Development and evaluation of real-time quantitative PCR assays for detection of Phellinus noxius causing brown root rot disease.

Brown root rot disease (BRRD) is a highly destructive tree disease. Early diagnosis of BRRD has been challenging because the first symptoms and signs are often observed after extensive tissue colonization. Existing molecular detection methods, all based on the internal transcribed spacer (ITS) region, were developed without testing against global Phellinus noxius isolates, other wood decay fungi, or host plant tissues. This study developed SYBR Green real-time quantitative PCR (qPCR) assays for P. noxius. The primer pair Pn_ITS_F/Pn_ITS_R targets the ITS, and the primer pair Pn_NLR_F/Pn_NLR_R targets a P. noxius-unique group of homologous genes identified through a comparative genomics analysis. The homologous genes belong to the nucleotide-binding-oligomerization-domain-like receptor (NLR) superfamily. The new primer pairs and a previous primer pair G1F/G1R were optimized for qPCR conditions and tested for specificity using 61 global P. noxius isolates, five other Phellinus species, and 22 non-Phellinus wood decay fungal species. While all three primer pairs could detect as little as 100 fg (about 2.99 copies) of P. noxius genomic DNA, G1F/G1R had the highest specificity and Pn_NLR_F/Pn_NLR_R had the highest efficiency. To avoid false positives, the cutoff Cq values were determined as 34 for G1F/G1R, 29 for Pn_ITS_F/Pn_ITS_R, and 32 for Pn_NLR_F/Pn_NLR_R. We further validated these qPCR assays using Ficus benjamina seedlings artificially inoculated with P. noxius, six tree species naturally infected by P. noxius, rhizosphere soil, and bulk soil. The newly developed qPCR assays provide sensitive detection and quantification of P. noxius, which is useful for long-term monitoring of BRRD status.

NICU Language, Everyday Ethics, and Giving Better News: Optimizing Discussions about Disability with Families.

The Neonatal Intensive Care Unit (NICU) has a language and culture that is its own. For professionals, it is a place of intense and constant attention to microdetails and cautious optimism. For parents, it is a foreign place with a new and unique language and culture. It is also the setting in which they are introduced to their child and parenthood for this child. This combination has been referred to as an emotional cauldron. The neonatal ethics literature mainly examines complex ethical dilemmas about withholding/drawing life sustaining interventions for fragile children. Rarely are everyday ethics or mundane ethics discussed. Microethics describe the mundane, discrete moments that occur between patients/families and clinicians. A key piece of these microethics is the language used to discuss patient care. Perception of prognoses, particularly around long-term neurodevelopmental outcome, is shaped with the language used. Despite this, clinicians in the NICU often have no specific training in the long-term neurodevelopment outcomes that they discuss. This paper focuses on the microethics of language used to discuss long-term neurodevelopmental outcomes, the developmental neuroscience behind language processing, and offers recommendations for more accurate and improved communication around long-term outcomes with families with critically ill neonates.

Distribution of Cronartium x flexili, an interspecific hybrid of two fungal tree rust pathogens, in subalpine forest ecosystems of western USA.

Interspecific hybridization plays a key role in the evolution of novel fungal pathogens, and when it occurs between native and invasive species, can lead to potentially serious consequences. In this study, we examined the temporal and spatial distribution of a recently detected hybrid (Cronartium x flexili) of two tree pathogens, invasive to North America Cronartium ribicola and native Cronartium comandrae. In total, 726 and 1452 aecia from 178 Pinus contorta ssp. latifolia and 357 Pinus flexilis trees were collected from 26 sites in four national forests in 2019-2021. Using morphological and molecular analyses, 71 aecia collected from 25 P. flexilis trees had intermediate morphology and contained heterozygous SNPs in two genomic regions. Population analyses revealed the presence of multiple hybrid genotypes randomly distributed among sites and years. No aecia from P. contorta ssp. latifolia were identified as hybrids suggesting unidirectional gene flow from native C. comandrae to invasive C. ribicola. Aeciospores from 2 hybrid aecia produced urediniospores on Ribes nigrum. Overall, these results suggest that, even though low in frequency, C. x flexili is persistent in the region and has pathogenic potential. Hybrid expansion into the large range of susceptible pines could have cascading impacts on forest health.

Genomic and Transcriptomic Comparisons of the Twig Blight Pathogen, Passalora sequoiae, with Mycosphaerellaceae Foliar and Conifer Pathogens.

Passalora sequoiae is a foliar pathogen to conifer tree species. In this study, we conducted whole-genome and transcriptome analyses on isolates of P. sequoiae collected from symptomatic Leyland cypress leaves from a Christmas tree farm in Mississippi. The objectives for this research were to elucidate the pathogenicity mechanisms of P. sequoiae by characterizing the genome and transcriptome and possibly identify unique and shared predicted genes in comparison with non-conifer/canker and foliar pathogens in the family Mycosphaerellaceae. P. sequoiae was found to be similar to other foliar Mycosphaerellaceae pathogens and likely represents a hemibiotrophic lifestyle based on comparisons across pathogens. The genome and in planta transcriptome highlighted some unique features of P. sequoiae: the significant presence of chitin synthases and fructose-degrading carbohydrate-degrading enzymes, trans-AT PKS genes, and antibiotic gene clusters that were unique to P. sequoiae compared with the other Mycosphaerellaceae species genomes. Several transcripts that were highly expressed in planta were identified as effectors, yet the functions were not characterized. These targets provide ample resources to continue to characterize pathogen-conifer host interactions in conifer foliar pathogens. Furthermore, this research helps build genomic resources for an important plant pathogen on Leyland cypress that will further our ability to develop novel management practices that could begin with breeding for resistance.

Population genomic analysis of an emerging pathogen Lonsdalea quercina affecting various species of oaks in western North America.

Understanding processes leading to disease emergence is important for effective disease management and prevention of future epidemics. Utilizing whole genome sequencing, we studied the phylogenetic relationship and diversity of two populations of the bacterial oak pathogen Lonsdalea quercina from western North America (Colorado and California) and compared these populations to other Lonsdalea species found worldwide. Phylogenetic analysis separated Colorado and California populations into two Lonsdalea clades, with genetic divergence near species boundaries, suggesting long isolation and populations that differ in genetic structure and distribution and possibly their polyphyletic origin. Genotypes collected from different host species and habitats were randomly distributed within the California cluster. Most Colorado isolates from introduced planted trees, however, were distinct from three isolates collected from a natural stand of Colorado native Quercus gambelii, indicating cryptic population structure. The California identical core genotypes distribution varied, while Colorado identical core genotypes were always collected from neighboring trees. Despite its recent emergence, the Colorado population had higher nucleotide diversity, possibly due to its long presence in Colorado or due to migrants moving with nursery stock. Overall, results suggest independent pathogen emergence in two states likely driven by changes in host-microbe interactions due to ecosystems changes. Further studies are warranted to understand evolutionary relationships among L. quercina from different areas, including the red oak native habitat in northeastern USA.

The role of stress factors in severity of Cytospora plurivora in greenhouse and field plantings of 13 peach (Prunus persica) cultivars.

Understanding the host-pathogen-environmental interactions in a pathosystem is essential for management of diseases and diminished crop yields. Abiotic stressors such as cold damage, water deficit, and high pH soils can be major limiting factors to tree fruit production. Along with decreased yields, these abiotic factors can have direct implications for disease severity within orchards. Cytospora plurivora is a ubiquitous fungal canker pathogen in western Colorado, USA and is a major focus in integrated pest management strategies. This research evaluated the influence of biotic and abiotic stress factors on peach tree health. Thirteen peach cultivars were placed under abiotic stress and inoculated with C. plurivora in greenhouse and field conditions. Under deficit irrigation, C. plurivora infections were significantly larger and more severe in both the greenhouse and field trials when compared with those under the full-irrigation controls. In controlled greenhouse conditions, a positive correlation between lesion size and water potential was evident, but no trend of cultivar tolerance was observed. Furthermore, increase in irrigation water pH, through additions of sodium carbonate and bicarbonate, in the greenhouse trials resulted in decreased leaf water potentials and increased pathogen necrotic tissue volumes (mm3). In field trials, there was no positive relationship between lesion size and water potential; trees with the most negative water potentials had the smallest lesions sizes that did not correspond to cultivar, suggesting that other abiotic or biotic factors may be shielding water stressed trees from increased pathogen aggression. This research highlights the importance of proper irrigation and soil pH management as tools for the management of Cytospora canker in peach orchards.

First draft genome and transcriptome of Cercosporidium personatum, causal agent of late leaf spot disease of peanut.

OBJECTIVE: Two main fungal leaf spot diseases occur in peanut, namely early leaf spot (ELS) and late leaf spot (LLS), these cause a yearly average of $44 million losses. Limited genetic information, 3534 bp of sequencing, exists about the causal agent of LLS, Cercosporidium personatum (syn. Nothopassalora personata, syn. Phaeoisariopsis personata). The extremely slow growth of this fungus, approximately 1 cm colony in 6 months, and challenges in nucleic acid extractions have hindered research on LLS. Our goal in this work is to provide a reference genome for research on this pathogen. RESULTS: Whole genome and transcriptome sequencing of the LLS fungus were obtained. A total of 233,542,110 reads of the genome were de novo assembled resulting in 1061 scaffolds, and estimated genome size 27,597,787 bp. RNA sequencing resulted in 11,848,198 reads that were de novo assembled into 13,343 contigs. Genome annotation resulted in 10,703 putative genes. BUSCO analysis of the genome and annotation resulted in 91.1% and 89.5% completeness, respectively. Phylogenetic dendrograms for 5442 bp and 4401 bp of RNA Polymerase II largest and second largest subunits, and for 5474 bp of the ribosomal RNA cistron of C. personatum are presented in relation to closely related fungi.

Genomic Comparisons of Two Armillaria Species with Different Ecological Behaviors and Their Associated Soil Microbial Communities.

Armillaria species show considerable variation in ecological roles and virulence, from mycorrhizae and saprophytes to important root pathogens of trees and horticultural crops. We studied two Armillaria species that can be found in coniferous forests of northwestern USA and southwestern Canada. Armillaria altimontana not only is considered as a weak, opportunistic pathogen of coniferous trees, but it also appears to exhibit in situ biological control against A. solidipes, formerly North American A. ostoyae, which is considered a virulent pathogen of coniferous trees. Here, we describe their genome assemblies and present a functional annotation of the predicted genes and proteins for the two Armillaria species that exhibit contrasting ecological roles. In addition, the soil microbial communities were examined in association with the two Armillaria species within a 45-year-old plantation of western white pine (Pinus monticola) in northern Idaho, USA, where A. altimontana was associated with improved tree growth and survival, while A. solidipes was associated with reduced growth and survival. The results from this study reveal a high similarity between the genomes of the beneficial/non-pathogenic A. altimontana and pathogenic A. solidipes; however, many relatively small differences in gene content were identified that could contribute to differences in ecological lifestyles and interactions with woody hosts and soil microbial communities.

Nutritional Profile and Ecological Interactions of Yeast Symbionts Associated with North American Spruce Beetle (Dendroctonus rufipennis).

To better understand functional ecology of bark beetle-microbial symbioses, we characterized yeast associates of North American spruce beetle (Dendroctous rufipennis Kirby) across populations. Seven yeast species were detected; Wickerhamomyces canadensis (Wickerham) Kurtzman et al. (Sachharomycetales: Saccharomycetaceae) was the most common (74% of isolates) and found in all populations. Isolates of W. canadensis were subsequently tested for competitive interactions with symbiotic (Leptographium abietinum, = Grosmannia abietina) and pathogenic (Beauvaria bassiana) filamentous fungi, and isolates were nutritionally profiled (protein and P content). Exposure to yeast headspace emissions had isolate-dependent effects on colony growth of symbiotic and pathogenic fungi; most isolates of W. canadensis slightly inhibited growth rates of symbiotic (L. abietinum, mean effect: - 4%) and entomopathogenic (B. bassiana, mean effect: - 6%) fungi. However, overall variation was high (range: - 35.4 to + 88.6%) and some yeasts enhanced growth of filamentous fungi whereas others were consistently inhibitory. The volatile 2-phenylethanol was produced by W. canadensis and synthetic 2-phenylethanol reduced growth rates of both L. abietinum and B. bassiana by 36% on average. Mean protein and P content of Wickerhamomyces canadensis cultures were 0.8% and 7.2%, respectively, but isolates varied in nutritional content and protein content was similar to that of host tree phloem. We conclude that W. canadensis is a primary yeast symbiont of D. rufipennis in the Rocky Mountains and emits volatiles that can affect growth of associated microbes. Wickerhamomyces canadensis isolates vary substantially in limiting nutrients (protein and P), but concentrations are less than reported for the symbiotic filamentous fungus L. abietinum.

Translocation of Fungicides and Their Efficacy in Controlling Phellinus noxius, the Cause of Brown Root Rot Disease.

Brown root rot disease (BRRD), caused by Phellinus noxius, is an important tree disease in tropical and subtropical areas. To improve chemical control of BRRD and deter emergence of fungicide resistance in P. noxius, this study investigated control efficacies and systemic activities of fungicides with different modes of action. Fourteen fungicides with 11 different modes of action were tested for inhibitory effects in vitro on 39 P. noxius isolates from Taiwan, Hong Kong, Malaysia, Australia, and Pacific Islands. Cyproconazole, epoxiconazole, and tebuconazole (Fungicide Resistance Action Committee [FRAC] 3, target-site G1) inhibited colony growth of P. noxius by 99.9 to 100% at 10 ppm and 97.7 to 99.8% at 1 ppm. The other effective fungicide was cyprodinil + fludioxonil (FRAC 9 + 12, target-site D1 + E2), which showed growth inhibition of 96.9% at 10 ppm and 88.6% at 1 ppm. Acropetal translocation of six selected fungicides was evaluated in bishop wood (Bischofia javanica) seedlings by immersion of the root tips in each fungicide at 100 ppm, followed by liquid or gas chromatography tandem mass spectrometry analyses of consecutive segments of root, stem, and leaf tissues at 7 and 21 days posttreatment. Bidirectional translocation of the fungicides was also evaluated by stem injection of fungicide stock solutions. Cyproconazole and tebuconazole were the most readily absorbed by roots and efficiently transported acropetally. Greenhouse experiments suggested that cyproconazole, tebuconazole, and epoxiconazole have a slightly higher potential for controlling BRRD than mepronil, prochloraz, and cyprodinil + fludioxonil. Because all tested fungicides lacked basipetal translocation, soil drenching should be considered instead of trunk injection for their use in BRRD control.

Phylogenetic analyses allow species-level recognition of Leptographium wageneri varieties that cause black stain root disease of conifers in western North America.

Leptographium wageneri is a native fungal pathogen in western North America that causes black stain root disease (BSRD) of conifers. Three host-specialized varieties of this pathogen were previously described: L. wageneri var. wageneri on pinyon pines (Pinus monophylla and P. edulis); L. wageneri var. ponderosum, primarily on hard pines (e.g., P. ponderosa, P. jeffreyi); and L. wageneri var. pseudotsugae on Douglas-fir (Pseudotsuga menziesii). Morphological, physiological, and ecological differences among the three pathogen varieties have been previously determined; however, DNA-based characterization and analyses are needed to determine the genetic relationships among these varieties. The objective of this study was to use DNA sequences of 10 gene regions to assess phylogenetic relationships among L. wageneri isolates collected from different hosts. The multigene phylogenetic analyses, based on maximum likelihood and Bayesian inference, strongly supported species-level separation of the three L. wageneri varieties. These results, in conjunction with previously established phenotypic differences, support the elevation of L. wageneri var. ponderosum and L. wageneri var. pseudotsugae to the species level as L. ponderosum comb. nov. and L. pseudotsugae comb. nov., respectively, while maintaining L. wageneri var. wageneri as Leptographium wageneri. Characterization of the three Leptographium species, each with distinct host ranges, provides a baseline to further understand the ecological interactions and evolutionary relationships of these forest pathogens, which informs management of black stain root disease.

Development of PCR-based markers for the identification and detection of Lophodermella needle cast pathogens on Pinus contorta var. latifolia and P. flexilis.

Morphological similarities and fastidious development of increasingly emerging fungal needle pathogens impede accurate disease diagnosis and early detection. This study analyzed the specificity and sensitivity of polymerase chain reaction (PCR)-based markers developed for emerging needle cast pathogens Lophodermella concolor and L. montivaga co-occurring on Pinus contorta var. latifolia, and Bifusella linearis and L. arcuata on P. flexilis. To design primers, we utilized sequences of the internal transcribed spacer (ITS) region and single-copy gene (RH_2175) of the TCP-1/cpn60 chaperonin family searched through genomes of related species. In addition to the DNA of target and non-target fungal species that were used for primer assays, environmental samples with next generation sequencing data were used to evaluate primer sensitivity. Direct amplification using ITS primer pairs generated 248-260 bp amplicons and successfully differentiated the needle pathogens used in this study. Nested amplification of single-copy gene RH_2175 primer pairs which produced 409-527 bp amplicons detected Rhytismataceae species and discriminated both Lophodermella pathogens on P. contorta var. latifolia, respectively. While ITS-based primers had higher sensitivity than the 2175-based primers, both primer sets for L. concolor and L. montivaga detected their respective pathogens in asymptomatic and symptomatic needles. These molecular tools can help monitor and assess needle diseases for forest management and phytosanitary regimes.

Transitions of foliar mycobiota community and transcriptome in response to pathogenic conifer needle interactions.

Profiling the host-mycobiota interactions in healthy vs. diseased forest ecosystems helps understand the dynamics of understudied yet increasingly important threats to forest health that are emerging due to climate change. We analyzed the structural and functional changes of the mycobiota and the responses of Pinus contorta in the Lophodermella needle cast pathosystem through metabarcoding and metatranscriptomics. When needles transitioned from asymptomatic to symptomatic, dysbiosis of the mycobiota occurred, but with an enrichment of Lophodermella pathogens. Many pathogenicity-related genes were highly expressed by the mycobiota at the necrotrophic phase, showing an active pathogen response that are absent in asymptomatic needles. This study also revealed that Lophodermella spp. are members of a healthy needle mycobiota that have latent lifestyles suggesting that other pine needle pathogens may have similar biology. Interestingly, Pinus contorta upregulated defense genes in healthy needles, indicating response to fungal recognition, while a variety of biotic and abiotic stresses genes were activated in diseased needles. Further investigation to elucidate the possible antagonistic interplay of other biotic members leading to disease progression and/or suppression is warranted. This study provides insights into microbial interactions in non-model pathosystems and contributes to the development of new forest management strategies against emerging latent pathogens.

Long evolutionary history of an emerging fungal pathogen of diverse tree species in eastern Asia, Australia and the Pacific Islands.

Emerging plant pathogens have been increasing exponentially over the last century. To address this issue, it is critical to determine whether these pathogens are native to ecosystems or have been recently introduced. Understanding the ecological and evolutionary processes fostering emergence can help to manage their spread and predict epidemics/epiphytotics. Using restriction site-associated DNA sequencing data, we studied genetic relationships, pathways of spread and the evolutionary history of Phellinus noxius, an emerging root-rotting fungus of unknown origin, in eastern Asia, Australia and the Pacific Islands. We analysed patterns of genetic variation using Bayesian inference, maximum-likelihood phylogeny, population splits and mixtures measuring correlations in allele frequencies and genetic drift, and finally applied coalescent-based theory using Approximate Bayesian computation (ABC) with supervised machine learning. Population structure analyses revealed five genetic groups with signatures of complex recent and ancient migration histories. The most probable scenario of ancient pathogen spread is movement from an unsampled population to Malaysia and the Pacific Islands, with subsequent spread to Taiwan and Australia. Furthermore, ABC analyses indicate P. noxius spread occurred thousands of generations ago, contradicting previous assumptions that this pathogen was recently introduced to multiple geographical regions. Our results suggest that recent emergence of P. noxius in eastern Asia, Australia and the Pacific Islands has probably been driven by anthropogenic and natural disturbances, such as deforestation, land-use change, severe weather events and/or introduction of exotic plants. This study provides a novel example of applying genome-wide allele frequency data to unravel the dynamics of pathogen emergence under changing ecosystem conditions.

Genetic and Phenotypic Characterization of the Fungal Pathogen Cytospora plurivora from Western Colorado Peach Orchards and the Development of a ddPCR Assay for Detection and Quantification.

Cytospora canker is one of the most important diseases affecting peach production in Colorado, yet previous efforts to characterize Cytospora species diversity in Colorado have relied exclusively on morphological traits. Recently, several new Cytospora species were described from peach orchards within the United States using molecular and morphological data, prompting the need to reexamine Cytospora spp. present on peach trees in Colorado. A total of 137 isolates of Cytospora spp. were collected from eight orchards in western Colorado. Isolates were sequenced at the internal transcribed spacer region and elongation factor 1-α and assessed with reference sequences in phylogenetic analyses. All isolates from western Colorado peach trees resolved with the newly described Cytospora plurivora. In addition to molecular characterization, temperature growth and virulence assays were conducted to assess phenotypic variation among the isolates from western Colorado. Variation across isolates was found both in growth at different temperatures and in virulence. Ancestral state reconstruction analyses resolved the most virulent (and most often collected) haplotypes together in a well-supported clade from which a single monophyletic origin of high virulence can be inferred. Finally, a droplet digital PCR assay was developed for use in ongoing and future studies to detect and quantify C. plurivora from field and laboratory samples.

Race, language, and neighborhood predict high-risk preterm Infant Follow Up Program participation.

OBJECTIVES: Infant Follow Up Programs (IFUPs) provide developmental surveillance for preterm infants after hospital discharge but participation is variable. We hypothesized that infants born to Black mothers, non-English speaking mothers, and mothers who live in "Very Low" Child Opportunity Index (COI) neighborhoods would have decreased odds of IFUP participation. STUDY DESIGN: There were 477 infants eligible for IFUP between 1/1/2015 and 6/6/2017 from a single large academic Level III NICU. Primary outcome was at least one visit to IFUP. We used multivariable logistic regression to identify factors associated with IFUP participation. RESULT: Two hundred infants (41.9%) participated in IFUP. Odds of participation was lower for Black compared to white race (aOR 0.43, p = 0.03), "Very Low" COI compared to "Very High" (aOR 0.39, p = 0.02) and primary non-English speaking (aOR 0.29, p = 0.01). CONCLUSION: We identified disparities in IFUP participation. Further study is needed to understand underlying mechanisms to develop targeted interventions for reducing inequities.

Soil fertility interactions with Sinorhizobium-legume symbiosis in a simulated Martian regolith; effects on nitrogen content and plant health.

Due to increasing population growth and declining arable land on Earth, astroagriculture will be vital to terraform Martian regolith for settlement. Nodulating plants and their N-fixing symbionts may play a role in increasing Martian soil fertility. On Earth, clover (Melilotus officinalis) forms a symbiotic relationship with the N-fixing bacteria Sinorhizobium meliloti; clover has been previously grown in simulated regolith yet without bacterial inoculation. In this study, we inoculated clover with S. meliloti grown in potting soil and regolith to test the hypothesis that plants grown in regolith can form the same symbiotic associations as in soils and to determine if greater plant biomass occurs in the presence of S. meliloti regardless of growth media. We also examined soil NH4 concentrations to evaluate soil augmentation properties of nodulating plants and symbionts. Greater biomass occurred in inoculated compared to uninoculated groups; the inoculated average biomass in potting mix and regolith (2.23 and 0.29 g, respectively) was greater than the uninoculated group (0.11 and 0.01 g, respectively). However, no significant differences existed in NH4 composition between potting mix and regolith simulant. Linear regression analysis results showed that: i) symbiotic plant-bacteria relationships differed between regolith and potting mix, with plant biomass positively correlated to regolith-bacteria interactions; and, ii) NH4 production was limited to plant uptake yet the relationships in regolith and potting mix were similar. It is promising that plant-legume symbiosis is a possibility for Martian soil colonization.

Molecular characterization and phylogenetic analyses of Lophodermella needle pathogens (Rhytismataceae) on Pinus species in the USA and Europe.

Increasing prevalence of conifer needle pathogens globally have prompted further studies on pathogen identification and a better understanding of phylogenetic relationships among needle pathogens. Several Lophodermella species can be aggressive pathogens causing needle cast in natural pine forests in the USA and Europe. However, their relationships with other Rhytismataceae species have historically been based on similarities of only limited phenotypic characters. Currently, no molecular studies have been completed to elucidate their relationships with other Lophodermella needle pathogens. This study collected and sequenced three gene loci, namely: internal transcribed spacer, large ribosomal subunit, and translation elongation factor 1-alpha, from five Lophodermella needle pathogens from North America (L. arcuata, L. concolor, L. montivaga) and Europe (L. conjuncta and L. sulcigena) to distinguish phylogeny within Rhytismatacaeae, including Lophophacidium dooksii. Phylogenetic analyses of the three loci revealed that all but L. conjuncta that were sampled in this study consistently clustered in a well-supported clade within Rhytismataceae. The multi-gene phylogeny also confirmed consistent nesting of L. dooksii, a needle pathogen of Pinus strobus, within the clade. Potential synapomorphic characters such as ascomata position and ascospore shape for the distinct clade were also explored. Further, a rhytismataceous species on P. flexilis that was morphologically identified as L. arcuata was found to be unique based on the sequences at the three loci. This study suggests a potential wider range of host species within the genus and the need for genetic characterization of other Lophodermella and Lophophacidium species to provide a higher phylogenetic resolution.

Desarmillaria caespitosa, a North American vicariant of D. tabescens.

Desarmillaria caespitosa, a North American vicariant species of European D. tabescens, is redescribed in detail based on recent collections from the USA and Mexico. This species is characterized by morphological features and multilocus phylogenetic analyses using portions of nuc rDNA 28S (28S), translation elongation factor 1-alpha (tef1), the second largest subunit of RNA polymerase II (rpb2), actin (act), and glyceraldehyde-3-phosphate dehydrogenase (gpd). A neotype of D. caespitosa is designated here. Morphological and genetic differences between D. caespitosa and D. tabescens were identified. Morphologically, D. caespitosa differs from D. tabescens by having wider basidiospores, narrower cheilocystidia, which are often irregular or mixed (regular, irregular, or coralloid), and narrower caulocystidia. Phylogenetic analyses of five independent gene regions show that D. caespitosa and D. tabescens are separated by nodes with strong support. The new combination, D. caespitosa, is proposed.

Whole genome analysis of the koa wilt pathogen (Fusarium oxysporum f. sp. koae) and the development of molecular tools for early detection and monitoring.

BACKGROUND: Development and application of DNA-based methods to distinguish highly virulent isolates of Fusarium oxysporum f. sp. koae [Fo koae; cause of koa wilt disease on Acacia koa (koa)] will help disease management through early detection, enhanced monitoring, and improved disease resistance-breeding programs. RESULTS: This study presents whole genome analyses of one highly virulent Fo koae isolate and one non-pathogenic F. oxysporum (Fo) isolate. These analyses allowed for the identification of putative lineage-specific DNA and predicted genes necessary for disease development on koa. Using putative chromosomes and predicted gene comparisons, Fo koae-exclusive, virulence genes were identified. The putative lineage-specific DNA included identified genes encoding products secreted in xylem (e. g., SIX1 and SIX6) that may be necessary for disease development on koa. Unique genes from Fo koae were used to develop pathogen-specific PCR primers. These diagnostic primers allowed target amplification in the characterized highly virulent Fo koae isolates but did not allow product amplification in low-virulence or non-pathogenic isolates of Fo. Thus, primers developed in this study will be useful for early detection and monitoring of highly virulent strains of Fo koae. Isolate verification is also important for disease resistance-breeding programs that require a diverse set of highly virulent Fo koae isolates for their disease-screening assays to develop disease-resistant koa. CONCLUSIONS: These results provide the framework for understanding the pathogen genes necessary for koa wilt disease and the genetic variation of Fo koae populations across the Hawaiian Islands.