Cherry Green Ring Mottle Virus Research Articles

Development of a multiplex RT-PCR and the occurrence characteristics of viral diseases in sweet cherry trees in the Shandong Province, China

Viral diseases have become a major threat to sweet cherry production worldwide. In this study, a broad survey on 17 target species of viruses was performed by RT-PCR detection of leaf samples with suspected symptoms of viral infection collected from 110 orchards in 13 cities of the Shandong Province, the largest sweet cherry production area in China. Eight viruses were identified, including little cherry virus 1, cherry green ring mottle virus, prunus necrotic ring spot virus, cherry virus A, cucumber mosaic virus, plum bark necrosis stem pitting-associated virus, prune dwarf virus and little cherry virus 2. To simultaneously detect different viruses infecting sweet cherry trees, a multiplex RT-PCR method for these 8 viruses was developed and performed. Based on the multiplex RT-PCR data, sweet cherry trees in most orchards were co-infected with multiple viruses, even some trees were co-infected with eight species of viruses. All of these viruses predominantly infected sweet cherry trees cultivars Hongdeng and Tieton. These viruses appear to be not evenly distributed among different regions in the Shandong Province, which Yantai and Tai'an cities represented two centers of sweet cherry virus diseases in the Shandong Province. These data will provide the basis for the detection and management of viral diseases in sweet cherry production.

Cherry green ring mottle virus. [Distribution map

Abstract A new distribution map is provided for Cherry green ring mottle virus. Tymovirales: Betaflexiviridae: Robigovirus. Hosts: Prunus spp. Information is given on the geographical distribution in Africa (South Africa), Asia (China, Beijing, Fujian, Hebei, Shandong, Xinjiang, Japan, Honshu, Lebanon, Pakistan, Korea Republic, Turkey), Europe (Belgium, Bulgaria, Czechia, France, Germany, Hungary, Italy, Montenegro, Poland, Russia, Serbia, Slovenia, Switzerland), North America (Canada, British Columbia, Ontario, USA, California, Idaho, Michigan, New York, Oregon, Washington, Wisconsin), Oceania (Australia, New Zealand), South America (Chile).

Cherry green ring mottle virus (cherry vein yellow spot)

Cherry green ring mottle virus (cherry vein yellow spot)

A Survey for Viruses and Viroids of Peach in Tennessee Orchards by RNA Sequencing

The prevalent viruses and viroids in a population of 100 peach trees grown in Tennessee (TN) orchards were investigated by RNA sequencing and analysis of a pool of ribosomal-depleted total RNA derived from these plants. The sequences of nine viruses (cherry green ring mottle virus; cherry necrotic rusty mottle virus; grapevine-associated ilarvirus, GaIV; nectarine virus M, NeVM; Prunus necrotic ringspot virus, PNRSV; tomato mosaic virus, ToMV; tomato ringspot virus, ToRSV; turnip vein-clearing virus, TVCV; and white clover mosaic virus, WClMV) and one viroid (peach latent mosaic viroid, PLMVd) were detected. The presence of ToMV, PNRSV, NeVM, ToRSV and PLMVd sequences were confirmed in the original samples by RT-PCR followed by Sanger sequencing. This study represents the first survey of viruses and viroids of peach trees in TN orchards. To the best of our knowledge, ToMV, NeVM, GaIV, TVCV, and WClMV have not been reported previously associated with peach trees elsewhere in the world.

First Report of Mume Virus A Infection of Prunus persica in China

Mume virus A, belonging to Capillovirus in the family Betaflexiviridae, was discovered recently from Japanese apricot (Prunus mume) and exhibited diffuse chlorotic spots on leaves (Marais et al. 2018). To date, Japanese apricot is the only known host of Mume virus A (MuVA) (Maliogka et al. 2018). Peach (Prunus persica) is a delicious and healthy summer fruit grown in most temperate regions of the world. More than 20 different viruses infecting peach trees have been reported (Jo et al. 2018). In April 2019, the leaves from four peach trees showing severe yellow mosaic and leaf curling were collected from peach orchards in Xinjiang, China. The pooled total RNA was extracted from these four trees leaves with TRIzol reagent (Life Technologies Corporation, Carlsbad, CA) and subjected to high-throughput sequencing (HTS) using a PE150 sequence strategy on an Illumina HiSeq2000 (Novogene Corporation, Tianjin, China). Approximately 39.3 million of 150-bp paired-end reads were obtained and used for de novo transcriptome assembly by SPAdes Genome Assembler (Bankevich et al. 2012). A total of 89,163 contigs with N50 of 866 bp were obtained and then were annotated by BLASTX analysis against the NCBI nr virus database (Altschul et al. 1997). Six known viruses (apple chlorotic leaf spot virus, Asian prunus virus 2, cherry green ring mottle virus, nectarine stem pitting-associated virus, peach associated luteovirus, and plum bark necrosis stem pitting-associated virus) and one known viroid (hop stunt viroid) were identified. Moreover, a 7,623-bp contig with a coverage of 84.4-fold was identified as capillovirus-related sequence. To classify the capillovirus accurately, pairwise genome alignments analysis was performed between all members of the Capillovirus genus using PASC online tool (Bao et al. 2014). The closest match was the genome of MuVA (NC_040568.1), sharing 78.36% nucleotide identity, followed by those of some isolates of cherry virus A, sharing less than 58.5% nucleotide identity. This result suggests that the capillovirus from peach belongs to the same species with the MuVA from apricot but should be a different isolate. We designated it as isolate pp, and its HTS-derived viral genome sequence was deposited in GenBank (accession no. MN412555). To confirm the HTS experiment results, from the same peach orchards, 23 peach leaf samples from different trees were collected, and total RNA was extracted with TRIzol reagent with standard procedures following the instructions. The RNA was subsequently used to test for pp isolate by reverse transcription polymerase chain reaction (PCR) using specific primers DT-MVAF5716 (5′-CTTCACATCGGAGCTGTCCT-3′) and DT-MVAR6277 (5′-TGCTGATTGCTTCAGACCCT-3′), targeting a portion of the open reading frame 2 (putative movement protein). Eleven samples produced the expected 560-bp PCR fragment. Three PCR products randomly selected were cloned into pMD19-T Vector (TaKaRa) for Sanger sequencing. All sequences of amplicons shared nearly 100% sequence identity with the corresponding region of the HTS-derived viral genome sequence. However, at least the other two viruses and viroid had been detected in all 11 MuVA-positive samples, so the symptoms of the initial sample cannot be explained by MuVA infection. To our knowledge, this is the first report of MuVA infecting peach trees. This report suggests that MuVA may have a broad host range in the genus Prunus.

Updating the Quarantine Status of Prunus Infecting Viruses in Australia.

One hundred Prunus trees, including almond (P. dulcis), apricot (P. armeniaca), nectarine (P. persica var. nucipersica), peach (P. persica), plum (P. domestica), purple leaf plum (P. cerasifera) and sweet cherry (P. avium), were selected from growing regions Australia-wide and tested for the presence of 34 viruses and three viroids using species-specific reverse transcription-polymerase chain reaction (RT-PCR) or polymerase chain reaction (PCR) tests. In addition, the samples were tested using some virus family or genus-based RT-PCR tests. The following viruses were detected: Apple chlorotic leaf spot virus (ACLSV) (13/100), Apple mosaic virus (ApMV) (1/100), Cherry green ring mottle virus (CGRMV) (4/100), Cherry necrotic rusty mottle virus (CNRMV) (2/100), Cherry virus A (CVA) (14/100), Little cherry virus 2 (LChV2) (3/100), Plum bark necrosis stem pitting associated virus (PBNSPaV) (4/100), Prune dwarf virus (PDV) (3/100), Prunus necrotic ringspot virus (PNRSV) (52/100), Hop stunt viroid (HSVd) (9/100) and Peach latent mosaic viroid (PLMVd) (6/100). The results showed that PNRSV is widespread in Prunus trees in Australia. Metagenomic high-throughput sequencing (HTS) and bioinformatics analysis were used to characterise the genomes of some viruses that were detected by RT-PCR tests and Apricot latent virus (ApLV), Apricot vein clearing associated virus (AVCaV), Asian Prunus Virus 2 (APV2) and Nectarine stem pitting-associated virus (NSPaV) were also detected. This is the first report of ApLV, APV2, CGRMV, CNRNV, LChV1, LChV2, NSPaV and PBNSPaV occurring in Australia. It is also the first report of ASGV infecting Prunus species in Australia, although it is known to infect other plant species including pome fruit and citrus.

Comparison of the incidence of viruses within germplasm, orchards and wild cherry trees in the Czech Republic

AbstractA survey targeting 19 viruses infecting sweet and sour cherry trees worldwide was conducted in germplasm, orchards and wild trees in the Czech Republic during 2014–2015. A total of 184 trees were screened (77 trees from a germplasm collection and 107 trees at 21 different localities in the South Bohemian region) using reverse transcription‐polymerase chain reaction and sequencing. The following 10 viruses were detected: apple chlorotic leaf spot virus (ACLSV), apple mosaic virus (ApMV), cherry green ring mottle virus (CGRMV), cherry leaf roll virus (CLRV), cherry necrotic rusty mottle virus (CNRMV), cherry virus A (CVA), little cherry virus 1 (LChV‐1), little cherry virus 2 (LChV‐2), prune dwarf virus (PDV) and prunus necrotic ringspot virus (PNRSV). ApMV and LChV‐2 were detected in the Czech Republic for the first time. CGRMV and CNRMV were found only in imported trees within the germplasm collection. CVA, CLRV and PNRSV were the most frequently identified viruses in the germplasm collection, and PNRSV and PDV were the most abundant in orchards and wild trees. Of the 108 symptomatic trees, 68 were infected with one or more of the 19 viruses tested. On the other hand, 45 of the 76 samples from trees with no obvious symptoms were infected at the time of collection. Moreover, we did not find any significant association between symptom manifestation and detected virus presence. Next‐generation sequencing of six selected samples yielded major genome portions (71.7–99.9%) of ACLSV, CGRMV, CLRV, CNRMV, CVA, LChV‐1, LChV‐2, PDV and PNRSV, which were deposited in GenBank. In addition, we obtained the first complete coding sequences of a European PNRSV isolate (CZ‐Cig).

Occurrence and detection of little cherry virus 1, little cherry virus 2, cherry green ring mottle virus, cherry necrotic rusty mottle virus, and cherry virus A in stone fruit trees in Poland.

To investigate the occurrence of little cherry virus 1 (LChV-1), little cherry virus 2 (LChV-2), cherry green ring mottle virus (CGRMV), cherry necrotic rusty mottle virus (CNRMV), and cherry virus A (CVA) in stone fruit trees in Poland, leaf samples were collected from sweet and sour cherry, peach, and apricot trees. Two sets of primers were used to increase the effectiveness of virus detection. The RT-PCR results indicated that the most frequently detected virus in all of the tested samples was CVA (60%), followed by CGRMV (13%), CNRMV (12%), LChV-1 (11%), and LChV-2 (4%). CVA and CNRMV were not detected in peaches. Mixed infections of these viruses were frequently detected. Keywords: little- cherry virus 1; little cherry virus 2; cherry green ring mottle virus; cherry necrotic rusty mottle virus; cherry virus A; RT-PCR.

Simultaneous infection of sweet cherry with eight virus species including a new foveavirus

The agent causing bud blight disease (BBD) of sweet cherry (Prunus avium), first reported in Yamagata Prefecture, Japan, in the 1990s is graft-transmissible. Previously, little cherry virus 1 (LChV-1), little cherry virus-2 (LChV-2), cherry necrotic rusty mottle virus (CNRMV), cherry green ring mottle virus (CGRMV), and/or cherry virus A (CVA) were all detected from sweet cherry trees with BBD. Here, RNA viruses in sweet cherry trees with BBD were reinvestigated by high-throughput sequencing. Illumina RNA-sequencing of double-stranded RNAs from leaves of diseased cherry trees (S1, S2, S3, and S4) indicated that all trees were infected with at least six known viruses, LChV-1, LChV-2, CNRMV, CGRMV, CVA, prune dwarf virus (PDV), and/or apple chlorotic leaf spot virus. Thus, cherry trees with BBD are all infected with multiple viruses. Moreover, we found an undescribed novel virus of the family Betaflexiviridae (tentatively named cherry virus B, ChVB) in tree S3. The complete genome sequence of ChVB comprises 8806 nt, in which five open reading frames were found similar to the viruses of the genus Foveavirus. Homology search and phylogenetic analysis indicated that ChVB is a new species of the genus Foveavirus.

Investigation of Virus Diseases and Molecular Detection of Little Cherry Virus 1 on Cherry Plants at Niğde Province

To investigate the virus infections of sour and sweet cherries, various locations of Niğde province were examined during 2017. Ninety sweet and sour cherry leaf samples showing suspicious virus symptoms were collected and screened with virus-specific primers: Little cherry virus 1 (LChV1), Cherry necrotic rusty mottle virus (CNRMV), Prune dwarf virus (PDV), Prune necrotic ring spot virus (PNRSV), Apple mosaic virus (ApMV), Cherry green ring mottle virus (CGRMV), Cherry leaf roll virus (CLRV), Cherry mottle leaf virus (CMLV), Plum bark necrotic stem pitting associated virus (PBNSPaV), Cherry twisted leaf virus (CTLV), Apple stem grooving virus (ASGV), Little cherry virus 2 (LChV2), Cherry rusty leaf virus (CRLV), Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV). Based on RT-PCR analysis, no amplification was observed except LChV1 amplifications, dsRNA analysis resulted in one suspicious profile. To validate those results, more sensitive TaqMan Real-Time PCR analysis and sequence analysis were conducted and LChV1 was detected on 7 samples. It can be concluded that only a low quantity of LChV1 infections was observed on some of the screened cherry samples.

First Report of Little cherry virus 1 and 2 in Sweet Cherry in Korea

HomePlant DiseaseVol. 102, No. 5First Report of Little cherry virus 1 and 2 in Sweet Cherry in Korea PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Little cherry virus 1 and 2 in Sweet Cherry in KoreaS.-Y. Cho, H. Kim, and S.-I. YiS.-Y. ChoSearch for more papers by this author, H. KimSearch for more papers by this author, and S.-I. Yi†Corresponding author: S.-I. Yi; E-mail: E-mail Address: seedin@korea.krSearch for more papers by this authorAffiliationsAuthors and Affiliations S.-Y. Cho H. Kim S.-I. Yi † , Seed Testing and Research Center, Korea Seed and Variety Service, Gimcheon, Republic of Korea. Published Online:9 Mar 2018https://doi.org/10.1094/PDIS-06-17-0783-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Sweet cherry, Prunus avium L. of the family Rosaceae, is a minor fruit crop in Korea whose cultivation area is steadily increasing through regional specialization. Recently, Apple chlorotic leaf spot virus, Apple mosaic virus, Cherry necrotic rusty mottle virus (CNRMV), Cherry virus A (CVA), and Cherry green ring mottle virus have been reported in sweet cherries in Korea (Cho et al. 2013, 2014). In July 2016, eight samples from eight Napoleon cherry trees showing virus-like symptoms, such as vein chlorosis, protruding veins, and reddened leaves, were harvested in Gyeongju-si, Gyeongsangbuk-do, Republic of Korea. Total RNA was extracted from these symptomatic leaves using TRI Reagent (Molecular Research Center, Cincinnati, OH) and the extracted total RNA were analyzed by next-generation sequencing using an Illumina HiSeq2500 (Theragen Bio Institute, Suwon, Korea) and raw sequence data were analyzed using SeqGenesis (Daejeon, Korea). BlastX and BlastN analyses of de novo assembled contigs confirmed the presence of Little cherry virus 1 (LChV-1, contigs of lengths of 3,521 and 725 bases), LChV-2 (362, 359, and 276 bases), CVA (555, 443, and 317 bases), and CNRMV (531, 377, and 339 bases). These contigs showed 74 to 99% nucleotide identity with reference genome sequences (accession nos. KR736335, AF416335, EU188438, and LN879389). Each sample was analyzed by reverse transcription (RT)-PCR using specific primers designed from the contig sequences obtained in this study, LChV-1 865F/1164R (5′-TGGCAAAGAGGTCCAGTGAC-3′)/(5′-TCCTTTCGAGCTAGTCGTATCA-3′), LChV-2 27F/276R (5′-GAGGGTCATACCGTCAGTAAAGT-3′)/(5′-ACCCGATCAATACATTGAAGCAC-3′), CVA TR10 F/R (5′-GGTCCACCACGCTCAACAAG-3′)/(5′-TTCCAAGGCCCCTTCTTATCTC-3′), and CNRMV 32F/331R (5′-GAACCTTCAAACTCGACGCTG-3′)/(5′-ATGTTTCAGACTCGGGCACC-3′). All samples were infected by one (CVA, 2/8 samples), two (LChV-1 and CVA, 2/8), three (LChV-1, CVA, and CNRMV, 2/8), or four viruses (LChV-1, 2, CVA, and CNRMV, 2/8). To amplify the full length of the LChV-1 coat protein (CP, 1,215 base pairs), specific primers were used for RT-PCR based on sequences deposited in GenBank (KR736335): LChV-1 CP NTR F/R (5′-AAACAGCTTGATATAAATTAATTTGTTTCT-3′)/(5′-TTTACTTGAATAGCATCGAAAGGTAC-3′). Sequences of the complete or partial viral genes were deposited in GenBank under the names LChV-1 CP isolate Gyeongju, and LChV-2 isolate Gyeongju (MF083705 and MF083706) and theses isolates showed 98 and 97% nucleotide identity with those of an LChV-1 isolate from China (KR736335) and LChV-2 isolate from Germany (AF531505). Crude leaf extracts of symptomatic virus infected leaves were mechanically inoculated into Nicotiana benthamiana, N. tabacum, N. glutinosa, and Chenopodium amaranticolor. All inoculated plants were tested by RT-PCR at 14 days postinfection with specific primer pairs (LChV-1 865F/1164R, LChV-2 27F/276R, CVA TR10 F/R, and CNRMV 32F/331R). Virus infections were confirmed in tested N. benthamiana (CNRMV and CVA), N. tabacum (LChV-1, CNRMV, and CVA), N. glutinosa (LChV-1, CNRMV, and CVA), and C. amaranticolor (LChV-1). LChV-2 was not detected in any tested plants, perhaps because LChV-2 is transmitted to plants by an insect (apple mealybug) (Raine et al. 1986). To our knowledge, this is the first report on the occurrence and sequences of LChV-1 and LChV-2 in sweet cherry trees in Korea.

First Report of Peach-associated luteovirus in Nectarine (Prunus persica) in Italy

Nectarines (Prunus persica) are among the most popular stone fruits grown in Italy. In particular, the Campania region produces an average of 80,000 metric tons of fresh fruits per year. In 2014, a survey was conducted in Eboli (Salerno province) in a 5-year-old nectarine (cv. Gartairo) orchard in which about 30% of the trees showed an intense pitting of the fruit surface. Leaves, branches, and trunk were symptomless, and the intensity of the pitting tended to increase with fruit ripeness. The fruit mesocarp was not altered, nor were the flavor and taste of the fruits. To investigate the possible etiology of the disease, a symptomatic sample was analyzed by high-throughput sequencing (HTS). Double-stranded RNAs were purified by CF11 cellulose chromatography as previously described (Candresse et al. 2013) from 2- to 3-mm-thick epicarp peel collected from a ripe fruit. The cDNA libraries were generated with SuperScript II reverse transcriptase (Invitrogen), polymerase chain reaction (PCR) amplified and sequenced on the Miseq Illumina platform using a 2 × 250-bp paired-end sequencing strategy. Raw data were trimmed and cleaned by removing adaptor sequences generating a total of 1,999,750 high-quality reads that were de novo assembled with CLC Genomic Workbench 9.0, generating a total of 1,663 contings. These contigs were annotated by BlastN and BlastX searches against the GenBank database, revealing the presence of several viral agents already reported as stone-fruit pathogens: Hop stunt viroid, Apple chlorotic leaf spot virus, and Cherry green ring mottle virus. In addition, two contigs (of respectively 648 and 510 bp, totaling 0.025% of trimmed reads) showing 97% nt identity with the corresponding sequence of Peach-associated luteovirus (PaLV) (KY635988) were also observed. To confirm the presence of PaLV and fill the sequence gap between the two contigs, total RNA was extracted and used in a reverse transcription (RT) PCR experiment carried out with specific primers (forward, 5′-GAAACGGTAAGTCGACGGACATCGCG-3′; reverse, 5′-GGAGGTTACACTGACAAGACAATGGA-3′) designed from the ends of the contigs. A PCR product of the expected size (672 bp) was obtained, purified, and directly sequenced, showing 96% nt identity with the PaLV corresponding sequence. The sequence of the completed scaffold (1,609 nt), integrating the HTS data and the sequence of the 451-nt gap determined from the PCR product, has been deposited in GenBank (accession no. MF990276). To our knowledge, this is the first report of PaLV infecting a stone fruit tree in Italy. Members of genus Luteovirus are aphid-transmitted and phloem-limited viruses that have been recently reported to infect nectarine (Bag et al. 2015), cherry (Lenz et al. 2017), and peach (Igori et al. 2017; Wu et al. 2017). Attempts to detect PaLV in other symptomatic nectarine trees of the same orchard by RT-PCR failed, thus indicating that the virus is most probably not associated with the symptoms. Given their recent discovery, a lot of information on the biology, symptomatology, distribution, and prevalence of fruit-tree-infecting luteoviruses is still missing. Further efforts to understand these agents and their potential significance are needed to reach scientifically motivated decisions as to the need to control them or to exclude them from Prunus propagation materials.

Cherry necrotic rusty mottle and Cherry green ring mottle viruses in Czech cherry germplasm

Using reverse transcription polymerase chain reaction, 160 sweet and sour cherry trees from a germplasm collection, orchards, and wild trees in the Czech Republic were screened for the presence of Cherry necrotic rusty mottle virus (CNRMV) and Cherry green ring mottle virus (CGRMV). The viruses were detected exclusively in sweet cherry trees in the germplasm collection, with CNRMV determined in two trees and CGRMV in four trees. Using next-generation sequencing, nearly complete genomic sequences (complete ORFs) were obtained for one CNRMV and three CGRMV isolates. Their relatedness to GenBank sequences of isolates from different countries together with negative results from screening outside of the germplasm collection suggests that the viruses had been imported with accessions.

Occurrence of Several Viruses Infecting Wild Growing Stone Fruit Trees in Central Bohemia

Abstract The phytosanitary status of wild growing stone fruit trees and shrubs was examined in surveys conducted in 2013 and 2014 in the region of Central Bohemia, Czech Republic. A total of 159 leaf samples were collected (42 cherries, 77 bird cherries, 10 cherry plums, 13 blackthorns, 2 round plums, 15 plums) and tested for the presence of Plum pox virus (PPV), Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Apple mosaic virus (ApMV), Apple chlorotic leaf spot virus (ACLSV), Cherry virus A (CVA), Cherry necrotic rusty mottle virus (CNRMV), Cherry green ring mottle virus (CGRMV), and Cherry leafroll virus (CLRV) using reverse transcription-polymerase chain reaction (RT-PCR). Totally 28.3% of the investigated trees and shrubs were infected by at least one monitored virus. Mixed infection occurred in 5 out of 159 trees (3.1%). PPV was the most widespread virus (13.2% of samples), followed by PDV (11.3%). Contrary to these two most relevant viral pathogens, the incidence of CLRV and CVA was negligible in individually growing trees of the genus Prunus and ApMV, ACLSV, CGRMV, and CNRMV were not detected at all.

INCIDENCE OF SWEET CHERRY VIRUSES IN SHANDONG PROVINCE, CHINA AND A CASE STUDY ON MULTIPLE INFECTION WITH FIVE VIRUSES

Leaves were collected from a total of 62 trees displaying virus-like disease symptoms such as rugose mosaic in leaf, irregular shaped leaf blades or small leaf blades in a survey of sweet cherry (Prunus avium L. cv Red Lamp) in Shandong Province, the largest cherry production area in China. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Little cherry virus- 2 (LChV-2), Cherry virus A (CVA) and Cherry green ring mottle virus (CGRMV) were present and that most of the samples (70%) were infected by at least two viruses. A case study in this survey of two selected trees revealed that a mixed infection with these five viruses simultaneously caused two types of field symptoms: late-maturity in fruits or fruit blast. Real-time quantitative RT-PCR analysis revealed that the relative proportion of the virus titers was distinct in the two sampled trees. The PNRSV titer was significantly higher than the four other viruses in the trees that exhibited fruit blast. Phylogenetic analysis of the PNRSV coat protein (CP) gene showed that the isolates HSY-4-1 and X-3, obtained from the two sampled trees separately, were classified into different subgroups of Group I isolates, which is characterized by a six-nucleotide insertion. A non-synonymous substitution was identified in the six-nucleotide insertion in the PNRSV CP gene in one of the two isolates. This is the first detailed study of the incidence of multiple sweet cherry viruses in China.

Genomic analyses of cherry rusty mottle group and cherry twisted leaf-associated viruses reveal a possible new genus within the family betaflexiviridae.

It is demonstrated that closely related viruses within the family Betaflexiviridae are associated with a number of diseases that affect sweet cherry (Prunus avium) and other Prunus spp. Cherry rusty mottle-associated virus (CRMaV) is correlated with the appearance of cherry rusty mottle disease (CRMD), and Cherry twisted leaf-associated virus (CTLaV) is linked to cherry twisted leaf disease (CTLD) and apricot ringpox disease (ARPD). Comprehensive analysis of previously reported full genomic sequences plus those determined in this study representing isolates of CTLaV, CRMaV, Cherry green ring mottle virus, and Cherry necrotic rusty mottle virus revealed segregation of sequences into four clades corresponding to distinct virus species. High-throughput sequencing of RNA from representative source trees for CRMD, CTLD, and ARPD did not reveal additional unique virus sequences that might be associated with these diseases, thereby further substantiating the association of CRMaV and CTLaV with CRMD and CTLD or ARPD, respectively. Based on comparison of the nucleotide and amino acid sequence identity values, phylogenetic relationships with other triple-gene block-coding viruses within the family Betaflexiviridae, genome organization, and natural host range, a new genus (Robigovirus) is suggested.

Survey of Cherry necrotic rusty mottle virus and Cherry green ring mottle virus incidence in Korea by Duplex RT-PCR.

The incidence of Cherry necrotic rusty mottle virus (CNRMV) and Cherry green ring mottle virus (CGRMV) have recently been occurred in Korea, posing a problem for sweet cherry cultivation. Since infected trees have symptomless leaves or ring-like spots on the pericarp, it is difficult to identify a viral infection. In this study, the incidence of CNRMV and CGRMV in sweet cherry in Gyeongbuk province was surveyed using a newly developed duplex reverse transcriptase polymerase chain reaction (RT-PCR) method that can detect both viruses in a single reaction. CNRMV and CGRMV co-infection rates were 29.6%, 53.6%, and 17.6%, respectively, in samples collected from three different sites (Daegu, Gyeongju and Gyeongsan) in Gyeongbuk province during 2012 and 2013. This duplex RT-PCR method offers a simple, rapid, and effective way of identifying CNRMV and CGRMV simultaneously in sweet cherry trees, which can aid in the management of viral infections that could undermine yield.

A multiplex RT-PCR assay for simultaneous detection of four viruses from sweet cherry

A multiplex reverse transcription polymerase chain reaction (mRT-PCR) assay was developed for simultaneous detection of four viruses: Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Little cherry virus-2 (LChV-2) and Cherry green ring mottle virus (CGRMV). Random hexamer primer was used for cDNA synthesis. Detection primers were designed against the genomic sequences of four viruses. The reaction conditions were firstly optimized by selecting primers combinations and standardizing the individual primer proportion and PCR protocols. Then, sensitivity of multiplex RT-PCR was evaluated. The assay could detect all four viruses in diluted cDNA (10−6, about 8.64×10−4ngμL−1) and RNA (10−1, about 0.2μg). The reliability of the assay was evaluated by cloning and sequence alignments. The developed multiplex RT-PCR method was then used to test virus infections from field samples of sweet cherry in this paper. It will be quite helpful for plant quarantine and certification programs. To our knowledge, it is the first report of the multiplex RT-PCR assay for simultaneous detection of PNRSV, PDV, LChV-2 and CGRMV.

First Report of Apple chlorotic leaf spot virus, Cherry green ring mottle virus, and Cherry necrotic rusty mottle virus on Peach in Montenegro.

The sanitary status of peach fruit trees was assessed in central and coastal regions of Montenegro during a survey in September and October of 2011 and 2012. Leaf samples were collected from 58 (2011) and 47 (2012) trees showing chlorotic rings and spots, mosaic, necrosis, leaf distortion, and stunting. Total RNAs was extracted from each sample by RNeasy Plant Mini kit (Qiagen, Germany) and used as a template in PDO (polyvalent degenerate oligonucleotides) nested reverse transcription (RT)-PCR for the detection of fruit tree viruses belonging to the genera Trichovirus, Capillovirus, and Foveavirus (family Betaflexiviridae). PDO primer sets PDO-F1i/PDO-R3i/PDO-R4i and PDO-F2i/PDO-R1i (2) were used in the first RT-PCR and nested PCR, respectively. Total RNAs obtained from Italian Apple chlorotic leaf spot virus (ACLSV)-infected isolate and healthy peach leaves were used as positive and negative controls, respectively. A nested set of primers amplified a 362-bp product from 6 samples collected in 2011 (10.3%) and 13 samples collected in 2012 (27.7%). Sequence analysis included three isolates (367/11, 133/12, and 168/12) chosen from different peach cultivars (Ritastar, Spring Belle, and Redhaven, respectively). Amplified products of expected size of the partial RNA-dependent RNA polymerase from three positive samples were cloned into p-GEM-T Easy Vector (Promega, Madison, WI) and sequenced (MWG-Biotech AG, Germany). Sequences were deposited in GenBank under accession nos. KF534757, KF534769, and KF534766, respectively. BLAST analysis showed that the sequence of isolate 367/11 (KF534757) shared high nucleotide similarity (78.9 to 87.2%) with ACLSV isolates from GenBank, showing highest identity with isolate PBM1 (AJ243438) from Germany. Sequence analysis of isolate 133/12 (KF534769) proved that it is 90.5 to 93.3% identical to Cherry green ring mottle virus (CGRMV) isolates reported from other parts of the world. In particular, the highest nucleotide similarity was showed with isolate P1C124 (AJ291761) from France. Finally, analysis of sequence from the isolate 168/12 (KF534766) revealed high degree of identity (86.1 to 96.1%) with the corresponding nucleotide sequences of the Cherry necrotic rusty mottle virus (CNRMV) isolates, showing highest similarity with isolate 120/86 (AF237816) from Switzerland. To confirm virus infectivity, according to the FAO/IPGRI Technical Guidelines (1), budwood from 367/11, 133/12, and 168/12 samples were grafted into seedlings of peach (GF305), Prunus serrulata (cv. Shirofugen) and P. avium (cv. Sam) then maintained in a greenhouse with controlled conditions. Six months post inoculation, GF305 indexed with 367/11 sample reacts with a green depressed mottle on leaves typical of ACLSV infection. Cherry tree of cv. Shirofugen indexed with sample 133/12 showed symptoms attributable to CGRMV such as epinasty, twisting and curling of leaves while a tree of cv. Sam indexed with 168/12 sample exhibited classical necrotic shot holes in leaves induced by CNRMV infection (1). Sequence analysis of PCR products obtained from indicator plants by RT-PCR as described above showed full nucleotide identity with KF534757, KF534769, and KF534766 sequences and confirmed the presence of previous described viral agents. To our knowledge, this is the first report of ACLSV, CGRMV, and CNRMV occurrence on peach in Montenegro. Due to the economic importance of this crop, sanitation measures should be adopted to improve the control of imported plants and the use of virus-tested propagation material in order to prevent spreading of these viruses.

First Report of Infection of Cherry Rusty Mottle Associated Virus in Portuguese Laurel (Prunus lusitanica) in Washington State.

During late spring of 2012 in Snohomish County of Washington State, chlorotic yellow leaf blotch symptoms suggestive of a virus infection were observed on Portuguese laurel (Prunus lusitanica) planted in a hedge row. Leaf samples from representative trees were initially tested for the presence of Cherry leaf roll virus (CLRV) and Plum pox virus (PPV) by ELISA with antibodies specific to CLRV and general potyvirus, respectively (Agdia, Inc., Elkhart, IN). The ELISA test yielded negative results for both viruses. Reverse transcription (RT)-PCR was pursued to detect other viruses known to infect Prunus spp., namely American plum line pattern virus (APLPV), Apple chlorotic leafspot virus (ACLSV), Cherry mottle leaf virus (CMLV), Cherry raspleaf virus (CRLV), Cherry virus A (CVA), Prune dwarf virus (PDV), and Prunus necrotic ringspot virus (PNRSV), as well as CLRV and PPV. None of these viruses were detected. However, RT-PCR with a generic primer pair Fovea2/AdPr (3) that amplifies the coat protein (CP) coding sequence and 3'-untranslated regions (3'-UTR) of several members of the family Betaflexiviridae yielded a 1.4-kb amplicon that was cloned into pCR2.1 (Invitrogen, Carlsbad, CA) and sequenced (GenBank Accession No. KF356396). The sequences from three clones were 99.8% identical to each other at the nucleotide level. Comparison of the consensus CP coding region with the nucleotide sequence database revealed 86 to 93% identity to Cherry rusty mottle associated virus (CRMaV), and only 73 to 75% identities to Cherry necrotic rusty mottle virus (CNRMV) (1) and 71 to 76% identities to Cherry green ring mottle virus (CGRMV) (4) isolates from sweet cherry (P. avium). This result suggested that the cloned fragment represents a strain of CRMaV. Prunus avium 'Bing' and 'Sam,' and P. serrulata 'Kwanzan' were grafted with bark patches from the symptomatic tree and observed for induction of cherry rusty mottle disease (CRMD) symptoms. Ninety days after grafting, symptoms typical of CRMD consisting of chlorotic yellow mottle appeared on 'Bing,' 'Sam,' and 'Kwanzan' indicators. Small necrotic spots also appeared on the leaves of the latter. Angular necrotic lesions on 'Sam' and epinasty of 'Kwanzan' that are diagnostic symptoms of cherry necrotic rusty mottle disease (CNRMD) and cherry green ring mottle disease (CGRMD), respectively, were absent from graft inoculated indicators. Further RT-PCR tests on the indicators using primers specific to CNRMV, CGRMV, and CRMaV (2) yielded negative results for CNRMV and CGRMV but showed positive amplification for CRMaV. The results of the woody indexing corroborate the presence of CRMaV but the absence of CNRMV and CGRMV in the symptomatic Portuguese laurel. To our knowledge, this is the first report of CRMaV in Portuguese laurel in the United States and the first description of symptoms associated with CRMaV in this host. As a potential reservoir of CRMaV, Portuguese laurel could play an important component in management of CRMD in cherry production areas where this ornamental cherry is also present.