Genetic diagnosis of phytopathogenic Agrobacterium tumefaciens isolates from Cydonia oblonga trees by PCR using 16SrRNA primers

A. Q. S. Khdeer; R. N. Gergees

doi:10.15421/0225005

A. Q. S. Khdeer Mosul University
R. N. Gergees Mosul University

Keywords: Agrobacterium, crown gall, Cydonia oblonga, biovar, 16SrRNA, pathogenicity.

Abstract

Agrobacterium is a plant pathogen causing crown disease and plant tumors in 140 or more dicotyledonous plant species. T u mor-causing agrobacteri a are members of the Rhizobiaceae family of nitrogen-fixing legume symbionts, but are harmful to plants. This study's initial goal was to isolate 25 bacterial isolates from crown tumors that developed on the stems of quince trees in various areas of Mosul. The colonies were selected for tests on the basis of the ir size, shape and color . From these , Gram-negative bacterial isolates were chosen and subjected to the following tests ; catalase, L-tyrosine utilization, citrate utilization, utilization of carboh y drates, lactose, mannitol, H 2 S production, pH range, g rowth at: 28, 35, 40 ºC, g rowth absence, ability to grow without biotine and thiamine, growth and pigmenentation in ferric ammonium citrate . Fi ve isolates (AtCo1, AtCo2, AtCo3, AtCo4, AtCo5) demo n strated their ability to form tumors on carrot discs for periods ranging from 22 to 26 days . Growth on the following selective media for their diagnosis produced positive results ; Mac C onkey agar, D1 medium, Luria- B ertani medium, glucose peptone agar, potato dextrose agar, AMM, chromogenic agar medium. These f ive isolates provided positive diagnostic signs and were designated with the symbols AtCo1, AtCo2, AtCo3 , AtCo4 and AtCo5 , in relation to the quince plant, which is the host plant from which the bact e ria were isolated. All exposures to A. tumefaciens formed tumors on carrot discs, according to the results of an artificial infection test used to determine the pathogenicity of the bacterial isolates. The outcomes of molecular diagnosis using agarose gel electroph o resis for the DNA of A. tumefaciens isolates corroborated these findings. The molecular diagnosis was achieved by amplifying the DNA using the 16S rRNA gene primer and the DNA p olymerase chain reaction (sPCR) technique. This allowed the clear separ a tion of single, shiny bands at the molecular size (1150 bp) in the five isolates, confirming their membership of the genus Agrobact e rium . Both of the Biovar I isolates (AtCo5 and AtCo2) from quince tumors were listed in the gene bank unde r the accession nu m ber PQ106659.

References

Aysan, Y., Sahin, F., Mirik, M., Donmez, M. F., & Tekman, H. (2003). First report of crown gall of apricot (Prunus armeniaca) caused by Agrobacterium tumefaciens in Turkey. Plant Pathology, 52(6), 793–793.

Bae, H., Kim, Y. B., Park, N. I., Kim, H. H., Kim, Y. S., Lee, M. Y., & Park, S. U. (2012). Agrobacterium rhizogenes-mediated genetic transformation of radish (Raphanus sativus L. cv. valentine) for accumulation of anthocyanin. Plant Omics Journal, 5(4), 381–385.

Bertolini, E., Olmos, A., López, M. M., & Cambra, M. (2003). Multiplex nested reverse transcription-polymerase chain reaction in a single tube for sensitive and simultaneous detection of four RNA viruses and Pseudomonas savastanoi pv. savastanoi in olive trees. Phytopathology, 93(3), 286–292.

Bivadi, V., Zakaria, R. A., Zare, N., & Yazdan, B. (2014). Effects of different tissue culture conditions in hairy roots induction in Hypericum perforatum L. International Research Journal of Applied and Basic Sciences, 8(5), 597–604.

Bouzar, H., Ouadah, D., Krimi, Z., Jones, J. B., Trovato, M., Petit, A., & Dessaux, Y. (1993). Correlative association between resident plasmids and the host chromosome in a diverse agrobacterium soil population. Applied and Environmental Microbiology, 59(5), 1310–1317.

Broothaerts, W., Mitchell, H. J., Weir, B., Kaines, S., Smith, L. M., Yang, W., Mayer, J. E., Roa-Rodríguez, C., & Jefferson, R. A. (2005). Gene transfer to plants by diverse species of bacteria. Nature, 433(7026), 629–633.

Bush, A. L., & Pueppke, S. G. (1991). A rapid and efficient new assay for determination of three biotypes of Agrobacterium tumefaciens. Applied Microbiology, 13(3), 126–129.

David, C., & Tempe, J. (1993). Transformation in Catharanthus species (Madagascar periwinkle). In: Bajaj, Y. P. S. (Ed.). Plant protoplasts and genetic engineering III. Springer-Verlag, Berlin, Heidelberig. Pp. 144–156.

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(1), 43–49.

Frank, J. A., Reich, C. I., Sharma, S., Weisbaum, J. S., Wilson, B. A., & Olsen, G. J. (2008). Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Applied and Environmental Microbiology, 74(8), 2461–2470.

Holt, J. G., Krieg, N. R., Sneath, P. H. A., Stanley, J. T., & William, S. T. (1994). Bergey’s manual of determinative bacteriology. Williams and Wilikins, Baltimore. Pp. 786–788.

Ibrahim, M. A., & Faisal, R. M. (2024). Molecular characterization of antibiotic resistance and virulence genes on plasmids of Proteus mirabilis isolated from urine samples of Hospitals in Mosul City, Iraq. Journal of Applied and Natural Science, 16(2), 830–841.

Islam, M. S., Akter, M. M., Rahman, M. A., Rahman, M. M., Akhtar, M. M., & Alam, M. F. (2010). Isolation of Agrobacterium tumefaciens strains from crown gall sample of dicot plants in Bangladesh. Current Research in Bacteriology, 3(1), 27–36.

Islam, M. S., Akter, M. M., Rahman, M. A., Rahman, M. M., Akhtar, M. M., & Alam, M. F. (2010). Isolation of Agrobacterium tumefaciens strains from crown gall sample of dicot plants in Bangladesh. Current Research in Bacteriology, 3, 27–36.

Kawaguchi, A., Sawada, H., Inoue, K., & Nasu, H. (2005). Multiplex PCR for the identification of Agrobacterium biovar 3 strains. Journal of General Plant Pathology, 71, 541–559.

Khalid, R., Zhang, X. X., Hayat, R., & Ahmed, M. (2020). Molecular characteristics of rhizobia isolated from Arachis hypogaea grown under stress environment. Sustainability, 12(15), 6259.

Kitahara, K., Yasutake, Y., & Miyazaki, K. (2012). Mutational robustness of 16S ribosomal RNA, shown by experimental horizontal gene transfer in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 109(47), 19220–19225.

Liaqat, F., & Eltem, R. (2016). Identification and characterization of endophytic bacteria isolated from in vitro cultures of peach and pear rootstocks. 3 Biotech, 6(2), 120.

Murugesan, S., Manoharan, C., Vijayakumar, R., & Panneerselvam, A. (2010). Isolation and characterization of Agrobacterium rhizogenes from root nodules of some leguminous plants. International Journal of Microbiological Research, 1(3), 92–96.

Nesme, X., Leclere, M. C., & Bard, R. (1990). PCR detection of an original endosymbiont: The Ti plasmid of Agrobacterium tumefaciens. National Dela Research in Agronomy, 1990, 47–50.

Ninkovic, S., Miljus-Dukic, J., Vinterhalter, B., & Neskovic, M. (2004). Improved transformation of alfalfa somatic embryos using a superbinary vector. Acta Biologica Cracoviensia, Series Botanica, 46, 139–143.

Paudel, D., Liu, F., Wang, L., Crook, M., Maya, S., Peng, Z., Kelley, K., Ané, J. M., & Wang, J. (2020). Isolation, characterization, and complete genome sequence of a Bradyrhizobium strain Lb8 from nodules of peanut utilizing crack entry infection. Frontiers in Microbiology, 11, 93.

Procópio, R. E., Araújo, W. L., Maccheroni, W., Jr, & Azevedo, J. L. (2009). Characterization of an endophytic bacterial community associated with Eucalyptus spp. Genetics and Molecular Research, 8(4), 1408–1422.

Puławska, J., & Sobiczewski, P. (2005). Development of a semi-nested PCR based method for sensitive detection of tumorigenic Agrobacterium in soil. Journal of Applied Microbiology, 98(3), 710–721.

Radomirka, N., Nevena, M., Slavica, N., & Mirjana, N. (2007). Efficient genetic transformation of Lotus corniculatus L. using a direct shoot regeneration protocol, stepwise hygromycin B selection, and a super-binary Agrobacterium tumefaciens vector. Archives of Biological Sciences, 59(4), 311–317.

Uchino, Y., Yokota, A., & Sugiyama, J. (1997). Phylogenetic position of the marine subdivision of Agrobacterium species based on 16S rRNA sequence analysis. The Journal of General and Applied Microbiology, 43(4), 243–247.

Wadhwa, Z., Srivastava, V., Rani, R., Tanvi, Makkar, K., & Jangra, S. (2017). Isolation and characterization of Rhizobium from chickpea (Cicer arietinum). International Journal of Current Microbiology and Applied Sciences, 6(11), 2880–2893.

Weller, S. A., Stend, D. E., & Mazzucchi, U. (2004). Crown and cane gall of a blackberry raspberry hybrid caused by Agrobacterium rhizogenes in Northern Italy. Journal of Plant Pathology, 86(2), 161–165.

Young, J. M., Kuykendall, L. D., Martínez-Romero, E., Kerr, A., & Sawada, H. (2001). A revision of Rhizobium Frank 1889, with an emended description of the genus, and the inclusion of all species of Agrobacterium Conn 1942 and Allorhizobium undicola de Lajudie et al. 1998 as new combinations: Rhizobium radiobacter, R. rhizogenes, R. rubi, R. undicola and R. vitis. International Journal of Systematic and Evolutionary Microbiology, 51(1), 89–103.

Younis, R. M., & Faisal, R. M. (2024). Plasposon mutagenesis in Pseudomonas aeruginosa isolates illustrates the role of ABC transporter in intrinsic resistance to antibiotics. Journal of Applied and Natural Science, 16(3), 1256–1264.

Zambryski P. (1988). Basic processes underlying Agrobacterium-mediated DNA transfer to plant cells. Annual Review of Genetics, 22, 1–30.

Zargar, M., Farahani, F., & Nabavi, T. (2010). Hairy roots production of transgenic Catharanthus roseus L. plants with Agrobacterium rhizogenes under in vitro conditions. Journal of Medicinal Plants Research, 4(21), 2199–2203.