Timothy D. Miles

Strobilurin (QoI) Resistance in Populations of Erysiphe necator on Grapes in Michigan

Powdery mildew, caused by Erysiphe necator, is the most common and destructive disease of grapes (Vitis spp.) worldwide. In Michigan, it is primarily controlled with fungicides, including strobilurins (quinone outside inhibitors [QoIs]). Within the United States, resistance to this class of fungicides has been reported in E. necator populations in some east coast states. Among 12 E. necator isolates collected from five Michigan vineyards in 2008, one carried the G143A single-nucleotide mutation responsible for QoI resistance. This isolate was confirmed to be resistant in a conidium germination assay on water agar amended with trifloxystrobin at 0.001, 0.01, 0.1, 1, 10, or 100 μg/ml and salicylhydroxamic acid (100 mg/liter). The mutant isolate was able to germinate on media amended with 100 μg/ml trifloxystrobin, whereas a representative wild-type isolate did not germinate at concentrations higher than 0.1 μg/ml. In 2009, 172 isolates were collected from a total of 21 vineyards (juice and wine grapes): three vineyards with no fungicide application history (baseline sites), six research vineyards, and 12 commercial vineyards. QoI resistance was defined as the effective concentration that inhibited 50% of conidial germination (EC50) > 1 μg/ml. Isolates from baseline sites had EC50 values mostly below 0.01 μg/ml, while isolates that were highly resistant to trifloxystrobin (EC50 > 100 μg/ml) occurred in five research and three commercial wine grape vineyards at frequencies of 40 to 100% and 25 to 75% of the isolates, respectively. The G143A mutation was detected in every isolate with an EC50 > 1 μg/ml. These results suggest that fungicide resistance may play a role in suboptimal control of powdery mildew observed in some Michigan vineyards and emphasizes the need for continued fungicide resistance management.

Systematic Development of Phytophthora Species-Specific Mitochondrial Diagnostic Markers for Economically Important Members of the Genus

The genus Phytophthora contains many invasive species to the U.S.A. that have the potential to cause significant damage to agriculture and native ecosystems. A genus and species-specific diagnostic assay was previously reported based on mitochondrial gene order differences that allowed for the systematic development of 14 species-specific TaqMan probes for pathogen detection ( Bilodeau et al. 2014 ). In this study, an additional 32 species-specific TaqMan probes for detection of primarily invasive species have been validated against 145 Phytophthora taxa as well as a range of Pythium and plant DNA samples. All validated probes were found to be species-specific and could be multiplexed with a genus-specific probe. The lower limit of linear detection using purified genomic DNA ranged from 1 to 100 fg in all assays. In addition, 124 unique TaqMan probes for Phytophthora spp. developed in silico are presented, which, if testing confirms they are species-specific, will provide diagnostic capabilities for approximately 89% of the genus. To enhance sensitivity of detection for several species that contained a single nucleotide polymorphism (SNP) in the reverse primer, a second primer was developed that is added in a small amount to the master mix. Furthermore, a PCR-RFLP system was developed that could be used to identify individual species when multiple species are present in a sample, without requiring cloning or sequencing. Several experiments were also conducted to compare various qPCR thermal cyclers and independent validation experiments with another research laboratory to identify possible limitations when the assays are used on a range of equipment in different labs. This system represents a comprehensive, hierarchal approach to increase the detection capability and provide tools to help prevent the introduction of invasive Phytophthora species.

Molecular approaches for biosurveillance of the cucurbit downy mildew pathogen, Pseudoperonospora cubensis

Abstract Globalization has allowed for rapid movement of plant pathogens that threaten food security. Successful disease management largely depends on timely and accurate detection of plant pathogens causing epidemics. Thus, biosurveillance of epidemic plant pathogens such as Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew, is becoming a priority to prevent disease outbreaks and deploy successful control efforts. Next Generation Sequencing (NGS) facilitates rapid development of genomics resources needed to generate molecular diagnostics assays for P. cubensis. Having information regarding the presence or absence of the pathogen, amount of inoculum, crop risk, time to initiate fungicide applications, and effective fungicides to apply would significantly contribute to reducing losses to cucurbit downy mildew. In this article, we discuss approaches to identify unique loci for rapid molecular diagnostics using genomic data, to develop molecular diagnostic tools that discriminate economically important pathogen alleles (i.e. mating type and fungicide resistance), and how to use molecular diagnostics with current and future spore trap strategies for biosurveillance purposes of important downy mildew pathogens. The combined use of these technologies within the already existent disease management framework has the potential to improve disease control.

Floral Scent Mimicry and Vector-Pathogen Associations in a Pseudoflower-Inducing Plant Pathogen System

Several fungal plant pathogens induce ‘pseudoflowers’ on their hosts to facilitate insect-mediated transmission of gametes and spores. When spores must be transmitted to host flowers to complete the fungal life cycle, we predict that pseudoflowers should evolve traits that mimic flowers and attract the most effective vectors in the flower-visiting community. We quantified insect visitation to flowers, healthy leaves and leaves infected with Monilinia vaccinii-corymbosi (Mvc), the causative agent of mummy berry disease of blueberry. We developed a nested PCR assay for detecting Mvc spores on bees, flies and other potential insect vectors. We also collected volatiles from blueberry flowers, healthy leaves and leaves infected with Mvc, and experimentally manipulated specific pathogen-induced volatiles to assess attractiveness to potential vectors. Bees and flies accounted for the majority of contacts with flowers, leaves infected with Mvc and healthy leaves. Flowers were contacted most often, while there was no difference between bee or fly contacts with healthy and infected leaves. While bees contacted flowers more often than flies, flies contacted infected leaves more often than bees. Bees were more likely to have Mvc spores on their bodies than flies, suggesting that bees may be more effective vectors than flies for transmitting Mvc spores to flowers. Leaves infected with Mvc had volatile profiles distinct from healthy leaves but similar to flowers. Two volatiles produced by flowers and infected leaves, cinnamyl alcohol and cinnamic aldehyde, were attractive to bees, while no volatiles manipulated were attractive to flies or any other insects. These results suggest that Mvc infection of leaves induces mimicry of floral volatiles, and that transmission occurs primarily via bees, which had the highest likelihood of carrying Mvc spores and visited flowers most frequently.

First Report of Leaf Rust on Southern Highbush Blueberry caused by Thekopsora minima in California

Leaf rust symptoms have been intermittently observed on southern highbush blueberry (Vaccinium corymbosum L. × V. darrowii Camp) in California. Rust symptoms were first seen in 2010 near Carpinteria, CA (M. Kong, personal communication) and then in July 2016 on leaves of three blueberry cultivars Emerald, Jewel, and Snow Chaser in a field trial in Watsonville. In early 2017, leaf rust was observed again in Watsonville on the same cultivars and on various 3-year-old plants. Symptoms were found primarily on older leaves. Purplish-brown necrotic spots were present on the adaxial leaf surface, while the abaxial leaf side exhibited orange to yellow colored uredinia. Upon closer inspection using a light microscope, the uredinia were clustered on the leaf surface, ovoid in shape, and approximately 100 to 400 μm in diameter. Certain rust species possess ostiolar cells projections near the ostiole of the uredinium, which aid in taxonomic identification; however, ostiolar cells were not observed. Each uredinium contained numerous ellipsoid urediniospores with spines that emanated from the spore walls. When measured, urediniospores were 17.5 to 30 × 17.5 to 20 μm (average 24 × 19 μm, n = 25). Leaf samples with rust signs and symptoms were taken and stored at 4°C for further taxonomic examination. Potential alternate hosts (i.e., Tsuga spp.) for blueberry leaf rust were not present. After morphological evaluation, the rust pathogen was identified as Thekopsora minima P. Syd. & Syd. and two specimens from different plants were sent to the U.S. National Fungus Collection (BPI 910346 and BPI 910349) (Sato et al. 1993; Schilder and Miles 2011). DNA was extracted from six samples and two loci amplified from each sample. A 1,410-bp fragment spanning 5.8S-ITS2-28S rDNA was amplified with Rust2inv and LR6 primers (fragment 1) and a 1,697-bp fragment spanning the 18S rDNA was amplified with Rust18S-R and NS1 primers (fragment 2) (Aime 2006). Fragment 1 shared 100% identity (1,007 of 1,007 bp) with a T. minima isolate (BRIP57654) that infected V. corymbosum from Australia; fragment 2 shared >99% identity (1,207 of 1,210 bp) with the same isolate (accession nos. KC763340 and KT199391, respectively). Sequences for fragment 1 and fragment 2 from isolate (001) were deposited in GenBank (KY991374 and KY991375, respectively). Although another rust, Naohidemyces vaccinii, has been reported on V. membranaceum Douglas ex Torr. in CA, recent reports of T. minima in Australia, China, Mexico, South Africa, and in several states in the U.S. (OR, MI, DE, NY) suggest that T. minima is the most commonly encountered pathogen causing leaf rust on northern and southern highbush blueberries (Schilder and Miles 2011; Wiseman et al. 2016). To our knowledge, this is the first report of T. minima on any plant species within CA. In 2017, another rust species was found in Rubus spp. that was unobserved in CA previously, which could be related to significant winter precipitation (Shands et al. 2018). Since the alternate host Tsuga spp. is outside the central coast region of CA, further research is necessary to understand the epidemiology of T. minima in CA.

Temporal occurrence and niche preferences of Phytophthora species causing brown rot of citrus in the Central Valley of California

Brown rot of citrus fruit is caused by several species of Phytophthora and is currently of serious concern for the California citrus industry. Two species, Phytophthora syringae and P. hibernalis, are quarantine pathogens in China, a major export market for California citrus. To maintain trade and estimate the risk of exporting a quarantine pathogen, the distribution and frequency of Phytophthora spp. causing brown rot of orange in major growing areas of California was investigated. Symptomatic fruit were collected from navel (winter to late spring) and Valencia (late spring to summer) orange orchards from 2013 to 2015. Species identification of isolates was based on morphological characteristics, random amplified polymorphic DNA banding patterns, and sequencing of the internal transcribed spacer and the partial cox2/spacer/cox1 regions from axenic cultures, or directly on DNA from fruit tissue using a multiplex TaqMan quantitative polymerase chain reaction assay. In winter samplings, the incidence of P. syringae based on the number of fruit with Phytophthora spp. detection ranged from 73.6 to 96.1% for the two counties surveyed. The remaining isolates were identified as P. citrophthora. In late spring or summer, only P. citrophthora was recovered. P. hibernalis and P. nicotianae were not detected in any fruit with brown rot symptoms. These results indicate that P. syringae is currently an important brown rot pathogen of citrus fruit in California during the cooler seasons of the year. In winter 2016 and 2017, P. syringae was recovered by pear baiting at a high incidence from leaf litter and from a small number of rhizosphere soil or root samples but not from living leaves on the tree. In contrast, P. citrophthora was rarely found in leaf litter but was commonly detected in the rhizosphere. Thus, leaf litter is a major inoculum source for P. syringae and this species occupies a distinct ecological niche.