Mikrobiol. Z. 2019; 81(4):118-130.
doi: https://doi.org/10.15407/microbiolj81.04.118

Destabilization of the Phage-Bacteria System during Bacterial Infections of Tree Plants

Tovkach F.I.1, Zhuminska G.I.2

1Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Akad. Zabolotny Str., Kyiv, 03143, Ukraine

2Odesa Mechnikov National University
2 Dvoryanskaya Str., Odesa, 65082, Ukraine

Despite significant progress in the understanding of the bacteriophage genome organization, the issues of their ecology and enormous diversity in nature remain under-explored. The aim of the work was to study the destabilization effect of witches’ broom disease symptom development in plant tissues on the pseudolysogenic endophytic bacterium Pantoea agglomerans resulting in accumulation of various lytic bacteriophages. Materials and methods. The subjects of the research were witches’ broom disease affected parts of willow plants that were collected at the Nizhne-Dnestrovsky National Nature Park (Odessa region) as well as in the suburb of Kyiv (Goloseevsky district) in the summer and autumn period. Endophytic bacteria were isolated from the crushed plant material by extraction into Luria-Bertani broth. The bacterial and bacteriophage quantity was estimated according to the standard techniques. Purification and TEM imaging of phage particles were performed with using of common approaches. Results. Observations show that witches’ broom disease of willow has massively epidemic character in Ukraine. For the first time for this disease, the main features of a three-component system: plant–epiphytic bacterium–bacteriophage were considered. It has been established that the transformation of plant tissues of Salix alba and Salix babylonica results in significant changes in the endophytic bacterial consortium with predominance of yellow-pigmented bacteria P. agglomerans (Pag). This in turn leads to the phage accumulation due to destabilization of the pseudolysogenic state of Pag in the binary system bacterium–phage. The number of viruses and their diversity depends on the bacterial host location in relation to the site of infection. Phages are less common in transformed willow tissues than at the periphery of the affected branches. In this work, the universal indicator system Pag g157 was proposed which determines up to 50% of specific bacteriophages. The morphology of negative colonies together with inability to lysogenize the sensitive bacterial cultures suggested that all isolated phages have an obligatory lytic lifestyle. A detailed study of six phage lines classified them as belonging to the Siphoviridae morphotype B1. The phage AF6 particles have the heads with the average diameter of 67.11±1.87 nm and the tails of 168.96±9.97 nm in length. Conclusions. Thus, a significant quantitative prevalence of P. agglomerans in the endophytic consortium together with the activation of pseudolysogeny in this bacterium can be a reliable indicator of local as well as systemic infections or mechanical damages of tree plants. In this work, approach for studying of the three-component system: plant–bacterium–bacteriophage has been proposed that may be helpful for our understanding of infectious processes occurring under interaction of biological objects with different complexity orders.

Keywords: Salix alba, Salix babylonica, Pantoea agglomerans, “witches’ brooms”, phage-bacterium system, destabilization of phage system, new lytic phages.

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