Investigation of the phenotypic and genetic diversity of Xanthomonas translucens pathovars, the causal agents of bacterial leaf streak of wheat and barley in parts of Iran

Document Type : Research paper-Persian

Authors

1 Gratuated M.Sc. student, Department of Plant Protection, Faculty of Agriculture, Shahid Chamran University of Ahvaz, Ahvaz, Iran

2 Associate Professor of the Iranian Research Institute of Plant Protection (IRIPP), Agricultural Research, Education and Extension Organization (AREEO) Tehran, Iran

3 Associate Professor, Department of Plant Protection, Faculty of Agriculture, Shahid Chamran University of Ahvaz, Ahvaz, Iran

4 Professor, Department of Plant Protection, Sari Agricultural Sciences and Natural Resources University, Sari, Iran

Abstract

Background and Objectives
Bacterial leaf streak of cereal was first reported from wheat and barley in the early twentieth century. This disease was observed in wheat and barley fields of Kerman province in 1987 and then isolated and identified from infected wheat and barley in different provinces of Iran, as Xanthomonas translucens pv. undulosa and X. t. pv. translucens respectively. In recent years, this disease became epidemic in Iran. For this reason, it was necessary to re-examine once again the different characteristics of the causal agents of this disease in different regions of Iran to determine if there is any possible genetic modification in these bacteria.
Materials and Methods
The phenotypic and genetic diversity of 12 bacterial strains isolated from wheat and barley (four isolates from wheat, six isolates from barley, one isolate from Phalaris sp. and one isolate from Lolium sp.) originated from Kerman, Kermanshah, and Fars provinces was studied. These isolates (selected from 67 isolates) were compared with three ancient Iranian standard strains (obtained from Unité de Phytopathologie, UCL, Louvain-la-Neuve, Belgium). The strains' pathogenicity test was performed on wheat and barley seedlings by spraying and injection methods. Then, the biochemical and physiological characteristics and SDS-PAGE profiles of the whole cell proteins of isolates were examined. Finally, using rep-PCR by BOX A1R, ERIC 1R, and ERIC 2 primers, the genetic diversity of isolates was performed.
Results
All strains were Gram, oxidase, and urease negative, and catalase positive. All strains produced xanthomonadin pigment. The strains could produce hydrogen sulfide from cystine and peptone. Negative results were obtained for arginine dihydrolase, urease, starch hydrolysis, nitrate reduction, acetoin and indole production, and methyl red reaction. All isolates produced acid from cellobiose, fructose, galactose, di-mannose, arabinose, xylose, trehalose, arabitol, but not from salicin, mannitol, melibiose, rhamnose, dulcitol, sorbitol, and mesoinositol. They utilized acetate, citrate, and lactate but not d-tartrate and inulin. Only barley isolates could utilize lactose and hydrolyze lecithin and starch. There are no discriminating bands among PAGE profiles of wheat, barley, and standard strains. Genomic fingerprinting of isolates was performed, and the data were analyzed using NTSYS V 2.2 software and the UPGMA cluster analysis method. All isolates of wheat, Phalaris sp., Lolium sp. and one isolate from barley along with UPB922 standard strain were formed one group consists of three subgroups. Isolates of this group could infect wheat and barley plants. The rest of barley isolates, which were pathogenic only on barley, separated from the first group at a similarity coefficient of 84% and formed the second group, including the standard strain UPB922. Furthermore, this group had three subgroups. This grouping was completely related to the host range from the isolates. Based on biochemical, physiological, pathogenicity (host range) characteristics of strains and their genetic diversity compared to the standard strains, the first group, and the second group isolates were identified as X. t. pv. undulosa and X. t. pv. translucens, respectively.
Discussion
It was found that the isolates are divided into two separate groups based on their host range. One group could contaminate wheat and barley plants, and another group infected only barley plants. Biochemical and physiological tests did not show much difference among the strains, but some tests were able to distinguish two pathovars, such as lactose utilization, the activity of lecithinase and lipase enzymes (hydrolysis of Tween 80), the tolerance of NaCl in the culture medium and the amount of slimes, produced on the nutrient agar medium containing glucose or sucrose. Rep-PCR tests could show the differences and variations within each pathovar. The grouping formed based on these tests showed a high correlation with the strain host range of pathovars. Using both BOX and ERIC primers showed that the genetic diversity of isolates was strongly related to the pathovars type and their host range. Since previous studies showed the same diversity in Iranian isolates, so it seems that there has not been a genetic change in the causal agent of this disease in Iran. The disease widespread occurred in recent years could be mainly due to environmental conditions and wheat and barley cultivars response to this disease.

Keywords


Adhikari, T.B., Hansen, J.M., Gurung, S., and Bonman, J.M. 2011. Identification of new sources of resistance in winter wheat to multiple strains of Xanthomonas translucens pv. undulosa. Plant Disease, 95: 582-588.
Alizadeh, A., and Rahimian, H. 1989. Bacterial Leaf Streak of Graminea in Iran. EPPO Bulletin, 19: 113-117.
Alizadeh, A., Arlat, M., Boucher, C.A., Barrault, G., and Sarrafi, A. 1997. RFLP Analysis of Iranian strains of Xanthomonas campestris from Cereals and Grasses. Plant Disease, 81: 31-35.
Alizadeh, A., Barrault, G., Sarrafi, A., Rahimian, H., and Albertini, L. 1995a. Identification of bacterial leaf streak of cereals by their phenotypic characteristics and host range in Iran. European Journal of Plant Pathology, 101: 225-229.
Alizadeh, A., Benetti, V., Sarrafi, A., Barrault, G., and Albertini, L. 1995b. Genetic analysis for partial resistance to an Iranian strain of bacterial leaf streak (X. campestris pv. hordei ) in barley. Plant Breeding, 113: 323-326.
Alizadeh, A., Dechamp-Guillaume, G., Sarrafi, A., Rahimian, H., and Barrault, G. 1996.Electrophoretic analysis of total and membrane protein of Xanthomonas campestris pathovars causing leaf streak of cereals and grasses in Iran. Journal of Phytopathology, 144: 97-101.
Alizadeh, A., Sarrafi, A., and Barrault, G. 1994b. Genetic variability for partial resistance to Xanthomonas campestris pv. hordei and pathogenicity variation of 13 pathovar's strains in barley. (Proccidings d'ANPP - 4. Conférence internationale sur les maladies des plantes (1994/12/06-08; Palais des congrès, Bordeaux, FRA), Tome I: 193-200.
Alizadeh, A., Sarrafi, A., and Barrault, G. 1995c. Genetic variability for partial resistance to Xanthomonas campestris pv. cerealis and pathogenicity variation of 13 pathovar's strains in wheat. Journal of Genetic and Breeding, 49: 309-312.
Azad, H., and Schaad, N.W. 1988. The relationship of Xanthomonas campestris pv. translucens to frost and the effect of frost on black chaff development in wheat. Phytopathology, 78: 95-100.
Charkhabi, N.F., Booher, N.J., Peng, Z., Wang, L., Rahimian, H., and ShamsBakhsh, M. 2017. Complete genome sequencing and targeted mutagenesis reveal virulence contributions of Tal2 and Tal4b of Xanthomonas translucens pv. undulosa ICMP11055 in bacterial leaf streak of wheat. Frontiers in Microbiology, 8: 1488.
Curland, R.D., Gao, L., Bull, C.T., Vinatzer, B.A., Dill-Macky, R., Van Eck, L., and Ishimaru, C.A. 2018. Genetic diversity and virulence of wheat and barley strains of Xanthomonas translucens from the Upper Midwestern United States. Phytopathology, 108: 443-453.
Fahy, P.C., and Hyward, A.C. 1983. Media and Methods for Isolation and Diagnostic Tests. In: Fahy, P.C. Persley (eds). Plant bacterial diseases: a diagnostic guide. Academic press, Sydney, Australia, 1983. Pp: 337-378.
Gardiner, D.M., Upadhyaya, N.M., Stiller, J., Ellis, J.G., Dodds, P.N., and Kazan, K. 2014. Genomic analysis of Xanthomonas translucens pathogenic on wheat and barley reveals cross‐kingdom gene transfer events and diverse protein delivery systems. PLOS ONE, 9: e84995.
Hersemann, L., Wibberg, D., Blom, J., Goesmann, A., Widmer, F., and Vorholter, F.J. 2017. Comparative genomics of host adaptive traits in Xanthomonas translucens pv. graminis. BMC Genomics, 18: 35.
Jaenicke, S., Bunk, B., Wibberg, D., Spröer, C., Hersemann, L., and Blom, J. 2016. Complete genome sequence of the barley pathogen Xanthomonas translucens pv. translucens DSM 1874T (ATCC 19319T). Genome Announcements, 4: e01334-e1416.
Khojasteh, M., Shah, S.M.A., Haq, F., Xu, X., Taghavi, S.M., Osdaghi, E., and Chen, G. 2020. Transcription activator-like effectors diversity in Iranian strains of Xanthomonas translucens. Phytopathology, 110: 758-767.
Khojasteh, M., Taghavi, S.M., Khodaygan, P., Hamzehzarghani, H., Chen, G., Bragard, C., Koebnik, R., and Osdaghi, E. 2019. Molecular typing reveals high genetic diversity of Xanthomonas translucens strains infecting small-grain cereals in Iran. Applied and Environmental Microbiology, 85: e01518-19.
Klement, Z., Rudolph, K., and Sands, D.C. (eds). 1990. Methods in Phytobacteriology. Akademiai Kiado, Budapest, 568 pp.
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685.
Langlois, P.A., Snelling, J., Hamilton, J.P., Bragard, C., Koebnik, R., Verdier, V., Triplett, L.R., Blom, J., Tisserat, N.A., and Leach, J.E. 2017. Characterization of the Xanthomonas translucens complex using draft genomes, comparative genomics, phylogenetic analysis, and diagnostic LAMP assays. Phytopathology, 107: 519-527.
Lelliot, R.A., and Stead, D.E. 1987. Methods for the Diagnosis ofBacterial Diseases ofPlants. Blackwell Scientific Publications. U. K. 215 pp.
Louws, F.J., Fulbright, D.W., Stephens, C.T., and de Bruijn, F.J. 1994. Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR. Applied and environmental microbiology, 60: 2286-2295.
Marefat, A., Ophel‐Keller, K., Scott, E., and Sedgley, M. 2006a. The use of ARMS PCR in detection and identification of xanthomonads associated with pistachio dieback in Australia. European Journal of Plant Pathology, 116: 57-68.
Marefat, A., Scott, E.S., Ophel‐Keller, K., and Sedgley, M. 2006b. Genetic, phenotypic and pathogenic diversity among xanthomonads isolated from pistachio (Pistacia vera) in Australia. Plant Pathology, 55: 639-649.
Mitkowski, N.A., Browning, M., Basu, C., Jordan, K., and Jackson, N. 2005. Pathogenicity of Xanthomonas translucens from annual bluegrass on golf course putting greens. Plant disease, 89: 469-473.
Mousavipour, S.H.B., and Najafipour, G. 2018. Genotypic characteristics of Xanthomonas arbaricola pv. juglandis, causal agent of walnut bacterial blight, based on Rep-PCR in Kohgiluye and Buyer- ahmad province. Plant Protection (Scientific Journal of Agriculture), 41: 73-87 (In Persian with English Abstract).
Ochiai, H., Horino, O., Miyajima, K., and Kaku, H. 2000. Genetic diversity of Xanthomonas oryzae pv. oryzae strains from Sri Lanka. Phytopathology, 90: 415-421.
Osdaghi, E., 2020. Xanthomonas translucens pv. translucens (bacterial leaf streak of barley). https://www.cabi.org/isc/datasheet/56978.
Peng, Z., Hu, Y., Xie, Z., Potnis, N., Akhunova, A., and Jones, J. 2016. Long read and single molecule DNA sequencing simplifies genome assembly and TAL effector gene analysis of Xanthomonas translucens. BMC Genomics, 17: 21.
Pesce, C., Bolot, S., Cunnac, S., Portier, P., Fischer‐Le Saux, M., and Jacques, M.A. 2015. High quality draft genome sequence of the Xanthomonas translucens pv. cerealis pathotype strain CFBP 2541. Genome Announcements, 3: e01574-e1614.
Pesce, C., Jacobs, J.M., Berthelot, E., Perret, M., Vancheva, T., and Bragard, C. 2017. Comparative genomics identifies a novel conserved protein, HpaT, in proteobacterial type III secretion systems that do not possess the putative translocon protein HrpF. Frontiers in Microbiology, 8: 1177.
Rademaker, J.L.W., Louws, F.J., Schultz, M.H., Rossbach, U., Vauterin, L., Swings, J., and De Bruijn, F.J. 2005. A comprehensive species to strain taxonomic framework for Xanthomonas. Phytopathology, 95: 1098-1111.
Rademaker, J.L.W., Norman, D.J., Forster, R.L., Louws, F.J., Schultz, M.H., and De Bruijn, F.J. 2006. Classification and identification of Xanthomonas translucens isolates, including those pathogenic to ornamental asparagus. Phytopathology, 96: 876-884.
Sapkota, S., Mergoum, M., and Liu, Z. 2020. The translucens group of Xanthomonas translucens: Complicated and important pathogens causing bacterial leaf streak on cereals. Molecular plant pathology, 21(3): 291-302.
Sapkota, S., Zhang, Q., Chittem, K., Mergoum, M., Xu, S.S., and Liu, Z. 2018. Evaluation of triticale accessions for resistance to wheat bacterial leaf streak caused by Xanthomonas translucens pv. undulosa. Plant Pathology, 67: 595-602.
Schaad, N.W., Joneas, J.B., and Chun, C. 2001. Laboratory Guide for identification of plant pathogenic bacteria. (Third edition) St Paul, Minnesota, APS press, 373 pp.
Sherafati, F., Khodaygan, P., Azadvar, M., Sedaghati, E., Saberi Riseh R., and Norolahi, K. 2014. Identification and comparative investigation into causal agent of citrus bacterial canker in Ilam province. Plant Protection (Scientific Journal of Agriculture), 36: 11-23 (In Persian with English Abstract).
Suslow, T.V., Schroth, M.N., and Isaka, M. 1982. Application of a rapid method for Gram differentiation of plant pathogenic and saprophytic bacteria without staining. Phytopathology, 72: 917-918.
Vauterin, L., Hoste, B., Kersters, K., and Swings, J. 1995. Reclassification of xanthomonas. International Journal of Systematic and Evolutionary Microbiology, 45: 472-489.
Versalovic, J., Koeuth, T., and Lupski, J.R., 1991. Distribution of repetitive DNA-sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Research, 19: 6823-31.
Versalovic, J., Schneider, M., de Bruijn, F.J., and Lupski, J.R. 1994. Genomic fingerprinting of bacteria using repetitive sequence based polymerase chain reaction. Methods in Molecular and Cellular Biology, 5: 25-40.
Wichmann, F., Vorholter, F.J., Hersemann, L., Widmer, F., Blom, J., and Niehaus, K. 2013. The noncanonical type III secretion system of Xanthomonas translucens pv. graminis is essential for forage grass infection. Molecular Plant Pathology, 14: 576-588.