بررسی تنوع فنوتیپی و ژنتیکی پاتووارهای xanthomonas translucens، عامل بیماری باکتریایی نواری گندم و جو در بخش‌هایی از ایران

تعداد 12 جدایه ازمیان 67 جدایۀ عامل بیماری باکتریایی نواری غلات، از گندم، جو، فالاریس و چچم از مناطق مختلف استان ‌های کرمان، کرمانشاه و فارس تهیه و خصوصیات باکتری ‌شناسی، دامنۀ میزبانی، ویژگی ‌های مولکولی و ژنتیکی آنها مورد بررسی قرار گرفت. کلیۀ جدایه‌ ها گرم منفی، اکسیداز و اوره‌ آز منفی و کاتالاز مثبت، دارای رنگ‌دانۀ زانتومونادین و قادر به تولید هیدروژن سولفوره از سیستئین و پپتون بودند. همۀ جدایه ‌ها از قندهای سلوبیوز، فروکتوز، گالاکتوز، دی ‌مانوز، آرابینوز، زایلوز، ترهالوز، آرابیتول و نمک‌ های استات، سیترات و لاکتات استفاده نمودند، اما از سالیسین، مانیتول، ملی‌ بیوز، رامنوز، دی‌ تارتارات، اینولین، آدونیتول، دلسیتول، سوربیتول و مزواینوزیتول استفاده نکردند. تنها جدایه ‌های جو قادر به استفاده از مالتوز و هیدرولیز لسیتین و نشاسته بودند. جدایه ‌ها، ازنظر مقایسۀ پروتئین محلول، دارای الگوی باندی مشابه‌ای بودند. انگشت نگاری ژنومی جدایه ‌ها به‌روش rep-pcr با استفاده از دو آغازگر eric و box انجام و داده ‌های به‌دست آمده با استفاده از نرم‌ افزار ntsyspc.2.2 و روش تجزیۀ خوشه‏‌ای upgma آنالیز شدند. نتایج حاصل نشان داد همۀ جدایه‎ های گندم، فالاریس، چچم و یک جدایه از جو، به‌ همراه جدایۀ استاندارد upb922 در یک گروه و سه زیرگروه و بقیۀ جدایه ‌های جو، که تنها روی جو بیماری‌زا بودند، به‌ همراه جدایۀ استاندارد upb922 با ضریب تشابه 84 درصد از گروه اول جدا و گروه دوم (با سه زیرگروه) را تشکیل دادند. براساس ویژگی ‌های بیوشیمیایی، فیزیولوژیکی، بیماری‌زایی (دامنۀ میزبانی)، و تنوع ژنتیکی، جدایه ‌های گروه اول به‏‌عنوان پاتووار x. translucens pv. undulosa و جدایه‌ های گروه دوم به ‌عنوان پاتووار x. translucens pv. translucens شناسایی شدند.

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

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 reexamine 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, LouvainlaNeuve, Belgium). The strains’ pathogenicity test was performed on wheat and barley seedlings by spraying and injection methods. Then, the biochemical and physiological characteristics and SDSPAGE 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, dimannose, arabinose, xylose, trehalose, arabitol, but not from salicin, mannitol, melibiose, rhamnose, dulcitol, sorbitol, and mesoinositol. They utilized acetate, citrate, and lactate but not dtartrate 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. RepPCR 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.