Gen za veliku subjedinicu šaperonina (groEL gen) kao dodatni molekularni marker za diferencijaciju fitoplazmi srodnih 'Candidatus Phytoplasma asteris'

Abstract

Na teritoriji Republike Srbije su u periodu od 2009. do 2011. godine sakupljeni uzorci biljaka sa simptomima karakterističnim za prisustvo fitoplazmi. Upotrebom PCR metode pomoću univerzalnih prajmera P1/P7 i R16F2n/R16R2 u njima su detektovane fitoplazme. Upotrebom restrikcionih enzima Tru1I, HhaI, RsaI i Tsp509I na R16F2n/R16R2 amplikone, ove fitoplazme su na osnovu dobijenih restrikcionih profila identifikovane kao Aster yellows fitoplazme (ribozomalna grupa 16SrI), podgrupe 16SrI-B, I-C i I-P, odnosno kao Stolbur fitoplazme (ribozomalna grupa 16SrXII). Upotrebom iste PCR-RFLP metode kod 19 sojeva Aster yellows fitoplazmi iz kolekcije i tri soja poreklom iz šargarepe iz Srbije, potvrĎena je pripadnost podgrupama 16SrI-A, I-B, I-C i I-F preuzeta iz literature. TakoĎe je utvrĎena pripadnost odreĎenoj podgrupi kod četiri soja iz kolekcije, koji se prvi put koriste u analizi i o kojima nema literaturnih podataka. Upotrebom novodizajniranih prajmera AYgroesF/AYampR i AYgroelF/AYgroelR umnoţen je groEL gen kod sva 34 testirana soja Aster yellows fitoplazmi. Analizom restrikcionih profila dobijenih pomoću Tru1I i AluI restrikcionih enzima na AYgroelF/R amplikone, utvrĎeno je postojanje šest, odnosno osam različitih profila na osnovu kojih su svi testirani sojevi svrstani u devet groELI podgrupa (groELI-I do groELI-IX). Na osnovu groEL gena podgrupe 16SrI-A i I-C su dalje diferencirane u dve groELI podgrupe, 16SrI-B u tri, dok podgrupe 16SrI-M i I-L nisu pokazale nikakve meĎusobne razlike u odnosu na jedan deo podgrupe 16SrI-B. Podgrupe 16SrI-F i I-P se razlikuju od ostalih podgrupa i na nivou groEL gena kao i na nivou 16S rDNK. TakoĎe je utvrĎeno da sedam novodetektovanih sojeva Aster yellows fitoplazmi iz Srbije pripada podgrupama groELI-III, I-VII i I-IX. Analizom tuf gena, dela rp operona i secY gena kod 22 odabrana od 34 testirana soja soja Aster yellows fitoplazmi moguće je testirane sojeve klasifikovati u šest tufI i sedam rpI i secYI podgrupa na osnovu MboI, Tsp509I i Tru1I restrikcionih profila za tuf gen, HhaI, AluI i Tsp509I za rp gen i Tsp509I i Tru1I za secY gen. Podgrupa 16SrI-A je dalje diferencirana u dve tufI podgrupe, odnosno 16SrI-A i I-B u po dve rpI i secYI podgrupe, dok podgrupe 16SrI-B, I-M i I-L nisu pokazale nikakve meĎusobne razlike. Podgrupe 16SrI-C, I-F i I-P se razlikuju od ostalih podgrupa i na nivou sva tri testirana gena kao i na nivou 16S rDNK

The plant samples with symptoms typical for phytoplasma infection were collected in Serbia from 2009 till 2011. In those samples phytoplasmas were detected by PCR technique with universal primers P1/P7 and R16F2n/R16R2 and identified according to their Tru1I, HhaI, RsaI and Tsp509I restriction profiles of digested R16F2n/R16R2 amplicons as Aster yellows phytoplasma (ribosomal group 16SrI), subgroups 16SrI-B, I-C and I-P or as Stolbur phytoplasma (ribosomal group 16SrXII). Applying the same PCR-RFLP method on 19 strains of Aster yellows phytoplasmas from collection and three strains from carrot from Serbia, it was confirmed that this strains belong to subgroups 16SrI-A, I-B, I-C and I-F as stated in literature. Also the subgroups were determined for four strains from collection that are used in analyses for the first time and that have no literature data. Using newly designed primers AYgroesF/AYampR and AYgroelF/AYgroelR, the groEL gene was successfully amplified in all 34 strains of Aster yellows phytoplasma tested. RFLP analyses of AYgroelF/AYgroelR amplicons with Tru1I i AluI restriction enzymes revealed existence of six and eight different restriction profiles, respectively, according to which all tested strains were classified in nine groELI subgroups (groELI-I till groELI-IX). On the basis of groEL gene, subgroups 16SrI-A and I-C were further differentiated into two groELI subgroups, 16SrI-B into three, while subgroups 16SrI-M and I-L showed no difference to some strains belonging to subgroup 16SrI-B. Subgroups 16SrI-F and I-P could be differentiated from other subgroups on the basis of groEL gene as on the basis of 16S rDNA. The seven newly detected Aster yellows strains from Serbia were affiliated to subgroups groELI-III, I-VII and I-IX. RFLP analyses with MboI, Tsp509I and Tru1I restriction enzymes of tuf gene, HhaI, AluI and Tsp509I of rp gene and Tsp509I and Tru1I of secY gene, classified 22 selected Aster yellows strains into six tufI subgroups and seven rpI and secYI subgroups. Subgroup 16SrI-A was further differentiated into two tufI subgroups and subgroups 16SrI-A and I-B were further differentiated into two rpI and secYI subgroups each. On the other hand subgroups 16SrI-B, I-M and I-L showed no mutual differences, while subgroups 16SrI-C, I-F and I-P could be differentiated from other subgroups on the basis of all three genes tested as on the basis of 16S rDNA. RFLP analysis of tuf gen with HpaII restriction enzyme showed that all 116 tested Stolbur phytoplasma strains from Serbia belong to tuf type II variant. For further analyses, 39 Stolbur strains were selected out of 116 detected and two strains from grapevine from Croatia were also included in analyses as tuf type I reference strains to gain a larger picture of variability of groEL gene in Stolbur phytoplasmas.