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PPV was first detected in Spain in 1984 in Japanese plum ( Prunus salicina Lindl) cv. Red Beaut and spread very quickly to other Japanese and European plums and apricot cultivars but left peach cultivars unaffected. In the years following the detection of PPV, the predominant aphid species visiting Prunus orchards in Mediterranean areas were Aphis gossypii followed by Aphis spiraecola, the latter being the main aphid species found at present. Both species are considered to be the main vectors of PPV in Spanish early Prunus growing areas. Spatial analysis of the spread of PPV-D in Japanese plum and apricot trees confirmed the lack of significant association between immediately adjacent trees. The observed spatial pattern of sharka suggests a lack of movement of PPV-viruliferous aphid vectors to immediately adjacent trees and indicates their preferential movement to trees several tree spaces away. PPV-D is the only type currently present in Spain, with the exception of a PPV-M outbreak that was detected in and successfully eradicated from Aragón in 2002. The short-distance spread of PPV-M infection occurred as far as 12 m along the rows of peach trees. However, PPV-D has not been observed to spread through peach cultivars, despite being grown in the vicinity of heavily infected plots of apricot or Japanese plum trees. [ABSTRACT FROM AUTHOR]

A Citrus tristeza virus ( CTV) isolate (L192 GR) naturally occurring in lemon trees of more than 100 years old in Greece was fully characterized. Virus-derived small interfering RNAs, induced by Dicer processing of ds RNAs formed during RNA virus replication, were isolated and used as targets for sequencing. Next-generation high-throughput sequencing using the Ion Torrent platform was performed. A total of 432 632 sequences, 94·05% of which corresponded to L192 GR, were determined. Subsequent bioinformatics analysis enabled the determination of the full-length 19 251 nt genome of the L192 GR isolate (GenBank no. ). Comparative analysis of complete genomes revealed molecular homology with CTV- VT isolate FS2-2 from Florida (GenBank no. ) with 98·2% nucleotide sequence identity. Recombination events were detected in L192GR and they probably contribute to its unique characteristics. Specifically, although most isolates of the CTV- VT group induce the seedling yellows syndrome and react positively with the monoclonal antibody MCA13, which is typically associated with severe CTV isolates, the MCA13-positive L192 GR gave very mild or even no symptoms in the seedling yellows indicator plants. Furthermore, experimental aphid transmissibility studies revealed a poor transmission efficiency of 20%. This is the first report of a CTV isolate originating from a lemon tree being fully characterized at biological, serological and molecular levels. The present study further confirms that, when the goal is the risk assessment associated with a new pathogen or isolate in a particular area, molecular data have to be combined with the biological properties of the pathogen. [ABSTRACT FROM AUTHOR]

A protocol for the specific detection and quantification of ' Candidatus Liberibacter solanacearum' in carrot seeds using real-time PCR was developed. The bacterium was detected in 23 out of 54 carrot seed lots from 2010 to 2014, including seeds collected from diseased mother plants. The average total number of ' Ca. L. solanacearum' cells in individual seeds ranged from 4·8 ± 3·3 to 210 ± 6·7 cells per seed from three seed lots, but using propidium monoazide to target live cells, 95% of the cells in one seed lot were found to be dead. Liberibacter-like cells were observed in the phloem sieve tubes of the seed coat and in the phloem of carrot leaf midrib from seedlings. The bacterium was detected as early as 30 days post-germination, but more consistently after 90 days, in seedlings grown from PCR positive seed lots in an insect-proof P2 level containment greenhouse. Between 12% and 42% of the seedlings from positive seed lots tested positive for ' Ca. L. solanacearum'. After 150 days, symptoms of proliferation were observed in 12% of seedlings of cv. Maestro. ' Candidatus Liberibacter solanacearum' haplotype E was identified in the seeds and seedlings of cv. Maestro. No phytoplasmas were detected in seedlings with symptoms using a real-time assay for universal detection of phytoplasmas. The results show that to prevent the entry and establishment of the bacterium in new areas and its potential spread to other crops, control of ' Ca. L. solanacearum' in seed lots is required. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) disease is seriously threatening and/or damaging the citrus industry worldwide. Accurate detection of the three species associated with HLB disease, 'Candidatus Liberibacter asiaticus', 'Candidatus Liberibacter africanus' and 'Candidatus Liberibacter americanus', is essential for the preventive control of the disease. Real-time PCR is a useful tool for bacterial detection. However, nucleic acid purification steps limit the number of samples that can be processed by PCR. Universal detection of 'Ca. Liberibacter' species was achieved by direct tissue-printing and spotting of plant leaf petiole extracts or squashing of individual psyllids onto paper or nylon membranes. Primers were designed and used with TaqMan chemistry for accurate detection of the bacterium in immobilized targets (prints of 10 overlapping leaf pedicels per tree, or squashed single vectors), by extraction with water and direct use for real-time PCR. This simplified method was validated and could detect HLB-liberibacters in 100% of leaves with symptoms and 59% of symptomless leaves collected from HLB-infected trees. The use of direct assays as template showed good agreement with use of purified DNA (κ = 0·76 ± 0·052). The squash assay allowed detection of the bacterium in 40% of mature Diaphorina citri that fed on leaves of HLB-infected trees with or without symptoms. A commercial ready-made kit based on this technology showed 96% accuracy in intra-laboratory performance studies. The simplified direct methods of sample preparation presented herein can be effectively adopted for use in rapid screening of HLB agents in extensive surveys, certification schemes or for epidemiological and research studies. [ABSTRACT FROM AUTHOR]

The application of horticultural mineral oil ( HMO) treatments has been reported as a possible control strategy to reduce Plum pox virus ( PPV) incidence in Prunus nurseries. The effect of Sunspray Ultrafine HMO at 1% on the natural viral spread was evaluated in experimental nursery plots of Nemaguard and Mariana GF8-1 Prunus rootstock blocks established under high natural inoculum pressure of the most prevalent PPV-types. Tests were conducted in experimental nursery plots in Plovdiv, Bulgaria ( PPV-M and PPV-Rec), in Bistrita, Romania ( PPV-D and PPV-Rec) and in Llíria, Spain ( PPV-D). Horticultural mineral oil treatments were applied weekly during the vegetative period from spring to fall (treatments were interrupted in the summer). Nursery plants were analysed yearly by double-antibody sandwich enzyme-linked immunosorbent assay with 5B-IVIA/ AMR monoclonal antibodies. The population dynamics of the aphids visiting plants in each experimental nursery plot was monitored by the sticky-shoot method and also by Moericke yellow water traps. At all three locations, the aphid population first peaked in the springtime. Furthermore, a variable second peak of aphid population was observed in Plovdiv and Bistrita in autumn. The treatments reduced PPV incidence in the three experimental locations and plots and in both assayed Prunus rootstocks grown under high PPV-inoculum pressure. A reduction from 10% to 20% of PPV-incidence between treated and control plants ( P < 0.05) in Plovdiv and Bistrita, respectively, was observed at the end of the tests. However, HMO treatments did not prevent PPV infection altogether, probably because of the high PPV prevalence in the area near the experimental nursery blocks. The control of PPV in nursery blocks based on HMO is presented as an environmentally friendly strategy based on the physical action of the treatments. [ABSTRACT FROM AUTHOR]

The control of Plum pox virus (PPV), the most important viral disease that affects stone fruit trees, requires the use of reliable detection methods. The effectiveness of spot real-time reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of PPV in samples collected from nursery blocks was compared with a validated PPV detection technique, the double antibody sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA) using the PPV-specific monoclonal antibody 5B-IVIA/AMR. In total, 5047 nursery plants were analysed by both techniques. The agreement between the techniques was almost perfect (Cohen's kappa index of 0·88 ± 0·01). The diagnostic parameters (sensitivity, specificity and likelihood ratios) of both techniques were simultaneously evaluated in 2473 nursery plants by latent class models using maximum likelihood functions and a Bayesian approach. The sensitivity and specificity of both techniques did not vary according to the latent model applied. Spot real-time RT-PCR was more sensitive while DASI-ELISA was more specific for PPV detection. In addition, the findings demonstrate that latent class models are a flexible and potent statistical method to estimate the accuracy of diagnostic tests for plant pathology. [ABSTRACT FROM AUTHOR]

The expression of engineered single-chain variable fragments specific to the NIb RNA replicase of Plum pox virus (PPV) (scFv2A) in transgenic plants was successfully used as a strategy to interfere with viral infection. Different scFv2A fusion proteins were constructed to target those subcellular compartments, such as the cytosol, endoplasmic reticulum (ER) membrane structures and the nucleus, where NIb protein presumably accumulates. Several transgenic lines of Nicotiana benthamiana plants expressing the scFv2A targeted to the cytosol (2A lines), ER (6K2 lines) and nucleus (NLS lines) were obtained. The protective effect of scFv expression was determined by mechanical virus inoculation in five 2A, three 6K2 and four NLS transgenic lines. The strongest resistance was afforded with the 2A-3 (six non-infected plants out of 10), 6K2-1 (17 out of 33) and NLS-11 (16 out of 19) transgenic lines. The success of this interference with PPV infection opens new possibilities for the control of this RNA virus and could be exploited not only to confer resistance in transgenic plants, but also to elucidate the role of the non-structural NIb protein in different cell compartments during viral infection. [ABSTRACT FROM AUTHOR]

Inesfly IGR FITO is an insecticidal paint containing chlorpyrifos and pyriproxyfen incorporated in a micro-encapsulated formulation that confers the advantage of releasing active ingredients slowly. In this study, a 15-cm band of Inesfly IGR FITO was painted around citrus trunks. The efficacy of this paint and a sticky barrier to exclude ants from foraging in citrus trees was evaluated in two citrus orchards during the season in two different ant communities, one dominated by Lasius grandis and the other by Linepithema humile. Field results demonstrated that a single application of Inesfly IGR FITO at the beginning of the season was highly effective in excluding ants from canopies throughout the season. Inesfly IGR FITO provides an efficient and more economical alternative than current ant exclusion strategies used in many perennial crops. Further studies should be performed to determine the effects of this strategy on other pests and on beneficial arthropods in citrus. [ABSTRACT FROM AUTHOR]

The use of rootstocks that are less susceptible or resistant to natural Plum pox virus (PPV) infection and/or the application of mineral oil treatments are two possible strategies to reduce viral incidence in nursery plots. We evaluated the susceptibility of Prunus rootstocks used in the Spanish stone fruit industry and the effect of mineral oil treatment (Sunspray Ultrafine at 1%) on the spread of the virus at two different localities in Valencia, Spain, under different natural PPV inoculum pressures (high inoculum pressure, 2006-08; low inoculum pressure, 2006-07). Samples from both plots were analysed by double-antibody sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA) and spot real-time RT-PCR. Under high inoculum pressure, the assayed rootstocks exhibited significant differences in their susceptibility to natural infection. The most susceptible rootstocks at the end of the experiment were Adesoto 101 and Mariana GF8-1. Cadaman and Garnem rootstocks presented the fewest PPV-infected plants; these infections could be detected only by spot real-time RT-PCR. No differences among the assayed rootstocks were found under low PPV inoculum pressure. Aphid species were monitored using Moericke yellow water traps and sticky-plant methods at both localities in May 2006 and 2007. Aphis spiraecola was the most abundant aphid species monitored by both methods at both localities. The average percentage of A. spiraecola carrying PPV PCR-amplifiable targets was 30.37% in the plot with high PPV inoculum pressure and only 7.98% in the plot with low inoculum pressure. We found significant differences in PPV incidence between Mariana GF8-1 plots that were treated with mineral oil and those that were not treated after one year under natural conditions and high PPV inoculum pressure. [ABSTRACT FROM AUTHOR]

Fifty-six tumorigenic Spanish grapevine strains of Agrobacterium spp. were tested for biovar classification, pathogenicity on several hosts, opine utilization, 16S rRNA gene sequencing and PCR amplifications using five primer sets targeting chromosomal and Ti plasmid genes. Fifty of them belonged to A. vitis (biovar 3), three to A. tumefaciens (biovar 1) and three to A. rhizogenes (biovar 2). All strains were tumorigenic on grapevines. Most A. vitis strains were also pathogenic on tomato and tobacco plants, while the three A. tumefaciens strains were only pathogenic on grapevine. Although most A. vitis strains used octopine, 12 utilized neither octopine nor nopaline. 16S rRNA gene sequencing clearly distinguished between strains belonging to the three species. Those of A. vitis could be further divided into three chromosomal backgrounds according to their 16S ribosomal RNA gene sequences. No universal primer pair was found for the detection of all three Agrobacterium species isolated from grapevine. DNA from all A. vitis strains was amplified with the chromosomally-encoded pehA primer pair. In both A. vitis and A. tumefaciens a correlation was observed between the amplifications obtained using the tmr and the virA Ti-plasmid-targeting primer pairs. Three types of Ti plasmid were found in A. vitis strains according to their PCR amplifications and opine utilization profiles. A given chromosomal background harboured only one type of Ti plasmid within the strains from each analysed sample, showing a strong association between chromosomal backgrounds and Ti plasmids in A. vitis. [ABSTRACT FROM AUTHOR]

Seven markers linked to QTL involved in CTV accumulation, leafminer resistance and apomictic reproduction were used to characterize 64 sour orange ( Citrus aurantium L.) accessions from three national collections in order to identify a representative core in which the resistance behaviour against two Citrus tristeza virus (CTV) isolates was studied. Different degrees of apomixis were found between the foreign collections. Most of the C. aurantium accessions fall into three main groups based on three multilocus genotypes. The haplotype diversity at three CTV accumulation QTL-linked markers was further studied by sequence analysis of alleles. Genotypic and allelic diversity at one of them, tightly linked to Ctv-R 2 in Poncirus trifoliata (L.) Raf. , match the plant–CTV interaction types reported among Poncirus and Citrus species. Only those selected accessions from the major group presented CTV resistance during 30 months of continuous growth, but later the resistance broke down in some plants. CTV tolerance appears related to slow growing genotypes. Certain micronutrients: Mn, and B depending on the accession, might play a relevant role in this host–pathogen interaction particularly in alkaline soils. [ABSTRACT FROM AUTHOR]

A protocol based on Co-operational PCR has been successfully applied to the detection of phytoplasmas. A triprimer reaction coupled with hybridization using general and specific probes permitted detection of ‘ Candidatus Phytoplasma mali’, ‘ Ca. Phytoplasma prunorum’ and ‘ Ca. Phytoplasma pyri’, and their identification as members of 16S ribosomal quarantine group X. The sensitivity of this method was at least one hundred times greater than conventional PCR and similar to that achieved by nested PCR and real-time PCR. The method was validated by testing field samples collected from Malus, Prunus and Pyrus spp. and Olea europaea and compared with seven phytoplasmas maintained in Catharanthus roseus. [ABSTRACT FROM AUTHOR]

Knowledge of the existence of strains or types of Plum pox virus (PPV), defined as monophyletic groups of closely related isolates sharing similar properties, has gradually emerged. Early attempts illustrated both the interest and the limits of biological properties for discrimination of isolates but in the late 1970s gel-immunodiffusion assays allowed the grouping of PPV isolates into two serogroups typified by the Dideron (D) and Marcus (M) isolates. Technical difficulties, however, limited the widespread use of this approach and only in the early 1990s, with the availability of more discriminating and higher throughput techniques, did it become clear that PPV isolates could be allocated into two major groups. This led to the description of the strains, or types, PPV-D and PPV-M. Following this, distant PPV isolates typifying other strains such as El Amar (PPV-EA), Cherry (PPV-C) or Winona (PPV-W) were identified, as well as the widespread group of recombinants known as PPV-Rec. Each of the six known strains or types can now be identified using a range of serological or molecular techniques. However, their evolutionary history and, in particular, the reason for the wide prevalence of the PPV-D, -M and -Rec types relative to PPV-EA, -C or -W is still a matter of speculation. [ABSTRACT FROM AUTHOR]

Tremendous progress has been made in the research and development of Plum pox virus (PPV) serological reagents and methods in recent years. Two facts have revolutionised the serological detection and characterization of the virus: the development of the ELISA method in 1977, and the later emergence of specific monoclonal antibody technology. The availability of commercial kits has popularised PPV diagnosis, now making diagnosis possible at large scale for quarantine purposes, eradication programmes and control of the disease in nurseries. The use of the universal monoclonal antibody 5B-IVIA, used in DASI-ELISA, is the most accurate system for routine PPV detection. Likewise, the use of typing monoclonal antibodies gives exact characterization of the main PPV types described: 4DG5 for PPV-D, AL for PPV-M, EA24 for PPV-EA, and TUV and AC for PPV-C. There is, in general, an excellent correlation between serological data obtained with PPV specific monoclonal antibodies and data obtained by molecular PCR based methods. ELISA using a single or a mixture of monoclonal antibodies will remain the preferred method for universal detection and routine screening of PPV for years to come. Today, other serological methods and reagents are also recommended in the EPPO Diagnostic Protocol, increasing the number of reliable tests available for PPV detection. These developments have helped to control sharka disease in recent years. International co-operation in this field has been crucial to the improvement and validation of serological tools for PPV detection and characterization. [ABSTRACT FROM AUTHOR]

Thirteen Spanish potato isolates of Dickeya chrysanthemi obtained from different cultivars, regions and years have been characterised by biochemical, physiological, serological, molecular and pathogenicity tests. Isolates studied have been classified into biovars 1, 3, 5 and 6 by a microtitre system that correlates with the conventional tests in tubes. This is the first description of the characteristics of D. chrysanthemi isolated from potato in Spain and of the development of a microtitre system for biovar determination. [ABSTRACT FROM AUTHOR]

Focuses on commercial detection and quantification of the backleg pathogen Erwinia carotovora subsp. atroseptica on seed potato tubers. Seed contamination level; Microbiological, immunological and molecular methods.

A citrus ringspot isolate from Star Ruby grapefruit (RS-SR) was mechanically transmitted to Chenopodium quinoa. RS-SR was partially purified by differential centrifugation, fractionation in a sucrose gradient, and agarose gel electrophoresis of selected fractions. Infectivity of concentrated extracts on C. quinoa was lost in individual fractions of the gradient, but it was recovered by combining a top and a bottom component. Both components contained a 48-kDa protein not found in similar preparations from healthy plants. After further purification the 48 kDa protein was detected at the top edge of the agarose gel. In the initial experiments a 38-kDa protein was found in the same fractions that later contained the 48-kDa protein. An antiserum obtained to the 38 kDa protein reacted in Western blots with both the 38- and the 48-kDa proteins, whereas another antiserum raised to the Florida isolate CRSV-4 (also containing a 48-kDa protein) did not re, act with the 38-kDa protein, indicating that the latter was probably a degradation product of the 48-kDa protein. Filamentous flexous particles were observed by serologically specific electron microscopy in crude extracts from RS-SR-infected C. quinoa plants. These results indicate that RS-SR is associated with a two-component virus similar to those associated with several psorosis and ringspot isolates, and serologically related to CRSV-4. [ABSTRACT FROM AUTHOR]

A survey of citrus tristeza virus (CTV) isolates was carried out in most citrus-growing areas in Spain. Twenty-two isolates were selected by geographical origin, cultivar of source tree, and symptoms observed on the host or in preliminary tests, and were biologically characterized. A wide range of variation in transmissibility by aphids and symptom intensity on nine different indicator species or scion-rootstock combinations was observed among CTV isolates. Mexican lime, Citrus macrophylla, and to a lesser extent citron were the most useful hosts for characterizing these isolates, and leaf symptoms and stem pitting were the most discriminating traits. Positive correlation was observed between symptoms induced on Mexican lime and C. macrophylla, but not between the symptoms induced on these indicators under greenhouse conditions and the homologous symptoms on plants grown in the screenhouse. Some of the traits studied enabled us to establish relatively well-defined groups of isolates, but in most cases a continuous range of variation was obtained and no clear group could be defined. [ABSTRACT FROM AUTHOR]

Since first being recorded in 1917–18 in Bulgaria, sharka (plum pox) disease has progressively spread via infected plant material to be present in most Prunus-growing nations today. The disease has serious agronomic and political consequences because it causes enormous economic losses. In countries in which sharka is endemic, a high percentage of apricot and European plum production is unmarketable because of the disease. To these figures should be added the costs of sanitary controls, surveys and eradication programmes against sharka virus. Estimated costs associated with sharka management worldwide in the last 30 years exceed 10 000 million euros. However, improvements in knowledge of the disease and in techniques used to identify the disease are significantly aiding disease control and management. [ABSTRACT FROM AUTHOR]

A description of plum pox (sharka) symptoms on European and Japanese plum, apricot, peach, sweet and sour cherry and almond is presented. Plum pox is a serious disease mainly because it produces severe symptoms on fruits of numerous cultivars of Prunus species, causing fruit to fall or making them unfit for consumption. [ABSTRACT FROM AUTHOR]

Focuses on the methods for detecting Phytophthora nicotianae. Description of physiological characters; Analysis of species-specific polymerase chain reaction characterization; Requirements for the agronomic impact of pathogens.

The article reports on the presence of Plum pox virus (PPV) in Spain. It was first detected on Japanese plum, Red Beaut which became an important source of inoculum and aphid vectors spreading PPV to other Japanese plum cultivars and apricots. Management of the disease includes production of certified virus-tested plants, control of mother plants producing standard material and the eradication of PPV-D infected trees in some regions. In addition, permanent surveys are also conducted.

The article presents the incidence of Plum Pox Virus in Kazakhstan in 2004. The article mentions the presence of the Plum Pox Virus (PPV) on plum and apricot trees, which corresponded to the PPV-D strains. The article also mentions a reference article "Detection and partial molecular characterization of two Plum pox virus isolates from plum and wild apricot in southeast Kazakhstan," by Spiegal S and others.

Focuses on the DIAGPRO project of the European Union for development of diagnostic protocols for quarantine pests. Detection of organisms harmful to plants; Inclusion of nematodes, bacteria, fungi, viruses and viroids; Importance of detection and identification of the organisms for plant protection.

An abstract of the article "First report of bacterial soft rot on onion caused by Dickeya sp. (ex Pectobacterium chrysanthemi ) in Spain," by A. Palacio-Bielsa, M. A. Cambra and M. M. López is presented.

The article presents the incidence of the plum pox virus outside the continent of Europe. The article mentions the detection of a mixed infection of PPV-D and PPV-Rec strains on apricot trees in the Baltistan District in the Northern Pakistan. The article also provides the details of reference material.