Viral Metagenomic-Based Screening of Sugarcane from Florida Reveals Occurrence of Six Sugarcane-Infecting Viruses and High Prevalence of Sugarcane yellow leaf virus.

A viral metagenomics study of the sugarcane virome in Florida was carried out in 2013 to 2014 to analyze occurrence of known and potentially new viruses. In total, 214 sugarcane leaf samples were collected from different commercial sugarcane (Saccharum interspecific hybrids) fields in Florida and from other Saccharum and related species taken from two local germplasm collections. Virion-associated nucleic acids (VANA) metagenomics was used for detection and identification of viruses present within the collected leaf samples. VANA sequence reads were obtained for 204 leaf samples and all four previously reported sugarcane viruses occurring in Florida were detected: Sugarcane yellow leaf virus (SCYLV, 150 infected samples out of 204), Sugarcane mosaic virus (1 of 204), Sugarcane mild mosaic virus (13 of 204), and Sugarcane bacilliform virus (54 of 204). High prevalence of SCYLV in Florida commercial fields and germplasm collections was confirmed by reverse-transcription polymerase chain reaction. Sequence analyses revealed the presence of SCYLV isolates belonging to two different phylogenetic clades (I and II), including a new genotype of this virus. This viral metagenomics approach also resulted in the detection of a new sugarcane-infecting mastrevirus (recently described and named Sugarcane striate virus), and two potential new viruses in the genera Chrysovirus and Umbravirus.

First Report of Cucumber mosaic virus Infection in Pachysandra in the United States.

Pachysandra terminalis Siebold & Zucc. (Japanese pachysandra, spurge) is widely used as a groundcover. In early 2012, Japanese pachysandra plants from Missouri, which originated in Pennsylvania, showed symptoms of light and dark green mosaic, leaf deformation, concentric ringspots, and stunting. Initial screening of symptomatic leaf tissue by transmission electron microscopy (TEM) using partially purified extracts confirmed the presence of spherical (~28 nm) and bacilliform (18-nm diameter, 35- to 58-nm length) virus particles. Immunosorbent electron microscopy (ISEM) using antisera to a clover isolate of Alfalfa mosaic virus (AMV) (PVAS 92) and to Cucumber mosaic virus (CMV) (ATCC PVAS-30) obtained from the American Type Culture Collection, Manassas, VA, confirmed the presence of AMV and CMV. No other type of virus-like particles were observed by TEM. After 6 months, nearly 20% of the 4,000 pachysandra cuttings exhibited the described symptoms. However, it is possible that more than 20% of the cuttings were infected with both viruses and not yet exhibiting symptoms. Reverse-transcription PCR (RT-PCR) was done using total RNA extracted with a Qiagen RNeasy kit and Ready-To-Go RT-PCR beads (GE Healthcare, UK Limited, UK). The primer pair CMV-1 (5'-GCCGTAAGCTGGATGGACCA) and CMV-2 (5'-TATGATAAGAAGCTTGTTTTCGCG) were used (3) to obtain a 502-bp amplicon from the coat protein (CP) region of CMV RNA 3. The product was ligated and cloned (pGEM-T Easy Vector System; Promega, USA). Three clones were sequenced (UMGC, USA), and the consensus sequence (Sequencher 5.1, Gene Codes Corp., USA) was deposited in GenBank (Accession No. JX227938). The sequence obtained had 100% identity with a homologous CP CMV sequence (AFQ94058) and 99% identity with several other homologous CP CMV sequences (CAX62443, CCK24369, and 15 others). It also contained an EcoRI site at nucleotides 332 to 337, characteristic of CMV Type II isolates (3). The primer pair AMV1F (5'-ATCCACCGATGCCAGCCTTA) and AMV1R (5'-TTCCGCCTCACTGCTGCTG) generated a 1,047-bp product from AMV RNA1 that was deposited in GenBank (JX227937). This product had 100% identity with a homologous AMV sequence (AFQ94057), and 99% identity with several other homologous AMV sequences (AGV15824, ADO85715, CBX36144). From the data presented here, it was concluded that the pachysandra had a mixed infection of AMV and a Type II isolate of CMV. Occurrence of AMV in pachysandra was first reported in New Jersey in 1982 (2) and reported for the first time in France and Germany in 2000 (1). The presence of CMV infection in pachysandra has not been reported in the present literature. Some of the symptoms associated with AMV infection in pachysandra in New Jersey (2) and Europe (1) were similar to the symptoms produced by pachysandra plants infected with both viruses (ring spots, mosaic, and line patterns). However, some symptoms were unique to the mixed infection in pachysandra by AMV and CMV (leaf deformation, stunting). A potential source of this co-infection could occur when plants are grown near alfalfa fields (AMV infection by aphids) and undergo vegetative propagation (CMV infection by contaminated tools). This is the first report of pachysandra co-infected by AMV and CMV in the United States. References: (1) L. Cardin and B. Moury. Plant Dis. 84:594, 2000. (2) D. E. Hershman and E. H. Varney. Plant Dis. 66:1195, 1982. (3) S. Wylie et al. Aust. J. Agric. Res. 44:41, 1993.

First Report of Catharanthus mosaic virus in Mandevilla in the United States.

Mandevilla (Apocynaceae) is an ornamental tropical vine popular for its bright and attractive flowers. During 2012 to 2013, 12 Mandevilla sp. samples from Minnesota and Florida nurseries were submitted for analysis at the University of Minnesota Plant Disease Clinic. Plants showed mosaic symptoms, leaf deformation, premature leaf senescence, and vine dieback. Filamentous virus particles with modal lengths 700 to 900 nm were observed by transmission electron microscopy (TEM) in partially purified preparations from symptomatic leaves. Partially purified virions were obtained using 30% sucrose cushion centrifuged at 109,000 gmax for 2 h at 10°C (5). No other virus particles were observed in these samples, nor were any observed in non-symptomatic samples. One sample was submitted as potted plant (Mandevilla 'Sunmandeho' Sun Parasol Giant White) and was kept under greenhouse conditions for subsequent analyses. Total RNA (Qiagen) was extracted from this sample, and Potyvirus was detected using the universal primers Poty S (5'-GGN AAY AAY AGY GGN CAR CC-3') and PV1 (5'-20(T)V-3') (1) by reverse transcription (RT)-PCR (3). The amplified product was the expected ~1.7-kb, corresponding to the partial nuclear inclusion body gene, the coat protein (CP) gene, and the 3' end untranslated region. The RT-PCR amplicon was cloned (NEB) and sequenced, and the 1,720-bp consensus sequence was deposited in GenBank (Accession No. KM243928). NCBI BLAST analysis at the nucleotide level revealed highest identity (83%) with an isolate of Catharanthus mosaic virus (CatMV) from Brazil (Accession No. DQ365928). Pairwise analysis of the predicted 256 amino acid CP revealed 91% identity with the CatMV Brazilian isolate (ABI94824) and 68% or less identity with other potyviruses. Two potyviruses are usually considered the same species if their CP amino acid sequences are greater than 80% identical (2). Serological analysis of the infected sample Mandevilla 'Sunmandeho' Sun Parasol Giant White using a CatMV specific antiserum (4) resulted in positive indirect ELISA reactions. CatMV has been previously reported in periwinkle (Catharanthus roseus) in Brazil (4). Based on the analyses by TEM, RT-PCR, nucleotide and amino acid sequence identities, and serological reactivity, we identify this virus as a U.S. Mandevilla isolate of CatMV. To our knowledge, this is the first report of Catharanthus mosaic virus both in the United States and in Mandevilla. References: (1) J. Chen et al. Arch Virol. 146:757, 2001. (2) A. Gibbs and K. Ohshima. Ann. Rev. Phytopathol. 48:205, 2010. (3) R. L. Jordan et al. Acta Hortic. 901:159, 2011. (4) S. C. Maciell et al. Sci. Agric. Piracicaba, Brazil. 68:687, 2011. (5) D. Mollov et al. Arch Virol. 158:1917, 2013.

First Report of Aster Yellows Phytoplasma in Soybean in Michigan.

During the summer of 2012, soybean plants in a commercial field in Clinton County, Michigan, exhibited symptoms characteristic of phytoplasmal diseases (1,2). Symptoms included extensive top dieback, stunting, purple stem surfaces, internal necrosis, leaf vein discoloration, and bud proliferation. Approximately 80% of plants in a half hectare along the southern edge of the field were symptomatic, although the majority of plants in the 4-ha field appeared symptomless. Total genomic DNA was extracted from one symptomatic and one asymptomatic leaf sample using a Qiagen DNeasy Plant Mini Kit (Qiagen, Germantown, MD) according to manufacturer's instructions. The DNA was used as template in direct PCR primed by the phytoplasma-universal primers P1/P7 followed by nested PCR primed by P1/AYint (3). Reactions containing template DNA from the symptomatic plant yielded ribosomal RNA gene amplicons of 1.8 kbp (P1/P7-primed) and 1.6 kbp (P1/AYint-primed), respectively. Reactions containing template DNA from the asymptomatic plant or water did not yield amplicons. The products of PCRs primed by P1/P7 were purified using PureLink PCR Purification kit (Life Technologies, Carlsbad, CA) and cloned in a pGem T-Easy vector system (Promega, Madison, WI). Three separate clones were sequenced using the sequencing primers M13For, M13Rev, SAYF nt 755, (5'-AAAGCGTGGGGAGCAAACAG), and SAYR nt 1159, (5'-TTTGACGTCGTCCCCACCTT). The sequences of all three clones were identical. A consensus (Sequencher 4.1, Gene Codes Corporation, Ann Arbor, MI) nucleotide sequence of 1,830 bp was deposited in GenBank under the accession number KF528320. A BLASTn similarity search revealed that the sequence shared 100% identity to rDNA of aster yellows phytoplasma (AF222063). Additionally, analysis of the 16Sr group/subgroup classification, based on in silico RFLP analyses using iPhyClassifier (4), indicated that the soybean phytoplasma is a member of subgroup 16SrI-B aster yellows phytoplasma subgroup. In a phylogenic tree deduced using the neighbor joining algorithm, the phytoplasma consensus sequence obtained from soybean in Michigan clustered with other group 16SrI (aster yellows phytoplasma) strains. While aster yellows phytoplasma has been previously reported in soybean in Wisconsin (2), to our knowledge, this is the first report of aster yellows in soybean in Michigan. References: (1) C. R. Grau et al. Compendium of Soybean Diseases, 4th ed. G. L. Hartman et al., eds. American Phytopathological Society, St. Paul, MN, 1999. (2) M. E. Lee et al. Can. J. Plant Pathol. 24:125, 2002. (3) C. D. Smart et al. Appl. Env. Microbiol. 62:2988, 1996. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

First Report of a 16SrI (Aster Yellows) Group Phytoplasma on Garlic (Allium sativum) in the United States.

During the growing season of 2012, 35 garlic plant samples were submitted to the University of Minnesota Plant Disease Clinic for disease diagnosis. Samples originated from multiple counties throughout Minnesota as well as Iowa, Wisconsin, and South Dakota. Symptoms first appeared at the time plants were starting to produce scapes. Symptoms included leaf discoloration that varied from yellow to purple, plant stunting, and leaf tip necrosis. In severe cases, the plants wilted and died. Bulbs of affected plants ranged from being soft and small to almost normal-looking. Symptoms were similar to those associated with phytoplasma infection in other plants. Total genomic DNA was extracted from 30 symptomatic samples and five asymptomatic leaf samples using a Qiagen DNeasy Plant Mini Kit (Qiagen, Germantown, MD) according to the manufacturer's instructions, and used with the universal phytoplasma primers P1/P7 in a direct PCR assay, and with P1/AYint in a nested PCR assay (2) to yield amplicons of 1.8 and 1.6 kb, respectively. Asymptomatic plants did not produce amplicons. Garlic cultivars displaying a range of symptoms tested positive for the presence of phytoplasma. These cultivars included: Susanville, Middle Eastern, Music, Ajo Rojo, Spanish Roja, Inchelium Red, Silver White, Asian Tempest, Chesnok Red, and Purple Glazer. The P1/P7 PCR products of 1,830 bp were purified using the PureLink PCR Purification kit (Life Technologies, Carlsbad, CA), and cloned in a pGem T-Easy vector system (Promega, Madison, WI). Sequences from a clone from each of Wisconsin, Iowa, and Minnesota were deposited in GenBank under the accession numbers KC000005, KC000006, and KC000007, respectively. A BLASTn similarity search revealed that the Wisconsin and Iowa isolates shared 99% homology to the sequences of 16SrI-A group phytoplasmas, aster yellows phytoplasma (AY389827), and aconitum proliferation phytoplasma (AF510323). The Minnesota isolate had 99% sequence homology to a 16SrI-B group phytoplasma, mulberry yellow dwarf phytoplasma (GQ249410). Also, the iPhyClassifier 16Sr group/subgroup classification based on similarity (3) analyses showed that the Wisconsin and Iowa phytoplasma isolates had 16S rDNA sequences in the 16SrI-A group with similarity coefficients of 0.97 and 1.00, respectively, to aster yellows witches'-broom phytoplasma AYWB (NC_007716). The same analysis revealed that the Minnesota phytoplasma isolate 16S rDNA sequence grouped with the 16SrI-B group onion yellows phytoplasma (NC_005303) with a similarity coefficient of 1.0. A phylogenic tree was deduced by the neighbor joining algorithm, clustering together the Iowa, Minnesota, and Wisconsin isolate sequences with a 16SrI group phytoplasma. Aster yellows phytoplasma has been reported in North America, but only in Canada (1). This is the first documented occurrence of 16SrI aster yellows group phytoplasma in garlic in the United States. The spring of 2012 was unusually warm, and high leafhopper pressure was observed throughout the Midwest; above average numbers of many ornamental crops and small grains were infected with phytoplasma. These events may have contributed to the phytoplasma infection in garlic. References: (1) A. H. Khadhair et al. Microbiol. Res. 157:161, 2002. (2) C. D. Smart et al. Appl. Env. Microbiol. 62:2988, 1996. (3) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

First Report of Garlic Rust Caused by Puccinia allii on Allium sativum in Minnesota.

High-quality garlic is an emerging crop grown in Minnesota for local markets, community supported agriculture, and select restaurants. In July 2010, Allium sativum cv. German Extra Hardy Porcelain plants showing foliar symptoms typical of rust infection were brought to the Plant Disease Clinic at the University of Minnesota by a commercial grower from Fillmore County, Minnesota. Infected leaves showed circular to oblong lesions (1 to 3 mm long), which ranged in color from yellow-orange (uredinia) to black (telia). Urediniospores collected from uredinia were globoid to ellipsoid, yellowish in color, and measured 18 ± 1 × 30 ± 2 µm with a wall thickness of 2.4 ± 0.5 µm. Teliospores were two celled, 18 ± 3 × 47 ± 10 µm, with a projected cross-sectional area (1) of 826 ± 87 µm2; cell walls were smooth, brown, 1.6 ± 0.3 µm (proximal cell) to 2.1 ± 0.5 µm (distal cell) thick, and 4.2 ± 0.8 µm at the apex. The pathogen was identified as Puccinia allii (2) and a sample was deposited in the U.S. National Fungus Collection (BPI 884132). DNA was extracted from infected leaf tissue and the nuclear ribosomal internal transcribed spacer region (ITS) and 5' end of the large subunit (LS) was amplified and sequenced as described by Anikster et al. (1). The 1,257-bp sequence from the sample collected in Minnesota (GenBank Accession No. JX402206) was identical to ITS/LS sequence of a sample of P. allii collected from garlic in California (GenBank Accession No. AF511077), with the exception that MN sequence contained nine "A"s rather than 10 in the hyper-variable area at the 3' end of the ITS region. P. allii has been shown to be a species complex comprising at least two different types, "leek type" and "garlic type" (1). Based on the ITS sequence and the projected cross-sectional area of the teliospores, the sample of P. allii from MN is consistent with the garlic type. Garlic rust occurred in localized foci late in the growing season and therefore did not cause significant loss to the 2010 crop. Reoccurrence of garlic rust was not reported in either 2011 or 2012 growing seasons in Minnesota. P. allii all but eliminated commercial garlic production in California in the late 1990s (1) and has the potential to cause significant negative impact to the emerging garlic crop in Minnesota. However, the epidemiology of garlic rust in the northern U.S. is not well understood and therefore predicting the risk of the Minnesota garlic crop to rust is difficult. References: (1) Y. Anikster et al. Phytopathology 94:569, 2004. (2) L. J. Szabo et al, Rust. Pages 41-44 in: Compendium of Onion and Garlic Diseases and Pests, Second Edition. H. F. Schwartz and S. K. Mohan, eds. APS Press, St. Paul, 2008.

First Report of Alfalfa mosaic virus Occurrence in Hydrangea in the United States.

In spring of 2012, a previously unrecorded virus-like disease characterized by conspicuous yellow leaf blotching (calico symptoms) was observed in plants of Hydrangea macrophylla in a single location in Southampton, NY. Bacilliform and spherical particles resembling those of Alfalfa mosaic virus (AMV) were observed by transmission electron microscopy (TEM) in partially purified extracts from symptomatic leaf tissue. The identity of the virus was confirmed by immunosorbent electron microscopy (ISEM) (4) using antiserum to AMV (ATCC PVAS 92) that both trapped and decorated the virions. Three primer pairs designed from available AMV RNA 1, RNA 2, and RNA 3 genomic sequences were used to generate amplicons from the hydrangea AMV isolate. Reverse-transcription (RT)-PCR was done using total RNA extracted from symptomatic hydrangea leaf tissue with a Qiagen RNeasy kit, and Ready-to-Go RT-PCR beads (GE Healthcare). Amplicons of 1,049, 1,013, and 658 bp were obtained using the primer pairs AMV1F (5'-ATCCACCGATGCCAGCCTTA)/AMV1R (5'-TTCCGCCTCACTGCTGTCTG), AMV2F (5'-GATCGCCGGAAGTGATCCAG)/AMV2R (5'-TCACCGGAAGCAACAACGAA), and AMV3F (5'-GCCGGTTCTCCAAAGGGTCT)/AMV3R (5'-CGCGTCGAAGTCCAGACAGA), respectively. The PCR products were cloned using a TOPO TA cloning kit (Invitrogen) and three clones of each were sequenced. The sequences obtained from the hydrangea AMV RNA 1 (JX154090), RNA 2 (JX154091), and RNA 3 (JX154092) had 95 to 98% nucleotide sequence identity to homologous genomic sequences of known AMV isolates. To our knowledge, this is the first report of AMV occurrence in H. macrophylla in the United States. This virus has been reported to occur in H. macrophylla in British Columbia (3), but in a previous survey its presence was not detected in hydrangeas in the United States (1). A report of possible AMV infection in H. macrophylla in Italy (2) was based solely on symptomatology and cross-protection tests and therefore cannot be verified. The AMV-infected hydrangea plants were found by ISEM to also contain low concentrations of Hydrangea ringspot virus (HRSV) and Hydrangea chlorotic mottle virus (HdCMV). However, based on previous evidence of single and mixed infections (3), it is unlikely that the calico symptoms observed were influenced by the presence of HRSV and HdCMV. This report is of interest both because AMV, unlike HRSV and HdCMV, causes foliar symptoms that would render hydrangea plant unmarketable, and because the disease can be spread by a number of common aphid species that transmit AMV. It will also serve to alert growers and diagnosticians to the potential threat posed by AMV infection. References: (1) T. C. Allen et al. Acta Hortic. 164:85, 1985. (2) G. Belli. Phytopathol. Mediterr. 7:70, 1968. (3) A. W. Chiko and S. E. Godkin. Plant Dis. 70:541, 1986. (4) B. E. L. Lockhart et al. Phytopathology 82:691, 1992.

First Report of Nerine yellow stripe virus in Amaryllis in the United States.

Ornamental flower bulbs (including true bulbs, bulbils, corms, tubers, and rhizomes) are increasingly important floriculture crops. Amaryllis is a small genus of flowering bulbs, with two species. The South African native, Amaryllis belladonna, also known as belladonna lily, Jersey lily, naked lady, Amarillo, or March lily, is one of numerous ornamental species with the common name "lily" due to their flower shape and growth habit. Amaryllis are popular for their 6- to 10-inch trumpet shaped colorful flowers that are borne on 1- to 2-foot stalks. In January, 2011, a home gardener in California observed mosaic symptoms on the leaves of A. belladonna growing in her garden. Leaf samples were sent to Agdia Inc. for testing. Samples tested positive for the presence of Potyvirus in a reverse transcription (RT)-PCR screen using universal potyvirus primers (2) yielding the expected ∼1,600-bp product corresponding to the partial nuclear inclusion body (NIb) gene, full-length coat protein (CP) gene, and 3' end untranslated region (UTR). Electron microscopy of symptomatic leaves confirmed the presence of filamentous potyvirus-like particles. The RT-PCR amplicon was cloned and sequenced (2); the 1,616-bp consensus sequence was deposited in GenBank (Accession No. JX865782). NCBI BLAST analysis of the consensus sequence revealed highest identities with isolates of Nerine yellow stripe virus (NeYSV; family Potyviridae, genus Potyvirus). Pair-wise analyses of the 261 amino acid sequence of the predicted CP had 88% sequence identity with a Stenomesson isolate reported from the Netherlands (EU042758); 87% identity with Hymenocallis and Nerine isolates, both also from the Netherlands (EF362622 and EF362621, respectively); and, 86% with two New Zealand isolates infecting Amaryllis or Vallota (FJ618537 and DQ407932, respectively). The five Netherlands and New Zealand isolates are more closely related to each other than to the U.S. isolate as they share 93 to 98% CP identity. When using viral genome sequence relatedness as a criterion for defining potyvirus species, isolates with CP amino acid identity greater than 80% are considered the same species (1). The predicted coat protein gene of the California isolate was sub-cloned into the bacterial expression vector pET44 EK/LIC. Serological analysis of coat protein expressing clones in ELISA and Western Blot analysis using a potyvirus broad-spectrum reacting monoclonal antibody PTY-2 (3) and a NeYSV-specific rabbit antiserum (Applied Plant Research, Lisse, The Netherlands) resulted in positive reactions. NeYSV has previously been reported in the United Kingdom, the Netherlands, Australia, and New Zealand. Based on the results of electron microscopy, RT-PCR, nucleotide and amino acid identity, and serological reactivity, we identify this virus as a U.S. isolate of NeYSV, NeYSV-US. To our knowledge, this is the first report of Nerine yellow stripe virus in the United States. Development of antisera specific to this U.S. isolate is in progress. References: (1) A. Gibbs and K. Ohshima. Ann. Rev. Phytopathol. 48:205, 2010. (2) R. L. Jordan et al. Acta Hortic. 901:159, 2011. (3) R. L. Jordan and J. Hammond. J. Gen. Virol. 72:1531, 1991.

First Report of Puccinia veronicae-longifoliae on Veronica spicata 'Royal Candles' in Minnesota.

In September 2008, Veronica spicata 'Royal Candles' plants showing foliar symptoms typical of a rust infection were brought to the Plant Disease Clinic at the University of Minnesota. Plants were grown in a local nursery in Dakota County, Minnesota. A dark brown discoloration was apparent on the upper surface of the leaf with lighter brown pustules on the underside. Teliospores collected from the pustules were 2-celled with smooth walls and 36.35 to 48.87 µm long, 11.96 to 18.28 µm wide, and had a wall thickness of 1.33 to 2.61 µm, which is in accordance with type specimen of Puccinia veronicae-longifoliae (4). Pathogen identity was confirmed by comparison of the DNA sequence of nuclear ribosomal RNA region containing the internal transcribed spacer regions 1 and 2, 5.8S and the 5' end of the 28S subunits between herbarium samples from the U.S. National Fungus Collection (BPI 841971/GenBank Accession JQ627617 and BPI 871789/GenBank Accession JQ627618) and the collected specimen (BPI 882886/GenBank Accession JQ627616). P. veronicae-longifoliae was first reported in the United States in 2004 from a commercial nursery in Michigan (2). Veronica rust has also been found in Michigan in 2005 and more recently in 2011 (1). The only other known report of Veronica rust in the United States occurred in Connecticut in 2007 (3). P. veronicae-longifoliae is not considered a quarantine pest by The Animal and Plant Health Inspection Service due to the limited host range, the host not being on the threatened or endangered list and the host being of little economic or environmental importance (2). References: (1) T. A. Dudek et al. MSU Extension News. Retrieved from http://msue.anr.msu.edu/news/veronica_rust_observed_this_season/ , 2011. (2) North American Plant Protection Organization's Phytosanitary Alert System. Retrieved from http://www.pestalert.org/oprDetail.cfm?oprID=129 , 2004. (3) Pundt, L. Floriculture Greenhouse Update. Retrieved from http://www.negreenhouseupdate.info/index.php/july/194-rust-on-veronica , 2007. (4) D. B. O. Savile. Can. J. Bot. 46:631, 1968.

First Report of Ditylenchus dipsaci on Garlic in Minnesota.

In the summer of 2011, two independent garlic samples from Morrison and Dakota counties and in 2012 one garlic sample from Carver county in Minnesota were submitted by commercial growers to the University of Minnesota Plant Disease Clinic for disease analyses. Symptoms of the above-ground plant parts were stunting and chlorosis. Symptoms of bulbs were necrosis, underdevelopment, and distortion. Upon microscopic examination, phytonematodes exuded into the surrounding water droplet. Nematodes were present in the protective leaves, abscission zone, and cloves in all submitted bulbs (n = 18) for analyses. Morphometric examination of females, males, and juveniles determined that they were Ditylenchus dipsaci. Nematodes extracted from garlic cloves were fixed in TAF (97 ml formalin [40%], 2 ml triethanolamine, and 91 ml dH2O). Morphological observations and measurements were made under an Olympus BX51 microscope equipped with a Nomarski differential interference contrast. Female (n = 6) measurements were: L = 1.411 to 1.636 mm, a = 38 to 44, b = 5.8 to 8.0, c = 14 to 17, stylet = 11.5 to 12.3 µm, V = 79 to 81%, and tail = 95 to 105 µm. The body was almost straight, when heat relaxed, lip region flattened, median bulb oval, and isthmus elongate and slender. The basal pharyngeal bulb overlapped the intestine. The post-vulval uterine branch was about half of vulva-anus distance. The tail was conoid with a pointed terminus. Male (n = 9) measurements were: L = 1.372 to 1.558 mm, a = 40 to 50, b = 6.5 to 7.0, c = 14 to 16, stylet = 11.5 to 12.3 µm, spicules = 22 to 27 µm, and gubernaculum = 9 to 10 µm. The bursa was leptoderan and spicules were curved with simple gubernaculum. Morphology and morphometrics of females and males of D. dipsaci from Minnesota generally fit the descriptions provided for the type and other populations by Hopper (1) and other authors. Several specimens were also taken for molecular identification. DNA extraction, PCR, and sequencing protocols were as described by Subbotin et al. (2). The TW81 and AB28 primers were used for amplification of ITS-rRNA region and the D2A and D3B primers were used for amplification of the D2-D3 expansion segments of 28S rRNA gene. Comparison of the ITS and D2-D3 of 28 rRNA gene sequences showed 100 and 99% identity with corresponding gene sequences of D. dipsaci published in the GenBank (2). The sequences were submitted in the GenBank under accession numbers JX123258 and X123259. This nematode problem has not been known to occur in either of these locations previously. The most likely source of introduction of D. dipsaci are imported garlic seed bulbs. To our knowledge, this is the first report of D. dipsaci affecting garlic or any other crops in Minnesota. The garlic produced in these locations was considered unmarketable and complete loss to the farmers. The presence of D. dipsaci could have a significant economic impact in the emerging multi-million dollar garlic industry in Minnesota. References: (1) D. J. Hooper. Ditylenchus dipsaci. CIH Descriptions of Plant-Parasitic Nematodes Set 1, No. 14, 1972. (2) S. A. Subbotin et al. Phytopathology 95:1308, 2005.

First Report of Bacterial Wilt in Mandevilla (= Dipladenia) splendens 'Red Riding Hood' in the United States Caused by Ralstonia solanacearum Biovar 3.

In November of 2007, 6-inch rooted cuttings of Mandevilla (= Dipladenia) splendens 'Red Riding Hood' were submitted from a greenhouse in Indiana to the Purdue Plant and Pest Diagnostic Lab. Plants exhibited leaf dieback, wilting, and reduced top growth. Microscopic observation revealed no fungal structures within the roots, stems, and leaves; however bacterial streaming was observed from the cut edge of stem and root tissue using ×100 magnification with phase contrast. A Ralstonia solanacearum ImmunoStrip test (Agdia Inc., Elkhart, IN) was used to determine that the samples (roots and stem) were positive for R. solanacearum, the causal agent of southern wilt. A bacterial suspension was prepared from infected tissue and streaked onto King's Medium B (KB). Gram-negative, nonfluorescent, oxidase-positive bacteria were consistently isolated from the diseased tissues and determined as R. solanacearum by BIOLOG (Hayward, CA) carbohydrate utilization. A culture of R. solanacearum and infected plant material were submitted to USDA APHIS PPQ as per select agent protocol. (3) CPHST NPGBL generated pure cultures and together with submitted plant materials they tested positive for R. solanacearum using Agdia ImmunoStrips. Culture and plant material tested positive for R. solanacearum and negative for biovar 2 using the Fegan conventional PCR (1) and the Central Science Lab (CSL, York, UK) real-time PCR (4). Pure cultures were determined to be negative for biovar 2 but positive for biovar 3 using the biovar carbohydrate utilization plate assay (2). On the basis of these results, the bacteria were identified as R. solanacearum biovar 3 and not as the select agent R. solanacearum race 3 biovar 2. Koch's postulates confirmed pathogenicity of the isolated bacteria on tomato, a susceptible host. Three 6-week-old plants were mechanically inoculated with a bacterial suspension of approximately 1 × 108 CFU/ml prepared from cultures grown on KB for 2 days at 28°C. Inoculum (0.1ml of bacterial suspension) was injected into stem axils with a 22-gauge hypodermic needle. Three 6-week-old control plants were inoculated with sterile water. Plants were kept at 24°C with supplemental 400W high-pressure sodium light. Within 5 days, all three inoculated plants exhibited wilt symptoms. No symptoms were observed in control plants. Bacteria were reisolated from symptomatic plants on KB medium as described above, and gram negative, nonfluorescent, oxidase-positive bacteria were obtained. Reisolated strains were identical to R. solanacearum using BIOLOG carbohydrate utilization testing, confirming the causal agent of the disease. Personal correspondence with other diagnosticians also confirms the presence of R. solanacearum biovar 3 in Mandevilla in Ohio, Michigan, and Minnesota. To our knowledge, this is the first documented report in the world of R. solanacearum biovar 3 on Mandevilla. References: (1) M. Fegan et al. Page 34 in: Bacterial Wilt Disease Molecular and Ecological Aspects. P. Prior et al., eds. INRA Editions-Springer. Verlag, Germany, 1998. (2) E. R. French et al. Fitopatologia 30:126, 1995. (3) USDA/APHIS/PPQ New Pest Response Guidelines. Ralstonia Solanacearum race3 biovar 2, from http://www.aphis.usda.gov/import_export/plants/manuals/emergency/ downloads/nprg-ralstonia.pdf , retrieved May 2008. (4) S. A. Weller et al. Appl Environ. Microbiol. 7:2853, 2000.

Goss's Bacterial Blight and Wilt of Corn Caused by Clavibacter michiganensis subsp. nebraskensis Occurs in Minnesota.

Goss's bacterial wilt and blight caused by Clavibacter michiganensis subsp. nebraskensis (Vidaver and Mandel) Davis et al. can be an economically significant disease of corn (Zea mays L.) (1). Corn hybrids with typical leaf and wilt symptoms of Goss's bacterial blight were observed in two western Minnesota fields in Chippewa and Stephens counties in August 2009. Disease incidence was estimated at 40% in one field and 90% in the other. Symptoms consisted of large, tan-to-gray, linear lesions with irregular margins parallel to the veins, with up to 50% of the leaf area symptomatic. Irregular, dark green-to-black, water-soaked spots occurred in the lesions and dried bacterial exudate was present on the lesions. Bacterial streaming from the cut edge of lesions was visible with light microscopy. Fungal structures were not observed in the lesions. Bacteria were isolated from infected leaves collected in both fields. Sections were cut from the margins of the lesions and placed in 0.02 mM phosphate buffer (PB). Bacterial suspensions were spread onto yeast glucose medium (YGM) (3) and incubated for 5 days at 22°C. All colonies were orange and similar in appearance to C. michiganensis subsp. nebraskensis reference strain CIC016 (= CN313.0). Single colonies were subcultured onto YGM and CNS media. Two gram-positive strains, CIC251 and CIC252, were orange, circular, and convex on CNS medium and used to demonstrate Koch's postulates on corn (2). Bacterial suspensions containing 2 × 108 CFU/ml were prepared in PB from 5-day-old cultures grown on YGM. For each of strains CIC251 and CIC252, six plants of the hybrid DKC51-45 were inoculated at the V3 growth stage by swabbing inoculum over the second and third youngest leaves with Carborundum. Three control plants were treated similarly with sterile PB. Plants were incubated in a greenhouse at 20 to 24°C. Linear, water-soaked lesions typical of Goss's wilt began to develop on all inoculated leaves 7 days after inoculation. No symptoms developed on control plants. Two leaf samples with lesions were collected per plant and bacteria isolated as described above. Colonies with characteristics of C. michiganensis subsp. nebraskensis were isolated from all lesions. Presumptive identification of strains CIC251 and CIC252 as C. michiganensis subsp. nebraskensis, as well as colonies isolated from inoculated plants, was validated by rDNA sequencing. Genomic DNA was extracted from 3-day-old colonies on YGM and the 16S region was amplified (~1,480 bp) by PCR assay using primers F27 and r1492 (4). Forward and reverse sequences were aligned and base calls confirmed using Sequencher 4.9. Consensus sequences for each strain were compared with the nucleotide database with BLAST to confirm a 99% match to C. michiganensis subsp. nebraskensis (NCBI GenBank AM410697.1 and U09763.1). This confirms, for the first time (to our knowledge), that Goss's bacterial leaf blight and wilt of corn occurs in Minnesota and could be a production and phytosanitary concern in that state. References: (1) M. Davis et al. Int. J. Syst. Bacteriol. 34:107, 1984. (2) M. Davis and A. Vidaver. Page 221 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (3) S. DeBoer and R. Copeman, Am. Potato J. 57:457, 1980. (4) S. Giovannoni. Page 177 in: Nucleic Acid Techniques in Bacterial Systematics. E. Stackebrandt and M. Goodfellow, eds. John Wiley & Sons, Chichester, New York, 1991.

First Report of Tobacco etch virus Infection in Coleus in the United States.

Virus-like disease symptoms consisting of foliar and veinal necrosis similar to those caused by Coleus vein necrosis virus (CVNV) (2) were observed in plants of coleus (Coleus blume Benth.) 'Rustic Orange' obtained from retail greenhouse outlets in Missouri and Minnesota. Flexuous, filamentous, 750 to 770 nm virus-like particles (vlps) were observed by transmission electron microscopy in negatively stained partially purified leaf tissue extracts from symptomatic 'Rustic Orange' leaf tissue. No other virus-like particles were observed and none were detected in extracts from asymptomatic leaves. These vlps were longer than those of CVNV (640 nm) (2) and were not detected by immunosorbent electron microscopy (ISEM) using antibodies to CVNV (2). Degenerate potyvirus primers PNIbF1 (5'GGBAAYAATAGTGGNCAACC3') and PCPR1 (5'GGGGAGGTGCCGTTCTCDATRCACCA3') (1) and total RNA extracted from 'Rustic Orange' leaf tissue with a Qiagen RNeasy Kit were used for reverse transcription-PCR with Ready-To-Go RT-PCR Beads (GE Healthcare). A 950-bp amplicon was obtained from total RNA from diseased but not from healthy leaf tissue. The nucleotide sequence of the amplicon (GenBank Accession No. GQ268818) had levels of identity to published Tobacco etch virus (TEV) sequences comprising portions of the nuclear inclusion body (NIb) and coat protein (CP) gene regions ranging from 89% (L38714) to 93% (M15239, M11458). The identity of the virus occurring in 'Rustic Orange' was further confirmed by ISEM. Virions were trapped and decorated by antibodies to TEV (ATCC PVAS 32). Systemically infected leaf tissue from Datura stramonium in which the coleus TEV isolate was propagated was used to mechanically inoculate Carborundum-dusted leaves of virus-free test plants of 'Rustic Orange' (Park Seed, Greenwood, SC). Inoculated plants developed foliar necrosis symptoms similar to those observed originally, and the presence of TEV was confirmed by ISEM and RT-PCR and nucleotide sequence analysis as described above. To our knowledge, this is the first report of a disease of coleus caused by TEV. Many of approximately 30 'Rustic Orange' plants in one nursery in Minnesota showed similar necrotic foliar symptoms and randomly selected plants tested positive for TEV by ISEM. This suggests that TEV infection in this variety may be spread by vegetative propagation from infected stock plants. References: (1) Y.-C. Hsu et al. J. Virol. Methods 128:54. 2005. (2) D. S. Mollov et al. Plant Dis. 91:754. 2007.