni of Photorhabdus luminescens MD;
WS : wet-strength;
CHAITER 1
INTRODUCTION
Much of the incnase in agricultural productivity over the past half century has
been due to more efficacious and economical pest control through the use of synthetic
chernical pesticides (Duke et al.. 1993; Hall, 1995; Pimentel, 1997). However, in ment
years there has been an increasing tendency to control pests by biological pesticides,
natural products and other environmentally benign measures (National Research Council,
1996; Whitten et al., 1996). This trend is due rnainly to increasing public concems about
the adverse effects of synthetic chemical pesticides on human health and environmental
safety and the increasing resistance of the pests to chemical pesticides (Rodgers, 1993;
Cross and Polonenko, 1996). Consequently, some pesticides have k e n deregistered or
their use restricted during the past decade, and many others are facing similar restrictions
(May, 1993; Cross and Polonenko, 1996). As an alternative, environmentdly acceptable
pest control measure, there has been a substantial investment into the exploration and
commercialization of some biological agents and naturai products from plants and
microorganisrns (Chitwd, 1993; Rodgers, 1993; Cross and Polonenko, 1996) that are
selective against the pests and are biodegradable.
Ail living organisms are subject to predation, parasitism or competition from
other organisms. Numerous scientific studies have shown that a wide variety of
microorganisrns and natural proâucts are capable of effectively controlling pests such as
weeds, insects, nematodes and bacterial and fungal disease causing agents. Research into
improving the efficacy of entomopathogenic nematodes has k o m e a major focus in
ment years. because of widespread interest in their commercial application to control
insect pests (Kaya et al., 1993; Wilkinson and Hay, 1997). As well, then is a growing
research interest in the bioactive properties of the secondary metabolites of their bacterial
symbionts, Xenorhabdus spp. and Photorhabdus spp., respectively (Webster et al., 1998;
Li et al., 1998). The insecticidal toxins isolated from cultures of the symbiotic bacterium,
P. luminescens, have the potential to be the next generation of micmbial insecticides
(Ensign et al., 1990; Bowen et al., 1998; Gou et al.. 1999). The antibiotic production by
the symbiotic bacteria may lead to the development of novel agrochemicals and dmgs
(Webster et al., 1998; Li et al., 1998) and these could have significant commercial
potential.
In preliminary tests, 1 observed that cultures of Xenorhabdus spp. were
nematicidal against the plant-parasitic nematode, Meloidogyne incognita. This discovery
of nematicidai activity against M. incognita was a surprise in view of the symbiotic
relationship of these bacteria with nematodes. The results were interesting also because
such naturally derived nematicidal agents are especially attractive to nsearchers at a time
when mosi chernical nematicides are king withdrawn from use for environmental
reasons (Noling and Becker, 1994). Further studies of this nematicidal phenornenon could
help us understand better the symbiotical relationship ktween the bacteria and their
nematode symbionts. It may help us also to control nematode pests at the same time as
controlling insect pests when entomopathogenic nematodes arc applied. Thus, the overall
biology and properties of these symbionts will be outlined as a prelude to defining my
research objective.
Xenorhabdus and Photorhabdus are members of the family Enterobacteriaceae
(Thomas and Poinar, 1979; Akhurst and Boemare, 1988; Boemare et al., 1993a). Five
species of Xenorhabdus and one species of Photorhabdus have been described and
accepted as valid (Font and Nealson, 1996; Boemare et al., 1997; Table 1).
The presence of symbiotic bacteria in entornopathogenic nematodes was first
postulated by Bovien (1937) and Dutky (1937). The first of these to be identified was
Achromobacter nematophilus (Poinar and Thomas, 1965), a syrnbiont of the DD-136
isolate of a Steinernema (=Neoaplectana) species, and these authors regarded the
association between the bactenum and the nematode as a fonn of mutualism (Poinar and
Thomas, 1966). The new bacterial genus, Xenorhabdus, was designated (Thomas and
Poinar, 1979) and later amended (Thomas and Poinar, 1983) following rejection of the
genus Achromobacter (Buchanan and Gibbons, 1974; Hendrie et al., 1974). Based on
extensive numerical analysis of 45 characteristics of 20 svains of Xenorhabdus spp.,
Akhunt and Boemare (1988) elevated four subspecies of X. nematophilus, narnely
nematophilus. poinarii, bovienii, beddingii, to the species level. After analyzing the
relative phylogenetic position of different strains and species of Xenorhabdus, using 16s
rRNA cataloping, Ehlen et al. (1988) found that X. nematophilus and X. luminescens
were not as closely related as had been thought previously, and they proposed that X.
luminescens be treated as a taxonomie unit equivalent to that of the farnily
Enterobacteriaceae in which it is cumntly placed. Subsequently, Boemare et al.(1993a),
based on DNA analysis, proposed that a new genus, Photorhabdur, accommodate
the luminescent bacteria associated with entomopathogenic nematodes, Heterorhabditis
Table 1. S p i e s of Xenorliobdus and Photorhabdus and their respective nematode
symbionts, Steinemema and Hetemrhabditis species
Bacteria Nematode symbionts Re ferences
X. bedding ii
X. bovien ii
X. japonicus
X. nematophilus
X. poinarii
Steinemema sp.
S. o#he
S. feltiae
S. intemedium
S. kraussei
S. kushidai
Xenorhabdus spp. S. abbusi*
S. arenarium
Se bicomuttun
S. caudatum
S. cerutuphotum
S. howaiiensis
Akhurst and Boemare, 1988
Steiner, 1923
Filipjev, 1934
Bovien, 1937
Poinar, 1985
Akhurst and Boemare, 1988
Marniya 1988
Nishimura et al., 1994
Weiser, 1955
Akhurst and Boemare, 1988
Steiner, 1929
Akhurst and Boemare, 1988
Mracek et al., 1994
Fischer et al., 1999
Elawad et al., 1997
Elawad and Hague, 1998
Artyukhovsky, 1967
Tallosi et al., 1995
Xu et al., 199 1
Iian et al., 1997
Gardner et al., 1994
to be continued
Table 1. (continued)
S. karii
S. long icaudum
S. monticolum
S. neoctrrtillae
S. oregonense
S. puertoncense
S. ra rum
S. riobrave
S. ritteri
S. siamkayui
S. scapterisci
Photorhabdus luminescens H. bacteriophoru
H. megidis
H. zealandica
Photorhabdus spp. H. argentinensis
H. brevicaudis
H. hawaiiensis
H. indicus
H. maraletus
Waturu et al., 1997
Shen and Wang, 1992
Stock et al., 1997
Nguyen and Srnart, 1992
Liu and Berry, 1996a
Roman and Figueroa, 1994
de Doucet, 1986
Cabanillas et al., 1994
de Doucet and Doucet, 1990
Stock et al., 1998
Nguyen and Smart, 1990
Poinar, 1976
Poinar et al., 1987
Poinar, 1990
Boemare et al., 1993a
Stock, 1993
Liu, 1994
Gardner et al., 1994
Poinar et al., 1992
Liu and Berry, 1996b
*: S. ubbari may not be associated with Xenorhabdus sp. Refer to reference for details.
spp. A ment study, comparing partial 16s rRNA gene sequences, found at least two
well-supported taxonomic groups within Photorhabdus which suggest that the genus may
be polyspecific (Liu et al., 1997). The identification of several isolates of P. luminescens
€rom a few human clinical specimens (Colepicolo et al., 1989; Farmer et al., 1989;
Akhurst et al., 1998) is unusual since al1 other isolates have been collected from
entomopathogenic nematodes in the soil. However, DNA-DNA hybridization has shown
that although these clinical isolates are closely related to each other they are sufficiently
different to consider them to be new species (Forst and Nealson, 1996).
The major discriminative characteristics between the genera Xenorhabdus and
Photorhabdus are listed in Table 2. Many taxonomic studies provide evidence for
separating these bacterial symbionts into the two genera (Wimpee et al., 1991; Boemare
et al., 1993a; Stackebrandt et al., !997). However, there continues to be some uncertainty
over the split within the genus Xenorhabdus (Rainey et al., 1995; Forst and Nealson,
1996; Liu et al. 1997; Stackebrandt et al., 1997). Part of the problem appears to be the
use of DNA-DNA hybridization methods. The technique is known to be useful for
species sepmtion but less so for delineation of genera (Forst and Nealson, 1996). The
inter- and intrageneric DNA-DNA similarity values are so low for the majority of the
species that this technique is not the optimum one to use (Stackebrandt et al., 1997).
Sirnilarly, thel6S rDNA similarity values and the majority of phylogenetic trees
generated €rom the sequence data did not indicate these two genera to be sister gmups
(Stackebrandt et al., 1997).
Xenorhabdus and Photorhabdus are considered atypical Enterobacteriaceae
(Boemare et al., 1997). because most of the Xenorhabdw and Photorhabdus species are
Table 2. Major characteristics distinguishing Xenorhubdus spp. h m Photorhabdus
--
Character Xenorhabdus Photorhabdus
Isolated fmrn Steinemema spp. + Isolated from Heterorhabditis spp. - Bioluminescence - Cat alase - Pigment unknown
Antibiotics** xenorhabdins, xenorxides
xenocoumacins, xenomins
nematophin, indoles
- + + + anthraquinones
hydrox ystilbenes
anthraquinones
genistein, Ap
furan derivative
*: Modified from Font and Neaison (1996). +: positive; -: negative.
**: Ap and a hiran derivative are novel antibiotics (Hu et al., unpubl.).
nitrate-reductase negative (similar to a few strains of Envinia and Yersinia species) and.
in addition, species of Xenorhabdus are catalase negative (similar to some strains of
Shigella dysenteria O group 1). Based on the results of whole-ce11 fatty acid patterns.
Janse and Smits (1990) suggested that the Xenorhabdus spp. may be sufficientiy
different from other enterobacteria to exclude them from the Enterobacteriaceae. The
taxonomic details that support this concept of them king separate from the
Enterobacteriaceae are still in question, as is the issue of whether Xenorhabdus and
Photorhabdus are themselves separate genera (Forst and Nealson, 1996: Stackebrandt et
al., 1997). As more isolates of both genera are ideniified. it seems likely that more species
will be established. and our overall view of the taxonomic status of this nmarkable group
of bacteria will change accordingly (Forst and Nealson, 1996; Stackebrandt et al.. 1997).
Xenorhabdus and Photorhubdw are chemoheterotrophic bacteria that use both
respiratory and fermentative metabolism. They are motile with peritrichous flagella,
nonsponilating, oxidase negative and Gram negative rods (2-10 x 0.3-2.0 p) (Boemare
et al., 1993% Font and Nealson. 1996), but filamentous f o m rnay occur under
conditions of low osmolarity (Krasomil-Osterfeld, 1995).
Al1 but one strain (4-614) of Photorhabdus are bioluminescent (Akhuat and
Boemare, 1986). However, a variant of strain Q-614 was recently recorded to =store the
luminescent property (Boemare, 1995). When an insect is infccted by nematodes carrying
P. luminescens the whole insect glows in the dark (Poinar et al., 1980b). Poinar et al.
(1980b) speculated that the glowing cadaver might attract other insects that could k
infected by the emerging infective juveniles (Us). An alternative hypothesis is that the
bioluminescence may be inhibitory to some invertebrate predators (Akhurst and Boemare,
1990). Bioluminescence is very weak dunng the U emergence phase of nematode
development, but is at its peak when the nematodes in the cadavers are in the multiplying
non-infective stage of development. Some invertebrate predators might be expected to be
photophobie and so avoid the glowing cadaver and its developing nematodes.
Both Xenorhabdus spp. and Photorhabdus spp. can be grown as free-living
organisms in microbiological media, such as tryptic soy broth (TSB), nutrient broth (NB)
and Luna Broth (LB) (Li et al., 1995a; Liu et al., 1997; Volgyi et al., 1998). As the
bacteria enter the stationary phase of their growth cycle, they secrete several extracel Iular
products, including lipase(s), phospholipase(s), protease(s), and several different broad
spectnim antimicrobial agents (Akhurst and Dunphy, 1993; Li et al., 1998). Cytoplasmic
inclusion bodies, composed of crystalline proteins. are produced by both bacterial genera
during the stationary phase (Couche and Gregson. 1987). In fresh plate cultures, the
bacterid colonies readily absorb dye such as bromothymol blue or neutral red added into
the medium (Boemare and Akhunt, 1988). The optimum in vitro temperature for growth
of most strains and species of Xenorhabdus and Photorhabdus is 2S°C (Thomas and
Poinar, 1979). Their doubling time varies from 0.8 to 3.0 h depending on the culture
medium, temperature and species of bactemm (Poinar et al., L980b; Dunphy et al.,
1985).
Xenorhabdus spp. and Photorhabdus spp. have not been isolated directly from the
soil (Akhurst and Boemare, 1990). It is generally believed that the bacterial symbionts
cannot survive in the soil environment outside of their nematode symbionts, even after
king introduced axenically into autoclaved soil (Poinar et al., 1980b; Chen et al., 1996).
However, there is speculation that the bacteria may exist as phase II cells or variants
outside of the Us and infected insect cadavers (Font and Nealson, 1996). Their presence
in soils or other environments rnay have ken missed, because the usual rnethod for their
identification relies heavily on those properties that are usually strongly expressed in
phase 1 rather than phase II cells (Hurlbert et al., 1989; Smigielski et al., 1994; Forst and
Nealson. 1996). in fact. Morgan et al. (1997) reported that a non-culturable but viable
population of cells was detected when X. nematophilus and P. luminescens were
inoculated into sterile river water. The results indicated that cells of Xenorhabdus spp.
and Photorhabdus spp. may survive longer than anticipated in the environment and
nmain undetectable using standard rnicrobiological methods. The above suggestion was
further supported by the observation (Bleakley and Chen, 1999) that P. luminescens,
either from nematodes or a human wound, survived in previously sterilized soil for 30 d.
It has been suggested that colony hybridizations with specific gene probes such as lu
genes, lipase genes, pigment genes, or others might be a good approach ta clarify the
presence of the bacterial spbionts in the soil (Forst and Nealson, 1996).
Poiymorphic foms of Xenorhabdur and Photorhabdus commonly occur, as they
do in many other bacterial species (Silverman and Simon, 1983). Most Xenorhubdus spp.
and Photorhnbdus spp. typically occur in two extreme colony fotms when cultured in
vitro. These fonns are referred to as phase 1 (primary fom) and phase II (secondary form)
(Akhurst and Boemare, 1990). Reversion from phase II to phase 1 is cornmon in
Xenorhubdw spp. However, reversion of Photorhabdus h m the secondary to the
primary form has not been detected in any solid medium culture (Gemtsen et al., 1992)
though nveaion rnay occur under certain conditions in Iiquid cultures (Krasomil-
Osterfeld, 1995).
Phase 1 and II differ in many characteristics. In general, phase 1 cells produce
proteases, phospholipases, lipases, and crystalline proteins, binds to specific dyes
(Akhurst, 1980; Boemare and Akhurst, 1988) and produce antimicrobial agents in culture
broth (Akhurst, 1982). Phase 1 cellular populations are pleomorphic, larger than phase Il
cells, contain rods (80-90%) and spheroplasts (IO-20%), are motile with pentnchous
flagella and s w m on appropriate aga- media (Givaudan et al., 1995). have
paracrystalline inclusions and fimbriae (Boemare et al., 1983; Brehelin et al., 1993). In
the case of Photorhabdus, phase 1 is luminescent (Poinar et al., 1980b; Boemare et al.,
1997). Phase II generally does not have the above properties typical of phase 1. Moreover,
phase iI occurs only under certain culture conditions, such as prolonged incubation or
lowssmolarity (Akhurst 1980; Krasomil-Ostetfeld, 1995). Analysis has shown that phase
1 and phase iI differ also in their ceIl surface properties, serology and in respiration
(Brehelin et al., 1993; Smigielski et ai., 1994; Gemtsen et al., 1995). As weli, phase 1,
the naturally occumng symbiont of the nematode, supports nematode propagation but
phase II does not (Akhurst and Boemare, 1990; Ehlers et al., 1990). However, both
phases are similar in king entomopathogenic and they commonly share vimially d i the
other bacteriological properties (Boemare, 1995).
Other pol ymorphisms, named colonylfonn variants, have been reported from
cultures of P. luniinescens (Hurlbert et al., 1989; Gemtsen et al., 1992). Some colony
variants were reponed as king similar to phase I while others were sirnilar to phase II in
ceIl size, colony fom, luminescence, pathogcnicity, antibiotic production, pigmentation
and dye adsorption. The variants can revert to each other and to their parental phase I or II
at a relatively high frequency.
Phase variation of Xenorhabdus has ken suggested to be a mechanism for
escaping the lytic activity of a bacteriophage that lyses phase 1 but not phase II (Poinar et
al., 1980a; Poinar et al., 1989). However, studies have since showed that phase II of some
Xenorhabdus can revert to phase 1 at a relatively high frequency (Akhurst, 1980), and no
diffennces has been detected in the plasmid patterns between phase 1 and II cells of
Xenorhabdus (Leclerc and Boemare, 199 1). Thaler et al. (1997) found that bacteriocins
occur naturally in low quantities in both phases of Xenorhabdus spp. Akhurst et al.
(1992) did extensive restriction fragment length polymorphism analysis and excluded the
possibility that phase variation may be a result of a rearrangement of the major DNA of
the bacteria. The occurrence of intermediate variants or colony variants suggests that the
mechanism of phase variation is more cornplex, and a variety of mechanisms may be
openiting at different levels (Forest and Neaison, 1996). Such mechanism(s) may also
differ between Xenorhabdus and Photorhabdus (Boemare, 1995).
The significance of fodcolony variants is unknown, but they and phase 1 and
phase II fonns may have evolved so as to give a suwival advantage to these bacteria
under changing environmental conditions (Hurlbea et al., 1989; Akhurst and Boemare,
199û; Gemtsen et al., 1992). The retention of the symbionts by Us that escape from the
insect cadaver is the beginning of a starvation p e n d for the bacteria whereas their release
into the hemolymph is the b e g i ~ i n g of an optimum growtb peciod. These opposite
environmental biotopes have piobably forceci adpptive responses and phase variation may
be a survival strategy (Boernace, 1995). Smigielski et al. (1994) speculateà that the phase
1 cells are better adapted to conditions in the insect and the nematode spbiont, whereas
phase II cells are better adapted to free-living conditions in the soil. Further nsearch is
necessary since Xenorhabdus spp. and Photorhabdus spp. have not k e n detected in soil.
The pathogenicity of the bacterium - nematode complexes to insects varies with
the nematode and insect species, the immunological and physiological state of the insect,
and on the nature of the interaction of the bacteral and nematode symbionts (Gotz et al.,
1985; Akhurst and Dunphy, 1993; Sambeek and Wiesner, 1999).
The pathogenicity of phase II Xenorhabdus cells toward Galleria mellonella is
similar to that of the phase 1 (Akhunt, 1980; Akhunt and Boernare. 1990). However,
Volgyi et al. (1998) demonstrated recently that the phase II of X. nematophilus AN6
(AN6m) was significantly less virulent than the phase 1 cells of X. nematophilus (AN6IZ)
to larval Manduca sextu, an insect with a more potent immune system than G. mellonella.
They showed also that the virulence of phase 1 and Ii is variable and can be influenced by
environmentai conditions.
Although the bacteria alone are usually pathogenic, they need to gain access to the
hemolyrnph and this is rhieved by the nematode symbionts acting as a vector. X.
nematophilus and Photorhabdus sp. are not pathogenic to G, mellonella when applied
orally or topically to the insect (Poinar and Thomas, 1967; Milstead, 1979). Lysenko and
Weiser (1974) suggested that the nematode did not merely hinction as "living syringes"
since when a highly virulent bacterium was injected into G. mellonella by the nematode.
it did not necessarily cause hi& percentage mortality of die G. mellonella lame or vice
versa Akhurst (1986) showed that both the nematade and its bacterid syrnbiont were
necessary for the X. poinarii - S. gfaseri complex to cause larval mortality of G.
mellonella as neither the bacterium nor the nematode alone caused insect death.
Taxonomic and experimental studies confirm that each species of
entomopathogenic nematodes has a specific natural association with only one
Xenorhabdus species though a Xenorhabdus sp. may be associated with more than one
nematode species (Akhurst and Boemare, 1990; Table 1). The nematode-bacterial
specificity appears to operate at two levels: the provision of essential nutrients for the
nematode by the bacterium and the retention of the bacterium within the intestine of the
Ws of the nematode (Akhurst and Boemare, 1990).
The requirement by the nematode of bacteria-produced nutrients does not impose
a high level of specificity for a particular bacteriai species, but nematodes generally
reproduce better on their specific bacterial symbiont (Akhurst, 1983; Akhurst and
Boemare, 1990; Aguillera et al., 1993). Apillera and Srnart (1993) reported that S.
scapterisci developed and npmduced on a number of bacterial species, including
Escherichia coli, Ochrobactemm unthropi, Paracoccus denitrifcans, Pseudomonas
aureofaciens, Pseudomonas fluorescens B iovar B. Xanthomonas maltophilia,
Xenorhabdus spp. and X. nematophilus, and that progeny production after 14 d was
significantly greater on X. nematophilus and P. fluorescens Biovar B than on the other
bacterial species. Akhurst (1983) hypothesized that since the association bctween the
nematode and bacteriai symbionts was not completely specific, it may be possible to
create a nematode - bacterium association that would be more effective against an insect
pest than any of the natural symbiotic associations.
S ymbiont transmission is indirect in entornopathogenic nematodes since Us
acquire bacteria from their immediate environment, i.e. the insect cadaver (Wilkinson and
Hay, 1997). The production of antibiotics and bacteriocin, which inhibit the growth of
other microorgnnisms (Akhurst, 1982; Thaler et al., 1997; Li et al.. 1998), result in a
transmission strategy which is hinctionally analogous to vertical transmission (the
nematodes acquire their bacterial symbionts from their immediate environment) and
therefore, specificity and recognition may not be important (Wilkinson and Hay, 1997).
Specificity is high in the S. carpocapsue-X. nematophilus association and these Us do not
ntain the syrnbiont of any other species (Akhurst, 1983). However, Us of S. feltiae
(=bibiunis) and of S. glaseri are able to cany the symbionts of some other Steinemema
spp., though usually some of them do not carry these bacteria or cany fewer bacterial
cells than they do their natural associate (Akhurst, 1983). Poinar (1986) indicated that S.
(=Neoaplectana) glaseri has a greater ability to tolerate and even benefit from other
bacterial species than do other Steinernerna species, and hypothesized that this
characteristic linked it to a less-evolved condition. In fact, although both Xenorhabdur
spp. and Photorhabdus spp. have been isolated exclusively from their respective freshly
harvested U symbionts in nature, many other bactecial species and genera have been
reported to be associated with Steinemema spp. and Heterorhabditis spp., especially if
the nematodes have been maintained in culture for a long p e n d (Weiser, 1962; Poinar
and Thomas, 1965; Lysenko and Weiser, 1974; Botmare et al., 1983; Apillera et al*,
1993; Jackson et al., 1995).
Uniike the Steinememu - Xenorhbdus association, spccificity appears to be
greater in Heterorhabditis - Photorhubdus complex in both nutrient cequirement and in
symbiont retention. Although various isolates of Heterorhabditis spp. have k e n cultured
on diets comprising bacteria derived from other heterorhabditid nematodes, many
experimental recombinations have been unsuccessful (Han et al., 1991; Gemtsen and
Smits, 1993, 1997). In some cases, even where growth and reproduction occumd, the Us
failed to retain the bacteria and the syrnbiosis has degenerated (Gerritsen and Smits,
1993).
The infection of an insect host by Us of the nematode is the beginning of the
tripartite nematode-bacterium-insect association (Fig. 1). The Us of the nematodes carry
the syrnbiotic bacteria in their intestine and use different foraging strategies to search for
a new insect host (Poinar et aL, 1980b; Poinar, 1990; Campbell and Gaugler, 1993). They
release the bacterial cells after entering the insect's hemocoel by way of natural openings
(spiracles, mouth and anus). In the case of Heterorhabditis, the Us may bore directly
through the insect cuticle. The syrnbiotic bacterial cells and nematode develop and
multiply, and together they kill the insect host usually within 24 - 48 h. The bacteria
provide nutrients for the nematode by breaking down insect tissue andor by acting as
food source themselves for the nematode, and by maintaining an optimal environment for
nematode development by producing different antimicrobial agents that rninimize the
competition from other micrmrganisrns (Dutky, 1959; Paul et al., 198 1). In this protected
environment, Steinemema continues to develop into amphimictic females and males
while Heterorhabdirs develops into a first generation of hermaphrodites (Poinar, 1990).
The subsequent, second generation of both Steinemenia and Heterorhabditis consists of
amphimictic females and males. Depending on the prevailing conditions in the insect
cadaver two or three generations of nematdes cm develop (Poinar. 1990). When the
Fig. 1. Generaîized life cycle of entomopathogenic nematdes,
Steinernema spp. and Heterorhabditk spp.
Us emerge frorn cadaver d
Us search for new host *
J 9
2nd andlor 3rd generation of Us enter host via nematodes fil1 the cadaver cuticle or openings
/ Bacterial symbiont
Host dies and both nematode and bacterial symbiont develop
nutrient conditions become lirniting, an alternative developmental pathway leads to the
development of Us of the nematode. It is this U stage that emerges from the insect
cadaver and carries the bacterial symbiont to a new host to start a new infection cycle
(Poinar, 1 990).
In soil. the successfÙl infection of an insect host by the nematode - bacterial
complexes depends on many non-biotic and biotic factors. The nematodes and their
bacteria are subject to predation. parasitism and competition before they reach the insect
host. Some bacteria. fungi and invertebrates may negatively affect the soil population of
this mobile dispersal phase (Kaya and Koppenhofer, 1996). Even after gaining entry io
the host, the nematode-bacterium complexes may still face intra- or interspecific
competition. The intra-spccific competition within the insect host affects progeny
production of the entomopathogenic nematodes (Kaya and Koppenhofer, 1996). Both
field surveys and in vivo experiments have shown that two or more species of
entomopathogenic nematodes may occur simultaneously in the insect where they compete
for nutrients (Kondo, 1989; Stuart and Gaugler. 1994). The successful colonization of the
host appears to depend on inoculum size, development time of the nematodes, bacterial
symbiont and host species (Alatome-Rosas and Kaya, 1991; Boemare et al., 1997;
Koppenhofer and Kaya. 1996). Two different steinemematid spccies CO-infecting a host
have a potential to co-exist and develop because some steinemematids utilize the
bacterial symbionts of other steinemematids (Akhurst, 1983; Aguillera and Smart, 1993;
Koppenhofer and Kaya, 1996). However, the speed of development of the nematode and
the lower degree of specificity with the bacmial symbiont appcars to determine which of
the nematode species colonizing a cadaver will be successful (Kondo, 1989; Koppenhofer
et al., 1995). When S. carpocapsue and H. bacteriophora CO-infected a host, S.
carpocupsae developed inside the cadavers in nearly d l cases (Alatorre-Rosas and Kaya,
1991). Perhaps heterorhabditid nematodes require mon time to release their symbiotic
bacteria than steinemematids (Alatorre-Rosas and Kaya, 199 1) or their bacterial symbiont
is inhibited by antibiotics or bacteriocins produced by bacterial symbionts of the
steinernematid (Akhurst, 1982; Boemare et al., 1993b; Thaler et al., 1997). Foraging
strategies of the Us influence the type of hosts encountered and therefore, influence inter-
specific competition between entomopathogenic nematodes. Although competition occurs
between entomopathogenic nematodes, two or more species of entomopathogenic
nematodes may successfully CO-exist in the soil by having different foraging strategies.
These strategies separate nematode species spatially and enable the nematodes to occupy
a different niche. This may result also in them having a clumped distribution (Kaya and
Koppenhofer, 1996).
These nematode - bacierium complexes interact with other nematode species in
the soil. Some mononchid and dorylaimid nematode species prey upon Steinemerna spp.
(Ishibashi and Kondo, 1986). These authon also reported that the addition of S. feltiae
DD-136 at 10,000 W l û û ml soil or S. glusen' at 2,500 IJsIlûû ml soil or in bark compost
samples caused a rapid decrease in the population density of the nematodes in the soil,
especially one week after application of the nematodes. However, these nematode
populations recovered or surpassed their initial levels within 1 to 8 weeks depending on
the nematode genus. Plant-parasitic nematodes, such as stubby-mot, ring, and spid
nematodes, were suppnssed significantly throughout the 8 week period, while rhabditids
increased several times above original level. The filtrate fkom a DD-136 nematode
suspension (5 x 10 Us150 ml incubated overnight) did not affect 12s of M. incognita.
Consequently, the authors suspected that competition for space or habitat contributed to
the observed population changes of these other soil-living nematodes. Bird and Bird
(1986) observed a similar inhibitory effect of S. gluseri on Meloidogyne javanica in
potted tornato seedlings. The authors (Bird and Bird, 1986) proposed that suppression of
M. javanica by S. glaseri was due io competition for space or habitat of the nematodes
because the Us of S. glasen are much larger and more active than the J2s of M. javanica
and both species cluster around the root tips. Similar inhibitory effects of Us of
Steinernema spp. and Heterorhabditis spp. on plant-parasitic and saprophagous
nematodes, such as M. javanica. Heterodera schachtii and Caenorhabditis elegans have
been reported (Richardson and Grewal, 1991; Gouge et al., 1994; Lopez-Robles, 1996).)
This inhibitory effect of the entomopathogenic nematode Us on populations of
plant-parasitic nematodes has been noted following inundative application of Us in the
field (Georgis and Kelly, 1997). The infection of turfgrass, banana or potato in the field
by Meloidogyne spp., Belonolaimus longicaudatus, Criconernella spp., Pratylenchus spp.,
Rodopholus similus and Heterodera spp., was significantiy deminished after an
inundative application of entomopathogenic nematodes (Georgis and Kelly, 1997).
Consequently, the potential of entomopathogenic nematodes for controlling plant-
parasitic nematodes, as well as insect pests, has been considered. The mechanism of the
observed inhibitory effects on other nematodes is not clear. Geocgi and Kelly (1997)
proposed that the inhibitory effect may be due CO cornpetition for habitat, enhanced pny-
predator response, or involvement of the bacterial secondary metabolites released from
infected insect cadavers.
In nature, many plants and rnicroorganisms produce substances toxic to
nematodes, and some of these substances also have other bioactivities (Chitwood, 1993;
Stadler et al., 1993; Betina, 1994; Anke and Sterner, 1997). A significant aspect of this
nematode - bacterium association of entomopathogenic nematodes is that the spbiotic
bacteria produce a variety of bioactive secondary metabolites, as has been shown in broth
cultures (Paul et al., 198 1 ; McInerney et al., 199 1 a, 199 1 b; Li et al., 1995a). The range of
these bioactive agents and their activities is summarized in Table 3.
The production of antibiotics by X. nemutophilus, a bacterial symbiont of
entomopathogenic nematode of the DD 136 strain of S. (=Neoaplectana) feltiae, was first
suggested by Dutky (1959). This antibiotic production is a cornmon property of
Xenorhabdus and Photorhabdus species. Most species of bacterial symbionts that have
been studied produce antimicrobial components in broth cultun that inhibit the growth of
a variety of bacteria, yeasts and fungi, many of which are of medicinal and agricultural
importance (Paul et al., 198 1; Akhurst, 1982; McInemey et al., 199 la, 199 1 b; Li et al.,
1995a, 1997a). It is generally believed that the antibiotics are important in maintainhg an
optimal, cornpetitor-free environment for the development of the nematode and bacterial
symbiont (Dutky, 1959; Paul et al., 198 1). Once killed, the infected insect host is a target
for fun@ and other decay-inducing organisms €rom the soi1 or the insect gut. The
presence of the various broad-spectrum antibiotics pmduced by the bacterial symbiont
appear to help maintain optimal nutrient conditions, in part by inhibiting the build up of
populations of cornpetitors (Paul et al., 1981; Thaler et al., 1997). However, linle is
known about the occurrence of these antibiotic metabolites in nematode-infected insects
(Maxwell et al., 1994; Iarosz, 1996). Recently, the hypotbesis of antibiotic inhibition was
Table 3. Bioactive agents 85sociated with or derived h m the bacterial symbionts,
Xenorhabdus spp. and Photorhabdus spp., of eotomopatbogenic nematodes
Bioactive agents Bacterial sources Bioactivi ties* References
Xenor habdins
Xenorxides
Xenocoumacins
indole derivatives
Nematophin
Genistein
S tilbene derivatives
An thraquinone
derivatives
Phages
X. nematophilus
X, bovienii
X. bovienii
X. nemutophilus
Xenorhabdus sp.
Xe bovienii
Xe nematophilus
P. luminescens
P. luminescens
P. luminescens
X. nematophilus
X. bovienii
X. beddngii
P, luminescens
Photorhabdus spp.
1,2,4,5 McInemey et al., 199 1 a
Li et al,, 1995a
1,2,5 Chen, 1996; Xu, 1998
L2,3 Mcbemey et al., 199 1 b
1,2 Paul et al., 1981;
Li et al., 1995a
4 2 Chen, 1996
Li et al., 1997a, 1997b
1 Sztaricskai et al., 1992
1,2 Paul et al., 198 1
Li et al., 1995b;
Hu et al., 1998
1 Richardson et al., 1988
Sztiuicskai et al., 1992
Li et al., 1995b
Hu et al., 1998
1 Poinar et al., 1980a
Boemare et al., 1992;
Baghdipian et al., 1993
to be continued
Table 3. (continued)
Bacteriocins X. beddingii
X. bovienii
X. nenratopophikcs
Xenorhabdicin X. nematophilus
Chitinases X, bovienii
X. nematophilus
P. luminescens
Protein crystai X, bovienii
Exo-and endotoxins Xenorhabdus spp.
Photorhabdus spp.
1 Boemare et al., 1992;
Baghdipian et al., 1993
Thder et al., 1997
1 Thaier et al., 1995
2 Chen et al., 1996
1 Yudina and Egorov, 1996
4 Dunphy and Webster, 1988
Ensign et al., 1990
Bowen and Ensign, 1998
Gou et al., 1999
-.
*: 1 =antibiotic, 2=antimycotic. 3=antiulcer, kinsecticidal, S=antineoplastic. Note that
not every denvative of a bioactive agent possesses al1 listed bioactivities of that agent.
questioned by Jarosz (1996). This author, based on a series of experiments, proposed that
a rapid and massive colonization of the insect body by symbiotic bacteria creates
unfavorable conditions for the growth and multiplication of bacterial (proteolytic)
contaminators, making the insect cadaver decay-resistant.
Our knowledge of the effect of the bacterial secondary metabolites on the insect
host (possibl y insecticidal), on competition among the prevailing bacterial species
(possibly antirnicrobial) and on the nutrition of the nematode spbiont has k e n
accumulating for decades. However, the possible involvement of these bacterial
metabolites in the symbiotic association between the nematodes and their associated
bacteria and in interactions between the nematode-bacterium complexes and other
organisms including soi1 nematodes is not fully known. In preliminary experiments, 1
showed that cultures of Xenorhabdw had nematicidal activity. Funher studies of this
nematicidal property were necessary in order to improve our understanding of the
symbiotic association between the nematodes and their bacterial symbionts and of the
interaction of the nematode-bacterium complexes with other organisms in the
environment. This could lead to improved in vitro production of the entornopathogenic
nematodes, better field efficacy when the nematode-bacterium complexes are applied as
biological control agents of insect pests and to the development of selective,
environmentally benign nematicides.
The objective of this study was to examine the nematicidai properties of the
metabolites of Xenorhabdus spp. and Photorhabdus spp. with particular refennce to their
chernical nature, nematicidal spectrum, in vitro and in vivo occurrence. biological roles
and their potential application.
CEINTER 2
GENEXAL MATERIAIlS AND METHODS
2.1 Sources of the nematdes and bacteria
Different species of entornopathogenic nematodes, Sleinemema spp. and
Heterorhabditis spp., were used in the snidy either for isolation of the symbiotic bacteria,
Xenorhabdus spp. and Photorhabdus spp., or for use in nematicidal and behavioral
bioassays. Al1 nematode and bacterial isolates and species used in the study were from the
laboratory collection of Dr. J. M. Webster (Department of Biological Sciences, Simon
Fraser University), and their initial sources are listed in Table 4.
2.2 Isolation, maintenance and culture of bacterial symbionts
Stock cultures of most bacteriai isolates fiom the entomopathogenic nematodes
that are listed in Table 4 were prepared previously by Dr. G. Chen (Welichem
Technology Corp., British Columbia) in Dr. J. M. Webster's laboratory. Photorhabdus
luminescens C9 was re-isolated fiom H. megidis 90 and P. luminescens MD was isolated
for the first time from Heterorhabditis sp. HMD.
To isolate the symbiotic bacteria P. luminescens C9 and P. luminescens MD, the
Us of their respective nematode symbionts, H. megidis 90 and Heterorhubditis sp. HMD
(Table 4), were collected separately fmm nematode-infected G. meilonella lame in Petri
dishes (Mracek and Webster, 1993). The Us were then allowed to pass through two layers
Table 4. Species and sources of nematodes and bacteria used in this study
Nematode Bacteria* Source of the
(species and isolates) (species and isolates) nematode isolates
Entomo~athogenic nematode-bacterium com~lexes
Steinernema carpocapsae Xenorhabdus nematophilus
B.J
DD136
Al1
BC 1
19
27
XQl
S. feltiae
A21
CH-S-MER
Dl
Al1
BC 1
19
27
ATCC 39497
X. bovienii
A2 I
Dr. H. Yang, Chinese Acad.
of Agriculture, Beijing, China
Dr. O. O. Poinar, Oregon
State University, USA
Biosys, Columbia, MD. USA
Soil sarnple: British
Columbia
Dr. W. M. Brooks, North
Camlina State University,
USA
Biosys, Columbia, MD, USA
American Type Culture
Collection, MD, USA
Soil sample: Memt, British
Columbia
Dr. J. M. Grunder, Swiss
Federal Research Station,
Waàenswil, Switzerland
to be continued
27
Table 4 (continued)
S. glaseri
NC19
S. glaseri
S. kushriiai
S. puertoricense
Heterorhabdilir sp.
HMD
Heterorhabditis sp.
Spain
H. bacteriophora
Oswego
H. mare la tus
H. megidis
90
Photorhabdus luminescens
MD
Photorhabdus sp.
Spain
P. luminescens
Oswego
Photorhabdus sp.
P. luminescens
c9
Dr. K. H. Kaya, University of
California, Davis, USA
Dr. K. H. Kaya
Dr. Yam;rkita, Forestry and
Forest Research Institute,
Tsukuba, Ibaraki, Iapan
Dr. J. Roman, Agiculturd
University of Puerto Rico,
Rio Piedras, Puerto Rico
Dr. R. Gordon, Mernorial
University, Newfoundland
Dr. 2. Mracek, Czech Acad.
of Science, Ceske
Budejovice, Czech Republic
Dr. 2. Mracek
Dr, E. S. Shields, Corne11
University, Ithaca, W. USA
Dr. R. E. Berry, Oregon State
University, Corvallis, USA
Soi1 sample: Sumerland,
British Columbia
to be continued
Table 4 (continued)
Bacteriai-feedine nematode
Caenorhabdiris elegans (wild type)
Fun~al-feedine; andor dant-parasitic nematodes
Ap helenchoides rhytium
Bursaphelenchus xylophilus
BC
BI mucronatus
French
Obliaate plant-oatasitic nematode
Meloidogyne incognita
Dr, D. L. Ballie, Simon
Fraser University, British
Columbia
Dr, R. V, Anderson,
Agriculture and Agri-food
Canada, Ottawa
Dr. R. V, Anderson, conifer
tree, British Columbia
Dr. R. V, Anderson,
conifer tree, Quebec
Dr, G. de Guiran,
Stade Res. de
Nematol. et de Genetique
Moleculaire des Invert.,
Antibes, France
Dr. I. W. Potter, Agriculture
and Agri-food Canada,
Vineland Station, Ontario
--
*: Spbiotic bacteria were isolated fiom their respective nematode symbionts.
29
of wet-strength (WS) paper tissue (Kimwipesa , Kimberly-Clark Corp., USA) to ensure
that only active Us were collected. They were surface sterilized by immersion in 0.2%
thimerosal for 2 h and washed four times with phosphate-bufîered saline (PBS) (Dunphy
and Webster, 1984). They were then homogenized, and the suspension was streaked ont0
tryptic soy agar (TSA) Oifco), containing 25 mg of the dye bromothymol blue pet liter
(TSAD), and incubated at 25OC in the dark. After 48 h, typical, isolated pnmary form
colonies of the bacteria on the plate were selected and colonially purified on TSAD
plates. The identity of the bacteria was confinned by their morphologicd, biochernicai
and physiological characteristics (Thomas and Poinar. 1979; Akhurst, 1980; Boemare and
Akhurst, t 988).
For long term storage of the bacterial cultures, 48 h-old prirnary form colonies of
P. luminescens C9 and P. luminescens MD grown separately on TSA plates were
removed and suspended in 128 sterilized sucrose solution, which was then freeze-dried
and stored at -20°C. Freeze-dry powders of other bacterial isolates used in the study were
prepared previously by Dr. G. Chen. For routine maintenance of the bacterial cultures, the
powder of the freeze-dried stock cultures of the bactena was inoculated separately ont0
TSA or TSAD plates, then subcultured weekly on TSA or TSAD plates. Cultures were
discarded once they had been subcultured eight tiws.
Broth cultures of the bacteria were prepared as described below. A loophil of a 48
h-old bacterial culture grown on a TSA or TSAD plate w u inoculated into an Erlenmeyer
flask containing tryptic soy broth (TSB) (Difco) (50 ml in 125 ml flask or 100 ml in 200
ml flask). The flask was shaken on a gyratory shaker at 150 rpm for 24 h in the dark
(25OC) to prduce the seed culture. Different quantities of the seed culhm broth (1 or 1 0
ml), depending on the experiments, were added to new flasks containing TSB, Luria
broth base (LB) (Miller, Sigma), or nutrient broth (NB) (Difco) (50 ml medium in 125 ml
flask or 8 0 ml medium in 2 L flask) and shaken as described above for 1 to 5 d.
To prepare the cell-free (CF) culture filtrate, the bacterial culture broth was
centrihiged (13,000 g, 10 min at 4OC) and the supernatant was then filter sterilized (0.2
pm pore size) (MiIlipore@, Millipore Products Division, Bedford, MA, USA).
Al1 bacterial tram fer and maintenance was conducted under standard, sterile
conditions, and the bacterial cultures either in solid or liquid medium were incubated at
25'C in the dark, unless otherwise stated.
2.3 Maintenance and culture of nematodes
Al1 entomopathogenic nematodes were maintained in the laboratory at room
temperature by passaging through last-instar G. mellonella larvae, which were supplied
by the Insectary of the Department of Biological Sciences, Simon Fraser University.
Infective juveniles of the nematodes were collected in a water trap in a Petri dish within 2
to 4 d of their emergence from the infected G. mellonella cadavers (Mracek and Webster,
1993). Only those active Us that passed through two layers of the WS paper tissue were
collected. These Us were washed several times with distilled water and the excess water
was removed with a pipette. The collected Us were either surface sterilized as described
above or not treated further. niey were then concentrated to the desind density in
sterilized distilled (SD) water or distilled water befon use, by nmoving excess water
with a pipette.
Bacterial-feeding nematodes, C. elegans, were cultured on freshly prepared
Escherichia coli culture growing on NGM medium (Sulston and Hodgkin, 1988) in Petri
dishes at room temperature. Fourth-stage juveniles (J4s) and adults of the nematode were
washed from the surface of the plates and rinsed thoroughly with SD water. They were
then concentrated to the desired concentration in sterilized M9 buffet (Sulston and
Hodgkin, 1988) by removing the excess buffer solution with a pipette.
Fungal-feeding andlor plant-parasitic nematodes, A. rhythm and Bursupitelenchus
spp., were cultured separately on freshly prepared fungal cultures of Botrytis cinerea
grown on potato dextrose agar (PDA) in Petri dishes (2S°C) (Ishikawa et al.. 1986).
Mixed populations (mainly J4s and adults) of the nematodes were washed from
condensation water on the covers of the Petri dishes, and passed through two layea of
WS paper tissue so as to collect only active nematodes. These nematodes were then
concentrated in distilled water by removing surplus water with a pipette before use.
The obligate plant-parasitic nematode, M. incognita, was maintained on potted
tomato plants, Lycopersicon esculentum (cv. Rutgers; seeds courtesy of Dr. K. R. Barker,
University of North Carolina. USA) in the greenhouse. Hand picked egg sacs of a golden
to light brown color were immersed in shallow water (-2 mm) at 2S°C in the dark, and
only those J2s that hatched from the eggs after the first 24 h and before the 8th d were
collected daily and rinsed thoroughly with distilled water or SD water before use.
Al1 nematode sarnples listed above were used in experiments immediately after
their appropriate preparation from their respective cultures.
2.4 Bioassays for nematicidai activity
2.4.1 Activity of cell-free culture filtrates
The test CF filtrate, either original strength or diluted with SD water, was added to
a small, sterile Petri dish (10 x 35 mm, ComingO) with 10 pi (5 pg/N ) streptomycin
sulfate (SMS, Sigma) (Otopro et al., 1988) solution, and into this was added a 20 pl
nematode (- 100 nematodes) suspension to make 1 ml of final test solution. The dish was
sealed with Parafilm and mortality of the nematodes was checked as described below
after 24 h incubation of the dish at 2S°C in the dark. The culture medium, either original
strength or diluted to a strength similar to that of the test filtrate, was adjusted to the same
pH as that of the test filtrate using 6N HCl or 6N NaOH. It was then filter sterilized and
tested similariy to serve as the control. The control also contained 10 pl (5 pg/pl ) SMS.
The above procedure was conducted under standard, sterile conditions.
2.4.2 Activity of metabolic compounds
Depending on the solubility of the test compound or mixture of compounds and
on the toxicity of the solvents to the test nematodes, the pure compound or crude mixture
was first dissolved in a selected type and quantity of a solvent, such as dimethyl
sulphoxide (DMSO), polyethylene glycol (PEG), ethanol or methanol, and diluted with
distilled water so as to form the stock solution. Different arnounts of the freshly pnpared
stock solution, depending on the experirnent, were added separately to the small Petri
dishes (10 x 35 mm) and further diluted with distilled water. Then, 20 pl of nematode
suspension (-100 nematodes) was added to the solution in the Petri dish to make 1 ml of
fuial test solution in each dish. The mortality of the test nematodes was determined as
described below after 24 h incubation of the dish at 2S°C in the dark. Control dishes
contained only the solvent at the highest concentration used in the test solutions.
2.4.3 Mortality of the test nematodes
Mortality of the nematodes in test solutions was determined under the
stereomicroscope (25 x). The nematodes that were immobile and did not response when
probed repeatedly with a fine bnstle were considered dead and those that were immobile
but responded when prokd were considered paralyzed with temporarily impaired
mobility (see also Mcleod and Khair, 1975; Birch et al., 1993; Atta-Ur-Rahman et al..
1997). These criteria were confirmed in preliminary tests where none of the
nonresponding nematodes revived following subsequent transfer to aerated water for up
to 24 h.
2.4.4 Antibacterial activity
An agar diffusion test (Hewitt and Vincent, 1989) was used during the process of
isolating the metabolites from the bacterial cultures to detect the antibiotic metabolites. A
100 pi spore suspension of Bacillus subtilis (3.3 x 109/ml) was pipetted ont0 a TS A plate,
and evenly spread over the surface of the plate with a stenle spreader. The plate, with the
lid open, was then dried in a sterile laminar-tlow hood for about 10 min. Four to six
wells (diameter of 5 mm eadi) were made in the inoculated agar medium in the plate
using a sterilized glass tuk, and the agar plugs wen nmoved Then 30 pl solution of the
test compound, which was fimi dissolved in methanol, was added to each well. The plate
was covered and incubated at 3S°C over night. The size of the clear zones around the
wells in the B. subtilis plates was a measure of the strength of the activity of the test
compound.
2.5 Statisticai analysis
Each expriment in this study had at least three replicates for each treatment and
the experiment was npeated at least once unless othewise stated. Experimental data
were expressed as means I standard errors. The data were analyzed using Proc Mixed
(SAS, SAS hstitute hc., Gary, NC) to determine whether there were significant
differences in treatments. and a Bonferroni correction was used to detect significant
diffennce (P < 0.05) between treatment means.
CHAPTER 3
NEMATICIDAL PROPERTIES
OF IN VITRO CüLTURES OF THE B A C T E U
3.1 Introduction
Xenorhabdus spp. and Photorhabdw spp. are well known for their ability to
produce insecticidal and antimicrobial substances in cultun broth (Table 3). These
metabolites are believed to play an important roie in the nematode-bacterium-insect
association (Dukty. 1959; Paul et al., 198 1; Bowen et al., 1998). In preliminary
experiments, 1 discovered that the culture broths of Xenorhabdus had nematicidai
properties (Chapter 1). Since nothing was reported about the nematicidal properties of the
metabolites produced by Xenorhabdus spp. and Photorhabdus spp., many questions
arose. For exarnple, is the nematicidal activity only an occasional or constant, cornmon
property of the in vitro bacterial cultures of Xenorhabdus spp. and Photorhabdus spp.?
How do the culture conditions affect nematicidal activity of the bacterid cultures? To
answer some of these questions and aiso to establish a foundation for further study of the
nematicidal properties of Xenorhabdus spp. and Photorhabdus spp., the following
experiments were done to investigate: 1) the nematicidal activity of in vitro cultures of
Xenorhabdus spp. and Photorhabdus spp.; 2) the infiuence of bacteriai culture conditions
on nematicidal activity of the bacterial cultures; 3) the nematicidai activity of the organic
and aqueous fractions of the bacterial cultures and 4) to investigate the nematicidal
activity of some known antibiotics produced by Xenorhabdus spp.
3.2 Materials and methods
3.2.1 Bacteria and nematodes
The following bacterial strains, X. nematophilus All, BCl, Dl, 19, 27 and ATCC
39497, X. bovienii A2 1 and P. iuminescens C9 and MD (Table 4), were used in a series of
experiments. The bacteria were maintained and cultured as described in section 2.2 or
section 3.2.2.
Two economically important species of plant-parasitic nematodes, root-hot
nematode. M. incognita, and pine-wood nematode, B. xylophilus BC, were used as test
nematodes in the nematicidai bioassays in this study. Meioidogyne incognita is an
obligate plant-parasitic nematode that has a wide host range and is a major cause of lower
crop yields worldwide (Sasser and Carter, 1985). Bursaphelenchus xylophilus is both a
plant-panisitic and hingal-feeding nematode that caused multimillion dollar loses to pine
forests, especially in some Asian countries (Mamiya, 1984; Sutherland and Webster,
1993). These test nematodes were cultured and prepared as described in section 2.3. They
were collected, washed thoroughly with SD water and concentrated to the appropriate
density (- 100 nematoded20 pi water) before use.
3.2.2 Preparation of cell-f~e filtrates
Seed cultures of each bacterial strain wen prepared separately by adding a loopful
of the respective, 48 hsld bacteriai culture grown on TSA or TSAD plates to a conical
flask (125 ml) containing 50 ml TSB. The flasks were shaken at 150 rpm on a gyratory
shaker at 25OC for 24 h in the dark. One rnilliliter of the seed culhire was then pipetted
into each of three new conicai flasks containing 50 ml TSB, and the flasks were shaken as
above for 4 d. The 4 d-old culture broth from each of the three flasks was centnfuged
(13,000 g, 4OC, 10 min) separately and filter sterilized to obtain the CF filtrates. The CF
filtrates of each strain were then tested, either undiluted or diluted with SD water, for
nematicidal bioassays.
Al1 experiments involving CF filtrates were conducted under standard. sterile
conditions, and 10 pl SMS solution was added to each test solution to prevent possible
bacterial contamination. Culture media containing the same quantity of SMS was tested
similarly and served as the control.
3.2.3 Nematicidal activity of bacterial strains and species
To investigate the nematicidal activity of different strains of Xenorhabdus spp.
and Photorhabdus spp. when cultured in vitro, the CF filtrates of each bacterial strain
were prepared as described above. They were tested either undiluted or diluted (114
strength of the original CF filtrates). In the undiluted tests, a 20 pl suspension (-100
nematodes) of J2s of M. incognita wûs added to 970 fl CF filtrate and 10 pl SMS
solution in a small Petri dish (35 x 10 mm). For the diluted test, 250 pi CF filtrate, 10 pi
SMS solution and 20 pi suspension of J2s of M. incognita were added to 720 p i SD water
in a small Petri dish to make 1.0 ml final test solution. The dishes were sealed with
Parafilm, incubated in the dark for 24 h, and the nematicidal activity was examined under
the ste~ornicroscope (25 X ) as described in section 2.4. Tryptic soy broth, containing
10 pl SMS solution and adjusted to the same pH as that of the test filtrates, was filter
sterilized and served as the control.
3.2.4 Nematicidal activity of the bacterial cultures against different nematode species
To evaluate any differential nematicidai effect on two species of nematodes,
diluted CF filtrates (112 or 114 strength of the original CF filtrates) of X. bovienii A21, X.
nematophilup BCI and P. luminescens MD were prepared as described above and tested
against J2s of M. incognita and J4s and adults of B. xylophilus BC. Tryptic soy broth,
containing 10 pi SMS solution and adjusted to the same pH as that of the test filtrates,
was filter sterilized and served as the control.
3.2.5 Influence of bacterial culture conditions on nematicidal activity of the culture
filtrates
3.2.51 Culture media
To investigate whether the type of cultun media used influence nematicidal
activity of the bacterial culture, 4 d-old CF filtrates of X. nematophilus BCI grown on
TSB, NB and LB media, three commonly used culture media for Xenorhabdus spp. and
Photorhabdus spp. (Thomas and Poinar, 1979; Li et al., 1995b). were prepared as
described in section 3.2.2. The nematicidal activity of these CF filtrates (at 112 and 114
strength of the original filtrates) was tested, as described in section 3.2.3, against J2s of
M. incognita and J4s and adults of B. xylophilus. Tryptic soy broth, NB and LB media,
containing 10 pi SMS solution and adjusted to the same pH as that of the test filtrates,
was filter sterilized and served as the controls.
3.2.5.2 Age of the bacterial culture
To determine whether the aematicidal activity is related to the age of the bacterial
culture, X. bovienii A2 I , X. nematophilus BC 1 and P. luminescens MD, were cultured as
described above in section 3.2.2 for up to 5 d. Three flasks of bacterial culture of each
species were taken randomly every day from the shaker, and the CF filtrates were
prepared separately. The diluted CF filtrates (114 strength of the original filtrates) were
tested against I2s of M. incognitcr as described in section 3.2.3. There were three
replicates for each treatment. Tryptic soy broth, containing 10 pl SMS solution and
adjusted to the sarne pH as that of the test filtrates, was filter sterilized and served as the
control,
Bacterial growth and pH change of the culture broths also were monitored daily.
At each sampling tirne 1 ml broth was taken from each of the above three flasks of each
bacterial species and diluted ten fold with TSB. The absorbance of the bacterial
suspensions was then measund immediately at 600 nm on a Milton 3000
spectrophotorneter. Cell-free culture filtrate, diluted ten fold from that of the culture
broth, served as the control. The pH of the broth in each of the three flasks of each
bacterial species was measured directly using a pH meter (mode1 32 1. Coming O).
3.2.5.3 pH of the culture broth
To investigate whether the pH of the culture broth influenced the nematicidal
activity of the CF filtrates against M. incognita, the pH of the 4 d-old CF filtrates of X.
bovienii A21 and X. nematophilus %Cl was adjusted with 6N HCl and/or 6N NaOH to
pH 5, 6, 7, 8 and 9, respectively. The filtrates were then filter sterilized and their
nematicidal activity against J2s of M. incognita were tested, as described in section 3.2.2.
TSB medium (pH 7.2, Difco) alone was adjusted similarly to pH 5, 6. 7, 8 and 9. filter
sterilized and semed as control.
To hirther investigate the effect of extreme pH of the test solution on 12s of M.
incognita and J4s and adults of B. nylophilus, buffer solutions at pH 10.0 (VWRB) and
pH 5.0 (Fisher@) were each diluted with distilled water to 1/30 of their original
concentration. The nematicidal activity of the diluted buffer solutions was tested against
the two nematode species as described in section 2.4. Buffer solution (pH 7.0, VWR@)
was diluted and tested similarly to serve as a control.
3.2.6 Nematicidal activity of organic and aqueous fractions of the culture filtrates
To provide information on the chernical nature of the nematicidal metabolites
mising from the culture filtrates, the organic and aqueous fractions of the bacterial culture
of each species were obtained as described below. The 4 d-old culture broths of X.
bovienii A2 1. X. nematophilus BC 1 and ATCC 39497 and P. luminescens C9 and MD
were prepared as described in section 3.2.2. The broth frorn the three flasks of each of the
five bacterial strains was cornbined into five batches and centrifuged (13.000 g, 10 min at
4OC) to obtain the respective supernatanu. nie filter-sterilized supmatant of each
bacterial strain was extracted separately in a separating hinnel with an equal volume of
ethyl acetate four times for 30 min each. The extracts of the same supernatant for each
bacterial culture were combined and dried under vacuum at 30°C on a rotary evaporator
to obtain the organic fraction for each culture. The remaining liquid fraction in each of
the separating funnels after ethyl acetate extraction was separately freeze-dned to obtain
the comsponding aqueous fraction.
The organic and aqueous fractions of each bacterial culture were tested for their
nematicidal activity agahst J4s and adults of B. xylophilus and JZs of M. incognitu. The
test solutions of the organic fractions were prepared by dissolving the extracts in either
DMSO and diluted with distiiled water to 2,000 pglml (5% DMSO, v/v), for the test
against B. xylophilus, or in DMSO and Triton X-100 and diluted with distilled water to
1,000 p g l d (1% DMSO plus 1% Triton X-100, v/v), for the test against M. incognita.
Into 980 pl of the test solution in a small Petri dish (35 x 10 mm) was added 20 pl
nematode suspension of J4s and adults of B. xylophilur BC or J2s of M. incognita to
make 1 ml test solution. There were three replicates for each treatment. The dishes were
sealed, incubated for 24 h and the mortality of the nematodes checked as described in
section 2.4.
To pnpare the test solution of the aqueous fraction. the freeze-dned material was
dissolved and diluted with distilled water to the sarne concentration of TSB as original
culture (27.5 mg /ml; Difco) in which the bacteria had grown. The solution was then filter
sterilized and tested against J2s of M. incognita and J4s and adults of B. xylophilus as
described in section 2.4. Tryptic soy broth that had the pH adjusted with 6 N HCI or 6 N
NaOH, comsponding to thot of the aqueous fractions, was filter sterilized and served as
control.
3.2.7 Nematicidal activity of some known antibiotics pmduced by Xenorhabdus spp.
A few of the antibiotics isolated from cultures of some other microorganisms
(e.g. Lechnm papyracewn and Penicillium brejèIdianum) have been found to be dso
nematicidal (Stadler et al., 1993; Anke and Stemer, 1997). Consequentiy, a test was done
to determine the possible nematicidal activity of some known antibiotics produced by
Xenorhabdw spp. and Photorhabdur spp. in broth cultures. The antibiotics tested were
xenorhabdins L and 3 (Xu, 1998), xenorxides 1 and 2 (Li et al., 1998), indole derivatives
1- 4 (Paul et al., 198 1 ; Li et al., 1995a) and nematophin (Li et al., 1997a). Xenorhabdins
1 and 3 were prepared by the method descnbed by Xu (1998) and the rest of the
antibiotics were kindly supplied by Dr. I. Li (JR laboratones uic., Btitish Columbia) and
Dr. G. Chen.
Test solutions of each of these antibiotics were prepared by dissolving each of
them in the specified solvent and diluting them with distilled water to a concentration of
200, 500 or 1,000 pgfml. For testing against J4s and adults of B. xylophilus, the solvent
was DMSO (5%, vfv) or DMSO (5%) plus Ttiton X-100 (ln, vfv). For the test against
J2s of M. incognito, the solvent was DMSO (146, v/v) plus Triton X-100 ( 1 %, v/v).
Nematicidal activity was examined as described in section 2.4. DMSO solution or the
solution of the DMSO plus Triton X-100 mixture at the same concentration as that in the
test solutions, served as controls.
3.3 Results
3.3.1 Nematicidal activity of bacterial strains and species
The CF filtrates of dl bacterial svains and species tested showed nematicidal
activity against J2s of M. incognito, but the activity varied depending on the
concentration of the CF filmte and on the strains and species of the bacteria from which
the CF filtrates onginated. DiIuted CF filtrates (diluted to haif strength) of the Merent
bacterial cultures caused 100% mortality of J2s of M. incognita. Howcver, when diluted
to 114 strength, the filtrates of different isolates and species of the bacteria caused
mortality ranging from 2.4 to 96.3% and paraiysis fiom 3.7 to 75.9% (Fig. 2). Higher
percentage mortality of M. incognita was caused by CF filtrates ( 1/4 strength) of BC 1, D 1
and 27 isolates than by other isolates tested (P < 0.05). Diluted TSB (diluted to 1/4
strength) did not cause any nematode rnortality or paralysis (Fig.2).
3.3.2. Nematicidal activity of different cultures against different nematode species
CF filtrates of X. bovienii A2 1, X. nematophilus BC 1 and P. luminescens MD,
respectively, showed nematicidal activity against both M. incognita and B. xylophilus
(Table 5). in general, the bacteriai culiures were more active against M. incognita than B.
xylophilus. As well, the culture filtrate of P. luminescens MD was more active against B.
xylophilus than that of Xenorhnbdus spp. (P < 0.05).
3.3.3. Influence of bacterial culture conditions on filtrate nematicidal activity
The media composition of the bacteriai cultures infiuenced the nematicidal
zictivity of the culture filtrate (Table 6). Of the three media used. the culture filtrates of X.
nematophilus BCl grown in TSB and LI3 showed significantly stronget activity against
M. incognita than that grown in NB (P < 0.05).
The percentage mortality of 12s of M. incognita increased gradually as the age of
the bacterial cultures increased over a 5 d period (Fig. 3). At u r h sampling time,
CF filtrates of X. nematophilus BC1 showed significantly stronger activity than did the
other filtrates against J2s of M. incognita and P. luminescens MD had the least activity.
Fig. 2. Percentage mortality and paralysis of second stage juveniles of Meloidogyne
incognita enrpod to diluted, cell-Cree culture filtrates (4 dsld filtnted culture
diluted to Y4 strength) of Xenorhubdus spp. and Photorhabdus luminescens. C:
control; Ml: isolate of Xenorhabdus bovienii; 19, 27, AU, ATCC, BC1 and Dl:
isolates of X. nemutophitus; C9 and MD: isolates of Phdorhabdus luminescens. n=6
except for Mates 19,27, Al1 and Dl where n d Bars represcnt standard errors of
the means.
Table 5. Percentage mortality of second stage juvenües of Melocdugyne incugnita
(MI) and fourth stage juveniies anà adults of Bursuphelenchus xgloplilus BC (BX)
exposeà to the düuted, celi-frec culture liltrates (4-d old filtrated culture and diluted
to i l 2 or î/4 strength) of Xenorhabdus spp. and Photorkabdus luminescens
Bactenalspecies Strains Dilution Mortality (95)
X. bovienii A2 i 112
X. nematophilur BC 1 112
P. luminescens MD 112
X. bovienii A2 L 114
X. nematophilus BC 1 1 /4
P. luminescens MD 1 /4
Controls (TSB)*
Data are expressed as mean f SE (n=6). Means in the same column followed by the same
letter are not significantiy different (P c 0.05).
*Diluted TSB (diluted to 112 or 114 of strength); pH of each control was adjusted to the
same pH as the corresponding CF filtrates of each bacterial culture.
Table 6. Percentage mortality of second stage juvedes of Melouiogyne incognita
(MI) exposed to the diluted, cell-free culture filtrates (4 dsld liltrated culture and
diluted to V2 or Y4 strength) of Xenorhubdus nemutophilus BC1 grown in tryptk
soy broth (TSB), Luria broth (LB) and nutrient broth (NB), respectively
Cell-free filtrates Dilutions Mortality (I)
TSB
LB
NB
TSB
LB
NB
TSB
LB
NB
- -
Data are expressed as mems f SE (n= 6). Means followed by the sarne letter are not
sipificantly different (P < 0.05).
*: pH adjusteci to the highest level(9.0).
Fig. 3. (a) Growth, (b) pH and (c) nematicidal activity against Meloidogyne
incognüu (second stage juveniles) of Xenorhabdus bovienii A21 (AZl), X.
nematophilus BC1 @Cl) and Photorhabdus luminescens MD (MD). Measurements
taken over time following the culture of these bacteria on tryptic soy broth. Bars
represent standard m o r s of the nieoas.
O 1 2 3 4 5 6
Age of the batcrial cultures (days)
X. nematophilus BCl and P. luminescens MD grew rapidly during the first 2 d but
thereafter the growth rate declined whereas X. bovienii A21 had a relatively low growth
rate (Fig. 3). Of the three bactena tested. X. nematophilus BCI had the pa t e s t ce11
density over the whole time period. The pH of al1 three bactehl cultures increased
gradually over 5 d (Fig 3), and the pH of the bacterial cultures at 5 d ranged from 8.5 to
8.9.
The nematicidal activity of the pH-adjusted bacterial filtrates increased with the
pH of the culture filtrates (Table 7). The higher the pH of the CF filtrates, the stronger the
nematicidal activity. However, when pH of the CF filhates were adjusted to below 7.0
there was no nematode monality of the filtrates, but there was still some percentage (- O -
30%) of nematode paralysis. Tryptic soy broth itself was not toxic to the nematode
whether its pH wu adjusted to 5.0 or 8.5. but it became toxic to the nematode when its
pH value was adjusted to 9.0 (Table 7).
The J2s of M. incognita and J4s and adults of B. xylophilus BC survived higher
(pH 10.0) or lower pH (pH 5.0) conditions in the buffer solutions for 24 h with no
monality, which indicates that higher pH itself was not a lethal factor against the
nematodes in CF filtrates or in the TSB controls that had been adjusted to pH 9.0.
3.3.4 Nematicidal activity of the organic and aqueous fractions of the bacterial cultures
The organic fractions of al1 five bacteriai cultures tested showed nematicidal
activity against J4s and adults of B. qdophilus BC but not against J2s of M. incognita and
dl aqueous fiactions that had been fmze-dried were inactive against both M. incognita
and B. xylophilus (Table 8).
Tabk 7. NematicidPI activity of the 4 d-old roll-free filtrates of Xenorhubdus
kvienii A21 (Ml) and X. nematophilus BCl @Cl) a g h t second stage juvedes of
MeIoidogyne inmgnita (MX) when the Ntrates were adjusted, usfng 6N NaOH or 6N
HCl, to pH values ranging from 5.0 to 9.0
Adjusted pH values
of the cell-free filtrates
A2 1 BC I TSB (control)
O* O O t O O I O
O * O O I O Of0
45.9I0.8 98.7f1.3 O f 0
100 * 0 100 f 0 Of0
nt nt O f O
100 f O l o f 0 1 O O I O
- - - -
Data are expressed as means i SE (n=3).
nt: not tested.
Table 8. Nematicidai activity of dried organic and aqueous fractions of
Xenorhabdus spp. and Photorhabdus luminescens against second stage juveniles of
Meloidogyne incognüa 0 and fourth stage j u v e n k and adults of
Bursuphelenchus xylophilus BC (BX)*
Bacterid cultures Nematicidal activity
Organic fractions Aqueous fractions
X. bovienii
A2 1 O + O O
X. nematophilus
BC 1 O ++ O nt
ATCC 39497 O + O O
P. luminescens
C9 nt + O O
MD nt + O O
-------- Controls
Solvents O O nt nt
TSB nt nt O O
*: The organic fractions were tested up to 1,000 pglml and aqueous fractions tested at
27,000 pg/d (=2.75% as in TSB control).
O: No activity. +: moderate nematicidai activity (percentage moriality < 50%). tt: strong
nematicidd activity (percentage mortaiity > 50%). nt: not tested n=3.
3.3.5 Nematicidal activity of some known antibiotics produced by Xenorhabdus spp.
None of the tested antibiotics showed nematicidal activity against 14s and adults
of B. xylophilus under the prevailing expecimental conditions (Table 9).
3.4. Discussion
The results demonstrate that nematicidal activity is a common properiy of
Xenorhabdus spp. and Photorhabdus spp. when they are cultured in vitro. Al1 tested
strains and species of the bacteria showed nematicidal activity against 12s of M. incognita
and J4s and adults of B. xylophilus. However, the activity varied with the bacterial strains
and species. CF filtrate of X. nemutophilus BCI, for example. caused nearly 100%
mortdity of J2s of M. incugnifa even when the filtrate was diluted four times, but the CF
filtrate of X. nematophilus 19 was not lethai when similariy diluted (Fig. 2).
Nematicidal activity of the bacterial CF Filtrate was significantly infiuenced by the
nature of the culture medium in which the bacteria had been grown. This difference in
nematicidal activity may result from differential bacterial growth, which may influence
qualitatively andlor quantitatively the secondary metabolites and, consequently, influence
the nematicidal activity of the bacterial cultures. The diffennce may also result from
differential activity of the metabolic pathways of the bacterium in different culture media.
It was reported that pH influences the nematicidal activity of test solutions in vitro
(Banage and Visser, 1965; Cayrol et al., 1989). The same is tme for bacterial cultures of
Xenorhabdus spp. and Photorhbdus spp. In general, for the same bacterid isolate the
higher the pH of the bacterial cultures, the stronger the nematicidal activity. The
Table 9. Nematicidal activity of some known antiblotics producd by Xenorhabdus
spp. when tested against fourth stage juveniles and adults of Bursuphelenchus
xy Ioph ilus
Bacteria
-
Antibiotics The maximum Mortality
concentration (%)
tested (pg/ml)
X. bovienii
A2 i
X. nematophilus
BC1
ATCC 39497
indole derivative 1 1,000
indole derivative 1 +2 1,000
indole denvative 3 1,000
indole derivative 4 1,000
xenomide I 500
xenomide 2 250
nematophin 1
xenorbabdin 1 500
xenorhabdin 3 500
relationship between the pH of the filtrates and their nematicidal activity was
demonstrated by the pH re-adjustment expriment. The nematicidal activity of the CF
filtrates against M. incognita decreased dramaticaily with decreasing pH of the filtrates,
especially at pH lower than 7.0. The results suggest that a major nematicidal metabolite is
either stable only under basic conditions or is itseif an alkaline substance.
The culture medium and pH may contribute to the total nematicidal activity of a
filtrate. When the pH of TSB was adjusted to 9.0, it became toxic to the nematodes
(Table 7), but neither TSB (pH 7.2 at original state) nor a buffer solution at pH 10.0
(section 3.3.3) caused any rnortality of the nematodes. The nsults indicate a synergistic,
toxic effect between high pH and TSB that contributed to the total nematicidal activity of
the filtrates.
The loss of nematicidal activity against M. incognita by both the organic fraction
and the freeze-dried aqueous fractions was puuling, because CF filtrates of Xenorhabdus
spp. and Photorhabdus spp. showed strong nematicidal activity against this species
(Tables 5 to 9; Figs. 2 and 3). The results imply thrt some active nemdcidal
metabolite(s) was lost or denatured dunng solvent extraction and the freeze-drying
process.
The results of these in vitro tests suggest that metabolites andfor factors that may
differ between species exist in the CF filtrates that are nematicidal. Firstly, the CF filtrate
of P luminescens MD showed stronger activity against B. xyhphilus than did that of X.
bovienii A21 or X. nematophilus BCI. though it had less activity than that of X.
nematophilus BCl against M. incognita. These observations support the concept of there
k i n g more than one nematicidal agent in the fütntcs and that they vary with bacterial
species. Secondly, CF filtrates of Xenorhabdus spp. were more active against J2s of M.
incognita than against the J4s and adults of B. xylophilus (Table 5). However, both the
organic and the freeze-dried aqueous fractions were inactive against J2s of M. incognita
but the organic fractions were still active against B. xylophilus under test conditions
(Table 8). These observations, together with pH related nematicidal activity and the
synergistic and toxic effect between pH and TSB, suggest that the overall nematicidal
activity of the CF filtrates is a result of complicated interactions involving multiple
metabolites andior factors.
In conclusion, the nematicidal activity of the cultures of Xenorhabdus spp. and
Photorhabdus spp was demonstrated. The activity was related to the type of the culture
media and to the age and pH value of the cultures. Of the three media used, the CF filtrate
of bacteria grown in TSB showed the strongest activity, and this activity increased
gradually over 5 d. Overall, the higher the pH of the cultures, the stronger the nematicidal
activity. Organic fractions of the bacterial cultures were active against B. xylophilus but
not M. incognita, and the f~eze-dried aqueous fraction were inactive against both B.
xylophilur and M. incognita. The fact that CF filtrates of Xenorhabdus spp. were more
active against nematodes under basic but not acidic conditions suggest that the major
active metabolite(s) is stable under basic condition or is an alkaline substance itself. As
well, the synergistic effect between pH and TSB at higher pH conditions and the loss of
the activity against M. incogrrita, but not against B. xyfophilus, by both fractions after
phase separation indicate that multiple nematicidai metabdites andor factors exist in the
CF filtrates, and that such metabolites a d o r factors may differ between bacterial species.
CRAPTER 4
ISOLATION, IDENTIFICATION AND IN VlTRO PRODUCTION
OF NEMATICIDAL METABOLITES FROM BACTEIUL CULTURES
4.1 Introduction
In Chapter 3 the nematicidal activity of the culture filtrates of different strains and
species of Xenorhabdus and Photorhabdus was demonstrated. The results showed that
the nematicidal activity was detectable in the ethyl acetate extracts of the bacterial
cultures rather than in the aqueous fraction of the bacterial cultures after the ethyl acetate
extraction. To hirther investigate the chemical nature of these nematicidal metabolites,
the chemical structure of each of the nematicidal metabolites produced in selected culture
broths of Xenorhabdus and Photorhabdus isolates was identified and the culture
conditions required for their in vitro production were determined.
4.2 Materials and methods
4.2.1 Bacteria and their broth cultures
The symbiotic bacteria, X. bovienii A2 1 , X. nematophilus BCI and ATCC 39497
and P. luminescens C9 and MD, were used.
To prepare bacterial cultures for isolation of nematicidal metabolites, a loopful of
the respective 48 h-old bacteria cuiture from a TSAD plate was added to a 200 ml
Erlenmeyer fiask containing 100 ml TSB. nie flask was shaicen on a gyratory shaker (150
rpm) for 24 h at 2S°C in the dark. The seed culture in the flask was then added totally to a
2 L Erlenmeyer fiask containing 800 ml TSB and shaken. as above, for 2 or 4 d. An
amount, generally more than 20 L, of the culture bmth of each of the above bacterial
strains was prepared. Harvested culture broth was processed immediately as described
below.
4.2.2 General procedures for isolation and identification of nematicidal metabolites
The general procedure for isolation and identification of nematicidal metabolites
from bacterial cultures is shown in Fig 4. The harvested culture broth was centrifbged
(13, 000 g, 4OC. 10 min) to obtain the cell-free supernatant. The supernatant was then
extracted three times with an equal volume of ethyl acetate in 2 L separating funnels. The
ethyl acetate extracts were combined, and the remaining aqueous fraction in the extracts
was removed by adding an appropriate amount of anhydmus sodium sulfate. The
resulting solution was filtered through glasswool in û glass funnel to remove the particles
of sodium sulfate from the solution. The filtrate was then dried under vacuum in a rotary
evaporator under 30°C to obtain the dry organic extract of the cell-free bacterial culture,
which was then subject to separation by gel column chromatography. Diffemnt
metabolites were eluted from the gel column and collected in 18 x 150 mm test tubes.
The collected solutions that contained mainly the sarne component wen combined and
dried as described above to obtain a single fraction. The resulting fractions were
subjected to further separation by gel column chromatography to obtain purified
metabolic compounds. Thin layer chmmatographic plates (TLC) (Kieselgel 60, F2!54,
Merck, Darmstadt, Germany) w e n used regularly to help in the separation process. Süica
Fig. 4. Fiowsbart showing the general isolation pcocess of the organic extracts from
broth cultures of Xenorhcrbdus spp. or Pliotorhabdus spp.
1 CELCFREE SUPERNATANT 1
1 ORGANIC EXTRACT 1
ethyl acetate extraction
1 ACTIVE FRACTIONS 1 1 INA- FRACTIONS 1
gel column chromatography
gel column
1 DETERMINATION OF CHEMICAL STIIUCïüRE(S) 1
chromatogiaphy
NMR, IR, MS, etc.
gel 60 @MO, EM Science, Darmstadt, Germany) was used in gel column isolation unless
otherwise stated, and al1 the solvents used were analytical standard. The isolation and
purification process described in section 4.2.3 was done jointîy with Dr. J. Li and the
chemical characterization of the purified compounds was done solely by Dr. J. Li.
Chernical structures were determined by NMR (nuclear magnetic resonance). IR (infra-
red spectrometry) and MS ( m a s spectra) analysis. Nematicidal bioassays using pine
wood nematode. B. xyhphilus BC. were conducted, as described in section 2.4, at each
isolation step to detect the nematicidally active fractions or compounds. Agar diffusion
tests, using B. subtilis as the test organism, were conducted. as described in section 2.4. in
order to detect possible antibiotic activity.
Due to the chemical complexity of the organic extracts, the detûiled isolation
procedure for the nematicidal metabolites produced by ench of the bacteria will be
described separately.
4.2.3 isolation and identification of nematicidal metabolites from cultures of
Photorhabdus luminescens and Xenorhobdus spp.
4.2.3.1 Isolation and identification of nematicidal metabolites produced by P.
luminescens C9
A total of 3.2 g of crude organic extract of P. luminescens C9 culture was
obtained, as described in section 4.2.1. The extract was loaded ont0 a silica gel column
(27.5 x 4.8 cm) and eluted with a mixture of ether and hexanes (6.0 : 4.0). One of the four
major fractions eluted showed nematicidal activity against B. xylophilus. The active
fraction (0.24 g) was hrther purified by gel column chtomatography (24.0 x 2.7 cm),
using a mixture of ethyl acetate and hexanes (1: 9) to obtain a white solid compound, ST
(0.18 g), which was both nematicidal and antibacterial, and its chemical structure was
further characterized. The remaining inactive fractions were not studied.
4.2.3.2 Isolation and identification of nematicidal metabolites produced by P.
luminescens MD
A totd of 7 g of crude organic extract of P. luminescens MD culture was obtained,
as described in section 4.2.1. The extract was separated by silica gel chrornatognphy
(column 41 x 4.8 cm) using a mixture of ethyl acetate and hexanes (1: 9) as the eluting
solvent (4 L). Five major fractions, A (0.73 g green-yellowish solid), B (0.4 g greenish
solid), C, D and E, were collected. Then, 100% methanol (1 L) was added to wash out
fraction F. Fractions A and B were determined by bioassay to be nematicidal against B.
xylophilus. The remaining fractions were inactive except for fraction C, which was shown
to be antibacterial and was studied later in a separate project.
Fraction A was further purified by gel chromatography (column 37 x 2.7 cm)
using a mixture of ethyl acetate and hexanes (0.8 : 9.2) as the eluting solution (2 L). One
major sub-fraction and a minor fraction were collected. The former gave a colorless
crystal, HD (0.7 g) and was confirmed as a nematicidal substance. The inactive minot
fraction was not analyzed further.
Fraction B was pucified by gel chromatography (column 28 x 2.7 cm), using a
mixture of ethyl acetate and hexanes (2.0 : 8.0) as the eluting solution (2 L). One major
and two minor sub-fractions were colîected and dcied under vacuum. The major sub-
fraction was a white miid compound, ST-1, and was confiied as a nematicidai and
antibacterial compound. The two minor sub-fractions were inactive and were not
analyzed further.
4.2.3.3 Isolation and identification of nematicidal metabolites produced by X.
nematophilus ATCC 39497
A total of 8.8 g of crude organic extract of X. nematophilus ATCC 39497 culture
was loaded ont0 the gel column (45 x 4.8 cm) and eluted initially with 40% ethyl acetate
in hexanes (2 L), then with 60% and 80% ethyl acetate in hexanes (2 L for each mixture),
respectively, and finally with 100% ethyl acetate (1 L). The column was then washed with
100% methanol(1 L). A total of 12 fractions were collected and their nematicidal activity
was tested, as described in section 2.4.
4.2.3.4 Isolation and identification of nematicidal metabolites produced by X.
nematophilus BC 1
A total of 2.0 g of crude organic extract of X. nematophilus BCl culture was
loaded onto the gel column (29 x 4.8 cm) and eluted initially with a mixture of ethyl
acetate and hexanes (2 : 8). The solvent was then changed to 100% ethyl acetate and
finally to 100% methanol. using 1 L of each solvent. Three fractions, A, B and C. were
collected. Fractions A and B were inactive and not studied further. The weakly active
fraction C was furcher separated in the gel colurnn (30 x 2.7 cm; C-18 reverse gel)
(BondapakB, Millipon Corp.. Milford USA;) and eluted initially with a mixture of
rnethanol and water (1 : 9, 400 ml). The eluate was changeci to 40% methanol in water
(400 ml), 70% methanol in water (400 ml) and f d l y to 100% methanol(400 ml) to get
the four major sub-fractions. The nematicidai activity of each of these sub-fractions was
then tested against B. xylophilus, as described in section 2.4.
4.2.3.5 Isolation and identification of volatile nematicidal metabolites produced by X.
bovienii A2 1 and P. luminescens C9
Prelirninary experiments indicated that a volatile metabolite from the bacterial
cultures was toxic to 12s of M. incognita. Consequently, the following experiments were
done to identify the volatile, nematicidai metabolites (VM) from the bacterial cultures.
In the collecting bottle of the evaporator was added 1 ml 6N HCl solution, and the
bottle was imrnersed in ice particles. A total of 200 ml of 4 d-old CF culture filtrate of X.
bovienii A2 1, which was cultured as described in section 3.2.2, was then dned on the
rotary evaporator at 35OC. The solution recovered in the collecting bottle was re-
evaporated to obtain a white solid (O. lg; the chloride salt of the VM).
The sarne volume of CF filmte from P. luminescens C9 culture, which was
cultured as described in section 3.2.2, was treated similarly as described above and a
similar while solid (O. 12g; the chloride salt of VM-1) was obtained.
The chloride salts were analyzed to determine their chernical structures. The
structures of volatile, nematicidal VM and VM-1 were detedned reversally based on the
chernical natures of their cornsponding white solid salts.
4.2.4 In vitro production of the nematicidal substances identified h m the bacterial
cultures
4.2.4.1 Establishment of standard curves for nematicidal metabolites VM, ST and HD
Standard curves of the three nematicidal metabolites, VM, ST and HD, produced
by Xenorhabdus spp. and Photorhabdus spp., were established using a Milton 3000
spectrophotometer. The standard curve of VM was detennined by a color reaction of VM
with Nessler's reagent (Gerhardt, 1981). A solution of VM in the cuvette reacted with a
known quantity of the agent to forrn a color cornplex. By measuring the W aborbance
(450 nm) of these complexes prepared from a series of standard solutions of VM and
comparing them with the concentrations of the VM test solutions, a standard curve was
established. For ST and HD, the standard curves were established by measuring directly
the UV absorbance of their respective standard solutions in cuvettes in relation with the
concentration of ST or HD. The detailed procedures are described below.
Standard curve of VM and its salt(s) was established by the Nessler Reaction
(Gerhardt, 1981). Stock solution of 100 pg (NH.,+)/ml was prepared by dissolving
anhydrous ammonium chloride in ammonium-free deionized distilled water and diluting
to give a series of standard solutions with concentrations of NH.,' ranging from 0.5, 1, 2,
4, 6 to 12 pg/mî. To 1 .O ml of each of the standard solutions in a 1.5 ml disposable UV
grade cuvette (VWRB, VWR Scientifc of Canada, Edmonton, Canada) was added 40 pl
of Nessler's nagent (Aldrich@, Aldrich Chernical Co., hc., Milwaukee, WI, USA) and
the solution was mixed thoroughly. The absorbance of each solution was measured at 450
nm with the spectrophotometer at 0.5 h. Based on the aborbance and the concentration of
W,,+ in the solution in the cuvette, the standard cuve could be determined. The
established standard curve of VM was Cw = 0.021 19 + 13.3387A (~*=0.992), where
CW was the concentration of VM and iu salt (NH,' pg/ml) in a test sample in the
cuvette and A (O - 1.0) was the absorbance of a sample in the cuvette at 450 nm.
The standard curve of ST was established sirnilarly to those described for VM. in
brief, standard solutions of ST in methanol, with concentrations ranging from 0.122,
0.244, 0.488, 0.977, 1.95, 3.91, 7.8 1 to 15.625 pg/ml, were prepared. Into a 1.5 ml
cuvette was added 0.8 ml of one of the standard solutions and the absorbance of the
solution at 315 nm, the wavelength of peak absorbance of ST, was measured. The
established equation of the standard curve of ST was Cm = -0.021 + 8.878A (R~=I),
when Cm was the concentration of ST (pglml) in a test sample in the cuvette and A (0.02
- 1.8) was the absorbance of a sample in the cuvette at 3 15 nm.
The standard curve of HD was established similarly to that of ST, except that the
standard solutions had concentrations from 1 S6. 3.125, 6.25, 12.5 to 25 pg/ml, and the
absorbance of the standard solutions in the cuvettes was measured in the
spectrophotometer at 271 nm, which is one of the wavelengths of maximum absorbance
for HD. The established equation of the standard curve of HD was CHD = -0.3636 +
20.9ûSA (R-1), where Cm was the concentration of HD (pg/ml) in a test sample in the
cuvette and A (0.09 -1.2) was the absorbance of a sample in the cuvette at 27 1 nm.
4.2.4.2 In vitro proâuction of VM
For quantitative detection of VM in the culture broth, 1 ml CF filtrate of the
bacterial culture was diluted LOO tirnes with deionized water. The pH of the diluted
filtrate was adjusted to 10.5 using 6N NaOH (Gerhardt, 1981). Into 1 ml pH-adjusted
filtrate in the cuvette was added 40 pl Nessler's nagent and the solution was mixed
thoroughly. The absorbance was measured at 450 nm at 0.5 h, as described above. TSB
was diluted and tested similarly to serve as the reference.
A. Production of VM and its salt(s) by different bacterial cultures
Four-day-old CF culture filtrates of the strains of A21 of X. bovienii, All, BCl,
D 1, 19. 27 and ATCC 39497 of X. nemutophilzu and C9 and MD of P. luminescens
grown in T S B were prepared. as described in section 3.2.2. The concentration of VM and
its salt(s) in the CF filtrates was deterrnined as descxibed above.
B. Production of VM by different bacterial culhues over time
X. bovienii A2 1, X. nematophilus BC 1 and P. luminescens MD were CU ltured for
up to 5 d as described in section 3.2.2. Three flasks of culture of each bacterial species
were taken randomly each day for 5 d and CF filtrates were prepared sepantely, as
described in section 3.2.2. The CF filtrates were then diluted and the concentration of VM
and its salt(s) was tested as described above.
C. Production of VM as influenced by aeration of the bacterial culture
To investigate the relationship between aeration and production of VM in the
culture broth, a 24 h-old seed culture of X. bovienii A2 1 grown in TSB was prepared, as
described in section 3.2.2. Into each of the tiuee flasks (125 ml) containing 12.25, 24.5,
49,73.5 or 98 ml TSB was added 0.25.0.5, 1 .O, 1.5 or 2.0 ml seed culture to make a final
volume of 12.5,25,50, 75 or 100 ml in each of the three flasks. The greater the volume
of TSB in the flask, the greater the volume of the seed culture was added so that the
volume ratios of seed c u l t u d S B wen the same in ail tnatments. The resulting cultures
filled 1/10, 115, US, 315 or 415 of the total capacity of the flasks, respectively. The flasks
were then sealed (air-proof) carefully with aluminum foil, and taped and cultured for 4 d,
as described in section 3.2.2. The aeration of the bacterid culture in each flask correlates
inversely with the volume of the culture under the experimental conditions. Bacterid
growth. pH value and concentration of VM and its sait(s) in the broth culture were
measured, as described in section 3.2.5.2.
D. Production of VM as influenced by culture medium
A 24 h-old seed culture of X. nematophilus BCl grown in TSB was prepared as
described above. One milliliter of the seed culture was then added to each of three flasks
containing one of the t h e media, TSB, LB and NB (50 ml medium/l25 ml flask), and
cultured for 4 d, as described in section 3.2.2. The pH of the 4 d-old culture broths were
measured directly using the pH meter. The CF filtrates of the cultures were prepared, and
the concentration of VM and its salt(s) was determined as described above.
4.2.4.3 In vitro production of ST and HD
A. Qualitative detection of Kû in the culture bmihs
To further clarib the variable production of HD by the same strain or species of
Xenorhabdus and Photorhabdus, X. bovienii A21, X. nematophilus BCI and ATCC39497
and P. luminescens C9 and MD were each cultured in TSB, LB and/or NB for 1,2 or 4 d
as described in section 3.2.2. The HD production in the culture broths was detected using
EMich's wthod (Holding and Colke, 1971). The experiments were repeated several
times.
B. Quantitative production of ST and HD by bacterial cultures over time
The procedure for extraction of ST and HD from the bacterial culture broth, which
was proven to have an extraction efficacy of about 95% in prelirninary experiments, is
descrikd below. Photorhabdus luminescens C9 and MD were cultured for up to 8 d as
described in section 3.2.2. Three flasks for each bacterial strain were taken randomly
from the shaker each day for 5 d and also on day 8. The harvested broth in each flask was
adjusted to pH 7.0 with 6N HCl. and 20 ml of it was taken and centrifuged (13.000 g, 10
min, 4OC). The CF supernatant from each of the flasks was poured into a separating
funnel, extracted with 20 ml ethyl acetate four times for 0.5 h each time, and the extracts
were combined. The sediment from the above broth, after centrifugation, was re-
suspended in 10 ml water, and also extracted four times with 20 ml ethyl acetate, as
described above. The nsulting extracts of the supernatant and sediment from the sarne
flask culture were combined and dned under vacuum on a rotary evaporator. The dried
rnaterial was re-dissolved in 1 ml methanol and subjected to TLC separation.
Since the above methanol solution contained many other metabolites in addition
to ST and HD, samples of the solutions were first developed in TU3 plates (9 x 2.5 cm)
to separate ST or HD from al1 other metabolites in the samples. This procedure is
described below .
A 10 pl sample of the solution was applied as a band 1 .O cm from the bottom of
the plate using a disposable 10 pl micropipette (Drurnmond Scientific, Broornall, PA,
USA). The plates were placed in a 250 ml beaker containing 20 ml of the developing
solvent that was a mixture of methmol - chloroform (0.2 : 9.8 or 0.15 : 9.85 for extracts
fiom P. luminescens MD and Cg, respectively). The beaker was sealed wiih aluminum
foi1 for TU3 development and the solvent was allowed to migrate to 1 .O cm from the top
of the TU3 plates. After development the bands of the metabolites on the plate was
visualized with a UV lamp (254 nm). The ST or HD band, which was cornpletely
separated from other bands. was cut off selectively, and then immersed irnmediately in 1
ml methanol in a 1.5 ml centrifuge tube. The resulting solution in each tube was put aside
in the dark for 1 h, stirred for 3 min on a mixer and centrifuged for 3 min (13,000 g) to
give a clear supernatant in each tube. The supematant was then transferred to a 1.5 ml UV
grade cuvette, and used directly for W absorbance measurement (3 15 nm for ST and 271
for HD) as described in section 4.2.4.1. Sometimes. the above supematant was diluted
with methanol first so as to fit the linear part of the standard curves. Based on W
absorbance and dilution of the test samples, the concentrations of ST or indole in the
original bacterial cultures can be determined.
4.3 Results
4.3.1 Nematicidal metabolites identified from cultures of Photorhabdus luminescens and
Xenorhabdus spp.
Three metabolites with nematicidal properties, nnmely arnrnonia, 3.5-dihydroxy4
isopmpylstilbene and indole, were identified €rom culture broths and organic extracts of
Xenorhabdus spp. and P. luminescens, and the results are summarized in Table 10.
After a series of NMR, IR and MS analyses of the nematicidal compounds
obtained, ST and ST-1, which were detected in bacterial cultures of P. luminescens C9
and P. luminescens MD, respectively, were identifKd to bc the sarne nematicidal
Table 10. Summ~ry of nematicidai metaboütes identified from cultures of different
isolates of Xenorhabdus spp. and Photorhabdus luminescens
X. bovien ii
A2 1
X. nematophilus
BC1
ATCC39497
P. luminescens
c9
MD
- ---
+: positive; -: negative.
nt: not determined.
compound, 3.5-dihydroxy4isopropylstilbene, and the HD, which was isolated in
cultures of P. luminescens MD. was confirmed to be indole (NMR, IR and MS data not
shown. because they are not novel compounds). The structures and UV spectra of these
compounds are shown in Fig. 5 and Fig 6, respectively.
The white solid cornpounds, which were obtained after acidification of the
condensed volatile metabolites of the cultures of X. bovienii A2 1 and P. luminescens Cg,
were identified to be the same compound, ammonium chloride. Consequently, the volatile
metabolites, VM and VM-1, from the cultures of X. bovienii A2 1 and P. luminescens C9 ,
respective1 y, were mmonia
Isolation of the nematicidal metabolites from the crude organic extracts of X.
nematophilus BCI and ATCC 39497 was unsuccessful, although the crude extracts of
both strains showed nematicidal activity against 14s and adults of B. xylophilus (Table 8) .
Fraction C of X. nematophilus BC1 showed weaker nematicidal activity than did the
crude extract and no activity was detected in sub-fractions of C. Twelve fractions had
been collected after separation of the crude organic extract of ATCC 39497, but none of
them showed nematicidal activity against B. xylophilur.
4.3.2 In vitro production of the nematicidal metabolites produced by Xenorhabdus spp.
and Photorhabdus spp.
4.3 -2.1 In vitro production of ammonia and its salt(s)
A. Production of ammonia and its salt(s) by different bacterial cultures
Arnmonia and its sdt(s) were pmduced by ai l the bactetial strains and species of
Fig. 5. Structures of (a) 3,s-dihydmxy-4-isopropylstilbene (ST) (R=CHd md (b)
indole.
Fig. 6. UV spectra of (a) 3$dhydroxy-4-isopropyIstilbene (ST) and (b) indole.
Xenorhabdus and Photorhabdus cultured in TSB, and the concentration varied with the
bacterial isolates and species (Table 1 1). X. bovienii A2 1 , X. nematophilas BC 1. D 1 and
ATCC 39497 produced significantly greater arnount of ammonia and its salt(s) in cultures
than X. nenuitophilus 19 (P < 0.05).
B. Production of ammonia and its sait (s) by different bacterial cultures over tirne
The concentration of ammonia and its salt in culture broths of X. nematophilui
BC1 and P. luminescens MD increased gradually over a 4d and 3d period, respectively,
before decreasing thereafter whereas it continued to increase in X. bovienii A21 culture
broth over a 5d petiod (Fig. 7).
C. Production of ammonia and its salt(s) as influenced by aeration of the bacterial
cultures
The concentration of ammonia and its salt(s), the bacterial growth and the pH of
the culture broth were influenced greatly by the degree of aeration of the bacterial culture
(Fig. 8). The smail volume of culture broth in the flasks allowed for better aeration of the
culture, better bacterial growth, a higher pH and a higher concentration of ammonia and
iis salt(s).
D. Production of arnrnonia as influenced by culture media of the bacterial culture
Culture media composition influenced the production of ammonia and its sait(s)
(Table 12). Of the three media used bacterial cultures grown in TSB and LB had
sipificantly greater amount of arnmonia and it salt(s) than did ihose grown in NB (P <
Table 11. Concentration of ammonia and its saIt(s) (N&+pg/mi) in 4 d-old culture
bmths of Xenorhabdus spp. and Pkotorhabdus luminescens grown in TSB
Bacteria Concentration Bacteria Concentration
(W+ pg/ml) (Mt+ c l g w
X. bovien ii X. nematophilus
A2 1 749.4 f 36. la 19 467.2 f 5.8bc
27 694.4 f 1 7 . 2 ~
AI1 593.0 I 6.7ab
P. luminescens BC 1 749.4 f 3 I .Sa
C9 70 1.2 i: 24.6ab DI 729.5 k 13.3a
MD 704.2 f 40.5ab ATCC 39497 766.9 f 4 1 S a
Data are expressed as mean f. SE ( n a except strains of 19,27, Al1 and Dl where n=3).
Means followed by the same letter are not significantly different (P < 0.05).
Fig. 7. Concentration of amnonia and its sdt(s) (N&* Wtd) in culture broths of
Xenorhabdus bovienii A21 (A21), X. nemutophilus BCl @Cl) and Photorhabdus
luminesceiis C9 (C9) grown in tryptic soy broth over 5 days.
Age of the bacterial cultures (days)
Fig. 8. (a) Bacterial gronth, @) pH and (c) concentration of -nia and its salt(s)
of Xenorhabdus buvienii A21 in tryptic soy broth as idïueaced by aeration of the
culture. Degree of aetation was baseà on the inverse relationship between the
volume of the bacterid cuI(Iates .ML the size of the llasks under the experimentiil
conditions. Y10, US, US, 3/S and 4/5 represent volume ratios of culture medium in
the flasks compareci with the totai capacity of the ~asks.
Volume ratios of the bacterial culture and flask
Table 12. pH and concentration of ammonia and its salt(s) of tryptic soy bmth
(TSB), Luria broth (LB) and nutrient broth (NB) in which Xenorhabdus
nemutophilus BC1 had ken grown for 4 d
Growth media Concentration
(NItf pgvml)
TSB
LB
NB
Data are expressed as mean f SE (n=6). Means in the sarne column followed by the sarne
letter are not significantly diffennt (P c 0.05).
0.05). Also, the pH of the bacterial cultures gown in LI3 was higher than that in TSB and
NB (P < 0.05). The nsults indicate that some alkaline metabolites other than arnmonia
and its salt significantly contributed to the high pH values of the bacterial culture grown
in NB.
4.3.2.2 In vitro production of ST and indole
A. Qualitative detection of indole in the culture broths
Indole production in the culture broths of Xenorhabdus spp. and Photorhabdus
spp. varied, even between the sarne strain or species of bacteria at replicate experiments
(Table 13). P. luminescens MD produced indole at a relative higher frequency in different
media tested. and X. bovienii A2 1 and X. nematophilus BC 1 produced no indole. As well,
it appears that the media composition affects indole production (Table 13) in that bacteria
grown in TSB and LB generally showed a higher frequency of indole production than did
those grown in NB.
B. Production of ST and indole in culture broths of P. luminescens C9 and MD over time
ST and indole weE detectable in the bacterial cultures of both C9 and MD
isolûtes of P. luminescens over a 8 d p e n d (Fig. 9). In general, ST increased gradually
duhg the first 2 or 3 d incubation and then maintained a relatively stable level before
decnasing gradually thereafter. Indole, however. increased rapidly and reached its peak at
2 d before decreasing themaiter.
Table 13. Occumnce of indole in broth cultums of Xendabdus spp. and
Photorhabdus luminescens grown in tryptic soy broth (TSB), nutrient broth (NB) or
Luria broth (LB) for 1,2 or 4 d
Bacteria Indole production in different media
(species and isolates)
TSB LB NB
Id 2d 4d 4d 4d
X. bovienii
A21 013 * X. nematophilus
BC 1 0/3
ATCC 39497 L /2
P. luminescens
C9 1 14
MD US
*: Number of positive tests1 total tests.
Fig. 9. Production over t h e of 3,5=àihydroxy4-isopropyIstNbene (ST) and indole
(HD) in culture broths of Photorhabdus luminescens C9 (C9) and P. luminescens
MD (MD) grown in tryptic soy broth. ST-MD and HD-MD represent production of
ST and EID by strain MD; ST-C9 and HD-C9 represent production of ST and HD
by strain Cg.
4 S T - k t D HD-MD
4.4 Discussion
Three nematicidal metaboli tes, amrnonia, ST and indole, have been iden ti fed
from bacterial cultures of Xenoriuzbdus spp. and Photorhabdus spp. The results confirm
and expand upon the observations described in Chapter 3 that multiple factors contributed
to the total nematicidal activity detected in the culture filtrates of the bacteria.
Ammonia and its salt(s), which are known for their toxicity and npelling activity
against plant-parasitic nematodes including J2s of M. incognita (Bishop. 1958; Vassallo,
1967; Rodriguez-Kabana., 1986; Castro et al.. 1991), was comrnonly pmduced in the
broth culture by d l the bacterial isolates tested. ST, however, was produced only by P.
iuminescens, whereas indole was produced by some species of both Xenorhabduî and
Photorhabdus.
Indole is a natural product of plants (Bannister, 198 1; Anderson, 1987; Kubo et
al., 1993) and microorganisms including Xenorhabdus spp. and Photorhabdus spp.
(Freeman, 1985; Fmer , 1995). It has a variety of effects on insects (Herbert et al., 1996;
Thûnabaiu et al., 1996), microorganisms (Kubo et al., 1993) and tumor cells (Kubo &
Morimitsu, 1995). ST has been identified earlier as an antimicrobial compound from
cultures of Photorhabduc spp. (Paul et al., 198 1; Li et al., 199%). In the present study,
both ST and indole were demonstrated for the first time to be nematicidai.
The isolation and identification of nematicidai metabolites h m cmde organic
extracts of X. nematophilus BCl and ATCC 39497 was unsuccessful, although the
extracts showed nematicidai activity against B. xylophilus (Table 8). Contrary to
expectation, the activity of Fraction C of the extract of X. nematophilus BCl d e r gel
column separation was weaker than the cmde extract, and activity was lost when fraction
C was further separated by gel column chromatography into subfractions. Similariy, the
crude organic extracts of X. nenultophilus ATCC 39497 showed nematicidal activity, but
no active metabolites were identified after gel column separation. The reason for this loss
of activity &ter gel column separation is not clear, but there are several possibilities.
Firstly, the active metabolites may be unstable and be denatured during the separation
process. Secondly, the quantity of the active metabolites may be too small, compared with
other nonactive metabolites, to be detected and collected using the methods descnbed.
Thirdly, it is possible that these active metabolites were bound strongly to the gel in the
column and were not eluted out dunng gel column chrornatography. Another possibility
is that a synergistic effect may occur among some metabolites, and the initially observed
nematicidal activity disappeared when the metabolites were separated into diffennt
factions. However, this latter option appears to be invalid, at least for ATCC 39497,
because no nematicidal activity was detected when dl the collected fractions were re-
combined into one sample, one by one, and tested each tirne for nematicidal activity until
al1 the fractions were combined
Fraction C of P. luminescens MD was not nematicidal, but it showed antibacterial
activity against B. subtilis in agar diffusion tests. Since only antibiotic stilbene derivatives
(Paul et ai., 1981; Li et al., 1995b). anhaquinone derivatives (Li et al., 1995b; Sztaricski
et al., 1992) and genistein (Sztacicski et ai., 1992) have been reported from cultures of P.
luminescens, the antibiotic in fraction C might cepresent a new antibiotic fkom P.
iuminescens. This finding led, subsequentiy, to the identification of another novel
antibiotic. a furan derivative, produced by P. lwninescens MD (Hu et al., unpubl.).
The loss of activity of the crude organic extracts of Xenorhabdus spp., following
separation procedures sirnilar to those of P. luminescens, indicated that the nematicidal
metabolites produced by Xenorhbdus spp. are quite different from those produced by
Photorhabdus spp.
The identification of ammonia from culture broths of Xenorhabdus spp. and
Photorhabdus spp. may partially explain the loss of activity of the bacterial cultures
against J2s of M. incognita when the cultures were acidified or separated into organic and
aqueous fractions (Tables 7 and 8). Ammonia should be in the aqueous fraction after
ethyl acetate extraction, and when the aqueous fraction was freeze-dried, ammonia should
evaporate cornpletely. This coincides with the fact that the pH values of the freeze-dried
aqueous fractions decreased significantly to nearly the same value as the TSB. However,
this does not necessarily mean that the higher pH values of the cultun filtrates could be
completely attributed to the occurrence of ammonia. Other metabolites also may be
involved. The cultures of X. nematophilus BCl grown in TSB. LB and NB had sirnilm
pH values, but concentrations of ammonia and its salt(s) in the cultures were significantly
different (Table 12). The results indicated that some alkaline metabolites, other than
ammonia and its salt(s), were present in the culture and contributed to the total pH of the
cultures, in particular in NB.
indole was not produced by al1 svains and species of Xenorhabdus and
Photorhabdus in this study (Table 13). Even for the sarne bacterial isolate, the occurrence
of indole was higbly variable under experimental conditions (Table 13). It was observed
that when the same bacterial strain was cultured in TSB h m the same batch of the
medium under virtually identical culture conditions but at different dates, indole may or
may not be produced. Moreover, it was repeatedly observed. but at variable frequency,
that indole occurrence was variable between replicate Basks that received the same seed
cultures. Supposedly indole is produced by microorganisms via tryptophan (Paul et al.,
198 1 ; Freeman, 1985). The media composition, especially the quantity of tryptophan,
probably directly influences indole production. However, the nason for the variable
indole production when al1 known factors were constant remains unclear.
The highly variable occurrence of indole rnay explain why indole was not isolated
from the organic extracts of the culture broths of ATCC 39497 and P. luminescens Cg,
although both strains produced indole in some instances (Table 13; Fig. 9). The only
exceptions in indole production were X. nematophih BC 1 and X. bovienii A2 1 , which
always gave a negative reaction during tests.
Al1 but one strain (Sztaricskai et al.. 1992) of P. luminescens have k e n reported
to produce ST in culture broths (Hu et al., 1998). It was proposed (Li et al., 1995b) that
prolonged incubation of that strain in the culture leads to the disappearance of ST. In the
present study it was shown that the concentration of ST that was produced in cultures by
both C9 and MD isolates declined graduaily after about 5 d incubation (Fig. 9). The
results support the hypothesis of ST production by Li et al. (1995b).
In conclusion, three nematicidal metaboiites, ammonia, ST and indole, were
identified from bacterial cultures of Xenorhabdus and Photorhabdus. The results confinn
and expand earlier observations described in Chapter 3 that in vitro cultures of the
bacteria are nematicidal and partially explain some of the results observed in Chapter 3.
Amrnonia was commonly produced by al1 the bacterial strains and species tested, and ST
was produced by only P. luminescens. Conversely, indole was produceci by some species
of both Xenorhabdus and Photorhabdus. However, occurrence of indole in TSB, LB and
NB was highly variable even for the same bacterial strain. The production of the
nematicidal metabolites was relaied to the bacterial strains and species and culture
conditions. Although no secondary metabolites, which are nematicidal against B.
xyfophilus, were identified from the organic extracts of the bacterial cultures of
Xenorhabdus spp., the results indicate that the nematicidal metabolites produced by
Xenorhabdus spp. are quite different from those produced by Photorhabdus spp.
CHAPTER 5
NEMATICIDAL PROPERTIES OF
33-DIHYDROXu-4-ISOPROPYLSTILBENE (ST) AND INDOLE
5.1 Introduction
Three secondary metabolites with nematicidal properties, arnmonia, ST and
indole, have been identified from broth cultures of Xenorhabdus spp. and Photorhabdus
spp. Ammonia and its salts are known to be toxic to many nematode species including
mot h o t nematodes, Meloidogyne spp. (Bishop, 1958; Castro et al., 1991). but ST and
indole have not been previously reported to be nematicidal. To better understand their
nematicidal properties, their potential application and to provide dues as to their possible
biological roles in the bacterium - nematode - insect interaction, a series of experiments,
using ST and indole, were done to investigate: 1) the nematicidal activity of ST and
indole against nematodes of different species including entomopathogenic nematodes; 2)
the effects of ST and indole on mobility, egg hatch and dispersal behaviour of nematodes
of different species; 3) the nematicidal activity of some indole derivatives and 4) to
determine the potential efficacy of indok against M. incognita in greenhouse tests.
5.2 Materiais and methods
5.2*1 Test nematodes
The following isolates and species of nematode were used. They are plant-
parasitic anaor fimgal-feeding nematode species, Aphelenchoides rhytium, B. xylophilus
BC and 41426, B. mucronarics France and M. incognita; a fne-living nematode,
Caenorhobditis elegans wild type; and entomopathogenic nematodes, Heterorhabditis
spp. HMD and Spain. H. bacteriophoru Oswego. if. murelatus, H. rnegidis 90,
Steinemema carpocapsae BJ, S. feltiae CH-S-MER, S. glaseri NC19. S. glaseri, S.
kushidai, S. puertoricense and S. riobrave (Table 4). The nematodes were cultured and
collected as described in section 2.3. and were used irnrnediately after collection from
their respective cultures.
5.2.2 Nematicidal activity of ST and indole against different nematode species
5.2.2.1 Effect of ST and indole on different nematodes in immersion tests
ST was dissolved in DMSO to form a stock solution. Different amounts of the
stock solution were diluted with distilled water plus 20 pl of nematode suspension (-100
nematodes of one of the nematode species) to give a final volume of 1 ml in each of the
small Petri dishes (35 x 10 mm) with concentrations of ST From 6.25 - 200 pg/ml
(DMSO 5 1%, vlv). However, in the test against C. elegans, ST was dissolved in ethanol
and diluted with M9 buffer to the desired concentrations (ethanol 5 2%, vlv). Similady,
indole stock solutions w e n prepared in PEG (S 2%, vlv). The dishes with the test
solutions and the nematodes were seded with Parafilm and incubated at 25°C in the dark.
Nematicidal activity was detennined as described in section 2.4. The experiment. using
each series of combinations of test substance and each nematode species were repeated at
least once with three replicates for each treatrnent.
The known nemeticidal compound, 2-stilben01 (Sigma) (Suga, 1994), which is a
stilbene derivative, was tested similady to serve as a reference.
5.2.2.2 Effect of indole on migration of J2s of M. incognita in a sand column
To test the mobility of the J2s of M. incognita in sand following exposure to low
concentrations of indole, indole solutions were prepared by dissolving it in PEG and
diluting it with distilled water to 50, 100,200 and 400 j,@ml (PEG S 1%, vlv).
River sand, supplied by the Greenhouse Facility of the Depariment of the
Biologicai Sciences, was screened to particle size 150 - 300 pm, washed thoroughly with
tap water, then air-dried. This sand was used to prepare a sand column, designed as
illustrated in Fig. 10. The glass column was closed at one end by taping a layer of WS
paper tissue over the end of the column. One milliliter of sand, prepared as described
above, was poured into the column, and the papered end was then immened in 2.5 ml of
indole test solution in a test tube. About 200 J2s of M. incognita in 30 pl distilled water
were added 10 min later ont0 the surface of the sand column. The test tube was then
sealed with Parafilm to rninimize evaporation, held vertically in a rack and incubated at
25OC in the dark.
After a 24 h incubation period the number of I2s remaining in the sand column
and those that had migrated down into the solution at fhe bottom of the test tube was
counted. To collect the J2s remaining in the sand column, the glass column was taken out
of the test tube, and the paper tissue and the sand inside the column were washed with
distilled water into a Petri dish (60 x 10 mm). The nematode suspension was decanted
from the dish into a glass via1 and the sand was washed ihus three times with 5 ml
distilied water for each wash. The nematode suspension in the glas via1 was allowed to
settle and the upper supernatant discardeci. The concentrated suspension of J2s was
examineci under the sien0 microscope (25 X) and the number of J2s counted. The J2s in
Fig. 10. Sand column useci in the migration tests of the second stage juvenües of
Meloidogyne incognito (length unit: cm).
rack
tape ,
, - test tube
L indole - solution
the test solution at the bottom of the test tube were counted by transfemng the nematode
suspension to a dish and counting them using a stereo microscope.
The percentage of J2s of M. incognita that had migrated into the indole solution at
the bottom of the test tube was calculated as follow: Migration (9%) = (No. of 12s that had
migrated to the test solution at the bottom of the test tube)/(Totd No. of J2s that had
migrated into the solvent control solution at the bottom of the test tube and those that
remained in the sand) x 100.
There were five nplicates for each treatment and the expriment was repeated
once. Both water and the solvent (1% PEG, v/v) were included in the experiments to
serve as controls.
5.2.2.3 Effect of ST and indole on egg hatch of the nematodes
A. Egg hatch of Meloidogyne incognita
Egg sacs of M. incognita were hand-picked from infected mots of tomato
seedlings grown in a greenhouse, as described in section 2.3. Three golden egg sacs of
equal size were immersed in a Petri dish containing 1 ml of ST solution at concentrations
from 6.25 to 200 pghl (DMSO S 1 %, vlv). The dishes wen sealed with Parafilm and
incubated at 25OC in the dark. After 5 d the hatched juveniles were counted and the egg
sacs in each dish washed with 5 ml distilled water thne times, before transfemng them to
a new dish containing 1 ml distilled water. The dishes were sealed and kept as above for
another 5 d, and then the hatched juveniles were again counted.
Experiments using indole were conducted similarly except that the solutions with
concentrations ranging from 25 to 200 pg/mI were prepared in PEG (S 146, v/v). The
above experirnents were repeated three times with t h e nplicates for each treatment. The
respective solvent (1% DMSO or 1% PEG, v/v) and distilled water served as control.
B. Egg hatch of Bursaphelenchus xylophilus BC
ST was dissolved in ethanol and diluted with distilled water to five different
concentrations in the range of 6.25 - 1 0 pghi (ethanol S 146, vlv). Its effect on egg
hatch of pine wood nematode B. xylophilus was tested as described below.
Eggs of B. lylophilus were obtained as described by Shuto et al., (1989). Gravid
B. xylophilus were suspended in 0.5% ethanol solution (- 5,000 - 7,000 nematodes/ml),
then 1 ml of the nematode suspension was poured into each small Petri dish (35 x 10
mm). After 4 h incubation (2S°C) to induce egg-laying, the suspension of the nematodes
was removed by decanting or sucking with a pipette. Each Petri dish was then washed
gent1 y three times with 1 ml distilled water each time to ensun complete removal of the
remaining nematodes while allowing most of the eggs to remain in the dish. Immediately,
1 ml of ST solution was added to each dish and the eggs were counted under a stereo
microscope (25 x). Dishes containing about LOO - 150 eggs were seded with P&ilm and
incubated in the dark (25°C). Egg hatching rate was recorded 24 h a€ter incubation.
The experîment was repeated three times, and ethanol solution (l%, vlv) md
distilled water served as controls.
5.2.3 In vivo effect of indole on Meloidugyne incognita
5.2.3.1 Sand application tests
The possible nematicidal effect of indole on M. incognita under in vivo conditions
was investigated. Indole solutions of different concentrations. 50, LOO, 200 pg/ml (PEG 5
1%. v/v), were prepared as described above. The concentrations of indole solution were
selected based on results from preüminary tests.
Tomato seedlings with one-pair of tme leaves that had been grown in autoclaved
sandy soil (3 parts sand and 1 part loam soil) were selected and their mots washed
thoroughly with tap water to wash away soil particles. Each of the seedlings was then
transplantcd into a plastic vial (diameter 2.9 cm and height 5.8 cm) containing 20 ml
sand, prepared as described above. Immediately, 7 mi indole test solution was added to
wet the sand. A k r 0.5 h about 300 J2s of M. incognita in 30 pi water was added onto the
surface of the sand in the vial. The vials were sealed at top with Cotton to minimize the
evaporation. The inoculated seedlings were kept in a growth chamôer (25'C) with a 14 h
light : 10 h dark daily regime and watered as required (3 ml each time). At 20 d post
inoculation the roots of the seedlings were stained as described below, examined under
the stereo microscope (25 x), and the number of galls, the total number of nematodes
inside the mots and the developmental stage of the nematode were recorded. The large
galls were dissected whenever necessary to obtain an accurate count of the nematodes
inside the galls.
To better observe the nematodes inside the rwts, the mts were stained using a
method modifieci from Byrd et al. (1983). Tomato mots were immersed in -5% sodium
hypochlorite for 20 min. washed in tap water, immersed in tap water for 30 min.
immersed in diluted (ln0 strcngth) acid-fuchsin-stwi solution. and heated to boiling for
30 sec. M e r the solution had cooled to room temperature, the roots were washed in
water and the nematodes counted under the steno microscope (25 x).
A commercial nematicide, oxamyl (DunpontB, 10% granular formulation;
courtesy of Dr. E. Riga, Vineland Station, Agriculture and A@-food Canada, Ontario),
was included in the expriment to serve as a reference. Solutions of oxamyl were
prepared by immersing 100 mg oxamyl granules in 20 ml 50% PEG solution, stimng and
homogenizing on a magnetic stirrer for 2 h and diluting with distilled water to 10 pg/ml
(1% PEG, v/v). There were 10 replicates for each treatment and the experiment was
repeated once. Distilled water and solvent (1% PEG, vfv) were prepared and tested
similarly to serve as the controls.
5.2.3.2. Foliage application tests
ùidole solutions of different concentrations, 50, 100,200 and 400 pg/rnl (PEG 'I
l%), were prepared as described above. Oxarnyl solutions were prepared as described
above except that 100 mg oxamyl granules wen immersed and homogenized in 4 ml 50%
PEG and the final concentration was adjusted with distilled water to 50 pg/rnl (1% PEG,
vlv)
Tomato seedlings with one-pair of true leaves were transplanted into the vials of
sand of the same type as those described above, watered immediately with 7 ml distilled
water and kept in the growth chamber as previously descniid. At 24 h p s t
transplantation, sand in the vials was re-wetted with distilled water (1.5 ml). The vials
were sealed with cotton at top, then the cotton was covered with a piece of aluminum foil.
Together, the cotton and the foi1 prevented any leaking of the solution into the via1 during
foliage spray. The seedlings were sprayed with the respective test solutions, prepared as
described above, using a hand sprayer until there was solution run-off from the leaves.
The aluminum foi1 tops and the cotton plugs were disassembled &ter about 2 h when
there were no liquid drops remaining on the leaves. The vials were then re-sealed with
new cotton and kept in the growth chamber, as described above. At 24 h post-spray about
300 12s of M. incognitu in 30 pi water were added to the surface of the sand in each vial.
The viais were again seaied with cotton and kept in the growth charnber as befon. The
seedlings were watered regularly to maintain sand moisture. At 20 d post inoculation the
roots of the seedlings were stained and examined using a stereo microscope. The number
of gdls, numbers of nematodes inside the root tissue and the number of each
developmental stage of the nematode were recorded.
There were eight replicates for each treatment. Both water and solvent control
(1% PEG, v/v) were included in the expriment. The experiment was repeated once but
with 10 replicates for each treatrnent, and the highest concentration of indole was
increased to 1,000 pg/ml and oxamyl to 100 pg/ml. As well, each treatment solution also
contained 0.05% (vfv) Tween 80 (Sigma@) to further promote leaf wetting (Marban-
Mendoza and Viglierchio, 1980).
5.2.4 Nematicidal activity of some indole derivatives
Several cornmercially available indole denvatives were purchased and their
activity against B. xylophilus BC was tested as descriid in section 2.4. The compounds
were dissolved in DMSO and diluted with distilled water to 12.5, 25, JO, 100,200,400,
600, 800 and 1,000 pg/ml (DMSO 5 5%, vfv). EC,, and LCm of each compound were
determined as described by Finney (197 1).
5.2.5 Chemosensory effect of ST and indole on different nematode spcies
A bioassay was developed to explore whether ST and indole influence behaviour
of the nematode symbionts of the bactena and other nematodes. Suspensions of J4s and
adults of B. xylophilus BC, J2s of M. incognita and Us of H. bacteriophoru Oswego, H.
marelatus, H. rnegidis 90, Heterorhabditis sp. HMD and Spain, S. carpocapsae BJ, S.
feltiae CH-S-MER, S. gluseri, S. glaseri NC19, S. kushidai, S. puertoricense and S.
riobrave were prepared as described in section 2.3. They were washed four times with SD
water and concentrated to about 250,000 nematodes/mi SD water. In a sterile, laminar-
flow hooâ, 10,000 nematodes in 40 pl stenlized water were added to the center of each
Petri plate (100 x 15 mm, plastic) contnining 10 ml of 1.5% agar. The plates were left
open and rotated frequently to ensure the evenness of the influence of air-flow on the agw
medium in the Petri plates. Stock solutions of ST of different concentrations, ranging
from 10 - 10,000 pg/ml methanol, were prepared befonhand. Ten microliter of one of the
concentrations of ST solution was pipetted onto a filter paper disc (diameter 0.6 mm,
Watennan No. 4. Dose of ST on each paper âisc was 0.1, 1, 10, and 100 pg, respectively),
w hich was allowed to just dry in the hood before placing on the agar surface of the plates.
The control discs containing 10 pl methanol alone were prepared similarly. When the
nematode suspension in the center of the plate was nearly dry in the laminar-flow hood
and the nematodes were beginning to actively crawl over the plate surface. the control
disc and three of the discs containing ST wen plred on the surface of the agar medium,
as shown in Fig. 1 I. The plates were sealed with Parafïlm and kept at room temperature
in the dark. The distribution patterns of the nematodes on the surface of the agar plates
were observed at 0.5, 1 and 2 h after sealing the plates. Tests using J2s of M. incognita
were conducted similarly, except that smaller plates (60 x 15 mm, plastic) were used, and
5,000 J2s 130 pi SD water wen inoculated ont0 the center of the plate.
Experiments using indole were conducted simiiarly and the concentrations of
indole tested were the same as those described above for ST.
The above procedures were conducted under standard, sterile conditions under
low light intensity (indole is light sensitive). Each test was repeated at least twice with
three replicates for each treatment. Careful preparation and handling of the plates was
necessary in order to avoid the effects of uneven drying and temperature gradient on
nematode distribution over the agar surface. A fine needle was sometimes used to help
spread the Us from the site of inoculation, where the Us often displayed clumping during
the drying process.
5.3 Results
5.3.1 Nematicidal activity of ST and indole
5.3.1.1 Effect of ST and indole on different nematode species in immersion tests
ST affectcd nematode species differenrly (Fig. 126. At 200 pg/mi, ST was toxic
to bacterial- and fimgal-feeding nematodes such as B. xylophilus. B. mucronatus, A.
rhythm and C. eleguns, but not to J2s of M. incognito or to Us of the entomopathogenic
nematode H. megidis 90. The mortality of the fint four species was proportional to the
Fig. 11. Arrangement of filter paper dises on the surface of an agar Petri àiih (100 x
15 mm) in relation to the point of introduction (O) of nematodes for chemosensory
tests. 1,2 and 3 represent the discs with different doses of indok or 3,5dihydroxy4-
isopropylstilbene (ST). The higbest dosage is nt disc 3, and C is the control disc.
S d e r dishes (60 x 15 mn) were uscd in tests for Meloidogyne inmgnith, where
both b and d were decreased to 0.9 cm but the diameter of the nematode inoculation
site (O) and the disa C, 1,Z and 3 rrmsined at 0e6 cm.
Fig. 12. Nematicidal activity of (a) 3,s-dlhydroxy4-isopropybtilbene (ST) and (b)
indole against nematodes of Merent species in test solutions in small Petri dishes.
AP: Aphelenchoidcs rhytium; BC: Bumapheienchus xylophilus; 41426: B.
xylophilus; France: B. nrucmnatus; CE: Caenorhabditis eleganr; MI: Meloidogyne
incognita; R90: Heterorhabditis megidis; HMD: tletemthbditis sp.
concentration of ST and reached 100% at the highest concentration tested. Higher
concentrations of ST were not tested against J2s of M. incognita and Us of H. megidis 90
due to its relative insolubility.
Indole was nematicidal against dl nematode species tested, including
entomopathogenic nematodes, at concentrations greater than 200 pglml (Fig. 12b).
However, Us of the entomopathogenic nematodes, H. megidis 90 and Heterorhabditis sp.
HMD, were more resistant to indole than were the other nematode species. Indole also
caused a high percentage of paralysis of M. incognita and Bursaphelenchus spp. at 100 -
300 ~g lrn l and Heterorhabditis sp. HMD at 400 - 800 pghl (Fig. 13).
2-stilbenol, a known nematicidal compound, was more toxic than was ST to B.
xyiophilus BC and 100% mortality was achieved at 6.25-12.5 pg/ml.
Mortality of the nematodes in dl the controls was less than 5%.
5.3.1.2 Effect of indole on migration of J2s of Meloidogyne incognita in sand column
Indole significantly inhibited the mobility of J2s of M. incognita at concentrations
equal to or higher than 50 pg/rnl (P < 0.05) (Fig. 14). The percentage inhibition was
proportional to increased concentrations of indole. At 200 pg/mi or higher none of the J2s
of M. incognita migrated through the sand column into the test solutions. In contmt,
mon than 95% 12s migrated down the columns in controls within 24 h of incubation. The
nsults parallel the observations in immersion tests that indole caused a high percentage
paralysis of J2s of M. incognita at low concentrations (Fig. 13).
Fig. 13. Percentage mortPlity and paralysis of (a) Bumphelenchus xylophilus BC
(juveniles and adults), (b) Meloidogyne incognüa (second stage juveniles) and (c)
Heterorhabditis sp. HMD (ideetive juveniles) fo11owing immersion in indole
solutions at different concentrations.
Mortality and paral ysis (96) of Heterorhabditis sp. HMD
Moatality and paralysis (%) of Mortality and paral ysis (%) of Bumphelenchur xylophilus
Fig. 14. Inhibitory effat of indok on mobiiity of second stage juveaües (529) of
Meloidogyne incognila in a sand column &et 24 h tmtment. Migration (%) = (No.
of 12s that migrated into the test soiution at the bottom of the test tube)/( Total No.
of 52s that migrated into the solution at the bottom of the test tube and those
remained in the sand) x 100. Bars with the same letter are not significantly difiecent
(P c 0.05).
wattr solven t 50 100 200 400
Concentration of indole (pghl)
5.3.2 Effect of ST and indole on egg hatch of the nematodes
Both ST and indole significantiy inhibited egg hatch of M. incognita (Table 14).
The egg hatch of M. incognita was inhibited at 100 and 200 pglrnl ST over 5 d cornpared
with the solvent control. but hatching resumed somewhat when the egg sacs were placed
subsequently in water. The total egg hatch over the 10 d period for eggs treated in 200
pglml ST was significantly lower than in the solvent conbols (P < 0.05) (Table 14).
Indole significantly inhibited egg hatch of M. incognita at concentrations equal or
higher than 25 pg/m1 (P < 0.05) (Table 14). It almost completely inhibited egg hatch at
100 pg/ml over 5 d. For those eggs treated previously at 200 pg./ml indole solution egg
hatch did not resume but did after lower concentrations although only to a smdl extent
after ûeatment with 100 pg/ml (Table 14).
ST significantly inhibited egg hatch of B. nylophilus BC at 50 and LOO pg/ml ( P c
0.05) (Table 15).
5.3.3 In vivo activity of indole on Meloidogyne incognita
5.3.3.1 Effect of indole on nematode infection via soi1 application
The results of the first expenment demonstrated that indole did not inhibit either
the percentage penetration of J2s of M. incognita into the tomato seedlings or the
development of the nematodes inside the mot system when tested at concentrations
between 50 - 200 pghi (Table 16). No signifcant difference was observed between
indole and control matments (both water and solvent controls) (P c 0.05). As well, by
20d post-inoculation more than 90% nematodes inside the mots had developed into
Table 14. Inbibitory eRect of 3$.dihydmxy4isopmpylstilbene (ST) and indole on
the percentage of egg hatch of Meloidogyne incognita over 5 d followed by
immersion in distilled water for anothsr 5 d
Concentration Hatch rate (%)* in Hatch rate (%) in
wp/d) ST water Final indole water Final
5 d 5 d 5 + 5 d 5 d 5 d S + 5 d
--______________-----YI - Solvent lûû(so1vent) 100 lûûa 1 OO(so1vent) 100 1 OOa
Water 86.O(water) 9 1.2 86.3ab 8 1.9(water) 1 15.1 98.3a
Data are means of the treatment (n=9). nt: not tested. Means in the sarne column
followed by the same letter are not significantly different (P < 0.05).
*: Hatch rate (%)=(No. of J2s hatched in test sample)/No. of J2s hatched in soivent
control sarnp1e)x 100.
Table 15. E f k t of J95dUiydroxy4-isoprnpyIstilbene (ST) on percentage of egg
hatch of Bu~~apkelenchus xyloplritus BC
Concentration
of ST (pg/ml)
6.25 82.6 f 1.4a
12.5 83.3 f 2.9a
25 86.0 L 0.7a
50 70.2 f 3.2b
LOO 41.4 f 3.5~
- Water 86.7 f 1.4a
1 % ethanol 90.9f l.la
Data are expressed as mean f SE (n=3). Means followed by the same letier are not
significantly different (P < 0.05).
Table 16. Effet of indole on infection of tomto seedlings by second stage juveniies
of Meloidogyne incognüa in sand application tests
Concentration Expriment I Expriment [I
of indole No. of galls No. of nernatodes No. of galls No. of nematodes
(W/ml) per seedling per seedling
50 48.2 i: 5.0a 80.4 I 7.4a 56.7 f 3.0ab 96.3 f 4.4a
1 0 0 44.1 L 3. la 75.6 I 5.0a 68.5 f 7.2a 108 f 9.6a
200 41.1f6.7a 78.2I12.9a 55 I 1 .Sab 88.2 st: 4.7a
- -II-
10 (Oxamyl) O I Ob O i O b O f Oc O +Ob
Water 43.5 f 3.2a 72.5 i 6.Sa 55.7 f 4.3ab 88.4 f 4.6a
Solvent 39.7 f 4.0a 80.9 I 7. la 49.9 i S.Ob 87.8 f 4.6a
Data are expressed as mean f SE (n=lO). Means in the same column foiiowed by the
same letter are not significantîy different (P < 0.05).
mature females with fully developed reproductive systems and about 5 - 10% of hem had
started egg laying.
In contrast. the commercial nematicide, oxamyl, cornpletely pnvented the
infection of the tomato seedlings by J2s of M. incognita at 10 pglml under the same test
conditions (Table 16). The above results were confirmed by a repeat experiment (Table
16).
5.3.3.2 Effect of indole on nematode infection via foliage application
The results of the first experiment demonstrated that indole did not inhibit
percentage petration of J2s of M. incognita into tomato seedlings or nematode
development inside the rwt system after the seedlings were treated at concentrations
between 50 to 400 pg/ml (Table 17). No significant difference was observed between
indole and control treatments (both water and solvent controls) (P c 0.05). The
developmental stages of the nematodes inside the rwt system of treated plants were
similar to those in the control plants.
Contrary to the nsuits in the soi1 application experiments, the commercial
nematicide, oxamyl, was inactive when sprayed onto foliage of the tomato seeâiings at 50
pghl under the same test conditions as indole and control treatments.
The above results were confumed in a repeat experiment where the highest
concentration of indole was increased to 1,000 pghl and that of oxamyl was increased to
1 ûû pg/d (Table 17).
Table 17. Effect of indole on infection of tomato seedîiigs by second stage juveniles
of Meloidogyne incogniki in foliage spray tests
-
Concentration Experiment 1 Expairnent IX
of indole No. of galls No. of nematodes No. of galls No. of nematodes
(iidd) per seedling per seedling
50 30.6 f 2.7a 72.5 f 6.8a nt nt
100 3 1.6 f 4.2a 76.8 * 9.2a nt nt
200 29.4 f 2-51 65 k 7.7a 34.7 f 1.9a 72.9 f 5.8a
400 43.6 k 6.3a 90.1 f 12a 42.2 f 2.3a 8 1.3 f 5.7a
1,m nt nt 42.4 f 4.4a 72.6 f 4.6a
-- H u u . . - " - H I I I - H - - - - - _ U U U _ _ U U U _ _ U U U _
50 (Oxarnyl) 34.1 f 4.0a 70.4 fi 7.2a nt nt
100 nt nt 40f1 .7a 81.3f5.8a
Water 34 i 4.6a 75.5 f 10.1 a 43.8 f 2.9a 88.6 f 8.4a
Solvent 33.Lf2.7a 72.2k7.Sa 36.2 f 2.9a 74.1 f 7.7a
Data are expressed as mean f SE (n=8 for experiment 1; n=10 for experirnent [D.
nt: not tested. Means in the same column followed by the same letter are not significantiy
different (P c 0.05).
5.3.4 Nematicidal activity of some indole derivatives
Several indole derivatives showed nematicidal activity against B. xylophilus BC
and their activities were structure-dependent (Table 18). An additional nitro group or a
chloide group on the benzene ring of indole, such as 5-nitroindole or 5-chlorideindole,
increased the nematicidal activity significantly compared with indole. Addition of other
groups on the benzene ring generally decreased activity. The location of the same
functional group on the benzene ring aiso influenced the activity. CMethoxylindole, for
example, had lower nematicidal activity than 6- methoxylindole. Most groups attached to
position 3 of the pyrrole ring of indole decrease the nematicidal activity of the
compounds. Addition of hydrogen atoms at positions 2 and 3 (indoline) of the pyrrole
ring of indole also decreased the nematicidal activity of the compound compared with
indole (Table 18).
The nematicidal effect of indole derivatives on B. xylophilus BC was similarly to
that of indole (Fig. 13). The nematodes were paralyzed at lower concentrations of the
compounds and were killed at higher concentrations. Consequently. the compounds had
lower ECs but relatively higher LC, values (Table 18).
5.3.5 Chemosensory effect of ST and indole on nematodes
Nematode species responded differentiy to ST and indole sources on the aga
surface of the plates. They either were repelled, paralyzed/killed or not affected when
exposed on an agar plate to paper discs containhg ST or indole (Fig 15; Table 19). The
Us of most, but not al1 species of Steinemema. were repelled by ST as shown by the clear
zones amund the paper discs of O. 1 pg ST or p a t e r (Fig. 15a; Table 19) at 0.5 - 2 h. The
Table 18. Nenistickln1 activity of some indole de rivatives aginst Bursaphelenchus
xyîophifus BC in immersion tests
indole
indoline
5-aminoindole
5-chloroindole
5- h ydrox y i ndole
Cmethox y lindole
5-methoxylindole
6-methoxy lindole
5-nitroindole
5-methylindole
indole-3-acetic acid
tryptophan
tryptophol
5-methoxy lindole-
3-acetic acid
None
None
L: 5-NH2
5-Cl
5-OH
4-WH3
5-WH3
6-0CH3
SNO2
5-CH3
R: 3-CH2COOH
3-CH2-CH(NH&COOH
3-CHrCH@H
R + L: S-OCH3 (R) and
3-CH2-COOH (L)
-- - -
*: R: side chah or group attached to the benzene ring of the indole skeleton. L: side chah
or group attached to the pyrrole ring of the indole skeleton.
ECm: concentration causing paralysis and mortality in 50% of test nernatodes.
&: concentration causing mortaiity in 50% of test nematodes.
Fig. 15. A diagrammatic represenbtion showing the influence of 3,S-dihydroxy-4-
isopropylstilbene (ST) and indole on dispersal behaviour of dinerent nematode
specks on Petri dishes. (a): Repelling effct: nematodes were repelled from a disc
containing the test substance. (b): Toxic eff'crt: nematodes that moved near a d i s
containing the test substance became inmobile or dead and they accumulatd
around the disr. (c): No effert: nematdes continued to move rondomly over the
plate.
Table 19. Chemosensory effcet of 3$~ibydmxy4-isopropylstilbene (ST) and indole
on dirrerent nematode species when tested at 0.1,1,10 and 100 Wdisc in 1.5% agar
plaîes.
Nematodes ST Effective bdole Effective
dosage dosage
R* T* N* (pg/disc) R T N (pgdisc)
Bursaphelenchus xylophilus
Meloidogyne incognita
Heterorhabditis sp. HMD
Heterorhabditis sp. Spain
H. bacteriophora Oswego
H. marelatus
H. megidis 90
Steinemema carpocapsae BI
S. feltiae CH-S-MER + S. glaseri NC 19 i
S. glaseri + S. kushidai + S. puertoricense
S. riobrave Rio
*: R: repelling effect; T: toxic effect; N: no effect (please refer to the text for details).
**: response observed under the category.
size of the clear area increased with time and the zones persisted for at least 24 h. ST is
apparently toxic to B. xylophilus BC as shown by the zone of increased numbers of
p d y z e d and/ or distorted nematodes near the paper discs of 10 pg ST. More nematodes
accumulated around the discs as they became imrnobilized and some of these nematodes
were dead after lh (Table 19; Fig. 15b). However, dispersal behaviour of J2s of M.
incognita, Us of al1 Heterorhabditis spp. and some Steinernema spp. tested was not
affected by ST (Table 19; Fig. 1 5c).
In contrast, indole had an effect on the Us of some species of both Heterorhabditis
and Steinemema (Table 19). It repelled the Us of Steinernema spp. tested at dosage of
1 00 pg/disc. Us of Heterorhabditis sp. HMD were repelled by indole at 0.1 pg/disc of
indoie at 0.5 - 2 h but H. megidis 90 was not affected at 100 pgidisc over 2 h. Unlike ST,
indole was toxic to both M. incognita and B. xyiophilus at 100 pgldisc (Table 19; Fig.
15b), but the mobility of these two species resumed foilowing gentle vibration or light
stimuli. Unlike ST, the effect of indole continued for more than 2 h, after which its effect
gradually diminished and eventuaily vanished.
5.4 Discussion
This series of experiments showed that both ST and indole are nematicidai against
a variety of nematode species in that the compounds diminished nematode viability,
mobility and egg hatch. In particular, the study demonstrated also that ST and indole
influenced the behaviour of IJs of Steinememu spp. and Heterorhabditis spp., the
respective syrnbionts of the bacteria that produced the ST and/or indole.
In the present study, ST, a stilbene derivative, was shown to be more active
against bacterial- and fungal-feeding nematodes, such as A. rhyrium, C. elegans and
Bursaphelenchus spp. than to the plant-parasitic nematode, M. incognita. or the
entomopathogenic nematode, H. megidis 90. The reason for the differential effect of ST is
not clear. The nematicidal activity of some other stilbene derivatives from such plants as
Cednrs deodara and Pinus massoniana, have been reported (Mohammad et al., 1992;
Suga et al. 1993; Suga, 1994). The mode of action of the nematicidal stilbene derivatives
is unknown, but it was shown (Suga et al., 1993) to be different from that of the
commercially available insecticides and nematicides including the organophosphorus and
the carbamoyl compounds which operate by inhibiting acetylcholinesterase (Opperman
and Chang, 1 990).
Indole caused a high percentage of paralysis of nematode species tested at lower
concentrations but of a high percentage of mortality at relatively high concentrations. The
inhibitory effect of indole on nematode mobility was confirmed by sand column
experiments where the J2s of M. incognita that were exposed to 100 pg/ml or higher
indole solution were not able to migrate through the column (Fig. 14). However, indole
failed to prevent the infection of the tomato seedlings by J2s of M. incognita when it was
applied at 200 pg/ml to the sand (Table 16). This Faiailure may possibly have been due to
indole's adsorption by sand particles, its rapid breakâown due to unstability, or to the
volatility of indole. In sand column tests, the nematodes were constantiy exposed to the
indole solution (Fig. 10). but in sand application tests. the nematodes' mobility following
initial paraiysis might have resumed following a decrease in indole concentrations due to
its sublimation. This was supported by the observation that the white Cotton plug of the
via1 containing the tomato seedling tumed orange-brown 2 d p s t indole application, an
indication of photochernical reaction of indoles exposed to ait and light (Remers, 1972).
Also, preliminary experiments indicated that J2s of M. incognita exposed to indole
solutions (100 - 200 pg/ml) in an immersion test for 24 h regained their rnobility after
king transferred to distilled water.
Indole did not prevent infection of the tomato seedlings by J2s of M. incognita as
a foliage spray, even when the concentration of indole solution was as high as 1,000
pg/d. This may have been due to result €rom poor uptake of indole by the leaflet of the
seedling or its unstability when exposed to light and air (Remers, 1972). The systemic
nematicide, oxamyl, was inactive too as a foliar application, although it completely
prevented the infection of M. incognita at 10 pg/d via sand application. The inactivity of
the foliar application of oxamyl is more likely to have been due to low concentration used
in this study, because Stephan and Tmdgill (1983) reported that foliar spray of oxamyl
solution (1,000 to 2,000 pglrnl) to tomato seedlings before inoculation of M. hapla
pmvided partial protection.
Several commercially available indole derivatives showed nematicidal activity
against B. xylophilus. and their activities were structure-dependent. The side chains or
groups when attached to both the benzene and pyrrole ring of indole may influence each
other and thus influence the activity of the compound. 5-Methoxylindole, for example,
has an 0CH3 group on the benzene ring of indole and had a ECSo of 243 pg/d. However,
when an acetic acid group was attached at position 3 of its pyrrole ring to form 5-
methoxylindole-3-acetic acid, the nematicidal activity of the latter disappeared (Table
18). Compared with indole, several indole derivatives were more potent against B.
xylophilus, and hither exploration of indole derivatives may help to develop nematicides.
Both ST and indole influenced nematode behaviour (Table 19; Fig. 15). but they
differ in several respects. Indole caused paralysis of M. incognita and B. xylophilus
around the high dosage discs in agar plates. This observation coincides with the fact that
both M. incognita and B. xylophilus were paralyzed by indole at lower concentrations in
the immersion tests (Fig. 13). Since the nematodes that moved closed to the disc were
paraiyzed and remained there while other nematodes continued to move forward from the
inoculation site, the nematodes accumulated around the disc and gave the false
impression of having been attracted. indole repelled Us of some species of both
Steinemema and Heterorhabditis. Both the repelling and toxic effects of indole on the
nematodes in the plates tended to diminish over time, probably due to its volatilization.
Unlike indole, ST repels only Us of some species of Steinemema, and the
effective dosage could be as low as 0.1 Wdisc. The results confinn the fact that ST is
produced only by Photorhubdus spp., the symbiont of Heterorhabditis spp., and it could
be expected that such nematicidal metabolites would not repel the respective nematode
symbiont.
In conclusion, both ST and indole were demonstrated to be nematicidal against
several nematode species especially bacterial- and hingal-feeding nematodes. ST and
indole were shown also to influence the behaviour of entomopathogenic nematodes. To
better understand the occumnce and biological d e s of these nematicidal metabolites in
the tripartite nematode-bacterium-insect association, hirther study of them under in vivo
conditions was necessary.
CHAFTER 6
IN V ' OCCURRENCE OF NEMATICIDAL METABOLITES IN
RELATION TO BACTERIAL GROIiVTH AND NEMATODE DEVELOPMENT
6.1 Introduction
Xenorhbdus spp. and Photorhabdus spp. produce in culture broth secondary
metabolites, such as ammonia, ST and indole, that have nematicidal properties. This
discovery is significant in that these bacteria are themselves symbiotically associated with
the nematodes, Steinernema spp. and Heterorhabditis spp., respective1 y. ST occurs in the
culture broths of several strains and species of Photorhabdus (Paul et al., 198 1; Li et al.,
1995b; Table 10). Indole is produced by several species of Xenorhabdus and
Photorhabdur under in vitro conditions (Farmer, 1995; Table 13). However, little
information is available about the production of ST and indole and other antibiotics by
different bacterial syrnbionts under in vivo conditions (Maxwell et al., 1994; Jarosz,
1996). The availability of such information may help increase our undentanding of their
possible biological role in the life history of these nematode - bacterial complexes.
Consequently, a series of expeciments were done to investigate the time course of
occurrence of ST and indole in nematode-infected lacval G. mellonella in relation to the
growth of Photorhabdus and development of Heterorhubditis.
6.2 Illilrrteriais and methods
6.2.1 G. mellonella larvae and entomopathogenic nematodes
Heterorhabditis bacteriophora Oswego, H. mareiatus, H. megidis 90,
Heterorhabditis sp. HMD and Heterorhabditis sp. Spain (Table 4) and G. mellonella
were cultured and collected as described in section 2.2.
6.2.2 Detection and identification of indole from nematode-infected larval cadavers of G.
mellonella
Infective juveniles of H. megidis 90 that haâ passed through two layers of WS
paper tissue were collected, washed. concentrated in distilled water, then applied to the
surface of filter papers (Waterman No.1) in Petri dishes (10 x 100 mm). Twenty-five last
instar larvae of G. mellonella (- 0.2 g/îarva) were placed on the surface of the filter
papers in each of two Petri dishes that had been inoculated with a suspension of
thousands of Us of H. megidis 90. The dishes were kept in an incubator at 25OC in the
darlc. At 4 d after incubation, al1 dead, nematode-infected cadavers had turned reddish-
brown. The cadavers from the two dishes were homogenized in acetone (10 ml each time)
using a small mortar. The homogenization and extraction processes were repeated severai
times with fresh solvent until the solvent extract was colorless. Al1 the extracts were then
combined, dned in a rotary evaporator under vacuum and re-extracted with 5 ml
methanol each time until the methanol extracts were colorless. The concentrated
methanol solution after evaporation (-10 ml) was useâ for both indole and ST detection
and identification, as described below.
Ten microliter of the concentrated methanol extract was applied to a TLC plate (3
x 9 cm) together with indole (Sigma@) as the refennce. The TU= plate was developed in
a 10 ml mixture of methanol and chloroform (1.5 : 985, v/v) in a 200 ml beaker that was
sealed with alurninum foil. The method for detection and identification of indole was the
sarne as that described in section 4.2.4.3.
Twenty-five, healthy larval G. mellonella were extracted and tested sirnilarly to
that described above, to serve as the control.
6.2.3 Detection of indole over time in larval cadavers of G. mellonella infected with P.
luminescens UD
An experiment was done to investigate whether indole is produced by the
bacterium in the insect cadavers in the absence of the nematode symbiont. To prepare the
bacterial inoculum, a single, 48 h-old primary form colony of P. luminescens MD grown
in TSAD plate was subcultured on a TSA plate for 48 h. The bacterial cells from the TSA
plate culture were then suspended in sterilized 0.8% NaCl solution, and the ce11 density
was adjusted to 5,000 celldpl. Into each of the selected 1st instar larvae of G. rnellonella
(-0.2 g/larva) 2 pi of the bacterial suspension was injected. The lame were then
incubated at 2S°C in the dark. At 0. 3, 6, 12, 24,48 and 72 h, then every altemate day
until 21 d and at 27 d, t h samples. each of five Iarvae, were chosen randomly from
among the bacteria-injected larvae and each sample was homogenized separately in a
small mortar with acetone, followed by methanol extraction, as described above. The
TU: and W spectmm analysis were performed as described in section 6.2.2. Five l ame
injected with only 0.8% NaCl were extracted and tested similady, to serve as the control.
To confirrn that the above prepared bacteid inonilum was indole-producing
under in vitro conditions, 1 ml of the above bacterid suspension was added into each of
two flaslcs (125 ml) containing 50 ml TSB and cultuced, as desc~lbed in section 2.2 for 2
or 4 d. Indole production in the culture broth was examined directiy, using Ehrlich's
reagent method (Holding and Collee, 1971). and indirectly, using TU= and W spectmm
analysis, after extracting the organic fraction of the broth (refer to section 4.2.4.3).
To further confirm that the bacteria inside the infected cadavers retained their
ability to produce indole, though they might not produce it, the bacterium was re-isolated
from the infected insect cadavers and cultured in TSB for indole detection, as described
below. To re-isolate the bacteria, three larvae were selected randomly from the above
injected larvae at each of 1,2,5,8, 10, 15 and 29 d post injection. At each sampling time
the cadavers' body surface was cleaned by washing three times with TSB, then the lame
were homogenized with 2 ml TSB in a small mortar. The macerated material containing
the cells of the bacterial syrnbiont was transferred to a glass vial and diluted with TSB.
The bacteria were transferred and inoculated ont0 TSAD plates using an inoculating loop.
The plates were sealed and incubated at Z0C in the dark. At 48 h, the primary form
colonies of the bacterium were inoculated into a 125 ml flask containing 50 ml TSB and
cultured for 2 dl as described in section 2.2. Indole production was examined using
Ehrlich's reagent method, and TU3 and UV spectrum analyses.
6.2.4 Isolation and identification of ST from infected larval cadavers of G. mellonella
To confirm the presence of ST in the extract prepared as described in section
6.2.2, HPLC analysis of the methanol extract was performed using a Waters 626 liquid
chromatograph. The mobile phase was acetonit.de and water, which was sparged prior to
use. The mobile phase was delivered at 1.2 mUmin to a 250 x 4.6 mm Nucleosil 5 C,,
column (Phenomenex, Rmcho Palos Verdes, CA, USA) using the following pro-
(prepared by Dr. J. Li): 15% acetonitrile in water for 1 min, followed by a linear gradient
to 62% acetonitrile in water for 20 min, and isocratic (62% acetonitrile in water) for 5
min. The eluate was passed through a Waters 484 tunable absorbante detector set at 3 15
nm. The results of the anaiysis were recorded with a Waters 746 data module. ST served
as a refennce in HPLC analysis. The metabolite that was collected had the same retention
time as that of the standard ST d u h g HPLC separation of the methanol extract. It was
dried on a rotary evaporator under vacuum and analyzed for MS data and its W
spectrum, as described in chapter 4.2.4.3.
To further vetify the antibacteriai property of the collected metabolite, an agar-
diffusing bioassay, as described in section 24, was performed.
6.2.5. Quantitative analysis of ST from nematode-infected larval cadavers of G.
melionella
6.2.5.1 Standard curve of ST for HPLC analysis
Standard solutions of ST w e n prepared by dissolving ST in methanol, then
diluting two-fold in methanol to give a series of ST solutions with concentrations of
1,000, 5 0 , 250, 125, 62.5, 3 1.25, 15.6, 7.8, 3.9, 1.95 pg/m.i. Twenty inicroliter of each
standard solution was injected for HPHP analysis, using the program as descnbcd in
section 6.2.4. The standard curve of ST, C n(wm = 0.150 + 4.494A (R2 = 0.9999). was
established for standard solutions with concentrations of ST from 1.95 pglrnl to 62.5
pglml where C ma is the comsponding concentration of ST @@mi) in the sample
injected into the HPLC system with a fmed volume of 20 pl and A is the area recorded
(recorded uniWlûû,ûûû, at 3 15 nm).
6.2.5.2 Selection of extracting solvent
To quantitatively analyze ST in infected insect cadavers, the appropriate solvent
had to be chosen that extracted as much ST as possible from the infected cadavers. Four
solvents, narnely acetone. ethyl acetate, methanol and diethyl ether, were tested for their
extraction efficacy following the sarne extraction procedure.
Infective juveniles of H. megidis 90 were collected and surfaced sterilized, as
described in section 2.2, and the nematode suspension was adjusted to 6,250 Us /ml of
PBS prior to injection into the insect larvae. Larval G. melloneh (-0.2 g/larva) were
each injected with about 25 Us in 4 pl PBS and kept at 2S°C in the dark. At 3 d post
injection, three sarnples, each of five cadavers, were selected randomly from the infected
cadavers for extraction by one of the solvents. Each of the three samples was immersed
separately in a mortar containing 3 ml of the solvent and homogenized. The resulting
liquid extract was transfemd into a 25 ml flask. The residues were recxtracted with 1 ml
of the same solvent four times and centrifbged at 13,000 g whenever necessary. Al1 the
extracts from the sarne sample were combined in the sarne 25 ml flask and dned under
vacuum below 30°C. The dried material was re-dissolved in 1 ml methanol, transferred to
a centrifuge tube (1.5 ml) and centrifuged (13,000 g). The supernatant was decanted.
diluted 100-times with rnethanol and 20 pJ of the diluted solution subjected to HPLC
analysis, as described in section 6.2.4, to detemine the recovery efficacy of the test
solvents on ST extraction. Based on the area recorded for the HPLC analysis of each
replicate, the concentration of the injected solution was calculated from the standard
HPLC curve for ST. The ST concentration in the original methanol solution was obtained
by multiplying by the number of dilution times for the sample for HPLC analysis.
6.2.5.3 Recovery efficacy of ST using acetone
Two standard ST solutions, with concentrations of 25 pg/N and 2.5 pg/pl, were
prepared by dissolving ST in DMSO. Each of five, healthy G. mellonella larvae was
injected with 5 pl of a standard ST solution and immediately irnmersed in a mortar
containing 3 ml acetone. Al1 the five larvae had been injected in less than 1 min and were
immediately homogenized together. They were then extracted using acetone as described
in section 6.2.5.2. The supernatant of the extract was first diluted with methanol50-times
(for lame injected with standard solution with a concentration of 25 pg/pl) or 10-times
(for the larvae injected with standard solution with a concentration of 2.5 pg/)rl) to fit the
linear range of the standard curve established for HPLC analysis, then analyzed to
determine the recovery efficacy. Twenty microliter of each diluted sample was injected
into HPLC each tirne. The study was repeated three times.
6.2.6 Occurrence of ST and indole in relation to the development of Heterorhabditis and
growth of Photorhubdus in l and G. rnellonella cadavers
The entomopathogenic nematodes used were H. megidis 90 and Heterorhabditis
sp. HMD, from which bacteriai symbionts, P. lminescens C9 and P. liainescens MD,
respectively, were isolated. Both P. luminescens C9 and P. luminescens MD were known
to pduce ST and indole in broth culturcs (Fig. 9).
6.2.6.1 G. mellonella - H. megidis 90 - P. luminescens C9 complex
A. Occurrence of ST and indole
Last instar larval G. mellonella (-0.2 g/iarva) were carehilly selected, and the
average weight (AW) of every €ive larvae was detemiined by weighing six groups of five
randornly selected Iarvae. Al1 the lame were than injected with surface sterilized IJs of
H. megidis 90 (-25 UsAarva ), carrying the symbiont P. luminescens Cg, and kept in an
incubator at 25OC in the dark. At each sampling tirne (0, 3, 6, I2, 24, 48 and 72 h, then
every altemate day until2 1 d, and also at 27 d after infection) three sarnples, each of five
randomly selected larvae were homogenized separately in a small mortar with acetone,
processed and reextracted in methanol following the extraction procedures as described in
section 6.2.5.2. The concentrations of ST and indole in the methanol in each of the three
sarnples at each sampling time were quantified using TU=-W methods, as described in
section 4.2.4.3. The concentration of ST or indole (Clglg wet insect tissue) at each
sampling time was determined by dividing the total arnount of ST or indole in each
sample with the AW, which was detennined at the beginning of the experiment.
Fifteen Iarvae injected with only PBS scrved as controls in the experiment. The
experiment was repeated once except that the sampies were collected at 1, 2, 3, 5.7, 12,
17,22 and 27 d after infection.
B. Development of H. megidis 90
To monitor the development of H. megidis 90 inside the infccted G. mellonella
larvae, three aâditional larvae selected nuidomly h m the above injected larvae were
dissected under the stereo microscope (25 X) at each sampling time and the
developmental stages of the nematodes recorded. The experiment w u cepeated once.
C. Population dynamics of P. luminescens C9
Last instar larval G. mellonella (-0.2 gAarva) were selected and the AW was
determined by weighing six groups of five, randomly selected larvae. The larvae were
each injected with 4 pl of PBS containing about 25 Us of H. megais 90 that were
collected, surface sterilized and concentrated in PBS buffer, as described in section 2.2,
then incubated at 2S°C in the dark. At 0,3, 6, 12,24,48 and 72 h after injection, then on
altemate days until day 27 after injection, five injected larvae were rûndomly collected,
their body surface washed clean three times with TSB, then the larvae were homogenized
with 2 ml of TSB in a small mortar. The macerated material was transferred to a
measuring bottle and adjusted to 10 ml with TSB. Standard dilution-plating methods were
followed, then the TSAD plates with bacterial cells were incubated. After 48 h
incubation, the CFü (colony-forming unit) of P. luminesceni per plate was recorded and
converted to CFUIg wet insect tissue based on the dilution times of the macerated
material and AW. The identity of the bacteria on the plates was confirmed by their
morphological, biochernical and physiological characteristics as defined by Thomas and
Poinar (1983), Boemare and Akhurst (1988) and Boemare et al. (1993a). The above
process of homogenization and dilution-plating was repeated for samples collected at
each sampling tirne. Five larvae injected with PBS alone served as a control. The above
process was perfonned under standard stede conditions, and the experiment was
repeated once.
D. pH changes of infected larval G. mellonella
The nematode-infected larval cadavers of G. mellonella were prepared, as
described above, and the samples were collected at 0,3,6, 12,24,48 and 72 h, then every
altemate day until 27 d. At each sarnpling tirne, three groups, each of three larvae, were
chosen randomly from among the infected larval cadavers and homogenized in 2 ml
distilled water. Immediately, the pH of the macerated material was measured using a pH
meter. The sarne process was repeated at each sarnpling time. Larvae injected with PBS
alone served as controls. The experiment was repeated once.
6.2.6.2 G. mellonelfa - Heterorhabditis sp. HMD - P. luminescens MD complex
Occurrence of ST and indole, development of Heterorhabditis sp. HMD and the
population dynamics of its bacterial symbiont, P. luminescens MD plus the pH of
nematode-infected larval cadavers of G. mellonella were investigated. The methods were
the same as those descnbed above for the G. mellonella - H. megidis 90 - P. luminescens
C9 complex.
6.2.7 Occurrence of ST and indole in larval G. mellonella cadaven infected by different
Photorhabdus spp. - Heterorhabditis spp. complexes
To detemine if ST and indole are pduced UI vivo by different bacterid
symbionts following the infection of G. mellonella by the respective Heterorhabditis
spp., Ils of H. maraietus, Heterorhabditis sp. Oswego, Heterorhabditis sp. Spain,
Heterorhubditiir sp. HMD and H. megidis 90 were collected and surface sterilized, as
described in section 2.2. They were concentrated in sterilized PBS buffer to 6,250 Uslm1
before use.
Last instar G. rnellonella larvae (-0.2 g/îarva) were each injected with 4 pl
nematode suspension containing about 25 Us of one of the four nematode species/isolates
listed above. Fifteen lame were injected with each nernatode speciedisolate. The
injected larvae were incubated at 2S°C in the dark until 7 d after injection. Cadaven
infected by each of the four nematode species were grouped randomly into three samples
with five larvae in each sample. Each sample of cadavea was weighed. homogenized.
extracted and concentrated in 1 ml methanol, as described in section 6.2.5.2. The
concentration of ST and indole in each sample was then detemiined using the TLC-UV
methods described in section 4.2.4.3.
6.3 Results
6.3.1 Detection of indole from larval cadavers of G. mellonella infefted by H. megidis 90
Several colorful and UV detectable metabolites showed on the TLC plates ;ifter
the plates, which were applied with extract from infected larval cadavea, had been
developed. However, no indole-like band, compared to indole sample (ceference). was
detectable on the TU3 plates. The control sample, pnpared fiom healthy larvae, did not
show any colorful or W detectable band on the TU: plates.
6.3.2 Detection of indole from larval cadavers of G. mellonella injected with P.
luminescens MD alone
The TU3 method failed to detect the occurrence of indole in the cadaver extracts
collected dunng the whole 27 d period p s t bacterial injection. However, the same
bacterial suspension that was left over after larval injection produced indole when it was
inoculated to TSB and cultured for either 2 or 4 d.
Al1 the bacterial cultures of P. luminescens MD, which were re-isolated from the
bacterial-injected larval G. mellonella cadavers at 2, 5 , 8 15 and 19 d post injection,
produced indole in TSB medium in flasks except the bacteria isolated from the cadaver
24 h post injection.
6.3.3 Isolation and identification of SI' from larval cadavers of G. mellonella infected by
H. megidis 90
ST had a retention time of about 24.2 min in the HPLC profile. One of the
metabolites from the extracts of larval G. rnellonella cadavers infected by H. megidis 90
had the same retention time under the same HPLC program (Fig. 16). Further MS and UV
spectmm analysis, as well as results from an antibacterial bioassay of the metabolite,
which was collected at this specific retention time, confirmed that the metabolite from
the cadavers was ST. This proved that ST was produced in the larvd cadavers of G.
mellonelh infected by H. megidis 90.
6.3.4 Quantitative analysis of ST from infected larval cadavers of G. mellonella
6.3.4.1 Selection of extracting solvent
The relative extraction efficacy of each solvent is summarized in Table 20.
Acetone extracted significantly more ST than did by other solvents tested (P < 0.05) and.
Fig. 16. Cornparison of HPLC c h r o m a t ~ r n s of a typ id test sample extracted
from GdleM rnellonetiu lawae infected with Heterorhabdia megidis 90, as detected
at two dEiferent wavelengths (254 am and 315 nm).
TlME (min)
Table 20. Extraction of 3$.dibydroxy4-isopropylsti1bene (ST), using dükrent
solvents, from cadavers of Guiferid mellonella infected by Heterorkabdilis megidis 90
- - - - -- - - -- - --
Solvent Wet weight (g) Area of HPLC Amount (pg) of Arnount of ST
of five cadaven anaiysis ST extracted (pg/g cadavers)
Diethyl ether 0.7 13 f 0.020 1.85 i 0.12 848 f 50 1187 I53d
Methanol 0.68 1 î 0.006 2.94 f 0.23 1337 I 104 1956 i 167c
Ethyl acetate 0.659 f 0.01 1 3.82 f 0.16 1732 I 7 1 2632 i 141b
Acetone 0.710f0.014 4.84f0.19 2192i 86 3085 f 65a
Data are expressed as mean f SE (na) . Means followed by the same letter are not
significantly different (P < 0.05).
therefore. was selected as the solvent for ST extraction in subsequent experiments.
6.3.4.2 Recovery efficacy of ST by acetone
The results showed that about 95% of ST that was injected into the healthy larvae
was recovered when acetone was used as the extracting solvent (Table 21). Therefore,
this extraction method was used in subsequent quantitative analysis of ST in nematode-
infected larval G. rnellonella cadavers.
6.3.5 Occurrence of ST in relation to the development of Heterorhabditis and growth of
Photorhabdus in lamal G. mellonella cadavers
6.3.5.1 G. mellonella - H. megidis 90 - P. luminescens C9 complex
A. Occurrence of ST over time
No indole was detected in any of the G. mellonella cadavers throughout the 27 d
pend. ST was not detectable in G. mellonella lame during the first 24 h of infection by
the H. megidis 90 - P luminescens C9 complex, but increased rapidly by 48 h to 5 d after
infection (Fig. 17). It remained at a relative constant level for 21 d (-3,000 pg/g wet
insect) before decreasing graduiùly thereafter. As well, it was found that metabolic
components in the extract of the insect cadavers diffend during the first few days of
infection (Fig. 1 8).
The results wen confiimed by a repeated expriment (Fig. 17).
B. Population dynamics of P luminescens C9
Al1 Iarvai G. nrellonella infected by H. megidis 90 died at 48 h post infection and
Table 21. Recovery of 3,s-dihydroxy4-isopropylstiIbene (ST) with acetone from
healthy Galle& nneltonelkà larvae mected with known amounts of ST
Total ST (pg) Area recorded by ST (pg) present in five Percentage of
injected into five the HPLC method larvae as detemined ST recovered
insect larvae (dilution) by the HPLC method
O (DMSO only) O O
62.5 1.3 1 f 0.03 (10) 60.43 f 0.89 96.7 f 1.4
625 2.61 I O . 11 (50) 593.67 =t 14.71 95.0 1 2.4
Data are expressed as mean f SE (n=3).
Fig. 17. (a) Occurrence of 3$.dihydmxy4-isopropylsti1bene (ST), (b) population
dynamics of Photorhabdus luminescens C9 and (c) pH of larval cadavers of Galleria
mebnello infixted by Heterorhabditis megidis 90 over Ume iii two repeat
experiments (Exp-1 and Exp-2).
Time &ter infection (days)
Fig. 18. TLC chrom~togram of 33-dihydroxy-4-isopropy~tilbene (ST) and of two
test samples extmted fmm Girrlle~ rnellonelh larvae inleetml with Heterorhabdiilis
rnegidis 90, 2 and 5 d (Zd and Sd) af'ter infection. TLC plate (9 x 5.3 cm) was
developed in a mixture of methanol- chlomform (15 : 98.5) in a b d e r seaied with
aluninum foi1 and visualized under UV lamp at (254 am).
tumed nddish brown. Within 24 h of infection the bacterial symbiont, P. lminescens
Cg, and one species of bacterial contaminant were readily isolated from the infected
insect cadavers. The contaminant was probably from the insect alimentary system since
they were present also in the control insects. The population of the bacterial contaminant
decreased rapidly by 24 h and was almost undeiectable at 48 h while P. luminescens
increased greatly to about 2.6 x log CFU/g wet insect at 48 h post infection. The peak
level of P. luminescens reached io 1-2 x 10'' ai about 7 - 9 d post infection before
decreasing gradually thereafter. Only the bactecial contarninants were isolated from
control insect larvae.
The results were confirmed by a repeat experiment (Fig. 17).
C. Development of H. megidis 90 inside the cadavea
It took about 14 d for the nematode to produce large quantities of new Us.
Population peaks of hermaphroditic females, arnphimictic females and new Us occurred
at about 5, 1 1 and 14 d, respectively.
D. pH change of the nematode-infected insect cadavea
The pH of the macerated infected larval cadavers of G. meltonell. dropped
slightly (-0.14.2) during the first 12 h, and then it increased gradually to its peak level at
about 7.6-7.7 at 5 - 7 d p s t infection before decnasing thenafter. The pH nmained
relatively stable (-7.0) fmm 13 - 23 d postinfection and decreased grPdually thereafter
(Fig 17).
6.3.5.2 G. mellonella - Heterorhabditis sp. HMD - P. luminescens MD complex
A. Occurrence of ST over time
The occurrence of ST and indole in infected G. mellonella cadavers showed a
similar pattern to that described for infection by H. megidis 90 - P. luminescens C9
complex. No indole was detected in any of the G. mellonella cadavers throughout the 27
d period. ST was not detectable in the insect cadavea during the first 24 h of infection by
the Heterorhabditis sp HMD - P. luminescens MD complex (Fig. 19), but increased
rapidly by 48 h and to 1,900 pglg wet insect at 5 d after infection. It remained at a
relatively stable level for 19 d (-1,700 Clglg wet insect) and decreased gradually
thereafter.
B. Population dynamics of P. lwninescens MD
After 48 h infection. al1 infected larval G. mellonella, except controls, died and
tumed orange-brown. Unlike P. luminescens Cg, described above, two distinct colony
types of P. luminescens MD, designated V p and Vsm. w e n detected for the bacterial cells
isolated from the nematode-infected larvae in addition to bacterial contarninants,
Bacterial cells of Vp colonies had al1 the characteristics of the primary form of P.
luminescens, but Vsm lost either completeiy or partly many of the primary form
properties, such as the ability to absorb dye and produce antibiotics (Table 22). The two
colony types showed also very different population dynamics from each other (Fig. 19).
Within 12 h of infection, Vp cells and bacterial contaminants wen nadily isolated hom
the infected insect larvae. At 24 h Vsm cells also were isolated from larvae but in smaller
numbers than those of Vp cells. By 48 h the number of Vp and Vsm cells was nearly
Fig. 19. (a) Occurrence of 3,5.dbydmxy4-i~propy~stilbene (ST), (b) population
dynamics of the primary fonn (Vp) and a sdl-colony variant (Vsm) of
Photorhubdus luminescens MD and (c) pH of Ional cadavers of Gollerià mellonella
infected by Hetemrhabdiîh sp. HMD over time in two repent expiments (Exp-1
and Exp-2).
O 4 8 12 16 20 24 28
T i i after infection Cdavs)
Table 22. Characteristics of Vp (primary t o m ) and Vsm (smnll-colony variant) of
Photorhabdus luminescens MD
Characteristic* VP Vsm
Gram stain
Ce11 size (p) (range)
Proteinaceous granules
Colony color on TSA
Colony size and f o m
on TSAD
Colony adhesion
Dye absorption
Bromothymol blue
Neutrd red on TSA
Neutra1 red on
MacConkey aga
Pigment difisible on TSA
Catalase
Antibiotic production
Luminescence
Negative
5.0 by 1.3
(3.0 by 1.0 to 8.0 by 1.8)
Yes
Yeilowish
Large; dark green center
with radial strips
Strong
Strong
Strong
Strong
Brown
Yes
Yes
Strong
-- --
Negati ve
2.1 by 0.9
( 1.5 by 0.8 to 3.0 by 1 .O)
No
Light gray
Small; light green and
homogeneous
None
Weak
Weak
Weak
None
Yes (weak)
No
Weak
- -- - - - -
*: Catalase activity was tested by immersing the bactecial mass of 48 h -old Vp and Vsm
cultures fmm TSA plates into 10% hydrogen peroxide and observing the release of
oxygen. Antibiotic production was determined by observing the clear, in hibitory zone
around the agar discs (diameter 6 mm) on Bocillus subtilis plates after incubation (36OC
for 24 h in the dark) (Hickey, 1986). The agar discs were taken separately from 3- to 12-
d s l d Vp and Vsm cultures on TSAD plates. The luainosity of the 2 - 3 ds ld bacterial
cultures was checked by eye in the dark rwm for up to 5 min.
equal. The number of Vsm cells increased dramatically from 4 x IO7 CFUIg wet insect
tissue at 24 h to 4 x 10' cells to 5 x 10' cells / g wet insect tissue at 2 d, but the number of
Vp cells decreased sharply from 4 x IO9 to 5 x IO9 C N l g wet insect tissue to 3 x 1O8 to 9
x 1P C m / g wet insect tissue in the same period of time. The Vsm count increased
gradually and reached a high level of 9 x 10' CFUl g wet insect tissue at day 5, while the
Vp count remained at a lower level during the period 2 - 3 d post infection before re-
gaining a high population of 4 x IO9 to 5 x IO9 C N I g wet insect tissue at 5 d after
infection. The population of Vsm in the larvae declined gradually after day 5, but the Vp
population remained relatively constant through to day 21 or day 25 postinfection before
decnasing rapidly thereafter. Bacterial contaminants, probably from the insect's
alimentary system or body surface of the insect. were readily detected during the fint 12
h, but their number decreased rapidly during the fint 24 h after infection, and few of them
could be detected thereafter. No Vp or Vsm was detected in the control insect larvae, but
bacterial contaminants were detected.
The resulu were confimed in a repeat expriment (Fig. 19).
C. Development of Heterorhabditis sp. HMD inside the cadavers
The symbiotic nernatoàe completed its life cycle about 9 d after infection of G.
mellonella larvae and produced new Us. Population peaks of hermaphroditic femaie,
amphimictic femde, and new Ils occumd at about 2,5 and 9 d, respectively.
D. pH change of the nematode-infected insect cadavers
The pH of the macesated G. mellonello caâavers dropped first from 7.05 at O h to
6.9 at 12 h after infection, and then increased gradually to a peak level at about 7.3 at 2 -
3 d postinfection. It then decreased gradually thereafter until27 d (Fig. 19).
6.3.6 In vivo production of ST by different Photorhabdus spp.
ST was produced in G. mellonella cadavers infected by al1 the nematode -
bacterial complexes tested by 7 d after nematode infection, but the quantity of ST varied
with bacterial species/isolate (Table 23). Greater arnount of ST was produced by H.
megidis 90 - P. luminescens C9 complex and H. murelatus - Photorhabdus sp. complex
than did by other complexes (P < 0.05). The H. bacteriophora Oswego - Photorhabdus
sp. Oswego pmduced the least amount of ST (655.2 pglg wet insect) and the H.
muretatus - Photorhabdirr sp. complex produced the greatest quantity of ST
(4182.1 pglg).
6.4 Discussion
Although antibiotic production in entomopathogenic nematode-infected insects
was noted as early as 1959 by Dutky (1959), and seved classes of antibiotics have since
k e n identified from in vitro cultures of Xenorhabdus spp. and Photorhabdus spp. (Li et
al., 1998), ]iule is knom about the qualitative and quantitative production of the
antibiotics inside the insect cadaver (Maxwell et al., 1994; Jarosz, 1996).
The nsults of the pnscnt study showed that ST, which is both an antibiotic (Paul
et al., 1981; Li et al.. 1995b) and a nematicide, was produced in nematode-infected G.
Tabk 23. Concentration of 3$-dihydmxy4-isopmpylstilbene (ST) produeed by
Hetetorhcrbdüis spp. - Photorhabdus spp. complexes in larval caàavers of GuIleriu
mellonella at 7 d postinfeetion
Complex ST (pgg wet insect)
H. bacteriophora Oswego - PhotorhaMus sp. Oswego 665.2 f 15 1 .Sc
H. murelatus - Photorhabdus sp. 4182.1 f 241.la
H. megidis 90 - P. luminescens C9 3729.4 f 2 14.4a
H. rnegidis 90 - P. luminescens Cg* 3857.3 f 176.9a
Heterorhabditis sp. HMD - P. luminescens MD 1697.2 k 83.0b
Heterorhabditis sp. Spain - Photorhabdus sp. Spain 153 1.7 f 1 56.2bc
Data are expressed as mean f SE (n=3). Means followed by the same letter are not
significantly diffennt (P c 0.05).
*: Repeated expenment.
mellonella cadavers in a much greater quantity and over a much longer period
postinfection compared with that produced in broth cultures (Table 23; Figs. 17 and 19).
However, another nematicidal compound, indole, which was identified from in vitro
cultures, was not detectable in any of the nematode-infected G. mellonella cadavers using
the TLC-UV methods. The injection of P. luminescens MD alone into larval G.
mellonella demonstrated that the absence of indole was not related to the presence or
absence of the nematode symbionts. As well, it was shown that the bacteria used for
injection and those re-isolated from injected larval cadavers were capable of producing
indole in TSB medium. The reason for the absence. or perhaps an undetectable level, of
indole under in vivo conditions is not clear. It is possible that the apparent lack of indole
in the larval cadavers was due to environmental factors, rather than to the bacteriurn
itself. Since indole is believed to be produced by microorganisms via tryptophan (Holding
and Collee, 1971 ; Freeman, 1985), the absence of indole may be due to the limited
quantity of tryptophan andfor the physiochemical conditions prevailing inside the
cadavers, or due partially to the TU=-UV methods for indole detection. It is known that
indole has two peaks of maximum W absorbance at 219 and 271 nm (Fig. 6). The
sensitivity of indole detection on TLC plates (or HPLC) would be increased by using a
W lamp with a wavelength of either 219 or 271 nm. The absence, or perhaps much
lower level, of indole is not surprising because indole is toxic to entomopathogenic
nematodes at higher concentrations (Figs. 12 and 13), and the data presented here show
that there wcre many developing nematodes in the infected cadavers.
ST was produced in infected insect Ianrae 24 h postinfection, which was when the
in- larvae were dying, and maintained a relatively high and constant level throughout
the infection cycle. That is, al1 developmental stages of the nematode symbiont were
virnially immersed in the nutrient environment with its high concentrations of ST. The
concentration of ST in nematode-infected larval G. mellonella was more than 1,000 pg/g
wet insect by 48 h infection (Figs. 17 and 19), which is many times greater then that
needed to inhibit the growth of several soil microorganisms under in vitro conditions (Li
et al., 1995b). The early production of ST may ôe triggered by bacterial contarninants
nleased from the rupnired alimentary system of the larvae due to the nematodes' andor
bacterial activity, and this production helps to maintain a suitable environment with
minimum cornpetition for the development of the nematode and bacterial symbionts.
Since ST is nematicidal, it dso might kill the bacterial-feeding nematodes that live in the
surrounding soil and that potentially could consume bacterial cells associated with the
insect cadaver.
The bacterial growth appears to be closely related to the development of the
nematode symbionts inside the cadavers, because peok population levels of the bacteria in
both H. megidis 90 - P. luminescens C9 and Heterorhabditis sp. HMD - P. luminescens
MD complexes appeared at about the same time that large numbea of amphimictic
female nematades were developing. Both bacterial species built up high population levels
(-IO9 CFü/ g wet insect tissue) inside the infccted larvae within 24 h of infection. The
increasing levels of the bacteria were accompanied by rapidly decreasing levels of the
bacterial contaminants. The rarely deteciable bacterial contarninants after 24 h maybe
due partially to the early production of ST. The population level of P. luminescens MD
primary fom cells, Vp, decrcaîed sharply by 48 h of infection. This sharp decnase of the
Vp ce11 level at 48 h of infection may perhaps be due to the increasing numbcr of Vsrn
cells that were competing for nutrients or the effect of Vsrn metabolites. The decreased
number of Vp cells at this stage of the infection might be beneficial to the nematode
symbionts, because then were only a few hermaphroditic females at this stage and more
food or food nserves could be used in the subsequent development and reproduction of
the large number of amphimictic females. In fact, the Vp cells regained high population
levels within the insect cadaver when there were huncûeds of amphimictic females and
males at 5 d after infection. Vsrn was rarely detectable in the cadavea after about 17 d of
infection presumably because the population of Vsrn was very low at the late stage of the
infection. In plate cultures, Vp and Vsrn were readily interchangable. However, it is not
clear whether the growth patterns of Vp and Vsrn in nematode-infected larval cadavers
are due to the relatively independent growth of these two forrns or to the interchange of
one form with another over the period of infection.
Polymorphism appears to be a cornmon property of Xenorhabdus spp. and
Photorhnbdus spp. in both the colonial and cellular levels of in vitro cultures (Akhurst,
1980; Boemare and Akhurst, 1988; Hurlben et al., 1989; Gemtsen et al., 1992). Its
significance is unknown, although there is speculation that both the secondary form
(phase ïI) and the small-colony variants may have a survival advantage for the species
(Hurlben et al., 1989; Gerritsen et al., 1992). Such cells do not produce secondary
metabolites, and so more energy could be diverted to ce11 division and growth (Gemtsen
et al., 1992; Smigielski et al., 1994). The pnsent study found chat a smalltolony variant,
Vsm, which is an intermediate type between the primary and secondary foms of the
bactecium occurred in both in vitro and, in particular, in vivo conditions (Table 22; Fig.
19). The prirnary form (Vp) and the Vsrn co-exist in infsted iasects and show very
different population dynamics. Nso, Vsm was demonstrated to be less prefemd by, and
less pathogenic to its nematode symbionts (data not shown). The question arises as to
why the Vsm variant should occw so early in the development of the nematode and be so
abundant in a newly-infected insect cadaver when, presumably the nutrient level is high.
Gemtsen et aL(1992) proposed that the nematode might prefer the primary form over the
smalltolony variants and so the presence of the small-colony variants might prevent the
nematode from nmoving al1 the bacterial cells in the cadaver during feeding. However,
almost al1 observations on colony variant were made under in vitro conditions, and
species/isolates of Photorhabdus, except for P. luminescens MD, are not known to have
colony variants under in vivo conditions. More strains and species of Xenorhabdus and
Photorhabdus should be studied under in vivo conditions in order to have a more
complete understanding of the biological roles of the small-colony variants and the
secondary fonn in the nematode-bacterium-insect association.
Unlike in vitro culture in TSB medium when the pH could be as high as 9.0, the
pH of macerated, nematode-infected G. mellonella was much lower king nearly neutrai
(-6.85 - 7.6) during the entire infection process (Figs. 17c and 19c). The difference
indicates a quantitative andor qualitative ciifference of the alkaline metabolites between
those under in vitro and those under in vivo conditions. Since extreme pH in the
environment is likely to be hannful to the entomopathogenic nematode, the results
suggest that it may be beneficid when mus-culhiring nematodes to control the pH
conditions in order to optimize nematode production.
Maxwell et al. (1994) reported that antibiotic metabolites released from
nematode-infected G. mellonella larval cadavers into the sumunding soi1 could
temporarily decrease the population levels of some soil bacteria. ST was produced in
larval cadavers at relatively high concentrations by al1 strains and species of
Photorhabdus studied (Table 23). The toxic effect of ST against hingal-feeding
nematodes or bacterial-feeding nematodes, such as C. eleganr, and its strong repelling
activity against Us of several Steinemema spp., but not those of Heterorhbditis spp.
tested (Table 19; Fig. 12), suggest that ST might not only help to maintain optimal
environmental conditions as an antibiotic inside the insect cadaver for the development of
the bacterium and its nematode symbiont, but might also play a role in decreasing
cornpetition for resources and habitat by imrnobilizing, killing or repelling other
nematode species within or outside the cadavers. The strong, nematode-repelling property
of ST also may be an advantage for Heterorhabditis spp., when it is nleased into the
surrounding soil during U emergence where it cm serve to repel cornpetitors from the
immediate foraging area while searching for a new host. Interestingly, in this regard, the
Us of S. glasen' and S. feltiae, two known cruiser foragers, were arnong the most sensitive
ones to ST in these experiments but S. carpocapsae, an arnbusher forager, was not
effected by ST (Table 19).
The finding in this study of a difference between in vitro and in vivo metabolitic
production has led to a separate research project in which two novel pigments (Hu et al.,
1998) and a novel antibiotic (Hu et al., unpubl.) were identified from P. luminescens Cg-
infected G. mellonellu cadaver extracts. As well, it was found in the present study that
there was distinct qualitative and quantitative difference in in vivo met abolites produced
by P. luminescens C9 following infection of lmal G. mellonellu, especially during the
f i t few days after infection (Fig. 18). Furtber study of these differences may help to
understand the metabolic process of the bacteria and the biological role of the
metabolites.
In conclusion, ST, but not indole, was identified from nematode-infected larvd
cadavers of G. rnellonella. ST was produceci in the cadavea by al1 the Photorhabdus spp.
tested but in variable quantities. In larval G. mellonella, infected by either H. megidis 90
or Heterorhabditis sp. HMD, ST was not detectable within the first 24 h of infection but
increased rapidly by 48 h to 5 d postinfection and remained ai a relatively high and
constant level even after the nematode symbiont had completed its reproduction. The
population dynamics of the bacteria under in vivo conditions were highly variable
depending on the bacterial isolates tested. However, bacterial growth appears to be related
to the development of the nematode symbionts in nematode-infected G. mellonella l w a e
in that the peak levels of the primary cells of the bactena and of arnphimictic fernales
occur simultaneously in both H. megidis 90 - P. luminescens and in Heterorhabditis sp
HMD - P. luminescens MD complexes. In nematode-infected l m a e pH of the macerated
larvae wen slightly higher than 7.0. The earl y production as well as the higher quantity of
ST, which has both antibiotic and nematicidal propcrties, suggests that it plays a
signifcant role in the symbiotic association between the nematodes and their respective
bacterial symbionts.
CHAITER 7
GENERAL DISCUSSlON
Entomopathogenic nematodes. Steinememo spp. and Heterorhabditis spp., and
their respective bacterial symbionts, Xenorhabdur spp. and Photorhabdu spp.. fom a
tripartite nematode-bacterium-insect association once the insect host is infected. The
symbiotic bacteria produce antimicrobial and insecticidal metabolites in broth culture.
These bioactive agents are generally believed to play an important role in this tripartite
association, such as in preventing competition from bacterial contarninants and in
weakening the defense response of the insect host (Dutky, 1959; Paul et al., 1981;
Mchemey et al., 1991a; Akhurst and Dunphy, 1993). The present study has demonstrated
that the bacterial symbionts also produce nematicidai metabolites under both in vitro and
in vivo conditions. This discovery provides new evidence on the important role of the
bacterial secondary metabolites in the nematode-bacterium-insect associations.
Unlike the insecticidal activity of the bacterial metabolites, which help to kill the
insect host (Ensign et al., 1990; Bowen et al., 1998). the role of the nematicidal and
antimicrobial substances appears to be to help minimize competition from other species
of nematodes and bacteria. This is in addition to the bacteria's mle in developing and
maintaining optimal growth conditions for the bacterial and nematode symbionts within
the insect cadavers. Together, the nematicidal, insecticidal and antimicrobial activities
represent three major biological contributions of the bacteria to the symbiotic
relationship with tôe entomopathogenic nematodes and to their mutual success in theK
tripartite association with the insect boa.
In the present study, three nematicidal metabolites, ammonia, 3,5-dihydroxy4-
isopropylstilbene and indole have been identified from cultures of Xenorhabdus spp.
andlor Photorhabdus spp.. Two important plant-parasitic nematodes, M. incognita and B.
xylophilus, were selected as the target nematodes in the routine bioassays. This selection
was based mainly on (i) the fact that M. incognita and B. xylophilw are representatives of
two distinctive nematode taxa, the Tylenchina and Aphelenchina; (ii) both nematode
species are commercially. very important worldwide pests in agriculture and forestry,
reyxtively (Sasser and Carter, 198s; Mamiya, 1984; Sutherland and Webster, 1993);
(iii) an inhibitory effect of the entomopathogenic nematode-bacterium complexes on
Meloidogyne spp. and other plant-parasitic nematodes has been reported (Bird and Bird,
1986; Ishibashi and Kondo, 1986; Georgis and Kelly, 1997), and (iv) a large quantity of
12s of M. incognita and J4s and adults of B. xylophilw were readily available in the
laboratory. In the present study, the occurrence of ST would properly have been rnissed if
only M. incognita had been used during the screening process, because J2s of M.
incognita are not affected by ST even at 200 pglml. Also. M. incognita is more sensitive
to solvents than is B. xylophilus. Consequently, the quantity of the solvents used in the
bioassay and, subsequently, the concentration of the crude, organic compounds screened
would have to be decreased. In other words, the sensitivity of the nematicidal bioassays
would be significantly decreased if only M. incognita had been used. It has ken ~po r t ed
that a significant factor in any nematicidal screening system is the choice of the bioassay
species, because the sensitivity of difîerent nematode species to test materials may Vary
significantly (Anke and Sterner. 1997). For example, the nematicidai compounds that
were pmduced by fungal cultures, such as ascomycetes and nematophagous fun@, and
detected by a bioassay using the free-living nematodes, Panagrellus redivivus, Rhabditis
spp. or C. elegans, were found not to be active against M. incognita (Anke and Sterner,
1997). The results of the present snidy emphasize the importance of selecting an
appropriate range of organisms for an effective bioassay.
Of the t h e nematicidal metabolites identified, ST and indole have not been
reported previously to ôe nematicidal. In the present study, both ST and indole &ected
egg hatch, and the viability, mobility and dispcrsal behaviour of a variety of nematode
species. Indole caused paralysis of nematodes at lower concentrations and was lethal to
al1 nematode species tested at relatively high concentrations in immersion tests. ST, on
the other hand, was active against bacterial-feeding nematodes, such as C. eleguns, and
fungal-feeding nematodes, such as Bursaphelenchus spp. and A. rhytium but not against
12s of M. incognita or Us of H. megidis 90. The differential nematicidal effect of ST is
important in the in vivo interaction between the bacterium and the nematode symbiont,
because it was shown in the present study that ail developmental stages of
Heterorhabditis spp. were immersed in relatively high concentrations (-600 - 4,000 pg/g
insect) of ST within the insect cadaver.
It was shown in the present study that culture filtrates of most bacteria were active
against J2s of M. Acognita, even the filtrates were diluted to 1/4 of the original strength.
Given the activities of the nematicidal metabolites identified from the filtrates and their in
vitro production, the nematicidal activity of the filtrates was apparcntly a combined effect
of nematicidal agents, including unidentified ones. Together, the identification of
ammonia ST and indole expands on and confirms the conclusion reached (Chapter 3)
that multiple factors involved and contributed to the total nematicidai activity detected in
the culture filtrates of Xenorhabdus spp and Photorhabdus spp.
It is a practical approach to screen the derivatives of a known bioactive compound
to find more active ones, and such a screening may sometimes result in more promising
agrochernicals and dmgs than the compound initially identified (Betina, 1994; Suga,
1994). ST and indole themselves oKer no potentiai application as demonstrated in the
present study. Firstly, ST was not active against M. incognita, one of the most
econornically important plant-parasitic nematode pests worldwide. Secondly, although ST
was active against Bursaphelenchus spp., its activity was lower compared with that of
certain stilbene derivatives reported by Suga (1994). Thirdly, indole is active against egg
hatch and JZs of M. incognitu, but it failed to pnvent infection of the nematode in
greenhouse tests. However, indole derivatives might be more effective. Since indole is
more active against M. incognita in vitro, several simple indole derivatives were tested
for their nematicidal activity. A few of them were more active than indole and their
activity is closely related with the type and position of the hinctional group(s) on the
indole skeleton. ST, a stilbene denvative, was not explond further, kcause dozens of
synthesized stilbcne derivatives had been studied or patented after identifying the
nematicidal property of a few natural occumng stilbene derivatives (Moharnmad et al.,
1992; Suga et al., 1993; Suga, 1994). The unidentified nematicidal metabolite(s) from
Xenorhobdus spp., especially X. nemtophilus BCI (Chapter 4). repnsents another kind
of nematicide(s) that is different fiom ST and indole, and remains to be identified and its
potential to be exploreci.
Large numbers of Us of entomopathogenic nematodes are required for the
successful control of insect pests in the field. Miller and Bedding (1982) showed that
about 6000 million S. feltiae (=Neoaplectuna bibionis) per hectare would be required to
effectively control stem borer, Synanthedon tipulifonnis, on black currants in the field,
and similar numbers per hectare of H. heliothidis (= H. bacteriophora) for black vine
weevil control on strawberry (Bedding, 1984). Consequently, the nematodes must be
mass-produced in very large numbers, at low cost and have a reasonable shelf life. The
present study showed that ammonia which is nematicidal, is commonly produced in in
vitro cultures of Xenorhabdus spp. and Photorhabdus spp. This suggests that improved
media formulation andlor cultural conditions that decrease the quantity of ammonia,
indole and other nematicidal metabolites, which are toxic also to entomopathogenic
nematodes, could enhance the eficacy of in vitro nematode production for these
commercial applications.
The possible production of nematode toxic metabolites by the secondary form of
the bacterial symbionts nmains unclear. Secondary forms of Xenorhabdus spp. and
PhotorhabduF spp. differ from the primary foms of the bacteria in several characteristics
(Akhurst, 1980). When both the pnmary and secondary forms are available as food
sources, the nematode symbionts prefer feeding on the pnmary rather than the secondary
fom (Gemtsen and Smits, 1997). In fact, the secondary form does not support the growth
and reproduction of the nematode symbionts as well as does the primary form (Akhurst,
1980; Aldiurst and Boemare, 1990; Gemtsen and Smits, 1997). Ehlers et al. (1990)
suggested that the secondary fom of P. luminescens produced a toxin that kills the
nematode symbionts. They found a negative effect of secondary form of P. luminescens
spp. on Heterorhabditis spp., but secondary form of Xenorhabdus spp. had no effect on
Steinemema spp. Later, Akhurst (1993) and Gemtsen and Smits (1997) considered that
the resuits were more likely nutrient reiated rather than the results of production of a
toxin. Further study on the possible production of nematode toxic metabolites by
secondary foms of Xenorhabdus spp. and Photorhabdus spp. is necessary, because it
might help explain more hlly the specific symbiotic association between the bacteria and
nematodes and help improve the in vitro production of the entomopathogenic nernatodes.
Metabolites of Xenorhabdus spp. and Photorhabdus spp. influence the nematode
symbionts in several ways. Grewal et al. (1997) suggested that symbiotic bacteria inside
the nematode-infected host are a source of volatile, infochemicals, which play an
important role in inter- and intra-specific nematode competition. The authors
hypothesized that Us may reduce competition by responding differently to the cues from
unparasitized hosts vs hosts parasitized by conspecific or heterospecific nematodes.
Glazer (1997) nported that the initial infection of an insect host by entomopathogenic
nernatodes induced the release of a substance that reduced the subsequent nematode
invasion and that such a decrease is nematode species specific. As well, Ehlers and
Iohnigk (1998) reported that the symbiotic bacteria excrete a signal which may change
the developmental pathway of the first stage juveniles. The nature of this signal is not
known yet.
One of the significant discoveries of the present study was that the nematicidal
metabdites prduced by Xenorhabdur spp. andlor Photorhabdus spp. dso infiuence the
behaviour of theK respective nematode symbionts. Interactions between Steinememu
spp., Heterorhabditis spp. and other nematode species have been reported (Bird & Bird,
1986; Ishibashi & Kondo, 1986; Robinson, 1995; Koppenhofer et al., 1996; Kaya &
Koppenhofer. 1996). However, the effect of metabolites from the bacterial symbionts
within the insect cadavers or when released into the surounding soil dunng U emergence,
previously has not k e n considered. ST was prduced in larval cadavers at relatively high
concentrations by dl strains and species of Photorhabdus studied (Figs. 16 and 18; Table
24). The toxic effect of ST against tùngal-feeding nematodes or bacterial feeding
nemntodes, such as C. eleguns, and its strong repelling activity against Us of several
Steinemema spp., but not those of the Heterorhabditis spp. tested, suggests that ST might
play a role in decreasing cornpetition for resources and habitat by immobilizing, killing or
repelling other nematode species within or outside the cadavers. In the soil, the bacterial
and nematode syrnbionts and the insect cadaver in the tripartite association may face
predation either individually or in total. Saprophytic nematodes are cornmon in the
midgut of insects and would if not controlled, continue to feed and reproduce on the
microflora of the Heterorhabditis infected cadavers. Some bacterial-feeding and
nematode-feeding mmatodes in the soil may be attmcted towards and feed on this
bbcontainer" of the bacteria and nematodes, the nematode-infected insect cadaver. ST may
help prevent such predation. The behaviour-influencing metabolites, including ST, may
dso play a role in intra- or inter-specific interaction between entomopathogenic
nematodes (Glazer, 1997; Grewai et al., 1997). The strong nematode-repelling property
of ST may be advantageous for Heterorhabditis spp., when it is released into the
surrounding soil d u h g D emergence where it could repel cornpetitors and protect the
nematode's habitulspace.
The inhibitory effect of the entomopathogenic nematode - bacterium complexes
on other nematode species has been noted in vivo (Bird and Bird, 1986; Ishibashi and
Kondo, 1986) and in the field (Georgis and Kelly, 1997). Consequently, the potential has
been considered for controlling plant-parasitic nematodes while applying
entomopathogenic nematodes against insect pests. The mechanism of action of the
inhibitory effect on plant-parasitic nematodes is not clear. Georgis and Kelly (1997)
suggested three possible mechanisrns that may be involved. Firstly. competition for space
and habitat between entomopathogenic nematodes and other nematode species. Secondly,
inundative application rnay enhance the predator-prey response in the field, since many
nematode-feeding organisms, such as protozoa, nematodes and fungi may consume
indiscriminately both entomopathogenic and plant-parasitic nematodes. Thirdly, bacterial
metabolites released into the surrounding soil from insect cadavers infected by
entomopathogenic nematode may adversely affect plant-parasitic nematodes and decrease
their populations.
The present study provides evidence to help clarify and stimulate further
speculation. The nematicidal metabolite, ST, is active against bacterial- and fungal-
feeding nematodes and is present in the nematode-infected larvai G. mellonella cadavers
at high concentrations throughout the life cycle of the nematodes. Maxwell et al. (1994)
reported that antimicrobial metabolites released from steinemernatid-infected larval G.
mellonella cadavers during Us emergence temporarily decreased population levels of soil
bacteria. The natural release of the contents of the cadavers during U emergence was
repeatedly confirmed in the present study. The nddish brown materials from the
heterorhabditid-infectecl cadaven stained the white filter paper in the Petri dishes during
Us emergence. As a result, the nematicidal metabolites released from the cadavers into
the surrounding soil may partially contribute to the observed inhibitory effect on soil
nematodes after inundative application of entomopathogenic nematodes. However, the
mrnaticidal effect alone appears to be limited in space and time compared with the
overall nematode inhibitory effect observed in the field, because the metabolites released
do not persist or spread widely because of biotic and/or non-biotic factors. Another factor
involved is the density of soil insects that are susceptible to these entomopathogenic
nematodes. If there are few of these insects, the density of cadavers infected by the
nematodes in the soil will be low and, subsequently, the quantity of antibiotic and
nematicidal compounds released into the soil will be relatively small. Ishibashi and
Kondo (1986) reported that application of the entomopathogenic nematodes inhibited the
populations of soil nematodes in potted soil and bark compost. It seems unlikely there
were many insect hosts in potted soil or bark compost and thus then would not be enough
nematicidal metabolites released from the cadaver. Consequently, the inhibited nematode
population in this particular case might be attributcd mainly to some other factors such as
enhanced prey-predator effect a d o r cornpetition for space.
Although it was proposed decades ago (Dutky, 1959) that the production of
antirnimbial substances in nematode-infected insects prevented putrefaction of the
cadavers, M e in vivo experimental data is available to support that speculation (Maxwell
et al., 1994; Jarosz, 1996). RecenUy, the hypothesis was questioned by Jarosz (1996). He
reported that a low antibiotic potency of a lirnited spectrum of antibacterial activity was
found during al1 the developmental stages of the nematode in G. mellunella infected with
S. carpocapsae or H. bacteriophora. Consequently, the author proposed that the lack of
putrefaction of the infected insect was rather a result of littie or no cornpetition for the
Xenorhabdus dunng rapid colonization of the insect body and this rapid growth prevented
secondary invasion of the insect cadaver.
In contrast to the results reported by Iarosz (1996), the pnsent study provides
new, chernical evidence of antibiotic production within nematode-infected insects and
supports the hypothesis of antibiotic inhibition (Dutky, 1959) at least in the
Heterorhabditis spp. - Photorhabdus spp.0 G. mellonella ttipartitate association. Firstly,
the nematicidal metabolite ST, which is dso an antibiotic (Paul et al., 1981; Li et al.,
1995b), was proven chemically to be produced by al1 five Heterorhabditis -
Photurhabduï complexes tested in l a r d G. mellonella cadavers, and at 7 d postinfection
it had a concentration of 665 - 4,182 pg/g wet insect (Table 24; Figs. 16 and 18). In Iarval
G. mellonella infected by either H. megidis 90 or Heterorhabditis sp. HMD. ST was
detectable after 24 h infection and maintained a relatively high concentration (-3,700
pg/g and 1,700 pglg wet insect respectively) throughout the Iife cycle of the nematode
symbiont within the cadavers (Figs. 16 and 18). These concentrations of ST are much
higher compared with those produced in broth cultures (Fig. 8) and are ten to hundnds of
times higher than the concentration necessary to inhibit most test microorganisms under
in vitro conditions (Li et al., 199%; Li et al., 1998). Secondly, it was repotted (Hu et al.,
1998) that a variety of anthraquinone derivatives besides ST and AT were pduced in
larval cadavers of G. mellonella infected by H. mgidis W. Some of the anthraquinone
derivatives have been shown to be antibacterial (Sztacicskai et al., 1992; Li et al., 199%).
Similar anthraquinone derivatives were dso produced by d l five Heterorhabditis spp. - Photorhabdus spp. complexes studied (see Chapter 6) in larval G. niellonellu cadavers.
Thirdly, Maxwell et al. (1994) reported that the antibiotic activity was detected after
demise of the insect whether infected by the nematode-bacterium complex or the bacterial
symbiont alone. The known antibiotics, xenocoumacins 1 and 2, were reported to be
produced at a 1:l ratio in larval G. mellonella infected by X. nematophilus subsp. dutki
(isolates GI and WU), and the total concentration of xenocoumacins 1 and 2 was 800
no00 mg (wet weight) of insect tissue for the GI isolate. Maxwell et al. (1994) noted
also that the levels of antibiotic activity was greater in extracts from nematode-infected
G. mellonella than in TSB broth. These results support Our observation that greater
amount of antibiotics were produced in vivo than in in vitro. Fourthly, the experimental
design and subsequent conclusion by Jarosz (1996) might be controversial. For example,
only aqueous extracts of the nematode-infected insects were tested. The results therefore
may be misleading, because dl known antibiotics produced in vitro by Xenorhabdus spp.
or Photorhubdus spp., such as indole denvatives, xenorhabdins, xenorxides, stilbene
derivatives, anthraquinone derivatives, nematophin (Table 3; Li et al., 1998) and two
novel abtibiotics (Ap and a furan derivative) (Hu et al., unpubl.) are soluble in organic
solvents. Only the xenocoumacins are water soluble (McInerney et al., 199 1 b). In fact, the
present study showed that ST, in addition to anthraquinone derivatives, was produced
(665.2 i 151.5 pg/g wet insect) in G. mellonella infected by H. bacteriophoru Oswego,
the same nematode species used by Jarosz (1996), and both ST and anthraquinone
derivatives would not be dissolved in the aqueous extract of the insect. Finally, it was
shown in the present study that a rapid and massive multiplication of the syrnbiotic
bacteria occumd in the nematode-infected larval G. mellonella within 24 h of infection
(Figs. 16 anci le), but antibiotics were s a 1 produced by the symbiotic bacteria &ter 24 h
of infection when the insect host was dying. The timing of antibiotic production appears
to be comlated with the mpture of the alimentary system of the host insect after
nematode infection. During the first few hours post nematode penetration of the host, the
non-symbiotic bacteria carried on the body surface of the nematodes are eliminated by the
insect's immune system, but the symbiotic bacteria are somewhat resistant to the insect's
immune system or are not recognized as nonself (Dunphy and Webster, 1988; Dunphy
and Thurston, 1990). Consequently, these symbionts multiply rapidly and begin to build
up high population levels (Figs. 16 and 18) within the fint 24 h of infection and, as a
result of the activity of the bacteria and its nematode syrnbiont. the insect host dies and its
tissues, including the p i , break down. This rupture of the host's digestive tract leads to
the release of bacterial contaminants into the hemocoel, which threatens the growth
conditions for the bacteria and the nematode syrnbiont inside the cadavers. However, the
production of the antibiotics, perhaps including bactenocins, by the symbiotic bacteria at
this stage diminishes such a risk. More in vivo studies, especially for the Steinetnema -
Xenorhabdus - insect host association, are necessary to clari@ the biological role of the
anti biotics.
in conclusion, this study has opened the gate to a new research area in the
tripartite association. The study has demonstrated for the first time the nematicidal
properties of Xenorhnbdus spp and Photorhabdus spp. Three nematicidal metabolites,
ammonia, 3,s-dihydroxyl4isopcopylstilbene and indole, were identified in broth cultures
and 3,5-dihydroxyl4isopropylsti1bene was shown to be aiso prduced by P. luminescens
at high levels in vivo. The nematicidal metabolites not only affect the viability, mobility
and egg hatch of a variety of nematode species but also Us' behaviour of
entomopathogenic nematodes, Steinemena spp. and Heterorhabditis spp. The study
provided evidence of the importance of the bacterial metabolites including antibiotics in
the tripartite association. However, many aspects of the role of these bacterial metabolites
in the nematode-bacterium-insect association are still to be revealed. For example, what is
the chernical nature of the unidentified, nematicidal metabolites produced by
Xenorhubdus spp. in culture in the present study? Do the secondary foms of
Xenorhubdus spp. and Photorhabdus spp. produce toxic metaboli tes that contribute
partially to the poor nematode production in vitro? Further studies on these topics would
help us undentand more completely these syrnbiotic associations and the biological roles
of the antibiotics and nematicidal metabolites in the tripartite interactions. Such
information would further enhance these nematodes as powerfd biological contro l agents
of insect pests and provide leading bioactive compounds for development of
agriculturally and medically important chemicals.
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