Seed Transmission and Management of White Leaf Spot and Light Leaf Spot Pathogens in Brassicas in the Pacific Northwest

Final report for GW16-055

Project Type: Graduate Student
Funds awarded in 2016: $15,675.00
Projected End Date: 04/30/2017
Grant Recipient: Washington State University
Region: Western
State: Washington
Graduate Student:
Major Professor:
Dr. Lindsey du Toit
Washington State University
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Project Information

Summary:

Carmody and du Toit – WSARE Annual Report 1-07-2017

Pyrenopeziza brassicae, cause of light leaf spot of brassicas, was first found in the USA in 2014 in the Willamette Valley of Oregon. Neopseudocercosporella capsellae, cause of white leaf spot of brassicas, occurred rarely in the Pacific Northwest prior to being found across the Willamette Valley in 2014. In this study, a 2016 survey of northwestern Washington, a primary region of biennial brassica vegetable seed production for the USA, revealed both diseases to be present in mustard (Brassica juncea) cover crops and on bird’s rape mustard (B. rapa) weeds, but not in cabbage (B. oleracea var. capitata) seed crops. Sexual crossing tests, pathogenicity tests, and DNA phylogenetic analyses (latter of the internal transcribed spacer region of ribosomal DNA, β tubulin gene, translation elongation factor 1-alpha gene, and mating type genes (MAT1-3 and MAT1-2); and multi-locus sequence analysis of the first three sequences) of P. brassicae isolates from the USA, European Union, New Zealand, and United Kingdom revealed that isolates from the USA likely represent a new species of Pyrenopeziza, hereafter referred to as P. cf. brassicae. P. cf. brassicae was demonstrated to be seedborne and seed transmitted on cabbage and mustard. Incubating infested seed on NP-10 agar medium at 4oC, followed by microscopic examination of the seed revealed P. cf. brassicae to be present on 12.50 to 19.75% of a mustard seed lot and <0.50% of a cabbage seed lot. Planting the infested mustard seed in a greenhouse resulted in a seed transmission rate of 0.1 to 5.3%. Seed treatment trials revealed that chlorine (1.2% NaOCl for 10, 20, 30, and 40 minutes), hot water (50oC for 15 and 30 minutes), steam (62.8, 65.6, 68.3, and 71.1oC for 90 seconds), and 10 fungicide seed treatments all reduced the incidence of mustard seed infected with P. cf. brassicae to <5%, and reduced seed transmission of the fungus from 3.4% for non-treated seed to <1%. Hot water and most of the steam treatments eradicated the pathogen from seed, but the hottest steam treatment was phytotoxic. The most efficacious fungicide seed treatments contained benzimidazole, a demethylation inhibitor, and/or strobilurin active ingredients.

Project Objectives:

The following research objectives were addressed:
1. Assess the potential for Pyrenopeziza brassicae (cause of light leaf spot) and Neopseudocercosporella capsellae (cause of white leaf spot) to be seedborne in brassica crops, and to understand if these pathogens can be introduced into new regions on infected seed. This was addressed by:

a. Establishing whether P. brassicae and/or N. capsellae can infest or infect brassica seed; and
b. Establishing whether brassica seed infected with P. brassicae and/or N. capsellae can lead to seed transmission.

2. Survey brassica crops, brassica weeds, and brassica seed lots to establish if the light leaf spot pathogen and/or white leaf spot pathogen are present in the primary region of biennial brassica vegetable seed production in northwestern Washington. This entailed:

a. Confirming pathogenicity of isolates collected from infected plants in this region, using Koch’s postulates; and
b. Determining differences between isolates collected in the PNW USA and isolates collected from other countries where P. brassicae and N. capsellae have established, such as the UK and New Zealand.

3. Identify seed treatments effective at eradicating P. brassicae and/or N. capsellae from infected brassica seed. This was accomplished by:
a. Evaluating the efficacy of organic seed treatments such as hot water, steam, and 1.2% NaOCl;
b. Evaluating fungicide seed treatments representing different Fungicide Resistance Action Committee (FRAC) groups to identify products effective at preventing seed transmission of P. brassicae and/or N. capsellae.

The research was aimed at developing a better understanding of the prevalence of light leaf spot and white leaf spot in northwestern Washington, and helping brassica growers and the brassica seed industry understand if the seedborne phase of either pathogens is of concern. This research is expected to provide brassica growers and the brassica seed industry with tools to detect the pathogens on infected seed, and to eradicate the pathogen from infected seed by treating infected seed lots using organic and/or conventional treatments.

Cooperators

Click linked name(s) to expand
  • Shannon Carmody
  • Prof. Lindsey du Toit (Researcher)
  • Dr. Cynthia Ocamb (Researcher)
  • Prof. Jonathan West (Researcher)
  • Dr. Kevin King (Researcher)

Research

Participation Summary

Educational & Outreach Activities

12 Consultations
2 Curricula, factsheets or educational tools
1 Published press articles, newsletters
2 Tours
6 Webinars / talks / presentations
2 Workshop field days

Participation Summary

50 Farmers
150 Ag professionals participated
Education/outreach description:

Abstracts for 2 posters presented at 2017 APS meeting:

  • Carmody, S.M., and du Toit, L.J. 2017. Seed treatments to eradicate Pyrenopeziza brassicae from infected mustard (Brassica juncea) seed. Poster 295-P, 2017 APS Annual Meeting, 5-9 Aug. 2017, San Antonio, TX.
  • Carmody, S.M., King, K.M., Claassen, B.J., Fraaije, B.A., West, J.S., Ocamb, C.M., and du Toit, L.J. 2017. Genetic analysis of Pyrenopeziza brassicae, cause of light leaf spot of brassicas, in the European Union, Oceania, and North America. Poster 536-P, 2017 APS Annual Meeting, 5-9 Aug. 2017, San Antonio, TX.

Abstract for oral presentation at 2016 APS PD meeting:

  • Carmody, S.M., Ocamb, C.M., and du Toit, L.J. 2016. Potential seed transmission of Pyrenopeziza brassicae and Mycosphaerella capsellae in brassicas in the Pacific Northwest USA. Paper presented at American Phytopathological Society (APS) Pacific Division Meeting, 28-30 Jun. 2016, La Conner, WA. Phytopathology 106:S4.196 (Abstr.)

Newsletter:

  • Carmody, S., and du Toit, L.J. 2016. Light leaf spot and white leaf spot – two new fungal diseases of brassicas in the PNW. Tilth Producers’ Quarterly 26(4):5,18,20.

Presentations at grower/commodity meetings:

  • du Toit, L.J. and Carmody, S.M. 2017. Brassica light leaf spot, white leaf spot, and black leg; onion neck rot; and bacterial blight of carrot. Invited presentation to Bejo Seeds production team, 7 Nov. 2017, Mount Vernon, WA. (30 people)
  • du Toit, L.J., and Carmody, S. 2017. Light leaf spot and white leaf spot on brassica crops in western Washington. Wilbur Ellis Growers’ Meeting, 2 Feb. 2017, La Conner, WA. (100 people)
  • Carmody, S.M. 2017. Light leaf spot and white leaf spot on brassica crops in western Washington. Puget Sound Seed Growers’ Association Annual Meeting, 13 Jan. 2017, Mount Vernon, WA. (75 people)
  • Carmody, S.M. 2016. Light leaf spot and white leaf spot on brassica crops in western Washington. Puget Sound Seed Growers’ Association Annual Meeting, 29 Jan. 2016, Mount Vernon, WA. (50 people)
  • American Seed Trade Association Vegetable Technical Subcommittee Meetings: Gave updates on WSDA Crucifer Quarantine and black leg, white leaf spot, and light leaf spot situation in the Pacific Northwest. 6 Jan., 12 Apr., & 8 Jun. 2016 (via conference call, 25 people at each meeting); prepared presentation on these topics for ASTA Vegetable & Flower Conference, Monterey, CA given by Philip Brown, Sakata America, 2 Feb. 2016. (75 people)
  • Diagnosing Plant Problems. S.M. Carmody prepared and presented a 4-hour plant diagnosis workshop (slides, handouts, and samples) for the “Growing Veterans” program and beginning Latino farmers in Skagit Valley, WA on 7 Nov. 2015, in collaboration with VIVA Farms. (25 people)

  • du Toit, L.J. Organic Seed Alliance Research Field Day, 14 Oct. 2015, Chimacum, WA. Discussion on black leg of crucifers, risk management, and pending WSDA quarantine regulation. (25 people)

  • du Toit, L.J. Black leg, light leaf spot, and white leaf spot of crucifers in the Pacific Northwest: Lessons learned from the 2014 Willamette Valley epidemic. Douglas Co. and Okanogan Co. Growers Meeting. Invited to help growers avoid introducing seedborne pathogens on crucifer cover crop and canola seed. 3 Mar. 2015, Waterville, WA. (25 people)

Presentations at field days:

  • du Toit, L.J., and Carmody, S.M. 2017. Light leaf spot and white leaf spot on brassica crops in western Washington. WSU Mount Vernon NWREC Field Day, 13 Jul. 2017, Mount Vernon, WA. (120 people)
  • Carmody, S.M. 2016. Light leaf spot and white leaf spot on brassica crops in western Washington. WSU Mount Vernon NWREC Field Day, 13 Jul. 2016, Mount Vernon, WA. (120 people)
  • Carmody, S.M. 2015. Light leaf spot and white leaf spot on brassica crops in western Washington. WSU Mount Vernon NWREC Field Day, Jul. 2015, Mount Vernon, WA. (100 people)

 

Project Outcomes

250 Farmers reporting change in knowledge, attitudes, skills and/or awareness
25 Farmers changed or adopted a practice
50 Farmers intend/plan to change their practice(s)
Did this project contribute to a larger project?:
Yes
Project outcomes:

Impacts

Refer to the PDF files of Shannon’s MS thesis, two posters presented at the 2017 annual meeting of the American Phytopathological Society (APS), and an oral presentation at the 2016 APS Pacific Division meeting. The project has demonstrated the presence of two new brassica diseases in Washington State, LLS and WLS, both in a primary region of brassica vegetable seed production. Of six brassica cover crop seed lots planted in the Skagit Valley of Washington in 2016, one was infected with the LLS pathogen. This seed lot of ‘Nemagon’, a white mustard cultivar was produced in the Willamette Valley of Oregon in 2015, demonstrating the potential for cover crop seed lots produced in areas where this disease has established to become a source of inoculum of this pathogen. The other seed lots were produced in the Columbia Basin of central Washington, where LLS has not been found.

The U.S. isolates of the LLS fungus from WA and OR differed molecularly (based on DNA sequences of each of 4 DNA regions, the ITS rDNA, beta-tubulin gene, and translation elongation factor 1-alpha gene, as well as the mating type locus), pathogenically (based on inoculating turnip plants of the cv. Hakurei, the US isolates caused much more severe chlorosis symptoms than the EU/UK isolates, and the EU/UK isolates produced small patches of white conidiomata on the leaves which were not observed on plants inoculated with any of the USA isolates), sexually (the US and UK/EU isolates of opposite mating type did not form the sexual stage apothecia with ascospores, whereas the sexual stage was observed when isolates of opposite mating type from the EU/UK were crossed on malt extract agar medium), and morphologically (conidia of the US isolates differed in size from the EU/UK isolates, and the US isolates tended to form a single septum in some of the spores whereas this was almost never observed with isolates from the UK or EU). All of this evidence suggests the US isolates of the LLS fungus are not the same species as P. brassicae, but represent a new species. The name P. cascadia is proposed. Molecular diagnostic tools are needed to differentiate the two species of this pathogen, and further research is needed to assess how isolates of the two species might differ in host range, cultivar susceptibility, epidemiology, etc. Further research is needed to assess the likely source of origin of the isolates introduced into the US.

Fungicide resistance genes to the benzimidazole and triazole families were identified in some EU/UK isolates but were not identified in any of the US isolates, demonstrating that the US population of the LLS fungus probably has not been exposed to much selection pressure for resistance to these classes of fungicides. These results indicate the LLS isolates introduced into the US probably did not originate from the EU/UK.

A seed lot of ‘Caliente 199’ mustard (B. juncea) infected with P. brassicae was used to assess the efficacy of chlorine (1.2% NaOCl for 10, 20, 30, and 40 minutes), hot water (50oC for 15 and 30 minutes), steam (62.8, 65.6, 68.3, and 71.1oC), and 10 fungicide treatments to manage seedborne P. brassicae. Each seed treatment was compared to non-treated seed, and fungicide treatments were also compared to seed treated with a polymer colorant (seed coating) added to each product. All treatments reduced the incidence of seed infected with P. brassicae, from an average of 13.5% for non-treated seed to 0 to 4.3%, based on seed health assays. Likewise, all treatments, including the seed colorant control treatment, reduced seed transmission of P. brassicae from an average of 3.4% for non-treated seed to 0 to 0.4%. Seed transmission was not observed for the hot water, steam, and six of the fungicide treatments (azoxystrobin, fludioxonil, iprodione, thiabendazole, pyracostrobin + boscalid, and difenoconazole + fludioxonil + mefenoxam + sedaxane + thiamethoxam). The hottest steam treatment reduced seed germination from 98.0% for non-treated seed to 90.0 and 93.8% in Trials 1 and 2, respectively. The results demonstrate there are effective organic and conventional seed treatments for management of P. brassicae.

Multiple presentations were made to stakeholders, including growers, across the PNW and to seed companies represented on the American Seed Trade Association Vegetable Technical Subcommittee, so that information generated by this project has been shared widely for the use and adoption by the seed industry.

Shannon defended her MS thesis on 14 June 2017. She was in a very severe car accident the afternoon of her defense exam, so the the edits to her thesis and submission of the final thesis had to be delayed to September 2017. Shannon is working with Dr. du Toit to prepare 2 journal articles to be published from her thesis.

Accomplishments

  1. Produced brassica seed lots infested with the LLS fungus by inoculating developing pods on plants of a yellow mustard and cabbage. Details of the plant inoculation process were provided in the 2015 and 2016 reports. Seed was harvested from these inoculated plants in late summer and fall of 2015. A seed lot of mustard (Brassica juncea) and a seed lot of cabbage (B. oleracea var. capitata) were each infested successfully with P. brassicae, demonstrating that the pathogen can be seedborne. This infested seed lot was used to accomplish the other research objectives related to seedborne infection. Infection of seed lots with the WLS pathogen, N. capsellae, was not accomplished.
  1. Develop seed health assay(s) to detect and quantify the LLS and WLS pathogens in infested seed lots. The LLS pathogen is new to North America and the seedborne phase of the pathogen is not well understood. Therefore, a method for detecting this pathogen on infected seed needed to be developed. Various methods of plating seed onto agar media, blotters, etc. were tested. Ultimately, the LLS fungus was detected on the greatest incidence of mustard and cabbage seed lots by plating the seed onto NP-10 agar medium, incubating the seed at 4°C in the dark for five weeks to facilitate development of the very slow-growing pathogen at a cold temperature that inhibits most other (faster-growing) fungi that can occur on brassica seed and examining the seed microscopically four, five, and six weeks after plating the seed. The fungus was detected on 12.5% of the mustard seed lot, and 0.4% of the cabbage seed lot.

3. Assess the rate of seed transmission of the LLS pathogen from infested seed lots planted under conditions conducive to LLS.  Seed transmission trials were carried out in greenhouses at the WSU Mount Vernon Northwestern Washington Research & Extension Center (NWREC). The infected mustard and cabbage seed lots were planted in a greenhouse under misters to mimic growing brassica plants under overhead irrigation with high relative humidity and extended durations of leaf wetness. Seed of the mustard lot were planted into 15 72-cell flats, with each set of three flats representing one replication of 216 seed planted, and five replications planted. Seed of the cabbage lot were planted into each of 5 200-cell flats, with each flat representing one replication. Once plants started emerging, micro-sprinklers were staked between the flats and set on a timer to mist the flats for 10 s every 30 min for the first week after emergence, and 10 s every 45 min for the subsequent 35 days (Figure 1). These damp conditions ensured a conducive environment for development of brassicae. The LLS fungus developed on 1.6% of the seedlings that developed from 1,080 seeds planted under these conditions, which represented a 20% seed transmission rate from the 12.5% infested seed in that lot (Figure 2). In contrast, P. brassicae was detected on only 1 of 1,000 seedlings grown from the infected cabbage seed lot. However, this represented a 25% seed transmission rate from the 0.4% infested seed in that lot. 

Most seed transmission studies focus on detecting the pathogen or symptoms of the disease on cotyledons as a measure of direct seed transmission. The LLS fungus, and symptoms of LLS, were very difficult to detect on mustard cotyledons as the symptoms typical of LLS, including venial browning and foliar chlorosis, were not distinct on cotyledons (cotyledons do not have distinct veins and senesce rapidly once true leaves start to form). Therefore, initial seed transmission tests necessitated focusing on symptoms of secondary infection, i.e., infection of the true leaves (Figure 2). The seed transmission trials demonstrated that P. brassicae readily is transmitted from infested seed to emerging seedlings. However, subsequent seed transmission trials completed in a different greenhouse, as part of the seed treatment Objective 4 described below, resulted in development of fairly distinct LLS symptoms on cotyledons prior to the first true leaves maturing, which provided a more direct measure of seed transmission and enabled seed transmission assays to be completed within 4 weeks. Even a very low incidence of seed transmission (<1%) can translate into a significant disease outbreak under conducive environmental conditions, given that >100,000 seed might be planted in some brassica crops (e.g., cover crops). The results reiterate the importance of growing or purchasing high quality, pathogen-free seed, particularly for organic production because of limited options for disease management once infection has occurred in a crop.

4. Assess seed treatments to reduce or prevent seed transmission of the LLS fungus. Using the brassicae infested seed lot generated for Objective 1, four types of seed treatments were assessed for reduction or prevention of seed transmission of this pathogen – hot water, steam, bleach, and conventional fungicides. Three assays were used to determine the efficacy and potential phytotoxicity of the seed treatments: 1) a seed germination test (four replications of 100 seeds tested/treatment), 2) a seed health assay (four replications of 100 seeds tested/treatment), and 3) a seed transmission assay (four replications of 216 seeds tested/treatment). Refer to the 2016 annual report as well as Shannon’s MS thesis for details of each of the treatments.

The four steam treatments and two hot water treatments were very effective at killing the LLS fungus on the infested mustard seed, as the pathogen was not detected in the seed health assay or on any seedlings in the seed transmission trial that developed from seeds subjected to these treatments, except for one seed subjected to one of the steam treatments on which the LLS fungus was detected in the seed health assay. Although all of the durations of 1.2% NaOCl treatment significantly reduced the incidence of seed on which the LLS fungus was detected, this disinfectant was not as effective as the steam or hot water treatments at preventing development of P. brassicae on mustard seed and seedlings. The ability to recover this pathogen on seed treated with 1.2% NaOCl for 40 minutes suggests that the infection is not just on the surface of the seed coat, but appears to be within the seed coat and possibly even in the embryo. The germination assay and seed transmission assay revealed that the hottest steam treatment (160oF) was phytotoxic as the mustard seed germination was delayed and reduced, with stunting of the seedlings that developed from seed steamed at this temperature. The same phytotoxicity was observed for this steam temperature in the repeat trial. In the first trial with the two hot water seed treatments, the seed was first warmed up for 10 minutes at 25oC prior to the 50oC treatment. With this protocol, the 30 minute duration of hot water treatment was slightly phytotoxic to the mustard seed as the incidence of normal seed germination was reduced slightly in both the seed germination assay and the seed transmission assay (data not shown). However, when this warming step was removed from the protocol in the repeat trial, the 30 minute duration of hot water treatment no longer was phytotoxic. Otherwise, results for the hot water, bleach, and steam seed treatment trials were similar in the repeat trials. In the fungicide trials, all treatments, including the seed colorant control treatment, reduced seed transmission of P. brassicae from an average of 3.4% for non-treated seed to 0 to 0.4%. Seed transmission was not observed for six of the fungicide treatments (azoxystrobin, fludioxonil, iprodione, thiabendazole, pyracostrobin + boscalid, and difenoconazole + fludioxonil + mefenoxam + sedaxane + thiamethoxam). The results demonstrate there are effective organic and conventional seed treatments for management of P. brassicae.

 5. Provide farmers, seed company representatives, consultants, extension educators, and other relevant stakeholders with educational materials and training opportunities on detection and management of the LLS and WLS pathogens, including the importance of purchasing high quality seed lots. A popular press article for farmers was written and published in the Washington Tilth Producers’ Quarterly: Carmody, S., and du Toit, L.J. 2016. Light leaf spot and white leaf spot – two new fungal diseases of brassicas in the PNW. Tilth Producers’ Quarterly 26 (4):5,18,20. Shannon gave a presentation on 29 January 2016 and again on 13 January 2017 on this project at the annual meeting of the Puget Sound Seed Growers’ Association in Mount Vernon, WA. Lindsey du Toit has presented updates on this project to the Vegetable Technical Subcommittee of the American Seed Trade Association during three conference calls in 2016 and again during 2017, and to various agricultural meetings in 2016-17. Seed company plant pathologists have requested regular updates because of the potential significance of these diseases. The Iowa State University Seed Science Center has indicated interest in offer the seed health assay Shannon developed for the light leaf spot pathogen to seed companies/producers.

In early April 2016, Shannon found LLS to be widespread on wild bird’s rape mustard (B. rapa) and mustard cover crops (B. juncea) in Skagit, Whatcom, and Snohomish Counties of northwestern Washington State. WLS also was detected in B. juncea cover crops and on bird’s rape mustard plants growing along the edges of fields in Skagit and Whatcom Counties. Neither disease had previously been found in Washington State, so this represented an important record for the region. Both LLS and WLS fungi can infect a wide range of genera and species in Brassicaceae, although their impact on diverse types of brassica crops grown in the Pacific Northwest remains to be determined. In the U.K., LLS has become the main cause of yield losses in winter rapeseed production. In the E.U., Canada, Australia, and the southeastern U.S., WLS has been demonstrated to impact the quality and value of fresh market brassica crops. The finding of both of these pathogens in western Washington lead to an additional objective for Shannon’s MS research project.

6. Conduct a survey for LLS and WLS in northwestern Washington. A limited survey in spring-summer of 2016 of brassica cover crops, weeds, and biennial seed crops revealed the presence of both the LLS and WLS pathogens in mustard cover crops and in bird’s rape mustard weeds growing alongside roads, ditches, and dikes in Skagit, Whatcom, and Snohomish Counties. Neither pathogen was found in surveys of a dozen cabbage seed crops in the summer of 2017. The survey was continued in November 2016 after planting and establishment of fall-sown cover crops and initiation of the fall rainy season, to assess when symptoms first appear in these cover crops and stands of bird’s rape mustard. Symptoms of LLS were observed on cover crops and birds’ rape mustard weeds in Skagit Co. in Nov. 2016, and additional isolates of the fungus collected from samples. Pathogenicity testing of isolates of brassicae and P. capsellae has been completed for all samples collected in spring 2016. Pathogenicity testing for isolates collected in western Washington entailed collecting symptomatic leaves or whole plants, isolating from the lesions, and developing single-spore isolates for long-term storage and further analyses. The single-spore isolates were inoculated onto healthy plants of mustard and/or turnip in a greenhouse to observe symptoms typical of each disease, and re-isolations completed from symptoms that developed. In addition, molecular (DNA-based) methods of species identification were used on each isolate, i.e., PCR assays with published primers to amplify the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) and the beta-tubulin DNA, which confirmed the genus and species identity of the original isolates and re-isolates from the pathogenicity tests. Interestingly, the ITS rDNA sequences only had 95% sequence similarity with the few ITS rDNA sequences available in GenBank for this species (mostly of isolates from the EU and UK), which typically is considered below the level of similarity for isolates of the same species.

 

7. Use the seed health assay developed in Objective 2 to survey brassica cover crop seed lots obtained from growers and seed companies that are planted in the Skagit Valley. To date samples of five brassica cover crop seed lots have been received and tested that were planted in Skagit Co., WA in 2016. Shannon detected the LLS pathogen on one seed lot of white mustard (Sinapis alba) cultivar Nemagon (at <1% incidence), but not on the four other seed lots assessed to date. The Nemagon seed lot was grown in the Willamette Valley of western Oregon, where the LLS spot pathogen was first documented to occur in North America in 2014, while the other seed lots were grown in eastern Washington where the pathogen has not been detected. This revealed the potential for movement of the LLS fungus on infested seed lots and, therefore, introduction of the pathogen into regions on cover crop seed.

8. Partner with researchers at Rothamsted, UK working with brassicae to examine genetic differences in isolates from the USA (Oregon and Washington) vs. isolates from the UK, EU, and New Zealand, to learn about the potential origin and population genetics of the pathogen in North America. Shannon completed sequencing of the ITS rDNA as well as the beta tubulin, and translation elongation factor (TEF) regions of the LLS isolates she has collected to date from Oregon and Washington. The sequences were used to complete a multi-locus sequence analysis (MLSA) of isolates of the LLS fungus obtained from infected plants in Oregon and Washington compared with isolates from the UK, EU, and new Zealand obtained from Drs. Jon West and Kevin King at Rothamsted Experimental Station in the UK. Drs. West and King have also examined the mating type genes of the US isolates we sent to them from Shannon’s collection, compared with UK, EU, and New Zealand isolates. They helped us establish that both mating types of P. brassicae, MAT1-1 and MAT1-2, are present in western Washington and Oregon, and the North American isolates are distinct genetically from the UK, EU, and New Zealand isolates. Their results suggest a different population of the LLS fungus was introduced into the Pacific Northwest USA than the UK, EU, and NZ isolates. Based on the interesting find of a different genetic grouping of isolates from the US, a grant proposal is being prepared to seek funding for further work in collaboration with Drs. West and King as well as researchers at Oregon State University.

The US (OR and WA) isolates of the LLS fungus differed molecularly (based on DNA sequences of each of 4 DNA regions, the ITS rDNA, beta-tubulin gene, and translation elongation factor 1-alpha gene, as well as the mating type locus), pathogenically (based on inoculating turnip plants of the cv. Hakurei, the US isolates caused much more severe chlorosis symptoms than the EU/UK isolates, and the EU/UK isolates produced small patches of white conidiomata on the leaves which were not observed on plants inoculated with any of the USA isolates), sexually (the US and UK/EU isolates of opposite mating type did not form the sexual stage apothecia with ascospores, whereas the sexual stage was observed when isolates of opposite mating type from the EU/UK were crossed on malt extract agar medium), and morphologically (conidia of the US isolates differed in size from the EU/UK isolates, and the US isolates tended to form a single septum in some of the spores whereas this was almost never observed with isolates from the UK or EU). All of this evidence suggested the US isolates are not the same species as P. brassicae, but represent a new species. The name P. cascadia is proposed. Molecular diagnostic tools are needed to differentiate the two species of this pathogen, and further research is needed to assess how isolates of the two species might differ in host range, cultivar susceptibility, epidemiology, etc. Further research is needed to assess the likely source of origin of the isolates introduced into the US.

Fungicide resistance genes to the benzimidazole and triazole families were identified in some EU/UK isolates but were not identified in any of the US isolates, demonstrating that the US population of the LLS fungus probably has not been exposed to much selection pressure for resistance to these classes of fungicides. These results indicate the LLS isolates introduced into the US probably did not originate from the EU/UK.

Lastly, a side project has developed during Shannon’s MS degree after she detected another fungal plant pathogen, Plectosphaerella cucumerina, on a turnip seed lot of the cultivar Barkant that was harvested from a seed crop grown in the Willamette Valley or OR in 2015. The seed crop had severe infections of LLS, WLS, and black leg in 2014-15. P. cucumerina was not considered a common pathogen of brassicas or to be seedborne in brassicas, even though the fungus is ubiquitous in many environments, can colonize plants endophytically, and has even been evaluated as a potential biocontrol agent for some fungi and nematodes. P. cucumerina was detected on >20% of the Barkant turnip seed lot, and Shannon demonstrated the fungus could be seed transmitted from that lot when planted under misters in a greenhouse using the seed transmission protocol described above. However, an average of only one in five isolates of P. cucumerina obtained from the Barkant seed lot was pathogenic when inoculated onto turnip foliage. Hannah Rivedahl, an MS student in Dr. Ken Johnson’s lab at Oregon State University, is studying isolates of this pathogen that she has obtained from wilted cucurbit plants (particularly squash) in the Willamette Valley. In her research on the host range of this fungus, Hannah isolated P. cucumerina from root and crown lesions of brassica crops. Shannon initiated collaboration with Hannah to test pathogenicity of cucurbit isolates of this fungus using both foliar inoculation methods and soil inoculation methods. Shannon found four isolates to be pathogenic on turnip using the foliar inoculation method, and has used PCR assays followed by sequencing of the ITS rDNA to confirm the genus and species identity of the P. cucumerina isolates. Interestingly, the ITS rDNA sequence of the isolates that are pathogenic on brassica foliage differed slightly from the sequences of the isolates that were not pathogenic on brassica foliage. Shannon was going to compare these with sequences of the Oregon cucurbit isolates from Hannah’s collection. However, the accident that occurred in June 2017 prevented Shannon from being able to pursue this project further.

Refer to the list of workshops, presentations, etc. given by Shannon Carmody and Lindsey du Toit for this project. Some of these were listed in the 2016 annual report. In addition, more presentations have been given in 2017 by Shannon and Lindsey. Shannon’s thesis defense seminar was presented to the WSU Department of Plant Pathology on 14 June 2017, followed by a successful defense exam. Her research was presented by Lindsey du Toit at the national meeting of the American Phytopathological Society in San Antonio, TX in August 2017 in the form of two posters, while Shannon was recovering from the car accident that occurred on 14 June. We anticipate two scientific journal articles from her thesis, to be submitted in winter 2017-18.

Knowledge Gained:

The survey Shannon Carmody completed of diverse agricultural stakeholders revealed the extent to which many are not aware of the risk of seedborne pathogens, and many do not assess if the seed lots they purchase have been tested for any potential seedborne pathogens that might be of risk to their region of production. Education and awareness of seedborne pathogens, particularly for lower value crops like cover crops, is needed as the ignorance on this topic is probably the greatest risk for moving pathogens on seed.

Success stories:

One farmer in northwestern Washington indicated that, prior to being made aware of these diseases on brassicas, he used to purchase the least expensive brassica cover crop seed he could find, including making purchases online (over the internet). This farmer produces brassica seed crops as well as other high value specialty crops, and uses cover crops to help rebuild soil between cash crops like potato that can be hard on the soil because of the extensive cultivation used with those crops. He acknowledged his ignorance of the need to be careful about potential seedborne pathogens, even for non-cash crops like cover crops. This project has changed his practice of where/how to purchase cover crop seed lots.

Recommendations:

Additional research is needed on light leaf spot and white leaf spot, as well as black leg, given the recent findings of black leg in >20 dryland canola production sites in eastern Washington as well as radish cover crops.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.