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survey was conducted in 2012 and 2013 to detect the presence and distribution of Alfalfa mosaic virus (AMV) in lavandin crops growing in continental parts of Croatia. A total of 73 lavandin samples from six crops in different localities were collected and analyzed for the presence of AMV and Cucumber mosaic virus (CMV) using commercial double-antibody sandwich (DAS)-ELISA kits. AMV was detected serologically in 62 samples collected at three different localities, and none of the samples tested positive for CMV. For further analyses, six selected samples of naturally infected lavandin plants originating from different localities were mechanically transmitted to test plants: Chenopodium quinoa, C. amaranticolor, Nicotiana benthamiana and Ocimum basilicum, confirming the infectious nature of the disease. Molecular detection was performed by amplification of a 751 bp fragment in all tested samples, using the specific primers CP AMV1/CP AMV2 that amplify the part of the coat protein (CP) gene and 3’-UTR. The RT-PCR products derived from the isolates 371-13 and 373-13 were sequenced (KJ504107 and KJ504108, respectively) and compared with the AMV sequences available in GenBank. CP sequence analysis, conducted using the MEGA5 software, revealed that the isolate 371-13 had the highest nucleotide identity of 99.5% (100% amino acid identity) with an isolate from Argentina originating from Medicago sativa (KC881010), while the sequence of isolate 373-13 had the highest identity with an Italian AMV isolate from Lavandula stoechas (FN667967) of 98.6% (99% amino acid identity). Phylogenetic analysis revealed the clustering of selected isolates into four molecular groups and the lavandin AMV isolates from Croatia grouped into two distinct groups, implying a significant variability within the AMV lavandin population.

Viruses are responsible for numerous epidemics in different crops in all parts of the world.As a consequence of their presence great economic losses are being incurred. In addition tothe development of sensitive techniques for detection, identification and characterization ofviruses, substantial attention has also been paid to biotechnological methods for their eliminationfrom plants. In this review article, the following biotechnological in vitro culture techniquesfor virus elimination from ornamental plants are presented: meristem culture, thermotherapy,chemotherapy, cryotherapy or a combination of these methods. The plant species,as well as the type of virus determine the choice of a most suitable method. The state ofthe art in investigation of virus elimination from Impatiens sp. in Serbia is summarized.

During 2010, Impatiens walleriana plants with symptoms of downy mildew were collected in a greenhouse in the vicinity of Mionica, Kolubara District. Disease incidence was extremely high, approaching 100%, and wilting and collapse of affected plants was very rapid, resulting in losses of more than 90%. White downy growth produced on the lower leaf surface consisted of hyaline, thin–walled sporangiophores with monopodial branching and numerous, ovoid and hyaline sporangia. Apical branchlets of sporangiophores were at right angles to the main axis, with no apical thickening. Pathogenicity tests included inoculation of young I. walleriana plants by spraying with a sporangial suspension, and downy mildew symptoms were observed after 13 to 15 days. The absence of well–defined spots on the infected impatiens leaves and straight sporangiophores indicated that the pathogen was P. obducens, which was further supported by molecular identification, the 5’–end of the nuclear DNA coding for the large ribosomal subunit (LSU rDNA) was amplified by PCR, using primers NL1 and NL4. A representative isolate, 28–10, was sequenced and phylogenetic analysis showed its grouping with other P. obducens isolates of different origin.Considering that impatiens downy mildew in Serbia is proved to be caused by P. obducens it is necessary to employ adequate phytosanitary measures to prevent further spread of the pathogen.

Three-year investigation of the presence and distribution of tobacco viruses in Serbia revealed that Alfalfa mosaic virus (AMV) appeared every year with different frequency in tobacco crops. During 2008, the presence of AMV was detected in most of the tested samples(58.82%) and it was the second most common compared to all other viruses which presence was confirmed in Serbia. In 2006 and 2007, AMV was detected in a significantly lower percentage (2.80% and 13.64%, respectively). This study showed that Alfalfa mosaic virus was more commonly found in multiple infections with two, three or even four detected viruses. Single infections were detected only in 2006, in one tobacco field in the locality of Futog. During this investigation, a rapid and simple protocol was optimized and developed for molecular detection of AMV in tobacco leaves, using primers CPAMV1/CPAMV2 and commercially available kits for total RNA extraction as well as for RT-PCR (reverse transcription - polymerase chain reaction). Using RT-PCR and these primers that flank the AMV coat protein gene, a DNA fragment of 751 bp was amplified, sequenced, and compared with the sequences available in GenBank database. The sequence of isolate 196-08 (GenBank Acc. No. FJ527749) proved to be identical at the nucleotide level of 99 to 93% withthose from other parts of the world. Phylogenetic analysis of 27 isolates based on 528 bp sequences of the coat protein gene did not show correlation of the isolates with their geographic origin or plant host and showed that these isolates fall into four molecular groups of strains. Serbian AMV isolate from tobacco belongs to group IV, the group that includes most of the isolates selected for phylogenetic analysis.

Cucumber mosaic virus (CMV) is considered one of the most economically importantplant viruses and has a worldwide distribution and a very wide host range including plantsfrom family Cucurbitaceae. In Serbia, on cucurbits CMV was detected in single and mixedinfections with Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus (WMV). Viruses,including CMV, are constantly present in cucurbit crops, but their frequency changesby year and locality. Surveys and sample collections were conducted in cucurbit crops inthe period from 2008 to 2009 at 15 localities in Vojvodina province, and sample testing wascarried out using the DAS-ELISA method and commercially available antisera for six economicallymost important cucurbit viruses. In 2008, a total of 51 samples were collected from13 cucurbit crops of oilseed pumpkin Olinka variety, squash, and bottle gourd and CMV wasdetected in a total of 55% of tested samples with symptoms of viral infection. The most commoninfectious type was mixed infection with ZYMV and WMV (35.3%), and then mixedinfection with ZYMV (17.7%) and WMV (2%). A total of 599 symptomatic samples of oilseedpumpkin Olinka variety, zucchini squash varieties Beogradska and Tosca, squash, and wintersquash were collected in 15 cucurbits crops in 2009. CMV was present in 4.4% of totalcollected samples, in single infections in 1.3%, and in mixed with WMV or ZYMV in 1.3%, and1.8%. Five CMV isolates were obtained by mechanical inoculations of N. glutinosa and oneof them was selected for further biological characterization. Test plants which were describedto be hosts of CMV expressed symptoms characteristic for those caused by CMV afterinoculations by isolate 115-08. CMV specific primers Au1u/Au2d were used to amplify an850 bp fragment using RT-PCR method. Amplified fragment encodes the entire viral coatprotein (CP) gene and partial 5’ and 3’ UTRs of two selected CMV isolates. Amplified fragmentswere sequenced and deposited in the NCBI, where they were assigned accessionnumbers, HM065510 (115-08) and HM065509 (151-08). The sequences of CMV isolates fromSerbia shared the highest nucleotide and amino acid identity with isolates from subgroupIA, from 99.5 to 97.4% and 99.1 to 97.4%, and the lowest identities were with the subgroup IIisolates from 66.9 to 64, 5%, from 75.8 to 74.1%.

As there is a growing frequency of viral plant diseases in epidemic proportions, the possibilities for successful control are constantly being explored. Despite the fact that integral and simultaneous employment of numerous control measures may contribute to thedecreasing amount of yield losses, especially concerning non-persistently aphid-transmitted viruses, these measures are often not efficient enough. Research into the basis of resistance to viral infection and principles of its inheritance, introduction of sources of resistance in susceptible genotypes, by conventional or genetic manipulations, are very intensive for cucurbit crops, especially pumpkins. Pumpkin crops are being endangered by a great number of different viruses, among which the Zucchini yellow mosaic virus, (ZYMV), Watermelon mosaic virus (WMV) and Cucumber mosaic virus (CMV) are present every year in Serbia, frequently causing epidemics.The majority of pumpkin cultivars are not resistant or tolerant to viral infections, but sources of resistance have been identified in various related species. So far, the identified sources of esistance to the ZYMV are found in Cucurbita moschata and Citrullus lanatus var.lanatus genotypes and consist of one or several major dominant genes of resistance. It is a similar case with WMV, although the sources of dominant major genes are identified in C. lanatus and C. colocynthis. The sources of resistance to CMV in the form of one dominant gene have been identified in the genotype C. moschata, although the introduction of this gene by conventional means proved to be very difficult. Besides the aforementioned, substantial efforts are being made in developing genotypes with multiple resistance against several viruses and even other pathogens, as well as genotypes with resistance to the most significant plant aphid species, through mechanisms of antixenosis or antibiosis. The other way of obtaining resistant genotypes includes genetic manipulation.Genetically modified resistant pumpkins have been among the first successfully developed crops. Genotypes with pathogen derived resistance can already be found in commercially grown pumpkins in some parts of the world, and they have been developed by introducingthe coat protein gene of one, two or all three viruses which are the most frequent,ZYMV, WMV and CMV. Yet, this approach to the control of pumpkin viral diseases is related to possible negative consequences, mostly through the already detected gene transferto wild plants and development of resistant transgenic weeds of unpredictable impact on the environment. Improved host plant genetic resistance to viral infections or biological vectors, developedby conventional or genetic engineering methods, represents the most dynamic and prominent field of research. It is economically and ecologically the most justified approach to the control of pumpkin and other plant diseases caused by viruses non-persistentlytransmitted by aphids.

Watermelon mosaic virus (WMV) is widespread in cucurbit crops, most commonly occuring in temperate and Mediterranean regions. In Serbia WMV has been detected in single and mixed infections with Zucchini yellow mosaic virus and Cucumber mosaic virus in field-grown pumpkin and squash crops. Among pumpkin-affecting viruses WMV is the most frequent one, both by the number of localities and its incidence at each location. During the growing season of 2009, samples from 583 plants of Cucurbita pepo cvs. Olinka, Belgrade zucchini and Tosca (Zucchini group), as well as from C. maxima and C. moschata showing symptoms of virus infection were collected from 12 commercial fields at eight localities and analyzed by DAS-ELISA using polyclonal antisera specific to six most important cucurbit viruses. Interestingly, WMV was detected at fewer sites and had lower ncidence rate than in two previous years. In single infections, WMV was found in 11% of tested plants in three fields; in mixed infections with ZYMV, it was recorded in 9.9% of plants in five fields and with CMV in only 0.2% in one field. The partial coat protein gene and 3’ non-translated region from two representativeisolates of WMV originating from different localities and host plant species were amplified by RT-PCR, sequenced, and compared with the sequences available in GenBank database. The PCR-amplified fragment of predicted size of approximately 1017 bp was obtained. The sequences of isolates 137-08 (Acc. No. GQ259958) and 159-08 (GU144020) proved to be 94-99% identical at the nucleotide level with those from other parts of the world. The sequences of these two isolates differed from each other only at two nucleotide positions, without any amino acid substitution. Phylogenetic analysis of 57 isolates based on 750 bp sequences of the coat protein gene showed no correlation between isolates and their geographic origin, and italso indicated that these isolates fell into three molecular groups of strains. Serbian WMV isolates belong to group I, the most widespread group of isolates of this virus in the world.