Antimicrobial activity of Xenorhabdus sp. (Enterobacteriaceae

107 Pages·2000·4.51 MB·English

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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ABSTRACT
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 I O ~ C F UofI w~e t 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-triacetyochitobioser, espectively. 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.C hen (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 ..................................... ... ......... 1 8
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. ............................................. 2 9
vii
3.2.2 Carbohydrate Metabolism .................................................... 30
3.2.3 Enzyme Production ............................................................ 3 0
3.3 Results .............................................................................................. 31
4.0 IN VIVO ANTlMlCROBlAL ACTlVlTY ........................................................... 37
4.1 Introduction ......................................................................................3. 7
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 .......................................................................................4 3
5.2 Materials and Methods .......................................................................4 4
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 .................................4 7
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 ........................................................................................ 7 2
7.0 REFERENCES ............................................................................................8 6
LIST OF TABLES
Table 1. Estimated 19 93 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. ..................................~~.................................................-......-.......1..4.. .
Table 5. Tested host insect species for Steinemema riobrave ....................-.-..1 6
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.. ...........................................................-...........-3 3
Table 10. Enzymatic activities of pn'mary form Xenorhabdus sp. RIO in
cornparison with those of three other Xenorhabdus species .................-.3 5
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) ............. ........... . ............