Degenerate primers facilitate the detection and identification of potyviruses from the northwest region of Iran

Potyviruses are accounted for 40% of viral diseases of various crops including vegetables and legumes. Potyviruses can be transmitted through plant sap, seeds and many aphid species. Due to ease of spread of these viruses detection of such viruses are crucial as to the control of the incited diseases. This study compared the efficiencies of two couples of primers in their detection of potyviruses infecting vegetables in North-West of Iran. To identify potyviruses infecting vegetables, total RNA preparations from leaves of diseased plants were subjected to reverse transcription [RT] with oligo d[T]15, M4T or a potyvirus-specific primer, followed by polymerase chain reactions [PCR] with two pairs of degenerate primers including Sprimer/M4 or NIb2F/NIb3R that would yield [tilde] 1700 or 350 bp fragments, respectively. Amplification was achieved from 7 samples when Sprimer and M4 were used, but non- specific fragments were also amplified. However, specific amplifications from 31 samples were achieved with NIb2F2 and NIb3R. PCR products resulting from the use of NIb2F/NIb3R were subjected for cloning and sequencing. BLAST analysis of the sequenced data revealed that the PCR-amplified fragments belonged to Bean common mosaic virus [green bean], Bean yellow mosaic virus [broad bean], Potato virus Y [potato], Watermelon mosaic virus [squash, muskmelon or melon], Zucchini yellow mosaic virus [squash], and Soybean mosaic virus [bean]. This study demonstrated the efficiency of NIb2F2/NIb3R in detection of potyviruses and revealed the extent of infections with diverse potyviruses species in the northwest part of Iran.

Characterization of the full length coat protein gene of Iranian Grapevine fanleaf virus isolates, genetic variation and phylogenetic analysis

The full-length coat protein gene of Grapevine fanleaf virus [GFLV] isolates from Iran was characterized by reverse transcription polymerase chain reaction [RTPCR] and sequencing. The expected 1515 bp coat protein [CP] gene amplicon was obtained for 16 isolates out of 89 that were identified by double antibody sandwich enzyme-linked immunesorbent assay [DASELISA] in a population of 330 symptomatic grapevine leaf samples. CP products of eight isolates were cloned and nucleotide sequences were determined. Parsimonious trees indicated that GFLV isolates from Iran formed a distinct cluster, suggesting an independent evolution

Isolation of the gene coding for movement protein from Grapevine fanleaf virus

A pair of degenerate primers, GMPF1 and GMPR1, was designed on the basis of alignment of previously reported Grapevine fanleaf virus [GFLV] movement protein [MP] nucleotide sequences from Iran and other parts of the world. cDNA was synthesized by the use of Oligo d[T]18 from total RNA extraction from each diseased grapevine leaf sample and subjected to polymerase chain reaction [PCR] with the degenerate primers under a range of annealing temperatures from 48 to 62°C. It was revealed that 55°C gave the best result in terms of producing exactly the expected fragment [1044 bp] from as many samples as possible although accompanied by few fade non specific fragments. However, by application of "hot-start" PCR and annealing at 60°C the specific fragment was amplified from 41 out of 86 samples. This was the first amplification of the precise MP cDNA from GFLVs in Iran which is very important as to preparation of recombinant anti- GFLV MP antibody to use in studying the GFLVgrapevine interaction, and also for generating pathogen-derived resistant vines