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 Al1 rights reserved. This work may not be reproduced in whole or in part, by photocopy or other means, without permission of the author.
Transcript
NEMATICIDAL PROPERTIES OF XENOREMBDUS SPP. AND
PHOTOiüX4BDUS SPP., BACTERIAL SYMBIONTS OF
ENTOMOPATHOCEMC NEMATODES
B.Sc., Northwestem College of Fonstry, Yangling, China, 1985 M.Sc., The Chinese Acaderny of Fonstry, Beijing, China, 1988
THESIS SUBMïlTED IN PARTIAL, FULFLMENT OF THE REQUlREMENTS FOR THE DEGREE OF
DOCTOR OF P W S O P H Y
in the Department of
Biological Sciences
OKAIJI HU 1999
SIMON FRASER UNIVERSïïY
Al1 rights reserved. This work may not be reproduced in whole or in part, by photocopy
or other means, without permission of the author.
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ABSTRACT
Nematicidal properties of some secondary metabolites of Xenorhnbdus spp. and
Photorhabdus spp. (Enterobacteriaceae), bacterial symbiunts of the entomopathogenic
nematodes, Steinernema spp. and Heterorhabdiris spp., respectively, were identified and
evaluated.
Cell-free (CF) culture filtrates of X. bovienii, X. nematophilus and P. luminescens
isolates were shown to be nematicidal against Meloidogyne incognita and
Bursaphelenchus xylophilus. The degree of activity varied with the bacterial
isolate/species and the culture conditions, such as media composition, a p , pH and
aeration of the cultures.
Three metabolites, namel y ammonia, 3,s-dihydroxy4isopmpylstil bene (ST) and
indole, were identified from CF filtrates as having nematicidal properties. Ammonia was
common to al1 bacterial cultures tested. but ST was produced (10 - 30 pg/ml) by only P.
luminescens and indole was produced (10 - 50 pg/ml) by some straindspecies of both
Xenorhabdus and Photorhabdus.
ST and indole affected viability, mobility, egg hatch and dispersal khaviour of
nematodes in vitro. ST was active against Aphelenchoides rhytium, Bursaphelenchus spp.
and Caenorhabditis eleguns, but was not lethal to infective juveniles (Us) of H. megidis
90. or second stage juveniles (J2s) of M. incognita at 200 pg/ml. Indole was active in
immersion tests against J2s (LOO - 400 pg/ml) of M. incognito, but failed to pievent
infection of tomato seedhgs by M. inmgnita foîlowing a soi1 (a0 pdml) or foüar
application (<1,000 pglml). Indole repelled Us of some species of both Steinememu and
Heterorhabditis whereas ST repelled only some species of Steinemema.
ST, but no< indole, was detected in variable quantities (-665.2 to 4,182 pg/g wet
insect) in larval Galleria mellonella infected with Heterorhabditis spp. ST was produced
after 24 h of infection (2S°C) of the larvae, increased rapidly in quantity by 48 h to 5 d,
and nmained at a relatively hi@ and constant level even after the nematode symbiont had
completed its reproduction. Bacterial symbionts built up high populations (-10' cellslg
insect) within 24 h of entenng G. meilonella lame, and increased the cadaver pH to 7.4-
7.7.
The early production and relatively large amount of ST in nematode-infected
insect hosts, and the antibiotic, nematicidal and nematode-repelling properties of ST
suggest that it play a significant role in the symbiotic nematode-bacterium association.
The potential commercial application of these nematicidal metabolites may be limited by
their relatively narrow spectnim and low activity.
1 would like to take this opportunity to express my heartfelt thanks for ail of the
people who kindly offered their thoughts and help during my research. 1 am deeply
grateful to Dr. J. M. Webster, rny senior supervisor, for his encouragement, guidance and
support throughout the course of this snidy. I would also like to thank Drs. J. R.
Sutherland and A. Plant for their helpful suggestions and comrnents during my research
and during the revision of the thesis. My thanks are also given to the following people
who offered their thoughts, encouragement and help during my research: Dr. J. Li for his
work on chemicai characterization and for his help and invaluable suggestions: Dr. G.
Chen and Mr. K. Ng for their discussion and help; Dm. V. L. Bourne and G. Gries, Mrs.
R. Gries and Mrs. M. SieWUnen as well as Mr. B. Leighton and Mr. M. Yang for their
technical support; Mr. Ian Bercovitz for statistical consulting; those mentioned in the text
for their kindness and generosity in providing some of the test materials (nematodes,
bacteria and plant seeds); finaily, my colleagues and friends for their discussion and help.
I acknowledge the financial support of five Graduate Fellowships, a President's
Ph.D. Research Stipend and a Prototype Developrnent Fund for Student Entrepreneurs
from Simon Fraser University and of support ihrough research gants to my senior
supervisor, Dr. J. M. Webster, from the National Science and Engineering Resemh
Council of Canada.
Finally, I would like to express my heartfelt gratitude to my wife and son for their
love, patience and support throughout the course of this study.
TABLE OF CONTENTS
2.4. Biossssys for nematiciàai actidty..... .............................................................. ...33
2.4.1. Activity of celi-free culture filtrates .............................. ............................ 33
2.4.2. Activity of metabolic compounds ........................ ..................................... ..33
.......... .......*.....**...*.......*..*.***.........*.... 2.4.3. Mortality of the test nematodes .... .34
CULTURES OF THE BA~RIAeoooiooem~omooaoooooooomoomooooo~emmoomm~oooooooooeoo o o o e o o o 0 0 3 6
3.1. htrod~~tl~~ooooooeooo~oooo~oooeooee~mooooeommmoommoeooomomoe~om~omooommooeoaooooooooooe 0 sommomoooeommooom 0 l 0 0 oo.36
3.2. Materials and ~ ~ t h o d s b m b m m b b b ~ ~ 0 b b ~ m b ~ b b 0 b b ~ b ~ b b b b b b b ~ b b ~ b ~ ~ ~ ~ ~ b m ~ b ~ ~ b ~ 0 ~ ~ b 0 b b ~ b ~ b ~ 0 ~ b ~ ~ b ~ b b b m m ~ 0 ~ 0 0 l *, b*bm m37
3.30 ~ e ~ ~ ~ ~ ~ o ~ ~ ~ ~ o ~ . o ~ ~ e ~ o o ~ ~ o b n m b o m ~ b b ~ o o ~ ~ m b ~ . b b ~ o o ~ b o m ~ b m m ~ m m b m b m m b m m m m m m b m b m b m m m m m o m m m b m m m m m m b m o m m l l memoa*mm l l l l l l l l e 4 3
3.3.1. Nematicidai activity of bacteriai strains and species. ... . . .. . . . ........................ .43
3.3.2. Nematicidal activity of different cultures against different nematodes
3m4m D k ~ ~ ~ m m o m m m m o m ~ ~ ~ ~ ~ o m m o e ~ o m o m m m m m ~ m m o o m m m o ~ w ~ m o m m ~ ~ m m m m m o m o m m o o m w m m m m m m m l l l l l l moS4
5.2.1. Test nematodes .....................~.......................................................................... 94
5.2.2. Nematicidal activiy of ST and indole against different nematode species ..... -95
viii
5.2.3. In vivo effect of indole on Meloidogyne incognita ............... .. .. ...*..... . . . . . . ... 100
5.2.4. Nematicidal activity of some indole derivatives ....................... .. ... . . ..... ... 103
5.2.5. Chemosensory effect of ST and indole on different mmatode species .... . . . . . . .104
53. R ~ ~ l f S m m o m m m m m o o m m m m * m m m m m m o m m o o m m m o m m o ~ m ~ m m m m m ~ o a m m o o m m o ~ m o o m m m o o m m m m ~ a m o m m m m m o m m m m m m l l 0 l *0 l a. 105
5.3.1. Nematicidai activity of ST and indole ............. .. .............................. . . . .. . . .. 105 5.3.2. Effect of ST and indole on egg hatch of the nematodes ............................... 1 15
5.3.3. In vivo activity of indole on Meloidogyne incognita. .. ... .. . .. . .. . . .. . . . . .. . . . . . .. 1 15
5.3.4. Nematicidal activity of some indole derivatives .............................. . . . . . . . . .. 12 1
5.3 .S. Chemosensory effect of ST and indole on nematodes .............. .... .... .. . . . . 1 2 1
5.40 D i ~ ~ ~ ~ ~ i ~ n m m m o o m o m m o m ~ m o m m m m m m o m m m m m m o m m m m m m ~ m m m ~ m m m o o m a o ~ m ~ m ~ m m m m m m m m m . m m m m m m m m m m ~ m ~ m m m m m ~ m m m m m m m m m m m m o o m l l l l l l l l l l 126
CHAPTER 6 m IN VIVO OCCURRENCE OF NEMATICIDAL
METABOLITES IN RELATION TO BACTERIAL GROWTH AND
NEMATODE D E V E L O P M E N T ~ m m m m m m m m m m m ~ m m m m m m m m m m m e m m m m m m m m m m mmmm.mmmammmmm.mmmmm l l l l l l l m a m l 3 0
60 1. I ~ t ~ d ~ ~ t i ~ ~ o m m m m . m m o m o ~ o m m m o m m m m m o m ~ e m m m m m m m m ~ m m m m o m m m m m m m m m m m m m m o m m o m m m m m m m m m o m m m m m m m m m m m m m m l 0 l l 0 l l l 0 130
6.2. Materials and ~ ~ t h o d s ~ ~ ~ m m ~ m m ~ m m m m m m m m m m m m o o m m m ~ ~ ~ ~ m ~ m o o m m o m m m m m m m m m l l l mm l l l 130
6.2.1. G. mellonella larvae and entomopathogenic nematodes .............. ... ..... . . . . .. 130
4.2.2. Detection and identification of indole from nematode-infected larval
cadavers of G. mell~nella*.~* .... +. ...... ....*.*.*eC*.**.*....* .... .,. .... *... 131
6.2.3. Detection of indole over time in larvai cadavers of G. mellonella infected
Data are expressed as mean f SE (n=3). Means followed by the same letter are not
significantly diffennt (P c 0.05).
*: Repeated expenment.
mellonella cadavers in a much greater quantity and over a much longer period
postinfection compared with that produced in broth cultures (Table 23; Figs. 17 and 19).
However, another nematicidal compound, indole, which was identified from in vitro
cultures, was not detectable in any of the nematode-infected G. mellonella cadavers using
the TLC-UV methods. The injection of P. luminescens MD alone into larval G.
mellonella demonstrated that the absence of indole was not related to the presence or
absence of the nematode symbionts. As well, it was shown that the bacteria used for
injection and those re-isolated from injected larval cadavers were capable of producing
indole in TSB medium. The reason for the absence. or perhaps an undetectable level, of
indole under in vivo conditions is not clear. It is possible that the apparent lack of indole
in the larval cadavers was due to environmental factors, rather than to the bacteriurn
itself. Since indole is believed to be produced by microorganisms via tryptophan (Holding
and Collee, 1971 ; Freeman, 1985), the absence of indole may be due to the limited
quantity of tryptophan andfor the physiochemical conditions prevailing inside the
cadavers, or due partially to the TU=-UV methods for indole detection. It is known that
indole has two peaks of maximum W absorbance at 219 and 271 nm (Fig. 6). The
sensitivity of indole detection on TLC plates (or HPLC) would be increased by using a
W lamp with a wavelength of either 219 or 271 nm. The absence, or perhaps much
lower level, of indole is not surprising because indole is toxic to entomopathogenic
nematodes at higher concentrations (Figs. 12 and 13), and the data presented here show
that there wcre many developing nematodes in the infected cadavers.
ST was produced in infected insect Ianrae 24 h postinfection, which was when the
in- larvae were dying, and maintained a relatively high and constant level throughout
the infection cycle. That is, al1 developmental stages of the nematode symbiont were
virnially immersed in the nutrient environment with its high concentrations of ST. The
concentration of ST in nematode-infected larval G. mellonella was more than 1,000 pg/g
wet insect by 48 h infection (Figs. 17 and 19), which is many times greater then that
needed to inhibit the growth of several soil microorganisms under in vitro conditions (Li
et al., 1995b). The early production of ST may ôe triggered by bacterial contarninants
nleased from the rupnired alimentary system of the larvae due to the nematodes' andor
bacterial activity, and this production helps to maintain a suitable environment with
minimum cornpetition for the development of the nematode and bacterial symbionts.
Since ST is nematicidal, it dso might kill the bacterial-feeding nematodes that live in the
surrounding soil and that potentially could consume bacterial cells associated with the
insect cadaver.
The bacterial growth appears to be closely related to the development of the
nematode symbionts inside the cadavers, because peok population levels of the bacteria in
both H. megidis 90 - P. luminescens C9 and Heterorhabditis sp. HMD - P. luminescens
MD complexes appeared at about the same time that large numbea of amphimictic
female nematades were developing. Both bacterial species built up high population levels
(-IO9 CFü/ g wet insect tissue) inside the infccted larvae within 24 h of infection. The
increasing levels of the bacteria were accompanied by rapidly decreasing levels of the
bacterial contaminants. The rarely deteciable bacterial contarninants after 24 h maybe
due partially to the early production of ST. The population level of P. luminescens MD
primary fom cells, Vp, decrcaîed sharply by 48 h of infection. This sharp decnase of the
Vp ce11 level at 48 h of infection may perhaps be due to the increasing numbcr of Vsrn
cells that were competing for nutrients or the effect of Vsrn metabolites. The decreased
number of Vp cells at this stage of the infection might be beneficial to the nematode
symbionts, because then were only a few hermaphroditic females at this stage and more
food or food nserves could be used in the subsequent development and reproduction of
the large number of amphimictic females. In fact, the Vp cells regained high population
levels within the insect cadaver when there were huncûeds of amphimictic females and
males at 5 d after infection. Vsrn was rarely detectable in the cadavea after about 17 d of
infection presumably because the population of Vsrn was very low at the late stage of the
infection. In plate cultures, Vp and Vsrn were readily interchangable. However, it is not
clear whether the growth patterns of Vp and Vsrn in nematode-infected larval cadavers
are due to the relatively independent growth of these two forrns or to the interchange of
one form with another over the period of infection.
Polymorphism appears to be a cornmon property of Xenorhabdus spp. and
Photorhnbdus spp. in both the colonial and cellular levels of in vitro cultures (Akhurst,
1980; Boemare and Akhurst, 1988; Hurlben et al., 1989; Gemtsen et al., 1992). Its
significance is unknown, although there is speculation that both the secondary form
(phase ïI) and the small-colony variants may have a survival advantage for the species
(Hurlben et al., 1989; Gerritsen et al., 1992). Such cells do not produce secondary
metabolites, and so more energy could be diverted to ce11 division and growth (Gemtsen
et al., 1992; Smigielski et al., 1994). The pnsent study found chat a smalltolony variant,
Vsm, which is an intermediate type between the primary and secondary foms of the
bactecium occurred in both in vitro and, in particular, in vivo conditions (Table 22; Fig.
19). The prirnary form (Vp) and the Vsrn co-exist in infsted iasects and show very
different population dynamics. Nso, Vsm was demonstrated to be less prefemd by, and
less pathogenic to its nematode symbionts (data not shown). The question arises as to
why the Vsm variant should occw so early in the development of the nematode and be so
abundant in a newly-infected insect cadaver when, presumably the nutrient level is high.
Gemtsen et aL(1992) proposed that the nematode might prefer the primary form over the
smalltolony variants and so the presence of the small-colony variants might prevent the
nematode from nmoving al1 the bacterial cells in the cadaver during feeding. However,
almost al1 observations on colony variant were made under in vitro conditions, and
species/isolates of Photorhabdus, except for P. luminescens MD, are not known to have
colony variants under in vivo conditions. More strains and species of Xenorhabdus and
Photorhabdus should be studied under in vivo conditions in order to have a more
complete understanding of the biological roles of the small-colony variants and the
secondary fonn in the nematode-bacterium-insect association.
Unlike in vitro culture in TSB medium when the pH could be as high as 9.0, the
pH of macerated, nematode-infected G. mellonella was much lower king nearly neutrai
(-6.85 - 7.6) during the entire infection process (Figs. 17c and 19c). The difference
indicates a quantitative andor qualitative ciifference of the alkaline metabolites between
those under in vitro and those under in vivo conditions. Since extreme pH in the
environment is likely to be hannful to the entomopathogenic nematode, the results
suggest that it may be beneficid when mus-culhiring nematodes to control the pH
conditions in order to optimize nematode production.
Maxwell et al. (1994) reported that antibiotic metabolites released from
nematode-infected G. mellonella larval cadavers into the sumunding soi1 could
temporarily decrease the population levels of some soil bacteria. ST was produced in
larval cadavers at relatively high concentrations by al1 strains and species of
Photorhabdus studied (Table 23). The toxic effect of ST against hingal-feeding
nematodes or bacterial-feeding nematodes, such as C. eleganr, and its strong repelling
activity against Us of several Steinemema spp., but not those of Heterorhbditis spp.
tested (Table 19; Fig. 12), suggest that ST might not only help to maintain optimal
environmental conditions as an antibiotic inside the insect cadaver for the development of
the bacterium and its nematode symbiont, but might also play a role in decreasing
cornpetition for resources and habitat by imrnobilizing, killing or repelling other
nematode species within or outside the cadavers. The strong, nematode-repelling property
of ST also may be an advantage for Heterorhabditis spp., when it is nleased into the
surrounding soil during U emergence where it cm serve to repel cornpetitors from the
immediate foraging area while searching for a new host. Interestingly, in this regard, the
Us of S. glasen' and S. feltiae, two known cruiser foragers, were arnong the most sensitive
ones to ST in these experiments but S. carpocapsae, an arnbusher forager, was not
effected by ST (Table 19).
The finding in this study of a difference between in vitro and in vivo metabolitic
production has led to a separate research project in which two novel pigments (Hu et al.,
1998) and a novel antibiotic (Hu et al., unpubl.) were identified from P. luminescens Cg-
infected G. mellonellu cadaver extracts. As well, it was found in the present study that
there was distinct qualitative and quantitative difference in in vivo met abolites produced
by P. luminescens C9 following infection of lmal G. mellonellu, especially during the
f i t few days after infection (Fig. 18). Furtber study of these differences may help to
understand the metabolic process of the bacteria and the biological role of the
metabolites.
In conclusion, ST, but not indole, was identified from nematode-infected larvd
cadavers of G. rnellonella. ST was produceci in the cadavea by al1 the Photorhabdus spp.
tested but in variable quantities. In larval G. mellonella, infected by either H. megidis 90
or Heterorhabditis sp. HMD, ST was not detectable within the first 24 h of infection but
increased rapidly by 48 h to 5 d postinfection and remained ai a relatively high and
constant level even after the nematode symbiont had completed its reproduction. The
population dynamics of the bacteria under in vivo conditions were highly variable
depending on the bacterial isolates tested. However, bacterial growth appears to be related
to the development of the nematode symbionts in nematode-infected G. mellonella l w a e
in that the peak levels of the primary cells of the bactena and of arnphimictic fernales
occur simultaneously in both H. megidis 90 - P. luminescens and in Heterorhabditis sp
HMD - P. luminescens MD complexes. In nematode-infected l m a e pH of the macerated
larvae wen slightly higher than 7.0. The earl y production as well as the higher quantity of
ST, which has both antibiotic and nematicidal propcrties, suggests that it plays a
signifcant role in the symbiotic association between the nematodes and their respective
bacterial symbionts.
CHAITER 7
GENERAL DISCUSSlON
Entomopathogenic nematodes. Steinememo spp. and Heterorhabditis spp., and
their respective bacterial symbionts, Xenorhabdur spp. and Photorhabdu spp.. fom a
tripartite nematode-bacterium-insect association once the insect host is infected. The
symbiotic bacteria produce antimicrobial and insecticidal metabolites in broth culture.
These bioactive agents are generally believed to play an important role in this tripartite
association, such as in preventing competition from bacterial contarninants and in
weakening the defense response of the insect host (Dutky, 1959; Paul et al., 1981;
Mchemey et al., 1991a; Akhurst and Dunphy, 1993). The present study has demonstrated
that the bacterial symbionts also produce nematicidai metabolites under both in vitro and
in vivo conditions. This discovery provides new evidence on the important role of the
bacterial secondary metabolites in the nematode-bacterium-insect associations.
Unlike the insecticidal activity of the bacterial metabolites, which help to kill the
insect host (Ensign et al., 1990; Bowen et al., 1998). the role of the nematicidal and
antimicrobial substances appears to be to help minimize competition from other species
of nematodes and bacteria. This is in addition to the bacteria's mle in developing and
maintaining optimal growth conditions for the bacterial and nematode symbionts within
the insect cadavers. Together, the nematicidal, insecticidal and antimicrobial activities
represent three major biological contributions of the bacteria to the symbiotic
relationship with tôe entomopathogenic nematodes and to their mutual success in theK
tripartite association with the insect boa.
In the present study, three nematicidal metabolites, ammonia, 3,5-dihydroxy4-
isopropylstilbene and indole have been identified from cultures of Xenorhabdus spp.
andlor Photorhabdus spp.. Two important plant-parasitic nematodes, M. incognita and B.
xylophilus, were selected as the target nematodes in the routine bioassays. This selection
was based mainly on (i) the fact that M. incognita and B. xylophilw are representatives of
two distinctive nematode taxa, the Tylenchina and Aphelenchina; (ii) both nematode
species are commercially. very important worldwide pests in agriculture and forestry,
reyxtively (Sasser and Carter, 198s; Mamiya, 1984; Sutherland and Webster, 1993);
(iii) an inhibitory effect of the entomopathogenic nematode-bacterium complexes on
Meloidogyne spp. and other plant-parasitic nematodes has been reported (Bird and Bird,
1986; Ishibashi and Kondo, 1986; Georgis and Kelly, 1997), and (iv) a large quantity of
12s of M. incognita and J4s and adults of B. xylophilw were readily available in the
laboratory. In the present study, the occurrence of ST would properly have been rnissed if
only M. incognita had been used during the screening process, because J2s of M.
incognita are not affected by ST even at 200 pglml. Also. M. incognita is more sensitive
to solvents than is B. xylophilus. Consequently, the quantity of the solvents used in the
bioassay and, subsequently, the concentration of the crude, organic compounds screened
would have to be decreased. In other words, the sensitivity of the nematicidal bioassays
would be significantly decreased if only M. incognita had been used. It has ken ~po r t ed
that a significant factor in any nematicidal screening system is the choice of the bioassay
species, because the sensitivity of difîerent nematode species to test materials may Vary
significantly (Anke and Sterner. 1997). For example, the nematicidai compounds that
were pmduced by fungal cultures, such as ascomycetes and nematophagous fun@, and
detected by a bioassay using the free-living nematodes, Panagrellus redivivus, Rhabditis
spp. or C. elegans, were found not to be active against M. incognita (Anke and Sterner,
1997). The results of the present snidy emphasize the importance of selecting an
appropriate range of organisms for an effective bioassay.
Of the t h e nematicidal metabolites identified, ST and indole have not been
reported previously to ôe nematicidal. In the present study, both ST and indole &ected
egg hatch, and the viability, mobility and dispcrsal behaviour of a variety of nematode
species. Indole caused paralysis of nematodes at lower concentrations and was lethal to
al1 nematode species tested at relatively high concentrations in immersion tests. ST, on
the other hand, was active against bacterial-feeding nematodes, such as C. eleguns, and
fungal-feeding nematodes, such as Bursaphelenchus spp. and A. rhytium but not against
12s of M. incognita or Us of H. megidis 90. The differential nematicidal effect of ST is
important in the in vivo interaction between the bacterium and the nematode symbiont,
because it was shown in the present study that ail developmental stages of
Heterorhabditis spp. were immersed in relatively high concentrations (-600 - 4,000 pg/g
insect) of ST within the insect cadaver.
It was shown in the present study that culture filtrates of most bacteria were active
against J2s of M. Acognita, even the filtrates were diluted to 1/4 of the original strength.
Given the activities of the nematicidal metabolites identified from the filtrates and their in
vitro production, the nematicidal activity of the filtrates was apparcntly a combined effect
of nematicidal agents, including unidentified ones. Together, the identification of
ammonia ST and indole expands on and confirms the conclusion reached (Chapter 3)
that multiple factors involved and contributed to the total nematicidai activity detected in
the culture filtrates of Xenorhabdus spp and Photorhabdus spp.
It is a practical approach to screen the derivatives of a known bioactive compound
to find more active ones, and such a screening may sometimes result in more promising
agrochernicals and dmgs than the compound initially identified (Betina, 1994; Suga,
1994). ST and indole themselves oKer no potentiai application as demonstrated in the
present study. Firstly, ST was not active against M. incognita, one of the most
econornically important plant-parasitic nematode pests worldwide. Secondly, although ST
was active against Bursaphelenchus spp., its activity was lower compared with that of
certain stilbene derivatives reported by Suga (1994). Thirdly, indole is active against egg
hatch and JZs of M. incognitu, but it failed to pnvent infection of the nematode in
greenhouse tests. However, indole derivatives might be more effective. Since indole is
more active against M. incognita in vitro, several simple indole derivatives were tested
for their nematicidal activity. A few of them were more active than indole and their
activity is closely related with the type and position of the hinctional group(s) on the
indole skeleton. ST, a stilbene denvative, was not explond further, kcause dozens of
synthesized stilbcne derivatives had been studied or patented after identifying the
nematicidal property of a few natural occumng stilbene derivatives (Moharnmad et al.,
1992; Suga et al., 1993; Suga, 1994). The unidentified nematicidal metabolite(s) from
Xenorhobdus spp., especially X. nemtophilus BCI (Chapter 4). repnsents another kind
of nematicide(s) that is different fiom ST and indole, and remains to be identified and its
potential to be exploreci.
Large numbers of Us of entomopathogenic nematodes are required for the
successful control of insect pests in the field. Miller and Bedding (1982) showed that
about 6000 million S. feltiae (=Neoaplectuna bibionis) per hectare would be required to
effectively control stem borer, Synanthedon tipulifonnis, on black currants in the field,
and similar numbers per hectare of H. heliothidis (= H. bacteriophora) for black vine
weevil control on strawberry (Bedding, 1984). Consequently, the nematodes must be
mass-produced in very large numbers, at low cost and have a reasonable shelf life. The
present study showed that ammonia which is nematicidal, is commonly produced in in
vitro cultures of Xenorhabdus spp. and Photorhabdus spp. This suggests that improved
media formulation andlor cultural conditions that decrease the quantity of ammonia,
indole and other nematicidal metabolites, which are toxic also to entomopathogenic
nematodes, could enhance the eficacy of in vitro nematode production for these
commercial applications.
The possible production of nematode toxic metabolites by the secondary form of
the bacterial symbionts nmains unclear. Secondary forms of Xenorhabdus spp. and
PhotorhabduF spp. differ from the primary foms of the bacteria in several characteristics
(Akhurst, 1980). When both the pnmary and secondary forms are available as food
sources, the nematode symbionts prefer feeding on the pnmary rather than the secondary
fom (Gemtsen and Smits, 1997). In fact, the secondary form does not support the growth
and reproduction of the nematode symbionts as well as does the primary form (Akhurst,
1980; Aldiurst and Boemare, 1990; Gemtsen and Smits, 1997). Ehlers et al. (1990)
suggested that the secondary fom of P. luminescens produced a toxin that kills the
nematode symbionts. They found a negative effect of secondary form of P. luminescens
spp. on Heterorhabditis spp., but secondary form of Xenorhabdus spp. had no effect on
Steinemema spp. Later, Akhurst (1993) and Gemtsen and Smits (1997) considered that
the resuits were more likely nutrient reiated rather than the results of production of a
toxin. Further study on the possible production of nematode toxic metabolites by
secondary foms of Xenorhabdus spp. and Photorhabdus spp. is necessary, because it
might help explain more hlly the specific symbiotic association between the bacteria and
nematodes and help improve the in vitro production of the entomopathogenic nernatodes.
Metabolites of Xenorhabdus spp. and Photorhabdus spp. influence the nematode
symbionts in several ways. Grewal et al. (1997) suggested that symbiotic bacteria inside
the nematode-infected host are a source of volatile, infochemicals, which play an
important role in inter- and intra-specific nematode competition. The authors
hypothesized that Us may reduce competition by responding differently to the cues from
unparasitized hosts vs hosts parasitized by conspecific or heterospecific nematodes.
Glazer (1997) nported that the initial infection of an insect host by entomopathogenic
nernatodes induced the release of a substance that reduced the subsequent nematode
invasion and that such a decrease is nematode species specific. As well, Ehlers and
Iohnigk (1998) reported that the symbiotic bacteria excrete a signal which may change
the developmental pathway of the first stage juveniles. The nature of this signal is not
known yet.
One of the significant discoveries of the present study was that the nematicidal
metabdites prduced by Xenorhabdur spp. andlor Photorhabdus spp. dso infiuence the
behaviour of theK respective nematode symbionts. Interactions between Steinememu
spp., Heterorhabditis spp. and other nematode species have been reported (Bird & Bird,
1986; Ishibashi & Kondo, 1986; Robinson, 1995; Koppenhofer et al., 1996; Kaya &
Koppenhofer. 1996). However, the effect of metabolites from the bacterial symbionts
within the insect cadavers or when released into the surounding soil dunng U emergence,
previously has not k e n considered. ST was prduced in larval cadavers at relatively high
concentrations by dl strains and species of Photorhabdus studied (Figs. 16 and 18; Table
24). The toxic effect of ST against tùngal-feeding nematodes or bacterial feeding
nemntodes, such as C. eleguns, and its strong repelling activity against Us of several
Steinemema spp., but not those of the Heterorhabditis spp. tested, suggests that ST might
play a role in decreasing cornpetition for resources and habitat by immobilizing, killing or
repelling other nematode species within or outside the cadavers. In the soil, the bacterial
and nematode syrnbionts and the insect cadaver in the tripartite association may face
predation either individually or in total. Saprophytic nematodes are cornmon in the
midgut of insects and would if not controlled, continue to feed and reproduce on the
microflora of the Heterorhabditis infected cadavers. Some bacterial-feeding and
nematode-feeding mmatodes in the soil may be attmcted towards and feed on this
bbcontainer" of the bacteria and nematodes, the nematode-infected insect cadaver. ST may
help prevent such predation. The behaviour-influencing metabolites, including ST, may
dso play a role in intra- or inter-specific interaction between entomopathogenic
nematodes (Glazer, 1997; Grewai et al., 1997). The strong nematode-repelling property
of ST may be advantageous for Heterorhabditis spp., when it is released into the
surrounding soil d u h g D emergence where it could repel cornpetitors and protect the
nematode's habitulspace.
The inhibitory effect of the entomopathogenic nematode - bacterium complexes
on other nematode species has been noted in vivo (Bird and Bird, 1986; Ishibashi and
Kondo, 1986) and in the field (Georgis and Kelly, 1997). Consequently, the potential has
been considered for controlling plant-parasitic nematodes while applying
entomopathogenic nematodes against insect pests. The mechanism of action of the
inhibitory effect on plant-parasitic nematodes is not clear. Georgis and Kelly (1997)
suggested three possible mechanisrns that may be involved. Firstly. competition for space
and habitat between entomopathogenic nematodes and other nematode species. Secondly,
inundative application rnay enhance the predator-prey response in the field, since many
nematode-feeding organisms, such as protozoa, nematodes and fungi may consume
indiscriminately both entomopathogenic and plant-parasitic nematodes. Thirdly, bacterial
metabolites released into the surrounding soil from insect cadavers infected by
entomopathogenic nematode may adversely affect plant-parasitic nematodes and decrease
their populations.
The present study provides evidence to help clarify and stimulate further
speculation. The nematicidal metabolite, ST, is active against bacterial- and fungal-
feeding nematodes and is present in the nematode-infected larvai G. mellonella cadavers
at high concentrations throughout the life cycle of the nematodes. Maxwell et al. (1994)
reported that antimicrobial metabolites released from steinemernatid-infected larval G.
mellonella cadavers during Us emergence temporarily decreased population levels of soil
bacteria. The natural release of the contents of the cadavers during U emergence was
repeatedly confirmed in the present study. The nddish brown materials from the
heterorhabditid-infectecl cadaven stained the white filter paper in the Petri dishes during
Us emergence. As a result, the nematicidal metabolites released from the cadavers into
the surrounding soil may partially contribute to the observed inhibitory effect on soil
nematodes after inundative application of entomopathogenic nematodes. However, the
mrnaticidal effect alone appears to be limited in space and time compared with the
overall nematode inhibitory effect observed in the field, because the metabolites released
do not persist or spread widely because of biotic and/or non-biotic factors. Another factor
involved is the density of soil insects that are susceptible to these entomopathogenic
nematodes. If there are few of these insects, the density of cadavers infected by the
nematodes in the soil will be low and, subsequently, the quantity of antibiotic and
nematicidal compounds released into the soil will be relatively small. Ishibashi and
Kondo (1986) reported that application of the entomopathogenic nematodes inhibited the
populations of soil nematodes in potted soil and bark compost. It seems unlikely there
were many insect hosts in potted soil or bark compost and thus then would not be enough
nematicidal metabolites released from the cadaver. Consequently, the inhibited nematode
population in this particular case might be attributcd mainly to some other factors such as
enhanced prey-predator effect a d o r cornpetition for space.
Although it was proposed decades ago (Dutky, 1959) that the production of
antirnimbial substances in nematode-infected insects prevented putrefaction of the
cadavers, M e in vivo experimental data is available to support that speculation (Maxwell
et al., 1994; Jarosz, 1996). RecenUy, the hypothesis was questioned by Jarosz (1996). He
reported that a low antibiotic potency of a lirnited spectrum of antibacterial activity was
found during al1 the developmental stages of the nematode in G. mellunella infected with
S. carpocapsae or H. bacteriophora. Consequently, the author proposed that the lack of
putrefaction of the infected insect was rather a result of littie or no cornpetition for the
Xenorhabdus dunng rapid colonization of the insect body and this rapid growth prevented
secondary invasion of the insect cadaver.
In contrast to the results reported by Iarosz (1996), the pnsent study provides
new, chernical evidence of antibiotic production within nematode-infected insects and
supports the hypothesis of antibiotic inhibition (Dutky, 1959) at least in the
Heterorhabditis spp. - Photorhabdus spp.0 G. mellonella ttipartitate association. Firstly,
the nematicidal metabolite ST, which is dso an antibiotic (Paul et al., 1981; Li et al.,
1995b), was proven chemically to be produced by al1 five Heterorhabditis -
Photurhabduï complexes tested in l a r d G. mellonella cadavers, and at 7 d postinfection
it had a concentration of 665 - 4,182 pg/g wet insect (Table 24; Figs. 16 and 18). In Iarval
G. mellonella infected by either H. megidis 90 or Heterorhabditis sp. HMD. ST was
detectable after 24 h infection and maintained a relatively high concentration (-3,700
pg/g and 1,700 pglg wet insect respectively) throughout the Iife cycle of the nematode
symbiont within the cadavers (Figs. 16 and 18). These concentrations of ST are much
higher compared with those produced in broth cultures (Fig. 8) and are ten to hundnds of
times higher than the concentration necessary to inhibit most test microorganisms under
in vitro conditions (Li et al., 199%; Li et al., 1998). Secondly, it was repotted (Hu et al.,
1998) that a variety of anthraquinone derivatives besides ST and AT were pduced in
larval cadavers of G. mellonella infected by H. mgidis W. Some of the anthraquinone
derivatives have been shown to be antibacterial (Sztacicskai et al., 1992; Li et al., 199%).
Similar anthraquinone derivatives were dso produced by d l five Heterorhabditis spp. - Photorhabdus spp. complexes studied (see Chapter 6) in larval G. niellonellu cadavers.
Thirdly, Maxwell et al. (1994) reported that the antibiotic activity was detected after
demise of the insect whether infected by the nematode-bacterium complex or the bacterial
symbiont alone. The known antibiotics, xenocoumacins 1 and 2, were reported to be
produced at a 1:l ratio in larval G. mellonella infected by X. nematophilus subsp. dutki
(isolates GI and WU), and the total concentration of xenocoumacins 1 and 2 was 800
no00 mg (wet weight) of insect tissue for the GI isolate. Maxwell et al. (1994) noted
also that the levels of antibiotic activity was greater in extracts from nematode-infected
G. mellonella than in TSB broth. These results support Our observation that greater
amount of antibiotics were produced in vivo than in in vitro. Fourthly, the experimental
design and subsequent conclusion by Jarosz (1996) might be controversial. For example,
only aqueous extracts of the nematode-infected insects were tested. The results therefore
may be misleading, because dl known antibiotics produced in vitro by Xenorhabdus spp.
or Photorhubdus spp., such as indole denvatives, xenorhabdins, xenorxides, stilbene
derivatives, anthraquinone derivatives, nematophin (Table 3; Li et al., 1998) and two
novel abtibiotics (Ap and a furan derivative) (Hu et al., unpubl.) are soluble in organic
solvents. Only the xenocoumacins are water soluble (McInerney et al., 199 1 b). In fact, the
present study showed that ST, in addition to anthraquinone derivatives, was produced
(665.2 i 151.5 pg/g wet insect) in G. mellonella infected by H. bacteriophoru Oswego,
the same nematode species used by Jarosz (1996), and both ST and anthraquinone
derivatives would not be dissolved in the aqueous extract of the insect. Finally, it was
shown in the present study that a rapid and massive multiplication of the syrnbiotic
bacteria occumd in the nematode-infected larval G. mellonella within 24 h of infection
(Figs. 16 anci le), but antibiotics were s a 1 produced by the symbiotic bacteria &ter 24 h
of infection when the insect host was dying. The timing of antibiotic production appears
to be comlated with the mpture of the alimentary system of the host insect after
nematode infection. During the first few hours post nematode penetration of the host, the
non-symbiotic bacteria carried on the body surface of the nematodes are eliminated by the
insect's immune system, but the symbiotic bacteria are somewhat resistant to the insect's
immune system or are not recognized as nonself (Dunphy and Webster, 1988; Dunphy
and Thurston, 1990). Consequently, these symbionts multiply rapidly and begin to build
up high population levels (Figs. 16 and 18) within the fint 24 h of infection and, as a
result of the activity of the bacteria and its nematode syrnbiont. the insect host dies and its
tissues, including the p i , break down. This rupture of the host's digestive tract leads to
the release of bacterial contaminants into the hemocoel, which threatens the growth
conditions for the bacteria and the nematode syrnbiont inside the cadavers. However, the
production of the antibiotics, perhaps including bactenocins, by the symbiotic bacteria at
this stage diminishes such a risk. More in vivo studies, especially for the Steinetnema -
Xenorhabdus - insect host association, are necessary to clari@ the biological role of the
anti biotics.
in conclusion, this study has opened the gate to a new research area in the
tripartite association. The study has demonstrated for the first time the nematicidal
properties of Xenorhnbdus spp and Photorhabdus spp. Three nematicidal metabolites,
ammonia, 3,s-dihydroxyl4isopcopylstilbene and indole, were identified in broth cultures
and 3,5-dihydroxyl4isopropylsti1bene was shown to be aiso prduced by P. luminescens
at high levels in vivo. The nematicidal metabolites not only affect the viability, mobility
and egg hatch of a variety of nematode species but also Us' behaviour of
entomopathogenic nematodes, Steinemena spp. and Heterorhabditis spp. The study
provided evidence of the importance of the bacterial metabolites including antibiotics in
the tripartite association. However, many aspects of the role of these bacterial metabolites
in the nematode-bacterium-insect association are still to be revealed. For example, what is
the chernical nature of the unidentified, nematicidal metabolites produced by
Xenorhubdus spp. in culture in the present study? Do the secondary foms of
Xenorhubdus spp. and Photorhabdus spp. produce toxic metaboli tes that contribute
partially to the poor nematode production in vitro? Further studies on these topics would
help us undentand more completely these syrnbiotic associations and the biological roles
of the antibiotics and nematicidal metabolites in the tripartite interactions. Such
information would further enhance these nematodes as powerfd biological contro l agents
of insect pests and provide leading bioactive compounds for development of
agriculturally and medically important chemicals.
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