Pd 2915 clustered separately from the strains

This preview shows 6 out of 8 pages.

PD 2915 clustered separately from the strains isolated from other Maloideae plant species. Its DNA fingerprint profile was more similar to the profiles showed by NCPPB 2292 and 2293, isolated from Rubus spp. By con- trast PD 103, isolated from Rubus sp., resulted more sim- ilar to the profile showed by the strains obtained from Maloideae. ERIC-PCR allowed the differentiation of NCPPB 2292 and NCPPB 2293. ARDRA To ascertain if ARDRA analysis also revealed the presence of different groups, with a subset of 40 representative E. amylovora strains (Table 1) including all strains from Rubus spp. and Amelanchier sp., the 16S rDNA was amplified and digested with Hin fI and Hae III restriction endonucleases. Only Hae III yielded a restriction pattern differentiating E. amylovora within the genus Erwinia (Bereswill et al. 1995). Using this endonuclease, fragments D. Barionovi et al. E. amylovora characterization ª 2006 The Authors Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 100 (2006) 1084–1094 1089
Image of page 6

Subscribe to view the full document.

of 204, 222 and 317 bp and a triplet containing fragments of 164, 165 and 170 bp were observed for all the 40 E. amylovora strains, and no grouping was possible. When compared with E. amylovora , E. pyrifoliae and B. nigriflu- ens , used as outgroup, gave rise to a clearly different banding patterns (Fig. 3). 16S rDNA sequencing and comparison of strains The 16S rDNA sequences of E. amylovora strains PD 103 and PD 2915, showing a different genomic pattern upon rep-PCR, were sequenced, and the 1506 nucleotides were compared by means of Clustal W algorithm and NJ clus- tering method with the corresponding sequences of E. amylovora BC199 and BC201, isolated from Rubus spp. (GenBank accession numbers AF141891 and AF141892 respectively), BC 204, isolated from Rubus fruticosus (GenBank accession number AF141895), Ea321 and NCPPB 683, isolated from P. communis (GenBank acces- sion numbers AF140337 and AF140341 respectively). The resulting cladogram is shown in Fig. 4. The E. amylovora strains isolated from P. communis clustered separately. Interestingly, E. amylovora PD 2915, obtained from Amel- anchier sp., was grouped with two strains isolated from Rubus spp. Sequence accession numbers The following nucleotide sequences were deposited in the EMBL GenBank nucleotide sequence database: 16S rDNA gene from E. amylovora PD 2915 and E. amylovora PD 103 (accession numbers AJ746201 and AJ746202 respect- ively). RFLP analysis of plasmid pEA29 and SSR With the sole exception of PD 103, isolated from Rubus sp., both Msp I and Sau 3A enzymes cleaved the amplified fragment of the plasmid pEA29 in all 93 E. amylovora strains tested. After the digestion with Msp I we found a greater variability in the larger fragment than expected according to the results of Lecomte et al. (1997). This was even more evident using Sau 3A restriction enzyme. In fact, besides a rather stable fragment of c. 180 bp, we found a great variability in the discriminative fragments between 200 and 400 bp. We found a sort of continuous variability in the larger fragment starting from around 250 bp of PD 2913 to 280–290 bp of PD 2915 (Fig. 5).
Image of page 7
Image of page 8
You've reached the end of this preview.
  • Spring '08
  • Devartanian
  • DNA, DNA sequencing, Restriction enzyme, Greece Greece Greece Greece Greece Greece Greece Greece Hungary Hungary Hungary Hungary Hungary Hungary Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy USA USA Italy Italy Italy Italy, E. amylovora

{[ snackBarMessage ]}

What students are saying

  • Left Quote Icon

    As a current student on this bumpy collegiate pathway, I stumbled upon Course Hero, where I can find study resources for nearly all my courses, get online help from tutors 24/7, and even share my old projects, papers, and lecture notes with other students.

    Student Picture

    Kiran Temple University Fox School of Business ‘17, Course Hero Intern

  • Left Quote Icon

    I cannot even describe how much Course Hero helped me this summer. It’s truly become something I can always rely on and help me. In the end, I was not only able to survive summer classes, but I was able to thrive thanks to Course Hero.

    Student Picture

    Dana University of Pennsylvania ‘17, Course Hero Intern

  • Left Quote Icon

    The ability to access any university’s resources through Course Hero proved invaluable in my case. I was behind on Tulane coursework and actually used UCLA’s materials to help me move forward and get everything together on time.

    Student Picture

    Jill Tulane University ‘16, Course Hero Intern