Isolation of Clavibacter michiganensis subsp. michiganenesis-specific bacteriophages from tomato fields in Turkey and their biocontrol potential

Bekircan Eski and Darcan Egyptian Journal of Biological Pest Control (2023) 33:71 https://doi.org/10.1186/s41938-023-00717-9 Egyptian Journal of Biological Pest Control Open Access RESEARCH Isolation of Clavibacter michiganensis subsp. michiganenesis‑specific bacteriophages from tomato fields in Turkey and their biocontrol potential Duygu Bekircan Eski1*   and Cihan Darcan2 Abstract Background Clavibacter michiganensis subsp. michiganensis (Cmm) is causing wilt and canker and significant economic losses in tomato production. Current control methods with conventional chemicals or antibiotics lost their effectiveness due to the development of bacterial resistance to this agent. Bacteriophages, viruses of bacteria, have been attracting increasing attention as an environmentally friendly means of controlling bacterial diseases. In this study, Cmm and Cmm-specific lytic bacteriophages were isolated from rhizosphere soil and phyllospheres of diseased tomato plants, and the biocontrol potential of bacteriophages was investigated. Results Eight isolates that showed typical symptoms of Cmm infection in biochemical and pathogenicity tests contained the virulence gene pat-1 in the genome. Twenty-two native bacteriophages were isolated from 90 samples. As a result of the host range tests performed on the local Cmm isolates obtained in the study, temperature and pH tolerance tests were performed with ten phages with a wide host range. Phage24, Phage33 and Phage41, which have higher temperature and pH tolerance than other phages, were used to kill curve assay. Although the combined applications of phages were performed to increase efficacy, it was found that the application of Phage33 alone was more effective. Conclusion Phage33 may prove to be a suitable agent to be used as part of the microbial control strategy of Cmm. In further studies, Phage33 should be formulated to protect it from adverse environmental conditions and tested under field conditions to confirm the results obtained in this study. Keywords Bacteriophages, Clavibacter michiganensis subsp. michiganensis, Biocontrol Background The tomato, Solanum lycopersicum L. (Solanaceae), is one of the most widely grown vegetables in the world, with an annual production of over 180 million tons (FAO 2021). *Correspondence: Duygu Bekircan Eski 1 Department of Biotechnology, Bilecik Seyh Edebali University, 11100 Bilecik, Turkey 2 Department of Molecular Biology and Genetics, Bilecik Seyh Edebali University, 11100 Bilecik, Turkey The large proportion of production has been affected by abiotic (temperature, pH) and biotic (microorganisms) factors that damage all parts of the plant (Elnaggar et al. 2018). One of the most important biotic factors is plant pathogenic microorganisms that cause diseases during plant growth and postharvest storage, leading to economic losses. More than 60 pathogens, including bacteria, fungi, viruses, and nematodes, are known to cause diseases in tomato (Jones et al. 2014). Clavibacter michiginensis subsp. michiganensis (Cmm) is a non-spore-forming, non-motile, gram-positive, and aerobic bacterium that © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Bekircan Eski and Darcan Egyptian Journal of Biological Pest Control causes bacterial canker and wilt disease of tomato (Gleason et al. 1993). Bacterial canker and wilt disease of tomato was first described in Michigan, USA, in 1909 (Smith 1910). Plant debris, plant tissue, and contaminated seeds allow the bacteria to spread over long distances. Even less than 1% infected seed can cause 60–70% of crop losses in the field (Shaker 2014). The pathogen can remain viable in the soil for 2–3 years. Infected tomato plants showed different symptoms depending on environmental conditions such as temperature and humidity, as well as the virulence of the pathogen and the susceptibility of the variety (de León et al. 2011). Although the disease is seed-borne, wounds and natural openings such as hydathodes and stomata are the main entry points for Cmm to penetrate host tissues. Once invaded, the pathogen spreads throughout the plant via the xylem vessels. As the disease progresses, wilting symptoms, stunting, decline in fruit yield, and early plant death occur (Tancos et al. 2013). Copper-based compounds such as copper hydroxide and copper sulfate are commonly used to control Cmm (Kasselaki et al. 2011). However, the excessive use of copper-based chemicals not only leads to the emergence of resistant pathogens, but also prevents seed germination and impairs plant growth through oxidative stress and a reduction in photosynthetic pigments (Raymaekers et al. 2020). On the other hand, bacteriophages, the viruses of bacterial cells, are highly specific to a single bacterial species, and may even be specific to a single strain of that species. In addition, they are self-replicating, can remain in the environment for a long time, and are safe to use as they are not infectious to non-target organisms such as humans, animals, and plants. Therefore, the use of bacteriophages has gained importance in recent years as a promising tool to control bacterial plant diseases (Svircev et al. 2018). There are important studies on phage-mediated biocontrol of Ralstonia solanacearum and Xanthomonas campestris, which are other important bacterial pathogens of tomato plants (Elnaggar et al. 2018). However, Cmm-specific bacteriophage was first described by Echandi and Sun (1973), and since then no studies have been carried out to isolate novel bacteriophages. In this context, the aim of the study was to isolate lytic bacteriophages against Cmm strains causing bacterial wilt and canker of tomato in Turkey and to determine their potential use as biocontrol agents. Methods Samples collection In the summer of 2021, soils and plant material were collected from different regions of İzmir, Bilecik, and (2023) 33:71 Page 2 of 10 Çanakkale in Western Turkey. Soil samples were collected from 30-cm root depth of diseased plants from the growing areas. For pathogen isolation, samples of the aboveground parts of tomato plants (leaves, fruits, and stems) were collected in Kraft bags fr (...truncated)