Contract number

J4-8228

Department:

Department of Food Science and Technology

Type of project

ARRS projects

Type of project

Basic research project

Role

Lead

Duration

01.05.2017 - 30.04.2021

Total

1.42 FTE

Project manager at BF

Štefanič Polonca

ABSTRACT

Background: Recently, we investigated soil bacterium, Bacillus subtilis for its ability to discriminate kin from non-kin strains in the context of swarming and for the first time identified kin groups of B. subtilis among natural isolates. Plant root co-colonization of kin strain pairs resulted in a mixed strain biofilm, whereas non-kin strains fought for the colonization of the plant root and eventually only one strain prevailed. Problem description: The project focuses on the study of ecological and evolutionary role of kin discrimination in the soil bacterium B. subtilis, which is unexplored. This microorganism is extremely important industrially and agriculturally. It is often used in formulations for support of agricultural plants against pathogens. New guidelines for the development of plant support inoculants are aimed at combining different bacterial strains. It is unknown whether antagonistic behaviour of non-kin B. subtilis strains on plant roots will affect the plant health or/and protect the plant from being attacked by pathogens? The aim of the study: The project aims to determine the relationship between kin discrimination and territoriality, horizontal gene transfer, and ecosystem function. This knowledge will be then used to better understand biocontrol and plant growth promoting activity of B. subtilis. We will test these activities on agricultural plants (tomato, tobacco) and in the presence/absence of plant deadly pathogen (Ralstonia) using mixtures of kin and non-kin strains. Comparative genomics on B. subtilis strains with a focus on how distribution of the antagonistic loci correlates with the phylogenetic distance, kin and pherotype groups will be performed. Originality of the results: B. subtilis is a soil organism with promising biocontrol activities. To this date it is unknown how antagonistic interactions between strains influence the plants. Given that antagonistic interactions between non-kin B. subtilis include the secretion of antimicrobial compounds these may protect the plant from pathogen invasion. Also, B. subtilis can protect the plant by competing for the plant root colonization area. These questions have never been addressed before in the context of kin discrimination and the results will give us valuable information about inter-species interactions that could potentially lead to a more efficient microbial biocontrol development and to commercialization of this research. Methods: Our laboratories have environmental Bacillus strains from two collections, namely soil and rhizosphere. We will construct fluorescently labeled strains in order to visualize biofilm formation, swarming and root colonization via fluorescent microscopy and perform bioinformatics analyses on genomes. Using total plant RNA we will analyse the expression of key genes involved in plant defence resulting from the interaction with different combinations of Bacillus strains. Results potential and relevancy: Only a few microbial species are able to distinguish kin from non-kin and we were the first to find this in B. subtilis. The proposed project is highly original and knowledge obtained will support 1) the development of new methods to follow kin and non-kin interactions using fluorescent microplate reader, fluorescent stereomicroscope and construction of fluorescently labeled strains; 2) deeper understanding of mechanisms behind kin and non-kin DNA exchange and the impact of this phenomenon on the ecology, sociality and evolution of this species; 3) improved understanding of inter-species interactions in the context of plant growth promotion and/or biocontrol activity. As the results will be new and exciting we will be able to publish our work in prestigious scientific journals reaffirming our group on a scientific map as a highly competent and productive in the field.


THE PHASES OF THE PROJECT AND THEIR REALIZATION

WP1: Kin recognition of B. subtilis strains isolated from tomato roots
Duration: 1-12th month
Objectives: In this WP we test the hypothesis predicting that kin strains co-exist and non-kin strains antagonize each other on plant roots. We have previously shown on a smaller set of strains that non-kin compete on plant roots. Here we extend this study by including broader range of strains to confirm that this is a general pattern. In addition, we test kin recognition patterns of these strains on agar, to obtain more robust data set, again testing the generality of the phenotype.

The strains from the tomato rhizosphere were classified into kin groups. During swarming of tomato isolates we observed the formation of a border line between swarms, whereas fusion of swarms was observed only in one combination of a rhizosphere strain with a B. subtilis strain, isolated from a sandy soil. In the case of root colonization, the presence of both strains on the roots was observed in the case of inoculation of the more closely related ('kin') isolates, while only one strain prevailed in the inoculation of non-kin isolates on the roots. We conclude that the dominant interactions between native strains are predominantly 'non-kin', which is reflected in the formation of the border line and root colonization, and that the origin of the strains (tomato plant 1 or 2) did not determine the interaction between the strains. In addition, we successfully implemented and optimized the method of cultivation of A. thaliana and the testing of root colonization with B. subtilis strains.

WP2: Relationship between kin recognition and territoriality of bacteria
Duration: 12-30th month
Objectives: In this WP we would like to test the hypothesis predicting that kin recognition has consequences for bacterial territoriality. Genetically heterogeneous and homogeneous swarms on semi solid B media will be examined and tested for swarm segregation, swarming speed and conquered territory. The results will give us new insights into the surface colonization of heterogeneous microbial communities and most importantly allow us to test whether cooperative interactions during swarming are stabilized by kin discrimination. So far, we have constructed several different mutants of B. subtilis soil isolates, which have been labeled with fluorescent proteins (YFP or mKate) under constitutive promoters (constantly expressed during the growth of the strain). We have optimized the fluormetric monitoring of the swarming on the surface and already started the experiments with a fluorescent microtiter plate reader and partially optimized data processing. We began investigating the influence of kin discrimination and mixing of strains on the swarming, root colonization and also in the formation of biofilms. The first results suggest that blends of 'kin' strains swarm together on the plate and that they can also help one another, as they can exchange public goods (e.g. surfactin). Conversely, 'non-kin' strains are excluded, usually a single strain swarms across the agar plate. Occasionally, segregation of swarms is observed, which is a visible spatially separated swarm of both strains, but not on the same surface.

WP3 The effect of kin discrimination on the horizontal gene transfer
Duration: 12-30th month
Objectives: The aim of WP3 is to test the hypothesis predicting the effect of kin discrimination on horizontal gene transfer, measured as a transformation frequency (DNA transfer) during swarming. We measure transformation frequency between kin and non-kin strain pairs at the point where two swarms collide. Non-kin swarm pairs usually form a border with each other. Since there are many dead cells on such borders we hypothesize that there will be a large amount of extracellular DNA present. Living bacteria on such borders can therefore acquire this DNA via horizontal gene transfer. We will also test whether the transformation frequency is greater when kin-DNA is acquired as compared to non-kin. The latter will be tested by adding exogenous DNA to semisolid B-media, on which bacteria tend to swarm, and by measuring the transformation frequency at different areas on swarm plate including boundary line.

Within this work package 14 constitutively labeled B. subtilis strains (of which 7 in the sacA gene and 7 in the amyE locus attached to a p43 promoter and marked with two different fluorescence markers: YFP and CFP) were prepared. Each strain also contains an antibiotic resistance gene (SpR or CmR). Using these strains, we determined the level of DNA exchange at the meeting point of two swarms. Different labeling of strains with antibiotic resistance genes allowed us to experimentally track horizontal DNA transfer between strains by selecting transformants resistant to both antibiotics. We hypothesized that the frequency of DNA exchange will be more frequent in the area of the boundary between non-kin strains compared to DNA exchange at the merging point of kin strains. We determined the transformation frequency in the area of the swarm of 4 pairs of kin strains and 3 pairs of non-kin strains, as well as the transformation frequency at the contact of swarms of isogenic strains with different constitutive indications. Our results showed that the DNA exchange is higher in between two non-kin strains.

WP4 Genomics
Duration: 12-34th month
Objectives: In WP4 we would like to analyse already available genomes in the project team (21 genomes from the unique soil microscale collection of B. subtilis) and sequence selected genomes of B. subtilis isolates (tomato collection), analyse them, determine relatedness of the isolates and compare genomes with each other in many different ways. The main goals are: 1) to compare core genes, e.g. genes present in all genomes, and based on these determine relatedness; 2) to compare unique genes, so called additional genes, in order to verify their distribution in kin groups or pherotypes. In this way, we will address a) whether it is the antagonism and/or quorum sensing (sexual isolation) that plays a significant role in modifying the genomes and evolution of this species and b) whether strain difference in antagonistic loci is the key determinant of kin discrimination. We predict that kin strains will share the same antagonistic and resistance genes, while non-kin will not. The milestones in this series are set to the second year of the project, however, the genomes of B. subtilis isolates of the Sava riverbank have already been assembled and annotated. We are currently in the process of acquiring knowledge and using tools that will enable us to analyze the genomes more thoroughly. 

WP5 Biocontrol and plant growth promotion
Duration: 16-36th month
Objectives: in WP5 we will verify the influence of kinship on plant support functions and through these define a combination of  B. subtilis strains, which could be further used for plant support (i.e. as plant growth promoting rhizobacteria (PGPR) and/or bio-control agents). In WP5 we will test various strains of B. subtilis (microscale soil and tomato collections) to promote growth of tobacco (Nicotiana tabacum), tomato (Solanum lycopersicum) and potato (Solanum tuberosum). We chose these plants because they are high value agricultural plants and also used routinely in the partner group (NIB) and they each bring a unique set advantages to test bacterial plant support. We will also examine bio-control characterisctics of B. subtilis strains from our collections against plant pathogen Ralstonia solanacearum.

As part of the WP5, we are currently in the process of characterization of strains isolated from the rhizosphere of chilies. We would like to identify those isolates that promote plant growth and/or provide bio-protecting against various bacterial plant pathogens, e.g. Ralstonia solanacearum. We have isolated 253 isolates from the rhizosphere collection, and will determine their phylogeny by sequencing of the housekeeping gyrA gene. We will also determine the PGP properties: the production of indoleacetic acid (IAA), ACC deaminase, solubilization of phosphates and the formation of siderophores.

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