بررسي خصوصيات و تنوع ژنتيكي Xanthomonas translucens عامل بيماري باكتريايي نواري گندم در استان كرمانشاه

Characterization and genetic diversity of Xanthomonas translucens, the causal agent of bacterial stripe of wheat in Kermanshah province, Iran

ﯿﻤﺎري ﺑﺎﮐﺘﺮﯾﺎﯾﯽ ﻧﻮاري ﯾﮑﯽ از ﻣﻬﻢﺗﺮﯾﻦ ﺑﯿﻤﺎريﻫﺎي ﮔﻨﺪم اﺳﺖ ﮐﻪ در ﺻﻮرت ﻣﻬﯿﺎ ﺑﻮدن ﺷﺮاﯾﻂ ﻣﺤﯿﻄﯽ ﺧﺴﺎرت ﻗﺎﺑﻞﺗﻮﺟﻬﯽ ﺑﻪ ﻣﯿﺰﺑﺎن ﺧﻮد در ﻣﻨﺎﻃﻖ ﻣﺨﺘﻠﻒ دﻧﯿﺎ وارد ﻣﯽﻧﻤﺎﯾﺪ. اﯾﻦ ﺑﯿﻤﺎري در ﺳﺎلﻫﺎي اﺧﯿﺮ در اﺳﺘﺎن ﮐﺮﻣﺎﻧﺸﺎه ﺷﯿﻮع ﭘﯿﺪاﮐﺮده و ﻣﻮﺟﺐ ﺧﺴﺎرت ﻗﺎﺑﻞﺗﻮﺟﻪ روي ﮔﻨﺪم ﺷﺪه اﺳﺖ. ﺑﻪﻣﻨﻈﻮر ﺑﺮرﺳﯽ ﺧﺼﻮﺻﯿﺎت ﻋﺎﻣﻞ ﺑﯿﻤﺎري، ﻃﯽ ﺳﺎلﻫﺎي 1398 و 1399 از ﺑﺬور و ﺑﺮگﻫﺎي داراي ﻋﻼﺋﻢ ﺑﯿﻤﺎري در ﻣﻨﺎﻃﻖ ﻋﻤﺪه ﮔﻨﺪمﮐﺎري اﺳﺘﺎن ﻧﻤﻮﻧﻪﺑﺮداري اﻧﺠﺎم ﺷﺪ. ﺟﺪاﺳﺎزي ﺑﺎﮐﺘﺮيﻫﺎ از ﺑﺎﻓﺖﻫﺎي ﮔﯿﺎﻫﯽ آﻟﻮده ﺑﻪ روش ﻣﻌﻤﻮل ﺗﻬﯿﻪ ﺳﻮﺳﭙﺎﻧﺴﯿﻮن از ﺑﺮگﻫﺎ و ﺑﺬور ﺧﺮدﺷﺪه در آب ﻣﻘﻄﺮ ﺳﺘﺮون و ﮐﺸﺖ روي ﻣﺤﯿﻂ ﮐﺸﺖ آﮔﺎر ﻏﺬاﯾﯽ )NA( ﺑﻪﺻﻮرت ﺧﻄﯽ اﻧﺠﺎم ﺷﺪ. ﭘﺲ از رﺷﺪ، 140 ﭘﺮﮔﻨﻪ زردرﻧﮓ ﺷﻔﺎف، ﺑﺮاق و ﮔﺮد ﺧﺎﻟﺺﺳﺎزي ﺷﺪﻧﺪ. ﺑﺮاي ﺗﻌﯿﯿﻦ ﺧﺼﻮﺻﯿﺎت ﻓﻨﻮﺗﯿﭙﯽ و ﺑﯿﻮﺷﯿﻤﯿﺎﯾﯽ ﺟﺪاﯾﻪﻫﺎ آزﻣﻮنﻫﺎي ﺑﺎﮐﺘﺮيﺷﻨﺎﺳﯽ ﻣﺮﺳﻮم اﻧﺠﺎم ﺷﺪ. ﺑﺮاي اﺛﺒﺎت ﺑﯿﻤﺎريزا ﺑﻮدن ﺟﺪاﯾﻪﻫﺎ و ﻧﯿﺰ ﺗﻌﯿﯿﻦ ﭘﺎﺗﻮوار ﻋﺎﻣﻞ ﺑﯿﻤﺎري، آزﻣﻮن ﺑﯿﻤﺎريزاﯾﯽ در ﮔﻠﺨﺎﻧﻪ روي ﭼﻬﺎر ﻣﯿﺰﺑﺎن اﺻﻠﯽ ﺷﺎﻣﻞ ﮔﻨﺪم، ﺟﻮ، ﭼﺎودار و ﺗﺮﯾﺘﯿﮑﺎﻟﻪ اﻧﺠﺎم ﺷﺪ. ﺑﺮاي ﺷﻨﺎﺳﺎﯾﯽ دﻗﯿﻖﺗﺮ ﻋﺎﻣﻞ ﺑﯿﻤﺎري، ﻧﺎﺣﯿﻪ ﺣﻔﺎﻇﺖﺷﺪه 16S rRNA در ﺟﺪاﯾﻪﻫﺎي ﻣﻨﺘﺨﺐ، ﺑﺎ آزﻣﻮن PCR و اﺳﺘﻔﺎده از آﻏﺎزﮔﺮﻫﺎي 27F و 1492R ﺗﮑﺜﯿﺮ و ﺗﻌﯿﯿﻦ ﺗﻮاﻟﯽ ﮔﺮدﯾﺪ. ﺗﻨﻮع ژﻧﺘﯿﮑﯽ ﺑﯿﻦ ﺟﺪاﯾﻪﻫﺎ ﺑﺎ اﺳﺘﻔﺎده از روش rep-PCR و ﺑﺎ آﻏﺎزﮔﺮﻫﺎي BOX و ERIC ﺑﺮرﺳﯽ ﺷﺪ. ﻃﺒﻖ ﻧﺘﺎﯾﺞ آزﻣﻮنﻫﺎي ﻓﻨﻮﺗﯿﭙﯽ و ﺑﯿﻮﺷﯿﻤﯿﺎﯾﯽ، ﺟﺪاﯾﻪﻫﺎ ﺑﻪ ﮔﻮﻧﻪ X. translucens ﺗﻌﻠﻖ داﺷﺘﻨﺪ. ﺑﺮ اﺳﺎس ﻧﺘﺎﯾﺞ آزﻣﻮن ﺑﯿﻤﺎريزاﯾﯽ، ﺟﺪاﯾﻪﻫﺎ در دو ﮔﺮوه ﻗﺮار ﮔﺮﻓﺘﻨﺪ. اﻋﻀﺎي ﮔﺮوه اول ﻓﻘﻂ روي ﮔﻨﺪم ﺑﯿﻤﺎريزا ﺑﻮدﻧﺪ و ﻋﻼﺋﻢ ﺧﻔﯿﻔﯽ در ﺑﺮگﻫﺎي ﺗﺮﯾﺘﯿﮑﺎﻟﻪ اﯾﺠﺎد ﮐﺮدﻧﺪ اﻣﺎ اﻋﻀﺎي ﮔﺮوه دوم در ﻫﺮ ﭼﻬﺎر ﻣﯿﺰﺑﺎن اﯾﺠﺎد ﺑﯿﻤﺎري ﮐﺮدﻧﺪ، ﺑﻪاﯾﻦﺗﺮﺗﯿﺐ اﻋﻀﺎي ﮔﺮوه اول ﺑﻪ ﭘﺎﺗﻮوار undulosa و اﻋﻀﺎي ﮔﺮوه دوم ﺑﻪ ﭘﺎﺗﻮوار cerealis ﺗﻌﻠﻖ داﺷﺘﻨﺪ. ﻧﺘﺎﯾﺞ ﺗﻌﯿﯿﻦ ﺗﻮاﻟﯽ 16S rRNA، ﺷﺒﺎﻫﺖ ﻧﻮﮐﻠﺌﻮﺗﯿﺪي 100% ﺟﺪاﯾﻪﻫﺎي ﻣﻨﺘﺨﺐ را ﺑﺎ ﮔﻮﻧﻪي MK356430 - CP043500) X. translucens( ﻣﻮﺟﻮد در ﭘﺎﯾﮕﺎه ژنﺑﺎﻧﮏ ﻧﺸﺎن داد. ﺑﺮرﺳﯽ ﺗﻨﻮع ژﻧﺘﯿﮑﯽ ﻧﺸﺎن داد ﺑﯿﻦ ﺟﺪاﯾﻪﻫﺎي ﻣﻨﺎﻃﻖ ﻣﺨﺘﻠﻒ اﺳﺘﺎن ﺗﻨﻮع زﯾﺎدي وﺟﻮد دارد، اﻣﺎ ارﺗﺒﺎﻃﯽ ﺑﯿﻦ اﯾﻦ ﺗﻨﻮع و ﻣﻨﺎﻃﻖ ﻧﻤﻮﻧﻪﺑﺮداري دﯾﺪه ﻧﺸﺪ. ﺿﻤﻦ اﯾﻨﮑﻪ rep-PCR ﻧﺘﻮاﻧﺴﺖ ﭘﺎﺗﻮوارﻫﺎي ﺷﻨﺎﺳﺎﯾﯽﺷﺪه را از ﯾﮑﺪﯾﮕﺮ ﺗﻔﮑﯿﮏ ﻧﻤﺎﯾﺪ

Background and Objectives Bacterial stripe caused by Xanthomonas translucens is one of the most important diseases of wheat worldwide. The disease was first reported on barley in America in 1917. In Iran, it was reported for the first time on wheat and barley in Kerman province in 1987. Based on the standards for naming pathovars, the currently accepted pathovars as pathogens infecting cereals include pv. cerealis, pv. translucens, pv. undulosa, and pv. secalis. Following climate changes, especially heavy rainfalls in recent years, and probably the transmission of infected seeds, the disease has shown significant outbreaks and damages on wheat in Kermanshah province, Iran. The present study was performed to (I) characterize the pathogen in Kermanshah province, (II) determine pathogen distribution, and (III) assess the genetic diversity of the strains. Materials and Methods Wheat seeds and leaves showing symptoms of the disease were collected from major wheat-growing areas in Kermanshah province during 2019 and 2020. In the laboratory, samples were rinsed with tap water and were macerated into a few drops of sterile distilled water. Two loops full of the resulting suspension were streaked on the nutrient agar medium. Plates were incubated at 25°C and pale yellowish colonies were purified. Physiological and biochemical tests were performed to identify and characterize 140 isolates. To determine the pathovar(s) of the pathogen, the pathogenicity of the strains was tested on wheat, barley, rye, and triticale in the greenhouse. Nearly complete 16S rRNA sequences were determined for two strains representatives of two pathovars distinguished by pathogenicity test using 27F and 1492R primers. Genetic diversity among the isolates was evaluated in rep-PCR utilizing BOX and ERIC primers. Results Based on physiological and biochemical tests, Xanthomonas strains from wheat in Kermanshah province were identified as X. translucens. Pathogenicity tests revealed two groups among the strains. Members of the first group induced disease symptoms on wheat and triticale exclusively and were identified as pv. undulosa, while the strains belonging to the second group infected wheat, barley, rye, and triticale, and were identified as pv. cerealis. The rep-PCR using ERIC and BOX primers demonstrated genetic variation among the strains. However, there was no significant difference between the results obtained by ERIC- and BOX-PCR. Nearly complete 16S rRNA sequences were determined as the representative strains of two pathovars distinguished by pathogenicity test and based on the comparison of sequences with the GenBank database. The two strains matched X. translucens with 100% similarity. The nucleotide sequences of the pathogen strains were deposited in the GenBank database under the accession numbers MW173461 and MW193070. Discussion According to the findings of this study, X. translucens strains collected from wheat in Kermanshah province belonged to pathovars undulosa and cerealis. Considering the wide host range of these pathovars, the disease could be found on other cereals and grasses belonging to the Poaceae in the province and this subject must be taken into consideration in future studies. Although rep-PCR showed significant diversity among the strains, there was no correlation between the rep-PCR groups and the pathovars identified in the pathogenicity test or the geographic origin of the strains. Biochemical and physiological tests, as well as rep-PCR and 16S rRNA sequencing, could not distinguish the two pathovars. This confirms that pathogenicity tests and host range are still the main methods for determining pathogen pathovar(s)