To be completed later.
The purpose of my research project is to assist growers in the management of tomato leaf mold through characterization of isolates collected between 2017-2019. The research will allow us to provide better recommendations to growers and extension specialists about the most resistant varieties of tomatoes to grow, and if the pathogen is surviving on-farm over winter.
1) Complete a final collection of tomato leaf mold samples from high tunnels throughout New York State in 2019. A final year of sample collection from across NYS State will contribute to an isolate collection that began in 2017. Samples will be received from growers and extension specialists and I will travel to collect samples in August in locations where tomato leaf mold has been a concern. Isolates will be obtained from lesions on foliage and single conidial isolates will be obtained in order to ensure that each isolate is genetically pure. I will have a total sample size of ~50 isolates.
2) Determine the race of Passalora fulva isolates collected between 2017-2019. To determine race, each isolate will be inoculated on a differential set of tomatoes containing a specific resistance gene. There will be three replications of each inoculation and negative water controls. Observations will be recorded based one whether disease symptoms are seen on any of the 11 tomato varieties carrying a different resistance gene after inoculation with each isolate. This protocol has been used to correctly determine the race of P. fulva isolates [7,8].
3)Evaluate the efficacy of currently available resistant tomato varieties. As we are determining the race of each isolate, each isolate will also be used to inoculate commercially available varieties that have been described as resistant and were found to be resistant in our previous trials using only a single isolate. Varieties include: Primo Red, Red Mountain, Red Deuce, BeOrange, Rebelski and Gereonimo. The susceptible grower favorite SunGold will also be included as a control. Because growers have reported some disease on tomato varieties thought to be resistant, testing each isolate against each tomato variety is critical.
The purpose of my research project is to better understand the population diversity of the tomato leaf mold pathogen Passalora fulva. Growers and extension specialists have wondered whether the pathogen population is diverse or not as they try to manage the disease in high tunnels. The high tunnel environment is conducive to the growth of the fungus. This is problematic because high tunnels have been an important way to extend the growing season on high value crops such as tomatoes. The disease can lead to defoliation and lower yields. Race determination and genetic analyses of the pathogen will have important implications for management and choosing resistant varieties of tomatoes to grow. If resistant tomato varieties are planted, fewer fungicides will be needed and high tunnel production of tomatoes will be more profitable.
In order to complete high tunnel experiments, tomato seed was obtained from the Centre for Genetic Resources in the Netherlands and C. M. Rick Tomato Genetics Resource Center in order to prepare a differential set of tomatoes. Tomatoes with no resistance genes (Cf-0) to Passalora fulva and tomatoes with one resistance gene including Cf-1, Cf-2, Cf-3, Cf-4, Cf-5, Cf-6, Cf-7, Cf-8, Cf-9, or Cf-11 were included in the differential set of 11 tomatoes. Plants were grown to maturity in the spring of 2019 and tomato seed was harvested over the course of several months. Seed was cleaned and sorted and stored for future experiments.
Once a differential set was prepared with adequate seed, high tunnel and inoculation preparation began. I covered the high tunnel with weed cloth to suppress weeds and then organized the tunnel so that blocks were evenly distributed in the high tunnel, one meter apart at the end of July 2019. Each block could hold 12, 1-gallon pots (equal to the number of plants in the differential set). Tomatoes were put in the high tunnel in pots in order to ensure easy clean-up and fast-turn around in between experiments. In past, we’ve planted tomatoes successfully in the high tunnels, but the potted experiment was necessary in order to get through many experiments during the growing season and also to ensure less possible cross-contamination of isolates.
The high tunnel fit 26 blocks, as well as a control block for a total of ~300 plants/experiment. Within each experiment, single isolates were used as inoculum for two blocks each, so 13 isolates in total could be tested at once for race determination assays. A random number generator was used to organize the blocks randomly for each isolate and also to organize the pots randomly within each block so different genotypes were distributed randomly within a block.
Meanwhile, additional isolates were also sent to us from extension specialists in NY and Vermont. Most of the isolations were successful (after a few years of struggling to isolate the casual fungus). Single conidial isolations were prepared for each isolate and afterwards, isolates were stored in 30% glycerol stocks for safe-keeping.
Seeds were planted in 50 cell flats in the greenhouse for 4 weeks. Once true leaves appeared, plants were fertilized to prevent nutrient deficiency like phosphorus deficiency, which can make ratings more challenging. At four weeks, plants were transplanted in 1-gallon pots and transported to the high tunnel. Plants were stabilized with string woven along the sides of pots and attached to stakes. Each block consisted of a line of pots. The recommended amount of slow-release osmocote (15-09-12 at ~15 grams/pot) was applied to each pot to ensure that plants were healthy during the time of data collection.
Inoculum was prepared in clean ½ gallon handheld pressure sprayers. Inoculum was dispersed onto tomatoes so that each leaf contained an even distribution of inoculum, with care to sanitize hands in between inoculations with different isolates. After 14 days, symptoms and signs appeared and three ratings were conducted over the course of 7-10 days. The disease was not seen evenly throughout the plants because disease begins on older leaves before progressing upward but clear signs and symptoms were seen on tomatoes that were susceptible, making ratings for presence and absence of disease straightforward. As an added control, the fungal pathogen was reisolated onto PDA to ensure that the pathogen was indeed present on symptomatic leaves.
If there were any discrepancies between replicated blocks, isolates were again used as inoculum in a subsequent experiment. Any results that seemed unusual were also repeated. Work will resume in spring and summer 2020 to complete the required race assays.
Data collection will continue until early summer 2020 and this section is a work in progress. During the course of the summer and fall of 2019, race assays were conducted on 35/50 isolates in our collection. Isolates collected in 2019 will be assayed in May 2020.
The results were showed that there was not much diversity between isolates. Plants containing Cf-0, Cf-1, Cf-2, and Cf-3 were most often susceptible following inoculation with all but three isolates from 2017 that were isolated from near or on Long Island (Suffolk County). When tomato blocks were inoculated with one of these three isolates, Cf-2 showed no disease symptoms, unlike with the rest of the isolates used as inoculum. These results will again be validated in the spring of 2020 in repeated experiments. Data will be collected to determine the race of the 2019 isolates. A second set of experiments will be completed in 2020 once it is warm enough and the environment is conducive to disease.
Results will be considered alongside the sequencing of avirulence genes/effectors. Only Avr2, 3, 4, 4e, and 5 have been cloned. Of this subset of genes of interest, we’re particular interested in the interaction between the resistance gene Cf-2 and Avr2, so attention will be placed on sequencing these genes and comparing the sequence results to some reference strains.
Education & Outreach Activities and Participation Summary
My data collection began in late summer 2019 and will be completed in early summer 2020. I will then prepare the bulk of my extension and outreach materials and talks. In the meantime, I’ve revised a tomato leaf mold factsheet that will be made available online and in print to provide information about tomato leaf mold and best management practices. I anticipate revising the factsheet further as my final results emerge. It is ADA-compliant and will be made available online after final revisions. Later this summer I’ll complete a journal article, a grower newsletter article, and share my results with growers and extension educators at a talk.
This is in progress. As part of my research, I began a collaboration with researchers at the University of Minnesota, including Lillian McGilp. The data collection continues for another season, but already I foresee being able to help breeders in the development of more resistant tomatoes. This in turn will directly impact growers and their ability to choose more resistant varieties of tomatoes.
The project is in progress. I have begun to understand the population diversity of Passalora fulva, the causal agent of tomato leaf mold. This will help inform management practices and efforts to breed more resistant tomato varieties. In turn growers will see less disease and fewer inputs in high tunnels will be necessary to manage the disease. To better understand diversity, I’ll be performing genotyping-by-sequencing of isolates that I collected during this project. This future work is independent of my race determination work funded by the SARE grant. I’ll be able to understand with more precision whether significant diversity exists across isolates and I’ll be able to use population genetics tools to better understand how the pathogen population may be changing over time.
This section is in progress.