Occurrence and enumeration of predator bacteria, Bdellovibrio, and their prey in plant rhizosphere and soil

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1 Symposium no. 11 Paper no Presentation: poster Occurrence and enumeration of predator bacteria, Bdellovibrio, and their prey in plant rhizosphere and soil MARKELOVA Natalya Y., KERZHENTSEV Anatoliy S. and HORNE James E. Institute of Basic Biological Problems, RAS, Pushchino, Moscow region Russia, , Russia In recent years considerable attention has been given to the functional activity of microbial populations in soil. Bacterial disease of plants is a problem of economic importance in agriculture. The wide use of chemicals against undesirable bacteria in agricultural practices has some drawbacks. Some uses of chemicals, like pesticides, are ecologically dangerous. Others, like antibiotics, may cause the selection of resistant forms with potential for adverse health effects. Therefore, alternative methods of plant protection are required. From this point of view the predator bacteria, Bdellovibrio, which are widely spread in various aquatic and soil ecosystems, are of great interest. The Bdellovibrios are predatory bacteria that prey upon other gram-negative bacteria and play a main role into controlling bacterial balance in the nature. Contact between a plant pathogens and host plant often start bacterial disease. Since contact takes place in the rhizosphere if predators are rhizosphere inhabitants, they might play a role in the control of pathogen densities and in protecting the plant against disease. The objective of this study was to enumerate, isolate and estimate of prey ranges of Bdellovibrio from soil and rhizosphere different plants in garden, hothouse and forest. Enumeration of prey was accomplished by plating, enumeration and isolation of Bdellovibrio was estimated according to the double-layer plague-forming technique. The prey range spectra was estimated by plague-forming technique, in liquid culture and in biofilm. In this study, Bdellovibrio was found in all rhizosphere samples (Froseria, Fabu, Licopersicon), ranging in number from 10 1 to 10 4 cells per gram of sample. Bdellovibrio as well as gram-negative prey bacteria are more abundant in the rhizosphere than in soil. Densities of Bdellovibrio in the rhizosphere were 1-2 logs higher than in soil. Their prey spectra included plant pathogens. It was demonstrated that Bdellovibrio multiply in biofilm and using Bdellovibrio for protecting plants from disease may be considered. The results obtained indicate the significance of these findings for a better understanding of the role of bacterial predators in soil and might be beneficial for discussing the possibility of using Bdellovibrio in protecting plants from microbial pathogens. Keywords: predator-prey interaction, microbial contamination Introduction Bacterial diseases in plants is a problem of economic importance in agriculture. The wide use of chemicals against undesirable bacteria in agricultural practices has some drawbacks. Some uses of chemicals, like pesticides, are ecologically dangerous. Others, like antibiotics, may cause the selection of resistant forms with potential for adverse health effects. Therefore, alternative methods of plant protection are required. From this

2 point of view the predator bacteria, Bdellovibrio genus, which are widespread in various aquatic and soil ecosystems, are of great interest (Stolp and Srarr, 1963). The bdellovibrios are predatory bacteria that prey upon other gram-negative bacteria and play a main role into controlling bacterial balance in nature. Contact between a plant pathogens and host plant is often the start of bacterial disease. Since contact takes place in the rhizosphere if predators are rhizosphere inhabitants, they might play a role in the control of pathogen densities and in protecting the plant against disease. Although numerous Bdellovibrio species isolated from various habitats have been described in literature, virtually no attention has been given to plant rhizosphere, which is abundantly populated by potential prey species and, therefore, can create favorable conditions for predators. In order to cope with fundamental and practical environmental problems, special attention should be given to this zone characterized by microorganism-dependent processes that are important for plants and involve soil pathogen-plant interactions (Yanagia, 1990). The Aims of this study were to enumerating the numbers of gram-negative bacteria and bacterial predators (Bdellovibrios) in soil and plant rhizosphere, obtaining a Bdellovibrio isolate from rhizosphere samples, identifying their, and evaluating the prey range on gram-negative bacteria various taxonomic groups, including pathogenic species. Materials and Methods Samples were taken from the soil and rhizosphere of the following plants: Lycopersicon esculentum var. vul-gare, Fabu vulgaris, Fragaria ananassa Duch (grown on a plot of land), Capsicum annum L. (grown in a greenhouse), and Convallaria majalis L. (from a forest). Isolation of Bdellovibrios from Capsicum annum L. rhizosphere was performed by direct inoculation (I), using Pseudomonas ftuorescens VKM B-22 as the host bacterium. The prey range of the new Bdellovibrio isolate was investigated by exposing 18 bacterial cultures from different taxonomic groups. Pseudomonas putida VKM B-31, Ps. aeruginosa VKM B-l, Alcatigenes faecalis VKM B-166, Erwinia carotovora VKM B-72, and Escheri-chia coli VKM B-l 15 were obtained from the All-Russian Collection of Microorganisms. E. coli 01157, Salmonella typhimurium, Shigella dysenteriae ATCC 13313, Vibrio cholerae, Serratia marcescens, Campy-lobacter jejuni 1, С. jejuni 2, С. jejuni 3, С. jejuni 4» С. jejuni 5, С. jejuni 33560, С. jejuni 63R. and Helicobacterpylory TX 30A were obtained from M. Shahamat (University of Maryland, United States). C. jejuni and H. pylory were cultivated on 5% blood agar. The others test bacteria were grown on tryptone-soybean agar (BBL M. S., Becton Dickinson.il United States). The prey range of the new Bdellovibrio isolate was determined on solid and liquid media as described earlier (Afinogenova et al., 1991) and by visual observation of twocomponent! prey-predator systems immobilized on the surface of transparent plastic carriers (12 mm in diameter; Fisher Scientific, United States). The two-components Bdelloviibrio sp. rh-p. ftuorescens system was immobilized follows: 0.5-mL drop of a one-day-old culture; (10 9 cells ml -1 ) was introduced into a sterile petri dish, and a sterile plastic carrier was placed on it for 5 min. To remove cells that were not firmly adsorbed, the carrier was washed with sterile distilled water for 1 mitt and placed in a sterile petri dish on an 0.5 ml drop containing Bdellovibrio sp. rh cells. After 30 min,

3 the carriers with immobilized cells were washed again, placed on an 0.5 ml drop of sterile distilled water in a sterile petri dish. After 2 or 24 h of predator-prey interactions, the carriers were taken out, stained with 0.05% acridine orange for 4 min, and examined under a luminescent microscope (Olympus Optical, Japan). All the immobilization studies were conducted at room temperature. Result and Discussion The numbers of gram-negative bacteria and Bdellovibrios in soil and plant rhizosphere samples was determined. Bdellovibrio cells were detected in all samples, their numbers ranging from 1 x 10 1 to 4.8 x 10 4 cells g -1 (Table 1). Table 1 Abundance of gram-negative bacteria and Bdellovibrios in soil and plant rhizosphere. Sampling site Sample Gram-negative bacteria, Bdellovibrio, cells g -1 sample cells g -1 sample Garden Soil 5.1 xl xl0 1 Fragaria 8.0 xl x10 2 (strawberries) Fabu (beans) 1.0 xl х10 1 Lycopersicon 3.3 x x10 2 (tomatoes) Greenhouse Soil 1.2 xl x10 3 Capsicum (pepper) 3.5 x X10 4 Forest Soil 8.0 x x 10 1 Bdellovibrio were more abundant in rhizosphere than in soil. Their numbers in the rhizosphere of all plants tested exceeded those in soil by 1-2 orders of magnitude. Strain Bdellovibrio sp. rh was isolated from Capsicum annum L. rhizosphere. It was characterized by an infectious cycle typical of Bdellovibrios, with the following stages: attachment to the prey cell wall; penetration into the periplasmic space; growth and development inside the infected cell; and division and release of progeny of Bdellovibrio cells accompanied by prey cell s lysis. Results on the surface interactions between Bdellovibrio sp. rh and Ps. fluorescens were obtained using transparent plastic material as the carrier for immobilization. Cell interactions on the surface were monitored visually by examining acridine orangestained preparations under a luminescent microscope. It is a relatively recent idea that Bdellovibrio is characterized by a surfaceassociated mode of life in nature (Afinogenova, 1992; Williams et al., 1995). Techniques for coimmobilizing the predator and prey on the surface of solid carriers were developed earlier (Markelova and Colwell, 1999). Using transparent carriers in this work enabled us to visualize the interactions. Surface-associated cells of Bdellovibrio sp. rh could exist and reproduce within the bacterial community, which is consistent with data of other researchers (Fletcher, 1990; Roszak and Colwell n.d.). The prey range of Bdellovibrio sp. rh was determined with respect to gram-negative bacteria in suspensions, on solid medium, and on the surface of a transparent plastic carrier. The results are given in Table

4 Pseudomonas aeruginosa cells were resistant to Bdellovibrio sp. rh. Interestingly, prey range was variable with regard to Campy lohacter strains. Bdellovibrio activity towards strains 4 and 5 on solid medium, in suspension, and on the carrier.surface. However, C. jejuni strains 1, 2, 3, 3350, and 63R were infected by bdellovibrios only on the carrier surface. The differences in the specificity of Bdellovibrio sp. rh-c. jejuni interactions on the surface and in suspension or on solid medium support the idea that the attachment of bacteria to the surfaces may inhibit the expression of sertain bacterial genes and affect bacterial behaviour (Costerton et al., 1994). Table 2 Prey range of Bdellovibrio sp. Rh. Gram-negative bacteria Plage forming efficiency Solid medium, PFU ml -1 Suspension, lysis Solid surface, interaction Pseudomonas fluorescens 64 x Pseudomonas putida 14xl Pseudomonas aeruginosa Alcaligenes faecalis 38X Ervinia carotovora 70xl Esherichia coli В 32xl Esherichia coli xl Salmonella typhimurium 17 x Shigella dysenteriae 82xl Vibrio cholerae 18 x Serratia marcescens 44xl Campilobacterjejuni (strains ,2,3,33560,63R) Campilobacter jejuni 4 80 x Campilobacterjejuni 5 loxlo Helicobacter pylori 27 x Note: The "+" sign signifies that the respective process occurs; the "-" sign signifies its absence. The changes in the specifity of Bdellovibrio sp. rh cells occurring upon their attachment to a surface suggest the existance of different mechanisms of predator-prey interaction of surface-associated cells and, accordingly, the existence of different control mechanisms within a bacterial community in nature. Based on the recent conclusion that Bdellovibrios are surface-associated organisms, they are likely to interact with other components of ecosystems, including plants. Bdellovibrios are predators or parasites of bacteria. For this reason, relevant research has therefore focused on various aspects of interactions in two-component or multicomponent bacterial systems. The issue of interactions of Bdellovibrio with plants has not yet been raised. Attempts to use Bdellovibrios for treating plant diseases were made for a long time (Sherrf, 1973); however, researchers used cell suspensions that soon lost their viability due to a high endogenous respiration rate of Bdellovibrio cells (Kurath and Morita, 1983). The discovery of importance of interfaces for the processes carried out by microorganisms in natural ecosystems, a new concept of the functional activities of

5 Bdellovibrio in natural habitats can be developed. This knowledge could be put into practice using bacterial predators to protect plants against bacterial diseases. Obviously, the father experimental evaluation of the role of bacterial predators in protecting plants in ecosystems against bacterial pathogens requires joint research by microbiologists and plant physiologists. References Afinogenova, A.V., S. Romai Penabad, S.M. Konovalova, L.G. Churkina and V.A. Lambina comparative characterization of Bdellovibrio strains isolated from river and waste water. Mikrobiologiya 50(2): Afinogenova, A.V Bdellovibrio: metabolism, physiology and ecology. Usp. Mikrobiol. 25: Costerton, J.W., Z. Levandovski, D. DeBeer, D. Caldwell, D. Korber and G. James Biofilms, the customized microniche. J. Bacterial.176(8): Fletcher, M Methods of studying adhesion and attachment to surfaces. Methods Microbiol. 22: Stolp, H. and M.R. Srarr Bdellovibrio bacteriovorus gen. et sp. n a Predatory, Ectoparasitic and Bacteriolytic Microorganism. Antonie van Leeuwenhoek. 29: Kurath, G. and Y. Morita Starvation-survival physiological studies of marine Pseudomonas sp. Appl. Environ. Microbiol. 45: Markelova, N.Y. and R.R. Colwell Two-component predator-prey bacterial system immobilized on the surface of transparent cover glasses is a promising model for investigation of the role of bacterial predators in ecosystems. Mikrobiologia 68(3): Roszak, D.B. and R.R. Colwell. n.d. Survival strategies of bacteria in the natural environment. Microbiol. Rev. 51(3): Yanagia, Т Natural Microbial Communities. Berlin, Jpn. Sci. Soc. Sherrf, R.H Control of bacterial blight of soybean by Bdellovibrio bacteriovorus. Phytopalhology 63(3): Williams, H.N., J.Y. Keley, M.L. Baer and B.-F. Tumg The Association of Bdellovibrios with surfaces in the aquatic environment. Can. J. Microbiol. 41: