Multi-Year Field Plantings Evaluating Boxwood Cultivars for Susceptibility to the Blight Pathogens (Calonectria spp.) in Northern Germany.

Two multiyear field trials were conducted to evaluate boxwood cultivars for their susceptibility to the blight pathogens Calonectria pseudonaviculata and C. henricotiae in northern Germany. Fifteen cultivars were included in the first trial from 2007 to 2012, and 46 cultivars were included in the second trial from 2014 to 2017. Both trials were done in a naturally infested field that was supplemented with infected plant tissue added to the soil before planting. Each cultivar had three replicate hedge sections with 10 plants per section, and they were assessed annually for blight severity expressed as proportion of leaves blighted and fallen. Blight severity varied significantly among years (P < 0.0001) and cultivars (P < 0.05) within each trial. In the first trial, mean severity ranged from 0.03 to 0.11 for the most resistant cultivars and 0.35 to 0.96 for the most susceptible ones. Similarly, in the second trial, mean severity ranged from 0.06 to 0.27 and 0.71 to 0.97 for the most resistant and susceptible cultivars, respectively. 'Suffruticosa' was consistently the most susceptible cultivar, followed by 'Marianne', 'Myosotidifolia', 'Raket', and 'Morris Midget'. 'Herrenhausen' was the most resistant cultivar, followed by B. microphylla var. japonica, B. microphylla var. koreana, 'Green Mound', 'Faulkner', and 'Winter Beauty'. This study provides field data showing the performance of boxwood cultivars under different levels of disease pressure in an area where C. henricotiae was dominant. This knowledge will help boxwood growers and gardeners to choose less susceptible cultivars and help plant breeders to select for disease resistance.

Comparative analysis of extracellular proteomes reveals putative effectors of the boxwood blight pathogens, Calonectria henricotiae and C. pseudonaviculata.

Calonectria henricotiae (Che) and C. pseudonaviculata (Cps) are destructive fungal pathogens causing boxwood blight, a persistent threat to horticultural production, landscape industries, established gardens, and native ecosystems. Although extracellular proteins including effectors produced by fungal pathogens are known to play a fundamental role in pathogenesis, the composition of Che and Cps extracellular proteins has not been examined. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatics prediction tools, 630 extracellular proteins and 251 cell membrane proteins of Che and Cps were identified in the classical secretion pathway in the present study. In the non-classical secretion pathway, 79 extracellular proteins were identified. The cohort of proteins belonged to 364 OrthoMCL clusters, with the majority (62%) present in both species, and a subset unique to Che (19%) and Cps (20%). These extracellular proteins were predicted to play important roles in cell structure, regulation, metabolism, and pathogenesis. A total of 124 proteins were identified as putative effectors. Many of them are orthologs of proteins with documented roles in suppressing host defense and facilitating infection processes in other pathosystems, such as SnodProt1-like proteins in the OrthoMCL cluster OG5_152723 and PhiA-like cell wall proteins in the cluster OG5_155754. This exploratory study provides a repository of secreted proteins and putative effectors that can provide insights into the virulence mechanisms of the boxwood blight pathogens.

Comparative Secretome Analyses of Toxigenic and Atoxigenic Rathayibacter Species.

Phytopathogenic Rathayibacter species are unique bacterial plant pathogens because they are obligately vectored by plant parasitic anguinid nematodes to the developing seedheads of forage grasses and cereals. This understudied group of plant-associated Actinomycetes includes the neurotoxigenic plant pathogen R. toxicus, which causes annual ryegrass toxicity in grazing livestock. R. toxicus is currently endemic to Australia and is listed as a plant pathogen select agent by the U.S. Department of Agriculture-Animal and Plant Health Inspection Service. The complex Rathayibacter disease cycle requires intimate interactions with the nematode vector and plant hosts, which warrants an increased understanding of the secretory and surface-associated proteins that mediate these diverse eukaryotic interactions. Here we present the first comparative secretome analysis for this complex, nematode-vectored Rathayibacter genus that compares the three agronomically damaging toxigenic and atoxigenic Rathayibacter species, R. toxicus, R. iranicus, and R. tritici. The exoproteomic comparison identified 1,423 unique proteins between the three species via liquid chromatography-tandem mass spectrometry, leading to the identification of putative pathogenicity-related proteins and proteins that may mediate nematode attachment. Of the uniquely identified proteins, 94 homologous proteins were conserved between the three Rathayibacter exoproteomes and comprised between 43.4 and 58.6% of total protein abundance. Comparative analyses revealed both conserved and uniquely expressed extracellular proteins, which, interestingly, had more similarities to extracellular proteins commonly associated with bacterial animal pathogens than classic plant pathogens. This comparative exoproteome analysis will facilitate the characterization of proteins essential for vector attachment and host colonization and assist in the development of serological diagnostic assays.

One Clonal Lineage of Calonectria pseudonaviculata Is Primarily Responsible for the Boxwood Blight Epidemic in the United States.

Boxwood blight caused by Calonectria pseudonaviculata and C. henricotiae is destroying cultivated and native boxwood worldwide, with profound negative economic impacts on the horticulture industry. First documented in the United States in 2011, the disease has now occurred in 30 states. Previous research showed that global C. pseudonaviculata populations prior to 2014 had a clonal structure, and only the MAT1-2 idiomorph was observed. In this study, we examined C. pseudonaviculata genetic diversity and population structure in the United States after 2014, following the expansion of the disease across the country over the past 5 years. Two hundred eighteen isolates from 21 states were genotyped by sequencing 11 simple sequence repeat (SSR) loci and by MAT1 idiomorph typing. All isolates presented C. pseudonaviculata-specific alleles, indicating that C. henricotiae is still absent in the U.S. states sampled. The presence of only the MAT1-2 idiomorph and gametic linkage disequilibrium suggests the prevalence of asexual reproduction. The contemporary C. pseudonaviculata population is characterized by a clonal structure and composed of 13 multilocus genotypes (SSR-MLGs) unevenly distributed across the United States. These SSR-MLGs grouped into two clonal lineages (CLs). The predominant lineage CL2 (93% of isolates) is the primary contributor to U.S. disease expansion. The contemporary U.S. C. pseudonaviculata population is not geographically subdivided and not genetically differentiated from the U.S. population prior to 2014, but is significantly differentiated from the main European population, which is largely composed of CL1. Our findings provide insights into the boxwood blight epidemic that are critical for disease management and breeding of resistant boxwood cultivars.

Presence of a Sexual Population of Puccinia graminis f. sp. tritici in Georgia Provides a Hotspot for Genotypic and Phenotypic Diversity.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici, is a re-emerging disease exemplified by recent epidemics caused by new virulent races. Understanding the sources and origins of genetic variations in the pathogen populations globally can facilitate the development of better strategies in disease management. We analyzed 68 wheat stem rust samples collected between 2013 and 2015 from Georgia where stem rust incidences are frequent and the alternate host, common barberry, is present. A total of 116 single-pustule isolates were derived and evaluated on stem rust differential lines to determine the virulence phenotypes and 23 races were identified, many of which were detected for the first time. Unique virulence combinations including, Sr22+Sr24 and Sr13b+Sr35+Sr37 were detected. These virulence combinations pose new challenges to breeding programs because many of these genes are used in breeding for resistance to the Ug99 race group. Sixty-one isolates were genotyped using a custom single-nucleotide polymorphism chip and 17 genotypes were identified. The 2013 isolates contained 11 multilocus genotypes compared with isolates of 2014 and 2015, with five and three genotypes, respectively. The higher levels of virulence and genotypic diversity observed in the 2013 samples strongly indicated that sexual recombination occurs in the Georgian P. graminis f. sp. tritici population, and that the Caucasus region of Eurasia may be an important source of new races.[Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Annotation and analysis of the mitochondrial genome of Coniothyrium glycines, causal agent of red leaf blotch of soybean, reveals an abundance of homing endonucleases.

Coniothyrium glycines, the causal agent of soybean red leaf blotch, is a USDA APHIS-listed Plant Pathogen Select Agent and potential threat to US agriculture. Sequencing of the C. glycines mt genome revealed a circular 98,533-bp molecule with a mean GC content of 29.01%. It contains twelve of the mitochondrial genes typically involved in oxidative phosphorylation (atp6, cob, cox1-3, nad1-6, and nad4L), one for a ribosomal protein (rps3), four for hypothetical proteins, one for each of the small and large subunit ribosomal RNAs (rns and rnl) and a set of 30 tRNAs. Genes were encoded on both DNA strands with cox1 and cox2 occurring as adjacent genes having no intergenic spacers. Likewise, nad2 and nad3 are adjacent with no intergenic spacers and nad5 is immediately followed by nad4L with an overlap of one base. Thirty-two introns, comprising 54.1% of the total mt genome, were identified within eight protein-coding genes and the rnl. Eighteen of the introns contained putative intronic ORFs with either LAGLIDADG or GIY-YIG homing endonuclease motifs, and an additional eleven introns showed evidence of truncated or degenerate endonuclease motifs. One intron possessed a degenerate N-acetyl-transferase domain. C. glycines shares some conservation of gene order with other members of the Pleosporales, most notably nad6-rnl-atp6 and associated conserved tRNA clusters. Phylogenetic analysis of the twelve shared protein coding genes agrees with commonly accepted fungal taxonomy. C. glycines represents the second largest mt genome from a member of the Pleosporales sequenced to date. This research provides the first genomic information on C. glycines, which may provide targets for rapid diagnostic assays and population studies.

Evolution of the U.S. Biological Select Agent Rathayibacter toxicus.

Rathayibacter toxicus is a species of Gram-positive, corynetoxin-producing bacteria that causes annual ryegrass toxicity, a disease often fatal to grazing animals. A phylogenomic approach was employed to model the evolution of R. toxicus to explain the low genetic diversity observed among isolates collected during a 30-year period of sampling in three regions of Australia, gain insight into the taxonomy of Rathayibacter, and provide a framework for studying these bacteria. Analyses of a data set of more than 100 sequenced Rathayibacter genomes indicated that Rathayibacter forms nine species-level groups. R. toxicus is the most genetically distant, and evidence suggested that this species experienced a dramatic event in its evolution. Its genome is significantly reduced in size but is colinear to those of sister species. Moreover, R. toxicus has low intergroup genomic diversity and almost no intragroup genomic diversity between ecologically separated isolates. R. toxicus is the only species of the genus that encodes a clustered regularly interspaced short palindromic repeat (CRISPR) locus and that is known to host a bacteriophage parasite. The spacers, which represent a chronological history of infections, were characterized for information on past events. We propose a three-stage process that emphasizes the importance of the bacteriophage and CRISPR in the genome reduction and low genetic diversity of the R. toxicus species.IMPORTANCERathayibacter toxicus is a toxin-producing species found in Australia and is often fatal to grazing animals. The threat of introduction of the species into the United States led to its inclusion in the Federal Select Agent Program, which makes R. toxicus a highly regulated species. This work provides novel insights into the evolution of R. toxicusR. toxicus is the only species in the genus to have acquired a CRISPR adaptive immune system to protect against bacteriophages. Results suggest that coexistence with the bacteriophage NCPPB3778 led to the massive shrinkage of the R. toxicus genome, species divergence, and the maintenance of low genetic diversity in extant bacterial groups. This work contributes to an understanding of the evolution and ecology of an agriculturally important species of bacteria.

Rathayibacter agropyri (non O'Gara 1916) comb. nov., nom. rev., isolated from western wheatgrass (Pascopyrum smithii).

Aplanobacter agropyri was first described in 1915 by O'Gara and later transferred to the genus Corynebacterium by Burkholder in 1948 but it was not included in the Approved Lists of Bacterial Names in 1980 and, consequently, is not recognized as a validly published species. In the 1980s, bacteria resembling Corynebacterium agropyri were isolated from plant samples stored at the Washington State Mycological Herbarium and from a diseased wheatgrass plant collected in Cardwell, Montana, USA. In the framework of this study, eight additional isolates were recovered from the same herbarium plant samples in 2011. The isolates are slow-growing, yellow-pigmented, Gram-stain-positive and coryneform. The peptidoglycan is type B2γ containing diaminobutyric acid, alanine, glycine and glutamic acid, the cell-wall sugars are rhamnose and mannose, the major respiratory quinone is MK-10, and the major fatty acids are anteiso-15 : 0, anteiso 17 : 0 and iso-16 : 0, all of which are typical of the genus Rathayibacter. Phylogenetic analysis of 16S rRNA gene sequences placed the strains in the genus Rathayibacter and distinguished them from the six other described species of Rathayibacter. DNA-DNA hybridization confirmed that the strains were members of a novel species. Based on phenotypic, chemotaxonomic and phylogenetic characterization, it appears that strains CA-1 to CA-12 represent a novel species, and the name Rathayibacter agropyri (non O'Gara 1916) comb. nov., nom. rev. is proposed; the type strain is CA-4T (=DSM 104101T;=ATCC TSD-78T).

Partial Proteome of the Corynetoxin-Producing Gram-Positive Bacterium, Rathayibacter toxicus.

Rathayibacter toxicus is a Gram-positive bacterium that is the causative agent of annual ryegrass toxicity (ARGT), a disease that causes devastating losses in the Australian livestock industry. R. toxicus exhibits a complex life cycle, using the nematode Anguina funesta as a physical vector to carry it up to the seed head of the host plant. ARGT is caused by a tunicamycin-like corynetoxin that is produced in R. toxicus-infected seed galls. We analyzed protein expression in R. toxicus under stationary growth phase conditions to obtain a more complete understanding of the biology of this organism and identify potential targets for immunoassay development. A total of 323 unique proteins were identified, including those with putative roles in secondary metabolism and pathogenicity. The proteome analysis for this complex phytopathogenic Gram-positive bacterium will facilitate in the characterization of proteins necessary for host colonization and toxin production, and assist in the development of diagnostic assays. Data are available via ProteomeXchange with identifier PXD004238.

Whole genome sequence of two Rathayibacter toxicus strains reveals a tunicamycin biosynthetic cluster similar to Streptomyces chartreusis.

Rathayibacter toxicus is a forage grass associated Gram-positive bacterium of major concern to food safety and agriculture. This species is listed by USDA-APHIS as a plant pathogen select agent because it produces a tunicamycin-like toxin that is lethal to livestock and may be vectored by nematode species native to the U.S. The complete genomes of two strains of R. toxicus, including the type strain FH-79, were sequenced and analyzed in comparison with all available, complete R. toxicus genomes. Genome sizes ranged from 2,343,780 to 2,394,755 nucleotides, with 2079 to 2137 predicted open reading frames; all four strains showed remarkable synteny over nearly the entire genome, with only a small transposed region. A cluster of genes with similarity to the tunicamycin biosynthetic cluster from Streptomyces chartreusis was identified. The tunicamycin gene cluster (TGC) in R. toxicus contained 14 genes in two transcriptional units, with all of the functional elements for tunicamycin biosynthesis present. The TGC had a significantly lower GC content (52%) than the rest of the genome (61.5%), suggesting that the TGC may have originated from a horizontal transfer event. Further analysis indicated numerous remnants of other potential horizontal transfer events are present in the genome. In addition to the TGC, genes potentially associated with carotenoid and exopolysaccharide production, bacteriocins and secondary metabolites were identified. A CRISPR array is evident. There were relatively few plant-associated cell-wall hydrolyzing enzymes, but there were numerous secreted serine proteases that share sequence homology to the pathogenicity-associated protein Pat-1 of Clavibacter michiganensis. Overall, the genome provides clear insight into the possible mechanisms for toxin production in R. toxicus, providing a basis for future genetic approaches.

Rathayibacter toxicus, Other Rathayibacter Species Inducing Bacterial Head Blight of Grasses, and the Potential for Livestock Poisonings.

Rathayibacter toxicus, a Select Agent in the United States, is one of six recognized species in the genus Rathayibacter and the best known due to its association with annual ryegrass toxicity, which occurs only in parts of Australia. The Rathayibacter species are unusual among phytopathogenic bacteria in that they are transmitted by anguinid seed gall nematodes and produce extracellular polysaccharides in infected plants resulting in bacteriosis diseases with common names such as yellow slime and bacterial head blight. R. toxicus is distinguished from the other species by producing corynetoxins in infected plants; toxin production is associated with infection by a bacteriophage. These toxins cause grazing animals feeding on infected plants to develop convulsions and abnormal gate, which is referred to as "staggers," and often results in death of affected animals. R. toxicus is the only recognized Rathayibacter species to produce toxin, although reports of livestock deaths in the United States suggest a closely related toxigenic species may be present. A closely related but undescribed species, Rathayibacter sp. EV, originally isolated from Ehrharta villosa var. villosa in South Africa, is suspected of producing toxin. Many of the diseases caused by Rathayibacter species occur in arid areas and the extracellular polysaccharide they produce is believed to aid in their survival between crops. For example, R. "agropyri" was isolated from infected plant material after being stored for 50 years in a herbarium. Similarly, the anguinid vectors associated with these bacteria form seed galls in infected plants and are capable of surviving for very long periods of time under dry conditions. The addition of R. toxicus to the list of Select Agents has raised concern over its potential introduction and a realization that current diagnostic methods are inadequate to distinguish among Rathayibacter species. In addition, little is known about the Rathayibacter species and their seed gall nematode vectors present in the United States.

Population genomic analyses reveal a history of range expansion and trait evolution across the native and invaded range of yellow starthistle (Centaurea solstitialis).

Identifying sources of genetic variation and reconstructing invasion routes for non-native introduced species is central to understanding the circumstances under which they may evolve increased invasiveness. In this study, we used genome-wide single nucleotide polymorphisms to study the colonization history of Centaurea solstitialis in its native range in Eurasia and invasions into the Americas. We leveraged this information to pinpoint key evolutionary shifts in plant size, a focal trait associated with invasiveness in this species. Our analyses revealed clear population genomic structure of potential source populations in Eurasia, including deep differentiation of a lineage found in the southern Apennine and Balkan Peninsulas and divergence among populations in Asia, eastern Europe and western Europe. We found strongest support for an evolutionary scenario in which western European populations were derived from an ancient admixture event between populations from eastern Europe and Asia, and subsequently served as the main genetic 'bridgehead' for introductions to the Americas. Introductions to California appear to be from a single source region, and multiple, independent introductions of divergent genotypes likely occurred into the Pacific Northwest. Plant size has evolved significantly at three points during range expansion, including a large size increase in the lineage responsible for the aggressive invasion of the California interior. These results reveal a long history of colonization, admixture and trait evolution in C. solstitialis, and suggest routes for improving evidence-based management decisions for one of the most ecologically and economically damaging invasive species in the western United States.

In situ hybridization for the detection of rust fungi in paraffin embedded plant tissue sections.

BACKGROUND: Rust fungi are obligate pathogens with multiple life stages often including different spore types and multiple plant hosts. While individual rust pathogens are often associated with specific plants, a wide range of plant species are infected with rust fungi. To study the interactions between these important pathogenic fungi and their host plants, one must be able to differentiate fungal tissue from plant tissue. This can be accomplished using the In situ hybridization (ISH) protocol described here. RESULTS: To validate reproducibility using the ISH protocol, samples of Chrysanthemum × morifolium infected with Puccinia horiana, Gladiolus × hortulanus infected with Uromyces transversalis and Glycine max infected with Phakopsora pachyrhizi were tested alongside uninfected leaf tissue samples. The results of these tests show that this technique clearly distinguishes between rust pathogens and their respective host plant tissues. CONCLUSIONS: This ISH protocol is applicable to rust fungi and potentially other plant pathogenic fungi as well. It has been shown here that this protocol can be applied to pathogens from different genera of rust fungi with no background staining of plant tissue. We encourage the use of this protocol for the study of plant pathogenic fungi in paraffin embedded sections of host plant tissue.

Kenyan Isolates of Puccinia graminis f. sp. tritici from 2008 to 2014: Virulence to SrTmp in the Ug99 Race Group and Implications for Breeding Programs.

Frequent emergence of new variants in the Puccinia graminis f. sp. tritici Ug99 race group in Kenya has made pathogen survey a priority. We analyzed 140 isolates from 78 P. graminis f. sp. tritici samples collected in Kenya between 2008 and 2014 and identified six races, including three not detected prior to 2013. Genotypic analysis of 20 isolates from 2013 and 2014 collections showed that the new races TTHST, TTKTK, and TTKTT belong to the Ug99 race group. International advanced breeding lines were evaluated against an isolate of TTKTT (Sr31, Sr24, and SrTmp virulence) at the seedling stage. From 169 advanced lines from Kenya, 23% of lines with resistance to races TTKSK and TTKST were susceptible to TTKTT and, from two North American regional nurseries, 44 and 91% of resistant lines were susceptible. Three lines with combined resistance genes were developed to facilitate pathogen monitoring and race identification. These results indicate the increasing virulence and variability in the Kenyan P. graminis f. sp. tritici population and reveal vulnerabilities of elite germplasm to new races.

Phenotypic and Genotypic Characterization of Race TKTTF of Puccinia graminis f. sp. tritici that Caused a Wheat Stem Rust Epidemic in Southern Ethiopia in 2013-14.

A severe stem rust epidemic occurred in southern Ethiopia during November 2013 to January 2014, with yield losses close to 100% on the most widely grown wheat cultivar, 'Digalu'. Sixty-four stem rust samples collected from the regions were analyzed. A meteorological model for airborne spore dispersal was used to identify which regions were most likely to have been infected from postulated sites of initial infection. Based on the analyses of 106 single-pustule isolates derived from these samples, four races of Puccinia graminis f. sp. tritici were identified: TKTTF, TTKSK, RRTTF, and JRCQC. Race TKTTF was found to be the primary cause of the epidemic in the southeastern zones of Bale and Arsi. Isolates of race TKTTF were first identified in samples collected in early October 2013 from West Arsi. It was the sole or predominant race in 31 samples collected from Bale and Arsi zones after the stem rust epidemic was established. Race TTKSK was recovered from 15 samples from Bale and Arsi zones at low frequencies. Genotyping indicated that isolates of race TKTTF belongs to a genetic lineage that is different from the Ug99 race group and is composed of two distinct genetic types. Results from evaluation of selected germplasm indicated that some cultivars and breeding lines resistant to the Ug99 race group are susceptible to race TKTTF. Appearance of race TKTTF and the ensuing epidemic underlines the continuing threats and challenges posed by stem rust not only in East Africa but also to wider-scale wheat production.

Draft Genome Sequence of Xanthomonas arboricola Strain 3004, a Causal Agent of Bacterial Disease on Barley.

We report here the annotated genome sequence of Xanthomonas arboricola strain 3004, isolated from barley leaves with symptoms of streak and capable of infecting other plant species. We sequenced the genome of X. arboricola strain 3004 to improve the understanding of molecular mechanisms of the pathogenesis and evolution of the genus Xanthomonas.

Disruption of Rpp1-mediated soybean rust immunity by virus-induced gene silencing.

Phakopsora pachyrhizi, a fungus that causes rust disease on soybean, has potential to impart significant yield loss and disrupt food security and animal feed production. Rpp1 is a soybean gene that confers immunity to soybean rust, and it is important to understand how it regulates the soybean defense system and to use this knowledge to protect commercial crops. It was previously discovered that some soybean proteins resembling transcription factors accumulate in the nucleus of Rpp1 soybeans. To determine if they contribute to immunity, Bean pod mottle virus was used to attenuate or silence the expression of their genes. Rpp1 plants subjected to virus-induced gene silencing exhibited reduced amounts of RNA for 5 of the tested genes, and the plants developed rust-like symptoms after subsequent inoculation with fungal spores. Symptoms were associated with the accumulation of rust fungal RNA and protein. Silenced plants also had reduced amounts of RNA for the soybean Myb84 transcription factor and soybean isoflavone O-methyltransferase, both of which are important to phenylpropanoid biosynthesis and lignin formation, crucial components of rust resistance. These results help resolve some of the genes that contribute to Rpp1-mediated immunity and improve upon the knowledge of the soybean defense system. It is possible that these genes could be manipulated to enhance rust resistance in otherwise susceptible soybean cultivars.

Gene expression and proteomic analysis of the formation of Phakopsora pachyrhizi appressoria.

BACKGROUND: Phakopsora pachyrhizi is an obligate fungal pathogen causing Asian soybean rust (ASR). A dual approach was taken to examine the molecular and biochemical processes occurring during the development of appressoria, specialized infection structures by which P. pachyrhizi invades a host plant. Suppression subtractive hybridization (SSH) was utilized to generate a cDNA library enriched for transcripts expressed during appressoria formation. Two-dimensional gel electrophoresis and mass spectroscopy analysis were used to generate a partial proteome of proteins present during appressoria formation. RESULTS: Sequence analysis of 1133 expressed sequence tags (ESTs) revealed 238 non-redundant ESTs, of which 53% had putative identities assigned. Twenty-nine of the non-redundant ESTs were found to be specific to the appressoria-enriched cDNA library, and did not occur in a previously constructed germinated urediniospore cDNA library. Analysis of proteins against a custom database of the appressoria-enriched ESTs plus Basidiomycota EST sequences available from NCBI revealed 256 proteins. Fifty-nine of these proteins were not previously identified in a partial proteome of P. pachyrhizi germinated urediniospores. Genes and proteins identified fell into functional categories of metabolism, cell cycle and DNA processing, protein fate, cellular transport, cellular communication and signal transduction, and cell rescue. However, 38% of ESTs and 24% of proteins matched only to hypothetical proteins of unknown function, or showed no similarity to sequences in the current NCBI database. Three novel Phakopsora genes were identified from the cDNA library along with six potentially rust-specific genes. Protein analysis revealed eight proteins of unknown function, which possessed classic secretion signals. Two of the extracellular proteins are reported as potential effector proteins. CONCLUSIONS: Several genes and proteins were identified that are expressed in P. pachyrhizi during appressoria formation. Understanding the role that these genes and proteins play in the molecular and biochemical processes in the infection process may provide insight for developing targeted control measures and novel methods of disease management.

Novel Phakopsora pachyrhizi extracellular proteins are ideal targets for immunological diagnostic assays.

Phakopsora pachyrhizi, the causal agent of Asian soybean rust (ASR), continues to spread across the southeast and midsouth regions of the United States, necessitating the use of fungicides by producers. Our objective in this research was to identify ASR proteins expressed early during infection for the development of immunodiagnostic assays. We have identified and partially characterized a small gene family encoding extracellular proteins in the P. pachyrhizi urediniospore wall, termed PHEPs (for Phakopsora extracellular protein). Two highly expressed protein family members, PHEP 107 and PHEP 369, were selected as ideal immunodiagnostic targets for antibody development, after we detected PHEPs in plants as early as 3 days postinfection (dpi). Monoclonal antibodies (MAbs; 2E8E5-1 and 3G6H7-3) generated against recombinant PHEP 369 were tested for sensitivity against the recombinant protein and extracts from ASR-infected plants and for specificity against a set of common soybean pathogens. These antibodies should prove applicable in immunodiagnostic assays to detect infected soybeans and to identify ASR spores from sentinel surveillance plots.

Molecular, ultrastructural, and biological characterization of Pennsylvania isolates of Plum pox virus.

Plum pox virus (PPV) was identified in Pennsylvania in 1999. The outbreak was limited to a four-county region in southern Pennsylvania. Initial serological and molecular characterization indicated that the isolates in Pennsylvania belong to the D strain of PPV. The Pennsylvania isolates were characterized by sequence analysis, electron microscopy, host range, and vector transmission to determine how these isolates related to their previously studied European counterparts. Genetically, Pennsylvania (PPV-Penn) isolates were more closely related to each other than to any other PPV-D strains, and isolates from the United States, Canada, and Chile were more closely related to each other than to European isolates. The PPV-Penn isolates exist as two clades, suggesting the possibility of multiple introductions. Electron microscopy analysis of PPV-Penn isolates, including cytopathological studies, indicated that the virions were similar to other Potyvirus spp. PPV-Penn isolates had a herbaceous host range similar to that of European D isolates. There were distinct differences in the transmission efficiencies of the two PPV-Penn isolates using Myzus persicae and Aphis spiraecola as vectors; however, both PPV-Penn isolates were transmitted by M. persicae more efficiently than a European D isolate but less efficiently than a European M isolate.