A new potyvirus isolated from Pennisetum alopecuroides with the potential to infect cereal crops

Pennisetum plants (Pennisetum alopecuroides L.), displaying a dwarfing phenotype along with delayed flowering and mosaic symptom on leaves, were found in Beijing, China. Flexuous filamentous particles with a size of approximate 15 × 850 nm were observed in symptomatic leaves via transmission electron microscopy. Deep sequencing of small RNAs (sRNA) from symptomatic leaves and analysis of sRNA populations were then conducted to determine the genome sequence of the viral agent in diseased plant tissues. It showed that the viral agent had one positive-sense and single-stranded RNA genome, which consisted of 9717 nucleotides (nts) excluding poly(A) tail. The complete viral genome contained a large open reading frame, encoding a polyprotein of 3131 amino acids (aa). Sequence comparison and phylogenetic analysis demonstrated that the viral agent belonged to the genus Potyvirus in the family Potyviridae. In the cladogram it was most closely related to johnsongrass mosaic virus, sharing 72% nt and 65% aa sequence identity. This viral agent was provisionally named pennisetum alopecuroides mosaic virus (PalMV). Subsequently, it was confirmed that PalMV is the causal agent of this new disease in P. alopecuroides by Koch’s postulates and reverse transcription-polymerase chain reaction analysis. Moreover, maize, millet, wheat, sorghum and rice plants were experimentally infected by PalMV via rub inoculation. Consequently, we proposed that PalMV could be a potentially dangerous virus threating a wide range of cereal crops.

Molecular Identification of Begomovirus Infecting Angled Luffa

Begomovirus was reported as one of the most aggressive and destructive viruses on several commercial crops, including cucurbits in Indonesia. Plants that infected with Begomovirus show the mosaic symptom on the leaves, change in leaf shape, stunts, change in color and shape of fruit. It was recently observed in cultivated angled luffa [Luffa acutangula (L.) Roxb] around Yogyakarta and Central Java. The aim of this research was to identify the virus by using Polymerase chain reaction (PCR). The result of Begomovirus amplification from the total DNA samples amplification using primer Krusty-Homer showed that DNA of Begomovirus from angled luffa was amplified at ~580bp. The DNA sequencing of angled luffa’s leaf isolate GD1 had 97.8% homology with SCLV-China isolate MC1. However, amplification of DNA seed samples using the same primer showed negative result. It was concluded that Begomovirus was not a seed borne virus. This is the first molecular report on the occurence of Begomovirus in angled luffa in Yogyakarta.

Genetic Diversity of Cucumber Mosaic Virus from Catharanthus roseus, Jasminum sambac, Patchouly, Cubeb and Java-tea

<p><em>Cucumber mosaic virus (CMV) symptoms are found in </em>Catharanthus roseus, patchouly (Pogostemon cablin), cubeb (Piper chaba), Jasminum sambac and Java-tea (Orthosiphon aristatus); however, their genetic characterization has not been studied.<em> The study aimed to molecularly characterize the CMV isolates from </em>Catharanthus roseus, patchouly, cubeb, Jasminum sambac and Java-tea.<em> </em><em>Disease plant samples showing mosaic and yellow mosaic symptom</em>s<em> were collected from Petak Pamer Garden, ISMCRI, Bogor. Molecular characterization was carried out by reverse transcription</em>-<em>polymerase chain reaction (RT-PCR) assay using a specific primer of CMV coat protein gene and DNA sequenced. Sequence analysis was performed using the BLAST, Bioedit, Genedoc, Mega 5 programs. The RT-PCR technique succeeded in amplifying a DNA band measuring 650 bp, according to the prediction of the primary design. BLAST analyses revealed that all of these CMV isolates belonged to subgroup IB. Nucleotide sequence homology of CMV from </em>C. roseus,<em> </em>patchouly, P. chaba, <em>and</em> J. sambac, <em>were more than 95.00%. Based on </em>the <em>phylogenetic tree, these four isolates were closely related to CMV isolate from Japan (AB070622). Homology of </em>the <em>nucleotide sequence of CMV from </em>Java-tea<em> </em><em>with </em>the <em>other four isolates w</em>as<em> below 95.00%. This isolate clustered with CMV isolate from Indonesia </em>(<em>AB042294</em>) <em>and</em> <em>was</em> <em>separated with another four isolates according to </em>the <em>phylogeny tree. In the amino acid sequence alignment, </em>Java-tea <em>isolates had five different amino acids compared to the other four isolates. This result indicates the poss</em>i<em>bility of CMV transmission between patchouly</em>, Java-tea, C. roseus <em>and</em><em> </em>J. sambac, <em>so it must be anticipated to prevent its spread. </em></p>

RNA-Seq Reveals Hawthorn Tree as a New Natural Host for Apple Necrotic Mosaic Virus, Possibly Associated with Hawthorn Mosaic Disease

Apple mosaic disease is widespread in the major apple-producing areas in China and is frequently associated with the presence of the newly identified Apple necrotic mosaic virus (ApNMV), belonging to subgroup 3 of Ilarvirus genus in the family of Bromoviridae. Mosaic symptoms were also observed in a hawthorn tree. Deep sequencing revealed the hawthorn tree with mosaic symptom was infected by ApNMV, which was confirmed by RT-PCR. The complete nucleotide sequences of RNA1 (3,378 nt), RNA2 (2,778 nt), and RNA3 (1,917 nt) of ApNMV from the hawthorn were obtained, sharing 93.8 to 96.8%, 89.7 to 96.1%, and 89.8 to 94.6% nucleotide identities with those from apples and crabapples, respectively. Two hypervariable regions were found, which showed 59.2 to 85.7% and 64.0 to 89.3% sequence identities at position 142 to 198 aa and at position 780 to 864 aa in the POL protein, respectively, between the hawthorn isolate and other isolates (apple, crabapple). A grafting test demonstrated that ApNMV was easily transmissible from hawthorns to apple trees, with severe chlorosis, yellowing, mosaic, curling, and necrosis. In addition, a total of 11,685 hawthorn trees were surveyed for the incidence of mosaic disease from five provinces in China, and only six were found showing typical mosaic symptoms. A total of 145 individual trees (six symptomatic, 68 asymptomatic, and 71 other symptoms) were tested for the presence or absence of ApNMV by RT-PCR. Among them, six symptomatic, four asymptomatic, and 10 other symptomatic trees tested positive for ApNMV. Taken together, these results demonstrated that the hawthorn tree was identified as a new natural host for ApNMV with a relatively low frequency (13.8%, 20 out of 145) in the main producing areas, and it was likely to be the causal pathogen of hawthorn mosaic disease.

Complete Genome Sequence of a Distinct Isolate of Cassava common mosaic virus (CsCMV) Infecting Cassava in Hainan, China

The complete genomic sequence of a Cassava common mosaic virus Linggao isolate (CsCMV-LG) was determined from cassava (Manihot esculenta Crantz) with mild leafy mosaic symptom to no symptom in China. Excluding the poly(A) tail, the CsCMV-LG genome (GenBank accession No. MT038420) is 6374 nucleotides (nts) in length, with five major open reading frames encoding a 1450-amino acids (aa) RNA-dependent RNA polymerase (RdRp), three triple gene block (TGB) proteins (231-aa, 110-aa and 95-aa), and a 229-aa coat protein (CP). Phylogenetic analysis indicated that the complete genome of the CsCMV-LG is closely related to that of CsCMV-Brazilian which has been assigned to the genus Potexvirus, but the sequence identity shared only 88.0%. Notable, the mild CsCMV-LG isolate can also infect Nicotiana benthamiana in laboratory through rub inoculation causing mild vein yellowing at 15-day post inoculation. This is the first full-length genome sequence of a distinct isolate of Cassava common mosaic virus (CsCMV) infecting cassava in Hainan, China.

Molecular characterization of Mosaic Virus from the cocoa trees showing mosaic symptoms in Yogyakarta, Indonesia

Abstact. Probowati W, Somowiyarjo S, Hartono S. 2019. Molecular characterization of Mosaic Virus From the cocoa trees showing mosaic symptoms in Yogyakarta, Indonesia. Biodiversitas 20: 3698-3704. Indonesia is the world's second-biggest producer of cacao after Ivory Coast with its cacao plantations spreads over 1,652 million hectares and mostly managed by smallholders. Cacao as a beneficial commodity can provide job opportunities for over 1.64 million people, with its contribution of more than US-$ 1.6 billion/year to national income. However, pest and disease infections are inevitably common constraints for the cacao cultivators. One current disease is caused by Cacao Swollen Shoot Virus (CSSV). Despite its noticeable symptoms on the cacao trees suffering from the disease, the knowledge on both the virus existence and the prevention steps in dealing with it is lacking. The information on the diversity of mosaic virus will help us to comprehend its epidemiologic development and the needed countermeasures, as well as its evolution. This research is intended to study the mosaic disease virus molecularly, the virus was obtained from DR1 clones from Kalibawang cacao plantation in Kulon Progo, Yogyakarta. The virus morphology was observed using a transmission electron microscope applying quick dipping method. The PCR analysis with conserved region ORF1 primers was conducted to detect the viral existence in the infected trees. The identification of CSSV molecular characters was undertaken using PCR sequencing analysis which was then examined using BioEdit and Mega5 programs to initiate a relationship dendrogram. The result showed that the DR1 cacao tree clones from Kalibawang were infected by mosaic virus with mild visual severity of leaf typical symptom. From the electronic microscope observation, a ±100 nm rod-shaped viral particle with a diameter of 15.3 mm was found. On the molecular level, the cause of mosaic symptom has CSSV amplified at conserved regions with size 375 bp. The results are the first report confirming that the molecular cause of cacao mosaic disease in Indonesia is CSSV. The molecular characters of CSSV in Yogyakarta are very different from those found abroad.

Characterization of a highly divergent Sugarcane mosaic virus from Canna indica L. by deep sequencing

Background Cannas are popular ornamental plants and widely planted for the beautiful foliage and flower. Viral disease is a major threaten to canna horticulture industry. In the city of Beijing, mosaic disease in canna was frequently observed, but the associated causal agent and its biological characterization is still unknown. Results After small RNA deep sequencing, 36,776 contigs were assembled and 16 of them shared high sequence identities with the different proteins of Sugarcane mosaic virus (SCMV) of the size ranging from 86 to 1911 nt. The complete genome of SCMV isolate (canna) was reconstructed by sequencing all cDNA clones obtained from RT-PCR and 5′\3′ RACE amplifications. SCMV-canna isolate showed to have a full RNA genome of 9579 nt in length and to share 78% nt and 85% aa sequence identities with SCMV isolates from other hosts. The phylogenetic tree constructed based on the full genome sequence of SCMV isolates allocated separately the canna-isolate in a distinct clade, indicating a new strain. Recombination analyses demonstrated that SCMV-canna isolate was a recombinant originating from a sugarcane-infecting isolate (major parent, acc. no. AJ310103) and a maize-infecting isolate (minor parent, acc. no. AJ297628). Pathogenicity test showed SCMV-canna could cause typical symptoms of mosaic and necrosis in some tested plants with varying levels of severity but was less virulent than the isolate SCMV-BJ. Field survey showed that the virus was widely distributed. Conclusions This study identified SCMV as the major agent causing the prevalent mosaic symptom in canna plants in Beijing and its genomic and biological characterizations were further explored. All these data enriched the knowledge of the viruses infecting canna and would be helpful in effective disease management in canna.

Complete Genomic Sequence of Noni mosaic virus (NoMV), a Novel Potyvirus Associated with a Mosaic Disease in Morinda citrifolia L.

An outbreak of a virus-like disease has caused severe damage to noni plants (Morinda citrifolia L.) in Xishuangbanna area of China's southwestern Yunnan province since 2015. The diseased plants displayed typical mosaic symptom with light and dark green patches on leaves. Flexuous filamentous virus particles of about 800 nm in length were observed from the leaf saps by transmission electron microscope. Illumina transcriptomic sequencing further revealed the presence of a potyvirus and its near complete genome was obtained from de novo assembly. The complete genome of 9,659 nts was obtained by Sanger sequencing of eight amplicons generate by RT-PCR and 5&rsquo; and 3&rsquo; RACE. BLASTp analysis of the polyprotein sequence showed that the virus was most closely related to Tobacco vein banding mosaic virus (TVBMV), but these two viruses only shared 50.7% amino acid sequence similarity. Both phylogenetic analyses of the polyprotein and CP amino acid sequences indicated that this virus is a member of genus Potyvirus. However, the low sequence homology with all known potyviruses established this virus as a new species in the genus, tentatively named as Noni mosaic virus (NoMV). Our field surveys showed that 100% of the symptomatic samples and 28.57% of the asymptomatic samples were infected with this novel potyvirus. Aphids collected from diseased leaves were also detected carrying the virus. In summary, our data indicated that a novel species of potyvirus, NoMV, is prevalent in Yunnan, China and is associated with an emerging mosaic disease on M. citrifolia.

Karakterisasi Molekuler Papaya ringspot virus tipe P pada Tanaman Mentimun di Jawa

Moleculer Characterization of Papaya ringspot virus type P on Cucumber in JavaInfection of Papaya ringspot virus (PRSV) on cucumber plants showing mosaic symptom was detected using specific antibody. Further investigation was conducted to determine molecular characters and status of PRSV infecting cucumber in Java. Infection of PRSV was detected from leaf samples collected from the field using dot immunobinding assay (DIBA). Disease frequency caused by PRSV infection reached 81.11%, 95.86%, 91.66%, and 92.3% in East Java, Central Java, Yogyakarta, and West Java, respectively. Characterization of PRSV isolates was conducted by reverse transcription polymerase chain reaction (RT-PCR) using specific primers for PRSV-P and PRSV-W, followed by cloning, and DNA sequencing. DNA fragment of 470 bp was successfully amplified using specific primers for PRSV-P from several samples from Nganjuk, Brebes, Kulon Progo, and Subang; but no amplification was achieved using specific primers for PRSV-W. Nucleotide and amino acid analysis showed high homology among PRSV-P isolates from Nganjuk, Brebes, Kulon Progo, and Subang, i.e. 98.6%-99.7% and 99.3%-100%, respectively. This is an indication of a low genetic variation among PRSV-P from Java. Further phylogenetic analysis indicated that PRSV-P isolate cucumber is in the same cluster with PRSV-P isolate papaya from Bali, Indonesia. This is the first report of PRSV-P infecting cucumber in Indonesia.