Zucchini Yellow Fleck Virus is an Emergent Virus on Melon in Sicily (Italy)

Since 2006, winter melon plants (Cucumis melo L. var inodorus) showing symptoms of pin‐point yellow spots were noticed in Sicily (Italy). Leaf samples were tested by enzyme‐linked immunosorbent assay to the most important viruses‐infecting cucurbits. Zucchini yellow fleck virus (ZYFV, genus Potyvirus) was the only virus detected. Surveys in 2007 and 2008 revealed an increasing number of sites in Sicily with ZYFV‐infected winter melon plants. To confirm the identity of the virus as ZYFV, two isolates from different locations were sequenced and shown to be approximately 85% identical to the published sequences of isolates previously identified in Italy and France. This is the first report of ZYFV occurring on melon in Italy.     

First Report of Tomato infectious chlorosis virus on Tomato in Bulgaria

Virus‐like chlorotic symptoms were observed on tomato plants, cv. Velocity, grown in a greenhouse, region of Plovdiv. Samples collected from the leaves with interveinal yellowing and with initial interveinal chlorosis were tested for virus presence. Only the samples collected from the upper leaves with slight interveinal chlorosis were positive for Tomato infectious chlorosis virus (TICV) in indirect ELISA. Further, RT‐PCR analysis with specific primers for Tomato chlorosis virus (ToCV) heat shock protein 70, for TICV heat shock protein 70 and for TICV minor capsid protein was positive for TICV in all tested samples. No signals were obtained with primers for ToCV. Phylogenetic analysis showed that the Bulgarian sequence of Hsp70 and a sequence of Greek isolate clustered together having the highest resampling score. Regarding CPm, the Bulgarian isolate was more relevant to the French isolate. The obtained results from phylogenetic analysis supported the idea of a close relationship between the Bulgarian and Greek isolates.     

Evidence of Cotton leaf curl Burewala virus Variant and its Associate Betasatellite Causing Yellow Mosaic of Eggplant (Solanum melongena) in Pakistan

Eggplant (Solanum melongena L.) plants with severe leaf mosaic and mottling were found in a kitchen garden near cotton fields in Pakistan. Rolling Circle Amplification products from six of the naturally infected eggplant plants, subjected to PCR, successfully amplified expected products of 2.8 and 1.4 kb using begomovirus and betasatellite‐specific primers, respectively. Based on 99% nucleotide sequence identity, the virus was identified as a variant of Cotton leaf curl Burewala virus (CLCuBuV) (GenBank Accession No. HG428709). Likewise, the sequenced betasatellite with a maximum of 97% nucleotide sequence identity was recognized as a new variant of Cotton leaf curl Multan betasatellite (CLCuMuB^Mul) (GenBank Accession No. HG428708). The symptomatic induction of Cotton leaf curl disease in CLCuBuV susceptible cotton genotype CIM‐496 by back‐indexing further confirmed the presence of CLCuBuV in eggplant. This is the first report of CLCuBuV and its associate betasatellite in naturally infected plants of eggplant.     

Orchid Fleck Virus Infecting Orchids in Paraguay: First Report and Use of Degenerate Primers for its Detection

The presence of Orchid fleck virus (OFV) in Paraguay was confirmed in orchid plants collected during a survey carried out in 2013. Leaves displayed ringspot and fleck symptoms, and in infected tissues, non‐enveloped, short, rodlike viral particles were observed. Partial OFV N and L genes were amplified using specific and degenerate primers, respectively; the nucleotide sequences demonstrated high identities (98%) with other OFV isolates. Degenerate primers for the L gene were designed considering conserved regions within all of the available OFV sequences and those from the closely related isolates citrus leprosis virus nuclear type (CiLV‐N) and citrus necrotic spot virus. Degenerate primers were also successfully used for the detection of CiLV‐N from infected citrus samples.     

Temporal and spatial spread of Lettuce mosaic virus in lettuce crops in central Spain: factors involved in Lettuce mosaic virus epidemics

Lettuce mosaic virus (LMV) is transmitted by aphid vectors in a nonpersistent manner as well as by seeds. The virus causes severe disease outbreaks in commercial lettuce crops in several regions of Spain. The temporal and spatial patterns of spread of LMV were studied in autumn 2002 in the central region of Spain. Symptomatic lettuce (var. Cazorla) plant samples were collected weekly, first at the seedling stage from the greenhouse nursery and later outdoors after transplantation. The exact position of symptomatic plants sampled in the field was recorded and then material was tested by enzyme‐linked immunosorbent assay to assess virus infection. Cumulative spatial data for infected plants at different growth stages were analysed using spatial analysis by distance indices. For temporal analysis, the monomolecular, Gompertz, logistic and exponential models were evaluated for goodness of fit to the entire set of disease progress data obtained. The results indicated that the disease progress curve of LMV epidemics in the selected area is best described by a Gompertz model and that the epidemic follows a polycyclic disease progression. Our data suggest that secondary cycle of spread occurs when noncolonising aphid species land on the primary infected plants (probably coming from infected seed) and move to adjacent plants before leaving the crop. The role of weeds growing close to lettuce fields as potential inoculum sources of virus and the aphid species most likely involved in the transmission of LMV were also identified.     

First Report of Cucumber mosaic virus Infecting Chinese Mallow in China

The virus in naturally infected, stunted Chinese mallow plants and mosaic leaves was identified as Cucumber mosaic virus (CMV). Six symptomatic plants and one symptomless plant were collected in Chongqing, China. DAS‐ELISA suggested CMV was likely associated with the diseased Chinese mallow. Double‐stranded RNA was extracted from the samples, analysed by RT‐PCR, and the coding sequences of their coat proteins (CPs) were sequenced. The results further confirmed CMV was the pathogen causing Chinese mallow stunted, mosaic disease. The isolate was named CMV‐DXC. The full sequence of CMV‐DXC CP was determined, and it had the highest nucleotide identity (99.4%) of those of CMV‐lily, CMV‐WSJ and CMV‐Hnt, respectively. Phylogenetic analysis shows that CMV‐DXC belongs to CMV subgroup II. To our knowledge, this is the first report of CMV infecting Chinese mallow in China.     

Detection and Molecular Characterization of Squash leaf curl virus (SLCV) in Jordan

Squash leaf curl virus (SLCV) was detected for the first time in Jordan using degenerated oligonucleotide primers. Two isolates of the virus, SLCV‐E and SLCV‐R, were detected using specific oligonucleotide primers in symptomatic Cucurbita pepo. SLCV was also found to occur naturally in Malva parviflora, which showed severe leaf curling, yellowing and stunting of the whole plants. The full‐length genomes of Squash leaf curl virus‐Malva (SLCV‐Malva) isolate were amplified using the bacteriophage Φ DNA polymerase enzyme. Nucleotide sequence analysis showed that SLCV‐Malva shared high nucleotide identity (98% and 97%) with SLCV‐EG and SLCV‐E from Egypt and USA, respectively. A survey using dot‐blot hybridization indicated that squash leaf curl disease occurred in all surveyed areas. The highest disease incidence (95%) was recorded in Dir Alla area, whereas disease incidence did not exceed 69% in squash samples collected from North Ghor.     

Molecular characterisation and relative incidence of bean‐ and soybean‐infecting begomoviruses in northwestern Argentina

Recent studies identified three begomoviruses infecting soybean and bean crops in northwestern (NW) Argentina, bean golden mosaic virus (BGMV), a virus with high capsid protein identity with Sida mottle virus (SiMoV) and a possible new viral species (isolate A). Analysis of complete DNA‐A sequences confirmed that isolate A represents a new viral species for which the authors propose the name soybean blistering mosaic virus (SbBMV), whereas the SiMoV‐like virus is actually an isolate of tomato yellow spot virus (ToYSV). Molecular hybridisation‐based detection of the three begomoviruses was accomplished using a general probe obtained by mixing full‐length DNA‐A clones of the three begomoviruses and specific probes comprising part of the common region of each viral genome. These probes were used to test samples collected in NW Argentina from 2004 through 2007. Fifty‐three percent of the bean samples were infected with BGMV, 11.5% with ToYSV and 9% with SbBMV. For soybean samples, 33% were infected with SbBMV and 18% with ToYSV. BGMV was not detected in soybean. ToYSV was also detected in the wild species Abutilon theophrasti.     

Rapid Detection of Banana Streak Virus by Loop‐mediated Isothermal Amplification Assay in South China

Banana streak virus (BSV) is a significant constraint to banana production and genetic improvement. It is necessary to develop and use BSV detection strategies that are both reliable and sensitive for the management of the virus. A loop‐mediated isothermal amplification (LAMP) assay was developed and evaluated for the detection of BSV. Four primers matching a total of six sequences of the conserved ORF III polyprotein genes were synthesized for developing a specific and sensitive LAMP for DNA extracts from field‐infected banana plants. LAMP assay could detect as low as 1 pg/μl template DNA. Test results of all field samples collected from different regions of South China showed that LAMP is more sensitive than PCR. This relatively simple and sensitive technique showed excellent potential with field‐collected samples and for routine screening of tissue culture materials in South China.     

Molecular characterization of a new badnavirus associated with streak symptoms on enset (Ensete ventricosum,Musaceae)

Electron microscopy of leaf samples displaying streak symptoms from enset (Ensete ventricosum), a banana‐like plant widely cultivated in Ethiopia, showed the presence of bacilliform shaped virions as known for badnaviruses. DNA extracts subjected to rolling circle amplification (RCA), polymerase chain reaction (PCR) and cloning and sequence analysis revealed that the virus has a circular double‐stranded DNA genome of 7,163 nucleotides encoding predicted proteins of 21.5 kDa, 14.5 kDa and 202.5 kDa, a genome organization known for badnaviruses. The virus is phylogenetically most closely related to Sugarcane bacilliform Guadeloupe D virus with a nucleotide sequence identity of 77.2% at the conserved RT/RNase‐H region and 73.6% for the whole genome. Following the current species demarcation criteria, the virus should be considered as an isolate of a new species in the genus Badnavirus for which the name Enset leaf streak virus (ELSV) is suggested. Leaf samples from enset and banana were screened using virus‐specific primers, and ELSV was detected in six of 40 enset but not found in any of 61 banana samples. On the other hand, Banana streak OL virus (BSOLV) was detected from the majority (60%) of symptomatic banana samples but not from enset samples. This paper reports the first full‐genome sequence of a putative new badnavirus species infecting plants in the genus Ensete. In addition, this is the first report of the occurrence of BSOLV in Ethiopia.     

Transmission of Pepino mosaic virus by the Fungal Vector Olpidium virulentus

Transmission of Pepino mosaic virus (PepMV) by the fungal vector Olpidium virulentus was studied in two experiments. Two characterized cultures of the fungus were used as stock cultures for the assay: culture A was from lettuce roots collected in Castellón (Spain), and culture B was from tomato roots collected in Murcia (Spain). These fungal cultures were maintained in their original host and irrigated with sterile water. The drainage water collected from irrigating these stock cultures was used for watering PepMV‐infected and non‐infected tomato plants to constitute the acquisition–source plants of the assay, which were divided into six different plots: plants containing fungal culture A (non‐infected and PepMV‐infected); plants containing fungal culture B (non‐infected and PepMV‐infected); PepMV‐infected plants without the fungus; and plants non‐infected either with PepMV and the fungus. Thirty‐six healthy plants grouped into six plots, which constituted the virus acquisition–transmission plants of the assay, were irrigated with different drainage waters obtained by watering the different plots of the acquisition–source plants. PepMV was only transmitted to plants irrigated with the drainage water collected from PepMV‐infected plants whose roots contained the fungal culture B from tomato with a transmission rate of 8%. No infection was detected in plants irrigated with the drainage water collected from plots with only a fungus or virus infection. Both the virus and fungus were detected in water samples collected from the drainage water of the acquisition–source plants of the assay. These transmission assays demonstrated the possibility of PepMV transmission by O. virulentus collected from tomato crops.     

First Report of Aster Yellows Phytoplasma in Goldenrain Tree (Koelreuteria paniculata) in Korea

Aster yellows phytoplasma was detected for the first time in goldenrain tree (Koelreuteria paniculata) growing in Sinpyeong‐myeon, Jeollabuk‐do, South Korea. DNA was extracted from the infected leaf samples and part of the 16S rDNA, rp operon and tuf gene were amplified using R16F2n/R2 and gene‐specific primers. The sequence analysis showed that the phytoplasma was closely related (99%) to members of the Aster Yellows (AY) group, and belonging to 16Sr I, subgroup B. Moreover, the 16S rDNA sequences of the isolate showed 88–96% identity with members of other 16Sr and undesignated groups. Based on the sequence identity and phylogenetic studies, it was confirmed that phytoplasma infecting goldenrain tree in South Korea belongs to the AY group.     

First detection of “Candidatus Phytoplasma asteris” - and “Candidatus Phytoplasma solani”-related strains in fig trees

In July 2017, a survey was conducted in a fig collection plot at Locorotondo (south of Italy) to investigate the possible presence of phytoplasmas in plants showing yellowing, deformed leaves, short internodes, mottling and mosaic. Samples were collected from symptomatic plants and tested by nested PCR assays using universal and specific primers to amplify the 16S rDNA of these prokaryotes. PCR results detected the presence of phytoplasma sequences in twenty plant samples that resulted clustering two phylogenetically distinct phytoplasmas, i.e., “Candidatus Phytoplasma asteris” and “Candidatus Phytoplasma solani” affiliated to 16SrI and 16SrXII ribosomal groups, respectively. The presence of phytoplasmas belonging to both ribosomal groups was confirmed with group specific quantitative PCR and RFLP assays on 16S ribosomal amplicons. Results of this study indicate for the first time the occurrence of phytoplasmas in fig; however, more work should be carried out to verify their association with the symptoms observed on diseased fig plants.     

First report on molecular detection of a stolbur phytoplasma (Group16SrXII-A) associated with kenaf (Hibiscus cannabinus L.) in India

Infection of stolbur phytoplasma was detected in kenaf (Hibiscus cannabinus) plants at CRIJAF research farm, Barrackpore, India. The infected plants formed profuse short branches at the top with bushy and bunchy top appearance. PCR with universal 16S rDNA phytoplasma primers P1/P7 yielded amplicons of 1.5 kb from all symptomatic leaf samples. Nested PCR with 16S-rDNA-specific nested primer pair R16F2n/R2 generated an amplicon of 1241 bp confirming the presence of a phytoplasma. The nested PCR products were sequenced and BALSTn analysis revealed 100% identity with 16S rRNA gene of phytoplasma. Phylogenetic analysis showed kenaf phytoplasma having 99% identity with both “Bois noir” stolbur phytoplasma 16SrXII group (Accession no: JQ181540). The RFLP data also supported the phylogenetic analysis. Multi-locus sequence characterisation assay was conducted by using different locus-specific primers viz. tuf, rpsC-rplV, rplF-rplR, map-SecY and uvrB-degV. The infected phytoplasma samples amplified only SecY gene and generated 1224 bp product which was deposited at NCBI (accession no: KC508636).