Little Cherry Virus 1 Research Articles

Developing a Management Strategy for Little Cherry Disease: The Case of Washington State.

Little cherry disease (LCD) threatens the long-term economic sustainability of the Pacific Northwest sweet cherry (Prunus avium) industry. Results from a series of partial budget analyses indicate that additional investments in monitoring, testing, spraying to control for insect vectors, and removing infected trees are lower than the reduced profit losses compared with the do-nothing scenario. Also, management can prevent or lessen the negative impacts of higher little cherry virus (Velarivirus little cherry virus 1, Ampelovirus little cherry virus 2) spread rates. Our findings illustrate the importance of prevention, correct identification, and controlling for insect vectors in preventing the dissemination of LCD, for which the only known treatment is tree removal.

High-Throughput Sequencing Assists Studies in Genomic Variability and Epidemiology of Little Cherry Virus 1 and 2 infecting Prunus spp. in Belgium.

Little cherry disease, caused by little cherry virus 1 (LChV-1) and little cherry virus 2 (LChV-2), which are both members of the family Closteroviridae, severely affects sweet (Prunus avium L.) and sour cherry (P. cerasus L.) orchards lifelong production worldwide. An intensive survey was conducted across different geographic regions of Belgium to study the disease presence on these perennial woody plants and related species. Symptomatic as well as non-symptomatic Prunus spp. trees tested positive via RT-PCR for LChV-1 and -2 in single or mixed infections, with a slightly higher incidence for LChV-1. Both viruses were widespread and highly prevalent in nearly all Prunus production areas as well as in private gardens and urban lane trees. The genetic diversity of Belgian LChV-1 and -2 isolates was assessed by Sanger sequencing of partial genomic regions. A total RNA High-Throughput Sequencing (HTS) approach confirmed the presence of both viruses, and revealed the occurrence of other Prunus-associated viruses, namely cherry virus A (CVA), prune dwarf virus (PDV) and prunus virus F (PrVF). The phylogenetic inference from full-length genomes revealed well-defined evolutionary phylogroups with high genetic variability and diversity for LChV-1 and LChV-2 Belgian isolates, yet with little or no correlation with planting area or cultivated varieties. The global diversity and the prevalence in horticultural areas of LChV-1 and -2 variants, in association with other recently described fruit tree viruses, are of particular concern. Future epidemiological implications as well as new investigation avenues are exhaustively discussed.

New sensitive and fast detection of Little cherry virus 1 using loop-mediated isothermal amplification (RT-LAMP)

Little cherry virus 1 (LChV-1) belongs to the genus Velarivirus, family Closteroviridae, is an economically important pathogen affecting mainly cherry around the world emphasizing the impetus for its efficient and accurate on-site detection. This study describes the development of a reliable diagnostic protocol of LChV-1 based on a one-step reverse-transcription loop-mediated isothermal amplification (RT-LAMP). The protocol detects LChV-1 isolates in less than 10 min by fluorescence monitoring using a mobile detection device and is most optimal when performed at 67 °C. Sharp melting curves and unique melting temperatures (Tm) were obtained for the positive samples. Both the RT-LAMP and classical RT-PCR methods are capable of specifically detecting LChV-1 in infected leaf tissues. In addition, the RT-LAMP has remarkable advantages in comparison to RT-PCR. It is at least hundred fold more sensitive, significantly faster (allowing on-field leaf-to-result diagnostic) and efficient at minimal cost. In conclusion, this innovative RT-LAMP approach can contribute to the implementation of sustainable integrated management strategies for detection of LChV-1 in commercial orchards or for horticultural research stations. It is also suitable for decision support in phytosanitary epidemiological programs.

Large-scale field survey reveals overall yield loss in Japanese apricot possibly caused by two ampeloviruses

Japanese apricot (Prunus mume Sieb. et Zucc.) is one of the most popular fruit tree species in Japan and is most densely cultivated in Wakayama Prefecture. Since the 1980s, graft-transmissible symptoms, such as leaf-edge necrosis and incomplete flower development, have been observed in Nanko, the major Japanese apricot cultivar in Wakayama. Two ampeloviruses, plum bark necrosis stem pitting-associated virus (PBNSPaV) and little cherry virus 2 (LChV-2) were detected in Nanko trees showing typical symptoms. However, there is currently insufficient epidemiological data to infer that these viruses cause the symptoms. In this study, we conducted a large-scale survey of symptoms and RT-PCR detection of the two viruses in midwestern districts, the major cultivation areas in Wakayama. Among 208 surveyed Nanko trees, two viral infections were significantly associated with incomplete flower development, low fruit bearing rate, and interveinal chlorosis. LChV-2 infection also affected Nanko fruit and stone sizes. Additionally, we showed that the two viruses were already widely distributed throughout the main cultivation area. These results imply that the two ampeloviruses cause overall yield loss in Japanese apricot in Wakayama Prefecture.

Development of a Multiplex RT-PCR for the Simultaneous Detection of Three Viruses in Cherry Plants

A multiplex RT-PCR (mRT-PCR) assay was developed for the detection of the recently reported viruses, Cherry virus A (CVA), Little cherry virus 1 (LChV-1), and Little cherry virus 2 (LChV-2), in cherry plants in Korea. Eight sets of primers were designed for each virus and their specificity was tested by using various combinations of mixed primer sets. From the designed primer sets, one combination was selected and further evaluated to estimate the optimum temperature and detection limits of the mRT-PCR. A newly developed mRT-PCR assay was also tested using 20 cherry samples collected in the field. This mRT-PCR assay may be a useful tool for field surveys of diseases and the rapid detection of these three viruses in cherry plants.

Small RNA NGS Revealed the Presence of Cherry Virus A and Little Cherry Virus 1 on Apricots in Hungary.

Fruit trees, such as apricot trees, are constantly exposed to the attack of viruses. As they are propagated in a vegetative way, this risk is present not only in the field, where they remain for decades, but also during their propagation. Metagenomic diagnostic methods, based on next generation sequencing (NGS), offer unique possibilities to reveal all the present pathogens in the investigated sample. Using NGS of small RNAs, a special field of these techniques, we tested leaf samples of different varieties of apricot originating from an isolator house or open field stock nursery. As a result, we identified Cherry virus A (CVA) and little cherry virus 1 (LChV-1) for the first time in Hungary. The NGS results were validated by RT-PCR and also by Northern blot in the case of CVA. Cloned and Sanger sequenced viral-specific PCR products enabled us to investigate their phylogenetic relationships. However, since these pathogens have not been described in our country before, their role in symptom development and modification during co-infection with other viruses requires further investigation.

Ampeloviruses associated with incomplete flower syndrome and leaf-edge necrosis in Japanese apricot

Japanese apricot (Prunus mume Sieb. et Zucc.) trees produce popular fruits and are also used in Japan as ornamental flowering trees. Since the 1980s, graft-transmissible symptoms (e.g., incomplete flowers and leaf-edge necrosis) have been observed on ‘Nanko’ trees in Wakayama Prefecture. We here describe the detection of viruses associated with these symptoms in Japanese apricot trees using DECS analysis (i.e., double-stranded RNA [dsRNA] isolation, exhaustive amplification, cloning, and sequencing). We isolated dsRNA from symptomatic flowers, including dsRNA of plum bark necrosis stem pitting-associated virus (PBNSPaV), little cherry virus 2 (LChV-2), and a Luteovirus-like sequence. The reverse transcription polymerase chain reaction results suggested that PBNSPaV and LChV-2 may induce the development of incomplete flowers and leaf-edge necrosis. In contrast, the Luteovirus-like sequence is apparently unrelated to these symptoms. Additional investigations are necessary to clarify the relationship between the symptoms and viral infections.

Development of a Real-Time RT-PCR for the Universal Detection of LChV1 and Study of the Seasonal Fluctuation of the Viral Titer in Sweet Cherry Cultivars.

Little cherry virus 1 (LChV1) is a sweet cherry pathogen which has lately been reported in other Prunus spp. LChV1 variability makes reliable detection a challenging undertaking. The objective of this work was to develop a rapid, sensitive, and reliable one-tube, real-time reverse-transcription polymerase chain reaction (RT-PCR) for the detection and quantification of LChV1. Primers and a TaqMan probe were designed, using conserved regions of the capsid protein gene. Detection range was evaluated using several divergent viral isolates. The amplification efficiency of the method was estimated at 96.7%, whereas the detection limit was about 100 RNA copies. The protocol was applied in the study of virus fluctuation within leaves and phloem tissue throughout the year and the best periods to test and plant tissues to sample were determined. Comparative analysis of this method with a previously published nested RT-PCR revealed the higher analytical and diagnostic sensitivity of the new test, making it a reliable tool that can be used in routine testing and certification programs.

First Report of Little cherry virus 1 in Chile

Little cherry disease (LChD), is one of the most important viral diseases of cherry worldwide. It is associated to two different viral species, Little cherry virus 1 (LChV-1) and Little cherry virus 2 (LChV-2). Productivity impact of LChD is reflected in the decrease in yield and fruit quality (Ruiz-Garcia et al. 2016). For LChV-1 (genus Velarivirus, family Closteroviridae) there is no known insect vector to date, thus, its presence worldwide is mainly associated to the propagation of infected plants. In 2015-2016, during southern hemisphere spring-summer vegetative season, a survey was carried out for virus and viroid detection in the main cherry producing regions in Chile. RT-PCR analyses did not show the presence of LChD-associated viruses using previously reported specific primer pairs (Rott and Jelkmann, 2001). Nineteen samples whose symptoms did not match with the detected viruses, were selected for NGS analyses. Small RNA extracts were prepared according to Giampetruzzi et al. (2012). Libraries wer...

High intra-host Cherry virus A population heterogeneity in cherry trees in Slovakia

Despite the absence of symptoms, next generation sequencing (NGS) of total RNAs from a wild cherry tree revealed a multiple virus infection. Besides prune dwarf virus (PDV) and little cherry virus 2 (LChV-2) the analysis revealed the presence of two genetically divergent cherry virus A (CVA) variants. Their complete genomes could be reconstituted, differing by 14% at the nucleotide level. The presence of these CVA variants in the original cherry tree was confirmed during three consecutive growing seasons by variant-specific RTPCR. The development of a polyvalent RT-PCR assay targeting the 3´ part of the CVA genome allowed the identification of the virus in several other cultivated, wild or ornamental cherries in Slovakia, and the analysis of CVA genomic variability in the amplified region. This has allowed regrouping of known CVA isolates into six major phylogenetic groups, some of which are only recently reported, further extending the known diversity of this virus. In addition, evidence was obtained supporting the recombinant nature of two Slovak isolates and for the presence of “non-cherry” group isolates of CVA in several cherry host plants. Further investigation of the intra-tree CVA variability in five unrelated cherry trees revealed a homogenous lineage in two trees (average nucleotide diversity 0.2-0.5%), while the CVA population in the other trees was composed of variants belonging to three to four different evolutionary lineages (average diversity 7.4-8.8%). The frequent presence of highly divergent molecular variants within the CVA population in single trees further highlights the complex and heterogeneous nature of viral populations infecting perennial hosts.

First Report of Little cherry virus 1 affecting European Plum (Prunus domestica) in Belgium

Little Cherry Disease (LChD), one of the major viral diseases of cherry worldwide, can be caused by two viruses (Little cherry virus 1 and 2), both Closteroviridae members. LChD has an important impact on both yield and fruit quality in commercial sweet and sour cherry (Prunus avium L. and Prunus cerasus L.) (Ruiz-Garcia et al., 2016). LChV-1 (genus Velarivirus) is known to be graft-transmissible and is spread via infected propagated plant material, but no vector has been identified. For LChV-2 (genus Ampelovirus) at least two species of mealybugs (Hemiptera, Pseudococcidae) are known to transmit the virus, namely the apple mealybug (Phenacoccus aceris Signoret) and grape mealybug (Pseudococcus maritimus Ehrhorn). During two growing seasons (2013-15), intensive surveys were conducted in Belgium to monitor the incidence of LChD in sweet and sour cherries and in ornamental Prunus spp., revealing widespread occurrence of both LChV-1 and 2 (De Jonghe et al. 2016). In the close vicinity of a sweet cherry (Prun...

Discovery ofAnagyrus schoenherri(Westwood, 1837) (Hymenoptera: Encyrtidae) in the Nearctic Region, a parasitoid of the apple mealybugPhenacoccus aceris(Signoret, 1875) (Hemiptera: Pseudococcidae) in Washington, U.S.A., with notes on the host

Phenacoccus aceris (Signoret, 1875), the apple mealybug, is a vector of little cherry virus 2 (LChV2), a causal pathogen of little cherry disease. Sweet cherry (Prunus avium Linnaeus) (Rosaceae) trees infected with LChV2 produce cherries of small size, poor color, and poor flavor, making the fruit unmarketable. Little cherry disease was first observed in British Columbia, Canada in 1933, where it had a dramatic impact on sweet cherry production, and was recently detected in Washington, U.S.A. cherry orchards. In 2014–2016, a natural infestation of P. aceris was monitored in an apple orchard located in Washington State University's Sunrise Orchard near Rock Island, Washington (47°18′45.14″N 120°04′08.31″W). Heavy parasitism was observed on overwintering nymphs and ovipositing female P. aceris, followed by a reduction in P. aceris crawlers. The emerged wasps were identified as the encyrtid Anagyrus schoenherri (Westwood, 1837). This is the first report of A. schoenherri parasitizing P. aceris in a Washington apple orchard, which represents an extension of the geographic distribution of A. schoenherri to the Nearctic Region. Reduction in P. aceris densities, one of the key vectors of LChV2, is potentially important for the sweet cherry industry in the Pacific Northwest. Historically, declining P. aceris populations in British Columbia were attributed to the establishment of a platygastrid parasitoid Allotropa utilis Muesebeck, 1939, suggesting that biological control could play a major role.

Pest categorisation of Little cherry pathogen (non-EU isolates).

The EFSA Panel on Plant Health performed a pest categorisation of non‐EU isolates of the Little cherry pathogen (LCP) for the European Union (EU) territory. LCP is now known to be in fact two distinct, well characterised viruses, Little cherry virus 1 (LChV1) and Little cherry virus 2 (LChV2) collectively referred to here as LChV. Efficient molecular detection assays are available for both viruses but not to discriminate EU and non‐EU isolates. LChV are transmitted by vegetative multiplication of infected hosts and, for LChV2, by mealybug vectors. LChV are reported from a range of countries, both outside and within the EU. Non‐EU isolates are not known to occur in the EU and therefore do not meet one of the criteria for being a Union regulated non‐quarantine pest. The host ranges of LChV are restricted to Prunus species, in particular cultivated and ornamental cherries. LChV non‐EU isolates are listed for some, but not all hosts, in Annex IIAI of Directive 2000/29/EC. LChV isolates are expected to be able to enter and establish in the EU. They have the potential to subsequently spread through plants for planting and, for LChV2, through the action of the Phenacoccus aceris vector, which is present in many EU MS. LChV are able to cause severe symptoms in some cherry varieties while others are less affected. Overall, non‐EU LChV isolates meet all the criteria evaluated by EFSA to qualify as Union quarantine pests. However, given the currently limited impact of EU LChV isolates, the impact of non‐EU isolates, if introduced, could be similarly limited. The main knowledge gaps and uncertainties concern (1) whether EU and non‐EU isolates of LChV might differ in their biology, epidemiology or symptomatology, (2) efficiency of natural spread by vectors under EU conditions and (3) extent of symptoms caused on many EU‐grown varieties.

FIRST REPORT OF LITTLE CHERRY VIRUS 2 ON PRUNUS CERASUS var. MARASCA IN CROATIA

Little cherry virus 2 (LChV-2) is considered an important pathogen of cherries and, as such, testing of plant material is required according to the EPPO certification scheme PM 4/29 for cherries. In Croatia sour cherry Marasca is a well-known native variety with total production of 1500-1800 tons of fresh fruit per year, used mainly for production of renowned liqueurs and juices. In June 2014, 19 trees were selected and total RNA was extracted from leaves with the RNeasy plant mini kit (Qiagen, Germany). RT-PCR for LChV-2 was performed using two sets of primers: LCHV2LO2/LCHV2UP2 (Rott and Jelkmann, 2001), amplifying a 438 bp fragment of the methyltransferase (MT) gene, and LC26L/LC26R (Eastwell and Bernardy, 2001), amplifying a 409 bp fragment of the RdRp gene. Six trees were positive, four with the first primer set and two with the second. However, none of the trees tested positive by both sets, suggesting the presence of significant sequence variability among LChV-2 isolates (Theilmann et al., 2004) and the presence of at least two virus variants in Croatia. One PCR product for each primer set was sequenced from both directions and sequences were compiled using MEGA6 software. BLAST searches indicated that the part of the MT gene of Croatian isolate M-79 (GenBank accession No. KT369315) shares 88% identity with the USA6b isolate of LChV-2 (AF531505), while isolate M-75 (KT369316) was closest to Canadian strain LC5 (AF416335), sharing 96% identity with part of the RdRp gene. During the 2015 harvest period, some of the LChV-2-infected trees displayed the characteristic symptoms of uneven ripening. To the best of our knowledge this is the first report of LChV-2 in Croatia.

Complete nucleotide sequence of little cherry virus 1 (LChV-1) infecting sweet cherry in China.

Little cherry virus 1 (LChV-1), associated with little cherry disease (LCD), has a significant impact on fruit quality of infected sweet cherry trees. We report the full genome sequence of an isolate of LChV-1 from Taian, China (LChV-1-TA), detected by small-RNA deep sequencing and amplified by overlapping RT-PCR. The LChV-1-TA genome was 16,932 nt in length and contained nine open reading frames (ORFs), with sequence identity at the overall genome level of 76%, 76%, and 78% to LChV-1 isolates Y10237 (UW2 isolate), EU715989 (ITMAR isolate) and JX669615 (V2356 isolate), respectively. Based on the phylogenetic analysis of HSP70h amino acid sequences of Closteroviridae family members, LChV-1-TA was grouped into a well-supported cluster with the members of the genus Velarivirus and was also closely related to other LChV-1 isolates. This is the first report of the complete nucleotide sequence of LChV-1 infecting sweet cherry in China.

Chemical Control of Apple Mealybug, 2014: Table 1

The purpose of this test was to determine the effects of various pesticides on AMB, a vector of little cherry virus 2 (LChV2), a cause of little cherry disease in sweet cherry. This test was conducted in a mixed “Delicious” apple block with “Golden Delicious” pollinizers in the Washington State University Sunrise orchard near Rock Island, WA. The trial took place in an apple orchard due to heavy infestation of AMB, but materials and timings simulated those available for use on cherry. A RCB design was used to assign four replications of each of the nine treatments and an …

Genomic detection and characterization of a Korean isolate of Little cherry virus 1 sampled from a peach tree.

A peach tree (Prunus persica) showing yellowing and mild mottle symptoms was analyzed using high-throughput RNA sequencing to determine the causal agent. A total of nine contigs similar to Little cherry virus 1 (LChV-1) were produced, and all the contigs showed nucleotide sequence identity (lower than 83 %) and query coverage (higher than 73 %) with LChV-1. The symptomatic peach sample was confirmed to be infected with LChV-1-like virus as a result of reverse transcription-polymerase chain reaction using primers designed based on sequences of the contigs. Occurrence of diseases caused by LChV-1 in Prunus species has been reported. Complete 16,931-nt genome of the peach virus composed of eight open reading frames was determined, and conserved domains including viral methyltransferase, viral helicase 1, RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homologue (HSP70h), HSP90h and closterovirus coat protein (CP) were identified. Phylogenetic trees based on amino acid sequence alignments between the peach virus and members in the family Closteroviridae showed that the virus was most similar to LChV-1. Pairwise comparisons based on amino acid sequence alignments of three genes (RdRp, HSP70h and CP) between the peach virus and LChV-1 isolates showed the highest amino acid sequence identities, with 84.32 % for RdRp, 85.48 % for HSP70h and 80.45 % for CP. These results indicate that this is the first report for the presence of LChV-1 in South Korea and may be one of the first reports of natural infection of peach by LChV-1. Although it is not clear if LChV-1 YD isolate was responsible for specific symptoms observed, detection and characterization of the peach tree-infecting LChV-1 in South Korea would be useful in terms of the epidemiology of LChV-1.

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.

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.

Insights into the genetic diversity and evolution of Little cherry virus 1

Little cherry virus 1 (LChV‐1), a member of the recently proposed genus Velarivirus, is a sweet cherry pathogen that has been recently reported to infect other Prunus species and is associated with various plant disorders. In this work the incidence of the virus on its putative hosts and possible mechanisms driving its evolution were investigated. Due to problems encountered with LChV‐1 detection, a new nested RT‐PCR assay was developed and applied. The virus was found to be prevalent in cherry plantations in Greece and only occasionally detected in other Prunus species. Sequences corresponding to the partial RNA‐dependent RNA polymerase (RdRp), heat‐shock protein homologue (HSP70h) and coat protein (CP) genes were determined from Greek LChV‐1 isolates originating from different hosts; these were analysed, along with published homologous genomic regions from other isolates. Phylogenetic analysis of the three genes revealed the segregation of four evolutionary distinct groups showing no host or geography‐based clustering. Mean genetic distances among the four groups were high with the CP region showing the highest divergence, although intragroup variability levels were low. Nevertheless, estimations of the mean ratio of nonsynonymous substitutions per synonymous site to synonymous substitutions per synonymous site (dN/dS) for the partial RdRp, HSP70h and CP indicated that these genomic regions are under negative selection pressure. Interestingly, a recombination event was identified at the 3′ end of RdRp on a Greek virus isolate, thus highlighting the role of this mechanism in the evolutionary history of LChV‐1.