It's a trap! An approachable guide to constructing and using rotating-arm air samplers.

An increasing number of researchers are looking to understand the factors affecting microbial dispersion but are often limited by the costs of commercially available air samplers. Some have reduced these costs by designing self-made versions, however there are no published sampler designs and there is limited information provided on the actual construction process. Lack of appropriate reference material limits the use of these self-made samplers by many researchers. This manuscript provides a guide to designing and constructing rotating-arm impaction air samplers by covering 1) environmental considerations; 2) construction materials and equipment; 3) the construction process; and 4) air sampler deployment. Information regarding how to calculate rotational velocity, motor speed, power supply requirements, and troubleshoot common issues is presented in an approachable format for individuals without experience in electronics or machining. While many of the components discussed in this guide may change in their availability or be updated over time, this document is intended to serve as a "builder's guide" for future research into air sampling technology for phytopathology research.

First report of Bretziella fagacearum infecting chestnut in Michigan.

In the summer of 2021, a 20-year-old 'Colossal' (Castanea sativa × C. crenata hybrid) tree in a commercial chestnut orchard in northwest Michigan suddenly declined. Until 2023, an additional 26 adjacent trees declined, suggesting the occurrence of root-graft transmission of the pathogen. The initial wilting of leaves progressed to complete tree death in about 10 days. Symptoms included wilting, and bronzing, followed by tanning starting at leaf apex and margins, with significant defoliation. Sometimes black-to-brown streaks of discoloration appear in the sapwood, with no signs of mycelial mat production on dead trees. Branches from symptomatic trees in two different areas of the orchard were submitted to Plant and Pest Diagnostics at Michigan State University. Bretziella fagacearum (Bretz) Z.W. de Beer, Marinc., T.A. Duong & M.J. Wingf. was detected in both samples using nested PCR (Wu et al. 2011) and qPCR (Bourgault et al. 2022). The products of the nested PCR were sequenced (GenBank accession nos. OR522695-OR522696) and BLASTn search results showed 100% identity to an ex-type strain of B. fagacearum (MH865866). Surface-sterilized discolored sapwood chips were plated on acidified potato dextrose agar (aPDA). Bretziella fagacearum was consistently recovered; colony and endoconidia morphology aligned with the description of the pathogen (De Beer et al. 2017). A pure culture (BF277) was obtained for inoculation experiments. To confirm pathogenicity, 10 'Colossal' chestnut seedlings (average stem diameter of 9 mm) were inoculated in the greenhouse with a 14-day old culture of BF277. Using a conical drill bit, two 0.4 mm diameter holes were drilled, one was 5 cm above the soil line at a 45° angle and the other was on the opposite side of the stem at least 10 cm above the soil line. A 50-µl conidial suspension (1 × 107 conidia per ml) was applied and the holes were sealed with Parafilm. Five 'Colossal' seedlings were inoculated with sterile water. Leaf epinasty with bent petioles was observed 14 days later. Leaf wilting and necrosis similar to natural infection in the orchard were observed at 24 and 34 days after inoculation, respectively. Water-inoculated control plants showed no symptoms. Bretziella fagacearum was reisolated from symptomatic plants by surface sterilizing leaf petioles with 75% ethanol (30 s), followed by 10% (v/v) bleach (1 min), and two rinses with sterile deionized water (>1 min). Petiole pieces (~1 cm) were plated on aPDA. The pathogen was reisolated from six symptomatic plants and detected using qPCR in the remaining four seedlings. Bretziella fagacearum was not detected in control plants. The identity of the recovered fungus was confirmed following the amplification of the internal transcribed spacer (ITS) from extracted genomic DNA, as described in Chahal et al. 2022. The resulting PCR product was sequenced and assembled into a consensus sequence using Geneious Prime. The consensus sequence (accession no. OR515809) revealed 100% identity to the ex-type of B. fagacearum (KU042044). This is the first record of B. fagacearum infecting chestnut trees in Michigan. Previously, B. fagacearum has been reported infecting Chinese chestnut (C. mollissima) in Missouri (Bretz and Long, 1950). Oak wilt is widely distributed in Michigan and is the predominant disease afflicting red oaks in the Midwestern U.S. Consequently, constant vigilance and monitoring are essential in chestnut orchards to promptly detect and effectively manage potential infections.

It's a trap! Exploring the application of rotating-arm impaction samplers in plant pathology.

Although improved knowledge on the movement of airborne plant pathogens is likely to benefit plant health management, generating this knowledge is often far more complicated than anticipated. This complexity is driven by the dynamic nature of environmental variables, diversity among pathosystems that are targeted, and the unique needs of each research group. When using a rotating-arm impaction sampler, particle collection is dependent on the pathogen, environment, research objectives and limitations (monetary, environmental, or labor). Consequently, no design will result in 100% collection efficiency. Fortunately, it is likely that multiple approaches can succeed despite these constraints. Choices made during design and implementation of samplers can influence the results and recognizing this influence is crucial for researchers. This article is for beginners in the art and science of using rotating-arm impaction samplers; it provides a foundation for designing a project, from planning the experiment to processing samples. We present a relatively non-technical discussion of the factors influencing pathogen dispersal and how placement of the rotating-arm air samplers alters propagule capture. We include a discussion of applications of rotating-arm air samplers to demonstrate their versatility and potential in plant pathology research as well as their limitations.

First report of the beech leaf disease nematode Litylenchus crenatae mccannii (Nematoda: Anguinidae) in Michigan.

The North American beech leaf disease (BLD) nematode, Litylenchus crenatae mccannii Handoo, Li, Kantor, Bauchan, McCann, Gabriel, Yu, Reed, Koch, Martin and Burke, 2020, is recognized as a newly emergent nematode species that causes BLD in beech trees (Fagus spp.) in North America (Carta et al. 2020; Kantor et al. 2022a). Since the first report of BLD on American beech (Fagus grandifolia Ehrh) within the Lake County, located at the north-eastern corner of the state of Ohio in 2012 (Carta et al. 2020), the disease has rapidly spread to other US states and a province in Canada (Erwing et al. 2018; Carta et al 2020; Marra and LaMondia 2020; Reed et al 2020; Kantor et al. 2022b). Currently, besides Ohio, this nematode has been reported in Pennsylvania, New York, Connecticut, Massachusetts, Maine, Rhode Island, New Jersey, West Virginia, and Virginia, as well as Ontario, Canada. Different life stages of L. crenatae mccannii were isolated from symptomatic American beech leaves from an isolated natural maple-beech stand in rural Saint Clair Cty., Michigan, US; presenting typical symptoms of beech leaf disease, i.e., swelling and darkening of interveinal leaf tissues. Samples were taken to the Forest Pathology Laboratory at Michigan State University where L. crenatae mccannii presence was confirmed in the leaves after which samples were sent to the Mycology and Nematology Genetic Diversity and Biology Laboratory (USDA-ARS) in Beltsville, Maryland for official confirmation. Nematodes were identified based on morphology and sequence analysis of the internal transcribed spacer (ITS), and the D2D3 region of the 28S large subunit ribosomal DNA. To validate the morphological identification two different ribosomal DNA loci were amplified, sequenced and the phylogenetic relationships were generated. The amplification yielded fragments of 784 and 741 bp flanked by the ITS (GenBank accession no. OP689654) and D2D3 (GenBank accession no. OP689710) primers, respectively. The sequences obtained for the specimens collected in Michigan revealed 100% similarity to L. crenatae mccannii sequences obtained from specimens collected from other geographical areas in the US, and therefore validating the morphological analyses as well. The ITS sequence shared a 99.75% similarity with the subspecies L. crenatae (GenBank accession no. LC383724.1), and 90.53% similarity to L. coprosma Zhao, Davies, Alexander and Riley, 2011 (GU727548.1). While the D2D3 sequences of both L. crenatae subspecies revealed a 100% similarity (versus LC383725.1), they revealed 95.35% similarity to L. coprosma (KY679564.1). Since the first confirmed detection of BLD in June 2022 in St. Clair Cty, BLD has been reported in Oakland and Wayne Ctys (7 reports total across the three counties), suggesting BLD spread in the SE of Michigan. BLD confirmation was based on either physical symptoms (leaf banding), and/or the presence of the beech leaf nematode by morphological or molecular confirmation. The presence of the beech leaf nematode in symptomatic leaves follow the results obtained by Carta et al. (2020) after inoculation of beech seedlings with L. crenatae mccannii. Based on both morphological and molecular analyses the specimens collected in the state of Michigan were identified as L. crenatae mccannii. To our knowledge, this is the first report of this species in conjunction with symptomatic F. grandifolia leaves in this state.

Clade-Specific Monitoring of Airborne Pseudoperonospora spp. Sporangia Using Mitochondrial DNA Markers for Disease Management of Cucurbit Downy Mildew.

Management of cucurbit downy mildew (CDM) caused by Pseudoperonospora cubensis, relies on an intensive fungicide program. In Michigan, CDM occurs annually due to an influx of airborne sporangia and timely alerts of airborne inoculum can assist growers in assessing the need to initiate fungicide sprays. This research aimed to improve the specific detection of airborne P. cubensis sporangia by adapting quantitative real-time polymerase chain reaction (qPCR) assays to distinguish among P. cubensis clades I and II and P. humuli in spore trap samples from commercial production sites and research plots. We also evaluated the suitability of impaction spore traps compared with Burkard traps for detection of airborne sporangia. A multiplex qPCR assay improved the specificity of P. cubensis clade II detection accelerating the assessment of field spore trap samples. After 2 years of monitoring, P. cubensis clade II DNA was detected in spore trap samples before CDM symptoms were first observed in cucumber fields (July and August), while P. cubensis clade I DNA was not detected in air samples before or after the disease onset. In some commercial cucumber fields, P. humuli DNA was detected throughout the growing season. The Burkard spore trap appeared to be better suited for recovery of sporangia at low concentrations than the impaction spore trap. This improved methodology for the monitoring of airborne Pseudoperonospora spp. sporangia could be used as part of a CDM risk advisory system to time fungicide applications that protect cucurbit crops in Michigan.

Independent Evolution Has Led to Distinct Genomic Signatures in Dutch Elm Disease-Causing Fungi and Other Vascular Wilts-Causing Fungal Pathogens.

Vascular wilts are important diseases caused by plant pathogenic fungi that result in the rapid death of their plant hosts. This is due to a systemic defense mechanism whereby the plant induces the compartmentalization of the infected vascular system in order to reduce the propagation of the fungus. The ascomycete class Sordariomycetes contains several species that cause vascular wilts in diverse plant hosts, and they can be classified into four taxonomic orders. The genetic mechanisms of pathogenesis have already been investigated in Fusarium and Verticillium species, but they have not yet been compared with other well-known wilt-causing species, especially fungi causing oak wilt or Dutch elm disease (DED). Here we analyzed 20 whole genome assemblies of wilt-causing fungi together with 56 other species using phylogenetic approaches to trace expansions and contractions of orthologous gene families and gene classes related to pathogenicity. We found that the wilt-causing pathogens evolved seven times, experiencing the largest fold changes in different classes of genes almost every time. However, some similarities exist across groups of wilt pathogens, particularly in Microascales and Ophiostomatales, and these include the common gains and losses of genes that make up secondary metabolite clusters (SMC). DED pathogens do not experience large-scale gene expansions, with most of the gene classes, except for some SMC families, reducing in number. We also found that gene family expansions in the most recent common ancestors of wilt pathogen groups are enriched for carbohydrate metabolic processes. Our study shows that wilt-causing species evolve primarily through distinct changes in their repertoires of pathogenicity-related genes and that there is the potential importance of carbohydrate metabolism genes for regulating osmosis in those pathogens that penetrate the plant vascular system.

Application of Target Enrichment Sequencing for Population Genetic Analyses of the Obligate Plant Pathogens Pseudoperonospora cubensis and P. humuli in Michigan.

Technological advances in genome sequencing have improved our ability to catalog genomic variation and have led to an expansion of the scope and scale of genetic studies over the past decade. Yet, for agronomically important plant pathogens such as the downy mildews (Peronosporaceae), the scale of genetic studies remains limited. This is, in part, due to the difficulties associated with maintaining obligate pathogens and the logistical constraints involved in the genotyping of these species (e.g., obtaining DNA of sufficient quantity and quality). To gain an evolutionary and ecological perspective of downy mildews, adaptable methods for the genotyping of their populations are required. Here, we describe a targeted enrichment (TE) protocol to genotype isolates from two Pseudoperonospora species (P. cubensis and P. humuli), using less than 50 ng of mixed pathogen and plant DNA for library preparation. We were able to enrich 830 target genes across 128 samples and identified 2,514 high-quality single nucleotide polymorphism (SNP) variants. Using these SNPs, we detected significant genetic differentiation (analysis of molecular variance [AMOVA], P = 0.01) between P. cubensis subpopulations from Cucurbita moschata (clade I) and Cucumis sativus (clade II) in the state of Michigan. No evidence of location-based differentiation was detected within the P. cubensis (clade II) subpopulation in Michigan. However, a significant effect of location on the genetic variation of the P. humuli subpopulation was detected in the state (AMOVA, P = 0.01). Mantel tests found evidence that the genetic distance among P. humuli samples was associated with the physical distance of the hop yards from which the samples were collected (P = 0.005). The differences in the distribution of genetic variation of the Michigan P. humuli and P. cubensis subpopulations suggest differences in the dispersal of these two species. The TE protocol described here provides an additional tool for genotyping obligate biotrophic plant pathogens and the execution of new genetic studies.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Population Structure of a Worldwide Phytophthora palmivora Collection Suggests Lack of Host Specificity and Reduced Genetic Diversity in South America and the Caribbean.

Phytophthora palmivora (Butler) is a highly destructive plant pathogen that infects tropical hosts worldwide, many of which are economically important crops. Despite the broad host range and wide distribution, the pathogen has displayed a considerable amount of variation in morphological characters, including virulence. However, the genetic variability at a global level, which is critical to understand the center of origin and the potential pathway(s) of introduction, was unclear. Here, we mapped the genetic variation of P. palmivora using isolates representing four regions, 15 countries, and 14 host species. We designed a large set of simple sequence repeat markers from the P. palmivora genome and picked 17 selectively neutral markers to screen 98 P. palmivora isolates. We found that P. palmivora populations from our collection generally did not cluster according to host; rather, some isolates from North America were generally distinct from all other populations. Isolates from South America and the Caribbean clustered and appeared to share ancestry with isolates from Asia. Populations from North America and Asia were the most genetically diverse, while the South American and Caribbean populations exhibited similar reduced genetic diversity. The isolates collected in various plantations in Colombia did not show host or geographic specificity. Our study brought a further understanding of this important plant pathogen, although the determination for hypothesized source of origin, spread, and evolution would need further sampling. The genomic resources developed in this study would facilitate further studies on P. palmivora diagnostics and management.

Characterization of Diaporthe spp. Associated With Spruce Decline on Colorado Blue Spruce in Michigan.

Since 2006 there has been a decline in Colorado blue spruce (CBS; Picea pungens) planted as landscape trees and for Christmas tree production throughout the Lower Peninsula of Michigan. This decline is characterized by a slow loss of needles in the lower portion of the tree starting at branch tips, followed by entire branch dieback, which progresses upward over several years. This dieback has been linked to shallow branch cankers visible in the phloem when the bark layer is removed. Isolates in the fungal genus Diaporthe have been consistently isolated from lesion margins on symptomatic branches. Before the initial reports of declining CBS in landscape and Christmas trees, Diaporthe was known only as a nursery disease of CBS. To determine the species of Diaporthe linked to the decline of CBS in Michigan, seven gene regions were sequenced from a collection of Diaporthe isolates collected in 2011 through 2018 from CBS and other coniferous hosts. Subsequent phylogenetic analyses indicated that Diaporthe eres and a novel Diaporthe clade were present on symptomatic CBS in Michigan. The new species D. brevicancria nov. is described, and Koch's postulates were confirmed for D. brevicancria nov. and D. eres. D. brevicancria nov. produced the largest cankers in greenhouse pathogenicity trials, and dual inoculations of D. brevicancria nov. and D. eres produced intermediate cankers.

Etiology of Halo Blight in Michigan Hopyards.

Michigan's hop acreage ranks fourth nationally, but the state's growers contend with unique disease challenges resulting from frequent rainfall and high humidity. In August 2018, a Michigan hop grower reported necrosis and blighting of foliage and shattering of cones resulting in yield loss. Irregular-shaped lesions developed on leaves, surrounded by a halo of chlorotic tissue, and cone bracts became brown. Pycnidia were observed in symptomatic tissue. The goal of this study was to identify and characterize the causal agent of symptoms in leaf and cone tissue. In symptomatic leaves, 15 of 19 isolates recovered had 96.4% internal transcribed spacer rDNA (ITSrDNA) homology with Diaporthe nomurai. Bayesian and maximum likelihood analyses were performed on a subset of isolates using ITSrDNA, histone H3, beta-tubulin, and elongation factor 1 alpha. Bootstrap and posterior probabilities supported a unique cluster of Diaporthe sp. 1-MI isolates most closely related to the Diaporthe arecae species complex, Diaporthe hongkongensis, and Diaporthe multigutullata. Diaporthe sp. 1-MI was pathogenic in detached leaf and whole plant assays. Single-spore isolates from pycnidia originating from cones and leaves shared 100% ITSrDNA homology with Diaporthe sp. 1-MI obtained from the lesion margins of leaves collected in 2018. The distribution of Diaporthe sp. 1-MI was widespread among 347 cones collected from 15 Michigan hop yards and accounted for >38% of fungi recovered from cones in three hop yards. Diaporthe sp. 1-MI causing halo and cone blight presents a new disease management challenge for Michigan hop growers.

Detection of Airborne Sporangia of Pseudoperonospora cubensis and P. humuli in Michigan Using Burkard Spore Traps Coupled to Quantitative PCR.

Cucurbit downy mildew (CDM), caused by the oomycete pathogen Pseudoperonospora cubensis, is a devastating foliar disease on cucumber resulting in reduced yields. In 2004, the pathogen re-emerged in the United States, infecting historically resistant cucumber cultivars and requiring the adoption of an intensive fungicide program. The pathogen cannot overwinter in Michigan fields but because of an influx of airborne sporangia CDM occurs annually. In Michigan, spore traps are used to monitor the presence of airborne P. cubensis sporangia in cucumber growing regions to guide the initiation of a fungicide program. However, Pseudoperonospora humuli sporangia, the causal agent of downy mildew on hop, are morphologically indistinguishable from P. cubensis sporangia. This morphological similarity reduces the ability to accurately detect P. cubensis from spore trap samples when examined with the aid of light microscopy. To improve P. cubensis detection, we adapted a qPCR-based assay to allow the differentiation between P. cubensis and P. humuli on Burkard spore trap samples collected in the field. Specifically, we evaluated the specificity and sensitivity of P. cubensis detection on Burkard spore trap tapes using a morphological-based and quantitative-PCR (qPCR)-based identification assay and determined whether sporangia of P. cubensis and P. humuli on Burkard samples could be distinguished using qPCR. We found that the qPCR assay was able to detect a single sporangium of each species on spore trap samples collected in the field with Cq values <35.5. The qPCR assay also allowed the detection of P. cubensis and P. humuli in samples containing sporangia from both species. However, the number of sporangia quantified using light microscopy explained only 54 and 10% of the variation in the Cq values of P. cubensis and P. humuli, respectively, suggesting a limited capacity of the qPCR assay for the absolute quantification of sporangia in field samples. After 2 years of monitoring using Burkard spore traps coupled with the qPCR in cucumber fields, P. humuli sporangia were detected more frequently than P. cubensis early in the growing season (May and June). P. cubensis sporangia were detected ∼5 to 10 days before CDM symptoms were first observed in cucumber fields during both years. This research describes an improved sporangial detection system that is key for the monitoring and management of P. cubensis in Michigan.

Genome-Enhanced Detection and Identification (GEDI) of plant pathogens.

Plant diseases caused by fungi and Oomycetes represent worldwide threats to crops and forest ecosystems. Effective prevention and appropriate management of emerging diseases rely on rapid detection and identification of the causal pathogens. The increase in genomic resources makes it possible to generate novel genome-enhanced DNA detection assays that can exploit whole genomes to discover candidate genes for pathogen detection. A pipeline was developed to identify genome regions that discriminate taxa or groups of taxa and can be converted into PCR assays. The modular pipeline is comprised of four components: (1) selection and genome sequencing of phylogenetically related taxa, (2) identification of clusters of orthologous genes, (3) elimination of false positives by filtering, and (4) assay design. This pipeline was applied to some of the most important plant pathogens across three broad taxonomic groups: Phytophthoras (Stramenopiles, Oomycota), Dothideomycetes (Fungi, Ascomycota) and Pucciniales (Fungi, Basidiomycota). Comparison of 73 fungal and Oomycete genomes led the discovery of 5,939 gene clusters that were unique to the targeted taxa and an additional 535 that were common at higher taxonomic levels. Approximately 28% of the 299 tested were converted into qPCR assays that met our set of specificity criteria. This work demonstrates that a genome-wide approach can efficiently identify multiple taxon-specific genome regions that can be converted into highly specific PCR assays. The possibility to easily obtain multiple alternative regions to design highly specific qPCR assays should be of great help in tackling challenging cases for which higher taxon-resolution is needed.

Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen.

Some of the most damaging tree pathogens can attack woody stems, causing lesions (cankers) that may be lethal. To identify the genomic determinants of wood colonization leading to canker formation, we sequenced the genomes of the poplar canker pathogen, Mycosphaerella populorum, and the closely related poplar leaf pathogen, M. populicola. A secondary metabolite cluster unique to M. populorum is fully activated following induction by poplar wood and leaves. In addition, genes encoding hemicellulose-degrading enzymes, peptidases, and metabolite transporters were more abundant and were up-regulated in M. populorum growing on poplar wood-chip medium compared with M. populicola. The secondary gene cluster and several of the carbohydrate degradation genes have the signature of horizontal transfer from ascomycete fungi associated with wood decay and from prokaryotes. Acquisition and maintenance of the gene battery necessary for growth in woody tissues and gene dosage resulting in gene expression reconfiguration appear to be responsible for the adaptation of M. populorum to infect, colonize, and cause mortality on poplar woody stems.

Use of the Genealogical Sorting Index (GSI) to delineate species boundaries in the Neofusicoccum parvum-Neofusicoccum ribis species complex.

Neofusicoccum is a recently described genus of common endophytes and pathogens of woody hosts, previously placed in the genus Botryosphaeria. Many morphological characteristics routinely used to describe species overlap in Neofusicoccum, and prior to the use of molecular phylogenetics, isolates from different hosts and locations were often misidentified. Two cryptic species Neofusicoccum ribis and Neofusicoccum parvum were initially described from different continents and recently another four species within this complex were described using fixed nucleotide polymorphisms for differentiation. In a survey of eucalypt cankers in eastern Australia, a collection of morphologically similar Neofusicoccum isolates were obtained. This collection was analysed within the framework of the morphological (MSRC), ecological (ESRC) and phylogenetic (PSRC) species recognition concepts. Morphological data based on spore measurements (MSRC), together with pathogenicity trials (ESRC) were considered alongside molecular analysis (PSRC), which included multiple gene phylogenies constructed from four nuclear gene regions. We also used the Genealogical Sorting Index method to provide objective evidence for the status of terminal taxa in the phylogenetic analysis. The isolates examined exhibited overlapping morphological and culture characteristics, similar pathogenicity to excised stems and shared hosts within the same locations. Phylogenetic analysis separated isolates into 8 clades corresponding to six described species: N. ribis, N. parvum, Neofusicoccum kwambonambiense, Neofusicoccum cordaticola, Neofusicoccum umdonicola, Neofusicoccum batangarum, and two new species. GSI support indicated combined phylogenetic data were monophyletic for all clades and all p-values were significant allowing us to reject the null hypothesis that all groups were from a single mixed group. Consequently the description of Neofusicoccum occulatum is presented.