Publications by authors named "Jo Anne Crouch"

46 Publications

, sp. nov., a new downy mildew species infecting the endangered Hawaiian plant var. .

Mycologia 2021 Mar 18:1-10. Epub 2021 Mar 18.

Mycology and Nematology Genetic Diversity and Biology Laboratory, Agricultural Research Service, United States Department of Agriculture, 10300 Baltimore Avenue, Beltsville, Maryland 20705.

var. is an endangered native Hawaiian plant, and part of the recovery plan includes repopulation using plants grown in a nursery. However, disease pressure from downy mildew is hindering repopulation efforts. The organism associated with the downy mildew was determined to be a species with brown, ellipsoid conidia measuring 21 by 16 µm on average, which was morphologically different from validly described species of that infect species, but it was morphologically similar to the invalidly published species (Art. 40.1). Comparison of mitochondrial cytochrome oxidase subunit I (1), mitochondrial cytochrome oxidase subunit II (2), nuclear internal transcribed spacer (ITS), and nuclear 28S rRNA D1-D2 (28S) loci revealed the unknown to be molecularly divergent from and , but very similar to from , the type host of . Phylogenetic trees inferred with maximum likelihood and Bayesian inference from a concatenated alignmaent and individual gene trees confirmed the divergence of the unknown from and and its similarity to . However, attempts to inoculate with the strain from var. were unsuccessful, which, in conjunction with divergence in ITS, suggests that the unknown is specific to var. . Herein, the strain on var. is described as the new species based on morphology, molecular phylogenetics, and host specificity. In addition, is described here to honor Nina Ivanova Gaponenko on the basis of her description of .
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http://dx.doi.org/10.1080/00275514.2021.1872869DOI Listing
March 2021

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

Biosci Rep 2021 Mar;41(3)

United States Department of Agriculture (USDA), Agricultural Research Service (ARS), Foreign Disease-Weed Science Research Unit, Fort Detrick, MD, U.S.A.

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.
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http://dx.doi.org/10.1042/BSR20203544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7937907PMC
March 2021

First report: Co-infection of (sweetbox) by and causes a foliar disease of sweetbox in Pennsylvania.

Plant Dis 2020 Dec 1. Epub 2020 Dec 1.

USDA-ARS Beltsville Area, 56741, Mycology and Nematology Genetic Diversity and Biology Lab, Beltsville, Maryland, United States;

Sweetbox () are high value ornamental shrubs susceptible to disease caused by () and () (Malapi-Wight et al. 2016; Salgado-Salazar et al. 2019). In July 2018, 18-month old sweetbox with leaf spots and defoliation were observed in a residential landscape in Lancaster County, Pennsylvania. Small tan leaf spots grew to cover half of the leaf, developing a concentric banding with dark brown rings and a yellow halo (Sup. Doc. 1: Sup. Fig. 1). The symptoms agreed with those of disease of sweetbox reported from Washington D.C. (Salgado-Salazar et al. 2019). Diseased plants were located ~1.5 m from with boxwood blight. Morphological and genetic characterization of isolated fungi and pathogenicity tests followed Salgado-Salazar et al. (2019) (Sup. Doc. 2). White to salmon pink spore masses developed on the abaxial leaf surface after humid chamber incubation. Two distinct fungal cultures were recovered (JAC 18-61, JAC 18-79) on potato dextrose agar (Fisher Scientific, Pittsburg, PA). JAC 18-61 presented cultural and morphological characteristics as described for (Crous et al. 2002). JAC 18-79 produced flat, filamentous, light salmon colonies with tan centers and white filiform borders containing pale pink sporodochia, verticillate and simple conidiophores (x̄: 61.8 ± 20.12 µm, N = 20) with lateral, cylindrical phialides (x̄ = 18.1 ± 5.83 x 2.4 ± 0.7 µm, N = 20), and ellipsoid, hyaline conidia without septa (x̄ = 15.2 ± 1.9 x 3.3 ± 0.7 µm, N = 20). Sexual structures and chlamydospores were not observed. The characteristics of JAC 18-79 agree with those reported for (Salgado-Salazar et al. 2019). Bidirectional sequencing of the ITS, beta-TUB, and RPB1 and RPB2 regions was performed as described (Salgado-Salazar et al. 2019). BLASTn comparisons against NCBI GenBank revealed JAC 18-61 sequences (MT318150 and MT328399) shared 100% identity with sequences (JX535321 and JX535307 from isolate CB002). Sequences from JAC 18-79 (MT318151, MT341237 to MT341239) were 100% identical to sequences (MH892596, MH936775, MH936703 from isolate JAC 16-20 and JF832909, isolate CBS 128674). The genome of JAC 18-79 was sequenced and yielded an assembly of 26.3 Mb (204 contigs > 1000 bases, N50 = 264.3 kb, 92x coverage, JABAHV0000000000) that contained the MAT1-2 mating-type idiomorph and shared 98.9% similarity with BPI910731. Isolate JAC 18-61 () caused lesions on wounded and unwounded sweetbox and boxwood leaves (Sup. Table 1). In general, JAC 18-79 () infected only wounded leaves of both hosts; however, in one trial, one unwounded sweetbox and two unwounded boxwood plants developed lesions, possibly due to the presence of natural wounds. Control plants did not develop symptoms. These results diverge to some degree from previous reports of infecting unwounded sweetbox and not infecting wounded boxwood (Salgado-Salazar et al. 2019). These results indicate that virulence variation among isolates might occur. Plating of symptomatic tissue and examination of spores fulfilled Koch's postulates for both pathogens. To our knowledge, this is the first report of blight on sweetbox in Pennsylvania, and the second U.S. report of the disease. This is also the first report of co-infection of and on diseased sweetbox foliage. Given the capacity of to infect both sweetbox and boxwood, inspection for on both hosts is advisable.
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http://dx.doi.org/10.1094/PDIS-06-20-1198-PDNDOI Listing
December 2020

Real-Time PCR Detection of spp., the Causal Agents of Dollar Spot in Turfgrasses.

Plant Dis 2020 Dec 15;104(12):3118-3123. Epub 2020 Oct 15.

Department of Plant Biology, Rutgers University, New Brunswick, NJ 08901, U.S.A.

Dollar spot is one of the most economically important diseases of turfgrasses. Recent taxonomic revisions have placed the dollar spot fungal pathogens in the new genus , with five species described. The main goal of this study was to develop a quantitative real-time PCR (qPCR) molecular detection assay based on the internal transcribed spacer (ITS) of the ribosomal RNA genes to quantify the abundance of spp. from environmental (field) samples. The qPCR assay was able to detect isolates of the four tested spp. but did not cross react with nontarget fungi, including closely related taxa, other turfgrass pathogens, or other fungal species commonly isolated from turfgrass. The assay is capable of detecting as little as 38.0 fg (3.8 × 10 g) of genomic DNA in 3 h. The qPCR assay detected spp. in both symptomatic and asymptomatic creeping bentgrass () foliar tissue. spp. were rarely detected in the thatch or soil, indicating that these pathogens are not widely distributed in these areas of the environment. The fact that the pathogen was detected in asymptomatic tissue suggests that creeping bentgrass may be able to tolerate a certain quantity of the pathogens in leaves before disease symptoms appear; however, further research is needed to validate this hypothesis.
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http://dx.doi.org/10.1094/PDIS-04-20-0726-REDOI Listing
December 2020

Population Genomics Trace Clonal Diversification and Intercontinental Migration of an Emerging Fungal Pathogen of Boxwood.

Phytopathology 2021 Jan 12;111(1):184-193. Epub 2020 Nov 12.

Mycology and Nematology Genetic Diversity and Biology Laboratory, United States Department of Agriculture-Agricultural Research Service, Beltsville, MD.

Boxwood blight was first documented in Europe, prior to its recent colonization of North America, where it continues to have significant negative impacts on the ornamental industry. Due to near genetic uniformity in the two sister species of fungal plant pathogens that cause boxwood blight, understanding historical disease emergence and predicting future outbreaks is limited. The goal of this research was to apply population genomics to understand the role of pathogen diversification and migration in disease emergence. Specifically, we tested whether the primary pathogen species has remained genetically isolated from its European-limited sister species , while diversifying into clonal lineages that have migrated among continents. Whole-genome sequencing identified 1,608 single-nucleotide polymorphisms (SNPs) in 67 isolates from four continents and 1,017 SNPs in 13 isolates from Europe. Interspecific genetic differentiation and an absence of shared polymorphisms indicated lack of gene flow between the sister species. Tests for intraspecific genetic structure in identified four genetic clusters, three of which corresponded to monophyletic phylogenetic clades. Comparison of evolutionary divergence scenarios among the four genetic clusters using approximate Bayesian computation indicated that the two genetic clusters currently found in the United States were derived from different sources, one from the first genetic cluster found in Europe and the second from an unidentified population. Evidence for multiple introductions of this pathogen into the United States and intercontinental migration indicates that future introductions are likely to occur and should be considered in plant disease quarantine regulation.
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http://dx.doi.org/10.1094/PHYTO-06-20-0219-FIDOI Listing
January 2021

Genome Resources for Seven Fungal Isolates That Cause Dollar Spot Disease in Turfgrass, Including and .

Plant Dis 2021 Mar 8;105(3):691-694. Epub 2021 Feb 8.

Center for Integrated Fungal Research, Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC 27695.

Fungi in the genus are widespread and destructive pathogens of grasses worldwide, and are best known as the causal agents of dollar spot disease in turfgrass. Here, we report genome assemblies of seven isolates, including ex-types of the two most widespread species, and . These datasets provide a valuable resource for ongoing studies of the dollar spot pathogens that include population diversity, host-pathogen interactions, marker development, and disease control.
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http://dx.doi.org/10.1094/PDIS-06-20-1296-ADOI Listing
March 2021

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

Phytopathology 2020 Nov 7;110(11):1845-1853. Epub 2020 Oct 7.

U.S. Department of Agriculture-Agricultural Research Service, Mycology and Nematology Genetic Diversity and Biology Laboratory, Beltsville, MD 20705.

Boxwood blight caused by and 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 populations prior to 2014 had a clonal structure, and only the idiomorph was observed. In this study, we examined 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 idiomorph typing. All isolates presented -specific alleles, indicating that is still absent in the U.S. states sampled. The presence of only the idiomorph and gametic linkage disequilibrium suggests the prevalence of asexual reproduction. The contemporary 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. 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.
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http://dx.doi.org/10.1094/PHYTO-04-20-0130-RDOI Listing
November 2020

Genome Sequence of the Chestnut Blight Fungus EP155: A Fundamental Resource for an Archetypical Invasive Plant Pathogen.

Phytopathology 2020 Jun 17;110(6):1180-1188. Epub 2020 Apr 17.

Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, U.S.A.

is the causal agent of chestnut blight, a fungal disease that almost entirely eliminated mature American chestnut from North America over a 50-year period. Here, we formally report the genome of EP155 using a Sanger shotgun sequencing approach. After finishing and integration with simple-sequence repeat markers, the assembly was 43.8 Mb in 26 scaffolds (L = 5; N = 4.0Mb). Eight chromosomes are predicted: five scaffolds have two telomeres and six scaffolds have one telomere sequence. In total, 11,609 gene models were predicted, of which 85% show similarities to other proteins. This genome resource has already increased the utility of a fundamental plant pathogen experimental system through new understanding of the fungal vegetative incompatibility system, with significant implications for enhancing mycovirus-based biological control.
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http://dx.doi.org/10.1094/PHYTO-12-19-0478-ADOI Listing
June 2020

Widespread Occurrence of a Pseudogene in .

Mycobiology 2020 23;48(1):44-50. Epub 2019 Nov 23.

Biology Department, Eastern Connecticut State University, Willimantic, CT, USA.

and . are two closely related fungal species responsible for boxwood blight disease of ornamental shrubs ( spp.) in the U.S. and Europe. A previous study has shown isolates of the latter species, which is restricted to Europe, to be less sensitive to tetraconazole, an azole fungicide. In this study, we have analyzed the paralogs for polymorphism in 26 genomes, representing geographically disparate populations of . ( = 19) and . ( = 7), from the U.S., Europe, Asia, and New Zealand. The presence of a pseudogene and lack of a functional paralog in all . genomes examined is a novel discovery for fungi and could have implications for the evolution of resistance to antifungal chemicals.
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http://dx.doi.org/10.1080/12298093.2019.1689600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048176PMC
November 2019

Prokaryotic taxa play keystone roles in the soil microbiome associated with woody perennial plants in the genus .

Ecol Evol 2019 Oct 26;9(19):11102-11111. Epub 2019 Aug 26.

Mycology and Nematology Genetic Diversity and Biology Laboratory U.S. Department of Agriculture, Agricultural Research Service Beltsville MD USA.

The microbiome associated with ornamental plants has largely been neglected, despite its potential for impacting plant health. This work characterized the composition, diversity, and microbial co-associations in the soil microbiome associated with species and cultivars of plant in the genus (common name boxwood), a group of woody perennial shrubs commonly used in residential landscapes and found in native ecosystems. Soil was collected from 82 individual curated boxwood accessions at the U.S. National Arboretum National Boxwood Collection. Amplicon libraries targeting archaea, bacteria, and fungi were generated and sequenced using the Illumina MiSeq platform. Identification of individual sequence variants resulted in 275 archaeal, 15,580 bacterial, and 7,525 fungal taxa. Neither spatial distance among samples nor association with different types of boxwood were significant predictors of soil microbiome composition. However, archaeal and bacterial diversity was significantly different in soil from distinct types of boxwood. Co-association networks indicated that archaea and bacteria show greater evidence of being keystone taxa than fungi. Overall, this work demonstrates the potential for targeting specific keystone taxa to shift the soil microbiome associated with these boxwood accessions and that planting different species or cultivars in the landscape may shift the diversity of prokaryotic microorganisms.
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http://dx.doi.org/10.1002/ece3.5614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6802073PMC
October 2019

Global distribution of mating types shows limited opportunities for mating across populations of fungi causing boxwood blight disease.

Fungal Genet Biol 2019 10 27;131:103246. Epub 2019 Jun 27.

United States Department of Agriculture, Agricultural Research Service, Mycology and Nematology Genetic Diversity and Biology Laboratory, Beltsville, MD, USA. Electronic address:

Boxwood blight is a disease threat to natural and managed landscapes worldwide. To determine mating potential of the fungi responsible for the disease, Calonectria pseudonaviculata and C. henricotiae, we characterized their mating-type (MAT) loci. Genomes of C. henricotiae, C. pseudonaviculata and two other Calonectria species (C. leucothoes, C. naviculata) were sequenced and used to design PCR tests for mating-type from 268 isolates collected from four continents. All four Calonectria species have a MAT locus that is structurally consistent with the organization found in heterothallic ascomycetes, with just one idiomorph per individual isolate. Mating type was subdivided by species: all C. henricotiae isolates possessed the MAT1-1 idiomorph, whereas all C. pseudonaviculata isolates possessed the MAT1-2 idiomorph. To determine the potential for divergence at the MAT1 locus to present a barrier to interspecific hybridization, evolutionary analysis was conducted. Phylogenomic estimates showed that C. henricotiae and C. pseudonaviculata diverged approximately 2.1 Mya. However, syntenic comparisons, phylogenetic analyses, and estimates of nucleotide divergence across the MAT1 locus and proximal genes identified minimal divergence in this region of the genome. These results show that in North America and parts of Europe, where only C. pseudonaviculata resides, mating is constrained by the absence of MAT1-1. In regions of Europe where C. henricotiae and C. pseudonaviculata currently share the same host and geographic range, it remains to be determined whether or not these two recently diverged species are able to overcome species barriers to mate.
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http://dx.doi.org/10.1016/j.fgb.2019.103246DOI Listing
October 2019

, Causal Agent of a New Foliar Blight Disease of .

Plant Dis 2019 Jun 8;103(6):1337-1346. Epub 2019 Apr 8.

1 Mycology and Nematology Genetic Diversity and Biology Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705.

Woody plants of the Buxaceae, including species of , , and , are widely grown evergreen shrubs and groundcovers. Severe leaf spot symptoms were observed on at the U.S. National Arboretum in Washington, DC, in 2016. Affected plants were growing adjacent to exhibiting Volutella blight symptoms. Fungi isolated from both hosts were identical based on morphology and multilocus phylogenetic analysis and were identified as (Nectriaceae, Hypocreales), causal agent of Volutella blight of species. Pathogenicity tests established that isolated from both hosts caused disease symptoms on and , but not on . Artificial inoculations with , causal agent of Volutella blight of , did not result in disease on or . Wounding enhanced infection by and on all hosts tested but was not required for disease development. Genome assemblies were generated for the Buxaceae pathogens that cause Volutella diseases: , . , and ; these ranged in size from 25.7 to 28.5 Mb. To our knowledge, this foliar blight of represents a new disease for this host and is capable of causing considerable damage to infected plants.
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http://dx.doi.org/10.1094/PDIS-09-18-1676-REDOI Listing
June 2019

Genome resources for the stem and bark canker pathogens Corinectria fuckeliana, Neonectria hederae and N. punicea.

Plant Dis 2019 Mar 9;103(3):389-391. Epub 2019 Jan 9.

1 U.S. Department of Agriculture, Agriculture Research Service (USDA-ARS), Mycology and Nematology Genetic Diversity and Biology Laboratory, 10300 Baltimore Avenue, Beltsville, MD, 20705, USA; and.

Corinectria fuckeliana, Neonectria hederae, and N. punicea are fungi in the family Nectriaceae that cause canker diseases of numerous hardwood trees, conifers, and woody perennials, often leading to plant mortality. Here, we report draft genome sequences for these three phytopathogenic fungal species. The genome sizes are consistent with those reported for other members of the Nectriaceae (28 to 43 Mb). These are the first genome resources available for C. fuckeliana, N. hederae, and N. punicea. These genome sequences may provide insights into the mechanisms of virulence and pathogenicity employed by these three destructive plant pathogens, and are resources suitable for the development of molecular markers that could be used for species identification, diagnostic tools and barcodes, and population studies.
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http://dx.doi.org/10.1094/PDIS-05-18-0904-ADOI Listing
March 2019

Visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH).

Plant Methods 2018 24;14:92. Epub 2018 Oct 24.

1Agriculture Research Service (ARS), Mycology and Nematology Genetic Diversity and Biology Laboratory, U.S. Department of Agriculture, 10300 Baltimore Avenue, Beltsville, MD 20705 USA.

Background: is the biotrophic oomycete responsible for impatiens downy mildew, a destructive disease of that causes high crop loss. Currently, there are no available methods for the microscopic detection of from leaves of impatiens, which may be contributing to the spread of the disease. Fluorescence in situ hybridization (FISH) is a sensitive and robust method that uses sequence-specific, fluorescence-labeled oligonucleotide probes to detect target organisms from the environment. To study this important pathogen, we developed and standardized a FISH technique for the visualization of from tissues using a species-specific 24-mer oligonucleotide probe designed to target a region of the rRNA internal transcribed spacer 2 (ITS2).

Results: Since cannot be propagated in vitro, we developed a custom expression vector that transcribes the rRNA-ITS target sequence (clone-FISH) for use as a control and to optimize hybridization conditions. The FISH assay could detect sporangiophores, sporangia and oospores, and hyphae from naturally infected leaves and stems. Cross-reactivity was not observed from plant tissue, and the assay did not react when applied to with self-ligated plasmids and non-target oomycete species.

Conclusions: This FISH protocol may provide a valuable tool for the study of this disease and could potentially be used to improve early monitoring of , substantially reducing the persistence and spread of this destructive plant pathogen.
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http://dx.doi.org/10.1186/s13007-018-0362-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199785PMC
October 2018

Genetic Variation of the Pathogen Causing Impatiens Downy Mildew Predating and Including Twenty-first Century Epidemics on Impatiens walleriana.

Plant Dis 2018 12 24;102(12):2411-2420. Epub 2018 Sep 24.

USDA-ARS, Mycology and Nematology Genetic Diversity and Biology Laboratory, Beltsville, MD 20705.

Impatiens downy mildew (IDM) of cultivated Impatiens walleriana has had a significant economic impact on the ornamental horticulture industry in the United States and globally. Although recent IDM outbreaks started in 2003, downy mildews on noncultivated Impatiens species have been documented since the 1880s. To understand the relationship between the pathogen causing recent epidemics and the pathogen historically present in the United States, this work characterized genetic variation among a collection of 1,000 samples on 18 plant hosts. Samples included collections during recent IDM epidemics and historical herbarium specimens. Ten major genotypes were identified from cloned rDNA amplicon sequencing and endpoint SNP genotyping. Three genotypes accounted for >95% of the samples, with only one of these three genotypes found on samples predating recent IDM outbreaks. Based on phylogenetic analysis integrating data from three markers and the presence of individual genotypes on multiple Impatiens species, there was some evidence of pathogen-specific infection of I. noli-tangere, but the distinction between genotypes infecting I. walleriana and I. balsamina was not upheld. Overall, this work provides evidence that the majority of rDNA genotypes recovered from recent IDM epidemics are different from historical U.S. genotypes, and that these genotypes can infect Impatiens spp. other than I. walleriana.
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http://dx.doi.org/10.1094/PDIS-01-18-0077-REDOI Listing
December 2018

Genome analysis of the ubiquitous boxwood pathogen .

PeerJ 2018 24;6:e5401. Epub 2018 Aug 24.

Mycology and Nematology Genetic Diversity and Biology Laboratory, US Department of Agriculture, Agriculture Research Service (USDA-ARS), Beltsville, MD, United States of America.

Boxwood ( spp.) are broad-leaved, evergreen landscape plants valued for their longevity and ornamental qualities. Volutella leaf and stem blight, caused by the ascomycete fungi , is one of the major diseases affecting the health and ornamental qualities of boxwood. Although this disease is less severe than boxwood blight caused by and , its widespread occurrence and disfiguring symptoms have caused substantial economic losses to the ornamental industry. In this study, we sequenced the genome of isolate ATCC13545 using Illumina technology and compared it to other publicly available fungal pathogen genomes to better understand the biology of this organism. A assembly estimated the genome size of at 28.7 Mb (425 contigs; N50 = 184,987 bp; avg. coverage 188×), with just 9,272 protein-coding genes. To our knowledge, has the smallest known genome within the Nectriaceae. Consistent with the small size of the genome, the secretome, CAzyme and secondary metabolite profiles of this fungus are reduced relative to two other surveyed Nectriaceae fungal genomes: JAC15-245 and Ph-1. Interestingly, a large cohort of genes associated with reduced virulence and loss of pathogenicity was identified from the dataset. These data are consistent with the latest observations by plant pathologists that and most likely are opportunistic, latent pathogens that prey upon weak and stressed boxwood plants.
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http://dx.doi.org/10.7717/peerj.5401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6110257PMC
August 2018

Downy Mildew: A Serious Disease Threat to Rose Health Worldwide.

Plant Dis 2018 Oct 15;102(10):1873-1882. Epub 2018 Aug 15.

Mycology and Nematology Genetic Diversity and Biology Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD.

Peronospora sparsa is a downy mildew-causing oomycete that can infect roses, blackberries, and other members of the rose family. During the last 70 years, this disease has become a serious problem for rose growers in the U.S. and worldwide. While much is known about the disease and its treatment, including significant research on molecular identification methods, as well as environmental conditions conducive to disease and the fungicides used to prevent it, significant knowledge gaps remain in our basic comprehension of the pathogen's biology. For example, the degree of genetic relatedness of pathogen isolates collected from rose, caneberries, and cherry laurel has never been examined, and the natural movement of genotypes from host to host is not known. Further work could be done to determine the differences in pathogen population structure over time (using herbarium specimens and fresh collections) or differences in pathogen population structure and pathogen environmental adaptation for specimens from different geographic regions. The oospore stage of the organism is poorly understood, both as to how it forms and whether it serves as an overwintering structure in nurseries and landscapes. In production greenhouses, the detection of the pathogen using infrared thermographic imaging and possible inhibition by ultraviolet light needs to be explored. Further work needs to be done on breeding using wild roses as new sources for resistance and using new methods such as marker assisted selection and RNAi technologies. As roses are one of the most economically important ornamental crops worldwide, a proper understanding of the disease cycle could allow for better use of cultural and chemical controls to manage rose downy mildew in landscapes and in greenhouse and nursery production areas.
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http://dx.doi.org/10.1094/PDIS-12-17-1968-FEDOI Listing
October 2018

Clarireedia: A new fungal genus comprising four pathogenic species responsible for dollar spot disease of turfgrass.

Fungal Biol 2018 08 23;122(8):761-773. Epub 2018 Apr 23.

Mycology and Nematology Genetic Diversity and Biology Laboratory, United States Department of Agriculture, Agricultural Research Service, 10300 Baltimore Avenue, Bldg. 010A, Beltsville, MD 20705, USA. Electronic address:

Dollar spot is one of the most destructive and economically important fungal diseases of amenity turfgrasses. The causal agent was first described in 1937 as the ascomycete Sclerotinia homoeocarpa. However, the genus-level taxonomic placement of this fungus has been the subject of an ongoing debate for over 75 y. Existing morphological and rDNA sequence evidence indicates that this organism is more appropriately placed in the family Rutstroemiaceae rather than the Sclerotiniaceae. Here we use DNA sequence data from samples of the dollar spot fungus and other members of the Rutstroemiaceae (e.g. Rutstroemia, Lanzia, Lambertella) collected throughout the world to determine the generic identity of the turfgrass dollar spot pathogen. Phylogenetic evidence from three nucleotide sequence markers (CaM, ITS and Mcm7; 1810-bp) confirmed that S. homoeocarpa is not a species of Sclerotinia; nor is it a member of any known genus in the Rutstroemiaceae. These data support the establishment of a new genus, which we describe here as Clarireedia gen. nov. The type species for the genus, Clarireedia homoeocarpa comb. nov., is described to accommodate the dollar spot fungus, and a neotype is designated. Three new species in this clade, Clarireedia bennettii sp. nov., Clarireedia jacksonii sp. nov., and Clarireedia monteithiana sp. nov. that also cause dollar spot disease are described. Clarireedia homoeocarpa and C. bennettii occur primarily on Festuca rubra (C3 grass) hosts and appear to be restricted to the United Kingdom. Clarireedia jacksonii and C. monteithiana occur on a variety of C3 and C4 grass hosts, respectively, and appear to be globally distributed. This resolved taxonomy puts to rest a major controversy amongst plant pathologists and provides a foundation for better understanding the nature and biology of these destructive pathogens.
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http://dx.doi.org/10.1016/j.funbio.2018.04.004DOI Listing
August 2018

Genome wide analysis of the transition to pathogenic lifestyles in Magnaporthales fungi.

Sci Rep 2018 04 12;8(1):5862. Epub 2018 Apr 12.

Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, New Jersey, 08901, USA.

The rice blast fungus Pyricularia oryzae (syn. Magnaporthe oryzae, Magnaporthe grisea), a member of the order Magnaporthales in the class Sordariomycetes, is an important plant pathogen and a model species for studying pathogen infection and plant-fungal interaction. In this study, we generated genome sequence data from five additional Magnaporthales fungi including non-pathogenic species, and performed comparative genome analysis of a total of 13 fungal species in the class Sordariomycetes to understand the evolutionary history of the Magnaporthales and of fungal pathogenesis. Our results suggest that the Magnaporthales diverged ca. 31 millon years ago from other Sordariomycetes, with the phytopathogenic blast clade diverging ca. 21 million years ago. Little evidence of inter-phylum horizontal gene transfer (HGT) was detected in Magnaporthales. In contrast, many genes underwent positive selection in this order and the majority of these sequences are clade-specific. The blast clade genomes contain more secretome and avirulence effector genes, which likely play key roles in the interaction between Pyricularia species and their plant hosts. Finally, analysis of transposable elements (TE) showed differing proportions of TE classes among Magnaporthales genomes, suggesting that species-specific patterns may hold clues to the history of host/environmental adaptation in these fungi.
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http://dx.doi.org/10.1038/s41598-018-24301-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5897359PMC
April 2018

Boxwood blight: an ongoing threat to ornamental and native boxwood.

Appl Microbiol Biotechnol 2018 May 2;102(10):4371-4380. Epub 2018 Apr 2.

Mycology and Nematology Genetic Diversity and Biology Laboratory, United States Department of Agriculture, Agricultural Research Service, 10300 Baltimore Avenue, Beltsville, MD, USA.

Boxwood blight is an emerging disease of ornamental and native boxwood plants in the family Buxaceae. First documented in the 1990s at a single location in England, the disease is now reported throughout Europe, Asia, New Zealand, and North America. To address the growing concern over boxwood blight, ongoing research focuses on multiple biological and genetic aspects of the causal pathogens and susceptible host plants. Characterization of genetic variation among the Calonectria fungi that cause boxwood blight shows that two unique sister species with different geographic distributions incite the disease. Studies of the pathogen life cycle show the formation of long-lived survival structures and that host infection is dependent on inoculum density, temperature, and humidity. Host range investigations detail high levels of susceptibility among boxwood as well as the potential for asymptomatic boxwood infection and for other plants in the family Buxaceae to serve as additional hosts. Multiple DNA-based diagnostic assays are available, ranging from probe-based quantitative PCR assays to the use of comparative genomics to develop robust diagnostic markers or provide whole genome-scale identifications. Though many questions remain, the research that continues to address boxwood blight demonstrates the importance of applying a multidisciplinary approach to understand and control emerging plant diseases.
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http://dx.doi.org/10.1007/s00253-018-8936-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5932091PMC
May 2018

Sarcococca Blight: Use of Whole-Genome Sequencing for Fungal Plant Disease Diagnosis.

Plant Dis 2016 Jun 22;100(6):1093-1100. Epub 2016 Mar 22.

USDA-ARS, Systematic Mycology and Microbiology Laboratory.

Early and accurate diagnosis of new plant pathogens is vital for the rapid implementation of effective mitigation strategies and appropriate regulatory responses. Most commonly, pathogen identification relies on morphology and DNA marker analysis. However, for new diseases, these approaches may not be sufficient for precise diagnosis. In this study, we used whole-genome sequencing (WGS) to identify the causal agent of a new disease affecting Sarcococca hookeriana (sarcococca). Blight symptoms were observed on sarcococca and adjacent Buxus sempervirens (boxwood) plants in Maryland during 2014. Symptoms on sarcococca were novel, and included twig dieback and dark lesions on leaves and stems. A Calonectria sp. was isolated from both hosts and used to fulfill Koch's postulates but morphology and marker sequence data precluded species-level identification. A 51.4-Mb WGS was generated for the two isolates and identified both as Calonectria pseudonaviculata. A single-nucleotide polymorphism at a noncoding site differentiated between the two host isolates. These results indicate that the same C. pseudonaviculata genotype has the ability to induce disease on both plant species. This study marks the first application of WGS for fungal plant pathogen diagnosis and demonstrates the power of this approach to rapidly identify causal agents of new diseases.
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http://dx.doi.org/10.1094/PDIS-10-15-1159-REDOI Listing
June 2016

LAMP Detection Assays for Boxwood Blight Pathogens: A Comparative Genomics Approach.

Sci Rep 2016 05 20;6:26140. Epub 2016 May 20.

United States Department of Agriculture, Agricultural Research Service (ARS), Systematic Mycology and Microbiology Laboratory, Beltsville, MD 20705, USA.

Rapid and accurate molecular diagnostic tools are critical to efforts to minimize the impact and spread of emergent pathogens. The identification of diagnostic markers for novel pathogens presents several challenges, especially in the absence of information about population diversity and where genetic resources are limited. The objective of this study was to use comparative genomics datasets to find unique target regions suitable for the diagnosis of two fungal species causing a newly emergent blight disease of boxwood. Candidate marker regions for loop-mediated isothermal amplification (LAMP) assays were identified from draft genomes of Calonectria henricotiae and C. pseudonaviculata, as well as three related species not associated with this disease. To increase the probability of identifying unique targets, we used three approaches to mine genome datasets, based on (i) unique regions, (ii) polymorphisms, and (iii) presence/absence of regions across datasets. From a pool of candidate markers, we demonstrate LAMP assay specificity by testing related fungal species, common boxwood pathogens, and environmental samples containing 445 diverse fungal taxa. This comparative-genomics-based approach to the development of LAMP diagnostic assays is the first of its kind for fungi and could be easily applied to diagnostic marker development for other newly emergent plant pathogens.
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http://dx.doi.org/10.1038/srep26140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4873745PMC
May 2016

SimpleSynteny: a web-based tool for visualization of microsynteny across multiple species.

Nucleic Acids Res 2016 07 3;44(W1):W41-5. Epub 2016 May 3.

Systematic Mycology and Microbiology Laboratory, U.S. Department of Agriculture (USDA), Agricultural Research Service (ARS), 10300 Baltimore Avenue, Building 10A, Beltsville, MD 20705, USA

Defining syntenic relationships among orthologous gene clusters is a frequent undertaking of biologists studying organismal evolution through comparative genomic approaches. With the increasing availability of genome data made possible through next-generation sequencing technology, there is a growing need for user-friendly tools capable of assessing synteny. Here we present SimpleSynteny, a new web-based platform capable of directly interrogating collinearity of local genomic neighbors across multiple species in a targeted manner. SimpleSynteny provides a pipeline for evaluating the synteny of a preselected set of gene targets across multiple organismal genomes. An emphasis has been placed on ease-of-use, and users are only required to submit FASTA files for their genomes and genes of interest. SimpleSynteny then guides the user through an iterative process of exploring and customizing genomes individually before combining them into a final high-resolution figure. Because the process is iterative, it allows the user to customize the organization of multiple contigs and incorporate knowledge from additional sources, rather than forcing complete dependence on the computational predictions. Additional tools are provided to help the user identify which contigs in a genome assembly contain gene targets and to optimize analyses of circular genomes. SimpleSynteny is freely available at: http://www.SimpleSynteny.com.
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http://dx.doi.org/10.1093/nar/gkw330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987899PMC
July 2016

Newly Emerged Populations of Plasmopara halstedii Infecting Rudbeckia Exhibit Unique Genotypic Profiles and Are Distinct from Sunflower-Infecting Strains.

Phytopathology 2016 Jul 18;106(7):752-61. Epub 2016 May 18.

First, second, and fourth author: U.S. Department of Agriculture-Agriculture Research Service (USDA-ARS), Systematic Mycology and Microbiology Laboratory, 10300 Baltimore Avenue, Beltsville, MD 20705; first and second authors: Department of Plant Biology and Pathology, Rutgers University, 59 Dudley Road, New Brunswick, NJ 08906; and third author: USDA-ARS, Sunflower and Plant Biology Research Unit, 1605 Albrecht Boulevard North, Fargo, ND 58102 (retired).

The oomycete Plasmopara halstedii emerged at the onset of the 21st century as a destructive new pathogen causing downy mildew disease of ornamental Rudbeckia fulgida (rudbeckia) in the United States. The pathogen is also a significant global problem of sunflower (Helianthus annuus) and is widely regarded as the cause of downy mildew affecting 35 Asteraceae genera. To determine whether rudbeckia and sunflower downy mildew are caused by the same genotypes, population genetic and phylogenetic analyses were performed. A draft genome assembly of a P. halstedii isolate from sunflower was generated and used to design 15 polymorphic simple sequence repeat (SSR) markers. SSRs and two sequenced phylogenetic markers measured differentiation between 232 P. halstedii samples collected from 1883 to 2014. Samples clustered into two main groups, corresponding to host origin. Sunflower-derived samples separated into eight admixed subclusters, and rudbeckia-derived samples further separated into three subclusters. Pre-epidemic rudbeckia samples clustered separately from modern strains. Despite the observed genetic distinction based on host origin, P. halstedii from rudbeckia could infect sunflower, and exhibited the virulence phenotype of race 734. These data indicate that the newly emergent pathogen populations infecting commercial rudbeckia are a different species from sunflower-infecting strains, notwithstanding cross-infectivity, and genetically distinct from pre-epidemic populations infecting native rudbeckia hosts.
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http://dx.doi.org/10.1094/PHYTO-12-15-0335-RDOI Listing
July 2016

Polymorphic SSR markers for Plasmopara obducens (Peronosporaceae), the newly emergent downy mildew pathogen of Impatiens (Balsaminaceae).

Appl Plant Sci 2015 Nov 10;3(11). Epub 2015 Nov 10.

USDA-ARS, Systematic Mycology and Microbiology Laboratory, Beltsville, Maryland 20705 USA.

Premise Of The Study: Simple sequence repeat (SSR) markers were developed for Plasmopara obducens, the causal agent of the newly emergent downy mildew disease of Impatiens walleriana.

Methods And Results: A 202-Mb draft genome assembly was generated from P. obducens using Illumina technology and mined to identify 13,483 SSR motifs. Primers were synthesized for 62 marker candidates, of which 37 generated reliable PCR products. Testing of the 37 markers using 96 P. obducens samples showed 96% of the markers were polymorphic, with 2-6 alleles observed. Observed and expected heterozygosity ranged from 0.000-0.892 and 0.023-0.746, respectively. Just 17 markers were sufficient to identify all multilocus genotypes.

Conclusions: These are the first SSR markers available for this pathogen, and one of the first molecular resources. These markers will be useful in assessing variation in pathogen populations and determining the factors contributing to the emergence of destructive impatiens downy mildew disease.
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http://dx.doi.org/10.3732/apps.1500073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4651634PMC
November 2015

Development of a greenhouse-based inoculation protocol for the fungus Colletotrichum cereale pathogenic to annual bluegrass (Poa annua).

PeerJ 2015 13;3:e1153. Epub 2015 Aug 13.

Systematic Mycology & Microbiology, USDA-ARS , Beltsville, MD , USA.

The fungus Colletotrichum cereale incites anthracnose disease on Poa annua (annual bluegrass) turfgrass. Anthracnose disease is geographically widespread throughout the world and highly destructive to cool-season turfgrasses, with infections by C. cereale resulting in extensive turf loss. Comprehensive research aimed at controlling turfgrass anthracnose has been performed in the field, but knowledge of the causal organism and its basic biology is still needed. In particular, the lack of a reliable greenhouse-based inoculation protocol performed under controlled environmental conditions is an obstacle to the study of C. cereale and anthracnose disease. Our objective was to develop a consistent and reproducible inoculation protocol for the two major genetic lineages of C. cereale. By adapting previously successful field-based protocols and combining with components of existing inoculation procedures, the method we developed consistently produced C. cereale infection on two susceptible P. annua biotypes. Approximately 7 to 10 days post-inoculation, plants exhibited chlorosis and thinning consistent with anthracnose disease symptomology. Morphological inspection of inoculated plants revealed visual signs of the fungus (appressoria and acervuli), although acervuli were not always present. After stringent surface sterilization of inoculated host tissue, C. cereale was consistently re-isolated from symptomatic tissue. Real-time PCR detection analysis based on the Apn2 marker confirmed the presence of the pathogen in host tissue, with both lineages of C. cereale detected from all inoculated plants. When a humidifier was not used, no infection developed for any biotypes or fungal isolates tested. The inoculation protocol described here marks significant progress for in planta studies of C. cereale, and will enable scientifically reproducible investigations of the biology, infectivity and lifestyle of this important grass pathogen.
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http://dx.doi.org/10.7717/peerj.1153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4558069PMC
September 2015

IMA Genome-F 4: Draft genome sequences of Chrysoporthe austroafricana, Diplodia scrobiculata, Fusarium nygamai, Leptographium lundbergii, Limonomyces culmigenus, Stagonosporopsis tanaceti, and Thielaviopsis punctulata.

IMA Fungus 2015 Jun 22;6(1):233-48. Epub 2015 Jun 22.

Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, P. bag x20, Pretoria 0028, South Africa.

The genomes of Chrysoporthe austroafricana, Diplodia scrobiculata, Fusarium nygami, Leptographium lundbergii, Limonomyces culmigenus, Stagonosporopsis tanaceti, and Thielaviopsis punctulata are presented in this genome announcement. These seven genomes are from endophytes, plant pathogens and economically important fungal species. The genome sizes range from 26.6 Mb in the case of Leptographium lundbergii to 44 Mb for Chrysoporthe austroafricana. The availability of these genome data will provide opportunities to resolve longstanding questions regarding the taxonomy of species in these genera, and may contribute to our understanding of the lifestyles through comparative studies with closely related organisms.
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http://dx.doi.org/10.5598/imafungus.2015.06.01.15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4500086PMC
June 2015

Draft Genome Sequence of Dactylonectria macrodidyma, a Plant-Pathogenic Fungus in the Nectriaceae.

Genome Announc 2015 Apr 16;3(2). Epub 2015 Apr 16.

Systematic Mycology and Microbiology Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland, USA

Dactylonectria macrodidyma is part of the Nectriaceae, a family containing important plant pathogens. This species possesses the ability to induce disease on grapevine, avocado, and olive. Here, we report the first draft genome of D. macrodidyma isolate JAC15-245. The assembled genome was 58 Mbp and contained an estimated 16,454 genes.
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http://dx.doi.org/10.1128/genomeA.00278-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4400431PMC
April 2015

IMA Genome-F 3: Draft genomes of Amanita jacksonii, Ceratocystis albifundus, Fusarium circinatum, Huntiella omanensis, Leptographium procerum, Rutstroemia sydowiana, and Sclerotinia echinophila.

IMA Fungus 2014 Dec 16;5(2):473-86. Epub 2014 Dec 16.

Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, P. Bag X20, Pretoria 0028, South Africa;

The genomes of fungi provide an important resource to resolve issues pertaining to their taxonomy, biology, and evolution. The genomes of Amanita jacksonii, Ceratocystis albifundus, a Fusarium circinatum variant, Huntiella omanensis, Leptographium procerum, Sclerotinia echinophila, and Rutstroemia sydowiana are presented in this genome announcement. These seven genomes are from a number of fungal pathogens and economically important species. The genome sizes range from 27 Mb in the case of Ceratocystis albifundus to 51.9 Mb for Rutstroemia sydowiana. The latter also encodes for a predicted 17 350 genes, more than double that of Ceratocystis albifundus. These genomes will add to the growing body of knowledge of these fungi and provide a value resource to researchers studying these fungi.
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http://dx.doi.org/10.5598/imafungus.2014.05.02.11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4329328PMC
December 2014

A Multiplex Real-Time PCR Assay for the Detection of Puccinia horiana and P. chrysanthemi on Chrysanthemum.

Plant Dis 2015 Feb;99(2):195-200

Systematic Mycology and Microbiology Laboratory, United States Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705.

Puccinia horiana, the cause of chrysanthemum white rust, is a regulated fungal plant pathogen in the United States, while P. chrysanthemi, the cause of chrysanthemum brown rust, is a widespread but less destructive pathogen. Accurate identification is essential to enforce quarantine measures, but the two species cannot be differentiated visually in the absence of mature spores or symptoms. A multiplex real-time PCR assay was developed to detect and discriminate between P. chrysanthemi and P. horiana. Species-specific hydrolysis probes labeled with different fluorescent dyes were designed based on the rDNA internal transcribed spacer region. Seven fresh samples and 270 herbarium specimens of chrysanthemum rust were tested with the assay with results confirmed using spore morphology. P. horiana and P. chrysanthemi were accurately detected from all fresh samples, and as little as 1 pg of template DNA was reproducibly detected. Of the herbarium specimens, 99% were positive for at least one species using the multiplex assay with 7% positive for both species. This multiplex assay can discriminate between P. chrysanthemi and P. horiana and provides an additional tool for identification of P. horiana to ensure appropriate application of quarantine measures.
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http://dx.doi.org/10.1094/PDIS-06-14-0632-REDOI Listing
February 2015