Molecular Identification of Weed hosts of Tomato yellow leaf curl virus in southeast of Kerman Province

Tomato yellow leaf curl virus, TYLCV belongs to the family Geminiviridae and Begomovirus genus (27). In recent years, extensive damage to tomatoes and cucurbits plants in the south and the southeast of Iran has arrived (23). This virus family have circular, and single-stranded DNA genome and are widespread in tropical and subtropical areas (30). They are infected several plant species with economic importance. Begomoviruses are dicot-infecting, whitefly-transmitted viruses with a genome comprised of one or two molecules DNA (5). Up to now, studies have been performed to evaluate the status of distribution, and identification of natural host and assess the genetic diversity, but there is not a comprehensive review about its weed hosts yet. In this research, The weeds from margins and inside greenhouses and farms of tomato and cucurbit in severely infected areas including Manoojan, Kahnooj, Faryab, Anbrabad and Jiroft to identify weed hosts of the virus in nature, were collected. Identification of collected samples were conducted by botanical specialists. Total DNAs were extracted from leaves according to the method of zhang et al. (1998) and stored at -20 oC. Identification of infected samples were carried out by PCR using degenerate primer pairs PCRv 181/Bc that direct the amplification of˷ 550 bp fragment of mono – and bipartite begomoviruses genome comprising the C-terminal portion of the intergenic region (IR) N-terminal portion of the CPgene. PCR were performed in 25 µl reaction volumes containing 1 µl of template DNA, o.5 µl of Taq DNA polymerase Sinaclon (IRAN), 1.2 µl MgCl2, 0.5 µl dNTPs. 1 µM of each forward and reverse primers, 4.3 µl of 10× reaction buffer and 15.5 distilled water. The amplification were performed using a peqSTAR 96x Termal Cycler (Peqlabe, Germany). PCR conditions consisted of initial denaturing 94 oC for 3 min followed by 30 cycles of denaturation at 94 oC for 50s, annealing at 55 oC for 60s, and extension at 72 oC for 1 min followed by 1 cycle at 72 oC for 10 min. Electrophoresis of polymerase chain reaction products on 1% agarose gel was performed and stained with DNA safe stain (sinaclon-IRAN). The polymerase chain reaction product were sequenced using automatic sequencer AB13730XL (Macrogen, Korea). The resulting sequences were looking similarity and after obtaining a degree of homology, 550 bp fragment of the coat protein gene of four isolates were ordinated by Bio Edit software. Looking for similar sequences were obtained and then achieved the 550 bp fragments of the coat protein gene homology four software isolates Bio edit (21) were ordination. To study the phylogenetic relationship of study strains, the phylogenetic tree was drawn with maximum likelihood way in the MEGA 5 software. Then percentage of similarity at the nucleotide and amino acid sequence with a genetic distance matrix was determined by using the software CLC Main work bench. The results showed that four weeds including Chrozophora tinctoria, Heliotropium annum, Malva neglecta and Chenopodium murale were infected with TYLCV. To compare the TYLCV isolates in infected weeds, 550 bp fragment of the coat protein gene in four different strains of the virus was sequenced. Assessment of the genetic similarity between study isolates and strains in the Gene Bank revealed that study isolates with isolates from Gene Bank have similarity in the range of 93/24-99/98% at the nucleotide level and in the range of 87/42-98/15% at the amino acid level. Sixty-six mutations at the nucleotide level in compared sequences in this study was also found. Drawn Phylogenetic tree was confirmed the results of the genetic distance matrix. The results showed that the virus has a wide host range, and identification of weed hosts to remove the maintenance of virus plays an important role in the epidemiology of the disease. and also it’s the management of this disease. This is the first report from Malva neglecta and Chenopodium murale as weed host of TYLCV in Iran. TYLCV causing major damage to the cultivation of tomatoes. Infection caused by it in the South and South East farms in Iran is usually very widespread. Because of the subtropical climate in these areas that is essential for the activity and proliferation of whitefly vector, this virus can cause a high damage in these areas (34). High genetic variability in begomovirus populations infecting different non-cultivated hosts has been reported (28, 35, 38). Conversely, begomovirus populations infecting cultivated hosts seem to have lower variability (6,14,41,43).Wild/non-cultivated plants from different botanical families can sustain a high species diversity of begomoviruses and can play an important epidemiological role serving as alternate/reservoir hosts, preventing local extinctions of the virus when the cultivated host is absent (1, 14). In these cases, whiteflies transmitting begomoviruses between cultivated and non-cultivated hosts contribute to virus evolution and disease epidemics (1, 34). Weeds and plant host of indigenous origin and imported ones can be by a large number of plant viruses including Begomoviruses infectedwhich are as reservoirs host of the virus, the primary focus for infection of crops, and play a pivotal role in the emergence of new viral strains (35). Identification and control of the weeds have an important role in disease management. Relatively high incidence of the virus in the weeds with a high density of whitefly population throughout the year, requires new management guidelines. The results showed that the virus has a wide host range, and identification of weed hosts to remove the maintenance of virus that play an important role in the epidemiology of the disease, in the management of this disease is important. This is the first report from Malva neglecta and Chenopodium murale as weed host of TYLCV in Iran.