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Ergot, caused by Claviceps purpurea, is a primary disease concern in irrigated cool-season grass seed production systems of Oregon. In order to better understand the genetic diversity, population structure, and the epidemiology of C. purpurea in grasses grown for seed, 226 isolates were obtained using a hierarchical sampling strategy from two fields each of Kentucky bluegrass (n = 102) and perennial ryegrass (n = 124) and characterized using 12 microsatellite markers. A total of 194 unique multilocus genotypes (MLGs) were identified in this study. There were moderate levels of genotypic diversity (H = 3.43 to 4.23) and gene diversity (Hexp = 0.45 to 0.57) within fields. After clone correction, analysis of molecular variance revealed that 66% of the genetic variation occurred between the two C. purpurea isolates collected from the same seed head of individual plants, indicating that many of the seed heads bearing multiple sclerotia were infected by ascospores rather than conidia. However, the majority of the clonal isolates obtained in this study were collected from the same seed head (i.e., the two isolates were identical MLGs), indicating a role of conidia (honeydew) in secondary infections within seed heads. Genetic differentiation was observed between populations from different hosts (22%) but was confounded by geography. The standardized index of association ranged from 0.007 to 0.122 among the four populations, suggesting potential outcrossing and differences in the relative contribution of ascospores and conidia to ergot among the fields. The results from this study provide insights into the epidemiology of ergot in cool-season grass seed crops of Oregon.

Eleven switchgrass cultivars (eight ornamental and three agronomic) were inoculated with 40 switchgrass rust isolates collected from the southeastern U.S. to study host resistance, rust virulence and host/pathogen interactions by measuring urediniospore germination percentage, latent period, and the number of uredia and urediniospores produced per cm2 of leaf surface. In general, ornamental switchgrass cultivars had reduced number of uredia and urediniospores produced per cm2 than did agronomic cultivars. Rust isolates were variable for virulence in culture (on grass blades in petri dishes); however they could not be segregated into groups based on collection locations or years. The results of this study will provide information concerning durable horizontal resistance in switchgrass for the ornamental industry. Index words:, Switchgrass, leaf rust, resistance. Species used in the study: switchgrass (Panicum virgatum L., rust (Puccinia emaculata).

Plants with crassulacean acid metabolism (CAM) generally utilize water 20%–80% more efficiently than non-CAM plants. The whole genomes of several CAM plants have been sequenced or are being sequenced. For effective genome characterization and genome editing of CAM plants, an efficient transformation system is essential. In this study, we developed an Agrobacterium-mediated transformation protocol for Kalanchoe laxiflora, an obligate CAM plant,by optimizing several factors affecting the transformation efficiency. Agrobacterium strains AGL1, C58, EHA105, and GV3101 were all suitable for K. laxiflora transformation. Fifty-nine percent of the leaf explants yielded kanamycin-resistant and GUS-positive shoots. Polymerase chain reaction and quantitative real-time PCR (qRT-PCR) using gusA-, gusPlus-, nptII- and hpt-specific primers confirmed that the transgenes were integrated into K. laxiflora genome and expressed. This efficient transformation system will allow effective functional characterization of genes through over- or down-expression, knockout, or genome editing. Keywords: Kalanchoe laxiflora, Agrobacterium-mediated transformation, CAM plants

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Protein kinases (PKs) have evolved as the largest family of molecular switches that regulate protein activities associated with almost all essential cellular functions. Only a fraction of plant PKs, however, have been functionally characterized even in model plant species. In the present study, the entire grapevine kinome was identified and annotated using the most recent version of the grapevine genome. A total of 1168 PK-encoding genes were identified and classified into 20 groups and 121 families, with the RLK-Pelle group being the largest, with 872 members. The 1168 kinase genes were unevenly distributed over all 19 chromosomes, and both tandem and segmental duplications contributed to the expansion of the grapevine kinome, especially of the RLK-Pelle group. Ka/Ks values indicated that most of the tandem and segmental duplication events were under purifying selection. The grapevine kinome families exhibited different expression patterns during plant development and in response to various stress treatments, with many being coexpressed. The comprehensive annotation of grapevine kinase genes, their patterns of expression and coexpression, and the related information facilitate a more complete understanding of the roles of various grapevine kinases in growth and development, responses to abiotic stress, and evolutionary history., Genetics: Mapping molecular switches in grape A comprehensive map of all the protein kinases (PKs) in grape is now available. PKs switch other proteins on or off by adding or removing a phosphate group. They regulate many cellular processes, coordinate plant growth, and trigger responses to various stresses. However, the number, relationships, and functions of PKs remain uncharacterized in most plants. Zong-Ming Cheng at Nanjing Agricultural University and the University of Tennessee mapped all the PKs in grape. Using the most up-to-date version of the genome, Cheng and coworkers identified over 1000 PKs, traced their evolutionary relationships, and identified which PKs are active in key growth phases. They also identified many PKs involved in response to stresses such as heat and drought. These results may help to improve stress tolerance in one of the world’s most economically important fruits.

In the past decade, black walnut (Juglans nigra) trees throughout western North America have suffered from widespread branch dieback and canopy loss, causing substantial tree mortality (2,3). The fungus, Geosmithia morbida, vectored by the walnut twig beetle (WTB), Pityophthorus juglandis, has been associated with this devastating disease known as Thousand Cankers Disease (TCD) (2,3). In August of 2012, branch samples from TCD symptomatic black walnut trees (5 to 10 cm in diameter and 15 to 30 cm long) were collected on the North Carolina side of the Great Smoky Mountain National Park (GRSM) in Cataloochee Cove (35°37.023′ N, 83°07.351′ W) and near the Big Creek Campground (35°45.290′ N, 83°06.473′ W), in Haywood County. Five symptomatic trees near the Big Creek Campground and three from Cataloochee Cove displayed typical TCD signs including progressive crown thinning, branch flagging, and branch dieback; however, insect holes were not observed. Samples were double bagged in Ziploc plastic bags, sealed in a 19-liter plastic bucket, and transported to the University of Tennessee. Outer bark was removed from the samples and small, elliptical, necrotic cankers were observed. Wood chips (3 to 4 mm2) from cankers were excised and placed on 1/10 strength potato dextrose agar amended with 30 mg/liter streptomycin sulfate and 30 mg/liter chlortetracycline HCL and incubated on a 12-h dark/light cycle at 22°C for 5 to 7 days. Fungal isolates were tentatively identified as G. morbida by using culture morphology, and characteristics of conidiophores and conidia (2). The isolated fungus from the Cataloochee Cove location was grown in 1/10 strength potato dextrose broth at room temperature for 2 weeks. Isolates from Big Creek Campground were contaminated and were not analyzed further. Fungal colonies were tan to light yellow. Conidia were tan, subcylindrical, and catenulate. Conidiophores were multibranched, verticillate, and verrucose. To verify the morphological data, DNA was extracted from fungal mycelia using DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's published protocol. Isolates from Cataloochee Cove were characterized using ITS1 and ITS4 universal primers (4). The putative G. morbida isolate (GenBank Accession No. KC461929) had ITS sequences that were 100% identical to the G. morbida type isolate CBS124663 (FN434082.1) (2). Additionally, fungal DNA from Cataloochee Cove was amplified using G. morbida-specific microsatellite loci (GS04, GS27, and GS36) (1). PCR products were analyzed with the QIAxcel Capillary Electrophoresis System (Qiagen) and were similar to those previously published (2). To date, all confirmed cases of TCD in the native range of black walnut have been in urban areas, along rural roadsides and/or fence rows. The report in North Carolina is the first finding of G. morbida, the causal agent of TCD, in a forest setting. References: (1) D. Hadziabdic et al. Conserv. Genet. Resources 4:287, 2012. (2) M. Kolarik et al. Mycologia 103:325, 2011. (3) N. Tisserat et al. Plant Health Progr. doi:10.1094/PHP-2011-0630-01-BR, 2011. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

The U.S. Pacific Northwest states of Oregon and Washington are major producers of cool-season grass seed. Ergot, caused by fungi in the Claviceps purpurea sensu lato group, is an important seed replacement disease of grass worldwide. Microscopic methods that are currently used to quantify airborne Claviceps ascospores captured by spore traps are not currently rapid enough to allow for detecting and reporting of spore numbers in a timely manner, hindering growers from using this information to help manage ergot. We developed a SYBR Green real-time quantitative polymerase chain reaction (qPCR)-based assay for fast and efficient detection and quantification of C. purpurea sensu lato ascospores from Hirst-type spore traps. Species-specificity of the qPCR assay was confirmed against 41 C. purpurea sensu lato isolates collected from six hosts and six other Claviceps spp. Significant relationships were observed between cycle threshold (Ct) values and standard curves of serial dilutions of DNA ranging from 1 pg to 10 ng (R2 = –0.99; P = 0.0002) and DNA extracted from a conidial suspension representing 8 to 80,000 conidia (R2 = –0.99; P = 0.0004). Ct values from qPCR were significantly correlated with results from microscopic examination of spore trap samples from the field (r = –0.68; P < 0.0001) and the procedure was able to detect a single ascospore from spore trap tape samples. The qPCR procedure developed in this study provided a means for quantifying airborne Claviceps ascospores that was highly specific and useful over a wide range of spore densities, and could be performed in a matter of hours instead of days. The qPCR assay developed in this study could be part of an integrated pest management approach to help grass seed growers make risk-based fungicide application decisions for ergot management in grass grown for seed.

The effect of pH at acidic pH range on the stability of hydroxypropyl cellulose (HPC) and casein complexes was studied. Results suggested the occurrence of clear and transparent appearance of HPC-casein complexes at pH 2.5 and 3. After storage for 3 weeks, the complexes maintained the excellent stability, as HPC molecules attached onto the surfaces of casein micelles through electrostatic interactions with strong positive charges resulted in an interparticle electrostatic repulsion, thereby preventing aggregation. The particle size, zeta potential analysis, turbidity, the protein concentration and viscosity confirmed the hypothesis. In addition, microscopic images revealed that the polysaccharide (HPC) particles adsorbed onto the surfaces of casein micelles, and casein-HPC complexes were uniformly dispersed in the system. Moreover, the effects of the ionic strength on the stability of the complex were investigated. It was indicated that the particle size of casein-HPC complexes at low concentration (2 mM) of NaCl remained the identical level (P > 0.05), while the particle sizes significantly were increasing at the relatively higher concentrations (50 mM and 100 mM) of NaCl (P

Figure S1. Phylogenetic classification of pineapple PKs. The phylogenetic tree was constructed with amino sequences of the kinase domain using FastTree 2.1.9 with maximum-likelihood method. Families were highlighted with different colors. (PDF 286 kb)

Figure S2. Chromosomal locations of pineapple PK genes. Figure S3. Subcellular localizations of pineapple PK genes in seven large groups predicted by CELLO. Figure S4. GO analysis of the 95 tandemly duplicated PK genes in pineapple. The size of each slice in the pie chart indicates the relative abundance of that GO term in the pineapple kinome. Figure S5. Heatmaps of the expression profiles of pineapple PK families in 14 different tissues with hierarchical clustering. The heatmaps were generated using R. Figure S6. Coexpression networks of pineapple PK families in 14 different tissues. Nodes indicated families and edges indicated significant coexpression between families. All of Pearson correlation coefficients of coexpression events were significant at the 0.01 significance level (p-value). Figure S7. Coexpression network analysis of PK genes in cluster 1 in green leaf tip and white leaf base during 24 h period. Nodes indicated genes, and edges indicated significant coexpression between genes. All of Pearson correlation coefficients of coexpression events were significant at the 0.01 significance level (p-value). Figure S8. Coexpression network analysis of PK genes which were generated by segmental (A) and tandem (B) duplication in green leaf tip and white leaf base during 24 h period. Nodes indicated genes, and edges indicated significant coexpression between genes. The absolute value of Pearson correlation coefficients > 0.9, and p

Alternaria blotch, caused by Alternaria alternata f. sp. mali, is a destructive defoliating disease that affects apple worldwide, especially in Asia. Despite previous efforts in characterizing this disease, few have reported on nucleotide binding sites (NBS)-encoding genes in response to pathogen infection. In this study, we characterized the morphological and gene expression response of ‘Golden Delicious’ and ‘Starking’ apples challenged by A. alternata. Inoculation of detached leaves of ‘Golden Delicious’ resulted in a rapid initiation of disease symptoms but a slow spread of the infection from the point of inoculation. In contrast, detached leaves of ‘Starking’ exhibited a delayed visibility of infection but a rapid spread of the infection once it was established. Based upon quantitative reverse transcription polymerase chain reaction data, we characterized the expression of 12 NBS-encoding genes in response to infection by A. alternata. We further characterized the structure, chromosomal location, and...

A genome-wide identification and cloning of CaM genes in pear was conducted and in compared withArabidopsisthat indicated a conserved expansion of CaM genes in pear, and PbCaMs and AtCaMs had a similar distribution of cis-elements and expressions in response to salt and osmotic stress. In particular, PbCaM1 and PbCaM3 were both significantly upregulated in response to salt and osmotic stress in pear.

A genome-wide identification and cloning of CaM genes in pear was conducted and in compared with Arabidopsis that indicated a conserved expansion of CaM genes in pear, and PbCaMs and AtCaMs had a similar distribution of cis-elements and expressions in response to salt and osmotic stress. In particular, PbCaM1 and PbCaM3 were both significantly upregulated in response to salt and osmotic stress in pear.

Basic leucine zipper (bZIP) genes are known to play a crucial role in response to various processes in plant as well as abiotic or biotic stress challenges. We have performed an identification and characterization of 50 bZIP genes across the woodland strawberry (Fragaria vesca) genome, which were divided into 10 clades according to the phylogenetic relationship of the strawberry bZIP proteins with those in Arabidopsis and rice. Five categories of intron patterns were observed within basic and hinge regions of the bZIP domains. Some additional conserved motifs have been found with the group specificity. Further, we predicted DNA-binding specificity of the basic and hinge regions as well as dimerization properties of leucine zipper regions, which was consistent with our phylogenetic clade and classified into 20 subfamilies. Across the different developmental stages of 15 organs and two types of fruits, the clade A bZIP members showed different tissue-specific expression patterns and the duplicated genes were differentially regulated, indicating a functional diversification coupled with the expansion of this gene family in strawberry. Under normal growth conditions, mrna11837 and mrna30280 of clade A showed very weak expression levels in organs and fruits, respectively; but higher expression was observed with different set of genes following drought and heat treatment, which may be caused by the separate response pathway between drought and heat treatments.

The infection process of Discula destructiva Redlin on Cornus florida L. leaves was studied using histological and microscopic techniques. Penetration of fungal hyphae through natural openings and wounds was not observed, while direct penetration without appressorium formation was demonstrated 3 days after inoculation (DAI). Leaves inoculated with D. destructiva developed symptoms of dogwood anthracnose after 7 to 8 days. At 8 DAI, hyphae were observed in aggregations located between the cuticle and epidermis and also growing intracellularly towards epidermal, palisade, parenchymal and spongy mesophyll cells. At 16 DAI, typical chlorotic and necrotic halos, with a red to purple external border, were formed on the inoculated leaves. Within leaf tissues, at 16 DAI, chloroplasts were intact but decompartmentalized and infection sites were clearly defined. Sporulation and ruptured acervuli (cuticle ruptured and spores released) were first detected at 20 DAI, and had fully developed to rupture the pla...

Summary Switchgrass (Panicum virgatum L.) is a leading candidate for a dedicated lignocellulosic biofuel feedstock owing to its high biomass production, wide adaptation and low agronomic input requirements. Lignin in cell walls of switchgrass, and other lignocellulosic feedstocks, severely limits the accessibility of cell wall carbohydrates to enzymatic breakdown into fermentable sugars and subsequently biofuels. Low-lignin transgenic switchgrass plants produced by the down-regulation of caffeic acid O-methyltransferase (COMT), a lignin biosynthetic enzyme, were analysed in the field for two growing seasons. COMT transcript abundance, lignin content and the syringyl/guaiacyl lignin monomer ratio were consistently lower in the COMT-down-regulated plants throughout the duration of the field trial. In general, analyses with fully established plants harvested during the second growing season produced results that were similar to those observed in previous greenhouse studies with these plants. Sugar release was improved by up to 34% and ethanol yield by up to 28% in the transgenic lines relative to controls. Additionally, these results were obtained using senesced plant material harvested at the end of the growing season, compared with the young, green tissue that was used in the greenhouse experiments. Another important finding was that transgenic plants were not more susceptible to rust (Puccinia emaculata). The results of this study suggest that lignin down-regulation in switchgrass can confer real-world improvements in biofuel yield without negative consequences to biomass yield or disease susceptibility.