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 PWSOPHY

in the Department of

Biological Sciences

OKAIJI HU 1999

SIMON FRASER UNIVERSïïY

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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, ap, pH and aeration of the cultures.

Three metabolites, namel y ammonia, 3,s-dihydroxy4isopmpylstilbene (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~oooooooooeoooo oeooo 0036

3.1. htrod~~tl~~ooooooeooo~oooo~oooeooee~mooooeommmoommoeooomomoe~om~omooommooeoaooooooooooe0 sommomoooeommooom 0 l0 0 oo.36 3.2. Materials and ~~thodsbmbmmbbb~~0bb~mb~bb0bb~b~bbbbbbb~bb~b~~~~~bm~b~~b~0~~b0bb~b~b~0~b~~b~bbbmm~0~00l *, 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~oo~~obnmbom~bb~oo~~mb~.bb~oo~bom~bmm~mmbmbmmbmmmmmmbmbmbmmmmmommmbmmmmmmbmommll memoa*mm ll l l l l ll e43

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 Dk~~~mmommmmom~~~~~ommoe~omommmmm~mmoommmo~w~momm~~mmmmmomommoomwmmmmmmmll 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...... dg CELIPTER 5. NEMATICIDAL PROPERTIES OF 33-DIEiYDROXY-4- ISOPROPYLSTILBENE (ST)AND INDOLE...... 94

5.1 CO...... 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~~lfSmmommmmmoommmm*mmmmmmommoommmommo~m~mmmmm~oammoommo~moommmoommmm~amommmmmommmmmmll 0 l *0 la. 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 Di~~~~~i~nmmmoomommom~mommmmmmommmmmmommmmmm~mmm~mmmoomao~m~m~mmmmmmmmm.mmmmmmmmmm~m~mmmmm~mmmmmmmmmmmmoomll l l l ll l ll 126

CHAPTER 6m IN VIVO OCCURRENCE OF NEMATICIDAL

METABOLITES IN RELATION TO BACTERIAL GROWTH AND

NEMATODE DEVELOPMENT~mmmmmmmmmmm~mmmmmmmmmmmemmmmmmmmmmmmmm.mmmammmmm.mmmmm ll l l l l l maml30

60 1. I~t~d~~ti~~ommmm.mmomo~ommmommmmmom~emmmmmmmm~mmmmommmmmmmmmmmmmmommommmmmmmmmommmmmmmmmmmmmml0 ll 0 ll l 0 130

6.2. Materials and ~~thods~~~mm~mm~mmmmmmmmmmmmoommm~~~~m~moommommmmmmmmmll l mm ll 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 lard 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. Species of Xenorhabdus and Photorhabdus and their respective nematode

symbionts, Steinernema and Heterorhabditis species ...... 4

Table 2. Major characteristics distinguisbing Xenorhabdus spp. from Photorhabdus

spp ...... 7

Table 3. Bioactive agents associated with or derived fmm the bacterial symbionts,

Xenorhabdus spp. and Photorhabdus spp., of entomopathogenic

nematodes...... ,...... 23

Table 4. Species and sources of nematodes and bacteria used in this study ...... 27

Table 5. 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

Table 6. 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 kengrown 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

hmhealihy Galleria rnellonella larvae injected with known arnounts of ST... ..o...... C...... 146

Table 22. Characteristics of Vp (primary form) and Vsm (small-colonyvariant)

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 melldla 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. 13. Percentage mortality and paral ysis of (a) Bursaphelenchus qlophilus BC

(juvenilesand 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-1and 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 chromatography ; 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 hydroxy4isopropylsti1bene; TLC: thin-l ayer chromatopphy; 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 ken 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 kome 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. Spiesof Xenorliobdus and Photorhabdus and their respective nematode symbionts, Steinemema and Hetemrhabditis species

Bacteria Nematode symbionts References

X. beddingii Steinemema sp. Akhurst and Boemare, 1988 X. bovien ii S. o#he Steiner, 1923 S. feltiae Filipjev, 1934 S. intemedium Bovien, 1937 S. kraussei Poinar, 1985 Akhurst and Boemare, 1988 X. japonicus S. kushidai Marniya 1988 Nishimura et al., 1994 X. nematophilus Weiser, 1955 Akhurst and Boemare, 1988 X. poinarii Steiner, 1929 Akhurst and Boemare, 1988 Mracek et al., 1994 Fischer et al., 1999 Xenorhabdus spp. S. abbusi* Elawad et al., 1997 Elawad and Hague, 1998 S. arenarium Artyukhovsky, 1967 Se bicomuttun Tallosi et al., 1995 S. caudatum Xu et al., 199 1 S. cerutuphotum Iian et al., 1997 S. howaiiensis Gardner et al., 1994

to be continued Table 1. (continued)

S. karii Waturu et al., 1997 S. long icaudum Shen and Wang, 1992 S. monticolum Stock et al., 1997 S. neoctrrtillae Nguyen and Srnart, 1992 S. oregonense Liu and Berry, 1996a S. puertoncense Roman and Figueroa, 1994 S. ra rum de Doucet, 1986 S. riobrave Cabanillas et al., 1994 S. ritteri de Doucet and Doucet, 1990 S. siamkayui Stock et al., 1998 S. scapterisci Nguyen and Smart, 1990

Photorhabdus luminescens H. bacteriophoru Poinar, 1976 H. megidis Poinar et al., 1987 H. zealandica Poinar, 1990 Boemare et al., 1993a Photorhabdus spp. H. argentinensis Stock, 1993 H. brevicaudis Liu, 1994 H. hawaiiensis Gardner et al., 1994 H. indicus Poinar et al., 1992 H. maraletus 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. hmPhotorhabdus

-- Character Xenorhabdus Photorhabdus

Isolated fmrn Steinemema spp. + - Isolated from Heterorhabditis spp. - + Bioluminescence - + Catalase - + Pigment unknown anthraquinones Antibiotics** xenorhabdins, xenorxides hydroxystilbenes xenocoumacins, xenomins anthraquinones nematophin, indoles 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 fom 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 hm 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 swm 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 di 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 pend for the bacteria whereas their release

into the hemolymph is the begi~ingof 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 1cells 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 ken 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 1cells 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. Symbiont 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 pend (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 ken 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, 1990).

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-infectinga 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 dl 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-existin 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-136at 10,000 Wlûû 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 1a, 199 1b; 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 1b; 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 hmthe bacterial symbionts, Xenorhabdus spp. and Photorhabdus spp., of eotomopatbogenic nematodes

Bioactive agents Bacterial sources Bioactivities* References

Xenor habdins X. nematophilus 1,2,4,5 McInemey et al., 199 1a X, bovienii Li et al,, 1995a Xenorxides X. bovienii 1,2,5 Chen, 1996; Xu, 1998 Xenocoumacins X. nemutophilus L2,3 Mcbemey et al., 199 1b indole derivatives Xenorhabdus sp. 1,2 Paul et al., 1981; Xebovienii Li et al., 1995a Nematophin Xe nematophilus 42 Chen, 1996 Li et al., 1997a, 1997b Genistein P. luminescens 1 Sztaricskai et al., 1992 Stilbene derivatives P. luminescens 1,2 Paul et al., 198 1 Li et al., 1995b; Hu et al., 1998 An thraquinone P. luminescens 1 Richardson et al., 1988 derivatives Sztiuicskai et al., 1992 Li et al., 1995b Hu et al., 1998 Phages X. nematophilus 1 Poinar et al., 1980a X. bovienii Boemare et al., 1992; X. beddngii Baghdipian et al., 1993 P, luminescens Photorhabdus spp.

to be continued Table 3. (continued)

Bacteriocins X. beddingii 1 Boemare et al., 1992; X. bovienii Baghdipian et al., 1993 X. nenratopophikcs Thder et al., 1997

Xenorhabdicin X. nematophilus 1 Thaier et al., 1995 Chitinases X, bovienii 2 Chen et al., 1996 X. nematophilus P. luminescens Protein crystai X, bovienii 1 Yudina and Egorov, 1996 Exo-and endotoxins Xenorhabdus spp. 4 Dunphy and Webster, 1988 Photorhabdus spp. 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 (possibly insecticidal), on competition among the prevailing bacterial species

(possibly antirnicrobial) and on the nutrition of the nematode spbiont has ken 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~athogenicnematode-bacterium com~lexes Steinernema carpocapsae Xenorhabdus nematophilus B.J Dr. H. Yang, Chinese Acad. of Agriculture, Beijing, China DD136 Dl Dr. O. O. Poinar, Oregon State University, USA Al1 Al1 Biosys, Columbia, MD. USA BC 1 BC 1 Soil sarnple: British Columbia 19 19 Dr. W. M. Brooks, North Camlina State University, USA 27 27 Biosys, Columbia, MD, USA XQl ATCC 39497 American Type Culture Collection, MD, USA S. feltiae X. bovienii A21 A2 I Soil sample: Memt, British Columbia CH-S-MER Dr. J. M. Grunder, Swiss Federal Research Station, Waàenswil, Switzerland

to be continued

27 Table 4 (continued)

S. glaseri NC19 Dr. K. H. Kaya, University of California, Davis, USA S. glaseri Dr. K. H. Kaya S. kushriiai Dr. Yam;rkita, Forestry and Forest Research Institute, Tsukuba, Ibaraki, Iapan S. puertoricense Dr. J. Roman, Agiculturd University of Puerto Rico, Rio Piedras, Puerto Rico Dr. R. Gordon, Mernorial University, Newfoundland

Heterorhabdilir sp. Photorhabdus luminescens HMD MD Dr. 2. Mracek, Czech Acad. of Science, Ceske Budejovice, Czech Republic Heterorhabditis sp. Photorhabdus sp. Spain Spain Dr. 2. Mracek H. bacteriophora P. luminescens Oswego Oswego Dr, E. S. Shields, Corne11 University, Ithaca, W.USA H. mare la tus Photorhabdus sp. Dr. R. E. Berry, Oregon State University, Corvallis, USA H. megidis P. luminescens Soi1 sample: Sumerland, 90 c9 British Columbia

to be continued Table 4 (continued)

Bacteriai-feedine nematode Caenorhabdiris elegans (wild type) Dr, D. L. Ballie, Simon Fraser University, British Columbia

Fun~al-feedine;andor dant-parasitic nematodes Ap helenchoides rhytium Dr, R. V, Anderson, Agriculture and Agri-food Canada, Ottawa Bursaphelenchus xylophilus BC Dr. R. V, Anderson, conifer tree, British Columbia Dr. R. V, Anderson, conifer tree, Quebec BI mucronatus French Dr, G. de Guiran, Stade Res. de Nematol. et de Genetique Moleculaire des Invert., Antibes, France

Obliaate plant-oatasitic nematode Meloidogyne incognita 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 wuinoculated 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 10 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 80ml 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 Istandard 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 BACTEU

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~efiltrates

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 pi 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 pgld (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 urh 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 ndBars 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 il2 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 morsof 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 patest 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) aghtsecond 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 OtO OIO O* O OIO Of0 45.9I0.8 98.7f1.3 Of0 100 * 0 100 f 0 Of0 nt nt OfO 100 f O lof 0 1OOIO

- -- - 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 juvenk 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 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 X. nematophilus BC1 nematophin 1 ATCC 39497 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

king 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-driedaqueous 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 adorfactors 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) wen 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

ethyl acetate extraction

1 ORGANIC EXTRACT 1

gel column chromatography

1 ACTIVE FRACTIONS 1 1 INA- FRACTIONS 1

gel column chromatogiaphy

NMR, IR, MS, etc.

1 DETERMINATION OF CHEMICAL STIIUCïüRE(S) 1 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 (mas 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 fdlyto 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 hm 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 1S6. 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 TSB 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 CUltured 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 cultudSB 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 themedia, 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~ryof 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 , respective1y, 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 ail 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+clgw

X. bovien ii X. nematophilus A2 1 749.4 f 36. la 19 467.2 f 5.8bc 27 694.4 f 17.2~ AI1 593.0 I6.7ab 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 1Sa

Data are expressed as mean f. SE (na 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 (NItfpgvml)

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 (Pc 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 pend (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 114 MD US

*: Number of positive tests1 total tests. Fig. 9. Production over the 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-MDand HD-MD represent production of

ST and EID by strain MD; ST-C9and HD-C9represent production of ST and HD by strain Cg. 4ST-ktD 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 dl 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 hmcmde 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 der 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 hm 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 ken 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 hot 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

(DMSO5 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 wen 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). , - test tube

tape ,

L indole rack - 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 thenplicates 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 - 10pghi (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 gent1y 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. Akr0.5 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. Mer 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 pst 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.

Sderdishes (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 ~glrnland 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 Moatality and paralysis (%) of Mortality and paral ysis (%) of Heterorhabditis sp. HMD 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 solvent 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+5d 5d 5 d S+5d

--______-----YI - Solvent lûû(so1vent) 100 lûûa 1 OO(so1vent) 100 1OOa 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. Efktof J95dUiydroxy4-isoprnpyIstilbene(ST) on percentage of egg hatch of Bu~~apkelenchusxyloplritus 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 I7.4a 56.7 f 3.0ab 96.3 f4.4a 100 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 I1 .Sab 88.2 st: 4.7a

- -II- 10 (Oxamyl) O I Ob OiOb OfOc O +Ob Water 43.5 f 3.2a 72.5 i 6.Sa 55.7 f 4.3ab 88.4 f4.6a Solvent 39.7 f 4.0a 80.9 I7. 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

1ûû 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

-- Huu..-"-HIII-H-----_UUU__UUU__UUU_ 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 pater (Fig. 15a; Table 19) at 0.5 - 2 h. The Table 18. Nenistickln1 activity of some indole derivatives aginst Bursaphelenchus xyîophifus BC in immersion tests

indole None indoline None 5-aminoindole L: 5-NH2 5-chloroindole 5-Cl 5- hydrox y i ndole 5-OH Cmethox y lindole 4-WH3 5-methoxylindole 5-WH3 6-methoxy lindole 6-0CH3 5-nitroindole SNO2 5-methylindole 5-CH3 indole-3-acetic acid R: 3-CH2COOH tryptophan 3-CH2-CH(NH&COOH tryptophol 3-CHrCH@H 5-methoxy lindole- R + L: S-OCH3 (R)and 3-acetic acid 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 dis 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 pdyzed 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. 15c).

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

100 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, 1990).

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 pst 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 0CH3group on the benzene ring of indole and had a ECSoof 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 des 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., respective1y. 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, thsamples. 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 lame 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~lbedin 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 duhg 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 wen prepared by dissolving ST in methanol, then diluting two-fold in methanol to give a series of ST solutions with concentrations of

1,000, 50, 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 land 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 hmthe 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 wu 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 pst 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 chromat~rnsof a typid 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 i0.12 848 f 50 1187 I53d Methanol 0.68 1 î 0.006 2.94 f 0.23 1337 I104 1956 i 167c Ethyl acetate 0.659 f 0.01 1 3.82 f 0.16 1732 I71 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. 18).

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 IO.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~togramof 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 bderseaied 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 pst 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 Vpand Vsm. wen 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 Tiiafter infection Cdavs) Table 22. Characteristics of Vp (primary tom) and Vsm (smnll-colony variant) of Photorhabdus luminescens MD

Characteristic* VP Vsm

-- -- Gram stain Negative Negati ve Ce11 size (p)(range) 5.0 by 1.3 2.1 by 0.9 (3.0 by 1.0 to 8.0 by 1.8) ( 1.5 by 0.8 to 3.0 by 1.O) Proteinaceous granules Yes No Colony color on TSA Yeilowish Light gray Colony size and fom Large; dark green center Small; light green and on TSAD with radial strips homogeneous Colony adhesion Strong None Dye absorption Bromothymol blue Strong Weak Neutrd red on TSA Strong Weak Neutra1 red on Strong Weak MacConkey aga Pigment difisible on TSA Brown None Catalase Yes Yes (weak) Antibiotic production Yes No Luminescence Strong 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- dsld Vp and Vsm cultures on TSAD plates. The luainosity of the 2 - 3 dsld 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 CNlg wet insect tissue to 3 x 1O8 to 9 x 1P Cm/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 CNI 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

ken 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 156.2bc

Data are expressed as mean f SE (n=3). Means followed by the same letter are not significantly diffennt (Pc 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

fitfew 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 lwae

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 lmae 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 ~ported

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 the 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 ken 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 duhg 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 adorcornpetition 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, Mein 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 lardG. 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 dl 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 1b). 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 sa1 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 pi, 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.

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