Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to
BREEDING OF DISEASE-RESISTANT CELERY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0230718
Grant No.
(N/A)
Project No.
CA-D-PPA-2165-H
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2012
Project End Date
Oct 1, 2017
Grant Year
(N/A)
Project Director
Epstein, L.
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Plant Pathology, Davis
Non Technical Summary
The overall goal of our project is to maintain sustained resistance to the major fungal pathogens in celery. In 2009, 1.9 billion pounds of celery were harvested from 26,600 acres in California with a total value of $389 million and consequently a gross value of $14,629/acre. Celery is an important vegetable in the U.S. diet with 5.6 pounds of retail celery per capita per year in 2009. Economically sustainable celery production is dependent on effective management particularly of the soil-borne fungus Fusarium oxysporum f. sp. apii (Foa), causal agent of Fusarium yellows. The disease was first observed in 1914. Foa infects through the roots, moves into the xylem, and causes severe stunting, yellowing, and eventually a crown and root rot. The only effective method of pathogen control requires disease-resistant cultivars. Fumigants are not particularly effective against the pathogen, are expensive for growers, and present environmental challenges. Because pathogen populations can change over time, it is important to monitor the pathogen population, to better understand the nature of the celery-pathogen interactions, and to continue to introduce new genes for resistance into breeding material. In addition to focusing on the biology and control of Foa, we also work on selecting and breeding for resistance for two other important plant pathogenic fungi: Septoria apiicola and Sclerotinia sclerotiorum, which are currently controlled by fungicides. We use a variety of methods for our research including collecting plant pathogenic fungi from celery in agricultural fields, performing selected DNA sequencing of those isolates to determine their diversity, screening the diversity of isolates in current celery breeding lines and in a collection of biodiverse relatives of celery to determine whether there are new sources of resistance, improving the disease screening process so that it can be performed in a faster and more quantitative manner, and incorporating resistance into current breeding lines. Our results will be communicated to the scientific community via peer-reviewed publications. Results also will be communicated to the celery community through the Celery Research Advisory Board, direct interactions with growers, and via the University of California Cooperative Extension. Importantly, all breeding lines produced by the project will be available for public release. The success of the project will be evaluated based on completion of peer-reviewed papers and release of information and materials that will allow sustained celery production that is healthful to the consumer, profitable for the grower, and with the minimal amount of fungicides.
Animal Health Component
(N/A)
Research Effort Categories
Basic
20%
Applied
60%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2124020110250%
2161430108025%
2161430116025%
Goals / Objectives
The overall goal of the research is to maintain and to improve sustainable disease resistance in celery, Apium graveolens var. dulce. Our major objective is to better understand the biology of Fusarium oxysporum f. sp. apii, which causes the potentially devastating disease Fusarium yellows, so that control with a disease-resistant cultivar can be sustained. Our specific objectives are the following: 1. To characterize the diversity of Fusarium oxysporum f. sp. apii (Foa) isolates from production fields and from the two field locations where celery is screened for resistance in order to determine if celery in the test plots is exposed to the full spectrum of Foa isolates in California; 2. To optimize the greenhouse assay for a quantitative assessment of virulence of Foa isolates and for resistance to Foa; 3. To continue to test and breed A. prostratum-derived progeny for resistance to Foa. 4. To collect California isolates of the late blight pathogen Septoria apiicola from celery production areas, to characterize the isolates by selected DNA sequence and by virulence tests, and to optimize the greenhouse assay for virulence of S. apii isolates and for resistance to S. apii; 5. To screen for Sclerotinia sclerotiorum resistance in the Apium diversity collection; and 6. To maintain the Apium diversity collection for future breeding. The expected outputs are the following: conducting and analyzing experiments, disseminating results yearly to the California Celery Research Board; interacting with growers and private seed company employees at the California Celery Research Board cultivar trials in Fusarium-infested soil in Oxnard and Santa Maria; and publishing results in a peer-reviewed journal.
Project Methods
Our methods will involve collection of plant pathogenic fungal isolates (particularly Fusarium oxysporum f .sp. apii but also Septoria apiicola and to a lesser extent Sclerotinia sclerotiorum) from celery (Apium graveolens var. dulce) in agricultural fields, to perform selected DNA sequencing of those isolates to determine their diversity, to screen the diversity of isolates in current celery breeding lines and in the Apium biodiversity collection to determine whether there are new sources of resistance, to improve the disease screening process so that it can be performed in a faster and more quantitative manner, and to introgress resistance into current breeding lines. Results will be communicated to the scientific community via peer-reviewed publications. Results also will be communicated to the celery community through the Celery Research Advisory Board, direct interactions with growers, and via the University of California Cooperative Extension. Importantly, all breeding lines produced by the project will be available for public release. The success of the project will be evaluated based on completion of peer-reviewed papers and release of information and materials that will allow sustained celery production that is healthful to the consumer, profitable for the grower, and with the minimal amount of fungicides.

Progress 10/01/12 to 10/01/17

Outputs
Target Audience:The target audience are primarily celery producers in California, the California Celery Research Advisory Board (CCRAB), which represents the celery producers, and celery breeders in the private sector that work, at least partly, in California. Secondarily, a larger national and international audience of celery producers can access our reports via the CCRAB. Thirdly, scientists can also access our results through the scientific literature (Epstein et al. 2017. Phytopathology 107:463-473. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two undergraduates were trained: Sabrina Loueiro and Edmond Line. Two visiting scientists were trained: Dr. Ruba Abuamsha from Palestine and Dr. Mei Li from China. The staff research associate Dr. Sukhwinder Kaur has had opportunities for professional development. How have the results been disseminated to communities of interest?Yes, there were Celery Field Days on Nov. 16, 2016 in Camarillo CA, and Sept 14 2017 in Santa Maria, CA. Results were presented orally to the California Celery Research Advisory Board on Sept. 14, 2017 What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? A highly aggressive and apparently new race of Fusarium oxysporum f. sp. apii (Foa), which we named race 4, was first observed in a celery field in Camarillo, CA in 2013; as far as we know, no commercially available germplasm is resistant to it. This past year, we continued to screen the UC Davis Apium germplasm collection for either tolerance or resistance to the new Foa race 4. To date, we have screened 231 accessions for resistance/tolerance to Foa race 4: 120 celery (Apium graveolens ssp.dulce); 66 celeriac (A. graveolens ssp. rapaceum); 25 smallage (wild-type A. graveolens); 5 celery x celeriac; and 15 miscellaneous Apium. Promising accessions were rescreened in an eight-week trial in the greenhouse in infested soil three times. Promising accessions were then tested in either field soil in the greenhouse and/or a field trial. We now have four accessions in further testing: one celery from China and three celeriacs from Turkey that has been crossed with cv. Challenger, which is resistant/tolerant to Foa races 1, 2, and 3. We made a total of eight crosses with Challenger (as male and female) with either accessions A0103, A0102, or A0134. F1 seeds were collected, screened and promising ones were selfed. The F2 seeds are now in process in order to 1) assess the number of genes involved in the resistance/tolerance and 2) for potential use in further breeding by backcrossing again to Challenger. We continued to monitor any spread of the new Foa race 4 in celery fields in California. As of Sept. 2017, we have detected Foa race 4 in 19 fields in Ventura County: 14 fields in the Camarillo area, 3 fields in Oxnard, and one each in Santa Paula and the community of Ventura. So far, based on the 628 bp from an intron-rich EF-1α and 640 bp from rDNA IGS, most fields have identical sequence. However, there is one Foa race 4 variant with one single nucleotide polymorphism. Using "2nd generation" Illumina DNA-sequencing, we demonstrated that Foa is polyphyletic, i.e., that Foa race 2 is in a different lineage (F. oxysporum species complex clade 3) than Foa races 1, 3, and 4, which are in F. oxysporum species complex clade 2. We used the sequences to develop PCR primers that can relatively rapidly identify whether a celery plant is infected with either Foa race 2, the most common celery pathogen in California, or Foa race 4. In order to better monitor the pathogen and to understand the origin of race 4, we have recently obtained DNA sequence using 3rd generation PacBio long-read sequences; based on DNA sequence, the extremely aggressive Foa race 4 is very similar to race 3, which is comparatively weakly virulent. To develop a sustainable control program, we have investigated alternate hosts of Foa. In general, strains of F. oxysporum cause disease on only a narrow host range, but can maintain their population in soil to varying extents either on root cortical cells of "non-hosts" or saprophytically. However, Foa (of what we would now call a group of race 1 strains) was historically considered to have a comparatively broad host range. In addition to celery, Foa race 4 is highly virulent on cilantro/coriander (Coriandrum sativum); cilantro is in the same family as celery (Apiaceae) and has been used as a rotation crop with celery in Ventura County. Cilantro is also susceptible to F. oxysporum f. sp. coriandrii (Foc), but Foc is not a pathogen of celery, and Foa race 2 is only a pathogen of celery.) In contrast, Foa race 4 (and Foa race 2) is not a pathogen of the single cultivars tested of lettuce, strawberry, bell peppers, cabbage, kale and lima beans. However, we have not yet determined to what extent these rotation crops may serve as hosts that are sufficient to maintain the population. We conducted bioassays of cv. Challenger planted in soil from nine fields that had had one or more incidents of Fusarium yellows caused by Foa race 4 between 2014 and 2016. In the bioassays, from 55 to 100% of the plants died, compared to none of the plants in the uninfested soil control. The results indicate that a celery-free period for three years is likely not sufficient to remove the threat of a Foa race 4 outbreak in a subsequent celery or cilantro crop. As with Fusarium yellows caused by Foa race 2, Foa race 4 is more severe in warmer weather. In Ventura county, transplanting in early to mid-August results in more severe disease than later transplants, with the best outcomes with plantings in late September or early October when maximum soil temperatures are below 75 °F. Our attempts to reduce disease severity with a variety of treatments in the potting mix (pre-transplanting) were unsuccessful, including the commercial biocontrol products Bio Tam and Root Shield Plus, and six different Trichoderma harzianum strains that were isolated from a celery field in Ventura County. A pre-transplant treatment with beta- aminobutyric acid also did not reduce disease severity of transplants into Foa race 4-infested soil. Attempts to reduce inoculum levels with applications in a greenhouse simulated "just after harvest" of crown debris were also unsuccessful; treatments included redworms, enchytriads, the biocontrol products Bio-Tam and Root Shield Plus mixed with On-Gard, and two non-pathogenic F. oxysporum strains that had been isolated from celery. In contrast to Foa race 2, which colonizes the roots and crowns of living celery but not the petioles, Foa race 4 colonizes the petioles; in the greenhouse and in the field. Foa race 4 also sporulates profusely on petioles, which may provide inoculum that can be spread, primarily within fields, by water, insects, or dust storms. We speculate that celery harvesters, which retain clods of soil and plant debris, may introduce infested soil into previously uninfested fields. Consequently, we recommend cleaning off harvesting and other equipment between fields. A newly infested field typically has a limited focus of infection, and removal of infected plants and roots may limit a wide-spread infection. Quantitative PCR data of cultivars grown in infested soil in the greenhouse indicates that while growth of Foa race 2 in crowns of susceptible cultivars reaches a maximum 0.5% of the total celery DNA content, Foa race 4 continues to grow at increasing density (5% of total celery DNA content) until the plants die.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Kaur S, Pham QA, Epstein L. High quality DNA from Fusarium oxysporum conidia suitable for library preparation and long read sequencing with PacBio. https://www.protocols.io/view/high-quality-dna-from-fusarium-oxysporum-conidia-s-inccdaw.


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audience are primarily celery producers in California, the California Celery Research Advisory Board (CCRAB), which represents the celery producers, and celery breeders in the private sector that work, at least partly, in California. Secondarily, a larger national and international audience of celery producers can access our reports via the CCRAB. Thirdly, scientists can also access our results through the scientific literature (see product below). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two undergraduates were trained: Quyen Pham and Hart Calligagan. One Chinese visiting scientist, Dr. Mei Li was trained. The staff research associate Dr. Sukhwinder Kaur has had opportunities for professional development. How have the results been disseminated to communities of interest?Yes, there were Celery Field Days on Nov. 20, 2015 in Camarillo CA, and Sept 19, 2016 in Santa Maria, CA. Results were presented orally and as a written document to the California Celery Research Advisory Board on Sept. 19, 2016. What do you plan to do during the next reporting period to accomplish the goals?We will test the remaining accessions in the UC Davis germplasm collection for resistance to Foa race 4, and will test the progeny of our current crosses for resistance or tolerance. Any promising lines will be backcrossed to cv. Challenger. We will continue to monitor any spread of the new Foa race 4 in celery fields in California and to determine whether each isolate is the dominant race 4 or the minor race 4 variant (only distinguishable by DNA sequence). We will determine if we can develop an economical strategy for increasing the size of the transplant plugs perhaps 1.5X of the size of current plugs with older transplants. Although we postulate that the "classic" Fusarium wilt on cilantro is not caused by either Foa race 4 or Foa race 2, we have determined that Foa race 4 is highly virulent on cilantro `Long standing.' Consequently, we will determine if Foa race 4 causes symptoms on other cultivars of cilantro, and whether Foa race 4 inoculum is either maintained or increases on cilantro cultivars. (We note that inoculum maintenance and/or increase may occur even in the absence of symptoms, as it does with Foa race 2 and celery cultivar Challenger, which has been called resistant, but should be called tolerant, i.e., asymptomatic but infected). Using quantitative PCR, we will evaluate the root inoculum in the following plants that were asymptomatic when grown in infested soil:' strawberry `Albion,' cabbage `Brunswick,' kale `Italian Lacinato,' romaine lettuce `Little Caesar,' lima bean `Fordhook no. 242', and sweet bell pepper 'Cal Wonder.' We propose to test feasible strategies to help decrease the inoculum of Foa race 4 or race 2 in soil. Immediately after removing the celery crop, producers could apply biocontrol agent(s) to the upper surface of the celery residue. In addition, between plantings, they could chop the residue, bury it to varying depths, and keep the soil wet or let it dry. In pot trials, we propose to produce celery residue with either Foa race 4 or Foa race 2, manipulate the residue with either a biocontrol agent, chopping/burying, or various irrigation regimes, and then after a 1 month period, to quantify the amount of remaining inoculum using Komada's selective medium followed by use of the race 2 and race 4-specific primers to differentiate between the Foa race 2, Foa race 4, and Fusarium spp. other than Foa race 2 or Foa race 4. In order to better understand the aggessiveness of Foa race 4, first, we are proceeding to use third generation sequencing of larger size inserts than we used previously in order to compare the DNA sequence of the highly virulent race 4 with the highly similar but weakly virulent race 3. We hope that this will give us insights about how new highly virulent strains arise, and consequently, how we might reduce the chances of another new strain arising. Foa race 2 and Foa race 4 are actually in different species, and consequently we will investigate some of the differences between the two strains that might have ramifications for the manifestation of the disease and its control; we are particularly interested in the optimal temperature for growth of Foa race 2 vs. race 4, and whether vascular infections are occurring at the root tips, at the points of lateral root emergence, or on the crown and if so where. We plan to use our race-specific PCR primers to quantify the growth and movement of the Foa race 4 and Foa race 2 in resistant vs. susceptible germplasm. The development of a hypothesis about Foa race 4 resistance could be critical for developing a rapid assay for selection of resistant vs. susceptible progeny obtained in our crosses.

Impacts
What was accomplished under these goals? We continue to screen the UC Davis Apium germplasm collection for either tolerance or resistance to the new race 4 of Fusarium oxysporum f. sp. apii (Foa race 4), and to initiate crosses with resistance or tolerance to cv. Challenger.To date, we have screened 225 accessions for resistance/tolerance to Foa race 4: 115 celery; 66 celeriac; 25 smallage; 5 celery x celeriac; and 14 miscellaneous Apium; we have almost finished the current UCD and USDA Apium collections. Promising accessions have been rescreened in an eight-week trial in the greenhouse in infested soil. Promising accessions are then tested in a field trial. The celery A0251 was put in the field trial, and then rejected as a potential source of resistance. So far, the celeriacs A0102 (PI 164944), A0134 (PI 181714) and A0103 (PI 165064) have performed the best in the field trials; A0103 was previously called a smallage, but it is actually a celeriac type. The most promising material to date has been crossed with Challenger. This year, we made eight crosses involving Challenger (as male and female) with either Smallage (A0103, PI165064, from Turkey), a celeriac (A0102), or another celeriac (A0134). Seeds were collected and are currently being screened for resistance/tolerance in order to 1) assess the number of genes involved in the resistance/tolerance and 2) for potential use in further breeding as F1for either selfing or for backcrossing to Challenger. We continue to monitor any spread of the new Fusarium oxysporum f. sp. apii (Foa) race 4 in celery fields in California. As of Sept. 2016, we detected Foa race 4 in twelve fields in Camarillo and one in neighboring Santa Paula. Based on the 628 bp from an intron-rich EF-1α and 640 bp from rDNA IGS, twelve fields (including the Santa Paula field) have identical sequence. However, one of the Foa race 4 has a slight variant. All appear to be equally virulent. This year we selected a Foa race 4 primer pair that does not amplify Foa race 3. To select potential primers, using genome sequences from either the Broad Institute's Fusarium oxysporum species complex (FOSC) comparative Fusarium genome project (FCGP), the NCBI's Genbank, or our own Illumina next generation sequences (NGS) FOSC discussed in our previous year's report, we selected 1 kb segments that appeared to be present in only Foa race 2 or race 4, and not in any of the other celery NGS, the 12 reference FOSC genomes, Genbank or FCGP. The similarity of Foa races 3 and 4 was further indicated by the fact that we ultimately only identified one amplicon for Foa race 4 primer design, in contrast to multiple options for Foa race 2 primers. We tested the specificity of the selected primers using 18 isolates from celery that were not in the NGS pool, and 12 other F. oxysporum forma specialis that were not used in selecting the primers. Of the 36 non-Foa race 2 or race 4-type tested, only 1, a relatively uncommon non-pathogenic isolate (249-1A) amplified with the Foa race 2 primer pair and only two relatively uncommon non-pathogenic isolates (273-2B and 273-1B) amplified with the Foa race 4 primer pair, indicating that there can be false positives. Nonetheless, despite some false positives, the primers are useful for relatively rapid (2-day) diagnosis of Foa race 2 and race 4 from infected plants in the field. We continue to develop an integrated control program for Fusarium yellows, particularly for race 4, but also to reduce inoculum levels of race 2 in soil. The California Celery Research Advisory Board trials clearly demonstrate that Foa races 2 and race 4 inoculum survives in soil between plantings, and we have started to determine whether inoculum increases, decreases or is maintained on rotation crops and weeds. The experiments are important because at least some strains of Fusarium oxysporum can infect and replicate to a certain extent in the cortical cells of plants in which it does not cause disease. Foa race 4 is a pathogen of the one celeriac cultivar tested (cv. Longstanding). In contrast, Foa race 4 (or Foa race 2) is not a pathogen of the single cultivars tested of lettuce, strawberry, bell peppers, cabbage, kale and lima beans. We have collected material, but have not yet determined whether there are detectable levels of inoculum in the crown and root tissue of these "non-hosts." In greenhouse experiments, we demonstrated that 3-month old transplants (90% survival at 35 days post-transplantation) survive significantly longer than 42-day old transplants (100% dead at 35 days post-transplantation). Growers could transplant older plugs. Although we can quantify Foa race 2 and race 4 biomass in celery crowns using quantitative PCR with the race-specific primers, we have been unable to quantify Foa race 2 and race 4 inoculum in soil, partly because the inoculum concentrations are diluted in soil compared to celery crowns, there are more PCR inhibitors in soil than in celery crowns, and the soil inoculum is aggregated and not uniformly dispersed in soil. We are now successfully using Komada's (Komada, 1976) selective medium to quantify the total Fusarium colony forming units in a commercial field soil that resemble Foa race 2 and race 4, followed by using the race 2- and race 4-specific primers to indicate the Foa race 2 and Foa race 4 inoculum concentration. For the second year in the CCRAB trial in Santa Maria, we showed that the highly susceptible Tall Utah and the moderately susceptible Sonora produced significantly more phenolics in the crowns than the tolerant Challenger. While there was more Foa race 2 in the Tall Utah crowns in both 2014 and 2015, than in either Sonora or the tolerant Challenger, there were indistinguishable amounts of Foa race 2 DNA in the crowns of Sonora and Challenger. Our hypothesis is that while phenolic production might be part of the resistance response, susceptible varieties over-produce phenolics and thereby damage the phloem, cambium and xylem parenchyma tissue. If the hypothesis that Fusarium yellows symptoms in celery are caused by an over-production of phenolics, there could be both non-GMO mutagenic and cisgenic means to select for cultivars with reduced phenolic production.

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: Epstein, L., Kaur, S., Chang, P., Carrasquilla-Garcia, N., Lyu, G., Cook, D., Subbarao, K., O'Donnell, K. 2017. Races of the celery pathogen Fusarium oxysporum f. sp. apii are polyphyletic. Phytopathology 107


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:The target audience are primarily celery producers in California, the California Celery Research Advisory Board (CCRAB), which represents the celery producers, and celery breeders in the private sector that work, at least partly, in California. Secondarily, a larger national and international audience can access our reports via the CCRAB. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Four undergraduates were trained: Quyen Pham, Hart Calligagan, Nancy Villalpando and Henry Teicheira. One Chinese visiting scientist, Dr. Lyu Guiyun was trained. How have the results been disseminated to communities of interest?Results were presented at two field days sponsored by the California Celery Research Advisory Board (CCRAB) on 29 June 2015 in Oxnard, CA and on 23 Sept. 2015 in Santa Maria, CA and results were presented orally and as a written document to the CCRAB on 23 Sept. 2015 in Santa Maria. What do you plan to do during the next reporting period to accomplish the goals?1. To continue to screen the UC Davis Apium germplasm collection for either tolerance or resistance to the new race 4 of Foa (Foa race 4), and to initiate crosses with resistance or tolerance to cv. Challenger. 2. To continue to monitor any spread of the new Foa race 4 in celery fields in California. 3. To characterize a putative race 4 variant that we isolated regarding its pathogenicity and DNA sequence. 4. To maintain the most valuable components of the UCD Apium germplasm collection for pathogen and pest resistance. 5. To develop an integrated control program for Fusarium yellows, particularly for race 4, but also to reduce inoculum levels of race 2 in soil. 6. To better understand the Foa-celery interaction and particularly the mechanism of resistance of cv. Challenger to race 2 and the lack of resistance in race 4. 7. As new technology and/or information becomes available, to develop an assay for determination of soil-borne inoculum concentration of the "classic" California Foa race 2 and the new race 4 in growers' fields

Impacts
What was accomplished under these goals? In 2013, a new Fusarium oxysporum f. sp. apii (Foa) race 4 was discovered in three celery production fields in Camarillo, California; Foa race 4 isolates were highly virulent on all celery cultivars including cv. Challenger in the CCRAB Camarillo/Oxnard 2013 trial. Two additional celery fields in Camarillo with Foa race 4 were identified in 2014. As of 20 Oct. 2015, we have identified Foa race 4 in six fields; all isolates are equally virulent, but based on a total of 1468 nucleotides, one of the isolates is a slight DNA-variant. This past year, we screened 101 germplasm accessions for either resistance or tolerance to Foa race 4 in the greenhouse and 5 accessions in the field. We are proceeding with crossing one acession from the field trial to Challenger, have one additional accession that has passed our second greenhouse screen and is ready for a field trial, and one accession that is being retested in the greenhouse. Based on our next generation sequencing (NGS), Foa race 4 is very closely related to Foa race 3, which was present in California in the 1980's, but has apparently not been collected since then. Foa races 3 and 4 are also related to Foa race 1, which appears to be a polymorphic group of strains which were first detected the early 1900's. The Foa races 1,3, and 4 are in a different ?clade"/species than Foa race 2, which is avirulent on cv. Challenger and other cultivars with the UC-derived resistance gene(s). That is, based on our NGS analysis of six Foa race 2, one isolate of Foa races 1,3 and 4, and 7 non-pathogenic strains isolated from symptomatic celery, Foa race2, which appeared in California in 1976, is relatively unrelated to the new Foa race 4, which appears to be a highly virulent variant of the comparatively weakly virulent Foa race 3. Based on our NGS in comparison to on-line bioinformatic resources at the Broad Institute and the NCBI GenBank, we identified Foa race 2-specific polymerase chain reaction (PCR) primers that we can be used for both rapid (one to two-day) diagnosis of infected plants and quantification of Foa race 2 in celery crowns. We also identified PCR-primers that recognize only Foa race 4 or race 3. We used the Foa race 2-specific PCR to quantify the amount of Foa race 2 biomass in crowns of cvs. Challenger, the moderately Foa race 2-susceptible Sonora and the highly susceptible Tall Utah in a field trial in Foa race 2-infested soil.. The results indicate that cv. Challenger is partly immune to Foa race 2; Challenger has significantly fewer crowns with Foa race 2 and significantly less Foa race 2 in the infected crowns than Tall Utah. However, cv. Challenger also appears to be partly tolerant to Foa race 2; Challenger has significantly fewer crowns with Foa race 2 than Sonora, but of those that are infected, Challenger has no less Foa race 2 than in the infected crowns in Sonora.

Publications


    Progress 10/01/13 to 09/30/14

    Outputs
    Target Audience: The target audence are primarily celery producers in California, the California Celery Research Advisory Board (CCRAB), which represents the celery producers, and celery breeders in the private sector that work, at least partly, in California. Secondarily, a larger national and international audience can access our reports via the CCRAB. Changes/Problems: Initially, we proposed to work more on Foa race 2 and on other celery pathogens including Septoria. With the emergence of Foa race 4, we will focus exclusively on Fusarium yellows, and primarily on management of race 4. What opportunities for training and professional development has the project provided? The following undergraduates have been trained for this project in this reporting period: Nancy Villalpando, Quyen Pham, Jonathan Vaccari, and Henry Teicheira. How have the results been disseminated to communities of interest? Results were presented at two field days sponsored by the California Celery Research Advisory Board (CCRAB) on 23 May 2014 in Oxnard, CA and on 15 Sept. 2014 in Santa Maria, CA and results were presented orally and as a written document to the CCRAB on 15 Sept. 2014 in Santa Maria. What do you plan to do during the next reporting period to accomplish the goals? 1. To continue to screen the UC Davis Apium germplasm collection for either tolerance or resistance to the new race 4 of Foa 2. To continue to monitor any spread of the new Foa race 4 in celery fields in California 3. To complete the next generation sequence analysis needed for Objective #4 of the 14 isolates from California celery with symptoms of Fusarium yellows 4. Using information from Objective #3, to develop a DNA-based assay for determination of inoculum concentration of the "classic" California Foa race 2 and the new race 4 in growers' fields

    Impacts
    What was accomplished under these goals? We identified a new race of Fusarium oxysporum f. sp. apii (Foa), which we call race 4 in four fields in Camarillo, California. Identification of the new race was based on virulent pathogenicity tests on celery cv. Challenger, Tall Utah and Golden Self Blanching. For reference, the historic race 1, which has never been identified in California, is only virulent on Golden Self Blanching. The historic isolate in California, called race 2, is virulent on Golden Self Blanching and Tall Utah, but Challenger and other cultivars derived from crosses with celeriac that were made by Orton and colleagues at the University of California in the 1980's are tolerant. In keeping with the literature by Puhalla from the 1980's, we call strains that are virulent on Tall Utah but avirulent on Golden Self Blanching and Challenger race 3; we identified an isolate with these characteristics in a culture collection, but do not isolate them currently. Except for the race 4 isolates collected 2013-2014 from Camarillo, all of our virulent isolates from celery with symptoms of Fusarium yellows are race 2. In the field and in greenhouse assays, race 4 seems extremely aggressive and appears to kill younger plants than race 2. Once infected, race 4 appears to more rapidly degrade the crown into a more watery rot than does race 2. In addition to characterization to race based on host response, we have determined DNA sequence at two loci (a total of 1267 base pair) that are used to identify Fusarium spp. and F. oxysporum: an intron-rich region of the elongation factor 1-alpha gene and a portion of the ribosomal DNA intragenic spacer (rDNA IGS). Race 2 and race 4 are readily distinguishable by DNA sequence and each is monomorphic, consistent with an asexually reproducing species. In addition to isolating either Foa race 2 or race 4 from symptomatic tissue, we typically isolate other F. oxysporum strains; these strains are clearly distinguishable in sequence from the race 2 and race 4 and are polymorphic. We selected 14 F. oxysporum isolates from symptomatic celery: five race 2 isolates selected from primarily two locations over the last twenty years; one race 4 isolate, six of the polymorphic non-pathogenic "clade 5" isolates that we detect in celery with Fusarium yellows but that don't cause symptoms; and two of the polymorphic non-pathogenic clade 1 isolates that we less frequently detect in plants with Fusarium yellows, but that don't cause symptoms. Illumina Mi-Seq was used to generate 10X coverage with approximately 200 bp paired-end reads. We did both a de novo assembly and assemblies against the full genome assemblies of ten F. oxysporum strains available from the Broad Institute. Based on comparisons to the NCBI and Broad comparative genome datasets, we have selected what appear to be Foa race 2 and race 4-specific primers for polymerase chain reaction (PCR); we are currently testing the primers for specificity and efficiency. After validation, we can use these primers for rapid diagnosis of race and hopefully for specific quantification of each race in soil and in plant tissue. The 2013 California Celecry Research Advisory Board trial in race 4 soil resulted in 100% loss of all commercial cultivars and all of the current University of California at Davis (UCD) breeding lines. Given that we had a new pathogen with no known resistance to Foa race 4, we immediately started to screen the UCD celery germplasm collection for tolerance or resistance, primarily in the greenhouse. Conclusions are as follows. 1) Although we are continuing to try to reduce trial-to-trial variability, our results are at least as reproducible as those historically obtained from field trials. 2) Finding resistance against race 4 will be a much greater challenge than finding resistance to race 2, i.e., a relatively high percentage of tested celeriac have resistance to race 2 --this is not true for race 4. 3) Nonetheless, we do have five accessions that are currently being field tested, and we are continuing to test the entire germplasm collection.

    Publications


      Progress 01/01/13 to 09/30/13

      Outputs
      Target Audience: The target audience are primarily celery producers in California, the California Celery Research Advisory Board (CCRAB), which represents the celery producers, and celery breeders in the private sector that work, at least partly, in California. Secondarily, a larger national and international audience can access our reports via the CCRAB. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? We have trained five undergraduate students to assist with the project during the past year: William Goss, Christopher Hart, Selene Bello, and Nancy Villalpando. How have the results been disseminated to communities of interest? Results were presented at two field days sponsored by the California Celery Research Advisory Board CCRAB (11 June 2013 in Oxnard, CA and 16 September 2013 in Santa Maria, CA) and results were presented orally and as a written document to the CCRAB on 16 September 2013 in Santa Maria. What do you plan to do during the next reporting period to accomplish the goals? We are performing next generation sequencing on 14 Fusarium oxysporum isolates from symptomatic California celery; the isolates were selected primarily to represent the isolate diversity based on DNA seqiemce at two loci, pathogenicity, and virulence. Our specific objectives for the next reporting period are the following. One, to determine if the unusually severe disease 2013 California Celery Research Advisory Board field trial in Oxnard was due to a clade 1 versus a clade 3 isolate, unusually high inoculum in the soil, and/or, to unusual environmental conditions. Two, to screen the University of California germplasm collection and breeding lines for resistance to the clade 1T isolate. Three, to develop a DNA-based assay for determination of F. oxysporum f. sp. apii (Foa) inoculum concentration in soil that will be useful to growers. Four, to continue to optimize a laboratory and greenhouse assay for resistance to Foa so that a) we can have multiple cycles of screening per year, b) we can increase the inoculum concentration of Foa and consequently can screen under more intense disease pressure than has typically occurred in field trials in most years, and c) we can have a quantitative measure of resistance to Foa so that we can more sensitively detect effects of different resistance genes and additive effects of quantitative resistance genes. Five, to determine if we can differentiate between resistance to Foa from the major dominant gene derived from celeriac and other sources of Fusarium-resistance.

      Impacts
      What was accomplished under these goals? We have determined the DNA sequence from two loci (an intron-rich portion of the translation elongation factor gene EF-1α and a portion of the IGS rDNA) of 209 Fusarium oxysporum isolates from symptomatic celery and tested the pathogenicity of most of them. Thirty eight of the isolates were collected before 2010, mostly by Krishna Subbarao between 1993 and 1996 with the earliest in about 1982. One eighty one of the isolates are from California, seven are from Michigan, six are from Ohio, 11 are from Wisconsin, and five are from Ontario, Canada. We also have access to DNA sequence of five previously identified as F. oxysporum f. sp. apii (Foa) isolates from a study by O'Donnell et al. from 2009 that examined two isolates from Germany, two isolates that were presumably from Europe, and a “T” isolate that was donated from the Smith-Snyder collection in 1981; these five isolates are from a highly diverse but related group that we call “clade 1.” Before 2013, all of the isolates from celery in our collection could be classified as either a single strain with invariant DNA sequence that generally cause discoloration in tissue (“clade 3”) or a diverse group of isolates that generally do not cause discoloration (“clade 2”). In 2013, in a field trial in Oxnard, California, in subsequent greenhouse experiments with the soil, and from two other fields in Oxnard, California, we isolated a highly virulent “clade 1” isolate with the same sequence as the “T” strain from the Smith-Snyder collection; this isolate was not recovered from either another fall Oxnard trial (in a different field) or in a field trial in Santa Maria, California in 2013. Thus, there are now at least two highly distinct strains that cause Fusarium yellows in celery on California: the monomorphic clade 3, which has been present since the 1980's, and an apparently monomorphic clade 1T, which may be limited to a relatively small geographic area in Oxnard, California. We have not found the clade 1T and the clade 3 in the same field. Celery lines derived from the University of California celeriac with resistance to clade 3 (e.g., Challenger) appear to be susceptible to clade 1T. We conclude the following: 1) if the isolates in the 2009 O'Donnell study are representative of those in celery fields in Europe, the agents of Fusarium Yellows in Europe and in most of California (between 1980 and 2013) have been very different. 2) In comparison to the clade 3 strain, the clade 1 “T” strain appears to be more virulent and the classic UC resistant lines such as Challenger appear to be more susceptible. 3) Internal symptomatic tissue in celery contains a community of F. oxysporum isolates dominated by members of a highly diverse “clade 2,” which cause little or no tissue discoloration, but which appear to have a range of virulence based on a newly developed laboratory seedling assay that we have recently developed. 4) The seed assay suggests that juvenile tissue is more susceptible to F. oxysporum f. sp. apii isolates and that these isolates may produce a toxin.

      Publications


        Progress 01/01/12 to 12/31/12

        Outputs
        OUTPUTS: Results of this work were shared on 25 September 2012 with the California Celery Research Advisory Board's Field Day in Santa Maria, CA and meeting in Guadalupe, CA. PARTICIPANTS: Dr. Sukhwinder Kaur and Mr. Vincent D'Antonio are Staff Research Associates working on the project at UC Davis. Four University undergraduates in the laboratory and greenhouse also have worked on the project. The California Celery Research Advisory Board supports the project. We collaborate with scientists at the USDA in Salinas, CA particularly with Dr. Krishna Subbarao and Dr. Beiquan Mou, and with Mr. Richard Hurstak in Crop Science Services in Paso Robles. TARGET AUDIENCES: The target audience are celery farmers, and celery breeders from either private seed companies or from the public sector (including Universities, USDA, etc.). PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

        Impacts
        Understanding the diversity of the pathogen population is important in both breeding and control programs because isolates that can cause disease in a "resistant" cultivar always have a selective advantage over isolates that cannot. Consequently, isolates can change over time, and it's important that the screening locations for disease resistance have the diversity of isolates present in California's major celery production areas. We assembled a collection of now 162 isolates from celery with Fusarium yellows symptoms (including 40 that Dr. Krishna Subbarao collected primarily between 1993 and 1996) and determined DNA sequence from two loci (an intron-rich portion of the translation elongation factor gene EF-1α and a portion of the IGS rDNA). We also had access to DNA sequence of five previously identified F. oxysporum f. sp. apii isolates from a study by O'Donnell et al. (2009) that examined two isolates from Germany, one isolate from California and two other isolates that are presumably from Europe. To summarize, 1) if the isolates in the O'Donnell study are representative of those in celery fields in Europe, the agents of Fusarium Yellows in Europe and in California are very different. 2) To date, all of the pathogenic (or more precisely all isolates that cause vascular discoloration in celery) isolates from our California celery collection are consistent with being clones, as one would expect from a member of the asexual species Fusarium oxysporum. The lineage has a unique DNA sequence and contains isolates that appear to have a range of virulences from highly virulent to avirulent. This pathogenic population apparently dominated since the mid 1990's in California and in other states in which we have samples, and continues to dominate now. 3) As far as we can tell, the two Fusarium trial sites in California do represent the pathogen(s) in growers' fields. 4) Internal symptomatic tissue in celery contains a community of F. oxysporum isolates including members of "clade 2," which do not appear to induce tissue discoloration or other symptoms when inoculated by themselves. 5) The non-pathogenic isolates in clades 1 & 2 (and the weakly pathogenic isolates in clade 2) are not "race 1" isolates, i.e., are not pathogenic on Self-Blanching Golden. In addition to working on the pathogen population of F. oxysporum f. sp. apii that causes Fusarium yellows, we are also continuing work from the previous breeding program on identifying and incorporating resistance from germplasm in the Apium collection at UC Davis: resistance to late blight caused by Septoria in Apium chilense, a wild species related to celery; celery mosaic virus (CeMV) resistance from a feral celery accession collected in Lompoc, CA; leafminer resistance from the wild celery relative Apium prostratum; and additional Fusarium yellows resistance in multiple accessions.

        Publications

        • No publications reported this period