A Method for Combining Isolates of Phytophthora sojae to Screen for Novel Sources of Resistance to Phytophthora Stem and Root Rot in Soybean.

Soybean cultivars with specific single resistance genes (Rps) are grown to reduce yield loss due to Phytophthora stem and root rot caused by the oomycete pathogen Phytophthora sojae. To identify novel Rps loci, soybean lines are often screened several times, each time with an isolate of P. sojae that differs in virulence on various Rps genes. The goal of this study was to determine whether several isolates of P. sojae that differ in virulence on Rps genes could be combined into a single source of inoculum and used to screen soybean lines for novel Rps genes. A set of 14 soybean differential lines, each carrying a specific Rps gene, was inoculated with three isolates of P. sojae, which differed in virulence on 6 to 10 Rps genes, individually or in a 1:1:1 mixture. Inoculum containing the 1:1:1 mixture of isolates was virulent on 13 Rps genes. The mixed-inoculum method was used to screen 1,019 soybean accessions in a blind assay for novel sources of resistance. In all, 17% of Glycine max accessions and 11% of G. soja accessions were resistant (≤30% dead plants), suggesting that these accessions may carry a novel Rps gene or genes. Advantages of combining isolates into a single source of inoculum include reduced cost, ability to screen soybean germplasm with inoculum virulent on all known Rps genes, and ease of identifying novel sources of resistance. This study is a precursor to identifying novel sources of resistance to P. sojae in soybean using RXLR effectors.

Temperature Affects Aggressiveness and Fungicide Sensitivity of Four Pythium spp. that Cause Soybean and Corn Damping Off in Iowa.

Damping off of soybean and corn, caused by Pythium spp., is favored by cool temperatures and wet soil conditions and is primarily managed using fungicide seed treatments. The goal of this research was to determine the effect of temperature on aggressiveness and fungicide sensitivity of Pythium spp. recovered from soybean and corn in Iowa. A total of 21 isolates of four of the most prevalent Pythium spp. in Iowa were screened. Seed and seedling assays were used to quantify the aggressiveness of P. lutarium, P. oopapillum, P. sylvaticum, and P. torulosum on soybean and corn at 13, 18, and 23°C. Isolates recovered from soybean or corn were equally pathogenic on both hosts. P. torulosum was more aggressive at 13°C compared with 18 and 23°C. Conversely, P. sylvaticum was more aggressive at 18 and 23°C than at 13°C. A plate assay was used to assess fungicide sensitivity to seven fungicides that are commonly used as seed treatments, and EC50 values at each of the three temperatures were determined and compared. EC50 values for P. torulosum were higher for all fungicides tested at 13°C, compared with 18 or 23°C, whereas EC50 values for P. sylvaticum were higher for all fungicides at 18 and 23°C compared with 13°C. These data contribute to our understanding of the effect of soil temperature on the risk of soybean and corn damping off, which may aid in the development of more effective management practices.

First Report of Pythium schmitthenneri Causing Maize Seedling Blight in Iowa.

In spring 2012, maize farmers in southeast and south central Iowa reported stand losses due to pre- and post-emergence damping-off, and many of the fields had to be replanted. Symptoms of the disease included rotted seed, or brown, rotted, water-soaked mesocotyls and root tips. Maize seedlings with severe root and mesocotyl symptoms were yellow and wilted, stunted, or dead. The disease occurred approximately 2 weeks after cool, wet conditions. Symptomatic mesocotyls and roots were washed for 30 min, rinsed with sterile distilled water, and blotted dry on sterile paper towels. Isolation of the pathogen was performed by aseptically cutting 2- to 3-mm sections of tissue from the edge of a lesion, placing the segments under corn meal agar (CMA) containing pimaricin, ampicillin, rifampicin, and pentachloronitrobenzene (PARP), and incubating at 22°C in the dark. Colonies that developed were putatively identified as Pythium species based on morphological characteristics and cultural features when compared to published descriptions (2,3). Characteristics of isolate IAC12F21-3 included spherical and smooth-walled oogonia 18 to 26 µm in diameter, monoclinous or usually diclinous antheridia 10 to 22 µm long and 5 to 10 µm wide with one or occasionally two per oogonium, and plerotic oospores 15 to 25 µm in diameter. Sporangia were globose to ellipsoidal, 22 to 41 µm in diameter, and zoospores were 7 to 10 µm long. Primers ITS1 and ITS4 were used to amplify the ITS region within clade E1 of 88 isolates. The resultant amplicons were sequenced and a BLAST search in GenBank confirmed isolate IAC12F21-3 as Pythium schmitthenneri based on 100% similarity with GenBank accession numbers JF836869 and JF836870. Pathogenicity testing was conducted using seed and seedling assays (1,4). Koch's postulates was performed by sampling pieces of symptomatic mesocotyl and root tissue from the inoculated pots, placing segments under CMA + PARP, and incubating at 22°C. Symptoms were similar to those observed in the field and P. schmitthenneri was re-isolated successfully. Non-inoculated control plants showed no symptoms. This is the first report of P. schmitthenneri causing seedling blight on maize in Iowa. Previously, P. schmitthenneri was reported as a pathogen on maize in Ohio (2). References: (1) K. Broders et al. Plant Dis. 91:727, 2007. (2) M. Ellis et al. Mycologia, 104:477, 2012. (3) J. Middleton. Memoirs of the Torrey Botanical Club 20:171, 1943. (4) A. Rojas et al. Phytopathology, 102(Suppl):S5.8, 2012.

Differential expression of alpha-2,3-sialyltransferases and alpha-1,3/4-fucosyltransferases regulates the levels of sialyl Lewis a and sialyl Lewis x in gastrointestinal carcinoma cells.

Sialyl Lewis x and sialyl Lewis a expression depends on sialyltransferases and fucosyltransferases. In this study, we screened for major variations of sialyltransferases and fucosyltransferases involved in the synthesis and regulation of sialyl Lewis x and sialyl Lewis a epitopes in gastrointestinal carcinoma cells. Our results show that expression of ST3Gal IV in several gastrointestinal cell lines is correlated with the expression of sialyl Lewis x at the cell surface. ST3Gal IV overexpressed in the gastric MKN45 cell line, showed exclusive enzymatic activity towards glycoproteins containing terminal Galbeta1-4GlcNAc structure. On the other hand, when ST3Gal III was overexpressed in MKN45, an increase in the expression levels of both sialyl Lewis epitopes was observed. ST3Gal III and ST3Gal IV lead to de novo synthesis of sialyl Lewis x determinant on different molecular weight glycoproteins of MKN45 cells suggesting that each enzyme used different substrates within the available glycoproteome. The final glycosylation step in sialyl Lewis x and sialyl Lewis a biosynthesis in MKN45 cell line was shown to be associated to FUT5, which efficiently fucosylated sialyl Lewis precursors on glycoproteins. Moreover we demonstrate that the expression of sialyl Lewis epitopes in the MKN45 was induced by cell confluence, which can be regarded as a model to study altered glycosylation during tumour progression. This increase was observed together with an increase in mRNA levels of ST3GAL3, FUT5 and FUT6, and a decrease in FUT4 transcript levels in MKN45 confluent cells, suggesting a possible control at the transcriptional level.

Interpreting peptide mass spectra by VEMS.

Most existing Mass Spectra (MS) analysis programs are automatic and provide limited opportunity for editing during the interpretation. Furthermore, they rely entirely on publicly available databases for interpretation. VEMS (Virtual Expert Mass Spectrometrist) is a program for interactive analysis of peptide MS/MS spectra imported in text file format. Peaks are annotated, the monoisotopic peaks retained, and the b-and y-ion series identified in an interactive manner. The called peptide sequence is searched against a local protein database for sequence identity and peptide mass. The report compares the calculated and the experimental mass spectrum of the called peptide. The program package includes four accessory programs. VEMStrans creates protein databases in FASTA format from EST or cDNA sequence files. VEMSdata creates a virtual peptide database from FASTA files. VEMSdist displays the distribution of masses up to 5000 Da. VEMSmaldi searches singly charged peptide masses against the local database.