Cherry Necrotic Rusty Mottle Virus Research Articles

Cherry necrotic rusty mottle virus (cherry necrotic rusty mottle disease)

Cherry necrotic rusty mottle virus (cherry necrotic rusty mottle disease)

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 cherry necrotic rusty mottle virus in sweet cherries in Slovenia

First report of cherry necrotic rusty mottle virus in sweet cherries in Slovenia

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.

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.

First Report of Cherry necrotic rusty mottle virus Infecting a Peach Cultivar in Korea

First Report of <i>Cherry necrotic rusty mottle virus</i> Infecting a Peach Cultivar in Korea

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.

Molecular evidence of natural occurrence of Apple stem grooving virus on bamboos

The present study identified natural infection of Apple stem grooving virus in five bamboo species. “Himalayan ‘maggar’ bamboo, widely distributed in the Western Himalayas, is abundantly found in and around the campus of CSIR-IHBT. Some of the plants near the apple germplasm and nursery field at this institute displayed virus-like symptoms such as chlorosis, mosaic, yellow streaks, leaf curling and necrotic spots. To understand whether this natural population carries Apple stem grooving virus (ASGV), an important and abundant virus in apple orchards, a total of 38 bamboo leaf samples (belonging to 10 different genera and 33 species), were collected and indexed for ASGV through DAS-ELISA, non-isotopic nucleic acid spot hybridization and RT-PCR. The presence of the virus was confirmed in 76 % of the samples belonging to 27 species and nine genera viz. Bambusa, Dendrocalamus, Fargesia, Arundinaria, Phyllostachys, Gigantochloa, Sasa, Guadua and variegated ornamental bamboo. Seven randomly selected samples were confirmed by partial sequencing of the replicase gene, characterized from the seven isolates of bamboo samples [Bam7(dd), Bam14(pp), Bam28(bn), Bam29(fs), Bam39(dh), Bam42(dh) and Bam44(dh)]. Further, coat protein gene was also characterized from all seven samples and in the phylogenetic analysis, five out of the seven isolates clustered with the apple isolate of the virus from India (LN627003) sharing 98–100 % sequence identity at amino acid (aa) level. While the other two isolates also clustered with apple isolates from India, (LN627002, LN627004 and LN901438, from apple germplasm field of CSIR-IHBT), sharing 98.7–100 % sequence identity at aa level. To the best of our knowledge, this is the first report of natural infection of ASGV on bamboos. After Bamboo mosaic virus and Cherry necrotic rusty mottle virus, ASGV happens to be only the third virus to be characterized from bamboo.

Cherry necrotic rusty mottle virus. [Distribution map

Abstract A new distribution map is provided for Cherry necrotic rusty mottle virus. Tymovirales: Betaflexiviridae. Hosts: sweet cherry ( Prunus avium ), apricot ( P. armeniaca ) and black cherry ( P. serotina ). Information is given on the geographical distribution in Europe (Czech Republic, Denmark, France, Germany, Greece, Hungary, Italy, Montenegro, Serbia, Switzerland, UK, England and Wales), Asia (China, India, Himachal Pradesh, Jammu and Kashmir, Japan, Honshu and Korea Republic), North America (Canada, British Columbia, USA, California, Montana, Utah and Washington), South America (Chile) and Oceania (New Zealand).

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.

Wild roses as natural reservoirs of Cherry necrotic rusty mottle virus

Cherry necrotic rusty mottle virus (CNRMV) is a graft transmitted virus, known to infect sweet cherries and some other Prunus species. Recently, this virus has been explored for its natural hosts and has emerged as a significant virus in India, infecting many species of bamboos with a high incidence of 65 %, thereby expanding its host range. A wild rose shrub (Rosa brunonii) growing near the already reported CNRMV infected bamboo, in the campus of CSIR-IHBT Palampur (India), was observed to display disease symptoms such as chlorosis and necrotic spots on the leaves. The plant was tested for CNRMV infection by reverse transcription polymerase chain reaction (RT-PCR) using the detection primers and was found to be infected. In order to widen the survey, 32 different samples of wild roses were collected from the floriculture division (CSIR-IHBT), where the institute maintains a germplasm of domesticated wild roses. The samples were tested by Double antibody sandwich-Enzyme linked immunosorbent assay (DAS–ELISA) and dot-blot hybridization and the results were validated by RT-PCR which confirmed the presence of CNRMV in 34 % of the tested samples, belonging to the species: R. brunonii, R. multiflora, R. cathayensis and R. alba. The complete coat protein (CP) gene of the virus was characterized from five positive samples. In the phylogenetic analysis it was seen that the three isolates (HPWRRA3, HPWRRC6 and HPWRRB31) were closely related to the Chilean sweet cherry isolate while two other isolates (HPWRRM2 and HPWR27) were closely related to the Indian bamboo isolate of the virus. The multiple sequence alignment revealed variation in the length of CP gene amongst the Indian isolates due to a missing triplet codon towards its 3′ end. The percentage identity matrix computed at nucleotide level revealed that the present wild rose isolates are 97.5 to 100 % identical to each other while the overall identity with other isolates of CNRMV characterized from different hosts and geographical regions was 85.1 to 100 %. To the best of our knowledge this is the first report of CNRMV infection in wild roses. Infected wild roses may act as reservoir of the virus during the dormant season and identification of new hosts will help us understand biological characteristics of the virus.

Himalayan wild cherry (Prunus cerasoides) as a new natural host of Cherry necrotic rusty mottle virus (CNRMV) and a possible role of insect vectors in their transmission

AbstractA Himalayan wild cherry (Hwc) tree (Prunus cerasoides) growing in the vicinity of an already reported Cherry necrotic rusty mottle virus (CNRMV) infected bamboo (Dendrocalamus hamiltonii) showed severe virus‐like symptoms on the leaves consisting of marginal chlorosis, mosaic, shot holes and necrotic spots. Reverse transcription‐polymerase chain reaction (RT‐PCR) using virus specific primers confirmed the presence of the virus. In order to assess the prevalence of this virus, 40 symptomatic and asymptomatic Hwc plants along with four different suspected insect vectors were analysed by double antibody sandwich‐enzyme linked immunosorbent assay (DAS‐ELISA), dot blot hybridisation and RT‐PCR. CNRMV was detected in 57% of the wild cherry samples, two aphid species Astegopteryx bambusae and Tinocalloides montanus, and in a delphacid of Bambusiphaga sp. Complete coat protein (CP) gene of CNRMV was sequenced from two randomly selected samples. The isolates characterised from Hwc shared 99% similarity to the already characterised isolates of CNRMV from India, suggesting a conserved nature of the CP gene. This study represents the first report of CNRMV infection in Himalayan wild cherry and its possible insect vectors.

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.

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.

Molecular evidence for bamboo as a new natural host of Cherry necrotic rusty mottle virus

SummaryHimalayan ‘maggar’ bamboo (Dendrocalamus hamiltonii), widely distributed in the Himalayas, was found to exhibit mild virus‐like symptoms, which included mosaic, chlorosis, necrotic spots and mottling. Leaf samples from symptomatic plants were subjected to reverse transcription polymerase chain reaction (RT‐PCR) for the detection of Cherry necrotic rusty mottle virus (CNRMV). Sequencing of the amplicon confirmed the presence of the virus. To further establish bamboo as a natural host of CNRMV, 35 varieties of bamboos maintained at CSIR‐IHBT were subjected to double‐antibody sandwich enzyme linked immunosorbent assay (DAS‐ELISA) and dot blot hybridization. Our results showed the presence of CNRMV in 21 species of bamboos belonging to five genera, viz. Bambusa, Dendrocalamus, Fargesia, Arundinaria and Melocanna. For further confirmation of the results, the coat protein (CP) gene of CNRMV was amplified from two different local ‘maggar’ plants. Sequencing of the CP gene confirmed the presence of the virus. The two isolates characterized from bamboo plants, shared 99.8% nucleotide sequence identity with sweet cherry isolate JK10 characterized from India. This study presents sequence information from two isolates of the virus. CNRMV infection in bamboo is being reported for the first time and only the second virus reported in bamboo after Bamboo mosaic virus (BaMV).