Molecular characterization of domestic and exotic potato virus S isolates and a global analysis of genomic sequences.

Five potato virus S (PVS) isolates from the USA and three isolates from Chile were characterized based on biological and molecular properties to delineate these PVS isolates into either ordinary (PVS(O)) or Andean (PVS(A)) strains. Five isolates - 41956, Cosimar, Galaxy, ND2492-2R, and Q1 - were considered ordinary strains, as they induced local lesions on the inoculated leaves of Chenopodium quinoa, whereas the remaining three (FL206-1D, Q3, and Q5) failed to induce symptoms. Considerable variability of symptom expression and severity was observed among these isolates when tested on additional indicator plants and potato cv. Defender. Additionally, all eight isolates were characterized by determining the nucleotide sequences of their coat protein (CP) genes. Based on their biological and genetic properties, the 41956, Cosimar, Galaxy, ND2492-2R, and Q1 isolates were identified as PVS(O). PVS-FL206-1D and the two Chilean isolates (PVS-Q3 and PVS-Q5) could not be identified based on phenotype alone; however, based on sequence comparisons, PVS-FL206-1D was identified as PVS(O), while Q3 and Q5 clustered with known PVS(A) strains. C. quinoa may not be a reliable indicator for distinguishing PVS strains. Sequences of the CP gene should be used as an additional criterion for delineating PVS strains. A global genetic analysis of known PVS sequences from GenBank was carried out to investigate nucleotide substitution, population selection, and genetic recombination and to assess the genetic diversity and evolution of PVS. A higher degree of nucleotide diversity (π value) of the CP gene compared to that of the 11K gene suggested greater variation in the CP gene. When comparing PVS(A) and PVS(O) strains, a higher π value was found for PVS(A). Statistical tests of the neutrality hypothesis indicated a negative selection pressure on both the CP and 11K proteins of PVS(O), whereas a balancing selection pressure was found on PVS(A).

First Report of Cucumber green mottle mosaic virus on Melon in the United States.

In July 2013, a melon (Cucumis melo var. Saski) field in Yolo County, California, was inspected as part of a phytosanitary inspection for seed production. The leaves of the plants showed mosaic, green mottle, and blotches. When plant sap was examined using a transmission electron microscope, rigid rod-shaped particles were observed. Melon plant samples were analyzed by both CDFA and USDA APHIS PPQ laboratories and tested positive using DAS-ELISA against Cucumber green mottle mosaic virus (CGMMV) (Agdia, Elkhart, IN). To confirm the presence of CGMMV, total RNA was analyzed by RT-PCR using primers CGMMV-F5370 5'-CTAATTATTCTGTCGTGGCTGCGGATGC-3' and CGMMV-R6390 5'-CTTGCAGAATTACTGCCCATA-3' designed by PPQ based on 21 genomic sequences of CGMMV found worldwide. The 976-bp amplicon was sequenced (GenBank Accession No. KJ453559) and BLAST analysis showed the sequence was 95% identical to MP and CP region of CGMMV isolates reported from Russia (GQ495274, FJ848666), Spain (GQ411361), and Israel (KF155231), and 92% to the isolates from China (KC852074), Korea (AF417243), India (DQ767631), and Japan (D12505). These analyses confirm the virus was CGMMV. To our knowledge, this is the first report of CGMMV in the United States. Based on our sequence data, a second set of primers (CGMMV-F5796 5'-TTGCGTTTAGTGCTTCTTATGT-3' and CGMMV-R6237 5'-GAGGTGGTAGCCTCTGACCAGA-3'), which amplified a 440-bp amplicon from CGMMV CP region, was designed and used for testing all the subsequent field and seed samples. Thirty-seven out of 40 randomly collected Saski melon samples tested positive for CGMMV, suggesting the virus was widespread in the field. All the melon samples also tested positive for Squash mosaic virus (SqMV) using DAS-ELISA (Agdia). Therefore, the symptoms observed likely resulted from a mixed infection. The melon field affected by CGMMV was immediately adjacent to fields of cucumber (Cucumis sativus var. Marketmore 76) and watermelon (Citrullus lanatus var. Sugar Baby) crops, both for seed production with no barrier between the crops. CGMMV was also detected from symptomatic plants from both fields. Seed lots used for planting all three crops were tested and only the melon seed was positive for CGMMV, suggesting the seed as the source of infection. The sequenced 440-bp RT-PCR amplicons from CGMMV-infected cucumber and watermelon plants and melon seeds were 99% identical to the CGMMV from the field melon. A cucumber plant infected with CGMMV but not SqMV was used for mechanical inoculation at the Contained Research Facility at University of California, Davis. Inoculated cucumber, melon, and watermelon plants showed green mottle and mosaic similar to that observed in the field. CGMMV is a highly contagious virus and damage by this virus on cucurbit crops has been reported in regions where CGMMV is present (2). CGMMV was detected on cucumber grown in greenhouses in Canada with 10 to 15% yield losses reported due to this virus (1). The three cucurbit crops in Yolo County were planted in an isolated area with no other cucurbits nearby. Measures, including destroying all the cucurbit plant material, have been taken to eradicate the virus. Use of CGMMV free cucurbit seed is necessary for prevention of this disease. References: (1) K.-S. Ling et al. Plant Dis. 98:701, 2014. (2) J. Y. Yoon et al. J. Phytopathol. 156:408, 2008.

First Report of Sorghum mosaic virus Causing Mosaic in Miscanthus sinensis.

Miscanthus is being evaluated as a bioenergy feedstock because of its potentially significant biomass production, perennial habit, and lack of major diseases and pests. It is also a valuable parent in sugarcane breeding programs as a source of cold tolerance. In May 2010, mosaic symptoms were observed on a clone of Miscanthus sinenesis Anderss. maintained at the USDA, ARS, Sugarcane Research Unit. All plants of the Miscanthus clone in our germplasm collection are from vegetative cuttings of the original infected plant and show mosaic symptoms. Leaves from the ratoon of a single plant tested positive in a reverse transcription-PCR with the Potyvirus Group PCR Test (Agdia, Inc., Elkhart, IN) with two primer sets, Poty-F1/Poty-R1 and Poty-F2/Poty-R2. After sequencing the potyvirus amplicons, a BLAST search in GenBank revealed that these sequences had the highest identities (81 and 69%) with Sorghum mosaic virus (SrMV) at the nucleic acid level and a 72 and 95% similarity at the amino acid level. Extracts from the Miscanthus clone prepared by the indirect extraction buffer (Agdia) containing sodium carbonate also tested positive for potyvirus by indirect ELISA with the 'universal' potyvirus monoclonal antibody, PTY1. To our knowledge, this is the first report of SrMV on Miscanthus. The only other member of the genus Potyvirus reported on Miscanthus is Sugarcane mosaic virus (1,2). Mosaic caused by SrMV could become an economically important disease of Miscanthus if this crop is grown for bioenergy feedstock on large areas. An SrMV-infected Miscanthus crop could pose a threat to established crops of susceptible sugarcane and sorghum since the virus is transmitted in a nonpersistent manner by several aphids, as well as, contributing to geographic shifts of the pathogen. References: (1) B. O. Agindotan et al. J. Virol. Methods 169:119, 2010. (2) D.-L. Xu et al. Arch. Virol. 153:1031, 2008.

Identification and full sequence of an isolate of Alternanthera mosaic potexvirus infecting Phlox stolonifera.

A potexvirus was isolated from creeping phlox (Phlox stolonifera) plants from a commercial nursery in Pennsylvania. The virus was serologically related to clover yellow mosaic virus, plantain virus X, potato virus X, and potato aucuba mosaic virus, and was most closely related to papaya mosaic virus (PapMV). The sequence of a PCR fragment obtained with potexvirus group-specific primers was distinct from that of PapMV; the coat protein (CP) gene and 3' untranslated region (UTR) were closely related to Alternanthera mosaic virus (AltMV), previously reported only from Australia. The host range was similar to that of the Australian isolate (AltMV-Au), and the phlox isolate reacted strongly with antiserum to AltMV-Au. The full sequence of the phlox isolate was more closely related to PapMV throughout the genome than to any potexvirus other than AltMV-Au, for which only the CP and 3'UTR sequences are available. The phlox isolate was therefore named AltMV-PA (for Pennsylvania), and the full 6607 nt sequence is presented(1). Additional AltMV isolates from creeping phlox (AltMV-BR and AltMV-SP) and trailing portulaca (Portulaca grandiflora; AltMV-Po) were also isolated, suggesting that AltMV may be widespread, and may have been mis-diagnosed in the past as PapMV. AltMV has the potential to spread to other ornamental crops.

First Report of Lolium latent virus in Ryegrass in the United States.

Initial reports of the presence of Lolium latent virus (LLV) in Lolium perenne L. and L. multiflorum Lam. breeding clones in Germany, the Netherlands, France (2), and recently the United Kingdom (3,4; described as Ryegrass latent virus prior to identification as LLV) prompted us to evaluate clonally propagated Lolium plants from the United States. Four genetically distinct plants (viz., MF22, MF48, MF125, and MF132) that have been maintained clonally for 5 years from a Lolium perenne × L. multiflorum hybrid population established in the United States exhibited either no symptoms or mild chlorotic flecking that coalesced to form chlorotic to necrotic streaking on the leaves. All four clonal plants tested positive using reverse transcription-polymerase chain reaction (RT-PCR) with the Potexvirus group PCR test (Agdia, Inc., Elkhart, IN), whereas all clones but MF48 tested positive using the Potyvirus group PCR test (Agdia, Inc.). No amplicons were obtained when the same plants were tested for tobamovirus, carlavirus, and closterovirus using appropriate virus group-specific primers. Cloning and sequencing of the potexviral amplicons revealed very high sequence identity with the comparable region of LLV-UK (GenBank Accession No. DQ333886), whereas those of the potyviral amplicons (GenBank Accession Nos. DQ355837 and DQ355838) were nearly identical with the comparable region of Ryegrass mosaic virus (RGMV), a rymovirus first reported from the United States in 1957 (1). Using indirect enzyme-linked immunosorbent assay (ELISA), extracts from all four Lolium clonal propagations tested positive for LLV using the antiserum raised to LLV-Germany (courtesy of Dr. Huth), whereas the potyvirus-positive results from RT-PCR of the three clones were confirmed using indirect ELISA with the broad spectrum potyvirus monoclonal antibody, PTY-1. LLV from singly or dually infected Lolium clones was transmitted to Nicotiana benthamiana Domin. but not to N. tabacum L. by mechanical inoculation. LLV was purified from infected N. benthamiana. Similar sized flexuous rods were observed using electron microscopy in leaf dip samples from Lolium clones and aliquots of the virions purified from N. benthamiana. References: (1) G. W. Bruehl et al. Phytopathology 47:517, 1957. (2) W. Huth et al. Agronomie 15:508, 1995. (3) R. Li et al. Asian Conf. Plant Pathol. 2:89, 2005. (4) C. Maroon-Lango et al. Int. Congr. Virol. 13:63, 2005.

Two unique US isolates of Pepino mosaic virus from a limited source of pooled tomato tissue are distinct from a third (European-like) US isolate.

Three strains of Pepino mosaic virus (PepMV) found in the US have been cloned and sequenced by RT-PCR using total RNA from infected tissue as template, and degenerate potexvirus- and PepMV species- and isolate-specific primers. Despite limited source material, the complete nucleotide sequences (6413 and 6410 nts, respectively) of two isolates, PepMV-US1 and PepMV-US2, were obtained and analyzed using total RNA from less than 0.2 g of a pooled infected tomato leaf sample from Arizona. Sequence of the 3'-end of the third isolate from infected fresh tomato fruits from Maryland (PepMV-US3) was also determined. The genome organizations of PepMV-US1 and US2 were typical of the genus Potexvirus, with the following reading frame order: ORF 1, encoding a putative replicase; ORFs 2-4, triple gene block proteins (TGBp) 1-3; and ORF 5, coat protein (CP). Gene-for-gene comparison between PepMV-US1 and US2 revealed the following amino acid identities: 91% in replicase, 89% in TGBp1, 92% in TGBp2, 85% in TGBp3, and 93% in the CP; with an overall nucleotide identity of 86%. Nucleotide sequence comparisons between US1 and US2 and the European isolates showed only 79-82% identity, whereas the identity among the European isolates was over 99%. Sequence comparisons and phylogenetic analysis indicate that PepMV-US1 and US2 are distinctly different from the European isolates, while the CP of PepMV-US3 is nearly identical to the European isolates. The results presented also suggest that TGBp1 and TGBp3 are more suitable than either the replicase or coat protein gene products for discriminating PepMV isolates.