Recognition between swarming isolates of Bacillus subtilis from tomato rhizosphere

Bakterije rodu Bacillus so ubikvitarne in raznolike po Gramu pozitivne bakterije, ki jih med drugim najdemo v rizosferi, kjer sobivajo z ostalimi bakterijami. V takem okolju je zelo pomembno poznati medsebojne interakcije med bakterijami. Te so lahko kooperativne ali antagonistične, o čemer odločajo medsebojno prepoznavanje in sorodstvena diskriminacija. Primer kooperativnega vedenja je rojenje, ki je večcelično gibanje bakterij z bički po poltrdni površini. Bakterije rodu Bacillus tvorijo tudi biofilme na koreninah rastlin. Namen magistrske naloge je bil ugotoviti, ali se roječi izolati Bacillus subtilis, izolirani iz rizosfere pradižnika na poltrdnem gojišču, prepoznajo kot sorodni (\u27\u27kin\u27\u27) ali manj sorodni (\u27\u27non-kin\u27\u27) in preveriti, ali pri izolatih, ki se med seboj prepoznajo kot manj sorodni (\u27\u27non-kin\u27\u27), pride do kompeticije pri kolonizaciji korenine in prevlade enega seva. Potrebno je bilo vpeljati in optimizirati metodo za testiranje kompeticije med izolati za kolonizacijo korenine navadnega repnjakovca (Arabidopsis thaliana). Ugotovili smo, da se večina izolatov med seboj prepozna kot \u27\u27non-kin\u27\u27, kar se odraža v nastanku mejne linije. Edina seva, katerih roja sta se zlila med seboj, sta bila T16-8 in PS-216. Ugotovili smo tudi, da časovni zamik inokulacije enega od sevov in razdalja med mesti inokulacije ne vplivata na nastanek mejne linije. Pri preverjanju vpliva pomanjkanja fosfata na rojenje in tvorbo mejnih linij smo opazili vidno razliko v rasti in morfologiji rojev, ki je bila ob pomanjkanju fosfata precej slabša in mejna linija je bila manj vidna. Pri kolonizaciji korenine smo v primeru inokulacije izolatov \u27\u27kin\u27\u27 opazili prisotnost obeh sevov na korenini, medtem ko je pri inokulaciji izolatov \u27\u27non-kin\u27\u27 na korenini prevladal le en sev. Uspešno smo vpeljali in optimizirali metodo za testiranje kompeticije za kolonizacijo korenine navadnega repnjakovca, ki bo zelo uporabna pri nadaljnjih raziskavah odnosov med bakterijami pri kolonizaciji korenin.Genus Bacillus contains ubiquitous, Gram positive bacteria which can be found in the rhizosphere coexisting with other bacteria. Research on the interactions between these bacteria in rhizosphere is very important. Interactions may be cooperative or antagonistic depending on how bacteria recognize and discriminate each other in a process called kin discrimination. An example of bacterial cooperative behaviour is swarming, a multicellular movement of flagellated bacteria over semi-solid surfaces. Bacillus species can also produce biofilm and colonize plant roots. The aim of this master’s thesis was to determine whether B. subtilis tomato rhizosphere isolates recognize each other as \u27\u27kin\u27\u27 or \u27\u27non-kin\u27\u27 on semi solid agar and to test whether \u27\u27non-kin\u27\u27 isolates, compete and exclude each other on the root surface. The root colonization assay on Arabidopsis thaliana was optimized to test the competition between the isolates. Our results showed that most of the isolates recognized each other as \u27\u27non-kin\u27\u27, which was visible as a boundary line between the swarms on semi-solid agar plate. The only \u27\u27kin\u27\u27 strain pair that merged was T16-8 and PS-216. Furthermore, the inoculation delay of one strain and the distance between the inoculation sites did not have an impact on the formation of the boundary line. In contrast, phosphate starvation caused a visible difference in bacterial growth and swarm morphology. Growth was significantly weaker with less visible boundary lines when no phosphate was present. Furthermore, when \u27\u27kin\u27\u27 strains were inoculated on the root in the colonization assay, we observed the presence of both strains, while only one strain prevailed when \u27\u27non-kin\u27\u27 strains we coinoculated on the plant root. We successfully optimized a method for root colonization assay of A. thaliana, which is important for the future studies of interactions between bacteria on the plant roots