Detection of Podosphaera macularis in Air Samples by Quantitative PCR.

Detection and quantification of pathogen propagules in the air or other environmental samples is facilitated by culture-independent assays. We developed a quantitative PCR assay for the hop powdery mildew fungus, Podosphaera macularis, for detection of the organism from air samples. The assay utilizes primers and a TaqMan probe designed to target species-specific sequences in the 28S large subunit (LSU) of the nuclear ribosomal rDNA. Analytical sensitivity was not affected by the presence of an exogenous internal control or potential PCR inhibitors associated with DNA extracted from soil. The level of quantification of the assay was between 200 and 350 conidia when DNA was extracted from a fixed number of conidia. The assay amplified all isolates of P. macularis tested and had minimal cross-reactivity with other Podosphaera species when assayed with biologically relevant quantities of DNA. Standard curves generated independently in two other laboratories indicated that assay sensitivity was qualitatively similar and reproducible. All laboratories successfully detected eight unknown isolates of P. macularis and correctly discriminated Pseudoperonospora humuli and a water control. The usefulness of the assay for air sampling for late-season inoculum of P. macularis was demonstrated in field studies in 2019 and 2020. In both years, airborne populations of P. macularis in hop yards were detected consistently and increased during bloom and cone development.

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 Colletotrichum fioriniae Infecting Hop (Humulus lupulus) in Michigan.

In July of 2020, a hop (Humulus lupulus L.) grower in Berrien County, Michigan submitted 'Chinook' leaf samples to MSU Plant & Pest Diagnostics. The leaves were covered in small, tan colored lesions, with a small chlorotic halo with an approximate diameter of 5 mm. The grower reported that foliar lesions were in the lower 2 m of the fully developed hop canopy. Disease incidence and severity were estimated at approximately 20% and 5 to 10%, respectively. After incubation at 100% relative humidity, acervuli with orange spore masses and a few setae were present. A pure culture was obtained from these sporulating lesions using water agar. The isolate was hyphal tipped onto potato dextrose agar (PDA) and stored in a glycerol-salt solution at -80o C (isolate CL001) (Miles et al. 2011). On PDA, cultures displayed gray growth on the top of the colony and a red color on the underside of the Petri dish. After 14 days, acervuli with no setae appeared exuding orange conidial masses on the surface of the culture. Conidia were hyaline, aseptate, smooth-walled and rounded at the ends and measured on average 15.89 µm (13.81 to 16.91 µm) × 7.26 (6.82 to 8.41 µm) (n = 20). The color and size of the conidia matched other descriptions of C. acutatum sensu lato (Damm et al. 2012). Four loci (ITS/515 bp - OQ026167, GAPDH/238 bp - OQ230832, CHS1/228 bp -OQ230830, and TUB2/491 bp - OQ230831) were amplified from isolate CL001 (using the primers ITS1/ITS4, GDF1/GDR1, CSH-79f/CHS-354R, and T1/Bt-2b, respectively) and had 100% pairwise identity with C. fioriniae 125396 (JQ948299, JQ948629, JQ948960, JQ949950, respectively, Damm et al. 2012). The GAPDH, CSH1, and TUB2 sequences from isolate CL001 were trimmed, concatenated and aligned with 31 different members of Colletotrichum acutatum sensu lato and C. gloesporioides 356878 (Damm et al. 2012; Kennedy et al. 2022). The alignment was then used to produce a maximum likehood phylogenetic tree using Geneious Prime (Biomatters Ltd.) with the PHYML add on using the HKY + G model (G = 0.34) (Guindon et al. 2010). Isolate CL001 had the closest similarity to C. fioriniae with a bootstrap value of 100. Pathogenicity tests were performed on 2 month-old 'Chinook' hop plants. Twelve plants were inoculated with 50 ml of a conidial suspension (7.95 x 106 conidia/ml) of isolate CL001 (n = 6) or water (n = 6) using a spray bottle until runoff. Inoculated plants were sealed in clear plastic bags and grown in a greenhouse at 21o C with a photoperiod of 14 h. After 7 days, lesions appeared on the hop plants inoculated with CL001, but no symptoms appeared on the water inoculated hop plants. Lesions with a chlorotic halo were observed but they were smaller than field lesions and no setae were present (approx. 1 mm in diam.). Leaves were surface sterilized (0.3% sodium hypochlorite solution for 15 s and then rinsed three times) and the leading margin of the lesions or healthy tissue (water control) were placed on 1% ampicillin amended PDA. Fungal isolates on PDA morphologically matched C. fioriniae were recovered from all CL001-inoculated plants. No C. fioriniae isolates were recovered from the water-inoculated plants. Based on conidial morphology, the four loci, and the phylogenetic tree, isolate CL001 was identified as C. fioriniae. This is the first report of Colletotrichum fioriniae (syn = Glomerella acutata var. fioriniae Marcelino & Gouli) infecting common hop and further investigation is needed to determine if management is needed for this pathogen.

Development of a PNA-LNA-LAMP Assay to Detect an SNP Associated with QoI Resistance in Erysiphe necator.

The repetitive use of quinone outside inhibitor fungicides (QoIs, strobilurins; Fungicide Resistance Action Committee [FRAC] 11) to manage grape powdery mildew has led to development of resistance in Erysiphe necator. While several point mutations in the mitochondrial cytochrome b gene are associated with resistance to QoI fungicides, the substitution of glycine to alanine at codon 143 (G143A) has been the only mutation observed in QoI-resistant field populations. Allele-specific detection methods such as digital droplet PCR and TaqMan probe-based assays can be used to detect the G143A mutation. In this study, a peptide nucleic acid-locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA-LAMP) assay consisting of an A-143 reaction and a G-143 reaction, was designed for rapidly detecting QoI resistance in E. necator. The A-143 reaction amplifies the mutant A-143 allele faster than the wild-type G-143 allele, while the G-143 reaction amplifies the G-143 allele faster than the A-143 allele. Identification of resistant or sensitive E. necator samples was determined by which reaction had the shorter time to amplification. Sixteen single-spore QoI-resistant and -sensitive E. necator isolates were tested using both assays. Assay specificity in distinguishing the single nucleotide polymorphism (SNP) approached 100% when tested using purified DNA of QoI-sensitive and -resistant E. necator isolates. This diagnostic tool was sensitive to one-conidium equivalent of extracted DNA with an R2 value of 0.82 and 0.87 for the G-143 and A-143 reactions, respectively. This diagnostic approach was also evaluated against a TaqMan probe-based assay using 92 E. necator samples collected from vineyards. The PNA-LNA-LAMP assay detected QoI resistance in ≤30 min and showed 100% agreement with the TaqMan probe-based assay (≤1.5 h) for the QoI-sensitive and -resistant isolates. There was 73.3% agreement with the TaqMan probe-based assay when samples had mixed populations with both G-143 and A-143 alleles present. Validation of the PNA-LNA-LAMP assay was conducted in three different laboratories with different equipment. The results showed 94.4% accuracy in one laboratory and 100% accuracy in two other laboratories. The PNA-LNA-LAMP diagnostic tool was faster and required less expensive equipment relative to the previously developed TaqMan probe-based assay, making it accessible to a broader range of diagnostic laboratories for detection of QoI resistance in E. necator. This research demonstrates the utility of the PNA-LANA-LAMP for discriminating SNPs from field samples and its utility for point-of-care monitoring of plant pathogen genotypes.

A Rapid Glove-Based Inoculum Sampling Technique to Monitor Erysiphe necator in Commercial Vineyards.

Information on the presence and severity of grape powdery mildew (GPM), caused by Erysiphe necator, has long been used to guide management decisions. While recent advances in the available molecular diagnostic assays and particle samplers have made monitoring easier, there is still a need for more efficient field collection of E. necator. The use of vineyard worker gloves worn during canopy manipulation as a sampler (glove swab) of E. necator was compared with samples identified by visual assessment with subsequent molecular confirmation (leaf swabs) and airborne spore samples collected by rotating-arm impaction traps (impaction traps). Samples from United States commercial vineyards in Oregon, Washington, and California were analyzed using two TaqMan qPCR assays targeting the internal transcribed spacer regions or cytochrome b gene of E. necator. Based on qPCR assays, visual disease assessments misidentified GPM up to 59% of the time with a higher frequency of misidentification occurring earlier in the growing season. Comparison of the aggregated leaf swab results for a row (n = 915) to the row's corresponding glove swab had 60% agreement. The latent class analysis (LCA) indicated that glove swabs were more sensitive than leaf swabs in detecting E. necator presence. The impaction trap results had 77% agreement to glove swabs (n = 206) taken from the same blocks. The LCAs estimated that the glove swabs and impaction trap samplers varied each year in which was more sensitive for detection. This likely indicates that these methods have similar levels of uncertainty and provide equivalent information. Additionally, all samplers, once E. necator was detected, were similarly sensitive and specific for detection of the A-143 resistance allele. Together, these results suggest that glove swabs are an effective sampling method for monitoring the presence of E. necator and, subsequently, the G143A amino acid substitution associated with resistance to quinone outside inhibitor fungicides in vineyards. Glove swabs could reduce sampling costs due to the lack of need for specialized equipment and time required for swab collection and processing.

First report of stem blight caused by Diaporthe eres on highbush blueberry (Vaccinium corymbosum) in Michigan.

The U.S. is the world's leading producer of highbush blueberries (Vaccinium corymbosum L.), and Michigan is ranked in the top five production states (USDA NASS, 2022). In June and July of 2021, 268 blueberry stem blight samples were collected for a pathogen survey across 22 total fields in Van Buren and Ottawa counties in Michigan. Current season stems with symptoms of necrosis and wilting were collected. Stems were cut just below the necrotic area and cross-sections (2-3 mm long) were surface disinfested in 10% bleach for 1 min, rinsed twice in sterile distilled water, and dried on sterile paper towels. Stem cross-sections were plated onto potato dextrose agar (PDA) amended with 100 µg/ml streptomycin sulfate and 50 µg/ml ampicillin. Plates were incubated at 21°C under a 12-h photoperiod for 5-6 days. Outgrowing fungi with morphology similar to Diaporthe spp. were transferred to new PDA plates 2 consecutive times after 7 days of similar incubation to ensure single colony isolation. After 7 days, colonies consisted of white and light brown mycelia that were mostly flat, with some isolates that had partially raised mycelia towards the center of the plate. After 3-4 weeks, colonies turned brown and gray and produced dark brown pycnidia. Aseptate, hyaline, fusiform to ellipsoid, biguttulate alpha conidia measuring 5.4 to 7.6 x 2.6 to 3.7 µm (n = 60) were produced. No beta conidia were observed. In total, 3 isolates, representing 3 different farms (37-95 km apart) and cultivars ('Duke', 'Jersey', and 'Bluecrop'), as well as 2 counties, were identified as Diaporthe through colony morphology (Gomes et al. 2013, Udayanga et al. 2014). Amplification and subsequent Sanger sequencing were performed for the internal transcribed spacer (ITS) region and portions of the translation elongation factor (TEF) 1-α, ß-tubulin (TUB), and histone H3 (HIS) genes using primers ITS5/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), T1/Bt-2b (Glass and Donaldson 1995), and CYLH3F/H3-1b (Glass and Donaldson 1995), respectively. Representative sequences were deposited in NCBI GenBank (accession no. OQ507870-OQ507872 for ITS, and OQ550272-OQ550278 for TEF, HIS, and TUB). BLASTn results revealed 97-100% identity for all 4 genes across other established D. eres isolates reported in Gomes et al. (2013). For example, JMK047 had 99.8% (577/578 bp), 99.7% (327/328 bp), 100% (701/701 bp), and 100% (439/439 bp) homology with ITS, TEF, TUB, and HIS sequences, respectively, of D. eres CBS 439.82 (accession no. KC343090, KC343816, KC344058, KC343574). Koch's Postulates were fulfilled via pathogenicity tests on 2-year-old potted 'Blueray' plants with 2 isolates. Stems were surface sterilized with 1% bleach then 8-mm long pieces of bark were removed using a sterile razor blade to expose the cambium. Plugs of sterile PDA (negative control) or mycelia from 7-day old cultures on PDA (5-mm diameter) were placed onto the cambium layer and sealed with Parafilm. Six stems on unique plants were inoculated per treatment. Plants were grown in a 20.5°C greenhouse with a 14-hr photoperiod. After 3 weeks, the stems inoculated with D. eres isolates showed similar stem blight symptoms to those observed in the field while control stems remained healthy. Re-isolation and sequencing of the ITS region of 3 replicates per treatment with the protocol described above confirmed symptoms correlated with D. eres isolates. This is the first report of D. eres associated with stem blight of highbush blueberry in Michigan, and the second report in the U.S. (Lombard et al. 2014). Increasing prevalence of D. eres in U.S. blueberries may affect disease management programs. References Carbone, I., and Kohn, L. M. 1999. Mycologia 91:553. 10.1080/00275514.1999.12061051. Glass, N. L., and Donaldson, G. C. 1995. Appl. Environ. Microbiol. 61:1323. 10.1128/aem.61.4.1323-1330.1995. Gomes, R. R., et al. 2013. Persoonia 31:1. 10.3767/003158513x666844. Lombard, L., et al. 2014. Phytopathol. Mediterr. 51(2):287. 10.14601/Phytopathol_Mediterr-14034. Udayanga, D., Castlebury, L. A., Rossman, A. Y., Chukeatirote, E., and Hyde, K. D. 2014. Fungal Divers. 67:203-229. 10.1007/s13225-014-0297-2. USDA NASS. 2022. Noncitrus Fruits and Nuts 2021 Summary. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, California, USA.

First report of halo blight on hop (Humulus lupulus) caused by Diaporthe humulicola in Minnesota.

Halo blight, caused by Diaporthe humulicola, is an emerging issue in hop production in the Upper Midwestern and Eastern North America. Reports of halo blight thus far have included Connecticut, Michigan, New York, and Quebec (Allan-Perkins et al.; Hatlen et al. 2022; Higgins et al. 2021; Sharma et al. 2022). In August 2020, brownish-gray necrotic foliar lesions and damaged cones were observed in an experimental hopyard consisting of a breeding population of hop plants grown at the University of Minnesota - Southern Research and Outreach Center in Waseca, MN. The foliar lesions consisted of necrotic concentric circles with some possessing chlorotic halos. Damage to the cones often appeared as reddish brown as bands around cone midsections, scattered on bracts and bracteoles, and in severe cases near entire cones. Disease incidence within the experimental hopyard was observed on >50% of hop plants. No pycnidia were observed on leaves or cones following collection of samples. A total of eleven samples were obtained from diseased leaves or cones. Symptomatic plant tissue was surface-sterilized and sections excised from the leading edge of lesions were plated onto potato dextrose agar (PDA). Fungal growth was hyphal tipped and incubated at 22° C under a 12-h photoperiod for a period of 21 days (Hatlen et al 2022). Culture characteristics on PDA included raised white to light gray mycelium with irregular pycnidia distribution over the surface. DNA was extracted from mycelia using the MagMAX Plant DNA Isolation Kit (Applied Biosystems, Foster City, CA). A representative isolate (M4N) was selected for DNA amplification and bi-directional Sanger sequencing using the following primers ITS1/ITS4 (ITS) for the internal transcribed spacer, CYLH3F/H3-1b for histone 3 (HIS), and Ef1728f/EF1-986R for translation elongation factor 1-α (TEF) (Carbone and Kohn 1999; Glass and Donaldson 1995; White et al. 1990). Following amplification and sequencing, reads were trimmed and assembled using Geneious Prime (Biomatters, New Zealand). BLASTn analysis revealed that the ITS (GenBank Accession OQ144379), HIS (GenBank Accession OQ256246), and TEF (GenBank Accession OQ256245) were 99 - 100% identical to D. humulicola sequences (MN152927, MN180213, MN180207) infecting hop in other US regions (Allan-Perkins et al. 2019; Hatlen et al. 2022). To complete Koch's postulates, two sets each of six 3-month old plants of the hop cv. 'Chinook' were inoculated with either 50 mL of conidial suspension (6 x 105 conidia/mL) derived from pycnidia harvested from 28-day old cultures or with water as a negative control. Following inoculation, plants were then grown in a greenhouse at 100% relative humidity at 22°C with a 14-h photoperiod. Light brown lesions with concentric circles appeared on the adaxial side of the leaf after 3 weeks but were not observed on mock-inoculated plants. We subsequently re-isolated D. humulicola from 100% of infected leaves which was identified based upon colony and conidial morphology using descriptions from Higgins et al. (2021). alpha-conidia (n = 20) averaged 10.96 µm ± 1.12 in length and 5.11 µm ± 0.67 in width, were unicellular and hyaline. No beta-conidia were observed, consistent with previous reports of this pathogen. No disease symptoms appeared on mock-inoculated plants, and D. humulicola was not recovered from mock-inoculated plants. There is significant concern regarding the increasing prevalence of D. humulicola as an emerging pathogen affecting hop production across the Midwestern and Great Lakes region of North America. Future research is needed to determine differences in hop varietal susceptibility and fungicide efficacy for management of this disease.

First report of Neofusicoccum ribis causing stem blight and dieback of blueberry (Vaccinium corymbosum) in Michigan.

In July 2020, a 3-year-old 'Envoy' northern highbush blueberry bush (Vaccinium corymbosum L.) from a commercial farm in Van Buren County, Michigan was submitted to the Plant & Pest Diagnostics laboratory at Michigan State University. Field disease assessments across the 2-acre planting were an incidence of 2-5% and a severity of 50-100%. Symptoms included red shoot flagging and dead shoots retaining dry leaves, shoots with light green leaves and necrotic margins, and brown-black cankers at the base of the symptomatic shoots. Shoot sections displaying wood discoloration were surface disinfested by dipping in 95% ethanol and flame sterilized. The internal discolored tissues (0.5 cm2) were plated onto 1% ampicillin and streptomycin, quarter-strength potato dextrose agar (PDA) and incubated at room temperature until fungal colonies were observed. A fungus with rapid growth, developing white and then dark gray mycelium, resembling species in the family Botryosphaeriaceae was isolated and subcultured. After DNA extraction and amplification, sequences of three loci were obtained: the internal transcribed spacer (ITS) region, ß-tubulin (Bt), and elongation factor 1-α (EF1) using primer pairs ITS1/ITS4, Bt2a/Bt2b, and EF1-728/EF1-986R, respectively (Slippers et al. 2004). The sequences showed 100% identity with Genbank numbers KF766205 (ITS region, 562 bp - OP588109), MT592721 (Bt, 436 bp - OP585548), and MT592229 (EF1, 306 bp - OP585547) of N. ribis (Slippers et al. 2013, Zhang et al. 2021). Sequences were identified using PopSet 1995604550 (Zhang et al. 2021). Pathogenicity was tested on 2-year-old 'Blueray' blueberries. Five plants, 3 shoots per plant (n = 15) were surface-sterilized with a 3% bleach solution by rinsing, wounded with razor blades using a scratching method in 'X' patterns across the length of the wound, and then inoculated using mycelium plugs (5 mm) from 7-day old cultures grown on full-strength PDA. Plugs were crushed and spread onto the wound and the wound was wrapped with parafilm. Control plant shoots (n = 9) were mock inoculated using sterile PDA plugs. Plants were maintained in the greenhouse at 23°C under a 14-hour photoperiod and imaged at 0-, 7-, and 12-days post inoculation (dpi). Symptoms began developing within 7 dpi. At 12 dpi, 9 of 15 inoculated shoots began displaying leaf necrosis and deep red or brown stem discoloration 6 cm above and below the wound, while controls remained healthy. Fungi morphologically identical to the original isolate were reisolated from sections taken from 2.5, 6.3, and 7 cm above and below the inoculation site. Species identity was confirmed by sequencing as described above. Neofusicoccum species are widespread and commonly associated with canker and dieback symptoms of blueberries (Flor et al. 2022). To our knowledge, this is the first report of stem blight and dieback caused by N. ribis in Michigan blueberry production. The species N. parvum and N. ribis have been reported on southern highbush blueberries in California (Koike et al. 2014) and Florida (Wright and Harmon 2010), but neither has been reported on blueberry in Michigan. Accurate diagnoses of Botryosphaeria fungal species in blueberries is critical for effective disease control and yield loss reduction.

Fungicide Resistance and Host Influence on Population Structure in Botrytis spp. from Specialty Crops in California.

Botrytis is an important genus of plant pathogens causing pre- and postharvest disease on diverse crops worldwide. This study evaluated Botrytis isolates collected from strawberry, blueberry, and table grape berries in California. Isolates were evaluated for resistance to eight different fungicides, and 60 amplicon markers were sequenced (neutral, species identification, and fungicide resistance associated) distributed across 15 of the 18 B. cinerea chromosomes. Fungicide resistance was common among the populations, with resistance to pyraclostrobin and boscalid being most frequent. Isolates from blueberry had resistance to the least number of fungicides, whereas isolates from strawberry had resistance to the highest number. Host and fungicide resistance-specific population structure explained 12 and 7 to 26%, respectively, of the population variability observed. Fungicide resistance was the major driver for population structure, with select fungicides explaining up to 26% and multiple fungicide resistance explaining 17% of the variability observed. Shared and unique significant single-nucleotide polymorphisms (SNPs) associated with host and fungicide (fluopyram, thiabendazole, pyraclostrobin, and fenhexamid) resistance-associated population structures were identified. Although overlap between host and fungicide resistance SNPs were detected, unique SNPs suggest that both host and fungicide resistance play an important role in Botrytis population structure.

Optimizing Molecular Detection for the Hop Downy Mildew Pathogen Pseudoperonospora humuli in Plant Tissue.

Downy mildew-free hop plantlets and rhizomes are essential to limit the introduction of this destructive pathogen, Pseudoperonospora humuli, into hopyards. The objective of this research was to determine which DNA-based diagnostic tools are optimal for P. humuli detection in plant tissue. Quantitative real-time PCR (qPCR) assays with TaqMan probes for nuclear (c125015.3e1) and mitochondrial (orf359) DNA loci were developed and tested side by side. A recombinase polymerase amplification (RPA) assay was designed based on the orf359 DNA locus. The mitochondrial qPCR assay had a 10-fold lower limit of detection (100 fg of genomic DNA) and was 60% more effective in detecting P. humuli in asymptomatic stems than the nuclear-based assay. Both qPCR assays had linear standard curves (R2 > 0.99) but lacked the quantitative precision to differentiate leaf infections beyond 1 day postinoculation. A wide range of Cq values (≥4.9) in standardized tests was observed among isolates, suggesting that the number of mitochondria and nuclear DNA targets can vary. The absence of P. humuli DNA in symptomatic rhizomes was explained, in part, by the detection of Phytophthora DNA. However, the Phytophthora-specific atp9-nad9 assay cross-reacted with P. humuli, leading to false positive amplification. Sensitivity in the RPA assay was reduced by crude plant DNA extract. Improvements to the objectivity of calling positive amplifications and determining the onset of amplification from RPA fluorescence data were realized by applying the first and second derivatives, respectively. The orf359 qPCR assay is specific and sensitive, making it well suited for P. humuli diagnostics in plant tissue.

Rotting Grapes Don't Improve with Age: Cluster Rot Disease Complexes, Management, and Future Prospects.

Cluster rots can be devastating to grape production around the world. There are several late-season rots that can affect grape berries, including Botrytis bunch rot, sour rot, black rot, Phomopsis fruit rot, bitter rot, and ripe rot. Tight-clustered varieties such as 'Pinot gris', 'Pinot noir', and 'Vignoles' are particularly susceptible to cluster rots. Symptoms or signs for these rots range from discolored berries or gray-brown sporulation in Botrytis bunch rot to sour rot, which smells distinctly of vinegar due to the presence of acetic acid bacteria. This review discusses the common symptoms and disease cycles of these different cluster rots. It also includes useful updates on disease diagnostics and management practices, including cultural practices in commercial vineyards and future prospects for disease management. By understanding what drives the development of different cluster rots, researchers will be able to identify new avenues for research to control these critical pathogens.

Identification of Putative SDHI Target Site Mutations in the SDHB, SDHC, and SDHD Subunits of the Grape Powdery Mildew Pathogen Erysiphe necator.

Succinate dehydrogenase inhibitors (SDHIs) are fungicides used in control of numerous fungal plant pathogens, including Erysiphe necator, the causal agent of grapevine powdery mildew (GPM). Here, the sdhb, sdhc, and sdhd genes of E. necator were screened for mutations that may be associated with SDHI resistance. GPM samples were collected from 2017 to 2020 from the U.S. states of California, Oregon, Washington, and Michigan, and the Canadian province of British Columbia. Forty-five polymorphisms were identified in the three sdh genes, 17 of which caused missense mutations. Of these, the SDHC-p.I244V substitution was shown in this study to reduce sensitivity of E. necator to boscalid and fluopyram, whereas the SDHC-p.G25R substitution did not affect SDHI sensitivity. Of the other 15 missense mutations, the SDHC-p.H242R substitution was shown in previous studies to reduce sensitivity of E. necator toward boscalid, whereas the equivalents of the SDHB-p.H242L, SDHC-p.A83V, and SDHD-p.I71F substitutions were shown to reduce sensitivity to SDHIs in other fungi. Generally, only a single amino acid substitution was present in the SDHB, SDHC, or SDHD subunit of E. necator isolates, but missense mutations putatively associated with SDHI resistance were widely distributed in the sampled areas and increased in frequency over time. Finally, isolates that had decreased sensitivity to boscalid or fluopyram were identified but with no or only the SDHC-p.G25R amino acid substitution present in SDHB, SDHC, and SDHD subunits. This suggests that target site mutations probably are not the only mechanism conferring resistance to SDHIs in E. necator.

First report of halo blight of hop (Humulus lupulus) caused by Diaporthe humulicola in Quebec, Canada.

Halo blight of hop caused by Diaporthe humulicola has recently been reported in Michigan and Connecticut (Higgins et al. 2021, Allan-Perkins et al 2020). In August 2020 growers in Quebec, Canada reported necrotic foliar lesions and desiccation of the hop strobile (cone) on Chinook and Nugget cultivars. The foliar lesions were dry concentric circles with a chlorotic halo surrounding the lesions; no pycnidia were observed on leaves or cones. Up to 100% of the infected bract tissue was dry and easily shattered, the grower estimated that more than 90% of the plants in the hopyard exhibited symptoms. Twenty-six isolates were obtained from surface-sterilized leaf and cone tissue by plating the leading edge of lesions on potato dextrose agar. Fungal isolates were hyphal tipped and were incubated at 22°C with a 12 h photoperiod. After 21-days, all cultures were white to beige with pycnidia. DNA was extracted from cultures using the MagMAX Plant DNA Isolation Kit (Applied Biosystems, Foster City, CA). DNA amplification of a representative isolate (CD6C) was performed with primers ITS1/ITS4 (White et al. 1990) for the internal transcribed spacer (ITS), CYLH3F/H3-1b (Glass and Donaldson 1995) for histone 3 (HIS), and Ef1728f/EF1-986R (Carbone and Kohn 1999) for translation elongation factor 1-α (TEF). Amplification primers were used for bidirectional Sanger sequencing, reads were assembled using Geneious Prime (Biomatters, New Zealand), and identified using NCBI BLAST. BLAST results showed that the sequences for TEF, ITS, and HIS all had 100% pairwise identity to Diaporthe sp. 1-MI (MT909101, MT909099, MT909093, OK001342, MZ934713, OK001341). Futhermore, BLAST results showed that ITS and HIS have 100% pairwise identity D. humulicola (MN152929, MN180214). The TEF sequence also had 99.7% pairwise identity to D. humulicola (MN180209). Koch's postulates were conducted by inoculating six 3-mo-old 'Chinook' plants with conidia harvested from 28-day-old cultures and spraying 50 ml of inoculum (6 x 105 conidia/ml) or water to each plant. Plants were then stored in a greenhouse at 100% relative humidity at 22°C with a 14-h photo period. Lesions appeared on the adaxial side of the leaf after 21 days. D. humulicola was re-isolated from all infected leaf tissue, but not from any water inoculated plants and identified by conidial morphology using descriptions from Higgins et al. (2021). So far, Diaporthe sp. 1-MI appears to be synonymous with Diaporthe humulicola, but currently two names are being utilized (i.e. Diaporthe leaf spot and halo blight). In Higgins et al., (2021) it was proposed that the name halo blight might be more appropriate because disease symptoms are not confined to the leaves and cause significant blighting of cones. Halo blight caused by D. humulicola appears widespread in Michigan and Canada and may become an issue in other eastern North American growing regions with humid conditions.

Population Genetic Analyses of Botrytis cinerea Isolates From Michigan Vineyards Using a High-Throughput Marker System Approach.

As sequencing costs continue to decrease, new tools are being developed for assessing pathogen diversity and population structure. Traditional marker types, such as microsatellites, are often more cost effective than single-nucleotide polymorphism (SNP) panels when working with small numbers of individuals, but may not allow for fine scale evaluation of low or moderate structure in populations. Botrytis cinerea is a necrotrophic plant pathogen with high genetic variability that can infect more than 200 plant species worldwide. A panel of 52 amplicons were sequenced for 82 isolates collected from four Michigan vineyards representing 2 years of collection and varying fungicide resistance. A panel of nine microsatellite markers previously described was also tested across 74 isolates from the same population. A microsatellite and SNP marker analysis of B. cinerea populations was performed to assess the genetic diversity and population structure of Michigan vineyards, and the results from both marker types were compared. Both methods were able to detect population structure associated with resistance to the individual fungicides thiabendazole and boscalid, and multiple fungicide resistance (MFR). Microsatellites were also able to differentiate population structure associated with another fungicide, fluopyram, while SNPs were able to additionally differentiate structure based on year. For both methods, AMOVA results were similar, with microsatellite results explaining a smaller portion of the variation compared with the SNP results. The SNP-based markers presented here were able to successfully differentiate population structure similar to microsatellite results. These SNP markers represent new tools to discriminate B. cinerea isolates within closely related populations using multiple targeted sequences.

The hop downy mildew pathogen Pseudoperonospora humuli.

Pseudoperonospora humuli is an obligate biotrophic oomycete that causes downy mildew, one of the most devastating diseases of cultivated hop, Humulus lupulus. Downy mildew occurs in all production areas of the crop in the Northern Hemisphere and Argentina. The pathogen overwinters in hop crowns and roots, and causes considerable crop loss. Downy mildew is managed by sanitation practices, planting of resistant cultivars, and fungicide applications. However, the scarcity of sources of host resistance and fungicide resistance in pathogen populations complicates disease management. This review summarizes the current knowledge on the symptoms of the disease, life cycle, virulence factors, and management of hop downy mildew, including various forecasting systems available in the world. Additionally, recent developments in genomics and effector discovery, and the future prospects of using such resources in successful disease management are also discussed. TAXONOMY: Class: Oomycota; Order: Peronosporales; Family: Peronosporaceae; Genus: Pseudoperonospora; Species: Pseudoperonospora humuli. DISEASE SYMPTOMS: The disease is characterized by systemically infected chlorotic shoots called "spikes". Leaf symptoms and signs include angular chlorotic lesions and profuse sporulation on the abaxial side of the leaf. Under severe disease pressure, dark brown discolouration or lesions are observed on cones. Infected crowns have brown to black streaks when cut open. Cultivars highly susceptible to crown rot may die at this phase of the disease cycle without producing shoots. However, foliar symptoms may not be present on plants with systemically infected root systems. INFECTION PROCESS: Pathogen mycelium overwinters in buds and crowns, and emerges on infected shoots in spring. Profuse sporulation occurs on infected tissues and sporangia are released and dispersed by air currents. Under favourable conditions, sporangia germinate and produce biflagellate zoospores that infect healthy tissue, thus perpetuating the infection cycle. Though oospores are produced in infected tissues, their role in the infection cycle is not defined. CONTROL: Downy mildew on hop is managed by a combination of sanitation practices and timely fungicide applications. Forecasting systems are used to time fungicide applications for successful management of the disease. USEFUL WEBSITES: https://content.ces.ncsu.edu/hop-downy-mildew (North Carolina State University disease factsheet), https://www.canr.msu.edu/resources/michigan-hop-management-guide (Michigan Hop Management Guide), http://uspest.org/risk/models (Oregon State University Integrated Plant Protection Center degree-day model for hop downy mildew), https://www.usahops.org/cabinet/data/Field-Guide.pdf (Field Guide for Integrated Pest Management in Hops).

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.

Fungicide Efficacy Against Pseudoperonospora humuli and Point Mutations Linked to Carboxylic Acid Amide Resistance in Michigan.

Hops have expanded as a niche crop in Michigan and other production areas in the eastern United States, but growers in these regions face annual downy mildew outbreaks incited by Pseudoperonospora humuli, exacerbated by frequent rainfall and high relative humidity. We evaluated the efficacy of foliar- and drench-applied fungicides against downy mildew and examined Michigan isolates for point mutations linked to carboxylic acid amide (CAA) resistance. Disease severity and density were assessed weekly in 2016 and 2017 in nontrellised research hop yards in Michigan. Area under the disease progress curve values for disease severity were significantly lower for plants treated with oxathiapiprolin, ametoctradin/dimethomorph, fluopicolide, cyazofamid, or mandipropamid (90.6 to 100% control) compared with those treated with fosetyl-Al (64.3 to 93.0% control) at both locations for both years. Drench treatments of fluopicolide and oxathiapiprolin/mefenoxam reduced disease density and severity at both locations but were only moderately effective (76.4 to 91.5% control). To assess CAA resistance, the cellulose synthase CesA3 gene was aligned using reference downy mildew species and primers designed to amplify the 1105 and 1109 amino acids. Point mutations conferring CAA resistance were not detected at these loci for sporangia from 42 symptomatic shoots collected from 11 commercial hop yards. These efficacy results for hop downy mildew are needed to guide disease recommendations in this expanding Michigan industry. The absence of resistant genotypes indicates that Michigan growers can continue to utilize CAA-containing commercial fungicides as part of an overall downy mildew management program.

Allele-Specific Detection Methods for QoI Fungicide-Resistant Erysiphe necator in Vineyards.

Grapevine powdery mildew (GPM), caused by the fungus Erysiphe necator, is a constant threat to worldwide production of grape berries, requiring repeated use of fungicides for management. The frequent fungicide applications have resulted in resistance to commonly used quinone outside inhibitor (QoI) fungicides and the resistance is associated with single-nucleotide polymorphisms (SNPs) in the mitochondrial cytochrome b gene (cytb). In this study, we attempted to detect the most common SNP causing a glycine to alanine substitution at amino acid position 143 (i.e., G143A) in the cytb protein, to track this resistance using allele-specific TaqMan probe and digital-droplet PCR-based assays. Specificity and sensitivity of these assays showed that these two assays could discriminate SNPs and were effective on mixed samples. These diagnostic assays were implemented to survey E. necator samples collected from leaf and air samples from California and Oregon grape-growing regions. Sequencing of PCR amplicons and phenotyping of isolates also revealed that these assays accurately detected each allele (100% agreement), and there was an absolute agreement between the presence or absence of the G143A mutation and resistance to QoIs in the E. necator sampled. These results indicate that the developed diagnostic tools will help growers make informed decisions about fungicide selections and applications which, in turn, will facilitate GPM disease management and improve grape production systems.