THESES OF THE PHD DISSERTATION OF GHAZALA FURGANI
Table of Contents
SUMMARY
INTRODUCTION
1. OBJECTIVES AND RATIONALE
2. MATERIALS AND METHODS
3. RESULTS
3.1.Antibiotics production of the EPB strains
3.1.1. Isolation EPB symbionts directly from INFECTIVE dauer JUVENILE (IJ) of Steinernema species
3.1.2. Antibiotics production of different Xenorhabdus and Photorhabdus strains tested on Bacillus cereus
3.1.3. The effect of antibiotic production ON other entomopathogenic bacteria
3.1.4. Effects of the Xenorhabdus antibiotics on Erwinia amylovora
3.1.4.1. Efforts to control the fire blight disease in apple orchards
3.1.4.2. Laboratory tests on Erwinia amylovora
3.2. Fermentative production, chemical identification and application of EMA antibiotics
3.2.1. Fermentation of the EMA strain and testing the antimicrobial activity of the fermentation soup
3.2.2. Isolation of compounds of antimicrobial activity from fermentor liquid culture of XenorhabdusBUDAPESTIENSIS (LENGYEL ET AL, 2005) EMA strain
3.3. CHARACTERIZATION OF THE Xenorhabditis szentirmaii (Lengyel et al., 2005) EMC strain
3.3.1. The phenotypic characterization of the EMC strain
3.3.1.1. Colony morphology and swarming behavior
3.3.2. Exo-crystal production
3.3.3. ANTIBIOTICS Production of EMC
3.3.4. ConcLUSIONS AND PERSPECTIVES
3.4.RESULTS OF GNOTOBIOLOGICAL ANALYSIS OF STEINERNEMA / XENORHABDUS SYMBIOSES
3.5. ATTEMPTS TO LABORATORY FERMENTATION OF A NEW STEINERNEMA ISOLATE
3.5.2. Gnotobiological testS
3.5.3. Laboratory fermentation of the Morocco strain
3.5.4. Conclusions AND PERSPECTIVES
3.6. Molecular, genetic and gnotobiological identification of new Steinernema isolates
4. SCIENTIFIC RESULTS
PUBLICATIONS BASED ON THE RESULTS OF THE DISSERTATION
5. REFERENCES
6. Acknowledgements
SUMMARY
The entomopathogenic nematode (EPN) / bacterium (EPB) symbiotic complexes have great agricultural potential with the key of success in bacterium. The bacterium produces toxins, killing the insect and produce antibiotics to protect the monoxenic EPN / EPB complex against other microorganisms. In my dissertation I aimed to answer some questions related to the taxon-specificity of symbiosis and the role of the antibiotics.
Several strains available in our stock were tested for antibiotic production. The data demonstrate that different compounds may play a role in the antibacterial action of an EPB and the competition with another EPB strain. Two Xenorhabdus strains, EMA and EMC produce antibiotics extremely effective against Erwinia amylovora (the cause of fire blight disease in apple orchard) both in laboratory and phytotron tests. These compounds are also effective against Phytophtora and Trichoderma. We are to concentrate and chemically characterize the antibiotics for liquid fermented culture of EMA. The EMC strain produces unusual exo-crystals of carbohydrate nature. The role and exact chemical nature of the crystals are yet to be answered.
In different gnotobiological studies it was confirmed that the Steinernema species can grow and propagate only their own symbionts or on symbionts of other nematode species. In this study we have compared large number of strains of Photorhabdus and Xenorhabdus species and we can conclude that the Photorhabdus strains are better source for more Heterorhabditis strains in vivo and in vitro than the Xenorhabdus strains for the Steinernema species. The Steinernema strains are more “picky” in terms of finding proper symbionts.
We have isolated the EPB symbiont of the Morocco strain and elaborated a 3.7-liter laboratory fermentation technique for growing them. The fermentation technique can be used for other entomopathogenic nematode strains as well. We are recommending a new molecular technique to identify new Steinernema isolates.TheGenePhore PCR-RFLP System might be useful for further molecular identification of unknown and known bacterial strains.
INTRODUCTION
Entomopathogenic nematode - bacterium complexes are of great potential as tools of biological control of harmful agricultural insect pests. The aim of this study is to exploit more of this potential. In the introduction of my dissertation I attempted to summarize what we know about the taxonomy and co-evolution of this type of bio-control symbiosis.
1. OBJECTIVES AND RATIONALE
This study is aiming at revealing some details of the nature and evolution of the developmentally controlled, intimate, and highly taxon-specific symbiosis of entomopathogenic nematodes (EPN) and bacterium (EPB) species. The ultimate goal is benefiting from this information for improved biological pest control in the agriculture. I intended to provide examples of using selected EPN strain against target insect pests and EPB strains against target microbial pests and focus on the potential of producing new EPN/EPB symbiotic complexes.
Biological control is characterized by the utilization of living microorganisms to control pests. Gnotobiology comprises a study of germ-free plants and animals to which specific microorganisms are coupled by experimental methods. When one or more known species of microorganisms are added experimentally to a germ-free animal, the host is no longer germ – free. Both the host and the introduced species are gnotobiological. Gnotobiological research seeks to explore the effects of microorganisms in natural diseases, to identify the specific causative agents in infectious diseases. Gnotobiology is a part of the microbial ecology, which studies the relationships between animals and their associated microbial populations. An axenic animal is an animal living and reproducing free from any microorganism.
Entomopathogenic nematodes are a welcome addition to the natural enemy pool of insects and can be integrated with various control measures for management of those target pests where individual tactics alone are inadequate. Entomopathogenic nematodes play a role underground reminiscent of that played by insect parasitoids. Like parasites or predators they have chemoreceptors and are motile. Like pathogens they are highly virulent, killing their hosts quickly and can be cultured easily in vivo or in vitro.
Entomopathogenic nematodes are among the best known of an otherwise poorly studied group of natural soil insect enemies. Interest in these beneficial organisms has increased rapidly in recent years and research is being conducted in many laboratories world-wide (Gaugler et al, 1997). New species are described every year and many more isolates are waiting for identification and study (Koppenhofer and Kaya, 1999).
The aims and goals of this study are:
- Screening the EPB bank of our laboratory to determine the antibiotic production of different Xenorhabdus and Photorhabdus strains, to compare the effects of the antibiotics on closely related strains and on taxonomically unrelated bacteria, such as E. coli, Erwinia amylovoraB. subtilis, and B. cereus.
- To determine the symbiotic partner range of a large number of Steinernema and Xenorhabdus strains (gnotobiological analysis).
- To identify new Steinernema isolates by using a molecular tool (GeneGel Excel kit), cross fertilization and gnotobiological studies.
- To establish a liquid fermentation technique of a new Steinernema isolate.
2. MATERIALS AND METHODS
My studied altogether 32 Steinernema / Xenorhabdus symbiotic complexes, including 10 S. feltiae, 4 S. carpocapsae, 4 S. glaseri, 1 S. anomali, 1 S. intermedia, 1 S. rarum, 1 S. bicornutum, 1 S. affine, 1 S. riobravum, 1 S. cubanum, 1 S. scapterisci, S. kraussei, 1 S. bibionis and 1 strains of 3 unidentified Steinernema species. The majority of the EPB symbionts I used have later been identified in our laboratory (Lengyel et al., 2005).
As for culturing bacteria I used conventional microbiological techniques and the nematodes were grown first in conventional lipid-agar (Woots) plate later on the media we have developed and called entomopathogenic nematode – growth agar media (ENGM, Fodor, Furgani, Jagdale, Mathe-Fodor, Klein and Grewal, submitted for publication). Nematodes were also cultured in liquid culture elaborated by the Ehlers group (Ehlers, 2002).
Symbiotic bacteria were isolated from infective dauer juveniles, as elaborated in our laboratory (Lucskai et al., (personal communication.)
In my gnotobiological experiments I seeded ENGM plates with bacterial symbiont of one strain and the inoculated the plates with surface sterilized infective dauer juveniles (IJs) of the other one. The growth and reproduction of the nematodes were monitored. If they produced infective dauer progeny we isolated the bacteria from them for taxonomic identification. The progeny IJs were also tested for infectivity by using Galleria mellonella wax moth larvae.
More precisely: The plates were carrying nematodes from one strain and bacterial symbionts from the other strain were scored as follows:
- EPN grew (+) or did not grow (-) in into fertile adults.
- In the progeny population IJ appeared (+) or did not (-) appear.
- The IJ proved (+) or did not prove to be pathogenic for insect hosts (4th instar larvae of Galleria mellonella).
- The new generation of IJs left (+) or did not leave (-) the insect cadaver on water traps.
- The bacteria isolated from the IJs could be identified as the “new” (+) or the original (-) symbiont.
3. RESULTS
3.1.Antibiotics production of the EPB strains
3.1.1. Isolation EPB symbionts directly from INFECTIVE dauer JUVENILE (IJ) of Steinernema species
I have adopted and established the technique of direct isolation EPB symbiont from a single dauer larva was originally elaborated by A. Lucskai. The method is considerably safer than isolating the symbionts from an infected insect, since the contamination inside the carcass can be eliminated.
3.1.2. Antibiotics production of different Xenorhabdus and Photorhabdus strains tested on Bacillus cereus
The antibiotics production of 103 strains was tested by the method described by Akhurst (Akhurst, 1980) and Bacillus cereus was used as indicator bacteria. The antibiotic productions of different Xenorhabdus strains are variable. The results show, that is in show a considerable variability according to strains (see Fig. 1).
LEGEND to Fig. 1. EPB bacteria were grown for 5 days in the center of the Petri dish and then the EPB was overlaid by the indicator strain resuspended in soft agar. The inhibition zone is shown as a ring around the EPB colonies. The strain EMC (left) produced excessive amount of antibiotics inhibiting the growth of the indicator strain almost completely. X. bibionis produced less antibiotics against B. subtilis, the inhibition zone is clearly visible on the plate.
In my antibiotics studies I retested 20 most productive strains on both B. cereus and Erwinia amylovora, but finally I focused on the two most powerful strains, EMA and EMC, (see 3.1.4) because they are the promising ones.
3.1.3. The effect of antibiotic production ON other entomopathogenic bacteria
I found that the sensitivity of different EPB strains to the antimicrobial compounds of the fermentation soups of different strains is different. I supposed that there is more than one antimicrobial compound produced by these bacteria. Antibiotics which are effective against microorganisms from other taxa are less effective against closely related species or strains. At the same time a competition between EPB strains is also existing, and it is also a “chemical warfare” between the strains. By other words: the Xenorhabdus strains produced antibiotics against Photorhabdus and Photorhabdus strains are also producing antibiotics against Xenorhabdus.
I found, that the antibiotics produced by the symbionts of S. scapterisci, S. bicornutum, S. rarum and S. intermedia were the most effective against the symbionts of H. bacteriophora. The symbionts of S. affine and S. kraussei produced effective antibiotics against most of the P. bacteriophora strains. The symbionts of S. feltiae and S. serratum did not produce antibiotics effective against the P. bacteriophora strains tested.
Data also show that different Photorhabdus strains of H. bacteriophora symbionts belonging to the presented subspecies of the P. luminescens species show a similar degree of sensitivity to the different Xenorhabdus antibiotics. Interestingly, the toxin-producing P. luminescens ssp. akhurstii strains (symbionts of H. indica) are very sensitive to the Xenorhabdus antibiotics.
3.1.4. Effects of the Xenorhabdus antibiotics on Erwinia amylovora
3.1.4.1. Efforts to control the fire blight disease in apple orchards
Fire blight is a destructive bacterial disease of apples and pears that kills blossoms, shoots, limbs, and, sometimes, entire trees. The disease is generally common throughout the mid-Atlantic region of the US and in Europe. The destructive potential and sporadic nature of fire blight, along with the fact that epidemics often develop in several different phases, make this disease difficult and costly to control.
Strains of the pathogen that are resistant to streptomycin are present in some orchards in the eastern USA and in Europe., and are widespread in most apple and pear regions of the western U.S. Biological control agents, although not widely used, have provided partial control of blossom infections. More effective biological agents are required if their use is to become widespread.
3.1.4.2. Laboratory tests on Erwinia amylovora
The majority of the tests were carried out in the Quarantine Laboratory of the Szent István University of Horticultural Sciences in Budapest, Hungary within a frame fruitful cooperation between Dr. Maria Hevesi and our laboratory.
The best results (antibiotic activity equivalent to 100-200 ppm streptomycin sulfate) were produced by Xenorhabdus strains EMA, EMC and N2-4, and by Photorhabdus strains IS5 and Az36. Other EPB strains proved to be also very effective against the Erwinia isolates. Other data show that antibiotics produced by 20 EPB strains were also effective against Agrobacterium, Clavibacter, Pseudomonas, and Xanthomonas species (M. Hevesi, personal communication). The Erwinia isolates were collected from different plants and geographical locations by M. Hevesi. Our results clearly indicate that the application of the EPB antibiotics might be a very important tool for fighting the fire blight disease and presents a novel tool for fighting other bacterial parasites as well.
3.2. Fermentative production, chemical identification and application of EMA antibiotics
3.2.1. Fermentation of the EMA strain and testing the antimicrobial activity of the fermentation soup
We have optimized the conditions for growing several Xenorhabdus strains in liquid culture. We used both shake cultures, a small (10 liters volume) bioreactor (G. Furgani, Cs. Sisak and A. Fodor) and a commercially available (35-liter) bioreactor (A. Szentirmai). The products of fermentation (fermentation cell free supernatant) were directly tested for antibiotics production in the laboratory and phytotron. The fractions of the fermentation soup were used to isolate and identify the biologically active compounds. The results of the tests are summarized in Figure 3. The figure shows that the antimicrobial activity of EMA (X. bicornutum) was extremely effective, the Erwinia test bacteria were almost completely killed. In the phytotron test the EMA strain produced antibiotics proved as effective as kasumycin or streptomycin when the infection and the treatment were performed at the same time. The project was completed in cooperation with M.Hevesi.
Fig. 3. EMA antibiotics tests on E. amylovora on Petri dishes, liquid culture and phytotron.
LEGENDS TO Fig.3. The antimicrobial activity of the fermentation soup of EMA (isolate 262). A: Antimicrobial activity of Xenorhabdus sp. EMA strain on E. amylovora agar plate. B: Phytotron test of EMA fermentation soup. The biological activity of the EMA antibiotics can be compared to those of streptomycin and kasumycin. With the treatment of kasumycin the length of the infected area is greatly decreased. The fermentation soup (FS) of EMA is effective against Erwinia and the length of the infected area is decreased in the case of application of the FS in diluted (1:1 and 1:10) and undiluted form. With dilution of FS the infection increases.
3.2.2. Isolation of compounds of antimicrobial activity from fermentor liquid culture of XenorhabdusBUDAPESTIENSIS (LENGYEL ET AL, 2005) EMA strain
By using our recently elaborated techniques we have been isolated active compounds from the fermentation soup of Xenorhabdus budapestiensis EMA cultures. The experiments were carried out in co-operation, within the framework of OTKA grants T 035010 (B.Sztaricskai, Batta, M. Szentirmai, A. Fodor,G.Furgani, 2003). The active compounds were adsorbed to charcoal from liquid cultures. The diluted compounds could be fractionated by paper chromatography. The biological activity was tested on B. subtilis. Both the supernatant and the cell fraction contained biologically active fractions. The material obtained from cell free supernatant could be separated into several ninhidrin-positive fractions by chromatography andonly one fraction (Compound 1, comprising about 3.2%) proved to be biologically active against B. subtilis. Biologically active compound was also obtained from the cell mass and separated by chromatography to several ninhidrin-positive fractions. One of them (Compound 2, comprising about a 2.6 %) proved to be biologically active, but its activity was significantly lower than that of the one originated from the cell-free supernatant. The two biologically active compounds were chemically very different. No synergism could be detected. The biological activity of Compound 1 was severely pH-dependent. Compound I was further purified by Kieselgel 60 [CHCl3-MeOH-NH4OH (8:2:0.25)] column chromatography. We finally found a ninhidrin-positive, UV-active compound of low biological activity. By using 1H-, and13CNMR-, as well as mass spectrum (EI) analysis it was identified as triptamin
The stability of the water solution of the charcoal isolated EMA antibiotics was also tested for two different isolates of the EMA strain and it was found that after 6 months storing more than 90% of the activity remained intact.
The rest of the biologically active compounds of unknown chemical structure were isolated by using an alcohol: hydrochloride elution. Both the pellet (Compound 2 about a 0.40 g) and the freeze-dried pellet (Compound 3, about 1.34 g) are active.
The data on biological activity of the compounds isolated by the charcoal and Dovex50 method on B. subtilis are presented in Fig. 10 A and 10 B, respectively. The purified material obtained by the Dowex technique was hydrolyzed with ical structurte yptamine fraction. the treatment was 6-12 hrs.ation 6 n HCl (105oC, 24 hrs). The identification of the 9 peptide fragments is in progress.