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

پژوهش های حفاظت گیاهان ایران

Article 4, Volume 38, Issue 4 - Serial Number 66, December 2025, Pages 351-359   

Document Type: Research Article

DOI: 10.22067/jpp.2024.88026.1188

Authors

kayvan Farri¹; Maryam Khezri^{* 2}; Masoud Abrinbana³; Mina Rastgou¹

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

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

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

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

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