نویسندگان

نویسنده

چکیده

توسکا یکی از مهم‌ترین گونه‌های درختی جنگل‌های شمال کشور بوده و از نظر اقتصادی به عنوان چهارمین درخت تجاری محسوب می‌شود. یک بیماری لکه برگی ناشی از گونه‌ای زانتوموناس در توسکا ییلاقی (Alnus subcordata subsp. Subcordata) در چند منطقه جنگلی استان مازندران گزارش شده است (Rahimian et al. 1383. 16th Iran. Plant Protec. Cong.,439). براساس یک بررسی ژنومی که در سال‌های اخیر انجام شده بود باکتری عامل بیماری بالاترین شباهت را به Xanthomonas arboricola داشت (Rahimian et al. 1387. 18th Iran. Plant Protec. Cong., 428). بررسی محدودی در زمینه همسانی جدایه‌ها و پراکندگی بیماری(Rahimian et al. 1383. 16th Iran. Plant Protec. Cong., 439) انجام گردید ولی اطلاع دقیقی از تنوع ژنتیکی جدایه‌های مناطق مختلف در دست نیست. بررسی حاضر به منظور ارزیابی همسانی یا تنوع ژنتیکی جدایه‌های به‌دست آمده از مناطق مختلف استان‌های مازندران و گلستان انجام گرفت. از نظر ویژگی‌های فنوتیپی جدایه بسیار شبیه هم بوده و فقط در مصرف -Lسرین، لوسین، دکسترین، کوئینات، تایروزین به عنوان منبع کربن اختلاف نشان دادند. تنوع ژنتیکی جمعیت باکتری عامل لکه زاویه‌ای توسکا با استفاده از BOX-PCR و REP-PCR انجام شد. DNA ژنومی جدایه‌ها به روش لیز قلیایی استخراج و PCR در شرایط توصیه شده ورسالوویچ و همکاران (Versalovich et al. 1991. Nucleic Acids Res, 24: 6823 -6831) ولی با کاهش زمان واسرشت (Denaturing) انجام شد. محصول PCR در ژل آگارز 2 درصد الکتروفورز و ژل با اتیدیوم بروماید رنگ آمیزی گردید. از ضریب تشابه جاکارد برای تعیین تشابه جدایه‌ها و از روش UPGAMA و نرم افزار NTSYS برای آنالیز خوشه‌ای استفاده شد. آنالیز خوشه‌ای نتایج به‌دست آمده با آغازگر BOXAIR نشان داد که در سطح 80 درصد جدایه‌ها به 24 گروه تقسیم شدند. این در حالی است که جدایه‌های توسکا با جدایه استاندارد Xanthomonas arboricola pv. coryli نیز همین میزان شباهت را نشان دادند. در سطح 63 درصد، جدایه‌های توسکا به 15 گروه تقسیم شده ولی با همین میزان شباهت با Xanthomonas arboricola pv. pruniهم خوشه بودند. در سطح 40 درصد هم ضمن تقسیم شدن جدایه‌ها به 7 گروه، همسانی مشابهی را با جدایه مرجع Xanthomonas arboricola pv. juglandisنشان دادند. با توجه به این یافته‌‌ها می‌توان نتیجه گرفت که جدایه‌های عامل لکه زاویه‌ای توسکا تنوع زیادی داشته و در عین حال BOX-PCR توانایی متمایز ساختن پاتووارهای متعلق به گونه Xanthomonas arboricola را ندارد. نتایج به‌دست آمده با آغازگر REP1R,REP2I نشان داد که در سطح 78 درصد جدایه‌ها به 14 گروه تقسیم شدند. در حالی که این جدایه‌ها با پاتوارهای X. arboricola pv. juglandisو X. arboricola pv. coryliنیز همین میزان شباهت را نشان دادند. در سطح تشابه 57 درصد، جدایه‌ها به 7 گروه تقسیم شده و در همین میزان شباهت با X. arboricola pv. pruni هم خوشه بودند. داده‌ها نشان می‌دهند جدایه‌های توسکا دارای تنوع زیادی بوده و REP-PCR کارایی بهتری برای گروه‌بندی جدایه‌های عامل لکه زاویه‌ای توسکا دارد.
 




 
 

 
 
Alder (Alnus subcordata subsp. Subcordata) is one of the most important tree species in northern forests of Iran and is economically considered as the fourth commercial tree. A leaf spot disease of alder caused by Xanthomonas sp. has been reported from several forest regions of Mazandaran province (Rahimian et al. 1383. 16th Iran. Plant Protec. Cong., 439). According to a recent genomic study, the bacterial agent of this disease has the highest similarity to Xanthomonas arboricola (Rahimian et al. 1387. 18th Iran. Plant Protec. Cong., 428). Though limited studies have been performed about the homology of isolates and the distribution of the disease (Rahimian et al. 1383. 16th Iran, Plant Protec. Cong., 439), detailed information is not available about the genetic diversity of isolates from different regions. This study was conducted to evaluate the genetic similarity or diversity among the isolates obtained from different regions of Mazandaran and Golestan provinces. Phenotypic characteristics of isolates were very similar and they were different only in utilization of carbon sources including L- Serin, Leucine Dextrin, Quinate, Tyrosine. The population genetic diversity analysis of the bacterial pathogen ofalder angular leaf spotwas performed using BOX-PCR and REP-PCR. Genomic DNA was extracted from isolates using alkaline lysis, and PCR was performed as recommended by Versalovich et al., but by reducing the deneturing time (Versalovich et al. 1991. Nucleic Acids Res, 24: 6823 -6831). PCR products were electrophoresed in 2 percent agarose gel and the gel was stained with ethidium bromide. Similarity, matrix of the isolates was obtained using Jaccard's coefficient and cluster analysis by employing the UPGAMA method and NTSYS software. Cluster analysis by BOXAIR primer showed that the isolates were divided into 24 groups at 80 percent level. However, the isolates of alder showed the same similarity with standard isolate Xanthomonas arboricola pv. coryli. At 63 percent level, alder isolates were divided into 15 groups, but with the same amount of similarity they were clustered with Xanthomonas arboricola pv. pruni. At 40 percent level, alder isolates were divided into seven groups and showed similar homology with reference isolate of Xanthomonas arboricola pv. juglandis. According to these findings, it can be concluded that isolates inciting alder angular leaf spot have high variability, while BOX-PCR was unable to differentiate X. arboricola pathovars. Results obtained with REP1R and REP2I primers showed that at 78 percent level, isolates were divided into 14 groups. In contrast, the isolates showed the same level of similarity with X. arboricola pv. juglandis and X. arboricola pv. coryli pathovars. At 57 percent similarity level, the isolates were divided into seven groups and were clustered with X. arboricola pv. pruni with the same amount of similarity. The data show that alder isolates have high variation and REP-PCR has a better efficiency for grouping causal agent of angular leaf spot of alder isolates.
 

عنوان مقاله [English]

Assessment of the genetic diversity of Xanthomonas the causal agent of alder angular spot in Mazandaran and Golestan provinces using BOX-PCR and REP-PCR

نویسندگان [English]

  • R. EBRAHIMI
  • H. RAHIMIAN
  • V. BABAIZAD
  • N. MOAREFZADE

چکیده [English]

Alder (Alnus subcordata subsp. Subcordata) is one of the most important tree species in northern forests of Iran and is economically considered as the fourth commercial tree. A leaf spot disease of alder caused by Xanthomonas sp. has been reported from several forest regions of Mazandaran province (Rahimian et al. 1383. 16th Iran. Plant Protec. Cong., 439). According to a recent genomic study, the bacterial agent of this disease has the highest similarity to Xanthomonas arboricola (Rahimian et al. 1387. 18th Iran. Plant Protec. Cong., 428). Though limited studies have been performed about the homology of isolates and the distribution of the disease (Rahimian et al. 1383. 16th Iran, Plant Protec. Cong., 439), detailed information is not available about the genetic diversity of isolates from different regions. This study was conducted to evaluate the genetic similarity or diversity among the isolates obtained from different regions of Mazandaran and Golestan provinces. Phenotypic characteristics of isolates were very similar and they were different only in utilization of carbon sources including L- Serin, Leucine Dextrin, Quinate, Tyrosine. The population genetic diversity analysis of the bacterial pathogen ofalder angular leaf spotwas performed using BOX-PCR and REP-PCR. Genomic DNA was extracted from isolates using alkaline lysis, and PCR was performed as recommended by Versalovich et al., but by reducing the deneturing time (Versalovich et al. 1991. Nucleic Acids Res, 24: 6823 -6831). PCR products were electrophoresed in 2 percent agarose gel and the gel was stained with ethidium bromide. Similarity, matrix of the isolates was obtained using Jaccard's coefficient and cluster analysis by employing the UPGAMA method and NTSYS software. Cluster analysis by BOXAIR primer showed that the isolates were divided into 24 groups at 80 percent level. However, the isolates of alder showed the same similarity with standard isolate Xanthomonas arboricola pv. coryli. At 63 percent level, alder isolates were divided into 15 groups, but with the same amount of similarity they were clustered with Xanthomonas arboricola pv. pruni. At 40 percent level, alder isolates were divided into seven groups and showed similar homology with reference isolate of Xanthomonas arboricola pv. juglandis. According to these findings, it can be concluded that isolates inciting alder angular leaf spot have high variability, while BOX-PCR was unable to differentiate X. arboricola pathovars. Results obtained with REP1R and REP2I primers showed that at 78 percent level, isolates were divided into 14 groups. In contrast, the isolates showed the same level of similarity with X. arboricola pv. juglandis and X. arboricola pv. coryli pathovars. At 57 percent similarity level, the isolates were divided into seven groups and were clustered with X. arboricola pv. pruni with the same amount of similarity. The data show that alder isolates have high variation and REP-PCR has a better efficiency for grouping causal agent of angular leaf spot of alder isolates.