Managing powdery mildew of wasabi, caused by Erysiphe cruciferarum, using biological control agents and organic fungicides. E. C. BETZ AND Z. K. PUNJA. Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada
Powdery mildew (PM) reduces photosynthesis and can affect yield of cultivated crops. Severe infections can result in chlorosis and defoliation. Disease surveys conducted over the 2016–2018 period indicate that PM is a prevalent foliar pathogen on wasabi (Wasabia japonica (Miq.) Matsum.) in British Columbia. Erysiphe cruciferarum Opiz ex L. Junell was confirmed as the causal agent using sequence analysis of the ITS1-5.8S-ITS2 region, along with conidia and conidiophore morphology. To evaluate management options for PM, the efficacy of four commercial products was assessed over three separate trials conducted in a commercial wasabi greenhouse. The products tested included two biological pesticides, Rhapsody® ASOTM (Bacillus subtilis strain QST 713, 1% v/v) and Actinovate® SP (Streptomyces lydicus strain WYEC 108, 0.5% v/v); an organic fungicide, Cueva® (Copper Octanoate soap, 1% v/v); and a plant extract, Regalia Maxx® (Reynoutria sachalinensis extract, 0.25% v/v). Natural infection by E. cruciferarum was allowed to progress on the plants. Treatments were made at 2-week intervals over 10–12 weeks. Leaves were assessed for PM biweekly based on percentage of leaf surface infected, which was then converted to area under the disease progress curve (AUDPC) for each treatment. Applications of Regalia Maxx® and Cueva® significantly (P < 0.05) reduced the progression of PM after multiple applications. Rhapsody® also reduced disease progression in some trials, but to a lesser extent. Nutrient analyses of wasabi leaves treated with Cueva® showed elevated levels (200–400 ppm) of copper. Total phenolic content assays performed on wasabi leaves treated with Regalia Maxx® showed a trend for increased phenolics compared with a control, but the increase was not significant. Currently, only Rhapsody® and Cease® (also Bacillus subtilis strain QST 713) are registered for use on wasabi in Canada for PM management.
Endophytic fungi and moulds associated with cannabis (Cannabis sativa) plants. D. C. COLLYER, S. LUNG, C. SCOTT, J. HOLMES AND Z. K. PUNJA. Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada
Moulds associated with marijuana (Cannabis sativa L.) plants can reduce quality of the crop under both indoor and outdoor production environments. These fungi may colonize the inflorescence (buds) during development or after harvest, or colonize the internal tissues as endophytes. Leaf, petiole, crown and pith tissues were surface-sterilized in 0.65% sodium hypochlorite for 20 min, followed by rinses in sterile distilled water and plated onto potato dextrose agar containing 100 mg L−1 streptomycin sulphate (PDA+S) to determine the types of fungi recovered. Airborne moulds were quantified by placing exposed PDA+S dishes in the growing environment for one hour and returning them to the laboratory. Sampling was repeated over time. Colonies were enumerated, subcultured and identified using a pair of universal eukaryotic primers that amplified the ITS1-ITS2 region of ribosomal DNA. A range of soil-colonizing and cellulolytic fungi, including species of Penicillium, Chaetomium, Trametes, Trichoderma, Beauveria, Lecanicillium, Simplicillium and Fusarium, were recovered from cannabis plant tissues. A range of unidentified bacteria, including Bacillus spp., were also present. Airborne moulds included Penicillium, Fusarium, Trichoderma, Cladosporium and unidentified yeasts. Plants grown in coconut fibre substrate revealed that a portion of the mycoflora could colonize the pith internally for distances of 30–35 cm from the crown. Fungal communities present in growing media such as coconut fibre, that include species of Penicillium and Aspergillus, are potential sources of mould contaminants on cannabis plants. Aerial distribution of spores and dissemination through vegetative propagation of cannabis plants can also result in spread, and entry through wound sites on roots and crowns facilitates establishment. Pruning sites on stems were shown to be colonized by isolates of Penicillium, Fusarium, Trichoderma and Chaetomium. A diverse range of fungi may therefore be present on or within cannabis plants, some of which are known pathogens and others are presumed endophytes with as-yet undetermined function.
Nanomaterials in our environment: challenges and opportunities. B. D. GATES. Department of Chemistry and 4D LABS, Simon Fraser University, Burnaby, BC V5A 1S6, Canada
Nanotechnology is a rapidly expanding field, pursued by global research and development efforts, and sought after for commercialization for the often unique and tunable properties of materials with nanoscale dimensions. Engineered nanomaterials, or those materials purposefully prepared in composition and nanoscale dimensions, are in many mainstream products. In this presentation, the importance of nanotechnology and the potential utility of these materials will be briefly discussed. There remain many opportunities and unknowns for most engineered nanomaterials, whether produced through more natural means or the result of built-for-purpose preparation methods. Some of these materials are known to have specific organ toxicities and other adverse health effects. Other materials are being utilized to provide new tools that enable applications in more efficient catalysis, higher contrast imaging, and tuned release of molecular species. This presentation will introduce some of these opportunities and challenges. We are just beginning to understand the various pathways that nanomaterials take within biological and ecological systems as we seek to understand their ultimate fate when encountered in either type of system. At the forefront of this challenge is the development of proper tools and techniques for assessing the fate and impact of these materials. Efforts are underway to understand the pathways for various engineered nanomaterials in biological and environmental systems. This presentation will discuss the challenges, concerns and recent progress in developing methodologies for a variety of engineered nanomaterials, and will highlight on-going efforts to establish the fate of these materials in the environment and opportunities for further development.
Elucidating the host-pathogen interaction between Phytophthora and woody plant species through dual RNA-sequencing. K. N. HRYWKIW, N. FEAU, A. DALE, A. UZUNOVIC, B. M. VAN DER MEER AND R. HAMELIN. The University of British Columbia, Forest and Conservation Science, 2424 Main Mall, Vancouver, BC V6T 1Z4, Canada; and (A.D., A.U.) FPInnovations, 2665 East Mall Vancouver, BC V6T 1Z4, Canada
Forests provide essential economic and ecosystem benefits but have become increasingly threatened by invasive pests and pathogens, which are often transported through the global plant trade. Phytophthora pathogens can cause various diseases in trees, including root and collar rot, trunk cankers and leaf blight, resulting in millions of dollars in forest losses annually. Each Phytophthora–host interaction is unique, some have devastating landscape level effects, whereas others go unnoticed; the genomic drivers of these differences remain unresolved. The goal of our research is to uncover the genomic factors that contribute to Phytophthora host range, aggressiveness and tissue preference, and host resistance and susceptibility. We are conducting Illumina NovaSeq dual RNA-sequencing to investigate conserved and unique transcripts pertaining to different Phytophthora–host interaction profiles. We performed artificial inoculations of eight Phytophthora on 10 forest species, generating a total of 80 different host–pathogen interactions. Infected tissues from stems and leaves were sampled at two time points. Each interaction was documented with photographs and analysed using a machine learning algorithm we developed to quantify the amount of infected tissue, from which we will assess host susceptibility and Phytophthora host range. Our preliminary bioinformatic analyses of the transcriptomes indicate that on average ~16% of Illumina reads align to the pathogen genome, suggesting that our inoculation approach was successful. Investigating the transcriptomes will increase our understanding of the genomic drivers of Phytophthora pathogenicity and host susceptibility, which will be used to develop tools to detect potentially harmful forest Phytophthora species, and help establish appropriate mitigation strategies.
Prediction of genetic resistance to blister rust (Cronartium ribicola) in natural western white pine (Pinus monticola Douglas ex D. Don) populations using a SNP genotyping tool. J.-J. LIU, A. ZAMANY, X.-R. LI AND S. GELLNER. Pacific Forestry Centre, Canadian Forest Service, 506 Western Burnside Road, Victoria, BC V8Z 1M5, Canada
Cronartium ribicola J.C. Fisch. is a rust fungus that causes white pine blister rust (WPBR) on five-needle pines. After accidental introduction into North America about 100 years ago, this invasive fungus has caused heavy mortality in western white pine (Pinus monticola), limber pine (Pinus flexilis), whitebark pine (Pinus albicaulis) and other native five-needle pines due to low genetic resistance in these trees across North America. Breeding by selection of rare resistant individuals of these species is time consuming; therefore, genomics-based tools are highly desirable to speed up the breeding process. Major gene resistance (MGR) has not been found in Canadian western white pine stands, however, offspring of MGR trees found in Oregon have been planted in BC at several locations since 1987. In past years, our laboratory has constructed a genetic map for the P. monticla Cr2 locus that confers MGR to WPBR, and identified DNA markers of single nucleotide polymorphisms (SNPs) within functional genes that were tightly linked to Cr2. In the present study, we developed a KASP SNP genotyping tool based on these Cr2-linked SNPs. Using this SNP genotyping tool, we analysed 46 P. monticla open-pollinated seed lots, randomly selected across BC’s landscape, with about 10 seeds per seed lot. SNP genotyping results showed that the majority of the tested seeds in each seed lot had homozygous genotype with the recessive cr2-linked allele, indicating that all 46 seed lots were cr2-susceptible. The Cr2-linked allele was detected only in ~10% of the total genotyped seeds with significant deviation of Hardy–Weinberg equilibrium (HWE) (P < 0.01), which may be caused by a population bottleneck resulting from disease, habitat loss or overharvesting. In particular, the Cr2-linked allele frequency was significantly higher in coastal regions than interior regions (10% vs. 5%, P < 0.05). These results demonstrate that the KASP SNP genotyping tool developed here is powerful for the prediction of Cr2-resistane in wild parental trees across BC’s landscape.
First report of ringspot and vein-clearing symptoms on Wasabia japonica plants associated with Wasabi mottle virus in North America. J. L. MACDONALD, E. C. BETZ, Y. Q. LI, Z. K. PUNJA, M. J. BOUTHILLIER, R. M. DEYOUNG AND M. G. BERNARDY. Summerland Research and Development Centre, Agriculture and Agri-Food Canada, 4200 Highway 97 Box 5000, Summerland, BC V0H 1Z0, Canada; and (E.C.B., Z.K.P.) Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada
Wasabia japonica (Miq) Matsum. is cultivated for its rhizome which is traditionally consumed with Japanese cuisine. In 2017, ringspot and vein-clearing symptoms were observed in a commercial greenhouse in British Columbia. Symptomatic leaves were ground in phosphate buffer and used to mechanically inoculate Nicotiana benthamiana, N. clevelandii, N. glutinosa, N. occidentalis, N. tabacum, Chenopodium quinoa, C. amaranticolor, Gomphrena globosa, Brassica perviridis and Tetragonia tetragonioides. After 4–11 days most hosts showed symptoms of necrotic lesions. N. benthamiana showed mild puckering, and B. perviridis showed mild puckering, vein banding and chlorosis. Transmission electron microscopy revealed the presence of ~250–300 nm long rod-shaped virions in inoculated N. occidentalis and N. clevelandii. RNA was extracted and purified from symptomatic wasabi tissue, and RT-PCR was conducted with six degenerate broad-spectrum primer sets for detecting the genera Tobamovirus, Nepovirus, Potyvirus, Ilarvirus and Cucumovirus, and the species Turnip ringspot virus and Alfalfa mosaic virus. One primer set, Ilar1F5 univ-F/Ilar1R7 univ-R, produced positive band sizes of ~300 and ~600 bp, which were sequenced. A MegaBLAST (NCBI) search identified Wasabi mottle virus (WMoV), a Tobamovirus, to be present. Two wasabi cultivars were inoculated as previously described: ‘Greenthumb’ developed ringspots and vein-clearing after 22–23 days in the greenhouse, while ‘Daruma’ were non-symptomatic. A PCR test confirmed the presence of WMoV in naturally infected and inoculated wasabi plants. A survey of a greenhouse facility conducted in June 2018 showed the frequency of diseased, typically non-symptomatic, plants to be 38%. Symptom development was more pronounced at higher temperatures (>30°C) than cooler temperatures (21–24°C). BC isolates showed 99% sequence identity to isolate ‘Alishan’ (GenBank accession no. KJ207375.1) and 98% identity to a Japanese strain (GenBank accession no. AB017504.1). The origin of WMoV is presumed to be through the importation of infected ‘Greenthumb’ plants from Taiwan, and subsequent propagation.
Efficacy of ActiveFlower, a foliar fertilizer containing boron, on the development of sclerotinia stem rot (Sclerotinia sclerotiorum) on canola (Brassica napus). L. NI, K. LEE AND Z. K. PUNJA. Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada
Canola (Brassica napus L.) is grown extensively in Canada and sclerotinia stem rot (Sclerotinia sclerotiorum Lib. De Bary) is one of the most destructive fungal diseases on this crop. The effect of Active Flower™ (AF), a foliar fertilizer containing 3% boron (B) plus 8:4:12 of N:P:K, in reducing disease severity was evaluated. Concentrations of AF at 0, 0.1, 0.3 and 0.5 mL 100 mL−1 were tested for inhibition of mycelial growth of S. sclerotiorum in potato dextrose broth. The most pronounced growth inhibition was observed at 0.5 mL 100 mL−1. Boric acid (BA), an important component of AF, was also tested against fungal growth at 0.25, 0.5, 0.75 and 10 mL L−1, and no significant effect was found. Applications of AF at 0.1, 0.3 and 0.5 mL 100 mL−1 were made weekly to canola plants grown under greenhouse conditions. Treatments of AF, formulated without B at the same concentrations as AF, and BA at 10 mL L−1 were also applied. After four applications, nutrient levels and enzyme activities were analysed on leaf tissues. Results showed that AF at 0.5 mL 100 mL−1 and BA at 10 mL L−1 enhanced B levels by five-fold and three-fold, respectively, compared with the control. These treatments significantly (P < 0.05) reduced mycelial development and lesion size of S. sclerotiorum on detached leaves. Levels of phenolic compounds in leaves treated with 0.5 mL 100 mL−1 AF were enhanced by two-fold compared with the control. There were no significant differences in lignin, peroxidase and polyphenol oxidase between the control and AF treatments. These results suggest that B had a positive effect on canola plants in enhancing tolerance to disease.
A high throughput genomic surveillance tool for the sudden oak death pathogen. R. RADHAMONY, A. CAPRON, N. FEAU, A. DALE AND R. HAMELIN. The University of British Columbia, Forest and Conservation Science, 2424 Main Mall, Vancouver, BC V6T 1Z4, Canada; and (A.D.) FPInnovations, 2665 East Mall Vancouver, BC V6T 1Z4, Canada
Invasive species such as Phytophthora ramorum, the pathogen that causes sudden oak death represent a threat to Canada’s trees and forests. Outbreaks of these invasive pathogens can cause substantial economic losses and result in irreversible damage to the environment. The key to reducing risk is vigilant biosurveillance to increase preparedness and facilitate interventions. A bioinformatic pipeline developed in our lab allowed broad comparisons of whole genome sequences and the identification of target regions that can differentiate between Phytophthora spp. at the species level and between P. ramorum lineages. Herein we have developed a high throughput and scalable assay that can be used to rapidly screen large numbers of samples for fast and precise identification as well as classification of Phytophthora taxa. Combining a targeted sequencing approach with the cost-effectiveness of high-throughput genome sequencing, we have developed two detection panels comprising 114 amplicons that can generate genome sequences that are polymorphic among species (panel I) or within species (panel II). We tested these panels on 28 samples and generated over 1000 single nucleotide polymorphisms. We have used variant calling, principal component analysis and phylogenetic assignment to accurately assign Phytophthora species to the proper clade and P. ramorum samples to the proper lineages. This approach is scalable since each panel can be augmented as needed, and high-throughput as 384 samples can be pooled in a single reaction. The assay is suitable for targeted sequencing during P. ramorum outbreaks to facilitate an understanding of its spread and to enable early detection and control.
Emerging diseases and their impact on young hazelnut orchards in the Fraser Valley, British Columbia. S. SABARATNAM AND B. DRUGMAND. Abbotsford Agriculture Centre, Ministry of Agriculture, 1767 Angus Campbell Road, Abbotsford, BC V3G 2M3, Canada
Hazelnut (Corylus avellana L.) production in the Fraser Valley, British Columbia is in decline due to eastern filbert blight (EFB), caused by Anisogramma anomala (Peck) Müller. As a result, infected orchards are being replanted with EFB-resistant varieties. In 2017 and 2018, 4- to 8-year-old orchards were examined for overall plant health and diseases caused by plant pathogens. Dieback and ‘flagging’ of branches due to enlarging cankers of brown to dark-brown lesions with light brown margins and, in some cases, sunken cankers with splitting margins and ‘V-shaped’ internal tissue discolouration were commonly observed on stems and tree trunks of ‘Jefferson’, ‘Yamhill’ and ‘Theta’. These cankers produced tiny, dark coloured pycnidia and pycniospores typical of a Phomopsis sp., and the pathogen was confirmed by DNA analysis. On some trees, symptoms of dieback and defoliation of branches resulting from bleeding cankers on tree-trunks were observed. These symptoms were suspected to be associated with bacterial canker (Pseudomonas avellanae (Psallidas) Janse et al.) or bacterial blight (Xanthomonas arboricola pv. corylina (Miller et al.)), warranting further confirmation. In poorly drained soils, root rot was commonly observed, resulting in weakening of foliage, chlorosis and tree decline. Pathogen Phytophthora sp. ‘hungarica’ was isolated from infected root tissues and confirmed by DNA analysis. Studies have been undertaken to confirm the species identity, pathogenicity and disease epidemiology of the pathogens affecting young hazelnut orchards.
Management of powdery mildew caused by Golovinomyces cichoracearum on Cannabis sativa. C. A. SCOTT AND Z. K. PUNJA. Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada
Powdery mildew (PM) caused by Golovinomyces cichoracearum can affect cannabis plants at all stages of growth, reducing overall yield and quality. If not managed properly, this pathogen can be especially problematic for producers as it can infect flower tissues (buds) making them unusable. Cannabis plants of a mildew-susceptible strain ‘Copenhagen Kush’ (CPH) were obtained as cuttings and potted in a 75% coir (Canna Coco) and 25% perlite mix. Cuttings were propagated for 14 days inside a humidity dome and then placed under two Sunblaster brand 54 watt 6400K T5H0 lights with a 24 h photoperiod. After 5–10 days, the CPH plants were initially assessed to determine the level of natural background mildew infection for each treatment group, after which they were allocated to their respective treatments. Plants, in groups of four, received four sprays, and five disease assessments, each on a weekly basis, over the span of 5 weeks. The experiment was conducted twice. The treatments applied included Actinovate® SP (Streptomyces lydicus strain WYEC 108, at 0.38 g L−1), MilStop® (85% potassium bicarbonate, at 2.4 g L−1), Neem Oil (52% neem oil, at 1 mL L−1), Regalia Maxx® (20% extract of Reynoutria sachalinensis, at 2.5 mL L−1), Rhapsody® ASO (Bacillus subtilis strain QST 713, at 15 mL L−1) and ZeroTol® (27% hydrogen peroxide, at 10 mL L−1). Assessments of final disease severity (percentage of leaf area infected) were converted to Area Under the Disease Progress Curve (AUDPC). The results showed that MilStop® and Regalia® were the two most effective treatments, with significantly (P < 0.05) lower AUDPC values (13.9 and 16, respectively) compared with the control (AUDPC value of 89.3). Rhapsody®, ZeroTol®, Neem and Actinovate® were less effective, with AUDPC scores of 39.5, 53, 55 and 73.6, respectively. These products therefore have varying efficacy at managing PM on cannabis. Currently, MilStop® and ZeroTol are registered for use on cannabis in Canada for PM disease control, while Actinovate® has been deregistered. Regalia® has potential for mildew control on cannabis.
Validation of microchip based real-time PCR kits for simultaneous detection of potato pathogens. M. SLYADNEV, S. GILL AND R. GILL. Lumex Instruments Canada Ltd, 7294 Fraserview Place, Mission, BC V4S 0A3, Canada
The proposed presentation deals with the validation of a novel microchip based real-time PCR technique invented with the aim for highly sensitive, selective, rapid and easy-to use detection and identification of economically important potato pathogens. Test-systems were developed for the microchip with miniaturized volume of microreactors (1.2 μL). An entire panel of six bacterial and one fungal pathogens of potato was developed in the microchip by optimizing unified amplification regime with a fast ramping PCR analyser AriaDNA. Similarly, a panel of six viral and one viroid pathogens of potato was developed in another microchip. In the test-systems, the mixture containing all the essential components of PCR along with optimized stabilizer was lyophilized in microreactors. Shelf-life stability of 6 months at ambient temperature was achieved to ensure freedom from cold chain transportation. The microchip kits were also tested for their sensitivity and specificity. The results obtained by microchip-based PCR were validated using positive controls and real potato samples, and were compared with ELISA tests, where ELISA suffered from false negative rate for not reporting some infections that were reported by the microchip PCR. Under optimized conditions, the microchip-based PCR completed the analysis in 38 min for the bacterial and fungal panel and 53 min for the viral panel. The data suggests that the microchip format of the chips may allow accurate, rapid and user-friendly pathogen screening and phytopathological studies not only for potato but also for other agricultural crops.
Plant disease suppression using vermicomposts on cucumber and radish plants. A. C. WYLIE AND Z. K. PUNJA. Simon Fraser University, Department of Biological Sciences, 8888 University Drive, Burnaby, BC V5A 1S6, Canada
Vermicomposts and their water extracts are used to suppress plant diseases in organic production systems, where fungicide use is limited. The past decade has seen a doubling in organic horticulture and a dramatic increase in vermicompost research. However, as disease suppression by vermicomposts has been inconsistent, research in this area requires standardization of methods, and compatibility with current biocontrol agents has not been assessed. We assessed recent developments in disease suppression using vermicomposts, tested the disease suppressive abilities of five vermicomposts with differing characteristics, and developed assays for compatibility with biocontrols. In vitro suppression of the pathogens Fusarium oxysporum f. sp. radicis-cucumerinum Vakalounakis, and Rhizoctonia solani Kühn as well as disease suppression on cucumber and radish plants, respectively, was assessed using vermicomposts incorporated into sterilized substrate and using aerated vermicompost water extract. We found a range of synergistic and antagonistic responses between the biocontrol agents Bacillus subtilis (Ehrenberg) Cohn strain QST 713 (Rhapsody®) and Clonostachys rosea f. catenulata Samuels, Seifert and Gams (syn, Gliocladium catenulatum) strain J1446 (Prestop®), and vermicomposts, depending on the host, pathogen and biocontrol agent. All vermicomposts in our experiments provided significant pathogen suppression in vitro as well as plant disease suppression. The mechanism for pathogen suppression was negated by autoclaving. The testing strategies investigated provide an efficient screen of vermicomposts for compatibility with existing biocontrol agents. With improved and consistent testing methods, vermicompost can be a reliable approach for plant disease management in organic agriculture.
Search for resistance to powdery mildew (Podosphaera macularis) in red raspberry (Rubus idaeus). J. D. ZURN, R. MEIERS, D. PALMER, N. V. BASSIL, W. MAHAFFEE AND M. DOSSETT. United States Department of Agriculture Agricultural Research Service (USDA-ARS), National Clonal Germplasm Repository, 33447 Peoria Road, Corvallis, OR 97333 USA; (R.M.) Wageningen University, Droevendaalsesteeg 4, 6708 PB, Wageningen, the Netherlands; (W.M.) USDA-ARS, Horticultural Crops Research Lab, 3420 NW Orchard Avenue, Corvallis, OR 97330 USA; and (M.D.) BC Berry Cultivar Development Inc., C/O Agassiz Research and Development Centre, 6497 Highway 7, Agassiz, BC V0M 1A0, Canada
Powdery mildew (Podosphaera macularis Wallr.) can be a serious disease of red raspberry (Rubus idaeus L.). Infection by powdery mildew is common in greenhouse or high-tunnel production, but it can also be problematic in the field, especially on susceptible cultivars. Identification of broad-spectrum, stable resistance to powdery mildew, such as that conferred by Mildew resistance locus O (MLO) mutants, would facilitate developing powdery mildew resistant raspberry cultivars that perform well across environments. This will become increasingly important as protected cultivation becomes more common. We identified the MLO orthologues in red raspberry likely to be involved in powdery mildew infection and sequenced their exons in 95 raspberry accessions at the United States Department of Agriculture’s National Clonal Germplasm Repository in Corvallis, Oregon, to identify those that might carry loss-of-function alleles. We also tested a detached-leaf inoculation assay on 30 accessions to phenotype for resistance. A total of 19 non-synonymous alleles were identified in two MLO homologues. Association analysis revealed that none of the non-synonymous alleles identified were associated with resistance in our preliminary screens. Future work is needed to identify MLO-mediated resistance.