Leaf Mottle Symptoms Research Articles

Next-Generation Sequencing Reveals a Novel Emaravirus in Diseased Maple Trees From a German Urban Forest.

While the focus of plant virology has been mainly on horticultural and field crops as well as fruit trees, little information is available on viruses that infect forest trees. Utilization of next-generation sequencing (NGS) methodologies has revealed a significant number of viruses in forest trees and urban parks. In the present study, the full-length genome of a novel Emaravirus has been identified and characterized from sycamore maple (Acer pseudoplatanus) – a tree species of significant importance in urban and forest areas – showing leaf mottle symptoms. RNA-Seq was performed on the Illumina HiSeq2500 system using RNA preparations from a symptomatic and a symptomless maple tree. The sequence assembly and analysis revealed the presence of six genomic RNA segments in the symptomatic sample (RNA1: 7,074 nt-long encoding the viral replicase; RNA2: 2,289 nt-long encoding the glycoprotein precursor; RNA3: 1,525 nt-long encoding the nucleocapsid protein; RNA4: 1,533 nt-long encoding the putative movement protein; RNA5: 1,825 nt-long encoding a hypothetical protein P5; RNA6: 1,179 nt-long encoding a hypothetical protein P6). Two independent NGS sequencing runs from the same symptomatic maple tree detected the same genome segments. For one of these sequencing runs the cDNA library was prepared using a primer targeting the conserved genome terminal region, known to be shared between emaraviruses genome segments. We suggest, therefore, that the six identified genome segments represent the complete genome of a novel emaravirus from maple, which we tentatively name maple mottle-associated virus (MaMaV). Phylogenetic and sequence homology analyses place this virus on the distinct “subgroup a” clade within the Emaravirus genus along with – among others – rose rosette virus, Actinidia emaravirus 2, and fig mosaic virus. Validation RT-PCR assays performed on symptomatic and non-symptomatic trees suggest that MaMaV may be the symptom-inducing virus in the diseased trees. To our knowledge, this is the first time an Emaravirus is described from maple and is fully genetically characterized. With the discovery of MaMaV, the genus Emaravirus comprising negative-sense single-stranded viruses with very divergent genomes – that were until recently overlooked – has substantially increased counting 22 established and putative members.

Molecular Characteristics of Jujube Yellow Mottle-Associated Virus Infecting Jujube (Ziziphus jujuba Mill.) Grown at Aksu in Xinjiang of China.

Chinese jujube (Ziziphus jujuba Mill.) is a native fruit crop in China. Leaf mottle and dapple fruit disease is prevalent in cultivated jujube plants grown at Aksu in Xinjiang Uygur Autonomous Region of China. Jujube yellow mottle-associated virus (JYMaV), a tentative member in the genus Emaravirus, was recently identified from mottle-diseased jujube plants grown in Liaoning Province in China, but its incidence and genetic diversity in China is unknown. In this study, the genome sequences of three JYMaV isolates from two jujube cultivars and one jujube variant were determined by high-throughput sequencing (HTS) for small RNA and rRNA-depleted RNA coupled with RT-PCR assays. Comparison of these sequences together with sequences of the viral RNA segments derived by primer set 3C/5H-based RT-PCR revealed that genetic diversity was present in the virus populations and high sequence variation occurred at the non-translational regions of each of the viral genomic segments. Field investigation confirmed the close association of the virus with leaf mottle symptoms of jujube plants. Furthermore, this study revealed that P5 encoded in the viral RNA5 displayed a nuclear localization feature differing from the plasmodesma (PD) subcellular localization of the virus movement protein (P4), and the two proteins could interact with each other in the BiFC assays. Our study provides a snapshot of JYMaV genetic diversity in its natural hosts.

A New Potyvirus sp. Infects Verbena Exhibiting Leaf Mottling Symptoms.

Verbena 'Taylortown Red' plants showed virus-like mottling symptoms. Virus purifications disclosed the presence of elongated and spherical particles, evidence of mixed virus infections, whereas double-stranded RNA analysis revealed the presence of several bands absent in healthy plants. After shotgun cloning, three viruses were identified in 'Taylortown Red': Broad bean wilt virus-1, Coleus vein necrosis virus, and a previously undescribed potyvirus. Given the importance of verbena to the ornamental industry, we studied the viruses found in 'Taylortown Red' and, in this article, we present our findings on the new potyvirus, provisionally named Verbena virus Y (VVY). VVY belongs to the Potato virus Y subgroup in the genus Potyvirus, has solanaceous plants, including potato, as alternative hosts, and can be transmitted by a ubiquitous pest in the ornamental industry, the green peach aphid.

First Report of 'Candidatus Liberibacter asiaticus' from Atalantia buxifolia in Guangdong, China.

Atalantia buxifolia (Poir.) Oliv., synonym Severinia buxifolia (Poir.) Ten. as commonly found in literature, is a common landscape plant and a popular Chinese medicinal herb known as Jiubingle or Dongfengjie. It remains unclear if this rutaceous plant could host 'Candidatus Liberibacter asiaticus', the pathogen of citrus Huanglongbing (HLB) in Guangdong, P. R. China. This information is important for HLB control in citrus because infected A. buxifolia could serve as a source of inoculum. In August of 1994, three A. buxifolia plants adjacent to a citrus experimental orchard of the South China Agricultural University at Guangzhou were found showing leaf mottle/yellowing symptoms. Two buds from each plant were grafted onto three mandarin trees (Citrus reticulata cv. Pongan) in a psyllid-proof screenhouse for indexing. By October of 1995, typical leaf mottle symptoms were observed in all three grafted trees compared with a healthy control. In March of 1996, one of the A. buxifolia plants was transferred to a screenhouse and has been maintained there. The leaf mottle/yellowing symptoms persisted but did not significantly affect plant growth. DNA was extracted from leaf samples in October 2006 by using the CTAB (cetyltrimethylammoniumbromide) method and assayed by nested-PCR using the general bacterial 16S rDNA primer set fDl/rD1 as the first round of amplification and primer set OI1/OI2c as second round amplification (1,3). After agarose gel electrophoresis and staining with ethidium bromide, an approximate 1.1-kb DNA band was detected in symptomatic samples but not healthy leaf samples of A. buxifolia and C. reticulata. XbaI digestion of the amplicons yielded approximate 500- and 600-bp fragments, characteristic of 'Ca. L. asiaticus'. Similarly, a standard PCR with primer set A5/J2 (3) yielded an approximate 700-bp DNA band characteristic of 'Ca. L. asiaticus' from symptomatic samples only. To our knowledge, this is the first report of graft transmission and PCR detection of 'Ca. L. asiaticus' from A. buxifolia in Guangdong, P. R. China. This work also confirms the findings from Taiwan (2) that A. buxifolia could serve as a source of 'Ca. L. asiaticus'.

New strain of Verticillium dahliae in North America

A new strain of Verticillium dahliae, the fungus causing Verticillium leaf mottle and wilt, was identified from northwestern Minesota in 2002. The new strain or biotype is characterized by its ability to overcome the single, dominant V-1 resistance gene employed in oilseed and confection hybrids. Samples collected in 2003 from diseased plants confirmed the new biotype also exists in Manitoba, Canada. Limited survey, conducted in September 2004, identified the new strain in roughly one-quarter of fields showing Verticillium wilt in North Dakota. In greenhouse trials in 2004/5 we tested 221 cultivars for reaction to the new Verticillium strain. One entry from the USDA Plant Introduction collection, developed in Russia as VNIIMK 8883, was immune and would be an ideal source of resistance. Other commercial oilseed hybrids from the U.S., Argentina and Europe, were also noted with ratings of 0.5 or lower on a 0 to 5 scale. On a related note, another fungus, Phialophora asteris F. sp. helianthi, was isolated from oilseed sunflower plants showing leaf mottle symptoms in Minot, North Dakota in 2002, and presumed to be due to Verticilliumdahliae. In greenhouse inoculations, Phialophora produced leaf mottle symptoms on sunflower lines with and without the V-1 gene, which were very similar to those incited by V. dahliae. Proper identification of this pathogen from plants displaying leaf mottle symptoms will be necessary to avoid confusion between the two pathogens.

First Report of "Candidatus Liberibacter asiaticus", the Agent of Citrus Huanglongbing (Ex-greening) in Bhutan.

Mandarin (Citrus reticulata) is the most important cash crop in Bhutan and plantations total approximately 1.8 million trees (Ministry of Agriculture, Thimphu, Bhutan, 2000). Most trees are a local mandarin variety. Seedlings trees are produced by local farmers or supplied by Druk Seed Nursery. Mandarin seedlings have also been introduced from India. In the mid-1990s, mandarin trees growing in Punakha Valley and Wangdue districts began showing symptoms of decline that included sparse yellow foliage and shoot die-back. After initial surveys in 2000, huanglongbing (HLB) was suspected as the cause of declining trees based on symptomatology and presence of the psyllid vector Diaphorina citri, but no confirmatory tests were carried out. In August 2002, we surveyed eight locations in the valley from Rimchu (North) to Kamichu (South). HLB-like leaf mottle symptoms were observed on declining mandarin trees at all locations at altitudes ranging from 700 to 1,450 m. Orchards around Punakha (1,350m) in the center of the valley were more severely affected. Symptoms were also observed on Mexican lime (Citrus aurantifolia), citron (Citrus medica), and on tangelo trees (Minneola, Seminola, and Iyo) introduced originally as certified HLB-free budwoods from Corsica, France and grafted onto Rangpur lime at the Wangdue Research Center (1,300m). Leaves were collected from symptomatic trees and three declining mandarin trees without characteristic leaf mottle symptoms. Two specific polymerase chain reaction (PCR) tests for the detection of HLB Liberibacter species (1,2) were carried out on 16 DNA samples extracted from leaf mid-veins of 10 mandarins, two Mexican limes, three tangelos, and one citron tree. "Candidatus Liberibacter asiaticus" was readily detected by both PCR assays in all but two samples (one mandarin with noncharacteristic symptoms and citron) and all sampled orchards. The presence in the Wangdue Research Center of liberibacter infected trees, propagated from certified HLB-free budwoods, suggests that natural spread of the HLB by D. citri is occuring, as the psyllid had been identified previously in the Punakha area by Bhutanese Entomologists. It is likely that the disease was originally introduced as infected planting material although its source has not been determined.

Cocksfoot mottle virus spreads to the South Island of New Zealand

In previous surveys of New Zealand cocksfoot (Dactylis glomerata L.) pastures, Cocksfoot mottle virus (CoMV) was detected at only one site near Taupo on the North Island. However, we have recently found CoMV at two sites near Dunedin on the South Island. Isolates from these sites, associated with mild and severe symptoms in cocksfoot (CoMV-Wa and CoMV-Ok, respectively), were partially characterised. In agarose gels, the virions of the two isolates had slightly different mobilities when subjected to an electric field, indicating a difference in net surface charge. However, both isolates produced similar leaf mottle symptoms in wheat, their capsid proteins had similar molecular weights (Mr = ca. 31 kDa) as determined by SDS-polyacrylamide gel electrophoresis and they were serologically indistinguishable in double-diffusion tests using 17 antisera to 10 sobemoviruses. CoMV-Ok and CoMV-Wa were serologically related to Lucerne transient streak virus but not to eight other sobemoviruses. Further surveys using DAS-ELISA showed variable incidences of CoMV (range 0–80%) at nine South Island sites. The beetle vectors of CoMV have not been introduced into New Zealand. At two sites where CoMV was found, we observed severe feeding damage from armyworms (Persectania aversa Walker). However, in growth-cabinet experiments this species failed to transmit the virus. Incidences were significantly higher at mown sites (39%) than at unmown sites (11%), suggesting that mechanical transmission is responsible for spread. In view of the increased use of cocksfoot on New Zealand farms, the impact of CoMV on cocksfoot productivity now needs to be assessed and current levels of resistance to CoMV in commercial cultivars need to be evaluated.