سبب‌شناسی و انتقال فیتوپلاسمای همراه با فیلودی لوبیا چشم بلبلی از استان آذربایجان شرقی

نوع مقاله : مقالات پژوهشی

نویسندگان

دانشگاه فردوسی مشهد

چکیده

در بررسی‌های انجام شده از مزارع لوبیا چشم بلبلی (Vigna unguiculata) استان آذربایجان شرقی در تابستان 1395، در برخی از بوته‌ها علائم ریز برگی، خشبی شدن برگ‌ها، فیلودی گل‌ها، ضخیم شدن و کتابی شدن ساقه، ازدیاد جوانه و کوتولگی بوته مشاهده شد. در گلخانه عامل بیماری به وسیله سس به پروانش انتقال داده شد و در پروانش مایه‌زنی شده علائم گل سبزی، برگسانی و کاهش فاصله میانگره‌ها ظاهر گردید. با استفاده از آغازگرهای عمومیP1/P7 و آغازگرهای آشیانه‌ای R16F2n/R16R2 در واکنش زنجیره‌ای پلی مراز (پی سی آر) از کلیه نمونه‌های دارای علائم قطعات 1830 جفت باز در پی سی آر عمومی و 1245 جفت باز در پی سی آر آشیانه‌ای تکثیر شد. در حالی‌که در نمونه‌های گیاهان سالم، قطعه‌ای تکثیر نگردید. محصول واکنش زنجیره‌ای پلی مراز پس از همسانه‌سازی، تعیین ترادف گردید. جستجو با برنامه Blast، آنالیز فیلوژنتیکی و بررسی چند شکلی طولی قطعات برشی (RFLP) محصول پی سی آر دو مرحله‌ای، با آنزیم‌های برشی HinfI, RsaI, AluI, TaqI, HpaII و مقایسه نقوش حاصله نشان داد که فیتوپلاسمای همراه با فیلودی لوبیا چشم بلبلی بیشترین نزدیکی را با اعضای گروه افژولش شبدر (Clover proliferation group, 16SrVI) دارد. همچنین آنالیز توالی‌ها با استفاده از نرم‌افزار iPhyClassifier و انجام RFLP مجازی با استفاده از 17 آنزیم برشی، علاوه بر تأیید نتایج آنالیزهای RFLP فیزیکی و تبارزایی نشان داد که فیتوپلاسمای مورد بررسی در گروه 16SrVI-A (با ضریب تشابه 1) قرار می‌گیرد.

کلیدواژه‌ها


عنوان مقاله [English]

Etiology and Transmission of Cowpea Phyllody Associated Phytoplasma in East Azarbaijan Province of Iran

نویسندگان [English]

  • Sara Gharouni Kardani
  • Elham Jamshidi
Ferdowsi university of Mashhad
چکیده [English]

Introduction: Phytoplasmas were first discovered in 1967 and named mycoplasma-like organisms or MLOs. They are wall-less prokaryotes of the class Mollicutes causing diseases in many economically important plant species. Phytoplasmas are transmitted by phloem-feeding leafhoppers and planthoppers. Plants infected by phytoplasmas exhibit a variety of symptoms suggesting profound disturbances to the normal plant physiology. Restriction fragment length polymorphism (RFLP) and sequence analyses of rDNA have been used for the taxonomic characterization and phylogenetic analysis of phytoplasmas. Because of the high economic losses, in particular those of cultivated crops, annuals and high-value vegetables, detection of phytoplasmas is very important. Cowpea (Vigna unguiculata) is an important vegetable crop cultivated in Iran. Progressive growth abnormalities and phyllody symptoms with high economic loss have been observed in cowpea fields of East Azarbaijan province. Causal agent of the disease is, however, unclear. This study was, therefore, carried out to identify pathogen associated with cowpea phyllody in East Azarbaijan province of Iran.
Materials and Methods: Infected cowpea plants were collected from fields in East Azarbaijan province of Iran in summer of 2016. The disease agent was transmitted by dodder to periwinkle plants under controlled greenhouse conditions. Leaf midribs from healthy control and naturally-infected hosts were used for DNA extraction. Total DNA was extracted from midrib of six symptomatic cowpeas, inoculated periwinkle plants using CTAB buffer as previously described by Zhang et al., 1998. The universal primer pair P1/P7 was employed in first round PCR to amplify an 1830 bp fragment of 16S rDNA. A 30-fold dilution of the first round PCR product was then used as template for nested PCR using primer pair R16F2n/R16R2 which amplified an internal fragment of 1245 bp from the 16S rDNA. PCR products were ligated into pTZ57R/T using InsT/AcloneTM PCR Product Cloning Kit and transformed to Escherichia coli DH5a cells. The recombinant plasmids were sequenced. The sequences were assembled using DNA baser V. 3 program. About 100 ng of DNA from R16F2n/R16R2 primed PCR product from each province was separately digested with 2.5 μL of AluI, HaeIII, HpaII, HinfI, RsaI and TaqI restriction enzymes according to the manufacturer’s instructions. Restricted DNA fragments were analyzed by electrophoresis through a 2% agarose gel. Phylogenetic tree was generated using neighbour-joining method with MEGA7 software. Acholeplasma laidlawii, a culturable mollicute that is phylogenetically related to the phytoplasmas, was designated as the out-group to root the tree. The reliability of the tree was assessed by bootstrap analysis with 1000 times replication. Further, the sequences of the 16S rRNA gene were subjected to virtual restriction fragment length polymorphism (RFLP) using iPhyClassifier software (http:// plantpathology. ba. ars. usda. gov/ cgi- bin/ resource/ iphyclassifier. cgi) to determine ribosomal group affiliation.
Results and Discussion: Cowpea plants collected from East Azarbaijan province showed disease symptoms similar to those caused by phytoplasmas. In infected cowpea plants, symptoms of little and thickened leaves, phyllody, proliferation of shoot, wrinkled and malformed leaves, stem fasciation and stunting were observed. Inoculated periwinkles showed little leaf, internode shortening, yellowing, virescence, phyllody, stem and floral proliferation and witches’ broom. Amplification of 1830 and 1245 bp fragments in PCR and nested-PCR, respectively, and Blast analysis of the fragment sequences confirmed phytoplasma infection of cowpea plant associated with phyllody symptoms. No band was found at healthy control. Consensus sequences of 1,830 bp of three isolates (Tabriz, Nazarlou, Shabestar) were submitted in the NCBI GenBank under accession numbers KC633092 to KC633094. Phylogenetic analyses and restriction fragment length polymorphism (RFLP) of 1245 bp fragment with 17 restriction endonucleases using iPhyClassifier software indicated that phytoplasma associated with cowpea phyllody belong to Clover proliferation group or 16SrVI A subgroup (similarity coefficient 1.00).
Conclusions: Tomato (Solanum lycopersicum), potato (Solanum tuberosum), cucumber (Cucumis sativus), cabbage (Brassica oleracea) and periwinkle (Catharanthus roseous) were reported as hosts for 16SrVI phytoplasma group in Iran. Recently, maize (Zea mays) has also been reported as a natural host of the 16SrVI group. In addition, different phytoplasma groups and subgroups were reported infecting cowpea crops from different countries over the globe. A 16SrXII-B strain of 'Ca. Phytoplasma australiense' associated with witches’ broom and small leaves of V. unguiculata var. sesquipedalis in Australia. A Vigna little leaf phytoplasma belonging to group 16SrV has also been reported from Australia. In India, different phytoplasmas associated with cowpea from 16S rDNA groups 16SrI-B, 16SrXIV-A and 16SrII-D, have been characterized. Identification of the causal agent of phyllody of cowpea in Iran should facilitate studies concerned with epidemic aspects of the disease, and could contribute to knowledge of the genetic diversity of phytoplasmas in Iran. To the best of the present study authors’ knowledge, it is the first report of the Vigna unguiculata infection with phytoplasmas related to the “Clover Proliferation Group” from East Azarbaijan province of Iran.

کلیدواژه‌ها [English]

  • Cowpea (Vigna unguiculata)
  • phylogenetic analyses
  • Phytoplasma
  • RFLP
1- Bertaccini A., Duduk B., Paltrinieri S., and Contaldo N. 2014. Phytoplasmas and phytoplasma diseases: a severe threat to agriculture. America Journal of Plant Science, 5:1763-1788.
2- De La Rue S., Padovan J., and Gibb K. 2001. Stylosanthes is a host for several phytoplasmas, one of which shows unique 16S 23S intergenic spacer region heterogeneity. Journal of Phytopathology, 149, 613–619.
3- Deeley J.W., Stevens A., and Fox R.T.V. 1979. Use of Dienes'stain to detect plant diseases induced by mycoplasma like organisms. Phytopathology, 69:1169-1171.
4- Esmailzadeh Hosseini S.A., Salehi M., Mirchenari S.M., Tarizeh D., and Gholampoor H. 2015. First report of a 16SrVI group related phytoplasma associated with cucumber phyllody in a greenhouse in Iran. New Disease Report, 32:35.
5- Fattahi M., Salehi M., Sharzehi A., and Esmailzadeh Hosseini S.A. 2016. Partial biological and molecular characteristics of a phytoplasma associated with Behshahr (Mazandaran) periwinkle phyllody. Iranian Journal of Plant Pathology 52(1), Pages 135-141. (In Persian with English abstract).
6- Gharouni Kardani S., and Jamshidi E. 2018. First report of 16SrVI group phytoplasma on cucumber (Cucumis sativus) in North Khorasan province of Iran. International conference on Agricaltural Science, Medicinal Plants and Traditional medicine. Mashhad. Iran (In Persian with English abstract).
7- Hosseini P., Bahar M., Madani G., and Zirak L. 2011. Molecular Characterization of a Phytoplasma Associated with Potato Witches-broom Disease in Iran. Journal of Phytopathology, 159:120–123.
8- Jamshidi E., Jafarpour B., Rouhani H., and Salehi M. 2014. Association of members of clover proliferation (16SrVI) and pigeon pea witches’broom (16SrIX) phytoplasma groups with tomato big bud disease in Iran. Iranian Journal of Plant Pathology, 50:79-89.
9- Kumar S., Singh V., and Lakhanpaul S. 2012. A 'Candidatus Phytoplasma asteris' isolate associated with bud proliferation disease of cowpea in India. New Disease Reports, 25, 28.
10- Kumar S., Stecher G., and Tamura K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33:1870-1874.
11- Lee I.M., Hammond R.W., Davis R.E., and Gundersen D.E. 1993. Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasma like organisms. Phytopathology, 83: 834-842.
12- Lee I.M., Gundersen-Rindal D. E., Davis R. E., and Bartoszyk I. M. 1998. Revised classification scheme of phytoplasmas based on RFLP analyses of 16S rRNA and ribosomal protein gene sequences. International Journal of Systematic Bacteriology, 48: 1153-1169.
13- Lee I.M., Davis R.E., and Gundersen-Rindal D.E. 2000. Phytoplasma: phytopathogenic mollicutes. Annual Review of Microbiology, 54, 221–55.
14- Lee I.M., Bottner K.D., Miklas P.N., and Pastor-Corrales M.A. 2004. Clover proliferation group (16SrVI) subgroup A (16SrVI-A) phytoplasma is a probable causal agent of dry bean phyllody disease in Washington. Plant Disease, 88:429.
15- Rao G.P., Madhupriya Kumar M., Tomar S., Maya B., Singh S. K., and Johnson J.M. 2018. Detection and identification of four 16Sr subgroups of phytoplasmas associated with different legume crops in India. European Journal of Plant Pathology, 150: 507-513.
16- Salehi E., Salehi M., and Masoumi M. 2016. Biological and molecular characterization of the phytoplasma associated with tomato big bud disease in Zanjan province, Iran. Iranian Journal of Plant Pathology, 52 (3):415-427. (In Persian with English abstract).
17- Salehi M., Izadpanah K., and Siampour M. 2007. Characterization of a phytoplasma associated with cabbage yellows in Iran. Plant Disease, 91: 625-630.
18- Saqib M., Bayliss K.L., and Jones M.G.K. 2006. Identification of sweet potato little leaf phytoplasma associated with Vigna unguiculata Var. sesquipedalis and Lycopersicon esculentum. Australasian Plant Pathology, 35, 293–296.
19- Schneider B., Seemuller E., Smart C.D., and Kirkpatrick B.C. 1995. Phylogenetic classification of plant pathogenic mycoplasma-like organisms or phytoplasmas. In: Razin S, Tully JG. (eds) Molecular and diagnostics procedures in Mycoplasmology, Vol I. San Diego, CA, Academic Press, pp 369–380.
20- Thorat V., Bhale U., Sawant V., More V., Jadhav P., Mane S.S., Nandanwar R.S., Tripathi S., and Yadav A. 2016. Alternative weed hosts harbors 16SrII group phytoplasmas associated with little leaf and witches’ broom diseases of various crops in India. Phytopathogenic Mollicutes, 6(1), 50–55.
21- Wei W., Lee I.M., Davis R.E., Suo X., and Zhao Y. 2007c. Virtual RFLP analysis of 16S rDNA sequences identifies new subgroups in the clover proliferation phytoplasma group. Bulletin of Insectology, 60:349-350.
22- Weintraub P.G., Jones P., (eds). 2010. Phytoplasmas: Genomes, Plant Hosts and Vectors CAB International. 348 pages.
23- Zhang Y.P., Uyemoto J.K., Kirkpatrick B.C. 1998. A small-scale procedure for extracting nucleic acids from woody plants infected with various phytopathogens for PCR assay. Virology Method, 71: 45-50.
24- Zhao Y., Wei W., Lee I.M., Shao J.X., and Davis R.E. 2009. Construction of an interactive online phytoplasma classification tool, iPhyClassifier, and its application in analysis of the peach X-disease phytoplasma group (16SrIII). International Journal of Systemic and Evolutionary Microbiology, 59: 2582-2593.
25- Zibadoost S., Rastgou M., Asghari S., Tazehkand S.A. 2016. Detection and molecular identification of ‘Candidatus phytoplasma trifoli’ infecting some cultivated crops and vegetables in West Azarbaijan province. Australasian Plant Disease Notes, 11: 3.