cmv1 is a gate for Cucumber mosaic virus transport from bundle sheath cells to phloem in melon

Cucumber mosaic virus (CMV) has the broadest host range among plant viruses, causing enormous losses in agriculture. In melon, strains of subgroup II are unable to establish a systemic infection in the near‐isogenic line SC12‐1‐99, which carries the recessive resistance gene cmv1 from the accession PI 161375, cultivar ‘Songwhan Charmi’. Strains of subgroup I overcome cmv1 resistance in a manner dependent on the movement protein. We characterized the resistance conferred by cmv1 and established that CMV‐LS (subgroup II) can move from cell to cell up to the veins in the inoculated leaf, but cannot enter the phloem. Immunogold labelling at transmission electron microscopy level showed that CMV‐LS remains restricted to the bundle sheath (BS) cells in the resistant line, and does not invade vascular parenchyma or intermediary cells, whereas, in the susceptible line ‘Piel de Sapo’ (PS), the virus invades all vein cell types. These observations indicate that the resistant allele of cmv1 restricts systemic infection in a virus strain‐ and cell type‐specific manner by acting as an important gatekeeper for virus progression from BS cells to phloem cells. Graft inoculation experiments showed that CMV‐LS cannot move from the infected PS stock into the resistant cmv1 scion, thus suggesting an additional role for cmv1 related to CMV transport within or exit from the phloem. The characterization of this new form of recessive resistance, based on a restriction of virus systemic movement, opens up the possibility to design alternative approaches for breeding strategies in melon.     

Dissection of the oligogenic resistance to Cucumber mosaic virus in the melon accession PI 161375

Resistance to Cucumber mosaic virus (CMV) in the exotic melon accession PI 161375, cultivar "Sonwang Charmi" (SC) had previously been described as oligogenic, recessive and quantitative, with a major QTL residing in linkage group XII (LGXII). We have used a collection of near isogenic lines (NILs) with introgressions of SC into the genome of the susceptible accession Piel de Sapo (PS) to further characterise this resistance. Infection of NILs carrying introgressions on LGXII showed that only NIL SC12-1 was resistant to CMV strains P9 and P104.82, but not to strains M6 and TL. Further mapping of this region showed that the resistance, named cmv1 maps in an area of 2.2 cM, between markers CMN61_44 and CMN21_55. Moreover, cmv1 confers total resistance to strains P9 and P104.82, indicating that in these cases it is not quantitative and that cmv1 is sufficient to confer full resistance to these CMV strains. Candidate gene mapping of ten translation initiation factors in the melon genome failed to find any of them in the interval between markers CMN61_44 and CMN21_55. All these results suggest that the resistance to CMV present in SC is oligogenic, where different loci confer resistance to different CMV strains, but not necessarily quantitative, since at least one of these genes (cmv1) confers total resistance, similar to that of the parental SC, and does not need the contribution of other loci.     

The complex resistance to cucumber mosaic cucumovirus (CMV) in the melon accession PI161375 is governed by one gene and at least two quantitative trait loci

The complex resistance to cucumber mosaic virus (CMV) present in the exotic melon accession Sonwang Charmi PI161375 (SC) has been studied using two populations, a near-isogenic line (NIL) collection and a doubled haploid line (DHL) collection, both generated from a cross between SC and the cultivar Piel de Sapo as resistant and susceptible parents, respectively. The NIL collection had previously allowed us to describe a single recessive gene, cmv1, which conferred full resistance to CMV strains P9 and P104.82. Screening of the whole DHL population followed by quantitative trait locus (QTL) analysis revealed that resistance to the strains M6 and TL, both non-responsive to cmv1, was quantitative and governed by at least three QTLs. One of them, cmvqw12.1, co-located with cmv1 in linkage group (LG) XII. The QTL analysis mapped another two QTLs in LGIII (cmvqw3.1) and LGX (cmvqw10.1) and showed interaction between cmvqw12.1 and cmvqw3.1. Progeny of crosses between resistant DHLs carrying the three main QTLs showed complete resistance to the strain M6, validating the accuracy of the QTL analysis. However, in our screening, there were resistant DHLs carrying only two QTLs, suggesting that there are other regions involved in resistance to M6 and required when one of the main QTLs is missing. Therefore, resistance to CMV in melon SC is qualitative for some strains and quantitative for the rest. For this late resistance, cmv1 is necessary and explains most of the phenotypic variance, but it is not sufficient, and needs the interaction with other loci.     

Resistance to Cucumber mosaic virus in Gladiolus plants transformed with either a defective replicase or coat protein subgroup II gene from Cucumber mosaic virus

Transgenic Gladiolus plants that contain either Cucumber mosaic virus (CMV) subgroup I coat protein, CMV subgroup II coat protein, CMV replicase, a combination of the CMV subgroups I and II coat proteins, or a combination of the CMV subgroup II coat protein and replicase genes were developed. These plants were multiplied in vitro and challenged with purified CMV isolated from Gladiolus using a hand-held gene gun. Three out of 19 independently transformed plants expressing the replicase gene under control of the duplicated CaMV 35S promoter were found to be resistant to CMV subgroup I. Three out of 21 independently transformed plants with the CMV subgroup II coat protein gene under control of the Arabidopsis UBQ3 promoter were resistant to CMV subgroup II. Eighteen independently transformed plants with either the CMV subgroup I coat protein or a combination of CMV subgroups I and II coat proteins were challenged and found to be susceptible to both CMV subgroups I or II. Virus resistant plants with the CMV replicase transgene expressed much lower RNA levels than resistant plants expressing the CMV subgroup II coat protein. This work will facilitate the evaluation of virus resistance in transgenic Gladiolus plants to yield improved floral quality and productivity.     

CMV甜瓜分离物外壳蛋白基因的克隆及植物表达载体的构建

从河南省临颖县采集的病毒感染的甜瓜样本经ELISA检测和接种分离获得黄瓜花叶病毒(Cucunbermosaicvirus,CMV)分离物。把该分离物接种西葫芦,从发病的叶片中提取总RNA,并以此为模板经RTPCR扩增获得CMV的外壳蛋白(cp)基因,将其克隆到pUCmT质粒上。经序列测定和分析,结果表明该cp基因由657个核苷酸组成,编码218个氨基酸。其核苷酸序列与黄瓜花叶病毒亚组I的分离物有较高的同源性,达92.2%～93.9%,与亚组II的同源性仅为76.8%～77.8%,与我国报道的CMV分离物的cp基因序列比较,除香蕉株系XB外核苷酸序列的同源性达91.8%～93.4%。根据这些分析,该CMV分离物属于亚组I。将cp基因通过中间载体pJIT163定向克隆到植物表达载体pBINPLUS中(重组双元载体质粒命名为pBCP),并经冻融法导入农杆菌中,经PCR及酶切鉴定,证实质粒已被导入。利用该植物表达载体对西瓜的遗传转化工作目前正在进行中。     

Extreme resistance to cucumber mosaic virus (CMV) in transgenic tomato expressing one or two viral coat proteins

For the production of broad commercial resistance to cucumber mosaic virus (CMV) infection, tomato plants were transformed with a combination of two coat protein (CP) genes, representing both subgroups of CMV. The CP genes were cloned from the CMV-D strain and Italian CMV isolates (CMV-22 of subgroup I and CMV-PG of subgroup II) which have been shown to produce severe disease symptoms. Four plant transformation vectors were constructed: pMON18774 and pMON18775 (CMV-D CP), pMON18831 (CMV-PG CP) and pMON18833 (CMV-22 CP and CMV-PG CP). Transformed R0 plants were produced and lines were selected based on the combination of three traits: CMV CP expression at the R0 stage, resistance to CMV (subgroup I and/or II) infection in growth chamber tests in R1 expressing plants, and single transgene copy, based on R1 segregation. The results indicate that all four vector constructs generated plants with extremely high resistant to CMV infection. The single and double gene vector construct produced plants with broad resistance against strains of CMV from both subgroups I and II at high frequency. The engineered resistance is of practical value and will be applied for major Italian tomato varieties. This revised version was published online in June 2006 with corrections to the Cover Date.     

Blockage of stylet tips as the mechanism of resistance to virus transmission by Aphis gossypii in melon lines bearing the Vat gene

Aphis gossypii is the main virus vector in muskmelon crops. The melon gene Vat confers resistance to non‐persistent virus transmission by this aphid. The mechanism of this resistance is not well understood, but no relationship has been detected between resistance and the probing behaviour of aphids on resistant plants. Results presented here suggest that temporary blockage of aphid stylet tips preventing virus particle release may explain the resistance conferred by Vat gene. We performed experiments in which viruliferous aphids were allowed to probe different sequences of resistant (Vat‐bearing) and/or susceptible melon plants. The results demonstrated that A. gossypii inoculates Cucumber mosaic virus (CMV) efficiently in susceptible plants having previously probed resistant plants, showing that the resistance mechanism is reversible. Furthermore, the infection rate obtained for susceptible plants was the same (25%) regardless of whether the transmitting aphid had come directly from the CMV source or had subsequently probed on resistant plants. This result suggests that virus is not lost from stylet to plant during probing of resistant plants, supporting the temporary blockage hypothesis. We also found that the ability of Myzus persicae to transmit CMV is noticeably reduced after probing on resistant plants, providing evidence that this aphid species also responds to the presence of the Vat gene. Finally, we also found that in probes immediately after virus acquisition M. persicae inoculates resistant plants with CMV more efficiently than susceptible plants, perhaps because the Vat gene product induces increased salivation by this aphid.     

Cucumber mosaic virus 2b proteins inhibit virus-induced aphid resistance in tobacco

Cucumber mosaic virus (CMV), which is vectored by aphids, has a tripartite RNA genome encoding five proteins. In tobacco (Nicotiana tabacum), a subgroup IA CMV strain, Fny-CMV, increases plant susceptibility to aphid infestation but a viral mutant unable to express the 2b protein (Fny-CMV∆2b) induces aphid resistance. We hypothesized that in tobacco, one or more of the four other Fny-CMV gene products (the 1a or 2a replication proteins, the movement protein, or the coat protein) are potential aphid resistance elicitors, whilst the 2b protein counteracts induction of aphid resistance. Mutation of the Fny-CMV 2b protein indicated that inhibition of virus-induced resistance to aphids (Myzus persicae) depends on amino acid sequences known to control nucleus-to-cytoplasm shuttling. LS-CMV (subgroup II) also increased susceptibility to aphid infestation but the LS-CMV∆2b mutant did not induce aphid resistance. Using reassortant viruses comprising different combinations of LS and Fny genomic RNAs, we showed that Fny-CMV RNA 1 but not LS-CMV RNA 1 conditions aphid resistance in tobacco, suggesting that the Fny-CMV 1a protein triggers resistance. However, the 2b proteins of both strains suppress aphid resistance, suggesting that the ability of 2b proteins to inhibit aphid resistance is conserved among divergent CMV strains.     

Rearrangements in the 5′ Nontranslated Region and Phylogenetic Analyses of Cucumber Mosaic Virus RNA 3 Indicate Radial Evolution of Three Subgroups

Cucumber mosaic virus (CMV) has been divided into two subgroups based on serological data, peptide mapping of the coat protein, nucleic acid hybridization, and nucleotide sequence similarity. Analyses of a number of recently isolated strains suggest a further division of the subgroup I strains. Alignment of the 5' nontranslated regions of RNA 3 for 26 strains of CMV suggests the division of CMV into subgroups IA, IB, and II and suggests that rearrangements, deletions, and insertions in this region may have been the precursors of the subsequent radiation of each subgroup. Phylogeny analyses of CMV using the coat protein open reading frame of 53 strains strongly support the further division of subgroup I into IA and IB. In addition, strains within each subgroup radiate from a single point of origin, indicating that they have evolved from a single common ancestor for each subgroup.     

Analysis of the coat protein gene and untranslated region of RNA3 of Cucumber Mosaic Virus isolates infecting various Lilium species and hybrids: association of the isolate infecting asiatic hybrid lily with subgroup II

The variability in the coat protein gene of Cucumber mosaic virus (CMV) isolates from various Lilium species and hybrids namely L. longiflorum, L. tigrinum, Asiatic hybrid and Oriental hybrid lilies was studied by sequence comparison of ～900 bp regions spanning the entire coat protein, intercistronic regions and 3′-UTR. CMV isolate characterised from Asiatic hybrid lily showed the highest homology with subgroup II isolates (94 – 97%), whereas 73 – 76% homology was observed with those belonging to subgroup I. Similarly, another three isolates showed 91 – 98% amino acid sequence homology with subgroup I and 74 – 76% sequence homology with subgroup II. Based on the criteria for classification of CMV isolates all the Indian isolates fall in subgroup I, except the one characterized from Asiatic Hybrid lily which falls into subgroup II. Other lily isolates from world were placed in subgroup II. This is the first case of Asiatic hybrid lily CMV isolate belonging to subgroup II.     

Differentiation of Bulgarian Isolates from Cucumber Mosaic Virus by Serological Methods

Cucumber mosaic virus (CMV) is of great importance to the Bulgarian economy and hence a detailed knowledge of its diversity under local geographic and climatic conditions is required. An extended study was carried out on CMV strains the currently occur in Bulgaria. Fifty-one isolates and strains found in different regions and various crops were biologically characterized and serologically differentiated into subgroups I and II using different variants of enzyme-linked immunosorbent assay (ELISA) [double antibody sandwich (DAS)-, antigen-coated plate (ACP)-, triple antibody sandwich (TAS)- with poly and monoclonal antibodies] and immunodiffusion tests. The ELISA modifications with monoclonal antibodies individually (ACP) or in combination with polyclonal antibodies (TAS-ELISA) are suitable for mass screening of CMV isolates. The hyperimmune sera against strains from CMV subgroups I and II were very efficient for use in isolate differentiation via gel double immunodiffusion. The results obtained correlated with the polymerase chain reaction and restriction fragment length polymorphism data reported by other authors. The majority of the isolates belonged to subgroup I, whereas 10, mainly from tomato and pepper, belonged to subgroup II. Most of the subgroup II isolates came from the north of Bulgaria. The results of the present study will help to clarify the virus epidemiology and to develop specific control measures.     

The groEL gene as an additional marker for finer differentiation of ‘Candidatus Phytoplasma asteris'‐related strains

Phytoplasma classification established using 16S ribosomal groups and ‘Candidatus Phytoplasma’ taxon are mainly based on the 16S rDNA properties and do not always provide molecular distinction of the closely related strains such as those in the aster yellows group (16SrI or ‘Candidatus Phytoplasma asteris'‐related strains). Moreover, because of the highly conserved nature of the 16S rRNA gene, and of the not uncommon presence of 16S rDNA interoperon sequence heterogeneity, more variable single copy genes, such as ribosomal protein (rp), secY and tuf, were shown to be suitable for differentiation of closely related phytoplasma strains. Specific amplification of fragments containing phytoplasma groEL allowed studying its variability in 27 ‘Candidatus Phytoplasma asteris'‐related strains belonging to different 16SrI subgroups, of which 11 strains were not studied before and 8 more were not studied on other genes than 16S rDNA. The restriction fragment length polymorphism (RFLP) analyses of the amplified fragments confirmed differentiation among 16SrI‐A, I‐B, I‐C, I‐F and I‐P subgroups, and showed further differentiation in strains assigned to 16SrI‐A, 16SrI‐B and 16SrI‐C subgroups. However, analyses of groEL gene failed to discriminate strains in subgroups 16SrI‐L and 16SrI‐M (described on the basis of 16S rDNA interoperon sequence heterogeneity) from strains in subgroup 16SrI‐B. On the contrary, the 16SrI unclassified strain ca2006/5 from carrot (showing interoperon sequence heterogeneity) was differentiable on both rp and groEL genes from the strains in subgroup 16SrI‐B. These results indicate that interoperon sequence heterogeneity of strains AY2192, PRIVA (16SrI‐L), AVUT (16SrI‐M) and ca2006/5 resulted in multigenic changes – one evolutionary step further – only in the latter case. Phylogenetic analyses carried out on groEL are in agreement with 16Sr, rp and secY based phylogenies, and confirmed the differentiation obtained by RFLP analyses on groEL amplicons.     

Expression of coat protein gene of Cucumber mosaic virus (CMV-subgroup IA) Gladiolus isolate in Nicotiana tabacum

The coat protein (CP)-mediated resistance against Cucumber mosaic virus (CMV) subgroup IA was developed in transgenic lines of Nicotiana tabacum cv. Petit Havana using Agrobacterium tumefaciens-mediated transformation. Ten independently transformed lines have developed, four of which were tested for resistance against CMV using virus challenge inoculations. The transgenic lines exhibiting complete resistance remained healthy and symptomless in their life span and showed reduced or no virus accumulation in their systemic leaves after virus challenge inoculation. These transgenic lines also showed resistance against CMV strains which are not closely related to CMV-Gladiolus strains. This is the first report of CP-mediated transgenic resistance against a CMV subgroup IA member isolated from India showing resistance to all CMV strains occurring in the same vicinity.     

Genetic Structure and Molecular Variability of Cucumber mosaic virus Isolates in the United States

Cucumber mosaic virus (CMV) has a worldwide distribution and the widest host range of any known plant virus. From 2000 to 2012, epidemics of CMV severely affected the production of snap bean (Phaseulos vulgaris L.) in the Midwest and Northeastern United States. Virus diversity leading to emergence of new strains is often considered a significant factor in virus epidemics. In addition to epidemics, new disease phenotypes arising from genetic exchanges or mutation can compromise effectiveness of plant disease management strategies. Here, we captured a snapshot of genetic variation of 32 CMV isolates collected from different regions of the U.S including new field as well as historic isolates. Nucleotide diversity (π) was low for U.S. CMV isolates. Sequence and phylogenetic analyses revealed that CMV subgroup I is predominant in the US and further showed that the CMV population is a mixture of subgroups IA and IB. Furthermore, phylogenetic analysis suggests likely reassortment between subgroups IA and IB within five CMV isolates. Based on phylogenetic and computational analysis, recombination between subgroups I and II as well as IA and IB in RNA 3 was detected. This is the first report of recombination between CMV subgroups I and II. Neutrality tests illustrated that negative selection was the major force operating upon the CMV genome, although some positively selected sites were detected for all encoded proteins. Together, these data suggest that different regions of the CMV genome are under different evolutionary constraints. These results also delineate composition of the CMV population in the US, and further suggest that recombination and reassortment among strain subgroups does occur but at a low frequency, and point towards CMV genomic regions that differ in types of selection pressure.     

Identification of quantitative trait loci associated with resistance to cucumber mosaic virus in Capsicum annuum

QTL analysis for resistance to cucumber mosaic virus (CMV) was performed in an intraspecific Capsicum annuum population. A total of 180 F3 families were derived from a cross between the susceptible bell-type cultivar Maor and the resistant small-fruited Indian line Perennial and inoculated with CMV in three experiments carried out in the USA and Israel using two virus isolates. Mostly RFLP and AFLP markers were used to construct the genetic map, and interval analysis was used for QTL detection. Four QTL were significantly associated with resistance to CMV. Two digenic interactions involving markers with and without an individual effect on CMV resistance were also detected. The QTL controlling the largest percentage (16–33%) of the observed phenotypic variation (cmv11.1) was detected in all three experiments and was also involved in one of the digenic interactions. This QTL is linked to the L locus that confers resistance to tobacco mosaic virus (TMV), confirming earlier anecdotal observations of an association between resistance to CMV and susceptibility to TMV in Perennial. An advanced backcross breeding line from an unrelated population, 3990, selected for resistance to CMV was analyzed for markers covering the genome, allowing the identification of genomic regions introgressed from Perennial. Four of these introgressions included regions associated with QTL for CMV resistance. Markers in two genomic regions that were identified as linked to QTL for CMV resistance were also linked to QTL for fruit weight, confirming additional breeding observations of an association between resistance to CMV originating from Perennial and small fruit weight. Received: 17 July 2000 / Accepted: 16 October 2000     

Resistance phenotypes of transgenic tobacco plants expressing different cucumber mosaic virus (CMV) coat protein genes

Tobacco plants expressing a transgene encoding the coat protein (CP) of a subgroup I strain of cucumber mosaic cucumovirus (CMV), I17F, were not resistant to strains of either subgroup I or II. In contrast, the expression of the CP of a subgroup II strain, R, conferred substantial resistance, but only towards strains of the same subgroup. When protection was observed, the levels of resistance were similar when plants were inoculated with either virions or viral RNA, but resistance was more effective when plants were inoculated with viruliferous aphids. Resistance was not dependent on inoculum strength and was expressed as a recovery phenotype not yet described for plants expressing a CMV CP gene. Recovery could be observed either early in infection (less than one week after inoculation) or later (4 to 5 weeks after inoculation). In plants showing early recovery, mild symptoms were observed on the inoculated leaves, and in some cases symptoms developed on certain lower systemically infected leaves, but the upper leaves were symptomless and virus-free. Late recovery corresponded to the absence of both symptoms and virus in the upper leaves of plants that were previously fully infected. Northern blot analyses of resistant plants suggested that a gene silencing mechanism was not involved in the resistance observed.     

A physical map covering the nsv locus that confers resistance to Melon necrotic spot virus in melon (Cucumis melo L.)

Melon necrotic spot virus (MNSV) is a member of the genus Carmovirus, which produces severe yield losses in melon and cucumber crops. The nsv gene is the only known natural source of resistance against MNSV in melon, and confers protection against all widespread strains of this virus. nsv has been previously mapped in melon linkage group 11, in a region spanning 5.9 cM, saturated with RAPD and AFLP markers. To identify the nsv gene by positional cloning, we started construction of a high-resolution map for this locus. On the basis of the two mapping populations, F₂ and BC1, which share the same resistant parent PI 161375 (nsv/nsv), and using more than 3,000 offspring, a high-resolution genetic map has been constructed in the region around the nsv locus, spanning 3.2 cM between CAPS markers M29 and M132. The availability of two melon BAC libraries allowed for screening and the identification of new markers closer to the resistance gene, by means of BAC-end sequencing and mapping. We constructed a BAC contig in this region and identified the marker 52K20sp6, which co-segregates with nsv in 408 F₂ and 2.727 BC1 individuals in both mapping populations. We also identified a single 100 kb BAC that physically contains the resistance gene and covers a genetic distance of 0.73 cM between both BAC ends. These are the basis for the isolation of the nsv recessive-resistance gene.     

Phenotypic traits,virulence‐associated gene profile and genetic relatedness of Edwardsiella tarda isolates from Japanese eel Anguilla japonica in Korea

Edwardsiella tarda is the predominant bacterium in farm‐cultured eel in Korea. Here, we evaluated the heterogeneity of 37 E. tarda isolates derived from Japanese eel with various origins (olive flounder, common carp and ornamental fish) between 2003 and 2010. Regardless of origins, the biochemical characteristics of E. tarda isolates were homogenous except hydrogen sulfide production, citrate utilization and mannitol fermentation. Based on the phylogenetic analysis of 16S rRNA, E. tarda isolates could be classified into two subgroups and displayed a close relation with Edwardsiella ictaluri and Edwardsiella hosinae lineages, suggesting that the subgroup I has been a predominant type in the Jeonnam and Jeonbuk provinces. I‐CeuI‐based pulsed‐field gel electrophoresis (PFGE) typing showed that the isolates from Japanese eels belonged to 11 pulsotypes, indicating that the presence of highly genomic diversity. Additionally, two isolates, ET‐060 and ET‐191, showed a high frequency of virulence genes (100%) and caused 90% and 60% mortality in Japanese eel, respectively. This finding suggests a substantial congruence of virulence gene profiles and pathogenicity. Our results demonstrate that the intraspecific diversity within E. tarda strains from Japanese eel has been in prior existence.

Significance and Impact of the Study

Based on the biochemical characteristics, the phylogenetic property of the 16S rRNA gene and PFGE types of Edwardsiella tarda, we could identify the intraspecific diversity of isolates from Japanese eel, Anguilla japonica in Korea. In addition, this study describes the strong congruence of virulence‐related genes and pathogenicity, suggesting that the virulence profile may be useful tool for prediction of pathogenicity.     

Characterization, Genetic Diversity, and Evolutionary Link of Cucumber mosaic virus Strain New Delhi from India

The genome of Cucumber mosaic virus New Delhi strain (CMV-ND) from India, obtained from tomato, was completely sequenced and compared with full genome sequences of 14 known CMV strains from subgroups I and II, for their genetic diversity. Sequence analysis suggests CMV-ND shares maximum sequence identity at the nucleotide level with a CMV strain from Taiwan. Among all 15 strains of CMV, the encoded protein 2b is least conserved, whereas the coat protein (CP) is most conserved. Sequence identity values and phylogram results indicate that CMV-ND belongs to subgroup I. Based on the recombination detection program result, it appears that CMV is prone to recombination, and different RNA components of CMV-ND have evolved differently. Recombinational analysis of all 15 CMV strains detected maximum recombination breakpoints in RNA2; CP showed the least recombination sites.