Developing Detection Methods for Tomato Diseases: Innovation to Support Food Availability

Developing Detection Methods for Tomato Diseases: Innovation to Support Food Availability.

INTRODUCTION. damage caused by pests and pathogens.

Clavibacter michiganensis subsp. michiganensis (CORBMI)[Photos]| EPPO Global Database.

Designing Primers tracing six tomA gene sequences from Cmm through the genebank database on the National Center for Biotechnology Information (NCBI ) alignment using Bioedit program and the regions showing conservations were selected as template of specific primers d esign of specific primer with Primer3Plus software a nalysis of specific primer candidates Validation of Primers Specificity s ynthesis of specific primer a mplification of the tomA gene using specific primer d etection of PCR product by electroforesis.

RESULTS AND DISCUSSION. Alignment sequences by Bioedit program tomA gene sequences with accession number KJ724012.1, KJ724011.1, KJ724010.1, KJ724009.1, KJ724008.1, and KJ724007.1, tomA gene sequences were obtained from Cmm consisting of 509 bp. Bioedit program performed that conserved region for these sequences was located on position 54 to 509 with 456 segment length. This 456 bp sequences was used for specific primer template..

Primer design using Primer3Plus software. Screenshot (370).

Primer 5 was appropriate to the criteria for good primer in amplification using PCR, including the primary length between 17-25 nucleotides, the primary GC composition (%) between 40 to 60% and the length of the amplicon obtained through the Primer3Plus3 program. Too long nucleotide primers may cause annealing temperatures to be less efficient. On the other hand, too short nucleotide primer may cause miss-priming (priming the primers in other places that are not desired). The right percentage of GC elements (%) in the primer design can produce more specific and stable bonds to perform the PCR method. Primer 5 do not have a sequence of GC base repeats more than four times (for example, AGCTGGGGGATCGGG) to prevent miss-priming. Primer 5 also produces melting temperature (Tm) at 58 °C, and 56.8 °C, respectively. The Tm in the primer should be the same or have a difference that is not too significant. Melting temperature (Tm) is used as a reference for determining or optimizing annealing temperature (Ta). High Ta produces in insufficient DNA primer-mold hybridization resulting in low PCR products. On the other hand, low Ta lead to nonspecific DNA amplification caused by primer attachment errors in the DNA template..

Primer Specifity Validation. + 232 bp A B c Fig. 2- Detection results ofPCRproducts- M is a marker benchtop ladder 100 bp; A is DNA undiluted; B is 5x dilution; C is IOX dilution; D is 20x dilution; E is SOX dilution-.

Conclusion. S pecificity of Primer 5 set can be applied for further specific detection of tomato diseases caused by Cmm . Timely and accurate diagnosis of plant diseases plays a major part in preventing loss in productivity and loss or reduction of agricultural products, in order to support food availability..

Thank You.