Insects Visiting Drippy Blight Diseased Red Oak Trees Are Contaminated with the Pathogenic Bacterium Lonsdalea quercina.

The focus of investigation in this study was to consider the potential of arthropods in the dissemination of the bacterium involved in drippy blight disease, Lonsdalea quercina. Arthropod specimens were collected and tested for the presence of the bacterium with molecular markers. The bacterium L. quercina was confirmed on 12 different insect samples from three orders (Coleoptera, Hemiptera, and Hymenoptera) and eight families (Buprestidae, Coccinellidae, Dermestidae, Coreidae, Pentatomidae and/or Miridae, Apidae, Formicidae, and Vespidae). Approximately half of the insects found to carry the bacterium were in the order Hymenoptera. Estimates of the insects that are contaminated with the bacterium, and likely carry it between trees, is conservative because the documented insects represent only a subset of the insect orders that were observed feeding on the bacterium or present on diseased trees yet were not able to be tested. The insects contaminated with L. quercina exhibited diverse life histories, where some had a facultative relationship with the bacterium and others sought it out as a food source. These findings demonstrate that a diverse set of insects naturally occur on diseased trees and may disseminate L. quercina.

Characterization of the Xanthomonas translucens Complex Using Draft Genomes, Comparative Genomics, Phylogenetic Analysis, and Diagnostic LAMP Assays.

Prevalence of Xanthomonas translucens, which causes cereal leaf streak (CLS) in cereal crops and bacterial wilt in forage and turfgrass species, has increased in many regions in recent years. Because the pathogen is seedborne in economically important cereals, it is a concern for international and interstate germplasm exchange and, thus, reliable and robust protocols for its detection in seed are needed. However, historical confusion surrounding the taxonomy within the species has complicated the development of accurate and reliable diagnostic tools for X. translucens. Therefore, we sequenced genomes of 15 X. translucens strains representing six different pathovars and compared them with additional publicly available X. translucens genome sequences to obtain a genome-based phylogeny for robust classification of this species. Our results reveal three main clusters: one consisting of pv. cerealis, one consisting of pvs. undulosa and translucens, and a third consisting of pvs. arrhenatheri, graminis, phlei, and poae. Based on genomic differences, diagnostic loop-mediated isothermal amplification (LAMP) primers were developed that clearly distinguish strains that cause disease on cereals, such as pvs. undulosa, translucens, hordei, and secalis, from strains that cause disease on noncereal hosts, such as pvs. arrhenatheri, cerealis, graminis, phlei, and poae. Additional LAMP assays were developed that selectively amplify strains belonging to pvs. cerealis and poae, distinguishing them from other pathovars. These primers will be instrumental in diagnostics when implementing quarantine regulations to limit further geographic spread of X. translucens pathovars.

Geosmithia associated with bark beetles and woodborers in the western USA: taxonomic diversity and vector specificity.

Fungi in the genus Geosmithia (Ascomycota: Hypocreales) are frequent associates of bark beetles and woodborers that colonize hardwood and coniferous trees. One species, Geosmithia morbida, is an economically damaging invasive species. The authors surveyed the Geosmithia species of California and Colorado, USA, to (i) provide baseline data on taxonomy of Geosmithia and beetle vector specificity across the western USA; (ii) investigate the subcortical beetle fauna for alternative vectors of the invasive G. morbida; and (iii) interpret the community composition of this region within the emerging global biogeography of Geosmithia. Geosmithia was detected in 87% of 126 beetle samples obtained from 39 plant species. Twenty-nine species of Geosmithia were distinguished, of which 13 may be new species. Bark beetles from hardwoods, Cupressus, and Sequoia appear to be regular vectors, with Geosmithia present in all beetle gallery systems examined. Other subcortical insects appear to vector Geosmithia at lower frequencies. Overall, most Geosmithia have a distinct level of vector specificity (mostly high, sometimes low) enabling their separation to generalists and specialists. Plant pathogenic Geosmithia morbida was not found in association with any other beetle besides Pityophthorus juglandis. However, four additional Geosmithia species were found in P. juglandis galleries. When integrated with recent data from other continents, a global pattern of Geosmithia distribution across continents, latitudes, and vectors is emerging: of the 29 Geosmithia species found in the western USA, 12 have not been reported outside of the USA. The most frequently encountered species with the widest global distribution also had the broadest range of beetle vectors. Several Geosmithia spp. with very narrow vector ranges in Europe exhibited the similar degree of specialization in the USA. Such strong canalization in association could reflect an ancient origin of each individual association, or a recent origin and a subsequent diversification in North America.

Virulence of Genetically Distinct Geosmithia morbida Isolates to Black Walnut and Their Response to Coinoculation with Fusarium solani.

Geosmithia morbida is well documented as the causal agent of thousand cankers disease of black walnut trees. However, it is not well understood how G. morbida strains differ in virulence and how their interactions with co-occurring pathogens contribute to disease severity. In this study, we systematically investigated virulence of genetically distinct G. morbida strains. Overall, we found varying degrees of virulence, although differences were not related to genetic groupings. Furthermore, the pathogen Fusarium solani is also commonly isolated from thousand canker-diseased trees. The degree of disease contribution from F. solani is unknown, along with interactions it may have with G. morbida. This research shows that coinoculation with these pathogens does not yield a synergistic response.

Erwinia iniecta sp. nov., isolated from Russian wheat aphid (Diuraphis noxia).

Short, Gram-negative-staining, rod-shaped bacteria were isolated from crushed bodies of Russian wheat aphid [Diuraphis noxia (Kurdjumov)] and artificial diets after Russian wheat aphid feeding. Based on multilocus sequence analysis involving the 16S rRNA, atpD, infB, gyrB and rpoB genes, these bacterial isolates constitute a novel clade in the genus Erwinia, and were most closely related to Erwinia toletana. Representative distinct strains within this clade were used for comparisons with related species of Erwinia. Phenotypic comparisons using four distinct strains and average nucleotide identity (ANI) measurements using two distinct draft genomes revealed that these strains form a novel species within the genus Erwinia. The name Erwinia iniecta sp. nov. is proposed, and strain B120T ( = CFBP 8182T = NCCB 100485T) was designated the type strain. Erwinia iniecta sp. nov. was not pathogenic to plants. However, virulence to the Russian wheat aphid was observed.

Virus-induced gene silencing of Arabidopsis thaliana gene homologues in wheat identifies genes conferring improved drought tolerance.

In a non-model staple crop like wheat (Triticum aestivumI L.), functional validation of potential drought stress responsive genes identified in Arabidopsis could provide gene targets for breeding. Virus-induced gene silencing (VIGS) of genes of interest can overcome the inherent problems of polyploidy and limited transformation potential that hamper functional validation studies in wheat. In this study, three potential candidate genes shown to be involved in abiotic stress response pathways in Arabidopsis thaliana were selected for VIGS experiments in wheat. These include Era1 (enhanced response to abscisic acid), Cyp707a (ABA 8'-hydroxylase), and Sal1 (inositol polyphosphate 1-phosphatase). Gene homologues for these three genes were identified in wheat and cloned in the viral vector barley stripe mosaic virus (BSMV) in the antisense direction, followed by rub inoculation of BSMV viral RNA transcripts onto wheat plants. Quantitative real-time PCR showed that VIGS-treated wheat plants had significant reductions in target gene transcripts. When VIGS-treated plants generated for Era1 and Sal1 were subjected to limiting water conditions, they showed increased relative water content, improved water use efficiency, reduced gas exchange, and better vigour compared to water-stressed control plants inoculated with RNA from the empty viral vector (BSMV0). In comparison, the Cyp707a-silenced plants showed no improvement over BSMV0-inoculated plants under limited water condition. These results indicate that Era1 and Sal1 play important roles in conferring drought tolerance in wheat. Other traits affected by Era1 silencing were also studied. Delayed seed germination in Era1-silenced plants suggests this gene may be a useful target for developing resistance to pre-harvest sprouting.

Susceptibility of Walnut and Hickory Species to Geosmithia morbida.

Thousand cankers disease (TCD) of walnut is a result of feeding in the phloem by the walnut twig beetle (WTB), Pityophthorus juglandis, and subsequent canker formation caused by Geosmithia morbida around galleries. TCD has caused extensive morbidity and mortality to Juglans nigra in the western United States and, in 2010, was discovered in the eastern United States, where the tree is a highly valuable timber resource. WTB and G. morbida also have been found in J. regia orchards throughout major production areas in California, and the numbers of damaged trees are increasing. We tested the susceptibility of walnut and hickory species to G. morbida in greenhouse and field studies. Carya illinoinensis, C. aquatica, and C. ovata were immune. All walnut species tested, including J. ailantifolia, J. californica, J. cinerea, J. hindsii, J. major, J. mandshurica, J. microcarpa, J. nigra, and J. regia, developed cankers following inoculation with G. morbida. J. nigra was the most susceptible, whereas J. major, a native host of the WTB and, presumably, G. morbida, had smaller and more superficial cankers. Canker formation differed among maternal half-sibling families of J. nigra and J. cinerea, indicating genetic variability in resistance to G. morbida. Our inoculation studies with G. morbida have corroborated many of the field observations on susceptibility of walnut and hickory species to TCD, although the ability of the WTB to successfully attack and breed in walnut is also an important component in TCD resistance.

Geosmithia morbida sp. nov., a new phytopathogenic species living in symbiosis with the walnut twig beetle (Pityophthorus juglandis) on Juglans in USA.

Widespread morbidity and mortality of Juglans nigra has occurred in the western USA over the past decade. Tree mortality is the result of aggressive feeding by the walnut twig beetle (Pityophthorus juglandis) and subsequent canker development around beetle galleries caused by a filamentous ascomycete in genus Geosmithia (Ascomycota: Hypocreales). Thirty-seven Geosmithia strains collected from J. californica, J. hindsii, J. major and J. nigra in seven USA states (AZ, CA, CO, ID, OR, UT, WA) were compared with morphological and molecular methods (ITS rDNA sequences). Strains had common characteristics including yellowish conidia en masse, growth at 37 C and absence of growth on Czapek-Dox agar and belonged to a single species described here as G. morbida. Whereas Geosmithia are common saprobes associated with bark beetles attacking hardwoods and conifers worldwide, G. morbida is the first species documented as a plant pathogen.

Genomics-Based Diagnostic Marker Development for Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola.

A computational genomics pipeline was used to compare sequenced genomes of Xanthomonas spp. and to rapidly identify unique regions for development of highly specific diagnostic markers. A suite of diagnostic primers was selected to monitor diverse loci and to distinguish the rice bacterial blight and bacterial leaf streak pathogens, Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola, respectively. A subset of these primers was combined into a multiplex polymerase chain reaction set that accurately distinguished the two rice pathogens in a survey of a geographically diverse collection of X. oryzae pv. oryzae, X. oryzae pv. oryzicola, other xanthomonads, and several genera of plant-pathogenic and plant- or seed-associated bacteria. This computational approach for identification of unique loci through whole-genome comparisons is a powerful tool that can be applied to other plant pathogens to expedite development of diagnostic primers.

Analysis of the Pythium ultimum transcriptome using Sanger and Pyrosequencing approaches.

BACKGROUND: Pythium species are an agriculturally important genus of plant pathogens, yet are not understood well at the molecular, genetic, or genomic level. They are closely related to other oomycete plant pathogens such as Phytophthora species and are ubiquitous in their geographic distribution and host rage. To gain a better understanding of its gene complement, we generated Expressed Sequence Tags (ESTs) from the transcriptome of Pythium ultimum DAOM BR144 (= ATCC 200006 = CBS 805.95) using two high throughput sequencing methods, Sanger-based chain termination sequencing and pyrosequencing-based sequencing-by-synthesis. RESULTS: A single half-plate pyrosequencing (454 FLX) run on adapter-ligated cDNA from a normalized cDNA population generated 90,664 reads with an average read length of 190 nucleotides following cleaning and removal of sequences shorter than 100 base pairs. After clustering and assembly, a total of 35,507 unique sequences were generated. In parallel, 9,578 reads were generated from a library constructed from the same normalized cDNA population using dideoxy chain termination Sanger sequencing, which upon clustering and assembly generated 4,689 unique sequences. A hybrid assembly of both Sanger- and pyrosequencing-derived ESTs resulted in 34,495 unique sequences with 1,110 sequences (3.2%) that were solely derived from Sanger sequencing alone. A high degree of similarity was seen between P. ultimum sequences and other sequenced plant pathogenic oomycetes with 91% of the hybrid assembly derived sequences > 500 bp having similarity to sequences from plant pathogenic Phytophthora species. An analysis of Gene Ontology assignments revealed a similar representation of molecular function ontologies in the hybrid assembly in comparison to the predicted proteomes of three Phytophthora species, suggesting a broad representation of the P. ultimum transcriptome was present in the normalized cDNA population. P. ultimum sequences with similarity to oomycete RXLR and Crinkler effectors, Kazal-like and cystatin-like protease inhibitors, and elicitins were identified. Sequences with similarity to thiamine biosynthesis enzymes that are lacking in the genome sequences of three Phytophthora species and one downy mildew were identified and could serve as useful phylogenetic markers. Furthermore, we identified 179 candidate simple sequence repeats that can be used for genotyping strains of P. ultimum. CONCLUSION: Through these two technologies, we were able to generate a robust set (approximately 10 Mb) of transcribed sequences for P. ultimum. We were able to identify known sequences present in oomycetes as well as identify novel sequences. An ample number of candidate polymorphic markers were identified in the dataset providing resources for phylogenetic and diagnostic marker development for this species. On a technical level, in spite of the depth possible with 454 FLX platform, the Sanger and pyro-based sequencing methodologies were complementary as each method generated sequences unique to each platform.

Geographic Distribution and Genetic Diversity of Three Ophiosphaerella Species that Cause Spring Dead Spot of Bermudagrass.

The distribution of three Ophiosphaerella spp. that cause spring dead spot (SDS) of bermudagrass was studied by systematically sampling two golf courses in Oklahoma and one in Kansas. O. herpotricha was isolated from all three locations and was the most abundant species. It was the only SDS pathogen found at Jenks, Oklahoma. O. korrae was isolated from Afton, Oklahoma, and Independence, Kansas, whereas O. narmari was only detected in samples from Afton. This is the first report of all three Ophiosphaerella species on bermudagrass at the same location. Amplified fragment length polymorphism (AFLP) marker analysis was used to investigate inter- and intraspecific genetic diversity of Ophiosphaerella isolates from North America and Australia. A majority of the O. herpotricha and O. narmari isolates from Afton were distinct haplotypes, suggesting that sexual recombination was occurring within the population. Conversely, the presence of multiple isolates of O. herpotricha and O. narmari with the same haplotype also indicated that asexual propagation was occurring. The genetic diversity among O. herpotricha isolates from Afton was not distinctly different from that of isolates collected throughout the southern United States. In contrast, O. narmari isolates from Afton were distinct from those collected in Australia. The genetic diversity in O. korrae was markedly different than that in the other Ophiosphaerella spp. The population at Afton was dominated by just a few haplotypes, and these were nearly identical to isolates collected from bermudagrass and Kentucky bluegrass throughout western, central, and northern North America. However, O. korrae isolates collected in the southeastern United States were only distantly similar to other North American isolates.

Genome Sequences of Strain ATCC 29281 and Pin and Northern Red Oak Isolates of Lonsdalea quercina subsp. quercina.

Two bacteria identified as Lonsdalea quercina subsp. quercina were isolated from oak trees showing symptoms of drippy blight. Here, we present their draft genome assemblies, as well as that of the type strain of this species. To our knowledge, these are the first published genome sequences of this subspecies of Lonsdalea quercina.

Population structure of Geosmithia morbida, the causal agent of thousand cankers disease of walnut trees in the United States.

The ascomycete Geosmithia morbida and the walnut twig beetle Pityophthorus juglandis are associated with thousand cankers disease of Juglans (walnut) and Pterocarya (wingnut). The disease was first reported in the western United States (USA) on several Juglans species, but has been found more recently in the eastern USA in the native range of the highly susceptible Juglans nigra. We performed a comprehensive population genetic study of 209 G. morbida isolates collected from Juglans and Pterocarya from 17 geographic regions distributed across 12 U.S. states. The study was based on sequence typing of 27 single nucleotide polymorphisms from three genomic regions and genotyping with ten microsatellite primer pairs. Using multilocus sequence-typing data, 197 G. morbida isolates were placed into one of 57 haplotypes. In some instances, multiple haplotypes were recovered from isolates collected on the same tree. Twenty-four of the haplotypes (42%) were recovered from more than one isolate; the two most frequently occurring haplotypes (H02 and H03) represented 36% of all isolates. These two haplotypes were abundant in California, but were not recovered from Arizona or New Mexico. G. morbida population structure was best explained by four genetically distinct groups that clustered into three geographic regions. Most of the haplotypes isolated from the native range of J. major (Arizona and New Mexico) were found in those states only or present in distinct genetic clusters. There was no evidence of sexual reproduction or genetic recombination in any population. The scattered distribution of the genetic clusters indicated that G. morbida was likely disseminated to different regions at several times and from several sources. The large number of haplotypes observed and the genetic complexity of G. morbida indicate that it evolved in association with at least one Juglans spp. and the walnut twig beetle long before the first reports of the disease.

Genome Sequence of Xanthomonas arboricola pv. Corylina, Isolated from Turkish Filbert in Colorado.

Previously, we reported the isolation of a bacterium producing leaf spots in Turkish filbert. Here, we present the draft genome assembly of the bacterium identified as Xanthomonas arboricola pv. corylina. To our knowledge, this is the first published genome of this pathovar of X. arboricola.

Comparative genomics reveals insight into virulence strategies of plant pathogenic oomycetes.

The kingdom Stramenopile includes diatoms, brown algae, and oomycetes. Plant pathogenic oomycetes, including Phytophthora, Pythium and downy mildew species, cause devastating diseases on a wide range of host species and have a significant impact on agriculture. Here, we report comparative analyses on the genomes of thirteen straminipilous species, including eleven plant pathogenic oomycetes, to explore common features linked to their pathogenic lifestyle. We report the sequencing, assembly, and annotation of six Pythium genomes and comparison with other stramenopiles including photosynthetic diatoms, and other plant pathogenic oomycetes such as Phytophthora species, Hyaloperonospora arabidopsidis, and Pythium ultimum var. ultimum. Novel features of the oomycete genomes include an expansion of genes encoding secreted effectors and plant cell wall degrading enzymes in Phytophthora species and an over-representation of genes involved in proteolytic degradation and signal transduction in Pythium species. A complete lack of classical RxLR effectors was observed in the seven surveyed Pythium genomes along with an overall reduction of pathogenesis-related gene families in H. arabidopsidis. Comparative analyses revealed fewer genes encoding enzymes involved in carbohydrate metabolism in Pythium species and H. arabidopsidis as compared to Phytophthora species, suggesting variation in virulence mechanisms within plant pathogenic oomycete species. Shared features between the oomycetes and diatoms revealed common mechanisms of intracellular signaling and transportation. Our analyses demonstrate the value of comparative genome analyses for exploring the evolution of pathogenesis and survival mechanisms in the oomycetes. The comparative analyses of seven Pythium species with the closely related oomycetes, Phytophthora species and H. arabidopsidis, and distantly related diatoms provide insight into genes that underlie virulence.

Carbohydrate-active enzymes in pythium and their role in plant cell wall and storage polysaccharide degradation.

Carbohydrate-active enzymes (CAZymes) are involved in the metabolism of glycoconjugates, oligosaccharides, and polysaccharides and, in the case of plant pathogens, in the degradation of the host cell wall and storage compounds. We performed an in silico analysis of CAZymes predicted from the genomes of seven Pythium species (Py. aphanidermatum, Py. arrhenomanes, Py. irregulare, Py. iwayamai, Py. ultimum var. ultimum, Py. ultimum var. sporangiiferum and Py. vexans) using the "CAZymes Analysis Toolkit" and "Database for Automated Carbohydrate-active Enzyme Annotation" and compared them to previously published oomycete genomes. Growth of Pythium spp. was assessed in a minimal medium containing selected carbon sources that are usually present in plants. The in silico analyses, coupled with our in vitro growth assays, suggest that most of the predicted CAZymes are involved in the metabolism of the oomycete cell wall with starch and sucrose serving as the main carbohydrate sources for growth of these plant pathogens. The genomes of Pythium spp. also encode pectinases and cellulases that facilitate degradation of the plant cell wall and are important in hyphal penetration; however, the species examined in this study lack the requisite genes for the complete saccharification of these carbohydrates for use as a carbon source. Genes encoding for xylan, xyloglucan, (galacto)(gluco)mannan and cutin degradation were absent or infrequent in Pythium spp.. Comparative analyses of predicted CAZymes in oomycetes indicated distinct evolutionary histories. Furthermore, CAZyme gene families among Pythium spp. were not uniformly distributed in the genomes, suggesting independent gene loss events, reflective of the polyphyletic relationships among some of the species.

The Comprehensive Phytopathogen Genomics Resource: a web-based resource for data-mining plant pathogen genomes.

The Comprehensive Phytopathogen Genomics Resource (CPGR) provides a web-based portal for plant pathologists and diagnosticians to view the genome and trancriptome sequence status of 806 bacterial, fungal, oomycete, nematode, viral and viroid plant pathogens. Tools are available to search and analyze annotated genome sequences of 74 bacterial, fungal and oomycete pathogens. Oomycete and fungal genomes are obtained directly from GenBank, whereas bacterial genome sequences are downloaded from the A Systematic Annotation Package (ASAP) database that provides curation of genomes using comparative approaches. Curated lists of bacterial genes relevant to pathogenicity and avirulence are also provided. The Plant Pathogen Transcript Assemblies Database provides annotated assemblies of the transcribed regions of 82 eukaryotic genomes from publicly available single pass Expressed Sequence Tags. Data-mining tools are provided along with tools to create candidate diagnostic markers, an emerging use for genomic sequence data in plant pathology. The Plant Pathogen Ribosomal DNA (rDNA) database is a resource for pathogens that lack genome or transcriptome data sets and contains 131 755 rDNA sequences from GenBank for 17 613 species identified as plant pathogens and related genera. Database URL: http://cpgr.plantbiology.msu.edu.

Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire.

BACKGROUND: Pythium ultimum is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on a broad range of crop and ornamental species. RESULTS: The P. ultimum genome (42.8 Mb) encodes 15,290 genes and has extensive sequence similarity and synteny with related Phytophthora species, including the potato blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86% of genes, with detectable differential expression of suites of genes under abiotic stress and in the presence of a host. The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions, although, surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes. A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host-specific oomycete species. Although we observed a high degree of orthology with Phytophthora genomes, there were novel features of the P. ultimum proteome, including an expansion of genes involved in proteolysis and genes unique to Pythium. We identified a small gene family of cadherins, proteins involved in cell adhesion, the first report of these in a genome outside the metazoans. CONCLUSIONS: Access to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage-specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae.