First Report of Potato spindle tuber viroid Naturally Infecting Greenhouse Tomatoes in North Carolina.

In spring 2012, a severe disease was observed on a limited number of tomato plants (Solanum lycopersicum L.) in a research greenhouse facility in western North Carolina. The first symptoms noted were downward curling of the terminal leaves accompanied by a rough puckered darker green texture. This was followed in time by greater distortion of the leaves with pale green on leaf margins. Older leaves with symptoms developed necrosis, with necrotic spots and streaks appearing on a few fruits. On some of these affected fruits, stems, peduncles, pedicels, and sepals also showed symptoms. Infected plants were badly stunted, and fruits in the upper parts of plants displaying severe symptoms remained very small. In just a few months, the disease spread to other tomato plants inside the greenhouse. A survey in May 2012 showed a disease incidence of 18% (156 symptomatic plants out of a total of 864) in this greenhouse. Initial screenings for possible viruses using ELISA (Agdia, Elkhart, IN), as well as a reverse transcription (RT)-PCR panel of 15 common tomato viruses in our laboratory were negative. Because of the symptoms and negative results for viruses, a viroid infection was suspected. Total plant RNA was prepared using TRIzol reagent (Invitrogen, Carlsbad, CA) from leaf tissues of eight diseased plants and one seed sample. Using real-time RT-PCR developed against Potato spindle tuber viroid (PSTVd) and some related pospiviroids (1), positive signals were observed with a mean Ct = 13.24 for leaf tissues and Ct = 19.91 for the seed sample. To obtain a full viroid genome, RT-PCR using two different sets of primers, one specific for PSTVd (PSTVd-F and PSTVd-R) (2), and a universal primer set for pospiviroids (MTTVd-F and MTTVd-R) (3) was performed. RT-PCR generated amplicons with expected size of ~360 bp from all eight leaf and one seed samples, but not from a healthy control. PCR products were cloned using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA). A total of 22 full genomic sequences were obtained. A multi-sequence alignment generated a consensus sequence of 360 nt, designated as NC12-01 (GenBank Accession No. JX280944). BLASTn search in the NCBI database revealed the highest sequence identity of 96.9% to Australian (AY962324) and UK (AJ583449) isolates of PSTVd and 95.9% identity to the tomato isolate of PSTVd-CA1 (HM753555). Similar disease symptoms were observed on two 'Rutgers' tomato plants 2 weeks post mechanical inoculation and the presence of PSTVd was confirmed by real-time RT-PCR (1). A mock-inoculated plant did not show any symptoms. In the U.S., natural infection of PSTVd on tomato was first identified in California in 2010 (3). To our knowledge, this is the first report of a natural occurrence of PSTVd on tomato in the eastern U.S. The diseased plants were contained, properly disposed of, and eradicated in this location. The broader geographic distribution of PSTVd on tomato in the U.S., and the potential latent infection in potato and a number of ornamentals (4), emphasizes the need for better plant and seed health tests for viroids on these plants. References: (1) N. Boonham et al. J. Virol. Methods 116:139, 2004. (2) H. Bostan et al. J. Virol. Methods 116:189, 2004. (3) K.-S. Ling and D. Sfetcu. Plant Dis. 94:1376, 2010. (4) R. A. Owens and J. Th. J. Verhoeven. The Plant Health Instructor. DOI: 10.1094/PHI-I-2009-0804-01, 2009.

Gene expression analysis in soybean in response to the causal agent of Asian soybean rust (Phakopsora pachyrhizi Sydow) in an early growth stage.

Asian soybean rust (ASR) caused by Phakopsora pachyrhizi Sydow is a potentially devastating disease posing a serious threat to the soybean industry. Understanding plant host response at the molecular level is certainly important for control of the disease. The main objective of this study was to perform a transcriptome profiling of P. pachyrhizi-exposed young soybean plants (V2 growth stage) using whole genome Affymetrix microarrays of soybean. Three-week-old soybean cv. 5601 T plants at the V2 growth stage were inoculated with P. pachyrhizi, and leaf samples were collected 72 h post inoculation with subsequent microarray analysis performed. A total of 112 genes were found to be differentially expressed from P. pachyrhizi exposure, of which 46 were upregulated, and 66 were downregulated. Most of the differentially expressed genes were general defense and stress-related genes, and 34 of these were unknown. Confirmational real-time reverse transcription-polymerase chain reaction was performed on a subset of 5 out of 112 differentially expressed genes. These results were congruent with the microarray analysis. Our results indicated that low and nonspecific innate response to the pathogen may account for the failure to develop rust resistance in the soybean variety studied. To our knowledge, this is the first microarray analysis of soybean in response to ASR.

Quantitative trait loci controlling sulfur containing amino acids, methionine and cysteine, in soybean seeds.

Soybean [Glycine max (L.) Merr.] is the single largest source of protein in animal feed. However, a major limitation of soy proteins is their deficiency in sulfur-containing amino acids, methionine (Met) and cysteine (Cys). The objective of this study was to identify quantitative trait loci (QTL) associated with Met and Cys concentration in soybean seed. To achieve this objective, 101 F(6)-derived recombinant inbred lines (RIL) from a population developed from a cross of N87-984-16 x TN93-99 were used. Ground soybean seed samples were analyzed for Met and Cys concentration using a near infrared spectroscopy instrument. Data were analyzed using SAS software and QTL Cartographer. RIL differed (P<0.01) in Met and Cys concentrations, with a range of 5.1-7.3 (g kg(-1) seed dry weight) for Cys and 4.4-8.8 (g kg(-1) seed dry weight) for Met. Heritability estimates on an entry mean basis were 0.14 and 0.57 for Cys and Met, respectively. A total of 94 polymorphic simple sequence repeat molecular genetic markers were screened in the RIL. Single factor ANOVA was used to identify candidate QTL, which were confirmed by composite interval mapping using QTL Cartographer. Four QTL linked to molecular markers Satt235, Satt252, Satt427 and Satt436 distributed on three molecular linkage groups (MLG) D1a, F and G were associated with Cys and three QTL linked to molecular markers Satt252, Satt564 and Satt590 distributed on MLG F, G and M were associated with Met concentration in soybean seed. QTL associated with Met and Cys in soybean seed will provide important information to breeders targeting improvements in the nutritional quality of soybean.