Evaluation of North American isolates of Soybean mosaic virus for gain of virulence on Rsv-genotype soybeans with special emphasis on resistance-breaking determinants on Rsv4.

Resistance to Soybean mosaic virus (SMV) in soybean is conferred by three dominant genes: Rsv1, Rsv3 and Rsv4. Over the years, scientists in the USA have utilized a set of standard pathotypes, SMV-G1 to SMV-G7, to study interaction with Rsv-genotype soybeans. However, these pathotypes were isolated from a collection of imported soybean germplasm over 30 years ago. In this study, 35 SMV field isolates collected in recent years from 11 states were evaluated for gain of virulence on soybean genotypes containing individual Rsv genes. All isolates were avirulent on L78-379 (Rsv1), whereas 19 were virulent on L29 (Rsv3). On PI88788 (Rsv4), 14 of 15 isolates tested were virulent; however, only one was capable of systemically infecting all of the inoculated V94-5152 (Rsv4). Nevertheless, virulent variants from 11 other field isolates were rapidly selected on initial inoculation onto V94-5152 (Rsv4). The P3 cistrons of the original isolates and their variants on Rsv4-genotype soybeans were sequenced. Analysis showed that virulence on PI88788 (Rsv4) was not associated, in general, with selection of any new amino acid, whereas Q1033K and G1054R substitutions were consistently selected on V94-5152 (Rsv4). The role of Q1033K and G1054R substitutions, individually or in combination, in virulence on V94-5152 (Rsv4) was confirmed on reconstruction in the P3 cistron of avirulent SMV-N, followed by biolistic inoculation. Collectively, our data demonstrate that SMV has evolved virulence towards Rsv3 and Rsv4, but not Rsv1, in the USA. Furthermore, they confirm that SMV virulence determinants on V94-5152 (Rsv4) reside on P3.

First Report of Leaf Spot and Necrotic Roots on Switchgrass Caused by Curvularia lunata var. aeria in the United States.

Curvularia lunata infects many grass species; however, switchgrass (Panicum virgatum L.) has not been reported as a host (2). In June 2009, small brown leaf spots and necrotic roots were observed on stunted 2-year-old 'Alamo' switchgrass on the University of Tennessee, Knoxville campus. Symptomatic leaf and root tissues were surface-sterilized in 95% ethanol for 1 min, 20% bleach for 3 min, and 95% ethanol for 1 min, and then air dried and placed on water agar amended with 10 mg/liter rifampicin (Sigma-Aldrich, St. Louis, MO) and 7.5 µl/liter Danitol (Valent Chemical, Walnut Creek, CA). Cultures were incubated at 25°C for 3 days. Hyphal tips were transferred to potato dextrose agar (PDA) and incubated at 25°C. Dark brown-to-black fungal colonies with black stromata formed. Conidiophores were dark brown, unbranched, septate, polytretic, sympodial, and geniculate at the apical region with rachis conidial ontogeny. Conidia were dark brown and cymbiform with three to four septations, with one or two central cells larger than the terminal cells. Spore size ranged from 17.5 to 30.0 × 8.8 to 12.5 µm (mean 21.6 × 10.8 µm). Morphological traits matched the description of C. lunata var. aeria (1). To test pathogenicity, fungal sporulation was optimized on PDA with pieces of sterile, moistened index card placed on each plate; cultures were incubated at 25°C with a 12-h photoperiod (3). After 14 days, conidia were dislodged in sterile water and the spore concentration adjusted to 8 × 104 conidia/ml. Ten pots, with about 15 plants per pot, of 6-week-old 'Alamo' switchgrass grown from surface-sterilized seed were inoculated with the spore suspension applied to the plant crown and surrounding soil with an aerosol sprayer. Prior to inoculation, roots were wounded with a sterile scalpel. Noninoculated plants (two pots), with roots also wounded, served as controls. To maintain high humidity, each pot was covered with a plastic bag and maintained in a growth chamber at 30°C with a 16-h photoperiod. Bags were removed after 3 days; plants were maintained as described for 6 weeks. Brown leaf spots and brown-to-black necrotic roots that matched symptoms on the naturally infected plants were observed in all inoculated plants; there were no symptoms of Curvularia infection on the controls. The fungus was reisolated from inoculated plants as described above. Genomic DNA was extracted from the original isolate and the reisolate from the pathogenicity test. PCR amplification of the internal transcribed spacer (ITS) regions from ribosomal DNA was performed with primers ITS4 and ITS5. PCR products of 503 bp were sequenced. There was 100% nucleotide identity for sequences of the original isolate and the re-isolate. The sequence was submitted to GenBank (Accession No. HQ130484.1). BLAST analysis of the fungal sequence resulted in 100% nucleotide sequence identity to the ITS sequences of isolates of C. affinis, C. geniculata, and C. lunata. On the basis of the smaller spore size and abundant stromata on PDA, the isolate was identified as C. lunata var. aeria. As switchgrass is developed as a biofuels crop, identification of new pathogens may warrant development of disease management strategies. References: (1) M. B. Ellis. Mycological Papers No. 106, CMI, Surrey, 1966. (2) D. F. Farr and A. Y. Rossman, Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , August 2011. (3) R. G. Pratt. Mycopathologia 162:133, 2006.

Occurrence of Alfalfa mosaic virus in Soybean in Tennessee.

Alfalfa mosaic virus (AMV), a member of the genus Alfamovirus in the family Bromoviridae, naturally infects a wide range of plant species (1). Soybean (Glycine max (L.) Merr.) has seldom been reported as a natural host of AMV and there are limited reports of detection of AMV in field-grown soybean plants (4). However, AMV incidence in soybean fields in the midwestern United States has been on the rise in recent years, which is partly attributed to the introduction of the soybean aphid (Aphis glycines) (1,4). In June 2009, soybean plants of cv. Lee68 exhibiting moderate leaf distortion, mottling, and stunting were observed at the East Tennessee Research and Education Center. Leaf samples from 18 symptomatic plants were collected and the sap was extracted and analyzed by antigen-coated indirect ELISA (3) with polyclonal antibodies against AMV, Soybean mosaic virus (SMV), and Bean pod mottle virus (BPMV). None of the samples tested positive for BPMV, but all were found to be infected with SMV. Sap extract from 1 of 18 plants tested positive for AMV and SMV. Sap from this infected plant ground in 10 mM phosphate buffer, pH 7.0, was mechanically inoculated to Carborundum-dusted unifoliate leaves of PI96983, which contains the dominant Rsv1-locus conferring functional immunity to a majority of SMV strains (2). AMV, not SMV, was detected by ELISA in the systemically infected trifoliolate leaves that exhibited moderate mottling, mild leaf distortion, and stunting 14 days postinoculation. Sap was extracted from the infected tissues and the virus was passaged four times through PI96983 before being inoculated to Phaseolus vulgaris cv. Blue Lake. A local lesion isolate was obtained following three successive passages in this host and the isolate was propagated in soybean cv. Williams82. The biologically purified isolate was capable of infecting soybean cvs. L78-379 (Rsv1), L81-4420 (Rsv1), L29 (Rsv3), V94-5152 (Rsv4), Lee68, and Colfax upon sap inoculation. The infected plants exhibited a range of systemic symptoms including mottling, leaf distortion, necrosis, chlorosis, and moderate stunting. To characterize the virus further, total RNA was extracted from infected Williams82 leaf tissues with the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). The RNA served as a template for cDNA synthesis in the presence of random primers. The resultant cDNA served as a template in a PCR assay with primers 1193 (forward) (5'-AGCTGAATTCATGAGTTCTTCACAAC-3') and 1858 (reverse) (5'-GCTAGCGGCCGCTCAATGACGATC-3') corresponding to nucleotides 1,193 to 1,210 and 1,858 to 1,840 of RNA3 from AMV-Kr (GenBank Accession No. AB126032), respectively. The amplified fragments were purified and directly sequenced bidirectionally using the same primers. BLAST analysis of the resultant nucleotide sequences showed 98% identity to an AMV isolate from a naturally infected soybean plant in Illinois (GenBank Accession No. HQ185569), and 97% identity to an isolate described from P. vulgaris in the United States (GenBank Accession No. AY340070.1). To our knowledge, this is the first report of natural infection of soybean by AMV in Tennessee. References: (1) J. Bol. Mol. Plant Pathol. 4:1, 2003. (2) M. R. Hajimorad and J. H. Hill. Mol. Plant-Microbe Interact. 14:587, 2001. (3) M. Malapi-Nelson et al. Plant Dis. 93:1259, 2009. (4) E. E. Mueller and C. R. Grau. Plant Dis. 91:266, 2007.

Genetic variability and phylogenetic analysis of hosta virus X.

Triple gene block 1 (TGB1) and coat protein (CP) sequences of 30 hosta virus X (HVX) isolates from Tennessee (TN), USA, were determined and compared with available sequences in GenBank. The CPs of all known HVX isolates, including those from TN, shared 98.3-100% and 98.2-100% nucleotide and amino acid sequence identity, respectively, whereas TGB1 shared 97.4-100% nucleotide and 97-100% amino acid sequence identity. TGB1 of TN isolates were all longer by one codon from that of a Korean isolate, which is the only sequence publicly available. Phylogenetic analysis of nucleotide and amino acid sequences of TGB1 and CP of all known HVX isolates, separately or combined, revealed a close relationship, suggesting that all of them are derived from a common ancestor. Phylogenetic analysis with the type member of each genus of the family Flexiviridae confirmed that HVX is a member of a distinct species of the genus Potexvirus.