بهینه‌سازی روش‌های مولکولی شناسایی نماتد عامل ریشه‌گره‌ای (Meloidogyne javanica)

نوع مقاله : مقالات پژوهشی

نویسندگان

1 رشته مهندسی کشاورزی علوم باغبانی، بیوتکنولوژی و ژنتیک مولکولی محصولات باغبانی، دانشگاه آزاد اسلامی واحد استهبان

2 بخش تحقیقات گیاه‌پزشکی، مرکز تحقیقات و آموزش کشاورزی و منابع طبیعی فارس، سازمان تحقیقات، آموزش و ترویج کشاورزی، زرقان، ایران

3 دانشگاه آزاد اسلامی واحد استهبان، گروه بیوتکنولوژی و ژنتیک مولکولی محصولات باغبانی، استهبان، ایران

چکیده

به منظور شناسایی سریع مراحل مختلف رشدی گونه Meloidogyne javanica که در ایران به عنوان یکی از خسارتزاترین نماتدهای گیاهی شناخته شده، تعداد 40 نمونه خاک و ریشه آلوده به نماتد ریشه‌گره‌ای از نقاط مختلف جالیزکاری استان فارس (داراب، جنت شهر، استهبان، نورآباد، کوار و شیراز) جمع‌آوری گردید. پس از خالص‌سازی به روش تک کیسه تخم روی ریشه گوجه فرنگی رقم Rutgers و شناسایی گونه M. javanica بر اساس ویژگی‌های ریخت‌شناسی (مورفولوژی) و ریخت‌سنجی ماده‌های بالغ و لارو سن دوم، DNA ژنومی کلیه مراحل رشدی از مرحله تخم، لارو سن دوم و ماده‌های بالغ نماتد، به سه روش ژانگ، سیلوا و لیوآ استخراج شد. DNA استخراج شده با کمک جفت آغازگرهای اختصاصی گونه Fjav/Rjav, Mj-MF/Mj-MR, Mj-DF/Mj-DR  تکثیر گردید. در کلیه مراحل رشدی گونه M. javanica یک قطعه 1650 جفت بازی توسط جفت آغازگر Mj-DF/Mj-DR ، یک قطعه 517 جفت بازی توسط جفت آغازگر Mj-MF/Mj-MR و یک قطعه 670 جفت بازی توسط جفت آغازگر Fjav/Rjav تکثیر شد ولی این قطعات با جفت آغازگر MI-F و MI-R، مربوط به نماتد M. incognita و آب که به عنوان کنترل منفی در آزمایش استفاده شده بودند تکثیر نشدند. لذا به نظر می‌رسد کاربرد این جفت آغازگرها در قیاس با خصوصیات ریخت‌شناسی در مراحل مختلف رشدی نماتد به تشخیص سریع تر گونه M. javanica منجر می‌شود. در بهینه‌سازی PCR برای تشخیص نماتد M. javanica بهترین دما برای آغازگرهای Mj-MF و Mj-MR، 56 درجه سانتی‌گراد برای Mj-DF و Mj-DR، 50 درجه سانتی‌گراد و برای Fjav و Rjav، 54 درجه سانتی‌گراد تعیین گردید، میزان DNA الگو حاصل از روش‌های مختلف استخراج در آزمون PCR در خصوص ماده کامل و کیسه تخم از یک میکرولیتر به دو میکرولیتر و لارو سن دوم از دو میکرو لیتر به چهار میکرولیتر بهترین نتیجه حاصل شد. در خصوص MgCl2 با افزایش میزان آن از 5/1 میلی‌مولار به 2 میلی‌مولار در واکنش 25 میکرولیتری PCR بهترین نتیجه حاصل گردید. بهترین روش استخراج که قادر به ردیابی تک لارو سن دوم نماتد بود روش لیوآ در نظر گرفته شد. بهترین آغازگرها که قادر به ردیابی نماتد  M. javanicaدر تمام مراحل رشدی بودند، جفت آغازگرMj-MF/Mj-MR و Fjav/Rjav بودند.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Improving Molecular Diagnosis of Root Knot Nematode(Meloidogyne javanica)

نویسندگان [English]

  • R. Keshavarz Kuhjerdi 1
  • A. Pak Niat Jahromi 2
  • M. Honarvar 3
  • M. Pak Niat jahromi 2
1 Agricultural Engineering Horticultural Sciences, Biotechnology and Molecular Genetics of Horticultural Products, Estahban Branch, Islamic Azad University, Estahban, Iran
2 Plant Protection Research Department, Fars Agricultural and Natural Resources and Education Center, Agricultural Research, Education and Extension Organization (AREEO), Zarghan, Iran
3 Department of Biotechnology and Molecular Genetics of Horticultural Products, Estahban Branch, Islamic Azad University, Estahban, Iran
چکیده [English]

Introduction: Root-knot nematodes are the most common and destructive plant-parasitic nematode group worldwide and adversely influence many economically important crops, ornamental plants, and fruit trees. They are obligate parasites of the roots of many plant species, including herbaceous and woody plants. Symptoms associated with Root-knot nematodes infection include root galls, shoot chlorosis, stunted growth, nutrient deficiencies, and secondary infections by other pathogens. A high level of damage can lead to serious crop loss. Due to the considerable morphological similarity between species of Meloidogyne and high intraspecies variation, it is difficult to differentiate species from each other based on the morphology of their perineal patterns alone. Molecular techniques are used for detection, species differentiation, and phylogenetic analysis of species. Polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail. The objective of this study was to improve molecular diagnosis of root knot nematode in vegetable crops in Fars province to enhance our diagnostic knowledge and allow species identification of root knot nematode through PCR by species-specific primers.
Materials and Methods: In 2012, in order to identify the different stages of growth of Meloidogyne javanica species, 40 soil and root samples infected with the nematode were collected from different cultivation sites of Fars province (Darab, Jannatshahr, Estahban, Nurabad, Qavar, and Shiraz). The roots of plants were flushed with water to remove soil and then washed with soap for 10 min. To obtain the pure population of each sample, a large egg bag containing more eggs was selected. The isolated egg bag was placed adjacent to Rutgers tomato transplant. After 70-60 days, the roots were removed from the pot and prepared to identify species of the nematode. After purification of nematodes using single egg method on Rutgers tomato root and diagnosis of them on the morphological basis, the DNA was extracted at different growth stage of nematode using three optimized methods of Zhang, 1998, Silva, 2000, and Liao, 2005. PCR products (5 µl) were separated on standard agarose gels. A DNA ladder (Sinagen 100bp-3000bp) was used to determine the molecular sizes of the bands. Band patterns were photographed under UV light using the GBOX document gel system. The PCR was optimized by varying the reaction components and cycling conditions. The annealing temperature was optimized separately for each primer based on the manufacturer's Tm and formula. The roots were flushed with water to remove soil and then washed with a 0.52% NaClO soap for 10 min. Individual root knots were obtained after three to five washes. Furthermore, DNA extraction was performed using four methods.
Results and Discussion: The root-knot nematodes were recovered from the vegetable crop soil samples. Species identification in this study was based on the PCR by species-specific primers. Specific primers of Fjav/Rjav, Mj-MF/Mj-MR and Mj-DF/Mj-DR could detect M. javanica with 670 bp, 517bp and 1650bp, respectively, proving their usefulness for PCR on root-knot nematodes. No band was detected using specific primer of M. incognita (MI-F/R primers) from the samples in PCR. During optimizing PCR method, it was revealed that the best annealing temperature is 54ºC for Mj-MF and Mj-MR primers, 56ºC for Mj-DF and Mj-DR primers, and 50ºC and for Fjav and Rjav primers. Adding the DNA extracted template from 1µ to 2µ for single female and single egg cist and from 2µ to 4µ for single juvenile made the best result for detection of root-knot nematode based on PCR. Changing the MgCl2 concentration from 1.5 mmol to 2 mmol in a 25µl PCR reaction made the best PCR result. The Liao optimized method was the best DNA extraction for monitoring M. javanica. Fjav/Rjav and Mj-MF/Mj-MR primers were the best primers in detecting M. javanica.
Conclusion: The results of this study showed a remarkable ability of DNA extraction methods, specific primers and the cases optimized in this study in identifying root knot nematode. Identifying M. javanica species based on morphological methods is time consuming and difficult, and requires high skills and also needs to be examined for a particular stage of nematode growth. In addition, identifying of this species based on biochemical methods is time consuming, affected by environmental conditions, requires relatively large amounts of living organism and a particular stage of the nematode's life cycle. Given these difficulties, DNA extraction methods, specific primers and the cases optimized in this study can be used to identify M. javanica species. However, as M. javanica and M. incognita primer pairs were not detected for a sample taken from Shiraz, more researches are needed to be carried out on this case.

کلیدواژه‌ها [English]

  • PCR optimizing
  • Root-knot nematode
  • Specific primersM. javanica
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