Antimicrobial activity of Xenorhabdus sp. (Enterobacteriaceae), symbiont of the entomopathogenic nematode, Steinernema riobrave (Rhabditida: Steinernematidae)
BY Peter 3. lsaacson
B. Sc., University of Victoria, 1994
THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF PEST MANAGEMENT
ln the Department
of
BIOLOGICAL SCIENCES
O Peter J. Isaacson 2000
SIMON FRASER UNIVERSITY
April2000
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The entomopathogenic nematode, Steinernema riobrave, releases its
bacterial symbiont, Xenorhabdus sp., into the haemocoel of an insect host
resulting in bacterial septicemia and death of the insect. Gallena mellonella
larvae infected with S. nobrave were killed within 72 h of infection by which time
the growth of the bactenal symbiont had reached 1.1 x IO~CFUI~of wet insect
tissue. These syrnbiotic bacteria praduced secondary metabolites that showed
antimicrobial properties. These metabolites helped prevent contaminating
microorganisms from becoming established in the insect cadaver and allowed
the bacterial and nematode symbionts to grow optimally. However, Gram
negative species other than Xenohabdus became established although no
additional antibiotic activity was detected. This indicates that the later stages of
nernatode development occur in a multixenic environment rather than a
monoxenic environment populated only by the nematode's symbiont.
The metabolites produced by the Xenorhabdus sp. showed significant
broad spectrum antibacterial and antimycotic activities against fungal and
bacterial species from different habitats. When tested against agnculturally
important fungi in agar diffusion plate assays. the cell free culture broth completely inhibited the growth of many plant pathogenic fungi including, Botrytis cinerea, Diddymella bryoniae, Fusarium solani and Pjdhium ultimum. The majority of this activity was caused by water soluble metabolites rather than ethyl acetate soluble compounds. Further analysis showed that sorne of this water soluble activity was a result of extracellular proteins including ex* and significant endochitinase activity as deterrnined by the release of p-nitrophenol from the artificial substrates pnitrophenyCN-acetyl-PD-glucosaminide and p-nitrophenyl-
P-D-N-N9-Nn-triacetyochitobiose,respectively. Gel filtration analysis of the water soluble proteinaceous fraction showed WO major activity peaks conesponding to
(1) large molecular weight proteins and broth components and (2) small molecular weight, peptide or nonproteinaceous. heat tolerant biomolecule(s).
Overall, the data showed that chitinases wntributed to but were not the dominant source of antimicrobial activity.
These findings suggest that secondary metabolites from Xenothabdus sp.
RIO may provide a source of novel compounds useful to the agncultural industry as pest management tools. FOR MY PARENTS ACKNOWLEDGMENTS
I am grateful for the guidance, encouragement and support of my senior
supervisor Dr. J. M. Webster who has persevered with me through the course of this project. Many thanks go to my committee member. Dr. J. E. Rahe for his
valuable suggestions during my research and in my writing of this manuscript.
The assistance of Drs. 2. Punja (Simon Fraser University), M. Moore (Simon
Fraser University), R. Utkhede (PARC-Agassiz), and S. Berch (Glyn Road
Research Station. Victoria) for supplying test cultures andfor laboratory facilities for my research was much appreciated. I would also Iike to thank Drs. J. Li and
G.Chen (Welichem Technology Corp.) for sharing their knowledge and providing guidance; E. Urquhart (Simon Fraser University) for his invaluable technical assistance; B. Leighton (Simon Fraser University) for rearing and supplying nematode cultures; the students of both Drs. J. M. Webster and J. E. Rahe for their great friendship and support; and the many people in the Department of
Biological Sciences who provided me with the opportunity to pursue this degree and who made my academic career much easier.
Special gratitude goes out to my fnends and family who showed much patience and understanding through the course of my work, and rnost of all to my wife Sophie who has stood behind me al the way. I am forever indebted for their support. TABLE OF CONTENTS
Approval ...... ii ... Abstract ...... III
Acknowledgrnents ...... vi .. Table of Contents ...... vil
List of Tables ...... ix
List of Figures ...... xi
1. 0 GENERAL INTRODUCTION ...... 1
2.0 GENERAL MATERIALS AND METHODS ...... 18
2.1 Source and Maintenance of Xenortrabdus ...... 18
2.2 Source and Maintenance of Test Organisms ...... 23
2.2.1 Test Bacteria ...... 23
2.2.2 Test Fungi ...... 23
2.3 Bacterial Fermentation ...... 24
2.4 Measurement of Antimicrobial Activity ...... 25
2.4.1 Bactenal Bioassays ...... 25
2.4.2 Fungal Bioassays ...... 26
2.5 Satistical Analysis ...... 27
3.0 CHARACTERISTICS OF XENORHABDUS SP. RIO ...... 28
3.1 Introduction ...... 28
3.2 Materials and Methods ...... 28
3.2.1 Morphological Characteristics...... 29
vii 3.2.2 Carbohydrate Metabolism ...... 30
3.2.3 Enzyme Production ...... 30
3.3 Results ...... 31
4.0 IN VIVO ANTlMlCROBlAL ACTlVlTY ...... 37
4.1 Introduction ...... 37
4.2 Materials and Methods ...... 38
4.2.1 In vivo Development of Microflora ...... 38
4.2.2 In vivo Development of Antimicrobial Activity ...... 39
4.3 Results ...... 39
5.0 CHARACTERIZATION OF ANTlMlCROBlAL ACTIVITY ...... 43
5-1 Introduction ...... 43
5.2 Materials and Methods ...... 44
5.2.1 h vitro Development of Xenorhabdus sp . RIO ...... 44
5.2.2 Spectnim of Antimicrobial Activity ...... 45
5.2.3 Fractionation of Antimicrobial Activity ...... 46
5.2.4 Analysis of the Exo-Enzymatic Activity ...... 47
5.2.5 Partial Purification of Chitinase and Bioactive Proteins ...... 50
5.2.6 Characteristics of Partially Purified Bioactive Proteins ...... -51
5.3 Results ...... 52
6.0 DISCUSSION ...... 72
7.0 REFERENCES ...... 86 LIST OF TABLES
Table 1. Estimated 1993 worid crop production and preharvest losses (in
millions of tones and percent of world production lost to diseases,
insects and weeds ...... 2
Table 2. Some biological control agents that are used for fungal disease
control ...... 6
Table 3. Entomopathogenic nematodes and their associated bacterial
symbionts ...... --...... -. 9
Table 4. Antimicrobial agents known from or derived from Xenorhabdus
spp...... ~~...... -...... -...... 14
Table 5. Tested host insect species for Steinemema riobrave ...... -.-..16
Table 6. Species and source of Xenorhabdus strains and their respective
nernatode symbiont used in this study ...... -...... -...... 19
Tabte 7. Test species used in this study and their known sources ...... 21
Table 8. Morphological characteristics of primary fom Xenorhabdus sp. RIO
in cornparison with those of three other Xenorhabdus species ...... 32
Table 9. Acid production by pnmary fom Xenorhabdus sp. RIO in
comparison with that of three other Xenohabdus species when tested
on carbhydrate sources...... -...... -33
Table 10. Enzymatic activities of pn'mary form Xenorhabdus sp. RIO in
cornparison with those of three other Xenorhabdus species ...... -.35 Table 11. Spectnim of antibiotic activity of different concentrations of freeze
dried, whole broth from in vitro cultures of the Xenorhabdus sp. RIO
measured as the radius of inhibition zones (mm) on agar diffusion
plate assays against eleven bacterial isolates...... 58
Table 12. Spectrum of antimycotic activity of different concentrations of
freeze dried whole broth from in vitro cultures of Xenorhabdus sp.
RIO as measured by radius of inhibition zones on agar diffusion plate
assays ...... 59
Table 13. Antibacterial and antimycotic activity of different supernatant
fractions expressed as the radius (mm) of inhibition zones in Petri
plate bioassays for Xenorhabdus sp. symbiont of Steinemema
nobrave ...... 62
Table 14. Cornparison of the final absorbance, pH, antibiotic activity,
antimycotic activity, total secreted protein concentration and exo-
and endochitinase activities in 72 h TSB cultures of two strains of
Xenorhabdus bovienii (AUS, B27), X. nematophilus (BJ), and Xenorhabdus sp. (RIO) ...... LIST OF FIGURES
Figure 1. Development of bacterial microflora and antimicrobial activity in
Gallena mellonella infected by Steinemema nobra ve as measured
by the number of cells/larvae and the size of the inhibition zone of
the test organism, respectively...... 40
Figure 2. Culture pH and growth curve (absorbance at 600nm) for
Xenorhabdus sp. RIO grown in TSB at 25OC ...... 53
Figure 3. Antibacterial and antimycotic activity of the cell-free culture broth
of Xenorhabdus sp. RIO as measured by the radius of the inhibition
zone ...... 55
Figure 4. Growth curve for Xenorhabdus sp. RIO showing total protein,
exochitinase and endochitinase development in TSB ...... 63
Figure 5. Profile of chitinase activity, protein concentration and antimycotic
activity in different fractions following separation of protein from
Xenorhabdus sp. RIO culture through Sephadex GlOO ...... -67
Figure 6. Profile of antimycotic activity before and after autoclaving the
Sephadex G100 protein fractions from Xenohabdus sp. RIO culture ..... 70 1.O GENERAL INTRODUCTION
Pests and pathogens of agro-forestry crops have caused major economic
loss and human misery throughout recorded history. Some epidemics have had
profound impacts on human society leading to malnutrition, starvation, migration
and death of people and livestock- Among the most devastating plant disease
epidemics was the 1845 Irish potato famine that followed the destruction of
potato crops by late blight fungus, Phytophthora infestans. Similar losses anse
today especially in underdeveloped wuntries where food is already in short
supply. In developed countries today, where food is more plentiful, plant
diseases in agriculture cause significant financial loss and an increase in
consumer prices. Insects, fungi and weeds are the major causes of loss of yield
in worfd agriculture (Table 1). There are an estimated 67,000 different pest
species that cause losses estimated at over 30% of the world's agricultural
output and of these some 50,000 species are fungal pathogens (Pimentel, 1997).
These fungal diseases account for world crop losses of approximately 12%
(James, 1981), which, in 1988. arnounted to a loss of over $9 US billion per year
in the United States alone (Agnos, 1997).
It is an irony of the times that one of the major reasons for the increased
incidence of plant disease is the increasing need for food by the rapidly
expanding world population. Through most of recorded history, the human
population increased at a rnodest 0.2% per year (Hewitt, 1998). However, in the
19" and 2omcenturies, a period of significant industrial, medicinal and agricultural advances, the human population has increased by as rnuch as 2% Table 1. Estimated 1993 world crop production and preharvest losses (in millions of tonnes) and percent of world production lost to diseases, insects and weeds
Actual Est. losses Potential Est. losses Total % Crop production to pests production to diseases % of crop lost to of crop lost
Millions of Tonnes Diseases lnsects Weeds
Cereals Potatoes Other root crops 8 t4 Sugar beets l Sugarcane Legumes Vegetables Fruits Coffee-wcoa-tea Oil crops Fiber crops Tobacco Natural rubber
Average percentages lost
Source: Agrios, 1997 and converted to metric tonnes per year. The cunent worid population is close to 6 billion and expected to climb to 10 to 16 billion by 2100 (Pimental, 1997). Augmented food production has, therefore, become the priority of world agricutturists as they endeavour to provide an adequate food supply for the world population. The increase in human population and in the global quality of life has resulted in a corresponding increase in food production that, in tum, has required (1) an increased need for agricultural land, (2) irnproved cultivation methods, (3) increased use of fertilizers, (4) improved crop varieties, (5) increased irrigation and (6) improved crop protection (Agrios, 1997). This 'green revolutionncame about through the
1960s and allowed mankind to overcome the challenge of feeding an increasing human population, at least initially. This led to large expanses of agricultural land being planted with virtual monocultures of high yielding, genetically similar varieties that required high levels of fertilization and extensive irrigation. This more intensive agriculture was prone to more frequent and damaging attacks by fungal, insect and nematode pests.
Concert& pest management methods began in the lgm century as agriculture became more of a commercial endeavour and subsistence farrning , although still widespread, became less important. This era saw the development of the first chemical pesticides including oil, sulphur, nicotine, rotenone, pyrethrum, Pans green, London purple, lead arsenate, Bordeaux mixture, copper sulphate and lime (Perkins and Patterson, 1997; Hewitt, 1998). The major expansion of chemical pesticide usage did not occur until the 1940s and 50s when advances in organic chemistry enabled the development of a wide range of synthetic chemical pesticides. The commercial potential of chemicals in crop protection and in maintenance of yield was realised through the availability of a wide range of relatively inexpensive, versatile and effective synthetic pesticide products. The agrochemical industry is now valued world-wide at approximately
$21 billion (US), which is spent on some 2.3 million tonnes of pesticide chemicals
(Pimentel and Greiner, 1997). Fungicides account for about 20% of the total agrochemical market with annual sales of close to $6.0 billion (US) (Hewitt,
1998).
During the last half of the twentieth century a great number of chemical fungicides were developed, new methods of application adopted, and new concepts on modes of action have evolved. The first chemical fungicides were non-systemic protectants that prevented infection by many fungal pathogens.
These products had to be applied many times through the growing season to ensure that the crop was protected from fungal attack. Great strides in the discovery of new fungicides occurred with the transition from protective to systemic fungicides in the 1960s. These products controlled fungi that had already infected the plants as well as provided, in some cases, season-long protection from further infection. It was estimated that application of such fungicides could decrease losses due to disease by as much as 10% (Hewitt,
1998). This, coupled with the development of high yielding ugreen revolution" cultivars and application of a high level of fertilizers. atlowed humans to maximise food production for an exploding worid population. However, it became increasingly apparent that there were costs associated with the widespread use of chemical pesticides through the development of strains of pests resistant to
the pesticides, by the occurrence of damage to non-target organisrns, including
humans and persistent negative environmental impacts such as contamination of
aquifers (Perkins and Patterson, 1997).
The now famous book 'Silent Springw,by Rachel Carson (Carson, 1962).
raised the public's awareness to the harmful effects of pesticides. This
publication raised the profile of the adverse environmental issues caused by
widespread indiscnminate use of pesticides and introduced them to the public
and political arena. Initially, Carson's argument was considered an extreme
perspective but eventually the scientific community came to realize the validity of
some of her concems. Subsequently, following extensive research and observation into these environmental problems, agnculturalists and scientists
have taken a "second lookwand have begun to develop methods to reduce the
hazards caused by these chemicals, such as use of alternative pest management practices. lntegrated pest management (Perkins and Patterson,
1997) and the use of pesticides from natural products (Hewitt, 1998) has become a necessary practice as the number of synthetic chemical pesticides has progressively diminished through legislative action. A number of biological agents have been developed or are in the process of being developed for controlling some important plant diseases (Table 2). However, the commercial use of these organisms has been slowed because the outcome of controlling a fungal disease was uncertain. Currently, the market for biopesticides is less than
1% or $100 million (US) of the total agrochemical Table 2: Some biological control agents that are used for fungal disease control.
Antagonist Target fungi Host crop
Ampelomyces quisquais Erysiphales Grape, apple, cucuhits Bacillus subti/is Phytophthora infestans Potato B. subtilis Pythium spp. Cotton Coniothynum minitans Sclerotinia scleratorum Vegeta bles Enterobacter cloacae Pythium spp. Seedlings Fusarium oxysporum FusanUm oxysporum Mushrooms Gliocladium roseum Botrytis cinerea Strawberry G. virens Pythium, Rhizoctonia sp p. Seedlings Peniophora gigantea Heterobasidion annosum Pine Pseudomonas Pythium, Rhizoctonia sp p. Cotton fluorescens P. fluorescens Gaeurnannomyces Wheat graminis Pythium oligandrum )ythium spp. Sugar beet Sphaerellopsis filum Melamspora spp. Willow Streptocyces griseovindis Fusarium oxysporum Ornamentals S. griseoviridis Altemana alternada Brassicae Trichoderma vin'dae AnniIlaria mellea Trees T. vindae Ceratocystis ulmi Elm T- vindae Chrondrostereum Fruit purpureum T- viridae Heterobasidion annosurn Pine Trichodema harzianum Botrytis cinerea Grape, apple T. harzianum Botrytis cinerea Strawberry T. harzianum Amiillaria mellea Trees Tnchodema spp. Botrytis, Pythium s pp. Fruit Trichoderma spp. Verticillium, Sclerotinia Vegetables SPP.
From Hewitt, 1998 market (Hewitt, 1998). These biologicals have been successful in some rnicroclimates, but their potential is greatest in integrated Pest management programs where they are used in conjunction with compatible chernical and other control methods.
There have been relatively few commercially successful biological organisms developed for controlling plant diseases, despite the natural antagonism between these biological agents and fungal pathogens. f he basis of control is often due to biochemical interactions, and these have provided opportunities also for the discovery and development of new, naturally derived, chemical controls. Natural products derived from living organisms show a tremendous chemical diversity and provide great potential for finding new classes of compounds with novel modes of action (Porter and Fox, 1993; Hewitt, 1998).
Many, naturallyderived, bioactive cornpounds have been discovered, but often they are not suitable for development as fungicides due to their environmental instability and/or phytotoxicity. Two groups of natural products that have gained some commercial success incfude strobilurins, produced by the basidiomycete
Strobilurus tenacellus. and phenylpyrroles which are derived from Pseudomonas pyrrocina (Hewitt, 1998). Secondary metabolites from rnicrobial fermentations offer a good source of bioactive cornpounds for controlling plant diseases. The
Japanese have used species of Streptomyces to produce and commercialize a number of secondary metabolites to control rice blast, including blasticidin (S. gnseochromogenes), kasugamycin (S. kasugaensis), polyoxins (S. cacaor'), validamycin (S. hygroscopicus) and natamycin (S. natalensis and S. chattanoogensis) (Hewitt, 1998)- Another organism that has received much attention recently for its production of novel, sewndary metabolites is the bacterium Xenorhabdus (Enterobacteriaceae), species which are symbionts of entomopathogenic nematodes of the genus Steinemema (Rhadbitida:
Steinemematidae).
Nematodes of the family Steinemematidae are obligate parasites of many insect species, and they have been developed cornmercially as biological control agents for a variety of agricultural and horticultural insect pests (Kaya and
Gaugler. 1993)- Their success is due to a number of favourable characteristics: they (1) have a broad host range, (2) actively seek out their host, (3) are highly virulent and rapidly kill their host, (4) have a high reproductive potential, (5) are relatively persistent in the environment, (6) can be mass produced, (7) are regarded as safe towards most non-target organisms, (8) are compatible with many existing chemical insecticides and (9) are easily applied with standard spray equipment (Kaya and Gaugler, 1993).
Steinernematids are lethal to the insects they infect as infective juveniles
(IJs). Each nematode species has a specific natural association with only one bacterial species, although some nematode species share the same bacterial species (Akhurst and Boemare, 1990). To date, there are 24 known
Steinernema species, each of which is symbiotically associated with a particular
Xenorhabdus bacterium (Table 3). It is from this association that the term
"entomopathogenic" arose. The bacteria are found associated with the intestinal lumen of the third stage, infective juvenile nematode, frequently temed the Table 3: Entomopathogenic nematodes and their associated bacterial symbionts
-- Nematode species Bacterial species Reference
Steinemema abbasi Fla vjmonas oryzihabitans Elawad et al., 1998 S. affine Xenorhabdus bovienii Akhurst and Boemare, 1990 S. arenanum Xenorhabdus sp. Akhurst and Boemare, 1990 S. bicomatum Xenorhabdus sp. Fischer-Le Saux et al., 1998 S. carpocapsae X. nematophilus Akhurst and Boemare, 1990 S. ceratophomm ND Jian et al., 1997 S. cubanum Xenorhabdus sp. Mracek et al., 1994 S. feltiae X. bovienii Akhurst and Boemare, 1990 S. glaserf X. poinarii Akhurst and Boemare, 1990 S. intemedium X. bovienii Poinar, 1985 S. karii ND Waturu et al., 1997 S. kraussei X. bovienii Akhurst and Boemare, 1990 S. kushidai X. japonicus Nishimuta et al., 1994 S. longicaudum Xenorhabdus sp. Kaya et al., 1993 S. monticolum Xenorhabdus sp. Stock et al., 1997 S. neocurtillae None reported Nguyen and Smart, 1992 S. oregoneme ND Liu and Berry, 1996 S. puertoricense Xenorhabdus sp. Fischer-Le Saux et al., 1998 S. rarum Xenorhabdus sp. Akhurst and Boemare, 1990 S. nobrave Xenorhabdus sp. Cabanillas et al., 1994 S. ritteri Xenothabdus sp. Kaya et al., 1993 S. scapterisci Xenorhabdus sp. Bonifassi et al., 1999 S. serratum Xenorhabdus sp. Bonifassi et al., 1999 S. siamkayai ND Stock et al., 1998 Steinemema sp. M X. beddhgii Akhurst and Boemare, 1990 Steinemema sp. N X. beddhgii Akhurst and Boemare, 1990
ND = not detennined "dauef stage. The life cycle and biology of entornopathogenic nematodes and their bacteria have been well documented (Akhurst and Boemare, 1990; Kaya and Gaugler, 1993). The IJs are attracted to and invade the host insect through natural body openings such as the rnouth, anus or spiracles. Once inside the insect the IJs exsheath and develop within the haemocoel. They evade the insect's immune systern possibly through possession of a nonreactive surface coat, heterophilic antigens or innate molecular mimicry (Akhurst and Dunphy,
1993) and release their symbiotic bacteria into the haemocoel. The bacteria grow rapidly and cause death of the insect through bacterial septicemia (Kaya and Gaugler, 1993). The nernatodes mature, mate and lay eggs to complete their life cycle in 3 to 4 d, depending on the temperature. This process may continue for several generations and, thereby, rapidly build up the nematode population in the insect cadaver. Within 7 to 10 days thousands of newly formed IJs exit the insect cadaver and migrate through the soi1 in search of new insect hosts to infect.
The Xenorhabdus symbionts are chemoheterotrophic bacteria with respiratory and fementative metabolisrns (Boernare et al., 1997). Poinar (1966) descnbed the species of bacteria inhabiting Sfeinemema carpocapsae (fomeriy
Neoaplectana carpocapsae) as Achrornobacter nematophilus
(Act-tromobacteriaceae: Eubacteriales). This species was later renamed as
Xenorhabdus nematophilus within the Enterobactenaceae (Thomas and Poinar,
1979). Through modem chemotaxonomic techniques and DNA analysis a number of Xenorhabdus spp. have been identified. These bactena are Gram negative, asporous, facultatively anaerobic rods (Akhurst and Boemare, 1990).
However, they are atypical among the Enterobactenaceae in being nitrate- reductase and catalase negative (Boemare et al., 1997). Currently, five species of Xenorhabdus have been described and each is associated with a particular
Steinemema species. The type species, Xenorhabdus nernatophilus, associated with the nematode S. carpocapsae, was described by Thomas and Poinar
(1979). Since then, four other species have been described including, X. beddingii, X. bovienii, X. poinani (Akhurst and Boemare, 1990) and X. japonicus
(Nishimura et al., 1994). The Xenohabdus species associated with S. nobrave has not yet been identified but it has characteristics similar to those of
Xenorhabdus nematophilus (Cabanillas et al., 1994).
Xenorhabdus spp. occur in two forms, termed Phase I and Phase II.
These foms are distinguished in vitro by the colony types produced on nutrient agar supplemented with bromothymol blue and triphenyîtetrazolium chloride
(NBTA) (Akhurst, 1980). Phase I bacteria can be isolated from the intestine of infective stage nematodes, but Phase II occurs almost exclusively in in vit/o cultures and rarely in the IJ's. Phase I bacteria differ from Phase II forms in the type of secondary metabolites that they produce. Some of these metabolites have antibiotic properties. As well, Phase 1 foms are pathogenic to insects
(Akhurst, 1980; Akhurst and Boemare, 1990; Smigielski et al., 1994). These metabolites help create an environment in the insect cadaver that is optimal for nematode reproduction (Akhurst and Boemare, 1990). It is unclear as to the function of the Phase II bacteria. However, there is some evidence that Phase II are capable of surviving much better in nutrient limiting conditions (Smigielski et al., 1994). This is supported by the fact that Phase I will at least partially convert to Phase II if exposed to adverse conditions (Smigielski et al., 1994). Such a phase shift rnay be a natural response by the bacterium when it is without a host in the soi1 environment. Under such conditions Phase II bacteria may be better suited for utilizing limited nutrient resources and competing with other free living microorganisms. However, it has not been proven that Phase II bactena occur freely in the soi1 or that they will revert back to the primary phase.
The symbiotic association between the nematodes and their specific bacteria is one of mutualism. The bactenum cannot survive outside of its host insect and cannot infect its target without being transporteci and then released by the nematode into the host haemowel. Axenic nematodes (without their bactenal syrnbiont) are capable of entering the host insect's haemowel, killing the insect and developing from 13's into adults but nematode reproduction has not been observed under such conditions (Jarosz et al., 1991). When the bacterial symbiont is artificially added to the axenic system, the nematodes reproduce successfully. lnitially, the bacterium prevents the growth of competing and saprophytic microorganisms in the cadaver (Maxwell et al., 1994), and as they grow they provide nutrients (Akhurst and Boemare, 1990; Akhurst and
Dunphy, 1993) for nematode development and reproduction.
lnsects killed by Steinemema spp. do not putrefy and bacteriological tests have shown that the bacterial symbionts are the predorninant microorganism in the cadaver, at least during the early stages of nematode development (Forst and Nealson, 1996; Boemare et al-, 1997). This implies the production of antimicrobial compounds by these bacterial symbionts. The lack of putrefaction could be due also to the fact that the bacterial symbionts reproduce so rapidly that they out-compete decomposers such as fungi and other bacterial species in the parasitized insect (Jarosz, 1996a and 1996b). It has been speculated that insect gut microflora and other invading microorganisms cannot establish in the insect body cavity because of the presence of the large, symbiont population and of its antimicrobial compounds (Boemare et al., 1997). Indeed, the antimicrobial secondary metabolite production is an important characteristic of Xenorhabdus spp. (Paul et al., 1981; Akhurst, 1982).
A number of antibiotic compounds produced by these bacteria have been identified. The first groups of antibiotics discovered from these bacteria were stilbenes and indole derivatives (Paul et al., 1981). Since then numerous studies have resulted in the identification of several novel, antimicrobial agents from different species of these bacteria (Table 4). These compounds are active against a broad spectnim of plant and animal pathogenic fungi under laboratory conditions (Chen et al., 1994; Li et al., 1995; Chen, 1996), and, for example inhibit economically important plant pathogenic fungi inciuding Botrytis cinerea,
Ceratocystis ulmiI Mucor pitifomis, Pythium coloratum, P. unimum, Phytophthora infestans and Rhizoctonia solani in Petri plate tests. Under similar in vitro conditions beneficial fungi such as the mycorrhizal fung i Suillus pseudobrevipes and Oidiodendron griseum and the insect pathogens Beauveria bassiana and
Metarhizium anisopilae are reported to be unaffected (Chen et al., 1994). Such Table 4: Antimicrobial agents known from or derived from Xenorhabdus spp.
Species Antimicrobial Properties' References Agent
X. nematophilus Phage Boemare et al., 1992 Xenocoumacin Mclnemey et al., 1991a Xenorhabdins Mclnemey et al., 1991 b; Li et al., 1995 Bacteriocin Boemare et al., 1992 Xenorhabdicin Thaler et al., 1995 Nematophin Li et al., 1997 Chitinase Chen et al., 1996
X. bovienli' Phage Boemare et al., 1992 lndole Paul et al., 1981 ; Li et al., derivat ives 1995 Xenorhabdins Mclnemey et al., 199 1b; Li et al., 1995 Xenomides Chen, 1996 Bactenocin Boemare et al., 1992 Chitinase Chen et al., 1996
X. beddingii Phage Boemare et al., 1992 Bactenocin Boemare et al., 1992
Xenorhabdus spp. Xenorhabdins Mclnemey et al., 1991a Xenocournacin Mclnemey et al., 1991b
1=antibiotic, 2= antimycotic, 3=antiulcer, 4=insecticidal
From Li et al., 1998 results imply that these compounds might be useful in controlling agricuiturally important plant diseases. Subsequently, Ng and Webster (1997) showed that soluble organic metabolites of X. bovienii controlled P. infestans on potted potato plants with little or no visible phytotoxicity. The study showed for the first time the potential of these products to control diseases of living plants aîthough the authors recognized that such cnide mixtures were unlikely to be registered for use in commercial agriculture.
Steinemema riobrave, a recently described entomopathogenic nematode species, was discovered infecting corn eanivorm, Helicoverpa zea, and fall amyworm, Spodoptera frugiperda, in the Lower Rio Grande Valley of Texas
(Cabanillas et al., 1994). This nematode has shown considerable promise in controlling insect pests due to its host finding capabilities and high insect rnortality (Cabanillas et ai., 1994). Steinemema nobrave appears to be naturally selected for biological control of lepidopteran pests such as H. zea and the pink bollwom, Pectjnophora gossypiella, from semi-and subtropical agricultural regions like Texas (Cabanillas et al., 1994) and Arkansas (Feaster and
Steinkraus, 1996). and it shows promise for the control of a variety of other pest organisms (Table 5). It has been suggested that this nematode species is able to survive extreme climatic conditions by vertical migration down through the soif
(Cabanillas et al., 1994). The effectiveness of this nematode in insect control is dependent, as with other entomopathogenic nematodes, on appropriate application and timing (Hayes et al., 1999), nematode concentration (Cabanillas Table 5: Tested host insect species for Steinemema riobrave
-
Species Name Common Name Reference
Carpophilus hemipterus Dnedfniit Beetle Vega et al., 1994 Cyclocephala hirta Western Masked Converse and Grewal, 1998 Chafer Cydia pomonella Codling Moth Lacey and Unnih, 1998 Delia radicum Cabbage Maggot Schroeder et al., 1996 Diabrotica Southem Corn Barbercheck and Wamck, Undecimpunctata Rootworzn 1997 howardii Diaprepes abbreviatus Root Weevil Schroeder, 1994 Frankliniella occidentalis Western Flower Chyzik et al., 1996 Thrips Helicoverpa zea Corn Earwom Cabanillas and Raulston, 1994a,b, 1995, 1996a,b; Feaster and Steinkraus, 1996 Pectinophora gossypiella Pink Bollworm Henneberry et al., 1995a; Henneberry et al., 1995b; Gouge et al., 1996 Plute lla xylostella Diamondback Moth Ratnasinghe and Hague, 1997 Spodoptera exiqua Beet Atmywom Henneberry et al., 1995a Spodoptera fnrgiperda Fall Armyworm Molina-Ochoa et al., 1999 Tnchoplusia ni Cabbage Looper Hennebeny et al., 1995a Ixodes scapulafis Black Legged Tick Hill, 1998 Meloidogyne ja vanica Root-knot Gouge et al., 1994 Nematode Meloidogyne sp., Root-knot, Sting, Grewal et al., 1997 Belonolaimus and Ring longicaudatus, Nematodes Criconemella sp. and Raulston, 1995) and unifonn distribution in the soi1 (Cabanillas and Raulston.
1996a; 1W6b; Hayes et al., 1999).
As more species and strains of Xenorhabdus are studied, the known spectrum of the antimicrobial activity of their metabolites increases (Li et al.,
1998). The d iversity of bioactive metabol ites produced by these bacteria and the sequestered environment in which these bacteria live suggest that these metabolites rnay be a potential source of new agrochernicals with novel modes of action. The recently described nematode S. riobrave and its Xenorhabdus sp. syrnbiont may provide researchers with a new array of bioactive metabolites.
The objective of this study was to begin characterizing the bacteria associated with S. riobrave and to evaluate the antimicrobial activity of their metabolites with particular attention to their antimywtic activity. GENERAL MATERIALS AND METHODS Source and Maintenance of Xenorhabdus
All species and strains of Steinemema (Steinemematidae: Rhabditida) and their associated bacterial symbionts, Xenorhabdus (Enterobacteriaceae),
used in this study were supplied by B. Leighton from the entornopathogenic
nematode collection in the research laboratory of J.M. Webster at Simon Fraser
University (SFU). Details of their identity and original source are given in Table
6. The three Steinemema species were maintained by passaging through late instar greater wax moth larvae, Galleria mellonella L., obtained from the
Department of Biological Sciences Insectary, SFU by methods described by
Dutky et al. (1964) and modified by Hui (1998). Nematode samples were sent to the International Institute of Parasitology (St. Albans, Herts, UK) and their taxonornic identification of was wnfirmed by molecular techniques.
Xenorhabdus bovienii AUS (ATCC39497) was obtained as a pure culture from the Amencan Type Culture Collection. The other bacterial species, BJ, 627 and RIO, were directly isolated from their respective nematode symbionts that infected G. mellonella lawae, and were identified through the specific association with their particular nematode symbiont. The bacteria were obtained by the following procedure. An aqueous suspension of 100-200 nematodes in about
1ml of sterite, distilied water was spread ont0 9 cm diameter plastic Petri dishes lined with three Whatman No. 1 filter paper sheets. Five late instar G. mellonella larvae (approximately 0.2 g each) were added and the Petri dishes were Table 6: Species and source of Xenorhabdus strains and their respective nematode symbiont used in this study
Strain Symbiotic Bacteria Nematode Source Code
RIO Xenorfiabdus sp. Steinemema riobrave Courtesy of Dr. R. Gordon, (Cabanillas, Poinar Mernorial University, and Raulston) Canada (original culture Texas, Rio Grande)
AUS X. bovienii S. feltiae (Filipjev) Bacteria obtained from (Akhurst and American Type Culture Boemare) Collection, ATCC39497
627 X.bovienii S. feltiae (Filipjev) Courtesy of BlOSYS Inc., (Akhurst and "BIOSYS" #27 Columbia, Maryland (circa Boemare) 1996)
BJ X. nematophilus S. carpocapsae Courtesy of Dr. H. Yang, (Poinar and (Weiser) Chinese Academy of Thomas) Agriculture, Beijing, China incubated at 24OC in darkness. After larval death, in approximately 48 h, each cadaver was dipped in 95% alcohol and flarne sterilized (Woodring and Kaya,
1988). The cadavers were then dissected under sterile conditions using forceps and dissecting needles, and haemolymph was streaked, using a sterile inoculating loop, ont0 Petri plates of tryptic soy agar (TSAD) supplemented with bromothymol blue (1.5% agar and 0.025 g bromothymol blue per litre) and incubated in the dark at 24OC. The Phase I form of Xenorhabdus was identified by its production of blue or green colonies (depending on the species of bacteria), caused by the bacterial uptake of bromothymol blue, as defined by
Chen (pers. comm.). The Phase II colonies of Xenorfiabdus and other bacterial species showed no such uptake (Akhurst and Boernare, 1990). To obtain pure cultures of Xenorhabdus bacteria, several primary form colonies were collected on the end of an inoculating loop and streak plated ont0 fresh TSAD plates.
The purified bacteria were maintained on TSAD and subcultured, using standard aseptic laboratory techniques, every 2 weeks to avoid phase shift to the secondary form. To ensure good quality, fresh stock cultures of new bacteria were isolated from infected Galleria larvae every 2-3 months. As well, bacteria that had been cultured on a gyrotatory shaker in tryptic soy broth (TSB) (Difco) for 48 h, centrifuged and resuspended in 2.75% TSB containing 15% glycerol
(v/v) were maintained in long-terni cold storage at -20°C until required, as described by Chen (1992). Table 7: Test species used in this study and their known sources
Test Species and Strains Source
Alcaligenes faecalis Microbiology Teaching Lab, Department of Biological Sciences, Simon Fraser University WU) Bacil/us cereus Microbiology Teaching Lab. ûept. 8io. Sci., SFU B. subfilis J.E. Rahe, Dept. Bio. Sci., SFU Escherichia coli Microbiology Teaching Lab, Dept. Bio. Sci., SFU Micrococcus luteus Microbiology Teaching Lab, Dept. Bio. Sci., SFU Pseudomonas aenrginosa Microbiology Teaching Lab, Dept. Bio. Sci., SFU Staphylococcus aureus Microbiology Teaching Lab, Dept. Bio. Sci., SFU
A cremonium strictum S. Berch, Glynn Road Research Station, Victoria, B.C. Aitemaria aitemaria Z. Punja, Dept. Bio. Sci., SFU Aspergillus fla vus J.M. Webster, Dept. Bio. Sci., SFU (originally ATCC 24133) A. fumigatus J.M. Webster, Dept. Bio. Sci., SFU (originally ATCC 13073) Beauveria bassiana USDA-ARS Collection of Entomopathogenous Fungi, Ithaca, New York Botrytis cinerea #1 J.M. Webster, Dept. Bio. Sci., SFU B. cinerea #2 Z. Punja. Dept. Bio. Sci., SFU B. cinerea #3 R. Utkhede. Pacific Agn-Food Research Centre (PARC), Agassiz, B.C. Candida parapsilosis M. Moore, Dept. Bio. Sci., SFU C. tropicales M. Moore, Dept. Bio. Sci., SFU Cylindrocarpon destructans J.E. Rahe, Dept. Bio. Sci., SFU Didymella bryoniae R. Utkhede. PARC, Agassiz, B.C. Fusarium oxysponrm #1 J.M. Webster, Dept. Bio. Sci., SFU F. oxysponrm #2 R. Utkhede, PARC, Agassiz, B.C.
Continued on next page Test Species and Strains Source
F. solani #1 J.M. Webster, Dept. Bio. Sci., SFU F. solani #2 R. Utkhede, PARC, Agassiz, B.C. Fusarium sp. JE. Rahe, Dept. Bio. Sci., SFU Metarhizium anisopliae USDA-ARS Collection of Entornopathogenous Fungi, Ithaca, New York Oidiodendron gnseum S. Berch, Glynn Road Research Station, Victoria, B.C. Phellinus sp. J.E. Rahe, Dept. Bio. Sci-, SFU Pythium aphalidermatum R. Utkhede, PARC, Agassiz, B.C. P. uitimum #t 2. Punja, ûept. Bk. Sci.,. SFU P. ultimum #2 R. Utkhede, PARC, Agassiz, B.C. Pythium sp. J.E. Rahe, Dept. Bio. Sci., SFU Rhizoctonia solani J.E. Rahe, Dept. Bio. Sci., SFU Sclerotium cepivotum J.E. Rahe, Dept. Bio. Sci., SFU Thielaviopsis basicola 2. Punja, Dept. Bio. Sci., SFU Trichodema sp. #1 2. Punja, Dept. Bio. Sci.,, SFU Trichodema sp. #2 J.E. Rahe, Dept. Bio. Sci., SFU 2.2 Source and Maintenance of Test Organisms
Several bacterial, yeast and fungal species were used to determine the
spectrum of bioactivity of the metabolites derived from Xenorhabdus sp. RIO.
The identity and sources of these microorganisms are given in Table 7.
2.2.1 Test Bacteria
Xenorhabdus bactena were maintained, as descnbed in section 2.1. Al1
other bacterial species were maintained on TSA, incubated in the dark at 24OC
and subcultured every 1-2 weeks. For long-terrn preservation of these bacteria
the following method was used. Several colonies were cut out from TSA plates
and placed in 250 ml Erlenmeyer flasks containing 100 ml sterile TSB. The
bacteria were then grown on a gyrotatory shaker (100 rpm) at 25OC for 48 h. The
bacterial culture was mixed with an equal amount of 24% sucrose, freeze dried
and then preserved at -20°C as a powder which could be reconstituted as
needed (Chen, 1996).
2.2.2 Test Fungi
The fungal and yeast species were maintained and subcultured biweekly on potato dextrose agar (PDA) (BDH Ltd.) and the insect pathogenic fungi were grown on sabouraud dextrose agar (SDA) (Difco Ltd.). The majority of test fungal species that produce mycelia were maintained by subcultunng mycelial
plugs from actively growing cultures ont0 the center of new PDA or SDA plates.
The test fungi that have little lateral growth, ie. Candida spp. and Aspergjllus spp. were maintained by streaking dry spores ont0 ?DA plates using a sterile wire
loop. For long terrn storage, fungal samples were inoculated ont0 PDA slants
rather than plates and allowed to grow for 2-3 d, covered with mineral oil and
stored at 4OC.
2.3 Bacterial Fermentation
The bacterial fermentation was done acwrding to procedures described
by Ng (1995) and Chen (1996). with some modifications. The antimicrobial
activity of the metabolites of Xenorhabdus spp. was determined using the ceil-
free culture filtrates of each species. Seed cultures of each species were
prepared by cutting out 34agar plugs with Phase I colonies from stock culture
plates and placing them into 250 ml Erlenrneyer flasks containing 100 ml sterile
TSB. The flasks were loosely covered with sterile, aluminum foi1 and incubated
in a controtled environment, gyrotatory shaker (100 rpm) at 25OC in darkness for
24-48 h, depending on the bacterial species. When the culture optical density at
600 nm was approximately 1.7 (mid-log phase), the bacterial cultures were
transferred under aseptic conditions into 2 L Erlenmeyer flasks, containing
900 ml sterile TSB, to make 1000 ml total under sterile conditions. These large culture fiasks were covered loosely with steRle alurninum foi1 and incubated in a gyrotatory shaker (100 rpm) at 25OC in darkness for 72 h. Following fermentation, the cultures were centrifuged (9000 rpm) for 20 m at 4°C in order to obtain the cell free culture broth. 2.4 Measurement of Antimicrobial Activity
2.4.1 Bacterial Bioassays
Bacillus subtilis was sefected as the initial indicator species for antibiotic activity because it is known to be very susceptible to antibiotics produced by
Xenorhabdus spp. (Maxwell et al., 1994; Chen, 1996). Spore suspensions of this bactenum were prepared, as described by Chen (1996). Agar diffusion plate assays (Hewitt and Vincent, 1989) were used throughout this study to determine the antibiotic activity of Xenorhabdus metabolites. TSA was autoclaved and
15 ml of the molten medium was added to 9 cm polystyrene Petri plates and allowed to cool. The plates were stored in sealed plastic bags at room temperature until needed. Agar plates were inoculated with 100 VIof B. subtilis spore suspension, and a stenle spreader was used to apply it evenly over the agar surface. The plates were allowed to dry in a larninar flow hood for 15 min.
An alcohol-flame sterilized cork borer was used to cut 3-5 wells, each 4 mm in diameter, around the perimeter of the inoculated plate and stenle forceps were used to remove the agar plugs. A Gilson@pipettor was used to place 25 pl of test material into each well, and the ptates recovered and incubated at 25°C for
24 h. After incubation, the plates were visually assessed for clear inhibition zones of no bacterial growth around the wells. The radius of these zones was then measured with hand-held calipers.
Bioassays that used other test bacteria (Table 7) followed the same basic procedures as above with slight modification since spore suspensions were not available for these bacteria. For each species, several colonies from stock plates were added to 100 ml sterile TSB in 250 ml Erlenmeyer flasks. The flasks were
loosely covered with sterile aluminurn foi1 and incubated in a controlled
environment gyrotatory shaker (100rpm) at 25OC in darkness for 5 d. The liquid
stock cultures were swirled gently to mix the bacteria evenly and 100 VIwas
pipetted ont0 fresh, sterile 9 cm Petri plates wntaining 15 ml of TSA and spread
with a sterile spreader. The test then proceeded as described above for B.
subtitis.
2.4.2 Fungal Bioassays
Botwis cinerea was used as the initial fungal test species because it is easily cultured and is highly susceptible to antimywtic cornpounds produced by
Xenorhabdus spp. (Chen, 1996). Antifungal activity was detemined using a modified version of the hyphal extension-inhibition assay (Roberts and
Selitrennikoff, 1988). Autoclaved PDA was poured into 9 cm polystyrene Petri plates in 15 ml amounts, allowed to cool and stored in sealed plastic bags at room temperature until needed. Mycelial plugs were cut out of actively growing B. cinerea culture plates with a sterile cork borer and placed in the center of a fresh
PDA plate. Then a 4 mm diameter sterile cork borer was used to cut 4-5 wells around the perimeter of the inoculated plate, and into each well was added 25 pl of test solution. The plates were recovered and incubated for 3 d at 25°C in darkness. The resulting hyphae grew outward from the mycelial plug and the circle of hyphal growth became inhibited as it grew into an area with an effective concentration of inhibitor. These arcs of inhibited growth were clearly identified usually within 3 d, and were measured with hand-held calipers.
2.5 Statistical Analysis
All tests done throughout this research were repeated three tirnes unless otherwise stated. The data are expressed as means t standard errors.
Cornparison of means was done using Tukey's Studentized Range Test with
JMP IN software (SAS lnstitute Inc., Cary, North Carolina). Levels of significance of P=0.05 was used in al1 analyses. 3.0 CHARACTERlSTlCS OF XENORHABDUS SP. RIO
3.1 Introduction
The taxonomic classification of bacteria, and of Xenorhabdus in particular, has conventionaliy been based on various morphological, biochemical and physiological characteristics (Thomas and Poinar, 1979; Akhunt and Boemare,
1988; Yamanaka et a/., 1992; Nishimura et al., 1994). Typical diagnostic tests range from general morphology and Gram staining to enzyme assays and tests of their carbohydrate metabolism. Cabanillas et al. (1994) isolated the symbiotic bacteriurn from S. nobrave and confirmed it as a Xenorhabdus sp. based on its absorption of bromothymol blue from NBTA and neutral red dye from MacConkey
Agar. Recent molecular studies based on PCR-ribotyping have confirmed this bacterium as Xenorfiabdus sp. (Fischer-Le Saux et al., 1998; Bonifassi et al.,
1999). Since then there has been no additional information concerning the taxonomic characteristics of this bacterium. For this reason, a series of observations were made and tests were done in order to charactenze the
Xenorhabdus sp. associated with S. riobrave.
3.2 Materials and Methods
All the bacterial diagnostic tests were perforrned under standard laboratory, sterile protocols. The primary form of each bacterial strain (see Table
6) was cultured and prepared according to section 2.4. Following measurements and observations the collected information was compared against known characte ristics for Xenorhabdus species published in Bergey's Manual (1994) and in research papers (Thomas and Poinar, 1979; Akhurst and Boemare, 1988;
Yarnanaka et al.. 1992; Nishimura et al., 1994) in order to identify this bacterial
isolate.
3.2.1 Morphological Characteristics
Cell shape of 72 h TSB bacterial cultures was observed under a Zeiss@
compound microscope (1000x magnification with oil immersion). Colony
pigmentation and bioluminescence was observed in 72 h cultures on tryptic soy
agar (TSA) (TSB + 1.5% agar). Dye uptake was rewrded as uptake of
bromothymol blue from TSAD plates creating blue or green colonies, depending on the natural colony pigmentation.
Cell motility was determined using semi-solid TSA medium (TSB + 0.5%
agar) according to MacFaddin (1976). Briefly, 5 ml aliquots of molten, semi-solid
TSA media were added to 10 ml disposable, borosilicate test tubes, autoclaved
(15 m at 121OC) then cooled in an upright position. A sterile inoculating needle was dipped into a well-mixed 72 h bacterial broth culture and then stabbed into the center of the semi-solid TSA medium to a depth of 2 cm. The inoculated test tubes were incubated at 25°C for up to 5 d. Positive tests were indicated by colonies migrating away from the stab lines and diffusing into the medium causing increased turbidity of the medium. Negative tests were indicated by the
presence of bacterial growth only along the stab line. 3.2.2 Carbohydrate Metabolism
The APIChSO carbohydrate test strip (Bio-Meneux Ltd.) allows for a
restricted but rapid carbohydrate metabolism analysis to assist in bacterial
diagnosis. Each strip contained 50 microtubules each with one of 49 different
carbohydrates, plus a control tubule. A special medium, AP150CHE (Bio-Merieux
Ltd.), designed for use with Enterobacteriaceae, and the Apt test strips were
used as per manufacture& instructions, with slight modification. Xenorhabdus
was grown in liquid TSB culture (see Section 2.4), and after 72 h of growth 1 ml
of bacterial suspension was added to 10 ml APISOCHE medium and thoroughly
rnixed. The medium/bacterial suspension was pipetted into each microtubule of
the stnp and covered with mineral oil. Finally, test stnps were incubated at 25°C
for 3 d in darkness and the results were recorded. During the incubation
carbohydrates are fermented to acids, which decreased the pH, and this was
detected by the colour change from red to yellow. Yellow was considered a
strong positive for bacterial metabolism of a particular carbohydrate, orange a
weak positive and red a negative result. For esculin detection the colour change
from red to black was considered positive.
3.2.3 Enzyme Analysis
In order to detemine the species specific enzymatic activities of
Xenorhabdus, the isolates were tested using APlZym test strips (Bio-Mérieux
Ltd.) as a semi quantitative micromethod to allow rapid and systematic study of
19 enzymatic reactions. Each strip was composed of 20 microtubules each of which contained the enzymatic substrate and appropriate buffer. Strips were
inoculated and read as per manufacturer's instructions. Briefly, two drops of a
72 h TSB culture of test bacteria were added to each tubule- After inoculation,
the strips were incubated at 25°C in darkness for 24 h, and then the colour
changes in the tubules were recorded.
Catalase activity was observed, as outtined by Srnibert and Kreig (1981),
where 4 day-old TSAD plates containing bacteria were flooded with 3% hydrogen
peroxide (VWR), and the liberation of oxygen signified a positive test for
catalase. Oxidase was measured by dipping cytochrome oxidase test sticks
(Unipath Ltd.. Hampshire, England) ont0 fresh, 4 day-old bacterial colonies and
observing a colour change to black. Urease was tested using Christensen agar
(MacFaddin, 1976).
3.3 Results
Tables 8. 9 and 10 show morphological, physiological and biochemical
characteristics of Xenorhabdus sp. RIO as compared with the three other
Xenorhabdus spp. used in this study. All primary forms of the genus
Xenorhabdus were Gram negative, rod-shaped bactena (Table 8). They
absorbed bromothymol blue from TSAD media and forrned blue or blue-green
colonies depending on the natural colony pigmentation, which varied according to species (Table 8). None of the bacteria showed bioluminescence (Table 8). Table 8. Morphological characteristics of primary form Xenofiabdus sp. RIO in cornparison with those of three other Xenorfiabdus species
Test bactenal strains
Characteristics BJ AUS 827 RIO
Gram reaction - - - - Cell shape rod rod rod rod Motility + + + + Pigmentation white yellow yellow white Dye uptake on TSAD + + + + Bioluminescence - - - -
+: positive; -: negative Table 9. Acid production by pnmary forrn Xenohabdus sp. RIO in cornparison with that of three other Xenorhabdus species when tested on carbohydrate sources
Bacterial strains
Carbohydrate BJ AUS 827 RIO
------
a-Methyl-D-Glucoside a-Methyl-D-Mannoside P-Methyl-O-Xyoside N-Acetyl-Glucosamine 2-Keto-Gluconate 5-Keto-Gluwnate Adonitol Amgdalin 0-Arabinose L-Arabinose 0-Ara bitol L-Arabitol Arbutin Cellobiose Dulcitol Erythritol Esculin Fructose D-Fucose L-Fucose Galactose Gentiobiose Gluconate Glucose Glycerol Glycogen lnositol lnulin Lactose D-Lyxose Maltose Mannitol Mannose
Continued on next page Bacterial strains
Carbohydrate AUS RIO
Melibiose Melezitose RafTinose Rhamnose Ribose Salicin Sorbitol Sorbose Starch Sucrose 0-Tagatose Trehalose 0-Turanose Xylitol 0-X ylose L-Xylose
+: positive; -: negative; + (w): weak positive Table 10-Enzymatic activities of prirnary form Xenorhabdus sp. RIO in cornparison with those of three other Xenorhabdus species
Bacterial strains
Enzyme BJ AUS 827 RIO
Phosphatase alkaline Esterase (C4) Esterase Lipase (CS) Lipase (C14) Leucine arylamidase Valine arylamidase Cysteine arylamidase Trypsin C hymotrypsin Phosphatase acid Naphthol-AS-01- phosphohydrolase a-galactosidase P-galactosidase P-glucuronidase a-glucosidase P-glucosidase N-acetyl-P-glucosaminidase a-mannosidase a-fucosidase Catalase Oxidase Urease
+: positive; -: negative; + (w): weak positive Table 9 shows acid production by the Xenorhabdus test strains on 49 different carbohydrates. All bacteria tested positive for fructose, glucose, glycerol, maltose, mannose, trehalose, 5-keto-gluconate and N-acetyl- glucosamine. The two X. bovienii strains were positive for ribose while both BJ and RIO were positive for inositol. RIO, AUS and 827 showed positive resutts for esculin. The AUS strain tested weakly positive for gluconate. 827 and RIO tested weakly positive for 2-keto-gluconate. All bacteria did not produce acid from a-methyl-D-glucoside, a-methyl-D-rnannoside, P-methyl-D-xyoside. adonitol, amgdalin, D-arabinose, L-arabinose, D-arabitol, L-arabitol, arbutin, cello biose, D-lyxose, dulcitoi, erythritol, D-fucose, L-fucose, galactose, gentiobiose, glycogen, inuiin, lactose, Dlyxose, mannitol, melibiose, melezitose, raffinose, rhamnose, salicin, sorbitol, sorbose, starch, sucrose, 0-tagatose, D- turanose, xylitol, L-xyfose and û-xylose.
All bacteria produced alkaline and acid phosphatase. esterase lipase (C8). leucine arylamidase, naphthol-AS-DI-phosphohydrolase, a-giucosidase and N- acetyl-P-glucosaminidase (Table 1O). Both X. bovienii strains tested weakly positive for esterase (C4). Negative resuhs were seen for lipase (C14), valine arylamidase, cysteine arylamidase, trypsin, chymotrypsin, a-galactosidase, P- glucuronidase, P-glucosidase, u-mannosidase, a-fucosidase, catalase, oxidase and urease (Table 10). 4.0 IN VIVO ANTIMICROBIAL ACTIVIW
4.1 Introduction
The production of secondary metabolites with antimicrobial properties is common to primary form Xenorhabdus sp. (Akhurst, 1982; Akhurst and Dunphy,
1993),as it is for many bactenal species. It is widely believed that the antibiotics produced by these bacteria help to maintain a relatively cornpetitor-free environment for Xenorhabdus within the host insect cadaver. They prevent and/or minimize secondary invasion by saprophytic organisms, which would otherwise cause the cadaver to putrefy and compromise the development of the nematode-bacterial complex (Akhurst, 1982; Akhurst and Boemare, 1990; Forst and Nealson, 1996; Boemare et al., 1997). These antibiotic substances inhibit, for example, the growth of bacteria released by the degradation of the insect gut, which occurs as the infection cycle progresses. Studies into the gut microflora of
G. mellonella showed that the dominant microorganism is the Gram positive coccus, Enterococcus faecalr's. Several other organisms including Gram positive and Gram negative bacteria and some yeast species have been identified but are much less frequently encountered (Boucher and Williams, 1967; Jarosz, 1979).
There is some disagreement as to whether or not the antimicrobial compounds maintain a monoxenic culture of Xenohabdus throughout the infection process and whether they alone are responsible for preventing putrefaction of the insect cadaver by other microorganisms (Maxwell et al.. 1994;
Jarosz, 1996a; Jarosz, 1996b). For these reasons a study was done to determine whether Xenorhabdus sp- RIO produces antimicrobial compounds in vivo and, if sol whether these compounds prevent development of secondary
microorganisms inside the infected G. mellonella larvae.
4.2 Materials and Methods
4.2.1 In Vivo Development of Microflora
Late instar G. mellonella larvae were infected with S. riobrave in 9 cm
Petri dishes (see Section 2.1). Sampling and monitoring of the resulting bacterial
population was adapted from Hu and Webster (1998). Five larvae were added
to each of 15 Petri dishes for a total of 75 larvae and incubated in the da& at
25°C. Five larvae were randomly collected frorn plates at 0, 3, 6, 12 and 24 h
after exposure to nematodes and every day thereafter up to 10 d post infection.
The larvae from each sampling tirne were flame sterilized in 95% alcohot and
then macerated together in 2 ml autoclaved TSB in a small, sterilized mortar.
Macerated matenal was transferred to 15 ml Falcon@tubes and adjusted to 10
ml with sterile TSB. Standard dilution plating methods were followed. the
materiat was spread ont0 TSAD plates, and plates were incubated in the dark at
25°C. After 48-72 h incubation, the number and type of colonies of
microorganisms were recorded. Controls with five uninfected larvae were
replicated and sampled once for each incubation time. The resulting bacterial colonies were identified and grouped according to Gram stain and whether or not they were Xenorhabdus type based on their uptake of bromothymol blue on
TSAD. 4.2.2 ln Vivo Oeveloprnent of Antimicrobial Activity
Undiluted macerated material (see section 4.2.1 ) for each sampling time was sterilized by passing through 0.2 pm syringe microfilters. To determine the bioactivity of the sterilized material, bioassays were done using B. subtilis as a test bacterium and B. cinerea as a test fungus, according to section 2.4.
4.3 Results
lnsect mortality was observed after 48 h of infection, and by 72 h, al1 of the insect larvae except for the controls had died. The nematode's symbiont,
Xenorhabdus sp., was first detected at 48 h, which was when the insects began to die. The number of bacteria increased rapidly from 1.O x 1o6 CFUIg to 1.1 x logCFUlg at 72 h pst-infection and remained at high levels throughout the course of the experiment (Figure 1). At this eariy stage of infection, the only other bactenum isolated from the larvae was a Gram positive coccus whose nurnbers declined from 6.9 x 106 CFUfg at the start of the experiment to an undetectable level atter 72 h. Based on previous studies, the predominant coccus was assumed to be E. faecalis. Several Gram negative bacteria began to appear at this time. and they slowly increased in numben hm1.7 x 10' CFUlg at 72 h to 1.9 x 10' CFUlg at the end of the experiment (240 h). By the ninth day
(216 h), the numbers of Xenorhabdus sp. and of the other Gram negative bacteria had plateaued. Neither Xenorhabdus sp. nor the other Gram negative bacteria were isolated from the mntrol insects. The only bacterial species Figure 1. Development of bacterial microfiora and antimicrobial activrty in Gallena mellonella infected by Steinemema nobrave as measured by the nurnber of cellsflarvae and the size of the inhibition zone of the test organism, respectively. +Entemcoccus faecalis -x- Xenorhabdus sp. +Other bacteria 3 '0 scn - - Q - - Antibacterial activity 2 0, - - Q - - Antimycotic activity a~ 1
O 24 48 72 96 120 144 168 192 216 240 Time (h) isolated from the controls was the Gram positive coccus. The experiment was repeated and the resuits were confimed.
The macerated insect tissue was tested for antimicrobial activity (Figure
1). Both antibactenal and antimycotic activities followed a similar trend and were first observed at 72 h post-infection at the time when al1 insects had died and
48 h after Xenorhabdus sp. first appeared in the larvae. Antimicrobial activity increased rapidly after 72 h, began to level off at 144 h and remained relatively constant thereafter. The Gram positive cocci disappeared from the system soon after the antimicrobial activity was detected and at the same time as the population of the other Gram negative bacteria were increasing rapidly. Neither
Xenorhabdus sp. nor the other Gram negative bacteria appeared to be affected by the presence of the antimicrobial activity. No antimicrobial activity was detected from the control insects. These results were confirmed by a repeat experiment. 5.0 CHARACTERKATION OF ANTIMICROBIAL ACTlVllY
5.1 Introduction
The nature of the antimicrobial activity of Xenorhabdus spp. from several
Steinemema species is quite diverse (Table 5). However, little is known about
the antimicrobial compounds produced by the Xenorhabdus sp. from S. riobrave
and, in particular, about the nature or role of the proteinaceous compounds
produced by the bacteria during the infection process.
Xenorhabdus species, including Xenorhabdus sp. RIO (see Section 3.0),
are known to produce a number of extracellular enzymes including proteases,
lipases, phospholipases and DNAses that are involved in providing nutrients for
both the bacteria and, most importantly, for the nematodes (Forst and Nealson,
1996)- It has been speculated that some of these may be associated with the
bactena's insecticidal action and/or rnay contribute to inactivation of the insect's immune system (Akhurst and Dunphy, 1993; Forst and Nealson. 1996). In fact, insecticidal protein toxins have been identified from Xenorhabdus as well as a related bacterium, Photohabdus luminescens (Bowen et al., 1998). Other proteins from Xenorhabdus spp. that have been identified are bactenocins with antibiotic activity (Thaler et al., 1997) and chitinases that display antifungal activity (Chen et al., 1996).
Chitin is a polysaccharide composed of a linear P-1,4 linked polymer of N- acetylglucosamine. It is a major structural component of many organisms, including fungi, insects, crustaceans and of nematode eggs (Punja and Zhang,
1993). The enzymes that cleave chitin polymers, the chitinases, include endochitinases that cleave the chitin chains randornly, releasing oligosaccharides, and exochitinases that hydrolyze individual sugar monomers from the terminal non-reducing ends of the chitin molecule. Another related enzyme, glucanase, serves to hydrolyze 9-glucan, another important structural molecule of fungal cell walls. Chitinases and glucanases are produced by a number of organisms including fungi, bacteria and higher plants. 80th enzymes are secreted by plants in response to fungal invasion (Sock et al., 1990; Punja and Zhang, 1993) and by a number of biological control agents (St. Leger et al.,
1991; Cotes et al., 1996). As such, these enzymes are thought to play a role in the control of fungal pathogens. Chen et al. (1996) detemined that nematophilus and X. bovienii produce both exo- and endochitinases and suggested that these enzymes may play an important role in protecting the dead or dying insect from fungal invasion. P-glucanase activity was not detected in these bacteria (Chen et al., 1996).
A series of experiments was initiated to begin to characterize the antimicrobial activity of the Xenorhabdus sp. RIO symbiont with particular focus on the bioactive proteins including possible chitinase and glucanase activity.
5.2 Materials and Methods
5.2.1 In Vitro Development of Xenorhabdus sp. RIO
A pure culture of Xenorhabdus sp. RIO was obtained from G. mellonella larvae that had been infected by the procedures described in section 2.1. In order to observe the in vitro growth characteristics of this bacterium a bacterial fermentation was cam'ed out (see section 2.3). Three, 1 L TSB cultures, each in
2 L Erlenmeyer flasks, were established and a 25 ml sample was rernoved from each culture fiask at 0, 3, 6, 9, 12, 18.24, 30, 36,48, 60, 72, 84, 96, 108, 120,
144 and 168 h. Each sample was analyzed for cell growth using a spectrophotometer (Phannacia LKB Novaspec II) set at 600 nm. The sample was then centrifuged (9,000 rpm, 20 m at 4°C) and the pH measured using a
Corning digital pH meter. Following this, in order to determine the initial the antimicrobial activity of the whole broth culture, the centnfuged samples were microfiltered, using a 0.2 Pm filtration unit, and tested using agar diffusion plate assays with B. subtilis and B. cinerea, as described in section 2.4. The entire experiment was repeated three times.
5.2.2 Spectrum of Antimicrobial Activity
To deterrnine the spectrurn of the antimicrobial activity of the secondary metabolites of Xenorhabdus sp. RIO these bacteria were cultured (see section
2.3), after which the cell-free culture broth was lyophylized (see section 5.2.2), and then reconstituted with potassium phosphate buffer to concentrations of 0.1,
1 .O, 10.0 and 100.0 pg/ml. Subsequently, the test solutions were bioassayed using agar diffusion plate assays (see section 2.4) against several species of fungi and bacteria (Table 7). Each reconstituted test solution was tested three times and the entire experiment was repeated twice. 5.2.3 Fractionation of Antimicrobial Activity
In order to detemine the chernical nature of the secondary metabolites that showed antimicrobial activity the cell-free broth was fractionated into several components using the following procedure. Cell free broth was obtained (see section 2.3) from 72 h TSB cultures of Xenorhabdus sp. RIO which had been centrifuged (9000 rpm, 20 m at 4°C) and filter sterilized through 0.2 Pm microfilters resulting in a whole broth fraction. Ethyl acetate was used to separate the organically soluble metabolites, which included the majority of non- proteinaceous bioactive metabolites of Xenorhabdus bacteria (Paul et al., 198 1;
Mclnemey et al., 1991a), from substances such as the water soluble xenocoumacins (Mclnerney et al., 1991b) and proteins. One hundreû milliliters of the whole broth fraction were combined with 100 ml ethyl acetate in a 250 ml separatory funnel and, subsequently, the organic fraction was separated from the aqueous fraction. This process was repeated twice more by wmbining two additional sequential amounts of 100 ml ethyl acetate to the aqueous fraction.
The organic fractions were combined and the remaining water was removed by shaking vigorously with anhydrous sodium sulphate. The ethyl acetate fraction was evaporated to dryness in a rotatory evaporator (Buchi Rotavapor-R), and the resulting powder was reconstituted in 5 ml methanol to create a 20-fold concentration that was bioassayed (see section 2.4).
In order to determine the possible des of the proteinaceous wmponent of the overall antimicrobial activity, 100 ml of the whole broth fraction was subjected to ammonium sulphate precipitation (Bollag et al., 1996). Ammonium sulphate was added to the sample to make up to 85% concentration (56.8 g per
100 ml broth). After al1 the salt had been added, the sample was agitated gently on a rnagnetic stir plate on ice for about 2 h. The material was then centrifuged
(9000 rpm, 20 m at 4°C) to separate the precipitated pmtein from the protein free broth. The supernatant was decanted and the pellet resuspended in an arnount of distilled water equal to double the pellet volume and recentrifuged. This procedure was repeated twice more in order to remove any insoluble, denatured, proteinaceous matenal (Bollag et a/.,1996). Ammonium sulphate salt was removed by dialysis using Spectra/PorB 1 dialysis tubing (6000-8,000 MW cut off). The remaining proteinaceous solution was frozen in liquid nitrogen. lyophylized to powder, reconstituted in 1OOml potassium phosphate buffer (pH
6.7) and bioassayed.
5.2.4 Analysis of the Exo-Enzymatic Activity
The enzyme activity in the culture broth of several Xenorhabdus spp. was obsewed. Bacterial cultures (72 h) were established, as outlined in section 2.3, and the powdered proteinaceous fraction from each bacterial isolate was prepared (see section 5.2.2). The culture broth was monitored for absorbance
(600 nm), pH, antibiotic activity and antimycotic activity (see section 5.2.1) as well as for totai protein, exochitinase, endochitinase, exoglucanase and endoglucanase activity (as outlined below).
The total protein concentration in the whole broth cultures and proteinaceous fractions was detemined using the procedure of Bradford (1976) with bovine serum albumin (BSA) as a standard. Briefly, 5 ml of Bradford
reagent was added to 15 ml disposable borosilicate test tubes, 100 pl of test solution was added and the test tube was gently vortexed for 10 s to mix thoroughly but gently so as to not cause foaming. The tube was incubated for
4 m at room temperature and then its absorbance (at 595 nrn) was measured. A reagent blank using distilled water and Bradford reagent was used as a wntroi.
The absorbance of the samples was compared with the standard curve generated with the BSA standards and the total protein concentration was determined. The Bradford reagent was prepared fresh every week and was stored in an amber bottle, in the dark at room temperature.
Chitinase activity of the culture broth was determined using a standard procedure involving the release of pnitrophenol from labeled substrates (Roberts and Sleitrennikoff, 1988; Tronsmo and Harrnan, 1993; Chen, 1996). Chitinase activity was determined by observing the release of p-nitrophenol from p- nitrophenyl-N-acetyf-P-D-glucosaminide (Sigma) for exochitinase and p- nitrophenyl-P-D-N-N'-Nu-tnacetyochitobiose (Sigma) for endochitinase activity.
The 1mM substrate solution was prepared by dissolving p-nitrophenyl-N-acetyl-P- glucosaminide (0.34 mglml) or p-nitrophenyl-P-0-N-N'-Nu-triacetyochitobiose
(0.75 mg/ml) into 50 mM potassium phosphate buffer (pH 6.7). Ten microlitres of test sample was added to a 1.5 ml Eppendorf tube with 60 pl of the 1.O mM substrate solution. The mixture was gently vortexed and incubated at 41 OC in a water bath for 30 m. After incubation the reaction was terminated with the addition of 1.43 ml of 0.4 M Na2C03 (Anachemia) to each tube. The concentration of p-nitrophenol released was measured using a spectrophotometer (410 nm). Enzyme activity was expressed in units where one unit represents the amount of enzyme that released 1 pmol of p-nitrophenol per minute under the given conditions.
1,3-P-glucanase activity was detemined by using laminarin (Sigma) as a substrate and obsewing the release of reducing sugars using existing procedures (Sock et al., 1990; Cotes et al., 1996) with some modifications. To determine 1.3-P-glucanase activity laminarin substrate was dissolved in 50 mM acetate buffer (pH 5.0) to a concentration of 4 mglml. The assay was perforrned by adding 0.5 ml substrate solution to 0.5 ml test solution in 10 ml disposable borosilicate test tubes and incubating for 1 h in a 40°C water bath. After incubation, the reaction was tenninated by boiling for 5 min. Enzyme activity was measured in units where one unit of activity represented the arnount of enzyme that released 1 pg glucose equivalents per hour. The total P-glucanase activity
(endo- and exo-) was measured using the Somogyi-Nelson method for detemining reducing sugars (Chaplin, 1994). Exo-1,3-P-glucanase activity releases onty glucose units so it was determineci by measuring the free glucose released using a glucose test kit #510-A (Sigma) as per the manufacturer's instructions. Endo-1.3-P-glucanase activity was estimated by subtracting the total 1.3-P-glucanase activity from the ex0-1,3-f3glucanase activity (Sock et al.,
1990; Cotes et al., 1996). 5.2.5 Partial Purification of Chitinase and Bio-Active Proteins
Gel filtration chromatography was used to determine the bioactive
components of the proteinaceous fraction. The dried proteinaceous fraction was
prepared as before (section 5.2.2)and dissolved in distilled water to a
concentration of 10 mglml. Ten miIliliters of solution was subjected to
chrornatographic separation using Sephadex G10Q.120 (Sigma). The column
(14 cm x 3.5 cm) was etuted with water at approximately 6 ml per minute and the
fractions were collected every minute in 15 ml disposable test tubes until al1
material had passed through the column (approxirnately 30 test tubes). Each
fraction was tested for a number of parameters. Total protein was measured
both by the Bradford assay and by measuring absorption at 280 nm using a
Milton Roy Spectrophotometer 3000. Exochitinase activity was monitored to
detennine the presence of chitinase. Antimicrobial activity was measured using
agar diffusion plate assays with B. subtilis and B. cinerea. In addition. tubes
were autoclaved to determine the heat tolerance of the active fractions. Heat
treated samples were subjected to antibiotic and antimycotic tests. A control,
using concentrated TSB, was done to detennine the activity of concentrated
broth. The concentration of broth was calculated as the amount of broth required
to make the equivalent amount of protein powder eluted through the column.
This fraction was treated the same as the proteinaceous fraction, dialyzed, freeze
dried and then passed through the column. This whole experiment was repeated three times. 5.2.6 Characteristics of Partially Purified Bio-Active Proteins
Following the analysis of the gel filtration column the fractions
corresponding to bioactive peaks were combined and freeze dried to powder.
The minimum inhibitory concentration (MIC) was detennined for each peak using the standard procedure for testing antibiotics (National Committee for Clinical
Laboratory Standards, 1990). Bacillus subtilis was used as the test bactenum and B. cinerea as the test fungus. Briefly, a 2 mglml stock solution of test
matenal and distilled water was filter sterilized with a 0.2 pm synnge microfilter.
The stock solution was serially diluted two fold and mixed gently but thoroughly with cooled, molten agar (45°C) to 100, 50,25, 12.5, 6.3, 3.2 and 1.6 pg of active ingredient. Ten rnilliliters of agar were then poured into Petri plates under sterile conditions, cooled and inoculated, as described in section 2-4. Control plates with no test solution were inoculated. All test plates were incubated in the dark at
25°C. The experiment was terminated at 24 h for the bacterial plates, and for the fungal plates, when the mycelium on one of the control plates reached the edge of the Petri plate. The MIC was taken as the concentration of active ingredient that showed no visible growth of the test organisrn. Each test concentration and the controls were replicated three times and this experiment was repeated twice.
Following the MIC experiment, mycelial plugs from each test plate were transferred to untreated PDA plates to assess whether the antimycotic substances were fungicidal or fungistatic. The newly inoculated plates were incubated in the dark at 25°C for 3 d. If the transferred mycelial plug in the untreated plate showed no fungal growth, the antimycotic substances were considered to be fungicidal at that concentration. If the transferred plug showed fungal growth, then the antimycotic substances were considered fungistatic at that concentration.
5.3 Results
Xenorhabdus sp. RIO showed strong growth in the first 24 h of culture
(Figure 2). Exponential growth was observed up to 9 h together with a corresponding drop in culture pH from 7.07 to 6.89. Figure 1 shows that exponential growth wntinued for a few hours after the pH started to rise, eventually slowing with the stationary phase being reached at 48 h. Culture pH continued to increase throughout the course of the experiment reaching 8.79 at
168 h. No pigmentation was obsewed in the culture at any stage of the fermentation.
This bacterium showed significant antimicrobial activity as it grew to stationary phase (Figure 3). The antibacterial activity was detectable from the beginning of the expriment and reached a maximum fevel at 48 h. The antirnycotic activity was first observed at 6 h after which it increased rapidly to 24 h and developed more slowly thereafter to reach a maximum at 72 h. After reaching maximum inhibition the antibacterial and antimycotic activity remained relatively constant throughout the remainder of the experiment. Neither antibacterial nor antimycotic activity was detected in the TSB controls. Figure 2. Cuiture pH and growth curve (absorbante at 600nm) for Xenorhabdus sp. RIO grown in TSB at 25°C X 0.0 O 24 48 72 96 120 144 Time (h) Figure 3. Antibacterial and antimycotic activity of the cell-free culture broth of
Xenohabdus sp. RI0 as measured by the radius of the inhibition zone. 72 96 120 Time (h) The effects of antibiotic substances produced by the Xenorhabdus sp. symbiont of S. riobrave varied based on the Gram stain of the bacteria tested
(Table 11 ). The Gram positive bacteria including Bacillus cereus, B. subtilis,
Micrococcus luteus and Staphylococcus aureus were strongly inhibited while the
Gram negative bacteria, with the exception of Escherichia coli, were weakly inhi bited . Pseudomonas aeruginosa was unaffected by the antibiotics as was
Xenorhabdus sp. RIO itsetf. The other species of Xenorhabdus bacteria tested were relatively unaffected. However, the highest concentration (100 mg/ml) did not prevent growth of the Xenorhabdus spp. but showed zones of poor bacterial growth.
The antimycotic substances exhibited a broad spectnim of activity on a range of fungi (Table 12). Several species of plant pathogenic fungi including
Attemaria aitemana, Botrytis cinerea, Cylindrocarpum destructans, Oidymella bryoniae, Fusarium solani, Pythium uitimum and Sclerotium cepivorum were strongly inhibited by these substances at a concentration of 100 mglml. Two strains of Botrytis cinerea were the most strongly affected being wmpletely inhibited at a concentration of 1O rng/ml. Some fungi including Aspergillus flavus,
A. fumigatus, Fusarium oxyspomm, Pythium aphalidennatum, Rhizoctonia solani and Thielaviopsis basicola a ppeared to be more resistant to these substances.
The insect pathogenic fungi Beauvena bassiana and Metarhizium anisopilae did not appear to be affected. Table 11: Spectrum of antibiotic activity of different concentrations of freeze dried, whole broth from in vitro cultures of the Xenorhabdus sp. RIO measured as the radius of inhibition zones (mm) on agar diffusion plate assays against eleven bacterial isolates.
Whole broth (rnglrnl)
Test bacteria 100 10 i 0.1
A lcaligenes faecalis Bacillus cereus Bacillus subtilis Eschenchia coli Micrococcus luteus Pseudomonas aetuginosa Staphylococcus aureus Xenorhabdus bovienii (AUS) Xenorhabdus bovienii (B27) Xenorhabdus nematophilus (BJ) Xenorhabdus sp. (RIO)
- Not detectable * Hazy zone of reduced growth (-2mm) ** Hazy zone of reduced growth (-4mm) Table 12: Spectnirn of antimycotic activity of different concentrations of freeze dried whole broth from in vitro cultures of Xenorhabdus sp. RIO as measured by radius of inhibition zones on agar diffusion plate assays
- -- -
Whole broth (mglrnl)
Test fungi 1O0 10 1 O- 1
Acremonium stnctum" Altemaria attemaria2 Aspergillus fla vus3 A. fumigatus3 Beauveria bassiana4 Botrytis cinerea5 B. cinerea3 B. cinerea6 Candida parapsilosis7 C. tropicales7 Cylindrocarpum destructans5 Diddyrnella bryoniae6 Fusarium oxyspond F. oxysporvm6 Fusarium solad F. solanr" Fusarium sp? Me tarhizium anisopliae4 Oidiodendron griseum' Phellinus sp? Pythium aphalidermatum6 P. ultimum5 P. ultimum6 Pythium spS5 Rhizoctonia solant
Continued on next page Whole broth (mglml)
Test fungi 100 10 1 O. 1
Sclerotium cepivord Thielaviopsis basicola2 Tnchodema sp.' Trichodema sp?
Four levels of antimycotic activity as measured by inhibition of fungal growth defined as: -, no inhibition; +++, clear inhibition zone that persisted for at least one week afier formation; ++, clear inhibition that was subsequently colonized by aerial hyphae or small clusters of hyphae after a few days; + a zone of poor fungal growth surrounding the test substance
Sources of fun al species: ' Shannon Berch (Glyn Road Research Station, Victoria, BC); 9 Dr. 2. Punja (Simon Fraser University); Dr. J.M. Webster (Simon Fraser University); USDA-ARS Collection of Entomo athogenous Fungi in Ithaca, New York; Dr. J. Rahe (Simon Fraser University); l' Dr. R. Utkhede (Pacific Agri-Food Reseanh Centre, Agassiz, 6.C); ' Dr. M. Moore (Simon Fraser University) All fractions of the cell-free culture broth of RIO including the whole broth (WB), ethyl acetate fraction (EAF), broth without protein (BWP) and proteinaceous (PR) fractions, showed antibacterial and antimycotic activity with the exception of undetected antimycotic activity from the EAF (Table 13). Of al1 the fractions, EAF showed significantly lower activity, particularly in view of the fact that it was tested at 20x concentration. After the proteins were rernoved from the WB, the
BWP showed a significant (P=0.05)decrease in both antibacterial and antimycotic activity as compared with that of the WB. The proteinaceous fraction showed strong antimicrobial activity with no significant difference (P=0.05) between the antibacterial and antimycotic activities. Antimicrobial activity was not detected from the TSB controls.
Xenorhabdus sp. RIO produced proteins including exochitinase and endochitinase in vitro (Figure 4). Chitinase activity was observed at 12 h when protein was first detected in the culture broth. Exochitinase activity increased rapidly after 12 h, reaching a maximum of 1156.8 enzyme units at 72 h, and remained at about this level throughout the course of the experiment.
Endochitinase activity developed more slowly being first detected at 1û-24 h, increased steadily to a peak of 896.1 units at 84 h and remained relatively constant thereafter. Throughout the course of the experiment, the bacteria exhibited more exochitinase activity than endochitinase activity. When antimywtic activity data (from Figure 2) was superimposed on this graph, it showed that the exochitinase activity closely followed that of the antimycotic Table 13. Antibacterial and antimycotic activity of different supernatant fractions expressed as the radius (mm) of inhibition zones in Petri plate bioassays for Xenorhabdus sp. RIO
Antimicrobial activity (mm)**
Fraction* Antibacterial Antimycotic
Whole broth (WB) 7.2 + 0.3 Aa Ethyl acetate fraction (EAF) 3.2 + 0.3 Ad Broth without protein (BWP) 5.7 I0.5 Bc Protein fraction of broth (PR) 6.5 + 0.4 Ab
'Whole broth: cell free culture broth; Ethyl acetate fraction: organic fraction tested at 20x concentration; Broth without protein: whole broth with protein component removed; Protein fraction of broth: protein component of broth only " Means with either the same capital letter in the same row or the same srnall letter in the same column are not significantly different (P=0.05) Figure 4. Growth curve for Xenorhabdus sp. RIO showing total protein. exochitinase and endochitinase development in TSB Exochitinase Endochitinase Total protein
72 96 Time (h) activity. No 1,3-9-glucanase activity was detected in the culture broth of any of the test bacteria. TSB alone did not show chitinase activity.
All four Xenorhabdus sp. bacterial species and strains were grown in vitro.
After 72 h the culture broth was tested for antimicrobial activity, total protein produced and exo- and endochitinase activity (Table 14). All bacterial strains showed similar growth and the culture bmths were of similar pH. The strain RIO had significantly higher antibiotic and antimycotic activity than did the other three bacterial strains. Protein production was greatest in the 827 strain and statistically similar among al1 the others. Each bacterial strain produced different fevels of exo- and endochitinase activity in the culture broth with the AUS and
827 strains producing the most exochitinase and 827 and RIO producing the most endochitinase.
Chitinase activity was detected from the 5m fraction to the 21" fraction with a peak at the grnfraction (Figure 5). Two protein peaks were observed including a sharp first peak at fraction 5, which coincided with the first peak in antimycotic activity, and a second lower broad peak around fraction 12. The major peak of antimycotic activity occurred at fraction 19 and did not correspond with chitinase or total protein levels. The chitinase activity occurred between the two protein and the two antimycotic peaks of maximum activity. Concentrated TSB, when run through the Sephadex column, showed a single peak of maximum antimycotic activity and total protein, which corresponded with the first protein peak and antimycotic activity peak in Figure 5 (data not shown). Table 14: Comparison of the final a bsorbance. pH. antibiotic activity, antimycotic activity. total secreted protein concentration and ex* and endochitinase activities in 72 h TSB cultures of two strains of Xenorhabdus bovienii (AUS, 827). X. nematophilus (BJ), and Xenohabdus sp. (RIO)
Bactenal strains'
Characteristics BJ AUS 627 RIO
Final absorbante (600nrn)
PH
Antibiotic activity (mm)"
Antimycotic activity (mm)"
Total protein (pglrnl)
Exochitinase (units)
Endochitinase (units)
means with the same letter in the same row are not significantly different (P=O.05) ** antimicrobial activity of the cell free culture broth rneasured as inhibition zone radius (mm) on agar diffusion plate assays Figure 5. Profile of chitinase activity. protein concentration and antimycotic activity in different fractions following separation of protein from Xenohabdus sp.
RIO culture through Sephadex G100. +Total protein *Chitinase Y
10 15 20 25 30 Fraction Number To test the effect of heat on the antimycotic activity profile, the fractions were autoclaved at 121°C (Figure 6). There was no significant difference in the antimycotic activities of the autoclaved and non-autoclaved fractions except at the mid-portion of the profile, which conespondeci with the large peak of chitinase active fractions.
Fractions containing the second bioactive peak were combined and the minimum inhibitory concentration (MIC) was deterrnined to be 12.5 pgfrnl active ingredient for 6. cinerea and 6.3 pglml for B. subtilis. The first peak was not analyzed due to the presence of additional bioactive broth components. All of the mycelial plugs from the MIC experiment showed growth when transferred ont0 clean agar plates with the exception of those exposed to the highest concentration (200 pglml) of bioactive compound. Figure 6: Profile of antimycotic activity before and after autoclaving the Sephadex
G100 protein fractions from Xenorhabdus sp. RI0 culture A Antimycotic activity o Autoclaved activity
O O 0 ood O OOOOQ O 5 10 15 20 25 30 Fraction Number 6.0 OISCUSSION
Dutky (1974) was the first to suggest that the bacterial symbionts of
Steinemematid nematodes produce antimicrobial substances. Since then a variety of antibiotic and antimycotic compounds have been found associated with these bacterial symbionts (Table 4). Steinemema riobrave, a newly discovered entomopathogenic nematode, shows much promise for use in the control of a variety of arthropod pests (Table 5) and is cunently available as a commercial biological control agent. However, little is known about the biology and antimicrobial metabolites of this nematode's symbiont, Xenorhabdus sp. RIO
This research has shown that Xenohabdus sp. RIO (1 ) shares similar characteristics with other Xenorhabdus species, (2) produces antimicrobial substances in vivo that, at least initially, prevent the growth of competing microorganisms in the insect cadaver and (3) produces a broad spectmm of antimicrobial activity in vitro which is due, in part, to bioactive, proteinaceous secondary metabolites. These findings will now be discussed in relation to existing information.
There are a number of distinguishing characteristics of the genus
Xenorhabdus: negative Gram reaction, rod shaped cells, motility, absence of bioluminescence, absorption of bromothyrnol blue, negative for catalase, cytochrome oxidase and urease, and producing acid from D-fructose, D-glucose,
D-maltose, D-mannose and D-trehalose (Yamanaka et al., 1992). Based on these criteria, the chemotaxonomic data (Table 8, 9 and 10) showed that the bacterial strains BJ, AUS, 827 and RIO are of the genus Xenorhabdus and not of other closely related genera such as Photorhabdus. RIO shared many characteristics with the other Xenorhabdus species tested, X. bovienii and X. nematophilus, but there were also some distinct difierences. For example, RIO tested positive for acid production from inostitol and the colony was not pigmented like X. nematophilus BJ. However, RIO tested positive for acid from
2-keto-gluconate, much like X. bovienii B27. The overall test results indicated that the symbiont of S. riobrave is of the genus Xenorhabdus. Nevertheless, due to the variability seen between species and strains of Xenorhabdus with these biochemical test results, it was not possible to clearly identify the bacterial symbiont of S. riobrave or differentiate it distinctly from the test species.
PCR-ribotyping studies have recently confirmed that the symbiotic bacterium of S. riobrave is a Xenorhabdus sp. and indicated that the syrnbiont may be a new species (Fischer-Le Saux et al., 1998; Bonifassi et al., 1999).
Species identification within the genus Xenorhabdus, using traditional chemotaxonornic methods, is difficult because the bacteria often provide variable results for many of these tests and most strains of Xenorfiabdus tend to be phenotypically very sirnilar. Additionally, the existing taxonornic status of these organisms is based on a few individuals from geographically distinct areas of the world (Fischer-Le Saux, 1998). Taxonomic studies based on phenotypical and biochemical characteristics (Thomas and Poinar, 1979; Akhurst and Boemare,
1988;Yamanaka et al., 1992; Nishimura et al., 1994) have shown taxonornic discrepancies between the tested bactena. There have even been discrepancies between PCR-ribotyping studies where the phylogenetic position, using portions of the 16s rRNA genotypes, of the symbiont of S. nobrave differed (Fischer-Le
Saux et a1.,1 998; Bonifassi et al., 1999). Fischer-Le Saux et al. (1998) suggested that the complete 16s rRNA genes should be sequenced in order to refine the phylogenetic tree of the Xenohabdus genus. Further analysis is warranted using both traditional taxonomic as well as contemporary molecular tools in order to discriminate effectively between the different species of
Xenorhabdus. Once a wmprehensive data base for this genus has been developed it should be possible to conclusively identify the bactenal symbionts of each Steinernematid species.
Xenorhabdus sp. RIO quickly wlonized the insect and produced antimicrobial compounds, which have antibacterial and antimycotic activity
(Figure 1). G. mellonella larvae infected by S. riobrave died within 72 h, presumably from the bacterial toxins released by the rapidly developing
Xenorhabdus sp. Antimicrobial activity appeared in the cadaver when the bacteria reached late growth and early stationary phase, as observed also by
Maxwell et al. (1994). These antimicrobials were probably the cause of the elimination of E. faecalis, which was released from the disintegrating insect gut, because the decline in E. faecalis coincided with the devefopment of antibacterial activity and this bacterium is known to be inhibited by the antimicrobial compounds produced by Xenomabdus sp. RIO on agar diffusion plate bioassays
(P. Isaacson, unpublished data). This aliowed the nematode-bacterial complex to grow without cornpetition frorn E. faecalis. It was assumed that the abundant Gram positive coccus found in the earty stages of infection was E. faecalis,
based on the report of Boucher and Williams (1967) and from comparing
phenotypic characteristics with those pubtished in Bergey's manual (Bergey,
1994).
These antimicrobial substances do not inhibit al1 organisms, as could be
seen from the presence in the cadaver of some Gram negative bacteria, probably
from the insect gut or nematode cuticular surface. These bacteria, which were
not Xenorhabdus, began to develop in the nutrient rich cadaver after
Xenorhabdus had become established and antibiotic activity detected. These
observations are not unexpected since it is known that the antibiotics produced
by Xenorhabdus spp. tend to be more effective against Gram positive than Gram
negative bacteria (Akhurst, 1982). As well, Maxwell et al. (1994) and Boemare et al. (1997) showed that the later stages of nematode development occur in a
multixenic environment rather than a monoxenic environment populated only by the nematode's symbiont (Akhurst and Boemare. 1990). The present data show that these bactena produce also antifungal activity in vivo. Although the Gram negative contaminants also produce secondary metabolites it has been shown in recent expenments that no increase in antimicrobial activity was detected that was specifically associated with these other bacteria (K.T. Walsh, pers. comm.).
These observations appear to support the long held hypothesis that the bacterial symbionts of entomopathogenic nematodes produce antimicrobial compounds to prevent putrefaction of the insect cadaver. However, an alternative hypothesis put forward by Jarosz (1996a and 1996b) cannot be disregarded. He postulated that a competition mechanism through rapid colonization by Xenorhabdus, rather than antirnicrobial production, prevents the establishment of contaminating bacteria and fungi. It is possible perse that both hypotheses apply because the symbionts have been shown both to produce antimicrobial substances in vivo and develop very rapidly at the beginning of the infection cycle ahead of any other competing microorganisms.
Further characterïzation and growth of the symbiont, Xenorhabdus sp.
RIO, was detemined in vitro using established laboratory protocols and to detect the large nurnbers of bacteria and the conespondingly high concentrations of secondary metabolites. The antibacterial and antifungal activity of the RIO culture broth was confimed when tested against B. subtilis and B. cinerea, respectively (Figure 3). This study confirms that the antibiotics of Xenorfiabdus sp. RIO are similar to that of other Xenorhabdus spp. in having activity against a wide range of bacteria (Akhurst, 1982), and that the Gram positive species are more strongly inhibited than Gram negative ones (Table 11). The whole broth of
RIO showed a broad spectrum also of antimycotic activity when tested against 29 different species and strains of fungi. Most fungi, including a nurnber of economically important plant pathogenic fungi, were completely inhibited by the highest concentration of whole broth (Table 12). However, the mycelial growth of
A. flavus, A. fumigatus, B. bassiana, F. oxysporum, M. anisopilae, P. aphalidennatum and T. basicola was less affected by these antimywtics than was that of other species of fungus. The present study wnfimis the resuîts of
Chen et al. (1994) who found that various Xenorhabdus spp. displayed broad spectrum antimycotic activity, which did not appear to be conelated with a particular taxonomic group of fungi. However, the bioactivity observed in the present study is directty attributed to compounds produced by Xenorhabdus sp.
RIO into the culture broth as the test was done on cell-free culture broth.
Although it is difficult to compare the two studies directly. it is interesting to note that in some cases the same test species showed relatively different susceptibilities to the antimycotic activity of the culture broth of different
Xenorhabdus bacteria. These differences could be due to different strains andlor culture histones of the Xenorhabdus spp. and/or the test fungi used, but could also imply the presence of different bioactive compounds in the cell free culture broth of RIO as compared with that of other species of Xenorhabdus.
For Xenorhabdus sp. RIO, the organically soluble metabolites in the EAF were not active against fungi and showed significantly lower antibacterial activity than did the PR fraction. Previously identified organically soluble antibiotics. such as xenorhabdins, indole compounds (Paul et al., 1981; Mclnemey et al.,
1991a; Li et al, 1995) and nematophins (Li et al., 1997). have been shown to be active against both bacteria and fungi. It is possible that some other compounds may be responsible for the activity in the EAF but the bioactivity against the bacteria, based on Gram stain, suggests a similar mode of action to that of the soluble organic compounds listed above. The lack of detectable antimycotic activity in the aqueous fraction of the present tests may have been due to the low sensitivity of the bioassays, particularly when testing substances with low activity, and the fact that these organic substances do not diffuse easily through an aqueous medium such as the agar diffusion pfates. Nevertheless, since previous studies have shown high activity among these organic compounds using similar bioassays (Ng. 1995; Chen, 1996), the present results indicate that although organic antibiotics are present they do not contribute significantly to the overall antimicrobial activity of RIO culture broth.
When proteins were removed from the culture broth there was a significant drop in bioactivity. This fact, and the low activity associated with the organic fraction, indicates that proteins in the aqueous fraction are an important contributor to the overall bioactivity of RIO. Since the level of antibacterial and antimycotic activity of the protein fraction were not significantly different from each other, this may indicate that a single protein or combination of proteinaceous components was active against both bacteria and fungi. The bioactivity of the proteinaceous fraction of other Xenorhabdus spp. has been attributed to hydrolytic enzymes such as proteases and chitinases as well as to some unidentified proteinaceous component(s) (Chen et al., 1996). Therefore, the proteinaceous component of RIO cutture broth was exarnined further.
Xenorhabdus sp. RIO showed growth in vitro similar to that of other
Xenorhabdus species grown in TSB (Ng , 1995; Chen, 1996). As with X. bovienii
(Ng, 1995) the culture broth pH of RIO decreased during the initial stages of growth and increased as the bacteria approached and reached stationary phase
(Figure 2). This pH increase may be the result of the bacteria metabolizing the broth contents into alkaline products such as arnmonia, which is known to be produced by Xenorhabdus spp. in TSB (Chen, 1996). Many of these secondary metabolites including extracellular enzymes are thought to increase dunng the late logarithrnic and early stationary phase of the bacterial growth cycle (Forst and Nealson, 1996). This is confirmeci in the timing and production of antimycotic activity (Figure 3), proteins and chitinase enzymes (Figure 4) in the culture broth. The strong correlation between antimycotic activity and pH
(~0.82)~total protein (r=0.88) and exochitinase activity (r=0.93) reinforces earlier observations (Chen, 1996) that the antimycotic activity may be associated with the production of proteinaceous secondary metabolites, such as chitinases.
Indeed, Xenorhabdus spp. are known to produce chitinases that show lysis of fungal cell walls (Chen et al., 1996). Conversely, antibiotic activity was detected immediately, and the correlation between antibiotic activity and total protein
(0.73) and exochitinase activity (~0.82)was not as strong. In fact, Chen (1996) speculated that the chitinases produced by Xenorfiabdus spp. are unlikely to have antibacterial activity due to the absence of lysozyme activity. It seems
Iikely, as conchded by Chen et al. (1996), that the antimicrobial activity associated with Xenorhabdus spp. is due at least in part to the presence of bioactive proteins. RIO showed significantly stronger antimicrobial activity than did the other Xenorhabdus isolates tested, and this bioactivity appeared to be dependent more on the proteinaceous than on the organic secondary metabolites (Table 13). Due to the antimycotic activity of chitinase enzymes and the correlation between antimycotic activity and exochitinase activity it is reasonable to assume that the chitinases wntributed to the antimycotic activity of the RIO products. The gel filtration chromatography column data (Table 5) supports this theory as the activity associated with the chitinase enzyme was significantly diminished upon heating, probably due to the denaturing of the chitinase enzymes (Figure 6). The initial activity peak corresponded to heat stable broth components as well as to the large molecular weight proteins produced by the bactena which tended to lose activity upon heating, although this decrease was not statistically significant. These symbionts are known to produce high molecular weight bioactive proteins, some of which have shown insecticidal activity (Bowen et a/., 1998). The second broad activity peak inhibited fungi at
12.5 pg/ml, but inhibition was not likely due to either chitinase or other proteins because it was highly heat stable and increased to maximum activity as the chitinase and protein levels declined to non-detectable levels. These findings contradict those of Chen et al. (1996) who found a similar profile with X. bovienii but obsewed that the second activity peak was not heat stable and assumed it to contain high concentrations of protein based on sample absorption at 280nm.
The Bradford assay used in this study is a more accurate measure for protein than ultra violet absorption since many molecules including proteins absort, at these wavelengths (Bollag et al., 1996).
Despite the strong chitinase activity, the majonty of bioactivity produced by
RIO is clearly not due to proteins and must be caused by different substances from those produced by bovienii. These bioactive molecules are Iikely small molecular weight compounds because they become trapped in the gel matrix and are eluted late from the column. Chitinases are typically moderately sized proteins and the chitinase identified from X. bovienii was found to be 38.8 KDa in size (Chen et al., 1996). The proteinaceous fraction in the present study was also subjected to dialysis (6-8000 MW cut off) to remove salts and it would be expected that small rnolecular weight antibiotics would be rernoved. Bacteriocins offer a possibility for this activity because they are of peptide origin, are of smafler molecular weight and tend to be heat stable (Sahl, 1994). Although these substances have been identified from Xenortiabdus (Thaler et al., 1997), they are unlikely to be responsible for this activity since they have limited action spectra and Gram negative bacteria typically produce bacteriocins over 20 KDa in size, which would elute out of the column along with the other proteins. It is likely that the bioactivity is due to some, as yet unidentified, peptide(s) since srnall molecular weight bioactive peptides are commonly produced by a vanety of organisms and these cornpounds offen display both antimycotic and antibiotic activity (Sahl, 1994; Bushnell et al., 1998).
Although chitinase enzymes were not a major component of the antimicrobial activity produced by Xenorhabdus sp. RIO they did almost certainly contribute to the overall antimicrobial activity- It is also likely that these enzymes along with other hydrolytic enzymes contributed to the nutritional requirements of the nematode-bacterial complex by digesting macromolecules in the insect cadaver. Although the egg shell is almost the only site of chitin in nernatode structure, chitinase enzymes have been identified from a variety of nematodes incl ud ing Onchocerca gibsoni, Haemonchus contortus, Bmgia malayi and from larval extracts of Wuchena banctani. Chitinases may play a role not only in nematode egg development and hatching but also during other stages of the nematode life cycle (Londershausen et al., 1996). This is plausible for
Steinemematid nematodes where these enzymes probably aid in egg hatch and in weakening the insect cuticle to allow egress of the IJs from the insect cadaver and to infect new hosts.
In recent years, chitinolytic enzymes have received much attention due to their potential as biopesticides, chernical defense proteins in transgenic plants and synergists for microbial control agents for the control of insect, nematode and fungal pests (Kramer and Muthukrishnan, 1997). Several groups of biological control agents produce chitinases including the entomopathogenic fungi, M. anisopliae, B. bassiana and Aspergillus flavus, to help facilitate the penetration of the host cuticle (St. Leger et al.. 1991 ) and in Trichodema sp. to aid in fungal pathogenesis (Cotes et al., 1996). Chitinases may act directly to kill pest organisms andlor may act to weaken fungal cell walls or insect cuticle and so increase the susceptibility of the pest organisms to other pathogens or they rnay function as to bioactive defense compounds of plants. Thus, exposure of pests to high concentrations of chitinases targeted at particular developrnental stages could be potentially useful in pest management. Unfortunately, bacterial chitinases are generally ineffective in bioassays against pest organisms because they produce pnmarily exochitinases, which are less effective at cleaving chitin than endochitinases (Kramer and Muthuknshnan, 1997). However, Xenorhabdus sp. RIO produces high levels of endochitinase in vitro, which raises the prospect of them or their source bacteria having biopesticide potential. Chitinase producing bacteria have shown promise in controlling soi1 boume fungal
pathogens (Cronin et al., 1997; Zhang and Yuen, 1997). but Xenorhabdus
bacteria are not good candidates for this application since they are not known to
survive in the soi1 without their nematode host (Chen, 1994).
A useful bioengineering technique has been the introduction of plant and
microbial chitinase genes into plants to enhance resistance of the host plant to fungal pathogens (Broglie et al., 1991) and insect pests (Ding et al., 1998).
Additionally, microbial chitinases are known to increase the potency of entomopathogenic rnicroorganisms Iike Bacillus thuringiensis and baculovirus
(Kramer and Muthukrishnan, 1997). Used in this way. these enzymes are thought to compromise the structural integrity of the insect midgut and facilitate the entry of toxins into the insect. It is logical to assume that this kind of synergism operates equally well with microbial agents that target plant pathogenic fungi although the antifungal activity of the metabolites of RIO would affect also some biocontrol agents like Trichodema sp.
The phenornenon of higher organisms utilizing their associated microflora for the production of beneficial secondary metabolites is not uncornmon (James et a/.,1996). In many cases, it is likely that compounds previously attributed to higher organisrns actually may be produced by symbiotically associated bacterial flora. Entomopathogenic nematodes are no exception and recent interest in the secondary metabolites of the genus Xenorhabdus have led to the discovecy of many novel cornpounds whose mle in nematode development is unclear. The initial stages of nematode infection of an insect host show a rapid increase in the concentration of the natural symbiont and of the antirnicrobial secondary metabofites. Probably the most critical time in the nematode Iifecycle is when it enters the insect haemocoel, releases its symbiont and begins to establish a favourable environment for the development of the nematode-bacterial complex.
After this initial 72 h and insect death, some contaminating bacteria begin to appear in the insect cadaver, likely from the insect gut as it begins to lose integrity- By this time the conditions inside the cadaver are favourable for nematode development and most saprophytic organisms including fungi are excluded and rapid breakdown of the cadaver, which would otherwise compromise the development of the nematode, is prevented. Due to the broad spectrum antimicrobial activity of these substances it is difficult to argue against the hypothesis that these substances aid in protecting the cadaver from saprophytic organisms. The Iimited effects on Gram negative bacteria is, in fact, advantageous to the nematode-bacterial complex as the symbiont, itself a Gram negative bacterium, is relatively unaffected by these substances. Hydrolytic enzymes produced by the bactena may play a role in providing nutrients, and protection for the parasite and perhaps in aiding its development.
Xenorhabdus sp. RIO produces broad spectrum antimicrobial activity and shows significant activity against a wide range of plant pathogenic fungi in the laboratory. Many of these are significant pests of agriculture, and recent studies have shown that antimycotic agents produced by X. bovienii show promise in the control of plant pathogens in pot expenments (Ng and Webster, 1997). The bioactivity of Xenorhabdus sp. RIO appears to be different from that of X. bovienii and may indeed provide a potential source of new agrochernicals with novel modes of action. Additionafly, recent in vivo studies on the entomopathogen,
Photohabdus luminescens, have found antibiotics of a more diverse nature and in higher concentrations than those found in culture broth (Hu et al., 1999).
Therefore, based on the research described here it would not be unreasonable to find that Xenorhabdus sp. RIO produces metabolites with different properties in vivo than those found in vitro or in other Xenorhabdus spp. The discovery of chitinase enzymes, especially of endochitinases, produced by these bacteria may provide another way of controlling plant pathogens through development of biopesticides, transgenic crops engineered to express the respective gene product andfor by synergistic effects with existing microbial biocontrol agents.
This research has only started to characterize the antimicrobial activity of
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