Molecular Identification of Gall-forming Bacteria in Stone and Pome Fruits using the recA Gene Sequence

Document Type : Research Article

Authors

• kayvan Farri ¹
• Maryam Khezri ²
• Masoud Abrinbana ³
• Mina Rastgou ¹

¹ Department of Plant Protection, Faculty of Agriculture, Urmia University, Urmia, Iran

² Assistant professor, Iranian Research Institute of Plant Protection, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran.

³ Department of Plant Protection, Faculty of Agriculture, Urmia University, Urmia, Iran.

10.22067/jpp.2024.88026.1188

Abstract

Introduction
Crown gall is an economically important plant disease that affects dicotyledonous and a few monocotyledonous plants in orchards, farms, and nurseries, worldwide. The disease is caused by Agrobacterium tumefaciens (Smith & Townsend, 1907) Conn 1942, a Gram-negative bacterium from the family of Rhizobiaceae in the class of AlphProteobacteria. This bacterium can survive in the soil as well as within host plants. The key characteristic of this bacterium is its ability to transform regular plant cells into tumor cells. Once this transformation is complete, the cells can continue to grow and divide independently of the bacterium. Molecular methods play a key role in the identification, classification and taxonomy of prokaryotes. Housekeeping genes are highly conserved protein-coding genes used to distinguish between different taxa of bacteria. In this study, the recA gene sequence was used to evaluate the efficiency of this gene in identifying and determining the phylogenetic relationships of tumor-forming Agrobacterium tumefaciens and Agrobacterium rubi (Hildebrand, 1940) Starr & Weiss, 1943 isolates.
Materials and Methods
The pathogenic isolates were isolated from tumors on the branches of cherry, plum, and apple trees in the orchards of Urmia, Naqadeh, Sardasht, and Khoy; located in West Azerbaijan province of Iran. In a previous study, phenotypic identification of the isolates was done, as well as the pathogenicity test with the gall formation on tomato crown. In this study, the recA gene sequence of four isolates (AT-U2, AT-K2, AT-N25 and AT-N15) was used for drawing the phylogenetic tree. After DNA extraction, polymerase chain reaction (PCR) was done using 91F/595R primers. Each PCR reaction was performed in a 25 µL PCR cocktail containing 12 µL of Taq DNA Polymerase 2x Master Mix RED (Amplicon, Denmark), 1 µL of each primer (10 pmol µL^-1), and 1 µL of template DNA (50 ng). The PCR amplification program was carried out under the following conditions; initial denaturation cycle at 94 °C (5 min), 35 cycles of denaturation at 94 °C (50 sec), annealing at 57 °C (50 sec) and extension at 72 °C (1.5 min), and a final extension at 72 °C for 10 min. The sequences of the studied isolates were compared with the sequences of the reference strains registered in the NCBI database. Sequence of the recA gene of A. tumefaciens and A. rubi strains, as well as strain AOL15 of Agrobacterium albertimagni Salmassi et al., 2002, as an outgroup strain, was obtained from GenBank. Phylogenetic relationships were inferred, by applying the Kimura-2-parameter model. The neighbor-joining (NJ) method and the adjacent method by MEGA 11 software were used for the phylogenetic tree and tested by bootstrap analysis with 1000 repetitions.
Results and Discussion
A 462 bp fragment was amplified in all bacterial isolates. Comparison of the recA sequence showed similarity of 99.95%-100%, with the reference strains of A. tumefaciens in GenBank. In the phylogenetic tree, sequence of the recA gene of studied isolates and reference strains of A. tumefaciens and A. rubi obtained from GenBank were used. The phylogeny tree included two main clads. The first clade included two subclades. AT-U2 placed in the first subclade and AT-K2, AT-N25 and AT-N15 placed in the second one. All the reference strains of A. tumefaciens from different countries were placed in the first clade, as well. In the second clade, A. rubi was placed separately from A. tumefaciens isolates. Currently, the analysis of housekeeping genes sequences is widely used to achieve higher accuracy in the phylogenetic relationships of different bacterial taxa. Among the bacterial conserved genes, sequence of the recA gene has been the interest of bacteriologist scientists (Costechareyre et al., 2010). Tumor-forming bacteria isolated from grapevine (Vitis vinifera L.), rose (Rosa sp.), red raspberry (Rubus idaeus L.), and cornelian cherry (Cornus mas L.) were classified in the Agrobacterium genus based on the 16S rRNA gene, but the result of phylogenetic analysis based on atpD, recA, and rpoB showed that some of the studied strains classified in Agrobacterium rosae Kuzmanovic et al., 2018, which is close to the species A. rubi and Agrobacterium skierniewicense (Puławska et al., 2012) Mousavi et al., 2015 (Kuzmanovic et al., 2018). A study of the recA gene sequence in 138 strains from 13 Agrobacterium species and genomospecies led to the differentiation between A. tumefaciens, Agrobacterium vitis Ophel & Kerr, 1990, A. rubi and Agrobacterium larrymoorei Bouzar & Jones, 2001 species (Costechareyre et al., 2010). In a multilocus sequencing study using rpoB, recA, gyrB, and atpD sequences, Agrobacterium isolates from various plants identified as A. larrymoorei and A. rubi (Mafakheri et al., 2019). The study of housekeeping genes has been proposed not only as a phylogenetic tool to clarify the relationships between prokaryote taxa but also as an alternative to previous methods, such as DNA-DNA hybridization and ITS sequence (Costechareyre et al., 2010). According to our results, A. tumefaciens strains were situated in a separated clade from A. rubi and A. albertimagni strains. Therefore, the recA gene sequence is a suitable tool to identify A. tumefaciens from other Agrobacterium isolates.
Conclusion
Results of the phylogenetic analysis revealed that using the recA gene sequence has enough efficiency in differentiating A. tumefaciens from A. rubi strains. This finding suggests that employing the sequences of the recA gene in phylogenetic relationship studies of Agrobacterium species provides accurate results in bacterial taxonomy.

Keywords

• Crown gall
• Housekeeping gene
• Taxonomy
• Tumorigenic bacteria

Main Subjects

• Bacteria

---

©2024 The author(s). This is an open access article distributed under Creative Commons Attribution 4.0 International License (CC BY 4.0), 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.

References

Aliu, E., Ji, Q., Wlazlo, A., Grosic, S., Azanu, M.K.,Wang, K., & Lee, K. (2024). Enhancing Agrobacterium-mediated plant transformation efficiency through improved ternary vector systems and auxotrophic strains. Frontiers in Plant Science, 15, 1429353. https://doi.org/10.3389/fpls.2024.1429353.

Bouzar, H., & Jones, J.B. (2001). Agrobacterium larrymoorei sp. nov., a pathogen isolated from aerial tumours of Ficus benjamina. International Journal of Systematic Microbiology, 51, 1023-1026. https://doi.org/10.1099/00207713-51-3-1023.

Conn, H.J. (1942). Validity of the genus Alcaligenes. Journal of Bacteriology, 44, 353-360. https://doi.org/10.1128/jb.44.3.353-360.1942.

Costechareyre, D., Rhouma, A., Lavire, C., Portier, P., Chapulliot, D., Bertolla, F., Boubaker, A., Dessaux, Y., & Nesme, X. (2010). Rapid and efficient identification of Agrobacterium species by recA allele analysis: Agrobacterium recA diversity. Microbial Ecology, 60, 862-872. https://doi.org/10.1007/s00248-010-9685-7.

Hildebrand, E.M. (1940). Cane gall of brambles caused by Phytomonas n. sp. Journal of Agricultural Research, 61, 685-696. https://doi.org/10.1038/s41598-018-27485-z.

Farri, K., & Khezri, M. (2021). Integrated management method of plant crown gall disease. Plant Pathology Science, 10, 116-127. https://doi.org/10.2982/PPS.10.2.116. (In Persian with English abstract)

Farri, K., Khezri, M., Abrinbana, M., & Rastgou, M, (2023). Identification of bacterial agents involved in tumor formation on plum, sweet cherry and apple trees. Applied Entomology and Phytopathology, 90, 171-181. https://doi.org/10.22092/jaep.2023.359240.1446. (In Persian with English abstract)

Ferdous, M.L., Hossain, M.N., Ali, M.O., Islam, M.S., & Yasmin, S. (2021). Morphological, biochemical and molecular identification of the wild strain of Agrobacterium tumefaciens from crown gall infected mango tree. Fundamental and Applied Agriculture, 6, 43-49. https://doi.org/10.5455/faa.136134.

Glaeser S.P., & Kampfer, P. (2015). Multilocus sequence analysis (MLSA) in prokaryotic taxonomy. Systematic and Applied Microbiology, 38, 237-245. https://doi.org/https://doi.org/10.1016/j.syapm.2015.03.007.

Kuzmanovic, N., Puławska, J., Prokic, A., Ivanovic, M., Zlatkovic, N., Jones, J.B., & Obradovic, A. (2015). Agrobacterium arsenijevicii sp. nov., isolated from crown gall tumors on raspberry and cherry plum. Systematic and Applied Microbiology, 38, 373-378. https://doi.org/10.1016/j.syapm.2015.06.001.

Kuzmanovic, N., Pulawska, J., Smalla, K., & Nesme, X. (2018). Agrobacterium rosae sp. nov., isolated from galls on different agricultural crops. Systematic and Applied Microbiology, 41, 191-197. https://doi.org/10.1016/j.syapm.2018.01.004.

Li, X., & de Boer, S.H. (1995). Selection of primers for polymerase chain reaction primers from an RNA intergenic spacer region for specific detection Clavibacter michiganensis subsp. sepidonicus. Phytopathology, 85, 837-842. https://doi.org/10.1094/Phyto-85-837.

Lim, S., Chun, S.C., & Kim, J.W. (2023). Cultivar resistance of Korean breeding cut-rose against crown gall by Agrobacterium tumefaciens evaluated by an in vitro inoculation. Plant Pathology Journal, 39(2), 220-227. https://doi.org/10.5423/PPJ.OA.07.2022.0095.

Lu, Y.L., Chen, W.F., Li, L. H, Wang, E.T., & Chen, W.X. (2009). Rhizobium alkalisoli sp. nov., isolated from Caragana intermedia growing in saline-alkaline soils in the north of China. International Journal of Systematic Microbiology, 59, 3006-3011. https://doi.org/10.1099/ijs.0.007237-0.

Mafakheri, H., Taghavi, S.M., Banihashemi, Z., Osdaghi, E., & Jay Ram, L. (2017). Pathogenicity, host range and phylogenetic position of Agrobacterium species associated with sugar beet crown gall outbreaks in Southern Iran. European Journal of Plant Pathology, 147, 721-730. https://doi.org/10.1007/s10658-016-1034-3.

Mafakheri, H., Taghavi, S.M., Pulawska, J., de Lajudie, P., Lassalle, F., & Osdaghi, E. (2019). Two novel genomospecies in the Agrobacterium tumefaciens species complex associated with rose crown gall. Phytopathology, 109, 1859-1868. https://doi.org/10.1094/PHYTO-05-19-0178-R.

McCarthy, R.R., Yu, M., Eilers, K., Wang, Y.C., Lai, E.M., & Filloux, A. (2019). Cyclic di-GMP inactivates T6SS and T4SS activity in Agrobacterium tumefaciens. Molecular Microbiology, 112, 632-648. https://doi.org/10.1111/mmi.14279.

Mousavi, S.A., Osterman, J., Wahlberg, N., Nesme, X., Lavire, C., Ludovic, V., Paulin, L., de Lajudie, P., & Lindstorm, K. (2014). Phylogeny of the Rhizobium–Allorhizobium–Agrobacterium clade supports the delineation of Neorhizobium gen. nov. Systematic and Applied Microbiology, 37, 208-215. https://doi.org/10.1016/j.syapm.2013.12.007.

Mousavi, S.A., Willems, A., Nesme, X., de Lajudie, P., & Lindstrom, K. (2015). Revised phylogeny of Rhizobiaceae: proposal of the delineation of Pararhizobium gen. nov., and 13 new species combinations. Systematic and Applied Microbiolog, 38, 84-90. https://doi.org/10.1016/j.syapm.2014.12.003.

Ophel, K., & Kerr, A. (1990). Agrobacteriurm vitis sp. nov. for strains of Agrobacteriurm biovar 3 from grapevines. International Journal of Systematic Bacteriology, 40(3), 236-241. https://doi.org/10.1099/00207713-40-3-236.

Puławska, J., Willems, A., De Meyer, S.E., & Sule, S. (2012). Rhizobium nepotum sp. nov. isolated from tumors on different plant species. Systematic and Applied Microbiology, 35(4), 215-220. https://doi.org/10.1016/j.syapm.2012.03.001.

Starr, M.P., & Weiss, J.E. (1943). Growth of phytopathogenic bacteria in a synthetic asparagine medium. Phytopathology, 33, 314-318. https://doi.org/10.1016/j.heliyon.2022.e09379.

Ren, da W., Chen, W.F., Sui, X.H., Wang, E.T., & Chen, W.X. (2011). Rhizobium vignae sp. nov., a symbiotic bacterium isolated from multiple legume species. International Journal of Systematic Microbiology, 61, 580-586. https://doi.org/10.1099/ijs.0.023143-0.

Rouhrazi, K., Khodakaramian, G., & Velazquez, E. (2016). Phylogenetic diversity of rhizobial species and symbiovars nodulating Phaseolus vulgaris in Iran. FEMS Microbiology Letters, 363(5), fnw024. https://doi.org/10.1093/femsle/fnw024.

Salmassi, T.M., Venkateswaren, K., Satomi, M., Newman, D.K., & Hering, J.G. (2002). Oxidation of arsenite by Agrobacterium albertimagni, AOL15, sp. nov., isolated from Hot Creek, California. Geomicrobiology Journal, 19, 53-66. https://doi.org/10.1080/014904502317246165.

Shahabi Mohammadabadi, M., Taghavi, M., & Djavaheri, M, (2012). Phenotypic and genotypic characteristics of Agrobacterium isolates from different hosts in Fars and Kohgiluye and Boyerahmad provinces. Iranian Journal of Plant Pathology, 48, 329-341. (In Persian with English abstract)

Smith, E.F., & Townsend, C.O. (1907). A plant-tumour of bacterial origin. Science, 24, 671e3. https://doi.org/10.1126/science.25.643.671.

Tamura, K., Stecher, G., & Kumar, S. (2021). MEGA11: Molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution, 38, 3022-3027. https://doi.org/10.1093/molbev/msab120.

Vargas Ribera, P.R., Kim, N., Venbrux, M., A´ lvarez-Pe´rez, S., & Rediers, H. (2024). Evaluation of sequence-based tools to gather more insight into the positioning of rhizogenic agrobacteria within the Agrobacterium tumefaciens species complex. PLoS ONE, 19(11), e0302954. https://doi.org/10.1371/journal.pone.0302954.

Wang, E.T., van Berkum, P., Beyene, D., Sui, X.H., Dorado, O., & Chen, W.X. (1998). Martinez-Romero E. Rhizobium huautlense sp. nov., a symbiont of Sesbania herbacea that has a close phylogenetic relationship with Rhizobium galegae. International Journal of Systematic Bacteriology, 48, 687-699. https://doi.org/10.1099/00207713-48-3-687.

Weisberg, A.J., Wu, Y., Chang, J.H., Lai, E.M., & Kuo, C.H. (2023). Virulence and ecology of Agrobacteria in the context of evolutionary genomics. Annual Review of Phytopathology, 61, 1-23. https://doi.org/10.1146/annurev-phyto-021622-125009.